Biological Activities Of Polyphenols From Stevia Rebaudiana(Bertoni):In Vitro And In Vivo Investigation

Food storage and process demand potential health-protecting factors such as antioxidants and antimicrobial agents to prevent food spoilage.Some synthetic phenolic antioxidants have been used in pharmaceuticals,as preservatives for cosmetics and to stabilize the fat,oil,and lipid in food,such as butylated hydroxyanisole(BHA),butylated hydroxytoluene(BHT),etc.However,excessive addition or incorrect use of these synthetic antioxidants may cause carcinogenic,cytotoxicity,and oxidative stress induction.Therefore,the demand for botanic antioxidants are being increased for their health benefits associated with their functionalities such as antioxidant and other potential activities.Stevia rebaudiana(Bertoni)is a well-known resource of natural sweetener steviol glycosides(SGs)that possess therapeutic potential against several diseases such as diabetes mellitus,candidiasis,hypertension,inflammation,obesity,and cancer.Apart from steviol glycoside,about 4% of the dried leaves is the polyphenol in Stevia leaves(PPS)have not been well studied and lack information on their bioactivities since most reported studies about the PPS which were based on the Stevia extract mixtures that contain abundant bioactive compounds and SGs.As an abundant byproduct during the production of steviol glycosides,considering their phenolic characters,the PPS would be a new antioxidative food resource or additives applied in foods and drugs with antidiabetic function.Therefore,the main objective of this research work is to produce natural antioxidants like polyphenol from Stevia waste generated from SGs manufactories.Through the research,to further understand the functional properties of real the PPS,support for the deep utilization of the PPS as an antioxidant,and also provide guidance for the use of other food additives.Firstly,the polyphenols in Stevia(PPS)were extracted comparing conventional and green extraction methods using Response surface methodology.Different antioxidant assays were used to measure the antioxidant activity of the PPS,with the comparison of some commercial antioxidants.And then,the stability of the PPS was compared with a synthetic antioxidant under various physical environments,and blending the PPS with some other antioxidants was studied to elucidate the synergistic effect of the PPS.In addition,the antimicrobial effect of the PPS on bacterial and fungal strains was studied by measuring the inhibition zone,MIC,and MBC/MFC.The inhibition effect of the PPS on four digestive enzymes was also contracted with epigallocatechin gallate(EGCG),and their kinetic analysis was categorized.In the last chapter,the antihyperglycemic effect of the PPS on a high-fat diet(HFD)induced diabetic rats was studied by measuring the body weight change,TG,TC,blood glucose,and Intraperitoneal glucose tolerance test(IPGTT).The main results were presented as follow:1.The efficiency of various solvents on the extraction of the PPS from Stevia waste was studied.It was found that methanol results in the highest PPS yield followed by ethanol and water.In addition,the extraction efficiency of the PPS using conventional and Ultrasoundmicrowave coextraction was studied.The key variables of the extraction process(temperature/microwave power,solvent concentration,p H,and time)were evaluated using response surface methodology.The optimum extraction conditions of methanol concentration,microwave power,p H,and extraction time for Ultrasound-microwave coextraction were 50%,100 W,p H4,and 5 min;and for conventional extraction,the optimum parameters were 70%,75°C,p H 2,and 120 min,respectively.Based on the obtained results,the Ultrasound-microwave coextraction increased the PPS yield rather than a maceration.The polyphenol content was26.9 mg /g DW for Ultrasound-microwave coextraction and 22.54 mg/g DW for maceration.In this way,Ultrasound-microwave coextraction declares a suitable extraction option due to the considerable reduction in time and solvent as well as the increase in the PPS production.2.The hydrophilic and hydrophobic antioxidant activity of real the PPS were firstly compared with other antioxidants namely α-Tocopherol,Trolox,Vitamin C and EGCG.After that,the stability of the PPS and Vitamin C under various physical environments(different temperature,p H,light(sunlight and UV),and their synergistic effect were studied.The HPLC results showed that the PPS was mainly composed of isochlorogenic acid A,B,and C.The PPS was found to possess antioxidant activity similar to that of epigallocatechin gallate and demonstrated stronger hydrophilic antioxidant activity but performed weakly in hydrophobic media.The ORAC(oxygen radical absorbance capacity)value of the PPS was significantly higher than that of other samples,approximately 1.5,3,and 5 times that of EGCG,VC,and α-tocopherol,respectively.It was observed that the PPS kept both of their TPC(total phenolic content)and antioxidant inhibition capacity about 98% in 12 h,while VC lost its DPPH(2,2-diphenyl-1-picrylhydrazyl)inhibition capacity entirely in 2h at the same temperature,but was less stable in the presence of potassium sorbate or sucrose.Moreover,the combination of the PPS and VC could increase the antioxidant stability of either VC or the PPS.Therefore,the PPS has the potential to be a new natural,inexpensive and abundant antioxidant that can be utilized in the pharmaceutical and cosmetic industries as well as natural plant-based products.3.The bioactivities of the PPS including the antimicrobial activity,the capacity to inhibit digestive enzymes,as well as the anti-inflammatory activity with the comparison of EGCG,were investigated.The results indicate that the PPS presented stronger antibacterial activity against E.coli,S.aureus,P.aeruginosa,and B.subtilis than its antifungal activity against Malassezia,A.niger.Depending on the microbial strains,the PPS has better antibacterial activity than the fungal one with a MIC range of 1.67 – 3.33 mg /m L on the bacteria and 6.67-13.3 mg /m L on the fungi.The inhibition of the PPS on digestive enzyme was about 60%against the porcine pancreas α-amylase,80% to the α-glucosidase from Saccharomyces cerevisiae,80% against the pancreatic lipase,90% to the trypsin,and presented similar IC50 with the comparison of EGCG,but weaker anti-inflammatory activity.Meanwhile,the PPS inhibited the four cancer cells Hep G2,MDA-MB-231,T47 D,MDA-MB-468 more than 60%on their viability in a dose-dependent manner.Therefore,the PPS could be recognized as a potential candidate for food,cosmetic and pharmaceutical ingredients.4.High-fat diet and excessive calorie intake are major risk factors for obesity and heart failure.The antihyperglycemic and hypolipidemic effects of the PPS against high-fat diet rat was examined.Rats were executed with the PPS of 25,50,and 100 mg/kg.The PPS at 50mg/kg was found to reduce body weight significantly(P ≤ 0.01)while it showed a significant reduction in blood glucose level in the obese rats at the same dose level only on 8 and 12 weeks of treatment.The administration of the PPS to HFD-induced obese rats reduced the fasting glucose level and improve the liver and muscle glycogen level compared with the positive controls.These results indicated that the PPS could be a functional component that contributed to the antiobesity,and might reduce the risk of type Ⅱ diabetes.