Common and lesser-known pulses from Northwestern Himalaya: A comparison study for quality traits

Legumes are the one of the richest and cheapest sources of proteins consequently an important part of the people’s diet in many countries. Among legumes, pulses are also good source of quality protein, amino acid, fatty acids, fibres, minerals and vitamins for the vegetarians, which constitute major population of the India (Singh, 2017). Recent researches have associated the consumption of pulses with a decreased risk for a variety of chronic diseases such as cancer, obesity, diabetes and cardiovascular diseases (Singh, 2017). Pulse grains are rich source of secondary metabolites (including lipophilic and hydrophilic compounds) possessing free radical scavenging and antioxidant capacity (Pal et al., 2017). Pulses contain a wide variety of non-nutritive bioactive components such as enzyme inhibitors, phytic acid and tannins. These non-nutritive bioactive compounds considered as anti-nutrients factors because of their activity to reduce protein availability, digestibility (Chung et al., 1998) and mineral bioavailability (Mathers, 2002). Phytic acid exhibits antioxidant activity and protects DNA damage (Phillippy, 2003). Trypsin inhibitors are proteins of low molecular weight capable of binding and inactivating some of the key digestive enzyme like trypsin whereas phytic acid has a strong binding affinity to important minerals. The binding of phytic acid with iron is more complex, although there certainly is a strong binding affinity bioavailability of minerals by forming compounds with minerals (Reddy et al., 1984). Tannins, forms complexes with proteins, which are reported to be responsible for low protein digestibility and decreased amino acid availability (Adsule and Kadam, 1989). The major pulses cultivated in India are kidney bean (Phaseolus vulgaris), chickpea (Cicer arietinum), cowpea (Vigna unguiculata) and lentil (Lens culinaris), consumed in various forms. Whereas some of the lesser known legume like rice bean (Vigna umbellata), horse gram [Macrotyloma uniflorum (Lam.) Verdc.] and black seed soybean [Glycine max (L.) Merrill] have been reported to be less accepted either due to their pungency or their antinutrient content. Rice bean [Vigna umbellata (Thunb.)] is a multipurpose grain legume crop mainly cultivated for food and fodder specifically by the resource poor farmers in the marginal areas of South and South East Asia. It is mainly cultivated in India, Nepal, Bangladesh, Thailand, Vietnam and China (Gautam et al., 2007). Horsegram is cultivated as a food, forage and green manure crop in India, Eastern and Southern Africa, Myanmar, Malaysia, West Indies and Australia. Black seeded soybean is a traditionally grown major food legume in Uttarakhand hills.
       
There are many studies that reported bioactive components and health benefits in terms of the antioxidant activities in common legumes and in some underutilized or lesser known legumes but available information on the comparison of well known pulses and these three lesser known pulses [rice bean, horse gram, soybean (black)] in respect of their phytochemicals (nutritional and antinutritional), antioxidant properties and fatty acid composition is rather scarce. Therefore,  the present study was undertaken for the simultaneous analysis for phytochemicals (nutritional and antinutritional), antioxidant properties and fatty acid composition in eight pulses comprising of five common pulses namely, rajma (kidney beans), cowpea (white), cowpea (black), lentil, chickpea and three lesser known pulses [rice bean, horse gram, soybean (black)].

Plant materials
 
The present study was conducted in the division of crop improvement at ICAR-Vivekananda Institute of Hill Agriculture, Almora, Uttarakhand. The pulse grain samples of rajma (kidney beans), cowpea (white), cowpea (black), lentil, chickpea, rice bean, horse gram, soybean (black) were obtained from pulse breeding programme of ICAR-Vivekananda Institute of Hill Agriculture, Almora, Uttarakhand, India. Rice bean, cowpea (white) and cowpea (black) were the collection from the Kalsi, Distt. Dehradun (UK) whereas the rajma (kidney beans), lentil, chickpea, horse gram and black seeded soybean were the released varieties from ICAR-VPKAS, Almora. Samples were cleaned to remove foreign material and damaged seeds prior to further analysis.
 
Chemical analysis
 
Total nitrogen amount was detremined by Kjeldhal Process (AOAC 2005) based on the theory that a plant protein contains 16 g of the protein in 100 g of total nitrogen. Total protein content was calculated using the formula, protein = nitrogen×6.25. Total sugar content (TSS) was determined spectrophotometrically while the starch content was estimated by anthrone method (Hedge and Hofreiter, 1962). Gravimetric method was used for determination of total fat content as described by Bligh and Dyer (1959). For tryptophan (Try) determination, the samples were first defatted by ether in soxlet apparatus and then the content was quantified in the samples by the methods as described by Dalby and Tsai (1975). Colorimetrically method described by Association of Official Analytical Chemists (AOAC) was used for quantify the tannin content (AOAC, 2005). For phytic acid (PA) contents determination, the samples were first defatted by ether and then the phytic acid was quantified in the samples by the methods as described by Haug and Lantzsch (1983). Calibration curve using phytate phosphorus salt of the range of 10–50 µg was used to calculate the content. Trypsin inhibitor activity (TIA) was determined colorimetrically using a spectrophotometer by the method as described by the (Smith et al., 1980).
 
Fatty acid profile
 
Oil from flour was extracted in hexane on soxhlet apparatus (Extraction unit, E-816, Buchi). Briefly, One g of dry and homogenized samples, 10 g sea sand and 200 ml petroleum benzine were extracted by Soxhlet for 7 hours. Then, the solvent was evaporated at 35°C using a rotary evaporator. Finally, the lipid content of samples was determined gravimetrically. Methyl esters were obtained by a two-step catalytic process according to the slightly modified method of Ghadge and Raheman (2005). Briefly, Oil (100-150 mg) was treated with 2% sulphuric acid in methanol (5ml) for 2 hr at 60°C. After the reaction, the mixture was allowed to settle for an hour and methanol-water mixture that separated at the top was removed. The second step product at the bottom was trans-esterified using 2 ml of 13% methanolic KOH for 30 minutes at 55°C. The organic phase was extracted with hexane and washed with water till neutral pH. The hexane was dried over anhydrous sodium sulphate and concentrated with nitrogen to get methyl esters.
       
Fatty acid composition was determined using an Agilent 7860A gas chromatograph (GC) equipped with a flame ionization detector (FID) and an auto sampler. Peak separation was performed on a DB-225 capillary column (Diameter-250 μm, Length-30 m, film thickness-0.25 μm) from Agilent Technologies. The carrier gas was nitrogen set to a constant gas flow of 1.2 ml/min at 160°C initial temperature. One micro liter of sample was injected at a 20:1 split ratio into the column with the following temperature conditions: 160°C for 2 min; raised from 150 to 220°C at 6°C /min. Both inlet and detector were set to 230°C. Fatty acid composition was determined by identifying and calculating relative peak areas percent by GC post run analysis EZChrom elite compact software.

HPLC-PAD analysis
 
For high performance liquid chromatography with pulsed amperometric detection (HPLC-PAD) fine powders of samples (1.0 g) were extracted in 85% methanol at 35°C for 12 h and filtered through Whatman filter paper No. 1. The extract solutions stored in amber bottles at 4°C served as the working solution (10 mg/ml) for determination of gallic acid and antioxidant activities. All samples were filtered through a 0.22 µm cellulose acetate filter (Axiva Sichem Biotech, India) before injection. The chromatographic system is provided by a 996 photodiode-array detector (Waters, Milford, MA, USA) and a column spherisorb ODS2 C18 (300×3.9mm). The mobile phase was methanol: acetonitrile: water (60:20:20 v/v/v). Gallic acid was detected at 280 nm by the maintaining the flow rate and injection volume were 1.0 ml/min and 10 µl, respectively. The chromatographic peaks of the analytes were confirmed by comparing their retention time and UV spectra with those of the reference standards. The standard gallic acid was procured from Sigma, USA. Methanol, acetonitrile and water were HPLC grade.
 
Determination of anti-oxidative properties
 
Scavenging effects on 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2-azobis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) free radicals by pulses methanolic extract was measured following standard methods of Brand-Williams et al., (1995) and Arnao et al., (2001), respectively. DPPH and ABTS radical scavenging activity was expressed in per cent inhibition. The total antioxidant activity (TA) of pulses methanolic extract was estimated using the phosphomolybdenum method of Prieto et al., (1999). The ferric reducing antioxidant power (FRAP) was assayed following the method of Benzie and Strain (1996). Total antioxidant activity and ferric reducing antioxidant power were expressed as µM trolox equivalent/gram dry weight (µM TE/g DW).
 
Statistical analysis
 
The analysis was carried out in three replicates for all determinations. An analysis of variance (ANOVA) were performed. significance of each group was verified with one-way analysis of variance followed by Duncan’s multiple range test (p<0.05). The results are presented as mean±standard deviation of the triplicate analyses of each measurement. A principal component analysis (PCA) was also conducted to generate a map of the quality and antioxidant characteristics of all fruit samples and also to determine the correlation between each quality characteristic. All statistical analyses were performed using XLSTAT-Pro 7.5  (version 2013, Addinsoft, Paris, France).

Nutritional characteristics
 
Nutrient composition of rajma (kidney beans), cowpea (white), cowpea (black), lentil, chick pea, rice bean, horse gram and soybean (black) flours was depicted in Table 1.The protein content varied from 17.61% (rice bean) to 40.84% (soybean). Significantly, higher amount of protein was recorded in black seed soybean than other pulses. The protein content of studied pulses is in line with Wallace et al., (2016) for pigeon peas, soybeans and some varieties of cowpea.
 

       
Total lipid content in legume seeds ranged from 1.20 to 7.30% (Table1). Concentration was found highest in black seeded soybean (7.30%), while the lowest was in rice bean (1.20%).  More lipids in black seeded soybean flour might be disadvantageous in terms of the shelf life and keeping qualities. However, this is an important property of soybean flour, enabling its wide utilization in food products. Higher fat in soybean (black) flour also enhances its ability to absorb and retain oil, improves binding of the structure, improves mouth feel and reduces moisture and fat losses of food products (Pal et al., 2016). The lower lipid content in rice bean and horse gram flour may be utilized as ingredients in weight restriction diets.
       
Cowpea (white) flour possessed the highest (24.55%) amount of total soluble sugars, followed by cowpea (black). Among the flours, total soluble sugars (8.92%) were lowest in soybean (black). These values are in accordance to the values reported for chickpea (href="#costa_2006">Costa et al., 2006) and horse gram (Sreerama et al., 2008). Cowpea (black), lentil, chickpea, horse gram, rajma (kidney beans), rice bean showed intermediate values. Wallace et al., (1995) also reported intermediate carbohydrate contents for these pulses. Total starch content of the eight legumes are shown in Table 1. Total starch (TS) ranged between 5.55 and 29.57% with minimum in soybean (black) and maximum in lentil, respectively. The lowest TS contents corresponded to soybean (black), assumes as a variant of soybean, most widely consumed legumes world-wide whereas all other pulses possessed the TS values in a range of 21.69-29.57%. Tryptophan being important essential amino acids is involved in various metabolic process and its deficiency resulted in various chronic diseases. All the pulses showed the presence of tryptophan and soybean (black) contained the highest (0.47%) amount of tryptophan whereas the lowest (0.22%) was recorded in lentil. Soybean (black) is native to the Uttarakhand and widely consumed in the northern part of India.
 
Anti-nutritional characteristics
 
Different antinutritional components may decrease protein digestibility, e.g. factors such as trypsin inhibitors, phytic acid and tannins (Usman et al., 2018).  Presence of high level of phytic acid lowers the availability of many essential minerals (Ghavidel and Prakash, 2006). The highest phytic acid content was recorded in rice bean (13.21%) followed by horse gram (12.54%) and the lowest (3.28%) content was recorded in soybean (black) (Table 2). These findings are in accordance with the report of Rasha et al., (2011).
 

       
Tannins are secondary plant metabolites distributed ubiquitously within legumes and have a property of binding to protein to form reversible and irreversible complexes due to the existence of a number of phenolic hydroxyl groups. Tannins ranged between 1.94 and 5.41 g/kg in cowpea (white) and lentil, respectively. Pal et al., (2017) also reported similar tannins content in pulses. They are main anti-nutritional factors in lentil. The cultivars without tannins, commonly known as zero-tannin or tannin-free lentil, were found to have a wide application for human. Protease inhibitors are the most commonly encountered class of antinutritional factors of plant origin. Trypsin inhibitors have the ability to inhibit the activity of proteolytic enzymes within the gastrointestinal tract of animals (Chunmei et al., 2010). Cowpea (white) was the pulse with the highest amounts of trypsin inhibitor, up to 36.13 mg/g followed by cowpea (black). Rice bean and chickpea showed same amount of trypsin inhibitor, about 28.27 mg/g, while the lowest value was recorded in soybean (black), with only 10.37 mg/g. Manzoor et al., (2016) also reported trypsin inhibitor content within these range of values.
 
Antioxidant metabolite and activities
 
Gallic acid content in different pulses was evaluated by HPLC and shown in Fig 1. Results were expressed as mg of gallic acid per gram of samples. It was found that horse gram had the highest content of gallic acid (10.80 mg GA/g), followed by lentil (10.50 mg GA/g) while lowest gallic acid content (4.75 mg GA/g) was recorded in cowpea (white). In the  present  study,  gallic acid content varied from 4.75 to 10.80 mg GA/g (Table 3). The similar result was also reported by many workers (Xu and Chang, 2007; Campos-Vega et al., 2010). The content of phenolic compound (gallic acid) could be used as an important indicator of antioxidant capacity. Several reports have convincingly shown a close relationship between antioxidant activity and phenolic contents (Pal et al., 2016).
 

 

       
The radical scavenging activity (RSA) of pulses was tested against the DPPH and ABTS. RSA varied from 21.52-50.57% for DPPH and 23.61-59.33% for ABTS and the results were similar to that reported by Xu and Chang (2007) and Campos-Vega et al., (2010). DPPH and ABTS free radical inhibition was higher for horse gram, rice bean and lentil in comparison to other pulses. Overall, it could be concluded that between pulses, the horse gram, rice bean and lentil had a better antioxidant activity in terms of scavenging activity on the DPPH and ABTS free radical. Due to presence of radical scavenging activity, consumption of horse gram, rice bean and lentil might be beneficial to protect human body against oxidative damage (Campos-Vega et al., 2010).
       
The phosphomolybdenum method of antioxidant activity usually detects antioxidants such as ascorbic acid, some phenolics, a-tocopherol, and carotenoids (Prieto et al., 1999). Total antioxidant activity was significantly higher in lentil (22.33 µM TE/g) followed by horse gram (20.04 µM TE/g) while rajma (kidney beans) and chickpea showed significantly lower total antioxidant activity i.e. 11.68 and 11.49 µM TE/g, respectively. FRAP value of the studied pulses were shown in Table 3. FRAP value was found significantly higher in lentil (178.66 µM TE/g DW) followed by horse gram (163.26 µM TE/g DW) while rajma (kidney beans), cowpea (white) and chick pea showed significantly lower total antioxidant activity i.e. 49.48, 55.42 and 44.52 µM TE/g DW, respectively. Results of present study were in agreement with the reports by other workers (Karthiga and Dorothy, 2013; Pal et al., 2016). This study could provide valuable information that these pulses are excellent source of antioxidants in human diet especially to the low-income community.
       
The fatty acid composition of the total seed lipids of pulses are given in Table 4. Fatty acid profiles of all studied pulses revealed that they are good source of the nutritionally essential linoleic and oleic acids. Linoleic (18:2 omega-6) and linolenic acids (18:3 omega-3) are the most important essential fatty acids required for growth, physiological functions and maintenance. All the presently investigated seed lipids are rich in unsaturated fatty acids. These values are nutritionally desirable. These results were comparable to the findings of Campos-Vega et al., (2010). Significant variation in fatty acid composition was observed among the eight different pulses. Linoleic (18:2 omega-6) was the major unsaturated fatty acids in all pulses (Table 4). Chick pea showed a significantly higher amount of linoleic acid (60.66 g/100g) than the other pulses, while rajma (kidney beans), lentil and soybean (black) contained the lower amount of linoleic acid i.e. 29.41, 32.08 and 32.36 g/100g, respectively. Lentil showed a significantly higher amount of oleic acid (41.27 g/100g) than the other pulses, while soybean (black) contained the lowest amount of oleic acid (18.26 g/100g). Oleic-to-linoleic ratio (O/L) is indicator of oil stability and shelf life in oil from legume seeds such as peanut oil (Alajaji and El-Adawy, 2006). Lentil showed the highest oleic-to-linoleic ratio (1.28) hence, it may have better oil stability and shelf life. The pulse seed samples contained palmitic acid (10.40-18.05%), stearic acid (1.55-5.37%) and linolenic acids (2.88-35.32%). The fatty acid composition of these pulses are in agreement with the results of previous investigation (Campos-Vega et al., 2010).
 
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Multivariate analysis
 
Principal component analysis (PCA) is statistical technique, to find out inter-relationships between the different variables (Mishra et al., 2013). In the present study, multifactorial comparisons using principal component analysis clearly indicated correlation between original variables (nutritional, antinutritional and antioxidant activities related factor) in a smaller number of underlying variables (principal component) in order to reveal the inter-relationships between the different variables and to find the optimum number of extracted principal components. The first principal component (PC) always describes sample variation and the following PC successively explains smaller parts of the original variance. This means that correlated variables are explained by the same PC and less correlated variables by different PC. The principal component analysis (PCA) and their correlation are shown in Fig 2.
 

       
The first factor F1 represents 38.01% of variability, while the second factor F2 represents 32.83% of variability among the data. Maximam biochemical parameters were occupied on the right side of the biplot and among the parameters the starch, total soluble sugars, linoleic acid, phytic acid, trypsin inhibitor activity and palmitic acid were observed on the right upper side of the biplot with high positive loading for both factors I and II, while stearic acid, oleic acid, DPPH, ABTS scavenging activity and O/L ratio were grouped together with slightly lesser positive loadings on the right side of the biplot. This suggests that oleic acid, DPPH, ABTS, O/L ratio, total antioxidant activity, ferric reducing antioxidant power, gallic acid and tannins are having positive correlation.
       
Trypsin inhibitor has negative correlation with antioxidant metabolites (gallic acid and tannins) and antioxidant activities while phytic acid has negative correlation with protein, total lipid, linolenic acid and tryptophan. Pal et al., (2016) also reported that the PCA analysis showed positive correlations among the total phenol, DPPH, FRAP and total antioxidant activity. The results also showed that horse gram and lentil contained higher gallic acid, DPPH and ABTS activity, O/L ratio, total antioxidant activity, ferric reducing antioxidant power, TPP (gallic acid), while black soybean shown with total lipid and protein content. Cowpea and chickpea showed significantly higher trypsin inhibitor activity compared to others pulses.

Pulses make a major share of the human diet in many locations of the world and play a significant role in the human nutrition, especially as source of protein, vitamins, minerals, dietary fibre and folic acid. Besides these important nutrients, the pulse grains also contain certain biologically active components including enzyme inhibitors, phytates, tannins and phenolic compounds. The present study showed that lesser-known legumes from North-Western Himalaya viz. horse gram, black seeded soybean and rice bean are the rich source of many nutritional compounds, excellent nutritional composition and unique medicinal significance. Lowest content of phytic acid and trypsin inhibitor were recorded in black seeded soybean. Results showed that horse gram and lentil had higher free radical scavenging and antioxidant activities. The fatty acid profiles revealed that, the studied pulse oil contained higher concentration of unsaturated fatty acids. Studied lesser-known pulses are comparable in nutritional quality with commonly grown pulses and these pulses may acts as a potential source of human nutrition.

The authors are grateful to Indian Council of Agricultural Research (ICAR), for financial support to carry out this work at Vivekanand Parvatiya Krishi Anusandhan Sansthan (VPKAS), Almora (Uttarakhand) 263601.