Red Cabbage Microgreens Lower Circulating Low-Density

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Red cabbage microgreen lower circulating LDL, liver cholesterol and inflammatory cytokines in mice fed a high fat diet Haiqiu Huang, Xiaojing Jiang, Zhenlei Xiao, Lu Yu, Quynhchi Pham, Jianghao Sun, Pei Chen, Wallace Yokoyama, Liangli (Lucy) Yu, Yaguang Luo, and Thomas T. Y. Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03805 • Publication Date (Web): 12 Nov 2016 Downloaded from http://pubs.acs.org on November 17, 2016

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Journal of Agricultural and Food Chemistry

1

Red cabbage microgreen lower circulating LDL, liver cholesterol and inflammatory

2

cytokines in mice fed a high fat diet

3 4

Haiqiu Huang ‡,† , Xiaojing Jiang †, Zhenlei Xiao §,∥, Lu Yu †, Quynhchi Pham ‡, Jianghao

5

Sun ¶, Pei Chen ¶, Wallace Yokoyama ⊥, Liangli (Lucy) Yu †, Yaguang (Sunny) Luo §,

6

Thomas T.Y. Wang ‡,*

7 8

‡

9

Research Center, Beltsville, MD 20705

USDA, ARS, Diet, Genomics and Immunology Lab, Beltsville Human Nutrition

10

†

11

20742

12

§

USDA, ARS, Food Quality Lab, Beltsville Area Research Center, Beltsville, MD 20705

13

∥

Department of Agriculture, Culinology and Hospitality Management, Southwest

14

Minnesota State University, Marshall, MN 56258

15

¶

16

Research Center, Beltsville, MD 20705

17

⊥ USDA,

18

Center, Albany, CA 94710

Nutrition and Food Science Department, University of Maryland, College Park, MD

Food Composition and Methods Development Laboratory, Beltsville Human Nutrition

ARS, Healthy Processed Foods Research Unit, Western Regional Research

19 20

* Contact Information of the Corresponding Author: Thomas T. Y. Wang, Ph.D.

21

Tel: 301-504-8459; Email: [email protected]

1 ACS Paragon Plus Environment


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Mailing Address: 10300 Baltimore Ave., USDA-ARS, BHNRC, Bldg. 307C, Rm 132,

23

Beltsville, MD 20705


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ABSTRACT

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Cardiovascular disease (CVD) is the leading cause of death in the United States

26

and hypercholesterolemia is a major risk factor. Population studies, as well as animal and

27

intervention studies, support the consumption of a variety of vegetables as a means to

28

reduce CVD risk through modulation of hypercholesterolemia. Microgreens of a variety

29

of vegetables and herbs have been reported to be more nutrient dense compared to their

30

mature counterparts. However, little is known about the effectiveness of microgreens to

31

affect lipid and cholesterol levels. The present study used a rodent diet-induced obesity

32

(DIO) model to address this question. C57BL/6NCr mice (n=60, male, five-week old)

33

were randomly assigned to six feeding groups: 1) low fat diet; 2) high fat diet; 3) low fat

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diet+1.09% red cabbage microgreens; 4) low fat diet+1.66% mature red cabbage; 5) high

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fat diet+1.09% red cabbage microgreens; 6) high fat diet+1.66% mature red cabbage. The

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animals were on their respective diets for eight weeks. We found microgreen

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supplementation attenuated high fat diet induced weight gain. Moreover, supplementation

38

with microgreens significantly lowered circulating LDL levels in animals fed the high fat

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diet, reduced hepatic cholesterol ester and triacylglycerol levels and expression of

40

inflammatory cytokines in the liver. These data suggest that microgreens can modulate

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weight gain and cholesterol metabolism, and may protect against CVD by preventing

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hypercholesterolemia.

43 44

Keywords: cholesterol, inflammation, microgreen, red cabbage, triacylglycerol



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INTRODUCTION

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Chronic diseases account for about 70% of deaths and more than 75% of total healthcare

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costs in the U.S. in 2010 1. In 2012, close to half of U.S. population were reported to have

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one or more chronic conditions 2, and cardiovascular disease (CVD) in particular,

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accounted for 30% of all U.S deaths 3. Epidemiological studies indicate that individuals

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with higher intakes of fruits and vegetables tend to have a lower occurrence of CVD 4.

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Therefore, increased consumption of fruits and vegetables has been recommended as a

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key component of a healthy diet for the prevention of chronic diseases, including CVD 4,

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5

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cruciferous vegetables, may be particularly beneficial for human health

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vegetables, such as red cabbage, have been reported to be protective against chronic

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diseases such as CVD and prostate cancer 7, 8.

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As part of the concerted strategy to promote health and prevent chronic diseases,

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development of new foods with enhanced function, i.e. functional foods, has received

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much attention. Microgreens, an exotic genre of edible greens (Figure 1), is one of those

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foods that has gained popularity in upscale markets and restaurants over the past few

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years 9, 10. Microgreens are tender immature plants produced from the seeds of vegetables

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(such as red cabbage) and herbs, having two fully developed cotyledon leaves with or

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without the emergence of a rudimentary pair of first true leaves. Microgreens can provide

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a large array of intense flavors, vivid colors and tender textures 10 and can be served as an

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ingredient in salads, soups, and sandwiches, to enhance their color, texture, and/or flavor.

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Microgreens also serve as an edible garnish to brighten up a wide variety of main dishes.

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More importantly, previous studies found that microgreens possess higher nutritional

. Moreover, there is growing evidence that certain groups of vegetables, such as


5, 6

. Cruciferous

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10

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densities when compared to the mature counterparts

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microgreens contained approximately 260-fold more β-carotene, over 40 times more

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vitamin E and 2.4-6-fold higher vitamin C levels than previously published for mature

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red cabbage 11.

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Although microgreens, such as those from red cabbage have been reported to possess

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more nutrients and perceived to be “healthier”, no known study has been conducted to

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evaluate whether consumption reduces CVD risk factors. Previous research has reported

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potential health promoting properties of red cabbage, such as antioxidant, anti-

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inflammation, and antibacterial effects, and amelioration of dyslipidemia induced by

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excessive cholesterol

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cruciferous vegetables, we hypothesized that red cabbage microgreen (RCMG) would

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reduce CVD risk factors in a DIO mouse model. The current study tested this hypothesis

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and our results supported that consumption of RCMG, at physiologically achievable

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levels,

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hypercholesterolemia and liver inflammation. Elevated levels of circulating lipid and

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cholesterol are known risk factors for CVD, therefore, DIO mouse model was employed

84

to access the effect of RCMG to ameliorate dyslipidemia and hypercholesterolemia

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induced by high fat diet 15-17. Additionally, possible molecular mechanisms for the health

86

promoting effects of RCMG were evaluated.

attenuates

12-14

. For example, red cabbage

. Based on the existing literature on the protective effects of

several

high

fat

diet-induced

87 88


risk

factors,

including


89

MATERIALS AND METHODS

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Sample preparation. Mature red cabbage samples (Brassica oleracea L. var.

91

capitata) were obtained from a local market. Red cabbage microgreens (B oleracea L. var.

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capitata) were provided by a commercial microgreens grower (Fresh Origins, San Marcos,

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CA, USA). Microgreens, 7 days after seeding, were harvested without roots, packaged in

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clamshell containers and shipped overnight in insulated containers with ice packs. When

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received, all the mature and microgreen samples were flash frozen in liquid nitrogen and

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lyophilized for 48-72 h (VirTis Freezemobile 35 ES Sentry 2.0 freeze-dryer, SP Scientific

97

Corp., Warminster, PA, USA). Lyophilized samples were then ground into powder and

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stored in sealed bags at -20 °C before use.

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Bioactive composition analysis. Major bioactive components (anthocyanin,

100

polyphenols, and glucosinolates) in the stock mature red cabbage and red cabbage

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microgreen to be used for the formulation of animal diets were analyzed. The extraction

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and analysis protocols are as follows.

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Anthocyanin and polyphenol extraction from microgreen and mature red cabbage.

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Freeze-dried and powdered microgreen and mature red cabbage samples (200 mg) were

105

extracted with 5.00 mL of methanol-water (70:30, v/v) using sonication for 60 min at

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room temperature. The extracts were then centrifuged at 5,000 g for 15 minutes (IEC

107

Clinical Centrifuge, Damon/IEC Division, Needham, MA, USA) and the supernatant was

108

filtered through a 17 mm (0.45 µm) PVDF syringe filter (VWR Scientific, Seattle, WA,

109

USA). Five µL of the filtered extract was used for UHPLC-PDA-ESI/HRMS/MSn

110

analysis described below.


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Extraction of glucosinolates. Glucosinolates in microgreen and mature red 18

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cabbage were isolated according to previously published methods.

Briefly, 100 mg

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freeze-dried and powdered microgreen and mature red cabbage samples were heated at

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75 ⁰C for 1 min, mixed with 5 mL methanol solution and then vortexed for 20 s. Sinigrin

115

solution (5 mM, 200 µL) was added as internal standard in one set of samples and 200 µL

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methanol in the other set as control. Extraction solutions were heated with boiling

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methanol for 20 minutes, filtered with 0.45 µm syringe filter, and supernatants were

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transferred to another tube for future use. Then 1 mL extract was added to a pre-treated

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DEAE Sephadex A25 resin mini-column and then add 100 µL of sulfatase solution,

120

leave to act overnight at ambient temperature and then eluted with two 1mL portions of

121

water. Five µL of the purified extract was used for UHPLC-PDA-ESI/HRMS/MSn

122

analysis described below.

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UHPLC-PDA-ESI/HRMS/MSn Conditions. UHPLC-PDA-ESI/HRMS/MSn was

124

used to determine anthocyanin, polyphenol, and glucosinolates amount and species. The

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UHPLC-HRMS system used consisted of an LTQ Orbitrap XL mass spectrometer with

126

an Agilent 1290 UHPLC with G4220A binary pump, 1316C column oven, and G4226

127

auto sampler with temperature control and a PDA detector (ThermoFisher Scientific, San

128

Jose, CA). Anthocyanin and polyphenol separation were carried out on a Hypersil Gold

129

AQ RP- C18 UHPLC column (200 mm × 2.1 mm i.d., 1.9 µm, ThermoFisher Scientific)

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with an UltraShield pre-column filter (Analytical Scientific Instruments, Richmond, CA)

131

at a flow rate of 0.3 mL/min. A gradient mobile phase was used in this study (96% A (1%

132

formic acid in water) and 4% B (1% formic acid in ACN) at 0 min, 80% A and 20% B at

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20 min, 65% A and 35% B at 40 min, 25% A and 75% B at 45 min, and 10% A, and 90% 7 ACS Paragon Plus Environment


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B at 50 - 55 min) with an injection volume of 5 µL, column temperature at 50 ºc and

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sample temperature at 4 ºC.

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Glucosinolates separation was carried out on an RRHD Eclipseplus C18 column

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(150 mm × 2.1 mm i.d., 1.8 µm, Agilent) with an UltraShield pre-column filter

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(Analytical Scientific Instruments, Richmond, CA) at a flow rate of 0.4 mL/min. A

139

gradient mobile phase was used in this study (98% A (1% formic acid in water) and 2%

140

B (1:1 v:v of 1% formic acid in ACN) at 0 min, 90% A and 10% B at 10 min, 50% A and

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50% B at 25 min, and 10% A, and 90% B at 30 min) with an injection volume of 5 µL,

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column temperature at 50 ºC and sample temperature at 4 ºC, and UV detection

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wavelength at 229 nm. For determination of anthocyanin, cyanidin 3-O-glucoside

144

reference standard was used for determination of total anthocyanins by assuming molar-

145

response factor as 1.00 (UV 520nm). Rutin (quercetin-3-O-rutinoside) reference standard

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was used for determination of other polyphenols by assuming molar-response factor as

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1.00 (UV 330 nm). 19 Identification of anthocyanin and other polyphenols were made by

148

comparing with the published literature by using high-resolution MS data, MS2-4

149

fragmentation and UV-vis data. 20 An LTQ Orbitrap XL mass spectrometer was used for

150

the confirmation of each individual glucosinolates. The optimized conditions were set as

151

follows: sheath gas at 70 (arbitrary units), aux and sweep gas at 15 (arbitrary units), spray

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voltage at 4.8 kV, capillary temp at 270 °C, capillary voltage at 15 V, and tube lens at 70

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V. The mass range was from 100 to 1000 amu with a resolution of 30,000, FTMS AGC

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target at 2e5, FT-MS/MS AGC target at 1e5, isolation width of 1.5 amu, and max ion

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injection time of 500 ms. Source CID was set at 20%. The most intense ion was selected

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for the data-dependent scan to offer their MS2 to MS3 product ions, respectively, with


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normalization collision energy at 35%. The type and amount of desulfo-glucosinolates

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(desulfo-GLs) were calculated using known concentrations of standard sinigrin and

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recommended relative response factors with PDA signal at 229 nm (Table 3)

160

(Supplemental Fig. 4). GLs were identified based on their specific accurate masses (as

161

desulfo form) and retention times.

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Animals and diets. Male C57BL/6NCr mice, aged 5 weeks (approximately 20 g),

163

were purchased from Charles River Laboratories (National Cancer Institute, Frederick,

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MD, USA). Mice were single-housed in a temperature-controlled room with a 12 h/12 h

165

day-night cycle. Prior to the experiment, they were fed a standard rodent chow and had

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free access to water for 1 week to acclimatize to the environment. For the experiment, a

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total of 60 mice were randomly divided into six groups (10 mice/group) and fed one of

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six different diets: 1) low-fat diet (LF) (10 kcal% fat diet); 2) low-fat diet supplemented

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with red cabbage microgreen powder (LFMG) (10 kcal% fat diet containing 10.9 g/kg

170

diet); 3) low-fat diet supplemented with mature red cabbage powder (LFRC) (10 kcal%

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fat diet containing 16.6 g/kg diet); 4) a high-fat diet (HF) (45 kcal% fat diet); 5) a high-fat

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diet supplemented with red cabbage microgreen powder (HFMG) (45 kcal% fat diet

173

containing 10.9 g/kg diet); 6) high-fat diet supplemented with mature red cabbage

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powder (HFRC) (45 kcal% fat diet containing 16.6 g/kg diet). The experimental diets

175

were formulated and pelleted by Research Diets (New Brunswick, NJ, USA). Animals

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(10 per group) were fed the respective diets for 8 weeks with water available ad libitum.

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Food intake and body weight were recorded once a week. All the low fat diets consisted

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of 20% protein, 70% carbohydrate, and 10% fat on a caloric basis and the high fat diets

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consisted of 20% protein, 35% carbohydrate, and 45% fat on a caloric basis. Red cabbage



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microgreens and mature red cabbage samples were added into diets in the form of dry

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powder and the amount supplemented into diets for the mice calculated based on the

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equivalence of 200 g vegetables/day/person (60 kg) using the Dose Translation Formula

183

(Reagan-Shaw, et al., 2007). Detailed information of diet compositions is shown in Table

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1. All animal experiments were conducted under an animal study protocol (Protocol #:

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14-006) that was reviewed and approved by the USDA, ARS, Beltsville Area

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Institutional Animal Care and Use Committee (IACUC).

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Plasma, tissue and fecal sample collection. Mice were subjected to a 12 h fast

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with water available ad libitum. Mice were anesthetized in a CO2 chamber and blood was

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collected by cardiac puncture with syringes previously rinsed with potassium EDTA

190

solution (15% w/v), and plasma was separated by centrifugation at 1,300 rcf for 10 min at

191

4 °C. Livers were collected and an aliquot of the tissue was immediately frozen in liquid

192

nitrogen for later analysis, the other preserved in RNA Stabilization Solution (Ambion,

193

Austin, TX, USA) and kept at -80 °C. At the end of the eighth week, 24 h fecal samples

194

were also collected from the bottom of the cage and stored at -80 °C.

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Plasma lipoprotein analysis. Plasma lipoprotein cholesterol concentrations were

196

determined by size exclusion chromatography as previously described (German, et

197

al.,1996). Briefly, an Agilent 1100 chromatograph was employed with a postcolumn

198

derivatization reactor, consisting of a mixing coil (1615-50 Bodman, Aston, PA, USA) in

199

a temperature-controlled water jacket (Aura Industrials, Staten, NY, USA). A Hewlett-

200

Packard (Agilent, Palo Alto, CA, USA) HPLC pump 79851-A was used to deliver the

201

cholesterol reagent (Roche Diagnostics, Indianapolis, IN, USA) at a flow rate of 0.2

202

mL/min. Bovine cholesterol lipoprotein standards (Sigma-Aldrich, St. Louis, MO, USA) 10 ACS Paragon Plus Environment

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were used to calibrate the signal on the basis of peak areas. Fifteen µL of plasma was

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injected via an Agilent 1100 autosampler onto a Superose 6HR HPLC column

205

(Pharmacia LKB Biotechnology, Piscataway, NJ, USA). The lipoproteins were eluted

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with a pH 7.0 buffer solution containing 0.15 M sodium chloride and 0.02% sodium

207

azide at a flow rate of 0.5 mL/min. Plasma lipoprotein concentration was calculated

208

based on a standard curve.

209

Hepatic lipid extraction. The lipid extraction method was modified from Folch

210

method (Ametaj, Bobe, Lu, Young, & Beitz, 2003; Folch, Lees, & Sloane Stanley, 1957).

211

Frozen liver (10 mg) was minced and transferred into a test tube, 600 µL of

212

chloroform/methanol (2:1, v/v) was then added, followed by homogenization at 6500 rpm

213

for 10 s. Samples were then centrifuged at 14,000 rpm for 10 min. After centrifugation,

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300 µL of organic layer were transferred into a clean tube and vacuum dry for 20 min.

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The dried lipid was weighed and redissolved in 200 µL 1× Reaction Buffer (5× Reaction

216

buffer: 0.5 M potassium phosphate, pH 7.4, 0.25 M NaCl, 25 mM cholic acid, 0.5%

217

Triton. X-100) provided by Amplex Red cholesterol assay kit (Invitrogen, Eugene, OR,

218

USA) and used for triacylglycerol and cholesterol analysis as described below.

219

Triacylglycerol, total cholesterol and free cholesterol in the liver. Hepatic

220

triacylglycerol (TG) was enzymatically determined using commercial kits (Triglyceride-

221

SL, Sekisui Diagnostics PEI Inc., Charlottetown, Canada). Total cholesterol and free

222

cholesterol were quantified by a fluorometric method using the Amplex red cholesterol

223

assay kit (Invitrogen, Carlsbad, CA, USA) following manufacturer’s protocols.

224

Fecal cholesterol extraction and analysis. Fecal cholesterol was extracted using a

225

modified protocol (Folch, 1957). Fecal samples were collected during the 24 h period on 11 ACS Paragon Plus Environment


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day 56 after initiation of the experiment. The samples were freeze-dried and weighed. A

227

dried fecal sample (~10 mg) was homogenized in 200 µL of chloroform/methanol (v/v

228

2:1), and vortexed for 2 min. The mixture was then centrifuged for 5 min at 14,000 rpm

229

in a microcentrifuge tube. The organic phase was transferred into a new clean tube, then

230

vacuum dried. The samples were redissolved in 200 µL 1× reaction buffer (5 × reaction

231

buffer: 0.5 M potassium phosphate, pH 7.4, 0.25 M NaCl, 25 mM cholic acid, 0.5%

232

Triton X-100). The fecal cholesterol content was quantified by a fluorometric method

233

using a commercial kit (Amplex red cholesterol assay kit, Invitrogen, NY. USA)

234

following the manufacturer’s protocol.

235

Fecal bile acid extraction and analysis. Fecal samples collected during the 24 h

236

period on day 56 after initiation of the experiment were lyophilized, pulverized, and

237

weighed. Twenty mg fecal powder was hydrolyzed in 0.5 mL of 2 M KOH at 50 °C for 5

238

hours. The cooled mixture was then extracted with two 3 mL portions of diethyl ether to

239

remove non-saponifiable components. Sodium chloride (0.5 mL, 20%) followed by

240

hydrochloric acid (0.1 mL, 12 M) were then added to the mixture. Acidified mixture was

241

extracted with two portions of 6 mL diethyl ether, evaporated under nitrogen, and

242

redissolved in 0.5 mL pure ethanol. Bile acid concentration was measured using a

243

commercial kit (Total Bile Acid Assays, Cell Biolabs, Inc., San Diego, CA) following the

244

manufacturer’s protocol.

245

Total RNA isolation, cDNA synthesis and gene expression analysis from liver and

246

adipose tissue. To determine changes in the gene expression, liver were cut into 0.1 to 0.2

247

g pieces and homogenized using a Precellys 24 by Bertin Technologies (Villeurbanne,

248

France). The RNeasy Mini Kit from Qiagen (Valencia, CA, USA) was used for total 12 ACS Paragon Plus Environment

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RNA isolation for the liver. AffinityScript cDNA Synthesis Kit from Agilent

250

Technologies (Santa Clara, CA, USA) was used to reverse transcribe complementary

251

DNA. Real-time PCR was performed using the TaqMan method as previously reported

252

according to the manufacturer’s protocol (Life Technology). Mouse TaqMan gene

253

expression assays used in this study were purchased from Life Technology and included:

254

Acetyl-CoA Acetyltransferase (ACAT) 1 and 3, Cytochrome p450 7A1 (CYP7A1),

255

Diacylglycerol

256

Reductase (HMGCR), Lecithin-Cholesterol Acyltransferase (LCAT), LDL receptor

257

(LDLR), Sterol O-Acyltransferase 1 (SOAT1), TATA-binding protein (TBP).

O-Acyltransferase

1

(DGAT1),

3-Hydroxy-3-Methylglutaryl-CoA

258

Statistical analysis. All end point assays for each sample were conducted in

259

triplicate, and the average was used for group analysis. Data for each treatment group

260

were presented as mean ± SEM. Statistics analysis was performed using GraphPad Prism

261

6 (2012, GraphPad Software, La Jolla, CA, USA). A significant level of differences in

262

the means was detected using one-way ANOVA follow by Fisher’s PLSD post hoc test.

263

Statistical significance was defined at p ≤ 0.05.

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RESULTS

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Comparison of phytochemical composition in mature red cabbage and red cabbage

267

microgreen.

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Major purported bioactive components, anthocyanin, polyphenols, and glucosinolates, in

269

mature red cabbage and red cabbage microgreen were compared 20-22. Total anthocyanin

270

concentration was determined to be 12.44 µmol/g dry weight in MG, and 33.36 µmol/g

271

dry weight in RC. Cyanidin 3-(sinapoyl)(sinapoyl)sophoroside-5-glucoside was the most

272

abundant anthocyanin in MG, followed by cyanidin 3-diferuloylsophoroside-5-glucoside

273

and cyanidin 3-(feruloyl)sophoroside-5-glucoside. By contrast, the major RC anthocyanin

274

is cyanidin 3-(feruloyl)sophoroside-5-glucoside (Table 2A). Concentrations of other

275

polyphenols range from 58.57 µmol/g dry weight in MG to 17.22 µmol/g dry weight in

276

RC

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trisinapoylgentionbiose are the top 3 polyphenols in MG. Kaempferol acyl-glucoside

278

being the major polyphenol in RC (Table 2B) (Supplemental Fig. 1-3). Progoitrin and

279

sinigrin were the most abundant GLs in both MG and RC. However, MG contains 3× and

280

4× more progoitrin and sinigrin per gram dry weight than that in RC, respectively.

281

Overall, MG (17.15 µmol/g) contained two folds more GLs than that in RC (8.30

282

µmol/g).

283

Effects of Red Cabbage Microgreen on Food Intake and Body Weight Gain

284

There were no significant differences in food intake amongst the diet treatments (Fig. 2A).

285

For all diet groups, body weights continued to increase through the end of the experiment

286

(week 8). Body weights were significantly increased in animals fed the HF diet,

was

detected.

Sinapoyl-hexose,

disinapoylgentiobiose,


and

1,2,2'-

Page 15 of 41


287

compared to LF diet group, beginning on week 5. Weight gain in animals fed HFMG and

288

HFRC was significantly less than HF fed animals (Fig. 2B). Interestingly, the rate of

289

body weight gain appeared to be higher for LFMG and LFRC, when compared to the LF

290

control.

291

Effect of Red Cabbage Microgreen on Plasma Lipoprotein Levels

292

Compared to the animals on LF diet, low-density lipoprotein (LDL) (166%), and high-

293

density lipoprotein (HDL) (27%) were significantly higher in HF-diet fed group (Fig. 3).

294

Animals on the HFMG diet showed significantly lower LDL than the HF diet group,

295

while HFRC has significantly lower HDL than the HF group. There were no differences

296

in VLDL between the groups. Animals fed the LFRC diet also had significantly elevated

297

LDL (56%) when compared to those of the LF diet group.

298

Effects of Red Cabbage Microgreen on Liver Cholesterol and Lipid Content

299

There were no differences in hepatic cholesterol content between LF- and HF-fed animals

300

(Fig. 4A, B, C). HF-diet-fed animals had significantly higher hepatic TG than those of

301

LF-fed animals (Fig. 4D). HFRC-fed animals had significantly lower total hepatic

302

cholesterol than those of HF-fed animals, with a trend for lower total hepatic cholesterol

303

in the HFMG vs. the HF (p = 0.098) (Fig. 4A). Similarly, LFMG and LFRC also had a

304

trend of slightly lower total hepatic cholesterol than LF with p values of 0.096 and 0.073,

305

respectively (Fig. 4A). Both HFMG and HFRC group had significantly lower hepatic

306

cholesterol ester levels than those of HF-fed animals (Fig. 4B). No differences were

307

observed in hepatic free cholesterols among any of the diet groups (Fig. 4C).

308

Additionally, HFMG but not HFRC had significantly lower hepatic TG levels than those 15 ACS Paragon Plus Environment


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of HF diet animals (Fig. 4D). However, LFRC group has significantly higher TG than LF

310

group (Fig. 4D).

311

Effects of Red Cabbage Microgreen on Fecal Bile Acid and Cholesterol Content.

312

Compared to the LF diet group, animals fed the HF diet excreted more bile acids in their

313

feces (55%) (Fig. 5A). However, neither MG nor RC supplements affected bile acid level

314

in their respective LF and HF diet groups (Fig. 5A). HF diet group excreted significantly

315

more cholesterol than animals fed LF diet (177%) (Fig. 5B). HFRC fed animals showed

316

reduced total fecal cholesterol (32%) when compared to animals in the HF diet group

317

(Fig. 5B).

318

Effects of Red Cabbage Microgreens on Genes Associated with Cholesterol and

319

Triacylglycerol Metabolism in Liver.

320

Transcriptional regulation of cholesterol and triacylglycerol metabolism-related genes

321

were examined as it correlates to the biochemical analyses to elucidate potential

322

mechanisms. Feeding an HF diet significantly up-regulated bile acid synthesis enzyme

323

CYP7A1, which is the rate-limiting step for cholesterol removal 23, 24, compared to the LF

324

diet group (Fig. 6A). No differences were observed on the expression of CYP7A1 in any

325

of the MG- or RC-supplemented diet groups with the HF or LF control diets. We also did

326

not observe an effect of MG or RC-supplementation on the cholesterol pathway enzyme,

327

HMGCR, or LDLR (data not shown). An animal fed the HF diet had increased

328

expression of ACAT1 and ACAT3 (Figure 6 B, C), enzymes involved in fatty acid

329

oxidation. HFMG- and HFRC-fed animals had significantly reduced expression of

330

ACAT1 and ACAT3 (Fig. 6B, C). No effects were observed for ACAT2 in any of the 16 ACS Paragon Plus Environment

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331

diet groups (data not shown). HFMG fed animals also had significantly lower SOAT1

332

mRNA level, an enzyme involved in cholesterol ester synthesis, than the HF-fed animals

333

(Figure 6D). HFMG fed animals had significantly lower mRNA levels for a gene

334

necessary for storage of triacylglycerols, DGAT1, than those of the HF-fed animals (Fig.

335

6E). Both MG and RC supplementation in the HF diet significantly lowered mRNA

336

levels of the lipoprotein synthetic enzyme, LCAT (Fig. 6F).

337

Effects of Red Cabbage Microgreen on Inflammatory Markers in the Liver.

338

Consuming HF diets can lead to hepatic inflammation

339

hepatic inflammatory markers was also analyzed. CRP and TNF-α expression levels were

340

shown to be elevated in the livers of the HF diet group compared to those of the LF diet

341

group (Fig. 6G, H). Animals supplemented with MG and RC had significantly lower CRP

342

and TNF-α mRNA levels (Fig. 6G, H). IL-6 mRNA was also analyzed but was not

343

detectable in any group under the same assay condition.

25, 26

344


. Therefore, expression of


345

DISCUSSION

346

This is the first report evaluating the potential health promoting properties of RCMG in a

347

rodent model. We observed a 1.09% RCMG supplementation in a high fat diet for eight

348

weeks attenuated body weight compared to the HF diet control. Elevated circulating LDL

349

is a known risk factor for CVD

350

profiles in blood by lowering LDL levels induced by high-fat diet and thus may reduce

351

the risk of CVD. High fat diet induced hypercholesterolemia and chronic inflammation

352

are also associated with the development of several chronic diseases, such as CVD,

353

obesity, cancer, etc.

354

hepatic cholesterol ester, triacylglycerol levels and expression of inflammatory cytokines

355

(CRP and TNF-α). Hence, the improvements in CVD risk parameters from microgreen

356

supplementation are consistent with the generally health promoting effects for RCMG.

357

Compared to the mature RC counterpart, the RCMG shared similarity as well as

358

differences in biological effects. Both RCMG and RC lowered body weight gain, hepatic

359

cholesterol, and expression of inflammatory markers induced by HF diets. HFRC- but not

360

HFMG-fed animals had significantly lower fecal cholesterol excretion, however, the

361

exact mechanism was not clear. Intestinal cholesterol excretion or gut microbiota may

362

play a role in the altered fecal cholesterol excretion, and further study is warranted to

363

elucidate the underlying mechanism. HFMG- but not HFRC-fed animals had

364

significantly lower plasma LDL and hepatic TG levels than those of the HF-fed animals.

365

These results suggest that RC and RCMG share general health promoting effects,

366

although the underlying mechanisms may differ. This notion is further supported, at the

367

molecular level, by the differential effects on gene expression of cholesterol and

15, 27

. Supplementation of RCMG improved lipoprotein

28-30

. Animals fed RCMG-supplemented diets showed reductions in


Page 18 of 41

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368

triacylglycerols metabolism enzymes in the liver. RCMG but not RC affected the

369

cholesterol ester synthesis enzyme SOAT1, which is consistent with the cholesterol ester

370

lowering effect of RCMG observed in the HF-fed animals. The triacylglycerol synthesis

371

enzyme DGAT1

372

terminal and only committed step in triacylglycerol synthesis by using diacylglycerol and

373

fatty acyl CoA as substrates

374

triacylglycerol-lowering effects in the liver of RCMG but not RC.

375

ACAT1, a mitochondrial enzyme that catalyzes the reversible conversion of two

376

molecules

377

oxidation/degradation pathway 33, 34. Consuming an HF diet induces this enzyme and is

378

consistent with the animal’s response to the HF diet by increasing fatty acid oxidation.

379

Both RCMG and RC supplementation led to attenuation of HF diet-induced increases in

380

ACAT1. The result may be a reflection of the overall effect of RCMG and RC on overall

381

lipid metabolisms altered by HF diet. How RCMG or RC may directly regulate ACAT1

382

or reduce the effect of HF remain unclear and further study is necessary to elucidate the

383

specific mechanisms.

384

Both RC and RCMG consumption led to lower LCAT expression in the liver as

385

compared to their respective controls. LCAT is known to be involved in extracellular

386

cholesterol esterification and cholesterol transport

387

with small but significant decreases in HDL levels in HFRC group and the decrease in

388

LDL levels in the HFMG group compared to those of the HF group. However, change in

389

LCAT also occurred in LFRC and LFMG groups relative to the LF diet group suggesting

390

dietary component(s) from RCMG and RC may play a role in the regulation of hepatic

of

31, 32

was down-regulated by RCMG but not RC. DGAT1 catalyzes the

acetyl-CoA

31

. Downregulation of DGAT1 is consistent with the

to

acetoacetyl-CoA,

35

is

involved

in

the

fatty

. Lowering of LCAT is consistent



391

LCAT activity. The consequence of the changes in expression remained less clear and

392

warrants further study. Additionally, recent studies suggested that lower LCAT may be

393

associated with an increase in obesity-related insulin sensitivity 36, 37. The effect of RC or

394

RCMG on insulin sensitivity was not examined in this study and warrants further

395

validation.

396

Diets high in fat content are known to induce a variety of physiological changes,

397

including chronic, low-grade inflammation in the liver 26. Our observation of inhibitory

398

effects of RC and RCMG on HF diet-induced increases in CRP and TNF-α expression,

399

inflammatory markers in the liver 38, suggested that supplementation of RC or RCMG in

400

HF diets may help alleviate hepatic inflammation. Therefore, the inclusion of RC or

401

RCMG in the Western style diet may contribute to the prevention of diet-related chronic

402

conditions in the U.S. population.

403

Given that food intake was similar in all groups, the effect on lowering weight gain

404

elicited by RCMG or RC in animals fed the HF diet was unexpected. The mechanism of

405

this novel finding is unclear. Since the movement, activity, and behavior of animals in

406

this study were not specifically monitored, it is possible that altered baseline energy

407

expenditure may be a contributing factor and warrant additional study. One possible

408

regulatory mechanism may involve UCP1 39, a protein involved in energy balance, which

409

is known to be affected by phytochemicals in the diet40-42. Analysis of adipose tissue

410

from different depots and UCP1 expression would be an important next step. The

411

increase in body weight in LFRC and LFMG groups compared to those of the LF group

412

was also unexpected. The animals were transitioning from juvenile to adulthood from the

413

start of the experiment (6 weeks old) toward the end (14 weeks old), so it is possible that 20 ACS Paragon Plus Environment

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414

both RC and RCMG may provide additional components that maximize their growth.

415

Future studies are needed to identify the active components.

416

Polyphenols and glucosinolates are known bioactive compounds in fruits and vegetables,

417

and major phytochemical components in RC.20-22 In this study, RCMG was determined to

418

contain more polyphenols (71.01 µmol/g) and glucosinolates (17.15 µmol/g) than RC

419

(50.58 µmol/g and 8.30 µmol/g, respectively). Polyphenols and glucosinolates have been

420

shown in previous studies to possess cholesterol-lowering,

421

inflammation 47-49 properties. Therefore, it is possible that these bioactive components in

422

RCMG and RC may contribute to the observed differential cholesterol-lowering and anti-

423

inflammation effects in this study. Anthocyanins give a red hue, and consistent with the

424

color differences between RC and RCMG (Fig. 1), RCMG contained about 1/3 of

425

anthocyanin than the RC. Hence, the compositions of RCMG and its mature counterpart

426

are quite different. However, the precise components from RC or RCMG that provide

427

health beneficial effect remain to be elucidated in future studies.

428

In summary, supplementing diets with 1.09% RCMG or 1.66% RC, the equivalence of

429

200 g vegetables/day/person, modulated body weight, plasma lipoprotein profiles, and

430

inflammatory status. In mice fed high fat diets supplementation with RCMG or RC had a

431

beneficial effect against lipid risk factors for the development of CVD. This study also

432

provided the first evidence that RCMG and its mature counterpart may regulate the lipid

433

and cholesterol metabolisms differently. One could speculate that other microgreens may

434

have similar effects, although additional studies are needed. Further delineation of the

435

mechanisms of action is also needed, especially as related to energy expenditure and lipid

436

metabolism. 21 ACS Paragon Plus Environment

43-45

antioxidant,

46

and anti-


437

ACKNOWLEDGMENT

438

The authors thank Dr. Naomi Fukagawa for her comments and suggestion on the

439

manuscript.

440

441

FUNDING SOURCES

442

This work was supported by USDA appropriated fund #8040-51530-056-00D.


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443

FIGURE LEGENDS

444

Figure 1. Red cabbage microgreen and red cabbage photo.

445

Figure 2. Food intake and body weight gain. Animals were divided into six diet groups

446

and fed respective diets for 8 weeks. Food intake and body weight gain were assessed as

447

described in the Materials and Methods. The diet groups were : LF, HF, LFMG, LFRC,

448

HFMG, and HFRC. Panel A. Food intake (g/week). Panel B. Weight gain (g/week).

449

Results are expressed as mean ± SE (n=10/group). Bars with different letters indicate

450

significant difference at p≤0.05.

451

Figure 3. Plasma lipoproteins levels. Animals were divided into six diet groups and fed

452

respective diets for 8 weeks. Plasma HDL, LDL, and VLDL levels measured as described

453

in the Materials and Methods. The diet groups were : LF, HF, LFMG, LFRC, HFMG and

454

HFRC. A. VLDL, B. LDL, C. HDL. Results expressed as mean ± SE (n=10/group). *

455

indicates a significant difference between respective groups at p≤0.05.

456

Figure 4. Liver cholesterol and triacylglycerol levels. Animals were divided into six diet

457

group and fed respective diets for 8 weeks. Liver cholesterol and triacylglycerol levels

458

measured as described in the Materials and Methods. The diet groups were : LF, HF,

459

LFMG, LFRC, HFMG and HFRC. A. Total cholesterol , B. Cholesterol ester, C. Free

460

cholesterol, D. Triacylglycerols. Results expressed as mean ± SE (n=10). * indicates a

461

significant difference between the respective groups at p≤0.05.

462

Figure 5. Fecal bile acid and cholesterol levels. Animals were divided into six diet

463

groups and fed respective diets for 8 weeks. Fecal samples collected on the day of

464

sacrifice and fecal bile acid, cholesterol levels measured as described in the Materials and 23 ACS Paragon Plus Environment


465

Methods. The diet groups were : LF, HF, LFMG, LFRC, HFMG and HFRC. A. Fecal

466

bile acid, B. Fecal total cholesterol. Results expressed as mean ± SE (n=10/group). *

467

indicates a significant difference between the respective groups at p≤0.05.

468

Figure 6. Cholesterol, triacylglycerol metabolism and inflammation-related genes

469

expression levels in liver. Animals were divided into six diet groups and fed respective

470

diets for 8 weeks. Liver samples obtained at the end of 8 weeks. RNA isolated and

471

expression of marker genes assessed using RT-PCR as described in the Materials and

472

Methods. The diet groups were : LF, HF, LFMG, LFRC, HFMG and HFRC. A.

473

CYP7A1 , B. ACAT1, C. ACAT3 , D. SOAT1, E. DGAT1, F. LCAT, G. CRP, H. TNF-

474

α. Results expressed as mean ± SE (n=10/group). * indicates a significant difference

475

between the respective groups at p≤0.05.

476


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Page 25 of 41


Table 1. Diet composition. Dieta LFa g% kcal% Protein 19 20 Carbohydrate 67 70 Fat 4 10 Total 100 Kcal/g 3.8 Ingredient g kcal Casein 200 800 L-Cystine 3 12 Corn starch 452.2 1809 Maltodextrin 75 300 Sucrose 172.8 691 Cellulose 50 0 Corn oil 25 225 Lard 20 180 Mineral mix 10 0 Dicalcium phosphate 13 0 Calcium carbonate 5.5 0 Potassium citrate 16.5 0 Vitamin mix 10 40 Choline Bitartrate 2 0 b MG powder RC powderb Total 1055 4057

477

478 479 480 481 482

LF + MGa g% kcal% 19 20 67 70 4 10 100 3.8 g kcal 200 800 3 12 452.2 1809 75 300 172.8 691 50 0 25 225 20 180 10 0 13 0 5.5 0 16.5 0 10 40 2 0 11.63 1066.63

4057

LF + RCa g% kcal% 19 20 66 70 4 10 100 3.8 g kcal 200 800 3 12 452.2 1809 75 300 172.8 691 50 0 25 225 20 180 10 0 13 0 5.5 0 16.5 0 10 40 2 0 17.81 1072.81

4057

g% 24 41 24

HFa kcal% 20 35 45 100

4.7 g 200 3 72.8 100 172.8 50 25 177.5 10 13 5.5 16.5 10 2

kcal 800 12 291 400 691 0 225 1598 0 0 0 0 40 0

858.1

4057

a

HF + MGa g% kcal% 23 20 41 35 23 45 100 4.7 g kcal 200 800 3 12 72.8 291 100 400 172.8 691 50 0 25 225 177.5 1598 10 0 13 0 5.5 0 16.5 0 10 40 2 0 9.46 867.56

4057

HF + RCa g% kcal% 23 20 41 35 23 45 100 4.6 g kcal 200 800 3 12 72.8 291 100 400 172.8 691 50 0 25 225 177.5 1598 10 0 13 0 5.5 0 16.5 0 10 40 2 0 14.49 872.59

4057

All the diets used in this animal study were formulated by Research Diet (New Brunswick, NJ, USA). LF, LF + MG, and LF + RC represent low fat diet, low fat diet supplemented with red cabbage microgreens powder, and low fat diet supplemented with mature red cabbage powder, respectively. Similarly, HF, HF + MG and HF + RC represent high fat diet, high fat diet supplemented with red cabbage microgreens powder, and high fat diet supplemented with mature red cabbage powder, respectively. b MG powder is the powder made from lyophilized red cabbage microgreens and RC powder is the powder made from lyophilized mature red cabbage.



Table 2A. Anthocyanin in microgreen and mature red cabbage Red Cabbage Microgreen Tentative Identification µmol/g cyanidin 3-diglucoside-5-glucoside 0.41 cyanidin 3-(sinapoyl)-diglucoside-5-glucosides 1.13 cyanidin 3-(glucosyl)(sinapoyl)(p-coumaroyl)sophorside-5glucoside 0.49 cyanidin 3-(glucosyl)(sinapoyl)(feruloyl)sophorside-5glucoside 0.57 cyanidin 3-diferuloylsophoroside-5-glucoside 0.59 cyanidin 3-(coumaroyl)sophoroside-5-glucoside 0.57 cyanidin 3-(feruloyl)sophoroside-5-glucoside 1.68 cyanidin 3-diferuloylsophoroside-5-glucoside 2.06 cyanidin 3-(sinapoyl)(feruloyl)sophoroside-5-glucoside 1.30 cyanidin 3-(sinapoyl)(sinapoyl)sophoroside-5-glucoside 3.65 Total 12.44 Mature Red Cabbage Tentative Identification µmol/g cyanidin 3-diglucoside-5-glucoside 2.42 cyanidin 3-(sinapoyl)-diglucoside-5-glucosides 0.63 cyanidin 3-(glucosyl)(sinapoyl)(p-coumaroyl)sophorside-5glucoside 0.82 cyanidin-3-(glucosyl)(sinapoyl)(feruloyl)sophorside-5glucoside 1.54 cyanidin 3-(coumaroyl)sophoroside-5-glucoside 0.83 cyanidin 3-diferuloylsophoroside-5-glucoside 1.27 cyanidin 3-(feruloyl)sophoroside-5-glucoside 22.96 cyanidin 3-(sinapoyl)(feruloyl)sophoroside-5-glucoside 0.74 cyanidin 3-(sinapoyl)(sinapoyl)sophoroside-5-glucoside 2.13 Total 33.36 483 484


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Page 27 of 41


Table 2B. Other polyphenols in microgreen and mature red cabbage Red Cabbage Microgreen Tentative Identification µmol/g citric acid 0.27 3-caffeoylquinic acid 2.40 caffeoyl glucose 1.62 hydroxyferuloylglucose 1.59 kaempferol 3-diglucoside 2.80 sinapoyl-hexose 27.80 disinapoylgentiobiose 12.00 1,2,2'-trisinapoylgentionbiose 8.36 unknown 1.72 Total 58.57 Mature Red Cabbage Tentative Identification µmol/g citric acid 0.36 3-caffeoylquinic acid 1.92 sinapoyl-hexose 0.99 kaempferol acyl-glucoside 11.89 kaempferol 3-sinapoylsophoroside-7-glucoside 0.17 kaempferol 3-hydroxyferuloylsophorotrioside-7-glucoside 0.69 kaempferol 3-sinapoylferuloylsophoroside-7-glucoside 0.48 kaempferol 3-disinapoyldiglucoside-7-glucoside 0.07 1,2,2'-trisinapoylgentionbiose 0.27 unknown 0.39 Total 17.22 485 486



487

Table. 3 PDA response factor of desulfo glucosinolate No. 1 2 3 4 5 6 7 8

Desulfo glucosinolate desulfosinigrin desulfoglucoraphanin Desulfo-4-hydroxyglucobrassicin Other desulfo- hydroxyglucobrassicin Desulfo-glucobrassicin Desulfo-4-methoxyglucobrassicin Other desulfo methoxyglucobrassicin Other glucosinolate

Response factor 1.00 1.07 0.28 0.28 0.20 0.25 0.25 1.00

488 489

Table. 4 Desulfo-glucosinolates in microgreen and mature red cabbage

1 2 3 4 5 6 7 8 9

progoitrin glucoraphanin sinigrin gluconapin 4-hydroxyglucobrassicin glucoerucin glucobrassicin 4-methoxylglucobrassicin neoglucobrassicin Total

Red Cabbage Microgreen (µmol/g dry weight) 6.87 4.80 6.12 1.31 1.96 1.16 1.15 0.51 0.15 17.15

Mature Red Cabbage (µmol/g dry weight) 2.29 0.88 1.53 1.33 0.39 0.37 1.26 0.26 N.D 8.30

490 491


Page 28 of 41

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492


Figure 1. Red cabbage microgreen and red cabbage.

493



Figure 2. Food intake and body weight gain.

H FR C

G H FM

LF R C

G LF M

H F

LF

Growth rate (g/week)

Food intake (g/week)

494

495 496 497 498 499


Page 30 of 41

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500


Figure 3. Plasma lipoproteins levels.

A. VLDL 6

VLDL (mg/dl)

5 4

LDL (mg/dl)

LF HF LFMG LFRC HFMG HFRC

3 2 1

Diets

C. HDL

* *

*

140


120 100 80 60 40 20

H FR C

H FM G

LF R C

LF M G

H F

0

LF

Diets

501 502 503


HF R C

R C

H FM G

LF

M G LF

H F

LF

H FR C

R C

H FM G

LF

M G LF

H F

LF

0


505 506


C FR FR

C

H

G FM G

H

FM

C H H

LF R R C LF

M G LF

F

G

F H H

LF M

LF LF

C FR H

C FR

FM

FM

G

H

H

H

G

C LF R

RC LF

G LF M

LF

M G

H

H

F

LF

F

Figure 4. Liver cholesterol and triacylglycerol levels.

LF

504

Page 32 of 41

Page 33 of 41

507


Figure 5. Fecal bile acid and cholesterol levels.

A. Fecal Bile Acid

*

Fecal Bile Acid ( mol CA equiv/mg)

10


8

6

4

2

H FR C

H FM G

LF RC

F H

LF M G

LF

0

Diets

B. Fecal Total Choleterol 2.0

*

Total Cholesterol (µg/mg)

*


1.5

1.0

0.5

H FR C

G H FM

C

F

G

LF R

LF M

H

LF

0.0

Diets

508 509



Figure 6. Cholesterol and triacylglycerol metabolism and inflammation-related genes expression

511

levels in liver.

C

G

H FR

RC

FM

LF

LF

H

F

G

H

M

LF

G

FR C H

R C

FM

LF

H

F

G M LF

H

LF

Relative mRNA Levels

510

D. SOAT1

*

1.4

*

1.2 1.0 0.8 0.6 0.4 0.2

C F+ R H

G

+R C LF

H

LF

F+ M

G

F H

+M

LF

C

G

HF R

C R LF

H FM

G M LF

LF

H F

0.0

Relative mRNA Levels

*

*

C HF R

FM H

LF

H. TNF-

3.0

G

C R

G

F H

M LF

C

G

HF R

FM

C R LF

H

LF

M

G

F H

LF

LF

Diets

* LF HF LFMG LFRC HFMG HFRC

2.5 2.0 1.5 1.0 0.5

H

FR C H

G FM

RC LF

G

F H

M LF

LF

C

G

H FR

FM

RC LF

H

LF

M

G

F H

LF

0.0

Diets

512 513


Page 34 of 41

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514

Supplemental information

515

Fig. 1 HPLC chromatogram of microgreen and red cabbage at 330 nm A: MG and B: RC RT: 0.00 - 60.00 NL: 1.02E6 nm=329.5330.5 PDA MG

10.01 1000000

A

900000 800000 700000

23.43

9.90

uAU

600000

26.94

500000 400000 23.06

300000 200000

12.04

100000

1.64 4.39

0 300000

8.83 8.94 7.56

4.63

14.36 17.53

19.72

21.46 20.15

15.21

B

24.89

29.19

32.11 34.16 35.22 37.34 39.44

43.72 45.66 48.35

50.07 51.26

51.52

58.00 NL: 3.01E5 nm=329.5330.5 PDA rc

20.19

250000

200000 13.74

14.71

23.10

150000

100000

19.86 4.64

50000 1.69

5.84

4.42

22.89

10.03 11.45 9.91 8.95

16.00 18.68

26.96

23.44

29.28 30.60

34.37 35.91

0 0

5

10

15

20

25

516 517

30 Time (min)

35

42.65

40.06 40

45.34 47.64 50.06 51.25 45

56.84 59.21

54.21

50

55

Fig. 2 HPLC chromatogram of microgreen and red cabbage at 520 nm A: MG and B: RC RT: 0.00 - 60.00 NL: 2.19E5 nm=519.5520.5 PDA MG

23.06

A

200000 180000 160000

uAU

140000 120000 100000

22.47

80000 60000 20.27

40000 11.33 20000 1.84 2.26 4.47

0

7.57

10.33

18.30

12.80

46.72 48.35 50.06 51.26

18.69 23.52

34.05 35.98 37.31 40.22 42.43 44.84 46.30

25.24

NL: 4.31E5 nm=519.5520.5 PDA rc

20.19

400000

B

350000 300000 250000 200000 19.85

150000

23.10

100000

28.68 30.61 1.77

5.12 6.84

34.23 35.91

59.21

22.50

7.58

50000

10.41

11.34

15.47

16.02

27.77

18.67

33.88

37.65

41.65 42.50 43.02

45.49

50.06

52.61 54.47 57.01

58.62

0 0

5

10

15

20

25

30 Time (min)

518

35

519 35 ACS Paragon Plus Environment

40

45

50

55

60

60


520

Page 36 of 41

Fig. 3 The HPLC chromatogram of desulfo glucosinolates in A: MG and B: RC

RT: 1.06 - 19.28 160000 150000

NL: 1.64E5 Channel A UV RCMG1A

A

140000 130000 120000 110000 100000

uAU

90000 80000 70000 60000 50000 40000 30000 20000 10000 0 2

3

4

5

6

7

8

9

10 Time (min)

521 522

11

12

13

14

15

16

17

18

19

RT: 0.11 - 19.41

110000

NL: 1.26E5 Channel A UV RCVP1A

B

100000 90000 80000 70000

uAU

60000 50000 40000 30000 20000 10000 0 -10000 1

2

3

4

5

6

7

8

9

523 524

10 Time (min)

11

12

525


13

14

15

16

17

18

19

Page 37 of 41

526


Fig. 4 Initial and final animal body weight (week 1 and week 8)

527

528 529 530



531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577

References 1. Milani, R. V.; Lavie, C. J., Health care 2020: reengineering health care delivery to combat chronic disease. The American journal of medicine 2015, 128, 337-343. 2. Ward, B. W.; Schiller, J. S.; Goodman, R. A., Multiple Chronic Conditions Among US Adults: A 2012 Update. Preventing Chronic Disease 2014, 11, E62. 3. CDC Chronic Disease Notes & Report; CENTERS FOR DISEASE CONTROL AND PREVENTION: Cultivating Healthy Communities 2009; p 49. 4. Hung, H.-C.; Joshipura, K. J.; Jiang, R.; Hu, F. B.; Hunter, D.; Smith-Warner, S. A.; Colditz, G. A.; Rosner, B.; Spiegelman, D.; Willett, W. C., Fruit and vegetable intake and risk of major chronic disease. J. Natl. Cancer Inst. 2004, 96, 1577-1584. 5. Slavin, J. L.; Lloyd, B., Health Benefits of Fruits and Vegetables. Advances in Nutrition 2012, 3, 506-516. 6. Minich, D. M.; Bland, J. S., A review of the clinical efficacy and safety of cruciferous vegetable phytochemicals. Nutr. Rev. 2007, 65, 259-267. 7. Sarkar, F. H.; Li, Y. W., Indole-3-carbinol and prostate cancer. J. Nutr. 2004, 134, 3493S-3498S. 8. Chan, R.; Lok, K.; Woo, J., Prostate cancer and vegetable consumption. Molecular Nutrition & Food Research 2009, 53, 201-216. 9. LeeChungSoo; Pill, W. G., Advancing Greenhouse Establishment of Radish, Kale and Amaranth Microgreens through Seed Treatments. Horticulture, Environment, and Biotechnology 2005, 46, 363-368. 10. Xiao, Z.; Lester, G. E.; Luo, Y.; Wang, Q., Assessment of Vitamin and Carotenoid Concentrations of Emerging Food Products: Edible Microgreens. J. Agric. Food Chem. 2012, 60, 7644-7651. 11. Singh, J.; Upadhyay, A.; Bahadur, A.; Singh, B.; Singh, K.; Rai, M., Antioxidant phytochemicals in cabbage (Brassica oleracea L. var. capitata). Scientia Horticulturae 2006, 108, 233-237. 12. Mizgier, P.; Kucharska, A. Z.; Sokół-Łętowska, A.; Kolniak-Ostek, J.; Kidoń, M.; Fecka, I., Characterization of phenolic compounds and antioxidant and anti-inflammatory properties of red cabbage and purple carrot extracts. Journal of Functional Foods 2016, 21, 133-146. 13. Demirdöven, A.; Karabıyıklı, Ş.; Tokatlı, K.; Öncül, N., Inhibitory effects of red cabbage and sour cherry pomace anthocyanin extracts on food borne pathogens and their antioxidant properties. LWT-Food Science and Technology 2015, 63, 8-13. 14. Al-Dosari, M. S., Red cabbage (Brassica oleracea L.) mediates redox-sensitive amelioration of dyslipidemia and hepatic injury induced by exogenous cholesterol administration. The American journal of Chinese medicine 2014, 42, 189-206. 15. Nordestgaard, B. G.; Chapman, M. J.; Ray, K.; Borén, J.; Andreotti, F.; Watts, G. F.; Ginsberg, H.; Amarenco, P.; Catapano, A.; Descamps, O. S., Lipoprotein (a) as a cardiovascular risk factor: current status. European heart journal 2010, ehq386. 16. Sowers, J. R., Obesity as a cardiovascular risk factor. The American journal of medicine 2003, 115, 37-41. 17. Hokanson, J. E.; Austin, M. A., Plasma triglyceride level is a risk factor for cardiovascular disease independent of high-density lipoprotein cholesterol level: a metaanalysis of population-based prospective studies. Journal of cardiovascular risk 1996, 3, 213-219.


Page 38 of 41

Page 39 of 41

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18. Rapeseed, I., Determination of glucosinolates content—Part 1: Method using high performance liquid chromatography. 9167-1: 1992 (E). International Standard Organization, Geneva, Switzerland 1992, 1-9. 19. Lin, L. Z.; Harnly, J.; Zhang, R. W.; Fan, X. E.; Chen, H. J., Quantitation of the Hydroxycinnamic Acid Derivatives and the Glycosides of Flavonols and Flavones by UV Absorbance after Identification by LC-MS. Journal of Agricultural and Food Chemistry 2012, 60, 544-553. 20. Sun, J.; Xiao, Z.; Lin, L.-z.; Lester, G. E.; Wang, Q.; Harnly, J. M.; Chen, P., Profiling Polyphenols in Five Brassica Species Microgreens by UHPLC-PDA-ESI/HRMSn. Journal of Agricultural and Food Chemistry 2013, 61, 10960-10970. 21. Finley, J. W., Proposed criteria for assessing the efficacy of cancer reduction by plant foods enriched in carotenoids, glucosinolates, polyphenols and selenocompounds. Annals of Botany 2005, 95, 1075-1096. 22. Gliszczyńska-Świgło, A.; Ciska, E.; Pawlak-Lemańska, K.; Chmielewski, J.; Borkowski, T.; Tyrakowska, B., Changes in the content of health-promoting compounds and antioxidant activity of broccoli after domestic processing. Food additives and contaminants 2006, 23, 1088-1098. 23. Cohen, J. C., Contribution of cholesterol 7 alpha-hydroxylase to the regulation of lipoprotein metabolism. Current Opinion in Lipidology 1999, 10, 303-307. 24. Jelinek, D. F.; Andersson, S.; Slaughter, C. A.; Russell, D. W., Cloning and regulation of cholesterol 7 alpha-hydroxylase, the rate-limiting enzyme in bile acid biosynthesis. Journal of Biological Chemistry 1990, 265, 8190-8197. 25. Giugliano, D.; Ceriello, A.; Esposito, K., The effects of diet on inflammation Emphasis on the metabolic syndrome. Journal of the American College of Cardiology 2006, 48, 677-685. 26. Wouters, K.; van Gorp, P. J.; Bieghs, V.; Gijbels, M. J.; Duimel, H.; Lutjohann, D.; Kerksiek, A.; van Kruchten, R.; Maeda, N.; Staels, B.; van Bilsen, M.; Shiri-Sverdlov, R.; Hofker, M. H., Dietary cholesterol, rather than liver steatosis, leads to hepatic inflammation in hyperlipidemic mouse models of nonalcoholic steatohepatitis. Hepatology 2008, 48, 474-86. 27. Lecerf, J. M.; de Lorgeril, M., Dietary cholesterol: from physiology to cardiovascular risk. Br. J. Nutr. 2011, 106, 6-14. 28. Aggarwal, B. B.; Shishodia, S.; Sandur, S. K.; Pandey, M. K.; Sethi, G., Inflammation and cancer: How hot is the link? Biochem. Pharmacol. 2006, 72, 1605-1621. 29. Bosma-den Boer, M. M.; van Wetten, M. L.; Pruimboom, L., Chronic inflammatory diseases are stimulated by current lifestyle: how diet, stress levels and medication prevent our body from recovering. Nutrition & Metabolism 2012, 9. 30. Reuter, S.; Gupta, S. C.; Chaturvedi, M. M.; Aggarwal, B. B., Oxidative stress, inflammation, and cancer How are they linked? Free Radic. Biol. Med. 2010, 49, 1603-1616. 31. Coleman, R. A.; Lee, D. P., Enzymes of triacylglycerol synthesis and their regulation. Progress in lipid research 2004, 43, 134-176. 32. Harris, C. A.; Haas, J. T.; Streeper, R. S.; Stone, S. J.; Kumari, M.; Yang, K.; Han, X.; Brownell, N.; Gross, R. W.; Zechner, R., DGAT enzymes are required for triacylglycerol synthesis and lipid droplets in adipocytes. Journal of lipid research 2011, 52, 657-667. 33. Chang, T. Y.; Chang, C. C. Y.; Ohgami, N.; Yamauchi, Y., Cholesterol sensing, trafficking, and esterification. Annual Review of Cell and Developmental Biology 2006, 22, 129-157. 34. Thompson, S. L.; Krisans, S., Rat liver peroxisomes catalyze the initial step in cholesterol synthesis. The condensation of acetyl-CoA units into acetoacetyl-CoA. Journal of Biological Chemistry 1990, 265, 5731-5735.



626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667

35. Glomset, J.; Janssen, E.; Kennedy, R.; Dobbins, J., Role of plasma lecithin: cholesterol acyltransferase in the metabolism of high density lipoproteins. Journal of lipid research 1966, 7, 639-648. 36. Li, L.; Hossain, M. A.; Sadat, S.; Hager, L.; Liu, L.; Tam, L.; Schroer, S.; Huogen, L.; Fantus, I. G.; Connelly, P. W., Lecithin cholesterol acyltransferase null mice are protected from diet-induced obesity and insulin resistance in a gender-specific manner through multiple pathways. J. Biol. Chem. 2011, 286, 17809-17820. 37. Li, L.; Naples, M.; Song, H.; Yuan, R.; Ye, F.; Shafi, S.; Adeli, K.; Ng, D. S., LCAT-null mice develop improved hepatic insulin sensitivity through altered regulation of transcription factors and suppressors of cytokine signaling. American Journal of Physiology-Endocrinology and Metabolism 2007, 293, E587-E594. 38. Lind, L., Circulating markers of inflammation and atherosclerosis. Atherosclerosis 2003, 169, 203-214. 39. Wanders, D.; Burk, D. H.; Cortez, C. C.; Van, N. T.; Stone, K. P.; Baker, M.; Mendoza, T.; Mynatt, R. L.; Gettys, T. W., UCP1 is an essential mediator of the effects of methionine restriction on energy balance but not insulin sensitivity. The FASEB Journal 2015, 29, 26032615. 40. Alvarez-Crespo, M.; Csikasz, R. I.; Martínez-Sánchez, N.; Diéguez, C.; Cannon, B.; Nedergaard, J.; López, M., Essential role of UCP1 modulating the central effects of thyroid hormones on energy balance. Molecular metabolism 2016, 5, 271-282. 41. Goldwasser, J.; Cohen, P. Y.; Yang, E.; Balaguer, P.; Yarmush, M. L.; Nahmias, Y., Transcriptional regulation of human and rat hepatic lipid metabolism by the grapefruit flavonoid naringenin: role of PPARα, PPARγ and LXRα. PloS one 2010, 5, e12399. 42. Kamiya, T.; Nagamine, R.; Sameshima-Kamiya, M.; Tsubata, M.; Ikeguchi, M.; Takagaki, K., The isoflavone-rich fraction of the crude extract of the Puerariae flower increases oxygen consumption and BAT UCP1 expression in high-fat diet-fed mice. Global journal of health science 2012, 4, 147. 43. Ngamukote, S.; Mäkynen, K.; Thilawech, T.; Adisakwattana, S., Cholesterol-lowering activity of the major polyphenols in grape seed. Molecules 2011, 16, 5054-5061. 44. Manach, C.; Mazur, A.; Scalbert, A., Polyphenols and prevention of cardiovascular diseases. Current opinion in lipidology 2005, 16, 77-84. 45. Saeed, S. A.; Manzoor, I.; Quadri, J.; Tasneem, S.; Simjee, S. U., Demystifying phytochemicals: an insight. International Journal of Pharmacology 2005, 1, 234-238. 46. Wiczkowski, W.; Szawara-Nowak, D.; Topolska, J., Red cabbage anthocyanins: Profile, isolation, identification, and antioxidant activity. Food research international 2013, 51, 303309. 47. Villarreal-García, D.; Jacobo-Velázquez, D. A., Glucosinolates from broccoli: Nutraceutical properties and their purification. Current Trends in Nutraceuticals 2016. 48. Traka, M., Health Benefits of Glucosinolates. Advances in Botanical Research 2016. 49. Gonzalez, R.; Ballester, I.; Lopez-Posadas, R.; Suarez, M. D.; Zarzuelo, A.; MartinezAugustin, O.; De Medina, F., Effects of Flavonoids and other Polyphenols on Inflammation. Critical Reviews in Food Science and Nutrition 2011, 51, 331-362.

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