ACUTE TOXICITY AND ANALGESIC ACTION OF THE ESSENTIAL OIL OF NEPETA AMETHYSTINA

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1 1 P a g e International Standard Serial Number (ISSN): International Journal of Universal Pharmacy and Bio Sciences 3(3): May-June 2014 INTERNATIONAL JOURNAL OF UNIVERSAL PHARMACY AND BIO SCIENCES IMPACT FACTOR 1.89*** Pharmaceutical Sciences ICV 5.13*** RESEARCH ARTICLE!!! ACUTE TOXICITY AND ANALGESIC ACTION OF THE ESSENTIAL OIL OF NEPETA AMETHYSTINA T.Belabda 1, 3, K.Alaoui 2, El H.Bouidida 2, 3, Y.Cherrah 2, M S.Taleb 4, M.Arahou 1, R.Hassikou 1 1 Laboratory of Botany, Mycology and Environment, Faculty of Sciences, University Mohammed V -Agdal, Rabat, Morocco. 2 Research Team of Pharmacodynamics RTP, Laboratory of Pharmacology and Toxicology, Faculty of Medicine and Pharmacy, University Mohammed V- Souissi, Rabat, Morocco. 3 National laboratory of Drugs Controlled, Direction of Medicine and Pharmacy, Ministry of Health, Rabat, Morocco. 4 Department of Botany and Plant Ecology, Scientific Institute, University Mohammed V, Rabat, Morocco. KEYWORDS: Nepeta amethystina, essential oil, acute toxicity, analgesic activity. For Correspondence: T.Belabda * Address: Laboratory of Botany, Mycology and Environment, Faculty of Sciences, University Mohammed V -Agdal, Rabat, Morocco.. Id: belabda2010@gmail.com ABSTRACT The ethno-pharmacological survey conducted in Morocco shows that the species Nepeta amethystina is used in traditional medicine for its tonic activities, carminative, sedative, antispasmodic, antiseptic, and against the bites of snakes and scorpions. Our goal is to study the acute toxicity and analgesic activity of the essential oil EO of N.amethystina compared with previously published results, at the end of studies in Morocco on acute toxicity and analgesic action of EO of Nepeta atlantica Ball and Nepeta tuberosa. L.ssp. reticulata (Desf.) Maire. The study of acute toxicity shows a LD 50 of this EO as 2000mg/kg IP. The result of studies of analgesic activities give this species a power analgesic peripheral higher than acetylsalicylic acid (200mg/kg IP) from 50 mg/kg IP, while the central analgesic action morphine-like appears at the dose of 25 mg/kg IP, and is maximal at 50mg/kg IP.

2 2 P a g e International Standard Serial Number (ISSN): INTRODUCTION: Gender NEPETA (family Lamiaceae) comprises about 400 species, most of which grow in Central and meridian Europe, North Africa, Central and South Asia 1, 2. A large number of species of this genus is used in traditional medicine for its antiseptic and astringent properties, as a topical remedy against skin rashes in children, and against the bites of snakes and scorpions 3, 4. Orally, these species are used as antitussive, antispasmodic, anti-asthmatic, antipyretic and diuretic 5, 6. The ethno-pharmacological survey we conducted among the local population, at the time of harvest of the species of Nepeta amethystina reveals that she is used in traditional medicine for its tonic activities, carminative, sedative, antispasmodic, antiseptic, and against the bites of snakes and scorpions. In a recent work, Bouidida et al. showed that the essential oils of Nepeta atlantica Ball and Nepeta tuberosa. L.ssp. reticulata (Desf.) Maire have a strong analgesic effect peripheral and a power central analgesic morphine like 7. These results have inspired this study of analgesic potency of essential oil (EO) of another species not yet studied; it is the species Nepeta amethystina. The goals of this work are: The determination of the 50% lethal dose or LD 50 of EO of Nepeta amethystina, The study of central and peripheral analgesics activities of EO of N. amethystina, The comparison of the LD 50 and central and peripheral analgesics activities of EO of N. amethystina with LD 50 and central and peripheral analgesics activities of EO of Nepeta atlantica Ball and Nepeta tuberosa. L.ssp. reticulata (Desf.) Maire published. 2. Materials and methods: 2.1. Plant material: The species chosen was harvested during the flowering period (June-July) in the Middle Atlas of Morocco, in the region of BOUIBLANE (TAMATROUCHTE); identification was performed by botanists of the Scientific Institute of Rabat (identification code: 78994). After harvesting, the whole plant is dried form spread away from sunlight at room temperature. It is regularly returned for an average of 10 days, this is necessary to increase the yield of secondary metabolites Obtaining essential oil: The plant material is coarsely cut at its aerial parts (leaves, stems, flowers and twigs) and then subjected to steam distillation of water for 5 hours. The recovered aqueous phase (containing EO) is subjected to

3 3 P a g e International Standard Serial Number (ISSN): liquid/liquid extraction using ethyl acetate as solvent (two cycles); EO is recovered after removal of all traces of solvent by passage in Rotavapor and then stored in vials tinted in the refrigerator Animals: Animals tested are from Central Pet shop of the Faculty of Medicine and Pharmacy of Rabat. The study was conducted in accordance with accepted principles outlined in the «Guide for the Care and use of Laboratory Animals» prepared by the National Academy of Sciences and published by the National Institutes of Health and efforts were made to minimize animal suffering and number of animals used Acute toxicity tests: The acute toxicity study was carried out on IOPS Ofa mice adult females, nulliparous and non-pregnant, aged 2 to 2.5 months, weighing 20 to 30g, these females, healthy, are fasting food for 4 h before test and 3 hours after Study of the peripheral analgesic activity: The study of the peripheral analgesic activity was carried out on IOPS Ofa mice in both sexes, in equal numbers, aged 2 to 2.5 months, weighing 20 to 30g, females are not pregnant, these animals are healthy and are fasting food for 18 hours before and 3 hours after administration of the product Study of the central analgesic activity: The study of the central analgesic activity was carried out on Wistar rats of both sexes, in equal numbers, aged 3 to 4 months, weighing 200 to 300g, females are not pregnant, these animals are healthy and are fasting food for 18 hours before and 3 hours after administration of the product Products : - Essential oil (EO) was tested as an aqueous solution at different concentrations, with adding a few drops of the TWIN 80 to obtain a homogeneous emulsion for concentrated solutions (2000mg/kg), aqueous solutions of EO being administered intraperitoneally (IP); - Glacial acetic acid 3% (300 mg/kg) was administered intraperitoneally (IP); - Acetylsalicylic acid (Aspirin) at 200 mg/kg was administered intraperitoneally (IP); - Morphine solution at 5 mg/kg was administered subcutaneously (SC) Acute toxicity tests: The use of animals was conducted in accordance with the guideline of the OECD (Organization for Economic Cooperation and Development) for testing chemicals - class method of acute toxicity, adopted December 17, 2001, which adopts a sequential process using three animals of a single sex per step 8.

4 4 P a g e International Standard Serial Number (ISSN): Housing conditions: Female mice are maintained under constant conditions of temperature (22 ± 3 C); the relative humidity is between 40 to 70%. Artificial lighting is used, the sequence being 12 hours light, 12 hours dark. Access to food and water ad libitum Choice of the initial dose: The OECD guideline of 17 December 2001 specifies that in the absence of data on the toxicity of the test substance, the starting dose is recommended for reasons related to the welfare of animals is 300 mg/kg. So we chose the dose of 300mg/kg as starting dose Choice of injection route: Given that recent work Bouidida et al. studied the acute toxicity of EO of other species of the genus NEPETA using the intraperitoneal route of administration as, to compare, we are required to use the same route of administration. To this end, we made 4 batches each comprising three female mice: - Each mouse of batch 1 receives a dose of 300mg/kg of EO of N.amethystina in a volume of 20 ml/kg IP; - Each mouse of batch 2 receives a dose of 300mg/kg of EO of N.amethystina in a volume of 20 ml/kg IP (dose confirmation); - Each mouse of batch 3 receives a dose of 2000mg/kg of EO of N.amethystina in a volume of 20 ml/kg IP; - Each mouse of batch 4 receives a dose of 2000mg/kg of EO of N.amethystina in a volume of 20 ml/kg IP (dose confirmation); The animals were kept under observation for 15 days, in which are recorded the changes in body weight, signs of toxicity and mortality. Determining the lethal dose LD 50 was made according to the decision tree in Annex 2c (test pattern with an initial dose of 300 mg/kg) of the guideline OECD of 17 December Study of the peripheral analgesic activity: The study of the peripheral analgesic activity is based on the Koster test seeking any protection against cramps induced by intraperitoneal injection of glacial acetic acid 3% (300mg/kg IP) with a maximum limit to 0.1ml for each mouse 9, 10. Four batches were formed, namely: 1 control batch: This sample of 6 mice received intraperitoneal injection of 3% glacial acetic acid (300 mg/kg) in a volume of 0.1ml for each mouse.

5 5 P a g e International Standard Serial Number (ISSN): Reference batch: This group of 6 mice given 30 min before the injection of glacial acetic acid 300 mg/kg IP, the reference substance is acetyl salicylic acid (Aspirin) to 200mg/kg by intraperitoneal injection, in a volume of 0.2 ml for each mouse. 2 batches trials: a test batch for each assay: o A batch of 6 mice receiving the EO of Nepeta amethystina in aqueous solution at 50mg/kg IP; o A batch of 6 mice receiving the EO of Nepeta amethystina in aqueous solution at 25mg/kg IP; These 2 batches are given 30 min after IP injection of aqueous solutions, the solution of glacial acetic acid 3% in the same conditions as the control and reference batches. It counts the cramps or abdominal twists for 20 minutes as soon as the IP injection of glacial acetic acid produced Study of the central analgesic activity: The study of the central analgesic activity morphine-like is based on the tail flick test, which consists of immersing the rat's tail in hot water (55 ± 2 C) and study before and after administration of the essential oil, the withdrawal reflex of the tail of the animal 11, Sort animals: It was established that the normal reflex time is 2 seconds. Therefore, only rats whose time reflex is less than or equal to two seconds will be retained for the experiment during a screening. To avoid heat adaptation during repetitive tests, rats are used for only one test. A tail withdrawal time equal or greater than 6 seconds shows an elevation of the pain threshold in the rat whether a central analgesic action of EO administered. Four batches were formed: 1 control batch: consists of a sample of 6 rats each receive 0.8 ml of a solution of distilled water administered intraperitoneally. 1 reference batch (Morphine): formed a sample of 6 rats each received 5 mg/kg SC of morphine. 2 batches tests: each consisting of 6 rats receiving EO of Nepeta amethystina respectively at 25 mg/kg IP and 50 mg/kg IP Statistical Analysis: The results are expressed as mean and standard deviation. Comparison of means was made using ANOVA single factor using SPSS for Windows 10 (SPSS.Inc) software.

6 6 P a g e International Standard Serial Number (ISSN): RESULTS: 3.1. Acute toxicity tests: Initial dose (300mg/kg): Intraperitoneal injection of EO of Nepeta amethystina at a dose of 300mg/kg induced after 10min a sedative effect in mice, which lasts about 30 min, clinical signs observed during the first 24 hours after injection of EO of Nepeta amethystina (300mg/kg IP) are summarized in Table 1. Table 1: Clinical signs observed during the first 24 hours after injection of EO of Nepeta amethystina (300mg/kg IP) Clinical Signs Mouse 1 Mouse 2 Mouse 3 Grouping of 3 mice x Sedative effect x x x Abdominal contractions x Immobility x x x Feeding _ Rapid breathing x Hind limb paralysis _ Legend: - = Absence of signs; x = Presence of signs; The mice suffered a loss of weight of 5 to 10% on average over five days, the weight recovery was watching from the 6th day of treatment for mice 1 and 2, and from the eighth day for mouse 3 (Figure 1).

7 7 P a g e International Standard Serial Number (ISSN): Figure 1: Evolution of the weight of 3 mice after injection of a dose of 300mg/kg IP of the solution of EO of Nepeta amethystina Weight of the Mouse 1 ( g) Weight of the Mouse 2 ( g) Weight of the Mouse 3 ( g) No deaths were recorded among the three mice Confirmation dose (300mg/kg): Intraperitoneal injection of EO of Nepeta amethystina at a dose of 300mg/kg for three mice of the second batch showed the same clinical signs observed in mice of the first batch, signs broken down in Table 2. Table 2: Clinical signs observed during the first 24 hours after injection of EO of Nepeta amethystina (300mg/kg IP) [dose confirmation] Clinical Signs Mouse 1 Mouse 2 Mouse 3 Grouping of 3 mice x Sedative effect x x x Abdominal contractions _ x _ Immobility x x x Feeding _ Rapid breathing _ x _ Hind limb paralysis _ The mice suffered a loss of weight of 5 to 10% on average, the weight recovery is watching from: - 5th day of treatment for the mice 1, - 6th day of treatment for the mouse 2, - 8th day of treatment for the mouse 3 (Figure 2).

8 8 P a g e International Standard Serial Number (ISSN): Figure 2: Evolution of the weight of 3 mice (2nd batch) after injection of a dose of 300mg/kg IP of the solution of EO of Nepeta amethystina Weight of the Mouse 1 ( g) Weight of the Mouse 2 ( g) Weight of the Mouse 3 ( g) No deaths were recorded among the three mice Third dose (2000mg/kg): Intraperitoneal injection of EO of Nepeta amethystina at a dose of 2000mg/kg caused a more pronounced sedative effect among three mice, causing the death of the mouse N 2 on the 3rd day (Table 3). Table 3: Clinical signs observed during the first 24 hours after injection of EO of Nepeta amethystina (2000mg/kg IP) Clinical Signs Mouse 1 Mouse 2 Mouse 3 Grouping of 3 mice x Sedative effect x x x Abdominal contractions x x x Immobility x x x Feeding _ Rapid breathing x x x Hind limb paralysis _ Mouse N 1 has suffered a weight loss of about 10% on average over 7 days, the weight recovery was watching from the 8th day of treatment; Mouse N 2 has suffered a weight loss of about 15% on average until his death on the 3rd day; Mouse N 3 has suffered a loss of weight of 10% on average over 8 days, the weight recovery was watching from the 9th day of treatment (Figure 3).

9 9 P a g e International Standard Serial Number (ISSN): Figure 3: Evolution of the weight of 3 mice after injection of a dose of 2000mg/kg IP of the solution of EO of Nepeta amethystina Fourth dose confirmation (2000mg/kg): Intraperitoneal injection of EO of Nepeta amethystina at a dose of 2000mg/kg caused a more pronounced sedative effect among three mice, causing the death of two mice: The mouse N 2 died on the 2nd day after the injection of the solution of EO of Nepeta amethystina (2000mg/kg IP); The mouse N 3 died on the 4th day after the injection of the solution of EO of Nepeta amethystina (Table 4). Table 4: Clinical signs observed during the first 24 hours after injection of EO of Nepeta amethystina (2000mg/kg IP) [dose confirmation] Clinical Signs Mouse 1 Mouse 2 Mouse 3 Grouping of 3 mice x Sedative effect x x x Abdominal contractions x x x Immobility x x x Feeding _ Rapid breathing x x x Hind limb paralysis _ x _ Mouse N 1 has suffered a loss of weight of about 10% on average over 6 days, the weight recovery was watching from the 7th day of treatment; Mouse N 2 has suffered a loss of weight of 15% on average until his death on the 2nd day after the injection of the solution of EO; Mouse N 3 has suffered a loss of weight of 15% on average until his death on the 4th day after injection of the solution of EO (Figure 4). Weight of the Mouse 1 ( g) Weight of the Mouse 2 ( g) Weight of the Mouse 3 ( g)

10 10 P a g e International Standard Serial Number (ISSN): Figure 4: Evolution of the weight of 3 mice (4th batch) after injection of a dose of 2000mg/kg IP of the solution of EO of Nepeta amethystina Weight of the Mouse 1 ( g) Weight of the Mouse 2 ( g) Weight of the Mouse 3 ( g) Determination of the Lethal Dose 50%: From the results obtained, and according to the decision tree in Annex 2c (test pattern with an initial dose of 300 mg/kg) of the OECD guideline of 17 December 2001, the LD 50 of EO of Nepeta amethystina is 2000mg/kg IP Study of the peripheral analgesic activity: The results of Koster test, summarized in Table 5 and Figures 5 and 6 show the average number of cramps due to glacial acetic acid recorded for a period of 20 min, the standard deviation and the percentage of protection (P) of the EO at the doses studied with: P = number of cramps of the control batch number of cramps of the batch trial number of cramps of the control batch 100. Table 5: Effects of essential oil of Nepeta amethystina on the number of cramps induced by glacial acetic acid in mice Control Reference ASA (200mg/kg IP) EO (N.amethystina ) 25mg/kg IP 17.83±0.75 Number of 60.50± ±0.75 cramps during 20 min % Protection % 70.52% 85.95% EO (N.amethystina ) 50mg/kg IP 8.5±1.04

11 11 P a g e International Standard Serial Number (ISSN): Figure 5: Number of cramps during 20 min induced by glacial acetic acid: : 70 Number of cramps during 20 min Control 30 Reference ASA (200mg/kg IP) EO (N.amethystina ) 25mg/kg IP EO (N.amethystina ) 50mg/kg IP 0 Control Reference ASA (200mg/kg IP) EO (N.amethystina ) 25mg/kg IP EO (N.amethystina ) 50mg/kg IP

12 12 P a g e International Standard Serial Number (ISSN): Figure 6: Percentage of protection against cramp induced by acetic acid glacial Control Reference ASA (200mg/kg IP) % Protection EO (N.amethystina ) 25mg/kg IP EO (N.amethystina ) 50mg/kg IP Control Reference ASA (200mg/kg IP) EO (N.amethystina ) 25mg/kg IP EO (N.amethystina ) 50mg/kg IP 3.3. Study of the central analgesic activity: The results of Tail Flick test grouped in the summary table 6 and illustrated in Figure 7, representing the centrally acting analgesic of the essential oil of Nepeta amethystina at doses of 25mg/kg and 50mg/kg. For each batch indicated the average time of withdrawal reflex of the tail and its standard deviation before the administration of the EO studying (normal reaction time) and 15, 30, 45, 60 and 120 min after administration of the EO. Table 6: Average time of withdrawal reflex of the tail (in seconds) Reaction time in sec Normal reaction time Control 1.28±0.39 Morphine 1.19±0.29 (5mg/kg) SC EO (N.amethystina ) (25mg/Kg) IP EO (N.amethystina ) (50 mg/kg) IP 1.31± ± min 30 min 45 min 60 min 120 min 2.91± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.72

13 13 P a g e International Standard Serial Number (ISSN): Figure 7: Average time of withdrawal reflex of the tail (in seconds) Control 6 Morphine (5mg/kg) SC 4 2 EO(N.amethystina ) (25mg/Kg) IP 0 Normal reaction time 15 min 30 min 45 min 60 min 120 min EO(N.amethystina ) (50 mg/kg) IP 4. DISCUSSION: The acute toxicity tests by the IP route show a LD 50 = 2000mg/kg for EO of Nepeta amethystina, this relatively high toxic value is higher than those found by Bouidida et al. [7] for EO of other species of the genus Nepeta, namely: Nepeta Atlantica (LD 50 = ± mg/kg) and Nepeta tuberosa L. ssp. Reticulata (LD 50 = ± 70.3 mg/kg). This reflects a low toxicity of essential oils of three species of the genus Nepeta. Furthermore, the observation of clinical signs in mice during the 24 hours following the injection of EO of Nepeta amethystina we found a dose-dependent sedative action which begins to manifest 5 min after injection of the EO and lasts 30 minutes to 4 hours after injection, the intensity and duration of the sedative effect is dose-dependent. Studies of psychotropic activities of that EO would be justified. The study of the peripheral analgesic activity shows that the number of acetic acid-induced cramps, recorded for 20 min in mice pretreated with EO of Nepeta amethystina intraperitoneally at doses of 25mg/kg and 50mg/kg, decreases significantly (p = <0.05) compared to control batch. Statistical analysis of these results (a single factor ANOVA) revealed that at the dose of 25mg/kg IP of the EO, fewer cramps, is not significant (p = 1.00> 0.05) relative to the reference batch (pretreated with acetylsalicylic acid at 200 mg/kg IP). So that at the dose of 50mg/kg of the EO, the fewer cramps, dosedependent, significant (p = <0.05) relative to the reference batch.

14 14 P a g e International Standard Serial Number (ISSN): At 25mg/kg IP of EO, the percentage of protection against cramps induced by glacial acetic acid in mice is 70.52%. It is just ready equal to that of mice pretreated with acetylsalicylic acid 200 mg/kg IP (69.68%), this percentage of protection reaches its maximum at the dose of 50mg/kg IP (whether 85.95%). The EO of Nepeta amethystina has a power analgesic peripheral higher than acetylsalicylic acid 200 mg/kg IP from the dose of 50mg/kg IP. Furthermore this EO is as active as EO of Nepeta Atlantica (percentage of protection = 84.53% at a dose of 30mg/kg IP) and Nepeta tuberosa L. ssp. Reticulata (percentage of protection = 90.82% at a dose of 30mg/kg IP) 7. The study of the central analgesic activity shows that at 25mg/kg IP, the maximum time of withdrawal reflex in rats tail does not exceed the threshold of pain inhibition at t = 45min, whether 6.06s±0.83 reflecting a weak central analgesic action. Reverse against 50 mg/kg IP, the EO of Nepeta amethystina has an increased time of withdrawal reflex of the tail exceeding the threshold inhibition of pain which reaches its maximum significant (p = <0.05), is 10.06s ± 0.90 at t = 45 min, whereas rats pretreated with morphine solution at 5mg/kg SC react in an average time of reflex equal to 8.50 s ± 0.66 at t = 45min. This reflects a central analgesic action stronger than morphine at 5mg/kg SC. The EO of Nepeta amethystina at 50 mg/kg IP has a central analgesic activity slightly higher than EO of Nepeta tuberosa L. ssp. Reticulata (maximum threshold of pain inhibition = 9.4s ± 0.28 to t = 45min at a dose of 30mg/kg IP) and Nepeta atlantica (maximum threshold of pain inhibition = 8.38s±0.07 to t = 45min at a dose of 30mg/kg IP) CONCLUSION: The results of acute toxicity tests intraperitoneally suggest that EO Nepeta amethystina is not toxic to the experimental doses (25mg/kg and 50mg/kg) with a dose-dependent sedative effect on the central nervous system. The result of studies of analgesic activities give this species a power analgesic peripheral higher than acetylsalicylic acid (200mg/kg IP) from 50 mg/kg IP, while the central analgesic action morphine-like appears weakly at the dose of 25 mg/kg IP, but clearly at the dose of 50mg/kg IP. The EO of Nepeta amethystina exerts central and peripheral analgesic activities substantially identical to those of EO of Nepeta tuberosa L. ssp. Reticulata and Nepeta atlantica. To bring these works to their point of development, it would be desirable to complete later by: - Phytochemical analysis, - Studies of psychotropic activities, joining sedation recorded during the acute toxicity tests,

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