The Anti-tumor Activity Of The Effective Ingredient From Ganoderma Lucidu Used Simultaneously And The Microemulsion Containing Them

Ganoderma lucidum has been used in the Chinese Medicine in the treatment of cancer for thousands of years.As we all known,the main effective ingredients of the fruits of Ganoderma lucidum are polysaccharides and triterpenoids.Maybe when polysaccharides and triterpenoids were used simultaneously,they can have better anti-tumor activity.So we study it first. Then we study the physico-chemical property and the characters of its absorption and metabolism with a four-site perfused rate intestinal modell. Purpose of this study was to offer a basement for new dosage forms.At last, microemulsion was chosen for the dosage of polysaccharides and triterpenoids.1 Firstly,we study the contribution to anti-tumor activity when polysaccharides and triterpenoids from the fruits of Ganoderma lucidum were used simultaneously,as the following two parts.（1）MTT assay was used to investigate the influence of polysaccharides, triterpenoids,and the mixture of them on antiproliferative effect of BEL-7402 and SPC-A-1 cancer cells in vitro.The results indicated that triterpenoids can inhibit the two cancer cells strongly,the IC₅₀ was 197.84μg crude drug·mL^-1 and 586.12μg crude drug·mL^-1 respectively,and the neutral part played more important role than the acidic part.Polysaccharides have no antiproliferative activity in vitro,and even can enhance the proliferative activity at certain concentration,so when polysaccharides and triterpenoids were used simultaneously in vitro,the antiproliferative activity was decreased,the IC₅₀ was 389.60μg crude drug·mL^-1 and 1253.85μg crude drug·mL^-1 respectively.（2）The mouse model bearing tumor was constructed by injecting subcontaneously Heps tumor cells to study the anti-tumor activity of the mixture of triterpenoids and polysaccharides in vivo.The results showed that polysaccharides and triterpenoids can inhibit the growth of tumor in mouse model significant.When given polysaccharides and triterpenoids at the concentration of 14 g crude drug·kg^-1·d^-1 respectively,they can inhibit the growth of tumor at 42.09%and 27.56%.And when given the mixture of polysaccharides and triterpenoids,it can inhibit the growth of tumor better. Spleen index and thymus index were 7.45±1.30、3.120±0.85 when useing polysaccharides and triterpenoids simultaneously,increased significant（P＜0.05） than that used singley.So,when polysaccharides and triterpenoids were used together,they have better anti-tumor activity.2.Study on condition for extraction and purification of triterpenoids and polysaccharides from fruits of Ganoderma lucidum.（1）The optimal extraction conditions of triterpenoids using supercritical fluid extraction were pressure 25 MPa,temperature 45℃,extraction time 1.5h, using ethanol as modifier carrier at the volume of 3 mL·g^-1.（2） The triterpenoids were then purified into acidic part and neutral part. The content of total triterpenoids was 315.00 mg·g^-1 extract,and the acidity part 110.62 mg·g^-1 extract,the neutral part 535.29 mg·g^-1 extract.The HPLC chromatograms revealed that the acidic part was distributed between 0～80 min, and the neutral part was distributed after 80 min.（3）The polysaccharides were extracted from the remainder of the fruits of Ganoderma lucidum extracted using supercritical fluid extraction with water at 100℃for 3h,15 folds water,and extracted two times.And then purified with 75%ethanol.3.Study on triterpenoids and polysaccharides for designing suitable dosage formThe apparent aqueous solubility of triterpenoids and polysaccharides was determined.And the apparent oil-water partition coefficients of triterpenoids were studied also.The results indicated that polysaccharides had good solubility in water,and triterpenoids had low solubility in water or PBS.The study about apparent oil-water partition coefficients of triterpenoids indicated that the neutral part had better absorption than the acidic part.And the rat intestinal model was used to study the absorption of triterpenoids.The neutral part had high rate of abscis.The apparent coefficient of permeability of ganoderic acid B in the acidic part was 0.38±0.15,0.60±0.12,4.62±0.85, 2.90±0.70 in duodenum,jejunum,ileum and colon respectively.4.Combination using single-factor and orthogonal experiment to screen microemulsion prescription containing triterpenoids and polysaccharidesUsing particle size as the main standard,through the single factor and orthogonal experimental,we screen the microemulsion containing triterpenoids and polysaccharides.The best prescription were OP 60 mg,Tween-20 300mg, isopropyl-alcohol 650 mg,oleic acid 60 mg,triterpenoids 40 mg, polysaccharides 56 mg,water 1 mL.5.To establish the quality control standards of microemulsion containing triterpenoids and polysaccharidesThe grain size of microemulsion was 32.43 nm,and the Zeta potential was 4.63 mV.We determined the contents of ganoderic acid B and ergosterol by HPLC.Their content in three batches of microemulsion was:0.974,0.965, 0.975 mg·mL^-1 and 1.691,1.696,1.694 mg·mL^-1 respectively.We also inspected stability of the microemulsion which was prepared in constant temperature.The result showed that the microemulsion was stability for one month in constant temperature.6.Pharmacodynamic studies of microemulsion containing triterpenoids and polysaccharidesHeps tumor-mice was chosen as the experimental animal model to study the anti-tumor activity of the microemulsion containing triterpenoids and polysaccharides.The results shown that microemulsion prescriptions can inhibit tumor growth in Heps tumor-mice better,it can inhibit the growth of tumor at 40.16%at the concentration of 14 g crude drug·kg^-1·d^-1 and has significant with that undealed with any prescription（P＜0.05）,the spleen index and thymus index were 7.61±1.82 and 3.52±0.89 respectively,which indicates than microemulsion has no damage to the immunity of organism.