INTRODUCTION:

Mangiferin is known as xanthanoid glycoside and it plays an important role as α-glucosidase inhibitors for diabetes management. Mangiferin promotes glucose utilization by increasing its cellular uptake¹. Mangiferin has more medicinal importance because of its strong anti-oxidant², potential glucokinase activator³, anti-diabetic^4,5,6 and anti-inflammatory⁷actions. Mangiferin is a C-glycosyl compound, consists of 1,3,6,7-tetrahydroxyxanthen-9-one with a beta-D-glucosyl residue at the 6-position. Mangiferin and its derived main molecule have proven its effectiveness as an antioxidant, analgesic, antidiabetic, antiproliferative, chemopreventive, radioprotective, cardiotonic, immunomodulatory and diuretic⁸.

Mangiferin is main chemical constituents of Mangifera indica but it is also found in major proportion in Salacia species. Salacia, is a genus of the family Celastraceae is one of the medicinal plant groups broadly used to treat many ailments viz. hypoglycemia, hypolipidemia, inflammation, diabetic etc⁹. Salacia is known as ‘Ponkoranti’ in Ayurveda. Salacia species are broadly distributed in India, China, Malaysia, Sri Lanka and Vietnam¹⁰.Various chromatographic methods like high-performance liquid chromatography with Ultra violate visible spectroscopy (HPLC-UV)¹⁰, reversed-phase high-performance liquid chromatography (RP-HPLC) ¹¹, Liquid chromatography-mass spectrometry(LC-MS)¹² and High performance thin layer chromatography (HPTLC)¹³ are available for estimation of mangiferin in different plant extracts, bioanalytical samples like rat plasma, urine etc. but HPLC, LCMS like methods are tedious, costly, take more time for preparation of samples and mobile phase and optimization of instrumental method variables which are associated with this type of chromatographic methods. HPTLC is a helpful tool for identification plant, different raw materials, their extracts and formulations¹⁴. HPTLC is a modern separation technique which is concede as an exceedingly flexible and cost-efficient method¹⁵. Quantification of active constituents by using modern analytical tool is necessary for establishing the authenticity, creditability and usage of herbal formulations¹⁶. So many tests for standardization described in ancient literature seems to be based on observations and appears as subjective without valid scientific support. Hence there is need for standardization and development of reliable quality protocols of Ayurvedic formulations using modern techniques of analysis¹⁷. HPTLC analysis approach is the most potent tool for the quality control of herbal medicines because of its accuracy and reliability with ease of estimation, economic and faster analysis^18,19. Therefore, an effort has been made to develop simple, rapid, economic and sensitive HPTLC method for quantification of mangiferin in two species of Salacia like Salacia oblonga, Salacia reticulata and Antidiabetic polyherbal formulation Saptrangyadi vati. These validated HPTLC method will beneficial to rapid detection of mangiferin containing herbal formulations.

MATERIALS AND METHODS:

Reagents and Standard:

Standard mangiferin was purchased from Yucca enterprises, Mumbai. All chemicals like Methanol, Ethyl acetate, Formic acid (98%), HPLC grade water was purchased from Merck Specialities Pvt. Ltd. (Mumbai, India). Polyherbal antidiabetic formulation Saptarangyadi vati was purchased from local market.

Plant Materials:

The plant materials Salacia oblonga (SO) root and Salacia reticulata (SR) stem were purchased from Sai Anandha Export, Chennai. The drugs SO and SR were identified and taxonomically authenticated by Raw Materials Herbarium and Museum (RHMD) at National Institute of Science Communication and Information Resources, New Delhi (NISCAIR) and Department of bioscience, Saurashtra University, Rajkot respectively. A voucher specimen of authenticated plant materials with reference no. SU/DPS/Herbs/67(SO) and SU/DPS/Herbs/69(SR) were preserved in Department of Pharmaceutical Sciences, Saurashtra University, Rajkot.

Preparation of Plant Extracts:

Dried Salacia oblonga root powder (10g) and Stem powder of Salacia reticualta (10 g) were macerated with Ethanol: Water (50:50) at room temperature for 24 h. The extracts were filtered through Whatman filter paper No. 1 and the filtrates were concentrated under reduced pressure at 40⁰C. The hydro-alcoholic extracts of Salacia oblonga root (HASO) and Salacia reticualta stem (HASR) were dried, weighed (0.26gm for HASO and 0.5gm for HASR) and stored at 4 ◦C in storage vials for experimental use.

Preparation of Standard mangiferin solution:

Accurately weighed 10 mg of mangiferin was dissolved in 10 ml volumetric flask using 10 ml of methanol (1mg/ml). 500μl was taken from it and diluted with 10ml methanol in 10 ml volumetric flask (50μg/ml).

Preparation of Test solution:

Accurately weighed 100 mg of HASO and HASR were dissolved in 10 ml methanol in 10 ml volumetric flasks separately (10mg/ml).

Preparation of poly-herbal formulation extract:

Weighed accurately 5 tablets of poly-herbal formulation and powdered it. Tablet powder was weighed equivalent to about 100 mg and transferred in to 10 ml volumetric flask containing 4-5 ml methanol. Sonicate it for 30 min and volume made up with methanol. Extract was filtered through Whatman filter paper No. 1

HPTLC apparatus and Condition:

A Hamilton microliter syringe (100 μl) was used to apply standard and samples bands (6 mm width) with under a controlled nitrogen gas using a CAMAG Linomat V sample applicator (Switzerland). As a stationary phase precoated TLC silica gel aluminium Plates 60 F254 (10 cm × 10 cm, 20cm x 10cm), (100-μm thickness, Merck, Darmstadt, Germany) were used. The slit dimension was kept at 4 mm × 0.30 mm and a scanning speed 20 mm/s. Method development was done in CAMAG twin trough a glass chamber (20 cm × 20 cm) saturated with the mobile phase. The mobile phase consisted of ethyl acetate: formic acid: water (4:0.5:0.5 v/v/v). Prior to development, the chamber was saturated with the mobile phase for 30 min at room temperature. Densitometry scanning was applicable over CAMAG TLC Scanner "Scanner_171010" operated using win CATS software (Version 1:4:6:2002). Visualization of developed plates was done in CAMAG TLC visualizer at 254 nm wavelength.

Estimation of mangiferin in Salacia species and Herbal formulation:

The test solutions of HASO, HASR and poly herbal formulation were spiked triplicate 10 μL respectively on precoated TLC silica gel aluminium Plates 60 F254 with the CAMAG Linomat V sample applicator. The plate was developed in mobile phase. The peak areas and absorption spectra were recorded. The amount of mangiferin in the HASO, HASR and herbal formulation were calculated for each solution by use of the calibration plot.

Method validation:

Method validation was done as per the International Conference on Harmonization (ICH) guidelines Q2 (R1)15. The method was validated for linearity, precision, accuracy, specificity, robustness, limit of detection (LOD) and limit of quantification (LOQ)²⁰.

Linearity:

Linearity of the method was studied by injection of six known concentrations of mangiferin in the range of 100-600ng/spot in triplicate. The calibration curve was obtained by plotting the peak areas versus the amounts of the standard. The result was expressed in terms of correlation coefficient.

Precision:

Precision studies were carried out to check reproducibility of the proposed developed method. Intraday precision study was carried out by applying six times 300 ng per band of same concentration. It can be analysing at three different times in a day for intraday precision and the same procedure was followed for three different days to determine interday precision. The precision was expressed as percentage of relative standard deviation (%RSD).

Accuracy:

In Accuracy recovery was evaluated. For Accuracy studies three level (50%, 100% and 150%) of known amount of test sample was added in the definite quantity of standard. Samples were prepared in triplicate.

Specificity:

Specificity is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present. So, for specificity evaluation of Mobile phase, dissolving solvent methanol, blank (no band application) for stationary phase, standard, and test solutions were applied on plate and find out purity of sample spot corresponding to Mangiferin in samples and formulations by superimposing the spectrum of standard and sample peaks.

Robustness:

Robustness was studied in triplicate at 300ng/band by done small variation in optimised method parameters such as variation in scanning wavelength and chamber saturation time.

Limit of detection (LOD) and limit of quantitation (LOQ):

LOD and LOQ were determining based on standard deviation (SD) and slope (S) of calibration curve using following formula.

LOD = 3.3(SD/S)

LOQ = 10 (SD/S)

RESULTS AND DISCUSSION:

HPTLC is also an important tool for identification and isolation of different plant markers in their extracts, different formulations^21,22 and adulteration²³. HPTLC has powerful potential as a substitute chromatographic model for approximate partitioning properties in reinforce of environmental, and combinatorial chemistry^24,25. A fast, accurate, precise and economic HPTLC technique was developed for the analysis of mangiferin in two species of Salacia and Poly herbal formulation. From several trials, the mobile phase ethyl acetate: formic acid: water (4:0.5:0.5 v/v/v) was finalised which gave good resolution and confirmed the presence of mangiferin at 0.38±0.01 Rf value observed under 254nm. The chromatogram of mangiferin standard and samples were shown in Fig. 1.

Fig. 1: HPTLC chromatogram of mangiferin standard and sample (a) Mangiferin (Standard), (b) Hydroalcoholic extract of Salacia oblonga root, (c) Hydroalcoholic extract of Salacia reticulata stem, (d) Antidiabetic polyherbal formulation

Mangiferin was present in 1.02% in Salacia oblonga root extract, 0.94% in Salacia reticulata stem extract and 0.42% in polyherbal formulation. Validation parameters such as Accuracy, Precision, Specificity, Robustness, LOD and LOQ have been studied. The calibration curve was constructed from the peak area versus the concentration of the standards and exposed that the method was linear across the range of 100-600 ng/band with r² = 0.99854 correlation coefficient (Table 1 and Fig.2)

Table 1: Method validation parameter for quantification of mangiferin by HPTLC

Validation parameter	Results
Method	Linear
Range	100-600ng/band
Correlation coefficient (r2)	0.99854
Regression Equation^#	Y=594.319+19.695*X
SD	2.98%
LOD	0.50ng/band
LOQ	1.51 ng/band

^#X is the concentration of Mangiferin in ng/mL; Y is peak area at 254 nm.

Fig. 2 Calibration curve of mangiferin

Fig. 3 Overlay graph of standard and sample peaks

Table 2: Intraday and interday Precision of HPTLC method

Intraday precision
Mangiferin concentration	Area^#±SD	%RSD
300 ng/band	6561.52±78.52	1.20
300 ng/band	6647.83±75.60	1.14
300 ng/band	6596.20±61.62	0.93
Interday precision
Mangiferin concentration	Area^#±SD	%RSD
300 ng/band	6561.52±78.52	1.20
300 ng/band	6594.17±64.58	0.98
300 ng/band	6272.52±76.64	1.22

^#Each value is mean of six observations

Table 3 Recovery study of Mangiferin

levels	Amount of samples in ng/band	Known amount of standard added (ng)	Theoretical total amount (ng)	Obtained concentration (ng) n=3 Mean±SD	% Recovery n=3
50%	200	100	300	318.64±2.5	105.64
100%	200	200	400	399.95±0.44	99.98
150%	200	300	500	519.30±0.87	103.86

Method precision was studied using the sample solution containing mangiferin 300ng. Percentage relative standard deviation (%RSD) values were found to be less than 2% (Table 2) showed the acceptable precision of the method. The accuracy of the method represented by recovery studies ranged between 99.98% and 105.64% (Table 3). The LOD and LOQ were found to be 0.50ng/band and 1.51ng/band respectively, indicating the high sensitivity of the method (Table 1). Overlay graph of standard and sample peaks showed that method is very specific for mangiferin analysis (Fig.3). Results of validation parameter (Table 1, 2 and 3) showed that the developed HPTLC method is accurate, robust, linear, rapid and specific for estimation of mangiferin.

CONCLUSION:

The present study demonstrates a simple, accurate and precise HPTLC method for the mangiferin estimation. In addition, the proposed method does not impose a complex treatment or sophisticated analytical elements which are usually required with high performance liquid chromatography and liquid chromatography with mass spectrometry studies. Finally, the proposed method was rapid, economic and specific. So, it can be advised to use this validated HPTLC method for the routine analysis of the mangiferin, either in bulk form or in their polyherbal dosage forms.

ACKNOWLEDGMENT

The authors are gratefully acknowledged the financial support provided by UGC-BSR meritorious fellowship.

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Received on 17.05.2022 Modified on 21.03.2023

Research J. Pharm. and Tech 2023; 16(11):5480-5484.

DOI: 10.52711/0974-360X.2023.00886

*Corresponding Author E-mail: jalpa.sanandia@gmail.com