Preparation And Anti-sepsis Activity Study Of 2', 5, 6', 7-tetrahydroxy-flavanonol (THF) From Scutellaria Baicalensis Georgi

Introduction Sepsis is defined as suspected or proven infection plus a systemic inflammatory response syndrome. It is the most common complication of burn, trauma and infectious diseases. An epidemiological study found that there were approximately 18 million cases of sepsis annually worldwide and the incidence was expected to increase 1% per year. Septic shock and multiorgan dysfunction resulting from sepsis are the leading cause of death in intensive care units, with the mortality rates between 50 and 60%.Lipopolysaccharide (LPS; known also as endotoxin) is an outer membrane component of gram-negative bacteria, and thought to be a major PAMP of sepsis. In the presence of lipopolysaccharide-binding protein (LBP), LPS interacts with monocyte/macrophage cells via CD14 and the two form the LPS-CD14 complex. Then the complex binds to TLR4 and triggers the TLR4-mediated signal transduction to induce the release of pro-inflammatory cytokines, including TNF-αand IL-6. Over expression of cytokines will trigger systemic inflammatory response syndrome, as a result, sepsis and septic shock take place. Although great attentions have been paid in studies of the pathophysiology and treatment of sepsis, there is currently no effective anti-sepsis drug in clinical use.Application of traditional Chinese medicines in septic therapy has a long history. Recent clinical trials and studies also demonstrated that a lot of herbs possess the properties of anti-LPS. However, herbs usually have a large number of complex constituents and thus extremely difficult to identify the anti-LPS ones, which limit their clinical uses. In previous studies, we successfully established an effective method to screen anti-LPS compound from traditional Chinese herbs using affinity biosensor technology. In this study, based on this technology platform, the aqueous extracts from traditional Chinese herbs were isolated by using macroporous adsorptive resins technology, ultra filtration membrane technology and high performance liquid chromatography (HPLC) technology in order to obtain anti-LPS monomer to be used for sepsis therapy.Methods First, the aqueous extracts from sixty traditional Chinese herbs were tested for detecting the binding activity to Lipid A using the affinity biosensor technology. Herb has high Lipid A-binding activity in the screening test was isolated by using macroporous adsorptive resins technology, ultra filtration membrane technology and HPLC technology. In the isolation proceedings of aqueous extract from the herb, the biosensor affinity technology has been used to detect Lipid A-binding activity for various kinds of elutes in order to screen the active site from them. Furthermore, we evaluated the anti-LPS activity of the elute fraction which has the highest binding activity to Lipid A among them in vitro and in vivo, As a result, the active compound against LPS was attained.Second, the active compound was purified by HPLC and the purified monomer with high Lipid A-binding activity was analyzed by infrared, mass spectrometry and NMR (nuclear magnetic resonance) in order to identify its structure.Finally, anti-sepsis activity of the monomer was studied in vitro and in vivo. In detail, the neutralization of the monomer to LPS was detected by kinetic turbidimetric limulus test. Meanwhile, ELISA and RT-PCR was used respectively to measure the release of cytokine (TNF-αand IL-6) and detect the expression of TLR4 mRNA in RAW264.7 cells exposure to LPS with or without pretreatment of the monomer. Furthermore, the protective effect on mice subjected to lethal dose of heat-killed E.coli challenge with or without treatment of the monomer was observed.Result Five herbs were found to possess high Lipid A-binding activity in the screening test of aqueous extracts from sixty traditional Chinese herbs (RU>200 arc second). They were Radix Paeoniae Rubra, Rhizoma Picrorhizae, Galla Chinensis, Scutellaria baicalensis Georgi and Fructus Chebulae. To weigh the merits and demerits, we selected Scutellaria baicalensis Georgi for further study. Subsequently, SbG-4 was isolated from the herb, which had high Lipid A-binding activity. Moreover, SbG-4 could significantly increase the survival rate of sepsis mice induced by heat-killed E.coli. Interestingly, the Chemical compound assessment of SbG-4 indicated it is a flavonoid compound. So, we selected SbG-4 further to isolate using ultra filtration membrane technology and HPLC technology. In this step, we collected five HPLC fractions and named from 5KL-1 to 5. Among them, 5KL-1 had the highest Lipid A-binding activity and significantly neutralizing LPS in vitro. In RAW264.7 cells, the supernatant TNF-αlevel was increased after the cells exposure to LPS, however, this effect was attenuated by the pretreatment with 5KL-1. Likewise, 5KL-1 could protect mice from heat-killed E.coli challenge. Collectively, 5KL-1 has anti-LPS activity and deserves further study.After purified by HPLC, 5KL-1 yielded three products and named from 5KL-1A to C. among them, 5KL-1B had the highest binding activity to Lipid A. So, we selected it for further study. HPLC analysis to show that 5KL-1B is a single peak and purity factor is 999.685. After the structure was analyzed by mass spectrometry, infrared and NMR, 5KL-1B was confirmed as 2',5 ,6',7-tetrahydroxy-flavanonol (THF), molecular formula is C15H12O7.A series of experiments were used to evaluate the anti-LPS activity of THF. In detail, THF could neutralize LPS and decrease the release of TNF-αand IL-6 in LPS-induced RAW264.7 cells in a dose-dependent manner. Furthermore, THF reduced the expression of TLR4 and TNF-αmRNA in RAW264.7 cells induced by LPS in a dose-dependent manner too. In vivo experiments, Escherichia coli 35218 (E.coli 35218) was used to induce sepsis in an animal model. We also found that THF could protect mice against a lethal challenge with heat-killed E.coli in a dose-dependent manner.Conclusion An active monomer, 2', 5, 6', 7-tetrahydroxy-flavanonol (THF) was attained from Scutellaria baicalensis Georgi. In vitro, THF could neutralize LPS and suppress the activating of RAW264.7 cells induced by LPS. In vivo, THF could significantly decrease the plasme LPS level in endotoxemia mice and protect mice against a lethal challenge with heat-killed E.coli. Collectively, THF has anti-sepsis activity.