Journal of Ethnopharmacology 142 (2012) 539–547 Contents lists available at SciVerse ScienceDirect Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep Garcinia buchananii bark extract is an effective anti-diarrheal remedy for lactose-induced diarrhea$ Paul A. Boakye a, Stuart M. Brierley c, Sofie P. Pasilis b, Onesmo B. Balemba a,n a Department of Biological Sciences, Australia Department of Chemistry, University of Idaho, Moscow, ID, USA c Nerve-Gut Research Laboratory, Discipline of Medicine, University of Adelaide, Australia b a r t i c l e i n f o abstract Article history: Received 28 October 2011 Received in revised form 17 March 2012 Accepted 20 May 2012 Available online 27 May 2012 Ethnopharmacological relevance: The extract from the stem bark of Garcinia buchananii trees is used as an anti-diarrhea remedy in sub-Saharan Africa. We tested the hypothesis that G. buchananii bark extract and its anti-motility fractions are effective treatments against lactose-induced diarrhea. Materials and methods: A high-lactose (35%) diet was used to induce diarrhea in Wistar rats, which were then treated with either G. buchananii bark extract (0.1, 0.5, 1.0 and 5.0 g bark powder), and its antimotility fractions isolated using preparative thin layer chromatography; termed PTLC1 (15 mg) and PTLC5 (3.8 mg) or loperamide (8.4 mg). Drug preparations were dissolved in 1 L except PTCL1 and PTLC5 that were dissolved in 100 mL tap water. Numerous parameters were measured in each condition including consistency, fluid and mucus content of feces, body weight, water and food consumption, urine production and bloating. Results: Diarrheic rats produced watery or loose, mucuoid, sticky, feces. Fluids constituted 86% of stool mass compared with only 42% for control rats fed standard chow. Compared with controls, diarrheic rats produced more urine, lost weight and had bloated ceca and colons. All doses of the extract, its anti-motility fractions and loperamide individually stopped diarrhea within 6–24 h of administration, whilst significantly reducing mucus and fecal fluid content, urine production and intestinal bloating. Rats treated with 0.1 g extract, PTLC1 and PTLC5 gained weight, whilst PTLC5 also increased water intake. Conclusions: Garcinia buchananii extract and its anti-motility fractions are effective remedies against lactose-induced diarrhea. The extract contains compounds that reverse weight loss, promote food and water intake, supporting the notion that characterization of the compounds could lead to new therapies against diarrheal diseases. Published by Elsevier Ireland Ltd. Keywords: Traditional medicine Plant extracts Intestinal motility Intestinal secretion 1. Introduction Diarrheal diseases kill more children, especially those under five years of age than AIDS, malaria, and measles combined (UNICEF/ Abbreviations: ext., Extract; G. buchananii, Garcinia buchananii; g/L, Gram/Liter; HCA, (  ) – Hydroxycitric acid; HLD, High lactose diet; h, Hours; 5-HT, 5hydroxytryptamine (serotonin); LD, Lactose-induced diarrhea; LP, Loperamide; mg/L, Milligram/liter; OD, Osmotic diarrhea; ORS, Oral rehydration solution; PTLC, Preparative thin layer chromatography; SD, Standard chow diet; UNICEF, United Nations Children’s Fund; vs., Versus; WHO, World Health Organization $ Author contributions: PAB was involved in all aspects of research, analyzed data and wrote the paper; SMB contributed to research design, critical review and intellectual content; SPP contributed to research design, supervision of research, critical review, intellectual content, and wrote the paper; and OBB was involved in research design, supervision of the study, data analysis, and interpretation and wrote the paper. n Corresponding author. Tel.: þ1 208 885 8023; fax: þ1 208 885 7905. E-mail address: obalemba@uidaho.edu (O.B. Balemba). 0378-8741/$ - see front matter Published by Elsevier Ireland Ltd. http://dx.doi.org/10.1016/j.jep.2012.05.034 WHO, 2009). Each year, 2.5 billion cases of acute infectious diarrhea occur in children below five years of age alone, and this accounts for over 1.5 million child deaths in low and middle-income countries, mainly in Africa and South Asia (Thapar and Sanderson, 2004; UNICEF/WHO, 2009). Furthermore, infectious diarrheal diseases are a significant cause of morbidity and mortality in HIV/AIDS patients, people displaced by disasters and wars and the elderly, (Nwachukwu and Okebe, 2008; Thielman and Guerrant, 1996) and a significant health care burden and loss of productivity around the world (Ocfemia and Taylor, 2004; UNICEF/WHO, 2009). During the past four decades, concerted efforts have been made to combat the high morbidity and mortality rates associated with diarrheal diseases through improved sanitation and treatments using oral rehydration solution (ORS), anti-secretory and antimotility agents, vaccinations, zinc supplements, antibiotics and other regimens (Guerrant et al., 2003; Kelly, 2011; UNICEF/WHO, 2009). These measures have succeeded in curbing the high mortality rates associated with diarrheal diseases (Guerrant et al., 2003; Kelly, 540 P.A. Boakye et al. / Journal of Ethnopharmacology 142 (2012) 539–547 2011; UNICEF/WHO, 2009). However, millions of people are still dying from diarrheal diseases, suggesting a critical need for novel and affordable anti-diarrheal drugs. The ultimate goal in diarrhea treatment is to prevent or reverse dehydration, gastrointestinal hyper-motility and fecal urgency, shorten the duration of the illness, reduce the pain or stress, and in some cases treat the infection and prevent nutritional complications (Brown, 2003; Field, 2003; Kelly, 2011; UNICEF/WHO, 2009). Key drawbacks to current treatment strategies are that they do not necessarily reduce the duration of the illness; as well as the fact that in developing countries 39% of the population have no access at all to modern anti-diarrhea therapies (UNICEF/WHO, 2009). It is therefore believed that over 80% of the population in developing countries depend on phytotherapy to treat diarrheal illnesses (Groombridge and Jenkins, 2002). Garcinia buchananii (G. buchananii), Authority Baker, Family Clusiaceae (Brown, 1894), a plant native to Eastern, Central and Southern Africa is used by the indigenous population to treat dysentery, abdominal pain, and a range of infectious diseases (Balemba et al., 2010; Chinsembu and Hedimbi, 2010; Kisangau et al., 2007). In native communities, patients can treat themselves by either chewing the dried stem and root barks, or grinding the bark into powder, which is then added to water or beverages for drinking (Balemba et al., 2010; Chinsembu and Hedimbi, 2010). Recently, we showed that the aqueous extract from the stem bark of G. buchananii trees is a non-opiate preparation, which reduces peristalsis by inhibiting neurotransmission (Balemba et al., 2010) and 5-HT3 and 5-HT4 receptors (Boakye et al., 2012). Furthermore, the extract has anti-inflammatory, and anti-nociception effects (Castro et al., 2011). The compounds having anti-motility properties appear to be flavonoids, or a combination of flavonoids with alkaloids or steroids (Boakye et al., 2012). Clearly, research aimed at defining the bioactive components and mechanisms of action as well as indigenous uses suggest that G. buchananii could be an effective anti-diarrhea medication and also a source of novel nonopiate anti-diarrheal compounds. Currently, the only drugs available that rapidly shorten the duration of diarrhea and alleviate pain are opiates (Ruppin, 1987; Riddle et al., 2008). These drugs cause constipation, drowsiness and are addictive. Consequently they are not recommended for children (Kelly, 2011; Riddle et al., 2008). This indicates the unmet need for new non-opiate antimotility compounds and the need for the formal testing of the efficacy of G. buchananii bark extract and its derivatives as treatments against diarrheal diseases. This also requires the use of diarrheal models. Ingesting large quantities of lactose (45%–87%) causes osmotic diarrhea through increased secretion in the small and large intestine of animals (Bueno et al., 1994; Lawrence et al., 1956; Liuzzi et al., 1998) and lactose causes diarrhea in humans with lactose-intolerance accounting for over 50% of the world population (Haemmerli et al., 1965; Lomer et al., 2008). A novel hypothesis suggests that diarrhea, flatulence, nausea, pain and other symptoms of lactose-intolerance arise from the effects of toxic bacterial metabolites such as alcohols, acids, ketones and methylgyoxal on gut effector tissues including the epithelium, muscle and nervous tissue (Campbell et al., 2010). It has been shown that a high-lactose diet induces severe and persistent diarrhea, intestinal damage and malnutrition in experimental animals (Arciniegas et al., 2000; Bueno et al., 1994; Fijlstra et al., 2010; Liuzzi et al., 1998; Norton et al., 2001). These effects of lactose-induced diarrhea are to some extent, similar to changes seen in children suffering from gastroenteritis or chronic diarrhea (Bueno et al., 1994). Interestingly, diarrhea due to lactose intolerance is a common complication of infectious diarrhea in children with malnutrition (Brown, 2003; Moore et al., 2010; Nyeko et al., 2010). The aims of this study were to investigate the effectiveness of G. buchananii stem bark extract in treating lactose-induced diarrhea in rats, determine the effective dose, and test the effectiveness of its anti-motility fractions PTLC1 and PTLC5 as anti-diarrheal agents. 2. Materials and methods 2.1. Inducing diarrhea in rats using a high-lactose diet The study was conducted in accordance with the regulations of the University of Idaho Institutional Animal Care and Use Committee (IACUC). Sixty two, 10 week old Wistar rats (389.2þ/  6.3 g) were obtained from Harlan Animal Research Laboratory (Hayward CA, USA). Rats were individually caged (23–24 1C; 12:12 h light-dark cycle) and quarantined for one week. Rats were fed a standard chow diet ad libitum (Animal Specialties, Hubbard, OR, USA) and had free access to water for four days prior to the inducement of diarrhea. A high-lactose diet (HLD) containing 35% lactose in place of starch (3.004 kcal; Purina Mills; Richmond, Indiana, USA) was fed to 52 rats. Diarrhea was induced within 24–48 h after consuming the diet. Rats were monitored for changes in consistency of pellets and stool mass, fecal fluid and urine production (mass; g and volume; mL). Rats were considered diarrheic if they produced watery stools, soft, yellowish stools compared to normal, pliable, soft, well-formed pellets as previously described by other researchers (Arciniegas et al., 2000; de Groot et al., 1995; Lawrence et al., 1956). Four days after introducing rats to a HLD, diarrheic rats were treated using varying doses of G. buchananii extract, its antimotility fractions, PTLC1 and PTLC5 (Boakye et al., 2012) and loperamide for standard comparison. Rats were maintained on a HLD during the entire treatment period. 2.2. Preparation of G. buchananii bark extract, PTLC1 and PTLC5 fractions Garcinia buchananii bark powder was prepared from stem barks collected from trees in their natural habitat in Karagwe, Tanzania, as described previously by Balemba et al. (2010). A sample can be found at the University of Idaho Stillinger herbarium (voucher 159,918). 0.1 g, 0.5 g, 1.0 g and 5.0 g G. buchananii bark powder were each suspended in 1 L of tap water, stirred for 30 min and filtered. The filtrate was then immediately used to treat rats against lactose-induced diarrhea. The anti-motility fractions were obtained from aqueous G. buchananii bark extract using a preparative thin layer chromatoghraphy (PTLC) separation method as described previously (Boakye et al., 2012). 2.3. Treating diarrheic rats with G. buchananii extract, PTLC1 and PTLC5 fractions Twenty nine rats on a HLD were randomly assigned and treated with varying doses of G. buchananii extract. In total, nine rats were treated with 0.1 g, seven rats with 0.5 g, six rats with 1.0 g, and seven rats with 5.0 g G. buchananii bark extract. A total of eight rats were treated with PTLC1 or PTLC5. Four rats per group were treated with either PTLC1 or PTLC5 at a dose of 15 mg and 3.8 mg in 100 mL tap water, respectively. For control treatments, seven rats were treated with loperamide (8.4 mg/L tap water), eight rats were left untreated (HLD control) and ten rats received control standard chow diets (SD). Rats were treated for a total of four days, receiving freshly made drugs every two days. 541 P.A. Boakye et al. / Journal of Ethnopharmacology 142 (2012) 539–547 2.4. Monitoring the effect of treatments Lactose (n = 5) 0.1 g (n = 5) 2.5. Data analysis Statistical analysis was performed using GraphPad Prism 5 (GraphPad Software Inc., San Diego, CA, USA). One-way ANOVA and the Newman-Keul’s multiple comparison post-hoc test were used to determine differences between treatments. Differences were considered statistically significant at Po0.05. 3. Results 3.1. Effect of a HLD on the form of feces, fecal fluid content and urine production We found that all rats fed a SD diet produced many wellformed, rounded, oblong fecal pellets (20þ/ 2 pellets; 5.82þ / 1.0 g (n = 5) % Fecal fluid content (fecal fluid weight/ fecal fresh weight) 100 Loperamide (n = 4) Chow (n = 3) 90 80 70 60 50 40 30 Inducement Treatment 20 1 2 3 4 5 6 7 Treatment days 8 9 10 11 Fig. 1. Aqueous G. buchananii bark extract prevents fluid loss via stools. The 35% HLD caused a dramatic increase in fecal fluid content during the inducement period (see days 2–6). Compared with non-treated rats (lactose), all doses of G. buchananii bark extract (0.1 and 1.0 g bark powder/L) significantly reduced fecal fluid content after one day (day 7; P o0.05 and Po 0.01, respectively). Similarly, loperamide significantly reduced fecal fluid content after 1 day (P o 0.01). 200 ♦ Total number of pellets (4 treatment days) We studied the impact of a HLD and all the treatments on the consistency, form and appearance of feces. Body weight, food and water intake were measured every two days for the entire period. Diarrheic rats treated with G. buchananii (0.1 g/L, 1.0 g/L) and loperamide (8.4 mg/L) were used to study fecal fluid content, stool mass and urine production compared with untreated rats on a HLD and SD control diets. To achieve this, pellets/stools and urine were allowed to drop on non-leaking, polyester plastic mats spread in metal trays placed beneath wired cages housing rats. Pellets/stools and urine of individual rats were collected every three hours, for 24 h, for a 12-day period. To separate watery stool from urine, yellowish fluid that did not contain traces of fecal matter was considered as urine. Disposable polyethylene transfer pipettes (5.5 mL) were used to collect urine. Pellets/stools were collected by using custom-made pieces of Inkjet transparencies. The samples were stored in disposable, polypropylene containers (100 mL) tightly capped to avoid loss of moisture due to vaporization. Polyester mats, polypropylene containers and transfer pipettes were obtained from VWR international LLC, WA, USA. Cumulative fresh weights of pellets/stools, and urine mass and volume were recorded every 6 h for 24 h. At the end of every 24-h period, pellets and stools were transferred into Pyrex glass beakers and heated in the oven (70 1C; 24-h) to obtain constant dry weight. Fecal fluid content was obtained by subtracting dry weight from fresh weight. To eliminate variations between animals, especially during diarrhea period, fecal fluid content was expressed as a fraction of fecal fluid weight divided by total fecal fresh weight. On the last day, all animals were euthanized by isoflurane inhalation and exsanguinated. Gross anatomical evaluation of the size and color of the small intestine, cecum, and colon as well as of the spleen, liver and kidney were recorded. Length and width of cecum were estimated using cotton threads and a ruler. For logistical reasons, the experiments for this study were conducted in four phases. In the first two trials, we studied the effect of G. buchananii extract, PTLC1 and PTCL5 on animal weight, food and water intake. Pellet counting was done during phase two and three while in the last two phases we measured fluid content of stool in addition to weight, food and water. For each phase, the number of rats in control treatments was either n¼3 or n¼2 for the standard chow diet and n¼ 2 for the high lactose diet. This was purposely done in order to use the minimum number of animals for this study. However, in analyzing weight loss, food and water consumptions, we used measurements from all animals in the control groups because these parameters were measured for each animal used in the experiment. Subsequently, the numbers of replicates of control animals are different for fecal fluid measurement (Fig. 1), pellet numbers (Fig. 2) and measurements involving weight, food and water consumption (Figs. 3 and 4). 160 120 60 40 n=4 n=5 n=5 n=4 n=5 n=4 SD HLD 1.0 g ext. 0.1 g ext. 0.5 g ext. 5.0 g ext n=5 20 0 Loperamide Fig. 2. Treatment of diarrheic rats with G. buchananii extract and loperamide did not increase the number of fecal pellets (defecation rate) in rats with diarrhea due to HLD. Diarrheic rats (HLD) had a significantly reduced number of pellets beyond that of control rats fed a standard chow diet (SD) (~ Po0.001). The numbers of pellets produced by rats treated with G. buchananii stem bark extract (0.1–5.0 g/L) were 2–3 times greater than those produced by untreated rats on the HLD and HLD rats.  0.15 g per day), whilst mucus was not readily observable by visual inspection. However, the consumption of a HLD caused diarrhea in 67% of rats within 24 h and all rats within 48 h. The majority of diarrheic rats (46%) had severe diarrhea characterized by profuse, watery stools visibly containing lots of mucus (Table 1). Twenty three percent (23%) of rats produced wet, mucuoid, loose stools. Twelve percent (12%) of rats produced lots of loose, wet, sticky, yellowish stools with or without structural form of pellet (Table 1). The HLD caused a significant increase in fecal fluid content as fluid content constituted 86% of stool mass compared with 42% in pellets produced by rats fed the SD (Fig. 1; HLD: 86.47 1.9% versus (vs.) SD: 42.072.3%; Po0.001). Furthermore, the HLD significantly increased urine production compared with SD rats (HLD: 9.32 71.09 g/d vs. SD: 4.6870.19 g/d; Po0.001). In summary, a HLD successfully induced diarrhea in rats and caused a significant loss of body fluid through diarrhea. 542 P.A. Boakye et al. / Journal of Ethnopharmacology 142 (2012) 539–547 * 10.0 10.0 Two days average weightgain or weight loss (g) Two days average weight gain (positive values) or weight loss (negative values; g) ∆ ∆ 7.5 5.0 2.5 * n=10 n=9 n=8 n=7 n=6 n=7 n=7 0.0 -2.5 -5.0 2.5 n=6 n=7 n=6 n=7 HLD PTLC1 PTLC5 0.0 -2.5 -5.0 -10.0 SD HLD 0.1 g ext. 0.5 g ext. 1.0 g ext. SD 5.0 g Lopeext. ramide ♦ ∆ 80 50 40 30 20 10 n=10 n=8 n=9 n=7 n=6 n=7 n=7 SD HLD 0.1 g ext. 0.5 g ext. 1.0 g ext. 5.0 g ext. Loperamide Two days average food consumption (g) * 60 40 20 0 100 90 80 80 70 60 50 40 30 20 10 10 0 n=6 n=7 n=4 n=4 SD HLD PTLC1 PTLC5 0 ♦ ∆ * 150 n=10 n=8 n=9 n=7 n=6 SD HLD 0.1 g ext. 0.5 g ext. 1.0 g ext. n=7 n=7 5.0 g Lopeext. ramide Fig. 3. (A)–(C) Summary data showing the effect of G. buchananii stem bark extract on body weight (A), food consumption (B) and water intake (C) of rats with lactose diet-induced diarrhea. (A) The HLD caused significant loss of weight compared with the standard chow diet (SD; ; Po 0.001). 0.1 g extract, reversed the HLD-induced weight loss and caused a weight gain (*; Po 0.05). All other treatments were without significant effects (D P40.05). (B) A HLD significantly reduced food consumption ( ; P o0.001). Likewise, food consumption was reduced in diarrheic rats treated with G. buchananii bark extract (all doses) and loperamide (D; P o0.001). (C) A HLD lower doses (0.1 g/L and 1.0 g/L) of G. buchananii bark extract did not significantly alter water intake. However, 5.0 g/L G. buchananii bark extract and loperamide significantly reduced water intake (~; Po 0.01). 3.2. Garcinia buchananii bark extract stopped diarrhea and stopped fluid loss Rats treated with G. buchananii extract (0.1 g/L) produced soft and poorly formed pellets (Table 1; Figs. 1 and 2), whilst pellets Two days average water intake (mL) Two days food consumption (g) 5.0 -7.5 ∆ Two days average water intake (mL) 7.5 125 100 75 50 25 n=6 n=7 n=4 HLD PTLC1 n=4 0 SD PTLC5 Fig. 4. (A)–(C) The effects of PTLC1 (15 mg/100 mL) and PTLC5 (3.8 mg/100 mL) on animal weights (A) food consumption (B) and water intake (C). (A) A HLD caused significant weight loss compared with SD alone ( ; P o0.001). PTLC1 and PTLC5 both reversed the HLD induced weight loss and promoted weight gain (*; Po 0.001; D; P o 0.001). (B) Compared with SD rats, a HLD significantly reduced rats food consumption ( ; P o0.001). PTLC1 and PTLC5 both reversed the effect of a HLD and increased food consumption in diarrheic rats (*; P o 0.001 and D; Po 0.01; respectively). PTLC1 increased food intake compared with SD (~; Po 0.01). (C) A HLD and PTLC1 did not affect water intake compared with SD rats (no bracket; P 40.05). PTLC5 significantly increased fluid consumption when compared with PTLC1 (*; Po 0.01) as well as SD and untreated (HLD) rats (D; Po 0.05 and Po 0.001; respectively). produced by rats treated with G. buchananii extract (1.0 g/L) were well-formed, soft, and pliable. Rats treated with G. buchananii extract (5.0 g/L) produced well-formed, rounded, oblong, and 543 P.A. Boakye et al. / Journal of Ethnopharmacology 142 (2012) 539–547 Table 1 Comparison of the effectiveness of varying doses of G. buchananii extract, PTLC1, PTLC5 and loperamide on treating high lactose diet-induced diarrhea in rats. Treatment Standard chow diet 35% lactose diet (HLD) HLD þ0.1 g/L extract HLD þ0.5 g/L extract HLD þ1 g/L extract HLD þ5 g/L extract HLD þPTLC1 HLD þPTLC5 HLD þ8.4 mg/L LP Form of stool after 24 h to the 4th day of treatment Watery diarrhea þ mucous (incontinence or profuse) Loose stools þ mucous (non-structural stool) Loose stool with structural form of pellet Soft formed pellets Hardened normal pellets  þþþ         þþ         þ þþ     þ  þþ  þþþ þþþ þþþ þ þþþ þþþ  þþþþ  þ þþ þþþ þþþþ þþþ þþ þþþþþþ Key: LP is the Loperamide. Qualitative assessment score or classification: ‘ ’ condition not observed, þ is the observed once/day; þþ is the observed twice/day, þþþ is the observed 3–4 times/day, þþþþ is the abundantly observed and þþþþþþ is the almost exclusively observed. relatively harder pellets. The hardness of these pellets was comparable to that of rats treated with loperamide (8.4 mg/L; Table 1). Compared with rats on a HLD alone, G. buchananii extract (0.1 g/L and 1.0 g/L) reduced fecal fluid content (Fig. 1; HLD: 86.471.9% vs. 0.1 g extract: 59.772.5% and 1.0 g extract: 51.873.9%; Po0.01 and Po0.001, respectively), with effects observed as early as 6–24 h after commencement of treatments. The effect of G. buchananii extract (GB; 1.0 g/L) was similar to that of loperamide (LP; 8.4 mg/ L; GB: 51.873.9% vs. LP: 43.876.5%; P40.05). In addition, G. buchananii extract (GB; 1.0 g/L) and loperamide reduced the HLD induced-increase in urine production (HLD: 16.5þ/1.7 mL vs. CH: 8.4þ/ 0.5 mL; Po0.01) back to normal SD levels (GB: 8.2þ / 0.8 mL and LP: 5.4þ/ 1.1 mL vs. CH: 8.4þ/ 0.5 mL; P40.05, respectively). In summary, the varying doses of aqueous G. buchananii extract were all effective at treating lactose-induced diarrhea, intestinal mucus secretion, and loss of body fluid through stool and urine. Based on the texture of feces, fluid content, fresh stool weight, and urine production, G. buchananii extract (1.0 g/L) was very effective at treating diarrhea and also promoting fluid retention. Overall, the effects of the extract were comparable to that of loperamide (8.4 mg/L). lower doses of G. buchananii extract (0.5–1.0 g/L) showed considerable trends towards reversing weight loss. 3.3. A lower dose of G. buchananii bark extract also reversed weight loss due to lactose diet-induced diarrhea 3.5. A HLD and 0.1–1.0 g/L G. buchananii extract did not alter water consumption A HLD caused loss of weight (indicated by negative number values) in rats compared with rats fed a SD, whilst the SD fed rats actually gained weight (Fig. 3A; HLD:  3.1270.58 g vs. SD: 7.5070.50 g; P o0.001). Compared with rats on a HLD alone, the HLD rats treated with G. buchananii extract (0.1 g/L) gained weight (Fig. 3A; HLD:  3.1270.58 g vs. GB: 0.8270.4 g; Po0.05). Although not significant, G. buchananii extracts (0.5– 1.0 g/L) showed a trend towards reversing the weight loss caused by ingesting a HLD (GB:  1.0571.27 g and  0.7870.81 g vs. HLD:  3.12 70.58 g; P40.05, respectively). In contrast, rats treated with G. buchananii extract (5.0 g/L) lost weight with the same magnitude as rats on a HLD (GB:  3.2071.87 g vs. HLD: 3.12 70.58 g; P40.05). Loperamide treatment showed a trend of reversing weight loss in the HLD rats by 1.78 g, which was not significant when compared with a HLD (LP:  1.3370.64 g vs. HLD:  3.1270.58 g; P 40.05). Although rats treated with a lower dose of G. buchananii (0.1 g/L) gained 2.15 g compared with rats treated with loperamide, the difference was not significant (GB: 0.8270.4 g vs. 1.33 70.64 g P40.05). Taken together, these observations show that a HLD caused weight loss in rats, whilst G. buchananii (0.1 g/L) reversed this weight loss. Other Rats fed a SD consumed the same amount of water as those on a HLD (Fig. 3C; SD: 76.8873.39 mL vs. HLD: 63.5573.55 mL; P4 0.05). No difference in water intake was apparent between rats on a HLD and rats treated with G. buchananii extract at a dose of 0.1 g/L (HLD: 63.3173.55 mL vs. GB: 68.9377.12 mL; P4 0.05), 0.5 g/L (HLD: 63.3173.55 mL VS. GB: 72.25710.95 mL; P40.05), and 1.0 g/L (HLD: 63.5573.55 mL vs. GB: 66.3278.13 mL; P40.05). However, G. buchananii extract at a dose of 5.0 g/L and loperamide decreased water consumption in the HLD rats when compared to the SD rats (GB: 44.0074.57 mL and LP: 43.9677.66 mL vs. SD: 76.8873.39 mL; Po0.05 and Po0.01, respectively). In conclusion, at lower doses, (0.1–1.0 g/L) G. buchananii extract did not significantly promote water intake in diarrheic rats. A higher dose (5.0 g/L) of extract and loperamide caused a decrease in water consumption. 3.4. Garcinia buchananii extract did not improve food intake in diarrheic rats A HLD reduced food intake in rats when compared with those on a SD (Fig. 3B; HLD: 34.3971.08 g vs. SD: 41.3771.59 g; Po0.01). There was no difference in food consumption between untreated HLD rats and rats treated with G. buchananii extract at doses of 0.1 g/ L (HLD: 34.3971.08 g vs. GB: 32.4772.21 g; P40.05), 0.5 g/L (HLD: 34.3971.08 g vs. GB: 29.373.2 g; P40.05), 1.0 g/L (HLD: 34.3971.08 g vs. GB: 28.4772.77 g; P40.05) or 5.0 g/L (HLD: 34.3971.08 g vs. GB: 27.9472.67 g; P40.05). Likewise, food consumption in the HLD rats was not improved by loperamide treatment (LP: 28.5073.58 g vs. HLD: 34.3971.08 g; P40.05). There was also no significant difference in food intake between rats treated with loperamide and those treated with G. buchananii extract (all doses; P40.05). Taken together, these observations show that a HLD caused a significant decline in food intake. Garcinia buchananii extract and loperamide failed to improve food intake in rats with a HLD-induced diarrhea. 3.6. The anti-motility fractions PTLC1 and PTLC5 treated lactoseinduced diarrhea and reversed weight loss Rats treated with PTLC1 or PTLC5 recovered from diarrhea within 24 h. One day after starting treatments, rats treated with PTLC1 produced round, oblong and well-formed pellets. PTLC5 544 P.A. Boakye et al. / Journal of Ethnopharmacology 142 (2012) 539–547 treated rats produced soft and poorly formed pellets, which gradually became transformed into well-formed and pliable fecal pellets after 2–3 day. There was a complete reversal of weight loss in the HLD rats treated with PTLC1, when compared with the untreated HLD rats (Fig. 4A: PTLC1: 5.91 71.39 g vs. HLD:  3.1270.58 g; Po0.001). Similarly, a complete reversal of weight loss was also apparent in diarrheic rats treated with PTLC5 (Fig. 4A: PTLC5: 5.91 70.54 g vs. 3.12 70.58 g; Po0.001), with no significant difference in magnitude of effect between PTLC1 and PTLC5 treatments (P 40.05). In summary, both PTLC1 and PTLC5 individually reversed the HLD induced weight loss, with a similar magnitude of effect observed with both treatments. vs. SD: 76.8873.39 mL; P40.05). Interestingly, treating HLD rats with PTLC5 significantly increased their fluid consumption beyond that of SD rats (Fig. 4C; PTLC5: 120.2079.83 mL vs. SD: 76.887 3.39 mL, Po0.001), untreated HLD rats (Fig. 4C; PTLC5: 120.207 9.83 mL vs. HLD: 63.3173.55 mL, Po0.001) and HLD rats treated with PTLC1 (Fig. 4C; PTLC5: 120.2079.83 mL vs. SD and PTLC1: 76.9676.82 mL, Po0.01). In summary, PTLC5 markedly increased fluid intake in rats with HLD-induced diarrhea, whereas PTLC1 had no effect on fluid intake. 3.9. Gross anatomical observations of intestine, liver, spleen and kidney Compared with untreated HLD rats there was a significant increase in food consumption in HLD rats treated with PTLC1 (Fig. 4B; HLD: 34.39 71.08 g vs. PTLC1: 55.93 75.56 g; Po0.001). Correspondingly, treating HLD rats with PTLC5 also significantly increased food consumption compared with untreated HLD rats (Fig. 4B; HLD: 34.39 71.08 g vs. PTLC5: 49.28 77.25 g; P o0.01). Interestingly, food intake of HLD rats treated with PTLC1 was significantly increased beyond that of SD rats (Fig. 4B; PTLC1: 55.93 75.56 g vs. SD: 41.37 71.59 g; Po0.01). There was no difference in food intake between rats treated with PTLC1 and PTLC5 (Fig. 4B; PTLC1: 55.93 75.56 g vs. PTLC5: 49.28 g77.25; P40.05) and between rats treated with PTLC5 and those on a SD (Fig. 4B; PTLC5: 49.28 77.25 g vs. SD: 41.3771.59 g; P 40.05). In summary, both PTLC1 and PTLC5 significantly improved food consumption in HLD induced diarrheic rats, whilst PTLC1 also increased food intake above normal control levels. All SD rats had relatively small ceca and colons and were less bloated with gas. Colons were filled with well-formed pellets. Rats on a HLD had distended distal ileum, ceca and colons. The colons were often empty of stools but bloated with gas (Fig. 5). HLD rats treated with G. buchananii extract (0.1–1.0 g/L) and loperamide also showed signs of distended ceca and colons but not as enlarged as those of untreated rats. There were fewer wellformed pellets in the colons of rats treated with G. buchananii extract (0.1–0.5 g/L) as well as a relatively more bloated ceca and colons in comparison to those treated with G. buchananii extract (5.0 g/L). Rats treated with aqueous G. buchananii extract (1.0 –5.0 g/ L) had less bloated colons and produced relatively well-formed fecal pellets in the colon (cecum maximum diameter: SD: 1.9þ/ 0.2 cm vs. HLD: 2.9þ/0.5 cm; Po0.01; HLD: 2.9þ/ 0.5 cm vs. 1.0 g/L extract: 2.1þ/ 0.3 cm; Po0.01 and HLD: 2.9þ/ 0.5 cm vs. LP: 2.0þ/ 0.2 cm; Po0.01; Fig. 5). PTLC1 and PTLC5 also reduced bloating. None of the rats used in the experiment showed signs of hemorrhage or necrosis in the intestine, spleen, liver or kidney. In summary, G. buchananii extract, its isolated anti-motility fractions and loperamide all reduced bloating. 3.8. PTLC5 increased fluid consumption of diarrheic rats 4. Discussion There was no significant difference in fluid consumption between untreated HLD rats and HLD rats treated with PTLC1 (Fig. 4C; HLD: 63.3173.55 mL vs. PTLC1: 76.9676.82 mL; P40.05). Fluid consumption in HLD rats treated with PTLC1 was almost identical to that of SD rats (Fig. 4C; PTLC1: 76.9676.82 mL; The purpose of this study was to examine the effectiveness of G. buchananii extract and its fractions with anti-motility actions as a treatment against diarrhea using a model of lactose-induced diarrhea. We provide evidence that G. buchananii extract and its anti-motility fractions are capable of treating HLD-induced 3.7. PTLC1 and PTLC5 increased the food consumption of diarrheic rats Fig. 5. Demonstration of the effects of G. buchananii extract and loperamide on HLD induced cecum distension. All doses of G. buchananii extract and loperamide reduced cecum distension (size). P.A. Boakye et al. / Journal of Ethnopharmacology 142 (2012) 539–547 545 diarrhea, whilst significantly increasing food and fluid consumption and significantly increasing body mass. The efficacy of G. buchananii bark extract treatment when used at 1.0–5.0 g/L were in all cases highly comparable to those of loperamide, but actually outperformed loperamide in terms of weight gain or food/fluid intake. When used in small doses, G. buchananii extract (0.1 g/L) reversed the HLD diarrhea-induced weight loss. This beneficial effect was augmented by both PTLC1 and PTLC5 individually. PTLC5 also significantly increased water intake. Garcinia buchananii bark extract and its fractions also reduced bloating. These new findings support the indigenous usage of the extract as a folk remedy against diarrheal diseases (Balemba et al., 2010; Chinsembu and Hedimbi, 2010; Kisangau et al., 2007) and the notion that G. buchananii extract holds great potential for the isolation of novel, non-opiate anti-diarrheal compounds (Balemba et al., 2010; Boakye et al., 2012). body fluid occurs in all forms of diarrheal diseases and it is the main cause of debilitation and death seen in infectious diarrheas (Field, 2003; Guerrant et al., 2003; Thapar and Sanderson, 2004; UNICEF/ WHO, 2009). Our observations suggest that G. buchananii contains compounds that reverse the increased mucus and fluid secretion associated with diarrhea due to lactose intolerance in rats. Whether the extract promotes intestinal fluid absorption needs to be confirmed by investigating the effect of the extract and its isolated compounds on mucosal fluid transport. Taken together, our observations suggesting that G. buchananii extract effectively reduced stool fluid content and bowel motility in a non-infectious model of diarrhea, sets the premise to test the extract against infectious diarrheas. 4.1. Garcinia buchananii bark extract treats lactose-induced diarrhea within 6–12 h Another interesting and novel finding of this study is that G. buchananii extract (0.1 g/L) and the anti-motility fractions, PTLC1 and PTLC5 reversed the weight loss induced by a HLD. The considerable decrease in the weight of HLD rats indicates malnutrition (Bueno et al., 1994; Fijlstra et al., 2010; Liuzzi et al., 1998; Norton et al., 2001), which is a key symptom of lactoseinduced diarrhea in humans (Brown, 2003; Lomer et al., 2008; Moore et al., 2010; Nyeko et al., 2010). In most incidences of diarrhea, intestinal injury, altered mucosal function and loss of appetite results in poor nutritional status (Brown, 2003; Moore et al., 2010; Nyeko et al., 2010; Thapar and Sanderson, 2004; UNICEF/WHO, 2009). Individuals with acute diarrhea consume less food, have reduced absorptive capacity than those who are healthy, which leads to the continuous decrease in weight (Brown, 2003; Moore et al., 2010; Nyeko et al., 2010; Thapar and Sanderson, 2004). In most cases of chronic diarrhea and malnourished patients, the intestinal mucosa is damaged. Subsequently, patients become more prone to new and longer episodes of diarrhea, which exacerbates their nutritional status and health (Brown, 2003; Guerrant et al., 1992; Moore et al., 2010; Nyeko et al., 2010; Thapar and Sanderson, 2004). Therefore, diarrhea in malnourished children is difficult to treat (Brown, 2003; Lima et al., 2000; Moore et al., 2010; Nyeko et al., 2010), whilst adequate nutrition is very critical to the treatment of diarrhea (Brown, 2003; Guerrant et al., 1992; Moore et al., 2010; Nyeko et al., 2010; UNICEF/WHO, 2009). Our data strongly suggest that G. buchananii has the potential to promote weight gain both at lower doses and as purified components (PTLC1 and PTLC5). The reversal of weight loss observed in the present study could be due to a reduction of body fluid loss and an increase in food and fluid consumption (PTLC5). Whether G. buchananii extract and its derivatives could promote weight gain in humans when used as anti-diarrhea medications, and indeed the mechanisms involved are important questions that remain to be addressed. The exciting prospect is that G. buchananii bark extract may contain specific compounds that affect appetite. In support of this idea, the extract from the fruit rind of Garcinia indica is traditionally used to stimulate appetite (Deore et al., 2011). Our findings that G. buchananii serves as an effective anti-diarrheal remedy whilst promoting food and water consumption is extremely promising, as increased nutrition is critical to maintaining energy homeostasis during the rapid loss of fluids, essential electrolytes and nutrients in diarrheic patients. The effectiveness and advantages of the extract and its fractions highlights the need to determine the efficacy and safety of these preparations, especially as a treatment of diarrhea in children. At a higher dose, G. buchananii extract did not alter HLD diarrhea-induced weight loss. Rather, there was a trend towards increasing weight loss. We speculate this could be attributable to The ingestion of a HLD resulted in the onset of diarrhea in less than 24 h. The signs of diarrhea and loss of body weight seen in this study correspond with findings from previous studies in which rats fed HLD had chronic diarrhea and became malnourished (Arciniegas et al., 2000; Bueno et al., 1994; Fijlstra et al., 2010; Lawrence et al., 1956; Liuzzi et al.,1998; Norton et al., 2001). Few animals with delayed onset diarrhea consumed less food indicating a correlation of disease severity with the amount of diet consumed. One of the new findings from this study was that G. buchananii extract, at all doses tested, treated lactose-induced diarrhea within 6–12 h of initiating treatment. The overall effectiveness of 1.0–5.0 g/L G. buchananii extract in treating diarrhea was evidenced by the reduction of fecal fluid content leading to pellet production instead of stool. This effect was highly comparable to the effect of loperamide (8.4 mg/L). However, rats treated with loperamide produced up to four times fewer pellets compared with G. buchananii extract (1.0–5.0 g/L) treatment suggesting that loperamide caused a greater anti-motility effect than the extract. Anti-motility agents, such as loperamide, increase the time required for substances to transit the bowel, enhancing the potential for the re-absorption of fluids electrolytes and nutrients (Ruppin, 1987). Loperamide is an opiate drug, which is commonly used to treat diarrhea and considered to be the most effective anti-diarrhea medication to reduce gastrointestinal transit. However, due to constipation and its ability to cause respiratory depression, it is not used to treat children and young infants (Kelly, 2011). We have previously shown that G. buchananii extract is a non-opiate preparation that dramatically reduces guinea-pig colonic motility through inhibiting synaptic transmission in the myenteric ganglia (Balemba et al., 2010) and the extracts’ actions involve inhibiting 5-HT3 and 5-HT4 receptors (Boakye et al., 2012). Together, our findings suggest that the G. buchananii extract is an effective anti-motility agent to treat lactose-induced diarrhea. We have shown for the first time, that G. buchananii extract causes a drastic decline in stool mucus and fluid content (fresh weight) in less than 24 h. These findings and the transformation of feces from a watery form to well-formed, pliable pellets suggest that G. buchananii extract potentially has compounds that reduce intestinal mucus and fluid secretion, in addition to having compounds with anti-motility effects as shown in our previous studies (Balemba et al., 2010; Boakye et al., 2012). The reduction of fecal mucus conforms with indigenous reports that an extract of the outer rind of the fruits of Garcinia indica is a folk remedy for dysentery and mucous diarrhea in India (Deore et al., 2011). The excessive loss of 4.2. Lower doses and anti-motility fractions of G. buchananii bark extract reverse weight loss associated with lactose-induced diarrhea 546 P.A. Boakye et al. / Journal of Ethnopharmacology 142 (2012) 539–547 (-)-hydroxycitric acid (HCA), a derivative of citric acid found in fruit rind form in Garcinia species (Pedraza-Chaverri et al., 2008). HCA is thought to cause weight loss by competitively inhibiting the enzyme adenosine triphosphatase-citrate-lyase and increasing serotonin release or availability in the brain, which leads to suppression of appetite (Ohia et al., 2002; Pedraza-Chaverri et al., 2008). If HCA is present in G. buchananii bark extract, its effects become apparent when the extract is used at higher doses (5.0 g/ L). Furthermore, G. buchananii bark and its extracts have a bitter taste at increasing concentrations (Balemba, O.B. personal observation). As such it is possible that the bitter taste made rats avoid consuming water, reducing overall fluid consumption at the higher dose of the extract. 4.3. Garcinia buchananii extract reduces intestinal bloating During most cases of lactose-induced diarrhea, there is an enlargement of the ileum, cecum and colon due to the accumulation of gas (Lawrence et al., 1956). In humans, GI symptoms of lactose intolerance include pain, bloating, diarrhea and/or constipation and flatulence (Lomer et al., 2008; Campbell et al., 2010; Eadala et al., 2011). All doses of crude G. buchananii extract, its fractions and loperamide reduced bloating in cecum and colon compared with untreated HLD rats. (Lawrence et al., 1956) showed that bloating in albino rats fed a HLD were associated with an increase in bacterial mass in the cecum, which may have also occurred in the HLD rats used in the current study. However, it remains unclear whether these changes were due to the effect of drugs on intestinal bacteria or intestinal structure and physiology, or both. Whether G. buchananii bark extract could benefit patients having symptoms of bloating and diarrhea such as patients with hypolactasia, foodintolerance, and irritable bowel syndrome (Campbell et al., 2010; Eadala et al., 2011; Lomer et al., 2008) needs to be assessed. Phytochemical analysis performed on G. buchananii extract and its fractions with anti-motility effects, PTLC1 and PTLC5 indicated the presence of flavonoids, tannins, alkaloids and steroids (Boakye et al., 2012). These phytochemicals are considered to be responsible for the anti-diarrheal medicinal properties of most plant extracts (Atta and Mouneir, 2005; Longanga Otshudi et al., 2000; Mehmood et al., 2010; Palombo, 2006; Pedraza-Chaverri et al., 2008; Schuier et al., 2005). They act by suppressing gut motility, which delays gastrointestinal transit, the common feature of botanical extracts with anti-diarrheal properties (Atta and Mouneir, 2005; Balemba et al., 2010; Boakye et al., 2012; Mehmood et al., 2010; Ojewole et al., 2009). It appears evident that flavonoids, tannins, alkaloids and steroids are likely to be responsible for the anti-diarrheal effects of G. buchananii extract, either singly or in various combinations. Support for this idea is the recent finding showing that G. buchananii stem bark is a rich source of biflavanones and biflavanone-c-glycosides (Stark et al., 2012). Fecal production in HLD rats treated with various doses of the extract and loperamide did not return to normal SD values. The reasons for this include feeding rats the HLD to maintain diarrhea, and a reduced food intake. The increase in urine output appears to be ‘counter-intuitive’ because such an observation has not previously been reported (de Groot et al., 1995). It is unclear why rats on a HLD had an increase in urine production given that the extract had either no effect or water consumption (see above). 5. Conclusions This study shows that G. buchananii is an effective remedy of lactose-induced osmotic diarrhea, with an efficacy comparable to loperamide. The complete reversal of weight loss, increase in food and fluid consumption are additional benefits that are required to effectively treat diarrhea. There is the need to establish the exact bioactive compounds and the mechanisms underlying the antidiarrheal effects of G. buchananii in order to better understand how the extract works and lay the basis to promote its use for broader human treatment. Conflict of interest There are none. Acknowledgments Drs. Sofie Pasilis and. Onesmo B. Balemba are supported by the University of Idaho College of Science. Dr. Stuart M. Brierley is supported by a National Health and Medical Research Council of Australia (NHMRC) Australian Biomedical Fellowship. We thank Drs. Patrick J. Hrdlicka, Andrzej Paszczynski, and Lee Deobald for using their laboratory facilities. References Arciniegas, E.L., Cioccia, A.M., Hevia, P., 2000. Effect of the lactose induced diarrhea on macronutrients availability and immune function in well-nourished and undernourished rats. Archives Latinoamericana de Nutrition 50, 48–54. Atta, A.H., Mouneir, S.M., 2005. 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