Sanguisorba Officinalis (L.) Extracts Inhibit Matrix Metalloproteinases And Influence Cell Morphology

MMPs are a family of zinc endopeptidase, and it is dispensable for the degradation process of extracellular matrix (ECM) and connective tissue proteins. Based on their substrate specificity and primary structure, the MMPs family can be subdivided into six groups: collagenases, gelatinases, stromelysins, matrilysins, membrane type (MT) and heterogeneous subgroup. MMPs can degrade ECM components, other proteases, clotting factors, chemotactic molecules, latent growth factors, growth-factor binding proteins, adhesion molecules, cell surface receptors etc. So it plays an important role in the physiological and pathological conditions. The expression and activation of MMPs are directly correlated with the growth, invasion and diversion of tumor. MMPs are often expressed excessively in the malignant tumor. Thus it is believed that the growth and metastasis can be controlled by MMP inhibitors (MMPIs) which are expected to be useful for the treatment of cancer. Nowadays, many of the inhibitors of matrix metalloproteinase have come into clinical trial, but to most of our disappointment, none was tested to be a candidate for further curing disease. The main problem of this is the incomplete understanding of the function of MMPs, non-specific and side effects of MMPs inhibitors. Along with the continuous deepening of the understanding of MMPs function, the researching and screening MMPs inhibitors with high specificity and low toxicity are becoming increasingly important. According to the reports, currently, some research institutions have isolated new and potentially beneficial compounds which have been shown to exhibit some degree of MMPI activities from herbs, plants, fruits, and other agriculture products, but they are far less potent and specific than the TIMP family. These natural compounds include long chain fatty acids, epigallocatechin gallate (EGCG) and other polyphenols, flavonoids, and a variety of other natural compounds. Kim et al. have found nearly 90 extracts of oriental medicinal herbs, which are used as prescriptions for cancer treatment, such as Baicalin, cinnamon, Euonymus alatus and Magnolia, which all have strong inhibitory effects on MMPs. Recently, they found a new matrix metalloproteinase inhibitor, caffeic acid, from methanol extracts of Euonymus alatus. In our previous study, we proved that one of the components of Qing-Kai-Ling, Fructus gardeniae, strongly inhibits MMP activity.Traditional Chinese medicine (TCM) has a long history of development and application. Over the past decades,Chinese herbal medicines (CHM) and their commercial derivatives are said to possess numerous properties and as a consequence are used to treat a variety of diseases including arthritis, cancer, asthma and allergies,and these medicines with anti-inflammation and anti-tumor properties are extensively used in Chinese clinical practice. However, the molecular mechanism of these herbal medicines has not been fully recognized due to the lack of definitive information of active ingredients. Therefore, it is of great significance to do such a work.The purpose of this thesis is based on Chinese herbal medicine and expression, purification and refolding of matrix metalloproteinase,we intend to apply enzymology methods, screen MMPs inhibitors with high specificity and low toxicity, develop new drug targeting to MMPs. wec purified three kinds of MMPs, which were MMP-3,MMP-13 and MMP-14. Then, we used DQ-gelatin as substrate to study the inhibitory activities of sanguisorba officinalis to MMPs by fluorescent assays in vitro. Then we investigated the inhibitory effects of a number of Chinese medicines on MMPs,and find that Sanguisorba extract significantly inhibits the activities of MMP-2, MMP-3, MMP-9 and MMP-13. To evaluate the effect of this aqueous extract on B16 cell viability and migration, we used the MTT cell viability assay and wound-healing assay in which cells was induced to migrate by physical wounding of cells plated on plastic plates. We found that cell viability was not affected in concentrations ranging from 0 to 200μg/ml by the aqueous extract. However, the migration ability of B16 cells treated by aqueous extract was inhibited as compared with untreated cells, and the quantitative data revealed that aqueous extract could inhibit B16 cells migration in a dose-dependent manner and the effect was significantly. Because aqueous extracts also can strongly inhibit MMPs (MMP-2, MMP-3, MMP-9 and MMP-13) activity, we surmise that MMP inhibition may change cell morphology, which may in turn lead to an alteration of cell behavior and function. Then we docked the major components of sanguisorba officinalis(ziyu-glycoside I,ziyu-glycoside II) to different MMPs. The results of molecular simulation and the enzymatic data are comparable, and have a high credibility of experimental results,However, ziyu-glycoside I, ziyu-glycoside II are just two main component of sanguisorba officinalis and its other known or unknown ingredients may also inhibit MMPs. The simulation results of this experiment for further studying the active ingredients of sanguisorba officinalis which inhibit MMPs activity provide a basis, and provide a help for the understanding of which sanguisorba officinalis inhibit mechanism of MMPs. In this paper, we found that Sanguisorba officinalis (L.) strongly inhibited MMPs activities, and had no affect on B16 cell viability but inhibited cell migration ability. So we may presume that its ability of anti-tumor is correlated with its ability of inhibiting the activities of MMPs. It explained that why Sanguisorba had the ability of anti-tumor in molecular level. This means that its extract contains effective MMP-inhibitory compounds. We may abstract MMP inhibitor from Sanguisorba officinalis (L.), and we can explore new traditional Chinese medicines targating to MMPs.