Phenolic compounds from Rosemary (Rosmarinus officinalis L.) attenuate oxidative stress and reduce blood cholesterol concentrations in diet-induced hypercholesterolemic rats
© Afonso et al.; licensee BioMed Central Ltd. 2013
Received: 27 September 2012
Accepted: 29 January 2013
Published: 2 February 2013
Phenolic compounds combine antioxidant and hypocholesterolemic activities and, consequently, are expected to prevent or minimize cardiometabolic risk.
To evaluate the effect of an aqueous extract (AQ) and non-esterified phenolic fraction (NEPF) from rosemary on oxidative stress in diet-induced hypercholesterolemia, 48 male 4-week old Wistar rats were divided into 6 groups: 1 chow diet group (C) and 5 hypercholesterolemic diet groups, with 1 receiving water (HC), 2 receiving AQ at concentrations of 7 and 140 mg/kg body weight (AQ70 and AQ140, respectively), and 2 receiving NEPF at concentrations of 7 and 14 mg/kg body weight (NEPF7 and NEPF14, respectively) by gavage for 4 weeks.
In vitro, both AQ and NEPF had remarkable antioxidant activity in the 2,2-diphenyl-1-picrylhydrazyl (DPPH●) assay, which was similar to BHT. In vivo, the group that received AQ at 70 mg/kg body weight had lower serum total cholesterol (−39.8%), non-HDL-c (−44.4%) and thiobarbituric acid reactive substance (TBARS) levels (−37.7%) compared with the HC group. NEPF (7 and 14 mg/kg) reduced the tissue TBARS levels and increased the activity of tissular antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase). Neither AQ nor NEPF was able to ameliorate the alterations in the hypercholesterolemic diet-induced fatty acid composition in the liver.
These data suggest that phenolic compounds from rosemary ameliorate the antioxidant defense in different tissues and attenuate oxidative stress in diet-induced hypercholesterolemic rats, whereas the serum lipid profile was improved only in rats that received the aqueous extract.
KeywordsHypercholesterolemia Oxidative stress Polyphenols Rosmarinus officinalis
Atherosclerosis is a chronic inflammatory disease initiated by the subendothelial retention of low density lipoprotein (LDL) particles followed by their subsequent oxidation. The chemical modification of LDL particles induces the expression of adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), on endothelial cells and smooth muscle cells, which, once activated, lead to cytokine secretion and contribute to the recruitment of monocytes and T cells to the arterial intima[1, 2]. Macrophage colony-stimulating factor (MCSF) induces monocytes entering the plaque to differentiate into macrophages that display scavenger receptors on their surface. These receptors promote modified LDL uptake in a process that is not regulated by the intracellular lipid content, which ultimately leads to foam cell formation. Therefore, hemodynamic stress and the accumulation of lipids initiate an inflammatory process in the artery wall[3, 4].
Moreover, the accumulation of cholesterol in erythrocytes, leukocytes, platelets and endothelial cells can lead to an increase in the concentration of reactive species[5, 6] and a reduction in the antioxidant defense systems, such as catalase (CAT) glutathione peroxidase (GPx) and superoxide dismutase (SOD) enzyme activities. This condition favors a disruption of the redox balance, contributing to the establishment of oxidative stress, which is involved in several metabolic disorders[8, 9]. Thus, substances that combine antioxidant and hypocholesterolemic activities are expected to prevent or attenuate the cardiometabolic risk because the oxidative modification of LDL and its uptake by macrophages present in the arterial wall contribute to cardiovascular disease.
Phenolic compounds have been proven to be successful in attenuating hypercholesterolemia[11–13]. Moreover, these substances are known by their capacity to increase antioxidant enzyme activity and reduce free radical formation, effects that have been of interest to researchers as possible protective agents in diseases involving oxidative stress[7, 10, 14]. Among the herbal extracts reported to have antioxidant activity, rosemary (Rosmarinus officinalis L.) is one of the most widely commercialized plant extracts; it is used as a culinary herb for flavoring and as an antioxidant in processed foods and cosmetics. Regarding its antioxidant and anti-inflammatory activities, studies have identified phenolic compounds obtained through several extraction methods, mainly carnosic (phenolic diterpene) and rosmarinic acids (caffeic acid and 3,4-dihidroxifenilactate ester)[16–18]. Although antioxidant compounds have been demonstrated to have beneficial effects on some parameters related to the cardiovascular risk, the role of rosemary phenolic compounds on hypercholesterolemia-induced oxidative stress has not been elucidated in the literature. Therefore, the aim of this study was to evaluate the effect of two different extracts, an aqueous extract and a non-esterified phenolic fraction, from rosemary (Rosmarinus officinalis L.) on the antioxidant status of the serum and tissues from diet-induced hypercholesterolemic rats.
Preparation of the aqueous extract and non-esterified phenolic fraction
Dried and powdered rosemary leaves (20 g) were extracted with distilled water (100 mL) at room temperature (23°C) for 1 h. The samples were centrifuged at 15,000 rpm for 10 min. The supernatant was filtered through qualitative filter paper, and distilled water was added to a total volume of 100 mL. The non-esterified phenolic fraction was obtained by homogenizing powdered, defatted rosemary leaves (1 g) with tetrahydrofuran (6 successive one-minute periods with 20 mL) at room temperature. The supernatant was filtered with sodium sulfate anhydrous and vacuum-evaporated to dryness at 30°C. The dried mass was then resuspended in 5 mL of methanol and filtered through qualitative filter paper into a 5 mL volumetric flask, and methanol was added to achieve the final volume of 20 mL. The solutions were kept at −20°C until further analysis.
Determination of total phenolic content
The total phenolics were determined by the Folin-Ciocalteau method. The total phenolic content was expressed as milligrams of gallic acid equivalent (GAE) per gram of leaf.
High performance liquid chromatography (HPLC) analysis
Rosmarinic and carnosic acids were identified and quantified by a modified version of the method described by Almela et al. using HPLC with UV/Vis detection and a reversed-phase Luna C18 column (250 x 4.3 x 10 μm, Phenomenex, USA). The mobile phase consisted of 0.2% metaphosphoric acid (A) and acetonitrile (B) using a gradient program of 0–20 min 80% A, 20–30 min 60% A, and 30–40 min 0% A. The flow rate was 1 mL/min. The peaks for rosmarinic and carnosic acids, which have detection limits of 1.58 μg/mL and 1.46 μg/mL, respectively, were detected at 330 and 230 nm, respectively. The peaks were identified by comparing their retention times with those of standards (Sigma Aldrich, St Louis, MO, USA), which were run individually. Carnosic and rosmarinic acid calibration curves (R2 = 0.999 for both) were used for quantification.
Determination of in vitro antioxidant activity using the β-carotene/linoleic acid system and DPPH● assay
The β-carotene/linoleic acid co-oxidation system was conducted as previously described. The antioxidant activity of the samples was also assessed by the 2,2-diphenyl-1-picrylhydrazyl (DPPH●) radical method. The results were expressed as μmol equivalents BHT/g of sample.
Animals and diet
Male Wistar rats (Rattus norvegicus, var. albinus) (101.3 ± 0.4 g) were obtained from the Animal Laboratory of the Faculty of Pharmaceutical Sciences at the University of São Paulo. A total of forty-eight rats (n = 8 per group, 4 weeks old) were kept in a room at an ambient temperature of 22 ± 2°C and a relative humidity of 55 ± 10% under a 12-h light/12-h dark cycle (lights on at 0700 h). All procedures performed on animals were approved by the Ethics Committee on Animal Experimentation of the Faculty of Pharmaceutical Sciences, University of São Paulo (protocol number 174/2008), according to the guidelines of the Brazilian College on Animal Experimentation. The animals were randomly distributed into six groups: C (chow diet, receiving distilled water), HC (hypercholesterolemic diet, receiving distilled water), AQ 70 and 140 (hypercholesterolemic diet, treated with 70 and 140 mg aqueous extract/kg body weight/d, respectively), and NEPF 7 and 14 (hypercholesterolemic diet, treated with 7 and 14 mg of non-esterified phenolic fraction/kg body weight/d, respectively).
Composition of the experimental diets (g/kg diet) a
Parameters (g/kg diet)
Extraction and esterification of hepatic lipids
Fatty acid profile of hepatic tissue
The samples were analyzed by gas chromatography (Shimadzu, GC 17A, Kyoto, JAP) using a fused silica capillary column (100 m × 0.25 mm, SP-2560). After a 5 minute isothermal period, the temperature of the column was increased from 140°C to 240°C at a rate of 4°C/min and then kept constant for 20 minutes. The injector and detector temperatures were 250°C and 260°C, respectively. Helium was employed as the carrier gas at a flow rate of 1 mL/min, and a sample volume of 1 μL was injected at a split ratio of 1:200. The peaks were identified by comparing their retention times with those of the standards (Supelco 18919, USA), which were run at a split ratio of 1:50.
Activity of tissue antioxidant enzymes
The tissular catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities were determined as described by Beutler, McCord and Fridovich and Sies et al., respectively. All analyses were conducted using a spectrophotometer, and absorbances were monitored every 60 seconds for 6 minutes.
A spectrophotometric method was used to determine the protein content in the serum and tissues analyzed.
Determination of thiobarbituric acid-reactive substances (TBARS)
The method described by Ohkawa et al. was employed. The absorbance was measured at 532 nm on a spectrophotometer. The TBARS concentrations were calculated using a standard curve for 1,1′,3,3′-(TEP) tetraethoxypropane (10-4 mol/L) and were expressed as μmol of malondialdehyde (MDA) per milligram of protein.
The serum total cholesterol (TC), triacylglycerol (TG), and high density lipoprotein cholesterol (HDL-c) levels were assessed using commercial enzymatic colorimetric kits (Labtest®, BRA). The non-high-density lipoprotein cholesterol (non-HDL-c) levels were calculated by subtracting the HDL-c levels from the total cholesterol levels.
Statistical analyses were conducted using GraphPad Prism 4.0 for Windows (San Diego, CA, USA). The collected data were subjected to the Kolmogorov-Smirnov test to check for symmetry. The data were expressed as the mean and standard error, and the significance level was set at 0.05. Student’s t test was used to compare two independent samples (the C and HC groups). Three or more independent samples (treated or non-treated HC groups) were compared using a one-way analysis of variance (ANOVA) and Tukey’s test (p < 0.05).
Characterization of the aqueous extract and non-esterified phenolic fraction from Rosmarinus officinalis
In vitro antioxidant activity of the aqueous extract and non-esterified phenolic fraction of rosemary
β-carotene/linoleic acid system
antioxidant activity μmol BHT/g sample
113.25 ± 4.55A
118.03 ± 3.88A
4601.29 ± 60.54A
Non-Esterified Phenolic Fraction
929.35 ± 49.88B
1035.00 ± 21.21B
4752.80 ± 190.09A
Body weight, food intake and weight gain
Weight gain, food intake, food efficiency ratio (FER) and lipid profile of the experimental groups
Weight Gain (g)
167.64 ± 13.41
179.21 ± 24.13ª
175.95 ± 16.22ª
174.06 ± 25.68ª
175.53 ± 17.75ª
179.67 ± 24.71ª
Food intake (g/day)
20.24 ± 2.82
21.85 ± 3.77ª
21.52 ± 3.38ª
21.32 ± 3.20ª
21.79 ± 3.53ª
22.05 ± 4.63ª
61.30 ± 6.46
178.54 ± 33.31ª*
107.47 ± 32.42b
128.85 ± 23.71ab
139.01 ± 38.25a
143.01 ± 37.92a
26.46 ± 1.76
152.00 ± 14.67ª*
84.47 ± 12.78b
103.70 ± 10.03ab
120.60 ± 17.46a
127.20 ± 18.59a
38.15 ± 6.49
21.36 ± 3.47ª*
25.04 ± 2.57ª
25.28 ± 2.24ª
21.42 ± 3.71ª
21.04 ± 2.10ª
25.34 ± 7.75
30.25 ± 7.76ª
26.25 ± 5.28ª
23.92 ± 4.68ª
26.13 ± 4.57ª
30.27 ± 4.63ª
Effect of the aqueous extract and non-esterified phenolic fraction on serum lipids
The TC and non-HDL-c levels were increased in the serum of rats submitted to a hypercholesterolemic diet (HC group) compared with the normocholesterolemic (C) group (p < 0.001). These results are similar to those reported in the literature using the same experimental model[10, 13]. Moreover, the HC group had lower serum levels of HDL-c than the C group (p < 0.001, Table 3). Although both concentrations of the NEPF did not have a significant effect on the serum lipid profile, the AQ at 70 mg/kg significantly reduced the serum TC and non-HDL-c levels by 39.8% (p < 0.01) and 44.4% (p < 0.05), respectively, compared with the HC group but showed no effects on the TG and HDL-c levels. The AQ at a higher concentration (140 mg/kg body weight) did not result in any substantial reduction in the serum TC and non-HDL-c levels (p > 0.05 AQ70 vs AQ140, Table 3).
Effect of the aqueous extract and non-esterified phenolic fraction on thiobarbituric acid reactive substances (TBARS)
In the brain and liver (Figure 1d and e, respectively), no difference was observed in the HC group compared with the C group. However, the AQ and NEPF groups notably showed lower TBARS levels compared with the HC group in these tissues.
Activity of antioxidant enzymes
Fatty acid profile of hepatic tissue
Fatty acid composition (g/100 g fatty acids) of liver
20.36 ± 1.96
13.77 ± 0.54ab*
13.84 ± 0.36ab
14.76 ± 1.52b
13.11 ± 0.51a
12.58 ± 0.59a
18.27 ± 2.22
7.20 ± 1.28*
6.66 ± 0.58
6.10 ± 1.00
6.22 ± 0.95
6.24 ± 0.84
39.87 ± 3.13
21.49 ± 1.67 a*
20.90 ± 0.73 ab
21.39 ± 1.63 a
20.59 ± 1.43 ab
19.14 ± 0.88 b
0.64 ± 0.37
3.57 ± 0.85ab*
3.17 ± 0.60a
2.74 ± 0.47a
3.76 ± 0.79ab
3.57 ± 0.61b
11.75 ± 4.20
25.12 ± 1.78*
25.47 ± 1.12
26.18 ± 1.66
25.50 ± 1.93
26.37 ± 1.73
12.48 ± 4.66
29.38 ± 2.48 *
29.14 ± 1.73
29.23 ± 1.77
29.76 ± 2.69
30.37 ± 1.96
20.29 ± 3.79
31.42 ± 0.85*
32.33 ± 0.77
32.08 ± 2.05
31.57 ± 1.47
32.30 ± 1.26
45.85 ± 6.55
47.54 ± 1.08
49.20 ± 1.19
48.59 ± 2.34
47.77 ± 1.89
48.05 ± 1.96
Total lipids (mg)
11.71 ± 2.51
25.06 ± 5.46 *
26.09 ± 4.62
28.13 ± 3.56
25.77 ± 4.93
26.49 ± 1.65
In this investigation, we performed in vivo experiments that showed that only AQ 70 was capable of significantly reducing the serum TC and non-HDL-c levels in diet-induced hypercholesterolemic rats, as demonstrated in Table 3. This effect may be attributed to the higher amount of total phenolic compounds found in AQ, which was almost twice as much as in NEPF, which represented a more purified fraction. This result suggests that other phenolic compounds, beyond those found in the NEPF, may be responsible for these responses. This effect on cholesterol reduction may be attributed to a decrease in the micellar solubilization of cholesterol in the digestive tract, to an increase in bile flow, bile cholesterol and bile acid concentration and to a subsequent increase in the fecal excretion of steroids, as previously described[33, 34].
This study showed that both extracts (AQ70, AQ140) obtained from rosemary decreased the tissue TBARS levels, lowering the susceptibility to the peroxidative damage elicited by a cholesterol-rich diet. This effect could be attributed to the antiradical activity of these extracts, as demonstrated by the DPPH● assay. In this context, AQ70 could contribute to a decrease in the CVD risk because it simultaneously reduced the TBARS levels and improved the plasma lipid contents.
This protection could be attributed to rosmarinic acid, which has been reported to inhibit the expression of inducible nitric oxide synthase (iNOS) and suppress the production of superoxide and 3-nitrotyrosine in Raw 264.7 macrophages.
Notably, oxidative stress is characterized by not only increased free radical production but also reduced antioxidant enzyme activities. In fact, we observed that animals submitted to a high cholesterol diet plus water presented lower catalase activity in their hepatic and renal tissues (Figures 2a and3a, respectively) compared with the animals receiving only a chow diet. An important observation from this study was that the phenolic compounds from rosemary were able to reestablish catalase activity, as observed with the AQ and NEPF in the liver and with the NEPF in the kidney. Data from the literature demonstrated that carnosic acid (found in NEPF) increased the antioxidant enzyme activities and inhibited lipid peroxidation in Caco-2 cells. In kidneys, AQ70 also increased the GPx activity, which could be an adaptive process to cope with the free radical production (Figure 1c). However, AQ140 promoted a significant increase in the SOD activity (p < 0.05), similar to that observed for phenolic extracts from olive mill wastewater in which the antioxidant properties were only observed when administered at a higher concentration.
Both extracts (AQ and NEPF) at different concentrations were not able to increase the antioxidant enzyme activities in the cardiac tissue of the animals studied. Moreover, NEPF (7 mg/kg body weight) reduced the SOD and CAT activities. Bouderbala et al. have also reported a reduction in SOD activity in the kidneys of hypercholesterolemic animals fed on a high cholesterol diet supplemented with Ajuga iva (0.5%). The observed effect was attributed to the lack of superoxide anion accumulation in these tissues. Extrapolating these results to our study, we can infer that the phenolic constituents of NEPF may have reduced the concentration of superoxide anions in the cardiac tissue, thereby reducing the need to activate the antioxidant enzymes.
The HC group had a significant decrease in the SOD and GPx activities, indicating oxidative stress in the brain. As observed for the kidneys, only the highest AQ concentration was able to increase the activities of these enzymes (p < 0.05), whereas NEPF was able to increase the SOD activity (14.95% for 7 mg/kg and 17.95% for 14 mg/kg, P < 0.05). A recent study showed that pre-treatment with an aqueous extract from a plant also belonging to the Lamiaceae family was able to reduce the cerebral infarct size and lipid peroxidation. These protective effects involve the permeability of phenolic compounds across the blood–brain barrier, which has been demonstrated in both in vitro and in situ models[39, 40].
Cholesterol-enriched diets also favor liver fat deposition, which plays a role in lipoprotein synthesis and metabolism and therefore in the cardiovascular disease risk[6, 41]. In the present study, the increase in the total lipids and modification of the lipid composition in the HC group may be attributed to the higher stearoyl-CoA desaturase (SCD) activity elicited by cholesterol- and cholic acid-containing diets. In fact, in our study all animals fed on a hypercholesterolemic diet exhibited a higher oleic-to-stearic-acid ratio compared with the control group, which suggests a higher SCD activity as demonstrated in another study performed with the same animal model. This effect may be a protective mechanism as SCD can convert saturated into monounsaturated fatty acids, which are preferred substrates for acyl-CoA:cholesterol acyltransferase (ACAT), an enzyme that catalyzes the esterification of hepatic free cholesterol to an inert cholesteryl ester. However, no significant differences between the treated and untreated hypercholesterolemic groups were observed with respect to hepatic lipids. In this context, the effects of the phenolic compounds from rosemary on the lipoprotein levels can be suggested to be especially related to intestinal cholesterol absorption.
In conclusion, our results suggest that phenolic compounds from Rosmarinus officinalis protect against hypercholesterolemia-induced oxidative stress, increasing the activities of antioxidant enzymes and reducing the amount of thiobarbituric acid reactive substances. The aqueous extract was also able to improve the serum lipid profile, contributing to cardiovascular disease reduction. Future analysis involving the bioavailability of rosemary phenolic compounds and their role on modulating the signaling pathways activated under oxidative stress-related diseases may help to clarify the role of these antioxidant compounds in cholesterol homeostasis and parameters of oxidative stress.
Thiobarbituric Acid Reactive Substances
Non-Esterified Phenolic Fraction
Gallic Acid Equivalent
High Performance Liquid Chromatography
This investigation was supported by grants 08/51333-1 (Afonso MS) and 08/54319-0 (Mancini-Filho, J) from the Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP), Brazil. We would like to thank Gabriela Castilho for helping with the language revision. All authors read and approved the final manuscript.
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