GC-MS Screening of Sungkai Leaves and Relation With Its Antioxidant Capacity

Sungkai (Peronema canescens Jack) is a wild plant that grows in Kalimantan and Sumatera, Indonesia, and is used as traditional medicine. This study aimed to determine the components of chemical compounds in young leaves, half-old leaves, and old leaves of sungkai and their antioxidant activity. Gas Chromatography-Mass Spectrometry analyzed chemical compounds (GC-MS), and antioxidant activity was evaluated by 1,1-diphenyl-2-picrylhydrazil (DPPH). The analysis of chemical compounds showed that the chemical compounds were almost similar in young, half-old, and old leaves of sungkai with different intensities. Antioxidant activity showed the strong activity with IC50 of young leaves of sungkai is 26.389 mg/L, half-old leaves 29.874 mg/L and old leaves 30.9183 mg/L.

Leaves are the part of the plant that synthesizes organic compounds by using light as a source of energy needed in the process of photosynthesis. The leaves also store many secondary metabolite products. The leaves will change color as the leaves age. The color of the leaves will change from light green to dark green. Young leaves have the color of a light green with a soft texture, half old leaves have the color a green with a hard texture, and old leaves have a dark green color with a tough texture. Different leaf colors indicate differences in the pigments contained in these leaves. In general, older leaves have a higher chlorophyll content. Older leaves are due to differences in chlorophyll levels at each level of leaf development [9]. The aim from this study is to determine the differences in the content of metabolites in sungkai leaves based on the leaf senencence and their effect on antioxidant activity.

Preparation of plant extracts
A sample of young Leaves, half old and old leaves sungkai, was obtained from Padang Panjang, West Sumatera, Indonesia. Sungkai leaves were air-dried, and the sample was mashed using a grinder. The extraction process was carried out with a 20 g sample using methanol solvents with an ultrasonicator. The temperature was kept not exceeding 40°C. Then the sample was left overnight. The sample was filtered, then the filtrate was evaporated using a rotary evaporator to obtain the compound extract.

Extraction and derivatization
The procces of extraction and derivatization is carried out on the samples by following the procedure as described previously with some modification [10]. 40 mg sample powders were put into a 1,5 mL tube. The sample was extracted using solvent mixture with ratio 5/2/2 (v/v/v) of methanol, aquabidest, and chloroform. Then 60 µL internal standar ribitol was added (0,2 mg/mL in aquabidest). Sample was vortexed for 5 min and then centrifuged at 4°C and 13.000xg for 10 min. Supernatant was taken as much 900 µL and transferred into new tube, and 400 µL aquabidest was added. The mixture was then vortexed for min and centrifuged at 4°C and 13.000 xg for 10 min. 200 µL of aqueous phase was taken and evaporated by nitrogen gas for 25 min. Methoxyamine hydrochloride was dissolved in pyridine (20 mg/mL) and 100 µL of solution was taken and put into the tube, incubated sample at 35°C and 150 rpm for 90 min for oximation. After adding 50 µL of MSTFA, the mixture was incubated at 35°C and 150 rpm for 30 min for trimethylsilylation.

GC-MS Analysis
Analysis was performed after the derivatization was completed using a GC-MSQP2010 (Shimadzu) and an AOC-20i as an autosampler. The column used was Rxi-5MS (30 m x 0,25mm i.d., 0,25µm). Helium was used as carrier gas. The column temperature was held at 60 °C for 1 min and increased by 10 °C/min to 230 °C. The injector and detector temperatures are 200 °C and 230 °C, respectively. Ions were generated by electron ionization at 70 eV. Mass range of m/z 45 -500. Identified compounds were compared with data from the National Institute of Standards and Technologies, Mass Spectra Libraries (NIST).

Antioxidant activity assay
DPPH (1,1-Diphenyl-2-picrylhydrazyl) method was used to antioxidant activity assay [11]. The positive control was used quercetin. The DPPH with concentration 0.1 mM was dissolved with methanol, and samples were made in various concentrations (5,10,20,30,40,50 mg/L). The 2 mL of blank, plant extract and positive control was mixed in 3 mL of DPPH and each sample was incubated for 30 min at dark room. Absorbance sample and positive control was measured at 517 nm using a spectrophotometer UV-VIS.

Preparation of plant extracts
The collected samples were dried air-dried at room temperature without being exposed to sunlight. Drying procces is done to reduce the water content to inhibits microbial growth [12]. Sample was extracted using methanol solvent with an ultrasonicator for 3 hours. Ultrasonicator is one way to optimize the procces of extracting, the cell wall of the material is broken down by ultrasonic vibration. So the content will come out easily and saving time [13]. The temperature was kept not exceeding 40°C to prevent damage to the compounds in the sample and then left overnight to make filtering more accessible. The extract of young sungkai leaves was 0.6881 mg, the half-old leaves extract was 0.6506 mg, and the old leaves extract 0.6279 mg. Based on the Figure 1, it can be concluded that leaf senencence will affect the intensity of the compounds contained in the plant. It can be seen from the chromatogram results of each leaf that the young, half-od, and old leaves have the same compound components, but the intensity is different. The major components in sungkai leaves can be seen in the

Antioxidant activities
The antioxidant activity of samples of young, half-old, and old leaves was carried out with DPPH. The sample was prepared with various concentrations of 5,10,20,30,40,50 mg/L. Quercetin was used compared with the concentration variations of 1,2,3,4,5,6 mg/L. The calculation of antioxidant activity is seen from the changes in the DPPH colors, expressed in percent inhibition. The percent of inhibition value can be calculated to get the IC 50 . The IC 50 value indicates that the concentration can inhibit free radical activity is as much as 50%. It happens because of a large number of hydrogen donors given samples to reduce free radicals. DPPH has an unpaired electron from the nitrogen atom. When a compound donates hydrogen to react with DPPH, it will transform DPPH into DPPH-H [14].

Figure 2. Antioxidant activity of sungkai leaves
Based on the Figure 2, it can be concluded that the young leaves of sungkai leaves have higher antioxidant activity than half-old and old leaves of sungkai. a compound is classified when the IC 50 value is ˂50 mg/L is very strong, when IC 50 is 50 -100 mg/L is strong, moderate when IC 50 101-150 mg/L and weak antioxidants when IC 50 is ˃150 mg/L [15]. The antioxidant activity from all samples of sungkai leaves showed strong activity because the IC 50 value was less than 50 mg/L. Compared to standard quercetin, which has a low IC 50 value, it showed strong antioxidants. The high antioxidant activity in young leaves could be due to differences in the content of phenolic compounds. This is same as previous studies, it showed that young leaves in Tenggulun leaf (Protium javanicum Burm.F.) have higher antioxidant than old leaves caused higher phenolic compounds. decreased antioxidant can also caused by the absence of biosynthesis of new secondary metabolites formed during ripening.
Most of this biosynthesis occurs in the early stages of plant growth so that the antioxidant activity become higher in young leaves [16].