Heating Effect on Phytochemical and Proximate Content of Cooked Aqueous Extract of Phaseolus Vulgaris (Kidney Beans)

Plant chemicals and nutrients abound in different parts of plants and in different compositions. Phaseolus vulgaris (kidney beans) is a nourishing leguminous food commonly eaten by both human beings and animals in the world due to its health benefits and risk reduction of diseases. It is however, indispensible for plants’ food stuff to retain their phytonutrients for maximum benefits. This study therefore investigated the effect of heat on the phytochemicals and proximate contents in cooked Phaseolus vulgaris (kidney beans). Qualitative and quantitative phytochemical analysis and proximate (nutrient contents) analysis were determined on fresh kidney bean (FKB) and cooked kidney bean (CKB). Results of phytochemical quantification revealed a significant (p˂0.05) increase of alkaloids and saponins in FKB than those in CKB, significant (p˂0.05) increase of flavonoids, glycosides and tannins in CKB than those in FKB. While proximate analysis of cooked sample (CKB) showed significant (p˂0.05) increase in protein content, crude ash content and carbohydrate content FKB. While moisture content, crude fibre and crude fat of FKB were significantly (p˂0.05) higher than those in CKB of Phaseolus vulgaris . Obviously, the increased concentrations of phytochemicals in fresh Phaseolus vulgaris may be due to the absence of heat action and the heating effect on cooked Phaseolus vulgaris could unleash the high rich nutrients value and could supply its antioxidants roles, thereby improving healthy life when eaten cooked. Results are Mean ± Standard deviation for duplicate analysis; the mean difference is significant at P<0.05. Results are Mean ± Standard deviation for duplicate analysis; the mean difference is significant at P<0.05.


INTRODUCTION
Kidney bean (Phaseolus vulgaris) derived its name from the visual resemblance of the kidney in shape and colour. However, the red colour of the kidney beans should not be confused with other beans such as the pinto beans and adzuki beans, which are also red in colour 1 . Phaseolus vulgaris beans plant strive best in subtropical and tropical weather. The worlds' largest producer of beans is Brazil, having approximately five million hectares of land cultivated, with the production of 2.2 -2.5 million tons 2 . Researchers have evidently reported the various nutrients in beans and their health benefits, sample recipes and preparation tips 3 . Beans contain several nutrients including mineral salts such as Mg, Ca, P, Fe and K; contain about 20 -25% proteins, 50-60% complex carbohydrates and vitamins 4 . Certain antinutrients are also found in beans which inhibit and limit absorption of other useful nutrients. They include phytic acid and oligosaccharides; they limit carbohydrate and protein absorption and trypsin, which inhibit

Preparation of Cooked Sample (CKB)
The cooked Phaseolus vulgaris (kidney beans) sample was prepared appropriately by hand picking to remove all foreign particles followed by washing and cooked with enough water until soft and without broth to prevent the loss of some phytochemicals in the bean broth. This was dried under mild sunlight for two weeks under strict supervision. Five hundred gram (500g) of the dried bean seeds was weighed grinded/homogenized into powder. After which it was stored in a clean grease free airtight container with proper labeling for proximate and phytochemical analysis.

Preparation of Dry Extract from Samples
From the powdered samples (CKB and FKB), 200g was weighed and soaked in 700ml of distilled water, carefully sealed and allowed to stand for 48 hours (for thorough extraction), before filtering with whatman filter paper. The filtrate was concentrated in water bath at temperature of 70ºC.

Qualitative Phytochemical Screening of Fresh Bean (FKB) and Cooked Kidney Bean (CKB) samples
Alkaloids, flavonoids, saponins, glycosides, phenols, steroids, tannins, reducing sugars and anthraquinones were identified in cooked and fresh kidney beans samples using standard methods described by 14,18 with some modifications.

Determination of Alkaloids
Alkaloids in the bean samples were determined by the method described by 14 . Five gram (5g) of the sample was weighed into a 250mls beaker and 200mls of 10% acetic acid in ethanol was added and it was covered and allowed to stand for 4 hours at room temperature, after which it was filtered. The filtrate was concentrated on a water bath to one quarter of its original volume by evaporation and treated with drop wise addition of concentrated aqueous ammonium solution until the alkaloid was precipitated. Alkaloid precipitated was received in a weighed filter paper (W1). It was washed with 1% NH 3 solution and dried in the oven at 80ºC. The filter paper and residue was cooled in a desiccator and recorded as (W2). The alkaloid content was calculated and expressed as a percentage of the weight of the sample.
Percentage alkaloid content was calculated using the formula: Alkaloid% = weight of filter paper + sample after dryingweight of filter paper

Determination of Flavonoid
Flavonoid in samples was determined by the method described by 15 . Ten gram (10g) of sample was weighed into a 250mls conical flask. 100mls of 80% aqueous methanol added, the mixture was shaken for 3 hours by the aid of electrical shaker. The mixture was filtered into a previously weighed beaker, and evaporated to dryness over a water bath and weighed to a constant.
Percentage flavonoid content was calculated using the formula: Flavonoid% = were W1 = weight of empty beaker and W2 = weight of residue (weight of empty beaker + sample after drying).

Determination of Saponin
Saponin in both samples was determined using 16 method. About 10g of grinded sample was put into a conical flask and 100mls of 20% aqueous ethanol was added. The mixture was thoroughly mixed with for about 20 to 30 minutes and it was immediately transferred into a 250mls conical flask. Then, the mixture was covered properly and heated over a hot water bath at 90 o C for 4 hours with continuous stirring. The mixture was filtered with whatman filter paper and the supernatant separated. The solid residue was mixed with 100mls of another 20% ethanol and heated in a similar way for 4hours. The solution was then filtered and mixed with the previously filtered solution. The combined filtered solution was placed on a hot water bath at 90ºC and heated to 20% of the original volume. The concentrated solution was then transferred into a 250mls separating funnel and 10mls of diethyl ether was added to it and vigorously mixed. The diethyl layer was discarded carefully after settling down the solution. The purification process was repeated again. 60mls of n-butanol was added and two layer was formed, then the bottom layer was discarded and the upper layer recovered. The combined n-butanol extract was washed twice with 10mls of 5% NaCl solution. The remaining solution (i.e upper layer) was heated in a water bath at 50 0 C until the solvent evaporated and the solution turned into semi-dried form.
Percentage saponin content was calculated using the formula: Were W1 = weight of empty beaker and w2 = weight of beaker + sample after drying

Determination of Glycosides
Glycosides contents in samples were determined by the method of 17 . Five gram (5g) of the sample was soaked into 100mls of distilled water in a 250mls conical flask and agitated for 3 hours. The sample was filtered and the total extract was measured and noted. Into a test tube was 2mls of the extract measured and 2mls of 10% DNS reagent was added. Then, the test tube was boiled for 20 minutes in a beaker of boiling water. The test tube was cooled in cold water. The absorbance was read at 540nm using UV-Vis Spectrophotometer, DHG-9101 machine.
Percentage glycoside content was calculated using the formula: % Glycoside =

Determination of Tannin
Tannin in the bean samples was determined using the method described by 17 with some modification. 0.5g of the sample was weighed into a conical flask and 50mls of distilled water was added. The flask was shaken for 1 hour and filtered. About 5ml of the filtrate was pipetted into a 50ml volumetric flask followed by addition of 5ml of 0.1% tannic acid. The blank was prepared using 5ml of distilled water in a 50ml volumetric flask. The three flasks were incubated for one and half hour at 20ºC using a water bath and the flasks were made up to 50ml mark with distilled water. The concentration was determined at 760nm using UV-Vis Spectrophotometer, DHG-9101.
The concentration of tannin was calculated using the formula: Tannin (mg/l) = were x = concentration of extract; y = concentration = standard (tannic); z = concentration of blank.

Proximate Analysis
The proximate analysis of the bean samples was done using standard prescription as described by 19 . The proximate composition determined include; the moisture content, ash content, crude fat content, crude protein content, crude fibre content and carbohydrate content of the samples.

Determination of Moisture Content
The moisture content of the samples was determined according to the standard prescription as described by 19 , with the use of hot air oven. The sample was thoroughly homogenized in a domestic mixer. About 2-10g of the homogenized sample was weighed in a clean dried petri-dish pre-dried at 98ºC for 60 minutes. The sample was dried by heating for a period of 2 to 3 hours to overnight in a hot air oven at 100ºC. The sample was weighed periodically until it reaches a constant weight. The percent moisture content was calculated from the difference between the initial sample weight (W I ) and the final sample weight after drying (W D ).

Determination of Crude Ash Content
Ash content was determined by the standard prescription as described by 19 , using muffle furnace. A platinum crucible was heated to 600ºC in a muffle furnace for 1 hour, and was cooled in a desiccator and weighed (W 1 ). About 2g of the dried sample was weighed (W 2 ) into a crucible and heated at low flame by keeping on a clay triangle to char the organic matter. The charred material was kept inside the previously set muffle furnace and heat for 6 to 8 hours to greyish white ash and the crucible was cooled in a desiccator and weighed (W 3 ). The crucible was heated again for further 30 minutes to confirm completion of ashing, cooled and weighed.
Percentage of ash content was calculated using the formula: % of ash content (g/100g) = Where, W 1 -Weight of crucible W 2 -Weight of dry matter with crucible taken for ashing W 3 -Weight of crucible with ash

Determination of Total Protein
The total protein of the samples was determined by the standard prescription as described by 19 , with the use of biuret. Series of dilution of 0.2, 0.4, 0.6, 0.8 and 1ml of the working standard was pipetted into different test tubes. About 0.5ml and 1ml of the sample extract was pipetted into two other test tubes. The test tubes were made up to 2mls with distilled water along with the blank tubes. About 3mls of the Biuret reagent was added in all tubes and were mixed properly and incubated at 37ºC for 15minutes. The colour complex was measured spectrophotometrically at 520nm.

Determination of Crude Fat
Crude fat of samples was determined by the standard prescription as described by 20,21 , using soxhlet apparatus. About 5-10g (W 1 ) of the dry sample was weighed into a thimble and a cotton plug was kept on top of it. The thimble was placed in a soxhlet apparatus and volumes of ether was added into a pre-weighed flat-bottom flask (W 2 ) and distilled for 16hours the apparatus was cooled and the solvent was filtered into a pre-weighed conical flask (W 2 ). The flask of the apparatus was rinsed with small quantities of ether and then added washings to the above flask.

Determination of Total Carbohydrate
The carbohydrate contents of the bean samples were determined by the standard prescription as described by 19 and by the nitrogen free extractive (NFE) method described by 20 . Series of dilution of 0.2, 0.4, 0.6, 0.8 and 1.0ml of the working standard were prepared and pipetted into different test tubes. About 0.1ml and 0.2 ml of the sample solution was pipetted into two separate test tubes and each of the test tubes was made up to 1ml with distilled water. A blank test tube was set with distilled water. About 1ml of phenol solution was added to each tube followed by 5ml of 96% Sulphuric acid and was mixed very well. After 10minutes, the contents in the test tubes was mixed and placed in a water bath at 25 to 30ºC for 20minutes. The colour was read at 490nm. The amount of carbohydrate present was calculated using a standard graph. The percentage of total carbohydrate present was calculated using the formula: Absorbance corresponding to 0.1ml of the test = X mg of glucose 100ml of the sample solution contains = 100 mg of glucose = % of total carbohydrate present

Determination of Crude Fibre
The crude fiber of the samples was determined by the prescription as described by 19 . Two gram portions of water and 25ml alcohol (Ethanol). The residue was removed and transferred to a pre weighed ashing dish (W 1 ) and was dried for 2 hours at 130 ± 2ºC. The dish was cooled in a desiccator and weighed (W 2 ). The dish was heated again for 30 minutes at 600 ± 15ºC, cooled in a desiccator and reweighed (W 3 ).
The percentage content of crude fibre in sample was calculated using the formula: % Crude fibre in sample =

Statistical Analysis
All the analyses were performed in triplicate and average values calculated were expressed according to required units. Data collected were analysed by analysis of variance (ANOVA) with the IBM statistical package for social sciences (SPSS) for Windows version 23. The Bonferroni post hoc test was used to identify the means that differ significantly at p<0.05. Results were expressed as mean ± standard deviation.
Reducing Sugar ND ND 8.

Results of quantitative phytochemical analysis of aqueous extracts of fresh kidney bean (FKB) and cooked kidney bean (CKB) samples.
The results of quantitative analysis of CKB and FKB in Table 2   Results are Mean ± Standard deviation for duplicate analysis; the mean difference is significant at P<0.05. Keywords: Fresh Kidney beans (FKB) and cooked kidney beans (CKB).

Results of proximate analysis of fresh kidney bean (FKB) and cooked kidney bean (CKD) samples
The proximate analysis of the bean samples is shown in Table 3   Keywords: fresh kidney beans (FKB) and cooked kidney beans (CKB).

DISCUSSION
Results of phytochemical quantification revealed that alkaloids and saponins in FKB were higher than those in CKB, flavonoids, glycosides and tannins in CKB were higher than those in FKB. While proximate analysis of cooked sample (CKB) showed that protein content, crude ash content and carbohydrate content were higher than those in fresh sample (FKB). While moisture content, crude fibre and crude fat of fresh sample (FKB) were higher than those in cooked sample (CKB) Phaseolus vulgaris. Qualitative and quantitative phytochemical results are shown in (tables 1 and 2). The results revealed higher percentages of flavonoids, glycosides and tannins in CKB than FKB and lower percentages of alkaloids and saponins in CKB than FKB. Higher concentrations flavonoids were found in the extract of cooked bean. This was consistent with 22 , who reported that cooking increases the concentrations of flavonoids fractions in Phaseolus vulgaris especially catechin, quercetin and quercetin-3-glucoside and kaempferol and kaempferol-3-rutinoside. Quercetin was reported to be very effective in the deterrence and treatment of cancer, inhibition of free radical generation by hydrogen peroxide, blockage of histamine generation and involved in the reversal of cognitive insufficiency 23 . On the contrary, 24 assert that cooking and soaking reduce the concentrations flavonoid in Phaseolus vulgaris. Cooking boosted the phenolic compounds' antioxidant ability of beans and increases its nutritive values 22 . It is reported that flavonoids are the most abundant secondary metabolites in the plant and their presence in beans are appreciated as they can enhance tast, color and flavor in most beans specie when they are well cooked 22 .
Cooking enhanced the concentration of glycosides in the cooked extract than in the fresh. The level of flavonol glycoside in Phaseolus vulgaris was reported to be high in cooked Phaseolus vulgaris 25 . Cyanogenic glycoside is classified as a significant antinutrient factor in plants which must be reduced or destroyed for human and animal food consumption 26 . The tannin concentration of cooked Phaseolus vulgaris is higher than the fresh sample. Like other dark colour legumes, Phaseolus vulgaris (red kidney beans) is known to have high content of tannins and phytic acid than white beans 27 . This could support the high level of tannins in the cooked sample, that even after cooking, the percentage was still high. However, 28 reported contrary reduced tannin content in Phaseolus vulgaris after cooking. Tannins are included as antinutrients in legumes which inhibit the digestion of protein but are destroyed by cooking to increase the functions of amylase and protease and better protein digestion 29 . Dietary tannins, flavonoids and saponins found in Phaseolus vulgaris like other plant sources, are known for their medicinal importance in the preparation of herbal formulation as composite active ingredients to partake in cancer prevention, function as antioxidants and possess anti-inflammatory ability 30,31 . Cooking reduced the content of alkaloids in cooked extract of Phaseolus vulgaris. Different toxic alkaloids are said to be present in beans especially lupines spp, such as pyrrolizidine and piperidine alkaloids 32 . However on the contrary, 33 reported that alkaloids applications in medicine are suited for their non toxicity for spectacular physiological functions. The low alkaloid content in the cooked extract of Phaseolus vulgaris was in agreement with a previous work reported by 32 , that after the treatment of soaking and cooking, the alkaloid content of Lupin Bean was significantly reduced. On the contrary, 34 reported high level of alkaloid in Balanites aegyptiaca kernel.
Proximate analysis of cooked sample (CKB) showed that protein content, crude ash content and carbohydrate content were higher than those in fresh sample (FKB). While moisture content, crude fibre and crude fat of fresh sample (FKB) were higher than those in cooked sample (CKB) Phaseolus vulgaris. It was observed that cooking increased the concentration of protein. By contrast, 35 reported that heat treatment of pressure cooking and boiling had reductive effect of amino acids including lysine, methionine and tryptophan in Vigna unguiculata and protein was reported to be significantly reduced by boiling in Vigna Sesquipedalis 36 . In a diet combination experiment, the rich protein content in beans was reported to induce the higher weight loss in an 8 week period among 35 obese men 37,3 . Protein consumption in legumes was found to be associated to decrease in blood pressure, total cholesterol, waist circumference and body fat mass 37 . Similarly, a slightly higher crude ash was found in the cooked extract of Phaseolus vulgaris.
This finding is in contrast to that reported by 35 , that boiling reduced the content of ash in beans.
The amount of mineral present in plant is attributed to its ash content, thus increased crude ash could suggest high mineral contents in Phaseolus vulgaris extract 36 . Higher amount of carbohydrate was found cooked extract than in fresh extract. This is in agreement with 36 , who reported that boiling for 40 minutes significantly increased the carbohydrate content of Phaseolus vulgaris 58.70 ± 0.7% to 63.37 ± 0.11% (8% increase), a value which is well lower than the one reported here, indicating an effect of cooking on carbohydrate content in Phaseolus vulgaris. Carbohydrates contain resistant starch and cooking of legumes in boiling water is reported to decreases the resistant starch content because of destruction of a huge part of the crystalline regions. However, due to great amount of amylose in legumes and a stable form of crystalline amylase, an infinitesimal amount of resistant starch is eventually destroyed by cooking legumes 38 . Because the metabolism of resistant starch does not occur in the small intestine, glucose are unconfined into the blood, therefore the need for insulin and food calorific density, which may tend to obesity are reduced 39 . Furthermore, resistant starch in beans increases satiety, reduced glycemic index, reduced systolic blood pressure, decreased risk of coronary heart disease and prevent rise in blood glucose level 40 . The moisture content of this finding was higher in fresh extract than in cooked extract of Phaseolus vulgaris. This is consistent with 36 , who reported that heat treatment by boiling and roasting significantly reduced the moisture content of Vigna Sesquipedalis but in contrast to 35 , who reported that boiling increased the moisture content of Vigna unguiculata. Reduced moisture content in legumes plays a role in food storage and preservation as it guarantees a microbial growth inhibition 10 . The moisture content was reduced possibly because of the high temperature subjection which favors evaporation of the cooked extract for the period it was cooked 35 . Also, preservation of cooked sample may be enhanced since reduced moisture content is associated with low free fatty acids and acid value 41 . Crude fiber of cooked Phaseolus vulgaris extract was found to be reduced. This is in agreement to 36 , who reported that boiling and roasting reduced crude fiber content in Vigna Sesquipedalis and 35 , were crude fiber was reported to be reduced after boiling. Beans generally are rich sources of both soluble and insoluble fiber which play a very important role in bowel movement function 38 . The presence of fiber in food helps to keep proper functioning of digestive system and provides satiety or fullness 42 . It was observed that cooking significantly reduced the crude fat. The work of 36 is in agreement with this finding as it was reported that boiling and roasting reduced the fat content as time increases in Vigna Sesquipedalis. Similarly, 35 reported reduced content of crude fat after boiling of Vigna unguiculata. Beans are reported to contain little or no total fat, trans-fat, sodium and cholesterol 43 . Little or no saturated fat content in cooked beans is recommended for the reduction of cardiovascular risk factors such as raised blood levels of triacylglycerol and low density lipoprotein cholesterol 3 .