Antibacterial activity of Brazilian propolis and fractions against oral anaerobic bacteria

$: Antibacterial activity of Brazilian propolis and fractions against oral anaerobic bacteria$
Review

Antibacterial activity of Brazilian propolis and fractions against oralanaerobic bacteria

F.A. Santos a,b, E.M.A. Bastos c, M. Uzeda b, M.A.R. Carvalho a, L.M. Farias a,E.S.A. Moreira a,*, F.C. Braga d

a Departamento de Microbiologia, Laboratorio de Biologia de Microrganismos, Instituto de Ciencias Biologicas da Universidade Federal de Minas

Gerais, Caixa Postal 486, Avenida Antonio Carlos 6627, CEP 31270 901, Belo Horizonte, MG, Brazilb Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

c Fundacao Ezequiel Dias-Belo Horizonte, Belo Horizonte, Minas Gerais, Brazild Faculdade de Farmacia, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil

Received 1 May 2001; received in revised form 24 December 2001; accepted 29 December 2001

Abstract

Propolis collected from a cerrado area in Minas Gerais State, Brazil, was subjected to chromatography on silica gel column and to

partition between immiscible solvents. Propolis aqueous-ethanolic extract and fractions obtained were tested for inhibitory activity

against periodontitis-causing bacteria. All of the assayed bacterium species were susceptible to propolis extract. The two

fractionation methodologies yielded fractions which were active against bacteria, with minimum inhibitory concentrations (MIC)

ranging from 64 to 1024 mg/ml. TLC and HPLC analyses of the extract and of active fractions showed the presence of phenolic

compounds of varied polarity. None of the assayed fractions was more active than the extract, suggesting that the antibacterial

activity is probably due to the synergistic effect of several compounds. # 2002 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Propolis; Antibacterial activity; Anaerobes; Phenolic compounds

1. Introduction

Propolis is a resinous material collected by bees from

plant buds and exudates, which is employed for

construction and repair of the honeycomb (Ghisalberti,

1979; Asis, 1991). Propolis has been used in folk

medicine for curing infections for hundreds, perhaps

thousands of years (Cheng and Wong, 1996). Several

biological activities have been described for propolis,

including antibacterial (Grange and Darvey, 1990;

Ikeno et al., 1991; Kujumgiev et al., 1993; Menezes et

al., 1997), antifungal (Valdes et al., 1987), antiprotozoan

(Scheller et al., 1977), antiviral (Amoros et al., 1992),

antitumor (Grunberger et al., 1988), immunomodula-

tion (Dimov et al., 1992) and antiinflammatory (Do-

browolski et al., 1991) activities, among others. Propolis

is a complex mixture of chemical constituents and its

composition is dictated by the constituents of the plant

material making up the native vegetation and by the

season of collection (Ghisalberti, 1979; Vanhaelen and

Vanhaelen-Fastre, 1979; Bankova et al., 1992; Mar-

cucci, 1995; Cheng and Wong, 1996; Oliveira and

Bastos, 1998; Bankova et al., 2000).

The development of new therapies for the treatment

of the diseases of the oral cavity is of great relevance,

since the systemic administration of antimicrobials has

been reported to cause the development of multiresistant

microorganisms, interbacterial transfer of resistance

determinants, and side effects (Walker, 1996).

Actinobacillus actinomycetemcomitans is a Gram-ne-

gative bacterium associated with a variety of infectious

diseases such as brain abscess, urinary tract infections,

periodontal diseases and special cases of localized

juvenile periodontitis (Slots, 1982; Kaplan et al., 1989).

Species of Fusobacterium belong to the human micro-

biota, which is normally found in the oral cavity, colon,

genital tract and upper respiratory tract. Fusobacterium

species increase in number in the gingival pockets as

periodontal disease progresses (Tanner et al., 1979;* Corresponding author. Fax: �55-31-3499-2730.

E-mail address: [email protected] (E.S.A. Moreira).

Journal of Ethnopharmacology 80 (2002) 1�/7

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0378-8741/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved.

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Bolstad et al., 1996). Several black-pigmented Gram-

negative anaerobes such as Porphyromonas gingivalis

and Prevotella intermedia , are usually isolated from the

gingival crevice of patients with periodontitis (Dahlen,1993; Rodrigues et al., 1999).

Despite increasing use of propolis worldwide (Mar-

cucci, 1995; Cheng and Wong, 1996) only few studies

have been carried out to determine the inhibitory effect

of propolis against anaerobic bacteria of odontological

relevance (Santos et al., 1999). Within this context, the

aim of the present work was to evaluate the activity of

propolis against nine periodontal disease-causing bac-teria. Some propolis fractions were also evaluated, in

order to verify whether they possess antibacterial

activity.

2. Material and methods

2.1. Propolis samples

Crude samples of bee (Apis mellifera ) propolis were

collected from an experimental apiary located in a

cerrado area, in the city of Cachoeira da Prata, Minas

Gerais State, Brazil. An aliquot of crude propolis (7 g)

was dissolved in 80% ethanol (3�70 ml) by shaking at

70 8C, for 30 min protected from light. The resultingaqueous-ethanol extract was filtered through a What-man 1 paper and concentrated at 50 8C, and theobtained resin was dissolved in 95% ethanol to a finalconcentration of 100 mg/ml (Menezes et al., 1997). Thisfinal solution was employed for the antimicrobialassays.

2.2. Column chromatography

A portion of crude propolis (7 g) was dissolved in 80%

ethanol (3�70 ml) at 70 8C for 30 min. This extractwas concentrated in a rotary evaporator under reducedpressure at 70 8C to approximately 50 ml and parti-tioned with CH2Cl2 (3�50 ml). The organic layer wasconcentrated in a rotary evaporator at 40 8C and theresulting residue was dissolved in CH2Cl2 (2 ml) and wassubmitted to chromatography on a silica gel column(Silica gel 60 Merck 0.2�/0.5 mm; 50�600 mm I.D.).Elution was performed with solvents of increasingpolarity and solvents were removed using a rotaryevaporator, at a temperature 50 8C: n -hexane (21; 60

mg), n -hexane/CH2Cl2 (1:1, 4 l, 120 mg), CH2Cl2 (3 l,

220 mg), CH2Cl2/EtOAc (1:1, 3 l, 1700 mg), EtOAc (2 l,

190 mg), EtOAc/MeOH (1:1, 4.5 l, 560 mg) and MeOH

(7 l, 170 mg).

Fig. 1. Fractionation of propolis aqueous-ethanolic extract by partition between immiscible solvents.

F.A. Santos et al. / Journal of Ethnopharmacology 80 (2002) 1�/72

2.3. Partition between immiscible solvents

A portion of crude propolis (7 g) was dissolved in 80%

EtOH (3�70 ml) at 70 8C for 30 min. This extract wasconcentrated in a rotary evaporator under reducedpressure, at 70 8C, to approximately 50 ml and parti-tioned with CH2Cl2 (3�50 ml). The organic layer wassuccessively partitioned with 5% NaHCO3 aqueoussolution (3�30 ml), 5% Na2CO3 aqueous solution(3�30 ml) and 10% NaOH aqueous solution (3�30ml), according to the sequence depicted in Fig. 1.Solvents were removed in a rotary evaporator, at amaximum temperature of 70 8C.

2.4. TLC analysis

Thin-layer chromatography (TLC) analysis of propo-

lis aqueous-ethanolic extract and its fractions were

performed on silica gel plates (Merck TLC plates).

Samples were dissolved in MeOH (100 mg/ml) and 5ml aliquots were applied to the TLC plates. Elution was

performed with CHCl3/EtOAc (60:40, v/v) and the

plates were visualized under visible and ultraviolet light

(254 and 360 nm), after spraying with 5% AlCl3 in

methanol solution (Wagner et al., 1984).

2.5. HPLC analysis

Propolis aqueous-ethanolic extract and its fractions

were analyzed by high-pressure liquid chromatography

(HPLC). Analyses were carried out in a Merck-Hitachi

apparatus (Germany). An ODS column (250�4.0 mm

I.D., 5 mm) was employed (Merck, Germany) attemperature of 30 8C, flow rate of 1.0 ml/min andwavelength of 254 nm. A linear gradient of H2O�0.1%H3PO4 (A) and CH3CN�0.1% H3PO4 (B) was em-ployed: 0 min, 90% A 10% B; 60 min, 10% A 90% B,followed by 5 min of isocratic elution. Solvents used

Table 2

Susceptibility of nine oral anaerobic strains to propolis fractions obtained from propolis aqueous-ethanolic extract (PAE) silica gel column

chromatography

Bacterial strainsa MIC (mg/ml)

PAE HEXb HEX/DCM DCM DCM/EtOAc EtOAc EtOAc/ MeOH MeOH

Aa FDC Y4 1024 �1024 �1024 �1024 1024 1024 �1024 �1024

Aa ATCC 29 523 1024 �1024 �1024 �1024 1024 1024 �1024 �1024

Fn ATCC 10 953 1024 �1024 �1024 �1024 1024 1024 �1024 �1024

Fne ATCC 25 386 256 �1024 �1024 256 64 64 64 64

Pg ATCC 33 277 256 �1024 �1024 512 512 1024 1024 1024

Pi ATCC 25 611 256 �1024 �1024 128 64 256 128 128

Pn ATCC 33 563 512 �1024 �1024 256 128 512 1024 1024

El ATCC 25 559 1024 �1024 �1024 �1024 �1024 �1024 �1024 �1024

Pa ATCC 27 337 128 �1024 �1024 256 64 128 64 256

a Aa, A. actinomycetemcomitans ; Fn, F. nucleatum; Fne, F. necrophorum ; Pg, P. gingivalis ; Pi, P. intermedia ; Pn, P. nigrescens ; El, Eubacterium

lentum ; Pa, P. anaerobius.b HEX, Hexane; HEX/DCM, Hexane/Dichloromethane; DCM, Dichloromethane; DCM/EtOAc, Dichloromethane/Ethyl acetate; EtOAc/MeOH,

Ethyl acetate/Methanol; MeOH, Methanol.

Table 1

Susceptibility of nine oral anaerobic strains to propolis fractions obtained from propolis aqueous-ethanolic extract (PAE) partition between

immiscible solvents

Bacterial strainsa MIC (mg/ml)

PAE Aqueous fraction NaHCO3

fraction Na2CO

3fraction NaOH fraction Final residue Meropenem Penicillin G

Aa FDC Y4 1024 �1024 �1024 1024 �1024 �1024 0.5 4

Aa ATCC 29 523 1024 �1024 �1024 512 �1024 �1024 0.5 4

Fn ATCC 10 953 1024 �1024 �1024 512 �1024 �1024 0.06 4

Fne ATCC 25 386 256 �1024 �1024 512 �1024 �1024 0.03 1

Pg ATCC 33 277 256 �1024 256 256 �1024 1024 1 0.03

Pi ATCC 25 611 256 �1024 256 256 �1024 512 2 0.06

Pn ATCC 33 563 512 �1024 256 512 �1024 512 2 0.03

El ATCC 25 559 1024 �1024 �1024 512 �1024 �1024 8 4

Pa ATCC 27 337 128 �1024 256 256 1024 512 8 4

a Aa, A. actinomycetemcomitans ; Fn, F. nucleatum ; Fne, F. necrophorum ; Pg, P. gingivalis ; Pi, P. intermedia ; Pn, P. nigrescens ; El, E. lentum ; Pa, P.

anaerobius .

F.A. Santos et al. / Journal of Ethnopharmacology 80 (2002) 1�/7 3

were of HPLC grade (Merck, Germany) and weredegassed by sonication before use. Samples were dis-solved in methanol to concentrations of 10 and 5 mg/ml,for propolis aqueous-ethanolic extract and fractions,respectively. The solutions were centrifuged at 10 000rpm before injection to HPLC.

2.6. Bacterial strains

A total of nine microbial strains were tested: A.

actinomycetemcomitans ATCC 29 523 and FDC Y4,

Fusobacterium nucleatum ATCC 10 953, Fusobacterium

necrophorum ATCC 25 386, P. gingivalis ATCC 33 277,P. intermedia ATCC 25 611 and Prevotella nigrescens

ATCC 33 563, Eubacterium lentum ATCC 25 559 and

Peptostreptococcus anaerobius ATCC 27 337. All strains

were kept in our laboratory and cryopreserved at

�86 8C. For experiments bacteria were inoculated inBrucella agar supplemented with 0.5% yeast extract,hemine (5 mg/ml), menadione (1 mg/ml) and 5% horseblood. Incubation was performed at 37 8C, in anaerobicconditions, for 5 days in Brewer-like anaerobic jars(90% N2, 5% CO2 and 5% H2).

2.7. Determination of the minimum inhibitory

concentration (MIC)

Tests were performed by the agar dilution method,

according to the NCCLS guidelines (NCCLS, 1997).For the antibacterial assays, propolis aqueous-ethanolic

extract and fractions were dissolved in 95% EtOH to a

final concentration ranging from 50 to 100 mg/ml. The

antimicrobials meropenem and penicillin G were used as

positive controls (concentrations from 0.03 to 32 mg/ml).

To increase concentrations of antimicrobials, propolis

aqueous-ethanolic extract and fractions were added to

the Brucella agar medium. The inoculum was standar-dized using the Steer replicator (ca. 105 CFU/ml). Plates

were incubated in anaerobic environment for 48 h.

Control plates contained no drug. For testing propolis

aqueous-ethanolic extract, control plates containing the

culture medium plus 2% of 95% EtOH were also

included. The results were expressed as MIC, i.e. the

minimum concentration which completely inhibited

bacterial growth. All experiments were performed induplicates. The data were submitted to analysis of

variance using ANOVA test.

3. Results and discussion

The in vitro antibacterial activity of Brazilian propolis

aqueous-ethanolic extract was evaluated against ninestrains of periodontitis-causing bacteria (Table 1). More

than 180 substances have already been identified as

propolis constituents and phenolic compounds are

regarded as responsible for propolis biological activities

(Marcucci, 1995; Bankova et al., 2000). Propolis aqu-

eous-ethanolic extract was fractionated employing two

different processes (chromatography on silica gel col-

umn and partition between immiscible solvents) and the

Fig. 2. TLC chromatogram developed in chloroform/ethyl acetate

60:40 visualized under ultraviolet light (365 nm) and sprayed with 5%

aluminum chloride. (A) Fractions obtained from propolis aqueous-

ethanolic extract partition between immiscible solvents: (1) propolis

aqueous-ethanolic extract, (2) aqueous solution, (3) acid-rich fraction,

(4) strong phenol-rich fraction, (5) weak phenol-rich fraction, (6) final

residue, (7) quercetin, (8) kaempferol; (B) fractions obtained from

propolis aqueous-ethanolic extract silica gel column chromatography:

(1) propolis aqueous-ethanolic extract, (2) hexane fraction, (3) hexane/

dichloromethane (1:1) fraction, (4) dichloromethane fraction, (5)

dichloromethane/ethyl acetate (1:1) fraction, (6) ethyl acetate fraction,

(7) ethyl acetate/methanol (1:1) fraction, (8) methanol fraction, (9)

quercetin, (10) kaempferol; (C) fractions that exhibited antibacterial

activity: (1) propolis aqueous-ethanolic extract, (2) acid-rich fraction,

(3) strong phenol-rich fraction, (4) dichloromethane/ethyl acetate (1:1)

fraction, (5) ethyl acetate fraction, (6) ethyl acetate/methanol (1:1)

fraction, (7) quercetin, 8-kaempferol.


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activity of the resulting fractions were determined

against the same strains (Tables 1 and 2).All the assayed bacterial species were susceptible to

propolis aqueous-ethanolic extract and fractions derived

from both fractionation processes (Tables 1 and 2). P.

anaerobius , P. gingivalis and P. intermedia were the

bacteria most sensitive to propolis as well as acid- and

strong phenol-rich fractions, with MIC values ranging

from 128 to 256 mg/ml. Medium polarity fractions

(CH2Cl2/EtOAc 1:1; EtOAc), obtained from the silica

gel column and acid- and strong phenol-rich fractions,

from the partition between immiscible solvents showed

the lowest MIC values (Tables 1 and 2). ANOVA

indicated no significant difference in the susceptibility

Fig. 3. HPLC chromatograms of propolis fractions that exhibited antibacterial activity. (A) Propolis aqueous-ethanolic extract; (B) Acid-rich

fraction; (C) Strong phenol-rich fraction; (D) Dichloromethane/ethyl acetate (1:1) fraction and (E) Ethyl acetate fraction. HPLC conditions: see

experimental.


profile of the most active fractions (Tables 1 and 2) in

comparison to propolis aqueous-ethanolic extract

(P 50.05). None of the assayed fractions was more

active than propolis aqueous-ethanolic extract, suggest-

ing that the antibacterial activity is probably caused by

the synergistic effects of different compounds, corrobor-

ating previously reported results (Marcucci, 1995; Ku-

jungiev et al., 1999). Propolis mechanism of action

appears to be complex and a simple analogy cannot be

made to the mode of action of classic antibiotics (Takisi-

Kikuni and Schilcher, 1994).

Among the fractions from the partition between

immiscible solvents (Table 1) the one obtained by

partition with sodium carbonate solution, correspond-

ing to the strong phenolic constituents, was the most

active and exhibited a MIC value similar to that of

propolis aqueous-ethanolic extract. The aqueous frac-

tions from this procedure exhibited no inhibitory

activity on the tested bacteria (Table 1).

Several fractions from the silica gel column showed

antibacterial activity and those eluted with CH2Cl2/

EtOAc (1:1) and EtOAc were the most active (Table 2).

The less polar fractions from this column (hexane and

hexane/CH2Cl2) had no effect on the assayed strains. A.

actinomycetemcomitans FDC Y4 and ATCC 29 523, F.

nucleatum and E. lentum were the species least suscep-

tible to the fractions from silica gel column (MIC values

range from 1024 to �1024 mg/ml).

Meropenem and penicillin G were employed as

positive controls and showed MIC values ranging

from 0.03 to 8 and 0.03 to 4 mg/ml, respectively. In

comparison to these values, the minimum inhibitory

concentrations of propolis aqueous-ethanolic extract

and its fractions appear to be not significant. It has to

be stressed, however, that the propolis antibacterial

activity is very significant, since many of the assayed

bacteria present resistance against antibiotics in clinical

use (Walker, 1996).Propolis antibacterial activity has been attributed to

phenolic compounds, especially flavonoids, phenolic

acids and their esters (Ghisalberti, 1979). Some pre-

nylated p -coumaric acids isolated from Brazilian pro-

polis have been shown to possess antibacterial activity

(Aga et al., 1994). Bankova et al. (1995, 1996, 2000)

reported the antibacterial activity of volatile compounds

and diterpenic acids from Brazilian propolis. Park et al.

(1998) showed the presence of different flavonoids and

the inhibitory effects of propolis on cariogenic bacteria.

In order to characterize the chemical profile, the

bioactive fractions of propolis were analyzed by TLC

and HPLC. The presence of phenolic compounds in the

active fractions was evidenced by the intense fluores-

cence produced under UV360 light after spraying the

TLC plates with aluminum chloride solution. All the

active fractions showed strong fluorescent spots of a

wide range of polarity (Fig. 2). Quercetin and kaemp-

ferol were used as reference compounds.

The HPLC profile of propolis aqueous-ethanolic

extract indicates its complex composition with severalpeaks of varied retention times (Fig. 3). The chromato-

grams obtained for the active fractions did not show

similar patterns of composition. All of them, however,

contained a great number of substances (Fig. 3).

In the present work we demonstrated the antibacterial

activity of propolis and its fractions against several oral

anaerobes, including A. actinomycetemcomitans , F.

nucleatum , P. gingivalis and P. intermedia , speciesfrequently associated with destructive periodontitis

(Slots, 1982; Kaplan et al., 1989; Dahlen, 1993; Rodri-

gues et al., 1999). Since these bacteria may be resistant

to several antibiotics, the antimicrobial activity reported

here is of relevance and propolis may constitute, in the

future, an alternative for treating these pathogens.

Acknowledgements

The authors would like to thank Gilvania Ferreira S.

Santos for technical help. This study was supported by

grants from CNPq and FAPEMIG.

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Journal of Ethnopharmacology 80 (2002) 9–13

Assessment of reversible contraceptive efficacy of methanol extractof Mentha ar�ensis L. leaves in male albino mice

Nidhi Sharma *, D. JacobReproducti�e Physiology Section, Department of Zoology, Uni�ersity of Rajasthan, C-118(B), Kunoria P.G. Girls College, Mangal Marg,

Bapu Nagar, Jaipur 302 004, India

Accepted 8 October 2001

Abstract

The present study was undertaken to assess the reversible contraceptive efficacy of methanolic extract of Mentha ar�ensis leaves.Aqueous solution of the extract (10 mg per day per mouse) when administered orally to male mice of proven fertility for 20, 40and 60 days caused inhibition of fertility while maintaining their normal sexual behaviour. With the increase in treatmentduration, there occurred a corresponding decrease in the mean weight of testis and accessory organs of reproduction. Spermconcentration, motility and viability in the cauda epididymis were also decreased. Spermatozoa with coiled tails also appeared inthe epididymal smear. However, all the induced effects returned to normalcy within 30 days following withdrawal of 60-daytreatment. Oral administration of the extract also did not affect the body weight of the mice and their blood cells count, packedcell volume, haemoglobin and blood/serum biochemistry. © 2002 Published by Elsevier Science Ireland Ltd.

Keywords: Mentha ar�ensis ; Contraceptive; Methanol extract; Epididymal spermatozoa; Male reproductive organs


1. Introduction

A number of plants have been identified and evalu-ated by various researchers for fertility regulation inmales (Deshpande et al., 1980; Qian et al., 1980; Chat-topadhyay et al., 1983; Choudhary, et al., 1990; Jacobet al., 1991; Upadhyay et al., 1993; Lohiya and Ansari,1999; Zhen et al., 1995; Melis, 1999; Naseem et al.,1998). The present study is also an ongoing attempt toinvestigate fertility regulatory substances of plantorigin. Mentha ar�ensis L., which belongs to familyLamiaceae, is a common edible aromatic perennialherb, whose leaves are universally used for flavouringfoods and beverages. Several medicinal properties havebeen ascribed to it (Chopra et al., 1956; Aswal et al.,1984) including abortifacient and antinidational proper-ties in various mammalian species (Bodhankar et al.,1971; Kanjanapothi et al., 1981; Satyavati et al., 1987;Garg and Jacob, 1994). Sharma and Jacob (1996) havereported that aqueous extract of M. ar�ensis leaves caneffectively suppress male fertility also, if administeredvia intramuscular injections just for 20 days.

This investigation was designed following the WHOprotocols to assess the alteration produced, if any, inthe reproductive system, fertility and behaviour of malemice after oral administration of methanolic extract ofM. ar�ensis leaves.


2.1. Animals

Coloney-bred adult Swiss albino male mice (Musmusculus) of proven fertility weighing between 28 and32 g, were utilised for the present investigation anddivided into five groups of nine animals each.

2.2. Test-substance

Fresh leaves of M. ar�ensis L. obtained locally wereair-dried under shade, and powdered. The powderedherb (100 g) was extracted with methanol (800 ml) at65 °C for 30 h. using a Soxhlet extractor. The totalextract thus obtained was concentrated in vacuo toconstant weight. The yield of thick viscous extract was3%. On testing, it was found to be soluble in water. The

* Corresponding author.E-mail address: dr–[email protected] (N. Sharma).

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required doses, therefore, were freshly prepared bydissolving it in the requisite quantity of water.

2.3. Treatment protocol

The methanolic extract was administered orally at adose of 20 mg per day per mouse for 20, 40 and 60days. Animals of control group were given water aloneas vehicle.

2.4. Body and reproducti�e organs weight

Body weight of control and experimental males wereweighed prior to and after the treatment. They wereautopsied under ether anaesthesia 24 h after the termi-nation of each experimental schedule. Testis and sexaccessories were removed, trimmed of adherent tissuesand weighed to the nearest microgram.

2.5. Fertility test

Fertility test of individual males was done aftercompletion of the treatment schedule. Three malesfrom each group were caged separately with cyclingfemales in the ratio 1:2 and their mating behaviourwas observed. Next morning, after the mating expo-sure, the presence of spermatozoa in the estrus vaginalsmear was taken as day 0 post coitum. Mated femaleswere laparotomized on day 15 post coitum, the num-ber of implantation and/or resorption sites, if any,were recorded and allowed to complete the term. Thenumber of females who delivered and the number oflitters born were also recorded (WHO Protocol MB-50, 1983).

2.6. Sperm count and motility

Motility and concentration of cauda epididymalspermatozoa in control and treated mice were assessedusing haemocytometric Neubaur’s chamber (Prasad etal., 1972). The motile spermatozoa were calculated perunit area and expressed as per cent motility. Spermconcentration is expressed as million per millilitre ofsuspension.

2.7. Sperm �itality and morphology

Assessment of sperm vitality (eosin–nigrosin stain-ing technique) and morphological studies (Papanico-laou staining technique) were done following theWHO methods (WHO, 1992).

2.8. Haematology

Mean values of red blood cell (RBC) and whiteblood cell (WBC) counts, packed cell volume and

haemoglobin were recorded in the blood samples col-lected directly from the heart at the time of autopsy(Lynch et al., 1969).

Quantitative estimation of blood urea (BerthelotMethod), serum acid phosphates (King’s Method),serum bilirubin (Jendrassic and Grob’s Method), totalserum protein (Biuret and BCG Dye BindingMethod), SGOT and SGPT (Reitman and Frankel’sMethod) was carried out in control and treated miceat each autopsy schedule using kits manufactured bySigma Diagnostics (India) Pvt Ltd, Baroda.

2.9. Reco�ery studies

Recovery of all the altered parameters was exam-ined following withdrawal of 60 day treatment.

2.10. Statistical analysis

Where applicable, data obtained were analysedstatistically by Student’s t-test. The level of signifi-cance for the difference between sample means wastaken as P�0.05. Values are expressed as mean�S.E.

3. Results

3.1. Fertility test

Daily oral administration of 20 mg crude methano-lic extract of M. ar�ensis leaves for 40 and 60 dayscaused complete inhibition of fertility. Twenty-daytreatment also abolished male fertility upto 83%. Inproestrus females, mated with 20 day treated males,the number of viable foetuses was reduced markedly,while dropping to zero after mating exposure to 40and 60 day treated male (Table 1).

3.2. Sperm count, motility, �itality and morphology

Along with progressive inhibition of fertility, therealso occurred statistically significant depression in thecauda epididymal sperm motility, counts and vitality.Alteration in the structure of spermatozoa was alsonoticed. Tails of most of the sperms in cauda epi-didymis, appeared coiled (Table 2).

3.3. Body and reproducti�e organs weight

The effect of methanol extract on body and repro-ductive organs weights is shown in Table 3. The datareveal that daily ingestion of the test substance did notcause inhibitory effects on body weight of the male

N. Sharma, D. Jacob / Journal of Ethnopharmacology 80 (2002) 9–13 11

mice, however, statistically significant (P�0.001, 0.01)decrease in testis and accessory sex organs was ob-served after 40 and 60 day treatment. Twenty daytreatment, also resulted in reduction (P�0.01 and

0.05) of testicular and caput epididymal weights, whilecauda epididymal, seminal vesicular and prostateweights exhibited statistically insignificant decreasewhen compared with those of control.

Table 1Number of implantations and percent fertility in females mated with the males treated with Methanol extract of M. ar�ensis leaves (20 mg perday per mouse)

Duration of Number of Number of PercentNumber of mated Number of implantation sites onfertilitytreatment (days) females deliveredday 15 post coitum litters bornmales/females

ResorptionsViable foetus

3/6 10065Control 612 00108 0 014

9 12 0 00 4 11 9 1 5 1720 3/61 0 0 0

0 14 000 0 9 040 03/6 0 00 0 0 00 1 4 11

60 00000003/60 2 140

0 0 0 08 10058600Recovery 3/6 14

11 4 0610 9 0 0

Table 2Sperm count, motility, vitality and normal morphology in the control and the extract-fed mice at the dose 20 mg per day per mousse (valuesexpressed as mean�S.E.)

Sperm count (106 /ml) Sperm motility (%)Duration of treatment (days) Viality (%) Normal morphology (%)

11.52�0.55 80.86�3.52Control 86.54�9.65 63.30�7.1365.73�11.0620 24.37�6.87b4.38�0.7a 36.64�3.95c

23.21�12.29b40 3.9�0.71a 13.83�8.43a 60.95�11.0614.93�1.43a 25.94�5.88b3.67�2.0a 36.53�10.18a6078.85�10.01 82.22�11.0211.09�0.2 70.63�8.46Recovery

a Significant in relation to control: P�0.001.b Significant in relation to control: P�0.01.c Significant in relation to control: P�0.05.

Table 3Effect of crude Methanol extract of M. ar�ensis leaves (20 mg per day per mouse) on reproductive organs weight (mg/100 of body weight) of themale albino mice (values expressed as mean�S.E.)

ProstateTestisDuration of treatment (days) Epididymes Seminal vesicle

CaudaCaput

145.58�5.47701.09�26.37 680.62�35.35Control 95.12�2.15 82.07�12.03736.16�25.77610.89�12.20a 76.27�6.99122.63�3.75b 91.71�3.0720

70.85�3.22c 603.33�10.86b 70.83�10.10b40 620.89�14.55a 112.41�5.42c

536.64�7.67c565.03�9.9c 64.53�5.78c102.01�10.22c 66.68�1.92c6091.38�4.43Recovery 148.06�6.33 87.89�8.52645.79�10.40712.09�15.54

a Significant in relation to control: P�0.01.b Significant in relation to control: P�0.05.c Significant in relation to control: P�0.001.

N. Sharma, D. Jacob / Journal of Ethnopharmacology 80 (2002) 9–1312

Table 4Serum/blood biochemical and haematological profile of control and experimental male mice: dose 20 mg per day per mouse, oral (values expressedas mean�S.E.)

Parameters Durations of treatmentControl Recovery 60 days

20 Days 40 Days 60 Days

SGOT (U/ml) 35.74�0.53 33.92�1.33 36.22�0.72 34.43�1.21 36.46�0.9424.46�0.12 24.3�0.27 23.86�0.98 24.72�0.26SGPT (U/ml) 24.8�0.230.82�0.17 1.08�0.031.15�0.06 1.15�0.08SACP (KA units) 1.21�0.11.05�0.68 0.97�0.11S. bilirubin (mg per 100 ml) 0.88�0.060.86�0.10 0.87�0.24.78�0.27 4.41�0.784.8�0.43 4.73�0.14S. protein (mg per 100 ml) 4.69�0.84

11.8�0.11Haemoglobin (g per 100 ml) 11.38�0.23 11.64�0.22 11.77�0.03 11.87�0.0138.04�0.85 38.07�0.8538.21�1.6 38.41�0.85PCV (%) 38.51�2.43

3.43�0.19RBC (106 /mm3) 3.61�0.2 3.42�0.17 3.50�0.18 3.56�0.264.73�0.12WBC (103 /mm3) 4.95�1.114.85�1.1 4.3�1.17 5.07�1.02

13.45�0.63 13.42�0.6313.54�0.52 13.42�0.55Blood urea (mg/dl) 13.64�0.3

3.4. Reco�ery

If, however, experimental males, in which treatmenthad ceased after 60 days, were mated with cyclingfemales, a normal number of implantation sites com-parable with control were obtained within 30 days(Table 1). Likewise, other altered parameters, e.g.sperm counts, motility, vitality and organs weights werefound to be within the normal range. Percentage ofcoiled tailed spermatozoa was also low, 30 days afterwithdrawal of treatment (Tables 1–3).

3.5. Serum/blood profiles and haematology

Table 4 summarises the values obtained for the ex-amined biochemical parameters in the serum/blood andhaematological parameters, showing unaltered generalphysiology of the extract fed mice.

4. Discussion

The present study with crude methanolic extract ofM. ar�ensis leaves demonstrates its inhibitory organsand fertility of the male mice. The number of females,impregnated with 20 day treated males, was highlyreduced, resulting in 83% inhibition of male fertility,while, sterile mating in each case confirmed by thepresence of spermatozoa in the vaginal smears of fe-males suggests that the mating behaviour of the extract-fed mice remained unaltered.

At the tried dose regimen fertility, inhibitory effectsin the male mouse could be attributed to the inherentestrogenicity of M. ar�ensis (Kanjanapothi et al., 1981;Garg and Jacob, 1994), which essentially simulatesother estrogens or estrogenic substances and suppressesthe testicular and/or the epididymal functional in-tegrity, possibly by inhibiting local androgen and/orgonadotrophin secretions. Androgen deprivation, not

only suppresses spermatogenesis, leading to low spermconcentration, but alters the epididymal milieu also,which renders it hostile for maturation and survival ofthe spermatozoa (Setty, 1979; Rao and Mathur, 1988;Rao and Shah, 1988). The findings of the present studyare consistent with the suggestions given by other au-thors also (Wu et al., 1973; Jacob et al., 1991). Thesperm motility, density, vitality and morphology in thecauda epididymis was, therefore, adversely affected af-ter administration of methanol extract of M. ar�ensis.Inadequate concentration and sluggishly motile or im-motile spermatozoa could not penetrate the cervicalmucus, and thus failed to fertilise the ova (Amelar etal., 1980; Sharma et al., 1999; Lohiya and Ansari,1999).

Further treatment with methanol extract might possi-bly inhibit the activity of adenosine triphosphatase inthe spermatozoa by uncoupling of oxidative phospho-rylation from the respiratory chain and prevent phos-phorylation of adenosine diphosphate to adenosinetriphosphate (Kalla and Vasudev, 1981) and thus ren-ders the spermatozoa immotile.

Statistically significant reduction in weights of thetestis and accessory sex organs of the albino mice,following ingestion of the extract again indicates lowlevel of androgen, which was not enough to maintainthe weight of the gonads and accessories. Nevertheless,libido or potency of the treated animals may remainunaltered (Vanithakumari et al., 1989; Gill-Sharma etal., 1993; Rao and Shah, 1988; Sharma and Jacob,2001), as observed in the present study also. The abilityof the extract-fed mice, to mate, might have beenmaintained owing to low anti-androgen concentration(Sondersten, 1979), or owing to the circulating plasmatestosterone, which was sufficient for normal matingbehaviour, but it was insufficient for the maintenanceof fertilising ability of the epididymal spermatozoa(Bhasin et al., 1988). All these factors, thus, broughtabout functional sterility in the extracted mice. How-

N. Sharma, D. Jacob / Journal of Ethnopharmacology 80 (2002) 9–13 13

ever, the induced infertility was completely reversed onwithdrawal of treatment within a period of 30 days.Also, no apparent abnormality was observed in thelitters delivered by the females mated with the males ofrecovery group.

Nontoxicity of methanolic extract of M. ar�ensisleaves is further supported by the data obtained afterexamination of clinical chemistry and haematologicalparameters, which remained unaltered. The results,thus, suggest that oral administration of crudemethanol extract of M. ar�ensis leaves can lead to areversible sterile state in the male mouse, due to inter-ference on the testicular androgen levels altering struc-ture, function, viability and concentration ofspermatozoa in the cauda epididymis.

Acknowledgements

We acknowledge the Indian Council of Medical Re-search for the award of Research Associateship to DrNidhi Sharma for the present study. We are thankful tothe Head, Department of Zoology for extending labo-ratory facility in carrying out this study.

References

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Chattopadhyay, S., Chattopadhyay, U., Mathur, P.P., Saini, K.S.,Ghosal, S., 1983. Effects of hippadine and Amryllidaceae alkaloidon testicular function in rats. Planta Medica 49, 252–254.

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Deshpande, V.Y., Mendulkar, K.N., Sadre, N.L., 1980. Antifertilityactivity of Azadirachta indica in mice. Journal of PostgraduateMedicine (Bombay) 26, 77–81.

Garg, P., Jacob, D., 1994. Mentha ar�ensis leaf: an effective antinida-tional substance in laboratory mouse. Journal of Advanced Zool-ogy 15, 32–36.

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Jacob, D., Yadava, L., Vyas, D.K., 1991. Inhibition of fertility andalterations in the reproductive structures of the male rat afteradministration of carrot seed extract. Proceedings of the NationalAcademy of Science India 61(B) (I), 29–34.

Kalla, N.R., Vasudev, M., 1981. Studies on the male antifertilityagent—gossypol acetic acid II. Effect of gossypol acetic acid on themotility and ATPase activity of human spermatozoa. Andrologia13, 95–98.

Kanjanapothi, D., Smitasiri, Y., Panthong, A., Taesotikul, T., Rathna-panone, V., 1981. Postcoital antifertility effect of Mentha ar�ensis.Contraception 24, 559–567.

Lohiya, N.K., Ansari, A.S., 1999. Male contraceptive agents. In: Joy,K.R., Krishna, A., Haldar, C. (Eds.), Comparative Endocrinologyand Reproduction. Narosa Publishing House, New Delhi, pp.260–277.

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Evaluation of immunomodulatory potential of Ocimum sanctumseed oil and its possible mechanism of action

P.K. Mediratta a,*, K.K. Sharma a, Surender Singh b

a Department of Pharmacology, Uni�ersity College of Medical Sciences and GTB Hospital, New Delhi 110095, Indiab College of Pharmacy, Uni�ersity of Delhi, Pushap Vihar, Sector-III, New Delhi 110017, India

Received 30 January 2001; received in revised form 27 September 2001; accepted 18 October 2001

Abstract

The present study investigates the effect of Ocimum sanctum seed oil (OSSO) on some immunological parameters in bothnon-stressed and stressed animals. An attempt has also been made to explore the possible mechanism of immunomodulatoryactivity. OSSO (3 ml/kg, ip) produced a significant increase in anti-sheep red blood cells (SRBC) antibody titre and a decrease inpercentage histamine release from peritoneal mast cells of sensitized rats (humoral immune responses), and decrease in footpadthickness and percentage leucocyte migration inhibition (LMI) (cell-mediated immune responses). Restraint stress (RS) produceda significant reduction in the anti-SRBC antibody titre, foot pad thickness and percentage LMI (% LMI). The effects of RS onhumoral as well as cell-mediated immune responses were effectively attenuated by pretreating the animals with OSSO.Co-administration of diazepam (1 mg/kg, sc), a benzodiazepine (BZD), with OSSO (1 ml/kg, ip) enhanced the effect of OSSO onRS-induced changes in both humoral and cell-mediated immune responses. Further, flumazenil (5 mg/kg, ip), a central BZDreceptor antagonist inhibited the immunomodulatory action of OSSO on RS-induced immune responsiveness. Thus, OSSOappears to modulate both humoral and cell-mediated immune responsiveness and these immunomodulatory effects may bemediated by GABAergic pathways. © 2002 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Ocimum sanctum seed oil; Humoral immune response; Cell-meditated immune response; Restraint stress


1. Introduction

Stress, an integral part of a human life has beenreported to produce several disease states (Wolff et al.,1950; Solomon and Amkraut, 1981) Alleviation of suchstressful situations by psychotherapy or drugs is the keyto the treatment of conditions whose etiological basisstems from stress. Physiological stress is known to bringabout a wide range of biochemical and behavioralchanges in the organism, which is now better known asSelye’s Stress Adaptation Syndrome (Kulkarni, 1989).During recent years much attention has been focusedon the immunological changes occurring during stressand various studies have reported that stressful situa-tions in fact alter humoral, as well as cell-mediatedimmune responses (Dantzer and Kelley, 1989; Medi-ratta, 1994; Mediratta and Sharma, 1997).

Millions of people around the world use traditionalsystems of medicine for developing immunity, resis-

tance against infections/diseases, to prevent or alleviatethe symptoms of the disease or cure it. The mainfactors that make natural products attractive candi-dates for human use include their ease of availability,cost effectiveness and presumed safety. Efforts are nowbeing made to unravel the mechanism of action of thesenatural products.

Ocimum sanctum Linn (OS) commonly known as‘Tulsi’ has been extensively used in Ayurvedic system ofmedicine for various ailments and has been shown topossess significant adaptogenic/anti-stress properties(Bhargava and Singh, 1981). Different parts of theplant are claimed to be effective in a number of diseases(Satyavati, 1987). A steam distilled extract of OS leaveshas been shown to enhance anti-sheep red blood cells(SRBC) and IgE antibody titre and to reduce antigen-induced histamine release from peritoneal mast cells(Mediratta et al., 1988). The fixed oil obtained from OSseeds is reported to possess significant anti-inflamma-tory, antipyretic, analgesic and antiarthritic activities(Singh and Majumdar 1995a,b, 1997; Singh et al.,* Corresponding author.

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1996). However, the effect of OS seed oil (OSSO) onstress-induced immune responsiveness has not beenstudied. The present study was, therefore, undertakento investigate the effect of OSSO on some humoral andcell-mediated immunological parameters in stressed ani-mals. Further, the effect of OSSO has also been studiedon these immunological parameters in non-stressed ani-mals. Also, attempts have been made to explore thepossible mechanism of immunomodulatory activity ofOSSO.

2. Materials and methods

2.1. Animals

Male Wistar rats (200–220 g), male Swiss albinomice (25–28 g) and guinea pigs (700–1000 g) were usedin the study. They were kept in 12-h light:12-h darkcycles (07:00–19:00 h) and temperature controlled(22�2 °C) condition. Food and water were availablead libitum unless otherwise indicated. The care of theanimals was as per the ‘Guidelines for the Care andUse of Animals in Scientific Research’ prepared byIndian National Science Academy, New Delhi (Anony-mous, 2000).

2.2. Plant material

The dried OS seeds were collected from MaidanGarhi, New Delhi, India and authenticated by a resi-dent botanist, Department of Genetics, Indian Councilof Agricultural Research. The voucher specimen hasbeen deposited in the Indian Agricultural ResearchInstitute (IARI), Pusa, New Delhi, India. The seedswere crushed and cold macerated in petroleum ether(40–60 °C) for 3 days. The extract was taken out andpetroleum ether evaporated. The oil thus obtained wasfiltered and then stored at room temperature in light-protected bottles for experimental use.

2.3. Experiments in non-stressed animals

2.3.1. Humoral immune responses

1. Haemagglutination titre to sheep red blood cells.The rats were immunized with SRBC (0.5×109

cells per ml per 100 g, ip) on day 0. The animalswere then divided in two groups, each group com-prising of eight rats. Animals in one group wereinjected with normal saline while the second groupreceived OSSO in a dose of 3 ml/kg per day, ip fromday 1 to 6. On day 7 the animals were lightlyanaesthetized with ether and blood was collectedfrom retroorbital plexus. The serum was separatedand the haemagglutination titre was estimated using

microtitre plates. Two-fold dilutions (0.025 ml) ofsera were made in the microtitre plates with saline.To each well 0.025 ml of 1% (V/V) SRBC wasadded. The plates were incubated for 1 h at 37 °Cand then observed for haemagglutination. Thehighest dilution giving haemagglutination was takenas the antibody titre, which was expressed in agraded manner, the minimum dilution (1/2) beingranked as 1. The mean ranks of different groupswere compared for statistical analysis.

2. Histamine release from peritoneal mast cells of sen-sitized rats. The animals were sensitized by subcuta-neously injecting 0.5 ml egg albumin (25 mg/ml)along with 0.5 ml Freund’s complete adjuvant andtriple antigen (0.5 ml containing 20 000×106 Bpertussis organisms) on day 0. The rats were dividedinto two groups of eight animals each. Group I wastreated with normal saline and group II with OSSO3 ml/kg/day, ip from day 1 to 13. On the 14th dayanimals were sacrificed by exsanguination and peri-toneal mast cells were extracted in heparinizedKreb’s Ringer solution and finally suspended in thenon-heparinized Kreb’s Ringer solution containing,in addition, 1 mg/ml of human serum albumin.Cells were usually pooled from two sensitized ratsand the cell suspension was divided into 8–10 sam-ples. The samples were incubated at 37 °C for 10min under gentle shaking followed by addition ofantigen, i.e. egg albumin (0.5 mg/ml) and incubatedfor another 10 min. The reaction was terminated byplacing the sample in ice-cold water. The releasedhistamine was bioassayed on atropinized guinea pigileum and the spontaneous release values were de-ducted from the results.

2.3.2. Cell-mediated immune responses

1. Leucocyte migration inhibition (LMI) test in rats.The animals were sensitized by subcutaneous injec-tion of 0.5 ml egg albumin (25 mg/ml) along with0.5 ml of Freund’s complete adjuvant on day 0. Thecontrol group was administered normal saline whilethe test group was injected with OSSO 3 ml/kg perday, ip from day 1 to 13. On day 14 all animalswere lightly anaesthetized with ether and the chestwas opened. About 5–6 ml of blood was collectedin a syringe containing 250 U of heparin, by directcardiac puncture and LMI test was performed(Mediratta and Sharma, 1997).

2. Footpad thickness test in mice. The animals wereimmunized with SRBC (1×108 cells, sc) on day 0.The mice were then divided into two groups, eachcomprising of 10 animals. Group I received normalsaline and acted as control. Animals in group IIwere injected OSSO in a dose of 3 ml/kg per day, ipfrom day 1 to 5. On day 5 all animals received

P.K. Mediratta et al. / Journal of Ethnopharmacology 80 (2002) 15–20 17

1×108 SRBC in the right hind paw and normalsaline in the left hind paw. The difference in thefootpad thickness of the two paws was measured24-h later by fluid displacement method.

2.4. Experiments in stressed animals

2.4.1. Humoral immune responseThe rats were sensitized by injecting SRBC (0.5×109

cells per ml per 100 g, ip) on day 0. On day 7 theyreceived the same dose of antigen, i.e. booster dose andthe animals were then divided into different groups ofeight rats each, On day 8 one group was administeredvehicle and kept in home cages; they were not subjectedto RS and acted as ‘no stress’ (NS) control, however,like stressed animals they were also deprived of foodand water. The 2nd, 3rd, 4th, 5th, and 6th groups wereinjected with vehicle, OSSO (3 ml/kg), diazepam (5mg/kg), diazepam (1 mg/kg)+OSSO (1 ml/kg) andflumazenil (5 mg/kg)+OSSO (3 ml/kg), respectively,and then subjected to RS in Plexiglas restrainers (Inco,Ambala) at room temperature (22�2 °C). During theperiod of stress animals were deprived of food andwater. Then, 24 h later all the animals were lightlyanaesthetized with ether, and blood was collected fromretroorbital plexus. Serum was separated and haemag-glutination titre was estimated as above.

2.4.2. Cell-medicated immune responses

1. LMI test. The rats were sensitized with 0.5 ml eggalbumin (25 mg/ml) and 0.5 ml Freund’s completeadjuvant given sc on day 0. The animals were thendivided into two main groups. The rats in onegroup were subjected to RS for 24 h at roomtemperature (22�2 °C) on day +1 and +13 (1stand 13th day after the day of sensitization, i.e. day0). Vehicle, OSSO (3 ml/kg), diazepam (5 mg/kg),diazepam (1 mg/kg)+OSSO (1 ml/kg) or flumaze-nil (5 mg/kg)+OSSO (3 ml/kg) was administeredjust prior to subjecting the animals to RS on day+1 and +13. During the period of stress, the ratswere deprived of food and water. Animals in theother group were not subjected to RS and acted asa ‘no stress’ (NS) control. However, like stressedanimals they were also deprived of food and water.On the 14th day all the animals were anaesthetizedwith ether and the chest was opened. About 5–6 mlof blood was withdrawn in heparinized syringe bycardiac puncture and LMI test was performed(Mediratta and Sharma, 1997).

2. Footpad thickness test; mice were immunized byinjecting 1×108 SRBC sc in the back and thendivided into different groups of 10 animals each.The non-stressed animals were injected vehicle fromday 1 to 5 and kept in home cages. The animals of

the stressed group were treated with vehicle, OSSO(3 ml/kg) diazepam (5 mg/kg, diazepam (1 mg/kg)+OSSO (1 ml/kg) or flumazenil (5 mg/kg)+OSSO (3 ml/kg) and then subjected individually toRS for 1 h every day in round pipe mouse restrain-ers from day 1 to 5 at room temperature (22�2 °C). On the 5th day, animals in all the groupswere challenged with SRBC 1×108 SRBC sc in theright hind foot pad, whereas normal saline wasinjected in the left hind paw. Increase in foot padthickness was measured 24 h after the challenge byfluid displacement method.


The data were analyzed using Mann–Whitney ‘U’test, Student’s t-test and �2-square test whenever ap-propriate. A ‘P ’ value of �0.05 was used as the levelof significance in all the statistical tests.

3. Results

3.1. Effects in non-stressed animals

3.1.1. Humoral immune responsesIn rats sensitized with SRBC on day 0, administra-

tion of OSSO (3 ml/kg per day, ip) from day 1 to 5produced a significant (P�0.05) increase in anti-SRBCantibody titre (Fig. 1). On the other hand OSSO (3ml/kg per day, ip) injection from day 1 to 13 signifi-cantly (P�0.01) inhibited antigen induced histaminerelease from the peritoneal mast cells of rats sensitizedwith egg albumin along with Freund’s complete adju-vant and triple antigen (Fig. 1).

3.1.2. Cell-mediated immune responsesOSSO (3 ml/kg per day, ip) produced a significant

reduction of the increase in paw volume in mice (P�0.05) and % LMI in rats (P�0.01) as compared withthe control normal saline treated group (Table 1).

3.2. Effects in stressed animals

3.2.1. Humoral immune responseRS produced a significant (P�0.05) reduction in the

anti-SRBC antibody titre. The antibody titre decreasedfrom 7.8�0.4 (mean�S.E.) in the NS group to 5.6�0.3 in the RS group. Pretreating the animals with OSSO(3 ml/kg) or diazepam (5 mg/kg) immediately prior tosubjecting them to stress effectively blocked the effectof RS on the anti-SRBC antibody titre. Coadministra-tion of low doses of diazepam (1 mg/kg) with OSSO (1ml/kg) potentiated the immunomodulatory action ofOSSO on RS-induced immunosuppression. Further,flumazenil, a specific central benzodiazepine (BZD) re-


Fig. 1. Effect of Ocimum sanctum seed oil (OSSO) on anti-SRBC antibody titre and % histamine release in non-stressed rats; *P�0.05;**P�0.001.

ceptor antagonist also attenuated the OSSO-inducedantagonism of reduction in the anti-SRBC antibodytitre produced by RS (Fig. 2, Table 2).

3.2.2. Cell-mediated immune responsesSimilar to the effect on humoral immune response,

RS caused a significant suppression of cell-mediatedimmunity, the % LMI and footpad thickness responseswere significantly reduced as compared with the NSgroup. Pretreating the animals with OSSO (3 ml/kg) ordiazepam (5 mg/kg) prior to stress procedure attenu-ated the effect of RS on both % LMI and foot padthickness. Like humoral immune response, co-adminis-tration of diazepam (1 mg/kg) with OSSO (1 ml/kg)potentiated the effect of OSSO on both footpad thick-ness and % LMI. The immunomodulatory effect ofOSSO on both parameters of cell-mediated immuneresponses was effectively blocked by pretreating theanimals with flumazenil (5 mg/kg) (Table 3).

4. Discussion and conclusion

Stress is an important environmental factor, whichcan affect a number of body functions. It is known thatstressful conditions that modify the susceptibility of anindividual to a variety of illnesses also influence theimmune processes (Solomon and Amkraut, 1981) andalleviation of such stressful situations by psychotherapyor drugs is the key to the treatment of conditions whoseetiological basis stem from stress. Keeping this in viewthe present study was carried out to investigate theeffect of OSSO on some immunological parameters inboth non-stressed and stressed animals. Pretreating therats with OSSO produced a significant increase in theanti-SRBC antibody titre and a decrease in the antigen-

induced histamine release from the peritoneal mast cellsof the sensitized non-stressed animals. Thus, OSSOexhibited a positive/beneficial effect on humoral immu-nity in naive non-stressed animals. In the case of cellu-lar immunity, both the studied parameters, i.e. foot padthickness and % LMI were significantly reduced afterOSSO treatment. During the cell-mediated immune re-sponse the sensitized T-lymphocytes, on being chal-lenged with the antigen secrete a number oflymphokines including LMI factor (Mustaffa, 1992).These lymphokines attract scavenger cells to the site ofreaction, which are then immobilized to promote defen-sive (inflammatory) reaction. The results of the preventstudy indicate that there was an inhibition of release oflymphokines such as LMI factor on OSSO administra-tion, allowing the inflammatory cells to move awayfrom the site of reaction. Thus OSSO, besides having adirect anti-inflammatory effect as reported earlier(Singh and Majumdar, 1997), may reduce inflammationby inhibiting cell-mediated immune response.

Stress has been reported to produce suppression ofboth humoral and cell-mediated immune responses

Table 1Effect of Ocimum sanctum seed oil (OSSO) on cell-mediated immuneresponses in non-stressed animals

Footpad thickness test in LMI test inTreatmentmice (increase in paw rats %(ml/kg)volume in mm) inhibitionc

(mean�S.E.)b

0.07�0.005 58Normal saline0.034�0.004aOSSO(3) 30a

n=8.a P�0001.b Student’s t-test.c �2-square test.

P.K. Mediratta et al. / Journal of Ethnopharmacology 80 (2002) 15–20 19

Fig. 2. Effect of restraint stress (RS) and its modulation by Ocimum sanctum seed oil (OSSO) on anti-SRBC antibody titre in rats; *P�0.05; NS,no stress.

(Dantzer and Kelley, 1989; Mediratta, 1994; Medirattaand Sharma, 1997). In consonance, the results of thepresent study also show that RS produced a reductionof antibody titre as well as footpad thickness and %LMI. Pretreating the animals with OSSO effectivelyblocked the immunosuppressive effect of RS on bothhumoral and cell-mediated immune responses. For an-tagonism of suppression of humoral immunity pro-duced by RS, its positive immunomodulatory activityas observed in non-stressed animals where OSSO ad-ministration produced an increase in anti-SRBC anti-body titre, may be working in the same direction tooppose the stress-induced humoral immunosuppression.However, the same may not be true for the cell-medi-ated immune responses where both % LMI and footpadthickness appeared to decrease in non-stressed animalsbut seemed to be stimulated in stressed animals, i.e.inhibition of the RS-induced inhibition of % LMI andchange in paw volume. This effect on cell-mediatedimmunity may be related to immunological allostasiswhich explains that an organism is capable of allostaticregulation of the immune responses to avoid immuno-logical dissonance which can lead to death from disor-ders such as excessive inflammatory response syndrome,or conversely, the compensatory anti-inflammatory re-sponse syndrome (McEwen, 1988; Bone, 1996). In thecontext of the present study if inflammatory or anti-inflammatory connotations are substituted for ‘stress’and ‘anti-stress’ conditions, the results on both humoraland cell-mediated immune response in stressed animalssuggest that OSSO has acted to maintain the immuno-logical allostasis.

To investigate the mechanism of immunomodulatoryeffect of OSSO, its interaction with diazepam, a BZD

which is known to exert anti-anxiety and anti-stressactivity (Mediratta, 1994; Mediratta and Sharma, 1997;Tripathi, 1991) was studied. BZDs produce their effectby augmenting GABA receptor mediated chloride ionconductance (Havoundjian et al., 1987; Tripathi, 1991).In the present study, diazepam potentiated the im-munomodulatory activity of OSSO on RS-induced im-munosuppression. Further, the effect of OSSO onstress-induced immunological parameters was signifi-cantly antagonised by flumazenil, a central BZD recep-tor antagonist (Brodgen and Goa, 1991). These resultsindicate that OSSO may be exerting its immunomodu-latory effect by modulating GABAergic activity. Thus,OSSO appears to influence both humoral and cell-me-diated immunological parameters in naive non-stressed,

Table 2Effect of restraint stress (RS) and its modulation by Ocimum sanctumseed oil (OSSO), diazepam and flumazenil on anti-SRBC antibodytitre

Anti-SRBC antibody titreTreatment(mean�S.E.)(mg/kg)

Control (NS) 7.8�0.4RS 5.6�0.3*a

OSSO (3 ml)+RS 8.2�0.6**b

Diazepam (5 mg)+RS 8.4�0.4**b

8.8�0.6**bDiazepam (1 mg)+OSSO(1 ml)+RS

Flumazenil (5 mg)+0550 6.2�0.4*c

(3 ml)+RS

NS, No stress; n=8; *P�0.05; **P�0.001 (Mann–Whitney ‘U ’test).

a Compared with control (NS) group.b Compared with RS group.c Compared with OSSO+RS group.


Table 3Effect of restraint stress (RS) and its modulation by Ocimum sanctum seed oil (OSSO), diazepam and flumazenil on cell-mediated immuneresponses

Footpad thickness test in mice (increase LMI test in rats %Treatment (ml–mg/kg)in paw volume in mm) (mean�S.E.)a inhibitionb

Control (NS) 46.00.067�0.003RS 20.0**c0.042+0.004*c

39.0**d0.062+0.003*dOSSO (3 ml)+RSDiazepam (5 mg)+RS 41.5**d0.061�0.002*d

0.072�0.003**d 61.5**dDiazepam (1 mg)+OSSO(1 ml)+RS

0.048+0.003*e 28.2**eFlumazenil (5 mg)+OSSO(3 ml)+RS

n=8–10; *P�0.05; **P�0.001.a Student’s t-test.b �2-test.c Compared with NS group.d Compared with RS group.e Compared with OSSO+RS group.

as well as stressed animals and these immunomodula-tory properties could be mediated via GABAergicpathways.

Acknowledgements

The authors are thankful to Haffman La Roche,New Jersey, USA for the generous gift of flumazenil.

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Brodgen, R.N., Goa, K.L., 1991. Flumazenil a reappraisal of itspharmacological properties and therapeutic efficiency as a benzo-diazepine antagonist. Drugs 42, 1061–1089.

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Efficacy of some nupe medicinal plants against Salmonella typhi :an in vitro study

C. Egwim Evans *, Aderotimi Banso, O. Adeyemo SamuelScience Laboratory Technology Department, Federal Polytechnic, P.M.B. 55, Bida, Niger State, Nigeria

Received 15 February 2001; received in revised form 4 September 2001; accepted 18 October 2001

Abstract

Different medicinal plants: Euphobia hirta (Eh); Citrus aurantifolia (Ca), Cassia occidentalis (Co), and Cassia eucalyptus (Ce),which are claimed by the Nupes of Bida in Niger State of Nigeria to be effective in the treatment of typhoid fever were collected.Ethanolic and water extracts were obtained by standard procedures. Preliminary phytochemical screening showed that alkaloidswere absent in all the parts of Eh, Co, and Ca studied, but present in all the parts of Ce studied. Saponins were absent in theleaves and florescence of Eh, Ca fruits, but present in little amounts in Eh and Co roots. Saponins were present in large amountsin all the parts of Ce studied. Tannins were in little amount in all the medicinal plants studied, except in Ca. Glycosides were notpresent in any of the medicinal plants studied. The in vitro and microbial analysis showed that only Ce showed inhibition toSalmonella typhi growth. Ca plus ‘Kanwa’ (a locally mined, alkaline salt) showed inhibition only at a high concentration of‘Kanwa’. The MIC and MBC of Ce are 1 and 2 mg/ml, respectively. The paper concludes that, of all the medicinal plants claimedby the Nupes to be effective against S. typhi, only Ce contains the natural compound that can be used in the treatment of typhoidfever. © 2002 Published by Elsevier Science Ireland Ltd.

Keywords: Nupes; Medicinal plants; Tannins


1. Introduction

Medicinal plants contain physiologically active prin-ciples that over the years have been exploited in tradi-tional medicine for the treatment of various ailments(Adebanjo et al., 1983). Proven medicinal plants areused in treatment of diseases either alone or in combi-nation with other plants. They are used as anti-infec-tious agents, anti-malarial, anti-tumoral agents,laxatives, cardiovascular and nerval remedies(Owonubi, 1988).

Several laboratories have reported the effectivenessof common indigenous herbs against Gram-positiveand Gram-negative microorganisms. Recently, Mo-mordica charantia and Alstonia boonei have beendemonstrated to be active against Salmonella typhi thecausative organism of typhoid fever (Irobi et al., 1995).Ikenebameh and Metitiri (1988) have also reported theefficacy of Cassia alata against a large spectrum offungi and bacterial that are the causative organisms of

several common diseases. Indeed indigenous medicinalplants have been the bedrock for modern medicine.

In India, the rhizomes of Stephania glabra, which hasbeen known among the natives to be antiasthatic, an-tidysentric and antipyretic has been popularized(Bhakumi and Gupa, 1982), Occimum �iride and Occi-mum bacillium are medicinal plants that have long beenknown among the natives of the coast of West Africa,particularly, in Nigeria and Ghana, to be effective intreatment of dysentery and diarrhea which are mainlycaused by microorganisms (Chiori et al., 1977;Omoregbe et al., 1995).

In Nigeria, as in many other countries, it is commonamong the natives to combine several herbs which theyclaim to have particular curative properties, whereas,only one or some of the combined herbs may becontaining the active ingredients for the particular dis-ease. For instance, it is a common practice to use acombination of plants like Azadaricta indica (neem),Lymbopogan citractus (lemon grass) and Psidium gua-janya (guava) leaves for the treatment of malaria fever.However, Owonubi (1988) has reported that only theNeem contains the active substance against plasmod-

* Corresponding author.E-mail address: [email protected] (C.E. Evans).



C.E. E�ans et al. / Journal of Ethnopharmacology 80 (2002) 21–2422

ium parasites, lemon grass acts as flavoring agent toreduce the bitter taste of Neem, while the guava, adiuretic, increases the rate of urine excretion to avoidthe accumulation of Neem in the body. In the samevain, the Nupes in Niger State, Nigeria believe that thedecoction of Euphobia hirta, (roots, leaves, stem andflorescence), Cassia occidentalis (roots only) and Cassiaeucalyptus (leaves, stem and florescence) when takenregularly are good remedies against typhoid fever.

It is the aim of the present work therefore to confirmwhich of these medicinal plants contains the activesubstance against S. typhi, the causative organism oftyphoid fever and the potency of such plant.


2.1. Sources of plant materials and bacteruem

The medicinal plants used were roots, leaves andinflorescence of E. hirta, the roots of C. occidentalis,and leaves, stem and inflorsence of C. eucalyptus. Thewhole plant samples were collected from Federal Poly-technic, Bida, Niger State, Nigeria. Citrus aurantifoliaand ‘Kanwa’ were purchased from the local market inBida.

The local way of preparing these herbs was simulatedviza:1. a decoction of each of the herbs: E. hirta, C.

occidentalis and C. eucalyptus were made.2. E. hirta, C. occidentalis and C. eucalyptus were each

extracted in absolute ethanol.3. The juice of C. aurantifolia was squeezed into hot

water and kanwa was dissolved in it in increasingconcentration.

The microorganism used is S. typhi obtained fromthe Microbiology unit of the Science Laboratory Tech-nology of the Federal Polytechnic.

2.2. Preparation of plant extract

Ethanolic or aqueous extracts of the plant materialswere prepared according to the method described byBoaky-Yiadon (1979). Five grams of the plant materialswere air-dried, crushed and blended into powder usingan electric blender (National Mx 491 IV, Matsushitaelectric). The blended material was transferred to abeaker and 10 ml of distilled water or ethanol added atroom temperature. The mixture was extracted by agita-tion on a rotary shaker. The extract obtained wasvacuum-dried and used for further test.

2.3. Antibacterial test

The antibacterial test was performed using the agarditch method of Boaky-Yiadon (1979) and zones ofinhibition measured after 24 h incubation at 37 °C.

2.4. Determination of minimum inhibitory concentration(MIC) of plant extract

Different concentrations of the plant extracts wereintroduced into different test tubes; each tube wasinoculated with an overnight culture of S. typhi dilutedto give a final concentration of 106 cells per ml. Thetubes containing the test organism were incubated at37 °C for 24 h. The least concentration of the plantextracts that did not permit any visible growth of theinoculated test organism in both cultures was taken asthe MIC in each case.

2.5. Determination of minimum bactericidalconcentration (MBC)

The MBC was determined according to the methodof Boaky-Yiadon (1979). The lowest concentration ofthe plant extracts that did not yield any colony growthon the solid medium after the incubation period wastaken as the MBC.

2.6. Preliminary phytochemical analysis

Alkaloids, saponins, tannins and glycosides were de-termined in all the medicinal plants studied as describedby UNIDO (1969).

3. Results and discussions

The phytochemical analysis of plant parts shown inTable 1, reveals that all the parts of E. hirta, C.occidentalis and C. aurantifolia fruits contained no alka-loids while all the parts of C. eucalyptus tested showedpresence of alkaloid. Fluck (1973) has reported thatclasses of alkaloids are among the major powerfulpoisons known. Apart from being poisonous, somealkaloids have also been proved to be useful in correct-ing some renal disorders (Konkwara, 1976). It there-fore, means that the alkaloid of C. eucalyptus, may bea poison that can be tried on lower or higher organism.It further indicates that C. eucalyputs, is a medicinalplant that may also be tried on some renal disorders.

The E. hirta root and C. occidentalis roots showedthe presence of saponins in small amount (Table 1);while all the parts of C. eucalyptus showed the presenceof saponins in higher amount. Saponins are a specialclass of glycosides which have soapy characteristics andfacilitate the resorption of foods and medicine (Fluck,1973). It has also been shown that saponins are activeantifungal agents (Sodipo et al., 1991). This thereforesuggests that the extracts of E. hirta root, C. occiden-talis roots and C. eucalyptus used in the present workmay be useful in the chemotherapy of mycotic infec-tions. All the plant parts used in the present study

C.E. E�ans et al. / Journal of Ethnopharmacology 80 (2002) 21–24 23

Table 1Phytochemical screening of extracts of common Nupe Medicinal

Plant partsSubstance

HER EHF COR CELEHL CEF CES CAF

−Alkaloid − − − + + + −+ − + ++− ++Saponin ++ −

+Tannin + + + + + + −− − ND ND ND ND NDGycoside −

ND, not determined; +, present (less); ++, present (much); −, absent; EHL, E. hirta leaves; HER, E. hirta root; EHF, E. hirta Florescence;COR, C. occidentalis root; CEL, C. eucalyptus leaves; CEF, C. eucalyptus florescence; CES, C. eucalyptus stem; CAF, C. aurantifolia fruit.

Table 2Comparison of S. typhi sensitivity to the different medical plant extract by agar ditch method

Plant extractsConcentration(mg/ml)

HER EHF COR CELEHL CEF CES CAFK

− − − − − −1 −−− − − −− −2 − −− − − +3 +− + −− − − +− +4 + −− − − + + + −5 −− − − +− +6 + −

−7 − − − + + + −− − − + +8 +− −− − − +− +9 + −

10 −− − − + + + +

+, Zone of inhibition present; −, one of inhibition absent; EHL, E. hirta leaves (ethanolic extract); HER, E. hirta root (ethanolic extract); EHF,E. hirta Florescence (ethanolic extract); COR, C. occidentalis root (decoction); CEL, C. eucalyptus leaves (decoction); CEF, C. eucalyptusflorescence (decoction); SES, C. eucalyptus stem (decoction); CAFK, C. aurantifolia (juice)+Kanwa.

showed the presence of tannins in small amounts (Table1) except in C. aurantifolia. Tannins have been reportedto prevent the development of microorganisms by pre-cipitating microbial protein and making nutritionalproteins unavailable for them (Fluck, 1973). It there-fore suggests that the medicinal plants used in thepresent study may have a general antimicrobial activity.

The anti-microbial test (Table 2) showed that only C.eucalyptus and C. aurantifolia plus Kanwa extractsexhibited activity against S. typhi. The result indicatesthat although, all these medicinal plants (E. hirta, C.occidentalis and C. eucalyptus) are combined in variousproportions for the treatment of typhoid fever by theNupes, only C. eucalyptus contains the active substanceagainst S. typhi, the causative organism of typhoidfever. From the phytochemical screening (Table 1),only C. eucalyptus contains an alkaloid which is notpresent in any part of the other medicinal plants; it,therefore, suggests that the alkaloid present in C. euca-lyptus may be the active substance against S. typhi. TheC. aurantifolia plus Kanwa exhibited activity against S.typhi only at a high concentration of Kanwa. TheKanwa is an alkali salt with varying mineral composi-tions depending on where it is mined from. The mecha-

nism by which Kanwa is able to inhibit the growth ofS. typhi at high concentration may therefore be that ofwithdrawing water from the organism by osmosis.

However, the use of Kanwa at such high concentra-tion may be toxic particularly as it may contain highlevels of lead and cadmium (Makanjuola and Beetle-stone, 1975).

Table 3Zone of inhibition of S. typhi by C. eucalyptus and C. aurantifolia(juice) plus Kanwa

Zone of inhibition (mm)Concentration(mg/ml)

C. aurantifoliaC. eucalyptus

3 1.92 NI3.204 NI5.005 NI5.406 NI

7 NI6.006.598 NI

9 7.10 NI10 8.00 5.00

NI, No inhibition.

C.E. E�ans et al. / Journal of Ethnopharmacology 80 (2002) 21–2424

Table 4Minimum inhibitory concentration and minimum bactericidal con-centration (mg/ml)

Plant extract MBC (mg/ml)MIC (mg/ml)

10E. hirta leaves NI(ethanolic extract)

E. hirta root 10 NI(ethanolic extract)

E. hirta florescence 10 NI(ethanolic extract)

10C. occidentalis root NI(decoction)

C. eucalyptus leaves 1 2(decoction)

1C. eucalyptus florescence 2(decoction)

1C. eucalyptus stem 2(decoction)

C. aurantifolia 9 10(juice)+Kanwa

NI, not tested.

that have not been investigated in the present work.The present work also observes that the use of C.aurantifolia juice can be effective against S. typhi onlyat high concentrations of Kanwa as used by traditionalmedicine practitioners. However, the safety levels ofKanwa may be worth studying.

References

Adebanjo, A.O., Adewumi, C.O., Essein, E.E., 1983. Anti-infectiveagents of higher plants. In: International Symposium of MedicinalPlants, fifth ed. University of Ife, Nigeria, pp. 152–158.

Bhakumi, D.S., Gupa, S., 1982. The alkaloids of Stephania glabra.Lioyda 45 (4), 412.

Boaky-Yiadon, I.K., 1979. Antimicrobial activity of two flavononesisolated from the cameronian plant Enythinna sigmoidea. PlantaMedica 54 (2), 126–212.

Chiori, C.O., Ezeiruaka, H.N., Ogad, F.A. 1977. A study of twoantiseptic properties of the oil from the fresh leaves of Occimium�irde and Cympogan citratus. Journal of Pharmaceutical MedicineScience, (6): 207–243.

Fluck, H., 1973. Medicinal Plants and Their Uses. W. Feulsham andComp Ltd, New York, pp. 7–15.

Ikenebameh, M.J., Metitiri, P.O., 1988. Antimicrobial effect of anextract of four Cassia alata. Nigeria Journal of Microbiology 8,12–22.

Irobi, O.N., Mor-young, M.J., Anderson, M.A., Daramola, S.O.,1995. Antimicrobial activity of bark extracts of Bridellia femiginea(Euphorbiacea). Journal of Ethanopharmacology 43, 155–190.

Konkwara, J.O., 1976. Medicinal plants of East Africa. LiteratureBurea, Nairobi, pp. 3–8.

Kurosaki, F., Nishi, A., 1983. Isolation and antimicrobial activity ofthe phytoalexin 6-methyllein from cultured carrot cells. Phyto-chemical 22 (3), 669–672.

Makanjuola, A.A., Beetlestone, J.G., 1975. Some chemical mineral-ogy notes on Kanwa (Trona). Journal of Mining and Geology 10,31–34.

Omoregbe, R.E., Ikuebe, O.M., Ihimire, I.G., 1995. Antimicrobialactivity of some medicinal plants extracts on Escherichia coli,Salmonella paratyphi and Shigella dysenteras. Africa Journal Med.3 (1), 6–10.

Owonubi, M.O., 1988. Use of local herbs for curing diseases.Pharma. Herbal Med. 4 (2), 26–27.

Sodipo, O.A., Akanji, M.A., Kolawole, F.B., Odutuga, A.A., 1991.Saponin is the active antifungal principle in Garcina kola, heckelseed. Biological Science Research Community 3 (1), 171.

UNIDO (1969). Methodology for Analysis of Vegetable Drugs.Bucharest of the joint UNIDO Romania Centre, English Ed.,B.N. Balacescu 16. 70121.

Table 3 shows the diameter of the zone of inhibitionof S. typhi by extracts of C. eucalyptus and C. auran-tifolia plus Kanwa. The result reveals that higher con-centrations of the extracts increase the inhibition of S.typhi. The present result agrees with previous reports(Boaky-Yiadon, 1979; Kurosaki and Nishi, 1983) thathigher concentration of antimicrobial substances showsappreciable growth inhibition.

The MIC and MBC of C. eucalyptus (Table 4) are 1and 2 mg/ml, respectively. This indicates that the ex-tracts of C. eucalyptus are more effective against S.typhi. It is important to note that the MIC of the plantextracts is lower than the MBC against the test organ-ism. No colony growth was observed on the solidmedium after the incubation period when the MBC ofthe plant extracts was used against S. typhi.

The present work confirms that the preparation of E.hirta, C. occidentalia and C. eucalyptus in differentcombinations as used by the Nupes, are effectiveagainst S. typhi, the causative organism of typhoidfever. However, only C. eucalyptus contains the activecompound. It therefore suggests that the other medici-nal plants may be serving other physiological purposes


Screening of African medicinal plants for antimicrobial andenzyme inhibitory activity

Jeannette Ndaya Tshibangu a, Kusamba Chifundera b, Ronald Kaminsky c,Anthony David Wright a, Gabriele Maria Konig a,*

a Institute for Pharmaceutical Biology, Nussallee 6, D-53115 Bonn, Germanyb Centre de Recherches en Sciences Naturelles Lwiro, Buka�u, Congo

c Swiss Tropical Institute, Socinstrasse 57, CH-4002 Basel, Switzerland

Received 20 May 2001; received in revised form 10 November 2001; accepted 23 November 2001

Abstract

Seven plant species, belonging to different families, were collected in the eastern part of the Republic of Congo (Kivu) basedon ethnopharmacological information. Their dichloromethane and methanolic extracts were tested for biological activity. Five ofthe seven collected plants exhibited antiplasmodial activity with IC50 values ranging from 1.1 to 9.8 �g/ml. The methanolic extractof Cissampelos mucronata was the most active one showing activity against chloroquine sensitive (D6) and chloroquine resistant(W2) Plasmodium falciparum strains with IC50 values of 1.5 and 1.1 �g/ml, respectively. Additionally, this extract significantlyinhibited the enzyme tyrosine kinase p56lck (TK). The dichloromethane extract of Amorphophallus bequaertii inhibited the growthof Mycobacterium tuberculosis with a MIC of 100 �g/ml and the methanolic extract of Rubus rigidus inhibited the activity of bothenzymes HIV1-reverse transcriptase (HIV1-RT) and TK p56lck. © 2002 Published by Elsevier Science Ireland Ltd.

Keywords: Antimicrobial; Medicinal plants; Inhibitory activity; Malaria


1. Introduction

Plant materials remain an important resource tocombat serious diseases in the world. Pharmacognosticinvestigations of plants are carried out to find noveldrugs or templates for the development of new thera-peutic agents (Konig, 1992). Among the more than250 000 species of higher plants, only about 5–10% arechemically investigated (Nahrstedt, 1996). Since manydrugs, e.g. quinine and artemisinin (Wright and Phillip-son, 1990), taxol and camptothecin (Debernardis et al.,1996) were isolated from plants, and because of in-creased resistance of many microorganisms, e.g.malaria parasites, towards established drugs, investiga-tion of the chemical compounds within traditionalplants is necessary (Phillipson, 1991).

Malaria is still one of the most important parasiticdiseases of mankind. The yearly statistics of the WorldHealth Organization, concerning mortality caused bytropical diseases, particulary from malaria, is frighten-ing. It is estimated that there are more than a million

deaths and up to 500 million clinical cases of malariaeach year (Bulletin of the World Health Organization,1999).

The Republic of Congo is a country entirely situatedin the high risk zone of endemic malaria. Many plantsin Congo are used traditionally to heal different dis-eases (Wome, 1982, 1985) including malaria; they playan important role in the medical system of the country.Generally, the traditional use of medicinal plants isaccompanied with certain rituals and incantations. Manis considered to be part of an intriguing system com-posed of the community he is living in, the naturesurrounding him and the ancestors. Thus, sickness isconsidered as the destruction of balance in a person,and between the person and nature, community andancestors. Similarly, as in ayuverdic medicine (Mazars,1998), the psychical and corporeal state of a person istaken into account. These considerations make theaccompanying rituals of treatment understandable.Some people (healers or not healers), however, simplyuse medicinal plants, without ritual. The knowledgeconcerning traditional plants and remedies is mostlyhanded down orally. Efforts to collect and write down* Corresponding author.


J. Ndaya Tshibangu et al. / Journal of Ethnopharmacology 80 (2002) 25–3526

this knowledge are being made in different parts of thecountry. There are publications concerning medicinalplants from the North-East of the country, from Kin-shasa, from Kasai occidental (Dhetchuvi and Lejoly,1989; Delaude et al., 1971; Mabika and Ramaut, 1982;Mabika, 1983; Bakana, 1984), about medicinal plantsused in case of diarrhoea in Kinshasa and Lomela area(Kambu et al., 1989; Tona et al., 1998; Longanga et al.,2000), and about antimalarial plants (Tona et al.,1999), just to mention a few.

The main aim of the present project was to collectinformation on plant species used traditionally for thetreatment of malaria. Extracts of these plants were tobe subjected to biological screening and their phyto-chemical investigation should yield the secondarymetabolites responsible for the observed activity. Ac-cordingly, seven species were collected from the regionof Kivu, in the eastern part of the Republic of Congo.The plants studied are mainly used against malaria andother similar tropical diseases. Referring to their tradi-tional usage, their extracts were investigated in a multi-tude of test systems, especially for antiplasmodialactivity. Antimicrobial and antitubercular activities aswell as other biological activities not indicated by thetraditional usage, such as antitrypanosomal effects andinhibition of HIV1-reverse transcriptase (HIV1-RT)and tyrosine kinase p56lck (TK) were also assessed.


2.1. Plant material

Plants were collected in the Kivu region, Republic ofCongo, between January and March 1996. Sampleswere identified by reference to the Herbarium of theDepartment of Biology, Laboratory of MedicinalPlants, Lwiro, Republic of Congo. The voucher speci-mens of the seven collected species, i.e. Amorphophallusbequaertii De Wilde (JNT 1996), Celosia trigyna L.(Christiansen 3681, 2340; Meurillon 796; Ntakiyimana472), Cissampelos mucronata A. Richard (CKL 359),Myrianthus arboreus P. Beauv. (Troupin 6256), Otio-phora puciflora Baker (Auquier 2157-753), Polysciasful�a (Hierns) Harms (Ntakiyimana 401; Pierlot 1529;Troupin 3234) and Rubus rigidus Smit (Bouxin 835;Liben 1069; Michel 5710; Troupin 8355, 10500, 14968)are preserved at the the above mentioned Herbariumand at the Department of Pharmaceutical Biology,University of Bonn, Germany. Concerning C. mu-cronata and A. bequaertii, the species identification wasconfirmed by the late Professor G. Troupin (personalcommunication, on January 23th 1997, rue Grady 2,B-4053, Emburg, Belgium). Plants were prepared by airdrying, grinding and finally storage in paper bags.

2.2. Extraction

Plant powder (50 g) was extracted using an Ultra-Turrax with 450 ml each (3×150 ml) ofdichloromethane, and subsequently methanol. C. mu-cronata was extracted also with ethyl acetate, and sub-sequently with 80% methanol due to the low yield ofthe dichloromethane extract. Solvents were removed invacuo at 25 °C. Residue of both lipophilic (DCM andEtOAc), and hydrophilic (MeOH) extracts were dis-solved in solvents required for the various test systemsand then used for the biological screening.

2.3. Biological screening

2.3.1. Antimalarial acti�ityCultures of Plasmodium falciparum (chloroquine-sen-

sitive strain D6 and chloroquine-resistant strain W2)were maintained in human erythrocytes [(A+ , Albu-max, (Invitrogen)] in vitro. The antimalarial activity ofextracts was assessed with an in vitro radio isotopeincorporation method (Desjardins et al., 1979). IC50

values of test extracts, reference and positive controlwere determined from logarithmic dose–responsecurves and were expressed as a percentage of inhibitedparasite specific 3H-hypoxanthine incorporation (Ridleyet al., 1996). Samples with IC50 values �10 �g/ml wereconsidered as active.

2.3.2. Antitubercular acti�ityMycobacterium tuberculosis was cultured under

anaerobic conditions, inoculated into vials of BACTECmedium with and without antimycobacterial agents. Inthe absence of mycobacterial inhibition, the mycobacte-ria grew and utilized 14C-labeled substrate present inthe medium. The growth of mycobacteria in themedium is directly proportional to the growth index(GI), which represents the quantity of 14CO2 producedwhen the substrate is decarboxylated (Konig et al.,2000; Collins and Franzblau, 1997). Samples showingany inhibition at a concentration of 100 �g/ml wereconsidered active.

2.3.3. Antitrypanosomal acti�ity

2.3.3.1. Acti�ity against African trypanosomes. Activityof compounds against T. brucei rhodesiense, thecausative agent of African sleeping sickness, was deter-mined according to Raz et al. (1997). Briefly, parasiteswere propagated axenically in culture medium supple-mented with horse serum (Kaminsky and Brun, 1993).Cultures in logarithmic growth phase were propagatedfor 3 days in the presence of various drug concentra-tions. Viability of parasites was quantified using the dyeAlamar Blue according to Raz et al. (1997). The drugsensitivity was regarded as positive when the MIC wasbelow 100 �g/ml.

J. Ndaya Tshibangu et al. / Journal of Ethnopharmacology 80 (2002) 25–35 27

2.3.3.2. Acti�ity against Trypanosoma cruzi. Activity ofcompounds against T. cruzi (Tulahuen strain), thecausative agent of Chagas disease, was determined ac-cording to Buckner et al. (1996). Briefly, a T. cruzistrain that expresses the Escherichia coli �-galactosidasegene, was propagated intracellularly in mammalianfibroblast cells in 96-well microtiter plates. Cultureswere propagated for 5 days in the presence of drug,before viability of T. cruzi amastigote forms was deter-mined. Transfected parasites catalyse a colorimetricreaction with chlorophenol red and �-D-galacto-pyra-noside as substrate. The reaction was quantified em-ploying an enzyme-linked immunosorbent assay reader.

2.3.4. Agar diffusion testThe test organisms for the agar diffusion assays were

the bacteria Bacillus megaterium de Bary (Gram posi-tive) and E. coli (Migula) Castellani and Chambers(Gram negative), the fungi Microbotryum �iolaceum(Pers.) Roussel and Eurotium repens Corda, and thegreen microalga Chlorella fusca Shih Krauss.

Samples were prepared by taking 50 �l (�450 �g) ofthe solution of plant extracts and pipetting them onto asterile antibiotic filter disc, which was then placed ontothe appropriate growth medium and sprayed with asuspension of the corresponding test organism, andsubsequently incubated. Zones of inhibition were mea-sured from the edge of filter discs. Samples displaying agrowth inhibition zone �3 mm and/or a completeinhibition zone �1 mm were considered as positive(Schulz et al., 1995; Holler, 1999).

2.3.5. Inhibition of HIV1-RTThe activity of recombinant HIV1-RT was measured

according to the ELISA protocol etablished by Eberleand Seibl (1992) using an ELISA reader. The concen-tration of the extracts was 66 �g/ml. A sample wasconsidered as active when the activity of RT was re-duced by 20% or more (Kirsch, 1999; Kirsch et al.,2000).

2.3.6. Inhibition of TKTK inhibition assay was carried out using T cell TK

p56lck and a commercial test kit, modified by Dr G.Kirsch (Institute for Pharmaceutical Biology, TechnicalUniversity of Braunschweig, Germany). The concentra-tion of the extract was 200 �g/ml. Extracts whichreduced activity by at least 50% were considered active(Wessels et al., 1999; Kirsch, 1999).

2.3.7. CytotoxicityCytotoxicity was tested towards KB (human epider-

moid carcinoma) cells by analyzing the protein contentof cells surviving the treatment (Likhitwitayawuid etal., 1993). Cells were plated at 5×104 cells/ml ofculture medium in 96-well plates and incubated with

the test samples dissolved in DMSO (final DMSOconcentration was 0.5%). Incubations were continuedfor 72 h after which time the quantity of cells survivingthe treatment was determined by analyzing proteincontent using a sulforhodamine B dye-binding spec-trophotometric method. Samples with an IC50 of �20�g/ml were regarded as inactive.

3. Results

3.1. Area of collection and storage of plant materials

Bushi (Bashi land) and the area around Irangi station(transition forest) are situated in the eastern part of theRepublic of Congo (Kivu, 1°30� S and 28°27� E). Bashiland is mainly covered with savanna grass, while thetransition forest is tropical (Dieterlen, 1978). The in-habitants of this area, and the people living towardsKisangani (Haut-Congo) are well known for theirknowledge of the use of medicinal plants. This was themain reason this region was chosen for ourinvestigations.

The plants selected for this study were picked basedon the outcome of interviews with healers and peoplewho are in contact with them or who have otherwiseexperience with traditional medicine. Questions askedconcerned the plant species used for treatment of sus-pected malaria or when malaria was clinically diag-nosed, plants used for treatment of headaches, fever,dizzyness and vomiting. Finally, the interviewed personidentified the plants in the field. Plants used alone, i.e.not mixed with additional plant or inorganic materials,were considered further. After literature searches, sevenplant species were chosen and collected in sufficientamounts to allow biological and chemical investiga-tions. Plant names are given in the native language andlisted in Table 1 together with the species assignmentand their traditional use. As indicated in Table 1, plantschosen are not only employed in case of malaria butalso against other diseases.

C. mucronata was on one side often confused withCissampelos pareira and on the other side consideredtogether with C. owariensis as a variety of C. pareira(Rhodes, 1975; Neuwinger, 1994). According to a re-vised taxonomic concept, however, C. mucronata, rep-resents a separate species, which can be differentiatedfrom C. pareira based on its morphology. Thus, C.mucronata has more oval pointed leaves, their basisheart shaped, the stalk is fixed 0.5–3 mm away fromthe base of the leaf. The inflorescence is racemose whilethe inflorescence of C. pareira is cymose and its leavesare more round, the stalk is 1–4 mm away from therounded base of the leaves (Raynal et al., 1981; Ben-venuto, 1974; Troupin, 1962). Moreover, among thethree species, C. pareira is not to be found in Congo(Troupin: personal communication).


Tab

le1

Pla

nts

colle

cted

and

thei

rtr

adit

iona

lus

e

Spec

ies

Tra

diti

onal

met

hod

ofpr

epar

atio

nV

erna

cula

rna

me

Pla

ntpa

rtF

amily

Tra

diti

onal

lyus

edto

trea

tco

llect

ed

Mal

aria

,fe

ver,

abdo

min

alpa

in,

snak

ebi

teE

xtra

ctth

egr

ound

tube

rw

ith

war

mw

ater

,fil

ter,

Tub

erM

band

akab

iri,

Am

orph

opha

llus

Ara

ceae

bequ

aert

iiD

eW

ilde

Ikom

aly

aka

biri

use

this

extr

act

for

rect

alin

ject

ion

Her

bal

part

sM

alar

ia,

feve

r,in

test

inal

wor

ms,

head

ache

,no

seA

mar

anth

acea

eU

seab

out

10le

aves

from

the

top

ofth

epl

ant,

Buk

arag

ata,

Cel

osia

trig

yna

L.

Mub

alal

ain

flam

mat

ion,

pain

duri

ngpr

egna

ncy,

uter

uspa

ingr

ind

them

,po

urso

me

drop

sin

nose

and/

orva

gina

,or

mix

the

rem

aini

ngju

ice

ofgr

ound

leav

esw

ith

bana

naju

ice

and

drin

k.A

ddit

iona

lly,

drin

kth

eju

ice

afte

rm

acer

atio

n,tw

ice

daily

.M

alar

ia,

feve

r,se

xual

lytr

ansm

itte

ddi

seas

es,

Men

ispe

rmac

eae

Cis

sam

pelo

sB

oil

the

grou

ndro

otin

wat

erab

out

10m

inan

dR

oot

snak

ebi

t,co

njun

ctiv

itis

drin

kth

efil

trat

e.In

case

oful

cer

orco

njun

ctiv

itis

,m

ucro

nata

A.

Ric

hard

put

som

edr

ops

ofju

ice

offr

esh

leav

eson

the

infla

med

orul

cere

dpa

rtB

ark

Mal

aria

,fe

ver,

coug

hB

oil

the

stem

bark

(and

leav

es)

inw

ater

and

drin

kM

yria

nthu

sar

bore

usM

orac

eae

Cam

ba,

Bya

mba

P.

Bea

uv.

the

filtr

ate

twic

eda

ilyH

erba

lpa

rts

Lul

erhe

lerh

a,M

alar

ia,

coug

h,ps

ycho

logi

cal

dist

urba

nces

,O

tiop

hora

pauc

iflor

aR

ubia

ceae

Dri

nkth

eju

ice

afte

rm

acer

atio

nof

aeri

alpa

rtof

Bak

erC

ifub

ula,

the

plan

ttw

ice

ada

yov

er2

days

.B

urn

the

drie

dab

orti

on,

topr

ove

wit

chkr

aft

plan

tsto

aci

nder

,ad

dsa

ltan

dlic

ktw

ice

ada

yto

Hin

yang

arha

trea

tco

ugh.

Inca

seof

wit

chcr

aft,

mix

apl

ant

wit

hle

aves

ofB

asel

laal

ba(B

asel

lace

ae),

crus

h,sq

ueez

eth

em,

add

two

drop

sof

this

juic

ein

boili

ngw

ater

:if

the

foam

pour

sou

t,it

mea

nsth

eac

cuse

dis

aw

itch

,or

inth

eot

her

case

,he

(or

she)

isin

noce

ntD

rink

the

infu

sion

agai

nst

mal

aria

and

feve

r.In

Nto

nge,

Lun

gaB

ark

Ara

liace

aeP

olys

cias

ful�

aM

alar

ia,

feve

r,m

enta

lill

ness

,co

nseq

uenc

eof

Ndo

nyi

(Hie

rn)

Har

ms

case

ofad

ulte

ry,

the

juic

eof

mac

erat

ion

will

bead

ulte

ryvo

mit

ed.

Pou

ra

drop

ofju

ice

ofm

acer

atio

nin

each

nost

ril

ever

yda

yto

heal

men

tal

illne

ssF

ever

(mal

aria

),oe

dem

a,sk

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ncer

,hi

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sD

rink

the

juic

eof

the

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ndro

otR

oot

Rub

usri

gidu

sSm

itR

osac

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Luk

erer

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brea

stfe

edin

g,di

fficu

lty

wit

hsw

allo

win

gIk

anga

hwa


3.2. Biological screening

Results of biological testing of lipophilic and hy-drophilic extracts of the collected plants are presentedin Table 2. According to O’Neill et al. (1985), a crudeextract having in vitro an IC50 value of �50 �g/ml forP. falciparum can be considered as active. In our pro-ject, however, we defined samples with IC50 values �10�g/ml as inactive. Among the 15 extracts tested, sevenwere found to be active. The hydrophilic extract of C.mucronata and the lipophilic extract of M. arboreusshowed the most promising activity against both strainsof P. falciparum, one being chloroquine sensitive (D6)and the other chloroquine resistant (W2). C. mucronatahad a slightly more potent effect towards thechloroquine resistant strain (W2), the IC50 values being1.8 �g/ml (EtOAc) and 1.1 �g/ml (MeOH), respectively;while the IC50 values against the chloroquine sensitivestrain were 2.9 �g/ml (EtOAc) and 1.5 �g/ml (MeOH).All other active extracts, including the dichloromethaneextract of M. arboreus (IC50 2.6 �g/ml), showedstronger antiplasmodial effects toward the chloroquinesensitive P. falciparum strain. The lipophilic extract ofC. trigyna with an IC50 of 5.0 �g/ml can be consideredas moderatly active against strain D6 of P. falciparum.

Three extracts were significantly active towards M.tuberculosis at a sample concentration of 100 �g/ml.These were the dichloromethane extracts of A. bequaer-tii, M. arboreus and R. rigidus. The dichloromethaneextract of A. bequaertii caused a 99% inhibition ofmycobacterial growth at a concentration of 100 �g/mlwhile the MeOH extract caused only a 2% inhibition.At the same concentration, there was no inhibition ofMycobacterium a�ium ; none of the plant extracts hadan activity against this microorganism. All antitubercu-lar activity was found in the lipophilic extracts.

The parasite responsible for Chagas disease, T. cruzi,was inhibited by the aqueous extract from A. bequaertiiat a minimal inhibitory concentration (MIC) of 11�g/ml, while the same extract showed a MIC of 33�g/ml for T. rhodesiense, the cause of sleeping sickness.Against this latter species, the dichloromethane extractof M. arboreus showed a relatively potent effect (MIC11 �g/ml), whereas towards T. cruzi, the MIC wasdetermined as 100 �g/ml. The ethyl acetate extract of C.mucronata, also inhibited the growth of T. rhodesiense,the MIC value being 11 �g/ml, with the MeOH extractcausing growth inhibition of T. cruzi with a MIC of 33�g/ml. The remaining extracts displayed MICs �100�g/ml against both Trypanosoma species, and were thusregarded as inactive.

In test systems determining inhibition of enzymaticactivity of HIV1-RT and TK p56lck the hydrophilicextract of R. rigidus caused 100% inhibition of theenzymes, while its dichloromethane extract had no ef-fect. The aqueous extract of A. bequaertii also appeared

to be similarily active against both enzymes, showing 33and 45% inhibition of the enzymes activity, respectively.Remarkable was also the effect of the methanolic ex-tract of C. mucronata towards the enzyme TK. At aconcentration of 200 �g/ml this extract inhibits TKactivity to 92%. This activity appears to be selectivesince HIV1-RT is not inhibited. The EtOAc extract alsoappeared to have selective inhibitory activity towardsTK causing an inhibition of 71%. According to ourthreshold values for activity against HIV1-RT (�20%inhibition) and TK (�50% inhibition), the remainingextracts were judged as not active.

The alga C. fusca, among all the microorganismsused in the agar diffusion assay appeared to be themost sensitive to the tested extracts, especially to theMeOH extracts of plants. A significant antialgal activ-ity was observed by the MeOH extracts of R. rigidus (5mm inhibition zone) and O. pauciflora (3 mm inhibitionzone). Another interesting effect was seen with theDCM extract of A. bequaertii against B. megaterium, aninhibition zone of 4 mm. A further result worthy ofmention was the activity of the MeOH extract of C.trigyna against Eurotium repens ; inhibition zone of 5mm. The activity of remaining extracts towards mi-croorganisms were either too low to be consideredrevelant or there was clearly no activity (Table 3).

None of the extracts displayed any significant activitytowards KB cells; they all had IC50s �20 �g/ml.

4. Discussion

The most promising antiplasmodial activity wasfound for the extracts of C. mucronata with IC50 valuesof 1.8 �g/ml (EtOAc) and 1.1 �g/ml (MeOH) againstthe chloroquine resistant strain (W2), and 2.9 �g/ml(EtOAc) and 1.5 �g/ml (MeOH) towards thechloroquine sensitive strain (D6). An in vitro activityagainst P. falciparum has already been reported(Gessler et al., 1994), as well as an anti-ulcer activity(Akah and Nwafor, 1999). Gessler et al. (1994) found invitro antimalarial activity with IC50 values of 1.3(ethanolic fraction), 8.0 (petroleum ether fraction), 0.38(ethyl acetate fraction), and 1.2 �g/ml (aqueous frac-tion) against the multidrug resistant strain K1. Thegenus Cissampelos contains, among other compounds,different alkaloids: protoberberin, aporphin, benzyliso-quinoline, saturated and unsaturated isobutyl-de-camides (Bhattacharji et al., 1956; Mukerji andBhandari, 1959; Bhakuni et al., 1987; Galinis et al.,1993; Barbosa-Filho et al., 1997; Rosario et al., 1996;Anwer et al., 1968). The alkaloid curine (Scheme 1),among other compounds, isolated from the related C.pareira, showed in vitro antiplasmodial activity (Anger-hofer et al., 1999; Liu and Xiao, 1983). Results of invitro and in vivo tests with some bisbenzylisoquinoline


Tab

le2

Bio

logi

cal

scre

enin

gof

plan

tex

trac

ts

Bio

assa

ysSa

mpl

e

Pla

ntm

ater

ial

M.

tube

rcul

osis

%in

hibi

tion

at10

0E

xtra

ctT

rypa

noso

ma

rhod

esie

nse

MIC

inP

.fa

lcip

arum

IC5

0(�

g/m

l)T

.cr

uzi

MIC

inM

.a�

ium

%in

hibi

tion

at10

0�g

/ml

�g/m

l�g

/ml

�g/m

l

W2

D6

0D

CM

9910

0�

100

�10

�10

A.

bequ

aert

ii(t

uber

)0

2�

100

�10

0�

10�

10M

eOH

3311

�10

0�

10H

2O

0 0D

CM

0�

100

�10

05.

0�

10C

.tr

igyn

a(h

erba

lpa

rt)

00

�10

0�

100

MeO

H�

10�

10 1.8

00

11�

100

C.

muc

rona

taE

tOA

c2.

9(r

oot)

�10

033

00

1.1

1.5

MeO

H

096

1110

02.

6M

.ar

bore

us(b

ark)

7.7

DC

M33

100

MeO

Hn.

t.9.

4n.

t�

10

0D

CM

0�

100

�10

08.

7�

10O

.pa

ucifl

ora

(her

bal

part

)n.

tn.

t.�

100

�10

0M

eOH

�10

�10

DC

M0

100

�10

09.

8�

10P

.fu

l�a

(bar

k)0

�10

0�

100

�10

0M

eOH

0�

10

DC

M44

100

n.t.

�10

�10

R.

rigi

dus

(roo

t)0 0

010

0�

100

MeO

H�

10�

10 6.3

Art

emis

inin

5.0

0.01

1M

elar

sopr

olB

enzn

idaz

ole

0.4

9993

Rif

ampi

cin

0.25

�g/m

l

D6,

P.

falc

ipar

umst

rain

,ch

loro

quin

ese

nsit

ive;

W2,

P.

falc

ipar

umst

rain

,ch

loro

quin

ere

sist

ant;

MIC

,m

inim

alin

hibi

tory

conc

entr

atio

n;D

CM

,di

chlo

rom

etha

neex

trac

t;M

eOH

,m

etha

nolic

extr

act;

EtO

Ac,

ethy

lac

etat

eex

trac

t;H

2O

,aq

ueou

sex

trac

t;n.

t.,

not

test

ed.


Tab

le3

Scre

enin

gof

plan

tex

trac

tsfo

ren

zym

ein

hibi

tory

and

anti

mic

robi

alac

tivi

ty

Sam

ple

Bio

assa

ys

Aga

rdi

ffus

ion

assa

ysc

TK

%in

hibi

tion

at20

0�g

/mlb

HIV

1-R

T%

inhi

biti

onat

66�g

/mla

Pla

ntm

ater

ial

Ext

ract

M.

�iol

aceu

mE

.re

pens

C.

fusc

aB

.m

egat

eriu

mE

.co

li

DC

M4

00

0n.

a.n.

a.A

.be

quae

rtii

(tub

er)

0 00

1.5

02

n.a.

n.a.

MeO

H0

00

045

330

H2O

DC

M0

00

0n.

a.n.

a.C

.tr

igyn

a(h

erba

lpa

rt)

00

MeO

H0

52

n.a.

n.a.

0

n.t.

n.t.

n.t.

n.t.

n.t.

71n.

a.E

tOA

cC

.m

ucro

nata

(roo

t)n.

t.n.

t.n.

t.n.

t.n.

t.M

eOH

n.a.

92

00

02

DC

M0

M.

arbo

reus

(bar

k)n.

a.n.

a.0

MeO

H0

00

n.a.

n.a.

0

DC

M0

00

0n.

a.n.

a.O

.pa

ucifl

ora

(her

bal

part

)0

00

30

MeO

H0

n.a.

n.a.

P.

ful�

a(b

ark)

0D

CM

00

n.a.

n.a.

00

00

10

n.a.

n.a.

1M

eOH

DC

M0

00

0n.

a.n.

a.R

.ri

gidu

s(r

oot)

0 02

00

510

0M

eOH

100

DC

M,

dich

loro

met

hane

extr

act;

MeO

H,

met

hano

licex

trac

t;E

tOA

c,et

hyl

acet

ate

extr

act;

n.a.

,no

tac

tive

;n.

t.,

not

test

ed.

aH

IV1-

RT

enzy

me.

bT

Kp5

6lck.

cIn

hibi

tion

zone

inm

m,

mea

sure

dfr

omth

eed

geof

the

filte

rdi

sc,

ata

sam

ple

conc

entr

atio

nof

450

�g/d

isc.


alkaloids, e.g. curine (1), against T. cruzi have also beenreported. After an oral treatment with curine (1) for 10days, parasitemia was negative in mice infected with theY or CL strain of T. cruzi (Fournet et al., 1997). Thissame compound (1) displayed an in vitro IC50 value of10 �M (Fournet et al., 1998) towards trypomastigoteforms of the Y strain of T. cruzi. Thus, the effect of C.mucronata against T. cruzi, MIC of 33 �g/ml (MeOHextract) in our study may be due to the presence ofbisbenzylisoquinoline alkaloids in this plant. Bisben-zylisoquinoline alkaloids may also be responsible forthe antiplasmodial activity of this plant. It is not clearwhich compounds may contribute to the potent inhibi-tion of TK by the MeOH/EtOAc extracts of C. mu-cronata. These results could, however explain the use ofthe plant in the traditional medicine. C. mucronata wasreported to be traditionally employed against cough,diarrhoea, stomach (abdominal) pain, complicationsduring pregnancy (Adjanohoun et al., 1986a,b,c,d,1989; Watt and Breyer-Brandwijk, 1962), sexuallytransmitted diseases, fever and malaria (Publications del’INEAC, 1951; Legrand and Wondergem, 1987; Ra-soanaivo et al., 1992) in a number of countries.

The observed inhibition of growth of T. cruzi and T.rhodesiense by the aqueous extract of A. bequaertii wasweak when compared to the reference drugs melarso-prol and benznidazole, which have MIC values of 0.011and 0.4 �g/ml, respectively. These compounds werearound 103 and 102 times more active than the extract.Polysaccharides were found to be the main constituentsof plants of the genus Amorphophallus (Wang et al.,1989; Zhang et al., 1988; Li et al., 1987; Maeda et al.,1980; Chawla and Chibber, 1976). It is unlikely that theobserved activity of the aqueous extract of A. bequaertiiwas due to the presence of polysaccharides in the plant.Cardellina et al. (1993), however described the in vitroantiviral effect of anionic polysaccharides in a cellbased assay system. Since a high percentage of extractsfrom various organisms showed this type of activity, itis generally regarded as unspecific.

Noteworthy is the activity of the lipophilic extract ofA. bequaertii towards M. tuberculosis, causing 99%growth inhibition at a concentration of 100 �g/ml, andalso the growth inhibition of B. megaterium. An extractfrom the stem of A. campanulatus was reported to beactive against M. tuberculosis (Gupta and Viswanathan,1955). Concerning the traditional use of A. bequaertiiagainst malaria, it can be speculated that the extracts ofthis plant may be immunomodulating due to theirpolysaccharide content or that they are active againstother stages of the development of the malaria parasitenot covered by our assay system.

While the dichloromethane (DCM) extract of R.rigidus showed no effect on HIV1-RT and TK enzymes,its hydrophilic extract completely inhibited the activityof both enzymes. Among the substances known toinhibit RT (Matthee et al., 1999), some contain apyrogallol moiety. Pyrogallol derivatives were isolatedfrom R. rigidus (Rwagabo, 1981), and may thus beresponsible for its enzyme inhibitory and also antifun-gal effects (Rwagabo, 1993). Esters of gallic acid, e.g.penta-O-galloyl-D-glucopyranose and similar tanninswere isolated from other species of Rubus (Haddock etal., 1982). The observed activity could thus also be dueto the presence of these substances. The effect of suchcompounds, however, seems to be unspecific. The effectof its DCM extract against M. tuberculosis may supportthe traditional use of this plant.

In Cameroun, M. arboreus is used in the traditionalmedicine, in particular against headaches and boils(Ngounou et al., 1990). In the Republic of Congo, inKahuzi Biega Park, it is observed that gorillas consumethe fruit, leaves and bark of this plants when they showsigns of illness (personal communication from K.Kaleme, A member of the research team on Gorillas,Laboratoire de Mammalogie, Departement de Biologie,C.R.S.N-Lwiro, Bukavu, Republic of Congo). The ob-served activity of the lipophilic extract on M. tuberculo-sis, with an inhibition of 96% at a concentration of 100�g/ml, and on T. rhodesiense, with an MIC of 11 �g/ml,could explain the use of this plant species in traditionalmedicine. The observed activity is probably due to thepresence of terpenoids, e.g. triterpene acids and penta-cyclic triterpenes in the plants (Ngounou et al., 1987a,b,1988, 1990; Ojinnaka and Kenne, 1985). Terpenoidswere found to be present in plants of the genus Celosia(Prakash et al., 1995; Fu et al., 1992; Behari and Shri,1986; Xu et al., 1986; Mehta et al., 1981). Additionally,saponins were reported from the genus Polyscias(Chaboud et al., 1995; Lutomski and Luan, 1992a,b;Lussignol et al., 1991; Gopalsamy et al., 1990; Paphas-sarang et al., 1989a,b,c). From the genus Otiophora nosecondary metabolites are described.

Of the seven plant species identified in field studies astraditional agents to treat malaria, five were shown tohave considerable antiplasmodial effects. Above all, C.Scheme 1.


mucronata deserves further phytochemical and pharma-cological investigations due to its antimalarial potentialdemonstrated in the current study. Similary, M. ar-boreus is most interesting due to its antiplasmodial,antimycobacterial and antitrypanosomal effects. Theaqueous extract of A. bequaertii also merits detailedinvestigations to identify the compounds responsible forenzyme inhibitory and antitrypanosomal effects.

Acknowledgements

Jeannette Ndaya Tshibangu thanks the KatholischeAkademische Auslander-Dienst (KAAD) for a scholar-ship. Thanks also go to the Deutsche Forschungsge-meinschaft (DFG) (Project Ko 902-31) for financialsupport, and to Dr G. Kirsch (Department of Pharma-ceutical Biology, Technical University of Braunschweig)and C. Dreikorn (Department of Pharmaceutical Biol-ogy, University of Bonn) for RT and TK testing. Weare greatful to Professor S.G. Franzblau (GWLHansen’s Disease Center, P.O. Box 25072, BatonRouge, Louisiana 70894, USA) for tests against My-cobacterium tuberculosis. Boehringer Mannheim isthanked for arranging the shipment of plant materialfrom the Republic of Congo to Germany and for somefinancial support.

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Assessment of the antihypertensive and vasodilator effects ofethanolic extracts of some Colombian medicinal plants

M.F. Guerrero a,c, P. Puebla b, R. Carron a, M.L. Martın a,*, L. Arteaga c, L.San Roman a

a Laboratorio de Farmacognosia y Farmacologıa, Departamento de Fisiologıa y Farmacologıa, Facultad de Farmacia, Universidad de Salamanca, E-

37007, Salamanca, Spainb Departamento de Quımica Farmaceutica, Facultad de Farmacia, Universidad de Salamanca, E-37007, Salamanca, Spain

c Departamento de Farmacia, Facultad de Ciencias, Universidad Nacional de Colombia, Bogota AA 11430, Colombia

Accepted 10 December 2001

Abstract

The antihypertensive and vasodilator effects of ethanolic extracts prepared from Calea glomerata Klatt, Croton schiedeanus

Schlecht, Curatella americana L., Lippia alba (Mill)n N.E.Br. and Lupinus amandus , which are medicinal plants used in Colombian

folk medicine for the treatment of hypertension, were assayed both in SHR and Wistar rats and in rat isolated aortic rings. At a dose

of 20 mg/kg, intravenous bolus administration of the ethanolic extracts, from C. schiedeanus , C. americana and L. amandus showed

significant antihypertensive activity in SHR, C. schiedeanus being the most active. C. schiedeanus elicited dose-dependent decreases

in mean arterial pressure and heart rate (5�/100 mg/kg, i.v.) in SHR but 200 mg/kg administered orally did not show any significant

effects, even after 3 h of observation. In intact rat aortic rings, ethanolic extracts from C. schiedeanus and Calea glomerata relaxed

the contractions induced by KCl (80 mM) and phenylephrine (10�6 M) in a concentration-dependent manner (10�6�/3�10�4 g/

ml), with IC50 of 6.5�10�5 (7.3�/5.8) g/ml and 7.1�10�5 (7.9�/6.4) g/ml, respectively. Bioguided phytochemical fractionation of

the ethanolic extract from C. schiedeanus was started. More than one active principle seems to be present, flavonoids and terpenoids

compounds were detected. # 2002 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Calea glomerata Klatt; Croton schiedeanus Schlecht; Curatella americana L.; Lupinus amandus ; Lippia alba (Mill)n N.E.Br.;

Antihypertensive; Vasorelaxant; SHR

1. Introduction

Calea glomerata Klatt, Croton schiedeanus Schlecht,

Curatella americana L., Lippia alba (Mill)n N.E.Br. and

Lupinus amandus are among the medicinal plants used

in Colombian folk medicine for the treatment of

hypertension (Garcıa, 1975; Correa and Bernal, 1992).

In a previous work, we reported the antihypertensive

and vasorelaxant activity of the aqueous fraction

obtained from C. schiedeanus (Guerrero et al., 2001).

Here, we studied the effect of the ethanolic extract from

C. schiedeanus and compared it with those obtained

with extracts from other medicinal plants used for the

same purpose. To date the hypotensive and vasorelaxant

effects of these extracts has not been studied.


2.1. Plant material and extraction

The plant material was collected from the region of

Tocaima, Cundinamarca, Colombia in November 1998.

Jose Luis Fernandez confirmed its identity and the

corresponding voucher specimens (numbers 432161�/

432165) were deposited in the Herbarium of the Natural

Sciences Institute of the National University of Colom-bia.

Aerial parts of the plants were dried in a forced-air

oven at 40 8C and triturated. The powder thus obtained

* Corresponding author. Tel.: �34-92-329-4530; fax: �34-92-329-

4515.

E-mail address: [email protected] (M.L. Martın).




PII: S 0 3 7 8 - 8 7 4 1 ( 0 1 ) 0 0 4 2 0 - 2


was macerated with 95% ethanol, filtered, concentratedunder reduced pressure and lyophilized. For in vitroexperiments, all extracts were dissolved in dimethylsulf-oxide (DMSO). For in vivo experiments, extracts weresuspended and subsequently filtered as follows: C.

americana and C. schiedeanus in 30% Polyethylenegly-

col-400 (PEG-30%); L. amandus, L. alba and C.

glomerata in 0.1% Tween-80/Carboxymethylcellulose

(TC 0.1%).

The C. schiedeanus ethanolic extract was partitioned

with chloroform, ethyl acetate and isobutanol to yield

three fractions, respectively. Subsequently, the chloro-formic fraction was treated with base to yield strong-

acid, neutral and weak-acid fractions.

The weak-acid and neutral fractions were subjected to

silica gel flash column chromatography, eluting with

hexane/ethylacetate (9:1, 8:2, 1:1) to yield 5 {A} and 17

fractions {B}, respectively. The vasorelaxant effects of

all of these fractions were assayed in isolated aortic

rings.

2.2. Measurement of blood pressure and heart rate

SHR (200�/450 g) was kept and supplied by the

Departmental Animal Facilities of the University of

Salamanca (P.A.E-SA001). The animals were anaesthe-

tized with intraperitoneal (i.p.) sodium pentobarbitone

(60 mg/kg) and diazepam (5 mg/kg). In the experiments

with anaestethized rats, the trachea was exposed andcannulated to facilitate spontaneous respiration. Two

catheters (PE-50) were implanted in the left carotid

artery and right jugular vein for recording of arterial

blood pressure and drug administration, respectively.

In experiments with conscious freely moving rats, the

arterial catheter was filled with polyvinylpyrrolidone-

saline solution (0.5 mg/ml) and was exteriorized at the

dorsal neck level. Postoperatively, the rats were housedindividually for at least 24 h when the experiments were

performed.

Blood pressure was monitored using a Letica pressure

transducer connected to a PRS 205 amplifier, and

displayed on one channel of a Letica Polygraph 4000.

Heart rate (HR) was measured by analysis of the blood

pressure data, using a CAR 1000 tachograph connected

to the same PRS 205 amplifier. Mean aortic pressure(MAP) was calculated as diastolic�[(systolic�diasto-

diastolic)/3].

After stable MAP and HR had been obtained, each

anaesthetized animal received a dose of 20 mg/kg (1 ml/

kg) of the ethanolic extracts and the maximal decreases

in MAP and HR were registered. Parallel experiments

were performed with the corresponding vehicle (1 ml/

kg).In another set of experiments, each anaesthetized

animal received a series of increasing bolus (1 ml/kg)

doses of ethanolic extract from C. schiedeanus (EECS)

(5, 10, 20, 50 and 100 mg/kg) or Polyethylenglycol-400

30% (PEG-400 30%) as control, via the intravenous (i.v.)

catheter. When subsequent doses were administered,

MAP and HR were first allowed to return to theirrespective baseline levels.

In experiments with conscious rats, measurements of

MAP and HR were performed just before and every 30

min over 3 h, after a single oral dose of EECS (200 mg/

kg) or 2 ml/kg of vehicle.

All provisions concerning the protection of animals

used for experiments stipulated by Spanish law and in

European Community (EEC 1986) specifications wereapplied.

2.3. In vitro studies in aorta rings

Male Wistar rats (320�/470 g) were given light

anaesthesia with ether and decapitated. The descending

thoracic aorta was dissected and placed in a oxygenated

Krebs solution with the following composition (in mM):

NaCl, 118.0; KCl, 4.75; CaCl2, 1.8; MgSO4, 1.2;

KH2PO4, 1.2; NaHCO3, 25; glucose, 11; and ascorbic

acid, 0.1.

Rings of thoracic aorta (5�/7 mm in length) werecarefully excised and submerged in Allhin organ cham-

bers containing 5 ml of Krebs solution of bathing

medium maintained at 37 8C and bubbled with a 95%O2 and 5% CO2 gas mixture (pH 7.4). The rings weremounted by means of two parallel L-shaped stainless-steel holders inserted into the lumen. One holder servedas an anchor while the other was connected to a force-displacement transducer (Harvard UF1) to measureisometric contractile force, which was recorded with aMacLab/8-computer system (A.D. Instruments Ltd,London, UK). A basal tension of 2 g was applied.Each preparation was allowed to equilibrate for 60�/90min in Krebs solution prior to initiation of experimentalprocedures and during this period the incubation mediawere changed every 15-min.

After equilibration, the aortic rings incubated inKrebs solution were exposed to 80 mM KCl or 10�6

M phenylephrine (PE) until the contractile response

reached a steady tension. Then, ethanolic extracts of C.

schiedeanus fractions (10�6�/3�10�4 g/ml) were added

cumulatively to the bath. In some rings, DMSO was

added as control.

2.4. Drugs and solutions

The following products were used: NaCl, CaCl2,

NaHPO4, NaHCO3, glucose (Panreac); KCl, dimethyl-

sulfoxide (Merck); MgCl2, KH2PO4 and MgSO4 (Pro-bus); pentobarbitone, phenylephrine (SIGMA);

diazepam, (Almirall Prodesfarma), polyvinylpyrroli-

done (GUINAMA).

M.F. Guerrero et al. / Journal of Ethnopharmacology 80 (2002) 37�/4238

2.5. Data analysis and statistics

For the blood pressure and heart rate experiments,

after each dose of the extracts maximal changes(expressed as percentages of baseline values) in MAP

and HR were used to construct a dose�/response curve.

The response of the aortic rings was expressed as a

percentage of the initial contraction to 80 mM KCl or

10�6 M PE. Dose�/response curves were analyzed to

give the logarithm of the concentration of ethanolic

extracts or fractions producing a 50% inhibition of the

maximal contractile response (IC50) by sigmoidal curve-fitting analysis.

All results are expressed as mean9standard errors of

the mean (S.E.M.). Differences in MAP, HR and aorta

concentration-response curves were analyzed by one

way analysis of variance (ANOVA) followed by the

Dunett post hoc test, or by an unpaired Student’s t -test

as required, with the criterion set for statistical signifi-

cance at P B0.05. EXCEL† 97 and SPSS

# 7.5 softwarewas used for data analysis.

3. Results

3.1. Effects on blood pressure and heart rate

In anaesthetized normotensive Wistar rats and SHR,

the mean baseline values of MAP were 9394 (n�20)and 17098 mmHg (n�26) and the mean baseline

values HR were 397910 and 355910 beats/min,

respectively. In conscious freely moving SHR (n�12),

mean baseline MAP and HR values were 15697 mmHg

and 352911 beats per min, respectively.

In normotensive Wistar rats, a significant decrease in

HR was induced only by the ethanolic extract of C.

schiedeanus (20 mg/kg i.v.) (Fig. 1a). In SHR, C.

schiedeanus was able to induce an important reduction

in MAP and a slight but significant decrease in HR (Fig.

1b). C. americana and L. amandus exerted some degree

(significant) of reduction in MAP (Fig. 1b). The effects

of i.v. administration (5�/100 mg/kg) of the C. schiedea-

nus extract was dose-dependent and higher in SHR than

in Wistar rats (Fig. 2). However, oral administration of

C. schiedeanus extract in freely moving SHR (200 mg/kg, single dose) did not elicit significant changes up 3 h

of observation (results not shown).

3.2. Effects on rat isolated aorta

Stimulation of aortic rings with 80 mM KCl and 10�6

M PE resulted in a sustained contraction (49099281

mg, n�73 and 50619342 mg, n�41, respectively). Thecumulative additions of the ethanolic extracts from C.

schiedeanus and C. glomerata caused the most pro-

nounced concentration-dependent relaxant response

(Fig. 3). The concentrations producing 50% inhibition

of the maximum contractile response (IC50, g/ml) were

6.5 (5.8�/7.3) �10�5 and 7.1 (6.4�/7.9) �10�5 against

KCl and 5.3 (4.9�/5.7)�10�5 and 1.2 (1.1�/1.2)�10�4

against PE, respectively. With higher doses (3�10�4 g/

ml) C. schiedeanus induced the greatest relaxation

(7993% against KCl and 7893% against PE).Among the chloroformic, isobutanolic and ethylace-

tate fractions obtained from C. schiedeanus ethanolic

extract, chloroformic fraction showed a greater relaxant

effect in KCl-contracted aortic rings (IC50�9.5 [20�/

4.6]�10�6 g/ml), being more potent than the ethanolic

extract from which it was derived The neutral, weak-

acid and strong-acid fractions obtained subsequently

from the chloroformic fraction displayed a different

response, depending on the agonist used. When the

aortic rings were precontracted with KCl, the neutral

fraction showed the best response (IC50�8.3 [21�/

3.2]�10�6 g/ml) whereas the strong-acid fraction

displayed the greatest effects against PE pre-contracted

aortic rings (IC50�1.8 [1.8�/1.7]�10�5 g/ml) (Fig. 4).

Fig. 1. Effects of ethanolic extracts obtained from: Calea glomerata

Klatt, C. schiedeanus Schlecht, Curatella americana L., Lupinus

amandus and Lippia alba (Mill)n N.E.Br. (20 mg/kg administered

intravenously) on basal mean arterial pressure (MAP, solid bars) and

basal heart rate (HR, open bars) in: (a) anaesthetized normotensive

Wistar rats, and (b) anaesthetized SHR. Each bar represents the

mean9S.E.M (n�20�/26). Baseline Wistar MAP and HR were 9394

mmHg and 397910 beats per min, respectively. Baseline SHR, MAP

and HR were 17098 mmHg and 355910 beats per min, respectively.

*P B0.05; **P B0.01; with respect to control values (ANOVA).

M.F. Guerrero et al. / Journal of Ethnopharmacology 80 (2002) 37�/42 39

Among fractions obtained subsequently by chroma-

tography, A-7476 (IC50�5.7 [6.0�/5.4]�10�5 g/ml) B-

167�/182 (IC50�2.4 [3.0�/1.9]�10�5 g/ml,) B-183�/200(IC50�4.2 [5.2�/3.5]�10�5 g/ml), B-2860 (IC50�3.1

[3.4�/2.8]�10�5 g/ml) and B-6390 (IC50�2.5 [4.6�/

1.4]�10�5 g/ml) yielded the greatest potency against

high K� (Fig. 5) but the response was never greater

than those of the chloroformic or neutral fractions.

4. Discussion

In a previous work we reported the antihypertensive

and vasorelaxant effects of intravenous administration

of the aqueous extract from C. schiedeanus Schlecht(Guerrero et al., 2001). Here we wished to study the

ethanolic extract compared with others obtained from

plants with the same ethnobotanical use in Colombia.

According to the results of the in vivo and in vitro

assays, the ethanolic extract of C. schiedeanus (EECS)

showed the greatest antihypertensive and relaxant

response.

Although C. americana and L. amandus elicitedsignificant blood pressure decreases in anaesthetized

SHR with the screening dose used (20 mg/kg), C.

schiedeanus exerted the most pronounced effects. Ad-

ditionally, EECS intravenously administered in anaes-

thetized rats showed a dose-dependent decrease in both

MAP and HR, being greater in SHR than in Wistarnormotensive rats. Thus, the effects of C. schiedeanus

seem to be more of the antihypertensive type than

hypotensive.

As regards our previous results with the aqueous

extract from C. schiedeanus, the ethanolic extract is

more active since its antihypertensive effect is significant

with a lower dose, 20 mg/kg i.v. with ethanolic extract

against 50 mg/kg with the aqueous extract (Guerrero etal., 2001). Its vasorelaxant potency was also higher (IC50

values 6.5�10�5 g/ml with the ethanolic extract vs.

9.1�10�4 g/ml with the aqueous extract in high K�

pre-contracted rings).

However, as with the aqueous extract, the activity of

the ethanolic extract lasts for only a few minutes (less

than 15 min) and does not seem to be active after oral

administration. Although issues of absorption andbiotransformation could be considered, it is important

to note that it is necessary to perform chronic oral

administration (not less than 4 weeks in SHR) for an

objective assessment of the antihypertensive effects of C.

Fig. 2. Effects of intravenous administration of ethanolic extract of C.

schiedeanus Schlecht (EELS) on: (a) basal mean arterial blood pressure

(MAP), and (b) basal heart rate (HR) in anaesthetized Wistar (open

circles) and spontaneously hypertensive rats (solid circles). Each point

represents the mean9S.E.M of 5�/8 rats. *P B0.05; ***P B0.001

(Student’s t -test).

Fig. 3. Effects of ethanolic extracts from Calea glomerata Klatt, C.

schiedeanus Schlecht, Curatella americana L, Lupinus amandus and

Lippia alba (Mill)n N.E.Br., in aortic rings from normotensive Wistar

rats, contracted with (a) 80 mM KCl; and (b) 10�6 M PE. Ordenate:

percentage of KCl or PE contraction. Abscissa, ethanolic extract

concentration. Each point represents the mean9S.E.M of 4�/5 rings.

*P B0.05; **P B0.01; ***P B0.001 with respect to DMSO contrac-

tion (ANOVA test).


schiedeanus. In fact, in clinical practice physicians

usually maintain administration of the antihypertensive

medication for at least one month in order to estimate

the true usefulness of the pharmacotherapy (Williams,

1998). Furthermore, C. schiedeanus was not only active

in decreasing blood pressure after intravenous adminis-

tration in SHR, but also failed to elicit reflex tachycar-

dia, a phenomenon associated with post-ischaemic

properties in patients with coronary artery disease

(Yeh et al., 2000).

Although the C. glomerata ethanolic extract was not

as effective as that of C. schiedeanus in lowering blood

pressure in SHR, it is worth noting that it exerted

important relaxant effects, comparable to those of C.

schiedeanus , in aortic rings pre-contracted with KCl and

phenylephrine. Pharmacokinetic aspects could be in-

voked to account for this difference, but C. glomerata isnot certainly devoid of interest for future investigations.

Also, C. americana and L. amandus are also worthy

of attention since they elicited significant although slight

antihypertensive effects in SHR in spite of their rather

poor vasorelaxant activity against KCl and PE. Since

both produced significant in vivo effects it is not

possible to discard some degree of vasorelaxant activity,

although this would be unrelated to the cellular signal-ing cascades pertaining to KCl and PE.

However, since the C. schiedeanus ethanolic extract

showed the most pronounced antihypertensive and

vasorelaxant activity, we selected it to run a phytochem-

ical bioguided fractionation. At least in part, the

antihypertensive activity of C. schiedeanus could be

due to direct vasodilatory effects on vascular smooth

muscle, although this extract was also able to inducesome degree of bradycardic effect. Thus, direct effects

upon chronotropic and inotropic cardiac activity cannot

be ruled out.

Concerning the first fractionation from C. schiedea-

nus , the chloroformic fraction was most effective. It

notably increased the relaxant efficacy (94%) and

lowered the IC50 as compared with the ethanolic extract

(64-fold more potent). Regarding the second fractiona-tion, it is interesting to note that neutral and weak-acid

fractions afforded greater relaxant effects in KCl-con-

tracted rings (IC50 8.3�10�6 and 1.8� l0�5 g/ml,

respectively), whereas, the strong-acid fraction gave

more response in PE-pre-contracted rings (IC50

1.8�10�5 g/ml).

With respect to the third fractionation, which gave

fractions A and B assayed in high K�-contracted rings,some fractions showed more relaxant efficacy and

potency responses than the ethanolic extract, although

none was more effective than the chloroformic and

neutral fractions from which they were derived. In other

Fig. 4. Effects of C. schiedeanus fractions against KCl- (80 mM) and

phenylephrine- (10�6 M) pre-contracted Wistar aortic rings: (a)

ethylacetate, chloroformic and isobutanol fractions against KCl; (b)

neutral, weak-acid and strong-acid fractions against KCl; and (c)

neutral, weak-acid and strong-acid fractions against PE. Ordenate:

percentage of KCl or PE contraction. Abscissa, fraction concentration.

Each point represents the mean9S.E.M of 4�/5 rings. *P B0.05;

**P B0.01; (ANOVA test).

Fig. 5. Effects of Croton schiedeanus fractions with the greatest

inhibitory concentration 50 (IC50

) responses. The ethanolic extract is

included as reference.

M.F. Guerrero et al. / Journal of Ethnopharmacology 80 (2002) 37�/42 41

words, partial purification of the substances tends to

decrease the relaxant activity.

From these results it may be assumed can that C.

schiedeanus could have more than one active principleand that its antihypertensive activity may be due to

synergistic properties. It is known that KCl acts through

activation of voltage-dependent calcium channels to

induce the activation of fast and slow voltage-dependent

Ca2� (Karaky and Weiss, 1979, 1984). Furthermore,

stimulation of a1 receptors by phenylephrine activates

Ca2� pathways through voltage-dependent channels

and receptor-operated channels (Bulbring and Tomita,1987). Thus, the vasorelaxant effects of C. schiedeanus

might well be related to inhibition of voltage-dependent

calcium channels although mechanisms related to the

signal transduction pathway coupled to a1 adrenocep-

tors should also be considered.

Flavonoid compounds, detected in the phytochemical

analyses appeared in chromatographic fractions from C.

schiedeanus . These compounds are known to havecardiovascular and vasorelaxant effects (Andriambelo-

son et al., 1999; Chan et al., 2000; Duarte et al., 1994,

2001; Morales and Lozoya, 1994). Quercetin, quercitrin

and ayanin have been identified from C. glabellus

(Novoa et al., 1985; Garcıa et al., 1986). Other Croton

species have been reported to exhibit antihypertensive

activity (Lahlou et al., 1999, 2000). In C. cajucara

diterpene compounds have been reported (Itokawa etal., 1989; Kubo et al., 1991) and this type of constituent

is also currently being studied in C. schiedeanus .

In conclusion, our results show that C. schiedeanus

Schlecht elicits antihypertensive and bradycardic effects

in SHR and vasorelaxant activity in rat isolated rings

pre-contracted with high KCl and PE. Its phytochemical

fractionation reveals the probable presence of more than

one active principle with synergistic activity amongthem. Blocking properties on Ca2� influx through

voltage-dependent calcium channels seem to be involved

and further bioguided phytochemical fractionation

experiments are presently underway, in order to com-

pletely isolate the active principles.

Acknowledgements

This work was supported by the project ALFA-

RELAPLAMED.2, the Iberoamerican Program of

Science and Technology for Development, Subprogram

X (CYTED), Universidad de Salamanca and Universi-dad Nacional de Colombia. We would like to thank the

Project ‘Busqueda de Principios Bioactivos en Plantas

Medicinales Colombianas’, Departamento de Farmacia,

Universidad Nacional de Colombia.

References

Andriambeloson, E., Stoclet, J.C., Andriantsitohaina, R. 1999. Me-

chanism of endothelial nitric oxide-dependent vasorelaxation

induced by wine polyphenols in rat thoracic aorta. Journal of

Cardiovascular Pharmacology 33 (2), 248�/254.

Bulbring, E., Tomita, T. 1987. Catecholamine action on smooth

muscle. Pharmacological Reviews 39, 49�/96.

Chan, E., Pannangpetch, P., Woodman, O. 2000. Relaxation to

flavones and flavonols in rat isolated thoracic aorta: mechanism

of action and structure�/activity relationships. Journal of Cardio-

vascular Pharmacology 35, 326�/333.

Correa, J., Bernal, H., 1992. Especies Vegetales Promisorias de los

Paıses del Convenio Andres Bello. T. VII, 314�/322. Santafe de

Bogota, DC, Colombia.

Duarte, J., Perez, F., Zarzuelo, A., Jimenez, J., Tamargo, J. 1994.

Inhibitory effects of quercetin and staurosporine on phasic con-

tractions in rat vascular smooth muscle. European Journal of

Pharmacology 262, 149�/156.

Duarte, J., Perez, R., Vargas, F., Angeles, M., Perez, F., Zarzuelo, A.,

Tamargo, J. 2001. Antihypertensive effects of the flavonoid

quercetin in spontaneously hypertensive rats. British Journal of

Pharmacology 133, 117�/124.

Garcıa, H., 1975. Flora Medicinal de Colombia. T. II, 31�/35; 87�/91;

173�/174; 206�/208; 320�/325. Santafe de Bogota, Colombia.

Garcıa, L., Guarın, D., Tobar, M. 1986. Obtencion de ayanina de las

hojas de Croton glabellus . Revista Colombiana de Ciencias

Quımico-Farmaceuticas 15, 95�/98.

Guerrero, M.F., Carron, R., Martın, M.L., San Roman, L., Reguero,

M.T. 2001. Antihypertensive and vasorelaxant effects of aqueous

extract prepared from Croton schiedeanus Schlecht in rats. Journal

of Etnopharmacology 75, 33�/36.

Itokawa, H., Ichihara, Y., Kojima, H., Watanabe, K., Takewa, K.

1989. Nor -clerodane diterpenes from Croton cajucara . Phytochem-

istry 28, 1667�/1669.

Karaky, H., Weiss, G.B. 1979. Alterations in high and low affinity

binding of 45Ca2� in rabbit aortic smooth muscle by norepinephr-

ine and potassium after exposure to lanthanum and low tempera-

ture. Journal of Pharmacology and Experimental Therapeutics 211,

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Karaky, H., Weiss, G.B. 1984. Calcium channels in smooth muscle.

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Kubo, I., Asaka, Y., Shibata, K. 1991. Insect growth inhibitory Nor -

diterpenes cis -dehydrocrotonin and trans -dehydrocrotonin from

Croton cajucara . Phytochemistry 30, 2545�/2546.

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tial oil of Croton nepetaefolius decreases blood pressure through an

action upon vascular smooth muscle: studies in DOCA-salt

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Lahlou, S., Leal-Cardoso, J.H., Caldas-Magalhaes, P.J., Coelho-de-

Souza, A.N., Duarte, G.P. 1999. Cardiovascular effects of the

essential oil of Croton nepetaefolius in rats: role of the autonomic

nervous system. Planta Medica 65, 553�/557.

Morales, M., Lozoya, X. 1994. Calcium-antagonist effects of quercetin

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flavonoide con actividad hipotensora obtenido de Croton glabellus.

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The effects of fruit essential oil of the Pimpinella anisum onacquisition and expression of morphine induced conditioned place

preference in mice

Hedayat Sahraei a,*, Hassan Ghoshooni a, Sayed Hossein Salimi b, Abutaleb MohseniAstani c, Bijan Shafaghi c, Mansoor Falahi c, Mohammad Kamalnegad c

a Department of Physiology and Biophysics, Baghyatollah (a.s.) University of Medical Sciences, P.O. Box 19395-6558, Tehran, Iranb Department of Psychology, Baghyatollah (a.s.) University of Medical Sciences, Tehran, Iran

c Department of Pharmacognosy, School of Pharmacy, Shaheed Beheshti University of Medical Sciences, Tehran, Iran

Received 15 July 2001; received in revised form 16 November 2001; accepted 17 December 2001

Abstract

The problem of drug dependence still remains unresolved. In the present study the effects of an essential oil of Pimpinella anisum

(Umbeliferae) on the expression and acquisition of conditioned place preference (CPP) induced by morphine in mice were

investigated. Subcutaneous (s.c.) injections of morphine (2�/5 mg/kg) produced place preference in a dose-dependent manner.

Furthermore, intraperitoneal (i.p.) injection of the essential oil of P. anisum (0.125�/0.5 ml/kg) induced conditioned place aversion

(CPA). The mice which have received the essential oil of the P. anisum (0.125�/0.5 ml/kg, i.p.) as well as the oil with morphine (5 mg/

kg, s.c.) reduced the morphine effect. Administration of the essential oil of P. anisum (0.125�/0.5 ml/kg, i.p.) on the test day did not

show any effect on morphine action. It appeared that pre-administration with bicuculline (GABAA receptor antagonist) (1.5 mg/kg,

i.p., 20 min before essential oil) diminished the effect of the essential oil of the P. anisum on morphine which induced CPP, but this

result was not found for the GABAB receptor antagonist, CGP35348 (200 and 400 mg/kg, i.p., 10 min before essential oil). In

conclusion, it appeared that the essential oil of the P. anisum may reduce the morphine effects via a GABAergic mechanism.

# 2002 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Pimpinella anisum ; Morphine; Mice; GABA receptors; Conditioned place preference

1. Introduction

Pimpinella anisum L., Umbelliferae, is an annual herb

indigenous to Iran, India, Turkey and many other warm

regions in the world (Zargari, 1989). In Iranian folk

medicine, the plant and especially its fruit’s essential oil

have been used for treatment of a wide range of diseases

including seizures and epilepsy and also tobacco (nico-

tine) dependence (Aviccenna, 1988). The aqueous ex-

tract of the collection of flowers, stems and leaves of P.

anisum has been reported to postpone the onset of

picrotoxin-induced seizures in mice (Abdul-Ghani et al.,

1987). However, there is no study to clarify the effects of

the essential oil of the fruits of this plant on the

rewarding properties of opioids. Chemical studies have

demonstrated the presence of eugenol (Monod and

Dortan, 1950), anethole (Fujita and Nagasawa, 1960),

methylchavicol and anisaldehyde (Wagner et al., 1984)

and estragole (Zargari, 1989) as the major compounds

of the fruit essential oil of P. anisum . Eugenol and

estragole have been reported to show anesthetic, hy-

pothermic, muscle relaxant and anticonvulsant activities

(Dallmeier and Carlini, 1981), as well as anethole

possesses muscle relaxant effects (Albuquerque et al.,

1995). The conditioned place preference (CPP) para-

digm was chosen in our research because it offers a

simple method to assess the reinforcing effects of drugs

(Carr and White, 1983). The present work was under-* Corresponding author. Fax: �98-21-283-0262.

E-mail address: [email protected] (H. Sahraei).




PII: S 0 3 7 8 - 8 7 4 1 ( 0 2 ) 0 0 0 1 2 - 0


taken to evaluate the effects of the fruit essential oil of P.

anisum on the expression and acquisition of CPP

induced by morphine in mice.


2.1. Plant material

Fruits of P. anisum were obtained from the local

market. The plant was authenticated by M. Kamaline-

jad (Department of Pharmacognosy, Faculty of Phar-

macy, Shaheed Behashti University of Medical Sciences,Tehran, Iran) and a voucher specimen coded P-544 has

been deposited at the herbarium of Department of

Pharmacognosy, Faculty of Pharmacy, Shaheed Be-

hashti University of Medical Sciences, Tehran, Iran.

2.2. Preparation of the essential oil

The fruits were processed by stem distillation over aperiod of 4 h in all glass apparatus, to obtain the

essential oil with 2% yield. The essential oil was diluted

with sesame oil to obtain the desired doses and was

immediately administered intraperitoneally (i.p.) to mice

expressed as millilitres of essential oil per kilogram body

weight.

2.3. Apparatus and training

The apparatus which has been previously described

(Carr and White, 1983) consisted of two large adjacent

compartments A and B (45�/45�/30 cm) were con-

nected by a communicating tunnel (23�/15�/30 cm)

attached to one side. The conditioning compartments (A

and B) were painted different colors (white and black).

Access to the tunnel could be blocked by a removablepartition. A number of pilot experiments showed that in

the particular experimental set-up used in the present

study the animals do not typically show an uncondi-

tioned preference for either of the conditioning com-

partments (white side�/3409/34 s, black side�/3109/52

s). Therefore the unbiased method was used in this study

(the drug and control compartments were randomly

assigned to each animal in a counterbalanced way). TheCPP procedure took place on 8 consecutive days. On

day 1 (preexposure), each mouse was placed separately

into the apparatus for 10 min, with free access to all

compartments (A, B and C). In the next 6 days, animals

received three trials (i.e. days 2, 4 and 6) in which they

experienced the effect of drug while confined in one

compartment for 30 min. On other days (i.e. days 3, 5

and 7) they received a normal saline (or vehicle)injection and were confined to the other compartment.

Access to compartment C (communicating tunnel) was

blocked on these days. On the last day (preference test)

the communicating tunnel was opened, and the mice

could access all compartments. The animals received

injections of normal saline, vehicle or other drugs and

were placed in the communicating tunnel. The relativeamount of time which each mouse spent in each of the

compartments during a 15 min period was determined

as a preference criteria.

2.4. Drugs

The following drugs were used: morphine sulfate

(TEMAD, Iran). Sesame oil was obtained from the

local market. CGS35348 (Ciba Geigy, Switzerland) and

Bicuculline (Sigma Chemical Co., USA). The drugs were

dissolved in normal saline, except for Bicuculline which

was dissolved in a drop of acetic acid and diluted with

normal saline. The vehicle control in respective caseswas acetic acid in normal saline. The drugs were given in

a volume of 10 ml/kg and were prepared immediately

before use.

2.5. Animals

Male albino Swiss�/Webster mice (20�/25 g, Razi

Institute, Tehran, Iran) were used (10 mice for each

experiment). The animals were housed 10 per cage with

12/12 h light-cycle with ad lib food and water available.


Conditioning scores represent the time spent in the

drug-paired place minus the time spent in the vehicle-

paired place, and are expressed as the mean9/SEM.One-way analysis of variance (one-way ANOVA) fol-

lowed by Newman�/Keuls was performed. Differences

with P B/0.05 were considered statistically significant.

3. Results

3.1. Effects of morphine on behavior in CPP paradigm

Injection of different doses of morphine sulphate

(0.5�/5 mg/kg; s.c.) to mice caused a significant increase

in time spent in the drug-paired compartment compared

to that spent in the normal saline-paired compartment

i.e. CPP. However, s.c. injection of normal saline to the

mice (saline control group) in the two conditioning

compartments did not produce any preference or

aversion for either place [F (4, 45)�/13, P B/0.0001](Fig. 1A). The maximum response of morphine was

obtained with 5 mg/kg of the drug and this dose was

chosen in subsequent experiments.

H. Sahraei et al. / Journal of Ethnopharmacology 80 (2002) 43�/4744

3.2. Effects of essential oil on behavior in CPP paradigm

Injection of different doses of essential oil (0.125�/0.5ml/kg; i.p.) to mice caused a significant increase in time

spent in the vehicle-paired compartment compared to

that spent in the essential oil-paired compartment, i.e.

conditioned place aversion (CPA). However, injection

of sesame oil (as a vehicle) to the mice (vehicle control

group) in the two conditioning compartments did not

produce any preference or aversion for either

place.[F (5, 54)�/2.62, P B/0.04] (Fig. 1B).

3.3. Effects of essential oil administration on the

expression of morphine induced CPP

When the essential oil (0.125�/0.5 ml/kg, i.p.) was

administered 30 min before the beginning of the test on

the 8th day of experiments, no effects on the expression

of morphine-induced CPP were found [F (5, 54)�/0.04,

P �/0.05] (Fig. 2A).

3.4. Effects of essential oil administration on the

acquisition of morphine induced CPP

When essential oil (0.0125�/0.5 ml/kg, i.p.) was

administered 30 min before the beginning of morphine

injection during the training days of experiments, a

significant reduction in morphine-induced CPP was

observed [F (5, 54)�/6.2, P B/0.0001] (Fig. 2B). The

maximum response of essential oil was obtained with

0.25 ml/kg of the drug and this dose was chosen in

subsequent experiments. However, the dose 0.5 ml/kg

failed to reduced morphine effects and this is difficult to

explain.

Fig. 1. CPP induced by morphine and fruit essential oil of P. anisum .

Animals received different doses of morphine (A: 0.5�/5 mg/kg, s.c.), or

fruit essential oil of P. anisum (B: 0.125�/0.5 ml/kg, i.p.). Each point is

the mean9SEM for ten mice, * P B0.05, ** P B0.01 different from

respective saline or vehicle control group.

Fig. 2. Effect of different doses of fruit essential oil of P. anisum on

acquisition or expression of morphine induced CPP. Animals received

different doses of fruit essential oil of P. anisum 30 min before the

beginning of morphine injection in the training days of experiments (A:

0.125�/0.5 ml/kg, i.p.), or fruit essential oil of P. anisum 30 min before

the beginning of the test in the 8th day of experiments (B: 0.125�/0.5

ml/kg, i.p.). Each point is the mean9SEM for ten mice, * P B0.05,

** P B0.01 different from respective saline or vehicle control group.

H. Sahraei et al. / Journal of Ethnopharmacology 80 (2002) 43�/47 45

3.5. Effects of bicuculline and CGP35348 on the

suppressive action of the essential oil

Pretreatment of animals with bicuculline (1.5 mg/kg,

i.p.) but not CGP35348 (200 and 400 mg/kg, i.p., 20 min

before the essential oil on the training days) significantlyreduced the suppressive effects of the essential oil on the

acquisition of CPP by morphine [F (3, 36)�/6.63, P B/

0.002] (Fig. 3).

4. Discussion

The present study demonstrates that the animals

exhibited a marked preference for an environment

associated with the administration of morphine. These

findings support the previous studies (Mucha and

Iversen, 1984) and demonstrate that the rewarding

effects of opioid receptor agonists can be conditioned

to environmental stimuli which have previously signaledtheir administration (Mucha and Iversen, 1984). In

addition, our findings showed that injection of essential

oil of P. anisum , may induce CPA in mice, that is, the

essential oil has some aversive effects as investigated by

place conditioning paradigm. In addition, this oil has

also a GABAergic effect. It appears that the previous

research has not been concerned with this matter and

our finding is a new fact which needs to be investigatedmore in the future. Because of several unknown

mechanisms, it seems difficult to have an appropriate

conclusion about the effects of the essential oil. Current

data indicate that the essential oil has an anticonvulsant

activity (Pourgholami et al., 1999) and the researchers

proposed that these effects may be due to a GABAergic

mechanism (Pourgholami et al., 1999). Therefore, onecan conclude that the essential oil may induce CPA by

activation of a GABAergic mechanism. Regarding this

fact, that is, morphine reward is based on the activation

of dopamine neurons in the ventral tegmental area

(VTA) which is under the tonic inhibition of the

GABAergic interneurons (Koob, 1992), one may pro-

pose that the essential oil inducing CPA via activation of

a GABAergic mechanism(s), is probably in the VTA(Koob, 1992). Acute injection of the essential oil of the

P. anisum on the test day to the animals that were

conditioned to morphine on the training days did not

alter the CPP induced by morphine, showing that the

essential oil was not effective for reduction of the

expression of the CPP induced by morphine. It should

be noted that the expression of CPP induced by

morphine is a complex phenomenon and is based onthe memory mechanisms that underlay the ability of

morphine for induction of memory (Tzschentke, 1998;

Koob, 1992). Morphine can activate the memory

mechanisms by activating the distinct area of the limbic

system including hippocampus after repeated adminis-

tration. This memory mechanism could be activated

when the animal is displaced in the same area and

produced the phenomenon which is called drug seekingbehavior and could be investigated by CPP paradigm

(Tzschentke, 1998). The results indicate that the essen-

tial oil failed to reduce the expression of the morphine

effects and thus it is ineffective for impairments of the

memory mechanisms activated by morphine. Adminis-

tration of essential oil on the training days with

morphine to the animals decreases the CPP induced by

morphine, indicating that chronic administration of theessential oil could abolish the acquisition of morphine

CPP. Inhibition of the suppressive effect of the essential

oil by preadministration of bicuculline (a GABA-A

receptor antagonist) but not CGP35348 (a GABA-B

receptor antagonist) highlights the fact that the essential

oil activates the GABAergic mechanism, especially the

one that depends upon the GABA-A receptor subtype.

In conclusion, the present study indicated that theessential oil of the P. anisum could suppress the

acquisition of the morphine CPP and the effect appears

to be due the GABAergic mechanism(s) that depend

upon GABA-A receptor activity. Finally, the disruption

of morphine-induced CPP could be caused by indirect

effects of the essential oil of P. anisum , and these

mechanism(s) have not been yet determined. It may be

a simple summative response between morphine-in-duced acquisition and CPA by the essential oil of P.

anisum . Finally, it may be a pharmacokinetic interaction

between morphine and essentail oil that resulted in

reduction of morphine-induced CPP.

Fig. 3. Effects of GABA receptor antagonists on suppressive action of

fruit essential oil of P. anisum on acqisition of morphine induced CPP.

Animals received bicuculline (1.5 mg/kg, i.p.) or CGP35348 (200 and

400 mg/kg, i.p.) 30 min before essential oil administration. Each point

is the mean9SEM for ten mice, * P B0.05 different from respective

vehicle control group.

H. Sahraei et al. / Journal of Ethnopharmacology 80 (2002) 43�/4746

Acknowledgements

Authors are thankful to Dr. Mohammad-Reza Zar-

rindast for his helpful discussions. This work wassupported in part by the grant from Department of

Research, Baghyatollah (a.s.) University of Medical

Sciences.

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H. Sahraei et al. / Journal of Ethnopharmacology 80 (2002) 43�/47 47

Herbal medicines for sexually transmitted diseases and AIDS

Kavita Vermani, Sanjay Garg *

Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, SAS Nagar, Punjab 160 062, India

Received 20 September 2001; received in revised form 13 October 2001; accepted 19 December 2001

Abstract

Sexually transmitted diseases (STDs) and acquired immunodeficiency syndrome (AIDS) are gaining significant importance at

present due to rapid spread of the diseases, high cost of treatment, and the increased risk of transmission of other STDs and AIDS.

Current therapies available for symptomatic treatment of STDs and AIDS are quite expensive beyond the reach of common man

and are associated with emergence of drug resistance. Many patients of STDs and AIDS are seeking help from alternative systems of

medicines such as Unani, Chinese, Ayurvedic, naturopathy, and homeopathy. Since a long time, medicinal plants have been used for

the treatment of many infectious diseases without any scientific evidence. At present there is more emphasis on determining the

scientific evidence and rationalization of the use of these preparations. Research is in progress to identify plants and their active

principles possessing activity against sexually transmitted pathogens including human immunodeficiency virus (HIV) with an

objective of providing an effective approach for prevention of transmission and treatment of these diseases. In the present review,

plants reported to possess activity or used in traditional systems of medicine for prevention and treatment of STDs including AIDS,

herbal formulations for vaginal application, and topical microbicides from herbal origin, have been discussed. # 2002 Elsevier

Science Ireland Ltd. All rights reserved.

Keywords: Sexually transmitted diseases; AIDS; Vagina; Microbicides; Herbal medicine

1. Introduction

In normal healthy women, vaginal cavity is inhabited

by a number of microorganisms, existing in a dynamic

microenvironment. Any disturbance to this ecosystem

leads to a number of infectious conditions and diseases.

Sexually transmitted diseases (STDs), also known as

venereal diseases are infections caused by a variety of

pathogens including bacteria (Neisseria gonorrhoea ,

Treponema pallidum , Haemophilus ducreyi , Gardnerella

vaginalis ), viruses (human immunodeficiency virus

(HIV), herpes simplex virus, human papilloma virus

(HPV)), Chlamydia (Chlamydia trachomatis ), and para-

sites (Trichomonas vaginalis , Giardia lambia ) (Hardin,

1996). Acquired immunodeficiency syndrome (AIDS),

genital herpes, genital warts, chlamydial genital infec-

tions, trichomoniasis, vaginitis and vulvovaginitis are

some of the sexually transmitted infections (STIs).

Sexual contact is the most common but not the only

means of transmission of these infections. It is now well

established that STDs (both ulcerative and non-ulcera-

tive) increase the risk of transmission of other STIs,

including AIDS because of changes in the normal

vaginal epithelium (Wasserheit, 1992).

Current therapies for AIDS and other STDs include

drug administration by various routes including oral,

parenteral, and topical (vaginal and rectal). Since sexual

mode of transmission is the most common cause of

occurrence of STDs, vaginal and rectal approaches are

becoming significant for prevention of their transmis-

sion. In the last decade, major advancements have been

reported in the field of ‘microbicides’, i.e. compounds or

formulations which when applied topically (vaginal or

rectal) can prevent the transmission of STDs including

AIDS (Forbes, 2000). These include a few from plant

sources such as gossypol derivatives, Praneem polyher-

bal preparations, and plantibodies.

Medicinal plants have a long history of use and their

use is widespread in both developing and developed

Abbreviations: HIV, human immunodeficiency virus; AIDS,

acquired immunodeficiency syndrome; STDs, sexually transmitted

diseases; HSV, herpes simplex virus; HPV, human papilloma virus.

* Corresponding author. Tel.: �91-172-214682�/87; fax: �91-172-

214-692.

E-mail address: [email protected] (S. Garg).




PII: S 0 3 7 8 - 8 7 4 1 ( 0 2 ) 0 0 0 0 9 - 0


countries. Herbal medicines provide rational means for

the treatment of many diseases that are obstinate and

incurable in other systems of medicine. These are

gaining popularity because of several advantages such

as often fewer side effects, better patient tolerance,

relatively less expensive and acceptance due to long

history of use. Medicinal effects of plants tend to

normalize physiological function and correct the under-

lying cause of the disorder (Murray and Pizzorno, 1999).

Medicinal plants are renewable in nature unlike the

synthetic drugs that are obtained from non-renewable

sources of basic raw materials such as fossil sources and

petrochemicals (Samanta et al., 2000). Cultivation and

processing of plants often is environment friendly unlike

the pollution by chemical industry. Cultivation of

medicinal plants can also be a source of income for

poor families. Many of the medicinal plants are locally

available, especially in developing and underdeveloped

countries. Also, plants are often less prone to the

emergence of drug resistance. Due to all these advan-

tages, plants continue to be a major source of new lead

compounds.

A large number of active agents are available for the

symptomatic treatment of STDs and AIDS. Emergence

of drug resistant strains and dose limiting toxic effects

has complicated the treatment of these infectious

diseases. These complications have necessitated the

search for new antimicrobial substances from various

sources. Extracts of plants and phytochemicals have

been shown to possess activity against sexually trans-

mitted pathogens and may be a good source of new

active agents. Several plants have been screened for

activity against STDs on the basis of ethnopharmaco-

logical data (Vlietinck and Berghe, 1991; Mekkawy et

al., 1995; Matsuse et al., 1999; Kambizi and Afolayan,

2001; Rajbhandari et al., 2001) and some of these

screening programs have yielded potential leads.

In Europe, the use of medicinal plants for sympto-

matic treatment of STDs dates back at least to 1574

when ‘sarsaparilla’ (Smilax officinalis , family Liliaceae)

was first introduced for the treatment of syphilis.

Sarsaparilla was a better alternative to mercury, the

standard medical treatment for syphilis during that

period. In clinical studies, sarsaparilla was observed to

be effective in about 90% cases of acute syphilis and 50%

chronic cases (Murray and Pizzorno, 1999). Since then,

medicinal plants have been used for the treatment of

STDs and AIDS without any scientific evidence in

traditional systems of medicine. In the last century

enormous efforts have been made to select the plants,

isolate the active principles and screen the crude extract/

fractions/compounds for activity against various sexu-

ally transmitted pathogens, and elucidate their mechan-

ism of action.

2. Acquired immunodeficiency syndrome

AIDS is a clinical syndrome resulting from infection

with HIV that causes profound immunosuppression. Itis a complex multifactorial disease associated with

immunodeficiency and autoimmune inflammation.

HIV produces gradual effects on the body’s defense

mechanisms thereby leading to cancers and opportunis-

tic infections involving multiple systems of the body

such as immune, gastrointestinal, genitourinary, endo-

crine, dermatologic, and nervous systems. Symptoms

associated with AIDS include persistent fever, nightsweat, weight loss (wasting syndrome), headache, lym-

phademopathy, skin rashes, diarrhea, thrush, recurrence

of varicella zoster virus infection, Kaposi’s sarcoma,

Pneumocystis carinii pneumonia, cryptococcal meningi-

tis, Candida esophagitis, Toxoplasma encephalitis, and

disseminated atypical mycobacterial infection (Kapus-

nik-Uner, 1996; Murray and Pizzorno, 1999).

In Europe, herbal treatments have been considered asthe most popular complementary medicine used by HIV

infected individuals (Ozsoy and Ernst, 1999). Substan-

tial amount of research has been done and a lot more is

in progress to isolate the active leads from plants for

prevention of transmission of HIV and treatment of

AIDS. These active principles may act by different

mechanisms, targeting critical steps within the replica-

tion cycle of HIV. Recently, a review (Yang et al., 2001)on natural products under development for anti-HIV

activity has been published by National Cancer Institute

(USA). Several natural products based anti-HIV sur-

face-active agents, reverse transcriptase inhibitors, non-

nucleoside reverse transcriptase inhibitors, integrase

inhibitors and protease inhibitors have been reported.

Vlietinck et al. (1998) have summarized many com-

pounds of plant origin that inhibit HIV during variousstages of life cycle. These include several alkaloids,

carbohydrates, coumarins, flavonoids, lignans, pheno-

lics, proteins, quinines/xanthones, phospholipids, tan-

nins, and terpenes from various plants. Several studies

have been conducted to screen the plants used in folk

medicine for anti-HIV activity. These include plants

from Panama (Matsuse et al., 1999), Indonesia (Otake et

al., 1995), Egyptian folk medicine (Mekkawy et al.,1995), folk medicine of Iberian Peninsula (Bedoya et al.,

2001), and Ayurvedic medicine (Kusumoto et al., 1995).

Table 1 summarizes the plants that have been shown to

possess activity against HIV, their active principles, the

models used for anti-HIV testing, and suggested me-

chanisms of action.

3. Genital herpes

Genital herpes is an acute inflammatory infection

caused by herpes simplex virus (HSV-1 and HSV-2).

K. Vermani, S. Garg / Journal of Ethnopharmacology 80 (2002) 49�/6650

Table 1

List of plants that possess anti-HIV activity, their active principles/extracts and mechanism of action

Species (family) Vernacular name and tradi-

tional uses

Indigenous to Active constituents/extracts

tested

In vitro/in vivo assay model Mechanism of action References

Achillea millefolium Yarrow �/ Quercetagetin �/ �/ Ono et al., 1990

Alexia leiopetala Sand-

with (Leguminoseae)

�/ Guyana, Vene-

zuala, Brazil,

Amazon Basin

Castanospermine, 6,7 diepi-

castanospermine, australine,

alexine

In vitro model for syncytium for-

mation CD4� cell line H9 infected

with HIV, in vivo mice model

Interferes with syncytium for-

mation and viral infectivity,

inhibition of a-glucosidase I

located in endoplasmic reticu-

lum

Nash et al., 1988

Ancistrocladus ab-

breviatus (Ancistrocla-

daceae)

�/ Cameroon Michellamine A and B In vitro, MT-2 and CEM-SS cell

lines

Inhibits HIV-1 during early

phases of viral infection of T-

lymphocytes

Manfredi et al.,

1991

Andrographis paniculata

Nees. (Acanthaceae)

Sambiloto, kalmegh Indonesia, In-

dia

Aqueous extract of leaves Inhibition of HIV-1 induced cyto-

pathogenicity in MT-4 cells

Inhibition of HIV protease and

reverse transcriptase

Otake et al., 1995

Anogeissus acuminata

Roxb. Ex DC. Guill.

and Perr. (Combreta-

ceae)

No known traditional uses Bangladesh,

India, Burma,

Thailand, Viet-

nam

Anolignan A HIV-1 reverse transcriptase assay Inhibition of HIV -1 reverse

transcriptase

Rimando et al.,

1994

Areca catechu Linn.

(Palmae)

Betel nut India, Eastern

Archipelago

Seed extract, procyanidins,

arecatannin B1

�/ HIV protease inhibition Kusumoto et al.,

1995

A. indica A. Juss. (Me-

liaceae)

Neem, margosa; used for anti-

bacterial, antipyretic, and anti-

inflammatory properties

India, Asia Seed and leaf extracts �/ �/ Talwar et al.,

1997, 2000

Bersama abyssinica Fre-

sen. (Melianthaceae)

Azamer, bersama, lolchisa; used

in rabies, ascariasis, ulcers,

diarrhoea, worm infestations,

and cholera

Ethiopia Methanol extract of leaf,

methanolic and acetone ex-

tract of root bark

In vitro, inhibition of viral cyto-

pathic effect in MT-4 cells

�/ Asres et al., 2001

Buchenavia capitata

Vahl Eichl. (Combreta-

ceae)

�/ Dominican Re-

public

O -dimethyl-buchenavianine In vitro, cultured human lympho-

blastoid CEM-SS cells

Inhibition of reverse transcrip-

tase

Beutler et al., 1992

Callophyllum inophyl-

lum Linn. (Clusiaceae)

�/ Malaysia Inophyllums coumarin deri-

vatives

�/ Inhibition of reverse transcrip-

tion

Patil et al., 1993

Callophyllum lanigerum

Miq. (Clusiaceae)

�/ Malaysia Calanolides coumarin deri-

vatives

Inhibition of in vitro replication of

HIV-1 and cytopathic effects in T

cell lines CEM-SS and MT-2 cells


tion, inhibition of DNA and

RNA dependent DNA poly-

merase activities of HIV-1 re-

verse transcriptase

Kashman et al.,

1992; Boyer et al.,

1993

Camellia sinensis Linn.

(Theaceae)

Used as anti-inflammatory

agent and cholerectic

China, Japan,

India

Epigallocatechin gallate,

epicatechin gallate


tase HIV-1 and HIV-2 and

cellular RNA and DNA

Nakane and Ono,

1990

Canavalia ensiformis �/ �/ Concanavalin A �/ Inhibition of syncytium forma-

tion

Hansen et al.,

1989

Castanospermium aus-

trale (Leguminoseae)

�/ �/ Castanospermine In vitro model for syncytium for-

mation CD4� cell line H9 infected

with HIV, in vivo mice model

Interferes with syncytium for-

mation and viral infectivity,

inhibition of a-glucosidase I

located in endoplasmic reticu-

lum

Gruters et al.,

1987; Sunkara et

al., 1987; Nash et

al., 1988; Ru-

precht et al., 1989

K.

Verm

an

i,S

.G

arg

/J

ou

rna

lo

fE

thn

op

ha

rmaco

log

y8

0(

20

02

)4

9�

/66

51

Table 1 (Continued )


tional uses


tested


Cephaelis ipecacuanha

Brotero A. Richard

(Rubiaceae)

Ipecac; used as emetic, expec-

torant, amoebicide and for

treatment of gout

Brazil Psychotrine O -methylpsy-

chotrine

In vitro Inhibition of reverse transcrip-

tase

Tan et al., 1991

Chassalia parvifolia

(Rubiaceae)

�/ �/ Circulin A and B In vitro, XTT based anti-HIV

assay

�/ Gustafson et al.,

1994

Combretum paniculatum

Vent. (Combretaceae)

Baye, gabai, shaga; used for

tonsillitis, cold, constipation,

rheumatism, and as haemostatic

Ethiopia Ether, dicholoromethane,

acetone and methanolic ex-

tracts of leaf

In vitro, inhibition of viral cyto-

pathic effect in MT-4 cells

�/ Asres et al., 2001

Conospermum incurvum

Lindley (Protreaceae)

�/ Western Aus-

tarlia

Conocurvon, organic ex-

tracts of stem, twigs, leaves

and flowers

In vitro, T lymphoblastic cell line

infected with HIV-1 CEM-SS cells

Mechanism not fully resolved,

inhibition of late phases of viral

replication cycle

Decosterd et al.,

1993; Dai et al.,

1994

Curcuma aeruginosa

Roxb. (Umbelliferae)

Temu, ireng Indonesia Aqueous extract of rhizome Inhibition of HIV-1 induced cyto-




Otake et al., 1995

C. longa Linn. (Umbel-

liferae)

Haldi; used as a spice, food

colorant and for various medic-

inal purposes

India Curcumin In vitro integrase, E. coli integrase

assay, HeLa H 12 cells, transacti-

vation assay

Inhibition of HIV-1 integrase,

inhibition of Tat-mediated

transactivation of HIV-1 long

terminal repeat

Barthelemy et al.,

1998

Detarium microcarpum �/ �/ Epicatechin, epicatechin-3-

O -gallate

�/ Irreversible interaction with

glycoprotein gp120

Mahmood et al.,

1993

Dianthus caryophyllus �/ �/ Antiretroviral proteins

(DAP 30 and DAP 32)

�/ Ribosome inactivating protein,

inhibition of transcription and

transactivation

Lee-Huang et al.,

1991

Dodonaea angustifolia

L.f. (Sapindaceae)

Kitkita, teramin, tasos; used in

wound dressing, and for treat-

ment of skin diseases, fever, sore

throat, rhinitis, sinusitis, influ-

enza, flu, and piles

Ethiopia Ether, dicholoromethane,

acetone and methanolic ex-

tracts of leaf

�/ �/ Asres et al., 2001

Eugenia caryophyllata

Thun. (Myrtaceae)

Clove; used as antiemetic China Tannins eugenin, casuaric-

tin, tellimagrandin, chro-

mones biflorin and

isobiflorin

�/ Inhibition of virus cell fusion,

inhibition of syncytium forma-

tion

Kim et al., 2001

Eugenia jambolona

Lam.

�/ �/ Extract of bark �/ HIV protease inhibition Kusumoto et al.,

1995

Euodia roxburghiana

Benth. (Rutaceae)

�/ Thailand, Asia,

Australia

Buchapine, 3,-3-methyl-2-

butenyl-4-[3-methyl-2-bute-

nyloxy]-21H-quinolinone

In vitro, inhibition of cytopathic

effect in cultured human lympho-

blastoid CEM-SS cells infected

with HIV-1


tase

McCormick et al.,

1996

Euphorbia kansui Liou.

(Euphorbiaceae)

Kansui; used for edema, asutes,

and cancer

China Ingenol, ingenol triacetate In vitro, MT-4 and MOLT-4 cells

infected with HIV-1 and HTLV-

IIIB

Inhibition of virus adsorption

to host cells

Fujiwara et al.,

1996

Euphorbia watanabei

(Euphorbiaceae)

�/ �/ Putranjivain A �/ Inhibition of HIV reverse

transcriptase

Mekkawy et al.,

1995

Fagara xanthoxyloides

Lam.

�/ �/ Fagaronine HIV-1 reverse transcriptase inhi-

bition assay

Inhibition of HIV reverse

transcriptase

Tan, 1991

K.

Verm

an

i,S

.G

arg

/J

ou

rna

lo

fE

thn

op

ha

rmaco

log

y8

0(

20

02

)4

9�

/66

52



tional uses


tested


Galanthus nivalis

(Amaryllidaceae)

Snowdrop �/ Mannose-specific aggluti-

nines lectins

In vitro, MT-4 cells infected with

HIV-1 and HIV-2

Interference with virus-cell fu-

sion, inhibition of syncytium

formation between HIV in-

fected and uninfected cells

Balzarini et al.,

1991

Gelonium multiflorum �/ �/ Antiretroviral protein (GAP

31)

�/ RIP, inhibition of transcription

and transactivation

Lee-Huang et al.,

1991

G. glabra Linn. (Legu-

minoseae)

Liquorice; used as demulcent

and expectorant

England, Spain Glycyrrhizin, Licochalcone

A, glycocoumarin, licopyra-

nocoumarin

HIV infected OKM-1 and MOLT-

4 cells

Inhibition of giant cell forma-

tion of HIV-infected cells, in-

terference with viral adsorption

and protein kinase C

Ito et al., 1987,

1988; Hatano et

al., 1988

G. radix (Legumino-

seae)

�/ �/ Glycyrrhizin, licopyranocou-

marin

�/ Interference with viral cell

binding

Ito et al., 1988;

Balzarini et al.,

1991

Gossypium spp (Malva-

ceae)

Cotton seed India, East In-

dies, China,

Egypt

Gossypol In vitro �/ Lin et al., 1989;

Polsky et al., 1989

Helicteras isora Linn. Kiules Indonesia Aqueous extract of fruit Inhibition of HIV-1 induced cyto-




Otake et al., 1995

Hippeastrum hybrid

(Amaryllidaceae)

Amaryllic �/ Mannose-specific aggluti-

nines lectins


HIV-1 and HIV-2





Balzarini et al.,

1991

Homoalanthus nutans

Forster Pax. (Euphor-

biaceae)

Diverse medicinal purposes Samoa Prostratin (a phorbol diter-

penoid)

In vitro, CEM and MT-2 cells Mechanism not well under-

stood, possible mechanisms

are*/down regulation of CD 4

expression in CEM and MT-2

cells, interference in protein

kinase C enzyme pathway

Gustafson et al.,

1992a

Hypericum perforatum

(Hypericeae)

Saint John’s wort; used in de-

pression and mental illness

�/ Hypericin and pseudohyper-

icin

�/ Interference with assembly of

virions and secondary spread,

interaction with proviral DNA

integration, interference with

viral infection, prevention of

virus spreading and budding

Meruelo et al.,

1988; Fanet et al.,

1998; Lavie et al.,

1989

Jacobinia suberecta �/ �/ Moranoline �/ Inhibition of HIV reverse

transcriptase

Ratner and Hey-

den, 1993

Lepidobotrys staudtii

Engl. (Lepidobotrya-

ceae)

�/ Cameroon 1,3,4,5-tetra-O -galloylquinic

acid

In vitro, CEM-SS cells Inhibition of reverse transcrip-

tase

Bokesch et al.,

1996

Listera ovata (Orchida-

ceae)

Twayblade �/ Mannose-specific aggluti-

nins, lectins


HIV-1 and HIV-2





Balzarini et al.,

1991

K.

Verm

an

i,S

.G

arg

/J

ou

rna

lo

fE

thn

op

ha

rmaco

log

y8

0(

20

02

)4

9�

/66

53



tional uses


tested


Loranthus parasiticus L.

Merr.

Benalu teh Indonesia Water extract of stem and

bark

HIV-1 infected MT-4 cells Suppression of syncytium giant

cell formation, protease inhibi-

tion, reverse transcriptase inhi-

bition

Otake et al., 1995

Macaranga sinensis �/ �/ Putranjivain A �/ Inhibition of HIV reverse

transcriptase

Mekkawy et al.,

1995

Maesa lanceolata For-

sskal (Myrsinaceae)

�/ �/ Maesasaponins (triterpenoid

saponins)

Microtray assay, colorimetric as-

say

�/ Apers et al., 2001

Mallotus japonicum

(Euphorbiaceae)

�/ �/ Mallotojaponin, mallato-

chromene


tase

Nakane et al.,

1991

Maprounea africana

Muell-Arg. (Euphorbia-

ceae)

�/ Central Afri-

can Republic,

Tanzania

Triterpenes of maprounic

acid/aleuritolic acid class

HIV-1 and HIV-2 reverse tran-

scriptase inhibition assay


tase

Beutler et al.,

1995; Pengsuparp

et al., 1994

Momordica charantia

Linn. (Cucurbitaceae)

Bitter melon, karela; used for

antiviral, antitumor and immu-

nopotentiating purposes and as

hypoglycaemic

China, India Antiretroviral protein (MAP

30)

�/ Inhibition of transcription and

transactivation, inhibition of

viral integrase

Lee-Huang et al.,

1990, 1995; Wang

et al., 1999

Morus spp . (Moraceae) �/ �/ Moranoline �/ Inhibition of HIV reverse

transcriptase

Ratner and Hey-

den, 1993

Myrica rubra and Myr-

ica nagi (Myriaceae)

�/ �/ Myricetin �/ �/ Ono et al., 1990

Omphalea diandra Linn.

(Euphorbiaceae)

�/ Panama Deoxynojirimicin, a-homo-

jirimicin, 1-deoxymannojiri-

micin

In vitro, microtiter infection assays

using MT-2 cells, syncytium inhi-

bition assay H-9/HTLV-IIIB cell

line

Inhibition of HIV infectivity by

the enzymes glycosidase and

mannosidase, blocks syncytium

formation

Kite et al., 1988;

Montefiori et al.,

1988

Papaver somniferum

Linn. (Papaveraceae)

Poppy, opium India, Asia Papaverine �/ Interference with expression of

HIV proteins especially envel-

ope precursor protein gp 120

Turano et al., 1989

Phyllanthus emblica

Linn. (Euphorbiaceae)

Amla; used in jaundice and viral

diseases

�/ Methanol extract, Putranji-

vain A

Reverse transcriptase inhibition

assay


transcriptase

Mekkawy et al.,

1995

Phytolacca americana

(Phytolaccaceae)

Poke root �/ Pokeweed antiviral protein In vitro, acutely and chronically

infected lymphocytes and macro-

phages

Conjugation to antibody spe-

cific to cell surface receptors

anti-CD 7 which facilitate cel-

lular internalization of antiviral

protein

Uckun et al., 1998

Plumeria rubra Linn.

(Apocyanaceae)

�/ �/ Fulvoplumierin HIV-1 reverse transcriptase assay Inhibition of HIV reverse

transcriptase

Tan, 1991

Pothomorphe peltata L.

Miq. (Piperaceae)

�/ Dominican Re-

public

Prenylated catechol dimers,

peltatols

�/ �/ Gustafson et al.,

1992b

Psidium guajava L. �/ �/ Procyanidin B2 In vitro enzyme reverse transcrip-

tase assay


tase

Kakiuchi et al.,

1991

Punica granatum Linn.

(Punicaceae)

Pomegranate, anar �/ Punicacortein D, Punicala-

gin, Punicalin

In vitro, H-9 lymphocyte cells

infected with HIV-1


transcriptase

Nonaka et al.,

1990

K.

Verm

an

i,S

.G

arg

/J

ou

rna

lo

fE

thn

op

ha

rmaco

log

y8

0(

20

02

)4

9�

/66

54



tional uses


tested


Quercus myrsinaefolia ,

Q. stenophylla , Quercus

pedunculata

�/ �/ Aqueous and Methanol ex-

tracts, 1,3,4-tri-O -galloyl-

quinic acid, 3,4,5-Tri-O -

galloylquinic acid

In vitro, H-9 lymphocyte cells

infected with HIV-1


transcriptase and HIV cell

growth

Mekkawy et al.,

1995

Rauwolfia serpentina

(Apocyanaceae)

Sarpgandha India, Paki-

stan, Burma,

Thailand, Java

Papaverine �/ �/ Turano et al., 1989

Rumex cyprius (Polygo-

naceae)

�/ �/ Aqueous and Methanol ex-

tract

�/ Inhibition of HIV reverse

transcriptase

Mekkawy et al.,

1995

S. indica Linn. (Legu-

minoseae)

Asoka �/ Extract of bark �/ HIV protease inhibition Kusumoto et al.,

1995

Schumanniophyton mag-

nificum

�/ �/ Schumannificine �/ Irreversible binding to gp 120 Houghton et al.,

1994

Sindora sumatrana Miq. Supratul Indonesia Aqueous extract of fruit Inhibition of HIV-1 induced cyto-




Otake et al., 1995

Swertia frachetiana �/ �/ Swertifrancheside �/ Inhibition of DNA polymerase

activity of HIV-1 reverse tran-

scriptase

Pengsuparp et al.,

1995

Symphonia globulifera

(Clusiaceae)

�/ Tanzania Guttiferone A In vitro, CEM-SS cells �/ Gustafson et al.,

1992b

Syzygium claviflorum

(Myrtaceae)

�/ �/ Betulinic acid, Platanic acid

Betulinic acid derivatives

In vitro, CEM-SS and MT-4 cells Interference with virus-cell fu-

sion, effect on glycoprotein

gp41

Nakashima et al.,

1992; Fujioka et

al., 1994

Terminalia arjuna Wight

et Arn. (Combretaceae)

Arjuna �/ Extract of stem bark �/ HIV protease inhibition Kusumoto et al.,

1995

Terminalia bellerica

Roxb. (Combretaceae)

Bahera �/ Aqueous and methanol ex-

tracts, chebulagic acid, pu-

nicalin, punicalagin, and

punicacortein


transcriptase, inhibition of viral

adsorption to cells

Nonaka et al.,

1990; Weaver et

al., 1992; Mekka-

wy et al., 1995

T. chebula Ritz (Com-

bretaceae)

Harida, myrobalan �/ Aqueous and methanol ex-



punicacortein



adsorption to cells

Nonaka et al.,

1990; Weaver et

al., 1992; Mekka-

wy et al., 1995

Terminalia horrida

Staud, (Combretaceae)

�/ �/ Aqueous and methanol ex-



punicacortein



adsorption to cells

Nonaka et al.,

1990; Weaver et

al., 1992; Mekka-

wy et al., 1995

Trichosanthes kirilowii

Maxim.

Used as anti-inflammatory

agent and detoxifier

China a-trichosanthin In vitro, VB cell line, macrophage

assays cells chronically infected, in

vitro with exogenous virus; and

cells infected in vivo i.e. culture of

macrophages isolated from blood

of HIV infected patients

Inhibition of transcription and

transactivation

Chow et al., 1990

Urtica diocia (Urtica-

ceae)

Stinging nettle �/ Acetylglucosamine-specific

lectin

�/ Interference with virus-cell fu-

sion

Balzarini et al.,

1991

K.

Verm

an

i,S

.G

arg

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tional uses


tested


Ximenia americana L.

(Oleaceae)

Enkoi, huda, mellau; used in

contagious diseases, stomach

complaints, and worm infesta-

tions

Ethiopia Methanol extract of stem

bark

�/ �/ Asres et al., 2001

Xylopia spp. (Annona-

ceae)

�/ Peru Xylopinic acid In vitro, CEM-SS cells �/ Fuller et al., 1996

K.

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Transmission of virus by direct contact of recipient’s

mucous membranes or skin with infected sexual partner

leads to development of primary genital herpes. The

primary symptoms of HSV infection include prodromalflu like syndrome with fever, headache, malaise, diffuse

myalgias followed by local symptoms consisting of

genital itching, tenderness, dysuria, lesions, painful

papules over genital regions and ulceration (Hardin,

1996; Murray and Pizzorno, 1999).

Acyclovir is the most commonly used drug for

treatment of HSV infections. A serious problem with

acyclovir is the drug resistance in patients. Therefore,there is a need of developing new anti-HSV drugs.

Various phytochemicals have been traditionally used for

the treatment of viral infections and have been shown to

possess in vitro and in vivo antiviral activity against

HSV. Some of the plants reported to possess antiviral

activity against HSV are summarized in Table 2.

A concentrated extract of Melissa officinalis (lemon

balm) is one of the most widely used topical prepara-tions in the treatment and prevention of herpes. Melissa

cream had been reported to interrupt the infection,

promote healing of symptoms, and prevent the recur-

rence of herpes (Wolbing and Leonhardt, 1994). An-

other popular topical preparation (Pompei et al., 1980)

for preventing and treating herpes outbreaks contains

glycyrrhetinic acid, a triterpenoid component of Glycyr-

rhiza glabra (liquorice root). Glycyrrhizin has beenfound to improve the resistance of thermally injured

mice to opportunistic infection of HSV-1 through

induction of CD4� contrasupressor T cells (Utsuno-

miya et al., 1995).

Inhibitory effects of various Ayurvedic, Panamanian

and South American medicinal plants on infection of

HSV-1 have been studied (Hattori et al., 1995; Abad et

al., 1999). Some of the plants found to be active againstHSV-1 are Eupatorium articulatum , Baccharis trinervis ,

Heisteria acuminata , Strychnos potatrum , Rhus acumi-

nata , and Saraca indica . Traditional herbal medicines

such as Kakkon-to, Kanzo-bushi-to, Shigyako-to etc.

have been used historically for the treatment of in-

fectious diseases in China. Efficacy of these traditional

medicines, in HSV-1 has been studied in vitro and in

vivo. Kakkon-to was found to induce strong delayedtype hypersensitivity in HSV infected mice, leading to

localization of skin lesions and reduction of mortality in

mice model (Nagasaka et al., 1995). Kanzo-bushi-to and

Shigyako-to (contains medicinal plant extracts from

Zingiberis siccatum rhizoma, Aconiti tuber and Glycyr-

rhiza radix ) have been found to increase the resistance

of thermally injured mice (infected with HSV-1) through

the activation of contrasupressor T cells and CD8� Tcells (Ikemoto et al., 1994; Matsuo et al., 1994). Some of

the traditional medicinal plants such as Rhus javanica

Linn, Geum japonicum Thunb, Syzygium aromaticum

Linn, and Terminalia chebula Retuz have been shown to

exhibit anti-HSV activity in mice and guinea pig models,

and potentiate the activity of acyclovir (Kurokawa et

al., 1995; Nakano et al., 1998).

In a study, several compounds were tested in vitro byplaque reduction assay and found active against HSV-2.

Among the active compounds, cineole, eugenol, and

curcumin prevented the transmission of HSV-2 in a

mouse model of intravaginal HSV-2 challenge. Eugenol

was also found to provide protection in guinea pig

model of HSV (Bourne et al., 1999).

4. Genital warts

HPV causes venereal infections known as genital

warts or condylomata acuminata. HPV are easily

transmitted during sexual intercourse. Condylomata

acuminata is frequently asymptomatic, with occasional

clinical symptoms including anogenital pruritis and

burning. Penis, anus, vagina, vulva and cervix are

common sites of genital warts (Hardin, 1996).Topical application of small amount of 10�/25%

solution of plant resin, podophyllotoxin in compound

tincture of benzoin has been the most common initial

treatment of warts. Podofilox (0.5% solution), the most

active component of podophyllotoxin, has been ap-

proved by U.S. FDA for treatment of external genital

warts (Hardin, 1996). Condylox (Oclassen Pharmaceu-

ticals, Inc.), a gel containing podofilox has beenapproved by FDA for treatment of anogential warts

including external genital warts and perianal warts

(http://www.fda.gov/cder/da/da.htm).

5. Chlamydial genital infections

C. trachomatis is also transmitted through sexualcontact and leads to diseases such as non-gonooccal

urethritis, cervicitis, pelvic inflammatory disease, and

lymphogranuloma venereum.

Berberine is effective in treatment of ocular C.

trachomatis and is expected to be equally effective in

genital chlamydia infections. Berberine containing

douches and vaginal depletion pack can be used for

local application in chlamydial infections. Tinctures,powdered dried root, fluid and solid extracts of Hy-

drastis canadensis , Berberine vulgaris , and Berberis

aquifolium can be used orally for treatment (Murray

and Pizzorno, 1999). A polyherbal formulation, Pra-

neem (contains purified extracts from Azadirachta

indica and saponins from Sapindus mukerrosi ) has

been reported to possess activity against Chlamydia in

clinical studies (Garg et al., 1994). Out of 28 patients ofchlamydial cervicitis, 22 patients recovered clinically and

microbiologically after 7�/21 days of application of

Praneem cream.

K. Vermani, S. Garg / Journal of Ethnopharmacology 80 (2002) 49�/66 57

http://www.fda.gov/cder/da/da.htm

Table 2

List of plants reported to possess anti-HSV activity

Species (family) Vernacular name and traditional uses Indigenous to Active constituent/fraction/extract In vitro or in vivo assay

model

References

A. catechu Linn. (Palmae) Betel nut India, China,

Asia

Methanol and aqueous extracts of seed Plaque inhibition assay of

HSV-1 in Vero cells

Hattori et al., 1995

B. trinervis Pers. (Com-

positae)

�/ �/ Aqueous extract In vitro HeLa cells infected

with HSV

Abad et al., 1999

Bauhinia vahlii Wight and

Arnott

Bhorla; used for treatment of cuts and

wounds

Nepal Methanol extract In vitro, Vero cells Taylor et al., 1996a

Camptotheca acuminata

(Nyssaceae)

�/ China 10-Methoxycamptothecin Plaque reduction assay Tafur et al., 1976

Carissa carandus Linn.

(Apocyanaceae)

Karondath; used in diarrhea and dysen-

try


D. caryophyllus �/ �/ Dianthin 32, ribosome inactivating protein Plaque reduction assay Tomasi et al., 1982

E. articulatum �/ �/ Aqueous extract In vitro, HeLa cells in-

fected with HSV

Abad et al., 1999

Gelonium multiforum �/ Himalaya Antiretroviral protein GAP31 HSV infection assay, Pla-

que reduction assay

Tomasi et al., 1982;

Bourinbaiar and Lee-

Huang, 1996

G. japonicum Thunb. �/ �/ Eugenin Plaque reduction assay on

Vero cells, murine infection

model

Kurokawa et al., 1995,

1998

G. glabra Linn. (Legumi-

noseae)

Liquorice; used as demulcent, expector-

ant

England, Spain Glycyrrhizin, glycyrrhizic acid Inhibition of viral growth

in HSV infected cell cul-

tures, mice model

Pompei et al., 1979;

Utsunomiya et al.,

1995

Gossypium spp (Malva-

ceae)

Cotton seed India, East In-

dies, Egypt

Gossypol, apogossypol �/ Wichmann et al., 1982

H. acuminata �/ �/ Ethanolic extract In vitro Abad et al., 1999

Holoptelia integrifolia

Plance (Ulmaceae)

Used in rheumatic swellings �/ Methanol extract of bark In vitro, Vero cells Rajbhandari et al.,

2001

Hypericum cordifolium

Choisy (Hypericeae)

Marmhendo; used in fever Nepal Methanol extract In vitro, Vero cells Taylor et al., 1996b

Limonium sinense Girard

Ktze

Used in fever, hemorrhage, and men-

strual disorders

China Ethanolic extract, flavonoids isodihydrosyrengetin,

(�)epigallocatechin-3-O -gallate, samarangenin,

myrecetin, gallic acid, (�)epigallocatechin

Plaque inhibition assay of

HSV-1 in Vero cells

Lin et al., 2000

Macaranga pustulata

King ex Hook f. (Eu-

phorbiaceae)

Malato, kala; used in topical treatment

of skin blemishes

Nepal Methanol extract In vitro, Vero cells Taylor et al., 1996b

M. lanceolata Forsskal

(Myrsinaceae)

�/ �/ Maesasaponins triterpenoid saponins Microtray assay, colori-

metric assay

Apers et al., 2001

Maesa macrophylla Wall.

A. DC. (Myrsinaceae)

Bhogati; used in tonsillitis Nepal Methanol extract In vitro, Vero cells Taylor et al., 1996b

Malotus philippensis Lam.

(Euphorbiaceae)

Sindure, Kamala; used in diarrhea and

dysentry

India, Pakistan,

East Indies, Ne-

pal

Methanol extract In vitro, Vero cells Taylor et al., 1996a

M. officinalis (Labiatae) Lemon mint balm; used as carminative,

antispasmodic, sedative

�/ �/ �/ Wolbing and Leon-

hardt, 1994

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model

References

Milettia extensa Bentham

Baker (Euphorbiaceae)

Gaujo; used in infected wounds and

scabies


M. charantia Linn. (Cu-

curbitaceae)

Bitter melon, karela; used for antiviral,

hypoglycaemic, antitumor, and immu-

nopotentiating purposes

China, India Antiretroviral protein MAP30 HSV infection assay, Pla-

que reduction assay

Tomasi et al., 1982;

Bourinbaiar and Lee-

Huang, 1996

Myristica fragrans Van

Houtt. (Myristaceae)

Nutmeg �/ Methanol extract of aril Plaque inhibition assay of

HSV-1 in Vero cells


Nerium indicum Mill.

(Apocyanaceae)

Used in swelling, skin infection �/ Methanol extract In vitro, Vero cells Rajbhandari et al.,

2001

P. americana (Phytolac-

caceae)

Poke root Tropical Ameri-

ca, South Africa

Pokeweed antiviral protein PAP-S Plaque reduction assay Tomasi et al., 1982

Pongamia glabra Vent.

(Leguminoseae)

�/ �/ Methanol extract of bark and roots Plaque inhibition assay of

HSV-1 in Vero cells


Potamogeton malaianus

Miq.

�/ �/ Potamogetonyde, potamogetonol, and potamogeto-

nin

Vero cell line kidney fibro-

blast of an African green

monkey

Kittakoop et al., 2001

Punica gratum Linn. (Pu-

nicaeae)

Pomegranate, anar �/ Methanol extract of pericarp Plaque inhibition assay of

HSV-1 in Vero cells


R. acuminata L.f. (Ana-

cardiaceae)

�/ �/ Aqueous extract of gall Balb/c mice model Hattori et al., 1995

R. javanica Linn. (Ana-

cardiaceae)

Used in treatment of chronic diseases

such as gastric and duodenal ulcers

China and Japan Aqueous extract, Moronic acid Plaque reduction assay,

guinea pig model, mouse

model


1997, 1999; Nakano et

al., 1998

Rumex hastatus D. Don

(Polygonaceae)

Annile; used in tonsillitis and sore throat Nepal Methanol extract In vitro, Vero cells Taylor et al., 1996a

S. indica Linn. (Legumi-

noseae)

Asoka �/ Aqueous extract, Methanol extract of bark Balb/c mice model, Plaque

reduction assay of HSV-1

on Vero cells


Sibbaldia micropetala

(Rosaceae)

Bhui pasari jhar; used in diarrhea and

dysentry

Nepal Methanol extract In vitro, Vero cells Taylor et al., 1996b

Stephania cepharantha �/ �/ Methanol extract of root and tubers, 13 bisbenzyli-

soquinoline 1 protoberberine, 2 morphinamine

In vitro plaque reduction

assay on Vero cells, in vivo

Balb/c mice model

Nawawi et al., 1999

Streblus asper Loureiro

(Moraceae)

Sehor; used in diarrhea and dysentry Nepal Methanol extract In vitro, Vero cells Taylor et al., 1996a

S. potatrum L.f. (Loga-

niaceae)

�/ �/ Methanol extract Plaque inhibition assay of

HSV-1 on Vero cells, Balb/

c mice model


S. aromaticum L. Merr. et

Perry (Myrtaceae)

Clove Molucca Eugenin Plaque reduction assay on

Vero cells, Mouse infection

model


1998

Terminalia alata Heyne ex

Roth (Combretaceae)

Saj; used in diarrhea and dysentry Nepal Methanol extract In vitro, Vero cells Taylor et al., 1996a

T. chebula Retz. (Com-

bretaceae)

Harida, myrobalan �/ Aqueous extract of fruit Plaque reduction assay,

Balb/c mice model


1997

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model

References

Tridex procumbens Linn.

(Asteraceae)

Kurkure; used in cuts and wounds Nepal Methanol extract In vitro, Vero cells Taylor et al., 1996a

Tripterygium wilfordii

Hook fil.

�/ �/ Triptofordin C-2 In vitro Hayashi et al., 1996

Withania somnifera L.

(Solanaceae)

Ashwagandha Israel Methanol extract Plaque inhibition assay of

HSV-1 in Vero cells


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6. Trichomoniasis

Trichomoniasis, caused by the flagellated, motile

protozoan T. vaginalis , is usually transmitted sexually.Clinical symptoms of the disease include malodorous

yellowish-green vaginal discharge, vaginal itching, red-

ness of the vulva and/or vagina, painful intercourse,

abdominal pain, and painful urination (Hardin, 1996;

Murray and Pizzorno, 1999). Up to 50% of the women

infected with trichomoniasis are asymptomatic. Tricho-

monas is known to degrade secretory leukocyte protease

inhibitor, a substance that is believed to protect the cellsof vaginal mucous membrane from HIV infection,

thereby increasing the risk of HIV transmission.

A 0.4% solution of Melaleuca alternifolia (tea tree) oil

in 1 l of water as daily vaginal douche was found to be

an effective treatment for trichomoniasis (Pena, 1962).

Dried roots, rhizomes, tincture and fluid extracts of

botanicals containing berberine such as H. canadensis ,

Echinacea angustifolia , and Angelica species are beingprescribed for treatment of trichomonal infections

(Murray and Pizzorno, 1999). In another study, extracts

of bark and leaves of Mikania cordifolia , leaves of

Neurolaena lobata and bark of Scutia buxifolia have

been reported to inhibit the growth of T. vaginalis in

vitro. Some essential oils including those obtained from

Mentha piperita and Lavandula angustifolia have also

been reported to possess strong antitrichomonal proper-ties (Jankov et al., 1968).

7. Vaginitis and vulvovaginitis

Vaginitis is one of the most common mixed vaginal

infections and may reflect symptoms of a more serious

underlying STD. Vaginal infections may increase the

risk of transmission of HIV and other sexually trans-

mitted pathogens. Bacterial vaginosis had been reported

to be associated with increased susceptibility to HIV

infection (Murray and Pizzorno, 1999).Major symptoms of vaginitis are vaginal discharge

with a foul odor, itching, burning and inflammation

(Murray and Pizzorno, 1999). Vaginitis may be caused

by:

a) hormonal changes in postmenopausal women (hor-

monal vaginitis),

b) physical or chemical agents which cause damage to

vaginal membranes (irritant vaginitis), and

c) disturbance of ecology of the healthy vagina (in-

fectious vaginitis).

Infectious vaginitis may be caused by the prevalence ofmicroorganisms such as Candida albicans (vaginal

candidiasis), T. vaginalis (trichomonal vaginitis), and

G. vaginalis (non-specific vaginitis). Less frequent causes

of vaginitis include N. gonorrhoea , herpes simplex virus

and C. trachomatis (Ruggiero, 1996; Murray and

Pizzorno, 1999).

Traditionally, herbal preparations have been used for

the treatment of various STDs. In Central America and

Caribbean, 101 plants are claimed to be used in

traditional medicine for treatment of gonorrhoea. In a

study, tinctures from 44 plants used for STDs in

Guatemala were screened for in vitro activity against

N. gonorrhoeae . Extracts of bark of Bixa orellana , fruits

of Parmentiera edulis , leaf of Diphysa robinioides ,

Eupatorium odoratum , Gliricidia sepium , Physalis angu-

lata , Piper aduncum and Prosopis juliflora , root of

Casimiroa edulis , and whole Clematis dioica were found

to be active against N. gonorrhoea (Caceres et al., 1995).

In another study, active substances from medicinal

plants of Rwanda (Central Africa) that were indigen-

ously used for gonorrhoea were screened for their

antimicrobial activity against N. gonorrhoea , N. menin-

gitidis , Streptococcus pyogenes , and Staphylococcus

aureus . Plants showing greatest activity against these

organisms include Hygrophila auriculata , Vernonia ae-

nulans , V. crudia , Euphorbia grantii , Cajanus cajan ,

Orthosiphon australis , Rumex abyssinicus and Lanatana

trifolia .

Allium sativum (garlic) possesses antibacterial, anti-

viral and antifungal properties. Douching solutions and

gauze containing garlic may be used as a tampoon/

suppository for most of the infectious vaginitis. G.

glabra contains isoflavonoids that are reported to be

effective against Candida. Water-soluble chlorophyll

can also be added to the douching solutions to provide

relief in vaginitis. Atrophic vaginitis due to lack of

estrogens may be treated by the use of phytoestrogens

obtained from plants such as Ribes nigrum , Foeniculum

vulgare , Illicium verum , Panax ginseng , Medicago

sativa , Trifolia repens and G. glabra (Murray and

Pizzorno, 1999).

Tea tree oil, an essential oil from Australian plant M.

alternifolia , has a wide spectrum of antimicrobial

activity with a minimal effect on commensal lactobacilli

in vagina (Hammer et al., 1999). An alcoholic extract of

M. alternifolia (tea tree) diluted with water has been

used as a douche combined with saturated tampoons in

the treatment of vaginitis (Pena, 1962). In addition to

extract, its vaginal pessaries have also been reported to

treat bacterial vaginosis (Blackwell, 1991). Praneem

polyherbal products have been reported to be effective

in treating patients with abnormal vaginal discharge due

to microbial infections (Mittal et al., 1995).

Ayurveda also prescribes some drugs that can be

applied vaginally for the treatment of vaginal disorders.

These include Subhakari vati, Somanath rus and

Soubhagya vardhana tel (Essential ayurvedic drugs for

dispensaries and hospitals, 2000).


8. Vaginal formulations of herbal origin

V-gel and PH 5 are examples of vaginal formulations

based on herbal extracts, available in Indian market. V-gel, a polyherbal formulation of The Himalaya Drug

Company (Bangalore, India), is indicated for vaginal

infections of varied etiology such as vaginitis, cervicitis,

vaginal candidiasis and vaginal discharge. It contains

the extracts of Emblica officinalis , Terminalia belerica ,

T. chebula , Rosa centifolia , Elletaria cardamomum ,

Boerhaevia diffusa , Parmelia perlata , Curcuma longa

and Vitex negundo. V-gel has been found effective intreating diseases caused by microorganisms such as G.

vaginalis , Moniliasis , T. vaginalis , Gonococcus vaginalis ,

C. albicans and other non-specific organisms. In clinical

studies, it was found that V-gel provides symptomatic

relief within 4�/5 days of application and complete

cessation of symptoms within 7�/14 days of treatment.

The formulation was found to be safe and can be used

by pregnant women, during pelvic inflammatory dis-ease, and in postnatal cases (Mitra et al., 1997; Umadevi

and Swarup, Dec1998�/Feb1999; Narmada and Va-

nitha, 1999).

PH 5 (Zoic Pharmaceuticals, Delhi) has been claimed

to restore normal vaginal pH, reduce leucorrhoea and

various vaginal discharges, and possess astringent, anti-

inflammatory, antiseptic and bacteriostatic effects. The

vaginal pessaries consist of herbal extracts enclosed insmall bags made of cloth. It contains the extracts of

Quercus infectoria , Sausurea lappa and Tamarix gallica.

A vaginal depletion pack (‘Vag pack’) is regularly

used and prescribed by naturopathic physicians for

treatment of various vaginal disorders over the last 50

years (Murray and Pizzorno, 1999). Efficacy of this pack

has not been studied in controlled clinical trials, but it

has a long history of use, which dates back to 19thcentury. The ‘Vag pack’ consists of a tampoon contain-

ing a mixture of H. canadensis tincture, Thuja occiden-

talis oil, M. alternifolia oil, bitter orange oil, anhydrous

magnesium stearate, vita minerals and glycerin.

Praneem polyherbal cream, tablets and suppositories

are under clinical development and possess wide spec-

trum antibacterial, antifungal and antiviral effects

against sexually transmitted pathogens (Talwar et al.,1995, 1997, 2000). Praneem contains purified extract of

A. indica (neem) and saponins extracted from Sapindus

mukerrossi (reetha). These have been reported to inhibit

the clinical isolates of different species of Candida (C.

albicans , C. tropicalis and C. krusei ), N. gonorrhoea

(including penicillin resistant strains), G. vaginalis , and

multi drug resistant Escherichia coli and S. aureus.

Intravaginal inoculation of these formulations pre-vented lesions and vaginal transmission of HSV-2 and

C. trachomatis in progestin-sensitized mice. In addition,

they have also been found to possess virucidal activity

against HIV at doses non-toxic to cells in culture.

Praneem polyherbal had completed phase I safety and

acceptability trials in India. It was found to be effective

in post-coital tests in women and produced a curative

effect in women with vaginal discharge as per studies

conducted in India, Egypt and Dominican Republic.Viracea, a proprietary formula of Destiny BioMediX

Corporation, is a topical microbicide consisting of

benzalkonium chloride and phytochemicals derived

from Echinacea purpurea . Viracea has been reported to

possess antiviral activity against Acyclovir resistant as

well as susceptible strains of HSV-1 and HSV-2

(Thompson, 1998).

9. Plantibodies as topical microbicides

An innovative approach to microbicide development

is the use of genetically engineered plants to produce

human monoclonal antibodies, ‘plantibodies’, active

against a range of STIs. Even though plantibodies are

not based on indigenous empirical knowledge, these are

briefly mentioned because of their potential activity

against STIs. With this technology, it is possible to

deliver anti-HIV antibodies directly to the vagina,

allowing them to combat pathogens before actual

infection occurs (Forbes, 2000). Mass production of

human antibodies using genetically engineered plants is

relatively inexpensive as compared to those pro-

duced by fermentation technology and transgenic ani-

mals (Harvesting Monoclonal Antibodies from Plants,

1999).

From a public health perspective, monoclonal anti-

bodies provide a promising approach for preventing

reproductive tract infections and are expected to play an

important preventive role in future emerging disease

epidemics (Zeitlin et al., 1999). Corn has been geneti-

cally engineered to produce human antibodies against

herpes and sperm. Research is in progress to produce

anti-HIV antibodies. Herpes antibody has been found to

protect mice against infection, and a sperm antibody has

prevented pregnancy in rabbits. Corn has been proposed

as a cheap and safe potential source of antibodies for

contraceptive antimicrobial activities. Plantibodies are

very potent, specific, and have the potential to be used in

novel ways such as personal lubricants, gels, or con-

trolled-release devices for vaginal insertion. Topical gels

containing plantibodies for HSV-1 and HSV-2 are under

preclinical development. Application of these antibodies

to the mice vagina has been shown to prevent the

infection with genital herpes. A more potent herpes

plantibody to prevent mother to child transmission of

herpes is under development (Harvesting Monoclonal

Antibodies from Plants, 1999).


10. Conclusion

Several plant extracts and their constituents possess

activity against sexually transmitted diseases indicatingtheir huge potential as an effective measure for preven-

tion and treatment of STDs including AIDS. Plant

derived microbicides and plantibodies are some of the

new approaches for prevention of HIV and other

sexually transmitted pathogens. Herbal medicines can

be developed as a safe, effective and economical alter-

native to drugs presently approved for symptomatic

treatment of STDs and AIDS.

Acknowledgements

The authors would like to acknowledge the supportfrom the Department of Biotechnology (DBT), Govern-

ment of India and Contraception Research and Devel-

opment Program (CONRAD), United States, under the

‘Indo-US collaborative Program in Contraceptive and

Reproductive Health Research’. The vews expressed by

the authors do not necessarily reflect the views of DBT

or CONRAD.

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Antiproliferative activity of the Netherlands propolis and its activeprinciples in cancer cell lines

Arjun H. Banskota, Takema Nagaoka, Lucia Yoshie Sumioka, Yasuhiro Tezuka,Suresh Awale, Kiyoshi Midorikawa, Katsumichi Matsushige, Shigetoshi Kadota *

Department of Natural Products Chemistry, Institute of Natural Medicine, Toyama Medical and Pharmaceutical University, 2630-Sugitani, Toyama

930-0194, Japan

Received 18 September 2001; received in revised form 25 October 2001; accepted 25 December 2001

Abstract

The MeOH extract of the Netherlands propolis showed promising antiproliferative activity toward highly liver-metastatic murine

colon 26-L5 carcinoma with an EC50 value of 3.5 mg/ml. Further, antiproliferative activity-guided purification of the MeOH extract

led us to isolate four flavonoids (1�/4), seven cinnamic acid derivatives (5�/11) and two new glycerol derivatives (12, 13), whose

structures were elucidated on the basis of spectral analysis. The isolated compounds were tested for their antiproliferative activity

against murine colon 26-L5, murine B16-BL6 melanoma, human HT-1080 fibrosarcoma and human lung A549 adenocarcinoma cell

lines. The benzyl (9), phenethyl (10) and cinnamyl caffeates (11) possessed potent antiproliferative activities with EC50 values of

0.288, 1.76 and 0.114 mM, respectively, toward colon 26-L5 carcinoma. These caffeates were considered to be active constituents of

the Netherlands propolis in their antiproliferative activity. The antioxidative activity of these caffeates may play an important role in

their antiproliferative activities. # 2002 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Propolis; Antiproliferative activity; DPPH radical scavenging activity; Benzyl caffeate; Phenethyl caffeate; Cinnamyl caffeate

1. Introduction

Propolis is a resinous hive product collected by

honeybees from various plant sources. It has a pleasant

aromatic odor and yellow�/green to dark brown color

depending on its source and age (Ghisalberti, 1979).

Propolis has a long history of being used in traditional

medicine dating back at least to 300 BC (Ghisalberti,

1979) and has been reported to have a broad spectrum

of biological activities, viz. anticancer, antioxidant,

antiinflammatory, antibiotic, and antifungal activities

(Banskota et al., 2001a; Burdock, 1998; Marcucci,

1995). It has recently gained popularity as a health

drink and is used extensively in food and beverages in

various parts of the world including Japan, the USA and

Europe, where it is claimed to improve health and

prevent diseases such as inflammation, heart disease,

diabetes and even cancer. Due to these facts there is

renewed interest in the composition and biological

properties of propolis.

Propolis mainly contains sticky plant substances,

collected by honeybees and mixed with bees wax and

other bee secretions, thus, the composition of propolis

other than wax absolutely depends on the vegetation of

the area from where it was collected. Propolis from

temperate zones contains predominantly phenolic com-

pounds including flavonoids and cinnamic acid deriva-

tives (Marcucci, 1995). Diterpenes and prenylated

compounds, which are virtually absent in temperate

propolis, on the other hand, were reported from the

tropical propolis of the South-American continent

together with lignans, flavonoids and other classes of

compounds (Bankova et al., 2000). The difference in the

composition of propolis from temperate and tropical

zones is mainly due to their different vegetations. Even

given this difference in their composition, propolis from

both regions possessed similar biological properties

(Burdock, 1998; Banskota et al., 2001a). In our previous

work, we reported the isolation of 31 different consti-

* Corresponding author. Tel.: �81-76-434-2281x2825; fax: �81-76-

434-5059.

E-mail address: [email protected] (S. Kadota).




PII: S 0 3 7 8 - 8 7 4 1 ( 0 2 ) 0 0 0 2 2 - 3


tuents from Brazilian propolis, of which three were new

and 15 were isolated for the first time from propolis

together with either their antiproliferative activity or

hepatoprotective activity (Banskota et al., 2001b, 2000a,1998; Basnet et al., 1996). Moreover, we also made a

comparative study of propolis from different continents

for their antiproliferative, antihepatotoxic and DPPH

free radical scavenging activities (Banskota et al.,

2000b). In the study, the MeOH extract of the Nether-

lands propolis showed promising antiproliferative activ-

ity. Thus, we conducted a chemical investigation of the

MeOH extract of the Netherlands propolis. In thispaper, we report the active principle of the Netherlands

propolis together with its antiproliferative activities

against different tumor cells.


2.1. Chemicals

RPMI and Eagle’s minimum essential medium

(EMEM) were purchased from Nissui Pharmaceutical

Co. Ltd. (Tokyo, Japan). 3-(4,5-Dimethylthiazol-2-yl)-

2,5-dimethyltetrazolium bromide (MTT) was purchased

from Sigma Chemicals (St. Louis, MO). Heat-inacti-

vated fetal calf serum (FCS) was from Gibco BRL

Products (Gaitherburg, MD). Coster polystyrene 96-

well polystyrene plates (Corning Incorporated, Corning,NY) were used for the antiproliferative assay. Phenyl

ethanol and cinnamyl alcohol were from Nacalai Tesque

(Kyoto, Japan). 1,1-Diphenyl-2-picrylhydrazyl (DPPH),

sodium bicarbonate, glutamine, benzyl alcohol a-toco-

pherol, ascorbic acid and caffeic acid were from Wako

Pure Chemical Industries (Osaka, Japan). 5-Fluorour-

acil was purchased from Tokyo Kasei Kogyo Co. Ltd.

(Tokyo, Japan) and doxorubicin HCl was from KyowaHakko Co. Ltd. (Tokyo, Japan). Column chromato-

graphy was performed on silica gel 60 (Nacalai Tesque,

Kyoto, Japan) and thin layer chromatography (TLC),

both analytical and preparative, was carried out on

percolated Merck Kieselgel 60 F254 and RP-18 F254

plates (0.25 or 0.50 mm thickness), respectively.

2.2. Instrumentation

UV absorptions were recorded on a Shimadzu UV-

160A UV�/visible spectrophotometer. Optical rotations

were measured on a JASCO DIP-140 digital polarom-

eter. IR spectra were measured with a Shimadzu IR-408

spectrophotometer in CHCl3 solution. FAB mass mea-

surements were carried out on a JEOL JMS-700T

spectrometer and glycerol was used as a matrix. 1H-,13C- and 2D-NMR were recorded on a JEOL GX-400

spectrometer with tetramethylsilane (TMS) as an inter-

nal standard.

2.3. Propolis

The Netherlands propolis was collected in the north-

east of the Netherlands in 1998, by scraping it off fromthe frames of beehives belonging to Honeybee Husban-

dry, Rutten, the Netherlands. The voucher specimen

(No. TMPW 19920) is preserved in the Museum for

Materia Medica, Institute of Natural Medicine, Toyama

Medical and Pharmaceutical University, Toyama, Ja-

pan, as a reference.

2.4. Extraction and Isolation

The powder of crude propolis (400 g) was extracted

with MeOH (3L�2) under reflux for 3 h to give a

MeOH extract (210 g, 52.5%). The residue was further

extracted with water (3L�2) at 80 8C for 3 h. Thefiltrate was lyophilized to give a water extract (3.6 g,0.9%) and the residue was discarded. The MeOHextract (150 g) was divided into ten fractions by silicagel column chromatography (50�7.5 cm) eluting with aCHCl3/MeOH gradient system (fraction 1, 21.2 g;fraction 2, 2.9 g; fraction 3, 18.4 g; fraction 4, 6.6 g;fraction 5, 3.1 g; fraction 6, 5.0 g; fraction 7, 17.8 g;fraction 8, 4.8 g; fraction 9, 23.1 g and fraction 10, 42.0g). Chrysin (1, 10.0 mg) and galangin 7-methyl ether (2,

12.5 mg) were obtained as precipitates from fractions 2

and 3, respectively. Further column chromatography

followed by preparative TLC of fraction 6 gave 2 (659mg), 5 (1.7 mg), 9 (11.0 mg) and 11 (86.3 mg), while

fraction 7 gave 3 (7.7 mg), 4 (6.5 mg), 6 (6.9 mg), 8 (2.9

mg) and 9 (3.79 g). Similarly, fraction 8 gave 2 (14.2

mg), 4 (7.5 mg), 7 (46.8 mg), 12 (47.6 mg), 13 (10.3 mg)

and a mixture of 9�/11 (770 mg) in a ratio of 3.5:2:1,

which were separated by reversed-phase preparative

TLC (H2O/MeOH/CH3CN, 1:1:1).

2.4.1. 2-Acetyl-1,3-dicoumaroylglycerol (12)

A colorless amorphous solid; IR (CHCl3) nmax 3400,

1700, 1600, 1510, 1110 cm�1; HRFABMS m /z 427.1355

[Calc. for C23H23O8 (M�H)�, 427.1393]: 1H NMR

(CD3OD) d 7.61 (2H, d, J�15.9 Hz, H-3?, 3ƒ ), 7.47

(4H, d, J�8.6 Hz, H-5?, 8?, 5ƒ, 8ƒ), 6.80 (4H, d, J�8.6

Hz, H-6?, 9?, 6ƒ, 9ƒ), 6.33 (2H, d, J�15.9 Hz, H-2?, 2ƒ),5.36 (1H, tt, J�6.0, 4.4 Hz, H-2), 4.47 (2H, dd,J�12.0, 4.1 Hz, H2-1, 3), 4.35 (2H, dd, J�12.0, 5.8

Hz, H2-1, 3), 2.08 (3H, s, COCH3); 13C NMR (CD3OD)

d 171.9 (COCH3), 168.6 (C-1?, 1ƒ), 161.3 (C-7?, 7ƒ), 147

(C-3?, 3ƒ), 131.3 (C-5?, 8?, 5ƒ, 8ƒ), 127.0 (C-4?, 4ƒ), 116.8

(C-6?, 9?, 6ƒ, 9ƒ), 114.4 (C-2?, 2ƒ), 71.0 (C-2), 63.3 (C-1,

3), 20.9 (COCH3).

2.4.2. 2-Acetyl-1-coumaroyl-3-feruloylglycerol (13)

A colorless amorphous solid; [a ]D25 �6.58 (c�0.03,

CHCl3); IR (CHCl3) nmax 3400, 1700, 1670, 1600, 1510,

1430, 1370, 1260, 1160 cm�1; HRFABMS m /z 457.1467

A.H. Banskota et al. / Journal of Ethnopharmacology 80 (2002) 67�/7368

[Calc. for C24H25O9 (M�H)�, 457.1499]; 1H NMR

(CDCl3) d 7.64 (2H, d, J�15.9 Hz, H-3?, 3ƒ), 7.40 (2H,

d, J�8.5 Hz, H-5?, 8?), 7.07 (1H, dd, J�8.3, 1.7 Hz, H-

9ƒ), 7.03 (1H, d, J�1.7 Hz, H-5ƒ), 6.91 (1H, d, J�8.3Hz, H-8ƒ), 6.83 (2H, d, J�8.5 Hz, H-6?, 9?), 6.31 (1H, d,

J�15.9 Hz, H-3ƒ), 6.28 (1H, d, J�15.9 Hz, H-3?), 5.42

(1H, tt, J�5.8, 4.4 Hz, H-2), 4.47 (2H, J�12.2, 4.4 Hz,

H2-1, 3), 4.40 (2H, dd, J�12.2, 5.8 Hz, H2-1, 3), 3.92

(3H, s, OCH3), 2.09 (3H, s, COCH3); 13C NMR

(CDCl3) d 171.9 (COCH3), 166.9 (C-1?, 1ƒ), 158.2 (C-

7?), 148.2 (C-7ƒ), 146.8 (C-6ƒ), 146.0 (C-3ƒ), 145.3 (C-3?),130.4 (C-5?, 8?), 126.8 (C-4ƒ), 126.7 (C-4?), 123.4 (C-8ƒ),115.9 (C-6?, 9?), 114.8 (C-5ƒ), 114.4 (C-2ƒ), 114.3 (C-2?),109.4 (C-9ƒ), 69.4 (C-2), 62.3 (C-1, 3), 56.0 (OCH3), 20.9

(COCH3).

2.5. Antiproliferative assay

Human HT-1080 fibrosarcoma (Rasheed et al., 1974),

human lung A549 adenocarcinoma (Giard et al., 1973)

and murine B16-BL6 melanoma (Hart, 1979) cell lineswere maintained in EMEM medium supplemented with

10% FCS, 0.1% sodium bicarbonate and 2 mM gluta-

mine. Murine colon 26-L5 carcinoma cell line (Ohnishi

et al., 1997), on the other hand, was maintained in

RPMI medium containing the same supplements as in

EMEM. These are all highly metastatic cell lines except

for A-549 carcinoma.

Cellular viability was determined using the standardMTT assay as reported previously (Banskota et al.,

2000b; Rubinstein et al., 1990). In brief, exponentially

growing cells were harvested and 100 ml of cell suspen-

sion containing 2000 cells was plated in 96-well micro-

titer plates. After 24 h of incubation to allow for cell

attachment, the cells were treated with varying concen-

trations of test samples in medium (100 ml) and

incubated for 72 h at 37 8C under 5% CO2. Threehours after the addition of MTT, the amount offormazan formed was measured spectrophotometricallyat 550 nm with a Perkin Elmer HTS-7000 plate reader.The test samples were first dissolved in DMSO and thendiluted with medium; the final concentration of DMSOwas less than 0.25%. Normal also had the same extentof DMSO. 5-Fluorouracil (5-FU) and doxorubicin HClwere used as positive controls, and EC50 values werecalculated from the mean values of data from 4 wells.

2.6. DPPH radical scavenging activity

DPPH radical scavenging activity was measured

according to the procedure described by Hatano et al.

(1989). In brief, each different extract dissolved in EtOH

or in water (500 ml) was mixed with an equal volume ofDPPH solution (60 mM). The resulting solution was

thoroughly mixed by vortex and the absorbance was

measured at 520 nm after 30 min. The scavenging

activity was determined by comparing the absorbance

with that of the blank (100%) containing only DPPH

and solvent.

3. Results

3.1. Extraction and isolation

The crude propolis was successively extracted with

MeOH and water under reflux. The MeOH extract,

having an interesting antiproliferative activity, was

further fractionated into ten fractions by silica gel

column chromatography. Chrysin (1, Wagner et al.,1976) and galangin 7-methyl ether (2) were obtained as

precipitates from fractions 2 and 3, respectively. Frac-

tions 6�/8, having the strongest antiproliferative activity,

were further subjected to chemical investigation. Three

flavonoids, namely galangin 7-methyl ether (2), pino-

banksin (3, Bohlmann et al., 1982) and pinobanksin 5-

methyl ether (4, Bankova et al., 1983) were isolated from

these fractions together with cinnamic acid (5), ferulicacid (6, Kelley et al., 1976), isoferulic acid (7, McCorkin-

dale et al., 1969), 3,4-dimethoxycinnamic acid (8, de

Silva et al., 1979), benzyl caffeate (9, Yamauchi et al.,

1992), phenethyl caffeate (10, Grunberger et al., 1988)

and cinnamyl caffeate (11) as shown in Fig. 1. The

Fig. 1. Structure of the compounds tested for antiproliferative activity.

A.H. Banskota et al. / Journal of Ethnopharmacology 80 (2002) 67�/73 69

spectral data of these known compounds were identical

to those in the literature or authentic samples. More-

over, two new glycerol derivatives, 2-acetyl-1,3-dicou-

maroylglycerol (12) and 2-acetyl-1-coumaroyl-3-feruloylglycerol (13), were also isolated from fraction

8, whose structures were elucidated by spectral analysis

including 2D NMR spectra.

3.2. Antiproliferative activity

The MeOH extract of the Netherlands propolis,

having interesting antiproliferative activity (EC50, 3.5

mg/ml), was divided into ten fractions by silica gel

column chromatography and eluted with a CHCl3/

MeOH gradient system. The antiproliferative effects of

these fractions were tested against murine colon 26-L5carcinoma, and the EC50 values of fractions 1�/10 were

20.7, 22.5, 37.5, 29.1, 7.1, 0.6, 0.8, 1.1, 6.6, 13.9 mg/ml,

respectively. The active fractions, 6�/8, gave 12 com-

pounds (2�/13), whose antiproliferative effects were

tested against four different tumor cell lines. The EC50

values of all the isolated compounds together with

benzyl alcohol (14), phenyl ethanol (15), cinnamyl

alcohol (16) and caffeic acid (17) are summarized inTable 1. Benzyl (9), phenethyl (10) and cinnamyl (11)

caffeates showed potent antiproliferative activity against

HT-1080, colon 26-L5 and B16-BL6 cell lines with EC50

values of less than 14 mM, falling within the range of the

potent cytotoxic agent (EC50B4 mg/ml) made by Geran

et al. (1972). These caffeates also possessed the strongest

antiproliferative effects toward A-549 among all the

compounds. Flavanone derivatives 1 and 2 also pos-

sessed moderate antiproliferative activities toward HT-1080, colon 26-L5 and B16-BL6 melanoma cells among

which the EC50 value of 1 toward colon 26-L5 carci-

noma 13.4 mM (3.4 mg/ml) fell below the range of the

potent cytotoxic agent made by Geran et al. (1972).

Flavanols (3, 4), on the other hand, showed only weak

antiproliferative activities and other compounds [simple

cinnamic acid derivatives 5�/8, caffeic acid (17) and

alcohols 14�/16] possessed no antiproliferative activity,except for 16 (EC50 value, 47.8 mM against colon 26-L5;

44.0 mM against B16-BL6 melanoma). The water extract

of the Netherlands propolis did not possess any

antiproliferative effects toward colon 26-L5 carcinoma

(Banskota et al., 2000b).

3.3. DPPH radical scavenging activity

The DPPH radical scavenging activity of benzylcaffeate (9), phenethyl caffeate (10) and cinnamyl

caffeate (11) was tested together with the well known

antioxidants ascorbic acid and a-tocopherol. All these

caffeates (9�/11) possessed strong scavenging effects

toward DPPH radical and their scavenging strengths

were nearly equal to that of a-tocopherol and stronger

than that of ascorbic acid (Fig. 2). Chrysin (1) and a new

compound 12, having mild antiproliferative effects,showed only weak DPPH radical scavenging activities

(Fig. 2).

4. Discussion

Propolis, a complex mixture of plant metabolites,

possesses a broad spectrum of biological activitiesincluding antibiotic, antioxidative, antiinflammatory

and anticancer activities (Banskota et al., 2001a; Bur-

dock, 1998; Marcucci, 1995). In our previous work, we

found that the MeOH extract of the Netherlands

propolis had interesting antiproliferative activity against

highly metastatic liver murine colon 26-L5 carcinoma

cells with an EC50 value of 3.5 mg/ml (Banskota et al.,

2000b). Chemical examination of the MeOH extract wasthus performed to identify the active components

responsible for the antiproliferative activity of the

Netherlands propolis. Eleven known compounds, either

cinnamic acid derivatives or flavonoids (1�/11) were

isolated from the MeOH extract together with two new

glycerol derivatives (12, 13) through an antiproliferative

activity-guided purification.

2-Acetyl-1,3-dicoumaroylglycerol (12) was isolated asa colorless amorphous solid with molecular formula

C23H22O9. The IR spectrum showed absorptions corre-

sponding to hydroxyl (3400 cm�1) and carbonyl groups

Table 1

Antiproliferative activity of the isolated compounds from the MeOH

extract of the Netherlands propolis (EC50

values are in mM)

Compounds Human Murine

HT-

1080

A-549 Colon 26-

L5

B16-

BL6

Chrysin (1) 17.3 �200 13.4 20.5

Galangin 7-methyl ether (2) 26.3 �200 30.3 20.8

Pinobanksin (3) 284 �368 �200 �200

Pinobanksin 5-methylether

(4)

�200 �200 �200 187

Cinnamic acid (5) �200 �200 �676 �200

Ferulic acid (6) �200 �200 �515 �200

Isoferulic acid (7) �200 �200 �515 �200

3,4-Dimethoxycinnamic acid

(8)

�200 �200 �200 �200

Benzylcaffeate (9) 13.3 18.9 0.288 2.03

Phenethyl caffeate (10) 13.7 44.0 1.76 3.16

Cinnamyl caffeate (11) 9.45 18.9 0.114 1.92

Compound 12 83.3 72.3 85.9 81.9

Compound 13 80.5 �200 75.5 66.0

Benzyl alcohol (14) �200 �200 �200 �200

Phenyl ethanol (15) �200 �200 �200 �200

Cinnamyl alcohol (16) �200 �200 47.8 44.0

Caffeic acid (17) �200 �200 167 �200

5-Fluorouracil (5-FU) 3.92 5.76 0.538 4.69

Doxorubicin HCl (DOX) 0.086 0.189 0.017 0.012


(1700, 1600 cm�1). The 1H and 13C NMR spectra of 12

showed the signals of two equivalent coumaroyl groups

together with two oxygenated methylenes, an oxyge-

nated methine and an acetyl group. The 1H�/1H COSY

and HMQC correlations, together with the HMBC

correlations depicted by arrows in Fig. 3, indicated

that 12 is a symmetric glycerol ester having an acetyl

group at C-2 and two p-coumaroyl group at C-1 and C-

3. Thus, the structure of 12 was determined to be 2-

acetyl-1,3-dicoumaroylglycerol.2-Acetyl-1-coumaroyl-3-feruloylglycerol (13) was also

isolated as a colorless amorphous solid with [a ]D25 �6.58

(c�0.03, CHCl3). The molecular formula of 13 was

calculated as C24H24O9 by FABMS, and its IR spectrum

indicated the presence of hydroxyl as well as carbonyl

groups. The 1H and 13C NMR spectra of 13 were found

to be similar to those of 12, except for a difference in one

of the benzene rings. These spectral data indicated the

presence of a feruloyl group at C-3 of 13 instead of two

p -coumaroyl group as in 12. This was confirmed by1H�/

1H COSY, HMQC and HMBC correlations (Fig.

3). Thus, the structure of 13 was determined to be 2-

acetyl-1-coumaroyl-3-feruloylglycerol.

Propolis from different continents was reported to

have cytotoxic and antitumor effects (Burdock, 1998;

Banskota et al., 2001a). The flavonoids, cinnamic acid

derivatives including CAPE and artepillin C, and some

diterpenoids were previously reported from propolis

with interesting antitumor activity (Banskota et al.,

2001a). In the present study, we further isolated

cinnamic acid derivatives (5�/13) and flavonoids (1�/4)

from the MeOH extract of the Netherlands propolis,

which were previously reported from propolis of other

regions (Marcucci, 1995). The benzyl (9), phenethyl (10)

and cinnamyl caffeates (11) showed potent antiproli-

ferative activities toward HT-1080 and B16-BL6 mela-

noma cell lines and most selectively toward murine

colon colon 26-L5 carcinoma. Neither caffeic acid (17)

nor its corresponding alcohols (14�/16) showed any

antiproliferative activity against the tested cell lines,

except for 16 having moderate antiproliferative activity

toward colon 26-L5 and B16-BL6 melanoma. These

results indicate that the caffeates as a whole seem to be

necessary for the antiproliferative effects. Moreover,

compounds 9�/11 were also found to be present in the

highest content in the active fractions of the MeOH

extract. Benzyl caffeate (9) was present at more than 3.7

g in fraction 7, while fraction 8 contained 770 mg of a

mixture of 9�/11 in the ratio of 3.5:1:2. From these facts,

it was concluded that benzyl (9), phenethyl (10) and

cinnamyl (11) caffeates are the active components

responsible for the antiproliferative activity of the

Fig. 2. The DPPH radical scavenging activity of the isolated compounds.

Fig. 3. The 1H�/1H COSY and HMBC correlations of the new

compounds.


Netherlands propolis. Of course, flavones 1 and 2 also

possessed relatively stronger antiproliferative activities

to all the cell lines other than A-549 cells, indicating that

they might also partly contribute to the antiproliferative

activity.

Phenethyl caffeate (10) is well know by the name

caffeic acid phenethyl ester (CAPE) and was reported to

have strong antitumor activity. In addition to in vitro

cytotoxic activity (Grunberger et al., 1988; Lee et al.,

2000), CAPE (10) has also been reported to decrease

tumor formation in C57BL/6J-Min/� mice bearing a

germ line mutation in the Apc gene (Mahmoud et al.,

2000). It has been reported to completely block the

activation of NF-kB by tumor necrosis factor (Natar-

ajan et al., 1996). In addition, CAPE (10) inhibited 5-

lipoxygenase and soybean 15-lipoxygenase and comple-

tely blocked the production of ROS in human neutro-

phils and in the cell-free xanthine/XOD system

(Mirzoeva et al., 1995). Thus, the antioxidative property

of CAPE (10) seemed to play an important role in

various biological systems. Benzyl caffeate (9) and

cinnamyl caffeate (11) have similar chemical structures

and possess stronger antiproliferative activities than

CAPE (10) toward all the tested cell lines. Moreover,

it is interesting to note here that both caffeates 9 and 11

showed stronger antiproliferative activities than 5-fluor-

ouracil toward murine colon 26-L5 carcinoma and B16-

BL6 melanoma and fell within the range of the potent

cytotoxic agent (EC50B4 mg/ml) made by Geran et al.

(1972). Benzyl caffeate (9) was previously isolated from

Chinese propolis and has been reported to have strong

antioxidative activity against autoxidation of methyl

linoleate (Yamauchi et al., 1992). In the present study,

we also observed an equal strength of scavenging

activities of 9�/11 toward DPPH radical to that of a-

tocopherol, a well known antioxidant. Furthermore,

these caffeates showed stronger DPPH radical scaven-

ging activities than ascorbic acid used as positive control

(Fig. 2). Thus, like CAPE (10), benzyl caffeate (9) as well

as cinnamyl caffeate (11), appeared to be potent

candidates for chemopreventive agents and their anti-

oxidative activity may be associated with their anti-

proliferative properties. Further studies on these

caffeates are in progress in our laboratory and will be

reported elsewhere.

Acknowledgements

We are thankful to Nihon Propolis Co. Ltd., Tokyo,

Japan for continues support for our propolis research.

Marieke Mutsaers, Honeybee Husbandry, the Nether-

lands is also acknowledged for supplying the Nether-

lands Propolis.

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Ethnophysiology and herbal treatments of intestinal worms inDominica, West Indies

Marsha B. Quinlan *, Robert J. Quinlan, Justin M. Nolan

Department of Anthropology, University of Missouri-Columbia, 107 Swallow Hall, Columbia, MO 65211, USA

Received 1 August 2000; received in revised form 1 December 2001; accepted 27 December 2001

Abstract

In rural Dominican ethnophysiology worms reside in a human organ called the ‘worm bag’. Unchecked, worms can cause illness

by growing in size and number, spreading out of the worm bag and into other organs. In this study of ‘bush medicine’, we use a

measure of cognitive salience in free-listing tasks, which reveals five plants commonly used to treat intestinal worms. These were

Ambrosia hispida (Asteraceae), Aristolochia trilobata (Aristlochiaceae), Chenopodium ambrosioides (Chenopodiaceae), Portulaca

oleracea (Portulacaceae), and Artemisia absinthium (Asteraceae). Bioactive compounds appear to be present in all of these plants.

The cognitive salience of these plant remedies coupled with evidence of biochemical properties suggest that they provide efficacious

treatments for controlling intestinal parasite loads. # 2002 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Ethnomedicine; Ethnobotany; Body image; Dominica; West Indies; Caribbean; Intestinal worms

1. Introduction

1.1. Scope of the paper

One common goal of ethnopharmacology and ethno-

botany is to understand how people�/plant interactions

influence the health of indigenous populations. Study of

traditional healing systems is most fruitful when it

examines local models of human anatomy and illness

alongside bioscientific perspectives (e.g. Browner and

Ortiz de Montellano, 1986; Browner et al., 1988).Similarly, researchers can better understand treatment

systems by adopting quantitative measures in tandem

with qualitative observations (e.g. Moerman, 1979,

1989; Trotter and Logan, 1986). In this paper, we

combine emic (view based on native knowledge) and

etic (scientifically grounded categories of an observer

(Winthrop, 1991)) approaches to investigate local con-

ceptions and botanical treatments of intestinal wormsamong rural Caribbean villagers.

Specifically, we offer three contributions. First, we

provide an overview of a Caribbean ethnomedical

system. Here we illustrate how Dominicans’ notions of

intestinal worms relate to the local model of health.

Second, we discuss the plants Dominicans use as worm

treatments and describe why locals consider themefficacious in controlling intestinal parasites. Last, we

illustrate the utility of an ethnoscientific technique*/the

salience index*/for identifying patterns of plant use.

1.2. Ethnopharmacological methods: why salience

matters

Indigenous people’s selection and use of plants

ultimately depends on ‘cultural constructions of effi-

cacy’ (Etkin, 1990: 28). Documenting culturally useful

plants is aided by quantitative methods to target themost efficacious species among local pharmacopoeias.

One assumption is that the most efficacious species are

well known to respondents. For example, Moerman

(1979, 1989) determined that plant species with higher

frequencies of use among culturally isolated Native

American groups generally contain higher levels of

secondary alkaloids than plants with lower frequencies

of use. In a study of Mexican�/American plant remedies,species reported by high proportions of respondents

show greater bioactivity than those reported by lower

proportions of respondents (Trotter and Logan, 1986).* Corresponding author.

E-mail address: [email protected] (M.B. Quinlan).




PII: S 0 3 7 8 - 8 7 4 1 ( 0 2 ) 0 0 0 0 2 - 8


Phillips and Gentry (1993) constructed a use value

statistic to assess the importance of a given species in

an ethnobotanical inventory. Use value of a plant is the

number of uses listed by a respondent summed anddivided by the number of interview sessions in which the

respondent provided information about the plant. Mean

use value for each plant in the inventory is the sum of

values for each plant listed by each respondent divided

by the number of respondents.

Although the approaches mentioned above are useful,

none of them takes into account the salience, or

psychological prominence, of plants listed by respon-dents. One way to determine salience of medicinal plants

is to calculate the salience of plant names respondents

give during free-listing tasks. Free-listing is an efficient

ethnographic tool used to determine the constituents of

a semantic domain (Bernard, 1994; Weller and Romney,

1988). This method is used in a number of ethnobota-

nical and ethnomedical studies to identify traditional

remedies (e.g. Crandon-Malamud, 1991; Hatfield, 1994;Trotter, 1981). Several analytical techniques can be

applied to free-list data, such as list length (Brewer,

1995) and frequency of mentions of items (Bernard,

1994; Weller and Romney, 1988). Salience, however, is

especially useful because it combines frequency of

mention with the order of mention (Smith, 1993;

Robbins and Nolan, 1997). Because respondents tend

to mention the most culturally important items first in alist, and efficacious plants are usually listed more

frequently (Trotter and Logan, 1986), salience is the

appropriate method for revealing which medicinal

plants deserve special attention in our research. Though

Martin (1995) and Cotton (1996) emphasize the im-

portance of the salience of plants in free-lists, few if any

ethnopharmacological studies use salience to determine

the likely efficacy of medicinal species. Here we demon-strate the potential of the method for ethnopharmacol-

ogy.

1.3. The study site

The Commonwealth of Dominica is a small, rural

island nation located between the French Departments

of Guadeloupe to the North and Martinique to the

South (158N, 618W). The island is relatively undeve-loped as it is extremely mountainous and withoutsubstantial agricultural or tourist industries. Dominica’spopulation of about 70 000 people is of mixed Island-Carib, African and European descent. Most Domini-cans are bilingual in Creole-English and French-Patois,though Patois is the language of folk medicine.

The study site is a village on Dominica’s East Coast

(i.e., the Atlantic, windward coast). This village isnestled at the crux of two 1400-foot mountain ridges

that slope steeply into the ocean. These ridges trap rain

blown in from the ocean. Consequently, the site gets

between 100 and 150 inches of rain per year. The

vegetation surrounding the village is mostly littoral

forest with pockets of lower montane rain forest (Beard,

1949).The village is primarily a subsistence agricultural

community with a population of about 650 full and

part-time residents. In addition to subsistence gardens at

the village periphery, most land within the village is

cultivated with fruit trees and other plantings. Many

families also maintain small house-gardens for condi-

ments (e.g. peppers) and herbs for cooking and medi-

cine.Outside economic opportunities are few. Average

annual income is approximately $5000 EC ($1850 US).

More than half of the households have electricity. None

has running water.

Remote even by Dominican standards, the village is

located about a 40-min drive from the main road, at the

end of a mostly unpaved lane. No villager owns a car.

However, regular motorized transports to Roseau, thecapital, leave the village at 05:30 h on weekday morn-

ings.

Residents have limited access to health care, in part

because of their remote location. The village shares a

small clinic with the neighboring village. According to

locals, the clinic is normally out of medications such as

analgesics and antibiotics, though it does offer some

first aid, immunizations, and medicine when available.The nearest pharmacy is 2 h away. A doctor is available

at the government health center, 45 min drive from the

village. In cases of emergencies, residents can call an

‘ambulance’*/actually a pick-up truck*/which is sent

from the health center and carries the patient back in the

truck-bed. Thus, for less urgent cases, villagers take care

of themselves with remedies made from either foraged

plants, or herbs grown around the house in smallgardens and containers. Locals call this type of medicine

‘bush medicine’ because it is based on ‘bush’ (or herbal

pharmacopoeia) and because it is the type of medicine

used by people who live ‘way out in the bush’.

2. Methods

Fieldwork for this project was conducted during fivetrips to the study site between 1993 and 1998. Ethno-

graphic data on worm treatments were collected using

informal key informant interviews (by M.B.Q. and

R.J.Q.), a village health survey, and free-list tasks

(M.B.Q.).

The informal interviews were conversational and

involved asking a representative sample of village adults

about their own experiences with worms. The healthsurvey involved asking every village mother (including

healers) a series of recall questions regarding the health

of family members. Women were asked about the

M.B. Quinlan et al. / Journal of Ethnopharmacology 80 (2002) 75�/8376

general health history and condition of all household

residents. They were asked to recall any illness or

injuries their family members had suffered in the past

week, past month, and past year. Each time a womanmentioned an illness event she was asked how the person

got sick to probe for the perceived etiology of the illness.

Women were next asked what, if anything, anyone did

to treat the sick person. If someone at home treated the

sick person (which was usually the case), the woman was

asked to describe the treatment.

The free-list interview was conducted with a quota

sample of 30 adult villagers. This is approximately onequarter of the adult population. The sample contained

five women and five men from each of the three major

village hamlets. In each hamlet, one woman interviewed

was in her twenties, one in her thirties, one in her forties,

one in her fifties, and one in her sixties or seventies. The

men were represented in the same manner. The quota

was thus stratified equally regarding age, sex, and village

location.For these interviews, we asked our respondents to list

all the names of plants that are used for treating

intestinal worms. Next, respondents were asked to

describe which part of the plant is used, how each plant

is prepared, the appropriate dosage, and the relative

efficacy of the treatment. This process of ‘successive

free-listing’ resulted in an exhaustive inventory of all

local flora known by informants for the treatment ofworms, and the corresponding mode of use for each

plant.

We determined the mean list length for all respon-

dents, the frequency of mention of each listed plant, and

the average salience value of each plant. To calculate

salience values, each listed plant was ranked according

to its order of mention, or relative list position. Next, the

ranks were summed by 30, namely, the total number ofinformants, which yielded a composite salience value for

each plant (Smith, 1993). The resulting scores revealed

which plants have the greatest cognitive and cultural

significance as botanical worm treatments among our

sample of respondents.

Key informants from the village (identified by their

reputations as plant experts and their interest in this

project) helped in the collection of voucher specimens ofeach species. The vouchers, as indicated in Table 1, are

housed in the herbarium at the University of Missouri�/

Columbia.

A literature search was conducted to identify studies

of (1) cross-cultural concepts and treatments of intest-

inal parasites; (2) pharmacological properties of the

plant species Dominicans use as worm treatments; and

(3) other populations’ use of plant species used asvermifuges in Dominica. A comprehensive electronic

search was performed in MEDLINE, AGRICOLA, and

the Social Sciences Citation Index. Search terms were

keywords: antihelminthic, intestinal parasites, vermi-

fuge, worms, and all species names in Table 1. The

electronic search was supplemented by a review of the

bibliographies of retrieved materials.


3.1. Ethnophysiology of worms

The concept of internal balance is a focus of rural

Dominican ethnophysiology. For optimal function, the

body should (1) maintain equilibrium regarding intake

of food and drink and elimination of waste, and (2)remain at a neutral, warm state of equilibrium regarding

hot and cold.

A version of the hot/cold humoral theory exists in

Dominica. Food, medicine, mental and physical states

are classified according to culturally ascribed ‘hot’ or

‘cold’ categories. The hot/cold humoral system has been

documented throughout the New World, particularly in

Latin America (for an overview, see Foster, 1994).In the Dominican humoral system, every living thing

has its own inherent degree of ‘hot’ or ‘cold’. For

example, some plants have an intrinsically ‘cold’ quality.

If a person ingests a ‘cold’ plant, as either food or

medicine, the plant will internally cool his body. Other

plants are ‘hot’. Ingesting them will heat one’s body.

Dominicans’ humoral beliefs differ from their Hispanic

neighbors in that, to Dominicans, most plants andanimals share the same humoral temperature as humans

(Foster, 1994). Most living things thus have a ‘neutral’

humoral quality. Many illnesses are also ‘neutral’ in

humoral terms because the bodily insult was created by

a condition outside of the hot/cold dimension.

A worm infestation is an example of a humorally

neutral condition. Dominicans allege that intestinal

worms, like most other animals (including humans)share the neutral humoral status. This is one reason that

humans make good hosts for the worms, as do their

dogs, cats, and livestock.

Rural Dominicans describe the human body as having

a ‘worm bag’, an organ specifically for worms. The

worms that occupy the bag come from dirt. Infants are

born with empty worm bags. They acquire their initial

worms by ingesting invisible worm eggs when they suckand chew on their dirty hands or other dirty objects. By

the time a baby is a year old, worm eggs have hatched in

his worm bag. Even a person who takes regular worm

treatments maintains a worm supply by inadvertently

ingesting small amounts of dirt (when gardening, on

food, etc.) throughout his life.

According to local ethnophysiology, after one swal-

lows food or drink, the substance passes from the mouthto the throat, behind the lungs, through the ‘throat pipe’

and down into the ‘belly’. The belly is not an organ, but

the general term for the abdominal cavity; or as one

M.B. Quinlan et al. / Journal of Ethnopharmacology 80 (2002) 75�/83 77

informant put it, ‘‘The belly is like a bag. It holds

everything under your lungs until your ass.’’ In the belly,

the throat pipe channels the food into the stomach. The

stomach is a bag that ‘pounds up all what you eat’ then,

bit by bit, squeezes portions of the food into the ‘worm

bag’.

From a Dominican viewpoint, all humans should

have some intestinal worms at all times. Humans and

worms have a symbiotic relationship. Worms function

in the digestive process to refine food, turning it into

rich blood, much the way that earthworms convert

composting material to rich soil. Rich blood that the

worms expel passes through pores in the worm bag and

into the belly. Blood collects at the bottom of the belly

in a funnel-like organ and then filters through the liver.

From the liver, good, clean blood goes to the heart, and

waste goes to the bowel.

There are some villagers who resent all worms as

continual menaces*/parasites that, despite creating

some blood, mostly rob the body of nutrition. Most

locals, however, assert that worms are generally bene-

ficial and only dangerous or painful if they grow or

multiply beyond the capacity of their worm bag. Outside

of the worm bag, they steal nutrients from the host and

begin to feed on other organs, primarily the stomach

and ‘tripe’ (intestines).People attribute several symptoms to worm infesta-

tion. One symptom is excessive gas. Worms, like

humans, produce gas as a by-product of digestion.

This gas, like blood, passes through the porous worm

bag walls and into the belly. Gas that collects in the belly

can exit upward, through the ‘throat pipe’ (throat) or

downward, through the anus, depending on which part

of the belly it was nearer to begin with. If a resident is

suffering from unusual flatulence, he suspects he has a

plethora of worms simultaneously digesting his food.

‘Gripes’ or abdominal cramps are another symptom

of worm infestation. Gripes result from too many

worms stretching the worm bag, or worms outside of

the worm bag feeding on other organs. Finally, seizures

are sometimes attributed to extreme cases of worm

infestation. Villagers call such a seizure a ‘fit’ or ‘fix’ of

worms.

Ingestion of certain substances causes an overabun-

dance of worms. Villagers suggest that dairy products,

in particular, cause worms to thrive. They point to two

phenomena as evidence for this claim. First, adults

frequently experience stomach discomfort after eating

dairy products, probably due to high levels of lactose

intolerance within the population. Second, worms most

often trouble young children who ingest plenty of milk,

and babies (under two) who drink milk almost exclu-

sively. Some residents say that sweet foods augment

worm growth resulting in ‘fits’ or ‘fixes’ of worms, which

actually may be diabetic seizures. Finally, eating dirt can

introduce more worms and cause existing worms to

grow and multiply. Hence, young children are at highest

risk for worm infestation because they ingest more milk

and tend to put dirty, possibly wormy things in their

mouths.

Locals explain that worm medicines work by either

‘killing’ the worms or making the worms ‘sleep’. Dead

and sleeping worms loose their jaws from their hosts

internal organs. Thus they can pass through the

remainder of their host’s digestive system.

The existing cross-cultural literature on the ethno-

physiology of worms is relatively scant. The Warao of

Venezuela (Wilbert, 1986), the Highland Maya of

Mexico (Berlin and Berlin, 1996), and the Luo of Kenya

(Geissler, 1998) recognize contaminated food or water

as the primary source for intestinal worms, as do

Dominicans. Geissler points out that this view may be

derived or reinforced by exposure to Western biomedi-

cine. Whereas dirt is the only perceived worm source for

Dominicans, the Warao (Wilbert, 1986) and Highland

Maya (Berlin and Berlin, 1996) also allow that worms

may appear through sorcery or spontaneously. A

Guatemalan K’ekchi Indian (Booth et al., 1993) and

rural Hondurans (Kendall et al., 1983) suggest that

worms can pass from a mother to her children in breast

milk. The Luo of Kenya (Geissler, 1998), the Manica of

Mozambique (Green et al., 1994) and rural Hondurans

(Kendall et al., 1983) suggest that some people are born

Table 1

Plants mentioned as worm treatments

Plant’s Patois name Species name Times mentioned % of informants Salience score Voucher specimen

Sime kontwa C. ambrosioides 30 100 0.912 MBQ23, MBQ122

Twef A. trilobata 27 90 0.621 MBQ14

Set vil A. hispida 22 73 0.44 MBQ37

Kupiye P. oleracea 18 60 0.266 MBQ74

Lapsent A. absinthium 12 40 0.148 MBQ129

Papay C. papaya 6 20 0.054 Sight record

Ti bom unidentified vine 3 10 0.06 MBQ71

Goamye wouj B. simaruba 3 10 0.036 MBQ56

Gwenabafei P. tenellus 3 10 0.045 MBQ48


with worms. Dominicans say that everyone (except

newborns) always has some intestinal worms, and that

worm presence alone does not indicate illness. Similar

views are found among Jamaicans (Sobo, 1993), andseveral African (see Geissler, 1998) and Latin American

peoples (Kendall et al., 1984). Kendall et al., (1983)

describe a remarkably similar notion of worms to that of

Dominicans. In rural Honduras (Kendall et al., 1983),

worms are said to live in a worm bag, or bolsa , where

they aid in digestion. Notions of human-worm symbio-

sis also occur in various African groups (Bierlich, 1995;

Geissler, 1998; Green, 1997; Zondi and Kvalsvig, 1996).In some Latin American populations, a human’s soul

allegedly resides inside one intestinal worm*/the

mother worm (Kendall et al., 1984; Booth et al.,

1993). People in these societies attempt to control the

offspring worms while not harming the mother. The

Ronga of Mozambique (Green, 1997) and the Kenyan

Luo (Geissler, 1998) maintain that worms react nega-

tively to ‘bad’ foods (which Geissler explains may berotten, taboo, or cursed). The Dominican view that

worms react to sweet food is mirrored by the Venezue-

lan Warao who associate worm attacks with eating

brown sugar (Wilbert, 1986). The Warao further attri-

bute adult tooth decay to worms. Dominican villagers

assert that consumption of sweets can trigger the

seizures or convulsions they call ‘fixes of worms’. In a

similar vein, Geissler (1998) found that Kenyan Luoattribute vomiting and convulsions (possibly caused by

malaria) to notions of internal worm migrations.

3.2. Ethnopharmacology of worms

Residents of the study site have a number of plants at

their disposal for treatment of worm infestation (Table

1). In this section, we review the most salient plant

species, their local preparation, and what is known fromthe literature of their chemical properties, geographical

distribution, and uses in other localities.

3.2.1. Sime kontwa

Sime kontwa (Chenopodium ambrosioides L., Cheno-

podiaceae) had the highest salience value in our study

(Table 1). Every informant included it in his or her free-

list, and most people (73%) mentioned sime kontwa first.Some English common names for this species are ‘worm

grass’, ‘Mexican tea’, and ‘fit weed’ (Simpson, 1962). In

the French-speaking Caribbean, it is called herbe a vers ,

or simen contra . Dominicans refer to it as sime kontwa

in Patois and English. Sime kontwa is a pantropical

(Nicholson, 1991), aromatic herb that occurs in the

forest and in cultivated zones throughout the study area.

However, residents often transplant bushes to their yardor house garden.

Villagers drink a ‘bush tea’ of sime kontwa fairly

regularly. They make the infusion by breaking and

dropping a branch about 25 cm long (this typically

contains about 15�/20 leaves), complete with twigs,

leaves and flowers, into approximately one l of boiling

water. They boil the herb until the water has achartreuse tint to it. This takes less than one min. Half

of the mothers in our study gave a small cup of sime

kontwa ‘tea’ to their children every morning (the

amount of liquid depends on the body size of the child).

Others prefer to give it once a week or so. More than

half of the informants could not say exactly how often

they drink an infusion of sime kontwa . They say they

drink it from time to time, sometimes in the morning,sometimes to relax in the afternoon. If they feel a little

stomach discomfort, or if they suspect that their worms

are troubling them, they make themselves a cup of sime

kontwa ‘bush tea’. A number of people related that they

drink sime kontwa ‘tea’ until the bush by their house is

nearly branchless. Then they drink other ‘bush teas’

while their plant rejuvenates. People who drink the

infusion regularly say that they never (or rarely) haveworms in their stool because the drink ‘keeps the worms

down’ constantly. People who only drink sime kontwa

when they feel worm symptoms say that dead and

‘sleeping’ worms come out with their feces after one or

two days.

Oil of chenopodium, which is distilled from C.

ambrosioides is a pharmaceutical product used against

tapeworms, roundworms, and hookworms. The activecomponent, terpene ascaridole, makes up about 65% of

the oil (Lewis and Elvin-Lewis, 1977). The whole plant

(Kapadia et al., 1978) and the extracted oil of cheno-

podium (El-Mofty et al., 1992) are carcinogenic to

animals.

This species, C. ambrosioides , is used to treat intest-

inal worms throughout the Caribbean (Honychurch,

1986). It is also a vermifuge in Mexico (Berlin andBerlin, 1996), Guatemala (Booth et al., 1993) and South

America (Kainer and Duryea, 1992; Schultes and

Raffauf, 1990).

3.2.2. Twef

The Patois twef, Aristolochia trilobata L. (Aristolo-

chaceae), is the second highest ranked worm remedy in

the community. Ninety-three percent of our informants

mentioned twef , and 20% mentioned it first. This vine isnative to Dominica (Nicholson, 1991) and grows in

bushy coastal areas and secondary forests throughout

the Caribbean and Southern Central America (Hony-

church, 1986). It climbs up trees in the Littoral Forest

around the study site. Villagers transplant the vines to

their yards where they typically grow up the sides of

homes and outhouses.

Villagers use twef for worms and to settle upsetstomachs, in the form of a leaf infusion. Village rum

shops also sell shots (or ‘shoots’) of ‘twef rum’ (a

tincture of approximately four twef leaves soaked in a


fifth of cask rum). Many men in the village say they

prefer to take their twef with rum. For the infusion,

villagers boil about half a twef leaf per cup of water for

a few minutes (until the water changes color), then let

the leaves steep in the water while the tea cools. Adults

drink one cup of the infusion. Children take less,

according to their body size. Villagers say that one

dose of twef (cup of the infusion or shot of the rum

decoction) usually kills all the worms that are ‘outside’

of the worm bag, although people who suspect they have

a particularly bad infestation may take a second dose on

the following day. Worms are said to appear in the feces

within one and a half days of the first dose. Children

under 5 years rarely drink twef because parents prefer to

give them something milder. Villagers say that one ‘must

add plenty of sugar’ to this tea and let it cool for a long

time. This way one can drink it quickly, ‘like a shot of

rum’. They say that even this way, twef tastes terrible.

Indeed, twef ‘bush tea’ is so awful that parents force

their children to drink it as a punishment for speaking

rudely.

Several villagers noted that this worm remedy worked

at least as well*/in fact more quickly*/than other

worm remedies. However, they prefer not to use it

because of its bitter taste. In addition, they say that

something that tastes so bad and works so quickly is

obviously very strong. They believe twef might make

one weak if taken often.

Aristolochic acids in this species are renal toxins in

animals and humans (Lajide et al., 1993). Aristolochic

acids extracted from Aristolochi albida (a Nigerian

species) shows ‘extremely strong’ feeding-deterrent and

larval growth-inhibition activity in bioassays (Lajide et

al., 1993). This lends credence to their potential efficacy

against parasites. However, aristolochic acids are carci-

nogenic, causing stomach cancers, and atrophy in the

lymph glands (DeSmet, 1992). For that reason, Aris-

tolochia species are banned in European medicines

because of the dangers associated with aristolochic acids

(Zhu and Phillipson, 1996).

Nicaraguan Miskitu and Ulwa Indians use A. trilo-

bata for digestive problems, as well as for bites,

respiratory disorders, and as a general tonic (Coe and

Anderson, 1999). On the island of Marie Galante, twef

is used as an emetic in case of poisoning (Honychurch,

1986). In Trinidad, this vine is used for diabetes,

hypertension, dysmenorrhea, as an abortifacient, and

for snakebites (Wong, 1976). It is also used for

snakebites in tropical South America (Millspaugh,

1892). In fact, almost all of the species in this genus

are used in the countries in which they grow as remedies

for poisonous snakebites. There are no poisonous

snakes in Dominica, however (Coborn, 1991).

3.2.3. Set vil

Set vil , or Ambrosia hispida Pursh (Asteraceae), is the

third most salient worm treatment in the present study.

Seventy-three percent of the informants mentioned it,but none mentioned it first. Common English names for

set vil are ‘worm wood’, ‘ragweed’, and ‘bay geranium’.

This perennial herb is native to Florida, the West Indies

and Central America (Hodge and Taylor, 1957), and is a

fairly common weed in the village. It grows wild in

sandy, disturbed areas and is common along sunny

roads and trailsides.

Other than as a worm treatment, residents use set vil

as a poultice for muscle strains, sprains and bruises. For

this purpose, they crush the whole herb and mix it with

water or, preferably, castor oil, to make a paste. For

worms, villagers steep the leaves and inflorescence of

one branch in 2�/3 cups of boiled water to make a ‘bush

tea’. They drink this infusion once a day for 2�/4 days,

until they no longer ‘feel cramps around the worm bag’,

or until they see worms in their feces.Although, this species tested positive for alkaloids

(Raffauf, 1996), little is known of its chemical proper-

ties. People in Marie Galante drink set vil tea for high

blood pressure (Honychurch, 1986). Island Caribs

(Hodge and Taylor, 1957) and Rastafarians (Caesar,

1997) elsewhere on Dominica also use this herb for

worms.

3.2.4. Kupiye

In the present study, kupiye (Portulaca oleracea L.,

Portulacaceae) was the fourth most salient herbal

treatment. Sixty percent of our informants mentioned

it, but no one mentioned it first. Kupiye (purslane or

portulaca in common English) generally goes by the

name pussly throughout the English speaking Carib-

bean (Honychurch, 1986; Wong, 1976; Ausprey and

Thornton, 1953). Dominicans, however, use the FrenchPatois name kupiye . This creeping succulent herb grows

as a weed worldwide. In the village, it typically grows

under the edge of stilted houses, by paving stones and in

and along stone retaining walls.

Dominicans eat kupiye raw as salad or cooked in

soups and stews. They also crush the plant to make a

poultice for backaches and dysmenorrhea. More com-

monly, Dominicans steep a crushed handful of the plantin two or three cups of boiled water to make a tea for

worms.

Our informants assert that this medicine works well as

a vermifuge. After eating kupiye or drinking kupiye tea

for two or three days, they claim that one begins to see

worms in the feces. They do not believe that kupiye is a

very strong medicine; hence, they consider it a good

treatment for babies with worms. Because kupiye is amild medicine, villagers suggest that it is safe to eat

between other types of worm treatments to keep the

worms in check or ‘calm’.


Kupiye is one of only seven species in the Portulaca-

ceae that tests positive for alkaloids (Raffauf, 1996).

Chemical analyses of purslane show that the methanol

extract of the plant’s non-root parts contain portulo-side-A (a monoterpene glucoside) which exhibits mod-

erate antimicrobial activity (Sakai et al., 1996). Kupiye

also has a high concentration of oxalate, which results in

oxalate toxicity when it is fed to animals. Oxalate

interferes with the absorption and metabolism of trace

minerals (Mohamed and Hussein, 1994). In addition,

kupiye is a nutritious vegetable, high in omega-3 fatty

acids, beta-carotene (Kesden et al., 1987) and potassium(Mohamed and Hussein, 1994).

The chemical properties of kupiye may account for its

use as a vermifuge in geographically distant regions. For

example, this species was used to kill parasites in

Ancient Greece (Kesden et al., 1987). Present day

populations in Trinidad use it to treat intestinal worms

(Wong, 1976), and the kupiye ’s seeds are used as a

vermifuge in Java (Burkill, 1966).

3.2.5. Lapsent

Lapsent (Artemisia absinthium L., Asteraceae)

showed marginal salience in this study. Twelve people

(40%) mentioned the plant; however, they consistently

mentioned it near the end of their free-lists. This plant

goes by the common names ‘wormwood’ (English), and

‘absinthe’ (French). Dominicans use the Patois name

‘lapsent’ . This introduced native Mediterranean peren-nial (Millspaugh, 1892) does not thrive in the study site.

As far as we are aware, there is only one lapsent plant in

the village, growing in a container near one family’s

house garden.

Residents have access to this treatment only because a

few individuals and village rum shop owners buy dried

lapsent in Roseau, the capital. They soak the lapsent in

rum. Some adults take a shot of lapsent tincture aboutonce a month as a worm preventative.

People of this village report that lapsent is an excellent

vermifuge, along the lines of sime kontwa and twef .

They like to use lapsent because they enjoy its licorice-

like flavor. However, lapsent is more difficult to obtain

and reportedly works no better than native plants that

grow in abundance around the village.

The plant is an effective vermifuge that was probablyused by colonial Europeans in the Caribbean. That this

herb is present in Dominica is not surprising. France

was once the leading producer of absinthe liquor, and

the last country to ban it in 1915 (Conrad, 1988).

Dominica is a former French colony and lies between

two French islands.

In Western Civilization, A. absinthium has been

renowned as antihelmintic since ancient times. Conrad(1988) found that references to this plant appear in

Egyptian papyri, early Syrian texts, and in the Bible.

Early European texts refer to applying decoctions of A.

absinthium externally for fleas and other vermin, and to

drinking the decoctions as a tonic and vermifuge

(Conrad, 1988). The herb is now widely used as a

vermifuge. For example, it is used among peoples ofArgentina (Spegazzini and Najera, 1984), British Co-

lumbia (Turner et al., 1980), and Kashmir (Singh, 1994).

3.2.6. Less salient treatments

In addition to the five species mentioned above,

Dominicans in the study population mentioned four

other plants. These are papay (Carica papaya L.,

Caricaceae), mentioned by six people; ti bom (unidenti-

fied species), mentioned by three people; goamiye wouj

(Bursea simaruba Sargent, Burseraceae), mentioned by

three people; and gwenabafei (Phyllanthus tenellus

Roxburgh, Euphorbiaceae) also mentioned by three

people.

Villagers stated that they chew the seeds of ripe

papay , or papaya fruit, for worms. Laboratory screen-

ing found these seeds to exhibit antiamoebic activity

(Tona et al., 1998). They may be effective against otherparasites.

The sap of the bwa goamiye wouj (Patois), or ‘worm

gum tree’ (English) has a smell reminiscent of turpen-

tine. One elderly woman explained that soaking a 2 cm

ball of congealed sap overnight in about 70 ml of cask

rum, then drinking the rum first thing in the morning

before eating or drinking anything else, will ‘kill plenty

of worms immediately’. Then, subsequent food anddrink will help push the dead worms through the worm

bag. People in Montserrat drink a similar tincture ‘to

ease stomach distress’ (Brussell 1997).

The people who mentioned using ti bom and gwena-

bafei said that they made ‘bush teas’ with the herbs by

steeping one handful of fresh plant (all parts but the

roots) in two cupfuls of hot water.

4. Conclusions

The potential antihelmintic properties of salient

Dominican worm treatments suggest a methodological

approach for medical ethnobotanists interested in iden-

tifying bioactive plants. Researchers may find it expe-

dient to identify long-term endemic maladiesaccompanied by well developed local conceptions of

physiologic processes. These are culturally and envir-

onmentally prominent illnesses of a population*/con-

ditions for which local people need effective treatments.

Culturally significant plant species may be more

efficacious than species collected randomly (Balick and

Cox, 1996; Khafagi and Dewedar, 2000). Because of

this, we suggest a two-step research technique. First,investigators should identify illnesses of community

concern. Then, free-lists can be used to find salient

medicinals. Salient treatments are those with the most


community consensus, and are therefore the plants with

the most cultural significance.

In this study, the five most salient worm treatments all

have chemical properties that may make them effectivevermifuges. Two of these species, A. trilobata and P.

oleracea (the second and fourth most salient species)

have not been documented as worm medicines until

now. We found no references to possible chemical

antihelmintics in the third most salient plant, A. hispida .

These lesser-studied treatments merit scientific consid-

eration as vermifuges for two reasons. First, this

population has a multi-generational history of relianceon these plants as worm treatments. Second, Domini-

cans regard A. trilobata , A. hispida and P. oleracea as

equally effective as C. ambrosioides and A. absinthium ,

two of the most common vermifuges in the ethnobota-

nical record.

Acknowledgements

We wish to thank the following people: Mark Flinn,

for introduction and project support in the study site;

Deborah Pearsall, Ruthbeth Finerman, and three anon-

ymous manuscript reviewers, for valuable comments.We thank villagers of the study community, especially

Edith Coipel, Juranie Durand, and the Warrington

family. Prior to interviews, individuals were informed

on the nature of this research and gave their verbal

consent to partake. Each participant was paid $1 EC for

his free-list.

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Pharmacological effects of lavandulifolioside from Leonurus cardiaca

Katarzyna Mil kowska-Leyck b, Barbara Filipek a,*, Halina Strzelecka b

a Laboratory of Pharmacological Screening, Faculty of Pharmacy, Jagiellonian University, 9 Medyczna St., 30-688 Krakow, Polandb Department of Pharmacognosy, Faculty of Pharmacy, Medical Academia, Warsaw 02097, Poland

Accepted 2 January 2002

Abstract

Lavandulifolioside was detected for the first time in Leonurus cardiaca var. vulgaris [Moench] Briquet (Lamiaceae ). The isolation

was performed from the butanolic extract of the aerial parts and the identification by NMR and MS. The pharmacological

properties of lavandulifolioside consist of significant negative chronotropism, prolongation of the P�/Q, Q�/T intervals and QRS

complex, and decrease of blood pressure. Contrary to the butanolic extract lavandulifolioside did not reduce the spontaneous

locomotor activity. In conclusion, the pharmacological pattern of lavandulifolioside did not explain the pharmacological effects of

L. cardiaca L. alone. # 2002 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Leonurus cardiaca var. vulgaris ; Lavandulifolioside; Isolated rat heart; Hypotensive activity; Locomotor activity

1. Introduction

Leonurus cardiaca L. has been used in the traditional

medicine against nervous and functional cardiac dis-

orders since the 15th century and now is described in

pharmacopoeias (DAB, 1999; Russian Pharmacopoeia,

1968; BHP, 1992) for producing sedative, hypotensive

and cardiotonic pharmacological effects. Indications are

for the treatment of neuropathic and functional cardiac

disorders. Many chemical constituents of the plant

(alkaloids, iridoids, saponins, flavonoids, cardenolid-

like glycosides and diterpenoids) have been detected and

isolated, but their relevance for medical use has not been

investigated completely.

Phenylpropanoid glycosides have been detected in

Leonurus glaucescens (Calis et al., 1991) and their

pharmacological activities have been shown (Jimenez

and Riguera, 1994). Among them verbascoside seems to

play a dominant role concerning pharmacological

activity and occurrence. We now report the isolation

and the pharmacological activity of lavandulifolioside, a

phenylpropanoid glycoside with putative pharmacolo-

gical activity from L. cardiaca L.


2.1. Plant material

L. cardiaca var. vulgaris [Moench] Briquet (Lamia-

ceae ) was collected in July 1997 near StarogardGdanski, Poland. The plant orginated from controlled

tillages; seeds for cultivations were delivered by Profes-

sor Dr Hab. Kozlowski from the Institute of Plants and

Herbal Products in Poznan, Poland. Voucher specimen

is deposited at the Herbarium of Pharmacognosy

Department, Faculty of Pharmacy, Medical Academia,

Warsaw, Poland.

2.2. Extraction and isolation

The air-dried aerial parts of the plant (2.5 kg) were

extracted exhaustively with chloroform and methanol.

The methanol extract was evaporated to dryness,

dissolved in H2O and fractionated into ethyl ether, ethyl

acetate and n -butanol. The butanol extract was chro-

matographed over polyamide eluting with H2O, fol-

lowed by increasing concentrations of methanol to yield

fraction A (H2O), B (10% MeOH), C (20% MeOH), D(30% MeOH). Fraktion D was chromatographed over

silica gel 60 eluting with CH3Cl�/MeOH�/H2O (40:10:1)

followed by CC on silica gel 60 with EtOAc�/MeOH�/

* Corresponding author. Tel.: �48-12-658-8224; fax: �48-12-657-

0262.

E-mail address: [email protected] (B. Filipek).




PII: S 0 3 7 8 - 8 7 4 1 ( 0 2 ) 0 0 0 1 6 - 8


H2O (100:5:4) to give 2.45 g compound 1 (lavandulifo-

lioside).

2.3. Identification of compound 1

1H and 13C NMR spectra were recorded on a Bruker

AMX spectrometer at 500.13 and 127.75 MHz, respec-

tively. The 2D NMR experiments: COSY, 1H�/13C

HETCOR and HMBC were performed using standard

Bruker software. LSI MS spectra were measured on a

AMD-604 spectrometer. 1� lavandulifolioside (Fig. 1)was identified by comparison of their spectral data with

the literature (Basaran et al., 1988; Calis et al., 1991).

2.4. Animals

The experiments were carried out on male Albino-

Swiss mice (body weight 18�/26 g) and male Wistar rats

(180�/250 g). The animals were housed in temperaturecontrolled rooms on a 12-h light cycle at 22�/24 8C and50�/60% humidity. Animals were fed a standard pelletdiet with free access to tap water. Control and experi-mental groups consisted of six to eight animals each.


The statistical significance was calculated using a one-

way ANOVA or Student’s t -test. Differences were

considered significant when P B0.05.

2.6. Non-working heart perfusion

Heart from thiopental-anesthetized (50 mg/kg, i.p.)rat was perfused according to the Langendorff techni-

que at constant pressure of 70 cm H2O (6.87 kPa) with

Chenoweth�/Koelle solution of the following composi-

tion (mmol/l): NaCl (120.0), KCl (5.6), MgCl2 (2.2),

NaHCO3 (19.0), CaCl2 (2.4), and glucose (10.0) and

continuously gassed with 95% O2 plus 5% CO2. The

effect of the investigated BuOH extract and lavanduli-

folioside at a dose-range of 1�/2000 mg on the coronaryflow (cardiac effluent), heart rate and electrocardiogram

(obtained by two stainless steel electrodes, one inserted

into the muscle of the ventricular wall and another

attached to the metal aortic cannula) was assessed after

15�/20 min of initial stabilisation (Langendorff, 1895).

2.7. Influence on blood pressure

Male Wistar normotensive rats were anesthetized with

thiopental (50�/75 mg/kg) by intraperitoneal injection.

The right carotid artery was cannulated with polyethy-

lene tubing filled with heparin in saline to facilitate

pressure measurements using a Datamax apparatus

(Columbus Instruments). Lavandulifolioside was in-jected in dose corresponding to 1/10�/1/20 LD50 iv into

the cadual vein after a 5-min stabilisation period in a

volume equivalent to 1 ml/kg.

2.8. Locomotor activity

The BuOH extract and lavandulifolioside were given

intragastrically in doses of 800 and 1600 mg/kg to mice.

Thirty minutes later the animals were placed in cageswith a photocell to register the numbers of movements

of the animals during the first 30 min. Control animals

received the equivalent volume of solvent.

2.9. Acute toxicity

The BuOH extract and lavandulifolioside, dissolved

in 0.9% saline and suspendend in 0.5% methylcelullose,respectively, were injected intravenously or given in-

tragastrically, in a volume of 10 ml/kg, into the mouse

caudal vein. Each dose was given to six animals. The

number of dead mice was assessed 24 h after the

injection. LD50 were calculated according to the method

of Litchfield and Wilcoxon (1949).

3. Results

3.1. The effect on the isolated rat heart

BuOH extract from L. cardiaca , given at doses of 1�/

20 mg, did not change the heart rate significantly (Table

1). Higher doses (50�/2000 mg) reduced the heart rate

significantly by 25�/49%. The decrease in the heart rateobserved after test extract had the character of sinus

bradycardia with a marked prolongation of the P�/Q

(31�/78%), and Q�/T (12�/29%) intervals. Doses above

200 mg also produced a prolongation of the QRS

complex (22�/52%). The BuOH extract of L. cardiaca

at doses of 100�/2000 mg decreased significantly the

coronary outflow by 21�/35%.

Table 2 summarizes the effects of lavandulifoliosideon the heart rate, ECG parameters and coronary

outflow. The test compound given at doses of 1�/100

mg non significantly decreased the heart rate (by 4�/Fig. 1. Lavandulifolioside (1).

K. Mil kowska-Leyck et al. / Journal of Ethnopharmacology 80 (2002) 85�/9086

Table 1

Effects of BuOH extract from L. cardiaca var. vulgaris on heart rate and ECG intervals in isolated non-working rat hearts

Parameters Dose (mg)

0 1 5 10 20 50 100 200 500 1000 2000

Heart rate (min�1) 183.7910.5 179.899.8 177.1910.2 165.398.7 160.0912.2 137.6910.5* 126.697.1** 112.996.3** 109.4912.2*** 100.999.7**** 93.897.4****

Coronary outflow (ml/min) 4.890.5 4.490.6 4.390.3 4.090.8 3.990.3 3.890.8* 3.690.6* 3.590.9** 3.590.5** 3.490.4** 3.190.7***

P�/Q (ms) 53.693.7 55.692.6 55.794.3 60.496.7 65.793.9 70.596.3 75.095.4* 78.293.6** 86.594.8*** 89.096.6*** 95.697.5****

QRS (ms) 22.693.0 22.693.0 23.095.2 23.696.8 24.093.5 24.594.9 25.294.9 27.693.0* 28.095.7** 29.294.0** 34.396.7**

Q�/T (ms) 159.093.2 161.795.9 171.693.5 173.594.3 175.699.6 178.699.3* 180.298.1** 186.797.8** 188.294.7** 195.696.4*** 205.397.9**

The data are expressed as mean9S.E.M. (n�6). Statistical analyses were performed using a one-way ANOVA test.* P B0.05.** P B0.02.*** P B0.01.**** P B0.001.

K.

Mil k

ow

ska

-Ley

cket

al.

/J

ou

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lo

fE

thn

op

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rmaco

log

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0(

20

02

)8

5�

/90

87

Table 2

Effects of lavandulifolioside on heart rate and ECG intervals in isolated non-working rat hearts

Parameters Dose (mg)

0 1 5 10 20 50 100 200 500 1000 2000

Heart rate (min�1) 230.798.2 221.696.7 214.398.5 206.494.9 198.6910.0 190.4911.3 186.797.8 179.496.4* 176.499.8** 170.7910.3*** 163.898.6***

Coronary outflow (ml/min) 5.390.8 5.290.6 4.990.5 4.790.6 4.790.9 4.590.7 4.190.5 3.690.8** 3.590.9** 3.190.4*** 3.190.6***

P�/Q (ms) 50.694.9 50.792.6 56.795.8 58.698.5 58.793.9 60.597.3 65.095.8 68.293.2* 73.495.3** 79.097.6** 79.496.2**

QRS (ms) 19.895.0 19.895.0 20.797.6 23.494.9 24.097.5 24.598.8 25.199.2 27.796.0* 28.397.2* 29.795.6** 32.195.9**

Q�/T (ms) 139.794.3 151.297.5 164.296.2 169.295.7 170.898.9 172.1910.3 174.296.8 178.895.3* 181.596.7** 180.697.7** 186.498.2**

The data are expressed as mean9S.E.M. (n�6). Statistical analyses were performed using a one-way ANOVA test.* P B0.05.** P B0.02.*** P B0.01.

K.

Mil k

ow

ska

-Ley

cket

al.

/J

ou

rna

lo

fE

thn

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0(

20

02

)8

5�

/90

88

19%), prolonged P�/Q, Q�/T intervals and QRS complex

(by 27�/28%) or reduced the coronary outflow (by 2�/

23%). Given at doses of 200�/2000 mg, lavandulifolioside

significantly decreased the number of cardiac beats perminute (23�/29%), produced a prolongation of P�/Q

(35�/57%), Q�/T (28�/33%) intervals, increased in the

QRS duration (40�/62%) and depressed the coronary

outflow (32�/41%).

3.2. Influence on the blood pressure

Lavandulifolioside administered at a single dose of 50mg/kg (1/20 LD50 iv) did not affect the pulse rate or

arterial blood pressure in normotensive anaesthetised

rats. An intravenous injection of 77 mg/kg (1/13 LD50 iv)

of the investigated compound produced a significant

decrease in systolic (by 13�/15%) and diastolic (by 16�/

19%) blood pressure in thiopental anaesthetised rats.

The duration of action was more than 60 min (Fig. 2).

3.3. Locomotor activity

The BuOH extract and lavandulifolioside, given

intragastrically at doses of 800 and 1600 mg/kg slightly,

but not significantly, suppressed the spontaneous loco-

motor activity of mice during the 30-min observation

period (12�/23%), while BuOH extract of L. cardiaca ata dose of 800 mg/kg depressed the spontaneous loco-

motor activity in mice by 65% (P B0.01) (Fig. 3).

3.4. Acute toxicity

Lavandulifolioside given intravenously showed mod-

erate toxicity (LD50 approximately 1000 mg/kg), whilethe toxicity of BuOH extract from L. cardiaca was

higher (LD50 400 mg/kg). Administered orally, both test

samples were less toxic (LD50�2000 mg/kg).

4. Discussion

Recently we described that aqueous extract from L.

cardiaca diminished the frequency of the isolated rat

heart, prolonged the P�/Q intervals, reduced the cor-

onary outflow and spontaneous locomotor activity in

mice (Henneberg et al., 1995, 1996, 1997).

In continuation of our investigation we now report on

the phytochemical and pharmacological screening of the

n -butanol extract (n -BuOH extract) from L. cardiaca .

The conducted preliminary studies showed that the

investigated extract reduced the heart rate, lengthened

the time of duration of P�/Q, Q�/T intervals and QRS

complex, and diminished the coronary outflow. Given

intragastrically, the BuOH extract decreased signifi-

cantly the spontaneous locomotor activity in mice.

Our results indicate that BuOH extract, similar to

aqueous extract, induced a negative inotropic effect,

prolonged the P�/Q interval and possessed potent

sedative properties. Contrary to an aqueous extract,

Fig. 2. Influence of lavandulifolioside (i.v.) on the blood pressure.

Fig. 3. Influence of test compounds on the locomotor activity.

K. Mil kowska-Leyck et al. / Journal of Ethnopharmacology 80 (2002) 85�/90 89

the investigated extract induced a dose dependent

prolongation of Q�/T intervals and caused a significant

widening of the QRS.

It is known from literature that L. cardiaca containsbufadienolic glycosides, which are supposed to possess

cardiotonic activity (Schulz and Haack, 1961). Usually

cardiac glycosides cause a dose-dependent fall in heart

rate, prolong P�/Q interval, and shorten Q�/T interval

(Hahn et al., 1982). The electrocardiographic changes

observed after BuOH extract administration were com-

parable to those reported for quinidine (Sakai and

Shiraki, 1977), but not for cardiac glycosides. Ourresults suggest that the BuOH extract contains yet

some not well known constituents, which mediated a

significant widening of the QRS complex with a

concomitant prolongation of the Q�/T interval.

On the basis of these results, we isolated a compound

from the BuOH extract that was identified as lavandu-

lofolioside (Fig. 1). Lavanduliofolioside has been de-

tected for the first time in this plant.Lavanduliofolioside was tested separately for its effect

on isolated rat heart, blood pressure and spontaneous

locomotor activity in mice. Lavanduliofolioside, con-

trary to verbascoside, mediated an inhibitory effect with

significant negative chronotropism and coronary out-

flow in the Langendorff rat heart. Moreover, it was

interesting to observe that this compound prolonged the

P�/Q, Q�/T intervals and QRS complex. The resultsobtained on isolated rat hearts with lavanduliofolioside

were similar to geniposidic acids, the active compound

isolated lately from methanolic extract of Eremophila

longifolia (Pennacio et al., 1996a,b). In this report we

also demonstrate that the compound showed a weak

hypotensive effect. This compound at higher dose

decreased systolic, diastolic and mean arterial blood

pressure (Fig. 2). The hypotensive effect was slightlyweaker than that of verbascoside (Ahmad et al., 1995).

The mechanism of hypotensive action of lavanduliofo-

lioside is not yet clear and demand further studies. In

contrast to butanolic and aqueous extract from L.

cardiaca , lavanduliofolioside did not reduce the sponta-

neous locomotor activity in mice (Fig. 3). Therefore, we

conclude that the pharmacological pattern of L. cardi-

aca cannot be explained by lavanduliofolioside alone,and that further studies should be undertaken.

Acknowledgements

The authors wish to thank Dr I. Calis (Department of

Pharmacognosy, Faculty of Pharmacy, Hacettepe Uni-

versity, Turkey) for a sample of lavandulifolioside, DrW. Daniewski (Institute of Organic Chemistry, Polish

Academy of Sciences, Warsaw, Poland) for NMR LSI-

MS measurements.

References

Ahmad, M., Rizwani, G.H., Aftab, K., Ahmad, V.U., Gilani, A.H.,

Ahmad, S.P. 1995. Acteoside: a new antihypertensive drug.

Phytotherapy Research 9, 525�/527.

Basaran, A.A., Calis, I., Anklin, C., Nisibe, S., Sticher, O. 1988.

Lavandulifolioside: a new phenylpropanoid glycoside from Stachys

lavandulifolia . Helvetica Chimica Acta 71, 1483�/1490.

British Herbal Pharmacopoeia Compendium (BHP), 1992. BHMA

Megaron Press, Bournemouth, vol. 1, p. 161.

Calis, I., Ersoz, T., Tasdemir, D., Ruedi, P. 1991. Two phenylpropa-

noids from Leonurus glaucescens . Phytochemistry 31, 357�/359.

Kommentar zum DAB 1999/2. Nachtrag (3. Lieferung), H16.

Deutscher Apotheker Verlag, Stuttgart, Govi-Verlag GmbH,

Frankfurt.

Hahn, A.B., Barkin, R., Oestreich, S.J.K. 1982. Cardiac drugs. In:

Manning, T.A. (Ed.), Pharmacology in nursing. Mosby Company,

St. Louis, MO, pp. 568�/604.

Henneberg, M., Kowalski, J., Stasiulewicz, M., Filipek, B., Czarnecki,

R., 1995. Preliminary ethnopharmacological study of some native

drugs of Lithuania. XII Congress of the Polish Pharmacological

Society, Bydgoszcz, 17�/20 September, p. 71.

Henneberg, M., Stasiulewicz, M., Kowalski, J., Filipek, B., Sapa, J.,

Librowski, T., Zduleczna, R., Radzikowski, C., Opolska, A.,

Piasek, A., 1996. Cardiotonic, psychotropic and cytotoxic activities

of some native plants used in ethnopharmacology of Vilnius

Region, 3rd Colloquio Europeo di Etnofarmacologia, 1st Confer-

enza Internazionale di Antropologia e Storia della Salute e delle

Malattie, Genova, Italia 20 maggio-2giugno, p. 264.

Henneberg, M., Stasiulewicz, M., Kowalski, J., Filipek, B., Sapa, J.,

Librowski, T., Zduleczna, R., Opolski, A., Radzikowski, C.,

Piasek, A., 1997. Cardiotonic, psycho-neurotropic and cytotoxic

activities of some native plants used in ethnopharmacology of

Vilnius region (Lithuania), Etnofarmacologia/Ethnopharmacol-

ogy, vol. 3, December 1997/January 1998.

Jimenez, C., Riguera, R. 1994. Phenylethanoid glycosides in plants:

structure and biological activity. Natural Products Reports 12, 591.

Langendorff, O. 1895. Untersuchungen am Uberlebenden Saugethier-

herzen. Pflugers Archives 61, 291�/332.

Litchfield, J.T., Wilcoxon, F. 1949. A simplified method of evaluating

dose-effect experiments. Journal of Pharmacology and Experimen-

tal Therapeutics 96, 99�/113.

Pennacio, M., Syah Yana, M., Ghisalberti, E.L., Alexander, E. 1996.

Cardioactive compounds from Eremophila species. Journal of


Pennacio, M., Alexander, E., Syah Yana, M., Ghisalberti, E.L. 1996.

The effect of verbascoside on cyclic 3?,5?-adenosine monopho-

sphate levels in isolated rat heart. European Journal of Pharma-

cology 305, 169�/171.

Russian Pharmacopoeia, 1968. Gosudarstwiennaja Farmakopea SSSR

(X), Medgiz, Moskwa, pp. 347�/348.

Sakai, K., Shiraki, Y. 1977. A device for recording left ventricular

contraction and electrocardiogram in non-working isolated per-

fused rat heart. The Japanese Journal of Pharmacology 28, 223�/

229.

Schulz, O.E., Haack, H.J., 1961. Isolierung und Versuche zur

Konstitutionsaufklarung von drei Bitterstoffglykosiden aus Leo-

nurus cardiaca . Arzneimittel Forschung 11, 830�/835 and 975�/978.

K. Mil kowska-Leyck et al. / Journal of Ethnopharmacology 80 (2002) 85�/9090

Short communication

Isolation and identification of antibacterial compounds fromVernonia colorata leaves

T. Rabe a, D. Mullholland b, J. van Staden a,*a Research Centre for Plant Growth and Development, School of Botany and Zoology, University of Natal Pietermaritzburg, Private Bag X01,

Scottsville 3209, South Africab Natural Products Research Group, School of Pure and Applied Chemistry, University of Natal, Durban 4041, South Africa

Received 4 June 2001; received in revised form 30 November 2001; accepted 17 December 2001

Abstract

Vernonia colorata (Compositae) is used throughout Africa for a variety of ailments. This prompted the screening of this plant for

biological activity. Previous experiments carried out in our laboratory revealed that the leaves possessed high antibacterial activity.

Through conventional chromatographic techniques and bioassay-guided fractionation, the following sesquiterpene lactones were

isolated and identified by spectroscopic data; vernolide (1), 11b, 13-dihydrovernolide (2) and vernodalin (3). Only 2 is a novel

compound, although its antibacterial activity is low compared to compounds 1 and 3 which had minimum inhibitory concentrations

of 0.1�/0.5 mg/ml against Gram-positive bacteria. # 2002 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Vernonia colorata ; Antibacterial activity; Sesquiterpene lactones; Bioassay-guided fractionation; Vernolide; 11b,13-Dihydrovernolide;

Vernodalin

1. Introduction

Vernonia colorata (Willd.) Drake, a member of the

Compositae, is a small tree or large shrub that is

widespread in west, central and south tropical Africa.

This species has a wide variety of applications in

traditional medicine, especially for the treatment of

fevers, coughs, diarrhoea, boils, and as a general tonic,

for which decoctions of the leaves are generally used

(Hutchings et al., 1996). The leaves of V. colorata have

shown high antibacterial activity (Kelmanson et al.,

2000) in the disc-diffusion bioassay and the antibacterial

compound vernodalin has been isolated from the leaves

(Reid et al., 2001). A number of sesquiterpene lactones

have been isolated from other African Vernonia species

(Toubiana and Gaudemer, 1967; Kupchan et al., 1969;

Asaka et al., 1977; Zdero et al., 1991), that possess

significant in vitro antischistosomal, plasmodicidal, and

leishmanicidal activities (Ohigashi et al., 1994).

Therefore this particular species of Vernonia was

studied further by bioassay-guided fractionation in

order to isolate and characterise the major compoundsresponsible for it’s antibacterial properties. The com-

pounds identified are sesquiterpene lactones (Fig. 1),

common to those already found in other Vernonia

species, and 11b,13-dihydrovernolide which has not

been reported from Vernonia species before. This is

also the first account of antibacterial activity for

vernolide.


2.1. Plant material

Leaf material was collected from a V. colorata shrub

growing in the Botanical Garden, University of Natal,

Pietermaritzburg. A voucher specimen (TR 51NU) was

deposited in the herbarium of the University of Natal,Pietermaritzburg. Plant material was dried in an oven at

50 8C for 48 h and stored at room temperature untilprocessing.

* Corresponding author. Tel.: �27-33-2605130; fax: �27-33-

2605897.

E-mail address: [email protected] (J. van Staden).




PII: S 0 3 7 8 - 8 7 4 1 ( 0 2 ) 0 0 0 1 0 - 7


2.2. Isolation and identification of active compounds

Dried leaf material (580 g) was finely ground and

extracted with distilled ethyl acetate (5 l) at room

temperature for 48 h. The extract, after solvent evapora-

tion under reduced pressure (20 g green oily residue) was

subjected to repeated vacuum liquid chromatography

using silica gel (Merck 230�/400 mesh), eluting with an

increasingly polar gradient of hexane:ethyl acetate. At

this stage, column fractions were assayed according to a

TLC (hexane:ethyl acetate, 1:1) bioautography bioassay

method described below. Similar active fractions were

combined from each chromatography column and in

this way three of the most active fractions were

identified; sample C (1.4 g) eluted with 45:55% hex-

ane:ethyl acetate, sample D (2.2 g) eluted with 40:60%

hexane:ethyl acetate, and sample E (0.5 g) eluted with

35:65% hexane:ethyl acetate.

Samples C, D, and E were re-chromatographed on a

Sephadex LH-20 column with cyclohexane:dichloro-

methane: methanol (7:4:1) to remove any chlorophyll

present and further purify the samples. From the

fractions obtained after sample C was chromato-

graphed, compound 1 (180 mg) and compound 2 (85

mg) crystallised out in a pure form to yield white

needles. Fractions from sample D were combined

according to activity with the bioautographic TLC

assay. Compound 3 (630 mg, colourless oil) was

obtained after purification of the fraction on a chroma-

totron (CHCl3:acetone 85:15).

Compounds were identified using spectral data such

as 1H NMR, 13C NMR, and additional 2D spectra

where needed, as well as EI-MS spectra, and by direct

comparison with published spectra and structures.

NMR spectra were recorded in CDCl3 using a Varian

Inova 500MHz NMR spectrometer at the University of

Natal, Pietermaritzburg and FAB mass spectrometry at

the Cape Technikon. The biological activity of these

compounds was verified using bioautographic TLC and

quantitatively assessed by determining their minimum

inhibitory concentrations (MIC’s).

2.3. Bioautography on TLC plates

The antibacterial activity of chromatographic frac-

tions and the purified compounds was visualised usingdirect bioautography on TLC plates (Merck Si-gel 60

F254, 0.25 mm thick) according to the method of

Slusarenko et al. (1989). An overnight culture of

Staphylococcus aureus (ATCC 12600) was centrifuged

at 3000 rpm for 10 min, the pellet resuspended in sterile

phosphate buffer, and added to a molten (48 8C)Mueller-Hinton agar (Biolab) overlay containing 1%(v/v) glycerol and 0.1% (w/v) 2,3,5-triphenyltetrazoliumchloride (TZC). This overlay was poured over the TLCplate on which fractions had been separated. This wascovered and incubated at 37 8C for 6�/12 h. Activecompounds on the bacterial plate showed up as whitespots (inhibition) on a pink/dark red background (livebacteria). Active compounds were identified by compar-ing the zones of growth inhibition to a duplicate TLCplate developed under identical conditions. The dupli-cate plate was visualised by using an anisaldehyde stain(2.6%) and heating at 120 8C for 8 min.

2.4. Minimum inhibitory concentration

A micro-dilution technique (Eloff, 1998) using 96-well

microplates and tetrazolium salts to indicate bacterial

growth was used to obtain MIC values of the pure

compounds. The following test bacteria were used; S.

aureus (ATCC 12600), Bacillus subtilis (ATCC 6051),

Escherichia coli (ATCC 11775), and Klebsiella pneumo-

niae (ATCC 13883). The compounds were serially

diluted with water in the wells of the microtiter plate,

and an equal volume of a 1/100 diluted overnight culture

of bacteria added to the wells to give a final volume of

200 ml. The plate was covered and incubated at 37 8Covernight. To indicate bacterial growth, 50 ml of a 0.2mg/ml p -iodonitrotetrazolium violet (INT) solution was

added to each well and incubated at 37 8C for a further30 min, after which inhibition of bacterial growth wasvisible as a clear well and the presence of growthdetected by a pink/red colour. Appropriate controlsincluded the solvent used to dissolve the test compound,Mueller-Hinton broth alone, and the antibiotic neomy-cin as a positive control. Each assay was repeated twice.


Compounds 1 and 3 were identified as vernolide and

vernodalin respectively by means of NMR spectroscopy

and structures were confirmed by comparison of physi-

cal and NMR data against literature values (Toubianaand Gaudemer, 1967; Kupchan et al., 1969; Ganjian et

al., 1983). The structure of vernolide has been confirmed

by X-ray crystallography (Pascard, 1970). There is

Fig. 1. Structures of compounds 1�/3.

T. Rabe et al. / Journal of Ethnopharmacology 80 (2002) 91�/9492

confusion in the literature between these two com-

pounds as vernolide has been incorrectly named as

vernodalin in one publication, although the correct

structure for vernolide was provided (Zdero et al.,

1991). The NMR spectra for compound 2 were found

to be very similar to those of vernolide. However,

resonances ascribable to the methylene protons at C-

13 in vernolide were replaced by a doublet at d 1.63

(J� 6.9 Hz) integrating to three protons which was

coupled to a multiplet integrating to one proton at d

2.65. This indicated hydrogenation of the 11,13-double

bond allowing identification of 2 as 11b,13-dihydrover-

nolide. This was in agreement with the FAB mass

spectrum which gave a [M�Na�] ion at m/z 387.1

corresponding to a molecular ion at 364 and molecular

formula of C19H24O7, two mass units more than in the

FAB mass spectrum for vernolide as expected. The

stereochemistry of H-11 could be determined as b from

the NOESY spectrum. NOESY correlations were seen

between H-7a, 3H-13 and H-5 on the a-side of the

molecule and between H-11 and H-8 and H-6 (weak

correlation) on the b-side of the molecule. The HMBC,

HSQC and NOESY spectra allowed for assignment of

the NMR spectra for vernolide and 11b, 13-dihydro-

vernolide (Table 2).

Antibacterial testing showed that 1, 3, and 2, to a

lesser degree, were active at inhibiting bacterial growth

(Table 1). All the compounds had a very low activity

against the Gram-negative bacteria E. coli and K.

pneumoniae , while they were most active against the

two Gram-positive bacteria S. aureus and B. subtilis .

This is the first report of antibacterial activity for

compound 1, while the antimicrobial activity of 3 was

previously established (Jisaka et al., 1993; Al Magboul

et al., 1997). Although these compounds were respon-

sible for the major antibacterial activity of V. colorata ,

their MIC values are not comparable with those of the

antibiotic neomycin. These sesquiterpene lactones are

known to possess in vitro cytotoxicity (Kupchan et al.,

1969), and this fact, together with their relatively low

activity, would not make them feasible candidates for

drug development.

Antibacterial activity has been reported in other

species of Vernonia , namely V. amygdalina (Jisaka et

al., 1993), V. fastigiata (Roos et al., 1998), V.

kotschyana (Deeni and Hussain, 1994), and V. nitidula

(Montanaro et al., 1996). The compounds responsible

for this activity in all these species have been sesqui-

terpene lactones. The work presented here has demon-

strated that the sesquiterpene lactones identified in V.

colorata have antibacterial activity as well and are

common to those found in other African species of

Vernonia. This confirms the importance of pursuing

medicinal plants for the isolation and identification of

bioactive natural products with a view to characterising

groups of compounds within a genus that may con-

tribute to the plant’s medicinal value.

Acknowledgements

This project was financially supported by the Foun-

dation for Research Development, Pretoria, and the

University of Natal Research Fund. We are grateful to

Table 1

MIC results (mg/ml) of Vernonia colorata compounds

COMPOUNDS BACTERIA TESTED

S. aureus B. subtilis E. coli K. pneumo-

niae

Dihydrovernolide

(2)

�8.0 4.0 �8.0 �8.0

Vernolide (1) 0.5 0.1 �8.0 �8.0

Vernodalin (3) 0.25 0.1 8.0 8.0

Neomycin 7.5�10�4 1.8�10�4 7.8�10�4 7.8�10�4

Table 2

NMR data for vernolide and 11b,13-dihydrovernolide (500 MHz,

CDCl3) (J in brackets)

Carbon 1H 1 13C 1 1H 2 13C 2

1 2.72 dd (4.5,11.5) 66.43 CH 2.70 dd (4.5,11.5) 66.32 CH

2 2.30 m 22.77 CH2

2.29 m 22.74 CH2

1.69 m 1.68 m

3 2.40(2H) m 33.47 CH2

2.37 (2H) m 33.56 CH2

4 �/ 143.79 C �/ 142.85 C

5 5.57 d (9.5) 128.68

CH

5.49 d (9.6) 129.23 C

6 5.25 t (10.1) 77.43 CH 5.12 t (9.9) 77.31 CH

7 3.05 m 51.93 CH 2.18 m 57.11 CH

8 5.74a 70.75 CH 5.66a 71.50 CH

9 2.65d (14.6) 41.28 CH2

2.62 d (14.2) 40.79 CH2

1.39 dd (14.2,

10.7)

1.30 dd (14.6,

10.6)

10 �/ 58.88 C �/ 58.89 C

11 �/ 134.85 C 2.65 m 39.93 CH

12 �/ 169.43 C �/ 177.54 C

13 6.35 d (2.66) 126.14

CH2

1.43 d (6.9) 16.72 CH3

5.95 d (2.66)

14 4.55 d (10.1) 99.29 CH 4.53 d (11.0) 99.47 CH

15 4.59 d (13.7) 64.33 CH2

4.57 d (13.7) 64.30

3.67 d (13.7) 3.67 d (13.7)

16 �/ 167.22 C �/ 167.96 C

17 �/ 135.76 C �/ 135.62

18 1.96 s 18.27 CH3

1.96 s 18.28 CH3

19A B 6.16 d (1.8) 127.25

CH2

6.14 d (1.8) 127.44

CH2

5.71 d (1.8) 5.67 d (1.8)

OH 4.91 d (10.1) �/ 5.21 d (11.0) �/

a Peak obscured.

T. Rabe et al. / Journal of Ethnopharmacology 80 (2002) 91�/94 93

Mr Martin Watson and Dr Philip Boshoff for providing

NMR and mass spectra.

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H., Kraus, W. 1998. New sesquiterpene lactones with antibacterial

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T. Rabe et al. / Journal of Ethnopharmacology 80 (2002) 91�/9494

Short communication

Pharmacological and phytochemical screening of two Hyacinthaceaespecies: Scilla natalensis and Ledebouria ovatifolia

S.G. Sparg, J. van Staden, A.K. Jager *

Research Centre for Plant Growth and Development, School of Botany and Zoology, University of Natal Pietermaritzburg, Private Bag X01, Scottsville

3209, South Africa

Received 4 June 2001; received in revised form 15 December 2001; accepted 17 December 2001

Abstract

Aqueous, ethanolic, dichloromethane and n -hexane extracts of Scilla natalensis Planch. and Ledebouria ovatifolia (Bak.) Jessop

bulbs (Hyacinthaceae) were screened for antibacterial, anti-inflammatory, antischistosomic, anticancer and anthelmintic activity.

Poor antibacterial activity against both Gram-positive and Gram-negative bacteria was shown with S. natalensis extracts. Good

antibacterial activity was shown by the ethanolic and dichloromethane extracts of L. ovatifolia against Gram-positive bacteria. In

the anti-inflammatory screening, the dichloromethane and hexane extracts of S. natalensis resulted in good inhibition against both

COX-1 and COX-2. Ethanolic extracts had the highest inhibitory effect against nematodes in the anthelmintic assays. Poor anti-

inflammatory and anthelmintic activity was found with L. ovatifolia . Aqueous extracts of S. natalensis had good activity against

Schistosoma haematobium , with a minimum inhibitory concentration of 0.4 mg ml�1. Aqueous extracts of fresh L. ovatifolia bulb

material were found to be lethal to S. haematobium at a concentration of 1.6 mg ml�1. The phytochemical screening of S. natalensis

bulbs revealed the presence of saponins and bufadienolides within the bulbs. Bulbs of L. ovatifolia contained bufadienolides.

# 2002 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Ledebouria ovatifolia ; Scilla natalensis ; Screening; Traditional medicine

1. Introduction

Traditional medicine is widely used by many people in

Africa who still incorporate herbal medicine in their

daily existence. The plants that are used in traditional

medicines are likely and in some cases already known, to

contain pharmacologically active compounds. For this

reason, medicinal plants have become the focus of

intense study in recent years to determine whether their

traditional uses are supported by actual pharmacologi-

cal effects or are merely based on folklore. With the

increasing acceptance by Western health-systems of

traditional medicine as an alternative form of health

care, there is an urgent need for an evaluation of

traditional methods of treatment. Considerable impor-

tance has been placed on the screening of medicinal

plants for active compounds.

Scilla natalensis Planch. is ranked as one of the most

popular plant species sold at many of the medicinal

markets in South Africa (Cunningham, 1988; Mander,

1997). Bulbs of S. natalensis are sold for the treatment

of gastro-intestinal ailments which includes stomach

aches, constipation, intestinal worms, diarrhoea, dysen-

tery, nausea and indigestion (Hutchings, 1989). The

bulb is also used in the treatment of sprains and

fractures. Dried bulb material is boiled in hot water

and used for boils, veld sores and eczema. Bulbs are

taken as purgatives or as an enema for internal tumors,

lumps or other swellings that may or may not be

cancerous (Watt and Breyer-Brandwijk, 1962; Hutch-

ings, 1989; van Wyk et al., 1997). There have also been

reports that S. natalensis is used gynaecologically in the

treatment of menstrual pains and to facilitate delivery

(Gerstner, 1941; Hutchings, 1989; Hutchings et al.,

1996). Decoctions are also taken as enemas for female

fertility and to enhance male potency and libido (van

Wyk et al., 1997). Like many other Hyacinthaceae

species different Scilla species have been reported to

* Corresponding author. Tel.: �27-33-2605130; fax: �27-33-

2605897.

E-mail address: [email protected] (A.K. Jager).




PII: S 0 3 7 8 - 8 7 4 1 ( 0 2 ) 0 0 0 0 7 - 7


contain cardiac glycosides (Kamano and Petit, 1974).

Kellermen et al. (1988) recorded electrocardiographic

changes similar to those of cardiac glycoside poisoning

in their work on S. natalensis . It is thought that S.

natalensis may contain heart glycosides of the bufadie-

nolide type, such as scillaren A. The presence of such

glycosides needs to be confirmed (van Wyk et al., 1997).

S. natalensis is also reported to possibly contain

saponins (Hutchings et al., 1996; van Wyk et al.,

1997). Jager et al. (1996) reported low anti-inflamma-

tory activity with S. natalensis in their screening for

prostaglandin-synthesis inhibitors. Crouch et al. (1999)identified two homoisoflavanones from S. natalensis .

Ledebouria ovatifolia (Bak.) Jessop, is a herbaceous

geophyte that occurs in many of the grasslands of the

Eastern Cape, KwaZulu-Natal, Northern Free State

and northern provinces. Many of the species in the

family Hyacinthaceae have resulted in overlapping

taxonomic classification. L. ovatifolia has been placed

under other species names. Synonyms of L. ovatifolia

are Scilla cicatricosa, S. climatocarpha, S. elevans and

most commonly S. ovatifolia . Known as ‘icubudwana’

or ‘ntangana’ to the Zulu speaking people, the bulbs are

reportedly used in enemas for gastro-enteritis. They are

also taken medicinally for influenza, backache and

rubbed on female breasts at puberty to make them

grow (Hutchings et al., 1996). Very little is known about

the pharmacological and phytochemical activity of thisspecies. Toxicity tests showed that 1.5 kg of fresh bulb

material is fatal to sheep within a few hours (Watt and

Breyer-Brandwijk, 1962).

The objective of this study was to investigate some of

the pharmacological effects of S. natalensis and L.

ovatifolia , and in so doing, substantiate its use in

traditional medicine. Both species were screened biolo-

gically for prostaglandin-synthesis inhibitors (anti-in-flammatory), antibacterial, antischistosomic, anticancer

and anthelmintic activity. They were also screened

phytochemically for the presence of alkaloids, saponins

and cardiac glycosides.


2.1. Plant material

Mature plants of S. natalensis Planch. and L.

ovatifolia (Bak.) Jessop were obtained from the Uni-

versity of Natal Botanical Garden, Pietermaritzburg.

The bulbs were shredded and dried completely at 50 8Cfor :/72 h. The dried material were then ground andstored in airtight containers at room temperature in thedark until extraction. Plant material was stored for nolonger than 5 months. Voucher specimens were made ofS. natalensis (Sparg22NU) and L. ovatifolia

(Sparg23NU) and are lodged in the University of Natal

Herbarium, Pietermaritzburg.

2.2. Extraction of plant material

Crude extracts were prepared from the same plant by

extracting 2 g dried bulb material with 10 ml distilled

water, ethanol, dichloromethane and n-hexane under

sonication for 30 min, respectively. Extracts were

filtered and dried under vacuum. The samples were

then air dried and redissolved to 50 mg ml�1 solution

for antibacterial and anticancer testing, 10 mg ml�1 foranti-inflammatory testing and 100 mg ml�1 for anti-

schistosomic and anthelmintic testing.

2.3. Biological screenings

2.3.1. Antibacterial activity

The bioassay used for antibacterial screening was the

microplate method for minimum inhibitory concentra-

tion (MIC) determination (Eloff, 1998). Cultures of thefollowing bacteria were used: Bacillus subtilis (ATCC

No. 6051), Escherichia coli (ATCC No. 11775), Kleb-

siella pneumoniae (ATCC No. 13883) and Staphylococ-

cus aureus (ATCC No. 12600). The dichloromethane

and hexane extract residues were redissolved with

acetone. Neomycin (100 mg ml�1) was used as a positive

control for each bacterium, with solvent and bacteria

free wells being included as negative controls. Resultsare the mean of three experiments.

2.3.2. Anti-inflammatory activity

Anti-inflammatory activity was assessed using the

cyclooxygenase-1 (COX-1) assay as described by Jager

et al. (1996) and cyclooxygenase-2 (COX-2) assay as

described by Noreen et al. (1998) with slight modifica-

tions (Zschocke and van Staden, 2000). Inhibition ofboth COX-1 and COX-2 was determined by detecting

the conversion of radiolabelled [14C] arachidonic acid to

labelled prostaglandins. The percentage inhibition refers

to the reduction of prostaglandin formation in compar-

ison to a control blank (20 ml water or 2.5 ml EtOH in

17.5 ml water). Extracts were tested at a concentration of

250 mg ml�1. The dried dichloromethane and hexane

extract residues were redissolved with ethanol (10mg ml�1). Positive control measurements were carried

out with 2.5 ml indomethacin (20 mm for COX-1 and

200 mm for COX-2) (Sigma) in 17.5 ml water, a reference

anti-inflammatory drug. Results for COX-1 and COX-2

are the mean of three experiments.

2.3.3. Antischistosomal activity

Aqueous extracts were tested using the antischistoso-mic assay (Sparg et al., 2000) which determines a lethal

concentration (LC) against schistosomula worms, the

human stage of schistosomiasis (bilharziosis). S. nata-

S.G. Sparg et al. / Journal of Ethnopharmacology 80 (2002) 95�/10196

lensis and L. ovatifolia was screened against the

trematode Schistosoma haematobium . The bilharzia

parasite was obtained from Bulinus africanus (Krauss)

snails collected from the Slangspruit, Campsdrift Pie-termaritzburg. The snails were induced to shed cercariae

by exposing the snails to light. These cercariae were

subjected to a shearing stress to promote the losing of

their tails and thereby transforming them into schisto-

somula worms. Two times serial dilutions of aqueous S.

natalensis and L. ovatifolia extracts were prepared.

Schistosomula worm survival was examined after in-

cubating the bioassay plates at 25 8C for 1 h. Resultsare the mean of three experiments. The minimum LCwas recorded. Praziquantel (Sigma) (1 mg ml�1) astandard antischistosomic drug was used as a positivecontrol.

2.3.4. Anticancer (DNA-damaging) activity

The Biochemical Induction Assay (BIA) was used to

investigate the potential of the plant extracts to damage

DNA. The BIA is a simple, sensitive and rapid methodwhich has been successfully employed in the screening of

microbial culture broths for anticancer agents. This

bioassay is a colorimetric spot assay, based on the

production of b-galactosidase by the test organism E

coli BR513 (ATCC 33312), as a response to DNA

damaging. An indication of DNA damaging is the

appearance of a red-violet zone of enzyme activity at

the site of sample application. The method described byWhite et al. (1986) was used. Water, ethanol, dichlor-

omethane and n -hexane extracts (10 ml of redissolved

extracts at 100 mg ml�1) were tested for activity. The

strong mutagen 4-nitroquinoline 1-oxide was used at

concentrations of 1, 5, 50 and 100 mg/spot as a positive

control. The appropriate solvents were also included as

negative controls.

2.3.5. Anthelmintic acxtivity

Extracts of S. natalensis and L. ovatifolia were

screened for anthelmintic activity using the method of

Rasoanaivo and Ratsimamanga-Urverg (1993) as de-

scribed by McGaw et al. (2000). The nematode Caenor-

habditis elegans var. Bristol (N2) was cultured on

nematode growth agar seeded with E. coli according

to the method of Brenner (1974). A standard concentra-tion of levamisole (Sigma) (5 mg ml�1) was used as a

positive control. Solvent blanks were included. Water,

ethanol, dichloromethane and n -hexane extracts (1

mg ml�1) were tested. The percentage of living worms

was estimated after 2 h at 25 8C in the dark, using adissecting microscope. The results are mean for threeexperiments.

2.3.6. Cytotoxicity testing

Aqueous extracts of S. natalensis were tested for

cytotoxicity using the tetrazolium salt reduction (MTT)

assay (van Rensburg et al., 1994) which is based on the

methods of Hussain et al. (1993). Secondary vervet

monkey kidney cells (VK) were treated with S. natalen-

sis extracts from 3.9 mg ml�1 up to 1000 mg ml�1.

2.4. Phytochemical screening

2.4.1. Alkaloids

Two grams of dried powdered bulb material was

ground with 2 ml 10% ammonia solution and then

mixed with 7 g basic aluminium oxide (activity grade I).

This mixture was then packed loosely into a glass

column and 10 ml CHCl3 was added. The alkaloidswere eluted with 5 ml CHCl3. The elute was collected

and evaporated down to 1 ml and used for TLC. The

spray reagent used for the detection was Dragendorff

reagent (Wagner and Bladt, 1984). Observations were

made for the development of red orange spots on the

plate indicating the presence of alkaloids.

2.4.2. Saponins

The haemolysis test (Luyt et al., 1999) and froth test

was used to screen for the presence of saponins in the

bulb extracts of S. natalensis and L. ovatifolia . Ob-

servation were made for clear zones of haemolysis in the

blood agar plates surrounding the extracts. These

haemolysis zones indicate the presence of saponins.

With the froth test, the presence of a dense froth layer

after vigorous shaking is recorded.

2.4.3. Cardiac glycosides

The procedure for extraction and testing for cardiac

glycosides was performed according to Jager and van

Staden (1995). The Kedde’s reagent (Wagner and Bladt,

1984) was sprayed to test for cardenolides and antimony

(III) chloride (Wagner and Bladt, 1984) was sprayed to

test for bufadienolide type cardiac glycosides. Proscil-larindin A (Sigma) was used as a standard.


The results for the general screening for antibacterial

activity are shown in Table 1. No inhibitory activity was

detected against the four test bacteria with the S.

natalensis n -hexane extracts. Poor inhibitory activity

was detected for both the water and dichloromethane S.

natalensis extracts. The ethanolic extract gave the high-

est inhibitory activity. No differences were noted

between the activity obtained against the Gram-positive

or Gram-negative bacteria. Preliminary studies done by

Matthe (1988, 1989) indicated that extracts from S.

natalensis have antimicrobial activity against a range ofpus-forming organisms in vitro. Two of the four L.

ovatifolia extracts showed good antibacterial activity.

These two extracts showed better activity against the

S.G. Sparg et al. / Journal of Ethnopharmacology 80 (2002) 95�/101 97

Gram-positive than the Gram-negative bacteria. The

results support the observation that Gram-negative

bacteria are more resistant, probably because of their

thick murein layer which prevents the entry of inhibitors

(Martin, 1995). The ethanolic L. ovatifolia extracts had

the highest inhibitory activity against S. aureus. The

dichloromethane extracts of L. ovatifolia had the highest

inhibitory activity against B. subtilis . It is not likely that

the traditional healers would be able to extract the

compounds responsible for the antibacterial activity

since dichloromethane is not easily accessible to them.

The aqueous and hexane extracts showed low activity

against both Gram-positive and Gram-negative bac-

teria.

Results for the anti-inflammatory screening using the

COX-1 inhibition assay are shown in Fig. 1. Both the

aqueous and ethanolic extracts of S. natalensis showed

no noteworthy activity. Only the lipophilic extracts

showed high levels of anti-inflammatory activity, with

80 and 71% inhibition for the dichloromethane and n -

hexane extracts, respectively. The lipophilic extracts

showed high levels of anti-inflammatory activity in

COX-2 assay (Fig. 1). The dichloromethane and n -

hexane extracts gave 91 and 82% inhibition, respectively.

No activity was shown by the water and ethanol

extracts. In general, plant material extracted with

ethanol usually shows higher activity than plant mate-

rial extracted with water (McGaw et al., 2000).

Although water is the most commonly used solvent,

ethanol is also available to many traditional healers.

Solvents like n-hexane and dichloromethane, which

showed the highest percentage inhibition are not easily

available to the majority of traditional healers. How-

ever, these solvents may also extract other compounds

in higher concentrations, causing the crude extracts to

be toxic. Dosage is important with regard to which

solvent is being used. If water is used, the dosage would

be higher. The same dosage using a dichloromethane

extraction may be potentially dangerous. Both the

aqueous and dichloromethane extracts of L. ovatifolia

showed no activity in relation to the indomethacin

Table 1

MIC against four common bacterial types and LC against S. haematobium of S. natalensis and L. ovatifolia extracts

Species Solvent Bacterial MIC Schistosomula LC

E. c K. p B. s S. a S. haematobium

S. natalensis Water 6.3 6.3 12.3 6.3 0.4b,c

Ethanol 1.6 1.6 1.6 1.6 �/

Dichloromethanea 3.1 6.3 6.3 6.3 �/

Hexanea �12.5 �12.5 �12.5 �12.5 �/

L. ovatifolia Water 6.3 12.5 12.5 6.3 1.6b 25.0c

Ethanol 1.6 1.6 1.6 0.8 �/

Dichloromethanea 1.6 1.6 0.8 1.6 �/

Hexanea 6.3 6.3 6.3 6.3 �/

Neomycin 6.3�10�2 1.6�10�2 1.6�10�2 3.1�10�2 �/

Praziquantel �/ �/ �/ �/ 1�10�3

E.c ; E. coli ; K.p ; K. pneumonia ; B.s ; B. subtilis ; S.a ; S. aureus ; (�/) could not test; all measurements in mg ml�1.a Resuspended in acetone.b Fresh material.c Dried material.

Fig. 1. (A.) Percentage inhibition of S. natalensis and L. ovatifolia

extracts (250 mg ml�1) against COX-1, and (B.) percentage inhibition

by S. natalensis extracts (250 mg ml�1) against COX-2.


standard. The ethanol and n -hexane extracts showed

low activity, of 56 and 66%, respectively. However, this

activity is too low to rationalize the use of L. ovatifolia

to treat inflammation. Therefore if this species is used

traditionally in the treatment of backache and fevers, it

must be used in a concoction or other mechanisms, as

alone it is not that active. The difference in n-hexane

and dichloromethane extracts may be the result of

inactivation by other compounds that are also extracted

with dichloromethane. The results obtained were similar

for all three experiments. However the reason for this

remains unexplained.

The results of the antischistosomic activity are shown

in Table 1. Aqueous extracts from both fresh and dry

bulb material of S. natalensis had high activity. The

results show the activity to be higher than any of the

plants screened previously for anti-antischistosomic

activity using this assay (Sparg et al., 2000; Mølgaard

et al., 2001). The LC obtained by Mølgaard et al. (2001)

was 0.6 mg ml�1 from Abrus precatorius root extracts.

S. natalensis extracts were lethal to S. haematobium at

0.4 mg ml�1. S. natalensis is not even reported to be

used traditionally in the treatment of schistosomiasis.

However, the results of the cytotoxicity testing showed

S. natalensis to be extremely cytotoxic to VK cells.

Visual examination of the monolayers of VK cells

treated with the extracts, showed gross morphological

changes and cell death, suggestive of severe cytotoxicity

at all concentrations of the extract tested (Table 2). If

this species is so cytotoxic, it would not be recom-

mended to be taken internally. Extracts from L.

ovatifolia were not as active as S. natalensis against S.

haematobium. Fresh L. ovatifolia bulbs were more active

than dry bulbs. Possibly some of the active compounds

are broken down during the drying process. However,

due to the possible toxicity of this species care should be

taken if this species is to be taken orally.

Ethanolic extracts of S. natalensis showed good

activity killing :/50% of the nematodes in the anthel-

mintic assay (Fig. 2), with the remaining 50% moving

slower than those of the control. Both the dichloro-

methane and the n -hexane extracts killed :/40% of the

nematodes while the water extract killed only 30% of the

nematodes. S. natalensis is used traditionally to treat

intestinal worms (Hutchings, 1989). Some solvents like

ethanol are sometimes used by traditional healers for

extraction. This means that the compounds found active

in this anthelmintic screening are also obtained by the

healers. All four of the L. ovatifolia extracts showed

poor anthelmintic activity, and killed very few of the

nematodes when resuspended to 1 mg ml�1. The

ethanolic and dichloromethane extracts killed 30% of

the nematodes while the aqueous extracts killed only :/

10%, which was slightly more than the negative control.

There are no reports of L. ovatifolia being used in the

treatment of intestinal worms, and this screening

explains why it is not used traditionally as an anthel-

mintic.

S. natalensis is regarded as a ‘soap-plant’, and is

thought to contain saponins (Hutchings et al., 1996; van

Wyk et al., 1997). The screening confirmed this with the

help of the froth and haemolysis test. Blood cells are

known to haemolyse when exposed to certain saponin

type compounds causing a clear zone around the plant

extract. Most saponins have haemolytic properties that

can be attributed to their interaction with sterols that

are found in the erythrocyte membrane (Bruneton,

1995). This interaction results in an increase in mem-

brane permeability and the loss of haemoglobin and

therefore a clear zone. A distinct zone of haemolysis was

noted around each extract. Saponins are known to froth

up when exposed to aqueous solutions. This was evident

when working with aqueous extracts from S. natalensis .

Saponins are reported to have antimicrobial, cytostatic

and anti-inflammatory activity (Lower, 1985). The

presence of saponins may account for much of the

activity observed in this study. L. ovatifolia material

showed no haemolytic activity with the haemolysis test

and did not froth when shaken in water. Therefore, it

Table 2

Survival of the secondary vervet VK cells in the presence of varying

concentrations of aqueous extracts of S. natalensis

Extract concentration (mg ml�1) Percentage (%) survival of VK cells

1000.0 15

500.0 16

250.0 16

125.0 17

62.5 18

31.3 42

15.6 59

7.8 62

3.9 66

Fig. 2. Percentage of nematodes still alive after being exposed to S.

natalensis and L. ovatifolia extracts for 2 h. Extracts tested were at a

concentration 1 mg ml�1.


can be concluded that this species does not contain

saponins.

Kedde’s reagent indicates the presence of cardeno-

lides. This test showed that this type of cardiac glyco-sides are not present in the bulbs of S. natalensis and L.

ovatifolia . It was suggested that S. natalensis may

contain bufadienolide type of cardiac glycosides (van

Wyk et al., 1997). In screening for cardiac glycosides

using antimony (III) chloride (SbCl3) reagent, it was

found that bulbs of S. natalensis and L. ovatifolia do

contain bufadienolides. TLC analysis revealed S. nata-

lensis and L. ovatifolia to possibly contain the bufadie-nolide proscillaridin A, and prehaps a number of other

bufadienolide type glycosides. The Proscillaridin A

reference compound had a Rf value of 0.55 which

matched a band in both of the extracts of the two plant

species. This indicates that there is a good chance that

the bulb of S. natalensis and L. ovatifolia contains the

bufadienolide Proscillaridin A, however, HPLC isola-

tion and NMR analysis is needed to confirm this.Extracts from S. natalensis and L. ovatifolia had no

significant activity in the BIA assay, nor were any

alkaloids detected in the bulbs (results not shown).

From the screenings it is clear that S. natalensis is

pharmacologically active. It also contains important

phytochemicals, however, from the toxicity test it is

clear that this species must be used with caution as a

herbal medicine. L. ovatifolia has the potential to beused more in traditional medicine especially with regard

to treating bacterial infections and even schistosomiasis.

However, due to its suspected toxicity, caution should

be taken when using it.

Acknowledgements

South African�/Netherlands Research Programme on

Alternatives in Development (SANPAD) is gratefully

acknowledged for providing financial assistance. Dr M.Taylor and D. G. Erasmus of the Department of

Medical Virology, Institute of Pathology, University of

Pretoria are acknowledged and thanked for the cyto-

toxicity testing.

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Antibacterial activity of Brazilian propolis and fractions against oral anaerobic bacteria

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