Antitumor Activity Of Arabinoxylan And Its Degrading Oligosaccharides From The Residue Of Astragalus Radix

RATIONALES: Radix Astragalus(RA)was first recorded in the "Shen Nong's Herbal Classic" and regarded as a top grade herb.Its medicinal history has been recorded more than 2,000 years.In clinical application,RA is normally contained in 80% Chinese herbal medicines prescriptions.With its importance,RA has been listed as 60 strategic key varieties of the state and 18 major varieties of Chinese medicinal materials in the Ministry of Commerce.Moreover,RA has been included in the list of national drug and food homology in 2018.At present,the annual production of residues of RA in China exceeds tens of thousands of tons,which is increasing year by year.Unlike the main components of cell wall hemicellulose of other higher plants,i.e.xyloglucan(no pharmacological activity),our previous studies showed that the main component of the cell wall hemicellulose of RA herb residues is arabinoxylan.At present,it also has been proved that the polysaccharide extracted from the seed coats of cereals has strong anti-tumor activity,but no side effects.And the amount of arabinoxylan accounts for about 10% of herb residues of RA,which is 1000 times higher than that of soluble arabinogalactan in the RA cytoplasm.The latter has been developed as RA polysaccharide for injection,which is a commercial drug.Therefore,the development of hemicellulose polysaccharide anti-tumor high value-added products in RA residue will be of great significance for the benign recycling of Chinese medicine RA resources.OBJECTIVE: The active arabinoxylan was screened by four different types of arabinoxylans from RA residue,and the antitumor activity of the active arabinoxylan was evaluated by cell experiments in vitro and animal experiments in vivo.Further active arabinoxylan was degraded to obtain oligosaccharides,and structural analysis and antitumor activity evaluation of oligosaccharides were carried out,and finally high-activity oligosaccharides were obtained.To lay the foundation for the development of anti-tumor highvalue-added products of arabinoxylan in RA residue and to achieve a virtuous cycle of Chinese medicine resources.METHOD&CONTENT: In vitro cell assay,MTT assay was used to detect the inhibitory effect of four different types of arabinoxylans from RA residue on proliferation of human lung adenocarcinoma A549 cells.The anti-tumor activity of active arabinoxylans was evaluated,including scratch test,cell cycle and apoptosis experiment,q RT-PCR and Western blotting experiments.In vivo animal experiments to study the anti-tumor effect of active arabinoxylan AX-I-3 on Lewis lung cancer mice,including ELISA to detect serum cytokines IL-2,IL-6,TNF-? levels,flow cytometry to detect changes in spleen T lymphocytes,HE staining was used to detect the pathological changes of tumor tissues.The expressions of P53,Bax,Bcl-2,Cyt-C and Caspase-3 were detected by q RT-PCR and Western blot.The active arabinoxylan was acid-degraded to obtain oligosaccharides,and the structure was analyzed.High-activity oligosaccharides were screened by MTT assay,and their antitumor activities were evaluated by cell cycle and apoptosis experiments,q RT-PCR and Western blot.RESULTS: The in vitro antitumor activity of four different types of arabinoxylan AX-I-1~AX-I-4 from RA residue was detected by MTT assay.It was found that the AX-I-3 component polysaccharide had a certain proliferation inhibition effect on A549 cells,and when the concentration was400 ?g/m L,it has a strong killing effect on A549 cells and a relatively small cytotoxic effect on BEAS-2B cells.Scratch test and migration-related protein detection found that AX-I-3 may not exert anti-tumor effect by inhibiting the migration of A549 cells.The results of flow cytometry showed that the AX-I-3 component mainly blocked A549 cells in the G1 phase of the cell cycle and promoted apoptosis.The results of q RT-PCR showed that AX-I-3could significantly promote the expression of P53,Bax,Cyt-C and Caspase-3m RNA in human lung adenocarcinoma A549 cells and inhibit the expression of Bcl-2 m RNA.Western blot analysis of apoptotic proteins revealed thatAX-I-3 can increase the expression of P53,Bax,Cyt-C and Caspase-3proteins,and decrease the expression of Bcl-2.It is concluded that the active polysaccharide AX-I-3 may exert an anti-tumor effect by inducing apoptosis through the mitochondrial pathway.The study found that the arabinoxylan AX-I-3 can improve the living conditions of Lewis lung cancer mice,and has a certain inhibitory effect on the transplanted tumor of Lewis lung cancer mice.Paraffin section staining also showed that AX-I-3 inhibited growth of tumor cells.The results of ELISA showed that AX-I-3 could improve the serum cytokines IL-2,IL-6and TNF-? levels in Lewis lung cancer mice,and the effect was better when combined with the half-dose DDP,which enhanced the immune function of the body.The results of spleen T lymphocyte assay suggest that AX-I-3 may improve the cellular immune function of mice by increasing the ratio of CD4+ T cells and CD8+ T cells.Western blot analysis showed that AX-I-3may induce apoptosis of tumor cells by decreasing the expression of Bcl-2protein and increasing the expression of P53,Bax,Cyt-C and Caspase-3proteins.The polysaccharide was degraded into oligosaccharides by enzymatic hydrolysis and acid hydrolysis.The optimal degradation conditions were 0.2mol/L TFA and the hydrolysis was carried out at 80 °C for 3 h.The results of repeatability and stability evaluation of the oligosaccharides degraded under this condition showed that the acid hydrolysis method had good repeatability and stability.Monosaccharide composition analysis and mass spectrometry analysis of different molecular weight oligosaccharides showed that OS-2may be linked by xylose and glucose through 1,4 glycosidic bonds.OS-3~OS-7 may be linked by arabinose and xylose via 1,4 glycosidic bonds.The results of MTT assay showed that oligosaccharides OS-3,OS-5 and OS-6 significantly inhibited the proliferation of human lung adenocarcinoma A549 cells,and OS-3 has a better inhibitory effect.Flow cytometry results showed that OS-3 mainly blocked A549 cells in the G1 phase of the cellcycle and promoted apoptosis.The results of q RT-PCR showed that OS-3could significantly promote the expression of P53,Bax,Cyt-C and Caspase-3m RNA in human lung adenocarcinoma A549 cells and inhibit the expression of Bcl-2 m RNA.Western blot analysis of apoptotic proteins showed that OS-3 can increase the expression of P53,Bax,Cyt-C and Caspase-3 proteins,and decrease the expression of Bcl-2.It is concluded that OS-3 may exert anti-tumor effects by inducing apoptosis through the mitochondrial pathway.CONCLUSION: In this study,the anti-tumor activity of four different types of arabinoxylans from RA residue was screened by MTT method.It was found that AX-I-3 polysaccharide had a certain proliferation inhibition effect on A549 cells;the anti-tumor effect of active arabinoxylan AX-I-3 in vitro and in vivo was studied.It is found that the anti-tumor mechanism of AX-I-3 may be related to the enhancement of immunity and activation of mitochondrial apoptotic pathway.In vivo experiments also found that the combination of AX-I-3 and halving dose of DDP enhanced the anti-tumor effect and reduced the toxic side effects of DDP.The oligosaccharides degraded by active arabinoxylan AX-I-3 were subjected to structural analysis and anti-tumor activity screening,and the anti-tumor effect of active oligosaccharide OS-3 was further studied in vitro,and its mechanism was related to apoptosis.This research is of great significance for the development of high value-added products of RA residue,the establishment of a recyclable industrial chain,and the sustainable use of ecological resources.