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Department of Environmental and Preventive Medicine, Shimane University School of Medicine, Izumo City, Shimane 693-8501 and * Shimane Institute for Industrial Technology, Matsue City, Shimane 690-0816, Japan
2To whom correspondence should be addressed. E-mail: shiwaku{at}med.shimane-u.ac.jp.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • mulberry • quercetin 3-(6-malonylglucoside) • LDL oxidation • atherosclerosis • LDLR–/– mice
Oxidative modification of LDL is believed to play a pivotal role in the development of atherosclerosis (1,2). Oxidized LDL is taken up by macrophages at an increased rate via the scavenger receptor (3) and subsequently leads to the formation of lipid-laden foam cells, the hallmark of early atherosclerotic fatty streak lesions (4,5). Antioxidants that prevent LDL from oxidation could attenuate the development of atherosclerosis. Epidemiologic studies indicated that dietary intake of antioxidant substances from plants is inversely associated with mortality from coronary heart disease (6,7).
Flavonoids are a large group of polyphenolic compounds ubiquitous in fruits, vegetables, and herbs; they have attracted much attention due to their potential antioxidant properties and probable role in the prevention of oxidative stress-associated diseases including atherosclerosis. Quercetin, a major representative of the flavonol group of flavonoids, was shown to have strong inhibitory effects on oxidative modification of human LDL in vitro (8,9). However, quercetin is found in plant foods in the glycosylated form, and accumulates as glucuronide and sulfate conjugates in the blood circulation (10). Recent studies demonstrated that these conjugates also exert a substantial antioxidant effect on oxidative modification of human LDL (11,12).
In a previous study (13), we screened for antioxidant activity in 52 kinds of edible plant products in Japan using 3 [LDL oxidation, 1,1-diphenyl-2-picrylhydrazyl (DPPH)3 radical scavenging, and Folin-Ciocalteu] assays. Of these plants, mulberry (Morus alba L.) showed relatively high antioxidant activity, and the highest activity was observed in the LDL oxidation assay. Previous studies on the constituents of mulberry leaves identified quercetin 3-glucoside (Q3G) (isoquercitrin) and kaempferol 3-glucoside (astragalin) as the main flavonoids (14,15). We recently found that antioxidant activity of mulberry leaves can be attributed mainly to quercetin 3-(6-malonylglucoside) (Q3MG) (Fig. 1), the most abundant flavonol glycoside in mulberry leaves, even greater (260 mg aglycone/100 g fresh weight) than that of onions (40–100 mg/100 g), which are considered to be one of the richest sources of quercetin (16).
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Animals and diets. A breeding colony was generated from homozygous LDLR–/– mice with a C57BL6/J x 129Sv background obtained from Jackson Laboratories. Mice were maintained under controlled environmental conditions (temperature 23 ± 2°C, relative humidity 55 ± 10%, 12-h light:dark cycle, air change 13–15 times/h) and consumed food and water ad libitum. At the age of 8 wk, male mice weighing 20.5 ± 0.8 g were randomly assigned to 4 (control, quercetin, Q3MG, and mulberry) groups, consisting of 10 mice each, with the exception of 8 mice in the Q3MG group because of a shortage of the purified compound. The control group was fed an atherogenic diet containing 3 g cholesterol and 15 g cocoa butter/100 g, which was prepared from a standard rodent diet obtained from Funabashi-Nojo. The diet contained 8.0% moisture, 20.8% crude protein, 4.8% crude fat, 3.2% crude fiber, 5.0% crude ash, 58.2% nonfiber carbohydrate as well as vitamins and minerals adequate to meet the nutritional needs of mice. The other experimental groups were fed the same atherogenic diet supplemented with 0.05 g quercetin dihydrate/100 g for the quercetin group, 0.05 g Q3MG/100 g for the Q3MG group, and 3 g dry mulberry-leaf powder/100 g for the mulberry group. The diets were fed for 8 wk. Food intake was recorded daily and body weight was measured once each week. This study and all procedures were approved by the Animal Care and Use Committee of Shimane University School of Medicine, Japan.
Biochemical analysis of plasma and lipoprotein fractions. After overnight food deprivation, the mice were anesthetized by an i.p. injection of sodium pentobarbital solution (50 mg/kg body weight). At the time of killing, blood was collected from hearts into tubes containing 1000 mg/L EDTA and centrifuged immediately at 1000 x g for 15 min at 4°C. Plasma was stored at –80°C before analysis. An antioxidant, BHT (4.4 mg/L), was added to plasma aliquots used for lipoprotein separation. Concentrations of total cholesterol (TC), free cholesterol (FC), HDL cholesterol (HDL-C), triglyceride (TG), and glucose were measured using enzymatic assay kits (Wako Pure Chemical; Cholesterol E test, Free cholesterol E test, HDL-cholesterol test, Triglyceride G test, and Glucose C II test); LDL cholesterol (LDL-C) was calculated by the Friedewald formula (22), and levels of non-HDL-C were estimated by the formula TC (mmol/L) – HDL-C (mmol/L) (23). Concentrations of insulin were measured using an Ultrasensitive Mouse Insulin kit (Mercodia). Homeostasis model assessment-insulin resistance (HOMA-IR) was calculated by the following formula: fasting plasma insulin (pmol/L) x fasting plasma glucose (mmol/L)/22.5 (24). The protein concentration in the LDL fractions was determined using a Protein Assay Rapid kit (Wako Pure Chemical).
Plasma lipoprotein separation. Plasma lipoproteins were separated for each mouse using a sequential ultracentrifugation method as described previously (25) except the initial plasma volume was reduced to 150 µL and all of the reagents were used proportionately. All fractions were stored at –80°C until analyzed. The purity of lipoprotein fractions obtained by sequential ultracentrifugation method was verified by a 2.4–15.2% polyacrylamide gradient gel electrophoresis with a Multigel-LP kit (Daiichi Pure Chemical).
LDL oxidation. The susceptibility of LDL to oxidative modification was assessed by determining the lag phase for conjugated diene formation as described (13). Briefly, LDL was desalted using a Centricon YM-30 (Millipore) and aliquots containing 8 mg protein/L from each mouse were mixed with CuSO4 solution (5 µmol/L final concentration) to initiate oxidation. The appearance of conjugated dienes was then measured by monitoring continuously at 234 nm for 7 h at 37°C using a spectrophotometer UV-1700 (Shimadzu) equipped with a 16-position automated sample changer.
In addition to the lag phase, the susceptibility of LDL to oxidative modification was evaluated by the thiobarbituric acid (TBA) test. TBARS were determined spectrofluorometrically as described (26) with slight modification. Briefly, 50 µL of LDL fraction, 100 µL of 3% SDS, 400 µL of 0.1 mol/L HCI, 50 µL of 10% (wt:v) phosphotungstic acid, and 200 µL of 0.7% (wt:v) 2-TBA were combined and placed at 95°C for 30 min. Samples were cooled on ice, mixed with 500 µL of 1-butanol and centrifuged at 1800 x g for 10 min at 4°C. An aliquot of the butanol phase was separated and analyzed spectrofluorometrically (excitation 510 nm and emission 555 nm) using a plate reader attachment. TBARS values were expressed as malondialdehyde (MDA) equivalents. MDA standard was prepared from 1,1,3,3-tetramethoxyprophane.
Assessment of atherosclerosis in the aorta. The aortic tree of each mouse was prepared as described (27). Then the aorta was removed and fixed in 10% (v:v) neutral buffered formalin until used for staining (21). Briefly, the entire aorta and heart were rinsed in 70% (v:v) ethanol and then immersed in 0.5% (wt:v) Sudan IV in 35% ethanol:50% acetone for 15 min with continuous shaking. The aorta was destained in 80% ethanol until the background color became clear and then washed with water. Images of the aortae were captured with a digital camera mounted on a light microscope DF PLAN 1 (Olympus) (Fig. 2), processed with Adobe Photoshop 6.0FE, and analyzed using Scion Image Analysis software. Atherosclerotic lesion of the aorta was expressed as a percentage of the total surface area.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Effects of quercetin, Q3MG, and mulberry on plasma biochemistry. Plasma TC, HDL-C, and non-HDL-C levels were significantly different among the groups (Table 1). Compared with the controls, HDL-C levels were lower in quercetin- (P = 0.015) and Q3MG-treated (P = 0.009) mice. Although values of FC (P = 0.060) and LDL-C (P = 0.051) also decreased noticeably in the Q3MG- and mulberry-treated mice, the differences were not significant. Plasma glucose concentration was reduced in the mulberry-treated mice compared with the controls (P = 0.009).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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We observed that mulberry and Q3MG treatments retarded the onset of plasma LDL oxidation, as evidenced by the prolongation of the lag phase for conjugated diene formation. However, the treatments did not inhibit the formation of the end product of the LDL oxidation, as measured by the TBARS level. By way of explanation, we surmise the following. Oxidation of LDL is a free radical-driven lipid peroxidation process, which can be divided chronologically into a lag phase, a propagation phase, and a decomposition phase (28). During the course of the lag phase, the PUFAs in LDL are protected from oxidation by the lipophilic antioxidants, particularly 
-tocopherol, which is the most abundant antioxidant in LDL (29). Similarly, flavonoids were shown to protect LDL from oxidation at the initial stage of lipid peroxidation by acting as free radical scavengers (30,31). In our previous study (13), mulberry demonstrated potent free radical scavenging activity as determined by the DPPH radical scavenging assay. Quercetin and other flavonoids were shown to bind to the surface of LDL particles via the formation of an ether bond (32), limiting the access by oxidants and their initial attack on the surface. These mechanisms are likely responsible for the delayed onset of LDL oxidation observed in the mice consuming quercetin glucoside-rich mulberry leaves or pure Q3MG. Moreover, in these 2 groups, the prolongation of the lag phase for conjugated diene formation was associated with a reduction in atherosclerotic lesion formation. Studies in humans also demonstrated the lag phase to be independently associated with the severity of coronary atherosclerosis (33).
During the propagation phase (the next course of lipid peroxidation), depletion of the intrinsic antioxidants and LDL-bound flavonoids leads to rapid oxidation of PUFAs, forming lipid peroxides, followed by the decomposition phase characterized by the formation of aldehydes including MDA. However, we observed no differences in TBARS formation among the 4 experiment groups. Some studies reported that the reduction of atherosclerotic lesion formation after dietary supplementation of flavonoids is associated with the inhibition of formation of both conjugated diene and TBARS (32,34), although the same protective effect against atherosclerosis may be solely the result of inhibition of the former (32). Results of our study suggest that inhibition of conjugated diene formation at the initial stage of lipid peroxidation (delay of the onset of LDL oxidation) plays an important role in the attenuation of early atherosclerotic lesion formation.
The lack of relation between the values of the lag phase and LDL-MDA points out the limitations of measuring the susceptibility of LDL to oxidation ex vivo. Although measurement of MDA is one of the most widely used analytical methods of lipid peroxidation, it has been criticized as unspecific and prone to artifacts during sample processing (35). The measurement of MDA by TBA-test reflects only the formation and decomposition of certain lipid peroxidation products. In addition, MDA is not derived exclusively from PUFAs; under certain environmental circumstances, amino acids, carbohydrates, peptides, and proteins can also release intermediates that can form a MDA-TBA adduct under the acid-heating conditions of the test (36). Due to these limitations of the TBARS assay, we considered the inhibition of LDL oxidation observed in the "lag phase" to be a more reliable indicator. Continuous monitoring of the formation of conjugated dienes through measurement of the increase in absorbance at 234 nm, which reflects the initiation stage of lipid peroxidation, reveals the exact oxidation lag time (28).
In this study, quercetin, a well-known in vitro antioxidant, had no protective effects against LDL oxidation and atherosclerotic lesion formation even though daily quercetin intake in the quercetin group was higher (2 mg/d) than that of the Q3MG and mulberry (1.2 and 0.4 mg/d, respectively) groups, indicating that the concentration in the diet is not material. Differences in the absorption processes of quercetin glucosides and its aglycone may be responsible for their different effects on LDL oxidation and atherosclerotic lesion formation. Absorption of onion quercetin glucosides, 45% of which is quercetin-4'-glucoside (37), was far superior to that of its aglycone (38). Moreover, bioavailability of Q3G was shown to be as high as that of quercetin-4'-glucoside (39). In a study by Hollman et al. (38), glucosides were transported into enterocytes by the intestinal glucose carrier-sodium dependent glucose transporter in the small intestine, and subsequently hydrolyzed by intracellular cytosolic ß-glucosidase (40). Another pathway involves lactase phlorizin hydrolase, a glucosidase of the small intestine brush border membrane, which catalyzes extracellular hydrolysis of certain glucosides, followed by diffusion of the aglycone across the brush border (41). Q3G and Q3MG are not substrates for cytosolic ß-glucosidase, but they were absorbed after hydrolysis by lactase phlorizin hydrolase (42,43). Once absorbed, quercetin glucosides produce a rapid, higher plasma peak level and have a very slow elimination half-life (44). Thus, the repeated daily supplementation of pure Q3MG and quercetin-glucoside-rich mulberry leaves likely led to a buildup of a sufficient concentration to protect LDL from oxidation. The retardation of the onset of the LDL oxidation and prevention of atherosclerosis progression in the mulberry group despite a lower daily intake of quercetin vis a vis the Q3MG group indicates the possible role of some other flavonoids and biological active constituents present in mulberry leaves.
In this study, the mulberry treatment inhibited the increase in body weight of mice compared with the controls. Adding dried mulberry-leaf powder to the atherogenic diet reduced overall energy density (energy content in a given weight of a food, e.g., kcal/g or kJ/g) due to its fiber content such as cellulose, hemicelluloses and lignin, all present in plant cell walls (45). Consumption of the same amount of less energy-dense food may have led to the reduction of body weight change in the mulberry group. Plasma glucose levels after food deprivation were also reduced in the mulberry-treated mice. It was suggested that this effect is related to polyhydroxylated alkaloids, including 1-deoxynojirimycin, which exerts potent inhibitory activity against -glucosidase, to fagomine, which potentiates glucose-induced insulin release, and to the increase in tissue uptake of glucose by the leaves (19,20,46).
In conclusion, dietary consumption of mulberry leaves attenuated atherosclerotic lesion development in LDLR–/– mice through enhancement of LDL resistance to oxidative modification, and these antioxidative and antiatherogenic protective effects were attributed mainly to Q3MG, the quantitatively major flavonol glycoside in mulberry leaves.
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FOOTNOTES |
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3 Abbreviations used: DPPH1,1-diphenyl-2-picrylhydrazyl; FC, free cholesterol; HDL-C, HDL cholesterol; HOMA-IR, homeostasis model assessment-insulin resistance; LDL-C, LDL cholesterol; LDLR–/–, LDL receptor-deficient; MDA, malondialdehyde; Q3G, quercetin 3-glucoside; Q3MG, quercetin 3-(6-malonylglucoside); TBA, thiobarbituric acid; TC, total cholesterol; TG, triglyceride.
Manuscript received 31 October 2004. Initial review completed 13 December 2004. Revision accepted 31 December 2004.
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