PHYTOCHEMICAL AND TRACE ELEMENT ANALYSIS OF VERNONIA AMYGDALINA ( BITTER LEAF ) IN DIFFERENT LOCATIONS IN NIGERIA

Aim: This study was to evaluate the phytochemical and mineral elements of Vernonia amygdalina leaf grown in Anambra, Imo, Delta and Edo States of


INTRODUCTION
Vernonia amygdalina, a member of the Asteaceae family, is a small perennial shrub that grows in tropical Africa particularly in Nigeria.It grows to a height of 2-5m.The leaves are elliptical and up to 20cm long with a rough back.It is commonly called bitter leaf in English because of its bitter taste (Ijike, 2011).African common names include onugbu (Igbo), ewuro (Yoruba), chusar-doki (Hausa), grawa (Amharic), etidot (Ibibio), ityuna (Tiv), oriwo (Edo), mululuza (Luganda), labwori (Acholi), olusia (Luo) and Ndolé (cameroon) (Egedigwe, 2010;Kokware, 2009).Aliyu et al., (2008) documented that the leaves are being used as a valuable source of food and medicine for the prevention of illness and maintenance of human health.Huffmna and Seifu (1989), documented that chimpanzees have been observed to ingest the leaves when suffering from parasitic infection.Challand and Willcoe (2009), also stated that the fresh leaves have been successfully used for the treatment of uncomplicated malaria.Vernonia amygdalina extracts have been known to possess potential pharmacological effects (Song et al., 2005;Sweeney et al., 2005).Its anti cancerous effects as well as influence on body estrogen have also been documented (Blanco et al, 2010;Opata and Izevbigie, 2006;Song et al., 2005;Izevbigie et al., 2004;Jisaca et al., 1993;Kupchan et al., 1969).Also its antioxidant activities as well as its effects on blood glucose and lipids have also been documented (Erasto et al., 2007;Erasto et al., 2006;Nwanjo, 2005).Erasto et al., (2006) described its medicinal use in the treatment of leech and bilharzia as well as pneumonia, cough and as a laxative.The importance of Vernonia amygdalina in medicine remains even the greatest relevance with the current global shift to obtain drugs from plant source, as a result of which attention has been given to the medical values of herbal remedies for safety, efficacy and economy (Glombitza et al., 1993;Mahabir and Gulliford, 1997).The World Health Organization in a number of resolutions emphasizes the need to ensure quality control of plant products by using modern techniques and applying suitable standards (WHO, 1992).Bitter leaf is continually being utilized as therapeutic agent in formulations for treating diseases in the traditional ethno medicinal system in southern and Eastern parts of Nigeria.However, environmental, atmosphere, pollution, soil, harvesting and handling are some of the factors which may play important roles in contamination of Veanonia amygdalina leaves by metals.It is therefore of major interest to evaluate the phytochemicals and some trace elements in the leaves of Veanonia amygdalina because variations or elevated levels of these metals may influence their use in medical practice.This study was designed to evaluate the phytochemical and mineral contents of V. amygdalina leaves grown in four (4) states of Nigeria.

Sample Collection
Fresh plants of bitter leaf (Vernonia amygdalina) were obtained (plucked) from four states of Nigeria namely; Anambra, Delta, Edo and Imo.They were properly labeled according to each of the states and air dried in the laboratory for 21days.The leaves were pulverized to powder; firstly with dried long cup and secondly with the short cup home blender.They were stored in airtight bottles, properly labeled according to state from which samples were obtained before analysis.
Test for Tannins 1g of sample was extracted with 25ml 80:20 acetone: 10% glacial acetic acid for 4hours.It was then filtered and measured at 500nm absorbance.The absorbance of the reagent blank was also measured.A standard graph with10, 20, 30, 40, 50mg/100g of tannic acid was made.The concentration of tannins was read taken into consideration of the dilution factor.
Test for Terpenoids 1g of sample was weighed into 250ml beaker and 10ml petroleum ether was added.It was allowed to extract for15 min and was filtered.The absorbance was then read at 420nm.

Test for Cardiac Glycosides
1g of sample with 40ml of water was extracted and placed in an oven at 100 0 Cfor 15 min.Then, to 1ml of the extract dissolved in 5ml 0f water was added 2ml of glacial acetic acid followed by one drop of iron chloride (Fecl 3 ) and 1ml of H 2 SO 4 .The absorbance was then measured at 410nm.

Tests or Flavonoids
1g of sample was extracted with 10ml of 80% methanol and left to stand for 2 hours.It was filtered through Whatman filter paper into a petri-dish, evaporated to dryness in an oven at40 o C and weighed.
Test for Alkaloids 1g of each sample (W) was extracted with20ml of 10% acetic acid in ethanol, mixed and allowed to stand for 4hours.The extract was filtered through Whatman filter paper.The filtrate was evaporated to about a quarter of its original volume and one drop of concentrated ammonia was added.The extract was filtered through weighed (W 1 ) Whatman filter paper.The filter paper was dried in the oven at 60 o C. The dried filter paper was weighed to a constant weight (W 2 ).
Test for Phenolics 2g of each sample was extracted with 20ml of acetone, 0.5% formic acid for 2minand was filtered.2ml of the extract was mixed with 0.5ml focin-ciocalteau reagent, mixed for 15seconds and allowed to stand at 40 o C for 30m into develop a colour.The absorbance was measured at 765nm and expressed as mg/g Gallic Acid Equivalent (GAE).
Test for Saponnins 1g of sample of each sample was dispersed in 15ml of 20% ethanol.The suspension was put inside the water bath at 55 0 C for 4hours.The mixture was filtered and the residue re-extracted with another 15ml of 20% ethanol twice.The extract was reduced to about 5ml in the oven.The concentrate was transferred into a 250ml separating funnel and 5ml of petroleum ether was added and mixed vigorously.The petroleum ether layer was discarded and 3ml of butanol was added to the aqueous layer.The extract was washed twice with 5ml of 5% sodium chloride.The remaining solution was poured into a weighed petri-dish, evaporated to dryness in the oven and the residue was weighed.

Elemental Analysis
The major trace elements comprising iron, manganese, copper, fluorine, chromium, iodine, selenium, molybdenum, cobalt and zinc were determined according to the method of Shahidi et al., (1999) with slight modification.The grounded samples were sieved with a 2mm rubber sieve and 2g of each of the samples were subjected to dry ash in a well cleaned porcelain crucible at 55 0 C in a muffle furnace.The resultant ash was dissolved in 5ml of HNO 3 /H 2 O 2 (1:1) and heated gently on hot plate until brown fumes disappeared.To the remaining material in the crucible, 5ml of deionized water was added and heated until a colourless solution was obtained.The mineral solution in each crucible was transferred into a 100ml volumetric flask by filtration through a Whatman filter paper and the volume the volume was made to mark with de-ionized water.This solution was used for elemental analysis by atomic absorption spectrophotometer (AAS) and the concentration of each element was calculated on percentage of dry matter.
Therefore their high concentration indicates potential source of useful drugs.The concentration of saponins in the leaves of Vernonia amylgdalina were equally high (1850mg/100g in Anambra, 1895 in Delta, 1795mg/100g in Edo and 1745 in Imo state in the order Delta>Anambra>Edo>Imo State).Saponin has the property of binding with cholesterol, bitterness (which is the characteristics of Bitter leaves) and hemolytic activity in aqueous solution (Sodipo et al, 2000).Alsotannins, terpenoids, cardiac glycosides and phenolics are found in tangible concentration (Tables 2 and 3).Many plants are used in traditional medicine for treatment of diseases, fever and cough (Mutandzi et al, 2012).The very and fairly high concentration of some analyzed phytochemicals in this work (Tables 1  and 3) suggest that Vernonia amylgdalina leaves might be very effective in the treatment of some vital diseases in both man and animals.
Nutritional valuable trace element analysis has shown that the Vernonia amylgdalina leaves are rich sources of chlorine (Cl -), iron (Fe 2+ ), copper (Cu ++ ), Zinc (Zn ++ ) and reasonable concentrations of iodine (I -), manganese (Mn 2+ ), selemiun (Se 2+ ), and cobalt (Co 2+ )while fluorine (F -) and molybdenum (Mo 2+ ) were not detected (Tables 1 & 4).These trace elements are very essential in enzymes metabolism particularly as they serve as co-factors for most enzymatic features in man and animals (WHO, 1996).Therefore, the addition of Vernonia amylgdalina leaves in the diet might prevent the occurrence of certain disorders such as acrodermatits enteropathica, Wilson's disease, Keshan disease, Kaschui-Beck disease, Vitamin-k deficiency, Parkinson's like disease, glucose intolerance and neuropathy (Crook, 2006).The concentration of these elements in Vernonia amylgdalina leaves are quite high and comparable to the concentration reported in certain medicinal plants (Korc, 1988;Vanghan and Judd, 2003) though with variable differences from the individual states as seen in this work.

CONCLUSION
The findings provide quantitative estimation of the phytochemicals as well as elemental analysis of Vernonia amylgdalina (bitter leaves) which are important in understanding the pharmacological and or toxicological actions of bitter leaves used as a medicinal plant.schemp) picrre Exbeille.Global J. Pure Appl.Sci., 83-87.
Song YJ, Lee DY, Kim SN, Lee KR, Lee HW, Han JW, Kang DW, Lee HY, Kim YK (2005)."Apoptotic potential of sequiterpene lactone ergolide through the inhibition of NF-κB signaling pathway.J. Pharmacol57 ( 12 Vaughan JG, Judd PA (2003).The Oxford book of health foods: a comprehensive guide to natural remedies 1 st Edition, Oxford University Press, New York.WHO (1992).World Health Organization: expert committee on specific action for pharmaceutical preparation report, Geneva.WHO Technical Report Series 823. Pg. 44-96. WHO (1996).Trace elements in human nutrition and health.

Table 1 :
The location and mean values of elemental components (mg/100g) in four states

Table 2 :
The Phytochemical concentration in mg/100g in the four states

Table 3 :
The Mean values obtained in phytochemicals analysis in four states

Table 4 :
The Mean values obtained in elemental analysis in four states