Collection of astringent D. kaki
In this study, three local astringent D. kaki cultivars (Gojongsi, Danseongsi, and Bansi) were collected from the Hamyang, Hamyang, and Miryang regions respectively, during three different seasons (stage 1, August 19th; stage 2, October 14th; and stage 3, January 20th). The specimens were identified at Korea Forest Environment Research Institute at Gyeongsangnam, Korea and were deposited at a publicly available herbarium in Korea Institute of Oriental medicine and given voucher specimen numbers KIOM201501015023- KIOM201501015025. The astringent D. kaki samples were collected according to regional characteristics and maturity and the stalks cut and classified as immature stage (collected from August to September), mature stage (collected from October to November), and dried stage (collected from December to January).
Extraction from stalks of astringent D. kaki
The 9 samples used in this experiment were 3 samples of immature stalks, 3 samples of mature stalks, and 3 samples of dry stalks obtained from Hamyang Gojongsi, Hamyang Danseongsi, and Miryang Bansi. The ethanol and methanol reagents used for the extraction process were purchased as high-performance liquid chromatography (HPLC)-grade (JT Baker Inc., Phillipsburg, NJ, USA). The solvents and samples were filtered with a 0.2 μm membrane filter (PALL Corporation, Ann Arbor, MI, USA) before use. For each of the 9 samples, 10 g of the dried ground sample was extracted using maceration method with 250 mL 70% ethanol for 72 h. The extracts were filtered using filter paper (ADVANTEC, 110 mm, Toyo Robni kaisha, Japan) and concentrated under a vacuum reduced pressure at 40 °C, RPM 70 using EYELA N-1200B (Tokyo Rikakikai Co. Ltd., Japan) efficient rotary evaporator. The concentrated extract was then vacuum dried. 50 mg/mL of solution was then obtained by separately dissolving each of the extracts from the different samples in 80% methanol. For HPLC analysis, samples were filtered using a 0.45 μm membrane filter (PALL Corporation, Ann Arbor, MI, USA) prior to injection and analysis.
Analysis of tannic acid contents using HPLC
An HPLC system (Alliance® HPLC, Waters Corporation, Manchester, UK) was used in this study. The column was a Phenomenex Luna C18 (2) (250 × 4.6 mm, 5 μm), the column temperature was 40 °C, and the sample temperature was 25 °C. The mobile phase consisting of HPLC grade water with 0.1% acid and HPLC grade Methanol with 0.1% acid was used. The calibration curves were analyzed using 5 concentrations (25, 50, 100, 150, and 200 μg/mL) of tannic acid standard (Sigma-Aldrich Co., Inc. FLUKA, Trace SELECT, USA). The flow rate of the mobile phase was set to 1.0 mL/min, the injection amount of the standard product was set to 10 μL, while the injection amount of the 9 samples was set to 20 μL. The analysis time for all samples was set to 30 min in total. The analytical wavelength was measured at 190 to 400 nm and analyzed at 220 nm. The UV spectrum of each component was measured. All HPLC-related experiments were repeated three times and the content analysis was completed with an average value.
Preparation of RAW 264.7 cells and cell viability assay (CCK-8 assay)
RAW 264.7 macrophages were cultured in RPMI 1640 medium (WelGENE, Daegu, Korea) containing 10% fetal bovine serum (FBS) and 1% antibiotics, at 37 °C in a 5% CO2 atmosphere. RAW 264.7 macrophages were treated with various stalk extract concentrations (0, 10, 50, 100, 200, 500, and 1000 μg/ml) with different maturities for 24 h. The proliferation of RAW 264.7 macrophage cells on the substrates was determined by cell counting kit assay (CCK-8, Dojindo, Kumamoto, Japan). All samples were placed in a 96-well plate and seeded with a density of 5 × 104 cells/well. Absorbance was measured at 450 nm using an ELISA plate reader.
NO assay and immunoblotting using western analysis
The NO level produced from the RAW 264.7 cell lines was determined using a commercially available NO detection kit (iNtRON, Inc., Seoul, Korea). RAW 264.7 cells were pretreated with stalk extracts (at 0, 10, 20, 50, and 100 μg/ml) for 1 h, followed by incubation with lipopolysaccharides (LPS, 1 μg/ml) for 24 h. All samples were placed in a 48-well plate and seeded with a density of 1 × 105 cells/well. After incubating for 24 h, 100 μL of supernatant was collected and added to wells in triplicate; 50 μL of N1 buffer was added to each well, and the plate was incubated at room temperature for 10 min. Then, 50 μl of N2 buffer was added and the final reaction was incubated at room temperature for 10 min. The absorbance was measured at 540 nm using a multi-plate reader (Lambda Bio-20; Beckman Coulter, Inc., Brea, CA, USA).
The nitrite concentration of the supernatant was determined using a nitrite standard curve. For the western blot analysis, RAW 264.7 cells were pretreated with stalk extracts (at 0, 10, 20, 50, and 100 μg/ml) for 1 h, followed by incubation with LPS (1 μg/ml) for 24 h. All samples were placed in a 6-well plate and seeded with a density of 1 × 106 cells/well. Cells were harvested with ice-cold phosphate-buffered saline (PBS), and lysed in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% v/v IGEPAL® CA-630, 1 mM PMSF, 1 mM sodium fluoride, and 10 μg/ml aprotinin and leupeptin). The cell lysates were collected by centrifuging at 13,000 rpm for 15 min at 4 °C. The protein concentration of the collected supernatants was determined using a BCA™ protein assay kit (Pierce, Rockford, IL, USA). All lysates were loaded with the same amount of protein on SDS-PAGE gel. Proteins were then transferred to a PVDF membrane (Millipore, Bedford, MA, USA). After blocking with 5% w/v skimmed milk in 1xPBST at 27 °C for 40 min, the membrane was incubated for 2 h with specific primary antibodies (1/1000 dilution in 5% w/v skimmed milk in PBST) and then incubated with the HRP-conjugated secondary antibodies (1/2500 dilution in 1x PBST) for 1 h at room temperature.
RNA isolation and quantitative real-time PCR
Total RNA was extracted from the RAW 264.7 cell lines using an RNeasy Mini Kit (Qiagen, Gaithersburg, MD, USA) according to the manufacturer’s instructions. Cells were plated at a density of 1 × 106 cells/well in a 6-well plate, and pretreated with stalk extracts at concentrations of 0, 10, 50, and 100 μg/ml for 1 h. Thereafter, cells were stimulated with LPS (1 μg/ml) for 24 h. After 24 h of incubation, the cells were washed with cold PBS and collected. Total RNAs were reverse-transcribed to first strand cDNAs using a ReverTra Ace® qPCR RT Kit (Toyobo Co, Osaka, Japan) and analyzed by real time PCR (Takara Bio Inc., Shiga, Japan) using an SYBR Green PCR Master Mix (Takara Bio Inc., Shiga, Japan) with specific primers. The mRNA expression level was calculated using GAPDH as a control. The primer sequences were as follows: inducible nitric oxide synthase (iNOS), forward 5′- GGCAGCCTGTGAGACCTTTG-3′ and reverse 5′- GCATTGGAAGTGAAGCGTTTC-3′; GAPDH, forward 5′-ATGCCTCCTGCACCACCA-3′ and reverse 5′- CCATCACGCCACAGTTTCC-3′.
Confocal microscopy for p65 localization
The effect of the stalk extracts on the nuclear translocation of nuclear factor κB (NF-κB) p65 was evaluated by immunofluorescence. For these experiments, RAW 264.7 cells were pretreated with 100 μg/ml stalk extracts for 1 h and harvested by treatment with LPS (1 μg/ml) for 30 min. Cells were plated on round glass cover slips in 12-well plates for 10 min. The samples were then washed with cold PBS and fixed in 4% formaldehyde for 15 min at room temperature, followed by permeabilization with 0.2% Triton X-100 in PBS for 15 min at 4 °C. Cells were blocked for 1 h with 5% BSA in PBS and incubated with anti-p65 primary antibody at 4 °C overnight. After three washes with PBS, the cells were incubated with Alexa Fluor 546-conjugated goat-anti rabbit IgG (Invitrogen, Carlsbad, California, USA) for 1 h at room temperature, and then washed again with PBS. The images were captured using an LSM510 confocal microscope (Carl Zeiss, Gottingen, Germany).
Statistical analysis
Data collected from each group (experimental and control) were expressed as mean ± SD. Unpaired t-tests (in the Prism program, Graph Pad Software, San Diego, CA; two-tails, P < 0.05) were used to analyze the differences between experimental and control groups.