Rajkumar Tulsawani

A dose dependent adaptogenic and safety evaluation of Rhodiola imbricata Edgew, a high altitude rhizome

To examine the dose dependent adaptogenic activity aqueous extract of Rhodiola imbricata root was orally administered in rats at different doses, 30 min prior to cold (5 degrees C)-hypoxia (428 mm Hg)-restraint (C-H-R) exposure. The maximal effective adaptogenic dose of the extract was 100 mg/kg body weight. The acute and sub-acute toxicity of the extract was also studied in rats. Sub-acute toxicity studies included administration of single oral dose of 1 g/kg and 2 g/kg of extract once daily for 14 days and maximal effective single oral dose of 100 mg/kg once daily for 30 days. At the end of each treatment period the biochemical parameters related to liver function, kidney function, lipids (triglycerides, cholesterol) and hematological parameters were estimated in serum and blood. Biochemical and hematological analysis showed no significant changes in any of the parameters examined in treated groups animal, in comparison to control animals. No significant change was observed in organ weight/body weight ratios, of any organ studied in comparison to control rats. The oral LD(50) of the extract was observed to be >10 g/kg, indicating an adequate margin of safety. No histopathological changes were observed in the vital organs studied of the treated animals. These results suggest that aqueous extract of R. imbricata root possess potent adaptogenic activity with no acute and sub-acute toxicity.

Anti-oxidative effect of Rhodiola imbricata root extract in rats during cold, hypoxia and restraint (C-H-R) exposure and post-stress recovery.

Anti-oxidative potential of Rhodiola imbricata root aqueous extract was examined in rats, administered orally at a dose of 100 mg/kg both in single and multiple doses, 30 min prior to cold (5 degrees C)-hypoxia (428 mmHg)-restraint (C-H-R) exposure. Lipid per-oxidation, anti-oxidant parameters and lactate dehydrogenase (LDH), were studied in blood, liver and muscle of rats on attaining T(rec)23 degrees C during C-H-R exposure and after recovery (T(rec)37 degrees C) from C-H-R induced hypothermia. The results of untreated control rats on attaining T(rec)23 degrees C showed a significant increase in blood, liver and muscle malondialdehyde (MDA) and LDH levels. Hepatic catalase (CAT) and muscle glutathione S-transferase (GST) also increased significantly. Administration of single dose of Rhodiola imbricata root aqueous extract significantly restricted rise in blood MDA, increased blood reduced glutathione (GSH) and superoxide dismutase (SOD) activity with restricted rise in blood, liver and muscle LDH; improved liver and muscle SOD on attaining T(rec)23 degrees C and T(rec)37 degrees C; liver CAT on attaining T(rec)23 degrees C and liver GST during recovery. Multiple doses treatment of the extract further increased blood, liver and muscle GSH and GST levels; restricted increase in LDH on attaining T(rec)23 degrees C and recovery; increased CAT during recovery. Results suggested the anti-oxidant potential of Rhodiola root extract during C-H-R exposure and post-stress recovery and it also maintained cell membrane permeability.

Cordyceps sinensis increases hypoxia tolerance by inducing heme oxygenase-1 and metallothionein via Nrf2 activation in human lung epithelial cells.

Cordyceps sinensis, an edible mushroom growing in Himalayan regions, is widely recognized in traditional system of medicine. In the present study, we report the efficacy of Cordyceps sinensis in facilitating tolerance to hypoxia using A549 cell line as a model system. Treatment with aqueous extract of Cordyceps sinensis appreciably attenuated hypoxia induced ROS generation, oxidation of lipids and proteins and maintained antioxidant status similar to that of controls via induction of antioxidant gene HO1 (heme oxygenase-1), MT (metallothionein) and Nrf2 (nuclear factor erythroid-derived 2-like 2). In contrast, lower level of NFκB (nuclear factor kappaB) and tumor necrosis factor-α observed which might be due to higher levels of HO1, MT and transforming growth factor-β. Further, increase in HIF1 (hypoxia inducible factor-1) and its regulated genes; erythropoietin, vascular endothelial growth factor, and glucose transporter-1 was observed. Interestingly, Cordyceps sinensis treatment under normoxia did not regulate the expression HIF1, NFκB and their regulated genes evidencing that Cordyceps sinensis per se did not have an effect on these transcription factors. Overall, Cordyceps sinensis treatment inhibited hypoxia induced oxidative stress by maintaining higher cellular Nrf2, HIF1 and lowering NFκB levels. These findings provide a basis for possible use of Cordyceps sinensis in tolerating hypoxia.

HYPHENATED HIGH-PERFORMANCE THIN-LAYER CHROMATOGRAPHY FOR PROFILING OF SOME INDIAN NATURAL EFFICIENCY ENHANCERS

The high-performance thin-layer chromatographic (HPTLC) profiling of sugars, sugar alcohols, amino acids, flavonoids, and phenolic acids, as well as bioactive and antioxidative compounds, is described for the first time in aqueous and hydroalcoholic extracts of some Indian herbs, namely, Hippophae rhamnoides, Valeriana wallichii, Triticum aestivum, and the fungus Cordyceps sinensis, all known for their natural performance enhancement capacity. For the analysis of bioactive compounds such as flavonoids and phenolic acids, the Automated Multiple Development (AMD2) system was employed. For the determination of the bioactivity of the extracts, chromatography was linked to a bioassay, and, therefore, the developed plate was automatically dipped into the luminescent Vibrio fischeri bacteria suspension. The presence of sugars could be confirmed in all aqueous and hydroalcoholic extracts, except for the hydroalcoholic extract of Cordyceps sinensis. Relatively high amounts of amino acids were found in the extracts of Triticum aestivum and Cordyceps sinensis. In terms of sugar alcohols, the highest amount was observed in aqueous and hydroalcoholic extracts of Cordyceps sinensis, in which mannitol was found. Xylitol was present to a high extent in all three extracts of Valeriana wallichii. A high content of flavonoids, phenolic acids, and other bioactive compounds was observed in the hydroalcoholic extract of Valeriana wallichii. Using the DPPH• radical reagent, the antioxidant potential of the four extracts was in the following order: Valeriana wallichii hydroalcoholic extract > Triticum aestivum hydroalcoholic extract > Valeriana wallichii aqueous extract > Triticum aestivum aqueous extract > Hippophae rhamnoides aqueous extract > Cordyceps sinensis aqueous extract ≥ Cordyceps sinensis hydroalcoholic extract.

Survival response of hippocampal neurons under low oxygen conditions induced by Hippophae rhamnoides is associated with JAK/STAT signaling.

Janus activated kinase/signal transducers and activators of transcription (JAK/STATs) pathway are associated with various neuronal functions including cell survival and inflammation. In the present study, it is hypothesized that protective action of aqueous extract of Hippophae rhamnoides in hippocampal neurons against hypoxia is mediated via JAK/STATs. Neuronal cells exposed to hypoxia (0.5% O2) display higher reactive oxygen species with compromised antioxidant status compared to unexposed control cells. Further, these cells had elevated levels of pro-inflammatory cytokines; tumor necrosis factor α and interleukin 6 and nuclear factor κappa B. Moreover, the expression of JAK1 was found to be highly expressed with phosphorylation of STAT3 and STAT5. Cells treated with JAK1, STAT3 and STAT5 specific inhibitors resulted in more cell death compared to hypoxic cells. Treatment of cells with extract prevented oxidative stress and inflammatory response associated with hypoxia. The extract treated cells had more cell survival than hypoxic cells with induction of JAK1 and STAT5b. Cells treated with extract having suppressed JAK1 or STAT3 or STAT5 expression showed reduced cell viability than the cell treated with extract alone. Overall, the findings from these studies indicate that the aqueous extract of Hippophae rhamnoides treatment inhibited hypoxia induced oxidative stress by altering cellular JAK1, STAT3 and STAT5 levels thereby enhancing cellular survival response to hypoxia and provide a basis for possible use of aqueous extract of Hippophae rhamnoides in facilitating tolerance to hypoxia.

Ninety day repeated gavage administration of Hipphophae rhamnoides extract in rats.

Hippophae rhamnoides, is a high altitude plant, possesses immunomodulatory, anti-oxidant, anti-bacterial and adaptogenic activity and is widely used in treatment of various diseases. The present study was designed to ascertain the safety of aqueous extract of H. rhamnoides fruit when administered by gavage to rats for 90 days. Four groups of animals, each consisting of 15 males and 15 females, were administered 0, 100, 250, or 500 mg/kg extract, in a single dose/day. There were no treatment related change in mean body weight, organ/body weight ratio, histological, hematological and biochemical parameters studied in rats of either sex administered with extract at any dose evaluated. However, a significant increase in plasma glucose levels was observed in animals supplemented with 250 or 500 mg/kg extract, which returned to normal after a 2-week withdrawal of treatment. These results indicate no adverse effects of extract at a dose of 100mg/kg body weight/day in rats administered for 90-days. Based on the findings of this study, the NOAEL was 100mg/kg body weight/day of aqueous fruit extract of seabuckthorn in rats.

Radiolabeling and dose fixation study of oral alpha-ketoglutarate as a cyanide antidote in healthy human volunteers

Context. Radiolabeling and dose fixation study of alpha-ketoglutarate (A-KG). Objective. A-KG is a potential oral antidote for cyanide poisoning. Its protective efficacy in animals was best exhibited at a dose of 2.0 g/kg body weight, which when extrapolated to human is very high. The objective of this study was to reduce the dose of A-KG in humans with concomitant increase in its bioavailability, employing pharmacoscintigraphic techniques to assess kinetics in man. Materials and methods. A-KG was radiolabeled with technetium-99m pertechnetate (Tc-99m) and its purity, labeling efficiency, and stability in vitro were determined by instant thin layer chromatography. Time-dependent bio-absorption of the drug in rats and rabbits was assessed by gamma scintigraphy after oral administration of a tracer dose of 99mTc-A-KG mixed with nonradioactive A-KG at a concentration of 0.1–2.0 g/kg in the presence or absence of aqueous dilution. Furthermore, scintigraphy and radiometry studies were performed in healthy human volunteers using 5–20 g of A-KG, given in single or split doses followed by different quantity of water. Drug bioavailability was estimated periodically. Results. High radiolabeling (>97%) of A-KG with a stability of 24 h in vitro was obtained. Less than 1% absorption of the drug occurred within 20 min after A-KG was administered in animals at a concentration of 2.0 g/kg body weight. One-tenth reduction in dose increased the bioavailability to 15%. Significant improvement in gastric emptying of the drug was achieved when the drug was administered along with 1–5 mL of water. In humans, two doses of 10 g A-KG given at an interval of 10 min, followed by 300 mL of water, increased the drug bioavailability to 40% as compared to a single dose of 20 g. Discussion. Significant reduction in A-KG dose was achieved in humans as compared to the recommended dose in animals. Conclusion. Aqueous dilution improves the bioavailability of A-KG in humans.

HPTLC Based Chemometrics of Medicinal Mushrooms

The current study presents application of chemometrics to high performance thin layer chromatography (HPTLC) fingerprints of Indian medicinal mushrooms, Cordyceps sinensis (CS) and Ganoderma lucidum (GL). These mushrooms possess numerous bio-active constituents that endow them with several medicinal properties. Five CS extracts using different compositions of water and ethanol and nine sequentially extracted GL extracts were prepared employing the accelerated solvent extraction technique. A rapid and convenient HPTLC method was developed for generating phytochemical profiles and simultaneous detection of antioxidant compounds: ascorbic acid, hesperidin, gallic acid, rutin, and quercetin. The presence of these known and some of selective unknown zones visualized at 254 nm or 366 nm were confirmed by HPTLC–ESI/MS (HPTLC–Electronspray Ionization/Mass Spectrometry). The most probable m/z value of selective unknown HPTLC zones was searched against public databases to find putative compounds. Chemometric methods such as cluster analysis and principal component analysis (PCA) were performed on HPTLC datasets acquired during HPTLC scanning at 254 nm and 366 nm. Data matrices consisting of maximum peak heights of HPTLC zones at a specific Rf (retention factor) values were used as input variables. Also, the quantitative patterns of metabolites across the extracts were represented using HeatMap. This study illustrated HPTLC assisted chemometric analysis of Cordyceps sinensis and Ganoderma lucidum.

Efficacy of aqueous extract of Hippophae rhamnoides and its bio-active flavonoids against hypoxia-induced cell death.

Objectives: To investigate the protective efficacy of aqueous extract of Hippophae rhamnoides against chronic hypoxic injury using primary rat hepatocytes. Materials and Methods: The extract was prepared using maceration method and characterized by its phenolic and flavonoid content and chemical antioxidant capacity using ferric reducing antioxidant power assay. Hepatocytes were maintained in hypoxia chamber (3% and 1% oxygen) for 72 h. The cells kept under normoxic condition served as control. The cells were treated with the extract and flavonoids; isorhamentin, kaempferol or qurecetin-3-galactoside. After the end of exposure period; cell survival, reactive oxygen species (ROS), leakage of lactate dehydrogenase (LDH), alanine aminotransferase (ALT), aspartate aminotransferase (AST), reduced glutathione (GSH), glutathione peroxidase (GPx), and superoxide dismutase (SOD) levels were measured. Results: The extract showed presence of high phenolic and flavonoid content with significant antioxidant activity in chemical assay. The cell exposed to hypoxia showed concentration dependent cell death and harbored higher reactive oxygen species. In addition, these cells showed significant leakage of intracellular LDH, ALT, and AST accompanied by the diminished levels/activities of GSH, GPx, and SOD. The treatment of cells with aqueous extract of H. rhamnoides reduced hypoxia-induced cell death and prevented increase in ROS levels and leakage of intracellular LDH, ALT, and AST from cells. Moreover, these cells maintained better levels/activities of GSH, GPx, and SOD in comparison to the respective controls. The major flavonoids present in aqueous extract of H. rhamnoides; quercetin-3-galactoside, kaempferol, and isorhamentin also prevented hypoxia induced cell injury individually or in combination, however, the protection offered by these compounds taken together could not match to that of the extract. Conclusions: Overall the findings reveal significance of aqueous extract of H. rhamnoides in controlling ROS-meditated hypoxic injury in cells and can be useful in many hepatic complications.

Heat stress-induced neuroinflammation and aberration in monoamine levels in hypothalamus are associated with temperature dysregulation

Heat Stress (HS) induces diverse pathophysiological changes, which include brain ischemia, oxidative stress and neuronal damage. The present study was undertaken with the objective to ascertain whether neuroinflammation in Hypothalamus (HTH) caused under HS affects monoamine levels and hence, its physiological role in thermoregulation. Rats were exposed to HS in a heat simulation environmental chamber (Ambient temperature, Ta=45±0.5°C and Relative Humidity, RH=30±10%) with real-time measurement of core temperature (Tc) and skin temperature (Ts). Animals were divided into two subgroups: Moderate HS (MHS) (Tc=40°C) and Severe HS (SHS)/Heat stroke (Tc=42°C). Rats with MHS showed an increase in Mean Arterial Pressure (MAP) and Heart Rate (HR) while fall in MAP and rise in HR was observed in rats with SHS. In addition, oxidative stress and an increase in pyknotic neurons were observed in HTH. High levels of Adrenocorticotropic-hormone (ACTH), Epinephrine (EPI), Norepinephrine (NE) and Dopamine (DA) in the systemic circulation and progressive increase in EPI and DA levels in HTH were recorded after the thermal insult. Moreover, a substantial increase in Glutamate (Glu) level was observed in HTH as well as in systemic circulation of heat stroke rats. We found a rise in NE whereas a fall in Serotonin (5-HT) level in HTH at MHS, without perturbing inflammatory mediators. However, rats with SHS exhibited significant elevations in NF-kB, IL-1β, COX2, GFAP and Iba1 protein expression in HTH. In conclusion, the data suggest that SHS induces neuroinflammation in HTH, which is associated with monoamines and Glu imbalances, leading to thermoregulatory disruption.