Hypercholesterolemia is a metabolic disorder characterized by a high concentration of cholesterol in the blood.
Hypercholesterolemia is a metabolic disorder characterized by an increase in the concentration of plasma cholesterol (above 200 mg/dL); it is considered the primary risk factor for developing cardiovascular disease (atherosclerosis) [
Ezetimibe is another popular hypolipidemic agent. This drug inhibits the intestinal absorption of cholesterol from dietary and biliary sources by impeding the transport of cholesterol across the intestinal wall [
Due to side effects of these drugs other therapeutic agents have been sought. Among them are the so-called medicinal plants. For example, the Malaysian medicinal plant
In the Mexican traditional medicine, there are two species,
Considering the above, we decide to evaluate the effectiveness of a hydroalcoholic extract of
Male CD1 mice (30 ± 2 g, 8–10 weeks old) were used (Harlan Laboratories, Mexico). Animals were housed in plastic cages in a temperature-controlled room with 12 : 12 h light-dark cycles and provided ad libitum with access to food and water. After one week of acclimatization, the animals were randomly divided into 7 experimental treatments groups (
Experimental groups.
Number | Experimental group | Treatment |
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(1) | Control | Standard diet (Laboratory Rodent Diet 5001) ad libitum |
(2) | Hypercholesterolemia | Hypercholesterolemic diet (1% cholesterol, 0.5% cholic acid) ad libitum |
(3) | Ezetimibe | Ezetimibe 10 mg/kg and hypercholesterolemic diet ad libitum (Merck/Schering-Plough Pharmaceuticals, USA) |
(4) | Hydroalcoholic extract | Extract of |
(5) | Hydroalcoholic extract | Extract of |
(6) | Hexa-O-acetyl-D-mannitol | Mannitol hexaacetate at a dose of 10 mg/kg and hypercholesterolemic diet ad libitum |
(7) | D-Mannitol | D-Mannitol at a dose of 10 mg/kg and hypercholesterolemic diet ad libitum |
Different treatments during the evaluation of
The other six groups were feeding with a diet enriched with 1% cholesterol and 0.5% cholic acid (hypercholesterolemic/lithogenic diet, Harlan Laboratories TD 03451, 1% cholesterol, 0.5% sodium cholate). The mice had free access to this diet for four weeks. After this period, we found that the mice were already hypercholesterolemic, compared with healthy mice. At this point, the different treatments were administered orally, every day at 15:00 h, for the rest of the four weeks along with the hypercholesterolemic diet. Animal care and procedures were conducted according to the guidelines approved by Norma Oficial Mexicana (NOM-062-ZOO-1999) and were subjected to experimental protocols approved by the Comité Interno Ético para el Uso y Cuidado de los Animales de Laboratorio (15-CIECUAL, February 2017), Instituto de Ciencias de la Salud, Universidad Autónoma del Estado de Hidalgo.
The chronic toxicity test was performed using female CD1 mice (30 ± 2 g in weight and approximate age of 8–10 weeks), which were given orally the alcoholic extract (ECHE), D-mannitol, or hexa-O-acetyl-D-mannitol under the same schedule as the hypercholesterolemia study.
After eight weeks, the animals of all groups were fasted, weighed, and anesthetized (sodium pentobarbital 70 mg/kg). The blood samples were drawn from the retroorbital vein; then the mice were sacrificed under anesthesia. Serum was obtained from blood samples by centrifugation, and the different biochemical parameters were measured. Biochemical analysis was carried into a Randox Daytona automate using a commercial kit (total cholesterol CH201, 6 × 100 mL; HDL cholesterol-direct clearance method, CH1383,
We reported total cholesterol, HDL, and non-HDL cholesterol concentrations (Enzymatic Methods, Accelab, Mexico). In chronic toxicity test, we quantified the concentration of aspartate transaminase (AST) and alanine transaminase (ALT) liver enzymes, as well as the total cholesterol concentration (Accelab, Mexico).
Livers were entirely randomly selected and fixed in 4% neutral buffered formaldehyde embedded in paraffin, and 5
The first 5 centimeters of the small intestine corresponding to the jejunum were used. We analyze this inner region because it is the site where cholesterol is absorbed. The intestinal tissue was homogenized by mixing it with a protease inhibitor, and the cells were subsequently lysed and centrifuged. The quantified protein concentration was performed by the Lowry method.
The expression of major intestinal cholesterol transporters in mice, Abcg5 and Abcg8, was analyzed by the standard western blot method. We perform 4 replicates of these molecular analyses. Antibodies were purchased from Santa Cruz Biotechnology, Inc., México: ABCG5 (H-300): sc-25796, rabbit polyclonal; ABCG8 (H-300): sc-30111, rabbit polyclonal and b-actin (C4); HRP: sc-47778, mouse monoclonal. We used 12% SDS-polyacrylamide gel and proteins were transferred to PVDF membranes. Membranes were incubated in blocking buffer. Primary antibodies were diluted as 1 : 100 in blocking buffer and incubated overnight at 4°C. Secondary antibody was diluted as 1 : 2000 and coupled with horseradish peroxidase. Proteins were visualized by chemiluminescence and our results were normalized with
NMR spectra were acquired in CDCl3 at room temperature on a Bruker-Fourier (A300 MHz) spectrometer and the chemical shifts are given as
Aerial parts of
The air-dried and ground aerial parts of
Treatment of ECHE (4 g) with MeOH (50 mL) afforded a solid and a solution. The solution was filtered and mixed with activated carbon and subsequently gently heated. The solution was filtered using a Büchner funnel with celite pad, and it was evaporated under reduced pressure to give a solid which was purified by crystallization method with MeOH to give 650 mg of D-mannitol. The identity of this metabolite was achieved by comparison of its spectroscopic and spectrometric data with those reported in the literature [
The residue insoluble in MeOH (510 mg) was dissolved in dry pyridine (3 mL) and Ac2O (3 mL) was added. The reaction mixture was heated at 60°C during 4 h. Usual work-up provided a residue which was chromatographed on a silica gel open column using
The statistical analyses were performed using a Sigma Plot program. Data were analyzed using one-way analysis of variance (one-way ANOVA) and Tukey’s post hoc test. Each point in the tables and figures represents the mean ± standard error.
Treatment of ECHE with methanol yielded both a methanol solution and a methanol-insoluble solid; the latter phase was the most abundant. D-Mannitol was isolated from the methanol soluble fraction by evaporation of the solvent under reduced pressure and treatment of the residue with methanol. The methanol-insoluble solid (510 mg) was dissolved in pyridine and treated with acetic anhydride to yield 670 mg of acetylation product. The chromatographic separation of this product yielded 135 mg of hexa-O-acetyl-D-mannitol. The identification of the D-mannitol and its acetylated derivative was achieved by comparison of its spectral and physical data with those in literature [
The mice showed no evident signs of toxicity (motor incoordination, piloerection, dilation of pupils, etc.) after being given both 100 and 500 mg/kg doses of the extract for 4 weeks. However, the hydroalcoholic extract caused liver toxicity in mice at doses of 500 mg/kg, as evidenced by an increase in the concentration of aspartate transaminase (AST) and alanine transaminase (ALT) compared to the control group (Table
Analysis of biochemical parameters in toxicity test.
Number | Experimental group | Total cholesterol (mg/dL) | ALT (IU/L) | AST (IU/L) |
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(1) | Control |
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(2) | ECHE (100 mg/kg) |
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(3) | ECHE (500 mg/kg) |
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(4) | Hexa-O-acetyl-D-mannitol (10 mg/kg) |
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(5) | D-Mannitol (10 mg/kg) |
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For eight weeks mice had free access to cholesterol-rich diet. However, by the fourth week, according to our analysis, the animals were already hypercholesterolemic. Considering the above, we decided to start the administration of the different treatments along with the cholesterol-rich diet to the hypercholesterolemic mice at the fourth week and finish it until the eighth week. The mice that received the high cholesterol diet showed an increase in both total cholesterol levels and the non-HDL fraction, as well as a significant decrease in HDL cholesterol compared with control mice (Table
Analysis of serum cholesterol.
Number | Experimental group | Cholesterol concentration (mg/dL) | ||
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Total | HDL | Non-HDL | ||
(1) | Control |
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(2) | Hypercholesterolemia |
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(3) | Ezetimibe (10 mg/kg) |
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(4) | ECHE (100 mg/kg) |
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(5) | ECHE (500 mg/kg) |
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(6) | Hexa-O-acetyl-D-mannitol (10 mg/kg) |
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(7) | D-Mannitol (10 mg/kg) |
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Histopathology results were compared between different experimental groups (Figure
Histopathological microscopic examination in mice liver tissues. Control (a). Hypercholesterolemia (b). Ezetimibe 10 mg/kg (c). ECHE, 100 mg/kg (d). ECHE, 500 mg/kg (e). Hexa-O-acetyl-D-mannitol 10 mg/kg (f). D-Mannitol, 10 mg/kg (g). There were no apparent histological changes in control group fed with a regular chow diet (Figure
Since ECHE (100 mg/kg) showed a similar hypocholesterolemic activity compared to ECHE (500 mg/kg) without the induction of hepatotoxicity, only ECHE (100 mg/kg) was analyzed by western blot technique. Expression of Abcg8 intestinal transporter increased after treatments with hydroalcoholic extract at 100 mg/kg dose and with ezetimibe at doses of 10 mg/kg (Figure
Western blot results. Western blot analysis of Abcg5 and Abcg88 in small intestine tissues of mice. Control (1), hypercholesterolemia (2), ezetimibe 10 mg/kg (3), ECHE 100 mg/kg (4), hexa-O-acetyl-D-mannitol (5), and D-mannitol (6). Total protein lysates were separated by 12–15% SDS-PAGE followed by western blot analysis using the indicated antibodies.
It has been proposed that two ATP-binding cassette (ABC) transporters, Abcg5 and Abcg8, restricted sterol absorption and promoted biliary sterol excretion in humans. Furthermore, it has been reported that increased expression of
Taking the above into account, we studied the possible participation of these proteins in the antihyperlipidemic activity of ECHE, D-mannitol, and its hexa-acetate derivative in hypercholesterolemic mice.
The effect of dietary cholesterol on the expression of Abcg5 and Abcg8 in the small intestine is controversial [
However, D-mannitol and the hexa-O-acetyl-D-mannitol not only hindered overexpression of ABCG5/ABCG8 but also inhibited the overexpression of these proteins induced by the hypercholesterolemic diet (Figure
Under physiological conditions, dietary cholesterol is efficiently solubilized by bile salt-phospholipid mixtures in the form of either mixed micelles or vesicles that formed in bile [
The latter result is a little surprising if we consider that the ECHE induced overexpression of the enzymes ABCG5/ABCG8. However, there are several reports about the isolates from plants that lowered plasma cholesterol by increasing fecal excretion of acidic and neutral sterols in mice fed a cholesterol-enriched diet accompanied with downregulation of gene expression of intestinal ABCG8 [
In conclusion, we showed that ECHE (100 mg/kg) and D-mannitol (10 mg/kg) exert, each, hypolipidemic effects in hypercholesterolemic mice, with similar activity compared to ezetimibe (10 mg/kg), without causing damage to liver and without affecting the levels of HDL cholesterol. The hypocholesterolemic effect of ECHE seems to be mediated by ABCG5/ABCG8 intestinal transporters.
The authors declare no conflicts of interest.
Ibrahim Guillermo Castro-Torres is a doctoral student from Programa de Doctorado en Ciencias Biomédicas, Universidad Nacional Autónoma de México (UNAM), and received fellowship 278442 from CONACYT (CVU: 488911, no. Becario 278442). The authors thank M. en C. Antonio Nieto Camacho and M. en C. María Teresa Obdulia Ramírez Apan, Unidad de Pruebas Biológicas, Instituto de Química, Universidad Nacional Autónoma de México. The authors also thank Miss Graciela Ruiz of the Basic Research Unit of the National Institute of Cancerology for her technical help.