Separation and Identification of Anthocyanin Extracted from Mulberry

Article pubs.acs.org/JAFC

Separation and Identification of Anthocyanin Extracted from Mulberry Fruit and the Pigment Binding Properties toward Human Serum Albumin Feng Sheng,†,‡ Yuning Wang,‡,§ Xingchen Zhao,‡,∥ Na Tian,† Huali Hu,§ and Pengxia Li*,§ §

Institute of Agro-product Processing, Jiangsu Academy of Agricultural Sciences, Nanjing, P. R. China College of Chemistry & Material Science, Shandong Agricultural University, Taian, P. R. China ∥ State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, P. R. China †

S Supporting Information *

ABSTRACT: Purple pigments were isolated from mulberry extracts using preparative high-speed countercurrent chromatography (HSCCC) and identified by ESI-MS/MS and high performance liquid chromatography (HPLC) techniques. The solvent system containing methyl tert-butyl ether, 1-butanol, acetonitrile, water, and trifluoroacetic acid (10:30:10:50:0.05; %, v/v) was developed in order to separate anthocyanins with different polarities. Cyanidin 3-O-(6″-O-α-rhamnopyranosyl-βgalactopyranoside) (also known as keracyanin) is the major component present in mulberry (41.3%). Other isolated pigments are cyanidin 3-O-(6″-O-α-rhamnopyranosyl-β-glucopyranoside) and petunidin 3-O-β-glucopyranoside. The binding characteristics of keracyanin with human serum albumin (HSA) were investigated by fluorescence and circular dichroism (CD) spectroscopy. Spectroscopic analysis reveals that HSA fluorescence quenched by keracyanin follows a static mode. Binding of keracyanin to HSA mainly depends on van der Waals force or H-bonds with average binding distance of 2.82 nm. The results from synchronous fluorescence, three-dimensional fluorescence, and CD spectra show that adaptive structure rearrangement and decrease of α-helical structure occur in the presence of keracyanin. KEYWORDS: mulberry pigment identification, anthocyanin separation, keracyanin, pigment−protein binding

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INTRODUCTION Anthocyanins are natural food colorants which as well hold potential use as dietary modulators of mechanisms for various diseases. Due to increasing demand for natural food colorants, their significance in the food industry is also increasing.1 Previous studies have demonstrated that anthocyanins are helpful in protecting against oxidative stress due to their antioxidant activity. It is reported that the antioxidant activity increases with increasing number of hydroxyl groups and decreases with glycosylation degree of anthocyanidins.2 As a result, the antioxidant activity for berry extracts, particularly in the commercial market, varies due to differences in species and extraction methods. Anthocyanins extracted from berries are powerful antioxidants and therefore possess health-promoting properties such as analgesic properties and neuroprotective and anti-inflammatory activities.3−5 To exert the beneficial effects, anthocyanins need to be transported in the circulation system and distributed into targeted organs, which is associated with binding of biomacromolecules.6 HSA is the most important transport protein in sanguis, taking about 60% of the total plasma protein. HSA has been widely studied in recent years due to its amazing ability to transport hormones, fatty acids, drugs, nutrients, and inorganic ions reversibly, to buffer pH, and to maintain osmotic pressure bind. By binding with the protein, these small molecules are greatly affected in terms of the biodistribution and metabolism in the circulation system. As anthocyanins are transported in the circulation system, the protein-bound small © 2014 American Chemical Society

molecules are exchanging with their free form in order to maintain equilibrium. Therefore, the binding parameters play a key role when considering the availability of anthocyanins for their in vivo transport.7,8 Although blood total anthocyanin concentration after human uptake is very low, ranging from 5 to 168 nM, its biological activity may be influenced to a large extent due to different binding ability to serum proteins.9−12 Although the mulberry (Morus alba L.) mainly supplies leaves to raise silkworms, mulberry fruit has long been used as a medicinal agent in traditional Chinese medicine to nourish the yin and blood, benefit the kidneys, and treat weakness, fatigue, anemia, and premature graying of hair. However, the components of the active ingredients (mainly anthocyanins) and their transportation in the circulatory system after absorption are still unknown. In this study, we report the extraction of anthocyanins (mainly keracyanin) from Morus alba L. fruit using chromatography, in particular HSCCC. The fractions were then identified by ESI-MS/MS, and the main component was selected to study its binding properties with HSA in simulated physical conditions using fluorescence and CD spectroscopy. Since HSA contains amino acid residues like Trp, Tyr, and Phe, it displays intrinsic fluorescence after being stimulated by ultraviolet light, and the sensitivity makes Received: Revised: Accepted: Published: 6813

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fluorescence spectroscopy suitable for the study of HSA− keracyanin interaction. Besides, CD technique was utilized to quantitatively evaluate the secondary structure alterations of HSA upon binding to keracyanin chloride.

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spectrometer (Thermo Fisher Scientific Inc., Waltham, MA). The MS/ MS spectra were obtained using positive mode, and the ESI voltage was set at 5000 V. The sheath, aux, and sweep gas flow rates were adjusted at 9, 5, and 0 arb, respectively. All the spectra were collected within a scan range of 50−1200 m/z. Spectroscopic Characterization of HSA−Keracyanin Interaction. Fluorescence experiments were carried out on a fluorimeter Hitachi F4500 (Hitachi, Tokyo, Japan). Quartz cuvettes with a size of 1 × 1 × 4 cm were used in all the experiments. Voltage of the photomultiplier was set at 700 V. Excitation and emission slit widths were both set at 5 nm. All the spectra were recorded under the speed of 1200 nm min−1. The fluorescence spectra were corrected for the inner filter effects (IFEs) using the following formula:

MATERIALS AND METHODS

Materials. HSA and keracyanin were purchased from SigmaAldrich Corp (St. Louis, MO). Mature mulberry fruits were bought from Nanjing, China, freeze-dried, and finally ground to powder. Organic solvents used for ESI-MS/MS and HPLC were chromatographic grade and were bought from Sinopharm Co., Ltd. (Beijing, China). All other chemicals (analytical grade) were bought from Sinopharm Co., Ltd. (Beijing, China). A 0.2 M stock solution of pH = 7.4 phosphate buffer was prepared by mixing NaH2PO4 and Na2HPO4 in ultrapure water. Ultrapure water was used throughout. Methods. Pigment Extraction Condition Screening and Identification. In order to obtain the optimal extraction liquor, mulberry freeze-dried powder was weighed in the test tubes (0.05 g for each), and 10 mL of extraction solvent was added immediately to each tube. The ten extraction solvents were ethyl acetate, 1-butanol, acetone, alcohol, alcohol solution (70%), alcohol solution (50%), alcohol solution (30%), water, HCl solution (pH = 1), and alcohol (50%)− HCl mixture solution (pH = 3), respectively. The samples were processed using ultrasonic vibration with a water bath for 30 min to ensure a well mixed extraction. Then the supernatant was filtered through the filter paper and was diluted with water to a final volume of 50 mL. The absorbance spectrum of these solutions was recorded in the range of 200 to 800 nm. For extraction efficiency comparison, the method from Lee13 was used to determinate the total anthocyanin content. We compared the extraction efficiencies of ten extraction solvents and chose the best one to do further analysis. We also weighed the dry residues of the extract using different liquors. Mulberry freeze-dried powder (2 g) was added to 30 mL of petroleun ether (lipid-removal solvent). Then the mixture was stirred constantly with 60 °C water bath for half an hour. Supernatant was removed after centrifugation at 2000 rpm, and repeated extraction was done 5 times, after which the solvent was evaporated. The sample was then put into the optimal pigment extraction liquor (70% alcohol solution) and was processed using ultrasonic vibration with water bath for 30 min 4 times. All the extracts were transferred to a 300 mL round flask and evaporated by a rotary evaporator (30 rpm, 500 Pa, and 50 °C) to obtain solid substance (the crude mulberry anthocyanin extract). The pigment extract obtained here was utilized for separation and further analysis. The TBE-300A HSCCC instrument was made by Shanghai Tongtian Co., Ltd. (Shanghai, China). For HSCCC experiment, a solvent system made up of methyl tert-butyl ether, 1-butanol, acetonitrile, water, and trifluoroacetic acid (10:30:10:50:0.05; %, v/ v) was used. The two phases were separated and prepared for use after mixing of 1000 mL of solvent in a separating funnel. The instrument was then rotated at 850 rpm for equilibration of the solvent. 220 mg of anthocyanin mulberry extract was first dissolved in 5 mL of mobile phase, after which aqueous mobile phase was injected at 1.5 mL/min. The eluates at different time points were detected using a UV−vis detector at 280 nm. HPLC analysis was performed on a Shimadzu LC-20AT liquid chromatography system (Shimadzu, Tokyo, Japan). The solvent system was composed of phase A (water:acetonitrile:formic acid = 96:3.3:0.55; %, v/v) and phase B (acetonitrile:water:formic acid = 55:44:0.55; %, v/v). Gradient conditions: 0−20 min, 6−20% A in B; 20−30 min, 20−40% A in B; 30−35 min, 40−60% A in B; 35−40 min 60−70% A in B. A reequilibration of 6 min was made afterward. The flow rate was 0.8 mL min−1, and 20 μL aliquots were injected into the column. Diluted extracts were trasferred into the Kinetex PFP 100A column (Phenomenex Inc., Torrance, USA), and anthocyanins were detected at 280 nm. During the chromatography analysis UV−vis absorption spectra were recorded. Samples separated from HSCCC were prepared for structural identification using a Thermo Fisher LCQ DecaXP MAX mass

Fideal(λex , λem) = Fobs(λex , λem)CFp(λex )CF( s λem) ≈ Fobs(λex , λem)10(Aem + Aex )/2

(1)

where CFp and CFs are the correction factors for excitation and emission light at λex and λem, respectively. Aex and Aem stand for the absorbance at λex and λem, respectively. Fobs represents the observed fluorescence intensity.14 In quest of the fluorescence quenching mechanism, the well-known Stern−Volmer formula was applied to analyze and calculate the fluorescence data: F0/F = 1 + Kqτ0[Q] = 1 + KSV[Q]

(2)

where F0 and F represent the fluorescence intensities of a protein in the absence and presence of the ligand, and Kq and KSV are the bimolecular quenching rate constant and the Stern−Volmer quenching constant, respectively. τ0 is the average fluorescence lifetime of the fluorophore in the absence of quencher, and it is 10 ns for biomacromolecules. [Q] is the molar concentration of the quencher (keracyanin). The binding information on the HSA−keracyanin system is determinated by the following formula:

lg

F0 − F = lg KA + n lg [Q] F

(3)

KA is the binding constant, and n is the average number of binding sites for one HSA molecule. By utilizing the Van’t Hoff equation, we calculated the corresponding thermodynamic parameters:

⎛K ⎞ ΔH ° ⎛ 1 1⎞ ln⎜ 2 ⎟ = ⎜ − ⎟ R ⎝ T1 T2 ⎠ ⎝ K1 ⎠

(3)

where K is the binding constant calculated from the above step, R is the universal gas constant with a value of 8.314 J·mol−1·K−1, ΔH° and ΔS° are the changes in enthalpy and entropy. Gibbs free energy change (ΔG°) associated with the interaction of HSA and keracyanin can be calculated from ΔG° = ΔH ° − T ΔS° = − RT ln K °

(4)

The energy transfer efficiency E is defined by the following equation: 6

E=1−

R0 F = F0 R 06 + r 6

(4)

where R0 is the Förster critical distance and r is the average distance between the donor and the acceptor. R0 is usually calculated from the following equation: R 0 6 = 8.8 × 10−25k 2N −4 ΦJ

(6)

where k is the orientation factor, N is the refractive index, and Φ is the quantum yield of the donor. The spectral overlap integral between the donor fluorescence spectrum and the acceptor absorbance spectrum is represented by J, and it is expressed as 2

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∑ F(λ) ε(λ) λ 4 Δλ ∑ F(λ) Δλ

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extract separation with 1000 mL of a solvent system comprising methyl tert-butyl ether, 1-butanol, acetonitrile, water, and trifluoroacetic acid (10:30:10:50:0.05; %, v/v) according to the literature. The HSCCC run yields four peaks that correspond to fractions I−IV (27.9%, 25.7%, 41.3%, and 5.1%), respectively. The shaded part of the peaks was obtained and concentrated. This isolation gives more than 95% purity for fractions II−IV based on HPLC analysis. Solvent systems with high polarity are necessary for preparative separation of monomeric anthocyanin glycosides using HSCCC. For routine isolation, the aqueous solvent was usually employed as mobile phase, and 0.05% trifluoroacetic acid is required for stabilization of anthocyanins. In this way, the elution was in “head to tail” mode, and stable signals can be detected. Because anthocyanins are generally covalently bound with polymers and polysaccharides, the multiple peaks at the HPLC profile of fraction I are due to these complexes. Fraction I was also subjected to HPLC and represented a complicated mixture of components (Figure S2A, Supporting Information). Similar fractions were acquired from earlier isolation of anthocyanins using HSCCC in “head to tail” mode.15−17 Confirmation of Anthocyanin Structure. In order to elucidate the precise structure of the compounds, the fractions from HSCCC analysis were submitted to the ESI-MS/MS. Fraction II produced a molecular ion peak at m/z 595 (100) and MS2 fragments with 287 (100) and 449 (37) U. The first fragment coincides with the cyanidin moiety, due to the loss of rutinose (308 U), and the second one at m/z 449 is due to the loss of the deoxyhexose (146 U) molecular fragment (Figure S3, Supporting Information). Fraction III reveals a molecular ion peak at m/z 595 (100) and fragment ions at m/z 287 (100) and 449 (26) U, indicating that it is a cyanidin derivative with similar structure of fraction II (Figure 2). Considering the elution order of the fractions from HSCCC, m/z values of the fragments, and previously published work,18−20 fractions II and III should be identified as cyanidin 3-O-(6″-O-α-rhamnopyranosyl-β-glucopyranoside) and cyanidin 3-O-(6″-O-α-rhamnopyranosyl-β-galactopyranoside), respectively. Fraction IV shows a major molecular ion m/z 479 (100) confirmed probably as petunidin 3-O-β-glucopyranoside, however with other unknown ion peaks at m/z 385 (47), 499 (42), 249 (32) U, etc. as impurities (Figure S4, Supporting Information). HPLC analysis shows two major peaks for this fraction (Figure S2, Supporting Information). Comparisons were made for fractions II and III, both of which show fine purity. HSA Fluorescence Quenching by Keracyanin and the Interaction Mechanism. HSA possesses three intrinsic fluorophores: Trp, Tyr, and Phe residues. However, most of the intrinsic fluorescence of HSA is contributed by Trp alone, taking up more than 95% of the fluorescence intensity.21,22 Thus, alterations of HSA fluorescence intensity should be due to the change of Trp residue. Fluorescence measurements give information concerning the intermolecular distance, binding constants, binding sites, and binding mode for small molecule− protein interaction.23−25 To elucidate the binding mechanism of keracyanin−HSA interaction, steady-state fluorescence spectra of HSA were recorded after addition of different amounts of keracyanin (Figure 3A). By exciting at 295 nm, the strong fluorescence emission band at 340 nm can be detected, which is due to the single Trp residue (No. 214 residue, shown in Figure S5, Supporting Information), which is located in the hydrophobic cavity.26−28 The addition of keracyanin causes significant decrease in HSA fluorescence intensity, showing that

(7)

where F(λ) stands for fluorescence intensity of the donor at wavelength λ, and ε(λ) is the molar absorption coefficient of the acceptor at wavelength λ. For synchronous fluorescence analysis, the spectra were collected by setting the differences of excitation and emission wavelengths at 15 or 60 nm (Δλ = λex − λem = 15 or 60 nm). The rest of the experimental conditions are the same with the steady-state fluorescence measurements. CD measurements were performed on a JASCO-815 CD spectropolarimeter (Jasco Co. Ltd., Tokyo, Japan). CD spectra of 2 × 10−7 M HSA with or without pigment were measured between 190 and 260 nm using a 1 × 1 × 4 cm quartz cuvette. All the samples were prepared in pH 7.4 phosphate buffer, and the spectra were recorded at 25 °C. The contents of secondary conformation of the protein were analyzed using online SELCON3 program. All the absorption spectra in this study were collected using a Shimadzu UV-2450 (Shimadzu, Tokyo, Japan) spectrophotometer equipped with a 1 × 1 × 4 cm quartz cell.

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RESULTS AND DISCUSSION Optimization of the Extraction Liquor. Absorption spectra of the extraction obtained using different solvents are shown in Figure S1 (Supporting Information). There are two typical peaks for anthocyanins, one at around 280 nm (corresponding to its aromatic ring structures) and the other at around 530 nm. The extraction obtained by alcohol solution (70%) tested by the two ways results in the highest weight of solid extract, showing the best yield of anthocyanins (Table S1, Supporting Information). HSCCC Separation. The mobile phase flow rate and the revolution speed were optimized prior to the separation of anthocyanins. The results indicate that fast flow rate resulted in poor separation, and the chromatogram peaks easily huddled. Besides, when the revolution speed was set in the range of 800−900 rpm, the separation was not apparently influenced. Based on these facts, 1.5 mL/min flow rate and 850 rpm revolution speed were chosen as the experimental conditions. Separation of natural products using HSCCC depends on the suitability of solvent systems to a great extent. Figure 1 shows the HSCCC chromatogram for 220 mg of mulberry crude

Figure 1. Chromatography of the crude ethanol solution extract of mulberry by preparative HSCCC using solvent system of methyl tertbutyl ether, 1-butanol, acetonitrile, water, and trifluoroacetic acid (10:30:10:50:0.05; %, v/v). Stationary phase: upper organic phase. Mobile phase: lower aqueous phase. 6815

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static mode in another way. Hence it can be safely considered that static quenching mechanism is dominant for HSA− keracyanin interaction. Binding constants of HSA−keracyanin interaction were calculated using the fluorescence intensity at 340 nm. The results at different temperatures are shown in Table 1. Generally speaking, high binding constants for protein ligand interactions are within the range of 106−108 M−1. The obtained binding constant of the HSA−keracyanin system is 2.49 × 104 M−1 at 298 K, which indicates a relatively weak interaction between keracyanin and HSA. The binding number for the interaction is about 1, suggesting that keracyanin binds the protein in 1 to 1 mode at 298 K. As lower (at 104 to 103 scales) binding constants and fewer binding sites are observed for high temperature, HSA may bind fewer keracyanin molecules at weaker forces at body temperature than room temperature, making keracyanin easier to be released from plasma protein to target organs. In general, the binding forces between ligands and proteins involve electrostatic forces, H-bonding, van der Waals interaction, and hydrophobic forces. From the viewpoint of Ross,30 if positive enthalpy change and entropy change implies hydrophobic interaction, then negative enthalpy change and positive entropy change suggests electrostatic force, and negative enthalpy change and entropy change reflects the van der Waals force or H-bonding. ΔS○ can be treated as a constant since the temperature did not change too much, and the values of ΔH○, ΔS○, and ΔG○ at 293 K, 298 K, and 303 K are listed in Table 1. ΔH○ and ΔS○ calculated in this work are all negative, suggesting that the dominating force is van der Waals force or hydrogen bonds. Secondary Structure of HSA on Binding to Keracyanin. CD has been long used for evaluating conformational alterations of protein when binding ligands. The far-UV CD spectrum of HSA in the absence and presence of keracyanin was hence recorded for secondary structural evaluation. HSA was structurally dominated by α-helix, showing two negative peaks at 208 and 222 nm.31,32 By adding keracyanin to HSA (Figure 4), the negative peaks decrease correspondingly, indicating decreased content of α-helix. However, the peak shape and position do not change with the ellipticity value, demonstrating that HSA managed to retain the overall structure. The decrease in β-sheet (from 7.4% to 6.7% to 5.9%) and α-helix (from 60.4% to 58.3% to 54.7%) contents and increase in random coil (from 23.4 to 24.5 to 25.6) structure are due to partial protein conformational changes in the HSA−keracyanin complexes. Loss of α-helical content also indicates destruction of H-bondings which maintain the special structure of α-helix. Combined with the fact that hydrogen bonds may form in the interaction, it can be concluded that keracyanin first perturbs the hydrogen bonds of HSA and then establishes new ones with the protein. Energy Transfer from Trp of HSA to Keracyanin. Fluorescence resonance energy transfer (FRET) is a powerful and sensitive method to evaluate the possibility of energy trasfer from Trp residue of HSA to ligands and to estimate the binding distance between them. From Förster’s nonradiative energy transfer theory, energy transfer occurs when the distance between the donor and the acceptor is less than 8 nm as calculated, and there is enough overlap area between the receptor’s absorption spectrum and the fluorescence emission spectrum of donor.33

Figure 2. (A) MS and (B) MS/MS spectra of fraction III in mulberry separated from HSCCC. (C) HPLC chromatograms of fraction III separated from HSCCC (UV spectrum).

the intrinsic fluorescence of HSA was quenched upon keracyanin binding. It also implies that the microenvironment of Trp-214 was altered after interacting with keracyanin. The quenching rate in the studied range shows good linearity after eliminating IFE (Figure 3B). In order to elucidate whether the quenching is dynamic or static, the Stern−Volmer quenching constant KSV and the bimolecular quenching rate constant Kq were calculated. Kq was found to be larger than the maximum diffusion collision rate constant (2.0 × 1010 M−1 s−1).29 Besides, increment of the quenching constants is apparently observed with decreasing temperature, demonstrating that the fluorescence quenching of HSA was attributed to 6816

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Figure 3. (A) Fluorescence emission spectra of HSA in the presence of keracyanin. CHSA, 1 × 10−6 M; Ckeracyanin (a−g), 0, 1, 2, 3, 4, 6, and 9 × 10−6 M; pH 7.4, T = 298 K. (B) Stern−Volmer and (C) Hill plots of fluorescence data at different temperatures.

Table 1. Quenching, Binding, and Thermodynamic Parameters for HSA−Keracyanin System Stern−Volmer quenching constants

binding params

thermodynamic params

T (K)

Kq (M−1 s−1)

KSV (M−1)

R

KA (M−1)

n

R

ΔG° (J·mol−1)

ΔS° (J·mol−1·K−1)

ΔH° (J·mol−1)

293 298 303

7.23 × 1012 5.18 × 1012 3.44 × 1012

7.23 × 104 5.18 × 104 3.44 × 104

0.9960 0.9986 0.9968

1.06 × 105 2.49 × 104 6.44 × 103

1.03 0.936 0.855

0.9969 0.9984 0.9981

−2.82 × 104 −2.51 × 104 −2.21 × 104

−621 −622 −621

−2.10 × 105

Figure 4. CD spectra of HSA−keracyanin system obtained at room temperature and pH 7.4 with secondary structural content shown. CHSA: 2 × 10−7 M. Ckeracyanin: 0, 5, and 10 × 10−7 M for 1−3.

It has been reported for HSA that Φ = 0.118, N = 1.336, and k2 = 2/3,34,35 and J in eq 6 was the integration of the UV−vis absorption spectrum of keracyanin and the fluorescence emission spectrum of HSA, as shown in Figure S6 (Supporting Information). According to eqs 4−6, the values of J, E, r, and R0 were calculated to be 2.43 × 10−15 cm3 M−1, 0.0967, 2.82, and 1.94 nm, respectively. These results show that the value for r is between 2 and 8 nm and is also on the 0.5R0−1.5R0 scale, indicating the existence of energy transfer between HSA and keracyanin. Besides, 2.82 nm is near enough for microenvironmental alteration of Trp-214 by keracyanin. Conformational Changes of HSA by Keracyanin. Characteristic information on the microenvironmental changes in the vicinity of fluorophores was investigated by synchronous fluorescence spectra. When Δλ is set at 15 nm, the fluorescence spectra give information for Tyr, and 60 nm for Trp.36 The synchronous fluorescence spectra of the HSA−keracyanin system are shown in Figure 5. While no obvious change is found when Δλ is set at 15 nm, the emission maxima

Figure 5. Synchronous fluorescence spectra of HSA in the presence of keracyanin at 298 K when Δλ = 60 or 15 nm.

experienced a red shift from 279 to 281 nm when Δλ = 60 nm. Thus, it can be concluded that the hydrophilicity around Trp214 increases compared with that of Tyr. Water molecules are sufficient to perturb the vicinity of Trp-214 with the help of keracyanin binding, making the microenvironment from nonpolar to slightly polar. The 3D fluorescence spectra for the HSA−keracyanin interaction system are presented in Figure 6 with peak information summarized in Table 2, where we can get the microenvironmental and conformational information on HSA upon keracyanin interaction. As we can seen in Figure 6, peak a results from the Rayleigh scattering of water (λex = λem), whereas the strong peak b mainly reflects the characteristics of chromophores (mainly Trp residue) of HSA, and peak c is related to the polypeptide backbone structure of HSA.37,38 As 6817

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a semipreparative scale. Anthocyanins from mulberry most probably include cyanidin 3-O-(6″-O-α-rhamnopyranosyl-βgalactopyranoside) (keracyanin), cyanidin 3-O-(6″-O-α-rhamnopyranosyl-β-glucopyranoside), and petunidin 3-O-β-glucopyranoside. Details on the interaction between keracyanin and HSA were revealed using spectroscopy. Intrinsic fluorescence of HSA has been quenched by keracyanin following static mechanism, which is induced by the formation of a certain kind of new complex. The binding of keracyanin to HSA is moderate and equimolar. Thermodynamic parameters indicate that the reaction is spontaneous, with H-bonding and van der Waals forces playing the major role. Synchronous fluorescence spectroscopic results demonstrate that keracyanin gets close to Trp residues whereas the microenvironment around Tyr residues is almost unchanged. We have discovered and interpreted the structural changes of HSA by keracyanin complexation from CD and three-dimensional fluorescence analyses. Energy probably transfers from Trp to keracyanin in the binding process, and the binding distance between the two was estimated to be 2.82 nm at 298 K. The research results are expected to provide useful information for not only natural product separation and identification but the interactions of the carrier proteins with anthocyanin, which will be helpful for the development of food chemistry, clinical medicine, and life sciences.

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ASSOCIATED CONTENT

* Supporting Information S

Absorption spectra of pigment extract, HPLC and MS spectral data of the fractions, structure of HSA, and spectral overlap of keracyanin-HSA. This material is available free of charge via the Internet at http://pubs.acs.org.

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Figure 6. Three-dimensional fluorescence spectra of HSA without (A) and with keracyanin addition (B). CHSA, 2 × 10−6 M; Ckeracyanin, 5 × 10−6 M; pH 7.4, T = 298 K.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax/tel: +86-025-84392062. shown in Figure 6 and Table 2, with the addition of keracyanin the fluorescence of both peaks b and c is quenched and the fluorescence maximum of peak c red shifts slightly from 337 to 341 nm. These phenomena suggest that keracyanin perturbs the microenvironment of Trp-214 upon interaction with HSA, inducing slight unfolding or adaptive rearrangement of the polypeptide backbone of the protein. Besides, the 3D structure of HSA is known to be composed of three homologous domains with two subdomains for each. The hydrophobic cavity, where Trp-214 locates, is in the center of the three domains. The binding site of keracyanin might be located in this pocket. The interaction of keracyanin causes changes of polarity of the hydrophobic microenvironment (also proved by synchronous fluorescence study), leading to protein conformational changes. HSCCC technique makes it possible to separate anthocyanin-rich natural products and yield some pure anthocyanins on

Author Contributions ‡

Authors F.S., Y.W., and X.Z. contributed equally to this work.

Funding

This work was supported by Special Fund for National Forestry Scientific Research in the Public Interest (201204402-1) and Science & Technology Development Project of Shandong Province (2013GZX20109). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Thanks are given to Research Center for Food Safety & Inspection Engineering of Shandong Province and Science & Technology Department of Shandong Province for their support.

Table 2. 3D Fluorescence Information of HSA−Keracyanin System HSA

HSA−keracyanin

peaks

peak position λex/λem (nm/nm)

Stokes Δλ (nm)

fluorescence intensity

peak position λex/λem (nm/nm)

Stokes Δλ (nm)

fluorescence intensity

a b c

275/275 278/335 227/340

0 57 43

3290 7973 8790

285/285 280/337 230/341

0 57 71

1615 5940 7337

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dx.doi.org/10.1021/jf500705s | J. Agric. Food Chem. 2014, 62, 6813−6819