| Medicinal property is a summarization of various natures and functions of medicines,and four natures and five flavors are the specific reflects of medicinal properties.Descriptions for the natures and flavors of Traditional Chinese Medicine (TCM) were first recorded in "Shen Nong’ Herbal Classic", herbal classics after that all described the natures and flavors of TCM. Four natures and five flavors are one of the most important parts of TCM theory. On the basis of clarifying the theory of natures and flavors of TCM systematically, this project applied various modern techniques and research methods to split chemical constituents of Rhizoma Anemarrhenae and study the pharmacological effects of respect split fractions to explore the relationship between natures and flavors and the constituents of TCM and verify a new hypothesis (one flavor and one nature, one medicine has X flavor and Y nature (Y<X)).Rhizoma Anemarrhenae, the dried rhizome of Anemarrhena asphodeloides Bge. (Lily),is bitter and sweet in flavor, cold in nature, attributive to the lung, stomach and kidney meridians. It has the effect of clearing the heat-fire and nourishing yin for moistening dryness, and is used clinically to treat exogenous febrile diseases, high fever with irritability and thirsty, dry cough caused by lung heat, intermittent fever, diabetes with internal heat and intestinal dryness with constipation. In this paper, the chemical constituents and pharmacological effects of Rhizoma Anemarrhenae were mutually verified, the material basis of its natures and flavors was classified. Two new compounds with special effects were isolated, polysaccharides from Rhizoma Anemarrhenae, which were rarely reported, were isolated and determined. The above results not only verified the theory of "one flavor and one nature, one medicine has X flavor and Y nature (Y≤X)", but also enriched the contents of TCM theories and provided a new idea for the research of split fraction and develop multifunction of one drug.In this paper, the development of the natures and flavors of TCM was systematically explained, literatures on the natures and flavors of TCM were summarized, and the TCM theory was enriched. A solid theoretical foundation was laid by researching traditional TCM theories for this project and provided a theoretical basis for further pharmacological research.The chemical constituents, pharmacological effects and clinical use of Rhizoma Anemarrhenae were reviewed comprehensively, ensuring a correct split of its chemical constituents and their attribution to natures and flavors.First, a literature research was carried out on natures and flavors of Rhizoma Anemarrhenae, from the first "Shen Nong’s Herbal Classic" to the latest "Chinese Pharmacopoeia".Great changes have occurred during the long history, from bitter and cold to bitter, sweet and cold. The major changes were the description on flavors,from single"bitter" to a completely opposite compound flavor "bitter and sweet",flavor "pungent" once appeared during this process.According to the fundamental theory of TCM and reports on the chemical constituents,pharmacological effects and the traditional clinical use of Rhizoma Anemarrhenae, guided by the effects correlated to the flavors, the water decoction of Rhizoma Anemarrhenae was alcohol precipitated, concentrated and purified by macroporous resin, extracted by ethyl acetate and n-butanol. The decoction was fractioned as polysaccharides, ethyl acetate fraction, n-butanol fraction and water fraction. Several methods, such as general chemical characters, TLC and HPLC, were applied to identify the chemical constituents of each fraction. Differences of the constitution of each fraction were also verified. Besides polysaccharides, ethyl acetate fraction was constituted by xanthones and n-butanol fraction steroid saponins, which are all the material basis of its effects.To ensure the repeatability of these fractions, give a better explanation to the material basis of its effects and provide foundation for further research, samples from different origins were collected, authenticated and tested. Only qualified samples were used. Uniform quality control was carried out for each fraction to ensure the repeatability by different operators. According to their respective characters, polysaccharide fraction, ethyl estate fraction and n-butanol fraction were analyzed by HPLC to control their quality. Fingerprint analysis was carried out to analyze the constituents of ethyl estate fraction with 8 peaks,represented by mangiferin. Characteristic chromatography was developed for n-butanol fraction, represented by timosaponin B Ⅱ and timosaponin B Ⅲ, 7 peaks were identified as common peaks. All the above work provided strong technical support for the reproducibility of the fractions and their effects.Repeated gel silica column chromatography,ODS reverse phase chromatography,gel chromatography and preparative HPLC were applied on n-butanol fraction to afford 21 pure compounds. Structures of 11 compounds were identified by chemical, physical and spectral analysis, and data were attributed. Two of these compounds, n-butyl-2-O-(L-pyroglutamic acid-N-6-)-α-D-fructofuranoside and n-butyl-2-O-(L-pyroglutamic acid-N-6一)-β-D-fructofuranoside were isolated and identified for the first time.Polysaccharides were water-soluble dialyzed, freeze dried and centrifuged, then tandem weak positive and weak negative ion exchange resins, ion exchange fiber, ion exchange agarose gel and several high resolution sephair conditioning unitryl were applied to isolate and purify this fraction. Four homogeneous polysaccharides were isolated from Rhizoma Anemarrhenae for the first time, and the isolated homogeneous constituents were all qualitatively studied by spectral and chromatographic analysis and types of their constitutional monosaccharides were determined.Pharmacological effects of each fraction were studied according to the natures and flavors. The results showed that polysaccharides fraction and saponin fraction both had antipyretic effect for the fever caused by dried yeast and 2, 4-dinitrophenol. Polysacharides fraction had anti-inflammation effect for dimethylbenzene-caused ear swelling.Polysaccharides fraction had significant immunosuppression effect and ethyl estate fraction had significant immunoenhancement effect. N-butanol fraction had diuretic effect on water loaded rats. Fractions except water fraction had significant hypoglycemic action on diabetic model mice by alloxan, the effect of ethyl estate fraction was especially strong. Ethyl estate fractions had Relieving cough effect.The pharmacological effects of each fraction and the natures and flavors of Rhizoma Anemarrhenae were attributed, and it was concluded that flavor bitter and nature cold were the foundation of the clearing the heat-fire. Clearing the heat-fire could be reflected as antipyretic effect, anti inflammation effect and diuretic effect, its effective material basis was polysaccharide and n-butanol fractions. Flavor sweet and nature cold were the basis of nourishing yin for moistening dryness and reflected as hypoglycemic action and myocardial cell protection in modern pharmacology. Its effective material basic was ethyl acetate fraction. In addition, Rhizoma anemarrhenae had immune adjustment caused by combination effect of flavor sweet and bitter. |
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