Quantitative and Optimization of Anthocyanin Extracted from Pomegranate ( Punica granatum ) Extract by High-Performance Liquid Chromatography ( HPLC )

Objective: P. granatum is one of the oldest edible fruits of tropical and subtropical regions. This fruit had high antioxidant contained by hydrolysable tannins and anthocyanin compounds that give many health benefit properties. This study aims to quantify and optimized anthocyanin from P. granatum extract. Methods: A total of 50g of the flesh was soaked into two different polar solvents; water and 50% ethanol within a ratio of 1:10; w/v for 24-hr. Then, three different methods of extraction were done and test each with HPLC analytical, respectively. Results: The validated method proved to be linear in the range of 5 – 30 ug/mL and with LOD and LOQ determined respectively for Cy3, Cy3, 5, Pg3, and Pg3, 5. The method also shows recovery (%) close to 100 when accuracy was accessed. For samples, blender water extract had a higher composition of Cy3, Cy3, 5 and Pg3, 5 (22.77 ± 8.82 mg/100 g e.p; 25.36 ± 9.95 mg/100 g e.p; 11.16 ± 5.85 mg/100 g e.p) content as compared to other. Conclusion: As a conclusion, the present methodology proved to be capable of detecting and quantifying Cy3, Cy3, 5, Pg3, Pg3, 5 in a single run. Also, comparatively the composition of each AC detected in blender water extract is significantly higher in value than the other methods. It should regard as a valuable source of antioxidant with the potential used for health benefits properties worldwide.


INTRODUCTION
Anthocyanin (AC) are members of a class of watersoluble plant pigments that classified chemically as flavonoids.AC have C6 (A-ring), C3 (C-ring) and C6 (B-ring) flavonoid skeleton and structurally glycosylated or acylated. 1In pomegranate, AC is one of two major types of polyphenols compounds which account for the majority of antioxidant activity of the fruit. 2 This activity is due to contain a number of phenolic hydroxyl groups attached to ring structures, which can exert antioxidant or radical scavenging activity through single electron or hydrogen atom donation. 3 has been associated with wide range of anti-neurodegenerative, 4 anti-inflammatory, anti-oxidative activities, 3 and reducing the risk of cardiovascular disease (CVD).These potential health benefits had provoked an increasing demand for these compounds.In the pomegranate juice, cyanidin 3-glucoside (Cy3), cyanidin 3, 5-diglucoside (Cy3, 5), pelargonidin 3-glucoside (Pg3), pelargonidin 3,5-diglucoside (Pg3,5), delphinidin 3-glucoside (Dp3), and delphinidin 3,5-diglucoside (Dp3,5) had identified as the main compounds responsible for the AC.Previous studies had observed AC identification during pre and post-storage, used two methods of juice extraction and others. 1 Although much attention has focused on AC in pomegranate juice, the quantification of AC from different methods of juice extraction in Malaysia using a reliable technique is not studied yet.Thus, the objective of this study is to quantify and optimized for anthocyanin from pomegranate (Punica granatum) extract by highperformance liquid chromatography (HPLC)

Extraction and Isolation of Punica Granatum
Fresh pomegranate fruits were purchased from available sources in market Kuala Terengganu,

Standards Validation
Recovery (%), Limits of Detection (LOD) and Limits of Quantification (LOQ) are terms used to describe the smallest concentration of a measure, and that can reliably measure by an analytical procedure. 7The parameters are to define the smallest concentration of an analyte that can be detected with no guarantee about the bias or imprecision of the result by an assay with LOD.Meanwhile, LOQ is the lowest concentration at which the analyte cannot be reliably detected but at which some predefined goals for bias and imprecision met. 7ased on Table 1, LOD and LOQ of each standard were assessed to determine the methodology sensitivity.Each standard was injected with 5, 10, 15, 20, 25 and 30 ug/mL for this analytical procedure.LOD values were 3.32 ug/mL, 11.36 ug/mL, 11.32 ug/mL, and 5.40 ug/mL for Cy3, Cy3, 5, Pg3 and Pg3, 5, respectively.While, LOQ values were 10.05 ug/mL, 34.43 ug/mL, 34.29 ug/mL, and 16.38 ug/mL for Cy3, Cy3,5, Pg3, and Pg3,5, respectively.This result was demonstrating that the method is sensible enough to determine Cy3, Cy3, 5, Pg3, and Pg3, 5 in the samples further.AC analysis were used a RP-18 column (25 cm x 0.4 cm i.e.: 5 um particle size; Merck, Germany) and UV-Vis detector.This detector allows analysis of one wavelength at one time which is 260 nm, as well verification of the chromatographic peak purity.The purity of interest AC in this study were 0.999 for Cy3, Cy3,5, Pg3, and Pg3,5, respectively.Meanwhile, the recovery (%) was 100.73 ± 4.77, 102.31 ± 10.84, 101.67 ± 8.58, and 100.78 ± 6.83 for Cy3, Cy3, 5, Pg3, and Pg3, 5, respectively.

High-Performance Liquid Chromatography AC Analysis Standard Compound Preparation
1 mg of standard AC (Cyanidin 3-glucoside, Cyanidin 3, 5-diglucoside, Pelargonidin 3-glucoside, Pelargonidin 3, 5-diglucoside) were weighed and dissolved in 100% methanol (HPLC grade) to give a stock solution of 100 μg/ml.The stock solution was stored in vial HPLC and stored in 4 o C until used for analysis.Peaks for AC composition were identified on the chromatogram by comparing the retention time and spiking test with AC standard.

Identification and Quantification of AC by HPLC
The method used by Miguel et al. 6 with modifications were adopted in this study.The characterization of AC was performed by HPLC with a System Gold Programmable Detector Module 166-UV-Vis (Beckman Coulter, USA), using a LiChroCART 100 RP-18 column (25 cm x 0.4 cm i.d.: 5 um particle size; Merck (Germany).The mobile phases used were 0.001% T-fluoro acetyl acid (TFA) and DI water (A), 100% methanol HPLC grade (B) and 100% acetonitrile HPLC grade (C).Cy3 and Cy3, 5 were set using gradient 3, isocratic run for 20 min.Pg3 and Pg3, 5 were set using isocratic run for 10 min.The flow rate was 0.3 mL/min.Chromatograms recorded at an absorbance of 260 nm.The concentrations of AC were calculated from standard curves of Cy3, Cy3, 5 Pg3, and Pg3, 5 at six different concentrations (5 ppm, 10 ppm, 15 ppm, 20 ppm, 25 ppm and 30 ppm).Injection volume was set at 15 uL using an injector with a 20 uL loop (Rheodyne, USA).AC in samples was identified by comparing the chromatograms and retention times (RTs) of AC standard (Cy3, Cy3, 5, Pg3, and Pg3, 5) (Sigma, Co. Chemical, St Louis, USA) and used as a reference for quantification.

Data Analysis
The SPSS 20.0 software was employed for statistical analysis.Data were expressed as mean ± SEM of three independent values. of Cy3, Cy3, 5, Pg3, Pg3, 5 in blender (aril + seed) water is higher in value than the other methods.AC provides the color of pomegranate fruit and juice, so this compound stability through juice processing is a major importance.It was reported previously in pomegranate juices; the diglucoside AC was more stable than the monoglucosides. 8This was parallel with present study and Dionex; 9 Cy3, 5 and Pg3, 5 compositions are higher than Cy3 and Pg3.However, this was contrary to Miguel et al. 6 in which there was a high decrease of Cy3, 5 than Cy3 composition.Also, the result of this study was parallel with Anahita et al. 10 that pomegranate seed + juice had the highest total antioxidant activity (47.00 ± 5.50) than two other parts (seed and juice), separately.On the other hand, Mphahlele et al. 11 Akhavan et al. 12 and Fischer et al. 13 had identified the individual AC in pomegranate extract, and the result was parallel with the present study.However, undetectable AC and its derivatives in the juices among those studies and present study are contrary to each other, and this may lead to several reasons.According to Akhavan et al. 12 different methods of extraction used may affect the AC content of pomegranate juices as different parts and pressure introduced.

CONCLUSION
The optimization of HPLC methodology allowed simple and fast AC determination.The results demonstrated that the method is for quantitative AC, besides being linear and accurate when determined standards.On the other hand, all samples using different juice extraction and solvents can identify the Cy3, Cy3, 5, Pg3 and Pg3, 5 compounds in pomegranate.The composition of Cy3, Cy3, 5, and Pg3, 5 are higher in water extract with the preferable method of juice extraction blender (aril and seed).Comparatively, the method of juice extraction using blender shows the preferable of high composition among AC standards than the others.

ACKNOWLEDGEMENT
The authors would like to thank the Ministry of Higher Education, Malaysia and Universiti Sultan Zainal Abidin (UniSZA) for the financial aid (UNISZA/2014/FRGS/RR078) and the Faculty of Health Sciences for providing the facilities.The authors would also like to acknowledge all

Table 1 : Validation of optimization method from anthocyanin standard in HPLC.
*Each value in the table was obtained by calculating the average of five experiments.

Table 2 : Quantification of anthocyanin in P. granatum extracts (mg/100 g e.p).
Data represent the mean ± SEM of three independent experiments.Statistical analysis was carried out using a One-Way ANOVA.Different letters in the same column means a significant difference (p≤0.05).