Journal of Chemical and Pharmaceutical Research, 2017, 9(10): Research Article

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1 Available online Journal of Chemial and Pharmaeutial Researh, 2017, 9(10): Researh Artile ISSN : CODEN(USA) : JCPRC5 Antidiarrhoeal, Antibaterial Ativities and Growth Stimulatory Effet on Some Probioti Organisms of Aqueous and Methanol Leaves Extrat of Dissothis Thollonii Tadjoua Thoumbou Herve 1, Ateufak Gilbert 1*, Shabana Usman Simjee 2, Dongmo Matafak Olivette Laure 3, Yousseu Nana William 1, Rukesh Maharjan 2, Muhammad Iqbal Choudhary 2 and Gatsing Donatien 4 1 Department of Animal Biology, Dshang University, Laboratory of Animal Physiology and Phytopharmaology, PO Box 67, Dshang, Cameroon 2 Hussain Ebrahim Jamal Researh Institute of Chemistry, International Center for Chemial and Biologial Sienes, University of Karahi, Karahi 75270, Pakistan 3 Department of Biohemistry, Dshang University, Laboratory of Biohemistry of Mediinal Plants, Food Sienes and Nutrition, PO Box 67, Dshang, Cameroon 4 Department of Biohemistry, Dshang University, Laboratory of Mirobiology and Antimirobial Substanes, PO Box 67, Dshang, Cameroon ABSTRACT Dissotis thollonii (D. thollonii) is traditionally used for the treatment of diarrhoea in Afria. The study aim to evaluate aqueous and methanol extrats of leaves of D. thollonii (Melastomataeae) for their antidiarrhoeal, antibaterial ativities as well as probiotis growth stimulation effet. Both extrats effets were evaluated using rat animal models for in vivo assay. Antimirobial ativities were also been evaluated. D. thollonii extrats at all doses (125, 250 and 500 mg/kg) signifiantly inhibited the mean volume of intestinal fluid in prostaglandin E2 indued enteropooling assay, with the treatment of both extrats similarly to that of the standard antidiarrhoeal drug (loperamide), as well as the signifiant redution of eletrolytes onentration in the intestinal fluid. There was no inhibition, but rather growth of all probiotis tested. Extrats inhibited growth of 3 referene baterial strains (Staphyloous aureus NCTC 6571, Baillus subtili ATCC and Enteroous feaalis ATCC 39532) with minimum inhibitory onentrations (MIC) of 1000, 500 and 250, orresponding to perentage inhibition of ± 0.47, ± 4.40, and ± 4.72 with methanol extrat respetively. We also observed inhibition of one linial isolate (Heliobater pylori) with both extrat at minimum inhibitory onentrations (MIC) of 2000, orresponding to the inhibition perentage of ± 0.60 and ± 0.66 respetively to the aqueous and methanoli extrat. In vivo, extrats provoqued at all doses, a signifiant derease (P<0.001) of the baterial load in the faees of rats, during all days of treatment ompare to those whih was treated with distilled water. Histologial examination of the aeum showed at the dose of 250 and 500 mg/kg of aqueous and methanol extrat respetively, no degradation of different layers ompare to neutral and positive ontrol (iprofloxain). These results attested the ethnopharmaologial used of D. thollonii leaves in the treatment of diarrhoea and gastrointestinal infetions. Keywords: Dissotis thollonii; Antidiarrhoea; Prostaglandin E2; Antimirobial; Probiotis 95

2 INTRODUCTION Diarrhoea is a major health burden espeially in developing ountries where sanitation is inadequate. Diarrhoea has led to a signifiant inrease in infant mortality and morbidity partiularly in low inome settlements [1]. Charaterized by more than three loose motions of stool per day, diarrhoeal diseases have remained astronomial in spite of the efforts provided by organizations to urb with. Nowadays, there is a great interest in herbal remedies for the treatment of a number of ailments like diarrhea. These plants an be extremely useful as leads for syntheti modifiation and optimization of biologial ativity. Thus, the World Health Organizaion enourages the development of the traditional mediine whih uses the mediinal plants partly [2]. Among the many exploited speies, D. thollonii is traditionally used in the treatment of several evils. Thus it is used by traditional healer in Senegal and in west region of Cameroon on ezematous wounds [3]. The leaves are reommended in therapy of the gastrointestinal disorders (obstrution, amoebiasis, diarrhoeas, vomiting, and onstipation) and ulers. The aim of this study was, therefore, to evaluate the antidiarrhoeal ativities of aqueous and methanoli leaves extrats of D. thollonii in experimentally indued-diarrhoea in rats, the in vitro antibaterial ativities of the same extrats over some enterobateria strains and to determine the effet of these extrats on benefiial intestinal miroflora. MATERIALS AND METHODS Colletion and Identifiation of Plant Material The fresh leaves of D. thollonii were olleted in September 2016 in the Bafou village, Menoua division, West Region of Cameroon. A sample was identified at the National Herbarium of Cameroon (Yaoundé) by omparison with existing vouher speimen No.13292/SRF Cam. Preparation of the Plant Material The olleted plant parts (leaves) were separated from undesirable materials. They were dried under shade, ground and stored in an airtight ontainer prior to extration. Preparation of Plant Extrats The aqueous extrat was prepared by boiling 800 g of powder in 5 L distilled water for 15 min as indiated by the traditional healer. The deotion was ooled during 1 h and filtered using filter paper (Whatman No.1), and the filtrate was evaporated in a regulated drying oven at 35 C to give 51.6 g of the aqueous extrat orresponding to an extration yield of 6.45% (w/w). The other portion of leaf powder (900 g) was maerated in 6.75 L of methanol for 72 h, filtered and the solvent removed from the extrat under redued pressure, using a Bühi (R-200) rotary evaporator at 65 C. This gave 63.8 g of the methanol extrat, orresponding to a yield of 7.09% (w/w). Experimental Animal Wistar Albino female rats of 2.5 to 3 months and weighing on average 180 g were used for infetious indued diarrhoea model whereas female (2 to 2.5 months) of the same rat strain, weighing on average 140 g, were used for the tests of prostaglandin E2 indued enteropooling. They were bred in the Animal House Faility, Panjwani Center for Moleular Mediine and Drug Researh (PCMD), International Center for Chemial and Biologial Sienes. Prior to experimental protool, the rats were alimatized for 48 h to laboratory onditions for minimizing any nonspeifi stress. The studies were onduted aording to the Protool Number#: as assigned by Institutional Animal Care and Use Committee (IACUC). Chemials and Reagents All reagents used in the study were of high purity. The hemials inluded isopropyl alohol 70%, 90% and100%, xylene, hematoxylin, 0.5% eosine, methanol, deonized water, distilled water, saline solution, MTT (3-[4,5- dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide), AlamarBlue, iprofloxain (Hilton Pharma, Korangi Industrial Area, Karahi, Pakistan), Doxyyline Hylate (Pfizer, S.I.T.E, Karahi, Pakistan), Mueller Hinton broth, Manitol Salt Agar, Tryptone Soy Agar, Brain Heart Infusion Broth, Brain Heart Infusion Agar, loperamide (Zadip Pharma NIG. LTD 488, Town planning Way, Hupeju, Lagos), Prostaglandin E2 (ZAFA Pharmaeutial Laboratory. A-46, S.I.T.E., North Karahi-Pakistan). 96

3 Miroorganisms The miroorganisms used in this study inluded 6 baterial strains [Esherihia oli ATCC (E. oli), Staphyloous aureus NCTC 6571 (S. aureus), Pseudomonas aeruginosei NCTC (P. aeruginosei), Baillus subtili ATCC (B. subtili), Salmonella typhi ATCC (S. typhi) and Enteroous feaalis ATCC (E. feaalis)], one linial isolate (Heliobater pylori) and 6 probiotis (Latobaillus latis, Pedioous aidilatisi, Latobaillus fermentum strain1, Latobaillus fermentum strain2, Enteroous durans and pedioous lolie LMG-25667). These miroorganism were hosen based on their linial and pharmaologial importane [4]. They were obtained from Hussain Ebrahim Jamal Researh Institute of Chemistry, International Center for Chemial and Biologial Sienes, Karahi, and were used for evaluating antimirobial ativity. The strains were maintained at 4 C on agar plates. In vitro Antimirobial Ativity Alamar blue assay: For this assay, a single olony of different strains of bateria grown in Tryptone Soy Agar (TSA) and in BHI Agar for Heliobater pylori, were inoulated in Mueller Hinton Broth (MHB) and inubated overnight at 37 C for baterial strains and for Heliobater pylori, inoulation was done in Urea Broth, then inubation during 48 h at 37 C at 10% CO 2. Fully grown turbid baterial ulture was then diluted to adjust with 0.5 Mfarland Turbidity Index ( CFU/ml). Stok solution (80 mg/ml) of different extrats was prepared in distilled water. 10 μl of extrats were plaed in tripliate in the first well of sterile 96-well miro titer plate exept the ontrol wells. This gives onentration of 4000 μg/ml of extrat, and a serial two fold dilutions was performed to obtain 7 onentrations, ranging from 4000 to 62.5 μg/ml for the extrats. Finally, 7 μl of baterial suspension were then added in eah well. Plates were sealed by parafilm and inubated 20 hr at 37 C. Next day, 10% AlamarBlue dye was added in eah well and inubated in shaking inubator at 80 rpm, 37 C for 2-3 hrs. The olor hange from blue to reddish pink shows there is growth of bateria [5]. The absorbane was read at 570 nm and 600 nm in spetrophotometer, and the % inhibition of different extrat was alulated [5]. MTT assay: In this assay, a single olony of different strains of Probiotis grown in Brain Heart Infusion Agar (BHIA) were inoulated in Brain Heart Infusion Broth (BHIB) and inubated overnight at 37 C. Fully grown turbid baterial ulture was then diluted to adjust with 0.5 Mfarland Turbidity Index ( CFU/ml). Stok solution (160 mg/ml) of different extrat was prepared in distilled water, and 5 μl, 3.75 μl and 2.5 μl respetively for the onentration of 4000 μg/ml, 3000 μg/ml and 2000 μg/ml of these extrats were plaed in well of flat bottom, polystyrene, sterile 96-well miro titer plate exept the ontrol wells, in final 200 μl solution. Eah onentration was plaed in tripliates. Finally, baterial suspension ( CFU/mL) was then added in eah well. Plates were sealed by parafilm and inubated hr at 37 C. Next day, 20 µl MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) dye was added in eah well and inubated in shaking inubator at 80rpm, 37 C for 2-3 hrs. The olor hange from yellow to blak shows there is growth of bateria. The absorbane was read at 570 nm in spetrophotometer, and the % inhibition of different extrats was alulated using the formula: % inhibition = 100 ((test absorbane/untreated ontrol absorbane) 100) In vivo Antidiarrhoeal Ativity Enteroous feaalis-indued diarrhea: The experimental animals were firstly deparasitized through an oral administration of 10 mg/kg of Doxyyline for 3 days [6]. These animals were further alimatized in fitted ages, where they were reeiving standard diet and water ad libitum. Furthermore, animals were left under sterile onditions during 4 days in order to maximize the omplete elimination of the deparasitizing substane. At the end of this period, rats were deprived from food for 18 h, but reeived water ad libitum and then orally inoulated with a Enteroous feaalis (E. faealis) suspension, oldly prepared at the 1.0 MFarland standard sale ( CFU/mL). Infeted rats were set out in groups and submitted to a treatment with three test doses (500, 250 and 125 mg/kg of body weight (b.w) of plant extrats. Group 1 (neutral) was made of non-infeted/non-treated rats. Group 2 (negative ontrol) reeived distilled water. The third group (positive ontrol) reeived iprofloxain at 2.5 mg/kg b was referene drug and. All the substanes (extrats plants and iprofloxain) were administered per os until omplete elimination of baterial load in faees. 97

4 These faees were olleted eah 2 days between 10 h and 11 h. The follow-up of the treatment was arried out by evaluating the load of E. feaalis in a ulture of 1g of faees on Manitol Salt Agar Petri dishes, inubated at 37 C for 24 h. At the end of treatment, animals were sarified. Liver, spleen and aeum were removed. 0.5 g of liver and all spleen were rushed by a homogenizer in 0.5 ml of saline and entrifuged at 4000 g for 15 min at 4 C for pelleting baterial [7]. 1 ml of saline solution were add in pellet and mixed by vortexing, then 0.25 ml was used for evaluated the E. feaalis load. Histologial Assessment One part of the aeums were preserved in formalin fixative (3.7%) solution and followed by tissue dehydrated with isopropyl alohol and xylene. Then, eah sample was embedded in paraffin wax, setioned at 6 μm in slides prior for staining. Haematoxylin and eosin 0.5% stain was used. The slides were examined under light mirosope and reorded with 10 BF. Prostaglandin-E2 Indued Enteropooling The effet of the extrats on Prostaglandin- E2 indued enteropooling was investigated using the method desribed by [8] with slight modifiation. Six groups of female wistar rats of six (6) animals eah ( ) were fasted for 18 hours before the experiment but had aess to water. Rats were treated orally with vehile (10 ml/kg), Loperamide (2.5 mg/kg p.o.) or aqueous and methanol extrats (125, 250 and 500 mg/kg body weight). Immediately after extrat administration, PGE2 (100 μg/kg) p.o. was administered. After 30 min following administration of PGE 2 eah rat was sarified by administering exessive dose of ketamine and the whole length of the intestine from pylorus to aeum was disseted out and weigh, its ontent olleted in measuring ylinder and volume measured, then the empty intestines was weigh and the differene between the full and empty intestines was alulated [9]. Eletrolytes Conentration in Intestinal Fluid The volume of intestinal ontent was entrifuged at 2000 g for 15 min at 4 C. the supernatant was olleted for determination of the onentration of 23Na, 24Mg, 43Ca, 44Ca and 39K using the method desribe by [10] with slight modifiation. 250 µl of eah sample were preleved and put eah in orresponding vials. A volume of 300 μl onentrated nitri aid (HNO 3 ) (Ultrex purity, Fisher), 200 μl onentrated hydrohlori aid (HCl) (Ultrex, Fisher), and 100 μl of non-stabilized 30% hydrogen peroxide (H 2 O 2 ) solution (Ultrex, Fisher) was added to eah vial. Deionized water was added to provide a final volume of 2.0 ml. Samples were then sealed, plaed in the mirowave, and digested following the program desribed in Table 1. After digestion, samples were homogenized with 5 ml of deonized water and soniated at 65 C, then diluted 100 times and run in indutively oupled plasma mass spetrometry (ICP-MS) with flow rate for organ gas of 15 l/min, eletron multiplier as detetor and detetion limits for eah sample like presented in Table 2. Statistial Analysis Table 1: Mirowave digestion program for study samples Max.Temperature Stir Ramp (min) Hold (min) Max. Pressure (PSI) Power (W) ( C) rate 200 7:00 9: Medium Table 2: ICP-MS analyze for eah element Elements Detetion limits 43 Ca Ca K Mg Na Values are presented as mean ± SEM (standard error of the mean). The measurement data were analyzed by Oneway and Two-way ANOVA, followed by Tukey-Kramer multiple omparison test if the overall differenes were signifiant (P<0.05) for prostaglandin E2 indued enteropooling and baterial load in spleen and liver, whereas the Bonferroni post-test (P<0.05) was used for the infetious diarrhea (baterial load in feaes). 98

5 RESULTS AND DISCUSSION Evaluation of Antibaterial Ativity Both plant extrats expressed antibaterial ativities on Staphyloous aureus NCTC 6571 and Enteroous feaalis ATCC 39532, whereas Baillus subtili ATCC show ativity only with methanol extrat. However, these ativity were more interesting with the aqueous extrat, whih presented higher inhibition on Enteroous feaalis ATCC (71.93% ± 0.18) and Staphyloous aureus NCTC 6571 (76.18% ± 4.48), all at the onentration of 4000, whereas methanol extrat presented inhibition at the same onentration with the same miroorganism at 65.47% ± 3.48 and 66.52% ± 3.07 respetively. Extrats also show good ativity with Heliobater pylori with the inhibition perentage of ± 0.82 and ± 0.78 respetively with aqueous and methanol extrat at the onentration of Results are showed in Table 3. Table 3: Effet of aqueous and methanol extrat of D. thollonii against different bateria by employing Alamar Blue assay and their % inhibition. Data are reported as the Mean ± SEM. All values were read in tripliate Compounds Con. E. oli ATCC S. aureus NCTC 6571 Pseudomon as aeruginosei NCTC Baillus subtili ATCC Salmonel la typhi ATCC Enteroous feaalis ATCC Heliobater Pylori ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.44 Aqueous ± ± ± ± 0.40 Extrat ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.57 Methanol ± ± ± ± 0.67 Extrat ± ± ± ± ± ± ± ± ± ± ± Ofloxain ± ± ± ± ± ± Amoxiilin +Metronidaz ol ± 0.20 Effet of Extrats on Intestinal Miroflora All extrats presented no inhibition and enhane growth of all probiotis tested at all the onentration, exept the aqueous extrat on Latobaillus latis sub sp. K98 whih present low inhibition (34.76% ± 4.09, 36.13% ± 0.1 and 39.53% ± 4.09) respetively at the onentration of 4000 μg/ml, 3000 μg/ml and 2000 μg/ml ompare to Doxyylin (80.23% ± 0.42) at the onentration of 4 μg/ml (Table 4). Table 4: Effet of aqueous and methanol extrat of D. thollonii on the inhibition perentage of probiotis, data are reported as the mean ± SEM all values were read in tripliate, AEDt: aqueous extrat of D. thollonii, MEDt: methanoli extrat of D. thollonii AEDt 4000 AEDt 3000 AEDt 2000 MEDt 4000 MEDt 3000 MEDt 2000 Latobaillus latis sub sp. K98 Pedioous aidilatisi Latobaillus fermentum strain1 Latobaillus fermentum strain2 Enteroous durans L.O. Pedioous lolie LMG ± (growth) 0 (growth) 0 (growth) 0 (growth) 0 (growth) ± ± (growth) 0 (growth) 0 (growth) 0 (growth) ± ± ± (growth) 0 (growth) 0 (growth) 7.39 ± (growth) 0 (growth) 0 (growth) 0.00 ± (growth) 7.01 ± (growth) 0 (growth) 0 (growth) 3.02 ± (growth) ± (growth) 0 (growth) 0 (growth) ± (growth) 99

6 Doxyylin ± ± ± ± ± ± 3.60 Enteroous Feaalis-Indued Diarrhoea in Rats The aqueous and methanol extrats of D. thollonii leaves, produed signifiant (P<0.001) derease in the baterial load in the fees of rats during day 2 until the end of treatment. The doses of 250 and 500 mg/kg of aqueous and methanol extrats respetively, exhibited a highly signifiant (P<0.001) effet from Day 6 for aqueous extrat and day 8 for methanol extrat, when ompared to negative ontrol group and similarly as the standard drug used. Results are illustrated by Figures 1 and 2. Extrats also showed a signifiant inhibition (P<0.001) of bateria load in liver and spleen at all the doses of aqueous extrat, 250 and 500 mg/kg of methanol extrat, when ompared to negative ontrol group (distilled water), and there is no signifiant differene (P 0.05) when ompared to positive ontrol group ( iprofloxain). Results are illustrated by Figures 3 and 4. Baterial load (nber of olonies / g of fees) Time (days) Biginning of treatment neutral ontrol negative ontrol positive ontrol Aqueous extrat 125 mg/kg Aqueous extrat 250 mg/kg Aqueous extrat 500 mg/kg Figure 1: Variation of the baterial load (Enteroous feaalis) in the fees of infeted rats treated with the aqueous extrat of D. thollonii. Eah point represent the mean ± SEM (n=6). β : signifiant differenes (P<0.01) ompared with positive ontrol (iprofloxain), C and γ: signifiant differenes (P<0.001) ompared with negative (distilled water) and positive ontrol (iprofloxain), respetively Baterial load (nber of olonies / g of fees) Time (days) Biginninig of treatment neutral ontrol negative ontrol positive ontrol Methanol extrat 125 mg/kg Methanol extrat 250 mg/kg Methanol extrat 500 mg/kg Figure 2: Variation of the baterial load (Enteroous feaalis) in the fees of infeted rats treated with the methanol extrat of D. thollonii. Eah point represent the mean ± SEM (n = 6). β : signifiant differenes (P<0.01) ompared with positive ontrol (iprofloxain), C and γ: signifiant differenes (P<0.001) ompared with negative (distilled water) and positive ontrol (iprofloxain), respetively 100

7 number of baterial olonie in spleen Neutral DW Cipro aqueous extrat (mg/kg) Methanol extrat (mg/kg) Figure 3: Number of baterial olonie (Enteroous feaalis) in the spleen of infeted rats treated with the aqueous and methanol extrat of D. thollonii. Eah point represent the mean ± SEM (n = 6). α : signifiant differenes (P<0.05) ompared with positive ontrol (iprofloxain), C : signifiant differenes (P<0.001) ompared with negative ontrol (distilled water) number of baterial olonie in 0,5 g of liver Neutral DW Cipro 125 aqueous extrat (mg/kg) Methanol extrat (mg/kg) Figure 4: Number of baterial olonie (Enteroous feaalis) in 0.5 g of liver of infeted rats treated with the aqueous and methanol extrat of D. thollonii. Eah point represent the mean ± SEM (n = 6). β: signifiant differenes (P<0.01) ompared with positive ontrol (iprofloxain), C : signifiant differenes (P<0.001) ompared with negative ontrol (distilled water) Histologial Assessment in Caeum Histologial examination of the aeum removed from animals that were treated with distilled water showed higher degradation of different layers (muosa, submuosa and musularis), while those who reeived aqueous and methanol extrat at the dose of 250 and 500 mg/kg respetively appear intat with all the different layers ompare to neutral and positive ontrol (iprofloxain). However, 125 mg/kg of aqueous and methanol extrat show higher degradation of muosa, while aqueous extrat 500 mg/kg show light degradation of this layer (Figure 5). 101

8 Figure 5: Histologial analysis of aeum. a: setion of a neutral ontrol rat: no degradation are seen and the layers are normal. b: negative ontrol (distilled water): there is denaturation of all layers. : histologial setion of a positive ontrol (iprofloxain) : no degradation are seen and the different layers are normal. d: treated with aqueous extrat (125 mg/kg): there is higher degradation of vilis. e: treated with aqueous extrat (250 mg/kg): all the different layers are present and normal f: treated with aqueous extrat (500 mg/kg): there is light degradation of vilis and non-regularity on musularis. g: treated with methanol extrat (125 mg/kg): there is higher degradation of muosa. h: treated with methanol extrat (250 mg/kg): all layers are present but, there is low degradation of villis. i: rat treated with methanol extrat (500 mg/kg): there is no degradation of different layers Effet of the Extrats on Prostaglandin- E2 Indued Enteropooling in Rats The aqueous and methanol extrats at doses of 250 and 500 mg/kg signifiantly inhibited prostaglandin E2-indued enteropooling in terms of volume of intestinal fluid and in a manner omparable to that of the standard antidiarrhoeal drug loperamide (2.5 mg/kg b.w). There is no signifiant different (P 0.05) between all the treated groups when ompare to untreated group in terms of mass of intestinal ontent (Table 5). Effet of Extrats on Eletrolytes Conentration in Intestinal Fluid The aqueous and methanol extrats show a redution of onentration of all eletrolytes at all the doses ompared to negative ontrol (distilled water). At the doses of 250 and 500 mg/kg of aqueous extrat and 500 mg/kg of methanol extrat, we observed higher redution of the onentration of 23Na ompared to those treated by loperamide. This higher redution of onentration is also observed with 24 Mg at the doses of 500 mg/kg of aqueous extrat, 125 and 250 mg/kg of methanol extrat ompare to those who were treated by loperamide. Both extrats at the doses of 250 and 500 mg/kg redued the onentration of 39 K omparably to positive ontrol (loperamide). The same observations are made with 43Ca at the doses of 250 and 500 mg/kg of methanol extrat (Table 6). 102

9 Table 5: Effet of aqueous and methanoli extrats of D. thollonii on prostaglandin E2- indued intestinal fluid aumulation in rat. Data are reported as the mean ± S.E.M. for n = 6 per group. ANOVA one way followed by Tureky s test ;). a p <0.05; p <0.001: signifiant differenes from the negative ontrol (distille water). AEDt : Aqueous Extrat of D. thollonii, MEDt : Methanol Extrat of D. thollonii Weight empty intestine 103 Parameters Mean % of inhibition Mean % of inhibition Treatments Weight full intestine Masse intestinal ontent Vol. intestinal fluid Distilled water 5.62 ± ± ± ± Loperam 2.5 mg/kg 5.46 ± ± ± ± AEDt 125 mg/kg 6.10 ± ± ± ± 0.69a AEDt 250 mg/kg 5.24 ± ± ± ± AEDt 500 mg/kg 5.28 ± ± ± ± MEDt 125mg/kg 6.00 ± ± ± ± 0.72a MEDt 250 mg/kg 5.84 ± ± ± ± MEDt 500 mg/kg 5.80 ± ± ± ± Table 6: onentrations of elements (eletrolytes) in intestinal fluid of rats treated with aqueous and methanol extrat of Dissotis thollonii (mg/ml). Data are reported as the mean ± S.E.M. for n = 6 per group. All samples were run in tripliate on ICP-MS. AEDt : Aqueous Extrat of D. thollonii, MEDt : Methanol Extrat of D. thollonii Elements Sample 23 Na 24Mg 39K 43 Ca 44 Ca Distilled water ± ± ± ± ± Loperamide (2.5 mg/kg) ± ± ± ± ± AEDt 125 mg/kg ± ± ± ± ± AEDt 250 mg/kg ± ± ± ± ± AEDt 500 mg/kg ± ± ± ± ± MEDt 125 mg/kg ± ± ± ± ± MEDt 250 mg/kg ± ± ± ± ± MEDt 500 mg/kg ± ± ± ± ± The major ausative agents of diarrhea in human beings inlude various enteropathogens like, Esherihia oli, Staphyloous aureus, and Enteroous feaalis. This physiopathology is haraterized by the frequent passage of liquid faees and it involves both an inrease in the motility of the gastrointestinal trat, along with inreased seretion and dereased absorption of fluid, and thus a loss of eletrolytes (partiularly sodium) and water [11]. For the treatment and management of diarrhea in developing ountries, several plant extrats are studied for this purpose. Methanol and aqueous leaves extrat of D. thollonii that have not been studied so far, were evaluated for their antimirobial ativities in vitro, antidiarrhoeal potential against infetious diarrhoea indued by Enteroous feaalis and non-infetious diarrhoea indued by prostaglandin-e2 in rat. The results of antimirobial ativities revealed that aqueous and methanol extrats presented no inhibition in all probiotis tested, while these extrats have inhibited pathogeni bateria like Staphyloous aureus NCTC 6571 (MIC: 500 and 1000 respetively ) and Enteroous feaalis ATCC ( MIC: 250 ). Seondary metabolites groups that have previously been desribed in this plant extrat (tannins, flavonoids, sterols, anthraquinones, phenols and polyphenols) [6] are known to possess antimirobial properties. This ativity ould be done by inhibiting the baterial protein synthesis, or by bloking the inhibiting effets of the baterial enzymes. It should be noted that the ative ingredients whih at on the wall of the baterium must find suitable reeptors for their fixation and ation, whereas those whih at inside the ell must not only ross the membrane, but they must also find target in the ell [6]. The ensus of pathogens revealed that E. oli, entero- invasive (EIEC) and entero-hemorragi (EHEC), Salmonella, Shigella, Vibrio holerae are the major baterial pathogens most often responsible for pandemi and epidemi diarrheal infetious diseases in developing ountries [12]. However, like Shigella flexneri, Enteroous feaalis is also known to serete verotoxins whih helps it to olonize the olon muosa and get inside the epithelial ells, induing inflammation and degeneration of the Lamina propria [13]. This ould be explaining the installation of the diarrhoea, assoiated to the febrility observed at Day 0 in infeted rats. Consequently, the desquamation and uleration of the muosa leads to the loss of blood and muus in the intestinal lumen, whih in turns hinders reabsorption. The redution in the baterial load observed from the 2 nd day in all infeted animals faees, treated with seleted dose extrats (500, 250

10 and 125 mg/kg) ould be due to the same presene of polyphenols and the flavonoids whih would have failitating the restoration of protein hannels-like strutures to support the flow of antimirobial ompounds towards intra baterial target sites [14] as well as bateriidal mehanisms desribed above, as demonstrated by [15]. All these bateriidal mehanisms of the extrat due to their seondary metabolites explain the good appearane of different layers of the histologial setion of aeum of rat treated with aqueous and methanol extrat at the dose of 250 and 500 mg/kg respetively. So, these extrats, while destroying the bateria, would have prevented the release of verotoxins, Thereby promoting inhibition of irritation and degeneration of the Lamina propria. In the human intestine the extensive metabolism of flavonoids has been attributed to the ation of intestinal miroflora [16]. So it was appropriate to ondut an experiment for evaluating the effiay of the leaves extrat of D. thollonii in promoting growth of some probiotis. Clinial effets of some probioti strains are observed in human s intestine. These inlude, for example, immunomodulation, modulation of intestinal flora, prevention of diarrhoeas, and lowering of feal enzyme ativities. To have an impat on the oloni flora it is important for probioti strains to show antagonism against pathogeni bateria via antimirobial substane prodution or ompetitive exlusion. Enormous researh efforts have foused on baterioin researh. Although probioti strains may produe baterioins, their role in the pathogen inhibition in vivo an only be limited, sine traditional baterioins have an inhibitory effet only against losely related speies [17]. However, low moleular weight metabolites (suh as hydrogen peroxide, lati and aeti aid, and other aroma ompounds) and seondary metabolites (polyphenols and the flavonoids) may be more important in the inhibition of pathogeni bateria and the potentialisation of some probiotis [18]. These onfirm the results obtain in our study whih shows an inrease in growth with the dose. The extrats also signifiantly inhibited the PGE2 indued intestinal fluid aumulation (enteropooling). It has been shown that E type of prostaglandins ause diarrhoea in experimental animals as well as human beings by inhibiting the absorption of Na+, Cl- and gluose whih auses the aumulation of intestinal fluid and leads to diarrhea [19,20]. Their mehanism has been assoiated with dual effets on gastrointestinal motility as well as on water and eletrolyte transport [20,21]. Both extrats signifiantly redued prostaglandin E2-indued enteropooling as evident from the higher redution in the volume of the intestinal ontents in a manner omparable to the referene drug, (loperamide) [22]. Loperamide has been reported to derease olon flow rate and attenuate transit in the small intestine [23]. It is possible that the aqueous and methanol extrats of D. thollonii produed their antidiarrhoeal ation by redution in the synthesis or ation of PGE2. Elsewhere, the anti-diarrheal properties of some mediinal plants have been attributed to their phytohemial onstituents like tannins and some flavonoids [24], whih have been shown to possess anti-diarrheal ativity attributable to their ability to inhibit intestinal motility and hydro-eletrolyti seretion. CONCLUSION The results of this investigation revealed that both extrats of D. thollonii ontains pharmaologially ative substane(s) with anti-diarrhoeal and antibaterial properties. These properties onfirm the use of the plant leaves as an anti-diarrhoeal and antibaterial drug by traditional healers in Cameroon. ACKNOWLEDGEMENTS The authors wish to express their gratitude to TWAS (Aademy of Siene of Developing Countries) and ICCBS (International Center for Chemial and Biologial Sienes) for Sandwih Postgraduate Fellowship Award to TADJOUA TCHOUMBOU Herve (FR number: ). FUNDING This manusript researh projet was supported by the TWAS (Aademy of Siene of Developing Countries) and ICCBS (International Center for Chemial and Biologial Sienes), University of Karahi. 104

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