A Preliminary Study On The Preparation Of Flavonoids From Uraria Crinita Root And Their Anticancer Activity In Vitro

Objectives To prepare flavonoids of Uraria crinita root and examine their anticancer effects in vitro. Methods Uraria crinita root was extracted refluently with 95% ethanol to prepare its crude extract and then the crude extract was purified by D-101 macroporous resin adsorbent. Select a method to determine the content of flavonoids in Uraria crinita root by examining UV-vis spectra of the crude extract and flavonoids in different chromogenic reaction with rutin as reference substance. The appropriate adsorbent for enrichment of flavonoids was selected by comparing the dynamic flavonoids adsorption and desorption rate of four types of macroporous resin adsorbent, then screen the purification process of flavonoids with the selected type of adsorbent to purity flavonoids. The effect of extracts from Uraria crinita root on the proliferation of tumor cell lines CA46, NB4, AGS and BEL-7404 was evaluated by MTT assay. Apoptosis of AGS cells was observed by Hoechst 33258 staining, DNA fragmentation analysis and the apoptotic rate was determined by TUNEL method. Results The spectral shape of crude extract and flavonoids extract of Uraria crinita root was similar to that of rutin with a absorption peak at 405 nm by aluminum trichloride-acetic acid- sodium acetate buffer colorimetry. Linear regression equation was D=24.338c+0.0043, r=0.9996, n=5 (D: absorbance, c: concentration(μg·mL-1)) and the calibration curve was linear when the concentration of rutin was in the range of 0.008～0.04 mg·mL-1. The repeatability precision RSD<3% (n=6); the absorbance of the complex was stable within 60 min with RSD<1% (n=6); the average recovery was 99.59%. ADS-17 macroporous resin showed good adsorption and desorption capacity, and the proper technological conditions were: sample concentration: 0.8 mg·mL-1, sample velocity: 2 mL·min-1, mobile phase: 30～90% ethanol, gradient elution. The flavonoids content of crude extract and the purified are 13.6% and 63.3%. Results of MTT assay showed that both the crude and the flavonoids extract from Uraria crinita root can inhibit proliferation of CA46, NB4, AGS and BEL-7404 cells. Compared with the crude extract group, the inhibitory rate of the flavonoids extract group increased obviously (P<0.01) and showed its effect in a concentration and time-dependent manner. AGS was the most sensitive of the four types of tumor cells. Flavonoids induced apoptosis in AGS cells. Compared with control group, the apoptotic rate of 12.8μg·mL-1 group increased significantly (P<0.05) while the 32, 80, 200, 500μg·mL-1 group increased very significantly (P<0.01). Conclusions The aluminum chloride-acetic acid- sodium acetate buffer colorimetry is suitable for determination of flavonoids in Uraria crinita root. Flavonoids extract purified by ADS-17 macroporous resin has inhibitory effect on tumor cells and can induce apoptosis in vitro. The material basis of anticancer effect in Uraria crinita root might be present in flavonoids extract.