Int J Pharm Pharm Sci, Vol 14, Issue 3, 40-46Original Article

GAS CHROMATOGRAPHY STUDY OF N-HEXANE AND CHLOROFORM FRACTIONS OF ETHANOL EXTRACT OF MELASTOMA MALABATHRICUM L. LEAVES: AN IN VITRO STUDY OF ANTIOXIDANT AND SPF VALUES

P. APRIDAMAYANTI1* , L. PRATIWI2 , R. SARI3

1,2,3Pharmacy Department, Faculty of Medicine, Tanjungpura University, Pontianak 78124, Indonesia
Email: apridamayanti.pratiwi@gmail.com

Received: 08 Dec 2021, Revised and Accepted: 25 Jan 2022

ABSTRACT

Objective: This research aimed to qualitatively determine the chemical compound constituents, antioxidant capacity, and sun protection factor (SPF) value of extract of the leaves of Melastoma malabathricum L. using GC-MS.

Methods: The detected compounds were identified by processing the raw GC-MS data using ChemStation software and comparing results with the NIST mass spectral database. Antioxidant capacity was measured using 1,2-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging and ferric-reducing antioxidant power (FRAP) assays. The SPF contained in the n-hexane and chloroform fractions separated from the ethanol extract of the leaves of Melastoma malabathricum L was identified.

Results: The GC-MS test results revealed that 21 and 29 peaks of separation occurred in the n-hexane and chloroform fractions. The highest peak values are hexadecanoic acid methyl ester (a fatty acid group also known as palmitic acid) (100%) in the n-hexane and chloroform fractions. The highest chemical content in n-hexane and chloroform fractions was obtained at 20 min. The thin-layer chromatography (TLC) results indicated that the n-hexane and chloroform fractions contained steroid and terpenoid compounds. The antioxidant capacity test using DPPH showed that the n-hexane and chloroform fractions of ethanol extract of the leaves of Melastoma malabathricum L had IC50 values ​​of 49.2±7.9 g/ml and 11.3±3.65 g/ml, respectively. Using FRAP, the IC50 values ​​of the n-hexane and chloroform fractions were identified as 365.2±133.6 g/ml and 80.8±8.9 g/ml, respectively, categorizing them as strong and weak antioxidants. SPF testing produced SPF values ​​for the n-hexane and chloroform fractions of ethanol extract of Melastoma malabathricum L leaves of 11.7±0.1 and 21±0.4, respectively.

Conclusion: The study results show that the antioxidant activity and the SPF values of the chloroform fraction are greater than those of the n-hexane fraction. In conclusion, the n-hexane and chloroform fractions are found to have antioxidant properties in the weak and solid categories and the ability to protect against sunlight in the vulnerable category.

Keywords: GC-MS, n-hexane fraction, Chloroform fraction, Antioxidant, SPF value

© 2022 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/)
DOI: https://dx.doi.org/10.22159/ijpps.2022v14i3.43801. Journal homepage: https://innovareacademics.in/journals/index.php/ijpps.

INTRODUCTION

Sunlight affects human skin via UV radiation, causing sunburn, skin cancer, skin pigmentation, and premature aging. It is, therefore, necessary to develop sunscreen formulations to protect against sunburn, skin cancer, and early skin aging [1]. UVR rays are divided into three main groups: UVA with a wavelength range of 320 to 400 nm; UVB: with the wavelength range of 280 to 320 nm; and UVC with the wavelength range of 100 to 280 nm [2]. Ultraviolet C emission (UVC) is the most dangerous UVC light that can cause mutagenicity and erythematous. UVB affects the stratum corneum of the skin and can trigger the release of inflammatory mediators such as histamine, serotonin, and prostaglandins that causes capillary dilation and the development of erythema and edema [3, 4]. UVA radiation can stimulate pigment production more than other UV radiation. UVA radiation penetrates the deeper layers of the dermis and impairs normal cell function, affecting blood vessels and collagen fibers. UVA light indirectly affects cellular DNA by generating reactive oxygen species [2].

Melastoma malabathricum L. found in Indonesia has several local names, including kluruk (Javanese), haredong (Sundanese), sak (Dayak), and kemanden (Maduran) [5]. Melastoma malabathricum L. is used as a herbal medicine in several Asian countries, including India, Malaysia, Indonesia, and Singapore. It has been known to have various medicinal benefits based on the traditional practices and beliefs of different communities and tribes [6]. Plants have evolved self-protection mechanisms to avoid damage because, in plants, exposure to sunlight is required for photosynthesis. Bioactive compounds such as phenol have antioxidant properties in plants [7, 8]. As a result, phytochemical analysis of plants has become an increasingly important method in plant medicine and drug research [9]. The study conducted by Amini et al., 2019 found a positive relationship between DPPH radical protection activity (IC50), total phenolic content, and total flavonoids, with SPF values with R2 values of-0.9603, 0.8999, and 0.8476, respectively. The results of Amini et al., 2019 research show that purified extract of Elaeagnus angustifolia has high phenol and flavonoid content (with an IC50 of 4.5µg/ml) and has the highest SPF value of 29.73±0.31 [10].

Research into the existence of compounds with sun protection properties in Melastoma malabathricum L has not yet been carried out, with the focus of previous studies being on antioxidant activity in extracts and parts of the plant. This study examined the chemical content of the separated compounds in ethanolic extract of the plant Melastoma malabathricum L. using different solvent polarities, namely n-hexane and chloroform. This Melastoma malabathricum L. content analysis study was carried out using the thin-layer chromatography (TLC) technique followed by GC-MS. In vitro examination of antioxidant activity was conducted using DPPH and FRAP to confirm antioxidant activity in the plant's constituent compounds. In vitro testing of the ability to protect against UV rays in terms of SPF can be a reference source in tracing sources of cosmetic raw materials derived from medicinal plants.

MATERIALS AND METHODS

Material

Oven (Modena), camera (Canon), rotary evaporator (Butchi), ultrasonicator (Branson 1510), spectrophotometer UV/Vis (Shimadzu), Agilent JandW GC column type DB-5MS (30 m x 0.25 mm), Agilent Gas Chromatography GC: 7890A (G3440A), Thin Layer Chromatography silica gel 60 F254 (Merck),Whatman (Merck).

Chemicals

Ethanol (Merck), ethanol (Brataco), n-hexane (Brataco), Ethyl acetate (Merck), Formic acid (Merck), Chloroform (Merck), HCl (Merck), AlCl3 (Merck), FeCl3 (Merck), metal Mg, H2SO4 (Merck), TPTZ (Merck) reagent, Citric acid (Merck), Ascorbic acid (Merck), 1,1-Diphenyl-2-picrylhydrazyl radical (DPPH) (Merck), and vanillin-sulfuric acid 10%.

Collection and authentication of plant samples

Melastoma malabathricum L. samples were collected in the Punggur Besar village, Kubu Raya Sub-district, Pontianak, West Borneo, Indonesia. Authentication was by the Biology Faculty of Tanjungpura University (26 Sep 2016).

Extraction

The dried powder of Melastoma malabathricum L was extracted with 78% ethanol by maceration method and rotary evaporator apparatus to reduce solvent and crude extract. The crude extract was then separated using a funnel with solvents of different polarities: n-hexane and chloroform [11]. Thus, we obtained n-hexane and chloroform fractions. The dried fraction was stored at room temperature.

Preliminary phytochemical screening and thin-layer chromatography

Preliminary phytochemical testing was carried out to investigate the presence of secondary metabolites. According to standard practice, phenol, flavonoid, alkaloid, steroid, anthraquinone, tannin, and saponin tests were carried out [12].

Confirmation testing of secondary metabolic content of n-hexane and chloroform fractions with TLC identification of stationary phase was carried out via silica gel 60 F254. The mobile phase is n-hexane: ethyl acetate: formic acid at 10 ml: 10 ml: 8 drops. Spot viewers used in this study were AlCl3, FeCl3, and H2SO4 10%, and vanillin-sulfuric acid 10% [13].

Preparation of GC-MS analysis

The preparation of GC-MS analysis in this study followed Prakash and Meena's method. Five milligrams samples of fractions were obtained by separating the ethanol extract of Melastoma malabathricum L, namely the n-hexane fraction and the chloroform fraction, which were then dissolved using 3 ml of chloroform. The solution was sonicated for 1 minute, after which chloroform was added to the 5 ml mark on the volumetric flask. GC-MS analysis was performed using an Agilent JandW GC column type DB-5MS (30 m x 0.25 mm). A total of 1L of sample solution was injected into Agilent Gas Chromatography GC: 7890A (G3440A) for 40 min, with the injection port temperature at 230 °C. Helium was used as the carrier gas. MS was operated in the electron impact mode with ionization energy of 70 eV. Full scan mass spectra were acquired from 30 to 380 m/z. Detected compounds were identified by processing the raw GC/MS data with ChemStation software and comparing results with the mass spectral database [14].

Antioxidant activities with DPPH and FRAP method

Antioxidant activity was tested using a 1,1-Diphenyl-2-picrylhydrazyl radical (DPPH) assay to identify the sample's free-radical-scavenging activity. The technique used in this study is following Brand-Williams et al., 1995 and Kikuzaki et al., 2002 [15, 16]. 1 ml of each sample with various concentrations (0.5–250 ppm) was added to 3 ml of DPPH 1 mmol and 1 ml methanol. This was then incubated in the dark for 15 min at room temperature, and absorbance was measured at 515.5 nm. Experiments were carried out in triplicate and ascorbic acid as comparison compounds in this study.

Antioxidant activity testing with FRAP was performed according to Syamsu et al., 2017. 30 µl of each sample with various concentrations of 1 µg/ml to 650 µg/ml were added to 30 µl FeCl3 solution (3 mmol in 5 mmol citric acid) and 240 µl TPTZ reagent (1 mmol in 0.05 M HCL) in 96 microplate wells. Incubation was for 20 min at room temperature, and absorbance was measured at 615 nm [17]. The results in this study are presented in triplicate using vitamin C (ascorbic acid) as a comparison compound.

Identification in vitro of SPF

The n-hexane and chloroform fractions that form the samples in this study were dissolved in ethanol at a 250 µg/ml concentration and scanned across the 290 to 320 nm range at five nm intervals. Screening of sun protection activity was measured by determination in vitro of SPF, based on the equation proposed by Mansur., 1986 [18].

Note: CF is the correction factor; EE is the erythema effect in the spectrum (nm); I is light intensity (nm), and Abs is the absorbance sample. The absorbance sample was measured three times and used for SPF calculation.

Statistical analysis

Data are presented as means±standard deviation. Statistical analysis was performed using SPSS software to analyze statistical significance, with a value of p<0.05 considered significant.

RESULTS

The percentage yield of extract and fraction

Details of the n-hexane and chloroform fractions of ethanol extract of Melastoma malabathricum L. obtained in this study are presented in table 1.

Table 1: Composition of ethanol 78% extract yield of Melastoma malabathricum L. leaves

Sample Percentage yield (%w/w)
Hexane fraction 3.16
Chloroform fraction 9.29

Preliminary phytochemical screening and thin-layer chromatography (TLC)

Initial phytochemical screening tests were carried out to determine the content of secondary metabolites. The results are shown in table 2. Confirmation tests of secondary metabolites of n-hexane and chloroform fractions of ethanol extract of Melastoma malabathricum L. were performed using TLC, as presented in fig. 1.

Table 2: Screening of secondary metabolite content in extracts and fractions from Melastoma malabathricum L. leaves

Secondary metabolite N-hexane fraction Chloroform fraction
Phenols - +++
Flavonoids - +
Alkaloids +++ +++
Terpenoids - -
Steroids +++ ++
Anthraquinone - -
Tannins - -
Saponins - -

Key: = absent; += present in small quantity; ++= present in moderate quantity; +++= present in large quantity

a. Spot sight with sulfuric acid 10% b. Spot sight with AlCl3 10% c. Spot sight with FeCl3 10% d. Spot sight with vanillin-sulfuric acid 10% after heating at 95 °C for 5 min
1 2 1 2

Fig. 1: TLC separation sample of mobile phase n-hexane: ethyl acetate: formic acid (10 ml: 10 ml: 8 drops) and stationary phase silica gel F254. Sample volume = 10 µl, Key: 1. N-hexane fraction; 2. Chloroform fraction

GC-MS analysis of n-hexane and chloroform fractions

The chemical compositions of the n-hexane and chloroform fractions of ethanol extract of Melastoma malabathricum L. were determined by GC-MS and are presented in tables 3 and 4.

The antioxidant activity identified using DPPH and FRAP methods

Investigating antioxidant activity in n-hexane and chloroform fractions of ethanol extract of Melastoma malabathricum L. leaves were carried out using the DPPH and FRAP methods. The DPPH method identifies the ability of a sample to donate hydrogen and act as a radical scavenger. Meanwhile, the FRAP method converts hydrogen peroxide into hydroxyl radicals as an iron-reducing antioxidant. Investigation results for the antioxidant properties of Melastoma malabathricum L. can be seen in table 5.

SPF value of Melastoma malabathricum L. fractions

Screening of sun protection activity was measured by determining in vitro SPF using the Mansur equation [18]. The result is presented in table 6.

Table 3: GC-MS results for the n-hexane fraction of Melastoma malabathricum L

No Retention time in min Name of compound Molecule formula Percent peak value Nature of compound
1 15.17 2(4H)-Benzofuranone, 5,6,7,7a-tetrahydro-4,4,7a-trimethyl C11H16O 2.58 Monoterpene
2 17.26 Tetradecanoic acid, 12-methyl, methyl ester C16H32O2 1.07 Fatty acid
3 18.56 Tetradecanoic acid, 12-ethyl, methyl ester C16H32O2 1.16 Fatty acid
4 18.84 2-Pentadecanone, 6,10,14-trimethyl C18H36O 11.75 Sesquiterpene
5 20.02 2-Piperidinone, N-[4-bromo-n-buthyl] C9H16NO 1.78 -
6 20.09 Hexadecanoic acid, methyl ester C17H34O2 100 Fatty acid ester
7 20.10 1-Hexadecen-3-ol, 3,5,11,15-tetramethyl C20H40O 2.06 Diterpene
8 21.04 Decosanoic acid, ethyl ester C23H46O2 3.54 Fatty acid
9 21.46 Heptadecanoic acid, methyl ester C18H36O2 1.21 Fatty acid
10 22.29 9,12-Octadecadienoic acid, methyl ester (E, E) C19H34O2 7.84 Fatty Acid
11 22.37 9,12-Octadecadienoyl cloride, (Z,Z) C18H31ClO 13.08 -
12 22.49 Phytol C20H40O 1.18 Diterpene
13 22.68 Methyl stearate C19H38O2 1.58 -
14 23.63 2-Hexadecen-1-ol, 3,7,11,15-tetramethyl, acetate C20H40O 3.69 Diterpene
15 24.79 2-Hexadecen-1-ol, 3,7,11,15, 17-pentamethyl, acetate C20H40O 2.22 Diterpene
16 25.01 2-Hexadecen-1-ol, 3,7,11,15-tetramethyl, acetate C20H40O 2.16 Diterpene
17 26.63 2-Hexadecen-1-ol, 3,7,11,15-tetramethyl, acetate C20H40O 5.07 Diterpene
18 29.09 2-Hexadecen-1-ol, 3,7,11,15-tetramethyl, acetate C20H40O 5.34 Diterpene
19 29.24 2-Hexadecen-1-ol, 3,7,11,15-tetramethyl, acetate C20H40O 6.91 Diterpene
20 32.71 1,2-Bis(trimethylsilyl)benzene C12H22Si2 9.73 -
21 34.18 1,2-Bis(trimethylsilyl)benzene C12H22Si2 7.58 -

Table 4: GC-MS results for the chloroform fraction of Melastoma malabathricum L

No Retention time Name of compound Molecule formula Percent peak value Nature of compound
1 5.95 Methane, dichloronitro CHCl2NO2 1.45 Diena
2 7.79 Butanedioic acid, dimethyl ester C6H10O4 1.92 Fatty acid
3 9.32 Butanedioic acid, hydroxy-dimethyl ester C6H10O5 4.77 Fatty acid
4 10.39 Butanedioic acid, hydroxy-dimethyl ester C6H10O5 1.12 Fatty acid
5 11.03 Butanedioic acid, hydroxy-dimethyl ester C6H10O5 1.49 Fatty acid
6 12.39 Oxalic acid, allyl hexadecyl ester C21H38O4 1.24 Fatty acid
7 15.17 Nonanedioic acid, dimethyl ester C11H20O4 7.41 Fatty acid
8 15.51 Phosphonofluoridic acid, (1-methyl ethyl)-cyclohexyl ester C16H14FO2P 1.36 Fatty acid
9 16.05 Phosphonofluoridic acid, (1-methyl ethyl)-cyclohexyl ester C16H14FO2P 1.27 Fatty acid
10 16.42 Phosphonofluoridic acid, (1-methyl ethyl)-cyclohexyl ester C16H14FO2P 1.81 Fatty acid
11 16.94 Phosphonofluoridic acid, (1,2-dimethyl ethyl)-cyclohexyl ester C16H14FO2P 2.28 Fatty acid
12 17.20 1.05 Fatty acid
13 18.00 Phosphonofluoridic acid, (1-methyl ethyl)-cyclohexyl ester C16H14FO2P 3.99 Fatty acid
14 18.17 1.51 Fatty acid
15 18.56 2.80 Fatty acid
16 20.02 Phosphonofluoridic acid, (1-methyl ethyl)-cyclohexyl ester C16H14FO2P 1.85 Fatty acid
17 20.10 Hexadecanoic acid, methyl ester C17H34O 100 Fatty acid
18 21.03 Hexadecanoic acid, ethyl ester C17H34O 7.81 Fatty acid
19 22.30 E-11, Z-13-Octadecatriene C18H32 5.45 Fatty acid
20 22.37 9,12,15-Octadecatrienoic acid, methyl ester (Z,Z,Z) C19H32O 15.57 Fatty acid
21 22.67 9,12,15-Octadecatrienoic acid, methyl ester (Z,Z,Z) C19H32O 4.25 Fatty acid
22 23.13 9,12,15-Octadecatrienoic acid, methyl ester (Z,Z,Z) C19H32O 1.20 Fatty acid
23 28.31 Cyclotrisiloxane, hexamethyl C6H18O3Si3 1.53 -
24 31.06 Cyclotrisiloxane, hexamethyl C6H18O3Si3 4.89 -
25 31.62 Cyclotrisiloxane, hexamethyl C6H18O3Si3 1.57 -
26 32.70 1,2 Bis(trimethylsilyl)benzene C12H22Si22 82.84 -
27 32.82 1,2 Bis(trimethylsilyl)benzene C12H22Si22 1.19 -
28 33.05 Cyclotrisiloxane, hexamethyl C6H18O3Si3 2.28 -
29 34.18 Cyclotrisiloxane, hexamethyl C6H18O3Si3 15.61 -

Table 5: Antioxidant activity of the n-hexane and chloroform fractions of Melastoma malabathricum L. and the standard compound

Sample Antioxidant activity (IC50) µg/ml
DPPH FRAP
N-hexane fraction 49.2±7.9c 365.2±133.6z
Chloroform fraction 11.3±3.65b 80.8±8.9y
Ascorbic acid 1.9±0.2a 4.13±0.86x

a,b,cData different significantly (p<0.05) when compared with each other within the DPPH radical scavenging column, x,y,zData different significantly (p<0.05) when compared with each other within the DPPH radical scavenging column

Table 6: SPF results for n-hexane and chloroform fractions of Melastoma malabathricum L

Sample SPF value (at 250 µg/ml)
N-hexane fraction 11.7±0.1e
Chloroform fraction 21.0±0.4f

e,fData different significantly (p<0.05) when compared with each other within the DPPH radical scavenging column

DISCUSSION

This study was performed to determine the qualitative content of the hexane and chloroform fractions in the ethanol extract of the leaves of Melastoma malabathricum L. with GC-MS analysis was used to identify and understand the critical properties of the active compounds. Active in medicinal plants; This study provides preliminary tests in the isolation of chemical compounds that have the potential to discover potential natural resources for sunscreen cosmetics. In vitro testing of antioxidant activity and the ability of compounds to protect the skin from UV rays was carried out to identify the potential of Melastoma malabathricum L as a source of natural cosmetics. A study conducted by Reddy et al., 2018 regarding new compounds as biological sources such as plants, minerals, and fish oil are known to have activity against solar radiation. They have antioxidant activity that can inactivate free radicals and show better protection than synthetic compounds because they are environmentally friendly. Research results that have been proven, such as the compound quercetin, Moringa oleifera, and Marcetia taxifolia have significant SPF activity when used as a sunscreen agent [19].

The first step in this research was to extract the Melastoma malabathricum L. leaf by maceration using 78% ethanol, obtaining a thick extract which was then separated using n-hexane and chloroform liquid-liquid extraction. The yields of n-hexane and chloroform fractions of Melastoma malabathricum L. are as presented in table 1. Phytochemical screening of the n-hexane and chloroform fractions, as shown in table 2, reveals that n-hexane and chloroform fractions of Melastoma malabathricum L. contain alkaloids, phenols, flavonoids, and steroids. A confirmation test of the chemical content of the samples was carried out by TLC using a mobile phase of n-hexane: ethyl acetate: formic acid (10 ml: 10 ml: 8 drops). Examination of terpenoids, steroids, and saponins using reagent spots by 10% vanillin and 10% sulfuric acid heated at 100 °C for 6 min produced blue-violet and red-violet spots [13].

The separation elution of the samples is shown in fig. 1. Confirmation tests on the n-hexane and chloroform fractions confirmed their terpenoid and steroid content. In this study, qualitative examination of the secondary metabolite content of the n-hexane and the chloroform fractions of ethanol extract of Melastoma malabathricum L. identified alkaloids, steroids, terpenoids, phenols, and flavonoids. Research conducted in line with the previous study found that the n-hexane extract of Melastoma malabathricum L. is a terpenoid and a quinone [14, 20].

A study of the chemical composition of the n-hexane and chloroform fractions was carried out using the GC-MS technique, with test results compared with the NIST mass spectra database. We diagnosed 21 chromatogram peaks inside the n-hexane fraction and 29 chromatogram peaks inside the chloroform fraction, with a separation time of five to 35 min. The results of chromatogram separation using GC-MS can be seen in fig. 2 and 3. Diterpenes and fatty acids dominate in the n-hexane and chloroform fractions, and the composition of peak values ​​can be seen in tables 3 and 4.

Fig. 2: GC-MS results for n-hexane fraction of Melastoma malabathricum L

Fig. 3: GC-MS results for chloroform fraction of Melastoma malabathricum L

The GCMS results of the study showed that the compounds with antioxidant, anti-inflammatory, anticancer, and anticancer activities in the hexane fraction were methyl esters of tetradecanoic acid, methyl esters of tetradecanoic acid, hexadecanoic acid, phytol isomer, these compounds belong to the groups of fatty acids and terpenoids. GC-MS revealed compounds with antioxidant, anti-inflammatory, and cancer prevention activities in the chloroform fraction: hexadecanoic acid-ethyl ester, cyclotrisiloxane-hexamethyl, and 1,2 Bis(trimethylsilyl)benzene [21]. These results are presented in tables 4 and 5.

The most abundant chemical compound in both the n-hexane and chloroform fractions was 1-hexadecanoic acid methyl ester, obtained at 20 min. The hexadecanoic acid methyl ester is a chemical compound belonging to the fatty acid methyl ester group (also known as palmitic acid) with antioxidant, antimicrobial, hypercholesterolemic, and hemolytic properties [21].

Diterpene compounds found in the n-hexane fraction comprise phytol isomer compounds known to have antimicrobial, anticancer, cancer preventive, anti-inflammatory, and antidiabetic biological properties [22, 23]. In in vitro testing, phytol is seen to reduce the production of free radicals by donating a hydrogen atom with an unpaired electron (H∙), converting free radicals into unreactive species. The antioxidant activity of phytol can be related to its chemical structure as unsaturated alcohol with branched chains. Its antioxidant properties are ascribed to the hydroxyl group (OH) present in the molecule [24]. A study by De Menezes Patrício Santos et al., 2013 showed that phytol could remove hydroxyl radicals (OH∙), thus exhibiting antioxidant activity and inhibiting cell damage caused by these radicals [26]. In this study, hexadecanoic acid methyl ester, the chemical compound with the highest content in the chloroform fraction obtained at 20 min, belongs to the fatty acid methyl ester group known as palmitic acid. It has antioxidant, antimicrobial, hypercholesterolemic, and hemolytic properties [21].

Antioxidant activity tests were carried out on the n-hexane and chloroform fractions using the DPPH and FRAP methods. The results of the antioxidant study obtained IC50 values ​​for the n-hexane fraction of the ethanol extract of Melastoma malabathricum L of 49.2 µg/ml and 365.2 µg/ml. The IC50 values ​​were 11.3 µg/ml and 80.8 g/ml in the chloroform fraction. The results are summarized in table 5. These results show that the n-hexane fraction has both strong and weak antioxidant properties. In contrast, the chloroform fraction has only strong antioxidant activity, at<100 µg/ml [26]. Vitamin C is a compound that is generally used as a comparison in antioxidant activity tests. The test results show that the order of antioxidant activity is vitamin c>chloroform fraction>n-hexane fraction with a confidence level of p<0.05. In line with the study of Jacob and John, 2020 plant Hypoesters phyllostachya extracted with the n-hexane solvent had the worst scavenging activity at a concentration of 500µg/ml–2500 µg/ml compared with chloroform and methanol extraction by DPPH method [25].

The difference in antioxidant activity between the DPPH and FRAP tests reflects the compound responsible for the sample's classification as a secondary antioxidant. The compound stabilizes hydroperoxides by inhibiting the decomposition of hydroperoxides into free radicals. The FRAP antioxidant test method is based on the ability of a compound to reduce Fe3+to Fe2+through Fe3+-triptidyltriazine (Fe3+-TPTZ) compounds so that an indicative color is formed as a result of the complex formed between the sample compound and Fe2+-TPTZ measured at the maximum absorbance wavelength. The decrease in absorbance is proportional to the concentration of antioxidants in the sample [27]. The mechanism of the FRAP method is different from the DPPH test. The concept of testing using the DPPH method is that compounds with antioxidant activity can donate hydrogen atoms, causing a reduction in the purple color of DPPH; thus, results are proportional to the number of compound molecules that have antioxidant activity [28]. The compound responsible has a mechanism for scavenging free radicals by rapidly donating or receiving hydrogen radicals by breaking the chain of radical reactions [29].

The antioxidant activity test results showed that the chloroform fraction had better antioxidative activity than the n-hexane fraction, with a p<0.05% confidence level. The content of fatty acid compounds in the chloroform fraction was more significant than in the n-hexane fraction (Tables 3 and 4). In the n-hexane fraction, six compounds found are classified as fatty acids, while in the chloroform fraction, 17 compounds classified areas fatty acids and two as linoleic-ester acids of the polyunsaturated fatty acid (PUFA) group, namely nonanedioic acid dimethyl ester and E-11, Z-13-Octadecatriene. It is known that fatty acids can act as antioxidants through oxidative stress protection pathways in cells [30].

A study conducted by Da Silva, 2005 found that antioxidant activity plays an essential role in UV protection [31]. Quercetin is a compound that has been tested and has the potential as a topical sunscreen factor able to protect against UVA and UVB rays in human beings [32]. This study tested the ability to protect against sunlight through the SPF test, a quantitative method for measuring the effectiveness of sun protection products. Protective effects must have the ability to absorb light at wavelengths in the range of 290 to 400 nm. The evaluation used in this study is the in vitro measurement of SPF value, this being the initial stage in screening potential products. The SPF test results for the n-hexane and chloroform fractions are 11.7 and 21.0, respectively, as shown in table 6. The results showed that the chloroform fraction had a higher SPF value than the n-hexane fraction with a p<0.05 confidence level. It is possible that the type of fatty acid compounds contained in the chloroform fraction can offer increased protection against sunlight. Previous studies show that fatty acids can protect the skin from UV rays through protective mechanisms against the effects of UV rays, such as erythema and epidermal lipid peroxidation, via photoprotective and anti-inflammatory properties [33–37]. In this study, it is identified that the chloroform fraction contains phytol compounds, and it is posited that there is the biological activity of phytols associated with sun protection. The antioxidant activity of phytols was investigated in a previous study, revealing a relationship among others between reducing the production of free radicals and the active compounds attributed to their structural features. Phytols are branched unsaturated chain alcohols, and their antioxidant properties may be related to the hydroxyl group (OH) present in the molecule. Several previous studies have shown that oxidative stress has a vital role in the nociceptive signaling involved in pain in the neuropathy and inflammation resulting from adverse effects of sunlight on the skin. As a result, phytols may play a role in protecting the skin from the sun [26].

CONCLUSION

The GC-MS test results found the highest number of peak values for hexadecanoic acid methyl ester (100%) in the n-hexane fraction and the fatty acid group known as palmitic acid in the chloroform fraction. The DPPH, antioxidant capacity test showed IC50 values ​​of 49.2±7.9 µg/ml and 11.3±3.65 µg/ml for the n-hexane and the chloroform fractions, respectively. The FRAP antioxidant capacity tests indicated IC50 values ​​of the n-hexane and chloroform fractions in the strong and weak antioxidants category of 365.2±133.6 µg/ml and 80.8±8.9 µg/ml, respectively. From SPF testing, the SPF values ​​for the n-hexane and chloroform fractions were found to be 11.7±0.1 and 21±0.4, respectively.

ACKNOWLEDGMENT

The authors gratefully acknowledge the financial support of the Ministry of Education, Culture, Research, and Technology, Indonesia, for this research. Thanks also go to the Rector of Tanjungpura University, Pontianak City, for providing laboratory facilities.

FUNDING

This research was fully funded by the Ministry of Education and Culture Research and Technology Indonesia under Contract No 2222/UN22.10/PG/2021.

AUTHORS CONTRIBUTIONS

All authors were contributed to the idea, designed the study, draft the article, review the data, and edit the article.

CONFLICTS OF INTERESTS

Authors declare no conflict of interest

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