Extraction,Purification And Biological Activities Of Polyphenols From Moringa Oleifera Leaves And Its Application In Edible Composite Film

Moringa oleifera is a tropical and subtropical plant that is found in Bangladesh,Afghanistan,Pakistan,and northeastern India.Moringa has good application prospects in the food industry and nutrition fields.Its various parts,including bark,roots,seeds and leaves,are rich in nutrients.M.oleifera leaves(MOL)have recently received an increasing attention.It was found that MOL has a variety of biological functions such as anti-proliferation,antiinflammatory,antihyperglycemic and improving the human intestinal microbial community.With the development of food analysis technology,studies have shown that certain phenolic compounds such as caffeoylquinic acid(CQAs),flavonoids and their derivatives in MOL may be the cause of these unique biological functions.It has been reported that the CQAs and flavonoids compounds have ability on inhibition ?-glucosidase,?-amylase and pancreatic lipase enzymes.Inhibitory of these enzymes is one of biological solutions to control in blood glucose level,considering therapeutic mechanisms for diabetes disease.Most of the current research on MOL has focused on the biological activity of plant extracts or isolated compounds,however,these studies have not considered whether these compounds have been degraded in the digestive system before reaching the area of action.In addition,there is a lack of applications research on active MOL extracts.Therefore,on the one hand,flavonoids compounds(FC)were extracted and isolated from MOL by an optimized method,and their inhibitory effect on ?-glucosidase activity was studied.On the other hand,the extraction and separation of caffeoylquinic compounds from MOL and the effect of extraction temperature on their activity were studied.The cytotoxic activity of MOL extract on cancer cells and its inhibitory activity on metabolic syndrome enzymes before and after in vitro gastrointestinal digestion and colonic fermentation,as well as its role in improving intestinal microbial community were also investigated in this study.In addition, MOL crude extract was added to chitosan to produce a film with excellent nutritional properties.The results are as follows:1.Optimized the ultrasonic-assisted extraction method of flavonoids in MOL using BoxBehnken Design(BBD).In addition,five macroporous resins were used to study the kinetics of flavonoids purification from MOL extracts.The results show that the optimal conditions for extracting flavonoids are as follows:the ratio of the extraction material to the liquid is the ratio of the aqueous ethanol solution to the raw material is 52 mL/g,the extraction time is 43 minutes,and the extraction temperature is 76?.In addition,the best conditions for purifying flavonoids with AB-8 resin are as follows:the flavonoid loading concentration is 20 mg/mL,the elution solvent is a 70%ethanol aqueous solution,and the elution flow rate is 1.5 mL/min.In addition,five flavonoids(rutin,kaempferol acetylglycoside,quercetin-3-glucoside,quercetin-3-acetylglucoside,and kaempferol 3-were identified by HPLC-ESI-MS.Glucoside).Antioxidant results showed that MOL flavonoids had a scavenging activity of 2,2-diphenyl-1pyridinohydrazinyl(DPPH)free radicals at a concentration of 100 ?g/mL at 85.99%at a concentration of 50 ?g/mL The lower scavenging activity for 2,2'-diaza-bis(3ethylbenzothiazole-6-sulfonic acid)(ABTS)free radical was 84.72%.The results show that flavonoids in MOL can be used for the development of functional foods.1.Ultrasonic assisted extraction of FC from MOL was optimized by Box-Behnken Design(BBD)methodology.In addition,five macroporous resins were used to study the kinetics of purification of FC from MOL extract.The results showed that the optimal conditions for FC extraction were as follows:the ratio of aqueous ethanol solution to raw material was 52 mL/g,the extraction time was 43 min,and the extraction temperature was 76?.Nevertheless,the optimal conditions for the purification of FC with ab-8 resin are as follows:the concentration of the sample of flavonoids crude extract is 20 mg/mL,the elution solvent is 70%ethanol solution,and the elution flow rate is 1.5 mL/min.Nevertheless,AB-8 resin was the most suitable resin for purification of FC with the following conditions:initial FC concentration 20 mg/mL,eluting solvent 70%aqueous ethanol solution and 1.5 mL/min eluting flow rate.Five flavonoids(rutin,kaempferol acetyl glycoside,quercetin-3-glucoside,quercetin-3-acetylglucoside and kaempferol 3-glucoside)were identified by HPLC-ESI-MS.The results of antioxidant experiments showed that the scavenging activity against 2,2-diphenyl-1pyridinazide(DPPH)radical of FC was 85.99%at a concentration of 100 g/mL,while the scavenging activity against 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid)(ABTS)radicals was 84.72%at 50 ug/mL.Our results suggest utilizing of purified FC from MOL on development of function.The results show that the FC in MOL can be used in the development of functional food.2.The inhibitory effect(IAC)of purified flavonoids(PF)from MOL on ?-glucosidase(?glu)activity before and after simulated gastric digestion.The combined effect of PF with chlorogenic acid(CGA)and acarbose(Aca)on the activity of ?-glu.The results showed that at 100 g/mL,the inhibition rates of PF and Aca on ?-glu were 54.41%and 69.17%,respectively.At PF concentration of 800 g/mL,the inhibition rate reached 99.01%.HPLC-DAD/MS analysis showed that the main flavonoids detected in PF were lutin,kaempferol acetyl glycoside,quercetin-3-glucoside,quercetin-3-acetyl-glucoside,and kaempferol 3-O-glucoside.After simulated gastric digestion,the PF in the presence of nitrate(SGM-N)were lower than those in the simulated gastric juice(SGL)alone.The highest flavonoids compounds stability were rutin and kaempferol 3-O-glucoside in SGL,quercetin-3-acetyl-glucoside and kaempferol 3-O-glucoside in SGL-N.IAC of PF at 50 ?g/100 mL decreased from 89.56%to 46.17%and 24.97%after SGL and SGL-N,respectively.In addition,PF is more effective when used in combination with Aca,reducing the Aca semi-inhibitory concentration(IC50)by 8.17 times.The results show that PF can be used to design special foods for diabetics.3.The effects of extraction temperature on the extraction rate and biological activity of the CQAs from MOL were investigated.The gradual increase of extraction temperature leads to the increase of mono-CQA and the decrease of 3,5-diCQA.When the extraction temperature was 80?,the extract had the highest antioxidant capacity(p<0.05),and the IC50 values of ABTS and DPPH were 53.56 and 56.9 g/mL,respectively.Compared with ?-glu and ?amylase,CQAs from MOL had the best inhibitory effect on pancreatic lipase(IC50=0.073 mg/mL),and was higher than orlislat(IC50=0.101 mg/mL).The cytotoxic activity of CQAs from MOL against HeLa and HepG2 increased with the increase of extraction temperature.Compared with the pathogenic bacteria of gram-negative Escherichia coli and Salmonella typhimurium,all the extracts had higher inhibitory effects on the growth of gram-positive Bacillus cereus and Staphylococcus aureus.The results showed that high temperature extraction(80?)was beneficial to obtain better bioactive CQAs from MOL.4.The effects of gastrointestinal digestion(GI)and colonic fermentation(CF)on MOL polyphenol extract(MPPE)were investigated.The results showed that compared with the undigested samples,the total phenolic compounds(TPC)in the samples after GI and CF treatment decreased 30%and 38.82%respectively,while the FC decreased 32.34%and 60.07%respectively.After GI treatment,the content of caffeic acid increased significantly(p<0.05),while the content of 3,5-diCQAs did not change significantly(p<0.05).After CF treatment,the content of polyphenol compounds decreased and two new compounds(pcoumaric acid and ferulic acid)were identified.The inhibitory activities of MPPE on pancreatic lipase and ?-glu decreased significantly by 5.63 and 14.20%after GI and by 27.41 and 34.01%after CF,respectively.Whereas,complete disappearance of the inhibitory activity against ?-amylase after GI and CF.A slight decrease observed in both antioxidant activity assays DPPH and ABTS after GI and CF,on the contrary,Ferric ion reducing antioxidant power(FRAP)showed increased after sequential digestion steps.Although the cytotoxic activity against HeLa cells and HepG2 decreased after GI and CF,antiproliferation activity against HepG2 improved after CF.The results of 16S rDNA sequencing showed that MPPE could shift the abundance of gut microbiota groups,MPPE increased Prevotella and significantly diminish the ratio of Firmicutes/Bacteroidetes,suggesting that MPPE has the potential to improve glucose metabolism,reduce energy intake and reduce the risk of obesity.5.Chitosan(Chs)polymer and MOL extract with different concentrations(0.5-4%)were incorporated to produce a novel edible film,and their physicochemical properties and biological functions were evaluated.MOL significantly improved thickness,solubility,swelling,and colour of Chs-films in a concentration-dependent manner(p<0.05).Incorporating 4%of MOL with Chs-film improved its water vapour permeability from 8.85 to 2.47 gm-1s-1Pa1,and increased the film surface heterogeneity observed by scanning electron microscope(SEM).The results of SEM showed that the inhomogeneity of the membrane surface increased with the addition of MOL extract.High concentration of MOL extract enhanced the mechanical properties(tensile strength and elongation at break)and decreased the thermal stability of Chs films.The scavenging abilities of DPPH,ABTS radicals and FRPA were increased by 74.84%,70.31 and 77.51%,respectively.In addition,4%MOL extract decreased the inhibition rate of Chs membrane to ?-glu,pancreatic lipase,and ?-amylase,and inhibited the growth of B.cereus and S.aureus in packed meat burger.These results indicated that MOL extract can be used to improve the physicochemical and biological properties of edible films and prolong the shelf life of packaged foods.In addition,Chs-MOL film also has potential application value in the treatment of type 2 diabetes.