Effects of W. Somnifera Extracts in the Treatment of Liver Diseases

Several studies have evaluated the hepatoprotective potential of W. somnifera in animal models of various hepatic disorders. Aqueous root extract of W. somnifera administered at a dose of 500 mg/kg by gavage 60 minutes after acetaminophen (APAP) dosing was found to significantly reduce the elevated hepatotoxicity biomarkers, decrease lipid peroxidation, and enhance glutathione, catalase, glutathione reductase, and glutathione peroxidase activity in APAP-treated mice (Malik and Pandey, 2013). In another study, the ethanol extract of W. somnifera at 100 mg/kg protected against γ radiation-induced hepatotoxicity in rats; decreased serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and γ-glutamyl transpeptidase; decreased hepatic levels of malondialdehyde and total nitrate/nitrite; and increased hepatic antioxidant enzymes, including superoxide dismutase and glutathione peroxidase in rats (Hosny Mansour and Farouk Hafez, 2012). The hepatoprotective activity of a methanolic extract of W. somnifera roots was examined in APAP-intoxicated rats, and a significant hepatoprotective activity was observed when W. somnifera extract was given 2 hours prior to APAP administration through alleviating inflammatory and oxidative stress, accompanied by an inhibitory effect on hepatic levels of proinflammatory cytokines, including tumor necrosis factor α (TNFα), interleukin-1β (IL1β), cyclooxygenase 2, and inducible nitric oxide (Devkar et al., 2016). Among the components of W. somnifera extracts, WA was the most extensively studied among the natural compounds isolated from W. somnifera, whereas other abundant chemical constituents of W. somnifera have been studied to a much lesser extent in the treatment of liver diseases. Hence, in the following section of this review, a summary of the literature related to the effects of WA on the treatment of liver diseases is presented.

Effects of WA in the Treatment of Liver Injury

WA (Fig. 1, compound 3) was recognized as the most important bioactive component isolated from W. somnifera. Beyond the classic antitumor role of WA, a growing list of recent reports have shown that WA has activity against the obesity-associated metabolic syndromes (Lee et al., 2016), diabetes (Tekula et al., 2018), and systematic inflammation and liver disease (Gu et al., 2020). Although the detailed mechanisms by which WA can achieve these activities remains elusive, several hypotheses have been proposed, including leptin sensitizer for obesity treatment, immunoregulator for inflammation and fulminant acute liver injury, and activating antioxidant response for treatment of APAP-induced liver injury dependent on the NF-E2-related factor-2 (NRF2) or carbon tetrachloride (CCl₄)-induced liver injury and fibrosis depending on sirtuin 3 (SIRT3). How WA may improve liver diseases are summarized in Fig. 2 for acute liver injury, Fig. 3 for chronic liver injury, and Fig. 4 for liver cancer.

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Fig. 2.

The role and mechanism of WA in the treatment of acute liver injury. In GalN/LPS-induced acute liver injury model, macrophage depletion using clodronate liposome were used to demonstrate that WA decreases the GalN/LPS-induced fulminant liver failure depending on its inhibitory effect toward macrophage activation, whereas global NLRP3 knockout mice (Nlrp3^−/− mice) were used to demonstrate that WA decreased GalN/LPS-induced liver injury partially by inhibiting the NRLP3 inflammasome activation. In the APAP-induced acute liver injury model, WA attenuates APAP-induced hepatotoxicity accompanied by hepatic NRF2 activation. Although hepatocyte-specific Nrf2-null mice were used to determine the contribution of NRF2 to the hepatoprotective effect of WA in APAP model, whether the hepatopretecive effect of WA in treating APAP-induced liver injury is dependent on NRF2 signaling still warrants further study due to the questioned experimental process (dashed line).

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Fig. 3.

The role and mechanism of WA in the treatment of chronic liver injury. (A) In the HFD-induced obesity model, WA decreases obesity-associated metabolic syndrome and fatty liver as a leptin sensitizer. In the NASH model, WA both prevents and therapeutically alleviates the obese and lean NASH in accompanied by decreasing ceramides and ER stress. In HFD- and HFHC-fed ob/ob mice, WA therapeutically improves the obesity and NASH independent of the presence of leptin signaling. In ethanol-induced alcoholic liver injury model, WA therapeutically improves the alcoholic liver injury via inhibiting the hepatic lipogenesis. (B) In the non-NASH-associated fibrosis model induced by CCl₄, bile duct ligation (BDL), or platelet-derived growth factor (PDGF), WA was demonstrated to activate SIRT3 to decrease the oxidative stress and liver fibrosis by using Sirt3^−/− mice.

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Fig. 4.

The role and mechanism of WA in the treatment of liver cancer. In HCC, WA inhibits proliferation, migration, and invasion of HCC cells (Hep3B, HepG2, Huh7, QGY-7703, SK-Hep1, MHCC97H, and MHCC97L cells) by elevating the levels of p90-ribosomal S6 kinase, ETS-like transcription factor-1, DR5, and ERK and activating liver X receptor α to inhibit nuclear factor κB (NF-κB) transcriptional activity. In orthotopic liver tumors, WA inhibits liver tumor invasion and angiogenesis by downregulating the expression of pyruvate kinase (PYK2), rho-associated coiled-coil containing protein kinase 1 (ROCK1) and vascular endothelial growth factor (VEGF).

D-galactosamine/Lipopolysaccharide-Induced Fulminant Hepatitis

Fulminant hepatitis is a life-threatening clinical syndrome worldwide. WA was recently reported to attenuate D-galactosamine (GalN)/lipopolysaccharide (LPS)-induced hepatotoxicity in mice, associated with attenuating the inflammatory response via targeting macrophage and NLR family, pyrin domain containing 3 (NLRP3), while largely independent of NRF2 signaling, autophagy induction, and hepatic AMPKα1 and hepatocyte inhibitor of nuclear factor κB (Iκκβ) signaling (Xia et al., 2021). In this GalN/LPS-induced acute liver injury model, WA was found to have both potent preventive and therapeutic effects when mice were dosed with a single intraperitoneal injection of WA at 0.5 hour before or 2 hours after GalN dosing. By using clodronate liposome pretreatment to deplete macrophage, the hepatoprotective effect of WA was abolished, but by further using global Nlrp3-null mice as well as NLRP3-deficient primary macrophages, the WA effects were partially lost, but not totally abolished (Xia et al., 2021). In this same study, by using gene knockout mouse strains, the hepatoprotective effect of WA was found to be independent of the presence of hepatocyte AMPKα1, NRF2, and Iκκβ (Xia et al., 2021). Given that macrophage depletion abolished the hepatoprotective effects of WA in this model and NLRP3 knockout could not totally, but only partially, abolish the hepatoprotective effect of WA, additional targets located in macrophage, such as macrophage-specific Iκκβ, were suspected to mediate the hepatoprotective effects of WA in this model.

The GalN/LPS-induced acute liver injury model is characterized with systematic inflammation and hepatocyte apoptosis, whereas WA was found to have no direct role in TNFα-induced hepatocyte apoptosis (Xia et al., 2021). Decreasing LPS-induced systematic inflammation, also known as “cytokine storms” (Noori et al., 2020), was inferred to play a dominant role in the hepatoprotective effect of WA. Thus, with WA as a chemical probe, the results suggest that a strategy targeting the immune system, such as macrophage, to treat acute liver injury is achievable. This study suggests WA as a potential immunoregulator to normalize the altered immune response for the treatment of acute liver injury. Future studies on the effects of WA as one potent herbal immunoregulators in the treatment of other types of systematic inflammatory disorders are promising.

APAP-Induced Acute Liver Injury

WA was reported to protect against APAP-induced liver injury in two reports, indicating that NRF2 activation plays an important role in mediating the hepatocyte protective effects of WA (Jadeja et al., 2015; Palliyaguru et al., 2016). In an earlier study, a single dose of 40 mg/kg WA administered via intraperitoneal injection at 1 hour after APAP dosing significantly rescued APAP-induced hepatotoxicity, compared with the control vehicle ethanol (2 µL/g) (Jadeja et al., 2015), which suggests an efficient therapeutic effect. APAP-induced Jun N-terminal kinase (JNK) activation, mitochondrial B-cell lymphoma-2 (BCL-2)-associated X protein translocation, nitrotyrosine production, and hepatic inflammation were reduced by WA, accompanied by upregulation of NRF2 target genes, including Nrf2, GcLc, and Nqo1. In addition, WA alleviated H₂O₂-induced hepatocyte death and oxidative stress in the AML12 cell line, a nontransformed normal hepatocyte-derived cell line, in vitro (Jadeja et al., 2015). To better evaluate the therapeutic effect of one drug in the APAP model, APAP-treated mice are usually administered drugs for 2 hours or longer after APAP dosing to evaluate the effects on late-stage liver injury (Abdullah-Al-Shoeb et al., 2020). Comparing the hepatoprotective activity of a drug candidate to the clinical drug N-acetyl cysteine (Dear et al., 2021) could help to further assess the translational significance. Therefore, whether WA has a superior hepatoprotective effect against APAP-induced acute liver injury compared with N-acetyl cysteine, and whether WA still has a therapeutic effect at the later stages of APAP-induced liver injury warrant future study. In addition, this work only described the NRF2 activation effect of WA in APAP-treated mice (Jadeja et al., 2015); however, whether WA improvement of APAP-induced acute liver injury depends on its effect on NRF2 activation remains unexplored.

In a later study, WA was found to have an NRF2-dependent protective activity toward APAP-induced hepatotoxicity (Palliyaguru et al., 2016). WA exhibited direct NRF2-inducing activity both in mice in vivo and in mouse embryonic fibroblasts generated from fibroblasts isolated from 13.5-day-old mouse embryos in vitro. WA induced NRF2 signaling in a kelch like ECH associated protein 1 (KEAP1)-independent and phosphatase and tensin homolog (PTEN)/phosphatidylinositol 3-kinase (PI3K)/AKT serine/threonine kinase 1 (AKT1)-dependent manner in vitro as revealed by using NRF2/kelch like ECH associated protein 1 (KEAP1) double-knockout mouse embryonic fibroblasts (Palliyaguru et al., 2016). More importantly, this study employed a hepatocyte-specific Nrf2-null mouse line to confirm that the ameliorating effect of WA on APAP-induced liver injury was dependent on the presence of hepatocyte NRF2 (Palliyaguru et al., 2016), thereby supporting the contribution of WA-induced NRF2 signaling to the hepatoprotective effect of this compound in APAP-dosed mice. However, a major limitation of this study is that 100 µL of DMSO or 7 mg/kg of WA was administrated to mice via oral gavage 22 hours before APAP dosing. Given that WA showed a short retention time in vivo in previous pharmacokinetic studies (Thaiparambil et al., 2011; Vanden Berghe et al., 2012; Devkar et al., 2015; Dai et al., 2019), it is doubtful that WA could still achieve a hepatoprotective effect if only dosed once via gavage 22 hours before APAP, considering the anticipated low plasma concentration of this compound. DMSO is not suitable as a control vehicle for drugs when studying the APAP model, since DMSO has a hepatoprotective effect against APAP-induced liver injury even when used at a very low dose (Yoon et al., 2006). Thus, the dose and route of administration need to be considered when interpreting results. More studies are required to determine the extent by which NRF2 is involved in mitigating APAP toxicity by WA treatment.

Although the parent drug APAP is not hepatotoxic, APAP is metabolized into a toxic metabolite N-acetyl-p-benzoquinone (NAPQI) in the liver, a reaction catalyzed by cytochrome P450 (CYP) 2E1 (Lee et al., 1996; Cheung et al., 2005) and to a lesser degree by CYP3A11 and CYP1A2 (Zaher et al., 1998; Yan et al., 2016). Any factors that inhibit the generation of NAPQI could contribute to decreasing APAP-induced hepatotoxicity. How W. somnifera and WA affect APAP metabolism has not been examined in the APAP model in vivo and in vitro. However, several studies reported no significant inhibitory effects of W. somnifera extract on the CYPs that are involved in NAPQI generation from APAP (Savai et al., 2013; Varghese et al., 2014; Dey et al., 2015; Savai et al., 2015), indicating that it is less likely that WA exerts hepatoprotective effect toward APAP-induced liver injury by inhibiting CYP-mediated APAP metabolic activation. However, W. somnifera extract was found to exhibit inductive effects on CYP3A4 and CYP1A enzymes (Kumar et al., 2021). Thus, further studies are warranted to determine the extent by which metabolic activation of APAP is involved in reducing APAP-induced hepatotoxicity after W. somnifera and WA treatment.

Bromobenzene-Induced Hepatotoxicity

In an earlier study, a protective effect of W. somnifera extract was found in treating bromobenzene-induced nephrotoxicity and mitochondrial oxidative stress in rats through a proposed mechanism of decreasing mitochondrial oxidative stress (Vedi et al., 2014). More recently, pretreatment with WA provided a significant protection against bromobenzene-induced liver damage by preventing mitochondrial dysfunction through increasing activities of mitochondrial enzymes and balancing the expression of Bcl-2 associated X/Bcl-2 in liver (Vedi and Sabina, 2016). WA in W. somnifera at least partially contributes to the hepatoprotective effect of W. somnifera in this bromobenzene-induced liver injury model.

WA in the Metabolic Diseases and Nonalcoholic Fatty Liver Disease

As one of the most prevalent chronic liver diseases worldwide, nonalcoholic fatty liver disease (NAFLD) affects 20%–25% of the adult population (Cotter and Rinella, 2020). NAFLD is a typical type of obesity-associated metabolic syndrome, which is frequently is associated with, but is not limited to, obesity (Younes and Bugianesi, 2019; Cotter and Rinella, 2020). NAFLD includes simple nonalcoholic fatty liver (NAFL) characterized with hepatic steatosis and mild inflammation and the progressive nonalcoholic steatohepatitis (NASH) characterized by severe liver inflammation and fibrosis, which could further progress to end-stage liver diseases, including cirrhosis and hepatocellular carcinoma (HCC) (Schwabe et al., 2020; Sheka et al., 2020; Francque et al., 2021). The role of WA in treating diet-induced NAFL and progressive NASH is diagrammed in Fig. 3A.

An initial study demonstrated that WA alleviated obesity-associated metabolic syndrome and hepatic steatosis by acting as a leptin sensitizer (Lee et al., 2016). In this study, 12-week WA (1.25 mg/kg/d, i.p.) treatment reduced the levels of proinflammatory cytokines including toll-like receptor, nuclear factor κB (NFκB), tumor necrosis factor-α (TNFα), chemokine ligand-receptor, and cyclooxygenase 2 in both the serum and liver of high-fat diet (HFD)-fed obese mice, along with protective activity against obesity, oxidative stress, and insulin resistance (Lee et al., 2016). Moreover, WA (6 mg/kg, 3 times per week, i.p.) was found to therapeutically improve insulin sensitivity, downregulate the inflammatory response, and decrease body weight and hepatic steatosis in established HFD-fed obese mice, which was suggested to be partly through upregulating peroxisome proliferator-activated receptor gamma phosphorylation-related downstream genes, including carbonic anhydrase 3, selenium binding protein 1, amyloid beta precursor-like protein 2, thioredoxin interacting protein, and adiponectin (Khalilpourfarshbafi et al., 2019). The antihepatic inflammation effect of WA was also observed and described in pre-existing HFD-induced obese mice in a therapeutic manner when mice were fed a HFD for 12 weeks and then dosed by oral gavage with WA at 1.25 mg/kg/d for 12 weeks (Abu Bakar et al., 2019). These studies support the view that WA has a therapeutic effect in treating obesity-induced metabolic syndrome that includes hepatic steatosis, inflammation, and diabetes. However, these HFD-induced obesity models may only represent the mild fatty liver phenotype, NAFL, while still at the early stage of NAFLD.

The effect of WA on the later stages of NAFLD, NASH, has also been examined. WA was demonstrated to have a potent therapeutic effect in decreasing NASH-associated symptoms in both obese and lean NASH models. In several widely used NASH models, WA had both preventive and therapeutic effects in improving NASH, with reduced serum aminotransferase levels, liver steatosis, inflammation, endoplasmic reticulum (ER) stress, and fibrosis in the WA-treated group compared with the vehicle-treated group, and this was correlated with normalized serum ceramides, hepatic ceramide catabolism, and ER stress (Patel et al., 2019). In this study, methionine-choline-deficient diet-fed wild-type C57BL/6N mice were used to induce a lean NASH model, the 40% high-fat and high-cholesterol (HFHC) diet-fed wild-type C57BL/6N mice were used to establish an obese NASH model, whereas 40% HFHC-fed ob/ob mice were employed to generate a leptin signaling-deficient NASH model (Patel et al., 2019). Beyond the preventive effects, WA improved all NASH symptoms in a therapeutic manner when dosed at the later stage of the established NASH models. Given that WA was found to improve methionine-choline-deficient-induced NASH, a lean NASH model, the hepatoprotective effect of WA against NASH was inferred to be at least partially independent of its antiobesity effect. Using ob/ob mice, WA was confirmed to produce an anti-NASH effect independent of leptin signaling (Patel et al., 2019).

WA in Non-NASH-Associated Liver Fibrosis

The effect of WA in non-NASH-associated fibrosis are summarized in Fig. 3B. In a bile duct ligation-induced liver fibrosis model, WA inhibited epithelial mesenchymal transition process by inhibiting the expression of enzymes such as MMP2, TIMP1, and LOXL2, and the transcriptional repressor SNAIL1, thus enhancing the expression of CDH1 leading to the reversal of epithelial mesenchymal transition (Sayed et al., 2019). In the liver fibrosis models induced by platelet-derived growth factor BB and CCl₄, WA attenuated liver fibrosis by inhibiting oxidative stress in a SIRT3-dependent manner as revealed by using SIRT3 knockout mice and Sirt3 silencing in JS1 cells, an immortalized mouse hepatic stellate cell line (Gu et al., 2020). These studies further extend the antifibrosis scope of WA in different types of hepatic fibrosis models. The direct effects and detailed mechanisms of WA in alleviating hepatic fibrogenesis, such as hepatic stellate activation, macrophage activation, and hepatocyte damage, warrant additional studies.

WA in Alcoholic Liver Injury

The hepatoprotective effect of WA in alcoholic liver injury was investigated in an acute-upon-chronic liver injury model (Hamada et al., 2022). In this binge model, WA significantly decreased binge ethanol-induced liver injury and hepatic lipid accumulation both in a preventive and therapeutic manner. Mechanistically, WA inhibits ethanol-induced lipogenesis in vivo as well as in primary hepatocytes in vitro. This study suggests the therapeutic potential of WA in the treatment of alcoholic liver injury (Hamada et al., 2022). The direct target of WA and the role of WA in long-term alcohol-induced alcoholic steatohepatitis and fibrosis warrants further study.

Metabolism of WA

Studies on the pharmacokinetics and bioavailability of WA are summarized in Fig. 5 and Table 2. WA was found to be impermeable in an in vitro absorption model using MDCK cells derived from canine kidney (Devkar et al., 2015). WA is readily transported across Caco‐2 cell plasma membranes in vitro, whereas WA had an oral bioavailability of 32.4 ± 4.8% in vivo, based on the studies of intravenous (5 mg/kg) and oral (10 mg/kg) dosing in male rats (Dai et al., 2019). Extensive first-pass metabolism of WA was further suggested by rat intestine‐liver in situ perfusion, where WA concentration in the media was rapidly decreased and only 27.1% remained within 1 hour (Dai et al., 2019). Pharmacokinetic studies on WA showed a rapid plasma clearance in mice administered a single dose of WA via intraperitoneal injection (Thaiparambil et al., 2011; Vanden Berghe, 2012). A study reporting pharmacokinetic evaluation of WA after oral administration demonstrated a short half-life with the value of 59.9 minutes for this constituent in mice administered a single oral dose of W. somnifera extracts (Patil et al., 2013). A previous report found a relatively short half-life (t_1/2 = 2.0 hours) of WA in mice after a single intraperitoneal injection of 5 mg/kg WA (Patel et al., 2019). Moreover, the observation that no detectable WA was found in plasma samples in humans orally administered a capsule formulation of WA at 72–216 mg/kg in two or four divided doses per day for at least 30 days as measured by high performance liquid chromatography with a limit of quantitation of 50 ng/mL (Pires et al., 2020). However, few studies evaluated the metabolic route of WA. Others found that WA was metabolized via hydroxylation, hydrogenation, and hydrolysis (Rosazza et al., 1978; Funska et al., 1985; Fuska et al., 1987), which was further supported by a more recent study where metabolites of WA generated from these pathways were found in rats (Dai et al., 2019). Due to the structural similarities of steroidal lactone classification between WA and endogenous steroids, it likely undergoes oxidative metabolism mediated by CYPs. More detailed future investigations are needed to characterize the metabolic pathway(s) as well as the major metabolizing enzymes responsible for WA metabolism in vivo.

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Fig. 5.

The proposed metabolism routes of WA. Seven metabolites, M1–M7, were identified via hydroxylation, hydrogenation, and hydrolysis of WA in rat or human liver microsomes, with M2, M3, and M7 specifically found in human liver microsomes.

Pharmacokinetic Insights into Future Directions

Poor pharmacokinetic behavior indicated that the hepatoprotective activity of WA may be attributed to its metabolites. Phase I reactions convert a parent drug to metabolites that, in some cases, could possess superior efficacy and be more biologically active and water-soluble than the parent compound by unmasking or inserting a polar functional group. The generated active metabolites, as “metabolized” molecules, can be less prone to first pass metabolism and thus have improved pharmacokinetic behaviors compared with the parent drugs (Sun and Wesolowski, 2021). Therefore, it is of great interest to compare the hepatoprotective effects of WA and its metabolites side by side to elucidate the effective substance basis of WA and explore new drug candidates in treating liver diseases.

On the other hand, it is also worth noting that decreased expression levels of phase I and II enzymes and drug transporters are often observed under pathologic states of the liver, including acute liver injury, the metabolic diseases and NAFLD, liver fibrosis, liver cancer, and systematic inflammation (Hanada et al., 2012; Wu and Lin, 2019; Bao et al., 2020; Feng et al., 2020), which may lead to reduced metabolism of WA. Thus, it is important that pharmacologists keep this in mind for designing appropriate doses of WA when using liver injury models. Whether WA is toxic is still controversial, and more toxicological evaluation is needed to determine the safety of WA. In addition, different formulations may need to be studied so that the adjusted pharmacokinetic properties (absorption, distribution, metabolism, and elimination) will yield optimal efficacy. Indeed, WA embedded in polycaprolactone implants was proposed to overcome the problems regarding its bioavailability and pharmacokinetics (Gupta et al., 2012), thereby allowing long-term systemic circulation for controlled treatment.

The exact mechanism by which WA alleviated liver diseases remains largely unknown, due in part to that fact that no receptor for this compound has been identified. Regarding the potentially extensive metabolism of WA in vivo, it is not clear whether WA, the parent drug, or its biologically active metabolite(s) may directly contribute to its hepatoprotective effects. Due to the short half-life of WA after gavage in vivo, the pharmacological effects achieved after oral intake of WA may be due in part to modulation of the host intestinal targets, gut microbiota, or other factors involved in the gut-liver axis. The specific intestinal targets with which WA interacts to produce its pharmacological effects on liver diseases after oral gavage warrant further investigation. Various methodologies, including such as RNA sequencing, proteomics, metagenomics, and untargeted metabolomics, are needed to flush out mechanistic clues. Pharmacokinetic studies are also needed to be performed to clarify the distribution of WA and its potential active metabolites, which could direct future mechanistic research to better select a target organ/cell for analyses.