Extraction And Pharmacokinetics Of Anthraquinones From Polygonum Cillinerve (Nakai) Ohwi

Polygonum Cillinerve (Nakai) Ohwi (P.Cillinerve) is one of the Chinese herbs and recorded in the "Bencaotujin". It has the function of heat-clearing and detoxifying, analgesia, hemostasis. Modern pharmacological studies have shown that P.Cillinerve possesses antibacterial and antivirus effects. It has extensive application on clinic. Traditional Chinese medicine (TCM) is a scientific summary of rich experiences of the Chinese nation's struggle against disease for thousands of years. With occurring the upsurge of savageness, nutrition and returning natural all over the world, the catchword of "modernization and internationalization of traditional Chinese medicine" has become a very stringent strategy and action in China. Supercritical fluid extraction (SFE) is presently considered as one of the most clean and highly effective technologies for processing TCM. Pharmacokinetics research of TCM has important significance for realizing the TCM modernization and moving towards to the international market. So this subject studied supercritical CO₂ extraction and pharmacokinetics of anthraquinones from P.Cillinerve. There are three chapters in this thesis:Chapter One: IntroductionThis introduction includes three parts: The first part is about the research survey of P.Cillinerve. It includes medicinal history, TLC identification, resource distribution, summary of chemical constituents and the progress of pharmacological and clinical study. Anthraquinones are the major components of P.Cillinerve. Emodin and physcion are researched in many papers. So P.Cillinerve has been widely used for the treatment of tonsillitis, acute gastritis, enteritis, dysentery, urinary tract infection, hematemesis, hematochezia, dysfunctional uterine bleeding and menoxenia. It can also be exterior used to treat the damage, hemorrhage and so on. The second part is about the research progress of anthraquinones. This part introduces existence, structure and property of anthraquinone in plants. And the traditional methods for extraction and separation of free anthraquinones and anthraquinone glycosides are also summaried. The determination methods of anthraquinones include colorimetry, TLC, TLCS, HPLC, HPCE and so on. In this study, we used the methods of colorimetry and HPLC. Finally, we introduce the progress of pharmacokinetics of anthraquinones; The third part indroduces the supercritical fluid extraction technology. It includes the concept and kind of supercritical fluid, the characteristics of supercritical CO₂ fluid, the influencing factors of supercritical CO₂ fluid extraction and the application in herb extraction. At last, we predict the trend.Chapter Two: Study on supercritical CO₂ extraction of free anthraquinones from P.Cillinerve.The supercritical CO₂ fluid extraction procedure of free anthraquinones from P Cillinerve was studied. The contents were determined by magnesium acetate-colorimetry. The influences of extraction temperature, extraction pressure, static extraction time, volume of entrainer, maceration, and the times of repeated extracting were investigated. The results showed that the plants (200mg) should be macerated in 10 mL water for 24 h before SFE. The optimal extraction parameters were temperature 45℃, pressure 35 MPa, 15 mL of ethanol as modifiers, static extracting 35 min and dynamic extracting 30 min, repeated extracting 3 times. Contents of free anthraquinones, emodin and physcion obtained by SFE were 3.98 %, 3.44 % and 0.38 %, respectively. Compared with sonication extraction, supercritical CO₂ extraction has the advantages of high yield, environment-friendly property and fewer residual solvents.Chapter Three: HPLC method for simultaneous determination of five anthraquinones in rat plasma and pharmacokinetics of emodin from P.CillinerveA method was developed for the quantitative determination of aloe-emodin, rhein, emodin, chryscophanol and physcion in rat plasma by HPLC-UV-FLD. Agilent HC-C₁₈ column (250 mm×4.6 mm id, 5μm), mobile phase: methanol -water (pH=2.6, pH was adjusted by 1 % H₃PO₄) = 80:20 (v/v) at a flow rate of 1.0 ml/min, excitation wavelength 445 nm, emission 525 nm, 20μL of standards and sample were injected, column temperature 30℃. The linear rangs of aloe-emodin, rhein, emodin, chryscophanol and physcion are 0.132-4.4, 0.32-6.4, 0.8-24, 0.14-7.2 and 0.1-1.5μg·mL^-1 , respectively. The correlation coefficients of aloe-emodin, rhein, emodin, chrysophanol and physcion are 0.9993, 0.9996, 0.9997, 0.9995 and 0.9997 respectively. The detect limits of aloeemodin,rhein, emodin, chrysophanol and physcion are 0.8, 0.1, 0.2, 0.05 and 0.04μg·mL^-1. The intra-precision and inter-precision (RSD) is lower than 9.2 %. The accuracy (RE) is lower than 7 %. The extraction recovery in rat plasma rings from 60 % to 90 %. The method recovery in rat plasma rings from 81 % to 107 %. The profile of concentration-time of emodin after a single oral dose of 1.85 g/kg P.Cillinerve's crude extracts fits a two-compartment open model in rat. The pharmacokinetic parameters of T_max, C_max, AUC_(0-t), T_1/2a, T_1/2B, CL_(s) was 1 h, 17.649 mg·L^-1, 224.15 mg·(L·h)^-1, 62.882 h, 69.315 h and 0.001 L·(h·kg)^-1, respectively. This proposed method is simple, fast and sensitive.