| Part one Study on the chemical constituents of dichloromethane extractions of Ailanthus altissima SwingleAilanthus altissima (Mill.) Swingle (also known as AILANTHUS tree) belongs to simaroubaceae and deciduous trees of the genus. Ailanthus altissima is named due to an odd odor send from bursting oil glands. It is widely distributed in all parts of China except Heilongjiang, Jilin and Hainan Provinces. In the 18th century, as an ornamental and medicinal plant it was introduced to North America and Europe, up to now, Ailanthus altissima has been widely distributed in Europe, as well as each state of the United State, and now it has become one of the seven control species. Ailanthus altissima bark, the dried bark or root barks, is a popular herb with the function of reducing fever, eliminating dampness, Haemostatic, anti-inflammatory, insecticides and other effects. It can be used for treatment of uterine bleeding, getting rid of phlemg-fire retention in the lung and stomach, enteritis, spleen, gonorrhea, dysentery and liver diseases such as inflammation of the urethra and bladder. Recent researches have shown that the bark extract contains chemical constituents against active anti-amoebas, therefore it can be used as insecticide, and it also has significant anti-cancer effects and can be used to treat colon cancer, cervical cancer and colorectal cancer and so on, which also has an anti-African lymphoma cell tumor viruses and anti-TB effect.The main chemical constituents of Ailanthus altissima bark are quassioides, alkaloids, volatile components, terpenes,etc. In while the most eye-catching constituent is the quassioides that is made up of tetracyclic diterpene lactone and five rings diterpene lactone, which is the characteristic constituent in the family Simaroubaceae. Genus Ailanthus contains the main constituents of quassioides ingredients, and is most widely studied medicinal plants. In this research, the chemical constituents of dichloromethane extraction of Ailanthus altissima bark has been studied.Objective:To select Ailanthus altissima bark as research object, to conduct systematic research on its chemical components (especially quassioides) by using various separation means, and to analyze and identify the plane structure and stereo structure of the obtained compounds by using spectroscopic methods, including:13C-NMR,’H-NMR, HMQC, COSY, HMBC, NOESY and so on, therefore to pave the way to find new lead compounds for drug development.Methods:50 kg of dry bark of Ailanthus altissima (Mill.)Swingle was extracted by thermal 95% ethanol to get the samples by concentration. The extracted sample in a saturated solution of sodium chloride was dispersed to extract the solution respectively with petroleum ether, dichloromethane, ethyl acetate and n-butanol in order to get the respective fraction, of which the dichloromethane fraction is 260 g. The chemical constituents in the dichloromethane fraction were isolated and purified by silica gel chromatography, sephadex gel preparation, preparative thin-layer chromatography and high performance liquid chromatography, The chemical results of the obtained compounds were identified by using modern analytical methods, such as 13C-NMR,’H-NMR, HMQC, COSY, HMBC and NOESYResults:Nine compounds have been isolated and identified in the bark of Ailanthus altissima Swingle as follows:Vanillin (1), stigmast-4-en-3-one (2), isoailanthone (3), shinjudilactone (4), ailanthone (5), shinjulactone B (6), 6a-tigloyloxychaparrin(7),2-dihydroailanthone (8),12-dihydroisoailanthone (9).Part two Antitumor active constituents in the dichloromethane extractions and compounds of Ailanthus altissima barkAilanthus altissima bark, as traditional Chinese medicine, was first documented in the Tang Materia Medica. In ancient China, Ailanthus bark referred to the dry bark of toona sinensis or Ailanthus alltissima as traditional Chinese medicines in which the bark of Ailanthus alltissima was the best. All dynasties in ancient China had records of the bark used medicinally:the Dietetic Materia Medica records its effects of curing diarrhoea and killing belly worms; the Activity on the Tree records its effects to treat dysentery, intestinal smooth skin, hemorrhoids spilled blood; Effective Prescriptions for Diseases of Women records a recipe of frying roasted Ailanthus altissima, the pepper, the spring onion to steam to treat postpartum hysteroptosis; frying with honey water to treat hemorrhoids, frying the bark water singly to treat sores. Modern pharmacological research found that, besides the above mentioned traditional effect, Ailanthus bark also has anti-tumor, anti-TB bacteria, anti-EB virus, anti-Amoeba, and anti-malaria role, especially anti-tumor role. The chemical constituents, quassioides, have the effects to treat colon cancer, cervical cancer, and rectal cancer, which becomes the focus of current pharmacological research. This research will study the antitumor activity of the bark on the basis of separation, purification and preparation of compounds in Ailanthus bark with the research principles "focus on application-oriented development".Objective:The extractions and the isolations, purification and preparation compounds from Ailanthus altissima bark were selected as the object of study to systematically screen antitumor activity by taking advantage of modern cell screening technology, with the indicator of tumor cell proliferation inhibition rate of the components, and study the antitumor mechanism by ailanthone and 2-dihydroailanthone.Methods:1 The antitumor activity of different extraction parts and compounds were determined by MTT assay. The experiment was divided into blank control group, DMSO solvent control group and the test drug group (the concentrations of the extracts were 1 μg/mL,10 μg/mL and 100 μg/mL respectively; the concentrations of the compound were 0.01 μg/mL,0.1 μg/mL, 1 μg/mL,10 μg/mL and 100 μg/mL respectively).2 The proliferate inhibition rate, the apoptosis, cell cycle of MCF-7 and U-251 were studied, Bax and Bcl-2 mRNAd were detected by RT-PCR and the Bax and Bcl-2 in MCF-7 and U-251 cells protein were detected by Western blot assay, in different concentrations of drugs.Results:1 The different fractions and compounds from dichloromethane extract parts acting on 13 tumor cells were tested and the results showed different antitumor activities, and compounds 2 and 4 were generally weak in high, moderate and low concentrations for 13 tumor cell lines.The IC50 values of the compound 1,3,5, and 6 were tested and the results showed that the IC50 in the four compounds to different tumor cells were quite different, the measurement data of IC50 on 13 kinds of tumor cells showed that the IC50 of compound 5,6 to 13 tumor cells were lower to the positive drug (Cisplatin), which implied that the inhibition effects to 13 tumor cells was superior to the positive control drug cisplatin; IC50 of compound 3 on U251, Hela, AsPC-1, HCT116, AGS, SMMC-7721, U-2OS, CCRF-CEM cells were less than positive control drug cisplatin. IC50 of compound 1 to U251, Hela, HCT116, AGS, SMMC-7721, U-2OS, CCRF-CEM cells were less than positive control drug cisplatin. Evaluation of results:When the IC50 of compounds was IC50<10 μg/mL or the plant extracts was IC50<20 μg/mL, the samples were determined in vitro tumor cell killing effect.2 Different concentrations of ailanthone and 2-dihydroailanthone worked for different time on MCF-7 and U251 cells, the survival rates of MCF-7 and U251 cell gradually decreased respectively, which indicated that the drugs significantly inhibited the growth of tumor cells, but also presented a time and concentration dependen.Ailanthone and 2-dihydroailanthone in different concentrations on MCF-7 and U251 cells apoptosis were detected with Flow cytometry for 48h, the results showed that the cell apoptosis rates were significantly higher than those in the control group, the difference was statistically significant (p<0.05), and the apoptosis rate significantly increased with drug concentration increasing. Ailanthone and 2-dihydroailanthone in different concentrations on cell cycle of tumor cell were detected with Flow cytometry for 48h, the results showed that the cell percentage of G0/G1 phase increased gradually, but the cell percentage of S phase and G2/M phase gradually decreased respectively, which indicated that cell block occurred in GO/G1 phase.Different concentrations of ailanthone and 2-dihydroailanthone on MCF-7 and U251 cells for 48h, the percentages Bax/gapdh increased gradually, but Bcl-2/gapdh gradually decreased respectively, the results indicated that the expression of Bax mRNA increased with the increase of drug concentration, the expression of Bcl-2 mRNA decreased with the increase of drug concentration, which implied a dose-dependent manner, indicating that drugs could increase the expression of Bax mRNA, inhibit the expression of Bcl-2 mRNA and had the dose-effect relationship.The results of protein expression of Bax and Bcl-2 were detected by Western blotting. Different concentrations of ailanthone and 2-dihydroailanthone on MCF-7 and U251 cells for 48h, the proteins expression of Bax and Bcl-2 were detected, the percentages of bax/β-actin increased gradually, but bcl-2/β-actin gradually decreased respectively. Experimental results showed that different doses of the drugs could reduce the expression of Bcl-2, but the expression of Bax increased gradually compared with control group, which indicated a dose-dependent relationship.Part there The preparation technology of dispersible tablets and quality standard research on dichloromethane extractions of Ailanthus altissima barkThe dispersible tablets have many advantages, such as quick-dissolution and disintegration, high bioavailability, good stability, easy-taking and carrying. It can be taken orally directly or dissolved into the water, especially suits for the patients who have difficulty in swallowing and can increase the patients’ compliance because of the good taste. So it has attracted more and more attention of patients.Objective:To take the active ingredients extracting by methylene chloride in Cortex Ailanthi as the research object and render it various characteristics of modernized drug form by using modernized pharmaceutics technology and methods of traditional Chinese medicine. To prepare ailanthone extract dispersible tablets with quick release and good stability, mainly to study the factors influencing the process of preparation and the optimal process, and to evaluate the quality of the dispersible tablets. Through the above pharmaceutics research, to make the ailanthone extract which can hardly soluble in aqueous into dispersible tablets, and then to lay the foundation for widely use of Cortex Ailanthi in clinic.Methods:Ultraviolet spectrophotometry was adopted to establish the analysis method and determine the content of ailanthone extract in vitro, laying the foundation of prescription screening, process research, quality control and analysis in vitro of ailanthone extract dispersible tablets. By using the single factor test and orthogonal design method, setting 12-hours cumulative dissolution as the index, the prescription of ailanthone extract dispersible tablets was studied and the best preparation method was screened.Results:Based on the results of prescription craft research, the prescription of ailanthone extract dispersible tablets were determined, and the small sample was test. Disperse uniformity and content and all the inspection items under tablets met the requirements of quality standards. The quality standard was established reference in the relevant provisions of the dispersible tablets in Chinese pharmacopeia 2010 edition partes secunda, and methodology of quantitative determination and dissolution were studied.Quantitative determination measured by ultraviolet spectrophotometry. Methodology results showed that the absorbance and the concentration of ailanthone presented a good linear relationship in the concentration range of 1.5-30 μg/mL. The linear correlation coefficient is 0.9997. Reproducibility and stability were good. RSD of precision experiment was 0.15%, which conformed to the requirements of the precision. The average recovery rate was 99.02%.Conclusions:1 In this study, the systematic study was conducted on dichloromethane in the chemical constituents of the bark of Ailanthus altissima Swingle to isolate and identify 9 compounds. Conferring to the NMR data obtained in this study and literature review, NMR pectroscopic characteristics of quassioide have been summarized to provide a basis for the rapid identification of these compounds.2 The research focused on the chemical components’antitumor activity of the dichloromethane in all the effective parts and monomer compounds in ailanthus altissima bark, and it showed that all effective parts and monomer compounds basically have antitumor activity, even some compounds’activity exceeded positive control drug (Cisplatin), and the antitumor mechanism was studied by ailanthone and 2-dihydroailanthone with the cell proliferation inhibition, cell apoptosis, cell cycle, RT-PCR and Western blotting methods. The results showed that ailanthone had inhibitory effect on MCF-7 and U251 cells, and promote cell apoptosis with increased bax and its mRNA and decreased bcl-2 and its mRNA, which provides the guiding principles for separation, purification and further pharmacy research.3 Based on the relevant provisions of the Chinese pharmacopoeia about dispersible tablets preparation, the prescription and preparation have been studied with ailanthone extract as model drug, the preparation and evaluation of ailanthone extract dispersible tablets have been completed, a quality control method of ailanthone extract dispersible tablets has been established, and the methodology and stability have been researched as well. All the indexes meet the dispersible tablets preparation requirements of Chinese Pharmacopoeia 2010 edition. |
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