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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 6  |  Issue : 4  |  Page : 248-257

Identification and estimation of vicine bioactive compound in Momordica charantia L. fruit and coded Ayurvedic formulation using HPLC method


1 Regional Ayurveda Research Institute, Gwalior, Madhya Pradesh, India
2 Central Council for Research in Ayurvedic Sciences, New Delhi, India
3 Captain Srinivasa Murthy Central Ayurveda Research Institute, Chennai, Tamil Nadu, India
4 Central Ayurveda Research Institute, Kolkata, West Bengal, India

Date of Submission23-Jul-2021
Date of Acceptance20-Jan-2022
Date of Web Publication17-May-2022

Correspondence Address:
Ajay Kumar Meena
Regional Ayurveda Research Institute, Gwalior, Madhya Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jdras.jdras_19_21

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  Abstract 

BACKGROUND AND OBJECTIVE: Momordica charantia L. is one of the important Ayurvedic drugs having anti-diabetic, anti-viral, anti-ulcerogenic, anti-tumor, immunomodulatory, and anti-lipolytic and hepatoprotective properties. This study was designed for comparative fingerprint profiling of vicine bioactive compound in M. charantia L. fruit extract and coded Ayurvedic formulation extract through high-performance thin layer chromatography (HPTLC) and for developing rapid high-performance liquid chromatography (HPLC) method for the determination of vicine in fruit extract and coded Ayurvedic formulation. MATERIALS AND METHODS: The dried powder of M. charantia L. fruit (10.3448 g) and powder of coded Ayurvedic formulation (13.4975 g) were extracted by using a Soxhlet apparatus for 24 h. For HPTLC finger printing, ethyl acetate: methanol: water: formic acid (7:3:1:0.5; v/v/v/v) was used as a solvent system. The quantitative analysis was performed by HPLC using acetonitrile and water (65:35 v/v) as mobile phase, with 1.0 mL/min flow rate at 275 nm, and the retention time of vicine was 3.688 min. RESULTS AND DISCUSSION: A band was obtained at 254 nm (green, Rf = 0.31) in standard and test solution tracks of M. charantia L. fruit extract and coded Ayurvedic formulation extract corresponding to vicine. The calibration plot showing linear relationship with concentration has been observed. A linear equation is y = 12.224x + 7.829, and the goodness of fit (r2) of 0.99 was obtained from the regression analysis, which showed proportional dependence between concentration and peak area. CONCLUSION: Results of the HPLC analysis showed that the coded Ayurvedic formulation extract and M. charantia L. fruit extract contains 0.0273% and 0.0603% of vicine biomarker compound, respectively.

Keywords: Alkaloid, anti-diabetic, coded Ayurvedic formulation, M. charantia, quantification, vicine


How to cite this article:
Meena AK, Singh A, Swathi KN, Ojha V, Dixit AK, Ilavarasan R, Srikanth N. Identification and estimation of vicine bioactive compound in Momordica charantia L. fruit and coded Ayurvedic formulation using HPLC method. J Drug Res Ayurvedic Sci 2021;6:248-57

How to cite this URL:
Meena AK, Singh A, Swathi KN, Ojha V, Dixit AK, Ilavarasan R, Srikanth N. Identification and estimation of vicine bioactive compound in Momordica charantia L. fruit and coded Ayurvedic formulation using HPLC method. J Drug Res Ayurvedic Sci [serial online] 2021 [cited 2022 Jun 29];6:248-57. Available from: http://www.jdrasccras.com/text.asp?2021/6/4/248/345394




  Introduction Top


Momordica charantia L. from Cucurbitaceae family is used as holistic folk medicine in the treatment of various ailments since ages [Figure 1]. Various pharmacological activities of M. charantia L. have been reported showing anti-fertility, anti-diabetes, anti-lipolytic, anti-ulcerogenic, antibacterial, hepatoprotective, anti-tumor, antioxidant, anti-mutagenic, anthelmintic, and anti-inflammatory potentials.[1],[2],[3] Cucurbitacins having cucurbitane skeleton are highly oxygenated, triterpenic, tetracyclic, bitter taste plant substances found in Cucurbitaceae family plants. Studies indicated that cucurbitane types are responsible for hypoglycemia and anti-diabetic activities in M. charantia L.[4] Gurmarin, a polypeptide which is present in fruit and leaf of M. charantia L., has similarity with bovine insulin and exhibits sugar-regulating efficacy by modulating neural responses to sweet taste stimuli.[5]
Figure 1: Plant materials of M. charantia L. fruit

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Heteropolysaccharide composed of glucose (Glu), galactose (Gal), rhamnose (Rha), arabinose (Ara), and mannose (Man) forms about 6% powder of bitter gourd.[6] The unsaturated fatty acid content in M. charantia fruit is relatively higher; the ratio of monounsaturated fatty acids to total fatty acids is about 20.1%, and of the total fatty acid, nearly 64.3% is polyunsaturated fatty acid.[7] Catechin, gallic acid, gentisic acid, chlorogenic acid, and epicatechin are important phenolic constituents in the M. charantia L. fruit extracts.[8] Cucurbitane compounds, i.e., momordicosides A, C, F1, I, and K; goyasaponins I, II, and III; goyaglycosides a, b, c, d, e, f, g, and h, were already reported in the methanol extract of M. charantia L. fruits.[9] The vicine content in seeds, leaves, and fruits of M. charantia L. was 0.524%, 0.0456%, and 0.115%, respectively.

Vicine (2,6-diamino-5-[(2S, 3R, 4S, 5S, 6R)-3, 4, 5-trihydroxy-6-(hydroxymethyl)-2-tetrahydropyranyl]oxy-1H-pyrimidin-4-one) is an alkaloid glycoside [Figure 2]. Studies reported that this pyrimidine nucleoside induced hypoglycemic effect in non-diabetic fasting rats. Vicine causes hemolytic anemia called favism in people with inborn glucose-6-phosphate dehydrogenase enzyme deficiency.[10]
Figure 2: Chemical structure of vicine

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In this study, we have selected an Ayurvedic coded formulation and M. charantia L. fruit, which is used to maintain diabetes. The selected coded Ayurveda formulation contains M. charantia L. and nearly 11 other plant crude drugs. The literature suggests various analytical methods for isolation and determination of vicine in M. charantia L. and in compound formulation.[11],[12],[13] Therefore, in this study, vicine, one of the most important active biomarkers having various physiological actions, was selected for the identification quantification in formulation and its ingredient.


  Materials and Methods Top


Coded Ayurvedic formulation, its ingredient, and M. charantia L. fruit were provided by the Central Council for Research in Ayurvedic Sciences (CCRAS), Ministry of Ayush, Government of India, New Delhi.

The chemicals, reagents, and solvents for the study were procured from E. Merck and of HPLC and AR grade. Bioactive marker was purchased from M/s Natural Remedies, Bengaluru, Karnataka, India.

HPTLC fingerprint profile of vicine in M. charantia L. fruit extract and coded Ayurvedic formulation extract

Extraction

The dried powder of M. charantia L. fruit (10.3448 g) and coded Ayurvedic formulation (13.4975 g) were extracted separately with water (200 mL) using a Soxhlet apparatus for 24 h, dried under reduced pressure, collected, weighed, and stored for studies. The obtained extracted residual weights of M. charantia L. fruit and coded Ayurvedic formulation were 4.2304 and 3.8044 g, respectively (Residue A and Residue B).[14],[15],[16],[17]

Standard solution

Standard vicine solution has been prepared by dissolving 4.2 mg of vicine standard in 10 mL of HPLC grade water to get a 0.42 mg/mL vicine stock solution.

Test solution

The test solution for HPTLC analysis and identification of vicine, was prepared by dissolving 50 mg of residue A and residue B in water and filtered by 0.22 μm membrane filters.

Solvent system

Ethyl acetate: methanol: water: formic acid were taken in the ratio of 7:3:1:0.5; v/v/v/v.

Procedure

An aliquot of 4 µL of coded Ayurvedic formulation extract and M. charantia L. fruit extract and vicine standard solution was applied separately on different tracks of silica gel 60F254-coated TLC plate (0.2 mm thickness) using automatic sample applicator and developed.

Detection and scanning

The TLC plate was observed at UV wavelengths of 254 and 366 nm by TLC visualizer having deuterium and mercury lamps, and photographs were recorded. The plate was again dipped in vanillin-sulfuric acid solution and heated at 105°C using hot air to get color spots and scanned at 540 nm and the photo was documented. WIN CATS software was used for fingerprints data.

Method development and optimization for quantitative estimation using HPLC

About 0.42 mg/mL (see the Standard solution section) vicine stock solution and 0.50 mg/mL (see the Test solution section) sample solution were taken for this analysis. To optimize the chromatographic conditions on a C18 (250 cm × 4.6 mm; 5 μm) column, a series of trails by changing the concentration of acetonitrile and water were performed. Acetonitrile and water buffer in the ratio 65:35 v/v at 1 mL/min flow rate, 10 μL injection volume, 10 min run time, with 30°C column temperature at 275 nm wavelength (λ) and DAD detector was observed to be the best suitable chromatographic condition for this study.[18],[19],[20],[21]


  Results and Discussion Top


HPTLC fingerprint profiling

Photos of the TLC plate were documented at 254 and 366 nm, after treatment with vanillin-sulfuric acid reagent, and Rf values were calculated [Figure 3] and [Table 2]. The TLC plate was scanned at 254, 366, and 540 nm [Figure 4][Figure 5][Figure 6].
Figure 3: HPTLC fingerprint profiling

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Table 1: Estimation of vicine biomarker compound

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Table 2: Rf values of test solutions and vicine reference standard

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Figure 4: HPTLC fingerprint profile at UV 254 nm

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Figure 5: HPTLC fingerprint profile at 366 nm

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Figure 6: HPTLC fingerprint profile at 540 nm

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A band at 254 nm (green, Rf = 0.31) corresponding to vicine is found in standard and test solution tracks of M. charantia L. fruit extract and coded Ayurvedic formulation extract, but a band of vicine is not visible in UV at 366 nm and white light.

Quantitative estimation of vicine by HPLC

In the HPLC analysis, vicine was eluted with symmetrical peak at retention time (RT) of about 3.688 min [Figure 7].
Figure 7: HPLC chromatogram of coded Ayurvedic formulation, Momordica charantia L. fruit, and vicine biomarker compound

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Calibration curve

Vicine stock solution (0.42 mg/mL) was diluted to get 0.105, 0.0525, 0.02625, 0.013125, 0.00656 mg/mL concentrations and run through HPLC system. Standard calibration curve was made [Figure 8].
Figure 8: HPLC of vicine standard and calibration curve

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Estimation of vicine

The quantification of vicine in coded Ayurvedic formulation extract and M. charantia L. fruit extract was done using HPLC. Samples and standard showed respective peaks of vicine at the same RT. Coded Ayurvedic formulation extract and M. charantia L. fruit extract were injected (10 μL) to HPLC, and chromatograms were analyzed to get the concentration from the standard calibration curve.

Similar to the vicine standard, the HPLC of M. charantia L. fruit extract and coded Ayurvedic formulation extract was also obtained at an RT of 3.688 min, at 275 nm wavelength. The vicine contents in coded Ayurvedic formulation and M. charantia L. fruit extract were presented in [Figure 7] and [Table 1].

The quantitative evaluation of vicine present in the coded Ayurvedic formulation extract and M. charantia L. fruit extract was 0.0273% and 0.0603%, respectively.


  Conclusion Top


Identification, separation and estimation of bioactive components in plants material are still considered as strenuous process as plant materials are multi-component mixture of different biomolecules and requires combination of several chromatographic techniques and various other purification methods to isolate. Standardization, optimization, method validation protocol of herbal formulations, and its bioactive constituents are of utmost importance to ensure quality, purity, efficacy, and safety. Presently, in Ayurvedic drug development modern and sophisticated analytical tools like HPLC and HPTLC are equally essential for quality control and quality assurance of the raw drug and formulation. This study showed the presence of vicine biomarker in coded Ayurvedic formulation, which reveals the presence of M. charantia L. in the formulation. Results of the present study have also established a successful HPLC and HPTLC method to identify and estimate vicine biomarker from M. charantia L., which can be conveniently employed for routine quality control analysis of the marker compounds in formulations with this ingredient.

Acknowledgments

I would like to thank all the people of scientists, New Delhi who contributed in some way to the work described in this study.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.





 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
 
 
    Tables

  [Table 1], [Table 2]



 

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