| High Performance Liquid Chromatography (HPLC), also known as high-pressure liquid chromatography, is a commodity inspection technology which is widely used in chemistry, medicine, industry, agriculture and so on. HPLC has great advantage in high column efficiency, high sensitivity, high separation efficiency, fast speed of analysis and so on.Chinese herbs and formula is a treasure of Chinese traditional medicine, which has a magical effect without sideefect, however, their composition are complex and diverse. Nowdays, HPLC has been widely used in the analysis of herbal medicines and traditional Chinese medicine because it’s many advantages above, which could. The HPLC can be applied in separating, analysing and testing the complex and diverse component of Chinese herbs and formula. The HPLC provide the scientific basis for clinical Chinese medicine production, application and treatment.Paenoiae Radix Rubra and Moutan Cortex are the most common Heat-clearing and Blood-cooling herbs in clinic, which main active ingredients are Paeoniflorin and Paeonol. Present study indicates that both the active ingredients have broad pharmacological effects, such as protecting the heart, brain and blood vessels, protecting nervous system and anti-tumor, regulating blood pressure and other effects, which have been used in the treatment in various diseases.Psoriasis is a common chronic inflammatory skin disease, also called "white Bi" in Chinese medicine, is unclear in etiology. Psoriasis is high in incidence and the treatment is different because of it’s stubborn refractory. It is well-known that’Blood-heat’is the most common reason for the pathogenesis of psoriasis, so many Heat-clearing and Blood-cooling herbs are appllied in the blood heat type psoriasis in clinical. A number of research have showed using Heat-clearing and Blood-cooling herbs in the treatment of psorisis have achieved high efficacy, which composition including Paenoiae Radix Rubra and Moutan Cortex.The Heat-clearing and Blood-cooling Formula (also called QuYin Granula) is an effective Chinese medicine from my advisor Professor Bai-Yanping, consist of cooling blood to remove pathogenic heat herbs, which has many component, such as Paenoiae Radix Rubra and Moutan Cortex. Using the Heat-clearing and Blood-cooling Formula in the treatment in psoriasis has good clinical efficacy has been confirmed by the preclinical and animal model studies. The previous study has indicated that the serum of Heat-clearing and Blood-cooling Formula from rat could inhibit the activation of the JNK2MAPK signaling pathway and decrease the expression of HBD-2by regulate TNF-a and IL-p in psoriasis vitro cytology model of HaCaT cell lines.Objective:Paeoniflorin and Paeonol contained in the Heat-clearing and Blood-cooling Formula are determined by HPLC technology, furthermore, both the contents in the extractum which was prepared in the hospital’s pharmacy department.Methods:Samples were extracted by ultrasound to prepare of the test solution. Paeoniflorin and Paeonol were added methanol formulated as a mixed standard solutions as a postive control groups. The negetive group were the formula expect Paenoiae Radix Rubra and Moutan Cortex after boiling. The Paeoniflorin and Paeonol were diteced by RP-HPLC.(1) Select chromatographic conditions:The UV detection wavelength of two components were determined by full wavelength scan and literature review. The optimum separation conditions were determined by0.1%phosphoric acid-ace tonitrile as the mobile phase at a different time-comparing the ratio gradient.(2) Draw the standard curve:Two components corresponding to the peak area were measured in vertical axis and the injection volume as the horizontal axis of standard curve, then to calculate the regression equation of the establishment for determining the test Paeoniflorin and Paeonol.(3) The test of precision:the same test solution was continuous injected6times then peak area was measured and the RSD were calculated.(4) The test of repetitive:Six copies of the test solutions were prepared and the peak area of each sample were measured and the RSD were calculated.(5) The test of stability:The same test solution was measured at different times of peak area and the RSD was calculated at room temperature.(6) The test of recovery:the same sample of known content was added a mixed standard solutions, then the contents was determined and the RSD was calculated.(7) Determination:Paeoniflorin and Paeonol of the Heat-clearing and Blood-cooling Formula were detected by the method above.Results:Paeoniflorin is in25.075-300.90ng, which has a good linear regression and the equation is y=1.4983x+6.3114(r=0.9995). The average recovery rate of Paeoniflorin was99.70%(RSD=2.09%, n=6). Paeonol is in the range of1.407-16.884ng, which has a good linear regression and the equation is y=5.9812x+2.2144(r=0.9999), the average recovery was98.30%(RSD=1.76%,n=6).Conclusions:The method established by HPLC has many advantages, such as simple, accurate and stable, which can be used in the determination and quality control in the Paeoniflorin and Paeonol of the Heat-clearing and Blood-cooling Formula. |
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