Nutritional characterization of an underutilized legume Entada rheedii Spreng. seeds and validation of its folklore uses

Abstract Entada rheedii seeds are a rich source of protein (23.99% ± 0.07), starch (42.04% ± 0.05) and potassium (1670.15 ppm ± 116.732). HPTLC–UV analysis (seeds) reveals galactose in considerable amount, that is, 2.60% ± 0.008. Additionally, the species is low in fat and anti-nutrient metabolites like tannin. Interestingly, the proximates in Entada seeds was found comparable with the commonly consumed legumes like cowpea, green gram, and so on. The species exhibits promising anti-radical, anti-inflammatory and anti-diabetic activity. Data advocates the folklore use of E. rheedii seeds and can be a promising alternative source of dietary nutrition, fortified with medicinal value. Standardization and validation of scientific knowledge will bring such underutilized, low-cost legumes into daily dietary intake and are a promising source for Protein-Energy-Malnutrition. Graphical Abstract


Introduction
Entada rheedii Spreng.(Fabaceae) is a perennial liana mainly found in the Eastern and Western Ghats of India and in the humid forests of Andaman Island (Brink and Achigan-Dako 2012).It is traditionally used for diarrhea, jaundice and mumps in Karnataka and Orissa states of India (Nzowa et al. 2010).The seeds are a rich source of carbohydrates and amino acids (Sugimoto et al. 2012) and also contain therapeutically active metabolites like glutamic acid, thioamides, saponins, phenyl propanoid glycosides, N-sulfonyl-L-tryptophan, 3-(N-sulfonylindolyl)-D-lactic acid, and 1-O-methylglucopyranoside (Okba et al. 2013).The seeds has alexiteric, febrifuge, narcotic, emetic, anti-periodic activity and is effective in rheumatoid arthritis and cerebral hemorrhage (Nzowa et al. 2010).
The use of E. rheedii seed as food is localized in its native place and need substantial popularization, with scientifically validated data for its commercial utilization.This will address the demand of cheaper and sustainable sources of nutrition, due to inclination of world population towards vegetarian diet.Therefore, the present study validates the traditional claims of Entada seeds for nutritional and medicinal benefits.

Botanical characterization of E. rheedii Spreng. seeds
The pharmacognostic standards of E. rheedii seeds were evaluated to regulate the quality of plant material.The anatomy of seed was done to reveal the cellular details (Figure S1).Transverse section of a seed showed an outermost, thick cuticle that covered the epidermal layer.Epidermis was made of radially elongated palisade cells, compactly associated with each other.This was followed by hypodermis, which consisted of sclerenchymatous cells without intercellular spaces.These observations are well corroborated with the previous study (Okba et al. 2013).The presence of bone-shaped cells in the hypodermal region was followed by hypodermal region which consists of thin-walled, flattened parenchymatous cells, upto the epidermal layer of cotyledon.The cells were filled with abundant starch granules, near to the peripheral region.Powder microscopy revealed the presence of creamy white seed kernels with brown coloured seed coat, having a characteristic odour.Powdered micrographs of whole seed revealed the fragmented structure of palisade tissue which forms hypodermis of the seed coat together with fractured sclereid cells, a bunch of fragmented parenchymatous cells filled with spherical shaped starch granules.The botanical studies will serve as a tool to identify specific cellular characters required for authentication of plant sample.The powdered plant material is more prone to adulteration (as it loses its morphological identity) and in such cases the identity of species can be preliminarily checked through the basic tool of powder microscopy (Alamgir 2017).
Physico and phytochemical parameters of seeds were analyzed and expressed (Table S1), the water-soluble extractive value was highest, that is, 14.48% ± 0.08 followed by alcohol and hexane soluble extractive value.Swelling and foaming index was 3.5 mL ± 0.131 and 125.63 mL ± 0.20, indicating that seeds are rich in mucilaginous substances and saponin(s).The total phenolic, flavonoid and tannin content was 2.01% ± 0.04, 1.26% ± 0.02 and 4.19% ± 0.06, respectively.Similar observations were recorded earlier (Siddhuraju et al. 2002) indicating that the phenolic acid was 2.55%.The phenolics, flavonoids and tannins are considered as anti-nutritional compounds because they obstruct protein metabolism (Siddhuraju et al. 2002) and seed with low anti-nutrient like Entada is a promising source of dietary protein.

Simultaneous quantification of galactose and sucrose
The identification of sucrose and galactose was done by comparing the UV absorption spectra and R f of markers with that in the sample.The markers were found at the R f 0.26 ± 0.03 and 0.33 ± 0.05 (Figure S4), respectively.Quantification of markers was based on regression equation of area versus concentration of standard markers and results were expressed in per cent dry weight basis (Table S2).Seeds of E. rheedii were found rich in galactose (2.61% ± 0.008).Therapeutically, galactose plays an important role in the functioning of brain, removing neurotoxic substances from the brain of patients suffering from hepatic encephalopathy (Waisbren et al. 2012;Coelho et al. 2015).Consumption of galactose rich diet may serve as an alternative source of energy as it metabolize into glucose and is transported to the brain.This kind of specialized diets would be beneficial for the patients suffering from dementia where glucose transportation is decreased (Berry et al. 1995).

of developed HPTLC method
The calibration curve at five variable dilutions of 0.2-1 µg/spot for both the markers was found linear, having statistically significant regression coefficient (R 2 ) of 0.994 and 0.996 for sucrose and galactose.Limit of detection (LOD) and limit of quantitation (LOQ) were calculated by the ratio of standard deviation to slope and is within the limit of acceptance (Table S3).The reproducibility of the developed method at repeated (n = 5) analysis of standard at a single level of 1 mg/ml, RSD (%) is in accordance with ICH guidelines, that is, found less than 5% (Table S4).An average recovery of 100.29% for sucrose and 98.44% for galactose was recorded (Table S5) and is within the acceptable limit.

Validation of bioactivity through in-vitro assays
The anti-radical activity of test extract was evaluated through DPPH radical scavenging and antioxidant potential assay.In DPPH assay, IC 50 value was 95 µg/mL ± 0.25 whereas in antioxidant potential assay, the IC 50 value was 137.60 µg/ml± 1.38 as compared to the standard, that is, IC 50 6.12 µg/mL ± 0.005) (Figure S5).In a previous study, IC 50 value of antioxidant activity of E. rheedii seeds was found 281.34, 555.22, 279.79, 233.72, and 321.88 µg/mL in methanolic, petroleum ether, chloroform, ethyl acetate, and water extract respectively (Sufian et al. 2015).Plants having high anti-inflammatory activity might contribute to lower the risk of various lives -threatening diseases like cancer, cardiovascular disease (CVD) and chronic inflammatory diseases.The anti-inflammatory activity of seed extract (1 mg/ml) showed 50% inhibition of albumin at a concentration of 94 µg/mL ± 0.14.In protease inhibition assay, 107.80 µg/mL ± 1.04 concentration of plant extract showed 50% inhibition of trypsin (Figure S5).

Experimental
The detailed experimental section is provided in supplementary file.

Conclusions
E. rheedii seeds are a rich source of essential nutrients like protein, minerals and galactose content which makes it a good dietary source of energy.Furthermore, the species is low fat and low anti-nutrient containing under-utilized legume which can be included in daily diet as an additional source of nutrition like Moringa, finger millet, and kodo millet.Moreover, the results validate the traditional claims of species in medicinal use.
The standardization pharmacognostic parameters of E. rheedii will aid in regulating the quality of raw material at industrial/commercial scale.The study advocated the utilization of this lesser known species as dietary supplement, not only to its native places but also in other part of the world.It can be commercially explored as an alternative source of protein fortification in flour, breads, bakeries, and so on, and may be a promising, low cost dietary alternative in the future.