MATERIALS AND METHODS

Drugs and reagents: The AMR was collected from Yuqian, Zhejiang, November, 2012 (Batch No. 20121101), where is the geo-authentic producing area and were identified by Professor Wang Bing (Liaoning University of Traditional Chinese Medicine) as rhizomes of Atractylodes macrocephala Koidz. Domperidone tablets (Batch No. 120913731, Xian-Janssen Pharmaceutical Ltd.). The P substance Elisa Kit (Batch No. 201310, Shanghai lianshuo Biological Technology Co., Ltd.) and Vasoactive Intestinal Peptide (VIP) Elisa Kit (Batch No.201310, Shanghai lianshuo Biological Technology Co., Ltd.). Other reagents were of analytical grade.

Animals: Male Kunming mice (18-22 g) were purchased from Liaoning Changsheng Biotechnology Co., Ltd [SCXK (Liao) 2010-0001]. All mice were maintained with free access to food and water in plastic cages at 22±2, relative humidity 50-60% and kept on a 12 h light/dark cycle. Animals were housed for one week prior to the experiments. The experimental protocols were approved by the Guide for the Care and Use of Laboratory Animals Published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). All studies were carried out in accordance with the ethics regulations of Liaoning University of TCM (131/2010).

Preparation of splitted fractions: One hundred gram of AMR was pulverized and immersed in water for 1 h, boiled twice and the filtrates were combined and concentrated in vacuum to 0.5 g crude drug mL^–1 and then stored in 4°C for the water decoction of AMR. Further, according to the polarity of components of AMR, the water decoction of AMR was split as reported in reference (Li et al., 2014). Briefly, the water decoction of AMR was concentrated and the 95% ethanol was added to regulate the alcohol concentration to 75%, standstill under 0~5°C overnight and then the supernatant was poured out to obtain the precipitate and the supernatant. The precipitate was washed three times by 95% ethanol and acetone respectively to furnish the Crude Polysaccharides Faction (CPF) after lyophilization. Then, the supernatant was evaporated on water bath at 50°C to eliminate the alcohol and the resulting water layer was extracted eight times with 60~90°C petroleum ether until there is no color in layer of petroleum ether to give Petroleum Ether Fraction (PEF). The resulting water layer was subjected to a column of macroporous adsorption resin D101, washing with distilled water, 60% ethanol and 80% ethanol successively. The water eluate was concentrated at 50°C and lyophilized to give Water Eluted Fraction (WEF) and the 60 and 80% ethanol elutes were collected and evaporated in vacuum to give the Alcohol Eluted Fraction (AEF).

Analysis of the main compounds in VOF, PEF, AEF and WEF: The GC-MS analysis was performed using an Agilent HP 5975 Series instrument combined with an Agilent HP 7890 Mass Selective Detector. Separation of VOF was performed using a polar capillary column (DB-5MS) with 30 m×0.25 mm×0.25 μm film thickness.Thecarrier gas was helium and the flow rate was 1.0 mL min^–1. The temperature program was optimized to separate quickly as follows: initial temperature 70°C, held for 2 min, increased at 10°C min^–1 to 280°C for 20 min. The injection volume was 1 μL and thesplit ratio was 1/50. The injector temperature was 260°C. The mass spectrometer was operated inthe electron impact ionization mode at 70 eV. The ionization source and transfer line temperature were kept at 230 and 260°C. Identification of the constituents of each sample was achieved by matching their MS and with the fragmentation pattern of their mass spectra with those in NIST 05.LIB.

Various chromatographic columns, including silica gel, ODS column and HPLC methods, were used to isolate and purify the chemical compounds of AMR. Thechemical compounds of AMR were identified by the analysis of their spectroscopic data in comparison with reference compounds. The fingerprint of each of the separated chemical fractions was established using an HPLC method. A HPLC Agilent system 1260, with a DAD detector and a KromasilC₁₈ (250×4.6 mm, 5 μm) column was used. Conditions were as follows: Column temperature 25°C, flow 1.0 mL min^–1, running time 70 min; the solvent were aacetonitrile and B water; gradient: 0-5 min, 3% A→5-10 min, 3-%-10% A→10-25 min, 10%-40% A→25-40 min, 40-60% A→40-50 min, 60-100% A→50-60 min, 100% A→60-70 min, 100-3% A. Detection at 242 nm provided the best level information for all compounds. The main peaksin every fraction were identified by the retention time of the individual compounds isolated.

Preparation of semi-solid paste: According to the literature method (Xing et al., 2003),10 g CMC-Na was dissolved with 250 mL distilled water and mixed with 10 g milk powder, 8 g sugar, 8 g starch and 2 g active carbon powder, respectively. About 300 g black semi-solid paste was obtained and stored at 4°C.

Effects of water decoction of AMR on GIP: The slight modification of earlier methods was carried out to evaluate the gastric residue and intestinal propulsion rates (Xing et al., 2003). Sixty Kunming mice, half male and half female, were randomly divided into six groups with 10 mice for each group. Each group was administered i.g for 7 days as follows. Control group, 0.2 mL/10 g of the purified water, Positive control group, 0.01 g kg^–1 domperidone, 0.1 g kg^–1 water decoction group, 0.1 g kg^–1 WD group, 0.1 g kg^–1 water decoction; 1 g kg^–1 water decoction group 1 g kg^–1 WD group, 1 g kg^–1 water decoction, 2 g kg^–1 water decoction group 2 g kg^–1 WD group, 2 g kg^–1 water decoction, 10 g kg^–1 water decoction group 10 g kg^–1 WD group, 10 g kg^–1 water decoction. Before the experiment, the mice were deprived of food but not water for 24 h. The mice were i.g with 0.4 mL/10 g semi-solid paste at day 8. Then the mice were sacrificed after 20 min and the gastric residue and intestinal propulsion rates were evaluated. The methods of evaluation as follow:

where, A is total gastric weight of mice, B is gastric weight, C is the weight of the administration semi-solid paste in mice, D is the length of black small intestine and F is the length of small intestine.

Effects of the fractions of AMR on GIP: The experiment protocol is similar to the water decoction of AMR and the mice were randomly divided into 14 groups with 10 mice for each group. Each group was orally administered i.g as follows. Control group: the purified water 0.2 mL/10 g, positive control group: 0.01g kg^–1 domperidone, 1g kg^–1 WD group, 1 g kg^–1 water decoction; the low dosage of 5 splitted fractions groups: corresponding extracts according to 1 g kg^–1 crude herbs; 10 g kg ^–1 WD group: 10 g kg^–1 water decoction and the high dosage of 5 splitted fractions groups: corresponding extracts according to 10 g kg^–1 crude herbs. Before the experiment, the mice were deprived of food but not water for 24 h. The mice were i.g with 0.4 mL/10 g semi-solid paste at day 8. Then the mice were sacrificed after 20 min and the gastric residue and intestinal propulsion rates were evaluated. The methods of evaluation as follow:

where, A is total gastric weight of mice, B is gastric weight, C is the weight of the administration semi-solid paste in mice, D is the length of black small intestine and F is the length of small intestine.

Determination of VIP and SP in gastric antrum and ileum: After the evaluation of fractions on GIP, gastric antrum and ileum of the mice were isolated and homogenized for the determination of VIP and SP levels.

Statistical analysis of data: All data were expressed as Mean±Standard Deviation (SD). The statistical analysis of the results was performed by one-way analysis using the Statistical Package for Social Science program (SPSS 20.0, Chicago, IL, USA). The p<0.05 was considered to indicate a statistically significant difference.

RESULTS

Analysis of the main compounds in VOF, PEF, AEF and WEF: In this study, 18 main compounds were isolated and analyzed as follow: atractylone which was obtained from VOF; Juniper camphor, atractylenolide, taraxeryl acetate, (4E, 6E, 12E)-tetradeca-4, 6, 12-trien-8, 10-diyne-1, 3, 14-triol, 3β-acetoxy-atractylenolide, stigmasterol, atractylenolide, Isoatractylenolide, sitosterin, atractylenolide, dibutyl phthalate and diisobutyl phthalate were obtained from PEF; 14. atractyloside A; 3 β-acetoxy-atractylenolide, caprolactam, 5-hydroxymethyl furfural ether and (4E, 6E, 12E)-3, 14-dihydroxytetradeca-4, 6, 12-trien-8, 10-diyn-1-yl acetate were obtained from AEF; 5-Hydroxymethyl furfural and small molecular sugar were obtained from WEF. In combination with the HPLC chromatogram, it is founded that VOF mainly contains atractylone, PEF mainly contains sesquiterpene lactone, AEF mainly contains polyacetylene and WEF mainly contains 5-hydroxymethyl furfural and small molecular sugar. In the Table 1, 37 compounds were identified and analyzed. The results indicated that the content of atractylone was highest than others and most of compounds belong to terpene and sesquiterpenoids.

Effects of water decoction (WD) of AMR on GIP in mice: Results (Fig. 1) indicated the gastric retention rate in 0.1 g kg^–1 WD group and 1 g kg^–1 WD group were significantly decreased than control group; the intestinal propulsionrate in 1 g kg^–1 WD group and 2 g kg^–1 WD groups was significantly increased than control group. However, the gastric retention rate in 10 g kg^–1 WD group was significantly increased than control group; the intestinal propulsionrate in 2 g kg^–1 WD group and 10 g kg^–1 WD group was significantly decreased than control group.

Table 1:	GC-MS analysis of VOF

Fig. 1:	Effects of water decoction of AMR on GIP in the mice. Cumulative values are reported as Mean±SD for 10 rats in each group. *p<0.05 and **p<0.01 compared to controls using LSD analysis

Fig. 2:	Effects of fractions of low dose of water decoction (1 g kg–1 water decoction) on GIP. Cumulative values are reported as Mean±SD, for 10 rats in each group. *p<0.05 and **p<0.01 compared to controls using LSD analysis

Effects of fractions of AMR on GIP in mice: Figure 2 indicated the gastric retention rates of mice in low dose of VOF (L-VOF) group were significantly decreased than control group; however, the gastric retention rates of mice in low dose of PEF (L-PEF) and low dose of AEF (L-AEF) groups were significantly increased. The intestinal propulsion rates of mice in low dose of VOF (L-VOF) group were significantly increased than control group, whereas low dose of PEF (L-PEF) group were significantly decreased.

Figure 3 indicated the gastric retention rates of mice in high dose of VOF (H-VOF), high dose of WEF (H-WEF) and high dose of CPF (H-CPF) groups were significantly decreased than control group; however, the gastric retention

Fig. 3:	Effects of fractions of high dose of water decoction (10 g kg–1 water decoction) on GIP. Cumulative values are reported as Mean±SD, for 10 rats in each group. *p<0.05 and **p<0.01 compared to controls using LSD analysis rates of mice in high dose of PEF (H-PEF) group were significantly increased.

Fig. 4:	Effects of fractions of low dose of water decoction (1 g kg–1 water decoction) on the level of SP in antrum and ileum. Cumulative values are reported as Mean±SD for 10 rats in each group, *p<0.05 and **p<0.01 compared to controls using LSD analysis

The intestinal propulsion rates of mice in high dose of VOF (H-VOF) and high dose of WEF (H-WEF) group were significantly increased than control group, whereas high dose of PEF (H-PEF) group were significantly decreased.

Effects of AMR and its fractions on the level of SP and VIP: Figure 4-7 showed that the VIP and SP levels of mice in 1 g kg^–1 WD and L-VOF groups were significantly increased than control group. The gastric antrum and ileum SP and ileum VIP levels in L-PEF group were significantly decreased than control group.

Fig. 5:	Effects of the fractions of low dose of water decoction (1 g kg–1 water decoction) on the level of VIP in antrum and ileum. Cumulative values are reported as Mean±SD for 10 rats in each group, *p<0.05 and **p<0.01compared to controls using LSD analysis

Fig. 6:	Effects of the fractions of high dose of water decoction (10 g kg–1 water decoction) on the level of VIP in antrum and ileum. Cumulative values are reported as Mean±SD for 10 rats in each group, *p<0.05 and **p<0.01 compared to controls using LSD analysis

The ileum SP and VIP levels in L-AEF and H-AEF group were significantly decreased than control group. The gastric antrum SP and ileum VIP levels in H-WEF and H-CPFgroups were significantly decreased than control group. Gastric antrum and ileum VIP levels in10 g kg^–1 WD group were significantly increased than control group. The gastric antrum and ileum VIP and gastric antrum SP levels in H-VOF group were significantly decreased than control group but ileum SP level was increased than control group. The gastric antrum SP and ileum VIP levels in H-PEF and H-AEF groups were significantly increased than control group.

Fig. 7:	Effects of the fractions of high dose of water decoction (10 g kg–1 water decoction) on the level of VIP in antrum and ileum. Cumulative values are reported as Mean±SD for 10 rats in each group, *p<0.05 and **p<0.01 compared to controls using LSD analysis

The gastric antrum SP, gastricantrum and ilium VIP levels in H-WEF groups were significantly increased than control group. The gastric antrum SP and VIP levels in H-CPF groups were significantly increased than control group.

DISCUSSION

Since the plant living organisms contains thousands of components, just a few individual compounds always could not represent the whole pharmacologic effects. In this study, we attempted to use splitted fraction obtained from the crude extract to overcome this disadvantages. In order to reflect the real effective material of AMR, in this study, the main components of the fractions were identified by HPLC and GC-MS chromatography. The results indicated that the main components of VOF were monoterpenes and sesquiterpenes; the main components of PEF were sesquiterpene lactone; the main components of AEF were polyacetylene; the WEF mainly contained 5-hydroxymethyl furfural and small molecular sugar. In the same time, the research (Lin et al., 2015) indicated inulin-type oligosaccharides were the main component of CPF.

Our results of GIP experiments indicated the low dose of water decoction exhibited promotion effects, moreover, the effects of 1.00 g kg^–1 water decoction were strongest than others; the high dose of water decoction exhibited inhibition effects, moreover, the effects of 10.00 g kg^–1 water decoction were strongest than others. Thus, AMR possessed bidirectional regulation effects on GIP. As for the dosage, in order to reflect the real effective material of AMR, in this study, effects of the fractions of 1.00 g kg^–1 water decoction and10.00 g kg^–1 water decoction on GIP were firstly explored. The results indicated that L-VOF, H-VOF, H-WEF and H-CPF exhibited promotion effect, however, L-PEF, H-PEF and L-AWF exhibited inhibition effect. And the effect order as follow: promotion effect: H-VOF>L-VOF, H-WEF>L-WEF, H-CPF>L-CPF; inhibition effect: L-PEF>H-PEF, L-AEF>L-AEF. It indicated that promotion effects of fractions were VOF, WEF and CPF and that VOF was most important; promotion effect of fractions were PEF and AEF and that PEF was most important. Moreover, the promotion effect of H-WEF and H-CPF and the inhibition effect of L-AEF were found for the first time; promotion effect of VOF and inhibition effect of PEF were consistent with the reference (Chen et al., 2009; Liu, 2009). In combination with components of fractions, we can know that the real effective material of promotion effect were monoterpenes, sesquiterpenes, 5-hydroxymethyl furfural, small molecular sugar and in ulin-type oligosaccharides; the real effective material of inhibition effect were sesquiterpene lactone and polyacetylene.

Further, in the study the effect of relative mechanism were explored. The GIP was mainly regulated by gastrointestinal nerve and brain-gut peptide (Ma, 2006). The SP, as an important gastrointestinal peptide, was widely distributed in the enteric nerve system and the gastrointestinal tract (Maake et al., 1999). Moreover, SP was the main excitatory neurotransmitter in the control of regulating GIP; when it exhibited excitatory effect on gastrointestinal tract, it showed double contraction effects on longitudinal gastrointestinal muscle and circular muscle and the effects contain direct-short term effects and indirect-long term effects (Lordal et al., 1997; Wheatley et al., 1999). In the same time, the research indicated that the effects of AMR were related to the SP levels (Zhao et al., 2008). The VIP, a non-adrenergic inhibition neurotransmitter, distributed in the gastrointestinal tract and the central nervous system, could inhibit gastrointestinal motility owing to its effects of relaxing gastrointestinal smooth muscle, increasing gastrointestinal fluid and electrolyte secretion(Grider, 1993; Ljung and Hellstrom, 1999). Thus, the SP and VIP in gastric antrum and ileum were selected as index to explored relative mechanism. In combination with the effects of WD and its fractions on GIP, WD, VOF and CPF had promotion effects on gastrointestinal function through up regulated SP and down regulated VIP; PEF had inhibition effects on gastrointestinal function through up regulated VIP; AEF had inhibition effects on gastrointestinal function through down regulated SP but promotion effects of WEF on gastrointestinal function had no relation to up regulated SP or down VIP. Thus, the effects of AMR maybe related to the level of SP and VIP and the fractions had different effect on the level of SP and VIP and that the effects of AMR were related significantly to VIP level for the first time. The AMR possessed bidirectional regulation effects on GIP. The real effective materials of promotion effect were monoterpenes, sesquiterpenes, 5-hydroxymethyl furfural, small molecular sugar and inulin-type oligosaccharides; the real effective materials of inhibition effect were sesquiterpene lactone and polyacetylene. The relative mechanism may be related to the SP and VIP levels. All the conclusions in the study have role significance in studying the effects of AMR on GIP at later.

ACKNOWLEDGMENTS

The research project was supported by grants from National Basic Research 973 Program of China (2013 CB53 1803) and Program for Liaoning Innovate Research Team in University (LT2013020).