Evaluation of Free Radical Scavenging Activity of Ethanolic Extract from Promising Accessions of Curcuma aeruginosa RoxB

This study evaluated the antioxidant activity through the determination of free radical scavenging activity of ethanolic extracts from 20 accessions of Curcuma aeruginosa, and it is to use for the development of varieties in future. The radical scavenging activity of the extract accessions was investigated with 2,2diphenyl-1-picrylhydrazyl (DPPH) radical. IC50 values for DPPH radical scavenging activity ranged from 89.81 to 505.65 μg mL -1 . Based on IC50 values, twenty accessions of C. aeruginosa can be divided into three groups: strong (two accessions); moderate (seventeen accessions); and low (one accession) of DPPH scavenger. Sukoharjo (SH) and Muara Bungo (MB) showed promising accessions for antioxidant potential, thus these accessions important to selection for the future breeding program in pharmaceutical products.


EXPERIMENTAL SECTION
C. aeruginosa rhizomes were collected in the month of February 2015 from different regions of Indonesia (Table 1).Most of the accessions were pooled and taken for identified by experts at Tropical Biopharmaca Research Center, Bogor Agricultural University (IPB).

Preparation of ethanolic extract
The rhizome materials were sun dried to moisture content < 10% and then powdered (100 mesh).The resulting powder was subjected to extraction with 70% ethanol (W Nurcholis et al., 2015).The ethanolic extract was concentrated, using a rotary evaporator (BUCHI, R-250, Switzerland) and stored at 4 °C until used.

Determination of free radical scavenging activity
The stable 2,2-diphenyl-1-picrylhy drazyl (DPPH) analysis was used to determine free radical scavenging activity of the ethanolic extracts (25,50,100,200,400 and 800 µg mL - 1 ) (W.J. Li et al., 2012).Ethanolic extract (200 µL) in DMSO (Merck, Germany) was added to 50 µL of a methanol solution of DPPH.Absorbance at 517 nm was determined using microplate readers or microplate photometers (Epoch Biotech, USA) after incubation in dark for 30 min at 37 °C, and the percentage inhibition activity was calculated from [ ] , where A 0 is the absorbance of the control, and A is the absorbance of the ethanolic extract.The inhibition curves were prepared and, IC 50 values were obtained.

Statistical analysis
The data were analyzed using one-way variance analysis.The comparisons among the difference of means were further analyzed using Duncan's multiple range test at p<0.05.Analyses were performed using SPSS 19 statistical computer software.

RESULTS AND DISCUSSION
Analysis of the free radical scavenging activity of the ethanolic extract from 20 C. aeruginosa accessions tested by a stable free radical, namely DPPH as a reagent.The DPPH method is a preferred method because it is a convenient and fast technique to evaluate free radical scavenging or antioxidant activity (J.Li et al., 2013).The DPPH radical is a deep purplish color, which is at its maximum wavelength at 515-520 nm and can change color from violet to yellow if received an electron or hydrogen from antioxidant molecules to become a stable DPPH molecule (Carmona-Jiménez, García-Moreno, Igartuburu, & Barroso, 2014;Pérez & Aguilar, 2013).Therefore, the discoloration of the DPPH radical reflects the free radical scavenging activity of the analyzed extract in 20 C. aeruginosa accessions (Figure 1).The   The free radical scavenging reported as IC 50 values that defined as the concentration of sample extract necessary to obtain an activity of 50% of the DPPH radicals (Nickavar, Alinaghi, & Kamalinejad, 2008).The lower IC 50 value indicates higher free radical scavenging activity.The IC 50 values in the ethanolic extract of C. aeruginosa accession ranged from 89.91 to 505.65 µg mL -1 .Accessions of Sukoharjo (SH) and Muara Bungo (MB) had a significantly higher free radical scavenging (p<0.05)compared to others accessions except with accessions of Solo (GD), Kendal (KN), Pacitan (PT) and Sragen (SG).According to IC 50 values, the free radical scavenging activity can be divided into three groups: (a) strong with IC 50 <100 µg mL - 1 ; (b) intermediate with IC 50 , 100-500 µg mL -1 ; and (c) weak with IC 50 >500 µg mL -1 (Bi et al., 2016) In literature, there are some reports regarding free radical scavenging of C. aeruginosa tested by the DPPH method.Based on our result, free radical scavenging capacity in ethanolic extract of twenty C. aeruginosa accessions are lower than essential oil extract with IC 50 values of 28 µg mL -1 (George & Britto, 2015) and 24 28 µg mL -1 (Theanphong, Mingvanish, & Kirdmanee, 2013).In another report, the free radical scavenging of the ethanol and oleoresins extracts were 437.07 µg mL -1 (W Nurcholis et al., 2015) and 450 µg mL -1 (Rajamma, Bai, & Nambisan, 2012), respectively.The ethanolic extract accessions of Sukoharjo (SH) and Muara Bungo (MB) were shown strong the free radical scavenging activity with IC 50 values of 89.81 and 90.87, respectively.Accessions of SH and MB had strong to potent antioxidant activity.This result provides important information for selecting high-quality C. aeruginosa rhizome in the future breeding program for antioxidant activity.

CONCLUSION
Among all the accession of C. aeruginosa, Sukoharjo (SH) and Muara Bungo (MB) showed promising accessions for antioxidant potential by free radical scavenging activity.Thus, these accessions important to selection for the future breeding program in pharmaceutical products.
percentage inhibitions of DPPH assay from the ethanolic extract of twenty C. aeruginosa promoting accession are given in Figure 2, and the IC 50 values are presented in Figure 3.The DPPH radical scavenging capacity was evaluated regarding percent reduction of the initial DPPH absorption.The percentages of inhibition of the Sukoharjo (SH) and Muara Bungo (MB) accessions demonstrated superior DPPH radicals scavenging activity than the others accessions at 25-800 µg mL -1 .

Figure 1 .Figure 2 .
Figure 1.The color change of DPPH: (A) from purple to yellow in graph (Pérez & Aguilar, 2013) and sample of C. aeruginosa in 96-micro plate well (B), when it exposed to antioxidant substance

Figure 3 .
Figure 3. Free radical-scavenging activity in an ethanolic extract of twenty C. aeruginosa promising accessions by DPPH assay.Each data represents the mean ± SD of three replicates.Values with different lowercase letters represent significant differences at p<0.05.

Table 1 .
Geographical collection sites of 20 C. aeruginosa accessions