Journal of Applied Botany and Food Quality 89, 150 - 155 (2016), DOI:10.5073/JABFQ.2016.089.018 1 The Key Laboratory of Biomedical Engineering Ministry of Education, Department of Biomedical Engineering, Zhejiang University, China 2 College of Basic Medical Science, Shandong University of Traditional Chinese Medicine, Jinan, China 3Changshu Qiushi Technology Co. Ltd., Changshu, China Phytochemical compositions, antioxidant and antimicrobial activities analysis of extracts from Vaccinium bracteatum Thunb. leaves Jin Hu1, Jing Wang2, Shouxin Li3, Bingxian Yang1, Minghua Gong2, Ximin Li3, Lin Zhang1, Jingkui Tian1,* (Received October 27, 2015) * Corresponding author Summary Vaccinium bracteatum Thunb. is an edible plant, which has been used for many food products and is also a resource of traditional Chinese medicine. In this study, the antioxidant and antimicrobial activities of ethanol extracts from its leaves were investigated. To study the characteristic compositions, twelve compounds of extracts accumulated by the D-101 macroporous adsorption resin (VBE) were identified by HPLC-DAD and HPLC-ESI/MS techniques, in- cluding chlorogenic acid and its isomers, and eight flavonoid com- pounds. The contents of total flavonoids, orientin and isoorientin in the accumulated part were 601.4, 44.7, and 96.1 mg/g, respectively, which were far more than that in the raw materials. Furthermore, the antioxidant activities were estimated by DPPH, ABTS, and FRAP assays, which showed that the high content accompanied with strong antioxidant activities. Besides, compared to the same type of bam- boo leaves (AOB), the accumulated part possesses better activities. At the last, the antimicrobial activities of VBE were assessed by a serial two-fold dilution assay, the results showed that it had good antimicrobial activities. Taken together, extracts from Vaccinium bracteaturn Thunb. leaves have better antioxidant activities, which can be used as a natural antioxidant. Introduction Antioxidants, as food additive, are used to keep the foods quality during their shelf life. At the present, the most commonly used anti- oxidants are chemical preservatives, such as butylated hydroxyani- sole (BHA), butylated hydroxytoluene (BHT), propyl gallate (PG), and tertbutylhydroquinone (TBHQ). However, some studies showed that the chemical preservatives exhibited certain toxicity, which can affect the respiratory enzymes activity, and cause teratogenesis and carcinogenesis (WETTASINGHE and SHAHIDI, 1999; LUO and FANG, 2008). Nowadays, the demand for food additives is increasingly high, and the utilization of natural antioxidants becomes a necessary research direction. A lot of research had found that the extracts of most plants possessed antibacterial and antioxidant effects (SUN et al., 2011; RYSZARD et al., 2004; ICHIKAWA et al., 2001). Vaccinium bracteatum Thunb. is mainly distributed in the regions south of the Yangtze River, which belongs to the genus Vaccini- um (family Ericaceae). The leaves were rich in proteins, vitamins and minerals, which contained abundant vitamin C (Vc), calcium, iron, selenium and manganese. With methionine (Met) and histidine (His) being the highest in them, seventeen amino acids were found (CHEN et al., 2008). Furthermore, it showed multiple advantages for Wufanshu being taken as a rootstock species of blueberry, the contents of potassium, sodium, iron, phosphorus, zinc, and calcium element in fruit of Wufanshu were significantly higher than those in none-grafted blueberry (XU et al., 2014). In China, a series of Vaccinium bracteatum food was also made of its extracts such as Vaccinium bracteatum beverage. Besides, making Wufan with rice was people’s custom, it had been developed into a local tourism food (HAO et al., 2010). Modern medical research found that it had many pharmacologi- cal activities, such as improving blood microcirculation and anti- inflammatory (LANDA et al., 2014). In recent years, some studies showed that the main constituents of Vaccinium bracteatum Thunb. leaves were flavonoids, polyphenols, and diterpenes. Additionally, it was reported that the flavonoids in extracts from plants had antioxi- dant activity (BERNONVILLE et al., 2011; CHUA et al., 2011; LI et al., 2009), and the mechanism of antioxidant activity was related to the structure of the flavonoids and the substituents of the heterocyclic rings (KONG et al., 2003). In our previous studies (LI et al., 2008; ZHANG et al., 2009), a sys- tematical investigation of the chemical components of Vaccinium bracteatum Thunb. leaves had been carried out. Twenty-five com- pounds were isolated from the leaves, and twenty-four of them were identified. Here, the antioxidant and antibacterial activities of extracts from leaves were performed. The results showed that the extracts from V. bracteratum leaves could be used as a natural anti- oxidant in food industry. Materials and methods Materials and chemicals 1,1-Diphenyl-2-picrylhydrazyl (DPPH) was purchased from the Wako Pure Chemical Company (Osaka, Japan). 2,4,6-Tris(2- pyridyl)-1,3,5-triazine (TPTZ), and 2,20-azino-bis (3-ethylbenzo- thiazoline-6-sulfonic acid) (ABTS) were purchased from the To- kyo Chemic Industry Company (Tokyo, Japan). Ethanol, methanol (HPLC-grade), acetonitrile (HPLC-grade), iron vitriol, ferric trichlo- ride, sodium acetate, and potassium persulfate were obtained from the Sinpharm Chemical Reagent Company (Shanghai, China). All solvents and chemicals were of analytical grade, unless otherwise specified. HPLC grade water was obtained from Milli-Q System (Millipore, Billerica, MA, USA). Extraction and accumulation of total flavonoids from Vaccinium bracteatum Thunb. leaves Vaccinium bracteatum Thunb. leaves were harvested in Zhejiang Province, China. The leaves were dried in the shade for a week, and then were ground to powder using a grinder. A portion (100 g) of powder was extracted with 70 % ethanol solution. One part of etha- nol extracts was dried in a rotary evaporator, which was the raw materials part. Another part of ethanol extracts was added into the column contained D-101 macroporous resin. The column was suc- cessively washed by water and 70 % ethanol. The eluted solution from the column was collected and dried, which was the macropo- rous adsorption resin part (VBE). All samples were packed in the bag and stored at room temperature until later analysis. The nutraceutical properties of Vaccinium bracteatum Thunb. leaves 151 HPLC analysis The extract (25 mg) was dissolved in 10 mL of 70 % (v/v) ethanol and filtered through a 0.45 μm cellulose acetate filter before HPLC analysis. The samples were analyzed by Shimadzu LC-10AT HPLC system (Shimadzu Co., JAPAN), equipped with CBM-20A Triton System Controller, SPD-M20A detector, SIL-20A auto sampler and CTO-10A column oven. The analytical column was a C18 column of Agilent TC-C18 (250 mm × 4.6 mm, 5 μm i.d., Agilent Techno- logies, USA) with a flow rate of 1 mL min-1 at 40 °C. The mobile phase consisted of 1 % acetic acid in water and 100 % methanol. The applied gradient program was: 0-10 min (5 % to 15 % methanol), 10- 40 min (15 % to 20 % methanol), 40-60 min (20 % to 30 % methanol), 60-100 min (30 % to 50 % methanol), 100-120 min (50 % to 95 % methanol), and 120-130 min (95 % methanol). 20 μL of the standard solutions and samples were used and all of the samples were analy- zed in triplicate. HPLC-ESI/MS analysis The chromatographic separation condition was described above. An Agilent 1100 analytical HPLC system was used, with a G1312 Bin- pump, G1314A variable-wavelength detector (VWD), model 7725 injector fitted with a 20 μL sample loop, and an Agilent chemsta- tion data system. LC/ESI-MS analyses were performed by using the Agilent HPLC system described above combined with a Bru- ker Esquire 3000 plus ion trap mass spectrometer (Bruker-Franzen Analytik GmbH, Bremen, Germany), which was equipped with an electrospray ionization. Instrument control and data acquisition were performed by using Esquire 5.0 software. The ion source tempera- ture was 270 °C, and the needle voltage was always set at 4.0 kV. Nitrogen was used as the drying and nebulizer gases at a flow rate of 10 L min−1 and a back-pressure of 30 psi. Total flavonoids content The total flavonoids content of the extracts was determined by the method described in the Chinese Pharmacopoeia (2010 edition). 100 mg of extract was dissolved in 100 mL of 70 % (v/v) ethanol sol- vent. 1 ml solution was taken, being mixed with 1 mL of 5 % NaNO2. 6 min later, 1 mL of 10 % AlCl3 was added, and then 10 ml of NaOH (1M) was added after the same time. Immediately, the mixture was constant volume to 25 mL and was kept in dark place to react for 15 min. With 70 % ethanol as a blank control, the absorbance (A) was measured at the wavelength of 510 nm. Rutin was used as standard compound and the concentration ranged from 0.01 to 0.80 mg ml−1. The total flavonoids content was expressed as mg of rutin, which was equivalent to per g of extracts. Orientin and isoorientin content The content of orientin and isoorientin in extracts was measured with HPLC method. The mobile phase consisted of 0.1 % phosphoric acid in water (A) and 2 % Tetrahydrofuran in methanol (B). The gradient profile was established as follows: the ratio of A and B was 77:23 (v/v) within 60 min. The flow rate was 1.0 mL min−1, and the co- lumn temperature was 40 °C. All of the samples were analyzed in triplicate. Assays for antioxidant activities DPPH assay The DPPH assay of the extracts was determined following the me- thod used by Shimada, Fujikawa, Yahara, and Nakamura (KAZUKO et al., 1992). DPPH was dissolved in ethanol and the reaction soluti- on concentration was 0.1 mM. 1 mL of the DPPH reaction solution was added to 5 mL of extract solution with being shaken vigorously. The mixture reacted for 30 min in the dark at room temperature, and then was measured at a wavelength of 517 nm using a UV spectro- photometer. Ascorbic acid and the antioxidant composition of bam- boo leaves (AOB) were used as control groups. All of the samples were analyzed in triplicate. The DPPH radical scavenging activity was calculated by the following equation: Scavenging activity % = [ 1 - Asample / Ablank ] × 100 % According to different concentrations of the clearance curve, the sca- venging activities were expressed as half maximal inhibitory con- centration (IC50). ABTS assay The ABTS assay was based on the method of RE et al. (RE et al., 1999) with some modifications. ABTS stock solution was dissolved in deionized water to a 7 mM concentration. The ABTS reagent so- lution was generated by 5 mL of ABTS stock solution with 88 μL of 140 mM potassium persulfate. This solution was stored at room temperature in the dark for 12-16 h. Before it was used, the ABTS reagent solution was diluted with PBS (pH 7.4) to an absorbance of 0.70 ± 0.02 at 734 nm. For the ABTS assay, 0.3 mL sample of dif- ferent concentrations was added to 6 mL of diluted ABTS solution to react in the dark at 30 °C for 6 min, and then the absorbance was measured at a wavelength of 517 nm. Ascorbic acid and AOB were used as control groups. All of the samples were analyzed in tripli- cate. The ABTS radical scavenging activity was calculated by the following equation: Scavenging activity % = [ 1 - Asample / Ablank ] × 100 % According to different concentrations of the clearance curve, the sca- venging activities were expressed as IC50. FRAP assay The FRAP assay was determined by the method of Benzie and Strain (BENZIE and STRAIN, 1996) with minor modifications. The FRAP reagent included the following solution: 300 mM acetate buffer; 10 mM TPTZ in 40 mM hydrochloric acid; and 20 mM FeCl3·6H2O. The FRAP reagent was prepared by mixing 25 mL acetate buffer, 2.5 mL of TPTZ solution and 2.5 mL of FeCl3·6H2O solution, and then warmed at water bath to 37 °C before using. 0.3 mL of sample solutions was added into 6 mL of FRAP solution, and reacted for 10 min. Absorbance was measured at a wavelength of 593 nm. As- corbic acid and AOB were used as control groups. All of the samples were analyzed in triplicate. Antimicrobial activities The antimicrobial activity of the extracts was determined by the serial 2-fold dilution method (CHARLES et al., 1979; SHADOMY and ESPINEL, 1980). Three bacterial strains were used in this study: Sta- phylococcus aureus (ATCC6538), Escherichia coli (8099), and Can- dida albicans (10231). All strains were obtained from the Zhejiang Academy of Medical Sciences, China. The bacterial strains were incubated on micrococcus, nutrient, and Yeast Maltose (YM) media then cultured at incubator at 35 °C for 24 h. VBE was dissolved in PBS at the highest concentration (100 μg/mL), then diluted to seri- al two-fold dilutions. Tetracycline was used as standard antibiotics. The minimum inhibitory concentration (MIC) was defined as the lowest concentration that inhibited microorganism growth, which was based on the degree of turbidity visually. Statistical Analysis All samples were measured in triplicate. SPSS statistical software was used for the statistical evaluation of results, which were presen- ted as the mean ± standard deviation (SD). Statistical analysis was done by Independent-Samples t-test. 152 J. Hu, J. Wang, S. Li, B. Yang, M. Gong, X. Li, L. Zhang, J. Tian Results and discussion HPLC analysis and identification compounds by HPLC–ESI/MS analysis In order to get the information of the VBE compounds, the analysis of HPLC and HPLC–ESI/MS was needed. The HPLC chromatogram of the VBE was showed in Fig. 1 (A), the separation of the com- pounds was excellent under the optimized conditions. As to the LC- MS information, the TIC negative and the TIC positive were shown in Fig. 1 (B), Fig. 1 (C), the TIC positive intensity was obvious- ly less than the TIC negative, it was related to noise level and the compound structure, especially flavonoid compositions (PETSALOA et al., 2006). Compared to previous research, the compounds 1-12 in Fig. 1 (A) were identified as shown in Tab. 1 which include eight flavonoids: (4) orientin; (5) isoorientin; (6) hyperoside; (7) quercetin-3- O-β-D-glucuronide; (8) quercetin-3-O-α-L-arabinopyranoside; (10) quercetin-3-O-α-L-rhamnoside; (11) quercetin-3-O-β-D-glucuronide methyl ester; (12) isolariciresinol-9-O-β-D-xyloside. Compared with the HPLC chromatogram of chlorogenic acid and the VBE, the result showed the existence of chlorogenic acid in the Vaccinium bractea- tum Thunb. leaves in Fig. 1 (D). 0 20 40 60 80 100 120 Time [min] 0.00 0.25 0.50 0.75 1.00 1.25 6x10 Intens . B 0 10 20 30 40 50 60 70 80 90 100 11 0 120 mi n -2 5 0 25 50 75 100 125 150 175 A 1 2 3 4 5 6 8 9 10 11 12 7 0 20 40 60 80 100 120 Time [min] 0 1 2 3 4 5 7x10 Intens . C 3-O-Chlorogenic acid VBE D Fig. 1: (A) HPLC chromatography of the VBE at the 254 nm; for peak iden- tification see Tab. 1. (B) The TIC negative; (C) The TIC positive; (D) Chromatography contrast between chlorogenic acid reference and the VBE HPLC. Tab. 1: Identification of 12 compounds. Peak Retention time Λmax (nm) m/z Identification 1 16.4 240, 324, 443 354 5-O-Chlorogenic acid 2 29.0 240, 324, 443 354 3-O-Chlorogenic acid 3 30.9 240, 324, 443 354 4-O-Chlorogenic acid 4 64.0 252, 266, 348 448 Orientin 5 67.6 255, 269, 348 448 Isoorientin 6 74.9 253, 267, 352 464 Hyperoside 7 75.5 255, 354 478 Quercetin-3-O-β-D- glucuronide 8 77.9 247, 443 434 Quercetin-3-O-α-L- arabinopyranoside 9 79.0 220, 247, 342 536 10-O-trans-p-coumaroyl- sandoside 10 82.9 246, 286, 321 448 Quercetin-3-O-α-L- rhamnoside 11 86.7 266, 344, 324 492 Quercetin-3-O-β-D- glucuronide methyl ester 12 90.0 248, 443 494 Isolariciresinol-9-O-β-D- xyloside Tab. 2: The content between the raw materials and the extracts. (Mean ± SD, n = 3) Sample Total flavonoid Orientin Isoorientin (mg/g) (mg/g) (mg/g) Raw materials 56.3 ± 0.27 5.31 ± 0.05 9.42 ± 0.04 VBE 601.4 ± 8.50 44.7 ± 0.60 96.1 ± 0.92 The total flavonoids, orientin and isoorientin content The contents of total flavonoids, orientin, and isoorientin were compared between the raw materials and the VBE. The results are shown in Tab. 2. Orientin and isoorientin have a typical flavonoid structure, so we choose the two standards as index compositions for measurements. The total flavonoids content was expressed as rutin equivalents in mg/g of extracts. Results indicated that the content of the total flavonoids in the extracts was 601.4 ± 8.50 mg/g, which was nearly ten times than that in the raw materials. Furthermore, the con- tents of orientin and isoorientin in the extracts were 44.7 ± 0.60 mg/g and 96.1 ± 0.92 mg/g, respectively, which were far more than that in the raw materials. The macroporous resins D-101 had good effect in enrichment of flavonoids, which was also confirmed in many studies (FANG et al., 2008; QI et al., 2007). As active ingredients, it would enhance the antioxidant activity and other activities by increasing flavonoids content. The nutraceutical properties of Vaccinium bracteatum Thunb. leaves 153 The antioxidant activities Antioxidant activities were evaluated by three methods, and the re- sults were displayed in Fig. 2 and Tab. 3. The results of DPPH assay found that all samples had scavenging action. The IC50 value was 42.2 ± 1.2 μg/ml for VBE, while 449.5 ± 10.6 μg/ml for the raw materials. Additionally, the results of ABTS assay showed that the IC50 value of the raw materials, ascorbic acid, VBE, and AOB were 381.8 ± 12.4, 48.7 ± 1.0, 71.1 ± 1.1, and 104.3 ± 2.2 μg/ml, respec- tively. Furthermore, using 0.3 mmol/L FeSO4 as the standard, the result of FRAP assay showed that the concentrations up to the same absorbance of the raw materials, ascorbic acid, VBE and AOB were 327.9 ± 7.5, 28.2 ± 0.5, 65.0 ± 1.8, and 101.7 ± 1.1 μg/ml, respec- tively. Antioxidant capacities varied among these three antioxidant assays, all the samples showed certain antioxidant ability. Compared to the extracts of the raw materials, the antioxidant activity of VBE was better (P < 0.05), which also showed that the high content accom- panied with strong antioxidant activities. As chemical antioxidant, ascorbic acid had the highest antioxidant ability, however, the activi- ty was significantly higher in VBE compared to AOB (P < 0.05). It is reported that all of the three bamboo extracts from different parts had antioxidant activities, which had approved the development of natural antioxidants (GONG et al., 2015). Besides, its main active ingredients were flavonoids, azalides, and phenolic acid, and four major flavonoids were orientin, isoorientin, vitexin and isovitexin. The VBE and AOB had the same compound ingredients, but the VBE flavonoids content was much higher and the antioxidant acti- vity of VBE was better than that of AOB. It was indicated that the Vaccinium bracteatum Thunb. leaves had large potential to be used as natural antioxidant. The antimicrobial activities of VBE The results of antimicrobial activities were presented in Tab. 4, which showed that the serial 2-fold dilution method was feasible. Some re- searches had found that chlorogenic acid had strong antimicrobial activity (ZHAO et al., 2010; CHAKRABORTY and MITRA, 2008), there were also many antimicrobial reports on flavonoids (MELLOU et al., 2005; SOUSA et al., 2009; SOHN et al., 2004). As representative com- positions of the flavonoids, there were many studies on the antimi- crobial activities of orientin and isoorientin (ZU et al., 2010; BECKER et al., 2005). Based on these researches reported previously, we can infer that the VBE has good antimicrobial activity. The results ob- tained showed that the VBE has the inhibition to the three kinds of bacteria, and the minimum inhibitory concentration was 12.5 mg/ml with high level of antimicrobial activity. Conclusions In this research, twelve compounds were identified by using HPLC- DAD and HPLC–ESI/MS data analysis, which contained chloro- genic acid and its isomers, and eight flavonoids. Furthermore, we quantitatively analyzed the total flavonoids, orientin and isoorientin content of the extracts from Vaccinium bracteatum Thunb. leaves. These substances provided the basis of chemical compositions to explain the antioxidant activities. The antioxidant activities of the extracts were estimated by DPPH, ABTS, and FRAP assays. The results suggested that the antioxidant activity of the VBE could be significantly stronger than the AOB. Finally, the antibacterial activi- ty of the VBE was determined by a serial twofold dilution method. Based on these results, the Vaccinium bracteatum Thunb. leaves could be a potential natural antioxidant. For further research, more works are needed to be carried out; the VBE will be added to the meat or the fried food as a readily accessible source of new natural food antioxidants. Fig. 2: Antioxidant activities of the raw materials, VBE, AOB and ascorbic acid. (A) DPPH radical scavenging assay; (B) ABTS radical scavenging assay; (C) FRAP-FeSO4 standard curve; (D) Different concentration samples absorbance curve in FRAP as- say. The value in the figure is average value, n = 3. 154 J. Hu, J. Wang, S. Li, B. Yang, M. Gong, X. Li, L. Zhang, J. Tian Tab. 3: The antioxidant activity results. (Mean ± SD, n = 3) IC50 (μg/ml) DPPH ABTS VBE 42.2 ± 1.2** 71.1 ± 1.1** 65.0 ± 1.8** Raw materials 449.5 ± 10.6** 381.8 ± 12.4** 327.9 ± 7.5** AOB 105.2 ± 3.4** 104.3 ± 2.2** 101.7 ± 1.1** Ascorbic acid 29.7 ± 0.5 48.7 ± 1.0 28.2 ± 0.5 a: Result was expressed as the concentration of antioxidants up to the same absorbance that of 0.3 mmol/L FeSO4. **: Significantly different at the 0.05 level (2-tailed). Sample FRAP assay (μg/ml) a Tab. 4: Antimicrobial activities of the VBE The concentration of VBE (mg/ml) 50 25 12.5 6.25 3.125 1.5625 0.78125 Staphylococcus aureus (ATCC6538) – – – + + + + + – Escherichia coli (8099) – – – + + + + + – Candida albicans (10231) – – – + + + + + – +: Instructions bacterial growth was positive; –: Instructions bacterial growth was negative. Negative control Positive control Bacterial strain HAO, J.J., FENG, J.Y., DING, Y.L., WANG, F.S., 2010: Dynamic changes of the nutritional compositions of Vaccinium bracteatum leaf from different provenances. (Chinese). 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Address of the author: Jin Hu, Bingxian Yang, Lin Zhang, Jingkui Tian, The Key Laboratory of Biomedical Engineering Ministry of Education, Department of Biomedical Engineering, Zhejiang University, Hangzhou 310027, China E-mail: tjk@zju.edu.cn Jing Wang, Minghua Gong, College of Basic Medical Science, Shandong University of Traditional Chinese Medicine, Jinan, 250355, PR China Shouxin Li, Ximin Li, Changshu Qiushi Technology Co. Ltd., Changshu, 215500, China © The Author(s) 2016. This is an Open Access article distributed under the terms of the Creative Commons Attribution Share-Alike License (http://creative- commons.org/licenses/by-sa/4.0/).