Pre-clinical Experimental Studies Of Yiru Tiaojing Granule

BackgroundHyperprolactinemia (HPRL) is a widely common disease caused by dysfunction of hypothalamus-pituitary-gonadal axis. When serum prolactin (PRL) level is consistently higher than the normal, it will be generally defined as HPRL. It mainly occurs in women. Large numbers of factors are known to affect the onset of HPRL. It is well known that pituitary tumors, primary hypothyroidism, idiopathic hyperprolactinemia, renal failure, gynecologic surgery, trauma, long-term use of interfering dopamine synthesis, metabolism or blocking dopamine receptor drugs, steroids including antipsychotics, antiemetic, estrogen and especially antipsychotics therapy will trigger HPRL. The elevated serum PRL disrupts the function of hypothalamus-pituitary-ovarian gonadal axis and the release of gonadotropin-releasing hormone. This dysfunction will trigger the reduction of secretion of FSH and LH which can lead to follicular maldevelopment, luteal phase and ovulation deficiency. Finally, these will cause reproductive dysfunction, menstrual disorders, infertility, sexual function decline, breast disease, hypogonadism, osteoporosis, and autoimmune diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis, etc. Therefore, we can take surgical removal or radiation treatment of pituitary tumors, dopamine receptor excited drugs, traditional Chinese medicine and other ways to restore sex gland and reproduction function in order to prevent HPRL.With the rapid development of traditional Chinese medicine (TCM), more and more researchers apply the diagnosis and treatment of TCM to HPRL owing to its multi-target, low toxicity, good patients’ compliance and affordable price compared with Western medicine. Currently, there is no promising traditional Chinese preparation to protect from HPRL on the market. Therefore, conducting clinical studies for HPRL combined with TCM theories according to its according to its pathogenesis is a good way to develop Chinese herbal preparations with more effective and fewer side effect, in order to maintain the economic interests of patients, obtain good social benefits.Since2001, our team has been working on protecting from HPRL. Moreover, we have already developed a Chinese herbal preparation for HPRL-Yiru Tiaojing Granule (YRTJ) which is composed of Curculigo Orchioides, Morinda Officinalis, Radix Paeoniae Alba, and Glycyrrhiza, and has the protective effects for liver function and nourishing function to liver and kidney. This subject will evaluate this preparation from quality control standard, long term toxicity, pharmacodynamics and pharmacological mechanism systematically and comprehensively. It aims to lay the reliable foundation for the study of further clinical research.ObjectiveTo optimize quality control standard for YRTJ, in order to improve quality standards and control the quality of this product effectively. To predict the long-time stability of the product by estimating the shelf life at specified storage conditions through accelerated storage test. To observe the chronic toxicity after long-term administration, provide the adverse reaction of target organs and the recovery after drug withdrawal. To establish animal models of hyperprolactinemia and explore the effect of YRTJ on experimental HPRL rats and its possible pharmacological mechanism, in order to provide primary data for the further study.Method 1. The quality standard of YRTJ1.1. Qualitative identification Identity the Morinda officinalis, Paeonia lactiflora and Glycyrrhiza in YRTJ by Thin layer chromatographic method (TLC method)1.2. Content determination The content of Paeoniflorin which is the effective substance in the preparation was determined by HPLC. The mobile phase of Paeoniflorin was a mixture of acetonitrile-0.1%phosphoric acid(18:82). Flow rate was1.0mL/min. The UV detective wavelength was230nm. Chromatographic column was Diamonsil C18(250mm×4.6mm,5μm). Column temperature was30℃. Under these conditions, the linear range was studied and the negative control test, precision test, recovery test, stability test and Sample determination of YRTJ were done.2. Studies on accelerated storage test of YRTJ According to requirements of Chinese Pharmacopoeia2010. The sample was stored under the condition of40℃±2℃, RH75%℃5%for6months, the test samples were randomly selected in0,1,2,3,6months to investigate the shape and size of granule, identity the Morinda officinalis, Paeonia lactiflora and Glycyrrhiza and determine the content of the YRTJ3. Studies on long-term toxicity test of YRTJ60Female SD rats were randomly assigned to receive different doses of YRTJ or saline, the dosages were corresponding to100,50,25times of human clinical equivalent dosage. These rats were administered intragastrically for60d. After24h of last drug administration,8rats were randomly selected to kill, the level of hematology and biochemistry of blood serum were analyzed respectively, and rats’tissue was examined histopathologically. The remained rats were observed for14d, and then killed them. 4. Pharmacodynamics’ research of YRTJ4.1. Studies on animal models of HPRL To screen the most optimizing animal model by comparing different establishing methods:anterior pituitary-grafted (APG), Diethylstilbestrol (DES), i.m., Metoclopramide (MPC), i.p..4.2. Effect of YRTJ on the experimental HPRL rats The HPRL rat models were made by continuously intraperitoneal injecting hydrochloride metoclopramide. Then the serum levels of PRL, LH, P, T, E2and FSH were detected in high, middle, low dose groups of YRTJ, the positive group of bromocriptine, after administration of30d and withdrawal of10d.4.3. Pharmacological mechanism of YRTJ The expression of PRL protein in pituitary was examined by Western blot method, and the expression of D2receptor in hypothalamus was examined immunohistochemicallyResult1. Morinda officinalis, Paeonia lactiflora and Glycyrrhiza can be identified by TLC method, and this method was simple, specific and reproducible. Moreover, the native control was little interferential.2. The regression equation is Y=4.3997×10-5X-0.9393622, R=0.9999956(n=8), the range of20.5-410μg/mL had a good linear relationship. The content of paeoniflorin were≥4.0mg/g. HPLC method was not interfered by other components and had a good precision, recovery rate and stability.Linearity range, limit of detection and recovery were met in compliance with established content determination requirements.3. After6months, YRTJ had no change of the shape and size. Loss on drying was met in requirements. Bacteria, mildew or Escherichia coliform were not existed in microbial limit tests. Morinda officinalis, Paeonia lactiflora and Glycyrrhiza could be identified and the content of paeoniflorin was≥4.0mg/g.4. Result of long-term toxicity test in YRTJ4.1. There were no apparent changes in morphological appearance, food intake and activities during the test. As the time went on, body weight gradually increased, and growth rate of the control group is the slowest. Neither pus blood stool, diarrhea, nor abnormal secretions were found.4.2. After60d, WBC of the high-dose group was increased significantly compared with the control group(P<0.01), RBC of the medium-dose group was decreased significantly compared with the control group(P<0.05), PLT level of the medium-dose group was increased. After withdrawal of14d, there was no significant difference in all groups.4.3. After60d, BUN of the high-dose, medium-dose and low-dose group was decreased significantly compared with the control group. After withdrawal of14d, there was no significant difference in all groups.4.4. After60d, ALT of the high-dose group was decreased significantly compared with the control group(P<0.05), TBIL of the medium-dose group were increased significantly(P<0.05). After withdrawal of14d, there was no significant difference in all groups.4.5. After60d, Spleen’s organ index of the medium-dose group was increased significantly compared with the control group(P<0.05),the organ index of other groups has no significant difference compared with the control group. The organ index of heart, liver, spleen, lungs, uterus in high dose group was increased compared with other groups, but the index of kidney was decreased. After withdrawal of14d, there was no significant difference in all groups.4.6. The histological and pathological examination showed that there was no apparent pathologic changes and toxic side effect in all groups.5. The result of pharmacodynamics study in YRTJ5.1. Four methods (APG, APG+MCP, DES, MCP) were selected to build HPRL model. Satisfactory results were obtained from all model tests. However, the PRL level of DES group only elevated144.47%, which is the lowest in the four groups. The deaths occurred in APG and APG+MCP groups.5.2. To select the proper MCP dose, we chosen four doses to make experimentations. After15d, all groups could make PRL level increased. Slightly peritoneal adhesion was found in50mg MCP (three times daily) group, but did not exist in75mg (two times daily) group. Thus, we have chosen the method to establish HPRL model of rats. HPRL model was built by intraperitoneal injection (75mg,2times daily) of MCP for10days.5.3. The YRTJ could significantly deceased the PRL level in HPRL rat model, and increase the E2, LH and Pg level. Thus, YRTJ could maintain the normal physiological functions. What should be of concern was that the high-dose group achieved a similar effect in regulation of hormonal levels compared with the bromocriptine group.5.4. YRTJ could reduce PRL expression in pituitary and D2receptor expression in hypothalamus; it achieved a similar effect compared with the bromocriptine. Thus we concluded that YRTJ might be the agonists of D2receptor, just like the bromocriptine, but the detailed mechanism need to be further studied.Conclusion1. The identification of YRTJ by TLC was characteristic and repeatable. The HPLC method was not interfered by other components, which has a good linear range, precision, recovery and stability, and can meet the determination of YRTJ.2. The stability tests show TRTJ granule is stable in quality during six months. According to the results of the influencing factor test and accelerated test. This research can provide an evidence for the long-term tests and predict the expiration date of YRTJ according to the results of the influencing factor test and accelerated test.3. YRTJ granule has no significant toxicity for long-term administration to rats. It infers that the drawing up doses in clinic will be safe.4. The animal model was built up by intraperitoneal injection (75mg,2times daily) of MCP for10days. It laid a good foundation for further study and application for HPRL.5. YRTJ achieved a similar pharmacological effect compared with the bromocriptine, which shows agonist D2receptor activities just like bromocriptine and can improve the disordered serum hormone level induced by Metoclopramide. What’s more, it can decrease PRL secretion in pituitary and serum levels of PRL in order to alleviate the clinical symptoms of HPRL. Thus YRTJ has a good treatment effect on experimental HPRL rat models.