Analysis of total phenolics and flavonoids content from methanol extract of Caesalpinia bonduc (L.) Roxb. seeds and antioxidant activity assay

: Caesalpinia bonduc (L.) Roxb. seed is known as gorek seed contain secondary metabolites such as alkaloids, tannins, flavonoids, and phenolics, due to the content of these secondary metabolites so that this plant has antioxidant activity. This study aimed to determine total phenolic and flavonoid contents of methanol extract of gorek C. bonduc seeds as well as antioxidant activity. Extraction of gorek C. bonduc seeds was carried out with methanol, then quantitative determination of total phenolic by the Folin-Ciocalteu method as Gallic Acid Equivalent (GAE)/gram of extract, flavonoid content by AlCl 3 method as Quercetin Equivalent (QE)/gram of extract, and in vitro antioxidant activity with DPPH (2,2-diphenyl-1-picrylhydrazyl) method was in IC50 (inhibition concentration). Based on the results of research, the phytochemical screening of methanol extract of gorek C. bonduc seeds showed alkaloids, flavonoids, saponins, tannins, and phenolics. The determination of the total phenolic and flavonoid content were obtained 0.55 ± 0.02 mgGAE/g and 0.50 ± 0.02 mgQE/g, respectively. The antioxidant activity of methanol extracts and ascorbic acid showed IC 50 values were 3770.77 ppm and 3.36 ppm respectively.


■ INTRODUCTION
Free radicals are intermediate products formed in various chemical reactions in the body and also from polluted environments such as cigarette smoke, vehicle fumes, pollutants and radiation [1]. Free radicals are highly reactive compounds and can bind or attack electrons in surrounding molecules such as carbohydrates, lipids, proteins, and DNA [2]. Imbalance occurs if the free radicals produced exceed the limit of their cellular antioxidant protection capability, the free radicals will affect the process of disease emergence [3][4]. Free radicals are one of the causes of several diseases, such as Parkinson disease, Alzheimer type dementia etc. Free radical production is effectively combated by the use of antioxidants [5].
Plants containing flavonoids and phenolics are known to possess strong antioxidant properties [6] and potent antioxidant activity leading therefore to various defensive and disease fighting properties. Phenolic compounds are plants secondary metabolites considered as very important plant constituents due to the presence of one or more hydroxyl groups on their aromatic ring. Those phenolic compounds being non harmful to human's health [7].
Caesalpinia bonduc (L.) Roxb. is a medicinal plant belonging to the family Caesalpiniaceae. It is a prickly shrub widely distributed all over the world especially in Indian tropical regions such as Kerala, Andaman and Nicobar Islands and Sri Lanka. It is popular in indigenous system of medicine in India. it has been considered as an important remedy for the treatment of several diseases. In Indonesia known as gorek plant. All parts of the plant have medicinal properties so it is a very valuable medicinal plant which is utilized in traditional system of medicine. The plant has been reported to possess antidiabetic, antiinflammatory, antimalarial, antimicrobial, antifungal, antitumor, and antioxidant [9][10]. The antioxidant activity of Caesalpinia bonduc (L.) Roxb. may be it is caused by the presence of flavonoid dan fenolic compound in it. Hence, This study aimed to determine total phenolic and flavonoid contents of methanol extract of gorek Caesalpinia bonduc (L.) Roxb. seeds as well as antioxidant activity as a basic information to develop the research about this plant in health, pharmacology, and medic.

Extraction
The extraction result of C.bonduc seeds with methanol as solvent can be seen in Table 1.

Phytochemical Screening
The methanol extract of C. bonduc seeds were obtained are then tested to determine the phytochemical components contained there in. Common phytochemical content of plants such as flavonoids, alkaloids, terpenoids, steroids, tannins, and saponins can be identified, can be seen in Table 2. Phytochemical analysis conducted by Manikandaselvi, et al. [18] that the seed powder of C. bonduc had alkaloids, phenols, flavonoids, tannins, lignins, carbohydrates, protein, lipids, and fiber.

Total Phenolic Content
The total phenolic content of C. bonduc seeds was determined by Folin-Ciocalteu method were reported as gallic acid equivalents (GAE) mg/g of extract. The gallic acid calibration curve can be seen in Figure 1 which shows the maximum absorbance at a wavelength 750 nm with the equation y = 0.055x + 0.006, R = 0.999. It is known that the total phenolic content in the methanol extract of C.bonduc seeds was 0.55 ± 0.02 mgGAE/g. As compared with other study from several references, the total phenolic content obtained in this study was lower.

Flavonoid Content
The flavonoid content of C.bonduc seed was determined by AlCl3 method were reported as Quercetin equivalent (QE) mg/g of extract. AlCl3 will cause a complex reaction with flavonoid compounds [21]. Quarcetin calibration curve can be seen in Figure 2 which shows the maximum absorbance at a wavelength 428 nm with y = 0.009×-0.001, R = 0.997. It is known that the flavonoid content in the methanol extract of C.bonduc seeds was 0.50 ± 0.02 mgQE/g. As compared with other study from several references, the flavonoid content obtained in this study was lower. Sembiring, et al. [19] reported the flavonoid content of the ethanolic extract of C. bonduc seeds were 31.05 ± 0.35 mgQE/g (leaf), 21.82 ± 0.46 mgQE/g (stem), 12.55 ± 0.08 mgQE/g (root), and 13.21 ± 1.35 mgQE/g (seed).

Antioxidant Activity
The antioxidant capacity of plants can be tested using a wide variety of methods. In the present study, the antioxidant activity of the fresh produce was evaluated in terms of their free radical scavenging capacity by DPPH assay. DPPH method is a simple antioxidant measurement method that does not require a lot of reagents like other methods. DPPH reagents are compounds that can counteract or reduce the negative impact of oxidants by donating one electron to compounds that are oxidants so that the activity of these oxidant compounds can be inhibited [1].
DPPH is a free radical that is stable at room temperature and accepts an electron or hydrogen radical to become a stable diamagnetic molecule. The DPPH radical is considered to be a model of lipophilic radical. A chain reaction in lipophilic radicals is initiated by lipid autooxidants [22]. Radical inhibitory activity is determined from the reduction reaction that occurs. The radical scavenging activity of sample was determined from the reduction in absorbance at 517 nm due to scavenging of stable DPPH free radical by antioxidants. A positive DPPH test indicates that the sample is a free radical scavenger [23].
In this study, antioxidant activity of C.bonduc seeds was measured by DPPH at wavelength 515 nm. Ascorbic acid with known antioxidant potential was used as a positive control. The antioxidant activity of the extract and positive control was shown in Figure 3 and 4. The antioxidant activity of the extract and positive control was determined according to the regression equation, y = 0.013x + 0.980, R² = 0.989 for the extract and y = 15.50x -2.019, R² = 0.998 for the control positive, so that the IC50 value can be calculated, it was 3770.77 ppm and 3.36 ppm, respectively. The ability of the methanol extract of C. bonduc seeds inhibiting DPPH radicals was lower than ascorbic acid. According to Matheos, et al. [24], vitamin C or ascorbic acid has two sites for the hydrogen release process which are connected internally so that there is a further release process after the first release. Ascorbic acid has high antioxidant activity because ascorbic acid has 2 hydroxyl groups which makes it easier to donate hydrogen. Antioxidant activity of C. bonduc seeds obtained in this study is relatively low. This is consistent with the low total phenolic and flavonoid content obtained. As it is known that the content of phenolic compounds may contribute to antioxidant activity through the ability of these compounds to donate hydrogen so that their levels in the extract can determine high or low antioxidant activity. Antioxidant properties of C. bonduc seeds had been reported by Sachan, et al. [20] that the chloroform extract of C. bonduc seed had an IC50 170±4.08 g/mL and a total phenolic content of 21.96±2.12 g/mL in 1000 g/mL, the water extract of shell C. bonduc had a value of IC50 350.638 μg/ml [25], and the ethanol extract of C. bonduc seed had IC50 74.73 μg/ml [4]. The variation might be caused by different phytogeographic region and plant nutrition, which could modify the secondary metabolites of the plant and due to the different method of extraction and solvents polarities [19].

■ CONCLUSION
The result showed that methanol extract of C. bonduc seeds contains alkaloids, phenols, flavonoids, saponins, and tannins. The total phenolic and flavonoid contents respectively were 0.55 ± 0.02 mgGAE g and 0.50 ± 0.02 mgQE/g, as well as antioxidant activity with IC50 3770.77 ppm, and ascorbic acid as positive control has a value of IC50 3.36 ppm.

Procedures
Sample Preparation C. bonduc seeds used in this research were obtained from Gusung Timur, Selayar, South Sulawesi, Indonesia. C. bonduc seeds were cut into small sizes. Then dried in an oven for 2 days at 40°C, then crushed in an electric grinder into powder [11].

Extraction
The extraction process was carried out based on Devi, et al. (2020) [12]. which has been modified. Powdered of C. bonduc seeds was weighed as much as 300 grams was extracted by maceration method using methanol as the solvent for 24 hours and repeated 3 times. The crude extract was filtered. The filtrate obtained was concentrated under vacuum in a rotary evaporator at 30-40°C for further use. The extract that obtained was continued to the phytochemical test. Phytochemical screening for alkaloid, phenolic, flavonoid, tannin, saponin, and terpenoid/steroid was performed according to the standard procedure described by Harbone, et al. [13].

Determination of Total Phenolic Content
The total phenolic content was determined by Folin-Ciocalteu method adapted from Chanwitheesuk, et al. (2004) [14] with slight modification.

Sample Preparation
The sample was weighed as much as 0.05 g, then added 10 mL of hot distilled water (80 o C). After that, it was allowed to stand for 10 minutes, then filtered to obtain a sample filtrate. Samples are diluted if necessary.

Determination of Total Phenolic Content
A total of 5 mL of sample was added 0.25 mL of 50% Follin reagent, then 0.5 mL of saturated Na2CO3 was added, then the mixture was left for 30 minutes. The absorbance of the mixture was measured at the maximum wavelength 750 nm using a UV-Vis spectrophotometer. Gallic acid was used as standard in various concentration 1,2,4,8,16 ppm were prepared in ethanol and distilled water was used as blank. Using a standard curve, the total phenolic compound content can be calculated and expressed as gallic acid equivalents (GAE) in mg/g of extract.

Determination of Flavonoid Content
Total flavonoid content was determined by aluminium chloride method adapted from Singleton and Rossi (1965) [15] with slight modification.

Sample Preparation
The sample was weighed as much as 0.05 g, then dissolved in 10 mL of ethanol. Then filtered if necessary to obtain a sample filtrate. Samples are diluted if necessary.

Determination of Total Flavonoid Content
Samples of 0.5 mL were added 3 mL of methanol, was added 0.2 mL of AlCl3 and 0.2 mL of 10% CH3COOK 1 M. Then volume was made up to 10 mL by adding 6.2 mL aquadest. The absorbance of the mixture was measured at the maximum wavelength 428 nm using a UV-Vis spectrophotometer. Quercetin was used as a standard in various concentration 2, 4, 8, 16, 32 ppm were prepared in methanol and methanol was used as a blank. By using a standard curve, the content of flavonoid can be calculated and expressed as quarcetin equivalents (QE) in mg/g extract.
Determination of Antioxidant Activity DPPH scavenging ability assay was used to evaluate the antioxidant activity of extract. Test was conducted in a a UV-Vis spectrophotometer according to Molyneux (2004) [16] with slight modification. The extract of C. bonduc seeds were made in concentrations of 300, 400, 500, 600 and 700 ppm. Ascorbic acid was used as positive control in different concentrations 1, 2, 3, 4, and 5 ppm. After that, the measuring series solution was added with 1 mL of 0.4 mM DPPH each solution and added methanol until a total volume of 5 mL was obtained. After 30 minutes incubation at room temperature in dark room, absorbance was read at 515 nm using using a UV-Vis spectrophotometer [17]. The scavenging ability (%inhibition) was calculated as follows: Concentration of samples resulting in 50% inhibition on DPPH (IC50 value) were calculated.

■ DECLARATION
There is no conflict of interest from authors for this research.

■ ACKNOWLEDGEMENT
Author wishing to acknowledge assistance or encouragement from parents, colleagues, special workers by technical staff such as the laboratory technicians in the affiliation and all parties who have helped and participated in this research.