Hypolipidemic and anti-atherogenic effect of aqueous extract leaves of Ficus glumosa (Moraceae) in rats

EXG-09560; No of Pages 10 Experimental Gerontology xxx (2014) xxx–xxx

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Experimental Gerontology journal homepage: www.elsevier.com/locate/expgero

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Article history: Received 25 July 2014 Received in revised form 26 December 2014 Accepted 30 December 2014 Available online xxxx

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Keywords: Ficus glumosa Hypolipidemic Anti-atherosclerotic Lipid peroxidation TBARS Histopathology

Department of Biological Sciences, Faculty of Science, University of Ngaoundéré, PO Box 454, Cameroon Department of Chemistry, Faculty of Science, University of Ngaoundéré, PO Box 454, Cameroon c Department of Animal Biology and Physiology, Faculty of Science, University of Yaoundé I, PO Box 812, Cameroon d Department of Food Science and Nutrition, National School of Agro-industrial Science, University of Ngaoundéré, PO Box 455, Cameroon e Department of Neuroscience, Faculty of Medicine, University of Montréal, 2960, Chemin de la Tour, Pavillon Paul-G. Desmarais, Montréal, Québec H3T 1J4, Canada f School of Wood, Water and Natural Resources, Faculty of Agriculture and Agricultural Sciences, University of Dschang, Ebolowa Campus, PO Box 786, Ebolowa, Cameroon b

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Ntchapda Fidele a,⁎, Djedouboum Abakar a, Talla Emmanuel b, Sokeng Dongmo Sélestin a, Nana Paulin f, Adjia Hamadjida e, Nguimbou Richard Marcel d, Bonabe Christian a, Gaimatakon Samuel a, Njintang Yanou Nicolas a, Dimo Théophile c

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Leaves of Ficus glumosa are used in northern Cameroon and southern Chad for the treatment of cardiovascular diseases, as food and as a stimulant for milk production in both women and animals. Atherosclerosis is a disease in which frequency increases with age. The first lesions appear at the young subject during adolescence. Atherosclerosis lesions appear very precociously and worsen with age. They interest the levels chronologically aortic, coronary then carotid. Age is a risk factor in that it reflects the exposure time of individual to the other risk factors. The frequency of the atherosclerosis increases with age because of the aging of the cells. This study was undertaken to evaluate the hypolipidemic and anti-atherosclerotic properties of aqueous extract of the leaves of F. glumosa in rats with hypercholesterolemia (HC). 60 male rats were fed for 4 weeks with a high-cholesterol diet (1%) and 3 doses (225, 300 and 375 mg/kg) of extract of F. glumosa were used in these experiments. The experiments were conducted under the same conditions with atorvastatin (1 mg/kg), as pharmacological reference substance. The effects of F. glumosa on weight gain, water and food consumption, levels of serum lipids and lipoprotein lipid oxidation and stress markers in the blood and liver were examined. The administration of F. glumosa extract prevented significant (P b 0.05) elevation in TC, LDL-c, VLDL-c, hepatic and aortic TG and TC. The atherogenic, triglyceride, and lipid peroxidation (TBARS) indexes were also decreased in the rats treated with the extract. F. glumosa favored the performance of fecal cholesterol. It also significantly inhibited the changes and the formation of aortic atherosclerotic plaques. These results revealed the hypolipidemic and antiatherosclerotic effects of F. glumosa extract and support the traditional use of the extract of this plant in the treatment of hypertension and diabetes. © 2014 Elsevier Inc. All rights reserved.

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Hypolipidemic and anti-atherogenic effect of aqueous extract leaves of Ficus glumosa (Moraceae) in rats

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48 47 49 Q21 1. Introduction

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Cardiovascular disease is the leading cause of death and disability in developed countries (Engeland et al., 2004). In developing countries some authors have observed an increase in the incidence of cardiovascular disease due to certain risk factors. The main risk factors include smoking, hypertension, diabetes and dyslipidemia (Touze, 2007; Verdier and Fourcade, 2007; Tiahou et al., 2010). Indeed, several epidemiological studies have shown that high concentrations of total cholesterol and/or LDL-c significantly increase coronary risk (Ducimetiere et al., 1977; Sans et al., 1997; Moarreaf, 2004). Framingham studies in the United States conducted in cohorts (Anderson et al., 1987) or the

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Section Editor: Holly M. Brown-Borg

⁎ Corresponding author. E-mail address: [email protected] (N. Fidele).

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PROCAM study in Europe (Assmann and Schulte, 1998) have established this relationship conclusively. Dyslipidemia is a major risk factor for the occurrence of atherosclerosis and cardiovascular disease (Tiahou et al., 2010). Atherosclerosis is a leading cause of death in industrialized countries. This inflammatory disease usually begins in childhood with the formation of plaque in the artery which compromises blood flow (Braunwald, 1997). Atherosclerosis is a disease in which frequency increases with age. The first lesions appear at the young subject during adolescence. They extend gradually and insidiously, and appear in general at a more or less late age (after 50 years on average) due to complications. The age of revelation depends on a very great number of factors and the disease is not dependent exclusively on age. Indeed, we see appearing accidents related to the atherosclerosis at an increasingly young age. The prevention of cardiovascular disease appears to be fundamental in public health, and the objective is to decrease or

http://dx.doi.org/10.1016/j.exger.2014.12.015 0531-5565/© 2014 Elsevier Inc. All rights reserved.

Please cite this article as: Fidele, N., et al., Hypolipidemic and anti-atherogenic effect of aqueous extract leaves of Ficus glumosa (Moraceae) in rats, Exp. Gerontol. (2014), http://dx.doi.org/10.1016/j.exger.2014.12.015

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2. Materials and methods

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2.1. Plant materials

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The leaves of F. glumosa were harvested in Ngaoundéré, Adamawa 109 region of Cameroon. The identification was confirmed by comparing 110 Q37 the harvested plant to reference specimen No 60695/HNC deposited at 111 the National Herbarium of Cameroon. 112

2.2. Animals

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Only Wistar male rats (155 ± 5 g) which were from Centre Pasteur in Yaoundé were used in this study. They were reared in the Laboratory of the Medicinal Plants, Health and Galenic Formulation of the Depart116 ment of Biological Sciences, Faculty of Science of the University of 117 Ngaoundéré. The animals were housed under controlled temperature 118 (24 ± 2 °C) and relative humidity (45% ± 10%). The animals had free ac119 cess to balance diet (pellets from LANAVET (Laboratory NVS)) and pota120 ble drinking water. The animal handling was under the control of 121 Q39 veterinary surgeons from the School of Veterinary Medicine and Sci122 ences of the University of Ngaoundéré. Experimental protocol and pro123 cedure were approved by Animal Ethics Committee of the University of 124 Q40 Ngaoundéré. The rats were not subjected to any suffering, pain or 125 distress.

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2.3. Preparation of the aqueous extract of leaves of F. glumosa Del. One thousand grams (1000 g) of fresh leaves of F. glumosa was

128 soaked in 1 L of distilled water for 12 h at room temperature. The mac129 Q41 erate was filtered through Whatman filter paper No 3, and the filtrate 130 was concentrated in a rotary evaporator at 40 °C for 24 h. This process 131 Q42 repeated several times yielded 112 g of crude oil extract was obtained. 132

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A total of 60 normocholesterolemic (NC) rats were divided into 6 groups of 10 rats. 5 groups were fed for 4 weeks with a diet consisting of 50% corn starch, 11.25% rice powder, 01% vegetable oil, 10% egg white, 08% fish meal, 19% cellulose, 0.125% mineral complex, 0.125% vitamin complex and 0.50% salt. For induction of hypercholesterolemia (HC), 1% of cholesterol was added in the feed of rats. The nutrient contents of the NC (g/100 g food) diet were: total lipid (19.70 ± 0.28); protein (32.95 ± 2.4); ash (0.02 ± 0.005); fiber (12.33 ± 1.50); and carbohydrates (35 ± 2.3) (Alam et al., 2011). The plant extract was administered to animals at the increasing doses of 225, 300 and 375 mg/kg. After 4 weeks of treatment 5 rats out of 10 were sacrificed, blood and organs were collected for biochemical analysis. The remaining 5 rats were sacrificed 4 weeks after the end treatment and blood were collected again for biochemical analysis. Results were later compared to the first group to confirm the anti-atherogenic properties of the leaf extract. Blood collected in heparinized tubes were centrifuged at 3000 rev/min for 10 min to extract plasma for the enzymatic determination of total cholesterol, HDL cholesterol and triglycerides and malondialdehyde. Blood pellet was used in the preparation of hemolysates while the liver samples collected were used to prepare liver homogenates for the determination of catalase, hydroperoxides and proteins. The enzymatic colorimetric method was used to determine the serum cholesterol and HDL-c. Cholesterol is measured after enzymatic hydrolysis and oxidation. The indicator quinoneimine was formed from hydrogen peroxide, amino 4 antipyrines and in the presence of phenol and peroxidase. The amount of quinoneimine formed was proportional to the concentration of cholesterol. The chylomicrons and the Very Low Density Lipoproteins (VLDLs) and Low Density Lipoproteins (LDLs) contained in the sample were precipitated by the addition of phosphotungstic acid in the presence of magnesium ions. The supernatant after centrifugation contained High Density Lipoprotein (HDL). Enzymatic cholesterol reagent was used to determine the level of cholesterol. The clear supernatant containing the HDL fraction was tested with the reagent Chronolab kit for determining the levels of HDL-c. Under the action of lipases, triglycerides were hydrolyzed to glycerol. The glycerol was then converted to hydrogen peroxide in the successive action of the glycerol kinase and glycerol-3-phosphate oxidase. Quinoneimine that served as indicator was formed as previously indicated. The serum concentration of LDL was determined by the method of difference according to the equation of Friedewald et al. (1972): LDL-c = TC − (HDL-c − triglycerides) / n. Carbonyl compounds like malondialdehyde reacted with Thiobarbituric Acid (TBA) to give pink chromophores absorbed at 532 nm. For 100 mL of reagent, trichloroacetic acid (TCA) 20% w/v; Thiobarbituric Acid (TBA) 0.375% w/v; Butylhydroxytoluene (BHT) 0.01% w/v; and Hydrogen chloride (fl) 375 mg of 1 N TBA, TCA 20 g, 0.01 g of BHT, 25 mL of 1 N HCl and 50 mL of distilled water were introduced into a beaker. The resulting solution was heated at 40 °C in a water bath until complete dissolution of the TBA, and was then transferred to a 100 mL flask and filled to volume with distilled water. TBARS concentration was determined using the molar extinction coefficient of the MDA (å = 1.53 · 105 M−1 cm−1). The results were expressed in mmol·L−1. Total proteins were determined by the method of Lowry et al. (1951) proteins in alkaline form with Cu2 + ions a hexa-coordinated complex ranging in color from pink to blue-violet purple depending on the amount of protein present in the medium. The complex of which the wavelength of maximum absorption is 540 nm is proportional to the amount of protein present. Briefly 100 μL sample or distilled water (blank) or BSA (Bovine Serum Albumin: 5 mg in 5 mL water) was introduced into a test tube with 1 mL reagent 1. The mixture was incubated at room temperature for 10 min. Then 100-μL of reagent 2 was added. The mixture was incubated at room temperature for 30 min. The optical density was read at 750 nm against the blank. Albumin was used as a standard.

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eliminate the risk factors. Atorvastatin (statins) drugs that contribute to the decreased levels of plasma LDL and a synthetic antioxidant 77 (probucol) are widely used to treat atherosclerosis. Unfortunately, 78 these drugs have adverse effects (Lankin et al., 2003). Therefore, there 79 is the need for alternative methods against atherosclerosis. Herbs or 80 Q35 foods are increasingly used as a remedy because of their fewer side ef81 Q36 fects (Cooke, 1998). Many studies such as using promoted the use of tra82 ditional medicine and food for the treatment of atherosclerosis such as 83 using Pistacia vera (Anarcadiaceae) (Marinou et al., 2010); Glycyrrhiza 84 glabra (Fabaceae) (Asgary et al., 2007); Pu-erh tea (Yan et al., 2009); 85 Syzygium aromaticum (Lauraceae); Foeniculum vulgare (Apiaceae) 86 (Fatiha et al., 2011) and Clerodendrum capitatum (Adeneye et al., 2008). 87 Ficus glumosa is a medicinal plant used in East Africa, Cameroon and 88 Senegal for the treatment of skin diseases and diabetes (Madubunyi 89 et al., 2012). In northern Cameroon and southern Chad the leaves and 90 bark of the plant are used in foods as a stimulant for milk production 91 in both women and animals (Tourneux and Yaya, 1998). They are also 92 used in the treatment of edema, hemorrhoid, and cardiovascular dis93 eases including hypertension (Arbonnier, 2000). Recently, Tanko et al. 94 (2012) showed that effects of methanol extract of the leaves of 95 F. glumosa affect gastrointestinal motility. Madubunyi et al. (2012) 96 also showed in vitro antioxidant and in vivo antidiabetic potential of 97 the methanol extract of F. glumosa Del. (Moraceae) stem bark in 98 alloxan-induced diabetic mice. Onoja et al. (2014) revealed subacute 99 antidiabetic and in vivo antioxidant effects of methanolic extract of 100 F. glumosa stem bark on alloxan-induced hyperglycemic rats. Nana 101 et al. (2012) showed evidence of ceramides and cytotoxic constituents 102 from F. glumosa extract. The main objective of this study therefore was 103 to examine in a hyperlipidemic rat model the effects of aqueous extract 104 of F. glumosa on blood lipids and lipoproteins and the progression of 105 atherosclerosis.

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The extract was stored at -20 °C until uses.

Please cite this article as: Fidele, N., et al., Hypolipidemic and anti-atherogenic effect of aqueous extract leaves of Ficus glumosa (Moraceae) in rats, Exp. Gerontol. (2014), http://dx.doi.org/10.1016/j.exger.2014.12.015

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Analytical tests for the identification of different families of metabolites in crude extracts of the leaves were made at IMPM (Institute of Me228 dicinal Plants for Medicinal research) Yaoundé-Cameroon. The 229 Q57 procedures similar to those described by Trease and Evans (1983) 230 were used for the detection of various chemical groups. Preliminary 231 tests of the phytochemical study were conducted in view of the identi232 fication of the chemical structure of the compounds responsible for the 233 diuretic activity (Ntchapda et al., 2014). 2.6. Statistical analyses

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The results were expressed as the means ± standard error of the means. Comparison of means was made using the Student's t-test and one-way ANOVA using the Origin Graph software (Microcal Origin 6.0, Microcal, MA USA) version 6.0. For *P b 0.05, **P b 0.01, and ***P b 0.001, the difference was considered significant.

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3. Results

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3.1. Phytochemical study

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During the treatment, F. glumosa extract caused a significant increase (P b 0.01) in water consumption (Table 1). At the dose of 375 mg/kg, the water intake increase was 33.69% and 52.94% respectively at week 1 and week 4. Four weeks after treatment, water consumption decreased in animals previously treated with the aqueous extract. At the dose of 375 mg/kg, water consumption decreased by 111.93 ± 1.11 and 99.55 ± 0.83 respectively in week 1 and week 4, leading to a decrease of 68.54% and 189.84% respectively when compared to the water consumption during the treatment at the same dose (Table 1).

2.5. Phytochemical study

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3.3. Effect of the extract of F. glumosa Del. on water consumption in rats

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10.01%, 18.63%, 23.96% and 35.57% respectively in the 1st, 2nd, 3rd and 4th weeks (Fig. 1). During the study, animals administered with daily dose of 225 mg/kg of the experimental plant extract experienced a relative decreased in their body weight of 7.88% and 12.39% respectively at the 1st week and 4th week. Animals, that received the extract at dose of 300 mg/kg, showed weight drop of 9.57% and 15 51% respectively in the 1st week and 4th week of treatment. Animals that were administered with the extract at a dose 375 mg/kg revealed body weight decrease from 238.26 ± 0.82 g/rat to 227.43 ± 13.76 g/rat in week 1 and from 227.43 ± 13.76 g/rat to 201.81 ± 9.32 g/rat in week 4 equivalent to decrease rates of 13.55% and 27.94% respectively. The increase in body weight observed in animals administered atorvastatin was also significant (P b 0.05) (Fig. 2). A significant body weight gain was observed in general in animals that were not treated with the experimental plant extract in this study (Fig. 2).

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The hydroperoxides (ROOH) were determined by the method described by Jiang et al. (1992). In an acidic medium, peroxide ion pro200 voked the oxidation of the ferrous ion (Fe2 +) to ferric ion (Fe3 +). 201 With orange xylenol a Fe 3 + -complex orange-xylenol was formed 202 whose optical density was read at 560 nm. The calibration of the re203 action was done with the standard hydroperoxides such as hydrogen 204 peroxide, linoleic hydroperoxide, tert-butyl hydroperoxide or 4 −1 205 Q55 cumene peroxides (å = 4.52·10 M cm− 1). FOX reagent was pre206 pared by mixing 88 mg of BHT, 7.6 mg of xylenol orange and 9.8 mg 207 of ammonium sulfate. 90 mL of methanol and 10 mL of 250 mM sul208 furic acid were then added. The resulting mixture was homogenized 209 and stored. 100 μL of hemolysate and homogenate was added to 210 900 μL of FOX reagent. After homogenization, the various tubes 211 were incubated in a water bath at 37 °C for 30 min. The absorbance 212 of the colored complex formed was read at 560 nm against the re213 agent blank. The concentration of hydroperoxides was expressed as 214 hydroperoxides μM (plasma) and hydroperoxides μM/100 g of tissue 215 (homogenate). Catalase (CAT) was determined by the method de216 scribed by Sinha (1972). This method is based on the fact that the 217 catalase present in the sample wanted reduce H2O2 to H2O and O2. 218 Q56 In 0.1 mL of sample or distilled water (blank) was added 250 μL of 219 phosphate buffer and 200 μL hydrogen peroxide and the reaction 220 was stopped after 30 s and 60 s by the addition of 1 mL of mixture 221 acid-dichromate acetic. All tubes were incubated in a boiling water 222 bath (100 °C) for exactly 10 min. They were then cooled and absor223 bance was read at 620 nm against the blank. The catalase activity 224 was expressed in mM H2O2 consumed/min/g of protein.

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3.4. Effect of the extract of F. glumosa Del. on food intake

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HC + distilled water group showed an increase of 46.97 ± 1.14% and 68.88 ± 1.36% respectively in the 1st week and in the 4th week in food consumption during treatment. During the 4 weeks of hypercholesterolemia treatment, food consumption significantly decreased (P b 0.05) in treated animals compared to HC + distilled water. At the dose of 375 mg/kg, there was a decrease in food intake of 40.22 ± 1.15% in the first week and 26.54 ± 1.31% in week 4 compared to HC + H20 (Table 2). Food intake increased significantly in all batches after the treatment of hypercholesterolemia. At the dose of 375 mg/kg, there was a significant increase (P b 0.05) in food intake of 48.22 ± 1.25% in the first week and 61.88 ± 1.55% at week 4.

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Phytochemical screening performed on crude extracts revealed the presence of several primary and secondary metabolites such as fatty 244 Q58 acids, anthraquinones, glycosides, saponins, tannins, coumarins and 245 triterpenes. Phenolic compounds and sterols were also present in the 246 extract. The presence of flavonoids and alkaloids was significant. 247 These initial results suggest that the aqueous extract of leaves of 248 F. glumosa contains several chemical compounds whose potential bio249 logical activity remains to be demonstrated. 250

3.2. Change in body weight of animals

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During induction period, the body weight of NC animals fed cholesterol-free diet, significantly increased (P b 0.05) from 9.62% in the 1st week to 24.34% in week 4. Animals fed with a diet rich in cholesterol showed significant increase with body weights (P b 0.05) of

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Fig. 1. Variation of rat body weight during induction after 4 weeks of observation. Values Q1 are means ± S.E.M., n = 5, *P b 0.05, significant difference compared to HC. NC: normocholesterolemic rats, HC: hypercholesterolemic rat.

Please cite this article as: Fidele, N., et al., Hypolipidemic and anti-atherogenic effect of aqueous extract leaves of Ficus glumosa (Moraceae) in rats, Exp. Gerontol. (2014), http://dx.doi.org/10.1016/j.exger.2014.12.015

N. Fidele et al. / Experimental Gerontology xxx (2014) xxx–xxx

rats HC

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After 4 weeks of treatment of hypercholesterolemia, there were no significant changes (P b 0.05) observed in relative weights of the liver, kidney, heart and the testis (Table 3).

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3.6. Effect of aqueous extract of F. glumosa Del. on lipid parameters during treatment

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3.6.1. The rate on total cholesterol (TC) The rate of TC increased significantly from 177 ± 17.13 mg/dL in the control group compared to 265.8 ± 8.92 mg/dL in the HC + distilled water rats. Daily administration of the aqueous extract of the leaves of F. glumosa for 4 weeks resulted in a significant decrease (P b 0.05) of serum cholesterol compared to HC rats (Table 4). At the dose of 375 mg/kg, the total cholesterol dropped from 265.8 ± 17.13 mg/dL to 169.42 ± 19.44 mg/dL in HC + distilled water rats, a decrease of 56.88%. The same trend was observed in the HC treated with atorvastatin (1 mg/kg); cholesterol reduced from 265.8 ± 17.13 mg/dL to 172 ± 25.53 mg/dL in HC + distilled water rats, a decrease of 54.53%. A significant increase in total cholesterol (50.16%) was observed in HC animals treated with distilled water when compared to NC.

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3.6.2. On triglycerides (TG) After 4 weeks of induction of hypercholesterolemia, TG levels in 315 HC + distilled water rats increased significantly (P b 0.05) (69.89%) 316 compared to NC. It was observed in animals treated with the aqueous 317 Q66 extract of F. glumosa (225, 300, 375 mg/kg) dose-dependent decrease

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During treatment

After treatment

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First week (mL/rat/week)

Second week (mL/rat/week)

Third week (mL/rat/week)

Fourth week (mL/rat/week)

HC + distilled water HC + extract

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106.89 ± 1.41 187.04 ± 1.23*a 141.68 ± 2.11**a 188.65 ± 2.33**a 45.95 ± 2.22b 75.83 ± 1.17** 93.62 ± 1.66** 111.93 ± 1.11** 46.85 ± 3.72

127.47 ± 1.02 149.38 ± 2.13*a 211.12 ± 1.12**a 258.65 ± 1.67**a 64.75 ± 2.13 78.69 ± 1.17** 83.62 ± 1.66** 98.93 ± 1.11** 77.75 ± 2.76

141.89 ± 1.21 222.04 ± 1.14*a 281.82 ± 2.13**a 279.65 ± 2.37**a 74.15 ± 1.33 88.69 ± 1.17** 93.62 ± 1.66** 99.93 ± 1.11** 68.75 ± 3.78

136.23 ± 1.12 213.39 ± 0.75*a 305.83 ± 1.21**a 288.54 ± 1.23**a 66.64 ± 1.11 8910 ± .1.31** 96.38 ± 0.35** 99.55 ± 0.83** 86.69 ± 1.03

HC + atorvastatin

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3.6.6. Ratio of LDL/HDL-C and TC/HDL-c LDL/HDL-c ratio decreased in a dose-dependent manner when compared with HC + distilled water rats. Rats treated with the extract at a dose of 375 mg/kg showed a reduction of LDL/HDL-c ratio of 313.01% when compared to the HC + distilled water rats. This reduction was also observed in the TC/HDL-c and the value decreased from 8.25 ± 2.50 in HC + distilled water rats to 2.89 ± 2.68 in rats treated with the extract at a dose of 375 mg/kg, equivalent to a reduction of 185.46%. In NC, the TC/HDL-c had a non-significant increase (P N 0.05), whereas LDL/HDL-c also showed no significant differences (P N 0.05). HC rat treated with atorvastatin showed a 406.72% reduction in LDL/ HDL-c against 210.15% in TC/HDL-c rate (Table 4).

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3.6.5. The concentration of VLDL-c VLDL-c significantly (P b 0.05) decreased in treated HC rats. The administration of the aqueous extract of F. glumosa (225, 300, 375 mg/kg) daily for 4 weeks significantly decreased (P b 0.05) VLDL-c in serum compared to HC + distilled water. At the dose of 375 mg/kg, VLDL-c decreased from 39.28 ± 6.63 mg/dL in HC + distilled water to 24.85 ± 2.40 mg/dL in the rat treated with extract, leading to a decrease of 58.06% (Table 4). This decrease was also observed in HC rats treated with atorvastatin (Table 4).

Table 1 Variation of water consumption.

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3.6.4. The concentration of LDL-c The LDL-c in HC rats was significantly increased (P b 0.05) by 59.30%. This level was reduced in animals treated with the extract at the doses of 225, 300 and 375 mg/kg (Table 4). Animals treated with atorvastatin (1 mg/kg) also showed a significant reduction (149.99%) (P b 0.05) of LDL-c when compared to the HC + distilled water. In animals treated with the plant extract at the dose of 375 mg/kg, LDL-c decreased from 194.32 ± 07.77 mg/dL in HC rats to 86.03 ± 30.10 mg/dL meaning a decrease of 55.72% (Table 4).

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3.6.3. The concentration of HDL-c HDL-c significantly (P b 0.05) decreased in treated HC rats. HDL-c went from 73.6 ± 4.39 mg/dL in NC rats to 41.40 ± 0.83 mg/dL in HC + distilled water rats. The administration of the aqueous extract of F. glumosa (225, 300, 375 mg/kg) daily for 4 weeks caused a significant increase (P b 0.05) in serum HDL-c. This increase was also observed in HC + distilled water rats treated with atorvastatin (Table 4). At the dose of 375 mg/kg, HDL-c increased from 32.20 ± 3.56 mg/dL in HC + distilled water rats to 58.54 ± 7.23 mg/dL, an increase of 44.99%. A significant decrease (P b 0.05) of 8.05% in HDL-C was observed in NC (Table 4).

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293 Q65 3.5. Effect of the extract of F. glumosa Del. on the relative organ weights of

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Fig. 2. Variation of rat body weight during the 4 weeks of treatment. Values are means ± S.E.M., n = 5, *P b 0.05, significant difference compared to the first week. NC: normocholesterolemic rats, HC: hypercholesterolemic rat. *a: Significant decrease compared with the first week. *b: Significant increase compared with the first week.

of triglyceride. At the dose of 375 mg/kg, triglyceride levels decreased by 58.06% from 196.40 ± 33.18 mg/dL in HC + distilled water rats to 124.25 ± 22.03 mg/dL. HC rats treated with atorvastatin showed a decrease of 35.76% of triglyceride; while HC rats treated with distilled water showed an increase of 3.63% of triglyceride (Table 4).

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Values are means ± S.E.M., n = 5, *P b 0.05, **P b 0.01 significant difference compared to HC + distilled water. HC: hypercholesterolemic rat. a Significant difference compared to HC + distilled water. b Significant difference compared to animals previously treated with aqueous extract during the treatment at the same dose.

Please cite this article as: Fidele, N., et al., Hypolipidemic and anti-atherogenic effect of aqueous extract leaves of Ficus glumosa (Moraceae) in rats, Exp. Gerontol. (2014), http://dx.doi.org/10.1016/j.exger.2014.12.015

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N. Fidele et al. / Experimental Gerontology xxx (2014) xxx–xxx Table 2 Variation of the food intake during treatment.

t2:3 t2:4 t2:5 t2:6 t2:7 t2:8 t2:9 t2:10 t2:11 t2:12

During treatment

Drugs

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First week (g/rat/week)

Second week (g/rat/week)

Third week (g/rat/week)

Fourth week (g/rat/week)

HC + distilled water HC + extract

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46.97 ± 1.14% 56.80 ± 1.12% 54.23 ± 1.45% 40.22 ± 1.15%*a 35.89 ± 1.24%***a 54.11 ± 1.56% 58.12 ± 1.22%*b 48.22 ± 1.25%*b 40.21 ± 1.14%*b

54.86 ± 1.29% 54.47 ± 1.14% 52.39 ± 1.09%*a 35.13 ± 1.12%*a 37.23 ± 1.27%**a 57.43 ± 1.22%*b 57.76 ± 1.33%*b 50.27 ± 1.20%**b 43.89 ± 1.31%*b

62.59 ± 1.33% 50.23 ± 1.24%*a 50.22 ± 1.19%*a 28.62 ± 1.23%***a 40.13 ± 1.08%**a 60.48 ± 1.43%*b 62.86 ± 1.33%*b 53.34 ± 1.13%**b 48.82 ± 1.34%*b

68.88 ± 1.36% 47.47 ± 1.32%**a 45.39 ± 1.23%**a 26.54 ± 1.31%***a 43.43 ± 1.16%**a 65.43 ± 1.21%**b 67.32 ± 1.25%**b 61.88 ± 1.55%***b 51.78 ± 1.31%**b

HC + atorvastatin After treatment

HC + extract HC + atorvastatin

Values are means ± S.E.M., n = 5, *P b 0.05, **P b 0.01, ***P b 0.001. HC: hypercholesterolemic rat. a Significant difference compared to HC + distilled water. b Significant difference compared to animals previously treated with aqueous extract during the treatment at the same dose.

364

3.7. Effect of aqueous extract of F. glumosa Del. on lipid parameters after treatment

372

373 374

E

C

T

3.7.2. On triglycerides The concentration of TG in animals treated with the extract at the 375 dose of 225 mg/kg went from 184.33 ± 13.14 mg/dL with HC + distilled 376 Q69 water observed 4 weeks after treatment without the abovementioned 377 diet to 144.98 ± 2.08 mg/dL during the 4 weeks of observation after 378 treatment, a significant decrease (P b 0.05) of 21.34%. At the dose of 379 375 mg/kg the decrease was 34.22%, less than the rate observed in HC 380 rats treated with the extract of F. glumosa at the same dose (Table 5). 381

3.7.3. On the concentration of HDL-c Animals treated with the doses (225, 300 and 375 mg/kg) of the extract of F. glumosa and observed 4 weeks after the treatment showed 384 significant differences (P b 0.05) in the level of HDL-c compared to 385 Q70 HC + distilled water without the abovementioned diet and observed 386 4 weeks after treatment. At the dose of 375 mg/kg, a significant decrease 387 (P b 0.05) of 57.29% in HDL-c was noted (Table 5).

N C O

R

R

382 383

388

3.7.4. The concentration of LDL-c The level of LDL-c in treated animals and quarantined for 4 weeks decreased compared to HC + distilled water rats without the 391 Q71 abovementioned diet. The decrease was 32.31% at the dose of 225 392 mg/kg, 46.00% at the dose of 300 mg/kg and 54.26% at the dose of 375 393 mg/kg 4 weeks after treatment (Table 5).

t3:1 t3:2

U

389 390

Table 3 Variation of the relative organ weights of rats HC.

3.7.6. The ratio LDL/HDL-c and TC/HDL-c There were significant changes in LDL/HDL-c and TC/HDL-c ratios in rats, 4 weeks after treatment. LDL/HDL-c ratio decreased from 4.68 ± 1.22 in HC + distilled water rats without diet consisting to 1.36 ± 2.21 in the rats treated with F. glumosa at the dose of 375 mg/kg after treatment. The TC/HDL-c also decreased from 6.76 ± 1.04 in HC + distilled water rats without diet consisting to 2.76 ± 2.55 in the rats treated with F. glumosa at the dose of 375 mg/kg after treatment (Table 5).

400 401

R O

370 371

3.7.1. The concentration of total cholesterol Rats treated with increasing doses of 225, 300 and 375 mg/kg of F. glumosa extract for 4 weeks showed a significant decrease (P b 0.05) of total cholesterol compared to HC + distilled water. At the dose of 375 mg/kg, the total cholesterol went from 259.12 ± 3.31 mg/dL to 166.65 ± 19.75 mg/dL 4 weeks after treatment, a significant decrease (P b 0.05) of 35.68% (Table 5).

P

368 369

394

D

366 367

3.7.5. The concentration of VLDL-c HC rats treated with the doses (225, 300 and 375 mg/kg) of the aqueous extract of F. glumosa observed 4 weeks after treatment showed a significant decrease (P b 0.05) in the level of VLDL-c. A significant decrease concentration (P b 0.05) of 31.54% in VLDL-c was however noted at the dose of 375 mg/kg (Table 5).

E

365

O

t2:13 t2:14 Q7 t2:15

F

t2:1 t2:2

5

395 396 397 398 399

402 403 Q72 404 405 406 Q73 407

3.8. Effects of the aqueous extract of F. glumosa Del. on cholesterol, 408 triglycerides and lipid peroxidation (TBARS) levels 409 Cholesterol, triglycerides and lipid peroxidation (TBARS) levels in both liver and aorta showed a significant (P b 0.05; P b 0.01) increase in rats treated with aqueous extract of the leaves of F. glumosa at the doses of 225, 300 and 375 mg/kg compared to HC + distilled water rats. Fecal cholesterol also increases in rats treated with aqueous extract of the leaves of F. glumosa at the doses of 225, 300 and 375 mg/kg compared to HC + distilled water rats (Table 6).

410 411 412 413 414 415 416

3.9. Effects of the aqueous extract of F. glumosa on markers of oxidative 417 stress 418 The analysis of the activity of catalase in the liver homogenates and in hemolysates showed a significant decrease (P b 0.05) in concentration of liver catalase. There was also a significant decrease (P b 0.05) in the concentration of hydroperoxide in liver homogenates of rats treated with aqueous extract of the leaves of F. glumosa at the doses of 225, 300 and 375 mg/kg compared to HC rats. In addition, there was a significant decrease (P b 0.001) in plasma malondialdehyde in rats treated with the extract of F. glumosa (Table 7).

419 420 421 422 423 Q74 424 425 426

t3:3

Drugs

Doses (mg/kg)

Liver (g)

Kidneys (g)

Heart (g)

Testicles (g)

3.10. Histopathological studies

427

t3:4 Q8 t3:5 t3:6 t3:7 t3:8 t3:9

Control NC Control HC HC + extract

0 0 225 300 375 1

3.16 ± 0.26⁎ 3.01 ± 0.71 2.55 ± 0.15 2.44 ± 0.91 3.01 ± 0.25 2.90 ± 0.18

0.61 ± 0.25 0.58 ± 0.35 0.47 ± 0.07 0.62 ± 0.53 0.54 ± 0.04 0.50 ± 0.06

0.30 ± 0.10 0.31 ± 0.07 0.28 ± 0.03 0.27 ± 0.07 0.31 ± 0.04 0.28 ± 0.07

1.05 ± 0.45 1.08 ± 0.44 0.85 ± 0.49 1.51 ± 3.41 1.03 ± 0.57 0.97 ± 0.15

Histopathological studies of aorta from HC (Fig. 3C) showed extensive atherosclerotic plaques covering almost the whole upper part of the excised aorta, which was not the case with of the normocholesterolemic rats (NC) (Fig. 3A). Rats treated with aqueous extract of the leaves of F. glumosa at the doses of 300 and 375 mg/kg showed less extensive lesions compared to NC (Fig. 3B and D). Histopathological results of rat aorta treated with aqueous extract of the leaves of F. glumosa showed vulnerable plaques with many foam cells, inflammation and remarkable

428

t3:10 t3:11 t3:12

HC + atorvastatin

Values are means ± S.E.M., n = 5. NC: normocholesterolemic rats, HC: hypercholesterolemic rat. ⁎ P b 0.05, significant difference compared to control.

Please cite this article as: Fidele, N., et al., Hypolipidemic and anti-atherogenic effect of aqueous extract leaves of Ficus glumosa (Moraceae) in rats, Exp. Gerontol. (2014), http://dx.doi.org/10.1016/j.exger.2014.12.015

429 430 Q75 431 432 Q76 433 434 435

6 t4:1 t4:2

N. Fidele et al. / Experimental Gerontology xxx (2014) xxx–xxx

Table 4 Effect of aqueous extract of F. glumosa Del. on lipid parameters during treatment.

t4:3

Drugs

Doses (mg/kg)

TC (mg/dL)

TG (mg/dL)

VLDL-c (mg/dL)

HDL-c (mg/dL)

LDL-c (mg/dL)

LDL/HDL-c

TC/HDL-c

t4:4 t4:5 t4:6 t4:7 t4:8 t4:9

NC HC + distilled water HC + extract

0 0 225 300 375 1

177 ± 17.13 265.8 ± 8.92 203.53 ± 19.26⁎ 180.04 ± 7.46⁎⁎ 169.42 ± 19.44⁎⁎ 172 ± 25.53⁎⁎

115.6 ± 38.03 196.40 ± 33.18 147.52 ± 1.31⁎ 134.31 ± 5.43⁎⁎ 124.25 ± 22.03⁎⁎ 144.66 ± 4.50⁎⁎

23.12 ± 7.6 39.28 ± 6.63 29.50 ± 0.26⁎ 26.86 ± 1.08⁎⁎ 24.85 ± 2.40⁎⁎ 28.93 ± 0.9⁎⁎

73.6 ± 4.39 32.20 ± 3.56 50.02 ± 2.04⁎⁎ 54.39 ± 3.93⁎⁎ 58.54 ± 7.23⁎⁎ 65.00 ± 5.00⁎⁎

79.08 ± 17.87 194.32 ± 7.77 124.03 ± 21.21⁎ 98.78 ± 4.61⁎⁎ 86.03 ± 30.10⁎⁎ 77.73 ± 20.21⁎⁎

1.07 ± 4.07 6.03 ± 2.18 2.47 ± 10.39⁎⁎⁎ 1.81 ± 1.17⁎⁎⁎ 1.46 ± 0.41⁎⁎⁎ 1.19 ± 4.04⁎⁎⁎

2.40 ± 3.90 8.25 ± 2.50 4.06 ± 9.44⁎⁎⁎ 3.31 ± 1.89⁎⁎⁎ 2.89 ± 2.68⁎⁎⁎ 2.66 ± 5.10⁎⁎⁎

HC + atorvastatin

Values are means ± S.E.M., n = 5. NC: normocholesterolemic rats, HC: hypercholesterolemic rat. TC: total cholesterol, TG: triglyceride, VLDL-c: Very Low Density Lipoprotein Cholesterol, HDL-c: High Density Lipoprotein Cholesterol, LDL-c: Low Density Lipoprotein Cholesterol. ⁎ P b 0.05 significant difference compared to HC. ⁎⁎ P b 0.01 significant difference compared to HC. ⁎⁎⁎ P b 0.001 significant difference compared to HC.

436 437

440 441

intimal thickening (Fig. 3B and D). Histological examination of the heart did not reveal significant changes between NC and rats treated with aqueous extract of the leaves of F. glumosa (data not shown). Histological examination of the liver did not reveal significant changes between NC and rats treated with aqueous extract of the leaves of F. glumosa (data not shown).

442

4. Discussion

445 id. Age is a risk factor since it reflects the duration of exposure of individ446 Q78 ual to the other risk factors. Physiologically, during oldness, the arterial 447 448

t5:1 t5:2

Table 5 Effect of aqueous extract of F. glumosa Del. on lipid parameters after treatment.

460 461 462 Q82 463 464 465 466 Q83 467 468 469 470 471

C

E

R

458 459

R

456 457

O

454 455 Q81

C

453

N

451 Q80 452

U

449 Q79 450

O

R O

P

T

472

wall is extended to the endothelial functions and the muscular smooth cells are modified with the following consequences: increase arterial rigidity and the loss of compliance and vasomotricity. With age, the endothelial cells can undergo and at least focal destruction which decreases the function of endothelial. The man has a risk of atherosclerosis much higher than the woman. This protection is attached to the beneficial influence of the natural estrogens on the lipidic profile, the sensitivity to insulin and on the blood pressure. This protection disappears 10 to 15 years after the menopause and explains the late age of occurred of the complications of the atherosclerosis at the woman. The use of plants in the treatment of dyslipidemia is common worldwide. The oxidation of LDL cholesterol in the artery walls by oxygen free radicals leads to the production of oxidized LDL (OLDL) that attracts the macrophage scavenger of the immune system. These macrophages accumulate in the arterial wall after ingestion of OLDL particle concentration is associated with atheromatous arteriosclerosis plaques. The increase in total cholesterol and LDL cholesterol, lower HDL cholesterol and to a lesser extent, the increased triglycerides are risk factors for atherosclerosis, the main complications include coronary heart disease, ischemic stroke and occlusive arterial disease of the lower limbs (Kim et al., 2008. Alam et al., 2011). The results of this study showed a decrease in body weight of HC rats during the 4 weeks of treatment. Also many studies suggested that the loss of body weight in HC rats can be explained by an increase in the catabolism of lipids and proteins due to a deficiency in carbohydrates (Sathishsekar and Subramanian, 2005). These

D

443 Atherosclerosis lesions appear very precociously and increase with 444 Q77 age. They interest the levels chronologically aortic, coronary then carot-

results are the consequence of reduced food intake. Indeed, consumption of food declined in rats treated with aqueous extract of the leaves of F. glumosa. However, there was an increase in water consumption. This increase is due to the diuretic activity of the extract of F. glumosa (Ntchapda et al., 2014). This study showed that the administration of the aqueous extract of the leaves of F. glumosa to HC animals resulted in a significant decrease (P b 0.05) and dose dependent levels of cholesterol and plasma triglycerides. This was not the case with HC animals treated with distilled water. This can be explained by an increase in fecal excretion of bile acids and neutral sterols with the consequent reduction of hepatic cholesterol because of its use for the biosynthesis of these bile acids; the extract also slowed the rate of diffusion through the intestinal mucosa and reduced the absorption of cholesterol and triglycerides (Lin and Lin-Shiau, 2006; Alam et al., 2011). Also these polysaccharides could finally be an excellent substrate for bacteria in the large intestine and fermentation in the colon leading to the formation of volatile fatty acids entering the blood stream via the portal vein leading to a suppression of cholesterol synthesis (Dall'Agnol and Poser, 2000). The decrease of the concentration of cholesterol was also observed in HC animals treated with atorvastatin (1 mg/kg), a selective, competitive inhibitor of HMG-CoA reductase, the enzyme responsible for controlling the level of biotransformation HMG-CoA to mevalonate, a precursor of sterols and particularly of cholesterol (Istvan and Deisenhofer, 2001). Unlike many studies on improving lipid parameters of HC subjects, the aqueous extract of the leaves of F. glumosa resulted in a significant increase in HDL cholesterol (Jain et al., 2007; Nnodim et al., 2011). However, this concentration is generally inversely correlated with the risk of cardiovascular diseases both in humans and in experimental animals, and this increase reduces the development of atherosclerosis (Oh et al., 2006). In a recent study (Hopkins et al., 2001), the concentration and the size of particle LDL, with age and sex, were the principal risk for the development of ACD (atheroselerotic coronary disease) among patients. The frequency of the atherosclerosis increases with age because of the aging of the cell. Several other studies showed that low plasmatic cholesterol-HDL levels represent a risk factor independent of ACD in the population (Real et al., 2001). In fact, it is not only the plasmatic level of cholesterol-HDL but also transport with wrong way in general, which would play a significant role in the predisposition to

E

438 439

F

t4:10 t4:11 t4:12 t4:13 t4:14

t5:3

Drugs

Doses mg/kg

TC (mg/dL)

TG (mg/dL)

VLDL-c (mg/dL)

HDL-c (mg/dL)

LDL-c (mg/dL)

LDL/HDL-c

TC/HDL-c

t5:4 t5:5 t5:6 t5:7

HC + distilled water After treatment HC + extract

0 225 300 375

259.12 ± 3.31 201.34 ± 17.37⁎ 178.00 ± 4.72⁎ 166.65 ± 19.75⁎

184.33 ± 13.14 144.98 ± 2.08⁎ 128.48 ± 3.64⁎ 121.24 ± 22.42⁎

35.41 ± 2.76 28.99 ± 0.41⁎ 25.69 ± 0.72⁎ 24.24 ± 4.48⁎

38.33 ± 3.17 50.72 ± 3.39⁎ 55.34 ± 5.18⁎ 60.29 ± 7.72⁎

179.54 ± 3.89 121.53 ± 16.97⁎ 96.95 ± 0.06⁎ 82.11 ± 17.08⁎

4.68 ± 1.22 2.39 ± 5.00⁎ 1.75 ± 0.01⁎ 1.36 ± 2.21⁎

6.76 ± 1.04 3.96 ± 5.12⁎ 3.21 ± 0.91⁎ 2.76 ± 2.55⁎

t5:8 t5:9 t5:10

Values are means ± S.E.M., n = 5. HC: hypercholesterolemic rat. TC: total cholesterol, TG: triglyceride, VLDL-c: Very Low Density Lipoprotein Cholesterol, HDL-c: High Density Lipoprotein Cholesterol, LDL-c: Low Density Lipoprotein Cholesterol. ⁎ P b 0.05, significant difference compared to HC + distilled water.

Please cite this article as: Fidele, N., et al., Hypolipidemic and anti-atherogenic effect of aqueous extract leaves of Ficus glumosa (Moraceae) in rats, Exp. Gerontol. (2014), http://dx.doi.org/10.1016/j.exger.2014.12.015

473 474 475 476 Q84 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 Q85 493 494 495 496 497 Q86 498 499 500 501 502 503 504 Q87 505 506 507 508 509 510 Q88 511

N. Fidele et al. / Experimental Gerontology xxx (2014) xxx–xxx t6:1 t6:2

7

Table 6 Effect of aqueous extract of F. glumosa Del. on the level of total cholesterol, triglycerides, lipid peroxide (TBARS) of liver and aorta and on fecal cholesterol.

t6:3

Cholesterol total (mg/g)

Triglycerides (mg/g)

Lipid peroxide (TBARS) nmol/g

t6:4

Treatments

Doses mg/kg

Liver

Aorta

Liver

Aorta

Liver

Aorta

t6:5 t6:6 t6:7 t6:8 t6:9

NC HC + distilled water HC + extract

0 0 225 300 375

9.00 ± 0.28 22.35 ± 1.62 12.33 ± 1.15⁎⁎ 13.11 ± 1.03⁎⁎ 14.26 ± 1.26⁎⁎

7.11 ± 0.65 19.90 ± 1.04 13.12 ± 0.33⁎⁎ 13.01 ± 0.13⁎⁎ 13.23 ± 0.92⁎⁎

8.32 ± 0.47 19.64 ± 1.11 13.23 ± 1.12⁎⁎ 14.11 ± 0.92⁎⁎ 15.40 ± 1.12⁎⁎

7.80 ± 0.86 17.19 ± 1.30 13.12 ± 1.15⁎ 12.52 ± 1.04⁎ 14.12 ± 1.34⁎

12.20 ± 1.51 38.61 ± 3.51 23.26 ± 1.32⁎⁎ 23.79 ± 1.66⁎⁎ 24.29 ± 1.89⁎⁎

11.18 ± 1.27 37.05 ± 3.64 20.21 ± 2.42⁎⁎ 20.36 ± 2.22⁎⁎ 21.31 ± 2.31⁎⁎

Values are means ± S.E.M., n = 5. NC: normocholesterolemic rats, HC: hypercholesterolemic rat, TBARS: Thiobarbituric Acid Reactive Substances. ⁎ P b 0.05, significant difference compared to HC + distilled water. ⁎⁎ P b 0.01, significant difference compared to HC + distilled water.

play significant roles in the development of atherosclerosis, in vitro studies suggest that LDL is not atherogenic. The oxidative modification of LDL could indeed play a significant role in the pathogenesis of the atherosclerosis (Heinecke, 2003).At the HC, an increase in the concentration of LDL was observed. Thus, more LDL will be exposed longer with modifications, leading to the formation of high negative changed oxidable that contributes to the increase of potential atherogenic activities (Sanchez-Quesada et al., 1999). The mechanisms by which oxidized particles LDL (LDLox) succeed in the development of the atherosclerosis are many. Among the most studied, the inability of the macrophages to degrade LDLox is associated to the capacity of inhibition and cytotoxicity that LDLox exerts on these same macrophages and thus, with a larger recruitment of monocytes on the level of the area under-intimale (Witztum and Steinberg, 1991). Administration of F. glumosa extract caused significant decrease of cholesterol and triglycerides levels in the liver and aorta showing a hypolipidemic effect. Feeding animals with cholesterol (1%) caused a significant accumulation of cholesterol and triglycerides in the liver and aorta. These results are similar to those found by Mehta et al. (2003). The evaluation TC of the aorta in this study suggests the severity of atherosclerotic HC rats (Nielson et al., 1993) (Table 8). Reduction of cholesterol by aqueous extract of F. glumosa is probably associated with a decrease in intestinal absorption of cholesterol resulting in increased fecal excretion of lipids (Purohit and Vyas, 2006). Significant increase in the level of lipid peroxide (TBARS) in the liver and aorta indicates an increased oxidative stress that hyperlipidemic rats which results in the development and progression of atherosclerotic lesions in the aorta (Prasad, 2005). The extract also caused a decrease in lipid peroxide (TBARS) in these tissues indicating an antioxidant activity that reduces the oxidative stress. These results indicate that the aqueous extract of the leaves of F. glumosa contains active components that reduces serum lipid profile and lowers the risk of atherosclerosis in HC rats. Other results show that F. glumosa had an effect on markers of stress. These results indicate that the leaves of F. glumosa have the property to significantly reduce the levels of hydroperoxides in homogenates,

R O

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E

U

N C O

R

R

E

C

T

the ACD. The risk factor of most significant to atheroselerotic coronary disease (ACD) was connected with the metabolism of lipoproteins. 514 The lipoproteins consist of center hydrophobic lipids (triglycerides 515 and cholesterol esters) surrounded by phospholipids and free cholester516 ol. The control of the cholesterol concentrations in cells also required 517 the HDL They are synthesized on the liver and are secreted in blood cir518 culation by exocytose. They have the capacity to collect the free choles519 terol of peripheral tissues through ATP binding cassette (Attie et al., 520 2001) to accomplish transport to the liver where it is excreted with 521 Q89 bile. The HDLs produced on the intestine or liver or by the lipolysis of 522 the lipoproteins rich in TG induce a cholesterol and phospholipid flow 523 on the level of the cellular membranes. The phospholipid transfer pro524 tein stimulates this process. When HDL is charged with free cholesterol, 525 it is transported to the liver so that cholesterol can be extracted by the 526 scavenger receptor, class B, type I (Acton et al., 1996). 527 There was a decrease in LDL-c/HDL-c and TC/HDL-c ratios. This de528 crease and LDL-c/HDL-c and TC/HDL-c ratios in the animals treated 529 with F. glumosa confirmed the anti-arteriosclerotic activity of the extract 530 and its importance in reducing the cardiovascular risk factors, as report531 ed by Bajaj et al. (1997). Therefore, the extract could possess anti532 arteriosclerotic effect for the doses considered. The extract of 533 F. glumosa caused a significant decrease in serum TC, LDL-c and VLDL534 c levels 4 weeks after treatment suggesting important modulatory influ535 ence on the metabolism of HDL-c. Higher HDL-c observed 4 weeks after 536 in rats treated with the extract could be a consequence of a higher pro537 portion of HDL-c which reduced atherogenic risk under the overturned 538 carriage increased cholesterol peripheral organs to the liver (Kinosian 539 et al., 1994; Hermansen et al., 2003). Higher levels of serum triglycer540 ides are often considered as independent risk factors for cardiovascular 541 diseases. Significant decrease in TG observed in rats treated with the ex542 tract four weeks after treatment supports the protective activity of 543 F. glumosa against cardiovascular disease. 544 Recently, a great interest was granted to the contribution of oxida545 tive stress in vascular diseases in general and the atherosclerosis in par546 ticular (Keaney et al., 2003). Although the high cholesterol-LDL levels

O

512 513

t7:1 t7:2

44.94 ± 2.63 81.14 ± 4.16 106.43 ± 3.83⁎⁎ 105.98 ± 3.44⁎⁎ 106.28 ± 3.71⁎⁎

F

t6:10 t6:11 t6:12

Fecal cholesterol (mg/g)

Table 7 Effects of the aqueous extract of F. glumosa on markers of oxidative stress.

t7:3

Homogenate

Plasma

Hemolysates

t7:4

Treatments

MDA (μM/100 g of tissue)

ROOH (μM/100 g of tissue)

CAT (mMH2O2/min/g of protein)

Protein (g/100 g of tissue)

MDA (μM/L)

ROOH (μM/L)

CAT (mMH2O2/min/g of protein)

Protein (g/L)

t7:5 t7:6 t7:7 t7:8

Control HC 225 mg/kg 300 mg/kg 375 mg/kg

9.96 ± 1.32 9.18 ± 2.07 8.94 ± 3.96⁎ 7.80 ± 0.86⁎

1.37 ± 0.09 0.93 ± 0.11⁎⁎ 0.87 ± 0.18⁎⁎ 0.76 ± 0.25⁎⁎⁎

0.02 ± 0.02 0.04 ± 0.01 0.05 ± 0.01 0.17 ± 0.06⁎

56.04 ± 0.52 51.24 ± 4.30⁎ 47.69 ± 4.26⁎⁎ 30.29 ± 5.18⁎⁎

20.96 ± 1.50 15.54 ± 0.66⁎⁎⁎ 12.33 ± 0.92⁎⁎⁎ 8.10 ± 3.12⁎⁎⁎

0.03 ± 0.01 0.04 ± 0.00 0.05 ± 0.01 0.06 ± 0.01⁎

0.01 ± 0.00 0.11 ± 0.04⁎ 0.12 ± 0.07⁎ 0.13 ± 0.03⁎⁎⁎

46.81 ± 8.01 47.82 ± 1.99 52.20 ± 9.8⁎ 54.81 ± 0.29⁎

t7:9 t7:10 t7:11 Q9 t7:12 t7:13

Values are means ± S.E.M., n = 5. HC: hypercholesterolemic rat. MDA: malondialdehyde, ROOH: hydroperoxyde, CAT: catalase. ⁎ P b 0.05, significant difference compared to HC. ⁎⁎ P b 0.01, significant difference compared to HC. ⁎⁎⁎ P b 0.001, significant difference compared to HC.

Please cite this article as: Fidele, N., et al., Hypolipidemic and anti-atherogenic effect of aqueous extract leaves of Ficus glumosa (Moraceae) in rats, Exp. Gerontol. (2014), http://dx.doi.org/10.1016/j.exger.2014.12.015

547 548 549 550 551 552 553 554 555 Q90 556 557 558 559 560 561 562 563 564 565 566 567 Q91 568 569 570 571 572 573 574 Q92 575 576 577 578 579 580 581

N. Fidele et al. / Experimental Gerontology xxx (2014) xxx–xxx

T

Q3

Fig. 3. Aortic intima cross-section in the studied groups. Arrows indicate atherosclerotic plaque formations. As depicted, the HC (C) (×400) exhibited extensive atherosclerotic plaques covering almost the whole upper part of the excised aorta. A (×400) (rat normocholesterolemic) did not show a vulnerable plaque with many foam cells. C (rat treated with cholesterol) shows a vulnerable plaque with many foam cells, inflammation and remarkable intimal thickening. Rat treated with F. glumosa at the doses 300 and 375 mg/kg (B and D) (×400) exhibited less extensive lesions and least lesion formation compared to NC (A). B (rat treated with F. glumosa at the dose 300 mg/kg) and D (rat treated with F. glumosa at the dose 375 mg/kg) show the above described lesion, but less extensive (intima/media ratio thickness is less than in A).

C

Q2

E

D

P

R O

O

F

8

582

E

the levels of malondialdehyde in plasma and the activity of catalase in homogenates and hemolysates. In reality, the assessment of lipid peroxidation in tissue involves the determination of certain stress markers 585 such as malondialdehyde and hydroperoxides which are byproducts de586 rived from the lipid peroxidation and certain enzymes such as catalase, 587 Q93 superoxide dismutase, and glutathione peroxidase that protect tissues 588 against the oxygen free radicals that damage membranes and biological 589 structures. More specifically, the catalase was responsible for the detox590 ification of significant quantities of hydrogen peroxide (H2O2) (Gupta 591 et al., 2008). 592 Since the treatment of animals by fractions causes a decrease in cat593 alase activity and a reduction of hydroperoxide and malondialdehyde 594 levels, these fractions could have the ability to block lipid peroxidation 595 and thus strengthen the defense system in vivo. Induced hyperlipidemia 596 often leads to increased production of oxygen free radicals, causing lipid 597 peroxidation (Oh et al., 2006). 598 Macroscopically plaque formation in rats treated with aqueous ex599 tract of the leaves of F. glumosa was less extensive compared to Control

U

N

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R

583 584

t8:1 t8:2

Table 8 Morphometric analysis on rat aortas. 2

t8:3

Group

Thickness (mm)

Surface area (mm /mm)

t8:4 t8:5 t8:6 t8:7

NC HC Extract 300 mg/kg Extract 375 mg/kg

0.163 ± 0.132 0.798 ± 0.178 0.479 ± 0.312a 0.367 ± 0.211a

0.245 ± 0.145 0.936 ± 0.168 0.785 ± 0.232b 0.665 ± 0.221b

t8:8 Q10 Values are means ± S.E.M., n = 10, P b 0.05, t8:9 NC: normocholesterolemic rats, HC: hypercholesterolemic rat. a t8:10 Significant difference for P b 0.05, compared to HC. b t8:11 Significant difference for P b 0.05, compared to control.

HC. The findings of this study are similar to other reports which observed that despite the increase of serum cholesterol after the administration of an extract, the pathological results of the aortic specimens may show an improvement of the progress of atherosclerosis (Prasad, 2007). This is in agreement with this study, which noted a significant beneficial effect of aqueous extract of the leaves of F. glumosa on the development of aortic atherosclerosis. Rats treated with aqueous extract of the leaves of F. glumosa showed significant inhibition of atherosclerotic plaques regarding both their thickness and extent within the aortic lumen. This may be attributed probably to major components, such as gallic acid and catechin that possess inhibitory activities as already indicated by Fassbender et al. (2008) and Kavantzas et al. (2006). The hypolipidemic and anti-atherogenic effects presented by the aqueous extract leaves of F. glumosa could be explained by the presence of naturally bioactive compounds. Hypolipidemic and anti-atherogenic properties of aqueous extract of the leaves of F. glumosa could be due to other active principles such as flavonoids, saponins and organic acids (Abed, and Benmrabet, 1981), the cumulative effects of several substances in the extract and/or to secondary active(s) metabolite(s) (Tanira et al., 1988). The present study of the aqueous extract of fresh leaves of F. glumosa (Moraceae) highlighted its hypolipidemic and anti-atherosclerotic effects. This could explain the use of the plant by traditional healers in the treatment of hypertension, cardiovascular diseases and diabetes.

Conflict of interests

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The authors declare that there is no conflict of interests regarding 625 the publication of this paper. 626

Please cite this article as: Fidele, N., et al., Hypolipidemic and anti-atherogenic effect of aqueous extract leaves of Ficus glumosa (Moraceae) in rats, Exp. Gerontol. (2014), http://dx.doi.org/10.1016/j.exger.2014.12.015

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The authors thank Prof Tchiégang Clergé of Food Biochemistry and Technology Laboratory, ENSAI, University of Ngaoundéré (Cameroon) and ALLARAMADJI Ndohortongar of N'Djaména hospital Le Bon Samaritain for their assistance in this project. The authors also thank the Laboratory of the Medicinal Plants, Health and Galenic Formulation of the Department of Biological Sciences.

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