| Gardenia jasminoides fruit and Dan dou chi (Fermented Soybean) are two component herbs of traditional Chinese formulae Zhi-zi-chi Decoction, which is a traditional Chinese formula and used to treat depression, agrypnia. In the present thesis, chemical constituents,quality control method, the constituents in rat plasma, pharmacokintics of the active constituents of zhi-zi-chi decoction and the improvement on depressive rats were studied.1. Phytochemical studies on the n-butanol extract of Gardenia jasminoides fruit and the EtAc extract of Dan dou chi with vacuum flash chromatography on silica gel/ODS and semi-preparative HPLC led to the isolation and purification of 14 compounds. On the basis of physicochemical properties and spectroscopic analysis including 1D, 2D NMR, MS, their structures were identified as follows: geniposide (1), gardenoside (2), deacetyl asperulosidic acid methyl ester (3), scandoside methyl ester (4), 6-methoxyl -scandoside methyl ester (5),jasminoside B (6), genipin-1-β-gentiobioside (7), 6"-O- [(E)- pcoumaroyl]gentio biosyl genipin (8), crocin-1 (9), crocin-3 (10), genistein (11), daidzein (12), genistin (13) and daidzin(14).2. A high-performance liquid chromatography coupled with Q-time-of-flight mass spectrometry was established to identify multi-components of Zhi-zi-chi decoction and the constituents in rat plasma after oral administration of Zhi-zi-chi decoction. The LC/Q-TOF fingerprints of the samples were established first in vitro and in vivo, and 47 compounds in Zhi-zi-chi decoction and 24 constituents in rat plasma after oral administration of Zhi-zi-chi decoction were identified. Of the 47 detected compounds in vitro, 15 were identified by comparing the retention time and mass spectrometry data with that of reference compounds and the rest were identified by mass spectrometry analysis and retrieving the reference literatures. Of the identified 24 compounds in rat plasma, 19 were the original form of compounds absorbed from the 47 detected compounds in vitro, 5 were the metabolites of the compounds existed in Zhi-zi-chi decoction. A high-speed and sensitive method has been developed and successfully utilized for screening the active ingredients of a Chinese medical formula, which provided helpful chemical information for further pharmacology and active mechanism research on Traditional Chinese Medicine.3. A simple and sensitive assay based on high performance liquid chromatography mass spectrometry (HPLC-MS) coupled to an electrospray ionization (ESI) interface for the simultaneous determination of 16 compounds in different compatibility of Gardenia jasminoides fruit and Dan dou chi (4:0, 2:1, 1:1, 1:2, 0:4). Separations were carried out with a Diamonsil C18 reversed-phase column (250 mm × 4.6 mm, 5 μm). The mobile phase consisted of methanol and 0.05% (v/v) aqueous formic acid. A gradient elution program was used. The flow rate was set at 0.8 mL·min-1 and aliquots of 5 μL were injected into the HPLC system for analysis. Mass data were acquired using a mono-quadrupole mass spectrometer coupled with an electrospray ionization source (ESI) in the positive ion mode with a curved desolvation line (CDL) temperature of 250 ℃, a heat block temperature of 200 ℃, a CDL voltage of 10.0 kV and a detector voltage of 1.75 kV. All the analytes showed good linearity(r > 0.9980) in a relatively wide concentration range. The precisions were evaluated by intra-and inter-day tests, and the relative standard deviation (RSD) values were within the range of 1.2～2.7 % and 1.3～2.8 %, respectively. The recoveries for the quantified compounds were observed over the range of 99.3～103.7 % with RSD values less than 4.6 %.The method developed was successfully applied for simultaneous quantification of the 16 compounds in different compatibility of Gardenia jasminoides fruit and Dan dou chi and our results showed that there are significant deviation about the contents of geniposide and the isoflavone in different compatibility.4. A sensitive and efficient liquid chromatography-mass spectrometry (LC-MS) method was developed and validated for the simultaneous determination of geniposide, scandoside methyl ester and genipin gentiobioside in rat plasma. After the addition of internal standard(I.S.) salidroside and acidification (0.1% formic acid), plasma samples were carried out by protein precipitation with acetonitrile and separated on a Kromasil C18 column (200 mm × 4.6 mm, 5 μm) within a runtime of 15.0 min. The linear ranges were 2-250 ng·mL-1 for both scandoside methyl ester and genipin gentiobioside and 2-2000 ng·mL-1 for geniposide,respectively. The lower limit of quantification (LLOQ) was 2 ng mL-1 for all the analytes. The validated method was successfully applied to the pharmacokinetics study of geniposide,scandoside methyl ester r and genipin gentiobioside in rats after oral administration of Zhi-zi-chi Decoction and the extract of Gardenia jasminoides fruit. The Cmax of geniposide,was 1484 ± 241,1441 士 239 ng·mL-1,AUC0-∞ was 7284 ± 1186, 6397 ± 2218 ng·h·mL-1,T1/2 was 6.4 士 2.3, 7.0 ± 5.6 h,Tmax was 0.75 ± 0.27, 1.41 ± 1.24 h; The Cmax of scandoside methyl ester was 131.3 ± 26,3,116.3 ± 23.2 ng·mL-1,AUC 0-∞ was 978 ± 251,772.8 ± 206.6 ng·h·mL-1,T1/2 was 5.2 ±0.9, 5.4 ± 0.3 h,Tmax was 2.8 ± 0.8, 3.2 ± 0.4 h; The Cmax of genipin gentiobioside was 216.5 ± 61.6, 171.4± 63.1 ng ·mL-1,AUC0-∞ was 1105 ± 369, 621.1 ± 183.4 ng·h·mL-1,T1/2 was 4.8 ± 0.4, 3.4 ± 2.2 h,Tmax was 1.0 ± 0.3,1.0 ± 0.5 h. The results indicated that the AUC0-∞ of geniposide, scandoside methyl ester and genipin gentiobioside after oral administration of Zhi-zi-chi Decoction are higher than the AUC0-∞ of them after oral administration of the extract of Gardenia jasminoides fruit.5. The improvement of Zhi-zi-chi decoction on depressive rats were studied with depressive rats induced with lonely feeding and the chronic moderate intensive unpredictable stimulation.The improvement of depressed rats were evaluated with the weight of the rats,sugar consumption, the monoamine transmitters and their metabolities in cerebral cortex and plasma.The results showed that the increasing of the weight of model group was slower than control group, Zhi-zi-chi decoction group and the Fluoxetine group and the weight of the model group showed significant difference(P < 0.05) from the other three group; the sugar consumption in control group showed significant difference(P < 0.05) from the model group,Fluoxetine group and Zhi-zi-chi decoction group after 35 days, but the sugar consumption in control group showed significant difference(P < 0.05) from the model group and showed not significant difference(P < 0.05) from Fluoxetine group and Zhi-zi-chi decoction group after oral administration of Zhi-zi-chi Decoction and fuxiting with 21 days; the levels of 5-HT and 5-HIAA in rat plasma and cerebral cortex of model group showed significant difference(P <0.05) from the control, Fluoxetine group and Zhi-zi-chi decoction group.But the levels of DA,DOPAC from the model group showed not significant difference(P < 0.05) from the other three group. |
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