Isolation,Identification And Characterization Of Phytochemicals And Their Antimicrobial Activities From Ampelopsis Grossedentata

Vine Tea(Ampelopsis grossedentata(Hand-Mazz.)is a medicinal and edible plant commonly,known as moyeam,is a species of plant in the Vitaceae family.It is mainly distributed in central and southern China,but can also be found in some south-east Asian countries.Plants were(and still)considered as paramount sources of novel phytochemicals,active compounds and therapeutics.It has been reported that vine tea contains many flavonoids which possess a number of biological activities,such as hypoglycemic,antioxidant,anti-thrombosis functions,anti-tumor,anti-inflammatory and antibacterial activities.Among the flavonoids,dihydromyricetin,myricitrin and myricetin have been confirmed as the main bioactive constituents of the herb.The purpose of this study was to explore new phytochemicals and their functional properties in vine tea,and provide the theoretical basis for developing new food additives and functional health foods.1.Isolation and characterization of antimicrobial metabolite from vine tea with mechanism against Bacillus cereus(AS1.1846)Vine tea as a Chinese traditional medicinal are found having a big role as a health promoting agent.In this study six compounds were isolated from vine tea extract using solvent extraction method and fractionated with the help of Sephadex LH-20 column.Furthermore,these compounds were detected,separated and purified using RP-HPLC,all these compounds were further tested on MALDI TOF and LC-MS/MS to detect their masses,molecular weight of isolated compounds 1-6(six compounds)were detected as,(1)Myricetin(C15H10O8),(2)Myricetin 3-O-rhamnoside(C21H20O12),(3)5,7,8,3',4'-Pentahydroxyisoflavone(C15H10O7),(4)Dihydroquercetin(C15H12O7),(5)6,8-Dihydroxykaempferol(C15H10O8)and(6)Ellagic acid glucoside(C2OH16O13).All these isolates were further tested against Gram positive and Gram negative and fungi,all compounds were found to have an antimicrobial activity against all Gram positive and Gram negative as well as fungi except compound(4).Furthermore,compounds 3,5 and 6 were the first time detected and reported in this plant.However,others compound 1,2,and 4 were already reported.Among these newly reported compounds,compound(3)was most efficient for its antimicrobial behavior.Therefore,the objective of the present study was to analyze the physicochemical properties of the compound 3(peak 3),evaluate their in vitro inhibitory effects against Bacillus cereus(AS 1.1846)and exploring their mechanism against Bacillus cereus(AS1.1846)and E.coli(ATCC 25922)through scanning electron microscope.Results showed that antimicrobial activity of isolated compound,5,7,8,3',4'-Pentahydroxyisoflavone against Bacillus cereus(AS 1.1846)was stronger with ZOI the lowest MIC and MBC values of 25.42±0.12(mm),32?g/ml and 32?g/ml,whereas,compare to the positive control(tetracycline in this case)was 14.12±0.67(mm),64?g/ml and 64?g/ml respectively.Furthermore,we had proposed that peak(3)interacts with the negatively charged phospholipid head groups of the cytoplasmic membrane by the electrostatic attraction of the positively charged guanidinium group and is inserted into the hydrophobic membrane core.Additionally,the hydrophobic residues of peak(3)align and bind to the hydrophobic membrane core to form a hydrophilic channel,leading to the leakage of essential cytoplasmic components(e.g.,proteins,nucleic acid,potassium ions).In addition to the formation of ion channels leading to trans-membrane pores and extensive membrane rupture.2.Inhibition and action mode of 5,7,8,3',4'-Pentahydroxyisoflavone isolated from vine tea,against Staphylococcus aureus ATCC 25923Previously,isolated novel antimicrobial compound was further tested to explore its physiological aspects.Whereas,and antibacterial properties of new aromatic dihydroxylated metabolite 5,7,8,3',4'-Pentahydroxyisoflavone isolated from vine tea extract against multi drug resistant bacteria Staphylococcus aureus ATCC 25923 was evaluated.The antibacterial activity was determined by measuring the zones of inhibition(ZOI),minimum inhibitory concentration(MIC),minimum bactericidal concentration(MBC),and the time-kill curve assay and antibacterial mechanism was determined by Scanning electron microscope assay,Crystal violet assay,oxygen consumption rate,Interaction between amino acids,Membrane damage assay,leakage of cell constituents,Isolation and quantification of the peptidoglycan,Results showed the preservative potential of new aromatic dihydroxylated metabolite against Staphylococcus aureus(S.aureus ATCC 25923)was excellent with ZOI the lowest MIC and MBC values of 22.20±0.17(mm),32?g/ml and 32?g/ml,respectively.The results also depicted the probable antibacterial mechanism of new aromatic dihydroxylated was the change in permeability and integrity of the cell membranes that leading to leakage of nucleic acids and loss of integrity,that leads towards the loss of essential nutrients for its survival and eventually cause the death of the cell.Furthermore,these finding was also confirmed and analyzed by the help of different experiments.i.e,Membrane damage assay was conducted with crystal violet,which poorly penetrates in the membrane,can easily enter to the damaged membrane so increases in the crystal violet assay up-to 3 times from 27%to>80%clearly represents the destruction of cell membrane because contact membrane will resist in the penetration of the crystal violet to the cell membrane.Oxygen consumption assay showed that the level of microbial count was significantly decreased so oxygen consumption rate was also decreases.In case of interaction between amino acids,there are some amino acids like tryptophan(Trp),tyrosine(Try)and phenylalanine(Phe)which can emit fluorescence in the membrane protein,the amino acids that reacted strongly with 5,7,8,3',4'-Pentahydroxyisoflavone to form a precipitate were the basic amino acids Lys,Arg,and His.but in this study we found that,5,7,8,3',4'-Pentahydroxyisoflavone does not interact with any of these fluorescence emitting amino acid(tryptophan,tyrosine and phenylalanine)which also confirm the rapturing mechanism of cell membrane protein,the concentration of cell constituents(OD 260nm)in suspensions increased by 6.5 times and 8.9 times at 1h,6.2 times and 9.1 times at 2h,and 5.7,times and 7.1 time at 3h,and 5.4 times and 6.3 times at 4h also represents the rapturing mechanism of bacterial cell wall which results in release of bacteria cell constituents,the peptidoglycan content was expressed in terms of Glucosamine hydrochloride content,5,7,8,3',4'-Pentahydroxyisoflavone showed decrease in peptidoglycan content with the time dependent manners.Finding and results of present studied depicted that it is necessary to consider that,this 5,7,8,3',4'-Pentahydroxyisoflavone,new aromatic dihydroxylated compound isolated from vine tea would be an ideal candidate to become a promising antibacterial additive for food preservation.3.Effects of extraction variables on pharmacological activities of Ampelopsis grossedentataThe extraction and optimization of these phytochemicals is very important to be studied and considered and the impact of these extraction variables on the physiological function of these phytochemicals is important.In this chapter,we have studied the area of research entitled because Vine tea(Ampelopsis grossedentata)may potentially perform multiple pharmacological roles,including antibacterial,anti-cancer,antioxidant,hepatoprotective and anti-hypertension functions.But the impact of these extraction variables like,extraction polarity,extraction time and temperature on the recovery of these bioactive compounds along their pharmacological activity of vine tea extract have not been reported yet.So,in this study,the importance of extraction variables for obtaining vine tea extract aiding in purification and isolation of functional and polyphenolic compound and their pharmacological activity from vine tea.The effects of extraction variables(time,temperature and polarity)on total phenolic content(TPC),total flavonoid content(TFA)and condensed tannin content(CTC)were evaluated using single factor experiment.Antiradical capacity assay and radical-scavenging capacity 2,2-diphenyl-l-picrylhydrazyl(DPPH)were tested for evaluating the antioxidant activities of vine tea crude extract.To analyze data,one-way analysis of variance(ANOVA)was established followed by the Tukey's test and to test the correlation,Pearson correlation coefficient was tested.Data explained that extraction polarity,extraction time and the temperature had a significant effect(p<0.05)on yield and their antiradical activities.The optimized extraction parameters for TFA and CTC were 40%aqueous ethanol at 45? while 50? for TPC for 180 min.Whereas,maximum yield was 40.01?g catechin equivalent per gram(CE/g)of dry weight(DW)for TFA,15.12?g Gallic acid equivalent per gram of DW(GAE/g)for TPC and 12.70 ?g CE/g DW for CTC.Ethanol concentration showed a significant effect(p<0.05)on extraction of phenolic compounds and their pharmacological activity especially antioxidant capacity and DPPH assay.This study can be used as preliminary and key information to design central composite rotatable design for response surface methodology(RSM).Moreover,this approach can be used to determine significant factors that influence functional compounds and their other pharmacological activities.4.Optimization of dihydromyricetin from Ampelopsis grossedentata,using the response surface methodology approachThis work provides an optimized extraction approach intended to maximize the recovery of dihydromyricetin(DHM)from Chinese vine tea,Ampelopsis grossedentata leaves.The presented approach adopts Box-Behnken design(BBD)as a response surface methodology(RSM)to understand the role and influence of specific extraction parameters including:time,temperature,and solvent composition/ethanol(%)on DHM final yield.Initially,single factor experiments were used to delineate the role of above factors(temperature,time,and solvent composition)before proceeding with three factors-three levels Box-Behnken design with 17 separate runs to assess the effect of multifactorial treatments on DHM recovery rates.The collected data showed that independent variables(solvent composition,time,and temperature)can significantly affect DHM recovery rates with a maximum yield resulting from a combined 60?,60%aqueous ethanol,and 180min treatments.From the empirical point of view,the above optimized extraction protocol can substantially enhance processing and profitability margins yet with a minimum need of interventions or associated cost.5.Structural identification of two flavonoids and its food preservative effect from vine teaBy analysis of ultraviolet-visible(UV)spectrometry,electrospray ionization mass spectrometry(ESI-MS),nuclear magnetic resonance(NMR)spectrometry and high-performance liquid chromatography(HPLC)-ESI/MS,the compound isolated by solvent extraction was detected as(+)-dihydromyricetin(DHM)while an isomer of(-)-dihydromyricetin(DHM)was collected from recrystallization.HPLC was used for quantification of these elements using external standards as a reference.Results depicted that the contents in leaves of(+)-dihydromyricetin(DHM)and an isomer of(-)-dihydromyricetin(DHM)were measured to be 37.69 ± 1.18%and 74:14±1.98%respectively.Various antioxidant tests(DPPH,reducing power,showed that(+)·dihydromyricetin(DHM)and an isomer of(-)-dihydromyricetin(DHM)were good antioxidants as compare to TBQH.Detection of the kinetics of E.coli ATCC25922 deactivation in situ showed that of the(+)-dihydromyricetin(DHM)and an isomer of(-)-dihydromyricetin(DHM)can be used as food preservative with reference of tetracycline.However,results also depicted that,the antioxidant activity and antimicrobial activity of an isomer of(-)-dihydromyricetin was better than that of the(+)-dihydromyricetin(DHM).These isolated compounds(+)-dihydromyricetin(DHM)and an isomer of(-)-dihydromyricetin(DHM)from vine tea leaves have a potential to be a new natural antibacterial agent with good antimicrobial activities without affecting the quality of food and have a great potential for the application in food industry.