The Preliminary Study Of Formononetin Used In The Treatment Of Non-small Cell Lung Cancer

Background: In2005, MD Anderson Cancer Center did a large sampleretrospective analysis, and found that eating foods rich in phytoestrogens cansignificantly reduce the risk of lung cancer, especially for men and non-smokers.However, due to the existence of recall bias and the retrospective study of defects, thesurvey was not perfect. In2010, Japan Public Health Center in a11-year-longprospective study of36,177male and40,484female, found that, for malenon-smokers, the phytoestrogen content in the diet is highly negatively correlatedwith the risk of lung cancer. And for female smokers, compared with weak negativecorrelation. In2011, Japan JPHC Study Group in a prospective study of24,127women found that the plasma concentration of genistein and female lung cancerincidence were negatively correlated. The above researches suggest a closerelationship between phytoestrogens and lung cancer. And the research ofphytoestrogens used in lung cancer is an important direction. Red clover isoflavonesis a plant estrogen mixture with a better anti-cancer effect, and includingformononetin, biochanin a, genistein, daidzein. In North America and Europe, it haslisted as a health care. To develop that used in the treatment of lung cancer, we mustclarify its monomer mechanism of action on lung cancer. However the separation andpurification of the monomer in red clover isoflavones is extremely difficult. Therefore,the separation and purification of Formononetin is the basis.Objective: Study the separation and purification method of phytoestrogens inred clover. Research Formononetin in serum pharmacology, and provide a theoretical basis in drug formulations choose; Used formononetin and formononetin incombination with cisplatin in the A549lung adenocarcinoma cell line, and observedthe inhibitory effect.Methods: Weighs2.5g of red clover isoflavones, soluble in methanol, add6gpolyamide powder (100mesh to200mesh) grinding mixed samples, dry. Weighspolyamide powder50g, add distilled water and stir until no bubbles, and put into thecolumn. Respectively with double distilled water and ethanol, leaching to noimpurities in the effluent by UV spectrophotometer. Adding samples, Using differentconcentrations of ethanol as the mobile phase with the flow rate of1ml/min. Puteffluent into bottles per30ml. Mix biochanin A solution which content>80%, andevaporate to dryness. Selection of the12weeks of clean grade SD rats24, weighingbetween200-250g, and randomly divide into six groups, fasting12hours. Gavage torats with Formononetin suspension (Formononetin480mg, ethanol6ml,1,2propanediol6ml, Twain-806ml, distilled water42ml) according to the standard of1ml/100g. The six groups of rats were blood through the aorta in1h,2h,3h,4h,5h,6h after the gavage, and each rat blood about5ml. After15min centrifugation ofblood in the speed of3000r/min, take the serum. Each serum plus two times volumeethyl acetate, set in warm water ultrasonically for15min extraction, and stand forseparation used the separatory funnel. Extract three times for each serum, mix ethylacetate solution, evaporated to dryness, using HPLC analysis. Cultured A549humanlung adenocarcinoma cell line with RPMI1640medium containing10%fetal serum,inoculated in100ml flasks, culture conditions of37°C,5%CO2. Cover96-wellplates with A549cell in trypsin digestion, add four groups of medium(Formononetinmedium group, cisplatin medium group, Formononetin cisplatin mixed medium groupand the control group) after24h, each group plus ten holes. Detect the absorbanceafter48h using the MTT method. The control group inhibition rate is0, and calculateinhibition rate of each group.Results: Finally purify88.7%Biochanin A0.05g,76%Biochanin A0.12g,71.52%Formononetin0.04g; Formononetin appeared in the serum, and the crest had a little rise over time, which suggested that Formononetin can be directly absorbedinto the blood. Formononetin crest is higher than genistein in Formononetinsuspension, but the difference between Formononetin crest and genistein crest issmall, which suggested the bioavailability of Formononetin may be relatively low,but the mechanism needs further study. The inhibition rate of Formononetin groupwas (20.4±0.67)%, cisplatin group was (49.7±1.23)%, the mixed drug group was(69.4±2.3)%.Conclusions: The polyamide powder (column chromatography,100-200mesh)has better separation and purification effect of red clover four isoflavones; The rat oralFormononetin can be directly absorbed into the bloodstream; But the bioavailabilitymay be lower, and the exact mechanism needs further study. The formononetin has abetter inhibition in A549human lung adenocarcinoma cell line, and has a synergisticinhibitory effect with cisplatin.