Characterization, Production And Bioactivity Of Flavonoids And Volatile Oils From Bamboo Leaves

Bamboo-leaf-flavonoids and bamboo-leaf-volatile oils are claimed to have bioactivities and be potential for use as functional components.The leaves of Phyllostachys heterocycla(Carr.) Mitford cv.Pubescens Mazel ex H.de leh., Pleioblastus amarus(Keng) Keng f.,Dendrocalamopsis oldhami(Munro) Keng f.and Acidosasa edulis Wen were collected monthly from the Nanping City of Fujian Province from Sep.2007 to Jun.2008.Electrospray ionization(ESI)-tandem quadrupole/orthogonal-acceleration time-of-flight(Q-TOF) mass spectrometer,in combination with high performance liquid chromatograph(HPLC) was used to identify the structures of trace,tiny or ordinary flavonoid compounds extracted from the four kinds of bamboo leaves.Total flavonoids' level and the main flavonoid compounds from the four kinds of bamboo leaves were quantified with the help of diode array high performance liquid chromatography(HPLC-DAD).GC/MS was used to confirm the volatile oil components of bamboo leaves collected from Phyllostachys heterocycla(Carr.) Mitford cv. Pubescens Mazel ex H.de leh.,Pleioblastus amarus(Keng) Keng f., Dendrocalamopsis oldhami(Munro) Keng f.and Acidosasa edulis Wen.The objectives of this study were to:(1) obtain the data of bamboo leaves flavonoids according to differences in growing seasons and bamboo's species;(2) establish to an optimized and unpolluted process to extract and purify bamboo's flavonoids;(3) evaluate the antioxidant activities of extracted bamboo-leaf flavonoids in vitro and in vivo according to obtained flavonoid's fingerprint data;(4) set up a quick GC internal standard quantitative method to accumulate the data of bamboo-leaf volatile oil according to the varying in growing seasons and species;and(5) deal with the antimicrobial activities of volatile oil from Phyllostachys heterocycla(Can'.) Mitford cv.Pubescens Mazel ex H.de leh., Pleioblastus amarus(Keng) Keng f.,Dendrocalamopsis oldhami(Munro) Keng f.and Acidosasa edulis Wen bamboo leaves in combination with volatile oils' fingerprint data.1.The structures identification of bamboo-leaf-fiavonoids 16 flavonoid's compounds were identified from the bamboo-leaves of Phyllostachys heterocycla(Carr.) Mitford cv.Pubescens Mazel ex H.de leh.,including 5 mono-C-glycosylflavones,3 O-glycosylflavones,1 flavonoid aglycones and 7 other flavonoids;24 from Pleioblastus amarus(Keng) Keng f.leaves,including 4 Di-C-glycosylflavones,1 mono-C-glycosylflavones,11 O,C-Diglycosylflavones,2 O-glycosylflavones,1 flavonoid aglycones and 5 other flavonoids;17 from Dendrocalamopsis oldhami(Munro) Keng f.leaves,including mono-C-glycosylflavones,2 O,C-Diglycosylflavones,3 O-glycosylflavones,2 flavonoid aglycones and 5 other flavonoids;and 25 from Acidosasa edulis Wen leaves,including 2 Di-C-glycosylflavones, 1 mono-C-glycosylflavones,7 O,C-Diglycosylflavones,1 O-glycosylflavones,1 flavonoid aglycones and 13 other flavonoids,respectively.2.Seasonal diversification of bamboo-leaf flavonoids(1) The contents of total flavonoids(TF),independent of bamboo's species,increased with the bamboo-leaf development and maturation throughout this study period,and showed an upward trending from the fall,winter,and spring to summer.(2) Like TF from autumn to summer,the levels of individual flavonoid compounds in leaves of the four bamboo species showed an increasement during the whole growing seasons,but variation in the contents of individual flavonoid compounds depended on the bomboo's species.3.Estiblishment of Bamboo-leaf flavonoids extraction technology(1) The yields of bamboo-leaf flavonoids(y) were greatly affected by ethanol concentration(X₁),extraction time(X₂),and liquid-solid ratio(X₃).A return mathematical model,i.e.,y=-12.6432 + 0.3114X₁+ 1.7292X₂+ 0.5018X₃- 0.0106X₁X₂+ 0.1115X₂X₃-0.0023X₁² -0.2808X₂²-0.0459X₃²,was very good in describing the relationships between the extracted yields of bamboo-leaf flavonoids and the 3 parameters.Extraction time, liquid-solid ratio,and ethanol concentration in important order were interactive in influencing the yields of flavonoids extracted from bamboo leaves.The concentration of extracting agent and the extraction time,like the extraction time and liquid-solid ratio, showed an significant interaction to the yields of extracted flavonoids,whereas no significant interaction between the concentration of extracting agent and liquid-solid ratio was found in extracting bamboo-leaf flavonoids.The optimized procedure to extract bamboo-leaf flavonoids was 59.86%of ethanol concentration,3.94 hrs of extraction time, and the liquid-solid ratio of 10:1.The range in error differences between the predicted yields of flavonoids in mathematical model and the real yields of flavonids obtained from experiment did not exceed 3%.(2) The crude extractions of flavonoids from the leaves of four bamboo species were collected using above-given optimized extraction parameters.TF contents ranged from 3.74%to 5.88%in crude extractions.Recoveries in TF were from 58.6%to 84.9%,and yields of the crude extractions in four bamboo's species were from 6.5%to 15.03%.The crude extractions of bamboo-leaf flavonoids were purified by column chromatography filled with HP-20 macroporons resin.HPLC determination showed that the contents of TF were increased from 4.76%to 20.78%after purfication.4.The antioxidant activities of bamboo-leaf flavonoids confirmed in vtiro and in vivo(1) Total antioxidant capacity in vitro,expressed as the equivalent quality of VC(g) per bamboo-leaf flavonoids extractions(g) was 0.102gVC/g for Phyllostaehys heterocycla(Corr.) Mitford cv.Pubescens Mazel ex H.de leh.,0.088gVC/g for Pleioblastus amarus(Keng) Keng f.,0.084gVC/g for Dendrocalamopsis oldhami(Munro) Keng f.and 0.068gVC/g for Acidosasa edulis Wen. Antioxidant capacity of flavonoid crude extracts obtained by purified-column in Phyllostachysheterocycla(Carr.) Mifford cv.PubescensMazel ex H.de leh.was 0.101gVC/g.Reduction ability,defined as the equivalent quality of quercetin(g) per bamboo-leaf flavonoids extractions(g),was 0.069g quercetin/g for Phyllostachys heterocycla(Carr.) Mitfordcv.PubescensMazel ex H.de leh.,0.068g quercetin/g for Pleioblastus amarus(Keng) Keng f.,0.074g quercetin/g for Dendrocalamopsis oldhami(Munro) Keng f.,and 0.062g quercetin/g for Acidosasa edulis Wen.Reduction ability of flavonoid crude extract by purified-column in Phyllostachys heterocycla(Carr.) Mifford cv.Pubescens Mazel ex H.de leh.was 0.131g quercetin/g.The ability of bamboo-leaf flavonoids to scavenge DPPH,defined by their IC50,was 2.26 mg/g for Phyllostachys heterocycla(Carr.) Mifford cv.Pubescens Mazel ex H.de leh.,2.46 mg/g for Pleioblastus amarus(Keng) Keng f.,2.22 mg/g for Dendrocalamopsis oldhami(Munro) Keng f.,and 2.67 mg/g for Acidosasa edulis Wen. DPPH scavenged by column purified flavonoids crude extracts from Phyllostachys heterocycla(Carr.) Mifford cv.Pubescens Mazel ex H.de leh.was 0.96 mg/g. Among four bamboo species,the flavonoids from Acidosasa edulis Wen leaves(calculated at 8mg/g) had the best performance in hydroxyl radical scavenging ability in vitro, followed by Dendrocalamopsis oldhami(Munro) Keng f.,Pleioblastus amarus(Keng) Keng f.and Phyllostachys heterocycla(Carr.) Mifford cv.Pubescens Mazel ex H.de leh.(2) Thus the flavonoid extracts from Dendrocalamopsis oldhami(Munro) Keng f.leaves was further selected to investigate their antioxidant activity in rat's model.These rats were divided randomizely into five groups,named as blank control,negative control,positive control,low dosage bamboo-leaf extracts(LDBE) and high level dosage bamboo-leaf extracts groups(HLBE),respectively.Statistically,each group had seven individuals.Rats in the blank control were fed with no cholesterol-based diet which was not supplemented with any bamboo-leaf extracts or Vit.E.Rats in the negative control were fed with cholesterol-based diet without any supplementation of bamboo-leaf extracts or Vit.E. 0.0025%Vit.E was added to the cholesterol-based diet of rats in positive control. 100mg/kg or 400mg/kg of freeze-dried bamboo-leaf extracts in rat's body weight was added to the diets of LDBE or HLBE group,espectively.After feeding rats for six weeks, tissues collection from liver and kidney showed that bamboo-leaf extracts significantly influenced enzyme's activities of superoxide dismutase(SOD),catalase(CAT), glutathione peroxidase(GSH-Px) and concentation malonaldehyde(MDA) levels in all tesed rats well-controlled of above mentioned activities.The higher antioxidant activity proportional to the dose of bamboo leaf extracts was observed in tested rats.5.The characteristics and activity of volatile oil compounds from bamboo leaves(1) The bamboos leaves collected from Phyllostachys heterocycla(Carr.) Mitford cv. Pubescens Mazel ex H.de leh.were pre-treated with four techniques,i.e.simultaneous distillation extraction(SDE),volatile oil distillation(VOD),supercritical fluid extraction (SFE) and Soxhlet's extraction method(SEM)) to extract volatile oils.The major composition of volatile oils when SDE or VOD was used were alcohols,carboxylic acids, and alkane hydrocarbons.If SFE technique was chosen to treat bamboo leaves,the main compounds of the volatile oils were alkane hydrocarbons and carboxylic acids.Use of SEM technique to treat bamboo leaves produced more alkane hydrocarbons from volatile oil.It was clear that differences in the profiles of the volatile oil of bamboo leaves existed due to the application of different extraction techniques.The present study indicated that the use of SDE or VOD to extract bamboo leaves would give more information on volatile oils composition than the other techniques.(2) The yields of volatile oils of four bamboo species collected by SDE were between 0.1% and 0.3%,with Pleioblastus amarus(Keng) Keng f.the largest,followed by Acidosasa edulis Wen,Dendrocalamopsis oldhami(Munro) Keng f.and Phyllostachys heterocycla(Carr.) Mitford cv.Pubescens Mazel ex H.de leh..The volatile oil compounds of Phyllostachys heterocycla(Carr.) Mifford cv.Pubescens Mazel ex H.de leh.were mainly alcohols,carboxySc acids and alkane hydrocarbons.Volatile oil composition of Acidosasa edulis Wen was very similar to that of Phyllostachys heterocycla(Carr.) Mitford cv.Pubescens Mazel ex H.de leh.The volatile oil compounds of Acidosasa edulis Wen and Dendrocalamopsis oldhami(Munro) Keng f. mainly composed of carboxylic acids,aldehydes and alkane hydrocarbon.(3) Internal standard quantitative method confirmed 7 volatile oil compounds,including Dodecanoic acid,6,10,14-trimethyl-2-Pentadecanone,tetradecanoic acid,tricosane, heptacosane,hexadecanoic acid,and 9-octadecanal.The content of hexadecanoic acid was the highest in all of bamboo-leaf volatile oil.The next was dodecanoic acid in Pleioblastus amarus(Keng) Keng f.and Phyllostachys heterocycla(Carr.) Mitford cv. Pubescens Mazel ex H.de leh.leaves.9-octadecanal ranked in the second position in Acidosasa edulis Wen or Dendrocalamopsis oldhami(Munro) Keng f.leaves. 6,10,14-trimethyl-2-Pentadecanone ranked as the third position in Pleioblastus amarus(Keng) Keng f.and Phyllostachys heterocycla(Carr.) Mitford cv. Pubescens Mazel ex H.de leh.leaves.Among volatile oil compounds determined, dodecanoic acid ranked in the third positon in Acidosasa edulis Wen and Dendrocalamopsis oldhami(Munro) Keng f.(4) Bacillus subtilis,Escherichia coli,2 Pseudomonas fluorescens strains SHL5 and SHL7, Flavobacterium SHL45 and three Saccharomyces cerevisiae strains Ja64,Tokay and Y8 were tested to evaluate growth inhibition of tesed organims bamboo-leaf volatile oils. Among those four bamboo species,the volatile oil from the leaves of Phyllostachys heterocycla(Carr.) Mitford cv.Pubescens Mazel ex H.de leh.showed strongest antimicrobial activity.The minimal inhibition concentration(MIC) toward Escherichia coli and Bacillus subtilis were 2.25mg/mL.In collusions,the present study proved that bamboo leaves contained large amount of flavonoids and volatile oils,with diversity in their composition.The levels of bamboo-leaf flavonoids or volatile oil depended on the species of bamboo and growing seasons, exhibiting good performances in antioxidant and antimicrobial activity.The collection of these data should be very useful in identifying the species of bamboo,and promoting fine production of bamboo leaves as food additives.