Food Sci. Biotechnol. 23(4): 1295-1303 (2014) DOI 10.1007/s10068-014-0178-1

RESEARCH ARTICLE

Antioxidant Activities of Sea Buckthorn Leaf Tea Extracts Compared with Green Tea Extracts Hyunnho Cho, Eunae Cho, Hana Jung, Hae Chang Yi, Bomee Lee, and Keum Taek Hwang

Received October 30, 2013; revised January 15, 2014; accepted January 23, 2014; published online August 31, 2014 © KoSFoST and Springer 2014

Abstract The polyphenol, flavonoid, and ascorbic acid contents of sea buckthorn leaf tea extracts, along with antioxidant activities, were compared with green tea extracts under different extraction conditions. Sea buckthorn leaf tea and green tea were extracted using water (SW, GW) and ethanol at room temperature (SE, GE), respectively, and at 80oC (SWH, GWH, SEH, and GEH, respectively). GEH, GWH, SE, and SEH contained more antioxidant compounds and higher activities, and SWH, SEH, GWH, and GEH had elevated antioxidant enzyme activity levels in H2O2-treated RAW264.7 cells. Cells treated with SWH and SEH showed elevated expression of nuclear factor (erythroid-derived 2)-like 2 and maintained the cell glutathione (GSH)/glutathione disulfide (GSSG) ratio at levels similar to H2O2-untreated controls. Keywords: Hippophae rhamnoides L., sea buckthorn, green tea, antioxidant activity, Nrf2

Introduction Sea buckthorn (Hippophae rhamnoides L., Elaeagnaceae) is a deciduous spiny tree widely distributed over Asia and Europe. It is a hardy plant, tolerant against drought and cold, and is useful for land reclamation and farmstead protection (1). The chemical composition of sea buckthorn berries has been extensively investigated. Carbohydrates,

Hyunnho Cho, Eunae Cho, Hana Jung, Hae Chang Yi, Bomee Lee, Keum Taek Hwang (
) Department of Food and Nutrition, and Research Institute of Human Ecology, Seoul National University, Seoul 151-742, Korea Tel: +82-2-880-2531; Fax: +82-2-884-0305 E-mail: [email protected]

proteins, organic acids, and ascorbic acid are abundant in sea buckthorn berries. Although the ascorbic acid content of berries varies depending on the species, geographical location, and degree of physiological maturity, the maximum content of ascorbic acid reported was 2,500 mg per 100 g of berries (2). Unlike other plants, both the pericarp and seeds of sea buckthorn are rich in linoleic and linolenic acids, as well as monounsaturated fatty acids, including palmitoleic, vaccenic, and oleic acids (3). This makes sea buckthorn a promising plant containing many dietary and medicinal compounds with potential beneficial applications in human health. The health-promoting properties of sea buckthorn have been well recognized since ancient times. The important pharmacological functions of sea buckthorn berries include prevention of gastric ulcers and hepatic fibrosis, reduction of blood glucose and cholesterol levels, relief of inflammation and heartburn, and anti-mutagenic and anti-tumor activities (4,5). Compared to the berries, study of sea buckthorn leaves is still in the initial stage. Clinical investigations of the medicinal effect of the leaves began in the 2000s. Sea buckthorn leaves have been shown to have anti-microbial, anti-viral, antioxidant, and anti-inflammatory properties both in vivo and in vitro (6,7). Sea buckthorn leaf extracts prevented chromium-induced oxidative damage in an in vivo model (6). In the present study, the antioxidant compounds and the antioxidant activities of sea buckthorn leaf tea produced in Korea were investigated. The antioxidant activities of sea buckthorn leaf tea were compared with the activities of green tea as a positive control. Effects of solvents and temperature on extractability and functional properties were also investigated. Information reported in this study will provide insight into the antioxidant activities and the mechanism of action of sea buckthorn leaves.

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Materials and Methods Chemical reagents Filter paper and nitrocellulose transfer membrane were obtained from Whatman International Ltd. (Kent, UK). Folin-Ciocalteu phenol reagent (2 N), gallic acid, (+)-catechin hydrate (15 mM), L-ascorbic acid (100 mM), DPPH, ABTS, potassium persulfate, TPTZ, sodium acetate, chelex 100 sodium form, Trolox, MTT, trichloroacetic acid, quercetin, quercetin-3-galactoside, isorhamnetin, kaempferol, and rutin were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Sodium hydroxide, dimethyl sulfoxide (DMSO), methanol and ethanol were from Samchun Chemical Co. (Pyeongtaek, Korea). H2O2, o-phosphoric acid, sodium nitrate, and aluminum chloride were from Junsei Chemical Co. (Tokyo, Japan). FeCl3 was obtained from Duksan Pure Chemical Co. (Pyeongtaek, Korea). HPLC grade acetonitrile and methanol were obtained from J.T. Baker Chemical Co. (Phillipsburg, NJ, USA). Dulbecco’s modified Eagle’s medium (DMEM, containing 4.5 g D-glucose/L, 4 mM Lglutamine, and 110 mg of sodium pyruvate/L), fetal bovine serum, and penicillin/streptomycin were obtained from Gibco BRL (Grand Island, NY, USA). Phosphate buffered saline (PBS), radioimmune precipitation assay (RIPA) buffer, loading bufer and protease inhibitor cocktail #6 were obtained from Biosesang, Inc. (Seongnam, Korea). West-Q chemiluminescent substrate kit was obtained from GenDEPOT, Inc. (Barker, TX, USA). A modified Lowry protein assay kit was obtained from Thermo Scientific (Rockford, IL, USA). All the other chemicals and reagents were obtained from Bio-Rad (Hercules, CA, USA). Superoxide dismutase (SOD) and glutathione peroxidase (GPx) assay kits were obtained from Cayman Chemical Co. (Ann Arbor, MI, USA). GSH/GSSG-Glo assay kit was obtained from Promega Co. (Madison, WI, USA). Triply distilled water, produced by Aquamax Water System (Younglin, Anyang, Korea), was used throughout the study. Plant materials Sea buckthorn leaf tea produced using young leaves harvested in June of 2010 was obtained from Hwacheon Vitamin Tree Farm (Hwacheon, Korea). Green tea produced using young tea leaves harvested on Jeju Island (Korea) in September of 2010 was purchased from a local market in Seoul, Korea in June of 2010. The teas were stored at 4oC until use. Preparation of leaf tea extracts Sea buckthorn leaf and green teas were ground by a grinder (Angelia; Angel Juicer Co., Busan, Korea), and then sieved through a size 50 mesh (Sungkwang Metal Co., Gimpo, Korea). Ground sea buckthorn leaf and green teas (5 g each) were extracted using water (100 mL) at room temperature (SW and GW, respectively), and at 80oC (SWH and GWH) and in 80%

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(v/v) ethanol at room temperature (SE and GE) and at 80oC (SEH and GEH). Extraction was done using a heating mantle attached to temperature controller and a condenser (MS-C406 and TC-200p; Misung Scientific Ltd., Seoul, Korea). Since green tea is commonly brewed at 80oC, this temperature was used to produce SWH, GWH, SEH, and GEH. Extracts were filtered through Whatman No. 2 filter paper (Whatman). Leaf extract filtrates were concentrated using a vacuum rotary evaporator (N-1000; Eyela Co., Tokyo, Japan) and freeze-dried using a freeze dryer (Bondiro; Ilshin Lab Co., Seoul, Korea). Equal weights of freeze-dried extracts were dissolved in a reaction solution or a medium to be used for antioxidant studies and cell cultures. Solvents are described below. Determination of polyphenol, flavonoid, and ascorbic acid contents The polyphenol contents of extracts were determined using the Folin-Ciocalteu colorimetric method (8). The total polyphenol content was expressed as gallic acid equivalents (GAE). For measurement of the flavonoid content, 500 µL of each extract diluted with water was added to 75 µL of 5% (w/v) sodium nitrate. After 5 min, 150 µL of 10% (w/v) aluminum chloride was added. After 5 min, 500 µL of 1 M sodium hydroxide was added and the solution was vortexed for 10 s. The absorbance of each sample was measured at 510 nm using a spectrophotometer (SpectraMAX 190; Bio-Rad Laboratories). The flavonoid content was expressed as catechin equivalents (CE). The ascorbic acid content in extracts was determined using the modified method of Kampfenkel et al. (9). An amount of 200 µL of each extract diluted with water was added to 0.8 mL of 10% (w/v) trichloroacetic acid followed by incubation for 5 min on ice and centrifugation (Mega 21R; Ilshin Lab Co.) at 273×g for 5 min at 4oC. After 0.5 mL of the supernatant was transferred to a new vessel, 1.5 mL of water and 0.2 mL of 0.2 N Folin-Ciocalteu phenol reagent were added. After 10 min of incubation at room temperature, the absorbance was measured at 760 nm. L-Ascorbic acid was used to construct a standard curve. Identification and quantification of polyphenols using HPLC A Waters 2695 separation module and a Waters 2996 photodiode array detector equipped with an Xbridge C18 column (4.6×250 mm, 5 µm; Waters, Milford, MA, USA) were used to identify and quantify polyphenols in extracts. Mobile phases were solvent A (water:o-phosphoric acid, 99.7:0.3) and solvent B (acetonitrile:methanol, 75:25), and the gradient was: 0-1 min, 99-87% A; 1-15 min, 8775% A; 15-30 min, 75-45% A; 30-31 min, 45-10% A; and 31-32 min, 10-99% A. The equilibration time was 5 min, the flow rate was 1 mL/min, and the injection volume was 20 µL. Freeze-dried extracts were dissolved in respective extraction solvents and filtered through 0.20 µm filters

Antioxidant Activities of Sea Buckthorn Leaf Tea

(Advantec, Tokyo, Japan) and injected. Standards were dissolved in methanol diluted to appropriate concentrations, and injected. DPPH and ABTS free radical scavenging activities and the ferric reducing antioxidant power (FRAP) The DPPH and ABTS free-radical scavenging activities of extracts were determined according to the methods of Brand-Williams et al. (10) and Re et al. (11), respectively. For the DPPH radical assay, 50 µL of extract was added to 2.95 mL of 0.1 mM DPPH diluted with 80% (v/v) methanol. After 30 min at room temperature in the dark, the absorbance was measured at 517 nm. To measure the ABTS radical scavenging activities, 100 mL of a 2.45 mM potassium persulfate solution was added to 100 mL of 7 mM ABTS and kept for 15 h at 30oC in the dark to produce the ABTS solution, which was then diluted using ethanol so that the absorbance ranged from 0.65 to 0.75 at 734 nm before analysis. An amount of 220 µL of each extract diluted with water was mixed with 2 mL of ABTS solution. After 5 min, the absorbance was measured at 734 nm. The DPPH and ABTS free-radical scavenging activities (%) were calculated as: DPPH or ABTS free-radical scavenging activity (%)=(1−sample absorbance/control absorbance)×100. The DPPH and ABTS values were calculated and expressed as the trolox equivalent antioxidant capacity (mM of trolox). A FRAP assay was carried out according to the method of Benzie and Strain (12). FRAP reagent was prepared by mixing 300 mM acetic acetate buffer (pH 3.6), 10 mM potassium persulfate, and TPTZ containing 40 mM HCl and 20 mM FeCl3 at a ratio of 10:1:1 (v:v:v) immediately before FRAP determination. The mixture was incubated for 15 min at 37oC. The water used to dilute the extracts and prepare the reagents for FRAP determination was deionized using chelex (2 g/L). Briefly, 270 µL of FRAP reagent was added to 30 µL of tea extract. After 15 min at 37oC, the absorbance was measured at 540 nm. A standard curve was prepared using different concentrations of Trolox. The FRAP value was expressed as the trolox equivalent antioxidant capacity. Cell culture and viability Murine RAW264.7 macrophages were obtained from the Korean Cell Line Bank (Seoul, Korea). Cells were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin and incubated at 37oC in 5% CO2. Cells in the culture medium (104 cells/well) were incubated in each well of a 96-well, flat-bottom plate in 5% CO2 at 37oC for 24 h. After incubation, the medium was replaced with new medium (100 µL) containing different concentrations of the freezedried extracts. The plates were then incubated for another 24 h. MTT (10 µL) was added to each well, followed by

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incubation at 37oC in 5% CO2 for 2 h. After removing the medium, DMSO was added to the wells to dissolve the formazan for 20 min at room temperature. The absorbance values of the plates were measured at 540 nm. Measurement of nuclear factor (erythroid-derived 2)like 2 (Nrf2) expression RAW264.7 cells were plated at a density of 1.5×106/mL cells in a 6×15 dish. After 24 h of incubation at 37oC in 5% CO2, followed by removing the medium, new medium containing 40 µg/mL of freeze-dried extracts was added to the dish, and after 4 h, 5 mM H2O2 was added to medium to cause oxidative stress for the cells. After the cells were incubated for another 4, 12, or 20 h, the medium in the dish was removed. The dish was washed with 1 mL of ice-cold PBS. Then, another 1 mL of ice-cold PBS was added to the dish and the cells were then scraped. The PBS with the cells was centrifuged (Micro 17R+; Hanil Electronics, Seoul, Korea) at 22,250×g for 1 min at 4oC, after which the PBS was removed, leaving the cells on the bottom. A lysis reagent was then prepared by mixing RIPA buffer (containing 1% of protease inhibitor cocktail) and PBS at a ratio of 1:1 immediately before cell lysis. Cells with the lysis reagent were vortexed for 10 s, incubated for 30 min on ice, and stored at 80oC for subsequent determination of the total protein content using the Lowry method (13). Cell lysates were adjusted to contain equal concentrations of proteins using RIPA buffer. Loading buffer was added to each lysate (1:4, v/v), which was heated in boiling water for 5 min. The samples were then separated using 10% SDS-polyacrylamide gel and transferred to nitrocellulose membrane. The membrane was blocked using a 5% skim milk solution and blotted with primary and secondary antibodies. Results were visualized using a chemiluminescent substrate kit. Relative protein levels were determined via scanning densitometry analysis using Quantity One software (Bio-Rad). Values are mean values (in arbitrary units) normalized to control values of at least 3 independent experiments. In vitro antioxidant activities SOD and GPx activities were measured according to the manufacturers’ instructions for the kits. Enzyme activity was expressed as unit/mg of proteins of the cell lysates. An amount of 1 unit of SOD activity was defined as the amount of enzyme needed to exhibit 50% dismutation of the superoxide radical. An amount of 1 unit of GPx activity was defined as the amount of the enzyme that oxidized 1.0 nmol of NADPH to NADP+ per min. The glutathione (GSH)/glutathione disulfide (GSSG) ratio was measured according to manufacturer’s instructions for the relevant kits. Statistical analysis All experiments were carried out in triplicate or quadruplicate and results were expressed as the

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mean±standard deviation (SD). Statistical analyses were performed using SPSS software (SPSS version 12.0; SPSS Inc., Chicago, IL, USA) using unpaired t-tests (p