Altered biliary flow rate and bile composition following consumption of ethanolic fruit extract of Dennettia in rats.

BACKGROUND: Dennettia tripetala is a tropical plant with some ethno-medicinal uses; it enhances uterine contraction/involution in pregnant and post-partum women, it is also a mild laxative, with anti-bacteria, antifungal properties etc.. AIMS: This study sought to elucidate the impact of D. tripetala fruit extract intake on biliary flow rate and bile composition in rats.
MATERIALS AND METHODS: A total of 18 albino Wistar rats were randomly assigned into three groups of 6 rats each and fed on normal rat chow + drinking water and/or 85 mg/kg, 170 mg/kg body weight of D. tripetala extract for 14 days.
RESULTS: The rate of bile secretion in the control, low dose (LD) and high dose (HD) D. tripetala extract treated rats was 4.40 ± 0.24 ml/h, 3.20 ± 0.20 ml/h and 4.60 ± 0.25 ml/h respectively. Showing a significant (P < 0.05) decrease in LD and an increase in HD. Na+ concentration increased significantly (P < 0.01) in the LD extract recipients, but was reduced in the HD. LD of the extract increased K+ significantly (P < 0.001) while HD decreased it (P < 0.05). Both low and HD of the extract reduced Cl(-) concentration significantly (P < 0.05 and P < 0.001 respectively). HCO3(-) increased significantly (P < 0.05) in the HD extract recipients. However, total cholesterol, total and conjugated bilirubin concentrations were not significantly altered by D. tripetala fruit extracts.
CONCLUSION: In conclusion, this study showed that LD of D. tripetala may reduce bile flow rate while HD may increase it without altering the saturation of cholesterol and bilirubin.

INTRODUCTION

A large number of plants herbs or crops are traditionally noted and recognized for medicinal purposes.[1] Pepper fruit (tripetala) is of these fruits. D. tripetala is a medium sized tropical plant of the family Annonaceae, it found in the mangrove and rainforest of West Africa. This plant flowers at the beginning of the rainy season in Nigeria where it is locally known by different names such as Ako, Nkarika, Nmini, Igeri or Imako. It is commonly used as a masticator, which when chewed produced a special peppery impact and a spicy taste that stimulate appetite.[2]

D. tripetala is also used as a flavor and seasoning, it is applied in the food of pregnant and post-partum women to aid uterine contraction and involution.[12] It is also used to treat cough, fever.[3] It is also a mild laxative,[4] a potent anti-bacteria, antifungal agent.[5]

Phytochemical screening of D. tripetala reveal the following components: Crude fiber (9.84%), fat and oil (3.47%), carbohydrate (62%), crude protein (15.31%), moisture (8.00%), caloric value (480.24%), hydrogen cyanide (HCN)(0.02%).[6] It is also rich in minerals such as iron, calcium, potassium, manganese etc.[6] and vitamin such as thiamine, riboflavin, niacin and ascorbic acid. It also contains essential oils and phenolic acids, ethanol, alkaloids, ethylacetate, flavonoids, tannis and glycosides. Indications show that the rich presence of a type of essential oil called oleoresins determines the aromatic flavoring, coloring and pungent properties of pepper fruits. It also contains a hexanolic constituent (n-hexane), which is toxic to larvae.[78]

Bile is greenish compound produced by the liver cells then concentrated and stored in the gall bladder.[9] Alterations in the composition of bile could serve as a useful index in assessing liver functions and the fate of the body in general, bearing in mind that alterations in bile composition may lead to gall stones, fat indigestion and altered bilirubin metabolism. Obviously, substances taken into the body are capable of altering the physiology of tissues and organs in one way or the other and D. tripetala is not an exception.

It has also been revealed that methods use by traditionalist in early diagnosis lack scientific basis[10] and there is a paucity in scientific literatures on the effect of consumption of D. tripetala on the gastrointestinal system, the first line of contact of food substances and drugs when consumed.

It is therefore the aim of this study to investigate the effect of consumption of D. tripetala fruit extract on rate of bile secretion and its biochemical composition in rats.

MATERIALS AND METHODS

Experimental animals

A total of 18 male albino Wistar rats were obtained from the animal house of the Department of Physiology, University of Calabar, Nigeria. The rats weighed between 210 and 250 g at the time of sacrifice. They were weighed before the commencement of the feeding experiment and thereafter were weighed daily. They were nursed under control environmental condition. The research was conducted in accordance with the internationally accepted principles for laboratory animal use and care as found in for example the European Community Guidelines.[11]

Experimental plant

4 kg of fresh leaves of the pepper fruit were purchased from a local market (Marian Market) in Calabar-North Local Government Area of Cross River State, Nigeria, during the rainy season and were identified and authenticated as D. tripetala by a botanist (Mr. Frank Adepoju) in the Department of Biological Sciences, University of Calabar, Nigeria.

Preparation of plant extract

D. tripetala fruits were rinsed with water to remove debris and sand. The fruits were cut and sun-dried for some days until they got brittled, they were then transferred into an Astell Hearson oven (1950 IRC) set at a temperature of 45°C. The dried fruits were the ground to powdered form using an electric blender. 520 g of the powder was percolated in 500 mL of ethanol (80% v/v). At 18 h later, the supernatant was filtered with satin material and then with Whatman No. 1 filter paper. The filtrate was then concentrated in the oven to a constant weight. This yielded 7.5% of the crude extract. The extract was reconstituted to an appropriate concentration before administration.

Experimental protocol

A total of 18 male albino Wistar rats were randomly assigned into three groups of 6 rats each. Group 1 (control) received normal rat chow + drinking water, group 2 low dose (LD) and group 3 in addition received 85 mg/kg and 170 mg/kg body weight respectively of D. tripetala p.o once daily. The feeding regimens lasted for 14 days. After which the animals were sacrificed.

Collection of bile

At the end of the feeding period, the rats were removed from the animal room to the laboratory after being starved for 18 h prior to the experiment. The rats were weight and anaesthetized with sodium thiopentone (6 mg/100 g body weight). They were fastened onto the dissecting board for tracheostomy to clear the airways.

An incision was made along the linea alba to expose the stomach. A laparotomy was performed and the liver lobes defleced anterolaterally to expose the common bile duct. The common bile duct was then cannulated with a portex cannula (0.5 mm in diameter) after a small incision was made. The cannula was fastened with a white cotton thread. The bile was collected for 3 h for each rat.[12]

Determination of biliary electrolytes concentrations

Sodium and potassium in bile were determined using a flame photometer (Model 410 C, Petra Court Ltd., England). The bile was sprayed into a non-luminous gas flame and absorbance read at 598 nm and 767 nm respectively for sodium and potassium.

Bile bicarbonate ion was measured by a modified method of Forrester et al.[13] Biliary chloride ions were determined by the principle of end point titration.[14]

Determination of biliary bilirubin concentration

Biliary bilirubin concentrations were determined by colorimetric method as describe by Jendrassik and Grof[15] and modified by Sherlock.[16]

Extraction of cholesterol from bile

Cholesterol was extracted from bile following the principle of esterification reaction.[17]

Statistical analysis

Data are presented as mean ± standard error of the mean Data were analyzed using one-way analysis of variance (ANOVA) then followed with post-hoc test (least square deviation) using computer software's (SPSS version 15.0 (SPSS Inc. Chicago, IL 60606-9653, Nov. 2006) and Excel for windows (Microsoft Corporation 2010, Washington.). P < 0.05 was declared as significant statistically.

RESULTS

Rate of bile secretion

The rate of bile secretion in the control, LD and high dose (HD) D. tripetala extract treated rats was 4.40 ± 0.24 ml/h, 3.20 ± 0.20 ml/h and 4.60 ± 0.25 ml/h respectively. It was significantly (P < 0.01) lower in LD compared with the control and HD [Figure 1].

Figure 1

Comparison of the rate of bile secretion in control and test groups. Values are mean ± standard error of the mean, n = 6 (**P < 0.01 vs. control b = P = 0.01 vs. low dose)

Bile electrolytes concentrations

The concentration of sodium ion in the LD group (140.60 ± 1.21 mmol/L) was significantly (P < 0.01) higher compared with control (135.60 ± 0.68 mmol/L) and HD (136.40 ± 0.75 mmol/L) groups [Table 1].

Table 1

Comparison of biliary electrolytes concentration in the different experimental group

The potassium ion concentration in the control, LD and HD treated groups were 3.62 ± 0.04 mmol/L, 4.28 ± 0.12 and 4.00 ± 0.11 respectively. It was significantly (P < 0.01) higher in the HD compared with control. No significant different was observed between low and HD groups [Table 1].

Chloride ions were significantly lower in LD (P < 0.05) and HD (P < 0.001) groups compared with control (103.20 ± 0.80 mmol/L). It was in turn significantly (P < 0.001) lower in the HD compared with LD group [Table 1].

The HD extract recipients had a significantly (P < 0.05) higher bicarbonate ion concentration compared with LD extract recipients. Treatment with the extract did not significantly alter the bicarbonate ion concentrations compared with control [Table 1].

Bile cholesterol concentrations

There were no significant differences in the biliary cholesterol concentrations among the different experimental groups [Figure 2].

Figure 2

Comparison of cholesterol levels in control and test groups. Values are mean ± standard error of the mean, n = 6

Bile concentrations of total, conjugated and unconjugated bilirubin

The concentrations of total bilirubin in the control, LD and HD treated groups were 53.82 ± 3.35 μmol/L, 47.26 ± 4.80 μmol/L and 56.70 ± 4.35 μmol/L respectively. No significant differences were observed among the different experimental groups [Table 2].

Table 2

Comparison of hypomagnesemic and normomagnesemic critically ill-patients

The concentrations of conjugated bilirubin did not differ significantly among the different experimental groups [Table 2].

Biliary concentrations of unconjugated bilirubin were significantly (P < 0.05) lower in LD extract recipients compared with control. However, levels were significantly (P < 0.01) higher in HD recipients than in the LD [Table 2].

DISCUSSION

Alterations in bile secretion and composition has been linked in different situations that results in the syndrome of cholestasis and fat metabolism. There is a paucity in scientific literatures on the impact of D. tripetela extract on bile secretion and composition.

The current study demonstrated that the rate of bile secretion was significantly decreased in rats treated with LD of fruit extract of D. tripetala extract when compared with control, whereas treatment with HD of the extract did not produce any significant alterations in bile secretion compared with control. D. tripetala has been reported to contain uvariopsin, an alkaloid which improves bile secretion and attenuates hepatic disorders.[18] The decrease in the rate of bile secretion observed in the LD extract recipients is not quit clear. The liver is the only gland that secretes bile in the body, hence, the decrease in bile secretion in the LD extract recipients may not be due to diminished synthesis but probably due to obstruction of the liver cannaliculi.

The extract was also observed to cause an increase in sodium and potassium ions concentrations of bile. Indicating that some components of the extract would have influenced the secretion of these electrolyte's in bile. This is not surprising because the extract is rich in mineral like iron, calcium, potassium, manganese etc.[6] and vitamin such as thiamine, riboflavin, niacin and ascorbic acid. Some of these vitamins function anti-oxidants which reduce oxidative stress and relieve hepatic disorders.

The extract also caused a reduction in the levels of biliary chloride ions, indicating enhancement of biliary reabsorption of chloride ions. The bicarbonate ion concentration of bile was not significantly altered following LD of the extract, but was significantly increased in the HD extract recipients. The increased bicarbonate observed in the HD recipient could be due to increase in secretion induced by HD of the extract. It has been reported that increased bile secretion due to secretagogues like secretin as seen after meals produces almost entirely secretion of bicarbonate rich watery solution by the epithelial cells of the bile ducts.[9]

D. tripetala did not significantly alter the concentrations of cholesterol, total and conjugated bilirubin in bile, whether in low or HD. However, it leads to a significant reduction in the levels of unconjugated bilirubin.

In conclusion, consumption of ethanolic extract of D. tripetala fruits at LD may increase biliary sodium and potassium ions while at HD it may reduce biliary sodium and chloride ions. LD of the extract may reduce rate of bile secretion while at HD may increase the rate the biliary secretion and bicarbonate ions concentrations without altering the saturation of cholesterol, conjugated and total bilirubin.