Moringa Study (20151151B)
Ibrahim, Kasimu Ghandi
Cleaned data from the experiement conducted with 21-day old male Sprague-Dawley rats . These rats(n=50) were randomized to six treatment groups (n=8-9) with unlimited access to commercial rat feed. Either plain water (C) or 20% fructose solution (Fr) were provided to drink. 400 mg.kg-1 M. oleifera methanolic leaf extract (Mo) or 100 mg.kg-1 fenofibrate were also administered daily for ten weeks. Growth, circulating metabolites, visceral and epididymal fat pads mass, hepatic lipids and general health markers were assessed. Liver samples were histologically examined using H&E stain. Analysis of the data was done with GraphPad Prism 5.0v for windows (GraphPad Software, Inc. CA), and the data were expressed as mean ± standard deviation and analysed by means of a one-way analysis of variance (ANOVA). Statistical significance was considered at P < 0.05.
Methods Preparation of Moringa oleifera leaves extracts Moringa oleifera fresh leaves (7.5 kg) were sourced from the Agricultural Research Council Research (ARC) Farm, Roodeplaat, Pretoria, South Africa. The plant was authenticated and a voucher specimen (Id: J 103704) deposited at the Moss Herbarium of the University of the Witwatersrand in Johannesburg, South Africa. The leaves were desiccated in an oven (Salvis®, Salvis Lab, Schweiz, Switzerland) set at 40ºC. The leaves were then pulverized using a blender (Waring Commercial Blender, HGB2WTG4, USA). Within a 24 hour period, the powder was extracted with absolute methanol (Merck (pty) Ltd, South Africa) (100 g in 400 ml) on a shaker. The mixture was then filtered through Whatmann No.1 filter paper, and concentrated using a rotary evaporator (Buchi Rotavapor-R, Buchi Laboratoriums Technik AG, Schweiz, Switzerland) at 40°C. The condensed extract was then oven-dried (Salvis®, Salvis Lab, Schweiz, Switzerland) at 40°C. A 6% yield of dried extract was obtained and then stored in tightly sealed dark bottles at -4°C until use. Design of the experiment The Animal Ethics Screening Committee (AESC) of the University of the Witwatersrand approved the study (Reference No: 2015/11/51/B) which was conducted according to the internationally accepted principles for laboratory animal use and care as stipulated in South African National Standard (SANS 10386:2008) and Animals Protection Act, 1962: Act No. 71. Fifty 21-day old weaned male Sprague-Dawley rats weighing between 40-60g were individually housed in a well ventilated room at a temperature of 26±2°C in the Central Animal Service, University of the Witwatersrand, Johannesburg. The animal room was maintained on a 12-hour alternating light and dark cycle (illumination turned off between 7pm-7am). Standard rat polycarbon cages which contained clean wood shavings for bedding were used to house the rats individually. The treatment intervention commenced after the rats were allowed two days of adaptation to the environment. Pups were randomly allocated to six treatment groups which all received commercially sourced rat chow [LabChef Rodent Breeder, Nutritionalhub (PTY) LTD, Stellenbosch, South Africa] formulated to meet the nutritional requirements of rats ad libitum for the 10 week period of intervention. Plain drinking water and plain gelatine cubes were given to Group I (C; n=9); 20% fructose solution as drinking fluid (Mamikutty et al. 2015) and plain gelatine cubes were given to Group II (Fr; n= 9) to induce metabolic dysfunction; 20% fructose solution as drinking fluid and 400 mg.kg-1 body weight of methanolic extract of M. oleifera (Muhammad et al. 2018) were given to Group III (Fr + Mo; n= 8) in which the prophylactic effects of the Moringa extracts were investigated against fructose induced metabolic dysfunction; 20% fructose solution as drinking fluid and 100 mg.kg-1 body weight of fenofibrate (Sigma-Aldrich, France) (Abd El-Haleim et al. 2016) were given to Group IV (Fr + Fn; n= 8) as the positive control group; 400 mg.kg-1 body weight of methanolic extract of M. oleifera and plain drinking water were given to Group V (Mo; n= 8) to investigate the effects of Moringa alone and 100 mg.kg-1 body weight of fenofibrate and plain drinking water were given to Group VI (Fn; n= 8) to investigate the effects of fenofibrate alone. Flavoured gelatine cubes (2 ml) were used to suspend the Moringa extracts and fenofibrate and were given one daily. The fructose solution and plain drinking water were provided ad libitum. Measurement of body mass The rats were weighed at induction, thereafter they were weighed twice a week to determine gain in body mass and monitor growth performance in order to ensure that the rats received appropriate doses of the treatments. Procedures performed at termination A day prior to termination, the rats were fasted of feed for 12 hours overnight with access to plain drinking water. The terminal body masses of the rats were obtained and thereafter two drops of blood were taken following a pin prick to the tail vein. The fasting blood concentrations of glucose and triglycerides were measured using a glucose meter (Contour Plus Bayer Health Care, Diabetes Care, Isando, South Africa) and a triglyceride meter (Accutrend, Roche Diagnostics, Germany) respectively. An intraperitoneal injection of sodium pentobarbitone (Centaur Laboratories, Johannesburg, South Africa) at 150 mg.kg-1 body weight was used to euthanase the rats. The ventral thorax and abdomen were incised along the midline, and intra-cardiac blood was drawn and transferred into heparinized vacutainers and then centrifuged (Rotofix 32A, Hettich Zentrifugen, Germany) at 3700 revolutions per minute for 15 minutes. The harvested plasma was stored at -20˚C for later use. The liver, visceral and epididymal fat were removed, weighed and expressed relative to terminal body mass. A section of each liver sample was preserved at minus 20˚C for lipid content determination while rest was fixed in 10% phosphate buffered formalin for histological evaluation. Hepatic lipid content determination The frozen stored liver samples were lyophilised, milled and pooled into a composite sample for each group. Hepatic lipid content was quantified in triplicate for each group by solvent (petroleum ether) extraction at an accredited laboratory of the Agricultural Research Council (Irene Analytical Services Laboratory) using Tecator Soxtec apparatus with standard protocols (Official Methods of Analysis of Analytical Chemists, 2005). Histological examination of the liver The fixed liver samples were processed with an automatic tissue processor (Microm STP 120, ThermoScientific, MA, USA), embedded in paraffin wax, sectioned at 5µm using a rotary microtome [Leica Instruments GmbH, (PTY) LTD, Germany] and stained with hematoxylin and eosin (H&E) or Masson’s trichrome and then cover slipped. The H&E slides were viewed under a light microscope (×100) to evaluate the progression of fatty liver. Similarly, the Masson’s trichrome stained slides were viewed under a light microscope (×40) to determine collagen deposition and fibrosis. General health profile markers A VetTest analyser (IDEXX VetTest® Clinical Chemistry Analyser, IDEXX Laboratories Inc., USA) was used to determine the plasma concentrations of cholesterol, blood urea nitrogen (BUN), creatinine and activity of alanine aminotransferase (ALT). Fasting serum insulin concentration (ng.ml-1) was ascertained using an Enzyme Linked Immuno-Sorbent Assay kit (Elabscience Biotechnology Co., Ltd), which was then expressed as (µU.ml-1) using the formula (Darby et al. 2001): 1 ng.ml-1 of insulin is equivalent to 0.02 µU.ml-1 The insulin resistance index was computed according to the Homeostasis Model of Assessment (HOMA-IR) using the following formula (Divi et al. 2012): “HOMA-IR = fasting insulin (µU.ml-1) × fasting glucose (mmol.l-1) ÷ 22.5” Statistical analysis Analysis of the data was done with GraphPad Prism 5.0v for windows (GraphPad Software, Inc. CA), and the data were expressed as mean ± standard deviation and analysed by means of a one-way analysis of variance (ANOVA). This was followed by a Bonferroni post hoc test for comparison of the means. Statistical significance was considered at P < 0.05.
Fructose, obesity, fatty liver, Moringa oleifera, male rats