Four Flavonoid Compounds from Phyllostachys edulis Leaf Extract Retard the Digestion of Starch and Its Working Mechanisms

Four Flavonoid Compounds from Phyllostachys edulis Leaf ExtractRetard the Digestion of Starch and Its Working MechanismsJun-Peng Yang,†,‡ Hao He,†,‡ and Yan-Hua Lu*,†,‡

†State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai200237, People’s Republic of China‡Shanghai Collaborative Innovation Center for Biomanufacturing Technology, 130 Meilong Road, Shanghai 200237, People’sRepublic of China

*S Supporting Information

ABSTRACT: Bamboo leaf extract as a food additive has been used for preventing the oxidation of food. In the present study, weinvestigated the influence of Phyllostachys edulis leaf extract on starch digestion. Orientin, isoorientin, vitexin, and isovitexin weredetermined as its α-amylase inhibitory constituents. An inhibitory kinetics experiment demonstrated that they competitivelyinhibit α-amylase with Ki values of respectively 152.6, 11.5, 569.6, and 75.8 μg/mL. Molecular docking showed the four flavonescan interact with the active site of α-amylase, and their inhibitory activity was greatly influenced by the glucoside linking positionand 3′-hydroxyl. Moreover, the results of starch−iodine complex spectroscopy, X-ray diffraction, and scanning electronmicroscopy indicated that P. edulis flavonoids retard the digestion of starch not only through interaction with digestive enzymes,but also through interaction with starch. Thus, P. edulis leaf extract can be potentially used as a starch-based food additive foradjusting postprandial hyperglycemia.

KEYWORDS: bamboo leaf, orientin, isoorientin, vitexin, isovitexin, diabetes, α-amylase, starch

■ INTRODUCTION

With the improvement of living standards and the alteration ofour eating habits, the prevalence of diabetes has increased inrecent decades. Data from 130 countries around the worldshow that, in 2013, about 382 million people suffered fromdiabetes, and this number is expected to be 592 million by2035.1 Postprandial hyperglycemia is considered the mostdangerous factor that causes the onset and gradual deterio-ration of diabetes. It can trigger the generation of free radicalsto damage islet cells. Moreover, it can activate a series ofintracellular signal mediators to block the signal transductionpathway of insulin and result in insulin resistance.2,3 Worse still,it will lead to the accumulation of advanced glycation endproducts (AGEs) which directly cause the complications ofdiabetes and the death of patients.4,5

Acarbose, a competitive inhibitor of digestive enzymes, hasbeen clinically used for delaying the expeditious generation ofpostprandial blood glucose, but it has been reported to causeliver disorders, diarrhea, flatulence, and abdominal pain.6,7

These side effects are the most common reason for thewithdrawal of acarbose in recipients.8 Therefore, the develop-ment of a safer drug is urgent. Natural products which havebeen proved relatively safer for humans might be analternative.9 In 2004, an antioxidant of bamboo leafabrown extract of bamboo leafpassed toxicity studies10,11

and was permitted as a new kind of food additive authorized bythe Ministry of Health, People’s Republic of China.12 Inaddition, bamboo leaf is easily accessible and of low cost. Incomparison with the other parts of bamboo, bamboo leaf isnearly an agricultural waste: more than 80% of it rots in farmseach year (Anji County, China, 2013). To date, bamboo leafextract has been reported to exhibit antidiabetic activities by

relieving lipotoxicity,13 improving insulin sensitivity,14 andretarding diabetes complications.15,16 However, whetherbamboo leaf extracts exhibit hypoglycemic activity throughaffecting the digestion of starch is still unclear. The digestion ofstarch is the major source of postprandial hyperglycemia.Additionally, bamboo leaf contains many kinds of flavonoidsand phenolic acids.17,18 These classes of polyphenols have beenreported to possess potential digestive enzyme inhibitoryactivity.19−23

The digestion of starch is also affected by the nature of thestarch itself. According to its digestibility, starch is classified asRDS (rapidly digestible starch), SDS (slowly digestible starch),and RS (resistant starch).24 Some phytochemicals such astannic acid, catechin,25 epicatechin dimethylgallate, rutin,26

genistein,27 and Citrus flavonoids28 have been reported to bindwith starch and affect the digestibility of starch. This bindingalso can affect the physical properties of starch, includingpasting, gelling, and retrogradation.29,30 However, to date, nostudy has focused on the binding between bamboo leaf extractand starch.In this study, first, we will determine the α-amylase inhibitory

constituents of Phyllostachys edulis leaves (a kind of bamboowhich is most widely planted in China). Second, these activeconstituents will be subjected to an inhibitory kinetics test andmolecular docking. Last, whether these active constituents caninteract with starch and affect its digestion feature will beinvestigated.

Received: April 22, 2014Revised: July 3, 2014Accepted: July 14, 2014Published: July 14, 2014

Article

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© 2014 American Chemical Society 7760 dx.doi.org/10.1021/jf501931m | J. Agric. Food Chem. 2014, 62, 7760−7770

pubs.acs.org/JAFC

■ MATERIALS AND METHODSChemicals. Porcine pancreas α-amylase, amylose, amylopectin, and

acarbose were purchased from Sigma-Aldrich Co., Ltd. (St. Louis,MO). α-(2-Chloro-4-nitrophenyl)-β-1,4-galactopyranosylmaltoside(Gal-G2-α-CNP) was purchased from Toyobo Co., Ltd. (Osaka,Japan). Vitexin, isovitexin, orientin, and isoorientin were purchasedfrom Shifeng Biological Technology Co., Ltd. (Shanghai, China), andtheir structures are shown in Figure 1. Soluble starch was purchased

from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 2-Chloro-4-nitrophenol (CNP) was purchased from Energy Chemical(Shanghai, China). Acetonitrile (HPLC-grade) was provided by TediaCo. Inc. (Fairfield, OH).Preparation of the Main Solutions. Iodine solution:26,31 1.5 g of

potassium iodide was dissolved to 25 mL of distilled water, and then0.635 g of iodine was added. Finally, the solution was adjusted to 50mL by distilled water. Starch suspension:26,31 10 mg of soluble starch

was suspended in 1 mL of boiling water and water bathed at 90 °C for30 min to make the starch swell. Then 7.5 mL of 50 mM KCl−HCl(pH 2.0) buffer was added followed 30 min later by 7.5 mL of 100mM sodium phosphate buffer (pH 7.1) (the final pH was 6.8). Theywere used to simulate the pH of the stomach and duodenum. Last, thesuspension was homogenized by a glass homogenizer. The preparationof amylose and amylopectin suspensions was the same as that ofsoluble starch suspensions.

Sample Preparation. P. edulis leaves were collected in AnjiCounty (the bamboo hometown of China) during the winter andidentified by Pei-xin Zhang of the Anji Forestry Bureau. Thepregrounded P. edulis leaves were extracted with 50% ethanol (1:10,w/v) with a 1 h sonic treatment (DT series, Xingzhi Co., Ltd., Ningbo,China) and intermittent stirring. The extract was concentrated to 1/10of the original volume by a rotary evaporator (RE-52A, ShanghaiJingke Co. Ltd., Shanghai, China). Then it was stored in a 4 °Crefrigerator for 12 h to flocculate and filter some macromolecularcompounds. After that, the solution was sequentially fractioned bypetroleum ether, EtOAc, and 1-butanol. All these extracts were driedto obtain the petroleum ether fraction, EtOAc fraction, 1-butanolfraction, and residual fraction. The 1-butanol fraction was subjected toan AB-8 macroporous adsorption resin (polystyrene resin, weakpolarity, 0.3−1.25 mm particle size) for further purification. Thepurification was carried out in a glass column (1.4 cm × 40 cm) wet-packed with washed AB-8 resin. The sample was dissolved in water.After all the sample was loaded, the column was first washed by 6 BV(bed volumes) of water to remove the impurities which could notadsorb tightly. Then the column was desorbed by 2 BV of 40% ethanol(v/v) solution at a speed of 2 BV/h.

Determination and Quantification of the Main BioactiveComponents. The Shimadzu HPLC system (Shimadzu Corp.,Tokyo, Japan) equipped with an Agilent Eclipse Plus C18 column(250 mm × 4.6 mm, particle size of 5 mm, Agilent Technologies Inc.,Santa Clara, CA) was used. The mobile phase was phosphoric acid−water (0.3:99.7, v/v; A) and acetonitrile (B). The gradient elutioncondition was 15−25% B in 0−35 min. The flow rate was set as 1.0mL/min. The signal was monitored at 330 nm. A Thermo LCQ-DecaHPLC-MS (Thermo Fisher Scientific Inc., San Jose, CA) fitting withan ESI (electrospray ionization) interface and negative ion mode wasalso used for determining the main bioactive components. Forquantitative analysis, the calibration curves of orientin, isoorientin,vitexin, and isovitexin were obtained at a concentration of 6.25−200μg/mL (n = 6).

Measurement and Kinetics Assay of α-Amylase InhibitoryActivity. The measurement of α-amylase inhibitory activity wascarried out by using the method described previously.32,33 Briefly, 20μL of P. edulis flavonoids at different concentrations (finalconcentrations were 9, 18, 36, and 72 μg/mL) were mixed with 10μL of 200 μg/mL α-amylase and 70 μL sodium phosphate buffer (pH6.8). After intensive shaking for mixing, 100 μL of 5 mM Gal-G2-α-CNP solution was added to start the reaction. After 10 min ofincubation at 37 °C, the absorbance of released CNP was monitored at405 nm by a Bio-Tek Power Wave XS2 microwell plate reader (Bio-Tek Instruments, Winooski, VT). To further explore their inhibitorycharacter, we performed kinetic analysis by using Lineweaver−Burkplots. The Ki value of competitive inhibition was obtained from theleast-squares regression line of the slopes of Lineweaver−Burk plotsversus the corresponding [I]. The derivation formulas are

=+ +

= + +⎛⎝⎜

⎞⎠⎟

( )V

V

K

VK

V K V1

[S]

1 [S]and

1 [I] 1[S]

1

K

max

m[I]

m

max i max

i

(1)

= + = +⎛⎝⎜

⎞⎠⎟

KV K

KV K

KV

slope 1[I]

and slope [I]m

max i

m

max i

m

max

(2)

Figure 1. Structures of four P. edulis leaf flavonoids. The parentnucleus of flavonoid is composed of A, B, and C rings.

Journal of Agricultural and Food Chemistry Article

dx.doi.org/10.1021/jf501931m | J. Agric. Food Chem. 2014, 62, 7760−77707761

− =K xintercept of eq 2 on axisi

Thermal Stability. First, the standard solutions of the fourflavonoids were mixed. Second, we placed a certain volume of themixed standard into a tube. The tube was sealed by plastic tape andheated at 100 °C for 2 h. Last, we detected the samples without andwith heat treatment by HPLC and calculated the concentrationalteration of our four flavonoids according to their peak areas.Molecular Modeling. The three-dimensional structures of

orientin, isoorientin, vitexin, and isovitexin were downloaded fromthe National Centre for Biotechnology Information PubChemcompound database (http://pubchem.ncbi.nlm.nih.gov/). The three-dimensional structure of substrate Gal-G2-α-CNP was constructed byusing ChemBio3D Ultra 12.0 software (Cambridge Soft Corp.,Cambridge, U.K.). The crystal structure of the pig pancreatic α-amylase protein complex (PDB code 1WO2) was retrieved from theRCSB Protein Data Bank (http://www.rcsb.org). Then docking wascarried out by using the Autodock v4.2 program (Scripps ResearchInstitute, La Jolla, CA). The Lamarckian Genetic Algorithm was usedto run the docking. First, we did a blind docking in which the grid boxcovers the whole receptor protein. Then a smaller grid box that mainlycovers the site of the best cluster of blind docking was set for doing anaccurate docking. After docking, cluster analysis based on the root-mean-square deviation (RMSD) value was performed. The toleranceof RMSD was set as 2.0. The lowest energy conformation of the mostpopulated cluster was considered as the most reliable solution. Theinteraction analysis was carried out by using Pymol 0.99 (DeLanoScientific LLC, San Carlos, CA), YasaraView v13.9.8 (YASARABiosciences, Vienna, Austria), and Ligplus v1.4.5 (EMBL-EBI,Cambridge, U.K.).Interaction between Flavonoids and Starch and Their

Influence on Starch Digestion. The interaction between P. edulisflavonoids and starch was studied by using the spectroscopic methoddescribed previously.26,31 Briefly, 25 μL of flavonoid was added to 0.9mL of soluble starch suspension (the final concentrations of flavonoidwere 9, 18, and 36 μg/mL). After vortexing, 0.1 mL of iodine solutionwas added to the suspension. Then the absorption spectrum of thestarch−iodine complex was measured from 500 to 900 nm by using aUV-1800 spectrophotometer (Shimadzu, Tokoyo, Japan). Theabsorption spectra of the amylose−iodine complex and theamylopectin−iodine complex were obtained in the same way as thatof the soluble starch−iodine complex. The digestion of starch was alsostudied by using the spectroscopic method.26,31 Briefly, 25 μL offlavonoid was added to 0.9 mL of starch suspension (the finalconcentrations of flavonoid were 9, 18, and 36 μg/mL), and then 10μL of 0.5 mg/mL α-amylase was added to the suspension. After thesuspension was incubated for 5 min at 37 °C, 0.1 mL of iodinesolution was added to terminate the digestion and color the residualstarch. The degree of digestion was estimated by the differencespectrum (ΔA) of the starch−iodine complex after and beforedigestion.X-ray Diffraction (XRD). The sample preparation and detection

parameters were the same as those described in previous reports.29,34

Soluble starch and resin-purified extract (10:1, w/w) were mixed first,and then deionized water was added (10:1, v/w, based on starch). Themixture was water bathed at 90 °C with vigorous stirring for 60 min.Then it was stored at 4 °C for 3 days (at room temperature more than10 days is needed to achieve the same degree of retrogradation) andfreeze-dried last. A D/MAX 2550 rotating anode X-ray powderdiffractometer (Rigaku, Tokoyo, Japan) equipped with a copper tubeat 40 kV and 100 mA with Cu Kα radiation (λ = 0.154 nm) was usedfor crystalline analysis. The diffractograms were detected in the anglerange from 5° to 50° (2θ) with a step size of 0.02°. The MDI Jade 5.0software (Materials Data Inc., Livermore, CA) was used to analyze thediffractograms. The relative crystallinity was calculated by using thefollowing equation: relative crystallinity (%) = 100Ac/(Ac + Aa), whereAc is the area of the crystal peak and Aa is the area of the amorphouspeak.Scanning Electron Microscopy (SEM). The sample was fixed on

a silver specimen holder using double-sided adhesive and sputtered

with gold in a vacuum evaporator to make the sample conductive.Then the sample was investigated with an S-3400N scanning electronmicroscope (Hitachi, Tokoyo, Japan) with a 15 kV acceleratingvoltage.

Statistical Analysis. All experiments were repeated three times.Statistical analysis was carried out by using one-way analysis ofvariance in SPSS 18.0 (SPSS, Chicago, IL); p < 0.05 was considered tobe statistically significant.

■ RESULTS AND DISCUSSIONα-Amylase Inhibition Ability and Yield of P. edulis

Leaf Extract Fractions. The α-amylase inhibition ability and

the yield of several P. edulis leaf extract fractions are listed inTable 1. The EtOAc fraction and 1-butanol fraction exhibitedstronger α-amylase inhibitory activity than the other fractions,

Table 1. α-Amylase Inhibition Ability and Yield of EachFraction

extract IC50 (μg/mL) yield (%)

ethanol extract 591.8 6.96 ± 0.33petroleum ether fraction >800 0.97 ± 0.04EtOAc fraction 318.1 0.33 ± 0.041-butanol fraction 354.1 0.93 ± 0.06residual fraction >800 4.36 ± 0.32resin-purified extract 206.7 0.63 ± 0.03

Figure 2. α-Amylase inhibitory activity of P. edulis leaf flavonoids,resin-purified extract, and acarbose. Values are normalized to controls(100%) and expressed as the mean ± SD of three experiments. Oneasterisk means p < 0.05, two asterisks mean p < 0.01, and threeasterisks mean p < 0.001 compared to the control group.



http://pubchem.ncbi.nlm.nih.gov/

http://www.rcsb.org

but the yield of the EtOAc fraction is just one-third of the yieldof the 1-butanol fraction and the EtOAc fraction is barelysoluble in water. When loading the resin column, the materialsmust be dissolved in water, so the 1-butanol fraction is moresuitable than the EtOAc fraction to be subjected to the AB-8macroporous adsorption resin column for further purification.The obtained resin-purified extract exhibited the strongest α-amylase inhibitory activity.Determination and Quantitation of the Main Bio-

active Components. From the HPLC spectrum of the resin-purified extract (Supporting Information), we found two peakswere significantly higher than the other peaks. The two peaksmight be the main α-amylase inhibitory components of theresin-purified extract. From the ultraviolet spectra (SupportingInformation) of the two peaks, we found their maximumabsorbances were near 270 and 340 nm, which suggested thatthese compounds might be flavonoids. According to thepublished data12,17 and by further comparing the retentiontimes, ultraviolet spectra, and m/z values with those of ourstandards, peaks 7, 8, 9, and 10 were identified as isoorientin

([M − H]−, m/z 447.0), orientin ([M − H]−, m/z 447.0),vitexin ([M − H]−, m/z 431.1), and isovitexin ([M − H]−, m/z431.0), respectively (Supporting Information). Using thecalibration curve, the concentrations of orientin, isoorientin,vitexin, and isovitexin were respectively 12.64, 87.15, 4.82, and9.33 mg/g in the resin-purified extract. We were going tofurther affirm their α-amylase inhibitory activity. However,there might be other compounds with potent α-amylaseinhibitory activity in the ethyl acetate fraction. We will try topurify them by silica gel column chromatography and SephadexLH-20 column chromatography in the future.

α-Amylase Inhibitory Activities of the Four Flavo-noids and Their Structure−Activity Relationships. Asshown in Figure 2, all four flavonoids can dose-dependentlyinhibit α-amylase, and the inhibitory effect of isoorientin andisovitexin is stronger than the inhibitory effect of many otherphytochemicals such as hesperidin, naringin, neohesperidin,nobiletin,28 andrographolide,35 cyanidin, cyanidin 3-glucoside,36

myricetin, quercetin, luteolin, kaempferol, and baicalein19 (thecomparison of their IC50 values is given in the SupportingInformation). However, their inhibitory effect is weaker thanthat of the positive drug acarbose.All the four flavonoids are flavone C-glycosides. Unlike

flavone O-glycosides, flavone C-glycosides will not be hydro-lyzed to their aglycon by β-glucosidase in the small intestine.37

Therefore, the four flavonoids can contact α-amylase which islocated in the intestine. According to the two HPLC spectrawithout and with heat treatment (Supporting Information), theconcentration alterations of isoorientin, orientin, vitexin, and

Figure 3. Lineweaver−Burk plots of P. edulis leaf flavonoids. The concentrations of P. edulis leaf flavonoids were respectively 0, 36, and 72 μg/mL.The concentrations of Gal-G2-CNP were respectively 1, 1.5, 2, 3, and 5 mM. The insets ([I] versus the corresponding slope of Lineweaver−Burkplots) were used to calculate the Ki value.

Table 2. Inhibitory Type, Ki, and IC50 of P. edulis LeafFlavonoids and Resin-Purified Extract against α-Amylase

ligand inhibitory type Ki (μg/mL) IC50 (μg/mL)

orientin competitive 152.6 >270isoorientin competitive 11.5 29.9vitexin competitive 569.6 >270isovitexin competitive 75.8 65.3resin-purified extract 206.7



isovitexin are respectively 6.6%, 1.8%, 9.3%, and 8.0% after heattreatment. This indicates that the four flavonoids will not beeasily destroyed during food processing. However, manyphytochemicals such as anthocyanin38 and hypericin39 areextremely unstable under heating, even under lighting.The Lineweaver−Burk plots of the four flavonoids are given

in Figure 3. They all have an intersection at the y axis whichindicates their inhibitory types are all competitive. Their Ki andIC50 values are given in Table 2. Their inhibitory sequence isisoorientin > isovitexin > resin-purified extract > orientin >vitexin, and the differences between their effects are verysignificant. This is an interesting phenomenon because thestructures of the four flavonoids are highly similar. Two

speculations can be elicited from this phenomenon: (1)Flavone C6-glycosides have stronger α-amylase inhibitoryactivity than flavone C8-glycosides (because the IC50 ofisoorientin is less than that of orientin and the IC50 ofisovitexin is less than that of vitexin). (2) The C3′-hydroxyl onthe B ring is important for the α-amylase inhibitory activity(because the IC50 of isoorientin is less than that of isovitexin).Speculation 2 can be supported by the previous work of

Tadera et al.19 They found that the inhibitory activity offlavonoid is positively related to the number of hydroxyls onthe B ring. For speculation 1, we have not found any literaturesupport, but we tried to explain it by using molecular dockingin the following subsection.

Molecular Docking between P. edulis Flavonoids andα-Amylase. Molecular docking was used to stimulate thebinding mode of the ligand and receptor according to the“lock” and “key” principle. Here we used molecular docking toanalyze why the four P. edulis flavonoids have highly similarstructures but exhibit greatly different α-amylase inhibitoryactivities.Figure 4 illustrates that all four flavonoids bind at the active

site of α-amylase, which is a large hydrophobic pocket on thesurface of α-amylase.40 They all occupy the binding position ofsubstrate Gal-G2-CNP more or less. Among them, isooreintin(B) and isovitexin (D) cover a large part of the binding positionof the substrate, but oreintin (A) and vitexin (C) cover asmaller part. Therefore, isoorientin and isovitexin can moreeffectively cause steric hindrance to hinder the binding of thesubstrate. This is consistent with our experimental result thatisoorientin and isovitexin exhibit much stronger inhibitoryactivity than orientin and vitexin. In fact, when the glucose linksat C8 (orientin, vitexin), a corner of less than 90° will existinside the flavonoid molecule and lead the three-dimensionalstructure of the flavonoid to look like a “V”. Molecules with a“V” structure (orientin and vitexin) are easily trapped in a smallhollow near the active site of α-amylase (which is veiled inFigure 4A,C, but is clear in Figure 4E,F). When the glucoselinks at C6 (isoorientin, isovitexin), the structure of theflavonoid is linear. Molecules with a linear structure are moresimilar to the substrate and fit the active site of amylase better.Additionally, we downloaded the X-ray crystal structure of theporcine pancreatic α-amylase−acarbose complex (Figure 4G)which was derived from an X-ray diffraction experiment and notin silico docking as ours was. We find that the positive drugacarbose almost fully occupies the binding position of thesubstrate. This is why its inhibitory effect is stronger than thatof our flavonoids.Besides geometry complementarity, the binding strength

between the ligand and receptor is also important.41 In Table 3,we summarize the H-bond, π−π, and hydrophobic interactions

Figure 4. Surface representations of docked ligands on α-amylase withthe superimposition of substrate Gal-G2-CNP. The big orangebackground is the surface of α-amylase (PDB code 1WO2), thedark blue molecule is synthetic substrate Gal-G2-CNP, the pinkmolecule is orientin, the green molecule is isoorientin, the light bluemolecule is vitexin, the yellow molecule is isovitexin, and the redmolecule is the positive drug acarbose.

Table 3. H-Bond, π−π, and Hydrophobic Interactions between Our Flavonoids and the Residues of α-Amylasea

hydrogen bond π−π interaction hydrophobic interaction

orientin Asp300, His299, Gly304, His305 Trp58, Trp59, His305 Glu233, Asp300, Trp58, Trp59, Tyr62, His299, Arg303, Gly304, His305,Asp356

isoorientin Asp197, Glu233,Gln63, Val163,Arg195

Trp58, Trp59, Tyr62, Arg195,His299

Asp197, Glu233, Asp300,Trp58, Trp59, Tyr62, Gln63, Val163, Arg195, Ala198, His299

vitexin Asp300, His299, Gly304, His305 Trp58, Trp59, His305 Glu233, Asp300,Trp58, Trp59, Tyr62, His299, Arg303, Gly304, His305,Asp356

isovitexin Asp197, Gln63, Val163, Arg195 Trp58, Tyr62, Arg195, His299 Asp197, Asp300,Val51, Trp58, Trp59, Tyr62, Gln63, Val163, Leu165,Arg195, His299

aThe bold-font residues (Asp197, Glu233, Asp300) are the residues which play the key role in cutting the starch chain.



between flavonoids and the residues of α-amylase. Theirinteracting graphs are also given in the Supporting Information(Figures S4−S6). We find that, due to the extra 3′-hydroxyl,isoorientin forms more H-bonds with α-amylase than isovitexin,and two of them are formed with key residues. (The active siteof α-amylase contains three key acidic residues, Asp197,Glu233, and Asp300. They work together to cleave the starchchain.40,42) This explains why isoorientin exhibited strongerinhibitory activity than isovitexin.P. edulis Leaf Extracts Bind with Starch and Affect Its

Digestibility. Some small molecular phytochemicals can affectthe digestibility of starch through binding with starch.25,26,28

Here we used a spectroscopic method26,31 to investigatewhether P. edulis extracts bind with starch and further affect itsdigestibility. The ΔA curve of Figure 5 was obtained throughsubtraction of the absorption curve of flavonoid + iodine fromthe absorption curve of starch + flavonoid + iodine (the controlis absence of flavonoid). Figure 5 shows that the ΔA curvesdecline with an increase of our flavonoids and resin-purifiedextract, but do not decline with an increase of acarbose. This

indicates that orientin, isoorientin, vitexin, isovitexin, and resin-purified extract can bind with starch, but the positive drugacarbose cannot. According to the percentages on the right sideof each curve, the binding strength order of our compounds isisoorientin ≈ orientin > vitexin ≈ isovitexin > resin-purifiedextract > acarbose.The ΔΔA spectrum at the left bottom of each picture was

obtained through subtraction of the ΔA curve of the controlfrom the ΔA curve of 36 μg/mL flavonoid. Unlike the ΔΔAcurves of naringin and neohesperidin28 which have a distinctpeak at 600 nm but are nearly zero at 500 nm, the ΔΔA curvesof our flavonoids do not have a distinct peak at 600 nm butshow a significant negative value at 500 nm. This indicates theymainly bind with amylopectin because the λmax of theamylopectin−iodine complex is between 500 and 540 nm butthe λmax of the amylose−iodine complex is between 540 and660 nm.43

The influence of P. edulis leaf extracts on the formation of theamylopectin−iodine complex and amylose−iodine complex wasalso tested (Supporting Information). The decline of the ΔA

Figure 5. Interactions between P. edulis leaf flavonoids, resin-purified extract, acarbose, and soluble starch. ΔA = A(soluble starch + compound +iodine) − A(compound + iodine): (A) orientin added, (B) isoorientin added, (C) vitexin added, (D) isovitexin added, (E) resin-purified extract(RPE) added, (F) acarbose added. The control for each panel is no addition of compound. Inset: ΔΔA spectrum, which is the curve of 36 μg/mLcompound minus the curve of the control. The percentage on the right side of each curve is the peak ΔA value of the curve divided by the peak ΔAvalue of the control curve.



curve of amylopectin−iodine is more significant than thedecline of the ΔA curve of amylose−iodine. This confirms theconclusion induced from the ΔΔA spectra of Figure 5 that P.edulis flavonoids mainly bind with amylopectin. Additionally,the amylopectin−iodine complex shows a peak around 535 nm,and the amylose−iodine complex shows a peak around 605 nm.The peak of the soluble starch−iodine complex is betweenthem (around 565 nm, Figure 5) due to soluble starch beingcomposed of amylopectin and amylose.The ΔA curve of Figure 6 was obtained through subtraction

of the absorption curve of the undigested starch−iodinecomplex from the absorption curve of the digested starch−iodine complex (addition of iodine after incubation with α-amylase). The less the starch was digested, the higher the ΔAvalue (negative). Figure 6 illustrates that the ΔA curve riseswith an increase of our compounds. This indicates that ourcompounds can inhibit the digestion of soluble starch. Theorder of their effect was acarbose > isoorientin > isovitexin ≈

orientin > vitexin > resin-purified extract. However, this isdifferent from the α-amylase inhibitory order we obtained fromFigure 2 (the substrate was Gal-G2-CNP, a small moleculewhich was used to exclude the influence of the interactionbetween starch and our compounds), that is, acarbose >isoorientin > isovitexin > resin-purified extract > orientin >vitexin, and the gap between orientin and isovitexin and the gapbetween acarbose and isoorientin are narrowed when starch isused as the substrate. If these compounds simply inhibited theactivity of α-amylase, whether using Gal-G2-CNP or starch asthe substrate, the orders of their inhibitory effect should be thesame. Therefore, we speculate that the effect of P. edulis extractson the digestion of starch is not only due to the inhibition of α-amylase. Maybe they can further affect the digestibility of starchthrough binding with it. Two studies can support ourspeculation: One study found that due to the binding ofrutin, epicatechin dimethylgallate, and fatty acids the glycemicindex of buckwheat flour is lower than that of wheat flour.26

Figure 6. Effect of P. edulis leaf flavonoids, resin-purified extract, and acarbose on the digestion of soluble starch: (A) orientin added, (B) isoorientinadded, (C) vitexin added, (D) isovitexin added, (E) resin-purified extract added, (F) acarbose added. ΔA = A(digested starch−iodine complex) −A(undigested starch−iodine complex). The control for each panel is no addition of compound. The percentage at the right side of each curve is thepeak ΔA value of the curve divided by the peak ΔA value of the control curve.



The other study found that the resistant starch content of thequercetine−starch complex was sharply higher than that ofcommon starch.44 We had known that the binding strengthbetween our compounds and starch was different: isoorientin ≈orientin > vitexin ≈ isovitexin > resin-purified extract >

acarbose. Therefore, the inhibitory order change when usingstarch as the substrate was due to the different bindingstrengths of these compounds with starch. Why can the bindingretard the digestion of starch? Many researchers hold the viewthat some compounds can bind with starch through occupation

Figure 7. X-ray diffractograms of starch: (A) resin-purified extract, (B) natural soluble starch, (C) retrograded starch, and (D) retrograded resin-purified extract−starch complex. The red jagged line is the original diffractogram. The green area represents the amorphous peak obtained by fitting.The other colored areas represent the crystal peaks obtained by fitting.



of the hydrophobic helical structure of starch. The occupationof the hydrophobic helical structure will disturb thecombination of starch and amylase.26−28

Influence of P. edulis Extract on the Physical Proper-ties of Starch. In the cooking process, well-ordered starchpolymers will be broken into free starch chains; this is calledgelatinization. During cooling, these free starch chains willreassociate and re-form crystalline domains; this is calledretrogradation. Retrogradation will markedly increase therigidity and shorten the shelf life of starch-based food.45 Tofurther prove the binding between P. edulis leaf extract andstarch, we investigated whether the resin-purified extract caninfluence the retrogradation of starch by using X-ray diffractionand scanning electron microscopy.Figure 7A is the X-ray diffractogram of the resin-purified

extract which does not have any crystal peak. This means thatthe resin-purified extract is amorphous. Figure 7B is the X-raydiffractogram of natural soluble starch (without heating andcooling treatment). It exhibits a typical B-type crystallinestructure (according to X-ray diffractograms, the crystallineform of starch can be classified as A, B, C, and V46) with peaksclose to 17.2°, 22.2°, and 24.0°. Parts C and D of Figure 7 arethe diffractograms of retrograded starch and the retrogradatedresin-purified extract−starch complex. We find three crystalpeaks in Figure 7C, but only one crystal peak (17.2°) in Figure7D. Their crystallinities are respectively 8.99% and 4.93%. Thisindicates that, once we added the resin-purified extract, theretrogradation of starch was retarded.

The mechanism of retrogradation is the reassociation ofstarch chains through hydrogen bonds. Phenolic compoundswhich contain many reactive hydroxyls can disturb theformation of H-bonds between starch chains through formationof H-bonds with starch.29,30 Researchers had confirmed that thegreater the number of OH groups in the plasticizer, the betterthe plasticizer reduces the crystallization of starch.47 Someother researchers hold the view that, during boiling, phenoliccompounds and starch can form covalent bonds which canfurther retard the crystallization of starch.48

Scanning electron microscopy was used to investigate themorphology difference between natural soluble starch,retrogradated starch, and the retrogradated resin-purifiedextract−starch complex. In Figure 8A1,A2,A3 (100×), theshape of natural soluble starch is an elliptic granule (A1) whosediameter is about 50 μm; the retrogradated starch (A2) andretrogradated resin-purified extract−starch complex (A3) areirregularly shaped agglomerates whose long axes respectivelyvary between 200 and 250 μm and betwen 50 and 150 μm.From Figure 8B1,B2,B3 (3000×), we find that the surface ofnatural starch is smooth (B1), but there are many apophyses onthe surface of retrogradated starch and the retrogradated resin-purified extract−starch complex (B2, B3). Parts C1, C2, and C3(5000×) of Figure 8 are the faultages of these starches. Thenatural starch showed a very dense faultage which indicates itscompact interior structure (C1), while on the faultages ofretrogradated starch and the retrogradated resin-purifiedextract−starch complex there are many irregular bulges (C2,C3). Inside the faultage of the retrogradated resin-purified

Figure 8. SEM images of starch. Panels A1, B1, and C1 are natural soluble starch (without gelatinization and retrogradation), and theirmagnifications are respectively 100×, 3000×, and 5000×. Pictures A2, B2, and C2 are retrograded starch (without addition of resin-purified extract),and their magnifications are respectively 100×, 3000×, and 5000×. Pictures A3, B3, and C3 are the retrograded resin-purified extract−starchcomplex, and their magnifications are respectively 100×, 3000×, and 5000×. B1, B2, and B3 are the outside surface. C1,C2, and C3 are the fracturesurface.



extract−starch complex (C3), we also find some cracks andcavities. The cracks and cavities illustrate the crystalline textureof the retrogradated resin purified extract−starch complex isless compact than that of retrogradated starch. This indicatesthat P. edulis leaf extract can interact with starch and retard theretrogradation of starch. This is similar to the result of anotherstudy that the gel matrix of a wheat starch−pomegranate peelextract complex is looser than that of common starch.30

In conclusion, our studies provide scientific support for thefurther use of P. edulis leaf extract as a functional food additivethat can adjust postprandial hyperglycemia and prolong theshelf life of starch-based food. Furthermore, our study on thestructure−activity relationship of flavone C-glycosides can beapplied to drug design. In the future, the influence of bambooleaf extract on the glucose transporter of small intestinal brushborder cells deserves further study, because it is the last step inthe generation of blood glucose.49 We hope our study canattract more attention to this bamboo leaf resource and benefitdiabetes research and hardworking bamboo farmers in thefuture.

■ ASSOCIATED CONTENT*S Supporting InformationComparison of the IC50 values of isoorientin, isovitexin, andother compounds (Table S1), HPLC spectrum (Figure S1) andHPLC−MS spectrum (Figure s2) of the resin-purified extract,HPLC spectra of the mixed standard without and with heattreatment (Figure S3), hydrogen bond interaction networks(Figure S4), π−π interaction networks (Figure S5), andhydrophobic interaction networks (two-dimensional) (FigureS6) between P. edulis flavonoids and α-amylase, andinteractions between P. edulis leaf flavonoids and amylopectin(Figure S7) and amylose (Figure S8). This material is availablefree of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*E-mail: [email protected]. Phone: +86-21-64251185.Fax: +86-21-64251185.FundingThis work was supported by the Fundamental Research Fundsfor the Central Universities and partially supported by theShanghai Leading Academic Discipline Project (Grant B505)and the National Special Fund for State Key Laboratory ofBioreactor Engineering (Grant 2060204).NotesThe authors declare no competing financial interest.

■ ABBREVIATION USEDGal-G2-CNP, α-(2-chloro-4-nitrophenyl)-β-1,4-galactopyrano-sylmaltoside

■ REFERENCES(1) Guariguata, L.; Whiting, D.; Hambleton, I.; Beagley, J.;Linnenkamp, U.; Shaw, J. Global estimates of diabetes prevalencefor 2013 and projections for 2035 for the IDF Diabetes Atlas. DiabetesRes. Clin. Pract. 2013, 103, 137−149.(2) Stumvoll, M.; Goldstein, B. J.; van Haeften, T. W. Type 2diabetes: principles of pathogenesis and therapy. Lancet 2005, 365,1333−1346.(3) Pekkanen, J.; Tuomilehto, J.; Qiao, Q.; Jousilahti, P.; Lindstrom,J. Glucose tolerance and mortality: comparison of WHO and

American Diabetes Association diagnostic criteria. Lancet 1999, 354,617−621.(4) Ahmed, N. Advanced glycation endproductsrole in pathologyof diabetic complications. Diabetes Res. Clin. Pract. 2005, 67, 3−21.(5) Peng, X.; Zheng, Z.; Cheng, K.-W.; Shan, F.; Ren, G.-X.; Chen,F.; Wang, M. Inhibitory effect of mung bean extract and itsconstituents vitexin and isovitexin on the formation of advancedglycation endproducts. Food Chem. 2008, 106, 475−481.(6) Hsiao, S.-H.; Liao, L.-H.; Cheng, P.-N.; Wu, T.-J. Hepatotoxicityassociated with acarbose therapy. Ann. Pharmacother. 2006, 40, 151−154.(7) Chiasson, J.-L.; Josse, R. G.; Gomis, R.; Hanefeld, M.; Karasik, A.;Laakso, M. Acarbose treatment and the risk of cardiovascular diseaseand hypertension in patients with impaired glucose tolerance: theSTOP-NIDDM trial. JAMA, J. Am. Med. Assoc. 2003, 290, 486−494.(8) Neuser, D.; Benson, A.; Bruckner, A.; Goldberg, R. B.; Hoogwerf,B. J.; Petzinna, D. Safety and tolerability of acarbose in the treatmentof type 1 and type 2 diabetes mellitus. Clin. Drug Invest. 2005, 25,579−587.(9) Yilmazer-Musa, M.; Griffith, A. M.; Michels, A. J.; Schneider, E.;Frei, B. Grape seed and tea extracts and catechin 3-gallates are potentinhibitors of α-amylase and α-glucosidase activity. J. Agric. Food Chem.2012, 60, 8924−8929.(10) Lu, B.; Wu, X.; Tie, X.; Zhang, Y.; Zhang, Y. Toxicology andsafety of anti-oxidant of bamboo leaves. Part 1: acute and subchronictoxicity studies on anti-oxidant of bamboo leaves. Food Chem. Toxicol.2005, 43, 783−792.(11) Lu, B.; Wu, X.; Shi, J.; Dong, Y.; Zhang, Y. Toxicology andsafety of antioxidant of bamboo leaves. Part 2: developmental toxicitytest in rats with antioxidant of bamboo leaves. Food Chem. Toxicol.2006, 44, 1739−1743.(12) Zhang, Y.; Jiao, J.; Liu, C.; Wu, X.; Zhang, Y. Isolation andpurification of four flavone C-glycosides from antioxidant of bambooleaves by macroporous resin column chromatography and preparativehigh-performance liquid chromatography. Food Chem. 2007, 107,1326−1336.(13) Panee, J.; Liu, W.; Lin, Y.; Gilman, C.; Berry, M. J. A novelfunction of bamboo extract in relieving lipotoxicity. Phytother. Res.2008, 22, 675−680.(14) Nam, J. S.; Chung, H. J.; Jang, M. K.; Jung, I. A.; Park, S. H.;Cho, S. I.; Jung, M. H. Sasa borealis extract exerts an antidiabetic effectvia activation of the AMP-activated protein kinase. Nutr. Res. Pract.2013, 7, 15−21.(15) Jung, S. H.; Lee, J. M.; Lee, H. J.; Kim, C. Y.; Lee, E. H.; Um, B.H. Aldose reductase and advanced glycation endproducts inhibitoryeffect of Phyllostachys nigra. Biol. Pharm. Bull. 2007, 30, 1569−1572.(16) Choi, Y.-J.; Lim, H.-S.; Choi, J.-S.; Shin, S.-Y.; Bae, J.-Y.; Kang,S.-W.; Kang, I.-J.; Kang, Y.-H. Blockade of chronic high glucose-induced endothelial apoptosis by Sasa borealis bamboo extract. Exp.Biol. Med. 2008, 233, 580−591.(17) Zhang, Y.; Bao, B.; Lu, B.; Ren, Y.; Tie, X.; Zhang, Y.Determination of flavone C-glucosides in antioxidant of bamboo leaves(AOB) fortified foods by reversed-phase high-performance liquidchromatography with ultraviolet diode array detection. J. Chromatogr.,A 2005, 1065, 177−185.(18) Ying, Z. Natural functional extract of bamboo leaves-bambooleaf anthoxanthin. China Food Addit. 2002, 3, 54−66.(19) Tadera, K.; Minami, Y.; Takamatsu, K.; Matsuoka, T. Inhibitionof α-glucosidase and α-amylase by flavonoids. J. Nutr. Sci. Vitaminol.2006, 52, 149−153.(20) Ali Asgar, M. Anti-diabetic potential of phenolic compounds: areview. Int. J. Food Prop. 2013, 16, 91−103.(21) Cao, H.; Chen, X. Structures required of flavonoids forinhibiting digestive enzymes. Anti-Cancer Agents Med. Chem. 2012, 12,929−939.(22) Tsujita, T.; Shintani, T.; Sato, H. α-Amylase inhibitory activityfrom nut seed skin polyphenols. 1. Purification and characterization ofalmond seed skin polyphenols. J. Agric. Food Chem. 2013, 61, 4570−4576.



http://pubs.acs.org

mailto:[email protected]

(23) Hanhineva, K.; Torronen, R.; Bondia-Pons, I.; Pekkinen, J.;Kolehmainen, M.; Mykkanen, H.; Poutanen, K. Impact of dietarypolyphenols on carbohydrate metabolism. Int. J. Mol. Sci. 2010, 11,1365−1402.(24) Perera, A.; Meda, V.; Tyler, R. Resistant starch: a review ofanalytical protocols for determining resistant starch and of factorsaffecting the resistant starch content of foods. Food Res. Int. 2010, 43,1959−1974.(25) Thompson, L. U.; Yoon, J. H. Starch digestibility as affected bypolyphenols and phytic acid. J. Food Sci. 1984, 49, 1228−1229.(26) Takahama, U.; Hirota, S. Fatty acids, epicatechin-dimethylgal-late, and rutin interact with buckwheat starch inhibiting its digestion byamylase: implications for the decrease in glycemic index by buckwheatflour. J. Agric. Food Chem. 2010, 58, 12431−12439.(27) Cohen, R.; Orlova, Y.; Kovalev, M.; Ungar, Y.; Shimoni, E.Structural and functional properties of amylose complexes withgenistein. J. Agric. Food Chem. 2008, 56, 4212−4218.(28) Shen, W.; Xu, Y.; Lu, Y. H. Inhibitory effects of Citrus flavonoidson starch digestion and antihyperglycemic effects in HepG2 cells. J.Agric. Food Chem. 2012, 60, 9609−9619.(29) Wu, Y.; Chen, Z.; Li, X.; Li, M. Effect of tea polyphenols on theretrogradation of rice starch. Food Res. Int. 2009, 42, 221−225.(30) Zhu, F.; Cai, Y.-Z.; Sun, M.; Corke, H. Effect of phytochemicalextracts on the pasting, thermal, and gelling properties of wheat starch.Food Chem. 2009, 112, 919−923.(31) Takahama, U.; Hirota, S. Inhibition of buckwheat starchdigestion by the formation of starch/bile salt complexes: possibility ofits occurrence in the intestine. J. Agric. Food Chem. 2011, 59, 6277−6283.(32) Morishita, Y.; Iinuma, Y.; Nakashima, N.; Majima, K.;Mizuguchi, K.; Kawamura, Y. Total and pancreatic amylase measuredwith 2-chloro-4-nitrophenyl-4-O-β-D-galactopyranosylmaltoside. Clin.Chem. 2000, 46, 928−933.(33) Kandra, L.; Gyemant, G.; Zajacz, A.; Batta, G. Inhibitory effectsof tannin on human salivary α-amylase. Biochem. Biophys. Res.Commun. 2004, 319, 1265−1271.(34) Liu, J.; Wang, M.; Peng, S.; Zhang, G. Effect of green teacatechins on the postprandial glycemic response to starches differingin amylose content. J. Agric. Food Chem. 2011, 59, 4582−4588.(35) Subramanian, R.; Asmawi, M. Z.; Sadikun, A. In vitro α-glucosidase and α-amylase enzyme inhibitory effects of Andrographispaniculata extract and andrographolide. Acta Biochim. Polym. 2008, 55,391−398.(36) Akkarachiyasit, S.; Charoenlertkul, P.; Yibchok-anun, S.;Adisakwattana, S. Inhibitory activities of cyanidin and its glycosidesand synergistic effect with acarbose against intestinal α-glucosidase andpancreatic α-amylase. Int. J. Mol. Sci. 2010, 11, 3387−3396.(37) Nmeth, K.; Plumb, G. W.; Berrin, J.-G.; Juge, N.; Jacob, R.;Naim, H. Y.; Williamson, G.; Swallow, D. M.; Kroon, P. A.Deglycosylation by small intestinal epithelial cell-glucosidases is acritical step in the absorption and metabolism of dietary flavonoidglycosides in humans. Eur. J. Nutr. 2003, 42, 29−42.(38) Gradinaru, G.; Biliaderis, C. G.; Kallithraka, S.; Kefalas, P.;Garcia-Viguera, C. Thermal stability of Hibiscus sabdariffa L.anthocyanins in solution and in solid state: effects of copigmentationand glass transition. Food Chem. 2003, 83, 423−436.(39) Wirz, A.; Meier, B.; Sticher, O. Stability of hypericin andpseudohypericin in extract solutions of Hypericum perforatum and instandard solutions. Pharm. Ind. 2001, 63, 410−415.(40) Buisson, G.; Duee, E.; Haser, R.; Payan, F. Three dimensionalstructure of porcine pancreatic alpha-amylase at 2.9 A resolution. Roleof calcium in structure and activity. EMBO J. 1987, 6, 3909−3916.(41) Lo Piparo, E.; Scheib, H.; Frei, N.; Williamson, G.; Grigorov,M.; Chou, C. J. Flavonoids for controlling starch digestion: structuralrequirements for inhibiting human α-amylase. J. Med. Chem. 2008, 51,3555−3561.(42) Rydberg, E. H.; Li, C.; Maurus, R.; Overall, C. M.; Brayer, G. D.;Withers, S. G. Mechanistic analyses of catalysis in human pancreatic α-

amylase: detailed kinetic and structural studies of mutants of threeconserved carboxylic acids. Biochemistry 2002, 41, 4492−4502.(43) Wang, J.; Li, Y.; Tian, Y.; Xu, X.; Ji, X.; Cao, X.; Jin, Z. A noveltriple-wavelength colorimetric method for measuring amylose andamylopectin contents. Starch/Sta rke 2010, 62, 508−516.(44) Zhang, L.; Yang, X.; Li, S.; Gao, W. Preparation,physicochemical characterization and in vitro digestibility on solidcomplex of maize starches with quercetin. LWTFood Sci. Technol.2011, 44, 787−792.(45) Karim, A. A.; Norziah, M.; Seow, C. Methods for the study ofstarch retrogradation. Food Chem. 2000, 71, 9−36.(46) Zobel, H. Starch crystal transformations and their industrialimportance. Starch/Sta rke 1988, 40, 1−7.(47) Smits, A.; Kruiskamp, P.; Van Soest, J.; Vliegenthart, J. Theinfluence of various small plasticisers and malto-oligosaccharides onthe retrogradation of (partly) gelatinised starch. Carbohydr. Polym.2003, 51, 417−424.(48) Takahama, U.; Tanaka, M.; Hirota, S. Proanthocyanidins inbuckwheat flour can reduce salivary nitrite to nitric oxide in thestomach. Plant Foods Hum. Nutr. 2010, 65, 1−7.(49) de la Garza, A. L.; Etxeberria, U.; Lostao, M. a. P.; San Roman,B.; Barrenetxe, J.; Martínez, J. A.; Milagro, F. n. I. Helichrysum andgrapefruit extracts inhibit carbohydrate digestion and absorption,improving postprandial glucose levels and hyperinsulinemia in rats. J.Agric. Food Chem. 2013, 61, 12012−12019.



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Four Flavonoid Compounds from Phyllostachys edulis Leaf Extract Retard the Digestion of Starch and Its Working Mechanisms