COMPARATIVE PHARMACOGNOSTIC AND BIOLOGICAL STUDIES ON THE LEAVES OF VERNONIA AMYGDALINA DEL. AND VERNONIA KOTSCHYANA SCH. BIP. (ASTERACEAE)
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ABSTRACT
Vernonia amygdalina Del. and V. kotschyana Sch. Bip. (Asteraceae) are respectively shrub or tree and annual herb found commonly in tropical and subtropical countries. They are employed in ethno-medicine in the treatment of many ailments namely stomach ache, gingivitis, rheumatism, schistosomiasis, fevers and cough, which are associated with pains and inflammations. Macroscopic, microscopic, chemo-microscopic, quantitative evaluative and thin layer chromatographic studies by using standard pharmacognostic methods of evaluations were carried out on both plant species. Elemental analysis by using Energy Dispersive X-ray Fluorescence (EDXRF) technique; analgesic and anti-inflammatory studies by respectively using acetic acid-induced writhing in mice and carrageenaninduced hind paw oedema in rats’ methods were carried out on the two plants. Macroscopically, leaves of the two plant species were found to have some similar and differing diagnostic features. Leaves of both plant species were alternately arranged with acute apices, symmetrical bases and pubescent surfaces. Leaves of V. amygdalina were relatively smaller (12.0 x 5.7 cm) than those of V. kotschyana (17.0 x 6.7 cm) and both were petiolate. Leaves of the former plant were lanceolate in shape with serrated margin while those of the latter one were elliptical in shape with dented margin. Organoleptically, leaves of V. amygdalina were more greenish in colour and bitter in taste than those of V. kotschyana, although, both plants had distinct odour. Microscopically, leaves of V. amygdalina had epidermal cells with slightly beaded wavy anticlinal walls while those of V. kotschyana had deeply wavy anticlinal walls. Numerous anomocytic stomata (abaxially and adaxially), unicellular and few uniseriate multicellular covering trichomes were identified in both plants. Few uniseriate multicellular glandular trichomes with warty surface were found only in V. kotschyana leaves. Transversely, the leaves of both plant species were dorsiventral and had multilacunar vascular bundles. Calcium oxalate crystals (prism and rosette), starch grains (oval-shaped), xylem vessels (reticulate and spiral) and fibres were found in the fresh and powdered leaves of both plants. - 8 - Quantitative-leaf microscopy revealed that, palisade ratio, vein-islet and veinlet termination number were higher in V. kotschyana, 5.2, 12.7 and 16.5, than those in V. amygdalina, 4.1, 6.2 and 10.7 respectively. Stomatal numbers and indices were higher in the latter plant: 265.2 and 19.1 (upper epidermis) and 312.1 and 38.1 (lower epidermis) than those in the former one: 118.3 and 17.1 and 227.3 and 28.3 respectively. Physical constants (%w/w) showed that V. kotschyana had higher moisture content (5.0%) than V. amygdalina (4.5%). However, the latter plant had higher total ash value (11.0%) than the former one (8.0%), although, both plants had similar acid-insoluble ash value (1.5%). Water-soluble ash, alcohol and water extractive values were higher in V. amygdalina: 4.5, 14.0 and 22.5% than those in V. kotschyana: 4.2, 13.5, and 18.5% respectively. Mineral element concentrations in milligram (mg) per 1.0 g of the leaf powders of the two plant species vary considerably. The V. kotschyana was found to contain more mineral elements than V. amygdalina. Uranium (0.007±LDL mg) was found only in the latter plant. Potassium, calcium, manganese and iron (although with varied concentrations) were of common occurrence in both plants. The yield of extracts per 800 g leaf powders of the two plants show that, the petroleum ether extract was more in V. kotschyana (98.72 g) than in V. amygdalina (92.61 g). However, the ethanol extract was more in the latter plant (221.94 g) than in the former one (195.25 g). Phytochemical investigation on the extracts of the plants showed that, both plants contained similar constituents namely tannins, flavonoids and saponins. Fixed oils and alkaloids were detected in V. kotschyana only. The acute toxicity study on the ethanol leaf extracts of the two plants show that V. amygdalina was relatively more toxic (288.5 mg/kg) than V. kotschyana (471.2 mg/kg) body weight i.p. to the experimental mice. - 9 - Fractionated crude flavonoids and saponins per 150.0 g of the ethanol leaf extracts of the two plants show that, V. amygdalina had higher percentage yield of crude flavonoids (1.43%) than V. kotschyana (1.35%). However, the latter plant specie had higher percentage yield of crude saponins (2.97%) than the former one (2.21%). The thin layer chromatographic analysis carried out on the crude ethanol extracts, flavonoids and saponins of V. amygdalina and V. kotschyana showed the presence of different types of flavonoids and saponins in both plant species. The crude extracts of both plants gave brown coloured spots when exposed to iodine vapour. The crude extracts and flavonoids fraction were found to have range of colour (yellow, purple, brown and green) spots and Rf values (0.55 – 0.95) when they were visualized by exposure to concentrated ammonia vapour. However, the crude extracts and saponins were found to have brown, purple, blue, yellow and green colours and Rf values (58 - 0.95) when sprayed with LiebermannBurchard’s reagent followed by heating at 105oC. The analgesic studies on the ethanol leaf extracts of both plants, show that V. kotschyana (V. K. E. E.) produced significantly (p<0.05) higher inhibition (74.6%) at 50 mg/kg than that (71.9%) of V. amygdalina (V. A. E. E.) at 100 mg/kg against acetic acid-induced writhing in mice when compared with the negative control (normal saline) group. The effects were comparable to that produced by ketoprofen (67.5%) at 10 mg/kg that was used as positive control group. The crude flavonoids of both plants show significant (p<0.05) and dose-dependant analgesic activities. The V. kotschyana crude flavonoids (V. K. C. F.) offered higher inhibition (99.0%) than that (97.0%) of V. amygdalina (V. A. C. F.) both at 100 mg/kg against acetic acid-induced writhing in mice when compared with the negative control group. The activities were comparable to that produced by the control ketoprofen (58.0%) group at 10 mg/kg. The significant (p<0.05) and dose-dependant analgesic activities produced by the crude saponins from the two plants show that, the crude saponins of V. kotschyana (V. K. C. S.) had higher inhibitory effect (97.0%) than that (78.0%) of V. amygdalina (V. A. C. S.) both - 10 - at 100 mg/kg against acetic acid-induced writhing in mice when compared with the negative control group. The control ketoprofen group produced (58.0%) inhibitory effect at 10 mg/kg. The anti-inflammatory studies on the ethanol extracts of the two plants show that V. kotschyana (V. K. E. E.) and V. amygdalina (V. A. E. E.) offered significantly (p<0.05) similar inhibition (45.5%) at 50 and 100 mg/kg, against carrageenan-induced oedema in rats when compared with the negative control (normal saline) group. The activity of both plant extracts was lower than that produced by ketoprofen (63.6%) at 10 mg/kg that was used as the positive control. The crude flavonoids of V. amygdalina (V.A.C.F) offered significantly (p<0.05) higher dose-dependant inhibitory effect (45.5%) at 100 mg/kg than that (36.7%) of V. kotschyana (V. K. C. F.) at 50 mg/kg against carrageenan-induced oedema in rats when compared with the negative control group. This effect was lower than that produced by the control ketoprofen group (50.0%) at 10 mg/kg. The crude saponins of V. amygdalina (V. A. C. S.) produced significantly (p<0.05) higher dose-dependant inhibition (50.0%) than that (40.9%) of V. kotschyana (V. K. C. S.) both at 100 mg/kg against carrageenan-induced oedema in rats when compared with the negative control group. The control ketoprofen group produced similar effect (50.0%) at 10 mg/kg. The various diagnostic features identified from the results of pharmacognostic studies on V. amygdalina and V. kotschyana leaves, would significantly aid in identifying and differentiating them. The significant analgesic and anti-inflammatory activities of the crude ethanol leaf extracts, flavonoids and saponins of both plant species had greatly supported the traditional claims of using the plant species in the management of tooth ache, stomach ache, rheumatism and gingivitis, which are associated with pains and inflammations. The two plant species could therefore, serve as potential sources of both analgesic and antiinflammatory drugs from medicinal plants.
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