Male ICR mice (MJ Co., Seoul, Korea) weighing 20~25 g were used for all the experiments. Animals were housed 5 per cage in a room maintained at 22±0.5℃ with an alternating 12 hr light-dark cycle. Food and water were available ad libitum. The animals were allowed to adapt to the laboratory for at least 2 hr before testing and were only used once. Experiments were performed during the light phase of the cycle (10:00~17:00).

Oral administration was performed with gage in a volume of 500µl/ 25 g bodyweight.

Opuntia ficus-indica var. saboten (300 g) was dissolved in 80% ethanol (1,500 ml) and extracted as refluxing for 3 hours, and then the extract was filtered for obtaining A. This process was repeated again once to obtain B from residue. A and B were mixed. This mixture was decompressed and dried for using as extract of Opuntia ficus-indica var. saboten.

Three animals of each group were dissected for Northern blot analysis. Every result was confirmed by three times independent experiments. Total cellular RNA was extracted from dissected pituitary gland and hypothalamus tissues using a rapid guanidine thiocyanate-watersaturated phenol/chloroform extraction procedure and subsequent precipitation with acidic sodium acetate (Chomczynski and Sacchi, 1987). Total cellular RNA in the aqueous phase was precipitated with ice-cold isopropyl alcohol. Isolated RNA samples were subjected to spectrophotometric analysis at 260 nm and 280 nm.

The cRNA probe for POMC (Civelli et al., 1982) was synthesized in vitro from linearized expression vectors which contained SP6 or T7 viral promoter. One microgram of linearized plasmid was mixed with RNA labeling mixture that contained ATP, CTP, GTP and DIG-labeled-UTP, transcription buffer, and SP6 or T7 RNA polymerase. After incubation at 37℃ for 2 h, the mixture was co-incubated with DNase I (RNase free) at 37℃ for 15 min, precipitated in ethanol containing lithium chloride at 70℃ for 30 min, and washed with 70% chilled ethanol.

Isolated RNA samples were dissolved in 40~50 ml water and a 400X diluted RNA solution was subjected to spectrophotometric analysis at 260 and 280 nm. Ten micrograms of total RNA and an equal volume of RNA loading buffer (50% glycerol, 1 mM EDTA) were denatured in 65℃ for 10 min and subsequently cooled on ice. The denatured RNA samples were electrophoresed on 1% agarose-formaldehyde gels under 6070 V and transferred to nylon hybond-N hybridization membrane sheets (Amersham, Buckinghamshire, UK). After UV cross-linking, the membranes were pre-hybridized at 68℃ for at least 1 h in a prehybridization buffer (5X SSC, 50% formamide, 0.02% SDS, 0.1% sodium N-lauroyl sarcosine, and 2% blocking reagent). The DIG-labeled POMC probe was added to the prehybridization buffer containing 50% formamide. The membranes were incubated overnight at 68℃ in a shaking waterbath, and washed twice for 10 min per wash in 2X SSC and 0.1% SDS at room temperature. Then the membranes were washed twice for 15 min per wash 0.1X SSC and 0.1% SDS. After equilibrating the membranes in Buffer I (100 mM maleic acid [pH 7.5] and 150 mM NaCl) for 1 min, the membranes were gently agitated in Buffer II (1% blocking reagent in Buffer I) for 30~60 min. The membranes were hybridized with the diluted anti-DIG-alkaline phosphatase (1:10,000 [75 mU/ml]) in Buffer II for 30 min. After washing the membranes twice for 15 min per wash in 0.3% Tween 20 (in Buffer I), the membranes were equilibrated in Buffer III (100 mM Tris-HCl [pH 9.5], 100 mM NaCl, and 50 mM MgCl₂) for 2 min. Diluted disodium 3-[4-methoxyspiro (1,2-dioxietane-3,2'-[5'-chloro] tricycle [3.3.1.1.3,7decan]-4-yl) phenyl phosphate (CSPD) (Boehringer Mannheim) (1:100 dilution in Buffer III) was spread over the surface of the membranes. After incubation of the membranes at 37℃ for 15~20 min, the membranes were exposed to Hyperfilm-ECL (Amersham) for detection of the chemiluminescent signal. For rehybridization, blots were washed for 20 min at room temperature in sterilized (Millipore) water. The membranes were washed overnight at 65℃ in 50 mM Tris-HCl (pH 8.0), 50% dimethylformamide and 1% SDS to remove the hybridized probe and rehybridized to the DIG-labeled rat cyclophilin cRNA probe, a gene encoding peptidyl-prolyl cis-trans isomerase which is constitutively constitutively expressed in most mammalian tissues with the exception of skeletal muscles (Danielson et al., 1988; Takahashi et al., 1989).

The blood sample was collected three times (30, 90 and 180 min) or 90 min after oral administration of the diluted-extract (1,000 and 2,000 mg/kg) or kaempferol (10, 25 and 50 mg/kg) or quercitrin (10, 25 and 50 mg/kg), and then the serum was obtained by centrifuge (4℃, 1,600×g, 15 min). We observed the serum β-endorphin level with ELISA kit (Phoenix pharmaceutical, INC). The immunoplaste in this kit is pre-coated with secondary antibody and nonspecific binding sites are blocked. The secondary antibody can bind to the Fc fragment of the primary antibody (peptide antibody) whose Fab fragment will be competitively bound by both biotinylated peptide and peptide standard or targeted peptide in sample. The biotinylated peptide is able to interact with streptavidin-horseradish peroxidase (SA-HRP) which catalyzes the substrate solution composed of 3,3',5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide to produce a blue colored solution. The enzyme-substrate reaction is stopped by hydrogen chloride (HCL) and the solution turns to yellow.

Stress treatment procedure has been described previously (Kim and Han, 2006). To deliver restraint, 8-week-old mice weighing 22~23 g were individually placed head-first into a well-ventilated 50-ml conical tube and plugged with a 3 cm long middle tube and finally the cap of the 50 ml tube. Mice were not able to move forward or backward in this device. This restraint stress was delivered to animals at set times daily from 10 AM. Control mice remained in their original cages and were left undisturbed in this home environment. After restraint stress administration, restrained animals were returned to their normal home environments by housing them in pairs. This procedure was repeated for 14 days unless otherwise indicated. After restraint, animals were kept in their normal home cages for additional fourteen days without any further stress, but with drugs. Mice were fed with a diet supplemented for 14 days and during the behavioral test period with kaempferol or quercitrin (Sigma) (30 mg/kg/day). Behavioral tests were performed in the sequence of TST, FST, and rota-rod tests.

Mice were suspended individually by their tail from a metal rod. The rod was fixed 50 cm above the surface of a table covered with soft cloth in a sound-isolated room. The tip of the tail was fixed using adhesive Scotch tape; the duration of the test was 6 min. The immobility time was determined by an observer, using a stopwatch, who was unaware of the strain (Steru et al., 1985; Bilkei-Gorzo et al., 2002).

Mice were placed in a Plexiglas cylinder (height: 27 cm, diameter: 15 cm) containing water at 24℃ and a depth of 14 cm so that they could neither escape nor touch the bottom. Mice were subjected to a 15-min pre swim. After a 15-min test, the animals were dried quickly with a towel and returned to home cages. One day later, mice were forced to swim for 6 min. The animals were habituated for the first 1 min and behavior was noted over the next 5 min. Immobility time was defined as the summed time during which animals remained floating with all limbs motionless. This scoring method has been described previously (Porsolt et al., 1977; Armario et al., 1988; 1991; Kim and Han, 2006).

Mice were evaluated on their motor activity using Rota-rod tested at 10 or 15 rpm. The time mice remained on the Rota Rod was recorded. Maximum time was adjusted to 5 min.

Kaempferol and quercitrin were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Extract of Opuntia ficus-indica var. saboten and quercitirin were dissolved in saline. Kaempferol was prepared following steps: (A) 1 g of kaempferol was dissolved in 0.5 ml of ethanol plus 0.5 ml of polyethylene glycol 400. (B) Separately, 100 mg of sodium carboxymethylcellulose was dissolved in 9 ml of distilled water. (C) Finally, Solution (A) and Solution (B) were vigorously mixed. This solution excluding kaempferol was used as vehicle control. All drugs were prepared just before use.

The Fig. 1 is structures of kaempferol and quercitrin.

ELISA data was presented as the mean±SEM. The statistical significance of differences between groups was assessed with one-way ANOVA with Bonferroni's post-hoc test using GraphPad Prism version 4.0 for Windows XP (GraphPad Software, San Diego, CA, USA); p<0.05 was considered significant. Behavior test data two-sample comparisons were carried out using the Student's t-test, and multiple comparisons were made using oneway ANOVA, followed by the Newman-Keuls multiple range test. All data are presented as mean±SEM, and statistical significance was accepted at the 5% level unless otherwise indicated.

Extract of Opuntia ficus-indica var. saboten (1 or 2 g/kg) was administered orally in mice, and hypothalamus was dissected or blood sample was collected at 30, 90 and 180 min after administration of extract of Ofuntia ficus-indica var. saboten. The control group was administered 0.9% normal saline orally instead of the extract. In the northern-blot assay, we found that POMC mRNA was increased in extract-treated group compared to control group at all times (Fig. 2A). In ELISA method, we found that plasma β-endorphin level was increased by extract of Opuntia ficus-indica var. saboten (Fig. 2B). In 30 min after oral administration, plasma β-endorphin level was increased in extreat-treated group of 2,000 mg/kg compared to control group. In 90 min after oral administration, plasma β-endorphin level was increased slightly in both extract-treated group, but not significantly. In 180 min after oral administration, plasma β-endorphin level was increased significantly in dose dependent manner in extract-treated group (1 and 2 g/kg) compared to control group.

Kaempferol or quercitrin (10, 25 and 50 mg/kg) was administered orally in mice, and blood sample was collected after 90 min. In ELISA method, we found that plasma β-endorphin level was increased by kaempferol and quercitrin (Fig. 3). Plasma β-endorphin level was increased significantly in dose dependent manner in kaempferol or quercitrin treated group compared to control group.

After restraint stress (2 hrs/day for 14 days), animals were kept in cage for another 14 days without any further stress, bet with drugs. Mice were fed with a diet supplemented for 14 days and during the behavioral test period with kaempferol or quercitrin (30 mg/kg/day). Behavioral tests were performed in the sequence of TST, FST, and rota-rod tests tests as described below (Fig. 4A). In TST test, the immobility time was increased in chronically restraint stress group compared to control group. Both kaempferol and quercitrin attenuated the total immobilization time to the control level. (Fig. 4B). In FST test, the immobility time was increased in chronically restraint stress group compared to control group, similar to the result shown in TST test. Both kaempferol and quercitrin attenuated the total immobilization time to the control level (Fig. 4C). In rota-rod test, the time of permanence in chronically restraint stress group was decreased significantly compared to control group during at the speed of 15 rpm. Both kaempferol and quercitrin reversed the time of permanence to the control level. However, during at the speed of 10 rpm, no different response was observed in all groups (Fig. 4D).