Weight reduction with improvement of serum lipid profile and ratios of S esamum radiatum leaves diet in a non-obese Sprague Dawley rats

Department of Anatomy, Lagos State University College of Medicine, P.M.B. 21266, Ikeja, Lagos, Nigeria. Department of Medical Microbiology and Parasitology, College of Medicine, University of Lagos/Lagos University Teaching Hospital, Idi-araba, Lagos, Nigeria. Department of Biochemistry, Obafemi Awolowo University, Ile-Ife, Osun State, Nigeria. Department of Chemical Pathology, College of Medicine/ Lagos State University, Teaching Hospital, Ikeja, Lagos Nigeria. Medical Microbiology Unit, Bolomedics Laboratories, Egbeda, Lagos, Nigeria. Diarrhea Unit, Microbiology Division, Nigerian Institute of Medical Research, Yaba, Lagos, Nigeria. Department of Microbiology, Drug Quality Control Laboratory/Lagos State University Teaching Hospital, Ikeja, Lagos, Nigeria. Department of Obstetric and Gynaecology, Lagos State University College of Medicine/ Lagos State University Teaching Hospital, Ikeja, Lagos, Nigeria.


INTRODUCTION
The development of new dietary adjuncts and novel natural anti-obesity agents with a reducing effect on the *Corresponding author.E-mail: drlukemanjoseph@yahoo.com.long-term complications associated with metabolic syndrome, thereby helping to reinstate a normal metabolic environment in the body have been expressed in recent times.Today, obesity has attained a pandemic healthneeds concern status affecting all age groups worldwide and recognized since ancient times to be a clinical syn-drome with multiple aetiology.However, it is a chronic complex metabolic syndrome with genetic determinants and associated with serious long term morbidity and mortality worldwide (Arner, 2000;Nanchanda et al., 2005).Also, recognized by World Health organization to be among the top 10 global health problem, such that over 1 billion adults are overweight and more than 300 billions are obese, worldwide with a rising incidence from country to country (Marx, 2003).This was corroborated with data from US National Health and Examination Survey in 2003 -2004, which revealed that 67% of adults are overweight or obese, with 17% of children and adolescents overweight (Ogden et al., 2006).
We know that as our body mass index (BMI) increases, the total and LDL-cholesterol increases while, the HDLcholesterol decreases.Thus, it is not surprising that coronary heart disease (CHD) is the leading cause of death worldwide and is strongly linked to overweight and obesity in adolescent.As obtained from report of the 1998 National Health Survey (NHS) in England, where excess body weight alone accounted for 47% of the CHD found in these high-risk men (United States Renal Data System, 1998).
Adipose tissue is now known to play a vital role in body metabolism (carbohydrates, proteins, and lipids) through the exclusive secretion of arrays of proteins such as adiponectin (also called Acrp30 or adipoQ in mice) (Scherer et al., 1995;Maeda et al., 2001).Other studies have shown that plasma concentrations of adiponectin are lower in high fatty diet, obesity and type 2 diabetes (Hotta et al., 2000;Weyer et al., 2001) and that adipose tissue Acrp30/adiponectin mRNA expression is equally reduced in obese ob/ob mice and obese humans (Hu et al., 1996).However, its impact on dietary fat or vice versa is unknown.Although, one potential pathway is the activation of nuclear receptor peroxisome proliferatoractivated receptor-(PPAR-), involved in peroxisomal oxidation of fatty acids.Such that, PPAR-can also be activated by fatty acids in itself (Green, 1995) and that pharmacologic activation with PPAR-agonists including hypolipidaemic agents has led to increased plasma adiponectin concentrations (Maeda et al., 2001;Miyazaki et al., 2004).However, increase in level of sesamin (phytoestrogenic-lignans) consumption in diets has been found to progressively increase the mitochondrial and peroxisomal fatty acid oxidation activity rate in rats (Jeng and Hou, 2005).
Study also showed that hyperlipidaemic treatment would reduce 25 -30% of CHD risks in patients and that free fatty acids are associated with rise in myocardial oxygen consumption (Nasiruddin and Ahmed, 2006).It is now known that certain nutritional factors present in our natural diets through certain mechanisms can reduce serum cholesterol level, these substances include dietary soluble fiber (Saltzman et al., 2001), plant sterols (Jones, 1999;Meguro et al., 2001;Maki et al., 2001) and phytoestrogens-lignans (Potter et al., 1998;Baum et al., Shittu et al. 24291998).More so, serum lignan concentrations has an inverse relationship with the risk of acute coronary heart disease as shown in a recent population based study (Vanharanta et al., 1999).Fujiyama-fujiwara et al. (1995) in a similar study showed that sesamin (0.5%, w/w) synergistically increases the liver weight and phospholipids contents in liver of rats fed with -linolenic acid-rich ( GLA) diet or -linolenic acidrich (ALA) diet (found abundantly in sesame plantleaves) for 4 weeks.Thus, the hypocholesterolemic effect of sesame plant could also be attributed to its -linolenic acid (ALA) and fiber mucilage components (Chen and Anderson, 1986;Brown et al., 1999).However, feeding of -linolenic acid-rich diet to rats lowers their serum cholesterol levels more effectively than a linoleic acid rich diet (Garg et al., 1989;Steinberger et al., 1995).Their mechanisms may be related to their effects on platelet functions as provided in a recent clinical trial (Renaud, 1990).
Sesame is an annual herbaceous plant, which belongs to the family-Pedaliaceae and genus-Sesamum (Purseglove, 1974).Sesame is reputed in folk medicine in Africa and Asia.Sesame plant especially the seed, oil and leaves are consumed locally as a staple food by subsistence farmers in South-West and Middle Belt areas of Nigeria (Akpan-Iwo et al., 2006) and this may account for the high fecundity among the adult male population in these areas (Shittu, 2006).Phytochemical study has shown that the sesame plant is rich in phenolic compounds (phenols, lignans and flavonoids), non-protein amino acids, cyanogenics glycosides, alkaloids, polyunsaturated fats and lipids, mucilage, phospholipids, vitamins B1, B2 and E, trace elements and minerals such as calcium, iron, magnesium, copper and phosphorus (Shittu, 2006;Shittu et al., 2006).
The high incidence of obesity, its mutifactorial nature and the scarcity of adequate therapeutics have fuelled the increase in anti-obesity drug related research in recent times.However, because of paucity of knowledge on the chronic toxicity effects of aqueous crude leaves extracts of Sesame radiatum on the lipids profile in a nonobese adult male Sprague Dawley rats, this study test the serum lipid response and dietary adaptation to recommended daily inclusion of sesame leaves in an otherwise regular diet and to give its possible mechanism of action.

Collection of plant materials
Sesame plants (S. radiatum, Schum and Thonn -Pedaliacaea family) were bought from a vendor in Agege market.The plant was authenticated by the herbarium section of Forestry Institute of Research (FRIN) with FHI # 107513 on the 5 th of August, 2005 (Shittu, 2006).Voucher specimens were deposited in Botany Departments of University of Ibadan and Lagos State University, Nigeria.

Preparation of aqueous extraction of sesame leaves
The leaves of the plants were air dried for 2 weeks and powdered.100 g of the powdered leaves was added to 1.0 litre of distilled water at a ratio of 1:10 in a beaker.This was allowed to boil to boiling temperature after intermittent stirring on a hotplate for one hour.The decoction was filtered into another clean beaker using a white sieve clothing material and the filtrate evaporated at 50 o C to dryness in a desiccator to produce a black shinning crystal residue form with a yield of 83% w/w of the extract.The crude extract was kept in the refrigerator (4 o C) before being reconstituted and used for the in vivo study (Nanchanda et al., 2005).

Ether-extracts preparation of sesame leaves for phytochemical assay
The leaves were air-dried and 100 g of the powdered leaves were extracted with 500 ml of 40% diethyl ether for 72 h with Soxhlet equipment as previously described in our study (Shittu et al., 2006).

Phytochemical screening using gas chromatography-mass spectral
Gas chromatography of crude ether extract of sesame leaves was performed using a Hewlett Packard GCD system (model 6890), equipped with a flame ionization detector and injector MS transfer line temperature maintained at 230°C respectively as described in our previous studies (Shittu et al., 2006;Bankole et al., 2007).Compound identification was accomplished by comparing the GC relative retention times and mass spectra to those of authentic substances analyzed under the same conditions, by their retention indices (RI) and by comparison to reference compounds and library searches (Shittu et al., 2006).

Animal
Thirty mature and healthy adult male Sprague Dawley rats weighing 120 to 216 g were procured from Lagos State University, College of Medicine, Ikeja, and housed in well-ventilated wire-wooden cages in the departmental animal house.They were maintained under controlled light schedule (12 h light : 12 h dark) at room temperature (28 o C) and constant humidity of 40 -50%.The animals were allowed to acclimatize for a period of 7 days before treatment.During this period, they were fed with standard rat chows/pellets supplied by Pfizer Nigeria Limited and water ad libitum.Individual identification of the animal was made by ear tags.

Experimental procedure
The rats were randomly divided into three groups (A to C) comprising of ten rats each.The group A served as the control while B and C constituted the treated groups.The animals in group A received equal volume of 0.9% (w/v) normal saline daily while group B (high dose) received aqueous extract of sesame leaves at 28.0 mg/kg body weight /day, the animals in group C (low dose) were given aqueous extract of sesame at 14.0 mg/kg body weight /day (half group B dose).All the doses were given via gastric gavage (oro-gastric intubation) daily for a period of 6 weeks.
All animals were observed for clinical signs of plant-induced toxicity (such as tremors, weakness, lethargy, refusal of feeds, weight loss, hair-loss, coma and death) throughout the duration of the experiment.
All procedures involving animals in this study conformed to the guiding principles for research involving animals as recommended by the Declaration of Helsinki and the Guiding Principles in the Care and Use of Animals (American Physiological Society, 2002) and were approved by the Departmental Committee on the Use and Care of Animals.

Animal sacrifice
The rats were anaesthetized at the time of sacrifice by being placed in sealed cotton wool soaked chloroform inhalation jar between 0900 and 1100 h done the following day after the termination of the experiment after post over night fasting of the animals.The weights of the animals were taken weekly and before the sacrifice.

Blood collection and serum preparation
Blood was collected via cardiac puncture from each animal as described in our previous study (Shittu, 2006).The blood were collected into sterile bottles and centrifuged at 4000 × g for 10 min using a MSE tabletop centrifuge machine (Minor Gallenkamp).The extracted serum of each rat was stored in the freezer at -20 o C for biochemical assay.

Determination of biochemical lipid parameters
The concentration of serum total cholesterol and triglyceride concentrations were measured enzymatically by using kits and standards supplied by (Sigma Diagnostics, St. Louis, MO).HDL cholesterol was measured by using an EZ HDL-Cholesterol Kit as previously described (Burstein et al., 1970).LDL-Cholesterol was calculated by using the formula of Friedewald et al. (1972).However, we calculate the non-HDL-Cholesterol concentration by subtracting HDL-Cholesterol from total cholesterol.The intra assay CVs were 2.2, 2.6 and 1.7% for total cholesterol, HDL Cholesterol and triglycerides, respectively.

Statistical analysis
The weight data were expressed in Mean ± S.D (standard deviation) while other data were expressed as Mean ± S.E.M (standard error of mean).Statistical analyses were done by using the student t-test and ANOVA as the case may be with input into SPSS 12 software Microsoft computer (SPSS, Chicago, Illinois).Statistical significance was considered at P 0.05.

RESULTS
No obvious signs of toxicity (such as tremors, weakness and lethargy, refusal of feeds, weight loss, hair-loss, coma and death) were observed in any of the animals throughout the duration of our observation.The GC/MS showed the presence of essential oils mainly the carboxylic phenolic groups such as (sesamol, sesamin) and other compounds such as thiazole, pyrroles, disulphide and aldehyde.Also, confirmed the presence of trace elements/minerals, vitamins and steroid such as adrostenedione among others.Significant (P < 0.05) evidence of weight gain was seen in all the treated animals.However, about 78% and 57% relative weight loss were found in the high and low dose groups respectively when compared to the control group in the adjusted weight as shown in Table 1.Also, the weight loss in low dose was increased by 27% over the high dose group as seen in Table 1.About 21 and 39% significant (P < 0.05) reduction in total cholesterol when compared to control for both high and low dose were observed respectively.Such that, cholesterol level in high dose was reduced by 47% over the low dose group as shown in Table 2. LDL reduces significantly (P < 0.05) by 94 and 82% in high and low dose respectively when compared to the control.Thus, the high dose is decreased by 13% over the low dose group as shown in Table 2. HDL significantly (P < 0.05) reduced by 19 and 48% in both high and low dose groups respectively, when compared to the control.However, the high dose increased by 60% over the low dose as shown in Table 2. VLDL significantly (P < 0.05) reduced by 9 and 13% for both high and low dose respectively when compared to the control.However, high dose mildly decreased by 31% over the low dose group as shown in Table 2.There were significant reduction in TG level about 2% in the treated than the control, but no significant difference exists between the low dose and high dose groups as shown in Table 2.

DISCUSSION
To our knowledge after extensive literature searches, this study appear to be the first that looked at the chronic toxicity of aqueous sesame leaves extract on the serum lipids profile and ratios in non-obese rats.The benefits derived from high intake of any fruits and vegetables on the various metabolic disease conditions such as diabetes mellitus, obesity, heart diseases and cancer might not always be due to the impact of their well characterized antioxidants (such as, beta-carotene, vitamins C and E only).But rather related to the presence of other antioxidants or non-antioxidant phytochemicals or by an additive action of the different compounds present in foods themselves such as alpha-linolenic acid (poly unsaturated fatty acids), various phenolic compounds (sesamin, sesamol) and fibres, which are also present in sesame leaves for example.
All the animals used in this study, irrespective of their initial aggressive nature, exhibited some degree of calmness (non-aggressive state) during treatment.More so, there was a general state of well-being observed among all the animals during the whole experimental period.This may reflect the positive effect of some of the active ingredients/agents present in the sesame plant on the neurophysiological pathway of the animals.
In this study, there was evidence of significant (P < 0.05) weight gain observed in all the animals.But, when adjustment was made for their relative weight, a significant (P < 0.05) weight reduction difference was observed in the treated groups when compared to the control and was dose dependent, such that the weight loss in the high dose was more than in the low dose group as shown in Table 1.
Findings in this study also showed evidence of decrease in the total cholesterol and the LDL-C when compared to control, which reflects the beneficial effect of sesame leaves.However, this effect is dose dependent and is much lower in the high dose than the low dose.Hence, in a way has favoured a significant lower LDL/HDL ratio in this group of animals as observed in Table 2.These findings are due to the fact that Apo A-1, the major HDL proteins inhibits LDL oxidation (Ohta et al., 1989).Other study by Gosh et al. (2006) show that low HDL-C levels with low ApoA-1 will increase LDL oxidation and hence, decrease the ratio of LDL-C: HDL-C, which is a better indicator of lipid perioxidation as seen in this study.It is now recognized that dietary LA also favours oxidative modification of LDL cholesterol (Abbey et al., 1993) and increases platelet response to aggregation (Renaud, 1990).However, HDL-C also has preventive role in CHD, by reducing endothelial incorporation of lysophosphatidy choline (Nasiruddin and Ahmed, 2006).
The effect of sesame lignans in this study was similar to that obtained with other types of lignans found in Flaxseed supplementation diet with a significantly lowered serum total cholesterol and non-HDL-cholesterol and Soy-based meal, which was effective in losing weight and reducing LDL-C beyond the expected level (Gregory, 2006).Diesters of arylnapthalene lignans (synthetic lignans) have been shown to be also effective in lowering serum total cholesterol and LDL cholesterol while increasing HDL cholesterol (Kuroda et al., 1997).Study has shown that LDL, which is a major component of total cholesterol is directly related to coronary artery disease (CHD) as a major atherogenic lipoprotein and hence, appears to be the main target of any lipid lowering agents including sesame leaves as reflected in this study.
Sesame leaves significantly (P < 0.05) lowered both the TG and VLDL, although, found to be within the same range for both the high and low dose.No doubt, the arteriosclerosis index (AI) for the animals will be reduced because of the higher HDL-cholesterol level in the high dose compared to the low dose; which can also be of beneficial importance to heart disease management.This significant reduction in TG levels may be attributed to an increasing lipid peroxidation activity (LPL), which will lead to an increase in the degredation of TG and/or reduction in VLDL which is usually produced in the liver and aids in lipolytic removal of TG-rich lipoproteins from the circulation (Wang et al., 2003;Megalli et al., 2006).Studies have shown that elevated VLDL-C is associated with a high TG, since VLDL-C carries the highest amount of TG in the body (Ghosh et al., 2006) as observed in this study.
Similar to our findings, Sirato-Yasumoto et al. ( 2001) observed that serum triacylglycerol concentrations were lower in rats fed with high dose sesame-lignans rich diets than Masekin (low dose) diet or control.They concluded that consumption of sesame rich in lignans diet have more profound physiological effects on hepatic fatty acid oxidation and serum triacylglycerol levels.Other study has also suggested that sesamolin and its metabolitessesamol and sesamolinol in the vivo system will strongly inhibit lipid peroxidation, hence preventing oxidative DNA stress damage and contribute to the antioxidative properties of sesame lignans (Kang et al., 1998).Studies in both humans and animals have suggested that the primary mechanism through which dietary soluble fiber present in sesame plant lowers cholesterol is through enhanced synthesis of bile acids and their fecal excretion.Such that the enhanced elimination of bile acids also results in increased hepatic cholesterol synthesis.However, in this study, the low cholesterol may be due to impairment of cholesterol synthesis or rapid eliminations by bile salt from the system.

Conclusion
This study has demonstrated that sesame-treated rats significantly showed evidence of weight loss and hypolipidaemia in a dose related manner via the combined effects of all the active ingredients mainly the rich lignans (sesamin, sesamolin and sesamol) and fatty acids (especially the oleic, linoleic, linolenic and palmitic acids) among others present in the plant leaves.Further study is aimed to isolate these already characterized active agents and evaluation of their individual effects on lipid profiles as whole.

Table 1 .
Average weekly body weight of animal.

Table 2 .
Lipids and lipoproteins profiles of sesame-treated animals.