Central properties of the essential oil and the crude ethanol extract from aerial parts of Artemisia annua L

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Central properties of the essential oil and the crude ethanol extract from aerial parts of Artemisia annua L Article in Pharmacological Research · December 2003 DOI: 10.1016/S1043-6618(03)00216-0 · Source: PubMed

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Pharmacological Research 48 (2003) 497–502

Central properties of the essential oil and the crude ethanol extract from aerial parts of Artemisia annua L. F.F. Perazzo a,b , J.C.T. Carvalho a,∗ , J.E. Carvalho c , V.L.G. Rehder c a b c

Laboratório de Fitofármacos, Universidade de Alfenas, Rod. MG 179, km 0, CP 23, CEP 37130-000, Alfenas, MG, Brazil Department of Pharmacology, Anesthesiology and Therapeutics, Piracicaba Dentistry School, UNICAMP, São Paulo, Brazil Centro de Pesquisas Qu´ımicas, Biológicas e Agr´ıcolas-Universidade de Campinas, UNICAMP-Campinas, São Paulo, Brazil Accepted 13 June 2003

Abstract The present study evaluated the central activity of the essential oil and the ethanolic extract from Artemisia annua L. in animals as a part of a psychopharmacological screening of this plant. The extract was prepared with fresh leaves in ethanol (AEE) and the essential oil (AEO) was obtained by hidrodestilation. The ED50 and the LD50 obtained for the essential oil were 470 mg/kg (correlation coefficient r = 0.97333 and linear regression y = −26.52x + 0.158) and 790 mg/kg, and for the extract, 450 mg/kg (correlation coefficient r = 0.99266 and linear regression y = −27.34 + 0.156) and more than 2 g/kg, respectively. The doses increased the latency time to convulsions induced by picrotoxin and pilocarpine but prevented the onset of pentylenotetrazol and strychnine induced seizures. In addition to, the products have caused marked inhibition in the Rota-rod assay. According to the results, the AEO has a high acute toxicity and a possible cholinergic action, and the AEE showed a possible central activity as dopaminergic and cholinergic agents, and did not present a significant acute toxicity. These differences should be due to chemical substances present in each product. These products had no significant effect as an anticonvulsant, while exhibited a strong depressant activity on the CNS. © 2003 Elsevier Ltd. All rights reserved. Keywords: Artemisia annua; CNS; Essential oil; Ethanolic extract; Rota-rod; Induced seizures

1. Introduction Artemisia annua L. (Asteraceae) has been used in the Chinese medicine for hundred of years. The infusion is used in the treatment of malaria disease and fever [1]. This plant contains an essential oil that is mainly composed of linalool, 1,8-cineol, p-cymene, thujone, and camphor [2]. This last compound produces excitation on the central nervous system (CNS), while others produce depression, reduce spontaneous activity, and increase the hypnotic action of pentobarbital [3]. When these compounds were studied to evaluate its potential on the CNS, they presented a facility to cross biological membranes due to its high liposolubility. Eugenia caryophyllata essential oil has produced depression on animal used to evaluate anticonvulsant drugs [4]. Occhiuto et al. [5] showed that the non-volatile residue of Citrus bergamia has a depressive action on the CNS. Elisabetsky et al. [6] conclude that linalool, a major monoterpenoid of several essential oils present in aromatic plants, have a high ∗ Corresponding

author. Fax: +55-21-353-299-3239. E-mail address: [email protected] (J.C.T. Carvalho).

1043-6618/$ – see front matter © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S1043-6618(03)00216-0

depressive activity, as galphimine B, a triterpenoid isolated from Galphimia glauca [7]. The essential oil obtained from Anacardium occidentale is a high CNS depressor when studied on behavioral basis [8]. Even A. annua is regarded as safe for Chinese medicine for hundred of years in the treatment of malaria disease and fever [9], all these data suggest that the compounds above might affect the CNS. This document reports the results of experiments carried out to investigate this possibility. Such activity has not been previously mentioned in such literature.

2. Material and methods 2.1. Plant material Leaves of A. annua L. (hybrid CPQBA 2/39 × PL5) were collected in April 2001 at Centro Pluridisciplinar de Pesquisas Qu´ımicas, Biológicas e Agr´ıcolas (CPQBA)– UNICAMP, from the experimental field located in Paul´ınia, SP, Brazil. A voucher specimen is deposited at CPQBA/ UNICAMP, under registration number CPQBA-12.46.

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2.2. Preparation of extract

2.6. Determination of ED50

Fresh leaves of A. annua (450 g) were submitted to dynamic maceration with ethanol (4000 ml, 99.0%) during 4 h. The macerate was filtered and this procedure was repeated twice. Concentration of the extracts under reduced pressure gave 51.07 g (yield 11.35%) of crude ethanolic extract (AEE). The crude dried extract was suspended in 3% Tween 80 0.9% saline solution (100 mg AEE/ml) [10].

The ED50 was determined based on the picrotoxin (6 mg/kg) induced convulsion described by Perazzo et al. [14]. Groups of mice (n = 6) were treated i.p. with AEO and AEE 30 min before the application of the stimulus. Several doses were administered (300, 400, 500, and 600 mg/kg) and the ED50 was determined from the curve for convulsion potentiation percentage, in function of the dose.

2.3. Extraction of essential oil The essential oil was obtained using the method I of the Brazilian Pharmacopoeia [11]. Fresh leaves of A. annua (500 g) were distilled for 3 h using a Clevenger apparatus, with 1.28% yielding. The obtained essential oil (AEO) was added to a 5% Tween 80 0.9% saline solution (100 mg AEO/ml) just before administration to animals. 2.4. Phytochemical analysis by combined gas chromatography–mass spectrometry (GC–MS) The essential oil was submitted to quantitative analysis in a Hewlett-Packard 5890 Model II automated gas chromatograph mass spectrometer data system with selective mass detector Hewlett-Packard 5971. GC conditions: carrier gas, helium at flow rate of 1.0 ml min−1 ; sample size, 2 ␮l injected using the splitless injection technique; fused capillary silica column HP-S (25 m × 0.20 mm × 0.33 ␮m). Temperatures: injector = 220 ◦ C, detector = 280 ◦ C, column = 60 ◦ C, 3 ◦ C min−1 , 240 ◦ C (7 min). The MS was taken at 70 eV. The main constituents were identified by comparison with the mass spectrums from Wiley and Nist 98 spectrum library [12]. The essential oil was also analyzed using a HewlettPackard 5890 Model II automated gas chromatograph with flame ionization detector. GC conditions: carrier gas, helium at flow rate of 1.0 ml min−1 ; sample size, 2 ␮l injected using the splitless injection technique; fused capillary silica column HP-S (25 m × 0.20 mm × 0.33 ␮m). Temperatures: injector = 220 ◦ C, detector = 280 ◦ C, column = 60 ◦ C, 3 ◦ C min−1 , 240 ◦ C (7 min). The identification of the main constituents was made by comparison of the obtained retention times (Rt ) [13] in comparison with previously injected known compounds. 2.5. Animals Male rats (Rattus norvegicus, Albinus, Wistar) and male mice (Mus musculus, Albinus, Swiss) were used, specific pathogen free, weighing 150–200 and 20–25 g, respectively. The animals were acquired from the Animal Experimental Center of Campinas State University. The animals were kept in five animal groups in polyethylene boxes, in a climatic environment (23 ± 2 ◦ C), air humidity control (53 ± 2%), in 12-h-dark/12-h-light control, with food and water “ad libitum,” for at least 7 days before the experiments.

2.7. Determination of LD50 Mice (n = 10) were given single doses at different concentrations (500, 750, 1000, 1250, 1500, and 2000 mg/kg) of A. annua essential oil and ethanolic extract to determine the median lethal dose (LD50 ). These animals were observed during a 48-h period. The number of animals, which died during this period, was expressed as a percentile, and the LD50 was determined by probit test using death percent versus dose’s log [15]. 2.8. Rota-rod The method used was the one described by Lima et al. [10]. Groups of eight rats each were previously selected for their ability to remain on the revolving bar of a Rota-rod apparatus over a 2-min period. These animals were treated with a dose of AEO (470 mg/kg) and AEE (450 mg/kg), or saline solution and tested on the Rota-rod with interval of 15 min in a total of a treatment of 3 h. The parameter registered was the time spent on the bar at different interval of time. 2.9. Chemically induced convulsions 2.9.1. Pentiylenotetrazol induced seizures The experiment used the method described by Goodman et al. [16]. In such experiment, groups of mice (n = 8) were treated with AOE (470 mg/kg), AEE (450 mg/kg), or saline solution (i.p. route) 30 min before the administration of pentylenotetrazol (85 mg/kg, i.p.). The time before the onset of clonic seizures was registered. 2.9.2. Picrotoxin induced seizures The method described by Abdul-Ghani et al. [17] was used. Groups of mice (n = 8) were treated with AEO (470 mg/kg), AEE (450 mg/kg), or saline solution (i.p. route) 30 min before the administration of the convulsing drug (picrotoxin 6 mg/kg, i.p.). The time before the onset of clonic seizures was registered. 2.9.3. Strychnine induced seizures The method described by Vohora et al. [18] was used. Groups of mice (n = 8) were treated with AEO (470 mg/kg), AEE (450 mg/kg), or saline solution (i.p. route) 30 min

F.F. Perazzo et al. / Pharmacological Research 48 (2003) 497–502

before the administration of the convulsing drug (strychnine 2 mg/kg, s.c.). The time before the onset of clonic seizures was recorded. 2.9.4. Pilocarpine induced seizures The methodology used was described by Lima et al. [10]. Three groups of mice were treated with AEO (470 mg/kg), AEE (450 mg/kg), or saline solution (i.p. route) 30 min before the administration of the convulsing drug (pilocarpine 300 mg/kg, i.p.). The time before the onset of clonic seizures was registered. 2.10. Statistical analysis The statistical analyses were done using ANOVA followed by Tukey–Kramer multiple comparison test [19]. The results with P < 0.05 were considered significant. The ED50 and LD50 methods determination were done by linear regression model I [19]. The data are expressed as mean ± S.D.

3. Results The chromatographic analysis of the AEO showed 15 terpenic compounds, including 1,8-cineol (20.42%), camphor (22.68%), linalool (3.82%), and p-cymene (12.21%) as major monoterpenic constituents. The major sesquiterpenic compounds identified were germacrene D (3.54%), and trans-caryophyllene (2.08%) (Table 1). Relative percentages were obtained from GC–MS analysis. The chromatogram should be seen in the Fig. 1. The AEE was analyzed to compare the composition of both extracts. The AEE analysis showed a few components for identification by GC. It was possible to iden-

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tify camphor (Rt 4.24 min), ␤-cubeben (Rt 10.7 min) and trans-caryophyllene (Rt 9.40 min). The major compound identified was the phytol (Rt 24.14 min). The treatment with AEO produced a dose-dependent effect (correlation coefficient r = 0.97333 and linear regression y = −26.52x+0.158). The ED50 value was 470 mg/kg. The LD50 determined for the essential oil was 790 mg/kg. The ethanolic extract showed an ED50 of 450 mg/kg (correlation coefficient r = 0.99266 and linear regression y = −27.34 + 0.156), and the LD50 was more than 2.0 g/kg. The picrotoxin administration has shown a latency time for the convulsion onset of the control group (18.05±1.55 s) greater than the latency time induced by AEO and AEE (14.27±1.75 s and 10.42±1.46 s, respectively). Both groups were statistically different. The pilocarpine treatment did not show difference between AEO and AEE (7.39 ± 0.55 s and 7.22 ± 0.67 s), but they were significant different from the control group (10.54 ± 0.65 s, P < 0.05). The same could be seen when PTZ was administered. The control group has presented a latency time for the onset seizures (1.59 ± 0.26 s) lower than the one caused by the A. annua extracts in this study (AEO 12.36 ± 1.18 s and AEE 11.17 ± 1.59 s). The strychnine induced seizures presented a difference among all groups. The control has shown a latency time (1.18±0.07 s) lower than the AEE (2.66±0.36 s). The AEO showed a higher latency time (3.41 ± 0.40 s) when all the groups were compared (P < 0.05). The Rota-rod assay showed that the group treated with AEO was different when compared to the control in all experimentation time (3 h). The animals treated with AEE began to show differences after 30 min of treatment, and it would be noticed by the end of the experiment. The treated groups began to show differences after 45 min of research.

Table 1 Main constituents of A. annua essential oil Number

Retention time (Rt )

Identification

RI

Relative percentage

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

7.557 8.045 8.835 8.980 9.337 10.987 12.069 13.739 15.873 17.900 25.489 28.388 29.844 31.053 31.286

␣-Pinene Canfene Sabibene ␤-Pinene p-Cymene 1,8-Cineol ␣-Terpinene Linalool Camphor ␣-Terpineol Eugenol trans-caryophyllene Farnesene Germacrene D Bicyclogermacrene Compounds not identified

934.4 949.5 974.0 978.6 989.8 1032.0 1057.9 1098.1 1146.5 1192.0 1359.4 1425.0 1458.8 1486.8 1492.0

3.67 5.31 5.44 0.51 12.21 20.42 1.28 3.82 22.68 0.78 0.20 2.08 1.90 3.54 0.19 15.97

Retention time (Rt ); identified compounds; retention index (RI); relative percentage of the compounds.

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Fig. 1. Gas chromatography chromatogram of A. annua essential oil.

4. Discussion and conclusion The treatment with increasing doses of AEO and AEE (i.p.) 30 min before the application of the convulsing stimulus (picrotoxin), produced a dose-dependent effect, in which the ED50 was 470 mg/kg for the essential oil and 450 mg/kg for the ethanolic extract. LD50 was determined in function of the i.p. administration of several AEO and AEE doses, and the occurrence of deaths observed during 48 h. The lethal dose of AEO was 790 mg/kg and more than 2000 mg/kg for the AEE. Under these doses, the animals showed the following symptoms: stereotypy, convulsion, and ataxia. The genesis of the convulsion originated due to picrotoxin action which involves the antagonistic effect of this drug in gabaergic receptors and they have an important role in convulsions [20,21]. The administration of picrotoxin increased the latency time of the onset convulsion in 21% for AEO and 42% for AEE (Fig. 2). When PTZ was administered, the drugs decreased the latency time, but did not reduce the death percentage (100%). This result should be seen in the Fig. 3. Browning and Nelson [22] showed the mechanism of action of this drug considering two distinct neural paths, one located in the forebrain, decreasing the gabaergic activity, which sends a signal to the respiratory center, this one is involved in the convulsion onset, and another, increasing

Fig. 2. Effect of i.p. administration of AEO (470 mg/kg) and AEE (450 mg/kg) on the convulsion induced by picrotoxin (6 mg/kg, i.p.). Each bar representing the mean ± S.D. (n = 8). a P < 0.05 significantly different from the control; b P < 0.05, Tukey’s test; significantly different from the control and AEO.

Fig. 3. Effect of i.p. administration of AEO (470 mg/kg) and AEE (450 mg/kg) on the convulsion induced by PTZ (85 mg/kg, i.p). Each bar representing the mean ± S.D. (n = 8). a P < 0.05, Tukey’s test; significantly different from the control.

dopamine liberation, this one is not related with convulsions. The potentiation of the picrotoxin action by AEO and AEE should not be related to GABA receptors, once there was interference in the PTZ onset, but might be done to central mechanisms not related with GABA, therefore the mechanisms together can make a decrease in the excitability threshold, producing the convulsion onset. The presence of camphor can be involved in this response. Anyway, A. annua extracts shall not present affinity by gabaergic receptors, without any interference in this pathway. When pilocarpine, an agonist for cholinergic receptors, was used as convulsing agent, it has decreased the onset of the convulsion of AEO and AEE (Fig. 4). Generally, pilocarpine administration induces to cholinergic symptoms in the CNS (tremor) and perifericaly (salivation), which could be noted in the animals treated with the drug in this research. Eugenia caryophyllata essential oil prevented the onset seizure induced by PTZ [4]. Lima et al. [10] has demonstrated that the hydroalcoholic extract of Artemisia verlotorum prevented the convulsion induced by PTZ, but increased the latency time for the seizures generated by pilocarpine, as happened with the products obtained from A. annua in this study. Thus, the results obtained for this assay suggested that the stimulant activity caused by AEO and AEE could be

F.F. Perazzo et al. / Pharmacological Research 48 (2003) 497–502

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ergic like mechanisms in the CNS, and showed a low LD50 . Acknowledgements We are grateful to Dr. Pedro Melillo Magalhães (Agrotechnology Division) for the samples used in this research. Fig. 4. Effect of i.p. administration of AEO (470 mg/kg) and AEE (450 mg/kg) on the convulsion induced by pilocarpine (300 mg/kg, i.p.). Each bar representing the mean ± S.D. (n = 8). a P < 0.05, Tukey’s test; significantly different from the control.

involved with cholinergic mechanisms, because the latency time induced by pilocarpine was decreased. The inhibition of Renshaw cells (interneurons) is related with convulsions when glicine receptors are blocked. Strychnine is a glicine receptors antagonist, and when these receptors are blocked, Renshaw cells do not regulate the inhibitory process, causing convulsion under any stimulus, including optical, sound, or physic. In cases of intoxication, glicine administration or restness can inhibit this process [23]. Considering that both AEO and AEE inhibited this convulsing process (Fig. 5), it is possible that these extracts do not have capacity of binding to glicine receptors as agonists, but as antagonists, increasing the convulsion onset. In another way, the increase of the latency time for the convulsion can be generated by a depressive activity on the CNS not related with GABA mechanisms. Tortoriello and Ortega [7] demonstrated a triterpenoid (galphimine B) with depressant activity, but not anticonvulsant when strychnine was used as stimulus. These data lead us to conclude both the essential oil and crude ethanolic extract of A. annua have a depressant activity, but not an anticonvulsant activity. It is possible to suggest that the AEO has a cholinergic action, as well as a high acute toxicity. The AEE can present cholinergic and dopamin-

Fig. 5. Effect of i.p. administration of AEO (470 mg/kg) and AEE (450 mg/kg) on the convulsion induced by strychnine (2 mg/kg, s.c.). Each bar representing the mean ± S.D. (n = 8). a P < 0.05 significantly different from the control; b P < 0.05, Tukey’s test; significantly different from the control and AEO.

References [1] Mueller MS, Karhagomba IB, Hirt HM, Wemakor E. The potential of Artemisia annua L. as a locally produced remedy for malaria in the tropics: agricultural, chemical and clinical aspects. J Ethnopharmacol 2000;73:487–93. [2] Carnat AP, Gueugnot J, Lamaison JL, Guillot J, Pourrat H. Annais des Pharmaceutiques Françaises 1985;43:397–405. [3] Robbers JE, Speedie MK, Tyler VE. Terpenoids. In: Pharmacognosy and pharmacobiotechnology. New York: Williams & Wilkins; 1996. p. 79–104, 375. [4] Pourgholami MH, Kamalinejad M, Javadi M, Majzoob S, Sayyah M. Evaluation of the anticonvulsant activity of the essential oil of Eugenia cariophyllata in male mice. J Ethnopharmacol 1999;64:167– 71. [5] Occhiuto F, Limardi F, Circosta C. Effect of the non-volatile residue from the essential oil of Citrus bergamia on the central nervous system. Int J Pharmacognosy 1995;33(3):198–203. [6] Elisabetsky E, Coelho de Souza GP, dos Santos MAC, Siqueira IR, Amador TA. Sedative properties of linalool. Fitoterapia 1995;LXV(5):407–14. [7] Tortoriello J, Ortega A. Sedative effect of Galphimine B, a nor-secotriterpenoid from Galphimia glauca. Planta Medica 1993;59:398– 400. [8] Garg SC, Kasera HL. Neuropharmacological studies of the essential oil of Anacardium occidentale. Fitoterapia 1984;LV(3):131–6. [9] Klayman DL. Qinghaosu (artemisinin): an antimalarial drug from China. Science 1985;228:1049–55. [10] Lima TCM, Morato GS, Takahashi RN. Evaluation of the central properties of Artemisia verloturum. Planta Medica 1993;59: 326–9. [11] Brazilian Pharmacopea, 2a ed.; 1959. [12] Juteau F, Masotti V, Bessière JM, Viano J. Compositional characteristics of the essential oil of Artemisia campestris var. glutinosa. Biochem Syst Ecol 2002;30(11):1065–75. [13] Adams RP. Identification of essential oil components by gas chromatography/mass spectroscopy, 1a ed. Illinois, USA: Allured Publishing Corp.; 1995. p. 1112. [14] Perazzo FF, Carvaiho JE, Rehder VLG. Efeito central do óleo essencial e extrato etanólico de A. annua. XVI Reunião Anual da Federação de Sociedades de Biologia Experimental, FeSBE 2001. [15] Thompson WR, Weil CS. On the construction of tables for moving-average interpolation. Biometrics 1952;8:51–4. [16] Goodman LS, Grewal MS, Brown WC, Swinyard EA. Comparison of maximal seizures evoked by pentylenotetrazol (metrol) and electroshock in mice, and their modification by anticonvulsants. J Pharmacol Exp Ther 1953;108:168–76. [17] Abdul-Guani AS, El-Lati SG, Sacaan AI, Suleiman MS, Amin RM. Anticonvulsant effects of some Arab medicinal plants. Int J Crude Drug Res 1987;1(25):39–43. [18] Vohora SB, Shaukat AS, Dandya PC. Central nervous system studies on ethanol extract of Acorus calamus rhizomes. J Ethnopharmacol 1990;28:53–62.

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[19] Sokal RR, Rohlf FJ. In: Freeman WH, editor. Biometry. San Francisco; 1995. p. 175–205, 404–86. [20] Leidenheimer NJ, Browning MD, Harris RA. GABAa receptor phosphorylation: multiple sites, actions and artifacts. Trends Pharmacol Sci 1991;12:84–7. [21] Gale K. GABA and epilepsy: basic concepts from preclinical research. Epilepsia 1992;33(Suppl 5):S3–S12.

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[22] Browning RA, Nelson OK. Modification of electroshock and pentylenetetrazol seizure pattern in rats after precollicular transection. Exp Neurol 1986;93:546–56. [23] Gilman AG, Limbird LE. The pharmacological basis of therapeutics, 9 ed. NY, USA: McGraw-Hill; 1996.

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