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Pharmacological activities on Ephedra nebrodensis TineoMauro Balleroa; Caterina Foddisa; Cinzia Sannaa; Paolo Scartezzinib; Ferruccio Polib; Valentina Petittoc;Mauro Serafinic; Alessandra Stanzionec; Armandodoriano Biancod; Anna Maria Serillid; Liliana Spinae;Rosanna Longonie; Sanjay Kasturef

a CoSMeSe, Department of Botanical Sciences, University of Cagliari, Italy b Department ofEvolutionary and Experimental Biology, University of Bologna, Italy c Department of BiologiaVegetale, University 'La Sapienza', Roma, Italy d CoSMeSe, Department of Chimica, University 'LaSapienza', Roma, Italy e Department of Toxicology, University of Cagliari, Italy f MGV's PharmacyCollege, Panchavati, Nashik, India

Online publication date: 23 June 2010

To cite this Article Ballero, Mauro , Foddis, Caterina , Sanna, Cinzia , Scartezzini, Paolo , Poli, Ferruccio , Petitto,Valentina , Serafini, Mauro , Stanzione, Alessandra , Bianco, Armandodoriano , Serilli, Anna Maria , Spina, Liliana ,Longoni, Rosanna and Kasture, Sanjay(2010) 'Pharmacological activities on Ephedra nebrodensis Tineo', Natural ProductResearch, 24: 12, 1115 — 1124To link to this Article: DOI: 10.1080/14786410802680902URL: http://dx.doi.org/10.1080/14786410802680902

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Natural Product ResearchVol. 24, No. 12, 20 July 2010, 1115–1124

Pharmacological activities on Ephedra nebrodensis Tineo

Mauro Balleroa, Caterina Foddisa, Cinzia Sannaa, Paolo Scartezzinib, Ferruccio Polib,Valentina Petittoc, Mauro Serafinic, Alessandra Stanzionec, Armandodoriano Biancod,Anna Maria Serillid, Liliana Spinae, Rosanna Longonie and Sanjay Kasturef*

aCoSMeSe, Department of Botanical Sciences, University of Cagliari, Italy; bDepartment ofEvolutionary and Experimental Biology, University of Bologna, Italy; cDepartment of BiologiaVegetale, University ‘La Sapienza’, Roma, Italy; dCoSMeSe, Department of Chimica, University‘La Sapienza’, Roma, Italy; eDepartment of Toxicology, University of Cagliari, Italy; fMGV’sPharmacy College, Panchavati, Nashik, India

(Received 10 October 2008; final version received 27 November 2008)

As a part of our endeavour to screen Mediterranean medicinal plants forvarious pharmacological activities, we evaluated antihistaminic, adaptogenic,anti-inflammatory, antinociceptive, hypotensive and locomotor properties,and antioxidant potential of Ephedra nebrodensis. 1H-NMR spectroscopy wascarried out to identify the plant metabolites, which confirmed the presenceof ephedrinic skeleton alkaloids. The ethanol : acetone (1 : 1) extract exhibiteddose-related antihistaminic, anti-inflammatory, antinociceptive, hypotensive,antioxidant and locomotor stimulant activity. The plant bears potential forfurther studies.

Keywords: Ephedra nebrodensis; pharmacological activities; antioxidant;adaptogenic

1. Introduction

Ephedra nebrodensis Tineo (EN) (Ephedraceae) grows extensively in central southernItaly, Sicily and Sardinia, with a fragmented distributional area (Pignatti, 1982).In Sardinia it grows abundantly in some areas of Mesozoic limestones of centraleastern Sardinia, namely Oliena, Dorgali, Orgosolo and Urzulei, where it was collecteduntil the 1960s to prepare drugs that produced effects similar to those obtained bysympathetic stimulation. Cottiglia et al. (2005) have isolated two phenolic glycosidesfrom EN Tineo, namely 4-hydroxy-3-(3-methyl-2-butenyl)phenyl �-D-glucopyranoside(nebrodenside A) and O-coumaric acid �-D-allopyranoside (nebrodenside B), along withO-coumaric acid glucoside, (�)-epicatechin and (�)-ephedrine from aerial parts andfound that (�)-epicatechin has weak antiviral activity against influenza A virus andvery weak cytotoxicity. Several researchers reported different activities of the Ephedragenus: Kim, Choi, Chang, and Verpoorte (2003), and Kim et al. (2005) have carriedout metabolomic analysis of Ephedra species using 1H-NMR spectroscopy andmultivariate data analysis, and they have shown that major differences in different

*Corresponding author. Email: kasture_sb@hotmail.com

ISSN 1478–6419 print/ISSN 1029–2349 online

� 2010 Taylor & Francis

DOI: 10.1080/14786410802680902

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species were due to benzoic acid analogues in the aqueous fraction and ephedrine-typealkaloids in the organic fraction. Floradal and Svensson (1992) have reported somehaemostatic effects of ephedrine. The plant has bronchodilator activity and is also usedfor treating myasthenia gravis (Farnsworth, 1995; Goodman & Gilman, 1993; WHO,1989), as an analgaesic, immune stimulant and antiviral agent (Kim, Yang, Hwang, &Park, 1991; Konno, 1979). Recently, Munhall and Johnson (2006) have reported thatephedrine releases dopamine in the substantia nigra. The phenolic compounds areknown to impart antioxidant activity to the plant extract. It has been reported thatmast cells generate intracellular reactive oxygen species in response to antigen challengeand this may be involved in histamine release (Matsui et al., 2000). In view of thisliterature support, we evaluated antihistaminic, anti-inflammatory, antinociceptive,hypotensive, antioxidant and locomotor stimulant activity of the ethanol : acetoneextract of the aerial parts of EN.

2. Results

2.1. Chemical analysis

The phytochemical characterisation of alkaloids from the EN ethanolic extract was carriedout using 1H-NMR spectroscopy. It allows rapid and simultaneous determination of theephedrine analogues according to Veerporte analysis (Kim et al., 2003). H-1 was selectedas a target signal in 1H-NMR because these signals of ephedrine analogues have separatedresonances and do not overlap with other signals from extract. The region of � 4.0–5.0 isconsidered when CDCl3 is used because no interference with other peaks is observed in the1H-NMR spectrum. The H-1 signal is most suitable for use as a target peak compared tothe other signals which appeared in the C-methyl region (�¼ 0.6–1.0) and in the N-methylregion (�¼ 2.0–2.4). Therefore, the diastereomers can be differentiated by the couplingconstants of chemical shifts. The compounds which have (S,S) configuration show a largercoupling constant than those of a (R,S ) configuration, because the dihedral angle betweentwo protons at C-1 and C-2 is quite different. The obtained chemical shifts are verified bydata in existing literature.

The chloroform fraction which was derived from the ethanolic extract of EN shows thepresence of ephedrine (H-1 �¼ 4.77, J¼ 3.9); pseudoephedrine (H-1 �¼ 4.19, J¼ 8.4),methylephedrine (H-1 �¼ 4.96, J¼ 3.8) and methylpseudoephedrine (H-1 �¼ 4.22, J¼ 6.9)were also detected. When the relative intensity of the characteristic signals of N–CH3 asa singlet was compared, the signal of pseudoephedrine to that of ephedrine was high.

Another signal at �¼ 4.52 (J¼ 4.5) is related to the H-1 of norephedrine which precedeephedrine in the biosynthetic process (Gunnar & Ian, 1994). All such conclusions wereconfirmed by data obtained from 13C-NMR spectra.

The ethyl acetate fraction contained a mixture of ephedrinic skeleton alkaloids. The1H-NMR signals for N–CH3 protons and for the C-3 methyl group confirmed the presenceof the alkaloids here reported (Figure 1).

2.2. Antihistaminic activity

The log dose response curve (DRC) showed that both the extract and diphenhydramineinhibited histamine-induced contraction significantly (p50.05), indicating presence of anantihistaminic principle in the ethanol : acetone extract (Figure 2).

1116 M. Ballero et al.

2.3. Adaptogenic activity

The subcutaneous injection of milk induced leukocytosis as well as eosinophilia in mice.EN extract as well as diazepam significantly decreased the leucocyte count. The extractand diphenhydramine significantly decreased the eosinophilia (Table 1), indicating theadaptogenic potential of EN.

2.4. Anti-inflammatory activity

In rats that received vehicle, the volume of oedema was 2.54mL at the third hour.Ibuprofen reduced paw oedema by 45.12%, whereas the extract of EN (100 and200mgkg�1) reduced oedema by 26.8 and 38.4%, respectively (Table 2).

2.5. Antinociceptive activity

In the vehicle-treated rats, the amount of writhing was 42.5� 8.2 after 30 min. EN(100mgkg�1, i.p.) reduced the number of writhes to 25.6� 6.5, whereas the higher dose ofthe extract (200mgkg�1, i.p.) reduced the amount of writhing to 19.2� 5.5. In rats thatreceived Ibuprofen, the amount of writhing was of 21.5� 4.7 (Table 3).

2.6. Blood pressure

A dose of 10mcg of extract of EN reduced blood pressure by 14mmHg, showinga hypotensive effect.

0

20

40

60

80

100

120

7.26 6.96 6.66 6.36

Log of molar concentration ofhistamine

Per

cent

age

cont

ract

ion Vehicle

ENDiphenhydramine

Figure 2. Effect of EN extract on histamine induced contraction of guinea pig tracheal chainpreparation.

N

OH

CH3

H

H R

CH3N

H

CH3

H

OH R

CH3NH2

OH

CH3

H

H

12

3

12

3

R=H, EphedrineR=CH3, Methylephedrine

R=H, PseudoephedrineR=CH3, Methylpseudoephedrine

12

3

Norephedrine

Figure 1. Ephedrinic skeleton alkaloids contained in the ethyl acetate fraction.

Natural Product Research 1117

2.7. Rat stereotyped and locomotor activity

The ethanol : acetone (1 : 1) extract (200mgkg�1, i.p.) as well as amphetamine(0.25mg kg�1, s.c.) significantly increased locomotor activity (p50.01 versus saline).The data is presented in Figure 3. A similar effect of EN and amphetamine was observedon rearing. Both the extract and amphetamine increased rearing and stereotypedbehaviour significantly (p50.05 versus saline) (Figures 4 and 5, respectively).

Table 1. Effect of EN extract on milk-induced leukocytosis and eosinophilia inmice.

Treatment WBC count (mean� SEM) Eosinophil count (mean� SEM)

Vehicle 2012� 223.0 120.2� 10.2EN (100) 1290� 56.0* 75.0� 8.35 *EN (200) 987.5� 34.5* 52.0� 5.8*Diazepam (1) 605� 43* –Diphen (1) – 14.2� 2.5 *

Notes: n¼ 5, EN=Ephedra nebrodensis. The values are the difference in WBCcount, before and 24 h after of drug administration. *p50.001, compared to milk-treated group (one-way ANOVA followed by Dunnett’s test).

Table 3. Effect of EN extract on acetic acid-induced writhing inmice.

TreatmentNumber of writhes in30min (mean� SEM)

Vehicle 42.5� 8.2EN (100) 25.6� 6.5*EN (200) 19.2� 5.5*Ibu (40) 21.5� 4.7*

Notes: n¼ 5, EN¼Ephedra nebrodensis, Ibu¼ Ibuprofen. All drugswere given orally 60 min before intraperitoneal injection of aceticacid. *p50.01 (ANOVA followed by Dunnett’s test).

Table 2. Effect of EN extract on carrageenan-induced paw oedema in rats.

Paw volume in mL (mean� SEM) at

Treatment 0 h 1 h 2 h 3 h% inhibition of paw

oedema at 3 h

Vehicle 0.9� 0.04 1.7� 0.03 1.9� 0.02 2.54� 0.06 –EN (100) 0.9� 0.03 1.3� 0.02 1.7� 0.05 2.1� 0.04 26.8*EN (200) 0.89� 0.05 1.2� 0.04 1.6� 0.04 1.9� 0.06 38.4*Ibu (40) 0.9� 0.05 1.4� 0.05 1.6� 0.03 1.8� 0.04 45.12*

Notes: n¼ 5, EN¼Ephedra nebrodensis, Ibu¼ Ibuprofen. All drugs were given orally 60minbefore subplantar injection of carrageenan. *p50.01 (ANOVA followed by Dunnett’s test).

1118 M. Ballero et al.

2.8. Antioxidant activity

The extract showed notable antioxidant activity but the IC50 was high when comparedwith the positive control, butyl-hydroxy-anisole (BHA). The ethanol : acetone extractshowed less potent antioxidant activity, with an IC50 value of 41.35mgmL�1, whereas theIC50 of BHA was 14.65� 0.08.

Locomotion

0

1000

2000

3000

4000

Saline Amphet. 0.25 mg EN

*

*

Tot

al c

ount

s (1

h)

Figure 3. Effect of amphetamine 0.25mgkg�1 s.c. (n¼ 4) and of the extract of EN 5g kg�1 o.s.(n¼ 5) on the motor activity. Data are mean� SEM of the photocell counts over a 1 h test. *p50.05as compared to saline controls (n¼ 4).

Rearing

0

25

50

Saline Amphet. 0.25 mg EN

*

Num

ber

(1h)

*

Figure 4. Effect of amphetamine 0.25mgkg�1 s.c. (n¼ 4) and of the extract of EN 5g kg�1 o.s.(n¼ 5) on the rearing. Data are mean� SEM of the number of items over a 1 h test. *p50.05as compared to saline controls (n¼ 4).

Stereotyped behaviour

0

1

2

3

Sco

re (

1h)

Saline Amphet. 0.25 mg EN

*

*

Figure 5. Effect of amphetamine 0.25mgkg�1 s.c. (n¼ 4) and of the extract of EN 5g kg�1 o.s.(n¼ 5) on the stereotyped activity. Data are mean� SEM of the score over a 1 h test *p50.05 ascompared to saline controls (n¼ 4).

Natural Product Research 1119

3. Experimental

Aerial parts of EN Tineo were collected at Piscina Urtaddala, Urzulei (east-central part of

Sardinia), and were botanically identified and registered with the specimen number 1656 at

the General Herbarium of the Botany Department of the University of Cagliari.

Pharmacological studies were carried out on the ethanol : acetone (1 : 1) extract of the dried

aerial parts of EN. The percent yield of the ethanol : acetone extract was 11.4%. The

extract was concentrated under reduced pressure and preserved in a refrigerator.

3.1. Extraction, purification and analysis

Extraction of the alkaloids of EN was performed according to the method of Carboni

(1940). Dried plant material (200 g) was exhaustively extracted with EtOH (96% v/v) at

room temperature. The extract was concentrated in vacuo at 40�C and 8.3 g of crude

extract was obtained. This extract was treated with 1N of HCl, filtered, and the pH was

adjusted to 8 with ammonia. The solution was extracted with CHCl3 (100 mL� 3) and

then with ethyl acetate (EtOAc, 70 mL� 3). The chloroform and the ethyl acetate extracts

were dried with anhydrous sodium sulphate (Na2SO4) and volatile materials were

evaporated in vacuo to obtain 275 and 42mg of dry residue, respectively. Dried samples

were dissolved in CDCl3 (chloroform extract) and acetone-d6 (ethyl acetate extract) and

the 1H-NMR profile was recorded for these two extracts.The utilised solvent was purchased from Carlo Erba reagents, while the deuterated

solvent was obtained from Sigma–Aldrich.

3.2. Pharmacological studies

Guinea pigs weighing 250–300 g and Albino Male Swiss mice (18–25 g) were housed in

groups of five each under the standard laboratory conditions (light period of 12 h per day,

temperature 25� 2�C and humidity 55� 5%) with access to food (standard pellets chow,

Lipton, India) and water ad libitum. Food but not water was withdrawn overnight and

during the experiment.To evaluate stereotyped locomotor activity, Sprague–Dawley male rats (175–200 g)

were used. Rats were individually housed in the polypropylene cages with sawdust in the

bottom and a metallic grill covering, and they were maintained at 25� 2�C room

temperature and humidity 55� 5%, with day and night cycles of 12 h each, and with free

access to food and water, though not during the experiments. The experimental procedures

were carried out in strict compliance with Institutional Animal Ethics Committee

regulations. All pharmacological tests in vivo were carried out using the ethanol : acetone

(1 : 1) extract.

4. Pharmacological evaluation

4.1. Antihistaminic activity

Guinea pig tracheal chain was prepared using the procedure described earlier by Nag

Chaudhari and Lahiri (1974). A DRC of histamine in the absence and presence of an

aqueous solution of EN (2.0mgmL�1 of bath volume) was obtained. Diphenhydramine

hydrochloride (2.0mgmL�1 of bath volume) was used as the reference standard.

1120 M. Ballero et al.

4.2. Adaptogenic activity

Mice were divided into four groups of five each. Group I served as the control and was

treated with vehicle and milk. Group II and III were treated with EN (100 and

200mgkg�1, i.p. each, respectively), and Group IV received diazepam (1mgkg�1, i.p.).

After 30 min of drug treatment each animal was injected with buffalo milk (4mLkg�1,

s.c.). Total leucocyte count was measured in each group before drug administration and

24 h after milk injection (Brekhman & Dardymov, 1969).The effect of the extract on milk-induced eosinophilia was evaluated using eosin

solution, using the same procedure. Diphenhydramine (1mg kg�1, i.p.) was used as the

reference standard. The difference in the eosinophil count before and 24 h after milk

administration was noted (Brekhman & Dardymov, 1969).

4.3. Anti-inflammatory activity

Male Albino rats divided in four groups of five each received vehicle (10mLkg�1 orally),

EN extract (100 or 200mgkg�1 orally) or ibuprofen 40mgkg�1 orally 60 min before

subplantar injection of carrageenan (0.1mL of 1% suspension). The paw volume was

measured using a plethismometer (Ugo Basile, Italy) at 0, 1, 2 and 3 h after carrageenan.

The percentage inhibition of oedema was determined (Winter, Risley, & Nuss, 1962).

4.4. Antinociceptive activity

On the previous day of testing, mice were injected with acetic acid and those showing

writhing were divided in four groups, each containing five mice. The groups received

vehicle (0.5% CMC), EN extract (100 and 200mgkg�1, p.o.) or ibuprofen suspension

(40mgkg�1, p.o.). After 60 min, the mice were injected intraperitoneally with 0.1mL of

1% acetic acid solution and writhes were recorded in a period of 30 min.

4.5. Effect on rat blood pressure

Rats were anaesthetised using urethane and blood pressure was measured using PowerLab

(AD Instruments, Australia). The animals were allowed to stabilise for 15min and a

change in diastolic blood pressure was recorded after administration of 10mcg of EN

extract in the femoral artery.

4.6. Stereotyped activity

Sprague–Dawley rats weighing 175–200 g were divided into four groups, each containing

five rats. The spontaneous motor activity was evaluated by the ‘test cage’, using a

particular cage provided with a recording system by photocell (Opto Varimex Mini,

Columbus, Ohio). Both spontaneous motor activity and total motor activity were

recorded.Stereotyped movements such as grooming, rearing, sniffing, licking and chewing were

recorded by observing rat behaviour every 10 min for a total time of 60 min. Only behaviour

persisting for at least 4 s were evaluated. Stereotyped movements were evaluated

in accordance with scores reported earlier by Spina, Longoni, Mulas, Chang, and

Natural Product Research 1121

Di Chiara (1998). Rats were individually housed in the cages for 60 min before experiments.After this time, Group I received ethanol : acetone extract of EN orally, dissolved inphysiologic solution (5 g kg�1); Group II saline solution; and Group III amphetamine(2.5mg kg�1, s.c). Motor and stereotyped activities were measured for the following 60 min.

4.7. Determination of the antioxidant activity

The antioxidant activity of extracts was assessed by their decolouration effect on theDPPH radical solution (1,1-diphenyl-2-picrylhydrazyl) (Poli et al., 2003; Wang et al.,1998). The tests were run in triplicate and averaged.

4.8. Statistical analysis

All observations are presented as mean� SEM. The data was analysed by Student’s t-testor one-way ANOVA followed by Dunnett’s test. p50.05 was considered as significant.

5. Discussion

The extract of EN obtained by extraction with ethanol : acetone (1 : 1) showed significantantihistaminic, adaptogenic, analgaesic, anti-inflammatory and hypotensive activities.The antihistaminic activity is complementary to the antiasthmatic activity due to thepresence of ephedrine, a sympathomimetic molecule acts on adrenergic �2 receptorsthereby reducing bronchial muscle tone. The extract showed a moderate adaptogenicactivity. These results are in line with those of Chinese ephedra (Ma-Huang), in which thealcoholic extract showed antiallergic effect in vitro (Leung & Foster, 2000). The observedanti-inflammatory activity of EN is in congruence with data obtained from Ma-Huang.The phytoconstituents present in Ma-Huang like ephedrine, pseudoephedrine, ephedrox-ane and pseudoephedroxane have anti-inflammatory effect on experimentally-inducedoedema (Kasahara, Hikino, Tsurufuji, Watanabe, & Ohuchi, 1985). Bruneton (1995) hasalso reported anti-inflammatory activity of ephedroxane and pseudoephedrine. Theinhibition of acetic acid-induced writhing suggests that the EN acts peripherally (Erdem &Kupeli, 2002).

Ephedradine, tiroxin, betaine and feruloistamine are hypotensive compounds ofEphedra species (Hikino, Ogata, Konno, & Sato, 1983a, 1983b) and in the total extractthey probably are contained in a greater quantity than ephedrine. The hypotensive effectof the ethanol : acetone extract of EN can be due to a prevalence of d-pseudoephedrineover l-ephedrine. This observation was confirmed previously by Van Hellemont (1986),who ascribes d-pseudoephedrine only to the Mediterranean variety of Ephedra. It wasdemonstrated that Ephedra contains flavonoids and proanthocyanidols that influenceabsorption of ephedrinic compounds, making their pharmacological activities less intensebut more enduring (Harada & Nishimura, 1991).

Results obtained from the tests on stereotyped activity showed an amphetamine-likeaction of the acetone : ethanol extract of EN. This observation is in congruence with thefindings of Munhall and Johnson (2006) that ephedrine releases dopamine from substantianigra. Thus the observed increase in locomotion can be explained on the basis of increaseddopamine release. Lida et al. (1990) have reported a dopamine D2 receptor-mediatedantioxidant and neuroprotective effect of ropinirole, a dopamine agonist. In this study we

1122 M. Ballero et al.

noticed antioxidant activity of the extract. Lohr, Kuczenski, and Niculescu (2003) havealso suggested a link between oxidative mechanisms and tardive dyskinesia, which willform the basis for further research. Therefore it is concluded that the ethanol : acetoneextract of EN Tineo collected in Sardinia has antihistaminic, adaptogenic, antinociceptive,anti-inflammatory and antioxidant activities.

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