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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; Sanjay Kasturef a CoSMeSe, Department of Botanical Sciences, University of Cagliari, Italy b Department of Evolutionary and Experimental Biology, University of Bologna, Italy c Department of Biologia Vegetale, University 'La Sapienza', Roma, Italy d CoSMeSe, Department of Chimica, University 'La Sapienza', Roma, Italy e Department of Toxicology, University of Cagliari, Italy f MGV's Pharmacy College, 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 Product Research, 24: 12, 1115 — 1124 To link to this Article: DOI: 10.1080/14786410802680902 URL: http://dx.doi.org/10.1080/14786410802680902
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Natural Product Research Vol. 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* a
CoSMeSe, Department of Botanical Sciences, University of Cagliari, Italy; bDepartment of Evolutionary and Experimental Biology, University of Bologna, Italy; cDepartment of Biologia Vegetale, University ‘La Sapienza’, Roma, Italy; dCoSMeSe, Department of Chimica, University ‘La Sapienza’, Roma, Italy; eDepartment of Toxicology, University of Cagliari, Italy; fMGV’s Pharmacy 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 for various pharmacological activities, we evaluated antihistaminic, adaptogenic, anti-inflammatory, antinociceptive, hypotensive and locomotor properties, and antioxidant potential of Ephedra nebrodensis. 1H-NMR spectroscopy was carried out to identify the plant metabolites, which confirmed the presence of ephedrinic skeleton alkaloids. The ethanol : acetone (1 : 1) extract exhibited dose-related antihistaminic, anti-inflammatory, antinociceptive, hypotensive, antioxidant and locomotor stimulant activity. The plant bears potential for further studies. Keywords: Ephedra adaptogenic
nebrodensis;
pharmacological
activities;
antioxidant;
1. Introduction Ephedra nebrodensis Tineo (EN) (Ephedraceae) grows extensively in central southern Italy, Sicily and Sardinia, with a fragmented distributional area (Pignatti, 1982). In Sardinia it grows abundantly in some areas of Mesozoic limestones of central eastern Sardinia, namely Oliena, Dorgali, Orgosolo and Urzulei, where it was collected until the 1960s to prepare drugs that produced effects similar to those obtained by sympathetic stimulation. Cottiglia et al. (2005) have isolated two phenolic glycosides from EN Tineo, namely 4-hydroxy-3-(3-methyl-2-butenyl)phenyl -D-glucopyranoside (nebrodenside A) and O-coumaric acid -D-allopyranoside (nebrodenside B), along with O-coumaric acid glucoside, ()-epicatechin and ()-ephedrine from aerial parts and found that ()-epicatechin has weak antiviral activity against influenza A virus and very weak cytotoxicity. Several researchers reported different activities of the Ephedra genus: Kim, Choi, Chang, and Verpoorte (2003), and Kim et al. (2005) have carried out metabolomic analysis of Ephedra species using 1H-NMR spectroscopy and multivariate data analysis, and they have shown that major differences in different
*Corresponding author. Email:
[email protected]
ISSN 1478–6419 print/ISSN 1029–2349 online ß 2010 Taylor & Francis DOI: 10.1080/14786410802680902 http://www.informaworld.com
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species were due to benzoic acid analogues in the aqueous fraction and ephedrine-type alkaloids in the organic fraction. Floradal and Svensson (1992) have reported some haemostatic effects of ephedrine. The plant has bronchodilator activity and is also used for 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 that ephedrine releases dopamine in the substantia nigra. The phenolic compounds are known to impart antioxidant activity to the plant extract. It has been reported that mast cells generate intracellular reactive oxygen species in response to antigen challenge and this may be involved in histamine release (Matsui et al., 2000). In view of this literature support, we evaluated antihistaminic, anti-inflammatory, antinociceptive, hypotensive, antioxidant and locomotor stimulant activity of the ethanol : acetone extract of the aerial parts of EN.
2. Results 2.1. Chemical analysis The phytochemical characterisation of alkaloids from the EN ethanolic extract was carried out using 1H-NMR spectroscopy. It allows rapid and simultaneous determination of the ephedrine analogues according to Veerporte analysis (Kim et al., 2003). H-1 was selected as a target signal in 1H-NMR because these signals of ephedrine analogues have separated resonances and do not overlap with other signals from extract. The region of 4.0–5.0 is considered when CDCl3 is used because no interference with other peaks is observed in the 1 H-NMR spectrum. The H-1 signal is most suitable for use as a target peak compared to the other signals which appeared in the C-methyl region ( ¼ 0.6–1.0) and in the N-methyl region ( ¼ 2.0–2.4). Therefore, the diastereomers can be differentiated by the coupling constants of chemical shifts. The compounds which have (S,S) configuration show a larger coupling constant than those of a (R,S ) configuration, because the dihedral angle between two protons at C-1 and C-2 is quite different. The obtained chemical shifts are verified by data in existing literature. The chloroform fraction which was derived from the ethanolic extract of EN shows the presence of ephedrine (H-1 ¼ 4.77, J ¼ 3.9); pseudoephedrine (H-1 ¼ 4.19, J ¼ 8.4), methylephedrine (H-1 ¼ 4.96, J ¼ 3.8) and methylpseudoephedrine (H-1 ¼ 4.22, J ¼ 6.9) were also detected. When the relative intensity of the characteristic signals of N–CH3 as a singlet was compared, the signal of pseudoephedrine to that of ephedrine was high. Another signal at ¼ 4.52 (J ¼ 4.5) is related to the H-1 of norephedrine which precede ephedrine in the biosynthetic process (Gunnar & Ian, 1994). All such conclusions were confirmed by data obtained from 13C-NMR spectra. The ethyl acetate fraction contained a mixture of ephedrinic skeleton alkaloids. The 1 H-NMR signals for N–CH3 protons and for the C-3 methyl group confirmed the presence of the alkaloids here reported (Figure 1).
2.2. Antihistaminic activity The log dose response curve (DRC) showed that both the extract and diphenhydramine inhibited histamine-induced contraction significantly (p50.05), indicating presence of an antihistaminic principle in the ethanol : acetone extract (Figure 2).
Natural Product Research H
OH
R
2
N
1
H CH3
OH 2
1
HO NH2
H CH3
H CH3
3
3
R=H, Ephedrine R=CH3, Methylephedrine
Norephedrine
R
H 2
1 3
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N
CH3
H CH3
R=H, Pseudoephedrine R=CH3, Methylpseudoephedrine
Percentage contraction
Figure 1. Ephedrinic skeleton alkaloids contained in the ethyl acetate fraction.
120
Vehicle EN Diphenhydramine
100 80 60 40 20 0
7.26
6.96
6.66
6.36
Log of molar concentration of histamine
Figure 2. Effect of EN extract on histamine induced contraction of guinea pig tracheal chain preparation.
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 extract and diphenhydramine significantly decreased the eosinophilia (Table 1), indicating the adaptogenic potential of EN.
2.4. Anti-inflammatory activity In rats that received vehicle, the volume of oedema was 2.54 mL at the third hour. Ibuprofen reduced paw oedema by 45.12%, whereas the extract of EN (100 and 200 mg kg1) 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 (100 mg kg1, i.p.) reduced the number of writhes to 25.6 6.5, whereas the higher dose of the extract (200 mg kg1, i.p.) reduced the amount of writhing to 19.2 5.5. In rats that received Ibuprofen, the amount of writhing was of 21.5 4.7 (Table 3).
2.6. Blood pressure A dose of 10 mcg of extract of EN reduced blood pressure by 14 mmHg, showing a hypotensive effect.
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WBC count (mean SEM)
Eosinophil count (mean SEM)
2012 223.0 1290 56.0* 987.5 34.5* 605 43* –
120.2 10.2 75.0 8.35 * 52.0 5.8* – 14.2 2.5 *
Vehicle EN (100) EN (200) Diazepam (1) Diphen (1)
Notes: n ¼ 5, EN=Ephedra nebrodensis. The values are the difference in WBC count, before and 24 h after of drug administration. *p50.001, compared to milktreated group (one-way 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
0h
1h
2h
3h
% inhibition of paw oedema at 3 h
Vehicle EN (100) EN (200) Ibu (40)
0.9 0.04 0.9 0.03 0.89 0.05 0.9 0.05
1.7 0.03 1.3 0.02 1.2 0.04 1.4 0.05
1.9 0.02 1.7 0.05 1.6 0.04 1.6 0.03
2.54 0.06 2.1 0.04 1.9 0.06 1.8 0.04
– 26.8* 38.4* 45.12*
Notes: n ¼ 5, EN ¼ Ephedra nebrodensis, Ibu ¼ Ibuprofen. All drugs were given orally 60 min before subplantar injection of carrageenan. *p50.01 (ANOVA followed by Dunnett’s test).
Table 3. Effect of EN extract on acetic acid-induced writhing in mice.
Treatment
Number of writhes in 30 min (mean SEM)
Vehicle EN (100) EN (200) Ibu (40)
42.5 8.2 25.6 6.5* 19.2 5.5* 21.5 4.7*
Notes: n ¼ 5, EN ¼ Ephedra nebrodensis, Ibu ¼ Ibuprofen. All drugs were given orally 60 min before intraperitoneal injection of acetic acid. *p50.01 (ANOVA followed by Dunnett’s test).
2.7. Rat stereotyped and locomotor activity The ethanol : acetone (1 : 1) extract (200 mg kg1, i.p.) as well as amphetamine (0.25 mg kg1, 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 observed on rearing. Both the extract and amphetamine increased rearing and stereotyped behaviour significantly (p50.05 versus saline) (Figures 4 and 5, respectively).
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Locomotion
Total counts (1h)
4000
*
3000
*
2000 1000 0 Saline
Amphet. 0.25 mg
EN
1
Figure 3. Effect of amphetamine 0.25 mg kg s.c. (n ¼ 4) and of the extract of EN 5g kg1 o.s. (n ¼ 5) on the motor activity. Data are mean SEM of the photocell counts over a 1 h test. *p50.05 as compared to saline controls (n ¼ 4). Rearing
*
Number (1h)
50
*
25
0
Saline
Amphet. 0.25 mg
EN
1
Figure 4. Effect of amphetamine 0.25 mg kg s.c. (n ¼ 4) and of the extract of EN 5g kg1 o.s. (n ¼ 5) on the rearing. Data are mean SEM of the number of items over a 1 h test. *p50.05 as compared to saline controls (n ¼ 4). Stereotyped behaviour *
3
Score (1h)
* 2
1
0
Saline
Amphet. 0.25 mg
EN
1
Figure 5. Effect of amphetamine 0.25 mg kg s.c. (n ¼ 4) and of the extract of EN 5g kg1 o.s. (n ¼ 5) on the stereotyped activity. Data are mean SEM of the score over a 1 h test *p50.05 as compared to saline controls (n ¼ 4).
2.8. Antioxidant activity The extract showed notable antioxidant activity but the IC50 was high when compared with the positive control, butyl-hydroxy-anisole (BHA). The ethanol : acetone extract showed less potent antioxidant activity, with an IC50 value of 41.35 mg mL1, whereas the IC50 of BHA was 14.65 0.08.
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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 42 mg 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 (2.0 mg mL1 of bath volume) was obtained. Diphenhydramine hydrochloride (2.0 mg mL1 of bath volume) was used as the reference standard.
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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 200 mg kg1, i.p. each, respectively), and Group IV received diazepam (1 mg kg1, i.p.). After 30 min of drug treatment each animal was injected with buffalo milk (4 mL kg1, 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 (1 mg kg1, 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 (10 mL kg1 orally), EN extract (100 or 200 mg kg1 orally) or ibuprofen 40 mg kg1 orally 60 min before subplantar injection of carrageenan (0.1 mL 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 200 mg kg1, p.o.) or ibuprofen suspension (40 mg kg1, p.o.). After 60 min, the mice were injected intraperitoneally with 0.1 mL 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 15 min and a change in diastolic blood pressure was recorded after administration of 10 mcg 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
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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 in physiologic solution (5 g kg1); Group II saline solution; and Group III amphetamine (2.5 mg kg1, 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 the DPPH 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-test or 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 significant antihistaminic, adaptogenic, analgaesic, anti-inflammatory and hypotensive activities. The antihistaminic activity is complementary to the antiasthmatic activity due to the presence of ephedrine, a sympathomimetic molecule acts on adrenergic 2 receptors thereby reducing bronchial muscle tone. The extract showed a moderate adaptogenic activity. These results are in line with those of Chinese ephedra (Ma-Huang), in which the alcoholic extract showed antiallergic effect in vitro (Leung & Foster, 2000). The observed anti-inflammatory activity of EN is in congruence with data obtained from Ma-Huang. The phytoconstituents present in Ma-Huang like ephedrine, pseudoephedrine, ephedroxane and pseudoephedroxane have anti-inflammatory effect on experimentally-induced oedema (Kasahara, Hikino, Tsurufuji, Watanabe, & Ohuchi, 1985). Bruneton (1995) has also reported anti-inflammatory activity of ephedroxane and pseudoephedrine. The inhibition of acetic acid-induced writhing suggests that the EN acts peripherally (Erdem & Ku¨peli, 2002). Ephedradine, tiroxin, betaine and feruloistamine are hypotensive compounds of Ephedra species (Hikino, Ogata, Konno, & Sato, 1983a, 1983b) and in the total extract they probably are contained in a greater quantity than ephedrine. The hypotensive effect of the ethanol : acetone extract of EN can be due to a prevalence of d-pseudoephedrine over l-ephedrine. This observation was confirmed previously by Van Hellemont (1986), who ascribes d-pseudoephedrine only to the Mediterranean variety of Ephedra. It was demonstrated that Ephedra contains flavonoids and proanthocyanidols that influence absorption of ephedrinic compounds, making their pharmacological activities less intense but more enduring (Harada & Nishimura, 1991). Results obtained from the tests on stereotyped activity showed an amphetamine-like action of the acetone : ethanol extract of EN. This observation is in congruence with the findings of Munhall and Johnson (2006) that ephedrine releases dopamine from substantia nigra. Thus the observed increase in locomotion can be explained on the basis of increased dopamine release. Lida et al. (1990) have reported a dopamine D2 receptor-mediated antioxidant and neuroprotective effect of ropinirole, a dopamine agonist. In this study we
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noticed antioxidant activity of the extract. Lohr, Kuczenski, and Niculescu (2003) have also suggested a link between oxidative mechanisms and tardive dyskinesia, which will form the basis for further research. Therefore it is concluded that the ethanol : acetone extract of EN Tineo collected in Sardinia has antihistaminic, adaptogenic, antinociceptive, anti-inflammatory and antioxidant activities.
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