| Bergamot,also known as five-fingered citrus and Fushou citrus is the dried fruit of Citrus medica L.Var.sarcodactylis Swingle,a citrus plant in the Rutaceae family.It is mainly produced in Guangdong,Guangxi,Sichuan,Zhejiang,Fujian,and other places in China.Its main active ingredients include polysaccharides,volatile oils,flavonoids,coumarins,and so on.Among them,bergamot polysaccharide has antioxidant,anti-tumor,immunomodulatory and hypoglycemic pharmacological activities.Parkinson’s disease(PD)is a common degenerative disease of the central nervous system,characterized by progressive death of dopaminergic neurons and the formation of intracellular vesicles.The pathogenesis of PD is extremely complex,and its main causes include aging,cell damage caused by environmental toxins,genetic mutations,and so on.At present,the commonly used PD treatment drug is levodopa,but with the prolongation of medication time and the increase of dosage,the toxic side effects generated are also increasing.Many active ingredients of traditional Chinese medicine have good preventive and therapeutic effects on Parkinson’s disease,and have the advantages of low toxicity and minimal side effects.Bergamot is a component of the anti-PD traditional Chinese medicine"Zhi Chan Fang",but the effective ingredients of its action are still unclear.Based on the above,this study extracted the effective components of bergamot polysaccharides and explored their protective effects and mechanisms against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine ion(MPP~+)induced damage to human neuroblastoma cells(SH-SY5Y),providing experimental basis for the prevention and treatment of PD and reference for the development and utilization of bergamot resources.The main research findings are as follows:1.Extraction,separation and purification of bergamot polysaccharideMethod:The hot water extraction method and composite enzyme extraction method were used to extract and separate bergamot polysaccharides.The process conditions of the hot water extraction method were:extraction temperature was 80℃,material liquid ratio was 1:20(g/m L),extraction time was 3 hours,extraction frequency was 2 times,and the process conditions of the composite enzyme extraction method were:1.5%enzyme dosage,material liquid ratio was 1:30(g/m L),extraction temperature was 50℃,extraction time was 2 hours,and protein was removed using the Sevag method,Activated carbon adsorption method is used to remove pigments,and finally,crude bergamot polysaccharides are obtained through ethanol precipitation.Purification of crude polysaccharides from bergamot using AB-8 macroporous resin,determination of polysaccharide content using phenol sulfuric acid method,and calculation of polysaccharide purity.Result:The yield of crude polysaccharides from water extracted bergamot was 2.74±0.43%,and the purity was 53.13±1.63%.The yield and purity of crude polysaccharides extracted from bergamot using composite enzymes were 4.86±0.50%and 44.46±4.64%,respectively.After purification with AB-8 macroporous adsorption resin,the purity of water extracted bergamot polysaccharides reached 73.80±3.87%,which increased by 20.7%.The purity of compound enzyme extracted bergamot polysaccharides reached 60.81±1.41%,which increased by 16.5%.Conclusion:The yield of polysaccharides extracted by hot water extraction method is 2.74±0.43%,while that extracted by composite enzyme extraction method is 4.86±0.50%.The yield of polysaccharides extracted by composite enzyme extraction is higher than that extracted by water extraction.In terms of purity,the purity of water extracted polysaccharides is 53.13±1.63%,and the purity of composite enzyme extracted polysaccharides is 44.46±4.64%.The purity of water extracted polysaccharides is higher than that of enzyme extracted polysaccharides.After purification with macroporous adsorption resin AB-8,the purity of water extracted bergamot polysaccharides reached 73.80±3.87%,an increase of 20.7%,and the purity of composite enzyme extracted bergamot polysaccharides reached 60.81±1.41%,an increase of 16.5%.2.Protective effect of bergamot polysaccharide on MPP~+inducedSH-SY5Y cell injuryMethod:Cells were cultured in vitro and an MPP~+induced SH-SY5Y cell injury model was established.The experiment was divided into a control group,an MPP~+model group,a water extracted bergamot polysaccharide group,and an enzyme extracted bergamot polysaccharide group.MTT method was used to detect cell viability,Hoechst33258staining method was used to observe cell morphology,2’,7’-2’,7’-dichlorofluorescein diacetate(DCFH-DA)fluorescent probe was used to detect the level of reactive oxygen species(ROS),JC-1 fluorescent probe was used to detect mitochondrial membrane potential,Western blot was used to detect phosphorylated protein kinase B(p-Akt),protein kinase B(Akt)Phosphorylated extracellular regulated protein kinases 1/2(p-ERK1/2)and cytochrome c(Cyt-c)expression levels.Results:Compared with the control group,the cell viability of the MPP~+-treated group was significantly reduced,the cells were broken and the nucleus was shrunk after Hoechst33258 staining.Meanwhile,the intracellular ROS was significantly increased,and the mitochondrial membrane potential was significantly decreased.Compared with the model group,the cell viability of the water-extracted polysaccharide group and the enzyme-extracted polysaccharide group were significantly increased,the cell morphology was significantly improved,the enhancement of ROS in the cells was inhibited,and the mitochondrial membrane potential was increased.The results of the Western blot showed that both kinds of bergamot polysaccharides could inhibit the decrease of p-Akt and p-ERK1/2 and the increase of Cyt-c caused by MPP~+.Conclusion:Both methods of extracting bergamot polysaccharides have a protective effect on MPP~+induced SH-SY5Y cell damage,which may be mediated by regulating the production of mitochondrial ROS and the release of cytochrome c,thereby maintaining mitochondrial homeostasis,activating the ERK and Akt signaling pathways,inhibiting cell apoptosis,and thus exerting a protective effect. |
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