INTRODUCTION:

Okra (Abelmoschus esculentus L. Moench) belong to Malvaceae family, known as the lady’s finger, is a vegetable plant that has the highest number of chromosomes among vegetable plants¹. The seeds of Okra, containing high fiber, nutrients, can be used as a vegetable and culinary medicine, rich in vitamins, calcium and minerals^2-5. Okra seeds contain 2.5mg/g of catechins seeds and flavonol derivatives of 3.4mg/g seeds. It also contains hydroxysamic and quercetin derivatives of 0.2 and 0.3mg/g of skin, while the seeds contains phenolic compounds⁴.

Antioxidants are compounds that play an important role in preventing and or curing diseases caused by free radicals.

Free radicals are chemicals that are highly reactive species produced in the body and have the potential to damage cells, organelles, DNA and other biomolecules^6-9. Many research results show that antioxidant compounds play important role in reducing the risk of chronic diseases such as cancer, premature aging, heart disease, asthma, diabetes and rheumatism^10-15. Antioxidants were mostly originated from natural ingredients including grains, seeds and vegetables. This is because the source of natural ingredients contains several important metabolites, such as vitamin C, vitamin E, phenolic acids, carotene and others. All of these compounds have the potential to reduce the risk of disease¹⁰.

The DPPH method (2,2-diphenyl-1-picrylhydrazy) is commonly known method to evaluate the antioxidant activity. This method is based on electron transfer, which is characterized by a change in color to violet in ethanol solution. In addition, these free radicals are also stable at room temperature, and are easily applied using the spectrophotometer method¹⁶.

MATERIL AND METHODS:

Sample Preparation:

The okra seed is obtained from a community garden in Kabangka Village, Kabangka District, Muna Regency, Southeast Sulawesi Province, Indonesia. Okra seed samples are cut into pieces and then dried in the sun covered with black cloth. The dried samples of okra were then powdered. The 2.5kg of okra seed powder was macerated using ethanol to obtain a concentrated extract, and subsequently fractionated with the solvents based on an increase in polarity. Figure 1 shows the extraction and fractionation steps of Okra seeds. The extracts and fractions obtained were concentrated using a rotary evaporator. Furthermore, ethanol extracts as well as fraction were tested for antioxidants, total phenolic and flavonoid contents.

Figure 1. Extraction and fractionation steps of Okra seed.

Antioxidant activity:

Determination of free antiradical activity was performed following Garcia et. al. (2012) work by slight modification. It consisted of 1ml of each sample concentration was added to 3 ml of ethanol and 1 ml of DPPH (2,2-diphenyl-1-picrilhydrazil) radical solution. They are then shaken until homogeneous. Sample was incubated at room temperature for 30 minutes. Sample absorbance was measured by using UV-VIS spectrophotometer at a wavelength of 513nm. The antioxidant strength of extracts and fractions in inhibiting DPPH radicals was calculated using the following equation:

A_c - A_S

% Inhibition = ––––––––– x 100 %

A_c

Where:

% Inhibition = percentage of inhibition of radical DPPH

A_c = control absorbance

A_s = sample absorbance

Percentage of inhibition was then plotted against concentration (µg/mL) to obtain regression linear equation¹⁷.

Total phenolic content:

Determination of total phenolic content of Okra seed extracts and fractions was carried out by the modified Folin-Ciocalteu reagent method according to John et. al. (2014). 1mL of each sample concentration series was added 0.4ml of the Folin-Ciocalteau reagent. They are then shaken, left for 8 minutes, and 4mL of 7% Na₂CO₃ solution was added and shaken until homogeneous. Water was added to achieve 10mL volume. Absorbance was measured using a UV-Vis spectrophotometer at a wavelength of 647nm. Phenolic content is expressed as gallic acid equivalents (GAE). Each sample was measured three times.

Determination of flavonoid contents was performed using the aluminum chloride calorimeter method¹⁹. 10 mg extract and fraction were dissolved with ethanol up to 10mL, then 1mL of them was added to 3mL ethanol, 0.2 of aluminum chloride, 0.2mL of potassium acetate 1 M, and solution was added by distilled water up to 10 %. Solution was left for 30 minutes, and absorbance was then measured at a wavelength of 415 nm. Measurement was done in 3 times and the content of flavonoids are expressed as quersetin equivalent (EQ)/g sample.

RESULTS AND DISCUSSION:

The use of DPPH radical (1,1-diphenyl-2-picrylhidrazyl) in free antiradical analysis has been well known because DPPH is the free radical that is employed to conduct initial tests that are used to test the antioxidant potential of plant extracts^20-22. In addition, DPPH radicals are stable, in which it receives hydrogen donors, which are characterized by losing their characteristic color, namely the change in color of the solution from dark purple to light yellow. This DPPH method is a popular, reliable and reproducible for testing antioxidants from natural ingredients with large numbers of samples in vitro^,16,23-24. In addition, this method requires simple and conventional laboratory equipment^25-26. The antiradical strength of the test compound is expressed as a 50% decrease in DPPH concentration²⁷.

Table 1 shows the IC₅₀ values of methanol extract and okra seed fraction with positive control of vitamin C. It shows that the extract and fraction of the IC₅₀ value is very low, which indicate that okra seed is very promising to be developed as an antioxidant. The lower IC₅₀ value indicate the stronger the antiradical properties of sample. The ethyl acetate fraction showed lowest IC₅₀ value, which indicated that its strongest antiradical activity. This is in line with several studies that report that ethyl acetate fraction has stronger antioxidant activity. Several examples were ethyl acetate fractions from rambutan skin extracts and fractions²⁸ and mangosteen peel^29-30.

Table 1. The IC₅₀ values of extract methanol dan fraction of okra seed.

Sample	IC₅₀ (µg/mL)			Mean IC₅₀±SD
Sample	I	II	III	Mean IC₅₀±SD
Ethanol extract	10.158	10.191	10.236	10.195±0.039
n-hexane fraction	13.079	13.152	13.125	13.119±0.037
Chloroform fraction	13.989	14.079	14.142	14.07±0.077
Ethyl acetate fraction	10.158	10.118	10.014	10.097±0.074
Water fraction	17.533	17.222	17.066	17.274±0.238
Vitamin C	7.635	7.694	7.704	7.678±0.037

Based on the Table 1 above which taken using ANOVA test using SPSS version 24 at significance 0.05, the antioxidant strength of the ethyl acetate fraction of okra seed was significantly different compared to other samples and it was comparable to Vitamin C as control.

The antioxidants activity is influenced by the content of polyphenol compounds This is because the polyphenol and flavonoid compounds have the ability to donate hydrogen radicals to neutralize radical molecules in the body. Therefore, in this study the relationship between phenolic and flavonoid levels found in extracts and fractions with scavenging free radicals in the body is determined. Tables 2 and 3 provide data on phenolic and flavonoid levels found in okra seed extracts and fractions. They show that the ethyl acetate fraction contains the highest levels of phenolic and flavonoid with a value of 61.412±0.196 gram of gallic acid equivalent and 97.933±0.222 gram of quercetin equivalent, respectively.

Table 2. Phenolic content of extract and fraction of Okra seed expressed as Gallic Acid Equivalence (GAE).

Sample	Phenolic content (Gallic Acid Equivalence)			Mean contents (GAE ± SD)
Sample	I	II	III	Mean contents (GAE ± SD)
Ethanol extract	58.765	58.667	58.569	58.667 ± 0.098
n-Hexane fraction	55.922	55.922	56.216	56.02 ± 0.169
Chloroform fraction	50.431	50.529	50.627	50.529 ±0.098
Ethyl acetate fraction	61.216	61.412	61.608	61.412±0.196
Water fraction	46.706	46.804	46.902	46.805 ± 0.098

Table 3. Flavonoid content of extract and fraction of Okra seed expressed as Quercetin Equivalent (QE).

Sample	Flavonoid content (Quercetin Equivalence)			Mean Contents (QE ± SD)
Sample	I	II	III	Mean Contents (QE ± SD)
Ethanol extract	37.267	37.489	37.933	37.563±0.339
n-Hexane fraction	34.6	35.044	35.489	35.044±0.444
Chloroform fraction	15.933	16.378	17.044	16.452±0.559
Ethyl acetate fraction	98.156	97.933	97.711	97.933±0.222
Water fraction	4.378	3.933	4.6	4.304±0.002

The linear relationship between phenolic and flavonoid content with antioxidant strength is illustrated in Figures 2 and 3.

Fig 2. Correlation between each phenolic (left) and flavonoid contents (right) with IC₅₀ of Okra seeds.

The IC₅₀ values of phenolic or flavonoid contents of extract and fraction of okra seed were depicted in Figure 2, in which linear equations of Y = −1.9239x + 79.602 with R² = 0.9281 and Y = −9.5299x + 161.68 with R² = 0.6229, respectively, were obtained. It was indicated that 92.81% and 62.29% of antiradical strength are influenced by phenolic and flavonoid compounds, respectively, while the rest are influenced by compounds other than flavonoids and phenolics including vitamins, alkaloids and other compounds. In addition, the most responsible compounds as free radical scavengers are the compounds contained in the ethyl acetate fraction. So that the ethyl acetate fraction has the opportunity to be developed as a functional food that has the potential as a natural antioxidant from okra seeds.

CONCLUSION:

Extract and ethyl acetate fraction of Okra seed had good radical inhibition properties, which indicated its potential for further development assays. Merr bark had an extremely strong inhibitory ability against DPPH, with the ethyl acetate fraction being the strongest. The ethyl acetate fraction contained higher phenolic and flavonoid content among other fractions. It indicated that isolation for active compound could be performed for ethyl acetate fraction to identify the compound structures responsible for the radical scavenging properties.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 31.01.2020 Modified on 29.05.2020

Research J. Pharm. and Tech. 2021; 14(4):2045-2048.

DOI: 10.52711/0974-360X.2021.00363