Four Flavonoid Compounds from Phyllostachys edulis Leaf Extract

Article pubs.acs.org/JAFC

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

State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, People’s Republic of China ‡ Shanghai Collaborative Innovation Center for Biomanufacturing Technology, 130 Meilong Road, Shanghai 200237, People’s Republic 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, we investigated the influence of Phyllostachys edulis leaf extract on starch digestion. Orientin, isoorientin, vitexin, and isovitexin were determined as its α-amylase inhibitory constituents. An inhibitory kinetics experiment demonstrated that they competitively inhibit α-amylase with Ki values of respectively 152.6, 11.5, 569.6, and 75.8 μg/mL. Molecular docking showed the four flavones can interact with the active site of α-amylase, and their inhibitory activity was greatly influenced by the glucoside linking position and 3′-hydroxyl. Moreover, the results of starch−iodine complex spectroscopy, X-ray diffraction, and scanning electron microscopy 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 for adjusting postprandial hyperglycemia. KEYWORDS: bamboo leaf, orientin, isoorientin, vitexin, isovitexin, diabetes, α-amylase, starch

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relieving lipotoxicity,13 improving insulin sensitivity,14 and retarding diabetes complications.15,16 However, whether bamboo leaf extracts exhibit hypoglycemic activity through affecting the digestion of starch is still unclear. The digestion of starch is the major source of postprandial hyperglycemia. Additionally, bamboo leaf contains many kinds of flavonoids and phenolic acids.17,18 These classes of polyphenols have been reported to possess potential digestive enzyme inhibitory activity.19−23 The digestion of starch is also affected by the nature of the starch itself. According to its digestibility, starch is classified as RDS (rapidly digestible starch), SDS (slowly digestible starch), and RS (resistant starch).24 Some phytochemicals such as tannic acid, catechin,25 epicatechin dimethylgallate, rutin,26 genistein,27 and Citrus flavonoids28 have been reported to bind with starch and affect the digestibility of starch. This binding also can affect the physical properties of starch, including pasting, gelling, and retrogradation.29,30 However, to date, no study has focused on the binding between bamboo leaf extract and starch. In this study, first, we will determine the α-amylase inhibitory constituents of Phyllostachys edulis leaves (a kind of bamboo which is most widely planted in China). Second, these active constituents will be subjected to an inhibitory kinetics test and molecular docking. Last, whether these active constituents can interact with starch and affect its digestion feature will be investigated.

INTRODUCTION With the improvement of living standards and the alteration of our eating habits, the prevalence of diabetes has increased in recent decades. Data from 130 countries around the world show that, in 2013, about 382 million people suffered from diabetes, and this number is expected to be 592 million by 2035.1 Postprandial hyperglycemia is considered the most dangerous factor that causes the onset and gradual deterioration of diabetes. It can trigger the generation of free radicals to damage islet cells. Moreover, it can activate a series of intracellular signal mediators to block the signal transduction pathway of insulin and result in insulin resistance.2,3 Worse still, it will lead to the accumulation of advanced glycation end products (AGEs) which directly cause the complications of diabetes and the death of patients.4,5 Acarbose, a competitive inhibitor of digestive enzymes, has been clinically used for delaying the expeditious generation of postprandial blood glucose, but it has been reported to cause liver disorders, diarrhea, flatulence, and abdominal pain.6,7 These side effects are the most common reason for the withdrawal of acarbose in recipients.8 Therefore, the development of a safer drug is urgent. Natural products which have been proved relatively safer for humans might be an alternative.9 In 2004, an antioxidant of bamboo leafa brown extract of bamboo leafpassed toxicity studies10,11 and was permitted as a new kind of food additive authorized by the Ministry of Health, People’s Republic of China.12 In addition, bamboo leaf is easily accessible and of low cost. In comparison with the other parts of bamboo, bamboo leaf is nearly an agricultural waste: more than 80% of it rots in farms each year (Anji County, China, 2013). To date, bamboo leaf extract has been reported to exhibit antidiabetic activities by © 2014 American Chemical Society

Received: Revised: Accepted: Published: 7760

April 22, 2014 July 3, 2014 July 14, 2014 July 14, 2014 dx.doi.org/10.1021/jf501931m | J. Agric. Food Chem. 2014, 62, 7760−7770

Journal of Agricultural and Food Chemistry

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Article

was suspended in 1 mL of boiling water and water bathed at 90 °C for 30 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 100 mM sodium phosphate buffer (pH 7.1) (the final pH was 6.8). They were used to simulate the pH of the stomach and duodenum. Last, the suspension was homogenized by a glass homogenizer. The preparation of amylose and amylopectin suspensions was the same as that of soluble starch suspensions. Sample Preparation. P. edulis leaves were collected in Anji County (the bamboo hometown of China) during the winter and identified by Pei-xin Zhang of the Anji Forestry Bureau. The pregrounded 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 1/10 of the original volume by a rotary evaporator (RE-52A, Shanghai Jingke Co. Ltd., Shanghai, China). Then it was stored in a 4 °C refrigerator for 12 h to flocculate and filter some macromolecular compounds. After that, the solution was sequentially fractioned by petroleum ether, EtOAc, and 1-butanol. All these extracts were dried to obtain the petroleum ether fraction, EtOAc fraction, 1-butanol fraction, and residual fraction. The 1-butanol fraction was subjected to an AB-8 macroporous adsorption resin (polystyrene resin, weak polarity, 0.3−1.25 mm particle size) for further purification. The purification was carried out in a glass column (1.4 cm × 40 cm) wetpacked 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 not adsorb 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 Bioactive Components. 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 elution condition was 15−25% B in 0−35 min. The flow rate was set as 1.0 mL/min. The signal was monitored at 330 nm. A Thermo LCQ-Deca HPLC-MS (Thermo Fisher Scientific Inc., San Jose, CA) fitting with an ESI (electrospray ionization) interface and negative ion mode was also used for determining the main bioactive components. For quantitative analysis, the calibration curves of orientin, isoorientin, vitexin, and isovitexin were obtained at a concentration of 6.25−200 μg/mL (n = 6). Measurement and Kinetics Assay of α-Amylase Inhibitory Activity. The measurement of α-amylase inhibitory activity was carried out by using the method described previously.32,33 Briefly, 20 μL of P. edulis flavonoids at different concentrations (final concentrations were 9, 18, 36, and 72 μg/mL) were mixed with 10 μL of 200 μg/mL α-amylase and 70 μL sodium phosphate buffer (pH 6.8). After intensive shaking for mixing, 100 μL of 5 mM Gal-G2-αCNP solution was added to start the reaction. After 10 min of incubation at 37 °C, the absorbance of released CNP was monitored at 405 nm by a Bio-Tek Power Wave XS2 microwell plate reader (BioTek Instruments, Winooski, VT). To further explore their inhibitory character, we performed kinetic analysis by using Lineweaver−Burk plots. The Ki value of competitive inhibition was obtained from the least-squares regression line of the slopes of Lineweaver−Burk plots versus the corresponding [I]. The derivation formulas are

MATERIALS AND METHODS

Chemicals. 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 purchased from Shifeng Biological Technology Co., Ltd. (Shanghai, China), and their structures are shown in Figure 1. Soluble starch was purchased

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

Vmax[S]

V=

(

Km 1 +

[I] Ki

) + [S]

and

K ⎛ 1 [I] ⎞ 1 1 = m ⎜1 + + ⎟ V Vmax ⎝ K i ⎠ [S] Vmax

from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 2Chloro-4-nitrophenol (CNP) was purchased from Energy Chemical (Shanghai, China). Acetonitrile (HPLC-grade) was provided by Tedia Co. 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 then 0.635 g of iodine was added. Finally, the solution was adjusted to 50 mL by distilled water. Starch suspension:26,31 10 mg of soluble starch

slope =

Km ⎛ [I] ⎞ ⎜1 + ⎟ Vmax ⎝ Ki ⎠

and

slope =

(1)

Km K [I] + m VmaxK i Vmax (2)

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


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− K i = intercept of eq 2 on x axis

with gold in a vacuum evaporator to make the sample conductive. Then the sample was investigated with an S-3400N scanning electron microscope (Hitachi, Tokoyo, Japan) with a 15 kV accelerating voltage. Statistical Analysis. All experiments were repeated three times. Statistical analysis was carried out by using one-way analysis of variance in SPSS 18.0 (SPSS, Chicago, IL); p < 0.05 was considered to be statistically significant.

Thermal Stability. First, the standard solutions of the four flavonoids were mixed. Second, we placed a certain volume of the mixed standard into a tube. The tube was sealed by plastic tape and heated at 100 °C for 2 h. Last, we detected the samples without and with heat treatment by HPLC and calculated the concentration alteration of our four flavonoids according to their peak areas. Molecular Modeling. The three-dimensional structures of orientin, isoorientin, vitexin, and isovitexin were downloaded from the National Centre for Biotechnology Information PubChem compound database (http://pubchem.ncbi.nlm.nih.gov/). The threedimensional structure of substrate Gal-G2-α-CNP was constructed by using ChemBio3D Ultra 12.0 software (Cambridge Soft Corp., Cambridge, U.K.). The crystal structure of the pig pancreatic αamylase protein complex (PDB code 1WO2) was retrieved from the RCSB Protein Data Bank (http://www.rcsb.org). Then docking was carried out by using the Autodock v4.2 program (Scripps Research Institute, La Jolla, CA). The Lamarckian Genetic Algorithm was used to run the docking. First, we did a blind docking in which the grid box covers the whole receptor protein. Then a smaller grid box that mainly covers the site of the best cluster of blind docking was set for doing an accurate docking. After docking, cluster analysis based on the rootmean-square deviation (RMSD) value was performed. The tolerance of RMSD was set as 2.0. The lowest energy conformation of the most populated cluster was considered as the most reliable solution. The interaction analysis was carried out by using Pymol 0.99 (DeLano Scientific LLC, San Carlos, CA), YasaraView v13.9.8 (YASARA Biosciences, 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. edulis flavonoids and starch was studied by using the spectroscopic method described previously.26,31 Briefly, 25 μL of flavonoid was added to 0.9 mL of soluble starch suspension (the final concentrations of flavonoid were 9, 18, and 36 μg/mL). After vortexing, 0.1 mL of iodine solution was added to the suspension. Then the absorption spectrum of the starch−iodine complex was measured from 500 to 900 nm by using a UV-1800 spectrophotometer (Shimadzu, Tokoyo, Japan). The absorption spectra of the amylose−iodine complex and the amylopectin−iodine complex were obtained in the same way as that of the soluble starch−iodine complex. The digestion of starch was also studied by using the spectroscopic method.26,31 Briefly, 25 μL of flavonoid was added to 0.9 mL of starch suspension (the final concentrations of flavonoid were 9, 18, and 36 μg/mL), and then 10 μL of 0.5 mg/mL α-amylase was added to the suspension. After the suspension was incubated for 5 min at 37 °C, 0.1 mL of iodine solution was added to terminate the digestion and color the residual starch. The degree of digestion was estimated by the difference spectrum (ΔA) of the starch−iodine complex after and before digestion. 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). The mixture 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 than 10 days is needed to achieve the same degree of retrogradation) and freeze-dried last. A D/MAX 2550 rotating anode X-ray powder diffractometer (Rigaku, Tokoyo, Japan) equipped with a copper tube at 40 kV and 100 mA with Cu Kα radiation (λ = 0.154 nm) was used for crystalline analysis. The diffractograms were detected in the angle range from 5° to 50° (2θ) with a step size of 0.02°. The MDI Jade 5.0 software (Materials Data Inc., Livermore, CA) was used to analyze the diffractograms. The relative crystallinity was calculated by using the following equation: relative crystallinity (%) = 100Ac/(Ac + Aa), where Ac is the area of the crystal peak and Aa is the area of the amorphous peak. Scanning Electron Microscopy (SEM). The sample was fixed on a silver specimen holder using double-sided adhesive and sputtered

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RESULTS AND DISCUSSION α-Amylase Inhibition Ability and Yield of P. edulis Leaf Extract Fractions. The α-amylase inhibition ability and Table 1. α-Amylase Inhibition Ability and Yield of Each Fraction extract

IC50 (μg/mL)

ethanol extract petroleum ether fraction EtOAc fraction 1-butanol fraction residual fraction resin-purified extract

591.8 >800 318.1 354.1 >800 206.7

yield (%) 6.96 0.97 0.33 0.93 4.36 0.63

± ± ± ± ± ±

0.33 0.04 0.04 0.06 0.32 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. One asterisk means p < 0.05, two asterisks mean p < 0.01, and three asterisks mean p < 0.001 compared to the control group.

the yield of several P. edulis leaf extract fractions are listed in Table 1. The EtOAc fraction and 1-butanol fraction exhibited stronger α-amylase inhibitory activity than the other fractions, 7762



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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−Burk plots) were used to calculate the Ki value.

([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/z 431.0), respectively (Supporting Information). Using the calibration curve, the concentrations of orientin, isoorientin, vitexin, and isovitexin were respectively 12.64, 87.15, 4.82, and 9.33 mg/g in the resin-purified extract. We were going to further affirm their α-amylase inhibitory activity. However, there might be other compounds with potent α-amylase inhibitory activity in the ethyl acetate fraction. We will try to purify them by silica gel column chromatography and Sephadex LH-20 column chromatography in the future. α-Amylase Inhibitory Activities of the Four Flavonoids and Their Structure−Activity Relationships. As shown in Figure 2, all four flavonoids can dose-dependently inhibit α-amylase, and the inhibitory effect of isoorientin and isovitexin is stronger than the inhibitory effect of many other phytochemicals such as hesperidin, naringin, neohesperidin, nobiletin,28 andrographolide,35 cyanidin, cyanidin 3-glucoside,36 myricetin, quercetin, luteolin, kaempferol, and baicalein19 (the comparison of their IC50 values is given in the Supporting Information). However, their inhibitory effect is weaker than that of the positive drug acarbose. All the four flavonoids are flavone C-glycosides. Unlike flavone O-glycosides, flavone C-glycosides will not be hydrolyzed to their aglycon by β-glucosidase in the small intestine.37 Therefore, the four flavonoids can contact α-amylase which is located in the intestine. According to the two HPLC spectra without and with heat treatment (Supporting Information), the concentration alterations of isoorientin, orientin, vitexin, and

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

inhibitory type

Ki (μg/mL)

IC50 (μg/mL)

orientin isoorientin vitexin isovitexin resin-purified extract

competitive competitive competitive competitive

152.6 11.5 569.6 75.8

>270 29.9 >270 65.3 206.7

but the yield of the EtOAc fraction is just one-third of the yield of the 1-butanol fraction and the EtOAc fraction is barely soluble in water. When loading the resin column, the materials must be dissolved in water, so the 1-butanol fraction is more suitable than the EtOAc fraction to be subjected to the AB-8 macroporous adsorption resin column for further purification. The obtained resin-purified extract exhibited the strongest αamylase inhibitory activity. Determination and Quantitation of the Main Bioactive Components. From the HPLC spectrum of the resinpurified extract (Supporting Information), we found two peaks were significantly higher than the other peaks. The two peaks might be the main α-amylase inhibitory components of the resin-purified extract. From the ultraviolet spectra (Supporting Information) of the two peaks, we found their maximum absorbances were near 270 and 340 nm, which suggested that these compounds might be flavonoids. According to the published data12,17 and by further comparing the retention times, ultraviolet spectra, and m/z values with those of our standards, peaks 7, 8, 9, and 10 were identified as isoorientin 7763



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speculations can be elicited from this phenomenon: (1) Flavone C6-glycosides have stronger α-amylase inhibitory activity than flavone C8-glycosides (because the IC50 of isoorientin is less than that of orientin and the IC50 of isovitexin is less than that of vitexin). (2) The C3′-hydroxyl on the 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 of flavonoid is positively related to the number of hydroxyls on the B ring. For speculation 1, we have not found any literature support, but we tried to explain it by using molecular docking in the following subsection. Molecular Docking between P. edulis Flavonoids and α-Amylase. Molecular docking was used to stimulate the binding mode of the ligand and receptor according to the “lock” and “key” principle. Here we used molecular docking to analyze why the four P. edulis flavonoids have highly similar structures but exhibit greatly different α-amylase inhibitory activities. Figure 4 illustrates that all four flavonoids bind at the active site of α-amylase, which is a large hydrophobic pocket on the surface of α-amylase.40 They all occupy the binding position of substrate Gal-G2-CNP more or less. Among them, isooreintin (B) and isovitexin (D) cover a large part of the binding position of the substrate, but oreintin (A) and vitexin (C) cover a smaller part. Therefore, isoorientin and isovitexin can more effectively cause steric hindrance to hinder the binding of the substrate. This is consistent with our experimental result that isoorientin and isovitexin exhibit much stronger inhibitory activity than orientin and vitexin. In fact, when the glucose links at C8 (orientin, vitexin), a corner of less than 90° will exist inside the flavonoid molecule and lead the three-dimensional structure of the flavonoid to look like a “V”. Molecules with a “V” structure (orientin and vitexin) are easily trapped in a small hollow near the active site of α-amylase (which is veiled in Figure 4A,C, but is clear in Figure 4E,F). When the glucose links at C6 (isoorientin, isovitexin), the structure of the flavonoid is linear. Molecules with a linear structure are more similar to the substrate and fit the active site of amylase better. Additionally, we downloaded the X-ray crystal structure of the porcine pancreatic α-amylase−acarbose complex (Figure 4G) which was derived from an X-ray diffraction experiment and not in silico docking as ours was. We find that the positive drug acarbose almost fully occupies the binding position of the substrate. This is why its inhibitory effect is stronger than that of 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 with the superimposition of substrate Gal-G2-CNP. The big orange background is the surface of α-amylase (PDB code 1WO2), the dark blue molecule is synthetic substrate Gal-G2-CNP, the pink molecule is orientin, the green molecule is isoorientin, the light blue molecule is vitexin, the yellow molecule is isovitexin, and the red molecule is the positive drug acarbose.

isovitexin are respectively 6.6%, 1.8%, 9.3%, and 8.0% after heat treatment. This indicates that the four flavonoids will not be easily destroyed during food processing. However, many phytochemicals such as anthocyanin38 and hypericin39 are extremely 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 which indicates their inhibitory types are all competitive. Their Ki and IC50 values are given in Table 2. Their inhibitory sequence is isoorientin > isovitexin > resin-purified extract > orientin > vitexin, and the differences between their effects are very significant. This is an interesting phenomenon because the structures of the four flavonoids are highly similar. Two

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

a

π−π interaction

hydrophobic interaction

orientin

Asp300, His299, Gly304, His305

Trp58, Trp59, His305

isoorientin vitexin

Asp197, Glu233,Gln63, Val163, Arg195 Asp300, His299, Gly304, His305

Trp58, Trp59, Tyr62, Arg195, His299 Trp58, Trp59, His305

isovitexin

Asp197, Gln63, Val163, Arg195

Trp58, Tyr62, Arg195, His299

Glu233, Asp300, Trp58, Trp59, Tyr62, His299, Arg303, Gly304, His305, Asp356 Asp197, Glu233, Asp300,Trp58, Trp59, Tyr62, Gln63, Val163, Arg195, Ala 198, His299 Glu233, Asp300,Trp58, Trp59, Tyr62, His299, Arg303, Gly304, His305, Asp356 Asp197, Asp300,Val51, Trp58, Trp59, Tyr62, Gln63, Val163, Leu165, Arg195, His299

The bold-font residues (Asp197, Glu233, Asp300) are the residues which play the key role in cutting the starch chain. 7764



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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/mL compound 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 ΔA value of the control curve.

between flavonoids and the residues of α-amylase. Their interacting 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 site of α-amylase contains three key acidic residues, Asp197, Glu233, and Asp300. They work together to cleave the starch chain.40,42) This explains why isoorientin exhibited stronger inhibitory activity than isovitexin. P. edulis Leaf Extracts Bind with Starch and Affect Its Digestibility. Some small molecular phytochemicals can affect the digestibility of starch through binding with starch.25,26,28 Here we used a spectroscopic method26,31 to investigate whether P. edulis extracts bind with starch and further affect its digestibility. The ΔA curve of Figure 5 was obtained through subtraction of the absorption curve of flavonoid + iodine from the absorption curve of starch + flavonoid + iodine (the control is absence of flavonoid). Figure 5 shows that the ΔA curves decline with an increase of our flavonoids and resin-purified extract, but do not decline with an increase of acarbose. This

indicates that orientin, isoorientin, vitexin, isovitexin, and resinpurified extract can bind with starch, but the positive drug acarbose cannot. According to the percentages on the right side of each curve, the binding strength order of our compounds is isoorientin ≈ orientin > vitexin ≈ isovitexin > resin-purified extract > acarbose. The ΔΔA spectrum at the left bottom of each picture was obtained through subtraction of the ΔA curve of the control from the ΔA curve of 36 μg/mL flavonoid. Unlike the ΔΔA curves of naringin and neohesperidin28 which have a distinct peak at 600 nm but are nearly zero at 500 nm, the ΔΔA curves of our flavonoids do not have a distinct peak at 600 nm but show a significant negative value at 500 nm. This indicates they mainly bind with amylopectin because the λmax of the amylopectin−iodine complex is between 500 and 540 nm but the λmax of the amylose−iodine complex is between 540 and 660 nm.43 The influence of P. edulis leaf extracts on the formation of the amylopectin−iodine complex and amylose−iodine complex was also tested (Supporting Information). The decline of the ΔA 7765



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Figure 6. Effect of P. edulis leaf flavonoids, resin-purified extract, and acarbose on the digestion of soluble starch: (A) orientin added, (B) isoorientin added, (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 the peak ΔA value of the curve divided by the peak ΔA value of the control curve.

curve of amylopectin−iodine is more significant than the decline of the ΔA curve of amylose−iodine. This confirms the conclusion 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 between them (around 565 nm, Figure 5) due to soluble starch being composed of amylopectin and amylose. The ΔA curve of Figure 6 was obtained through subtraction of the absorption curve of the undigested starch−iodine complex from the absorption curve of the digested starch− iodine complex (addition of iodine after incubation with αamylase). The less the starch was digested, the higher the ΔA value (negative). Figure 6 illustrates that the ΔA curve rises with an increase of our compounds. This indicates that our compounds can inhibit the digestion of soluble starch. The order of their effect was acarbose > isoorientin > isovitexin ≈

orientin > vitexin > resin-purified extract. However, this is different from the α-amylase inhibitory order we obtained from Figure 2 (the substrate was Gal-G2-CNP, a small molecule which was used to exclude the influence of the interaction between starch and our compounds), that is, acarbose > isoorientin > isovitexin > resin-purified extract > orientin > vitexin, and the gap between orientin and isovitexin and the gap between acarbose and isoorientin are narrowed when starch is used as the substrate. If these compounds simply inhibited the activity of α-amylase, whether using Gal-G2-CNP or starch as the substrate, the orders of their inhibitory effect should be the same. Therefore, we speculate that the effect of P. edulis extracts on the digestion of starch is not only due to the inhibition of αamylase. Maybe they can further affect the digestibility of starch through binding with it. Two studies can support our speculation: One study found that due to the binding of rutin, epicatechin dimethylgallate, and fatty acids the glycemic index of buckwheat flour is lower than that of wheat flour.26 7766



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Figure 7. X-ray diffractograms of starch: (A) resin-purified extract, (B) natural soluble starch, (C) retrograded starch, and (D) retrograded resinpurified 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.

acarbose. Therefore, the inhibitory order change when using starch as the substrate was due to the different binding strengths of these compounds with starch. Why can the binding retard the digestion of starch? Many researchers hold the view that some compounds can bind with starch through occupation

The other study found that the resistant starch content of the quercetine−starch complex was sharply higher than that of common starch.44 We had known that the binding strength between our compounds and starch was different: isoorientin ≈ orientin > vitexin ≈ isovitexin > resin-purified extract > 7767



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Figure 8. SEM images of starch. Panels A1, B1, and C1 are natural soluble starch (without gelatinization and retrogradation), and their magnifications 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−starch complex, and their magnifications are respectively 100×, 3000×, and 5000×. B1, B2, and B3 are the outside surface. C1,C2, and C3 are the fracture surface.

The mechanism of retrogradation is the reassociation of starch chains through hydrogen bonds. Phenolic compounds which contain many reactive hydroxyls can disturb the formation of H-bonds between starch chains through formation of H-bonds with starch.29,30 Researchers had confirmed that the greater the number of OH groups in the plasticizer, the better the plasticizer reduces the crystallization of starch.47 Some other researchers hold the view that, during boiling, phenolic compounds and starch can form covalent bonds which can further retard the crystallization of starch.48 Scanning electron microscopy was used to investigate the morphology difference between natural soluble starch, retrogradated starch, and the retrogradated resin-purified extract−starch complex. In Figure 8A1,A2,A3 (100×), the shape of natural soluble starch is an elliptic granule (A1) whose diameter is about 50 μm; the retrogradated starch (A2) and retrogradated resin-purified extract−starch complex (A3) are irregularly shaped agglomerates whose long axes respectively vary between 200 and 250 μm and betwen 50 and 150 μm. From Figure 8B1,B2,B3 (3000×), we find that the surface of natural starch is smooth (B1), but there are many apophyses on the surface of retrogradated starch and the retrogradated resinpurified extract−starch complex (B2, B3). Parts C1, C2, and C3 (5000×) of Figure 8 are the faultages of these starches. The natural starch showed a very dense faultage which indicates its compact interior structure (C1), while on the faultages of retrogradated starch and the retrogradated resin-purified extract−starch complex there are many irregular bulges (C2, C3). Inside the faultage of the retrogradated resin-purified

of the hydrophobic helical structure of starch. The occupation of the hydrophobic helical structure will disturb the combination of starch and amylase.26−28 Influence of P. edulis Extract on the Physical Properties of Starch. In the cooking process, well-ordered starch polymers will be broken into free starch chains; this is called gelatinization. During cooling, these free starch chains will reassociate and re-form crystalline domains; this is called retrogradation. Retrogradation will markedly increase the rigidity and shorten the shelf life of starch-based food.45 To further prove the binding between P. edulis leaf extract and starch, we investigated whether the resin-purified extract can influence the retrogradation of starch by using X-ray diffraction and scanning electron microscopy. Figure 7A is the X-ray diffractogram of the resin-purified extract which does not have any crystal peak. This means that the resin-purified extract is amorphous. Figure 7B is the X-ray diffractogram of natural soluble starch (without heating and cooling treatment). It exhibits a typical B-type crystalline structure (according to X-ray diffractograms, the crystalline form of starch can be classified as A, B, C, and V46) with peaks close to 17.2°, 22.2°, and 24.0°. Parts C and D of Figure 7 are the diffractograms of retrograded starch and the retrogradated resin-purified extract−starch complex. We find three crystal peaks in Figure 7C, but only one crystal peak (17.2°) in Figure 7D. Their crystallinities are respectively 8.99% and 4.93%. This indicates that, once we added the resin-purified extract, the retrogradation of starch was retarded. 7768



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extract−starch complex (C3), we also find some cracks and cavities. The cracks and cavities illustrate the crystalline texture of the retrogradated resin purified extract−starch complex is less compact than that of retrogradated starch. This indicates that P. edulis leaf extract can interact with starch and retard the retrogradation of starch. This is similar to the result of another study that the gel matrix of a wheat starch−pomegranate peel extract complex is looser than that of common starch.30 In conclusion, our studies provide scientific support for the further use of P. edulis leaf extract as a functional food additive that can adjust postprandial hyperglycemia and prolong the shelf life of starch-based food. Furthermore, our study on the structure−activity relationship of flavone C-glycosides can be applied to drug design. In the future, the influence of bamboo leaf extract on the glucose transporter of small intestinal brush border cells deserves further study, because it is the last step in the generation of blood glucose.49 We hope our study can attract more attention to this bamboo leaf resource and benefit diabetes research and hardworking bamboo farmers in the future.

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American Diabetes Association diagnostic criteria. Lancet 1999, 354, 617−621. (4) Ahmed, N. Advanced glycation endproductsrole in pathology of 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 its constituents vitexin and isovitexin on the formation of advanced glycation endproducts. Food Chem. 2008, 106, 475−481. (6) Hsiao, S.-H.; Liao, L.-H.; Cheng, P.-N.; Wu, T.-J. Hepatotoxicity associated 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 disease and hypertension in patients with impaired glucose tolerance: the STOP-NIDDM trial. JAMA, J. Am. Med. Assoc. 2003, 290, 486−494. (8) Neuser, D.; Benson, A.; Brückner, A.; Goldberg, R. B.; Hoogwerf, B. J.; Petzinna, D. Safety and tolerability of acarbose in the treatment of 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 potent inhibitors 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 and safety of anti-oxidant of bamboo leaves. Part 1: acute and subchronic toxicity 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 and safety of antioxidant of bamboo leaves. Part 2: developmental toxicity test 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 and purification of four flavone C-glycosides from antioxidant of bamboo leaves by macroporous resin column chromatography and preparative high-performance liquid chromatography. Food Chem. 2007, 107, 1326−1336. (13) Panee, J.; Liu, W.; Lin, Y.; Gilman, C.; Berry, M. J. A novel function 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 effect via 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 inhibitory effect 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 glucoseinduced 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 liquid chromatography with ultraviolet diode array detection. J. Chromatogr., A 2005, 1065, 177−185. (18) Ying, Z. Natural functional extract of bamboo leaves-bamboo leaf anthoxanthin. China Food Addit. 2002, 3, 54−66. (19) Tadera, K.; Minami, Y.; Takamatsu, K.; Matsuoka, T. Inhibition of 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: a review. Int. J. Food Prop. 2013, 16, 91−103. (21) Cao, H.; Chen, X. Structures required of flavonoids for inhibiting digestive enzymes. Anti-Cancer Agents Med. Chem. 2012, 12, 929−939. (22) Tsujita, T.; Shintani, T.; Sato, H. α-Amylase inhibitory activity from nut seed skin polyphenols. 1. Purification and characterization of almond seed skin polyphenols. J. Agric. Food Chem. 2013, 61, 4570− 4576.

ASSOCIATED CONTENT

S Supporting Information *

Comparison of the IC50 values of isoorientin, isovitexin, and other compounds (Table S1), HPLC spectrum (Figure S1) and HPLC−MS spectrum (Figure s2) of the resin-purified extract, HPLC spectra of the mixed standard without and with heat treatment (Figure S3), hydrogen bond interaction networks (Figure S4), π−π interaction networks (Figure S5), and hydrophobic interaction networks (two-dimensional) (Figure S6) between P. edulis flavonoids and α-amylase, and interactions between P. edulis leaf flavonoids and amylopectin (Figure S7) and amylose (Figure S8). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +86-21-64251185. Fax: +86-21-64251185. Funding

This work was supported by the Fundamental Research Funds for the Central Universities and partially supported by the Shanghai Leading Academic Discipline Project (Grant B505) and the National Special Fund for State Key Laboratory of Bioreactor Engineering (Grant 2060204). Notes

The authors declare no competing financial interest.

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ABBREVIATION USED Gal-G2-CNP, α-(2-chloro-4-nitrophenyl)-β-1,4-galactopyranosylmaltoside REFERENCES

(1) Guariguata, L.; Whiting, D.; Hambleton, I.; Beagley, J.; Linnenkamp, U.; Shaw, J. Global estimates of diabetes prevalence for 2013 and projections for 2035 for the IDF Diabetes Atlas. Diabetes Res. Clin. Pract. 2013, 103, 137−149. (2) Stumvoll, M.; Goldstein, B. J.; van Haeften, T. W. Type 2 diabetes: principles of pathogenesis and therapy. Lancet 2005, 365, 1333−1346. (3) Pekkanen, J.; Tuomilehto, J.; Qiao, Q.; Jousilahti, P.; Lindström, J. Glucose tolerance and mortality: comparison of WHO and 7769



Article

(23) Hanhineva, K.; Törrönen, R.; Bondia-Pons, I.; Pekkinen, J.; Kolehmainen, M.; Mykkänen, H.; Poutanen, K. Impact of dietary polyphenols on carbohydrate metabolism. Int. J. Mol. Sci. 2010, 11, 1365−1402. (24) Perera, A.; Meda, V.; Tyler, R. Resistant starch: a review of analytical protocols for determining resistant starch and of factors affecting the resistant starch content of foods. Food Res. Int. 2010, 43, 1959−1974. (25) Thompson, L. U.; Yoon, J. H. Starch digestibility as affected by polyphenols and phytic acid. J. Food Sci. 1984, 49, 1228−1229. (26) Takahama, U.; Hirota, S. Fatty acids, epicatechin-dimethylgallate, and rutin interact with buckwheat starch inhibiting its digestion by amylase: implications for the decrease in glycemic index by buckwheat flour. 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 with genistein. J. Agric. Food Chem. 2008, 56, 4212−4218. (28) Shen, W.; Xu, Y.; Lu, Y. H. Inhibitory effects of Citrus flavonoids on 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 the retrogradation of rice starch. Food Res. Int. 2009, 42, 221−225. (30) Zhu, F.; Cai, Y.-Z.; Sun, M.; Corke, H. Effect of phytochemical extracts on the pasting, thermal, and gelling properties of wheat starch. Food Chem. 2009, 112, 919−923. (31) Takahama, U.; Hirota, S. Inhibition of buckwheat starch digestion by the formation of starch/bile salt complexes: possibility of its 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 measured with 2-chloro-4-nitrophenyl-4-O-β-D-galactopyranosylmaltoside. Clin. Chem. 2000, 46, 928−933. (33) Kandra, L.; Gyémánt, G.; Zajácz, Á .; Batta, G. Inhibitory effects of 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 tea catechins on the postprandial glycemic response to starches differing in amylose content. J. Agric. Food Chem. 2011, 59, 4582−4588. (35) Subramanian, R.; Asmawi, M. Z.; Sadikun, A. In vitro αglucosidase and α-amylase enzyme inhibitory effects of Andrographis paniculata 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 glycosides and synergistic effect with acarbose against intestinal α-glucosidase and pancreatic 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 a critical step in the absorption and metabolism of dietary flavonoid glycosides 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 copigmentation and glass transition. Food Chem. 2003, 83, 423−436. (39) Wirz, A.; Meier, B.; Sticher, O. Stability of hypericin and pseudohypericin in extract solutions of Hypericum perforatum and in standard solutions. Pharm. Ind. 2001, 63, 410−415. (40) Buisson, G.; Duee, E.; Haser, R.; Payan, F. Three dimensional structure of porcine pancreatic alpha-amylase at 2.9 A resolution. Role of 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: structural requirements 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 three conserved carboxylic acids. Biochemistry 2002, 41, 4492−4502. (43) Wang, J.; Li, Y.; Tian, Y.; Xu, X.; Ji, X.; Cao, X.; Jin, Z. A novel triple-wavelength colorimetric method for measuring amylose and amylopectin contents. Starch/Stärke 2010, 62, 508−516. (44) Zhang, L.; Yang, X.; Li, S.; Gao, W. Preparation, physicochemical characterization and in vitro digestibility on solid complex of maize starches with quercetin. LWTFood Sci. Technol. 2011, 44, 787−792. (45) Karim, A. A.; Norziah, M.; Seow, C. Methods for the study of starch retrogradation. Food Chem. 2000, 71, 9−36. (46) Zobel, H. Starch crystal transformations and their industrial importance. Starch/Stärke 1988, 40, 1−7. (47) Smits, A.; Kruiskamp, P.; Van Soest, J.; Vliegenthart, J. The influence of various small plasticisers and malto-oligosaccharides on the retrogradation of (partly) gelatinised starch. Carbohydr. Polym. 2003, 51, 417−424. (48) Takahama, U.; Tanaka, M.; Hirota, S. Proanthocyanidins in buckwheat flour can reduce salivary nitrite to nitric oxide in the stomach. Plant Foods Hum. Nutr. 2010, 65, 1−7. (49) de la Garza, A. L.; Etxeberria, U.; Lostao, M. a. P.; San Román, B.; Barrenetxe, J.; Martínez, J. A.; Milagro, F. n. I. Helichrysum and grapefruit 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

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