In vitro binding of bile salt to rhubarb stalk powder

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ELSEVIER

Research. Vol. 18, No. 5. pp. 893-903.1998 Copyright 0 1998 Elsevier Science Inc. F’rinted in the USA. All rights reserved 0271-5317/98 $19.00 + .CKl

PI1SO271-5317(98)00074-S

IN VITRO BINDING OF BILE SALT TO RHUBARB STALK POWDER Vinti Goel Ph.D’, St.&hinder K. Cheema Ph.D*, Luis B. Agellon Ph.D*, Buncha Ooraikul Ph.D’, Michael I. McBumey Ph.D’,Tapan K. Basu Ph.D’* ‘Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Canada.*Lipid and Lipoprotein Research Group and Department of Biochemistry, University of Alberta, Edmonton, Canada.

ABSTRACT

A new fiber source was developed by blanching, drying and grinding the fresh stalks of rhubarb, ah underutilized and universal crop. The product was found to contain 74% total dietary fiber (66% insoluble and 8% soluble dietary fiber), on dry weight basis. Preliminary studies involving experimental animals and hypercholesterolemic subjects revealed that this fiber source is potentially hypolipidemic, though the underlying mechanism still remains unknown. To investigate its mechanism of action, this study was undertaken to determine its potential to complex with bile salts in v&o. Comparison of various fiber sources showed that the rhubarb fiber had the maximum ability to bind taurocholate, and bound 11 and 2.5 fold more bile salt than cellulose and wheat bran respectively. The binding increased linearly with increasing rhubarb fiber concentration. Increasing the bile salt concentration also increased the binding in a dose dependent manner and showed a saturation at a concentration greater than 1OmM. The rhubarb fiber had a binding capacity of 40 pm01 of taurocholate per gram. Cholate competed taurocholate for binding indicating that conjugation of bile salt was not a mandatory requirement for the binding . Based on the chemical composition of rhubarb stalk powder, the bile salt binding property appears to be due to a combination of factors present in this product such as its high fiber content (insoluble with pectin), hydrophilic nature and high ash content. The ability to bind bile salts might be responsible for its hypocholesterolemic action observed in 0 1998ElscvierScienceInc. experimental animals and humans. Key Words : Cellulose, Corn bran, Rice bran, Wheat bran, Rhubarb stalk powder, Colestipol, Bile acids, In vitro-binding capacity.

*To whom correspondence and reprint request should be addressed. Phone : (403) 492492 1; Fax : (403) 492-9 130.

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V. GOEL et al. INTRODUCTION

Dietary fiber consists of a diverse group of substances that can vary widely in chemical and morphological properties (1). Soluble fibers are generally considered as better hypocholesterolemic agents than insoluble types of fibers (2,3). Viscosity (4) and fermentability (5) are considered to be the major attributes of dietary fiber responsible for these effects. However the physiological relevance of these properties is a subject of discussion since some feeding trials using highly viscous fibers, such as methyl cellulose, could not produce any effects on plasma cholesterol concentrations (6). In addition short chain fatty acids at physiological concentrations (7) have been shown to have no effect on plasma cholesterol levels. Except for fibers, such as barley and oat fibers (8,9), which contain P-glucans and phytosterols, fibers that have proved effective in lowering plasma cholesterol concentrations have the ability to affect bile acid metabolism. In this regard most of these fibers have been shown to accelerate fecal bile acid losses (lo), change biliary bile acid composition (11) or upregulate cholesterol 7cc-hydroxylase activity (12). Some of these fibers, both soluble and insoluble have also elicited ability to complex with bile salts in vitro, and therefore mimic ion exchange resins in vivo (4). A new fiber source has been developed in our laboratory by blanching, drying and grinding the stalks of rhubarb plants, that grow widely in North America and belong to a buck wheat family, polygonaecae. Fresh rhubarb stalks have traditionally been used for household consumption in products such as juices, pies and tarts, and in the wine making industry (13). The rhubarb stalk powder is a valuable source of dietary fiber containing 74% dietary fiber (66% insoluble and 8% soluble ), on dry weight basis. Experimental studies using hypercholesterolemic mice (14) and clinical trials on hypercholesterolemic men (15) have shown that dietary supplementation of the fiber source lowers appreciably the plasma concentrations of cholesterol. The underlying mechanism by which these effects were achieved is unknown. Since rhubarb stalk powder contains a small fraction of soluble fiber and has been shown to have moderate fermentability (Unpublished observation), it is unlikely that viscosity or fermentation could be the major attributes responsible for the hypocholesterolemic effects. To investigate a probable mechanism of action, the ability of rhubarb fiber to form complexes with bile salts, such as taurocholate or cholic acid, was determined in vitro and compared to that of other commonly used fiber sources including cellulose, and corn, rice or wheat bran and a cholesterol lowering agent Colestipol, which is an ion exchange resin.

RHUBARB FIBER AND BILE ACID BINDING

MATERIALS

AND METHODS

Rhubarb fiber Fresh rhubarb stalks, were obtained from growers and food stores in Edmonton, Alberta. The stalks were cut into 3 cm length and steamed for 10 minutes and then squeezed with a manual screw press to remove the juice. The pressed stalks were leached twice in about 10 times its weight of water at 70°C for 15 minutes, squeezed, and then dried in a fluidized bed dryer at 85’C for about 1 h 40 minutes. Subsequently, the dried stalks were ground finely. The ground product was sifted through a 16 mesh sieve and was analyzed for chemical composition (Table 1). The water holding capacity was also determined by measuring the amount of water retained by the dry powder (16). Binding studies The assay for in vitro binding of bile salt was done using a method described previously (17). Briefly, 200 mg samples of various fibers and colestipol (Colestid, Upjohn Pharmaceuticals) were incubated with 10 mM [24-14C] taurocholic acid (sodium salt, specific activity 2000 dpm/pmol) in 5 ml phosphate buffer (pH 7.0) at 37’C for 2 hr. Following the incubation, 3 ml mixture was centrifuged at 30,OOOxg for 10 minutes. A 0.5 ml aliquot was counted for radioactivity in liquid scintillation counter. The amount of bile salt bound was calculated as the difference between the amount of bile salt added and that recovered in the supernatant. The concentration dependent effect of rhubarb stalk fiber on bile salt binding was also investigated through a separate experiment. The various amounts of rhubarb stalk powder or cellulose (100 - 300 mg) were incubated with 1OmM of labeled taurocholate and the amount of bile salt bound was calculated as described above. The effect of bile salt concentration on the ability of rhubarb fiber to bind bile salts was also determined. Rhubarb or cellulose fiber (200 mg) was incubated with 2 to 10 mM of labeled taurocholate and the amount of bile salt bound was calculated as stated above. To investigate if the conjugation of bile salt was essential for binding to rhubarb fiber, the binding of 1OmM of labeled taurocholate and increasing amounts of cholic acid to rhubarb or cellulose fibers (200mg) was tested. The ability of cholic acid to compete the binding of taurocholate to the fiber sources was calculated as described earlier.

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V. GOEL et al. TABLE 1 Chemical Composition of Rhubarb Stalk Powder**’ -.Protein Ash Oxalic Acid Malic Acid Insoluble dietary fiber Soluble dietary fiber Total dietary fiber

%, -- Dry w&ht 5.6 5.6 5.7 3.2 65.9 8.2 74.1

*Values are means of 2-4 replicates. #Insoluble and Soluble dietary fiber contents were estimated by the method of Prosky et al. (18). Oxalic acid and malic acid were determined by the procedure of Kok et al. (19).

RESULTS

Table 1 gives the chemical composition of rhubarb stalk powder. The major component of the product was dietary fiber comprising 74% of the total dry weight. Insoluble fiber contributed for the majority of the fiber (66%) though an appreciable amount of soluble fiber (8%), mainly as pectin was also present. The product also contained considerable quantities of protein, oxalic acid, malic acid and ash. The rhubarb stalk powder absorbed 20 times its weight of water. Table 2 summarizes the bile salt binding capacity of rhubarb stalk powder relative to colestipol, an anion exchange resin and bile acid sequestrant, and several commonly used fiber sources such as wheat bran, corn bran, rice bran and cellulose. Colestipol bound more taurocholate than any of the fibers tested. Among the fibers tested, rhubarb fiber exhibited the highest capacity for binding. It bound 2 to 3 fold as much bile salt than corn bran, rice bran or wheat bran, and 11 fold more bile salt than cellulose. The effect of increasing fiber concentration on binding to taurocholate was also evaluated. As the concentration of rhubarb fiber increased, the binding increased linearly in a concentration dependent fashion (FIG 1). In contrast, cellulose did not show any appreciable increase in binding even at the highest concentration.

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RHUBARB FIBER AND BILE ACID BINDING

TABLE 2 In vitro binding capacities of taurocholate by various fibers Fiber

Taurocholate bound (ltmoY2OOmg) -.41.77 f 0.10” 1.89 _+0.87b 6.86 + o.6gc 6.93 f 0.65’ 4.52 + 0.96’ 12.34 + 1.09d

Colestipol Cellulose Corn Bran Rice Bran Wheat Bran Rhubarb Stalk Fiber

.._-.

Values are means f SEM of 5 independent determinations. Values not sharing a common superscript letter are significantly different at p
25

rz

Cellulose

0 100

Fiber (mg)

200

FIG 1 An in vitro dose-response curve of either rhubarb stalk powder or cellulose binding to taurocholate. All values are means 4 SEM of 5 independent determinations and are significantly different at p
300

V. GOEL et al. Addition of increasing amounts of taurocholate to a fixed amount of rhubarb stalk powder showed a linear increase in binding up to a concentration of 1OmM (FIG 2). No further increase in binding was observed beyond this concentration. Thus, the rhubarb stalk powder has a binding capacity of 50 PM per gram. Cellulose, exhibited a small increase in binding with increasing concentrations of taurocholate but to a much lesser extent than rhubarb fiber. In order to investigate if the conjugation of bile salt was essential for the binding, the effect of cholic acid on the binding of taurocholate by rhubarb stalk powder or cellulose was examined. As shown in FIG 3, cholate competed effectively with taurocholate for binding. At equimolar concentrations of both conjugated taurocholate and unconjugated cholic acid, the amount of taurocholate bound was reduced by 50%. Thus, conjugation of bile salt did not appear to be a prerequisite for binding to rhubarb fiber.

12

I Cellulose

4 Taurocholate

6

8

concentration P-W

FIG 2. Effect of taurocholate concentration on its binding by rhubarb stalk powder or cellulose. All values are means + SEM of 5 independent determinations and are significantly different at p
RHUBARB FIBER AND BILE ACID BINDING

899

Cellulose

acid concentration

15 (mM)

20

FIG 3. Effect of cholic acid on the binding of taurocholate by rhubarb stalk powder or cellulose. All values are means k SEM of 5 independent determinations and are significantly different at ~~0.05.

DISCUSSION

Degradation of cholesterol to bile acids is one of the major pathways by which cholesterol is eliminated from the body. Bile acid sequestrants such as cholestyramine and colestipol have for many years been considered to be a useful drug therapy for patients with hypercholesterolemia (20). The sequestrants work by binding the bile salts in the gut lumen therefore inhibit their reuptake by the intestinal ileocytes and thus facilitate their fecal excretion. The bile salt binding ability of these agents has been demonstrated in vitro (21) as well as in vivo (22). However, because of the undesirable side effects such as constipation and hypertriglyceridemia, associated with their continuous use (20) and their low in viva potency (23) , dietary modifications such as lowering the intake of fat and increasing the intake of dietary fiber have emerged as the first choice of therapy to manage mild to moderate hypercholesterolemia (24).

900

V. GOEL et al. Many fibers especially the soluble and viscous fibers such as psyllium (25) and pectin (26) have proved effective as hypocholesterolemic agents. Since the physiological effects of dietary fibers vary greatly depending on their solubility, fermentability and viscosity, fibers having both soluble and insoluble fractions are considered more desirable (1). Compositional analysis of rhubarb stalk powder revealed that the new fiber source contains both soluble and insoluble fractions. In vitro fermentation experiments have shown that rhubarb fiber has moderate fermentability, in between the non fermentable alphacel and highly fermentable pectin fibers (Unpublished observation). Despite being predominantly insoluble, the fiber has also elicited pronounced hypocholesterolemic effects in experimental and clinical trials. The novel ability of rhubarb fiber to bind bile salts illustrated in this study could be responsible for its consistent lipid lowering effects. Compared to the other fibers tested here, rhubarb fiber is second only to colestipol in ability to bind taurocholate. This binding capacity is significant considering that the natural source was not specifically modified for this purpose. It was noteworthy that although fiber sources such as corn bran (27) and rice bran (28) are also mixed fibers and have exhibited cholesterol lowering effects in experimental and clinical trials, their potency to bind taurocholate was much lower than rhubarb fiber. The relative ineffectiveness of wheat bran and cellulose fibers in binding with bile salts is consistent with the previous observations (29) and these fibers are also not associated with any cholesterol lowering effects (2,30). The specific component in rhubarb powder responsible for bile salt binding, although, is not known, this appears to be an effect of unique combination of factors present in rhubarb powder. The fiber source contains predominantly insoluble type of fibers. Although cellulose, the most abundant insoluble polysaccharide did not show any appreciable binding, other insoluble noncellulosic polysaccharides and nonpolysaccharides such as hemicelluloses (3 1) and lignin (4) have shown appreciable adsorption capacity for bile salts. Additionally, the rhubarb fiber’s high hydration capacity and high pectin content (8%) could have also led to the entrapment of bile salt in the interstitial space of the fiber matrix or in the gelatinous mass formed by pectin. Some highly viscous fibers such as guar gum (32) also show the ability to adsorb bile salts and thus increase fecal bile salt excretion (10). The rhubarb stalk powder also contains appreciable amounts of minerals (5.6%). It is possible that some of these minerals especially the divalent cations (such as calcium) may participate in sequestering bile salts (33). In VZ’POstudies have revealed that the bile salt structure is an important determinant of bile salt affinity to the sequestrants (17). Sorbents such as cholestyramine possess greater affinity for taurine conjugated bile salts because of the ionic interaction between the ammonium groups of resin and acidic sulfonic groups of taurine (2 1). However, binding assay revealed that rhubarb powder had an equal affinity for both conjugated (taurocholate) and nonconjugated (cholate) bile salts. Thus there is limited involvement of taurine in binding.

RHUBARB FIBER AND BILE ACID BINDING

901

In conclusion, this study shows that rhubarb stalk powder exhibits the ability to bind bile salts. This property could be responsible for its consistent hypocholesterolemic effects in animals (14) and humans (15).

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for

publication

February

9,

1998.

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