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

Background : Tampoi ( Baccaurea macrocarpa) is a tropical rainforest plant 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. Methods : The bark of Tampoi was extracted with methanol and 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. Results : Phytochemical evaluation results showed that the methanol 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 50 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 50 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. Conclusion : The results obtained in this research demonstrated that the bark of Tampoi ( B. macrocarpa) has potential as an antioxidant. Copyright:

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. contain toxic 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 and bark, fruits and leaves of several species are used for medicine such as B. motleyana (Rambai) for stomachache and sore eyes, B. brevipes for the regulation of menstruation, and B. lanceolata against stomach-ache [3, 4]. The B. angulata has been reported as a potential functional food with effective antioxidant [5], anti-inflammatory, anti-atherogenic, and hypocholesteromia activities [6]. 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 [7].

The 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. Tampoi fruit skin has high antibacterial inhibitory effects on the growth of S. aureus and E. coli., and it was toxic to Artemia salina [8, 9]. 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 compounds contained.

Methods

Extraction

Extraction was carried out as described previously by Erwin et al. (2014) [10]. The bark of Tampoi ( B. macrocarpa) was dried for one 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 [11]. The presence of secondary metabolites was identified by observing the changing color of the extract. These evaluations were performed as follows:

1 mg of extract was inserted into a test tube and then diluted in 1 mL methanol. Then a few drops of H 2SO 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.

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.

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.

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 2SO 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.

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 [12]. 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 Probit analysis.

Antioxidant assay

The antioxidant activity of the extract was evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging method, as described previously [11, 13– 15]. 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: 2, 4, 6, and 8 ppm, respectively. Determination of antioxidant activity or DPPH scavenging effect (%) of extract and vitamin C were carried out in triplicates using equation as follow.

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.

Table 1.

Phytochemical evaluation of the methanol extract of bark of Tampoi ( Baccaurea macrocarpa).

Secondary metabolites	Bark
Alkaloids	+
Steroids	+
Triterpenoids	+
Flavonoids	+
Phenolics	+
Saponins	˗

(+): Presence; (-): Absence

The result of toxicity test against Artemia salina larvae of the methanol extract of bark of Tampoi ( B. macrocarpa) can be seen in Table 2.

Table 2.

Toxicity test of methanol extract of bark of Tampoi ( B. macrocarpa).

Average of three replicates performed for each concentration
Concentration (ppm)	Log Concentration	Average of total larvae	Average of mortality	% Mortality	Probit	LC 50 (ppm)
500	2.6989	10,3	2.3	22.3	4.23	1577.89
250	2.3979	10,7	2.7	25.2	4.33
125	2.0969	10,3	3.3	32.0	4.53
62.5	1.7959	10,3	1	9.7	3.66
31.2	1.4948	10	4.3	43	4.82
15.6	1.1938	9,7	0	0	0
7.8	0.8928	9,3	2.7	29	4.45

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 3.

Table 3.

Antioxidant activity of the methanol extract of bark of Tampoi ( Baccaurea macrocarpa).

Average of three replicates performed for each concentration.

Sample	Concentration (ppm)	Absorbance		Inhibition	Percentage of inhibition (%)	IC 50 (ppm)
		Sample	Blank
Bark	20	0.2190	0.4150	0.47229	47.229	11.15
	40	0.0560		0.88193	88.193
	60	0.0490		0.86506	86.506
	75	0.0305		0.92651	92.651
Vitamin C	2	0.5470	0.6700	0.18360	18.360	3.28
	4	0.1530		0.77160	77.160
	6	0.0450		0.93280	93.280
	8	0.0340		0.94930	94.930

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 4, respectively.

Figure 1.

GC chromatogram of methanol extract of bark of Tampoi ( Baccaurea macrocarpa).

Table 4.

Composition of compounds from methanol extract of bark of Tampoi ( B. macrocarpa).

Peak	Retention Time (min)	% Peak Area	Molecule Formula	Molecular Weight	Compounds
1	9.479	0.76	C 8H 8O 3	152	Methylparaben
2	14.877	1.32	C 14H 26	194	Cyclohexane, 1-(cyclohexylmethyl)-2-methyl-, cis
3	19.329	9.91	C 17H 34O 2	270.	Methyl palmitate
4	20.034	16.14	C 16H 32O 2	256	palmitic acid
5	20.227	0.72	C 16H 32O 2	256	palmitic acid
6	20.300	3.08	C 34H 65F 3O 2	562	Dotriacontyl trifluoroacetate
7	20.432	3.18	C 34H 65F 3O 2	562	Tricosyl trifluoroacetate
8	21.234	1.40	C 18H 36O 2	284	Methyl 7-methylhexadecanoate
9	22.481	4.23	C 19H 34O 2	294	9,12-Octadecadienoic acid (Z,Z)-, methyl ester
10	22.597	8.46	C 19H 36O 2	296	9-Octadecenoic acid, methyl ester
11	22.811	0.62	C 29H 60O	424	Eicosyl nonyl ether
12	23.069	7.05	C 19H 38O 2	298	Heptadecanoic acid, 16-methyl, methyl ester
13	23.334	3.34		336	Undec-10-ynoic acid, undecyl ester
14	23.431	0.29	C 18H 32O	264	9,17-Octadecadienal, (Z)-
15	23.485	0.07	C 24H 48O 2Si	396	cis-Vaccenic acid
16	23.730	1.19	C 18H 34O 2	282	Oleic Acid
17	23.774	1.15	C 15H 24O	220	(2S,3S,6S)-6-Isopropyl-3-methyl-2-(prop-1-en-2- yl)-3-vinylcyclohexan one
18	23.794	0.78	C 15H 28	208	7-Pentadecyne
19	24.592	0.67	C 18H 35ClO 2	318	2- Chloropropionic acid, pentadecyl ester
20	26.520	2.77	C 21H 42O 2	326	Methyl 18-methylnonadecanoate
22	26.733	3.58	C 20H 42	282	Eicosane
23	27.207	0.87	C 36H 65F 7O 2	662	Dotriacontyl heptafluorobutyrate
24	27.255	0.08	C 54H 108Br 2	917	Tetrapentacontane, 1,54-dibromo-
25	28.234	0.74	C 28H 58	394	Octacosane
26	28.286	1.48	C 47H 94	659	Pentatriacontane, 13-docosenylidene-
27	28.374	2.31	C 19H 36	264	1H-Indene, 5-butyl-6-hexyloctahydro-
28	28.403	2.33	C 21H 39F 3O 2	380	Nonadecyl trifluoroacetate
29	28.941	1.68	C 29H 52	400	Nonacos-1-ene
30	28.963	0.31	C 22H 41F 3O 2	394	Eicosyl trifluoroacetate
31	28.980	0.34	C 23H 46	322	9-Tricosene, (Z)-
32	29.192	1.32	C 18H 36	252	1-Octadecene
33	29.224	1.10	C 26H 52	364	1-Hexacosene
34	29.708	7.09	C 23H 46O 2	354	Methyl 20-methyl-heneicosanoate
35	29.829	0.10	C 18H 36	252	1-Octadecene
36	29.878	0.29	C 29H 52	400	Nonacos-1-ene
37	29.907	0.28	C 35H 70	490	17-Pentatriacontene

Click here for additional data file.

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 [16, 17], 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 [18]. Based on Table 3, 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 not toxic because it displayed LC 50 value above 1000 ppm [12].

According to the results of GC-MS analysis, the chromatogram showed 37 peaks (compounds). The profile of the compounds showed that the main components were fatty acids and fatty acid esters. Total content of unsaturated fatty acids and esters with a peak area of 19.88% including 9,12-octadecadienoic acid (Z,Z)-, methyl ester (peak area 4.23), 9-octadecenoic acid, methyl ester (peak area 8.46), undec-10-ynoic acid, undecyl ester (peak area 3.58), undec-10-ynoic acid, undecyl ester (peak area 3.346), cis-vaccenic acid (peak area 0.07), and oleic acid (peak area 0.19). It was been reported that unsaturated fatty acid compounds and unsaturated fatty acid esters have significant antioxidant properties [19– 21].

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 [22]. Furthermore, ArO will react with a radical (ArO . or other radical) to form a stable compound [23, 24].

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 9.479 min and peak area of 0.76% showed the characteristic of methylparaben (Molecular formula=C 8H 8O 3; Molecular weight=152).

Methylparaben is widely used as a preservative in cosmetic products, medicines or pharmaceutical products and food ingredients [25, 26]. and the antibacterial activity of methylparaben is stronger than benzoate acid [27]. Methylparaben does not show negative effects on male mouse reproduction [28], but it was shown to have androgen antagonistic activity, to act as inhibitors of the sulfotransferase enzyme and to possess genotoxic activity. Paraben is allegedly able to trigger breast cancer in women [29].

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 [23]. As the prediction of the previous reaction mechanism [11, 23], the prediction of the reaction mechanism between DPPH radical and methyl paraben can be seen in Figure 2.

Figure 2.

Prediction of DPPH radical scavenging mechanism by methylparaben.

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.

Data availability

The data referenced by this article are under copyright with the following copyright statement: Copyright: © 2019 Erwin E et al.

Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).

F1000Research: 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 LC 50 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 [30]

This manuscript reported the information of phytochemical and antioxidant activity of barks of Baccaurea macrocarpa.

The results of evaluation on phytochemicals of barks of B. macrocarpa showed that the methanol extract consisted of alkaloids, steroids, triterpenoids, flavonoids and phenolic compounds. But the profile of GC-MS showed only fatty acids, fatty acid esters and methyl paraben. The authors should use LC-MS to investigate chemical constituents of methanol extract instead of GC-MS.

Check the scientific name through the whole manuscript. When the scientific name appears first time in manuscript, it is full written both Genus and species, and then use the abbreviated binomial form for the following times.

In methods section, Phytochemical evaluation of alkaloids: “H 2SO 4 1M” must correct to “1M H 2SO 4”

In methods section, Phytochemical evaluation of alkaloids: Please correct your statement “The formation of orange on filter paper….”

In methods section, Steroids and triterpenoids: “green or purple precipitation/red precipitation” must correct to “green or purple precipitate/red precipitate"

In methods section, the details of GC and MS conditions must clearly clarify.

In results section, the results should express your statistical analysis.

In results section, Table 2: It is essential to set the concentration of sample for calibration range to less than 50% mortality and greater than 50% mortality, and the calibration curve is linear. The results should express your statistical analysis.

In results section, Table 3: Check the data and calculation in Table 3. The results should express your statistical analysis.

In results section, Table 4: Data of peak no. 13 does not complete.

In discussion section, the data of GC-MS analysis (page 7) does not match the data in Table 4.

In discussion section, correct the equation of DPPH• and ArOH on page 7.

In discussion section, correct “benzoate acid” to “benzoic acid”.

To improve the quality of English, the authors must correct grammatical errors and English improvement throughout the manuscript.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

I respectfully reject this manuscript as following reasons:

This manuscript contains insufficient data although the authors tried to explain that this is a screening study.

In addition to my previous comments I have following notifications:

Table 2,  the LC 50 value which is 1577.89 ppm was not in the concentration range (7.8 -  500 ppm) used in the study.

Table 3, Inhibition should be rewritten.

Table 3 , an IC 50 value (11.15 ppm) of bark was suspicious as the lowest concentration used was 20 ppm.

There is some typing error of DPPH reaction as AOH supposed to be ArOH.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

Approved.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

This manuscript reported the new information of phytochemical and antioxidant activity of barks of Baccaurea macrocarpa.

The authors used only DPPH assay to evaluate antioxidant activity. Antioxidant activity should be investigated using various assays to present antioxidant capacity of the extracts.

The results of evaluation on phytochemicals of barks of B. macrocarpa showed that the methanol extract consisted of alkaloids, steroids, triterpenoids, flavonoids and phenolic compounds. But the profile of GC-MS showed only fatty acids, fatty acid esters and methyl paraben. The authors should use LC-MS to investigate chemical constituents of methanol extract instead of GC-MS.

In results section, the authors did not mention on the toxicity test of the extract using brine shrimp lethality test. And the results should express yours statistical analysis.

In discussion section, the third paragraph, the authors need to rewrite the total content and composition of fatty acids. GC chromatogram in Figure 1 was not related to the data of composition of compounds in Table 3 such as retention time, % peak area. For example, peak at retention time 19.329 showed high intensity on GC chromatogram but it expressed low % peak area just 0.91. The peak at retention time 14.877 showed low intensity on GC chromatogram but it expressed % peak area 1.32. Moreover, some compounds presented low matching percentage from the library searching. In Figure 2, structure of methyl paraben was wrong.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Reviewer comment #1

The authors used only DPPH assay to evaluate antioxidant activity. The antioxidant activity should be investigated using various assays to present antioxidant capacity of the extracts. The results of evaluation on phytochemical of barks of B. macrocarpa showed that the methanol extract consisted of alkaloids, steroids, triterpenoids, flavonoids and phenolic compounds. But the profile of GC-MS showed only fatty acids, fatty acid esters and methyl paraben. The authors should use LC-MS to investigate chemical constituents of methanol extract instead of GC-MS.

Author response #1

This study is an initial screening of the antioxidant activity of Tampoi bark. The next project if we get the funding, we will use another antioxidant test and other instruments as you suggest. We will also carry out isolation and purification to obtain active compounds from Tampoi bark. Thank you for your valuable suggestion

Reviewer comment #2

In the results section, the authors did not mention on the toxicity test of the extract using brine shrimp lethality test. And the results should express your statistical analysis

Author response #2

Toxicity test data have been added in the revised article

Reviewer comment #3

some compounds presented low matching percentage from the library searching

Author response #3

It has been fixed

Reviewer comment #4

In Figure 2, the structure of methylparaben was wrong.

Author response #4

The structure of methylparaben has been fixed

Reviewer comment #5

In the discussion section, the third paragraph, the authors need to rewrite the total content and composition of fatty acids. GC chromatogram in Figure 1 was not related to the data of composition of compounds in Table 3 such as retention time, % peak area. For example, peak at retention time 19.329 showed high intensity on GC chromatogram but it expressed a low % peak area just 0.91. The peak at retention time 14.877 showed low intensity on GC chromatogram but it expressed % peak area 1.32.

Author response #5

The percentage (%) peak area of GC chromatogram has been fixed

Introduction: Methods: Discussion:

In first paragraph line 5, please correct your statement on natural ingredients “ do not contain chemicals” that only can be found in modern medicines, as all things in this world is formed from chemical constituents. Please change to “contain toxic chemicals”.

Last paragraph line 5, the statement “kinds of isolated compounds contained” are not correct as you don’t isolate the compound. Please remove the word “isolated”.

DDPH assay alone can’t express the antioxidant properties of sample, so we suggest you to add other antioxidant assay such as ABTS and FRAP.

GCMS result indicated that the main constituent in Baccaurea macrocarpa extract is fatty acid, this is because the GCMS can only detect the volatile compounds. To identify other compounds that are responsible in the antioxidant activity of Baccaurea macrocarpa, we suggest you to run your sample using LCMS.

Methylparaben is familiar compound. Can you give literature which supported your claim that methylparaben is responsible to the antioxidant of Baccaurea macrocarpa extract?

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Reviewer comments #1

In first paragraph line 5, please correct your statement on natural ingredients “do not contain chemicals” that only can be found in modern medicines, as all things in this world is formed from chemical constituents. Please change to  “contain toxic chemicals”

Author response #1

Thank you for your suggestion, It has been fixed

Reviewer comments #2

The statement “kinds of isolated compounds contained” are not correct as you don’t isolate the compound. Please remove the word “isolated”

Author response #2

The word isolated has been removed

Reviewer comments #3

Methods:

DPPH assay alone can’t express the antioxidant properties of the sample, so we suggest you add other antioxidant assays as ABTS and FRAP

Author response #3

This study is an initial screening of the antioxidant activity of Tampoi bark.

Reviewer comments #4

Discussion:

GCMS result indicated that the main constituent in  Baccaurea macrocarpa extract is a fatty acid, this is because the GCMS can only detect the volatile compounds. To identify other compounds that are responsible for the antioxidant activity of  Baccaurea macrocarpa, we suggest you to run your sample using LCMS.

Author response #4

The next project if we get the funding, we will use another antioxidant test and other instruments as you suggest. We will also carry out isolation and purification to obtain active compounds from Tampoi bark.

Reviewer comments #5

Methylparaben is a familiar compound. Can you give literature which supported your claim that methylparaben is responsible for the antioxidant of  Baccaurea macrocarpa extract?

Author response #5

I could not find in the literature that discusses the antioxidant properties of parabens in vegetation, but methylparaben is preservative and antioxidant in cosmetic products, medicines or pharmaceutical products, and food ingredients. Based on GC-MS data, methylparaben is most likely to be antioxidants

According to the GC chromatogram the peak that was claimed to be methyl paraben was detected at 9.479 min but when identified with the MS the peak at 9.467 min was identified instead. This might be wrong interpretation.

Why the peak with high intensity detected at 19.329 min of methyl palmitate was not considered as the major compound or whether it was contributed to the antioxidant effect?

Due to there are many antioxidant mechanisms, therefore, only DPPH scavenging activity is not sufficient.

Cytotoxicity result was not shown and sufficiently discussed in correlation with the antioxidant activity.

The statistical analysis should be mentioned in the method section.

Based on the insufficient information and evidence, this manuscript needs more experimentation and well written regarding the method, results and discussion.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

The study design and the methodology sounds good. Still more details on the plant  B. macrocarpa need to be included in the introduction. Even though the authors try to substantiate Methyl paraben as a non-toxic compound, it is a universally known fact usage of Methyl paraben is strongly discouraged in all human usage food and cosmetics as preservative. The Environmental Working Group (EWG) lists methylparaben as being a low to moderate Health Hazard. Parabens are potential endocrine disruptors due to their ability to mimic estrogen. Studies demonstrate that at sufficient concentrations, parabens can increase cell proliferation in human breast cancer MCF-7 cells, which are often used as a sensitive measure of estrogenic activity. Applying personal care product containing parabens—especially methylparaben—can lead to UV-induced damage of skin cells and disruption of cell proliferation (cell growth rate). These are evidenced reports on the Methyl paraben. Nevertheless, it is available in the natural source from the plant in meagre quantity. The authors can check for other compounds in GC-MS and state its importance in the manuscript. The GC-MS can be repeated. or HPLC can be performed using an aqueous extract. A simple TLC becomes handy for compound prediction, Then a column chromatography will be useful to check if there are any useful compounds.

The authors fail to include the ill effects of Methyl paraben in the literature. Is there any reason for avoiding such inclusions? The authors should weigh the importance of other compounds in the plant. Is there any traditional/ancient usage of the fruit mentioned in literature must be included in Introduction section.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Reviewer comment #1

The authors can check for other compounds in GC-MS and state its importance in the manuscript

Author response #1

We have re-checked the 37 peaks which appeared to be methylparaben most likely to be antioxidants

Reviewer comment #2

The GC-MS can be repeated or HPLC can be performed using an aqueous extract. A simple TLC becomes handy for compound prediction, Then a column chromatography will be useful to check if there are any useful compounds.

Author response #2

This study is an initial screening of the antioxidant activity of Tampoi bark. The next project if we get the funding, we will use other instruments as you suggested. We will also carry out isolation and purification to obtain active compounds from Tampoi bark. We appreciated your suggestion

Reviewer comment #3

The authors fail to include the ill effects of Methyl paraben in the literature

Author response #3

We have added additional literature about the side effects of methyl paraben. Ref. no: 27

Reviewer comment #4

Is there any traditional/ancient usage of the fruit mentioned in literature must be included in the Introduction section

Author response #4

We did not find any use of tampoi as traditional medicine, but several other species of the genus Baccuarea were used as traditional medicine. In addition, there are several preliminary studies on Tampoi's bio-activity. Ref. no: 3,4,8, and 9