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Horsegram

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horsegram
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What is horse gram
Horsegram non-nutrient bioactive compounds
Anti-nutritional factors
Flatulence factors
Phytic acid
Phenolic compounds
Proteinase or enzyme inhibitors
Horsegram nutrient bioactive compounds
Protein and amino acid
Starch, soluble sugar and dietary fiber
What are uses of horsegram?
Horsegram potential health benefits
Antioxidant
Lowers high cholesterol
Anti-bacterial activity
Antihelmintic activity
Analgesics and anti-inflammatory effect
Anti-diabetic activity
Anti-gallstone activity
Anti-kidney stone activity
Anti-histamine activity
Diuretic activity
Hepatoprotective activity
Hemolytic activity

What is horse gram

Horse gram (Macrotyloma uniflorum or Dolichos biflorus) also known as kulattha, kulthi, kollu, kulath, muthera, gahat, hurali and Madras gram, is an underutilized and inexpensive food legume due to the presence of antinutrients like lectin and trypsin inhibitors, which limit protein digestibility and availability 1. In addition, horsegram is also rich in certain anti-nutritional factors such as oxalic acid and phytic acid, which have the tendency to bind with calcium and iron to form an insoluble salt and hence decrease their bioavailability 2. Horsegram is normally consider as a poor man’s pulse as it offers a relatively cheap source of proteins for human consumption and livestock production 3. Horsegram is considered as a good source of protein (17.9–25.3 %), carbohydrates (51.9–60.9 %), essential amino acids, energy, low content of lipid (0.58–2.06 %) 4, molybdenum 5, phosphorus, iron and vitamins such as carotene, thiamine, riboflavin, niacin and vitamin C 4. Horsegram seeds are the edible part of the plant and consumed as a whole (boiled) seed, as sprouts, as a curry or as whole meal in Asia, popular especially in southern Indian states. Procedures such as de-husking, germination, cooking, and roasting can be undertaken to enhance the nutritional quality of horse gram 6. The details of major nutrient available in horse gram are presented in Table 1 below. Considering the nutritional and antinutritional aspects of horse gram, it has the greatest potential for further utilization as nutraceuticals, forage and food for malnourished areas of the world 7.

Horsegram is relatively high in iron, but the availability of the iron is reduced by the phytates, tannins, and oxalic acid it contains. Horsegram is also a good source of calcium too. However, the oxalic acid content is high in horsegram which combines with calcium and iron to form an insoluble salt, rendering the calcium and iron unavailable for absorption 8. Hull of horsegram contains maximum amount of tannins (commonly referred to as tannic acid) are water-soluble polyphenols, which can be decreased successfully by dehulling 3. Whereas, soaking and germination (widely used as traditional technology) are the simplest, effective and the most common methods to enhance the nutritional quality and to reduce the anti-nutritional factors of horse gram. Soaking is a domestic technological treatment of hydration of seeds in water for few hours to allow the horse gram seeds to absorb water 9. Thus, it decreases and eliminates the anti-nutritional factors present in the horse gram 10. Soaking is done prior to a number of treatments including germination, cooking and fermentation 11. Several studies have reported that 12–18 hours soaking time is the most effective to reduce the levels of phytic acid and proteolytic enzyme inhibitors in legumes which are partly or totally solubilized in soaked water 12. Soaking is followed by sprouting or germination, where the soaked seeds are left in moist conditions until they germinate in order to improve their nutritional value 13. It increases the concentration of bioactive compounds 3, improves the sensory characteristics 14 and reduces the anti-nutritive compounds at the same time. Different researchers have reported that 24–48 hours germination can successfully result in the enhancement of nutritional properties and decreasing the anti-nutritional factors of legume, whereas, too long duration may adversely affect the composition of germinated seeds (i.e., loss of nutrients and moisture) 15. Physical conditions during germination, such as the presence of light or dark situation plays an important role in the nutritional composition of legumes 14, as the presence of light during germination increases the metabolic changes thereby increasing the nutritional value of legumes 15.

The origin of the horse gram is not known. However, the region of maximum genetic diversity is considered to be in the Old World Tropics, especially in India and Himalayas 16 and has diploid chromosome numbers of 2n = 20, 22, 24 17. Horse gram has been used as a food item for millennia. Archaeological investigations revealed that horse gram used as food around 2000 BC 17. Horse gram is tolerant to drought 18, salinity 19 and heavy metal stresses 20. Horse gram (Macrotyloma uniflorum) is native to African countries such as Angola, Ethiopia, Kenya, Namibia, Somalia, South Africa, Tanzania; Asian countries such as Bhutan, China, India, Nepal, Pakistan, Philippines, Sri Lanka and Taiwan and Australasian countries such as Australia 6. Horse gram is mainly grown in India, Africa, Australia, Burma, Malaysia, Mauritius, and the West Indies 21 under low soil fertility status with few inputs 22. Horsegram is regularly cultivated in India, Malaysia, Myanmar, Nepaland Mauritius, and Sri Lanka as minor crop. In Australia and Africa, it is used as a fodder crop 23. Horsegram is adapted to wide range of temperature regimes (Smartt 1985) where other crops invariably fail to survive. In India, it is generally sown late in the rainy season by resource-poor farmers in marginal and drought-prone condition. However, sowing of seeds on the first fortnight of August and September recorded higher grain and straw yields than those sown on the first fortnight of October 24, even some report indicated that delayed sowing beyond 28 August substantially reduced crop yield 25. Horsegram is a short day and day neutral plant, matures in 120–180 days after planting 17.

Table 1. Major nutrients in different milled fractions of horsegram (percent of dry matter)

ConstituentCotyledon*Embryonic axe*Seed coat*
Moisture5.8 ± 0.31b8.4 ± 0.21a3.9 ± 0.05c
Protein22.6 ± 1.23a18.6 ± 0.90b9.1 ± 0.35c
Fat1.8 ± 0.06b2.6 ± 0.04a0.6 ± 0.02c
Ash2.9 ± 0.02b2.2 ± 0.04b3.8 ± 0.05a
Crude fiber1.6 ± 0.02c11.2 ± 0.26b21.8 ± 1.6a
Total carbohydrate a66.9 ± 2.6b68.2 ± 1.9b82.6 ± 1.1a
Soluble sugars6.4 ± 0.15a4.8 ± 0.19b0.96 ± 0.06c
Reducing sugar (mg/100 g)538.3 ± 16.2a211.7 ± 6.3b108.6 ± 5.1c
Non reducing sugar5.86 ± 0.15a4.6 ± 0.08b0.85 ± 0.03c
Dietary fiber16.7 ± 0.27c22.6 ± 0.18b36.4 ± 0.90a
Soluble1.32 ± 0.04b3.1 ± 0.04a3.9 ± 0.05a
Insoluble15.38 ± 0.16c19.5 ± 0.28b32.5 ± 1.1a

Footnotes: * Values are mean ± standard deviation of three independent determinations. Means with the same letter (a, b, c) within the same row do not differ.

a By difference as 100 – (moisture + protein + ash + fat)

[Source 26 ]

Table 2. Horsegram seeds nutritional value (100g of dry seeds) 

Macro Nutrients
Protein 24.24%
Carbohydrates 37.15 %
Fat 1.10 %
Unsaturated fat 72.49%
Saturated fat 27.51%
Starch 31.86%
Sugar 5.81%
Crude fiber 5.63 %
Ash 3.34%
Moisture 8.9%
Micro Nutrients
Calcium 0.34 %
Iron 72%
Manganese 37%
Zinc 0.28%
Phosphorus 0.27%
Magnesium 0.17%
Copper 19 %
Vitamin A 2.1%
Vitamin C 1.4%
Ascorbic acid 0.7%
Niacin (Vitamin B3) 1.5%
Ribloflevin (Vitamin B2) 0.09%
Thiamin (Vitamin B1) 0.42%
[Source 6 ]

Table 3. Horsegram amino acid and fatty acid composition

Amino acids
Arginine 8.80%
Cysteine 1.96%
Histidine 3.15%
Isoleucine 6.14%
Leucine 8.96%
Lysine 8.63%
Methionine 1.16%
Phenylalanine 6.31%
Threonine 3.82%
Tryptophane 1.16%
Valine 6.47%
Fatty acids
Linoleic acid 40.3 – 45.6%
Linolenic acid 11.6 – 14.3%
Oleic acid 8.9 – 16.8%
[Source 6 ]

Table 4. Raw horsegram composition of the dietary fiber fractions (g/100 g dry matter)

Dietary fiber fractionsInsoluble dietary fiber (IDF)Soluble dietary fiber (SDF)Total dietary fiber (TDF)
Fucosendndnd
Arabinose4.14 ± 0.080.17 ± 0.034.30 ± 0.05
Xylose1.93 ± 0.10nd1.93 ± 0.10
Mannose0.11 ± 0.010.22 ± 0.050.33 ± 0.04
Galactose0.41 ± 0.020.11 ± 0.030.52 ± 0.04
Glucose7.46 ± 0.530.11 ± 0.017.57 ± 0.52
Total neutral sugars14.06 ± 0.780.60 ± 0.0814.65 ± 0.73
Uronic acids1.94 ± 0.100.26 ± 0.032.20 ± 0.08
Klason lignin5.61 ± 0.39–5.61 ± 0.39
Total dietary fiber21.61 ± 0.050.86 ± 0.0322.47 ± 0.07

Abbreviation: nd = not detected

[Source 5 ]

Table 5. Phytochemicals isolated from horsegram

CategoryPhytoconstituents
Anthocyaninscyanidin, delphinidin, malvidin, petunidin
Flavonoidsdaidzein, genistein, kaempferol, myricetin, quercetin
Phenolic acids(benzoic acid derivatives)gallic acid, protocatechuic acid, p-hydroxybenzoic acid, syringic acid, vanillic acid
Phenolic acids (cinnamic acid derivatives)caffeic acid, chlorogenic acid, ferulic acid, p-coumaric acid, sinapic acid
Enzyme sourceamylase, a and b glucosidase, b- N-acetylglucosaminidase, urease
Hemagglutininsagglutinin and lectins
Tannins
Phytic acid

Footnotes: Phytochemical screening studies reveal the existence of flavonoids, urease, glycosides, lenoleic acid, polyphenols, beta Sitosterol, amino acids- glycine, alanine, cysteine, serine, isoflavones, genistein, isoferririn,cumesterol, psoralidin, galactosidase, glucosides and streptogenin 27. The seeds of Macrotyloma uniflorum contain extractable total phenolics and tannins. Dry heated samples were found to have considerable amounts of phenolics and tannins than in raw samples 28. Phenolic acids are isolated from the ethanolic extract of the seeds of Macrotyloma uniflorum by reversed phase HPLC. There were eight phenolic acids components and the most abundant was p-coumaric acid and p-hydroxy benzoic acid. The successive extracts of root, seeds of Macrotyloma uniflorum have revealed the presence of alkaloids, flavonoids, glucosides, lignins, phenols, saponins, tannins and sterols. Alkaloids are the lead molecules of curative importance from Macrotyloma species.

[Source 6 ]

Horsegram non-nutrient bioactive compounds

Pulses contain various molecules that can exert a broad range of biological actions in the human body, including both favourable and undesirable ones. The undesirable ones are to be considered as antinutrients or anti-nutritional factors (or compounds) 29. The antinutritional compounds found in pulse crops can be categorized as protein antinutritional compounds (ANCs) and non-protein ANCs 30. Non-protein antinutritional compounds (ANCs) include alkaloids 31, phytic acid, phenolic compounds such as tannins 32 and saponins 33. The presence of antinutritional factors, such as phenols, tannins, phytic acid and flatulence-causing oligosaccharides are now being considered as potential antioxidants. These antinutrients causes a number of positive health effects such as a decrease risk of intestinal diseases (gallstones, diverticulosis, constipation and colon cancer), coronary heart disease, prevention of dental caries and treatment of diabetes 34. Saponins and another common class of antinutrients compounds have been reported to show hypocholesterolemic as well as anticarcinogenic effects 35.

Anti-nutritional factors

Anti-nutritional factors reduce the bioavailability of nutrients 36. Horsegram flour also contains such factors such as trypsin inhibitor (9246±18 TIU/g), phytic acid (10.2±0.4mg/g), polyphenols (14.3±0.4mg GA/g) and oligosaccharides (26.8mg/g) 37. The consumption of horse gram as a human food is limited due to existence of high level of enzyme inhibitors, hemagglutinin activities, oligosaccharides, tannins, polyphenols and phytic acid compared to the other legumes 38. Conventional processing methods such as de-husking, germination, soaking, germination, steaming, fermentation, cooking, roasting and microwave heating have been shown to produce beneficial effects by decreasing the content of undesirable components which results in enhanced acceptability and nutritional quality in addition to optimal utilization of horse gram as human food 39: A legume to reckon with for health and nutrition.)), 40.

Horsegram plant is relatively high in iron, but the availability of the iron is reduced by the phylates, tannins and oxalic acid it contains. However these generally considered antinutritional compounds like phytic acid, phenols and tannins are now being measured as potential antioxidants having health promoting effects. The phytic acid has now been shown to possess rich antioxidant, anticarcinogenic and hypoglycaemic activities. Hence depending upon consumer preferences retaining or elimination of these compounds could be facilitated 41.

Table 6. Concentration of antinutritional factors in different milled fractions of horsegram (percent of dry matter)

Antinutritional factorCotyledon * Embryonic axe * Seed coat *
1. Phytic acid (mg/g)8.42 ± 0.41a3.81 ± 0.11b1.02 ± 0.09c
2. Flatulence factors (mg/g)
a. Raffinose 6.35 ± 0.26a2.86 ± 0.09b0.96 ± 0.03c
b. Stachyose 14.84 ± 0.91a6.38 ± 0.38b0.70 ± 0.05c
c. Verbascose 3.75 ± 0.26a1.38 ± 0.04b1.05 ± 0.04c
3. Enzyme inhibitors (units/g)
Buffer extract
a. Trypsin inhibitor activity 9,856 ± 16.1a2,018 ± 12.9b1,134 ± 8.2c
b. α-amylase inhibitor activity 56.9 ± 1.1a12.3 ± 0.4b4.1 ± 0.2c
Methanol extract
a. Trypsin inhibitor activity 2,474 ± 11.8c3,663 ± 13.1b10,434 ± 21.4a
b. α-amylase inhibitor activity 632.2 ± 2.4b639.5 ± 2.0b937.3 ± 2.7a

Footnote: * Results are mean ± standard deviation of triplicate determinations. Mean values bearing different letters (a, b, c) in the same row are significantly different on application of Duncan’s multiple-range test.

[Source 26 ]

Flatulence factors

Raffinose family oligosaccharides (raffinose, stachyose and verbascose) α-galactosyl derivates of sucrose, a low molecular weight causes accumulation of gas, discomfort, diarrhea, pain and cramps after digestion 42. The formation of flatus in human is due to absence of an enzyme α-galactosidase in the digestive tract 43. These oligosaccharides eventually fermented in the large bowel by the action of anaerobic bacteria of clostridia group to produce gases (CO2, H2, CH4, H2S and NH2) and short-chain fatty acids 44. However, these oligosaccharides can be used as sources of non-digestible carbohydrates, which not only promote several beneficial physiological effects but can also selectively stimulate the growth of the colon microflora and act as prebiotics 45. In fact, many prebiotic i.e. non-digestible food ingredient, stimulate the growth and/or activity of bacteria in the digestive system such as ingredients in various food products like soft drinks, cookies, cereals, candies and infant foods 46.

The major flatulence factors in horse gram are presented in Table 6. The cotyledon fractions contain higher concentrations of oligosaccharides (raffinose, stachyose, and verbascose), accounting for 39 % of the total soluble sugars. Among the oligosaccharides, stachyose is recorded in higher amounts in both cotyledon and embryonic axe fractions, whereas, verbascose was the major oligosacharide in seed coat fractions (1.05 mg/g) in horse gram) 26. However, substantial amounts of raffinose and verbascose were also found in cotyledon and embryonic axe fractions.

The presence of flatulence causing raffinose family oligosaccharides limits their biological value and acceptance as a regular food item 43. However, it is well-known fact that the balance of intestinal bacterial flora is important for human health, especially bifidobacterium, which is facilitated by galacto-oligosaccharides 47 and thus invigorate human health.

Phytic acid

Phytic acid or inositol hexaphosphate (IP6) is a simple ringed carbohydrate with six phosphate groups attached to each carbon (a bioactive sugar molecule) and a major form of phosphorylated inositol 48. Phytic acid exists in the form of free acid, phytate, or phytin and all of these forms are interchangeable 49. It is widely distribute in legume seeds and it accounts for about 78 % of the total phosphorus in pulses 50. The concentration of phytic acid in horse gram (see Table 6) revealed a significant quantity in embryonic axe fractions, whereas, most of it concentration located in the cotyledon fractions. Phytic acid is considered as an anti-nutrient because it inhibit the digestibility of proteins, ‘hard-to-cook’ 51 and also reduces the bioavaibility of minerals such as calcium, zinc, iron, and magnesium 52. On the other hand, it has potential antioxidant 53, anticarcinogenic 54, reduces the rate of cell proliferation, augmenting the immune response 55 and hypoglycemic or hypolipidemic 56 activities. Phytate may have a stronger ability to quench free radicals because of its metal-chelating ability, which renders the prooxidant metal iron unavailable to participate in the Fenton reaction and to catalyze hydroxyl radical formation in vitro 57. Thus, phytate may prevent oxidative damage, such as lipid peroxidation 58 and may thereby decrease the formation of atherosclerotic lesions.

Phenolic compounds

Phenolic compounds have a greatest beneficial interest on human health due to presence of antioxidant property, such as protection of oxidative damage 59. Natural polyphenols can range from simple molecules, such as phenolic acids, to highly polymerized compounds like tannins. Phenolic content of legumes varied in the range of 0.325–6.378 mgGAE (gallic acid equivalent)/g 60. Marathe et al. 60 reported that the horse gram is grouped under high phenolic acid content group (3.579 ± 0.072 mgGAE/g). The principal phenolic compound (Table 7) of horse gram seed are flavonols (flavanoids) such as quercetin, kaempferol, and myricetin, vanillic, ρ-hydroxybenzoic, and ferulic acids 61. The phenolic acids are a large family of secondary metabolites having either derivatives of benzoic acid (e.g. gallic, syringic and vanillic acid) or of cinnamic acid (e.g.caffeic, ferulic, sinapic and ρ -coumaric acid), which are commonly found as esters of caffeic and quinic acids 62 and are responsible for various beneficial effects in a multitude of diseases 63. Sundaram et al. 59 reported 0.101 g/100 g tannin in horse gram seed. Tannins are oligomeric, higher molecular weight polyphenolic compounds, occurring naturally in plants. Synthetic antioxidants are widely used to reduce oxidative damage, but due to safety and toxicity concern much attention has been focused on the use of natural antioxidants to protect the human body by free radicals 64.

Phenolic acids are believed to work synergistically to promote human health through a variety of different mechanisms such as impacting cellular processes associated with apoptosis, platelet aggregation, blood vessel dilation, enzyme activities associated with starch, protein, and/or lipid digestion, carcinogen activation, and detoxification 65. The phenolic compounds have also been associated with color, sensory qualities 66 and organoleptic properties (flavor, astringency, and hardness) of foods 67.

There is growing evidence that polyphenols found in plants have potential health benefits 68. Phenolic compound have antimicrobial 69, antimutagenic 70, antiviral 71, anticarcinogenic 72, anti-inflammatory (Dos Santos et al. 2006), antiproliferative and vasodilatory actions 73 and have potential preventive properties against cardiovascular diseases, hormonal related cancers 74. In addition, to their antioxidant properties, isoflavones and lignans exert a weak oestrogenic activity 75, which may be implicated in several mechanisms protecting the human body and also have been suggested to reduce the risks of cancer and to lower serum cholesterol 75. Some phenolic compounds, like the flavonoids, known to have antioxidant activities 76 and are used as antibiotics, anti-diarrheal, anti-ulcer and anti-inflammatory agents, and also for treatment of diseases such as hypertension, vascular fragility, allergies, and hypercholesterolemia 77. Furthermore, these flavonoids are known to possess antioxidant, anticancer, antiallergic and gastroprotective properties 26. The dietary antioxidants are capable of blocking neuronal death in vitro and many therapeutic properties of neurodegenerative diseases, including Alzheimer’s and Parkinson’s diseases 78. The antioxidant activity of dietary polyphenols is considered to be much greater than that of the essential vitamins 79. Hence, the evaluation and exploitation of phyto-nutrient compounds particularly phenolic acids, flavonoids and high molecular weight tannins of legumes assumed great significance 80.

Table 7. Phenolic compounds in different milled fractions of horse gram a

Horse gram fractions
CotyledonEmbryonic axeSeed coat
Concentration (μg/g on dry weight basis)
Flavonoids
 Quercetin9.7 ± 0.55c113.4 ± 6.0b129.5 ± 11.3a
 Kaempferol6.0 ± 0.25c67.4 ± 3.7b117.2 ± 10.5a
 Myricetin2.4 ± 0.07b32.9 ± 3.3a35.5 ± 5.2a
 Daidzein4.1 ± 0.08b22.2 ± 1.3a0.94 ± 0.03c
 Genisteinnd44.7 ± 3.22nd
Phenolic acids
Benzoic acid derivatives
 Gallic acid26.9 ± 1.3a19.8 ± 0.8b5.5 ± 0.06c
 Protocatechuic acid39.0 ± 2.0a11.8 ± 0.423.1 ± 1.2b
 ρ-hydroxybenzoic acid20.1 ± 0.8b13.1 ± 0.5c28.8 ± 1.4a
 Vanillic acid58.4 ± 3.3a53.2 ± 5.1a42.4 ± 3.6b
 Syringic acid18.4 ± 1.0andb4.5 ± 0.02b
 Subtotal162.897.9104.3
Cinnamic acid derivatives
 Caffeic acid88.9 ± 5.0a61.5 ± 4.9b10.6 ± 0.6c
 Chlorogenic acid160.8 ± 9.8a26.8 ± 2.1b22.5 ± 1.1 b,c
 Ferullic acid70.1 ± 5.1a31.4 ± 2.4c37.5 ± 2.5b
 Sinapic acid9.7 ± 0.09and3.7 ± 0.02b
 ρ-coumaric acid40.9 ± 3.2a21.4 ± 1.3c24.5 ± 1.8b
 Subtotal370.4141.198.8
 Total533.2239203.1

Footnotes: a Data are expressed as mean (standard deviation of three independent determinations. Means with the same letters (a, b, c) within the same row do not differ significantly. b Below detection limit.

Abbreviation: nd = not detected

[Source 61 ]

Proteinase or enzyme inhibitors

Horse gram habitually contains inhibitors of proteases that reduce the digestibility of dietary proteins. Protease inhibitors form irreversible trypsin enzyme and trypsin inhibitor complex in the intestine. These protease inhibitors resemble other Bowman-Birk protease inhibitors and characterized by low molecular weight, high disulfide content with low content of aromatic amino acids. They can bind as well as inhibit trypsin and chymotrypsin either independently or simultaneously 81. Trypsin inhibitor activity is significantly higher in horse gram flour (9246 TIU/g) as compare to chickpea (6452 TIU/g) and cowpea (6981 TIU/g) flour 37. In germinated seeds trypsin inhibitor activity is 16% less than that of un-germinated horse gram seeds (950 x 103 TIU/g seed) 82. As germination induces changes in the Bowman-Birk type proteinase inhibitors in both qualitative and quantitative ways, it facilitates protein hydrolysis for utilization in germination process 83. Trypsin inhibitors are thermo-labile and their inhibitory activity can be reduced noticeably by thermal treatment 84.

More recently Bowman-Birk inhibitors have been acknowledged to be helpful for human health, by their ability to suppress carcinogenesis 85 due to intrinsic ability of Bowman-Birk inhibitor to inhibit serine proteases which involved in carcinogenesis 86. The Bowman-Birk inhibitors have potential anti-inflammatory activity, against obesity and several degenerative and autoimmune diseases 80. Bowman-Birk inhibitor concentrate recently been used as treating ulcerative colitis 87 and multiple sclerosis 88. Horse gram having higher trypsin inhibitor activity (9,856 TIA/g) could be used as functional food ingredients similar to soybean Bowman-Birk inhibitor concentrate 89.

The major Bowman-Birk inhibitor associated with horsegram is HGI-III that has 76-amino acid single-chain poly peptide with two independent inhibitory domains directed toward trypsin and chymotrypsin 90. HGI-III, though a single polypeptide of low molecular weight (∼8,600 Da) and like other Bowman-Birk inhibitors it undergoes self-association and exists as a dimer in solution 91. In contrast to HGI-III, the three inhibitors of germinated horse gram seeds (HGGIs) are single polypeptides of 6,500–7,200 Da that exist as monomers and exhibit no such self-association 92. HGI-III like other legume Bowman-Birk inhibitors is stable at cooking temperatures 93 and also exhibits remarkable stability to high temperature (95 °C) as well as extremes of pH from 2 to 12 90. HGI-III contains seven inter-weaving disulfides and among these seven disulfides, four occur in the trypsin and three in the chymotrypsin reactive site domain, respectively 86. Proteinase inhibitors in horse gram (Table 6) revealed that buffer extracts showed higher proteinase inhibitors in cotyledon fractions than that of embryonic axe and seed coat fractions but methanol extracts showed higher proteinase inhibitors in seed coat fractions than that of cotyledon and embryonic axe fractions 26. Horse gram having higher trypsin inhibitor activity (TIA) (9,856 TIA/g) could be used as functional food ingredients, similar to soybean Bowman-Birk inhibitor concentrate 89.

Horsegram nutrient bioactive compounds

Protein and amino acid

Grain legumes are important sources of food proteins and often they represent a necessary supplement to other protein sources 30. Protein content in horse gram seed varies from 18.5 to 28.5 % 94. Seed fraction of legume consist of cotyledon, seed coat and embryonic axe, which represent on an average of 89, 10 and 1 % of the total seed weight, respectively 95. The protein content of milled fraction of horse gram (see Table 1) varies widely from 22.6 % in cotyledon to 9.1 % in seed coat fraction, whereas, embryonic axe contain higher protein than seed coat and lower than cotyledons 61.

Proteins are classified on basis of their solubility as water-soluble albumins, salt-soluble globulins, alcohol-soluble prolamins, and acid- or alkaline-soluble glutelins 96. Digestibility of raw and cooked legume protein varies from15–80 to 50–90 %, respectively, as compare to cereal seed protein (75–90 %) 97. The nutritional value of high protein foods are based on both protein quantity and quality.

In legume seeds, the major proteins are globulins (50–90 %) 96, glutelins (10–20 %) and albumins (10–20 %) 80. In horse gram, out of the total protein content, albumin-globulin protein fraction contributes from 75.27 to 78.76 %, while glutelin and residual protein varies from 9.93–17.52 to 6.96–11.30 %, respectively 98. It also has high lysine content, an essential amino acid (0.52 g g−1 of nitrogen) as compared to blackgram (0.40 g g−1 of N) and pigeonpea (0.48 g g−1 of N) 99. The other major amino acid found in horse gram seed are arginine, histidine, lysine, valine, leucine etc. but have primary limitation in methionine and tryptophan (sulfur-containing amino acids) 100.

Proteins and its peptides have been shown to inhibit the angiotensin-converting enzyme (ACE), antimicrobial activity, antioxidant activity, anti-carcinogenic activity, hypocholesterolemic effect, reduced serum triglycerides, increased lean muscle mass, protection against pathogens, regulation of blood glucose levels, and satiety effects 101. The branched-chain amino acids, leucine, isoleucine and valine participate in a variety of important biological functions in the brain, including those from tryptophan and tyrosine 102. There are many other types of protein found in legumes including various enzymes, protease inhibitors and lectins, which are collectively known as antinutritional compounds (ANCs).

Starch, soluble sugar and dietary fiber

The most abundant carbohydrates in legume seeds are starch and non-starch polysaccharides (dietary fiber), with smaller but significant amounts of oligosaccharides 103. Starch is considered as partly digestible 5 and those starches which are not digested in small intestine reaches the large intestine, where it is fermented by the colonic microflora and is known as resistant starch 104. Resistant starch, a non-digestible carbohydrate account 43.4 % of total carbohydrate in horse gram 5. Fermentation of resistant starch yields relatively high amounts of butyrate and it is believed to exert a protective effect against colorectal cancer 105. This butyrate may reduce the risk of malignant changes in cells 106, increase in fecal bulking 107 and lower fecal pH 108. Resistant starch enriched food have beneficial for management of diabetes due to reduced postprandial glycemic and insulinemic response 109. Horse gram contains both soluble and non-soluble fiber. Horsegram seeds contain more insoluble dietary fiber than kidney bean 110. Crude fiber content was higher in seed coat fractions than in embryonic axe and cotyledon fractions. Furthermore, seed coat fractions of legumes with high fiber and low protein may be useful in food product formulations to improve gastrointestinal health and satiety changes 61.

In human being, fibers primarily act on gastrointestinal tract, affecting different physiological effects like, alteration of the gastrointestinal transit time, satiety changes, influence on the levels of body cholesterol, after-meal serum glucose and insulin levels, flatulence and alteration in nutrient bioavailability 111. The main bioactive functions that have been attributed to dietary fibers are reduce constipation, modulation of blood glucose level 112, cholesterol reduction, prebiotic effects, prevention of certain cancers 113, cardiovascular diseases, diverticulosis, obesity 113 and lowers blood pressure 114. Similarly, Sharma and Kawatra 115 also reported that soluble fiber also decreases serum cholesterol, reducing the risk of heart attack and colon cancer. Insoluble dietary fiber is required for normal, lower intestinal function in humans 116.

What are uses of horsegram?

Various parts of the horse gram (Macrotyloma uniflorum) plant are used in medical systems such as Ayurveda, Siddha and Unani for thousands of years for various ailments both internally and externally 6.

The soup extract from horse gram called yusa, was consumed commonly during the Sutra period (c. 1500–800 BC) 117. Horse gram is widely grown for human food as a pulse and fodder crop for livestock, as well as green manure and medicinal crop 17. In rural areas, seeds of horsegram are consumed after parched followed by boiling or frying along with cooked rice, sorghum or pearl millet 118. Sprouted seeds, having high nutritional quality, are widely consumed by the indigenous tribal peoples 5. Even now, in addition to its nutritive value, the consumption of sprouted seed become increasingly popular due to the excellent source to reduce the risk of various diseases and exerting health promoting effects 119. In Indian traditional medicine, seeds of horse gram are used for treatment of urinary stones 120, urinary diseases and piles 121, regulate the abnormal menstrual cycle in women 95, act as astringent, tonic 122, and also used to treat calculus afflictions, corpulence, hiccups, and worms 123. Furthermore, the cooked liquor of the horse gram seeds with spices is considered to be a potential remedy for the common cold, throat infection, fever and the soup said to generate heat 124.

Different parts of the horse gram plants are used for the treatment of heart conditions, asthma, bronchitis, leucoderma, urinary discharges and for treatment of kidney stones 125. A literature survey showed that Dolichin A and B, pyroglutaminylglutamine along with some flavonoids were isolated from horse gram 126. The ethanolic seed extract of Macrotyloma uniflorum (Lam.) showed potential free radical scavengers (antioxidant) with significant scavenging activity of 64.01 % ± 1.78 at 500 μg/ml in Nitric Oxide radical Scavenging Assay, 74.42 % ± 2.37at 1,000 μg/ml in Hydroxyl method and 92.59 % ± 2.05at 250 μg/ml in Phosphomolybdate method as compared to that of standard 120. Moreover, phytochemical studies revealed that Kaempferal-3-O-β-D-glucoside, β-sitosterol,stigmasterol 127 and phenolic compounds were isolated from horse gram have the cytotoxicity and antimicrobial activities 62.

Horsegram potential health benefits

Antioxidant

The unprocessed horsegram seed is loaded in polyphenols, flavonoids and proteins, the major anti-oxidants which are also available in fruits and other food materials. The greater part of anti-oxidant properties is limited to the outer coat of seed and its removal would eliminate these properties. Singh, et al. 128 reported the in-vitro antioxidant activity of ethanolic seed extracts of horsegram (Macrotyloma uniflorum). As per Ravishankar, et al. 129 administration of horsegram extract to rabbits with high-fat diet induced oxidative stress, showed improvement in anti-oxidant enzymes such as superoxide dismutase, catalase and increased glutathione concentration.

Siddhuraju, et al. 130 revealed that among the various extracts, 70% acetone extracts of dry-heated samples of brown variety of horsegram as well as raw and dry-heated samples of black variety exhibited significantly higher hydroxyl radical-scavenging activity. Generally all extracts showed good antioxidant activity (53.3-73.1%) against the linoleic acid emulsion system but were significantly lower than the synthetic antioxidant, BHA (93.3%) 130.

Lowers high cholesterol

Kumar, et al. 131 demonstrated that horsegram (Macrotyloma uniflorum) extracts have strong activities against high blood cholesterol (hypercholesterolemia) and obesity. Antihypercholesterolemic effect of M. uniflorum extract is examined in rats by assessing its effects on food consumption, weight gain, serum lipid profile, serum glutamate oxaloacetate transamianse (SGOT), serum glutamate pyruvate transaminase (SGPT) and body fat 131. Researchers reported that the consumption of ethanol and water extract of the horsegram plant for 5 weeks resulted a significant decrease of total cholesterol (TC), triglycerides, low-density lipoprotein (LDL also called “bad” cholesterol), very low density lipoprotein (VLDL), serum glutamate oxaloacetate transamianse (SGOT) and serum glutamate pyruvate transaminase (SGPT) levels 6. There was a significant increase in high-density lipoprotein (HDL) (also called “good” cholesterol). They also discovered ethanol extract-treated group has shown a significantly higher fecal excretion of cholesterol level than those treated with water extract. Body weight of rats in the water extract-treated group was significantly lower than that in the ethanol extract-treated group.

Anti-bacterial activity

According to Kawsar, et al. 132, Ram, et al. 133 and Gupta, et al. 134 extracts from horse gram seeds had shown significant activity against Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa 132.

Antihelmintic activity

Horsegram seeds have anthelmintic activity which can be beneficial in eliminating worms 135. Philip, et al. 135 tested he alcohol extracts of M. uniflorumseeds for their anthelmintic activity. These extracts exhibited potent anthelmintic activity against Pheretima posthuma and its activity was comparable with that of the standard, albendazole 136.

Analgesics and anti-inflammatory effect

Giresha et al., assayed the aqueous extracts of horse gram (Macrotyloma uniflorum) coat and pulp by in-vitro method for inhibition of human secretory phospholipase A2 (sPLA2) as a function of anti-inflammatory activity. The extract effectively neutralized indirect hemolytic activity and showed similar potency in neutralizing the in vivos PLA2 induced mouse paw edema 137.

Anti-diabetic activity

Horse gram has slowly digestive starch which is considered tohave low postprandial glucose reaction upon its utilization by individuals experiencing diabetes 138. Gupta et al. 139, investigated the antidiabetic effect of α-amylase inhibitor isolated from horse gram seeds in streptozotocin- nicotinamide induced diabetic mice. They have determined the biochemical parameters such as serum total cholesterol, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels 139. Horse gram (Macrotyloma uniflorum) amylase inhibitor hindered the two mice pancreatic and human salivary α-amylases. Purwar, et al. 140, found that horsegram α-amylase inhibitor (MUAI) inhibited both the mouse pancreatic and human salivary α-amylase. MUAI reduced the serum glucose level in the treated diabetic mice. Histological findings revealed minimum pathological changes in the treated diabetic mice as compared to the diabetic control 139. Furthermore, it was discovered that the methanolic concentrate of the horse gram legumes alongside the methanolic concentrate of green tea leaves (at acombined portion of 200 and 100 mg/kg individually) was fundamentally successfulin decreasing serum glucose level and in treating streptozotocin in instigated diabetic hyperlipidemia in rats after seven weeks of treatment 141. Fasting glucose levels diminished altogether upon horse gram treatment (300 mg/kg body weight/day for 30 days) and the serum triglycerides and absolute cholesterol levels were likewise diminished fundamentally. In this manner, antidiabetic effects of horse gram were proposed, and it was prescribed to utilize horse gram as a day by day diet adjuvant against diabetes mellitus and related issues 142. In view of the suggestion made, including crude horse gram (not their sprouts) to the everyday diet of diabetic people might behelpful 143.

Anti-gallstone activity

Bigonia, et al. 144 found that horsegram seed exerted antilithogenic influence by decreasing he formation of lithogenic bile in mice. Both the methanolic and acetone extracts were capable of decreasing cholesterol hyper-secretion into bile and increasing the bile acid output. The maximum effect was found in the acetone extract as it decreased the papillary proliferation of gallbladder and hepatic fatty degeneration. Antioxidant property of polyphenol and tannin in acetone extract may provide its potential antilithogenic effect 144.

Anti-kidney stone activity

Horsegram (Macrotyloma uniflorum) was found to be effective in preventing the deposition of the stones in experimental rats. Chaitanya, et al. 145 reported the antiurolithiatic activity of aqueous and alcohol extracts of horsegram seed on ethylene glycol induced urolithiasis in albino rats. Das et al. 146 noticed an excessive urinary excretion of oxalate, calcium and phosphate was resulted after the feeding of ethylene glycol. As per their findings of Atodariya, et al. 147 and Bijarnia et al. 148, horsegram seeds are endowed with significant antiurolithiatic activity and the alcoholic extract of horsegram showed better anti urolithiatic activity than aqueous extract.

Anti-histamine activity

Suralkar et al. 149, screened the ethanolic extract of horsegram seeds for their anti-histaminic activity by using histamine induced contraction on goat tracheal chain preparation and histamine induced bronchoconstriction in Guinea pigs. Histamine induced contraction of isolated goat tracheal chain preparation was significantly subdued by the ethanolic extract of horsegram seeds. The guinea pigs were significantly protected against histamine induced bronchospasm as indicated by delay in the preconvulsivedyspnoea time following the exposure of histamine aerosol 149.

Diuretic activity

Ravishankar, et al. 150 explored the diuretic effect of ethanolic horsegram seed extracts in albino rats. The urine volume, Sodium, Potassium, Chloride and Bicarbonate contents were measured after the oral administration of extracts at doses of 200mg/kg and 400mg/kg. Diuretic effect was significant in experimental animals treated with of horsegram extracts compared to the control, Furosemide (5mg/kg) 150.

Hepatoprotective activity

Parmar, et al. discovered the significant hepatoprotactive properties of horsegram seeds against D-Galctosamine and paracetamol induced hepatotoxicity in rats 151.

Hemolytic activity

The 1-butanol horsegram extract showed the significant hemolytic activity by mouse erythrocytes. Kawsar, et al. 152 reported the presence of compounds such as methyl ester of hexadecanoic, ethyl ester of hexadecanoic acid mixture and n-hexadecanoic could be constituted a possible chemotaxonomic marker.

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