Structural Identification Of Quinoa Polysaccharides And Its Inhibitory Mechanism On The Proliferation And Differentiation Of 3T3-L1 Preadipocytes

Quinoa(Chenopodium quinoa Wild.)is a highly nutritious,dicotyledonous crop of the family Amaranthaceae that can be grown in different agro-ecological zones and is resistant to frost,salinity and drought.The nutritional factors in quinoa have various biological activities such as antioxidant,anti-inflammatory,and hypolipidemic.It is an effective way to use these characteristics to develop a variety of functional quinoa foods and extend the quinoa industry to increase added value.In this study,two different extraction methods were used to extract quinoa polysaccharides,and the quinoa polysaccharide with higher purity was obtained by separation and purification.3T3-L1 cells were used as the research model and the mechanism of its inhibition of cell differentiation was analyzed.Transcriptome sequencing technology was also used to comprehensively analyze the regulation of intracellular gene expression before and after treated with two kinds of quinoa polysaccharides to explore the regulatory mechanism of inhibiting cell proliferation.The following experimental progress was made:1.Component structure identification of quinoa polysaccharides: The quinoa polysaccharides were extracted by hot water and alkaline solution.Then crude polysaccharides were preliminarily separated and purified by ion exchange chromatography and gel filtration chromatography.The two components,Separated quinoa water-extracting polysaccharides(SQWP-1)and Separated quinoa water-extracting polysaccharides SQAP-1,have purities of 99.32% and 99.17%,respectively.SQWP-1 is linked by four glycosidic bonds Glc-(1→;Fru-(2→;→4)-Glcp-(1→;→4,6)-Glcp-(1→ with a molar ratio of0.05:0.17:0.56:0.22.SQWP-1 is linked by three glycosidic bonds Glc-(1→;→4)-Glcp-(1→;→4,6)-Glcp-(1→ with molar ratio of 0.2:0.6:0.2 The glycosidic bond on the main chain of SQWP-1 is →4)-α-D-Glcp-(1→ and the end group α-D-Glcp-(1→ and Fru-(2→ was connected to the main chain through O-6 bond.The main chain of SQAP-1 is connected by a glycosidic bond of →4)-α-D-Glcp-(1→,and the end group α-D-Glcp-(1→is connected to the main chain through the O-6 bond of →4,6)-α-D-Glcp-(1→.2.Study on the inhibition mechanism of quinoa polysaccharides components on the differentiation of 3T3-L1 preadipocytes: 3T3-L1 preadipocytes were cultured in vitro,and the results of oil red staining showed that SQAP-1 was more effective than SQWP-1 in inhibiting adipocyte differentiation.The polysaccharide isolated from quinoa did not cause excessive accumulation of triglycerides in 3T3-L1 preadipocytes.When the sample concentration was 4 mg/m L,the triglyceride contents of the SQAP-1and SQWP-1 groups were 0.71 mmol/L and 0.75 mmol/L,respectively.When the sample concentration was 2 mg/m L,the triglyceride contents of the SQAP-1 and SQWP-1 groups were 1.05 mmol/L and 1.21mmol/L,which were significantly lower than the 1.51 mmol/L triglyceride content of the Adipocyte group.SQWP-1 can down-regulate the m RNA and protein expressions of differentiation-related genes PPARγ,C/EBPα,C/EBPβ,LPL and AP2,and up-regulate the m RNA and protein expressions of FAS and C/EBPδ;SQAP-1 can down-regulate differentiation-related genes PPARγ,C/EBPα,C/EBPβ,FAS,LPL,AP2 m RNA and protein expression,up-regulated C/EBPδ m RNA and protein expression.3.Study on the inhibition mechanism of quinoa polysaccharide components on the proliferation of3T3-L1 preadipocytes: Intracellular gene expression before and after quinoa polysaccharides treatment was comprehensive analysis using transcriptome sequencing technology.Differentially expressed genes were screened.GO and KEGG enrichment analysis was performed to explore the anti-obesity regulatory molecular mechanism of quinoa polysaccharides.GO enrichment analysis showed that quinoa polysaccharide treatment was significantly enriched in terms of “Extracellular region”,“Extracellular matrix” and “Cellular response to chemical stimulation”.KEGG enrichment analysis showed that quinoa polysaccharide was significantly enriched in “PI3K-Akt signaling pathway”,“ECM-receptor interaction”,“DNA replication” and other pathways.After quinoa polysaccharide treatment,the intracellular PI3K-Akt signal and DNA replication pathway was significantly inhibited,and the apoptosis pathway was significantly activated.It is speculated that quinoa polysaccharides can prevent cell mitosis by inhibiting PI3K-Akt signal and DNA replication and stimulate cells through its unique chemical structure to inhibit cell proliferation.