Secondary Metabolites and Antioxidant Activity of Methanol Extract of Castanopsis costata Leaves

Castanopsis costata (C.costata) leaves were empirically used by people in North Sumatera, Indonesia, as an antioxidant dietary supplement. However, its pharmacological effect has not been scientifically explored. The purpose of this study was to identify the secondary metabolites and antioxidant activity of C. costata. The leaves were cold extracted with methanol. The secondary metabolites were determined using thin layer chromatography. Its antioxidant activity was investigated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) method. The results showed that methanol extract of C. costata leaves consisted of alkaloids, flavonoids, glycosides, anthraquinone glycosides, tannins and triterpenoids. The methanol extract of C. costata leaves showed comparable antioxidant activity with vitamin C, IC50 35.56 μg/ ml and 14.17 μg/ml, respectively.


Introduction
Free radicals reactive oxygen species are responsible for development of degenerative diseases such as atherosclerosis, coronary heart disease, stroke, cancer, and kidney failure.[3] Endogoneous antioxidants, such as catalase enzymes bound to Fe, glutathione peroxidase, glutathione S-transferase binding to Se, superoxide dismutase binding to Cu, Zn and Mn are responsible to diminish free radical activities.
However, an imbalance between free radical production and antioxidant can result in oxidative stress.Exogenous antioxidants are necessary to prevent harm caused by oxidative stress. 4tioxidant compounds can be either from synthetic or natural compounds.Several studies showed that synthethic antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate (PG), and tert-butylhydroquinone (TBHQ) can cause several side effects. 5It encourages investigation to find new natural compounds as natural antioxidants which are safer for human health.
Natural compunds can be used as new sources of antioxidant.Major plant antioxidants were resulted from shikimic acid pathway and phenyl-propanoid metabolism.One of the plant that is empirically used as dietary antioxidant supplement is Castanopsis costata (C costata) with the local name cepcepan plant.However, its pharmacological effect has not been scientifically explored yet.Thefore, this study was conducted to identify the secondary metabolites and antioxidant activity of C. Costata.

Antioxidant activity
Quantitative antioxidant activity determined by DPPH method was expressed as IC50, i.e., the concentration required to inhibit 50% of DPPH free radicals. 9Antioxidant activity of C. Costata methanol extract was performed at different concentrations.We found that the higher the concentration of the extract showed the higher of the percentage value of the inhibition.We observed a decrease in the absorbance of DPPH by the addition of combination of C. Costata and vitamin C. The higher concentration of the extract yielded the greater reduction of absorbance (Table 2, Figure 1).
We found that antioxidant activity of C. Costata leaves was comparable with that of vitamin C, which was in the category of very strong activity (Table 3).Polar substances such as polyphenol (flavonoid), cinnamic acid derivatives, coumarin, and tocopherol in C. Costata might contribute in its antioxidant activity. 9,10lar substances are more easily dissolve in polar solvent such as methanol, ethanol, and water.Methanol has a polarity index of 5.1.
Most polar compunds can be extracted using methanol and certain group of non polar compounds are fairly soluble in methanol.
Therefore, methanol is generally preferred for the extraction of bioactive compounds.[12][13][14] This is the first study investigating C. costata leaves antioxidant activity.This analysis could provide insight into the variations in the antioxidant profiles between different concentration of C. Costata.Further research might be necessary to examine the mechanism of its antioxidant activity.

Conclusion
The methanol extract of C. costata leaves had comparable antioxidant activity with vitamin C, which showed the IC 50 value of 35.56 ppm and 14.17 ppm, respectively.Secondary metabolites of C. Costata leaves consisted of alkaloids, flavonoids, glycosides, anthraquinone glycosides, tannins and triterpenoids.
8ntioxidant activityDetermination of antioxidant activity was performed using DPPH.8DPPH200 µg/ml stock solution was made by dissolving 20 mg of DPPH to 100 ml methanol.Sample solution was obtained by dissolving 25 mg of viscous extract with 25 ml of methanol.It was then diluted into 10, 20, 30, 40, and 50 µg/ml.It was mixed until homogeneous and incubated at 37°C for 30 minutes.The antioxidant activity