Moringa oleifera leaf meal as a hypocholesterolemic agent in laying hen diets

T S Olugbemi, S K Mutayoba and F P Lekule

Department of Animal Science and Production, Sokoine University of Agriculture, P.O. Box 3004, Morogoro, Tanzania
olugbemitaiye1@yahoo.com

Abstract

Moringa oleifera has being studied in human nutrition because of its nutritional benefits. However, its hypocholesterolemic potential, as attested to by traditional medical practitioners has not been extensively studied.   Due to controversies as to the role of eggs in cardiac related diseases, research has continued to focus on ways in reducing the cholesterol content.  The potential of Moringa oleifera leaf meal (MOLM) as a hypocholesterolemic agent was therefore investigated using layers fed cassava based diets over a 90 day period.  Eighty layers were assigned to four dietary treatments containing MOLM at 0, 5, and 10% (treatments 2, 3 and 4) levels with cassava chip constituting 20% of each diet and a control diet (treatment 1) containing neither cassava nor Moringa. A completely randomized design was employed.  The effect of the dietary treatments on serum and yolk cholesterol was determined. 

Serum cholesterol levels in  treatments 2, 3 and 4 declined by 14.2%, 19.8% and 22.0 %, respectively, while yolk cholesterol levels declined by 6.55%, 7.45% and 12.1%, respectively.

Results of the study indicate that Moringa oleifera possesses hypocholesterolemic properties and its inclusion in layers diets could facilitate reductions in egg cholesterol content.

Keywords: cholesterol, eggs, feed ingredients, heart

Introduction

There is a general assumption that high dietary cholesterol levels result in high serum cholesterol and consequently a higher risk of arteriosclerosis and coronary heart disease (Grundy 1990).  There has been much controversy as to the role of eggs in blood cholesterol levels however, eggs are undisputedly the highest source of dietary cholesterol.  An egg can contain up to 208mg of cholesterol (Holden et al 1989) and even more depending on its size, hence the long lasting debate as to the risk in consuming an egg a day.  Consuming one egg a day (about 200 mg cholesterol) increases the ratio of total to HDL cholesterol concentrations by 0.040 units, thereby increasing the risk of myocardial infarction (Weggemans et al 2001).  Such reports amongst others have contributed to the “egg scare” and may have created a negative influence on consumers’ attitudes toward eggs (Chen et al 2005).  However, another school of thought asserts that eggs in themselves are not a threat to normal health but for those with raised blood cholesterol levels (above 5.2 mmol/L), reducing the amount of high-cholesterol foods such as eggs will help  reduce blood cholesterol levels (Weggemans et al 2001).  

While the fact remains that the human body produces cholesterol in the absence or presence of dietary cholesterol and less cholesterol is produced to compensate for dietary cholesterol, blood cholesterol levels still increase when dietary cholesterol is increased (Weggemans et al 2001).  Attention has recently focused on identifying ingredients or production methods that can facilitate a reduction in egg cholesterol. Genetic selection of hens for lowered cholesterol has not been successful in lowering the egg cholesterol content and research into lowering egg cholesterol has therefore centered mostly on diet and pharmacological intervention (Jacob and Miles 2000).  Cholesterol reducing measures have been by Chromium supplementation, lowering of the energy consumption of hens (Jacob and Miles 2000), production of omega 3 rich eggs and raising chickens free range (Linden 2008).

Moringa oleifera belongs to the single genus monogeneric family Moringaceae and is well distributed in Africa and Asia.  Apart from being a good source of vitamins and amino acids, it has medical uses (Makkar and Becker 1999; Francis et al 2005).  Moringa oleifera, otherwise regarded as a “miracle tree”, is reputed to have many medicinal properties, although many have not been scientifically substantiated.  It has been used in the treatment of numerous disease conditions (Pal et al 1995; Makonnen et al 1997; Ghasi et al 2000 and Matthew et al 2001), including heart disease and obesity due to its hypocholesterolemic property (Ghasi et al 2000).  This study therefore aimed at evaluating the potential of Moringa oleifera leaf meal (MOLM) as a cholesterol reducing agent in cassava based feeds for layers. 

Materials and methods 

Preparation of leaf meal

Moringa oleifera leaves were harvested from an orchard on the premises of the Department of Animal Science and Production farm unit of the Sokoine University of Agriculture, Tanzania.  The cut branches were spread out on a floor and allowed to dry for a period of 3-4 days under shady and aerated conditions.  The leaves were separated from the twigs before milling in a hammer mill to produce the leaf meal.  The proximate composition of MOLM is shown in Table 1.

Preparation of experimental diets

Four iso-nitrogenous and iso-caloric diets (Table 2) were formulated incorporating Moringa oleifera Leaf Meal (MOLM) at 0, 5, and 10% levels (treatments 2, 3 and 4) with cassava chip constituting 20% of each diet and a control diet (treatment 1) containing neither cassava nor Moringa. 

Representative samples from all the feed ingredients were obtained for determination of the proximate chemical analysis according to AOAC 1990 methods.  Apart from the cassava chip, which was fed whole, all the other feed ingredients were ground in a hammer mill before being mixed.

Management of experimental animals

Eighty (80) layers were randomly allocated to one of four treatments in a completely randomized design (CRD).  Each treatment comprised of 2 replicates with 10 birds per replicate kept in pens.  Feed and water were provided ad libitum and all medications and managerial practices applied when required.  The birds were group feed twice daily (9.00 and 15.30 h).  The birds were given the experimental diets for 90 days.

Cholesterol determination

Serum cholesterol

Blood samples were obtained from five birds per replicate, hence ten per treatment, by inserting a new sterile needle into the wing vein of the birds and extracting 1 ml of blood which was placed inside vacutainer test tubes containing Ethylene diamine tetra acetic acid (EDTA). The samples were properly shaken to mix with the EDTA in order to prevent coagulation of the blood and within two hours of collection were taken to the laboratory for serum cholesterol analysis.  The vacutainer tubes were placed in a centrifuge and centrifuged at 3000 r.p.m. for 10 minutes in order to separate the serum.  A commercial diagnostic cholesterol reagent kit (Erba Diagnostic Mannheim GmbH) was used for cholesterol determination using the following procedure.  1000µl of a working reagent (supplied in the kit) was diluted with 10µl of distilled water and 10µl of the aliquot was then placed in a cuvette and inserted into a spectrophotometer set at 505nm in order to take the blank absorbance reading.  1000µl of the working reagent was again mixed with 10µl of the calibrator and a 10µl aliquot taken and put in a cuvette before placing in the spectrophotometer to obtain the absorbance of the calibrator. Lastly, 1000µl of the working reagent was mixed with 10µl of the serum from each sample and incubated for about 10 mins at room temperature, after which an aliquot of 10µl was taken and put in a cuvette and the absorbance read. The addition of the serum sample to the reagent resulted in plain and purple colours in various degrees depending on the amount of cholesterol in the sample.  The absorbance of the test sample was then recorded.

Cholesterol was determined using the following formula:

Yolk cholesterol

Ten eggs per treatment (five per replicate) were broken and yolks carefully separated into a container from which 1ml of the yolk was taken with a pipette and placed into a beaker, after which it was diluted with 1ml buffer pH 7.4 (10 mmol sodium phosphate/l, 100 mmol NaCl/l).  10µl of the test sample was taken and mixed with 1000µl of the working reagent and the absorbance read after incubating for 10 minutes at room temperature.  The absorbance of the blank and calibrator were determined as for the serum cholesterol.  The yolk cholesterol was calculated using the formula used for the calculation of serum cholesterol. 

Statistical analysis

The data obtained were analysed by General Linear Model (GLM) of Statistical Analysis Systems (1995) and the following model was used:

Y_ij = µ + T_i + e_ij

Where :

Y_ij = Observation on the j^th bird on the i^th treatment

µ  = General mean effect

T_i = Effect due to the i^th dietary treatment

e_ij =  Random error

Results  

The influence of the treatments on the serum and yolk cholesterol is shown in Table 3 and figure 1.

There was a progressive decline (P<0.05) in serum cholesterol among treatments (181 – 141 mg/dl). However, only Treatment 1 differed significantly (P<0.05) from the rest.   Progressive, though insignificant (P>0.05) declines were similarly observed for yolk cholesterol (282 – 248 mg/dl) as MOLM levels increased.  Serum and yolk cholesterol levels from birds on Treatment 2 (cassava based diet) were numerically lower (P>0.05) than those from treatment 1 (maize based control diet) and there were also progressive declines (P>0.05) in the measured parameters with increase in the inclusion of MOLM from 5% to 10%. Serum cholesterol levels of birds on Treatments 2, 3 and 4 were 14.2%, 19.8% and  22.0 % lower than of birds on Treatment 1, while  Treatments 3 (5% MOLM) and 4 (10% MOLM) showed reductions of 6.50% and 9.02%, respectively, compared to Treatment 2.  Yolk cholesterol levels from birds on Treatments 2, 3 and 4 declined by 6.55%, 7.45% and 12.1%,  respectively, compared to  Treatment 1 while levels on Treatments 3 and 4 were 0.96% and 5.89% lower, respectively than for Treatment 2. 

Discussion  

Results obtained from this study show that MOLM inclusion in layer diets was instrumental in cholesterol reduction in serum and yolk.  The fact that inclusion of MOLM resulted in a decrease in cholesterol levels, affirms its potential as a hypocholesterolemic agent.  This is in agreement with the report of Ghasi et al (2000).  The relatively low reductions in this study compared with that of Ghasi et al (2000) might be due to differences in the form and quantity of Moringa oleifera included in the diets, as well as the animal type used, as crude extracts of the leaf were used in their rat study as compared with the raw meal used in the present study.  In a related study we conducted with broilers, abdominal fat was observed to be lower in birds on MOLM diets.  Higher cholesterol contents are an indication of higher fat deposition (Tewe and Bokanga 2001) which is not desirable in layers.  Reductions at these low percentages are however still significant, as a 25% reduction of the total cholesterol in plasma can reduce the incidence of coronary events by nearly 50% (Lipid Research Clinics Program 1984a and b) especially as eggs are highest major source of dietary cholesterol.

The mechanism of cholesterol reduction is thought to be through the lowering of plasma concentrations of LDL by B-sitosterol, the bioactive phytoconstituent isolated from Moringa oleifera (Saluja et al 1978; Kane and Malloy 1982 and Ghasi et al 2000).  Although there were no significant effects of replacing maize by cassava chips in the present study, the use of cassava as a supplement to maize in layer diets has been shown to lower cholesterol levels, probably due to its fibre content (Charina 2006).  During digestion in the intestine, cholesterol is the main component of bile acids secreted.  The fibre coats the bile acids in the intestine and is excreted in the body, subsequently causing the body to draw cholesterol from the blood to form bile acids, and thus lowering blood cholesterol level (Charina 2006). 

Results of this study attest to the hypocholesterolemic properties of MOLM.  The main thrust however in maximizing the potential of Moringa most likely lies in the form of the leaf.  Its inclusion in the form of powder might enhance its hypocholesterolemic properties. 

Acknowledgement 

The authors are grateful to the African Network of Scientific and Technological Institutions (ANSTI) for providing funds to conduct this research.

References 

AOAC 1990 Association of Official Analytical Chemists 1990 Official analytical methods.  15^th Edition Volume 1, AOAC Washington DC

Charina J 2006 RUMD FNRI-DOST S&T Media Service 2006   Food and Nutrition Research Institute. Department of Science and Technology, General Santos Avenue, Bicutan, Taguig City

Chen Y C, Nakthon C and Chen T C 2005 Effects of Chicory Fructans on Egg Cholesterol in Commercial Laying Hen.  International Journal of Poultry Science 4 (2): 109-114

Francis G, Makkar H P S and Becker K  2005  Products from little researched plants as aquaculture feed ingredients.  Retrieved February 24, 2005 from   http://www.fao.org/DOCREP/ARTICLE/AGRIPPA/551_EN.HTM#TopOfPage

Ghasi S, Nwobodo E and Ofili J O 2000  Hypocholesterolemic effects of crude extract of leaf of Moringa oleifera Lam in high fat diet fed wistar rats.  Journal of Ethnopharmacology 69(1):21-25 

Grundy S M 1990 Cholesterol and coronary disease: future directions.  Journal of American Medicine 265: 3053-3059 

Holden J, Exler J, McCharen C and Lockard J 1989 A nationwide study of the cholesterol, vitamin and mineral levels in large eggs.  Federation of American Societies for Experimental Biology 3:A658

Jacob J and Miles R 2000 Designer and Specialty Eggs.  Fact sheet PS-51, Department of Animal Sciences, Florida Cooperative Extension Service, Institute of Food and Agricultural Sciences, University of Florida. November 2000. http://edis.ifas.ufl.edu/pdffiles/PS/PS04800.pdf

Kane J P and Malloy M J 1982 Treatment of hypercholesterolemia.  Medical Clinics of North America 66:537-550 

Linden J 2008 Free-range eggs with a boost of omega-3 fatty acids.  Poultry International  January 2008 WATT, Rockford, Illinois, U.S.A pp.18-19

Lipid Research Clinics Program 1984a The lipid research clinics coronary primary prevention trial results I. Reduction in incidence of coronary heart disease. Journal of American Medical Association 251:251–364

Lipid Research Clinics Program 1984b The lipid research clinics coronary primary prevention trial results II. The relationship of reduction in incidence of coronary heart disease to cholesterol lowering Journal of American Medical Association 251:365–374

Makkar H P S and Becker K 1999 Plant toxins and detoxification methods to improve feed quality of tropical seeds - Review.  Asian-Australian Journal of Animal Science (3): 467- 480

Makonnen E, Hunde A and Damecha G 1997 Hypoglycaemic effect of Moringa stenopetala aqueous extracts in rabbits.  Phytotherapy Research 11: 147 - 148

Matthew T, Matthew Z, Taji  S A and Zachariah S  2001  A review of Viricidal Ayurvedic Herbs of India for Poultry Diseases.  Journal of American Holistic Veterinary Medicine Association 20(1): 17 – 20

Pal S K, Mukherjee P K and Saha B P 1995 Studies on the anti-nuclear activity of Moringa oleifera leaf meal extract on gastric ulcer models in rats.  Phytotherapy Research 9: 463 - 465.

Saluja M P, Kapil, R S and Popli S P 1978  Studies in medicinal plants part VI.  Chemical constituents of Moringa oleifera Lam (hybrid variety) and isolation of 4-hydroymellein.  Indian Journal of chemistry B 16B 11:1044-1045

SAS Institute Inc 1995  SAS users Guide version 8.  SAS Institute, Inc., Cary, NC

Tewe O O and Bokanga M 2001 Cost effective cassava-plant based rations for poultry and pigs.  Proceedings of the 8^th International Society for Tropical Root Crops-Africa Branch symposium, Ibadan, Nigeria pp 229-234  

Weggemans R M, Zock  P L and Katan M  B 2001 Dietary cholesterol from eggs increases the ratio of total cholesterol to high-density lipoprotein cholesterol in humans: a meta-analysis^.  American Journal of Clinical Nutrition 73(5): 885-891

Received 19 January 2009; Accepted 18 March 2010; Published 1 April 2010

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