Article pubs.acs.org/JAFC In Vitro Bile Acid Binding Capacities of Red Leaf Lettuce and Cruciferous Vegetables Isabelle F. Yang, Guddadarangavvanahally K. Jayaprakasha,* and Bhimanagouda S. Patil* Vegetable and Fruit Improvement Center, Department of Horticultural Sciences, Texas A&M University, 1500 Research Parkway, Suite A120, College Station, Texas 77843, United States *S Supporting Information ABSTRACT: In the present study, we tested the bile acid binding capacity of red leaf lettuce, red cabbage, red kale, green kale, and Brussels sprouts through in vitro digestion process by simulating mouth, gastric, and intestinal digestion using six bile acids at physiological pH. Green and red kale exhibited significantly higher (86.5 ± 2.9 and 89.7 ± 0.9%, respectively) bile acid binding capacity compared to the other samples. Further, three different compositions of bile acids were tested to understand the effect on different health conditions. To predict the optimal dose for bile acid binding, we established a logistic relationship between kale dose and bile acid binding capacity. The results indicated that kale showed significantly higher bile acid binding capacity (82.5 ± 2.9% equivalent to 72.06 mg) at 1.5 g sample and remained constant up to 2.5 g. In addition, minimally processed (microwaved 3 min or steamed 8 min) green kale showed significantly enhanced bile acid binding capacity (91.1 ± 0.3 and 90.2 ± 0.7%, respectively) compared to lyophilized kale (85.5 ± 0.24%). Among the six bile acids tested, kale preferentially bound hydrophobic bile acids chenodeoxycholic acid and deoxycholic acid. Therefore, regular consumption of kale, especially minimally processed kale, can help excrete more bile acids and, thus, may lower the risk of hypercholesterolemia. KEYWORDS: Brassica oleracea, kale, red leaf lettuce, bile salt, in vitro digestion 1. INTRODUCTION membranes and enhance the defense against oxidative stress, 12 Bile acids are amphipathic molecules that function as thus protecting the liver. Bile acid sequestrants prevent bile emulsifiers to assist in digestion and absorption of lipids in acid absorption and promote synthesis of primary bile acids from cholesterol, which leads to a reduction of serum the gastrointestinal tract. Cholesterol can be biosynthesized 13 into bile acids, including cholic acid (CA), chenodeoxycholic cholesterol. Some foods can function like bile acid acid (CDCA), lithocholic acid (LCA), deoxycholic acid (DCA), sequestrants; for instance, guar gum can reduce the glycochenodeoxycholic acid (GCDCA), glycocholic acid concentration of serum bile acids by binding them in the 1 gastrointestinal tract, which relieves the symptoms associated (GCA), and glycodeoxycholic acid (GDCA). The two primary 14 bile acids, CA and chenodeoxycholic acid, are synthesized by with intrahepatic cholestasis of pregnancy and reduces plasma cholesterol levels.15 the liver, stored in the gallbladder, and then secreted into the fi small intestine, when food is being digested.2 Human intestinal Beyond their functions in digestive emulsi cation and bacteria can convert CA and chenodeoxycholic acid to reduction of cholesterol, bile acids are also associated with secondary bile acids, such as DCA and LCA, though glucose metabolism. Because bile acids can activate farnesoid X dehydroxylation. After assistance in lipid digestion, bile acids receptor and G-protein coupled bile acid receptor 1, which are are reabsorbed through the ileum and transferred back to the involved in glucose metabolism, the control of bile acid 1,2 absorption can be used as a potential novel therapy for some liver. Up to 95% of bile acids are reabsorbed, which limits the 2,14 synthesis of new bile acids from cholesterol. Some bile acids metabolic diseases, such as type 2 diabetes. In fact, one of can escape the enterohepatic circulation and reach the colon, the bile acid sequestrants, colesevelam hydrochloride, has been 1 approved by the U.S. Food and Drug Administration (FDA) in where gut bacteria form more DCA and LCA. LCA, DCA, and 16 chenodeoxycholic acid are hydrophobic bile acids, which are 2008 as a medicine for treatment of type 2 diabetes. In considered more toxic to liver and colon cells, and may induce addition, bile acid binding has recently emerged as a supporting 3−5 fi approach to strengthen the standard treatments used to control colon cancer. However, dietary ber increases the excretion 17 of bile acids in the stool,6 which can potentially prevent colon plasma glucose in type 2 diabetic patients. Several studies 7 fi suggested that bile acid sequestrants reduced the fasting plasma cancer and lower cholesterol levels. Besides dietary ber, 17−20 vitamin D and calcium supplements also have a protective glucose level in type 2 diabetic patients. Therefore, a function against colon cancer.8 Hence, binding bile acids can vegetable with good bile acid binding capacity may also lower reduce intestinal cancer and reduce plasma cholesterol the plasma glucose level of type 2 diabetic patients. levels.9,10 When hydrophobic bile acids are conjugated with taurine or Received: June 2, 2017 glycine, they become more hydrophilic and much less toxic to Revised: August 7, 2017 hepatic and intestinal cells.11 Some conjugated bile acids, such Accepted: August 16, 2017 as tauroursodeoxycholic acid, can even stabilize hepatocyte Published: August 16, 2017 © 2017 American Chemical Society 8054 DOI: 10.1021/acs.jafc.7b02540 J. Agric. Food Chem. 2017, 65, 8054−8062 Journal of Agricultural and Food Chemistry Article Figure 1. Schematic representation of in vitro bile acid binding capacity of different vegetables and extraction of bound bile acids. Previous studies have examined the bile acid binding amylase. The samples were vortexed and then placed in a shaking capacities of various vegetables, but few studies have examined water bath (37 °C and 180 rpm) for 5 min. During the gastric the binding preferences for individual bile acids by specific digestion phase, the mixture from the oral digestion phase was 21 adjusted to pH 2.0 with 1 N HCl, followed by the addition of 600 μL vegetables. According to previous research, collard greens, ff μ kale, mustard greens, broccoli, Brussels sprouts, spinach, green of pepsin bu er (200 g of pepsin in 1 mL of 0.1 M HCl), vortexed and incubated in a shaking water bath (37 °C and 180 rpm) for 1 h. bell peppers, and cabbage have good bile acid binding 21 For the intestinal phase, chyme from the gastric digestion phase was capacity. However, whether hydrophobic or hydrophilic bile first adjusted to pH 6.8 with 1 N NaOH. The sample was then mixed acids are preferentially bound by these vegetables has yet to be with 5 mL of pancreatin (6.25 mg/mL in 50 mM phosphate buffer) tested. Whether the different compositions of bile acid mixtures and 4 mL of bile acid mixture solution prior to incubation in a shaking − can influence the in vitro binding capacity also remains water bath, shaking at 37 °C and 180 rpm, for 3 h.22 25 The bile acid unknown. This study compares the in vitro bile acid binding mixture contained 10 mM cholate, 10 mM deoxycholate, 10 mM capacities of different vegetables, including red cabbage, red leaf glycochenodeoxycholate, 10 mM glycocholate, and 10 mM cheno- lettuce, Brussels sprouts, red kale, and green kale. The most deoxycholate in potassium phosphate buffer (pH 6.8). After ff incubation with the bile acids, the in vitro digestion was terminated active vegetable was used to test di erent bile acid ° compositions and optimal amounts for bile acid binding. by inactivating enzymes in a 78 C water bath for 7 min. The analyses were conducted using triplicate samples of freeze-dried red cabbage, red kale juice, green kale, red leaf lettuce, and Brussels sprouts (2.0 g 2. MATERIALS AND METHODS each). Digestion chymes were centrifuged at 3600 rpm for 30 min, and fi fi 2.1. Materials. Fresh green kale (Brassica oleracea var. acephala), supernatants were collected and ltered through Whatman lter paper. red cabbage (Brassica oleracea var. capitata f. rubra), red kale (Brassica The residue was washed with 20 mL of nanopure water by mixing on a shaking water bath for 3 h and centrifuged at 800g for 30 min. Both of oleracea convar. acephala var. sabellica), Brussels sprouts (Brassica fi oleracea var. gemmifera), and red leaf lettuce (Lactuca sativa) were the supernatants were ltered, combined, and concentrated under ° purchased from a local supermarket. All vegetables were cut into small vacuum by rotary evaporation at 40 C to obtain 7 mL and passed μ fi pieces, lyophilized, powdered, and stored at −20 °C until further use. through 0.45 m cellulose lters for high-performance liquid 2.2. Chemicals. Sodium glycocholate (G7132), sodium cholate chromatography (HPLC) analysis. In Vitro ff (C1254), sodium glycochenodeoxycholate (G0759), sodium glyco- 2.4. Digestion of Di erent Bile Acid Compositions. ff deoxycholate (G9910), sodium chenodeoxycholate (C8261), sodium Bile acid composition di ers in various individuals depending upon deoxycholate (D6750), ammonium nitrate, potassium dihydrogen their health conditions and gender. To understand that the binding fl phosphate, potassium chloride, potassium citrate, uric acid sodium salt, capacity is in uenced by the composition of the bile acid mixture, we ff urea, lactic acid sodium salt, porcine gastric mucin, α-amylase, pepsin, used three di erent compositions equivalent to male patients with and pancreatin were obtained from Sigma-Aldrich Co. (St. Louis, MO, gallstones (BAC-1), healthy females (BAC-2), and males with type 2 26,27 U.S.A.). diabetes (BAC-3). Mixtures with three different bile acid 2.3. Screening of Different Vegetables for Bile Acid Binding compositions (BACs) were prepared as shown in Table S2 of the Ability. The in vitro digestion was performed to mimic digestion in Supporting Information. The BAC-1 bile acid mixture contained the mouth, stomach, and small intestine (Figure 1). Simulated saliva 33.3% CA in both conjugated and unconjugated forms, 24.9% DCA, fluid (SSF) was prepared by dissolving the reagents (Table S1 of the and 41.8% chenodeoxycholic acid.