| Lycium barbarum polysaccharide (LBP) is one of the important active ingredients in Chinese wolfberry which is considered as a famous traditional Chinese herbal medicine. Pharmacodynamic studies show that LBP has a good effect of reducing blood sugar. However, compared with its pharmacodynamics, the metabolic dynamics of LBP in vivo is not yet clear. Therefore, to study the structure characteristics, absorption and metabolism processes of LBP or other polysaccharides is the key to further clarify the active effects of the LBP and other polysaccharieds. So the chemical composition and basic structure of LBP were discussed in this study based on the previous research, the pharmacokinetics, tissue distribution and excretion of LBP in rats were also studied to elucidate the process of the absorption, distribution and excretion of LBP in rats, meanwhile, the changes of metabolic profiling in rats after LBP in oral administration were studied by using the method of metabolism, which provided a new way to study the mechanism of hypoglycemic effect of LBP, and provided scientific basis for further research and utilization of LBP. The main research works of this paper are as follows:1. Studies on the chemical composition and structure analysis of LBPLBP were extracted from the Ningxia wolfberry fruit by the method of warm water extraction and membrane separation, the crude LBP was preliminary purified by the processing of Sevage method to deproteinization, dialysis water and freeze-drying, and further purified by chromatography of DEAE cellulose column and Sephadex G-150 to get the sub-fraction of LBP3. The high performance permeation chromatography (HPGPC) analysis showed that the average molecular weight (Mw) of LBP3 was about 4.9 kDa. Monosaccharide composition analysis by PMP-HPLC revealed that the LBP3 was comprised of D-mannose, L-rhamnose, D-glucose, D-galactosamine and D-xylose with a molar ratio of 5.52:5.11:28.06:1:1.70.The preliminary structure features and morphology of LBP3 were investigated by Ultraviolet spectrum scanning (UV), Fourier Transform Infrared Spectroscopy (FT-IR), Nuclear magnetic resonance spectroscopy (NMR) and Scanning electron microscope (SEM), the results indicated that LBP3 is a proteoglycan which molecular structure containing amino or imino groups, mainly composed of alpha-D-pyranose residues, the content of beta configuration is low. So it is speculated that there are eight kinds of linear structure of LBP3 combined with the above analysis.2. The flurocescent labeling of LBP3 and their biodegradable stability in biological samplesLBP3, similar to other polysaccharides such as glucosan, possesses neither light nor chromophore groups structurally, so derect detection is impossible with UV or flurocescence method, and Mass spectra method is unsuitable to quantification analysis of polysaccharides either. Therefore, LBP3 was labeled by fluorescein of FITC with carbon acylation reaction to obtain LBP3-FITC. The maximum excition and emission wavelength of LBP3-FITC is 495 nm and 518 nm. The labeling efficiency was evaluated by HPLC-FD method and the labeling rate of FITC in LBP3-FITC molecular was calculated as 1.3% according to the calibration curve based on the FITC standards; The average molecular weight of LBP3-FITC was not significant different from that of LBP3.The stability of LBP3-FITC in vitro and in vivo was investigated using HPLC-FD and fluorescence spectrum analysis method. After 0,1,6,12,24h incubation in phosphate buffer, rat blank plasma and rat blank urine under the condition of 37℃ and 60 r/min, the molecular weight and fluorescence intensity were not changed, likewise, the same results were obtained by LBP3-FITC in the lysozyme and alpha amylase solution after 2,6 and 12h incubation under the conditions of 37℃,60 r/min. Which showed LBP3-FITC was stable in vitro. After oral administration of LBP3-FITC to rats, LBP3-FITC was in its original form in rat plasma urine and feces, which demonstrated LBP3-FITC metabollized in its original form.3. The pharmacokinetics study of LBP3-FITCFirstly, the fluorescence quantitative analysis method of LBP3-FITC in plasma, urine, feces and tissues of rats was established. The results showed that the fluorescence intensity of LBP3-FITC was good in the range of 0.2-20 g/mL concentration in rat plasma, the inter-day CV% at 0.2,2.0 and 20 μg/mL of LBP3-FITC was between 1.97%-7.28%, the intra-day CV% at the above concentrations was between 2.62%-7.75%; The stability of plasma samples was not changed after the treatment of placing at room temperatue 24h, repeating freezing and thawing for 3 times,-20℃ frozen 15d. The inter-group recovery at 0.2,2.0 and 20 μg/mL of LBP3-FITC was between 84.6%-104.0%, the intra-group recovery at the above concentrations was between 98.3%-100.2%. In addition, LBP3-FTIC in rat urine, feces and tissues of the linearity, precision, stability and recovery were all in line with the analysis of biological samples in vivo quantitative analysis, showed that the established method is suitable for LBP3-FITC pharmacokinetics studies.The pharmacokinetics of LBP3-FITC in rats was studied by the method of fluorescence quantitative analysis. The results showed that after single oral administration of 100,50 and 25 mg/kg LBP3-FITC, the AUC0-t was 151.09±15.10,141.25±12.02,128.21±27.64 mg/L.h, the AUC(0-∞) was 230.49±73.26,236.18±35.08,242.57±64.09 mg/L.h, the Cmax was 7.44±0.72,6.56±0.51,5.27±0.44 mg/L respectively. The average elimination half-life of the drug in the three doses was 38.41±9.55 h, which indicated that LBP3-FITC had a long half-life and elimination period.LBP3-FITC was widely distributed to tissues of most organs in rats 1h after oral administration of LBP3-FITC, such as stomach, intestine, liver, kidneys and heart. The concentration in intestine and stomach was the most highest in all tissues, the concentration in liver, kidneys and heart was higher than other tissues. The concentration in stomach and intestine degraded rapidly, and increased rapidly in liver, kidneys and large intestine 6h after oral administration of LBP3-FITC, which showed that LBP3-FITC was excreted mainly by kidney after absorption, for all tissues, the concentrations of LBP3-FITC had significantly decreased at the time point of 24h after oral administration.The cumulative excretion of LBP3-FITC in urine and feces of rat after oral administration were 0.094±0.036% and 92.18±3.609% respectively. Drug which was excreted totally in urine and feces in 72h after an oral administration of LBP3-FITC was equal to 92.274% of given dose. LBP3-FITC was mainly excreted from feces after oral administration.4. Study on the metabolism of LBP3 in serum, urine and liver of ratsThe gas chromatography-time of flight mass spectrography (GC-TOF MS) was developed for rat serum metabolite profiling study of control group (NC group), diabetes model group (DM group) and drug intervention group (LBP3 group). Principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were used for their clustering and biomarker searching. The result demonstrated that the three groups were successfully separated and serum alanine, thymidine were significantly decreased in type 2 diabetic group. After one month intervention by LBP3, the results indicated that the LBP3 can increase the level of alanine, thymidine in serum of type 2 diabetic rats, which may be associated with their amino acid and nucleotide metabolism.The gas chromatography-time of flight mass spectrography (GC-TOF MS) was developed for rat urine metabolite profiling study of NC group, DM group and LBP3 group. PCA and OPLS-DA were used for their clustering and biomarker searching. The result demonstrated that the three groups were successfully separated and methylmalonic acid, benzoic acid and galactose levels in urine were significantly increased in type 2 diabetic group while the levels of 2-hydroxybutyric acid purine riboside were decreased. The method was applied to the action mechanism study of LBP3. The results indicated that LBP3 can improve the metabolism of these metabolites in urine of type 2 diabetic rats, which may be associated with their anti-oxidative stress function, amino acid and nucleotide metabolism as well as gut microflora metabolism.The gas chromatography-time of flight mass spectrography (GC-TOF MS) was developed for rat liver tissue metabolite profiling study of NC group, DM group and LBP3 group. PCA and OPLS-DA were used for their clustering and biomarker searching. The result demonstrated that the three groups were successfully separated and myoinositol, hydroxybutanoic acid and pantothenic acid levels in liver tissue were significantly decreased in type 2 diabetic group while the levels of L-malic acid,3,6-anhydro-D-galactose and arachidic acid were increased. The method was applied to the action mechanism study of LBP3. The results indicated that LBP3 can improve the metabolism of these metabolites in liver tissue of type 2 diabetic rats, which may be associated with their three carboxylic acid cycle (TCA), insulin sensitivity and fat metabolism. Conclusions1. LBP3, as sub-fraction of LBP, was obtained by column chromatography purification, it mainly consisted of five kinds of monosaccharide residues, and there were amino or imino groups in LBP3 molecular, LBP3 was mainly composed of a-D-pyranose residues, the content of P configuration was low.2. FITC was successfully labeled on LBP3 molecular by the covalent coupling interaction, and the solubility and molecular weight of them were not changed significantly, LBP3-FITC showed good stability in vivo and in vitro.3. The fluorescence quantitative analysis method of LBP3-FITC determination in biological samples was established and comfirmed for the first time to obtain the pharmacokinetic data of LBP3-FITC by oral administration.4. LBP3-FITC had a long half-life and a slow elimination in animals, and mainly excreted in the form of a prototype from urine and feces.5. GC-TOF MS was established for the study of the metabolites in serum, urine and liver tissues of rats, multivariate statistical analysis revealed that the serum, urine and liver tissue in DM group were significantly changed compared with the NC group, which suggested that there might be an abnormality of amino acid, nucleotide, three carboxylic acid cycle as well as fat metabolism and intestinal flora metabolism. After a month intervention of LBP3, the same biomarkers in the serum, urine and liver tissues of DM rats were changed to normal levels, thus LBP3 is beneficial to promote the recovery of amino acids, nucleotides, three carboxylic acid cycle, fat metabolism and intestinal flora metabolism of DM rats. The results of this study would be helpful to hypoglycemic mechanism study of LBP, also be benefit to the development and utilization of LBP. |
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