Anti-microbial efficacy of a scientifically developed and standardized herbal-alcohol sanitizer.

Hands are the primary mode of transmission of microbe-based infections, as they harbor normal microbiota and pathogenic microbes. SARS-CoV-2 has endangered lives worldwide, and WHO has recommended good hygiene practices, especially hand hygiene. In addition, other infectious diseases like diphtheria, measles, tuberculosis, HIV, malaria, etc. are spreading in the shadow of the COVID-19 pandemic. The anti-microbial efficiency of two in-house developed herbal-alcohol based hand sanitizers containing Azadirachta indica, Citrus limon, Zingiber officinale, and Aloe vera (HS1) and Zingiber officinale replaced with Ocimum sanctum (HS2) was evaluated. HS1, with Zingiber officinale, and HS2, with Ocimum sanctum, herbal sanitizers showcased in-vitro anti-viral activity on MDCK cells using the reference strain of influenza A virus, A/PR/8/34 (H1N1), and reduced 99.99% of microbial load within 30 s of contact time, estimated by the Antimicrobial Susceptibility Testing Method. On volunteers, HS1 and HS2 were more effective than alcohol-based WHO sanitizers. Moreover, HS2 sanitizer is more effective against viruses and has better efficiency and hedonic qualities in volunteers than HS1. These sanitizers don't irritate or dry up the skin and have a longer shelf life. Overall, findings reveal that herbal-alcohol-based sanitizers are promising hand hygiene products with the capability of reducing microbial load.

Efficacy of Aqueous Extract of Chinese Caterpillar Mushroom Ophiocordyceps sinensis (Ascomycota) Against Simulated Altitude Stress and Subacute Toxicity Studies.

Exposure to high altitude severely impacts performance of unacclimatized individuals and contraindications associated with synthetic drugs ascertain the need for development of herbal drugs. Thus, the present study investigated the adaptogenic potential of Ophiocordyceps sinensis aqueous extract (CSAQ) using simulated altitude stress models such as severe hypoxia (SH) in hermetic vessel, cold restraint (CR) at 4°C, and hypobaric hypoxia (HBH) at 7,620 meter, ~ 282 mm Hg. To further address safety limits of extract, subacute toxicity studies were conducted in rats orally administered with CSAQ (0, 100, 500 and 1000 mg/kg) in a single dose/day for 28 days. Results revealed that animals administered with CSAQ increased convulsion time and core body temperature during SH and CR stress. CSAQ modulated thermogenic response by upregulating uncoupling protein 1 and maintaining metabolic homeostasis. Further, CSAQ improved antioxidant status (glutathione and 2,3-diposhphoglycerate), attenuated pro-inflammatory cytokine NF-κB, and augmented hypoxia inducible factor and nuclear erythroid 2 related factor 2 in HBH exposed animals. Toxicity studies revealed no observed adverse effect level with 1000 mg/kg extract in body weight gain, organ/body weight ratio, hematological variables, biochemical parameters and histoarchitecture of vital organs. In conclusion, CSAQ initiated dose dependent adaptive response and exhibited high safety margins, which strongly suggests the therapeutic potential of CSAQ in mitigating high altitude maladies.

Dual Targeting by Inhibition of Phosphoinositide-3-Kinase and Mammalian Target of Rapamycin Attenuates the Neuroinflammatory Responses in Murine Hippocampal Cells and Seizures in C57BL/6 Mice.

Emerging evidence suggests the association of seizures and inflammation; however, underlying cell signaling mechanisms are still not fully understood. Overactivation of phosphoinositide-3-kinases is associated with both neuroinflammation and seizures. Herein, we speculate the PI3K/Akt/mTOR pathway as a promising therapeutic target for neuroinflammation-mediated seizures and associated neurodegeneration. Firstly, we cultured HT22 cells for detection of the downstream cell signaling events activated in a lipopolysaccharide (LPS)-primed pilocarpine (PILO) model. We then evaluated the effects of 7-day treatment of buparlisib (PI3K inhibitor, 25 mg/kg p.o.), dactolisib (PI3K/mTOR inhibitor, 25 mg/kg p.o.), and rapamycin (mTORC1 inhibitor, 10 mg/kg p.o.) in an LPS-primed PILO model of seizures in C57BL/6 mice. LPS priming resulted in enhanced seizure severity and reduced latency. Buparlisib and dactolisib, but not rapamycin, prolonged latency to seizures and reduced neuronal loss, while all drugs attenuated seizure severity. Buparlisib and dactolisib further reduced cellular redox, mitochondrial membrane potential, cleaved caspase-3 and p53, nuclear integrity, and attenuated NF-κB, IL-1ß, IL-6, TNF-α, and TGF-ß1 and TGF-ß2 signaling both in vitro and in vivo post-PILO and LPS+PILO inductions; however, rapamycin mitigated the same only in the PILO model. Both drugs protected against neuronal cell death demonstrating the contribution of this pathway in the seizure-induced neuronal pyknosis; however, rapamycin showed resistance in a combination model. Furthermore, LPS and PILO exposure enhanced pAkt/Akt and phospho-p70S6/total-p70S6 kinase activity, while buparlisib and dactolisib, but not rapamycin, could reduce it in a combination model. Partial rapamycin resistance was observed possibly due to the reactivation of the pathway by a functionally different complex of mTOR, i.e., mTORC2. Our study substantiated the plausible involvement of PI3K-mediated apoptotic and inflammatory pathways in LPS-primed PILO-induced seizures and provides evidence that its modulation constitutes an anti-inflammatory mechanism by which seizure inhibitory effects are observed. We showed dual inhibition by dactolisib as a promising approach. Targeting this pathway at two nodes at a time may provide new avenues for antiseizure therapies.

Ganoderma lucidum Induces Myogenesis Markers to Avert Damage to Skeletal Muscles in Rats Exposed to Hypobaric Hypoxia.

Jatwani, Arti, and Rajkumar Tulsawani. Ganoderma lucidum induces myogenesis markers to avert damage to skeletal muscles in rats exposed to hypobaric hypoxia. High Alt Med Biol. 16:000-000, 2021. Background: Hypobaric hypoxia (HH) has been reported to induce skeletal muscle loss and impair myogenesis. Aqueous extract of G. lucidum (AqGL) contains bioactive metabolites attributed to various pharmacological effects. In this study, protective effect of AqGL in ameliorating muscle mass loss following acute HH has been reported. Materials and Methods: Male Sprague-Dawley rats were divided into following five groups of six rats in each group: unexposed control (Group 1), 6 hours of HH exposure (Group 2), 6 hours of HH exposure+AqGL extract 50 mg/kg body weight (BW) (Group 3), 6 hours of HH exposure+AqGL extract 100 mg/kg BW (Group 4), and 6 hours of HH exposure+AqGL extract 200 mg/kg BW (Group 5). Experimental animals from all groups, except Group, 1 were exposed to HH, simulated altitude of 25,000 ft for 6 hours. After exposure period, gastrocnemius muscle was collected, weighed, and morphological, biochemical, and molecular markers were analyzed. Results: HH-exposed rat muscle showed significant (p < 0.05) increase in oxidative stress markers (reactive oxygen species & malondialdehyde), which was concomitant with decrease in its mass compared to controls. AqGL treatment significantly (p < 0.05) prevented muscle oxidative stress, restored reduced glutathione content, reduced protein carbonyl content and advanced oxidation protein product, and restored muscle mass loss at effective dose of 100 mg/kg BW. Furthermore, AqGL supplementation enhanced Myf5 (p < 0.01), MyoD (p < 0.01), MyoG (p < 0.05), and Mrf4 (nonsignificantly), brain-derived neurotrophic factor (p < 0.01), and interleukin 6 (p < 0.01) expression along with restoration of tumor necrosis factor alpha (p < 0.001) and myostatin (p < 0.05) in hypoxia-exposed muscle, evidencing induction of myogenesis markers. Moreover, histological analysis showed increased myocyte number; nuclei shifted toward the periphery in the treatment group supporting muscle regeneration. Conclusion: AqGL supplementation attenuates muscle mass loss by preventing oxidative stress and inducing modulation in myogenesis markers under HH environment.

Effects of Extraction Temperature on Efficacy of Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes), Aqueous Extract against Oxidative Stress.

This study was designed to understand the effect of extraction temperature, i.e., room temperature (GLRT), 50°C (GL50), 100°C (hot water; GL100), and 200°C (GL200) on antioxidant and biological activity of G. lucidum. The % yield obtained was 5.3%, 7.6%, 10.7%, and 13.2% at various extraction temperatures; room temperature, 50°C, 100°C and 200°C, respectively. Similarly, phenolic content (51.6, 57.9, 82.9, and 93.1 mg/g extract) and flavonoid content (18.8, 23.2, 34.3, and 36.3 mg/g extract) were observed to be increased with rise in extraction temperature. However, extraction temperature resulted in loss of antioxidant activities above 100°C as evident by chemical assays such as DPPH, FRAP, ABTS, and TRP conducted on extracts. In contrast, three bioactive compounds, i.e., adenine (3.26, 3.48, 2.16, and 1.45 mg/g extract), uracil (3.99, 3.21, 2.51, and 1.47 mg/g extract), and adenosine (5.92, 5.62, 2.22 and 0.7 mg/g extract), quantified by high performance thin layer chromatography showed decrease in their content with increasing extraction temperature. Extract prepared at room temperature and 50°C prevented loss of cell viability and generation of reactive oxygen species resulted after hydrogen peroxide exposure; however, cytoprotective efficacy was not significant at 100°C and 200°C The order of cytoprotective effects observed by these extract were in the following order: room temperature ≥ 50°C > 100°C > 200°C. Overall, the optimal temperature conditions for the efficient extraction of G. lucidum with water retaining bioactive compounds and biological activity was found to be below 100°C.

Ganoderma lucidum aqueous extract prevents hypobaric hypoxia induced memory deficit by modulating neurotransmission, neuroplasticity and maintaining redox homeostasis.

Oxidative stress due to hypobaric hypoxia at extreme altitudes causes severe neuronal damage and irreversible cognitive loss. Owing to contraindications of current drug therapies, the aim of the study was to investigate memory enhancing potential of aqueous extract of Ganoderma lucidum (GLAQ) and underlying neuroprotective mechanism using rat hypobaric hypoxia test model. Rats exposed to hypobaric hypoxia showed deranged spatial memory in morris water maze test with hippocampal damage and vasogenic cerebral edema. All these changes were prevented with GLAQ treatment. Blood and biochemical analysis revealed activation of hypoxic ventilatory response, red blood cells induction, reversal of electrolyte and redox imbalance, and restoration of cellular bioenergetic losses in GLAQ treated animals. Notably, GLAQ treatment ameliorated levels of neurotransmitters (catecholamines, serotonin, glutamate), prevented glucocorticoid and α-synuclein surge, improved neuroplasticity by upregulating CREB/p-CREB/BDNF expression via ERK1/ERK2 induction. Further, restoration of nuclear factor erythroid 2-related factor with stabilization of hypoxia inducible factors and inflammatory markers were evidenced in GLAQ treated rats which was additionally established in gene reporter array using an alternative HT22 cell test model. Conclusively, our studies provide novel insights into systemic to molecular level protective mechanism by GLAQ in combating hypobaric hypoxia induced oxidative stress and memory impairment.

Acute exposure of 532 nm laser differentially regulates skin tissue transcription factors.

High energy laser, particularly 532 nm, is widely used in defense and medical applications and there is need to address its occupational safety. Thermal and non-thermal effects of 532 nm high energy laser on skin are cause of concern. This study indicates impact of 532 nm laser on rat skin and first of its kind of attempt to understand transcriptional activation of genes as an early response following laser exposure. Skin of experimental rats were exposed to 532 nm radiance at 0.1, 0.25 and 0.50 W/cm2 for 10 sec. Thermographic changes of skin exposed to 532 nm laser exhibited increased Tmax temperature in radiance dependent manner. After thermal imaging, skin of experimental rats was collected 1 h post laser exposure for studying differential gene expression. The skin exposed to lower power density (0.1 W/cm2) did not show significant changes in expression of gene pathways studied. At moderate radiance (0.25 W/cm2), predominantly canonical wnt/B-catenin pathway genes notch1, axin2, ccdn1, wnt5a and redox homeostasis genes; txn1, nqo1 and txnrd1 were expressed. At higher radiance (0.5 W/cm2), significant repression of genes related to wound healing process particularly notch/wnt pathway viz. hes5, wnt1, wn3b with higher expression of dab2 was recorded. The data obtained from these studies would help in drawing safety limits for skin exposure to 532 nm laser. Further, genes expressed at moderate and high level of radiance exposure to skin were distinct and differential and provide new avenue to configure pathway to counteract laser induced delay in tissue injury and hair follicular damage.

Cysteine becomes conditionally essential during hypobaric hypoxia and regulates adaptive neuro-physiological responses through CBS/H2S pathway.

Brain is well known for its disproportionate oxygen consumption and high energy-budget for optimal functioning. The decrease in oxygen supply to brain, thus, necessitates rapid activation of adaptive pathways - the absence of which manifest into vivid pathological conditions. Amongst these, oxygen sensing in glio-vascular milieu and H2S-dependent compensatory increase in cerebral blood flow (CBF) is a major adaptive response. We had recently demonstrated that the levels of H2S were significantly decreased during chronic hypobaric hypoxia (HH)-induced neuro-pathological effects. The mechanistic basis of this phenomenon, however, remained to be deciphered. We, here, describe experimental evidence for marked limitation of cysteine during HH - both in animal model as well as human volunteers ascending to high altitude. We show that the preservation of brain cysteine level, employing cysteine pro-drug (N-acetyl-L-cysteine, NAC), markedly curtailed effects of HH - not only on endogenous H2S levels but also, impairment of spatial reference memory in our animal model. We, further, present multiple lines of experimental evidence that the limitation of cysteine was causally governed by physiological propensity of brain to utilize cysteine, in cystathionine beta synthase (CBS)-dependent manner, past its endogenous replenishment potential. Notably, decrease in the levels of brain cysteine manifested despite positive effect (up-regulation) of HH on endogenous cysteine maintenance pathways and thus, qualifying cysteine as a conditionally essential nutrient (CEN) during HH. In brief, our data supports an adaptive, physiological role of CBS-mediated cysteine-utilization pathway - activated to increase endogenous levels of H2S - for optimal responses of brain to hypobaric hypoxia.

Pharmacological effects of Ganoderma lucidum extract against high-altitude stressors and its subchronic toxicity assessment.

Acclimatization is a major pathophysiological concern during ascent to high altitude and may cause mortality in unacclimatized individuals. Absence of target drugs, especially prophylactics, emphasizes the need for development of herbal agents. Present study revealed that animals pre-administered with aqueous extract of Ganoderma lucidum (GLAQ) dose dependently (50, 100, 200 mg/kg) delayed onset of convulsion following severe hypoxia (SH) and restored rectal temperature post-cold restraint (CR) and hypobaric hypoxia (HBH). The compromised antioxidant status (MDA, GSH, SOD, GPx), biochemical (ALT, AST, glucose, triglycerides, cholesterol, urea), and hematological parameters (red blood cells, white blood cells) were ameliorated with GLAQ treatment. Further, extract modulated inflammatory and thermogenic response by attenuating pro-inflammatory cytokines (NFĸB, TNFα, IL6) and restoring UCP1, SIRT1, respectively. Notably, extract did not produce any noxious effects subchronically in rats of both sexes with GLAQ administered at 100, 500, and 1,000 mg/kg in a single dose/day for 90 days, deeming it fit for therapeutic purpose. PRACTICAL APPLICATIONS: GLAQ exhibited better efficacy compared to internal control (gallic acid) suggest that array of bioactive compounds in extract might contribute toward efficacy. Further, antistress properties of GLAQ against multiple stressors including SH, CR, and HBH demonstrate its therapeutic potential for inducing rapid acclimatization and preventing mountain sickness. Conclusively, the present study based on Ganoderma lucidum extract intents to fill the lacunae behind development of nontoxic therapeutic agent for controlling high altitude-related maladies.

p38 MAPK pathway-dependent SUMOylation of Elk-1 and phosphorylation of PIAS2 correlate with the downregulation of Elk-1 activity in heat-stressed HeLa cells.

Stress-activated and mitogen-activated protein kinases (MAPKs) regulate gene expression by post-translational modifications of transcription factors. Elk-1, a transcription factor that regulates the expression of immediate early genes, is amenable to regulation by all the three mammalian MAPKs. In the present report, using inhibitors specific for different MAPK pathways, we show that during exposure of HeLa cells to heat stress, Elk-1 is SUMOylated with SUMO1 by p38 MAPK pathway-dependent mechanisms. Elk-1-phosphorylation levels were significantly reduced under similar conditions. We also show that transcriptional activity of Elk-1 as assessed by luciferase reporter expression and qPCR estimation of the expression of genes regulated by Elk-1 was downregulated upon exposure to heat stress; this downregulation was reversed when heat exposure was performed in the presence of either SB203580 (p38 MAPK inhibitor) or ginkgolic acid (inhibitor of SUMOylation). Elk-1 induced transcription is also regulated by PIAS2 which acts as a coactivator upon the activation of extracellular signal-regulated kinases (ERKs) and as a corepressor upon its phosphorylation by p38 MAPK. Since heat stress activates the p38 MAPK pathway, we determined if PIAS2 was phosphorylated in heat-stressed HeLa cells. Our studies indicate that in HeLa cells exposed to heat stress, PIAS2 is phosphorylated by p38 MAPK pathway-dependent mechanisms. Collectively, the results presented demonstrate that in heat-stressed HeLa cells, p38 MAPK pathway-dependent SUMOylation of Elk-1 and phosphorylation of PIAS2 correlate with the downregulation of transactivation by Elk-1.

Role of Hydroalcoholic Extract of Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes), in Facilitating Cellular Acclimatization in a Low-Oxygen Microenvironment.

Ganoderma lucidum is known to exert many health benefits including effects to improve oxygen utilization. Therefore, this study was designed to evaluate the role of a hydroalcoholic G. lucidum extract in providing tolerance to HT22 cells grown under hypoxic conditions. HT22 cells were exposed to 0.5% O2 in the presence or absence of the extract for 24 hours. At the end of the exposure period, we performed cell viability assays, cell cycle analysis, and biochemical and protein expression studies. The extract-treated cells revealed less cell death, minimized caspase 3 and reactive oxygen species levels, and relieved G0/G1 cell cycle arrest compared with hypoxic cells cultured without the extract. Further, extract-treated cells showed improved expression of Nrf2, heme oxygenase 1, and metallothionein and stabilized levels of hypoxia-inducible factor 1α. Moreover, lower levels of nuclear factor-κB and tumor necrosis factor a were evident in extract-treated cells. Overall, the G. lucidum extract reduced hypoxia-induced cell death and augmented transcription factors (HIF-1α and Nrf2), conferring tolerance to hypoxia.

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-1ß, 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.

Screening of Indian Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes): A UPC2-SQD-MS Approach.

Oriental medicinal mushroom Ganoderma lucidum has been widely used for the promotion of health and longevity owing to its various bioactive constituents. Therefore, comprehending metabolomics of different G. lucidum parts could be of paramount importance for investigating their pharmacological properties. Ultra-performance convergence chromatography (UPC2) along with mass spectrometry (MS) is an emerging technique that has not yet been applied for metabolite profiling of G. lucidum. This study has been undertaken to establish metabolomics of the aqueous extracts of mycelium (GLM), fruiting body (GLF), and their mixture (GLMF) using ultra-performance convergence chromatography single quadrupole mass spectrometry (UPC2-SQD-MS). Aqueous extracts of G. lucidum prepared using an accelerated solvent extraction technique have been characterized for their mycochemical activities in terms of total flavonoid content, 1,1-diphenyl-2-picryl-hydrazyl scavenging activity, and ferric ion reducing antioxidant power. The UPC2-SQD-MS technique has been used for the first time for metabolite profiling of G. lucidum on a Princeton Diol column (4.6 × 250 mm; 5 µm) using supercritical CO2 (solvent) and 20 mM ammonium acetate in methanol (co-solvent). In the present study, UPC2-SQD-MS was found to be a rapid, efficient, and high-throughput analytical technique, whose coupling to principal component analysis (PCA) and phytochemical evaluation could be used as a powerful tool for elucidating metabolite diversity between mycelium and fruiting body of G. lucidum. PCA showed a clear distinction in the metabolite compositions of the samples. Mycochemical studies revealed that overall GLF possessed better antioxidant properties among the aqueous extracts of G. lucidum.

Chromatographic analysis of wheatgrass extracts.

AIM: Wheatgrass (WG) is the shoot of Triticum aestivum Linn. belongs to the family Gramineae, and possess high chlorophyll content and essential vitamins, minerals, vital enzymes, amino acids, dietary fibers etc., It has been shown to possess anti-cancer, anti-ulcer, antioxidant, and anti-arthritic activity due to the presence of biologically active compounds, and minerals. Therefore, in the present study, high-performance thin layer chromatography (HPTLC), and high-performance liquid chromatography (HPLC) methods for qualitative and quantitative analysis have been proposed, which will help in quality evaluation of wheat grass extract. MATERIALS AND METHODS: Samples for analysis were prepared in methanol and water simply by sonication. These were applied on pre-coated silica plate and chromatograms were developed using toluene: Ethyl acetate: Formic acid. HPLC analysis was done on Waters HPLC system using water, methanol, and acetonitrile as mobile phase. Merck C18 column has been used. RESULTS: HPTLC finger printing of alcoholic extracts of WG was carried out and found 10-11 spots at different wavelengths 254, 366, and 435 nm. HPLC fingerprinting produced 22 peaks at 256 nm. Quantitative HPTLC analysis was done to determine the gallic acid content, and was found to be 0.077% w/w in aqueous extract. By HPLC, the content of gallic acid and rutin was found to be 0.07%, and 0.04% w/w in aqueous extract of WG. CONCLUSION: The developed HPLC and HPTLC fingerprinting method can be used for the quality control, and standardization of WG and its extracts used as nutritional supplement.

Determination of nucleosides in Cordyceps sinensis and Ganoderma lucidum by high performance liquid chromatography method.

BACKGROUND: Nucleosides are supportive in the regulation and modulation of various physiological processes in body, they acts as precursors in nucleic acid synthesis, enhance immune response, help in absorption of iron and influence the metabolism of fatty acids. Cordyceps sinensis and Ganoderma lucidum are well-known for its use in traditional medicine of China, Nepal and India. They are rich in nucleosides such as adenine, adenosine, cordycepin, etc. Hence, a simple, economic and accurate high-performance liquid chromatography (HPLC) analytical method was proposed for determination of adenine and adenosine for the quality control of plants. MATERIALS AND METHODS: Chromatographic experiments were conducted on YL9100 HPLC system (South Korea). Reversed-phase chromatography was performed on a C18 column with methanol and dihydrogen phosphate as the mobile phase in isocratic elution method at a flow rate of 1.0 mL/min. Detection was carried out at 254 nm, which gives a sharp peak of adenine and adenosine at a retention time of 6.53 ± 0.02 min and 12.41 ± 0.02, respectively. RESULTS AND DISCUSSION: Linear regression analysis data for the calibration plot showed a good linear relationship between response and concentration in the range of 25-200 µg/mL for adenosine and 100-800 µg/mL for adenine with regression coefficient of 0.999 and 0.996, respectively. The adenine was found 0.16% and 0.71% w/w in G. lucidum and in C. sinensis, respectively, and adenosine was found to be 0.14% w/w in G. lucidum whereas absent in C. sinensis. CONCLUSION: The developed HPLC method for the quantification of adenosine and adenine can be used for the quality control and standardization of crude drug and for the different herbal formulations, in which adenine and adenosine are present as major constituents. The wide linearity range, sensitivity, accuracy, and simple mobile phase imply the method is suitable for routine quantification of adenosine and adenine with high precision and accuracy.

Protective Efficacy of the Caterpillar Mushroom, Ophiocordyceps sinensis (Ascomycetes), from India in Neuronal Hippocampal Cells against Hypoxia.

This study demonstrated the protective efficiency of extracts of the Indian variety of Ophiocordyceps sinensis (=Cordyceps sinensis) (CSEs) in HT22 (murine hippocampal) cells under hypoxic conditions. Various parameters such as cell viability, reactive oxygen species, levels of endogenous antioxidants, inflammatory cytokines, transcription factors, and oxidation of macromolecules were analyzed. In addition, the radical scavenging abilities of hydroxyl radicals, nitric oxide, and superoxide radicals were also studied. Antioxidant compounds, ascorbic acid, hesperidin, and rutin were quantified by high-performance thin-layer chromatography. The information acquired from high-performance thin-layer chromatography profiling was subjected to principal component analysis for data clustering. Findings of this research revealed that ascorbic acid and rutin were highest in aqueous CSE, whereas the maximum amount of hesperidin was found in 25% alcoholic CSE. In vitro studies showed that all the CSEs protected HT22 cells well by upregulating the level of endogenous antioxidants and preventing the oxidation of lipids and proteins. These extracts also reduced the amount of hypoxia-induced inflammatory cytokines and transcription factors on par with the normoxic control with more or less equal protection in the cells under hypoxia, and indicated significant radical scavenging potential.

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.

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.

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 NF κ B (nuclear factor kappaB) and tumor necrosis factor- α observed which might be due to higher levels of HO1, MT and transforming growth factor- ß . 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, NF κ B 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 NF κ B levels. These findings provide a basis for possible use of Cordyceps sinensis in tolerating hypoxia.

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.