GC-MS profile, total phenolic, and DPPH radical scavenging activity of Saurauia minahassae stembark ethyl acetate extract

Several species of the genus Saurauia have been traditionally used as remedies. However, Saurauria minahassae, as an endemic plant, is still understudied. This study aims to reveal the total phenolics, GC-MS profile, and bioactivity as DPPH free radical scavengers of ethyl acetate extract of S.minahassae collected from Banggai Island, Indonesia. Determination of Total Phenolic Content (TPC) was conducted using the colorimetry method by spectrophotometer. Thin layer chromatography (TLC) and TLC-bioautography performed analysis of secondary metabolites and qualitative DPPH free radical scavenging activity, respectively. GC-MS analysis was conducted to identify the chemical compounds of the extract. The extract’s IC50 value and Antioxidant activity index (AAI) were performed by serial microdilution method on the 96-microwell plate. The results showed that TPC was 432.33 mg GAE/g extract. TLC analysis revealed several metabolites, and TLC-bioautography also revealed several compounds active as DPPH free radical scavengers. The ethyl acetate extract of S. minahassae is categorized as a very strong DPPH free radical scavenger or potent antioxidant with an IC50 value of 3.29 ug/ml and AAI of 9.32. GC-MS profile of the ethyl acetate extract of S.minahassae stembark contained several compounds with antioxidant activity. The potential antioxidant activity of S. minahassae stembark extract was contributed by phenolic content and several compounds with antioxidant activity in the extract. This extract could be a good source of natural antioxidants, and S. minahassae sustainability as an endemic plant should be maintained.


Introduction
Oxidative stress has gained attention globally in the last two decades due to its association with several diseases [1].It relates to several pathophysiological conditions, including cancer and inflammatory diseases [2][3], renal disorders, and cardiovascular [4].Oxidative stress occurs due to an imbalance of production and accumulation of reactive oxygen species (ROS) [5].It exceeds the antioxidant capacity within the cells [6] and, in the long term, can cause various oxidative stress-related diseases, such as Alzheimer's disease, cardiovascular diseases, cancer, and diabetes [7].ROS could be in superoxide radicals, hydrogen peroxide, singlets oxygen, and hydroxyl radicals, metabolic by-products from biological systems [8][9].Exogenous free radicals could be generated from exposure to environmental pollutants, cigarette smoke, heavy metals, alcohol, and radiation [6].
Free radicals are highly reactive, unstable molecules that can damage DNA and cause mutations [10].It also can damage cell structures [11].The harmful effects of free radicals could be counteracted by antioxidant molecules [12].The human body has several mechanisms against free radicals and oxidative stress using endogenous antioxidants [5].Besides endogenous antioxidants, there are several sources of exogenous antioxidants.One of them is chemical compounds produced by plants with antioxidant activity.
Saurauia belongs to the Actinidiaceae family, and some species are widely used as traditional herbal medicines to treat various diseases [13].Several biological activities of Saurauria species also have been documented.S. elegans and S. sparsifolia possess radical scavenging activity with an IC50 of 86.40 ppm [14].Lowena et al. 2018 reported that water extract of S. vulcani leaves has a strong antioxidant activity with an IC50 value of 22. 92±1.32 µg/ml.The chloroform extract of S. vulcani inhibited the growth of S.aureus [15].
One of the endemic Saurauia species, native to Sulawesi, Indonesia, was S.minahassae [16].The information about S. minahassae is very limited.To the best of our knowledge, there is still no information on the chemical compounds and biological activity of S. minahassae.Therefore, this study was designed to determine the chemical compounds by GC-MS, total phenolic content, and the DPPH free radical scavenging activity or antioxidant of ethyl acetate extract of S. minahassae stem bark.

Sample collection and preparation of extraction
Stembark of Saurauia minahassae was collected from Tinangkung village, Banggai Island, Central Sulawesi, Indonesia.The stem bark is cleaned of dirt, chopped into small pieces, and dried.After drying, the stembark is ground.

Extraction
Bark powder (95 g) was macerated with ethyl acetate for 24 hours, three times.The ethyl acetate extract was concentrated with an evaporator to obtain a concentrated extract.The remaining solvent was evaporated with nitrogen.The extract was stored at -4C for use in further analysis.

Analysis of secondary metabolite and qualitative DPPH radical scavenging using Thin-Layer Chromatography
Ethyl acetate extract of S. minahassae (10 µL at 10 mg/mL concentration) was transferred on a silica TLC plate (Merck F254), followed by eluting in the mobile solvent of dichloromethane: methanol (10:1).After elution, the plate was observed under short UV light (254 nm) and long UV light (366 nm).The plates were also sprayed with stained reagents, i.e., cerium sulfate and vanillin sulfate.Qualitative DPPH radical scavenging activity was carried out by spraying the eluted plate with methanolic DPPH solution and then incubating it in the dark at room temperature for 30 minutes.

Determining the total phenolic content (TPC)
68 µl of the extract (1 mg/mL) were mixed with 50% Folin-Ciocalteu reagent in a vortex for one minute.Following homogenization, 1,364 µl of 2% sodium carbonate (Na2CO3) was added to the mixture, which was then left at room temperature for 30 minutes.Gallic acid at 0.031-0.250mg/mL was used to make a calibration curve.UV-Vis spectrophotometry determined the absorbance of the standard solution (gallic acid) and the extract at 750 nm.Gallic acid equivalent (mgGAE/g extract) was used to express the extract's total phenolic content (TPC).

Determining IC50 value and Antioxidant Activity Index (AAI)
Serial microdilution on a 96-well microplate in triplicate was carried out to determine the IC50 value and AAI of the extract for antioxidant activity.195 µL methanol p.a was transferred to each well on the 1 st row, adding 5 µL extract (10.240 µg/ml) and homogenized.After homogenization, 100 µL was taken from the 1 st and transferred to the second row in the same column.The same procedure was applied in the following row.In the last row, 100 µL was taken and discarded.After dilution, every well was added with 100 µL of methanolic DPPH solution (61.5 μl/ml).The final concentration of DPPH was 30.75 μl/ml.The plate was then incubated for 90 minutes at room temperature under dark conditions.After incubation, the absorbance was determined at 517 nm using a Vario Scan Flash microplate reader (Thermo Scientific).The following equation was used to calculate the concentration inhibition: IC (%) = (ADPPH 100% − Asample) × 100/Asample IC: inhibition concentration ADPPH: Absorbance of DPPH Asample: Absorbance of sample IC50 value was the concentration needed to scavenge 50% of DPPH free radicals.It was determined by linear regression curve of concentration of extract against inhibitory percentage.

Qualitative analysis using TLC-bioautography for DPPH free radical scavenging activity
Ten microliters of 10 mg/ml extracts were spotted on the TLC silica plate (Merck F254), then the spot of the extract was developed using the mobile phase of dichloromethane: methanol (10:1).After the elution, the plate was sprayed with DPPH solution in methanol (0.2%).The spots or bands with whiteyellowish color indicate that spots or bands have DPPH free radical activity or antioxidants.

Analysis and identification of chemical compounds in the S.minahassae by Gas-Chromatography and Mass-Spectrometry
Chemical compounds analysis of the extract of S.actinidiifolia was carried out using a Gas Chromatograph (Agilent 19091S-433UI:93.928coupled with a Mass Spectrophotometer (MS) and equipped with a capillary column of 5% phenyl methyl siloxane, 30 m x 250 µm x 0.25 µm.The mobile phase or carrier gas was Helium with a 1.0 ml/min flow rate) column velocity flow.The injection volume was 1µL.The initial oven temperature was 40 °C to 300°C for 4 min at 10°C/min.Compound identification was done by comparing their mass spectrum with the mass spectra available in the computer library (NIST database).

Secondary metabolite analysis using TLC and qualitative DPPH free radical scavenging activity
Secondary metabolites of the ethyl acetate extract of S.minahassae were analyzed by Thin Layer Chromatography using the mobile phase of dichloromethane: methanol (10:1) .The DPPH free radical scavenging activity of the extract was presented in Fig. 1e.Compounds in the ethyl acetate stembark extract of Saurauia minahassae were detected using shortwave UV light (254 nm), long-wave UV light (366 nm), stain reagents of cerium sulfate and vanillin sulfate.UV light visualizes the compounds with a chromophore [17] as they strongly absorb UV.Shortwave UV light visualizes the compounds as unsaturated with conjugated double bonds, possessing aromatic rings, and long-wave UV light visualizes long-chain conjugated double bonds [18].Stain reagent cerium sulfate is used to visualize alkaloids that appear with a brown color, while vanillin sulfate is appropriate in detecting hydroxyl and carbonyl compounds [19] (Fathoni et al. 2022).
The TLC bioautography method performed a qualitative assay to detect DPPH free radical scavenging or antioxidant activity.This method can identify the active chemical compound on the eluted chromatogram from the crude extract.It is also an easy, quick, and straightforward method.The active chemical compounds as free radical DPP scavengers were shown by yellowish-white dots or bands.The color's intensity may indicate the activity's power [20].The yellowish-white color on a purple background after spraying DPPH was due to the antioxidant compounds in the extract reducing the free radical DPPH as diphenyl picryl hydrazine [21].The antioxidant compound donates the electrons that cause purple DPPH free radicals to turn yellowish-white.

Total Phenolic Content (TPC) and quantitative antioxidant activity
Plant phenolics are considered an essential dietary component for humans and show remarkable antioxidant activity and other health advantages [22].Several previous studies showed a direct correlation between reducing the power of plant extracts and antioxidant activity [23].The result showed that the TPC of the stembark ethyl acetate extract of S. minahassae was 432.33 mg GAE/g extract (Table 2.).Assessing TPC content is crucial in determining the antioxidant of plant extract.It is due to the ability of phenolic to donate electrons [24] and decrease the absorption of the solution [25].The antioxidant capacity of the sample is proportional to the degree of color intensity [26].Plant phenolic antioxidants could act as reducing agents [27] and free radical scavengers [28].
Further analysis showed that the IC50 value and AAI were 3.2 µg/ml and 9.32, respectively.Based on these values, the ethyl acetate extract of S. minahassae was classified as having very strong antioxidant activity [29].The classification of antioxidant activity based on the AAI values was as follows: weak (low) < 0.05 < moderate < 1.00 < strong (significant) < 2.00 < very strong activity as an antioxidant agent.The very strong antioxidant activity of the extract might be attributed to TPC content and other chemical compounds in the extract.
Conclusion of this study that the evaluation of bioactivity and chemical compounds of ethyl acetate extract of S. minahassae stembark possessed very strong antioxidant activity, i.e., DPPH free radical scavenger with an IC50 value and AAI of 3.29 µg/ml and 9.32, respectively.The antioxidant activity is due to the relatively high TPC content (432.33 mg GAE/g extract) and several other compounds, such as octadecane and tetracontane, in the extract.The ethyl acetate extract of S. minahassae stembark may be a potential source of natural antioxidants.Due to limited study on the biological activity of S. minahassae, further study to reveal other biological activity and the chemical compounds responsible for the biological activity need to be further studied.

Figure 2 .
Figure 2. GC-MS chromatogram of ethyl acetate extract of stembark of S. minahassae

Table 2 .
Chemical compounds of stembark ethyl acetate extract of S. minahassae identified by GC-