Bioactivity Assessment of Aqueous Extract of Ashwagandha and Liquorice on HT29 and MCF-7 with a Focus on Chemotherapy-induced GI Toxicityand LC-MS Profiling

Introduction: Chemotherapy with 5-fluorouracil (5-FU) is a cornerstone treatment for various cancers but often induces severe gastrointestinal (GI) toxicity, myelotoxicity, and reduces Quality of Life (QoL). This study aimed to evaluate the bioactivity, antioxidant potential, and protective effects of aqueous extracts of Withania somnifera (WS; Ashwagandha) and Glycyrrhiza glabra (GG; Liquorice) against 5-FU-induced GI toxicity, using in vitro cell lines (MCF-7 breast cancer and HT-29 GI), antioxidant assays, LC-MS profiling, and an in vivo anti-fatigue model. Methods: Aqueous extracts of WS and GG were prepared and characterized via HPLC-ESIMS/ MS for active compounds (withaferin A and glycyrrhizic acid). Cytotoxicity was assessed using an MTT assay on MCF-7 cells (IC50 determination). Antioxidant activity was measured using DPPH radical scavenging (EC50). GI protective bioactivity was evaluated using proliferation assay on HT-29 cells post-H2O2/UVC exposure (EC50). In vivo efficacy was tested in a rotarod anti-fatigue model in 5-FU-treated albino mice (n=6/group; doses: 100, 200, 400 mg/kg blend; p.o., 7 days). Data were analyzed using ANOVA with post-hoc Tukey test (mean ± SD; significance: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Result: MTT assay revealed potent cytotoxicity of WS (IC50: 36.38 ± 0.8 μg/mL) and GG (IC50: 34.32 ± 0.7 μg/mL) on MCF-7 cells, indicating selective anti-proliferative effects on breast cancer cells with minimal toxicity at low doses (viability >75% up to 1.25 μg/mL). DPPH assay demonstrated strong free radical scavenging, with GG outperforming WS (EC50: 99.75 ± 0.8 μg/mL vs. 134.4 ± 0.3 μg/mL), suggesting robust antioxidant defense against oxidative stress from chemotherapy. Proliferation assay on HT-29 cells confirmed GI cytoprotection, with comparable EC50 values (WS: 76.78 ± 0.3 μg/mL; GG: 77.90 ± 0.6 μg/mL), restoring viability post-oxidative insult (*p*<0.05 at low concentrations). LC-MS/MS verified key bioactives: withaferin A (m/z 476.4) in WS and glycyrrhizic acid (m/z 822.6) in GG, matching standards. In the rotarod model, the 200 mg/kg WS:GG blend (2:1) significantly mitigated 5-FU-induced muscle fatigue (fall latency increase: ****p*<0.0001 vs. disease control; ##p*<0.01 vs. vitamin C standard), without dosedependency, implying non-linear protective mechanisms via flavonoids and probiotics. Discussion: The presented data underscore the therapeutic promise of Withania somnifera (WS) and Glycyrrhiza glabra (GG) extracts as adjuvant agents in breast cancer chemotherapy, evidenced by robust cytotoxicity (IC50 values of 36.38 ± 0.8 μg/mL for WS and 34.32 ± 0.7 μg/mL for GG on MCF-7 cells; t-test comparison: p > 0.05, indicating non-significant difference). Antioxidant efficacy via DPPH scavenging highlights GG's superiority (EC50: 99.75 ± 0.8 μg/mL vs. WS: 134.4 ± 0.3 μg/mL; p < 0.001 by unpaired t-test), and LC-MS/MS confirmation of withaferin A and glycyrrhizic acid aligns with prior spectral libraries. Conclusion: WS and GG extracts exhibit promising anti-cancer cytotoxicity, antioxidant efficacy, and GI mucosal protection, corroborated by bioactive confirmation and in vivo anti-fatigue effects. These findings support their potential as safe adjuvant therapies to alleviate 5-FU-induced GI toxicity, enhance QoL, and warrant clinical translation for chemotherapy support.