Extraction,Identification And In Vitro Activity Of Phenolic Compounds From Coconut Pericarp

Coconut(Cocos nucifera L.),belongs to the coconut genus in the Palmaceae,widely grows in tropical and subtropical regions,It is an important monocotyledonous plant and one of the three major economic forest trees in Hainan Province,but the coconut pericarp is used as a waste,has not been efficiently developed and utilized.A small number of studies have shown that the non-edible parts of coconuts(such as the coconut pericarp),are rich in active ingredients like polyphenols,flavonoids,and tannins.In order to deeply study and evaluate the content of phenolic compounds,main compounds in coconut pericarp,especially coconut mesocarp(CM)and coconut endocarp(CE),and whether they have potential value in preventing and inhibiting Alzheimer's disease and diabetes.Firstly,the total phenol content in CM and CE was compared with a variety of high activity plant materials.Secondly,the extraction process of soluble phenol in CM and CE was optimized.Then,through acid and alkali treatment,the soluble phenol and insoluble phenol(12 components)in CM and CE were further subdivided,and analyzed by HPLC-MS.Finally,the inhibitory activities of these components and their characteristic compounds on acetylcholinesterase(ACh E),butyrylcholinesterase(BCh E),?-amylase and?-glucosidase were determined,and the mechanism of action was analyzed by Auto Dock.The main research contents and results were as follows:(1)The total phenolic contents of CM and CE were compared with those of 11 woody plants such as Dalbergia retusa and Cupressaceae,we tried to screen out plant materials with high activity.The results showed that among the 13 plant materials,the total phenolic content(166.85 mg GAE/g dw)of CM was higher,which provided a reliable basis for in-depth research on CM.(2)In order to make better use of the phenolic compounds in CM,the extraction process of polyphenols carefully studied by screening seven solvent systems,optimizing the assisted ultrasonic process by response surface methodology,and comparing the four process of Ultrasound,Homogenization,Ultrasound+Homogenization,and Homogenization+Ultrasound.The results showed that the polyphenol content of CM was about 13 times that of CE,Using 60%acetone as extraction solvent,solid-liquid ratio of 1:5 g/m L,ultrasonic temperature of 80 min,ultrasonic power of 225 W,and then homogenizing at 10,000 rpm for10 min,the total flavonoid content of CM reached the maximum 551.99±12.69 mg Rutin/g dw,while the total phenolic content reached the maximum of 289.48±4.41 mg GAE/g dw at10,000 rpm for 5 min,which may be related to the oxidative degradation of polyphenols caused by the increase of polyphenol oxidase with the extension of homogenization time.This study provides a technical guarantee for the further utilization of phenolic substances in CM.(3)To make full use of phenols in CM,the soluble phenols(free phenols,esterified phenols and glycosylated phenols)and insoluble phenols(insoluble bound phenols,insoluble esterified phenols and insoluble glycosylated phenols)in CM and CE were extracted by acid and alkali treatment.The total phenols,antioxidant activity and inhibitory activity of acetylcholinesterase(ACh E)of the 12 phenolic components were determined.The results showed that the soluble phenols of CM and CE accounted for 66.90%and 52.94%of the total phenols,respectively,and the insoluble phenols accounted for 33.10%and 47.06%of the total phenols,respectively.There was a correlation between the antioxidant activity,anti-ACh E activity and polyphenol content of 12 components.In addition,10 flavonoids and 12 phenolic acids were identified by HPLC-MS.According to the identification results,19 typical phenolic compounds and luteolin,quercetin and vanillic acid with similar structural characteristics were screened,and their inhibition on ACh E was determined.It was found that quercetin(71.25%)and kaempferol(64.84%)had the highest inhibitory activity on ACh E.The inhibition of flavonoids on ACh E had a high correlation with antioxidant activity(FRAP)(R=0.9221),while the correlation of phenolic acids was weak(R=0.6018).Therefore,these results are conducive to the comprehensive evaluation and utilization of phenolic compounds in CM and CE.(4)To inhibitors of natural anti-Alzheimer and anti-diabetic activity with small side effects were developed,9 typical flavonoids,7 compounds with similar structural characteristics and3 standard substances(galantamine)in CM and CE were further determined for anti-acetylcholinesterase(ACh E),anti-butyrylcholinesterase(BCh E),anti-?-glucosidase and anti-?-amylase activities.The inhibitory effects were analyzed by molecular docking.The results showed that myricetin had the highest anti-ACh E activity(IC_{50-ACh E}=34.46?g/m L)and anti-?-amylase activity(IC_50-?-A=46.52?g/m L),quercetin had the highest anti-BCh E(IC_{50-BCh E}=32.23?g/m L)and anti-?-glucosidase activities(IC_50-?-G=13.34?g/m L).The binding sites of these three flavonoids in enzymes were further studied by molecular docking.According to Auto Dock calculations,the binding energies of myricetin-ACh E,quercetin-BCh E,quercetin-?-glucosidase and myricetin-?-amylase were–5.64 kcal/mol,–6.74 kcal/mol,–8.86 kcal/mol and–7.40 kcal/mol,respectively.Among them,both the methoxyl group and glycosylation in flavonoids reduce the inhibitory activity of the enzyme,while the hydroxyl group increases the inhibitory activity of the enzyme.Therefore,CM and CE waste were rationally utilized can not only solve environmental problems,but also serve as natural inhibitors of cholinesterase,?-glucosidase and?-amylase for the treatment and prevention of Alzheimer's disease and Type II diabetes.