ABSTRACT
We investigated whether a standardized saw palmetto extract (SP, mixture of supercritical extract and ethanol extract at a ratio of 9.5 to 0.5) can relieve the symptoms of andropause, including metabolic syndrome, and decreases in muscle endurance and spermatogenesis, in old rats. Twenty-four-week-old male Sprague Dawley rats received oral supplementation of SP at 40, 80, and 160 mg/kg body weight (bw) for 4 weeks. We found that SP supplementation reduced body weight gain by decreasing visceral and epididymal fat weights and the levels of serum triglycerides, total cholesterol, and low-density lipoprotein/very low-density lipoprotein cholesterol. In addition, SP supplementation increased muscle endurance, sperm counts, and testosterone biosynthesis through hormonal regulation. In Leydig cells under hydrogen peroxide-induced oxidative stress, SP treatment directly induced testosterone biosynthesis by activating the mRNA expression of the genes encoding 17,20-desmolase and 3ß-hydroxysteroid dehydrogenase 4. In conclusion, our results suggest that supplementation of SP may be useful for alleviating the symptoms of andropause via direct and indirect regulation of testosterone biosynthesis.
Subject(s)
Plant Extracts , Spermatogenesis , Animals , Male , Plant Extracts/pharmacology , Rats , Rats, Sprague-Dawley , Serenoa , TestosteroneABSTRACT
BACKGROUND: Withania somnifera is an Indian medicinal herb known for the multipotential ability to cure various therapeutic ailments as described in the ayurvedic system of medicine. OBJECTIVE: In the present study, we have evaluated the antiproliferative activity of a standardized W. somnifera root extract (Viwithan) against different human and murine cancer cell lines. MATERIALS AND METHODS: The cytotoxicity of Viwithan was determined using thiazolyl blue tetrazolium blue assay and crystal violet staining. The apoptotic changes in B16F1 cells following treatment with Viwithan were observed by acridine orange/ethidium bromide (AO/EB) staining and DNA fragmentation assay. The binding affinity of withanolides in Viwithan with antiapoptotic proteins B-cell lymphoma 2, B-cell lymphoma-extra large, and myeloid cell leukemia 1 (MCL-1) were studied using in silico approach. RESULTS: The half maximal inhibitory concentration (IC50) values of Viwithan against liver hepatocellular carcinoma, Henrietta Lacks cervical carcinoma cells, human colorectal carcinoma cell line, and Ehrlich ascites carcinoma cells were 1830, 968, 2715, and 633 µg/ml, respectively. Interestingly, Viwithan was highly effective against B16F1 cells with an IC50 value of 220 µg/ml after 24 h treatment. The morphological alterations of apoptotic cell death were clearly observed in the AO/EB-stained cells after treatment with Viwithan. Viwithan induced late apoptotic changes in treated B16F1 cells as evident by the ladder formation of fragmented DNA in a time-dependent manner. The findings of molecular docking showed that withanolides present in Viwithan have a more binding affinity with the antiapoptotic proteins, particularly MCL-1. CONCLUSION: We have reported for the first time that Viwithan with 5% withanolides has a potent cytotoxic effect, particularly against B16F1 murine melanoma cells among the different cancer cell lines tested. SUMMARY: The present study reports for the first time that Viwithan, a standardized 5% Withania somnifera root extract, has potent cytotoxicity against B16F1 murine melanoma cellsWe have investigated the in vitro cytotoxicity of Viwithan in different human and murine cancer cells. Interestingly, we found that Viwithan was particularly very effective against B16F1 melanoma cells with a half maximal inhibitory concentration value of 220 µg/mlThe microscopic observations following acridine orange/ethidium bromide staining and DNA fragmentation assays clearly indicated that Viwithan might initiate late apoptosis in B16F1 cellsThe binding affinity of withanolides in Viwithan with antiapoptotic proteins of B-cell lymphoma 2 family was predicted using AutoDock tool. The results from in silico studies indicated a plausible synergistic effect of withanolides attributing to the Viwithan-induced apoptosis through suppression of intrinsic pathway for carcinogenesis. Abbreviations used: MTT: Thiazolyl blue tetrazolium blue; DMSO: Dimethyl sulfoxide; BSA: Bovine serum albumin; DMEM: Dulbecco's minimum essential medium; NCCS: National Centre for Cell Science; PBS: Phosphate-Buffered Saline; HepG2: Liver hepatocellular carcinoma; HeLa: Henrietta Lacks cervical carcinoma cells; HCT-116: Human colorectal carcinoma cell line; EAC: Ehrlich ascites carcinoma cells; IC50: Half maximal inhibitory concentration; AO/EB: Acridine orange/Ethidium bromide; BCL-2: B-cell lymphoma 2; BCL-XL: B-cell lymphoma-extra large; MCL-1: Myeloid cell leukemia 1; PDB: Protein Data Bank; ANOVA: Analysis of variance.
ABSTRACT
OBJECTIVE: The present study was investigated to provide a documentary evidence for the determination of rutin, isoquercetin, and quercetin flavonoids from the flora of Nelumbo nucifera by reversed-phase high-performance liquid chromatography (RP-HPLC). MATERIALS AND METHODS: RP-HPLC analysis was performed by gradient elution with a low-pressure gradient using 0.5% acetic acid: acetonitrile as a mobile phase with a flow rate of 1.0 ml/min. The separation was done at 26°C using a Kinetex XB-C18 column as stationary phase and the detection wavelength at 356 nm. The proposed method was validated as per International Conference on Harmonisation guidelines with respect to specificity, linearity, precision, accuracy, limit of detection (LOD), and limit of quantification (LOQ). RESULTS: The validated results were within the acceptable limits. In specificity, the retention time of rutin, isoquercetin, and quercetin peak in the sample was matched with the reference standard peak and showed good resolution. An excellent linearity was obtained with correlation coefficient (r) higher than 0.999. In precision, the repeatability and intermediate showed <1.0% of % relative standard deviation of peak area percentage indicating high precision and accurate. The recovery rate for rutin, isoquercetin, and quercetin was between 99.85%-101.37%, 101.90%-103.24%, and 101.74%-106.73%, respectively. The lower LOD and LOQ of rutin, isoquercetin, and quercetin enable the detection and quantitation of these flavonoids in N. nucifera at low concentrations. CONCLUSION: The developed analytical method is convenient for the determination of flavonoids content in herbal drugs.
ABSTRACT
Mangiferin, a polyphenolic xanthone glycoside from Mangifera indica, is used as traditional medicine for the treatment of numerous diseases. The present study was aimed to develop and validate a reversed-phase high-performance liquid chromatography (RP-HPLC) method for the quantification of mangiferin from the bark extract of M. indica. RP-HPLC analysis was performed by isocratic elution with a low-pressure gradient using 0.1% formic acid: acetonitrile (87:13) as a mobile phase with a flow rate of 1.5 ml/min. The separation was done at 26°C using a Kinetex XB-C18 column as stationary phase and the detection wavelength at 256 nm. The proposed method was validated for linearity, precision, accuracy, limit of detection, limit of quantification, and robustness by the International Conference on Harmonisation guidelines. In linearity, the excellent correlation coefficient more than 0.999 indicated good fitting of the curve and also good linearity. The intra- and inter-day precision showed < 1% of relative standard deviation of peak area indicated high reliability and reproducibility of the method. The recovery values at three different levels (50%, 100%, and 150%) of spiked samples were found to be 100.47, 100.89, and 100.99, respectively, and low standard deviation value < 1% shows high accuracy of the method. In robustness, the results remain unaffected by small variation in the analytical parameters, which shows the robustness of the method. Liquid chromatography-mass spectrometry analysis confirmed the presence of mangiferin with M/Z value of 421. The assay developed by HPLC method is a simple, rapid, and reliable for the determination of mangiferin from M. indica. SUMMARY: The present study was intended to develop and validate an RP-HPLC method for the quantification of mangiferin from the bark extract of M. indica. The developed method was validated for linearity, precision, accuracy, limit of detection, limit of quantification and robustness by International Conference on Harmonization guidelines. This study proved that the developed assay by HPLC method is a simple, rapid and reliable for the quantification of the mangiferin from M. indica. Abbreviations Used:M. indica: Mangifera indica, RP-HPLC: Reversed-phase high-performance liquid chromatography, M/Z: Mass to charge ratio, ICH: International conference on harmonization, % RSD: Percentage of relative standard deviation, ppm: Parts per million, LOD: Limit of detection, LOQ: Limit of quantification.