Anti-inflammatory and Analgesic Activities of the Extract from Garcinia mangostana Linn.

N. Pongphasuk and W. Khunkitti Department of Pharmaceutical Technology Faculty of Pharmaceutical Science Khon Kaen University, Khon Kaen Thailand M. Chitcharoenthum Department of Pharmacology Faculty of Medicine Khon Kaen University, Khon Kaen Thailand

Keywords: acute toxicity, Clinacanthus nutans, Cyperus rotundus, Nyctanthes arbortristis, sub-chronic toxicity, Thunbergia laurifolia Abstract The anti-inflammatory activity of extracts from Garcinia mangostana Linn., Nyctanthes arbortristis Linn., Cyperus rotundus L. and Clinacanthus nutans (Burm.f.) Lindau were tested in mice using carrageenan induced paw edema. All extracts (5 g/kg, p.o.) had anti-inflammatory activity at 3 h (P<0.01) and 6 h (P<0.001). Among all the extracts studied, the extract of G. mangostana showed the highest antiinflammatory activity with 45% inhibition at these two time points. The result of dose response study (1.0, 2.0, 4.0 g/kg, p.o.) showed dose-dependent activity. Analgesic activity was also tested using heated glass. None of the extracts had marked analgesic activity. Sub-chronic toxicity study of G. mangostana extract showed low safety (TI!2, LD50= 9.37 g/kg). Mice fed this extract at 1, 2, 4, 8 g/kg/d, showed death rates of 0, 15, 17, 43% respectively during 30 d. T. laurifolia extract had anti-inflammation efficacy !2-folds greater than G. mangostana extract (ED50 T. laurifolia = 2.51 g/kg, ED50 G. mangostana = 5.51 g/kg). In the toxicity study of T. laurifolia extract in mice at 1, 2, 4, 8 g/kg/d, no mice died during 30 d. T. laurifolia extract was the most effective and safe. INTRODUCTION Arthritis is one of the most distressing and disabling syndromes encountered in medical practice. In modern medicine, steroidal and non-steroidal (NSAIDs) drugs are used to alleviate the symptoms of arthritis. However, these drugs have the risk of peptic ulcer, and additionally the steroidal drugs suppress bone marrow and adrenal gland. At the present time, scientists are in search of better drugs and medicinal plants. Thailand has numerous medicinal plants with anti-inflammatory activity. Plants of interest are Clinacanthus nutans (Burm.f.) Lindau, Nyctanthes arbor tristis Linn., Cyperus rotundus L., Garcinia mangostana Linn., and Thunbergia laurifolia Linn. G. mangostana extract also has antibacterial activity which maybe useful for the treatment of infectious inflammation (Sindermsuk et al., 1989). Earlier reports (Satayavivad et al., 1996; Saxena et al., 1984; Gupta et al., 1980; Charuumanee et al., 2001; Meunwongyat, 1994) indicate antiinflammatory activity of ethanol (C. nutans, N. arbor tristis, and C. rotundus), and boiling water (G. mangostana) extracts. Since the anti-inflammatory activity and toxicity of these 5 extracts has not been compared previously, we compared the anti-inflammatory activity, efficacy and toxicity of these plant extracts. The extract with greater activities and safety will be chosen for further development as topical antiinflammatory preparation for arthritis. MATERIALS T. laurifolia was collected in October 2002. G. mangostana, C. nutans, N. arbor tristis, and C. rotundus were collected during three months from November 2001. These plants were collected from three different places, G. mangostana from Chumporn, C. nutans from Nakhonratchasima, and the others from Khonkean.

Proc. WOCMAP III, Vol.6: Traditional Medicine & Nutraceuticals Eds. U.R. Palaniswamy, L.E. Craker and Z.E. Gardner Acta Hort. 680, ISHS 2005

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Animals Albino mice (40-50 g) of either sex, in equal number were kept in air-conditioned room, maintained at 23C, 70% relative humidity, 12 h dark-light cycle. Food and water were given ad libitum, food was deprived for 6h before oral administration of any extracts. Chemicals Carrageenan Lambda Type IV (Sigma, St Louis, MO, USA), and 95% Ethanol BP (Excise Department of Thailand). METHODS Extraction Coarse powders of C. nutans leaves, N. arbor tristis leaves, C. rotundus rhizome, T. laurifolia leaves were macerated separately in 95% ethanol (1:4 kg/L for C. nutans, N. arbor tristis, and C. rotundus, and 1:50 kg/L for T. laurifolia) for 7 d. Powder of G. mangostana pericarp was extracted with boiling water. All extracts were filtered through filter papers No.1 (Whatman) and dried in oven at 60C. Study of Pharmacological Activities 1. Anti-inflammatory Activity. Albino mice were divided into 5 groups with six animals in each group (3 males, 3 females). Mice were orally fed 5 g/kg (0.5 mL), either G. mangostana, N. arbor tristis, C. rotundus, C. nutans extract or 0.9% saline. One hour later, 0.025 mL of 1% Carrageenan in 0.9% saline was injected into the subplantar of one hind paw (Winter et al., 1962). The paw volume was measured by microgauze (Grain measure, Kett Co. Ltd.) at 0, 3, 6 h after the injection. % Swelling and % Inhibition were calculated using the following formulas 1 and 2: Formula 1 % Swelling = (V-Vi) " 100 Vi

V = Paw thickness after carrageenan, Vi = Paw thickness at 0 time Formula 2

% Inhibition = #1- (% Swelling of drug-treated group / % Swelling of control group)$ "100

2. Analgesic Activity. Each mouse from anti-inflammatory study was placed individually in a glass jar of known temperature-time equation, example Y= 0.2912X+26.645, X is time in second of response and Y is temperature (C) (Chicharoenthum and Khunkitti, 1997). The glass jar was immersed in 72C water bath about 1 inch deep. Jumping response-time was tested at 0, 3, 6 h after carrageenan. 3. Anti-inflammatory Dose-response of G. mangostana and T. laurifolia Extracts. Albino mice were divided into 9 groups with six animals in each group (3 males, 3 females). Mice were orally fed 1, 2, 4, 8 g/kg (0.5 mL) either G. mangostana or T. laurifolia extracts or 0.9% saline. One hour later, 0.025 mL of 1% Carrageenan in 0.9% saline was injected into the subplantar of one hind paw as given by Winter et al. (1962). The paw volume was measured by microgauze (Grain measure, Kett Co. Ltd.) at 0, 3, 6 h after the injection. % Swelling and % Inhibition were calculated by using formulas 1 and 2, respectively. ED50 was estimated by linear regression analysis (Wallace Hayes, 1989). The Study of Toxicities 1. Acute and Subchronic Toxicities of G. mangostana Extract.

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Acute Toxicity. Albino mice were divided into 5 groups with 7 animals in each group (males). Mice were fed 2.5, 5, 10, 20 g/kg (1.0 mL) either G. mangostana extract or 0.9% saline. Mortality was observed at 1, 2, 3, 6, 12, 24, 48, and 72 h. Sub-chronic Toxicity. Mice from acute toxicity were continually fed 1, 2, 4, 8 g/kg/d (0.5 mL) of G. mangostana extract for 30 d. Body weight was measured everyday. At the end of the experiment, animals were sacrificed by neck dislocation. Stomach, liver and kidney were removed and weighed. LD50 was estimated by linear regression analysis (Wallace Hayes, 1989). 2. Subchronic Toxicity of T. laurifolia Extract. Albino mice were divided into 5 groups with 7 animals in each group (males). Mice were orally fed 1, 2, 4, 8 g/kg/d (0.5 mL) of T. laurifolia extract for 30 d. Body weight was measured everyday. At the end of the experiment, the animals were sacrificed by neck dislocation. Stomach, liver and kidney were removed and weighed. LD50 was estimated by linear regression analysis (Wallace Hayes, 1989). Statistical Analysis Results were expressed as mean % SE. One way analysis of variance (ANOVA) and t-test were used to compare the differences between control and drug-treated animals. Paired t-test was used to compare initial and final body weight (p<0.05). ED50 and LD50 were calculated by linear regression analysis (Wallace Hayes, 1989). RESULTS Extraction The yield for the extracts were (yield % + variance) G. mangostana (63.66%0.97), N. arbor tristis (27.42%0.91), C. rotundus (16.98%0.64), C. nutans (9.87%0.14), T. laurifolia (6.50%0.41). C. rotundus extract was yellow, G. mangostana extract was brown and the rest three extracts were green. The Study of Pharmacological Activities 1. Anti-inflammatory Activity of Extracts. The study of anti-inflammatory activity compared C. nutans, C. rotundus, N. arbor tristis and G. mangostana at the highest dose 5 g/kg. All extracts had anti-inflammatory activity with different efficacy (Fig. 1). The greatest reduction of 45% was from G. mangostana extract (p<0.05, at 6 h), the other extracts reduced edema less than 35% (Table 1). 2. Analgesic Activity. Glass jar increased temperature according to time. Mice responded to the increasing temperature in the jar by climbing, padding and finally jumping in 40 sec. The response temperature was calculated from temperature-time equation. The control group before carrageenan induced edema responded at 41&C but responded at a lower temperature 40 and 38&C after 3 and 6 h carrageenan. The extract-treated groups showed a similar pattern of the response and were not different from control group. All the extracts had low or no analgesic activity (data not shown). 3. Anti-inflammatory Dose-response of G. mangostana and T. laurifolia Extracts. T. laurifolia extract and G. mangostana extract at 1, 2, 4 g/kg reduced edema in a dose dependent manner. All the doses of T. laurifolia extract reduced the edema about 30-70% at 3 and 6 h after carrageenan, while G. mangostana extract reduced edema about 5-35%, especially at the highest dose (4 g/kg) (Fig. 2, Table 2). ED50 calculated from the linear regression curve, was 2.51 g/kg (R2=0.87) for T. laurifolia extract and was 5.51 g/kg (R2=0.99) for G. mangostana extract. T. laurifolia extract was more potent than G. mangostana extract. The Study of Toxicities 1. Acute and Subchronic Toxicities of G. mangostana Extract. Acute Toxicity. Mice that received G. mangostana extract at single oral doses of 2.5, 5, 10, 20 g/kg, did not die during 72 h. The volume and high viscosity of the extract posed a

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problem of feeding. The volume was reduced to 0.5 mL and the extract was diluted for easier oral feeding in subchronic toxicity test. Subchronic Toxicity. The weight of mice treated with G. mangostana extract at doses of 8, 4, 2 g/kg/day decreased in a dose dependent manner. The final weight of mice fed at 8 g/kg was 12% lower than the initial weight but was not statistically significant. Each group of mice that received the extract began to die at 14, 20, 24 d of treatment. At 30 d, 57.14, 83.33, 85.71% of the mice survived respectively (data not shown). The stomach and liver weight of extract-treated groups died before 30 d and after neck dislocation were not different from control group but the kidney weight were heavier than the control about 2 folds. The number of mice with enlarged kidney were 28.6, 16.7, 14.3%, respectively. LD50 calculated from linear regression curve was 9.37 g/kg. 2. Subchronic Toxicity of T. laurifolia Extract. Mice that received T. laurifolia extract at 1, 2, 4, 8 g/kg/d, did not die during 30 d and the body weight did not alter. The organ weight, stomach, liver, kidney weight were not different from control group. DISCUSSION The results of this study helped to compare the known anti-inflammatory activity of G. mangostana, N. arbor tristis, C. rotundus and C. nutans. The activity of aqueous G. mangostana extract was about 1.5 folds more than the other extracts. Besides, percentage yield of this extract was more than the others, about 2, 3, and 6 folds, respectively. Therefore, this extract has the potential for formulation as topical anti-inflammatory preparation. However, subchronic toxicity study revealed that G. mangostana extract had greater safety (LD50= 9.37 g/kg, ED50= 5.51 g/kg). Mice fed this extract at doses 1, 2, 4, 8 g/kg/d died before the end experiment at about 0, 15, 17, 43%, respectively. They had enlarged kidney, which may be the cause of death. The study by Saxena (1984) had reported the anti-inflammatory activity of N. arbor tristis extract without any reported adverse reaction. However, this study showed that anti-inflammatory activity of N. arbor tristis extract was secondary to G. mangostana, and also caused diarrhoea and hypothermia. T. laurifolia extract was another herb which had efficacious anti-inflammation more than G. mangostana extract by about 2 folds (ED50 of T. laurifolia = 2.51 g/kg, ED50 of G. mangostana = 5.51 g/kg). T. laurifolia extract had percentage yield less than G. mangostana extract by about 10 folds. The yields are from use of different solvents, T. laurifolia was extracted with ethanol, and G. mangostana was extracted with boiling water. However, in this study percentage yield of T. laurifolia extract was higher at 6.5% vs. 4.9% which was reported earlier (Charuumanee et al., 2001). Subchronic toxicity test of this extract at orally doses 1, 2, 4, 8 g/kg/d showed that no mice died during 30 d. The body weight, stomach, liver, kidney weight were not different from control group. In conclusion, T. laurifolia extract had the greatest anti-inflammatory activity and safety, suitable for developing a topical anti-inflammatory preparation for arthritis. ACKNOWLEDGEMENTS The authors are grateful to the Khon Kaen Universitys Graduate Research Fund, 2002. Literature Cited Charuumanee, S., Vejabhikul, S., Taesotikul, T., Netisingha, W., Sirisaard, P. and Leelapornpisit, P. 2001. Topical anti-inflammatory preparations from Thunbergia Lauriforia Linn. p.20-23. In: Pharma Indochina II, Honoi-vietnam. Chitcharoenthum, M. and Khunkitti, W. 1997. A modified hot plate method. Thai J. Pharmacol. 19(1):15-22. Gupta, M.B., Nath, R., Srivastava, N., Shanker, K., Kishor, K. and Bhargava, K.P. 1980. Antiinflammatory and antipyretic activities of '-sitosterol. Planta medica. 39:157-163. Meunwongyat, P. 1994. Samupai kua mai. T.P. prints, Bangkok. Satayavivad, J., Bunyapraphatsata, N., Kitisiripornkul, S. and Tanasomwang, W. 1996.

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Analgesic and anti-inflammatory activity of extract of Clinacanthus nutans (Burm.f.) Lindau. J. Phytopharmacy 3(1):7-17. Saxena, R.S., Gupta, B., Saxena, K.K., Singh, R.C. and Prasad, D.N. 1984. Study of antiinflammatory activity in the leaves of Nyctanthes arbortristis Linn. J. Ethanopharm. 11:319-330. Sindermsuk, J., Deekitsermphong, S. and Jaruprechachan, V. 1989. Comparision of the Efficiency in diarrhoeal treatment between the leaf of guava and the fruit hull of mangosteen. Mahidol Univ. J. Pharm. Sci. 16(2):32-34. Wallace Hayes, A. 1989. Principles and methods of toxicology. Raven Press, New York. Winter, C.A., Risley, E.A. and Nuss, G.W. 1962. Carrageenin induced edema in hind paw of the rats as an assay for anti-inflammatory drugs. Proc. Soc. Exper. Biol. Med. 111:544-547.

Tables Table 1. Effect of oral administration of herbal extracts at dose 5 g/kg on carrageenaninduced paw edema in mice at 3 and 6 h. Each group consisted of 6 animals. The extract 3h G. mangostana N. arbor tristis C. rotundus C. nutans 45.72 % 17.91 37.65 % 13.13 22.88 % 13.39 17.73 % 8.93 % Inhibition % SE 6h 43.09 % 12.67 32.63 % 6.53 20.02 % 11.6 36.47 % 12.49

Table 2. Effect of oral administration of T. laurifolia and G. mangostana extract at dose 1, 2, 4 g/kg on carrageenan-induced paw edema in mice at 3 and 6 h. Each group consisted of 6 animals. Extract Dose (g/kg ) 4.0 2.0 1.0 4.0 2.0 1.0 % Inhibition % SE 3h 6h 57.47 % 6.62 50.91 % 14.85 38.47 % 12.49 35.20 % 13.68 16.36 % 8.63 5.85 % 3.23 68.04 % 13.94 60.59 % 12.95 31.82 % 13.14 23.02 % 17.48 30.83 % 14.85 10.59 % 6.31

T. laurifolia

G. mangostana

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Figures

Control

C.nutans

C. rotundus

N. arbor

G.mangostana

6 0 5 0
%Swelling

4 0 3 0 2 0 1 0 0
3 Time (hrs) 6
*

Fig. 1. Effect of various herbal extracts on carrageenan-induced edema at 3 and 6 h (*p<0.025, n=6).

70 60 50

% S we lling

40 30 20 10 0
l nt ro 3 1 2 co

* * * * * * *
4 l co nt ro 6 1

* * * *
2 4

* * *

T .la urifo lia ( g /k g )

G .ma ng o s ta na (g /k g )

T.laurifolia ( g/kg

T ime (hrs )

Fig. 2. Effect of G. mangostana and T. lauriforia extracts on carrageenan-induced paw edema at 3 and 6 h (****p<0.05, ***p<0.025, **p<0.005, *p<0.001, n=6).

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