Methanolic root extract of Codonopsis clematidea prevents hypoxia induced procoagulant state by inhibition of GPIb receptor regulated Lyn kinase activation.

BACKGROUND: The prevalence of procoagulant state under prolonged hypoxic exposures and the complications and lack of specificity associated with use of existing anti-thrombotic agents have necessitated the search for safer and natural therapeutics. Codonopsis, a widely studied medicinal herb, has been reported to decrease whole blood viscosity but the bioactive ingredients involved, and their mechanism of action therein however remain to be investigated. PURPOSE: The present study aimed at evaluating the efficacy of C. clematidea root extract and mechanism of action of its bioactive constituent flavonoid, Kaempferol, in ameliorating hypobaric hypoxia induced procoagulant state. METHODS: Fingerprinting analysis of methanolic extract of C. clematidea root was performed by RP-HPLC. In vitro toxicity study was conducted using HUVEC cell line and in vivo acute and sub-acute toxicity were done according to OECD guidelines (section-4, number-420 and 407 respectively). Adult male Sprague-Dawley rats weighing 230-250 g were exposed to global hypoxia simulating an altitude of 7600 m (282 mmHg), in animal decompression chamber for 3, 7, 14 and 21 days for in vivo studies. Dose optimisation of the extract was done by quantification of Thromboxane A2 in the serum of hypoxic rats. C. clematidea root extract was also evaluated for its in vitro and in vivo antioxidant properties. Procoagulant changes were studied by biochemical plasma coagulation assays and expression analysis of the signalling molecules of the platelet activation cascade like vWF, platelet activation marker CD41, GpIb-IX-V (CD42), Lyn kinase, p-PI3K, p-ERK and p-PLCγ were conducted to investigate C. clematidea mediated signalling mechanisms. RESULTS: Methanolic extract of C. clematidea root showed improved antioxidant status and improvement in bleeding time and in vitro coagulation assays like pT, aPTT, INR. Decreased concentrations of D-Dimers along with that of platelet activation marker CD41 and serum concentration of Thromboxane A2 were observed in C. clematidea root extract supplemented hypoxic animals. Phosphorylation of Lyn kinase, was reduced despite increase in concentration of activating ligand vWF. CONCLUSION: C. clematidea root extract was effective in preventing hypoxia induced platelet activation and resultant procoagulant state by inhibiting Lyn kinase, a serine threonine kinase effector of vWF signalling cascade.

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.

Acute and sub-acute toxicological evaluation of lyophilized Nymphaea x rubra Roxb. ex Andrews rhizome extract.

Nymphaea x rubra Roxb. ex Andrews (N. rubra) has been widely reported for immunomodulatory properties and treatment of piles, bleeding nose and dysentery in traditional medicinal systems. However, its in-vitro and in-vivo toxicity studies have never been investigated. So, the present study was designed to investigate in-vitro and in-vivo toxicity of methanolic extract of N. rubra rhizome in rats. In-vitro cytotoxicity studies were conducted for different doses of extract in N2a cell lines. For in-vivo toxicity studies, SD rats were divided into three groups and administered with 0, 300 and 2000 mg/kg b. w., p. o., of N. rubra extract respectively. In acute toxicity studies, female animals after extract administration animals were sacrificed for hematological profiling and gross necropsy. In sub-acute toxicity studies, both male and female animals were administered with extract daily for 14 days and were sacrificed for hematological, biochemical and histological examination. Body weight and food water intake was measured daily and animals were observed for visual toxic effects, behavioral changes and mortality. During in-vivo toxicity studies, none of the animals showed signs of toxicity and mortality during toxicity studies. The present findings suggest its safety and NOAEL of N. rubra rhizome extract to be > 2000 mg/kg b. w.

Terminalia arjuna bark extract improves diuresis and attenuates acute hypobaric hypoxia induced cerebral vascular leakage.

ETHNOPHARMACOLOGICAL RELEVANCE: Terminalia arjuna (Roxb. ex DC.) Wight & Arn. (T. arjuna) has been widely used in the traditional ayurvedic system of medicine as a cardioprotectant and for acute and chronic renal diseases supporting its ethnopharmacological use. AIM OF THE STUDY: The present study aimed at evaluating the diuretic action of an alcoholic extract of T. arjuna and its possible use as a prophylactic to prevent vascular leakage during acute mountain sickness at high altitude. MATERIALS AND METHODS: Rats were exposed to hypobaric hypoxia simulated to an altitude of 27,000 ft. in a decompression chamber for 12h. T. arjuna bark extract was administered at a single dose of 150 mg/kg (p.o.) to male Sprague Dawley rats (200 ± 20 g) 30 min prior to exposure. Total urine volume was measured during exposure to hypobaric hypoxia. The animals were then investigated for cerebral vascular leakage and serum concentration of sodium, potassium, renin, angiotensin-II, aldosterone and atrial natriuretic peptide (ANP). RESULTS: T. arjuna ameliorated acute hypobaric hypoxia induced decrease in glomerular filtration rate (p<0.5), increased total urine output (p<0.5) and prevented cerebral vascular leakage in hypoxic rats. T. arjuna treated animals also showed decrease in serum levels of renin (p<0.001) and angiotensin-II (p<0.5) as compared to placebo treated animals. Administration of T. arjuna attenuated acute hypobaric hypoxia induced oxidative stress, improved aldosterone levels and altered electrolyte balance in animals through ANP dependent mechanism. CONCLUSION: Results of the present study indicate towards diuretic potential of hydro-alcoholic extract of T. arjuna bark and provide evidence for its novel application as a prophylactic to attenuate acute hypobaric hypoxia induced cerebral vascular leakage through ANP mediated modulation of renin-angiotensin-aldosterone system.