| ObjectiveTo study the absorption model and effective factor of polysaccharides from vinegar-baked Bupleurum radix(VBCP).MethodsPart Ⅰ.Immune activity screen of different parts of VBCP1.The isolation and the screening of the immune activity of different molecular weight of VBCP:1)Membrane separation method was used to separate the polysaccharide from vinegar Bupleurum Chinese into 5 parts with different molecular weight ranges.2)The mice model of delayed type hypersensitivity was established by applying 50 μ L of 1%2,4-dinitrofluorobenzene(DNFB)on the abdominal skin of mice for two days,and applying 10 μL 1%DNFB on the right ear of mice 7 days later.Before modeling,the mice were administrated with polysaccharides(100mg·kg-1·d-1)of VBCP for 3 consecutive days,and then continued to be administered for 7 days during modeling.After 10 days,the degree of ear swelling,the thickness of abdominal skin as well as the organ index,the number of lymphocytes and the morphological changesof the Peyer’s patches in mice were determined.2.Isolation and characteristics determination of homogeneous VBCP:The column chromatography method was used to separate and purify the active VBCP.The 3-hydroxybiphenyl colorimetric method,high performance liquid chromatography(HPLC)method with PMP pre-column derivatization,high-performance gel permeation chromatography(HPGPC)method,and infrared spectroscopy(IR)detection were used to determine the basic properties of the homogeneous VBCP,such as uronic acid content,polysaccharide content,monosaccharide composition,molecular weight and so on.3.Fluorescent labeling of homogeneous VBCP:homogeneous VBCP was labeled with fluorescein isothiocyanate(FITC),which prepared for the subsequent detection of oral absorption of the active polysaccharide.4.Vitro transport model establishment:Caco-2 cells(5×104/mL)were evenly seeded in Transwell plates and cultured in a cell incubator(37℃,5%C02)for 16 days to establish the Caco-2 monolayer cell model.The integrity of Caco-2 cell monolayer was determined by measuring the cell transmembrane resistance value TEER(Ω·cm2),the alkaline phosphatase activity on both sides of the cell layer,the transmembrane transport of FITC,and the morphological observation of the cell layer.The M cell model was established by co-culture of Caco-2 cells and Raji B cells:at the first 11 days,Caco-2 cells were cultured according to the method of establishing a Caco-2 monolayer cell model,and from about the 12th day,Raji B cells(1×105/mL)was inoculated into the lower chamber of the Transwell plate,and co-cultured the two cells to induce Caco-2 cells to differentiate into M cells.At the same time,the changes in the cell transmembrane resistance TEER(Ω·cm2)of the M cell model was observed.5.The transportation study of FITC-labeled homoacetic polysaccharide in cell models:Each fluorescently labeled homogeneous polysaccharide of vinegar Bupleurum Chinese was set in three concentration groups of low(250 μ g/mL),medium(500 μ g/mL)and high(1000 μg/mL).Multifunctional microplate reader was used to measure and calculate the transport amount and apparent permeability coefficient of FVBCP1-4,FVBCP3-4 and FVBCP5-1 in Caco-2 monolayer cell model and M cell model with different concentrations.In addition,a normal control group(37℃)and a low temperature group(4℃)are set.The same detection method was used to determine and compare the transport amount of FVBCP1-4,FVBCP3-4 and FVBCP5-1 in two cell models at the same concentration(1000 μg/mL)at different temperatures.Result1.The isolation and the screening of the immune activity of different molecular weight of VBCP:1)Five polysaccharides of different molecular weights VBCP were obtained and named VBCP1,VBCP2,VBCP3,VBCP4 and VBCP5.2)Screening of the immune activity of different molecular weight of VBCP:The sequence of immunoregulatory activities in different parts of VBCP was:VBCP5=VBCP4>VBCP1>VBCP3>VBCP2,Comparing with the model control group,VBCP1 significantly reduced the spleen index,the ear swelling,and the skin thickness(P<0.05 or P<0.01).VBCP4 significantly reduce spleen index,the ear swelling and the skin thickness(P<0.05 or P<0.01),VBCP5 significantly reduce ear swelling and skin thickness(P<0.05 or P<0.01),while VBCP3 and VBCP2 significantly reduced the skin thickness(P<0.01).The observation and comparison found that the morphological changes of the Peyer’ s patches in each group were not obvious.In addition,compared with the normal group,the number of lymphocytes of the Peyer’s patches in the model group was significantly increased.Compared with the model group,except for the VBCP2 group,the lymphocytes in the other polysaccharide groups were significantly reduced.2.Isolation and characteristics determination of homogeneous VBCP:Three uniform polysaccharides VBCP1-4,VBCP3-4 and VBCP5-1 were isolated respectively from the active polysaccharides VBCP1,VBCP3 and VBCP5.Among them,VBCP1-4 and VBCP3-4 are pyranose with similar monosaccharide composition,and mainly composed of rhamnose,galacturonic acid,galactose,arabinose and an unknown component.The polysaccharide content and uronic acid content of VBCP1-4 were>76.80%and 67.74%,respectively.Its average molecular weight is greater than 2400Kd,and the molar ratio of known components in the monosaccharide composition is(Rha:Gal A:Gal:Ara=1.05:4.20:1.67:4.09,and a small amount of Glc).The average molecular weight of VBCP3-4 is 845.46Kd,the polysaccharide content and uronic acid content are>89.02%and 64.32%,respectively.The molar ratio of known components in the monosaccharide composition is(Rha:Gal A:Gal:Ara=0.80:9.39:0.39:1.34).In addition,VBCP5-1 is furanose,and the molar ratio of its monosaccharide composition is(Man:Gal-acid:Glc:Gal:Ara=0.25:0.24:4.72:1.02:0.83),its average molecular weight is 30.78Kd,and the polysaccharide content is 50.06%,while the content of uronic acid is only 2.40%.3.Fluorescent labeling of homogeneous VBCP:A method for the fluorescence synthesis of VBCP was established,and FITC fluorescently labeled homogeneous polysaccharides FVBCP1-4,FVBCP3-4 and FVBCP1-5 were prepared.The fluorescence substitutions of them were 0.50%,0.39%and 0.38%,respectively.4.Vitro transport model establishment:By measuring cell transmembrane resistance,alkaline phosphatase activity,FITC transport amount,and observing cell layer morphology,a Caco-2 monolayer cell model and M cell model with good integrity were successfully established.5.The transportation study of FITC-labeled homoacetic VBCP in cell models:In the Caco-2 monolayer cell model,the apparent permeability coefficients of the three kinds of homogeneous polysaccharides of VBCP at different concentrations were all shown as Papp<10-7(means their absorption<1%).While In the M cell model,the Papp values of them were between 10-7~10-6(means the absorption were 1%~100%).At the same time,the transport amount of these three kinds of polysaccharides at 37℃ was obviously higher than that at 4℃.All these suggested that the polysaccharides FVBCP1-4,FVBCP3-4 and FVBCP5-1 of VBCP may be absorbed through the active transport(endocytosis)of M cells in the intestine.In addition,the transport amounts of VBCP in the same concentration but different molecular weight in the M cell model were basically the same,which suggesting that the oral absorption and transportation of polysaccharides from vinegar Bupleurum Chinese may not have a great relationship with their molecular weight.Conclusion1.The polysaccharides from different parts of vinegar Bupleurum Chinese have different levels of anti-inflammatory immune activity,but their activity may not have much relationship with their molecular weight.2.The oral absorption of polysaccharides from vinegar Bupleurum Chinese with different molecular weights may be achieved through the endocytosis of M cells in the intestine,but its absorption may not be related to the molecular weight of polysaccharides.3.Absoption of polysaccharides may be related with the content of uronic acid,monosaccharide composition and sugar configuration,which one is the main influencing factor needs to be further explored. |
PDF Full Text Request