The Journal of Nutrition Nutrition and Disease
An Anthocyanin-Rich Extract from Black Rice Enhances Atherosclerotic Plaque Stabilization in Apolipoprotein E–Deficient Mice1 Xiaodong Xia, Wenhua Ling,* Jing Ma, Min Xia, Mengjun Hou, Qing Wang, Huilian Zhu, and Zhihong Tang Department of Nutrition, School of Public Health, Sun Yat-sen University (Northern Campus), Guangzhou 510080, P. R. China
Abstract Black rice and its pigment fraction may have antiatherogenic activity, but the exact component contributing to the beneficial effect remains unclear. The aim of the present study was to investigate the influence of the anthocyanin-rich extract from black rice on the vulnerability of advanced plaques in apolipoprotein (apo) E-deficient mice. Using LC-MS, the deficient mice (n ¼ 30; 30 wk old) were randomly divided into 3 groups: a control group (fed the AIN-93G diet), the simvastin group [simva; fed the AIN-93G diet containing simvastatin, 50 mg/(kgd)], or the anthocyanin-rich extract group [antho; fed the AIN-93G diet supplemented with anthocyanin-rich extract from black rice, 300 mg/(kgd)]. After 20 wk of intervention, the plaque area that developed in the brachiocephalic artery of mice in the antho group was smaller than that of the control mice. Both the antho and simva groups had lower frequencies of the large necrotic core and thin fibrous cap in plaques than the control group. Collagen I was increased and matrix metalloproteinase-1 contents were reduced in the brachiocephalic lesion of both the antho and simva groups compared with the control group. Furthermore, mRNA levels of tissue factor and inducible nitric oxide synthase in aortae were decreased in the antho and simva groups. Supplementation of anthocyanin-rich extract improved the lipid profile by decreasing serum triglyceride, total cholesterol, and non-HDL cholesterol. These results suggest that chronic diet intake of anthocyanin-rich extract from black rice may enhance plaque stabilization in old apoE-deficient mice. The underlying mechanism is related mainly to inhibiting proinflammatory factors and improving the serum lipid profile. J. Nutr. 136: 2220–2225, 2006.
Introduction Acute coronary syndromes (ACS),2 including unstable angina, myocardial infarction, and sudden coronary death, are the major causes of sudden cardiovascular diseases (1,2). Clinical studies demonstrated that the occurrence of ACS always correlates with some vulnerable atherosclerotic plaques and subsequent thrombosis. Those plaques seldom cause severe luminal narrowing, but they are predisposed to rupture, erosion, or calcification and are prone to thrombosis formation (3–5). In terms of atherosclerotic disease prevention and treatment, a major task is to take measures to increase the stability of the plaques and to inhibit thrombosis formation in the case of plaque rupture. An abundance of studies revealed that the 1
Supported by grants from the National Natural Science Foundation of China (30371215) and the National Natural Science Foundation of Guangdong province (2005). 2 Abbreviations used: ACS, acute coronary syndromes; antho, anthocyanin-rich extract; HDL-C, HDL cholesterol; HE, hematoxylin and eosin; HMG-CoA, hydroxy-3-methylglutaryl coenzyme A; iNOS, inducible nitric oxide synthase; MMP, metalloproteinase; simva, simvastatin; TC, total cholesterol; TF, tissue factor; TG, triglyceride. * To whom correspondence should be addressed. E-mail:
[email protected]. edu.cn.
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underlying cause of vulnerable plaque progression is related to inflammation (6). At present, many drugs are applied to stabilize plaque, including anti-inflammatory drugs, lipid-lowering drugs, angiotensin-converting enzyme inhibitors, receptor blockers, and metalloproteinase (MMP) inhibitors (7). Among these drugs, the statins and the hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors are the most effective agents currently available for the treatment of atherosclerosis and vulnerable plaque. In addition to lowering lipids, these drugs exhibit potent anti-inflammatory activities and prevent degradation of the extracellular matrix by decreasing the levels of matrix MMP (6). However, the effects of these drugs are generally limited, and some of the mechanisms underlying their effects remain unclear. Early studies showed that polyphenols such as plant anthocyanins are beneficial to cardiovascular health (8,9). Our laboratory identified the antiatherogenic effects of black rice and its pigment fraction in a series of studies (10,11). In rabbits, diet supplementation of black rice improved the lipid profile and increased glutathione peroxidase activity (10). Supplementation of black rice pigment fraction to the diet significantly inhibited atherosclerotic plaque formation in rabbits and in apolipoprotein (apo)E-deficient mice (11,12). These studies also revealed
0022-3166/06 $8.00 ª 2006 American Society for Nutrition. Manuscript received 7 March 2006. Initial review completed 14 March 2006. Revision accepted 19 April 2006.
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anthocyanin-rich extract from black rice was identified as containing cyanidin-3-glucoside and peonidin-3-glucoside. ApoE-
that the antiatherosclerotic properties of black rice do not arise from the dietary fiber or vitamin E components. The anthocyanin components of black rice may be responsible for the antiatherosclerotic effect; however, it requires clarification. In further cooperative work, 2 major anthocyanins were extracted and identified in black rice, cyanidin-3-glucoside, and peonidin3-glucoside, which showed antioxidative and anti-inflammatory activities in vitro (13). However, whether anthocyanin-rich extract from black rice possesses the antiatherosclerotic property in vivo remains uncertain. In recent years, several groups reported that old apoEdeficient mice had vulnerable plaques in the brachiocephalic arteries. These lesions recapitulate some of morphologic features of human advanced atherosclerotic lesions, making this a useful model for investigating interventions that may alter plaque composition and increase plaque stability (14–16). In this study, we used apoE-deficient mice as a model to investigate the effects of anthocyanin-rich extract from black rice on plaque vulnerability and serum lipid level, as well as total serum antioxidant level. Furthermore, the antiatherosclerotic effect of anthocyaninrich extract from black rice is compared with simvastatin to clarify the protective efficacy of an anthocyanin-rich extract against atherosclerosis.
Preparation of anthocyanin-rich extract. The aleurone layer of black rice was removed by an abrasive dehuller (Satake) at 10% yield; this layer was extracted with 60% ethanol containing 0.1% HCl. All of the extracts were concentrated with a rotary evaporator until all alcoholic residues were removed and partitioned against petroleum ether. Then the aqueous extract was purified by an Amberlite XAD-7 column. The eluted anthocyanin fraction was concentrated and, finally, the aqueous residue was lyophilized. Characterization and quantification of anthocyanins present in the extract from black rice were conducted by HPLC (a 250 3 4.6 mm i.d., 5mm Hypersil GOLD C18 column, Waters) followed by further LC-MS analysis (17,18). Anthocyanins were identified by both retention time and mass profile in comparison with authentic standards. The proteins, lipids, carbohydrates, and other components of the anthocyanin-rich extract were analyzed by routine laboratory techniques. The protein content was measured by the Kjeldahl method. The method of phenol-sulfate acid was used to determine the polysaccharide content. Rutin was used as a reference gauge to determine spectrophotometrically the total flavonoids. Animals and diets. Male apoE deficient mice (n ¼ 30; C57BL/6J background) were purchased from Jackson Laboratories. Mice were fed a laboratory diet based on the AIN-93G formula (19) to 30 wk of age to establish the vulnerable plaque model. Then the mice were randomly divided into 3 groups matched for body weight, each including 10 mice. The 3 groups of mice were fed one of the following diets: AIN-93G diet (control group), AIN-93G diet containing simvastatin [50 mg/(kgd)], or the AIN-93G diet supplemented with anthocyanin-rich extract [300 mg/ (kgd)]; all mice were fed for another 20 wk. During the experiment, mice had free access to food and water. This study was approved by the Animal Care and Use Committee of Sun Yat-sen University. Tissue preparation. After 20 wk of intervention, mice were anesthetized (pentobarbital sodium, 50 mg/kg) and killed by exsanguination from the retro-orbital plexus. The mice were then perfused via the abdominal aorta with PBS at a pressure of 100 mm Hg. The aortae were excised from the aortic arch to the bifurcation of the femoral arteries, and the peripheral fat and adventitia were removed carefully. The aorta were immediately snap-frozen in liquid nitrogen and stored at 280C until further use. After infusion with 10% formalin in situ, brachiocephalic arteries were removed together with pieces of the right subclavian artery and right common carotid artery to help orientation in the
Serum lipid profile and total antioxidant capacity. Blood samples from the retro-orbital plexus were centrifuged to obtain serum. Serum total cholesterol (TC) and HDL cholesterol (HDL-C) levels were determined using cholesterol esterase and cholesterol oxidase assays (20). The level of non-HDL-C was calculated as total cholesterol minus HDL-C. The total antioxidant capacities of serum were measured according to the ability to scavenge the 2, 2#-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) radical cation (10). Plaque composition and plaque size. Two independent investigators who were unaware of the study protocol evaluated each section for the frequency of features characteristic of plaque instability. These features included: thickness of the fibrous cap (thin fibrous cap was defined as #3 cell layers) and size of the necrotic core (a large necrotic core was defined as occupying .50% of the volume of the plaque). These were recorded as binary outcomes and the frequency for each mouse was determined (21). Plaque size was quantified after HE staining. Cross-sectional areas of the plaque and the whole artery were calculated separately by computer-assisted morphometry (Image Pro Plus; Media Cybernetics). The relative plaque area was defined as the percentage of the vessel area that was occupied by plaque. The frequencies of a large necrotic core and thin fibrous cap in plaques were determined as described previously (21). Immunohistochemistry. The MMP-1 and collagen I contents in the plaque were determined by immunohistochemistry using rabbit anti-mouse MMP-1 antibody (BA1270, Boster, 1:100 dilution) and rabbit anti-mouse collagen I antibody (BA0325, Boster, 1:200 dilution). The secondary antibodies, biotinylated goat anti-rabbit immunoglobulins (BA1003, Boster, 1:200 dilution), were then applied followed by incubation with horseradish peroxidase–labeled streptavidin solution. Sections were developed with a commercial DAB kit (Boster), counterstained with Harris Hematoxylin, and mounted. The sizes of the MMP-1 and collagen I positive area within the plaques in the brachiocephalic arteries were determined by computer-assisted morphometry (Image Pro; Media Cybernetics). Measurements of TF and iNOS mRNA in aortas. Tissue factor (TF) and inducible nitric oxide synthase (iNOS) expression were analyzed at the mRNA level by semiquantitative RT-PCR. Total RNA was extracted from the aortae with Trizol reagent (Invitrogen). b-Actin gene was used as an internal control. Specific primers for TF, iNOS, and b-actin were as follows: for TF, sense, 5#-CCA CCATCT TTATCATCC-3# and anti-sense, 5#-TTC CCA CTT TAC TGT TCT AC-3#, which afforded a 473-bp fragment; for iNOS, sense, 5#-AGA CAT GGC TTG CCC CTG G-3# and anti-sense, 5#-GAT CAG GAG GGA TTT CAA AGA CCT-3#, which produced a 959bp fragment; for b-actin, sense, 5#-GGA CTC CTA TGT GGG TGA CGA GG-3# and anti-sense, 5#-GGG AGA GCATAG CCC TCG TAG AT-3#, which gave a 366-bp fragment. PCR products were visualized and quantified by electrophoresis in 2% agarose gels stained with ethidium bromide. Statistical analysis. Results are expressed as means 6 SD. For analysis of plaque morphology, groups were compared by x2 test. Other data were analyzed by 1-way ANOVA coupled with the LSD/t multiple comparison test. Differences were considered significant when P , 0.05. SPSS version 10.0 was used for all statistical analysis.
Results Anthocyanin profile. HPLC and LC-MS analysis demonstrated that the 2 major anthocyanins in the extract from aleurone layer of black rice were cyanidin-3-glucoside and Plaque stabilization by anthocyanin-rich extract
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Materials and Methods
following process. Brachiocephalic arteries were sampled at the site of atherosclerotic plaque with the use of a dissecting microscope, and then were embedded in paraffin; 5-mm sections were cut every 30 mm along the artery. Four vessel cross-sections of each specimen were stained with hematoxylin and eosin (HE) and quantified to determine the mean plaque area and presence or absence of vulnerable features in each mouse. Two sections were immunostained for MMP-1 and Collagen I content measurement, respectively.
peonidin-3-glucoside. Total anthocyanin content in the extract was 43.2%; other component6s of the extract were as follows: carbohydrate, 21.6%; protein, 4.9%; flavonoids, 16.6%; water, 5.5%; others, 8.2%. Plaque size and morphology. Advanced atherosclerotic lesions were observed in the brachiocephalic arteries of 3 groups of mice. Image analysis of sections stained with HE showed that the relative plaque size was decreased by 18% in the anthocyanin-rich extract (antho) group (P , 0.01) and 13% in the simvastin (simva) group (P ¼ 0.12) compared with the control group (Fig. 1). The frequencies of a large necrotic core and thin fibrous cap were lower in both the antho and simva groups than in the control group (P , 0.05) (Fig. 2). Immunohistochemistry. Immunostaining for MMP-1 revealed that the percentage of MMP-1–positive area in the brachiocephalic artery plaque was smaller in both the antho (P , 0.05) and simva (P , 0.05) groups than in the control group (Fig. 3). In contrast, the collagen I–positive area was larger in the antho (P , 0.01) and simva (P , 0.05) groups than in the control group (Fig. 3).
Serum lipid profile and total antioxidant capacity. After 20 wk of intervention, mice in the antho group had lower serum levels of TG, TC, HDL-C, and non-HDL-C than those in the control group (Table 1). Compared with the control group, serum TC and non-HDL-C was increased, but HDL-C was decreased in the simva group (Table 1). TG did not differ between the simva group and the control group (Table 1). Compared with the antho group, the simva group had higher concentrations of TC and non-HDL-C, but lower HDL-C. Total serum antioxidant capacity did not differ among the 3 groups (Table 1).
Discussion
vulnerable plaque in old apoE-deficient mice. The potency and effectiveness of the extract containing anthocyanin in this well-characterized animal model were comparable to those of simvastatin, a classic cholesterol-lowering and anti-inflammatory agent. The extract is composed of different kind of nutrients (protein and carbohydrate) and phytochemicals, including mainly anthocyanin (43%) and flavonoids (16%). On the basis of previous results from our group, the phytochemicals rather than the nutrients contained in the black rice were responsible for the atheroprotective effect (11). Anthocyanins were recently considered as important phytochemicals with potential healthpromoting activities, such as antioxidation and anti-inflammation (22). The high amount of anthocyanin contained in the extract might be the major component responsible for the extract-induced plaque-stabilizing effects in the present study. The extract also contains a small amount of flavonoids, which were reported to possess atheroprotective effects in apoE-deficient mice (23,24). Nevertheless, flavanoids, even in an amount higher than that consumed by the mice in the present study, did not lower the serum cholesterol level of the mice (23,24). In this study, however, supplementation of anthocyanin-rich extract significantly lowered serum TG, TC, HDL-C, and non-HDL-C. These results implied that the flavanoids contained in the extract may not be the determining factor for the improvement of lipid profile, although it cannot be ruled out that flavonoids in the extract may contribute partially to other plaque-stabilizing effects observed in this study. The old (26–40 wk) apoE-deficient mice were considered to be a good animal model of vulnerable plaque (14–16,21). The mice used in the present study were a fed normal laboratory
The present study showed that anthocyanin-rich extract from black rice has atheroprotective activity by inhibiting atherosclerotic plaque progression and increasing the stability of the
Figure 1 Relative atherosclerotic plaque area in the brachiocephalic arteries of apoE-deficient mice fed the control, simva or antho diets. Values are means 6 SD, n ¼ 10. Means without a common letter differ, P , 0.05.
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Figure 3 Immunohistochemical analysis of MMP-1 content and collagen I content within lesions in the brachiocephalic artery. The percentage of the positive area within the lesion was determined by computer-assisted image analysis. Values are means 6 SD, n ¼ 10. Means without a common letter differ, P , 0.05.
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Aortic expression of TF and iNOS. RT-PCR analysis was performed with total mRNA extracted from the aorta of each of the apoE-deficient mice. After 20 wk of anthocyanin-rich extract or simvastatin intervention, there was a significant reduction in TF mRNA in the antho and simva groups compared with the control group (P , 0.05) (Fig. 4). Similar to TF mRNA, iNOS mRNA in aortae was also decreased in the antho and simva groups compared with the control group (P , 0.05) (Fig. 4).
Figure 2 Frequencies of thin fibrous cap and large necrotic core in the atherosclerotic lesions in the brachiocephalic artery of simvastatin-treated, anthocyanin-rich extract–treated and control apoE-deficient mice. Values are means 6 SD, n ¼ 10. Means without a common letter differ, P , 0.05.
Figure 4 Anthocyanin-rich extract from black rice reduced TF and iNOS expression in the aortae of apoE-deficient mice. The mRNA level was measured by RT-PCR, and data were normalized to b-actin expression. Values are mean 6 SD, n ¼ 10. Means without a common letter differ, P , 0.05.
TABLE 1
Serum lipid concentrations and total antioxidant capacity in apoE-deficient mice fed the control, simva, or antho diet1,2 Group
Analysis TG, mmol/L TC, mmol/L HDL-C, mmol/L non-HDL-C, mmol/L TAOC, kU/L
Control
Simva a
1.34 6 0.45 6.94 6 1.28b 0.77 6 0.32a 6.17 6 0.57b 306.98 6 42.75
Antho ab
0.97 6 0.21 9.40 6 0.87a 0.29 6 0.05b 9.11 6 0.34a 334.73 6 41.40
0.72 6 0.14b 2.64 6 1.37c 0.44 6 0.08b 2.20 6 0.60c 319.06 6 41.59
1 Values are means 6 SD, n ¼ 10. Means in a row with superscripts without a common letter differ, P , 0.05. 2 TAOC, total antioxidant capacity.
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diet for 30 wk before the intervention. These mice should share the common characteristics of vulnerable plaque. Vulnerable plaque is generally composed of an atrophic fibrous cap, a lipidrich necrotic core, dystrophic mineration, and accumulated inflammatory cells (25). The larger the lipid core and the thinner the fibrous cap, the greater the risk of plaque rupture (26). The common measure to increase the stability of a vulnerable plaque is to alter the composition of the plaque (27,28). In this study, compared with the control mice, mice treated with anthocyaninrich extract had lower frequencies of a large necrotic core and thin fibrous cap in the brachiocephalic artery plaque. The fibrous cap consists mainly of collagen I, proteoglycan, and smooth muscle cells (26). Collagens in plaque are secreted mainly by smooth muscle cells and contribute primarily to the stability of plaque (29,30). Many studies demonstrated that certain cells in plaque such as macrophages can accelerate the degradation of collagen through the production of proteolytic enzymes such as MMPs (31,32). Immunostaining for collagen I showed that positive areas were found mainly in the fibrous cap (31,32). The plaques in both the antho and simva groups contained a much higher content of collagen I than those of the control group; this is likely attributable to the lower content of MMP-1 in the antho and simva groups because MMP-1 is considered to have an important role in the degradation of the extracellular matrix, leading to weakness of the fibrous cap. Biological studies have yielded insights into the pathogenesis of atherosclerosis, an inflammatory process appearing integral to the transition of plaque from stable to unstable. Therefore, inflammatory factors are considered to play an important role in the formation of vulnerable plaque (33). TF and iNOS are both
important proinflammatory factors during the development and progression of atherosclerotic lesions, and they were expressed in all stages of atherosclerotic plaques from various arterial sites (34,35). TF is a potent initiator of the coagulation cascade situated within the vessel wall and is highly exposed to the blood after plaque rupture (36). In addition to its function as an initiator of coagulation, TF plays an important role in inflammation. Expression of TF on the cell surface and its appearance as a soluble molecule are characteristic features of chronic inflammation in conditions such as atherosclerosis (37). TF also cleaves cell-surface receptors that summon proinflammatory cytokines such as IL-6 and IL-8 into the circulation (37). iNOS is involved in multiple biological functions and promotes the progression of atherosclerosis as a proinflammatory agent (38– 40). It was reported that upregulation of iNOS induced IL-8 expression (40). Our results demonstrated that chronic intake of anthocyanin-rich extract from black rice, similar to simvastatin, induces significant inhibitory effects on TF and iNOS mRNA expressions in the aortae of old apoE-deficient mice. The reduction of inflammatory agents of TF and iNOS may thus contribute in part to the enhancement of plaque stability and the reduction of plaque area in apoE-deficient mice. Anthocyanins are considered to have certain antioxidant activities in vitro (41,42). Nevertheless, there was no difference in serum antioxidant activity among the 3 groups, suggesting that the in vivo beneficial effects of anthocyanin-rich extract from black rice are probably unrelated to the antioxidant properties of anthocyanins in this animal model. On the other hand, it remains possible that the anthocyanin-rich extract may enhance antioxidant levels inside the plaque, which we did not measure in the current study. Increased lipid levels are considered to be important risk factors during the development and progression of plaque. Previous studies demonstrated that hyperlipidemia induced the inflammatory response in aortic tissue and subsequently promoted the instability of plaque, whereas lipid lowering by dietreduced MMP expression increased the plaque stability (43). In the present study, anthocyanin-rich extract caused a marked (;60%) reduction in serum TC and non-HDL-C in the antho group compared with the control group. It is likely that the improvement in plaque stability and inflammatory responses seen in the antho group was due largely to the improvement in the lipid profile by the reductions in TC and non-HDL-C. The statin class of drugs inhibits HMG-CoA reductase, the rate-limiting enzyme of sterol synthesis, and lowers plasma cholesterol levels (44). For many years, all of the beneficial effects of the statins were attributed to their cholesterol-lowering capabilities. Nevertheless, several recent studies revealed that simvastatin did not consistently lower the plasma cholesterol of apoE-deficient mice, whose lipid metabolism differs from that of human beings (45,46). Statins exert direct cardiovascular effects that are clearly independent of their cholesterol-lowering capabilities and are not directly attributable to a reduction in serum cholesterol levels (45,46). In the simva group, similar changes in plaque stability were observed, yet TC and non-HDL-C were increased. The mechanism underlying the action is not yet understood, and further research may lead to a new understanding of the actions of statins in old apoE-deficient mice. The improved stability of the plaque by simvastatin in apoE-deficient mice in this study may be attributed to its anti-inflammatory activity. Mice in the antho group had lower TC and non-HDLC, but higher HDL-C than mice in the simva group. The greater improvement in the lipid profile found in the antho group than in the simva group indicates that the anthocyanin-rich extract may
have greater potential against atherosclerosis progression or vulnerable plaque. In conclusion, the present study provided the first experimental evidence that an anthocyanin-rich extract from black rice enhances plaque stability in an apoE-deficient mouse model. The mechanism of this action is related to its lipid-lowering property and anti-inflammatory activity. These observations suggest that an anthocyanin-rich extract may provide a new accessible therapeutic means with which to fight vulnerable plaque in patients and prevent plaque disruption and subsequent thrombus formation. Acknowledgments We thank Ms. Huiqun Wu and Dr. Yingjie Liang for their expert technical assistance.
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Plaque stabilization by anthocyanin-rich extract
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