Peanut (PoongAn cultivar) and its sprout were freeze-dried, powdered, and extracted with 40 volumes of 80% (v/v) ethanol solution as a solvent, along with agitation at 70℃ for 90 min. Extracts were then filtered, concentrated using a rotary evaporator, and freeze-dried prior to use as a test sample. Extraction yields were 2.7 and 23.4% for peanut and its sprout, respectively. Loperamide (Youngil Pharmaceutical Co.) was used to induce constipation at a dose of 2 mg/kg bw per day.

Male SD rats (8-weeks-old) weighing 200-250 g were purchased from Samtako Co. Ltd (Osan, Korea). Animals were tested for their general health condition and allowed to acclimate for 1 week before use. All animals were housed at a controlled room temperature and humidity (20-24.0℃, RH 50 ± 5%) under a 12/12 h light/dark cycle. Food pellets (Samtako Co. Ltd., Osan, Korea) prepared based on AIN-76 and tap water were provided ad libitum. All animal experiments were approved by the "Animal Ethic Committee of Wonkwang University (permission number WKU12-47)".

Fifty SD rats were divided into five groups with 10 animals per group: (1) normal, (2) negative control, (3) PS (100 mg/kg bw), (4) PS (300 mg/kg bw), (5) PS (1 g/kg bw). Constipation was induced by force-feeding with loperamide (2 mg/kg bw, Youngil Pharmaceutical Co., Seoul) twice a day for 7 days after orally administration of PSE for 4 weeks. Before induction of constipation, diet and water intakes were measured once a day.

The frequency, weight, and water content of stools from each rat were measured at 1-week intervals before constipation induction as well as every day after constipation induction. The water content and dry weight of stools were measured once a day. The moisture content of stools was measured by drying stools at 70℃ for 24 h.

Rats were maintained on experimental diets for 4 weeks, after which constipation was induced through oral forced feeding of loperamide (Young-Il Pharmaceuticals Co. Ltd, 2 mg/kg bw) twice a day for 7 days.

Total phenolics were determined using Folin-Ciocalteu reagent [9]. Briefly, 30 mg of sample was extracted for 18 h with 50 mL of 75% ethanol at room temperature on an orbital shaker set at 200x rpm. The mixture was filtered through Whatman filter paper and used for total phenolics assay at a total volume of 50 mL. Four hundred microliters of extract was mixed with 50 µL of Folin-Ciocalteu reagent (previously diluted 5-fold with distilled water) and 100 µL of saturated sodium bicarbonate solution and then allowed to stand at room temperature for 1 h. The absorbance was measured at 750 nm. Results are expressed as gallic acid equivalents.

The concentrations of resveratrol and polydatin were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) as previously described, with some modification [10]. In brief, 100 mg of sample was dissolved in dimethylsulfoxide. This solution was diluted with methanol to obtain a final concentration of 10 mg/mL, after which 0.5 µL of the solution was analyzed in a Thermo TSQ Vantage Triple-stage quadrupole mass spectrometer (ThermoFishcher Scientific, San Jose, CA, USA) equipped with an electrospray ionization interface. Resveratrol and polydatin were separated on a Luna C18 column (2.0 mm i.d. × 100 mm, 2.1 µm particle size; Phenomenex). Gradient elution was carried out with acetonitrile containing 0.1% formic acid as mobile phase A and water containing 0.1% formic acid as mobile phase B. A non-linear gradient profile was applied as follows: 0-2 min, 20% A; 2-5 min, 20-60% A; and 5-8 min, 60% A. The column was re-equilibrated for 5 min in order to return to 20% A. The mobile phase was eluted using a Thermo Accela HPLC system (Thermo Fishcher Scientific, San Jose, CA, USA) at a flow rate of 0.2 mL/min. The mass spectrometer was operated in negative ionization mode. The operating conditions were as follows: capillary temperature, 350℃; vaporizer temperature, 300℃; and ionization voltage, 3,000 V. The collision gas was nitrogen at a nominal pressure of 1.5 mTorr, and the collision energy was set to 20 eV. Quantitation was performed by selected reaction monitoring (SRM) of the precursor ion as well as the related product ion for resveratrol and polydatin using an external standard to establish peak area ratios. The mass transitions used for quantitation of resveratrol and polydatin were m/z 227→143 and 389→227, respectively. The retention times for resveratrol and polydatin were 6.1 and 4.6 min, respectively.

Values are presented as the mean ± SEM (standard error of the mean). Measurement data were analyzed by one-way ANOVA, followed by Duncan's multiple range post hoc test if overall differences were significant (P < 0.05). All statistical analyses were performed using SPSS 12.0 statistical software (SPSS Inc., Chicago, IL), and a difference was considered significant at P < 0.05.

The dried peanut sprout was powdered and subjected to extraction with 80% (v/v) aqueous ethanol solvent, with the yield of 20.3 ± 3.1% on a dry basis. The content of total phenolics present in peanut sprout extract was 390 ± 20 mg/g (Table 1). This value is much higher than the total phenolic content of peanut (1.52 mg/g), which is consistent with the other report [5].

Body weight changes among experimental groups were not significantly different during the 4-week experimental period. There were no significant differences in diet or water intake among the experimental groups both before and after constipation induction.

To examine the laxative effects of PSE on frequency and wet weight of stools in normal male rats, PSE (100, 300, and 1,000 mg/kg) was orally administered once daily for 4 weeks. The frequency and wet weight of stools were measured every week before constipation induction and daily after constipation induction. Stool frequency was not significantly different among the experimental groups before constipation induction. However, stool frequency decreased after constipation induction in all groups. Whereas the control group showed the lowest fecal number, rats administered 300 and 1,000 mg PSE/kg bw showed gradual increases in stool frequency starting from the first day of constipation induction (Fig. 1).

Meanwhile, fecal weight was not significantly different among the experimental groups before constipation induction. However, fecal weight significantly increased in groups fed a diet containing 300 or 1,000 mg PSE/kg bw (Fig. 2). The fecal water contents of these groups were significantly higher than that of the control (Fig. 3). However, fecal water content in the colon was not significantly different among the experimental groups.

Although there was no significant difference in small intestinal length among the experimental groups, the constipation-induced group fed a diet of 300 mg PSE/kg bw showed faster intestinal movement of BaSO₄ than the negative control, whereas groups fed a diet of 100 or 1,000 mg PSE/kg diet showed no difference in transit time of the indicator (Fig. 4). However, colon length was not statistically different among the experimental groups although it tended to be longer in the 1 g PSE diet group than the others. Further, there was no significant difference in stool number among the experimental groups.