Comparative Study of Withaferin A and Withanolide A in Different Cultivators of Withania Somnifera by RP-HPLC Method

2020 ◽  
Author(s):  
Saikishore Gajula ◽  
P. Sri Devi ◽  
Bhagavan Raju
Keyword(s):  
Comparative Study ◽  
Withania Somnifera ◽  
Hplc Method ◽  
Withaferin A ◽  
Rp Hplc ◽  
Withanolide A

Development and validation of an RP-HPLC method for the simultaneous determination of total withanolide glycosides and Withaferin A in Withania somnifera (Ashwagandha)

2020 ◽  
Vol 7 (2) ◽  
pp. 106-120
Author(s):  
Benny Antony ◽  
Merina Benny ◽  
Binu T. Kuruvilla ◽  
Anu Sebastian ◽  
Anu Aravind Aravindakshan Pillai ◽  
...  
Keyword(s):  
High Performance Liquid Chromatography ◽  
Quantitative Determination ◽  
High Performance ◽  
Withania Somnifera ◽  
Reversed Phase ◽  
Hplc Method ◽  
Withaferin A ◽  
Rp Hplc ◽  
Ich Guidelines

Background:: Withanolide glycosides in Ashwagandha (Withania somnifera), are important metabolites attributed with widely acclaimed therapeutic potential for which validated methods for quantitative determination are limited. Objective:: The primary objective was to develop and validate a Reversed-Phase High Performance Liquid Chromatography (RP-HPLC) method for simultaneous quantification of total withanolide glycosides (WG), withanoside IV and withaferin A present in ashwagandha extract.The study also aimed to identify various other constituents present in the extract. Materials and Methods: Aqueous methanol extract (AME) of Ashwagandha was prepared and fractionated into two viz. flavonoid rich fraction (FF) and withanolide rich fraction (WF). RP-HPLC method was developed and validated for the estimation of total WG in ashwagandha extract according to ICH guidelines. Preparative HPLC based purification of major compounds from WF fraction was carried out and constituents were identified using spectroscopic techniques. HPLC chemical profiling of WF before and after acid hydrolysis under controlled conditions was carried out to further confirm the glycosidic compounds. Results and Discussion: The RP-HPLC method gave a precise differentiation of flavonoids, withanolides and WG present in ashwagandha extract. The method demonstrated good reliability and sensitivity, and can be conveniently used for the quantification of total WG, withanoside IV and withaferin A present in ashwagandha extracts. According to this method, a purified fraction (WF) prepared from roots and leaves of Ashwagandha comprise 35% of total WG, 3.27% of withanoside IV and 2.40% of Withaferin A. The method was also applied to different products prepared from Ashwagandha with total withanolide glycosides ranged from 1.5% to 60%, and the results were found to be reproducible. Identification of the individual chemical constituents as well as the acid hydrolytic pattern of the extract further supported the reliability of the developed method for the quantitative determination of total WG. This study also reported a new withanolide glycoside named, cilistol V-6’-O-glucoside (Aswanoside) along with some other known withanolide glycosides. Conclusion:: A Reversed-Phase High Performance Liquid Chromatography (RP-HPLC) method was developed and validated for the quantitative determination of total WG, withanoside IV and withaferin A present in ashwagandha extract according to ICH guidelines. This study also reported a new withanolide glycoside named, cilistol V-6’-O-glucoside (Aswanoside) along with some other known WG.

Extraction Optimization for Phenolic- and Withanolide-Rich Fractions from Withania somnifera Roots: Identification and Quantification of Withaferin A, 12-Deoxywithastromonolide, and Withanolide A in Plant Materials and Marketed Formulations Using a Reversed-Phase HPLC–Photodiode Array Detection Method

2018 ◽  
Vol 101 (6) ◽  
pp. 1773-1780 ◽  
Author(s):  
Satyanshu Kumar ◽  
Raghuraj Singh ◽  
Narendra Gajbhiye ◽  
Tushar Dhanani
Keyword(s):  
Withania Somnifera ◽  
Phenolic Content ◽  
Hplc Method ◽  
Withaferin A ◽  
Total Phenolic ◽  
Photodiode Array Detection ◽  
Withanolide A ◽  
Photodiode Array ◽  
Array Detection ◽  
Identification And Quantification

Abstract Background: Both the roots and leaves of Withania somnifera are products of commerce. They contain active compounds of therapeutic value and mostly different withanolides. Several pharmacological activities of W. somnifera have links to one or more withanolides. The presence of phenolic compounds in extracts could play a vital role in the reduction of blood glucose levels in diabetic subjects. Objective: The present study was carried out for the selection of a solvent to prepare extracts rich in phenolics, withaferin A (WA), 12-deoxywithastromonolide (12WD), and withanolide A (WDA). A simple, rapid HPLC method was also developed for the identification and quantification of WA, 12WD, and WDA. Methods: The extraction efficiency of aqueous alcoholic solvents including hexane, chloroform, ethyl acetate, and methanol were compared for three selected withanolides and total phenolic content. The contents of WA, 12WD, and WDA and total phenolics were determined in the extracts. The quality of nine formulations containing W. sominfera were also compared in terms of the content of WA, 12WD, and WDA and total phenolics. Results: The maximum extract yield and the total withanolide and phenolic content were obtained from aqueous alcoholic compositions at 50:50 (v/v), 70:30 (v/v), and 100:0 (v/v), respectively. In the case of organic solvents, chloroform and ethyl acetate yielded the highest concentrations of phenolics and three withanolides, respectively. The total phenolic content in formulations was in the range of 1.84–3.13%, and total withanolide content showed wide variability. Conclusions: The outcome of the present investigation could be utilized for the selection of extraction solvents to prepare W. somnifera–enriched extracts and their quality monitoring by using the developed and validated HPLC-Photodiode array detection method. Highlights: A process for preparation of phenolics and withanolides (withaferin A, 12-deoxywithastramonolide and withanolide A) enriched extracts of Withania somnifera. Simple and rapid HPLC method was also developed and validated as per the ICH guidelines for identification and quantification of three major withanolides. The developed HPLC method was applied to analyze the quality of extracts and marketed herbal products (mono, as well as poly constituents). Optimized extraction process could be utilized for upscaling process development in preparation of enriched extracts from Withania somnifera, crop improvement, bio-prospection studies and quality control.

Traditional Horticulture Practices Increase the Production of Selected Withanolides in Withania Somnifera (L.) Dunal—A RP-UFLC Analysis

2020 ◽  
Vol 58 (10) ◽  
pp. 899-906
Author(s):  
Gireesh M Ankad ◽  
Sandeep R Pai ◽  
Jagadishchandra Hiremath ◽  
Harsha V Hegde
Keyword(s):  
Withania Somnifera ◽  
Farmyard Manure ◽  
Simultaneous Detection ◽  
Withaferin A ◽  
Reverse Phase ◽  
Ancient India ◽  
Enhanced Production ◽  
Plant Life ◽  
Multiple Comparison Test ◽  
Withanolide A

Abstract The study evaluates the effect of two traditional horticulture treatments mentioned in Vrikshayurveda, a text from ancient India on the science of plant life, namely Kunapa jala (KJ) and Pancha gavya (PG) on the production of Withaferin A (WFA), withanolide A (WIA) and Withanolide B (WIB) in Withania somnifera (L) Dunal. Leaves and roots of W. somnifera were collected from different treated groups viz. control, KJ, PG, farmyard manure (FYM) and inorganic fertilizer (NPK). Reverse phase ultra-flow liquid chromatography (RP-UFLC) method was developed, validated for simultaneous detection of WFA, WIA and WIB. Statistical analysis of data was performed by ANOVA and tested for significance by the Dunnett multiple comparison test and data were expressed as mean ± standard deviation (SD). Results revealed, leaves possessed highest WFA content and roots possessed highest content of WIA and WIB. PG treated leaves were observed highest WFA (18.29 mg/g) and roots were observed highest WIA (19.63 mg/g) and WIB (1.36 mg/g). Conclusively, RP-UFLC method for simultaneous detection of withanolides has been developed and validated to evaluate the effect of traditional horticulture treatments. It is concluded that the enhanced production of withanolides can be achieved by the application of PG when compared to NPK application.

scholarly journals COMPARATIVE STUDY OF RP-HPLC METHOD VERSUS FOURIER TRANSFORM CONVOLUTION CHEMOMETRIC METHODS; AN APPLICATION ON PHARMACEUTICAL BINARY MIXTURES OF CANDESARTAN CILEXETIL-PITAVASTATIN CALCIUM AND CLOPIDOGREL BISULFATE-ROSUVASTATIN CALCIUM

2016 ◽  
Vol 8 (10) ◽  
pp. 125 ◽  
Author(s):  
Marwa K. El Jamal ◽  
Azza A. Gazy
Keyword(s):  
Fourier Transform ◽  
Comparative Study ◽  
Binary Mixtures ◽  
Candesartan Cilexetil ◽  
Hplc Method ◽  
Dihydrogen Phosphate ◽  
Clopidogrel Bisulfate ◽  
Rp Hplc ◽  
Pitavastatin Calcium ◽  
Rosuvastatin Calcium

<p><strong>Objective: </strong>A comparative study of smart spectrophotometric chemometric assisted techniques and RP-HPLC for the determination of candesartan cilexetil (CAN)-pitavastatin calcium (PIT) and clopidogrel bisulfate (CLO)-rosuvastatin calcium (ROS), binary co-administered drugs were developed and validated.</p><p><strong>Methods: </strong>The spectrophotometric chemometric assisted methods included two simple techniques, namely Fourier transform convolution (FF) and ratio spectra of Fourier transform convolution (FFR) methods. FFR is considered as a hybrid divisor ratio spectra method where Fourier functions are applied to divisor ratio signals. The RP-HPLC method involves a rapid separation on a C<sub>18 </sub>column using a mobile phase consisting of acetonitrile: sodium dihydrogen phosphate (adjusted to pH 2.6 using orthophosphoric acid) in the ratio of 70:30 v/v at a flow rate of 1 ml/min in isocratic mode. CLO and ROS were monitored at 220 nm however CAN and PIT were monitored at 238 nm.</p><p><strong>Results: </strong>The spectrophotometric chemometric assisted methods proved their ability to quantify each of the studied drugs in their binary mixtures, where excellent percentage recoveries were obtained. FF and FFR method proved to be linear over the concentration range of 10-50 µg/ml for CLO, 4-20 µg/ml for ROS, 8-20 µg/ml for CAN and 2-10 µg/ml for PIT. The RP-HPLC method was able to separate the drugs in the study; retention times were found to be 3.9 min and 14.4 min for ROS-CLO, 4.2 min and 14.5 min for PIT-CAN respectively. The RP-HPLC method was found to be linear in the concentration range of 0.1-0.5 µg/ml for CLO, 0.04-0.2 µg/ml for ROS, 0.5-1 µg/ml for CAN and 0.05-0.1 µg/ml for PIT. System suitability parameters proved that peaks were well resolved from each other.</p><p><strong>Conclusion: </strong>The spectrophotometric and chromatographic methods were validated according to ICH guidelines. Recovery was found to be in the range of 95.9 %-100.5 % in synthetic laboratory mixtures. The suggested spectrophotometric methods have the advantage over other methods that they do not require a preliminary separation. Statistical analysis between the suggested spectrophotometric chemometric assisted and RP-HPLC methods, using student’s t-and F-test revealed that there is no difference between the applied methods.</p>

Determination of Withaferin-A in twoWithania Speciesby RP-HPLC method

2006 ◽  
Vol 68 (2) ◽  
pp. 253 ◽  
Author(s):  
Shaila Dalavayi ◽  
SM Kulkarni ◽  
RajyaLakshmi Itikala ◽  
S Itikala
Keyword(s):  
Hplc Method ◽  
Withaferin A ◽  
Rp Hplc

scholarly journals Comparative study of phenolic acids from underground parts of Rheum palmatum L., R. rhaponticum L. and R. undulatum L.

2011 ◽  
Vol 74 (4) ◽  
pp. 275-279 ◽  
Author(s):  
Helena Danuta Smolarz ◽  
Ewa Medyńska
Keyword(s):  
Comparative Study ◽  
Ferulic Acid ◽  
Phenolic Acids ◽  
Hplc Method ◽  
Rheum Palmatum ◽  
Rp Hplc ◽  
Rheum Undulatum

In three species of <em>Rheum</em> L. genus growing in Poland the composition of phenolic acids was determined. By 2D-TLC method the following acids were identified: ellagic, chlorogenic, gallic, protocatechuic, homoprotocatechuic, caffeic, α-resorcilic, p-hydroxyphenylacetic, p-hydroxybenzoic, p-coumaric, syringic, vanillic and ferulic. There are no substantial qualitative differences among the complex of phenolic acids in the investigated species. The RP-HPLC method was used for quanitative determination of phenolic acids. The amount of individual phenolic acids ranged between 2.2 µg/g and 147.8 µg/g in air-dry rhizome. The content of ferulic acid is the highest in all the examined cases. The total amount of the tested phenolic acids in <em>Rheum undulatum</em> L., <em>R. palmatum</em> L., and <em>R. raponticum</em> L. was respectively 346.4 µg/g, 229.8 µg/g, and 195 µg/g.

Development and Validation of a Novel, Rapid Gradient HPLC Method for Simultaneous Estimation of Bioactive Marker Compounds in a Mixture of Convolvulus pluricaulis, Withania somnifera and Bacopa monnieri Extracts

2019 ◽  
Vol 57 (10) ◽  
pp. 920-930 ◽  
Author(s):  
Sunayna Choudhary ◽  
Indu Pal Kaur ◽  
Jai Malik
Keyword(s):  
Withania Somnifera ◽  
Therapeutic Option ◽  
Herbal Medicines ◽  
Hplc Method ◽  
Bacopa Monnieri ◽  
Withaferin A ◽  
Simultaneous Estimation ◽  
Bacoside A ◽  
Convolvulus Pluricaulis ◽  
Development And Validation

Abstract The use of herbs as medicine is an ancient form of healthcare known to mankind. Standardization of herbal medicines is however a challenging task and is the major bottleneck in their acceptance as the primary therapeutic option. The aim of this study was to develop and validate a simple, rapid HPLC method for standardizing the mixture of extracts of three Medhya Rasayanas (neurotonic), Convolvulus pluricaulis, Withania somnifera and Bacopa monnieri. Simultaneous estimation of the respective bioactive markers of these plants viz., scopoletin, withaferin A, bacoside A 3, bacopaside II, jujubogenin and bacosaponin C has been reported for the first time. The method was developed using Waters Hybrid X-Bridge shield with BEH technology 2.5 μm, 4.6 × 75 mm column and validated according to ICH guidelines. The 20 minutes run time makes the method eco-friendly. The method was linear over a range of 12.5–400 ng/10 μL for scopoletin and 62.5–2,000 ng/10 μL for withaferin A, bacoside A 3, bacopaside II, jujubogenin and bacosaponin C with detection limits of 8.0, 48.3, 30.4, 40.7, 15.6 and 18.9 ng/10 μL and quantification limits of 24.5, 146.5, 92.2, 123.4, 47.4 and 57.4 ng/10 μL, respectively. The correlation coefficient for each analyte was &gt;0.999. The intra-day and inter-day precision was &lt;2%. These results confirmed the precision, accuracy and robustness of the proposed method.

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