Antioxidant Activity of Alkaloid Compounds from Litsea cubeba Lour

Litsea cubeba (Lour.) heartwood. was extracted by maceration. Ethanol extract was fractionated with liquid-liquid extraction using n-hexane, chloroform at pH 3, 7, 9 and 11 to obtained alkaloid fractions. Isolation of alkaloid compounds were isolated with preparative thin layer chromatography. Antioxidant activity for fractions and isolates were determined with DPPH and ABTS assay. The highest IC 50 were given by chloroform fractions at pH 7 and compound III. They IC 50 were 23.81 ± 0.01; 3.12 ± 0.02 μg/mL for DPPH assay and 56.43 ± 0.06; 8.62 ± 0.02 μg/mL for ABTS assay. Compound III was showed maximum absorbance at (302.8; 280.8; 206 nm) and wave number (3286.70; 3109.25; 2939.52; 2835.36; 1589.34; 1462.04; 1365.60; 1246.02; 1018.41; 879.54 cm-1) which indicate its an alkaloid compound with hydroxyl, methyl, methylene and methoxy group. The results reveal that alkaloid fractions and compounds of Litsea cubeba heartwood has very strong antioxidant potential.


INTRODUCTION
Oxidation is an important process (normally) in living organisms, free radicals producing from metabolism pathway process or enviromental sources that interact sustainable with biological system.Reactive species are molecules which have an electronic unstability and most reactive.Reactive oxygen species (ROS) are the biggest sources of primary catalyst which initiate process of oxidation in vivo and in vitro and produce oxidative stress.Oxidative stress products when reactive forms of oxygen are produced faster than they could be safely neutralized with antioxidant mechanisms and/or from a decrease in antioxidant defense.The uncontrolled production of oxygen free radicals and the unrateabled system of antioxidant capability in protection results in the cause of many diseases, such as cancer, diabetes, heart diseases, Alzheimer's, and aging [1][2][3][4][5][6] .

DPPH radical scavenging activity
The DPPH assay was tested based on previously method 11 , 200 µM solution of DPPH• in methanol was added to various concentrations.After 60 min.absorbance was measured at 516 nm and percentage of inhibition was calculated by comparing the absorbance values of the control and test samples 2,5 .

ABTS radical anion scavenging activity
The ABTS radical was produced from reaction between 5 mL of 14 mM ABTS solution and 5 mL of 4.9 mM pottasium persulfate (K 2 S 2 O 8 ) solution, stored in the dark at temperature room stand for 16 h, Before use, solution was diluted with phospate buffer saline (PBS) to obtain an absorbance of 0.700 ± 0.020 at 734 nm.The fractions and isolates with various concentrations with 1 mL of ABTS solution was homogenized and its absorbance was recorded at 734 nm, PBS blanks were run in each assay and all of measurements were done after at least 6 min.Similarly the reaction mixture of standard group were obtained using quercetin, ABTS scavenging ability was expressed as IC 50 (µg/mL) [5].

Statistical analysis
Data was expressed as mean ± SD which were analyzed using the SPSS 21 software.

Antiradical activity
Antiradical activity of the plant was measured in term of hydrogen donating ability using DPPH which is a stable, nitrogen-centered free radical and produces deep purple colour in methanol solution, antioxidants either transfer an electron or a hydrogen atom to DPPH, thus neutralizing its free radical character 12 .Antioxidant assay with DPPH and ABTS which is based on the ability of DPPH and ABTS, the stable free radical, to decolorize in the presence of antioxidants, is a direct and reliable method for determining radical scavenging action 13 and has been largely used as a quick, reliable and reproducible at in vitro antioxidant acitivity assay 14 .The reducing capacity of compounds could serve as marker of potential antioxidant activity [15][16][17][18] .Alkaloids are compound which contain OH and NH functional group, they are coud be donating their hydrogen to DPPH and ).The O-H groups were interpreted by wave number between 3000 -3500 cm -1 (3394.72 and 3286.70 cm -1 ).The N-H group was showed at 3109.25 cm -1 .The C-H streching groups were showed at (2800 -2950 cm -1 ) and C-H bending groups were showed at (2800 -2950 cm -1 ).Many researchers were reported that alkaloid compounds have major functional groups O-H, N-H and C-H streching and bending groups 19,20,21 .

CONCLUSION
The result of this study showed that alkaloid fractions and compounds of Litsea cubeba heartwood have antioxidant activity.