| Paeonia is a cool temperature, it tastes pungent and acid , which is the dry root of Paeonia lactifora Pall. It is the TCD into liver and spleen load and had the capable of liver flatting, analgesia, blood enrichment, menstruation regulation, lian yin and anti-sweats. In clinical, it is used to treat headache dizziness, flank pain, abdominal pain, limbs pain, blood deficiency and irregular menstruation, spontaneous perspiration, perspire during sleep etc. Modern pharmacological studies have shown that the active ingredients of paeoniflorin in Radix Paeoniae Alba have the effect of sedative analgesic, antipyretic and antispasmodic effect and immune regulation etc.Compatibility applications of hurbs are the main form of the traditional Chinese medicine (TCM) treatment on the disease. TCM through the compatibility can not only enhance expanding the range of treatment and adapt to the complex disease condition, but also can reduce the adverse reactions and prevent poisoning. Shang Han Lun recorded compatibility of Bupleurum and Paeonia, two herbs combination, can regulate liver-qi and converges liver, lift Yang and converges Yin, benefit the body of liver and regulate the liver, so that liver-qi was sparse, hepatic blood may fill, soothing and nourishing the liver, combining static and dynamic to play the liver's function of blood storage and catharsis, can treat emotions depression, liver-qi stagnation due to depression or irritability, chest and hypochondrium suffering full, two threatened and breast tenderness, irregular menstruation and so on. Paeonia compatibility of common herbs into liver meridian can be widely used in the treatment of many diseases. The Paeonia's clinical effects are related to the active ingredients in Paeonia--Paeoniflorin have been report- ed from detailed investigation of documents and studies of international and domestic scholars.Hyperplasia of mammary glands (HMG) is the common and frequently illness occurring in the young women, in traditional Chinese medicine it falls within the category of"Breast Accumulation (Ru Bi)". TCM think that it's source is liver depression, and it's occurrence related to the liver pulse, spleen pulse, stomach pulse, blunt pulse and Ren channel, especially related to the liver pulse and it mostly related to liver depression and more use of "wood-work up" therapeutic method with curative effect. In modern society, because acceleration of the paces of life and increase of work pressure, so the stasis of liver-qi stagnation HMG was the most common in clinic.Therefore, this experiment revealed the content of the form of compatibility applications of TCM for clinical applications scientific basis through observing the Bupleurum, Angelica and Rose compatibility of Paeonia effects of its main components Paeoniflorin, by using paeoniflorin as an important indicator. However, the content of paeoniflorin and clinical effects of compatibility of its Rougan, nourishing and analgesic exists a positive correlation or not has not been reported, so further studies on animal experiments is necessary.So, this study combined traditional Chinese medicine on the etiology and pathogenesis of this disease awareness, use of the method of restraints braking plus continuous and from time to time forced swimming, to reproduction the animal model, which belonged to the stasis of liver-qi stagnation hyperplasia of mammary glands, and use Paeonia ,having the effect of nourishing blood and analgesic, as the main drug, through Paeonia compatibility of Bupleurum, or Chinese angelica, or Roses, to research the mechanism of the treatment of hyperplasia of mammary glands in Rats, further exploration of the correlation of the paeoniflorin and the nourishing, analgesic and soften the liver effects of Paeonia compatibility of traditional Chinese medicines, Rougan. So we can provide scientific data for future clinical application.Part One The Effect of Paeoniflorin's Dissolubility by Compatibility of Bupleurum, Angelica or Rose in Paeonia In this study, Identification of Paeoniflorin by using thin-layer chromatography(TLC), and quantitative determination by high-performance liquid chromatography (HPLC) of single herb and Paeonia with the Bupleurum, Angelica, Roses compatibility, the paeoniflorin content changes, and thus the scientific of traditional Chinese medicine compatibility could be confirmed.Objective: To observe the Paeoniflorin's Dissolubility by Compatibility of Bupleurum, Angelica or Rose in Paeonia.Methods:(1)Studies on identification methods: To use TLC, selecting suitable extraction solvents and methods, with chloroform: methanol (4:1) as mobile phase, 5% vanillin sulfuric acid solution as chromatography reagent, paeoniflorin standard as the reference substance, in Paeonia decoction, Paeonia decoction with Bupleurum, peony root decoction with angelica, Paeonia decoction with Roses, Peony compatibility of Bupleurum,Angelica and Rose solution were qualitative identified with TLC.(2)The assaying of Paeoniflorin:①Chromatographic conditions: Choose appropriate fixed phase, adjust different formulation and proportion of mobile phase and regulate flow rate and wavelength in order to separate the peak of Paeoniflorin from others well.②Preparation of standard curve: Prepare a series of the reference solution, examine peak area, then the regress equation was obtained with the content of Paeoniflorin as abscissa, the relevant peak area as ordinate.③Precision: Inject Paeoniflorin reference substance solution into the chromatograph for five times, , under the conditions of the chromatographic conditions, measure the Precise amount of take responses of Paeoniflorin, and then calculate the precision.④Stability: Prepare the test solution to measure the relevant responses at different time respectively in 48 hours, and then determine the peak area.⑤Reproducibility: Prepare the test solution five shares in the same way respectively to determine the content of Paeoniflorin.⑥Recovery: Use recoveries method of determining, transfer a portion of contents of sample solution, add a certain volume of reference solution of Paeoniflorin to it, and mix. Injections of samples were obtained in the above determination of the peak area under the chromatographic conditions to calculate the recovery rate.⑦Sample Determination: The content of Paeonifl- orin in sample solution was analyzed under above-mentioned conditions.Results: Identification: (1) TLC methods were established for the identification of Paeoniflorin. The principal spot in the chromatogram obtained from the test solution was similar in position, color and intensity to the principal spot in the chromatogram obtained from the reference solution. But the negative control was non-interference. (2)The assaying of Paeoniflorin:①Chromatographic conditions: Diamonsic-C18 column(200×4.6mm,5μm)was used for HPLC. The mobile phase was a mixture consisted of Methanol-Water (40:60) at a flow rate of 1.0 ml·min-1 and the detective wavelength was set at 230nm.Column temperature was 25℃; Sample size was 10μl.Under the system, the peaks of samples were separated well with the resolution of not less than 1.5.The retention time is about 8 min. The theoretical plates should not be less than 2000.②Preparation of standard curve: The liner range for Paeoniflorin was 0.0974～0.5846μg.Regress equation was Y=1452.3X+12.614 (R2=0.9998, n=5).③Precision: The precision instrument is fine. The RSD value in the precision test was 0.24%(n=5).④Stability: The sample was stable in 48 hours. The RSD value of stability was 0.43%(n = 6).⑤Reproducibility: This method has good reproducibility. The RSD value of reproducibility was 0.15%(n=5).⑥Recovery: Average recovery is 99.55%and the value of RSD was 0.56%(n=9). That explained it had a higher recovery rate, and the method was accurate and reliable.⑦Sample Determination: The content of Paeoniflorin in Peony root decoction with the Bupleurum equal parts boiled were 29.0mg/g, compare with the single herb Paeonia (19.8mg/g), significantly increased 46% or more; The content of Paeoniflorin in Peony root decoction with the Angelica equal parts boiled were 24.6mg/g, compare with the single herb Paeonia, significantly increased 21%; The content of Paeoniflorin in Peony root decoction with the Roses equal parts boiled were 33.4mg/g, compare with the single herb Paeonia, significantly increased 69%; The content of Paeoniflorin in Paeonia decoction with Bupleurum,Angelica and Roses equal parts boiled were 32.0mg/g, compare with the single herb Paeonia, significantly increased 62%; All that explained the content of Paeoniflorin were higher than the single herb Peony improved significantly in Peony compatibility of Bupleurum,Angelica,Rose.Conclusion: The method of TLC is simple, rapid, accurate and suitable for qualitative identification of Paeoniflorin; the method of High performance liquid chromatography (HPLC) has good accuracy and precision, assay results of Paeoniflorin show that the commonly used drugs Bupleurum, Angelica, rose compatibility with Paeonia can significantly increase the amount of Paeoniflorin.Part two Experimental Study on the mechanism of hyperplasia of mammary glands in rats with the compatibility of different drugsIn this study, with the method of plus a continuous braking of restraint and forced swimming from time to time to copy the animal model of liver-Qi stagnation hyperplasia of mammary glands , combined with traditional Chinese medicine on the etiology and pathogenesis of this disease, common characteristics of drugs and the awareness of physiological and pathological changes of modern medicine, we use radix paeoniae with the function of analgesic, nutrition blood and liver as the main drug, with the compatibility of the Bupleurum, or Chinese angelica, or roses to the experimental observation, and to further explore the effects of compatibility on the function of analgesic, nutrition blood and liver of radix paeoniae , in order to better to reveal the relevance between the compatibility of its original role of traditional Chinese medicine and its active ingredient.Objective: To explore the compatibility of radix paeoniae with commonly used drugs (Bupleurum, Angelica, roses)and the mechanism of action on the treatment of hyperplasia of mammary glands in rats.Methods: In this study, we use the method of plus a continuous braking of restraint and forced swimming from time to time to copy the animal model of liver-Qi stagnation hyperplasia of mammary glands. The rats were placed in the self-made meshy gauze bags, then placed in water of 25℃and forced to swim 5~10min, 1 times/d, irregular, intermittent to 30d, a total of 25 times, copy the rats model of liver-Qi stagnation hyperplasia of mammary glands. Compared with the normal group, the diameter of the rats'second and third pairs nipple were significantly increased (P<0.05 or P<0.01), the model-making was success. After making a successful model, rats were randomly divided into A (blank) group, B (model) group, C (treatment) group, including the C1 (radix paeoniae + Bupleurum), C2 (radix paeoniae + angelica) group, C3 (radix paeoniae + Rose) group and the C4 (radix paeoniae + Bupleurum + Angelica + Rose) group. In addition to the blank group and model group were fed with the appropriate amount of saline, the treatment groups were fed with appropriate drugs, a daily dose of 16g/kg (equivalent to 10 times the dose for adults), each group 1time/d, continuous 30d. During the experiment, rats were given free access to water and common fodder. after 30days, rats were decapitated ,take the blood , determine the 6 levels of reproductive hormones in serum, using the vernier caliper measure diameter of root of the second and third pairs nipple of rats, and then remove the full breast tissue of the third pairs, with 4% poly-formalin-fixed, paraffin-embedded, HE stained, in the light microscope observation, do pathological analysis.Results: (1) Observation of general condition in rats: Blank rats'Fur were gloss, responsive, appetite and faces were normal, and weight gain were normal, no death; model rats dull coat, drooping mustache, unresponsive, welt crowding together, activities, drinking water and food consumption were decreased, the feces sometimes rare, sometimes soft, body weight loss or weight gain is slow, no death; the treatment rats'fur can still be, the diet and faces were normal, often fighting, body weight was significantly higher than the model group, no one died. (2)①The effect on the diameter of second pairs of nipple: Compared with the A group, B group rats were significantly increased in nipple diameter (P<0.01), it shows that a successful modeling. Compared with the B group, every ctreatment group's rats have a reduced in nipple diameter except C1 group(P<0.05; P<0.01), which reduced most significant were the nipple diameter of C4 ,C5 groups;②the effect on the diameter of third pairs of nipple: Compared with the A group, B group were significantly increased in rat nipple diameter (P<0.05), shows that a successful modeling. Compared with the B group, every ctreatment group's rats significantly reduced in the nipple diameter (P <0.05; P<0.01), which reduced most significant were the nipple diameter of C3, C4, C5 groups. (2) Effects on the 6 levels of reproductive hormones in serum: Compared with the A group, B group rats'E2, PRL levels in serum were significantly increased (P<0.01), while the P content was significantly lower (P<0.01), shows that modeling success. Compared with the B group, every ctreatment group's rats'E2 in serum were decreased except C1 group (P<0.05; P<0.01), every ctreatment group's rats'PRL in serum were decreased (P<0.05; P<0.01), in which C2, C3, C4 rats'PRL levels in serum significantly lower (P<0.01) and C5 rats'E2, PRL levels in serum significantly lower (P<0.01); every ctreatment group's rats'P content in serum were increased (P<0.05; P<0.01), in which C5 group P was significantly higher (P<0.01). (3) pathological observation of breast tissue: the control group rats were not lobular hyperplasia, the number of glands was very small, acinar cells were not expansion, in a quiescent state; model rats have shown hyperplasia: the majority of breast lobular were hyperplasia, the majority of acinar significant expansion, increased levels of epithelial hyperplasia, acinar and duct have more secretions; the treated rats hyperplasia, but significantly reduced compared with the model group; C2, C3, C4 group Most of lobular were hyperplasia, a small number of acinar slight expansion, increased levels of epithelial hyperplasia, acinar and duct have secretions inside; the number and size of C5 rats'acinar and duct tissue were reduced in mammary gland, close to the normal group rats'mammary tissue structure state. (4) after Radix paeoniae compatible with Bupleurum, Chinese angelica and rose ,the fry out of paeoniflorin were significantly increased than the single herb Radix paeoniae, of which the most significant group were the radix paeoniae + Rose group and the radix paeoniae + Bupleurum + Angelica + Rose group, and the C4 (radix paeoniae + Rose) groups and the C5 (radix paeoniae + Bupleurum + Angelica + Rose) groups treat the mammary gland hyperplasia of liver-Qi stagnation of rats better compared with Bupleurum, Angelica groups, indicating the amount of paeoniflorin boiled associated with the compatibility of Radix paeoniae cure rat mammary gland hyperplasia of liver stagnation type.Conclusions: (1) The treatment group have shown improvement and effect on diameter of nipple, E2, P, PRL in serum of the liver-Qi stagnation type mammary gland hyperplasia in rat; of which C4, C5 groups were the most remarkable to treat liver-Qi stagnation type mammary gland hyperplasia in rat; its mechanism of improve the mammary gland hyperplasia in rats may be the regulating of sex hormone levels. (2) the amount of paeoniflorin boiled, radices peonies'compatibility the effect of nutrition blood rougan and acesodyne ,and the treatment of liver-Qi stagnation type mammary gland hyperplasia in rat was positively correlated.
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