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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
http://www.informaworld.com
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.
References
Brekhman, I.I., & Dardymov, I.V. (1969). New substances from plant origin which increase non-
specific resistance. Annual Review of Pharmacology, 9, 419–430.Bruneton, J (1995). Pharmacognosy, phytochemistry, medicinal plants. Paris: Lavoisier.Carboni, S. (1940). Sulle efedre della Sardegna. La Ricerca Scientifica, XVIII, 754–757.Cottiglia, F., Bonsignore, L., Casu, L., Deidda, D., Pompei, R., Casu, M., et al. (2005). Phenolic
constituent from Ephedra nebrodensis. Natural Product Research, 19(2), 117–123.Erdem, Y., & Kupeli, E. (2002). Berberis crataegina DC. root exhibits potent anti-inflammatory,
analgesic and febrifuge effects in mice and rats. Journal of Ethnopharmacology, 79,
237–248.Farnsworth N.R. (Ed). (1995) NAPRALERT database. Chicago: University of Illinois at Chicago.
Flordal, P.A., & Svensson, J. (1992). Hemostatic effects of ephedrine. Thrombosis Research, 68,
295–302.Goodman, L.S., & Gilman, A. (1993). Goodman and Gilman’s pharmacological basis of therapeutics
(8th ed., pp. 213–214). New York: MacMillan.Gunnar, G.-S., & Ian, D.S. (1994). Biosynthetic route to the Ephedra alkaloids. Journal of American
Society, 116, 6195–6200.
Harada, M., & Nishimura, M. (1991). Contribution of alkaloid fraction to pressor and
hyperglycemic effect of crude Ephedra extract in dogs. Journal of Pharmacobio-Dynamics, 4,
691–699.
Hikino, H., Ogata, K., Konno, C, & Sato, S. (1983a). Structure of feruloylhistamine, a hypotensive
principle of Ephedra roots. Planta Medica, 48(6), 108–110.Hikino, H., Ogata, K., Konno, C., & Sato, S. (1983b). Hypotensive actions of ephedradines,
macrocyclic spermine alkaloids of Ephedra roots. Planta Medica, 48(4), 290–293.Kasahara, Y., Hikino, H., Tsurufuji, S., Watanabe, M., & Ohuchi, K. (1985). Antiinflammatory
actions of ephedrines in acute inflammations. Planta Medica, 51, 325–331.
Kim, H.K., Choi, Y.H., Erkelens, C., Lefeber, A.W.M., & Verpoorte, R. (2005). Metabolic
fingerprinting of Ephedra species using 1H-NMR spectroscopy and principal component
analysis. Chemical & Pharmaceutical Bulletin, 53, 105–109.
Kim, H.K., Choi, Y.H., Chang, W.T., & Verpoorte, R. (2003). Quantitative analysis of ephedrine
analogues from Ephedra species using 1H-NMR. Chemical and Pharmaceutical Bulletin, 51,
1382–1385.
Kim, T.H., Yang, K.S., Hwang, E.Z., & Park, S.B. (1991). Effect of Ephedra herba on the immune
response in mice. Korean Journal of Pharmacognosy, 22, 183–191.Konno, C. (1979). Ephedroxane, anti-inflammatory principle of Ephedra herbs. Phytochemistry, 18,
697–698.Leung, A.Y., Foster, S. (2000). Enciclopedia delle piante medicinali utilizzate negli alimenti, nei
farmaci e nei cosmetici. Roma: Aporie.
Lida, M., Miyazaki, I., Tanaka, K., Kabuto, H., Ichikawa, E., & Ogawa, N. (1990). Dopamine D2
receptor mediated antioxidant and neuroprotective effect of ropinirole, a dopamine agonist.
Brain Research, 838, 51–59.Lohr, J.B., Kuczenski, R, & Niculescu, A.B. (2003). Oxidative mechanisms and tardive dyskinesia.
CNS Drugs, 17, 47–62.
Natural Product Research 1123
Matsui, T., Suzuki, Y., Yamashita, K., Yoshimaru, T., Suzuki-Karasaki, M., Hayakawa, S., et al.(2000). Diphenyleneiodonium prevents reactive oxygen species generation, tyrosine phos-phorylation, and histamine release in RBL-2H3 cells. Biochemical and Biophysical ResearchCommunications, 276, 742–748.
Munhall, A.C., & Johnson, S.W. (2006). Dopamine-mediated actions of ephedrine in the ratSubstantia nigra. Brain Research, 1069, 96–103.
Nag Chaudhari, A.K., & Lahiri, S.C. (1974). Use of goat trachea for an isolated tracheal chain
preparation. Indian Journal of Pharmacology, 6, 149–151.Pignatti, S. (1982). Flora d’Italia. I, II, III. Edagricole: Bologna.Poli, F., Muzzoli, M., Sacchetti, G., Tassinaro, G., Lazzaroni, R., & Bruni, A. (2003). Antioxidant
activity of supercritical CO2 extracts of Helichrysum italicum (Roth) Don (Asteraceae).Pharmaceutical Biology, 41, 379–383.
Spina, L., Longoni, R., Mulas, A., Chang, K.J., & Di Chiara, G. (1998). Dopamine-dependent
behavioural stimulation by non-peptide delta opioids BW373U86 and SNC 80: 1.Locomotion, rearing and stereotypies in intact rats. Behavioural Pharmacology, 9, 1–8.
Van Hellemont, J. (1986). Compendium de phytotherapie. APB: Bruxelles.Wang, M., Li, J., Rangarajan, M., Shao, Y., LaVoie, E.J., Huang, T.C., et al. (1998). Antioxidative
phenolic compounds from sage (Salvia officinalis). Journal of Agricultural and Food Chemistry,46, 4869–4873.
Winter, C.W., Risley, E.A., & Nuss, G.W. (1962). Carraggeenin-induced edema in hind paw of the
rat as an assay for antiinflammatory drugs. Proceedings of the Society for ExperimentalBiology and Medicine, 111, 544–547.
World Health Organization. (1989).Medicinal plants of China (WHO Regional Publications Western
Pacific Series, No. 2). Manila: WHO.
1124 M. Ballero et al.