Study On The Preparation Process、Quality Control And Antitumor Effect Of Neutral Triterpenoid From Ganoderma Lucidum

Objective To optimize the extraction and purification technology of neutral triterpenes from Ganoderma lucidum(NTG). To establish the quality control standard of NTG. To evaluate the anti-tumor effect of NTG in vivo and in vitro.To study the security of NTG and lay the foundation for the research of Ganoderma lucidum triterpenes anti-tumor drugs.Methods1. The extraction and purification of NTG(1) Single factor and orthogonal tests was used to optimize the extraction and purification technology of Ganoderma lucidum triterpenes.(2) AB-8 macroporous resin was used to gathering and purification of NTG.2. Primary study on the quality control of NTG(1) The determination method of Ganoderma lucidum triterpenoid was established by UV-vis method.(2) The fingerprint of NTG was established through RP-HPLC technology.3. Research on the antitumor effect of NTG(1) The cytotoxicity of NTGs which preparation by two different methods(solvent method and resin method) to K562 and HL60 cell was evaluated by MTT assay in vitro.(2) The solvent-melting method was developed to produce the solid dispersions of NTG.(3) Establishing solid tumor and ascites tumor models of H22 and S180, observing the effect of NTG on the inhibition rate of solid tumor and the life extension rate of ascites tumor, to evaluate the antitumor activity of NTG in vivo.4. The general pharmacolog study of the NTGOpen field method, metal stick method and tilting plate method were used to examining the locomotor activity and point coordinated function of mice; recording the sleep rate and sleep time of mice to describ NTG with different dose of sodium pentobarbital induced hypnosis; the blood pressure, respiratory rate and heart rate of anesthetized rat were recorded by physiological function recorder.Results1. The extraction and purification of NTG(1) The optimal extractive process was that extracting three times using alcohol(95%) with the ratio of 1:6(m/v) for 1.5 h.(2) The condition of using AB-8 macroporous resin to enrichment and purification NTG is that adsorption capacity was 4ml Ganoderic triterpenes(1 ml is equivalent to 0.35 g crude drugs) per milliliter resin under volume flow at 1 BV/h, the optimum elution condition was decided as four times resin column volumes’ eluent as follows: saturated Na HCO3 solution, water, 60% and 95% ethanol, and with the velocity of flow 2 BV/h, then collected 95% ethanol elution fluid, vacuum drying.2. Primary study on the quality control of NTG(1) The determination method of Ganoderic lucidum triterpenoid was established by UV-vis method.(2) The separation effect of NTG’s fingerprint was very well, and the fingerprint contained 11 strong absorption and feature obvious common peaks.3. Research on the antitumor effect of NTG(1) It display inhibitory activity on K562 and HL60 cancer cell of the two kinds of NTG, the resin method and solvent method’s IC50 values to K562 for 48 h were120.36 μg/ml and 149.3 μg/ml, 30.63 μg/ml and 24.49 μg/ml were belong to HL60.(2) The best preparation method of NTG solid dispersions was that: F68 and 44/14 as the carrier(1:1, g/g), and the proportion of carrier and drug was 8:1.(3) The three experimental group of NTG solid dispersions(resin method) were significantly inhibit the growth of H22 and S180’s solid tumor(P<0.05), the inhibition rate of 500 mg/kg group were 55.73% and 53.83%; NTG soliddispersions of solvent method can significantly inhibit the growth of H22 and S180’s solid tumor(P<0.05), but the inhibitory rate of its 500 mg/kg dose were21.22% and 31.61%; the none solid dispersions’ NTG also can significantly inhibit the growth of H22 and S180’s solid tumor(P<0.05), but the inhibitory rate of its500 mg/kg dose were 28.28% and 23.31%; the three experimental group of NTG solid dispersions(resin method) could prolong H22 and S180 ascites tumor-mice’s life, the middle and high dose group can significantly improve the life extension rate(P<0.05), the 500 mg/kg group’s life extension rate were 35.4% and 43.4%;NTG solid dispersions of solvent method can significantly prolong H22 and S180 ascites tumor-mice’s life(P<0.05), but the ife extension rate of its 500 mg/kg dose were 27.1% and 32.8%; the none solid dispersions’ NTG also can significantly prolong H22 and S180 ascites tumor-mice’s life(P<0.05), but the ife extension rate of its 500 mg/kg dose were just 15.1% and 20.5%.4. The general pharmacolog study of the NTGNTG solid dispersions(250~1000 mg/kg)made no effect on locomotor activity,coordinated function and mice’s sleep; NTG solid dispersions(125~500 mg/kg)had no influence on respiratory and cardiovascular system of the anesthetized rat.Conclusion1. The extraction and purification of NTG(1) The optimal extracting method was stabilized, which could be used for the extraction of Ganoderma lucidum triterpenes.(2) There were many advantages by using AB-8 macroporous resin, which could be used for the purification and enrichment of NTG.2. Primary study on the quality control of NTG(1) The UV method could be used as NTG’s quantitative control method in the quality control.(2) The fingerprint of NTG which constructed by RP-HPLC could be operated and repeated easily, this method could be used in NTG’s quality controlling.3. Research on the antitumor effect of NTG(1)It revealled significant inhibition activity on K562 and HL60 cancer cell of thetwo kinds NTG with an obvious dose-response relationship, and the two kinds of NTG which were made by different preparation methods has no obvious difference on the inhibition of tumor cells in vitro.(2) NTG solid dispersions can improve the dispersion and water-soluble of triterpenes, the optimized method can be used for the preparation of NTG solid dispersions.(3) NTG solid dispersions hold strong activity on S180 and H22, both solid tumor and ascites tumor; accoding to the results, solid dispersions could improve NTG’s bioavailability; the NTG made by resin method had higher activity in vivo than the solvent method NTG under the same dose.4. The general pharmacolog study of the NTGNTG solid dispersions has no obvious effect on nervous system,respiratory-system and cardiovascular-system of the experimental animal, it is suggested that NTG has higher safety and reliability within effective range.