Withania somnifera: Progress towards a Pharmaceutical Agent for Immunomodulation and Cancer Therapeutics.

1. Introduction

Cancer is only second to heart disease as the leading cause of mortality globally. In 2022, the American Cancer Society reported about 1,918,030 new cases of cancer and 609,360 cancer-related mortalities in the United States alone [ 1 ]. Over the past three decades, significant progress in the detection and treatment of cancers has altered early diagnosis, prevention, and therapeutic strategies resulting in a decline in the mortality rate. The cancer mortality rate reduced by 27% between 2001 and 2020. Male cancer death rates decreased by 30% and female death rates decreased by 25%, although male cancer death rates remained higher (170.3 deaths per 100,000 people) than female death rates (124.5 deaths per 100,000 people) [ 2 ]. Chemotherapy, radiation therapy, surgery, immunotherapy, and targeted therapy are being used to treat cancer. However, many chemotherapeutic methods are linked with severe side effects and resistance within a few months of therapy. Commercially available Food and Drug Administration (FDA)-approved drugs mostly target a single gene or pathway [ 3 , 4 , 5 , 6 ]. As a result, there is a growing need to find new anticancer molecules/medicines that could target multiple targets in cancer while having minimal adverse effects. Recently, many plant-based agents such as alkaloids, have demonstrated pronounced anti-cancer activity, either as a single agent or as a combination regimen with standard anti-cancer agents [ 7 ]. The majority of characterized phytochemicals have exhibited chemo- and radio-sensitizing activity in chemo and radio-resistant tumor cells [ 7 ]. About 40% of the FDA-approved drugs in the market are based on plant products, 74% of which are anticancer drugs [ 7 , 8 , 9 ]. Moreover, the majority of these phytochemicals actively target tumor cells specifically and pose minimal toxicity to healthy cells.

Withania somnifera (WS) is a woody, evergreen shrub, roughly 0.5 to 2.0 m tall, and in English it is called “Winter cherry” or “Indian Ginseng”, in Sanskrit “Ashwagandha”, in Hindi “Asgandh”, and in Urdu “Asgand” [ 10 , 11 ]. The name “Ashwagandha” originates from the plant’s roots, which have the distinctive smell of a wet horse (“ashwa” means horse and “gandha” means smell). For more than 5000 years, the complete plant or various portions of WS have been employed in India’s Ayurvedic and Unani medical systems for medicinal and therapeutic purposes, and the plant was reported as an official drug in the Indian Pharmacopoeia-1985 [ 10 ].

WS is cytotoxic to a variety of tumor cells but has minimal effect on healthy human cells, indicating its specific effects on cancer cells [ 12 ]. WS has been demonstrated to have anxiolytic, antiangiogenic, antidepressive, anti-metastatic, anti-tumoral, cytotoxic, genotoxic, antibacterial, antifungal, and antidiabetic properties in several in vitro and in vivo experiments [ 13 , 14 , 15 ]. WS modulates cytotoxicity in cancer cells by accumulating intracellular reactive oxygen species (ROS) [ 16 , 17 , 18 ]. WS inhibits several aberrant pathways implicated in inflammation and proliferation (e.g., IL-6, TNF-α, and cycloxygenase-2 (COX-2)), angiogenesis and metastasis (e.g., VEGF, MMP9, TWIST, NF-κB, and STAT), cell survival (e.g., Bcl-2, Bcl-xL, survivin, and cIAP1/2), and regulation of the cell cycle (e.g., cyclin A, cyclin D1, Cdks, p21, and p53) [ 12 , 13 , 14 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 ]. Furthermore, WS is an adaptogenic Ayurvedic plant, which is often used to counteract and relieve stress, ultimately improving overall well-being, and numerous studies have shown the use of WS for stress resistance [ 29 , 30 , 31 ]. A high-concentration full-spectrum WS root extract enhances an individual’s resistance to stress and, as a result, their self-assessed quality of life [ 31 , 32 ]. In this review, we discuss the chemical properties, pharmacokinetics, anticancer potential, toxicity, and pharmacological significance of WS. We also highlight the molecular pathways of WS contributing towards anticancer activity, combinatorial therapy, and the chemo/radio-sensitizing effects. The phytochemical properties of WS would yield novel insights and establish the basics for clinical investigations to develop WS as an anti-cancer medication.

2. Biological and Chemical Properties of Withania somnifera

2.1. Sources and Chemical Properties of Withania somnifera

The 23 known Withania species are widely distributed in the arid regions of India, Baluchistan, Afghanistan, Sri Lanka, Congo, South Africa, Egypt, Morocco, and Jordan [ 33 ]. WS plant organs such as the root, leaf, fruit, and seed possess adequate bio-active chemicals that have been implicated in pronounced anticancer, anti-microbial, cardioprotective, and neuroprotective mechanisms [ 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 ].

The presence of withanolides, a group of steroidal lactones, is thought to be responsible for the pharmacological activity of WS roots [ 43 ]. WS has demonstrated non-medicinal properties, such as enhanced memory and cognition, mood elevation, and rejuvenation [ 29 ]. WS also serves as an energy-boosting tonic called Medharasayana, meaning “enhanced memory and learning.” Laboratory studies have shown that the roots of WS contain about 35 phytoconstituents [ 44 ]. The main physiologically active chemical molecules present are alkaloids (isopellertierine and anferine), steroidal lactones (withanolides, withaferins saponins with additional acyl group (sitoindoside VII and VIII), and withanoloides with C-27 linked to glucose (sitonidoside XI and X). A chemo assessment of Ashwagandha revealed that its primary ingredients are alkaloids and steroidal lactones. Withanine is the most abundant out of all the numerous alkaloids. Others include somniferine, pseudo-withanine, tropine, pseudo-tropine, 3-a-gloyloxytropine, choline, anaferine, anahydrine, etc. Withanolides present in leaf possess a C28 steroidal nucleus, a C9 side chain, and a hexagonal lactone ring. WS has been investigated for its twelve alkaloids, 35 withanolides, and seven sitoindosides. The majority of Ashwagandha’s pharmacological action has been ascribed to two major withanolides, Withaferin A and Withanolide D [ 45 , 46 ]. The fresh plant of WS is rich in fatty acids, fatty alcohols, volatile oils, and hydrocarbons, including myristic acid, palmitate, linoleic acid, and the straight chain hydrocarbon hexatriacontane [ 47 ]. The roots of ashwagandha include alkaloids (wide variation of 0.13–0.31%), starch, reducing carbohydrates, hentriacontane, glycosides, dulcitol, withaniol acid, and a neutral molecule. In addition, the leaves contain free amino acids including aspartic acid, glycine, tyrosine, alanine, proline, tryptophan, glutamic acid, and cystine. Fruits of Ashwagandha harbor a milk-coagulating enzyme, two esterases, free amino acids, fatty oil, essential oils, and alkaloids. The bioactive chemicals extracted from WS is listed in Figure 1 and their anticancer functions are presented in Table 1 .

2.2. Toxicity of Withania somnifera

Due to low cost and abundance, medicinal plants have been of considerable pharmacological interest, particularly in cancer prevention. Various studies have shown that natural bioactive drugs with minimal toxicity possess immense therapeutic potential. However, safety issues have often been highlighted and must be addressed before they are utilized as immunomodulatory and anticancer agents. Traditional chemotherapies pose several side effects, including those that impair the functioning of numerous organs, such as the heart, liver, and kidneys. Research reports have emphasized the preventative activity of Withaferin A against bromobenzene-induced liver and kidney breakdown in mice [ 48 ]. This was evident from the reduced levels of liver and kidney biomarkers, lipid peroxidation, and cytokines (TNF-α and IL-1), after a 10 mg/kg pre-treatment dose of Withaferin A administered in mice. In the Withaferin A pre-treatment mice group, there were low levels of cytokines with reduced oxidative stress; mitochondrial impairment was prevented and the equilibrium between Bax/Bcl-2 was restored [ 48 ].

According to a recent study, Withaferin A increased the capacity of H9c2 cells to survive against simulated ischemia/reperfusion (SI/R) or hydrogen peroxide (H 2 O 2 )-induced cell death in myocardial ischemia reperfusion (MI/R) damage, as well as inhibiting the increased oxidative stress caused by SI/R [ 49 ]. In addition, Withaferin A effectively inhibited H 2 O 2 -induced overexpression of SOD2, SOD3, and Prdx-1, which improved cardiomyocyte caspase-3 activity in an Akt-dependent way [ 49 ]. Furthermore, Withaferin A reduced cerulein-induced acute pancreatitis due to oxidative stress and inflammation [ 50 ]. Increased tissue malondialdehyde (MDA), NO, and myeloperoxidase and nitrotyrosine expression in the parameters assessed contribute to the notion that oxidative stress and inflammation play a role in acute pancreatitis [ 50 ]. Furthermore, Withaferin A also decreased the acetaminophen-mediated liver damage in mice in an Nrf2-dependent manner, typical of a stress-responsive transcription factor and a well-established chemoprevention target. Withaferin A increased nuclear factor-erythroid factor 2-related factor 2 (Nrf2) signaling in a non-canonical Keap-independent, Pten/PI3k/Akt-dependent way in this research [ 51 ]. Studies indicate that Withaferin A performs an antifibrotic action in scleroderma by inhibiting pro-inflammatory fibrosis involving Transforming growth factor-b1(TGF-β)/Smad signaling and substantially reducing fibroblast conversion to myofibroblasts. Also, the FoxO3a-Akt-dependent nuclear factor-kappa B (NF-κB)/IKK-regulated inflammatory cascade, which is a key signaling mechanism in fibrogenesis, is modulated by Withaferin A [ 52 ]. In a recent research study, tumor-targeting silver nanoparticles (Ag NP) were used to produce NP-related macrophage toxicity. When nanoparticles (NPs) were given in combination with WS root extract (35 mg/kg), the toxic effects in rats were significantly reduced [ 53 ]. Despite these fundamental and mechanistic investigations, the potential of WS extracts as dietary supplements has only been explored in one study, which included 100 breast cancer patients undergoing chemotherapies (used as complementary). It was shown that the tiredness associated with treatment reduced and the overall quality of life improved after supplementation with WS extracts [ 54 ].

2.3. Pharmacokinetic Studies and Bioavailability of Withania somnifera

The major drawbacks of biological agents are rapid metabolism, quick excretion, and poor bioavailability, which reduce their potential as anticancer agents [ 104 ]. It is critical to know a drug’s bioavailability before recommending it to treat a disease. WS bioavailability has been shown in preclinical tests to be acceptable [ 105 , 106 ]. In a recent assessment, the ideal oral pharmacokinetics of Withaferin A was determined in male rats and the in vitro screening of absorption factors by liquid chromatography–mass spectrometry (LC–MS/MS) and quadrupole trap mass spectrometry (Q-TRAP) analysis [ 105 ]. Male rats were given Withaferin A intravenously (5 mg/kg) and orally (10 mg/kg), and the oral bioavailability was found to be 32.4 ± 4.8%. Furthermore, in vitro findings revealed that Withaferin A was readily transported through Caco-2 cells and Withaferin A did not seem to be a substrate for P-glycoprotein.

The stability of Withaferin A in male rat or human intestinal microflora was assessed as drugs given orally always interact with a significant population of intestinal microflora in the digestive tract and Withaferin A is susceptible to bacterial degradation. There were no significant differences in the stability of Withaferin A in male rats and humans when tested in formulated gastric fluid (stable), in intestinal microflora solution (gradual reduction), and in liver microsomes (swift expenditure with a half-life of 5.6 min). As a result, the initial metabolism of Withaferin A was confirmed using rat intestine-liver in situ perfusion, which showed that Withaferin A quickly dropped and remained at 27.1% in the first hour, while the level of the three key metabolites (M1, M4, and M5) detected by Q-TRAP analysis increased.

Patil et al. [ 106 ] identified Withaferin A and Withanolide A in mouse plasma using high-performance liquid chromatography-tandem mass spectrometry. In this study, plasma samples were pretreated with tert-butyl methyl ether and the simple liquid–liquid extraction method was performed. Here, a Hypurity C18 column using methanol and ammonium acetate (95:5, v / v ) is used as a mobile phase to partition the analytes and identified by electrospray ionization in the multiple reaction monitoring mode. The mass transition ion-pair was m / z 437.2 →292.2 for tianeptine (IS); m / z 471.3 → 281.2 for Withaferin A; and m / z 315 → 9270 for clonazepam (IS) and m / z 488.3 → 263.1 for Withanolide A. Furthermore, this technique demonstrated good linearity (r2 > 0.997) across the concentration dosage of 0.476–116.050 ng/mL for Withanolide A and of 0.484–117.880 ng/mL for Withaferin A. The lower bounds of quantification (LLOQs) for Withanolide A and Withaferin A were determined to be 0.476 ng/mL and 0.484 ng/mL, respectively, which is less than the C max /20 ratio, indicating that the technique has sufficient sensitivity to detect these withanolides in plasma samples. The optimum precision (% CV) and accuracy (% bias) were recorded between 3.7–14.3% and −14.4–4.0%, respectively. This proven technique was effectively used in pharmacokinetic research to estimate Withaferin A and Withanolide A in mice plasma after oral dosage of WS root aqueous extract.

The withanolides are rapidly absorbed after oral administration and withaferin A is relatively nearly twice as bioavailable as Withanolide A [ 106 ]. WS has been documented to have efficient pharmacological activities, including anticancer activity in vitro and in vivo. The WS can selectively target and kill cancer cells by activating various apoptosis-related molecular and cellular pathways. Various findings on the antitumor potential of WS revealed its regulatory impact on various erratic signaling pathways implicated in cancer establishment and progression, such as NF-κB, COX-2 and phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) Figure 2 . The in vitro cytotoxicity and targeted and regulatory aberrant mechanisms of WS are summarized in Table 2 .

3. Role of Withania somnifera in Cancer

3.1. Lung Cancer

Lung cancer is the predominant cause of cancer-associated mortalities globally [ 1 ]. A combination therapy of paclitaxel and WS (400 mg/kg body weight) extracts in treating benzo(a)pyrene-induced lung carcinogenesis in Swiss albino mice shielded the mice from reactive ROS-induced damage through antioxidant activity, restored immune activity, and decreased cell viability [ 107 , 108 ]. It has also been shown that Withaferin A suppressed the binding of U937 monocytic cells with A549 cells stimulated by tumor necrosis factor-α (TNF-α) via deregulation of vascular cell adhesion molecule 1 and intracellular adhesion molecule 1 expression, blockade of Akt phosphorylation, and shunted nuclear factor kappa B (NF-κB) activity [ 25 ]. Additionally, Withaferin A has demonstrated potent activity against TNF-α-induced epithelial–mesenchymal transition (EMT) and TGF-β in A549 and H1299 non-small cell lung cancer (NSCLC) cell lines and has also induced apoptosis and cell cycle arrest by inhibiting the PI3K/Akt pathway [ 109 , 110 ]. These findings encourage the testing of efficacy of WS as a single agent or with other chemotherapeutic agents for lung cancer therapy.

3.2. Breast Cancer

Breast cancer is the leading form of cancer in females [ 1 , 111 ]. The heterogeneity of breast malignancies greatly influences their aggressiveness and ability to naturally metastasize [ 112 ]. Fluorescence microscopy studies on breast cancer cell lines demonstrated the efficacy of Withaferin A in initiating mitotic arrest in MDA-MB-231 and MCF-7 cell lines as well as the Ser10 residue-targeted phosphorylation of H3 histone [ 113 ]. Five-week intraperitoneal supplementation of 4 mg WA/kg body weight in female nude mice injected with MDA-MB-231 cells exhibited significantly reduced tumor growth while there was Bim-dependent and FOXO3a-induced apoptosis in the same cells in in vitro [ 20 ]. Furthermore, a recent study has evinced a novel apoptosis induction mechanism using Withaferin A, in which levels of survivin proteins, the cellular inhibitor of apoptosis-2 (cIAP-2), and the X-linked inhibitor of apoptosis (XIAP) all decreased after about 6 h of WA treatment; in in vivo, only survivin proteins were inhibited by Withaferin A [ 114 ]. Withaferin A has been shown to inhibit oxidative phosphorylation in breast tumors and induce the apoptotic death of cells through ROS [ 115 ].

Thaiparambil et al. [ 56 ] demonstrated the ability of Withaferin A to elevate phosphorylation of vimentin at its Ser56 residue, which indicates disassembly of vimentin, and thereby enhanced anti-metastatic and anti-invasive phenotypes in in vitro and in vivo. Withaferin A can potentially augment the distinct Cys328 vimentin residue in Human Umbilical Vein Endothelial Cells (HUVECs) covalently, resulting in vimentin denigration in in vivo and inhibition of neovascularization [ 116 ]. Furthermore, Withania root extracts have been shown to influence the EMT in breast cancer in in vitro and in xenograft mouse models supplemented with MDA-MB-231 cells [ 117 , 118 ]. The immunohistochemistry studies demonstrated that methylnitrosurea-induced mammary malignancies in female Sprague-Dawley rats caused a reduction in the proliferating cell nuclear antigen marker and Ki67 expression after Withaferin A root extract treatment [ 119 ]. Interestingly, transfection of MDA-MB-231 cells with ER-α inhibited Withaferin A-induced apoptosis but failed to obstruct the Withaferin A-induced cell cycle arrest at the G2/M phase. Withaferin A demonstrated anti-estrogenic tendencies by halting the growth of the estrogen receptor (ER)-positive T47D and MCF7 cells [ 120 ].

There have also been reports of anti-proliferative propensities of Withaferin A under various experimental settings [ 121 ]. For instance, Withaferin A suppressed the phosphorylation of Jak2 and signal transducer and activator of transcription-3 (STAT-3) [ 122 ]; inhibited NF-κB [ 123 ]; activated Notch-2 and Notch-4 upregulated [ 124 ]; and induced the overexpression of Elk-1-mediated Death Receptor five [ 125 ]. Research on the epigenetic properties of Withaferin A elucidated the potential of Withaferin A to methylate or demethylate numerous genes implicated in Triple-Negative Breast Cancer and inhibit their specific features of mildly aggressive luminal breast cancer with enhanced therapeutic sensitivity and response [ 126 ]. In conclusion, these studies highlight the exceptional therapeutic potential of active constituents of WS against breast cancer through anti-proliferative and anti-invasive molecular modes of action.

3.3. Prostate Cancer

Prostate cancer is the second most frequent cancer in men globally and accounts for 3.8% of cancer-related mortalities in men worldwide [ 1 , 111 ]. The anticancer activity of WS constituents has been fairly documented, such as the induction of prostate apoptosis response-4 (Par-4) dependent apoptosis in prostate cancer cell lines and regression of PC-3 xenografts in nude mice after a combination treatment of Withaferin A and other anti-androgens [ 73 ]. Furthermore, Withaferin A resulted in a dose-dependent inhibition of cell viability and led to the accumulation of Weal in the G2/M phase of the cell cycle [ 127 ] and facilitated vimentin denigration, which has been previously documented in breast malignancies, stimulated ROS generation, and decreased c-FLIP levels [ 56 , 81 ].

A 3-azido derivative of WA (3-azidoWA) restricted the cell invasion, mediated by extracellular Par-4-dependent inhibition of matrix metalloproteinase-2 (MMP-2) in PC-3 and HeLa cells [ 93 ]. Additionally, in vivo studies demonstrated a decreased expression of p-ERK and p-Akt and inhibited angiogenesis in mice [ 93 ]. 3-azidoWA also exhibited anticancer potential against prostate cancer by stimulating ER stress and improved chemosensitivity by influencing the shift from autophagy to apoptotic death in prostate cancer cells. Recent reports have shown the ability of WS root extracts to suppress lipogenesis in 22Rv1 cells, most likely by decreasing the expression levels of p-Akt and c-Myc, and this possibly indicates the mechanism of fatty acid metabolism in malignant cells and a novel strategy of inducing antitumor activity in prostate cancer [ 101 ].Kunimasa et al. [ 128 ] found that Withaferin A combined with glucose metabolism focused therapy might be an effective treatment for cancer cells resistant to tyrosine kinase inhibitors (TKI). Drug tolerance per sisters (DTPs) were formed in EGFR mutant lung cancer cell lines were treated with gefitinib and characterized by increased senescence (CD133 low) and stemness (marked by CD133 high population). Senescent cells exhibit the SASP (senescence-associated secretory phenotype) phenotype and may connect with other cells through secreted substances that have been SASP treated with gefitinib conditioned medium enhanced CD133 high in CSCs. The researchers recommended combining glucose metabolism targeting treatment with Withaferin A to target CSCs (as senescent CD133 low cells have enhanced glucose metabolism).

3.4. Colon Cancer

Colon cancer ranks third globally incidence wise and ranks second in mortality cases among the various cancers [ 1 , 111 ]. In Swiss albino mice, it was observed that ethanolic extracts of WS evinced immunoregulatory tendencies in azoxymethane-induced colon cancer [ 129 ]. Withaferin A has also demonstrated anticancer activity against colon cancer by targeting and downregulating Notch-1 signaling via targets such as Hey-1 and Hes-1 and concurrently suppressing crosstalk between Notch-1 and Akt/mTOR pathways. This makes the Notch-Akt-mTOR axis an attractive therapeutic target in colon cancer therapy [ 130 ]. Furthermore, there was observed dose-dependent apoptotic induction in three colon cancer cell lines as seen by the upregulated expression of apoptotic markers such as Poly ADP ribose polymerase (PARP) and caspase-3, as well as upregulated phosphorylation of c-Jun and JNK [ 130 ]. Moreover, Withaferin A caused cell cycle arrest at the G2/M phase of the cell cycle as a result of spindle assembly checkpoint blockade that invariably results in mitotic disruption, and proteasomal denigration of Mad2 and Cdc20, which ultimately results in chromosomal instability [ 82 ].

Other reports indicate that Withaferin A is capable of inhibiting migration and the IL-6 mediated the stimulation of STAT-3 in HCT116 cells [ 131 ]. Additionally, Withaferin A treatment of HCT116 xenograft tumors in Balb/c nude mice showed a pronounced decrease in tumor weight and volume [ 131 ]. A drastic reduction in tumor progression, volume, polyp size, and adenomas in Withaferin A-treated mice relative to controls highlights the need for active investigation into clinical application of Withaferin A [ 24 ]. A recent report indicates a combination regimen of 5-fluorouracil with Withaferin A inhibited colorectal cancer cell viability and stimulated the ER-stress-mediated induction of apoptotic cell death and autophagy, while causing cell cycle arrest at the G2/M phase [ 132 ]. Additionally, the induction of apoptosis was mediated by the PERK axis of ER stress and was non-toxic to healthy colon cancer cells [ 132 ].

3.5. Leukemia

Withaferin A has demonstrated potent anticancer activity against solid tumors. In this section, its efficiency in inhibiting hematological malignancies would be discussed. Initial research reports demonstrated that Withaferin A-mediated inhibition of cell proliferation in several malignant lymphoid and myeloid cells, cell cycle arrest at the sub-G0 phase, and apoptotic induction via the p38/MAPK signaling pathway [ 71 ]. Furthermore, L-Asparaginase isolated and purified from WS fruit demonstrated anti-proliferative tendencies in acute lymphoblastic leukemia cells obtained from leukemia patients [ 87 ]. Conclusive studies on leukemia cell lines such as U937 and others directly correlated Withaferin A with improved ionizing radiation-mediated cell death via ROS stimulation, cell cycle disruption at the G2/M phase and simultaneous upregulation of JNK signaling, and deregulated Akt phosphorylation [ 57 , 133 , 134 ]. Moreover, WS has demonstrated various anti-leukemic tendencies such as the ability of WS root extracts to improve ROS generation, induce cell cycle arrest, pump intracellular calcium, and denigrate DNA structure of T-lymphoblastoid cell lines [ 135 ].

3.6. Other Cancers

The anticancer activity of WS has been fairly documented in other carcinomas. In melanoma, it was observed that Withaferin A augured ROS production that resulted in mitochondrial-mediated cell death in melanoma cells with a range of IC 50 values between 1.8 to 6.1 mM and caused DNA damage. In Swiss albino mice models, WS root extracts therapy caused pronounced weight gain and decreased skin lesions [ 136 ]. Also, in human osteosarcoma cells and gastric and oral cancer cells, WA led to the G2/M phase cell cycle arrest [ 137 , 138 , 139 ]. However, there is a paucity of comprehensive research of antitumor effects of Withaferin A in such cancers, though in oral cancer, studies have identified selective degradation of oral cancer cells as a result of oxidative stress and depolarized mitochondrial membrane potential as well as DNA fragmentation [ 137 ].

In pancreatic cancer (PanCa), a combination regimen of Withaferin A and oxaliplatin resulted in the intracellular accumulation of ROS, which correlated with downregulation of Akt and apoptotic cell death. This provided the strongest evidence yet of effective antitumor activity of a combination therapy of Withaferin A and oxaliplatin in PanCa therapy [ 140 ]. Similar evidence was seen in a combination therapy of doxorubicin and Withaferin A in ovarian cancer which led to improved ROS generation and stimulated autophagy [ 141 ]. In vivo studies in mice showed a 70–80% reduction in tumor mass upon combination treatment of Withaferin A and doxorubicin relative to the control or single drug treatments [ 141 ].

Another study demonstrated therapeutic efficacy of Withaferin A alone or Withaferin A plus cisplatin in downregulating Notch-1 signaling and repressing metastasis in nude mice [ 142 ]. Immunohistochemistry (IHC) and proteomic analysis showed pronounced decrease in cancer stem cell biomarkers (CCD117, CD34, Oct-4, CD44, and CD-24) and metastatic biomarkers such as Notch-1. Interestingly, the administration of cisplatin alone in xenograft mice had the opposite effect, highlighting the efficacy of the Withaferin A-cisplatin combination treatment regimen in drug-resistant ovarian cancer [ 142 ]. Withaferin A-treatment of SKOV3 and CaOV3 cells attenuated cell viability and clonogenicity, initiated apoptosis, and caused cell cycle arrest at the G2/M phase [ 55 ].

Withaferin A demonstrated potent anti-proliferative effects in cervical cancer cells as documented by Munagala et al. [ 67 ]. These studies showed that Withaferin A suppressed most tumor characteristics including CaSki cell viability (IC50 = 0.45 mM), deregulated HPV E6/E7 oncoproteins, decreased STAT-3 phosphorylation, and upregulated p21 and p53 proteins [ 143 ]. In vivo studies in athymic nude mice also yielded a pronounced decrease in tumor mass [ 67 ]. A dose-dependent inhibition of TGF-β induced Akt phosphorylation and decreased MMP2 and MMP9 expression in Withaferin A-treated CaSki and SK-Hep1 cells, indicating anti-invasiveness of WA [ 143 ]. Withaferin A has demonstrated potency against renal carcinoma as well. Withaferin A induced apoptosis in CaSki cells, suppressed JAK-2 activation, IL-6-mediated STAT-3, Akt, and Bcl-2 phosphorylation, and upregulated the expression of glucose-regulated protein (GRP)-78 and CAAT/enhancer-binding protein-homologous protein as well as the stimulation of ROS-dependent expression of the endoplasmic reticulum (ER) stress markers such as the phosphorylation of eukaryotic initiation factor-2α and X-box binding protein 1 (XBP1) splicing [ 21 , 144 , 145 ]. Interaction between Withaferin A and the Cys179 residue found in the catalytic site of IKKβ also suppressed NF-κB activity in HEK293T cells [ 146 ].

3.7. Chemosensitization and Synergistic Actions of Withania somnifera

The development of resistance to standard chemotherapy indicates that single drugs may not be enough for cancer treatment. Combination therapy has received attention in recent years as a novel cancer treatment approach [ 147 ]. Traditional chemotherapeutics combined with therapy with phytochemicals like WS may enhance the therapeutic effectiveness in cancer treatment. WS has a chemo-sensitizing impact on many cancer types by altering numerous signaling pathways, such as MAPK/ERK, PI3K/AKT, and NF-κB. Interestingly, many in vitro and in vivo investigations have shown the therapeutic potential of WS as a combinatorial anticancer medicine. In this section, we discuss the synergistic effects of co-treatment with chemotherapeutic agents and WS in inhibiting various carcinogenic pathways. Chemo-sensitization and synergistic effects of WS are thought to enhance intracellular concentrations of chemotherapeutic drug(s) in cancer cells as well as at the tumor site.

Kyakulaga et al. [ 148 ] have shown synergistic effects of paclitaxel and Withaferin A against human NSCLC. In H1299 and A549 cells, paclitaxel, and WA co-treatment reduced cell proliferation, colony formation, migration, invasion, and enhanced apoptosis. Contrary to expectations, the synergy of paclitaxel and Withaferin A was increased when cells were pretreated with Withaferin A, suggesting an anti-chemosensitivity effect. On the other hand, Withaferin A inhibited both paclitaxel-susceptible (TS-A549) and paclitaxel-resistant (TR-A549) cells in in vitro and in vivo. Withaferin A suppresses NSCLC cell growth via oxidizing thiols. In doxorubicin-sensitive K562 and doxorubicin-resistant K562/Adr cells, Withaferin A alone can negate attenuated caspase activation and apoptosis, while quercetin-mediated caspase regulation and apoptosis is just delayed. However, only Withaferin A lowers intracellular protein levels of Bcl2, Bim, and p -Bad, while increasing PARP cleavage, caspase 3 activation, and apoptosis, perhaps through thiol oxidation [ 149 ]. Withanolide D (C4β-C5β,C6β-epoxy-1-oxo-,20β, dihydroxy-20S,22R-witha-2,24-dienolide; Withanolide D) is isolated from WS initiates leukemic apoptosis by the upregulating activation of neutral sphingomyelinase-ceramide cascade, facilitated by synergistic activation of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase [ 97 ]. This study shows that Withanolide D may raise ceramide levels in myeloid (K562) and lymphoid (MOLT-4) cells and enhance JNK and p38MAPK phosphorylation downstream of ceramide. In addition, N-SMase 2 is a major mediator of Withanolide D-induced apoptosis, and N-SMase 2 siRNA and N-SMase inhibitor (GW4869) reduced Withanolide D-induced ceramide production and MKK4 and MKK3/6 phosphorylation but not MKK7 in leukemic cells. The inhibitor GW4869 also protected these cells against Withanolide D-mediated mortality and reduced apoptosis, while Fumonisin B1, a ceramide synthase inhibitor, had no impact. Also, Withanolide D efficiently triggered apoptosis in newly separated lymphoblasts from patients, through JNK and p38MAPK activation [ 97 ].

In prostate cancer cells, PAWR-regulated the suppression of Bcl-2 influences shift from 3-azido Withaferin A induced autophagy to apoptosis [ 150 ]. As a result, many MAP1LC3B and EGFP-LC3B puncta accumulated, and SQSTM1 gradually degraded. Higher toxic doses of 3-azido Withaferin A increased CaP cell ER stress, resulting in the activation of apoptosis by increasing PAWR expression, which inhibited Bcl2 and BECN1l expression, both of which are involved in autophagy. Overexpressed PAWR inhibits BECN1 in CaP cells, causing the Bcl2-BECN1 connection to be disrupted. Furthermore, with the lethal concentrations of 3-azido Withaferin, pawr-KO MEFs showed extensive autophagy signals, demonstrating the importance of PAWR in the transition from autophagy to apoptosis. Finally, overexpression of EGFP-LC3B and DS-Red-BECN1 in CaP cells resulted in a delay in the apoptotic turnover at greater 3-AWA concentrations. Another benefit was that it increased chemosensitivity by making prostate cancer cells more susceptible to apoptosis, which is why it has therapeutic promise [ 150 ].

Furthermore, sub-toxic concentrations of 3-azido Withaferin A suppressed cancer cell motility and invasion in wound healing and the Boyden chamber invasion by inhibiting MMP-2 activity in gelatin zymography, which is a significant barrier in chemo-sensitivity. An external activation of the tumor suppressor candidate Par-4 protein by 3-azido Withaferin A generated a new mechanism, and an immunoblot analysis revealed an associated significant decrease in pAkt/pERK signaling. This study also found that 3-azido Withaferin A inhibited MMP-2 through secretory Par-4, which is consistent with our zymography findings. MMP-2 gelatinase activity was not restored by 3-azido Withaferin A apoptotic suppression. In addition, 3-azido Withaferin A inhibited neovascularization in mice using the Matrigel plug test when administered in a dose-dependent manner [ 93 ].

3.8. Clinical Trials

WS actively inhibits a variety of oncogenic signaling molecules and warrants further clinical investigations. There is, however, a dearth of studies in this field that is centered on cancer, with only three clinical cancer-related investigations out of 11 total studies on WS. There are about 29 clinical trial studies reported on clinicaltrials.gov ( https://clinicaltrials.gov/ct2/results?cond=&term=Withania+somniera&cntry=&state=&city=&dist= accessed on 3 March 2022). In an open-access nonrandomized comparative study on 100 patients with breast cancer; WS root extract was given to patients in the study group at a dose of 2 g every 8 h throughout the duration of chemotherapeutic treatment. It was observed that WS root extract exhibited therapeutic potential against cancer-related fatigue and improved the quality of life [ 54 ]. In another study, 24 participants were recruited to assess the efficacy of curcumin formulation and Ashwagandha extracts on advanced osteosarcoma. The conclusion of this study, however, has not been properly stated [ 158 ].

A prospective, randomized double-blind, placebo-controlled study conducted by Chandrasekhar et al. [ 32 ] evaluated the pharmacological profile of a highly concentrated full spectrum isolate of Ashwagandha roots in decreasing stress-induced anxiety and ameliorating the health of stressed participants. The extract was found to be safe, tolerable, and effective at decreasing stress and anxiety. However, another study reported the WS-mediated improvement of NK cell activity after the consumption of tea infused with WS-active herbal drugs [ 159 ]. Zwickey et al. [ 160 ] also investigated the effects of Ashwagandha on stress, inflammation, and immune cell activation in 25 participants. The root extract may be utilized as an adjuvant treatment in cancer patients to alleviate stress and anxiety. Table 3 outlines Withaferin A clinical studies on different disorders. Further studies are required to examine Withaferin A’s therapeutic potential in cancer. A randomized, double-blind, placebo-controlled study by Chengappa et al. [ 161 ] demonstrated the benefits of WS, including its safety in patients with recent schizophrenic inflammation.

3.9. Immunomodulatory Activity and Hematopoiesis Actions of Withania somnifera

Withania somnifera is an immunostimulant herbal remedy that is used to boost general health and prevent illness in the elderly [ 15 , 162 , 163 , 164 ]. Toll-like receptors, transcription factors, and inflammasomes all have a role in regulating inflammatory cytokines and chemokines [ 165 ]. NF-κB is the most researched transcription factor for modulating inflammatory cytokines in a variety of cell types [ 166 ]. Constitutive NF-κB activation has been demonstrated to increase the expression of NF-κB-related genes such as inflammatory cytokines/chemokines like CCL20/MIP-3 and granulocyte-macrophage colony stimulating factor (GM-CSF) [ 167 ]. WA’s ability to suppress NF-κB has been investigated in a variety of cell types and with a variety of triggering events [ 168 ]. A recent study by Kaileh et al. [ 169 ] showed that WA suppressed NF-κB activation by directly inhibiting IKKβ activity through thioalkylation, which are steroid lactones produced from WS, such as Withanolide A and 12-deoxywithastramonolide, are significantly less potent. The administration of a methanolic extract of the WS plant roots (1–256 g/mL) to mice macrophages resulted in an increase in nitric oxide generation due to nitric oxide synthase activation.

A recent study shows that WS has significant cytotoxic and cytostatic potential and induced immunogenic cell death (ICD) in human T leukemia cells [ 135 ]. In in vitro, WA inhibited mitogen-induced T-cell and B-cell growth without causing cell death. The upregulations of activation markers on T-cells (CD25), B-cells (CD80, CD86, and MHC-II), and the generation of Th1 and Th2 cytokines were also inhibited by WA. In microglial cells, WA reduced LPS-induced COX-2 and prostaglandin E-2 (PGE2) synthesis, while inhibiting TNF-α and IL-1β production in mononuclear cells [ 170 , 171 ]. The immunomodulatory effects of an alcoholic extract of the plant roots were studied in mice using cyclophosphamide, azathioprine, or prednisolone myelosuppression models. The extract enhanced the quantity of blood cells, the cellularity of bone marrow, and the number of α-esterase positive cells [ 172 , 173 , 174 ]. A study reports that Withaferin A suppressed iNOS expression and nitric oxide generation by Akt activations and downregulated LPS-induced NF-κB in RAW264.7 cells [ 175 ]. Dubey et al. show that Withaferin A treatment of THP-1 cells prevents NF-kB from translocating to the nucleus, resulting in lower levels of cytokine release [ 27 ]. Withaferin A inhibits caspase-1 activation by altering the nigericin-induced co-localization of NLRP3 and ASC proteins [ 27 ].

4. Nanotechnology-Based Strategies for the Delivery of WS

Multiple in vitro and in vivo studies have shown that WS exhibits anticancer properties. However, due to its poor water solubility, poor biodistribution, and multi-targeting capability, it may cause inevitable systemic toxicity. Nanotechnology approaches may help to reduce such uninvited adverse effects and improve clinical translation. Nanotechnology has gained a lot of attention in recent times because of its improved payload delivery to specific therapeutic locations, as well as its potential to alter cellular permeability, absorption, and pharmacokinetic profiles [ 176 , 177 , 178 , 179 ]. In recent years, the bioinspired production of NPs employing various biological systems, such as microbes and plants, has gained prominence [ 180 , 181 , 182 , 183 ]. Plant-based NPs production has attracted attention because of plant availability, tolerance, and eco-friendly NPs synthesis [ 180 , 181 , 182 , 183 ]. The phytoconstituents in the extract reduce and stabilize the generation of non-toxic NPs [ 180 , 181 , 182 , 183 ]. Among the many nanoparticle carriers (iron oxide, silicone material, and quantum dots), AuNPs are preferred owing to their high biocompatibility, quenching efficiency, ease of production, numerous functions, and adjustable optical nature [ 184 , 185 , 186 ]. According to Grand View Research, Inc., the nanomedicine industry is expected to be valued at USD 350.8 billion by 2025 [ 187 ].

Nanomedicines are divided into two categories ( Figure 3 ): organic nanoparticles (such as polymeric, liposomes, etc.) and micelles and inorganic nanoparticles (such as gold, silica, and iron oxide, etc.).

Inorganic nanoparticles have been utilized in several applications, including lymph node imaging, hyperthermia, and anemia therapy, and a section of them have been successful in preclinical research and clinical studies. Organic-based nanoparticles, like lipid and polymer nanoparticles, have successfully entered the clinical phase and are now available on the market for a variety of applications such as immunization, microbial infection, and cancer.

4.1. Inorganic Nanoparticles

Various inorganic nanomaterials utilized in bioimaging and therapeutics include metals, metal oxides, semiconductors, and lanthanide-laced NPs [ 188 , 189 , 190 , 191 , 192 , 193 , 194 ]. In bioimaging, inorganic NPs have been used as imaging probes that improve imaging methods like as computed tomography, magnetic resonance imaging, and optical imaging owing to their magnetic, X-ray attenuation, and optical characteristics [ 195 , 196 , 197 ]. Inorganic NPs have demonstrated immense potential in cancer disease therapy as whole drugs or drug delivery systems [ 198 , 199 , 200 , 201 ]. Furthermore, smart inorganic nanotherapeutics, which are stimuli responsive and target specific, have been generated to yield precise cancer treatment [ 202 , 203 , 204 ]. Some inorganic NPs that have gained clinical approval for disease therapy include the iron oxide NPs, Dexferrum, Feraheme, Infed, Feridex, Ferrlecit, Venofer, and Nano-therm [ 205 ]. Metallic NPs have been synthesized using aqueous extracts of various plant parts such as seeds, roots, leaves, stems, and fruits.

4.1.1. Gold Nanoparticles (AuNPs)

AuNPs offer unique qualities such as a huge surface area, the capacity to bind with different molecules, high stability, outstanding biocompatibility, and minimal toxicity, as well as the ability to control drug release [ 206 , 207 ]. AuNPs can easily be tagged with ligands for selective targeting because they can make bonds with amine and thiol groups. Because of their nano conjugation capacity, phytochemicals have been employed to develop Au nanocarrier-based conjugation for active-targeting drug delivery. AuNPs have been assessed for clinical applications as a result of their unique physicochemical properties. Tabassam et al. [ 208 ] recently assessed the anti-cancer potential of Withanolide-A with 20 nm AuNPs conjugates against SKBR3 breast cancer cell lines. The AuNPs can be generated by several in vitro and in situ methods, but only limited techniques can yield uniformly spherical particles.

A chemical synthesis method was utilized in the preparation of spherical AuNPs as well as conjugation of 10 μg/mL of Withanolide-A (1) with spherical AuNP solution, based on the same principle [ 96 ], and the phytochemical gold nanoconjugates was assessed through various analytical techniques, UV-visible spectroscopy, dynamic light scattering (DLS), and transmission electron microscopy (TEM). The absorption peak of AuNPs was λmax at 523.5 nm, similar to results from previous findings. DLS data showed that the presence of a slightly positively charged single hydrogen atom on an Au surface changed the zeta potential value from −44.3 ± 0.86 mV before conjugation to −20 ±0.1 mV after conjugation. A slight variation of the PDI from 0.285 ± 0.02 to 0.3 ± 0.009, which was less than 0.3, indicates non-uniformity in aggregation and size of the particles. The hydrodynamic size increased from 25.35 ± 0.61 nm to 29.73 ± 0.65 nm, which indicates the attachment of a single molecule on Au surface [ 209 ]. TEM analysis of the size and distribution of synthesized AuNPs recorded an average size of 20 nm on micrograph before and after conjugation. Moreover, Au nanoconjugates with Withanolide-A efficiently inhibited the growth of SKBR3 cells at half maximal concentration in contrast to pure Withanolide-A [ 208 ].

4.1.2. Titanium Oxide Nanoparticles (TiO 2 NPs)

The phytomediated synthesis of TiO 2 NPs has a lot of potential for killing bacteria, viruses, fungus, and cancer cells, and it can also be used to treat malignant tumors as a catalyzer [ 210 , 211 ]. Due to its extreme hydrophilicity, low toxicity, strong thermal conductivity, good optical absorption, and chemical and thermal durability in in vivo, TiO 2 NPs could be a promising candidate for biomedical applications as agents in converting photon energy into heat in the PTT method [ 212 , 213 ]. The biomediated production of TiO 2 NPs has been employed in disease therapy, surgical product manufacturing, photocatalysis, agriculture and tissue engineering [ 214 , 215 ].

Poly(ethylene glycol) (PEG) could be added to the surfaces of TiO 2 NPs to improve their biocompatibility. Several plants and plant organs have been utilized in TiO 2 NPs including roots, leaves, peel, flower, seeds, and pollen [ 216 , 217 , 218 , 219 ]. TiO 2 NPs are synthesized by various methods such as chemical vapor deposition, hydrothermal and reversed micellar methods, and the sol-gel process [ 220 , 221 , 222 , 223 ]. Titanium dioxide is a high-quality photocatalysts [ 224 , 225 , 226 ] with a wide bandgap of 3.2 eV and frequently used in optoelectronic gadgets and dye-sensitized solar cells [ 227 , 228 , 229 , 230 ]. Al-Shabib et al. synthesized phytomediated green TiO 2 NPs from Withania somnifera root extract, tested their broad-spectrum biofilm inhibitory efficacy against bacterial and fungal pathogens, and assessed HepG2 cytotoxicity [ 231 ]. The synthesized NPs significantly reduced the viability of HepG2 in in vitro and may be useful in the treatment of liver cancer. In another study, Maheswari and colleagues noted the antitumor and antibacterial properties of hydrothermally synthesized bio-modified TiO 2 nanoparticles with WS on KB oral cancer cell lines [ 230 ]. Bio-modified TiO 2 nanoparticles show dose-dependent activity in KB oral cancer cell lines. When compared with the modified bio-modified TiO 2 nanoparticles, pure TiO 2 nanoparticles had a higher viability percentage, proving that plant dopants treated with TiO 2 are effective anticancer agents [ 230 ].

4.1.3. Silver Nanoparticles (AgNPs)

AgNPs are among the most often used nanomaterials because of their antimicrobial characteristics, easily modified surface, controllable size and shape, strong electrical conductivity, and optical features [ 232 ]. AgNPs silver nanoparticles have been employed in a wide range of applications, including biosensors, electrical compounds, antimicrobials, and pharmaceuticals [ 233 ]. AgNPs can be synthesized through various methods such as physical (e.g., Turkevich), chemical (e.g., citrate or NaBH 4 ), and biological methods (e.g., plants, fungi, algae and other organic sources) with remarkable stability [ 232 , 234 ]. Biodegradable compounds and polymers can be added to the surface of AgNPs to improve their biocompatibility. Alternatively, these nanoparticles can be integrated into hybrid systems [ 235 ].

Tripathi et al. developed AgNPs through the reduction of silver nitrate solution using an in vitro-produced leaf extract of Withania coagulans Dunal (WcAgNPs) and evaluated anticancer activity with SiHa cell lines) [ 236 ]. WcAgNPs had a size of 14 nm and a spherical form with a face-centered cubic structure. WcAgNPs have excellent in vitro cytotoxicity in cervical cancer cells SiHa and induced apoptosis at 48 hrs. Gaurav et al., synthesized silver nanoparticles with root extract of WS (AgNPs-REWS) and tested for anticancer activity in in vitro [ 237 ]. A stable AgNPs made from Rhodiola imbricata root extract (RIW) and WS (RIWS-AgNPs) have been shown to have prospective uses in biomedicine and agriculture as phytostimulant, antioxidant, and anticancer agents [ 238 ]. Furthermore, RIWS-AgNPs have potent cytotoxic action against the HepG2 cancer cell line in a dose-dependent manner (cell viability: 9.51 ± 1.55%) [ 238 ].

4.1.4. Zinc Oxide Nanoparticles (ZnO NPs)

ZnO NPs are showing diverse medical applications and great promise in cancer treatment due to their high potency and selectivity for cancer cells [ 239 , 240 ]. As a possible substitute for photothermal therapy (PTT), zinc oxide (ZnO) has excellent chemical stability and minimal toxicity, as well as optical, electrical, and anticancer characteristics [ 241 ].The ROS and protein activity disequilibrium may be responsible for the cytotoxic action of ZnO NPs [ 242 ]. ZnO NPs are effective nanocarriers for the administration of several medications, such as DOX, paclitaxel, CUR, and baicalin as they have minimal toxicity and are biodegradable [ 242 ]. Kumar et al. show the immunomodulatory and protective effect of WS extract and Withaferin A supplementation on zinc oxide nanoparticles mediated the toxicity in a mouse model [ 243 ]. When ZnO NPs were delivered in vivo, a dose-dependent decrease in phagocytosis, an increase in NO generation, and an up-regulation of the TLR6 arginase gene were found to be significant. In the presence of WS and Withaferin A, however, ZnO NPs toxicity was reduced, with decreased TLR6 overexpression and restoration of phagocytic activity.

4.2. Organic Nanoparticles

Liposome-based nanomedicines employ drug encapsulation inside the phospholipid bilayer to improve its pharmacokinetics and biodistribution. Liposomes are globular vesicles surrounded by a phospholipid bilayer [ 244 , 245 ] and drug delivery mostly operates by passive targeting [ 205 , 246 ]. The advantages of liposomal drug delivery systems are as follows: (i) amphiphilic nature of liposomes enable them to deliver both hydrophobic and hydrophilic drugs; (ii) liposomal nanotherapeutics demonstrate enhanced accumulation and superior pharmacokinetics compared with non-liposomal agents at wound sites, resulting in decreased off-target toxicity and improved therapeutic efficacy; (iii) liposomes offer drug protection and stability and improves circulation half-life; and (iv) the functional coating of liposomal surfaces can yield targeted drug delivery systems [ 244 , 247 , 248 , 249 , 250 , 251 ]. However, the reticuloendothelial system (RES) and the mononuclear phagocytic network regulate liposomal clearance [ 252 , 253 , 254 ]. As such, PEG has been employed in liposomal modification to prolong the circulation half-life of liposomes [ 79 , 205 , 255 , 256 ].

Liposomal drug delivery systems are being assessed in clinical trials, including the liposomal nano formulations of docetaxel, paclitaxel, irinotecan, and cisplatin [ 257 , 258 , 259 , 260 ]. For instance, EndoTAG-1, a cationic liposome-based formulation of paclitaxel geared at PanCa therapy, liver metastases, and triple negative breast cancer has completed phase II clinical trials. Similarly, liposome-based nanomedicines of WS can be used for the cancer. Off-target toxicity and scale-up are the major obstacles in the way of the clinical translation of liposomal nano formulations. Recent liposomal formulations seek to improve precise disease targeting, such as ligand-functionalized liposomes, and have shown promising preclinical outcomes [ 261 , 262 ]. The disadvantages of polymeric micelles include minimal efficiency for intracellular drug release as well as possible off-target delivery. To navigate these limitations, stimuli-responsive polymeric micelles have been generated to facilitate sustained drug release in response to fluctuating environmental stimuli such as temperature or pH [ 263 , 264 ].

It is important to note that at the current time not all types of organic nanoparticles, such as self-assemblies, polymeric nanoparticles, dendrimers, and protein nanoparticles, have been utilized for the delivery of WS. However, future research may include the use of such clinically relevant carriers for efficient delivery of WS.

5. Future Perspective of WS Delivery

Phytochemicals have significant potential as anticancer agents. WS has anticancer properties through inhibiting pro-cancer mechanisms such as angiogenesis, migration, proliferation, invasion, and metastasis. WS promotes apoptosis through ROS generation, DNA damage, and regulation of oncogene and tumor suppressor gene expression. So far, there are no documented reports of toxicity of WS in humans. WS co-treatment may re-sensitize resistant cancer cells to chemotherapeutics and radiation. Despite several preclinical studies indicating WS cytotoxic potential against various cancers, physicians have not yet recognized its therapeutic value in treating cancer patients. Moreover, extant research on WS has barely evaluated its oral bioavailability, and investigation of these aspects of a new drug is essential before initiating clinical trials. Indeed, poor water solubility and biodistribution may restrict WS efficacy as an anticancer drug in somatic settings, and the dearth of extensive clinical investigations into WS as an anticancer agent may be due to these factors.

Nano formulation of this compound may help overcome barriers associated with natural phytochemicals like WS. Because phytochemicals constitute a varied collection of substances, it is critical to tailor nanoparticle formulations to the desired therapeutic agents’ physical and chemical characteristics. Fortunately, nanoparticles may be constructed from different substances, including lipids and polymers. In animals, formulations may be rationally planned as well as experimentally optimized. Passive targeting is quite simple to implement and may be readily applied in clinical practice. Liposomal vincristine, liposomal paclitaxel, and paclitaxel polymeric micelles are current examples of available nano-formulations. Actively focused drug delivery systems can outperform passively targeted nanocarriers with EPR effects. Smaller systems like antibody-drug conjugates (ADC) fall under this category. The market has several ADCs and many more are in different phases of trials. Many variables influence active targeting, including receptor expression and systemic circulation accessibility. As a result, WS must be carefully designed and optimized in terms of formulation. Targeted nanoparticles, we believe, will be the next step in the therapeutic evolution of WS. In conclusion, further extensive preclinical and clinical research of WS is required to better understand and enhance its anticancer activity. We anticipate that a new generation of nanocarriers will significantly advance the clinical use of WS by using the rapidly developing expertise in this sector.

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Author Contributions

V.K.K., M.M.Y. and S.C.C. conceived the idea and wrote the majority of manuscript. The manuscript was also written and edited by G.P.-D., A.D. and M.J. All authors have read and agreed to the published version of the manuscript.

Funding

The authors acknowledge the start-up research support from the Department of Immunology and Microbiology, School of Medicine, University of Texas Rio Grande Valley given to MJ, MMY, and SCC. This work was supported by the National Institute of Health/National Cancer Institute’s funding R01 CA210192, R01 CA206069, and SC1GM139727.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

References

1. Siegel R.L. Miller K.D. Fuchs H.E. Jemal A. Cancer Statistics, 2021 CA Cancer J. Clin. 2021 71 7 33 10.3322/caac.21654 33433946

2. The Centers for Disease Control and Prevention, an Update on Cancer Deaths in the United States Available online: https://www.cdc.gov/cancer/dcpc/research/update-on-cancer-deaths/index.htm (accessed on 3 March 2022)

3. Banik K. Harsha C. Bordoloi D. Lalduhsaki Sailo B. Sethi G. Leong H.C. Arfuso F. Mishra S. Wang L. Kumar A.P. Therapeutic potential of gambogic acid, a caged xanthone, to target cancer Cancer Lett. 2018 416 75 86 10.1016/j.canlet.2017.12.014 29246645

4. Kunnumakkara A.B. Banik K. Bordoloi D. Harsha C. Sailo B.L. Padmavathi G. Roy N.K. Gupta S.C. Aggarwal B.B. Googling the Guggul (Commiphora and Boswellia) for Prevention of Chronic Diseases Front. Pharmacol. 2018 9 686 10.3389/fphar.2018.00686 30127736 PMC6087759

5. Manu K.A. Shanmugam M.K. Ramachandran L. Li F. Siveen K.S. Chinnathambi A. Zayed M.E. Alharbi S.A. Arfuso F. Kumar A.P. Isorhamnetin augments the anti-tumor effect of capecitabine through the negative regulation of NF-κB signaling cascade in gastric cancer Cancer Lett. 2015 363 28 36 10.1016/j.canlet.2015.03.033 25827070

6. Manu K.A. Shanmugam M.K. Ramachandran L. Li F. Fong C.W. Kumar A.P. Tan P. Sethi G. First evidence that γ-tocotrienol inhibits the growth of human gastric cancer and chemosensitizes it to capecitabine in a xenograft mouse model through the modulation of NF-κB pathway Clin. Cancer Res. 2012 18 2220 2229 10.1158/1078-0432.CCR-11-2470 22351692

7. Seca A.M.L. Pinto D. Plant Secondary Metabolites as Anticancer Agents: Successes in Clinical Trials and Therapeutic Application Int. J. Mol. Sci. 2018 19 263 10.3390/ijms19010263 PMC5796209 29337925

8. Shanmugam M.K. Lee J.H. Chai E.Z. Kanchi M.M. Kar S. Arfuso F. Dharmarajan A. Kumar A.P. Ramar P.S. Looi C.Y. Cancer prevention and therapy through the modulation of transcription factors by bioactive natural compounds Semin. Cancer Biol. 2016 40–41 35 47 10.1016/j.semcancer.2016.03.005 27038646

9. Patel S.M. Nagulapalli Venkata K.C. Bhattacharyya P. Sethi G. Bishayee A. Potential of neem ( Azadirachta indica L.) for prevention and treatment of oncologic diseases Semin. Cancer Biol. 2016 40–41 100 115 10.1016/j.semcancer.2016.03.002 27019417

10. Singh N. Bhalla M. de Jager P. Gilca M. An overview on ashwagandha: A Rasayana (rejuvenator) of Ayurveda Afr. J. Tradit. Complement. Altern. Med. 2011 8 208 213 10.4314/ajtcam.v8i5S.9 22754076 PMC3252722

11. Dhuley J.N. Effect of ashwagandha on lipid peroxidation in stress-induced animals J. Ethnopharmacol. 1998 60 173 178 10.1016/S0378-8741(97)00151-7 9582008

12. Mehta V. Chander H. Munshi A. Mechanisms of Anti-Tumor Activity of Withania somnifera (Ashwagandha) Nutr. Cancer 2021 73 914 926 10.1080/01635581.2020.1778746 33949906

13. Rai M. Jogee P.S. Agarkar G. dos Santos C.A. Anticancer activities of Withania somnifera : Current research, formulations, and future perspectives Pharm. Biol. 2016 54 189 197 10.3109/13880209.2015.1027778 25845640

14. Dutta R. Khalil R. Green R. Mohapatra S.S. Mohapatra S. Withania somnifera (Ashwagandha) and Withaferin A: Potential in Integrative Oncology Int. J. Mol. Sci. 2019 20 5310 10.3390/ijms20215310 31731424 PMC6862083

15. Kashyap V.K. Dhasmana A. Yallapu M.M. Chauhan S.C. Jaggi M. Withania somnifera as a potential future drug molecule for COVID-19 Future Drug Discov. 2020 2 Fdd50 10.4155/fdd-2020-0024 33269342 PMC7702281

16. Siddiqui M.A. Farshori N.N. Al-Oqail M.M. Pant A.B. Al-Khedhairy A.A. Neuroprotective Effects of Withania somnifera on 4-Hydroxynonenal Induced Cell Death in Human Neuroblastoma SH-SY5Y Cells Through ROS Inhibition and Apoptotic Mitochondrial Pathway Neurochem. Res. 2021 46 171 182 10.1007/s11064-020-03146-4 33052512

17. Peng S.Y. Wang Y.Y. Lan T.H. Lin L.C. Yuan S.F. Tang J.Y. Chang H.W. Low Dose Combined Treatment with Ultraviolet-C and Withaferin a Enhances Selective Killing of Oral Cancer Cells Antioxidants 2020 9 1120 10.3390/antiox9111120 33202766 PMC7697686

18. Heidari Z. Mahmoudzadeh-Sagheb H. Sarbishegi M. Gorgich E.A.C. Withania coagulans extract attenuates oxidative stress-mediated apoptosis of cerebellar purkinje neurons after ischemia/reperfusion injury Metab. Brain Dis. 2021 36 1699 1708 10.1007/s11011-021-00745-0 33970396

19. Zhang X. Mukerji R. Samadi A.K. Cohen M.S. Down-regulation of estrogen receptor-alpha and rearranged during transfection tyrosine kinase is associated with withaferin a-induced apoptosis in MCF-7 breast cancer cells BMC Complement. Altern. Med. 2011 11 84 10.1186/1472-6882-11-84 21978374 PMC3198756

20. Stan S.D. Hahm E.-R. Warin R. Singh S.V. Withaferin A causes FOXO3a-and Bim-dependent apoptosis and inhibits growth of human breast cancer cells in vivo Cancer Res. 2008 68 7661 7669 10.1158/0008-5472.CAN-08-1510 18794155 PMC2562581

21. Yang E.S. Choi M.J. Kim J.H. Choi K.S. Kwon T.K. Withaferin A enhances radiation-induced apoptosis in Caki cells through induction of reactive oxygen species, Bcl-2 downregulation and Akt inhibition Chem.-Biol. Interact. 2011 190 9 15 10.1016/j.cbi.2011.01.015 21256832

22. Sinha P. Ostrand-Rosenberg S. Myeloid-derived suppressor cell function is reduced by Withaferin A, a potent and abundant component of Withania somnifera root extract Cancer Immunol. Immunother. 2013 62 1663 1673 10.1007/s00262-013-1470-2 23982485 PMC11028968

23. Kim S.H. Singh S.V. Mammary cancer chemoprevention by withaferin A is accompanied by in vivo suppression of self-renewal of cancer stem cells Cancer Prev. Res. 2014 7 738 747 10.1158/1940-6207.CAPR-13-0445 24824039 PMC4083822

24. Chandrasekaran B. Pal D. Kolluru V. Tyagi A. Baby B. Dahiya N.R. Youssef K. Alatassi H. Ankem M.K. Sharma A.K. The chemopreventive effect of withaferin A on spontaneous and inflammation-associated colon carcinogenesis models Carcinogenesis 2018 39 1537 1547 10.1093/carcin/bgy109 30124785 PMC6314334

25. Oh J.H. Kwon T.K. Withaferin A inhibits tumor necrosis factor-α-induced expression of cell adhesion molecules by inactivation of Akt and NF-κB in human pulmonary epithelial cells Int. Immunopharmacol. 2009 9 614 619 10.1016/j.intimp.2009.02.002 19236958

26. Garg S. Huifu H. Kumari A. Sundar D. Kaul S.C. Wadhwa R. Induction of Senescence in Cancer Cells by a Novel Combination of Cucurbitacin B and Withanone: Molecular Mechanism and Therapeutic Potential J. Gerontol. Ser. A 2019 75 1031 1041 10.1093/gerona/glz077 31112603

27. Dubey S. Yoon H. Cohen M.S. Nagarkatti P. Nagarkatti M. Karan D. Withaferin A Associated Differential Regulation of Inflammatory Cytokines Front. Immunol. 2018 9 195 10.3389/fimmu.2018.00195 29479354 PMC5811468

28. Tewari D. Chander V. Dhyani A. Sahu S. Gupta P. Patni P. Kalick L.S. Bishayee A. Withania somnifera (L.) Dunal: Phytochemistry, structure-activity relationship, and anticancer potential Phytomedicine 2022 98 153949 10.1016/j.phymed.2022.153949 35151215

29. Choudhary D. Bhattacharyya S. Bose S. Efficacy and Safety of Ashwagandha ( Withania somnifera (L.) Dunal) Root Extract in Improving Memory and Cognitive Functions J. Diet Suppl. 2017 14 599 612 10.1080/19390211.2017.1284970 28471731

30. Choudhary D. Bhattacharyya S. Joshi K. Body Weight Management in Adults Under Chronic Stress Through Treatment With Ashwagandha Root Extract: A Double-Blind, Randomized, Placebo-Controlled Trial J. Evid. Based Complement. Altern. Med. 2017 22 96 106 10.1177/2156587216641830 PMC5871210 27055824

31. Salve J. Pate S. Debnath K. Langade D. Adaptogenic and Anxiolytic Effects of Ashwagandha Root Extract in Healthy Adults: A Double-blind, Randomized, Placebo-controlled Clinical Study Cureus 2019 11 e6466 10.7759/cureus.6466 32021735 PMC6979308

32. Chandrasekhar K. Kapoor J. Anishetty S. A prospective, randomized double-blind, placebo-controlled study of safety and efficacy of a high-concentration full-spectrum extract of ashwagandha root in reducing stress and anxiety in adults Indian J. Psychol. Med. 2012 34 255 262 10.4103/0253-7176.106022 23439798 PMC3573577

33. Kulkarni S.K. Dhir A. Withania somnifera : An Indian ginseng Prog. Neuropsychopharmacol. Biol. Psychiatry 2008 32 1093 1105 10.1016/j.pnpbp.2007.09.011 17959291

34. Dar N.J. Hamid A. Ahmad M. Pharmacologic overview of Withania somnifera , the Indian Ginseng Cell Mol. Life Sci. 2015 72 4445 4460 10.1007/s00018-015-2012-1 26306935 PMC11113996

35. Gorelick J. Rosenberg R. Smotrich A. Hanuš L. Bernstein N. Hypoglycemic activity of withanolides and elicitated Withania somnifera Phytochemistry 2015 116 283 289 10.1016/j.phytochem.2015.02.029 25796090

36. Gupta A. Singh S. Evaluation of anti-inflammatory effect of Withania somnifera root on collagen-induced arthritis in rats Pharm. Biol. 2014 52 308 320 10.3109/13880209.2013.835325 24188460

37. Mohanty I.R. Arya D.S. Gupta S.K. Withania somnifera provides cardioprotection and attenuates ischemia-reperfusion induced apoptosis Clin. Nutr. 2008 27 635 642 10.1016/j.clnu.2008.05.006 18620784

38. RajaSankar S. Manivasagam T. Sankar V. Prakash S. Muthusamy R. Krishnamurti A. Surendran S. Withania somnifera root extract improves catecholamines and physiological abnormalities seen in a Parkinson’s disease model mouse J. Ethnopharmacol. 2009 125 369 373 10.1016/j.jep.2009.08.003 19666100

39. Bhatnagar M. Goel I. Roy T. Shukla S.D. Khurana S. Complete Comparison Display (CCD) evaluation of ethanol extracts of Centella asiatica and Withania somnifera shows that they can non-synergistically ameliorate biochemical and behavioural damages in MPTP induced Parkinson’s model of mice PLoS ONE 2017 12 e0177254 10.1371/journal.pone.0177254 28510600 PMC5433711

40. Chandrasekaran S. Dayakar A. Veronica J. Sundar S. Maurya R. An in vitro study of apoptotic like death in Leishmania donovani promastigotes by withanolides Parasitol. Int. 2013 62 253 261 10.1016/j.parint.2013.01.007 23416156

41. Sachdeva H. Sehgal R. Kaur S. Studies on the protective and immunomodulatory efficacy of Withania somnifera along with cisplatin against experimental visceral leishmaniasis Parasitol. Res. 2013 112 2269 2280 10.1007/s00436-013-3387-2 23519426

42. Reuland D.J. Khademi S. Castle C.J. Irwin D.C. McCord J.M. Miller B.F. Hamilton K.L. Upregulation of phase II enzymes through phytochemical activation of Nrf2 protects cardiomyocytes against oxidant stress Free Radic. Biol. Med. 2013 56 102 111 10.1016/j.freeradbiomed.2012.11.016 23201694

43. Budhiraja R.D. Sudhir S. Review of biological activity of withanolides J. Sci. Ind. Res. 1987 46 488 491

44. Rastogi RP M.B. Compendium of Indian Medicinal Plants Central Drug Research Institute New Delhi, India 1998 6

45. Bone K. Clinical applications of Ayurvedic and Chinese herbs Monograph for the Western Herbal Practitioner Phytotherapy Press Warwick, QLD, Australia 1996 137 141

46. Elsakka M. Grigorescu E. Stănescu U. Stănescu U. Dorneanu V. New data referring to chemistry of Withania somnifera species Rev. Med. Chir. Soc. Med. Nat. Iasi 1990 94 385 387 2100857

47. Srivastava D.N. Deshpande S.M. Gas chromatographic identification of fatty acids, fatty alcohols, and hydrocarbons of Convolvulus pluricaulis (Chois) J. Am. Oil Chem. Soc. 1975 52 318 319 10.1007/BF02637735 1176757

48. Vedi M. Sabina E.P. Assessment of hepatoprotective and nephroprotective potential of withaferin A on bromobenzene-induced injury in Swiss albino mice: Possible involvement of mitochondrial dysfunction and inflammation Cell Biol. Toxicol. 2016 32 373 390 10.1007/s10565-016-9340-2 27250656

49. Yan Z. Guo R. Gan L. Lau W.B. Cao X. Zhao J. Ma X. Christopher T.A. Lopez B.L. Wang Y. Withaferin A inhibits apoptosis via activated Akt-mediated inhibition of oxidative stress Life Sci. 2018 211 91 101 10.1016/j.lfs.2018.09.020 30213729

50. Tiruveedi V.L. Bale S. Khurana A. Godugu C. Withaferin A, a novel compound of Indian ginseng ( Withania somnifera ), ameliorates Cerulein-induced acute pancreatitis: Possible role of oxidative stress and inflammation Phytother. Res. 2018 32 2586 2596 10.1002/ptr.6200 30307087

51. Palliyaguru D.L. Chartoumpekis D.V. Wakabayashi N. Skoko J.J. Yagishita Y. Singh S.V. Kensler T.W. Withaferin A induces Nrf2-dependent protection against liver injury: Role of Keap1-independent mechanisms Free Radic. Biol. Med. 2016 101 116 128 10.1016/j.freeradbiomed.2016.10.003 27717869 PMC5154810

52. Bale S. Pulivendala G. Godugu C. Withaferin A attenuates bleomycin-induced scleroderma by targeting FoxO3a and NF-κβ signaling: Connecting fibrosis and inflammation Biofactors 2018 44 507 517 10.1002/biof.1446 30367690

53. Anwar M.F. Yadav D. Rastogi S. Arora I. Khar R.K. Chander J. Samim M. Modulation of liver and kidney toxicity by herb Withania somnifera for silver nanoparticles: A novel approach for harmonizing between safety and use of nanoparticles Protoplasma 2015 252 547 558 10.1007/s00709-014-0701-5 25248758

54. Biswal B.M. Sulaiman S.A. Ismail H.C. Zakaria H. Musa K.I. Effect of Withania somnifera (Ashwagandha) on the development of chemotherapy-induced fatigue and quality of life in breast cancer patients Integr. Cancer Ther. 2013 12 312 322 10.1177/1534735412464551 23142798

55. Zhang X. Samadi A.K. Roby K.F. Timmermann B. Cohen M.S. Inhibition of cell growth and induction of apoptosis in ovarian carcinoma cell lines CaOV3 and SKOV3 by natural withanolide Withaferin A Gynecol. Oncol. 2012 124 606 612 10.1016/j.ygyno.2011.11.044 22188785 PMC3278582

56. Thaiparambil J.T. Bender L. Ganesh T. Kline E. Patel P. Liu Y. Tighiouart M. Vertino P.M. Harvey R.D. Garcia A. Withaferin A inhibits breast cancer invasion and metastasis at sub-cytotoxic doses by inducing vimentin disassembly and serine 56 phosphorylation Int. J. Cancer 2011 129 2744 2755 10.1002/ijc.25938 21538350

57. Oh J.H. Lee T.-J. Kim S.H. Choi Y.H. Lee S.H. Lee J.M. Kim Y.-H. Park J.-W. Kwon T.K. Induction of apoptosis by withaferin A in human leukemia U937 cells through down-regulation of Akt phosphorylation Apoptosis 2008 13 1494 1504 10.1007/s10495-008-0273-y 19002588

58. Widodo N. Kaur K. Shrestha B.G. Takagi Y. Ishii T. Wadhwa R. Kaul S.C. Selective killing of cancer cells by leaf extract of Ashwagandha: Identification of a tumor-inhibitory factor and the first molecular insights to its effect Clin. Cancer Res. 2007 13 2298 2306 10.1158/1078-0432.CCR-06-0948 17404115

59. Widodo N. Takagi Y. Shrestha B.G. Ishii T. Kaul S.C. Wadhwa R. Selective killing of cancer cells by leaf extract of Ashwagandha: Components, activity and pathway analyses Cancer Lett. 2008 262 37 47 10.1016/j.canlet.2007.11.037 18191020

60. Yang H. Shi G. Dou Q.P. The tumor proteasome is a primary target for the natural anticancer compound Withaferin A isolated from “Indian winter cherry” Mol. Pharmacol. 2007 71 426 437 10.1124/mol.106.030015 17093135

61. Samadi A.K. Mukerji R. Shah A. Timmermann B.N. Cohen M.S. A novel RET inhibitor with potent efficacy against medullary thyroid cancer in vivo Surgery 2010 148 1228 1236 10.1016/j.surg.2010.09.026 21134556 PMC3088305

62. Yang H. Wang Y. Cheryan V.T. Wu W. Cui C.Q. Polin L.A. Pass H.I. Dou Q.P. Rishi A.K. Wali A. Withaferin A inhibits the proteasome activity in mesothelioma in vitro and in vivo PLoS ONE 2012 7 e41214 10.1371/journal.pone.0041214 22912669 PMC3422308

63. Yu Y. Hamza A. Zhang T. Gu M. Zou P. Newman B. Li Y. Gunatilaka A.A. Zhan C.G. Sun D. Withaferin A targets heat shock protein 90 in pancreatic cancer cells Biochem. Pharmacol. 2010 79 542 551 10.1016/j.bcp.2009.09.017 19769945 PMC2794909

64. Manoharan S. Panjamurthy K. Menon V.P. Balakrishnan S. Alias L.M. Protective effect of Withaferin-A on tumour formation in 7,12-dimethylbenz[a]anthracene induced oral carcinogenesis in hamsters Indian J. Exp. Biol. 2009 47 16 23 19317347

65. Hahm E.R. Lee J. Singh S.V. Role of mitogen-activated protein kinases and Mcl-1 in apoptosis induction by withaferin A in human breast cancer cells Mol. Carcinog. 2014 53 907 916 10.1002/mc.22050 24019090 PMC3859703

66. Devi P.U. Sharada A.C. Solomon F.E. Kamath M.S. In vivo growth inhibitory effect of Withania somnifera (Ashwagandha) on a transplantable mouse tumor, Sarcoma 180 Indian J. Exp. Biol. 1992 30 169 172 1512021

67. Munagala R. Kausar H. Munjal C. Gupta R.C. Withaferin A induces p53-dependent apoptosis by repression of HPV oncogenes and upregulation of tumor suppressor proteins in human cervical cancer cells Carcinogenesis 2011 32 1697 1705 10.1093/carcin/bgr192 21859835

68. Xia S. Miao Y. Liu S. Withaferin A induces apoptosis by ROS-dependent mitochondrial dysfunction in human colorectal cancer cells Biochem. Biophys. Res. Commun. 2018 503 2363 2369 10.1016/j.bbrc.2018.06.162 29966656

69. Chang E. Pohling C. Natarajan A. Witney T.H. Kaur J. Xu L. Gowrishankar G. D’Souza A.L. Murty S. Schick S. AshwaMAX and Withaferin A inhibits gliomas in cellular and murine orthotopic models J. Neurooncol. 2016 126 253 264 10.1007/s11060-015-1972-1 26650066 PMC5597337

70. Li X. Zhu F. Jiang J. Sun C. Zhong Q. Shen M. Wang X. Tian R. Shi C. Xu M. Simultaneous inhibition of the ubiquitin-proteasome system and autophagy enhances apoptosis induced by ER stress aggravators in human pancreatic cancer cells Autophagy 2016 12 1521 1537 10.1080/15548627.2016.1191722 27308733 PMC5082778

71. Mandal C. Dutta A. Mallick A. Chandra S. Misra L. Sangwan R.S. Mandal C. Withaferin A induces apoptosis by activating p38 mitogen-activated protein kinase signaling cascade in leukemic cells of lymphoid and myeloid origin through mitochondrial death cascade Apoptosis 2008 13 1450 1464 10.1007/s10495-008-0271-0 18987975

72. Malik F. Kumar A. Bhushan S. Khan S. Bhatia A. Suri K.A. Qazi G.N. Singh J. Reactive oxygen species generation and mitochondrial dysfunction in the apoptotic cell death of human myeloid leukemia HL-60 cells by a dietary compound withaferin A with concomitant protection by N-acetyl cysteine Apoptosis 2007 12 2115 2133 10.1007/s10495-007-0129-x 17874299

73. Srinivasan S. Ranga R.S. Burikhanov R. Han S.S. Chendil D. Par-4-dependent apoptosis by the dietary compound withaferin A in prostate cancer cells Cancer Res. 2007 67 246 253 10.1158/0008-5472.CAN-06-2430 17185378

74. Abutaha N. In vitro antiproliferative activity of partially purified Withania somnifera fruit extract on different cancer cell lines J. Balkan Union Oncol. 2015 20 625 630 26011359

75. Mohan R. Hammers H.J. Bargagna-Mohan P. Zhan X.H. Herbstritt C.J. Ruiz A. Zhang L. Hanson A.D. Conner B.P. Rougas J. Withaferin A is a potent inhibitor of angiogenesis Angiogenesis 2004 7 115 122 10.1007/s10456-004-1026-3 15516832

76. Ahmed W. Mofed D. Zekri A.-R. El-Sayed N. Rahouma M. Sabet S. Antioxidant activity and apoptotic induction as mechanisms of action of Withania somnifera (Ashwagandha) against a hepatocellular carcinoma cell line J. Int. Med. Res. 2018 46 1358 1369 10.1177/0300060517752022 29392963 PMC6091842

77. Yu Y. Katiyar S.P. Sundar D. Kaul Z. Miyako E. Zhang Z. Kaul S.C. Reddel R.R. Wadhwa R. Withaferin-A kills cancer cells with and without telomerase: Chemical, computational and experimental evidences Cell Death Dis. 2017 8 e2755 10.1038/cddis.2017.33 28425984 PMC5477593

78. Mayola E. Gallerne C. Esposti D.D. Martel C. Pervaiz S. Larue L. Debuire B. Lemoine A. Brenner C. Lemaire C. Withaferin A induces apoptosis in human melanoma cells through generation of reactive oxygen species and down-regulation of Bcl-2 Apoptosis 2011 16 1014 1027 10.1007/s10495-011-0625-x 21710254

79. Hsu J.H. Chang P.M. Cheng T.S. Kuo Y.L. Wu A.T. Tran T.H. Yang Y.H. Chen J.M. Tsai Y.C. Chu Y.S. Identification of Withaferin A as a Potential Candidate for Anti-Cancer Therapy in Non-Small Cell Lung Cancer Cancers 2019 11 1003 10.3390/cancers11071003 PMC6678286 31319622

80. McKenna M.K. Gachuki B.W. Alhakeem S.S. Oben K.N. Rangnekar V.M. Gupta R.C. Bondada S. Anti-cancer activity of withaferin A in B-cell lymphoma Cancer Biol. Ther. 2015 16 1088 1098 10.1080/15384047.2015.1046651 26020511 PMC4622669

81. Nishikawa Y. Okuzaki D. Fukushima K. Mukai S. Ohno S. Ozaki Y. Yabuta N. Nojima H. Withaferin A Induces Cell Death Selectively in Androgen-Independent Prostate Cancer Cells but Not in Normal Fibroblast Cells PLoS ONE 2015 10 e0134137 10.1371/journal.pone.0134137 26230090 PMC4521694

82. Das T. Roy K.S. Chakrabarti T. Mukhopadhyay S. Roychoudhury S. Withaferin A modulates the Spindle assembly checkpoint by degradation of Mad2-Cdc20 complex in colorectal cancer cell lines Biochem. Pharmacol. 2014 91 31 39 10.1016/j.bcp.2014.06.022 24995417

83. Vaishnavi K. Saxena N. Shah N. Singh R. Manjunath K. Uthayakumar M. Kanaujia S.P. Kaul S.C. Sekar K. Wadhwa R. Differential Activities of the Two Closely Related Withanolides, Withaferin A and Withanone: Bioinformatics and Experimental Evidences PLoS ONE 2012 7 e44419 10.1371/journal.pone.0044419 22973447 PMC3433425

84. Sari A.N. Bhargava P. Dhanjal J.K. Putri J.F. Radhakrishnan N. Shefrin S. Ishida Y. Terao K. Sundar D. Kaul S.C. Combination of Withaferin-A and CAPE Provides Superior Anticancer Potency: Bioinformatics and Experimental Evidence to Their Molecular Targets and Mechanism of Action Cancers 2020 12 1160 10.3390/cancers12051160 32380701 PMC7281427

85. Sundar D. Yu Y. Katiyar S.P. Putri J.F. Dhanjal J.K. Wang J. Sari A.N. Kolettas E. Kaul S.C. Wadhwa R. Wild type p53 function in p53Y220C mutant harboring cells by treatment with Ashwagandha derived anticancer withanolides: Bioinformatics and experimental evidence J. Exp. Clin. Cancer Res. 2019 38 103 10.1186/s13046-019-1099-x 30808373 PMC6390572

86. Choudhary M.I. Hussain S. Yousuf S. Dar A. Mudassar Attaur R. Chlorinated and diepoxy withanolides from Withania somnifera and their cytotoxic effects against human lung cancer cell line Phytochemistry 2010 71 2205 2209 10.1016/j.phytochem.2010.08.019 21044792

87. Oza V.P. Parmar P.P. Kumar S. Subramanian R.B. Anticancer properties of highly purified L-asparaginase from Withania somnifera L. against acute lymphoblastic leukemia Appl. Biochem. Biotechnol. 2010 160 1833 1840 10.1007/s12010-009-8667-z 19448978

88. Leyon P.V. Kuttan G. Effect of Withania somnifera on B16F-10 melanoma induced metastasis in mice Phytother. Res. 2004 18 118 122 10.1002/ptr.1378 15022162

89. Bani S. Gautam M. Sheikh F.A. Khan B. Satti N.K. Suri K.A. Qazi G.N. Patwardhan B. Selective Th1 up-regulating activity of Withania somnifera aqueous extract in an experimental system using flow cytometry J. Ethnopharmacol. 2006 107 107 115 10.1016/j.jep.2006.02.016 16603328

90. Zhao J. Nakamura N. Hattori M. Kuboyama T. Tohda C. Komatsu K. Withanolide derivatives from the roots of Withania somnifera and their neurite outgrowth activities Chem. Pharm. Bull. 2002 50 760 765 10.1248/cpb.50.760 12045329

91. Wadhwa R. Singh R. Gao R. Shah N. Widodo N. Nakamoto T. Ishida Y. Terao K. Kaul S.C. Water Extract of Ashwagandha Leaves Has Anticancer Activity: Identification of an Active Component and Its Mechanism of Action PLoS ONE 2013 8 e77189 10.1371/annotation/b7059f27-5970-4734-8601-9913adcce984 24130852 PMC3795014

92. Jayaprakasam B. Zhang Y. Seeram N.P. Nair M.G. Growth inhibition of human tumor cell lines by withanolides from Withania somnifera leaves Life Sci. 2003 74 125 132 10.1016/j.lfs.2003.07.007 14575818

93. Rah B. Amin H. Yousuf K. Khan S. Jamwal G. Mukherjee D. Goswami A. A novel MMP-2 inhibitor 3-azidowithaferin A (3-azidoWA) abrogates cancer cell invasion and angiogenesis by modulating extracellular Par-4 PLoS ONE 2012 7 e44039 10.1371/journal.pone.0044039 22962598 PMC3433490

94. Wang H.-C. Tsai Y.-L. Wu Y.-C. Chang F.-R. Liu M.-H. Chen W.-Y. Wu C.-C. Withanolides-Induced Breast Cancer Cell Death Is Correlated with Their Ability to Inhibit Heat Protein 90 PLoS ONE 2012 7 e37764 10.1371/journal.pone.0037764 22701533 PMC3365124

95. Wang H.-C. Hu H.-H. Chang F.-R. Tsai J.-Y. Kuo C.-Y. Wu Y.-C. Wu C.-C. Different effects of 4β-hydroxywithanolide E and withaferin A, two withanolides from Solanaceae plants, on the Akt signaling pathway in human breast cancer cells Phytomedicine 2019 53 213 222 10.1016/j.phymed.2018.09.017 30668401

96. Yadav D.K. Kumar S. Saloni Singh H. Kim M.H. Sharma P. Misra S. Khan F. Molecular docking, QSAR and ADMET studies of withanolide analogs against breast cancer Drug Des. Dev. Ther. 2017 11 1859 1870 10.2147/DDDT.S130601 28694686 PMC5491705

97. Mondal S. Mandal C. Sangwan R. Chandra S. Mandal C. Withanolide D induces apoptosis in leukemia by targeting the activation of neutral sphingomyelinase-ceramide cascade mediated by synergistic activation of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase Mol. Cancer 2010 9 239 10.1186/1476-4598-9-239 20836852 PMC2949798

98. Issa M.E. Wijeratne E.M.K. Gunatilaka A.A.L. Cuendet M. Withanolide D Exhibits Similar Cytostatic Effect in Drug-Resistant and Drug-Sensitive Multiple Myeloma Cells Front. Pharmacol. 2017 8 610 10.3389/fphar.2017.00610 28943850 PMC5596074

99. Kataria H. Shah N. Kaul S.C. Wadhwa R. Kaur G. Water extract of ashwagandha leaves limits proliferation and migration, and induces differentiation in glioma cells Evid.-Based Complement. Altern. 2011 2011 267614 10.1093/ecam/nep188 PMC3096473 20007262

100. Widodo N. Priyandoko D. Shah N. Wadhwa R. Kaul S.C. Selective Killing of Cancer Cells by Ashwagandha Leaf Extract and Its Component Withanone Involves ROS Signaling PLoS ONE 2010 5 e13536 10.1371/journal.pone.0013536 20975835 PMC2958829

101. Kim S.-H. Singh K.B. Hahm E.-R. Lokeshwar B.L. Singh S.V. Withania somnifera root extract inhibits fatty acid synthesis in prostate cancer cells J. Tradit. Complement. Med. 2020 10 188 197 10.1016/j.jtcme.2020.02.002 32670813 PMC7340880

102. Setty Balakrishnan A. Nathan A.A. Kumar M. Ramamoorthy S. Ramia Mothilal S.K. Withania somnifera targets interleukin-8 and cyclooxygenase-2 in human prostate cancer progression Prostate Int. 2017 5 75 83 10.1016/j.prnil.2017.03.002 28593171 PMC5448731

103. Henley A.B. Yang L. Chuang K.-L. Sahuri-Arisoylu M. Wu L.-H. Bligh S.W.A. Bell J.D. Withania somnifera Root Extract Enhances Chemotherapy through ‘Priming’ PLoS ONE 2017 12 e0170917 10.1371/journal.pone.0170917 28129345 PMC5271386

104. Kunnumakkara A.B. Harsha C. Banik K. Vikkurthi R. Sailo B.L. Bordoloi D. Gupta S.C. Aggarwal B.B. Is curcumin bioavailability a problem in humans: Lessons from clinical trials Expert. Opin. Drug Metab. Toxicol. 2019 15 705 733 10.1080/17425255.2019.1650914 31361978

105. Dai T. Jiang W. Guo Z. Wang Z. Huang M. Zhong G. Liang C. Pei X. Dai R. Studies on oral bioavailability and first-pass metabolism of withaferin A in rats using LC-MS/MS and Q-TRAP Biomed. Chromatogr. 2019 33 e4573 10.1002/bmc.4573 31062367

106. Patil D. Gautam M. Mishra S. Karupothula S. Gairola S. Jadhav S. Pawar S. Patwardhan B. Determination of withaferin A and withanolide A in mice plasma using high-performance liquid chromatography-tandem mass spectrometry: Application to pharmacokinetics after oral administration of Withania somnifera aqueous extract J. Pharm. Biomed. Anal. 2013 80 203 212 10.1016/j.jpba.2013.03.001 23584079

107. Senthilnathan P. Padmavathi R. Banu S.M. Sakthisekaran D. Enhancement of antitumor effect of paclitaxel in combination with immunomodulatory Withania somnifera on benzo (a) pyrene induced experimental lung cancer Chem.-Biol. Interact. 2006 159 180 185 10.1016/j.cbi.2005.11.003 16375880

108. Senthilnathan P. Padmavathi R. Magesh V. Sakthisekaran D. Chemotherapeutic efficacy of paclitaxel in combination with Withania somnifera on benzo (a) pyrene-induced experimental lung cancer Cancer Sci. 2006 97 658 664 10.1111/j.1349-7006.2006.00224.x 16827807 PMC11159414

109. Cai Y. Sheng Z.-Y. Chen Y. Bai C. Effect of Withaferin A on A549 cellular proliferation and apoptosis in non-small cell lung cancer Asian Pac. J. Cancer Prev. 2014 15 1711 1714 10.7314/APJCP.2014.15.4.1711 24641396

110. Kyakulaga A.H. Aqil F. Munagala R. Gupta R.C. Withaferin A inhibits Epithelial to Mesenchymal Transition in Non-Small Cell Lung Cancer Cells Sci. Rep. 2018 8 15737 10.1038/s41598-018-34018-1 30356176 PMC6200817

111. Bray F. Ferlay J. Soerjomataram I. Siegel R.L. Torre L.A. Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries CA Cancer J. Clin. 2018 68 394 424 10.3322/caac.21492 30207593

112. Arpino G. Milano M. De Placido S. Features of aggressive breast cancer Breast 2015 24 594 600 10.1016/j.breast.2015.06.001 26144637

113. Stan S.D. Zeng Y. Singh S.V. Ayurvedic medicine constituent withaferin a causes G2 and M phase cell cycle arrest in human breast cancer cells Nutr. Cancer 2008 60 51 60 10.1080/01635580802381477 19003581 PMC2597540

114. Hahm E.-R. Singh S.V. Withaferin A-induced apoptosis in human breast cancer cells is associated with suppression of inhibitor of apoptosis family protein expression Cancer Lett. 2013 334 101 108 10.1016/j.canlet.2012.08.026 22935676 PMC3530008

115. Hahm E.-R. Moura M.B. Kelley E.E. Van Houten B. Shiva S. Singh S.V. Withaferin A-induced apoptosis in human breast cancer cells is mediated by reactive oxygen species PLoS ONE 2011 6 e23354 10.1371/journal.pone.0023354 21853114 PMC3154436

116. Bargagna-Mohan P. Hamza A. Kim Y.-e. Ho Y.K.A. Mor-Vaknin N. Wendschlag N. Liu J. Evans R.M. Markovitz D.M. Zhan C.-G. The tumor inhibitor and antiangiogenic agent withaferin A targets the intermediate filament protein vimentin Chem. Biol. 2007 14 623 634 10.1016/j.chembiol.2007.04.010 17584610 PMC3228641

117. Yang Z. Garcia A. Xu S. Powell D.R. Vertino P.M. Singh S. Marcus A.I. Withania somnifera root extract inhibits mammary cancer metastasis and epithelial to mesenchymal transition PLoS ONE 2013 8 e75069 10.1371/journal.pone.0075069 24069380 PMC3771884

118. Khazal K.F. Hill D.L. Withania somnifera extract reduces the invasiveness of MDA-MB-231 breast cancer and inhibits cytokines associated with metastasis J. Cancer Metastasis Treat. 2015 1 94 100 10.4103/2394-4722.157601 28090590 PMC5228601

119. Khazal K.F. Samuel T. Hill D.L. Grubbs C.J. Effect of an extract of Withania somnifera root on estrogen receptor-positive mammary carcinomas Anticancer Res. 2013 33 1519 1523 23564793 PMC3675906

120. Hahm E.R. Lee J. Huang Y. Singh S.V. Withaferin a suppresses estrogen receptor-α expression in human breast cancer cells Mol. Carcinog. 2011 50 614 624 10.1002/mc.20760 21432907 PMC3129407

121. Lee J. Hahm E.R. Marcus A.I. Singh S.V. Withaferin A inhibits experimental epithelial-mesenchymal transition in MCF-10A cells and suppresses vimentin protein level in vivo in breast tumors Mol. Carcinog. 2015 54 417 429 10.1002/mc.22110 24293234 PMC4039625

122. Lee J. Hahm E.-R. Singh S.V. Withaferin A inhibits activation of signal transducer and activator of transcription 3 in human breast cancer cells Carcinogenesis 2010 31 1991 1998 10.1093/carcin/bgq175 20724373 PMC2966554

123. Mulabagal V. Subbaraju G.V. Rao C.V. Sivaramakrishna C. DeWitt D.L. Holmes D. Sung B. Aggarwal B.B. Tsay H.S. Nair M.G. Withanolide sulfoxide from Aswagandha roots inhibits nuclear transcription factor-kappa-B, cyclooxygenase and tumor cell proliferation Phytother. Res. Int. J. Devoted Pharmacol. Toxicol. Eval. Nat. Prod. Deriv. 2009 23 987 992 10.1002/ptr.2736 19152372

124. Lee J. Sehrawat A. Singh S.V. Withaferin A causes activation of Notch2 and Notch4 in human breast cancer cells Breast Cancer Res. Treat. 2012 136 45 56 10.1007/s10549-012-2239-6 22965833 PMC3474857

125. Nagalingam A. Kuppusamy P. Singh S.V. Sharma D. Saxena N.K. Mechanistic elucidation of the antitumor properties of withaferin a in breast cancer Cancer Res. 2014 74 2617 2629 10.1158/0008-5472.CAN-13-2081 24732433 PMC4009451

126. Vel Szic K.S. Declerck K. Crans R.A. Diddens J. Scherf D.B. Gerhäuser C. Berghe W.V. Epigenetic silencing of triple negative breast cancer hallmarks by Withaferin A Oncotarget 2017 8 40434 10.18632/oncotarget.17107 28467815 PMC5522326

127. Roy R.V. Suman S. Das T.P. Luevano J.E. Damodaran C. Withaferin A, a steroidal lactone from Withania somnifera , induces mitotic catastrophe and growth arrest in prostate cancer cells J. Nat. Prod. 2013 76 1909 1915 10.1021/np400441f 24079846 PMC4144448

128. Kunimasa K. Nagano T. Shimono Y. Dokuni R. Kiriu T. Tokunaga S. Tamura D. Yamamoto M. Tachihara M. Kobayashi K. Glucose metabolism-targeted therapy and withaferin A are effective for epidermal growth factor receptor tyrosine kinase inhibitor-induced drug-tolerant persisters Cancer Sci. 2017 108 1368 1377 10.1111/cas.13266 28445002 PMC5497794

129. Muralikrishnan G. Dinda A.K. Shakeel F. Immunomodulatory effects of Withania somnifera on azoxymethane induced experimental colon cancer in mice Immunol. Investig. 2010 39 688 698 10.3109/08820139.2010.487083 20840055

130. Koduru S. Kumar R. Srinivasan S. Evers M.B. Damodaran C. Notch-1 inhibition by Withaferin-A: A therapeutic target against colon carcinogenesis Mol. Cancer Ther. 2010 9 202 210 10.1158/1535-7163.MCT-09-0771 20053782 PMC3041017

131. Choi B.Y. Kim B.-W. Withaferin-A inhibits colon cancer cell growth by blocking STAT3 transcriptional activity J. Cancer Prev. 2015 20 185 10.15430/JCP.2015.20.3.185 26473157 PMC4597807

132. Alnuqaydan A.M. Rah B. Almutary A.G. Chauhan S.S. Synergistic antitumor effect of 5-fluorouracil and withaferin-A induces endoplasmic reticulum stress-mediated autophagy and apoptosis in colorectal cancer cells Am. J. Cancer Res. 2020 10 799 32266092 PMC7136917

133. Yang E.S. Choi M.J. Kim J.H. Choi K.S. Kwon T.K. Combination of withaferin A and X-ray irradiation enhances apoptosis in U937 cells Toxicol. In Vitro 2011 25 1803 1810 10.1016/j.tiv.2011.09.016 21964475

134. Okamoto S. Tsujioka T. Suemori S. Kida J. Kondo T. Tohyama Y. Tohyama K. Withaferin A suppresses the growth of myelodysplasia and leukemia cell lines by inhibiting cell cycle progression Cancer Sci. 2016 107 1302 1314 10.1111/cas.12988 27311589 PMC5021033

135. Turrini E. Calcabrini C. Sestili P. Catanzaro E. de Gianni E. Diaz A.R. Hrelia P. Tacchini M. Guerrini A. Canonico B. Withania somnifera Induces Cytotoxic and Cytostatic Effects on Human T Leukemia Cells Toxins 2016 8 147 10.3390/toxins8050147 27187469 PMC4885062

136. Prakash J. Gupta S.K. Dinda A.K. Withania somnifera root extract prevents DMBA-induced squamous cell carcinoma of skin in Swiss albino mice Nutr. Cancer. 2002 42 91 97 10.1207/S15327914NC421_12 12235655

137. Chang H.-W. Li R.-N. Wang H.-R. Liu J.-R. Tang J.-Y. Huang H.-W. Chan Y.-H. Yen C.-Y. Withaferin A induces oxidative stress-mediated apoptosis and DNA damage in oral cancer cells Front. Physiol. 2017 8 634 10.3389/fphys.2017.00634 28936177 PMC5594071

138. Lv T.Z. Wang G.S. Antiproliferation potential of withaferin A on human osteosarcoma cells via the inhibition of G2/M checkpoint proteins Exp. Ther. Med. 2015 10 323 329 10.3892/etm.2015.2480 26170956 PMC4487066

139. Kim G. Kim T.H. Hwang E.H. Chang K.T. Hong J.J. Park J.H. Withaferin A inhibits the proliferation of gastric cancer cells by inducing G2/M cell cycle arrest and apoptosis Oncol. Lett. 2017 14 416 422 10.3892/ol.2017.6169 28693185 PMC5494816

140. Li X. Zhu F. Jiang J. Sun C. Wang X. Shen M. Tian R. Shi C. Xu M. Peng F. Synergistic antitumor activity of withaferin A combined with oxaliplatin triggers reactive oxygen species-mediated inactivation of the PI3K/AKT pathway in human pancreatic cancer cells Cancer lett. 2015 357 219 230 10.1016/j.canlet.2014.11.026 25444914

141. Fong M.Y. Jin S. Rane M. Singh R.K. Gupta R. Kakar S.S. Withaferin A synergizes the therapeutic effect of doxorubicin through ROS-mediated autophagy in ovarian cancer PLoS ONE 2012 7 e42265 10.1371/journal.pone.0042265 22860102 PMC3408484

142. Kakar S.S. Ratajczak M.Z. Powell K.S. Moghadamfalahi M. Miller D.M. Batra S.K. Singh S.K. Withaferin a alone and in combination with cisplatin suppresses growth and metastasis of ovarian cancer by targeting putative cancer stem cells PLoS ONE 2014 9 e107596 10.1371/journal.pone.0107596 25264898 PMC4180068

143. Lee D.H. Lim I.-H. Sung E.-G. Kim J.-Y. Song I.-H. Park Y.K. Lee T.-J. Withaferin A inhibits matrix metalloproteinase-9 activity by suppressing the Akt signaling pathway Oncol. Rep. 2013 30 933 938 10.3892/or.2013.2487 23708780

144. Um H.J. Min K.-j. Kim D.E. Kwon T.K. Withaferin A inhibits JAK/STAT3 signaling and induces apoptosis of human renal carcinoma Caki cells Biochem. Biophys. Res. Commun. 2012 427 24 29 10.1016/j.bbrc.2012.08.133 22982675

145. Choi M.J. Park E.J. Min K.J. Park J.W. Kwon T.K. Endoplasmic reticulum stress mediates withaferin A-induced apoptosis in human renal carcinoma cells Toxicol In Vitro 2011 25 692 698 10.1016/j.tiv.2011.01.010 21266191

146. Heyninck K. Lahtela-Kakkonen M. Van der Veken P. Haegeman G. Berghe W.V. Withaferin A inhibits NF-kappaB activation by targeting cysteine 179 in IKKβ Biochem. Pharmacol. 2014 91 501 509 10.1016/j.bcp.2014.08.004 25159986

147. Tripathi S.K. Pandey K. Panda M. Spinella M.J. Rengasamy K.R.R. Biswal B.K. The potential of retinoids for combination therapy of lung cancer: Updates and future directions Pharmacol. Res. 2019 147 104331 10.1016/j.phrs.2019.104331 31254665 PMC7066582

148. Kyakulaga A.H. Aqil F. Munagala R. Gupta R.C. Synergistic combinations of paclitaxel and withaferin A against human non-small cell lung cancer cells Oncotarget 2020 11 1399 1416 10.18632/oncotarget.27519 32362998 PMC7185067

149. Suttana W. Mankhetkorn S. Poompimon W. Palagani A. Zhokhov S. Gerlo S. Haegeman G. Berghe W.V. Differential chemosensitization of P-glycoprotein overexpressing K562/Adr cells by withaferin A and Siamois polyphenols Mol. Cancer 2010 9 99 10.1186/1476-4598-9-99 20438634 PMC2873443

150. Rah B. ur Rasool R. Nayak D. Yousuf S.K. Mukherjee D. Kumar L.D. Goswami A. PAWR-mediated suppression of BCL2 promotes switching of 3-azido withaferin A (3-AWA)-induced autophagy to apoptosis in prostate cancer cells Autophagy 2015 11 314 331 10.1080/15548627.2015.1017182 25803782 PMC4502794

151. Grogan P.T. Sleder K.D. Samadi A.K. Zhang H. Timmermann B.N. Cohen M.S. Cytotoxicity of withaferin A in glioblastomas involves induction of an oxidative stress-mediated heat shock response while altering Akt/mTOR and MAPK signaling pathways Investig. New Drugs 2013 31 545 557 10.1007/s10637-012-9888-5 23129310 PMC3677827

152. Grogan P.T. Sarkaria J.N. Timmermann B.N. Cohen M.S. Oxidative cytotoxic agent withaferin A resensitizes temozolomide-resistant glioblastomas via MGMT depletion and induces apoptosis through Akt/mTOR pathway inhibitory modulation Investig. New Drugs. 2014 32 604 617 10.1007/s10637-014-0084-7 24718901 PMC4380174

153. Sun G.Y. Li R. Cui J. Hannink M. Gu Z. Fritsche K.L. Lubahn D.B. Simonyi A. Withania somnifera and Its Withanolides Attenuate Oxidative and Inflammatory Responses and Up-Regulate Antioxidant Responses in BV-2 Microglial Cells Neuromolecular Med. 2016 18 241 252 10.1007/s12017-016-8411-0 27209361

154. Kataria H. Wadhwa R. Kaul S.C. Kaur G. Withania somnifera water extract as a potential candidate for differentiation based therapy of human neuroblastomas PLoS ONE 2013 8 e55316 10.1371/journal.pone.0055316 23383150 PMC3561198

155. Kataria H. Kumar S. Chaudhary H. Kaur G. Withania somnifera Suppresses Tumor Growth of Intracranial Allograft of Glioma Cells Mol. Neurobiol. 2016 53 4143 4158 10.1007/s12035-015-9320-1 26208698

156. Ting L.-L. Chou A.S.-B. Hsieh C.-H. Hsiung S.-C. Pang S.-T. Liao S.-K. Withaferin A targeting both cancer stem cells and metastatic cancer stem cells in the UP-LN1 carcinoma cell model J. Cancer Metastatis Treat. 2016 2 29 40

157. Cohen S.M. Mukerji R. Timmermann B.N. Samadi A.K. Cohen M.S. A novel combination of withaferin A and sorafenib shows synergistic efficacy against both papillary and anaplastic thyroid cancers Am. J. Surg. 2012 204 895 900 10.1016/j.amjsurg.2012.07.027 23231932

158. Pilot Study of Curcumin Formulation and Ashwagandha Extract in Advanced Osteosarcoma (OSCAT) Available online: https://clinicaltrials.gov/ct2/show/results/NCT00689195?term=Withania+somnifera&cond=cancer&draw=2&rank=3 (accessed on 3 March 2022)

159. Bhat J. Damle A. Vaishnav P.P. Albers R. Joshi M. Banerjee G. In vivo enhancement of natural killer cell activity through tea fortified with Ayurvedic herbs Phytother. Res. 2010 24 129 135 10.1002/ptr.2889 19504465

160. Ashwagandha: Effects on Stress, Inflammation and Immune Cell Activation Available online: https://clinicaltrials.gov/ct2/show/NCT00817752?term=Withania+somnifera&cond=cancer&draw=2&rank=2 (accessed on 3 March 2022)

161. Chengappa K.N.R. Brar J.S. Gannon J.M. Schlicht P.J. Adjunctive Use of a Standardized Extract of Withania somnifera (Ashwagandha) to Treat Symptom Exacerbation in Schizophrenia: A Randomized, Double-Blind, Placebo-Controlled Study J. Clin. Psychiatry 2018 79 22496 10.4088/JCP.17m11826 29995356

162. Mandlik Ingawale D.S. Namdeo A.G. Pharmacological evaluation of Ashwagandha highlighting its healthcare claims, safety, and toxicity aspects J. Diet Suppl. 2021 18 183 226 10.1080/19390211.2020.1741484 32242751

163. Khanal P. Chikhale R. Dey Y.N. Pasha I. Chand S. Gurav N. Ayyanar M. Patil B.M. Gurav S. Withanolides from Withania somnifera as an immunity booster and their therapeutic options against COVID-19 J. Biomol. Struct. Dyn. 2021 10.1080/07391102.2020.1869588 33459174

164. Chandran U. Patwardhan B. Network ethnopharmacological evaluation of the immunomodulatory activity of Withania somnifera J. Ethnopharmacol. 2017 197 250 256 10.1016/j.jep.2016.07.080 27487266

165. Turner M.D. Nedjai B. Hurst T. Pennington D.J. Cytokines and chemokines: At the crossroads of cell signalling and inflammatory disease Biochim. Biophys. Acta (BBA)-Mol. Cell Res. 2014 1843 2563 2582 10.1016/j.bbamcr.2014.05.014 24892271

166. Liu T. Zhang L. Joo D. Sun S.-C. NF-κB signaling in inflammation Signal Transduct. Target. Ther. 2017 2 17023 10.1038/sigtrans.2017.23 29158945 PMC5661633

167. Ghosh S. May M.J. Kopp E.B. NF-kappa B and Rel proteins: Evolutionarily conserved mediators of immune responses Annu Rev. Immunol. 1998 16 225 260 10.1146/annurev.immunol.16.1.225 9597130

168. Vanden Berghe W. Sabbe L. Kaileh M. Haegeman G. Heyninck K. Molecular insight in the multifunctional activities of Withaferin A Biochem. Pharmacol. 2012 84 1282 1291 10.1016/j.bcp.2012.08.027 22981382

169. Iuvone T. Esposito G. Capasso F. Izzo A.A. Induction of nitric oxide synthase expression by Withania somnifera in macrophages Life Sci. 2003 72 1617 1625 10.1016/S0024-3205(02)02472-4 12551750

170. Min K.J. Choi K. Kwon T.K. Withaferin A down-regulates lipopolysaccharide-induced cyclooxygenase-2 expression and PGE2 production through the inhibition of STAT1/3 activation in microglial cells Int. Immunopharmacol. 2011 11 1137 1142 10.1016/j.intimp.2011.02.029 21396905

171. Singh D. Aggarwal A. Maurya R. Naik S. Withania somnifera inhibits NF-kappaB and AP-1 transcription factors in human peripheral blood and synovial fluid mononuclear cells Phytother. Res. 2007 21 905 913 10.1002/ptr.2180 17562568

172. Russo A. Izzo A.A. Cardile V. Borrelli F. Vanella A. Indian medicinal plants as antiradicals and DNA cleavage protectors Phytomedicine 2001 8 125 132 10.1078/0944-7113-00021 11315755

173. Davis L. Kuttan G. Immunomodulatory activity of Withania somnifera J. Ethnopharmacol. 2000 71 193 200 10.1016/S0378-8741(99)00206-8 10904163

174. Ziauddin M. Phansalkar N. Patki P. Diwanay S. Patwardhan B. Studies on the immunomodulatory effects of Ashwagandha J. Ethnopharmacol. 1996 50 69 76 10.1016/0378-8741(95)01318-0 8866726

175. Oh J.H. Lee T.J. Park J.W. Kwon T.K. Withaferin A inhibits iNOS expression and nitric oxide production by Akt inactivation and down-regulating LPS-induced activity of NF-kappaB in RAW 264.7 cells Eur. J. Pharmacol. 2008 599 11 17 10.1016/j.ejphar.2008.09.017 18838070

176. Chauhan D.S. Dhasmana A. Laskar P. Prasad R. Jain N.K. Srivastava R. Jaggi M. Chauhan S.C. Yallapu M.M. Nanotechnology synergized immunoengineering for cancer Eur. J. Pharm. Biopharm. 2021 163 72 101 10.1016/j.ejpb.2021.03.010 33774162 PMC8170847

177. Shetty A. Nagesh P.K.B. Setua S. Hafeez B.B. Jaggi M. Yallapu M.M. Chauhan S.C. Novel Paclitaxel Nanoformulation Impairs De Novo Lipid Synthesis in Pancreatic Cancer Cells and Enhances Gemcitabine Efficacy ACS Omega 2020 5 8982 8991 10.1021/acsomega.0c00793 32337462 PMC7178800

178. Samanta K. Setua S. Kumari S. Jaggi M. Yallapu M.M. Chauhan S.C. Gemcitabine Combination Nano Therapies for Pancreatic Cancer Pharmaceutics 2019 11 574 10.3390/pharmaceutics11110574 31689930 PMC6920852

179. Massey A.E. Sikander M. Chauhan N. Kumari S. Setua S. Shetty A.B. Mandil H. Kashyap V.K. Khan S. Jaggi M. Next-generation paclitaxel-nanoparticle formulation for pancreatic cancer treatment Nanomedicine 2019 20 102027 10.1016/j.nano.2019.102027 31170509 PMC6705422

180. Kim B. Park J.E. Im E. Cho Y. Lee J. Lee H.J. Sim D.Y. Park W.Y. Shim B.S. Kim S.H. Recent Advances in Nanotechnology with Nano-Phytochemicals: Molecular Mechanisms and Clinical Implications in Cancer Progression Int. J. Mol. Sci. 2021 22 3571 10.3390/ijms22073571 33808235 PMC8036762

181. Salama L. Pastor E.R. Stone T. Mousa S.A. Emerging Nanopharmaceuticals and Nanonutraceuticals in Cancer Management Biomedicines 2020 8 347 10.3390/biomedicines8090347 PMC7554840 32932737

182. Granja A. Frias I. Neves A.R. Pinheiro M. Reis S. Therapeutic Potential of Epigallocatechin Gallate Nanodelivery Systems BioMed Res. Int. 2017 2017 5813793 10.1155/2017/5813793 28791306 PMC5534279

183. Khan T. Gurav P. PhytoNanotechnology: Enhancing Delivery of Plant Based Anti-cancer Drugs Front. Pharmacol. 2018 8 1002 10.3389/fphar.2017.01002 29479316 PMC5811929

184. Wilhelm S. Tavares A.J. Dai Q. Ohta S. Audet J. Dvorak H.F. Chan W.C.W. Analysis of nanoparticle delivery to tumours Nat. Rev. Mater. 2016 1 16014 10.1038/natrevmats.2016.14

185. Khan S. Setua S. Kumari S. Dan N. Massey A. Hafeez B.B. Yallapu M.M. Stiles Z.E. Alabkaa A. Yue J. Superparamagnetic iron oxide nanoparticles of curcumin enhance gemcitabine therapeutic response in pancreatic cancer Biomaterials 2019 208 83 97 10.1016/j.biomaterials.2019.04.005 30999154 PMC13521448

186. Nagesh P.K.B. Chowdhury P. Hatami E. Boya V.K.N. Kashyap V.K. Khan S. Hafeez B.B. Chauhan S.C. Jaggi M. Yallapu M.M. miRNA-205 Nanoformulation Sensitizes Prostate Cancer Cells to Chemotherapy Cancers 2018 10 289 10.3390/cancers10090289 30149628 PMC6162422

187. Nanomedicine Market Is Estimated To Be Valued At $350.8 Billion By 2025: Grand View Research, Inc Available online: https://www.grandviewresearch.com/press-release/global-nanomedicine-market (accessed on 3 March 2022)

188. Anselmo A.C. Mitragotri S. A Review of Clinical Translation of Inorganic Nanoparticles AAPS J. 2015 17 1041 1054 10.1208/s12248-015-9780-2 25956384 PMC4540735

189. Zhang C. Yan L. Gu Z. Zhao Y. Strategies based on metal-based nanoparticles for hypoxic-tumor radiotherapy Chem. Sci. 2019 10 6932 6943 10.1039/C9SC02107H 31588260 PMC6676466

190. Ju Y. Dong B. Yu J. Hou Y. Inherent multifunctional inorganic nanomaterials for imaging-guided cancer therapy Nano Today 2019 26 108 122 10.1016/j.nantod.2019.03.006

191. Hess K.L. Medintz I.L. Jewell C.M. Designing inorganic nanomaterials for vaccines and immunotherapies Nano Today 2019 27 73 98 10.1016/j.nantod.2019.04.005 32292488 PMC7156029

192. Tian G. Zhang X. Gu Z. Zhao Y. Recent advances in upconversion nanoparticles-based multifunctional nanocomposites for combined cancer therapy Adv. Mater. 2015 27 7692 7712 10.1002/adma.201503280 26505885

193. Kim T. Hyeon T. Applications of inorganic nanoparticles as therapeutic agents Nanotechnology 2013 25 012001 10.1088/0957-4484/25/1/012001 24334327

194. Erathodiyil N. Ying J.Y. Functionalization of inorganic nanoparticles for bioimaging applications Acc. Chem. Res. 2011 44 925 935 10.1021/ar2000327 21648430

195. Na H.B. Song I.C. Hyeon T. Inorganic nanoparticles for MRI contrast agents Adv. Mater. 2009 21 2133 2148 10.1002/adma.200802366

196. Liu J. Zheng X. Yan L. Zhou L. Tian G. Yin W. Wang L. Liu Y. Hu Z. Gu Z. Bismuth sulfide nanorods as a precision nanomedicine for in vivo multimodal imaging-guided photothermal therapy of tumor ACS Nano 2015 9 696 707 10.1021/nn506137n 25561009

197. Tian G. Gu Z. Zhou L. Yin W. Liu X. Yan L. Jin S. Ren W. Xing G. Li S. Mn2+ dopant-controlled synthesis of NaYF4: Yb/Er upconversion nanoparticles for in vivo imaging and drug delivery Adv. Mater. 2012 24 1226 1231 10.1002/adma.201104741 22282270

198. Du J. Gu Z. Yan L. Yong Y. Yi X. Zhang X. Liu J. Wu R. Ge C. Chen C. Poly(Vinylpyrollidone)- and Selenocysteine-Modified Bi(2) Se(3) Nanoparticles Enhance Radiotherapy Efficacy in Tumors and Promote Radioprotection in Normal Tissues Adv. Mater. 2017 29 1701268 10.1002/adma.201701268 28714110

199. Samia A.C. Chen X. Burda C. Semiconductor quantum dots for photodynamic therapy J. Am. Chem. Soc. 2003 125 15736 15737 10.1021/ja0386905 14677951

200. Son S.J. Bai X. Lee S.B. Inorganic hollow nanoparticles and nanotubes in nanomedicine Part 1. Drug/gene delivery applications Drug Discov. Today 2007 12 650 656 10.1016/j.drudis.2007.06.002 17706547

201. Zhang C. Yan L. Wang X. Dong X. Zhou R. Gu Z. Zhao Y. Tumor Microenvironment-Responsive Cu(2)(OH)PO(4) Nanocrystals for Selective and Controllable Radiosentization via the X-ray-Triggered Fenton-like Reaction Nano Lett. 2019 19 1749 1757 10.1021/acs.nanolett.8b04763 30773886

202. Gu Z. Zhu S. Yan L. Zhao F. Zhao Y. Graphene-Based Smart Platforms for Combined Cancer Therapy Adv. Mater. 2019 31 e1800662 10.1002/adma.201800662 30039878

203. Du Z. Zhang X. Guo Z. Xie J. Dong X. Zhu S. Du J. Gu Z. Zhao Y. X-Ray-Controlled Generation of Peroxynitrite Based on Nanosized LiLuF(4): Ce(3+) Scintillators and their Applications for Radiosensitization Adv. Mater. 2018 30 e1804046 10.1002/adma.201804046 30260520

204. Chen H. Gu Z. An H. Chen C. Chen J. Cui R. Chen S. Chen W. Chen X. Chen X. Precise nanomedicine for intelligent therapy of cancer Sci. China Chem. 2018 61 1503 1552 10.1007/s11426-018-9397-5

205. Bobo D. Robinson K.J. Islam J. Thurecht K.J. Corrie S.R. Nanoparticle-based medicines: A review of FDA-approved materials and clinical trials to date Pharm. Res. 2016 33 2373 2387 10.1007/s11095-016-1958-5 27299311

206. Lim Z.-Z.J. Li J.-E.J. Ng C.-T. Yung L.-Y.L. Bay B.-H. Gold nanoparticles in cancer therapy Acta Pharmacol. Sin. 2011 32 983 990 10.1038/aps.2011.82 21743485 PMC4002534

207. Muddineti O.S. Ghosh B. Biswas S. Current trends in using polymer coated gold nanoparticles for cancer therapy Int. J. Pharm. 2015 484 252 267 10.1016/j.ijpharm.2015.02.038 25701627

208. Tabassam Q. Mehmood T. Raza A.R. Ullah A. Saeed F. Anjum F.M. Synthesis, Characterization and Anti-Cancer Therapeutic Potential of Withanolide-A with 20nm sAuNPs Conjugates Against SKBR3 Breast Cancer Cell Line Int. J. Nanomed. 2020 15 6649 6658 10.2147/IJN.S258528 32982224 PMC7498930

209. Wang D. Naydenov N.G. Dozmorov M.G. Koblinski J.E. Ivanov A.I. Anillin regulates breast cancer cell migration, growth, and metastasis by non-canonical mechanisms involving control of cell stemness and differentiation Breast Cancer Res. 2020 22 3 10.1186/s13058-019-1241-x 31910867 PMC6947866

210. Devanand Venkatasubbu G. Ramasamy S. Ramakrishnan V. Kumar J. Folate targeted PEGylated titanium dioxide nanoparticles as a nanocarrier for targeted paclitaxel drug delivery Adv. Powder Technol. 2013 24 947 954 10.1016/j.apt.2013.01.008

211. Yamaguchi S. Kobayashi H. Narita T. Kanehira K. Sonezaki S. Kudo N. Kubota Y. Terasaka S. Houkin K. Sonodynamic therapy using water-dispersed TiO 2 -polyethylene glycol compound on glioma cells: Comparison of cytotoxic mechanism with photodynamic therapy Ultrason. Sonochem. 2011 18 1197 1204 10.1016/j.ultsonch.2010.12.017 21257331

212. Wang R. Hashimoto K. Fujishima A. Chikuni M. Kojima E. Kitamura A. Shimohigoshi M. Watanabe T. Light-induced amphiphilic surfaces Nature 1997 388 431 432 10.1038/41233

213. Yin Z.F. Wu L. Yang H.G. Su Y.H. Recent progress in biomedical applications of titanium dioxide Phys. Chem. Chem. Phys. 2013 15 4844 4858 10.1039/c3cp43938k 23450160

214. Xu P. Wang R. Ouyang J. Chen B. A new strategy for TiO 2 whiskers mediated multi-mode cancer treatment Nanoscale Res. Lett. 2015 10 94 10.1186/s11671-015-0796-4 25852390 PMC4385221

215. Liu E. Zhou Y. Liu Z. Li J. Zhang D. Chen J. Cai Z. Cisplatin loaded hyaluronic acid modified TiO 2 nanoparticles for neoadjuvant chemotherapy of ovarian cancer J. Nanomater. 2015 16 275

216. Kulshrestha S. Qayyum S. Khan A.U. Antibiofilm efficacy of green synthesized graphene oxide-silver nanocomposite using Lagerstroemia speciosa floral extract: A comparative study on inhibition of gram-positive and gram-negative biofilms Microb. Pathog. 2017 103 167 177 10.1016/j.micpath.2016.12.022 28027944

217. Rajkumari J. Magdalane C.M. Siddhardha B. Madhavan J. Ramalingam G. Al-Dhabi N.A. Arasu M.V. Ghilan A.K.M. Duraipandiayan V. Kaviyarasu K. Synthesis of titanium oxide nanoparticles using Aloe barbadensis mill and evaluation of its antibiofilm potential against Pseudomonas aeruginosa PAO1 J. Photochem. Photobiol. B 2019 201 111667 10.1016/j.jphotobiol.2019.111667 31683167

218. Madadi Z. Bagheri Lotfabad T. Aqueous Extract of Acanthophyllum laxiusculum Roots as a Renewable Resource for Green synthesis of nano-sized titanium dioxide using Sol-gel Method Adv. Ceram. Prog. 2016 2 26 31 10.30501/acp.2016.70015

219. Kashale A.A. Gattu K.P. Ghule K. Ingole V.H. Dhanayat S. Sharma R. Chang J.-Y. Ghule A.V. Biomediated green synthesis of TiO 2 nanoparticles for lithium ion battery application Compos. B Eng. 2016 99 297 304 10.1016/j.compositesb.2016.06.015

220. Bischoff B.L. Anderson M.A. Peptization Process in the Sol-Gel Preparation of Porous Anatase (TiO 2 ) Chem. Mater. 1995 7 1772 1778 10.1021/cm00058a004

221. Reinke M. Ponomarev E. Kuzminykh Y. Hoffmann P. Combinatorial Characterization of TiO 2 Chemical Vapor Deposition Utilizing Titanium Isopropoxide ACS Comb. Sci. 2015 17 413 420 10.1021/acscombsci.5b00040 26036755

222. Sakai H. Kawahara H. Shimazaki M. Abe M. Preparation of Ultrafine Titanium Dioxide Particles Using Hydrolysis and Condensation Reactions in the Inner Aqueous Phase of Reversed Micelles: Effect of Alcohol Addition Langmuir 1998 14 2208 2212 10.1021/la970952r

223. Chen H. Wang Y. Dong S. An Effective Hydrothermal Route for the Synthesis of Multiple PDDA-Protected Noble-Metal Nanostructures Inorg. Chem. 2007 46 10587 10593 10.1021/ic7009572 17999489

224. Leshuk T. Parviz R. Everett P. Krishnakumar H. Varin R.A. Gu F. Photocatalytic activity of hydrogenated TiO 2 ACS Appl. Mater. Interfaces 2013 5 1892 1895 10.1021/am302903n 23465766

225. Schneider J. Matsuoka M. Takeuchi M. Zhang J. Horiuchi Y. Anpo M. Bahnemann D.W. Understanding TiO 2 photocatalysis: Mechanisms and materials Chem. Rev. 2014 114 9919 9986 10.1021/cr5001892 25234429

226. Anderson C. Bard A.J. Improved Photocatalytic Activity and Characterization of Mixed TiO 2 /SiO 2 and TiO 2 /Al 2 O 3 Materials J. Phys. Chem. 1997 101 2611 2616 10.1021/jp9626982

227. Benavides J.A. Trudeau C.P. Gerlein L.F. Cloutier S.G. Laser Selective Photoactivation of Amorphous TiO 2 Films to Anatase and/or Rutile Crystalline Phases ACS Appl. Energy Mater. 2018 1 3607 3613 10.1021/acsaem.8b00171

228. Carta D. Salaoru I. Khiat A. Regoutz A. Mitterbauer C. Harrison N.M. Prodromakis T. Investigation of the Switching Mechanism in TiO 2 -Based RRAM: A Two-Dimensional EDX Approach ACS Appl. Mater. Interfaces 2016 8 19605 19611 10.1021/acsami.6b04919 27409358

229. Bai Y. Mora-Seró I. De Angelis F. Bisquert J. Wang P. Titanium Dioxide Nanomaterials for Photovoltaic Applications Chem. Rev. 2014 114 10095 10130 10.1021/cr400606n 24661129

230. Maheswari P. Harish S. Navaneethan M. Muthamizhchelvan C. Ponnusamy S. Hayakawa Y. Bio-modified TiO(2) nanoparticles with Withania somnifera , Eclipta prostrata and Glycyrrhiza glabra for anticancer and antibacterial applications Mater. Sci. Eng. C Mater. Biol. Appl. 2020 108 110457 10.1016/j.msec.2019.110457 31924033

231. Al-Shabib N.A. Husain F.M. Qais F.A. Ahmad N. Khan A. Alyousef A.A. Arshad M. Noor S. Khan J.M. Alam P. Phyto-Mediated Synthesis of Porous Titanium Dioxide Nanoparticles From Withania somnifera Root Extract: Broad-Spectrum Attenuation of Biofilm and Cytotoxic Properties Against HepG2 Cell Lines Front. Microbiol. 2020 11 1680 10.3389/fmicb.2020.01680 32849352 PMC7399045

232. Xu L. Wang Y.-Y. Huang J. Chen C.-Y. Wang Z.-X. Xie H. Silver nanoparticles: Synthesis, medical applications and biosafety Theranostics 2020 10 8996 9031 10.7150/thno.45413 32802176 PMC7415816

233. Abbasi E. Milani M. Fekri Aval S. Kouhi M. Akbarzadeh A. Tayefi Nasrabadi H. Nikasa P. Joo S.W. Hanifehpour Y. Nejati-Koshki K. Silver nanoparticles: Synthesis methods, bio-applications and properties Crit. Rev. Microbiol. 2016 42 173 180 10.3109/1040841X.2014.912200 24937409

234. Mousavi S.M. Hashemi S.A. Ghasemi Y. Atapour A. Amani A.M. Savar Dashtaki A. Babapoor A. Arjmand O. Green synthesis of silver nanoparticles toward bio and medical applications: Review study Artif. Cells Nanomed. Biotechnol. 2018 46 S855 S872 10.1080/21691401.2018.1517769 30328732

235. Kim D. Amatya R. Hwang S. Lee S. Min K.A. Shin M.C. BSA-Silver Nanoparticles: A Potential Multimodal Therapeutics for Conventional and Photothermal Treatment of Skin Cancer Pharmaceutics 2021 13 575 10.3390/pharmaceutics13040575 33920666 PMC8073043

236. Tripathi D. Modi A. Narayan G. Rai S.P. Green and cost effective synthesis of silver nanoparticles from endangered medicinal plant Withania coagulans and their potential biomedical properties Mater. Sci. Eng. C Mater. Biol. Appl. 2019 100 152 164 10.1016/j.msec.2019.02.113 30948049

237. Gaurav I. Singh T. Thakur A. Kumar G. Rathee P. Kumari P. Sweta K. Synthesis, In-Vitro and In-Silico Evaluation of Silver Nanoparticles with Root Extract of Withania somnifera for Antibacterial Activity via Binding of Penicillin-Binding Protein-4 Curr. Pharm. Biotechnol. 2020 21 1674 1687 10.2174/1389201021666200702152000 32614743

238. Kapoor S. Sood H. Saxena S. Chaurasia O.P. Green synthesis of silver nanoparticles using Rhodiola imbricata and Withania somnifera root extract and their potential catalytic, antioxidant, cytotoxic and growth-promoting activities Bioprocess Biosyst. Eng. 2022 45 365 380 10.1007/s00449-021-02666-9 34988733

239. Bisht G. Rayamajhi S. ZnO Nanoparticles: A Promising Anticancer Agent Nanobiomedicine 2016 3 9 10.5772/63437 29942384 PMC5998263

240. Zhang Y. Nayak T.R. Hong H. Cai W. Biomedical applications of zinc oxide nanomaterials Curr. Mol. Med. 2013 13 1633 1645 10.2174/1566524013666131111130058 24206130 PMC3838497

241. Kołodziejczak-Radzimska A. Jesionowski T. Zinc Oxide-From Synthesis to Application: A Review Materials 2014 7 2833 2881 10.3390/ma7042833 28788596 PMC5453364

242. Vasuki K. Manimekalai R. NIR light active ternary modified ZnO nanocomposites for combined cancer therapy Heliyon 2019 5 e02729 10.1016/j.heliyon.2019.e02729 31844691 PMC6895759

243. Kumar J. Mitra M.D. Hussain A. Kaul G. Exploration of immunomodulatory and protective effect of Withania somnifera on trace metal oxide (zinc oxide nanoparticles) induced toxicity in Balb/c mice Mol. Biol. Rep. 2019 46 2447 2459 10.1007/s11033-019-04705-x 30847852

244. Ventola C.L. Progress in nanomedicine: Approved and investigational nanodrugs Pharm. Ther. 2017 42 742 PMC5720487 29234213

245. Ventola C.L. The nanomedicine revolution: Part 1: Emerging concepts Pharm. Ther. 2012 37 512 PMC3462600 23066345

246. Fenske D.B. Cullis P.R. Liposomal nanomedicines Expert. Opin. Drug Deliv. 2008 5 25 44 10.1517/17425247.5.1.25 18095927

247. Min Y. Caster J.M. Eblan M.J. Wang A.Z. Clinical translation of nanomedicine Chem. Rev. 2015 115 11147 11190 10.1021/acs.chemrev.5b00116 26088284 PMC4607605

248. Zhang C. Yan L. Wang X. Zhu S. Chen C. Gu Z. Zhao Y. Progress, challenges, and future of nanomedicine Nano Today 2020 35 101008 10.1016/j.nantod.2020.101008

249. Sercombe L. Veerati T. Moheimani F. Wu S.Y. Sood A.K. Hua S. Advances and challenges of liposome assisted drug delivery Front. Pharmacol. 2015 6 286 10.3389/fphar.2015.00286 26648870 PMC4664963

250. Guo X. Szoka F. Steric stabilization of fusogenic liposomes by a low-pH sensitive PEG− diortho ester− lipid conjugate Bioconjugate Chem. 2001 12 291 300 10.1021/bc000110v 11312691

251. Bibi S. Lattmann E. Mohammed A.R. Perrie Y. Trigger release liposome systems: Local and remote controlled delivery? J. Microencapsul. 2012 29 262 276 10.3109/02652048.2011.646330 22208705

252. Woodle M.C. Lasic D.D. Sterically stabilized liposomes Biochim. Biophys. Acta (BBA)-Rev. Biomembr. 1992 1113 171 199 10.1016/0304-4157(92)90038-C 1510996

253. Allen T.M. Hansen C. Rutledge J. Liposomes with prolonged circulation times: Factors affecting uptake by reticuloendothelial and other tissues Biochim. Biophys. Acta Biomembr. 1989 981 27 35 10.1016/0005-2736(89)90078-3 2719971

254. Hua S. Wu S.Y. The use of lipid-based nanocarriers for targeted pain therapies Front. Pharmacol. 2013 4 143 10.3389/fphar.2013.00143 24319430 PMC3836271

255. Kraft J.C. Freeling J.P. Wang Z. Ho R.J. Emerging research and clinical development trends of liposome and lipid nanoparticle drug delivery systems J. Pharm. Sci. 2014 103 29 52 10.1002/jps.23773 24338748 PMC4074410

256. Bulbake U. Doppalapudi S. Kommineni N. Khan W. Liposomal formulations in clinical use: An updated review Pharmaceutics 2017 9 12 10.3390/pharmaceutics9020012 PMC5489929 28346375

257. Liu D. He C. Wang A.Z. Lin W. Application of liposomal technologies for delivery of platinum analogs in oncology Int. J. Nanomed. 2013 8 3309 10.2147/IJN.S38354 PMC3767488 24023517

258. Ko A.H. Tempero M.A. Shan Y.S. Su W.C. Lin Y.L. Dito E. Ong A. Wang Y.W. Yeh C.G. Chen L.T. A multinational phase 2 study of nanoliposomal irinotecan sucrosofate (PEP02, MM-398) for patients with gemcitabine-refractory metastatic pancreatic cancer Br. J. Cancer. 2013 109 920 925 10.1038/bjc.2013.408 23880820 PMC3749576

259. Koudelka Š. Turánek J. Liposomal paclitaxel formulations J. Control. Release 2012 163 322 334 10.1016/j.jconrel.2012.09.006 22989535

260. Deeken J.F. Slack R. Weiss G.J. Ramanathan R.K. Pishvaian M.J. Hwang J. Lewandowski K. Subramaniam D. He A.R. Cotarla I. A phase I study of liposomal-encapsulated docetaxel (LE-DT) in patients with advanced solid tumor malignancies Cancer Chemother. Pharmacol. 2013 71 627 633 10.1007/s00280-012-2048-y 23274395 PMC3703246

261. Belfiore L. Saunders D.N. Ranson M. Thurecht K.J. Storm G. Vine K.L. Towards clinical translation of ligand-functionalized liposomes in targeted cancer therapy: Challenges and opportunities J. Control. Release 2018 277 1 13 10.1016/j.jconrel.2018.02.040 29501721

262. Sapra P. Tyagi P. Allen T.M. Ligand-targeted liposomes for cancer treatment Curr. Drug Deliv. 2005 2 369 381 10.2174/156720105774370159 16305440

263. Cheng C.-C. Chang F.-C. Kao W.-Y. Hwang S.-M. Liao L.-C. Chang Y.-J. Liang M.-C. Chen J.-K. Lee D.-J. Highly efficient drug delivery systems based on functional supramolecular polymers: In vitro evaluation Acta Biomater. 2016 33 194 202 10.1016/j.actbio.2016.01.018 26796210

264. Hassan S. Prakash G. Ozturk A.B. Saghazadeh S. Sohail M.F. Seo J. Dokmeci M.R. Zhang Y.S. Khademhosseini A. Evolution and clinical translation of drug delivery nanomaterials Nano Today 2017 15 91 106 10.1016/j.nantod.2017.06.008 29225665 PMC5720147