Anti-inflammatory agents from Sandoricum koetjape Merr.

Summary

The anti-inflammatory activity of the stem extracts of Sandoricum koetjape was investigated on topical administration using the TPA (tetradecanoylphorbol acetate)--induced mouse ear inflammation model. Bioassay-guided chromatographic fractionation of active fractions led to the isolation 3-oxo-12-oleanen-29-oic acid and katonic acid as the bioactive principles responsible for the anti-inflammatory acitivity. The percentage of inhibition exhibited by 3-oxo-12-oleanen-29-oic acid was almost equivalent to indomethacin.

Key words: Sandoricum koetjape, Anti-inflammatory activity, 3-oxo-12-oleanen-29-oic acid, katonic acid

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Introduction

Inflammation is known to occur via a series of complex pathophysiological pathways, influenced by various mediators such as prostaglandins and leukotrienes. These mediators can cause oedema such as heat, pain, disturbed tissue function, reddening and swelling. Assay models which focus on blocking the production or the action of these mediators have been used in evaluating extracts and purified compounds for anti-inflammatory properties. A general topical method to assay for anti-inflammatory activity of plant samples is the 12-O--tetradecanoylphorbol-13 acetate (TPA) induced mouse ear oedema model (Carlson et al. 1985, Bird et al. 1986). The model proved useful in determining the in vivo activities of non-steroidal anti-inflammatory drugs (NSAIDs), mixed lypoxygenase (LO)/cyclooxygenase (COX) inhibitors or 5-LO inhibitors (Carlson et al. 1985).

In the course of a random screening of plants for potential anti-inflammatory properties using the above model, the methanolic stem extract of Sandoricum koetjape Merr (Meliaceae), demonstrated significant activity. Sandoricum koetjape is known locally as 'sentul'. It is a medium sized tree, native to South East Asia, including Malaysia and the Philippine Islands. The fruit of the species is edible and makes an excellent jelly or candy, popular among locals (Burkill, 1966). The bark constitutes one of the ingredients of "rempah ratus", a traditional Malay preparation used as protective medicine after childbirth. Previous chemical studies on S. koetjape included reports of triterpenoids from the fruit hulls (Sim et al. 1972) and stems (Kaneda et al. 1992), as well as limonoids from the seeds (Powell et al. 1991). There are relatively few reports on the bioactivity and pharmacological properties of S. koetjape, thus its high anti-inflammatory makes the plant an interesting candidate for further study. We now report our investigation on the in vivo anti-inflammatory activity of S. koetjape and the bioassay-guided isolation of its bioactive constituents.

Materials and Methods

Sandoricum koetjape was collected during the month of October, 2000, from the Fruit Tree Arboretum of Forest Research Institute Malaysia (FRIM) in Selangor. A voucher specimen (FR0057) has been deposited at the Institute's Herbarium for reference.

Extraction and fractionation

Powdered, dried stems (1 kg) of S. koetjape were extracted, successively, with hexane, dichloromethane, ethyl-acetate and methanol, each for over a period of 12 hours. The extracts were concentrated under reduced pressure to provide the dry extracts. The methanol extract was (20 g) was further partitioned between hexane, dichloromethane, ethyl-acetate methanol and water fractions. All fractions were then concentrated in vacuo and stored at -20[degrees]C prior to testing. All fractions were evaluated for anti-inflammatory activity on the TPA-induced mouse ear oedema assay. The active hexane (5 g) and dichloromethane (5 g) fractions of S. koetjape were further subjected to bioassay-guided isolation of the active compounds using silica gel chromatography, the eluants for each fraction being CH[Cl.sub.3]-MeOH (9:1) and CH[Cl.sub.3]-EtOAC (4:1), respectively. In each case, active fractions were mixed and subjected to further column chromatography and preparative thin layer chromatography (TLC) to yield 3-oxo-12-oleanen-29-oic acid (10 mg), katonic acid (96 mg) and koetjapic acid (63 mg) and [beta]-sitosterol (35 mg). Each of these compounds were tested for their anti-inflammatory activities. Alloaromadendrene and caryophyllene, were also detected by GC/MS in the active fractions but these were isolated in such small amounts and thus were not tested for activity.

[FIGURE 1 OMITTED]

Structure determination

The identities of the isolated compounds were determined by comparison of their physical ([.sup.1]H NMR, [.sup.13]C NMR and MS) data with published values (Kaneda et al. 1992). [beta]-sitosterol was confirmed by comparison with an authentic sample.

Animals

Groups of six female BALB/c mice weighing 17-22 g were purchase from the Animal Resource Centre, Institute for Medical Research (IMR), Kuala Lumpur. The animals were housed at 23 [+ or -] 4[degrees]C with a 12 h lighting schedule. The animals were fed on pellet chow and water.

Chemicals

12-O-tetradecanoylphorbol-13-acetate (TPA) was purchased from Wako Pure Chemical Industries Ltd. Japan and indomethacin from Sigma.

TPA--induced mouse ear oedema

Anti-inflammatory activity of S. koetjape was evaluated using a modification of the methods of Gschwendt et al. (1984) and Hirota et al. (1990). A 25 [micro]g/ml stock solution of 12-0-tetradecanoylphorbol-13-acetate or TPA was prepared in acetone. Each mouse was then treated with 20 [micro]l (0.5 [micro]g/ear) of TPA on the inner surface of both ears. Test extracts, pure compounds and the standard drug were each prepared in acetone and applied topically to the right ear (2 mg/ear) 40 minutes before or after TPA application. The left ear (control) received the same volume of acetone. After 6 hrs, the mice were killed by cervical dislocation and a 7-mm diameter section of both ears were obtained and weighed.

The swellings induced by TPA was assessed in terms of the increase in the weight of the right ear punch biopsy over that of the left ear. The inhibitory effects (IE%) of each extract were then calculated as the ratio of the weight increase of the ear sections, according to the following formula:

Inhibitory effect (IE%) = [[L - R]/[L - C]] X 100 = [[L - R]/[L - (L/2.41*)]] X 100

where L -- weight of left ear which is treated with TPA only

R -- weight of right ear which is treated with TPA plus tested extract

C -- calculated weight of untreated ear. (* treating with 0.5 [micro]g TPA resulted in a 2.41 times increase in weight of the ear)

Results and Discussion

The effect of the different extracts, fractions and purified compounds of S. koetjape are shown in Table 1. The initial crude methanolic extract induced a 94% reduction in the TPA-induced oedema and further fractionation revealed that the activity was distributed over the hexane (90%), dichloromethane (100%), ethyl acetate (64%) and methanol (77%) fractions. The notably high topical activity of the hexane and dichloromethane justified their further subjection to a bioassay-guided fractionation to yield the pure compounds.

Koetjapic acid (1), katonic acid (2) and 3-oxo-12-oleanen-29-oic acid (3) were the major triterpenes isolated from the active fractions of S. koetjape. Both 3-oxo-12-oleanen-29-oic acid and katonic acid showed significant reduction in TPA-induced oedema with percentage inhibition of 94% and 81%, respectively. The activity shown by 3-oxo-olean-12 en-29-oic acid was almost equivalent to that shown by the reference standard, indomethacin which gave 97% inhibition. In contrast, koetjapic acid was considerably inactive, inhibiting oedema only by 13%. In koetjapic acid, ring A of the triterpenic structure is opened to form the secostructure, whereas the ring is intact in both of the related but active triterpenes. This seems to suggest that an intact ring A is required for anti-inflammatory activities of these compounds.

Table 1. Anti-inflammatory effect of the crude extract (5 mg/ear),
fractions (5 mg/ear) and purified compounds (0.5 mg/ear) from Sandoricum
koetjape, on mouse ear oedema.

Sample                         Oedema inhibition (%)

Crude methanolic extract        94 [+ or -] 7
Hexane fraction                 90 [+ or -] 11
Dichloromethane fraction       100 [+ or -] 5
Ethyl-acetate fraction          64 [+ or -] 10
Methanol fraction               77 [+ or -] 8
Water fraction                  14 [+ or -] 4
3-oxo-olean-12 en-29-oic acid   94 [+ or -] 8
Katonic acid                    81 [+ or -] 12
Koetjapic acid                  13 [+ or -] 12
[beta]-sitosterol               26 [+ or -] 16
Indomethacin                    97 [+ or -] 3

Each value represented the mean [+ or -] SEM obtained from 6 mice.

Acknowledgements

The work was supported by IRPA Project No.09-04-01-0155. The authors thank Mohd Rizal Abdul Karim for technical assistance.

References

Bird J, Kim HP, Lee HJ (1986) Topical anti-inflammatory activity of esters of steroid 21-oic acid. Steroid 47: 35-40.

Burkill LH (1996) A dictionary of the Economic Products of the Malay Peninsula. Ministry of Agriculture and Cooperatives, Kuala Lumpur. Volume I & II. 2444 pages

Carlson RP, O'Neill-Davis L, Chang J, Lewis AJ (1985) Modulation of mouse ear oedema by cyclooxygenase and lipoxygenase inhibitors and other pharmacologic agents. Agents Actions 17: 197-204

Gschwendt M, KittsteinW, Furstenberger G, Marks E (1984) The mouse ear oedema: a quantitatively evaluable assay for tumor promoting compounds and for inhibition of tumor promotion. Cancer letter 25: 175-185

Hirota M, Mori T, Yoshida M, Iriye R (1990) Suppresion of tumor promoter-induced inflammation of mouse ear by ursolic acid and 4,4-dimethylcholestane derivatives. Agriculture Biological Chemistry 54: 1073-1075

Kaneda N, Pessuto JM, Kinghorn D, Farnsworth NR (1992) Plant anticancer agents. I. Cytotoxic triterpenes from Sandoricum koetjape stems. J Nat Prod 55: 654-659

Powell KL, Mikolajczak KL, Zilkowski BW, Mantus EK, Cherry D, Clardy J (1991) Limonoid antifeedants from seed of Sandoricum koetjape. J Nat Prod 54: 241

Sim KY, Lee HT (1972) Triterpenoid and other constituents from Sandoricum indicum. Phytochemistry 11: 3341

M. A. Rasadah (1), S. Khozirah (2), A. A. Aznie (1), and M. M. Nik (1)

(1) Forest Research Institute Malaysia, Kepong, Kuala Lumpur, Malaysia

(2) Chemistry Department, Faculty of Science and Environment, University Putra Malaysia, Serdang, Selangor, Malaysia

Address

Mat Ali Rasadah, Forest Research Institute Malaysia, Kepong, 52109 Kuala Lumpur, Malaysia

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