Standards for Collection, Preservation, and Transportation of Fecal Samples in TCM Clinical Trials.

Background: Unlike chemical drugs with a single or a few kinds of active compounds, traditional Chinese medicines (TCMs)uses herbal formulas composed of numerous kinds of chemical constituents. Therefore, TCM clinical trials require unique and stricter standards for collecting, preserving, and transporting fecal samples than those used for chemical drugs. Unfortunately, there are no special standards for processing fecal samples in TCM clinical trials. Methods: We invited interdisciplinary experts within TCM clinical trials and gut microbiome research to help formulate this standard. After more than a year's in-depth discussion and amendments, we achieved a standard via expert interviews, literature research, questionnaire surveys, and public opinion solicitation. This standard has been reviewed and approved by the Standards Office of China of the Association of Chinese medicine. Results: We established a sample information processing method prior to TCM clinical sample collection, which is adapted to the unique features of TCM. The method formulates detailed processing requirements for TCM information in addition to the factors that may disturb the gut microbiome. We also constructed a set of methods for collecting, preserving, and transporting fecal samples that meet the characteristics of TCM. These methods formulate detailed operating specifications on the collection approaches, storage conditions, transportation requirements, and management of fecal samples. Conclusions: This standard guides the information processing prior to sample collection and the standard operating procedures for the collection, preservation, and transportation of fecal samples in TCM clinical trials, which also can be used as a reference by clinicians and researchers in modern medicines.

[Identification of active components in Xuefu Zhuyu Decoction based on targets of blood-activating function].

As a classic prescription for promoting blood circulation to remove blood stasis, Xuefu Zhuyu Decoction(XFZYD) is widely used in clinical practice and has notable curative effect. Based on the key targets of activating blood circulation, this study identified the active components of XFZYD to reveal the material basis. The components of XFZYD were collected from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP). The molecular docking models were built for the blood-activating targets obtained from the previous study with the components of XFZYD. The top five active components with measurability for each target were selected as the potential blood-activating components in the prescription. The efficacy of the prescription can embody key pharmacological and high-content components. In this study, anti-platelet aggregation activity was used to characterize the effect of activating blood, and the in vivo experiments were conducted to verify the accuracy of the active components. A total of 210 chemical components of XFZYD were screened out from TCMSP and docked with the key targets with the function of activating blood. Ligustrazine, acteoside, naringin, etc. were selected as the potential active components for activating blood in XFZYD. The anti-platelet aggregation activity of the combination of Chuanxiong Rhizoma, Rehmanniae Radix, Aurantii Fructus, Glycyrrhizae Radix et Rhizoma, and Carthami Flos was 9.82%±5.11%. Compared with that in the control group, the platelet aggregation induced by adenosine diphosphate(ADP) was significantly inhibited in the test group(P<0.01), which verified the accuracy of the active components. This study can guide the research on the material basis of XFZYD and provide insights into the development and utilization of the classical prescription.

The Effect of Dietary Vitamin K1 Supplementation on Trabecular Meshwork and Retina in a Chronic Ocular Hypertensive Rat Model.

Purpose: The pathophysiologic relationship between vitamin K and glaucoma remains largely unknown. The aim of the study was to explore the effect of dietary vitamin K supplementation in a rat glaucoma model. Methods: Rats were randomly divided into two groups: standard diet and high vitamin K1 (VitK1) diet (300 mg VitK1/kg diet). Induction of chronic ocular hypertension by episcleral vein cauterization was performed on the right eye. The left eye with sham operation served as controls. Rats received standard or high VitK1 diets for 5 weeks after surgery until the end of experiment. Immunohistochemistry analyses of the retina and trabecular meshwork were performed. The change in coagulation function and IOP were evaluated. Results: We observed a significant declined IOP at 2 weeks after surgery in the high VitK1 group compared with the control group. High VitK1 showed no significant effect on the body weight, rat phenotypes, or coagulation function. High VitK1 significantly inhibited the loss of retinal ganglion cells in the retina and increased the expression of matrix gla protein. High VitK1 also ameliorated the collapsed trabecular meshwork structure and increased collagen staining in the trabecular meshwork. Conclusions: High VitK1 intake inhibited the loss of retinal ganglion cells during glaucomatous injury, probably by increasing the expression of matrix gla protein. A transient decrease in the IOP was observed in the high VitK1 group, implying a potential effect of VitK1 on aqueous outflow. Retinal ganglion cells protection by high VitK1 supplementation may be due to the IOP-lowering effects as well as neuroprotective effect. Further research is required to delineate these processes.

Modified Cortex Mori Capsules improving the successful rate of functional filtering blebs after reclinical glaucoma filtering surgery.

BACKGROUND: The major reason for filtering bleb failure or scarring of the bleb site is due to excessive scarring after glaucoma filtration surgery in the clinic. Traditional Chinese medicine has preeminence in the prevention of fibrosis formation through the regulation of systemic circulation and improvement of the properties of the inflammatory cells in the blood. AIM: To examine the clinical efficacy of using the Modified Cortex Mori Capsules (MCMC; Chinese name: Jiawei Sangbaipi Capsules) in the success rate of functional filtering blebs after glaucoma filtering surgery in clinical patients. METHODS: Sixty resurgery glaucoma patients were randomly divided into two groups: 30 patients in surgery with the placebo group and 30 patients in surgery with the MCMC group. Patients took either the placebo or the MCMC 2 wk before and after surgery. Postoperative morphology and function filtering bleb, visual acuity, intraocular pressure, postoperative complications, the success rate of filtration surgery and clinical efficacy were observed. RESULTS: Fifty patients completed the study. The percentage of functional filtering blebs in the surgery plus MCMC group was 84% at 6 mo after surgery, which was higher than surgery plus placebo group (64%, P < 0.05). The surgical success rate in the MCMC and placebo groups were 79% ± 8.3% and 57% ± 10.6% respectively (P < 0.05). The visual acuity, intraocular pressure and the postoperative complications in the two groups had no significant differences. CONCLUSION: Glaucoma filtering surgery while taking MCMC not only reduced excessive scar formation and increased the success rate of functional filtering blebs but also improved the success of glaucoma filtration operations.

[Multi-target neuroprotective effect of Xixian Tongshuan Preparation against cerebral ischemia injury].

In this paper, Xixian Tongshuan Preparation was used as the research object, and all the chemical components of the 13 traditional Chinese medicines were collected. The target finding technique was used to obtain the key targets of the neuroprotective effect of Xixian Tongshuan Preparation, including 5 glutamate receptors, TGFR-1 and VEGFR-2. Molecular docking technology was used to screen out the potential active components of the above targets and to analyze their mechanism of action. It was found that single component, such as neo-complanatoside and neo-carthamin, in Xixian Tongshuan Preparation could simultaneously act on different targets. The chemical constituents in Ligusticum chuanxiong, Angelica sinensis, Carthamus tinctorius, and Panax pseudo-ginseng could simultaneously act on different neuroprotective-related targets, which reflected the application of multi-components to multi-targets. Point and multiple sites played a key role in protecting neurons against cerebral ischemic injury. This study explains the multi-target mechanism of anti-cerebral ischemic injury in neuroprotection at the molecular level, and provides a certain direction for the clinical application and experimental research of Xixian Tongshuan Preparation.

[Anti-inflammatory mechanism of Xixian Tongshuan Preparation based on molecular simulation methods].

Inflammatory response is caused by exogenous and endogenous stimuli,resulting in a non-specific resistance reaction.After acute ischemic cerebral infarction,inflammatory factors gather and adhere in the ischemic area of leukocyte infiltration,and the released inflammatory factors causes the injury cascade,aggravate the brain tissue damage and the symptoms of neurological deficits,and hinder the repair of brain neurons and the recovery of nerve function. In this paper,the key targets in the arachidonic acid metabolic pathway were studied. The Hiphop pharmacophore model of s PLA2-ⅡA and COX-2 inhibitors was built. According tothe two previously constructed 5-LOX and LTA4 H target inhibitors,the pharmacophore model was used to initially screen out the composition database of all of 13 traditional Chinese medicines in Xixian Tongshuan Preparation. The molecular matching study was carried out by selecting the matching value greater than 0. 6,and the component with the CDOCKER score greater than 80% of the original ligand score was used as the potential active inhibitor of the target. Considering the pharmacophore matching value,the molecular docking score and the interaction between the components and the target,one Chuanxiong component and one safflower component were selected as potential inhibitors of s PLA2-ⅡA; two Chuanxiong components,two Panax notoginseng,one safflower component,one angelica component,one valerian component were taken as a potential inhibitor of COX-2; two Gentiana components,one safflower component,one valerian component,one P. notoginseng component and one Angelica component were taken as potential inhibitors of 5-LOX; and two Gentiana components,two Chuanxiong components,and two safflower components were taken as potential inhibitors of LTA4 H. This study screened out the potential inhibitors of the four targets in a high-efficiency and low-cost manner,and explained that Xixian Tongshuan Preparation showed an effect in the treatment of inflammatory responses caused by ischemic stroke by acting both LOX pathway and COX pathway in the metabolic pathway.

[Anti-platelet aggregation mechanism of Xixian Tongshuan Preparation based on molecular simulation methods].

The thrombus is a deposit that is formed on the surface of the endovascular or at the site of repair,and known as the main complication of cardiovascular disease and the cause of death. At the same time,thrombus is mainly treated by the following three ways: anticoagulation,anti-platelet aggregation and thrombolysis. In this study,the chemical constituents of seven traditional Chinese medicines in the Xixian Tongshuan Preparation were collected to construct a component database. Subsequently,the pharmacophore were used to screen out the component database,and molecular docking was used to screen out the results of pharmacophore for explaining the material basis and mechanism that Xixian Tongshuan Preparation exerts anti-thrombotic activity by inhibiting platelet aggregation. First of all,P2 Y12,GPⅡb/Ⅲa and PAR1 were selected as study vectors,the optimal models of inhibitors were obtained respectively through verification and evaluation of the pharmacophore models. Afterwards,the component database was screened out by the optimal pharmacophore models of PAR1,P2 Y12 and GP Ⅱ b/Ⅲ a,and the molecular docking method was used to further refine the screening results. The screening results indicated that the anti-platelet aggregation effect of Xixian Tongshuan Preparation was correlated with the inhibition of P2 Y12,PAR1 and GPⅡb/Ⅲa expressions with saffower yellower,hirudin and candidin and notoginseng triterpenes,folinic acid,respectively. The material basis and mechanism of anti-platelet aggregation of Xixian Tongshuan Preparation provided a theoretical basis for the clinical use of the preparation and the lead compounds for the development of anti-platelet aggregation drugs.

[Lipid regulation mechanism and dosage form selection of Xixian Tongshuan Preparation based on molecular simulation methods].

Atherosclerosis is the main cause of stroke, and dyslipidemia is the most important risk factor for atherosclerosis. In this paper, pharmacophore and molecular docking models of eight key lipid-lowering targets, namely NPC1 L1, HMG-CoA reductase, SQS, MTP, CETP, PPARα, LXRα and LXRß, were used to screen out the small molecular database of traditional Chinese medicine(TCM), which was made up of ingredients of thirteen Chinese herbal medicines contained in Xixian Tongshuan Preparation. The screening results indicated that the preparation could showed an effect in regulating lipid on target NPC1 L1, HMG-CoA reductase, LXRß and SQS through four groups of potential active compounds, namely prupersin A in peach kernel and suffruticoside A in gastrodiaelata, limocitrin-ß-D-glucoside in Ligusticum chuanxiong, 2'-(2,3-dihydroxybenzoyl)-sweroside in Pinellia ternate and quercitrin in Panax notoginseng, 4-tert-butyl-2-[(5-tert-butyl-2-hydroxy-phenyl)methoxy-methyl]-6-(hydroxymethyl)phenol in Gastrodia elata. Moreover, the properties and extraction process of the most potentialactive compounds were consistent with the preparation process of Xixian Tongshuan Capsules, which indicated that the capsule had more advantages than the pill in the existing two dosage forms of Xixian Tongshuan Preparation. This study analyzed the pharmacodynamic basis and mechanism of Xixian Tongshuan Capsules in regulating lipid for treating stroke, and provided evidence for its further research and clinical application.

In Silico Analysis of the Association Relationship between Neuroprotection and Flavors of Traditional Chinese Medicine Based on the mGluRs.

The metabotropic glutamate receptors (mGluRs) are known as both synaptic receptors and taste receptors. This feature is highly similar to the Property and Flavor theory of Traditional Chinese medicine (TCM), which has the pharmacological effect and flavor. In this study, six ligand based pharmacophore (LBP) models, seven homology modeling models, and fourteen molecular docking models of mGluRs were built based on orthosteric and allosteric sites to screening potential compounds from Traditional Chinese Medicine Database (TCMD). Based on the Pharmacopoeia of the People's Republic of China, TCMs of compounds and their flavors were traced and listed. According to the tracing result, we found that the TCMs of the compounds which bound to orthosteric sites of mGluRs are highly correlated to a sweet flavor, while the allosteric site corresponds to a bitter flavor. Meanwhile, the pharmacological effects of TCMs with highly frequent flavors were further analyzed. We found that those TCMs play a neuroprotective role through the efficiencies of detumescence, promoting blood circulation, analgesic effect, and so on. This study provides a guide for developing new neuroprotective drugs from TCMs which target mGluRs. Moreover, it is the first study to present a novel approach to discuss the association relationship between flavor and the neuroprotective mechanism of TCM based on mGluRs.

[Discover potential inhibitors of 5-LOX and LTA4H from Rhei Radix et Rhizoma, Notopterygii Rhizoma et Radix and Genitana Macrophyllae Radix based on molecular simulation methods].

5-lipoxygenase (5-LOX) and leukotriene A4 hydrolase (LTA4H), as the major targets of 5-LOX branch in the arachidonic acid (AA) metabolic pathway, play an important role in the treatment of inflammation. Rhei Radix et Rhizoma, Notopterygii Rhizoma et Radix and Genitana Macrophyllae Radix have clear anti-inflammation activities. In this paper, the targets of 5-LOX and LTA4H were used as the research carrier, and Hiphop module in DS4.0 (Discovery studio) was used to construct ingredients database for preliminary screening of three traditional Chinese medicines based on target inhibitor pharmacophore, so as to obtain 5-LOX and LTA4H potential active ingredients. The ingredients obtained in initial pharmacophore screening were further screened by using CDOCKER module, and the screening rules were established based on the score of initial compound and the key amino acids to obtain 12 potential 5-LOX inhibitors and 7 potential LTA4H inhibitors. To be more specific, the potential 5-LOX inhibitors included 6 ingredients in Rhei Radix et Rhizoma, such as procyanidins B2-3,3'-O-double gallate and revandchinone 2; four ingredients in notopterygium, such as dodecanoic acid and so on. On the other hand, potential LTA4H inhibitors included revandchinone 1, revandchinone 4 in Rhei Radix et Rhizoma, tridecanoic acid, tetracosanoic acid and methyl eicosanoate in Notopterygii Rhizoma et Radix, montanic acid methyl ester and N-docosanoyl-O-aminobenzoate in Genitana Macrophyllae Radix and so on. The molecular simulation methods were highly efficient and time-saving to obtain the potential inhibitors of 5-LOX and LTA4H, which could provide assistance for discovering the chemical quality indicators of anti-inflammatory efficacy of three Chinese herbs, and may be helpful to promote the whole-process quality control of three Chinese herbs.

Effects of matrine on proliferation and apoptosis of cultured retinoblastoma cells.

BACKGROUND: Extracted from the traditional Chinese medicine of Kushen, matrine is an alkaloid with potential anti-neoplastic and anti-inflammatory effects. Here, we examined the effect of matrine on proliferation and apoptosis of cultured retinoblastoma cells. METHODS: The retinoblastoma cell lines Y79, WERI-RB1 and SO-RB50 were treated with matrine in increasing concentrations from 0.2-1.1 mg/ml for 24 hours, and the cell proliferation rate was measured. The cells were exposed to matrine at 50% inhibition concentration (IC50) for 12, 24 and 48 hours. Cell cycle was analyzed by flow cytometry, concentration of proteins regulating cell cycle and apoptosis was determined by Western blot, apoptosis rate was measured by TUNEL staining, and cell morphology was assessed by electron transmission microscopy. RESULTS: The retinoblastoma cell lines Y79, WERI-RB1 and SO-RB50 showed an increased inhibition of cell proliferation with increasing matrine concentrations. Applying the IC50 concentration of matrine, the alteration of the cell cycle, including a reduced percentage of the S phase, was significantly (P < 0.01) associated with a longer treatment time by matrine. Correspondingly, the cell-cycle-associated proteins P21 and P27 were up-regulated and the protein cyclinD1 was down-regulated. The apoptosis-associated protein Bcl-2 was down-regulated, and Bax was up-regulated. In a similar manner, the apoptosis rate was significantly increased with longer treatment time. CONCLUSIONS: Matrine added to cultures of immortalized retinoblastoma cells led to a reduced tumor cell proliferation, decreased rate of mitosis and an increased tumor cell apoptosis, paralleled by corresponding changes in the proteins regulating the cell cycle or apoptosis.