Antioxidants · 7 authors, 4 centres
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This study analyzed 18 commercial ashwagandha samples and found that aqueous extracts had higher total phenolic content and antioxidant activity than hydromethanolic extracts, while hydromethanolic extracts showed stronger acetylcholinesterase inhibition. The results highlight significant variability in chemical composition and bioactivity among over-the-counter products, emphasizing the need for quality control.
**Background:** *Withania somnifera* (Ashwagandha) is a medicinal plant used in Ayurveda for over 3000 years, with reported immunomodulatory, neuroprotective, and anti-inflammatory effects. While withanolides and alkaloids have been extensively studied, data on phenolic composition and antioxidant activity of commercial products are limited. This study aimed to assess the phenolic profile, antioxidant, antibacterial, and cholinesterase inhibitory activities of 18 commercial samples in both aqueous and hydromethanolic extracts.
**Methods:** Eighteen commercial samples (capsules, tablets, powders) were purchased in Poland. Hydromethanolic extracts (80:20 methanol-water) and aqueous infusions were prepared. Total phenolic content (TPC), total flavonoids (TF), total phenolic acids (TPA), and L(+)-ascorbic acid (ASA) were measured spectrophotometrically. Individual phenolic compounds (gallic acid, catechin, vanillic acid, caffeic acid, p-coumaric acid, ferulic acid, sinapic acid, rutin, quercetin, naringenin) were quantified by HPLC-UV. Antioxidant activity was evaluated using DPPH, ABTS, and FRAP assays. Antibacterial activity (MIC/MBC) was tested against Gram-positive and Gram-negative bacteria using broth microdilution. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibition were measured by Ellman's method. Statistical analysis included ANOVA, Pearson correlation, and PCA.
**Key Results:** Aqueous extracts had significantly higher TPC (range 1.73–5.95 mg GAE/g DW), TF (1.44–3.60 mg QE/g DW), TPA (0.82–8.16 mg CAE/g DW), and ASA (13.97–60.42 mg/g DW) compared to hydromethanolic extracts (TPC: 0.65–6.96 mg GAE/g DW; TF: 0.16–0.64 mg QE/g DW; TPA: 0.11–2.71 mg CAE/g DW; ASA: 2.46–35.90 mg/g DW). In hydromethanolic extracts, catechin (up to 6.54 mg/g) and quercetin (up to 2.16 mg/g) were most abundant; in aqueous extracts, catechin (up to 11.93 mg/g) and gallic acid (up to 1.95 mg/g) were highest. Antioxidant activity was higher in aqueous extracts: DPPH ranged 93.66–281.92 mg TE/100 g DW (vs. 19.48–91.17 in hydromethanolic), ABTS 21.31–90.04 mg TE/g DW (vs. 16.54–124.84), FRAP 13.17–40.01 µmol Fe2+/g DW (vs. 3.87–45.57). Antibacterial testing showed MIC values from 0.25 to >32 mg/mL; *Streptococcus pyogenes* was most sensitive (MIC 0.25 mg/mL for samples 1, 3, 4, 6). AChE inhibition was higher in hydromethanolic extracts (mean 76% of control activity vs. 64% for aqueous), while BChE inhibition was higher in aqueous extracts (mean 78% vs. 40%). PCA clearly separated aqueous and hydromethanolic extracts based on chemical profiles.
**Clinical Implications:** The significant variability in phenolic content and bioactivity among commercial ashwagandha products underscores the need for standardized quality control to ensure consistent therapeutic effects. Aqueous extracts (as tea infusions) may be preferable for antioxidant and antibacterial benefits, while hydromethanolic extracts may be more suitable for neuroprotective applications targeting AChE inhibition. The study provides a foundation for optimizing extraction methods and selecting raw materials for nutraceutical formulations.