Antidiarrheal effect of Alpinia oxyphylla Miq. (Zingiberaceae)in experimental mice and its possible mechanism of action

Sheng Wang a,b,1, Yang Zhao c,1, Junqing Zhang d, Xiaoxing Huang a,b, Yifei Wang a,b,Xiaotao Xu a,b, Bin Zheng a,b, Xue Zhou a,b, Huajie Tian a,b, Li Liu a,b,n, Qibing Mei a,b,nn

a China State Institute of Pharmaceutical Industry, Shanghai 200040, Chinab State Key Laboratory of New Drug & Pharmaceutial Process, Shanghai Institute of Pharmaceutial Industry, Shanghai 200437, Chinac Vascular Biology Program, Centenary Institute, The University of Sydney, Shanghai 2042, NSW, Australiad Hainan Provincial Key Laboratory of Research and Development of Tropical Medicinal Plants, Hainan Medical University, Haikou 571199, China

a r t i c l e i n f o

Article history:Received 12 September 2014Received in revised form12 February 2015Accepted 30 March 2015Available online 7 April 2015

Keywords:AntidiarrhealFructus Alpinia oxyphylla Miq. (AOM)Chronic diarrheaGastrointestinal motilityGastrointestinal peptide

a b s t r a c t

Ethnopharmacological relevance: The fructus Alpinia oxyphylla Miq. (AOM) has been used for treatingdiarrhea with spleen deciency and gastralgia for thousands of years. A number of traditional Chinesemedicine formulae provide AOM as an alternative herbal treatment for diarrhea, but the scientic basisfor this usage has not been well dened.Aim of the study: In this study, we tried to investigate the antidiarrheal activity and possible mechanismsof Fructus AOM, aiming to enrich our understanding to the scientic meanings and theoreticalsignicance of Fructus AOM in clinical practice.Materials and methods: The fructus of AOM collected from Hainan province in China were macerated inthe 95% ethanol to obtain the crude 95% ethanol extract, followed by subjected to chromatographicseparation over a Diaion HP20 column to obtain 90% and 50% ethanol eluted fractions. The activities ofthe crude extract and fractions on castor oil induced acute diarrhea, rhubarb induced chronic diarrhea,gastrointestinal transit (GIT) in mice, and contractions of isolated guinea-pig ileum were evaluated.Additionally, nitric oxide (NO), gastrointestinal peptides gastrin (GAS), motilin (MTL) and somatostatin(SS) levels that related to gastrointestinal motilities were detected to demonstrate the potentialmechanisms. Ultimately, LCMS/MS method was utilized to ensure the chemical consistency.Results: The 95% ethanol extract and 90% ethanol eluted fraction signicantly delayed the onset time anddecreased the wet faeces proportion compared with control group in the castor oil induced acutediarrhea mice. In terms of further evaluation of antidiarrheal activity, the 95% ethanol extract and 90%ethanol elution displayed signicant inhibition of the intestinal propulsion at the two highest oral dosesof 20 g crude drug/kg and 1 g/kg. Moreover the 95% ethanol extract (10 and 20 g crude drug/kg) and 90%ethanol elution (0.5 and 1 g/kg) could signicantly inhibit the GIT, which was partially attributed to theincrease in NO and SS levels, and the decreased MTL. In vitro spontaneous contractions of the isolatedguinea pig ileum induced by carbachol, neostigmine and histamine were attenuated by both the extractand elution. Phytochemical analysis of 95% ethanol extract and its fractions identied the presence ofdiphenylheptanes, sesquiterpenes, and avones as the major components.Conclusions: Our in vivo and in vitro data could partly support and justify the traditional usage of FructusAOM on the treatment of diarrhea in traditional medicine.

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Abbreviations: AOM, Alpinia oxyphylla Miq.; GIT, gastrointestinal transit; NO, nitric oxide; GAS, gastrin; MTL, motilin; SS, somatostatin; 5-HT, 5-hydroxytryptaminen Correspondence to: State Key Laboratory of New Drug & Pharmaceutial, Shanghai Institute of Pharmaceutial Industry, China State Institute of Pharmaceutical Industry,

No. 1 Zhongshan North Road, Hongkou District, Shanghai 200437, China. Tel.: 86 21 55514600; fax: 86 2155514600.nn Correspondence to: State Key Laboratory of New Drug & Pharmaceutial, Shanghai Institute of Pharmaceutial Industry, China State Institute of Pharmaceutical Industry,

No. 1 Zhongshan North Road, Hongkou District, Shanghai 200437, China. Tel.: 86 21 65449361; fax: 86 2165449361.E-mail addresses: wang1986sheng@163.com (S. Wang), 183034355@qq.com (Y. Zhao), a1986bc@126.com (J. Zhang), 30695140@qq.com (X. Huang),

279695361@qq.com (Y. Wang), 594045591@qq.com (X. Xu), 443749358@qq.com (B. Zheng), 547979179@qq.com (X. Zhou), 892782662@qq.com (H. Tian),liuli1129@hotmail.com (L. Liu), qibingmei@126.com (Q. Mei).

1 These authors contributed equally to this study and share rst authorship.

Journal of Ethnopharmacology 168 (2015) 182190

1. Introduction

Diarrhea disease, a common gastrointestinal problem withpotentially fatal implications, characterized by an increased fre-quency of bowel, wet stool and abdominal pains, is a seriouspublic health challenge (Ezekwesili et al., 2004). The disease is oneof the major health threats to people in tropical and subtropicalcountries. The World Health Organization (WHO) estimates that35 billion cases occur each year, and it is responsible for about5 million deaths annually, of which 2.5 millions are malnourishedchildren of less than 5 years especially in developing countries(Page et al., 2011). According to the National Institutes of Health,the average adult can expect about four bouts of acute diarrhea peryear. Children in the United States will have had 7 to 15 episodes ofdiarrhea by age 5 (Gerald et al., 2006).

The studies of diarrhea disease in human and animal modelssuggested that many different factors may trigger diarrhea, includ-ing infections, food intolerances, intestinal disorders and medica-tions (Pimentel et al., 2010; Al Jarousha et al., 2011; Barnoy et al.,2011; Young and Schmidt, 2004). Impaired intestinal absorption isa major mechanism for diarrhea and is generally accompaniedwith improvement of the gastrointestinal motility (ShuangYu etal., 2011; LaiFu et al., 2008). Accordingly, NO is believed to be aninhibitory neurotransmitter that mediates gastrointestinal motilityin physiological and certain pathophysiologic states (Takahashi,2003; Wirthlin et al., 1996). It has been reported that NO couldregulate prostaglandin (PG) synthesis to inuence gastric mucosalintegrity (Kato et al., 1997a, 1997b). On the other hand, NO has alsobeen shown to be involved in the inhibition of smooth musclecontraction, resulting in various aspects of the pathophysiology ofgastrointestinal mucosae (Jimenez et al., 2002). Moreover, theincrease in gastrointestinal motility and changes in the plasmaconcentrations of a series of gastrointestinal peptides, includingmotilin (MTL), gastrin (GAS) and somatostatin (SS), are commonlyobserved in the patients with chronic diarrhea (Jiang et al., 2013;Mondal et al., 2012).

Nowadays, a wealth of the traditional medicines from plants havebecome increasingly signicant in modern medicine, and lots ofscholars are payingmore attention tomedical plants. WHO has createda Diarrheal Disease Control (CDD) Programme that studying traditionalmedicine for the management of diarrhea disease. Interestingly,numerous experienced clinicians incline to use holistic remedies fordiarrhea disease given the complexity of the symptoms (Sanger, 2007).Alpinia oxyphyllaMiq. (Zingiberaceae), including 250 species and about48 species grown in the subtropical parts of China (Xu et al., 2012),mainly containing Sesquiterpenes, diterpenes, avonoids, and diaryl-heptanoids (Shi et al., 2014; Koo et al., 2004; Li et al., 2013), isextensively used as a traditional Chinese medicine in curing diarrhea,intestinal disorders and dieresis (Chinese Pharmacopoeia Commission,2010; Zhang et al., 2013). Previous studies indicated A. oxyphyllashowed a wide range of biological activities, and it has been mainlyused for treating diarrheawith splenic cold in traditional folk medicine,but reports on antidiarrhea effect of A. oxyphyllawere relatively limitedin recent 5 years (Zhang et al., 2011; Jianguang et al., 2012; He et al.,2010). Additionally, although a host of traditional Chinese medicinecompounds provide AOM as an alternative herbal treatment fordiarrhea, there was very few scientic report on the antidiarrhealactivity and underlying mechanisms of actionwere not clear. Thus, thepresent study was focused on the evaluation of the antidiarrhealactivity of the crude extract and fractions from AOM using castor oilinduced acute diarrhea, rhubarb induced chronic diarrhea in vivo andcontractions of isolated guinea-pig ileum in vitro. Furthermore, NOlevel and a series of gastrointestinal hormone including GAS, MTL andSS were detected to explore the potential mechanisms underlyingAOM exerted the antidiarrhea effect. Subsequently, LCMS/MS methodwas applied to ensure the chemical consistency.

2. Materials and methods

2.1. Plant material

The fructus of AOM were collected from Hainan province, Chinain 2013. The plant was identied by the locals and traditionalhealers in the implemented areas. After botanical identication bycomparative macroscopic and microscopic studies by A/Prof.Jianping Tian (Department of Pharmacognosy, Hainan MedicalUniversity), the voucher specimens were identied as A. oxyphyllaMiq. (Zingiberaceae) and the voucher specimen (No. 20130518)was deposited in the herbarium of the Department HerbalResources of Hainan Medical University. The fructus were driedat room temperature and evaporated to powder.

2.2. Preparation of extract and fractionations

5 kg of dried fructus were separated, shade dried, and pow-dered mechanically in an electrical blender. The 95% (v/v, 3000 ml)ethanol was added, and the mixture was sonicated in an ultrasonicbath at 60 1C for 1 h. The mixture was ltered and the residue wasextracted two more times. Subsequently, the three ltrates werecombined, concentrated in a rotary evaporator at 67 1C (yield:13.8%) (EYEL4 SB-1100). The obtained crude extract was used forthe evaluation of antidiarrheal activity and preparation of fractions(90% and 50% ethanol elution). Half part of the 95% ethanol extractwas subjected to chromatographic separation over Diaion HP 20(10 kg) with H2O, 50% and 90% EtOHH2O successively until theefuent was colourless (Jun et al., 2006).

2.3. Castor oil induced acute diarrhea in vivo

The method described by Joseph was followed for this inves-tigation (Shelton et al., 2007). ICR (Impriting control region) mice(weighing 1822 g, 68 weeks) were randomly divided intonegative control, positive control and test groups, ten mice foreach group. Positive drug was dissolved in distilled water andtested samples (95% crude extract, 90% ethanol elution and 50%ethanol elution) were dissolved in distilled water and Polyethyleneglycol (PEG400, nal concentration of 20%).

Group 1: Negative control group, distilled water and PEG400(20%) was orally administrated (20 ml/kg);Group 2: Positive control group, smecta (montmorillonitepowder, B0174), orally administrated by gavage (6 g/kg);Group3: Test group, the 95% ethanol extract (14.4 g crude drug/g),by gavage (5 g crude drug/kg);Group 4: Test group, the 95% ethanol extract, by gavage (10 gcrude drug/kg);Group 5: Test group, the 95% ethanol extract, by gavage (20 gcrude drug/kg);Group 6: Test group, the 90% ethanol elution (40.1 g crude drug/g), by gavage (0.25 g/kg);Group 7: Test group, the 90%ethanol elution, by gavage (0.5 g/kg);Group 8: Test group, the 90%ethanol elution, by gavage (1 g/kg);Group 9: Test group, the 50%ethanol elution (26.9 g crude drug/g),by gavage (0.25 g/kg);Group 10: Test group, the 50%ethanol elution, by gavage (0.5 g/kg);Group 11: Test group, the 50%ethanol elution, by gavage (1 g/kg).

Thirty minutes after the respective treatments, acute diarrheawas induced by oral administration of castor oil (0.2 ml/mouse).Following the delivery of castor oil, the animals, placed in separatecages over clean white papers and replaced the white papers everyhour, were inspected up to ve hours for the presence of thetypical diarrhea. The following parameters were recorded: the

S. Wang et al. / Journal of Ethnopharmacology 168 (2015) 182190 183

time elapsed between the administration of the cathartic agentand the excretion of the rst diarrhoeic faeces, the total number ofwet faeces excreted by the animals in 5 h, the weight of wet faecesand the total weight of faecal output (Nsaka et al., 2012).

The diarrhoeic faeces proportion % the weight of wet faecesthe total weight of faecal output

100%

Throughout the experiments, mice were maintained in plasticcages at 2172 1C, on a 12 h light/dark cycle and with free access towater. Animal welfare and experimental procedures were carriedout in accordance with the Guide for the Care and Use ofLaboratory Animals, and were approved by the animal ethicscommittee of Genetics and Cytology of Shanghai Institute ofPharmaceutial Industry. The registration number of animal ethiccommittee is 2013235HM and the approved date of animal ethicsDecember 23, 2013. All possible efforts were made to minimize theanimals suffering and to reduce the number of animals used.

2.4. Rhubarb induced chronic diarrhea in vivo

ICR mice (weighing 1822 g, 68 weeks), were randomlydivided into normal, negative control, positive control and testgroups with ten animals per group and housed in separate cages.Chronic diarrhea was induced by oral administration of rhubarbdecoction (1 g crude drug/ml, 0.2 ml/10 g weight) for 21 days.After the administration of rhubarb for 14 days, normal andnegative control groups received a vehicle (0.2 ml/10 g weight).The positive control group received atropine (Xinxiang Changlepharmaceutical LTD, 1108185) dissolved in distilled water at thedose of 0.3 mg/kg orally administrated by gavage, test groupsdissolved in distilled water and Tween 80 received the 95% ethanolextracts (14.4 g crude drug/g) and the ethanol elution of 90%(40.1 g crude drug/g), separately by gavage (95% ethanol extract atthe doses of 5, 10 and 20 g crude drug/kg and 90% ethanol elutionat the doses of 0.25, 0.5 and 1 g/kg). Following the respectivetreatments for 7 days and 1 h after the last administration, 0.2 mlof a 10% suspension of charcoal meal (charcoal:CMCNa2.5:1,50 ml) was given to each mouse by intragastric gavage, the micewere then sacriced by cervical dislocation after 30 min and thesmall intestine and the stomach were isolated immediately. Thedistance traveled by the charcoal meal as well as the total length ofthe intestine were measured. Intestinal propulsion rate wasexpressed as the percentage of the distance traveled by thecharcoal meal relative to the total length of the small intestine(Jing et al., 2009). Besides, gastric residual rate was dened as thepercentage of the weight of stomach contents to the total weightof charcoal meal administered for each mouse.

2.5. Determination of plasma levels of NO and gastrointestinalpeptides

Thirty minutes after oral administration of 10% charcoal meal tothe ICR mice that bearing rhubarb induced chronic diarrhea asmentioned above, the inferior vena cava of each mouse waspunctured and the blood was collected in tubes using a universalanticoagulant (heparin) before the mice were sacriced. Thesamples were centrifuged at 3000 rpm for 10 min to obtain theplasma. Plasma levels of NO, MTL, SS and GAS were detected bymouse ELISA kits following the manufacturers instructions.

2.6. Isolated tissue experiments

Locally bred Guinea pigs of both sexes were fasted for 24 h, thenanesthetized with sodium pentobarbital. The Guinea pigileum wasdissected out, immersed in Tyrodes solution and cleaned off mesen-teries. Segments of Guinea pig ileum approximately 2 cm long were

maintained in Tyrodes solution bath at 37 1C and continuouslybubbled with a mixture of 95% O2 and 5% CO2. A preload of 0.5 g oftension was adjusted to each tissue segment and spontaneouscontraction was recorded. The tissues were left to equilibrate for40 min before the additions of any drugs. After the equilibrium,80 mM KCl was added to the bath and the contractionwas consideredas the reference response. Ileum segments were allowed to equili-brate for 30 min based on the KCl response. Aiming at elucidating ofthe bioactivities of 95% crude extract and 90% ethanol elution,carbachol (National Institute for Food and Drug Control, 200301),Neostigmine (Xiya Regent, 201209152), Histamine (kayon, A0166) and5-HT (kayon, Q2156) were used as spasmodic agents, producingsustained contractions that enable to determine the inhibitory acti-vities. The tested drugs and positive drug Atropine (Aladdin,A109524) were incubated with ileum segments for 5 min, followedby adding the spasmodic agents to obtain the concentration-dependent inhibitory responses. The relaxation of isolated tissuepreparation was expressed as the percentage of response caused bytested drugs to the control response mediated by the spasmodicagents added to the tissue bath.

2.7. Phytochemical analysis

LCMS/MS method was applied to analyze the 95% ethanolextract and fractions. Liquid chromatography Chromatographicanalysis was performed on a Shimadzu LC-20AD UPLC system(Shimadzu Corp., Tokyo, Japan), and a Shim-Pack XR-ODS column(100 mm2.0 mm, 2.2 m) was employed. Mass spectrometrydetection was conducted using a 4000 plus Triple Quadrupole MS(Applied Biosystems Incorporation, Foster, CA, USA) equipped withan electrospray ionization source (ESI). Operation conditionsreferred to the methods (Zhang et al., 2010).

2.8. Statistical analysis

The data obtained from at least three independent experimentswere presented as means7SD, and statistical comparisonsbetween groups were performed using one-way ANOVA followedby Students t-test with P values of o0.01 (nn) or o0.05 (n)compared with negative control.

3. Results

3.1. Effects of AOM on castor oil induced acute diarrhea in mice

The yield of crude 95% ethanol extract was 690 g from 5 kg offresh AOM. Half part of the 95% ethanol extract was subjected tochromatographic separation over Diaion HP20 with H2O, 50% and90% EtOH, yielding 92.9 g of 50% ethanol elution and 61.6 g of 90%ethanol elution.

In the present study, mice in control group receiving only vehicle(20 ml water/kg) produced intense diarrhea, with 104.13718.17 minof onset time, and characterized by 5.2571.75 wet faeces and76.1577.53% of wet faeces proportion 5 h after the administrationof castor oil. In the normal group, the increases of stool mass andstool liquidity, the primary hallmarks of diarrhea, were not observed.However, the oral administration of positive drug Smecta showed adiarrhea inhibition with a signicant increase of onset time anddecreasing the wet faeces proportion compared with control group(Po0.01). The results indicated that both the 95% ethanol extract and90% ethanol elution signicantly prolonged the onset times ofdiarrhea and reduced the wet faeces proportions of animals indosedependent manners when orally administered 95% ethanolextract at doses of 10 and 20 g crude drug/kg body weight and 90%ethanol elution at doses of 0.5 and 1 g/kg (Table 1). However, the 50%

S. Wang et al. / Journal of Ethnopharmacology 168 (2015) 182190184

ethanol elution at all oral doses had no obvious inhibitory effects ondiarrhea. All doses of tested drugs were referred to the literature andtoxicity experiments (90% ethanol elution, LD5020 g/kg).

3.2. Effect of AOM on gastrointestinal transit in rhubarb inducedchronic diarrhea

Our data regarding the effects of AOM on acute diarrheademonstrated that 95% crude extract and 90% ethanol elutionmight contain certain bioactive sites. Further studies concerningthe bioactive sites revealed that the increase of stool mass andstool liquidity were observed on day 12 after the administration ofrhubarb decoction. As shown in Fig. 1, intestinal propulsion ratewas dramatically increased in the control group (59.0720.4%)compared with that in normal group (42.8711.6%). Consistently,0.3 mg/kg of positive drug atropine delayed the intestinal propul-sion signicantly compared with control group (Po0.05). Treat-ments with 5 g crude drug/kg and 10 g crude drug/kg of 95%ethanol extract and 0.25 g/kg and 0.5 g/kg of 90% ethanol elutionshowed no changes on intestinal propulsion rates compared withcontrol group. However, the administrations of 20 g crude drug/kgof 95% ethanol extract and 1 g/kg of 90% ethanol elution reducedthe intestinal propulsion rate to 35.9711.7 (Po0.05) and38.679.0% (Po0.05), respectively. Fig. 2 illustrated that gastricresidual rate was decreased signicantly in the control groupcompared with that in normal group (Po0.05). The oral admin-istration of positive drug atropine exhibited a signicant increaseof gastric residual compared to control group with 27.8714.3% ofgastric residual rate (Po0.05). The gastric residual rates of miceafter oral administrations of 10 g crude drug/kg and 20 g crudedrug/kg of 95% ethanol extract increased to 46.479.8% and47.9714.1%, respectively (each Io0.05). Furthermore, the 90%ethanol elution dose-dependently boosted the gastric residuals,with either 0.5 g/kg or 1 g/kg dosage signicantly increasing to51.5724.0% (Po0.05) and 58.9725.4% (Po0.01) compared withcontrol group.

3.3. Effects of AOM on plasma NO and gastrointestinal peptides

In terms of plasma level of NO (Fig. 3A), the NO level of controlgroup (1.7770.03 M) was signicantly reduced compared withthat of normal group (Po0.01). 5 g crude drug/kg and 10 g crudedrug/kg of 95% ethanol extracts treated groups signicantlyincreased the levels of NO (Po0.05) compared with control group,but the high dosage (20 g crude drug/kg) had no obvious effect.The mice in treatments of 0.5 g/kg and 1 g/kg of 90% ethanolelutions and the mice received 0.3 mg/kg of positive drug atropineshowed dramatically increase of NO level (1.8170.02 M,Po0.05) compared with that in response to rhubarb. Fig. 3Bshowed the level of MTL (0.3570.01 pg/ml) in the control groupwas enhanced signicantly compared with that of normal group(Po0.05). MTL levels of mice received 20 g crude drug/kg of 95%ethanol extract and 0.5 g/kg or 1 g/kg of 90% ethanol elution weredropped signicantly (each Po0.05) compared with that ofcontrol group. The plasma levels of SS in the control group, asshown in Fig. 3D, reduced signicantly to 0.3570.03 pg/mlcompared with that in normal group. 20 g crude drug/kg of 95%ethanol extract treated group increased the level of SS (Po0.05)dramatically compared with control group, and 90% ethanolelution at either 0.5 g/kg or 1 g/kg dosage increased the amounts

Table 1Antidiarrhea activity of samples from AOM against castor oil -induced diarrhea(AV7SD, n10).

Samples Oral dose(g/kg)

Onset time(min)

Total number ofwet faeces

%wet faecesproportion

Control 104.13718.17 5.2571.75 76.1577.53Smecta 6 128.75713.22** 3.8870.99 60.1713.38**

95% Ethanolextract

5 103.38714.20 4.7571.16 70.3879.99

95% Ethanolextract

10 121.50712.41* 4.8872.03 65.35710.95*

95% Ethanolextract

20 134.13720.99** 4.3871.41 58.23711.31**

90% Ethanolelute

0.25 104.88715.87 4.6371.60 70.30712.74

90% Ethanolelute

0.5 125.13718.91* 4.5071.41 71.42710.14

90% Ethanolelute

1 128.13710.00** 4.6371.92 62.50711.89*

50% Ethanolelute

0.25 104.25718.40 4.8871.46 77.47711.26

50% Ethanolelute

0.5 109.13711.22 4.8871.36 76.8878.92

50% Ethanolelute

1 108.38717.16 4.6371.51 66.88714.38

n Po0.05 vs. control; Students t-test.nn Po0.01 vs. control; Students t-test.

Fig. 1. The 95% ethanol ectract and 90% ethanol elution inhibited intestinalpropulsions in rhubarb induced chronic diarrhea. Normal and control groupsreceived 20 ml/kg of vehicles orally. Positive control group received Atropine at adose of 0.3 mg/kg orally, and the 95% ethanol extract treated groups received thedoses of 5, 10, and 20 g crude drug/kg orally. Additionally, the 90% ethanol elutiongroup received the doses of 0.25, 0.5, 1 g/kg. Values are means7SD (n10).nPo0.05 compared with control group; #Po0.05 compared with normal group.

Fig. 2. The 95% ethanol ectract and 90% ethanol elution increased gastric residualsin rhubarb induced chronic diarrhea. Values are means7SD (n10). nPo0.05,nnPo0.01 compared with control group; #Po0.05compared with normal group.

S. Wang et al. / Journal of Ethnopharmacology 168 (2015) 182190 185

of SS dose-dependently. No signicant differences of GAS levelwere observed between all groups, as shown in Fig. 3C.

3.4. Effects of AOM on contractions of isolated guinea pig ileum

Accordingly, in vitro studies suggested that carbachol, neostig-mine and histamine had strong spasmodic effects, reaching max-imum levels at 5 M (Fig. 4a), 6.25 M (Fig. 4c) and 12.5 M(Fig. 4b), respectively. However, 5-HT had little impact on thecontraction of guinea pig ileum in vitro (Fig. 4d). To characterizethe inhibitory effects of 95% ethanol extract and 90% ethanolelution, tissues were pretreated with extract and fractions, fol-lowed by spasmodic agents carbachol (5 M), neostigmine(6.25 M) and histamine (12.5 M) were added. The spasmodiceffects of carbachol (5 M) diminished signicantly (Po0.05) inthe presence of 25 g/ml and 50 g/ml of 95% ethanol extracts.Additionally, the presence of 90% ethanol elutions at 25 g/ml,50 g/ml and 100 g/ml doses reduced (Po0.01) the carbacholeffects dose-dependently (Fig. 5A). Fig. 5B showed that 100 g/mlof 95% ethanol extract antagonized (Po0.05) the effect of neos-tigmine to 20.3711.9%, the effects of neostigmine were droppedto 29.3717.3% and 21.1710.1% in reponse to 50 g/ml and100 g/ml of 90% ethanol elution, respectively (each Po0.05).The effect of histamine was reduced by 100 g/ml of 95% ethanolextract (Po0.05), and diminished in the presence of 50 g/ml and100 g/ml of 90% ethanol elutions (Po0.01) (Fig. 5C).

3.5. Phytochemical analysis

The results of LCMS/MS exhibited the diphenylheptanes,sesquiterpenes, and avones were the main components of 95%ethanol extract, with 10.611.4 min of retention time (Fig. 6A).Representative compounds of the diphenylheptanes includedya-kuchinone A, yakuchinoneB and oxyphyllacinol. The avonescontained tectochrysin and izalpinin, and nootkatone belongs tosesquiterpenes. All these three types of main components weredetected in the 90% ethanol elution (Fig. 6B), but extremely lowamounts were found in the 50% ethanol elution (Fig. 6C).

4. Discussion and conclusions

A. oxyphylla Miq., a widely distributed medical plant in Hainan,China, recorded in Chinese Pharmacopoeia 2010, has been used forthe treatment of diarrhea for several centuries (Chinese Pharm-acopoeia Commission, 2010). In the present study, we investigatedthe crude extract and fractions of AOM on the antidiarrheal andgastrointestinal modulatory effects to rationalize the traditional uses.Diarrhea is generally divided into acute and chronic types, acutediarrhea lasts from a couple of days up to a week while chronicdiarrhea can be dened in several ways, but almost all last more thanthree weeks. Pathophysiologic mechanisms of acute diarrhea (e.g.,Giardia lamblia infection, antibiotic use) are different from those ofchronic diarrhea (e.g., secretory dysfunction from thyrotoxicosis,

Fig. 3. The 95% ethanol ectract and 90% ethanol elution increased plasma levels of NO, SS and decreased MTL levels in rhubarb induced chronic diarrhea. Values aremeans7SD (n10). nPo0.05, nnPo0.01 compared with control group; #Po0.05, ##Po0.01 compared with normal group.

S. Wang et al. / Journal of Ethnopharmacology 168 (2015) 182190186

Fig. 4. The concentration dependent spasmodic effects on isolated Guinea pig ileum preparations of carbachol, neostigmine, histamine and 5-HT.

Fig. 5. The relaxant effects of 95% ethanol extract and 90% ethanol elution of Fructus Alpinia oxyphylla. 25 and 50 g/ml of 95%ethanol extract signicantly reduced carbacholinduced contraction (Po0.05) and 100 g/ml of 95% ethanol extract signicantly inhibited neostigmine and histamine induced contrations. 50 and 100 g/ml of 90% ethanolelution inhibited carbachol, neostigmine and histamine induced contractions dose-dependently. The values are represented as mean7SD from 4 independent experiments.nPo0.05, nnPo0.01 compared with control group (one-way ANOVA followed by Students t-test).

S. Wang et al. / Journal of Ethnopharmacology 168 (2015) 182190 187

lactose or fructose intolerance) (Talal and Murray, 1994), thus twotypes of diarrhea animal models were carried out to investigate theeffects of AOM in this study.

Castor oil, hydrolyzed in the upper small intestine to ricinoleicacid, which can stimulate uid secretion, inhibit water andelectrolyte absorption, reduce active Na and K absorption,and decrease Na , K-ATPase in the small intestine and colon(Leng-Peschlow, 1993), was given to each mouse by intragastricgavage as a very effective laxative to induce acute diarrhea ofmouse. Our results indicated that both the 95% ethanol extractand90% ethanol elution signicantly prolonged the onset time and

reduced the wet faeces proportion in dose-dependent manners,while the 50% ethanol elution at all oral doses, showed noinhibitory effects on acute diarrhea (Table 1). It is well knownthat the action mechanism of castor oil induced diarrhea isassociated with gastrointestinal motility (Uddin et al., 2005). Sincethe 95% ethanol extract and the 90% ethanol elution of AOMsuccessfully inhibited the castor oil induced diarrhea, they mightexert the effect by antimotility mechanism. Subsequently, rhubarbinduced chronic diarrhea in mice was used to performa furtherstudies on the antidiarrhea activity and mechanisms. Rhubarb, themost traditional and widest application is the treatment of

Fig. 6. Representative schematic diagram of LCMS/MS: the 95% ethanol extract of Fructus AOM (A); the 90% ethanol elution of Fructus AOM (B); the 50% ethanol elution ofFructus AOM (C).

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constipation due to its effective purgative activity. Anthraquinonesare usually considered as the chemical basis of purgative activity ofrhubarb and the mechanism refers to promoting intestinal peri-stalsis and accelerating colonic transit (Qin et al., 2011). In thepharmacological model used in this study, the administration ofeither 20 g/kg of 95% ethanol extract or 1 g/kg of 90% ethanolelution signicantly reduced the intestinal propulsion rate(Po0.05), and the 95% ethanol extract and 90% ethanol elutionsignicantly increased the gastric residual rates in dose-dependentmanners. These ndings clearly suggested that 95% ethanol extractand 90% ethanol elution largely affected the gastrointestinalmotilities, which was in good agreement with the hypothesis.

In this study, the results showed that the 95% ethanol extractand the 90% ethanol elution could signicantly reduce castor oilinduced diarrhea as well as inhibit the gastrointestinal motilities.NO and gastrointestinal peptides haves been reported to playimportant roles in gastrointestinal motility. NO is the mostimportant non-adrenergic, non-cholinergic inhibitory neurotrans-mitter in the gut and plays a critical role in several majorphysiologic processes of gastrointestinal tract such as motility,secretion, digestion, absorption and elimination (Kochar et al.,2011). Interestingly, we found that 95% ethanol extract and 90%ethanol elution increased the plasma NO levels in the rhubarbinduced chronic diarrhea, implying that the antidiarrheal effect ofFructus AOM was partially mediated by NO level. Of note, GASinhibits contractions in the proximal stomach while MTL aug-ments proximal gastric contraction and speeds gastric emptying ofliquids. The contractions of the distal stomach can be stimulatedby GAS and MTL. Besides, the stimulatory effect of motilin on smallintestinal contractions is greatest in the duodenum (Thomas et al.,1979). Extensive functional and morphological studies havedemonstrated the pivotal roles of SS in the regulation of a widevariety of gastrointestinal activities particularly its profound inhi-bitory effects on gastrointestinal motility (Op den bosch et al.,2009). In the present study, the results showed that the 95%ethanol extract and the 90% ethanol elution could signicantlyreduce the MTL plasma levels and increase SS levels in diarrhea,but they had no effects on the plasma levels of GAS. These resultsdemonstrated that Fructus AOM might exhibit its antidiarrheaeffect partially by affecting the levels of NO, gastrointestinalpeptides MTL and SS.

Based on the effect of Fructus AOM on gastrointestinal moti-lities in vivo, we further performed a study on the spasmolyticactivity using isolated guinea pig ileum in vitro. Nervous control ofgastrointestinal motility is extremely complex, is mainly regulatedby the enteric system and modulated by extrinsic nerves (Hansen,2003). This system covers its multiplicity of transmitters andreceptors, including cholinergic, adrenergic, dopamine, histamine,5-hydroxytryptamine (5-HT), opioid and gamma-aminobutyricacid (GABA) (Demoi et al., 1989). Surprisingly, the in vitro resultsdisplayeded that carbachol, neostigmine and histamine decreasedtheir stimulant effects on contractions of isolated guinea pig ileumin the pretreatments of the 95% ethanol extract and 90% ethanolelution, indicating the antidiarrheal activity of Fructus AOM waspossibly mediated by the cholinergic, adrenergic and histamine.The data from the phytochemical analysis revealed diphenylhep-tanes, sesquiterpenes, and avones that rarely detected in the 50%ethanol elution were clearly detected in the samples of the 95%ethanol extract and 90% ethanol elution. The presence of thesecompounds in both 95% ethanol extract and 90% ethanol elutionfrom Fructus AOM may largely contribute to and justify theantidiarrheal activity.

Based on the present study, it could be concluded that thereported studies could partly support the claim about the traditionalusage of Fructus AOM for the cure of diarrhea in folk medicine. The95% ethanol extract and 90% ethanol elution possessed antidiarrheal

activities due to the inhibition of gastrointestinal motilities, possiblymediated by affecting the levels of NO and a series of gastrointestinalpeptides. The phytochemical investigations will continue in order toisolate and identify compounds responsible for the observed activ-ities. Further studies are needed to clarify the exact mechanisms anddene the chemical compounds that contribute to the traditionalcurative effects.

Conict of interest

The authors declare no conicts of interests.

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S. Wang et al. / Journal of Ethnopharmacology 168 (2015) 182190190

Antidiarrheal effect of Alpinia oxyphylla Miq. (Zingiberaceae) in experimental mice and its possible mechanism of actionIntroductionMaterials and methodsPlant materialPreparation of extract and fractionationsCastor oil induced acute diarrhea in vivoRhubarb induced chronic diarrhea in vivoDetermination of plasma levels of NO and gastrointestinal peptidesIsolated tissue experimentsPhytochemical analysisStatistical analysis

ResultsEffects of AOM on castor oil induced acute diarrhea in miceEffect of AOM on gastrointestinal transit in rhubarb induced chronic diarrheaEffects of AOM on plasma NO and gastrointestinal peptidesEffects of AOM on contractions of isolated guinea pig ileumPhytochemical analysis

Discussion and conclusionsConflict of interestReferences