Study On Pharmacokinetics Of Polysaccharide From Lentinus Edodes And Its Mechanism Of Absorption And Metabolism In Vivo

Lentinus edodes,named as xianggu and shiitake,is the world's second largest artificially grown medicine and food homologue.It is delicious and extremely nutritious.The theory of traditional Chinese medicine believes that the Lentinus edodes has the effect of "replenishing the vitality of life and strengthening the spleen,restoring genuine qi and expelling illness,and prolonging life".Modern medical research shows that Lentinus edodes contain a variety of effective medicinal ingredients,chief of which is lentinan(LNT).LNT injection has been widely used in the adjuvant treatment of tumors in clinic and has been proven to have obvious curative effect.Oral preparations of LNT,such as lentinan tablets and lentinan capsules,have begun to be used in the adjuvant treatment of digestive tract tumors and immune regulation.As a health care product,the oral preparation of lentinan is popular in Europe and the United States.At present,various pharmacological activities of lentinan are still being developed,for example,it has been confirmed to have significant effects in anti-diabetes,anti-fatigue,anti-radiation,anti-colitis and so on.However,the pharmacokinetic information of LNT has rarely been reported,which severely limited the further promotion and security of lentinan as a drug in clinic.Therefore,this subject follows the extraction method determined early by our laboratory,improves the purification process,extracts and purifies LNT from the Lentinus edodes of Fangxian County.LNT is traced by radionuclide labeling and fluorescent labeling.Systematic study of pharmacokinetics after oral and intravenous administration of LNT.The intestinal absorption mechanism of LNT was studied using the Ussing Chamber and Caco-2 cell monolayer model.The liver metabolic degradation mechanism of LNT was studied by in vitro liver microsome incubation.It aims to systematically and deeply explore the pharmacokinetics of LNT in vivo.Part ?.The isolation and purification of the lentinanThe crude polysaccharide of Lentinus edodes was obtained by water extraction and alcohol precipitation method.The water-soluble refined LNT was obtained by H2O2 decolorization,ultrafiltration,medium pressure column separation and freeze drying.The sugar content was 97.8±2.9%by phenol-sulfuric acid method.The molecular weight was determined by high performance gel permeation chromatography(HPGPC)and size exclusion chromatography(SEC).UV scanning suggested that lentinan contained almost no nucleic acid and protein.The results showed that the lentinan extracted and purified in this experiment has high purity and good molecular weight uniformity,which is suitable for subsequent experimentsPart II.Establishment of qualitative and quantitative detection methods in biological samples by labeling of lentinanA diethylenetriaminepentaacetic acidic(DTPA)derivative of LNT(D-LNT)was synthesized first to allow labelling with 99m-technetium(99mTc).The infrared spectrum of D-LNT showed that the absorbance intensity at 1735 cm-1 was weak but obvious.In addition,the dramatic increase in nitrogen from 0.05%in LNT to 0.57%in D-LNT indicated that the esterification reaction had occurred.The curve of y intensity from the PD-10 column was basically consistent with the curve of LNT concentration.In addition,the Rf of Na99mTcO4 and 99mTc-LNT in chromatographic separation assays was 0.70 and 0.09,respectively,indicating that the labelling process was successful.The negligible differences in retention time and peak shape between LNT and D-LNT indicated that our labelling process had almost no effect on the molecular size and distribution of LNT Congo red experiment results show that D-LNT still maintains the triple helix structure of LNTThe stability analysis results showed that the radiochemical purity was 92.25 ± 2.38%and 91.28 ± 1.39%at 24 h in PBS and serum,respectively.The calibration curves for all the biosamples showed good linearlty(r2>0.998)over the concentration ranges tested.The precision was less than 6.12%and the accuracy ranged from 95.01%to 104.51%for QC samples.The total recovery in mice was 90.46 ± 4.77%.To characterize the specificity of 99mTc-LNT,a controlled study comparing the uptake of radioactivity from unbound and bound 99mTc was carried out.99mTc-LNT was mainly concentrated in the liver and spleen,whereas free 99mTc was mostly distributed in the stomach.These results showed acceptable stability,precision,accuracy,specificity and total recovery.Lentinan was labeled with water-soluble fluorescein 5-DTAF by reacting in a carbonate buffer of pH=9.5 at 25 ? and 4 ?,respectively.FLNT was characterized by infrared spectroscopy,elemental analysis,UV spectroscopy,fluorescence spectroscopy,and HPGPC fluorescence detection.Similarly,HPGPC differential detection results and Congo red experiments confirmed that the fluorescent labeling process had no significant effect on the molecular weight and spatial structure of LNT.The effects of protein removal conditions and 37 ? incubation conditions on the peaks of FLNT were investigated.The specificity of FLNT fluorescence peaks in biological samples was confirmed,and the qualitative detection of LNT in biological samples was established.In this part,we combined with the advantages of radiolabeling and fluorescent labeling to establish a qualitative and quantitative detection method for LNT in biological samples,which provided a basis for the study of pharmacokinetics of LNT in vivo.Part III.Pharmacokinetics of LNT after oral administrationThe blood concentration,tissue distribution and excretion of 99mTc-LNT after oral administration were explored in this part.The results showed that LNT had obvious absorption after oral administration,and reached the maximum blood concentration at 1 h.LNT was still detected significantly in the blood at 8 h.There was a certain distribution in each tissue,and the LNT content of orally absorption-related gastrointestinal tissue distribution is the most,followed by the liver.99mTc-LNT is mainly excreted with feces after oral administration,but there is still a significant part of excretion with urine.Part IV.Study on oral absorption mechanism of LNTThe absorption mechanism of LNT was explored by Ussing Chamber technique and Caco-2 cell monolayer model.Ussing Chamber results showed that LNT was significantly absorbed in the intestinal tract.The Papp values of FLNT in the duodenum,jejunum,ileum and colon were 1.08×10-6 cm/s,1.62×10-6 cm/s,4.06×10-6 cm/s and 0.70×10-6 cm/s,respectively.The ileum is the dominant intestinal segment absorbing LNT.The uptake of FLNT by Caco-2 cells was observed by laser confocal microscopy.It was confirmed that the uptake of FLNT by Caco-2 cells was obvious and evenly distributed in the cytoplasm.Flow cytometry was used to semi-quantitatively analyze the uptake of FLNT by Caco-2 cells and to study the transmembrane transport mechanism of FLNT(1)In the FLNT uptake experiment,it was found that NaN3 and low temperature environment(4 ?)can inhibit the uptake of FLNT by Caco-2 cells;(2)Endocytosis inhibitors Chloropromazine,Dynasore,Ly294002 and Cytochalasin D significantly inhibited the uptake of FLNT by Caco-2 cells,while Genistein and ?-CD had no effect on FLNT uptake;(3)Intracellular transport inhibitors Bafilomycin A1 and Thapsigargin significantly inhibited the uptake of FLNT by Caco-2 cells,while Chloroquine,Vinblastine and Monensin had no effect on FLNT uptake(4)In the exocytosis experiment of FLNT,it was found that Bafilomycin A1 and Vinblastine had significant inhibitory effects on FLNT outgrowth,while Chloroquine,Thapsigargin and Monensin had no effect on the exocytosis of FLNTThe Caco-2 cell monolayer model was constructed by transwell chamber,and the transport mechanism of FLNT in the epithelial cell monolayer was investigated by adding various endocytic pathway inhibitors.The results showed that transmembrane transport rates were reduced by 26.89%,16.83%,22.30%and 34.65%,respectively,after addition of Dynasore,Chloropromazine,Ly294002 and Cytochalasin D.However,there was no significant change in transmembrane transport rate after adding Genistein and ?-CD.This result is basically consistent with the results of flow cytometry to detect the uptake of FLNT by Caco-2 cellsIt is confirmed by the results of this part that FLNT can be trans-membrane transported by clathrin-mediated pathway and macrophage pathway,which may be one of the mechanisms of oral absorption of LNTPart V.Pharmacokinetics study after intravenous administration of LNTThe pharmacokinetic characteristics of LNT after intravenous administration were systematically studied by combining with SPECT/CT in vivo imaging.The results of blood concentration showed that 99mTc-LNT was biphasic elimination after intravenous administration,and the dose of LNT had no effect on the pharmacokinetic characteristics after intravenous administration at the dose range of 0.5?8 mg/kg.The results of distribution showed that 99mTc-LNT was rapidly accumulated in the liver after intravenous administration,followed by the spleen.Excretion results showed thatit was mainly excreted by the kidney with urine after intravenous administration of 99mTc-LNT,but fecal excretion also occupied a certain proportion.SPECT/CT imaging confirmed that the strongest part of the 99mTc-LNT signal was the liver,and the ? signal was still strong at 4 h.SPECT dynamic imaging visually showed the real-time dynamic change of the body within 1 h after intravenous administration of 99mTc-LNT,and its change trend was consistent with the results of previous blood drug concentration,body distribution and excretionPart ?.Study on the mechanism of LNT liver degradation and metabolismThe mechanism of metabolic degradation of LNT liver was studied by liver microsome incubation.Based on the qualitative detection method in FLNT biological samples,the optimal conditions for the incubation of FLNT with liver microsomes were optimized.The degradation degree of FLNT was judged by the ratio of H2/H1(degradation peak intensity/primary peak intensity)as a degradation parameter.The results showed that two subtypes of CYP2D and CYP2C,as well as epoxide hydrolase,were involved in the metabolic degradation of liver LNT.