Isolation, characterization, and evaluation of anxiolytic bioactive compounds from the seed of Vigna radiata (L.) R. Wilczek in mice

Abstract Recent therapy for managing anxiety disorders is linked with a wide range of adverse effects. The conventional practice of the use of plant extract may indicate an important and new approach to the anxiolytic agent. Seeds of V. radiata belonging to the family Fabaceae is commonly employed to treat several diseases. However, no data is available to screen its viable neuropharmacological effect regardless of its famous use. Hence, the objective of the present study was to isolate the anxiolytic bioactive compound from seeds of V. radiata. Pure bioactive Compounds SU1 and SU2 were obtained from bioactive fraction F9.3 and fraction F9.5 using the bioactivity-guided fractionation method. The current investigation found that 4 mg/kg (o.p.) of kaempferol and γ-aminobutyric acid exhibit significant anxiolytic action in mice that is statistically comparable to diazepam (2 mg/kg.i.p). This study validates the ethnopharmacological use of V. radiata seeds in the management of anxiety disorders. Graphical Abstract


Introduction
Anxiety is a common sensitive experience in the human population, occurring in response to physiological and environmental issues.Besides, it can be a root of uproar in our daily life.Benzodiazepines and antidepressant drugs are generally used for anxiety disorders (Yende et al. 2016).These treatments showed clinical efficacy but had several side effects.Therefore, there is a demand for the development of new anxiolytic drugs from plant sources that may be free from these side effects.
V. radiata plants are considered legumes and belong to Family-leguminosae or Fabaceae.It has been appreciably used as a conventional food on the whole Planet.Besides, seeds have several pharmacological activities and are lesser in cholesterol, saturated fat, and sodium (lopes et al. 2018).Regardless of its well-known use, no data on its viable neuropharmacological effect is available.So, the anxiolytic compounds have been isolated from seeds of V. radiata using chromatographic techniques in the current investigation.Further, an elevated plus-maze model (ePM) and mirror chamber model were used to assess anxiolytic effects.Diazepam is used as a standard drug in the present research (Colla et al. 2015).

Results and discussion
In the present study, all successive extracts like petroleum ether, chloroform, ethanol, and water were prepared by the triple cold maceration method.The elevated plus maze test is one of the most frequently used tests to gauge anxiety-like behaviour.The test is based on mice's innate aversion to elevated, open spaces as well as on their spontaneous natural inquisitive tendency in unfamiliar surroundings as well as rodents without an anxiety-like phenotype may also be keen to explore in closed arms.Thus, this measure is not very useful for determining anxiolytic behaviour.Also, the mirror chamber has been proposed as a specific and quantitatively/qualitatively distinct measure of anxiety from all of that used in other behavioural models such as the elevated plus-maze (lamberty 1998).Based on ePM and mirror chamber model results have been evaluated and the methodology is represented in Figure S1.The water extract of V. radiata seeds was found anxiolytic in Swiss albino mice in the elevated plus-maze model at 150 mg/kg (Table S1).As well, water extract did not give any mortality even at the topmost dose i.e. up to 1000 mg/kg.Further, bioactive water extract 350 g was subjected to column chromatography to get ten sub-fractions F1 (4.10 g), F2 (1.85 g), F3 (1.76 g), F4 (4.12 g), F5 (13.4 g), F6 (8.73 g), F7 (8.25 g), F8 (14), F9 (90 g), F10 (20 g) that have presented in Table S4.Outcomes of the anxiolytics assessment of ten sub-fractions showed that F9 at 20 mg/kg doses reveals significant anxiolytic action on mice in ePM (Table S2).The yield of five column fractions has been found as F9.1 (1.41 g), F9.2 (0.42 g), F9.3 (1.27 g), F9.4 (0.85 g), and F9.5 (2.53 g) and are indicated in Table S8.F9.3 and F9.5 fractions have shown remarkable anxiolytic activity in mice using the ePM model at 10 mg/kg (Table S3).F9.3 & F9.5 gave a positive test for amino acids and flavonoids respectively.After drying, a pure compound viz., SU1 and SU2 were isolated from F9.3 and F9.5 respectively.Finally, the anxiolytic action of mung bean seeds is determined using a mirror chamber test, and found that both isolated compounds have significantly improved the number of entries and time spent in the mirror chamber model at a dose of 4 mg/kg as compared to diazepam (2 mg/kg) and results have been indicated (Tables S4-S7).Figure S2 indicated a graphical representation of all anxiolytic studies of mung beans that have been performed in the present research.
Compound SU2: Colour-White amorphous powder, melting point −202 °C to 204 °C, UV-280nm, IR(KBr)V max cm −1 : 3400,3060,1720,1695,1675,1414,1390,1367,1341,1200, MS: M + at m/z 108.Molecular formula-NH2 (CH2)3COOH.So, SU2 was found to match the literature with γ-aminobutyric acid (lY Wang et al. 2013).Thus, SU2 has been characterized as γ-aminobutyric acid (Figure S4).This is evidence of the presence of Kaempferol and γ-aminobutyric acid (GABA) in V. radiata seeds.Column fractions F9.3 and F9.5 have remarkable anxiolytic effects and resulted in the isolation of Kaempferol and γ-aminobutyric acid which potentially exhibited potent antianxiety activity at 4 mg/kg, which was comparable to that of diazepam (2 mg/kg).As per the literature survey, in vivo studies, oral administration of Kaempferol and γ-aminobutyric acid (GABA) exhibit several pharmacological properties.However, an incipient area of curiosity is the activation of GABA receptors by flavonoid-like chemical constituents in the absence of GABA (DD de Oliveira et al. 2020).GABA binds with GABA-A receptors and indicates anxiolytic effect and also works together with another neurotransmitter like serotonin (Ramos-Ruiz et al. 2018).Also, flavonoids responsible for anxiolytic effect and its CNS effect as an antidepressive is mediated by 5-HT1A receptors (Chatterjee et al. 2011).Thus, literature supports to anxiolytic effect of V. radiata seeds.

Conclusion
Oral administration of V. radiata seeds exhibited an anxiolytic effect in mice.This study validates the ethnopharmacological use of V. radiata seeds in the management of anxiety disorders.Isolated constituents Kaempferol and γ-aminobutyric acid are responsible for antianxiety activity in Mung bean seeds.Since herbal drugs may be an inspiration for new anxiolytic compounds with fewer side effects compared to synthetic medication and strongly suggest that these natural products are interesting prototype compounds in the study of new anxiolytic drugs.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
The author(s) reported there is no funding associated with the work featured in this article.