In Vitro Bile Acid Binding Capacities of Red Leaf Lettuce and

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In vitro bile acid binding capacities of red leaf lettuce and cruciferous vegetables Isabelle Yang, Guddadarangavvanahal Jayaprakasha, and Bhimanagouda S. Patil J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02540 • Publication Date (Web): 16 Aug 2017 Downloaded from http://pubs.acs.org on August 17, 2017

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Journal of Agricultural and Food Chemistry

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In vitro bile acid binding capacities of red leaf lettuce and cruciferous

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vegetables

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Isabelle F. Yang, Guddadarangavvanahally K. Jayaprakasha*, and Bhimanagouda S. Patil*

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Vegetable and Fruit Improvement Center, Department of Horticultural Sciences,

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Texas A&M University, 1500 Research Parkway, Suite# A120, College Station, TX 77843, USA

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Short title: In vitro bile acid binding of vegetables

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*Corresponding authors

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Bhimanagouda S. Patil Vegetable and Fruit Improvement Center, Department of Horticultural Sciences, 1500 Research Parkway, Suite# A120, College Station, TX 77845, USA Tel.: +1 979 862 4521; Fax: +1 979 862 4522. E-mail: [email protected]

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G.K. Jayaprakasha Vegetable and Fruit Improvement Center, Department of Horticultural Sciences, 1500 Research Parkway, Suite# A120, College Station, TX 77845, USA Tel.: +1 979 845 3864; Fax: +1 979 862 4522 E-mail: [email protected] ; [email protected]

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ABSTRACT: In the present study, we tested the bile acid binding capacity of red leaf lettuce,

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red cabbage, red kale, green kale, and Brussels sprouts in vitro digestion process by simulating

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mouth, gastric, and intestinal digestion using six bile acids at physiological pH. Green and red

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kale exhibited significantly higher (86.5±2.9 and 89.7±0.9%) bile acid binding capacity

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compared with the other samples. Further, three different compositions of bile acids were tested

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to understand the effect on different health conditions. To predict the optimal dose for bile acid

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binding for each condition, we established a logistic relationship between kale dose and bile acid

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binding capacity. The results indicated that kale showed significantly higher bile acid binding

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capacity (82.5±2.9% equivalent to 72.06 mg) at 1.5 g sample and remained constant up to 2.5 g.

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In addition, minimally processed (microwaved 3 min or steamed 8 min) green kale showed

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significantly enhanced bile acid binding capacity (91.1±0.3 and 90.2±0.7%) as compared with

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lyophilized kale (85.5±0.24%). Among the six bile acids tested, kale preferentially bound

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hydrophobic bile acids chenodeoxycholic acid and deoxycholic acid. Therefore, regular

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consumption of kale, especially minimally processed kale, can help excrete more bile acids and

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thus may lower the risk of hypercholesterolemia.

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KEYWORDS: Brassica oleracea, kale, red leaf lettuce, bile salt, in vitro digestion

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1.

INTRODUCTION

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Bile acids are amphipathic molecules that function as emulsifiers to assist in digestion and

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absorption of lipids in the gastrointestinal tract. Cholesterol can be biosynthesized into bile

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acids, including cholic acid (CA), chenodeoxycholic acid, lithocholic acid (LCA), deoxycholic

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acid (DCA),

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glycodeoxycholic acid (GDCA).1 The two primary bile acids, cholic acid and chenodeoxycholic

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acid, are synthesized by the liver, stored in the gallbladder, and then secreted into the small

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intestine, when food is being digested.2 Human intestinal bacteria can convert cholic acid and

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chenodeoxycholic acid to secondary bile acids such as deoxycholic acid and lithocholic acid

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though dehydroxylation. After assisting in lipid digestion, bile acids are reabsorbed through the

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ileum and transferred back to the liver. Up to 95% of bile acids are reabsorbed,1, 2 which limits

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the synthesis of new bile acids from cholesterol. Some bile acids can escape the enterohepatic

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circulation and reach the colon, where gut bacteria form more deoxycholic acid and lithocholic

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acid.1 Lithocholic acid, deoxycholic acid, and chenodeoxycholic acid are hydrophobic bile acids,

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which are considered more toxic to liver and colon cells, and may induce colon cancer.3-5

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However, dietary fiber increases the excretion of bile acids in the stool,6 which can potentially

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prevent colon cancer and lower cholesterol levels.7 Besides dietary fiber, vitamin D and calcium

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supplements also have a protective function against colon cancer.8 Hence, binding bile acids can

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reduce intestinal cancer and reduce plasma cholesterol levels. 9, 10

glycochenodeoxycholic acid

(GCDCA),

glycocholic acid (GCA), and

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When hydrophobic bile acids are conjugated with taurine or glycine, they become more

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hydrophilic and much less toxic to hepatic and intestinal cells.11 Some conjugated bile acids,



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such as tauroursodeoxycholic acid, can even stabilize hepatocyte membranes and enhance the

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defense against oxidative stress, thus protecting the liver.12 Bile acid sequestrants prevent bile

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acid absorption and promote synthesis of primary bile acids from cholesterol, which leads to a

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reduction of serum cholesterol.13 Some foods can function like bile acids sequestrants; for

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instance, guar gum can reduce the concentration of serum bile acids by binding them in the

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gastrointestinal tract, which relieves the symptoms associated with intrahepatic cholestasis of

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pregnancy,14 and reduces plasma cholesterol levels.15

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Beyond their functions in digestive emulsification and reduction of cholesterol, bile acids

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are also associated with glucose metabolism. Because bile acids can activate farnesoid X

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receptor and G-protein coupled bile acid receptor 1, which are involved in glucose metabolism,

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the control of bile acid absorption can be used as a potential novel therapy for some metabolic

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diseases, such as type 2 diabetes.2,

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hydrochloride, has been approved by FDA in 2008 as a medicine for treatment of type-2

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diabetes.16 In addition, bile acid binding has recently emerged as a supporting approach to

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strengthen the standard treatments used to control plasma glucose in type-2 diabetic patients.17

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Several studies suggested that bile acid sequestrants reduced the fasting plasma glucose level in

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type-2 diabetes patients.17-20 Therefore, a vegetable with good bile acid binding capacity may

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also lower the plasma glucose level of type-2 diabetic patients.

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In fact one of the bile acid sequestrants, colesevelam

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Previous studies have examined the bile acid binding capacities of various vegetables, but

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few studies have examined the binding preferences for individual bile acids by specific

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vegetables.21 According to previous research, collard greens, kale, mustard greens, broccoli,

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Brussels sprouts, spinach, green bell peppers, and cabbage have good bile acid binding

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capacity.21 However, whether hydrophobic or hydrophilic bile acids are preferentially bound by 4 ACS Paragon Plus Environment

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these vegetables has yet to be tested. Whether the different compositions of bile acid mixtures

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can influence the in vitro binding capacity also remains unknown. This study compares the in

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vitro bile acid binding capacities of different vegetables, including red cabbage, red leaf lettuce,

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Brussels sprouts, red kale, and green kale. The most active vegetable was used to test different

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bile acid compositions and optimal amounts for bile acid binding.

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2. MATERIALS AND METHODS

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2.1 Materials. Fresh green kale (Brassica oleracea acephala), red cabbage (Brassica

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oleracea capitata rubra), red kale (Brassica oleracea acephal, sabellica), Brussels sprouts

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(Brassica oleracea gemmifera), and red leaf lettuce (Lactuca sativa) were purchased from a local

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supermarket. All vegetables were cut into small pieces, lyophilized, powdered and stored at -

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20°C until further use.

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2.2. Chemicals. Sodium glycocholate (G7132), sodium cholate (C1254), sodium

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glycochenodeoxycholate

(G0759),

sodium

glycodeoxycholate

(G9910),

sodium

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chenodeoxycholate (C8261), sodium deoxycholate (D6750), ammonium nitrate, potassium

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dihydrogen phosphate, potassium chloride, potassium citrate, uric acid sodium salt, urea, lactic

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acid sodium salt, porcine gastric mucin, α-amylase, pepsin, and pancreatin were obtained from

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Sigma-Aldrich Co (St. Louis, MO, USA).

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2.3. Screening of Different Vegetables for Bile Acid Binding Ability. The in vitro

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digestion was performed to mimic digestion in the mouth, stomach, and small intestine (Figure

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1). Simulated saliva fluid (SSF) was prepared by dissolving the reagents (Table S1) in pH 6.8

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phosphate buffer.22, 23 In the oral digestion phase, 2.0 g lyophilized vegetable was mixed with an 5 ACS Paragon Plus Environment


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equal weight of water and 10 mL of the SSF containing 0.31 mg α-amylase. The samples were

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vortexed and then placed in a shaking water bath (37°C and 180 rpm) for 5 min. During the

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gastric digestion phase, the mixture from the oral digestion phase was adjusted to pH 2.0 with 1

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N HCl, followed by the addition of 600 µL pepsin buffer (200 µg pepsin in 1 mL 0.1 M HCl)

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vortexing and incubation in a shaking water bath (37°C and 180 rpm) for 1 hour. For the

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intestinal phase, chyme from the gastric digestion phase was first adjusted to pH 6.8 with 1 N

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NaOH. The sample was then mixed with 5 ml pancreatin (6.25 mg/mL in 50 mM phosphate

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buffer), 4 mL bile acid mixture solution prior to incubation in a shaking water bath shaking at

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(37°C and 180 rpm) for 3 h.22-25 The bile acid mixture contained 10 mM cholate, 10 mM

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deoxycholate, 10 mM glycochenodeoxycholate, 10 mM glycocholate, and 10 mM

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chenodeoxycholate in potassium phosphate buffer (pH 6.8). After incubation with the bile acids,

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the in vitro digestion was terminated by inactivating enzymes in a 78°C water bath for 7 min.

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The analyses were conducted using triplicate samples of freeze dried red cabbage, red kale juice,

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green kale, red leaf lettuce, and Brussels sprouts (2.0 g each). Digestion chymes were centrifuged

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at 3600 rpm and supernatants were collected and filtered through Whatman filter paper. The

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residue was washed with 20 mL of nanopure water by mixing on a shaking water bath for 3 h

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and centrifuged at 800 g. Both the supernatants were filtered, combined, and concentrated under

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vacuum by rotary evaporation at 40°C to obtain 7 mL, and passed through 0.45 µm cellulose

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filters for HPLC analysis.

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2.4. In vitro Digestion of Different Bile Acid Compositions. Bile acid composition differs

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in various individuals depending on their health and gender. To understand the binding capacity

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is influenced by the composition of bile acid mixture, we used three different compositions

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equivalent to male patients with gallstones (BAC-1), healthy females (BAC-2), and males with 6 ACS Paragon Plus Environment

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type 2 diabetes (BAC-3).26, 27 Mixtures with three different bile acid compositions (BAC) were

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prepared as shown in Table S2. The BAC-1 bile acid mixture contained 33.3% cholic acid in

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both conjugated and unconjugated forms, 24.9% deoxycholic acid, and 41.8% chenodeoxycholic

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acid. The BAC-2 mixture contained 33.1% cholic acid, 17% deoxycholic acid, and 49.9%

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chenodeoxycholic acid. The BAC-3 mixture contained 33.3% cholic acid, 39.3% deoxycholic

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acid, and 27.4% chenodeoxycholic acid. Lyophilized green kale was used for digestion steps

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according to the previously described in vitro digestion protocol and during intestinal digestion

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phase, 4 mL of the bile acid mixture with different compositions was used. Except the bile acid

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mixture, other steps of the in vitro experiment are same as shown in Figure 1. After in vitro

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digestion, the supernatant was concentrated by rotary evaporation and filtered through a cellulose

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filter for HPLC analysis.

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2.5. Impact of Different Kale Parts on Bile Acid Binding Capacity. Lyophilized kale

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stem, kale leaf, and whole kale were tested for in vitro bile acid binding capacity, through oral

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digestion, gastric digestion, and intestinal digestion phases, according to the above protocol

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except during the intestinal digestion phase, the bile acid mixture containing taurocholate (4.84

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mM), cholate (13.9 mM) and deoxycholate (6.16 mM) was used. After the in vitro digestion, the

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supernatant was concentrated to achieve the minimal volume under vacuum, and passed through

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cellulose filter for HPLC analysis.

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2.6. Optimization of Different Amounts of Kale for Bile Acid Binding. Kale was found

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to have the highest bile acid binding capacity among the tested vegetables, and therefore, to find

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the minimal kale amount needed to bind the maximum amount of bile acids, different weights of

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kale (0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0, and 2.5 g) were examined in the in vitro experiment. For



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this experiment, in the intestinal digestion phase, the bile acid mixture BAC-3 was used. All

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other procedures were the same as those described before.

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2.7. Minimal Processing of Green Kale for Bile Acid Binding. To understand the

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impact of mild processing of kale and bile acid binding ability, two minimal processing

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techniques were used as follows:

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a). Microwave processing. Fresh-cut kale (18.3 g equivalent to 2.0 g of dried sample) was

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weighed into three 150 mL beakers and 20 mL of water was added to the vegetables and

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microwaved for 1, 2, and 3 min separately, homogenized to a paste for 2–3 min. All samples

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were processed in triplicate and used for bioassays.

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b). Steaming. Fresh-cut kale (18.3 g) was taken in a 150 mL beaker and steaming was performed

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for 8 and 10 min at 95 °C using a water bath and homogenized to obtain a paste. Analysis of both

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the time points were conducted in triplicate and used to test the bile acid binding capacity.

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2.8. Extraction of Bound Bile Acids. Digested residue was extracted with

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methanol:water:formic acid (80:19:1), followed by 2 min homogenization, 2 h sonication at

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60°C, centrifuged for 30 min and filtered through Whatman filter paper. The residue was re-

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extracted with the same solvent, using 10 mL of solvent as mentioned above, and combined and

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concentrated by rotary evaporation at 40 °C under reduced pressure to obtain 5–10 mL and

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analyzed by HPLC

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2.9. Quantification of Bile Acids by HPLC. The HPLC system consisted of an Agilent

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HPLC 1200 series (Foster City, CA) with a degasser, quaternary pump, auto-sampler, column

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oven, and photodiode array detector. Elution of bile acids was carried out with gradient mobile

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phases of (A) 0.3 M phosphoric acid in water and (B) acetonitrile with a flow rate of 0.8 mL/min 8 ACS Paragon Plus Environment

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at 30 °C using Gemini C18 (Phenomenex, Torrance, CA) column. Bile acids were separated

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using the gradient, 75% to 45% A from 0 to 10 min, 45% to 10% in 10–20 min, 10% to 75% in

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20–25 min, maintained isocratic flow for 5 min. Data were processed using the

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CHEMSTATION software (Agilent, Foster City, CA). All six bile acids at different

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concentrations were injected to obtain the area. The calibration graphs were prepared by area

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versus different concentration (31.25 to 1000 ppm) of bile acids and regression equations are

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presented in Table 1. Bound bile acid samples were injected and the presence of bile acids in

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each sample was examined by comparing the retention times with those of the standard bile

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acids. This assay was conducted in triplicate with three independent experiments and results

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were averaged.

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2.10. Identification of Bile Acids by LC-MS. Bile acids were identified by ultra-high

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performance liquid chromatography time of flight-mass spectrometry (LC QTOF–MS) (Maxis

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Impact, Bruker Daltonics, Billerica, MA). Bound bile acid samples were separated on a Zorbax

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Eclipse Plus C18 rapid resolution column (1.8 micron partial size, 100 × 2.1 mm) using an

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Agilent 1290 UHPLC instrument (Agilent, Waldbronn, Germany). The separation was carried

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out at 65°C with a flow rate of 0.2 mL/min using gradient elution with increasing strength of

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acetonitrile in 0.1% formic acid. Mass spectral analyses were performed using ESI-Q-TOF mass

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spectrometer equipped with an electrospray ionization source in positive ion mode. Capillary

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voltage was maintained at 2.9 kV, source temperature was set at 65°C and nitrogen was used as

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the desolvation gas (12 L/min).

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2.11. Fiber analysis. Lyophilized green kale, red kale, red cabbage, red leaf lettuce, and

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Brussels sprouts were analyzed for total dietary fiber, soluble fiber, and insoluble fiber according

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to AOAC protocols at Medallion labs, Minneapolis. 9 ACS Paragon Plus Environment


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2.12. Statistical Analysis. All results were expressed as means ± SD. The data were

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evaluated by JMP Pro 12 and probability of P ≤0.05 was considered as statistically significant.

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All experiments were conducted in triplicate with two independent experiments.

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3. RESULTS AND DISCUSSION

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3.1. Bile Acid Binding Capacities of Leafy Vegetables. Binding bile acids has multiple

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health benefits.21, 23, 25 Although certain vegetables have been tested for in vitro bile acid binding

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capacity, individual bile acids were not quantified separately.21, 28, 29 It would be desirable to

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include human fecal bacteria in the in vitro model however, the intestinal microbiome diversity

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differs in different parts of the world, varies from individual to individual, and can be altered by

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diet.30-32 Thus, understanding bile acid binding using human intestinal bacteria will be

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challenging for large-scale screening studies. The present in vitro study simulated the digestion

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from mouth to small intestine and provides segmental results, which can be a reference for

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further studies with human fecal bacteria involved in the digestion for the most-active samples.

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The present study compared the bile acid binding capacities of different vegetables to test

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various compositions of bile acids. Figure 2 shows that green and red kale exhibited the highest

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total bile acid binding capacities, 87% and 90%, respectively. The bile acid binding capacity of

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kale and Brussel sprouts were higher than the results previously reported.21 This may be due to

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the low amount (0.1g) of samples used in the digestion process, as well as the sensitivity of the

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colorimetric assay used for the quantification of bile acids. Among the five tested bile acids, only

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cholic acid showed the lowest binding capacity by kale. The rest of the vegetables, such as red

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leaf lettuce (78%) exhibited significantly higher binding than Brussels sprout (62%) and red

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cabbage (58%). Deoxycholic acid and chenodeoxycholic acid are the more hydrophobic bile 10 ACS Paragon Plus Environment

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acids, and may induce colon cancer and liver stress,33, 34 but cholic acid is more hydrophilic and

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its liver toxicity is milder than that of the hydrophobic bile acids. The decreasing order of bile

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acid binding capacities of five vegetables were found to be red kale

In Vitro Bile Acid Binding Capacities of Red Leaf Lettuce and

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