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.

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.

Validation of traditional claims of anti-arthritic efficacy of trans-Himalayan snow mountain garlic (Allium ampeloprasum L.) extract using adjuvant-induced arthritis rat model: A comparative evaluation with normal garlic (Allium sativum L.) and dexamethasone.

ETHNOPHARMACOLOGICAL RELEVANCE: Snow Mountain Garlic (SMG) (Allium ampeloprasum L.) is a wild trans-Himalayan member of the genus Allium, valued for its anti-inflammatory and anti-arthritic properties in the mountain folk medicinal system (Sowa-Rigpa). Despite its age-old medicinal usage by traditional therapists and the native population for various ailments including rheumatism, there is no scientific validation of its phyto-pharmaceutical merits. AIM OF THE STUDY: The present pre-clinical study compared the in-vivo anti-arthritic effects of SMG with reported efficacy doses of normal garlic (Allium sativum L.) extract and dexamethasone in a complete Freund's adjuvant (CFA)-induced arthritis rat model. MATERIALS AND METHODS: The female Wistar rats were immunized by the subplannter injection of CFA into the right hind footpad. Aqueous extracts of SMG and normal garlic were administered orally at a dose of 250 mg/kg and 500 mg/kg for 28 days. Dexamethasone was used as positive control drug. Behavioral parameters including paw markers, arthritis index, joint stiffness, body weight change, etc. were measured. Also, the changes in histopathological indices, hematological profile, inflammatory mediators, and serum cytokines level was determined. RESULTS: Treatment of rats with SMG extracts significantly (p < 0.001) prevented the reduction in body weight and hematological changes as well as ameliorated clinical symptoms such as arthritic index, joint stiffness, arthritis score, edema, hyperalgesia, and histopathological indices. This was associated with a significant reduction in the serum levels of RF, CRP, anti-CCP, and proinflammatory cytokines exhibiting strong anti-arthritic potential. SMG extracts could also significantly down regulate the NF-κB, COX-2, and iNOS expression in the ankle joint tissues. CONCLUSIONS: The present study is the first attempt to validate the phyto-pharmaceutical efficacy of this folk garlic variety from the trans-Himalayan region. Overall, SMG extract showed remarkable preventive anti-inflammatory and anti-arthritic activities which were closely comparable to therapeutic effects of dexamethasone and at par or even better than normal garlic w.r.t. several study parameters.

A comparative study of in-vitro and in-silico anti-candidal activity and GC-MS profiles of snow mountain garlic vs. normal garlic.

AIM: The study aimed to profile the volatile phytocomposition of snow mountain garlic (SMG) compared to normal garlic and investigate the anti-Candida efficacy against clinically relevant multi-drug resistant isolates of Candida species. METHODS AND RESULTS: Herein, SMG has shown significantly superior fungicidal power at 2x-MIC dose against C. albicans and C. glabrata in killing kinetic evaluation unlike the fungistatic effect of normal garlic. GC-MS headspace-based profiling of SMG showed 5 unique volatile compounds and a 5-fold higher content of saponins than normal garlic. In an in-silico analysis, cholesta-4,6-dien-3-ol,(3-beta) was uniquely identified in SMG as a potential inhibitor with high binding affinity to the active site of exo-1,3-betaglucan synthase, an established anti-candida drug target crucial for the biofilm matrix formation, thus suggesting a plausible anti-Candida mechanism. CONCLUSION: The in-vitro and in-silico studies have demonstrated the Candida-cidal and anti-biofilm activities of SMG, distinguishing it from the Candida-static efficacy of normal garlic. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report that identifies several phytochemical signatures of SMG along with a potential anti-Candida compound, that is cholesta-4,6-dien-3-ol,(3-beta)-, which appears worthy of detailed studies in the future to explore the utility of SMG as a fungal phytotherapy agent, especially against drug-resistant Candida sp.

Green synthesis of silver nanoparticles using Rhodiola imbricata and Withania somnifera root extract and their potential catalytic, antioxidant, cytotoxic and growth-promoting activities.

This study presents the development of a sustainable production process of environmentally benign silver nanoparticles (AgNPs) from aqueous root extract of Rhodiola imbricata (RI) and Withania somnifera (WS) for mitigating environmental pollution and investigating their potential applications in agriculture and biomedical industry. RIWS-AgNPs were characterized using several analytical techniques (UV-Vis, DLS, HR-TEM, SAED, EDX and FTIR). The antioxidant and anticancer activity of RIWS-AgNPs were estimated by DPPH and MTT assay, respectively. UV-Vis and DLS analysis indicated that equal ratio of RIWS-extract and silver nitrate (1:1) is optimum for green synthesis of well-dispersed AgNPs (λmax: 430 nm, polydispersity index: 0.179, zeta potential: - 17.9 ± 4.14). HR-TEM and SAED analysis confirmed the formation of spherical and crystalline RIWS-AgNPs (37-42 nm). FTIR analysis demonstrated that the phenolic compounds are probably involved in stabilization of RIWS-AgNPs. RIWS-AgNPs showed effective catalytic degradation of hazardous environmental pollutant (4-nitrophenol). RIWS-AgNPs treatment significantly increased the growth and photosynthetic pigments of Hordeum vulgare in a size- and dose-dependent manner (germination (77%), chlorophyll a (12.62 ± 0.07 µg/ml) and total carotenoids (7.05 ± 0.04 µg/ml)). The DPPH assay demonstrated that RIWS-AgNPs exert concentration-dependent potent antioxidant activity (IC50: 12.30 µg/ml, EC50: 0.104 mg/ml, ARP: 959.45). Moreover, RIWS-AgNPs also confer strong cytotoxic activity against HepG2 cancer cell line in dose-dependent manner (cell viability: 9.51 ± 1.55%). Overall, the present study for the first time demonstrated a green technology for the synthesis of stable RIWS-AgNPs and their potential applications in biomedical and agriculture industry as phytostimulatory, antioxidant and anticancer agent. Moreover, RIWS-AgNPs could potentially be used as a green alternative for environmental remediation.

Enhanced Production of Phenolic Compounds in Compact Callus Aggregate Suspension Cultures of Rhodiola imbricata Edgew.

Rhodiola imbricata is a rare medicinal plant of the trans-Himalayan region of Ladakh. It is used for the treatment of numerous health ailments. Compact callus aggregate (CCA) suspension cultures of Rhodiola imbricata were established to counter extinction threats and for production of therapeutically valuable phenolic compounds to meet their increasing industrial demands. The present study also investigated the effect of jasmonic acid (JA) on production of phenolic compounds and bioactivities in CCA suspension cultures. CCA suspension cultures established in an optimized Murashige and Skoog medium supplemented with 30 g/l sucrose, 3 mg/l NAA, and 3 mg/l BAP showed maximum biomass accumulation (8.43 g/l DW) and highest salidroside production (3.37 mg/g DW). Upon 100 µM JA treatment, salidroside production (5.25 mg/g DW), total phenolic content (14.69 mg CHA/g DW), total flavonoid content (4.95 mg RE/g DW), and ascorbic acid content (17.93 mg/g DW) were significantly increased in cultures. In addition, DPPH-scavenging activity (56.32%) and total antioxidant capacity (60.45 mg QE/g DW) were significantly enhanced upon JA treatment, and this was positively correlated with increased accumulation of phenolic compounds. JA-elicited cultures exhibited highest antimicrobial activity against Escherichia coli. This is the first report describing the enhanced production of phenolic compounds and bioactivities from JA-elicited CCA suspension cultures of Rhodiola imbricata.

Influence of light quality on growth, secondary metabolites production and antioxidant activity in callus culture of Rhodiola imbricata Edgew.

Rhodiola imbricata is a rare medicinal herb well-known for its adaptogenic and antioxidant properties due to the presence of a diverse array of secondary metabolites, including phenylethanoids and phenylpropanoids. These secondary metabolites are generating considerable interest due to their potential applications in pharmaceutical and nutraceutical industries. The present study investigated the influence of light quality on growth, production of industrially important secondary metabolites and antioxidant activity in callus cultures of Rhodiola imbricata. Callus cultures of Rhodiola imbricata were established under different light conditions: 100% red, 100% blue, 100% green, RGB (40% red: 40% green: 20% blue) and 100% white (control). The results showed that the callus cultures grown under red light accumulated maximum amount of biomass (7.43 g/l) on day 21 of culture, as compared to other light conditions. Maximum specific growth rate (0.126 days-1) and doubling time (132.66 h) was observed in callus cultures grown under red light. Reverse phase-high performance liquid chromatographic (RP-HPLC) analysis revealed that the callus cultures exposed to blue light accumulated maximum amount of Salidroside (3.12 mg/g DW) on day 21 of culture, as compared to other light conditions. UV-Vis spectrophotometric analysis showed that the callus cultures exposed to blue light accumulated maximum amount of total phenolics (11.84 mg CHA/g DW) and total flavonoids (5.53 mg RE/g DW), as compared to other light conditions. Additionally, callus cultures grown under blue light displayed enhanced DPPH free radical scavenging activity (53.50%). Callus cultures grown under different light conditions showed no significant difference in ascorbic acid content (11.05-13.90 mg/g DW) and total antioxidant capacity (27.37-30.17 mg QE/g DW). The correlation analysis showed a positive correlation between total phenolic content and DPPH free radical scavenging activity in callus cultures (r = 0.85). Taken together, these results demonstrate the remarkable potential of light quality on biomass accumulation and production of industrially important secondary metabolites in callus cultures of Rhodiola imbricata. This study will open new avenues and perspectives towards abiotic elicitation strategies for sustainable growth and enhanced production of bioactive compounds in in-vitro cultures of Rhodiola imbricata.

S1P prophylaxis mitigates acute hypobaric hypoxia-induced molecular, biochemical, and metabolic disturbances: A preclinical report.

Sphingosine-1-phosphate (S1P) is emerging to have hypoxic preconditioning potential in various preclinical studies. The study aims to evaluate the preclinical preconditioning efficacy of exogenously administered S1P against acute hypobaric hypoxia (HH)-induced pathological disturbances. Male Sprague Dawley rats (200 ± 20 g) were preconditioned with 1, 10, and 100 µg/kg body weight (b.w.) S1P (i.v.) for three consecutive days. On the third day, S1P preconditioned animals, along with hypoxia control animals, were exposed to HH equivalent to 7,620 m (280 mm Hg) for 6 h. Postexposure status of cardiac energy production, circulatory vasoactive mediators, pulmonary and cerebral oxidative damage, and inflammation were assessed. HH exposure led to cardiac energy deficit indicated by low ATP levels and pronounced AMPK activation levels, raised circulatory levels of brain natriuretic peptide and endothelin-1 with respect to total nitrate (NOx), redox imbalance, inflammation, and alterations in NOx levels in the pulmonary and cerebral tissues. These pathological precursors have been routinely reported to be coincident with high-altitude diseases. Preconditioning with S1P, especially 1 µg/kg b.w. dose, was seen to reverse the manifestation of these pathological disturbances. The protective efficacy could be attributed, at least in part, to enhanced activity of cardioprotective protein kinase C and activation of small GTPase Rac1, which led to further induction of hypoxia-adaptive molecular mediators: hypoxia-inducible factor (HIF)-1α and Hsp70. This is a first such report, to the best of our knowledge, elucidating the mechanism of exogenous S1P-mediated HIF-1α/Hsp70 induction. Conclusively, systemic preconditioning with 1 µg/kg b.w. S1P in rats protects against acute HH-induced pathological disturbances. © 2016 IUBMB Life 68(5):365-375, 2016.

Exogenous sphingosine 1-phosphate protects murine splenocytes against hypoxia-induced injury.

Sphingosine-1-phosphate (S1P), a biologically active pleiotropic lipid, is involved in several physiological processes especially in the area of vascular biology and immunology encompassing cell survival, angiogenesis, vascular tone, immune response etc. by interacting with specific cell surface receptors. Hypoxia, a condition common to innumerable pathologies, is known to lethally affect cell survival by throwing off balance global gene expression, redox homeostasis, bioenergetics etc. Several molecular events of cellular adaptations to hypoxia have been closely linked to stabilization of hypoxia inducible factor-1α (HIF-1α). Signalling functions of S1P in physiological events central to hypoxia-induced pathologies led us to investigate efficacy of exogenous S1P in preconditioning murine splenocytes to sustain during cellular stress associated with sub-optimal oxygen. The present study recapitulated the pro-survival benefits of exogenous S1P under normobaric hypoxia. Results indicate a direct effect of S1P supplementation on boosting cellular adaptive responses via HIF-1α stabilization and, activation of pro-survival mediators ERK and Akt. Overwhelming anti-oxidative and anti-inflammatory benefits of S1P preconditioning could also be captured in the present study, as indicated by improved redox homeostasis, reduced oxidative damage, balanced anti/pro-inflammatory cytokine profiles and temporal regulation of nitric oxide secretion and intra-cellular calcium release. Hypoxia induced cell death and the associated stress in cellular milieu in terms of oxidative damage and inflammation could be alleviated with exogenous S1P preconditioning.