Anti-inflammatory Activity of a Protease Extracted from the Fruit Stem Latex of the Plant Artocarpus heterophyllus Lam.

Indranil Chanda^1*, Smriti Rekha Chanda¹and Sadhan Kr Dutta²

¹Department of Pharmacy, Girijananda Chowdhury Institute of Pharmaceutical Science, Hathkhowapara, Azara, Guwahati – 781017, Assam, India

²Department of Pharmacy, Gupta College of Technological Sciences, Asansol- 713301 West Bengal, India

ABSTRACT

The anti-inflammatory activity of the new protease fraction Artocarpain-H extracted from the fruit stem latex of the plant Artocarpus heterophyllus Lam. was evaluated using Carrageenan induced rat paw oedema and Cotton pellet-induced granuloma model. The Artocarpain-H dose dependently inhibited carrageenan induced rat paw edema. It also showed reduction on the granuloma weight in the cotton pellet granuloma method.. In the present investigation, as the test drug is effective in both models of inflammation, there is a possibility that these drugs may be effective in acute and chronic inflammation.

KEY WORDS: Protease, Artocarpain-H, rat paw oedema, granulom.

INTRODUCTION

Artocarpus heterophyllus Lam. (Moraceae) tree is handsome and stately, 30 to 70 ft tall, with evergreen, alternate, glossy, somewhat leathery leaves, oval on mature, wood sometimes oblong or deeply lobed on young shoots. All parts contain sticky, white latex. It is also known as ‘Jackfruit’ in English and ‘katahal’ in Hindi.^{1, 2} Jackfruit is native to India, Bangladesh, and Nepal. It is cultivated at low elevations throughout India, Burma, Ceylon, southern China, Malaya, East Indies and Philippines.³ Traditionally the dried latex of the plant is used to yields artostenone, a compound with marked androgenic action .The juice of the plant is applied externally to glandular swelling and abscesses to promote suppuration.^{3, 4} The reported medicinal properties of the plant are Antibacterial,⁵ Scavenger and Antioxidant,⁶ Antiinflammatory,⁷ sperm –agglutinating.⁸ In this study, we investigated the analgesic and anti-inflammatory activities of the extracted protease fraction ‘Artocarpain-H’ from the fruit stem latex of the plant.

MATERIAL AND METHODS:

Plant material:

The Latex of was collected from Mayurbhanj district (Orissa, India) in February 2006. The plant was authenticated at Botanical Survey of India, Howrah, West Bengal (India). A voucher specimen was deposited at the Central National Herbarium of Botanical Survey of India. Latex was collected from the stem of the fruit by incision.

Extraction of Protease:

Latex was collected from the immature fruit stem of Jackfruit into a flask containing 0.1% EDTA solution and 10% sodium metabisuphite as antioxidant. The collected crude latex was kept overnight in cold condition in refrigerator. The latex was then filtered first through cotton and then through ordinary filter paper to remove the white gummy material. To the clear crude extract 65% saturated ammonium sulphate solution was added slowly with constant shaking. The solution was kept overnight in a refrigerator (2-8°C) and then centrifuged (5000 rpm).The precipitate was washed with acetone and dried.^9,
10 Chemical test for protein¹¹ and protease assay were carried out using standard procedures.¹²

Table1: Effect of Artocarpain-H on carrageenan induced rat paw edema (Mean paw volume)

Drug	Mean paw volume [ml]
Drug	1h	2h	3h	4 h
Control (10ml/kg)	0.82 ± 0.05	0.91± 0.07	0.98± 0.04	0.98± 0.06
Indomethacine (10mg/kg)	0.21± 0.02**	0.18± 0.03**	0.17± 0.02**	0.16±. 04**
Artocarpain-H (50mg/Kg)	0.58± 0.07	0.51± 0.04	0.45± 0.06*	0.45± 0.08*
Artocarpain-H (100 mg/Kg)	0.51± 0.05	0.32± 0.08*	0.31± 0.02*	0.30± 0.04**
Artocarpain-H (200 mg/Kg)	0.46± 0.04	0.24 ±0.04**	0.22± 0.01**	0.20± 0.02**

Values expressed as mean ± SEM; (n=6); *(p<0.01), **(p<0.001) control vs. treated groups

Table2: Effect of Artocarpain-H on carrageenan induced rat paw edema (Percentage of inhibition)

Drug	Percentage (%)inhibition
Drug	1h	2h	3h	4h
Control	-	-	-	-
Indomethacine (10mg/kg)	74.39	80.22	82.65	83.67
Artocarpain-H (50mg/Kg)	29.27	43.96	54.08	54.08
Artocarpain-H (100 mg/Kg)	37.80	64.83	68.37	69.39
Artocarpain-H (200 mg/Kg)	43.90	73.63	77.55	79.59

Animals:

Swiss albino mice of both sexes weighing between 20–25 gm and Wistar albino rats (150–175 g) were used for the test of LD₅₀ value and anti-inflammatory activity respectively. They were obtained from the animal house, S.I.P.S., Jharpokharia, Orissa. They were maintained under standard environmental conditions and were fed with standard pellet diet supplied by Hindustan Lever Ltd., Kolkata, India and water ad libitum.

Toxicity study:

The extracted protease fraction (named as ‘Artocarpain-H’) was dissolved in glass distilled water and used for the toxicity study. The experiment was carried out taking 10 groups of animals each group containing 10 mice. The LD₅₀ value was determined by the method Litchfield and Wilcoxon.¹³ The animals were administered with Calotropain-G intraperitoneally, starting from the dose of 400 mg/kg up to 1200 mg/kg bodyweight. From the observed value three dose levels of 50, 100 and 200 mg/kg were selected for the estimation of antinflammatory activity.

Anti-inflammatory activity:

Carrageenan induced rat paw oedema:

Acute anti-inflammatory activity of Artocarpain-H was evaluated using carrageenan induced oedema in rats described by Winter et al.¹⁴ Five groups of albino rats (n = 6) were randomly distributed in control, standard and test (50mg/kg, 100 mg/kg and 200 mg/kg of Artocarpain-H ) groups. The initial paw volumes of each animal were measured by means of a mercury plethysmometer. The standard group was treated with Indomethacine (10 mg/kg). Artocarpain-H at the concentration of 50, 100 and 200 mg/kg and normal saline (10 mL/kg) were given respectively to the test and control groups. Thirty minutes after treatment, 0.1 mL of 1% carrageenan solution was injected in the plantar region of the left hind paw of rats. The paw volume was measured at 1, 2, 3, 4h after carrageenan injection using Plythesmograph. The acute difference in oedema volume was calculated in each control, test and standard group and compared with the control group for determination of the percentage of inhibition of the paw oedema.

Table 3: Effect of Artocarpain-H on cotton pellet induced granuloma in rats

Treatment	Weight of granuloma (mg)	Percentage (%) inhibition
Control	36.43 ± 2.3	-
Indomethacine (10mg/kg)	20.21 ± 1.6^**	44.52
Artocarpain-H (50mg/Kg)	31.83±1.1^*	12.63
Artocarpain-H (100 mg/Kg)	27.31 ±0.98^**	25.03
Artocarpain-H (200 mg/Kg)	23.19 ±1.09^**	36.34

Values expressed as mean ± SEM; (n=6); * (p<0.01), ** (p<0.001) control vs. treated groups

Cotton pellet-induced granuloma:

Wistar albino rats (170 -200 gm) of either sex were divided into 4 groups of 6 animals in each group. Cotton pellets weighing 30±1mg were autoclaved and implanted subcutaneously into both sides of the groin region of each rat as described by D’Arcy etal.¹⁵ Group I served as control and received the vehicle (1% CMC). Group II animals received Indomethacine as reference standard at a dose of 10 mg/kg orally for 7 days. Artocarpain-H at concentrations of 50, 100 and 200 mg/kg were administered orally respectively for Group III, IV and V to animals for same period. On the 8^thday the animals were sacrificed and the pellets together with the granuloma tissues were carefully removed, dried in an oven at 60^oC, weighed and compared with control.

RESULT:

The extracted compound Artocarpain-H gave positive test for protein. Artocarpain-H also showed significant activity in protease assay. The LD₅₀ value of Artocarpain-H when given through intraperitoneal route in mice was found to be 676.08 mg/kg body weight of mice.

In acute inflammation model, the carrageenin induced paw oedema was significantly reduced by the standard and test drugs when compared to control (Table 1and 2).

This anti-inflammatory activity was dose-dependant and found to be statistically significant at the higher concentration, 200 mg/kg body weight.

In the model of sub acute inflammation, the weight of the granulation tissue was significantly reduced by treatment with Artocarpain-H and Indomethacine. There was also dose dependant reduction in granular tissue formation in Artocarpain-H and indomethacin treated rats (Table 3). The activity was found to be statistically significant for the dose ranges used.

DISCUSSION:

Carageenan induced inflammation is a useful model to detect oral action of anti-inflammatory agents.¹⁶ The development of oedema in the paw of the rat after the injection of Carageenan is due to release of histamine, serotonin and prostaglandin like substances.¹⁷ The significant anti-inflammatory activity of the extract (Artocarpain-H) and standard drug observed in the present study may be due to inhibition of the mediators of inflammation such as histamine, serotonin and prostaglandin. The Carageenan assay is a good method for the comparative bioassay of anti-inflammatory agents. The present results indicate the efficacy of Artocarpain-H as an efficient therapeutic agent in acute anti-inflammatory conditions.

In the cotton pellet granuloma model, inflammation and granuloma develops during the period of several days. The cotton pellet granuloma method has been widely employed to assess the transudative, exudative and proliferative components of chronic inflammation. Inflammation involves proliferation of macrophages, neutrophils and fibroflasts, which are basic sources of granuloma formation. The fluid absorbed by the pellet greatly influences the wet weight of the granuloma. Hence, the decrease in the weight of granuloma indicates that the proliferative phase was effectively suppressed by the test drug Artocarpain-H.

ACKNOWLEDGEMENT:

The authors are thankful to the Principal and the Management of Seemanta Institute of Pharmaceutical Sciences, Jharpokharia, Mayurbhanj, Orissa for providing necessary facilities to carry out the present research work.

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