JPM Vol. 28, No. 1 August 1992:15-19

A Sensitive and Relevant Model for Evaluating Anti-Inflammatory Activity-Papaya Latex-Induced Rat Paw Inflammation

O. P. Gupta, N. Sharma, and D. Chand

Department of Pharmacology, Regional Research Laboratory, Jammu-Tawi, India

A new model employing latex of papaya as an inflammagen has been developed for testing anti-inflammatory activity. The latex (exudate) was harvested from the unripe papaya fruit, which had been dried under vacuum. The latex was then suspended in 0.05 M sodium acetate buffer. This suspension when injected in rat hind paw produced concentration- dependent inflammation. Of the 0.25% of this suspension, 0.1 ml was found ideal for evalu- ating anti-inflammatory activity of test drugs. This concentration produced 70%-100% inflammation lasting for about 5 hr with a maximum effect at h 3. The test drugs employed were prednisolone, aspirin, indomethacin, phenylbutazone, ibuprofen, piroxicam, chlo- roquine, levamisole, and a mixture of boswellic acids. For comparison, these drugs were also tested against carrageenan-induced inflammation. All the test drugs--steroidal, aspi- rin, and non-aspirin-like--showed anti-inflammatory activity against latex-induced inflam- mation. The activity of chloroquine, levamisole, and boswellic acids was significantly more against latex as compared with that of the carrageenan model. The inflammation caused by latex may be attributed to both its hydrolytic enzymes--papain and chymopapain--and glutathione, the activator of these enzymes. These enzymes seem to act like lysosomal enzymes that are released in inflammatory disease processes which mediate inflammation by stimulating the synthesis of prostaglandins. The papaya latex-induced inflammation model appears to be a sensitive, broad-based, and relevant one likely to prove useful for discovering new and effective drugs against inflammation and rheumatoid arthritis.

Keywords: Papaya latex; Hydrolytic enzymes; Rat paw; Anti-inflammatory evaluation; Preference over carrageenan.

Introduction

The success in discovering new clinically effective drugs depends mainly on the appropriateness of the animal model employed for testing. The search for a drug useful in the treatment of rheumatoid disorders is handicapped for lack of an ideal experimental model, as the very etiology of these disease processes is not known. The remission-inducing or disease-modifying properties of drugsmlevamisole, chloroquine, gold salts, o-penicillamine, and cyclophosphamidemin ar- thritis were discovered, not as a result of studies with known experimental models, but by serendipidity as- sociated with clinical observations. These remission-

Address reprint requests to Dr. O. P. Gupta, Department of Phar- macology, Regional Research Laboratory, Jammu-Tawi 180 001, (J&K), India.

Received June 1991; revised and accepted May 1992.

Journal of Pharmacological and Toxicological Methods 28, 15-19 (1992) © 1992 Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, New York, NY 10010

inducing drugs have been reported to show little or no activity in animal models of inflammation and arthritis developed so far (Lewis et al., 1985).

Inflammation itself is a complex biological phenom- enon that defends the host against a noxious agent. It constitutes a cascade of events without a clear percep- tion. The chemical mediators known to be involved are histamine, serotonin (5-HT), chemotactic factors, bradykinin, prostaglandins, leukotrienes, and others (Ward, 1974). The invasion of inflammed tissue by polymorphonuclear (PMN) leukocytes, phagocytosis, and the release of lysosomal enzymes, followed by the release of arachidonic acid from phospholipids, and the generation of prostaglandins has been reported (Fer- reira and Vane, 1979).

Drugs effective against acute inflammation are ef- fective in combating rheumatic disorders. Therefore, an ideal anti-inflammatory testing model was devel-

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16 JPM Vol. 28, No. 1 August 1992:15-19

oped by employing an agent that mimicked lysosomal enzymes in nature and action. To accomplish this, we chose the hydrolytic enzyme papain, which is obtained from papaya latex; however, upon testing it showed only mild activity. Papaya latex was tried with the hope that delayed absorption of papain from latex at the site of injection may enhance papain's effect. Once tested, it was found to cause marked inflammation. A review of the literature revealed that, aside from papain, latex also contains chymopapain and glutathione, the activa- tor of these enzymes (Ganapathy and Sastry, 1938). Thus the inflammation caused by latex may be attrib- uted to their combined action.

This study discusses the induction of inflammation with papaya latex, and the response of various anti- inflammatory drugs acting by different mechanisms against this inflammation. An attempt is also made to compare the efficacy of this model with the popularly employed carrageenan model.

Materials and Methods

Inflammation Induction and Anti- Inflammatory Evaluation

Male albino Charles-Foster rats (150-200 g) were used in this study. Test drugs were prepared as a finely homogenized suspension in 2% w/v gum acacia. These were administered orally (PO) to rats 45 rain before the 0. l-mL injection of inflammagen-papaya latex or carrageenan. Paw volume was measured at 0 hr and every hour for 5 hr after inflammagen injection, using volume differential meter, model 7101 UGO Basile.

Inflammagens Papaya latex. This was obtained by making longitu-

dinal incisions one-eighth of an inch deep on the sur- face of unripe, green Papaya fruit while it was still hanging on the tree. The exudate was collected in glass petri dishes and dried under a vacuum. The product obtained was creamy white and was stored in an air- tight container at 4°C. Stock suspension of the latex (1%) was prepared in 0.05 M sodium acetate buffer (pH 4.5). The stock solution was diluted with buffer before being injected in rat hind paw.

Carrageenan. An acqueous suspension (1%) of this product obtained from Springfield, MA, U.S.A. was used.

Papain. This was obtained from Sigma Chemical Co., St. Louis, MO, U.S.A. A suspension of different concentrations in sodium acetate buffer was employed.

Test Drugs The following drugs were used:

Jammu-Tawi, India), indomethacin aspirin (RRL, (Merck Sharp

Dohme), phenylbutazone (SG Pharmaceuticals), ibu- profen (Cadila Labs. Ltd.), piroxicam (Pico Labs. Pvt. Ltd.), chloroquine phosphate (IDPL), levamisole hy- drochloride (British Pharmaceutical Laboratories), prednisolone (Wyeth Laboratories Ltd.), boswellic acids (RRL, Jammu), cyproheptadine (Albaetra Labo- ratories Pvt. Ltd.), and phenergan (May & Baker Ltd.).

Results

Rat Paw Inflammation with Papaya Latex and Papain

Papaya latex tested in 0.25%, 0.5%, and 1% concen- trations injected in 0.1-mL volume in rat hind paw pro- duced dose-related increases in paw volume; this was monitored every hour for 5 hr. The profile of inflamma- tion during 5 hr is that of a curvature. The increase in paw volume with above concentrations is about 70%-130% from initial level and is well maintained from hr 1 to 5. After 14 hr, 75%-80% of inflammation subsides. Papain tested in different concentrations showed weak inflammagenic activity. This activity when recorded even with a 5% solution of papain was of a lesser degree as compared with that recorded with a 0.25% suspension of papaya latex, however, the pro- file of inflammation in both cases was similar.

Effect of Cyproheptadine and Phenergan Pretreatment on Latex-Induced Inflammation

There was no significant effect on degree or profile of inflammation caused by papaya latex after cypro-

tO0

8(

I

2

W e , ,

t l . l Z

Figure 1. Comparison between raw paw inflammation in- duced by carrageenan 0.1 ml, 1% (1) and that of papaya latex 0.1 ml, 0.25% (2). Number of animals in each group = 40. Vertical bars represent SE _+ mean.

! i a I

2 3 4 5

TIME ( H O U R S )

GUPTA ET AL. 17 EVALUATING ANTI-INFLAMMATORY ACTIVITY-PAPAYA LATEX-INDUCED RAT PAW INFLAMMATION

heptadine and phenergan treatment (1 mg and 2.5 mg/ kg PO, respectively).

Comparison with Carrageenan- Induced Inflammation

Figure 1 shows the degree and profile of rat paw inflammation caused by papaya latex and carrageenan. As compared with curvature-type profile observed with papaya latex, the carrageenan-induced inflamma- tion is of ascending nature during the first 3 hr, fol- lowed by its maintenance at maximum in next 2 hr.

Evaluation and Comparison of Anti- Inflammatory Effect of Drugs Against Latex- and Carrageenan-lnduced Inflammation (Table 1)

Aspirin-like anti-inflammatnry drugs. Indomethacin 5 mg/kg i.p. This drug showed a

sharp increase in inhibition of inflammation up to hr 4, followed by steady state up to hr 5. Against carra- geenan, there was a sharp increase up to hr 3, followed by a decrease.

Peroxicam 5 mg/kg PO. This showed a sharp in- crease in inhibition of inflammation up to hr 4, followed

by steady state up to hr 5. Against carrageenan, a maxi- mum effect was reached by hr 2, then maintained be- tween hrs 2 and 3, and followed by a steady decrease.

lbuprofen 100 mg/kg PO. The inhibition of inflam- mation increases up to hr 3, which continues at its max- imum up to hr 5. Against carrageenan, a sharp rise up to hr 2 was followed by a steady decrease.

Aspirin 100 mg/kg PO. The inhibition of inflamma- tion reflects that of an ascending and descending order with a pattern such as that of a curvature. Against car- rageenan, the maximum effect is at hr 2, followed by a sharp decline.

Anti-inflammatnry drugs other than aspirin-like drugs.

Prednisolone 5 mg/kg PO. Steep inhibition of in- flammation up to hr 3, followed by maintenance of inhi- bition up to hr 5. Against carrageenan, the pattern is almost similar.

Levamisole 20 mg/kg PO. The inhibition of inflam- mation shows a steady increase up to hr 5. Against carrageenan, the inhibition observed is much less.

Boswellic acids 100 mg/kg PO. The inhibition ob- served reflects a gradual rise and fall. Against carra- geenan, this inhibition is negligible.

Chloroquine phosphate 25 mg/kg PO. Maximum in- hibition of inflammation is at hr 2, followed by a slow

Table 1. Comparison of Inhibition of Papaya Latex- and Carrageenan-Induced Inflammation in Rat Paw by Anti- Inflammatory Drugs

Case Number

% Inhibition of Inflammation Compared With Control Group

Drug (mg/kg/PO Inflammagen 1 hr 2 hr 3 hr 4 hr 5 hr

A. Aspirin-Like

Average of 5 hr

1. lndomethacin (5) a

2. Piroxicam (5)

3. Ibuprofen (100)

4. Aspirin (100)

P 29 ± 2.0 46 ± 2.5 56 ± 3.5 61 -*- 4.0 60 ± 4 50.4 C 26 ± 3.5 62 ± 2.5** 69 ± 5.5 59 • 4.5 52 ± 4 53.6 P 28 ± 2.5 41 __. 3.0 49 ___ 3.0 55 -* 4.0 56 __- 4.0 45.8 C 51 ± 3.5 66 ± 3.0** 65 ___ 3.5** 52 ± 3.0 48 ± 3.0 56.4 P 25 ± 2.0 39 ± 2.5 44 ± 3.5 44 ± 3.0 43 _+ 3.5 39.0 C 49 - 2.5 59 __- 3.0** 46 ± 4.0 39 ± 3.5 32 ± 3.5* 45.0 P 16 ± 1.5 29 ± 2.5 31 ± 2.5 26 _ 2.5** 18 - 2.0 24.0 C 36 ± 3.5** 40 ± 3.5* 28 ± 4.0 16 ± 2.5 12 ± 2.5 26.4

B. Non-Aspirin-Like

1. Prednisolone (5)

2. Levamisole (20)

3. Boswellic acids (100)

4. Chloroquine phosphate (25)

P 17 "¢- 2.5* 37 ± 2.5 47 ± 3.0** 48 ± 3.5 48 - 3.0 39.4 C 9 "¢" 3.0 30 ± 2.5 35 ± 2.5 39 - 3.5 39 ± 4.0 35.3 P 33 ± 2.5** 37 ± 2.0** 40 - 2.0** 42 *" 3.0 45 - 2.0 30.0 C 14 -~ 2.5 21 ± 3.0 15 ± 2.5 11 --. 4.0 10 ± 3.0 14.2 P 30 - 2.5** 39 --- 3.0** 41 _ 2.5** 37 ± 3.0** 28 - 2.5** 35 C 11 - 1.0 8 --. 2.0 8 _ 1.0 0 0 5.4 P 29 ± 2.5** 38 ± 2.0** 33 __+ 2.5** 29 ± 3.0** 27 ± 2.5** 31.2 C 15 ± 2.0 19 ± 2.0 17 --- 3.5 15 ± 4.0 11 ± 3.5 15.4

Note: Number of animals in each group = 15. P, papaya latex; C, carrageenan.

Administered i.p. * p = 0.05. **p = 0.01.

18 JPM Vol. 28, No. 1 August 1992:15-19

decrease. Against carrageenan, the inhibition observed is much less.

Discussion

From the results of the present study, it is evident that the latex of papaya induces inflammation in the rat paw, which responds well to steroidal, aspirin, and non-aspirin-like drugs. Furthermore, this inflammation appears to mimic that of inflammatory disease pro- cesses, and the response of remission-inducing drugs, that is, chloroquine and levamisole, effective clinically in rheumatoid arthritis, has been found to be significant by this model.

The inflammation induced by papaya latex is dose related and remains well maintained for 5 hr. Com- pared with this, inflammation caused by carrageenan is of ascending nature in the first 3 hr, followed by its maintenance at maximum during the next 2 hr. Whereas the inflammation caused by carrageenan is attributed to the release of histamine, 5-HT, kinin, and prostaglandins (Di Rosa et al., 1971), histamine and 5- HT do not seem to be involved in the latex-induced inflammation, as cyproheptadine and phenergan pre- treatment had no effect on the genesis and course of this inflammation. This is a favorable feature as antago- nists of histamine and 5-HT are known to have little or no effect in inflammation.

The anti-inflammatory effect recorded with aspirin- like drugs against latex-induced inflammation follows a uniform pattern as compared with that observed with carrageenan, but the average anti-inflammatory effect recorded in both cases during the course of 5 hr is almost the same (Table 1). The anti-inflammatory ef- fect recorded with levamisole, chloroquine, and bo- swellic acids is significantly more and sustained against latex as compared with carrageenan-induced inflam- mation. This is an important observation as these drugs found clinically effective in rheumatoid arthritis have been reported to show little or no activity against known animal models of inflammation and arthritis (Lewis et al., 1985).

The inflammation caused by papaya latex may be attributed to its hydrolytic enzymes--papain and chy- mopapain--and their activator glutathione (Ganapathy and Sastry, 1938). These enzymes seem to be acting like lysosomal enzymes, which are involved in inflam- matory disease processes. The release of hydrolytic enzymes from lysosomes in response to inflammagenic agents is considered one of the important responses in the development of inflammation (Rainsford, 1987). These enzymes are released following either the death of cells after interaction with cytotoxic agents or in a selective manner where release occurs by secretion from viable cells including macrophages (Dean, 1979;

Crowder et al., 1969; Hawkins and Peeters, 1971; Hen- son, 1971; Cardella et al., 1974; Cohn and Wiener, 1963). These enzymes, aside from causing autolytic re- actions, are also reported to participate or initiate in- flammatory reactions (Rainsford, 1987). Included among these enzymes are phospholipases responsible for the release of arachidonic acid from phospholipids of cell membrane which lead to the production of in- flammatory prostanoids, hydroxy fatty acids, and leu- kotrienes (Rainsford, 1987). It is likely that, similar to phospholipases, enzymes present in latex mediate in- flammation through prostanoids and leukotrienes. This inference is supported by the observations made with anti-inflammatory drugs on latex-induced inflam- mation.

The aspirin-like group of drugs is known to act on inflammation through the inhibition of prostaglandin synthesis (Vane, 1971). The effectiveness of these drugs against latex-induced inflammation is indicative of the fact that this inflammation is mediated through prostaglandins. Also, chloroquine, which showed a significant effect against this inflammation and is clini- cally effective in rheumatoid arthritis, has been re- ported to act through the inhibition of phospholipase A2 (Arrigoni-Martelli, 1985) and by direct influence on prostaglandins (Famaey and Fontaine, 1977). Like- wise, boswellic acids, found clinically effective in rheu- matoid arthritis (unpublished data) and reported to act by the inhibition of PMN leukocyte migration (Sharma et al., 1989), have showed significant anti-inflamma- tory effect against latex-induced inflammation. Be- cause the presence of leukocytes at the inflammation site leads to phagocytosis of inflammatory stimulus, release of lysosomal enzymes, activation of membrane phospholipases, release of arachidonic acid, and syn- thesis of prostaglandins, this anti-inflammatory effect can be attributed again to the inhibition of prostaglan- din synthesis because of the inhibition of leukocyte migration to the inflammation site. Levamisole, re- ported to act as a result of its immunonormalizing ac- tivity (Talal and Hadden, 1985) in rheumatoid arthritis, has also been found to be significantly effective against papaya latex-induced inflammation. This could be at- tributed to its reported direct action on mobility, secre- tion, and proliferation of lymphocytes, macrophages, and granulocytes, augmenting or depressing in its ef- fect, depending on the dose and timing of administra- tion (Talal and Hadden, 1985). The effectiveness of prednisolone against latex-induced inflammation is again explicable based on inflammation induced by latex, which is mediated through prostaglandins, as re- ported by several authors who state that glucocorti- coids act on inflammation through the inhibition of prostaglandin synthesis (Kantrowitz et al., 1975; Lewis

GUPTA ET AL. 19 EVALUATING ANTI-INFLAMMATORY ACTIVITY-PAPAYA LATEX-INDUCED RAT PAW INFLAMMATION

and Piper, 1975; Blackwell et al., 1980; Flower and Blackwell, 1979).

Inflammation associated with rheumatoid arthritis is attributed to the combination of an antigen with an antibody and complement leading to the local release of chemotactic factors, leukocytes attraction, phago- cytosis of antigen, antibody and complement complex, and release of lytic enzymes. It is also reported that under certain conditions, selective release of hy- drolases continues over a long period of time, thus ex- plaining the possible cause of tissue damage, degrada- tion, and remodelling seen during chronic inflamma- tory processes (Davies and Allison, 1976).

In view of the role of lysosomal enzymes in inflam- matory disease processes, a resemblance of enzymes present in papaya latex with lysosomal enzymes for mediating inflammation possibly through prostaglan- dins, and the response of this inflammation to steroidal, aspirin, and remission-inducing drugs--chloroquine and levamisole--effective clinically in rheumatoid ar- thritis but displaying little or no effect in the known models of inflammation and arthritis, and the fact that all these features are associated with latex-induced in- flammation, it can be concluded that papaya latex-in- duced inflammation is a sensitive, broad-based, and relevant model ideal for discovering new and effective anti-inflammatory and anti-arthritis drugs.

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