Analysis Of IR Spectral Of Agrimonia And Study On The Anti-cancer Of Tannin Components Of Agrimonia

Agrimony (Agrimonia Pilosa Ledeb) is the Rosaceae perennial herb agrimony of the whole plant. The traditional major treatment is for the kinds of bleeding. The modern researches show that agrimony was used for the anti-tumor, reducing blood glucose, boosting pressure and so on. The tannin is abundant in the agrimony, and the study on tannin is thought more and more important in internal and abroad scientific researchers day by day. In this paper, Fourier Transform Infrared Spectroscopy (FT-IR) was the fist time used for the analysis of agrimony, and investigating its tannin components of anti-tumor effect in vitro and vivo and exploring its mechanisms.The results of FT-IR showed through the FT-IR analysis the Mao ershan agrimony had the four tannin characteristic peaks in 1659 cm-1,1569 cm-1,1168 cm-1 and 989 cm-1, and there were tannin characteristic peak near the 1643 cm-1,1512 cm-1 and 1470 cm-1. The results illustrated the agrimony had tannin components. Through the analysis of the multi-steps infrared maro-fingerprint method showed that the ethyl acetate and the n-butanol abstraction from the acetone extraction of seven habitats agrimony contained a certain amount of tannin component and the content was different. We could distinguish them by using the origin Chinese medicinal materials and the extracts of the agrimony from different habitats. Through the analysis of the one-dimensional infrared spectroscopy and the second derivative infrared spectroscopy of the ethyl acetate layer and the n-butanol layer of the different extractives, the ethyl acetate layer and the n-butanol layer of the different extractives both had the tannin component and the content was different. The tannin was extracted adequately only by using the acetone. The comparative analysis between the ethyl acetate layer, the n-butanol layer and tannin through one-dimensional infrared spectroscopy and second-derivative infrared spectra. After the purification, the purity of the ethyl acetate layer and the n-butanol layer were raised on the different degree. The average tannin contents were 19.91% and 14.87% of the ethyl acetate and the n-butanol layer of agrimony acetone extracts by casein method. So, Fourier Transform Infrared Spectroscopy was the useful method on the identification of different habitats agrimony. And this was the foundation of the finger print of agrimony by FT-IR.The vivo anti-tumor results showed that the ethyl acetate layer and the n-butanol layer could inhibit the growth of S180 entity-tumor mice, as compared with the control group, the administration groups were significant difference(P<0.05). The inhibitory rate was 50.44% by 200 mg/kg of the ethyl acetate layer, and the inhibitory rate was 57.52% by 250 mg/kg of the n-butanol layer. Both the ethyl acetate layer and the n-butanol layer increased the thymus index and spleen index of S180 entity-tumor mice in different level, and had a good protective effect for the two immune organs in entity-tumor mice. The ethyl acetate and the n-butanol layer of agrimony acetone extracts enhanced the antioxidant enzymes SOD, CAT and GSH activity, reduced lipid peroxidation MDA content.So the ethyl acetate extract layer and the n-butanol layer significantly increased the antioxidant functions of the system of the entity-tumor mice, inhibited lipid peroxidation, cleaned the of excess oxygen free radicals, and played an anti-tumor effect, which might be the one of the mechanisms in vitro anti-tumor effect.Through the vitro anti-tumor results, we could see that the ethyl acetate layer and the n-butanol layer both inhibited the growth of BEL-7402 cells and HepG2 cells significantly, and the effect was enhanced by the increasing of concentration apparently. The BEL-7402 cell's IC50 was 89.28μg/mL and the HepG2 cell's IC50 was 127.85μg/mL of the ethyl acetate layer; The BEL-7402 cell's IC50 was 107.54μg/mL and the HepG2 cells's IC50 was 234.41μg/mL of the n-butanol layer.The ethyl acetate layer was better obviously than the n-butanol layer. The ethyl acetate layer and the n-butanol layer could induce the BEL-7402 cells and the HepG2 cells apoptosis by flow cytometry (FCM) further, and had a good effect. Through the laser confocal microscopy, we saw that the tumor cells significantly reduced by different concentrations of the ethyl acetate layer and the n-butanol layer on the BEL-7402 cells and HepG2 cells after 48 h, and there were nuclear cracking, apparent apoptosis shape, and there was a clear green fluorescence. These showed that the overloading of tumor cells elevated levels of intracellular free Ca2+ might be increased. The different concentrations of the ethyl acetate layer and the n-butanol layer effected on the BEL-7402 cells and HepG2 cells after 48 h, the intracellular reactive oxygen species (ROS) were increased with the concentration increased by FCM. As compared with the control group, there was significant differences (P<0.05). Therefore, the increasing of tumor cells intracellular free Ca2+ and the ROS might induce the tumor cells apoptosis, this might be the one of the mechanisms in vivo anti-tumor effect.In summary, FT-IR was the fist time used for the analysis of agrimony and created the foundation of the agrimony's finger print, and there were tannin component in the ethyl acetate and the n-butanol layer of agrimony acetone extracts, but the content was differnt. Through the pharmacology researches determined that the tannins of agrimony had anti-cancer effect, and its mechanisms might be enhancing the antioxidant functions and inducing the tumor apoptosis.