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Agricultural Science Research Journal Vol. 5(11): 153 - 165, November 2015 Available online at http://resjournals.com/journals/agricultural-science-research-journal.html ISSN: 2026 –6073 ©2015 International Research Journals

Full length Research Paper

Effect of moist heating and drying processing on the proximate and phytochemical composition of Vernonia

amygdalina and Gongronema latifolium leaves

Obeta, Nwamaka Alexandra

Department of Food Science and Technology, Micheal Okpara University of Agriculture, Umudike, P. M B 7267 Umuahia, Abia State, Nigeria

Email: amybeta2@gmail.com, Tel.: +234 (0)7060787491

Abstract Vernonia amygdalina and Gogronema latifolium leaves are popularly used in southern Nigeria for soup, sauce and spices. Their proximate and phytochemical compositions are known, but how processing affect their composition are not well known. These vegetables were subjected to boiling (100 °C) at time intervals of 0, 5, 10, 15, 20, 25 and 30 min. Another portion of the leaves were dried under the sun, in oven and using solar drier all at the temperature between 45°C and 50°C. The result showed that unprocessed leaves of both vegetables had high protein content V. amygdalina (25.82%) G. latifolium (27.24%) which was not affected by drying but significantly reduced (p<0.05) by 14% and 12 % for V.amygdalinaand G. latifolium respectively after boiling for 5min. Boiling reduced (p<0.05) fibre content (from 12.00% to 10.09 % and from 12.80 % to 10.35 % for V.amygdalina and G. latifolium respectively) and ash content (from 8.29 % to 7.77 % and from 8.13 % to 7.80 % for V. amygdalina and G. latifolium respectively). All the phytochemicals examined were reduced (p<0.05) by boiling but drying reduced alkaloid and flavonoid content but increased tannin, saponin, phytate and steroid. G. latifolium had higher anthocyanin and flavonoid content than V. amygdalina but V. amygdalina had higher saponin, phytate and steroid. The result further showed that 5min boiling did not reduce the food nutrients much but drastically reduced the anti -nutrients. Therefore boiling for 5min and oven drying are recommended formoist and drying treatments of the vegetables. Key words: - Vernonia amygdalina, Gogronema latifolium, proximate and phytochemicals. Introduction Leafy vegetables are included in the meal mainly for nutritional value. In Nigeria they are consumed as cooked complements, sauces, spices, and flavouring agents to major staples like cassava, cocoyam, corn, rice etc. Indeed, most of such meals are considered incomplete without a generous serving of cooked green leafy vegetables (Oguntona, 1998). The varieties of green leafy vegetables so utilized are as diverse as the different meals consumed and the localities. It has been estimated that over 60 species of green leafy vegetable families of plants are so used in Nigeria alone and a lot

of unknown species are yet to be discovered (Okoli et al., 1988).

In addition to the nutritive role played by these vegetables in human and animal nutrition, is the rediscovery of the connection between plants and health which is responsible for the recent emphasis on chemical and phytochemical compositions of plant materials (Svoboda, 2002; Akinjogunla et al., 2011). Phytochemicals are natural bioactive compounds found in plant foods which produce definite physiological action on the human body (Akpaso et al., 2011; Igwe et al., 2012). The knowledge of the proximate, micronutrients (vitamins and fibre)and phytochemicals

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composition of the vegetables is fundamental to the understanding of the phytochemicals modes and mechanism of action in the body against diseases. They have complementary and overlapping mechanism of action in the body including antioxidant effect, modulation of enzyme functions, stimulation of the immune system, modulation of hormone metabolism, anti-bacterial and antiviral effect. Some of these phytochemicals are considered as anti-nutrients (capable of precipitating deleterious effects in man and animal and reducing nutrient bioavailability) at certain levels but some exhibit health promoting activities in managing chronic diseases (Chinma & Igyor, 2007). Their protective effect is attributed probably to their high content of polyphenols and antioxidants (Karou et al., 2005). According to Aletor and Adeogun (1995), phytochemicals serve dual purposes, reducing some essential nutrients and protecting the body against a number of biochemical and metabolic disorders. They act as antioxidants with mechanism involving both free radical scavenging and metal chelating activities. Vegetables have been recognised to be more effective antioxidants than vitamins E and C. This led to the conclusion that vegetables provide the best and useful polyphenols against the development of chronic disease (Ames, 1998; Amicet al., 2003). The phytochemicals are easily absorbed to provide the maximum health benefits unlike supplements or pills which contain large doses of only one or two phytochemicals and some nutraceuticals with poor absorption rates, resulting in nutrients being disposed on the body without providing any nutritional or medicinal benefit.

Some of the most important bioactive phytochemical constituents are alkaloids, flavonoids, tannins, saponins, phenolic compounds, essential oil and many more (Odom et al 2013).

Alkaloids are nitrogenous compounds of plant origin which are physiologically active. The pharmacological action of alkaloids varies widely, some act as analgesics and narcotics, while others act as central stimulants. Some are mydriatics whereas others are myotics. Some will cause a rise in blood pressure but others will produce a fall in excessive hypertension (Swain, 1963). Generally, alkaloids work on the nervous systems of the human body as analgesic because they are capable of relieving pain. They have bactericidal and antispasmodic effects and can be used in the manufacture of sedatives (Okeke and Elekwa, 2006).

Flavonoids are found in fruits and vegetables; they act as antioxidants in vitro. Free radical damage is believed to contribute to a variety of health problems including cancer, heart disease and aging (Ahn et al.,1996). Even though flavonoids are good antioxidant in vitro, they are poorly absorbed being quickly metabolized and excreted (Lotito and Frei, 2006). Flavonoids also have the ability to lower the cholesterol in the body (Okoye and Ebeledika, 2013; Yusuf and Obiegbuna, 2015).Tannins are astringent, bitter plant polyphenol that either bind and precipitate or shrink proteins.They combine with the precipitated proteins rendering them resistant to enzymes. This is known as ―astringency‖ and forms the basis for the therapeutic application of tannins. The astringency from the tannins is what causes the dry and puckery feeling that is ―furriness‖ in the mouth following

the consumption of tannin containing food.Many tannin bearing drugs are employed in medicine as astringents, both in the gastrointestinal track and on skin abrasions (Kraus et al., 2003). Tannin is antiseptic in action; it prevents damage by insects and fungi in growing plants. It is finally deposited as end product of metabolism in certain dead tissues of the mature plants. In the treatment of burns, the proteins of the exposed tissues are precipitated forming a mildly antiseptic, protective coat under which the regeneration of new tissue may take place. In the treatment of alkaloidal poisoning, tannin solutions are extremely valuable for inactivating alkaloid by the formation of insoluble tannate. The phenolic groups of tannins are responsible for their astringent and antiseptic actions as well as their colouration with iron salts.

They prevent oxidation by breaking down fat molecule in the blood system. Tannins in the form of proanthocyanidins, suppress peptide product that is responsible for hardening arteries in the body. They act as antiviral, antibacterial, anti-parasite, inhibitor of HIV replication as reported by Akinmoladun et al., (2007). They are used for checking chronic diseases, decreasing blood lipids, lowering blood glucose response, antidote against acute lead poisoning etc (Maikai et al.,2008).Some damages attributed to tannin include intestinal track toxicity and interference with the absorption of iron Butler (1989); Urquiaga and Leighton (2000).

Saponins have been shown to lower blood cholesterol levels by binding to bile salt and cholesterol in the intestinal tract (Francis et al., 2002). Bile salts form small micelles with cholesterol facilitating its absorption while Saponins cause a reduction of blood cholesterol by preventing its re-absorption. Saponins have been associated with inhibition of cancer cells‘ growth in the body without killing the normal cells. Saponins seem to react with the cholesterol rich membranes of cancer cells thereby limiting their growth and viability (Shi, et al., 2004). It has been found that saponins may help to prevent colon cancer and as shown in the article ―saponins as anti-carcinogens‖. Saponins are immune boosters. Plant saponins are used to fight infections like parasites. When ingested by humans, saponins also seem to help the immune system and to protect against viruses and bacteria. It stops toxic materials from forming, a natural antibiotic used in fighting fungal infections, microbes and viruses (Araghi-Niknam et al.,1996; Shi, et al., 2004). Studies with ovariectomized rats have shown that some saponins such as the steroidal saponins from Anemarrhena asphodeloides chinese herb have a protective role on bone loss (Araghi-Niknam et al., 1996; Anonymous, 2008a).

The non-sugar parts of saponins also have a direct antioxidant activity, which may result in reduced risk of cancer and heart diseases. Saponins possess specific physical, chemical and biological activities that make them useful as drugs. Some of these biological properties include antimicrobial, anti-inflammatory, anti-deodant and haemolytic effects (Okafor, 1989). It has been established that saponins strengthen contraction of the heart muscle and makes they work more efficiently in human heart disease control, though it can be toxic in high doses (Francis et al., 2002).Phytate has anti-

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nutritional factor which is known to interfere with metabolic processes such that growth and bioavailability of nutrients are negatively influenced. Phytate has the ability to bind proteins and form chelates with di-and tri-valent metallic ions such as Cd, Mg, Zn, Ca, and Fe to form poorly soluble compounds that are not readily absorbed from the gastrointestinal tract thus decreasing their bioavailability (Eka, 1977). Phytate inhibits the functions of some digestive enzymes but phytate-degrading enzymes can improve the nutritional value of plant-based foods. This is done through enhancing protein digestibility and mineral availability through phytate hydrolysis during digestion in the stomach or during food and feed processing. Certain myo-inositol phosphates have novel metabolic effects, such as amelioration of heart disease by controlling hypercholesterolaemia, atherosclerosis and prevention of renal stone formation (Konietzny and Greiner, 2002; Agbaire and Emoyan, 2012). All steroids have the same fundamental structure of four (tetracyclic) carbon rings called the steroid backbone or steroid nucleus form different steroidal compounds such as sex hormones (progesterone and testosterone), the anti-inflammatory steroid cortisone, and the cardiac steroidal glycosides (Francis, 2002).

Among the vegetables popularly consumed in southern Nigeria are Vernonia amygdalina and Gongronema latifolium. Vernonia amygdalina (VA) (Onugbu---Igbo; Ewuroejijij---Yoruba; Shiwakaor Chusa-dikiHausa) is a perennial plant that belongs to the Compositae family (Akpaso et al., 2011) and is planted in homes in villages as a fence post and pot herb. It grows in many zones in Africa and is drought tolerant (Bonsi et al., 1995; Akpaso et al., 2011). It contains bitter pigment necessitating much squeezing, washing, several soakings in water or boiling before cooking and consumption (Burkill, 1985; Eyong et al., 2011). It is cherished in Nigeria for its distinctive flavour on dishes in which it is a component. Gongronema latifolium (Utazi –Igbo Arokeke--Yoruba) is a tropical rain forest plant from Ascelepindaceae family.Gongronema latifolium (GL) is also known to have antioxidant, hypoglycaemic, hypolipidemic and anti-inflammatory properties (Morebise et al., 2002).

Information on their composition, effect of processing on the composition etc are documented. Seasonality of production and need for preservation to extend their shelf life, ensure safety and wholesomeness leads to postharvest treatments (Oboh 2003; Yusuf and Obiegbuna 2015). These postharvest treatments may cause changes or loss in the phytonutrients composition (Morris et al., 2004), hence the need for this work.identify most appropriate processing method that will achieve the desired effect with least adverse effect on the phytonutrients. The effect of these processing treatments (boiling and drying) on the proximate and selected phytochemicals content of Vernonia amygdalina (VA) and Gongronema latifolium (GL) were investigated to identify most appropriate processing method that will achieve the desired effect with least adverse effect. Materials and Methods

Sample collection Fresh leaves of Vernonia amygdalina and Gongronema latifolium were bought from Nsukka (in Enugu State Nigeria) main market and were authenticated at the Department of Botany, University of Nigeria, Nsukka Nigeria. All chemicals which were purchased were of analytical grade. Sample preparation Vernonia amygdalina and Gongronema latifolium twigs were destalked, washed and drained. The washed leaves were divided into eight portions and subjected to different processing treatments. Treatment 1: Boiled leaf samples Fresh VA and GL each was divided into 14 portions/lots (100g per portion). The samples/lots were boiled each with 200ml of de-ionized water at a time interval of 0,5,10,15,20,25 and 30mins after which each lot was drained, homogenised and designated as boiled leaf samples. Treatment 2: Dried samples Three portions of 200g each of VA and GL was sun dried, solar dried (using drier fabricated by National Energy Centre, UNN) and oven dried (Fulton, England, NYC-101-Oven) between 45

oC and 50

oC ( 4hours for

3days in case of sun and solar drying but for oven drying only 4hours). The dried leaves were ground into powder using laboratory mortar and designated as sun dried, solar dried and oven dried samples respectively.

All the samples were analysed for proximate and phytochemicals contents. In all, the treatments raw unprocessed sample (homogenised) was used as control. Analyses Proximate analysis Moisture, protein, ash, fat, and fibre were carried out using the methods described by AOAC (1990). Carbohydrate was determined by difference as others parameters determined were reliable (Ihekoronye and Ngoddy, 1985) Phytochemical Studies Qualitative tests were determination of phytochemicals Alkaloid A 20ml volume of 3% sulphuric acid in 50% ethanol was added to 2g each of the macerated raw vegetable and heated in a boiling water bath for 10mins, cooled and filtered. The filtrate (2ml) was tested with a few drops of Dragendorff‗s reagent (bismuth potassium iodide).The remaining filtrate was placed in separating funnel and

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added dilute ammonia solution. The aqueous alkaline solution was separated and extracted with two 5ml portions of dilute sulphuric acid. The extract was tested with a few drops of Dragendorff‘s reagents and it gave brick red precipitate indicating presence of alkaloids (Harborne, 1973; Sofowora, 1993). Flavonoid Ethyl acetate (10ml) was added to 0.2g of each macerated VA and GL samples. The ethyl acetate sample mixture was heated on a waterbath for 2mins and filtered. Four millilitres of filtrate was shaken with 1ml of dilute ammonia solution and then allowed to stand for 20mins during which ammonical layer separated. The yellow colour observed in the separated ammonical layer indicated the presence of flavoniods (Trease and Evans, 1983). Tannins One gram of each macerated raw VA and GL leaves was boiled with 20ml of water for 10mins and filtered. Three millilitre of each filtrate was added few drops of ferric chloride (FeCl3) and formation of a greenish black precipitate indicated the presence of tannins. To another 2ml of filtrate from each sample was added 3drops of lead acetate solution and development of a reddish colour indicated the presence of tannins (Trease and Evans, 1983; Sofowora, 1993). Anthocyanins (glycosides) Dilute sulphuric acid (3ml) was added to 0.1g of each macerated leaves of VA and GL in a test tube. Each sample was boiled for 15min on a water bath, cooled and neutralized with 2ml of 20% potassium hydroxide solution. A 10ml volume of a mixture of equal parts of Fehling‘s solution 1 and 11was added and the mixture was boiled for 5min. Development of a more dense brick red precipitate indicated the presence of glycoside (Sofowora, 1993). Saponin Distilled water (20ml) was added to 0.25g of each macerated leaves of VA and GL and each sample was boiled on a hot water bath for 2min. The mixture was filtered while hot and then allowed to cool. Following cooling, 5ml of filtrate was diluted with 15ml of distilled water with vigorously shaking. Formation of a stable froth (foam) upon standing was taken to indicate the presence of saponins. Formation of an emulsion when the frothing solution was added 2 drops of olive oil with vigorous shaking was used as a confirmation test for the presence of saponins (Trease and Evans,1983; Sofowora, 1993). Phytate The method described by Harland and Oberleas (2001) was used. A filter paper was dipped into water extract of each of the leaves (VA & GA) and another filter paper (control) was dipped into distilled water. Both filter

papers were sprayed with alcoholic solution of sulfosalicylic acid. On the paper without the extract was developed colour complex of ferric sulfosalicylate while the paper with the extract did not form any coloured complex because it formed complex with phosphate or phosphate ester. Steroids Ethanol (9ml) was added to 1g each of macerated leaves of VA and GL. Each mixture was refluxed for a few minutes and filtered. The filtrate was concentrated to 2.5 ml in a boiling water bath. A 5ml volume of hot distilled water was added to the concentrated solution and the solution was allowed to stand for 1hour after which the waxy matter was filtered off. The filtrate was extracted with 2.5ml of chloroform using separating funnel. To each 0.5ml of the chloroform extract in a test tube was carefully added 1ml of concentrated sulphuric acid to form a lower layer. Development of a reddish brown interface indicated the presence of steroids (Trease and Evans 1983; Sofowora 1993). Quantitative Determination of phytochemicals Alkaloid This was done by the alkaline precipitation gravimetric method by Harborne, (1973) described by Onyeka and Nwambekwe (2007). A known weight (1mg) of the sample was dispersed in 10ml of 10% acetic acid solution in ethanol to form a ratio of 1:10 (10%). The mixture was allowed to stand for 4h at 28°C. It was later filtered with a Whatman No 42 grade filter paper. The filtrate was concentrated to one quarter of its original volume by evaporation and treated with drop wise addition of concentrated aqueous NH4OH until the alkaloid was precipitated. The alkaloid precipitated was filtered, received in a weighed filter paper, washed with 1% ammonia solution dried in the oven at 80ºC for 30min. Alkaloid content was calculated as the weight difference between weight of the precipitate and the weight of sample analyzed. Flavonoid This was determined according to the method of Harborne (1973) as described by Onyeka and Nwambekwe (2007). A 5g weight of each sample was boiled in 50ml of 2M HCl solution for 30min under reflux. It was allowed to cool and then filtered using Whatman No 42 filter paper. A known volume (5ml) of the extract was treated drop wise with equal volume of ethyl acetate. The flavonoid precipitated was recovered by filtration using weighed filter paper. The weight difference gave the weight of flavonoid in the sample. Tannins Tannin content was determined by the Folin-Denis colorimetric method described by Kirk and Sawyer (1991). A 5g weight of each sample was dispersed in 50ml of distilled water, shaken and allowed to stand for 30min at 28°C before it was filtered through whatman

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No.42 grade filter paper. A 2ml volume of each sample extract was dispensed into a 50ml volumetric flask and mixed with 2ml standard reagent and 2.5ml of saturated Na2CO3 solution. After mixing, the content of each flask was made up to 50ml with distilled water and allowed to incubate at 28°C for 90min. The respective absorbance was measured in a spectrophotometer at 260nm. The reagent blank was used to calibrate the instrument while the absorbance values of the samples were plotted to determine tannin content against the weight of the sample. Anthocyanins (glycosides) This was done gravimetrically by the method of Harborne (1973) as described by Onyeka and Nwambekwe (2007). Five gram of each test sample was hydrolyzed by boiling in 100ml of 2M HCl solution for 30min. The hydrolysate was filtered using whatman No 42 filter paper. The filtrate was transferred into a separation funnel, mixed with equal volume of ethyl acetate and allowed to separate into two layers. The ethyl acetate layer (extract) was discarded while the aqueous layer was recovered, transferred into a crucible and evaporated to dryness on a steam bath. The dried extract was rehydrated with concentrated amyl alcohol in an evaporating dish of known weight and evaporated to dryness. It was further dried in the oven at 30ºC for 30min and cooled in a desiccator. The weight of anthocynanin was determined and expressed as percentage of the original sample. Saponin The method of Trease and Evans,(1983) with modifications was used. Distilled water (20ml) was added to 0.25g of each macerated leaves of VA and GL and each sample was boiled on a hot water bath for 2min. The mixture was filtered while hot and then allowed to cool. Following cooling, 5ml of filtrate was diluted with 15ml distilled water with vigorous shaking. Formation of a stable froth (foam) upon standing was taken to indicate the presence of saponins. Formation of an emulsion when the frothing solution was added 2 drops of olive oil with vigorous shaking was used as a confirmation test for the presence of saponins. The emulsion formed from froth and oil in the qualitative test was centrifuged (Falcon 6/300R, England, CEK- 243-010J) at 10,000 x g for 15min and the quantity of residue was used to estimate the saponin content. The process was repeated with standard saponin and compared with the samples. Phytate The method of Thompson and Erdman (1982) was used. A 2g weight of each macerated vegetable in a flask was added 100ml of 1.2% HCl and 20ml of 10% Na2SO4. The flask was shaken for 2hours on a mechanical shaker (Gallenkamp England, BKS-350-010Q). The extract was filtered using vacuum filter through Whatman filter paper No1. The filtrate (10ml) was put into a centrifuge tube and 10ml deionised water, 12ml of ferric chloride (Fe2Cl3) solution (2.0g FeCl3.6H2O

+16.3ml Conc. HCl/L) added and mixed, heated for 75min in a boiling waterbath and cooled. Thereafter the tube was centrifuged at 10,000×g for 15min.The supernatant was discarded and the pellet was thoroughly washed 3times with a solution of 0.6% HCl and 2.5% Na2S04. After each washing the contents were recovered by centrifugation at (10,000× g for 10min). The pellets were washed again with 3ml of conc. HNO3 into a 400ml beaker. Four drops of H2SO4 were added to the pellet in the beaker and heated for approximately 30min on a hot plate. Following heating 5ml of 30% hydrogen peroxide (H2O2) was added to the mixture and heating continued until bobbling ceased. The residue was dissolved in 15ml of 3N HCl and heated at 80

oC for

15min. The solution was made up to 100.0ml volume and analysed for iron using Atomic Absorption Spectro-photometer (Buck, England Model 205). Steroids This was determined following the method of Okeke and Elekwa (2003). A 2g weight of each sample was dispersed in 100ml of freshly prepared distilled water and homogenized with a laboratory blender (Philip England/China HR 1727). The homogenate was filtered and the filtrate was eluted with normal ammonium hydroxide solution (pH 9).The elute (2ml) of each sample was put in a test tube, mixed with 2ml of chloroform, 3ml of ice-cold acetic anhydride and then 2 drops of concentrated H2SO4 was cautiously added to the cold mixture. Standard sterol solution was prepared and treated as described above. The absorbance of standard and samples were measured in a spectrophotometer (Jen way USA JY/6705AP)at 420nm and the steroid content of the sample determined from the standard curve. Results and Discussion Effect of boiling and drying on the proximate composition of boiled V. amygdalina (VA) and G. latifolium (GL) vegetables. Figs 1and 2 show the effect of boiling time and Figs 3 and 4 the effect of drying on the proximate composition of V. amygdalina (VA) and G. latifolium (GL) leaves, respectively. The mean moisture contents of the unprocessed VA and GL were 81.93% and 78.27% respectively, which were similar to earlier reports by Oboh and Masogje (2009) for VA and Chinma and Igyor (2007) for leafy vegetables in South east of Nigeria, but higher than the values reported by Okafor et al. (1996) for VA (76.67%). This disparity could be attributed to the time and season of harvest. Vegetables harvested during rainy season usually contain high moisture due to high relative humidity and low temperature. Boiling did not significantly (p>0.05) increase the moisture content of VA and GL even though an apparent softening effect was observed. This was due probably to the collapsing of the tissue integrity and consequent expulsion of trapped air which ordinarily would have allowed more space for water absorption.

It is observed in Figs 4 and 5 that different drying methods reduced (p<0.05) the moisture content of fresh unprocessed VA and GL by 93 % - 94 % and 92 % - 93

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% respectively. The percentage moisture loss was in agreement with the report of Oguntona (1998) who noted that 72 % to 93 % moisture loss occurred in vegetable during drying. The residual moisture content of dried GL was higher (p<0.05) than that of VA for all the drying methods employed. However, sun dried samples had the least mean moisture value of 4.18 % and 5.25 % for VA and GL, respectively. This was

unexpected; however the prolonged drying time under direct sun relative to other drying methods may have caused the low mean moisture value. Lower values were observed in this study than those reported by Aliero and Abdullahi (2009) for oven dried (20.23 %), solar dried (23.70 %) sun dried (14.61 %) VA. Also Okafor et al. (1996) reported a similar value for sundried GL (5.89 %).

Fig 1: Effect of boiling time on proximate composition of Vernonia amygdalina (VA).

Fig 2: Effect of boiling time on proximate composition of Gongronema latifolium (GL)

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Fig 3: Effect of different drying methods on the proximate composition of Vernonia amygdalina.

Fig 4: Effect of different drying methods on proximate composition ofGongronema latifolium

Several factors like age, freshness and weather may have contributed to the disparity in the observed moisture contents reported in this work relative to literature values. The low moisture value for sundried samples may be associated to fermentation of the leaves during drying. Variation in moisture contents with the drying methods was not significant (p>0.05). Among all the drying methods used in this work oven drying showed a faster drying rate and shorter time (4 hours) than the sun and solar drying ( 4 hours for 3 days). Similar observation was made by Bolaji et al., (2008) who reported on the advantage of oven drying method over other methods. From this result, all drying methods reduced moisture content and increased dry matter.

Both VA and GL showed high levels of crude protein content with raw GL having the highest value (27.24% on moisture free weight basis), followed by raw VA (25.82%). Eleyinmi et al., (2008) recorded similar results for protein of GL (27.20%) and VA (21.69 %). However Atangwho et al., (2009) reported a slightly lower protein value (25.55%) for GL and VA (23.25%). Modupe and Babayemi, (2009) had crude protein value as low as 21.8% for VA while Alabi et al., (2005) reported a lower

value of 20.2% for VA. Boiling reduced (p<0.05) the levels of crude protein in both vegetables. Five minutes boiling reduced crude protein content of VA by 14% and that of GL by 12%. This suggested that a substantial percentage of the vegetable protein leached into the boiling water. Increasing the boiling time to 25min in GL showed higher (p<0.05) reduction in protein on GL than in VA suggesting that GL has more soluble proteins than VA. Drying and drying methods did not affect the protein content of GL, while for VA, the protein content of the unprocessed sample differed (p<0.05) from that of the oven dried samples. The protein values obtained in the present study agreed with the values (31.7—34.6 g/100g) reported by Aletor et al., (2002) for similar vegetables. The mean protein value (25.82 %) for unprocessed VA was higher than the mean value (20.20 %) reported by Alabi et al. (2005) but similar to the value (27.20 %) reported by Aliero & Abdullahi (2009). Based on these results, it may be suggested that VA and GL should be taken without processing (raw) to get the most of the inherent proteins. Proteins in vegetables are usually low but they are of high quality (Ihekoronye and Ngoddy, 1985). However considering the observation of

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Morris et al., (2004) that moisture removal by heating increases protein availability, digestibility, palatability, improves shelf life, increases the concentration of nutrients and minimises food borne diseases it does seem evident that heat treatment of vegetables may not be avoided completely but the heating time may be reduced.

Boiling significantly (p<0.05) decreased the fibre content of raw VA from 12.00 % to 10.09 % and that of GL from 12.80 % to 10.35% due probably to thermally induced hydrolysis of fibre or leaching of soluble fibre. This result is in agreement with the report of Eleyinmi et al., (2008) who reported a decrease (p<0.05) in crude fibre of VA and GL with boiling. Crude fibre contents of unprocessed VA and GL (12.00 % and 12.80 % respectively) were not in agreement with the values reported by other workers. Aletor et al., (2002) reported a lower value (9.75 %) for VA than the value observed in the present study. The crude fibre content of VA was lower than that of GL in all the drying methods. Drying methods (sun, solar and oven) did not affect (p>0.05) the fibre content of VA and GL. The percentage fibre content of VA observed in this study was higher than the value reported by Adetuyi et al., (2008) for VA. High fibre content has been shown to have a positive effect in the management of diabetes and hyperglycaemia.

Boiled samples showed significant (p<0.05) increase in the fat contents of VA and GL. The higher the time of boiling, the higher the fat content, probably as other proximate constituents were decreased with boiling time. The low fat content obtained in the unprocessed VA (4.15 %) and GL (6.67 %) leaves were similar to the values reported by Atangwho et al., (2009) for VA (3.53 %) and GL (6.13 %). There was no significant difference (p>0.05) in the fat content of the samples dried with the different drying methods. The fat content ranged from 5.13% to 5.80% and 6.53% to 7.45% for VA and GL, respectively. Carbohydrate content increased with drying due to moisture loss. The VA oven dried sample had significantly higher (p<0.05) carbohydrate value. This was followed by the solar dried sample.

Vernonia amygdalina had higher ash content than GL. The ash content decreased significantly (p<0.05) with boiling time, the decrease was from 8.29 % to 7.77% and 8.13 % to 7.80% in VA and GL respectively. The values for the unprocessed VA (10.15 %) and GL (8.90%) were close to the ash contents reported by Okafor et al., (1996) for VA (10.93 %) and GL (10.94 %). The ash values for each of the leaves did not differ significantly (p>0.05). Sundried VA had the highest mean ash value (11.25 %) while solar dried had the least mean ash value (8.13 %) among the dried samples. The high ash content of sundried sample could be attributed to impurity like dust when exposed to long hours of drying. This result is in agreement with the report of Aliero & Abuldullahi (2009) on ash content of VA (11.60 %). The slight increase in ash content with drying may be as a result of concentration of the dry matter.

Carbohydrate content was low because vegetables are poor sources of carbohydrate. The value was significantly (p<0.05) lower in the unprocessed leaves than in the boiled leaves probably due to reduction of other nutrients with boiling. The carbohydrate content of

boiled leaves did not differ (p>0.05) with boiling time. The increase in carbohydrate value with boiling may be as a result of reduction of other nutrients with boiling. Also slight moisture loss of the unprocessed with boiling resulted in increased nutrients of which carbohydrate is one of them.

For GL, the highest carbohydrate content was observed in solar dried sample and the least content in the sun dried sample. The carbohydrate content of the dried samples were not significantly (p<0.05) different from those of the unprocessed in GL leaves. The report of Aliero and Abdullahi (2009) showed carbohydrate contents that ranged from 66 to 45.5% for VA which was similar to that observation in this work. Effect of boiling time on the phytochemical composition of Vernonia amygdalina and Gongronema latifolium Figures 5 and 6 shows the effect of boiling time while Figures 7 and 8 show effect of drying methods on phytochemical constituents of VA and GL. All the phytochemical constituents examined reduced (p<0.05) with boiling in both leaves.but drying reduced (P<0.05) alkaloid and flavonoid content, increased tannin, saponin, phytate and steroid contents but had no effect (p>0.05) on anthocyanin.

The alkaloid in both leaves decreased progressively from 5 min to 25 min of boiling. Boiling for only 5min reduced the alkaloid content by 30% and 44% in VA and GL respectively, while increasing the boiling time to 25min lead to 88% and 81% reduction respectively in VA and GL. The high level of alkaloids in both raw and boiled VA and GL were in agreement with the values reported in literature. Onyeka and Nwambekwe (2007) reported that boiling drastically reduced the alkaloid content of VA and GL. Alkaloid content showed significant ( p<0.05) reduction in VA from 1.73g/100g in the raw leaves to 1.02g/100g in the sundried, 1.01g/100g in solar dried and 1.13g/100g in oven dried leaves. In GL alkaloid was reduced from 0.86g/100g to 0.79g/100g (in sun dried), 0.82g/100g (in solar dried and) and 0.79g/100g (oven dried leaves). The reduction was higher in VA than in GL due probably to low alkaloid level in GL. This low alkaloid level in GL, may account for why it can be eaten without boiling or any heat treatment in such dishes like African Salad. Alkaloids work on the nervous system and are used as analgesic because they are capable of relieving pains (Okeke and Elekwa 2003, Akinmoladun et al., 2007).

Boiling for 5min reduced (p<0.05) the flavonoid content of the raw leaves from 0.07g/100g and 0.12g/100g to 0.05g/100g and 0.08g/100g for VA and GL, respectively. At 20min and 30min boiling, flavonoid was no longer detected in both VA and GL. According to Okwu (2004) flavonoids are water-soluble and are easily destroyed by boiling. Drying reduced the flavonoid content (0.07 g/100g) to the same mean value of 0.05 g/100g, in sun, solar and oven dried VA while in GL the value (0.09g/100g) was reduced to 0.062g/100g, 0.060g/100g and 0.664g/100g in sun, solar and oven dried samples respectively. Del-Rio et al. (1977) noted that flavonoid represent the most common and widely distributed group of plant phenolic compounds which often occur as

161

glycosides and glycosylation renders the molecule more water soluble and less reactive toward free radicals. As phenolic compounds, they act as antioxidants with mechanism involving both free radical scavenging and metal chelating. They have ideal structural chemistry for free radical scavenging activities and have been shown to be more effective antioxidants in vitro than Vitamins E and C on a molar basis (Rice-Evan et al., 1997).

Vernonia amygdalina had higher tannin content (0.23g/100g) than GL (0.118g/100g) in the raw leaves (Fig 5 and 6). Chinma and Igyor (2007) reported a range of 0.01g/100g to 0.25g/100g for raw vegetables in south East Nigeria. Boiling as one of the traditional processing methods, reduced tannin content after 30min boiling by 70% and 66% in VA and GL respectively. The reduction of tannin with boiling was observed to vary with length of boiling time. Tannin content of both leaves increased (p<0.05) with drying irrespective of the drying method. The mean tannin value in VA dried samples increased by 14%, 15% and 19% and in GL samples by 14%, 22.5% and 19.1% for sun, solar and oven dried respectively. The level of tannin in VA recorded in this work agreed with the value reported by Teguia et al., (1993) but higher than the value reported by Chinma and Igyor (2007) for GL (0.01g/100g). Tannins account for the astringent bitter taste of VA and has been

associated with binding and precipitation of proteins, which also decreased with boiling. This observation agreed with the report of Okafor, (1983).

Anthocyanin was very low (0.01g/100g - 0.09g/100g) in both leaves but unprocessed GL had 67% higher anthocyanin than unprocessed VA. Ten minutes boiling reduced the anthocyanin content of VA and GL leaves by 67 % and 56 % respectively and increasing boiling time showed no further significant reduction. In VA, there was no significant (P>0.05) difference in the anthocyanin content of raw and dried samples but in dried GL the anthocyanin content was significantly lower than in the unprocessed sample. Onyeka and Nwambakwe (2007) also reported low level of anthocyanin (0.02g/100g) in VA. According to Dewanto et al., (2002) anthocyanin occurs in higher concentration in fruits than in leaves. Raw VA showed 30% higher saponin level than raw GL. Five minutes boiling reduced the saponin level in VA by 63% and in GL by 58%. Increasing boiling time to 30min reduced the saponin to values of 1.49g/100g and 1.20g/100g respectively for VA and GL. Saponin increased (P<0.05) with drying in both leaves. Saponin content of raw VA (8.02 g/100g) and GL (5.64 g/100g), agreed with the value reported by Nwanjo et al., (2006).

Fig.5: Effect of boiling time on phytochemical contents of Vernonia amygdalina.

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Fig 6: Effect of boiling time on phytochemical contents ofGongronema latifolium.

Fig 7: Effect of drying methods on phytochemical contents of Vernonia amygdalina.

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Fig 8: Effect of drying methods on phytochemical contents of Gongronema latifolium

Unprocessed VA and GL showed phytate levels that ranged from 1.46g/100g to 1.25g/100g but boiling for 5min reduced the phytate content to 1.24g/100g and 1.17g/100g in VA and GL, respectively. There was a progressive decrease (p<0.05) in phytate level as the boiling time increased and by 30min boiling, phytate loss of 23% and 58% had occurred in VA and GL respectively. Phytate content significantly increased with drying in both leaves due probably to increase in concentration and complex formation of phytate with drying. In VA, the phytate content increased from 1.46 g/100g to 3.02 g/100g and 1.25 g/100g to 2.33 g/100gin GL. Chinma and Igyor (2007) recorded phytate content of 2.89 g/100g for GL which was higher than the value obtained in this work. The high phytate content observed in the vegetables suggest that absorption of minerals like Cu, Fe, Mg and Zn in the vegetables may be impaired since phytate has been implicated in chelating of divalent cationic mineral. Thompson and Erdman (1982) reported that heating vegetable may not directly improve the mineral bioavailability but Oboh et al.(2005) observed that many anti nutritional factors can be minimized by proper heat treatment. This shows that heating is a good method of reducing anti-nutrients in foods (Obeta and Ani, 2015).

Drying significantly increased the steroid content in both leaves from 0.275 g/100g in the unprocessed to 0.39-0.41 g/100g in the dried VA (Fig 7) and from 0.24g/100g (raw) to 0.32-0.385g/100g in dried GL (Fig 8). Raw GL had lower steroid content (0.24g/100g) than VA (0.275g/100g) which was in agreement with previous works (Thane and Reddy 1997). Boiling for 5min reduced (p<0.05) the steroid content in VA but GL showed no significant (p>0.05) decrease until after 15min boiling resulting in 33% and 31% reduction in VA and GL respectively. Onyeka and Nwambekwe (2007) also reported a decrease in steroid content of GL with boiling. Conclusion

Vernonia amaygdalina was observed to contain higher moisture, fibre, ash, alkaloids, tannin, saponin, phytate and steroid content than Gongronema latifolium. Drying in both leaves reduced alkaloids, flavonoids contents but increased tannin, saponin, phytate and steroids contents. Nutritionally these two leaves contain appreciable nutrients especially proteins (25.82 % -27.24 %). Their high content of phytochemicals explains why they are used for health management. Boiling for 5min and drying with solar drier at 45 -50

oC were

considered the best processing methods for Vernonia amygdalina and Gongronema latifolium leaves for nutrients retention. Also boiling for 5min was observed to have drastically reduced most of the phytochemicals especially the anti-nutrients to a very low level that may not be toxic. References Adetuyi, F.O. Ajala, L. and Dada, I. B. O. (2008). Effect of

processing methods on the chemical composition and antioxidant potentials ofstructumspaejanophora (ewuro-odo) and Vernonia amygdalina (Ewuro). World Food Journal 26(1):130-136.

Adjanahoun, E. Ahiyi, M.R.A. Ake A.L. Dramane, K. Elewude, J. A. Fadoja, S.O. Gbile, Z.O. Goudote, E. Johnson, C.L.A. Keita, A. Morakinye, O. Ojewole J. A.O. Olafunji, O.A. and Sofowora, E.A. (1993).Traditional medicine and pharmacopoeia contribution to ethnobotanical and floristic studies in western Nigeria. O.A.U Scientific Technical and Research Commission Lagos. Pp 1-5.

Agbaire, P.P. and Emoyan,O.O. (2012). Nutritional and antinutritional levels of some local vegetables from Delta State, Nigeria.African Journal of Food Science 6(1) :8 – 11.

Ahn, D., Putt, D., Kresty, L., Stoner, G.D., Fromm, D and Hollenberg, P.F. (1996).The effects of dietary ellagic acid on rat hepatic and esophageal mucosal cytochromes P450 and phase II enzymes. Carcinogenesis.17:821-828.

Akinjogunla O.J.; Eka O.H. and Odeyemi, A.T. (2011). Phytochemical screening and invivo antibacterial assessment of aqueous leaf extracts of Vernonia amygdalina (Asteraceae) and Ocimumgratissimum (Lamiaceae on

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

ph

yto

che

mic

al c

on

ten

ts(g

/10

0g)

Phytochemical

unprocessed

sundried

solardried

ovendried

164

myxifloracin resistant ascherichia coli isolatedfrom chemical and environmental samples. Nature and Science 9(7) :42-52.

Akinmoladun, A C. Ibukun, E.O. Afor E. Akinrinlola B.E. Onibon, T. Akinboboye, A.O. Obuofor, E.M. and Farombi E.O. ( 2007). Chemical constituents and antioxidant activity of Alstoniabvonei. African Journal of Biotechnology 6(10):1197—1201.

Akpaso, M. I.Atangwho,I. J. Akpantah, A. Fischer,V. A. Igiri, A. O. and Ebong, P. E.(2011). Effect of Combined Leaf Extracts of Vernonia amygdalina(Bitter Leaf) and Gongronema latifolium (Utazi) on the Pancreatic β-Cells of Streptozotocin-

Induced Diabetic Rats.British Journal of Medicine and Medical Research 1(1): 24-34.

Alabi, D. A. Onuibudo, M. and Amusa, N. A. (2005).Chemical and Nutritional composition of botanicals with fungi toxic properties. World Journal of Agricultural Science1(1): 84-88.

Aletor, O. Oshodi, A.A. and Ipinmoroti, K (2002). Chemical composition of common leafy vegetable and functional properties of their leaf protein concentrates. Food Chemistry 78 (1): 63-68.

Aliero, A.A and Abdullahi, L. (2009).Effect of drying on the nutrient composition of Vernonia amygalina leaves. Journal of phytology 1 (1): 28-32.

Ames, B. N. (1998): Micronutrients prevent cancer and delay aging. Toxicol Letters 102:103-105.

Amic, D. Davidovic-Amic, D., Bestlo, D.andTrinajstic, N. (2003).Structure radical scavenging activity relationship of flavonoids. ACTA 76(1): 55—61.

Anonymous,(2008a). Phytochemicals. http://www.phytochemicals.info/phytochemicals/saponins.php.

Accessed 2nd

June, 2008. Anonymous,

(2011).Phytochemicals.http://www.phytochemicals.info/htm. AOAC, (1990) Association of Official Analytical Chemists

Official Methods of Analysis, 17th

edition.Washington D.C Araghi-Niknam, M., Chung, S., Nelson- white, T. and Watson,

R.K. (1996). Antioxidant activity of Dioscorea and dehydroepiandrosterone (DHEA) in older humans. Life Science 59:133-139.

Asawalam, E.F and Hassanali, A. (2006).Constituents of the essential oil of Vernonia amygdalina as maize weavil protectants. Tropical and subtropical agro- ecosystem 6:95-102.

Atangwho, I. J; Ebong, P. E; Egbung, G. E and Ani, I. F. (2009).Effect of co-adminstration of extracts of Vernonia amygdalina and Azadirachtaindicaon serum electrolyte profile of diabetic and nondiabetic rats. Australian Journal of Basic and Applied Sciences, 3: 2974-2978.

Bonsi, M.L.K., Osuji, P.O., Tuah, A.K. and Umunna, N.N. (1995). Vernonia amygdalina as a supplement to left straw fed to Ethiopian sheep. Agroforestry systems 31(3) :229—241.

Boyer, J. and Liu, R.H. (2004). Apple phytochemicals and their health benefits: Nutrition Journal 3(5): 1-5. http:// www.Nutrition.com./content 3/1/5.Accessed 12

th Dec 2010.

Burkill, H.M., (1985): The useful plants of West Tropical Africa. 2nd Edition.Volume 1, Families A–D.

Burkill, H. M. (1985). The Useful Plants of West Tropical Africa: Review. Dalziel‘s J.M Royal Botanic Gardens, Kew.2nd Edition.

Butler, L.G. (1989). Effects of condensed taninin in animal

nutrition.In ―Chemistry and significance of condensed tannin‖ Eds R.W. Hemingway and J.J. Karchesy. Plenum press New York pp 391-402.

Chinma, C.E. and Igyor, M.E. (2007).Micro nutrients and anti- nutritional contents of selected tropical vegetable grown in south East Nigeria. Nigerian Food Journal 25(1) :111—117.

Del-Rio,A., Obdulio, B.G., Castillo,J., Martin R.R. and Ortuno,A. (1977). Uses and properties of Citrus flavonoids.Agriculture and Food Chemistry. 45:4505-4515.

Dewanto, V. Wu, X. and Liu, R. (2002). Processed sweet corn has higher antioxidant activity. Agriculture and Food Chemistry. 50 (17):4959-4964.

Eka, O.U. (1977). Studies on the level of oxalic acid and phytic

acid in traditional foods of Northern Nigeria.West African Journal of Boilogy and Applied Chemistry. 20:26—30

Eleyinmi, A.F. Sporns, P. Bressler, D. C. (2008).Nutritional composition of Gongronema latifolium and Vernonia amygdalina. Journal Nutrition and Food Science 38(2):99-109.

Erdman, J.W., Balentine, D., Arab, L., Beecher, G., Dwyer, J.T., Folts, J., Harnly, J., Hollman, P., Keen, G.L., Mazza, G., Messiana, M., Scalbelt, A., Vita, J., Williamson, G. and Burrowers, J. (2007). Flavonoid and Heart Health. Journal of Nutrition 137(3): 7185 – 7375.

Eyong, E.U; Agiang, M.A and Atangwho, P.E. (2011). Phytochemicals and micronutrients composition of root and stem bark extraction of Vernonia Amygdalina Del. Journal of

medicine and medical science2(6): 900-903. Francis, C; Gorge,G; Zoher,K; Harinder, P.S; Makhar, L.M.

and Klaus, B. (2002). The biological action of sapponins in animals system: A Review. British Journal of Nutrition 5 (2) 166-168.

Harborne, J.B.(1973). Phytochemical methods: A guide to modern technique of plant analysis. Chapman and Hall Ltd. London pp 49-188.

Harland B.F. and Oberleas D. (2001). Newer methods for phytate Analysis: Handbook of dietary fiber in human nutrition. 3

rded.Edited by Gene A spiller. CRC press Pp113-

126. http://www.crcnetbase.com/doi/asb/10.1201/9781420038514.ch3.7. Accessed Feb,19

th 2009.

Igwe, C.U, Ojiako A.O, Emejula A.A. and Iweke A. V, (2012).Phytochemical analysis of plants traditionally used in malaria treatment in southeastern Nigeria. Journal of Research in Biochemistry 1:015-022.

Ihekoronye, A.I. and Ngoddy, P.O. (1985).Integrated Food Science and Technology for the Tropics. Macmillan Pub, London. Pp 293-307.

Karou, D. Dicko, M. H. Simpore J, Alfred S. T. (2005). Antioxiant and antibaterialativities of polyphenols from

ethnomedicinal plants of Burkina Faso. African Journal of Biotechnology 4 (8): 823-828.

Kirk, R.S. and sawyer, R (1998).Pearson composition and Analysis of Foods. 9

th Ed. Longman Scientific and Technical

England Pp 607-617. Konietzny, U. and Greiner, R. (2002).Molecular and catalytic

properties of phytate- degrading enzymes (phatases). International Journal of Food Science 6(1): 8-11.

Kraus, T.E., Yu, Z., Preston, C.M., Dahlgren, R.A.andZasoski, R.J.(2003). Linking chemical reactivity and protein precipitation to structural characteristics of foliar tannins. Journal of ChemEcol 29(3): 703-730.

Lotito, S.B. and Frei, B. (2006). "Consumption of flavonoid-rich foods and increased plasma antioxidant capacity in humans: cause, consequence, or epiphenomenon?".Free Radical Biological Medicine 41(12): 1727–46.

doi:10.1016/j.freeradbiomed.2006.04.033. PMID 17157175. Maikai, V.A.Kobo, P.I. and Adaudi, A.O. (2008). Acute toxicity

studies of aqueous stem bark extract ofXimeniaamericana. Journal of Biotechnology 7 (10) :1600-1603.

Modupe, O. D. and Babayemi, O. J. (2009).Utilization of some Edge-row plants and forage in Nigeria.Pakistan Journal of Nutrition 8(8): 1269-1274.

Morebise, O., Fafunso, M.A., Makinde, J.M., Olajide, O.A.and Awe,E.O. (2002). Inflammatory property of the leave of Gongronema latifolium.Phytother Research 16:75-77.

Morris, A., Barnet,A. and Barrowsa, O. (2004). Effect of processing on Nutrient content of Foods.Cajanus 37:160-164.

165

Nwanjo, H.U, Okafor, M.C. and Oze, G.O.(2006). Anti-lipid peroxidative activity of Gongronema latifolium in streptozotocin induced diabetic rats. Nigeria Journal of physiological Science. 21(1-2): 61-65.

Oberley, L.W.(1988). Free radicals and diabetes. Free radical Biological Medicine. 5:113-124.

Obeta, N. A. and Ani, J.C. (2015). The hpoglyceamic and hypoglycaemic potentials of raw and boiled Vernonia amygdalina leaf extract on normal, diabetic induced and high fat feed male abino rats. Journal of Natural Science Research 5(7): 30-38.

Oboh, F.O.J. and Masogje (2009).Nutritional and anti-microbial properties of Vernonia amygdalina leaves. International Journal of Biomedical and health Sciences 5(2): 51-56

Oboh, G. (2005a). Effect of blanching on the antioxidant property of some tropical green leafy vegetable.Food Science and Technology. 38(5):513-517.

Odom, T.C. Udensi , E.A. and Ogbuji, C.A.(2013). Qualitative and Quantitative phytochemical analysis of Mucunacochichinensis unripe Carica papaya and unripe musaparadisiaka. European Journal of Biology and Medical Science Research 1(1): 15-22.

Oguntona, T. (1998).Green leafy vegetables. In nutritional quality of plant food.,Eds A.U. Osagie and O.U. Eka. Post Harvest Research Unit. University of Benin. Benin city. pp 120-133.

Okafor, J. C. (1983). Horticulturally promising indigenous wild plants species of the Nigeria forest zone. ACTA Horticulturae 123:165-176.

Okafor, J.C. (1989). Some useful tropical plants in health-care delivery. Guest lecture delivered at the 6

th Annual General

Meeting of the Nigeria society of pharmacognosy. University of Nigeria, Nsukka held from 9

th to 11

th July, 1989.

Okafor, J., Okolo, H.C. and Ejiofor, M.A.N. (1996). Strategies for enhancement of utilization potential of edible woody forest species of south-eastern Nigeria. In the biodiversity of African Plants.Ed. L.J.G Van der measen.kluwer Netherlands. Pp 684-695.

Okeke, C.U. and Elekwa,1.(2003). Phytochemical study of the extract of GongronemaLatifoluimBenth . Journal of Health

and visual science 5 (1): 47-55. Okeke, C.U and Elekwa, I. (2006). Comparative

hypoglycaemic effects of three Nigeria vegetables spices,

Gongronema latifolium Benth Allium sativumlinnand Ocimumgratissimum, L on Alloxan – induced diabetic rats. Nigerian Journal of Botany 19(1) :138-146

Okoli, E.C. Nmorka,O.O and Unaegbu, M.E. (1988). Blanching and storage of some Nigerian Vegetables.International Journal of Food Science and Technology 23:639-641.

Okoye, E.I. and Ebeledike A.O. (2013). Phytochemical constituents of piper guneense (uziza) and other health implications on some micro-organisms. Global Research Journal of Science 2:42-46.

Okwu, D.E. (2004). Phytochemical and vitamin content of indigenous spices of south Eastern Nigeria. Journal Sustain Agricultural Enviroment 6:30-34.

Onyeka, E.U. and Nwambekwe, I.O. (2007). Phytochemical profile of some green leafy vegetable in South East Nigeria. Nigeria Food Journal 25(1): 67-77

Rice-Evans, C.A. Miller, N.J. and Paganga, G. (1997).Antioxidant properties of phenolic compounds. Trends in Plant Science 2:152-159.

Sofowora, A. (1993). Recent trends in research into African medicinal plants. Journal of Ethnopharmacology 38:197-208.

Svoboda, K.P.(2002). Herbs, spices and condiments- In encyclopedia of plant and crop science Ed. R. M. Goodman and M.Dekkar, USA pp559-563.

Swain, T. (1963). Chemical plant Taxonomy Academic Press London. In www.ephamacognosy com/classificationofalkaloids.htm accessed 7

th July 2012.

Teguia, A.Tchoumboue, J. Mayaka, B.T. and Tinakou,C.M. (1993). The growth of broiler chickens as affected by the replacement of graded levels of maize by sweet potato leaves (Ipomoea batatas) or Ndole (vernonia spp.) in finisher

diet. Animal Feed Science and Technology 40:233-237. Thompson, D.B. and Erdman (JR), J.W (1982). Phytic acid

determination in soybeans. Journal of Food Science 47:513 -514.

Trease, G.E and Evans, M.C. (1983).Pharmacognosy 12th

edition BailliereTindall London pp 343-383. Yusuf, M.I. and Obiegbuna, J.E (2015).Effect of processing

methods on the phytochemical content and consumer acceptability of two selected green leafy vegetables. Agricultural Science Research Journal 5(7):105-110.