Abstract
Snow mountain garlic (SMG) is a trans-Himalayan medicinal plant used in the traditional medicine system for several ailments, including inflammatory arthritis. Research studies are insufficient to validate its folk medicinal applications. In the present study, the comparative abundance of its key bioactive phytocompounds, viz., S-allyl-L-cysteine (SAC), alliin, and S-methyl-L-cysteine (SMC) against normal garlic were assessed using the LC–MS/MS-MRM method. In addition, the study also explored the antioxidant and anti-inflammatory potency of crude extract of SMG and purified signature phytocompounds (i.e., SMC, SAC, and alliin) in comparison with normal garlic and dexamethasone in LPS-stimulated RAW264.7 macrophage cells. The LC–MS/MS-MRM study revealed significant differences among SMG and normal garlic, viz., alliin 22.8-fold higher in SMG, and SMC could be detected only in SMG. In the bioassays, SMG extract and purified signature phytocompounds significantly downregulated oxidative damage in activated macrophages, boosting endogenous antioxidants’ activity. SMG extract-treated macrophages significantly suppressed NF-κB expression and related inflammatory indicators such as cytokines, COX-2, iNOS, and NO. Notably, the observed anti-inflammatory and antioxidant bioactivities of SMG extract were comparable to signature phytocompounds and dexamethasone. In addition, SAC being uniformly found in SMG and normal garlic, its comparative pharmacokinetics was studied to validate the pharmacodynamic superiority of SMG over normal garlic. Significantly higher plasma concentrations (Cmax), half-life (t1/2), and area under curve (AUC) of SAC following SMG extract administration than normal garlic validated the proposed hypothesis. Thus, the abundance of bioactive phytocompounds and their better pharmacokinetics in SMG extract might be underlying its medicinal merits over normal garlic.
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Acknowledgements
The authors are thankful for financial support from DIHAR, Defence Research and Development Organization (DRDO), Ministry of Defence, Government of India.
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The study is supported by Senior Research Fellowship (SRF) provided by Defence Research and Development Organization (DRDO), Ministry of Defence, Government of India.
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BK: conceptualization, methodology, data curation, formal analysis, writing—original draft. NK: resources, formal analysis. LK: resources, formal analysis. APG: resources, formal analysis. RS: formal analysis. KC: investigation, resources, writing—review and editing. SS: conceptualization, methodology, supervision, investigation, writing—review and editing, funding acquisition.
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: Figure S1. Total ion chromatogram (TIC) of alliin (a), and MS-MRM spectra of alliin showing parent ion transition to major product ion (b). The parent ion (m/z 178) and fragmented product ion (m/z 87.9) are used in MRM screening to quantify the alliin in the aqueous extracts of SMG and normal garlic. Figure S2. Standard calibration curve of alliin (a), LC-MS/MS MRM chromatogram of alliin (b). Figure S3. Total ion chromatogram (TIC) of S-allyl-L-cysteine (SAC) (a), and MS-MRM spectra of SAC showing parent ion transition to major product ions (b). The precursor ion (m/z 161.9) and fragmented product ion (m/z 120.0) are used in MRM screening to quantify the SAC in the aqueous extracts of SMG and normal garlic. Figure S4. Standard calibration curve of SAC (a), LC-MS/MS MRM chromatogram of SAC (b). Figure S5. Total ion chromatogram (TIC) of s-methyl-l-cysteine (SMC) (a), and MS-MRM spectra of SMC showing parent ion transition to major product ions (b). The precursor ion (m/z 136.1) and fragmented product ion (m/z 73.0) are used in MRM screening to quantify the SMC in the aqueous extracts of SMG and normal garlic. Figure S6. Standard calibration curve of SMC (a), LC-MS/MS MRM chromatogram of SMC (b). Figure S7. Effect of different concentrations of LPS-stimulation on the cell viability of RAW 264.7 macrophage cells treated for 24 hours. Cell viability was assessed by MTT. The values are presented as mean±SD of triplicate experiments, and p-values ≤0.05 were considered significant. The percentage of cell viability in the LPS-treated groups was expressed relative to that of control RAW 264.7 cells (LPS non-treated). ***p-value<0.001, control group compared to the LPS-treated groups. Statistical significance was determined by one-way ANOVA followed by Turkey’s multiple comparison post hoc tests. Figure S8. Cytotoxic evaluation of aqueous extracts of both types of garlic, standard phytocompounds, and dexamethasone on RAW 264.7 macrophage cells treated for 24 hours without LPS-stimulation. Cells were treated with dexamethasone 50 μM (III), SMG 10 μg/mL (IV), SMG 100 μg/mL (V), SMG 1000 μg/mL (VI), NG 10 μg/mL (VII), NG 100 μg/mL (VIII), NG 1000 μg/mL (IX), SAC 10 μM (X), SAC 25 μM (XI), SAC 50 μM (XII), SMC 10 μM (XIII), SMC 25 μM (XIV), SMC 50 μM (XV), alliin 10 μM (XVI), alliin 25 μM (XVII), alliin 50 μM (XVIII), and untreated normal cells serves as control (I). The cytotoxicity was measured by MTT assay. The values are expressed as mean±SD (n=6). Figure S9. Calibration curve and HPLC chromatogram of plasma spiked SAC.
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Kaur, B., Kumar, N., Kumari, L. et al. In-vitro antioxidant and anti-inflammatory potential along with p.o. pharmacokinetic profile of key bioactive phytocompounds of Snow Mountain Garlic: a comparative analysis vis-à-vis normal garlic. Inflammopharmacol (2024). https://doi.org/10.1007/s10787-024-01435-w
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DOI: https://doi.org/10.1007/s10787-024-01435-w