Isolation,Purification,Structure Analysis And Biological Activity Screening Of PaoTianXiong(A Processed Product Of The Tuber Of Aconitum Carmichaeli Debx.)

ObjectivePaoTianXiong(PTX)is one of the processed products of the tuber of Aconitum carmichaeli Debx(Fuzi in Chinese).The Aconitum carmichaeli Debx has to be processed for use as herbal medicine because processing can reduce the toxic alkaloids.Processing is an ancient Chinese pharmaceutic technique to facilitate the use of Chinese herbal medicines for a specific clinical need.Polysaccharide is the main active component of PTX.The processing process of traditional Chinese medicine is usually accompanied by complex physical and chemical changes,which may cause changes in the properties and activities of polysaccharides.Therefore,the polysaccharides of PTX(PTXP)are different from the polysaccharides in Aconitum carmichaeli Debx.Since no one has reported the structure and biological activity of PTXP,it is of great significance to study the structure of PTXP and screen its biological activity.Methods1.The PTXPs were extracted by ultrasonic-assisted extraction,cellulase assisted-extraction,diluted water extraction and hot water extraction,and purified preliminarily.By comparing the extraction yield,monosaccharide composition,molecular weight and other structural information of PTXPs extracted by different extraction methods,as well as scavenging rate of DPPH and ABTS free radical,the best extraction method of PTXP was screened and to be optimized by response surface methodology.2.Crude PTXP was extracted.After extracted by hot water extraction method,alcohol precipitation,sevage method to remove protein,purification by anion exchange chromatography and gel column chromatography,purified PTXP was obtained.And then infrared spectrum scanning,molecular weight determination,monosaccharide composition determination,methylation experiment,nuclear magnetic resonance analysis methods were used to study the structure of PTXP.3.Cell experiments were conducted to investigate whether PTXP has immunomodulatory activity.The model of H9c2 cardiomyocytes damaged by H2O2 was used to explore the myocardial protective effect of PTXP.To investigate the anti-tumor activity of the refined polysaccharides,the inhibitory rate of PTXP on adenocarcinoma,human alveolar basal epithelial cells,human colon cancer cells,human nasopharyngeal carcinoma cells and other tumor cells was detected.4.The sulphate derivatives of PTXP were prepared by the method of sulfur trioxide-pyridine.The immunomodulatory,anti-inflammatory and anti-tumor activities of the sulphate derivatives of PTXP were investigated by the experiment of promoting the proliferation of spleen lymphocytes from mice,LPS-induced macrophage RAW264.7 inflammatory model,and experiment of inhibiting the proliferation of HepG2 cells.Results1.Different extraction methods were used to extract PTXPs.Hot water extraction method had the highest extraction yield and little effect on the structure and antioxidant activities of PTXP.Cellulase extraction method was more likely to have an impact on the structure of PTXP.In addition,the monosaccharide composition of PTXPs extracted by different extraction methods were the same.The scavenging ability of PTXPs extracted by different extraction methods on DPPH and ABTS free radicals was different,especially the antioxidant activity of crude PTXPs and purified PTXPs was significantly different.In general,hot water extraction method was considered as the best extraction method for PTXP.The optimal extraction process was as follows:the extraction temperature was 90?,the extraction time was 2 h 10 min,the solid-liquid ratio was 1:29,the number of extraction was 2.2.After extraction,alcohol precipitation,deproteinization and lyophilization,the crude polysaccharide was separated into two parts by anion exchange chromatography:water elution part and 0.05 mol/L NaCl solution elution part.The purified polysaccharide PTXP-W was isolated from the water eluted part,the purified polysaccharides PTXP and PTXP-N were isolated from the elution part of 0.05 mol/L NaCl solution.And PTXP-W,PTXP and PTXP-N were all a-glucan,which were easily soluble in water.We studied PTXP in-depth.The protein content of PTXP was 0.68%,and the content of uronic acid was 2.47%.PTXP is mainly composed of 1?4 and 1?6 glycosidic bonds.3.PTXP could promote the proliferation of spleen lymphocytes from mice.At the same time,PTXP could promote the proliferation of spleen T lymphocytes induced by ConA and B lymphocytes induced by LPS.Flow cytometry showed that PTXP could increase the CD4+/CD8+value of splenic lymphocytes induced by ConA,which indicated that PTXP had certain immunomodulatory ability.In addition,PTXP showed no direct antitumor activity.PTXP did not play a protective role in H9c2 cardiomyocyte injury induced by H2O2.4.Three derivatives,PTXP-S1,PTXP-S2 and PTXP-S3 with different degree of substitution were prepared.The infrared absorption peaks of these three derivatives at 1220 cm-1 and 810 cm-1 showed that the sulfate group had been successfully introduced on structure of PTXP-S1,PTXP-S2 and PTXP-S3.In addition,PTXP-S1,PTXP-S2 and PTXP-S3 all showed stronger ABTS radical scavenging ability than PTXP,but there was no significant difference in DPPH radical scavenging ability.The effect of PTXP-S1,PTXP-S2 and PTXP-S3 on the proliferation of splenic lymphocytes from mice was observed by microscope.PTXP-S2 had the best activity.CCK8 assay showed that PTXP-S2 could promote the proliferation of splenic lymphocytes from mice.In addition,PTXP-S2 could also alleviate the LPS-induced morphological changes of macrophages.PTXP-S2 did not show any inhibitory effect on HepG2 tumor cells,suggesting that sulfate modification could not directly enhance the antitumor activity of PTXP-S2.Conclusion1.Different extraction methods were used to extract PTXPs,and the obtained PTXPs showed different scavenging ability of DPPH and ABTS free radicals.In this experiment,the cellulase extraction method could affect the structure of PTXP,and the hot water extraction method had the highest extraction yield.In addition,considering the extraction yield of PTXPs and the effects on the structure and activity of PTXPs,the best extraction method is the hot water extraction method.2.Based on the method of hot water extraction method,the purified polysaccharides PTXP-W,PTXP-N and PTXP were isolated.The three polysaccharides were white spongy solid,soluble in water,and they were all made of glucose,belonging to a-pyranose.The main difference between the three is the difference in average molecular weight.Methylation experiments of PTXP showed that PTXP was mainly composed of 1?4 and 1?6 glycosidic bonds.PTXP-W,PTXP and PTXP-N were obtained from PTX for the first time.3.PTXP could promote the proliferation of spleen lymphocytes from mice,which indicated that it had certain immunomodulatory ability.PTXP has no direct antitumor activity,but it could not be ruled out that it plays an antitumor role by activating the immune system.And PTXP could not alleviate the myocardial injury of H9c2 cells caused by H2O2.4.Sulfation could have a certain effect on the structure and activity of PTXP.PTXP-S2,a sulfate derivative of PTXP could promote the proliferation of splenic lymphocytes from mice and inhibit the LPS-induced morphological changes of macrophages,suggesting that PTXP-S2 may have immunomodulatory effects.