| At present, the lung cancer is the fastest growing, highest morbidity and mortality of cancer.It is the biggest threat to human life and health of malignant tumor. Natural antineoplastic drugs development, especially to come from the study of antitumor active ingredient in higher plants, has become the hot topics in the study of anticancer drugs. Hcordata stems from saururaceae plants of the genus houttrynia cordata thunb,has good qingrejiedu, abscess carbuncle effect. Volatile oil from Houttuyniae Herba(HHO) is the main effective fraction. Early studies show that the HHO can significantly inhibit the growth of A549 cell reproduction.In this paper,we studied on HHO ’s antitumor effect in lung cancer both in vivo and in vitro,to provide reliable scientific basis for the development and clinical application of HHO as a new kind of antineoplastic drugs for lung cancer.1. Study on apoptosis of A549 cells induced by HHOWe observed the inhibition rate induced by HHO on A549 cells by MTT method, the cell cycle change by single PI staining method, the apoptosis rate by FITC Annerxin V/PI double staining method, the cell morphology by optical microscope. The MTT results show that HHO has obvious inhibitory effect and have time and concentration dependence on A549 cells. Differerent concentration(0.25, 0.3125, 0.375, 0.3125, 0.375 μL/m L) of HHO induced A549 cells.The 24 h inhibition rate was 73.01%, and the IC50 was 0.3987μL/m L;The48 h inhibition rate was 84.99%, and the IC50 was 0.3498μL/m L;The 72 h inhibition rate was 94.01%, and the IC50 was 0.2729μL/m L.The difference between groups was statistically significant(P < 0.05). The cell cycle results showed that with the increase of drug concentration, proportion of G1 phase cells gradually reduced, G2 phase and S phase cell proportion increased gradually, and the differences between groups with statistical significance(P < 0.05). Apoptosis detection results showed that the cell apoptosis rate after 48 h were(12.63 ±1.27)%,(17.23±1.44)%,(48.50±2.71)%,(84.97±2.64)%,(76.15 ± 2.38)%,(52.66±2.04)% and(49.59 ±1.45)%, and the necrosis rate was(2.74±0.58)%,(2.52±1.08)%,(6.04±0.78)%,(2.79±1.57)%,(19.62±2.11)%,(40.16±2.89)% and( 41.24±1.68)%, when A549 cells induced by HHO with the concentrations of 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35μL/m L.Different concentrations of HHO induced A549 cells after 48 h, we can observe visible membranes shrivel and vesicular,cells turn smaller and round and the decrease in the number by optical microscope. The results show that HHO can induce A549 programmed cell death, and blocked the A549 cells in G2 phase and S phase, prompt the mechanism of HHO induced apoptosis of A549 cells may be related to blocking the RNA and protein of G2 phase and DNA of S phase.2.Preparetion of HHO-Hydroxypropyl-β-cyclodextrin inclusion compoundThe HHO-Hydroxypropyl-β-cyclodextrin inclusion compound was prepared by aqueous solution mixing method. The orthogonal tests designed by inclusion rate and drug loadings, the final optimum formulation for: HHO with HPCD proportion in 25(m L/g), HPCD and water ratio of 1:2(g/m L), inclusion temperature 50 ℃, stirring speed 420 r/min, the closure time of 80 min.The compound prepared by this prescription has uniform color, loose powder, good solubility, high stability, the inclusion rate of 77.35% and the drug was 4.48%.Experements of thin layer chromatography(TLC), gas chromatography, infrared spectrum(IR) and differential scanning calorimetry(DSC) proved that the formation of inclusion compound. Using TLC and GC, we found that HHO inclused before and after did not change its chemical composition.Using IR and DSC, we found that volatile oil inclused by HPCD is not simple mixed, but generates new substances, changing the original phase of both HHO and HPCD.3.Study on the pharmacokinetics and tissue distribution of HHO and HHO-HPCD in vivo and tissue distribution of HHO.This expriment used the GC method to detect blood drug concentration of HHO in vivo, when drawing the plasma concentration-time curves, small animal in vivo imaging method was applied to detect tissue distribution of HHO in vivo.HHO was lavaged with the dose of 240 mg/kg and tail intravenous injectied with the dose of 160 mg/kg,the HHO-HPCD inclusion compound was lavaged with the equivalent of HHO dose of 240 mg/kg and tail intravenous injectied with the equivalent of HHO dose of 160 mg/kg.The results showed that the 2-Undecanone can be detected in the plasma in 5min after HHO be lavaged, around 60 min to the absorption peak 5.0438μg/m L, HHO can be quickly absorpted into the bloodstream after intravenous injection, absorption peak was 4.2947μg/m L,the 2-Undecanone can be detected in the plasma in 5min after HHO-HPCD inclusion compound be lavaged, around 30 min to the absorption peak 4.6508μg/m L,the absorption peak was 3.4805μg/m L after the compound be absorpted into the bloodstream, showing that HHO can be quickly absorbed in the bloodstream and reach the peak blood drug concentration.The time to the peak and the residence time in the bloodstream of HHO-HPCD inclusion compound were shoter than that of HHO, explaining that the HHO-HPCD inclusion compound has the function which accelerates the HHO release.In vivo imaging results show that HHO can be quickly absorbed to the whole body after lavaged, increasing to its peak in 4 hour,and enriched more in the liver and kidney. HHO can be quickly absorbed into the blood and distributed of the entire body after tail vein injected,we can observe the enrichment of drugs in the trachea after 5 minutes, the enrichment of drugs in the lungs after 1 hour, a small amount of drug concentrated in the bladder, the enrichment of drugs in the liver and kidney in 2~4 hours and eventually excreted.The tissue imaging results show that the drugs mostly enriched in the liver, kidneys, lungs and trachea after lavaged for 12 hours, the heart, liver, spleen, lung, kidney and trachea were enriched after tail vein injected for 12 hours, suggesting that HHO was excreted by the liver and kidney and has the lungs and trachea targeting property.4. Study on anti lung cancer pharmacodynamics of HHOBuild a tumor-burdened lewis lung cancer tumor model and divided into 5 groups: negative control group, HHO 80mg/kg group, HHO 160mg/kg group, HHO 240mg/kg and cisplatin 1 mg/kg group. We observed the mice life state during treatment, drawed a tumor-burdened mice survival curves and detected the tumor inhibitory rate. The cell morphologic changes of the tumor tissue were observed by hematoxylin and eosin(HE) staining.It showed that the inhibitory rate of HHO80mg/kg group, HHO160mg/kg, HHO240mg/kg group were36.96%, 45.37% and 52.65% respectively. Compared with negative groups, HHO has obviously inhibited the lung cancer cell growth with dose and time dependent.Compared with cisplatin group and negative group tumour biopsies, the HE staining slice of tumor showed that HHO could kill cancer cells, significantly increase the clearance between cells and edge fiber connective tissue around and suppress the growth of lung cancer. Each groups of gut HE staining slice in mice showed that HHO has certain toxicity on the liver, increasing the clearance between cells and edge fiber connective tissue around. Low concentration of HHO has not obvious toxicity in heart, spleen, lung and kidney, but when the concentration increased, the HHO showed a certain toxicity in spleen and kidney. Cisplatin has obvious toxicity both in liver and kidney.The results showed that HHO could induce lung cancer cell apoptosis and inhibit tumor growth and has less side effects. With HPCD inclusion, it can significantly increase the solubility and stability of HHO, and make HHO quick release. If the inclusion compound were nebulized, control the appropriate size,it should reach the lung lesions directly to avoid the first effect and improve the bioavailability of HHO. Therefore, HHO has a very good application prospect to develop into the lung cancer drugs. |
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