| Efficient extraction that preserves or improves biological activities of polyphenols from different plant materials has been a major research focus.Different contemporary extraction techniques have been used with varying degrees of success.However,a lot more needs to be done to understand the mechanisms and the effects of some of these techniques on the yield,extraction kinetics and bioactivities of polyphenols.Being used for several centuries in China and the whole of Asia,Tartary buckwheat（Fagopyrum Tataricum L.Gaerth）has been highly rated for its nutritional and medicinal properties.Based on the high correlation between the biological properties and its high polyphenol content,to obtain polyphenols with high biological activity,the effects of green extraction methods,such as ultrasonic assisted extraction（UAE）,subcritical water extraction（SWE）and their combination（UA–SWE）,on the yield of polyphenols were systematically studied,and the purification,identification and biological activities（antioxidation,antiproliferation and cytotoxicity）of these polyphenols on human hepatoma cells Hep G2 were also studied.These studies may provide theoretical foundation and scientific evidence for the effective extraction of polyphenols from Tartary Buckwheat and its application in pharmaceutical,health care products,cosmetics and medical industry.The main research contents and results are as follows:（1）Optimization of ultrasound-assisted extraction and subcritical water extraction of Tartary buckwheat polyphenols for maximum yield and antioxidant activityThe ultrasonic and subcritical extraction of polyphenols from Tartary buckwheat meal according to the Box Behnken design of the response surface methodology（RSM）were optimized.The Folin Ciocalteu method（FCM）and ABST assay were used to respectively measure total phenol content（TPC）and antioxidant activity（AA）.All predictors[time（minutes）,frequency（k Hz）,power（Watts）and ratio of liquid to solid(m L?g–1)]showed significance in RSM models generally.The optimum extraction conditions were 20 min,20 k Hz,150 W and 26 m L.g-1 at 50oC,yielding TPC of 6.947 mg GAE.g-1.dw-1 and AA of 63.469%.Liquid to solid ratio positively influenced TPC but amplified a negative influence when it interacted with all other factors.For the AA model,power played a directly proportional role but as well amplified the inverse relationship when it interacted with other factors.This showed that liquid to solid ratio needed to be carefully observed in order to efficiently extract polyphenols from plant materials when temperature is kept constant at 50oC and water is used as the only extraction solvent.For SWE,the predictors were time,temperature and liquid to solid ratio and the response factors were TPC and AA.The optimized conditions were 48 min,220oC and 60 m L.g-1 ratio,yielding TPC of 9.20 mg GAE.g-1.dw-1 and AA of 74.12%.All terms of the model positively influenced TPC as single factors and as interactions.Only the quadratic terms show negative relationship with TPC because of the concave nature of the curve.It means all terms are crucial to successful SWE of polyphenols from Tartary buckwheat.The AA model terms followed a similar trend as the TPC model.All single factors positively influenced AA as well as their interactions.However,according to the model coefficients,temperature（β=12.09）influenced AA the most,followed by liquid to solid ratio（β=4.46）and time（β=3.94）.For interactive terms,time*ratio had the highest influence（β=4.50）.This showed that although all terms are significant,important attention should be focused on temperature and its interaction with time.The approach used showed an innovative way of easily deciding on the most influential variable during SWE/UAE of polyphenols from buckwheat.（2）Comparison of extraction yield,antioxidant activity and cytotoxicity of free Tartary buckwheat polyphenols extracted by ultrasound-,subcritical water-and ultrasound assisted subcritical water treatmentsThe effects of UAE,SWE and UA–SWE treatments on Tartary buckwheat polyphenol yield,composition,antioxidant activity（AA）and cytotoxicity in Hep G2 cells were also studied.The highest polyphenol yield was obtained by SWE(53.3 mg?g–1.dw–1),followed by UA–SWE(31.8 mg?g–1?dw–1),UAE(19.3 mg?g–1dw–1)and hot water extraction（HWE）(4.2 mg?g–1?dw–1).Also,SWE had the highest TPC(7.9 mg GAE?g–1?dw–1).UAE and UA–SWE showed no differences with total phenol content（TPC）being 6.6 and 6.8 mg GAE?g–1?dw–1（p<0.05）,respectively.The control method（HWE）had the largest number of phenolic compounds identified（25）,followed by UAE,SWE and UA–SWE which had 20,13 and 11 phenolics respectively.Beside phenolic acids,all treatments extracted a number of flavonoids such as flavan–3–ols（catechin–7–O–glucoside,epigallocatechin–3–gallate,epigallocatechin,epicatechin）,flavonols（kaempferol–3–O–glucoside,kaempferol,kaempferol–3–rutinoside,rutin,quercetin,quercetin–3–O–glucuronide hyperin）,flavones（vitexin,isovitexin,orientin,isoorientin）and anthocyanins（cyanidin–3–O–rutinoside,Cyanidin 3–O–galactoside,cyanidin–3–O–glucoside）.SWE gave the highest AA for all tests.The IC50 of AA were 270.8±21.3,198.1±16.0,97.9±13.5,and 150.4±12.8μg extract?m L–1,respectively for HWE,UAE,SWE and UA–SWE.Generally,SWE and UA–SWE showed the highest cytotoxic activities,followed by UAE,with HWE being the lowest.SWE has shown the best results and is a promising method for polyphenol extraction and its combination with ultrasound（UA-SWE）should be further exploited for high free polyphenol yield and conservation of extract AA and cytotoxicity.（3）Effects of pretreatment and type of hydrolysis on the composition,antioxidant potential and Hep G2 cytotoxicity of bound polyphenols from Tartary buckwheat hullsThe effects of UA–SWE,SWE,UAE and HWE pretreatments and acid（AH）and alkaline（Alk H）hydrolyses on the phenolic composition,antioxidant potential and cytotoxicity of Tartary buckwheat hull extracts were investigated.Results showed that UA–SWE gave the best AH yield of phenolics(128.45 mg?g–1),followed by SWE(85.82 mg?g–1)and UAE(64.70mg?g–1),compared to the control,HWE(35.82 mg?g–1).The same trend was observed for phenols extracted using Alk H.UA–SWE had the highest antioxidant activity,followed by SWE and UAE regardless of hydrolytic method used.Cytotoxicity followed a similar trend with UA–SWE and SWE being the most cytotoxic to liver cancer cells,followed by UAE,with the least being HWE.The findings suggested that plant materials such as Tartary buckwheat hulls can be pretreated with UA–SWE,SWE and UAE prior to hydrolytic recovery of bound polyphenols.Also,AH was more efficient than Alk H for phenol extraction,and gave extracts with higher antioxidant activity and cytotoxicity in Hep G2 liver carcinoma cells.This application allows for beneficial usage of agricultural biomass and help diversify income sources and products for industry.（4）Ultrasound–induced lipid peroxidation:Kinetics and effects on phenols,flavonoids and antioxidant activity of Tartary buckwheat water extractLipoxidation caused by ultrasound treatment was investigated.Its effects on TPC,total flavonoid content（TFC）,AA and phenols extraction kinetics in Tartary buckwheat extracts were also studied.Malondialdehyde（MDA）levels and peroxide value（PV）were used to measure lipoxidation.Kinetic modeling of phenol extraction was also done according to Fick’s second law of diffusion.Increasing treatment time increased TPC,TFC and AA significantly（p<0.05）and increments in MDA and PV were significant after 45 min.Increases in MDA and PV correlated with reductions in TPC,TFC and AA.Phenol diffusion followed a kinetic curve that agrees with the classical solid–liquid extraction models,with a rapid initial phase and a slow phase at the end.The effective diffusion coefficients（De）,both De–rapidand De–slow,for phenols positively correlated with time（5 to 60 min）and temperature（20 to 80 oC）.TPC increased when extraction temperature and time increased with De–rapidfrom 3.30 to 7.97×1011m2?s–1 from 20 to 80 oC and 2.88 to 7.47×1011 m2?s–1 from 5 to 60 min.The De–slow phase also increased from 2.02 to 5.21×1012 m2?s–1 and 2.85 to 4.33×1012 m2?s–1,respectively,for the same range of extraction temperatures and times.It is recommended that ultrasound assisted extraction of phenols from polyunsaturated fatty acid–rich sources are done at temperatures below 65 oC and not for extended periods to preserve bioactivity and minimize lipoxidation. |
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