Introduction

Mangiferin obtained from the dried stem bark and leaves of Mangifera indica Linn. and having a molecular formula C ₁₉ H₁₈ O₁₁ and  molecular weight  422 . The major bioactive constituent of Mangifera indica is a xanthone c-glucoside mangiferin. The other constituents are triterpenen viz. mangiferolic acid, isomangiferolic acid and several cycloartenod derivatives a flavonoide amantoflavone.

Dried mango flowers, containing 15% tannin, serve as astringents in cases of diarrhea,   chronic dysentery, catarrh of the bladder and chronic urethritis resulting from gonorrhea. The bark contains Mangiferin and is astringent and employed against rheumatism and diphtheria in India. The resinous gum from the trunk is applied on cracks in the skin of the feet and on scabies, and is believed helpful in cases of syphilis.^1-10

Experimental

Chromatographic condition

The following HPLC condition was maintained through out the experiment.

Mobile Phase

( Buffer 85% Acetonitrile 15%)

Buffer

I weigh accurately 1.36 gm. of Potassium dihydrogen orthophosphate in 900 ml of HPLC grade water; adjust the P^H to 2.5 to 2.8 with dilute orthophosphoric acid   than make up the volume to 1000ml.  (Solvent A)

II) Acetonitrile (solvent B)

Detector: UV Detector.

Wavelength: 254 nm.

Flow rate: 1.0ml/min

Injection volume: 20µl

Run Time: For Standard: – 15 min; For Sample: – 15min

Data acquisition: Using suitable software CFR-21 Part 11 Compliant

Record the Chromatogram: Record the chromatogram with the following

Details: Retention time, Peak area, Peak height, % Area, Assigning the name to the Peaks.

Integration parameters: Integrate peaks base to base.

For calculating concentration, Mean, Standard Deviation, Relative Standard  Deviation, Relative retention time, Response factor, Theoretical plate, Resolution, Asymmetry, Tailing factor by EXCELL or any other suitable Software can be used.

Plotting the Graph: EXCELL and PRISUM software.

Standard solution

Weigh accurately 10.089 mg of Mangiferin in the 10ml volumetric flask; add 1ml of   D.M.F and make up the volume 10 ml by using methanol i.e. dilution I, Dilution II, III, IV, V, VI, VII were made consecutively by dissolving 5ml to 10ml  in sufficient quantity of methanol in a standard flask. The Peak area & Retention time for individual standard are tabulated in the table no. 02 and 03 respectively.

The concentration of analyte in 10 ml is; Mangiferin = = 9.7358 mg.

Sample solution 🙁 for raw material) The sample solution was prepared by weighing the required quantity of sample (coarse powder) and transferred to a 100 ml beaker. Extract with 10 ml of DMF (Dimethyl form amide) and 80 ml of HPLC grade methanol sonicate for 5 minutes, by warming on water bath for about 20 min. discard the supernatant liquid (extract) to a 250 ml beaker. Repeat the procedure 4-5 times till the raw material was completely extracted or till the extract is colorless. Collected all the extract in a 250ml beaker and concentrated it to below 100ml volumetric flask and made up the volume to 100ml with methanol, mixed well and filtered through 0.45µl membrane.

Procedure

Take required quantity of Mangifera indica extract or raw material such as leaves or bark of Mangifera indica,(After extraction) weighed and transfer into 100ml volumetric flask, dissolved in 10ml dimethyl form amide and 25ml of methanol of HPLC grade, sonicated for 5 minutes. Warmed on steam water bath for 5 minutes, cool and make up the volume to 100ml with Methanol. Inject 20µLof sample and record the chromatogram. Inject 3 times and calculated the results.

Graph 1:  Linear Graph of Mangiferin.   Click here to View figure

Graph 2: Graph showing retention time of Mangiferin.   Click here to View figure

Graph 3: Overlay spectra of Mangiferin by using different concentration.   Click here to View figure

Table 1:

Correlation coefficient (r2) for Mangiferin was found to be 0.9994 indicating that the method is linear between the concentration 15.212 to 973.589 µg mL-1.

Table 2: For Peak Area of Mangiferin.

Table 3: For Retention Time of Mangiferin.

Average of retention time = 6.7204 minutes

Results and Discussion

The main constituents present in Mangifera indica is mangiferin. Quantitative determination of mangiferin from Raw material and extracts of Mangifera indica was done.

The Present method was done on HPLC, using Isocratic pump, C₁₈ column with UV detector. As mangiferin is a polar molecule, its analysis was carried out using reverse phase HPLC method. The column used for separation of mangiferin was Lichrospher® C₁₈ with a particle size 5 µl. The mobile phase used was (Buffer 85% Acetonitrile 15%). The mangiferin have Absorption maxima at 254 nm. Therefore, the wavelength was chosen as 254 nm. From the typical chromatograms the Retention time for mangiferin was found to be 6.7204 min. The method is validated in terms of Linearity, precision, repeatability, accuracy, range, Quantification and specificity. A good linear relationship was observed for mangiferin with correlation coefficient (r²) of more than 0.9994 the chromatograms indicating that the method is free from interference. The repeatability and reproducibility indicated that the proposed method is precise and reproducible. The percentage recovery of different batches of extract and raw material were found to be in between 95-110%. This demonstrates that the developed HPLC method is simple, precise and accurate

Acknowledgements

The authors are grateful to Natural Remedies Private Limited, Bangalore for providing gift samples of Extract and Raw materials and for providing laboratory facility and constant encouragement.

References

1. The Ayurvedic Pharmacopoeia of India, by government of india Ministry of health and family welfare department of ISM and H, 1st Edition, Part -1, vol. –III, 9.
2. Molecular formula and molecular weight of mangiferin. (Source: http://www.made-in-china.com/showroom/byro 2000/product-list/Sap-Extract-1.html).
3. Clasificación of Mangifera indica. (Source :http://en.wikipedia.org/wiki/Mango)
4. Parts used for the estimation of mangiferin in Mangifera indica. (Source: http://www.rain-tree.com/mango.htm)
5. Ivan Ross A, Medicinal plants of the world, Published by Humana Press Totowa, New Jersey, 197.
6. Rastogi RP, Mehrota BN, Compendium of Indian Medicinal Plants, Central Drug Research Institute Lucknow; India, 1991; 265-6.
7. Rastogi RP, Mehrota BN, Compendium of indian Medicinal Plants, Central Drug Research Institute Lucknow; India, 1993; 407-9.
8. Rastogi RP, Mehrota BN, Compendium of indian Medicinal Plants, Central Drug Research Institute Lucknow; India, 1995; 456.
9. Rastogi RP, Mehrota BN Compendium of indian Medicinal Plants, Central Drug Research Institute Lucknow; India, 1998; 524.
10. Trease and Evans, A Textbook of Pharmacognosy, 14^th Edition, 249.