“Benifuuki” Extract Reduces Serum Levels of Lectin-Like Oxidized Low-Density Lipoprotein Receptor-1 Ligands Containing Apolipoprotein B: A Double-Blind Placebo-Controlled Randomized Trial

(1) Background: Arteriosclerosis is associated with high levels of low-density lipoprotein (LDL) cholesterol. O-methylated catechins in “Benifuuki” green tea are expected to reduce cholesterol levels, although there is limited research regarding this topic; (2) Methods: This trial evaluated 159 healthy volunteers who were randomized to receive ice cream containing a high-dose of “Benifuuki” extract including 676 mg of catechins (group H), a low-dose of “Benifuuki” extract including 322 mg of catechins (group L), or no “Benifuuki” extract (group C). Each group consumed ice cream (with or without extract) daily for 12 weeks, and their lipid-related parameters were compared; (3) Results: A significant reduction in the level of lectin-like oxidized LDL receptor-1 ligand containing ApoB (LAB) was detected in group H, compared to groups L and C. No significant differences between the three groups were detected in their levels of total cholesterol, triglycerides, and LDL cholesterol; (4) Conclusions: “Benifuuki” extract containing O-methylated catechins may help prevent arteriosclerosis.


Introduction
Lifestyle changes, such as overeating and lack of exercise, have caused global increases in the rates of obesity, hypertension, dyslipidemia, and cardiovascular diseases [1]. As hyperlipidemia is one of the most important risk factors for cardiovascular disease, reductions in serum low-density lipoprotein (LDL) cholesterol levels can help prevent cardiovascular disease [2]. This is because oxidized LDL injures the vascular endothelium, which promotes platelet aggregation and thrombus or a desire to become pregnant in the near future, lactation, and other conditions that were judged by the physician to preclude inclusion (e.g., the use of dietary supplements). Based on the inclusion and exclusion criteria, a total of 159 individuals were considered eligible for inclusion (56 men and 103 women who were 23-80 years old). The mean age was 53.7 ± 10.4 years and the median age was 53 years. All participants were recruited between late August of 2014 and September of the year. All participants lived in Osaka, Kyoto, or Hyogo (Japan), and underwent baseline evaluations between September 2014 and December of the year. The participants were followed for 12 weeks after the baseline evaluation.

Randomization
The participants were randomized 1:1:1 using a computer-generated randomization sequence, with the block size kept constant. The groups were assigned non-specific identifiers, which corresponded to group H, high-dose "Benifuuki" extract containing 676 mg of catechins (314 mg of epigallocatechin gallate (EGCG) and 66 mg of O-methylated catechins) and 66 mg of caffeine in ice cream; group L, low-dose "Benifuuki" extract containing 322 mg of catechins (138 mg of EGCG, 32 mg of O-methylated catechins) and 33 mg of caffeine in ice cream; and group C, no "Benifuuki" extract in ice cream. All participants received individually wrapped packages of ice cream with or without the extract, which were provided by an unrelated third party and were only labelled with the corresponding group identifier. Thus, the investigators and participants were blinded to the group assignments and amounts of extract in the ice cream packages.

Sample Size
Previous studies have evaluated 22-240 participants (group sizes of 22-120 participants) to determine whether daily consumption of catechins was associated with significant reductions in LDL cholesterol and oxidized LDL cholesterol [14][15][16]. Based on the results of those studies, we set the target sample size to 150 participants.

Restrictions during the Study Period
The participants were instructed to avoid excessive overeating and overdrinking starting at 1 week before the baseline evaluation, and not to modify their lifestyle (e.g., dietary habits, smoking, and exercise). Furthermore, the participants were instructed not to consume unregulated drugs and supplements that could influence their serum lipid levels. Moreover, the participants were instructed to fast starting at 10 PM on the nights before the study measurements, although they were allowed to drink water during the fasting period. Finally, the participants were instructed to refrain from smoking and extreme exercise until completion of the study, and to record their physical condition and compliance eating the ice cream (daily life record).
A limitation of our study is that polyphenols contained in chocolate, wine, and certain other foods were not evaluated in the Food Frequency Questionnaire because only the consumption of polyphenols present in tea and coffee were restricted.
The participants' body weights were measured using bioelectricity impedance analysis (InnerScan ® 50V BC-621-SS; Tanita Corporation, Tokyo, Japan). Blood pressure was measured twice, before and after a 3-min rest, using a sphygmomanometer (HEM-7200 ® ; Omron Corporation, Kyoto, Japan). Biochemical tests were performed by SRL Inc. (Tokyo, Japan). The participants also completed questionnaires regarding their health, lifestyle, food intake, type of green tea that they consumed, and the frequency of green tea consumption. Intake of tea catechins was evaluated using the food frequency questionnaire from the Osaki National Health Insurance cohort study [7]. We show the study's protocol in Figure 1. The participants' body weights were measured using bioelectricity impedance analysis (InnerScan ® 50V BC-621-SS; Tanita Corporation, Tokyo, Japan). Blood pressure was measured twice, before and after a 3-min rest, using a sphygmomanometer (HEM-7200 ® ; Omron Corporation, Kyoto, Japan). Biochemical tests were performed by SRL Inc. (Tokyo, Japan). The participants also completed questionnaires regarding their health, lifestyle, food intake, type of green tea that they consumed, and the frequency of green tea consumption. Intake of tea catechins was evaluated using the food frequency questionnaire from the Osaki National Health Insurance cohort study [7]. We show the study's protocol in Figure 1.

Ice Cream and Intervention
Ice cream packages (125 mL (110 g), 138 kcal/package) with no extract, as well as packages with high or low levels of "Benifuuki" extract, were manufactured by MORINAGA & Co., Ltd. (Tokyo, Japan). The nutritional profile and the content of catechins and caffeine of the test ice cream is shown in Table 1. The analysis of catechins and caffeine in "Benifuuki" extract was performed as previously described [17]. All participants received packages of ice cream that corresponded to their blinded group assignment and were instructed to consume one package per day for 12 weeks.

Ice Cream and Intervention
Ice cream packages (125 mL (110 g), 138 kcal/package) with no extract, as well as packages with high or low levels of "Benifuuki" extract, were manufactured by MORINAGA & Co., Ltd. (Tokyo, Japan). The nutritional profile and the content of catechins and caffeine of the test ice cream is shown in Table 1. The analysis of catechins and caffeine in "Benifuuki" extract was performed as previously described [17]. All participants received packages of ice cream that corresponded to their blinded group assignment and were instructed to consume one package per day for 12 weeks.

Statistical Methods
Differences between groups H, L, and C were evaluated based on the intention-to-treat principle and using the Kruskal-Wallis test, the Steel-Dwass test, the Steel test, and the paired t-test, as appropriate. All analyses were performed using JMP software (version 11.2.1; SAS Research Institute Corporation, Carey, NC, USA). Differences were considered statistically significant at a p-value of <0.05.

Participants and Follow-Up
The 159 participants were allocated to group H (n = 53, 17 men and 36 women), group L (n = 53, 21 men and 32 women), and group C (n = 53, 18 men and 35 women), although ten participants were subsequently excluded from the analysis. At the baseline evaluation, two participants from group L were disqualified because they had developed anemia. During the study period, three participants in group H were excluded (withdrawal for personal reasons, gastric pain, and treatment of a previous illness). One participant in group L was excluded because of gastric pain. One participant in group C was disqualified because they could not tolerate the daily ice cream consumption, two participants withdrew because of personal reasons, and one participant withdrew because of gastric pain. Thus, 149 participants (53 men and 96 women) completed the study, with 50 participants in group H (16 men and 34 women), 50 participants in group L (21 men and 29 women), and 49 participants in group C (16 men and 33 women). We show the flowchart of the study in Figure 2.

Statistical Methods
Differences between groups H, L, and C were evaluated based on the intention-to-treat principle and using the Kruskal-Wallis test, the Steel-Dwass test, the Steel test, and the paired t-test, as appropriate. All analyses were performed using JMP software (version 11.2.1; SAS Research Institute Corporation, Carey, NC, USA). Differences were considered statistically significant at a p-value of <0.05.

Participants and Follow-Up
The 159 participants were allocated to group H (n = 53, 17 men and 36 women), group L (n = 53, 21 men and 32 women), and group C (n = 53, 18 men and 35 women), although ten participants were subsequently excluded from the analysis. At the baseline evaluation, two participants from group L were disqualified because they had developed anemia. During the study period, three participants in group H were excluded (withdrawal for personal reasons, gastric pain, and treatment of a previous illness). One participant in group L was excluded because of gastric pain. One participant in group C was disqualified because they could not tolerate the daily ice cream consumption, two participants withdrew because of personal reasons, and one participant withdrew because of gastric pain. Thus, 149 participants (53 men and 96 women) completed the study, with 50 participants in group H (16 men and 34 women), 50 participants in group L (21 men and 29 women), and 49 participants in group C (16 men and 33 women). We show the flowchart of the study in Figure 2.

Baseline Data and Outcomes
The overall compliance rate for the ice cream consumption was 96.0 ± 6.1%, and there was no significant inter-group difference in the compliance rates (group H, 96.2 ± 5.5%; group L, 94.5 ± 7.8%; group C, 97.5 ± 4.2%). No significant differences were observed between the three groups in their baseline values for weight, BMI, abdominal circumference, and γ-glutamyltransferase levels. By the

Baseline Data and Outcomes
The overall compliance rate for the ice cream consumption was 96.0 ± 6.1%, and there was no significant inter-group difference in the compliance rates (group H, 96.2 ± 5.5%; group L, 94.5 ± 7.8%; group C, 97.5 ± 4.2%). No significant differences were observed between the three groups in their baseline values for weight, BMI, abdominal circumference, and γ-glutamyltransferase levels. By the paired t test, a significant decrease in group H's LAB level was detected at the 12-week follow-up, compared to the baseline value in that group. However, no meaningful reductions were observed in the week 12 values for LAB in groups L and C, compared to their respective baseline values. And, by the Steel-Dwass test, a significant reduction in the level of LAB was detected in group H, compared to both groups L and C, at the 12-week follow-up ( Figure 3) ( Table 2).  The Steel-Dwass test shows significant differences in the Δ value at 12 weeks between group H and group L, and between group H and group C. Data are shown as mean ± SD, * p < 0.05. Black bar: High-dose "Benifuuki" extract ice cream group (group H). Gray bar: Low-dose "Benifuuki" extract ice cream group (group L). White bar: No "Benifuuki" extract ice cream (control) group (group C).  The Steel-Dwass test shows significant differences in the ∆ value at 12 weeks between group H and group L, and between group H and group C. Data are shown as mean ± SD, * p < 0.05. Black bar: High-dose "Benifuuki" extract ice cream group (group H). Gray bar: Low-dose "Benifuuki" extract ice cream group (group L). White bar: No "Benifuuki" extract ice cream (control) group (group C).
No significant inter-group differences were observed in the levels of total cholesterol and LDL cholesterol when we compared the baseline and week 12 values (Table 2).
In addition, the Steel-Dwass test showed significant differences in the ∆ value of GA at 12 weeks between group H and group L, and between group H and group C, but showed no significant differences in the ∆ value of HbA1c (Table 2).

Ancillary Analysis
A subgroup analysis was performed between the two participant groups with (n = 105) and without (n = 44) a habit of daily tea drinking. In the both groups, no endpoints had any significant change, suggesting that the division of the study population weakened the detection power.
On the other hand, the differences adjusted for age and sex between groups H, L, and C were evaluated using the analyses of covariance (ANCOVA) test. The analyses adjusted for age and sex showed significant differences in the ∆ value of LAB level at 12 weeks between three groups. We show the result of the analyses in Table 3.

Adverse Events
None of the participants reported experiencing adverse events that could be attributed to the ice cream or catechins, although a previous report has indicated that green tea catechin consumption can be associated with liver damage or low levels of iron [18].

Discussion
The present study revealed that consumption of "Benifuuki" extract in ice cream was associated with a significant reduction in LAB levels. The LAB levels reflect various types of modification (e.g., oxidization, malondialdehyde modification, acetylation, carbamylation, and glycation) of LDL [19]. Progression of arteriosclerosis is driven by the binding of modified LDL to LOX-1 in the vascular endothelium. This promotes endothelial injury through apoptosis, release of inflammatory cytokines, production of active oxygen, and reduction of nitric oxide levels [20]. In addition, LOX-1 is expressed in smooth muscle cells, macrophages, and platelets, where it can cause smooth muscle cell proliferation, foam cell formation, and aggregation of activated platelets [21]. In this context, LAB levels are suggested to be a new biomarker for evaluating the progression of arteriosclerosis and related diseases. For example, recent studies have suggested that LAB and the carotid intima-media thickness were significantly and positively correlated [22,23]. In addition, a recent study reported that LAB showed significant positive correlations with history of smoking, waist circumference, triglycerides [24]. In the present study, the ∆ value of LAB at 12 weeks showed significant positive correlations with the ∆ value of body weight, the ∆ value of BMI, and the ∆ value of triglyceride by the Spearman's rank correlation coefficient test. However, the ∆ values of body weight and BMI at 12 weeks were not significantly different between the three groups, suggesting that no significant correlations exist between the ∆ value of LAB and the ∆ value of body weight and BMI. Significantly positive correlations between the ∆ value of triglyceride and the ∆ value of LAB at 12 weeks were detected only in group C, and was not detected either in group H or L (the data shown in Supplementary material Table S1). However, the ∆ value of triglyceride at 12 weeks had no significant difference between the three groups, suggesting that a significant decrease in group H's LAB level at the 12 weeks was not related with triglyceride.
On the other hand, consumption of "Benifuuki" extract in ice cream was not associated with a reduction in LDL cholesterol levels. One possible explanation is that significant changes in cholesterol levels were not clear, in the participants without lipid abnormalities and without overweight. Alternatively, the doses of catechin may have been insufficient to affect cholesterol levels in such participants. Finally, all of the participants had a pre-existing habit of drinking green tea, which might have obscured any effects of the catechins in the ice cream.
In addition, by the result of this study, the Steel-Dwass test showed significant differences in the ∆ value of GA at 12 weeks between group H and group L, and between group H and group C, but showed no significant differences in the ∆ value of HbA1c. These results suggested that high-dose "Benifuuki" extract might have some influences on the participants' glycemic control, compared to both low-dose "Benifuuki" extract and Control. However, the influence could be too minimal or short-lasting to significantly change HbA1c level, and may have no clinical significance, because GA is a useful clinical biomarker for glycemic control and reflects a short-term glycemic control, compared to HbA1c, which reflects long-term glycemia.

Conclusions
In the present study, daily consumption of "Benifuuki" extract (676 mg of catechins per day) was associated with a significant decrease in serum LAB levels, compared to the group that received less extract (322 mg of catechins per day) or no extract. We attribute this effect to the "Benifuuki" extract containing O-methylated catechins, which may help suppress the progression of arteriosclerosis.