Biosci. Biotechnol. Biochem., 69 (4), 693–699, 2005

Myricitrin Degraded by Simulated Digestion Inhibits Oxidation of Human Low-Density Lipoprotein

y Atsushi YOKOMIZO and Masamitsu MORIWAKI

Fundamental Research Division, San-Ei Gen F.F.I. Corporation, 1-1-11 Sanwacho, Toyonaka, Osaka 561-8588, Japan

Received August 10, 2004; Accepted November 30, 2004

The inhibitory effects of on the oxidation oxidation. can act as potent inhibitors of of human low-density lipoprotein were investigated LDL oxidation via such mechanisms as metal-ion before and after its degradation by simulated digestion. and free- scavenging.2,3) Myricitrin strongly inhibited the low-density lipoprotein has been reported to have a strong oxidation induced by either 2,20-azobis (2-amidinopro- inhibitory effect on LDL oxidation.1,4) In previous pane) dihydrochloride or CuSO4 in a concentration- studies, only the effect of the aglycon of myricetin dependent manner. Myricitrin was very stable under an was investigated, although myricetin is unstable and acidic condition (pH 1.8) corresponding to the gastric usually contained in plants in the form of the more stable environment, but it was easily degraded under an glycoside, myricitrin. Furthermore, recent findings have alkaline condition (pH 8.5) corresponding to the intes- suggested that flavonoids were unstable under a mildly tinal environment. However, degraded myricitrin also alkaline condition.5) Typical pH values for human had a strong inhibitory effect on the oxidative degrada- plasma, bile, intestinal juice and pancreatic juice are tion of -tocopherol, cholesterol and apolipoprotein B- 7.4, 7.1–8.5, 8.3 and 7.0–8.5, respectively, and unstable 100 in low-density lipoprotein. Our study revealed that flavonoids may be degraded in a mildly alkaline myricitrin was degraded into many components under a biological milieu.6) mildly alkaline condition, but the degraded myricitrin Myricitrin is a rhamnose glycoside of myricetin still retained the free radical-scavenging and - contained in various plants.7–9) However, there have chelating activities toward low-density lipoprotein. only been a few reports on myricitrin, regardless of its strong antioxidative effect.10) Moreover, there have been Key words: myricitrin; flavonoid; low-density lipopro- no reports on the relationship between the stability under tein; oxidation; a biogenic condition and the antioxidative activity of myricitrin toward LDL. In this present study, the Atherosclerosis is characterized by an accumulation antioxidative activity of myricitrin toward LDL was of arterial foam cells mainly derived from oxidized low- investigated before and after its degradation by simu- density lipoprotein (LDL)-loaded . Macro- lated digestion. In addition, the antioxidative effect of phage-scavenging receptors recognize oxidized LDL, degraded myricitrin on -tocopherol, cholesterol and but not native LDL. LDL containing cholesterol, fatty ApoB was also investigated in the presence of the 2,20- acid, phospholipids and apolipoprotein B-100 (ApoB) is azobis (2-amidinopropane) dihydrochloride (AAPH) easily oxidized by factors such as free radicals and metal radical. ions. Hence, inhibiting LDL oxidation may play an important role in preventing the development of athero- Materials and Methods sclerosis. There are many reports that a dietary intake of Reagents. Myricitrin was purchased from Funakoshi flavonoids prevents LDL oxidation. The total amount of Co. (Tokyo, Japan) and , myricetin, , flavonoid intake has been reported to be 10 mg to 1 g per , and were purchased from day and is mainly from tea, onions, apples, and red Sigma Chemical Co. (St. Louis, MO, U.S.A.). Figure 1 wine.1) Those diets with an antioxidative potential are shows the structures of the individual flavonoids. LDL enriched with various types of flavonoid such as (5 mg protein/ml, from human plasma) was purchased catechin and quercetin. However, it is still unclear from Sigma Chemical Co. and stored at 4 C in the dark which flavonoid is the most effective in preventing LDL until its use. AAPH, CuSO4, 1,1-diphenyl-2-picrylhy-

y To whom correspondence should be addressed. Fax: +81-6-6333-3531; E-mail: mmoriwaki@saneigenffi.co.jp Abbreviations: LDL, low-density lipoprotein; ApoB, apolipoprotein B-100; AAPH, 2,20-azobis (2-amidinopropane) dihydrochloride; DPPH, 1,1- diphenyl-2-picrylhydrazyl; TEP, 1,1,3,3-tetraethoxypropane; TBA, thiobarbituric acid; TBARS, thiobarbituric acid-reactive substance; BHT, butylated hydroxytoluene; MDA, malondialdehyde; HPLC, high performance liquid chromatography; TFA, trifluoroacetic acid; DM, degraded myricitrin; MS, mass spectrometry; PAGE, polyacrylamide gel electrophoresis 694 A. YOKOMIZO and M. MORIWAKI DPPH radical-scavenging activity. The free radical- scavenging activity was determined by using the stable DPPH radical according to the method of Blois with a modification.14) A 2-ml sample of a methanol solution at each concentration was added to 2 ml of a 300 mM DPPH methanol solution, mixed vigorously and kept for 30 min at 37 C in the dark. The radical-scavenging activity was quantified by the decolorization of DPPH measured at 517 nm. A A DPPH scavenging (%) ¼ control sample 100 Acontrol

Copper-chelating ability. The copper-chelating ability of myricitrin was determined by the method of Nardini et al.15) The spectral shift of myricitrin in the 200– Fig. 1. Structure of the Flavonoids Used in This Study. 600 nm range after adding an equimolar copper solution were monitored by spectrophotometry. The reaction mixture consisted of 2 ml of 100 mM myricitrin and 2 ml drazyl (DPPH) and 1,1,3,3-tetraethoxypropane (TEP) of 100 mM CuSO4 prepared with a 0.01 M phosphate were purchased from Wako Pure Chemicals Co. (Osaka, buffer (pH 7.4). The effect on the spectral shift of Japan). adding equimolar EDTA to the myricitin–copper com- plex was also investigated. LDL oxidation. The EDTA-containing human LDL solution was dialyzed against a 100-fold volume of Stability of myricitrin at various pH values. The 0.01 M phosphate buffer (pH 7.4, 0.16 M NaCl) which stability of myricitrin was examined at various pH had been freed from oxygen by vacuum degassing.11) values (1.8–8.5).6,16) A 100 mM myricitrin solution was The buffer was changed four times. This EDTA-free prepared with simulated gastric juice (a 0.24% hydro- LDL stock solution was used for all the oxidation chloric acid 0:2% sodium chloride solution at pH 1.8), studies. The stock solution was stored at 4 C, but not for a 0.05 M phosphate buffer (pH 5.0, 6.0, 7.0, 7.4 or 8.0), longer than 24 h. The protein content was determined by or simulated intestinal juice (a 1.5% sodium hydrogen the Lowry method, using a DC protein assay kit (Bio- carbonate solution at pH 8.5), each solution being Rad Co., Hercules, CA, U.S.A.).12) The EDTA-free LDL incubated at 37 C while stirring. An aliquot of the stock solution was diluted to 100 mg protein/ml with incubated solution was periodically analyzed by high oxygen-saturated 0.01 M phosphate buffer (pH 7.4), and performance liquid chromatography (HPLC, Agilent the subsequent LDL oxidation was initiated by adding 1100, Agilent technologies Co., Palo Alto, CA, U.S.A.) aqueous a 10 mM AAPH or 12 mM CuSO4 solution at with a TSKgel ODS-120T reversed-phase column 37 C. After the incubation, the oxidation degree of LDL (4:6 250 mm, Tosoh Co., Tokyo, Japan). Mobile was measured by the thiobarbituric acid (TBA) method. phase A consisted of 0.05% trifluoroacetic acid (TFA) in water and phase B consisted of 0.05% TFA in Thiobarbituric acid-reactive substance (TBARS). methanol. The flow rate was 0.8 ml/min, and the linear TBARS produced from LDL by the oxidation reaction gradient of phase B changed from 0 to 100% in 30 min. were measured by the modified TBA method of Ohkawa Myricitrin was monitored by HPLC with diode array et al.13) A 0.6-ml aliquot of the LDL solution was mixed detection at 363 nm. with 0.5 ml of a 35% trichloroacetic acid solution, 1 ml of a 0.5% TBA solution, 0.05 ml of a 0.22% butylated Degraded myricitrin (DM). Myricitrin digestion was hydroxytoluene (BHT) solution, and 0.05 ml of a simulated essentially as described by Yoshono et al.16) 0.5% SDS solution in a test tube. The tube was tightly After incubating 100 mM myricitrin at 37 C for 60 min in closed with a screw cap and placed in a water bath at simulated gastric juice (a 0.24% hydrochloric acid 100 C for 30 min. After cooling in an ice bath, 0.5 ml of 0:2% sodium chloride solution at pH 1.8), the solution acetic acid and 1 ml of chloroform were added, and the was adjusted to pH 8.5 by adding sodium carbonate and test tube was shaken vigorously for 30 s. After centri- incubated for another 60 min. After this simulated fugation at 3,000 rpm for 10 min, the absorbance of the digestion, degraded myricitrin (DM) was neutralized, upper layer at 532 nm was measured with a spectropho- and the antioxidative activity of DM was investigated at tometer (V-550, Jasco Co., Tokyo, Japan). In this study, a concentration equal to the myricitrin concentration malondialdehyde (MDA) produced from TEP was used before degradation. as a reference standard, the results being expressed as an MDA equivalent in nmol. Measurement of -tocopherol. The consumption of - Degraded Myricitrin Inhibits LDL Oxidation 695 tocopherol in LDL (200 mg protein/ml) by oxidation were less effective than myricetin and quercetin for with 1 mM AAPH was monitored by HPLC.17) An inhibiting the LDL oxidation induced by AAPH and 4) aliquot of the LDL reaction solution (2 ml) was periodi- CuSO4. McAnlis et al. have compared the antioxida- cally sampled and chilled on ice. Before the measure- tive effect of each flavonoid toward LDL in the presence ment, 100 ml of a 0.5% BHT ethanol solution was added of AAPH and CuSO4, and found that the effect was in as an , and -tocopherol was immediately the order of quercetin > rutin > myricetin > kaemp- extracted with 5 ml of n-hexane. The n-hexane layer was ferol > apigenin.1) However, this order varied with the then evaporated under a gentle stream of nitrogen, and experimental conditions. We compared the inhibitory the resulting extract was redissolved in 200 mlof effect of myricitrin and other flavonoids on the LDL ethanol. The amount of -tocopherol in the ethanol oxidation induced by the AAPH radical with the TBA extract solution was analyzed by HPLC/mass spectrom- method. The antioxidative activity of a flavonoid is said etry (MS) with an Alliance ZQ2000 LC/MS system to be derived from its metal ion-chelating and radical- (Waters Co., Milford, MA, U.S.A.), using a reversed- scavenging activity. In this study, to investigate the phase XTerra MS C18 column (2:1 150 mm, Waters effects of the radical-scavenging activity of flavonoids, Co.). -tocopherol was eluted with methanol at a flow we mainly used the AAPH radical to oxidize LDL rather rate of 0.8 ml/min and monitored with a diode array than CuSO4, and found that 77.9% of the oxidation detector at 292 nm. The initial concentration of - induced by 10 mM AAPH was inhibited by adding 50 mM tocopherol in the final incubation solution of LDL was myricitrin, as shown in Fig. 2. Myricitrin and myricetin 2.69 mM. each showed strong antioxidative activity, but the effects of kaempferol and apigenin were rather weak. The Measurement of total cholesterol. Total cholesterol in number and location of hydroxyl groups are important LDL (200 mg protein/ml) after degradation with 10 mM for the radical-scavenging activity of a flavonoid.18) We AAPH was enzymatically measured with a Determiner found that the antioxidative activity of myricitrin is L LDL-C kit (Kyowa medics Co., Tokyo, Japan), using slightly decreased by rhamnose glycosilation at the 3- cholesterol esterase and cholesterol oxidase. The initial position of a hydroxyl group of myricetin. Myricetin is concentration of total cholesterol in the LDL solution more lipophilic than myricitrin; hence, myricetin can was 72.42 mg/dl. protect LDL from oxidation more easily than myricitrin. Salah et al. have also reported that the radical-scaveng- Polyacrylamide gel electrophoresis (PAGE) for meas- ing activity of rutin was weaker than that of the aglycon, uring ApoB. ApoB fragmented from LDL (200 mg quercetin, because of glycosilation at the 3-position of protein/ml) by the treatment with 10 mM AAPH was the hydoroxyl group.19) detected by SDS–PAGE. The LDL reaction solution The inhibitory effect of myricitrin at various concen- (0.1 ml) was mixed with the same volume of a sample trations on the LDL oxidation induced by 10 mM AAPH buffer containing 1% SDS, 20% glycerol, 1% 2-mercap- toethanol and a 0.05 M Tris–HCl buffer (pH 6.8). This mixture was heated in a water bath at 100 C for 3 min. A sample (10 ml) and molecular weight marker (High SDS– PAGE standard, Bio-Rad Co.) were loaded on to 3–10% gradient gel (PAGEL NPG310L, Atto Co., Tokyo, Japan), and 20 mA was applied for 75 min. The gel was then stained with Quick CBB ( R 250, Wako Pure Chemicals Co.).

Statistical analysis. Each data point represents the mean of 3–6 replicated samples and a result is expressed as the mean SD. Differences between means were compared by using one-way ANOVA. Two-way AN- OVA for repeated measurements was used to compare data in the time-course experiments. A post-hoc analysis for significant mean differences between the effects of Fig. 2. Inhibitory Effect of Various Flavonoids on the LDL Oxida- various was done by using the Tukey- tion Induced by the AAPH Radical. Kramer HSD test and Dunnett test. An LDL solution (100 mg protein/ml) was incubated at 37 C for 180 min in a 0.01 M phosphate buffer (pH 7.4) containing 10 mM Results and Discussion AAPH and 50 mM of various flavonoids. The oxidation degree was measured by the TBA method. MDA produced from TEP was used as a reference standard, and TBARS are expressed as nmol MDA Antioxidative activity of myricitrin equivalents. Control values were obtained without the flavonoid. Many flavonoids have strong antioxidative activity for Each data point shows the mean SD (n ¼ 5). Values with different LDL. According to Zhu et al., kaempferol and letters are significantly different from each other (p < 0:05). 696 A. YOKOMIZO and M. MORIWAKI Table 1. Inhibitory Effect of Myricitrin at Various Concentrations on the LDL Oxidation Induced by the AAPH Radical and CuSO4

Myricitrin TBARS (nmol MDA/mg LDL protein) (mM) AAPH-oxidation Cu2þ-oxidation Control 36:5 2:6a 59:9 3:4a 5 30:9 3:0b 11:7 2:6b 10 29:7 1:9b 4:7 0:5c 20 19:3 1:8c 4:6 0:5c 50 7:6 0:6d 3:3 0:7c 100 4:1 0:7d 2:7 0:5c

An LDL solution (100 mg protein/ml) was incubated in a 0.01 M phosphate buffer (pH 7.4) containing 10 mM AAPH or 12 mM CuSO4 and myricitrin at 37 C for 180 min. The oxidation degree was measured by the TBA method. MDA produced from TEP was used as a reference standard, and TBARS are expressed as nmol MDA equivalents. Control values were obtained without myricitrin. Each data point shows the mean SD (n ¼ 5{6). Values with different letters within a column are significantly different from each other (p < 0:05). Fig. 3. Effect of Copper and EDTA on the Myricitrin Spectrum. The spectrum of 50 mM myricitrin in a 0.01 M phosphate buffer (pH 7.4) was monitored just after adding 50 mM CuSO4 and after Table 2. DPPH-Radical Scavenging Effect of Myricitrin incubating for 60 min. To another 50 mM myricitrin solution, 50 mM of EDTA was added 5 min after adding 50 mM CuSO4, and the Concentration DPPH Scavenging (%) spectrum was monitored after incubation for 60 min. (mM) Myricitrin Rutin 1 4:9 1:2a 3:4 1:5a 5 20:3 1:9b 16:7 0:7c 10 40:1 0:4d 31:2 0:9e 20 78:1 0:7f 65:6 0:7g

Myricitrin was incubated in methanol with 150 mM DPPH for 30 min at 37 C. Each data point shows the mean SD (n ¼ 4). Values with different letters are significantly different from each other (p < 0:05).

and by 12 mM CuSO4 was investigated (Table 1). With both oxidation systems, myricitrin prevented the oxida- tive formation of TBARS in a concentration-dependent manner. The addition of 100 mM myricitrin prevented 88.8% AAPH radical-induced and 95.5% copper-in- duced TBARS formation. Myricitrin was more effective in inhibiting the oxidation induced by CuSO4 than that induced by the AAPH radical. Furthermore, myricitrin showed strong concentration- dependent scavenging activity, even toward the stable Fig. 4. Stability of Myricitrin at Various pH Values. free radical, DPPH (Table 2). Myricitrin (100 mM) was prepared in solution at various pH values, before being incubated at 37 C while stirring. The decrease in Copper chelation myricitrin concentration was monitored by HPLC, using a reversed- phase column. Each data point shows the mean of triplicate The spectrum of a flavonoid solution is characteristi- measurements. cally changed by the presence of metal ions.2,3,15) The interaction between myricitrin and copper in an LDL- free phosphate buffer was investigated (Fig. 3). The spectrum of the myricitrin–copper complex did not peaks of the spectrum were shifted rapidly by adding change for 60 min (data not shown), implying that the equimolar copper to the myricitrin solution (from 363 equimolar myricitrin–copper complex was stable in a to 407 nm, and from 269 nm to 253 nm). Equimolar phosphate buffer (pH 7.4). This indicates that myricitrin addition of EDTA to the myricitrin–copper complex inhibits LDL oxidation by the chelating activity of the solution recovered the spectral shift of myricitrin. The copper ion. addition of copper to the myricitrin solution formed a copper–myricitrin complex, but a strong chelator such as Stability of myricitrin EDTA chelated the copper, preventing the formation of Myricitrin solutions (100 mM) prepared at various pH copper-myricitrin. This result suggests that the ability of values were incubated at 37 C to investigate the myricitrin to combine with the copper ion was weaker stability of myricitrin under biogenic pH conditions than that to combine with EDTA. Moreover, the shifted (Fig. 4). At pH 1.8, corresponding to the pH of human Degraded Myricitrin Inhibits LDL Oxidation 697 gastric juice, myricitrin was very stable and did not different from that of native myricitrin, although by degrade. However, under a mildly alkaline solution of only a little. This suggests that DM still retained its basic pH 7.4 to 8.5 corresponding to that in intestinal juice, structure of myricitrin after modification under the myricitrin was unstable and many components were mildly alkaline condition. produced depending on pH the value. This result We investigated the inhibitory effect of DM on the suggests that myricitrin would be stable in the stomach, LDL oxidation induced by AAPH and CuSO4 (Table 3). but unstable and degraded in human intestinal juice. The antioxidative activity of DM toward LDL gradually To investigate the antioxidative activity of myricitrin decreased with the progress of degradation. However, after its absorption in the intestines, we prepared the inhibitory effect of DM on AAPH-induced LDL degraded myricitrin (DM) by incubating a myricitrin oxidation was only 25.8% less than that of myricitrin. solution at pH 8.5 after being at pH 1.8, which corre- Although DM contained a small amount of undegraded spond to the gastric and intestinal pH values (Fig. 5). myricitrin, it showed stronger antioxidative activity than Myricitrin was greatly degraded by 60 min incubation at the remaining amount of myricitrin. This result suggests pH 8.5, the peak area of myricitirn remaining after this that myricitrin was degraded to many components in a incubation being only 2.4% of the original peak area of mildly alkaline intestinal environment; however, the myricitrin. DM did not contain myricetin derived from degradation products still maintained an inhibitory effect alkaline degradation of myricitrin. on LDL oxidation. The spectrum of the main peaks in DM (P1–P3 in Fig. 5) was monitored by HPLC with a diode array -Tocopherol consumption and cholesterol degrada- detector (Fig. 6). The shape of the spectrum was tion The presence of -tocopherol in LDL is known to play an important role in inhibiting LDL oxidation.20)

Fig. 5. HPLC Profile of Myricitrin after Incubating under Simulated Gastric and Intestinal Conditions. Myricitrin (100 mM) was incubated in simulated gastric juice for 60 min at pH 1.8, and then in intestinal juice at pH 8.5 for 60 min, Fig. 6. Spectrum of Each Peak in Degraded Myricitrin (DM) before being analyzed by HPLC with a reversed-phase column. Generated under Mildly Alkaline Conditions.

Table 3. Changes in the Inhibitory Effect of Myricitrin on AAPH-Radical, and CuSO4-Induced LDL Oxidation under Simulated Gastric and Intestinal Conditions

Myricitrin residual ratio TBARS (nmol MDA/mg LDL protein) Treatment (%) AAPH-oxidation Cu2þ-oxidation Control 34:1 2:8a 60:6 4:8a Myricitrin 100.0 7:4 0:9b 3:7 0:5b 60 min at pH 1.8 98.2 7:5 0:6b 3:8 0:6b 15 min at pH 8.5 66.9 7:9 1:4b 3:5 0:8b 30 min at pH 8.5 36.1 8:4 1:8b 4:4 0:8b 45 min at pH 8.5 19.2 10:3 1:5c 4:5 1:1b 60 min at pH 8.5 (DM) 2.4 14:3 2:1c 8:0 1:8b

A myricitrin solution incubated under a simulated gastric condition (pH 1.8) and simulated intestinal condition (pH 8.5) was periodically sampled. After neutralization, 50 mM of each sample was added to the LDL solution (100 mg protein/ml) in a 0.01 M phosphate buffer (pH 7.4) containing 10 mM AAPH or 12 mM CuSO4 at 37 C for 180 min. The oxidation degree was measured by the TBA method. MDA produced from TEP was used as a reference standard, and TBARS is expressed as nmol MDA equivalents. The myricitrin residual ratio indicates the peak area ratio of myricitrin in all peak area of myricitrin incubation solution at 363 nm. Control values were obtained without myricitrin. Each data point shows the mean SD (n ¼ 5{6). Values with different letters within a column are significantly different from each other (p < 0:05). 698 A. YOKOMIZO and M. MORIWAKI

Fig. 7. Inhibitory Effect of DM on the AAPH Radical-Induced Fig. 8. Inhibitory Effect of DM on the AAPH Radical-Induced Consumption of -Tocopherol in LDL. Degradation of Total Cholesterol in LDL. An LDL solution (200 mg protein/ml) was incubated with 10 mM An LDL solution (200 mg protein/ml) was incubated with 1 mM AAPH and DM at 5, 10, or 50 mM in a 0.01 M phosphate buffer AAPH and DM at 5, 10, or 50 mM in a 0.01 M phosphate buffer (pH 7.4) at 37 C. The -tocopherol content in LDL was monitored (pH 7.4) at 37 C. The total cholesterol content in LDL was at 292 nm by HPLC, using a reversed-phase column after extraction enzymatically measured with a kit. The initial concentration of total with n-hexane. The initial concentration of -tocopherol in the LDL cholesterol in the LDL incubation solution was 72.42 mg/dl. Control values were obtained without DM. ( ) control, ( )5mM DM, ( ) solution was 2.69 mM. Control values were obtained without DM. 10 mM DM, ( )50mM DM. Each data point shows the mean SD ( ) control, ( )5mM DM, ( )10mM DM, ( )50mM DM. Each data point shows the mean SD (n ¼ 4). Significant difference (n ¼ 4). Significant difference from the control (*p < 0:05, from the control (*p < 0:05,**p < 0:01). **p < 0:01).

The effect of DM on the -tocopherol content in LDL About 71% of total cholesterol was oxidized and degrad- was measured by HPLC in the presence of 1 mM AAPH ed within 9 h in 10 mM AAPH, but this degradation was at 37 C (Fig. 7). The content of -tocopherol in LDL retarded by the concentration-dependent addition of DM, rapidly decreased in the absence of DM and reached the addition of 50 mM DM preventing the degradation by zero after 2 h, but this decrease was significantly about 39%. This result suggests that DM also prevented retarded by adding DM to the incubation solution, even the oxidative degradation of cholesterol in LDL. 5 mM DM being effective. After a 2-h incubation in 5 mM DM together with 1 mM AAPH, the content of - Apolipoprotein B-100 (ApoB) fragmentation tocopherol was reduced by 66% compared with the LDL constitutes about 21% of the lipoprotein mass, control, but in the presence of 50 mM DM, it was reduced and more than 95% of this consists of apolipoprotein by only 28%. Thus, DM concentration-dependently B-100 (ApoB). During the oxidation of LDL, the amino prevented the AAPH-induced decrease of -tocopherol acids of ApoB are oxidized and ApoB is fragmented to in LDL. In a 1 mM AAPH solution containing 10 and polypeptides. The inhibitory effect of DM on the 50 mM DM, the content of -tocopherol in LDL rapidly oxidative fragmentation of ApoB induced by AAPH and respectively decreased during the first 15 and was investigated by SDS–PAGE (Fig. 9). The spot for 30 min, but it slightly increased again after 1.5 h. Such ApoB protein completely disappeared when LDL was regeneration of -tocopherol observed when incubated incubated with AAPH alone. The addition of 5 mM DM in some flavonoids other than DM. According to Zhu et slightly inhibited the fragmentation of ApoB, and 10 mM al., -tocopherol was regenerated in human LDL by DM suppressed it markedly. Myricitrin was also as adding 5 to 30 mM of catechins.17) This result effective as DM in this respect. DM thus inhibited the suggests that DM prevented LDL oxidation by its oxidative fragmentation of ApoB induced by AAPH hydrogen-donating ability to regenerate -tocopherol concentration-dependently. from the -tocopheryl radical.21) Oxidized cholesterol is closely associated with heart In conclusion, our experiments demonstrated that disease as well as atherosclerosis. LDL consists of 38% myricitrin and its alkaline degradation products (DM) cholesterol ester and 8% unesterified cholesterol.22) The prepared under the same mildly alkaline conditions as Polyunsaturated groups of esterified cholesterol in LDL those in the human intestines had a strong inhibitory are susceptible to oxidation. The amount of total choles- effect on the LDL oxidation induced by radical terol in LDL incubated in an AAPH solution containing scavenging and metal-ion chelation. These results DM at various concentrations was determined by an suggest that dietary myricitrin was degraded under enzymatic method, using cholesterol oxidase (Fig. 8). biogenic mildly alkaline conditions during digestion, but Degraded Myricitrin Inhibits LDL Oxidation 699 Agric. Food Chem., 50, 1700–1705 (2002). 7) Gluchoff-fiasson, K., Fenet, B., Leclerc, J., Reynaud, J., Lussignol, M., and Jay, M., Three new flavonol malonylrhamnosides from Ribes alpinum. Chem. Pharm. Bull., 49, 768–770 (2001). 8) Slacanin, I., Marston, A., and Hostettmann, K., Isolation and determination of flavonol glycosides from Epilobi- um species. J. Chromatogr., 557, 391–398 (1991). 9) Paul, B. D., Rao, G. S., and Kapadia, G. J., Isolation of myricadiol, myricitrin, taraxerol, and taraxerone from Myrica cerifera L. root bark. J. Pharm. Sci., 63, 958–959 (1974). 10) Washino, T., Sankabousizai tositeno yamamomo chu- shutubutsu. Gekkan Food Chemical (in Japanese), 9, 51– 56 (1998). 11) Esterbauer, H., Striegl, G., Puhl, H., and Rotheneder, M., Continuous monitoring of in vitro oxidation of human Fig. 9. Effect of DM on the AAPH Radical-Induced ApoB Frag- low-density lipoprotein. Free Rad. Res. Comms., 6, 67– mentation. 75 (1989). An LDL solution (200 mg protein/ml) was incubated with 10 mM AAPH in a 0.01 M phosphate buffer (pH 7.4) at 37 C for 120 min. 12) Lowry, O. H., Rosebrough, N. J., Farr, A. L., and The ApoB fragments in LDL were detected by SDS–PAGE. The gel Randall, R. J., Protein measurement with the folin was stained with Coomassie Brilliant Blue R250. (Lane 1) standard phenol reagent. J. Biol. Chem., 193, 265–275 (1951). molecular weight marker, (Lane 2) native LDL, (Lane 3) LDL 13) Ohkawa, H., Ohishi, N., and Yagi, K., Assay for lipid incubated with AAPH, (Lane 4) LDL incubated with AAPH and peroxides in animal tissues by thiobarbituric acid 5 mM DM, (Lane 5) LDL incubated with AAPH and 10 mM DM, reaction. Anal. Biochem., 95, 351–358 (1979). (Lane 6) LDL incubated with AAPH and 50 mM DM, (Lane 7) LDL 14) Blois, M. S., Antioxidant determinations by the use of a incubated with AAPH and 10 mM myricitrin. stable free radical. Nature, 181, 1199–1200 (1958). 15) Nardini, M., D’Aquino, M., Tomassi, G., Gentili, V., that the degraded product was still effective in prevent- Felice, M. D., and Scaccini, C., Inhibition of human low- ing LDL oxidation. A further study will be necessary to density lipoprotein oxidation by caffeic acid and other identify the component of DM that inhibited LDL hydroxycinnamic acid derivatives. Free Radical Biol. oxidation in human plasma. The results of the present Med., 19, 541–552 (1995). 16) Yoshino, K., Suzuki, M., Sasaki, K., Miyase, T., and study suggest that the intake of foods containing Sano, M., Formation of antioxidants from ()-epigallo- myricitrin would be effective for lowering the risk of catechin gallate in mild alkaline fluids, such as authentic atherosclerosis caused by LDL oxidation mediated by intestinal juice and mouse plasma. J. Nutr. Biochem., 10, free radicals or metal ions. 223–229 (1999). 17) Zhu, Q. Y., Huang, Y., Tsang, D., and Chen, Z. Y., References Regeneration of -tocopherol in human low-density lipoprotein by green tea catechin. J. Agric. Food Chem., 1) McAnlis, G. T., McEneny, J., Pearce, J., and Young, 47, 2020–2025 (1999). I. S., Dietary flavonoids protect low-density lipoprotein 18) Bors, W., Heller, W., Michel, C., and Saran, M., from oxidative modification. Spec. Publ. R. Soc. Chem., Flavonoids as antioxidants: determination of radical- 215, 116–119 (1998). scavenging efficiencies. Methods Enzymol., 186, 343– 2) Brown, J. E., Khodr, H., Hider, R. C., and Rice-Evans, 355 (1990). C. A., Structural dependence of flavonoid interactions 19) Salah, N., Miller, N. J., Paganga, G., Tijburg, L., Bolwell, with Cu2þ ions: implications for their antioxidant G. P., and Rice-Evans, C., Polyphenolic flavanols as properties. Biochem. J., 330, 1173–1178 (1998). scavengers of aqueous phase radicals and as chain- 3) Afanas’ev, I. B., Dorozhko, A. I., Brodskii, A. V., breaking antioxidants. Arch. Biochem. Biophys., 322, Kostyuk, V. A., and Potapovitch, A. I., Chelating and 339–346 (1995). free radical scavenging mechanisms of inhibitory action 20) Esterbauer, H., Dieber-Rotheneder, M., Striegl, G., and of rutin and quercetin in lipid peroxidation. Biochem. Waeg, G., Role of in preventing the oxidation Pharmacol., 38, 1763–1769 (1989). of low-density lipoprotein. Am. J. Clin. Nutr., 53, 314– 4) Zhu, Q. Y., Huang, Y., and Chen, Z. Y., Interaction 321 (1991). between flavonoids and -tocopherol in human low 21) Miura, S., Watanabe, J., Tomita, T., Sano, M., and density lipoprotein. J. Nutr. Biochem., 11, 14–21 (2000). Tomita, I., The inhibitory effects of tea 5) Record, I. R., and Lane, J. M., Simulated intestinal (flavan-3-ol derivatives) on Cu2þ mediated oxidative digestion of green and black teas. Food Chem., 73, 481– modification of low-density lipoprotein. Biol. Pharm. 486 (2001). Bull., 17, 1567–1572 (1994). 6) Zhu, Q. Y., Holt, R. R., Lazarus, S. A., Ensunsa, J. L., 22) Shen, M. M. S., Krauss, R. M., Lindgren, F. T., and Hammerstone, J. F., Schmitz, H. H., and Keen, C. L., Forte, T. M., Heterogeneity of serum low-density lip- Stability of the flavan-3-ols epicatechin and catechin and oproteins in normal human subjects. J. Lipid Res., 22, related dimeric procyanidins derived from cocoa. J. 236–244 (1981).