Article International Journal of Modern Biology and Medicine, 2013, 3(2): 69-77 International Journal of Modern Biology and Medicine Journal homepage: www.modernscientificpress.com/journals/ijbiomed.aspx ISSN: 2165-0136 Florida, USA Phytochemical Screening, Analgesic and Anti-inflammatory Properties and Median Lethal Dose of Ethanol Leaf Extract of Wild Species of Eryngium foetidum L. on Albino Rats Joseph Okon 1, *, Emmanuel Edet 2, Godwin Esenowo 1, Nse Umoh 1 1 Department of Botany and Ecological Studies, University of Uyo, P. M. B. 1017, Uyo, Akwa Ibom State, Nigeria 2 Department of Microbiology, University of Uyo, P. M. B. 1017, Uyo, Akwa Ibom State, Nigeria * Author to whom correspondence should be addressed; E-Mail: joesplendid@yahoo.com; Tel.: +2348038811626. Article history: Received 28 January 2013, Received in revised form 18 February 2013, Accepted 20 February 2013, Published 23 February 2013. Abstract: The present study was carried out to investigate the phytochemical screening, analgesic and anti-inflammatory properties of ethanol leave extract of Eryngium foetidum. Median lethal dose (LD 50 ) of crude extract of Eryngium foetidum was examined inthraperitoneally. For evaluation of analgesic, acetic acid induced writhing response model was used. In anti-inflammation, xylene-induced ear oedema and egg albumin were used in Swiss albino mice. In both cases, leaves extract were administered and the obtained effects were compared with commercially available analgesic and anti-inflammatory drug acetyl salicylic acid. The results of phytochemical screening revealed the presence of saponins, flavonoids, alkaloids, tannins and anthraquinones, cardiac glycosides and terpenes. The LD 50 value for Eryngium foetidum leaves extract was 1649.24 mg/kg. In analgesic bioassay, oral administration of the ethanol leaves extract reduced the writhing response in dose-dependent manner when compared with positive and the negative control i.e. 50%, 53% and 0% respectively. The ethanolic extract of Eryngium foetidum decreased the inflammation induced in both xylene and egg albumin. The degree of inhibition of leaves extract was not significant when compared to the effect of standard inflammatory drug, acetyl salicylic acid (53.26%). These results indicate that the extracts could possess analgesic and anti-inflammatory properties. All these effects and the changes in the behavioural activities could be suggested as contributory effects to the use of Eryngium foetidum leaf in the management of inflammation and painful conditions. Keywords: Eryngium foetidum; analgesic; inflammation; phytochemical screening.
70 1. Introduction Plants are one of the most important sources of medicines. India is known as the Emporium of Medicinal plants due to availability of several thousands of medicinal plants in the different bioclimatic zones. The inflammatory diseases including rheumatoid arthritis are still one of the main health problems of the world s population. Several modern drugs are used to treat these disorders but, their prolonged use may cause severe adverse side effects (Gil, 2002), the most common being gastrointestinal bleeding and peptic ulcers (Hemamalini et al., 2010). Consequently, there is a need to develop new anti-inflammatory agents with minimum side effects. The use of natural remedies for the treatment of inflammatory and painful conditions has a long history, starting with Ayurvedic treatment, and extending to the European and other systems of traditional medicines. Plant drugs are known to play a vital role in management of inflammatory diseases. Eryngium foetidum L., Spiny Coriander also known as Culantro is an aromatic plant, grown as a leafy vegetable belongs to the family Umbelliferae (Saikia and Shadeque, 1996). Culantro is one of the rare, biennial indigenous species distributed in Continental Tropical America, Coastal West Africa and West Indies (Adams, 1971). Eryngium foetidum has essential oil which contained about 40 compounds, and has high economic value in international and domestic markets (Wong et al., 1994). E. foetidum is use as a condiment for culinary purposes, widely use in perfumery and cosmetic industry (Inacimuthu et al., 1999; Pino et al., 1997). A perusal of the literature revealed that although Eryngium foetidum is widely used in traditional medicine as an anti-inflammatory and analgesic agent, these properties have not been scientifically evaluated. Therefore, the present study is an attempt to investigate the phytochemicals, median lethal dose, anti-inflammatory, and analgesic properties of the ethanolic extract of Eryngium foetidum leaves in experimental animals. 2. Materials and Methods 2.1. Collection and Identification of Plant Material The fresh leaves of Eryngium foetidum were collected from forest of Itak, Ikono Local Government Area of Akwa Ibom State on September, 2012. The plant was identified and authenticated by Dr. (Mrs.) M. E. Bassey, a plant taxonomist in the Department of Botany and Ecological studies, University of Uyo, Akwa Ibom State. 2.2. Preparation of Plant Extract The Eryngium foetidum leaves samples were shade dried and pulverized to powder form and was macerated with 1000 ml of 70% ethanol for 72 h. The solution was filtered using glass funnel
71 packed with cotton wool. The filtrate was evaporated to dryness by heating in a water bath at 40 o C to give a yield of 58 g of semi-dry extract with brownish colour. The extract was stored in the refrigerator at -4 o C for further use. 2.3. Collection and Maintenance of Animals Healthy male and female albino rats weighing 150 250 g were obtained from the animal house of the Department of Pharmacology and Toxicology, Faculty of Pharmacy, University of Uyo, Uyo and maintained according to the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals. The animals were fed with standard pellets (Guinea Feed Nigeria Ltd., Benin) for two weeks to acclimatize. They were allowed for free access to water and feed ad libitum. 2.4. Phytochemical Screening The experiment was carried out in the Department of Pharmacognosy and Natural Medicine, University of Uyo, Uyo. The phytochemical screening involves the simple chemical test to detect the presence of secondary metabolites. The methods of Trease and Evans (2009) were used for phytochemical screening. 2.5. Determination of Median Lethal Dose (LD50) Swiss albino mice weighing 25-32 g were dose by the intraperitoneal (i.p.) route using the method of Lorke (1983). The animals were administered with 2500 mg/kg, 2000 mg/kg, 1000 mg/kg and 500 mg/kg of Eryngium foetidum extract in 5 groups of 5 mice each. The animals were observed for manifestation of physical signs of toxicity and the number of death within 24 h was recorded. The LD 50 was calculated as the geometric mean of the maximum dose producing 0% mortality and the minimum dose producing 100% mortality. Food was withdrawn for 18 h before the onset of the experiment according to methods of Amresh et al. (2008). where, a = Maximum dose producing 0% mortality, b = Minimum dose producing 100% mortality. 2.6. Analgesic Activity Acetic acid-induced writhing in mice The 25 Swiss mice were divided into six groups of five animals each. Each group of animals was treated orally with morphine (1.5 mg/kg), The LD 50 values of Eryngium foetidum extract were use
72 as a working doses (164.92, 329.84 and 494.76 mg/kg) respectively. One hour after administration of the test drugs, animals were injected intraperitoneally with 1% acetic acid (1 ml/100 g body weight). The number of writhing responses such as contortions and stretching were recorded at 5 min interval for 30 min. The results were evaluated by calculating the mean number of contortions per treated group and results compared to results obtained from control animals. Percentage pain inhibition was calculated as follows. where, Nc = mean number of contortions of the control group, Nt = mean number of contortions per treated group. 2.7. Anti-inflammatory Activity 2.7.1. Xylene-induced ear edema Anti-inflammatory activity of Eryngium foetidum was assessed by xylene-induced ear edema method. Rats were divided into 5 groups (5 animals in each group). Animals of all the groups were injected with 0.1 ml of 1% xylene in 0.9% normal saline at the inner surface of the right ear and left to act for 1 min. Eryngium foetidum extract at the doses of 164.92, 329.84 and 494.76 mg/kg were administered orally to each group as well as the standard drug (4.0 mg/kg dexamethasone) and distilled water (10.0 mg/kg) 1 hour before induction of inflammation. The anti-inflammatory activity was determined as the percentage of inhibition of inflammation after it was induced by xylene by taking volume of inflammation in control group as 100%. The percentage inhibition was calculated by using the formula: where, M1 = Mean of control groups, M2 = Mean of test groups. 2.7.2. Egg albumin-induced inflammation in mice Inflammation was induced in mice by injecting the egg albumin (0.3 ml, of 3% in normal saline) into the plantar surface of the right hind paw. The linear paw circumference was measured in 5 min interval for 5 hours using vernier callipers. Eryngium foetidum extract at the doses of 164.92, 329.84 and 494.76 mg/kg and acetyl salicylic acid (100 mg/kg) were administered orally, control group received 10 ml/kg of distilled water 1 hour before induction of inflammation. The above
73 formula was used to calculate the percentage inhibition. 2.8. Statistical Analysis Results were expressed as mean ± SEM and statistical significance was obtained by the Student s t test using SPSS. 3. Results and Discussion 3.1. Results The phytochemical screening of the ethanolic extract of Eryngium foetidum leaves revealed the presence of saponins, flavonoids, alkaloids, tannins, anthraquinones, cardiac glycosides and terpenes (Table 1). The LD 50 value of the mice treated intraperitoneally with a single dose of 0.03-4.00 g/kg of Eryngium foetidum extracts after being starved for 18 hours was calculated to be 1649.24 mg/kg. The results presented in Table 2 showed that Eryngium foetidum extract at doses of 164.92, 329.84 and 494.76 mg/kg decreased the number of writhes by 3.14 0.92, 3.10 0.45 and 3.00 0.10 respectively. The reference drug acetyl salicylic acid recorded 3.74 0.53 and negative control had 6.90 0.12. Although three doses were used, the higher dose (494.76 mg/kg), did not elicit a greater protection from acetic acid induced abdominal writhing as would have been expected. The extract showed non-significant anti-inflammatory activity against acute inflammation induced by egg albumin and xylene-induced ear oedema. This reduction was in a dose-dependent manner with the highest dose (494.76 mg/kg) of extract comparable with the standard antiinflammatory drug acetyl salicylic acid (Tables 3 and 4). 3.2. Discussion This studies shows that flavonoids and terpenes are moderately present while tannins in ferric chloride test are present in abundant. It has been reported that the leaves and barks of Anogeissus acuminata contains flavonoids, tannins, terpenes in appreciable amount and oleo resins. These compounds are used in anti-inflammatory and analgesic agents in modern medicine. Preliminary phytochemical studies indicated the presence of gum resins of Anogeissus acuminata. The volatile oils, resins, flavonoids and terpenoids isolated form plant extracts are known to produce anti-inflammatory and analgesic effects (Hemamalini et al., 2010).
74 Table 1. Phytochemical screening of ethanolic extract of Eryngium foetidum leaves Test Inference Alkaloids Dragendoff s test Mayer s test Picric acid test ++ ++ Saponins Frothing test Fehling test ++ Tannins Ferric chloride test Bromine water test + Flavonoids Shinoda s test ++ Cardiac glycosides Lieberman s test Keller Kiliani s test Salkowski s test ++ Terpenes Lieberman s Burchards test + Anthraquinones Free anthraquinone Combined anthraquinones Note: - = Absent, + = Trace, ++ = Moderate, = Abundance. Table 2. Effect of ethanolic extract of Eryngium foetidum leaves on acetic acid induced writhing response in Wistar Albino mice Groups Treatment Dose Writhing Count Inhibition (%) Negative control Distilled water 10.0 (ml) 81.00 4.15 _ Positive control Acetylsalicylic acid 100.0 (mg/kg) 37.86 1.26 53.26 Treated Low dose 164.92 (mg/kg) 49.86 1.87* 38.44 Treated Middle dose 329.84 (mg/kg) 43.57 1.54*** 46.21 Treated Hugh dose 494.76 (mg/kg) 39.11 2.007* 50.96 Note: All values are given in mean ± SD (n = 5), ANOVA *p < 0.05, **p < 0.01, ***p < 0.001, when compared to control group.
75 Table 3. Effect of extract of Eryngium foetidum leaves on xylene-induced ear edema in Wistar Albino mice Groups Treatment Dose Ear edema (mm 3 ) Inhibition (%) Negative Control Distilled water 10.0 (ml) 0.06 0.01 - positive Control Dexamethasone 4.0 (mg/kg) 0.02 0.50 66.67 Treated Low dose 164.92 (mg/kg) 0.05 0.11** 16.67 Treated Middle dose 329.84 (mg/kg) 0.04 0.02* 33.33 Treated Hugh dose 494.76 (mg/kg) 0.02 0.13*** 66.67 Note: All values are given in mean ± SD (n = 5), ANOVA *p < 0.05, **p < 0.01, ***p < 0.001, when compared to control group. Table 4. Anti-inflammatory effect of ethanolic extract of Eryngium foetidum leaves on egg albumininduced edema in Swiss Albino mice Treated Dose Reaction Time Groups 0 min 30 min 1 hr 2 hrs 3 hrs 4 hrs 5 hrs NA 10.0 (ml) 4.17 0.32 6.93 1.18 6.58 0.61 5.95 0.37 6.26 0.30 6.13 0.68 6.90 0.12 ASA 100 (mg/kg) 3.86 0.03 6.46 0.15 5.92 0.32 5.93 0.12 5.39 0.71 4.06 0.24 3.75 0.53 EF 164.92 (mg/kg) 3.49 0.43 6.72 0.52* 6.41 0.54 5.76 0.52 5.42 0.19 4.98 0.91 3.14 0.92* EF 329.84 (mg/kg) 4.03 0.32 6.42 0.69 6.09 0.49 5.21 0.24* 5.54 0.42* 4.74 0.34* 3.10 0.45* EF 494.76 (mg/kg) 3.60 0.15 6.44 0.24** 5.86 0.41* 5.55 0.37* 4.69 0.45* 4.69 0.33** 3.00 0.10** Note: All values are given in mean ± SD (n = 5), ANOVA *p < 0.05, **p < 0.01, when compared to control group. NA = normal saline, ASA = acetyl salicylic acid, EF = Eryngium foetidum. The observations of present study are shown in Table 2. It indicates that the ethanolic extract of Eryngium foetidum (EF) possesses analgesic and anti-inflammatory effects which are comparable to that of standard drug. Among the doses, EF (494.76 mg/kg) higher dose was found to be more effective than EF (164.92 mg/kg) lowest dose. In these studies, there was a decrease in analgesic and inflammation in dose-related fashion but was not significant. Report of Atta and Afolabi (1997) showed that the aqueous extract of Solanum torvum induced dose-dependant analgesic effect against the writhing syndrome indicating its peripheral effect. In peripheral tissues, prostaglandins and kinines would seem to play an important role in the pain process (Hajare et al., 2000) and writhing induced by chemical substances injected intraperitoneally is said to be the consequence of sensitisation of the chemosensitive nociceptors by prostaglandins (Maria et al., 1997). Their results suggest that the pain killing effect of this plant may be by the prostaglandins synthesis inhibition. This test also confirms the peripheral action of Solanum torvum for the treatment of inflammatory conditions (Rang et al., 1995). It is worth highlighting that a high dose of Eryngium foetidum exhibited an equipotent effect to an
76 equivalent dose of anti-inflammatory drug acetyl salicylic acid. This finding is important because the extract shows the same effect as the pure reference compound. It is noteworthy that Eryngium foetidum and acetyl salicylic acid displayed equipotent effects at equivalent doses (10 mg/kg); however, major doses of the extracts did not display significant increase effects. This anti-inflammatory effect could be attributed to the cyclomargenol and β-sitosterol in the extracts. Some sterols and cycloartane-type triterpenes have been reported as anti-inflammatory compounds (Maldonado et al., 2010) and cancer chemopreventive compounds by Kikuchi et al. (2007). Additionally, there are reports that establish this method implies two phases in which several mediators are involved. The mechanism of action of the most important non-steroidal anti-inflammatory drugs in actual therapy is inhibition of this phase throughout dismiss CO X enzymes activity. Eryngium foetidum extract presented inhibition of oedema from 2 to 5 hours (second phase) which suggest that the extract is probably associated to inhibition of enzyme cyclooxygenase (CO X enzymes) leading to inhibition of prostaglandin synthesis. Oxidative species has been related to the last phase of inflammation, Eryngium foetidum extract presented antioxidant capacity. These findings justify conventional use of this plant in the treatment of pain and other inflammatory conditions and validate its claim of being used for the said purpose in folklore medicine. 4. Conclusions The findings of the present study have demonstrated that Eryngium foetidum has potent antiinflammatory and analgesic activity and justify the folkloric use of the plant among the rural people of Uruan, Itu, Ibiono and Ikono Local Government Area of Akwa Ibom State of Nigeria to treat inflammatory and painful conditions. The results also furnish evidence that the beneficial effects of this plant may be due to its free radical scavenging activity. However, further work is advocated to elucidate other therapeutic values of Eryngium foetidum. Acknowledgements The authors gratefully acknowledge the Department of Pharmacognosy & Natural Medicine, Department of Pharmacology & Toxicology for providing the laboratory space. They also appreciated the technical assistance of Mr. Bala D. N. and Mr. Nsikan M. References Adams, C. D. (1971). Flowering Plants of Jamaica. University of West Indies Press, Mona, Jamaica WI, p256-264. Amresh, G.; Paras, N. S. and Chandana, V. A. (2008). Toxicological screening of traditional medicine
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