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Keywords:

  • diarrhea;
  • Garcinia;
  • herbal remedy;
  • intestinal motility;
  • serotonin and 5-hydroxytryptamine

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. Author Contributions
  10. Conflicts of Interest
  11. References

Background Garcinia buchananii bark extract is an anti-motility diarrhea remedy. We investigated whether G. buchananii bark extract has components that reduce gastrointestinal peristaltic activity via 5-HT3 and 5-HT4 receptors.

Methods  Aqueous G. buchananii extract was separated into fractions using preparative thin layer chromatography (PTLC), and major chemical components were identified using standard tests. The anti-motility effects of the extract and its fractions (PTLC1-5) were studied through pellet propulsion assays using isolated guinea-pig distal colons.

Key Results  Anti-motility (PTLC1 & PTLC5) and pro-motility (PTLC2) fractions were isolated from the extract. Flavonoids, steroids, alkaloids, tannins, and phenols were identified in the extract and PTLC1&5. The potency of the extract applied via the mucosal surface was reduced by 5-HT, 5-HT3 receptor agonist RS-56812, 5-HT4 receptor agonists cisapride and CJ-033466, 5-HT3 receptor antagonist granisetron, and 5-HT4 receptor antagonist GR-113808. The anti-motility effects of the aqueous extract and PTLC1&5 when applied serosally were reversed by RS-56812, cisapride, and CJ-033466. The 5-HT3 receptor antagonists, granisetron and ondansetron, reduced the effects of the extract to an extent and completely reversed the anti-motility effects of PTLC1&5. GR-113808 inhibited the actions of the extract during the initial 10 min, but enhanced the extracts’ anti-motility effects after 15 min. GR-113808 augmented the anti-motility activities of PTLC1 and PTLC5 by 30%.

Conclusions & Inferences  These results indicate that the anti-motility effects of G. buchananii aqueous extract are potentially mediated by compounds that affect 5-HT3 and 5-HT4 receptors. Identification and characterization of the bioactive compounds within G. buchananii could lead to the discovery of new non-opiate anti-diarrhea formulations.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. Author Contributions
  10. Conflicts of Interest
  11. References

Diarrhea is the second leading cause of malnutrition and death among children under 5 years old in developing countries, causing 20% of all child deaths and a total of over 1.7 million deaths each year.1,2 In addition, diarrheal diseases cause debilitation, morbidities, and mortalities among persons displaced by humanitarian crisis and natural disasters, HIV/AIDS patients, and the elderly.1–4 Within developed countries, the episodes of diarrhea are relatively high, although only approximately one of six people seek medical treatment.5–7

Diarrhea is a protective response of the gastrointestinal (GI) tract that manifests itself with excessive mucosal fluid secretion and increased propulsive motility to remove the insulting agents, such as pathogens, toxins, drugs, or allergens, from the bowel. As such, it is often associated with a net loss of body fluids due to intestinal hyper-secretion, powerful hyper-motility, and cramping pain.1,6,8,9 Therefore, treatments are directed to combat these symptoms, the causative agents, and inflammation.1–3,6,8,9 Treatment options include the use of low-osmolarity oral rehydration salts (ORS) supplemented with zinc to counter intestinal fluid loss and, in children, continuous breast feeding to provide nutrients and enhance child immunity. The regimen also includes anti-motility agents, opiates, somatostatin analogs, absorbents, anti-secretory medicines, vaccinations, and antibiotics.1–3,6,8,9 However, recommended ORS therapy alone does not treat all underlying causes of diarrheal symptoms, or shorten the duration of illness. In developing countries, approximately 60% of children with diarrhea are from poor families and do not use ORS or synthetic drugs. Approximately 80% of the population use herbal remedies to treat all forms of diarrheal diseases.10–13

Botanical extracts have historically played key roles as precursors to modern drugs and as remedies for a host of diseases, including diarrhea and dysentery.12,14 Scientific data and indigenous knowledge suggest that Garcinia species can be employed as natural remedies for a variety of illnesses, including diarrheal diseases.10,12,13,15 Stem and root bark extracts of G. buchananii are currently used in Africa to treat diarrhea, dysentery, abdominal pain, and a range of infectious diseases.10,16 Recently, we demonstrated that the aqueous extract from the stem bark of G. buchananii reduces peristalsis, neurotransmission, and smooth muscle activity in the guinea-pig distal colon.16,17 However, the bioactive components and mechanism of action are not well understood.

Serotonin (5-hydroxytryptamine, 5-HT) signaling plays a critical role in the regulation of intestinal secretion and motility.18–20 All forms of diarrhea are characterized by the hyperplasia of enterochromaffin cells and increased serotonin release.18,20 In the gut, 5-HT is a major neuromodulator, and 5-HT3 and 5-HT4 receptors play a key role in regulating propulsive motility.19,21–24 Studies of anti-diarrheal tannic and phenolic components of wood creosote,25,26 gamma-mangostin, a xanthone from Garcinia cambogia fruit extract,27 and pro-motility components of Daikenchuto28 have shown that botanical remedies can inhibit 5-HT receptors. Therefore, the aim of the present study was to determine whether the anti-motility action of G. buchananii extract involves 5-HT dependent mechanisms, operating through 5-HT3 and 5-HT4 receptors.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. Author Contributions
  10. Conflicts of Interest
  11. References

Preparation of G. buchananii extract and fractionations using preparative thin layer chromatoghraphy (PTLC)

Garcinia buchananii stem bark was collected from plants in their natural habitats in Karagwe, Tanzania, and processed as described previously by Balemba and colleagues.16 A sample of bark powder was deposited at the University of Idaho Stillinger herbarium (voucher # 159918). An aqueous extract for isolating PTLC fractions was prepared by suspending 10 g G. buchananii bark powder in a 50 mL ethanol/water (70 : 30) mixture, sonicating for 20 min, and filtering using Whatman filter paper no. 4 (Fisher Scientific, Pittsburgh, PA, USA). The filtrate was extracted with 40 mL of hexane. The aqueous fraction was collected in a round bottom flask, and the solvent was removed using a rotary evaporator (150 rpm at 50 °C). The sample was then freeze-dried for 24 h. 10 g bark powder produced 0.9 ± 0.2 g of freeze-dried sample. The sample was reconstituted using15 mL of 70% ethanol/water mixture to obtain an aqueous extract (0.06 g mL−1 freeze-dried sample) for chromatographic separations.

Chromatographic separations were performed using 20 × 20 cm, silica gel GF preparative thin layer chromatoghraphy (PTLC) plates (1000 μm thickness; Analtech, Newark, USA). Eight microliters of the aqueous extract was applied as 13 mm band to the silica gel plates using a CAMAG Linomat five sample applicator equipped with a 100 μL syringe. A total of 72 μL of the aqueous extract (nine bands) was applied onto each PTLC plate. Separation was accomplished in a glass chromatography chamber using toluene/ethyl acetate/formic acid (30 : 20 : 5) as the mobile phase. Plates were viewed under a UVLS-28 EL Series UV Lamp (UVP LLC; Upland, CA, USA) using 365 nm UV light. Five major fractions were identified. Each fraction was scraped into a 50 mL centrifuge tube. Scrapings from 19 PTLC plates were pooled together for each individual fraction. 30 mL of ethanol was added to each fraction. Samples were sonicated for 20 min and centrifuged at a speed of 4838.4 g for 5 min. The supernatant was emptied into a 50 mL round bottom flask and solvent removed using a rotary evaporator. The weight of each collected fraction was determined. Approximately 1368 μL (roughly containing 82.08 mg) of purified freeze-dried reconstituted aqueous extract was applied to the 19 plates used to collect scrapings for isolating PTLC1-5 fractions.

Phytochemical screening

Phytochemical screening was carried out using standard chemical methods.29,30 Flavonoids were detected by heating 5 g of G. buchananii bark powder in 10 mL ethyl acetate for 3 min. The presence of flavonoids was confirmed by a yellow coloration when the filtrate was mixed with 1 mL of 1% ammonia solution. The presence of tannins was confirmed by the appearance of a blue-black coloration after mixing 1% FeCl3 with 10 mL of 0.5 g G. buchananii powder in distilled water. Steroids were confirmed by the appearance of a reddish-brown ring after mixing 0.5 mL each acetic acid anhydride with 0.5 g G. buchananii powder in methanol, cooling in ice, and mixing with both 0.5 mL chloroform and 1 mL of conc. H2SO4. Phenols were confirmed by the appearance of a bluish-green coloration after mixing 1 mL aqueous extract (5 g G. buchananii bark powder/30 mL water) with 2 mL of FeCl3 solution. Alkaloids were confirmed by the appearance of an orange color after adding a drop of picric acid to 2 mL aqueous extract (5 g G. buchananii bark powder/30 mL water). These procedures were used to characterize the compound classes found in PTLC1 (15 mg) and PTLC5 (3.8 mg).

Gastrointestinal motility assays

Animals and solutions: All studies were approved by The University of Idaho Animal Care and Use Committee. Male adult guinea-pigs weighing 250–450 g (Elm Hill Breeding Labs, Chelmsford, MA, USA) housed in metal cages with soft bedding were used in these studies. Animals were maintained at 23–24 °C on a 12 : 12 h light-dark cycle and provided free access to food and water. Midline laparotomy and distal colon collection was performed after isoflurane anesthesia and exsanguinations of individual animals. Colons were stored in ice-chilled Krebs solution (mmol L−1: NaCl, 121; KCl, 5.9; CaCl2, 2.5; MgCl2, 1.2; NaHCO3, 25; NaH2PO4, 1.2; and glucose 8; all from Sigma, St. Louis, MO, USA; aerated with 95% O2/5% CO2).

A segment (approximately 10–12 cm long) of distal colon was pinned on either end in a Sylgard-lined 50 mL organ bath being continuously perfused with oxygenated Krebs solution (rate: 10 mL min−1) and maintained at temperatures between 36 and 37 °C. Nail polish-coated guinea-pig pellets were used to determine propulsive velocities using the Gastrointestinal Motility Monitoring system (Med-Associates Inc., Saint Albans, VT, USA) for filming pellet propulsion as described by Hoffman and others (2010).31 Velocity was calculated as described previously by Balemba and colleagues.16

For intraluminal studies, the aqueous extract was prepared by suspending 2 g of G. buchananii bark powder in 100 mL Krebs, whereas 1 g G. buchananii bark powder prepared in the same manner was used for bath (serosal) applications. The extract-Krebs mixture was continuously stirred for 30 min and filtered using analytical filter paper (Schleicher and Schuell filter paper 589/3; 0.2 μm retention). Garcinia buchananii extract and test compounds were delivered to isolated colons in Krebs solution, and evaluated for effects on pellet propulsion during 20–25 min time intervals. Intraluminal drug deliveries (350 μL min−1 Krebs solution) were performed using polyethylene tubing (PE 205; outside diameter 9.5 mm) inserted approximately 1.5 cm into the oral end of the segments. In bath deliveries, G. buchananii extract and test compounds were superfused into the organ bath. Studies involving PTLC fractions were performed using the organ bath delivery method only. In initial pellet propulsion assays, we found that PTLC1 and PTLC5 obtained by collecting scrapings from 19 PTLC plates produced enough PTLC1 and PTLC5 fractions to significantly reduce pellet propulsion. Therefore, scrapings from 19 PTLC plates were used for each pellet propulsion assay to roughly maintain their proportionality relative to the bark powder and the aqueous extract fraction, used for chromatographic separations (see above).

Data analysis

To reduce variability between trials and between experiments, pellet propulsion velocity (mm s−1) for each trial was expressed as a percentage of normalized baseline velocity as described previously by Balemba and others.16 Statistical analysis was performed using GraphPad Prism 5 (GraphPad Software Inc., San Diego, CA, USA). Normalized control data (Krebs vehicle) passed the Shapiro–Wilk normality test (w = 0.93 and P = 0.20 for intraluminal applications and w = 0.96 and P = 0.27 for bath applications). 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 < 0.05.

Drugs

Serotonin hydrochloride (5-HT), CJ-033466, GR-113808, RS-56812 hydrochloride, and granisetron hydrochloride were purchased from Tocris Bioscience (Ellisville, MO, USA). Cisapride and ondansetron hydrochloride were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Stock solutions (1–5 mmol L−1) were prepared by dissolving 5-HT, ondansetron, and granisetron in water. Other drugs were dissolved in DMSO. The final dilution of DMSO in Krebs was <1 : 10 000.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. Author Contributions
  10. Conflicts of Interest
  11. References

Phytochemical composition of aqueous G. buchananii extract and its anti-motility fractions

Using fluorescent color indexes, we identified five distinct regions after PTLC separation of G. buchananii extract. These regions were designated as PTLC1-5 (Fig. 1A). Upon further separation using high performance thin layer chromatography, each PTLC fraction revealed 3–5 sub-fractions (Fig. 1B). Initial screening for the classes of compounds commonly found in Garcinia species29 revealed that aqueous G. buchananii extract contains flavonoids, sugars, glycosides, alkaloids, tannins, phenols, and steroids (Table 1). These compound classes were also found in PTLC1 and PTLC5, the G. buchananii extract fractions that inhibited pellet motility (Table 2; Fig. 2). Flavonoids and sugars were detected in both fractions, phenols were found only in PTLC1, whereas alkaloids, tannins, and steroids were exclusively found in PTLC5. PTLC2, 3, and 4 were not subjected to chemical testing, as they did not have anti-motility effects (Table 2; P > 0.05).

image

Figure 1.  Chromatographic separation of aqueous G. buchananii extract into fractions. (A) Preparative thin layer chromatography (PTLC) separation of aqueous G. buchananii extract and fraction identification based on color indexes. Extract sample indicates the region where 100 μL of extract was applied using a CAMAG Linomat 5 sample applicator. PTLC1- 5 designate the five fractions isolated from aqueous G. buchananii extract. PTLC silica shows a region used to collect silica for control experiments. (B) Separation of aqueous G. buchananii extract (extract) and further separation of PTLC1-5 fractions using high performance thin layer chromatography. Each PTLC fraction contained a minimum of three distinct bands.

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Table 1.   Phytochemical analysis of G. buchananii extract and anti-motility fractions PTLC1 and PTLC5
CompoundG. buchananii Aqueous extractPTLC1PTLC5
  1. Qualitative assessment of the classes of natural chemical compounds found in G. buchananii bark extract and PTLC fractions having anti-motility effects. ‘+ indicates that the compound was present and ‘− depicts that the compound was absent. ‘N/A’ refers to an inability to perform the desired chemical test. ‘ND’ refers to not done as the component was not found in the extract.

  2. Reaction intensity scores: + = low intensity; ++ = medium intensity, +++ = high intensity and ++++ = very high intensity.

Flavonoids+++++++++
Glycosides+++N/AN/A
Sugars++++++++
Alkaloids++++++
Tannins++++++
Phenols+++N/AN/A
Steroids+++++++
AnthraquinonesNDND
PhlobatanninsNDND
TerpenoidsNDND
SaponinsNDND
Table 2.   Bath application: The effect of 1 g G. buchananii extract and PTLC1 – PTLC5 fractions on pellet propulsion (expressed as a percent of velocity due to treatment, divided by normalized baseline pellet velocity)
Duration (min.)Vehicle (Krebs; n = 6)1 g G. buchananii extract (n = 5)PTLC silica (1 mg; n = 6)PTLC1 (15 mg; (n = 8)PTLC2 (28 mg; n = 7)PTLC3 (10 mg; n = 7)PTLC4 (0.9 mg; n = 5)PTLC5 (3.8 mg; n = 7)PTLC1 +  PTLC5; (n = 4)
  1. P-values were obtained using One-way anova and the Newman–Keul’s multiple comparison post hoc test. Top P-values indicate comparisons between vehicle with 1 g G. buchananii extract and PTLC silica, and PTLC silica compared with PTLC1- PTLC5. Lower P-values (*P) indicate comparison between 1 g extract with PTLC silica and PTLC1-PTLC5.

5102.1 ± 2.1%42.7 ± 16.4% P < 0.001106.3 ± 4.7% P > 0.05 *P < 0.00189.3 ± 4.5% P > 0.05 *P < 0.00198.3 ± 4.7% P > 0.05 *P < 0.00188.5 ± 8.4% P > 0.05 *P < 0.00192.6 ± 6.7% P > 0.05 *P < 0.00191.7 ± 6.4% P > 0.05 *P < 0.00199.6 ± 3.6% P > 0.05 *P < 0.001
10100.3 ± 1.9%39.2 ± 24.1% P < 0.001101.4 ± 2.5% P > 0.05 *P < 0.00178.9 ± 4.8% P < 0.01 *P < 0.001106.7 ± 5.2% P > 0.05 *P < 0.001106.3 ± 2.4% P > 0.05 *P < 0.00195.5 ± 4.1% P > 0.05 *P < 0.00182.5 ± 2.7% P < 0.05 *P < 0.00192.8 ± 3.7% P > 0.05 *P < 0.001
20102.5 ± 2.5%11.0 ± 11.0% P < 0.00197.5 ± 2.2% P > 0.05 *P < 0.00176.4 ± 4.0% P < 0.05 *P < 0.001117.9 ± 8.5% P < 0.05 *P < 0.001100.5 ± 8.1% P > 0.05 *P < 0.00195.3 ± 4.7% P > 0.05 *P < 0.00179.3 ± 3.2% P < 0.05 *P < 0.00174.1 ± 9.0% P < 0.05 *P < 0.001
image

Figure 2.  Characterization of the effect of aqueous G. buchananii extract fractions on guinea-pig colon motility using pellet propulsion assays in isolated distal colons. Summary data (20 min application to the serosal surface) showing that PTLC fractions contain components with anti-motility and pro-motility effects. PTLC1, PTLC5, and a combination of PTLC1 + PTLC5 (*; P < 0.05; see Table 1) significantly inhibited pellet propulsion. The effects of PTLC1 and PTLC5 were not additive. PTLC2 increased pellet propulsion (Δ; P < 0.05). Compared with control (PTLC silica), PTLC3 and PTLC4 did not alter propulsive velocity (P > 0.05).

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The aqueous G. buchananii bark extract contains anti-motility and pro-motility components

Organ bath applications of the PTLC fractions were used to study the effect of PTLC fractions on pellet propulsion in the guinea-pig distal colon in an effort to understand the variety of anti-motility components present in G. buchananii extract. The bath application approach was chosen because previous studies showed that G. buchananii exerts anti-motility effects with greater potency when applied in this manner.16Garcinia buchananii extract (1 g extract) reduced pellet velocity by 55% (Table 2; P < 0.001) after 5 min, 60% (P < 0.001) after 10 min, and 90% (P < 0.001) after 20 min. Similar to vehicle, application of PTLC silica (1 mg) for 20 min did not alter pellet propulsion (Table 2; Fig. 2; P > 0.05). Compared with PTLC silica, fractions from aqueous G. buchananii extract (PTLC1-5) had variable effects on colon motility (Table 2; Fig. 2). PTLC3 (10 mg) and PTLC4 (0.9 mg) did not affect pellet propulsion (P > 0.05). Somewhat surprisingly, given that the overall effect of G. buchananii extract is to inhibit motility, PTLC2 (28 mg) increased pellet velocity (P < 0.05). Conversely, PTLC1 (15 mg) and PTLC5 (3.8 mg) decreased pellet propulsion (each P < 0.05). Compared with PTLC silica, a combination of PTLC1 and PTLC5 inhibited pellet propulsion (P < 0.05). The magnitude of this inhibition was similar to that observed with either PTLC1 or PTLC5, when they were applied individually (P > 0.05). These results suggest that G. buchananii extract contains anti-motility and pro-motility components. However, as we chose to focus on the anti-motility effects of G. buchananii extract, only PTLC fractions having anti-motility effects were subjected to investigation concerning how they affect 5-HT3 and 5-HT4 receptor activities.

5-HT4 and 5-HT3 receptor agonists and antagonists inhibit anti-motility effects normally elicited by mucosal surface application of G. buchananii extract

To determine the roles of 5-HT3 and 5-HT4 receptors on G. buchananii extract activity, numerous compounds were applied intraluminally, either alone or in combination with the aqueous G. buchananii extract (2 g extract). These compounds were: the endogenous 5-HT agonist, 5-HT (0.5 μmol L−1); the partial 5-HT3 receptor agonist, RS-56812 (50 nmol L−1); and the 5-HT4 receptor agonists, cisapride (100 nmol L−1) and CJ-033466 (300 nmol L−1). Given the variations of the selectivity of 5-HT4 agonists at 5-HT4 receptors,32–34 cisapride and CJ-033466 were employed to test the effect of 5-HT4 agonists against the actions of the extract. Garcinia buchananii extract did not alter the pH of Krebs (7.38 ± 0.05). Pellet velocity obtained with intraluminal superfusion of Krebs solution was 3.04 ± 0.15 mm s−1 (n = 26), which was similar to organ bath Krebs application (2.76 ± 0.12 mm s−1; n = 34; P > 0.05). Compared with vehicle, intraluminal application of 2 g extract had no effect for the first 5–10 min (Table 3A; Fig. 3A; P > 0.05; 5 min), but inhibited pellet propulsion by 25% after 15 min (P < 0.05) and 90% after 20 min (P < 0.001).

Table 3.    (A) Intraluminal delivery: Control experiments of treatments using G. buchananii extract, 5-HT3, and 5-HT4 receptor agonists and antagonists. (B) Intraluminal delivery of G. buchananii extract in combination with 5-HT3 and 5-HT4 agonists and antagonists
(A)
Duration (min)Vehicle (Krebs; n = 7)2 g G. buchananii extract (n = 8)5-HT (n = 5)Cisapride (n = 4)CJ-033466 (n = 6)GR 113808 (n = 4)RS 56812 (n = 4)Granisetron (n = 4)
5101.9 ± 2.9%97.0 ± 4.3% P > 0.05 *P > 0.05124.3 ± 6.2% P < 0.05 *P < 0.05115.6 ± 4.9% P < 0.05 *P > 0.05132.7 ± 7.1% P < 0.05 *P < 0.001109.3 ± 6.1% P > 0.05 *P > 0.05119.7 ± 5.2% P > 0.05 *P > 0.0599.3 ± 4.2% P > 0.05 *P > 0.05
15101.2 ± 1.7%76.2 ± 11.1% P < 0.05116.5 ± 8.0% P > 0.05 *P < 0.05109.6 ± 9.3% P > 0.05 *P < 0.05112.1 ± 6.7% P > 0.05 *P < 0.00193.2 ± 3.6% P > 0.05 *P > 0.05119.6 ± 5.4% P > 0.05 *P < 0.0592.4 ± 1.4% P > 0.05 *P > 0.05
20100.6 ± 1.6%7.8 ± 6.0% P < 0.001118.3 ± 7.3% P < 0.05 *P < 0.001122.0 ± 8.2% P < 0.05 *P < 0.001116.6 ± 5.6% P < 0.05 *P < 0.00191.7 ± 3.1% P > 0.05 *P < 0.001126.7 ± 9.2% P < 0.0001 *P < 0.00186.5 ± 3.6% P < 0.05 *P < 0.001
(B)
Duration (min)Vehicle (Krebs; n = 7)2 g extract (n = 8)2 g extract + 5-HT (n = 5)2 g extract + cisapride (n = 5)2 g extract + CJ-033466 (n = 7)2 g extract + GR-113808 (n = 4)2 g extract + RS-56812 (n = 4)2 g extract + GRAN (n = 4)
  1. P-values were obtained using One-way anova and the Newman–Keul’s multiple comparison post hoc test. Top P-values denote comparisons between vehicle with all other treatments. Lower P-values (*P) indicate comparison between 2 g extract with 5-HT3 and 5-HT4 receptor agonists and antagonists.

  2. P-values were obtained using One-way anova and the Newman–Keul’s multiple comparison post hoc test. Top P-values refer to comparisons between vehicle with 2 g extract alone, 2 g extract + 5-HT3, and 5-HT4 agonists as well as 2 g extract + 5-HT3 and 5-HT4 antagonists. Lower P-values (*P) indicate the comparison between 2 g extract with the various 5-HT3 and 5-HT4 agonist and antagonists combined with 2 g extract. Gran, granisetron.

5101.9 ± 2.9%97.0 ± 4.3% P > 0.05104.3 ± 16.1% P > 0.05 *P > 0.0599.2 ± 4.9% P > 0.05 *P > 0.0597.9 ± 8.0% P > 0.05 *P > 0.05101.6 ± 5.8% P > 0.05 *P > 0.05115.5 ± 13.2% P > 0.05 *P > 0.0586.3 ± 4.8% P > 0.05 *P > 0.05
15101.2 ± 1.7%76.2 ± 11.1% P < 0.05111.0 ± 4.7% P > 0.05 *P > 0.0598.4 ± 19.4% P > 0.05 *P > 0.0578.7 ± 9.3% P > 0.05 *P > 0.0589.7 ± 14.9% P > 0.05 *P > 0.0589.3 ± 20.6% P > 0.05 *P > 0.0566.7 ± 16.4% P < 0.05 *P > 0.05
20100.6 ± 1.6%7.8 ± 6.0% P < 0.00141.1 ± 12.4% P < 0.05 *P < 0.0566.5 ± 20.6% P < 0.01 *P < 0.0150.2 ± 13.1% P < 0.05 *P < 0.0576.2 ± 15.7% P < 0.05 *P < 0.00162.7 ± 19.9% P < 0.05 *P < 0.00159.2 ± 15.1% P < 0.001 *P < 0.001
image

Figure 3.  The anti-motility effects of aqueous G. buchananii bark extract elicited via mucosal surface application depend on 5-HT4 and 5-HT3 receptors. (A) Summary data (20 min after application) showing that compared with vehicle, G. buchananii extract (2 g bark powder/100 mL Krebs) significantly inhibited pellet velocity (*P < 0.001). Serotonin hydrochloride (5-HT; 0.5 μmol L−1), CJ-033466 (300 nmol L−1), cisapride (CISA; 100 nmol L−1), and RS-56812 (50 nmol L−1) all individually increased pellet velocity beyond that of vehicle (Δ; each P < 0.05) and 2 g extract alone (inline image; each P < 0.001). Compared with G. buchananii extract alone (2 g ext.), the endogenous 5-HT agonist 5-HT, 5-HT4 receptor agonists cisapride (CISA) and CJ-033466, and 5-HT3 receptor agonists RS-56812 significantly reduced the potency of the extract to inhibit pellet propulsion (♦; each P < 0.05). (B) Summary data (20 min after application) showing that compared with vehicle, 2 g bark powder/100 mL Krebs) significantly reduced pellet velocity (*; P < 0.001). Furthermore, intraluminal application of the 5-HT3 receptor antagonist granisetron (GRAN; 2 μmol L−1) and GRAN +2 g extract significantly reduced pellet velocity (Δ; each P < 0.05). In contrast, the 5-HT4 receptor antagonist (GR-113808) and 2 g extract plus GR-113808 did not reduce pellet velocity. Compared with G. buchananii extract (2 g ext.) alone, a combination of 2 g extract with granisetron and GR-113808 significantly reduced the effectiveness of G. buchananii extract to inhibit motility (*; P < 0.05).

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5-HT, cisapride, CJ-033466, and RS5612 all increased pellet propulsion when compared with vehicle (Table 3A; Fig. 3A; P < 0.05–P < 0.001; 20 min) and 2 g extract alone (P < 0.001; 20 min). When 5-HT, cisapride CJ-033466, and RS-56812, each were applied in combination with the extract, they reduced the anti-motility potency of G. buchananii extract (Table 3B; Fig. 3A; each P < 0.05; 20 min). In summary, 5-HT, 5-HT3, and 5-HT4 receptor agonists reduced the anti-motility actions of the extract elicited from the mucosal side, and there was no difference between the effects of these agonists.

In another set of experiments, we found that 5-HT3 and the 5-HT4 receptor antagonists granisetron and GR-113808 also reduced the efficacy of G. buchananii extract. Compared with vehicle, when applied for 20 min in the absence of the extract, granisetron (2 μmol L−1) reduced pellet propulsion by 15% (Table 3A; Fig. 3B; P < 0.05), whereas GR-113808 (5 μmol L−1) did not significantly reduce pellet propulsion (P > 0.05). Propulsive velocity was significantly reduced when the extract was combined with granisetron (P < 0.05), whereas the effect of the extract plus GR-113808 was similar to that of the vehicle. Nonetheless, compared with G. buchananii extract, combining granisetron with G. buchananii extract reduced the potency of the extracts’ anti-motility effects by 50% (Table 3B; Fig. 3B; P < 0.001), whereas GR-113808 reduced the efficacy of G. buchananii extract anti-motility effects by 70% (P < 0.001). In summary, the 5-HT4 antagonist, GR-113808 and the 5HT3 receptor antagonist, granisetron, inhibited the actions of the extract elicited from the mucosal side.

5-HT3 and 5-HT4 receptor agonists reverse the anti-motility effects of G. buchananii extract elicited during serosal surface application

When compared with vehicle, organ bath application of the 5-HT3 receptor agonist RS-56812 and the 5-HT4 agonist CJ-033466 for 20 min did not affect pellet velocity (Fig. 4A; 101.7 ± 2.5%vs 103.3 ± 4.7% and 102.8 ± 6.5%; each P > 0.05), whereas the 5-HT4 agonist cisapride reduced it by 15% (101.7 ± 2.5%vs 85.0 ± 5.5%; P < 0.05). Combining 1 g G. buchananii extract with RS-56812 or cisapride completely reversed the anti-motility effects of the extract (Fig. 4A; 104.3 ± 6.9% and 107.4 ± 16.7%vs 11.0 ± 11.0%; P < 0.05), while combining G. buchananii extract with CJ-033466 actually increased pellet velocity beyond that of vehicle (129.8 ± 12.6%vs 101.7 ± 2.5%; P < 0.05) and cisapride alone (129.8 ± 12.6%vs 85.0 ± 5.9%; P < 0.05). Furthermore, when RS-56812 and cisapride were tested together with the extract, pellet velocity was increased beyond that of vehicle (Fig. 4B; 132.5 ± 31.4%vs 101.7 ± 2.5%; P < 0.05). In summary, the 5-HT3 and 5-HT4 receptor agonists reversed the extract’s anti-motility actions elicited from serosal side. There was no difference between the effects of the agonists tested.

image

Figure 4.  5-HT3 and 5-HT4 receptor agonists reverse the anti-motility effects elicited by G. buchananii bark extract when applied to the serosal surface. (A) Summary data (20 min after application) showing that compared with vehicle, 1 g G. buchananii (1 g ext.) significantly inhibited pellet motility by 90% (*P < 0.001) and the 5-HT4 receptor agonist, cisapride (CISA: 100 nmol L−1) inhibited pellet propulsion by 15% (Δ; P < 0.05). Unlike intraluminal applications (see Fig. 3A), the 5-HT3 receptor agonist RS-56812 (50 nmol L−1) and the 5-HT4 receptor agonist CJ-033466 (CJ, 300 nmol L−1) did not alter pellet velocity (P > 0.05). In addition, the extract (1 g ext.) did not inhibit pellet propulsion in the presence of 5-HT3 receptor agonist, RS-56812 and 5-HT4 receptor agonists cisapride and CJ-033466 (♦; P < 0.001). Interestingly, the combination of G. buchananii extract with CJ-033466 increased pellet velocity when compared with vehicle and cisapride (inline image; P < 0.05 and P < 0.01, respectively). (B) Summary data (20 min after application) showing that compared with vehicle, G. buchananii extract (1 g ext.) inhibited pellet propulsion (*P < 0.001). A mixture of 5-HT3 agonist RS-56812 and 5-HT4 agonist cisapride (CISA) increased pellet velocity (Δ; P < 0.05). Compared with extract alone, when colons were treated with 1 g G. buchananii extract combined with cisapride and RS-56812, the extract failed to reduce pellet motility (★; P < 0.001). Instead, the combination increased pellet velocity beyond that of vehicle alone (♦; P < 0.05). (C) In serosal applications (20 min after application), 5-HT3 and 5-HT4 receptor antagonists did not alter the anti-motility effect of G. buchananii. G. buchananii extract (1 g ext.) inhibited pellet propulsion (*P < 0.001). A combination of G. buchananii extract with the 5-HT3 antagonists ondansetron (ONDAN, 0.5 μmol L−1) and granisetron (GRAN, 1 μmol L−1) and 5-HT4 receptor antagonists GR-113808 (GR-113808, 5 μmol L−1) inhibited pellet propulsion with similar magnitude to that of the extract alone (Δ; P < 0.05). GR-113808 appeared to augment the extract’s actions, whereas 5-HT3 receptor antagonists tended to inhibit the effects of the extract by 5–10%. (D) A combination of the 5-HT3 receptor antagonist, ondansetron (ONDAN; 0.5 μmol L−1) with 5-HT4 receptor antagonist GR-113808 (5 μmol L−1) did not affect pellet propulsion for 20 min (P > 0.05). However, mixing ondansetron and GR-113808 with G. buchananii extract (1 g ext.) reduced the anti-motility potency of G. buchananii extract by 40% (♦; P < 0.05).

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5-HT4 and 5-HT3 receptor antagonists combined affect the anti-motility action of G. buchananii extract normally elicited during serosal surface application

Compared with vehicle, in organ bath deliveries, 5-HT3 antagonists granisetron (1 μmol L−1) and ondansetron (0.5 μmol L−1) did not alter pellet propulsion for up to 20 min (Fig. 4C; 101.7 ± 2.5%vs 101.4 ± 7.0% and 111.3 ± 9.8%; P > 0.05). Contrary to the intraluminal findings, G. buchananii extract reduced pellet propulsion in the presence of ondansetron and granisetron with magnitudes that were similar to that of the extract alone (Fig. 4C; 12.8 ± 12.7% and 25.6 ± 12.5%vs 11.0 ± 11.0%; P > 0.05). In a manner similar to the 5-HT3 antagonists, 5-HT4 receptor antagonist GR-113808 did not alter pellet propulsion. Compared with vehicle, a mixture of GR-113808 and 1 g extract did not affect pellet propulsion during the initial 5–10 min (Fig. 4C; 101.7 ± 2.5%vs 129.7 ± 22.8%; P > 0.05; 5 min). However, after 20 min, the velocities had dropped to levels slightly below those elicited by G. buchananii alone (4.2 ± 4.0%vs 11.0 ± 11.0%; P > 0.05). Compared with vehicle, combining ondansetron (0.5 μmol L−1) and GR-113808 (5 μmol L−1) did not affect pellet velocity (Fig. 4D; 101.7 ± 2.5%vs 104 ± 7.3%; P > 0.05). Combining ondansetron and GR-113808 with the extract reduced the efficacy of the extract’s anti-motility effects by 40% (Fig. 4D; 50.5 ± 29.6%vs 11.00 ± 11.0%; P < 0.01). In summary, the 5-HT3 receptor antagonists did not affect the actions of G. buchananii extract. 5-HT4 receptor antagonist GR-113808 inhibited the extract for 5–10 min. Combining 5-HT3 and 5-HT4 antagonists reduced the efficacy of the extract.

5-HT3 and 5-HT4 receptor agonists reverse the anti-motility effects normally elicited from serosal surface by PTLC1 and PTLC5 fractions

We investigated whether RS-56812, cisapride, and CJ-033466 affected the anti-motility effects of PTLC1 (15 mg) and PTLC5 (3.8 mg). Compared with PTLC silica, application of PTLC1 and PTLC5 for 20 min did not reduce pellet propulsion in the presence of CJ-033466 (Fig. 5A,B; 97.5 ± 2.2%vs 99.4 ± 4.5%; P > 0.05), which is similar to G. buchananii extract. Cisapride inhibited the anti-motility effects of PTLC1 (Fig. 5A; 97.5 ± 2.2%vs 90.3 ± 5.4; P > 0.05), but failed to inhibit anti-motility effects of PTLC5 (Fig. 5B; 97.5 ± 2.2%vs 83.2 ± 5.9%; P < 0.05). RS-56812 failed to inhibit anti-motility effects of PTLC1 (Fig. 5A; 97.5 ± 2.2%vs 65.3 ± 22.3%; P < 0.05), but completely reversed the effects of PTLC5 (Fig. 5B; 97.5 ± 2.2%vs 93.8 ± 6.5%; P > 0.05). In summary, CJ-033466 inhibited the effects of PTLC1 and PTLC5, whereas cisapride inhibited PTLC1, but not PTLC5. The 5HT3 receptor agonist, RS-56812, inhibited the effect of PTLC5, but not PTLC1.

image

Figure 5.  5-HT3 and 5-HT4 receptor agonists and antagonists affect the anti-motility effectiveness of PTLC1 and PTLC5 fractions elicited from serosal side of guinea-pig distal colon. (A) Summary data showing that compared with PTLC silica, PTLC1 (15 mg per 100 mL Krebs) inhibited pellet propulsion by 25% after 20 min (*P < 0.05). As with G. buchananii extract, in the presence of the 5-HT4 agonists cisapride (CISA; 100 nmol L−1) and CJ-033466 (300 nmol L−1), PTLC1 did not alter pellet propulsion (P > 0.05). In contrast, PTLC1 inhibited pellet propulsion by 35% in the presence of the 5-HT3 receptor agonist RS-56812 (50 nmol L−1) (♦; P < 0.01). (B) Summary data showing that compared with PTLC silica PTLC5 (3.8 mg per 100 mL Krebs) inhibited pellet propulsion by 20% after 20 min (*;P < 0.05). Unlike PTLC1, PTCL5 inhibited pellet propulsion in the presence of the 5-HT4 receptor agonist cisapride (CISA; 100 nmol L−1) (Δ;P < 0.05). As with PTLC1 and G. buchananii extract, in the presence of the more specific 5-HT4 receptor agonist CJ-033466 (300 nmol L−1), PTLC5 did not alter pellet propulsion (P > 0.05). The pellet propulsion velocity obtained in the presence of PTLC5 combined with CJ-033466 was higher than that of PTLC5 alone (♦; P < 0.05). Furthermore, PTLC5 did not reduce pellet propulsion in the presence of the 5-HT3 receptor agonist RS-56812 (50 nmol L−1; P > 0.05), which contrasts with PTLC1. (C) Summary data (20 min application) showing that compared with PTLC silica, PTLC1 alone (15 mg per 100 mL Krebs) inhibited pellet propulsion by 25% (*P < 0.05) and by 58% in the presence of 5-HT4 antagonist GR-113808 (Δ; P < 0.001). GR-113808 augmented the effects the fractions by 30%. In contrast, PTLC1 did not inhibit pellet propulsion in the presence of 5-HT3 receptor antagonists, granisetron and ondansetron (each 1 μmol L−1; P > 0.05). Instead, combining PTLC1 with ondansetron increased propulsive motility beyond that of PTLC silica, PTLC1 alone (inline image; P < 0.05). Combining PTLC1 with granisetron significantly reduced the anti-motility potency of PTLC1 when compared with a mixture of PTLC1 with the 5-HT4 antagonist GR-113808 (♦; P < 0.05). (D) Summary data (20 min application) showing that compared with PTLC silica, PTLC5 alone inhibited pellet propulsion by 20% (*; P < 0.05) and by 50% in the presence of 5-HT4 antagonist GR-113808 (Δ;P < 0.001). As with PTLC1, GR-113808 augmented the effects the PTLC5 by 30%. Similar to PTLC1, PTLC5 did not inhibit pellet propulsion in the presence of 5-HT3 receptor antagonists, ondansetron and granisetron (each 1 μmol L−1; P > 0.05). Pellet propulsion velocity obtained when PTLC5 was applied in combination with granisetron was significantly greater than that of PTLC5 alone and PTLC5 mixed with GR-113808 (♦; P < 0.05 and P < 0.001, respectively). PTLC5 mixed with GR-113808 caused a significantly reduced propulsion velocity when compared with PTLC5 plus odansetron (inline image; P < 0.001).

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5-HT3 and 5-HT4 receptor antagonists affect PTLC1 and PTLC5 anti-motility effects elicited from serosal surface

When compared with PLTC silica, serosal application (20 min) of PTLC1 in the presence of ondansetron (0.5 μmol L−1) and granisetron (1 μmol L−1) did not affect pellet propulsion (Fig. 5C; 97.5 ± 2.2%vs 116.9 ± 7.9% and 90.2 ± 8.8%; P > 0.05). The combination of ondansetron and PTLC1 actually increased pellet propulsion (Fig. 5C; 97.5 ± 2.2%vs 116.9 ± 7.9%; P < 0.05). In contrast, PTLC1 reduced pellet velocity with greater potency in the presence of 5-HT4 receptor antagonist GR-113808 (Fig. 5C; 97.5 ± 2.2%vs 52.4 ± 22.4%; P < 0.001). Like PTLC1, PTLC5 did not affect pellet propulsion in the presence of ondansetron and granisetron (Fig. 5D; 100.4 ± 8.5 and 110.7 ± 5.7%vs 97.5 ± 2.2%; P > 0.05). In addition, PTLC5 inhibited pellet propulsion with greater efficacy in the presence of GR-113808 (97.5 ± 2.2%vs 49.1 ± 35.6%; P < 0.001). In summary, the 5-HT4 receptor antagonist augmented the actions of PTLC1 and PTLC5, whereas the 5HT3 receptor antagonists entirely inhibited the anti-motility actions of both fractions.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. Author Contributions
  10. Conflicts of Interest
  11. References

The goals of this study were to determine whether the anti-motility effect of G. buchananii extract involves 5-HT3 and 5-HT4 receptors, and whether the extract can be separated into fractions that have similar effects. The extract was initially separated into five fractions: two with anti-motility effect, one with pro-motility effect, and two without any effect. This suggests that G. buchananii may be a valuable source of novel anti-motility and pro-motility compounds, or their derivatives. The ability of G. buchananii extract and its fractions to inhibit guinea-pig colonic motility was reversed by 5-HT3 and 5-HT4 receptor agonists and variably affected by antagonists of these receptors. This indicates that the anti-motility bioactive components in the extract affect 5-HT3 and 5-HT4 receptor signaling either directly or indirectly. G. buchananii aqueous extract had a greater effect on 5-HT4 receptors, especially during mucosal application, whereas the isolated fractions showed higher efficacy with 5-HT3 receptors compared with 5-HT4 receptors. Overall, our findings suggest that G. buchananii extract contains several anti-motility compounds that exert different effects on 5-HT3 and 5-HT4 receptors through mechanisms that remain to be determined.

Garcinia buchananii is a potential source of anti-motility and pro-motility compounds

There is a great deal of clinical and scientific evidence regarding major breakthroughs in treating diarrheal diseases using ORT, synthetic drugs, especially opiate- and enkephalin-based formulations, and vaccinations.1–3,8,9 However, current therapies do not necessarily shorten the duration of diarrhea or lessen abdominal pain. Most synthetic drugs are limited by their side effects, mainly constipation, dependency, and safety for children and pregnant women.1–3,8,9 The alteration in enteric neurotransmission is a major contributor to increased enteric secretion, hypermotility, and cramping pain observed in diarrheal diseases.1,6,9,20 Therefore, there is the critical need for novel anti-diarrheal agents that target enteric neurotransmission. Botanical products and their derivatives have historically led to the discoveries of effective modern drugs and have the potential to fulfill this requirement.12,14 Currently, preparations from Garcinia are used by a large subpopulation of people in developing countries to treat diarrheal diseases.10,12,13,15,16,29 Our findings support indigenous medicine practices, and the idea that G. buchananii bark extract is a potential source of new anti-diarrhea drugs. This notion is supported by our previous findings that G. buchananii bark extract is a non-opiate preparation that reduces propulsive motility by inhibiting synaptic transmission in the myenteric ganglia.16,17 Additional support comes from the observations of this study that the extract’s anti-motility effect may involve inhibition of 5-HT3- and 5-HT4- receptors. On the basis of the current study, it would be beneficial to identify and characterize the bioactive compounds, determine how these compounds affect enteric neurotransmission, and also their interaction with 5-HT3- and 5-HT4- receptors.

Individual extract fractions (PTLC1 and PTLC5) with anti-motility effects produced <25% reduction of propulsive motility and their effects were not additive, whereas the extract alone inhibited motility by >70%. The reasons for this disparity remain unclear at the present time. It is possible that the extract contains larger levels of bioactive compounds than PTLC1 and PTLC5, as increasing the amount of PTLC1 or PTLC5 caused greater inhibition of motility (Boakye and Balemba personal observations). Alternatively, despite the overall anti-motility effects of PTLC1 and PTLC5, these fractions do contain numerous sub-fractions, which may have pro-motility properties, which could reduce the overall degree of motility inhibition evoked by PTLC1 and PTLC5.

5-HT3 and 5-HT4 receptors play a crucial role in the anti-motility effect of bioactive components of G. buchananii extract

The key finding of this study is that G. buchananii extract contains unknown compounds that probably interact with 5-HT3 and 5-HT4 receptors to inhibit pellet propulsion in the guinea-pig distal colon. Although our study did not address the exact mechanisms, it is evident that 5-HT3 and 5-HT4 agonists reversed the effects of the extract and its fractions. Similarly, the 5-HT3 and 5-HT4 receptor antagonists inhibited the anti-motility actions of G. buchananii extract and its fractions. The observations of the trend toward augmenting the effects of the extract and its fractions by the 5-HT4 receptor antagonist GR-113808 suggests that this compound may significantly enhance the actions of purified compounds. These initial studies show that the crude extract and fractions contain multiple bioactive components, suggesting that the mechanisms of action of the crude extract and fractions on the effector tissues of colon motility are complex.16,17,35 Overall, our study indicated that the bioactive compound(s) have a serotonergic effect, and provides the basis for future experiments using purified compounds. Our findings are similar to observations that a botanical preparation of wood creosote stopped stress-induced diarrhea by inhibiting 5-HT3 and 5-HT4 receptors in rat colon.25 The observation that G. buchananii bark extract had a significant anti-motility effect supports the notion that concurrent inhibition of these receptors drastically reduces colonic motility.36 Agents that inhibit 5-HT4 receptors or both 5-HT3 and 5-HT4 receptors reduce both the ascending and descending peristaltic reflexes.21–23,37 Furthermore, anti-emetic drugs act by inhibiting 5-HT3 and 5-HT4 receptors,18–20,38 and 5-HT3 receptor antagonists are used as medications for functional bowel disorders and diarrhea-predominant IBS.6,18–20 Therefore, G. buchananii bark extract could also be a potential source of new compounds for the treatment of nausea and vomiting,38 functional bowel disorders, and diarrhea-predominant IBS.6,18–20 Also, the extract may be beneficial in pain management associated with these conditions.39 It has been shown that steroids have anti-inflammatory, anti-motility, and anti-secretory effects in patients with collagenous colitis.40 Steroids have been shown to contribute to the anti-inflammatory activity of Garcinia extracts.13,41 Whether the steroids found in G. buchananii extract and PTLC5 have anti-inflammatory actions and also reduce motility currently remains unclear.

The effect of the bioactive components of G. buchananii extract on 5-HT3- and 5-HT4- receptor signaling in the guinea-pig distal colon appears unique. The reasons for this claim are that the effects of G. buchananii extract and its fractions were inhibited by 5-HT3 and 5-HT4 receptor agonists and antagonists, or to some extent augmented by 5-HT4 antagonist or a combination of 5-HT3 and 5-HT4 antagonists. The findings using PTLC1 and PTLC5 suggest differences in the relative contribution of 5-HT3 and 5-HT4 receptors to the anti-motility effect of the fractions, with the 5-HT3 receptors being the major effector. In contrast, 5-HT4 receptors appeared to be the primary receptors affected by crude G. buchananii extract. As the bioactive compounds have not been characterized, the interpretation of these results is difficult. It is possible that PTLC1 and PTLC5 fractions contain different bioactive compounds, or that PTLC fractionation altered biologic activity of the active compounds, thus changing how they affect 5-HT receptors. Regardless of these differences, our results bolster the idea that the efficacy of bioactive compounds in G. buchananii extract involved in reducing colon motility depends on an inhibition of 5-HT3 and 5-HT4 receptors.

Gamma-mangostin, a xanthone from G. cambogia fruit extracts, interacts with 5-HT2A receptors.27 Herein, we demonstrate for the first time that G. buchananii extract contains compounds that inhibit intestinal motility via 5-HT3- and 5-HT4-receptors. Botanical compounds that have been shown to inhibit 5-HT3- and 5-HT4- receptors are flavonoids,42 phenolics, creosol, guaiacol, and 4-ethylguaiacol.25 The compounds in G. buchananii extract that actively interact with the receptors therefore appear to be flavonoids and/or phenols. Whether steroids, alkaloids, and tannins are involved should also be considered.

We have previously proposed that the rebound of pellet propulsion during washout following treatment of isolated guinea pig colon with G. buchananii extract is due to pro-motility components in the extract.16 This study provides preliminary evidence to support the existence of pro-motility compounds in G. buchananii bark extract. It is possible that GABA receptors16 have a role in the pro-motility effect of PTLC2.

Intraluminal and serosal deliveries of 5-HT3 and 5-HT4 agonists show several differences. The agonists augmented motility, whereas antagonists inhibited propulsion during intraluminal perfusion. In contrast, with the exception of cisapride, agonists and antagonists did not affect pellet velocity when applied to the serosal side. The difference between CJ-033466 and cisapride suggests that cisapride acted via a mechanism that involves inhibition of 5-HT3 and 5-HT2 receptors.32,34 Pharmacological studies have shown that, compared with cisapride, CJ-033466 has greater selectivity at the 5-HT4 receptor.32–34 The observations of differences between the effects of cisapride and CJ-033466 on the anti-motility potency of G. buchananii extract and PTLC1 and PTLC5 fractions, when applied via serosal surface could be due to differences in selectivity at the 5-HT4 receptor.32–34Garcinia buchananii bark extract inhibits pellet propulsion rapidly from the serosal side. It is believed that this effect is due to the inhibition of synaptic neurotransmission in the myenteric ganglia, presumably by inhibiting presynaptic release of acetylcholine, or by other mechanisms and neurotransmitters.16,17 While the findings of this study suggest serotonergic effects, a clear interpretation of the 5-HT3 and 5HT4 agonists ability to reverse extract inhibition will require further studies using purified compounds.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. Author Contributions
  10. Conflicts of Interest
  11. References

Garcinia buchananii bark extract contains bioactive compounds that interact with 5-HT3 and 5-HT4 receptors to inhibit peristaltic activity. Although the bioactive compounds have not been characterized, and the mechanisms of action still remain unclear, these findings re-enforce the idea that G. buchananii extract is a potential source of novel non-opiate anti-diarrheal compounds.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. Author Contributions
  10. Conflicts of Interest
  11. References

Dr. Sofie Pasilis and Dr. 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. The authors are thankful to Ms. Marjorie Newman for her assistance with motility assays, and Drs. Patrick J. Hrdlicka, Andrzej Paszczynski, Lee Deobald, and Matt Morra for using their laboratory facilities.

Author Contributions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. Author Contributions
  10. Conflicts of Interest
  11. References

PAB was involved in all aspects of research, analyzed data, and wrote the paper; CS performed research and wrote the paper; YB performed research; MDH performed research; SMB contributed to research design, critical review, and intellectual content; SPP contributed to research design, supervision of the study, critical review, and intellectual content; and OBB was involved in research design, supervision of the study, data analysis, interpretation, and wrote the paper.

Conflicts of Interest

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. Author Contributions
  10. Conflicts of Interest
  11. References

None of the authors have competing financial interests or other issues to declare.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. Author Contributions
  10. Conflicts of Interest
  11. References
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