Antioxidant Activity of Ethanol Extract of Polygonum pulchrum Blume

The use of antioxidants from natural resources has attracted increasing interest. One of the plant that was empirically used as an antioxidant dietary supplement was Polygonum pulchrum Blume (P. pulchrum Blume). This study aimed to investigate antioxidant activity of roots, stems, leaves and flowers ethanol extract of P. pulchrum Blume. The extract was obtained by maceration method using ethanol solvent. Antioxidant activity was determined with 1,1-diphenyl-picrylhydrazyl (DPPH) method. We found that ethanol extracts of P. pulchrum Blume roots and stems had strong antioxidant activity with IC50 values of 25.2 mg/l and 43.26 mg/l, respectively. Ethanol extract of flowers had the weakest antioxidant activity with IC50 value of 202.96 mg/l. Vitamin C had very strong antioxidant activity with IC50 value of 3.97 mg/l. In conclusion, this study revealed that ethanol extract of P. pulchrum Blume roots and stems had strong antioxidant activity, therefore, this plant might be potential as an excellent source for natural antioxidant agents for medical application.


Introduction
The use of antioxidants derived from plants have widely improved around the world.Many plants contain various phytochemicals which possess antioxidant activity to protect cells from damaging effects of reactive oxygen species (ROS), such as superoxides, peroxyl radicals, hydroxyl radicals and peroxinitrins. 1,2 human, production of ROS as the result of biochemical process might increase with several factors, including toxins and chemicals in food, pollutants, radiations, etc. Antioxidants compounds are needed to tackle this problem.However, synthetic antioxidant compounds such as butylated hydroxytoluene and hydroxyanisole might cause various side effects. 3This encourage researchers to find alternative treatments, particularly from natural source.
One of the plants that has a potential to be developed as a natural antioxidant is Polygonum pulchrum Blume (P.pulchrum Blume).This plant is widely spread around the Asian-African continents, including India, China, Malaysia, Myanmar, Srilanka, Thailand and Indonesia. 4This plant is known as ketapan or kumpai air in Indonesia.
6][7] However, limited information was available regarding biological activity of P. pulchrum Blume.Therefore, this study was performed to investigate antioxidant activity of root, stems, leaves and flowers ethanol extracts of P. pulchrum Blume.

Phytochemical screening using thin layer chromatography (TLC)
The phytochemical screening test with TLC was performed using silica gel plate GF254.

Antioxidant activity
Preparation of DPPH reagent 40 mg/ml 2 mg of DPPH were weighed and dissolved in ethanol until all dissolved.It was then added with 50 ml ethanol.The solution was shaken until homogeneous.

Preparation of blank solution
The preparation of the blank was performed by reacting 1ml of 40 mg/l DPPH and 3 ml ethanol.The solution was shaken until homogeneous and was incubated at 37 °C for 30 minutes.

Extraction
Maceration of plants was performed in a closed container for 3x24 hours using ethanol Preparation of ethanol extract 1000mg/l 50 mg of roots, stems, leaves and flowers viscous extract were dissolved in 50 ml of ethanol.Dilution was performed on 1000 mg/l sample to obtain 12.5, 25, 50, 100, and 200 mg/l of samples.The solutions were shaken until homogeneous and were incubated at 37 °C for 30 minutes.The absorption test was performed at 515.5 nm.

Calculation
All of the inhibition concentrations (IC 50 ) of DPPH radical from each sample was calculated using this formula:

Extraction
The extract was obtained using maceration with ethanol solvent.The yield of each extract was 3.6%, 5.1%, 3.24%, and 2.3% for roots,

Phytochemical screening
Phytochemical screening was performed to identify the secondary metabolites in the extract.We found that alkaloids, flavonoids, tannins, saponins, and triterpenoid were found in different parts of the plant (Figure 1, Table 1).Figure 2 shows the result of phytochemical screening indentification after reagent markers were sprayed into the TLC plate.

Antioxidant activity
Antioxidant activity testing aimed to determine the inhibiton concentration value (IC 50 ) of the extract using DPPH method.
This method is based on the DPPH radicals color change which caused by the reaction between free radical of DPPH and one electron or hydrogen atom released from the compound contained in the extract to form a DPPH compound whose color is yellow.We compared the antioxidant activity of the extracts with vitamin C (Table 3).
Antioxidant activity increased in accordance with the rise of concentration.This was marked by the fading colour of DPPH and the greater percentage of inhibition value.
After obtaining the inhibition percentages, the graphs between the sample concentration (x) and the percentage of inhibition (y) was made.The results can be found in Figure 3-7.The IC 50 values can be determined using the linear regression equations.

Conclusion
This study revealed that ethanol extract of P. pulchrum Blume roots and stems had strong antioxidant activity, therefore, this plant might be potential as an excellent source for natural antioxidant agents for medical application.

Table 3 . % inhibition of ethanol extract of P. pulchrum Blume
value of 202.96 mg/l.Vitamin C had very strong antioxidant activity with an IC 50 value of 3.97 mg/l.A compound can be said to be a very powerful antioxidant if the IC 50 is less than 50 mg/l, strong antioxidant if IC 50 is between 50-100 mg/l, moderate antioxidant if IC 50 is between 100-150 mg/l, weak antioxidant if IC 50 value is between 150-200 mg/l, and very weak antioxidant effect if IC 50 value is more than 200 mg/l. 9igh antioxidant activity in the ethanolic extract might be due to the presence of flavonoids, alkaloids, and triterpenoids.[10][11][12]