Quick Identification And The Mechanism In The Release And Transformation By Fermentation Of Active Phenolics Components In Guava Leaves

As an important natural product resource in folk,guava leaves?GL?extracts have extremely remarkable effects in anti-oxidant activity,anti-hypoglycemic,anti-microbial activity and anti-inflammatory effect.Due to its unique flavor,taste and bio-activity,GLs has been processed into the functional tea products with anti-hypoglycemic effect in China and other countries.However,the series of problems restricts its promotion in the market,such as its instability of efficacy,a lack of methods for quality assessment,the active efficacy ingredients are not clear and the low utilization rate of effective ingredient.In this study,HPLC fingerprinting technology was firstly used to assess the quality of GL;An efficient method was established to screen the core efficacy compositions with the antioxidant activity and anti-hypoglycemic effect in the GLs extracts,respectively.The high efficacy fermentation strains with good compatibility were selected,and the high efficient co-fermentation system was constructed to promote the release of polyphenols compounds and bio-converion of flavonoid aglycone,and further improved the efficacy and the utilization of the core active compounds in GLs,and the enzymatic action mechanism in releasing of soluble phenolics and the orientation bio-transformation of flavonoid aglycones was preliminary elucidated.Some key factors for controlling the fermentation degree of guava leaves has been found,which will have an important guiding meaning for the quality control of fermented natural products.The main conclusions are:?1?The GLs samples collected in different regions were grouped into cluster I and cluster II by the HPLC fingerprinting and chemo-metric methods:The samples in cluster I were collected from Hangzhou?Zhejiang province?,Shaoguan,Panyu,Jiangmen,Dongguan and Meizhou?Guangdong province?,Taipei and Tainan?Taiwan province?and Nanping?Fujian?;The samples in cluster II were from Bozhou and Hefei?Anhui province?,Baoding?Hebei province?,Hengyang,Yueyang and Chenzhou?Hunan province?.The results showed significant differences of the flavonoid compositions and contents of GLs among different regions.However,no significant difference was found in germplasm resources from the same or adjacent regions.Fingerprint-activity relationships showed that quercetin glycosides and aglycones?quercetin and kaempferol?were the key factors in controlling the quality of GLs.?2?Eleven major anti-oxidantive components were successfully identified from guava leaves?GL?extracts by the established off-line HPLC-free radical scavenging activity detection?HPLC-FRSAD?method,and the main antioxidant phenolic components in the GL extracts were identified as gallic acid,procyanidin B3,quercetin,and kaempferol.The structure-activity relationship analysis results showed that the number and position of hydroxyl groups in phenolic compounds had very important influences on the free radical scavenging capacity;the hydroxylation of flavonoids could increased the antioxidant activity,whereas the glycosylation of hydroxyl structure reduced its antioxidant capacity.?3?The GL extracts(IC₅₀=19.37±0.21?g/mL)exhibited a stronger inhibitory potency against?-glucosidase than did acarbose?positive control?at IC₅₀=178.52±1.37?g/mL,which confirmed that the GL has strong hypoglycemic activity in vitro.An approach based on bio-affinity ultrafiltration combined with HPLC-ESI-TOF/MS?UF-HPLC-ESI-TOF/MS?was developed.The optimal affinity ultrafiltration conditions were as:10 U/mL?-glucosidase concentration and a 30 kDa membrane pore size filter.Twelve active molecules with strong affinity degree?AD?to?-glucosidase was successfully identified by the UF-HPLC-TOF/MS method in GL extract,among which quercetin?AD=18.86%?and procyanidins B3?AD=8.54%?had the highest affinity for?-glucosidase.Quercetin(IC₅₀=4.51±0.71?g/m L)and procyanidin B3(IC₅₀=28.67±5.81?g/mL)were determined to be primarily responsible for the anti-hyperglycemic effect,which further verified the established screening method.Structure-activity relationship analysis revealed that the number and position of polyphenols and flavonoids in natural products significantly affected their inhibitory activity on?-glucosidase;hydroxylation of flavonoids could increase their inhibitory effect on?-glucosidase.Glycosylation of flavonoids would reduce its inhibitory effect on?-glucosidase;the proanthocyanidins had a strong inhibitory effect on ?-glucosidase.?4?The contents of flavonoid compounds in the GLs samples collected from different seasons in the same region were greatly different.The contents of flavonoid compounds in GLs increased by the microbial fermentation;The HPLC fingerprints combined with HCA and PCA analysis showed the natural GLs had the similarity between from 0.837 to 0.927.However,Monascus anka-S.cerevisiae-fermented GLs had the similarity higher than 0.993.Consequently,microbial fermentation can improve the GLs quality consistency and promote the efficacy stability significantly.?5?Four cellulase-producing strains were successfully screened and identified as Arthrobacter AS1,two strains of Bacillus sp.BS2 and BS3,and Alcaligenes AS4.Bacillus sp.BS2 and Monascus anka co-fermentation increased the soluble phenolics content of GLs.The optimal fermentation conditions was as:the inoculation of Monascus anka and Bacillus BS2was maintained at 2:1;the water content of fermentation substrates was maintained at 60%;the fermentation time was set at 8 days.The released soluble phenolics content in GLs reached a maximum of 53.37 mg GAE/g DM.The phenolics compositions of GLs were successfully identified by HPLC-ESI-TOF/MS method.Most of individual phenolics contents were significantly increased after microbial fermentation,but the content of gallic acid was decreased significantly.The results also confirmed that the co-fermentation of Monascus and Bacillus significantly enhanced the antioxidant capacity and the protection effect of DNA damage of GLs,and the level of citrinin was not detected.?6?Co-fermentation of Monascus and Bacillus significantly improved the content of soluble phenolics of GLs.While the content of insoluble-bound phenolics decreased at the same time.The trend of these enzymes was consistent with the contents of soluble phenolics,and reached the maximum in an 8 day fermentation:total cellulase activity reached 31.32 U/g;the?-amylase activity reached 83 U/g;the xylanase activity was 5.2 U/g;the?-glucosidase activity was only 0.19 U/g.With the increase of the fermentation time,the activities of these enzymes decreased significantly.A significant positive correlation was found between the concentrations of cellulase,?-amylase and?-glucosidase and the release of total soluble phenolics from GLs.The R were 0.8878,0.8089 and 0.8428?p<0.01?,respectively.However,the R between xylanase and the release of total soluble phenolics was only 0.5192?p<0.5?.Treatment with a low concentration of complex enzymes?<50 U/g?had no sufficient effect on release of soluble phenolics from GLs,while a high concentration of complex enzymes?>300 U/g?could significantly promote the release of soluble phenolics from GLs.However,the released total soluble phenolics content?42.54 mg GAE/g DM?was significantly lower than the fermentation treatment group?53.08 mg GAE/g DM?.?7?Three representative HPLC fingerprints of phenolic compositions from GLs can be used for indicating the differences of fermentation degrees.Four characteristic compounds,including gallic acid,quercetin-3-O-?-L-arabinopyranoside,quercetin,and kaempferol,that significantly responded to the changes of bio-activity in the fermented GLs were identified as the marker components by HPLC fingerprint and principal component analysis.High levels of the four marker components in the HPLC fingerprints are an indication that the fermented GLs are mature.In addition,the correlation analysis confirmed that the total phenolic,total flavonoid,and the quercetin and kaempferol contents strongly correlated with its bioactivities.?8?Fermentation and enzymatic hydrolysis?FE?processing can further promote the release of soluble phenolics and directed biotransformation of flavonoid aglycones from GLs.Compared to un-treated GLs,the total soluble phenolics,flavonoids,quercetin,and kaempferol levels increased by 2.1,2.0,13.0 and 6.8-fold,respectively.FE processing also significantly improved the scavenging activity of DPPH,ABTS⁺,and NO₂^-radical and reducing power of GLs extract.Additionally,compared with the fermented GLs(IC₅₀=9.5?g/mL)and the un-fermented GLs(IC₅₀=14.5?g/mL),GLs extracts following FE processing exhibited the inhibitory efficacy towards?-glucosidase(IC₅₀=5.9?g/mL).