Study On Antiendotoxin Constituents From Garcinia Mangostana L.

Endotoxin （lipopolysaccharide, LPS） is the major component of outer membrane of gram-negative bacteria. The recognition of LPS by the innate immune system via a set of pattern recognition receptors （PRRs） results in the activation of inflammatory cells, it subsequently induce the overproduction of proinflammatory mediators （e.g., tumor necrosis factor-alpha and interleukin-6） and eventually cause sepsis, which is also known as systemic inflammatory response syndrome （SIRS） and has become the leading cause of death among patients in intensive care units （ICUs）. However, the incidence and mortality rate are high, and there are still few effective therapeutic drugs available in clinical practice. The excessive inflammatory response will be ameliorated if the agent could enable the neutralization of LPS and then block the recognition of LPS by the receptors. It, therefore, has great significance to screen and research the LPS-neutralizing lead compounds for the research and development of anti-sepsis drug.Based upon the potential anti-LPS activity of the extract of Garcinia mangostana L. （mangosteen）, a traditional natural medicine, the bioactivity-guided isolation of anti-LPS compounds, structure elucidation by spectral analysis, quantitative determination and fingerprint by high performance liquid chromatography （HPLC）, determination of affinity constant by utilizing affinity sensor, neutralization of LPS assayed by limulus amebocyte lysate test and inhibition of TNF-αrelease from LPS-induced RAW264.7 cells determined by enzyme-linked immunosorbent assay （ELISA） were studied in this thesis. The results are as follow:1. Isolation and structure elucidation of anti-LPS ingredients from mangosteen.Six compounds were obtained by combined use of macroporous adsorptive resins, polyamide column chromatography, silica gel column chromatography, preparative thin layer chromatography and preparative liquid chromatography. They were identified as catechin, epicatechin, procyanidin dimer B2, procyanidin trimer,α-mangostin andγ-mangostin, respectively, by spectroscopic data （ultraviolet spectrum, electrospray ionization-mass spectrometry, nuclear magnetic resonance） analysis.2. Quality control of anti-LPS ingredients from mangosteen.A new method was developed for the simultaneous quantitative determination of catechin, epicatechin, procyanidin dimer B2 and procyanidin trimer in the pericarp of mangosteen by HPLC. Separation was performed on a reverse phase octadecyl silane column. The mobile phase was acetonitrile/water （12:88）, the column temperature was kept constant at 30℃, the flow rate was 1 ml/min and the wavelength was 230 nm. A good linear relationship was established in the range 50²50 ng for catechin with the regression equation y=1.9662x^-5.17, r=0.9992; 250¹250 ng for epicatechin with the regression equation y=2.1752x-113.07, r=0.9993; 250¹250 ng for procyanidin dimer B2 with the regression equation y=0.8531x+6.81, r=0.9995; 250¹250 ng for procyanidin trimer with the regression equation y=0.7504x-98.64, r=0.9993. It is simple and reliable for the quantitative determination and quality control of procyanidins of mangosteen.The fingerprint of total xanthones of mangosteen was determined by HPLC. All the separations were performed on a reverse phase octadecyl silane column. The mobile phase was a gradient methanol/water elution, the column temperature was kept at 30℃, the flow rate was 1 ml/min and the wavelength was 320 nm. HPLC fingerprint method was established by usingα-mangostin as reference substance. It could be used for the quality control of xanthones of mangosteen;3. Assessment of anti-LPS activity of ingredients from mangosteen in vitro.The results of in vitro test demonstrated that catechin, epicatechin, procyanidin dimer B2 and procyanidin trimer could bind to lipid A. The dissociation equilibrium constants （KD） of catechin, epicatechin, procyanidin dimer B2 and procyanidin trimer were 2.96×10^-4, 5.32×10^-4, 1.58×10^-5 and 9.72×10^-6 M, respectively. Catechin, epicatechin, procyanidin dimer B2 and procyanidin trimer could neutralize LPS. Catechin, epicatechin, procyanidin dimer B2, procyanidin trimer,α-mangostin andγ-mangostin could selectively inhibit LPS-induced TNF-αrelease from RAW264.7 cells. They possessed potential anti-LPS activity. It revealed that catechin, epicatechin, procyanidin dimer B2 and procyanidin trimer are novel anti-LPS ingredients in traditional natural medicine mangosteen.