| Objective: Paeoniae alba is capable of liver flatting, analgesia, blood enrichment, menstruation regulation, lian yin and anti-sweats. In clinical, it was used to treat headache dizziness, flank pain, abdominal pain, limbs pain, blood deficiency and chloranemia, menoxenia, spontaneous sweats, night sweats etc. TGP injection is made from TGP, the effective part of paeoniae alba. TGP is multifunctional, such as anti-inflammation, anti-oxidation, immune regulation, anti-liver damage, anti-liver fibrosis. In order to improve the utilization of medicinal herbs, we adopted the single factor and orthogonal test for optimization of TGP in the extraction process, by using column chromatography technology in separation and purification in order to acquire TGP semi-finished products with high purity. Furthermore, in conjunction with the powder characteristics, we achieved freeze-dried powder of TGP by prescription design and optimization of preparation process.Methods:1 Studies on the measurement methodsWe performed HPLC methods to establish the measurement methods of TGP and paeoniflorin, as well as the methodology.2 Studies on the process of extraction, separation, purificationWe used the content of TGP and paeoniflorin as indicators to select the best extraction solvency, through single factor extraction solvency inspection. We adopted four factors and three levels orthogonal design to optimize the extraction process of TGP, furthermore, in the combination of the actual extraction process for the production with the verification test, through the verification test, we identified the process of TGP. We compared four kinds of different proportion of the water condition impact on paeoniflorin, TGP, and dried cream yield as 1:1, 1:2, 1:3, 1:4, as well as water precipitation time to find the best water conditions.Macroporous resin was used to separate and purify TGP. With static and dynamic adsorption method, respectively, we inspected different models and macroporous resin from various manufacturers, and selected the one for TGP isolation and purification. We then inspected the impact factors of macroporous resin absorption and elution rate: the pH of the liquid, the concentration of liquid, column Drive / column high proportion, elution ethanol concentration, eluting solvent amount, sample flow rate, the largest sample volume and elution velocity, from which we got the process conditions by macroporous resin to separate and purify TGP. Through the comparison of three removing tannin methods: the modified gelatin removing tannin, alcohol solution to adjust pH removing tannin, polyamide absorption removing tannin, we identified the removing tanning method in the preparation.3 Studies on the forming process of injection of TGP By comparison of different frames of the freeze-dried and direct freeze-drying liquid, such as mannitol, glucose, sodium chloride and lactose, we determined the prescription of TGP freeze-dried powder. Through inspection of some factors which affect freeze-dried powder's quality , such as liquid's pH, the method of removing pyrogen, solution concentration and sterilization process, we gained the final freeze-drying process.Results:1 The HPLC methods for the determination of TGP and paeoniflorin were established.1.1 HPLC method for the determination of TGPChromatographic conditions and system suitability test: 18 alkyl silane bonded silica as filler and acetonitrile -0.1% phosphoric acid (25:75) as the mobile phase, the detection wavelength is 230nm, flow rate is 1ml/min. Theoretical plate number calculated by benzoic acid should be no lower than 4000.Reference standard solution preparation: precisely take appropriate amounts of benzoic acid reference substance and put it into water to make it containing 10μg benzoic acid per ml solution.Test solution preparation: precisely draw paeoniae extracting solution (approximately 0.2g herbs), put it into a pan, and make it dry, add up 10 ml 1% NaOH solution to solve the residue, precisely draw 0.5 ml and put it into 10ml measuring flask, add up acetonitrile -0.1% phosphoric acid (25:75) 8 ml, shake up evenly, use 1mol / L HCl to adjust pH close to 3-6 , add up with acetonitrile-0.1% phosphoric acid (25:75 ) to dilute to scale, and then shake up evenly.Determination: draw the test solution and reference substance solution 10μl, respectively, inject into HPLC to determine the contents .TGP content was calculated as follows:TGP content (mg) = acid content (mg) *480.27/122.121.2 HPLC method for the determination of paeoniflorinChromatographic conditions and system suitability test: 18 alkyl silane bonded silica as filler and acetonitrile-0.1% phosphoric acid (14:86) as the mobile phase, the detection wavelength is 230nm, flow rate is 1ml /min. Theoretical plate number calculated by paeoniflorin should not be lower than 3000.Reference standard solution preparation: precisely take appropriate amounts of paeoniflorin reference substance and put it into MeOH to make it containing 60μg paeoniflorin per ml solution.Test solution preparation: precisely draw paeoniae extracting solution (approximately 0.1g herbs), put it into a 50ml measuring flask, add up with water to dilute to scale, and then shake up evenly.Determination: draw the test solution and reference substance solution 10μl respectively, inject into HPLC to determine the content.2 The studies findings of the process of extraction, separation and purification:2.1 The studies findings of the process of extractionTo optimize TGP extraction process by adopting the single factor inspection and orthogonal test: add 70% ethanol (10 times), extract twice, 2 hours per time. By inspecting water precipitation proportion and time, water precipitation condition is to condense liquid to 1ml liquid containing 1g herbs, the proportion of liquid and water is 1:2, 72 hours below 4℃.2.2 The studies findings of the process of separation and purificationTo screen out the optimum macroporous resin (HPD300) by adopting static and dynamic adsorption methods to separate and purify TGP. Its absorption capacity and washout rate is higher than others. The process to purify TGP by HPD300 was defined as: add 7 times water of the volume of liquid to dilute to per ml medicine liquid containing 0.1g herbs, adopt 4BV/h through HPD300 macroporous resin column (resin column Drive/high is 1:11), use five times volume of water to wash first, following by seven times volume of 50% ethanol to wash and the elution velocity is 4BV/h, collect the elution liquid, standby.3 The studies findings of the forming process of injection of TGPTo set up freeze-drying process through research on injection of TGP: put liquid medicine into the bottles and placed in refrigerator for pre-freeze at -40℃, 12 hours; then put the samples into freeze-drier, 24 hours. cover the samples with rubber plugs and aluminum caps. The uniform and osteoporosis appearance of the products with less than 3 percent water show that the process is stable and feasible.Conclusions: The results show that with reasonable extraction, separation and purification technology on paeoniae alba, more than 80% of the TGP content and 50% paeoniflorin content are available, which can be made into TGP freeze-dried powder according to the requirements of freeze-dried powder.
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