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International Journal of Food Science and Nutrition 1 International Journal of Food Science and Nutrition ISSN: 2455-4898 Impact Factor: RJIF 5.14 Received: 02-04-2019 Accepted: 04-05-2019 www.foodsciencejournal.com Volume 4 Issue 4 July 2019 Page No. 01-05 Phytochemical composition and antioxidant potential of itching beans Mucuna pruriens var. pruriens L. DC: A less-known food and medicinal legume V Smitha 1 V Vadivel 2 1-2 PG Research Department of Botany V.O. Chidambaram College Tuticorin Tamil Nadu India 2 Manonmaniam Sundaranar University Abhishekapatti Tirunelveli Tamil Nadu India Abstract Five accessions of itching beans Mucuna pruriens var. pruriens L. DC were collected from five different locations in Western Ghats and Eastern Ghats in South India. They were analysed for their proximate and mineral composition amino acid profiles of total seed protein in vitro protein digestibility IVPD and certain antioxidants. Crude protein ranged from 25 to 34.5 crude lipid 5.7-8.6 crude fibre 5.5-7.8 ash 3-4.8 and carbohydrates 48.9-58.9. Mineral profiles viz. sodium potassium calcium magnesium phosphorus iron zinc and manganese ranged from 31-107.7 723.8-1599 304.5-780 68.5- 639.6 119.6-640.2 10.7-20 1.2-3.9 and 1-4.3 mg 100g -1 seed flour respectively. The essential amino acid profile of total seed proteins compared favourably with FAO/WHO requirements except that there were deficiencies of threonine lysine and sulphur containing amino acids. The IVPD of the accessions ranged from 65.64 to 74.66. Antioxidant substances like phenols tannins and L-DOPA 3 4-dihydroxyphenylalanine were also investigated. Keywords: itching beans Mucuna pruriens var. pruriens chemical composition amino acid antioxidants 1. Introduction Pulses are good source of nutritional components they are low in fat and rich in complex carbohydrates vegetable protein and minerals 1 . Researchers have found a positive correlation between the anticarcinogenic effects and various components present in pulses including dietary fiber 2 and folate 3 . The major polyphenolic compounds of pulses are tannins flavonoids and phenolic acids. These compounds are the products of secondary metabolism of plants they have a good antioxidant activity 4 . Itching bean Mucuna pruriens var. pruriens L. DC with the vernacular name Naikuruna is found wild in the forests of South India. It is a hardy herbaceous vigorous and twining annual which forms a thick soil covering and smothering the growth of weeds. It can be grown on almost every type of soil in tropical and subtropical plains 5 . Mature seeds seeds from unripe pods and young pods of itching beans are soaked and boiled / roasted and eaten as such or mixed with salt by the tribes of North-East India: Khasi Naga Kuki Jaintia Chakma and Mizo 6 tribes of North-Western part of Madhya Pradesh: Abujh-Maria Maria Muria Gond and Halba 7 tribes of South India: Mundari Dravidian Kani Kader and Muthuvan 8 and Savera Jatapu Gadebe and Kondadora 9 . To make this less-known legume palatable tribal people follow a special processing method of continuous boiling and draining for about eight times until the boiled water changes from black to milky white 9 . Janardhanan and Lakshmanan 10 have attributed the presence of a non-protein amino acid L- DOPA 3 4-dihydroxyphenylalanine which is pharmacological active and is known to cure Parkinson’s disease. The seed powder of M. pruriens has a faster hypothermic and anti-parkinsonian activity than synthetic L-DOPA 11 . The seed powder increases the sexual activity of male albino rats considerably and L-DOPA also is reported to arouse sexual desire in the patients suffering from Parkinson’s disease 12 . Vigorex-SF capsule an Ayurvedic herbomineral formulation comprising M. pruriens is found to have adaptogenic effect to improve one’s libido disturbed due to psychological fear and emotional imbalance and other allied ailments 13 . Alcohol extracts of leaf and fruit trichomes of M. pruriens are found to increase the pain threshold and decrease body temperature. The extracts also showed anti- inflammatory activity as they are able to inhibit carrageenin-induced edema 14 . Seeds of this plant species are widely used for treating male sexual dysfunction in Unani medicine 15 . The blocking effect of King cobra venom at the neuromuscular junction is removed by the aqueous extract of M. pruriens seeds 16 . Rhinax an herbal formulation comprising M. pruriens possesses anti- hepatotoxic activity 17 . The tribe Garos of Meghalaya India consume the seeds for increasing potency and the hairs of the pod are used as vermifuge 18 . In Nigeria powdered hairs on pods are administered with honey for expelling intestinal worms 19 . The roots are used as tonic stimulant diuretic purgative and emmenagogue. An ointment prepared from the root is applied for elephantiasis. The leaves of the plant are applied to ulcers 20 . Besides the potential use of itching bean flour in high protein biscuits is highlighted 21 . Despite the potential of this species as a source of less- known food and medicine to our knowledge meagre information is available on the germplasm collection from South India and their evaluation for chemical composition. In this context five accessions of itching beans collected from five locations of South India have been investigated phytochemically and the results are discussed with earlier findings on cultivated food legumes.

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International Journal of Food Science and Nutrition 2 2. Materials and methods 2.1. Sources of seed Five accessions each 5kg of itching beans were gathered as mature pods from natural stands in five locations. Locality district and state of collection are given in Table 1. The accessions were botanically identified by using the botanical key of Wilmot-Dear 22 . The mature pods were collected from tropical rain forests of Western Ghats three accessions and deciduous forests in Eastern Ghats two accessions. After thoroughly drying in the Sun the pods were thrashed to remove seeds. After thorough clearing and removal of broken seeds and foreign materials seeds were stored in airtight plastic containers at room temperature 25°C±2°C. The air-dried seeds nearly 50g from each accession were powdered in a Wiley Mill to pass a 60- mesh screen and stored in screw-capped bottles at room temperature for further analysis. Table 1: Information on location of collection of five accessions of itching bean seeds Locality District State Aliyar Western Ghats Coimbatore Tamil Nadu Arunooli Western Ghats Trichur Kerala Elagiri hills Eastern Ghats Vellore Tamil Nadu Karwar Western Ghats Uttarkannada Karnataka Salem Eastern Ghats Salem Tamil Nadu 2.2. Proximate composition The moisture content of the seed was estimated by taking 50 transversely cut seeds at a time and the weight was taken before and after incubation in a hot-air-oven at 80 C for 24h followed by cooling in a desiccator. Nitrogen content in the powdered seed samples was estimated by the micro- Kjeldahl method 23 and crude protein was calculated N x 6.25. The recommended methods of Association of Official Analytical Chemists 24 were used for the determination of ash crude lipid and crude fibre. Ash content was determined by incineration of 2g of sample in a muffle furnace kept at 550 C for 6h. Crude lipid was determined by exhaustively extracting 2g of sample with petroleum ether using a Soxhlet apparatus. Crude fibre was determined by acid and alkaline digestion methods. The carbohydrate content was calculated by subtracting the total of the percentages of crude protein crude lipid crude fibre and ash on moisture- free basis from 100. The energy value of the seed was estimated in kJ by multiplying the percentages of crude protein crude lipid and carbohydrates by the factors 16.7 37.7 and 16.7 respectively. All these constituents were analysed in triplicate. All the results were expressed on a dry weight basis. 2.3. Mineral analysis Five hundred milligrams of the ground legume seed was digested with a mixture of 10ml concentrated nitric acid 4ml of 60 perchloric acid and 1ml of concentrated sulphuric acid. After cooling the digest was diluted with 50ml de-ionised distilled water filtered with Whatman no. 42 filter paper and filtrates made up to 100ml in a glass volumetric flask with de-ionised distilled water. All the minerals except phosphorus were analysed from triple acid digested samples by using an atomic absorption spectrophotometer 25 . Phosphorus content in the triple acid digested extract was colorimetrically analysed 26 at 650nm using a spectrophotometer. 2.4. Amino acid analysis The total seed protein was extracted by a modified method of Basha et al. 27 The ethanol treatment was omitted to retain the prolamin fraction. The extracted proteins were purified by precipitation with cold 20 trichloroactic acid TCA. A protein sample of 30mg was hydrolysed by 6N HCl 5ml in an evacuated sealed tube which was kept in air oven maintained at 110 C for 24h. The sealed tube was broken and the acid removed completely by repeated flash evaporation after the addition of de-ionised distilled water. Dilution was effected by means of citrate buffer pH 2.2 to such an extent that the solution contained 0.5mg protein ml - 1 . The solution was passed through a millipore filter 0.45µm and derivatized with O-phthaldialdehyde by using an automated pre-column OPA. Amino acids were analysed by a reversed-phase HPLC Model 23250 ISCO Lincoln NE USA fitted with a spherisorp C 18 column 4.6 x 250mm and ISCO-dual pump. The flow rate was 1.5ml min -1 and a fluorescence detector excitation 305-395nm emission 430-470nm was used. The cystine content of protein samples was obtained separately by the Liddell and Saville 1959 28 method. For the determination of tryptophan content of proteins aliquots containing known amounts of proteins were dispersed into glass ampoules together with 1ml 5M NaOH. The ampoules were flame sealed and incubated at 110ºC for 18h. The tryptophan contents of the hydrolysates were determined colorimetrically using the method of Rama Rao et al. 29 . The contents of the different amino acids were expressed as g 100g -1 proteins. 2.5. Determination of in vitro protein digestibility IVPD Protein digestibility was assayed by the in vitro method described by Hsu et al. 30 . The enzymes used for IVPD were purchased from Sigma Chemical Co. St. Louis MO USA. Calculated amounts of the control casein and sample were weighed out hydrated in 10ml of distilled water and refrigerated at 5 C for 1h. The samples containing protein and enzymes were all adjusted to pH 8.0 at 37 C. The IVPD was determined by the sequential digestion of the samples containing protein with a multi-enzyme mixture trypsin porcine pancreatic trypsin–type IX with 14190 BAEE unites per mg protein -chymotrypsin bovine pancreatic chymotrypsin–type II 60 units per mg powder and peptidase porcine intestinal peptidase–grade III 40 units per g powder at 37 C followed by protease type IV from Streptomyces griseus at 55 C. The pH drop of the samples from pH 8.0 was recorded after 20min of incubation. The IVPD was calculated according to the regression equation Y 234.84 – 22.56 X where Y is the digestibility and X the pH drop. 2.6. Quantification of antioxidants The antioxidant compounds phenols 31 tannins 32 and the non-protein amino acid L-DOPA 3 4- dihydroxyphenylalanine 33 were quantified. 2.7. Statistical analysis Analysis of Variance ANOVA and Duncan’s Multiple Range Test DMRT were used for analysis MSTAT–‘C’ software version 1.4.1 Michigan State University MI USA of any significant difference in chemical compositions among the five accessions collected from five

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International Journal of Food Science and Nutrition 3 locations. Significance was accepted at p  0.05. 3. Results and discussion The proximate compositions of five accessions of itching bean are shown in table 2. The crude protein content of the itching bean accessions ranged from 25.0 to 34.5. This range is higher than those reported for and black gram horse gram and pigeon peas 9 . These legumes are used extensively in typical Indian diets and are expected to play a significant role in improving protein nutrition in India and Asia. The significant p ≤ 0.01 difference in protein content was probably due to different growing conditions for the plants from which the seeds were collected 34 . The fat content range is higher than that of black gram and green gram 35 . The range in ash content of this wild legume Table 2 would be important to the extent that it contains the nutritionally important mineral elements shown in Table 3. It appears that itching bean has a high range of carbohydrate probably because of their low lipid content. The range in calorific values exceeds the energetic values of cowpea green gram horse gram moth bean and peas 36 which are in the range of 1318-1394 kJ 100g -1 DM. Table 2: Proximate composition of five accessions of itching bean g 100g -1 seed flour † Component Location CD 5 Aliyar Arunooli Elagiri hills Karwar Salem Moisture 3.2 ± 0.2 d 8.4 ± 0.2 a 6.2 ± 0.2 c 7.4 ± 0.2 b 7.9 ± 0.1 ab 0.6042 Crude protein 25.0 ± 0.1 d 32.4 ± 0.5 b 28.0 ± 0.1 c 34.5 ± 1.3 a 33.0 ± 0.1 ab 1.9500 Crude lipid 5.9 ± 0.1 d 5.7 ± 0.2 d 7.3 ± 0.2 b 6.4 ± 0.1 c 8.6 ± 0.1 a 0.3854 Crude fibre 6.5 ± 0.1 b 7.8 ± 0.3 a 6.0 ± 0.1 bc 5.5 ± 0.2 c 5.6 ± 0.2 c 0.6341 Ash 3.7 ± 0.1 b 3.5 ± 0.2 b 4.8 ± 0.2 a 4.6 ± 0.2 a 3.0 ± 0.2 c 0.5388 CHO 58.9 ± 0.1 a 50.6 ± 0.6 c 54.0 ± 0.1 b 48.9 ± 1.0 c 50.0 ± 0.6 c 1.8720 Energy kJ 100g -1 DM 1623.3 ± 0.1 b 1601.7 ± 11.4 c 1643.4 ± 7.0 b 1635.3 ± 2.7 b 1706.7 ± 5.0 a 20.4304 Mean values in the row sharing common letters are not statistically significant according to Duncan’s Multiple Range Test DMRT. Significant at 1 level † Mean of three replications expressed on dry weight basis  S E CHO- Carbohydrate The mineral elements analysed and presented in Table 3 are important nutritionally. Potassium as in most legumes is the predominant macro-mineral and sodium levels are low. The low amounts of sodium in the legume seeds is good for health because of the relationship that low sodium diet has to hypertension in humans 37 . Among the micro-minerals iron concentration ranged between 10.7 and 20mg 100g-1. In general all the five accessions are found to contain higher levels of sodium compared to chick peas kidney beans peas and cowpeas 38 higher potassium and magnesium contents compared to cowpeas 39 and higher calcium and iron contents compared to chickpea pigeonpea black gram and green gram 40 . The variability in the content of minerals for the same species may be related to genetic origin geographical source level of soil fertility and the efficiency of uptake from the soil 34 . Table 3: Mineral composition of five accessions of itching beans mg 100g-1 seed flour † Component Location CD 5 Aliyar Arunooli Elagiri hills Karwar Salem Sodium 104.5 ± 3.4 a 47.8  6.8 b 42.6  1.8 bc 107.7  1.4 a 31.0  3.2 c 12.0848 Potassium 1588.0 ± 2.4 a 835.1  5.7 c 1599.0  2.4 a 723.8  3.4 d 1326.0  4.3 b 12.2045 Calcium 780.0  3.8 a 304.5  7.4 d 568.5  1.1 c 316.0  4.7 d 695.2  8.8 b 18.3780 Magnesium 68.5  4.7 e 209.0  4.4 d 639.6  3.5 a 241.3  4.8 c 417.1  6.5 b 15.3502 Phosphorus 633. 8  3.3 a 119.6  3.8 d 408.6 ± 7.8 c 423.7  3.4 b 640.2  2.0 a 14.1959 Iron 17.3 ± 1.4 ab 16.4 ± 0.4 b 10.7 ± 0.8 c 20.0 ± 0.9 a 10.8 ± 0.6 c 2.7271 Zinc 1.8 ± 0.5 b 2.3 ± 0.5 ab 3.9 ± 0.3 a 2.6 ± 0.8 ab 1.2 ± 0.5 b 1.6437 Manganese 1.0 ± 0.6 b 1.8 ± 0.4 b 4.3 ± 0.3 a 1.0 ± 0.5 b 1.1 ± 0.2 b 1.3130 Mean values in the row sharing common letters are not statistically significant according to Duncan’s Multiple Range Test DMRT. Significant at 5 level Significant at 1 level † Mean of three replications expressed on dry weight basis  S E The essential amino acid profile of total seed proteins compared favourably with FAO/WHO 41 requirements except that there were deficiencies of threonine lysine and sulphur containing amino acids Table 4. Table 4: Amino acid profiles of five accessions of itching bean g 100g -1 protein Amino acid Aliyar Arunooli Elagiri hills Karwar Salem FAO/WHO 41 requirement pattern Aspartic acid 9.54 10.53 8.96 10.24 13.21 Glutamic acid 10.18 9.84 10.51 12.47 15.10 Alanine 2.00 2.55 2.91 3.83 4.72 Valine 9.74 8.57 5.32 6.75 7.86 3.5 Glycine 8.83 10.73 6.53 7.54 5.12 Arginine 3.91 7.14 5.76 4.87 6.12 Serine 4.08 5.75 4.58 3.83 4.14 Cystine 0.85 0.65 0.98 0.84 0.93 2.5 Methionine 0.91 1.27 0.51 0.83 0.74

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International Journal of Food Science and Nutrition 4 Threonine 2.40 2.58 2.02 2.78 2.21 3.4 Phenylalanine 3.97 3.38 3.89 4.83 4.83 6.3 Tyrosine 6.01 5.27 5.03 5.54 5.60 Isoleucine 3.66 3.96 3.08 3.14 3.98 2.8 Leucine 8.86 7.28 7.40 7.57 8.40 6.6 Histidine 2.57 4.27 2.30 2.85 2.80 1.9 Lysine 3.51 5.06 4.51 4.85 3.30 5.8 Tryptophan 1.34 1.72 1.22 1.52 1.32 1.1 Proline N.D N.D N.D N.D N.D N.D- Not Detected The in vitro protein digestibility IVPD range of itching bean Table 5 was higher than that of black gram 42 and green gram 43 . Data on antioxidants such as phenols tannins and L-DOPA were shown in Table 5. Among the five accessions the Salem accession had the highest percentage of phenolics and tannins. In the present study L- DOPA range Table 5 seems to be higher as compared to earlier reports in M. utilis 44 . The high range of L-DOPA is encouraging from the point of view of pharmacological industries. Cultivar differences and accession variations are known to exist in the L-DOPA content of Mucuna beans 44 . Table 5: In vitro protein digestibility IVPD and antioxidant contents of itching beans Component Location CD 5 Aliyar Arunooli Elagiri hills Karwar Salem IVPD ‡ 74.66 72.41 65.64 70.78 70.16 -- Phenols † 5.29 ± 0.13 b 5.96 ± 0.11 a 5.34 ± 0.22 b 3.89 ± 0.06 c 6.39 ± 0.29 a 0.5702 Tannins † 0.14 ± 0.01 b 0.05 ± 0.01 c 0.06 ± 0.01 c 0.08 ± 0.01 c 0.47 ± 0.03 a 0.0521 L-DOPA † 7.62 ± 0.13 a 8.37 ± 0.39 a 7.78 ± 0.68 a 7.54 ± 0.52 a 7.82 ± 0.14 a n.s Mean values in the row sharing common letters are not statistically significant according to Duncan’s Multiple Range Test DMRT. Significant at 1 level and n.s. means not significant † Mean of three replications expressed on dry weight basis ± SE ‡ Mean of two independent determinations. 4. Conclusion To conclude it is suggested that the less-known food and medicinal legume itching beans might be useful as a source of crude protein crude fat carbohydrate certain essential amino acids and some minerals and antioxidants such as phenolis tannins and L-DOPA. In vitro protein digestibility of itching bean is also found to be higher than that of certain common legumes. 5. References 1. Janardhanan K Vadivel V Pugalenthi M. Biodiversity in Indian underexploited / tribal pulses. In: Improvement Strategies for Leguminosae Biotechnology. Jaiwal PK Singh RP Eds.. Kluwer Academic Publishers London Great Britain 2003 353-405. 2. Mathers JC. Pulses and carcinogenesis: Potential for prevention of colon breast and other cancers. British Journal of Nutrition. 2002 88:S272-S279. 3. Rampersaud GC Bailey LB Kauwell GPA. Relationship of Folate to Colorectal and Cervical Cancer: Review and Recommendations for Practitioners. Journal of the American Dietetic Association. 2002 102:1273-1282. 4. Xu BJ Chang SKC. A comparative study on phenolic profiles and antioxidant activities of legumes as affected by extraction solvents. Journal of Food Science. 2007 72:S159-S166. 5. Vadivel V Janardhanan K. Genetic resources of some South Indian tribal pulses. IPGRI Newsletter Asia Pacific Oceania. 1998 26:21-22. 6. Arora RK. Native food plants of the northeastern India. In: Contributions to Ethnobotany of India. Jain SK Ed. Scientific Publishers Jodhpur India 1991 pp.137-152. 7. Sahu TR. Life support promising food plants among aboriginal of Baster M.P. India. In: Ethnobiology in Human Welfare Jain SK Ed. Deep Publications New Delhi India 1996 26-30. 8. Radhakrishnan K Pandurangan AG Pushpangadan P. Ethnobotany of wild edible plants of Kerala India. In: Ethnobiology in Human Welfare. Jain SK Ed. Deep Publications New Delhi India 1996 48-51. 9. Rajyalakshmi P Geervani P. Nutritive value of the foods cultivated and consumed by the tribals of South India. Plant Foods for Human Nutrition. 1994 46:53- 61. 10. Janardhanan K Lakshmanan KK. Studies on the pulse Mucuna utilis: Chemical composition and antinutritional factors. Journal of Food Science and Technology. 1985 22:369-371. 11. Rajendran V Joseph T David J. Reappraisal of dopamineric aspects Mucuna pruriens and comparative profile with L-DOPA on cardiovascular and central nervous system in animals. Indian drugs. 1996 33:465- 472. 12. Ananthakumar KV Srinivasan KK Shanbhag T Rao SG. Aphrodisiac activity of the seeds of Mucuna pruriens. Indian Drugs. 1994 31:321-327. 13. Shaw BP Bera CH. A preliminary clinical study to evaluate the effect of Vogorex-SF in sexual debility patients. International Journal of Internal Medicine. 1993 3:165-169. 14. Iauk L Galati EM Kirjavainen S Forestieri AM Trovato A. Analgesic and antipyretic effects of Mucuna pruriens. International Journal of Pharmacognosy. 1993 31:213-216. 15. Amin KMY Khan MN Zillur-Rehman S Khan NA.

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