Exploring the industrial importance of a miracle herb Withania somnifera (L.) Dunal: Authentication through chemical profiling, in vitro studies and computational analyses

Highlights

• This study unveils metabolic profile and biological activities of Withania somnifera.

• The Chloroform (WS-C) fraction showed high phenolic contents and bioactivities.

• UHPLC-MS analysis of WS-C fraction leads to identify 100 secondary metabolites.

• Docking studies validated the enzyme inhibition activities of the extract.

• Multivariate statistical analysis established relationship between phenolics and bioactivities.

Abstract

Withania somnifera (L.) Dunal has been identified as a miracle herb, thus the present study was carried out to investigate W. somnifera growing in Cholistan Desert for its secondary metabolic profile and various biological activities. The WS-C fraction of methanolic extract (WS-M) of W. somnifera was found rich in phenolics (39.96 ± 0.90 mg GAE/g extract), and it exhibited significant DPPH (30.69 ± 0.78 mg TE/g extract) and ABTS (113.60 ± 2.41 mg TE/g extract) free radical inhibitory activities, cupric (Cu⁺²) and ferric (Fe⁺³) reducing potential as 157.51 ± 2.10 and 124.60 ± 0.34 mg TE/g extract. The same fraction also exhibited strong total antioxidant capacity (1.39 ± 0.01 mmol TE/g extract) in phosphomolybdenum assay. Other fractions displayed relatively lower potential. In metal-chelating assay, WS-M, WS-E and WS-B showed nearly equal potential with values of 22.35 ± 0.62, 20.96 ± 0.12 and 22.10 ± 0.75 mg EDTAE/g of the extract, while in AChE, BChE and tyrosinase inhibitory assay, again WS-C fraction was most active (7.50 ± 0.21, 8.01 ± 0.53 mg GALAE/g extract and 36.81 ± 0.20 mg KAE/g extract, respectively). In α-amylase and α-glucosidase inhibitory assay, WS-C showed significant potential with values as 0.74 ± 0.01 and 1.14 ± 0.004 mmol ACAE/g extract, respectively. UHPLC-MS analysis of WS-C fraction leads to identify 100 secondary metabolites of phenolic, withanolide, terpenoid, steroid, lignin, flavonoid and limonoid classes of compounds. This profile makes W. somnifera a fascinating plant. These findings were validated by computational studies, which revealed that selected compounds exhibited high binding free energy and inhibition constants with the enzymes tested. Pearson correlation analysis clearly established that the phenols in the tested extracts were the main players as antioxidants and enzyme inhibitors, which was also substantiated through Principal Component Analysis (PCA). These results suggest that chloroform might be suitable for preparing further applications with W. somnifera extracts, and this plant can be placed in the list of top-selling herbs, and may become source of commercial assets.

Introduction

Withania somnifera (L.) Dunal (Family: Solanaceae) has been listed among the top-rated medicinal plants. World Health Organization (WHO) has also recognized this plant as a precious medicinal herb [1]. National Medicinal Plant Board of India has listed W. somnifera among top 32 prime herbs due to its huge demand [2]. It is an important component of folk medicinal system being used as natural healer. People have used W. somnifera for thousands of years to relieve stress, increase energy level, and improve concentration [3], [4]. It is also a well-known herb in Pakistan, Africa and Middle East, where it is known as Ashwagandha or Asgandh; it is also called as Indian ginseng or winter cherry [5].

Powdered root extract has been reported to treat various health conditions like anxiety, fertility issues, inflammation, fatigue, pain, skin diseases, diabetes, arthritis and epilepsy or as a general tonic to increase energy and to decrease ageing process [6]. In addition to above conditions, it also improves sleep quality and bipolar disorder [7], [8], [9], [10], [11], [12]. It is further reported to boost the immune system, increase the production of vital fluids, lymph blood, semen, cells, memory power and possesses immunomodulatory, hemopoietic, rejuvenation and antineoplastic effects [13]. W. somnifera enhances human physical performance, including strength and oxygen use during exercise [14], [15], [16]. The food supplements comprising extracts of this plant have been shown to increase male fertility by increasing testosterone level, sperm concentration, semen volume, and sperm motility [17], [18]. Extract of W. somnifera has also been reported to reduce inflammatory effects in COVID-19 patients when given as an Ayurvedic drug [19]. Another striking feature of this plant is that it exhibits chemo-preventive effects [20]. Visweswari and other research groups have identified wide range of secondary metabolic classes through chemical testing that includes phenols, flavonoids, tannins, saponins, alkaloids, steroids, terpenoids, glycosides and reducing sugar, which they held responsible for anti-inflammatory, anti-spasmodic, anti-analgesic, neuroprotective and diuretic effects of this plant [21], [22]. Anil Bhatia and co-workers analyzed fruit extract of W. somnifera through GC-MS and NMR, and have reported 82 compounds of fatty acid, aliphatic and aromatic amino acid, polyol, sugar, sterol, tocopherol, phenolic acid and withanamides classes of secondary metabolites [23]. In another analytical study through LC-MS/MS, 32 withanamides, 22 withanolides, 3 steroidal saponins, 2 lignanamides, feruloyl tyramine, methoxy feruloyl tyramine and a diglucoside of hydroxyl palmitic acid were identified [24]. Alam and his co-workers have studied methanolic extracts of various parts of W. somnifera for its antioxidant and antibacterial bioactivities [25]. In Pakistan, W. somnifera is popular with name of Asgandh as an evergreen bush growing widely along the road sides and in fields. However, despite of its importance in folk medicine system, it is still an ignored herb in Pakistan, and has not got the status of cultivated plant on commercial grounds. The above literature revealed that this plant is intensively investigated in several capacities, however, it is known fact that environmental changes lead to change in metabolic profile of a plant species. Therefore, the aim of the present study was to offer a deep insight in to the chemical and pharmaceutical properties of W. somnifera growing in Cholistan Desert of Pakistan. For this purpose, various extracts of W. somnifera were investigated for their bioactive contents, complete metabolic picture and several in vitro and computational analyses. Most of these studies have been carried out first the time for this top-rated herb.