Withanolides as Prospective Drug Candidates: Production and Therapeutic Applications–A Review

Keywords

withanolides - therapeutic potential and effectiveness - production enhancement - Withania somnifera - Solanaceae - endophytic fungi

Introduction

Withanolides (WL) are steroidal lactones mainly isolated from the Withania somnifera. The other species of ‘Withania’ and other families like Solanaceae (Acnistus, Datura, Dunalia, Jaborosa, Lycium, Nicandra, and Physalis), Fabaceae, and Taccaceae can also produce them [1]. Apart from plants, the other source of WL production is soft coral (Paraminabea acronocephala [2], Sinularia brassica [3], and Minabea sp. [4]). Withanolide type and content depend upon the chemotype of Withania; for example, Indian chemotypes (AGB002 and AGB025) are rich in withaferin A (WA) and withanone. The Israel chemotypes I, II, and III are rich in WA, WL-D, and -E, respectively, while chemotype I of South Africa is rich in WA and withaferin D [5], [6]. The first isolated WL were WA obtained from the W. somnifera [7] and Acnistus arborescens [8] in 1965.

WL possess different types of activities such as neuroprotective [9], [10], [11], hepatoprotective [12], [13], antiproliferative [14], antidiabetic [15], antimicrobial [14], anti-inflammatory [16], and osteoblastosis [17]. These compounds also exhibit immunomodulatory [18], [19], muscle cell differentiation [20], antiviral against COVID [21], antiadepogenic [22], and anti-psoriasis activities [23]. WL help in the management of chronic diseases such as kidney diseases [24] and hyperlipidemia [25]. The preclinical and clinical findings in recent years demonstrate the significant therapeutic potential of withanolides. Phase-1 clinical trial studies of WA on osteosarcoma patients revealed that the formulation is safe and well tolerated but has very low oral bioavailability. The side effects included skin rash, increased liver enzymes, fever, fatigue, and diarrhoea [26]. Targeting various cancer hallmarks by WA may improve patient therapeutic outcomes by preventing or overcoming drug resistance [27]. The administration of 6 capsules having 5 g/daily of W. somnifera root in 100 infertile male patients revealed improvements in sperm parameters without any side effects [28]. W. somnifera capsules were administered in human volunteers, indicating the absorption of withanosides and withanolides from the stomach, its high oral bioavailability, and an optimum half-life, which aid in significant pharmacological activity. A dose of 500 mg of AgeVel/Witholytin was safe and well tolerated with no adverse reactions when administrated orally [29].

Various trials revealed chondroprotective, anti-inflammatory, and analgesic characteristics of withanolides in human joints without significant adverse effects. There are no FDA (Food and Drug Administration)-approved GMP (good manufacturing process) facilities for the purification and production of withanolides for clinical usage. However, other facilities produce plant capsules and extracts having withanolides that the FDA does not regulate. The recent clinical trials involving the use of W. somnifera extract revealed a significant improvement in anxiety, stress relief, and psychomotor functions in patients with anxiety, bipolar disorder, and schizophrenia. Therefore, clinical and preclinical trials provided safe and efficacious treatment for autoimmune/inflammatory disorders, abnormal cell proliferation, neurodegenerative, and neurobehavioral/psychiatric diseases [30].

The market value of the roots of this plant in 2008 – 2009 was 2 USD/kg, and its turnover was 0.74 M USD/year. W. somnifera extracts are available in the market in various formulations, such as tablets, powders, tonics, and health drinks [31]. The analogue of WA, i.e., 2,3-di-hydro-WA-3B – O-sulfate, is the only WL orally bioavailable as it is water soluble [32].

Previously published reviews mainly focus on WAʼs therapeutic effects and mechanism of action. Limited articles emphasised non-plant-based production approaches, innovative drug delivery systems, or newly discovered WL derivatives. An association of synthetic biology, computational techniques, and biosynthetic pathway elucidation is also briefly discussed. In contrast, this review aims to overcome these gaps by providing a comprehensive perspective on the WL research landscape. Also, this review emphasises the chemical and structural diversity, current sources, mechanism of action, biosynthetic pathways (genes) of withanolides, importance in therapeutics, and current approaches to enhance production. In silico and nano-techniques for better use of WL globally to meet the market demand, plant-host mechanism, and importantly, utilisation of endophytic fungi as an alternative sustainable source for WL are also discussed. This article provides an overview of structural diversity and therapeutic potential of withanolides and their derivatives. The different sources (apart from plants), such as soft-coral and endophytic fungi, for the production of WL, along with their enhancement strategies for production, are described.

Description of Literature Search

A systematic review of the production and therapeutic potential of withanolides has been presented in this study. For this, databases such as Google Scholar, PubMed, and Science Direct were explored. The search strings used in these databases were ‘Withania somnifera’, ‘Withania’, ‘Ashwagandhaʼ, ‘withanolidesʼ, and ʼwithaferin A’. The literature published up to 2024 in English was studied and utilised for this article. The inclusion criteria were solely based on the production, sources, and optimisation for enhanced production of withanolides and their therapeutic potential. On the other hand, the exclusion criteria were steroidal lactones other than withanolides and the therapeutic potential of ‘Withania’ extracts.

Structural Characteristics and Chemical Synthesis of Withanolides

The main structural characteristic of WL is that they contain trans-hydrindane dehydro-d-lactone [33]. Generally, WL are considered 22-hydroxy ergostane-26-oic acid 26, 22-lactones [34]. The backbone of WL is polyhydroxy C-28 steroidal lactone with nine carbon side chains and polyoxygenated C-28 ergostane skeleton-type steroids ([Fig. 1]) [35]. It has five rings, namely A, B, C, D, and E, out of which ring E is the lactone ring [36]. It has a fundamental oxidation characteristic at C-1, C-22, C-23, and C-26 [1], [37]. It is divided into two groups based on oxidation as oxidation at C-22 and C-26 grouped as δ-lactone/δ-lactol-type WL while oxidation at C-23 and C-26 grouped as γ-lactone/γ-lactol-type WL [1]. Mainly, they belong to the δ-lactone/δ-lactol-type WL [37]. The structure of WL is highly diverse because of its side-chain modifications [38]. Substitutions in the side chain of WL correspond to aglycone WL like WA, WL-A, and withanone. On the other hand, its side-chain linkage to glycosides leads to the formation of withanolide glycosides such as sitoindosides, withanoside IV, and physagulin D [35], [39]. WA is an essential bioactive withanolide, chemically known as 5β, 6β-epoxy-4β, 27-dihydroxy-1-oxowitha-2, 24-diexolide. It is also called an oxygenated ergostane-type steroid, having a 2-en-1-one system in ring A in addition to the lactone ring [6].

Fig. 1 General chemical structure of withanolides.

Withanolides can be semi-synthesised chemically using precursors; however, the process involves many time-consuming steps and raises the production cost. Precursors like C-sterol-3 – 28-b,24j-dihydroxy-ergosta-5,25-dienolide are generally used for producing WL [40]. WA was first synthesised from commercially available 3β-hydroxy-22,23-isnorchol-5-enoic acid and further synthesised through several steps from α, β-unsaturated δ-lactone [41]. WL-A is semi-synthesised using the starting material of pregnenolone, a steroid precursor [42]. In addition to this, WA and WL-D are semi-synthesised by the reaction of triphenylphosphine and iodine in dichloromethane, acetylation of acetic anhydride in pyridine, oxidation of manganese dioxide in chloroform, and ethyl acetate mixture, as well as reduction of a complex of sodium borohydride-cerium chloride in methanol [43]. Several reports from the past few decades revealed that Michael addition or nucleophilic edition, as well as modification of hydroxyl groups, leads to the design of various mono-, di-, and tri-substituted WL, which showed high therapeutic potency and bioavailability and less toxicity [14].

Mechanism of Action

The structural diversity of the WL makes them promising pharmacological candidates for curing various diseases [38]. Chemical modifications like adding a side chain or functional group affect their pharmacological properties [39]. The five pharmacological relevant functional moieties are (i) α, β-unsaturated ketone group, (ii) at C-4, a secondary hydroxyl group, (iii) between C-5 and C-6 an epoxide ring, (iv) at C-27, a primary hydroxyl group, and (v) a 6-membered lactone ring associated with α, β-unsaturated carbonyl group ([Fig. 2]). The structurally diverse WL play a vital role in antiproliferative activity by targeting various factors and enzymes responsible for abnormal cell proliferation. Like unsaturated lactone, epoxide, 1-keto-2-ene, 4-hydroxy-5,6-epoxy-2-en-1-one, and 5β,6β-epoxide moieties are responsible for cytotoxic effects. Dissociation of the double bond at C-24,25 and the removal of the hydroxyl group at C-27 (structural modifications) do not significantly diminish the antiproliferative activity of WL [1]. Its AB rings can be modified, but the delta lactone of the CD rings is responsible for its pharmacological properties [32]. Inserting the 16β-OAc group into 4,27-di-O-acetyl-WA increases the cytotoxic effect [43].

Fig. 2 Pharmacological functional moieties of withaferin A.

In WL, a hydroxyl group and another carbonyl group at C-4 disrupt the activity of heat shock protein 90 (HSP90), and the epoxide group at C-5 and C-6 induces the cytotoxic effect against leukaemia [1]. The epoxide ring in the WA interacts with the cysteine and disrupts the HSP90-cdc37 complex [44]. WA is an electrophile, and its susceptible active sites for nucleophile attack are C-3 (unsaturated A ring), C-5 (epoxide), and C-24 (E-ring). Therefore, these sites bind to the HSP90′ cysteine group covalently by Michael addition alkylation reactions, thus causing the loss of target protein activity. Among all the functional groups, the hydroxyl group at C-27 has no significant role in the pharmacological potential. However, this hydroxyl group can be linked to biotin to form biotinylated WA, which further helps in the identification of target HSP90. Likewise, the 2-en-1-one moiety is a pharmacophore at C-3 of WA and gets attached to a cysteine residue to target the cells [27], [45]. Apart from the cytotoxic effect, adding sugars at C-3 by enzyme SGT (sterol glycosyltransferases) showed the antifungal potential of the WL [46]. On the other hand, some modifications in the structural moieties of WL may lead to decreased therapeutic effects. Double bond dissociation at C-2,3 leads to reduced cytotoxic effects in derivatives of WL. Substitution and removing epoxide at C-5 or C-6 nullify the antiproliferative activity of WA [1]. The cytotoxicity effect decreased when the 16β-OAc group was inserted into 4-O-acetyl-WD [43].

Withanolides from Plant Sources

WL can be produced from the various plant genuses Acnistus, Ajuga, Aureliana, Datura, Cassia, Dioscorea, Discopodium, Deprea, Dunalia, Eriolarynx, Eucalyptus, Exodeconus, Hyoscyamus, Iochroma, Jaborosa, Mandragora, Nicandra, Physalis, Salpichroa, Solanum, Tacca, Tubocapsicum, Vassobia, Withania, and Witheringia as discussed in Table 1S (Supporting information). The most common plant source for withanolide production is W. somnifera. The taxonomical classification for this plant is Kingdom–Plantae, Sub-kingdom–Tracheobionta, Superdivision–Spermatophyta, Division–Magnoliophyta, Class–Agnoliopsida, Subclass–Asteridae, Order–Solanales, Family–Solanaceae, Genus–Withania, and Species–somnifera [47]. The botanical name for W. somnifera is Ashwagandha (in Sanskrit), which is commonly recognised as Indian Winter Cherry, Indian Ginseng (in English), poison gooseberry [48], Asgand (in Urdu) [49], Samm Al Ferakh, Kanaje (in Hindi), Ajagandha [50], horse smell, Kaknaje [28], and Sattvic Kapha Rasayana. The word “somnifera” is a Latin word that denotes the “sleep inducers”, and “Ashwagandha” indicates the odour of the horse because the roots of this plant smell like a horseʼs sweat. W. somnifera is also known as Asgandnagori in the Unani traditional and Tibetan systems of medicine [48]. Morphologically, this plant is an evergreen, erect, greyish, and hairy shrub of 30 – 150 cm in height with flashy taproots. The roots are aromatic, fleshy, tuberous, and whitish brown. The leaves are sub-opposite or alternate, round-oval, 5 – 10 cm long, and 2.5 to 7 cm wide. Orange-red berries of this plant surrounded by green calyx carry yellow kidney-shaped seeds ([Fig. 3]) [47], [51]. The suitable conditions required for planting W. somnifera are a height of 1500 m from the sea level, annual rainfall of 500 – 800 mm, 20 – 38 °C temperature, light red or loamy soil, and shade from the sun [48].

Fig. 3 W. somnifera and its parts.

There are various methods to extract WL from the plants, like maceration, microwave-aided extraction, subcritical water extraction, soxhlet extraction, and solvent extraction. Among these, subcritical water extraction yielded more WL than other extraction methods [52]. The alcoholic extract of W. somnifera mainly contains WA, withanone, and sitoindosides [53]. The WL such as WA, WL-A, B, and withanoside IV, V can be extracted from the water extract of W. somnifera at static condition after an incubation time of 10 – 30 min [54].

Biosynthetic Pathway for the Production of Withanolides

Although the whole series of genes and enzymes involved in the biosynthetic pathway of genus “Withania” for withanolide production is still under exploration, [Fig. 4] represents the essential genes and enzymes involved in the withanolide biosynthetic pathway reported to date. Synthesis of WL occurs in the cytosol by the MVA (mevalonic acid pathway) and plastids by the MEP (methylerythritol phosphate pathway), which is also called the DOXP (deoxyxylulose pathway) [55]. Agarwal et al. studied the effect of upregulation and downregulation of biosynthetic pathway genes in W. somnifera on the synthesis of WL. Isopentenyl-pyrophosphate is the intermediate compound formed by acetyl Co-A pyruvate with the help of the MEP/MVA pathway. For this, pyruvate generated by glycolysis of acetyl Co-A pyruvate interacts with glyceraldehyde (in the presence of DXS) to form 1-Deoxy D-xylulose 5-phosphate (DOXP). This DOXP is converted to 2C-Methyl D-erythritol 4-phosphate (MEP) with the help of an enzyme encoded by gene DXR. Then, MEP was catalysed to CDP-ME (by catalysing unit CDPMES), and CDP-ME was converted to CDP-MEP with the help of CDPMEK. CDP-MEP is converted into MECP via MECPS and then MECP to HMBPP by the enzyme encoded by the gene HDS. Finally, the intermediate isopentenyl-pyrophosphate was obtained from the conversion of HMBPP with the help of the catalysing unit HDR [56].

Fig. 4 Biosynthetic pathway to produce withanolides.

On the other hand, acetyl Co-A pyruvate conversion (via the MVA pathway) into isopentenyl-pyrophosphate involves various steps. These steps include converting acetyl Co-A pyruvate to acetyl Co-A with gene AACT. Then, this acetyl Co-A is converted to hydroxymethylglutaryl phosphate (HMG Co-A) with the help of the gene HMGS. HMG Co-A is converted to MVA via gene HMGR, and MVA is converted to MVA-5-phosphate with the help of gene MK. MVA-5-phosphate is converted to mevalonic diphosphate with PMK and ultimately converted to intermediate isopentenyl-pyrophosphate [56].

Thus, the intermediate produced (isopentenyl-pyrophosphate) via the MEP/MVA pathway is converted to GPP with the help of gene GPPS. GPP is then converted to farensyl pyrophosphate with the help of the gene FPPS. Farensyl pyrophosphate is converted to squalene by gene SQS. Squalene is converted to 2,3-oxidosqualene by gene SQE, and 2,3-oxidosqualene is catalysed to cycloartenol by gene CAS. After this, cycloartenol conversion into 24-methylenecycloartinol takes place via gene SMT-1. Following which, 24-methylenecycloartinol is converted to cycloeucalenol by gene SMO1. Then, cycloeucalenol is converted to obtusifoliol by gene CEC1, and obtusifoliol conversion into delta-8,14-sterol takes place by gene ODM. Delta-8,14-sterol is catalysed into 4α-methyl-fecosterol by the gene FK, and 4α-methyl-fecosterol conversion into 24-methylenelophenol is carried out by gene HYD1. Then, this 24-methylenelophenol is converted to episterol with the help of gene SMO2. Episterol is catalysed into 5-dehydroepisterol with the help of gene STE1, and 5-dehydroepisterol is converted into 24-methylene-cholesterol by the gene DWF-5. Thus, the key compoundʼs, 24-methylene-cholesterolʼs, conversion into withanolides takes place with the help of a catalysing enzyme encoded by gene DWF-1 [56].

The withanolides can be produced by various interlinked conversion steps whose catalysing units are unknown to date and are represented as dashed arrows in [Fig. 4]. Likewise, delta-8,14-sterol can also be converted to 24-methylenelophenol, and then 24-methylenelophenol is converted to 24-methylene-cholestrol and ultimately to withanolides. Also, 24-methylenelophenol is converted to sitosterol, then to stigmasterol and ultimately to withanolides. Another interlinked conversion is from 24-methylene-cholesterol to campesterol and, finally, withanolides [56]. The description of genes and their catalysing units are discussed in [Table 1].

Table 1 Genes and their catalysing unit of the withanolide biosynthetic pathway.

Sr. No.	Gene	Catalysing unit
1.	Acetyl Co-A acetyltransferase (AACT)	Acetyl Co-A to Acetoacetyl Co-A
2.	Hydroxymethyl glutaryl Co-A synthase (HMGS-1,2)	Acetoacetyl Co-A to Hydroxymethyl glutaryl phosphate
3.	3-Hydroxy-3-methyl glutaryl Co-A reductase (HMGR-3)	Hydroxymethyl glutaryl phosphate to mevalonic acid
4.	Mevalonate kinase (MK)	Mevalonic acid to mevalonic-5-phosphate
5.	Phosphomevalonate kinase (PMK-1,2)	Mevalonic-5-phosphate to mevalonic diphosphate
6.	Mevalonate diphosphate decarboxylase (MDD)	Mevalonic diphosphate to isopentenyl-pyrophosphate
7.	1-Deoxy-D-xylulose-5-phosphate synthase (DXS-1,2)	Pyruvate and glyceraldehyde to 1-Deoxy-D-xylulose-5-phosphate
8.	1-Deoxy-D-xylulose-5-phosphate reductoisomerase (DXR)	1-Deoxy-D-xylulose-5-phosphate to 2C-Methyl D-erythritol 4-phosphate
9.	2-C-methyl-D-erythritol 4-phosphate cytidylyl transferase (CDPMES)	2C-Methyl D-erythritol 4-phosphate to 4-diphosphocytidylyl-2-C-methyl-D-erythritol
10.	4-diphosphocytidylyl-2-C-methyl-D-erythritol kinase (CDPMEK)	4-Diphosphocytidylyl-2-C-methyl-D-erythritol to 4-diphosphocytidylyl-2-C-methyl-D-erythritol-2-phosphate
11.	4-Hydroxy-3-methylbut-2-enyldiphosphate synthase (HDS)	4-Diphosphocytidylyl-2-C-methyl-D-erythritol-2-phosphate to hydroxy-2-methyl-2-(E) butenyl-4-diphosphate
12.	4-Hydroxy-3-mrthylbut-2-enyldiphosphate reductase (HDR)	Hydroxy-2-methyl-2-(E) butenyl-4-diphosphate to isopentenyl-pyrophosphate
13.	Geranyl diphosphate synthase (GPPS)	Isopentenyl-pyrophosphate to geranyl pyrophosphate
14.	Farnesyl diphosphate synthase (FPPS-1,2)	Geranyl pyrophosphate to farnesyl pyrophosphate
15.	Squalene synthase (SQS)	Farnesyl pyrophosphate to squalene
16.	Squalene epoxidase (SQE)	Squalene to 2 – 3-oxidosqualene
17.	Cycloartenol synthase (CAS)	2 – 3-oxidosqualene to cycloartenol
18.	1-Cycloartenol C-24 methyl transferase (SMT1)	Cycloartenol to 24-methylene cycloartenol
19.	1-Sterol 4α-methyl oxidase 1 (SMO1)	24-methylene cycloartenol to cycloeucalenol
20.	Cycloeucalenol cycloisomerase (CEC1)	Cycloeucalenol to obtusifoliol
21.	Obtusifoliol 14-α-demethylase (ODM/CYP51G1)	Obtusifoliol to delta 8,14 sterol
22.	Sterol delta-14 reductase (FK)	Delta 8,14 sterol to 4α-methyl fecosterol
23.	Sterol C-7,8 isomerase (HYD1)	4α-methyl fecosterol to 24-methylenelophenol
24.	Sterol 4α-methyl oxidase 2 (SMO2)	24-methylenelophenol to episterol
25.	Sterol C-5 desaturase (STE1)	Episterol to 5-dehydroepisterol
26.	1-Sterol delta-7 reductase (DWF5)	5-dehydroepisterol to 24-methylene cholesterol
27.	1-Sterol delta-7 reductase (DWF1)	Methylene cholesterol to withanolides

Therapeutic Potential of Withanolides

Withanolides are the major bioactive compounds in W. somnifera, and of all WL, WA is the most crucial pharmaceutical compound [1]. [Fig. 5] describes the therapeutic biomarker for WL, [Fig. 6 a – d] depicts the structure of some bioactive WL, and Table 2S (supporting information) represents the list of withanolides with their bioactive potential.

Fig. 5 Withanolides and their bioactive markers. U*–Upregulate/increase/enhance/trigger; D*–Downregulate/decrease/inhibit/block

Fig. 6 Structures of bioactive withanolides.

Fig. 6 (continued)

Cytotoxic Effect

The most common hallmarks of cancer are cell death resistance, angiogenesis induction, activating invasion and metastasis, evading growth suppressors, sustained proliferative signalling, deregulating cellular energetics, avoiding immune destruction, tumour-promoting inflammation, genomic instability, mutation, and replicative immortality. WA, the most common withanolide, has been extensively explored for its cytotoxic potential, utilising various experimental models. Exploring WA mainly focuses on molecular targets, in vivo efficacies, and their pharmacokinetic behaviour for cytotoxicity [1]. It is a potential cytotoxic compound that functions as an antiproliferative, anti-migration, pro-apoptosis, and anti-invasive. It is safe as a cytotoxic compound as it is resistant to radiosensitivity and chemosensitivity [6].

Various cancer cell lines have been studied for the effectiveness of WL, including breast, colorectal, pancreatic, cervical, oral, prostate, ovarian, and lung cancers. Among these, 27-deoxy-24,25-dihydroWA, 27-O-glucopyranosylviscosalactone B, diacetyl WA, physagulin D, viscosalactone B, WA, withanoside IV, and 4,16-dihydroxy-5 h, and 6 h-epoxyphysagulin D inhibited the cell proliferation and viability by downregulating the COX-2 enzyme in the lung (NCI-H460), colon (HCT-116), nervous system (SF-26), and breast (MCF-7) cancer cell lines. 3-Azido-WA inhibited motility, invasion, and neovascularisation by downregulating the MMP-2 enzyme in the prostate (PC-3) and cervical cancer (HeLa) cell lines. WA and withanolide analogues also induced the cell cycle arrest by upregulating the apoptosis, G1-phase arrest and G2/M phase, Bim-s, Bim-l, Bim-EL, and β-tubulin, as well as downregulating the 4-HW, PI3K/Akt, XIAP, cIAP, ER-α, IL-6, STAT3, and HSP90 in breast cancer cell lines (MCF-7, SK-Br-3, BUS, and MDA-MB-231). These compounds also increase cytotoxicity by enhancing antioxidant activity, upregulating caspase 3, 8, and 9, and downregulating TNF factor in hepatocellular carcinoma cell lines (Hep G2). WA causes DNA damage, inducing cytotoxicity by promoting telomerase dysfunction and apoptosis by downregulating ROS and Bcl-2 factors in melanoma cells. WA exhibited antiproliferative activity by downregulating the EGFR, ICAM-1, VCAM-1, Akt, and NF-κB in lung cancer cell lines (A549, CL141, CL97, CL152, H441, H1975, and H1299). WA reduced cell survival in various cancer cell lines by modulating key signalling pathways. In lymphoma cell lines (U-937, MDS1, HL-60, THP-1, MOLT-4, and REH), WA downregulated critical kinases such as HSP90 and NF-κB, while upregulating p38 and JNK. In malignant glioma cell lines (U-87, U-251, and GL26), WA inhibited cell proliferation by upregulating apoptotic markers and HSPs. In colorectal cancer cell lines (HCT-116, SW-480, and SW-620), WA promoted cell death due to upregulating c-Jun N-terminal kinase (JNK) and downregulating Notch-1, Akt, NF-κB, Bcl-2, Mad2, and Cdc20, contributing to G2/M arrest and disruption of the spindle assembly checkpoint. Additionally, WA decreased cell viability in renal cancer cell lines (Ca Ski) by downregulating Bcl-2 and promoting apoptosis via increased caspase-3 activity, PARP cleavage, and accumulation of cells in the sub-G1 phase [1].

LSD1 (lysine-specific demethylase 1) acts as a demethylase of lysine 4,9 & histone 3 and can be a prospective target for cancer therapy. In silico and in vitro studies revealed the effectiveness of WA against the MDA-MB-231 cell line via inhibiting activity of LSD1 [57]. In combination with withanone, WA upregulated p53 and p21 in osteosarcoma (U2OS and TIG) and hepatocarcinoma cell lines (HUH-6 and HUH-7), respectively. This combination also downregulated hnRNP-K, VEGF, and metalloprotease in metastatic cancer cell lines (A-172, YKG-1, MCF-7, U2OS, HT1080, and IMR-32) and hence induced cell death. WA, combined with caffeic acid phenethyl ester, downregulated the mortalin and upregulated the p53 in ovarian (SKOV-3 and SOKV-18) and cervical (SKG-II, SKG-III b, ME-180, and HeLa) cancer cell lines. WA induces cancer cell death by downregulating the survivin, IL-6, TNF-α, COX-2, p-Akt, Notch1, and NF-κB in breast and colon cancer animal models. It also inhibited tumour growth in prostate cancer animal models by downregulating the Par-4, NF-κB, P13K/Akt, and survivin, and upregulating the apoptosis [1].

WA is effective against neuroblastoma by inactivating glutathione peroxidase 4 [58]. It is active against hepatocellular carcinoma by affecting Akt signalling and mediated cell death by the FOXO3a-Par-4 and p38 pathways, as well as by increasing the phosphorylation of ERK [59]. WA decreased the proliferation of pancreatic cell lines (PANC-1, MIA-PaCa-2, and BxPC-3) by disrupting the HSP90-Cdc37 complex [44]. It inhibits cell proliferation in MDA-MB-231/SUM-159 (by blocking the notch 2 pathway) and in MDA-MB-231, T-47D, A549, and H1299 cell lines [60], [61]. WA and 5FU showed synergistic effects against colorectal cancer cell lines (HCT-116, HT29, and SW480) [62]. WA initiates the process of ferroptosis, in which the iron level is increased due to heme oxygenase-1 activation and Gpx4 depletion, which helps fight neuroblastoma [63]. It showed antitumour activity against colon cancer cell line HCT-116 by inhibiting the STAT3 signalling pathway in balb/c mice at 2 mg/kg [64]. The two-pore potassium channel (TASK-3) is the new target for cancer treatment in oncology. Pharmacological obstruction, overexpression, gene downregulation, patch-clamp, and molecular docking studies revealed that WA could inhibit the TASK-3 in a dose-dependent manner. The docking studies showed that mutation in the residues of TASK-3 affects the binding of WA. It decreases the expression of TASK-3 when treated on breast cancer cell line MDA-MB-231 [65].

WA disrupts the interaction of HSP90-cdc37, adversely affects the chaperonʼs function, and inhibits IκB kinase [66]. It depletes MGMT and induces apoptosis in glioblastoma cells by Akt pathways. WA downregulates EGFR and PI3K in cancer cell lines including A549, CL97, CL141, CL152, H441, H1299, H1975, and MIA-PaCa-2. In xenograft mouse models, WA reduces tumour growth by downregulating Bcl-2, Hey1, Hes1, Notch1, and XIAP [67]. Additionally, WA inhibits targets such as β-tubulin, chymotrypsin, aldehyde dehydrogenase 1, HSP90, the STAT3 pathway, MMP-9, ATR, and Notch1 signalling. It also upregulates tumour-suppressive or regulatory genes including p53, PP2A, PPP2R1A, Bcl-2, Par-4, BRMS-1, and DR5 [39]. WA inhibits breast cancer cell lines by affecting the mitochondriaʼs complex III and its dynamics [45]. It is helpful in various DNA damage repair mechanisms; it degrades FANCA proteins and repairs the DNA double-strand break repair pathway [68]. Diabetes can also induce cancer by producing RAGE (receptors of advanced glycation end products). WA can inhibit the RAGE products so that diabetes-mediated cancer will not occur [15]. A study revealed that WA kills the cancerous cells and protects the normal lymphocyte cells when exposed to IR-radiation treatment by activating miceʼs ERK/Nrf-2/HO-1 axis. It decreases DNA damage (due to IR-radiation exposure) to bone marrow and lymphocytes by activating the Nrf-2 pathway (cytoprotection) and inhibits the activation of caspase [69]. The IC₅₀ dose for WA against various cancers is 1.8 – 6.1 µM. This compound exhibited IC₅₀ of 500 nM against vimentin inhibitors and showed no toxicity at even a higher dose of 2000 mg/kg, although it showed poor oral availability [32]. WA is mostly used to treat glioblastoma as it inhibits vimentin by binding with cys328 in the α-helix region [70].

In addition, molecular docking studies revealed that WA has an affinity to bind with various cancer targets and enzymes that help cure cancer [1], [35]. It has an excellent binding relationship with the ABL kinase domain, Bcl-2, Bcl-XL, EGFR, VEGFR, MDM2, mTOR, p53, estrogen, and progesterone receptors [1]. WA and withanone N are supposed to be effective against lung cancers driven by EGFR. It showed similar antiproliferative activities as drugs like erlotinib and poziotinib [71]. The characteristic feature of prostate cancer is its high oxidative stress and high metabolic rate, which attains resistance to cancer treatments. Therefore, a novel therapy is required to increase oxidative stress and inhibit the G3BP1 (cytoprotective stress granule protein). The SILAC-based approaches in proteomics revealed that WA targets the G3BP1 protein and upregulates genes for oxidative stress, which predicts the use of WA for the treatment of prostate cancer in a non-resistive manner [72].

In silico studies have predicted that WA and their derivatives are effective against breast cancer stem cells. The cancer stem cells are the root cause of the reappearance of cancer and its metastasis. Therefore, targeting cancer stem cells is essential to protect against further reoccurrence. The breast cancer stem cell markers (BRCA1, HSP70, HSP90, and NF-κB) were studied to interact with WA and its acetate. These showed a high binding affinity in the range of − 55.241 to − 40.250 kcal/mol and an IC₅₀ value of 1.476 µM against MCF-7 cancer cell lines. Molecular simulating and docking studies revealed that disruption of HSP90-cdc37 by WA is thermodynamically dependent [44]. However, further validation studies are required to determine the efficacy of WA for breast cancer stem cells [73]. WL-D, E, G, withasomniferol B, C, and 27-hydroxy withanone are potent therapeutic molecules for the HAT oncogene (KAT6A) [74].

WA is engaged in clinical trials to determine its impact on various cancer types [75]. Phase-1 trial studies on osteosarcoma patients were performed using a 3 + 3 design in which four doses (72, 108, 144, and 216 mg) were selected. The studies revealed that the formulation is safe and well tolerated but has very low or no oral bioavailability. The side effects included skin rash, increased liver enzymes, fever, fatigue, and diarrhoea [26]. Only a few studies have evaluated the cytotoxic potential of WL (other than WA) derived from W. somnifera [1]. Thus, there is a further need for clinical studies to establish the efficacy and safety of WL to cure cancer.

Neurological

The most common brain disorders are Alzheimerʼs, addiction, anxiety, bipolar disorder, depression, autism spectrum disorder, attention deficit hyperactivity disorder, dyslexia, Huntingtonʼs disease, Parkinsonʼs disease, amyotrophic lateral sclerosis, and schizophrenia. WA at a concentration of 27.1 µM and 2 µM (IC₅₀) induced the cytoprotective pathway (PI3K/mTOR) and disrupted aggregation of α-synuclein and beta-amyloid, respectively. WL-A, B, and WA showed anti-addictive effects at a concentration of 5, 10, and 20 mg/kg, respectively, when administered intraperitoneally in male albino mice [48]. WA is helpful in treating brain stroke by upregulating P13K/Akt protein and can cure neuroinflammation in the spinal cord of a rat model [9], [10].

WA and WL A, D – P can treat Huntingtonʼs disease [11]. WL-A plays a significant role in Huntingtonʼs disease by increasing the antioxidant mechanism via increasing the levels of antioxidant enzymes such as CAT, Gpx, and SOD and helps in recovering from ischemia-reperfusion injury. Withanoside IV is beneficial in Alzheimerʼs disease by curing synaptic damage due to aggregation of beta-amyloid [49]. WL such as WL-A, B, and withanoside IV, V were found to reduce the accumulation of β-amyloid (1 – 42) as revealed by in vitro studies. IC₅₀ values for the cytotoxic activity against the SK-N-SH cell lines were 28.61 ± 2.91 µM (WL-A), 14.84 ± 1.45 µM (WL-B), 18.76 ± 0.76 µM (withanoside IV), and 30.14 ± 2.59 µM (withanoside V) [76]. Utilising in vitro studies, the author revealed that withanoside IV isolated from W. somnifera showed inhibition of β-amyloid peptide (25 – 35), a causative factor for Alzheimerʼs disease [77].

In silico studies predicted that 27-hydroxy withanolide B and withanone are effective against Alzheimerʼs disease, and WL (E, F, G, J, M, N) inhibit gelatinase enzymes. Also, WL-B, G, stigmasterol, and WA showed a high binding affinity towards PARP-1 and inhibited both nNOS (eNOS and iNOS). WL-A showed reduced neurodegeneration in a corticosterone-dependent manner and retrieved memory defects in Sprague–Dawley rats and male ddY mice, respectively, at 10 µmol/kg concentration. It regulates neuronal cell markers and regenerates neuritis and synapses. Withanoside IV and V are reported to inhibit AChE and BChE in a non-competitive manner and linear mixed types, respectively. Glycowithanolides are helpful in stress, memory learning power, anxiety, and depression [48]. In silico studies showed that WL-A, B, and withanoside IV, V bind with the LVFFA region (β-amyloid_1 – 42 inhibitory site) [76]. Autodock Vina software predicted that withanone and 27-hydroxy WL-B were effective against Alzheimerʼs disease after binding with monoamine oxidase, beta-secretase 1, and phosphodiesterase [78]. Molecular docking studies predicted that WL-G can be a potential inhibitor of ROCK2 (Rho-associated kinase 2) and prevents cerebral stroke [79]. These studies indicate promising applications of WL to treat various neurological disorders.

Anti-Inflammatory

Inflammation generally affects innate immunity and is regulated by inflammasomes. WA triggers expression of caspase-1 protein, and AIM2 activates NF-κB, inhibits IL-1β;18, and suppresses the release of TGF-β. It reduces inflammasome activation (NLRP3 mediated) and modulates the polarisation of macrophages [80]. It downregulates NF-κB and facilitates the release of pro-inflammatory cytokine and production of IL-18, IL-1β, caspase-1, PGE2, and COX2 expression [66]. It prevents inflammation triggered by Helicobacter pylori by downregulating IL-8 [81]. WL at 200 mg/kg showed anti-inflammatory activity in rat models [82]. Withanone protects male Wistar rats from anti-inflammatory proteins and oxidative stress at 10 – 20 mg/kg [48]. In silico analysis predicted that withanoside IV, V, WL-A, B, and sitoindoside IX exhibited inhibition against the NLRP₉ factor (associated with inflammation) [83].

Anti-Viral (COVID)

WL and withanosides are reported to have potent antiviral activities. The Serum Institute of India and the University of Pune use WL as a vaccine adjuvant, which increases the effectiveness of the vaccine against COVID-19 in a dose-dependent manner when administered parenterally, and this invention has been granted a US patent [84]. The steroidal lactones like WA, WL-A, D, 12-deoxywithastramonolide, sitoindosides, and withanoside V managed COVID-19 by improving cell-mediated immunity. These WL can control the synthesis of α-2 macroglobulin during inflammation and lower oxidative stress [21]. Withanone, WA, withanoside IV, and V interrupt the electrostatic interaction of viral RBD with host cell ACE2, inhibit Mpro, and hence block entry of SARS-CoV-2 [84], [85]. Withanone and WA (at a concentration of 20 – 40 µM) catalytically change serine protease residue (TMPRSS2), essential for SARS-CoV-2 entry. On the other hand, withanone, withanoside V, and methoxy WA inhibited the activity of TMPRSS2 (transmembrane protease serine 2) and Mpro [71]. WL-G, M, and I modulate the patientʼs Th-1/Th-2 immune system and have a high affinity for PLpro, spike proteins, and 3CLpro, respectively [86]. Withanoside II showed anti-COVID activity by binding with RBD-ACE2, Mpro, TMPRSS2, PLpro, NSPIS, RdRp, and NSP12 receptors as revealed in silico studies [87]. The two main targets of SARS-CoV-2 are NSP15 and RBD proteins, as shown in silico analysis [84].

Others

T-cell motility plays an essential role in autoimmune reactions and adaptive immune responses. WA at a concentration of 0.3 to 1.25 µM disrupts the migration of T-cells by interacting with 273 proteins in T-cells; therefore, it could potentially prevent autoimmune pathologies [18]. Transplantation of bone marrow sometimes leads to acute graft-versus-host disease, which must be controlled before its occurrence. In a study by Mehta et al., WA given to donors before bone marrow transplantation reduced damage in recipients (caused by the acute graft-versus-host disease) [19]. It helps treat bone injuries by inducing osteoblastogenesis. It degrades Runx2 and Smurf2 proteins responsible for osteoclastogenesis via the MAPK pathway, overcomes bone loss by inhibiting NF-κB signalling, and inhibits osteoclastogenesis [17], [88]. It helps treat chronic kidney diseases resulting from stress in the endoplasmic reticulum. It increases endoplasmic reticulum proteins, i.e., ATF4, caspase12, CHOP, GRP78, GRP94, and phosphorylated eIF2α [24].

WA is also helpful in lipid metabolism and bile acid circulation. It significantly decreased lipid levels in serum, bile acid uptake, and reductase activity of HMG-CoA and increased Cyp7a1 hepatic protein [25]. It can cure miceʼs liver inflammation, injury, and necrosis [89]. It prevents liver toxicity at a concentration of 7 mg/kg (induced by APAP) in C57BL/6 J mice by activating Nrf2 proteins [12], [90]. It showed an antileishmanial effect by inhibiting the PTR1 enzyme [90]. This compound is also helpful in curing male reproductive problems, such as reproductive problems linked to endocrine, oligozoospermia, erectile dysfunction, and semen quality [28], [91].

WA synergistically showed activity (in vivo and in vitro) against inflammation and antibacterial (against bacteria Staphylococcus aureus) [92]. Novel WL, such as withasilolides G, H, and I, suppressed lipid droplet enlargement. These WL at a concentration of 25 µM decreased the expression level of markers for adipocytes (Adipsin and FabP4). These compounds also upregulate the expressions of lipolytic genes (ATGL and HSL), as well as downregulate the expressions of the lipogenic gene (SREBP1) [22]. WL can be a promising drug candidate for treating psoriasis as it suppresses the STAT3, ERK1/2, and P38 signalling pathways in imiquimod-stimulated HaCat cells [23]. WL-rich extract, especially withanone, modulates the differentiation of muscles like myoblasts into myotubes, autophagy, hypoxia activation, and disaggregation of protein aggregates. These findings are helpful in the repair and improvement of muscles in Parkinsonʼs disease models (Drosophila). It is useful in differentiating muscle cells in C2C12 muscle cell lines by differentiating myoblast into myotubes, disintegrating metal, heat stress-induced protein aggregates, autophagy, and hypoxia pathway activation [20]. In investigations using in silico studies, the author predicted that WL-A exhibits antidiabetic activity by inhibiting α-amylase and α-glucosidase. This compound also possesses better pharmacological properties than standard acarbose, like minimal toxicity, higher bioavailability, good absorption, and metabolism [93]. In silico studies showed that the main target of WA for disruption of T-cell migration is binding to zeta-chain-associated protein kinase 70 (ZAP70) at cysteine residues 560 and 564. The Western blotting and bait-pulldown method further confirms it. In silico studies by Autodock Vina 4.5 software showed the potential of WA in treating cardiovascular diseases by binding with HMG-CoA, angiotensinogen converting enzymes, and β1-adrenergic receptors [78]. Hamada et al. studied the therapeutic effect of WA on an alcoholic liver disease C57BL/6 J mice model (in vivo) and ethanol-treated primary hepatocytes (in vitro). WA significantly reduced the levels of hepatic lipogenesis genes in both the cases in vivo and in vitro. Thus, this compound can be considered a hepatoprotective agent, especially in ethanol-related lipid accumulations [13]. The anti-obesity properties of WA were studied in C57BL/6 mice, and they showed that it increases energy expenditure and diminishes obesity. The mechanism of action behind the treatment of obesity is the induction of AMPK and activation of the MAPK pathway, which triggers the biogenesis of mitochondria [94].

Apart from WLs, other phytoconstituents help in managing diabetes, as diabetes is becoming the seventh leading cause of death worldwide. Biguanides do not depend upon the bodyʼs insulin, as exhibited in an enhanced pancreatic spectrum of action. Peripheral glucose utilisation, increased muscle sensitivity to insulin action, and reduced intestinal glucose absorption was stimulated. Another compound, thiazolidinediones, binds with the peroxisome proliferator-activated γ-receptor, expanding the muscle glucose uptake and reducing endogenous glucose production. Agents related to the above compounds, like pioglitazone and rosiglitazone, are commercially available and used worldwide. Insulin is a well-known compound used for the treatment of diabetes, but it is orally ineffective. The first orally effective agent against diabetes was synthalin-A, which was found to be toxic later. After that, sulphonamides came into the picture with a blood-glucose-lowering effect, and since then, a lot of sulfonylureas have been utilised as an oral antidiabetic compound. Also, phenformin, a biguanide, was used as an effective, less toxic oral antidiabetic drug. Other compounds like curcumin (isolated from turmeric) and S-allylcysteine (derived from garlic) lower blood glucose levels in diabetic rats. The mechanism of action of S-allyl cysteine involves glucose uptake and metabolism promotion and inhibition of hepatic gluconeogenesis, which showed an insulin-like effect on the peripheral tissues [95].

Therapeutic Effectiveness

Despite WL being reported to have potent biological activities, there is a need to improve therapeutic efficacy as they have poor bioavailability, water solubility, and permeability. Hence, therapeutic efficacy can be increased by fabricating WL using phospholipid complexation (naturosomal delivery). This complex molecule increases the overall solubility of WL by 14-fold and aqueous solubility by 1.3-fold and enhances the stability and bioavailability of WL by more than 95%. Therefore, WL entrapped in the naturosome can be an alternative delivery system to enhance bioavailability, solubility, and permeability [96].

WA is effective as a cytotoxic compound but has significantly less or poor solubility in water. That is why there is a need for the conjugates of WA, which are water-soluble. The WA-polymer conjugate was prepared by grafting using a technique in which the OH group at C-27 of WA was modified with CTA (chain transfer agent). This is done to polymerise RAFT (reversible addition-fragmentation chain transfer) via an ester bond (degradable). The polymerisation of N, N-dimethyl acrylamide (hydrophilic), and RAFT results in a WA conjugate that is water soluble. Although this conjugate was tested for cytotoxicity activity, it did not give significant results because WA conjugates require ester hydrolysis for their action [97]. In the thin film hydration method, PEGylated nano liposomal WA was synthesised with high stability, having a particle size of 125 nm and 83.65% encapsulation efficiency. In vitro studies revealed that encapsulated WA showed increased sustained release and significantly reduced cell growth in DLA and EAC cell lines compared to free WA [98]. WA gold nanoparticles decreased cell growth in the breast cancer cell line (SKBR3) [1]. Exosomes loaded with WA showed improved treatment for human breast cancer (T47D, MDA-MB-231) and lung cancer cell lines (H1299, A549) compared to free WA [99]. Also, when WA was encapsulated in mannose liposomes, it was found to treat arthritis in rats compared to free WA [100]. A liposomal-mediated WA drug delivery system was highly effective against pancreatic cancer compared to free WA. For this study, WA was encapsulated in C16-lipopeptide, which inhibits cell proliferation in CD13 receptor-mediated pancreatic cancers, as well as in angiogenic endothelial cells. The C-16-lipopeptide complex was prepared by conjugation of (NGR) homing peptide with C-16 aliphatic twin chain lipid with the help of K (lysine) spacer [101].

Inexpensive cervical polymeric implants were synthesised, mushroom-shaped, containing 2 – 4% WA, and inserted in the cervix of non-pregnant adult goats (Boer). On the implantation site, there was an accumulation of WA at a concentration of 12 – 16 ng/tissue, but in distal areas, no WA was accumulated. This study may help treat various cancers in target-based administration of WA [102]. Niosome is a novel drug delivery formulation that is mainly considered a promising approach for treating cancers. A cholesterol-based system and a non-ionic surfactant were used proportionately to prepare the niosome. The potential steroidal lactone WA was formulated as a niosome in the nanovascular system and tested against HeLa cell lines. Its cytotoxic effects increased three times compared to free WA. W. somnifera leaf extract–magnesium oxide nanoparticles were synthesised using the green combustion technique. The synthesised nanoparticles were further screened for their characteristic features and bioactive potential [103]. The WA gold nanoparticles and their conjugation with glucocorticoid lead to the delivery of WA to the target tissue, inhibiting tumour progression [32].

Nanoemulsion of W. somnifera leaf extract was administered to rats having penconazole-induced neurotoxicity and observed for neuroprotective activity. After 6 weeks, improvement in performance related to behaviour, anti-oxidative enzymes, and anti-inflammatory cytokines was observed. This improvement is due to the downregulation of the GFAP, APP, vimentin, TGF-β1, Bax, and Smad2 expression. This also effectively crosses the blood-brain barrier and shows neuroprotection [104].

Ashwagandha is formulated with cow ghee. This Ashwagandha ghrita (AG) formulation was predicted to be safe up to 2000 mg per kg of body weight, as revealed by molecular docking and network pharmacology. It predicted a dose-dependent increase in intromission and mount frequency, anogenital sniffing, and genital grooming at 150 and 300 mg per kg of body weight, revealing aphrodisiac activity. Experimental studies showed the corpus cavernosal smooth muscle relaxation at all the concentrations of AG in a dose-dependent manner. Thus, AG possessed an aphrodisiac effect and was supposed to be a traditional ʼVajikarana Rasayanaʼ [105].

Boswellic acid (pentacyclic triterpenes) produced from Boswellia serrata was reported to treat rheumatoid arthritis. However, its shortcomings were poor water solubility and low oral absorption. Therefore, the naturosomal formulation of boswellic acid was prepared to enhance the bioavailability, water solubility, and colloidal stability. The studies showed colloidal stability and 16× improvement in the water solubility with the naturosomal-formulated boswellic acid. The recovery rate of boswellic acid from the naturosomal formulation is > 99%. In vivo studies in rodents (with arthritis) revealed reduced paw thickness, paw volume, and TNF-α [106].

The naturosomal formulation is used to enhance the therapeutic effectiveness. In a study, the Bacopa naturosome formulation and Centella naturosome formulation were prepared using the solvent evaporation method. For this, the formulations and process variables were optimised using a central-composite design. The prepared formulation was evaluated and confirmed by SEM (scanning electron microscopy), FTIR (Fourier transformed infrared spectroscopy), photomicroscopy, PXRD (powder X-ray diffraction), and DSC (differential scanning calorimetry). Both naturosome formulations exhibited enhanced water solubility, permeation rate (> 90%), and > 97% recovery rate of Bacopa, as well as a 99.2% recovery rate of Centella from the formulation. Thus, the naturosomal delivery system exhibited a promising strategy for the enhanced solubility of bioactive phytoconstituents [107], [108]. In vivo studies of Centella naturosome formulation revealed improved spatial learning and memory in elder mice [107].

Antidiabetic compoundsʼ potency and efficacy mainly depend on their chemical structures. Therefore, future research must modify the lead compoundʼs chemical structures to produce effective antidiabetic compounds. The SAR study helps in the development of effective antidiabetic compounds by improving bioavailability and potency with lesser/no side effects [95].

The quantitative structure–activity relationship (QSAR) in silico model was used to check the toxicity, as well as the pharmacokinetic and physiological properties, of 75 withanolides. Due to high molecular weight, pKa value, lipophilicity, and low aqueous solubility, some WL showed high human plasma protein binding, jejunal permeability, extensive metabolism, hepatic uptake, renal excretion, and tissue partitioning. The toxicity studies of oral administration of WL in rats revealed that for 2/3rd WL, the lethal dose was found to be below 100 mg/kg [35].

Approaches to Enhance the Production of Withanolides

Biotechnological approaches like culture condition optimisation, chemical induction, epigenetic modification, co-culture, and fermentation technology are essential in producing potent pharmaceutical compounds [109]. There is an inadequate amount of WL, so it is challenging to meet the market demand. Therefore, it is necessary to optimise and develop a technique that industries can quickly adopt to produce WL to meet market demand cost-effectively [75]. Elicitors are the substances or factors that help plants to enhance the production of secondary metabolites to ensure their competitiveness, persistence, and survival. In vitro plants or plant cells show morphological and physiological responses to various abiotic and biotic elicitors [110]. Genome manipulation of the source can produce WL more significantly [111]. Table 3S (supporting information) represents the list of factors that enhanced the production of withanolides.

Abiotic and Biotic Elicitors

The exogenous treatment of elicitors is the most efficient technique for increasing the production of secondary metabolites in plants [75]. Some elicitors induce the production of various WL in hairy root cultures of W. somnifera. These elicitors are triadimefon (100 mg/L), salicylic acid (150 µM, 100 µM, and 1 µM), chitosan (100 mg/l), methyl jasmonate (20 µM), salacin (750 µM), copper sulphate (100 µM), aluminum chloride, picloram (1 mg/l), and potassium nitride (0.5 mg/l), which are responsible for the induction of WL and also increase its content. In the medium, carbohydrate sources such as fructose, glucose, and maltose, as well as sucrose (2% and 6%) and nitrogen sources such as adenine sulphate, ammonium nitrate, L-glutamine (20 mg/l), potassium nitrate, and sodium nitrate, positively affect the production of WL in plants by 1.14 – 1.18-fold [32], [110], [112]. Treating the leaves (covered with polybags) of the plant with 20 µM methyl jasmonate for 4 h increases WL content by 2.2-, 1.9-, 2-, and 2.2-fold of WA, WL-A, D, and withanone, respectively [75]. WL is mainly derived from ergostane using isoprene as a plant precursor [39]. Zinc-silver nanoparticles enhance the yield of the WL in the W. somnifera plant compared to other nanoparticles like nickel nanoparticles and cadmium selenium nanoparticles [113]. Adding cholesterol, squalene, and mevalonic acid enhances the WL content in the plant cell culture. This squalene enhances the WL content by 1.37-, 1.17-, 1.35-, 1.27-, 2-, 1.63-, and 1.2-fold in WL-A,B, WA, withanone, 12 deoxy withanstramonolide, withanoside IV and withanoside V, respectively. Isoprenogenesis is the crucial step for the biosynthesis of WL, which involves using precursor isoprenoids. Slight physical changes in cultural conditions may vary the production of WL. The red laser light of 632.8 nm wavelength and at a concentration range of 10, 15, 20, and 25 J/cm² was irradiated on the cell culture of W. somnifera. It was found to increase WL content at a concentration of 15 (0.52 µg/mg) and 20 (0.60 µg/mg) J/cm² [114]. Slightly acidic pH (pH-6) and sonication for 15 seconds at 41 °C for 15 min. trigger increased production of WL-A. Cell culture in 2.5 – 5 l bubble column reactors yielded maximum production of WL compared to shake flask [110].

Several studies have been reported on enhanced WL content in plants by upregulating WL biosynthetic genes using endophytes [115]. Mishra et al. reported enhanced production of WL in plants by upregulating its biosynthetic genes with the help of bacterial endophytes (Bacillus amyloliquefaciens and Pseudomonas fluorescens) associated with W. somnifera. These bacterial endophytes modulate WL content, reduce the pathogenic effect of Alternaria alternata in plants, and lessen the production cost of expensive WL [116]. Homogenate culture (3%) of Piriformospora indica enhances WL content (WL-A: 1.7-fold, WA: 1.5-fold, and withanone: 1.5-fold) in plants by upregulating their biosynthetic genes [117]. Homogenate of Aspergillus niger, seaweed extract of Gracilaria edulis, and Verticillium dahaliae extract, Sargassum wightii, increases WL content [110]. It can also be semi-synthesised by biotransformation with the fungus Cunninghamella echinulata [43]. The content of WA can be improved by inoculating arbuscular mycorrhizal fungi (Rhizophagus irregularis) along with the cell culture medium [118].

Upregulation of Central Genes of the Biosynthetic Pathway

Although factors such as abiotic and biotic affect the production of WL, there is also a need to develop a strategy for the consistent, stable, and high output of WL; therefore, upregulation of the central gene of the biosynthetic pathway is required [1]. WL are diversified due to glycosylation carried out by the SGT enzyme. This enzyme uses steroidal alkaloids as a substrate for further modifications commonly present in Solanaceous species. Squalene synthase and squalene monooxygenase are responsible for triterpenoid biosynthesis, and genes encoding DXS, DXR, and HMGR are involved in the biosynthesis of isoprenoids [110]. The other precursors of WL production are 24-methylene cholesterol and 24-methyl desmosterol. The genomic and transcriptomic data revealed a key enzyme, sterol Δ²⁴ isomerases (24ISO), for WL production encoded by the DWF1 gene. This enzyme catalyses 24-methylene cholesterol into methyl desmosterol (a characteristic step for WL production). Thus, the DWF1 gene plays a significant role in producing WL and its derivatives on a large scale [38]. The 24-methylene cholesterol is the main intermediatory compound for WL synthesis in its biosynthetic pathway [36]. Biosynthesis of WL from 24-methylene cholesterol occurs by modifications, desaturation, epoxidation, hydroxylation, and glycosylation. The expression of two genes, STE and DWF-5, have been studied, indicating the different expression levels in other tissues. The expression of these genes is higher in young leaves and chemotypes treated with methyl jasmonate and salicylic acid. The DWF genes are localised in the endoplasmic reticulum. Two precursors (dimethyl allyl pyrophosphate and isopentenyl pyrophosphate) exist in the biosynthesis of WL [56].

The upregulation of the squalene synthase gene increased the yield of WL (WA, WL-A, B, and withanone) by 1.08 – 1.25% in the hairy roots of a plant. It regulates the pathway of WL synthesis in the plant. It produces WL by forming farnesyl pyrophosphate using NADPH in various chemical reactions. On the other hand, the upregulation of steroidal glucosyltransferase-4 enhances the production of WL-A in hairy root cultures [119]. Also, the overexpression of Δ²²-desaturase leads to the increased accumulation of WL and stigmasterol [120]. Methyl jasmonate also increased the expression level of squalene synthase, cycloartenol synthase, squalene epoxidase, and 3-hydroxy-3-methylglutaryl coenzyme A reductase genes, essential genes in the biosynthetic pathway of WL [75]. Using methyl jasmonate and salicylic acid increased the expression of squalene epoxidase, squalene synthase, and WsCPR2 [111].

Many genes, like ASAT, CAS, CYP710A, DWF1, PSAT, SMT, SSR2, and SGT, play a significant role in the production of phytosteroids via biosynthetic pathways in plants. SGT genes help plants to combat infections (caused by A. alternata and Spodoptera litura) and stress [121]. Gene silencing of the CAS gene (cycloartenol gene; biosynthetic gene of WL synthesis pathway) via RNAi increases WL content [122]. The SMT1 gene plays a vital role in plantsʼ biosynthesis of WA [32]. The cytochrome P450 enzyme plays an essential role in forming WL as it is one of the critical enzymes in its biosynthetic pathway. In a study, gene silencing and induced gene expression of three genes of cytochrome P450 enzyme such as CYP71B10, CYP76, and CYP749B1 were analysed for WL production. When treated with methyl jasmonate, all three genes have the highest induced gene expressions in leaves. Gene silencing of CYP450 genes leads to an increase in WA and reduces WL-A and B content. On the other hand, overexpression of CYP450 genes leads to a rise in WL-A, B and reduces 12-deoxywithastramonolide content. Thus, these findings indicated that CYP450 genes are crucial in WL production [123]. The in silico modelling studies revealed that the SGT gene in W. somnifera has the highest specificity for stigmasterol. This gene is also specific for 27β-hydroxyl steroidal lactones in plants. Biotic and abiotic stress in plants leads to different ratios of sterols and glucosidal sterols based on variations of SGT enzyme numbers. This enzyme also helps the plant to combat heating effects. The overexpression of the SGT gene leads to higher production of steroidal lactones in plants [46].

The 27-β-hydroxy glucosyltransferase enzyme isolated from plant leaves showed specificity to UDP-glucose. This enzyme added the β-OH group to C-17 (provided α-OH group at C-17) and C-2, C-27, and glucose to the alkanol present at position C-25, although it did not add an OH group at C-3. This enzyme resembles the glucuronosyltransferase analysed by peptide fingerprinting. This enzyme showed upregulation in the presence of salicylic acid [124]. Isoprenogenesis leads to the production of WL via non-mevalonate and mevalonate pathways in the ratio of 25 : 75. The exact mechanism of production of WL from 24-methylene cholesterol or 24-methylene lophenol or campesterol or intermediates of the triterpene pathway, as well as analogues of lanosterol, is still unknown. Therefore, dimethyl allyl pyrophosphate, IPP, and isopentenyl diphosphate are considered critical intermediates for the production of WL via MVA (in the cytosol) and the DOXP/MEP (in plasmid) pathway [34]. The mono-oxygenase (P450) enzymes help in metabolising the core structure of WL. The two P450s (WsCYP98A and WsCYP76A) were studied for their expression in Escherichia coli BL21 (DE3) by the vector pGEX4T-2 in different parts of the plant and also after the treatment with the elicitors. The expression level after performing RT-PCR showed a high expression of WsCYP98A in stalks and of WsCYP76A in roots. The use of elicitors is also helpful in increasing expression levels, indicating that the stress conditions may lead to the increased accumulation of WL [111].

Endophytic Fungi as a Promising Source of Withanolide Production

The primary source of withanolide production is plants, but this has become challenging for making the production of withanolides sustainable. For example, the production of roots of Ashwagandha at present is 1500 tonnes, but todayʼs need is 7000 tonnes [47]. The production of W. somnifera is only 24%, which is one-fourth of the required global demand. Plant tissue culturing (tissue and organ culture, bioreactor technology, shake flask method, and agro-mediated hairy root production) meets the rest of the worldwide demand. These techniques have limitations as they are more expensive, tedious, require expertisation, and are time-consuming [125]. Despite having high therapeutic potential, W. somnifera (wild and cultivated) is generally susceptible to fungal pathogens like A. alternata, Myrothecium roridum, and Fusarium oxysporum. Among them, A. alternata is the leading cause of leaf spot disease, which affects metabolite production and causes lung infection in humans.

Moreover, fungicide spray leads to resistance against A. alternata, and residual fungicide in the roots leads to toxicity; hence, the consumption of infected plant tissue is prohibited by the WHO. This plant disease also affects other physiological activities like photosynthesis and the carbon-to-nitrogen ratio, reducing WL content by 76.3% [116]. Another fungus, Alternaria solani, is also considered a pathogenic fungus for W. somnifera [126]. The root parasite Orobanche cernua Loefl decreased the biomass, root yield, and major WL by 82%, 68%, and 38%, respectively, after 145 days [127]. The overexploitation of plants for WL production, land deterioration, and environmental degradation may lead to the extinction of plants. This is a threatened act, according to the IUCN (International Union for Conservation of Nature and Natural Resources) [75]. Also, the seed germination percentage of Withania is less [114]. Therefore, due to insufficient WL content, extensive harvesting of host plants, and increasing demand for WL, there is a need to explore a sustainable alternative source for WL production [125]. In plants, the approximate content of WA is only 0.5% of the dry weight [75]. Moreover, the production and accumulation of secondary metabolites varies with species, weather, geographical location, developmental stages of tissues, and gene regulations. These parameters may significantly limit WLʼs drug development and industrial production [56].

Nowadays, it has been widely acknowledged that plants and their associated endophytes produce similar bioactive compounds due to the genetic recombination of endophytes with the host plant during evolution. This concept helps in preserving biodiversity and preventing the extinction of the host plant. Endophytes are used to produce bioactive compounds similar to those produced by their host plant, which is an eco-friendly, easily accessible, and sustainable source. As a source of WL production, endophytes can be cultured in large volumes, scaled up by improved fermentation technology, and easily preserved. The yield can be enhanced by optimising the endophytic biosynthetic pathway of WL [115]. In the literature, there are various reports that suggest that endophytic fungi produce secondary metabolites as produced by their host plants like taxol (genera like Pestalotia, Pestalotiopsis, Alternaria, Seimatoantlerium, Sporormia, Fusarium, Trichothecium, Tubercularia, Pithomyces, Monochaetia, Penicillium, and Truncatella are endophytic fungi isolated from the Taxus plant), podophyllotoxin (endophytes like Phialocephala fortinii and F. oxysporum), and camptothecin (Trichoderma atroviride LY357, Aspergillus sp. LY355, and Aspergillus sp. LY341 were isolated from Camptotheca acuminata) [128].

Although fewer studies reported on the biodiversity of endophytic fungi associated with W. somnifera, [Fig. 7] shows the biodiversity of endophytic fungi in W. somnifera. Khan et al. [51] studied the biodiversity of the endophytic fungi of W. somnifera and revealed that they vary significantly with geographical regions and climatic conditions. High-altitude plants have fewer endophytic fungi than those growing in low-altitude areas. The old leaves colonise a large number of endophytic fungi as compared to young ones [126]. The isolated endophytic fungi were taxonomically identified as Aspergillus awamori, Aspergillus auricomus, Aspergillus flavus, Aspergillus terreus, Aspergillus pulvinus, A. niger, Aspergillus terricola, Aspergillus thomii, A. alternata, Chaetomium bostrycodes, Cladosporium cladosporioides, Drechslera australiensis, Eurotium rubrum, Fusarium moniliforme, Fusarium semitectum, Melanospora fusisora, Melanospora roridum, Penicillium corylophilum, and Phoma sp. The endophytic fungi associated with leaves from the Panchmarhi Biosphere Reserve, M. P., were found to have dominancy only over Hyphomycetes (A. alternata, A. flavus, and A. niger) [129]. Thirty-eight endophytic fungi were isolated from the roots, ten from the stems, and five from the leaves, mainly belonging to Aspergillus sp., Fusarium sp., and Mucor sp. [130]. Fusarium solani, Penicillium chrysogenum, Corynespora cassicola, Penicillium setosum, A. terreus, Penicillium oxalicum, Sarocladium kiliense, and Colletotrichum gloeosporoides were also isolated [115]. Endophytic fungi Fusarium chlamydosporum was also isolated from W. somnifera of Saudi Arabia [131]. Fungal endophytes Penicillium sp., Trametes versicolor, A. terreus, and Sarocladium implicatum were obtained from the leaves of W. somnifera. From roots, P. oxalatum, Ceratobasidium sp., Colletotrichum capsici, Aspergillus brasiliensis, Colletotrichum truncatum, and Hypocrea lixii were isolated. Endophytic fungal genera like Colletotrichum, Fusarium, Talaromyces, Aspergillus, Nigrospora, Mucor, and Alternaria were also isolated from different parts [115]. Endophytic fungi associated with W. somnifera are organ-specific, but A. alternata is the most predominant endophytic fungi among all plant parts [51], [129], [132].

Fig. 7 Biodiversity of endophytic fungi in W. somnifera.

Apart from biodiversity, reports in the literature revealed the use of crude extracts of endophytic fungi (associated with W. somnifera) for their bioactive potential. Likewise, a crude extract of F. solani, an endophytic fungus, showed significant antiproliferative activity against the cervical cancer cell line HeLa. Antibacterial effect against Bacillus subtilis, S. aureus, E. coli, and Klebsiella pneumoniae, as well as antioxidant activity, was also exhibited by the extract [109]. The crude ethyl acetate extract of Alternaria sp. and P. setosum showed antibacterial activity against S. aureus and E. coli and immunomodulatory activities by phagocytosis. The biological silver nanoparticles of F. oxysporum showed antibacterial activities against K. pneumonia, S. aureus, Salmonella typhi, Pseudomonas aeruginosa, and E. coli. Bio-silver nanoparticles of C. gloeosporoides and F. solani showed antiproliferative activity against Si Ha, MDA-MB, and L929 fibroblast cancer cell lines, respectively [115]. Enzyme L-asparaginase isolated from endophytic fungi A. alternata showed chemotherapeutic activity [132].

The endophytic fungi Taleromyces pinophilus and Taleromyces trachyspermus were isolated from leaves of W. somnifera, having the ability to produce WL. The WL structure was confirmed by NMR and LC-MS [115], [133]. Another recent study by Gupta and Vasundhara in 2024 showed the production of withanolides from the endophytic fungus Penicillium oxalicum associated with the W. somnifera [134]. Nigrospora oryzae isolated from the plant Bacopa monnieri is reported to produce WA and WL-A, and another endophytic fungus A. alternata, of this plant produced WL-B [135].

The production of withanolides from endophytic fungi was also associated with challenges like strain-to-strain variability in metabolite production, scalability issues, and regulatory considerations. Likewise, endophytic fungi T. pinophilus, P. oxalicum, N. oryzae, and A. alternata yielded 360 mg/L [133], 219 mg/L [134], 480 µg/L, and 1024 µg/L [135] of withanolides, respectively. This signifies the variation in the production of withanolides with different fungal strains.

Even though withanolide content varies with the use of different fungal strains, there is a need to scale up the fermentation processes for efficient and cost-effective production of withanolides. However, scaling up is associated with various challenges like optimising the culture conditions, availability of raw materials, and production cost. Scale-up from laboratory to pilot requires financial resources, logistics, timelines, and end goals. These requirements can be assessed by process hazards analysis (PHA) processes. The phases of scale-up processes are pre-, in-, and post-production, and they have their own sets of hurdles. Before overcoming the challenges of PHA, pre-, in-, and post-production processes, the gap between research carried out in a laboratory and process development optimisation (PDO) must be bridged. Among these approaches, PDO is found to be a time saver, but the drawback of this approach was significant economic losses and poor product performance with poor yield in the long term [136]. The DBTL (design-build-test-learn) cycle framework can also overcome the hurdles of scaling up. This DBTL cycle framework helps in phenotyping methodologies, encompassing genetic manipulation, each cycle phase optimisation, knowledge integration to iterative improvement, and data interpretation with the help of computational tools [137].

Among bacteria, fungi, and yeast systems, bacterial systems are considered cost-effective, sustainable, and genetically manipulative, while fungal and yeast systems are diverse and show versatility [137]. To our knowledge, scaling up the production of plant endophytes in a bioreactor requires different design phases, which are still under-explored because of growth and intracellular lifestyle within the host plant. As endophytes are biological entities, the main challenges of scaling up processes are the upstream and downstream optimisation processes. As in the bioreactor, there are a lot of control systems; despite that, the cellular properties (physico-chemical, thermodynamical, and molecular properties) of the fungi may vary during adaptation processes in the new fermentation conditions [136].

For example, penicillin production has increased significantly and cost effectively by improving strains and optimising production processes. Over that time, Penicillium chrysogenum has been considered ideal for penicillin production, and various optimisation approaches helped in the elevation of production titre. For fully scaling up the production, a computer framework, metabolically structured kinetic model, and mathematical model were highly recommended to elevate the flow field of large-scale bioreactors. These will help in the prediction of changes during the entire period of fermentation and in optimising the penicillin production [138].

The research on products with microbial origin that helps in bringing them to the market must pass the regulatory stage. Although many of the microbial cell factories were commonly found in the natural environment, their evaluation for regulatory approval is still needed for commercial use without significant problems. Regulatory agencies such as the FDA (Food and Drug Administration) and EMA (European Medicines Agency) govern the therapeutic products produced from microorganisms. Before launching into the market, the producers must ensure the efficacy and safety of the products by performing preclinical and clinical trials to navigate the regulatory frameworks. Also, optimising the microbiota for commercial use can limit the regulatory hurdles. For example, naturally occurring bacteria used as probiotics have the potential to compete against the available ecologically similar microbiota and are therefore approved by the regulatory agencies for commercial use. Live micro-organisms as therapeutic products comply with GMP (good manufacturing practices) standards for market production, storage, and distribution. The manufacturing and QC (quality control) procedures require strict compliance with GMP standards for product purity, uniformity, and stability. To develop a microbe-based therapeutic product, authorities must recognise the associated challenges, and producers must acknowledge the relevant production guidelines. Also, post-marketing research programs track the safety and long-term efficacy of the product by gathering microbiome dynamics and patient outcomes [139].

As discussed earlier, the production of WL varies with different sources, such as plants, soft coral, and fungal strains. Different fungal strains produce varied amounts of withanolide content. In addition, optimising growth culture conditions like media, pH, temperature, and incubation period enhances the withanolide content. The upregulation of key genes in the biosynthetic pathway also enhances the withanolide production. Also, using various elicitors enhances the production yield of withanolides [140]. Based on these studies, further exploration can be undertaken to obtain bioactive WL. Endophytic fungi can be a good source for producing WL, and [Fig. 8] describes the general approach for extracting WL from the endophytic fungi.

Fig. 8 General approach to isolate withanolides from endophytic fungi of W. somnifera.

Challenges and Future Prospects

Despite many pharmacological studies, the development of WL into clinically feasible therapeutics remains restricted by various challenges. The foremost challenge is the low yield of WL from W. somnifera and its susceptibility to biotic and abiotic stresses, which limits the feasibility of large-scale production of WL. Another source reported for WL production is soft coral (P. acronocephala, S. brassica, and Minabea sp.), though it has little significance compared to the plant sources. Since endophytic fungi can produce secondary metabolites like their host plants, they could be explored for WL production. Although endophytic fungi associated with W. somnifera are diverse, they have yet to be examined for WL production. After screening and developing endophytic fungi as a source of WL production, an appropriate approach is required for stable and enhanced production of WL either by media optimisation, use of elicitors, precursors, or co-culture techniques. Targeted metabolic engineering approaches for the sustainable development of microbes producing a high yield of WL are hindered due to incomplete detailed information on biosynthetic pathways. There is a need to study unknown steps in converting WL from 24-methylene cholesterol. This study may help to enhance WL content and ease the screening of endophytic fungi that can produce WL. There is a requirement for merging new genetic techniques with a conventional approach for the repository of W. somnifera genomic data so that manipulation of bioresources becomes easy by identifying genes responsible for synthesising WL. With the help of transcriptomics, genes/enzymes responsible for WL and, further, their pathways can be engineered for rapid and enhanced production. Gene overexpression or silencing, gene mining, and bioinformatics for the metabolic engineering of bioresources to improve the production of WL are much needed [125].

The reported studies in the literature are mainly based entirely on WA, while structurally diverse WL other than WA are underexplored in terms of their pharmacodynamics, biosynthesis, and clinical applications. Moreover, WL have pharmacokinetic limitations like limited bioavailability, poor aqueous solubility, and selective tissue distribution, which lead to reduced therapeutic efficacy. Additionally, standardised protocols for the extraction, quantification, and biological evaluation of the WL are lacking. These challenges collectively address the need for interdisciplinary and innovative strategies to progress the WL from preclinical to clinically marketed approved drugs.

Strategies like synthetic biology and metabolic engineering, multi-omics integration and pathway elucidation, computational and AI-assisted drug discovery, drug delivery and formulation innovations, and preclinical and translational frameworks are much needed to overcome the challenges. Synthetic biology and the metabolic engineering approach will help in reconstructing WL biosynthetic pathways in microbial models. These employ CRISPR/Cas9, regulatory engineering, and pathway optimisation to increase production yield. This approach can lead to exploring the endophytic fungi from W. somnifera as cell factories. The multi-omics integration and pathway elucidation technique utilises transcriptomics, metabolomics, and proteomics to identify the key regulatory genes in WL biosynthesis. The computational and AI-assisted drug discovery approach involves pharmacophore screening, molecular docking, and AI-driven SAR modelling for synthesising derivatives with drug-likeness and optimal target specificity. This approach also utilises a machine learning algorithm to predict ADMET profiles of novel drug candidates. Drug delivery and formulation innovation approaches help in developing the nano-carrier systems like polymer-based micelles, solid lipid nanoparticles and liposomes for targeted delivery, and the improvement of pharmacokinetics. Preclinical and translational frameworks help in the establishment of standardised in vivo and in vitro models for evaluating the safety and efficacy of the compound. They also facilitate multicentre collaborative studies to advance promising drug candidates into early clinical trials.

The interdisciplinary collaborations between plant genomics and synthetic biology help in identifying the biosynthetic gene clusters and their transfer into engineered microbes. Pharmacology and computational chemistry are approaches that accelerate lead optimisation, target validation, and toxicity prediction. Material science and drug delivery systems co-developed responsive formulations that overcome the limitations related to compound-specific pharmacokinetics. Collaborations of academia and industry partnerships overcome the gap between commercialisation and discovery via the sharing of resources and expertise. Hence, the future of WL research lies beyond the focus on WA and comprises an integrated approach for discovering, producing, and delivering novel compounds with effective therapeutic potential. The long-term barriers in the new era of WL-based drug discovery and development can be overcome by collaborating with computational tools, biotechnological innovations, and multi-omics technologies.

Conclusions

Withanolides are a group of steroidal lactones predominantly present in the plant W. somnifera. Among all withanolides, withaferin A is the most common and abundantly present in the leaves and roots of the W. somnifera. This compound possesses various biological activities and has entered clinical trials for cancer therapy. Although promising preclinical and clinical research studies have been published, the therapeutic potential of WL is compelled by challenges like sustainable and large-scale production. As W. somnifera is susceptible to pathogens, it is becoming an endangered species owing to slow growth and produces a limited natural yield of WL. Therefore, an urgent need is to explore an alternative source to produce WL that can offer high yield, cost, and scalability efficiency. In the literature, endophytic fungi are supposed to mimic the biological pathways of their host plants that help them in producing metabolites similar to those produced by their host plants. Despite the high endophytic fungal diversity associated with W. somnifera, only a few reports have been published on exploring their ability to produce WL. Hence, future investigations must focus entirely on isolation, systematic screening, and characterisation of endophytic fungal strains that can produce WL. Also, there is a requirement to develop metabolic and genetic engineering approaches to enhance the cost-effective production of WL. A detailed study of key genes and enzymes involved in the biosynthetic pathway of WL production offers a platform to elucidate the key genes that produce the withanolides with increased yield. Simultaneously, bioprocess-engineering and fermentation process optimisation approaches are essential to support the production of WL commercially. The nanotechnology-based drug-delivery systems have advanced the clinical utilisation of WLs by overcoming the challenges related to off-target effects, stability, and bioavailability. Additionally, target-based in silico modelling studies accelerate the development of WL derivatives with improved safety profiles and efficacy. In conclusion, this review stresses the importance of biotechnological interventions for producing WL. Future research must integrate system biology, clinical pharmacology, and biotechnological innovations to overcome the current challenges in WL production. These integrated systems help in enabling the translation of withanolides and their derivatives into accessible and effective therapeutics.

Contributorsʼ Statement

Data collection, analysis, compilation, writing original draft, drawing chemical structures: A Gupta Conceptualization, design of the study, analysis and interpretation of the data, critical revision: M Vasundhara

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