Metabolite profiling and protein quantification to a large library of 96 horsegram (Macrotyloma uniflorum) germplasm.

The present study was framed for the assessment of metabolic diversity of 96 diverse horsegram lines derived from 700 germplasm accessions of Pan India. The nutritional component of horsegram germplasm was studied using nuclear magnetic resonance spectroscopy (1H NMR) and micro-Kjeldal method. Differential range of protein content was found 13-40% in the selected panel. The related wild species Macrotyloma sar-gharwalensis contained highest protein content (40%), and the lowest protein content (13%) was found in IC-120837 and TCR-1439. Seed based 1H NMR spectroscopy of horsegram discovered 45 different metabolites (17 amino acids, 7 flavonoids, 10 organic acids/phenolic acids/sugar alcohols/steroids, 7 carbohydrades/sugars, and 4 vitamins). The number of total metabolites quantified among diverse horsegram panel ranged from 25-44. The lowest metabolites number was 26 in PLKU-38, while, IC-280031 and IC-139356 lines showed the presence of highest number of metabolites (44). Lines IC-280031 and IC-139356 were found most nutritive amongst selected panel of horsegram germplasm by containing maximum number of quantifiable metabolites during 1H NMR spectroscopy study. Further, the NMR based data of 96 germplasms was subjected for statistical analysis (PCA, Matrix plot, stacked charts) that revealed the similarities and variations among the whole germplasm. The methionine, sucrose, maltose, riboflavin and myricetin were observed as differential chemo-markers that help to differentiate the horsegram lines of selected panel. This information will further aid in the selection of nutritionally efficient cultivars for proteomics and genomics studies and these lines can be used as nutraceutical food for the prevention of various health ailments including kidney stone.

Nuclear magnetic resonance-based chemical signature and nutritional discrimination of Macrotyloma uniflorum accessions from the alpine Himalayas.

BACKGROUND: Macrotyloma uniflorum (horsegram) is cultivated in the alpine Himalayas for its nutritional benefits. The chemical diversity of M. uniflorum has been explored by several research groups, but none has reported the chemical signature to monitor the identity and quality. To overcome quality control problems and provide more accurate chemotype information, proton nuclear magnetic resonance (1 H-NMR)-based chemical signature and tentative metabotype discrimination was studied. RESULTS: Metabolites were identified by characteristic chemical shifts and their correlations in one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) experiments. The identifications were confirmed by reference standards, with in-house databases, and with public databases. From the analysis of seed samples from eight locations, NMR detected 46 diversified metabolites, including 18 amino acids, nine carbohydrates, three vitamins, seven flavonoids, and nine miscellaneous molecules (fatty acids/choline/phenolics/organic acids/sterols). These metabolites generated a single snapshot chemical signature for M. uniflorum. Greater content of identified metabolites was found at high elevations of 1829 m (55.78 g kg-1 ) followed by 1000-1150 m (19.30-17.23 g kg-1 ) and 1150-1450 m (7.33-13.41 g kg-1 ). Principal component analysis of samples revealed that chemotypes grouped into three distinct clusters. 'Cluster-3' was positively correlated with metabolites and found the best nutritionally enriched chemotype. CONCLUSIONS: A proton nuclear magnetic resonance-based M. uniflorum chemical signature was generated for the first time. The environmental conditions of 1829 m were most suitable for obtaining nutritionally enriched M. uniflorum crops. Overall, this information could help in the conservation, quality control, product development, and agriculture inventions of M. uniflorum. © 2021 Society of Chemical Industry.

Comprehensive nutritional profiling and activity directed identification of lead antioxidant, antilithiatic agent from Macrotyloma uniflorum (Lam.) Verdc.

Macrotyloma uniflorum (Lam.) Verdc. (family Fabaceae) is traditionally important pulse used for kidney stone degradation, cold,cough, asthma and in bronchial issues. It is well reported for the antilithiatic activity and nutritional profiling but no attempt has been made to find out the mechanism and nutrient responsible for the activity in its seed decoction. Therefore, lead high altitude variety responsible for the highest nutrient content and antilithiatic activity was selected for the study. Boiled spray dried extract (M1), lead fractionation (Spray dried residual water fraction: M2), and crude powder extract (M3) were nutritionally profiled using proximate composition analysis, Atomic absorption spectroscopy (minerals and trace elements), Ion exchange chromatography (carbohydrate analysis) and NMR spectroscopy (polar and non polar metabolite analysis). These extracts were also screened for the antilithiatic activity and found significantly important. M1 showed more activity than M3. Further, boiled extract was fractionated with different polarity of solvents (hexane, chloroform, n-butanol, residual water extract). All fractions and reference standard (cystone) were screened for the antilithiatic activity. M2 showed highest activity followed by ethyl acteate while hexane and chloroform did not show any activity in comparison to standards. Thus, lead fraction and market reference standard (cystone) were validated for the presence of nutritional components. Moreover, the selected nutritional components were individually and in combinations screened for the activity which revealed that kidney stone degradation was due to the synergetic action of components than the individual. The results clearly pave a way that Macrotyloma uniflorum is best for combating with malnutrition and kidney stone related problems.

Assessment of calcium oxalate crystal inhibition potential, antioxidant activity and amino acid profiling in horse gram (Macrotyloma uniflorum): high altitude farmer's varieties.

Seeds of Macrotyloma uniflorum collected from different Himalayan regions of Himachal Pradesh, India were screened for inhibition potential of calcium oxalate crystals along with their antioxidant and amino acid profiling. Aqueous, ethanol, 50% ethanol extracts were tested using agar-gel overlay, nucleation, and artificial urine methods in comparison to marketed products. The aqueous extract of Sundernagar and Rampur seeds showed highest inhibition potential (9.0 ± 0.81 mm) than alcohol and hydroalcohol. Sundernagar seeds (aqueous extract) also showed maximum inhibition (45 ± 1.2 and 24 ± 0.88%) in nucleation and artificial urine assay, respectively. The Tris-buffer (pH. 8.0) extract of dry seeds showed more inhibition with low protein than high protein germinated seeds. The findings revealed that horse gram protein is not alone responsible for CaOx-crystals inhibition but its secondary metabolites may also contribute. Among all samples, buffer and aqueous extracts showed maximum activity, followed by hydroalcohol and alcohol. Further, TLC and UPLC method were used to monitor the presence of amino acids in aqueous and Cystone extracts. Sundernagar seeds contain 11 amino acids with additional arginine, aspargine, and methionine. Moreover, higher antioxidant potential was observed in seeds of high altitude, which relates the factors of altitudinal variation in secondary metabolism of plants. Therefore, this study will help in the development of economical and more reliable plant based nutraceuticals for kidney stone problems and related health issues.