Molecular property–affinity relationship of the interaction between dietary polyphenols and bovine milk proteins

Abstract

The relationship between the molecular properties of dietary polyphenols and their affinities for bovine milk proteins (BMP) was investigated. The affinities of polyphenols for BMP were determined by means of fluorescence titration. The affinities of polyphenols for BMP increased with increasing partition coefficient and decreased with increasing hydrogen bond acceptor number of the polyphenol. From this point, the hydrophobic force played an important role in the binding interaction between polyphenols. It was found that the topological polar surface area value decreases with increasing binding constant of the polyphenol for BMP, which illustrates that the glycosylation of hydroxyl groups in polyphenols weakens their binding affinity for BMP. A strong correlation between Mulliken electronegativity and binding affinity was found (R = 0.64626), and Mulliken electronegativity values were found to increase with increasing binding constant of polyphenols for BMP. This illustrates that electrostatic interactions play a key role in binding dietary polyphenols to BMP.

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Introduction

Polyphenols are the most abundant antioxidants in the human diet, and are the most common and widespread constituents in plants.^1–5Polyphenols contain at least one aromatic ring with one or more hydroxyl group, in addition to other substituents. The most important polyphenol classes are phenolic acids, such as compounds with one C₆ aromatic ring of hydroxybenzoic acids like hydroxytyrosol, tanins and gallic acid, those with a C₆–C₃ structure of hydroxycinnamic acids like caffeic acid and coumaric acid, those with the C₆–C₂–C₆ structure of stilbenes such as resveratrol, and those with the C₆–C₃–C₆ structure of flavonoids.^6–9

Flavonoids are the most important polyphenols in plant sources.^10–13 Their structures are represented by a benzene ring (A) condensed with a heterocyclic six member pyran or pyrone ring (C), which in the 2- or 3-position carries a phenyl ring (B) as a substitute. Over 10 000 flavonoids have been separated and identified from plants, most of which are divided into subclasses, including anthocyanidins, flavanones, flavonols, flavones and isoflavones.^10–13

Milk proteins are natural vehicles that evolved to deliver essential micronutrients (e.g.calcium and phosphate) and immune system components (e.g. immunoglobulins, and lactoferrin).¹⁴ Bovine milk proteins (BMP) consist of caseins (2.4–2.8%), β-lactoglobulin (0.2–0.4%), α-lactalbumin (0.1–0.15), bovine serum albumin (BSA, 0.01–0.04%), immunoglobulins (0.06–0.1%), etc.¹⁵

Many of the structural and physicochemical properties of BMP, such as excellent surface and self-assembly properties, and superb gelation properties, facilitate their functionality in the binding of ions and small molecules.^14,16,17 Individual bovine milk proteins such as BSA, β-lactoglobulin and γ-globulin are reported to bind with many small molecules, such as dietary polyphenols.^18–23 There are several techniques, such as capillary electrophoresis, electrospray mass spectroscopy, high-performance affinity chromatography, NMR spectroscopy, fluorescence quenching and multi-spectroscopic methods, that have been developed to characterize polyphenol–protein interactions.²⁴ The fluorescence quenching method is a simple and popular weapon to investigate the nature of polyphenol–protein interactions.^19–23 In studying protein–polyphenol interactions, many physicochemical properties of polyphenols (lipophilicity, ionic charge, molecular weight, hydrophobic bonding, water solubility and specific surface area of particles) have been taken into consideration. These physicochemical properties, especially lipophilicity and hydrogen bonding potential, are essential parameters for food applications of dietary polyphenols.

Few reports, however, have focused on the molecular property–affinity relationships of dietary polyphenols on their affinities for BMP. The present work concerns the relationship between the molecular properties of dietary polyphenols and their affinities for BMP. Fifty-five polyphenols (Table 1) were studied.

Table 1 The affinities of polyphenols for BMP

Polyphenols	lgKa	n	Polyphenol	lgKa	n
Flavone	4.81	1.05	Naringenin	3.94	0.86
7-Hydroxyflavone	5.09	1.05	Naringin	3.76	0.83
6-Hydroxyflavone	6.02	1.16	Narirutin	3.64	0.82
6-Methoxyflavone	5.63	1.10	Hesperitin	5.14	1.05
Chrysin	5.19	1.04	Hesperitin-7-O-rutinose	4.80	0.99
Baicalein	6.14	1.17	Dihydromyricetin	5.07	1.04
Baicalin	4.76	0.99	Flavanone	3.58	0.84
Apigenin	5.82	1.14	7-Hydroxyflavanone	5.59	1.12
Luteolin	5.82	1.13	6-Hydroxyflavanone	4.87	1.08
Hispidulin	5.29	1.07	6-Methoxyflavanone	4.77	1.06
Wogonin	5.13	1.07	Silibinin	5.28	1.07
Tangeretin	5.76	1.12	Alpinetin	5.43	1.17
Nobiletin	5.69	1.11	GCG (2,3-trans)	4.98	1.02
Galangin	5.40	1.08	EGCG (2,3-cis)	4.54	0.97
Kaempferide	5.36	1.05	ECG (2,3-cis)	4.96	1.01
Kaempferol	5.57	1.06	EC (2,3-cis)	3.01	0.79
Kaempferitrin	4.99	1.03	EGC (2,3-cis)	2.05	0.65
Quercetin	5.72	1.12	C (2,3-trans)	2.29	0.66
Quercitrin	5.65	1.09	Resveratrol	4.94	1.02
Myricetin	6.04	1.17	Polydatin	4.54	0.95
Fisetin	5.40	1.07	Gallic acid	5.06	1.18
Rutin	3.98	0.87	Methyl gallate	5.71	1.20
Formononetin	4.12	0.91	Ethyl gallate	5.58	1.18
Genistein	4.76	1.01	Propyl gallate	5.50	1.15
Daidzein	5.29	1.04	Tectorigenin	5.47	1.11
Daidzin	5.21	1.09	Puerarin	4.21	0.90
Genistin	4.65	1.04	Sophoricoside	3.39	0.79
Biochanin A	4.79	0.96

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Materials and methods

Apparatus and reagents

Fluorescence spectra were recorded on a JASCO FP-6500 fluorimeter (Tokyo, Japan). pH measurements were carried out on a Cole-Parmer PHS-3C Exact digital pH meter (IL, USA). Biochanin A, genistein, apigenin, puerarin, catechin, epicatechin (EC) and luteolin (99.0%) were purchased from Aladin Co., Ltd. (Shanghai, China). Flavone, chrysin and baicalein (99.5%) were obtained commercially from Wako Pure Chemical Industries (Osaka, Japan). Flavanone, 7-hydroxyflavanone, 6-hydroxyflavanone, 6-methoxyflavanone, 6-hydroxyflavone and 6-methoxyflavone were purchased from TCI Chemical Industries (Tokyo, Japan). Others polyphenol standards were obtained commercially from Shanghai Tauto Biotech Co., Ltd. (Shanghai, China). Working solutions of the polyphenols (1.0 × 10⁻³ mol L⁻¹) were prepared by dissolving each polyphenol in methanol. Pure bovine milk was obtained from Guangming Co. (Shanghai, China). It contains (per 100 mL) 3.0 g protein, 3.2 g fat and 4.7 g carbohydrate. The working solution of BMP (1 : 100) was prepared using double-distilled water and stored in a refrigerator prior to use. All other reagents and solvents were of analytical grade, and all aqueous solutions were prepared using freshly double-distilled water.

Fluorescence spectra

A 3.0 mL working solution of BMP (1 : 100) was transferred to a 1.0 cm quartz cell. It was then titrated by the successive addition of the 3.0 μL polyphenol solution (1.0 × 10⁻³ mol L⁻¹). Titrations were performed manually using trace syringes. After each titration, the fluorescence spectrum was collected with the working solution of BMP (1 : 100). The results of the time course experiments for the equilibration are not shown here. The fluorescence emissions of these polyphenols within the range 300–400 nm were not observed under the excitation wavelength of 280 nm. The polyphenols were stable during the fluorescence measurements, as shown by HPLC analyses (not shown here). Each fluorescence intensity determination was repeated and found to be reproducible within experimental error.

Results and discussion

The binding constants (K_a) and the number of binding sites (n)

As representative examples, the fluorescence spectra of BMP after the addition of 7-hydroxyflavanone (A) and 6-hydroxyflavone (B) are shown in Fig. 1 (the fluorescence spectra of BMP quenched by the other polyphenols are not shown here). The maximum λ_em of BMP was obviously red-shifted in the presence of 7-hydroxyflavanone (A) and 6-hydroxyflavone (B) (Fig. 1). These results suggest that there was a change in the immediate environment of the tryptophan residues of BMP, and that 7-hydroxyflavanone and 6-hydroxyflavone were situated in close proximity to the tryptophan residues for quenching to occur.^25–28 In the present study, the information about other amino acid residues was not understood. The buried indole group of tryptophan residues could be re-deployed in a more hydrophobic environment after the addition of polyphenols.

Fig. 1 The quenching effect of 7-hydroxyflavanone (A) and 6-hydroxyflavone (B) on MP fluorescence spectra at 300.15 K. λ_ex = 280 nm; BMP (1 : 100); a–j: 0.00, 1.00, 2.00…8.00 (× 10⁻⁶ mol L⁻¹) of polyphenols.

Moreover, in these and all other cases, the fluorescence intensities of BMP decreased with increasing concentration of polyphenol. However, the quenching degree of each differs depending on the structure of the polyphenol (data not shown here). For example, 6-hydroxyflavone (B) showed a stronger quenching effect on BMP fluorescence than that of 7-hydroxyflavanone (A). 6-Hydroxyflavone resulted in a quenching of about 62.62% of the BMP fluorescence; however, 7-hydroxyflavanone only quenched about 43.32% of the BMP fluorescence.

The binding constants were calculated according to the double-lgarithm equation:^25–28

lg (F₀ − F)/F = lgK_a + nlg[Q] (1)

where F₀ and F represent the fluorescence intensities of MP in the absence and presence of the polyphenol, K_a is the binding constant, n is the number of binding sites and [Q] is the concentration of polyphenol. Table 1 summarizes the correspondingly calculated results according to eqn (1). The values of lgK_a were found to be proportional to the number of binding sites (n) (Fig. 2), which indicates that eqn (1) used here is suitable to study the interaction between polyphenols and BMP.^29,30

Fig. 2 The relationship between the affinity (lgK_a) and the number of binding sites (n) between polyphenols and BMP.

Structure–affinity relationship of flavonoid–BMP interactions

As shown in Fig. 3, some of the structural elements that influence the affinity of polyphenols for BMP are as follows: (1) The methylation and methoxylation of flavonoids decrease or little affect the affinity for BMP. (2) Hydroxylation on rings A and B of flavones and flavonols slightly enhances the interaction. Hydroxylation on ring A of flavanones significantly improves the affinity. However, the hydroxylation on the ring C of flavones hardly influences the binding affinity, and hydroxylation on ring A of isoflavones reduces or little affects the affinity for BMP. (3) The glycosylation of flavonoids weakens the affinity by 1–2 orders of magnitude. (4) The hydrogenation of the C2=C3 double bond of flavonoids decreases the binding affinity. (5) The galloylation of catechins significantly improves the binding affinity. (6) The glycosylation of resveratrol decreases its affinity for BMP. (7) The esterification of gallic acid increases its binding affinity.

Fig. 3 The structural elements that influence the affinities of polyphenols for BMP. The up arrows represent increasing the binding affinity, the down arrows represent decreasing the binding affinity.

Relationship between the partition coefficient and the affinity for BMP

The lipophilicity of the compounds under study was assessed by their partition coefficient values (XlgP₃) according to PubChem public chemical database. There is a relationship between the XlgP₃ values and the lgK_a values for polyphenols (Fig. 4). The linear regression equation using Origin 7.5 software was XlgP₃ = 5.5864 − 0.8704 lgK_a (R = 0.2920). The affinities of polyphenols for BMP increased with increasing partition coefficient. From this point of view, the hydrophobic force played an important role in the binding interactions between the polyphenols and BMP. To further investigate whether or not the hydrogen bond force plays an important role in binding polyphenols to BMP, the relationship between the hydrogen bond acceptor/donor number (N, data from the PubChem public chemical database) of flavonoids with the affinity for BMP are shown in Fig. 5. The affinity for BMP obviously decreases with increasing hydrogen bond acceptor number of the polyphenol. These results illustrate that the hydrogen bond force is not the main force binding polyphenols to BMP.

Fig. 4 Relationship between the apparent binding constant (lgK_a) and the partition coefficient (XlgP₃) of flavonoids. The partition coefficient values (XlgP₃) are taken from the PubChem public chemical database.

Fig. 5 Relationship between the hydrogen bond acceptor/donor number of flavonoids (N) and their affinity for BMP. The hydrogen bond acceptor/donor numbers are taken from the PubChem public chemical database.

Relationship between the topolgical polar surface area and the affinity for BMP

The topolgical polar surface area (TPSA) is defined as the sum of the surfaces of polar atoms in a molecule. TPSA has been shown to be a very good descriptor that characterizes drug absorption, including intestinal absorption, bioavailability, Caco-2 permeability and blood–brain barrier penetration. Compounds with a high TPSA are transported while those with a low TPSA are not. A strong correlation between TPSA and transport properties (K_m) is also found. In the present study, the relationship between TPSA and the binding affinity of flavonoids for MP was studied. TPSA values were obtained from the PubChem public chemical database. It is found that TPSA values decrease with the increasing lgK_a values of flavonoids for BMP (Fig. 6). This result also illustrates that the glycosylation of hydroxyl groups in polyphenols weakens their binding affinity for BMP (Table 1).

Fig. 6 Relationship between TPSA and the affinity of flavonoids for BMP. TPSA values were obtained online (http://www.molinspiration.com/cgi-bin/properties).

Relationship between Mulliken electronegativity and the affinity of flavonoids for BMP

Electronegativity, symbol χ, is a chemical property that describes the tendency of an atom or functional group to attract electrons (or electron density) towards itself and thus the tendency to form negative ions. Mulliken electronegativity is simply the average of the first ionization energy and electron affinity. Unlike Pauling electronegativity, Mulliken's equations are absolute and need no starting reference point.³¹ Mulliken electronegativity is widely used in structure–activity relationships (SAR) of polyphenols.^32,33 In the present study, the relationship between Mulliken electronegativity and the binding affinity of flavonoids for BMP was investigated. A strong correlation between Mulliken electronegativity and binding affinity (lgK_a) was found (R = 0.64626). As shown in Fig. 7, Mulliken electronegativity values were found to increase with increasing lgK_a of flavonoids for BMP. The pH value of normal milk ranges from 6.6 to 6.8. In this pH range, caseins have negative charges and are not solubilized as salts with a stable colloidal form.¹⁵Polyphenols with a higher electronegativity will form stronger attractions with caseins. It illustrated that the electrostatic interaction plays a key role in binding polyphenols to BMP.

Fig. 7 Relationship between Mulliken electronegativity and the affinity of flavonoids for BMP. The Mulliken electronegativity values were obtained from refs. 32 and 33.

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