|Year : 2013 | Volume
| Issue : 1 | Page : 18-22
Increased bile flow rate and altered composition of bile induced by ethanolic leaf extract of Azadirachta indica (neem) in rats
Ofem E Ofem1, Daniel E Ikpi1, Nsima M Essien2
1 Department of Physiology, College of Medical Sciences, University of Calabar, Calabar, Nigeria
2 Department of Biochemistry, College of Medical Sciences, University of Calabar, Calabar, Nigeria
|Date of Web Publication||30-Dec-2013|
Ofem E Ofem
Department of Physiology, College of Medical Sciences, University of Calabar
Source of Support: None, Conflict of Interest: None
Background: Azadirachta indica (neem) is an ever green tropical plant with ethno-medicinal uses; it is a very potent anti-malaria plant. There is a paucity of the scientific literature on the impact of A. indica on the biliary flow rate and bile composition, considering that alterations in bile composition may lead to gall stone. Aim: This study therefore sought to elucidate the impact of A. indica leaves extract on biliary flow rate and bile composition in rats. Materials And Methods: Eighteen (18) albino Wistar rats were randomly assigned into three groups of six rats each and fed on normal rat chow and/or 150 mg/kg, 300 mg/kg body weight of A. indica extract for 21 days. Results: The rate of bile secretion is in the control, low dose (LD) and high dose (HD) A. indica extract treated rats was 3.0 ± 0.02 ml/h, 5.60 ± 0.46 ml/h and 5.38 ± 0.32 ml/h respectively, showing a significant (P < 0.001) increase in LD and HD compared with control. Na + concentration increased significantly (P < 0.05) in the HD extract recipients compared with control. LDs of the extract increased K + significantly (P < 0.001) compared with control and HD. HDs of the extract increased Cl− concentration significantly (P < 0.05) compared with LD. HCO3− did not alter significantly among these groups. LDs of the extract significantly (P < 0.01) increased total cholesterol, total and unconjugated bilirubin concentrations, HDs reduced it. Conclusion: Hence, A. indica leaves extract increases bile flow rate, LDs of the extract increases cholesterol and bilirubin saturations while HDs reduces it.
Keywords: Azadirachta indica (neem), bile composition, rat, secretion
|How to cite this article:|
Ofem OE, Ikpi DE, Essien NM. Increased bile flow rate and altered composition of bile induced by ethanolic leaf extract of Azadirachta indica (neem) in rats. Niger J Exp Clin Biosci 2013;1:18-22
|How to cite this URL:|
Ofem OE, Ikpi DE, Essien NM. Increased bile flow rate and altered composition of bile induced by ethanolic leaf extract of Azadirachta indica (neem) in rats. Niger J Exp Clin Biosci [serial online] 2013 [cited 2018 Mar 18];1:18-22. Available from: http://www.njecbonline.org/text.asp?2013/1/1/18/123958
| Introduction|| |
From time immemorial, humans have relied on herbs for the treatment of various ailments. Extracts of roots, barks, leaves and seeds of different plants provide a ready source of relief for various seemingly incurable diseases.  Among these plants, which is used in this traditional medicine is Azadirachta indica. ,,,
A. indica commonly called neem or margosa in English language is an evergreen tropical plant of the family meliaceae, it has a garlic-like odor and a bitter taste. It is native to India where it is known as the "Village Pharmacy" because of its healing versatility, it has been used in Ayurvedic medicine for more than 4000 years due to its medicinal properties.  Neem trees occupy above 3500 hectares of land in Kebbi, Sokoto, Borno and Zamfara in Northern Nigeria, with a density of about 1200 trees/hectares.  In Nigeria, neem is known as "Dogonyaro". Traditional health practitioners have used the bark, seeds, fruits, gums and leaves of neem to treat a number of ailments such as skin diseases, inflammatory disorders and fever. , Neem has also demonstrated anti-bacterial, anti-diabetic, hypoglycemic and anti-inflammatory effects. Neem is also used to manage fertility problems. It also possesses some anti-viral, anti-fungal, anti-oxidant and anti-cancer effects.  Neem extract inhibits gastric acid secretion, relieves gastritis and duodenal ulcers. ,,
Neem is commonly used in Nigeria to treat malaria; this property has been found due to the presence of quercetin in neem extracts. Both water and alcoholic extract of neem has been effective against the parasite Plasmodium falciparium.  Small doses of neem have been observed to reduce anxiety and stress. 
Phytochemical analysis of neem reveals the presence of alkaloids, limonoids (a terpenoid) such as azadirachtin, salanin, nimbin, nimbidin, nimbidol, nimbolinin, gedunin, azadirone, amoorastatin, vepinin and vilasinin. Other components of neem are sodium nimbinate, quercetin (a flavonoid) and nimbosterol (β-sitosterol), triterpenes. , Other constituent of neem leaves include protein (7.1%), carbohydrates (22.9%), minerals, calcium, phosphorus, vitamin C, carotene, etc. Others include glutamic acid, tyrosine, aspartic acid, alanine, praline, glutamine and cystine like amino acids and several fatty acids (dodecanoic, tetradecanoic, elcosanic, etc.). ,
The liver is the largest organ in the human body; it helps in glycogen storage, decomposition of red blood cells, plasma protein synthesis and detoxification. It produces bile. Bile is a greenish-yellow, thick, produced in the liver and stored in the gall bladder, its main function is in the digestion of food and sticky fluid elimination of waste products.  Since the liver detoxifies all compounds introduced into the body,  it is obvious that intake of neem may affect the liver synthesis of bile and its composition in one way or the other. However, there is paucity in the scientific literature on the impact of neem extract on bile flow rate and its composition.
This study therefore aims to investigate the impact of neem extract on bile flow rate and bile composition in rats.
| Materials and Methods|| |
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 g and 250 g at the time of sacrifice. The rats were weighed before the commencement of the feeding experiment and thereafter were weighed daily. All the animals were nursed under controlled environmental condition of 12 light and 12 dark cycles in a well-ventilated room. The research was conducted in accordance with the internationally accepted principles for laboratory animal use and care, European Community guidelines. 
A total of 4 kg of fresh leaves of neem 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 A. indica by a botanist (Mr. Frank Adepoju) in the Department of Biological Sciences, University of Calabar, Nigeria. Voucher number (VN = UCDB 1246).
Preparation of Plant Extract
A. indica fruits were rinsed with water to remove debris and sand. The fruits were cut and sun-dried for some days until they got brittle. The sliced fruits were then transferred into an Astell Hearson oven (1950 IRC) set at a temperature of 45°C.  The dried fruits were then 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. 
A total of 18 male albino Wistar rats were randomly assigned into three groups of six rats each. Group 1 (control) received normal rat chow + drinking water, Group 2 (low dose [LD]) and Group 3 (high dose [HD]) in addition received 150 mg/kg and 300 mg/kg body weight respectively of A. indica orally (p.o.) once daily. The feeding regimens lasted for 21 days.
Collection of Bile
After 21 days of feeding, the rats were starved for 18 h. Thereafter, the rats were weighed and anesthetized with sodium thiopental (6 mg/100 g body weight). They were fastened onto the dissecting board for tracheostomy to clear the reduced dead space.
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. 
Determination of Biliary Electrolytes Concentrations
Sodium and potassium ions in bile were determined using a flame photometer (Model 410C, 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.  Phosphoenol pyruvate carboxylase catalyses the reaction between phosphoenol pyruvate and carbondioxide (bicarbonate) to form oxaloacetate and phosphate ion with simultaneous oxidation of an equimolar amount of reduced nicotinamide adenine dinucleotide (NADH) to NAD + . The reaction is catalyzed by malate dehydrogenase. This results in decrease absorption at 340 nm that is directly proportional to CO 2 concentration in the sample.
Into 250 ml conical flask was placed 5 ml of CO 2 - free distilled water. Zero point 2 ml (0.2 ml) of plasma or serum, two drops of indicator and 2 ml N/100 H 2 SO 4 or HCl were added and mixed. The mixing and agitation was done for 90 s and quickly titrated with N/100 NaOH using 1 ml automatic burette until pink end-point appears.
Biliary chloride ion was determined by the principle of end point titration.  2 ml of buffer solution was placed in a conical flast and 0.2 ml of bile was added and mixed. Four drops of diphenyl carbazine indicator was added. This was titrated with mercuric nitrate from a 2 ml micropipette. The end point was indicated by a violet color. The standard solution (0.2 ml) was added to 2 ml of buffer solution with the indicator and similarly titrated.
Biliary chloride concentration was obtained from the following calculation and expressed in mEq/L:
Determination of Biliary Bilirubin Concentration
Biliary bilirubin concentrations were determined by colorimetric method as described by Jendrassik and Grof  and modified by Sherlock.  Total bilirubin is the sum of conjugated and unconjugated bilirubin. Conjugate bilirubin reacts with diazotized sulfanilic acid in alkaline medium to form the blue color complex. The total bilirubin is determined in the presence of caffeine which releases albumin bound bilirubin by reacting with diazotized sulfanilicacid. The color produced is read spectrophotometrically at 530-560 mm.
Extraction of Cholesterol from Bile
Cholesterol was extracted from bile following the principle of esterification reaction. 
Data are presented as mean ± standard error of the mean data were analyzed using one-way analysis of variance then followed with post-hoc test (least square deviation) using computer software's (SPSS version 15.0 and Excel for windows) SPSS Inc., 233 South Wacker Drive, 11 th Floor, Chicago, USA IL 60606-6412. Patent No. 7,023,453. Copyright © 2006 by SPSS Inc. P < 0.05 and was considered to be statistically significant.
| Results|| |
Rate of Bile Secretion
The rate of bile secretion is in the control, LD and HD A. indica extract treated rats was 3.00 ± 0.02 ml/h, 5.60 ± 0.46 ml/h and 5.38 ± 0.32 ml/h respectively. It was significantly (P < 0.001) higher in the extract treated groups compared with control [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 < 0.001 versus control|
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Bile Electrolytes Concentrations
The results for electrolytes composition of bile is shown in [Table 1].
|Table 1: Comparison of biliary electrolytes concentration in the different experimental group|
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The concentration of sodium ion in the HD group (139.20 ± 1.83 mmol/L) was significantly (P < 0.05) higher compared with control value of 134.60 ± 0.24 mmol/L. Sodium ion increased slightly in the LD extract group (with actual measure of 135.20 ± 0.37 mmol/L) compared with control.
The potassium ion concentration in the control, LD and HD treated groups were 3.54 ± 0.02 mmol/L, 4.66 ± 0.10 and 3.44 ± 0.16 respectively. It was significantly (P < 0.001) higher in the LD compared with control and HD groups.
Chloride ion concentrations of the control, low and HD groups were 98.60 ± 2.04 mmol/L, 94.40 ± 0.24 mmol/L and 98.80 ± 1.85 mmol/L respectively. Chloride ion concentration was significantly (P < 0.05) higher in HD extract group compared with the LD group. No significant differences were observed between the control and test groups.
The bicarbonate ion concentrations of the low and HD extract groups did not differ significantly from the control. The concentration of bicarbonate ions were 26.60 ± 1.08, 25.00 ± 0.32 and 27.00 ± 0.89 respectively for control, LD and HD groups.
Bile Cholesterol Concentrations
Mean concentrations of bile cholesterol were 1.04 ± 0.02 mmol/L, 1.30 ± 0.05 mmol/L and 0.88 ± 0.04 mmol/L in control, low and HD groups respectively, as shown in [Figure 2]. The mean bile cholesterol concentration was significantly (P < 0.01) raised in the LD compared with control. However the HD group had a significantly (P < 0.01) lower concentration of bile cholesterol compared with control and LD.
|Figure 2: Comparison of cholesterol concentrations in control and test groups. Values are mean ± standard error of the mean, n = 6. P < 0.01 versus control; C = P < 0.001 versus low dose|
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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, 83.68 ± 2.59 μmol/L and 46.04 ± 3.98 μmol/L respectively. It was significantly (P < 0.001) higher in the LD extract group compared with control and HD groups [Table 2].
|Table 2: Comparison of total, conjugated and unconjugated bilirubin concentration of bile in the different experimental group|
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There was a significant reduction in concentrations of conjugated bilirubin in the HD extract group compared with control (P < 0.05) and LD (P < 0.01) groups. Conjugated bilirubin concentrations for control, low and high does extract groups were 29.18 ± 1.15 μmol/L, 32.22 ± 1.31 μmol/L and 20.16 ± 3.04 μmol/L respectively, as shown in [Table 2].
Biliary concentrations of unconjugated bilirubin were 24.64 ± 4.50 μmol/L, 51.46 ± 2.35 μmol/L and 25.88 ± 4.96 μmol/L for control, LD and HD groups respectively. It was significantly higher in LD extract recipients compared with control (P < 0.001) and HD (P < 0.01), as shown in [Table 2].
| Discussion|| |
The effect of ethanolic extract of neem has been widely studied. It has been found to have several functions, it has been effective in the treatment of malaria, Bacteria infections, fungi infections, diabetes, arthritis and neem extract is also used as a spermicide. ,, The activity of neem has been attributed to the presence of some active bio-chemicals like alkaloids, nimbin, nimbidin, nimbidol, sodium nimbinate, quecertin, salanin, triterpenes, azadirachtin and limonoidas. ,
Results obtained from this study indicate that extracts of neem leaves increased the rate of bile secretion in rats. The increase in the rate of bile secretion may imply an increase in the degree of contraction of the gall bladder to release bile into the circulation (as a cholagogue) or an increase in synthesis of bile by the liver (as a choleretic).  The extract may be acting more like a cholagogue rather than a choleretic, enhancing bile flow via contraction of gallbladder probably through stimulation of cholecystokinin secretion, since the composition of bile was not severely altered. Neem has fats as one of its constituents and fats are known cholagogue. It has also been reported that cholagogue increase bile flow rate by stimulating the secretion of cholecystokinin, a powerful constrictor of the gallbladder. 
In the HD extract group, the concentrations of sodium and chloride ions increased significantly, this may be attributed to the high rate of bile secretion from the gall bladder, which did not allow time for reabsorption of these electrolytes in the gall bladder. Normally, reabsorption of electrolytes (except potassium and calcium) and cholesterol occurs in the gallbladder, making these substances more concentrated.  Potassium ion concentration was increased in the LD extract group, while bicarbonate ion was not altered significantly following extract administration.
Concentrations of bile cholesterol, total and unconjugated bilirubin were significantly raised in the LD extract group. However in the HD extract group, the concentration of cholesterol and conjugated bilirubin were significantly reduced. These results indicate that the extract preserved the integrity of the liver and bile duct.
| Conclusion|| |
Leaves extract of A. indica (neem) increases bile flow rate. LDs of the extract increase bile cholesterol, total and unconjugated bilirubin concentrations, while HDs reduce bile cholesterol and conjugated bilirubin concentrations in rats. This indicates that leaves extract of A. indica (neem) could help to improve the integrity of the liver and gallbladder when ingested.
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[Figure 1], [Figure 2]
[Table 1], [Table 2]
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