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Relieving visceral hyperalgesia effect of Kangtai capsule and its potential mechanisms via modulating the 5-HT and NO level in vivo.

ARTICLE INFO

Keywords:

Kangtai capsule

Fingerprint

Visceral hypersensitivity

Neonatal maternal separation

5-HT

NO

ABSTRACT

Kangtai capsule (KT) is one type of traditional Chinese medicine preparation derived from the proved recipe, which was frequently applied as an effective clinical treatment of IBS. However, there still lack the reasonable and all-round analytical approach and the scientific studies on its underlying mechanisms. Therefore, our study aimed to develop the novel method for evaluating its quality as well as to interpret the potential mechanisms. In our study, high performance liquid chromatography (HPLC) fingerprint was applied to provide a chemical profile of KT. The neonatal maternal separation (NMS) on Sprague-Dawley pups was employed to evaluate the therapeutic effect of KT by virtue of various parameters including visceral hyperalgesia, serum nitric oxide (NO) level, and tissue 5-hydroxytryptamine (5-HT) level. Consequently, a chromatographic condition, which was carried at 30[degrees]C with a flow rate of 0.5 ml/min on AQUA 3[micro], C18 column with mobile phase of acetonitrile and water--phosphoric acid (100:0.1, v/v), was established to give a common fingerprint chromatography under 254 nm with a similarity index of 0.963 within ten batches of KT samples. On the NMS model, KT markedly elevated the pain threshold of NMS rats. Furthermore, KT at three doses significantly decreased 5-HT content from distal colon of visceral hyperalgesia rats induced by NMS, while the significant decrease of 5-HT content in serum was only observed in the group with KT at high dose. However, compared with that in NMS rats without KT, there was no apparent difference of 5-HT level from brain issue in the rats with various doses. Besides, KT could substantially elevate the concentration of NO in the serum. The results showed our study developed the simple, rapid, accurate, reproducible qualitative and quantitative analysis by HPLC fingerprint for the quality control for KT. Data from the pharmacological investigation suggested that the curative effect of KT to the visceral hypersensitivity may be concerned with the level of 5-HT and NO in vivo, promising its potential in irritable bowel syndrome treatment.

Crown Copyright [c] 2012 Published by Elsevier GmbH. All rights reserved.

Introduction

The irritable bowel syndrome (IBS) is prevalent functional bowel disorder described firstly by Powell (1818), associated with a combination of chronic abdominal pain, discomfort diarrhea and/or constipation (Rey and Talley 2009; Rivkin 2003) and affects around 3-15% of the world's population according to the definitions set by International Congress of Gastroenterology in Rome (Cremonini and Talley 2005). Then IBS occupies around 3% of all general practice consultation and soars to as high as 40% of gastrointestinal referrals (Thompson et al. 2000), which causes the extensive social expenditure. The organic cause and the relevance of the criteria in clinical practice, however, are still unclear (Rey and Talley 2009; Bommelaer et al. 2004; Gilkin 2005). Traditionally, IBS has been considered a disorder of brain-gut-axis, and the potential underlying mechanisms of IBS associated with functional gastrointestinal disorders are multi-factorial and some hypotheses have been proposed to illustrate them, including abnormal motility, visceral hypersensitivity, inflammation and infection, neurotransmitter imbalance, and psychosocial factors (e.g., anxiety, depression, somatisation) (Rivkin 2003; Gilkin 2005). Among these, visceral hypersensitivity is defined as low threshold of stimuli perception from GI and currently regarded as a critical patho-physiological mechanism, bringing about various symptoms of IBS (Greenwood-van 2007). It is reported that visceral hypersensitivity has been described in majority of patients with IBS, especially the rectal hypersensitivity, which could be detected in 95% of patients with IBS (Azpiroz et al. 2007; Mertz et al. 1995). Hence, rectal hypersensitivity could be thought a biological marker of the syndrome. Besides, both Serotonin (5-hydroxytryptamine, 5-HT) and Nitric oxide (NO) have been considered to contribute to onset of visceral hypersensitivity depending on the level of them in vivo (Rivkin 2003; Sikander et al. 2009; Kuiken et al. 2006; Tjong et al. 2011a,b).

Kangtai capsule (KT) is a Chinese herbal medicine compound preparation, based on a famous proved recipe proposed by Prof. Fusheng Zhou, including Bai Shao (Radix Paeoniae Alba, RPA), Mu Xiang (Radix Aucklandiae, RA), Fang Feng (Radix Saposhnikoviae, RS), Yan Hu Suo (Rhizoma Corydalis, RC), Bai Zhu (Rhizoma Atractylodis Macrocephalae, RAM), and Shou Wu Teng (Caulis Polygoni Multiflori, CPM). KT recipe had clinical efficacy of reinforcing spleen and stomach, tonifying spleen to resolve dampness, promoting qi circulation to relieve flatulence and was frequently applied as effective clinical treatment of IBS by Prof. Fusheng Zhou (Huang et al. 2007; Zhou et al. 2004). All these raw herbs were frequently used for composing prescriptions, which were applied for the treatment of Gland psychological disorder in the clinic, for example To ngxieyao-fang including RAM, RPA and RS (Sun et al. 2004; Bian et al. 2006), Weichangan Wan including RAM and RA (Hu and Tang 2009; Hu et al. 2010), Shuganjieyu Decoction including RAM, RPA, RS, CPM, RC (Chen et al. 2010).

It is well known that the traditional Chinese medicines (TCM) generally exert their curative effects through the multiple bioactive constituents rather than single specie as synthetic drugs, which are vulnerable to quality of its raw herbs, depending on the cultivation areas and climatic conditions and so on. Thus, chromatographic fingerprint is quite imperative to be introduced as a more effective and reliable method so as to synthetically evaluate the quality of TCM. The major bioactive constituents of KT preparations are glucosides, lactones and alkaloids. Therefore, in our study, eight relevant compounds: paeoniflorin in RPA, dehydrocostus lactone in RA, prim-O-glucosylcimifugin and 5-0-methylvisammioside in RS, tetrahydropalmatine in RC, atractylenolide II and atractylenolide III in RAM, 2,3,5,4'-tetrahydroxystilbene-2-O-[beta]-D-glucoside in CPM were selected for analysis through the HPLC technology. Meanwhile, HPLC was also adopted as an analytical tool of fingerprint. Since then, we have successfully established the HPLC fingerprint profile for the assessment of the quality of KT by HPLC equipped with PDA.

Furthermore, in order to elucidate possible mechanisms of action of KT, visceral hypersensitivity was chosen as a biological marker to evaluate the rat model processed by neonatal maternal separation (NMS), a well-established early-life stress model that mimic human IBS symptoms, and then examined the content of 5-HT and NO. After that, the data analysis was adopted to confirm whether KT exerts its efficacy of IBS symptoms by modulating the 5-HT and NO level.

Materials and methods

Animals

For the neonatal maternal separation model, all maternal and neonatal Sprague-Dawley rats were provided by Guangdong Medical Experimental Animal Center. All the animals were maintained under environmentally controlled conditions of 22[degrees]C and 12h light/12 h dark cycle. The animals received humane care in accordance with the guide for the care and use of laboratory animals, published by the US National Institution of Health (NIH Publication, revised in 1985). All animal experimental procedures were approved by our institutional animal research ethics committee in reference to the European Community guidelines for the use of experimental animals.

Chemicals and reagents

Paeoniflorin, dehydrocostus lactone, prim-O-glucosylcimifugin, 5-O-methylvisammioside, tetrahydropalmatine, atractylenolide 11, atractylenolide III and 2,3,5,4'-tetrahydroxystilbene-2-O-[beta]-D-glucosicle were purchased from National Institutes for Food and Drug Control (Beijing, China). Atractylenolide II and Atractylenolide III were kindly provided by Shanghai R&D Center for Standardization of Traditional Chinese Medicines (Shanghai, China). 5-HT was provided by SIGMA Inc. (Lot29F0438). Cysteine (CYS) and Ortho-phthalaklehyde (OPT) were purchased from Mbchem Technology Group Co., Limited. The Acetonitrile (LC grade) was bought from Honeywell Inc. (U.S.A). Ultra-pure distilled water, prepared from special laboratory ultra-pure water machine, was used. Phosphoric acid (Shantou, China), methanol (Shantou, China), ethanol (Shantou, China) and other reagents were all of analytical grade.

KT consists of six medicinal plants as shown in Table 1. All raw herbs were purchased from Guangxi Yifang Chinese Herbal Medicine Department and authenticated by Professor Chen Jiannan, pharmacognosist of School of Chinese Materia Medica, Guangzhou University of Chinese Medicine. All of these accord with the demand in 2010 edition of China Pharmacopoeia.

Table 1 Recipe of Kangtai Capsule (KT) formulation.

Components                                                        Ratio

1. Bai Shao (Paeonia lactiflora Pall., root)                          3

2. Mu Xiang (Aucklandia lappa Decne., root)                           2

3. Fang Feng (Saposhnikovia divaricate (Turcz.) Schidchk., root)      2

4. Yan Hu Suo (Corydalis yanhusuo W. T. Wang, tuber)                  3

5. Bai Zhu (Atractylodes macrocephala Koidz., rhizome)                3

6. Shou Wu Teng (Polygonum Multiflorum Thunb., lianoid stern)         6


KT was provided by our department and prepared as follow. In brief, RAM, RPA, RS, CPM and RC were mixed and refluxed three times (for 1 h each) with 41, 3.41 and 3.41 of 70% ethanol, successively. After cooling to room temperature, the combined extract was filtered and condensed by rotor evaporation under reduced pressure. And then, concentrated extract was dried to obtain powder by spray-drying before adding the fine power of RA. Finally, the mixture was mixed with an amount of medical starch and the loaded into capsules. And the extract of single herb was prepared according the procedure above without being filled into capsules.

Preparation of sample solution

The contents of KT were carefully taken out from KT. The contents or dried power of single herb were weighted accurately and extracted by ultrasonic extraction with 25 ml of 70% methanol for 30 mm. Then, the extract solution was filtered through a 0.45 [micro]m filter membrane before analysis.

Apparatus and chromatographic conditions

The HPLC analysis was performed on DIONEX SUMMIT HPLC system equipped with a PDA-100 detector, a P680 pump, an ASI-100 automatic sampler, and a STH585 thermostatic column compartment. The chromatographic separation was carried at 30[degrees]C with a flow rate of 0.5 ml/min on an AQUA 3[micro], C18 125A (250 mm x 4.60 mm, 311, Phenomenex Inc., USA). The linear gradient elution, composed of solvent A (acetonitrile) and B (water--phosphoric acid, 100:0.1, v/v), was used for separation. The elution program was optimized and conducted as follow: a linear gradient of 5-16% A (0-10 min), a linear gradient of 16-24% A (10-50 min), a linear gradient of 24-32% A (50-70 min), a linear gradient of 32-75% A (70-90 min), a linear gradient of 75-80% A (90-110 min), a linear gradient of 80% A (110-115 min), a linear gradient of 80-5% A (115-130 min). The effluents from column were detected at 254 nm with PDA detector.

Evaluation of HPLC fingerprint analysis

Data analysis was performed by the Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (Version 2004A), which was composed by Chinese Pharmacopeia Commission and recommended by State Food and Drug Administration (SFDA) of China. The software was employed for calculating the correlative coefficient of different samples and comparing the similarities of different chromatograms with the mean chromatogram among the samples detected.

Neonatal maternal separation (NMS)

All Sprague-Dawley pups grouped to 12 pups per dam. Pups were assigned at random to group NMS: neonatal maternal separation and group N: inseparated control in accordance with the protocol. Briefly, the entire pups in group NMS were separated from darn for 3 hiday (between 09:00 and 12:00) between day 2 and 21 (the date of birth is set as day 0) and were allowed to remain with their dams till weaning. During separation, each pup was placed into a clean cage (5 cm x 15 cm), maintained at 22 C. While the pups in group N were left inseparate with the dam in standard cage. All pups were weaned on day 24 and housed in isolated cages without their dams, accessed freely to food and water.

Evaluation of the threshold of abdominal withdrawal reflex (AWR)

The colonic distension (CRD) study was implemented at 5th week after cessation of stress. The rats were anesthetized with ether to be inserted the inflatable balloon approximately 5 cm into anus and fix it, which was made of a latex glove finger connected to a Rigiflex balloon dilator via a Y connector to a syringe and a sphygmomanometer. The rats were placed into the specially designed cage where they cannot turn the body and were admitted to recover for 15 min prior to the CRD. The pressure threshold of abdominal elevation and extrados of rats was recorded through pumping air into anus. The pressure threshold was tested again at 7th weeks.

The treatment of ICT and sample collection

After the successful modeling (10th week), the treatment with KT at low dose (1.52 g/kg), middle dose (3.04 g/kg), high dose (6.08 g/kg) was started by gavage once daily and lasted 2 weeks. Then evaluation of the threshold of AWR was performed again following the method described above. After the CRD study, rats were anesthetized and the blood was collected from the ophthalmic venous plexus and stored at 4[degrees]C for examining content of NO and 5-HT respectively. The distal colon and brain tissue was dissected in ice tray after sacrifice, and immediately frozen and stored at--80[degrees]C for 5-HT content determine.

Measurement of NO content

The blood was stored at 4[degrees]C for 211 at first and centrifuged at 2000 x g for 10 min. The supernatant was collected for test. The NO content of 100 [micro]l of the serum sample was analyzed by Nitrate Reductase kit in accordance with the instructions of the manufacturer (Nanjing jiancheng Bioengineering institute, Nanjing, China). The ultraviolet spectrophotometer was adopted to measure the NO content of sample at 550 nm.

Measurement of 5-HT content

5-HT was extracted from blood (0.5 ml) with acidic butane (4.5 ml) for 5 min and centrifuged at 2000 x g for 15 min. The supernatant (3 ml) was gathered and washed with n-heptane (3 ml) and 0.1 N HCI (1 ml), followed by centrifugation at 2000 x g for 15 min. The lower solution was pooled as sample. Blank tube (B) used distilled water as a replacement of serum and was prepared according to the description above. The detail of reagents was added into each group as showed in Table 2. Then the samples were heated on boiling water bath for 10 min and cooled down with ice water. The 5-HT content of serum in each was detected by LS-55 fluorescence spectrophotometer (PerkinElmer, Massachusetts, USA) under the condition of [[lambda].sub.ex] = 350 nm, [[lambda].sub.em] = 477 nm.

Table 2 The detail of reagent addition for testing the 5-HT
content in serum.

Reagent (ml)        Test cube   Blank cube 1  Standard  Blank cube 2
                          (T)    ([B.sub.1])   cube (S)  ([B.sub.2])

Lower solution              1              1         -             -

5-HT (100 ng/ml)            -              -         1             -

HCI (0.1 N)                 -              -         -             1

CYS (82.4 mmol/l)         0.1            0.1       0.1           0.1

OPT (447.8                1.5            1.5       1.5           1.5
[micro]mol/l)

5-HT, CYS and OPT, respectively, are the standard solution
of 5-HT, Cysteine and Ortho-phthalaldehyde.


Frozen samples of the brain tissues were homogenized in 10 times volume of butanol and centrifuged at 2000 x g for 15 min at 4 C. The supernatant (1 ml) was transferred to a cube containing n-heptane (2 ml) and 0.1 N HCI (2 ml), followed by centrifugation at 2000 x g for 15 min. The aqueous phases were used for the estimation of 5-FIT level employing the fluorospec-trophotometry (PerkinElmer, Massachusetts, USA), following the condition of [[lambda].sub.ex] = 350 nm, [[lambda].sub.em] = 477 nm. Blank tube (B) used distilled water as a replacement of serum and was prepared according to the description above. Then the detail of reagent addition followed Table 3 shown. The frozen distal colon was isolated, weighed, prepared and tested according to the method of brain tissues.

Table 3 The detail of reagent addition for testing the
5-HT content in issues.

Reagent (ml)        Test cube   Blank cube 1  Standard  Blank cube 2
                   (T)         ([B.sub.1])    cube (S)  ([B.sub.2])

Lower solution              1              1         -             -

5-HT (100 ng/ml)            -              -         1             -

HCI (0.1 N)                 -              -         -             1

CYS (82.4 mmol/l)         0.1            0.1       0.1           0.1

OPT (447.8                  3              3         3             3
[micro]mol/l)

5-HT, CYS and OPT, respectively, are the standard solution
of 5-HT, Cysteine and Ortho-phthalaldehyde.


The content of 5-HT was calculated by the following formula:

5-HT (ng/ml) = (FT-FBI)/(FS-FB2) x 5/3 x 1.2/1 x 2 x 100

Statistical analysis

The data were analyzed using a statistical package (SPSS 10.0) and expressed as [bar.X] [+ or -] SD. Statistical significances were performed by one-way ANOVA with post hoc multiple comparison followed by Dunnett's test and differences were considered significant at [rho] <0.05.

Results

Optimization of HPLC condition

To find out an optimal HPLC condition for good separation of as many peaks as possible within a short analysis time, variable chromatographic factors were optimized respectively, including HPLC instruments, columns, mobile phase and column temperature. After comparing different HPLC instruments (Shimazu LC solution-20A and DIONEX SUMMIT HPLC system), columns (Kromasil 100-5[C.sub.18] Dimensions (250 mm x 4.6 mm, 5 [micro]m, Akzo Nobel Inc. Netherlands)), AQUA 3[micro] [C.sub.18] 125A (250 mm x 4.60 mm, 3[micro], Phenomenex Inc., USA)), mobile phase systems (acetonitrile-water, methanol-water, acetonitrile-0.1% phosphoric acid solution, methanol-0.1% phosphoric acid solution), temperatures (20[degrees]C, 30[degrees]C, 40[degrees]C), a sufficiently large number of peaks on the chromatogram were achieved on AQUA 3[micro] [C.sub.18] column at 30[degrees]C with acetonitrile-0.1% phosphoric acid solution within 130 min and the flow rate was 0.5 ml/min. Result indicated that no more compounds could be observed at other detection wavelengths than 254 nm. Therefore the most appropriated wavelength was set as 254 nm. Due to the separation of samples on DIONEX SUMMIT HPLC system was better than that on Shimazu LC solution-20A, so we chose the DIONEX SUMMIT HPLC system for analysis.

Chromatographic fingerprint analysis

By using the proposed chromatography condition, HPLC chromatograms of different batches of KT sample were acquired. HPLC fingerprint of single herb was shown in Fig. 1 and KT was shown in Fig. 2. All assigned peaks were identified by co-injection test with authentic samples and compared with UV spectral data. The relative retention time (RRA) and relative peak area (RPA) of all common peaks were calculated with reference to this substance and the values of them were both less than 3.6%, which indicated good stability and reproducibility of the fingerprint analysis by HPLC. Similarity Evaluation System was employed to attain the similarity indexes of ten batches of KT samples. The result demonstrated the similarity indexes were higher than 0.963 and showed good correlation among the samples (Fig. 3).

Formation of visceral hyperalgesia in NMS rats

As was shown in Fig. 4, compared with the group N, both the pain threshold pressure of abdominal elevation and extrados were significantly decreased in the NMS rats. In detail, the pain threshold pressure of abdominal elevation and extrados were 35.45 [+ or -] 1.36 mmHg and 47.12 [+ or -] 1.82 mmHg, decreasing by 19.69% and 28.03% (N = 10, [rho] < 0.01) respectively in the 5th week. Turning to the 7th week, the pain threshold pressure of abdominal elevation and extrados were descended by 24.46% and 27.59%, to 36.45 [+ or -] 1.47 mmHg and 48.14 [+ or -] 1.72 mmHg (N=10, [rho] < 0.01) respectively. Meanwhile, both in the 5th and 7th week, the pain threshold pressures of rats in the administration groups were substantially descended before given treatment with KT. According the data above, it suggested the visceral hyperalgesia model was successfully formatted.

Alleviation of NMS-induced visceral hyperalgesia in response to CRD

Visceral hyperalgesia was induced by neonatal maternal separation (NMS). NMS Veh rats were more sensitive to distension than the N rats, as shown in Fig. 5. On detail, the pain threshold pressure of abdominal elevation was 51.18 [+ or -] 2.12 mmHg and 36.28 [+ or -] 1.63 mmHg in N and NMS Veh rats, and the pressure of extrados was 66.93 [+ or -] 2.06 mmHg and 48.12[+ or -] 2.08 mmHg, respectively (N= 10, [rho] < 0.01). Preparation of KT at dose of low dose (1.5 g/kg), middle dose (3.04 g/kg) and high dose (6.08 g/kg) increased the pain threshold of abdominal elevation and extrados by 19.5% and 16.6% (N= 10, [rho] <0.01), 28.4% and 29.9% (N= 10, [rho] <0.01) and 32.6% and 27.8% (N=10, [rho] <0.01), respectively, when comparing with Veh group.

KT increase NO content in the serum of NMS rats

The NO level from the serum was significantly decreased in the NMS Veh rats in comparison to the N rats (33.45 [+ or -] 6.87 timol/I versus 49.12[+ or -] 10.45[micro]mol/1, N=10. [rho] < 0.05). In contrast, the NMS group pretreated with KT at high dose of 6.08 g/kg has a substantially elevation effect on concentration of NO comparing with NMS Veh group, and increased by 39.40% (N=10, [rho] < 0.01). The NO level of rats in middle and low dose group was significantly elevated by 22.96% and 19.94% respectively to 41.13 [+ or -] 8.10 iimol/I (N=10, [rho] <0.05) and 40.12 [+ or -] 5.94[micro]mo1/1 (N=10, p <0.05) (Fig. 6).

KT had different effects on 5-HT content from serum, brain issue and distal colon in the NMS rats

There was a significant change between N group rats and NMS Veh group rats (1762.13 [+ or -] 198.63 ng/ml verse 2301.36[+ or -] 156.32, N=10, [rho] < 0.05). The KT at high dose substantially decreased 5-HT the concentration in the serum of NMS rats by 20.8% (N= 10, [rho] <0.01), compared with Veh animals in the NMS rats. However, there were no significant changes among N and KT at low and middle dose groups (Fig. 7A).

The neonatal maternal separation evidently increased 5-HT content in brain issue from 512.32 [+ or -] 157.58 ng/ml (N rats group) to 896.14 [+ or -] 175.36 ng/ml (NMS Veh group)(N= 10, [rho] <0.05). However, there were no evident changes among NMS and KT at low, middle and high dose groups (Fig. 7B).

The 5-HT level from distal colon was substantially elevated in the NMS Veh rats (1347.28 [+ or -] 185.44 ng/ml) when comparing with that of N rats (835.47 [+ or -] 176.28 ng/ml) (N=10, [rho] <0.01). Meanwhile, la at low, middle and high dose have a marked effect on reduction of 5-HT content to 1020.75[+ or -] 123.75, 1085.78 [+ or -] 205.36, 945.41 [+ or -] 198.10 ng/ml (N = 10, [rho] < 0.01) in the distal colon in the NMS rats respectively (Fig. 7C).

Discussion

Chromatographic fingerprint is one of the most effective approaches of assessing the quality of the raw herbs and its preparations and adopted frequently all over the globe. And there are a set of chromatographic techniques, such as high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), gas chromatography (GC), capillary electrophoresis (CE) and thin layer chromatography (TLC) and so forth, are usually applied for chromatographic fingerprint. Compared with the conventional analytical approaches, chromatographic fingerprint pays attention to the integrity of analytes and can provide the comprehensive characterization so as to evaluate the quality rather than only several markers or active constitutes. In 1991, the WHO published 'Guidelines for the assessment of herbal medicines' on the evaluation of quality, safety, efficacy and intended use of herbal products, and the acceptance of chromatographic fingerprinting as a tool (World Health Organization 1991). Therefore, Chromatographic fingerprint was accepted and recommended for the quality control of herb drugs as an analytical tool by the EMEA, FDA and SFDA (European Medicine Evaluation Agency 2010; Food and Drug Administration 2004; Chinese State Food and Drug Administration 1993).1n our study, this was the first time to report HPLC fingerprint analysis for KT by HPLC-PDA. Meanwhile, the effective and reliable analytical method was established for the qualitative and quantitative analysis of KT manufactured during different period. This novel appraisal approach was based on the integrity of analytes, for the sake of the synergistic effects of various constituents in the sample, and could overcome drawbacks of conventional methods frequently applied for quality control of TCM, such as single or several markers or active constitutes evaluation.

In addition, the present study showed that traditional Chinese medicine KT significantly reduced visceral pain in NMS rats in a dose-dependent manner. The IBS patients usually have an enhanced sensitivity of visceral organ throughout the GI tract (Gilkin 2005). Although the organic causes and mechanisms of IBS are still unclear, the early-life stress is a key predisposing factor to the functional GI disorders including visceral hypersensitivity (O'Mahony et al. 2008). In our present study, we observed that the pain threshold experienced a significantly decrease in NMS rats, which corresponded with previous studies reported by others (Tjong et al. 2011a,b; Yang et al. 2012). Meanwhile, our study proved that the KT can increase the pain threshold pressure induced by CRD and the efficacy exhibits a dose-dependence tendency. Therefore, KT may be an effective TCM for modulating the visceral pain of IBS patients.

Serotonin (5-hydroxytryptamine, 5-HT) is 3-([beta]-aminoethyl)-5-hydroxyindole. It is found, as a neurotransmitter, in the gastrointestinal (GI) tract and central nervous system (CNS) and contributes to disorder of motility, secretion and visceral hypersensitivity (Rivkin 2003; Sikander et al. 2009; Gershon and Tack 2007; Farmer and Aziz 2009). Approximate 95% of body 5-FIT is found in the GI tract, 90% is in enterochromaffin cells (EC cell) and the surplus 10% in serotonergic neurons of myenteric plexus (Sikander et al. 2009; Tjong et al. 2011a,b). If the serotonergic pathways is disrupted, it may ultimately result in the symptoms of IBS, as it has been reported that in IBS patients the level of 5-HT significantly increased, so did in the rat models (Gilkin 2005; Tjong et al. 2011a,b; Yang et al. 2012). Therefore, the 5-HT signaling pathways have significant roles at the development of IBS. In our study, we found that the 5-HT levels in the serum and distal colon were significantly elevated in the NMS rats in accordance with previous similar result in clinical study (Moskwa et al. 2007), which suggests that 5-FIT maybe a possible factor in the pathological mechanism of IBS. It is reported that 5-HT was released by EC cell in response to mucosal stimuli, exerting its function of modulating the local excitation and inhibition via activating the submucosal primary afferent neuron (Pan and Gershon 2000; Kidd et al. 2008). The excess of 5-HT could contribute to abnormal intestinal motility and visceral hypersensitivity through altering the signaling pathway for gut motility and pain transmission (Kidd et al. 2008; Spiller 2008; Sanger 1996). Our study shows that KT therapy markedly attenuated the 5-HT concentration of serum and distal colon in NMS rats, with significant elevation of the pain threshold, suggesting that KT may elevate the pain threshold and eliminate visceral hypersensitivity by decreasing the 5-HT content from serum and distal colon. However, we did not observe any significant change of 5-HT level in brain of NMS rats pretreated with KT, although the difference of 5-HT content between N group and NMS group, which suggested KT could not exert its action through modulating content of 5-1-IT in brain.

As an important signalling molecule in GI tract, nitric oxide (NO) is a non-adrenergic, non-cholinergic (NANC) inhibitory neurotransmitter modulating the blood flow, gut motility, secretory and immunological function (Cho 2001; Sobko et al. 2005; Groneberg et al. 2011). It is synthesized and released from the myenteric plexus of the distal colon and exerts its action through Guanosine 3',5'-Cyclic Monophosphate pathway (cGMP) (Tjong et al. 2011a,b). It is reported that NO plays a role in the physiology and pathophysiology of the GI and may facilitate descending pain modulatory systems related to maintaining visceral hypersensitivity (Kuiken et al. 2006; Tjong et al. 2011a,b). In addition, the level of NO must be reasonably controlled, for both NO excess and NO insufficiency would elicit tissue injury and disease (Thomas and Darley-Usmar 2000). The present study showed that the NO content from serum was substantially descended associating the visceral hypersensitivity in NMS rats, identifying with the previous clinical study that the insufficiency of NO causes the decrease of pain threshold and elevation of visceral perception in IBS patients in clinical study (Mertz et al. 1995). By the treatment of KT, we obviously observed that the NO concentration in serum was significantly increased in NMS rats and the elimination of visceral hypersensitivity at the same time, suggesting that KT exerted its therapeutic effects of IBS may relate with the elevation of NO level in vivo.

In conclusion, our study developed the qualitative and quantitative analysis by the HPLC fingerprint for the quality control for MT. The potential mechanisms of curative effect of KT to the visceral hypersensitivity induced by NMS are preliminarily interpreted, which may be concerned with the level of 5-HT and NO in vivo. The further studies are warranted to focus on how MT affects the 5-HT and NO content and what specific physiological pathways participate the process.

Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgements

This work was supported by grants from Dongguan University Research Institutions and Medical Health Technology Project (No. 2008108102130), Guangzhou Science Research Project (No. 2006Z3-E5241) and Guangdong Province Universities and Colleges Pearl River Scholar Funded Scheme (2011).

* Corresponding author at: Institute of Digestive, Guangzhou University of Chinese Medicine, Guangzhou 510006 China.

** Corresponding author at: School of Chinese Materia Medica, Guangzhou University of Chinese Medicine, Guangzhou 510006, China. Fax: +86 20 3935 8390.

E-mail addresses: zhoufsh@gzucm.edu.cn (F.-S. Zhou), suziren@gzhtcm.edu.cn (Z.-R. Su).

(1.) These authors equally contributed to the current study and should be considered co-first authors.

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Yun-Long Chen (a), (1) Xiao-Qi Huanga (a), (1), Shi-Jie Xu (b), Jin-Bin Liao (a), Ru-Jun Wang (b), Xiao-Feng Lu (a), You-Liang Xie (a), Fu-Sheng Zhou (b), (c), *, Zi-Ren Su (a), (c), **, Xiao-Ping Lai (a), (c)

(a) School of Chinese Materia Medial. Guangzhou University of Chinese Medicine, Guangzhou 510006, China

(b) institute of Digestive, Guangzhou University of Chinese Medicine, Guangzhou 510006, China

(c) Dorigguan Mathematical Engineering Academy of Chinese Medicine, Guangzhou University of Chinese Medicine, Dongguan 523808, China
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Title Annotation:5-hydroxytryptamine; nitric oxide
Author:Chen, Yun-Long; Huang, Xiao-Qi; Xu, Shi-Jie; Liao, Jin-Bin; Wang, Ru-Jun; Lu, Xiao-Feng; Xie, You-Li
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9CHIN
Date:Feb 15, 2013
Words:6172
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