Biodiversity, Biochemical Profiling, and Pharmaco-Commercial Applications of Withania somnifera: A Review

Withania somnifera L. Dunal (Ashwagandha), a key medicinal plant native to India, is used globally to manage various ailments. This review focuses on the traditional uses, botany, phytochemistry, and pharmacological advances of its plant-derived constituents. It has been reported that at least 62 crucial and 48 inferior primary and secondary metabolites are present in the W. somnifera leaves, and 29 among these found in its roots and leaves are chiefly steroidal compounds, steroidal lactones, alkaloids, amino acids, etc. In addition, the whole shrub parts possess various medicinal activities such as anti-leukotriene, antineoplastic, analgesic, anti-oxidant, immunostimulatory, and rejuvenating properties, mainly observed by in vitro demonstration. However, the course of its medical use remains unknown. This review provides a comprehensive understanding of W. somnifera, which will be useful for mechanism studies and potential medical applications of W. somnifera, as well as for the development of a rational quality control system for W. somnifera as a therapeutic material in the future.


Introduction
The World Health Organization (WHO) stated that approximately 30% of drugs contain plant-derived compounds, and approximately more than half of the global population depends upon plant-derived medicines, including those of Ayurveda, the traditional plant-based medicine of India [1,2]. The Botanical Survey of India (BSI) reported that approximately 7500 of the 15,000 plant species in India are used for medicinal purposes [3]. A list of 32 therapeutically important plants of India prioritized by the National Bank for Agriculture and Rural Development (NABARD) includes Withania somnifera, regularly called as ashwagandha. The National Medicinal Plant Board (NMPB), India, has also described Withania species as a valuable therapeutic plant in great demand in domiciliary and global markets [4,5].
W. somnifera, indigenous to the Indian sub-continent, is used as indigenous medicine throughout Southeast Asia. Its roots, seeds, and leaves have been used for over 3000 years for multiple health purposes, and are included in Ayurvedic, Allopathic, Unani, homeopathic, and other medical systems [6]. Due to its superlative therapeutic properties, it is also known as the Queen of Ayurveda or a Rasayana herb. Withania spp. are members of the Solanaceae family (Bentham and Hooker, 1862-1883), also known as the deadly nightshade family. The Solanaceae family embraces 84 genera, comprising 3000 species, distributed all over the globe [6]. The genus Withania contains 26 species, only 2 of which, species, distributed all over the globe [6]. The genus Withania contains 26 species, only 2 of which, W. somnifera and W. coagulans (known as Paneer booti, Ashutosh booti or rishyagandha), are economically and medicinally important in many regions [7]. Other potentially important species are W. simonii, W. adunensis, and W. riebeckii.
Two subspecies have also been reported-W. somnifera Dunal and W. somnifera Kaul [8]. Both species of Withania, i.e., W. somnifera and W. coagulans, are cultivated on a large scale in India [9]. In Sri Lanka, two cultivars of W. somnifera are used, but the Indian cultivar is more suitable for drug development due to its starchy nature, whereas the local cultivar has fibrous roots that are difficult to transform into powder [10]. Its common name, ashwagandha, is a combination of two Sanskrit words, ashwa and gandha, and means "horse's smell", referring to the sweaty horse-like smell of the roots [9,11]. Other names include winter cherry, poison gooseberry [12,13], or "Indian ginseng", due to its restorative properties.
However, the mechanism of drug use by W. somnifera has not been elucidated. Accordingly, there are unreasonable points in the current W. somnifera quality management system. In fact, its medicinal value has received less attention, despite its long history of clinical application. In this review, information regarding various aspects of W. somnifera was collected from peer-reviewed journals for the period 2000-2022. PubMed, Shod Ganga, Web of Science, Google Scholar, etc., was used to retrieve electronic information. Based on this information, we performed a full review of W. somnifera aiming to provide information to better understand the pharmacological mechanism and potential medicinal applications of this species, and to develop a reasonable quality control system for W. somnifera as medicinal material in the future.

Leaves
The aerial part of the plant is covered with minute trichomes. The branches are round and leaves are up to 30-80 × 20-50 mm, narrowed into 5-20 mm long petioles. Morphologically, leaves are simple, exstipulate, oblique, petiolate, broadly ovate to oblong, entire, shiny smooth, dull green in color, globose, and elliptical. Leaves are alternate on vegetative shoots and opposite on floral shoots, having margins entirely to slightly wavy. The leaves are almost hairless above, densely hairy below, and bitter in taste. Its leaves have many advantages as shown in Table 2 [5,27]. Table 2. Different biological compounds present in leaves and their activities.

Roots
Roots are long tap roots, 15-25 cm in length, stout and fleshy, tapering, and brownishwhite or light-yellow-colored root with a horse-like smell and bitter taste. Its roots possess many valuable biological activities as shown in Table 3 [5,27].

Flowers and Fruits
Flowers are inconspicuous, solitary, green or dull yellow in color, 4-6 mm in diameter, bisexual, pedicellate, highly protogynous, small, and bell-shaped. Cross-pollination is carried out by ants and bees [32]. Inflorescences are axillary umbellate cymes. The calyx is gamosepalous with valvate aestivation. The corolla is greenish-yellow, valvate, campanulate, and has 5-8 mm lobes. Stamens (5) are polyandrous and epipetalous. Anthers are dithecous, basifixed, and introse. The ovary is superior, bilocular, and has axile placentation. Fruit are orange-red berries containing very light seeds, which are small, smooth, lens-shaped, or kidney-shaped, 2-2.5 mm length × 1.5-2 mm width × 0.5 mm height. Its flowers and fruits are having many phytoconstituents which helps in anti-microbial activities as well as works as a milk thickening agents as shown in Table 4 [33]. Table 4. Different biological compounds present in fruits and flowers, and their activities.

Biochemical Profiling of W. somnifera
Withania somnifera contain many important phyto-chemicals that perform various antimicrobial actions against pathogens. Different metabolic constituents such as secondary metabolites may differ with the tissue type, growth conditions, and variety of W. somnifera. These variations leading to unpredictable medication and health-promoting properties of various commercially available preparations developed with the help of Withania spp. [39]. In a multi-component therapeutic system, this makes standardization of the composition of herbal preparations and commercial exploitation of this plant difficult, because different components can influence health effects through complex multi-target interactions.
It has been reported that 62 major minor primary and secondary metabolites are present in the leaves, and 48 are present in roots, 29 of which are found in both the leaves and roots [40]. Among the bio-active constituents of Withania somnifera reported in the literature are steroidal flavanol glycosides, glycowithanolides, steroidal lactones, and phenolics as shown in Table 5 and Figure 2 [40,41].   As per the literature, 12 alkaloids, 40 withanoloids, and several sitoindosides have been isolated from the Withania species [48]. Several amino acids, alanine, aspartic acid, glycine, glutamic acid, cysteine, tyrosine, chlorogenic acid, glycosides, glucose, condensed tannins, iron, and flavonoids have also been reported [49].

Withanolides
Withanolides are unique classes of steroidal lactones. Over 130 withanolides are present in 15 genera of the Solanaceae family. As per some studies, withanolides are As per the literature, 12 alkaloids, 40 withanoloids, and several sitoindosides have been isolated from the Withania species [48]. Several amino acids, alanine, aspartic acid, glycine, glutamic acid, cysteine, tyrosine, chlorogenic acid, glycosides, glucose, condensed tannins, iron, and flavonoids have also been reported [49].

Withanolides
Withanolides are unique classes of steroidal lactones. Over 130 withanolides are present in 15 genera of the Solanaceae family. As per some studies, withanolides are isolated from some other Leguminosae and Labiatae families, as well as from some marine organisms [5].
W. somnifera plants fabricate the largest number of withanolides comprising highly diversified functional group. Withanolides are key biochemicals, ranging from 0.001% to 0.5% dry weight concentration in the leaves and roots of Withania somnifera. Withanolides are a 28-carbon-containing natural steroidal lactone built on an ergostane skeleton, in which C-22 and C-26 are oxidized to form six-or five-membered lactone rings. Withanolides are also known as 22-hydroxy ergostan-26-oic acid-26, 22-lactone. Its characteristic feature is that the C8 or C9 side chains exhibit a lactone or lactol ring. However, lactone ring may be either five-or six-membered and may be fused with the carbocyclic moiety of the molecule via a C-C bond or through an O-bridge.
Many structures are also isolated from W. somnifera, which are the modifications or structural variants of withanolides which cause modifications to the carbocyclic backbone or side chains, such as withaferin A, withanolide A and D, withanone. These compounds are polyoxygenated and oxidize all C atoms of the steroid nucleus [5]. A new dimeric thiowithanolide also isolated from roots from W. somnifera, is named ashwagandhanolide as shown in Figure 3.
Molecules 2023, 28, 1208 7 of 28 are a 28-carbon-containing natural steroidal lactone built on an ergostane skeleton, in which C-22 and C-26 are oxidized to form six-or five-membered lactone rings. Withanolides are also known as 22-hydroxy ergostan-26-oic acid-26, 22-lactone. Its characteristic feature is that the C8 or C9 side chains exhibit a lactone or lactol ring. However, lactone ring may be either five-or six-membered and may be fused with the carbocyclic moiety of the molecule via a C-C bond or through an O-bridge. Many structures are also isolated from W. somnifera, which are the modifications or structural variants of withanolides which cause modifications to the carbocyclic backbone or side chains, such as withaferin A, withanolide A and D, withanone. These compounds are polyoxygenated and oxidize all C atoms of the steroid nucleus [5]. A new dimeric thiowithanolide also isolated from roots from W. somnifera, is named ashwagandhanolide as shown in Figure 3.  Withanolides act as a precursor hormone, in that it can be transform physiology as needed [7]. W. somnifera is also sometimes referred to as I because it structurally resembles ginsenosides (the active constituents of In addition, Withanolide A also possesses anti-dementia activities, and ca for neuronal regeneration.
The biosynthesis starts with the formation of cholesterol by the a co-enzyme A. Acetyl-co A is combined and converted into two types of m only the R-type is used in triterpene biosynthesis, the S-type being inert lowing the reduction of a C-atom, the R-type is transformed into isopente phate (IPP) which, together with its isomer 3,3-dimethyl allyl pyrophosph undergoes condensation to form geranyl pyrophosphate (GPP) and, aft densation, farnesyl pyrophosphate (FPP). Two molecules of FPP conden ence of NADPH and squalene synthase to generate squalene which, a forms a squalene 2,3-epoxide undergoes ring closure to form lanosterol, a of various steroidal triterpenoid structures. The formation of 24-methy has still not been determined, and it is unknown whether it is a precurs lactones or not. Nevertheless, it has been observed that hydroxylation a δ-lactonization between C22 and C26 of 24-methylenecholestrol are par anolide pathway as shown in Figure 4. Withanolides act as a precursor hormone, in that it can be transformed into human physiology as needed [7]. W. somnifera is also sometimes referred to as Indian Ginseng because it structurally resembles ginsenosides (the active constituents of Panax ginseng). In addition, Withanolide A also possesses anti-dementia activities, and can thus be used for neuronal regeneration.
The biosynthesis starts with the formation of cholesterol by the action of acetyl coenzyme A. Acetyl-co A is combined and converted into two types of mevalonic acid; only the R-type is used in triterpene biosynthesis, the S-type being inert in nature. Following the reduction of a C-atom, the R-type is transformed into isopentenyl pyrophosphate (IPP) which, together with its isomer 3,3-dimethyl allyl pyrophosphate (DMAPP), undergoes condensation to form geranyl pyrophosphate (GPP) and, after further condensation, farnesyl pyrophosphate (FPP). Two molecules of FPP condense in the presence of NADPH and squalene synthase to generate squalene which, after oxidation, forms a squalene 2,3-epoxide undergoes ring closure to form lanosterol, an intermediate of various steroidal triterpenoid structures. The formation of 24-methylenecholesterol has still not been determined, and it is unknown whether it is a precursor of steroidal lactones or not. Nevertheless, it has been observed that hydroxylation at C-22 and the δ-lactonization between C22 and C26 of 24-methylenecholestrol are part of the Withanolide pathway as shown in Figure 4.

Withaferins
4β,27-dihydroxy-1-oxo-5β, 6β-epoxywitha-2-24-dienolide, also known as Withaferin A, was first isolated from the leaf of South Asian W. somnifera, in 0.13-0.31% concentra- Withaferin A and withanone ( Figure 5) act as angiogenesis inhibitors and have anticancer properties [54,55]. Withaferin A also exhibits antibiotic activity due to its unsaturated lactone ring [56], and is applied to cure ulcers and skin carbuncles, inhibit tumor growth, exhibit radio-sensitizing effects in cancer treatment, and induce immune-suppressive activities on B-and T-lymphocytes. It also shows other activities such as antibacterial, antitumoral, anti-arthritic, and immunomodulating actions [43]. Withaferin A also ceases the growth of some bacteria and fungi [57]. tion of dry weight. Quantitative analysis of Indian chemotypes of W. somnifera by TLC densitometry showed that withaferin A was completely absent in whole plants, but present in leaves by 1.6% concentration [53].
Withaferin A and withanone ( Figure 5) act as angiogenesis inhibitors and have anti-cancer properties [54,55]. Withaferin A also exhibits antibiotic activity due to its unsaturated lactone ring [56], and is applied to cure ulcers and skin carbuncles, inhibit tumor growth, exhibit radio-sensitizing effects in cancer treatment, and induce immune-suppressive activities on B-and T-lymphocytes. It also shows other activities such as antibacterial, antitumoral, anti-arthritic, and immunomodulating actions [43]. Withaferin A also ceases the growth of some bacteria and fungi [57].

Withanamides
Withanamides A-I ( Figure 6) are purified from the methanolic extract of fruits of W. somnifera. Its structure contains a 5-hydroxy tryptamine (serotonin) base with hydroxy fatty amide and diglucosidic moieties. The difference between the structural variation in Withanamide A and Withanamide C is only in the fatty acid side chain, because Withanamide A has two double bonds in its side chain compared with Withanamide C.
Withanamide A and Withanamide C help neutralize the toxicity of β-amyloid protein (BAP) and protect the cells from cell death. Withanamide A is more effective in treating Alzheimer's disease. It also exerts anti-oxidant properties, because it has the ability to inhibit lipid peroxidation (LPO). It inhibits tumor cell proliferation because it has selective cyclooxygenase (COX-1 and -2) enzymes [58,59].

Withanamides
Withanamides A-I ( Figure 6) are purified from the methanolic extract of fruits of W. somnifera. Its structure contains a 5-hydroxy tryptamine (serotonin) base with hydroxy fatty amide and diglucosidic moieties. The difference between the structural variation in Withanamide A and Withanamide C is only in the fatty acid side chain, because Withanamide A has two double bonds in its side chain compared with Withanamide C.

Sitoindosides
Sitoindoside is a type of withanolide which contains glucose molecule at carbon 27, isolated plants of W somnifera. From its roots, two acyl glucosides sitoindoside VII and VIII, and two glycowithanoloids, sitoindoside IX and X are isolated. These phytochemicals possess anti-stress activity and also help in retaining the memory [47,48]

Alkaloids
As per the literature, in addition to steroidal compounds, many alkaloids are re- Withanamide A and Withanamide C help neutralize the toxicity of β-amyloid protein (BAP) and protect the cells from cell death. Withanamide A is more effective in treating Alzheimer's disease. It also exerts anti-oxidant properties, because it has the ability to inhibit lipid peroxidation (LPO). It inhibits tumor cell proliferation because it has selective cyclooxygenase (COX-1 and -2) enzymes [58,59].

Sitoindosides
Sitoindoside is a type of withanolide which contains glucose molecule at carbon 27, isolated plants of W somnifera. From its roots, two acyl glucosides sitoindoside VII and VIII, and two glycowithanoloids, sitoindoside IX and X are isolated. These phytochemicals possess anti-stress activity and also help in retaining the memory [47,48].

Alkaloids
As per the literature, in addition to steroidal compounds, many alkaloids are reported from the W. somnifera which are named somniferin (bitter in taste), somniferinine, somine, cuscohygrine, nicotine, pseudo-withanine, withananinine, tropine, 3α-trigloyloxytropane, anaferine, anhygrine, choline, withananine, and Withanine. The total alkaloid content present in roots varies from 0.13% to 0.31%, although it elevates up to 4.3% as was reported from different regions/countries, whereas there are five unidentified alkaloids present in leaves which yield approximately 0.09% (Figure 7). The differences in the yield of alkaloids may be because of some factors such as the method of isolation, environmental interactions, or variability in genotypes. These alkaloids show sedative and hypotensive effect [60].

Other
Other phytochemicals have been reported, such as Ipuranol, tannins, resin, fatty acids, organic acids, amino acids, reducing sugars, phytosterol, and saturated and unsaturated acids. The roots are reported to contain starch, reducing sugars, glycosides, dulcitol, withanicil, hentriacontane, and peroxidases, whereas in leaves, flavonoids, withanone, chlorogenic acid, calystegines (N-polyhydroxylated heterocyclic compounds), and tannin have been reported; in the berries, amino acids have been reported. Several amino acids have also been isolated from the root, including alanine, aspartic acid, cysteine, glycine, glutamic acid, and tyrosine [60].

Extraction of Phytochemicals
Extraction methods generally include traditional techniques such as digestion, infusion, maceration, decoction, infusion, infiltration, and Soxhlet extraction [61]. Microwave-assisted solvent extraction (MASE) techniques, ultrasound-assisted solvent extraction (UASE) techniques, and supercritical fluid extraction (SPE) techniques are the most common approaches used in the extraction of bioactive compounds of Withania somnifera [62,63]. The UASE and MASE methods have special advantages over the amount of solvent and extraction time and achieve higher yield than traditional extraction techniques. Fractionation and purification of the photochemical is performed using a variety of chromatographic techniques such as high-performance liquid chromatography (HPLC), gas chromatography (GC), paper chromatography, and thin-layer chromatography (TLC) [63]. The extraction of secondary metabolites from Withania spp. Is affected by many factors, such as the nature, its origin, degree of processing, moisture content, and particle size of the plant material. The results will differ according to the type of extraction technique and variables such as temperature and time. The phytochemicals in the

Other
Other phytochemicals have been reported, such as Ipuranol, tannins, resin, fatty acids, organic acids, amino acids, reducing sugars, phytosterol, and saturated and unsaturated acids. The roots are reported to contain starch, reducing sugars, glycosides, dulcitol, withanicil, hentriacontane, and peroxidases, whereas in leaves, flavonoids, withanone, chlorogenic acid, calystegines (N-polyhydroxylated heterocyclic compounds), and tannin have been reported; in the berries, amino acids have been reported. Several amino acids have also been isolated from the root, including alanine, aspartic acid, cysteine, glycine, glutamic acid, and tyrosine [60].

Extraction of Phytochemicals
Extraction methods generally include traditional techniques such as digestion, infusion, maceration, decoction, infusion, infiltration, and Soxhlet extraction [61]. Microwaveassisted solvent extraction (MASE) techniques, ultrasound-assisted solvent extraction (UASE) techniques, and supercritical fluid extraction (SPE) techniques are the most common approaches used in the extraction of bioactive compounds of Withania somnifera [62,63]. The UASE and MASE methods have special advantages over the amount of solvent and extraction time and achieve higher yield than traditional extraction techniques. Fractionation and purification of the photochemical is performed using a variety of chromatographic techniques such as high-performance liquid chromatography (HPLC), gas chromatography (GC), paper chromatography, and thin-layer chromatography (TLC) [63]. The extraction of secondary metabolites from Withania spp. Is affected by many factors, such as the nature, its origin, degree of processing, moisture content, and particle size of the plant material. The results will differ according to the type of extraction technique and variables such as temperature and time. The phytochemicals in the extract may also be affected by the type of solvent and its concentration and polarity [64]. Thus, the extraction first depends on the type of plant material used, and whether it is fresh or dry, because different parts of the plants will vary in chemical composition according to their water content. The solvent choice depends on the polarity of target compounds. These should be non-toxic, evaporate at a low temperature, and not interfere with the assay. Many types of solvent, such as methanol, ethanol, ether, and water (traditionally used) [65], are used to extract anthocyanins, starches, tannins, saponins, terpenoids, polypeptides, lecithin, etc. (Table 6). "+" shows presence and "-" shows absence.

Biological Activity
W. somnifera possesses many vital biological activities because of the presence of valuable phytoconstituents in it. Some of its activities are as shown in Figure 8. "+" shows presence and "-" shows absence.

Biological Activity
W. somnifera possesses many vital biological activities because of the presence of valuable phytoconstituents in it. Some of its activities are as shown in Figure 8.

Anti-Stress and Anti-Anxiety/Psychotropic Activity.
Withanolide glycosides show anti-stress properties and having positive effects on boosting memory [69]. They help treat stress-induced conditions such as gastric ulceration, cognitive deficit, irregular glucose homeostasis, sexual dysfunction, changes in plasma corticosterone levels, and immunosuppression [7].
Withania somnifera root and seed extracts show anti-stress activity. An in vivo study was performed in mice to determine the anti-stress activity of the plant. An alcoholic extract of roots and seeds was prepared in normal saline and a single dose (100 mg/kg) was administered intraperitoneally to 20-25 g mice to test their swimming performance in water at 28′-30′ C. The experiment showed a promising result; the swimming rate was almost double that of the control [70].
Research conducted at University of Texas Health Science Center showed that Withania somnifera extract slows down the brain by reducing neuron excitability and inhibiting nerve transmission, closely related to the primary inhibitor Gama-Aminobutyric acid (a neurotransmitter) [71].
Glycowithanolides of Withania somnifera, sitoindoside IX and sitoindoside X at a concentration of 50-200 mg/kg, and glycosides (sitoindosides VII and VIII) at a concentration of 50-200 mg/kg orally, showed significant anti-stress activity in albino mice and rats and resulted in increased memory retention [8].
Double-blind, placebo-controlled studies were performed to determine the anti-stress activity of W. somnifera in human trials. Sixty adults were randomly assigned to take 240 mg of a standardized ashwagandha extract (Shoden) once daily. The results were measured using the Depression, Anxiety, Stress Scale-21 (DASS-21), hormonal changes in cortisol, dehydroepiandrosterone-sulphate (DHEA-S), the Hamilton Anxiety Rating Scale (HAM-A), and testosterone levels. It was found that ashwagandha extract was associated with greater reductions than placebo for mean mHAM-A score, morning cortisol C-reactive protein, pulse rate, and blood pressure, and increased significantly for mean serum DHEAS and hemoglobin [72][73][74].

Anti-Stress and Anti-Anxiety/Psychotropic Activity
Withanolide glycosides show anti-stress properties and having positive effects on boosting memory [69]. They help treat stress-induced conditions such as gastric ulceration, cognitive deficit, irregular glucose homeostasis, sexual dysfunction, changes in plasma corticosterone levels, and immunosuppression [7].
Withania somnifera root and seed extracts show anti-stress activity. An in vivo study was performed in mice to determine the anti-stress activity of the plant. An alcoholic extract of roots and seeds was prepared in normal saline and a single dose (100 mg/kg) was administered intraperitoneally to 20-25 g mice to test their swimming performance in water at 28 -30 C. The experiment showed a promising result; the swimming rate was almost double that of the control [70].
Research conducted at University of Texas Health Science Center showed that Withania somnifera extract slows down the brain by reducing neuron excitability and inhibiting nerve transmission, closely related to the primary inhibitor Gama-Aminobutyric acid (a neurotransmitter) [71].
Glycowithanolides of Withania somnifera, sitoindoside IX and sitoindoside X at a concentration of 50-200 mg/kg, and glycosides (sitoindosides VII and VIII) at a concentration of 50-200 mg/kg orally, showed significant anti-stress activity in albino mice and rats and resulted in increased memory retention [8].
Double-blind, placebo-controlled studies were performed to determine the anti-stress activity of W. somnifera in human trials. Sixty adults were randomly assigned to take 240 mg of a standardized ashwagandha extract (Shoden) once daily. The results were measured using the Depression, Anxiety, Stress Scale-21 (DASS-21), hormonal changes in cortisol, dehydroepiandrosterone-sulphate (DHEA-S), the Hamilton Anxiety Rating Scale (HAM-A), and testosterone levels. It was found that ashwagandha extract was associated with greater reductions than placebo for mean mHAM-A score, morning cortisol C-reactive protein, pulse rate, and blood pressure, and increased significantly for mean serum DHEAS and hemoglobin [72][73][74].
Studies suggest that the stress-relieving property of ashwagandha is due to modulation of the HPA axis. The HPA axis is an important hormonal response system which controls stress. This axis ensures that the body quickly responds to nerve-racking moments and returns to a normal state.

Anti-Spasmodic Activity
The alkaloids present in the plant have relaxant and antispasmodic effects against many spasmogens in uterine, intestinal, bronchial, tracheal, and blood vascular muscles. For example, Ashwagandholine exerts relaxant and antispasmodic effects [75].

Anti-Inflammatory Activity
W. somnifera possesses anti-inflammatory properties in both acute and chronic types of inflammation. Cultures of cartilage from patients with osteoarthritis and rheumatoid arthritis have been used to demonstrate its protective effects on chondroblasts [76]. Related effects on cytokines, transcription factors, and the suppression of nitric oxide (NO) have also been observed [77]. A decoction of root and leaf extracts containing alkaloids and withanolides proved effective against the denaturation of protein in vitro. Additionally, anti-inflammatory properties have been described for constituents such as withaferin A and 3-b-hydroxy-2,3-dihydrowithanolide F [78]. Thus, W. somnifera also exerts anti-arthritis effects and is used to treat osteoarthritis. It may be taken together with other supplements such as Articulin-F. This also works as an analgesic and helps to soothe the nerve system from pain.

Anti-Microbial Activity
W. somnifera has anti-microbial components in leaves and roots [79], which are effective against human pathogenic bacteria, fungi, and viruses [80,81]. The leaves of W. somnifera have anti-bacterial properties, and are effective against bacteria such as Pseudomonas aeruginosa and Staphylococcus aureus. Its antibacterial phytochemicals identified so far include withaferin A and 3-b-hydroxy-2,3-dihydrowithanolide F. The plant contains many alkaloids and other polar compounds with anti-bacterial activities which activate the immune system of the host [82]. Withaferin A inhibits the growth of various bacteria and pathogenic fungi; it is also active against Micrococcus pyogenes, partially active against Bacillus subtilis, and inhibits Ranikhet disease, vaccinia virus, and Entamoeba histolytica [43]. W. somnifera helps to provide protection against systemic Aspergillus infection by activating the macrophage function, which increases phagocytosis and the intracellular killing of peritoneal macrophages [83], while W. somnifera shows anti-fungal activity against Helminthosporium sativum [84]. Some glycoproteins derived from W. Somnifera inhibit the growth of phytopathogenic fungi (Aspergillus flavus and Fusarium verticilloides) by prohibiting their spore formation and the growth of hyphae [79]. Withaferin A has been found to inhibit infectious bursal disease virus [85], herpes simplex virus [86], HIV infection [87], and coronavirus [19].

Insecticidal Activity
Phytochemicals from W. somnifera have insecticidal activities and can protect from many insects such as Callosobruchus chinensis, Sitophilus oryzae, Triboliumcastaeneum [88,89], and Spodoptera litura, mosquito vectors such as Anopheles stephensi, Aedes aegypti, and Culex quinquefasciatus [90], and termites [91]. They also show herbicidal activity against the noxious weed Parthenium hysterophorus. 5.6. Anti-Diabetic Activity W. somnifera acts as an anti-diabetic, reducing blood sugar and cholesterol levels [29]. It decreases streptozocin, which is particularly toxic to insulin-producing beta cells of the pancreas and induces hyperglycemia. The anti-diabetic effect of the plant may arise from pancreatic islet free radical scavenging activity. The hyperglycemic activity of streptozocin results from reduced pancreatic islet cell superoxide dismutase (SOD) activity, which leads to the accumulation of degenerative oxidative free radicals in islet beta cells [92].

Hepatoprotective Activity
Histopathological studies confirm that the alcoholic extract of W. somnifera leaves inhibits CCl 4 -induced alterations in transaminase activity and pentobarbitone sleeping time, indicating hepatoprotective properties [93]. This also inhibits ochratoxin A, which causes liver inflammation and suppresses macrophage chemotaxis [83].

Anticarcinogenic Activity
Various studies have demonstrated the anticarcinogenic effects of W. somnifera secondary metabolites in animals and cell cultures, especially withaferin A and withanone [94]. Due to a broad spectrum of cytotoxic and tumor-sensitizing actions, the plant has potential applications in novel complementary therapies for integrative oncology. Effects against the HL-60 leukemia cell line, myeloid leukemia, and bladder, breast [95], prostate, colon, kidney, gastric, and lung cancer, have been described [96]. The mechanisms of W. somnifera-derived anticarcinogenic activity include antiproliferative effects [97], apoptosis [98], radio-sensitization, mitotic arrest, antiangiogenics, and the enhancement of cell defense mechanisms [99]. Leaf extracts have been used for the selective killing of cancer cells. Withaferin A is more effective than doxorubicin in inhibiting breast and colon cancer cell growth [58]. Due to these functions, W. somnifera bioactive agents have the potential to fight cancer by reducing tumor cells [100,101]. W. somnifera displays activity against urethaneinduced lung tumors in mice and radio-sensitizing actions. Withaferin A, 3-hydroxy-2,3dihydrowithanolide F, and withanolides D and E all showed anti-tumor activity in vitro against human epidermoid nasopharynx carcinoma and in vivo against Ehrlich ascites carcinoma, sarcoma, and mammary adenocarcinoma. They also acted as mitotic poison against human larynx carcinoma cells at the metaphase. The effect of Withaferin A also increased with the help of Methyl-thiodeacetyl colchicine. W. somnifera has many phytochemicals with unsaturated lactones in the side chain attached to the allelic primary alcohol group at C25 and highly oxygenated rings at the other end of the molecule, which may afford them cariostatic properties [43]. This also helps to reverse the harmful effects of urethane on lymphocytes, improve the leukocyte count, increase body weight, and reduce mortality [70]. Fibroid tumors of the uterus are also treated and help to reduce uterine bleeding [102].
Withania somnifera exhibits anticancerous property due to its phytochemicals (Withanolide and withaferin). Withanolide suppresses the inducible and constitutive expression of the "NF-kappa B" signaling pathway involved in extensive cancer development [103]. In addition, Withaferin induces apoptosis in tumor cells and prevents their spreading [104].

Cardiotonic Activity
W. somnifera helps to purify blood. It is used to treat heart weaknesses and blood disorders [29]. The effects of digoxin (a drug used to treat irregular heartbeat and heart failure) are similar to this plant. W. somnifera helps to reduce blood pressure by blocking the action of autonomic ganglia and exerting myocardial depressant, as well as positive inotropic and chronotropic effects [70]. W. somnifera also exhibits hypercholesteremic, hypolipidemic, and anti-atherogenic activities. It works against atherogenesis and vascular intimal damage. It inhibits lipid peroxidation, platelet aggregation, delays the plasma recalcification time, and improves the release of the lipoprotein lipase enzyme. It reduces body weight and increases high-density lipoprotein cholesterol levels [105]. The root powder has anti-aging effects, as it increases red blood cells and hair melanin, and decreases serum cholesterol [58,71,78].

Central Nervous System (CNS)
W. somnifera helps to treat depressive disorders and exhibits a tranquilizing effect. Animal studies suggest that the plant components affect the central nervous system, including the modulation of acetylcholinesterase and butyrylcholinesterase activity, the inhibition of calcium ion influx, blockade of gamma-aminobutyric acid receptors, modulation of 5-HT 1 and 5-HT 2 receptors, and the regeneration of neurites. [70,106] Glycowithanolides, withaferin-A, and sitoindosides VII-X obtained from the roots reduce the effect of ibotenic acid, which induces cognitive defects. Roots of W. somnifera are used as a sedative, a treatment for insomnia, and nervine tonic. They are useful in cases of fainting, dizziness, and insomnia. A well-known adaptogen, W. somnifera helps to protect the body from damaging effects and promotes normal physiological functioning. It also works on the neuroendocrine system [7] and is thought to help the body cope with stress, boost the whole nervous system, and improve attention and concentration power. They are used in the treatment of Parkinson's, Alzheimer's, neurotic atrophy, synaptic loss, and other neuro-degenerative diseases because of its radical quenching properties [107].

Thyrotropic Effect (Hypothyroidism)
Research has revealed that W. somnifera has thyrotropic effects [108]. Its constituents decrease thyroid activity and lipid peroxidation in the liver homogenate and affect cellular antioxidant systems by increasing catalase activity while T3 levels remain constant. Additionally, urine volume and sodium are increased, and serum cholesterol and triglycerides are reduced.

Immunomodulatory Activity
W. somnifera works as an immunomodulator in indigenous medicines [75], and its phytochemicals, such as withaferin A, 3-b-hydroxy-2,3-dihydrowithanolide F, glycowithanolides and sitoindosides IX and X have proven immunomodulatory properties. The activities are similar to some immunosuppressive drugs such as cyclophosphamide, azathioprine, and prednisolone. For example, spleen cell proliferation is inhibited by withanolides. Murine macrophages and phagocytosis are activated by withanolide glycosides, which also increase lysosomal enzymatic activity secreted by the macrophages. W. somnifera helps to increase hemoglobin concentration, white and red blood cells, platelets, and body weight, and reduces leukopenia induced by cyclophosphamide (CTX) or sublethal doses of gamma radiation. Moreover, it helps to slow hypersensitivity reactions. It increases the cytotoxic effect of nitric oxide synthase of macrophages, which works against microorganisms and tumor cells [70,71,108].

Antioxidant Activity
W. somnifera contains many powerful antioxidant phytochemicals such as polyphenols, [16] sitoindosides VII-X, withaferin A, and glycowithanolides [70,71]. The brains and nervous systems of humans are rich in lipids and iron; therefore, they are particularly susceptible to damage by reactive oxygen species (ROS). Our brain uses approximately 20% of the total oxygen supply of the organism, and free radical damage to the nervous system causes heavy neuronal losses [69].

Anti-Peroxidative Activity
Root extracts of W. somnifera regulate the effects of lead toxicity and the lipid peroxidative process in liver and kidney tissues [109]. This treatment enhances hepatic and renal lipid peroxidation, decreases lipid peroxidation, and increases the activities of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT), thereby helping to maintain normal oxidative status of the tissues [110].

Male Infertility
W. somnifera helps in maintaining fertility in men by improving the quality of sperm but not the sperm count [29,70]. The root extract, taken as an aphrodisiac, increases the sex hormones testosterone and cortisol [111]. It also increases semen volume, sperm concentration, and sperm motility [31,112]. Overall, it works against stress and infertility [113]. W. somnifera is also known as sukrala, i.e., it helps to increase semen, and is regarded as an aphrodisiac. The powdered plant mixed with ghee, sugar and milk is taken to treat puerperal backache and leucorrhea caused by endometritis [28,29].

Applications in Medical and Food Systems
W. somnifera is rapidly advancing and is used for wide range of applications in medicinal and food sectors. Its potential is widely studied and can be used tradionally to treat number of health problems. The Table 7 shows some of its functions in different modes of uses which are as follows. Treat fevers [5,8,78] Decoctions Treat sore eyes, boils, swellings in limbs and piles [29,118] Ointment Serves as an insecticide to kill lice infesting the body, and is also useful to treat bed sores, wounds, and cuts [29,37]

Roots
Milk boiled with fresh roots To leach out undesirable waste from the body [28,37] Paste Treats swellings and ulcers [28,37]

Tonic
Treat sexual deficiency in males [29,37,119] Powder To slow the growth of tumors, and improve male sperm count [37] Decoctions To treat sterility in females, chest complaints, scrofula, and colds [5,37] Black ashes To heal blisters and swellings [5] Others To treat rheumatism, loss of appetite, cough, dropsy, dyspepsia, joint pain, leukoderma (a skin disorder), inflammation, nervous breakdown, senility, debility, loss of memory, goiter, lower back pain, sciatica, boost immunity, increase white blood cells in the body, treat insomnia, the regulation of blood sugar, and reduction in bad cholesterol [5,8,29,37] 3. Seeds N/A To expel parasites out of the body, to treat chest problems, to thicken milk [29,37,78] 4.

Berries N/A
To coagulate milk Sedative, emetic, blood-purifying, febrifuge, and diuretic Treats dyspepsia, to promote growth, treats liver problems, asthma, and biliousness [5,8,29,37]  Withania somnifera have wide range of pharmacological activities and numerous medicinally important products in the form of capsules, tablets, gummy, and powder have been developed by different manufactures, as shown in following Table 8.

Food Applications
The clinical role of Withania somnifera has been thoroughly explored, but little knowledge is available concerning food applications [134]. Its leaves and roots are good sources of dietary fiber [135]. Leaves and roots of incorporated products of W. somnifera can be used as extrudates, juices and beverages, sweet products, bakery and cereal products, dairy products, etc. as shown in Table 9 [134,136]. Numerous therapeutically important compounds have been observed to possess various biological activities. These compounds could play major roles in the dietary system.

Juices and Beverages
Withania-somnifera-fortified beverage blends could be utilized for its nutritional quality in functional fruit beverages and the developed stored product was stable and acceptable for 90 days at room temperature [137].

Sweets Products
Shrikhand sweets are said to contain 0.5-0.6% ashwagandha powder and improve stability and self-life up to 52 days at refrigerator temperature [138].

Bakery and Cereal Products
Ashwagandha powders, including many products, are evaluated at different concentrations, such as 5% in Namakpara, Pap Chakal, and Muruk, and up to 10% in Missy Roti and Chutney powders. Containing up to 5% of Withania somnifera root powder, biscuits are rich in energy, minerals, fiber, and protein, enhancing their medicinal benefits [139]. Ashwagandha root powder at 2% in baked goods such as Thepla and breads reduces diabetes [140].

Future Perspectives
W. somnifera could be used as potential biomolecules for developing pharmaceutical products and formulations. In addition to developing a fine rational management for W. somnifera as a miraculous plant in the future, it may be useful for inspecting process and prospective of its remedial uses and food packages of W. somnifera.

Conclusions
In summary, W. somnifera has antiquity uses to treat various diseases. This review indicates that many pharmacologically important compounds are present in it which performs crucial role in natural medicinal systems, and also lays foundation for next generation.
W. Somnifera (Ashwagandha), a potential medicinal herb, has promising therapeutic and pharmacological properties due to its diverse phytochemicals. Many studies on this medicinal herb have shown antidiabetic, antistress, anti-inflammatory, anti-cancerous, anti-COVID-19, immunomodulator, antimicrobial, and hepatoprotective activity. Importantly, Withaferins induces apoptosis in tumor cells and prevents their spreading. Glycoproteins derived from W. Somnifera inhibit the growth of phytopathogenic fungi. The literature reveals that anti-anxiety properties are mainly because of glycowithanolides and glycosides.
Value-added products from Withania spp. include capsules, herbal tea, syrups, powder, root powder, root extract, strips, juice, tablets, oil, tonic, decoction, poultice, herbal beer, traditional drugs, and health drinks. Furthermore, there should be assessments on the quantitative improvement and exploration of its value in commercial sectors and herbal sectors. Thus, this medicinal plant is a good target for future studies in the fields of phytomedicines, drug development, and herbal sectors.
In a nutshell, in vivo and in vitro research should be conducted to determine the modes of action and molecular mechanism of phytochemicals for drug development and design. Phytochemicals exhibiting antiviral activity will also be potential candidates for developing antiviral medicines against COVID-19. However, more clinical trials should be conducted to prove its therapeutic and pharmacological properties.
In this review, we have compiled the detailed biochemical profiling of phytochemicals W. Somnifera, their extraction, and its value-added products and therapeutic and pharmacological properties. In summary, many pharmacologically important compounds are present, as illustrated in natural medicinal systems, as well as laying the foundation for the next generation.