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Preparation of Folic Acid-Chitosan Complex (Eco-Yellow) and Its Application for Adsorption of Mutagens and Carcinogens Hee-Kyung Jeon1, Youn-Jung Kim2 & Jae-Chun Ryu2 1

Busan Development Institute, #273-20 Yangjung-2dong Busanjin-gu, Busan 614-052, Korea 2 Cellular and Molecular Toxicology Laboratory, Korea Institute of Science and Technology (KIST), #39-1 Hawolgok-dong Seongbuk-gu, Seoul 136-791, Korea Correspondence and requests for materials should be addressed to J. C. Ryu ([email protected]) Accepted 22 November 2008

Abstract Folic acid, a pteridine derivative, is abundant in liver and green plants and is a growth factor for some bacteria. Chitosan is a unique polysaccharide derived from chitin and is utilized as adsorbents of organic trace pollutants in environmental water sample. In this study, we have found that chitosan was suitable as a solid support to carry folic acid. And it was examined that folic acid could form an insoluble material when mixed with chitosan, a polyglucosamine, and the solid chitosan-folic acid complex (eco-yellow) thus prepared could efficiently trap polycyclic mutagenic compounds. The adsorption of the test compounds was observed using UV/Visible spectrometry. The order of extent of removal by eco-yellow was B[a]P (62.0%)¤2-AA (56.2%)¤QC (55.9%)¤Trp-p-2 (40.2%). From these results, eco-yellow may be expected as a useful adsorber for several polycyclic mutagens and carcinogens. Keywords: Folic acid-chitosan complex, Adsorption, Polycyclic mutagens, Carcinogens

Introduction Folic acid (CAS 59-30-3, Figure 1A), also known as folate or folacin, is one of the B vitamins which is found naturally in orange juice, other citrus fruits and juices, leafy green vegetables, beans, peanuts, broccoli, asparagus, peas, lentils and whole grain products. Synthetic (manufactured) folic acid is added to certain grain products, including flour, rice, pasta, cornmeal,

bread and cereals. It is needed for energy production and in the formation of red blood cells. It is also vital to healthy cell division and replication, and to tissue growth. Most important, folic acid deficiency can cause neural tube defects (NTDs), which are birth defects of the baby’s brain (anencephaly) or spine (spina bifida)1-3. If women have enough of it in their bodies before pregnancy this vitamin can decrease the risk for NTDs. For this reason, the Food and Drug Administration (FDA) recommends that all women who could possibly become pregnant get 400 μg of folic acid every day. This could prevent up to 70% of some types of serious birth defects. Furthermore, vitamins such as folic acid, ascorbic acid and nicotinic acid have been shown to be potently antimutagenic against various mutagens including polycyclic aromatic hydrocarbons4, heterocyclic amines5 and aflatoxin B16 in the Salmonella/reversion assay using Salmonella typhimurium TA 98 and TA 100, and micronucleus test in mouse bone marrow. Dietary inhibitors of mutagenesis and carcinogenesis are of particular interest because they may be useful for human cancer prevention. Chitosan (poly (1 4)2-amino-2-deoxy-β-D-glucan, Figure 1B) is a unique polysaccharide derived from chitin (poly (1 4)2-acetamide-2-deoxy-β-D-glucan, Figure 1C), a main constituent of crustacean shell. Several attempts have been made to use this biopolymer in biomedical field, for example, the development of hemodialysis membranes, artificial skin, drug targeting and other applications7. This biodegradable and biocompatible chitosan may be used in substituting or regenerating the blood/tissue interfaces8. Also, chitosan was used in water treatment, photographic emulsions and improving the dye ability of synthetic fiber and fabrics. Chitin and chitosan, which are animal dietary fibers, are also utilized as adsorbents of organic trace pollutants in environmental water sample9. Therefore, we have selected that chitosan is suitable as a solid support to carry folic acid. Previously, we reported the preparation and absorption activity for several potent mutagens and carcinogens10 and heavy metals11 of chitosan-phthalocyanine complex (eco-blue). In this study, we established the process of preparation of the folic acid-chitosan complex (eco-yellow) and evaluated the usefulness of eco-yellow for the

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(A)

HO

Table 1. Adsorption of polycyclic mutagens to eco-yellow N

OH

N

O

NH2 N

O NH NH O

(B) HOCH2

HO

NH

O C CH3

O O

O

H n

O

HO

O NH C CH3 CH2OH

HOCH2

QC PhIP NH2

HO O

O

O O HO

NH2

B[a]P 2-AA Trp-p-2 Trp-p-1 NH 9-AA 2-AF IQ MS

230 nm 260 nm 264 nm 264 nm 348 nm 260 nm 282 nm 265 nm 301 nm

5 3 3 3 3 3 3 3 3

62.0 56.2 40.2 36.0 29.5 27.0 26.4 17.8 12.5

4.6 4.7 0.2 5.1 2.1 2.1 4.0 1.8 1.8

2 2

55.9 2.9 16.1 0.6

19.0 22.7 6.4 4.4 14.6 2.1 29.2 11.6 10.6

3.1 0.7 0.7 1.2 2.7 0.5 0.6 0.6 1.0

Compounds with 2 rings

(C)

HO

Abs.

Compounds with 3 or more fused rings

HO

HO

Mutagen

N

No. of rings Mean (%) SD in the molecular Eco-yellow Chitosan structure

H n

255 nm 271 nm

85.7 2.8 37.0 2.0

9-Aminoacridine (9-AA), 2-Aminoanthracene (2-AA), 2-Aminofluorene (2-AF), Benzo[a]pyrene (B[a]P), 2-Amino-3-methyl-3Himidazo[4,5-f]quinoline (IQ), 2-Amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP), 3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole acetate (Trp-p-1), 3-Amino-1-methyl-5H-pyrido[4,3-b]indole acetate (Trp-p-2), 8-Methoxypsoralen (MS), Norharmane (NH), Quercetin (QC)

CH2OH

Figure 1. Structure of (A) folic acid, (B) chitin and (C) chitosan.

Results

twice), water (300 mL, twice), methanol (300 mL, 4 times), water (500 mL, twice), 0.05 N NaOH (300 mL, twice) and water repeatedly untill the solution became colorless. Finally, it was washed with methanol and diethyl ether, dried in a desiccator and ground into powder. The yields were 3.0 0.5 g (mean SD, 6 experiments). This material is hereafter referred to as ‘eco-yellow’.

Preparation of Folic Acid-chitosan Complex (Eco-yellow) To a solution of chitosan (2 g) in distilled water (190 mL) was added 1 N HCl (ca. 10 mL) with continuous mechanical stirring of the solution, to adjust the pH to 3. For folic acid due to its low solubility in neutral water, folic acid (8.82 g) was dissolved in 70 mL of 0.5 N NaOH (alkalic status). After complete dissolution, the distilled water (130 mL) was added with stirring. 1 M EDAC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) was prepared by dissolving 3.84 g of EDAC in 20 mL of distilled water and incubated for 30 min before use. Folic acid (0.1 M) was mixed with the solution of chitosan and added 0.5 N of NaOH to adjust the pH to 6. After than 1 M EDAC was added to the solution and gently swirled in the reaction flask for 12 hr. The resulting precipitate was collected by centrifuging at 3,000 rpm for 10 min. The precipitate was washed with 10% NaCl (300 mL,

Adsorption of Polycyclic Mutagenic Compounds to Eco-yellow By mixing chitosan and folic acid, each in an aqueous solution, an insoluble yellow precipitate was formed. When suspended in water, the dry powdery ecoyellow did not precipitate, but floated. In order to ‘wet’ the material, a treatment with 5% Na2SO4 was found to be effective. The adsorption was achieved simply by a batch process, shaking the mutagen solution with the eco-yellow added. This adsorption took place in neutral aqueous solutions. According to Arimoto-Kobayashi et al. method, contents of adsorption were determined by UV/Visible spectrophotometry at maximum absorbance of compounds12. The adsorption of various mutagens to eco-yellow was measured and the results were given in Table 1. The order of extent of removal by eco-yellow was benzo[a]pyrene (B[a]P)¤2-aminoanthracene (2-AA) ¤quercetin (QC)¤3-amino-1-methyl-5H-pyrido[4,3

absorption of the mutagenic and carcinogenic compounds having polycyclic structures.

Adsorption of Mutagens Using Folic Acid-chitosan Complex

Table 2. Elution of polycyclic mutagens to eco-yellow. Mutagen

Abs.

No. of rings Mean (%) SD in the molecular Eco-yellow Chitosan structure

Compounds with 3 or more fused rings B[a]P 2-AA Trp-p-2 Trp-p-1 NH 9-AA 2-AF IQ MS

230 nm 260 nm 264 nm 264 nm 348 nm 260 nm 282 nm 265 nm 301 nm

5 3 3 3 3 3 3 3 3

ND 9.9 1.2 36.6 4.1 22.2 0.0 14.4 0.0 82.3 9.1 17.4 4.8 57.7 0.0 11.3 0.0

2 2

20.8 8.2 63.2 8.0

28.0 14.9 9.1 22.7 2.0 23.2 5.4 7.8 2.7

3.0 2.4 1.4 1.2 2.3 2.3 1.2 4.5 1.9

Compounds with 2 rings QC PhIP

255 nm 271 nm

0.4 0.8 3.8 6.6

9-Aminoacridine (9-AA), 2-Aminoanthracene (2-AA), 2-Aminofluorene (2-AF), Benzo[a]pyrene (B[a]P), 2-Amino-3-methyl-3H-imidazo [4,5-f]quinoline (IQ), 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole acetate (Trp-p-1), 3-Amino-1-methyl-5H-pyrido[4,3-b]indole acetate (Trpp-2), 8-Methoxypsoralen (MS), Norharmane (NH), Quercetin (QC)

-b]indole acetate (Trp-p-2). For the 11 compounds having 2 or more fused rings, extensive adsorptions were observed. The highest adsorption level tested in this study was determined for B[a]P (62.0%). Although these compounds showed non-specific adsorption to chitosan, these adsorptions of other compounds except for QC, 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP) and 2-aminofluorene (2-AF) were significantly smaller than those to eco-yellow.

Elution of Test Compounds from Eco-yellow Whether the adsorbed compounds were able to elute by treatment with phosphate buffer was tested. Table 2 shows the results of experiments in which the elution was compared between eco-yellow and chitosan of equal masses. The extents of elution to eco-yellow were greater than chitosan except for 2-AA and 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole acetate (Trp-p-1). Most of the compounds were eluted from the eco-yellow with phosphate buffer. Thus, it appears that these mutagens can be released into the buffer, even though once adsorbed to eco-yellow.

Discussion Many organic polycyclic compounds have mutagenic and carcinogenic properties. They are found in

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the environmental and also in protein-rich foods (e.g. meat and fish) as a result of their thermal treatment13. In general, PhIP is quantitatively most important in beef and fish, while 2-amino-3-methyl-3H-imidazo [4,5-f]quinoline (IQ) is usually only a minor constituent14. IQ, however, warrants special attention since it is among the most potent mutagenic and carcinogenic heterocyclic amines15. We present structures of polycyclic mutagens used in this study (Figure 2). Folic acid used in this study, is known as potently antimutagenic against various polycyclic mutagens and interested as dietary inhibitors of mutagenesis. Also chitosan was selected because it is suitable as a solid support to carry folic acid. In the previous studies, chitosan was clearly demonstrated that it can be used as an effective coagulating agent for organic compounds, as a chelating polymer for binding toxic heavy metals16 and as an adsorption medium for dyes and small concentrations of phenols present in various industrial wastewaters. Accordingly, folic acid and chitosan as its solid support formed potent synthetic product and it was expected to remove efficiently of mutagenic compounds having polycyclic fused rings. For the mutagenic compounds having 3 or more fused rings, the extent of removal by eco-yellow was higher than it by non-specific chitosan. These results suggest that folic acid in eco-yellow can complex with aromatic polycyclic compounds having planar surfaces of 3 fused-ring areas. However, in the case of 2-ring compounds tested, the extent of removal by eco-yellow was lower than it by non-specific chitosan. These 2-ring compounds against those with a 3-ring system particularly have a planar surface area having of the resonating character of dihydroxyphenyl ring with the benzopyran portion. In addition, to test the strength of adsorption of ecoyellow, the elution assay of eco-yellow was carried out. Most of test compounds adsorbed by eco-yellow were not remained to adsorbed form. Although once adsorbed to eco-yellow, these compounds can be readily released into aquatic media. From these results, the eco-yellow may be expected to be a valuable adsorbent for several polycyclic mutagens and carcinogens but still need to make improvement of the efficiency as adsorbent.

Methods Chemicals Chitosan (poly (1 4)2-amino-2-deoxy-β-D-glucan) used in this experiment was products of Jakwang Co. (Korea). Folic acid (pteroylglutamic acid) and EDAC (1-ethyl-3-(3-dimethylaminopropyl)carbodi-

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Toxicol. Environ. Health. Sci. Vol. 1(1), 64-68, 2009

(B)

(A)

(C)

(D)

NH2 NH2

NH2 N

(E)

(F)

NH2

(G)

(H)

CH3

N N

N CH3 O

O

O N H

OCH3

N

NH2

N H

N

CH3

OH

(I)

(J)

CH3

OH

(K) HO

N N H

O

CH3 N NH2

OH N

N

NH2

OH

O

Figure 2. Structures of polycyclic mutagens (A) 2-AA, (B) 9-AA, (C) 2-AF, (D) B[a]P, (E) IQ, (F) MS, (G) NH, (H) Trp-p-1, (I ) Trp-p-2, (J) PhIP, (K) QC.

imide hydrochloride) were obtained from Sigma-Aldrich Inc. (St. Louis, MO, USA). Folic acid was yellowish-orange crystals and light sensitive. The purity of it was found to be ¤98%. 9-Aminoacridine (9AA), 2-aminoanthracene (2-AA), 2-aminofluorene (2AF) and benzo[a]pyrene (B[a]P) were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA). 2-Amino3-methyl-3H-imidazo[4,5-f]quinoline (IQ), 8-methoxypsoralen (MS), norharmane (NH), 2-amino-1methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), quercetin (QC), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b] indole acetate (Trp-p-1) and 3-amino-1-methyl-5Hpyrido[4,3-b]indole acetate (Trp-p-2) were obtained from ICN biomedicals Inc. (Aurora Ohio, USA). All other reagents were of the highest quality available.

Procedure for Adsorption of Test Compounds to Eco-yellow The eco-yellow (10 mg) was soaked in 5% Na2SO4 (5 mL) by shaking at 37 C for 30 min for the purpose of wetting. The mixture was centrifuged at 3,000 rpm for 5 min and the supernatant was discarded. 0.1 mM Mutagen (2 mL; 0.2 μmol) in 20 mM Na phosphate-20 mM KCl buffer, pH 7, was added to the precipitate and the mixture was shaken vigorously at room temperature for 10 min. The mixture was centrifuged (3,000 rpm, 10 min) and the compound in the supernatant was

quantified spectrophotometrically (UV/Vis spectrophotometer, Varian co.). For B[a]P, due to its extremely low solubility in water, a stock solution (50 mM) was prepared by dissolving B[a]P in ethanol-Tween 80 (2 : 1). The adsorption was done in 2.5 mM Na-phosphate-2.5 mM KCl at pH 7. For measuring non-specific adsorptions to chitosan, as controls, the same treatments were done using raw chitosan at an equivalent amount. All these adsorptions were done in duplicate, and the data reported here are the average values of these measurements.

Procedure for Elution of Test Compounds from Eco-yellow After the adsorption test, eco-yellow was washed with water, the mixture was centrifuged and the supernatant was removed. Elution of mutagen from the precipitate with a 20 mM Na phosphate-KCl buffer (2 mL) was done for 10 min with shaking, as in the adsorption experiments. The amount of a mutagen found in supernatant obtained upon centrifugation of the mixture represents the degree of elution.

Acknowledgements This subject is supported by the Korea Research

Adsorption of Mutagens Using Folic Acid-chitosan Complex

Foundation grants from Korea Ministry of Environment as “The Eco-technopia 21 project”, KIST CoreCompetence Program and Korea Research Council of Fundamental Science and Technology to Ryu, J. C. of the Republic of Korea.

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