| Sequelae of pelvic inflammatory disease (SPID), which is orsh called pelvic iInflammatory disease, characterized by longer duration, stubborn and refractory illness and high recurrence rate, are common disease of obstetrics and gynecology and seriously affect the life quality of patients. Combination of two or more antibiotics is usually used for treatment of PID in western medicine, however, which displays poor curative effect for the symptoms of SPID such as intra-abdominal tissue destruction, extensive adhesion, proliferation, and hyperemia and scar formation. In recent years, TCM has achieved great improvement in the prevention and treatment of SPID, and the clinical results are obvious.Jingangteng Capsules (JC) is the recommended drug for treatment of SPID by the Association of Chinese Medicine and Chinese Medicine Gynecology Branch. With the function of clearing away heat, detoxifying, removing dampness and subsiding swelling, JC is usually used for the treatment of vaginal discharge that is thick and yellow and increases in volume due to downpour of damp-heat; and its clinical efficacy has been recognized for the treatment of pelvic inflammatory disease or inflammatory masses with above syndromes. However, in the long-term clinical use, it is found that JC is of weak effect in the treatment of chronic patients with physically weak, pain and symptoms. Therefore, to improve its indications and create more suitable new drugs for the treatment of complex conditions of SPID, this paper carried out a series of studies including selection of TCM prescription, determination of preparation process route, establishment of quality assessment system and taking the pharmacodynamics evaluation and mechanism research under the support of enterprise, and developed a traditional Chinese medicine (Compound Prescription Jingangteng Capsules, CPJC) with clear effective material, clear action mechanism, safety and effectiveness and stable quality control. PartⅠScreening of TCM prescription for the CPJCTo further expand the indications of JC, some appropriate medicine should be selected to develop into a compound prescription with SCR. Under the guidance of Chinese medicine theory and the principles of medicine matching and based on the premise of keeping the amount of SCR (90g) unchanged, the composition the orthogonal test is carried out to screen the four drugs that often used with SCR according to relevant pharmacological targets. The results shows that the anti-inflammatory activity of the four drugs (anti-paw swelling, anti-granuloma) ranks in decreasing order as peony> AR> CR> Salvia and peony> CR> AR> Salvia, and the order of analgesic effect of the four drugs is in the following decreasing order: CR> peony> AR> Salvia. As for the immune enhancement activity, the decreasing order of the four drugs is CR> AR> peony> Salvia. Since peony and AR have the same function of strengthen the host defense system, so we choose AR and CR to synergize the activity of SCR.Then, the anti-inflammatory activity, immune enhancement activity and analgesic effects of the new compound were validated and proved that its analgesic and immune-enhancing effects were significantly stronger than JC (P<0.01); and its clinical efficacy for the treatment of SPID were verified with total effective rate 83%, which is more favorable than JC. Finally, the prescription for the compound CPJC is determined with SCR 90g, CR 20g and AR 20g.PartⅡStudy of the preparation process of herbal raw materialsTo develop the prescription of CPJC into a Chinese Patent Medicines, based on two evaluation systems, i.e., transfer rate of the chemical composition and pharmacodynamics, this paper adopts orthogonal and single factor experiments, respectively, to carry out the research of preparation routes of raw materials, which includes extraction, refining, purification and drying process. After repeated laboratory test and pilot amplification verification, the preparation route is determined, which is that the three drugs are extracted, purified and dried respectively.The preparation of SCR extraction is as follows:the SCR pieces are reflux extracted with 60% ethanol for 2 times, then the filtrated solvent are combined and evaporated to no alcohol taste (relative density 1.00-1.01,60℃), and then filtrated again; after adding distilled water to form solution with a concentration of 0.2g/mL, the solvent was put in the AB-8 macroporous resin column (resin:medicine=1:1.8, ratio of diameter to height=1:6) and put aside for 2-3h before washing with 5-6BV of distilled water,6BV of 20% ethanol and eluting with 3-4BV of 70% ethanol, successively; the resulted eluting solvent is collected and evaporated to liquid density 1.08-1.10 (70℃), and finally spray dried.Preparation of CR extraction is as follows:the CR pieces are reflux extracted with 60% ethanol for 2 times, then the filtrated solvent are combined and evaporated to no alcohol taste (relative density 1.03,25℃), and then filtrated again; after adding distilled water to form solution with a concentration of 0.4g/mL, the solvent was put in the AB-8 macroporous resin column (resin:medicine=1:4, ratio of diameter to height=1:6) and put aside for 2-3h before washing with 2-3BV of distilled water,2BV 10% ethanol and eluting with 4.5-5BV of 70% ethanol, successively; the resulted eluting solvent is collected and evaporated to liquid density 1.003-1.015 (70℃), and then 5%(g/mL) dextrin is add, finally spray dried.Preparation of AR extraction is as follows:after adding distilled water and heating, the AR pieces are extracted for 2 times, then the filtrated solvent are combined and evaporated to a concentration slightly greater than 0.2g/mL (measured relative density 1.005,70℃), and then filtrated again; after adding distilled water to form solution with a concentration of 0.2g/mL, the solvent was put in the AB-8 macroporous resin column (resin:medicine=1:6, ratio of diameter to height=1:6) and put aside for 2-3h before washing with 4.5-5BV of distilled water, and eluting with 3.5-4BV of 70% ethanol, successively; the resulted eluting solvent is collected and evaporated to liquid density 1.08-1.10 (70℃), and finally spray dried. PartⅢStudy of preparation and formation of CPJCThe match-ratio of the amount of extract is studied in this part. With uniform design experiment, the pharmacodynamic effects of the mixture of AR, SCR and CR with different match-ratio are studied and compared, the results show that if the amount of the extracts are calculated based on the amount of the corresponding herbal raw material, that is, in accordance with the Chinese prescription (SCR 90g, CR 20g, AR 20g), the regression equation in the uniform design will give the tip that the prescription is in the best ratio range. The daily dose is determined as SCR 2.7g, AR 0.438g and CR 0.1814g (100301). The result once again demonstrates the reasonableness of the medicine amount of TCM prescription, and also validated the whole treatment process of the raw materials.Preparation and formation process is as follows. According to the amount and nature of herbal raw materials and clinical requirement, the medicine type is determined as capsule formulation. Single factor experiment is used to screen the type and amount of excipients, and dry granulation process parameters are optimized. After repeated laboratory test and pilot production verification, the preparation route is determined as:take a certain amount of mixed extract (62% of prescription preparations), mix them with appropriate amount of MCC (22%), CaCO3 (5%), SiO2 (1%), CMS-Na (3%), and dry granulate at 1.5 MPa pressure, then add appropriate amount of CCMC-Na (5%) and SiO2 (2%) and mix and put into the capsule.Part IV Study of the quality standard of CPJCQuality control is taken in the pilot production of CPJC. TLC is used to qualitatively identify SCR and AR, and the results show that the spots are clearly visible and easy to distinguish; HPLC method is used to detect the content of resveratrol, and tentatively the content of resveratrol in each piece should be not less than 1.96mg. Method verification for the content determination method is carried out at the same time, and the results show that the test method is specific, simple and accurate. According to Chinese Pharmacopoeia (appendix IL, partⅠ,2010 edition), the load difference, disintegration time, water content and microbial limit are examined for capsule, and the results are in line with regulations. The organic residues of the resin in CPJC are detected by using the headspace gas chromatography, residues such as n-hexane, benzene, toluene, xylene, styrene and divinyl benzene are not detected in the samples, and residues limit are determined according to the regulation issued in 2007 by the Drug Evaluation Center of State Food and Drug Administration, i.e., technical requirements for the separation and purification of Chinese medicine extract by using the big hole resin (trial).Part V Pharmacodynamic and mechanism research of CPJCPharmacodynamic study shows that CPJC with dose of 0.630g/kg and 1.260g/kg dose (1,2 times the clinical equivalent dose, respectively) can significantly improve the SPID lesions in rats, mainly including uterine swelling, pelvic adhesions and the lesions of organizational structure of uterine cavity (P<0.05). With dose of 0.315g/kg, 0.630g/kg and 1.260g/kg (0.5,1,2 times the clinical equivalent dose, respectively), CPJC can inhibit the inflammatory substances induced rat paw edema (P<0.05 or P< 0.01); with dose of 0.450g/kg,0.900g/kg,1.800g/kg (0.5,1,2 times the clinical equivalent dose), CPJC can inhibits inflammation induced ear swelling in mouse (P <0.05), proving its role against early inflammation; with dose of 0.450g/kg,0.900g/kg, 1.800g/kg dose (0.5,1,2 times the clinical equivalent dose, respectively), CPJC can reduce the migration of leukocytes to sites of inflammation (P<0.05 or P<0.01), proving its role against the meddle-stage inflammation; and with a dose of 1.260g/kg dose (2 times the clinical equivalent dose), it can inhibit the proliferation of granulation tissue (P<0.05), prove its role against the late-stage inflammation.As for the anti-inflammatory effect, CPJC is superior to JC in at the same dose. With dose of 0.900g/kg,1.800g/kg, CPJC displays a significant analgesic effect (P <0.01 or P<0.05), and significantly better than JC (P<0.01), so CPJC have clinical therapeutic value to relief SPID inflammation induced pain. With dose of 0.450g/kg, 0.900g/kg,1.800g/kg, CPJC can significantly increase peritoneal macrophage index in mouse (P<0.01 or P<0.05), and significantly better than JC (P<0.01 or P<0.05), proving the immune enhancement of CPJC. Therefore, CPJC can be used in the clinical treatment to address both the symptoms and root cause of SPID.Based on the understanding of the pharmacodynamic activity of CPJC, a preliminary study on the possible mechanism of action is taken. NF-κB signal transduction pathway is an important inflammatory cell signaling pathway. This study shows that CPJC can significantly reduce the TNF-αand IL-1βlevels in serum of rats, inhibit the activity of NF-κB (including protein and nucleic acid level) of uterine tissue in rats and reduce the release of downstream effectors iNOS and COX-2. Therefore, it is possible that the therapeutic role of CPJC is expressed by inhibiting the excessive activation of NF-κB, decreasing the expression of many pro-inflammatory mediators and reducing pathologic inflammation.PartⅥSummaryThis part overviews the chemical composition, pharmacology and quality control of the three traditional Chinese medicines that comprise CPJC. |
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