Chemical profiles and antioxidant properties of Bruguiera gymnorrhiza fruit extracts from Central Sulawesi, Indonesia

Bruguiera gymnorrhiza is one of the mangrove species that a source of antioxidants. Antioxidant substances are able to protect cells from oxidative stress and other related diseases. This study aimed to evaluate the chemical profiles, predicted biological activity, and antioxidant activity of the B. gymnoriza fruit extracts. The research methods included sampling, extraction (maceration with MeOH:DCM), identification of chemical profiles (GCMS spectra analysis), assaying for computational analysis (PASS server and ADMET), antioxidants (DPPH radical scavenging), and total phenolic content (Folin - Ciocalteu). Bruguiera gymnorrhiza fruits was collected from Central Sulawesi, Indonesia. The chemical profiles detected in the B. gymnorrhiza fruit extracts, namely isopimaradiene (64.20%); 4 - (2 - Aminopropyl) phenol (19.06%); dimethylamino - dimethylphosphene oxide (9.40%); 3 - amino - 2 - benzylbutanoic acid (5.46%); and 1,4 - dideuteriooctane (1.89%). PASS server analysis showed that the five compounds detected from B. gymnorrhiza fruit have the potential as an NF - E2 - related factor (Nrf2) stimulant and oxygen scavenger. The ADMET analysis results indicated that B. gymnorrhiza fruits could be developed as folk medicine and nutraceutical products.


Sampling location
Bruguiera gymnorrhiza fruits were collected from the coastal area of Tomini Bay in Laemanta, Parigi Moutong, Central Sulawesi, Indonesia (-0.1847 S and 120.0088E) (Figure 1) in April 2020.Mangrove fruits were randomly taken from a mangrove tree B. gymnorrhiza followed by identification based on Noor et al. (2006).After collection, the B. gymnorrhiza fruits were washed with water to eliminate the presence of contaminants.

Preparation of Bruguiera gymnorrhiza extracts
Before extractions, 500 g of B. gymnorrhiza fruits were dried at 50-60 o C using an oven (Finco OV50) for 15-20 hrs.After drying, the samples were pounded into flour.The crude extract of B. gymnorrhiza (100 g) was obtained by extraction with the combination of MeOH and DCM (1: 1) for 48 hrs (Hsiao et al., 2015;Putra et al., 2016).The filtrate obtained by the extraction was filtered with filter paper (Whatman No. 42).The filtrate was vacuum-concentrated at 40-45°C using a rotary evaporator (EYELA N-1100) to obtain the crude extracts.The extraction process was carried out three times.Then, the extracts were weighed and dissolved in 30% MeOH then stored at 4-5 o C for further analysis.

Chemical profiles screening using GCMS
The chemical components of B. gymnorrhiza fruits extract were profiled using Gas Chromatography-Mass Spectrometry (GCMS).The analysis was performed on an HP 6890 GCMS system (Hewlett-Packard, California, USA) equipped with a capillary column (Agilent 19091S -433 HP-5MS; 30 m x 250 µm i.d.; Santa-Clara, California, USA).The carrier gas was helium at a flow rate of 1 mL/min.The oven temperature was set at 325°C . The pre-oven temperature was 150°C held at 2°C/ min.It ran for 10°C/min and was then increased to 240°C , holding time for 11 mins.The total running time was 24 mins.The scanning range was 50 -550 amu.Structural assignments were based on analysis of mass spectral fragmentation patterns and compared with mass spectra in the National Institute of Standards and Technology (NIST) and Wiley's compound profile database (Tanod et al., 2019).

Prediction of biological activity
The chemical profiles from B. gymnorrhiza fruits extract by GCMS were predicted through Prediction of Activity Spectra for Substances (PASS)-way2Drug server (http://www.pharmaexpert.ru/passonline/index.php).PASS server is used to predict the chemical compounds' biological activity (Aisiah et al., 2020;Riyadi, Romadhon, Anggo et al., 2020).The structural target compounds obtained from the National Centre for Biotechnology Information (https:// pubchem.ncbi.nlm.nih.gov/) in the form of canonical SMILE were used to predict their biological activity.The chemicals profile structure was also used to estimate their ADMET (absorption, distribution, metabolism, excretion, and toxicity) through AdmetSAR (http:// lmmd.ecust.edu.cn/admetsar2).

Determination of total phenol contents
Total phenolic content (TPC) of B. gymnorrhiza fruits extract was determined by the Folin-Ciocalteu (FC) reagent method (Blainski et al., 2013 andLamuela-Raventós, 2017).The gallic acids (1 mg/mL) were used as a standard by diluting ethanol: distilled water (1: 1).Gallic acid solutions were diluted in serial dilution with concentrations 5, 20, 40, 60, 80, and 100 μg/mL.From each dilution concentration, 1 mL of gallic acid was taken, and 9 mL of distilled water was added.Then those aliquots were mixed with 1 mL of FC reagent (homogenization) and incubated for 8 mins at room temperature.After incubation, 3 mL of 20% Na 2 CO 3 was added, and the mixture was allowed to stand at room temperature for 2 hrs.The absorbance was measured at 750 nm.The standard curve for gallic acid was prepared with gallic acid concentration (μg/mL) with an absorbance value.
The aliquot (1 mg/mL extract) was added with 10 mL of distilled water + 1 mL of FC reagent and incubated as described above.Then TPC content was measured at 750 nm.B. gymnorrhiza fruits extract's total phenolic content was then calculated based on a gallic acid standard curve and expressed as mg of gallic acids equivalent (GAE)/g extract.

DPPH radical scavenging activity
DPPH is a free radical, stable at room temperature, which produces a violet solution in ethanol.Antioxidants' presence will reduce the colour and change into uncoloured ethanol solution (Clarke et al., 2013).The total free radical scavenging capacity of Free radical scavenging capacity was assessed according to the previously reported method using a stable DPPH (Molyneux, 2004;Dewanto et al., 2019).The extract was expressed as the IC 50 , which is the antioxidant concentration required to quench 50% of the initial DPPH under the experiment treatment.Vitamin E, known as the strong DPPH radical scavenger (Yu et al., 2002), was used as the positive control.The IC 50 value was converted to the vitamin E equivalent.Each evaluation was performed in triplicate.
Bruguiera gymnorrhiza fruit extracts (200 µg/mL) in ethanol solution was prepared and diluted into several concentrations (20,40,60,80, 100 µg/mL). 2 mL aliquot of each concentration was reacted with 50 μM DPPH ethanol solution.The mixtures were homogenized and stand for 30 mins in the dark at room temperature before reading against a blank at 517 nm (UV-VIS spectrophotometer T90 + PG Instruments Ltd).
The DPPH radical scavenging effect in percentage (%) was calculated according to the following equation: Where A sample is the absorbance of a sample solution and A blank is the absorbance of the blank solution (containing all reagents except the test sample).
The half of maximum concentration in reducing initial DPPH was calculated from the graph by plotting inhibition concentrations against initial DPPH to obtain a linear regression equation, y = a + bx.Each measurement was carried out in triplicates.

Sample identification
Identification was carried out according to Noor et al. (2006).The samples included in the B. gymnorrhiza based on the leaves' shape and tip, the types of roots, fruit, and flowers.BG's bark has lenticels, and the surface is smooth to rough, dark gray to brown.BG roots are like planks extending to the side at the tree's base and developing several knee roots.Characteristics of BG leaves are skinned green on top and yellow on bottom.The BG leaves shape is elliptical to elliptical-lanceolate and had average length and width 4.5 to 7 cm and 8.5 to 22 cm, respectively.Characteristics of BG fruit are straight, blunt, and dark green-purple when fresh with length (12-30 cm) and diameter (1.5-2 cm) (Figure 2).

Chemical profiles of Bruguiera gymnorrhiza fruit extracts
The GCMS analysis showed the presence of 5 compounds from B. gymnorrhiza fruit extracts (Table 1).The chemical profiles were identified through mass spectrometry attached with GC.The total ion chromatogram (TIC) of the GCMS analysis confirms that five compounds and their different retention time (4,700 to 22,824 mins) are presented in Figure 3

Biological activity prediction of fruits extracts
The prediction of biological activity was performed with chemical profiles in B. gymnorrhiza fruits extracts and presented in Table 2.The biological activity prediction was used the PASS server.The PASS server could predict compounds' biological activity based on their formula with 95 % accuracy (Filimonov and Poroikov, 2008;Riyadi, Tanod, Wahyudi et al., 2020).
The biological activity prediction value of the B. gymnorrhiza fruits extracts' chemical profiles was expressed as a "probability to be active" (Pa).The chemical profiles are probable to act as antioxidants, free radical scavengers, oxygen scavengers, and NF-E2related factor (Nrf2) stimulant properties (Figure 4).The PASS server was analysed and dominated by Nrf2 stimulant and oxygen scavenger, which play a role in protecting oxidative stress.
ADMET analysis is an important parameter to determine whether a natural product will reach the body's target when administered orally (Dong et al., 2018;Natesh et al., 2021).The ADMET properties of compounds are important to the development process of nutraceutical and pharmaceutical products (van de Waterbeemd and Gifford, 2003;Guan et al., 2019).The utilization of B. gymnorrhiza fruits extracts for human health benefits needs to be satisfied with the ADMET properties before further applications.Therefore, we evaluated the ADMET properties of B. gymnorrhiza fruits extracts chemical profiles by using admetSAR (Table 2).
The chemical profiles of B. gymnorrhiza fruit extracts showed good human intestinal absorption.However, only dimethylamino-dimethyl phosphene oxide did not show well on the caco-2 permeability (Table 2).Interestingly, none of the selected chemical profile functions is acted as both P-glycoprotein (P-gp) substrate and inhibitor.Furthermore, 4-(2-Aminopropyl) phenol), isopimaradiene, and 3-amino-2-benzyl butanoic acid from the B. gymnorrhiza fruits extracts act as substrate of CYP450 2D6, CYP450 3A4, and CYP450 2C9, respectively.In contrast, none of the chemical profiles acts as inhibitors of CYP450 enzymes, which  In addition, all chemical profiles showed a positive response as oral bioavailability, water-soluble, and could bind to proteins in the blood.

Total phenol content
Phenolic compounds are mainly found in plants.Phenolic compounds have a protective role in human health and are beneficial in food and pharmaceutical products due to their benefits (Salar et al., 2017).The present study evaluated the quantitative estimation of phenolic compounds in BGF extract collected from North Sulawesi, Indonesia.The TPC value of BGF extract was determined by the FC method reported by Blainski et al. (2013) and Lamuela-Raventós (2017).Gallic acid was used as a standard for the calibration curve to calculate the TPC with equation y = 0.0101x -0.0318.The calculated result of TPC was accounted for 27539.60±35.01mg GAE/100 g dry extract.The results indicated that B. gymnorrhiza fruit extracts contained a high level of phenolic compounds.

Antioxidant activity analysis using the DPPH method
The DPPH method has been widely used for antioxidant evaluation due to a stable free radical to evaluate radical scavenging activity (Liu et al., 2004).The level of discoloration exhibits the potency of antioxidants to scavenge free radicals.These phenomena due to the reaction between the 2,2-Diphenyl-1-Picrylhydrazyl and antioxidant compound to form the hydrazine diphenyl picrite (Huliselan et al., 2015;Tanod et al., 2019).DPPH could reduce the exposure of radical proton scavengers (Yamaguchi et al., 1998).The antioxidants reduced the stable DPPH radicals, resulting in decreased absorbance at 517 nm (Behgar et al., 2011).The present study evaluated B. gymnorrhiza fruit extracts' antioxidant activity using the DPPH radical scavenging method with more than one concentration and compared the results with vitamin E (Figure 5).
The B. gymnorrhiza fruit extracts were also

Discussion
Bruguiera gymnorrhiza, a medicinal food plant, is widely distributed in Indonesia (Kusmana, 2014).Bruguiera gymnorrhiza has been used for several purposes, such as vegetables, firewoods (Kusmana et al., 2018), and folk medicine (Ahmed et al., 2007).The present study reported the characteristics of chemical profiles and antioxidants properties of B. gymnorrhiza fruit extracts.This study also predicted the biological activity related to antioxidant and ADMET properties of chemical profiles in B. gymnorrhiza fruit extracts.The major chemical profiles identified by GCMS from the B. gymnorrhiza fruit extracts were dominated by Isopimaradiene (terpenoids component) and 4-(2-Aminopropyl) phenol (phenolics component) (Figure 3 and Table 1).Plant phenolics denote a major group of compounds with antioxidants properties (Adesegun et al., 2009;Pandey and Rizvi, 2009;Lin et al., 2016).Phenolics reported have the ability to strong reactive oxygen species (ROS)-scavenging activity, dropping nitrite levels, and downregulated cyclooxygenase-2 (COX-2) expression, which can play a vital role in human health (Lin et al., 2009;Rodríguez-López et al., 2020).In addition, terpenoids are also reported as antioxidant potential (Mohandas and Kumaraswamy, 2018).Terpenoids have a hydroxyl group that may act as an antioxidant due to their relatively complex cyclic structure (consisting of alcohol, aldehyde, or carboxylic acid) (Dewanto et al., 2019).
The predictive of B. gymnorrhiza fruit extract chemical profiles by PASS-Server of the major compounds are related to antioxidant properties.Isopimaradiene, a diterpenoid, has been reported to have anti-oxidative and anti-inflammatory properties (Kim and Kim, 2009;Tungcharoen et al., 2019) and antibacterial activity (Rijo et al., 2009).Meanwhile, 4-(2-Aminopropyl) phenol is exerted antioxidant activities, free radical scavenger, O 2 scavenger, and NF-E2-related factor 2 (Nrf2) stimulant.Nuclear factor erythroid 2related factor 2 (Nrf2) plays an important role in regulating the antioxidant proteins to protect against oxidative damages (Hwang and Jeong, 2010;Barajas et al., 2011;Ma, 2013;Cui et al., 2016).Under oxidative stress conditions, activated Nrf2 translocate into the nucleus and modulates antioxidant-related genes' expression at the transcriptional level (Johnson et al., 2008).Oxygen scavengers could prevent oxidation and quality foodstuff changes (Byun et al., 2011;Lalpuria et al., 2012;Mu et al., 2013).This compound has one phenol ring and one NH 2 bond.H atoms in the phenol and NH 2 ring act as electron donors to react as free radicalizers such as reactive oxygen species (ROS) (Lobo et al., 2010).
The present study has been found that B. gymnorrhiza fruit extracts possessed higher antioxidant potential and phytochemical content.The antioxidant potential is comparable with synthetic antioxidant vitamin E. The results revealed that B. gymnorrhiza fruit extracts exhibited strong antioxidant activity (IC 50 70.45).This activity is higher than methanol extract of B. gymnorrhiza fruit extracts collected from the Odisha coast, India (Rout et al., 2015) and B. gymnorrhiza extract collected from Bangladesh (Hosen et al., 2020).Some factors are related to the different antioxidant activity of BG, e.g., the polarity of solvents (Jacoeb et al., 2013), the maturity of fruits (Sudirman et al., 2014), geographical locations (Rout et al., 2015;Hosen et al., 2020).
Our data showed a high level of phenolic content.This level is considerably higher than ethanol: methanol (1: 1) of B. gymnorrhiza fruit extracts (21.9 mg GAE/g powder) (Hosen et al., 2020).The extract contains 4-(2-Aminopropyl) phenol and -amino-2-beunzylbutanoic acid with an aromatic ring structure with a hydroxyl group (typical of phenolic compounds).None of the selected chemical profiles functions as P -glycoprotein (P-gp) substrate and inhibitors.P-gp transporter acts as a protective barrier to keep toxins out of the body and as a cellular defence system to protect the accumulation of foreign materials in the human body (Marzolini et al., 2004;Szakács et al., 2008;Quan et al., 2012;Cloos et al., 2018).After absorption in the intestine, compounds flow to the blood then metabolized in the liver.The liver CYP450 enzyme family could break down the compounds and eliminate them from the human body (Natesh et al., 2021) phenol, isopimaradiene, and 3-amino-2-benzyl butanoic acid from the B. gymnorrhiza fruit extracts act as a substrate of CYP450 2D6, CYP450 3A4, and CYP450 2C9, respectively.Possibly, they will metabolize through those enzymes in the liver.Interestingly, all compounds are not showing any toxicity in the liver.Compound No. 1 -3 showed class II toxicity, while compounds No. 4-5 exhibited class 3 toxicity.According to the GHS United Nations 2015, the toxicity of any compounds taken orally was classified into six categories based on the LD 50 ranges.Class 1-fatal (LD 50 ≤5), class 2-fatal (5 <LD 50 ≤50), class 3-toxic (50 <LD 50 ≤300), class 4harmful (300 <LD 50 ≤2000), class 5-may be harmful (2000 <LD 50 ≤5000), and class 6, LD 50 >5000 is considered as non-toxic (Drwal et al., 2014).

Conclusion
It can be concluded that B. gymnorrhiza fruit extracts from Central Sulawesi could be developed as a folk medicine, nutraceutical, and functional food ingredient with antioxidant properties in a certain amount that is safe for consumption.

Figure 4 .
Figure 4. Probability to be active from Bruguiera gymnorrhiza fruit extracts profiles with PASS Server

Table 2 .
Admet profile of Bruguiera gymnorrhiza fruit extracts Figure 5. DPPH scavenging effect from Bruguiera gymnorrhiza mangrove fruit extracts FULL PAPER evaluated for the inhibitory effect of DPPH radicals for IC 50 determination (Table3).The B. gymnorrhiza fruit extracts possessed antioxidant activity because they could donate hydrogen atoms/electrons to react with DPPH radicals.The results showed a B. gymnorrhiza fruit extracts concentration dependent on free radical scavenging and comparable with synthetic antioxidants (vitamin E).The results indicated that B. gymnorrhiza fruit extracts have strong antioxidant activity (IC 50 : 70.45±0.49μg/mL), however its activity lower than vitamin E (IC 50 : 13.72±1.02μg/mL).

Table 3 .
. According to the IC 50 of Bruguiera gymnorrhiza mangrove fruit extracts using the DPPH Radical Scavenging, compared with vitamin E.