Phytochemical composition, total phenolic content and antioxidant activity of Anadara granosa (Linnaeus, 1758) collected from the east coast of South Sumatra, Indonesia

: Anadara granosa is a species of the class bivalve commonly found on the east coast of South Sumatra as a fishery commodity. This species has not been widely studied as a source of new bioactive compounds that have antioxidant abilities. This study aims to analyze the antioxidant ability of A. granosa against DPPH radicals and its phytochemical profile qualitatively. Samples were taken at the fishing port of Sungsang Village, South Sumatra, Indonesia. Furthermore, the samples were extracted using ethanol as a solvent and tested for antioxidants against DPPH radicals, total phenol analysis, and preliminary phytochemical test. Based on the antioxidant test results, the IC 50 value of the ethanolic extract of A. granosa was 85 g/ml with ascorbic acid 2 g/ml as a comparison. Then, the ethanol extract contained a total of 10.7057 mgGAE/g phenol and the results of the phytochemical test contained bioactive compounds of alkaloids, steroids, flavonoids, saponins, and tannins. The ethanolic extract of A. granosa contained bioactive compounds, which were reported to have potent antioxidant activity. The results of this study were expected to be important information in the latest report of the antioxidant activity of A. granosa species and contributed to the development of marine natural products.


Introduction:
Benthic communities are found in the ecosystem of the east coast of South Sumatra. The east coast area dominated by mangrove ecosystems becomes a good habitat for benthic community life 1 . The diversity and distribution of benthic organisms are supported by the high food sources and the availability of living places on the surface of vast mud subsrates 2 . The environment on the east coast of South Sumatra has a wide muddy landscape because it is influenced by the dynamics of the waters of major rivers such as the Musi River and Banyuasin River and the sea waters of the Bangka Strait which tend to carry a considerable sedimentation factor. The area is overgrown by dense mangroves with fluctuating environmental physical-chemical dynamics 3,4 . Natural fluctuations in the coastal environment cause only benthic organisms, especially certain mollusks, to survive such as marine shellfish and marine gastropods organisms.
Anadara granosa is a group of the family Arcidae, a class of bivalves, mollusk phylum known for a long time 5 . Marine mollusks have been reported to have biological activity because they contain a variety of bioactive compounds, such as gastropod groups in mangrove vegetation 6 and coral reef ecosystem 7 , and the marine bivalves group 8 . Bioactive compounds produce essential biological activities in the form of antioxidant abilities that are attracting attention today. Antioxidants aim to capture free radicals that enter the body's systems 9,10 , while free radicals can cause common diseases to cancer 11 . Some bioactive compounds have good antioxidant abilities, such as alkaloids, terpenoids, and phenols 12,13 . Phenols are a group of compounds that have properties as potent antioxidants 14 . The components of phenolic compounds released from the extract are responsible for antioxidant activity. The mechanism of action of these antioxidants is done by trapping free radicals and metal ions 15 .
A. granosa has been consumed by the general public as healthy seafood. This species is a high-protein food because it contains several minerals such as protein and zinc 16 . Some of the benefits of A. granosa are believed to cure and prevent some types of diseases. The biological activity research of A. granosa is still focused on its antimicrobial capabilities 17 . However, the antioxidant abilities of this species are still not well reported. The application of bioactive compounds with antioxidant activity in food products and medicines can have health effects on the body. Natural compounds such as flavonoids and phenolics can be used in food additives as nutritional enhancers, cytostatic drugs, and beauty products in pharmaceutical products.
The study aims to analyze the antioxidant abilities of A. granosa extracted with ethanol solvents. In addition, qualitative assessment of phytochemical content and quantitative assessment of phenol content were also measured. This study is significant to report the antioxidant activity of ethanol extract of A. granosa against the free radical 2,2-diphenyl-1-picrylhydrazyl.

Sample collection
A sample of A. granosa was taken in March 2021. The samples were obtained from Sungsang Village, South Sumatera caught from the Banyuasin waters. Next, the sample was carried out using a cooling box during the trip to the laboratory. The sampling location is presented in Fig. 1.

Preparation of extractions
A. granosa was taken from Sungsang Village, which came from fishers. The sample collected was cleaned using flowing water. The samples were separated between the shell and meat. The sample was put in the oven at 40 °C ± 30 min so that the meat was not too wet. The sample dried with the heat of the sun. After drying, the sample was smoothed using a blender. Maceration was carried out for two days and filtered using filter paper. Maceration required each of 200 g of meat.
Maceration used ethanol solvents with a ratio of 1: 4 (b/v). The sample was concentrated using a rotary evaporator until it became a paste or crude extract at 40 °C. The next step was to make a parent solution for the concentration dilution used in the antioxidant test. Preliminary phytochemical Qualitative tests of extract phytochemicals referred to 18 , included alkaloids, flavonoids, saponins, tannins, and steroids tests. Extract of 2.5 mg was prepared for each test and then mixed in each test solution formula. Preparation of phytochemical tests using several different solutions in each test included, the alkaloids test preparing the meyer and dragendoff reagents, flavonoids test using a mixture of 2% NaOH in alkaline reagent test, saponins test mixing HCl solution in foam test, Tannins test using 1% FeCl solution, and steroids test prepared acetic anhydride solution (CH3CO)2O and H2SO4. The phytochemical test was classified as a conventional qualitative test based on the color change in the liquid mixture.

Determination of total phenolic content
Total phenol of extract analysis was using the Folin-Ciocalteau method was remarked in detail in a previous study 19 .

Determination of antioxidant activity
Antioxidant activity was analyzed by the diphenyl-1-picrylhydrazyl (DPPH) method 9,20 . Extract weighed as much as 0.2 g and added 100 ml of ethanol, so that parent solution can be produced with the concentration of 2000 μg/ml. DPPH 0.1 mM solution was made by weighing 0.002 g of DPPH crystals into 50 mL of ethanol and obtaining a solution with a concentration of 40 μg/ml, then shaken to dissolve the DPPH powder. The test concentrations used were 200, 400, 600, 800, and 1000 μg/ml diluted from a parent solution of 2000 μg/ml. Next, as many as 1.5 ml of test solution and comparison was reacted by 1.5 ml solution DPPH 0.1 mM in the test tube.
DPPH solution mixed with ethanol and extracted was homogeneous and incubated for 30 min in a dark place. After that, absorbance measurements were taken using a UV-Vis spectrophotometer at a wavelength of 517 nm. Levels of antioxidant strength with DPPH method based on the strong criteria 50 -100 μg/ml, very strong < 50 μg/ml, moderate 101 -250 μg/ml, weak 251 -500 μg/ml, inactive > 500 μg/ml 21 .

Morphology of A. granosa
A. granosa had a thick, rough, and contorted shell on the surface. The shape of the shell was round, slightly oval, consisting of symmetrical shell, had a pallial line on the complete inner shell and a striped outer pallial line, while the inner shell had a smooth texture with shiny white color. The primary color of the clam was brownishred, and the flesh was blood red. The largest measurement of the individual length of A. granosa was obtained at 5.97 cm long and 4.36 cm wide, while the smallest individual had a length of 4 cm with a width of 3.42 cm.

Extraction of A. granosa
Extraction resulted in a yield of crude extract. The higher the percentage value of the extract yield, the more compounds contained in the extract. Crude extract A. granosa was blackish brown. It happened because the sample in the form of meat came from animals. The fishy smell contained in a sample was due to the sample derived from shellfish meat. The measurement process of each frish weight (FW), dry weight (DW), smooth weight (SW), extract weight (EW), and percentage of the water content of A. granosa is presented in Table 1. Sample A. granosa had taken frish weight 1500 g, and after a few days of drying, the dry weight of A. granosa became 227 g with a percentage of the decreased water content of 84.8 %. The sample was smoothed by 220 g, and after the extraction process was obtained, the extract weight was 29.46 g.

Preliminary Phytochemical of A. granosa extract
Identification of phytochemical content was helpful for grouping bioactive compounds in A. granosa extracts. Phytochemical screening of A. granosa ethanol extract obtained in this study showed that flavonoid compounds, saponins, tannins, alkaloids, steroids, and tannins were present in polar solvent testing. Phytochemical tests were conducted to determine the compounds contained in the extract so that the group of compounds that caused antioxidant activity. The result of the phytochemical test is presented in Table 2. Based on Table 2, a group of bioactive compounds analyzed on phytochemical tests was declared active in extracts. In ethanol extract, A. granosa contained alkaloids, steroids, flavonoids, saponins, and tannins. Some of these bioactive compounds were known to have a working system as good antioxidants.

Total phenolic determination of A. granosa extract
Determination of the content of phenolic compounds in ethanol extract was a quantitative measurement. Measurement of total phenol used the method of adding Folin-ciocalteu reagent to the test solution as shown in Table 3. In this study, the total phenol content of the ethanol extract of A. granosa was tested in the form of green crude extract. Based on Table 3, the analysis of the total phenol content in the ethanol extract was 10.7057 mgGAE/g. This result reported that A. granosa had quite good phenolic compounds. In addition, this measurement was quite useful for quantitatively validating the presence of a group of bioactive derivatives of phenolic compounds as reported in the qualitative results in Table 2.

Determination of antioxidant activity
Antioxidant potential was quantitatively expressed by the percentage value of free radical inhibition and IC50 value from A. granosa extract. The percentage of inhibition expressed the percentage value of the extract in inhibiting free radicals at a given concentration. IC50 value expressed the concentration of extract in inhibiting free radicals by 50%. The percentage results of inhibition and average absorbance extract A. granosa and ascorbic acid were presented in Table  4. Based on the calculation results of the percentage of inhibition obtained results as presented in Table 4. Extract of A. granosa had a percentage value of inhibition > 50 %, which could be interpreted that extract A. granosa had the potential as an antioxidant. Ascorbic acid was an antidote to radicals so well that it was used as a control and as a comparison. Antioxidant activity expressed in value IC50. The small IC50 indicated that its antioxidant activity improved, while a sizeable IC50 value indicated its antioxidant activity was lower. The value of the linear regression equation and IC50 of A. granosa extract and ascorbic acid can be seen in Table 5.

Discussion:
The size of the shellfish found in this study was included in the size taken for commercial use. Shellfish shells had colors and shapes that varied depending on the type, food, and habitat 3,22 . A. granosa lived by immersing themselves in sandy and muddy beaches 23 . A. granosa was found on a muddy substrate at the estuarine with sloping coastal topography up to a depth of 20 m. A. granosa was an infauna that was living by immersing itself in shallow waters under the surface of mud 24 .
The resulting extract was good enough to become a paste or crude extract 25 . Extraction was done at a low temperature so that the sample could not be too hot and the compound was not damaged. Polar compounds would only dissolve in polar solvents such as ethanol, methanol, butanol, and aqueous. Non-polar compounds would also dissolve in non-polar solvents such as ether, chloroform, and hexane 26 . Polar solvents tend to be able to extract more bioactive compounds.
The antioxidant activity was affected by the content of its bioactive components, namely flavonoids, alkaloids, and phenols 12,13 . Alkaloids could function as an antibacterial, were the most significant secondary plant compound 27,28 . Steroids compounds tend to have antibacterial and antiinflammatory functions 29 . Saponins were a glycoside form of sapogenin so that they would be polar. Saponins compounds were surface-active and could cause foam if shaken in water 30 . Tannins were secondary metabolite compounds that were efficacious as astringents, antidiarrheal, and inhibit free radicals 31 . Flavonoids had functioned as antioxidants 32 . Flavonoids act as antioxidants that could prevent cardiovascular disease 33 . Flavonoids could act as exogenous antioxidants that could dampen the activity of free radicals derived from sun exposure and air pollution. Phenol compounds such as flavonoids were the most potent compounds that played an active role as antioxidants 34 .
The total phenol obtained was directly proportional to the results of antioxidants. High phenol content could produce good antioxidant activity 35 . Phenol content was commonly found in plants, mollusks, and marine organisms. The content of phenols in some types of shellfish came from microalgae and other tiny organisms that were filtered or entered the digestive system of these shellfish 36 .
DPPH radicals included organic nitrogen compounds that were widely commercialized to assess antioxidant abilities in warding off free radicals. DPPH radicals could accept hydrogen atoms (electrons) to become more stable molecules with simple mechanisms 37 . In this study, the percentage of radical inhibition of A. granosa was measured using ethanol extract. Based on comparisons between three types of polar solvents; methanol, ethanol, and water, ethanol extracts were more significant than water and less significant than methanol 38 . However, ethanol solvents were safer from the safety element than methanol solvents because they contained methane elements.
Antioxidant activity using DPPH radicals in Table 5 indicated that the IC50 value from A. granosa extract was 85 μg/ml. The antioxidant activity of A. granosa extract was relatively strong but not more vital when compared to the antioxidant activity of ascorbic acid as a comparison, which was 2 μg/ml, classified as an antioxidant with potent inhibition activity was adjusted to the criteria 21 .
Some of the biological activities of the shellfish group had been evaluated by researchers. The antioxidant activity of the species P. viridis in the same solvent produced an IC50 of 154.3 μg/ml 38 . The razor clams of the family group Solenidae once measured their antioxidant activity by resulting in 489.56 μg/ml of IC50 with weak categories 39 . The antioxidant activity found from other bivalve species showed no powerful antioxidants. That indicates that the presence of bioactive compounds in A. granosa was better as an antioxidant. Apart from being a food source, variations in the biochemical content in marine organisms could be influenced by geographic location, marine environmental conditions, water quality, and extraction techniques also significantly affect the content of bioactive organisms [40][41][42] .
This study aims to investigate the capabilities of one of the commercial bivalves, A. granosa. The ability of antioxidants shown by A. granosa through ethanol extract in this study could be the latest and essential report as an evaluation material for utilizing bivalve species in the pharmaceutical field.

Conclusion:
The IC50 value of ethanol extract of A. granosa showed a value of 85 μg/ml and belonged to the strong antioxidant category. Based on preliminary phytochemical results, A. granosa extract contained several bioactive compounds such as alkaloids, steroids, flavonoids, saponins, and tannins. The results of the total phenol test analysis of 10.7057 mgGAE/g indicate the good antioxidant ability of A. granosa.