THE EFFECT OF EXTRACTION CONDITION ON THE POLYPHENOL CONTENT AND ANTIOXIDANT ACTIVITY OF Curcuma zedoaria (Christm.) ROSCOE RHIZOME

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Indo. J. Phar. Scie. Tech. Vol. 4, No. 2, 57-63 (2017).

Lia Marliani, Wempi Budiana, dan Yonara Anandari

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Sekolah Tinggi Farmasi Bandung, Bandung, Indonesia
Korespondensi : l.marliani.pharm@gmail.com (Lia Marliani)

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Abstrak/Abstract

Kata Kunci: Antioksidan, Curcuma zedoaria (Christm.) Roscoe, ekstraksi, polifenol

Introduction

White turmeric (Curcuma zedoaria (Christm.) Roscoe) is one of family Zingiberaceae that usually used as one of Indonesian herbal medicine. Its rhizome has antioxidant, anti-inflammatory, anti- cancer and anti-microbial activity 1 . White turmeric contains bioactive compounds such as flavonoids, polyphenols, curcuminoid, terpenoids and essential oils. There are approximately 60 essential oil components and oleoresin in white turmeric with curzerenone, germacrone, camphor, curcumenol as the largest component 2 .

The one of antioxidant compound of white turmeric is polyphenols. Polyphenols has been known as potent antioxidant. Polyphenols can scavenge radical species or inhibit some enzymes or chelating trace metals which involve in free radical formation 3 .

The extraction process aims to obtain maximum active compounds and the best quality in activities. The yield of extraction is influenced by the type of extraction method, solvent, extraction time, and temperature 4,5,6. There is no universal extraction method for all phenolic compounds because their solubility and physical characteristics varying as well as varying form of phenolic compounds from simple to highly polymerized substances 3 . Total phenol content and total flavonoid content of the extract are varying depend on extraction solvent 7 . The higher amounts of phenolic compound often extracted by more polar organic solvent such as methanol and ethanol. The amount of polyphenols extracted are influenced by selecting the right solvent 8 . According to Wissam et al, 2012 9 , an increasing temperature of extraction will increases the extraction efficiency by increases cell permeable, solubility and diffusion coefficients of the compounds.

The antioxidant activities are depending on the level of antioxidant compound was extracted. The purpose of this study was to determine the significant extraction factors that influence the polyphenol content and antioxidant activity of white turmeric (Curcuma zedoaria (Christm.) Roscoe).

Materials and Methods

Materials
White turmeric (Curcuma zedoaria (Christm.) Roscoe) rhizome were collected from Purwakarta, West Java, Indonesia, at Februari 2016. The specimen were identified at Herbarium Biology Department, Padjadjaran University, Bandung, Indonesia. Sample was dried by oven at temperature 500C for 2 days. Dry material grinded and was sieved by mesh 20 sieve.

Extraction
Sample was extracted by dynamic maceration method using 23 variable factor (two levels of three factor) includes variation of solvent (ethanol 96% and water) in 200 mL, temperature (25oC and 70oC) and time (6 hours and 24 hours). The extraction process was done in an orbital shaker at stirring speed of 30 rpm. Each extract was concentrated using Buchi® Rotavapor® R-215 to obtain spissum extract.

Determination of total phenol content
Total phenolic compounds contents were determined by the Folin-Ciocalteau method 10. The 0.5 ml extract samples were mixed with 5 ml Folin Ciocalteu reagent for 5 minute and Na2CO3(4 ml, 1 M) then added. The mixture was allowed to stand for 15 min and the phenols were determined by colorimetry at 765 nm. The standard was 20, 40, 50, 60, 80, and 100 μg/ml gallic acid. Total phenol values are expressed in terms of gallic acid equivalent (μg/ml of dry mass), which is a common reference compound 

Determination of antioxidant activity

The free radical scavenging activity of extract was determined using the stable radical DPPH (1,1‐diphenyl‐2‐ picrylhydrazyl) 10,11. Different concentration of each extract (50-250 μg/ml for ethanol 96% extract and 500- 1000 μg/ml for water extract) were added at equal volume (1:1) of freshly prepared DPPH solution (60 μg/ml) in methanol in each test tube and mixed. The absorbance was measured after 30 min incubation at 516 nm (λmax of DPPH solution). The capability to scavenge the DPPH radical was calculated using the following equation:

DPPH scavenged (%) = [(Ac – As)/Ac]x100

Ac is the absorbance of the control (DPPH solution) and As is the absorbance of the extracts. The antioxidant activity of the extract was expressed as IC50. The IC50 value was determined from linear regression analysis with Concentrations of extract as X axis and DPPH scavenged (%) as Y axis.

Data analysis
The results were analyzed by Factorial analysis. ANOVA/ Response Surface Methodology (RSM) on the experimental data was performed using using Design-Expert® Software Version 10 to determine which factors of solvent, temperature and time that influence the total phenols content, and antioxidant activity.

The mathematical models for each response were evaluated using a multiple regression method. The response function applied was a linear polynomial equation, given by equation (1):

In equation (1), Y is the dependent variable; β0 is the constant term; k number of variables; βi represents the coefficients of linear parameters; βij represents the coefficients of interaction parameters.

The significance of the equation parameters for each response variable was analyzed by F-test. Only the factors with significance higher than or equal to 5% (p≤0.05) were considered. The model adequacy was checked accounting for the coefficient of determination (R2 ). The response and result of data analysis by Design-Expert® Software Version 10 was showed by Interaction plot graphic (2D).

Results and Discussion

The result of total phenol content determination showed that the highest content was obtained by extraction using ethanol 96% at 24 hours and temperature 70oC (see Table 1).

This is accordance with Do et al. research 7 which its result showed the extract obtained by 100% ethanol has highest phenolic content than other solvent and the water extract showed the lowest phenolic content. Rhizome may contain varying phenolic compounds from simple to polymerized subtances. The other compound such as carbohydrates and proteins may also associated with phenolics compound 3 . The complex polyphenols are more soluble in organic solvent, such as ethanol than water. The solubility of nonphenol compounds like carbohydrate and protein are higher in water than in ethanol 12. The water can extract other compound of white turmeric such as amylum that also abundant in rhizome. This maybe the reason why ethanol 96% extract gave the highest the total phenol content.

Temperature and extraction time are enhancing the solubility and diffusion coefficient of solute 13. The result showed that at 700C and 24 hour extraction, both water and ethanol extract gave the highest phenolic compound. The chance of denaturation and oxidation of phenolics is increased by high temperature and long extraction which decrease the yield of phenolics in the extracts7 . The result showed that at 700C and 24 hour extraction, phenolic compound of white turmeric (Curcuma zedoaria (Christm.) Roscoe) rhizome may not be denaturated or oxidized.

Although at different temperature and time of extraction showed different phenol content, the data analysis by Design-Expert® showed only solvent variation ((significance higher than 5%, p p≤0.05) could affecting the total phenol content and ethanol 96% gave the highest total phenol content (figure 1).

The result of antioxidant activity that determined by free radical DPPH scavenging activity showed the lowest IC50 value also was obtained by extraction using ethanol 96% at 6 hours and temperature 70oC (see Table 2).

The lowest IC50 value determine the more potent antioxidant activity. This result (Table 1 and 2) showed that the high total phenol content gave the most active antioxidant. It is also determined the role of polyphenol as antioxidant compound of white turmeric (Curcuma zedoaria (Christm.) Roscoe). Although at different temperature and time of extraction showed different phenol content, but they are not significant.

The data analysis by Design- Expert® determines that only solvent variation (significance higher than 5%, p≤0.05) could influence the antioxidant activity (figure 2). The potent antioxidant showed by extract which obtain using organic solvent (ethanol 96%).

Conclusion

This study showed that solvent is significant extraction factor for polyphenol content and antioxidant activity of white turmeric (Curcuma zedoaria (Christm.) Roscoe) rhizome than temperature and time of extraction.

Acknowledgement
This research was supported by an Internal Grant from Bandung School of Pharmacy, Bandung, Indonesia.

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