In Vitro Cytotoxicity of Reproductive Stage Withania somnifera Leaf and Stem on HepG2 Cell Line

Background The ayurvedic plant Withania somnifera, a member of the Solanaceae family, has been used as a remedy for diverse health problems, including cancer. Objectives The objective of this investigation was to conduct a comparative analysis of the in vitro cytotoxic properties of methanolic extracts derived from the leaf, stem, and root of W. somnifera on HepG2 and L929 cell lines. Methods Methanolic extracts were obtained using the Soxhlet extraction method. To assess the in vitro anticancer action on the HepG2 and L929 cell lines, an MTT assay was performed. Changes in cell morphology were observed using an inverted microscope. Results The MTT assay results indicated that the leaf, stem, and root methanolic extracts of W. somnifera showed significantly higher in vitro cytotoxicity in HepG2 cells, with IC50 values of 43.06 ± 0.615, 45.60 ± 0.3, and 314.4 ± 0.795 μg/mL than in L929 cell lines with 78.77 ± 0.795, 90.55 ± 0.800, and 361.70 ± 0.795 μg/mL, respectively. The leaf methanolic extract was the most effective, followed by the stem methanolic extract in the HepG2 cell line. Conclusion The results of our study have confirmed that the methanolic extracts of both the leaf and stem of W. somnifera exhibit significant in vitro cytotoxicity in HepG2 cell lines, while displaying no significant cytotoxicity in the L929 cell line. Furthermore, the data obtained from the MTT assay indicate that the leaf methanolic extract possesses a more potent cytotoxic activity than the stem methanolic extract with respect to the HepG2 cell line. Further studies on the identification and isolation of bioactive metabolites are required to explore the mechanisms underlying their in vitro cytotoxicity.


Introduction
Ayurvedic medicines have long been used with various medicinal plants and their parts in several medicinal formulations [1].Withania somnifera is an important herb that belongs to the Solanaceae family.It is also known as Indian ginseng, winter cherry, or ashwagandha [2].Indians have been using this herb for therapeutic purposes since time immemorial [3].However, systemic scientifc research on this plant began in the 1950s only [4].Initially, it was used to treat fertility and reproductive health issues, but it is now used to prevent aging, boost important body fuids such as blood cells, lymph secretion, semen, and nourish various body organs [5].
Despite advancements in medicine, cancer remains the main cause of mortality worldwide [6].Among noncommunicable diseases, cancer is the second leading cause of death [7].Uncontrolled cell proliferation, metastasis, invasion, programmed cell death, and angiogenesis are hallmarks.Cancer can afect any organ in the human body, although it most frequently afects the breast, lung, liver, colon, prostate, kidney, and ovaries [8].Approximately 29% of all malignancies are lung cancers [9].Breast cancer is the major cause of morbidity in women, whereas prostate cancer is the major cause of morbidity in men.Liver cancer ranks third in terms of cancer mortality [10].Te rate of death from cancer is constantly increasing [11].Te lack of efcient anticancer drugs is a major burden on the healthcare system, and there is an urgent need for such drugs [12].
Despite the availability of many chemoprotective medications for cancer treatment, these are expensive and have numerous side efects [13].Terefore, it is critically important to search for cost-efective and promising natural medications with minimal side efects to reduce cancer morbidity rates.Herbal medicines are considered the most viable ways to cure cancer [14].A variety of plants, such as W. somnifera, are used for drug development.Tese are more efective and have fewer adverse efects.Tus, there is a pressing demand for natural medications to stop cancer progression and spread throughout the body [15].
W. somnifera has received a lot of attention recently for its anticancer studies [16].W. somnifera extracts have been investigated from various plant parts for therapeutic value in treating cancers of various origins [17].In one study, a decrease in mammary carcinomas in mice was observed with W. somnifera root extract [15].In another study, human umbilical vein endothelial cells (HUVECs) were treated with W. somnifera root extract, and withaferin A reduced cell proliferation [18].W. somnifera leaf extracts have been employed as a treatment during research investigations exploring its potential anticancer efects [15].
Previous studies have shown that W. somnifera extracts reduce the proliferation of MCF-7, pancreatic, prostate, kidney, and fbrosarcoma cells [19].Consequently, it can be concluded that substances extracted from W. somnifera have strong antineoplastic activity and could potentially be used as chemotherapeutic agents.Moreover, several investigations have demonstrated the anticancer activities of W. somnifera during the course of research on the anticancer activities of this plant for the development of herbal-based drugs [20].Te 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric method is a rapid and efective assay for assessing cellular metabolic activity, cytotoxicity, and proliferation [21].In light of the comprehensive assessments documenting the anticancer attributes of W. somnifera [15], this article presents in vitro comparative investigations into the anticancer potentials of distinct constituents of W. somnifera, specifcally the leaf, stem, and root, in the selected cell lines HepG2 and L929.

Materials and Methods
2.1.Chemicals.All chemicals and media were procured from Hi-Media and Sigma-Aldrich, respectively.

Cell Lines.
Te human liver cancer cell line HepG2 and the mouse fbroblast noncancerous cell line L929 were acquired from the National Centre for Cell Sciences (NCCS), Pune.Te cell lines were later propagated in Dulbecco's Modifed Eagle Medium (DMEM), supplemented with 10% (v/v) fetal bovine serum (FBS), an antimycotic antibiotic, and maintained with a continuous supply of 5% CO 2 at 37 °C.

Plant Material
Collection.Te W. somnifera seeds were bought from Zooqa Herbs, Chennai, Tamil Nadu.Te seeds were sown and matured under natural soil and light conditions at the Department of Genetics and Biotechnology, Osmania University, Hyderabad.Te plant materials were collected in the months of July-October 2022, during the monsoon season.Te collected plant material was thoroughly washed three times with running water.Ten, the plant material was rinsed once with sterile distilled water and weighted.Te plant material was air dried in the shade at room temperature and weighed again.Te fresh sample weight to dried sample weight ratios 37.02 : 23.82 of leaf, 28.72 : 16.92 of stem, and 28.31 : 14.71 of root, respectively, were used to coarsely grind and pulverise.

Plant Authentication.
Te authentication of W. somnifera was carried out by Dr. A. Vijaya Bhaskar Reddy, Botany Department, Osmania University, Hyderabad.Te plant was deposited in the herbarium of the Botany Department, Osmania University, Hyderabad, with voucher number GEN/OU/001-2018-HY.

Plant Extraction.
Te leaf, stem, and root parts of W. somnifera were grounded coarsely and extracted with methanol in a Soxhlet for 24 hours and then air-dried.A viscous semisolid mass was produced using a rotary evaporator to concentrate the extract under reduced pressure at 40 °C.

Morphological Study.
In this study, cells in 6-well plates were used to examine the morphology of the cell lines in vitro under an inverted microscope.

In Vitro Evaluation of Cell Viability and Cytotoxicity.
Cell viability and in vitro cytotoxicity were evaluated using an MTT assay [21][22][23].In order to ascertain the impact of the methanolic leaf, stem, and root extracts of W. somnifera on HepG2 and L929 cells, an MTT assay was conducted.In 96well plates, 100 μL of each cell line was seeded at a density of 10,000 cells per well.Te plates were then incubated for 48 h at 37 °C in a 5% CO 2 environment.After incubation, the cells were examined in a half-confuent monolayer.Next, 20, 50, 100, 200, 500, 1000, 1500, and 2000 μg/mL of W. somnifera leaf, stem, and root methanolic extracts were treated in triplicate, as presented in Figure 1.Te cells were then incubated at 37 °C and 5% CO 2 for 24 h.
Te untreated cell lines served as the negative control, and the cells treated with the anticancer drug doxorubicin served as the positive control.Cell lines treated with the extracts served as test samples.After 24 hours, the cells were observed using an inverted microscope to check for any morphological changes or cell death.After observation, the culture medium was removed, and 100 µL of fresh medium was added along with 10 µL of MTT reagent (5 mg/mL).Te plates were then placed in an incubator with 5% CO 2 at 37 °C for 4 h.Subsequently, the medium containing MTT was 2 Evidence-Based Complementary and Alternative Medicine removed, the formazan purple precipitate was solubilized, and 100 µL of DMSO was added.Te plates were then incubated for 1 hour at 37 °C in a 5% CO 2 incubator.Te absorbance at 570 nm was measured using a Multiskan SkyHigh Plate Reader by subtracting the absorbance at 630 nm from the background after the purple formazan crystals completely dissolved.Using GraphPad Prism Version 8.0, a log graph of the log test item concentration vs. cell survival percentage was plotted and the half-maximal inhibitory concentration (IC 50 ) values were calculated.[21,23].
Percent cell survival (%) � Absorbance of Test Absorbance of Control X 100. (1) 2.9.Statistical Analysis.All statistical analyses were carried out using the SPSS ® statistical software package for Win- dows ® , version 15.0 of SPSS Inc. (Chicago, IL, USA).Te results are presented as means ± SD, and p ≤ 0.05 was used to determine whether treatment diferences were signifcant.

Results
Te MTT assay is widely used to assess cell viability, cell proliferation, cytotoxicity testing, and drug screening [21].
In drug screening, the MTT assay is used to test the cytotoxicity of various compounds, such as drugs, natural products, or experimental molecules, on cell lines or primary cells [21].It measures the reduction of the MTT reagent to formazan, a purple-colored product indicating the metabolic activity of viable cells and the intensity of which is directly proportional to the number of viable cells in the sample [22].Tus, it helps to identify compounds that inhibit cell growth or induce cell death.In this study, the comparative evaluation for anticancer potential of W. somnifera leaf, stem, and root methanolic extracts was carried out to determine their cytotoxic efect on the selected cancer cell line HepG2 and normal cell line L929 viability.

In Vitro Evaluation of Morphological Changes in HepG2
and L929 Cells.HepG2 and L929 cells were treated with W. somnifera leaf, stem, and root methanolic extracts of 20, 50, 100, 200, 500, 1000, 1500, and 2000 μg/mL for 24 h.Figures 2 and 3 show the in vitro morphological profles of HepG2 and L929 cells, respectively, under an inverted microscope.Te treatments were compared to controls (a negative sample without a test sample and a positive sample with the standard drug doxorubicin).Cell morphology revealed signifcant morphological changes in HepG2 and L929 cells.Te HepG2 cells showed adhesion to the well wall with W. somnifera methanolic extracts of root at 20 μg/mL, leaf at 20 μg/mL, and stem at 50 μg/mL.On the other hand, L929 cells showed adherence to the well plate wall with W. somnifera methanolic extracts of root at 100 μg/m L, leaf at 100 μg/mL, and stem at 200 μg/mL.However, at higher concentrations, the HepG2 and L929 cells appeared to be in a state of splitting, detaching from the substrate, and increasing the number of suspended cells.

In Vitro Evaluation of HepG2 and L929 Cell Lines
Viability.Te viability of HepG2 and L929 cells was evaluated using the MTT assay.A considerable reduction in cell viability was observed in HepG2 (Tables 1-3) in a concentration-dependent manner.At the highest 2000 μg/mL concentration of W. somnifera leaf, stem, and root methanolic extracts, MTT assay results indicated that 100% viability in control HepG2 cells decreased to 10.19 ± 0.015%, 9.93 ± 0.051%, and 9.82%, respectively (Tables 1-3).Moreover, the viability of HepG2 cells at the lowest concentration of 20 μg/mL W. somnifera methanolic leaf, stem, and root Evidence-Based Complementary and Alternative Medicine extracts was observed to be 81.94 ± 0.046%, 86.09 ± 0.020%, and 95.99 ± 0.025%, respectively.Tese values were much lower than the viability of the control cells (untreated cells plus media), which ranged between 99 and 100%.
Similarly, at the highest 2000 μg/mL concentration of W. somnifera leaf, stem, and root methanolic extracts, the viability of L929 cells decreased from 100% in controls to 26.53 ± 0.045%, 25.94 ± 0.026%, and 26.87 ± 0.058%, respectively (Tables 4-6).In contrast, at the minimal concentration of 20 μg/mL W. somnifera leaf, stem, and root methanolic extracts, the viability of L929 cells was 72.37 ± 0.030%, 71.97 ± 0.052%, and 77.96 ± 0.030%, respectively.Tese values were greater than the values of HepG2 cell viability.However, these values were much lower than the 99-100% viability of the control cells (untreated cells + media).Accordingly, the decrease in viability of HepG2 cells was signifcant at p ≤ 0.05, indicating the effective cytotoxicity of the W. somnifera leaf and stem methanolic extracts.Interestingly, the extracts did not considerably afect L929 cells.

IC 50 Evaluation of Methanolic Leaf, Stem, and Root
Extracts.Te in vitro cytotoxicity of W. somnifera leaf, stem, and root methanolic extracts was assessed using the HepG2 and L929 cell lines, with the aim of determining their IC 50 values (Figures 4 and 5).Te IC 50 values for the methanolic extracts of W. somnifera leaf and stem were determined to be      Evidence-Based Complementary and Alternative Medicine extracts in HepG2 were less than 50.00 μg/mL and greater than 50.00 μg/mL in the L929 cell line.Te IC 50 values obtained for the root were greater than 100 μg/mL in both cell lines, HepG2 and L929.By calculating "p" values, the signifcance of diference between the observed value and the hypothesized mean of IC 50 was determined for leaf, stem, and root methanolic extracts of W. somnifera in HepG2 and L929 cells (Table 7).Te p values for IC 50 of W. somnifera leaf, stem, and root methanolic extracts were found to be 0.0014, 0.0007, and 0.9999 in HepG2 and 0.9993, 0.9994, and 0.9993 in L292 cell lines, respectively.
Human hepatocellular carcinoma (HCC), the most prevalent cancer with no efective treatment, is a malignant tumor that develops from hepatocytes [34].Globally, it is the ffth most common cause of cancer and the second most common cause of cancer-related deaths [35].In a previous study, HepG2 cells (a cell line obtained from hepatocellular carcinoma) were known to be arrested in the S phase of the cell cycle [36].Te MTT assay is a useful method to compare the cytotoxic activity of cancer cell lines to that of a normal cell line, L929 (mouse fbroblast normal cell line) [37].Nevertheless, there exists a gap in the comparative analysis of the anticancer efects of W. somnifera on HepG2 and L929 cell lines, focusing on plant organ-based research.Tis gap persists despite the extensive utilization of W. somnifera in various studies pertaining to anticancer properties.Hence, in this study, the anticancer activities of W. somnifera leaf, stem, and root methanolic extracts were evaluated using the MTT assay in HepG2 and L929 cells.
Te inverted microscope images of HepG2 and L929 cells (Figures 2 and 3) revealed signifcant dosedependent morphological changes.Te results of the experiment indicate that after a 24-hour treatment with methanolic extracts derived from the stem, leaf, and root of W. somnifera, HepG2 cells exhibited minimal morphological alterations at lower concentrations ranging from 20 to 50 μg/ mL, while L929 cells displayed negligible changes at higher concentrations ranging from 100 to 200 μg/mL.Tis   Evidence-Based Complementary and Alternative Medicine observation is in agreement with earlier fndings that W. somnifera extracts exhibited less cytotoxicity against normal cell lines, such as L929, than against cancerous cell lines [38].Tis may be attributed to the presence of certain phytochemicals in the extracts that selectively target cancer cells while sparing normal cells.
In both HepG2 and L929 cell lines at higher concentrations of W. somnifera stem, leaf, and root methanolic extracts, signifcant cell changes were observed, such as an increase in suspended cells, a decrease in cell density, reduced cell volume, detachment from the substrate, and cytoplasmic shrinkage.Tis result confrms earlier fndings on HepG2 cells exhibiting characteristics of apoptosis via morphological changes when treated with W. somnifera extracts at diferent concentrations for 24 hours [39].Tis suggests that W. somnifera stem and leaf methanolic extracts have anticancer properties, which may be due to bioactive compounds such as favonoids, withanolides, and alkaloids that induce apoptosis [26].Te proportional morphological alterations observed in HepG2 cells upon exposure to increasing concentrations of methanolic extracts derived from the leaf, stem, and root of W. somnifera were found to be consistent with previous research, which demonstrated a dose-dependent inhibition of cell proliferation and associated morphological changes [38,40].Te increased suppression of cellular proliferation observed at higher concentrations can be attributed to the existence of phytochemicals, which possess the capability to afect the structure and functionality of cancer cells.Tis impact may lead to morphological changes in HepG2 cancer cells, ultimately resulting in their death or hindering their ability to multiply and spread.
When treated with methanolic extracts of W. somnifera leaf and stem, the evaluation of viable cells using the MTT assay revealed a signifcant reduction in HepG2 cell viability in a dose-dependent manner.2).On the other hand, HepG2 exhibited cell viability of 9.93 ± 0.05 and 95.99 ± 0.025, 87.44 ± 0.015, 80.52 ± 0.020, 72.06 ± 0.017, 31.69 ± 0.017, 14.27 ± 0.025, 10.28 ± 0.035, and 9.82 ± 0.125 at 20, 50, 100, 200, 500, 1000, 1500, and 2000 μg/mL concentrations with root methanolic extracts (Table 3).It has been determined that the methanolic extract derived from the leaf exhibited the highest level of activity followed by stem in inhibiting the viability of HepG2 cells, surpassing the methanolic extracts obtained from the root.Tis result supports previous fndings that HepG2 cell viability decreases signifcantly as the W. somnifera leaf methanolic extract concentration increases [36].Te aforementioned data suggest a heightened efcacy of the methanolic extract derived from W. somnifera leaf in inhibiting the proliferation of the Hep G2 cell line.
In contrast to that of HepG2 cells, the viability of L929 cells was insignifcant when compared in a dosedependent manner.Te L929 cell line exhibited cell viability of 99.99 ± 0.005, 72.37 ± 0.030, 55. 39 5 and 6) at the 20, 50, 100, 200, 500, 1000, 1500, and 2000 μg/mL concentrations with stem and root methanolic extracts, respectively.Tis fnding indicates that the leaf methanolic extracts of W. somnifera exhibit anticancer properties compared to the stem and root methanolic extracts of the HepG2 cell line.Tis may be ascribed to certain bioactive compounds such as the phenolic compounds 1,2-bis (trimethylsilyl) benzene, the ester compound boric acid, trimethyl ester, and steroid amines such as dextroamphetamine [41] in the methanolic extract of W. somnifera leaf, which have been shown to inhibit the growth of cancer cells in the previous fnding.
Te fndings from the cytotoxicity evaluations conducted using the MTT assay revealed that the IC 50 values of methanolic extracts obtained from the leaf, stem, and root of W. somnifera exhibited signifcant potency in HepG2 cells.Te IC 50 values of W. somnifera leaf, stem, and root methanolic extracts in HepG2 cells were found to be 43.06 μg/mL, 45.60 μg/mL, and 314.4 μg/mL (Figure 4), and L929 78.77 μg/mL, 90.55 μg/mL, and 361.70 μg/mL (Figure 5), respectively.Te IC 50 value in pharmacological research is generally considered an indicator of a drug's efectiveness at its half-inhibitory concentration.It provides  [42].IC 50 values greater than 50 μg/mL are considered noncytotoxic [42,43].In this study, the IC 50 values were found to be less than 50 μg/mL for the leaf and stem methanolic extracts of HepG2 cells, which were found to be much lower than the IC 50 values for L929 cells.An IC 50 value of less than 50 μg/mL is usually considered to have signifcant cytotoxicity [43].Te MTT assay fndings of the methanolic leaf and stem extracts of W. somnifera indicate a signifcant degree of cytotoxicity on HepG2 cells, ranging from high to moderate, while demonstrating only weak cytotoxicity on the L929 cell line [9,29,43].Tese results support earlier fndings that W. somnifera leaf methanolic extracts are cytotoxic to HepG2 cells [44].Te methanolic extracts derived from the root of W. somnifera were found to lack satisfactory cytotoxic activity against both HepG2 and L929, as evidenced by IC 50 values exceeding 100 μg/mL.Tis observation is consistent with prior research indicating that W. somnifera root extract has a minimal cytotoxic impact on L929 cells [7,9,45].Tis selective cytotoxic efect on cancer cells may be due to bioactive compounds in the methanolic extracts of W. somnifera that specifcally target cancer cell lines, such as HepG2.Statistical analysis showed a signifcant efect of W. somnifera leaf and stem methanolic extracts on HepG2 cells (p ≤ 0.05).Te methanolic root extracts of W. somnifera did not exhibit any signifcant impact on either HepG2 or L929 cell lines, as indicated by the p value of ≥0.05 (Table 7).Similarly, leaf and stem methanolic extracts showed no signifcant efect on the L929 cell line (p ≥ 0.05).Tus, the p values further confrmed that the methanolic extracts of W. somnifera leaf and stem had signifcant cytotoxicity in HepG2 cells and weak cytotoxicity in the L929 cell line.

Conclusion
W. somnifera is a reliable source of herbal products.Research has revealed an array of anticancer phytochemicals present in the stem, leaf, and root.Tis study was conducted using the MTT assay to compare the cytotoxic efects of methanolic leaf, stem, and root extracts of W. somnifera on HepG2 and L929 cells.Te results of this investigation demonstrated that the methanolic leaf and stem extracts of W. somnifera exhibited signifcant cytotoxicity in HepG2 cells, while no signifcant cytotoxicity was observed in L929 cells.Furthermore, the IC 50 values indicated that the leaf methanolic extracts possessed the highest cytotoxic activity, followed by the stem methanolic extract in the HepG2 cell line.Hence, further studies on the identifcation and isolation of bioactive metabolites are required to explore the mechanism of cytotoxicity.

Figure 2 :Figure 3 :
Figure 2: In vitro morphological profle of HepG2 cell observed under an inverted microscope at various concentrations compared to the controls (without the test sample and with the standard drug doxorubicin), after 24 hours of treatment with W. somnifera leaf, stem, and root methanolic extracts.

Figure 4 :
Figure 4: IC 50 values in the HepG2 cell line treated with W. somnifera methanolic extracts from (a) leaf, (b) stem, and (c) root.

Table 1 :
IC 50 value in the HepG2 cell line treated with diferent concentrations of W. somnifera leaf methanolic extract.
4 Evidence-Based Complementary and Alternative Medicine 43.06 ± 0.615 μg/mL and 45.60 ± 0.3 μg/mL, respectively, in HepG2 cell lines.In the L929 cell lines, the IC 50 values were found to be 78.77± 0.795 μg/mL and 90.55 ± 0.800 μg/mL for the leaf and stem extracts, respectively.On the other hand, IC 50 values with root were 314.4 ± 0.795 and 314.4 ± 0.795 μg/mL in the HepG2 and L929 cell lines, respectively.Overall, the IC 50 values obtained for W. somnifera leaf and stem methanolic

Table 2 :
IC 50 value in the HepG2 cell line treated with diferent concentrations of W. somnifera stem methanolic extract.
IC 50 value in bold indicates signifcance at p ≤ 0.05.

Table 3 :
IC 50 value in the HepG2 cell line treated with diferent concentrations of W. somnifera root methanolic extract.
IC 50 value in bold indicates not signifcant at p ≤ 0.05.

Table 4 :
IC 50 value in the L929 cell line treated with diferent concentrations of W. somnifera leaf methanolic extract.

Table 5 :
IC 50 value in the L929 cell line treated with diferent concentrations of W. somnifera stem methanolic extract.

Table 6 :
IC 50 value in the L929 cell line treated with diferent concentrations of W. somnifera root methanolic extract.
IC 50 value in bold indicates not signifcant at p ≤ 0.05.

Table 7 :
[29]istical analysis of IC 50 p values obtained with W. somnifera leaf, stem, and root methanolic extract treatments in HepG2 and L929 cell lines.Based Complementary and Alternative Medicine antagonist drug potency by demonstrating the amount of drug required to block 50% of the biological process[29].Moreover, as per the GERAN Protocol and the American National Cancer Institute (NCI), IC 50 values for strong cytotoxic properties are defned as under 21 μg/mL, moderate cytotoxic properties between 21and 200 μg/mL, and weak cytotoxic properties between 201 and 500 μg/mL