A sample of D. candidum was purchased from Shanghai Pharmacy Co., Ltd. (Shanghai, China). The D. candidum was stored at −80°C and freeze-dried to produce a powder. A twenty-fold volume of boiling water was added to the powdered sample and the sample was extracted twice by stirring overnight. The aqueous extract was evaporated and concentrated using a rotary evaporator (N-1100; Eyala, Tokyo, Japan).

Seven-week-old female ICR mice (n=100) were purchased from the Experimental Animal Center of Chongqing Medical University (Chongqing, China). The mice were maintained in a temperature- and humidity-controlled (temperature 25±2°C, relative humidity 50±5%) facility with a 12-h light/dark cycle and free access to a standard rat chow diet and water. All experimental procedures carried out in the present study were approved by the Ethics Committee of Chongqing Medical University

Dried D. candidum was refluxed and extracted 3 times with a 10-fold quantity of ethyl acetate. An ethyl acetate extract was obtained after 1 h for every reflux extraction and concentrated by evaporation under reduced pressure. The combined ethyl acetate extracts were extracted with anhydrous ethanol 3 times. The ethanol extract was suspended in water, and extracted by petroleum ether, chloroform and butanol extraction, respectively. The ethyl acetate extract was treated by gradient elution in a silica gel (Shanghai Xinhuo Silica Gel Factory, Shanghai, China) column with a petroleum ether-ethyl acetate solvent system. Then, the chloroform extract was treated by gradient elution in a silica gel column with a petroleum chloroform-methanol system. The butanol extract was treated with water with ultrasonic irradiation, and the extracting solution was isolated after filtering. The extract was then eluted using an HP2MGL macroporous resin (Mitsubishi Chemical Corporation, Tokyo, Japan) column with water, 10% ethanol, 30% ethanol and 60% ethanol, respectively. After elution, the various solvents contained different compounds, and their compositions were determined by NMR (Varian Inova 400; Varian Inc., Palo Alto, CA, USA). NMR was conducted using the following settings: ¹H frequency, 300 MHz; temperature, 25°C; pulse length, 8 μsec; spin speed, 20 Hz; and scan number, 64 times. The ¹H NMR spectra were recorded using a standard high-resolution magic angle spinning probe with magic-angle gradient.

To investigate the preventive effects of D. candidum against activated carbon-induced constipation, the animals were divided into five groups with 20 mice in each. The experimental design was as follows: the normal and control groups were fed a normal diet for 9 days; the high and low concentration D. candidum groups received 400 and 200 mg/kg body weight of the aqueous extract orally in a volume of 2 ml; and the drug cure group mice were treated with a 100 mg/kg dose of bisacodyl dissolved in water for 9 days. The control and treatment groups received an oral administration of activated carbon (0.2 ml of 10% activated carbon, w/w; activated carbon dissolved in 10% arabic gum) at 18:00 from the sixth to ninth day to induce constipation (11). The body weight, stool weight and stool moisture content were determined at 09:00 every day.

This measurement was performed to determine whether the prokinetic action of D. candidum was capable of propagating a prokinetic signal along the length of the GI tract. The excreted fecal pellets of individual mice were collected daily at 09:00 for the duration of the experiment. The total number, weight and water content of the pellets were determined. The water content was calculated as the difference between the wet and dry weight of the pellet. After 16 h, the mice in the control and treatment groups received 10% activated carbon and the normal group was administered 10% arabic gum by intragastric gavage. The animals were then placed individually in small transparent cages and allowed access to food and tap water ad libitum. The length of time from carbon meal administration to the appearance of darkened feces was recorded. Feces were collected, counted, weighed and their water content was evaluated.

Mice were fasted for 16 h from the ninth day at 18:00; however, they were not deprived of water. After 16 h, the mice in the control and treatment groups received an oral administration of 10% activated carbon while the mice in the normal group received an oral administration of 10% arabic gum. After 30 min, the mice were sacrificed by cervical dislocation under anesthesia with diethyl ether. A total of 10 mice in each group were dissected and the small intestine from the pylorus to the cecum was carefully removed. The GI transit of each mouse was calculated as the percentage of the distance traveled by the activated carbon meal relative to the total length of the small intestine. The following equation was used to calculate GI transit: GI transit (%) = distance traveled by the activated carbon/total length of the small intestine ×100. The remaining 10 mice of each group were used to measure the time until the first defecation of the black stool following the oral administration of 10% activated carbon.

The levels of MTL, Gas, ET, SS, AChE, SP and VIP in the serum were determined using radioimmunoassay kits (Beijing Puer Weiye Biotechnology Co., Ltd., Beijing, China). The serum was collected from the heart following surgery.

Data are presented as mean ± standard deviation (SD). Differences between the mean values for individual groups were assessed by one-way analysis of variance (ANOVA) with Duncan’s multiple range test. P<0.05 was considered to indicate a statistically significant difference. SAS version 9.1 (SAS Institute Inc., Cary, NC, USA) was used to conduct the statistical analyses.

Eleven compounds were isolated and identified in D. candidum leaf. Compound 1 was obtained as a clear crystal; the ¹H-NMR spectrum of this compound exhibited peaks at δ 6.92 (2H, d), 6.62 (2H, d), 6.06 (2H, s), 6.03 (1H, s) and 2.65 (4H, m), consistent with this compound being dihydro-resveratrol. Compound 2 was obtained as a white powder; the ¹H-NMR spectrum of this compound exhibited peaks at δ 6.98 (2H, d), 6.74 (2H, d), 6.62 (1H, s), 6.47 (1H, d), 4.83 (1H, d), 4.63 (1H, d), 3.1–3.8 (12H), 3.73 (3H, s), 3.69 (3H, s) and 2.74 (4H, m), indicating that it was dendromoniliside E. Compound 3 was obtained as a black red needle; the ¹H-NMR spectrum of this compound exhibited peaks at δ 11.00 (1H, s), 8.15 (1H, d), 6.06 (2H, s), 8.07 (1H, d), 6.95 (1H, s), 6.83 (1H, s), 6.15 (1H, s), 3.96 (3H, s) and 3.93 (3H, s), indicating that it was denbinobin. Compound 4 was obtained as a colorless needle; the ¹H-NMR spectrum of this compound exhibited peaks at δ 4.72 (2H, m), 3.85 (1H, d), 6.06 (2H, s), 2.53 (1H, d), 2.49 (1H, t), 2.39 (1H, dd), 2.21 (1H, dd), 1.64 (1H, m), 1.35 (3H, s), 1.03 (3H, d) and 0.95 (3H, d), which suggests that this material was aduncin. Compound 5 was obtained as a white needle; the ¹H-NMR spectrum of this compound exhibited peaks at δ 8.25 (1H, s), 8.10 (1H, s), 5.90 (1H, d), 4.66 (1H, dd) and 3.5–4.2 (4H, m), and it was confirmed that material this was adenosine. Compound 6 was obtained as a white powder; the ¹H-NMR spectrum of this compound exhibited peaks at δ 7.95 (1H, d), 5.85 (1H, d), 5.66 (1H, d) and 3.2–4.3 (5H, m), confirming that the material was uridine. Compound 7 was obtained as a clear crystal; the ¹H-NMR spectrum of this compound exhibited peaks at δ 10.60 (1H, s), 7.92 (1H, s), 6.45 (2H, s), 5.66 (1H, d), 3.4–4.4 (5H, m), indicating that the material was guanosine. Compound 8 was obtained as a white powder; the ¹H-NMR spectrum of this compound exhibited peaks at δ 7.65 (1H, d), 7.41 (2H, d), 6.85 (2H, d), 6.33 (1H, d), 4.17 (2H, t), 1.69 (2H, m), 1.25 (54H, m) and 0.85 (3H, t), indicating that this material was defuscin. Compound 9 was obtained as a white powder; the ¹H-NMR spectrum of this compound exhibited peaks at δ 7.45 (2H, d), 6.82 (2H, d), 6.81 (1H, d), 5.83 (1H, d), 4.16 (2H, t), 1.67 (2H, m), 1.23 (54H, m) and 0.88 (3H, t), indicating that this material was n-triacontyl cis-p-coumarate. Compound 10 was obtained as a white powder; the ¹H-NMR spectrum of this compound exhibited peaks at δ 2.35 (2H, t), 1.62 (2H, m), 1.25 (24H, m) and 0.88 (3H, t), consistent with the material being hexadecanoic acid. Compound 11 was obtained as a white powder; the ¹H-NMR spectrum of this compound exhibited peaks at δ 3.85 (2H, t), 1.75 (2H, m), 1.45 (2H, m), 1.22 (54H, m) and 0.85 (3H, t), and was identified to be hentriacontane.

The body weights of the control mice with activated carbon-induced constipation were significantly decreased after six days. As shown in Fig. 1, following the initiation of activated carbon-induced constipation, the body weights of the mice in the D. candidum-treated groups were significantly lower compared with those of the normal mice and the bisacodyl-treated group, but higher than those of the control mice with activated carbon-induced constipation. The high dose of 400 mg/kg D. candidum alleviated the weight loss of the mice to a greater extent than the lower 200 mg/kg dose.

From the first to the sixth day, defecation weight, particle counts of defecation and water content of defecation in each group were not significantly different, although the defecation weight and particle counts of defecation in the bisacodyl group were slightly greater than those in the other groups (Table I). When constipation was induced, from the seventh day to the ninth day, the defecation weight, particle counts of defecation and water content of defecation were decreased to 0.31 g, 16 pieces and 18%, respectively, in the control group. Particle counts were decreased to 0.78 g (32 pieces), 0.58 g (22 pieces) and 0.67 g (27 pieces), respectively, and the water content of defecation was decreased to 41, 26 and 35% in the bisacodyl and 200 and 400 mg/kg D. candidum dose groups, respectively. These results demonstrated that D. candidum was able to relieve constipation and had a good effect in the treatment of constipation.

The time taken for the first defecation of a black stool in each group of mice following the administration of activated carbon, which demonstrates the constipation-inhibiting effect of the different treatments, is shown in Fig. 2. The defecation time was the shortest (84±6 min) in the normal group and the longest (202±12 min) in the control group; the defecation time in the bisacodyl group was 126±3 min, higher only than that of the normal group. The times taken for the first defecation of a black stool for the mice treated with 200 and 400 mg/kg D. candidum were 161±9 and 142±6 min, respectively. According to the defecation time, D. candidum demonstrated a strong effect as an inhibitor of constipation.

The constipation-inhibiting effects of the treatments were determined by GI transit in mice following the administration of activated carbon (0.2 ml/mouse, 10% activated carbon). In the bisacodyl-treated group, the mean GI transit was 90.2±4.8%, which was higher than that of the control group (48.8±6.1%; Fig. 3). The GI transits of the mice in the 200 and 400 mg/kg D. candidum treatment groups were 57.7±4.6 and 74.6±2.8%, respectively. D. candidum increased the GI transit compared with that of the control, reduced constipation and had an increased functional effect on GI transit.

Normal mice showed the highest MTL, Gas, ET, AChE, SP and VIP levels; however, these levels in the control mice were significantly decreased (P<0.05, Table II). The levels of these analytes in the bisacodyl-treated mice were most similar to those of the normal mice. In the D. candidum-treated mice, these analyte levels were also comparable with those in the normal mice and higher than those of the control mice. Additionally, the mice treated with 400 mg/kg D. candidum exhibited higher levels than did the mice treated with 200 mg/kg D. candidum. The higher dose helped to promote these analyte levels to similar values as were observed in the normal and bisacodyl-treated mice. The SS levels in mice showed the opposite tendency.