OBJECTIVE

2

To optimize the extraction technology of total coumarin from

Viola yedoensis

and to evaluate its

in vitro

antioxidant and anti-tumor activities of its purified product.

METHODS

2

On the basis of single factor experiments， the extraction technology of total coumarin from

V. yedoensis

was optimized by the response surface methodology， using yield of total coumarin from

V. yedoensis

as response value， solid-liquid ratio， extraction time and ethanol volume fraction as variables. The verification tests were conducted. The antioxidant activity of purified product of total coumarin from

V. yedoensis

was evaluated by 1，1-diphenyl-2-picrylhydrazyl （DPPH） method， 2，2′-azino-bis（3-ethylbenzothiazoline-6-sulfonic） acid ammonium salt （ABTS） method and 2-phenyl-4，4，5，5-tetramethylimidazoline-3-oxide-1-oxyl （PTIO） method. CCK-8 assay was used to determine the inhibitory effect of it on human cervical cancer cells HeLa， human intrahepatic bile duct cancer cells HCCC-9810， human hepatocellular carcinoma cells HepG2， human breast cancer cells MDA-MB-231 and human non-small cell lung cancer cells A549. The apoptosis morphology of HCCC-9810 cells was detected by Hoechst 33258 staining， and lactate dehydrogenase （LDH） and superoxide dismutase （SOD） content were determined.

RESULTS

2

The optimal extraction technology included solid-liquid ratio was 25∶1 （mL/g）， extraction time was 1.9 h， ethanol volume fraction was 68%. Average yield of total coumarin from

V. yedoensis

was （21.10±0.17） mg/g， and RSD was 0.80%（

n

＝3）， relative error of which with predicted value （20.79 mg/g） was 1.49%. The median inhibitory concentrations （IC

50

） of purified product of total coumarin from

V. yedoensis

to DPPH and ABTS free radicals were 21.54 and 17.11 μg/mL， which were slightly higher than that of ascorbic acid （15.21， 12.45 μg/mL）. The IC

50

to PTIO free radicals was 1 422.01 μg/mL， which was far higher the that of ascorbic acid （37.20 μg/mL）. The IC

50

values of total coumarin from

V. yedoensis

to above 5 cells were （113.18±15.97）， （51.17±1.72）， （134.15±11.75），（171.83±15.27） and （93.38±4.65）μg/mL， respectively. Total coumarin from

V. yedoensis

induced the apoptosis of HCCC-9810 cells and increased the content of LDH， as well as decreased the content of SOD.

CONCLUSIONS

2

The extraction technology of total coumarin from

V. yedoensis

is optimized successfully； the purified product of obtained extract has good antioxidant and anti-tumor activities.