jfbc_253 808..820

DOI: 10.1111/j.1745-4514.2009.00253.x

CHARACTERIZATION AND ANTIOXIDANT ACTIVITY OF FLAVONOID-RICH EXTRACTS FROM LEAVES OF GROSSEDENTATA

JIANHUA GAO1, BENGUO LIU2,3, ZHENGXIANG NING1, RUIXIANG ZHAO2, AIYUAN ZHANG1 and QIONG WU1

1College of Light Industry and Food Science South University of Technology Guangzhou, China

2School of Food Science Henan Institute of Science and Technology Hualan Street, Xinxiang 453003, China

Accepted for Publication March 11, 2008

ABSTRACT

Leaves of Ampelopsis grossedentata have been consumed in southern China as health tea and medicine. In this study, two kinds of flavonoid- rich extracts from leaves of A. grossedentata were prepared by traditional solvent extraction (product A) and recrystallization (product B). By analysis of ultraviolet-visible (UV) spectrometry, infrared (IR) spectrometry, electrospray ionization mass spectrometry (ESI-MS), nuclear magnetic resonance (NMR) spectrometry and high-performance liquid chromatography (HPLC)-ESI/MS, the major flavonoid in two products was identified as (+)-dihydromyricetin (DMY). Also, by HPLC, the contents of DMY in leaves, product A and product B were measured to be 32.96 Ϯ 1.18%, 77.15 Ϯ 1.98% and 95.12 Ϯ 2.86%, respectively. Various antioxidant tests (1,1-diphenyl-2-picrylhydrazyl radical scavenging, reducing power, antioxidant activity in linoleic acid system) showed that the antioxidant activity of the flavonoid-rich extracts could be comparable with that of tertiary butylhydroquinone. The flavonoid-rich extracts from A. grossedentata leaves have a promise to be a new natural antioxidant with potential application in food industry.

PRACTICAL APPLICATION

Leaves of Ampelopsis grossedentata are consumed in southern China as health tea (Tengcha) and . In this study, the flavonoid-rich

3 Corresponding author. TEL: 86-373-3040674; FAX: 86-373-3040709; EMAIL: [email protected]

Journal of Food Biochemistry 33 (2009) 808–820. 808 © 2009, The Author(s) Journal compilation © 2009, Wiley Periodicals, Inc. FLAVONOID-RICH EXTRACTS FROM LEAVES OF AMPELOPSIS GROSSEDENTATA 809 extracts from leaves of A. grossedentata, which were rich in (+)- dihydromyricetin, were prepared by traditional solvent extraction and recrystallization. Also, the antioxidant activity of the extracts can be compa- rable with that of tertiary butylhydroquinone. The results obtained in this study suggest that leaves of A. grossedentata may have potential as a new source of natural antioxidant and health food with great commercial interest in the food and phytopharmaceutical market.

INTRODUCTION

Many studies have shown that natural antioxidants from sources can effectively inhibit oxidation in food and reduce the risk of age-dependent diseases (Candan et al. 2003; Valentão et al. 2003; Škerget et al. 2005). Fla- vonoids, which are widespread in fruits, vegetables, teas and medicinal , have received the greatest attention and have been studied extensively, as they are a kind of highly effective antioxidants with less toxicity than synthetic antioxidants, such as butylated hydroxyanisole, butylated hydroxytoluene, which are suspected of being carcinogenic and causing liver damage (Hollman et al. 1996; Watanabe et al. 1997). It is reported that higher intake of antioxidant-rich food is associated with decreased risk of many degenerative diseases, particularly cardiovascular diseases and cancers (Burda and Oleszek 2001). Ampelopsis grossedentata is a wild plant, which spreads mainly in southern China. Its leaves have been consumed as health tea (Tengcha) and herbal medicine for hundreds of years, which are reported to have many curative effects, such as lowering of blood pressure, as well as antibacterial, antioxidant and analgesic properties (Zhang et al. 2003; Li et al. 2006). But so far, the flavonoid-rich extracts from leaves of A. grossedentata have not been fully investigated, and few studies have been conducted to investigate the antioxidant activity of this medicinal plant. Therefore, in this study, two kinds of flavonoid-rich extracts from leaves of A. grossedentata were prepared by traditional solvent extraction (product A) and recrystallization (product B). Also, their antioxidant activi- ties were evaluated by the most commonly accepted antioxidant tests (such as 1,1-diphenyl-2-picrylhydrazyl [DPPH] assay, reducing power) based on characterization of the main flavonoids in the extracts by ultraviolet-visible (UV) spectrometry, infrared (IR) spectrometry, mass spectrometry (MS), nuclear magnetic resonance (NMR) spectrometry and high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI/MS). 810 J. GAO ET AL.

MATERIALS AND METHODS

Materials and Chemicals Leaves of A. grossedentata (2006 production, moisture content 8.53%) for this study were purchased from a local herbal medicine market in Guangzhou, China. (+)-Dihydromyricetin (DMY) (>99%) was available as a gift from Professor Yousheng Zhang in Guangdong Academy of Agricultural Sciences. DPPH was purchased from Sigma Chemicals Co. (St. Louis, MO). Linoleic acid (>99%) was available form AMRESCO Inc. (Solon, OH). HPLC grade methanol was supplied by DIKMA (Lake Forest, CA). Other chemicals were of analytical grade and were used as received.

Preparation of Product A Leaves of A. grossedentata (10.08 g) were extracted with 250 mL of ethanol in a water bath at 80C for 5 h and then filtered. The filtrate was concentrated under vacuum at 45C and freeze-dried (Alpha 1-4, Christ, Germany) to obtain about 3.894 g of product A.

Preparation of Product B Leaves of A. grossedentata (200 g) were extracted with 3,500 mL of boiling water for 1.5 h and then filtered. The filtrate was left to stand for 24 h at ambient temperature and then filtered through a porous filter. The filtered precipitate (41.87 g) was collected and purified to provide the white crystal by recrystallizing from water for five times. After drying, about 3.90 g of product B was obtained.

Identification of the Major Flavonoid in Product B UV analysis was performed on a TU-1810PC UV-visible spectrophotometer (Purkinje, China) and the data were recorded and pro- cessed by UV Win software ver. 5.0.5 (Purkinje, Beijing, China). IR analysis was performed on a VECTOR 33 infrared spectrophotometer (Bruker, Ettlingen, Germany). 13 1 C and H NMR spectra were recorded in DMSO-d6 using an AVANCE Digital 400 MHz NMR spectrometer (Bruker) at 400 MHz. ESI-MS analysis was taken on an Esquire HCT PLUS electrospray ionization mass spectrometer (Bruker) in the negative-ion mode with a flow rate of 0.1 mL/h. The data were recorded and processed by Bruker Daltonics Data Analysis software ver. 3.3. FLAVONOID-RICH EXTRACTS FROM LEAVES OF AMPELOPSIS GROSSEDENTATA 811

HPLC-ESI/MS Analysis of Product A HPLC-ESI/MS analysis was performed on a Waters ZQ 2000 mass spec- trometer equipped with a Waters 1525 binary HPLC pump and a Waters 2487 dual l absorbance detector (Waters, Milford, MA). Separation was on a reversed-phase column, Diamonsil C18 column (4.6 ¥ 250 mm, 5-mm particle size, DIKMA). The mobile phase consisted of 1% acetate acid in water and acetonitrile (9:1). The flow rate was 1 mL/min. The wavelength for detection was set at 290 nm. Because of the acidic nature of flavonoids, the mass spectrometer was operated in the negative electrospray mode to obtain the best result with capillary voltage of 3 kV. Deprotonated molecular ions were observed at cone voltage of 40 V. Nitrogen was used as nebulizing gas with cone gas flow at 50 L/h, desolvation gas flow at 350 L/h. Source and desolva- tion temperatures were set at 100 and 250C, respectively. The scan range was set from 200 to 800 m/z. The data were recorded and processed by Masslynx software ver. 4.1 (Waters).

Quantitative Analysis of DMY in the Leaves, Product A and Product B In a Soxhlet extractor, 1.016 g of dried powdered leaves was placed, and refluxed at 80C for 10 h with 150 mL methanol, and then the extract was collected and diluted to 500 mL with methanol. After being filtered through 0.45-mm millipore filter, a 10-mL volume of the solution was injected for HPLC analysis. To produce stock solution of 1.05 mg/mL, 105 mg of DMY standard was dissolved in methanol. For the calibration curve, the stock solution was diluted with methanol in the concentration range from 0.0525 to 1.05 mg/mL. All the solutions were stored at 4C. About 50 mg of product A and product B were respectively dissolved in methanol for HPLC analysis. Also, the methanol solution of the leaves obtained in the previous sector was diluted for HPLC analysis. The samples were separated on a reversed-phase column, Diamonsil C18 column (4.6 ¥ 250 mm, 5-mm particle size) made by DIKMA. The mobile phase consisted of 1% acetate acid in water and acetonitrile (9:1) with a flow rate of 1.0 mL/min. The HPLC system consisted of a Waters 1525 binary HPLC pump and a Waters 2487 dual l absorbance detector. The injection volume was 10 mL and the wavelength for detection was set at 290 nm. Before HPLC analysis, all samples must be passed through a 0.45-mm millipore filter. The quantitative analysis of DMY in the samples was based on an external standard. The chromatographic data were recorded and processed by Breeze System software ver. 3.3 (Waters). 812 J. GAO ET AL.

DPPH Radical-Scavenging Assay DPPH radical-scavenging assay was done according to the published method (Sun and Ho 2005). Briefly, 2 mL of DPPH solution (0.2 mmol/L, in ethanol) was mixed with different concentrations of product A, product B and tertiary butylhydroquinone (TBHQ). The reaction mixture was shaken and incubated for 30 min at room temperature in the dark. Also, the absorbance was read at 517 nm against ethanol. Controls containing ethanol instead of the antioxidant solution and blanks containing ethanol instead of DPPH solution were also made. The inhibition of the DPPH radical by the samples was calculated according to the following formula:

DPPH scavenging activity ()% Abs... of control−−() Abs of sample Abs offblank = ×100% Abs. of control

Also, the percentage of DPPH radical-scavenging activity was plotted against the sample concentration to obtain the IC50, defined as the concentra- tion of sample necessary to cause 50% inhibition.

Reducing Power Assay The reducing power of the samples was determined according to the method of Strivastava et al. (2006). In addition, 0.5 mL of the extract in ethanol was mixed with phosphate buffer (2.5 mL, 0.2 M, pH 6.6) and potassium ferricyanide (2.5 mL, 1%). The mixture was incubated at 50C for 20 min. A portion (2.5 mL) of trichloroacetic acid (10%) was added to the mixture, which was then centrifuged at 3,000 rpm for 10 min. The upper layer of solution (2.5 mL) was mixed with distilled water (2.5 mL) and FeCl3 (0.5 mL, 0.1%), and the absorbance was measured at 700 nm. Increased absor- bance of the reaction mixture indicated reducing power.

Determination of Antioxidant Activity in a Linoleic Acid System The total antioxidant activities of the samples were carried out by use of a linoleic acid system (Zou et al. 2004). The linoleic acid emulsion was prepared by mixing 0.28 g of linoleic acid, 0.28 g of Tween 20 as emulsifier and 50 mL of phosphate buffer (0.2 M, pH 7.0), and then the mixture was homogenized. A 0.5 mL ethanol solution of the samples (0.5 mg/mL) was mixed with linoleic acid emulsion (2.5 mL, 0.2 M, pH 7.0) and phosphate buffer (2 mL, 0.2 M, pH 7.0). The reaction mixture was incubated at 37C in the dark to accelerate the peroxidation. The levels of peroxidation were deter- mined according to the thiocyanate method (0.1 mL, 30%), sample solution FLAVONOID-RICH EXTRACTS FROM LEAVES OF AMPELOPSIS GROSSEDENTATA 813

(0.1 mL) and ferrous chloride (0.1 mL, 20 mM in 3.5% HCl). After the mixture was left to stand for 3 min, the peroxide value was determined by reading the absorbance at 500 nm on a spectrophotometer.

Determination of Differential Scanning Calorimetry (DSC) Curve of DMY The DSC curve of DMY was obtained using a TA Q100-DSC thermal analyzer (TA Instruments, New Castle, DE). About 1.5–2.0 mg of samples (TBHQ and DMY) were accurately weighed into aluminum pans, and the pans were hermetically sealed and heated from 25 to 350C at a rate of 10C/min. A sealed empty pan was used as a reference. The DSC curves of samples were recorded and analyzed by Universal Analysis 2000, Version 4.1D (TA Instruments-Waters LLC).

Statistical Analysis The data obtained in this study were expressed as the mean of three replicate determinations and standard deviation. Statistical comparisons were made with Student’s t-test. P values of <0.05 were considered to be significant.

RESULTS AND DISCUSSION

Identification of Product B Product B obtained from leaves of A. grossedentata was identified as (+)-dihydromyriectin (Fig. 1) by UV, IR, ESI-MS and NMR analysis, based on the following characteristics: UV, lmax nm (MeOH) 293; IR bands (KBr disc): 3293(-OH), 1642 (-C=O), 1599, 1542, 1505, and 1471 (-Ar) cm-1; negative ESI-MS m/z (relative intensity): 639.0 (13.5) [2M-H]-, 355.0 (18.9) [M+Cl-]-, 319.1 (100) [M-H]- (Fig. 2); 13C NMR: 197.847 (C-4), 167.187 (C-7), 163.735 (C-5), 162.901 (C-9), 146.107 (C-3′ and C-5′), 133.875 (C-4′), 127.553 (C-1′), 107.388 (C-2′ and C-6′), 100.902 (C-10), 96.384 (C-6), 95.379 (C-8), 83.663 1 (C-2), 72.073 (C-3); H NMR: 11.877 (s, C5-OH), 10.819 (s, C7-OH), 8.903 (s, C3′-OH and C5′-OH), 8.201 (s, C4′-OH), 6.395 (s, C2′-H and C6′-H), 5.897 (d, J = 1.95 Hz, C8-H), 5.855 (d, J = 1.95 Hz, C6-H), 5.753 (d, J = 6.06 Hz, C3-OH), 4.900 (d, J = 10.83 Hz, C2-H), 4.411 (dd, J = 10.84 Hz, J = 6.06 Hz, C3-H).

HPLC-ESI/MSn Analysis of Product A Under the HPLC conditions mentioned in experimental section, good separation of product A is obtained in 60 min with UV detector recorded at 814 J. GAO ET AL.

OH

3' OH 4' 2'

8 HOO 1 5' 9 1' OH 7 2 6' 3 6 10 OH 5 4

OH O

FIG. 1. CHEMICAL STRUCTURE OF (+)-DIHYDROMYRICETIN

FIG. 2. ELECTROSPRAY IONIZATION MASS SPECTROMETRY SPECTRUM OF PRODUCT B

290 nm, as shown in Fig. 3. Also, two main compounds were detected by UV and MS. According to analysis of ESI-MS (Fig. 4) and comparison with DMY standard, Compound 1 eluted at 34.89 min in HPLC-UV chromatograph was identified as DMY, which coincided with identification of product B and what Du et al. reported (Du et al. 2002). To our surprise, Compound 2 eluted at 48.39 min in HPLC-UV chromatograph had nearly the same mass spectrum with Compound 1, which could be one of the isomers of DMY as there were two chiral centers (C-2 and C-3) in its molecular structure.

HPLC Analysis of DMY in Leaves, Product A and Product B According to analysis of product A and product B, the main flavonoid in two products was identified as DMY, which coincided with Du et al.’s report (Du et al. 2002). By using HPLC, the contents of DMY in leaves, product FLAVONOID-RICH EXTRACTS FROM LEAVES OF AMPELOPSIS GROSSEDENTATA 815

FIG. 3. HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY AND TIC PROFILES OF PRODUCT A

Compound 1

Compound 2

FIG. 4. ELECTROSPRAY IONIZATION MASS SPECTROMETRY SPECTRA OF COMPOUND 1 AND 2

A and product B were determined as 32.96 Ϯ 1.18%, 77.15 Ϯ 1.98%, 95.12 Ϯ 2.86% (n = 3), respectively. To this day, more than 4,000 kinds of flavonoids have been identified or synthesized, but few of them can be widely used in fields of food and medicine. The reason for this is that although flavonoids exist ubiquitously in plants, few kinds of plants can contain enough 816 J. GAO ET AL.

flavonoids to achieve large-scale production. The results of HPLC analysis showed that leaves of A. grossedentata are a flavonoid-rich plant source. The content of DMY in A. grossedentata leaves was so high that the content of DMY in leaves raw extract (product A) was more than 75%. Although the content of DMY in leaves of A. grossedentata was high, it is difficult to obtain DMY with high purity. Du et al. (2002) reported that DMY at a high purity of over 99% could be obtained by purifying the raw extract with a preparative triple-column countercurrent chromatograph, but this method is difficult to put to industrial use. The HPLC analysis of product B indicated that DMY with a purity of about 95% could be obtained by recrystallizing for five times from water, which was a useful technology for industrialization.

DPPH Radical-Scavenging Activity The DPPH radical is a stable organic free radical with adsorption band at 517 nm. It loses this adsorption when accepting an electron or a free radical species, which results in a visually noticeable discoloration from purple to yellow. Because it can accommodate many samples in a short period and is sensitive enough to detect active ingredients at low concentrations, it was used for evaluating the antioxidant potential of product A and B. In this study, a high DPPH radical-scavenging activity was observed in both product A and product B in a concentration manner (Fig. 5). Product B (IC50, 7.5 mg/mL) was more active than TBHQ (IC50, 9.4 mg/mL) and product A (IC50, 10.5 mg/mL), which should attribute to the high content of DMY processing six hydroxyl groups.

Reducing Power The reducing power of product A and product B, which may serve as a significant reflection of antioxidant activity, was determined using a modified Fe (III) to Fe (II) reduction assay, the yellow color of the test solution changes to various shades of green and blue depending on the reducing power of samples. The presence of antioxidants in the samples causes the reduction of the Fe3+/Ferricyanide complex to the ferrous form. Therefore, the Fe2+ can be monitored by measurement of the formation of Perl’s Prussian blue at 700 nm (Zou et al. 2004). Figure 6 showed the reducing power of product A and product B compared with that of TBHQ. All samples showed some degree of reducing power. Also, the reducing power of product A and product B was superior to that of TBHQ, which is known to be a synthetic antioxidant. In this study, the reducing power of the samples linearly increased with increasing concentration, and the correlation coefficients (R2) of product A, product B and TBHQ were 0.9994, 0.9993 and 0.9984, respectively. The reducing power of the samples followed the following order: product B > product A > TBHQ. FLAVONOID-RICH EXTRACTS FROM LEAVES OF AMPELOPSIS GROSSEDENTATA 817

-᭜- Product B, - - TBHQ, -᭿- Product A

FIG. 5. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) SCAVENGING ACTIVITY OF PRODUCT A, PRODUCT B, TERTIARY BUTYLHYDROQUINONE (TBHQ)

Antioxidant Activity in Linoleic Acid System In this study, the antioxidant activity of the samples was evaluated by peroxidation of linoleic acid using the thiocyanate method at 37C. During the linoleic acid peroxidation, peroxides are formed and these compounds oxi- dized Fe2+ to Fe3+, the latter Fe3+ ion forms a complex with SCN-, which has a maximum absorbance at 500 nm. High absorbance is an indication of high concentration of peroxide during the emulsion incubation. The antioxidant activity of the samples exhibited an amount-dependent manner, as can be seen from Fig. 7. The antioxidant activity of the samples followed the following order: TBHQ > product A > product B.

DSC Analysis of DMY Figure 8 showed the DSC curves of TBHQ and DMY. It could be con- cluded that the melting point of DMY (about 250C) was obviously higher than that of TBHQ (about 129C), which indicated that thermal stability of DMY 818 J. GAO ET AL.

-᭜ - Product B, - - TBHQ, -᭿ - Product A

FIG. 6. REDUCING POWER OF PRODUCT A, PRODUCT B, TERTIARY BUTYLHYDROQUINONE (TBHQ)

-᭹ - Control, -᭜- Product B, - - TBHQ, -᭿ - Product A

FIG. 7. ANTIOXIDANT ACTIVITY OF PRODUCT A, PRODUCT B, TERTIARY BUTYLHYDROQUINONE (TBHQ) IN LINOLEIC ACID SYSTEM FLAVONOID-RICH EXTRACTS FROM LEAVES OF AMPELOPSIS GROSSEDENTATA 819

FIG. 8. DIFFERENTIAL SCANNING CALORIMETRY CURVES OF TERTIARY BUTYLHYDROQUINONE (TBHQ) AND PRODUCT B (DMY) was superior to that of TBHQ. Thus, DMY from leaves of A. grossedentata could be used as antioxidant in cooking oil and baked food.

CONCLUSION

Although flavonoids ubiquitously exist in plants, few kinds of plants can contain enough flavonoids to achieve large-scale production. The results obtained in this study suggest that leaves of A. grossedentata are rich in DMY, and that the antioxidant activity of the flavonoid-rich extracts from A. grossedentata leaves can be comparable with that of TBHQ. Leaves of A. grossedentata will become a new source of natural antioxidant and health food with great commercial interest in the food and phytopharmaceutical market.

ACKNOWLEDGMENTS

The financial support provided by Science and Technology Plan of Guangdong province, China (No.2005A20303002) and Science and Technology Plan of Guangzhou, China (No. 2006J1-C0251) is appreciated. 820 J. GAO ET AL.

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