Anti-Ulcerogenic and Membrane Stabilization Effect of Ethanol Extract of Coconut (Cocos nucifera)

 

 

*Anosike Chioma Assumpta, Obidoa Onyechi, Nwodo Okwesilieze Fredrick C. and Joshua Parker Elijah.

Department of Biochemistry, Faculty of Biological Sciences, University of Nigeria, Nsukka, Enugu State, Nigeria

 

ABSTRACT:

The effects of the ethanol extract of coconut on indomethacin – induced gastric ulcer in Wistar rats and on Hypotonicity induced haemolysis of human red blood cells were studied. Ulcer was induced in the rats by post oral (p.o.) administration of indomethacin (50mg/kg) using standard procedures. The percentage ulcer inhibition was taken as the measure of the cytoprotection offered by the coconut extract. The extracts reduced the gastric erosions induced by indomethacin in a dose dependent manner with 100mg/kg dose having an ulcer inhibition of 65.4%; 200mg/kg gave 67.9% inhibition while 400mg/kg caused a 70.1% reduction in the ulcerations formed. These results were comparable to the 91% reduction recorded for ranitidine, the standard anti-ulcer drug. The effect of the coconut extract on haemolysis induced by distilled water was evaluated by incubating various concentrations of the extract with red blood cells and distilled water. The effect of the standard anti-inflammatory drug, indomethacin was determined as a positive control. Changes in absorbance were used to assess the extent of haemolysis, hence membrane stabilization. From the results obtained, coconut extract gave a dose dependent reduction in the haemolysis induced by distilled water. This suggests that the extract has a stabilizing effect on lysosomal membranes.

 

KEYWORDS: Coconut extract, gastric erosions, cytoprotection, RBCs haemolysis, distilled water.

 

INTRODUCTION:

The coconut palm (Cocus nucifera L.) locally referred to as kwa kwa in Hausa, Aku oyibo in Ibo, Agbon in Yoruba and Isip oyon in Efik, is a member of the family Arecaceae (palm family). It is the only specie of the genus cocos which grows throughout the tropical countries including Nigeria. The coconut palm is very large, growing as tall as 30 meters, with pinnate leaves 4-6m long. Every part of coconut is useful and medicinal to mankind, hence it is known as the “Tree of life” 1,2. In traditional medicine around the world, the coconut is used to treat a wide variety of health problems.

 

Its water (coconut water) is used as an intravenous fluid to counteract the effects of drug overdose, poisoning and adverse drug reactions[3, 1]. Coconut water is also a source of quick energy, boosts energy and endurance. It is used in place of dextrose\glucose in medical emergencies. During World War II, young coconut water was used as an emergency room glucose supply in the absence of sterile glucose1. Coconut water also relives the symptoms associated with Crohn’s disease, an illness in which the intestines are infected4. It has been reported to be antihelmintic, antidotal, antiseptic, astringent, bactericidal, diuretic, purgative, vermifuge, stomachic and supportive5. Indigenous people of tropical countries use young coconut juice in the treatment of stomach upsets, diarrhea and dysentery, ulcerative colitis and stomach ulcers5.


The most abundant nutrient in the coconut is fat, which makes up more than a third of its mature weight. The lauric acid content of coconut endows it with antimicrobial properties. As such, coconut is useful in the treatment of digestive tract infections6. It is also used to expel intestinal parasites like tapeworms and Helicobacter pylori, which are responsible for indigestion and ulcer7.

 

The oil can be processed and extracted as an organic product which can be employed in the cosmetic industry for skin care to moisturize the skin, relieve dryness, flaking and prevent stretch marks. It is used for wounds, bruises, burns, rashes, eczema, and dermatitis. It also supports the natural chemical balance of the skin and provides protection from the damaging effects of ultraviolet radiation from the sun7. Coconut, due to its contents of caprylic acid, which is fungicidal, is used in the treatment of fungal skin infections such as athlete’s foot, thrush, ringworm and candidiasis8. In modern medicine, coconut is used as an immune system boaster in infants6. It improves digestion and absorption of other nutrients such as vitamins, minerals and amino acids; prevents obesity and overweight problems by increasing metabolic rate, regulates thyroid function, boosts energy and fights fatigue9. Coconut is used in the treatment of mal-absorption of fat such as cystic fibrosis and enteritis. It improves insulin secretion and enhances the utilization of blood glucose. This forms the basis for its use in the management of diabetes10. Coconut is effective in the treatment and prevention of heart disease, chronic fatigue syndrome, osteoporosis, gall bladder disease, Crohn’s disease, prostate enlargement and cancer because of its composition of high and medium chain fatty acid[7]. It also reduces inflammation and allergic reactions due to its anti-histamic effect11.

 

The coconut palm has a multitude of industrial uses. It provides raw materials for industries such as the wood, furniture and food industry. Its products includes: timber, food, fermented and unfermented drink, alcohol, vinegar, thatching material, splint fibres for making baskets, masts rope, hats, brushes and broom. The palm leaves are used as a source of fuel and shelter12. It produces utensils for household use such as cups, bowls; oils for food, illumination, soap and margarine production and ointment12. The residue after extraction is used in feeding domestic animals and as fertilizer. Nearly one third of the world’s population depends on coconut to some degree for their food and their economy.

 

Several food uses also exist for coconut products. Coconut is highly nutritious; rich in fibre, vitamins, and minerals. It is classified as a “functional food” because it provides many health benefits beyond its nutritional content2. This work was aimed at investigating the cytoprotective effect of the ethanol extract of coconut on the gastric mucosal damage induced by indomethacin on Wistar albino rats and the membrane stabilization ability of the extract on hypotonicity induced haemolysis of red blood cells.

 

MATERIALS AND METHODS:

Animals:

Twenty five adult rats of either sex of weight 120-200g obtained from the animal house of the Faculty of Biological Sciences, University of Nigeria, Nsukka were used for the study. They were divided into five experimental groups of five rats each, housed in separate cages and acclimatised for seven days before the experiment. They were maintained ad libitum on water and growers mash (Pfizer Feeds, Aba) bought from Nsukka market. The research was conducted in accordance with the ethical rules on animal experimentation approved by the ethical committees of the Faculty of Biological Sciences, University of Nigeria, Nsukka.

 

Plant materials:

Matured coconut seeds purchased from the Nsukka local market was cracked and the fresh nuts chopped into tiny bits and sun-dried. The dried coconut was ground with a mechanical grinder and macerated in absolute ethanol for 24hrs and filtered with a white cloth. The filtrate was concentrated using a rotary evaporator at an optimum temperature of 40o-50oC.

 

Preparation of blood sample:

Fresh blood samples (5ml) were collected from apparently healthy donors (without drug treatment for at least 2 weeks) into plastic tubes containing 1ml of 3.8% sodium citrate. These test tubes were centrifuged at 300rpm for 10mins. The red cell pellets were collected and re-suspended in normal saline equal to 2 times the volume of the supernatant.

 

Reagents/Chemicals:

All chemicals in this study were of analytical grade and products of May and Baker, England; British Drug Home (BDH), England and Merck, Darmstadt, Germany. Reagents used for all the assays were commercial kits and products of Randox, USA; QCA, Spain; Teco (TC), USA; Biosystem Reagents and Instruments, Spain.

 

Phytochemical analysis:

Preliminary Phytochemical tests were carried out on the ethanol extract of coconut using the methods of Harborne13 and Trease and Evans14.

 

Indomethacin induced ulcer:

This assay was carried out using the method of Urushidani et al.15. Twenty five adult rats randomly divided into 5 groups of 5 rats each were deprived of food for 18hrs and treated per orally with normal saline and varying doses of the plant extract. The extracts and drugs used were freshly prepared as a suspension in normal saline and administered per orally to the animals in 5ml/kg doses. Group 1 (normal control) was administered normal saline (5ml/kg). Groups II, III and IV were treated with 100mg/kg, 200mg/kg and 400mg/kg of the coconut extract respectively. Group V (reference group) was administered 100mg/kg of ranitidine (standard anti ulcer drug).

 

 

 

Table 1: Effect of coconut extract on indomethacin induced gastric ulcer in rats

Treatment

Dose (mg/kg)

Ulcer index

% Ulcer inhibition

Control (normal saline)

5ml/kg

3.18 ± 1.81

 

Extract

100

1.1 ± 0.54*

65.4

Extract

200

1.02 ± 0.68*

67.9

Extract

400

0.95 ± 0.81*

70.1

Ranitidine

100

0.27 ± 0.09*

91.5

Values shown are mean ± SD. (N= 5). *Significantly different from control at p<0.05. N= 5


Table 2: Effect of coconut extract on hypotonicity induced haemolysis of red blood cells. (Membrane stabilization effect of the extract)

Treatment

Extract volume (ml)

Normal saline (ml)

Distilled water (ml)

RBC (blood) (ml)

Absorbance

% inhibition of haemolysis

Isotonic solution

-

2.4

-

0.1

0.05

--

Hypotonic solution

-

1.9

0.5

0.1

0.75

--

Test sample

0.1

1.8

0.5

0.1

0.67

11

Test sample

0.2

1.7

0.5

0.1

0.27

69

Test sample

0.4

1.5

0.5

0.1

0.09

87

Test sample

0.6

1.3

0.5

0.1

0.08

89

Test sample

0.8

1.1

0.5

0.1

0.075

89

Test sample

0.9

1.0

0.5

0.1

0.055

92

0.2ml indomethacin

-

1.7

0.5

0.1

0.065

91

 

 


Thirty minutes later, 50mg/kg of indomethacin was administered (p.o) to the rats. After 8hrs, each animal in the groups was sacrificed by chloroform anaesthesia and the stomach removed and opened along the greater curvature, pinned flat on a board, examined and scored for ulcer. Erosions formed on the glandular portions of the stomach were counted and the ulcer index calculated as described by Main and Whittle16. The ulcer was counted and scored as 0 = no ulcer; 1 point = superficial ulcer; 2 points = deep ulcer and 3 points = perforations.

 

Determination of membrane stabilization effect of coconut extract on hypotonicity induced haemolysis of red blood cells:

This assay was carried out by a modified method of Shinde et al.17. The effect of the ethanol extract of coconut on haemolysis induced by distilled water was evaluated by incubating various concentrations of the extract with 0.1ml of the sodium citrate treated blood, normal saline and distilled water in a test tube for 1hr at 37oC in a water bath. After the incubation, the test tubes were centrifuged at 300rpm for 10mins to terminate the reaction. The absorbance of the supernatants collected was read at 418nm. These experiments were done in triplicates and mean absorbance values taken. The effect of the standard anti-inflammatory drug, indomethacin was determined as a positive control. Changes in absorbance were used to assess the extent of haemolysis; hence membrane stabilization. Percentage inhibition of haemolysis by the extract was calculated thus:

% Inhibition of Haemolysis =

Where OD1 = Absorbance of isotonic solution

OD2 = Absorbance of test sample

OD3 = Absorbance of hypotonic solution.

 

Statistical analysis:

The results were expressed as mean ± SD and test of statistical significance were carried out using one-way ANOVA, correlation and T–test. The statistical package used was statistical package for social sciences (SPSS) version 15.0

 

RESULTS:

Phytochemical analysis of the ethanol extract of coconut shows the presence of phtochemicals such as alkaloids, resins, steroids, flavonoids, terpenoids and glycosides, and macronutrients like fats and oil, reducing sugar, carbohydrate and proteins.

 

Data from Table 1 show that indomethacin induced gastric ulcer in all the experimental groups. Groups administered with 100mg/kg, 200mg/kg and 400mg/kg of coconut had significant reductions (P<0.05) in the ulcer indices as compared to control, and with high percentage level of ulcer inhibition which was comparable with that of the standard drug, ranitidine.

 

Data from Table 2 show that the coconut extract exhibited high membrane stabilization effect against hypotonicity induced haemolysis of the red cells. This inhibition of haemolysis was found to be dose dependent, increasing with increased concentration of the extract in the medium and was comparable with that of indomethacin, a standard anti-inflammatory drug.

 

DISCUSSION:

In the present study, significant protection against indomethacin induced gastric mucosal ulceration was observed when the animals were administered with 100mg/kg, 200mg/kg and 400mg/kg of the coconut extract. 100mg/kg caused a 65.4% reduction in ulcer index; 200mg/kg caused a 67.9% reduction while 400mg/kg caused a 70.1% reduction comparable to the 91% reduction recorded for ranitidine, the standard anti-ulcer drug. This report is consistent with that of Fife7 which reported the use of coconut oil in the treatment of stomach ulcers. Stomach ulcer results from an injury or damage to the gastric mucosal lining of the stomach which could be as a result of excess or an overproduction of hydrochloric acid, an acid normally present in the digestive juices of the stomach or due to complications resulting from an infection with Helicobacter pylori18. It also results due to excess intake of non- steroidal anti-inflammatory drug (NSAIDs) such indomethacin, aspirin and ibuprofen.  Gastric ulcers induced by these drugs is related with the inhibition of cyclooxgenase 2 enzyme which synthesizes prostaglandin needed to maintain the integrity of the gastric lining of the stomach19. In this study, the ethanol extract of coconut exhibited anti-ulcerogenic effect against indomethacin induced gastric ulcer, which is comparable with that obtained for ranitidine, an antiacid used to neutralize intraluminal acid, improve gastric microcirculation and reduce the absorption and concomitant adverse drug interactions of many NSAIDs21. This result supports that of Nneli and Woyike22 which showed that coconut milk and water exhibited anti-ulcerogenic effect against indomethacin induced ulcer and suggests that coconut may act by reducing the intestinal absorption of indomethacin and its resulting drug interactions.

 

The effect of the coconut extract on haemolysis induced by distilled water evaluated by incubating various concentrations of the extract with red blood cells and distilled water showed a reduction in the haemolysis of the red cells with the incubation of coconut in a dose dependent manner (Table 2). During inflammation, there are lyses of lysosomes which release their component enzymes which produce a variety of disorders22. Since human red blood cell (RBC) membranes are similar to lysososmal membrane, human RBC stabilization was therefore used as a method to study the mechanism of action of anti-inflammatory agents23.

 

Hypotonicity induced haemolysis of red blood cells occurs due to water uptake by the cells and leads to the release of haemoglobin which absorbs maximally at 418nm. Hence, the reduced optical density at 418nm obtained for the various coconut test samples is a reflection of the stabilization of the red cell membrane caused by the extract. Iwu[11] reported that coconut reduces inflammation and allergic reactions due to its anti-histamic effect. The stabilizing effect of coconut extract on lysosomal membranes as seen in this study suggests a possible mechanism of action for the anti-inflammatory effect of coconut by reduction in edema which occurs during inflammation.

 

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11.   Iwu, M. (1993). Handbook of African Medicinal Plants. CKC Press Boca Raton, Boca, pp: 156-157.

12.   Ohler, J. G. (1999). Modern coconut management. Food and Agriculture Organization (FAO), Rome, pp: 458.

13.   Harborne, J. B. (1984). Phytochemical Methods. A Guide to Modern Technique of Plant Analysis. 2nd Edn., Chapman and Hall, London, pp: 282.

14.   Trease, G. E. and Evans, W. C. (1989). Phenols and Phenolic glycosides. In: Textbook of Pharmacognosy. 12th Edn., Balliese, Tindall and Co Publishers, London, pp: 343-383.

15.   Urushidani, T., Kasuya, Y. and Okabe, S. (1979). The mechanism of aggravation of indomethacin–induced ulcers by gastric adrenalectomy in the rat. Jpn J. Pharmacol., 29: 775-780.

16.   Main, I. H. M. and Whittle, B. J. R. (1975). Investigation of vasodilator and anti-secretory role of prostaglandins in the rat gastric mucosa by use of non-steroidal anti-inflammatory drugs. Br. J. Pharmacol., 53: 217- 224.

17.   Shinde, U. A., Phadke, A. S., Nair, A. M., Mungantiwar, A. A., Dikshit, V. J. and Sarsf, M. N. (1989). Membrane stabilization activity- a possible mechanism of action for the anti-inflammatory activity of Cedrus deodara wood oil. Fitoterapia, 70: 251-257.

18.   Marshall, B. J. and Warren, J. R. (1984). Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet, 1: 3111-1315.

19.   Wallace, J. M. (2002). Nutritional and botanical modulation of the inflammatory cascade: eicosanoids, cyclooxygenase and lipoxygenase - as an adjunct in cancer therapy. Integr Cancer Ther., 1: 7-37.

20.   Derle, O.V., Guijar, K. N. and Sagar, B. (2006). Adverse Effects Associated with the Use of Non-Steroidal Anti-inflammatory Drugs: An Overview. Indian J. Pharm. Sci., 68: 409-414.

21.   Nneli, R. O. and Woyike, O. A. (2008). Antiulcerogenic effects of coconut (Cocos nucifera) extract in rats. Phytother. Res., 22: 970-972.

22.   Goodman, G. and Gilman, G. (2000). The Pharmacological basis of therapeutics. 10th Edn., Joel G. Hardman. The Mcgraw-Hill companies, USA, pp: 686.

23.   Murgesh, N., Vembar, S. and Damadarana, C. (1981). In: Gandludasann, R., Thamasalchew, A. and Baburay, S. (1990). Anti- inflammatory actions of Lannea coromandelica by HRBC membrane stabilization. Fitoterapia, 5: 81-83.

 

Received on 03.01.2010

Accepted on 05.02.2010   

© A&V Publication all right reserved

Research Journal of Pharmacognosy  and Phytochemistry. 2(1): Jan.-Feb. 2010, 88-88

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