Indonesian Journal of Chemical Research http://ojs3.unpatti.ac.id/index.php/ijcr Indo. J. Chem. Res., 8(2), 15-20, 2020 DOI: 10.30598//ijcr. 2020.8-yun 15 Stability Indicating RP-HPLC for Quantification Mangiferin in Extract of Three Species Mango Leaves Yuni Retnaningtyas*, Nia Kristiningrum, Hidayah Dwi Renggani, Indah Purnama Sary 1 Departement of Chemistry, Faculty of Pharmacy, University of Jember Jl. Kalimantan 37 Kampus Tegal Boto Jember * Corresponding Author: ifir_retnaningtyas@yahoo.co.id Received: 2019-12-20 Received in revised: 2020-4-29 Accepted: 2020-5-28 Available online:2020-5-31 Abstract The stability indication of Reversed Phase-High Performance Liquid Chromatography (RP-HPLC) method was validated for quantitative determination of mangiferin on three species mango leaves (Mangifera odorata Griff, Mangifera foetida Lour, and Mangifera indica L.). The samples were extracted by maseration method using methanol and concentrated using rotary evaporator. The method carried out on stationary phase a purospher RP-18 endcapped (25 cm × 4.6 mm i.d., 5 µm) column with a mobile phase consisting of methanol: phosphoric acid 0.1% (v/v) (31:69); flow rate:0.8 mL/min; solvent methanol, detection was carried out at 258 nm. The analytical performace this measurement is good with the value of linearity (r 2 =0.998), precision (%RSD=0.649%), and accuration (10.67%). The forced degradation studies were carried out according to the International Conference on Harmonization (ICH) guidelines. The results indicating that the complete separation between degradation products and mangiferin peak occured. The degradation limit of mangiferin 5–20% (according to the guideline of ICH) except in basic condition (100%). The method was succesful applied to determine of the mangiferin in pakel (Mangifera foetida), kweni (Mangifera indica) and kopyor (Mangifera odorata) extract. The mangiferin content was obtained are pakel (9.95%), kopyor (7.40%) and kweni (Mangifera odorata) (2.49%) respectively. Keywords: Mangiferin, Mango leaf, Mangifera odorata Griff., Mangifera foetida Lour, Mangifera indica L., RP-HPLC, validation INTRODUCTION The Mango was easily found in Indonesia and distributed in all region of Indonesia. Indonesia was one of the country with the highest production of mango (Husen et al., 2012). But, until now only the fruit have been used while the leaves not yet. Mango leaves (Mangifera indica L.) from Anarcadiaceae family contains many chemical compounds such as phenol, β-carotene, flavonoid, tannin, saponin, alkaloid and steroid (Palafox-Carlos et al., 2012 ; Pino et al., 2011). One of the phenolic compounds that found in mango was mangiferin. Mangiferin can be found in all parts of mango plants as peel, pulp, seed kernel (Luo et al., 2012) , bark (García-rivera et al., 2011) and leaves (Jutiviboonsuk et al., 2010). The mangiferin was a phenolic compound that has poten antioxidant activity, and multifactorial pharmacological effects, including antidiabetic, antitumor, lipometabolism regulating, cardioprotective, anti-hyperuricemic, neuroprotective, anti-inflammatory, antipyretic, analgesic, antibacterial, antiviral and immunomodulatory effects (Mirza et al., 2013; Du et al., 2018). Mangiferin is c- glucosyl xanthone and its structures (Figure 1) 2-C-D- glucopyranosyl-1,3,6,7-tetrahydroxy xanthone, fulfill to Lipinski rules (Campa et al., 2012). Based on chemotaxonomy, mangiferin can be found in another mango species; more related the plant so the chemical compound will be more similar (Subha et al., 2007). But, the amount of the compound can be affected by some factors such as the location of cultivation, variety, and stage of maturity. Figure 1. Structure of Mangiferin Mangiferin was quantified only in one species of mango (Mangifera indica L.) while there are 62 species of mango (Pracaya, 2005). Quantification of mangiferin can be done using High-performance thin layer chromatography (HPTLC) (Subha, et al., 2007), Yuni Retnaningtyas et al. Indo. J. Chem. Res., 8(2), 15-20, 2020 DOI: 10.30598//ijcr. 2020.8-yun 16 liquid chromatography-mass spectrometry (LC-MS) (Ilango, et al., 2014) and High-performance liquid chromatography (HPLC) (Zhang, et al., 2014; Eddy, et al., 2014). So, the aim of present study was to develop a stability-indicating RP-HPLC assay method for mangiferin. The developed HPLC assay method was validated as per ICH guidelines Q2 (R1) (Anonim, 2005) and to determine the highest contents of mangiferin in three species of mango leaves so can be used as traditional medicines. METHODOLOGY Materials and Instrumentals Mangiferin standard was purchased from Sigma- Aldrich, India. Methanol used for HPLC was analytical grade and purchased from Sigma-Aldrich, Germany. Water used for HPLC was purchased from WIDA WITMUnicap. Another material used in this analysis were technical methanol, technical phosphoric acid, and membrane filter (0.22 µm). Kweni, pakel and kopyor leaves that were used in this study must had dark green color and taken from the plagiotrop branch and cultivated on January 2019. All the species used had been identified by Faculty of Science University of Jember. The research was carried out at the laboratory for analytical chemistry at Departement of Chemistry Faculty of Pharmacy Jember University. Methods Instrumentation and analytical condition The chromatographic analysis was performed using HPLC Shimadzu Prominence integrated with UV detector, on a purospher® STAR RP-18 end capped with 5 μm particle size. 4.6 mm internal diameter and 250 mm lenght (Merck, Darmstadt, Germany) column with flow rate 0.8 mL/min, wavelength 258 nm, injection volume 20 µL and optimum concentration 10 µg mL -1 . Mobile phase consists of methanol: phosphoric acid 0.1% (v/v) (31:69) with isocratic elution technique. Preparation of mobile phase The water amount 31 mL of was added by 10% of phosphoric acid (Solution 1). 69 mL of methanol and solution 1 were mixed using Erlenmeyer and filtered through 0.22 µm membrane filter. Extraction of Kweni, Pakel, and Kopyor leaves Leaves were washed and dried at room temperature for seven days. The leaves were heated with the oven at 50 o C for 30 minutes and blend to make the smaller size. Leaves powder weighed 100 mg and extracted by maceration method using methanol 500 mL for 24 hours. Then sample concentrated using rotary evaporator at 50 o C under pressure (Irmawan, et al., 2018) Preparation of standard stock solution The standard stock solution was prepared by weighing 1 mg of mangiferin standard and transferred to 10 mL clean dry volumetric flask. The volume was made up with methanol to obtain 100 µg/mL of mangiferin. The solutions were further diluted with the same solvent to obtain the needed concentration of mangiferin standard. Preparation of sample solution The sample solutions were prepared by weighing 1 mg of extract of kweni, pakel and kopyor leaves and transferred to 10 mL clean dry volumetric flask. 4 mL of solvent added and sonicated for 30 min in cold water. Finally, the volume was made up using methanol to obtain 100 µg/mL of the extract kweni leaves. The solutions were further diluted with the same solvent to obtain the concentration of extract kweni leaves 10 µg/mL. Validation method Validations method was performed using kweni leaves extract. The method of analysis was validated using recommendation of ICH for parameters like system suitability, linearity, accuracy, precision, detection limit, and quantitation limit. 1. System suitability The system suitability was determined by injected standard solution of mangiferin 10 μg/mL six times into system and chromatograms were recorded. The system suitability was determined by %RSD (Relative Standard deviation) of retention time and peak area, theoretical plates and tailing factor. 2. Specificity The blank solution, standard solution, and sample solution were injected simultaneously into the system and chromatograms were recorded. Specifity was determined of analyzing the cromatogram of sample in comparison with those obtained for mangiferin standard solution and blank solution aiming at confirming that none of the matrix interfere with the quantitation of the extract. 3. Linearity Linearity was determined by a least-square linear regression routine using the compound peak area and concentration of the working standard Yuni Retnaningtyas et al. Indo. J. Chem. Res., 8(2), 15-20, 2020 DOI: 10.30598//ijcr. 2020.8-yun 17 solutions prepared at seven concentration levels (6.08; 8.1; 10.1; 12.1; 16.2; 18.2 and 20.2 μg/mL) were prepared from working standard. 20μL each concentrations were independently injected into HPLC system. The linearity of the method was evaluated by calculation of correlation coefficient of calibration curves according to the ICH. 4. The limit of detection (LOD) and limit of quantitation (LOQ) The limit of detection (LOD) and limit of quantitation (LOQ) were calculated using following formula: LOD= 3.3(SD)/S and LOQ= 10 (SD)/S, where the SD=standard deviation of response (peak area) and S= average of the slope of the calibration curve (Pangabean, et al., 2016). 5. Intraday and interday precision The precision was assessed at intraday and interday time. The intraday precision was determined by measuring kweni leaves extract 100 μg/mL injected six times on the same day. The intermediate (interday) precision was estimated by injecting kweni leaves extract prepared at the same concentrations on three different days. Results were reported in terms of relative standard deviation (RSD) (Napitupulu, et al., 2019). 6. Accuracy The accuracy of the method was determined by calculating percentage recovery using standard addition method. The concentration of the standard had been added was 30, 45, and 60% of the concentration of the kweni leaves extract. Procedure for forced degradation studies Forced degradation studies were carried out to provide some information about the standard mangiferin and sample solutions stability during analysis. Those solutions were analyzed over a period of 24 h at room temperature. The force degradation studies were conducted by exposing the standard and sample solution with various degradation conditions such as acidic (2 N HCl for 30 min at 60 °C), basic (2 N NaOH for 30 min at 60 °C), neutral (refluxing the extract in water for 6 hours at 60 °C), oxidative (20 % H2O2 for 30 min at 60 °C), thermal (105 °C for 6 h ), and photolytic (UV chamber for 7 days ) (Naim, et. al., 2018, Bandla, et al., 2018). RESULT AND DISCUSSION The research indicated that the system suitability parameters were obtained with the mobile phase containing methanol 0.1% (v/v) of phosphoric acid (31:69 % v/v). The mobile phase eluted the extract at retention times 18.939 min. The suitability parameters like resolution, tailing factor, theoretical plate count and % RSD for peak area of five replicate injections of the standard are within limits. The corresponding chromatogram was shown in Figure 2 and the data are presented in Table 1. Figure 2. Typical sample chromatogram The calibration curve for mangiferin content was found to be linear over the range of 6.08-20.20 µg mL -1 . The linier regression equation obtained was y = -62737.69 + 88267.09x, where 𝑦 is the peak area and 𝑥 is the standard solution concentration. The correlation coefficient (r 2 ) 0.998171. The data of regression analysis of the calibration curve is shown in Table 2 and Figure 3. Table 1. System Suitability parameters No. Parameters Mangiferin 1 Tailing factor (Tf) 0.785 2 Resolution (Rs) 9.690 3 Retention time (Rt) 18.939 4 Theoretical plates (N) 1116.367 Table 2. Data of linearity Results Method Linierity Probability 95% Number of data 7 Equation Y = -62737.69 + 88267.09X Correlation coefficient 0.9982 The % RSD value were less than 2.0% for all concentrations tested and confirmed the suitable intraday and interday precision of the method. The results obtained for the intraday and interday precision are shown in Table 3. The Rt of mangiferin of standard solution and sample solution are identical. Moreover, the blank solution doesn't produce any peak. Hence the proposed analytical method is specific for estimation of mangiferin. The LOD for mangiferin was found to be 0.544 μg mL - while LOQ was 1.633μg mL - , respectively. The mean recoveries Yuni Retnaningtyas et al. Indo. J. Chem. Res., 8(2), 15-20, 2020 DOI: 10.30598//ijcr. 2020.8-yun 18 were found to be 101 to 102.5%. These results demonstrate accuracy for the determination of mangiferin in kweni leaves extract. The accuracy test parameters are summarized in Table 4. Figure 3. Curve of calibration The method was continued for testing at the stability of the samples under various stress conditions. Solution of mangiferin standard was exposed toward, acid (2.0 N HCl for 30 min at 60 °C), base (2.0N NaOH for 30 min at 60 °C), oxidizing agent (20% H2O2 for 30 min at 60 °C), thermal (105 o C for 6 h),UV Light (keeping the standard solution in UV chamber for 7 days). Degradation of drug substances between 5 and 20% has been accepted for validation of chromatographic assays (Taylor et al., 2012). Table 3. The result of precision test Precision (% RSD) Intraday Interday* n=6 First day Second day Third day 0.649% 0.649% 1.295% 1.212% RSD: Relative Standard Deviation, *Average of six determination Table 4. The result of accuracy test Addition of Standards *Theoretical Mass (mg) *Experiment Mass (mg) #%Recovery % RSD 30% 0.1652 0.1669 101.0 1.20 45% 0.1770 0.1791 101.2 1.55 60% 0.1982 0.2036 102.8 1.60 *Average of three estimation of extract, #Average of three estimation at each level The method was able to detect 18.14 % of decomposition in neutral hydrolysis condition. The chromatograms observed from samples, subjected to various stress conditions, are shown in Figures 4a to 4f. The amount of drug decomposed at various stress conditions are shown in Table 5. From the results, it was found that mangiferin was degraded 100 % in basic condition. In case of acid hydrolysis, the degradation was below the limit for mangiferin. It makes us choosing the pH of buffer in acidic region. 4(a) 4(b) 4(c) 4(d) 4(e) Yuni Retnaningtyas et al. Indo. J. Chem. Res., 8(2), 15-20, 2020 DOI: 10.30598//ijcr. 2020.8-yun 19 4(f) Figure 4. HPLC Chromatogram (a) acid degradation, (b) basic degradation, (c) neutral degradation (d) peroxide degradation, (e) thermal degradation, (f) photo degradation Table 5. Degradation Study of Mangiferin % Degradation Acid hydrolysis Base hydrolysis Neutral hydrolysis Oxidat ion Heat UV 3.79 100 18.14 2.19 11.81 7.90 Table 6. Quantification results of mangiferin in kweni, pakel and kopyor Samples *Weight (mg) *Mass of analyte (mg) *Content (%w/w) %RSD Pakel 5.150 0.5493 9.95 2.13 Kopyor 5.419 0.4014 7.40 1.41 Kweni 5.18 0.1287 2.49 0.65 The next step using the chromatographic analysis is the quantification of mangiferin in kweni, pakel and kopyor. The results of the quantification can be seen in Table 6. The highest concentration of mangiferin found in the pakel leaves extract. We hope there is some research in the future about pakel leaves used as traditional medicines. CONCLUSION The proposed stability indicating RP-HPLC method was found to be simple, sensitive, rapid, economical and useful for routine analysis of Mangiferin in the extract of Kweni leaf (Mangifera odorata Griff.), Pakel (Mangifera foetida Lour.), and Kopyor (Mangifera indica L.). The statistical parameters and recovery studies were carried out and reported. The obtained results were satisfactory according to the ICH guidelines. The study showed the leaves of three mango species contain mangiferin with the highest concentration was species pakel. REFERENCES Husen, S., Kuswanto, Ashari, S., and Basuki, N., 2012. Induction of Flowering and Yield of Mango Hybrids Using Paclobutrazol. J Agril. Food Tech., 2(9), 153–158. Palafox-Carlos, H., Yahia, E.M., and González- Aguilar, G. A., 2012. Identification And Quantification Of Major Phenolic Compounds From Mango (Mangifera indica, cv. Ataulfo) Fruit by HPLC-DAD-MS/MS-ESI and Their Individual Contribution To The Antioxidant Activity During Ripening, J. Foodchem, 135(1), 105–111. Pino, J. A., and Queris, O., 2011. Analysis Of Volatile Compounds Of Mango Wine, J. Foodchem, 125(4), 1141–1146. Luo, F., Qiang L.V., Yuqin, Z., Guibing, H., Guodi, H., Jiukai, Z., 2012, Quantification And Purification of Mangiferin From Chinese Mango (Mangifera indica L.) Cultivars and Its Protective Effect On Human Umbilical Vein Endothelial Cells Under H2O2-Induced Stress, Int. J. Mol. Sci., 13(9), 11260-11274. García-rivera, D., Delgado, R., Bougarne, N., Haegeman, G., and Vanden, W., 2011. Gallic Acid Indanone And Mangiferin Xanthone Are Strong Determinants of Immunosuppressive Anti-Tumour Effects of Mangifera indica L . Bark In MDA-MB231 Breast Cancer Cells, J. Canlet, 305(1), 21–31. Jutiviboonsuk, A., and Chanchai, S., 2010. Mangiferin In Leaves Of Three Thai Mango (Mangifera Indica L.) Varieties, Isan J. Pharm. Sci., 6(3), 123-129. Mirza, R.H., Nan, C., and Yuling, C., 2013. Therapeutic Potential Of The Natural Product Mangiferin Metabolic Syndrome, J. Nutr. Ther., 2(2), 74-79. Du, S., Liu, H., Lei, T., Xie, X., Wang, H., He, X., and Wang, Y., 2018. Mangiferin: An Effective Therapeutic Agent Against Several Disorders (Review), Mol. Med. Rep., 18(6), 4775–4786. Campa, C., Laurence, M., Arsene, R., Luc, P.R.B., Annick, G., and Couturon, E., 2012. A Survey Of Mangiferinand Hydroxynnamic Acid Ester Accumulation In Coffee (Coffea) Leaves: Biological Implications and Uses, Ann. Bot., 110, 595. Subha, R., Madan, P., and Ajay R., 2007. A New Convenient Method for Determination Of Mangiferin, An Anti-Diabetic Compound in Mangifera indica L., J. Planar. Chromat., 20(5), 317-320. Yuni Retnaningtyas et al. Indo. J. Chem. Res., 8(2), 15-20, 2020 DOI: 10.30598//ijcr. 2020.8-yun 20 Pracaya, 2005. Bertanam Mangga, Penebar Swadaya, Jakarta. Ilango, K., Ananth, K. K., Mohan, K.R., Agarwal, A., and Dubey, G.P., 2014. LC-MS Quantification of Mangiferin inhydroalcoholic extract of Salacia oblonga, Salacia roxburghii and polyherbal formulation, Int. J. Phytopharm., 4(1), 11-15. Zhang, X., Benwei, S., Jing, L., Yonghua, L., Dong, L., and Kaixin, Z., 2014. Analysis by RP-HPLC of Mangiferin Component Correlation between Medicinal Loranthus and their Mango Host Trees, J Chromatogr Sci, 52(1), 1-4. Eddy, C.G., Rolando, G.H., Lauro, N.P., and Jos, H., 2014. Determination of mangiferin in Mangifera indica L. Stem Bark Extract (Vimang®) and Pharmaceuticals By Liquid Chromatography, Emir, J. Food Agric. 26(7), 592-601. International Conference on Harmonisation, 2005. Validation of Analytical Procedures: Text and Methodology Q2(R1), Inter. Conf. on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, Geneva, 1-13. Irmawan, M., Mandey, F.W., and Dali, S., 2018. Identification, Characteriterization, an Toxicity Essay of Non-PolarSecondary Metabolite Fraction from Ageratum conyzoides L., Indo. J. Chem. Res., 6(1), 1-5. Panggabean, A.S., and Rachman, A., 2016. Validasi Metode X-Ray Fluorescence untuk Analisis Ion Fe dalam activated Methyl Diethanol Amine(aMDEA), Ind. J. Chem. Res., 3(2), 302- 307 Napitupulu, R.M., Julia, D., and Panggabean, A.S., 2019. Validation Method on The Determination of Mn In Lubricating Oil by Direct Dilution Method Using Atomic Absorption Spectrometer Indo. J. Chem. Res., 6(2), 94-100 Naim, M., and Ahmed, A., Gj, K., 2018. Stability Indicating Reverse-Phase High-Performance Liquid Chromatography Method Development And Validation For Simultaneous Estimation Of Telmisartan And Benidipine Hydrochloride In Pharmaceutical Dosage Form, Asian J Pharm Clin Res, 11(5), 342-350. Bandla, J., and Ganapaty, S., 2018. New Stability- Indicating Ultra Performance Liquid Chromatography Method Development And Validation Of Lenvatinib Mesylate In Bulk Drug And Pharmaceutical Dosage Forms, Asian J Pharm. Clin. Res, 11(9), 140-143. Taylor, P., Bonfilio, R., Cristina, E., Cazedey, L., Araújo, M. B. De, Bonfilio, R., and Salgado, N., 2012. Analytical Validation of Quantitative High-Performance Liquid Chromatographic Methods in Pharmaceutical Analysis: A Practical Approach, Crit Rev Anal Chem, 42(1), 87–100.