Anti-inflammatory actions of tannins isolated from the bark of Anacardwm occidentale L

Journal ofEthnopharmacoZogy, Elsevier Scientific Publishers

13 (1985) Ireland Ltd.

ANTI-INFLAMMATORY BARK OF ANACARDIUM

M.L.R.

MOTA,

Laborat6rio Joa’0 Pessoa, (Accepted

G. THOMAS*

20,

289

ACTIONS OF TANNINS ISOLATED OCCIDENTALE L.

and J.M.

de Tecnologia FarmacButica, Paraiba (Brazil)

March

289-300

BARBOSA

FROM THE

FILHO

Universidade

Federal

de Paraiba,

58.000,

1985)

Summary

A mixture of tannins (hydrolysable and non-hydrolysable) obtained from the bark of Anacardium occidentale L., on i.p. injection, demonstrated apparent anti-inflammatory activity in carrageenan- and dextran-induced rat paw oedemas, cotton pellet granuloma test and adjuvant-induced polyarthritis in rats. At higher doses orally administered tannins also had activity in carrageenan paw oedema and adjuvant arthritis experiments. The tannins i.p. also inhibited acetic acid-induced “writhing responses” in mice and were found to antagonise the permeability-increasing effects in rats of certain mediators of inflammation and to inhibit the migration of leucocytes to an inflammatory site. While not appearing to act by the release of adrenal hormones, tannins may produce effects in a non-specific manner by their astringent properties on cell membranes thus affecting cell functions. The above results should be considered while studying the anti-inflammatory actions of plant extracts which contain tannins.

Introduction Anacardium occidentale Linn (Anacardiaceae) commonly known as the cashew tree grows in many tropical countries including Brazil. In addition to its well known industrial and nutritional applications, parts of the tree are also used in Brazil for their diuretic (Braga, 1960), antidiabetic (Correa, 1969; Lewis and Elvin-Lewis, 1977) and anti-inflammatory properties (Coimbra, 1958; Gomes, 1973). In laboratory animals, extracts of the bark of the tree have been reported to reduce blood sugar levels (Aguiar et al.,

*To whom

all correspondence

0378-8741/85/$04.55 Published and Printed

o 1985 in Ireland

should

be addressed.

Elsevier

Scientific

Publishers

Ireland

Ltd.

290

1959) and anacardic acid obtained from the cashew nut shell has been reported to have antibacterial and antipyretic activities (Eichbaum, 1949). In earlier studies an aqueous fraction obtained from the eth~~olic extract of the trunk bark of A. occidentde was found to have anti-infl~mato~~ action in dextran- and carrageenan-induced rat paw oedemas when administered in doses of 10-80 mg/kg i.p. 300-900 mg/kg p.o., respectively (Mota and Thomas, 1981). As the phyto~hemic~ screening of this fraction demonstrated the presence of large quantities of tannins, a group of polyphen~ls extensively distributed in the plant kingdom, it was considered wo~hwhile to investigate whether these agents were responsible for the observed effects. Materials and methods Plant

material

The trunk bark was collected, after identification by the botany depart ment, from mature trees growing around the University Campus during January-June, 1982. Voucher specimens are preserved in the herbarium of the biology department of the University (catalog no. JPB 4.177).

The method described by IIaslam (1966) was employed. Briefly, it involved maceration of small pieees of freshly cut bark with a solution of 10% acetone in water for 72 h. The resulting extract was subjected to liquidliquid extraction with ethyl acetate and the ethyl acetate fraction was evaporated to dryness under reduced pressure using a rota-evaporator, The residue obtained was pulverized and mixed with silica gel. The mixture was placed on a ~hromato~aphic column (70 cm X 4 cm) packed with silica gel in methanol/water (60 : 40) and eluted with the solvent mixture. The first 8 fractions (50 ml each) were found to be tannins when analysed using paper chromato~aphy. The fractions were joined and evaporated to dryness as before to obtain a reddish brown residue, the nature of which was confirmed by generally used chemical (Rosenth~er, 1930; Salem et al., 1969; Costa, 1972) and physical methods (Arshad et al., 1969) for the identification of tannins.

Male albino mice and rats bred and maintained in the animal house attached to the laboratory were used. Free access to food and water were allowed prior to and during the experiments. Acute toxicity studies Mice (20-30

g) and rats (126-150

g) in groups of 10 were administered

291

i.p. or p.o. with different doses of tannins. The animals were observed for 24 h and the number of deaths noted. LD5,, values and their 95% confidence ranges were calculated from log-dose/mortality (probit) graphs using the methods of Litchfield and Wilcoxon (1949). Carrageenan-induced

paw oedema

Rats weighing 120-150 g in groups of 6 were used. Doses of tannins were administered i.p. or p.o. 30 or 60 min, respectively, before the S.C. injections of 0.1 ml of a 1% suspension of carrageenan into the plantar region of one of the hind paws of each animal. Percent inhibition of oedema relative to a control group receiving 0.9% saline was calculated as described by Winter et al. (1962). In a separate experiment, the inhibitory activity of tannins in carrageenan oedema was compared in sham-operated and adrenalectomized rats. The animals were operated and maintained with the usual precautions for 7 days before the test. Dex tran paw oedema

The method described above for carrageenan was used except that a 0.1 ml solution of 1% clinical dextran was the oedema-inducing agent and that the anti-inflammatory action was assessed 2 h after pedal injection. Cotton pellet granuloma

test in rats

The method of D’Arcy et al. (1960) with minor modifications was followed. Sterilized cotton wool pellets of known weights (10 + 1.5 mg) were implanted S.C. in groups of 7 rats (120-150 g) under ether anaesthesia. Two pellets, one in each groin were placed in each animal. Tannins were administered daily i.p. or p.o. at various doses for 7 days with the first dose given 1 h before the implantation. On the eighth day the rats were killed and the pellets dissected free of extraneous tissue and dried at 60°C to constant weights. The weight of granulation tissue in each pellet was calculated and the mean values for treated groups were compared with that of the salinetreated group. For both the control and tannin-injected (50 mg/kg i.p.) groups, the weights of adrenal and thymus glands were determined at the end of the experiment. Adjuuant-induced

polyarthritis

in rats

Polyarthritis was induced in rata (130-150 g) by i.d. injections of 0.05 ml of a 5 mg/ml suspension of heat-killed Mycobacterium tuberculosis in liquid paraffin into the plantar surface of the hind paws (Newbould, 1963). The secondary lesions in the ears, tail and paws (more or less established by the 15th day) were assessed on an arbitrary scale (O-3) according to the method of Rosenthale (1970) with slight changes. Animals with pronounced arthritis on the last day were separated into groups of 7, so that each group had approximately similar mean arthritic scores. Oral or i.p. administration of tannins were made daily from this day for the next 7 days. Mean arthritic

292

scores obtained for each day for treated groups were compared with appropriate scores of the control group. Writhing test in mice Tannins (i.p. and p.o.) or acetylsalicylic acid (i.p.) were given 30 min before i.p. injections of a 0.75% acetic acid solution (0.1 ml/10 g) to groups of 5 mice (18-24s). The number of abdominal constrictions produced in each group for the succeeding 15 min were counted and compared with the response in the control group as described by Koster et al. (1959). Vascular permeability studies Groups of 7 rats (150-200 g) had their backs depilitated 48 h before the experiments. Various doses of tannins were administered i.p. 30 min before the animals were anaesthetized with ether followed by i.v. injections of Evan’s blue solution (20 mg/kg). Intradermal injections of histamine (25 pg), 5-hydroxytryptamine (100 ng), bradykinin (5 pg) and prostaglandin E, (1 pg) in dose volumes of 0.1 ml in Tyrode’s solution were then made into the shaved areas. Forty five minutes later the rats were killed and the amount of plasma protein-bound dye leaked/site was measured spectrophotometrically (Harada et al., 1971). In each rat the quantity of dye exuded after i.d. injection of 0.1 ml Tyrode’s solution was subtracted from all the other values before calculations were made. The effect of tannins on agonistinduced dye leakage was assessed by comparing the responses in control and treated groups. The doses of agonists were such that these caused approximately similar (15-20 pg/site dye exudation) submaximal responses in preliminary studies. However, prostaglandin El was used at 1 pg/site, the dose which produced the maximum response of 8-12 pg/site of dye leakage obtained by this agent. Leucocy te migration studies The method described by Ford-Hutchinson et al. (1975) was followed. Sterilized-preweighed polyvinyl sponges (1.0 X 1.0 X 0.3 cm, 10 f 1 mg) were implanted S.C. into the dorsal region of Wistar rats (6/group) under ether anaesthesia. The animals received i.p. injections of various doses of tannins or saline 30 min before the implantation. Five hours later, the rats were killed and the sponge from each rat was placed in a 5-ml plastic syringe, the fluid squeezed gently into a volumetric flask and the process repeated after successive additions of 0.5-l .O ml Turk’s solution into the syringe to make the final volume of 5 ml. Total and differential leucocyte counts of the exudate were made using standard histological techniques. Inhibitory activities of tannins were assessed by comparing the mean number of cells present in the exudates of control and treated groups of rats. Prostaglandin synthesis inhibitory activity Rat fundic strip preparation was set up according to the method of Vane

293

(1957). The bathing fluid (Kreb’s) contained antagonists such as atropine, mepyramine and methylsergide, all at a concentration of 10 ng/ml. Various concentrations of arachidonic acid were added into the bath to obtain a concentration which would produce a submaximal contraction (60--80% of the maximal response) of the tissue. The selected concentration of arachidonic acid was repeated in the absence and presence of various concentrations of tannins to observe whether these would inhibit the responses to arachidonic acid. Indomethacin was used as the standard drug for comparison. When tested, both tannins and indomethacin were allowed to act for 5 min before the addition of arachidonic acid. Results Isolation

and identification

of tannins

From the freshly cut trunk bark, the extraction procedure gave a yield of 1.4% tannins. It was a mixture of hydrolysable (14%) and non-hydrolysable (86%) types. Acute

toxicity

tests

The LD,,, values in mice for tannins were 118.8 (95% confidence range 73.5-192.9) and 944.1 (95% confidence range 810.9-1099.5) mg/kg for i.p. and p.o. administrations, respectively. The value in rats was 245.0 (95% confidence range 187.5-320.3) mg/kg after i.p. dosing. As only 10% deaths occurred with the highest attempted dose of 4.0 g/kg p.o. the LDSOvalue could not be determined for this route. Carrageenan

and dex tran paw oedemas

A summary of the results obtained with tannins in these tests is presented in Table 1. When given i.p. the tannins were highly active in inhibiting the carrageenan oedema, the ED,,, value calculated from dose-response curve (method of least squares analysis) being 11.2 mg/kg (95% confidence range 6.2-20.2). A much greater dose of 1.0 g/kg had to be given orally to produce significant inhibition. Tannins injected i.p. also caused significant dosedependent suppression of dextran oedema, the inhibitions were, however, less than that for carrageenan. Oral dosing of tannins up to 1.0 g/kg was inactive in this test. Adrenalectomy did not appear to reduce the tannin effect. Cotton pellet granuloma

test

Tannins administerd daily for 7 days in doses of 6.25-50 mg/kg i.p. produced dose-dependent reduction of the granuloma tissue formation in rats.

294

TABLE 1 INHIBITORY ACTIONS OF TANNINS INDUCED PAW OEDEMAS -_________.._ Dose ;z:y,

Rats _-

ON CARRAGEENAN-

___.

___~_

Normals Normals Normals Normals Normals Normals Normals Normals Sham-operated control Adre~ectomized control -___ -.

AND DEXTRAN-

Percent inhibition of oedema * S.E.M. l_l_..... ____-

_--~_-.

Carrageenan

Dextran 7.4 12.6 29.7 31.0 40.4 NDa ND 0.0

_._._

3.12 6.25 12.50 25.00 50.00 200.00 500.00 1000.00

i.p. i.p. i.p. i.p. i.p. p.0. p.0. p.0.

35.7 51.8 65.4 68.4 80.4 7.3 23.9 38.1

t f f i f * * i

6.25 25.00 6.25 25.00

i.p. i.p. i.p. i.p.

47.5 70.0 42.0 65.5

k 5.0* 1: 6.1** * 5.0* i 6.51

_--.

5.8* 4.9** 4.6** 5.1** 5.7*** 1.6 2.8 3.6*

- --_II_._---

_

k 1.8 * 1.9 f 2.8* i 3.8* f 3.6*

ND ND ND ND

aND, Note done. Significance relative to untreated animals with pedal edemas *P C; 0.05; **f ***p < 0.001.

.< 0.01;

However, only the highest inhibition of 43.3% produced by the 50 mg/kg dose was statistically significant (P< 0.05). While the mean (+S.E.M). adrenal and thymus weights of control rats were 23.0 + 1.6 mg and 441.0 f 23.6 mg, respectively, the corresponding values in the tannin-treated group (50 mg/kg i.p.) were 26.0 f 2.1 and 420 f 30.0 mg, respectively. The results of the test group were not statistically different from those of the control group. No inhibitory activity was observed when tannins were given orally in daily doses of 500 mg/kg for 7 days. Studies on polyarthritic

rats

The results presented in Table 2 show that tannins injected i.p. or p.o. at a higher dose, reduced the severity of secondary lesions in rats with established arthritis. Statistically significant (P < 0.05) inhibitions of the severity of the lesions in test groups when compared with appropriate scores in the control group were obtained after a few day’s treatment with tannins. While control animals on average lost 2 g/rat during the experimental period, tannin-treated groups gamed between 4-8 g/rat, the gain depending on the route and doses administered. ~~r~thing test in mice Tannins given i.p. in graded doses of 6.25,12.5 and 25.0 mg/kg inhibited the writhing response by 34.2%, 42.6% and 59.2% respectively. The EDs0

ON ~OL~~T~R~~C

RATS

* 1.3 f 1.6 f 1.4 1: 1.6

-._~

i.p. 13.2 13.5 13.0 12.8

15

13.7 13.5 X2,5 12.9

16 +_1.3 f 1.7 f 1.4 f 1.6 14.0 13.8 12.0 11.9

17 * s f i

1.5 1.7 1.1 1.0

14.6 13.5 11.0 11.5

18 * r * *

1.3 1.0 1.0” 1.0

14.8 13.0 11.0 11.2

IQ + 1.4 f 0.8 f. l.O* * l.O*

15.0 11.6 10.0 10.7

20

I___-

t 1.4 f 0.7* I O.Q** +-0.8**

15.0 X1.6 9.6 10.0

21 t 1.4 * 0.6* ). 0x$*** * 0.8*”

15.0 f 1.4 11.0 f ox* 9.2 i 0.8*** 9.9 2 0.8***

22

--

*P < 0.05, **P< 0.025, ***P < 0.01 when compared with the mean arthritic score of corresponding days in control rats.

lOml/kg, 12.5 i.p. 25.0 i.p. 500 p-o.

Significance

Saline Tannins Tannins Tannins

Route

Tannins were administered daily from days 15-21. -. -I_ Mean arthritic score f S.E.M. for days Agent DOSt! (mg/kgI,

EFFECT OF TANN~S

TABLE 2

value was calculated (method of least squares analysis) as 15.8 mg (95% confidence range 7.4-33.7) while in the same series of experiments, the i.p. ED,, for aspirin was found to be 177.8 mg/kg (95% confidence range 92.3342.7). Tannins were without effect up to 300 mg/kg p.o. Permeability

studies

Previous i.p. administration of tannins in rats caused dose-related inhibition of the increased dye leakage induced by several mediators of inflammation. The antagonism was found to be non-specific as the effects of the agonists were reduced approximately to a similar degree even though the action of bradykinin appeared to be somewhat less affected (Table 3). Effect

on leucocyte

migration

Approximately 90% of the cells present in the 5 h sponge exudates were polymorphonuclear leucocytes (PMNL), and further, the number of mononuclear cells present varied widely among different samples. Therefore, only the effects of tannins on total leucocyte counts were determined. While the mean (*S.E.M.) total cell count for the control group was 8.1 t 1.2 millions, tannins injected i.p. in doses of 6.25, 12.5, 25.0 or 50 mg/kg significantly (P < 0.05 or less) reduced the number of cells by 30.0%, 49.5%, 77.0% and 81.2%, respectively, the ED5,, being 12.6 mg/kg (95% confidence range 8.718.3). Prostaglandin

synthetase

inhibitory

activity

Depending on the preparations, additions of l-8 @g/ml of arachidonic acid in the bath produced submaximal tissue contractions of the required magnitude. Presence of tannins in concentrations of up to 100 pg/ml did not affect the response to arachidonic acid. However, indomethacin (l-10 ng/ ml) produced concentrationdependent inhibitions or arachidonic acid-

TABLE

3

EFFECTS OF 30 min PREMEDICATION OF TANNINS i.p. ON DYE LEAKAGE INDUCED BY i.d. INJECTIONS OF HISTAMINE (H), 5-HYDROXYTRYPTAMINE (5-HT), BRADYKININ (BK) AND PROSTAGLANDIN E,, IN RATS Dose of tannin

Mean percent

(mgkg) --

H

6.25 12.5 25.0

18.2 35.7 66.2

Significance

relative

inhibition

r S.E.M.

5-HT * 2.3 f 3.0* i 5.0** to untreated

30.2 45.4 58.0 animals:

of dye leakage

-___ * 3.8* t 3.9* f 4.0** *P < 0.05;

induced

BK 14.3 33.3 42.8

by

PGE,

+z2.6 f 2.7* r 3.5*

**P < 0.01.

35.1 50.9 66.7

f 4.0* i 6.1** * 8.5**

297

induced stimulation of the fundic strips. These concentrations of indomethatin failed to modify prostaglandin E2 caused contractions of the preparations. Discussion The anti-inflammatory activities of plant constituents such as flavonoids, triterpenoids, coumarins, have been recently reviewed (Gabor, 1979). In addition, colchicine, cryogenine, tomatine and other alkaloids have also been reported to have anti-inflammatory or antirheumatic properties (Shen, 1974). Tannins are extensively distributed in the plant kingdom and tea tannin has been shown to inhibit rat paw oedemas induced by formalin or hyaluronidase, when administered along with vitamin C, but having no effect on its own (see Gabor, 1979). The present results suggest that the tannins from Anacardium occidentale apparently have some anti-inflammatory activity as these agents given i.p. inhibited the acute inflammatory responses of carrageenan and dextran paw oedemas and demonstrated activity in chronic inflammatory models such as cotton pellet-granuloma test and adjuvant-induced arthritis in rats. In higher oral doses, they also were effective against carrageenan-induced pedal edema and adjuvant-induced polyarthritis. In the carrageenan test tannins were approximately equiactive in normal and adrenalectomized rats and in the cotton pellet test the adrenal and thymus weights of animals were not different in control and treated groups. These results indicate that the action of the tannins is not mediated through the release of adrenal hormones. Preliminary studies with the rat fundic strip suggest that tannins may have no prostaglandin synthesis inhibitory activity. It is probable that the anti-inflammatory actions of these agents are due to an effect on leucocyte migration and antagonism of the phlogistic actions of mediators of inflammation as observed in these studies. Tannins may do so by their well known astringent properties causing precipitation of cell membrane proteins, thus affecting cell functions, for example, cell movements and membrane permeability changes. The analgesic action in the acetic acid-induced writhing test may also be explained in a similar manner as a non-specific effect as tannins in preliminary studies were inactive when heat was used to induce pain (unpublished results); further, throughout the pharmacological studies, dosing with tannins never demonstrated the presence of analgesia in the animals. There are many reports that irritant substances inhibit experimental inflammation by a counter-irritant mechanism (Laden et al., 1958; Benitz and Hall, 1963; Robinson and Robson, 1964; Jori and Bernardi, 1966) the nature of which is still unresolved (Atkinson and Hicks, 1971; FordHutchinson et al., 1973; Doherty and Robinson, 1976; Maguire and Wallis, 1977). To minimize the possibility of counter-irritation from parenteral injection Benitz and Hall (1963), originally recommended oral dosing for anti-inflammatory testing as the gastrointestinal tract was found to be less

298

sensitive to irritation than the peritoneal cavity. Since then others have continued to emphasize oral administration for anti-inflammatory screening of agents (Kaplan et al., 1967; Malone and Trottier, 1973). The present results with tannins demonstrating considerably higher activity by the i.p. than by the oral route also indicate that the irritant properties of these substances might have contributed to their anti-inflammatory actions. It would appear from these studies that the popular use of the decoction of the bark of Anacardium occidentale for rheumatic diseases may be due to its tannin content, which use must be discouraged as the toxic effects of tannin in animals (Glick and Joslyn, 1969; Peaslee and Einhelling, 1973; Pradhan et al., 1974; Kapadia et al., 1976) and humans (Krezanoski, 1966; Eschar and Friedman, 1974) are well documented. As water-soluble substances, tannins are likely to be present in aqueous or aqueous-ethanol extracts of plants and produce non-specific effects including anti-inflammatory and analgesic actions. This may present further difficulties in addition to the general problems already discussed by others (Farnsworth and Bingel, 1976; Malone, 1980) in evaluating the biological actions of plant extracts. Acknowledgement We thank Prof. Delby F. Medeiros for his encouragement, Prof. F. Agra for identifying and supplying the plant materials, Mr. J.C. Duarte, Mrs. C.G. de Oliveira and Mr. Gilmario Moreira Lima for technical help. Financial assistance was provided by CEME and CNPq. References Aguiar, F.J.C., Cardoso, J.V. and Azoubel, R. (1959) Novas considera@es sobre o efeito hipoglicemiante do Anacardium occidentale L. Anais da Faculdade de Medicina da Universidade do Recife 19, 353-367. Arshad, M., Beg, A. and Siddiqui, Z.A. (1969) Infrared spectroscopic investigation of tannins. Angewandte Makromolekulare Chemie 7, 67-78. Atkinson, D.C. and Hicks, R. (1971) The possible occurrence of endogenous antiinflammatory substances in the blood of injured rats. British Journal of Pharmacology 53, 85-91. Benitz, K.F. and Hall, L.M. (1963) The carrageenin-induced abscess as a new test for antiinflammatory activity of steroids and nonsteroids. Archives Internationales de Pharmacodynamie 144,185-195. Braga, R. (1960) Plantas do Nordeste, Especialmente do Ceara, 2nd edn., Imprensa Official, Fortaleza, Brazil, pp. 104-110. Coimbra, R. (1958) Notas de Fitoterapia, Laboratorio Clfnico Carlos da Silva Araujo S.A., Rio de Janeiro, Brazil, p. 85. Correa, P.M. (1969) Dictionario das Plantas cteis do Brazil e Exbticas Cultivadas, Vol. 1, Ministerio da Agricultura, IBDF, pp. 400-401. Costa, A.F. (1972) Farmacognosia, 2nd edn., FundacXo Calouste Gulbenkian. Lisboa, Portugal, pp. 638-641. D’Arcy, P.F., Howard, E.M., Muggleton, P.W. and Townsend, S.B. (1960) The antiinflammatory actions of griseofulvin in experimental animals. Journal of Pharmacy and Pharmacology 12, 659-665.

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