Materials
2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS), potassium persulfate, vitamin C, α-tocopherol, catechin, 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), sodium bicarbonate, streptomycin, ferrous sulfate (FeSO4), hydrogen peroxide (H2O2), dimethyl sulfoxide (DMSO), penicillin, amyloid β protein (Aβ25-35, A4559), 3-[4,5-dimethythiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay kit, lactate dehydrogenase (LDH) assay kit were purchased from Sigma Chemical Co (St. Louis, MO, USA). All solvents used were of analytical grade. RPMI 1640 medium and fetal bovine serum was obtained from Gibco BRL (Grand Island, NY, USA).
Sample preparation
Aged garlic (Allium sativum) was supported by Namhae Garlic Research Institute (Namhae, Korea), in December 2010 and was authenticated by Dr. JH Shin (Namhae Garlic Research Institute). Extraction of aged garlic was prepared by pouring 80% ethanol 3.5 L into the bottle containing 1 kg of aged garlic, and the mixture was steeped at room temperature (20°C) for 7 days[13], and filtrated. The extract was then concentrated under reduced pressure at 60°C using vacuum rotary evaporator. Ethanol extract was sequentially partitioned with n-hexane, chloroform, ethyl acetate and water. Four solvent fractions were collected and concentrated with vacuum rotary evaporator. The fractions were frozen and lyophilized. Finally, the fractions were placed in a glass bottle and stored at -20°C until used.
PC12 cell culture
Neuron-like PC12 cell line was derived from a transplantable rat pheochromocytoma. The cells respond reversibly to nerve growth factor (NGF) by induction of the neuronal phenotype. Cells (KCLB 21721, Korea Cell Line Bank, Seoul, Korea) were propagated in RPMI 1640 medium containing 10% fetal bovine serum, 50 units/mL penicillin and 100 μg/mL streptomycin.
ABTS radical-scavenging activity
ABTS was dissolved in water to make a concentration of 7 mM. ABTS was produced by reacting the ABTS stock solution with 2.45 mM potassium persulfate (final concentration) and allowing the mixture to stand in the dark at room temperature for 12-16 h before use. For the study of samples, the ABTS stock solution was diluted with phosphate-buffered saline 5 mM, pH 7.4 to an absorbance of 0.70 at 734 nm. After the addition of 980 μL of diluted ABTS to 20 μL of sample, the absorbance reading was taken 5 minutes after the initial mixing[14]. This activity is given as percent ABTS scavenging that is calculated as:
Malondialdehyde (MDA) assay using mouse whole brain homogenates
This assay was carried out to the method described by Jeong et al[15]. The brain of young adult male institute of cancer research (ICR) mice were dissected and homogenized in ice-cold Tris–HCl buffer (20 mM, pH 7.4) to produce a 1/10 homogenate. The homogenate was centrifuged at 12,000 × g for 15 minutes at 4°C. 1 mL aliquots of the supernatant were incubated with the test samples in the presence of 10 μM FeSO4 and 0.1 mM vitamin C at 37°C for 1 h. The reaction was terminated by addition of 1.0 mL trichloroacetic acid (TCA) (28%, w/v) and 1.5 mL thiobarbituric acid (TBA) (1%, w/v) in succession, and then the solution was heated at 100°C. After 15 minutes, the color of the MDA-TBA complex was measured at 532 nm. (+)-Catechin, a well-known antioxidant, was used as a positive control. The inhibition ratio (%) was calculated as follows:
Measurement of cellular oxidative stress
Levels of cellular oxidative stress were measured by 2',7'-dichlorofluorescein diacetate (DCF-DA). PC12 cells were pretreated with various concentrations of aged garlic for 48 h. Freeze-dried ethyl acetate fraction from aged garlic of various concentrations was dissolved in deionized distilled water. Cells were then treated with or without Aβ25-35 for 2 h. At the end of the treatment, cells were incubated in the presence of 50 μM DCF-DA for 50 min. After incubation, 2',7'-dichlorofluorescein (DCF) was quantified using a fluorometer (infinite F200, TECAN, NC, USA) with a 485 nm excitation filter and a 535 nm emission filter. The results were expressed as percent relative to the oxidative stress level of the control cells, which was set to 100%[3].
Determination of cell viability
MTT reduction assay was determined using the in vitro toxicology assay kit. Neuronal PC12 cells were plated at a density of 105 cells/well on 96-well plates in 100 μL of RPMI. Synthetic Aβ25-35 was prepared at a concentration of 1 mM in PBS. The cells were pre-incubated with various fractions obtained from aged garlic for 48 h before adding of 80 μM Aβ25-35. The cells were treated with or without Aβ for 3 h. MTT reduction was initiated by adding 10 μl MTT stock solution per well. Plates were incubated at 37°C. After 3 h incubation, the reaction was stopped by adding 100 μl of DMSO. The amount of cellular MTT formazan product was determined by measuring absorbance using a microplate reader (680, Bio-Rad, Tokyo, Japan) at a test wavelength of 570 nm and a reference wavelength of 690 nm[16].
Neuronal PC12 cells were precipitated by centrifugation at 250 × g for 4 minutes at room temperature, 100 μL of the supernatants was transferred into new wells, and LDH was determined using the in vitro toxicology assay kit. Damage of the plasma membrane was evaluated by measuring the amount of the intra-cellular enzyme LDH released into the medium[3].
Animals
ICR mice (male, 4 weeks old) were obtained from Samtako Co. (Osan, Korea). The mice were housed two per cage in a room maintained with a 12 h light–dark cycle, 55% humidity, and 23-25°C temperature. All animals and experimental procedures were approved by the guidelines established by the 'Institutional Animal Care and Use Committee (IACUC) of Gyeongsang National University (certificate: GNU-120409-M0009), and in accordance with the Ethical Committee of Ministry of Health and Welfare, KOREA. Freeze-dried ethyl acetate fraction from aged garlic extract was mixed in water at concentrations of 5, 10, and 20 mg/kg of body weight. Mice were divided into five groups: (I) control (n = 8), (II) Aβ (n = 8), (III) Aβ + sample 5 mg/kg (n = 8), (IV) Aβ + sample 10 mg/kg (n = 8) and (V) Aβ + sample 20 mg/kg (n = 8). The mice were allowed free access (ad libitum) to water, in which the fraction had been dissolved for 3 weeks. Aβ25-35 was administered by intracerebroventricular (ICV) injection to induce memory impairment. Aβ was dissolved in 0.85% (v/v) sodium chloride solution. Each mouse was injected at the bregma with a Hamilton microsyringe (depth, 2.5 mm; injection volume, 5 μL; dose, 410 pmol per mouse)[3].
Y-maze test
Recording spontaneous alternation behavior in the Y-maze test was used to assess the immediate working memory performance. The Y-maze test was performed 2 days after the ICV Aβ injection. The maze was made of black-painted plastic, and each arm of the maze was 33 cm long, 15 cm high and 10 cm wide, and was positioned at a constant angle. Each mouse was placed at the end of one arm and allowed to move freely through the maze for 8 min. The series of arm entries was recorded visually, and arm entry was considered to have been completed only when the hind paws of the mouse were placed completely in the arm of the maze. Alternation is defined as successive entries into the three arms in an overlapping triplet set. The percentage alternation was calculated as the ratio of actual to possible alternation (defined as the total number of arm entries minus two), multiplied by 100[3, 17].
Passive avoidance test
The passive avoidance test box was divided into two compartments, one illuminated and one dark, with a grid floor. The mice were allowed to move freely through a circular tunnel between the two compartments. During the training trial (3 days after the ICV Aβ injection), each mouse was placed in the lighted compartment: as soon as it entered the dark compartment, an inescapable electric shock was provided (0.5 mA, 1 sec). In the probe trial (4 days after the ICV Aβ injection), which was given 1 day after the training trial, the mouse was again placed in the lighted compartment, and the time until it re-entered the dark compartment was measured (the step-through latency maximum testing limit was 300 sec)[3].
Identification of active compounds
Sulfur compounds in fractions obtained from aged garlic were measured at 195 nm by a photo diode array detector (Ultimate 3000 series, Dionex, CA, USA). Separation was achieved with a Shiseido C18 column (250 mm × 4.6 mm id, 5 μm, Shiseido Co., Tokyo, Japan). The mobile phase consisted of water (A) and acetonitrile (B). The linear gradient as reverse phase condition was 0-60% (B) in 20 min, at a flow rate of 0.5 mL/min and the injection volume was 20 μL. Main sulfur compounds in aged extract were identified by comparison of their retention time (RT) values and UV spectra of known standards.
Statistical analysis
All data were expressed as mean ± SD. Each experimental set was compared with one-way analysis of variance (ANOVA) and Duncan’s multiple-range test (P < 0.05) using SAS program (SAS Institute, Cary, NC, USA).