INTRODUCTION:

Antioxidant is a compound that is able to inhibit the oxidation process caused by the influence of reactive oxygen species^1–3. An antioxidant compound stabilizes the polymer products of chemicals, food ingredients, cosmetics, and drugs. It is involved in an organism’s defense against free-radical-induced pathologies^4–7. The ability of antioxidant compounds to inhibit free radicals is directly proportionate to the number of hydroxy groups (-OH) bonded to the aromatic ring. Most secondary-metabolites of natural compounds have aromatic rings. In general, antioxidant activity is generated by the secondary metabolites or active compounds in plants like flavonoids, phenols, tannins, and anthocyanins⁸.

Therefore, compounds from natural sources are better at using as antioxidants than synthetic ones⁹. The community’s awareness of the side effects of synthetic antioxidants also drives researchers to conduct studies in natural antioxidants sourced¹⁰. One of the plants that used empirically as medicinal and food ingredient or flavoring is Dracontomelon dao (Blanco) Merr. This plant which is better known as Raghu belongs to Anacardiaceae and grows in lowland and ricefield areas across South Asia¹¹. By the people in East Kalimantan (Indonesia) and China, the bark of Dracontomelon dao (Blanco) Merr is often used for treating skin infections, skin ulcers, diarrhea, abscesses, and inflammations^12,13. Based on chemical analysis, Dracontomelon dao (Blanco) Merr contains alkaloid, sterol, tannin, terpenoids, and flavonoid compounds¹⁴. The presence of the flavonoid in the plant, along with its relevant properties in empirically used, shows that the plant carries antioxidant effect. Based on that, we think it is necessary to carry out research study to test the antioxidant activity and to determine the flavonoid and phenolic content of the extract and fractions of the plant’s bark.

MATERIAL AND METHODS:

Sample Preparation:

The sample used in this research was the Raghu bark (Dracontomelon dao (Blanco) Merr) from Laende-Muna, Southeast Sulawesi, Indonesia. The Dracontomelon dao (Blanco) Merr bark was sorted and dried. The dried sample was cut into small pieces and powdered.

Extract and Fraction Preparation:

Six hundred grams of Dracontomelon dao (Blanco) Merr powder bark were extracted with methanol solution for 3 × 24 hours, with changing of solution every 1 × 24 hours. The filtrate was collected and concentrated with rotary evaporator. One hundred grams of the methanol extract was diluted with 2 L of distilled water and partitioned with n-hexane and ethyl acetate. Extract Fractions obtained were n-hexane, ethyl acetate, and water. Fractions of n-hexane and ethyl acetate were concentrated with rotary evaporator at 40^oC, while the water fraction was concentrated with oven at 50^oC.

Phytochemical Screening:

Methanol extract and the three fractions (n-hexane, ethyl acetate, and water) of Dracontomelon dao (Blanco) Merr bark were subjected to phytochemical screening for secondary metabolite. The phytochemical screening performed was alkaloid, flavonoid, tannin, saponin, and terpenoid tests.

a. Alkaloid Test:

The extract and fractions of Dracontomelon dao (Blanco) Merr bark were inserted separately into 1mL test tubes and added with three drops of Dragendorff’s reagent. The formation of brown precipitate indicated presence alkaloid¹⁵.

b. Flavonoid Test:

The extract and fractions of Dracontomelon dao (Blanco) Merr bark were inserted separately into test 1 mL tubes at and added with 0.2grams of magnesium powder and 2mL of concentrated HCl. The formation of red, orange, and green solutions indicated the flavonoid presence¹⁶.

c. Terpenoid Test:

The extract and fractions of Dracontomelon dao (Blanco) Merr bark were inserted separately into 1mL test tubes and added with 0.5mL of acetic acid anhydride and 2mL of concentrated sulfuric acid. The formation of green, bluish, and brown solutions indicated the terpenoid presence¹⁷.

d. Tannin Test:

The extract and fractions of Dracontomelon dao (Blanco) Merr bark were inserted separately into 1mL test tubes and added with 1mL of 1% Fe (III) chloride solution. The formation of blue to black solution indicated the presence of tannin¹⁸.

e. Saponin Test

The extract and fractions of Dracontomelon dao (Blanco) Merr bark were inserted separately into 1mL test tubes and added with 2mL of hot water, then cooled and shaken for 10 seconds. It was declared positive for saponin if the fume generated stabilized in less than 10 minutes¹⁹.

Determination of Antioxidant Activity:

The determination of antioxidant activity was referred to the assay method applied by Garcia et al²⁰. The reference solution and the sample solution were each pipetted 1 mL and added with methanol 3 mL and 1 mL of 0.6 mM DPPH. The solutions were shaken until homogene and were incubated at operating time at room temperature. The absorbance of each solution was measured at the maximum wavelength of DPPH. The ability of extracts and fractions to scavenge the DPPH radical was calculated using the equation:

A_control- A_sample

Radical scavenging (%) =------------------- X 100

A_control

Where;

A_control is Absorbance of Control

A_sample is Absorbance of Sample

The percent antioxidant activity was plotted again the sample (ppm) to obtain IC₅₀ which defined as the concentration of the sample the necessary to cause 50% scavenging of DPPH radical, calculated by an equation generated for linear regression^21,22.

Determination of Total Flavonoid Content:

The determination of flavonoid content was performed by the aluminum chloride colorimetric method²³ with some modification. Ten mg of extract and fractions were solved with methanol 10mL, 1mL of the solution was added with methanol 3mL, 0.2mL of 10% aluminum chloride and 0.2mL potassium acetate 1M and add with distilled water to 10mL. The absorbance was measured at wavelength of 415nm in triplicate. The flavonoid content obtained was expressed in quercetine equivalent (QE)/g sample following the formula^24,25.

C x V x fp

C_F -----------------------

Where;

C_F is Concentration of sample

V is Volume of dilution

fp is Factor of dilution

m is weight of sample

Determination of Total Phenolic Content:

The determination of total phenolic content of the extract and fractions of Dracontomelon dao (Blanco) Merr bark was accordanced to Folin-Ciocalteu method²⁶. Each concentration series was taken 1mL, added with 0.4mL of Folin-Ciocalteu reagent, shaken, and left for 8 minutes. It was then added with 4mL of 7% Na₂CO₃, 10mL of distilled water and shaken until homogene. The absorbance was measured at 750nm. The phenolic content obtained was expressed in gallic acid equivalent (GAE). The total phenolic content obtained from the absorbance of each sample was then plotted on a standard curve of gallic acid. The value obtained was multiplied by the total volume of the sample and compared with the weighing weight as follows^24,25.

C x V x fp

C_F -----------------------

Where;

C_F is Concentration of sample

V is Volume of dilution

fp is Factor of dilution

m is weight of sample

RESULTS AND DISCUSSION:

The results showed that the extract and the two fractions (ethyl acetate and water) were positive for alkaloid, flavonoid, terpenoid, and tannin compounds but negative for saponin. This is presented in Table 1.

Table 1. Results of phytochemical screening of the extract and fractions of Dracontomelon dao (Blanco) Merr bark

Testing	Result
Alkaloid	+
Flavonoid	+
Terpenoid	+
Tanin	+
Saponin	-

The antioxidant activity test was performed by the DPPH method because this method is the most frequently used for radical scavenging testing on plant samples extract^27–30. Besides, the DPPH method is easy to conduct because DPPH is abundant and its commercial availability eliminates the need for reagents. DPPH radical will also decrease when added to compounds with the ability to donate radical hydrogen like phenolic and flavonoid compounds, earning them the name antioxidant compounds. The decrease in color intensity is observed with the decrease in absorbance value³¹. In this research, the solvent used was methanol because it has higher sensitivity than other polar solvents.

Table 3. Antioxidant activity of Dracontomelon dao (Blanco) Merr bark

Sample	IC₅₀(ppm)
Metanol Extract	7.62
Ethyl acetate fraction	7.11
Water Fraction	8.16
Vitamin C	4.92

Table 3 presents the IC₅₀ values of the extract and fractions of Dracontomelon dao (Blanco) Merr bark, with the IC₅₀ values of the ethyl acetate fraction, methanol extract, and water are 7.11ppm, 7.62ppm, and 8.16ppm, respectively. Meanwhile the vitamin C used as reference was vitamin C with 4.29 ppm for IC₅₀. The comparison can be seen in Figure 1.

Fig 1. IC₅₀ values (ppm) of the extract and fractions of Dracontomelon dao (Blanco) Merr and reference vitamin C

Based on the data above, the ethyl acetate fraction of Dracontomelon dao (Blanco) Merr bark was better able to inhibit free radicals than were the methanol extract and water fraction. This is in line with other research studies. One study on antioxidant activity of the ethanol extract and fractions of Crescentia cujete bark reported that ethyl acetate fraction exhibits stronger antioxidant activity than do chloroform fraction, water fraction, and crude extract of ethanol³². Similar results were also reported by study on Acacia arabica bark³³.

Figure 2 shows the relationship between concentration and percent inhibition of the methanol extract and fractions of Dracontomelon dao (Blanco) Merr bark. According to the data reported, the ethyl acetate fraction of Dracontomelon dao (Blanco) Merr bark can be subjected to a further research study in which the compounds in the plant’s bark that are responsible for antioxidant activity can be isolated.

Fig 2. (a) Relationship of the total flavonoid of the extract and fractions of Dracontomelon dao (Blanco) Merr bark to IC₅₀ value, (b) Relationship of the total phenolic of the extract and fraction of Dracontomelon dao (Blanco) Merr bark to IC₅₀ value

It is known that the compounds most responsible for warding off free radicals are phenols and flavonoids as these compounds will donate their protons to bind to free radicals, causing the free radicals penetrating into the body to stabilize through the following reaction ³⁴.

Where;

FL-OH = Flavonoid

FL-O* = Less reactive of Flavonoids radical

Hence, the phenolic and flavonoid content of natural sources is correlated with antioxidant activity strength ^31,35. According to the data presented in Table 4, the ethyl acetate fraction contained higher flavonoid and phenolic content than did the methanol extract, n-hexana fraction, and water fraction. Phenolic content was expressed in g GAE/100g sample, and the reference was gallic acid²¹.

Meanwhile, flavonoid content was expressed in g QE/100g sample, and the reference was quercetine²³. The data in Table 4 show that the highest phenolic and flavonoid content in Dracontomelon dao (Blanco) Merr bark was found in the ethyl acetate fraction at 58g GAE/100g sample and 75.89g QE/100g sample, respectively.

Table 4. Flavonoid and phenolic content of the extract and fractions of Dracontomelon dao (Blanco) Merr bark

Extract/ Fraction	Flavonoid content (g QE/100 g sample)	Phenolic content (g GAE/100 g sample)
Methanol extract	73.97	21.89
Ethyl acetate fraction	75.89	58
Water fraction	70.44	4.95

The relationship of free radical inhibitory ability to the phenolic and flavonoic compounds of the extract and fractions of Dracontomelon dao (Blanco) Merr bark is displayed in Figures 2 and 3, with R² values representing the contribution of phenol and flavonoid to the radical scavenging³⁶. The R² values obtained were 0.8126 and 0.6651, suggesting that the flavonoid was able to radical scavenging at 81.26% and the phenol was at 66.51%. These correlation data show that flavonoid and phenolic compounds have a considerable contribution to radical scavenging.

CONCLUSION:

The methanol extract and fractions of Dracontomelon dao (Blanco) 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 than did the methanol extract and water fraction. Flavonoid compound has the most significant contribution to radical scavenging. Isolation can be performed on ethyl acetate fraction to figure out which compound is the most responsible for radical scavenging.

ACKNOWLEDGEMENTS:

Gratitude is due to the Rector of Universitas Halu Oleh, who has funded this research through DIPA Halu Oleo University in Basic Research Program, 2019.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 26.09.2019 Modified on 11.11.2019

Research J. Pharm. and Tech 2020; 13(5): 2335-2339.

DOI: 10.5958/0974-360X.2020.00420.5