Effects of flavonol-rich green tea cultivar (Camellia sinensis L.) on plasma oxidized LDL levels in hypercholesterolemic mice

Abstract

To examine the possible benefits of tea flavonols, we compared anti-atherogenic effects between common and flavonol-rich tea cultivars. The tea infusion made from a flavonol-rich cultivar, but not a common cultivar, significantly decreased the plasma oxidized low-density lipoprotein level in mice fed a high-cholesterol diet. The result suggests that tea flavonols have the potential to protect against cardiovascular diseases.

Key words:

• green tea cultivars
• flavonol glycosides
• plasma oxidized LDL
• hypercholesterolemic mice

Tea plant, Camellia sinensis L., has been cultivated for thousands of years, and its leaves utilized for medical purposes. Epidemiological studies highlight the association between heavy consumption of green tea and the apparent risk reduction in cardiovascular diseases,^1,2) and laboratory studies suggest that catechins are primarily responsible for the preventive effects.³⁾ However, the effects of tea flavonols have been examined infrequently in spite of plentiful data on anti-atherogenic properties of quercetin,⁴⁾ one of the major flavonols in green tea. We previously measured the flavonol glycosides in tea infusions from various cultivars and identified the cultivars with high flavonol contents.⁵⁾ In this study, to examine whether tea flavonols have benefits for ameliorating cardiovascular health, we attempted to clarify the varietal differences in anti-atherogenic effects between a common and a flavonol-rich tea cultivar. Decreasing effects of green tea consumption on the circulating markers for atherosclerosis-related cardiovascular diseases, plasma oxidized low-density lipoprotein (OxLDL),⁶⁾ and soluble lectin-like OxLDL receptor-1 (LOX-1),⁷⁾ were investigated using a mouse model of hypercholesterolemia.

“Sofu” green tea leaves (Flavonol-rich Tea: TeaF) and control “Yabukita” tea leaves (Control Tea: TeaC) were obtained in Japan. The processed green tea leaves were boiled in water, and the filtrates were frozen as the stock solutions. Flavonol glycoside levels in the tea infusions were measured by LC/MS.⁵⁾ Catechin and caffeine levels were also measured by HPLC.⁸⁾ We investigated the effects of TeaC and TeaF using a mouse model of hypercholesterolemia, according to the previous study.⁹⁾ Briefly, ICR male mice (7 weeks old, Oriental Yeast Co., Tokyo, Japan) were divided into four groups (n = 9 or 10 per group) and hypercholesterolemia was induced in three of the groups by the consumption of high-cholesterol diet (HC, D12336, Research Diets, Inc., New Brunswick, NJ, USA). At the same time, these three groups were given one of the following drinking fluids, water (Water-HC), TeaC (TeaC-HC,) or TeaF (TeaF-HC). The remaining group was fed with control pellets (Cont, D12337, Research Diets, Inc.) and water (Water-Cont). The Cont and the HC diets contained 0.03 and 1.25% (w/w) cholesterol, respectively. The composition of the Cont diet was 20%kcal protein, 69%kcal carbohydrate, and 11%kcal fat, while that of the HC diet was 20%kcal protein, 45%kcal carbohydrate, and 35%kcal fat. The daily food intake was similar among all four groups (Water-Cont: 15.74 ± 0.36, Water-HC: 16.35 ± 0.31, TeaC-HC: 16.56 ± 0.40, TeaF-HC: 16.71 ± 0.34 kcal/day). After 4 weeks, the mice were fasted overnight and anesthetized. Blood samples were collected into heparinized tubes and were immediately centrifuged to separate the plasma samples. Plasma total cholesterol and high-density lipoprotein (HDL)-cholesterol levels were enzymatically measured with Cholesterol E-test and HDL Cholesterol Test Wako kits (Wako Pure Chemical Industries, Ltd., Osaka, Japan), respectively. Plasma OxLDL and LOX-1 levels were determined by enzyme-linked immunosorbent assay (ELISA) kits (Mouse OxLDL ELISA Kit: Cusabio Biotech Co., Ltd., Hubei, P.R.China; LOX1/OLR1 Mouse ELISA Kit: Abcam PLC., Cambridge, UK). Hepatic homogenates were prepared, and their protein contents were measured using a Pierce™ BCA Protein Assay Kit (Life Technologies Corp., Carlsbad, CA). Total lipid fractions were extracted from the homogenates by the ordinary method of Folch et al.¹⁰⁾ Cholesterol levels in hepatic lipid fractions were determined using Test Wako kits. This study was approved by the Ethical Committee on Animal Experiments in NARO Institute of Vegetable and Tea Science (No.H26-04), and all animal experiments were conducted according to Law No. 105 and Notification No. 6 of the government of Japan.

In reference to our previous study,⁵⁾ we determined the concentrations of two myricetin, five quercetin, and three kaempferol glycosides in the tea infusions (Table 1). Total aglycone contents were higher in TeaF than in TeaC for all of the aglycones measured (myricetin, quercetin, and kaempferol). The total amounts of catechins and caffeine in TeaF were similar to those in TeaC. Daily intake of total flavonol aglycones in TeaC-HC and TeaF-HC groups was approximately 650 and 1300 μg, respectively. Daily intake of total catechins and caffeine was approximately 9.57 and 1.65 mg, respectively, in both groups. As shown in Table 2, there was no significant difference in the body weights between the Water-Cont and the Water-HC groups at the end of the experimental period. The TeaC-HC and the TeaF-HC groups showed slightly lower, although not significantly, body weights than the Water-HC group. The Water-HC group showed lower HDL cholesterol level and higher non-HDL cholesterol level compare to the Water-Cont group, indicating that the Water-HC group would have greater risk factors for atherosclerosis. However, these cholesterol levels were similar among Water-HC, TeaC-HC, and TeaF-HC groups. The Water-HC group showed greater liver weight but lower protein content compare to the Water-Cont group. There were no significant differences in liver tissue weights and protein contents among Water-HC, TeaC-HC, and TeaF-HC groups. The liver cholesterol level of the Water-HC group was remarkably higher than that of Water-Cont group. The increased cholesterol level in the Water-HC group was slightly reduced in the TeaF-HC group, although not significantly. The plasma OxLDL level of the Water-HC group was distinctly higher than that of the Water-Cont group (Fig. 1). In the TeaF-HC group, but not the TeaC-HC group, the OxLDL level was significantly lower than that in the Water-HC group. The plasma level of sLOX-1, reported to be a surrogate marker of LOX-1 expression in the aorta, was also higher in the Water-HC group than in the Water-Cont group. The increased sLOX-1 level in the Water-HC group was slightly reduced in the TeaC-HC and the TeaF-HC groups, although no significant differences were observed among the groups.

Table 1. Flavonol glycoside concentrations in tea infusions.

	TeaC	TeaF
Flavonol glycosides (μg/ml)
Myricetin-3-O-glucoside	11.5	19.1
Myricetin-3-O-galactoside	25.6	40.7
Quercetin-3-O-glucoside (Isoquercitrin)	0.8	4.2
Quercetin-3-O-galactoside (Hyperoside)	5.7	14.4
Quercetin-3-O-rutinoside (Rutin)	1.0	13.5
Quercetin-3-O-glucosyl-rhamnosyl-glucoside	54.9	192.7
Quercetin-3-O-glucosyl-rhamnosyl-galactoside	49.4	59.8
Kaempferol-3-O-rutinoside	1.8	7.0
Kaempferol-3-O-glucosyl-rhamnosyl-glucoside	21.7	56.4
Kaempferol-3-O-glucosyl-rhamnosyl-galactoside	57.5	46.3
Total aglycones (μg/ml)
Myricetin	24.6	39.6
Quercetin	45.6	117.6
Kaempferol	30.8	42.2

Notes: Control Tea (TeaC) was made from a common tea cultivar, and Flavonol-rich Tea (TeaF) was made from a flavonol-rich tea cultivar. Total aglycone contents were calculated from the concentrations of the corresponding glycosides.

Table 2. Body weight and cholesterol levels in the plasma and liver.

	Cont diet	HC diet
	Water	Water	TeaC	TeaF
Body weight (g)	40.26 ± 0.89	41.19 ± 1.07	38.36 ± 1.09	37.43 ± 0.99
Plasma
Total cholesterol level (mg/dl)	127.54 ± 6.48	155.71 ± 8.60	158.40 ± 13.54	160.37 ± 9.76
HDL cholesterol level (mg/dl)	107.15 ± 2.92^a	86.17 ± 4.18^b	75.48 ± 7.86^b	82.26 ± 6.45^b
Non-HDL cholesterol level (mg/dl)	21.06 ± 4.94^a	69.54 ± 4.92^b	82.92 ± 9.40^b	78.11 ± 8.35^b
Liver
Tissue weight (g)	1.40 ± 0.03^a	2.33 ± 0.19^b	2.15 ± 0.11^b	1.92 ± 0.09^b
Protein content (mg/g tissue)	2.53 ± 0.04^a	2.28 ± 0.03^b	2.23 ± 0.02^b	2.28 ± 0.02^b
Cholesterol content (mg/mg protein)	2.04 ± 0.18^a	27.45 ± 2.66^b	31.44 ± 2.95^b	20.52 ± 2.83^b

Notes: Non-HDL cholesterol level was calculated by subtracting the level of HDL cholesterol from that of total cholesterol. All values are mean ± SEM (n = 9–10). Comparisons of means among the groups were performed using Scheffe’s post hoc test, following one-way factorial analysis of variance (ANOVA). When data were not normally distributed, the Kruskal–Wallis test was applied. Values within a row with different letters (a and b) are significantly different, p < 0.05. There were no significant differences in body weights and plasma total cholesterol levels among the groups.

Fig. 1. Effects of tea infusion prepared from a flavonol-rich cultivar on plasma oxidized LDL (OxLDL) and soluble LOX-1 (sLOX-1) levels in hypercholesterolemic mice.

Notes: All values are mean ± SEM (n = 9–10). Comparisons of means among the groups were performed using Kruskal–Wallis test. Columns with different letters (a and b) are significantly different, p < 0.05. There were no significant differences in plasma sLOX-1 levels among the groups.

In this study, TeaF, but not TeaC, significantly decreased the plasma OxLDL level in the HC-fed mice. Previous studies showed that oral administration of a quercetin glycoside reduced lipid peroxidation in rat plasma in a dose-dependent manner,¹¹⁾ and a dietary quercetin glycoside decreased the susceptibility of plasma LDL to oxidative modification in atherogenic mice.¹²⁾ Therefore, a double dose of flavonol glycoside in TeaF may contribute to the lowering effect on plasma OxLDL in this study. Further studies should be necessary to obtain direct evidence of, e.g., higher levels of flavonols and their derivatives and lower oxidative stress indices such as lipid peroxide levels in plasma by consumption of flavonol-rich teas.

Authors contribution

S. N. and M. M. designed the study and wrote the manuscript. S. N., M. M., K. E., A. M., M. M-Y., and H.·H. performed the experiments and interpreted the data.

Disclosure statement

No potential conflict of interest was reported by the authors.

Acknowledgments

We would like to express our cordial gratitude to Drs. Atsushi Nesumi and Katsuyuki Yoshida of the NARO for providing the tea leaf samples.