| The research direction of this thesis is the measurement of the bioactive ingredients and fingerprint analysis of traditional Chinese medicine. Taken one kind of traditional Chinese medicine—Herba Cistanche as the research object, several methods had been built up to determine effective constituents in this herb, and the fingerprint of Herba Cistanche was researched by HPLC. The thesis mainly includes the following four parts: 1 Determination of mannitol and carbohydrates in herba Cistanche by capillary electrophoresis with electrochemical detection; 2 Monosaccharide composition analysis and its content determination in polysaccharide extract from Herba Cistanche; 3 Determination of betaine in Herba Cistanche by high performance liquid chromatography coupled with evaporative light-scattering detection; 4 Study on the Fingerprint of Herba Cistanche by high performance liquid chromatography.In chapter1, an easy method, based on capillary electrophoresis (CE) with electrochemical detection (ED), for simultaneous determination of mannitol, sucrose, glucose and fructose in Herba Cistanche, was developed. The effects of several factors such as the applied potential to the working electrode,concentration of the running buffer, the separation voltage, and the injection time were studied to find the optimum conditions. The optimum CE-ED conditions were as fellows: 0.65 V (vs. SCE) as the applied potential to the working electrode, 0.1 mol/L NaOH as the running buffer, the separation voltage at 21 kV and 6s as the injection time. Under the optimum conditions, the analytes were base-line separated within 22 min and excellent linearity was obtained in the concentration range from 1×10-5 mol/L to 1×10-3 mol/L. The analytes in Herba Cistanche were also determined with recoveries between 98.8 % and 101.4 %, which showed the assay results were satisfactory. From the results, it was found that the contents of mannitol, sucrose, glucose and fructose were obviously different in Herba Cistanche collected from different regions of growth. The content of mannitol is higher in the herb grown in Gansu, and the wild herb grown in Xinjiang has a plenty of fructose. The experiment demonstrated that the contents of active ingredients in Herba Cistanche are different depending on the region of cultivation. This method was also suitable for the determination of content of mannitol in compound medicine including Herba Cistanche.In chapter 2, monosaccharide composition in polysaccharides from Herba Cistanche was studied by capillary electrophoresis (CE) with electrochemical detection (ED). Then Monosaccharide compositions were compared among different species of Herba Cistanche. The crude polysaccharide of Herba Cistanche was extracted with hot water, and precipitated by ethanol. At 100℃water bath, the crude polysaccharide was hydrolyzed with diluent hydrochloric acid. Monosaccharides in the hydrolysate were studied by capillary electrophoresis (CE) with electrochemical detection (ED). They were mainly consisted of glucose, isodulcitol, galactose, arabinose and fructose. The detection limits of these five monosaccharides ranged from 3.7×10-7 g/mL to 1.1×10-6 g/mL .The analytes in hydrolysate were also determined with recoveries between 99.0% and 105.2%. Mmonosaccharide composition of Herba Cistanche polysaccharides from different species was obviously dissimilar, and the difference was existed between wild and cultivated samples.In chapter 3, a method for the determination of betaine in Herba Cistanche by high performance liquid chromatography coupled with evaporative light-scattering detection (HPLC-ELSD) was developed. Effects of several factors, such as the separation column, the mobile phase and the parameters of ELSD were investigated to find the optimum conditions. Separation was carried out in Waters Spherisorb S5NH2 (250 mm×4.6 mm i.d, 5μm) column eluted with the mobile phase 0.1% TFA (A) and methanol (B) (A:B=15:85) at flow rate of 0.6 mL/min in temperature of 25℃. The parameters of ELSD were the drift tube temperature of 90℃, the nebuilzer temperature of 45℃and the gas pressure of 16 psi. The method was successfully used for assay of betaine in Herba Cistanche, and the average recovery of 99.2 % indicated that the experimental results were satisfactory. Additional, experimental results showed that betaine was not existed in C.tubulosa(schenk)R.Wight, which belongs to the same genus as Cistanche deserticola Y. C. Ma, but different species. Therefore, betaine was recommended as the marker for identification C. deserticola Y. C. Ma from C.tubulosa(schenk)R.Wight.In chapter 4, the fingerprint of Herba Cistanche from different origins was established by high performance liquid chromatography. The chromatographic conditions were optimized as follows: chromatogram column: NUCLEODUR 100-5 C18 (250 mm×4.6 mm i.d, 5μm)column; temperature: 25℃;the mobile phase: A, 0.1% TFA; B, methanol; velocity of flow: 0.6mL/min; drift tube temperature:90℃;the nebuilzer temperature: 45℃; The N2 pressure: 16psi. There were 23 common peaks in the fingerprint of five batches of Herba Cistanche. The data of fingerprints of 5 batches of Herba Cistanche were processed with two kinds of mathematic methods including correlation coefficient and cosine value of vectorial angle to validate their similarities, and most of samples of Herba Cistanche were consistent. The similarity values of Herba Cistanche were larger than 0.92 except samples from Neimeng province. Compared to the pattern of Herba Cistanche fingerprint, the similarity of Cynomorium songaricum Rupr was smaller than 0.55, showed that the fingerprint of Herba Cistanche and Cynomorium songaricum Rupr were definitely different. This method was accurate, reliable and could be used to distinguish Herba Cistanche from Cynomorium songaricum Rupr.
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