Translated Abstract
Malignant tumor has been one of the important reasons for threatening to human’s health, and the number of people who died of cancer has been increasing in all over the world. At present, many kinds of tumors have drug resistance to radiotherapy and chemotherapeutics. As a significant natural anti-tumor drug, gossypol is able to bind selectively with Bcl-2 proteins (resisiting apoptosis protein), and to inhibit their translation, which is responsible for the inhibition of tumor cells growth and the decrease of their drug resistance. Gossypol is promising to be a small molecular anti-cancer drug. However, gossypol has some obvious shortcomings such as higher cost, lower stability, water-insoluable character and noticeable toxcity. Therefore, it is very important to find out a new compound with prominent anti-tumor effects by preparing cheaper gossypol, separating its chiral isomer and modifying its structure.
In the present dissertation, a new process for recovering gossypol acetate from cottonseed oil stocks is established to decrease in its production cost. The process of separating chiral gossypol is studied for preparing (-)gossypol with high purity and bioactivity. The analysis and optimization of conditions for synthesizing small molecular Schiff’s base gossypol deriverate is carried out to raise its yield and to decline in its toxcity. The exploration of soluable-water gossypol prodrug promoted the biological availablity of gossypol and offered controlled release effect in vivo. The anti-tumor activity of gossypol and its derivarates is tested to find a novel anticancer drug inhibiting the growth of malignant tumor, strengthening the sensitivity of tumor cells to chemotherapeutics and lowering cytotoxicity. The main contents are as follows:
(1) Ultrasonic extration and crystallization are used to recycle gossypol acetate from cottonseed oil soapstocks. The single factor test and orthogonal test of extracting gossypol acetate from cottonseed oil soapstocks by ultrasound were carried out, and it is found that maximal recovery (1.243 g) about gossypol acetic acid from cottonseed oil soapstocks (100 g) is obtained just as ultrasonic extracting time 25 min, refluxing time 90 min, acid concentration 1.4 mol/L, ultrasonic crystallizating time 25 min. The chemical structure of gossypol acetic acid extracted by ultrasound is further confirmed by UV, IR and NMR spectra. MTT tests in vitro are shown that cheaper gossypol acetate sample can markedly inhibit the growth of prostatic carcinoma cell PC-3 and mammary cancer cell MDA-MB-231.
(2) (-)Gossypol from racemic gossypol is separated by derivatization, column chromatography, acid hydrolysis and ultrasonic purification. Then TLC, specific rotation, UV, IR and NMR are utilized for trailing the separating process of (-)gossypol. While many conditions including 40 min of derivatization, acetic ether as eluting solvent and ether as hydrolytic solvent are met, separated (-)gossypol and its chiral amine derivate have high optical purity and definite structure.
(3) A stable small molecular Schiff’s base gossypol (SDG) is synthesized by means of condensation reaction between Dimethylaminopropylamine (DMAPA) and gossypol. Three factors effecting on yield of SDG are studied and optimized. It is shown that highest yeild is made only to apply reasonable conditions i.e. DMAPA concentration 150 μmol/mL, react temperature 30 ℃ and react time 3 h. After IR, 1H-NMR and 13C-NMR analyzed, it is found that SDG is made according to the ratio of one to two between gossypol and DMAPA. SDG is able to inhibit prostatic tumor cells PC-3 and LNCaP distinguishedly according to the MTT test resutls.
Lastly, UV and IR spectra are used to investigate whether gossypol may react with gelatin, chitosan and PEG-(NH2)2 respectively. It is shown that PEG-(NH2)2 is the best water-soluble carrier of gossypol. The synthetic process of PEGGC is monitored by UV spectrum. Three significant factors effecting on the yield of PEGGC are reaction time, reaction temperature, molar ratio between gossypol and PEG-(NH2)2. And when they are 240 min, 40 ℃ and 2:1 respectively, the yield reachs peak (96.8%). The change in UV spectrum of PEGGC aqueous solution demostrates its stablity to acid or base and storage. It is found that PEGGC is sensitive to acid or base environment, and liable to release gossypol for the hydrolysis in acid solution and to transform for the oxidation in base solution. MTT assay shown that PEGGC from gossypol and LPEGGC from (-)gossypol have bioactivity to inhibit the growth of prostatic carcinoma cells.
Based on above studies, some conclusion could be drawn as follows:
(1) Ultrasonic extraction and ultrasonic inducing crystallization technic are introduced to recover gossypol acetate from cottonseed oil soapstocks (industrial residue). This technology has many advantages. Firstly, it makes the purity of gossypol high, the production cost low, production cycle short and anti-tumor activity kept. In addition, it can eliminate the risk in food and feeds safety from traditional process of preparing gossypol. More importantly, it resolves problem from enviorment pollution in processing cottonseed oil through turning waste to valuable drug.
(2) An improved process for separating (-)gossypol is established. Cheap chiral amine as resolution agent and prepared gossypol acetate as raw material are used to fast separate (-)gossypol with high purity by derivatization and ultrasonic crystallization. This process declines in production cost of (-)gossypol, increasing in its production efficiency and added value. Separated (-)gossypol can inhibit tumer growing in vivo, but it is not ignored that it is toxic to animal liver and intestinal tract. This is worth to be refered in clinical usage of (-)gossypol.
(3) Based on the mechanism of affinity-addition reaction, SDG is synthesized by DMAPA and gossypol extracted in this study. Compared with gossypol, it has the similar activity for inhibiting tumor and less toxicity. Unfortunately, SDG is unsoulable in water.
However, SDG has still the difficulty dissolving in water.
(4) Compared with gelatin and chitosan, PEG-(NH2)2 is the best water-soluable carrier of gossypol. PEGGC, a kind of gossypol derivate, which is synthesized by PEG-(NH2)2 and gossypol, is easy to dissolving in water. And the sensitivity of PEGGC to acid and base in water may be utilized to control the effective release of drug on the basis of therapeutic regimen. Neverthless, sealing and removeing oxygren are essential to avoid its oxydation during the long-time store. In comparision with racemic gossypol, PEGGC has slightly lower activity of anti-tumor in vitro, however, free gossypol is released due to the acid or enzymic hydrosis. Consequently, as a water-soluble gossypol predrug, PEGGC will be an novel promising anticancer drug in the future
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