Journal of Food Science and Engineering 1 (2011) 348-353

The Binding of Cholic Acid by Hemicellulose and Pectin of Yard-Long Bean [Vigna sesquipedalis (L.) Fruhw]
L. Yuanita, Suyono and I. G. M. Sanjaya
Chemistry Department, Faculty of Mathematics and Natural Sciences, The State University of Surabaya, Ketintang Campus, Surabaya 60231, Indonesia Received: July 18, 2011 / Published: October 20, 2011. Abstract: The aim of the study was to describe the binding of cholic acid by hemicellulose and pectin of yard-long bean at gastrointestinal system (in vitro), through variation of acidity (pH 3, 5 and 7) and boiling time (raw/0, 5, 20 and 35 minutes) of yard-long bean. Two-way Anova ( = 5%) and functional groups analysis by infrared spectrophotometer were applied for the binding description. The results of the study showed: (1) The highest binding percentage of cholic acid by dietary fiber of yard-long bean at pH 3-raw treatment (76.603%); (2) Cholic acid binding via C = O/ester or acid of hemicellulose, C-O-C/cyclic ether of pectin, while C = O/ester or acid of pectin through iron. Key words: Cholic acid, hemicellulose, pectin, yard-long bean.

1. Introduction
A high diet in dietary fiber has a positive physiological effect such as preventing hypercholesterolemia which is one of the causes of coronary heart disease. One possible mechanism for the hypocholesterolemia effect of dietary fiber is its ability as a binder of bile acids and to be excreted, thus preventing reabsorption at the ileum on enterohepatic circulation. About 95% of excreted bile acids are secondary bile acids, but the secondary bile acids formed from primary bile acids. Primary bile acids (e.g., cholic acid) are more polar than the secondary bile acids (e.g., deoxycholic acid). The presence of additional hydroxyl group causes an increased degree of polarization of the bile acids steroid ring structure. Among of the dietary fiber components, lignin has the highest binding capacity to bile acids [1]. However, soluble dietary fiber (pectic substance, hemicellulose) has metabolic effect and beneficially lowering blood cholesterol levels [2]. Hemicellulose and pectic
Corresponding author: L. Yuanita, Ph. D., research field: food science. E-mail: yuanita@sby.dnet.net.id.

substance having carboxyl groups thus promote polar properties. This shows there was the indication of binding possibility between cholic acid with hemicellulose and pectic substance, which helps in decreasing blood cholesterol levels. Yard-long bean [Vigna sesquipedalis (L.) Fruhw] is a vegetable high in dietary fiber and consumed either cooked through various processing methods or uncooked (raw). The approximate content of yard-long bean hemicellulose, pectic substance, cellulose and lignin are 23.572, 6.909, 14.206 and 5.318% dry matter respectively, while cooked (25 minutes in boiling water) yard-long bean had hemicellulose, pectic substance, cellulose and lignin 22.478, 6.712, 20.052 and 5.728%. Such increase is possibly due to the formation of resistant starch and lignin substance which were measured as cellulose and lignin [3]. Varying acidity (pH) of the medium would affect the composition and chemical structure of dietary fiber components, which result in change in physico-chemical properties of dietary fiber; therefore,

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influencing the physiological effect of dietary fiber along the digestive tract, including the binding of bile acids by the dietary fiber. During the cooking process, plant cell wall components changed: (1) hydrolysis of glycosidic bond of polysaccharides and degradation of pectic substance at neutral pH [4], and (2) hydrolysis of glycosidic bond of hemicellulose and pectic substance at acidic medium [5]. Food processing may change physico-chemical properties of dietary fiber, especially water holding capacity and adsorption other organic components [6]. According to Furda in the Ref. [7], Nagyvary stated, the hypothetical structure of bile acid binds towards polyuronate of hemicellulose or pectin via the bridge of polivalent cations. Similarly, the addition of iron (Fe) increased the binding of deoxycholic acid to all dietary fiber used, except low-methoxy pectin [8]; on the contrary, Eastwood and Hamilton suggested that the binding of bile acids by dietary fiber was not influenced by the presence of divalent ions [1]. As of the study was to describe the mechanism on the maximum binding of cholic acid by hemicellulose and pectin of yard-long bean via the wavenumbers shift of infrared absorption band spectra of hemicellulose and pectin functional groups at pH and boiling time treatment.

2. Materials and Methods

The study was done in two stages. First is to determine the percentage of cholic acid binding by dietary fiber of yard-long bean (the highest percentage is used in second stages). Second is to determine the hemicellulose and pectin functional groups of yard-long bean which play a role in cholic acid binding by dietary fiber. The factorial design was applied in the first stage, variation of pH and boiling time; the second stage is a post test only control group design. The samples of yard-long bean super green variety were randomly collected and cut into 4 cm length, and then were subjected to various pH (3, 5 and 7) and BT (raw/0, 5, 20 and 35 minutes). All samples were lyophilised and milled to pass a sieve of 100 meshes [9].

Citrate buffer solution are on pH 3 and 5 [10], while aqua dest is on pH 7. Cholic acid and porcine pancreatin were purchased from Sigma-Aldrich (St. Louis, MO). Cholic acid solution contained 31.25 mol/mL in 0.1 M phosphate buffer, pH 6.3. Porcine pancreatin was dissolved in 0.01M, pH 6.3, phosphate buffer to yield a concentration of 10 mg/mL. An in vitro digestion process was applied according to the method given in the previous study [11-13]. Defatted sample (0.1 gram) was digested in 1 mL of 0.01 N HCl for one hour and in 37 C shaker bath. After this acidic incubation which simulated gastric digestion, the sample pH was adjusted to 6.3 with 0.1 mL 0.1 N NaOH. To each test sample, 4 mL of cholic acid and 5 mL pancreatin was added. Incubate for one hour at 37 C in shaker bath. Mixtures were transferred to 10 mL centrifuge tube and centrifuged at 26.890 g for 10 minutes. An additional 5 mL of 0.1 M of phosphate buffer (pH 6.3) was used to rinse out the incubation tube and added to centrifuge tube, which was vortexed and centrifuged as before. Supernatant was removed and combined with the previous supernatant. A 5 mL aliquot of supernatant was mixed with 5 mL of 70% sulphuric acid. Two minutes later 1 mL of 25% furfural solution was added. A pink colour appeared and it took 5 min to develop to maximum intensity. Reading of absorbance was made at a wavelength of 510 nm. Hemicellulose and pectin were isolated from the yard-long bean sample with highest binding [14, 15], and being used for the binding of cholic acid. The binding results of cholic acid by functional groups of hemicellulose and pectin were analyzed with an infrared spectrophotometer. The role of divalent cations (i.e., Fe), is determined by the addition of a number of 2,400 ug FeSO4 7H2O/62.5 mg dietary fiber [8]. The percentage of bile acid binding by yard-long bean dietary fiber was analyzed by two-way Anova ( = 5%); while the infrared absorption band spectra of pectin and hemicellulose functional groups on the binding with cholic acid was analyzed by quantitative descriptive.

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3. Results and Discussion

3.1 The Percentage of Bound Cholic Acid by Yard-Long Bean Dietary Fiber The results of two-way ANOVA for the effect of pH and boiling time to bound cholic acid percentage by dietary fiber yard-long bean are cited in Table 1. There were no significant effect of pH and boiling time (P > 0.05) on percentage of bound cholic acid by yard-long bean dietary fiber (Table 1). The highest percentage of bound cholic acid at pH 3-raw/without boiling (76.603%), and the lowest at pH 7-boiling time 35 minutes (50.962%). The percentage of bound cholic acid decreased for pH 3, 7, 5 consecutively. The pKa of cholic acid is 4.98 0.05. The binding occurs at pH 6.5 of gastrointestinal tract system condition, but variations in pH medium will also affect the dietary fiber component ionization. The highest binding at pH 3 is very possible due to lack of cholic acid ionization and insoluble dietary fiber of yard-long bean are higher than soluble dietary fiber content [3], resulting in a hydrophobic interaction between dietary fiber component with cholic acid. At pH 7 treatment, the pH of yard-long bean matrix is 6.8. Cholic acid is more likely ionized at the conditions above pKa, resulting in lower hydrophobic interaction of dietary fiber with cholic acid compare with pH 3 treatment. The pH 5 treatment support ionization and formation of bile salts; since about half of the molecules are in a protonated state and bile salts form at the pKa value. This means that about half of the bile acids might play a role in bonding interactions directly with dietary fiber, as the binding of the lowest percentage (68.709%). In addition to the
Table 1 The effect of pH and boiling time to percentage of bound cholic acid. Boiling time ( minute) F; P 0 5 20 35 3 76.603 71.474 60.577 75.641 F = 1.460 5 63.568 51.068 66.453 56.838 P = 0.246 7 65.598 61.645 72.756 50.962 F = 0.429; P = 0.734 F = 0.728; P = 0.630 pH

pH 5 treatment-without boiling, the yard-long bean dietary fiber has a low anionic nature [16], therefore, the bond of dietary fiber and cholic acid is to be dominated by hydrophobic interactions. It was then concluded, the maximum adsorption when the acidic groupings on the fiber are unionized, in an acidic medium. As shown in Table 1, the decline trend in the binding of cholic acid were identifying on the increase of boiling time. The boiling process resulted in brittleness of cell wall which leads to the collapse of the fiber matrix and a consequent decrease at the total available surface area for adsorption. A negative influence on the binding capacity may be observed [17]. It has also been suggested that there was a positive correlation between surface area and adsorption capacity [9]. Besides, the effect of three hydroxyl groups of steroid ring structure of cholic acid would promote less adsorption. 3.2 Binding of Bile Acids by Hemicellulose and Pectin The highest binding of cholic acid by yard-long bean dietary fiber is on the treatment of pH 3-boiling time 0 minute. In this research to compare the results of infrared spectra analysis: (1) hemicellulose or pectin, (2) hemicellulose or pectin + pancreatin medium, (3) hemicellulose or pectin + pancreatin medium + cholic acid, (4) hemicellulose or pectin + pancreatin medium + cholic acid + Fe. Hemicellulose is a heteropolysaccharides consisting of mixed polymers of sugar units with side units commonly consisting of galactose, arabinose, and uronic acid units, usually methylated. The shift of wavenumbers of infrared absorption band spectra of hemicellulose at pH 3-boiling time 0 minutes treatment on cholic acid binding are shown in Figs. 1-4 and to be concluded in Table 2. Pectic substance form a complex group of polysaccharides consisting of -1.4 glycosidic-linked D-galacturonic acids as the major constituent with variable degrees of esterification with methyl or acetyl groups and the side chains consisting of galactose, arabinose, xylose, rhamnose, or glucose. Pectic substance

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Fig. 4 The spectra of hemicellulose + pancreatin medium + cholic acid + Fe. Fig. 1 The spectra of hemicellulose.

Fig. 2 The spectra of hemicellulose + pancreatin medium.

Fig. 3 The spectra of hemicellulose pancreatin medium + cholic acid.

are classified in three groups, pectic acid, protopectin and pectin. In pectin, carboxyl esterified at C6. The shifts of wavenumbers of infrared absorption band spectra of pectin at pH 3, and boiling time 0 minute treatment on cholic acid binding are shown in Figs. 5-8 and to be concluded in Table 3. From Tables 2-3, it is suggested that binding of cholic acid by dietary fiber components will increase the wavenumber of C = O stretching/ester or acid of pectin, whereas the wave number just to decrease at the addition of iron. An increasing wavenumber is possible due to the interaction of pancreatin with solvent molecules then reduced the interaction between solvent with dietary fiber component molecules or intermolecular dietary fiber component. However, the addition of iron has the effect of decreasing the wavenumber. This shows the role of iron in the binding of cholic acid by pectin, as being possible through the O-Fe-O bond between the C = O groups of pectin with cholic acid molecules. Also noted, bile acid binding to dietary fiber occurs through salt bridge of cell wall pectic [18]. Another research, disclose that the addition of Fe with or without Ca will increase the binding of cholic acid to dietary fiber (except for low-metoxyl pectin) [8].

Table 2 The shift of wavenumbers of infrared absorption band spectra of hemicellulose at pH 3, and boiling time 0 minute treatment on cholic acid (CA) binding. Assignment C = O st/ester or acid

Wavenumber (cm-1) Hemicellulose Hemicellulose + media Hemicellulose + media + CA Hemicellulose + media + CA + Fe 1,640.1 1,632.3 1,630.8 1,630.7

st = stretching.

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Fig. 5 The spectra of pectin.

Fig. 8 The spectra of pectin + pancreatin medium + cholic acid + Fe. Table 3 The shift of wavenumbers of infrared absorption band spectra of pectin at pH 3 and boiling time 0 minute treatment on cholic acid (CA) binding. Assignment Wave number (cm-1) Pectin + Pectin + Pectin + Pectin media + CA + media media + CA Fe 1,706.5 915.2

C = O st/ester 1,730.5 1,739.5 1,741.9 or acid C-O-C st/ 949 867.7 864.3 cyclic ether

st = stretching.

Fig. 6 The spectra of pectin + pancreatin medium.

shows the presence of binding between either hemicellulose or pectin with cholic acid. A statement supporting the above, the hydroxyl and carboxyl on hemicellulose able to react with iron to form Fe-hemicellulose complexes with high stability[19]; however, within this study found no effect of wave number shift of C = O stretching/ester or acid of hemicellulose on the addition of iron.

4. Conclusions
Fig. 7 The spectra of pectin + pancreatin medium + cholic acid.

The findings from this study show that there was a wavenumbers shift reduction of C-O-C stretching/cyclic ether hemiacetal of pectin, C = O stretching/ester or acid of hemicellulose. It is very possible due to the induction effect of hydrogen bonds between the pectin and hemicellulose with cholic acid, which causes a decline in O-H bond strength. This

pH and boiling time have no effect on the percentage of cholic acid binding by dietary fiber. The highest percentage of bound cholic acid is for pH 3-raw/without boiling treatment (76.603%). Dietary fiber binds to cholic acid via the C = O/ester or acid of hemicellulose, C-O-C/cyclic ether of pectin, while the C = O/ester or acid of pectin through iron. The results suggested another analysis in the determination of the mechanism of cholic acid binding

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by insoluble dietary fiber.

[9]

Acknowledgment
This research was supported by the DP2M DIKTI under Fundamental Research Grant.
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