| Hepatic Cell Carcinoma (HCC) is one of the common malignant tumors with a highmortality rate in our country, and the incidence of HCC was in an increasing trend inrecently years. Traditional Chinese Medicine (TCMs) has become an important part in thetreatment of HCC; TCMs have certain advantages in improving patient symptoms andimproving the survival rate, and have been growing attentions and concerns. Qinggansanjiedecoction was the primary anti-hepatoma prescription basic the long-term clinical practiceexperience against liver cancer in the department of integrative traditional Chinese andwestern medicine treatment, Eastern Hepatobiliary Surgery Hospital. It was formed byfourteen Chinese medical herbs including Actinidia valvata Dunn, Chinese Sage Herb,Herba Hedyotis and Herba Scutellariae Barbatae, etc, with the role of improving liverfunction, relieveing the patient’s symptoms, inhibition growth of hepatocellular carcinoma,resistance to tumor metastasis and recurrence, and improve survival of patientexpectations.TCMs were the main form in the treatment of desease, which was based on dialecticalideas, in accordance with the formula principle of monarch, minister, assistant and guide,choose the appropriate Chinese Herbal Pieces, and according to to a certain proportion ofprescription compatibility, was concrete manifestation of the theory of traditional Chinesemedicine. The effect of material basic research of TCMs was the role of Chinese medicinesand compound prescription, and the base and core of quality control to ensure the safe,effective and stable quality of TCMs and their preparations. In order to clarify the effectivein antitumor and control the quality of Qinggansanjie prescription and the preparation,hepatoma cell model of HepG2，SMMC-7721with fluorouracil as a positive control wereused to evaluate the effect in inhibiting the growth of liver cancer cells after oraladministration of Qinggansanjie extract, oleanolic acid, ursolic acid and the two mixtures.The IC50in HepG2cells were9.255mg/mL,7.53μg/mL (16.51μM),34.16μg/mL (74.91μM)and36.63μg/mL, respectively. The IC50in SMMC-7721cells were8.970mg/mL,15.11μg/mL (33.13μM),55.06μg/mL (120.7μM) and32.23μg/mL, respectively. The resultsshowed that the Qinggansanjie decoction has significant anti-cancer effect rather than thecompound of the single administration of oleanolic acid, ursolic acid and the two mixtures,oleanolic acid and ursolic acid were better than fluorouracil for treatment of HCC, thus itproved the effect of Qinggansanjie prescription in clinical applications with the strong experimental data in vitro.We established a constituent database of known chemical compositions been reportedand data in Chinese medicine ingredient database, including more than thousand ofcomponds in the herbs to compose Qinggansanjie prescription. With six times the amountof70%ethanol by ultrasonic extraction of the chemical composition in Qinggansanjieprescription and combined with LC-TOF/MS, the complex chemical composition in theextracts were qualitative analysis rapid with automatically and manually match to thedatabase in positive and negative mode. We found163chemical ingredients in the extractand its source of medicinal materials of belonging were interpreted. There were types oftriterpene acids, flavonoids and saponins with higher content relatively, mainly derivedfrom monarch herbs including Actinidia valvata Dunn, Chinese Sage Herb, HerbaHedyotis and Herba Scutellariae Barbatae, Radix Scutellariae and Chinese Thorowax Rootof assistant and Radix Glycyrrhizae of guide, the qualitative results of constituents in theextract clarified the chemical material base and provide basis for quality control ofQinggansanjie prescription.Because of the complex chemical compositions in Qinggansanjie prescription, thepolarity of compounds distribution were in a wide range, and the content of them weredefferent, many components in the chromatography have similar retention behavior. Wedesign an orthogonal experimental to optimize the impact of seven influential factorsincluding the extraction solvent, extraction temperature, static extraction time, extractionvolume, number of cycles, flush volume and purge time in the extraction procession ofQinggansanjie prescription by accelerated solvent extraction, and the total content of the11components in the higher levels were observed to conclude the parameters of extraction.AnLC-TOF/MS method was established to rapid separate and quantity of the complexchemical composition in the extracts, the content of30chemical compositions weredetermined as follows: protocatechuic55.65±1.80μg/g, Bergenin830.5±6.82μg/gcatechins11.74±0.43μg/g, chlorogenic acid174.6±8.41μg/g, gallic acid catechin ester27.40±0.52μg/g, epicatechin5.413±0.1μg/g, glycyrrhizin55.91±1.02μg/g, scutellarin174.±3.68μg/g, naringin54.34±2.29μg/g, rosmarinic acid38.430±1.73μg/g, isoliquiritin7.727±0.32μg/g, glycyrrhizin3.633±0.16μg/g, baicalin4440±193.17μg/g, quercetin24.83±0.64μg/g, naringenin9.995±0.25μg/g, luteolin24.79±0.77μg/g, kaempferol4.425±0.19μg/g, saikosaponin C7.298±0.26μg/g, wogonoside1161±47.64μg/g, apigenin29.66±0.76μg/g, baicalein669.0±31.35μg/g, wogonin208.6±4.89μg/g, glycyrrhizic acid188.9±4.58μg/g, tsaikosaponin A131.1±4.63μg/g, asiatic acid148.2±6.68μg/g,saikosaponin B21.871±0.07μg/g, saikosaponin D79.60±1.63μg/g, oleanolic acid227.4±7.82μg/g and ursolic acid428.2±13.26μg/g, glycyrrhetinic acid was not detected.The method was easy, good in repeatability, high separation and detection sensitivity, easyto automation, which has obviously advantages for quantitive analysis themulticomponents in TCMs simultaneously.According to the determination by LC-TOF/MS, the chemical compositions with highcontent relatively were chosen to pharmacokinetic study in rats. Using organic solventextraction combined with LC-MS determination method, the concentration of oleanolicacid and ursolic acid in rat plasma were measured after oral administration of differentcombinations extraction of Qinggansanjie prescription (monarch; monarch and minister;monarch, minister and assistant; compound recipe), and its pharmacokinetics parameters inrats were investigated. We found that the T1/2of oleanolic acid and ursolic acid weredifferent after administration of different combination. The T1/2of oleanolic acid andursolic acid in four groups was8.94±2.19h and4.78±1.07h,17.14±6.6h and7.31±3.70h,18.56±13.67h and9.52±4.41h,15.03±7.36h and8.64±1.36h, respectively. There weresignificant differences by statistical test, indicating that T1/2were prolonged to double afteradding the monarch, minister and guide group. The method of precipitation by organicsolvent combined with LC-MS/MS was used to simultaneously determine theconcentration of bergenin, chlorogenic acid, scutellarin, baicalin, wogonoside and wogoninafter administration of extratct of Qinggansanjie prescription. The results ofpharmacokinetic study shown that bergenin and chlorogenic acid have a typical disposalprocess of extravascular administration, but the flavonoids had double peak in the curve ofconcentration vs time, Tmax2of scutellarin was8.00±3.79h, baicalin was9.67±2.66h,wogonoside was16.33±8.62h, wogonin was9.00±2.54h, respectively. It was later than thereports in the literatures, which indicated that the complex chemical composition in theextract of Qinggansanjie prescription could prolong metabolic transformation process ofthe flavonoids in the intestine, result in secondary absorption time been delayed, theseflavonoids ingredients still maintain high concentration in12h rat plasma afteradministration, and the bioavailability were improved.Based in the effect material foundation study of Qinggansanjie prescription, weestablished a high performance liquid chromatography to determinate the content oftriterpene acids (oleanolic acid and ursolic acid) and flavonoids (scutellarin, baicalin, wogonoside, baicalein and wogonin) in Qinggansanjie granules, orthogonal design wasused to optimize the parameters in the process of extraction by ultrasonic. The content ofoleanolic acid, ursolic acid, baicalin, wogonoside, scutellarin, baicalein and wogoninin inQinggansanjie granules were7.29±0.18μg/g,16.37±0.47μg/g,3798±1.93μg/g,829.9±0.33μg/g,180.5±0.05μg/g,158.9±0.04μg/g and72.6±0.06μg/g, respectively. Itprovides a simple, rapidly, accurate, reliable, easy to popularize method for the qualitycontrol of Qinggansanjie granules. |
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