Simultaneous estimation of five biomarkers of neuroprotective herb Ashwagandha NMITLI-118R AF1 in rat plasma and brain using LC-ESI-MS/MS: Application to its pharmacokinetic and stability studies.

Withania Somnifera (WS) is a popular nutritional supplement in the USA, Europe, and Asia, known for its pharmacological effects on neurological disorders. However, the bioanalytical method development, validation, and pharmacokinetics of WS NMITLI-118R AF1 biomarkers Withanolide A (WLD A), Withanone (WNONE), Withanolide B (WLD B), Withaferin A (WF A), and 12 Deoxywithastramonolide (12 DEOXY) in rats have not been comprehensively explored. This study aimed to develop and validate a sensitive and selective LC-ESI-MS/MS method for these biomarkers in male Sprague Dawley rats plasma and brain matrix. Rats were divided into eight groups, each containing five rats. A plant extract of NMITLI-118R AF1 at 50 mg/kg was orally administered to the rats for in-vivo pharmacokinetic investigation. All the analytes had a linear calibration curve (r2 > 0.999), and intra-day and inter-day precision (%) were found in the range of 2.46 - 13.71% and accuracy were within the acceptable range (±15%). The biomarkers of NMITLI-118R AF1 were found stable in in-vitro plasma and simulated gastro-intestinal fluids. The observed (Cmax) and (Tmax) values for the biomarkers in the systemic circulation were WLD A (5.59 ± 0.34 ng/mL, Tmax 1.00 ± 0.00 h), WNONE (6.28 ± 0.41 ng/mL, Tmax 0.95 ± 0.11 h), WLD B (6.45 ± 2.87 ng/mL, Tmax 0.95 ± 0.11 h), WF A (6.50 ± 0.27 ng/mL, Tmax 1.00 ± 0.00 h), and 12 DEOXY (5.68 ± 0.39 ng/mL, Tmax 1.00 ± 0.00 h). In contrast to the old method, our approach exhibits a lower limit of quantification (LLOQ), shorter run time (less than10 min), and enables the detection of WF A and WNONE in fresh rat plasma by other quantitative analysis of mass spectrometry (m/z) [M]+. Shows high sample volumes for both, larger plasma volumes, costlier sample collection techniques dried blood spot (DBS), more expensive solid phase extraction techniques (SPE) and longer analysis time 14 min. Moreover, our method requires a smaller sample volume 10 µL, offers faster analysis time 4 min, and achieves a higher sensitivity 1 ng/mL. This is the first report of a comprehensive study on in-vitro and in-vivo pharmacokinetics of NMITLI-118R AF1 biomarkers, which may aid in further pre-clinical and clinical trial investigations.

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.

Evaluation of mutagenic, cytotoxic, mitochondrial dysfunction, apoptotic activity, and acute toxicity of ethanolic extract of Cissus quadrangularis.

Recent studies have focused on exploring the efficacy of Cissus quadrangularis extract (EECQ) against various metabolic disorders involving the liver as the prime target organ, suggesting a considerable threat of hepatotoxicity in the person encountering it. Consequently, the current study was aimed to unravel the mutagenic, cytotoxic, mitochondrial dysfunction, apoptotic activity in HepG2 cells, and acute toxicity of EECQ. MTT, SRB, trypan blue dye exclusion, and lactate dehydrogenase (LDH) assay were performed in HepG2 cell lines to determine the cytotoxicity of the extract. The mutagenic potential was determined by the Ames test using various strains of Salmonella typhimurium. Acute toxicity was done at a dose of 2000 mg/kg in Sprague Dawley rats. MTT and SRB cytotoxicity assays demonstrated dose-dependent cytotoxicity of extract. The three highest noncytotoxic doses from the above assay, investigated by trypan blue dye exclusion and LDH assay, did not reveal cytotoxicity. Besides, mitochondrial dysfunction was determined by measuring cellular and mitochondrial ROS, ATP, NAD, mitochondrial membrane potential, Bax/Bcl2 ratio, mitochondrial and cytoplasmic cytochrome c, and apoptosis-inducing factor, were found to be equivalent in both extract exposed and unexposed cells. Moreover, the apoptotic cell morphology and the expression of pro-apoptotic mRNAs and proteins were equivalent in both the group. In acute toxicity, EECQ in rats did not cause any significant change in body weight, liver index, and liver function test. All-encompassing, the present study unraveled that EECQ is not mutagenic, cytotoxic, nor apoptotic in human hepatic cells, as well as neither acute toxicity.

Polyphenolic-rich Cissus quadrangularis extract ameliorates insulin resistance by activating AdipoR1 in peri-/post-menopausal rats.

Insulin resistance (IR) is a significant complication in menopausal women, which predisposes them to cardiovascular disorder, obesity, and diabetes. Cissus quadrangularis is a polyphenolic plant rich in nutrients and is used as an edible vegetable in Nigeria. Previously, we investigated that C. quadrangularis extract (EECQ) treatment ameliorates IR, hyperlipidemia, and overweight in diabetic rats. Accordingly, in the current study, we further evaluated the adiponectin mimetic activity of EECQ in peri-/post-menopausal rats. Perimenopause was induced by High-fat diet/4-vinylcyclohexenediepoxide/(HFD-VCD), while postmenopause was by HFD/bilateral ovariectomy (HFD-OVX). Both the menopausal rats demonstrated an abnormal level of sex hormones, IR, hyperlipidemia, increased fat mass, and abnormal weight gain. Nevertheless, EECQ treated group revealed protection from these untoward complications. Furthermore, the docking score of major constituents of EECQ on adiponectin receptor 1 (AdipoR1) depicted a strong binding affinity, which was comparable to the ligand adipoRon. Besides, AdipoR1 expression determined by RT-PCR, Western blotting, and immunohistochemistry was downregulated in peri-/post-menopausal rats. Similarly, the expression of AdipoR1 downstream marker APPL1 and insulin sensitivity markers, including IRS1, Akt1, and GLUT4, were also dysregulated in menopausal rats. However, EECQ treated rats manifested restoration of normal expression of APPL1, IRS1, Akt1, and GLUT4 by upregulating AdipoR1. Altogether, the current study promulgated the adiponectin mimetic activity of EECQ, which is substantial to mitigate IR in menopausal conditions.

Ethanolic extract of Cissus quadrangularis improves vasoreactivity by modulation of eNOS expression and oxidative stress in spontaneously hypertensive rats.

BACKGROUND: Endothelial dysfunction is related to the reduced bioavailability of nitric oxide (NO) and plays a significant role in developing hypertension. The intake of a diet rich in antioxidants decreases the threat of hypertension. Cissus quadrangularis possesses antioxidant, anti-inflammatory, and hypocholesterolemic activities. However, to date, no studies have been performed to explore this plant's antihypertensive and vasorelaxant activity. Herein, we investigated the chronic effect of C. quadrangularis on blood pressure as well as vascular function in hypertensive rats. METHODS: Male spontaneously hypertensive rats (SHR) were randomly divided into two groups. Normotensive Wistar rats were taken as the control group. The treatment was done using ethanolic extract of C. quadrangularis (EECQ) at a dose of 200 mg/kg. RESULTS: The administration of EECQ for six weeks reduced the systolic blood pressure, mean arterial blood pressure, and heart rate. It also alleviated the cardiac and renal hypertrophy indices. Supplementation of EECQ improved the endothelium-dependent aortic vasodilation induced by acetylcholine. It restored the NO level and endothelial NO synthase expression in the aorta. Subsequently, the extract alleviates the oxidative stress and inflammatory markers in SHR rats. CONCLUSION: Thus, in the present study, the chronic treatment of EECQ to genetically hypertensive rats improved endothelium-dependent relaxation in addition to its antihypertensive effect by eNOS activation and inhibition of ROS production, inflammation.

A butanolic fraction from the standardized stem extract of Cassia occidentalis L delivered by a self-emulsifying drug delivery system protects rats from glucocorticoid-induced osteopenia and muscle atrophy.

We recently reported that a butanol soluble fraction from the stem of Cassia occidentalis (CSE-Bu) consisting of osteogenic compounds mitigated methylprednisone (MP)-induced osteopenia in rats, albeit failed to afford complete protection thus leaving a substantial scope for further improvement. To this aim, we prepared an oral formulation that was a lipid-based self-nano emulsifying drug delivery system (CSE-BuF). The globule size of CSE-BuF was in the range of 100-180 nm of diluted emulsion and the zeta potential was -28 mV. CSE-BuF enhanced the circulating levels of five osteogenic compounds compared to CSE-Bu. CSE-BuF (50 mg/kg) promoted bone regeneration at the osteotomy site and completely prevented MP-induced loss of bone mass and strength by concomitant osteogenic and anti-resorptive mechanisms. The MP-induced downregulations of miR29a (the positive regulator of the osteoblast transcription factor, Runx2) and miR17 and miR20a (the negative regulators of the osteoclastogenic cytokine RANKL) in bone was prevented by CSE-BuF. In addition, CSE-BuF protected rats from the MP-induced sarcopenia and/or muscle atrophy by downregulating the skeletal muscle atrogenes, adverse changes in body weight and composition. CSE-BuF did not impact the anti-inflammatory effect of MP. Our preclinical study established CSE-BuF as a prophylactic agent against MP-induced osteopenia and muscle atrophy.

Antioxidant and Hepatoprotective Potential of Swaras and Hima Extracts of Tinospora cordifolia and Boerhavia diffusa in Swiss albino Mice.

BACKGROUND: In Ayurveda, five basic extraction procedures are mentioned in order of their decreasing potency. Swaras is considered as the most potent followed by, kalka, kwatha, fanta and hima. OBJECTIVE: Present study was carried out to investigate the antioxidant and hepatoprotective potential of swaras and hima extracts of T.cordifolia and B. diffusa. MATERIALS AND METHODS: Swaras and hima extracts of T. cordifolia and B. diffusa were prepared. Phytochemical screening and in vitro antioxidant activities was carried out using standard methods. Hepatoprotective efficacy of extracts were carried out in Swiss albino mice using paracetamol induced hepatotoxicity. Animals were administered with swaras and hima extracts of both plants at 200 mg/kg BW dose for 7 days and on 8th day hepatotoxicity was induced by intraperitoneal injection of paracetamol at 500 mg/kg BW. The degree of liver protection was determined by measuring the levels of liver enzymes followed by histopathology. RESULTS AND DISCUSSION: The results of phytochemical, antioxidant and hepatoprotective activities showed that there were no significant difference between swaras and hima extracts. Both the extract of T. cordifolia were equally potent in reducing SGOT (P < 0.01) and ALP level (P < 0.001). Similar effects were observed with the Swaras and hima extracts of B. diffusa. Both the extracts reduced SGOT and ALP (P < 0.01). Histopathological findings among all the extracts were also more or less similar in lowering the paracetamol mitigated necrosis. CONCLUSION: The present study suggested that T. cordifolia and B. diffusa possess potential hepatoprotective activity irrespective of the extraction procedure. SUMMARY: Aqueous extracts of Tinospora cordifolia and Boerhavia diffusa exhibited significant antioxidant and hepatoprotective activitiesAqueous extracts of both the plants were extracted using different extraction procedures mentioned in AyurvedaSwaras and hima extracts of both the plants significantly reduced the deleterious effects of paracetamol, suggesting that both the plant extracts are equipotentAcute toxicity of both the plant extracts did not produce any toxic effects. Abbreviations used: TC swaras: T. cordifolia swaras; TC hima: T. cordifolia hima; BD swaras: B. diffusa swaras; BD hima: B. diffusa hima; BW: Body weight; LDL: Low-density lipoprotein; HDL: High-density lipoprotein; SGOT: Serum glutamate oxaloacetate transminase; SGPT: Serum glutamate pyruvate transminase; ALP: Alkaline phosphatase; I.P: Intraperitoneal; TAC: Total antioxidant capacity; DPPH: 2,2-diphenyl-1-picrylhydrazyl; TCA: Trichloro acetic acid; NO: Nitric oxide; TPC: Total phenolic content; NAPQI: N-acetyl-p-benzoquinone imine; PCM: Paracetamol.