Simultaneous Estimation of Quercetin and trans-Resveratrol in Cissus quadrangularis Extract in Rat Serum Using Validated LC-MS/MS Method: Application to Pharmacokinetic and Stability Studies.

Cissus quadrangularis is a nutrient-rich plant with a history of use in traditional medicine. It boasts a diverse range of polyphenols, including quercetin, resveratrol, ß-sitosterol, myricetin, and other compounds. We developed and validated a sensitive LC-MS/MS method to quantify quercetin and t-res biomarkers in rat serum and applied this method to pharmacokinetic and stability studies. The mass spectrometer was set to negative ionization mode for the quantification of quercetin and t-res. Phenomenex Luna (C18(2), 100 A, 75 × 4.6 mm, 3 µ) column was utilized to separate the analytes using an isocratic mobile phase consisting of methanol and 0.1% formic acid in water (82:18). Validation of the method was performed using various parameters, including linearity, specificity, accuracy, stability, intra-day, inter-day precision, and the matrix effect. There was no observed significant endogenous interference from the blank serum. The analysis was completed within 5.0 min for each run, and the lower limit of quantification was 5 ng/mL. The calibration curves showed a linear range with a high correlation coefficient (r2 > 0.99). The precision for intra- and inter-day assays showed relative standard deviations from 3.32% to 8.86% and 4.35% to 9.61%, respectively. The analytes in rat serum were stable during bench-top, freeze-thaw, and autosampler (-4 °C) stability studies. After oral administration, the analytes showed rapid absorption but underwent metabolism in rat liver microsomes despite being stable in simulated gastric and intestinal fluids. Intragastric administration resulted in higher absorption of quercetin and t-res, with greater Cmax, shorter half-life, and improved elimination. No prior research has been conducted on the oral pharmacokinetics and stability of anti-diabetic compounds in the Ethanolic extract of Cissus quadrangularis EECQ, making this the first report. Our findings can provide the knowledge of EECQ's bioanalysis and pharmacokinetic properties which is useful for future clinical trials.

Pancreastatin deteriorates hepatic lipid metabolism via elevating fetuin B in ovariectomized rats.

Hepatic steatosis is an important mstetabolic complication in women encountering postmenopausal phase of life. Pancreastatin (PST), has previously been investigated in diabetic and insulin resistant rodents. The present study highlighted the role of PST in ovariectomized rats. Female SD rats were ovariectomized and subsequently fed high fructose diet for 12 weeks. PST inhibitor peptide was intraperitoneally administered for 14 days and further examined for insulin resistance, glucose intolerance development, body mass composition, lipid profile detection and hepatic fibrosis. Gut microbial alterations has also been investigated. Results showed development of glucose intolerance in high fructose fed ovariectomized rats with reduced level of reproductive hormones including estradiol and progesterone. Enhanced lipid production was detected in these rats as they showed increased triglycerides, lipid accumulation in liver tissue (determined by HE staining, Oil Red O staining, Nile Red staining). Sirius Red and Masson's trichome analysis depicted positive results for fibrosis development. We also found gut microbiota alterations in fecal samples of these rats. Furthermore, PST inhibition decreased the expression of hepatic Fetuin B and resumed gut microbial diversity. PST deregulates hepatic lipid metabolism which leads to altered expression of Fetuin B in liver and gut dysbiosis in postmenopausal rats.

Evaluation of the Pharmacokinetics of the Pancreastatin Inhibitor PSTi8 Peptide in Rats: Integration of In Vitro and In Vivo Findings.

PSTi8 is a pancreastatin inhibitory peptide that is effective in the treatment of diabetic models. This study investigates the pharmacokinetic (PK) properties of PSTi8 in Sprague Dawley rats, for the first time. In vitro and in vivo PK studies were performed to evaluate the solubility, stability in plasma and liver microsomes, plasma protein binding, blood-plasma partitioning, bioavailability, dose proportionality, and gender difference in PK. Samples were analyzed using the validated LC-MS/MS method. The solubility of PSTi8 was found to be 9.30 and 25.75 mg/mL in simulated gastric and intestinal fluids, respectively. The protein binding of PSTi8 was estimated as >69% in rat plasma. PSTi8 showed high stability in rat plasma and liver microsomes and the blood-plasma partitioning was >2. The bioavailability of PSTi8 after intraperitoneal and subcutaneous administration was found to be 95.00 ± 12.15 and 78.47 ± 17.72%, respectively, in rats. PSTi8 showed non-linear PK in dose proportionality studies, and has no gender difference in the PK behavior in rats. The high bioavailability of PSTi8 can be due to high water solubility and plasma protein binding, low clearance and volume of distribution. Our in vitro and in vivo findings support the development of PSTi8 as an antidiabetic agent.

Pancreastatin induces hepatic steatosis in type 2 diabetes by impeding mitochondrial functioning.

AIMS: Mitochondrial dysfunction is among the key factors for the advancement of hepatic steatosis to NAFLD and NASH. Pancreastatin (PST: human ChgA250-301) is a dysglycemic hormone, previously reported to promote steatosis and inflammation in various animal models of metabolic disorders. Recently, we observed PST deregulates energy expenditure and mitochondrial functioning in perimenopausal rats. In the current study, we aimed to decipher the role of PST instigated altered mitochondrial functioning in hepatic steatosis. MAIN METHODS: The HepG2 cells were PST exposed and the Chga gene was knocked down using siRNA and lipofectamine. Parallelly, type 2 diabetes (T2D) was developed in C57BL/6 mice by HFD feeding and administered PST inhibitor (PSTi8). KEY FINDINGS: The PST exposed cells and HFD fed mice depicted: enhanced CHGA expression detected by IF/IHC, WB, and ELISA; dysregulated cellular ROS, mitochondrial ROS, oxygen consumption rate, mitochondrial membrane potential, ATP level, and NADP/NADP ratio; enhanced apoptosis determined by MTT, TUNEL, Annexin-V FITC, and WB of Bax/bcl2 and caspase 3; hepatic lipid accumulation upon Nile Red, Oil Red O, H&E staining, and the expression of SREBP-1c, FAS, ACC, and SCD; inflammation based on expression and circulatory level of IL6, IL-1ß, and TNF-α. However, Chga knocked down HepG2 cells and PSTi8 treated mice unveiled protection from all the above abnormalities. SIGNIFICANCE: Collectively, the aforementioned data suggested the alteration in mitochondrial function induced by PST is responsible for hepatic steatosis in T2D.

Pancreastatin mediated regulation of UCP-1 and energy expenditure in high fructose fed perimenopausal rats.

AIMS: Pancreastatin (PST) is a crucial bioactive peptide derived from chromogranin A (CHGA) proprotein that exhibits an anti-insulin effect on adipocytes. Herein, we investigated the effects of PST on brown adipose tissues (BAT) and white adipose tissue (WAT) in connection with uncoupling protein-1 (UCP-1) regulated energy expenditure in high fructose diet (HFrD) fed and vinylcyclohexenediepoxide (VCD) induced perimenopausal rats. MATERIAL AND METHODS: We administered VCD in rats for 17 consecutive days and fed HFrd for 12 weeks. After 12 weeks estradiol and progesterone levels were detected. Furthermore, detection of glucose tolerance, insulin sensitivity, and body composition revealed impaired glucose homeostasis and enhanced PST levels. Effects of enhanced PST on UCP-1 level in BAT and WAT of perimenopausal rats were further investigated. KEY FINDINGS: Reduced serum estradiol, progesterone, and attenuated insulin response confirmed perimenopausal model development. Furthermore, enhanced PST serum level and its increased expression in BAT and WAT downregulated the UCP-1 expression. Subsequently, impaired ATP level, NADP/NADPH ratio, citrate synthase activity, enhanced mitochondrial reactive oxygen species (ROS) generation and perturbed mitochondrial membrane potential, further exacerbated mitochondrial dysfunction, cellular ROS production, and promoted apoptosis. Interestingly, PST inhibition by PST inhibitor peptide-8 (PSTi8) displayed a favorable impact on UCP-1 and energy expenditure. SIGNIFICANCE: The aforementioned outcomes indicated the substantial role of PST in altering the UCP-1 expression and associated energy homeostasis. Hence our results corroborate novel avenues to unravel the quest deciphering PST's role in energy homeostasis and its association with perimenopause.

Pancreastatin induces islet amyloid peptide aggregation in the pancreas, liver, and skeletal muscle: An implication for type 2 diabetes.

Recent findings suggest that the accumulation of misfolded aggregates of islet amyloid peptide (IAPP) plays an essential role in pancreatic damage and type 2 diabetes (T2D). Pancreastatin (PST), a chromogranin derived peptide, instigates insulin resistance (IR) and promotes T2D. Here, we aimed to investigate whether PST induces IAPP aggregation in the pancreas, liver, and skeletal muscles. Foremost, we unraveled kinetics of fibril formation by ThT kinetic assay, ANS binding, turbidity, and far UV-CD. Subsequently, we checked the microarchitecture of fibril by TEM. Moreover, the PST action on IAPP expression was examined by immunocytochemistry, immunohistochemistry, western blotting, and real-time PCR. The outcome of spectral analysis and TEM demonstrated the fibril formation in the alone IAPP group but not in the alone PST; however, PST with IAPP produced stronger fibril. Moreover, PST was found to stimulate IAPP aggregation and expression more prominently in PANC1 and HepG2 cells, and pancreas and liver tissues than in L6 and skeletal muscle. Subsequently, pancreastatin inhibitor manifested a decline in the extent of the IAPP fibril formation and its expression. To conclude, PST upon combination induces the aggregation of IAPP in the pancreas, liver, and skeletal muscle, which may have the potential to generate IR and cause T2D.

Withania somnifera in Neurological Disorders: Ethnopharmacological Evidence, Mechanism of Action and its Progress in Delivery Systems.

BACKGROUND: The underlying cause of major neurodegenerative disorders remains a healthcare mystery. The thoroughly investigated causes include oxidative stress, inflammation, environmental factor, mitochondrial dysfunction, and irregular neuronal protein aggregation. Withania somnifera has been used for more than 2500 years as a useful medicinal plant to improve disease defense, prevent aging, rejuvenate the body in a vulnerable situation, and generate a feeling of mental well-being. However, a persuasive paper emphasizing its neuroprotective nature is missing. OBJECTIVE: In the current review, we have delineated the protective role of W. somnifera against various neurological disorders and its progress in delivery systems. METHODS: The database used in the retrieval of data were PubMed, Scopus, Science direct, and SciFinder. The keywords used were W. somnifera, Ashwagandha, neuroprotective activities, etc. The principal source of the data retrieval includes research articles, review papers, and short communications from reputed publishers, including the New England Journal of Medicine, Elsevier, Nature, Springer, and Taylor & Francis. RESULTS: After an extensive literature review, we found that W. somnifera mitigates various neurological disorders, including Parkinson's disease, Alzheimer's disease, Huntington disease, tardive dyskinesia, stroke, and anxiety. Furthermore, natural compounds in nano sizes range possess better neuroprotective activity. Consequently, polymeric nanomicelles, nanoparticles, and nanofibers of natural products are used in the treatment of neurodegenerative diseases. CONCLUSION: The current review substantially deciphered the protective role of W. somnifera against various neurological disorders. However, future studies are further required better to understand the molecular mechanisms behind their neuroprotective nature.

Combination of Pancreastatin inhibitor PSTi8 with metformin inhibits Fetuin-A in type 2 diabetic mice.

In the preceding study, we delineated that high-fat diet (HFD) consumption in mice increases the circulatory level of pancreastatin (PST), which additionally enhances the free fatty acid (FFA) concentration in circulation. Consequently, the aggravated FFA activates Fetuin-A, which facilitates hepatic lipid accumulation, insulin resistance (IR), and culminates in type 2 diabetes (T2D). Metformin (Met) is a widely known first-line drug for the treatment of T2D. We previously unveiled PSTi8, an inhibitor of PST, comprising antidiabetic property. Hence, we hypothesized that combination therapy of Met and PSTi8, at reduced therapeutic doses, would mitigate HFD-induced IR by inhibiting hepatic Fetuin-A in mice model of T2D. C57BL/6 mice were fed HFD for 12 weeks, followed by treatment with Met, PSTi8, and its combination for 10 days. Glucose and insulin tolerance tests were conducted. Circulatory levels of PST, Fetuin-A, and lipid markers were determined. Also, the mRNA and protein expression of Fetuin-A was assessed by qPCR, western blotting, and immunofluorescence. Moreover, the energy expenditure was measured by comprehensive laboratory animal monitoring system (CLAMS). Combination therapy displayed improved PST, Fetuin-A, and lipid profile in plasma. We also found reduced hepatic Fetuin-A, which reduced inhibitory phosphorylation of IRS and increased phosphorylation of AKT. Consequently, ameliorated hepatic lipogenesis, gluconeogenesis, and inflammation. Also, combination treatment attenuated Fetuin-A expression, lipid accumulation, and glucose production in palmitate-induced HepG2 cells. Altogether current study promulgates the beneficial effect of combination therapy of Met and PSTi8 (comparable to alone higher therapeutic doses) to ameliorate Fetuin-A activation, hepatic lipid accumulation, insulin resistance, and associated progressive pathophysiological alterations in T2D.

Pancreastatin inhibitor PSTi8 protects the obesity associated skeletal muscle insulin resistance in diet induced streptozotocin-treated diabetic mice.

Pancreastatin (PST), a chromogranin A (CHGA) derived peptide connects obesity with insulin resistance by inducing inflammation. Previously, we have evaluated potential activity of PST inhibitor (PSTi8) in liver and adipose tissue in type 2 diabetic mice model. In this study we further explore the therapeutic effect of PSTi8 on glucose metabolism in skeletal muscle cells/tissue and its effect on energy homeostasis in diet induced diabetic mice model. In in-vitro studies, we found that PSTi8 increases glucose uptake via enhanced GLUT4 translocation in L6 cells. This positive effect of PSTi8 led us to proceed with in-vivo studies in diabetic mice. C57BL/6 mice were fed HFD or HFrD diet for 12 weeks along with single STZ induction at 4th week followed by PSTi8 treatment. We found that HFD and HFrD model showed increased fat mass, caused glucose intolerance and insulin resistance, with accompanying proinflammatory effect on epididymal white adipose tissue (eWAT) together leading to skeletal muscle insulin resistance. Administration of PSTi8 protects from diet induced inflammatory response and enhances glucose tolerance and insulin sensitivity. PSTi8 improves circulating adipokine and lipid parameters, along with switch in macrophage polarisation from M1 to M2 in stromal vascular fraction of adipose tissue. In addition, treatment of PSTi8 also improves energy homeostasis, decreases circulatory non-esterified fatty acids level and inhibits ceramide deposition in muscle tissue. Overall this increased muscle insulin sensitivity is mediated via AKT/AS160/GLUT4 pathway activation. Our results reveal that PSTi8 inhibits the obesity mediated inflammation which enhances glucose disposal in skeletal muscle.

LC-ESI-MS/MS assay development and validation of a novel antidiabetic peptide PSTi8 in mice plasma using SPE: An application to pharmacokinetics.

PSTi8 is a 21 amino acid pancreastatin inhibitory peptide that demonstrated potent antidiabetic activity in insulin resistant rodent models. The goal of the current work is to establish and validate the LC-ESI-MS/MS bioanalytical assay of PSTi8 in mice plasma in order to unveil its pharmacokinetic (PK) behaviour for the first time. The MS detection of PSTi8 and diprotin A (internal standard, IS) was conducted with Q1/Q3 SRM transitions at 607.80 ([M+4 H]4+)/771.20 and 342.20/229.10, respectively using positive ESI. Phenomenex Aqua 5µ 125A (250 × 4.6 mm) column was utilized to separate PSTi8 and IS with a mobile phase consists of MeOH-0.1 % formic acid (1:1, v/v) using 0.4 mL/min flow rate. SPE using medium anion exchange cartridge (Oasis MAX) was used for the extraction of analyte and IS from the mice plasma and the extraction recovery was found to be >55 %. PSTi8 displayed good linearity across the 5-1000 ng/mL concentrations range. The intra- and inter- day accuracy was observed between 99.44-110.20 % and 99.66-110.93 %, respectively. The intra- and inter- day precision was observed between 2.61-4.03 % and 2.90-7.16 %, respectively. The intra-day and inter-day accuracy and precision data was within the 100 ±â€¯15 % nominal values recommended by the United States Food and Drug Administration bioanalytical guidance. The LC-MS/MS assay was validated effectively to investigate the PSTi8 plasma concentrations following intravenous and intraperitoneal PK studies in mice. The absolute bioavailability of PSTi8 was 52.74 ±â€¯13.50 %.

PSTi8 with metformin ameliorates perimenopause induced steatohepatitis associated ER stress by regulating SIRT-1/SREBP-1c axis.

AIMS: Hepatic steatosis in women confronting menopause is the manifestation of substantial fructose consumption and forms a positive feedback loop to develop endoplasmic reticulum (ER) stress. Previously pancreastatin inhibitor peptide-8 (PSTi8) and Metformin (Met) combination effectively ameliorated hepatic lipid accumulation in high fructose diet (HFrD) fed diabetic mice models at reduced doses. Moreover, SIRT-1 plays a crucial role in the regulation of SREBP-1c. Hence we hypothesized that Met and PSTi8 in combination (at therapeutic lower doses) could mitigate hepatic steatosis linked ER stress by activating SIRT-1 and precluding SREBP-1c in HFrD fed 4-Vinylcyclohexenediepoxide (HVCD) induced perimenopausal rats. MAIN METHODS: HVCD rats were fed HFrD for 12 weeks, accompanied by 14 days of treatment with Met, PSTi8, and combination. We confirmed model establishment by estrus cycle study, estradiol level, and intraperitoneal glucose tolerance test. Plasma lipid profile and liver function were determined. Also, mRNA and protein expressions were examined. Moreover, distribution of SIRT-1 and SREBP-1c was detected in HepG2 cells by immunofluorescence staining. KEY FINDINGS: HVCD group displayed augmented insulin resistance (IR), lipogenesis, and ER stress in the liver. Combination therapy improved the estrus cyclicity, estradiol, and lipid profile of HVCD rats. Met and PSTi8 combination reduced hepatic SREBP-1c and triggered SIRT-1 expression in high fructose-induced insulin-resistant HepG2 cells; consequently, combination therapy attenuated ER stress. SIGNIFICANCE: Succinctly, present research promotes impetus concerning the remedial impact of Met with PSTi8 at lower therapeutic doses to ameliorate hepatic IR, steatosis, and associated ER stress by revamping the SIRT-1/SREBP-1c axis in perimenopausal rats.

Pancreastatin inhibitor PSTi8 attenuates hyperinsulinemia induced obesity and inflammation mediated insulin resistance via MAPK/NOX3-JNK pathway.

Pancreastatin (PST), a chromogranin A derived peptide has anti-insulin effects and plays a significant role in obesity-induced insulin resistance. In obesity and type 2 diabetes mellitus, both insulin and PST level are elevated, but it is not clearly understood how anti-insulin effect of PST get regulated in hyperinsulinemic state. Simultaneously we have explored pancreastatin inhibitor PSTi8 against the native PST in the same hyperinsulinemic state. In in-vitro studies, we found that PST treatment increases lipid droplets and reactive oxygen species production in 3T3L1 adipocyte cells and theses effects of PST was found synergistic with chronic-insulin treatment. Treatment of PSTi8 in 3T3L1 adipocytes attenuates PST effect on lipid droplet formation and reactive oxygen species production. We further validated these findings in epididymal white adipose tissue of C57BL/6 mice, implanted with mini-osmotic insulin pump with and without PSTi8 for 4 weeks. We found that chronic hyperinsulinemia enhanced PST levels in circulation which in turn induces expression of various pro-inflammatory cytokines and oxidative stress. In addition, it also stimulated the expression of lipogenic genes, fat mass and body weight gain through the regulation of circulating adiponectin level. The change in PST mediated inflammatory and lipogenic parameters were attenuated by PSTi8 treatment, leading to enhanced insulin sensitivity and improved glucose homeostasis. PSTi8 rescue from PST mediated insulin resistance in adipose via inhibition of MAPK and NOX3-JNK stress signalling pathway which stimulates GLUT4 expression through activation of AKT-AS160 pathway. Thus PSTi8 may be a novel therapeutic agent for the treatment of hyperinsulinemia induced obesity and inflammation mediated insulin resistance.

Pancreastatin inhibitor, PSTi8 ameliorates metabolic health by modulating AKT/GSK-3ß and PKCλ/ζ/SREBP1c pathways in high fat diet induced insulin resistance in peri-/post-menopausal rats.

Increase in the prevalence of insulin resistance (IR) in peri-/post-menopause women is mainly due to hormone deficiency and lifestyle. PSTi8 (PEGKGEQEHSQQKEEEEEMAV-amide) is a pancreastatin inhibitor peptide which showed potent antidiabetic activity in genetic and lifestyle induced type 2 diabetic mice. In the present work, we have investigated the antidiabetic activity of PSTi8 in rat models of peri-/post-menopausal IR. 4-vinylcyclohexenediepoxide treated and ovariectomized rats were fed with high fat diet for 12 weeks to develop the peri-/post-menopausal IR. PSTi8 peptide was administered after the development of peri-/post-menopausal IR rats. PSTi8 (1 mg/kg, i.p) improved the glucose homeostasis which is characterized by elevated glycogenesis, enhanced glycolysis and reduced gluconeogenesis. PSTi8 suppressed palmitate- and PST- induced IR in HepG2 cells. PSTi8 treatment enhanced energy expenditure in peri-/post-menopausal IR rats. PSTi8 treatment increased insulin sensitivity in peri-/post-menopausal IR rats, may be mediated by modulating IRS1-2-phosphatidylinositol-3-kinase-AKT-GSK3ß and IRS1-2-phosphatidylinositol-3-kinase-PKCλ/ζ-SREBP1c signaling pathways in the liver. PSTi8 can act as a potential therapeutic peptide for the treatment of peri-/post-menopausal IR.

Pancreastatin inhibitor activates AMPK pathway via GRP78 and ameliorates dexamethasone induced fatty liver disease in C57BL/6 mice.

AIMS: To investigate the role of pancreastatin inhibitor (PSTi8) in lipid homeostasis and insulin sensitivity in dexamethasone induced fatty liver disease associated type 2 diabetes. MAIN METHODS: Glucose releases assay, lipid O staining and ATP/AMP ratio were performed in HepG2 cells. Twenty four mice were randomly divided into 4 groups: Control group (saline), DEX (1 mg/kg, im) for 17 days, DEX+PSTi8 (acute 5 mg/kg and chronic 2 mg/kg, ip) for 10 days. The glucose, insulin and pyruvate tolerance tests (GTT, ITT and PTT), biochemical parameters and Oxymax-CLAMS were performed. Further to elucidate the action mechanisms of PSTi8, we performed genes expression and western blotting of biological samples. KEY FINDINGS: We found that PSTi8 suppresses hepatic glucose release, lipid deposition, oxidative stress induced by DEX, stimulates the cellular energy level in hepatocytes and enhances GRP78 activity. It reduces lipogensis and enhances fatty acid oxidation to improve insulin sensitivity and glucose tolerance in DEX induced diabetic mice. The above cellular effects are the result of activated AMPK signalling pathway in liver, which increases Srebp1c and ACC phosphorylation. The increased ACC phosphorylation suppresses protein kinase C activity and enhances insulin sensitivity. The increased expression of UCP3 in liver elicits fatty acid oxidation and energy expenditure, which suppress oxidative stress. SIGNIFICANCE: Thus the activation of AMPK signalling through GRP78, improves lipid homeostasis, enhances insulin sensitivity via inhibition of PKC activity. PSTi8 suppresses inflammation associated with incomplete fatty acid oxidation. Hence, PSTi8 may be a potential therapeutic agent to treat glucocorticoid-induced fatty liver associated type 2 diabetes.

Determination of permeability, plasma protein binding, blood partitioning, pharmacokinetics and tissue distribution of Withanolide A in rats: A neuroprotective steroidal lactone.

Preclinical Research & Development Withanolide A (WA), a steroidal lactone is a major bioactive constituent of Withania somnifera (L.) with remarkable neuropharmacological activity. In this study, we investigated the permeability, plasma protein binding (PPB), blood partitioning, intravenous (i.v.), and oral pharmacokinetics as well as i.v. tissue distribution (TD) of pure WA in a rat model. The PPB, RBCs partitioning, and permeability of WA were determined by Ultra Performance Liquid Chromatography (UPLC) method. However, the pharmacokinetics and TD of WA were evaluated by validated and sensitive liquid chromatography coupled mass spectrometry (LC-ESI-MS/MS) method. The PPB and permeability of WA were determined by equilibrium dialysis and parallel artificial membrane permeability assay method, respectively. The results demonstrated that WA has high PPB and passive permeability. Furthermore, WA was found to have fast equilibration between RBCs and plasma. Following i.v. (2 mg/kg) and per-oral (25 mg/kg) administration of WA, the max concentration (Cmax ) in plasma was found as 85.53 ± 6.54 and 48.04 ±5.78 ng/mL, respectively. The TD study results indicated that WA has a rapid and wide TD. The maximum concentration in various tissues was found in following order: Clung > Cliver > Ckidney ≈ Cspleen > Cheart > Cbrain . The preclinical in vitro, as well as pharmacokinetics and TD results, are anticipated to support the future preclinical and clinical application of WA.

Discovery of pancreastatin inhibitor PSTi8 for the treatment of insulin resistance and diabetes: studies in rodent models of diabetes mellitus.

Pancreastatin (PST) is an endogenous peptide which regulates glucose and lipid metabolism in liver and adipose tissues. In type 2 diabetic patients, PST level is high and plays a crucial role in the negative regulation of insulin sensitivity. Novel therapeutic agents are needed to treat the diabetes and insulin resistance (IR) against the PST action. In this regard, we have investigated the PST inhibitor peptide-8 (PSTi8) action against diabetogenic PST. PSTi8 rescued PST-induced IR in HepG2 and 3T3L1 cells. PSTi8 increases the GLUT4 translocation to cell surface to promote glucose uptake in L6-GLUT4myc cells. PSTi8 treatment showed an increase in insulin sensitivity in db/db, high fat and fructose fed streptozotocin (STZ) induced IR mice. PSTi8 improved the glucose homeostasis which is comparable to metformin in diabetic mice, characterized by elevated glucose clearance, enhanced glycogenesis, enhanced glycolysis and reduced gluconeogenesis. PST and PSTi8 both were docked to the GRP78 inhibitor binding site in protein-protein docking, GRP78 expression and its ATPase activity studies. The mechanism of action of PSTi8 may be mediated by activating IRS1/2-phosphatidylinositol-3-kinase-AKT (FoxO1, Srebp-1c) signaling pathway. The discovery of PSTi8 provides a promising therapeutic agent for the treatment of metabolic diseases mainly diabetes.

Evaluation of anti-hypertensive activity of Ulmus wallichiana extract and fraction in SHR, DOCA-salt- and L-NAME-induced hypertensive rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Ulmus wallichiana Planchon (Himalayan Elm), a traditional medicinal plant, used in fracture healing in folk tradition of Uttarakhand, Himalaya, India. It is also used as diuretic. U. rhynchophylla, native to China, known as Gou Teng in Chinese medicine, is used for hypertension (WHO). U. macrocarpa has antihypertensive and vasorelaxant activity. However, no detailed studies related to hypertension have been reported previously, so we have explored the antihypertensive activity of U. wallichiana. AIM OF THE STUDY: To investigate the pharmacological effect of ethanolic extract (EE) and butanolic fraction (BF) of U. wallichiana in hypertensive rats. MATERIALS AND METHODS: SHR, DOCA-salt- and L-NAME-induced hypertension models were used. Treatment was performed by oral administration of EE and BF of U. wallichiana (500mg/kg/day and 50mg/kg/day) for 14 days. Then blood pressure was measured by non-invasive blood pressure (NIBP) measurement technique. Invasive blood pressure (IBP) was also reported to support the NIBP data. Concentrations of plasma renin, angiotensin II (Ang II), nitrate/nitrite (NO), cGMP were estimated. Angiotensin-converting enzyme (ACE) activity and ROS activity were also estimated. RESULTS: Blood pressure was significantly higher in SHR as compared to normotensive wistar group (170.59±0.83mmHg vs 121.54±1.24mmHg, respectively). SBP was increased in DOCA-salt induced group compared to their control (132.77±3.90mmHg vs 107.85±5.95mmHg, respectively) and L-NAME-induced group compared to their control (168.55±5.07mmHg vs 113.03±4.13mmHg, respectively). The treatment of extract and fraction of U. wallichiana significantly decreased the blood pressure in SHR+EE (151.26±1.85mmHg, p<0.001), SHR+BF (140.44±1.16mmHg, p<0.001); DOCA+EE (113.43±5.44mmHg, p<0.05), DOCA+BF (105.09±5.12mmHg, p<0.05) and L-NAME+EE (119.76±4.39mmHg, p<0.001), L-NAME+BF (117.50±7.27mmHg, p<0.001) compared to their respective diseased control groups. The plasma renin, Ang II and ACE activity were also significantly decreased and augmented the NO and cGMP levels. It also down regulated the expression of Renin, ACE, NOS3 and TGF-ß1 at mRNA levels. CONCLUSIONS: The EE and BF probably reducing the BP via Renin-angiotensin-aldosterone system and NO/cGMP signaling pathway. The decrease in blood pressure may be due to presence of quercetin analogue flavonoids (2S,3S)-(+)-3',4',5,7-tetrahydroxydihydroflavonol-6-C-ß-D-glucopyranoside; 6-Glucopyranosyl-3,3',4',5,7-pentahydroxyflavone; 6-Glucopyranosyl-4',5,7-trihydroxyflavanone and (2S,3S)-(+)-4',5,7-trihydroxydihydroflavonol-6-C-ß-D-glucopyranoside, may be due to its antioxidant activity. Thus EE and BF of U. wallichiana found to have the potential ability to be used as herbal medicament to treat hypertension.

Pharmacokinetics and bioavailability assessment of Miltefosine in rats using high performance liquid chromatography tandem mass spectrometry.

Miltefosine (MFS) is the first effective oral drug for treatment of visceral, mucosal and cutaneous leishmaniasis. In this study, liquid chromatography coupled mass spectrometry (LC-MS/MS) method of MFS was validated in rat plasma and its practical utilization to pharmacokinetic studies in rats for the first time. A rapid, selective and sensitive LC-MS/MS method for MFS in rat plasma was linear over the calibration range of 1-500ng/mL. MFS and Phenacetin (internal standard) were separated on Phenomenex Luna 3µ HILIC 200A (150×4.6mm) column under isocratic condition using methanol: 0.1% formic acid in triple distilled water, 90:10 (v/v) mobile phase at a flow rate of 0.8mL/min. The total chromatographic run time was 4.0min. The intra- and inter-day assay accuracy was observed between 99.45-102.88% and 99.92-101.58%, respectively. The intra- and inter-day assay precision was observed between 2.68-5.54% and 2.35-5.94%, respectively. The validated assay was practically applied to determine the plasma concentrations after oral and intravenous administration of MFS to rats. After oral administration, MFS showed Cmax (3200.00±95.39ng/mL) was observed at 12.00h (tmax) and t1/2 was 102.36±16.65h. The absolute bioavailability of MFS was 60.33±2.32%.

Formulation optimization of Docetaxel loaded self-emulsifying drug delivery system to enhance bioavailability and anti-tumor activity.

Poor bioavailability of Docetaxel (DCT) arising due to its low aqueous solubility and permeability limits its clinical utility. The aim of the present study was to develop DCT loaded self-emulsified drug delivery systems (D-SEDDS) and evaluate its potential ability to improve the oral bioavailability and therapeutic efficacy of DCT. D-SEDDS were characterized for their in vitro antitumor activity, in situ single pass intestinal perfusion (SPIP), bioavailability, chylomicron flow blocking study and bio-distribution profile. The D-SEDDS were prepared using Capryol 90, Vitamin E TPGS, Gelucire 44/14 and Transcutol HP with a ratio of 32.7/29.4/8.3/29.6 using D-Optimal Mixture Design. The solubility of DCT was improved upto 50 mg/mL. The oral bioavailability of the D-SEDDS in rats (21.84 ± 3.12%) was increased by 3.19 fold than orally administered Taxotere (6.85 ± 1.82%). The enhanced bioavailability was probably due to increase in solubility and permeability. In SPIP, effective permeability of D-SEDDS was significantly higher than Taxotere. D-SEDDS showed 25 fold more in vitro cytotoxic activity compared to free DCT. Chylomicron flow blocking study and tissue distribution demonstrated the intestinal lymphatic transport of D-SEDDS and higher retention in tumor than Taxotere. The data suggests that D-SEDDS showed desired stability, enhanced oral bioavailability and in vitro antitumor efficacy.

Cardioprotective Effect of Ulmus wallichiana Planchon in ß-Adrenergic Agonist Induced Cardiac Hypertrophy.

Ulmus wallichiana Planchon (Family: Ulmaceae), a traditional medicinal plant, was used in fracture healing in the folk tradition of Uttarakhand, Himalaya, India. The present study investigated the cardioprotective effect of ethanolic extract (EE) and butanolic fraction (BF) of U. wallichiana in isoprenaline (ISO) induced cardiac hypertrophy in Wistar rats. Cardiac hypertrophy was induced by ISO (5 mg/kg/day, subcutaneously) in rats. Treatment was performed by oral administration of EE and BF of U. wallichiana (500 and 50 mg/kg/day). The blood pressure (BP) and heart rate (HR) were measured by non-invasive blood pressure measurement technique. Plasma renin, Ang II, NO, and cGMP level were estimated using an ELISA kit. Angiotensin converting enzyme activity was estimated. BP and HR were significantly increased in ISO group (130.33 ± 1.67 mmHg vs. 111.78 ± 1.62 mmHg, p < 0.001 and 450.51 ± 4.90 beats/min vs. 347.82 ± 6.91 beats/min, respectively, p < 0.001). The BP and HR were significantly reduced (EE: 117.53 ± 2.27 mmHg vs. 130.33 ± 1.67 mmHg, p < 0.001, BF: 119.74 ± 3.32 mmHg vs. 130.33 ± 1.67 mmHg, p < 0.001); HR: (EE: 390.22 ± 8.24 beats/min vs. 450.51 ± 4.90 beats/min, p < 0.001, BF: 345.38 ± 6.79 beats/min vs. 450.51 ± 4.90 beats/min, p < 0.001) after the treatment of EE and BF of U. wallichiana, respectively. Plasma renin, Ang II, ACE activity was decreased and NO, cGMP level were increased. The EE and BF of U. wallichiana down regulated the expression of ANP, BNP, TNF-α, IL-6, MMP9, ß1-AR, TGFß1 and up regulated NOS3, ACE2 and Mas expression level, respectively. Thus, this study demonstrated that U. wallichiana has cardioprotective effect against ISO induced cardiac hypertrophy.