Lactic acid-based deep eutectic solvent: An efficient green media for the selective extraction of steroidal saponins from Trillium govanianum

Highlights

• Lactic-acid-based deep eutectic solvents efficiently extract steroidal saponins from Trilium govanianum.

• A total of 21 NADESs were synthesized, choline chloride-lactic acid (1:1; NADES 1) being the most effective among 15 tested NADESs to extract steroidal saponins from Trilium govanianum.

• The extraction efficiency of NADES 1 was found to be 53.72 % higher than that of conventional hydroethanolic extraction media.

• UHPLC-ELSD method was used for the quantification of seven steroidal saponins in the NADES 1 based extract from Trilium govanianum.

• The steroidal saponins-enriched extract obtained by using NADES 1 significantly inhibits acetylcholinesterase activity.

Abstract

Natural deep eutectic solvents have a high solvation and food-grade characteristics, which make them a preffered choice for extracting bioactives to develop nutraceutical and cosmetic formulations. This research aimed to investigate the systematic effects of several NADESs on the ultrasonic-assisted extraction of steroidal saponins from the rhizomes of Trillium govanianum Wall. ex D.Don, which is utilized in traditional Ayurvedic medicine. After screening of fifteen different NADESs, choline chloride: lactic acid (1:1) (NADES 1) was found most effective eutectic solvent for the extraction of steroidal saponins performed by the three-round micro-extraction (1 mL). Compared with hydro-ethanolic solvent-based extraction, lactic acid-derived NADESs showed higher extractability of steroidal saponins. The extraction efficiency of bioactive steroidal saponins e.g. borassoside E and protodioscin was also enhanced with NADES 1. The effects of extraction variables such as S:L ratio, working duration, and temperature on the extraction of steroidal saponins by NADES 1 was also studied and optimized.

Furthermore, optimized parameters were validated by performing 100 mL NADES 1-based extraction to recover phytomolecules and NADES 1 by using Diaion HP20 resin column chromatography. As a proof of concept, this study suggested that lactic acid-derived NADESs are effective media for the extraction of steroidal saponins from biomass. The cytocompatibility assay of NADESs with NRK-52E and IEC-6 cells revealed the safe nature of NADESs to normal animal cells. The steroidal saponins-enriched extract obtained with NADES 1 has shown promising antagonistic effects on acetylcholinesterase activity.

Introduction

The global demand for natural products has increased considerably in the past few years, especially in the nutraceutical, pharmaceutical, and cosmetic industries, over their synthetic counterparts [1], [2]. Several scientific investigations have validated and proved the potency of natural products for the treatment and management of several physiological malfunctions as a medicine or food [3]. Botanicals are the prime source of bioactive natural products. However, due to improper processing, extraction, solubility issues, and use of non-food-grade organic solvents, it isn't easy to get the quality and quantity of enriched extract to develop nutraceutical products. In addition, there are various limitations associated with the conventional extraction techniques for value-added natural products, including low extractability, time consumption, high energy inputs, and use of toxic organic solvents. Nowadays, the isolation and purification of natural products have been mainly executed by using aqueous-medium with organic solvents such as n-hexane, chloroform, acetone, n-butanol, and methanol. These organic solvents have intrinsic toxicity, high volatility, and flammability, with harmful impacts on human health and the environment [4]. Thereby, the utilization of Generally Recognized as Safe (GRAS) solvents such as natural deep eutectic solvent (NADES) to extract natural products from biomass as an alternative to organic solvents is highly desirable. The chemical components of NADES are edible, non-toxic, biocompatible, and biodegradable [5]. NADES is a food-grade solvent with a wide range of solvation properties from simple to complex biomolecules [6]. Thereby, the NADES has immense potential to extract the highest possible quantity of natural products from biomass to develop food-grade edible extracts.

Trillium govanianum Wall. ex D.Don is a Himalayan endemic medicinal plant, traditionally used to treat several inflammations and pain-related ailments, dysentery, and reproductive disorders [7]. Phytochemically, the rhizomes of this herb are rich in steroidal saponins [8], [9], [10], [11], [12] and scientifically evaluated for their antifungal [8], cytotoxicity [13], [14], antibacterial [15], infertility [16], anti-inflammatory [17], [18], [19], anti-diabetic [20], and insecticidal [21] activities. Aglycone skeleton of steroidal saponins reported from T. govanianum belongs to spirostane, furostane, and pregna-type [22]. The rhizomes of T. govanianum contains ∼7.8% of steroidal saponins [18]. To date, only fifteen phytomolecules are isolated from this plant including, ten steroidal saponins [8], [10], four steroids [8], [9], and one polyhydroxy fatty acid [9]. Therefore, there is an immense possibility to investigate chemically attractive steroidal scaffolds from this herb to decipher their therapeutic or nutraceutical function. Chemically diverse steroidal saponins of this herb may be useful as futuristic raw material for the industrial production of steroidal hormones, steroidal anti-inflammatory drugs, and neuro-active steroids. However, NADES-derived extraction of bioactive steroidal saponins may also be used to develop food-grade nutraceutical formulations and therapeutic syrups.

There are some reports on the deep eutectic solvent-based extraction of saponins [23], [24], [25], [26], [27], indicating that NADES is a promising and safe replacement for conventional extraction media. In addition, NADESs have also been utilized as potential extraction media for other bioactive, including aesculin and aesculetin from Cortex fraxini [28], anthocyanins and pectin from Myrciaria cauliflora fruit by-product [29], phenolics from Moringa oleifera L. leaves [30], rutin from Sophora japonica [31] and tartary buckwheat hull [32], astaxanthin from shrimp byproducts [33], and curcuminoids from Curcuma longa [34]. Eutectic solvents are the supramolecular network of hydrogen bonding which solubilize wide range of chemical scaffolds [35] and thereby applied for the extraction of phytomolecules from the botanicals. In continuation of our study on the phytopharmacological investigation of T. govanianum [10], [11], [12], [18], [19], [20], [21], [22], we report a NADES-based green method to extract steroidal saponins from T. govanianum. Besides this, the cytocompatibility of all prepared NaDESs, antagonistic effects of steroidal saponins enriched extract obtained by using NaDES 1, and pure steroidal saponins on acetylcholinesterase activity were also studied.