Phytochemical and antioxidant activity evaluation of the bark of Tampoi ( ) Baccaurea macrocarpa

Tampoi ( ) is a tropical rainforest plant Background: Baccaurea macrocarpa that produces edible fruit and is native to Southeast Asia, especially East Kalimantan, Indonesia. Previous research showed that Tampoi potentially can be developed as a drug. It was reported that the extract of Tampoi fruit displayed antioxidant activity, which was correlated with its phenolic and flavonoid substances. There is no information about the antioxidant activity of other parts of this plant, such as the bark, which might also have this kind of activity. Therefore, the aim of this study was to evaluate the phytochemical, toxicity, and antioxidant activity of the bark of Tampoi. : The bark of Tampoi was extracted with methanol and Methods concentrated using rotary evaporator to obtain the methanol extract of the bark. Secondary metabolites of this extract was determined using phytochemical analysis. Afterward, the methanol extract was tested for its toxicity using brine shrimp lethality test and antioxidant activity using the 2,2-diphenyl-1-picrylhydrazyl method. Phytochemical evaluation results showed that the methanol Results: extract of bark of this plant contains several secondary metabolites including alkaloids, flavonoids, phenolics, steroids, and triterpenoids. The toxicity test displayed no toxic property due to a LC value above 1000 ppm. For antioxidant activity, the result exhibited that the methanol extract of bark of this plant could be categorized as an active extract with IC value of 11.15 ppm. Moreover, based on gas chromatography-mass spectrometer analysis, there are 37 isolated compounds from the bark, one of which is methylparaben, a phenolic predicted to act as an antioxidant. : The results obtained in this research demonstrated that the Conclusion bark of Tampoi ( ) has potential as an antioxidant. B. macrocarpa


Introduction
Indonesia is a mega-diverse country in terms of biodiversity that is flanked by the Indian and Pacific Oceans.Indonesia's biodiversity encompasses the diversity of living things both on land and sea 1 .Indonesia, especially East Kalimantan, has very extensive tropical rainforest, which is a habitat for much biodiversity.Various types of plants have long been utilized by the community as traditional medicines.The utilization of natural products as an alternative medicine is increasing because natural ingredients are believed to be safer than synthetic substances, i.e. do not contain chemicals that only can be found in modern medicines, which are linked to toxicity 2 .
Among plants, the genus of Baccaurea have interesting biological activities.For example, B. angulata has been reported as a potential functional food with effective antioxidant 3 , anti-inflammatory, anti-atherogenic, and hypocholesteromia activities 4 .Other research has also investigated the biological activity of other species of this genus, i.e.B. lanceolata and B. macrocarpa.It was reported that the fruits of B. macrocarpa exhibited the highest antioxidant activity compared with B. lanceolata, which significantly correlated with the phenolic and flavonoid contents 5 .
B. macrocarpa is one of the typical plants of East Kalimantan, Indonesia and the edible fruits is a source of additional nutrients and known as Tampoi.Until now, the information about the antioxidant activity of other parts of this plant such as the bark of Tampoi has not been reported yet.Hence, the present research was conducted to investigate the phytochemical, toxicity, and antioxidant activity of the bark of Tampoi (B.macrocarpa).Furthermore, the gas chromatography-mass spectrometer (GC-MS) analysis was performed to obtain information about the kinds of isolated compounds contained.

Extraction
Extraction was carried out as described previously by Erwin et al. (2014) 6 .The bark of Tampoi (B.macrocarpa) was dried for 1 week at room temperature and ground to a powder.The powder was extracted using a maceration method by soaking in methanol for 24 hours at room temperature, which was repeated three times.Afterwards, the extract solution was filtered by filter paper and the solvent was evaporated under vacuum using a rotary evaporator (Buchi R II) at 45°C and 1 atm, to obtain the methanol extract of bark of Tampoi.

Phytochemical evaluation
Phytochemical evaluation was performed to investigate the secondary metabolites contents of the methanol extract of bark of Tampoi (B.macrocarpa), including alkaloids, flavonoids, phenolics, steroids, triterpenoids, and saponins, as described previously 7 .The presence of secondary metabolites were identified by observing the changing color of the extract.These evaluations were performed as follows: Alkaloids. 1 mg of extract was inserted into a test tube and then diluted in 1 mL methanol.Then a few drops of H 2 SO 4 1M was added.Afterwards, a few drops of Dragendorff reagent was added into the mixture.The formation of orange on filter paper indicated the presence of alkaloids.
Flavonoids. 1 mg of extract was inserted into a test tube and diluted in 1 mL methanol.A few 2 mg of Magnesium powder was added followed by a few drops of concentrated HCl.The presence of flavonoids was identified by the formation of pink or red color.
Phenolics. 1 mg of extract was introduced into a test tube and dissolved in methanol.Then a few drops of 1% FeCl 3 were inserted.The formation of green, red, purple, dark blue or black indicated the presence of phenolics.
Steroids and triterpenoids. 1 mL of methanol and 1 mg of extract were inserted into a test tube, stirred until homogeneous, then 2 drops of anhydride acetate and 1 drop of H 2 SO 4 were added (Liebermann Burchard reagent).The formation of green or purple precipitation showed a sample containing steroids, and red precipitation displayed the presence of terpenoids.
Saponins. 1 mg extract was put into a test tube and then dissolved in distilled water, and shaken strongly.The presence of saponins is characterized by the formation of durable foam on the surface of the liquid.Foam that remains stable after the addition of a few drops of concentrated HCl indicated the presence of saponins.

Toxicity test
The toxicity test of extract was performed using brine shrimp lethality test (BSLT), as described previously 8 .Methanol extract of bark of Tampoi (B.macrocarpa) (1 mg) was dissolved using 100 µL of 1% DMSO (dimethyl sulfoxide) and homogenized.The samples were diluted using 150 µL of distilled water until the total of volume reached 250 µL, and then pipetted 200 µL and diluted again using 600 µL of distilled water until the total of volume was 800 µL, so that the sample concentration was 1000 ppm.Samples with a concentration of 500, 250, 125, 62.5, 31.2, 15.6, and 7.8 ppm were made from sample dilutions of a concentration of 1000 ppm.The control solution was made with the same treatment as the sample without the addition of extract.
The toxicity test was carried out using several standard micro plates.About 100 µL seawater containing 8-13 shrimp larvae was added to each diluted sample so that the sample volume was 200 µL (with a concentration of 500, 250, 125, 62.5, 31.2, 15.6, and 7.8 ppm).The number of dead shrimp larvae was calculated for 24 hours after treatment.Each sample was treated in triplicate.The data obtained was recorded and the value of LC 50 calculated (Lethal Concentration 50%) using SAS Probit analysis.

Antioxidant assay
The antioxidant activity of the extract was evaluated using 2,2diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging method, as described previously 7,[9][10][11] .Briefly, the extract of bark of Tampoi (B.macrocarpa) was prepared in a solution with a concentration of 25, 50, 75 and 100 ppm, respectively.1 mL of extract and 1 mL of DPPH (0.024 mg/mL) were put into a test tube, which was incubated for 30 min at 37°C before being measured by Spectrophotometer UV Thermo Scientific Evolution 201 (measurements were carried out at a wavelength of 515 nm).Vitamin C was used as a positive control with variations in concentration were 2, 4, 6, and 8 ppm, respectively.Determination of antioxidant activity or DPPH scavenging effect (%) of extract and vitamin C were carried out in triplicate using equation as follow.

Absorbance of blank Absorbance of sample percentage of antioxidant activity
Absorbance of blank Then, the value of IC 50 (Inhibitory Concentration 50%) was determined using linear regression.

GC-MS analysis
In order to obtain the information of the kinds of compounds in methanol extract of bark of Tampoi, an analysis using GC-MS 5977 was performed.Specification of column that used in this research was HP-5MS with length 30 m, diameter 0.25 mm, thick of film 0.25 µm.The identification of the compound was compared to NIST standard data (https://webbook.nist.gov).

Results
The secondary metabolites found in the methanol extract of the bark of Tampoi (B.macrocarpa) are presented in Table 1.
To evaluate the antioxidant activity of the methanol extract of the bark, DPPH method was performed.The results of the antioxidant test can be seen in Table 2.
Furthermore, the methanol extract was analyzed using GC-MS analysis.The chromatogram and it compound contents of this extract is shown in Figure 1 and Table 3, respectively.

Discussion
Based on the phytochemical evaluation, the results showed that the methanol extract of bark of Tampoi (B.macrocarpa) contains several secondary metabolites including alkaloids, flavonoids, phenolics, steroids, and triterpenoids.Several secondary metabolites including alkaloids, steroids, triterpenoids, flavonoids, and phenolics are known to have antioxidant properties.These antioxidant compounds wield their activities through different mechanisms, for example by inhibiting hydrogen abstraction, radical scavenging, binding transition metal ions, disintegrating peroxides 12,13 , and one of the most important factors influencing antioxidant activity is the ability of the compounds to donate electrons.
Furthermore, in the present study the antioxidant activity of the Tampoi extract was determined by DPPH method.This method was used because it is simple, efficient, quick, more practical, and relatively inexpensive 14 .Based on Table 2, it is known that the methanol extract of bark of Tampoi (B.macrocarpa) can be categorized as an active extract in an antioxidant assay with  IC 50 value of 11.15 ppm.In addition, the results of the toxicity test using the BSLT method showed that the extract was toxic because it displayed LC 50 value above 1000 ppm.
It can be seen that only a small part of those are aromatic compounds.However, aromatic compounds are compounds that have the ability to stabilize high free radicals.The mechanism of phenolics as antioxidants is started by the formation a bond between free radical (DPPH radical) and hydrogen atom from OH-phenolics (ArOH) to form ArO .radical.Hydrogen atom will easier to be released because of the presence of electron withdrawing group which is bound at ortho-or para-positions 18 .Furthermore, ArO will react with a radical (ArO .or other radical) to form a stable compound 19,20 .It has been reported that methylparaben does not show negative effects on male mouse reproduction 21 .Methylparaben is widely used as a preservative in cosmetic products, medicines or pharmaceutical products and food ingredients 22,23 , and the antibacterial activity of methylparaben is stronger than benzoate acid 24 .
Methylparaben is a phenolic group that can reduce free radicals because it contains aromatic groups, -OH clusters and carbonyl groups.The presence of -COOCH 3 substituent at para-position in methylparaben makes this compound act as an electron withdrawing group.The bond dissociation energy (BDE) of the O-H bond is a main factor to investigate the action of antioxidant, due to the weaker OH bond the reaction of the free radical will be easier 19 .As the prediction of the previous reaction mechanism 7,19 , the prediction of the reaction mechanism between DPPH radical and methyl paraben can be seen in Figure 2.

Conclusion
The results of the study showed that the bark of Tampoi (Baccaurea macrocarpa) has antioxidant activity with an IC 50 value of 11.15 ppm.
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Dataset 1 .
Sheet 1, raw data of the results of phytochemical evaluation for alkaloids, flavonoids, phenolics, steroids, triterpenoids, and saponins by observing the changing of colors; Sheet 2, raw data of the observation of the mortality numbers of Artemia salina Leach and calculation of LC50 value in toxicity test using brine shrimp lethality test; Sheet 3, raw data for antioxidant activity by DPPH method, including the measurement of absorbance using spectrophotometer in triplicate, the calculation of percentage of antioxidant activity, and the value of IC50; Sheet 4, raw data of GC-MS analysis https://doi.org/10.5256/f1000research.16643.d227222
DPPH .+ AOH → DPPH-H + ArO .DPPH .+ ArO .→ DPPH-OAr or DPPH .+ R .→ DPPH-R According to identification of the compound in the methanol extract of bark of Tampoi (B.macrocarpa) using NIST database (DRUGBANK accession number, DB14212), it is known that the compound is identified as methylparaben.Based on the NIST database, peak at retention time at 9.467 min and peak area of 0.76% showed the characteristic of methylparaben (Molecular formula=C 8 H 8 O 3 ; Molecular weight=152).

Table 2 . Antioxidant activity of the methanol extract of bark of Tampoi (Baccaurea macrocarpa).
Average of three replicates performed for each concentration.