Study On Anti-colon Cancer Pharmacodynamics Of Herba Houttuyniae Volatile Oil And Preparation Of Its Thermosensitive In Situ Gel For Intestines

Colon cancer is a common malignant tumor of the digestive tract.The incidence rate ranks third among the nine major malignant tumors.It is located after lung cancer and gastric cancer.In China,along with the development of social economy,people’s life is getting richer and richer.The number of colon cancer patients is increasing year by year.The incidence of the disease in Europe,North America and Oceania is 5 times-10 times that of Africa,southern Asia and the middle of the United States.From the perspective of current medical technology,radiotherapy and chemotherapy are still the main way to treat colon cancer,but because the cells are highly resistant to drugs,and most of them have certain toxicity and side effects,the result of chemotherapy is not ideal.In recent years,some anti-tumor drugs have been discovered and become a hot spot of research.Chinese medicine can not only enhance efficacy and reduce toxicity,enhance body resistance,but also speed up postoperative recovery.It can not only regulate the body,but also increase the effect of radiotherapy and chemotherapy,and improve the quality of life of patients.Therefore,it is necessary to develop a highly effective and low toxic Chinese medicine to treatment colon cancer.Herba Houttuyniae is the fresh grass of Saururaceae Houttuynia cordata Thunb.and a traditional Chinese medicine that is both medicinal and edible.It is used for allergy,cough,cancer and virus infection.Among them,Herba Houttuyniae volatile oil（HHO）is one of the effective sites for anti-tumor.Our previous research shows that HHO could inhibit the proliferation of colon cancer SW480 cells.Based on this,this article on the HHO induced SW480 cell apoptosis and HHO anti-colon cancer in vivo pharmacodynamics,preparation and characterization of HHO-HPCD-Gel,HHO and HHO-HPCD-Gel pharmacokinetics of four aspects research,and it provides a reference for the development of anti-colon cancer new drugs by HHO.1.The study of HHO induced apoptosis in SW480 colon cancer cellsThe inhibition rate of cell growth was detected by MTT.The cells morphological changes of HHO after SW480 treatment were observed under optical microscope.AO/EB double fluorescent staining method was used to determine the cell metabolic activity.PI single staining and Annexin FITC/PI double staining were used to detect cell cycle and apoptosis.The effect of HHO on SW480 cells was investigated from the above five aspects.The results showed that when HHO concentration was 0.05μL/mL-0.45μL/mL,HHO showed significant time and concentration dependence on SW480cells.When the concentration of HHO was 0.45μL/mL,compared with the negative control group,the inhibition rates of 24 h,48 h and 72 h could reach 77.99%,89.31%and 92.70%respectively.The IC₅₀ values at 3 times points were 0.29±0.10μL/mL,0.24±0.06μL/mL,0.19±0.02μL/mL.After SW480 cells were treated with different concentrations of HHO,the cells were gradually smaller and shrunk,and fell off the bottle wall.After AO/EB staining,the living cells were dyed green,and the dead cells were dyed red.With the increase of HHO concentration,the number of dyed red cells was more and more,the number of G1 phase cells was more and more,S phase was less and the number of G2 phase cells did not change significantly,the total apoptosis rate was gradually increasing and there was statistical significance among all groups.2.Study on the pharmacodynamics of HHO in anti-colon cancer40 BALB/c-nu mice were randomly divided into 5 groups:negative control group,hydroxycamptothecine group,HHO 240 mg/kg group,HHO 160 mg/kg group and HHO 80 mg/kg group.The way of intraperitoneal injection was given.Dosing once at the same time every day for 10 days,and the mice were weighed every day to measure the tumor and calculate the tumor suppressor rate.The five organs（liver,heart,spleen,lung,kidney）of the mice were dissected and pathologically sectioned and photographed.The experimental results showed that compared with the negative control group,the tumor inhibition rates of hydroxycamptothecine group and HHO high,medium and low concentration group were 72.14%,52.86%,40.71%,and 30.00%,P<0.01,with statistical significance.Tumor tissue biopsy showed that compared with the negative control group,the cells in the drug group were loosely arranged and the cell outline was not clear,showing obvious apoptosis status.The viscera tissue section showed that compared with the negative control group,the HHO group and the hydroxycamptothecin group increased the interstitial space of the liver and kidney,increased the connective tissue and produced the lesions,and the renal toxicity of hydroxylcamptothexine was more obvious than HHO.3.Preparation and characterization of HHO-HPCD-GelHHO-HPCD-Gel was prepared by using cold dissolution method with the gel matrix of the poloxamer 407 and poloxamer 188.Using central composite design response surface methodology（CCD-RSM）and the phase transition temperature(T_Gel)as the inspection index,the content of P407 and P188 was fitted with binomial fitting,and the CCD-RSM method was used to optimize and verify the prescription.The dissolution rate of HHO-HPCD-Gel was measured by membrane dissolving method.Gas chromatography（GC）was used to determine the content of 2-Undecanone in HHO as the index.The appearance and characteristics of gel were investigated by drug stability test,and the content of pH and its content were determined.The optimized HHO-HPCD-Gel prescription:the content of P407 was 20.61%,and the content of P188 was 3.03%,PH was 6.93,HHO concentration 86.72μg/mL.The results of in vitro dissolution showed that the dissolution of HHO-HPCD-Gel was higher than that of HHO-Gel at 30 min-150 min,and the cumulative erosion rates of HHO-HPCD-Gel and HHO-Gel tended to be the same between 150 min-210 min.4.Study on pharmacokinetics of HHO and HHO-HPCD-GelThe accuracy of the method was verified by the experiments of precision,stability and repeatability.HHO was administered by 240 mg/kg rectum in rats.The same dose of HHO-HPCD-Gel was also administered in the same way,so that HHO entered the blood circulation through the middle,inferior veins and anus veins of rectum.After 12 times of administration,blood was collected from the orbit of rats,combined with GC,using 2-Undecanone as index ingredient.Excel draw a drug-time curve,calculation of HHO and HHO-HPCD-Gel pharmacokinetic parameters in rats the medicine with DAS2.0 software.The results of the methodological test show that the method is feasible.Both HHO and HHO-HPCD-Gel can detect 2-Undecanone in the blood after the administration of 5 min.After the HHO-HPCD-Gel was given to the rectum,the half-life was 2.46 times longer than that of HHO.The above four parts showed that HHO could inhibit the proliferation and induce apoptosis of SW480 colon cancer cells in vivo and in vitro,and HHO-HPCD-Gel had a sustained release effect.