Management of Secretory Diarrhea

“Diarrhea is the passage of 3 or more loose or liquid stool per day, or more frequently than is normal for the individual. It is usually a symptom of gastrointestinal infection, which can be caused by a variety of bacterial, viral and parasitic organisms, infection is spread through contaminated food or drinking-water, or from person to person as a result of poor hygiene” (WHO). Diarrheal diseases affect all races, sexes, ages and geographic areas, has high impact on mortality and morbidity worldwide, an estimated 2-4 billion episodes of infectious diarrhea occurred each year and are especially prevalent in infants (Hodges and Gill 2010; Farthing 2002). In 2005, 1.8 million people died worldwide from diarrheal diseases (WHO, 2007). In Mexico, in the past 6 years, the gastrointestinal infection has been a serious health problem and was the second cause of morbidity among all age groups (SS, 2008).


Introduction
"Diarrhea is the passage of 3 or more loose or liquid stool per day, or more frequently than is normal for the individual. It is usually a symptom of gastrointestinal infection, which can be caused by a variety of bacterial, viral and parasitic organisms, infection is spread through contaminated food or drinking-water, or from person to person as a result of poor hygiene" (WHO). Diarrheal diseases affect all races, sexes, ages and geographic areas, has high impact on mortality and morbidity worldwide, an estimated 2-4 billion episodes of infectious diarrhea occurred each year and are especially prevalent in infants (Hodges and Gill 2010;Farthing 2002). In 2005, 1.8 million people died worldwide from diarrheal diseases (WHO, 2007). In México, in the past 6 years, the gastrointestinal infection has been a serious health problem and was the second cause of morbidity among all age groups (SS, 2008).

Pathophysiology classification of diarrhea 
Osmotic; is caused by poorly absorbable solutes (eg. sorbitol, magnesium salts) remaining in the gastrointestinal lumen retain water and electrolytes resulting in reduced water reabsorption  Altered Motility; caused slowing of the motor function of the small intestine as with narcotic use, scleroderma, diabetic autonomic neuropathy and amyloidosis  Exudative; the intestinal epithelium's barrier function is compromised by loss of epithelial cells or disruption of tight junctions (eg. E. coli, Salmonella, Shigella, Yersinia, Campilobacter, Mycobacteryum tuberculosis, Clostridium difficile y Entamoeba histolytica), inflammatory disease process as in ulcerative colitis and Crohn's disease  Secretory; is caused by an increase in water and electrolytes (Chloride or bicarbonate) movements to the intestinal lumen, the final effect is the increase of secretion and decrease of absorption of net sodium and water (Navaneethan and Giannella, 2010).

Secretory diarrhea
Secretory diarrhea occurs when the balance between absorption and secretion in the small intestine is disturbed by excessive secretion caused by bacterial enterotoxins, is a net movement from mucous intestinal to lumen, the volume exceed 10 mL/Kg/day, and the osmolarity is similar with plasma. It is the leading cause of death in infants in developing  Amstrong and Cohen, 1999).

Vibrio cholerae enterotoxin
Vibrio cholerae enterotoxin is an oligomeric protein which is composed by two subunits, A subunit of 27.2 KDa composed as A1 and A2 subunits, and B subunit composed by five subunits B of 11.6 KDa each one, AB 5 complex Fig 1. (Sixma, 1991)

Vibrio cholerae enterotoxin mechanism
Mechanisms proposed to secretory diarrhea caused by V. cholera enterotoxin involves the union of subunits B to the oligosaccharide portion of the receptor GM1, present in the apical surface of enterocytes, this union lend the entrance of A subunit of toxin to the enterocyte for acidic endosomes, which pick up the golgi apparatus and endoplasmic reticulum, inside of enterocyte disulphure bond between A1-A2 is dissolved by protein disulfide isomerase, it causes release of A1 which is capable of binding NAD and catalyzing the NADP-ribosylation of G s , a GTP-binding regulatory protein associated with adenylate cyclase. A1 subunit stimulates increasing of 1000 times production of cAMP second messenger, cAMP active protein cinase A, which phosphorile and activate transmembranal chloride channels of the enterocytes located on intestinal crypts, it causes massive secretion of water and electrolytes to intestinal lumen, in villus cell there is a decrease of absorption of Na + and Clions (Kopic 2010). Fig 2. shows the proposed mechanism to the action of Vibrio Cholerae enterotoxin. At 5 to 10 minutes to the exposure of V. cholera toxin cause intestinal hypersecretion of water and electrolytes for several hours (Thiagarajah, 2005;Spangler, 1992). The symptoms are manifested as severe cramp and the copious "rice water" diarrhea characteristic of the disease.

Dehydration
During diarrhea there is an increased loss of water and electrolytes (sodium, chloride, potassium and bicarbonate) in the liquid stool; dehydration occurs when these losses are not replaced adequately and a deficit of water and electrolytes develops. The degree of dehydration is graded according to signs and symptoms that reflect the amount of fluid lost:  In the early stages of dehydration, there are no signs or symptoms  As dehydration increases, signs and symptoms develop. Initially these include: thirst, restless or irritable behavior, decreased skin turgor, sunken eyes, and sunken fontanel (in infants).  In severe dehydration, these effects become more pronounced and the patient may develop evidence of hypovolemic shock, including: diminished consciousness, lack of urine output, cool moist extremities, a rapid feeble pulse, low or undetectable blood pressure, and peripheral cyanosis.  Death follows soon if rehydration is not started quickly (WHO, 2005).

Secretory diarrhea treatment 2.4.1 Oral rehydration
To control diarrhea disease, a sufficient hydration of the patient should be procure and provide the necessary ions to maintain electrolyte balance, the treatment of choice is oral  rehydration solution (ORS), it has reduced the levels of mortality in children and elderly by dehydration, but not morbidity (Turvill et al., 2000), the treatment is based by active absorption of glucose by smooth intestine, during the intestinal infection lend to the cotransport of Na + ions and water absorption. WHO and UNICEF guidelines recommend their use, is important to notice that ORS, is useful to treat dehydration caused by diarrhea, but it not decrease the amount and duration at the same. Depends of severity of diarrhea, in some cases ORS is not enough and antibiotic, spasmolytic, and antiprotozoal drugs should be used. WHO recommended use of secure and effective drugs to the pediatrics (Marion et al., 2010).

Drugs used to treat secretory diarrhea
To treat the secretory diarrhea there are some drugs which reduce the intestinal movement such as codeine (1), loperamide (2), diphenoxylate (3), lidamidine (4), bismuth subsalicylate (5), racecadotril (6) and clonidine (7) Table 1.; which are capable to stimulate absorption direct and reduce secretion of water and electrolytes in gastrointestinal tract, to decrease propulsion, contact time of intestinal content with mucosal surface increase, it favors the absorption. They act not premised release of prostaglandins too (Marion et al., 2010;Martindale 2009).

Structure Effect
Codeine (1) Has a high antidiarrheal action but, produces secondary effects such as nauseas, dizziness and acts against central nervous system, it can be used carefully in children, continuous use can induce physical dependence and addiction Loperamide (2) Decrease intestinal motility and present antisecretory effect by activation of calmodulin, increase the water and electrolytes absorption to the intestinal lumen. It should not be administrated to children under six years old, patients with constipation, atony or intestinal obstruction, should avoid its use on bacterial infective severe and in acute dysentery. Frequent adverse reactions induced are hypersensibility reactions (cutaneous eruption), gastrointestinal disorders (constipation, colic, abdominal distention, nauseas and vomit), fiver and dry mouth, is a non-prescription drug for children because can cause CNS depression diphenoxylate (3) Inhibits intestinal propulsion and fecal excretion velocity, cause decrease of intestinal transit, to therapeutic doses it induces adverse reactions in central nervous system (confusion, sedation, depletion, cephalea), allergic reactions (anaphylaxis, prurite) on gastrointestinal apparatus (toxic megacolon, paralytic ileum, vomit, nauseas and abdominal pain) It can cause euphoria and has analgesic effect. Difenoxilate is contraindicated in children younger than 2 years old.
Lidamidine (4) Improve the absorption of water and electrolytes in intestinal velocities and reverse their secretion to level on intestinal crypts.
Racecadotril (6) Decrease intestinal hipersecretion of water and electrolytes to intestinal lumen, inhibits release of encephalinse endogenus witch act on opiaceus receptors  decreasing cAMP level (decrease water and electrolytes secretion), cause some adverse reactions such as hypokalemia, bronchospasm, fever, vomit and otitis.
Clonidine (7) Stimulates sodium and chloride absorption and inhibits chloride secretion by interaction with its receptor on enterocyte, causes an alteration of gut motility with effect on intestinal transport, it causes hypotension  (8), chlorpromazine (9), nicotinic acid (10), indomethacin (11), somatostatin (12) and ethacrinic acid (13) but they were not developed as antidiarrhoeal drugs (Fedorack and Field, 1987). Thus, the research for new antisecretory agents that should be effective and safe to treat diarrhea is still a necessary goal.

Potential target areas to design therapeutic agents on Vibrio cholerae toxin
During the last two decades there has been a continuous research of drugs that inhibit the secretory process in the enterocyte to help to the control of diarrhea, but only a few candidates have emerged, and none has found a place in the routine management of secretory diarrhea. Particularly to cholera toxin its mechanism of action revel several potential target areas to design therapeutic agents such as: a. The inhibition of adenylate cyclase b. The blockage of the active site of the enzyme located in the A subunit c. The disruption of the assembly of the holotoxin by interrupting the A 2 -B interaction d. The interception of the receptor binding to the bottom of the B pentamer (Guangtao Z., 2009).
e. Inhibitors of enkephalinase and of the cystic fibrosis transmembrane conductance regulator (Thiagarajah, 2005). f. Inhibition of transport proteins involved in cAMP activated chloride secretion The study of medicinal plants with the propose to provide pharmacological evidence that may explain its therapeutic use There are some in vitro models such as isolated ileum of guinea pig, isolated jejune of rabbit, ileum and duodenum isolated of rat or rabbit and in vivo reduction of intestinal motility using charcoal meal, Castor oil, PGE 2 , MgSO 4 induced diarrhea, and Enteropooling models. Antispasmodic activity has been demonstrate for some flavonoids such as quercetin, quercitrin, genistein, sakuranetin, rutin and bisabolol; terpenoids such as himachalol, coleonol, -damascenone, -fitol, capsidiol, -eudemol, hinesol, huatriwaico acid, camaldulin and tymol; essential oils such as, 1,8-cineol, eugeol, timol, carvacrol, estragolanetol,  y  pinenes, nonanal, and linalool; alkaloids such as himbacine, protopine, coptisine, cantleyine, mitraginine, vertine, retuline, cavidine and metuenine (Astudillo et al., 2009). There are a great number of natural remedies for diarrhea control, historically Papaver somniferum preparations are efficient and powerful against diarrhea, as the derivative codeine, alkaloids are ones of major substances explored form natural products and they give to pharmaceutical industry a big number of patents, another class of compound explored therapeutically are flavonoids and has been used as complement in treatment of cancer, heart diseases, venous insufficiency, venous ulcers, hemorrhoids and diarrhea. (Martindale 2009)

Antisecretory compounds isolated from medicinal plants
Some studies have been performed in order to find antisecretory compounds from several plants used in traditional medicine to treat several kinds of diarrheas. In this sense the extracts from Croton urucurana, C. lechleri, Berberis aristata and Guazuma ulmifolia were studied against intestinal secretion caused by V. cholera toxin, in the cases of C. lechleri, B. aristata and G. ulmifolia the isolated compounds were oligomeric proantocyanidins and berberine, respectively. From C. urucurana saponins, steroids, alkaloids, antocianidins and catechins have been isolated. Prontocianidins and catechins probably can be associated with their antisecretory activity. Steviol (29) and dihydroisosteviol (30)  Crofelemer (32) is a proanthocyanidin oligomer obtained from Croton lechleri (dragon's blood), the sap has been used to treat diarrheas including dysentery and cholera, pharmacological studies have shown that it reduces fluid secretion in cell culture and mouse models (Gabriel et al., 1999), it has been reported that the antisecretory mechanism of action of crofelemer involves dual inhibition of The cystic fibrosis transmembrane regulator conductance (CFTR), a cAMP stimulated Cl-channel, and calcium-activated Clchannels (CaCC) at the luminal membrane of enterocytes ( preliminary studies showed that crofelemer (32) may reduce watery stool output in patients with infectious diarrhea such as cholera. But it needs further Phase 3 clinical trials are still necessaries (Crutchley et al., 2010).
Penta-m-digalloyl-glucose (PDG) (31) Crofelemer (32) We continue with the research of compounds with antisecretory activity useful to treat diarrhea. Medicinal plants used in Mexican traditional medicine to treat gastrointestinal disorders could be a source of compounds with therapeutic utility. In México, the use of medicinal plants to treat gastrointestinal disorders such as diarrhea and dysentery is widespread (Aguilar et al., 1994). However most of these plants have not been investigated from a pharmacological point of view to demonstrate their antisecretory properties, which could lead to support their use as antidiarrheal and anti-dysenteric drug in traditional medicine. We screened aqueous and methanol extracts from 26 Mexican medicinal plants to assess their antisecretory activity using the cholera toxin-induced intestinal secretion in rat jejunal loops model. None of this species or their isolated compounds has been previously evaluated as antisecretory agents (Velázquez et al., 2006).

Plant materials
The plants used in that study were collected from different regions in Mexico: Mexico City, States of Hidalgo, Mexico, Sinaloa, Guanajuato and Yucatan, all of them were selected according to their use in Mexican traditional medicine to treat gastrointestinal disorders. Voucher herbarium specimens were deposited in Herbarium IMSSM of Instituto Mexicano del Seguro Social and were authenticated by MS Abigail Aguilar.

Preparation of crude extracts
The air-dried plant material (20g) was extracted by maceration with 300 mL of MeOH for 1 week. Then the macerate was filtered and concentrated under reduced pressure at 40°C. For aqueous extracts, 20 g of air-dried plant material were extracted by decoction with 100 mL of distilled water for 30 min, the solution was filtered and lyophilized.

Cholera toxin
Lyophilized powder (1mg) of Cholera toxin (SIGMA) containing approximately 220,000 units/mg of protein was suspended in 1 mL of sterile water. Aliquots of the toxin solution were dissolved in a 1x PBS (NaCl 8g, KCl 0.2 g, Na2HPO4.7H2O o.115 g, KH2PO4 0.2 g/L) solution with 1% bovine serum albumin (SIGMA) to obtain a concentration of 3 g/mL.

Antisecretory assay
The antisecretory activity of the extracts was tested using a method described by Torres et al., in 1993. Briefly, male Sprague-Dawley rats (200-250 g) were obtained from the animal house of the IMSS. The experimental protocols were in accordance with the official Mexican norm NOM 0062-ZOO-1999 entitled technical specifications for the production, care and use of laboratory animals (SAGARPA 2001). The antisecretory effect of the extract was studied on intestinal secretion indirectly by measuring the fluid accumulation in the intestine following cholera toxin administration to rats. Two jejuna loops were prepared in the rats and inoculated with 3 g/mL of cholera toxin dissolved in 1x PBS with 1 % bovine albumin. Rats (n=4 per group by duplicated) were treated orally with each extract (300 mg/Kg in 1 mL of a 2 % DMSO solution in water). Loperamide (10 mg/Kg) was used as antidiarrhoeal drug. After 4 h, the animals were sacrificed using ethyl ether. The antisecretory activity of the extracts was measured as the fluid accumulation in the loops and expressed in percentage of inhibition. Values are expressed as mean ± SEM. Statistical significance was determinate using Mann-Whitney U-test. Values with p<0.05 were considered significant.

Results
We tested 56 aqueous and methanol crude extracts obtained from 26 medicinal plants used in Mexican traditional medicine for the treatment of gastrointestinal disorders. The antisecretory activity was tested using the cholera toxin-induced intestinal secretion in rat jejunal loops model. Only the principal antisecretory activity of the extracts tested is shown in Table 2, the full list is showed in Velázquez et al., 2006. In traditional medicine since infusions or decoctions are usually taken three times per day when diarrhea occurs, our results can be related with their traditional use because the used dose is approximately one cup of plant tea which is recommended by Mexican people to treat gastrointestinal disorders (Aguilar et al., 1994).We found that both extracts from Chiranthodendron pentadactylon, Hippocratea excelsa and Ocimum basilicum were the most active with inhibition values ranging from 68.0 to 87.6% at 300 mg/kg. Methanol extract of Geranium mexicanum (aerial parts) and the aqueous extract of Bocconia frutescens were active too with inhibition values of 93.4 and 86.0%, respectively. On the other hand, the methanol extract of Chenopodium ambrosioides green variety (aerial parts), Lygodium venustum, Punica granatum and Ruta chalepensis, the aqueous extracts of Aloysia triphylla, Chenopodium ambrosioides green variety (aerial parts), Dorstenia contrajerva and Schinus molle shown inhibitory activity with values ranging from 43.4 to 79.5%. The 87% of the extracts tested showed inhibitory activity of the intestinal secretion; only seven extracts did not show any antisecretory activity. In general, among the researched extracts, the methanol extracts exhibited the highest antisecretory activity.

Antisecretory study of Chiranthodendron penthadactylon
We selected Chiranthodendron pentadactylon Larreat (Sterculiaceae) to perform bio-guided assay fractionation. C. pentadactylon know in Mexico as "flor de manita"has been used in Mexican traditional medicine since Aztecs ancient times to treat heart illness, epilepsy, diarrhea and dysentery (Linares et al., 1988, Argueta et al., 1994. This study lend to the isolation of some compounds with in vivo antisecretory activity (Velázquez et al., 2009).

Isolation of active compounds
The flowers of C. pentadactylon were ground and extracted by maceration at room temperature with methanol, the extract was suspended in 10 % MeOH-water and successively partitioned with CH 2 Cl 2 and EtOAc, the aqueous residual layer was lyophilized. The fractions were tested for antisecretory activity at doses of 50 mg/Kg. The most active fraction was AcOEt with 88.2 % of Inhibition. In order to isolate the active compounds, it was subjected to column chromatogaphy on Sephadex (Pharmacie) using CHCl 3 in EtOH, MeOH and Water to give eight secondary fractions, further chromatography lend to the isolation of tiliroside (33), astragalin (34), isoquercitrin (35), (+) catechin (36), and (-) epicatechin (37). All the isolated compounds were identified by comparison of spectroscopic data ( 1 H and 13 C NMR, UV, IR, [], and TLC and HPLC with authentic samples available in our laboratory (Kuroyanagui et al., 1978;Lee et al., 1992;Lui et al., 1999;Calzada et al., 2007).

Conclusion
Some of the medicinal plants tested showed antidiarrheal activity in the model used. Both extracts of Annona cherimola, Chiranthodendron pentadactylon, Hippocratea excelsa, Ocimum basilicum, Geranium mexicanum (aerial parts), methanol extract of Ruta chalepensis, Lygodium venustum, Punica granatum, and the aqueous extract of Bocconia frutescens, Aloysia triphylla, Dorstenia contrajerva and Schinus molle showed better antisecretory activity than loperamide. The active extracts found in this study will be an option to develop novel phytodrugs useful to treat fluid loss in diarrhea. These results allows to propose these species as a potential sources of antisecretory compounds and should be therefore subjected to further bioassayguided phytochemical studies to obtain their active principles, the antisecretory compounds isolated from medicinal plants combined with ORS might be useful in decreasing the mortality caused by dehydration. The properties previously described of (-)-epicatechin suggest that it may be a leading compounds in the development of novel antidiarrheal agents. The results obtained give some scientific support to the use of some medicinal plants tested for the treatment of gastrointestinal disorders such as diarrhea.