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in: III WOCMAP Congress on Medicinal and Aromatic Plants - Volume 4: Targeted Screening of Medicinal and Aromatic Plants, Economics and Law

The chorio-allantoic membrane (CAM) and hen’s egg test (HET-CAM) assays are based on neo-vascularisation (angiogenesis) in fertilised hens’s egg embryo. Therefore inhibition of angiogenesis is a prime target for solutions to afflictions such as growth of solid tumours, arthrithis, chronic inflammations. Natural products still represent an untapped source of interesting leads for drug development against these diseases. Serial dilutions of the methanolic extract of Alchornea cordifolia seeds were assayed on CAM, HET-CAM and 2,2-diphenyl-1picrylhydrazyl hydrate (DPPH) assays. The results revealed an inhibition of angiogenesis at 0.9 score and 86% inhibition of inflammation at 250 μg/pellet on the CAM and HET-CAM respectively. It was also very effective in the DPPH assay for non specific hydrogen atom or electron donating activity (IC50: 0.68 μg/ml). Subsequent fractionations, revealed the ethyl acetate fraction (Ea) as the most active against others in a dose response trend. Interestingly, no side effects such as embryotoxic effects and others were recorded. The findings support the folkloric uses of the plant against wounds, piles and cancer and therefore have implications on the quality of herbal drugs dispensed by the traditional medical practitioners (TMP) in Africa. INTRODUCTION In animal experimental models, the observation that tumour promoters recruit inflammatory cells to the site of application and in return release reactive oxidative species (ROS) is a clear indication of a close relationship between inflammation and cancer. These ROS, when released beyond the antioxidant capacity of a biological system give rise to oxidative stress, which is fundamental in the pathogenesis of a variety of human afflictions such as atherosclerosis, hypertension, inflammation, cancer and AIDS (Gutteridge and Halliwell, 1994). The neolignans magnosalin, magnoshinin and isoliquiritin isolated from Magnolia salicifolia and Licorice root respectively have been shown as potential inhibitors of these processes (Paper, 1998). However, more potent inhibitors are still needed for solutions to chronic afflictions. The different organs of Alchornea cordifolia (Euphorbiaceae) are traditionally used to cure wounds, yaws, ulcers, bronchitis, inflammations and skin infections (Iwu, 1993). The anti-microbial, the smooth muscle relaxant activities as well as the chemistry of extracts from leaves have been studied by several authors (Lamikanra et al., 1990; Ogungbamila and Samuelsson, 1990; Iruka et al., 1999). Routine anti-oxidant screening revealed the seed to contain the most active principles. However this organ is yet to be investigated both for anti-inflammatory and or anti-angiogenesis activity which is a logical justification of this study. MATERIALS AND METHODS Collection, Extraction of Plant Materials The different organs of Alchornea cordifolia (Schum and Thonn) Muell. Arg. were collected (June, 2001) in Uyo local government of Akwa-Ibom State, Nigeria and were identified by the taxonomist of the Department of Pharmacognosy of the University Proc. WOCMAP III. Vol. 4: Targeted Screening of MAPs, Economics & Law Eds. C. Franz, A. Mathe, L.E. Craker and Z.E. Gardner Acta Hort. 678, ISHS 2005 92 of Uyo, where a voucher specimen is deposited. 300 g of each fresh plant organs: [leaves (Lvs), stembark (Sb), rootbark (Rb) and seeds] were extracted cold in methanol (100%) by percolation for 48 h. The brown organic phase was filtered through Whatman paper No 1, concentrated in vacuo and freeze dried. These extracts were analysed for the presence or otherwise of bioactive ingredients using standard methods (Harborne, 1984) and assayed. The methanol extract of the seed was selected for successive fractionation in n-hexane (He), chloroform (Ch), ethylacetate (Ea), n-butanol (Bu) and aqueous (Aq) to yield different fractions for further assays. Antioxidant Activity: Rapid-TLC Screening for Anti-oxidant Activity The freeze-dried powder from different organs of the plant were dissolved in methanol 100% and spotted on silica gel sheets, developed in methanol:ethylacetate (2:1; v/v). The plates were air-dried and sprayed with 0.2% solution of the stable DPPH (2,2diphenyl-1-picrylhydrazyl hydrate) radical (Kirby and Schmidt, 1997) and visualised for the presence of whitish spots, indicating anti-oxidant activity. DPPH Assay The DPPH assay was carried out as described by Kirby and Schmidt, 1997. 50 μg of various dilutions from the extract of different organs were mixed with 5 ml of a 0.004% methanol solution of DPPH, after an incubation period of 30 min, the absorbancy of the sample was read at 512 nm using a spectrophotometer. Ascorbic acid (Vitamin C) was used as a positive control. The freeze dried seed extract was later selected and the anti-oxidant activity of its fractions was evaluated as earlier described. HET-CAM/CAM Assays The modified method of Marchesan et al. (1998) was used. Fertilised hens’ eggs were incubated for 75 h at 37°C and a relative humidity of 80%. The eggs were placed in horizontal position and rotated several times. They were opened on the snub side and prior to this, 10 ml albumen were sucked off through a hole pierced down by the side and sealed. Then a round piece of shell (3-4 cm diameter) was removed from the top of the blunt end and the eggs were sealed with laboratory film and incubated for further 75 h. The pellets consisting of 10 μl gelled 2.5% agarose solution were used as vehicle and sodium dodecyl sulphate (SDS) as inflammatory inducer. They were dissolved or suspended in 60% “warm” liquid agarose solution before gelling pellets with or without test drug in the presence or absence of SDS were used. 10 eggs were used per drug to be tested. The results were evaluated under the stereomicroscope. An anti-inflammatory activity exists if the irritation of the membrane induced by SDS decreases (i.e. the starlike picture around the granuloma) and the blood vessel net appears normal. The number of eggs with a positive effect is given in per cent and indicates the measure of the antiinflammatory activity of the drug tested (Marchesan et al., 1998). The anti-angiogenic activity was evaluated by using a score system (0-2). Suramin (50 μg/pellet) was tested as positive control. As blank, CAMs was treated only with agarose solution (score 0). Score < 0.5, no anti-angiogenic effect; score ≥ 0.5, weak to strong anti-angiogenic effect (Marchesan et al., 1998). Statistical Analysis The data are expressed (Table 2 and 3) as mean ± SD and the statistical significance between groups was analysed by means of an analysis of variance (ANOVA), followed by student – New man – Keul’s test. P values less than 0.01 was considered as indicative of significance. RESULTS AND DISCUSSION Processing of Plant Materials and Phytochemical Screening The plant materials used in this research were processed accordingly, all the

Tridax procumbens , is a common annual weed in the West African sub-region and other tropical zones of the world and is known as “coat buttons”. Traditional medical practitioners (TMPs) and the native peoples of these areas use the leaves of the plant as a remedy against several ailments ranging from conjunctivitis, diarrhoea, and dysentery to wound healing and related inflammatory conditions. Preliminary radical scavenging screenings proved positive and prompted further investigations into its anti-oxidant and anti-inflammatory activities. This, then necessitated the use of DPPH (2,2-diphenyl-1-picrylhydrazyl hydrate) and HET-CAM (Hen\'s egg chorioallanthoic membrane) assays to investigate the radical scavenging and anti-inflammatory potentials of the plant. The results indicated that the chloroform (CHL) and the aqueous (AQU) fractions had the highest and the lowest 50 % inhibitory concentration of free radical oxidative species (FROS) at 1.92 and 4.22μg/mL in the DPPH assay. The HET-CAM (Hen\'s eggs test on chorioallanthoic membrane) assay, a novel assay for anti-inflammatory evaluation revealed the ethylacetate (ETA) fraction as the most active: 96%: “very strong effect” in terms of restoration of an induced inflammatory condition, while the ethanol extract (ETH) showed 92%: “strong effect”. The aqueous (AQU), the Hexane (HEX) and the chloroform (CHL) fractions displayed a below 25 % inhibition. Serial dilutions of the most active fraction: the ethylacetate fraction (ETA) revealed a dose dependent trend. Chemical analysis of the whole plant showed the presence of flavonoids and alkaloids in the chloroform (CHL) and ethylacetate (ETA) fractions. Interestingly, these classes of natural products have been implicated in radical scavenging and inflammation activities in other studies and it is therefore probable that, they may be playing similar functions in Tridax procumbens and consequently would have justified its uses in traditional medical practices. Key words: Anti-oxidant, anti-inflammatory, HET-CAM, DPPH assay, Asteraceae, Tridax procumbens. Nigerian Journal of Physiological Sciences Vol.18(1-2) 2003: 39-43