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.