Extraction,Purification And Structural Identification Of Polysaccharides From Hericium Erinaceus Fruiting Bodies And Its Anti-colon Cancer Activities In Vitro

Colorectal cancer(CRC),one of the most common malignancies in the digestive tract,is the fourth leading frequent cause of cancer-related deaths worldwide.The main treatment method of CRC easy to produce drug resistance and strong toxic and side effects.Therefore,natural bioactive components,which can be used as alternative drugs or complementary functional food ingredient,are in great need for the therapy of CRC.Hericium erinaceus,a traditional Chinese food use and medicinal mushroom with well-known health-promoting effects,is believed to have the functions of strengthening the stomach healthy,replenishing deficiency and tonifying kidney in traditional Chinese medicine.Its fruiting bodies have been used in preventing and treatment of digestive system disorder-related diseases in China for over2,000 years.Previous studies have revealed various medicinal functions of the H.erinaceus,including anti-tumor,immune regulation,anti-gastric ulcer,anti-oxidation and liver protection activities.And macromolecular substances of the H.erinaceus,especially polysaccharides,have become one of the hot topics in current research.Up till now,there is rare information about the effect of HEPs on anti-colorectal cancer activities.Here,the effects of two key factors,ultrasound and deproteinization,on the quality of extracting polysaccharides(HEFPs)from H.erinaceus fruit-body were investigated using water extraction and alcohol precipitation method.The obtained polysaccharides(HEFP₂)were further isolated and purified,and the physicochemical property of the main components,named HEFP₂-?-2 and HEFP₂-?-2 were identified.Finally,we investigated the mechanism of HEFP₂ anti-colorectal cancer in terms of apoptosis and oxidative stress in vitro.Therefore,elucidating the molecular mechanism of HEFP₂-induced apoptosis and its underlying relationship with ROS generation might provide a promising avenue for seeking potential novel mushroom-derived polysaccharides as functional foods and cancer associated therapeutic drugs.And the main conclusions are as follows:(1)The result was that the extraction rate of HEFPs could be improved by ultrasound-assisted treatment,showing significant effect on the purity of HEFPs.Among three deproteinization methods,the method of combining ferrocyanide-zinc acetate with Sevage achieved the highest rate of protein removal,however,the polysaccharide retention rate was the lowest.The potassium ferrocyanide-zinc acetate method was simple and effective in protein removal and polysaccharide retention,but it could reduce the content and recovery rate of partial polysaccharide HEFP-?-2 component.The Sevage method had the lowest deproteinization efficiency and comparatively higher retention rate of HEFPs,showing highest main peak under elution of Sephacryl-S400 gel column,and it was suitable for protein removal when the bioactive components of polysaccharides were unknown.The data provide reference for extraction of macromolecular bioactive substances and further studies in exploring the related products of fruiting bodies of macrofungi.(2)The date of structural identification showed that the monosaccharide composition of HEFP₂ was determined to be mainly consisted of arabinose,galactose,glucose,and mannose in a molar ratio of8.99:11.15:1.2:1.97.SEM images showed that the surface morphology of HEFP₂ was rough and massive,with a dense honeycomb structure.Meanwhile,the aqueous solution exhibited typical shear thinning pseudo-plastic fluid properties and HEFP₂ had good thermal stability.FT-IR and NMR showed that the monosaccharide compositions of HEFP₂-?-2 were similar to HEFP₂-?-2.HEFP₂-?-2 and HEFP₂-?-2 were both composition of ?-D-arabinose,?-1,6-D-glucose,?-galactose and a small amount of ?-mannose with the molar ratios of 10.28: 6.57: 3.07: 1.57 and 3.23: 9.88: 3.82: 3.82,respectively.The number average molecular weight(Mn)of HEFP₂-?-2 and HEFP₂-?-2 is 2.886 × 10⁴ Da and 1.833 × 10⁴ Da,respectively.The surface of HEFP₂-?-2 was rough and lamellar,but HEFP₂-?-2 had a flat smooth shape in lamellar style.(3)In the study,we also found HEFP₂ had significant the inhibitory activities of the colony formation and apoptosis-inducing activities in HCT-116 and DLD1 cells with dose-dependent manners and IC₅₀ was0.587 ± 0.092 and 0.695 ± 0.073 mg/mL,respectively.However,the cytotoxicity of HEFP₂ was negligible within a certain concentration range.Subsequent results demonstrated that HEFP₂ could induce apoptosis of HCT-116 and DLD1 cells via Caspase-9-depedent intrinsic mitochondrial pathway by mediating the collapse of MMP and regulating the expression of the Bcl-2 family members(Bax and Bcl-2).Of interest,the data of this study indicated that HEFP₂ greatly enhanced the levels of reactive oxygen species(ROS)in HCT-116 and DLD1 cells treated with HEFP₂ along.In addition,in the present of antioxidant N-acetyl-L-cysteine(NAC),no obvious changes of ROS generation were observed in HCT-116 and DLD1 cells.Consisted with this,detailed molecular studies indicated that HEFP₂ could not modulate the expression of Bcl-2 and Bax,nor could they induce the loss of MMP and activation of Cleaved-caspase-9 and Cleaved-caspase-3.Taken together,these finding suggested ROS generation acts an upstream signal of HEFP₂-induced apoptosis via triggering a series signal cascades of Caspase-9 and Caspase-3-dependent intrinsic mitochondrial pathway in HCT-116 and DLD1 cells.And elucidating the molecular mechanism of HEFP₂-induced apoptosis and its underlying relationship with ROS generation might provide a promising avenue for seeking potential novel mushroom-derived polysaccharides as functional foods and cancer associated therapeutic drugs.