| Objective:1. To have a serum pharmacochemistry study on Atractylodes japonica.2. HPLC method is used to study the fingerprint of rats after oral administration of Atractylodes japonica medicine, and to have a preliminary study on the constituents absorbed into blood of rats after oral administration with the water extract of Atractylodes japonica; Gas purge microsyringe extraction combined with gas chromatographic-mass spectrometric (GP-MSE-GC-MS) was used for analyzing the volatile and semivolatile compounds of blood of rats after oral administration with the water extract of Atractylodes japonica. This study provides experimental basis for the quality control of Atractylodes japonica.3. HPLC method is used to have a preliminary study on the constituents absorbed into blood of Atractylodes japonica、Rhizoma Atractylodis Macrocephalae and Rhizoma atractylodis extract. To compare the similarity of three species in Atractylodes DC. This study could provide experimental basis for the reasonable development and utilization of the plants in Atractylodes DC.Methods:1. High Performance Liquid Chromatographic analyze of Serum containing the Atractylodes japonica medicine.To choose the relatively important factors in the serum pharmacochemistry experiment of Atractylodes japonica, each factor set some different experimental conditions for the HPLC analysis, compared the serum HPLC profiles with the presence and absence of water extract of Atractylodes japonica, the experimental method which has the good peak shape, transitional components the most is the one that this experiment adopt. Choose the best detection wavelength from the four wavelengths:240 nm、254 nm、270 nm and 340 nm; choose the different ratio of the methanol, acetonitrile and water as different elution condions for experiments, explore the best mobile phase; compare the different rats delivery way:A(once a day)、 B(twice a day)、C(three times/d)、D(twice a day,2 days) and E(twice a day, A total of3 days), to determine the best dosing method; choose the best dosing solution from four different solutions:the water extrat,the methanol extract, the supercritical extract and the powder suspension of Atractylodes japonica; choose the best method from the following serum processing methods:the perchlorate method、the methanol precipitation method and the freeze drying-methanol precipitation method.2 Gas purge microsyringe extraction combined with gas chromatographic-mass spectrometric (GP-MSE-GC-MS) analyze of Serum containing the Atractylodes japonica medicine.The freeze drying method is used to dry serum containing drug and the blank serum, take the serum powder after freeze drying, the volatile and semivolatile compounds was extracted by using gas purge microsyringe method. According to the following conditions, to detect, analyze and compare the volatile compounds of the Atractylodes japonica medicine.GC-MS analyze condition:DB-5MS capillary column (30 m×0.25 mm ×0.25 mm) was used. Helium of high-purity was used as the carrier gas at a flowrate of 1.0 mL/min. Splitless injection. was conducted and the injection volume is 2 μL.The injector temperature was 280℃. The GC oven temperature was programmed as follows:the initial oven temperature was 45℃ and held for 1 min, and then to 250℃ at a rate of 4℃/min and finally to 280℃ at a rate of 6℃/min and held for 5 min. EI for the ion source and the temperature is 200 ℃, electron bombardment energy is 70 eV.3 Have a preliminary study on the constituents absorbed into blood of Atractylodes japonica、Rhizoma Atractylodis Macrocephalae and Rhizoma atractylodis extractHigh Performance Liquid Chromatographic analysis method had been used to compare the differences of the chromatographic peak of the blank serum, serum containing drug and the water extract, to disguss the constituents absorbed into blood of rats after oral administration with the water extract of Atractylodes japonica, Rhizoma Atractylodis Macrocephalae and Rhizoma atractylodis given to rats.Results:1. High Performance Liquid Chromatographic analyze of Serum containing the Atractylodes japonica medicine.Water is the best extraction solvent; To fill the stomach of rats for 3 days (twice a day) is the best delivery times; after freeze drying, methanol precipitation method is the best method of the treatment of serum; 340nm and 240nm are the suitable detection wavelength; the gradient elution of the mixture of water(A) and acetonitrile (B) is suitable for the experiment. When 340nm is the detection wavelength, the gradient elution is:0～10 min 40%B ～ 50%B,10～ 15 min 50%B ～ 50%B, 15～40 min 50%B～65%B,40～50 min 65%B～95%B,50～60 min 95% B ～100%,60 ～ 65 min 100%B～40%B; When 240nm is the detection wavelength, the gradient elution is:0～8 min 40%B～50%B,8～28 min 50% B～75%B,28～40 min 75%B～90%B,40～60 min 90%B-100%B, 60-65min 100%B～40%B.Through the experiment, serum fingerprint of Atractylodes japonica was established on the detection wavelength of 340nm and 240nm, compared to the blank serum, there are seven new peaks and six new peaks on 240nm and 340nm.2. Gas purge microsyringe extraction combined with gas chromatographic-mass spectrometric (GP-MSE-GC-MS) analyze of the Atractylodes japonica medicine.Compared to the blank serum, there was one, four and four new peaks into the serum of rats after oral administration with the water extract, the methanol extract and the powder suspension of Atractylodes japonica. According to the mass spectrometry NISI 08 database retrieval and refer to the relevent literature, conclude the preliminary identification that GC-MS total ion chromatograms of serum of rats of the water extrat, the methanol extract and the powder of the Atractylodes japonica has one similar ingredient:Cystine, the methanol extract identified two ingredients:Benzofuran, 6-ethenyl-4,5,6,7-tetrahydro-3,6-dimethyl-5-isopropenyl-, trans-and solavetivone. The others are not identified. On the medicine GC-MS total ion chromatograms, Benzofuran,6-ethenyl-4,5,6,7-tetrahydro-3,6-dimethyl-5-isopropenyl-, trans-and solavetivone are detected.3. To have a serum pharmacochemistry study on the blood transitional components between Atractylodes japonica, Rhizoma Atractylodis Macrocephalae and Rhizoma atractylodis extract.There was one similar ingredient into the blood of Atractylodes japonica and Rhizoma Atractylodis on 240nm. There was one similar ingredient into the blood of Atractylodes japonica, Rhizoma Atractylodis Macrocephalae and Rhizoma atractylodis medicine on 340nm. And there were two similar ingredients into the biood of Atractylodes japonica, Rhizoma Atractylodis medicine on 340nm.Conclusion:1. HPLC method of serum containing the Atractylodes japonica that this experiment established is simple and accurate, this method can be used to analyze and study the constituents absorbed into blood of the Atractylodes japonica.2. The constituents absorbed into blood of the Atractylodes japonica mainly come from prototype components and metabolites, and the stress product by the stimulus of Atractylodes japonica.3. The performance of the constituents absorbed into blood of three species in Atractylodes DC. Show that the constituents absorbed into blood of Atractylodes japonica is more close to that of Rhizoma atractylodis. |
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