Background
Hyperlipidemia and oxidative stress are major risk factors for atherosclerosis, and all three are among the most important risk factors for cardiovascular diseases and conditions [
1,
2]. The cardiovascular diseases constitute one of the absolutely largest public health problem in the world. According to the World Health Organization statistics [
3], they are responsible of more than 17 million deaths annually. The cardiovascular diseases are associated to several cardio-metabolic risk factors such as hypercholesterolemia, diabetes, high blood pressure, obesity and sedentarity [
4]. Dyslipidemia is a very frequent metabolic disorder which is characterized by an increase of the rates of triglycerides (TG), total cholesterol (CT), cholesterol of the low density lipoprotein (LDL-c) and a reduction of the cholesterol high density lipoprotein (HDL-c) [
4]. A huge body of population based and experimental evidence shows that high levels of plasma low density lipoprotein (LDL-c) cholesterol and total cholesterol considerably increase the risk for developing atherosclerosis and associated arterial hypertension [
5,
6]. Other changes in lipid parameters associated with atherosclerosis include decreases in high density lipoprotein (HDL-c) cholesterol and increases in triglycerides. It is well documented that hypercholesterolemia contributes to the development of the atherosclerosis, with hypertension and the renal failure [
7]. The assumption of responsibility of the hypercholesterolemia in reduction of mortality as well as which has occurred of the events cardio/neurovasculaires, this via the reduction in the blood concentration of cholesterol related to the lipoproteins of low density (LDL-c). The low-density lipoprotein cholesterol (LDL-c) reduction is correlated with the magnitude of cardiovascular risks reduction.
For many decades medicinal plants have been used to prevent or treat various diseases. They are used throughout the world, for their hypoglycemia, hypolipidemia or antioxidant activities [
8,
9].
Cassia occidentalis Linn. (Caesalpiniaceae) is a diffuse shrub (usually annual), with loosely spreading branches (60–150 cm long), and can grow up to an altitude of 1500 m [
10]. Different parts of this plant have been reported to possess anti-inflammatory, antihepatotoxic [
11], antibacterial [
12], antiplasmodial [
13] and antidiabetic [
14] activities. They possess purgative, tonic, febrifugal, expectorant, and diuretic properties. The plant is also used to cure sore eyes, hematuria, rheumatism, typhoid, asthma, hemoglobin disorders and it is also reported to cure leprosy. A wide range of chemical constituents isolated from
C. occidentalis include sennoside, anthraquinone glycoside [
15], fatty oils, flavonoid glycosides, galactomannan, polysaccharides, and tannins [
16]. Although leaves aqueous extract of
C. occidentalis were reported to possess diuretic effects [
17], no data on the effect of this medicinal plant on cardiovascular diseases and conditions are available. The present study therefore aimed at evaluating the anti-dyslipidemic, antioxidant, and anti-atherogenic effects of
C. occidentalis leaf aqueous extracts and potential mechanisms driving its putative protective and therapeutic effects.
Methods
Plant material
Fresh leaves of
C. occidentalis used in this study were harvested in Mora 60 Km from Maroua, the largest city in the Far North Region, Cameroon in July 2013. They were identified by experts of the National Herbarium of Cameroon and a sample was deposited (specimen N
0 21057/SFR/CAM). Leaves of
C. occidentalis were extracted as described previously [
17].
Preparation of leave aqueous extract
Fresh leaves of C. occidentalis were soaked in distilled water (1000 g for 1 L at room temperature) for 12 h. The macerate was filtered through Whatman filter paper No 3, and the filtrate concentrated in a rotary evaporator at 40 °C for 24 h. This process was repeated until an oily paste extract was obtained (130 g), which represented the concentrated crude extract of C. occidentalis leaves. The extract was stored at −20 °C until use. The solution of C. occidentalis extract with the highest concentration tested was prepared by dissolving 800 mg of the concentrated crude extract obtained previously in 10 ml of distilled water (80 mg/mL concentration). The other solutions used in the study were 4:5, 3:5, 2:5, and 1:5 dilutions of this solution in distilled water. Solutions were given per os in a volume of 5 ml/kg body weight, thus, the increasing doses of aqueous extract of C. occidentalis tested were 80, 160, 240, 320, and 400 mg/kg.
Preliminary qualitative phytochemical analysis
In order to identify the chemical structure of the compounds responsible for the antioxidant and anti-atherosclerogenic activity, preliminary tests of the phytochemical study were conducted following the procedures described by Trease and Evans [
18]. Briefly, Essential oils from the aqueous extract of
C. occidentalis were extracted with hexane. These extracts were then stitched onto plates of thin layer chromatography on silica, the first disclosure was obtained by ultraviolet radiation (254 nm and 365 nm) and then with vanillin. Analytical tests for the identification of different families of metabolites in crude extracts of the leaves were performed at the national Institute of Medicinal Plants for Medicinal research (IMPM, Cameroon).
Animals
Sixty normo-cholesterolemic (NC) male Wistar rats (178.35 ± 1.46 g) were purchased from Yaounde (Cameroon) Pasteur Institute and acclimated to the Laboratory of Medicinal Plants, Health and Galenic Formulation of the Department of Biological Sciences, University of Ngaoundere (Cameroon). Animals were housed under controlled room temperature (24 ± 2 °C) and had ad libitum access to food [National Veterinary Laboratory (LANAVET), Garoua, Cameroon] and tap water. Animals were monitored for signs of general toxicity, under the supervision of a veterinarian. The number of animal per group approved in the experiments by the institutional committee of ethics was five. All experimental procedures were approved by the institutional Ethical committee of Department of Biological Science of the university of Ngaoundéré (ECDBSUN 15/01/2015/UN/FS/DSB).
Experimental procedures
Normo-cholesterolemic (NC) (60 rats) were divided into 6 groups of 10 rats. Five 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 [
19,
20]. 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); carbohydrates (35 ± 2.3) [
21]. The plant extract was administered to animals at the increasing dose of 240, 320 and 400 mg/kg. After 4 weeks of treatment 5 rats out of 10 were sacrificed, blood samples, aorta, liver, and fresh faecal were collected and processed for biochemical tests. 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 first group to confirm the anti-atherogenic properties of the leaves extract. Blood collected in heparinized tubes, were centrifuged at 3000 rev/min for 10 min; the supernatant (plasma) was used for the enzymatic determination of total cholesterol, HDL-c and triglycerides and malondialdehyde. Blood pellet was used in the preparation of hemolysates while the portion of the liver collected was used to prepare liver homogenates for the dosage of catalase, hydroperoxides and proteins. The experiments were conducted under the same conditions with Atorvastatin
® (1 mg/kg), as pharmacological reference substance.
Body temperature monitoring
Body temperature of treated rats was monitored daily 5 h after treatment using a rat rectal thermometer. It was inserted at a distance of approximately 2 mm in the anus.
Statistical analysis
Data obtained from the different experimental groups were compared by one-way ANOVA followed by LSD test for post hoc analysis, using Origin software (Origin Lab, Northampton, MA, USA). Test groups were compared to normal, disease, and positive control groups. Differences with P < 0.05 were considered significant. Data are presented as mean ± SEM.
Discussion
The aqueous extract of
C. occidentalis reduced dose dependently and significantly the triglyceride and cholesterol levels in the rats. Ajayi
et al. [
22], reported that drugs with anticholesteremic properties are also antioxidant. This suggests aqueous extract of
C. occidentalis may have antioxidant properties. This property could justify the use of the maceration of
C. occidentalis in traditional medicine to treat hypertension and to reduce the triglyceride and cholesterol levels in blood [
23,
24]. In our experiment, animal body weights were significantly increased compared to the controls. This increase could have resulted in the increase in the food and water intakes of the rats. Physiologically, the increase in the appetite could be due to orexine, the stimulative hormone of appetite (Balkan, 2002). Animals fed with a feeding diet rich in cholesterol, have seen their body weights increased significantly, thus developed obesity [
25,
26]. This increase in body weight is due to the increase of fat tissue deposit much more on the level of the hip. We also noted that body weight in rat fed with HC + dH
2O is significantly increasing in rat fed with NC before treatment in Fig.
1. This result means that the well nourished animals can take weight if they are in good health. But what we noted, it is with the difference of 1% of cholesterol giving in HC + dH
2O, the Body weight in rat fed with HC + dH
2O is significantly increasing than NC rat. We know that cholesterol induces the hormone synthesis such as cortisol, the aldosteron, the testosteron and the oestrogens which are the sex hormones may be the increasing in cholesterol in the HC + dH
2O rats would have contributed to increase the rate of these hormones and induce significantly increasing of body weight in rat fed with HC + dH
2O.
The treatment with the aqueous extract of
C. occidentalis, in the rat fed with a diet rich in cholesterol and triglyceride compared with the untreated rat and normal rats, induced a reduction in the contents of VLDL-c. This reduction of cholesterol is found on the level of the TC, the LDL-c and Triglycerides. These results are similar with those of several works completed with other plant extracts, such as the aqueous extract of
Dunaliella salina [
27] and the ethanolic extract of
Crataegus pinnatifida [
28], in rats subjected to a feeding regime enriched with lipids. Considering that abnormal lipid profiles constituting the hallmark of HC-induced metabolic syndrome were also prevented in the liver by the extract concomitantly with a marked increase in total cholesterol excreted, we hypothesized that hypolipidemic activity of the extract may be mediated by reducing or inhibiting intestinal cholesterol absorption and increasing reverse cholesterol transport, as observed with agents inducing comparable hypolipidemic effects together with antioxidant effects such as Ezetimibe [
29,
30] and bile acid sequestering cholestyramine [
22,
31]. This reduction did not reach the normal rate after 4 weeks of treatment and the 4 weeks without treatment what allow us to say that the treatment during 4 weeks could be insufficient, but we noticed a reduction in the rate of VLDL-c during the 4 weeks without treatment, it could be that the extract of
C. occidentalis continued to react and to show the effectiveness of the aqueous extract leaves. This effective action could be explained by the presence of the various chemical families present in the aqueous extract leaves. we limited our research to the treatment in 4 weeks without any time to reassure our self if after the treatment the formation of the atherom could continue and that the endothelium could find its integrity. The interest of this study consisted in checking that after 4 weeks of treatment, the extract would always act.the aim would be to highlight vasodilators which can reconstitute the level of integrity of the endothelium and the production and the diffusion of the oxide nitrite. The ratios TC/HDL and HDL/LDL are indexes of the coronary risk [
32]. The ratios of atherogenicity TC/HDL and HDL/LDL of dyslipidemic rats treated with the extract of
C. occidentalis were significantly reduced. These results reflect a lipidic profile antiatherogenic, and let suggest a protective effect of the extract with respect to the hypercholesterolemy induced by the mode enriched out of cholesterol. At the rats hyperlipidemic, saponins, steroid; especially saponins derived from the spirostanol, seem to be responsible for the reduction in total cholesterol. It was noted in all the cases a reduction in LDL-c and sometimes an increase in HDL-c. Saponins would act by formation of a complex with cholesterol or would have a direct effect on the metabolism of cholesterol. Several possible mechanisms of exercise-induced atheroprotective effects have been proposed such as increased HDL-c, decreased TC, and decreased oxidized LDL-c levels [
33]. In the present study, endurance exercise and/or switching from the high fat to the normal diet improved lipid profiles by lowering the atherogenic plasma levels of total and LDL-c. However, the marked change in lipid profile observed concerned the plasma levels of the anti-atherogenic HDL-c. They were significantly increased in the exercise trained groups of rat (independently of the diet used) and not in the sedentary rats which were switched from the high fat to the control diet. Consequently, the atherogenic index was less in the exercise trained than in the rats with modified diet. Concerning plasma triglycerides, studies have also shown that they are strongly correlated with the prevalence and incidence of metabolic syndrome and cardiovascular diseases [
34].
Many experimental studies showed the effectiveness of the medicinal plants or their extracts in the improvement of the activities of the enzymes implied in the metabolism of cholesterol [
35]. Li et al. [
36] showed that the increase in the activity of the Lecithin: Cholesterol Acyltransferase Activity (LCAT) and probably the reduction in that of hydroxy-methyl-glutary-coenzyme A reductase (HMG-CoA reductase), both ensuring the homeostasis of cholesterol, could be regarded as persons in charge for the reduction in the cholesterolemy, in the rat subjected to a feeding regime enriched with cholesterol treated with 5% by the extract by
Coriandrum sativum during 75 days [
36]. Moreover, Sudhop et al. [
37] noted a rise in the activity of the LCAT and an inhibition of HMG-CoA reductase, in the rat made hypercholesterolemic treated with 500 mg/kg of an extract leaves of
Symplocos cochinchinensis, during 28 days [
38]. For this reason
C. occidentalis could have an effect in the increase of the activity of the LCAT and in the reduction of hydroxy-methyl-glutary-coenzyme A reductase. it is very difficult to make a difference between efficacy of
C. occidentalis with the efficacy of other plant extract without knowledge in the content of each plant extract. For the moment we explore the ways that
C. occidentalis act and tried to explain the effect of the extract.
Our results showed in this model of hypercholesterolemy that several biomarkers of the oxydative stress faded in the rats subjected to the diet rich in cholesterol. The reduced glutathion is the most abundant endogenous antioxydant which interacts with activated oxygenated species, thus preventing the oxidation of the organic substrates (proteins, ADN, fatty acids). It is used as substrate of the glutathion peroxidase. The glutathion is also a trapper of radicals superoxydes and it protects the thiol groups from proteins against oxidation [
39]. Our results showed a reduction in glutathione levels in the blood of the hypercholesterolemic rats. The determination of the specific activity of the superoxyde dismutase (SOD), enzyme which catalyses the dismutation of the anion superoxyde (O
2
−) out of water and hydrogen peroxide [
40]. Faraci and Didion [
40] revealed a decrease of the activity of the SOD in blood of the hypercholesterolemic rats. In response to the oxydative stress, the SOD is controlled in two different ways. In the event of moderate oxydative stress one observes a sur-expression of the SOD. If the oxydative stress persists the SOD is destroyed and its expression decreases. Paradoxically, an excessive concentration of the SOD can be dangerous because, in this case, it is the base of a hydrogen peroxide overproduction [
41], would be secondary with the increase in the production of O
2
− [
42]., Malondialdehyde (MDA) is used as index of the lipidic peroxidation resulting from the reaction of the active species oxygenated with the membrane fatty acids [
43]. In this study, the levels of MDA in blood was significantly increased in hypercholesterolemic rats. In treated group, the aqueous extract of
C. occidentalis substantially prevented the decrease of GSH and the increase of MDA levels. The treatment also reduced the activity of the SOD. The polyphenols present in the aqueous extract of
C. occidentalis [
44], could also explain the antioxidant activity of this aqueous extract.
Gupta et al. [
45], revealed that; treatment of rats with aqueous extract of
Annona squamosa induced a decrease of VLDL-c, LDL, TG levels at the serum and liver and activity of HMG-CoA reductase. Moreover, this extract increased the rate of HDL-c, the activity of the LCAT and the synthesis of the biliary acids at the hepatic level, which involves a rise in the faecal excretion of cholesterol in rat subjected to a feeding regime enriched with cholesterol (2%) during 75 days. In the current study, it was observed a decrease in the TC, LDL-c, TG levels after treatment with
C. occidentalis aqueous extract. However, the reduction of those parameters did not reach the normal and could be explained by the short treatment period (4 weeks).
The results of this study showed a significant increase in the faecal excretion of cholesterol, in the rats dyslipidemic or hyperlipidemic treated with aqueous extract of
C. occidentalis compared with the untreated rats, which suggest an increase in the activity of the 7 α-hydroxylase, enzyme implicated in the transformation of cholesterol into biliary acids. These results are similar with those obtained with hypercholesterolemic rats, treated with aqueous extract of
Globularia alpum, which observed an increase in the synthesis of the biliary acids at the hepatic level and an increase in faecal cholesterol [
46].
The aqueous extract of C. occidentalis has an hypocholesterolemia effect and could then act effectively against the transport of cholesterol by the increase in the activity of the LCAT, thus resulting in enrichment of the HDL-c out of cholesterol esters.