Ajay Kumar Meena1*, Sadhna Chaturvedi2, Sreekala3
1,2,3Regional Ayurveda Research Institute, CCRAS, Amkhoh, Gwalior, Madhya Pradesh, India.
Department of Biotechnology, School of Sciences, ITM University, Gwalior, Madhya Pradesh, India.
*Corresponding Author E-mail: ajaysheera@gmail.com
ABSTRACT:
Species of Withania genus is most important medicinal plant in Ayurvedic, Siddha, Unani (ASU) and indigenous system of medicine, Withania species is help to support physical and mental health of human health. Withania coagulans and Withania somnifera were the two most esteem species of Withaniagenusit’s having high medicinal significance. These species were natural source of Withaferin A (steroidal lactones) is used as variety of diseases. The present study to compare the Withaferin A content is present in the Withaniacoagulans and Withania somnifera fruit using HPLC technique. The separation was achieved on C18 Eclipse, XDB, 4.6mm × 150mm, 5μm particle size column using mobile phase consisted of Acetonitrile and Buffer (35:65 v/v) in isocratic elution with flow rate 1.5ml/min. The detection of analytes at 227nm was carried out by using Diode Array Detector (DAD) for Withaferin A reference standard and test samples. The quantitative evaluation and comparison of Withaferin-A present in fruit part of Withania coagulans was 0.0121% and Withania somnifera was 0.0075%, respectively. The WithaferinA content was observed higher amountin Withania coagulans fruit compare to Withania somnifera.
KEYWORDS: Ayurveda, Withania, Withaferin A, HPLC, Quality control.
INTRODUCTION:
The genus Withania (Solanaceae) is an important Ayurvedic medicinal plant. In this genus Twenty-three species were identified andreported. Out of these 23 species two species Withania coagulans Dunal and Withania somnifera Dunalare more important and easily available in India1. Withania coagulans and Withania somnifera are known by the same names in most of the regional languages in India and no distinction is made between the berries obtained from Withania coagulans and Withania somnifera in the market2.
Withania coagulans universally called vegetable rennet, paneer-bandh, or Indian cheese maker,ithas multiple medicinal properties3. Native of the Withania coagulansis India, Afghanistan and Nepal regions, In India it occurs in dry places of Punjab, Rajasthan, Shimla, Kumaun and Garhwal4. Withania coagulans fruits arechief ingredient in many traditional/herbal formulations used formanagement of Liver disorder, Asthma, Biliousness, Antimicrobial,Hepato-protective, Anti-hyperglycaemic, Cardiovascular, Immuno-suppressive, Free radical scavenging, Central nervous system, Depressant activities, Anthelmintic, Antifungal, wound healing and Hypoglycaemic activities of the plant have been reported3-5. Withania coagulans fruits contain Withaferin A6, Withanolides7, Esterases, Lignan, Alkaloids, Free amino acids, Fatty oils, Essential oils8 and Volatile oil9. W. As a result of somnifera's value in Ayurvedic medicine, it has been used and grown for generations in India.
Withania somnifera Linn. Dunal, (Syn. Physalisflexuosa) is usually called as Ashwagandha, Indian ginseng, winter cherry, it is an important medicinal plant in Ayurvedic, Siddha, Unani (ASU) and indigenous system of medicine for over 3,000 years10,11. This plant grows widely in all dry parts and subtropical parts of Indiaas well as other parts of the world. It occurs in Madhya Pradesh, Uttar Pradesh, Punjab, Gujarat and Rajasthan 12. It is mainlycultivated in Madhya Pradeshand Rajasthan13. It is also found in Congo, South Africa, North America,Egypt, Morocco, Jordan, Sri Lanka, Iraq, Iran, Syria, Turkey, Pakistan and Afghanistan14-16. Bruised leaves and fruits are locally applied to tumours and tubercular glands, carbuncles and ulcers17,18. The fruits of Withania somnifera contain amino acids, aproteolytic enzyme, condensed tannins and flavonoids. It contains high ratio of free amino acids which are proline, valine, tyrosine, alanine, glycine, hydroxy proline, aspartic acid, glutamic acid, cysteine and cysteine19. Withania somnifera plant used as a traditional medicine of several countries as narcotic, anti-epileptic, against female sterility, hypotonic, for stomach ache, ulcers, colds, rashes, gonorrhoea, sedative, anxiety, depression, chronic stressand its antiseptic properties20-22. The fruit of Withania somnifera used in folk medicine as febrifuge, diuretic and antirheumatic under the name ‘Morgan’ in Egypt22,23. Withaferin Ais a dominated bioactive compound in Withania somnifera fruit given in Figure 1. Earlier studies this compound, reported the anti-inflammatory, anticonvulsive, antitumor and antioxidant properties12,24-27.
Figure 1. Structure of Withaferin A Compound
Quality control is a major problem for raw materials or crude drugs and their herbal formulations. Herbal plants and herbal products are subject to extensive differences in their phytochemical profile due to their diversity, environmental or climatic conditions, maturity of plant or plant parts, seasons of harvest, post-harvest processing, storage conditions, stabilityand other factors. Various bioactive compounds play a vital role in therapeutic activity, according geographical change presence of bio active marker constituent present in plant material differ, these differences have an effect on the therapeutic activity28.The biological activities of withanolides, particularly of the dominant withaferin-A, have been extensively studied. Notable activities reported for this compound include anti-inflammatory, anticonvulsive, antitumor, and antioxidant properties. The roots, leaves, and preparations thereof are traditionally used as tonics, hypnotics, sedatives, and diuretics.29-40 Due to the wide therapeutic prominence, it is worthwhile to obtain various qualitative and quantitative standards of drug to prevent its adulteration.Based on the reported therapeutic activities ofWithania coagulans and Withania somnifera, fruits, Withaferin A was used as a standard bioactive compound to develop the quality protocol for standardisation of plant parts and formulations. To support these studies a better analytical strategy is main demand to ensure efficacy, safety and consistency of herbal plants and plant products.Therefore,it is necessary to assure the repeatability and reliability of determining most of the major marker compounds in herbal products and their ingredients for standardisation and quality control.
In the current study, a quantitative assessment of the therapeuticallyactive bio compound Withaferin A content present in the two species of Withania genusi.e.,Withania coagulans and Withania somnifera fruit plant materials were carried out by usingHPLC technique.
MATERIALS AND METHODS:
Procurement of plant material:
The Withania coagulans and Withania somnifera,fruits raw materials were used in the present study was procured from local market of Chennai, Tamilnadu, India and authenticated by Botany/Pharmacognocy Section of CSMRADDI. The dried raw materialsamples were powdered and used for study.
Standard and Reagents:
Analytical grade and HPLC gradechemicals and solvents for this study were procured from E-Merck/Fine chemicals, Mumbai, India. Withaferin A reference standard was procured from M/S Natural Remedies Pvt. Ltd., Bengaluru, India.
Extraction Procedure (Sample Preparation):
The dried powdered fruit part materials of Withania coagulans (10.17g) and Withania somnifera (3.22g) were extracted with 200ml of chloroform by using soxhlet for 24hrs. The extracts were evaporated to dryness under reduced pressure. The obtained residue weights for the above extractions were 0.63g and 0.46g.
Test solution:
The residues obtained from chloroform extracts of Withania coagulans and Withania somnifera fruit were weighed in triplicate and dissolved in appropriate volume of methanol, filtered through 0.22μ membrane filters and used for HPLC analysis.
Standard solution:
Accurately weighed 1.7mg of Withaferin-A was transferred into a 10mlvolumetric flaskdissolved in methanol, sonicated and make up the volume upto the mark using methanol this solution contains 0.17mg/ml of WithaferinA (stock solution). This stock solution was further diluted as per requirement for the preparation of working standard. This solution was appropriately diluted further to get a concentration of 0.17, 0.085, 0.0425, 0.0212 and 0.0162 mg/ml of Withaferin A. Each of the standard solution was run through the HPLC and records the respective peak areas. Calibration curve was established for peak area vs concentration of Withaferin A applied.
HPLC Instrumentation and Chromatographic Conditions:
The Standard and extracts were analysed using an Agilent 1200 series High Performance Liquid Chromatographic system equipped with Agilent 1100/1200 quaternary pump, Agilent 1100/1200 Column Thermostat, manual sample injector (20µl) using Chemstation HPLC software. Separation was achieved on Eclipse XDB-C18 column (150 × 4.6mm, 5𝜇m, Agilent). All samples and standards were filtered through 0.22μm (Millipore) filters. The mobile phase consisted of Acetonitrile: Buffer (35:65 v/v) in isocratic elution with flow rate 1.5ml/min. Inject 10μl of the Standard and test sample (triplicate) to HPLC system. The column temperature was kept 25oC. The detection of analytes at 227nm was carried out by using 1200 Diode Array Detector.
Mobile phase
Solution A: Acetonitrile
Solution B (Buffer): Dissolve 0.136g of anhydrous potassium dihydrogen orthophosphate (KH2PO4) in 900 ml of HPLC grade water, add 0.5ml of Orthophosphoric acid and make up to 1000mL with HPLC grade water, filter through 0.45m membrane and degas for 3 minutes using a sonicator.
RESULTS AND DISCUSSION:
The present study is to develop perceptive, selective and accurate HPLC method for determination and comparison of Withaferin A in Withania coagulans and Withania somnifera fruits samples.
Preparation of test solution:
Withania coagulans and Withania somnifera fruits chloroform extract residue was weighed and dissolve according required volume of methanol. Each sample weighed in triplicate and make up the volume and finally inject the sample solution (10µl) into HPLC manual injector
Method Development:
Good separations and suitable retention time of Withaferin A were obtained in isocratic elution using Eclipse XDB-C18 column (150 × 4.6mm, 5𝜇m particle size, Agilent) with mobile phase consisting of acetonitrile and buffer. Various mobile phases have been trailed to get the best chromatographic separation among them are methanol and buffer, methanol and water, acetonitrile and water, acetonitrile and buffer. The use of the last mobile phaseacetonitrile and buffer system gave the finest chromatographic resolution with sharp symmetric peaks. Simultaneously determination of wavelength range also tried but good signal was observed when wavelength set at 227nm for Withaferin A.
Method development was achieved on Eclipse XDB-C18 (150 × 4.6mm, 5𝜇m particle size, Agilent) column using Acetonitrile and Buffer(35:65) in isocratic method, Flow rate 1.5ml/min, Detection at 227nm. Peak obtained at retention time 5.069 minutes for Withaferin A standard. The same conditions were followed for the separation of Withaferin A present in Withania coagulans and Withania somnifera fruit of chloroform extractsat retention time 4.969 and 5.085 minutes respectively. The calibration curve for Withaferin A was found to be linear from the concentration range 0.0106-0.17mg/ml; regression coefficient (R2) is 0.9998 for the compound studied shownin Figure 2.
Figure 2: Calibration Curve and Regression Coefficient of Withaferin A
The HPLC chromatogram of Withania coagulans fruit corresponding to standard Withaferin-A was showed at a retention time of 4.969min and Withania somnifera at 5.085min given in Figure 3. The variation in retention time of peak of Withaferin A in chromatograms of Withania coagulans and Withania somnifera may be due to the presence of other chemical constituents. The quantitative evaluation and comparison of Withaferin A present in fruit part of Withania coagulans was 0.0121 % and 0.0075% in Withania somnifera respectively. The Withaferin A content percentage was observed higher amount in Withania coagulans fruit compare to Withania somnifera fruits.
Table 1: Estimation of Withaferin A content present in Withania coagulans and Withania somnifera fruits.
|
S. No |
Name of the Plant |
Weight of sample (g) |
Weight of residue obtained (g) |
Concentration of test solution (mg/ml) |
Amount of Withaferin A present (mg/ml) |
Percentage of Withaferin A |
Mean of Withaferin A (%) |
|
1. |
Withania coagulans |
10.17 |
0.63 |
3.29 |
0.0072 |
0.0135 |
0.0121 |
|
3.92 |
0.009 |
0.0142 |
|||||
|
5.2 |
0.0076 |
0.009 |
|||||
|
2. |
Withania somnifera |
3.22 |
0.46 |
1.09 |
0.0006 |
0.0078 |
0.0075 |
|
17.35 |
0.096 |
0.0079 |
|||||
|
20.25 |
0.0098 |
0.0069 |
|
[Std.] |
|
[WS] |
|
[WC] |
|
Figure 3. HPLC Chromatograms of Withaferin A reference standard (Std.), Withania somnifera (WS) and Withania coagulans (WC) fruits extract |
CONCLUSION:
In this study, concludes the comparative estimation of Withaferin A from fruit parts of the two species of Withania genus samples using HPLC technique. The comparative studies done for variations of bioactive marker compound Withaferin A present in Withania coagulans and Withania somnifera fruit of chloroform extracts through HPLC method. Method development of Withaferin A consists of isocratic method mobile phase using Acetonitrile and Buffer (35:65), Flow rate 1.5ml/min, Detection at 227 nm, at retention time 5.069 minutes for Withaferin A standard. The same method was followed for the separation of Withaferin A present in Withania coagulans and Withania somnifera fruit of chloroform extractsat retention time 4.969 and 5.085 minutes respectively. Withania coagulans fruit sample contains higher percentage of Withaferin A (0.0121%) compare to Withania somnifera (0.0075%) fruit. This study can be used to develop quality control protocols for standardisation of Withania species on the basis of Withaferin A reference standard as a marker compound. It is useful for scientific as well as commercial applications of pharmaceutical industries.
CONFLICT OF INTEREST:
The authors have no conflicts of interest regarding this investigation.
ACKNOWLEDGEMENT:
The authors are very grateful to Director General and Deputy Director General, Central Council for Research in Ayurvedic Sciences, Ministry of AYUSH, New Delhi for their encouragement and providing facilities to conduct this study.Authors are also thankful to Dr. C. Arunachalam, RO (Botany), Department of Botany/ Pharmacognocy, CSMRSDDI, Chennai for providing support in authentication of plant materials.
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Received on 24.08.2022 Modified on 30.12.2022
Accepted on 10.06.2023 © RJPT All right reserved
Research J. Pharm. and Tech 2023; 16(9):4193-4198.
DOI: 10.52711/0974-360X.2023.00686