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Host–guest interaction between brazilin and hydroxypropyl-β-cyclodextrin: Preparation, inclusion mode, molecular modelling and characterization
Accepted Manuscript Host–guest interaction between brazilin and hydroxypropyl-β-cyclodextrin: Preparation, inclusion mode, molecular modelling and characterization Li-Juan Yang, Qing Chang, Shu-Ya Zhou, Yun-Han Yang, Fu-Ting Xia, Wen Chen, Minyan Li, Xiao-Dong Yang PII:
S0143-7208(17)32012-0
DOI:
10.1016/j.dyepig.2017.12.010
Reference:
DYPI 6416
To appear in:
Dyes and Pigments
Received Date: 24 September 2017 Revised Date:
3 December 2017
Accepted Date: 4 December 2017
Please cite this article as: Yang L-J, Chang Q, Zhou S-Y, Yang Y-H, Xia F-T, Chen W, Li M, Yang X-D, Host–guest interaction between brazilin and hydroxypropyl-β-cyclodextrin: Preparation, inclusion mode, molecular modelling and characterization, Dyes and Pigments (2018), doi: 10.1016/ j.dyepig.2017.12.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Graphical Abstract
H-3 HO
HP¦ÂCD
O H
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D HO H-5
OH
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B
C
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H H
OH 5' H Brazilin
Brazilin/HPβCD Complex
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H-5
Su-Mu
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Host–guest interaction between brazilin and
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hydroxypropyl-β-cyclodextrin: Preparation, inclusion mode,
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molecular modelling and characterization
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Li-Juan Yang a,*, Qing Chang a, Shu-Ya Zhou a, Yun-Han Yang a, Fu-Ting Xia a,
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Wen Chen b, Minyan Li c, Xiao-Dong Yang b,*
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Functional Materials of Yunnan, Yunnan Minzu University, Kunming 650500, P. R.
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China
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School of Chemistry & Environment, Engineering Research Center of Biopolymer
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Key Laboratory of Medicinal Chemistry for Natural Resources, Ministry of Education, School of Chemical Science and Technology, Yunnan University,
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Kunming, 650091, P. R. China
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Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104-6323, United States
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* Corresponding author. E-mail addresses: [email protected] (L.J Yang), [email protected] (X.D. Yang)
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ABSTRACT: Brazilin, the major constituent from the heartwood of Caesalpinia sappan, has been
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widely used as a natural red color dye or food ingredient and drug. However, the
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application of brazilin is greatly restricted by its low stability. In this study, the
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preparation, characterization, inclusion mode, stability and solubility of brazilin with
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hydroxypropyl-β-cyclodextrin (HPβCD) were studied for the first time in both the
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solid state and solution by means of XRD, DSC, SEM, 1H and 2D NMR and UV–vis
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spectroscopy. The molecular modelling demonstrated the most stable inclusion model
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of brazilin/HPβCD complex. The results illustrated that the stability of brazilin were
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improved in the inclusion complex with HPβCD. This satisfactory characterization
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and stability of the brazilin/HPβCD complex should be possibly useful for its
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utilization as dye, food or drug.
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Keywords:
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Brazilin; Hydroxypropyl-β-cyclodextrin; Inclusion mode; Molecular modelling;
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Characterization
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1. Introduction Caesalpinia sappan L. (Chinese name Su-Mu) is a plant that has been used as a
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natural red color dye for textile dyes, inks, paints, varnish tints and wood stains, as
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well as a traditional ingredient of food in Southeast and East Asia [1]. The heartwood
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of C. sappan has been used as an analgesic and anti-inflammatory drugs in Chinese
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folk medicine [2]. A lot of research has verified that its heartwood contains abundant
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homoisoflavones and several phenolics and chalcones. Among these homoisoflavones,
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brazilin (7,11b-dihydrobenz[b]indeno-[1,2-d]pyran-3,6a,9,10(6H)-tetrol, Figure 1) is
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regarded to be one of the most important active constituents due to its many
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pharmacological activities [3]. Scientific studies have demonstrated that brazilin
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has a wide range of biological activity including antioxidant, antibacterial, anticancer,
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anti-inflammatory, hypoglycemic, vasorelaxant and hepatoprotective properties
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[2,4–9]. In dye and food industry, brazilin has been widely used as a natural colorant
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and preservative agent [1]. However, the application of brazilin as a natural dye or
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food ingredient and drug is largely restricted by its low stability [1,10]. Brazilin is
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easily oxidized by air and light to produce brazilein [11]. Although much effort has
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been made to improve its stability by introducing some lyophillization or
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nanodispersion techniques [12-13], it is still not possible to sufficiently enhance the
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stability, which prevents its usage for applications in dye and food. As a result, it is
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significant to find an efficient carrier for brazilin with the aim of its further
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application.
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At the same time, cyclodextrins (CDs) are truncated-cone polysaccharides chiefly
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ACCEPTED MANUSCRIPT comprised of 6 to 8 D-glucose monomers connected by α-1,4-glucosidic bonds. CDs
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possess a hydrophobic interior cavity and a hydrophilic exterior surface, which can
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encapsulate model molecules to construct supramolecular complexes [14–16]. These
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properties enable them to be effectively used as substrate carriers and generally
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improve poor stability and aqueous solubility of the encapsulated drug
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in medical industry [17–22]. The similar results have been confirmed in the dye and
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pigment industry [23–30]. For example, CDs not only increased the stability of
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natural colorant laccaic acid A, but also improved its water solubility and dyeing
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effect [23]. The thermal stability of dope-dyed polyurethanes were improved by using
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disperse orange 31 as the dye and β-CD as the additive [25]. Hydroxypropyl-β-
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cyclodextrin (HPβCD, Figure 1) is a hydroxyalkylated β-CD derivative, which has
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quite high water solubility, low toxicity and suitable inclusion capacity [31,32].
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Recently, our group reported that the host–guest complexes of CDs with natural
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products such as hesperetin, pinocembrin, naringenin, crassicauline A and nimbin,
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remarkably improved the water solubility and stability of the products [33–37]. As a
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continuation of our research on natural products/CD inclusion complexes, a
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host–guest system of homoisoflavones (brazilin) with HPβCD was investigated. To
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the best of our knowledge, so far no scientific research on the inclusion behavior of
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brazilin/CD complexes has been reported.
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In the present study, we investigated firstly the preparation and characterization of
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brazilin/HPβCD host–guest system, as well as the inclusion mode and molecular
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modelling of the supramolecular complex, which will present a valuable method for
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getting new natural brazilin-based products of dye, food or drug.
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2. Materials and methods
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2.1. Materials
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Brazilin (FW = 286, PC>98%) was isolated from heartwood of Caesalpinia sappan
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L. in Yunnan Province, P.R. China. HPβCD (average FW = 1380) was obtained from
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Sigma-Aldrich Co. LLC. All reagents used were A. R. grade. All aqueous solutions
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were prepared using ultrapure water.
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2.2. Preparation of brazilin/HPβCD inclusion complex
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Brazilin (0.03 mM, 8.6 mg) and HPβCD (0.01 mM, 13.8 mg) were completely
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dissolved in a mixed solution of ultrapure water/ethanol (ca. 10 ml, V:V = 4:1), and
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the mixture was stirred at room temperature for 5 days in the dark. After evaporating
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the ethanol from the mixture, the uncomplexed brazilin was removed by filtration.
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The filtrate was evaporated under reduced pressure to remove the solvent and dried in
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vacuum to obtain the brazilin/HPβCD complex (yield 80%).
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2.3. Preparation of brazilin and HPβCD physical mixture
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The physical mixture was prepared by grinding together a mixture of brazilin and
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HPβCD (in 1:1molar ratio) in an agate mortar for 5 min.
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2.4. Job's plot
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The stoichiometry of inclusion complex was determined by the continuous
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variation Job's method [38]. The Job's plot was processed by the UV-Vis Spectra data
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recorded in an ultrapure water/ethanol (V:V = 4:1, pH 3.0) mixed solution. The total
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ACCEPTED MANUSCRIPT molar concentration of brazilin and HPβCD was held at 5.6×10-5 M and the molar
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fraction of brazilin ([brazilin]/([brazilin]+[HPβCD])) changed from 0.1 to 0.9. The
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absorbance was run on at 287 nm in UV at 37 oC. And the absorption of each brazilin
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solution in the presence and absence of HPβCD were also carried out in the same
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condition.
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2.5. Determination by UV Spectra
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Absorption spectra measurements were recorded on an Agilent UV 8453 using a
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conventional 1cm (1cm×1cm×4cm) quartz cell in a thermostated compartment. The
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concentration of brazilin was held constant at 0.058 mM. A suitable amount of
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HPβCD was added with the final concentrations varied from 0 to 4.000 mM (0, 0.231,
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0.471, 0.960, 1.372, 1.960, 4.000 mM). The absorption spectra measurement was
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carried out after 1 h. Given the poor water solubility of brazilin, an ultrapure
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water/ethanol (V:V = 4:1) solution was used in the spectral measurements.
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2.6. 1H and 2D NMR
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H NMR and 2D NMR experiments for HPβCD and brazilin/HPβCD complex were
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recorded on a Bruker Avance DRX500 spectrometer at 298 K. Tetramethylsilane
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(TMS) was used as a standard. All NMR experiments were conducted in D2O.
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2.7. Powder X-ray diffraction (XRD)
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XRD patterns were performed by a D/Max-3B diffractometer with Cu Kα radiation
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(100 mA, 40 kV) and a scanning rate of 5°/min. Samples were sett on a vitreous
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sample holder and scanned with a range of 2θ = 0.02° between 2θ = 3° and 50°.
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2.8. Thermal analyses
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2960 SDT V3.0F instrument or NETZSCH STA 449F3 with a heating rate of 10
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ºC/min from room temperature to 400 ºC in a nitrogen atmosphere.
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2.9. Scanning electron microphotographs (SEM)
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SEM pictures were run on a FEL QUANTA 200. The samples were fixed on metal
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stubs with double-sided adhesive tapes. The photographs were taken at an excitation
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voltage of 15, 20 or 30kV and a magnification of 1080, 1200, 1400 or 2000×.
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2.10. Solubilization test
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An excess amount of complex was added in ultrapure water (2 mL, ca. pH 6.0), and
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the solution was stirred for 24 h at room temperature in the dark. The mixture is
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filtered on a 0.45 µm cellulose acetate membrane. The absorbance of the filtrate was
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measured at 287 nm in UV and the residue was dosed by the standard curve of
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brazilin.
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2.11. Molecular Modelling
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Molecular modelling calculations were performed using Gaussian 03 program
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(Gaussian Inc., Wallingford, USA) [39]. The initial geometry of brazilin and HPβCD
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were constructed with Chembio3D ultra (Version 10.0, Cambridge Soft.com., USA)
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and then fully optimized by PM3 method without any symmetry constraint. The
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glycosidic oxygen atoms of HPβCD were placed onto the XY plane; their center was
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defined as the origin of the coordinate system. Then, the brazilin molecule was placed
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on the Z-axis, Which approaches and passes through the HPβCD cavity from +5 Å to
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-5 Å with a stepwise 1 Å. For each step, the geometry of the complex is fully
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possible models were investigated. In order to improve the accuracy of the results, the
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ONIOM2 (B3LYP/6-31G*:PM3) and (HF/6-31G*:PM3)] methods were used to
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calculated the most stable structure of the two models. All quantum theory
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calculations were performed at 1 atm and 298.15 K in vacuum. The structure with
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lowest heat energy at all positions was obtained as the optimal complex structure and
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then established in PyMol.
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3. Results and discussion
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3.1. Stoichiometry (Job's plot)
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The Job's plot is a typical and useful method to analyze the stoichiometry in
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host-guest molecules [31]. A Job's plot was used to confirm the stoichiometry of
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brazilin and HPβCD using the UV–vis spectroscopy. The method of continuous
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variation to generate Job's plots was used by preparing different mixtures of brazilin
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covering the whole range of molar fractions of HPβCD but keeping constant the total
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concentration of the solutions. In Figure 2, x-coordinate is the mole fraction of
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brazilin, and y-coordinate is the relative absorbance intensity of brazilin in the
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presence and absence of HPβCD. The stoichiometric ratio of inclusion compound can
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be judged from the highest point of the curve. If the abscissa corresponding to the
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highest point of the curve is 0.5, the stoichiometric ratio of the inclusion compound is
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1:1; if the highest point is 0.67 or 0.75, the corresponding stoichiometric ratio is 1:2 or
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1:3. In the Figure 2, the relative absorbance intensity was the largest when the value
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of the mole fraction of brazilin was 0.5, which clearly suggested the 1:1 inclusion
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stoichiometry of the brazilin/HPβCD complex. 3.2. Spectral titration
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Quantitative study of the inclusion complexation behavior of HPβCD with brazilin
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was performed in an ultrapure water/ethanol (V:V = 4:1) (v:v = 4:1) solution using a
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spectrophotometric titration method because of the quite low water solubility of
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brazilin. As shown in Figure 3, the absorbance intensity of brazilin increased steadily
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with the stepwise addition of HPβCD. In the control experiment, the pH of the
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solution did not vary obviously during the experimental process. These results
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suggested that the inclusion behavior rely mostly on the individual structural features
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of HPβCD and brazilin. Assuming a 1:1 stoichiometry for the brazilin/HPβCD
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inclusion complex, the inclusion complexation of brazilin with HPβCD could be
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expressed as Equation 1. Then, the stability constant (Ks) can be calculated from
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Equation 2, in which [Brazilin/CD], [brazilin], [CD], [Brazilin]0 and [CD]0 were the
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equilibrium concentration of the brazilin/HPβCD inclusion complex, the equilibrium
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concentration of brazilin, the equilibrium concentration of HPβCD, the original
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concentration of brazilin, and the original concentration of HPβCD, respectively, and
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∆ε was the differential molar extinction coefficient of brazilin in the absence and
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presence of HPβCD. Based on Lambert-Beer Law, the concentration of the
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brazilin/HPβCD complex was equal to ∆A/∆ε (Equation 2). We can obtain Equation 3
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from Equation 2. At last, Ks can be got from the investigation of the sequential vary of
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absorption (∆A) at HPβCD concentrations, by a non-linear least squares method
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Equation 1
Brazilin / CD
[Brazilin / CD] ∆Α / ∆ε = [Brazilin ][CD] ([Brazilin ]0 − ∆Α / ∆ε )([CD]0 − ∆Α / ∆ε )
∆Α = {∆ε ([Brazilin
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∆ε 2 ([Brazilin
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Equation 2
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From a nonlinear least squares curve-fitting method [40], the stability constant (Ks) could be derived. Figure 3 (inset) displayed a representative curve-fitting plot for the
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titration of brazilin with HPβCD, which showed the good fit between the calculated
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and measured data and the 1:1 stoichiometry of the brazilin/HPβCD inclusion
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complex. In repeated experiment, the Ks value can be reproducible within a 5%
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margin of error. The Ks and −∆Gº (Gibbs free energy change) of the brazilin/HPβCD
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inclusion complex were shown in Table 1.
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3.3. 1H and 2D NMR analysis
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To investigate the possible inclusion mode of the brazilin/HPβCD complex, the 1H
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NMR spectra of brazilin in the absence and presence of HPβCD were compared
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(Figure 4). Due to its poor water solubility, brazilin is transparent to 1H NMR under
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most conditions when D2O is used as a solvent. Investigation of the brazilin complex
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by 1H NMR obviously confirmed the presence of the protons of the brazilin, in
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accordance with the great solubilization. As shown in Figure 4, the most of brazilin
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protons exhibited chemical shifts at δ 6.0–7.5 ppm, which were different from the
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HPβCD protons (usually at δ 1.0–5.0 ppm). By contrastive analysis of the integration
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area of brazilin protons with that of the HPβCD’s H-1 protons, we calculated the
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inclusion stoichiometry of the brazilin/HPβCD complex, that is, 1:1 for the brazilin
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and HPβCD. To further explore the inclusion mode, the chemical shifts of HPβCD protons in
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the absence and presence of brazilin were investigated (Table 2). Inclusion
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complexation with brazilin had an insignificant effect on the δ values of the H-4, H-5
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and H-6 protons of HPβCD (∆δ = 0.01–0.03 ppm). On the contrary, those values of
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the H-2 and H-3 protons showed relatively weak but significant changes (∆δ =
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0.05–0.11 ppm). It is noteworthy that the H-3 protons shifted largely (∼0.11 ppm),
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but the H-5 protons changed relatively weak shifts (∼0.03 ppm) after inclusion
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complex formation. Since both H-3 and H-5 protons are located in the interior of
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HPβCD cavity, and H-3 protons are near the wide side of cavity while H-5 protons
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near the narrow side, this phenomena may suggest that brazilin should enter the
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HPβCD cavity from the wide side of HPβCD cavity (near H-3 protons).
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Two-dimensional (2D) NMR spectroscopy provides important information about
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the spatial proximity between host and guest atoms by observation of intermolecular
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dipolar cross-correlations. Two protons closely located in space can produce a nuclear
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overhauser effect (NOE) crosscorrelation in NOESY or ROESY. To get additional
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conformational information, 2D ROESY spectra of the inclusion complex of brazilin
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with CDs were carried out. The ROESY spectrum of the brazilin/HPβCD complex
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(Figure 5) displayed significant correlations of the H-5/H-8 protons (D ring) and H-5'
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proton (A ring) of brazilin with the H-5/H-6 and H-3 protons of HPβCD (peaks A), as
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well as key correlations between the H-9 protons (B ring) of brazilin and the H-3
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protons of HPβCD (peaks B). These phenomenons suggested that brazilin should
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penetrate the wide side of HPβCD cavity from the D ring of brazilin. Based on these observations, together with the 1:1 stoichiometry deduced by the
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experiments of NMR spectra, Job's plot and UV–vis spectral titration, the possible
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inclusion mode of brazilin with HPβCD was illustrated in Figure 6.
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3.4. XRD analysis
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The lack of crystallinity is an evidence for the formation of inclusion complex. The
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XRD patterns of brazilin, HPβCD and their inclusion complex were exhibited in
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Figure 7A. As shown in Figure 7A, the XRD patterns of brazilin was in crystalline
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form (Figure 7A(a)), wihch displayed sharp and highly intense peak at 8.15°, 13.03°,
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16.06°, 16.46°, 17.66°, 18.34°, 18.58°, 19.10°,19.84°, 22.03° and 24.42°, while
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HPβCD was amorphous (Figure 7A(b)). It was noteworthy that XRD pattern of the
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brazilin/HPβCD complex (Figure 7A(c)) did not presented the crystalline pattern of
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brazilin, in which the sharp diffraction peaks at 21.01°-32.66° of HPβCD partly
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disappeared, the peaks shape are widened and the intensity of the peaks are
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significantly reduced. The results further suggested that brazilin had been included
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into the HPβCD and presented as the amorphous structure. In other words, the
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brazilin/HPβCD inclusion complex has been formed.
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3.5. Thermal analysis
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The DSC themogram presented the thermal properties of the brazilin/HPβCD
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complex. As exhibited in Figure 7B, the DSC curve of brazilin showed an
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endothermic peak at 108.5 0C (Figure 7B(a)). On the contrary, the DSC curve of
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maximum at 83 oC (Figure 7B(b)), suggesting a dehydration process. Nevertheless,
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the DSC curve of the brazilin/HPβCD complex showed an endothermic peak at about
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brazilin/HPβCD complex, and the thermal stability of brazilin in inclusion complex
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was improved obviously compared with free brazilin, which will be helpful for the
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application of brazilin.
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3.6. SEM analysis
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The SEM photographs of HPβCD, brazilin, their physical mixture and inclusion
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complex were exhibited in Figure 8. Typical SEM photograph of HPβCD showed
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amorphous spherical particles with cavity structures (Figure 8a) and free brazilin
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showed irregularly shaped crystal particles (Figure 8b). Then, the physical mixture of
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brazilin and HPβCD showed a comparable morphology with free compounds (Figure
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8c). In contrast, the brazilin/HPβCD inclusion complex displayed a compact and
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homogeneous plate-like structures and was rather distinct from the sizes and shapes of
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free brazilin and HPβCD (Figure 8d), which verified the formation of the
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brazilin/HPβCD complex.
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3.7. Molecular Modelling
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Molecular modelling calculations were carried out to further elaborate the
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complexation mechanism of brazilin and HPβCD. There are two models for brazilian
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entering HPβCD cavity (Figure 9). Their molecular structures were established using
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quantum theory calculation to accurately exemplify the formation of the
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ACCEPTED MANUSCRIPT brazilin/HPβCD inclusion complexes. The binding energy curves plot of model A and
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model B computed using PM3 method is shown in Figure 10. Semi-empirical PM3
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method is widely used to study the inclusion pattern of drugs and cyclodextrins
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[41-43]. The energy minimized complex of model A and model B is named Complex
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1 and Complex 2, respectively. The binding energy changes (∆E), enthalpy changes
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(∆H), Gibbs free energy changes (∆G) and entropy changes (∆S) of the
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brazilin/HPβCD inclusion complexes were calculated from Equations 4-7. Energetic
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features, dipolemoments and thermodynamic parameters energy calculations of
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Complex 1 and Complex 2 were listed in Table 3. Generally, the more negative ∆E is,
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the stronger the interaction is between brazilin and HPβCD molecules. As a result, the
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two stable patterns for two possible brazilin/HPβCD inclusion models were in the
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following order: Complex 1 > Complex 2 (Figure 10). Negative values of the ∆H and
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∆E show that the preparation of brazilin/HPβCD complex was exothermic processes.
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Meanwhile, Negative values of the ∆H and ∆S suggested that these processes were
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enthalpy-entropy cooperative processes.
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In order to improve the accuracy of the PM3 method, Complex 1 and Complex 2
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were calculated by ONIOM2 [(B3LYP/6-31G*:PM3) and (HF/6-31G*:PM3)]
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methods, and relative energies of the complexes calculated at ONIOM2 in vacuum
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were shown in Table 4. The total optimized energies of Complex 1 were -625657.99
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(-625656.45 and -621961.01 kcal/mol). These results follow the same trend as the
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PM3 method optimizations. In other word, the Complex 1 is more stable than
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Complex 2. The ONIOM2 energy is described as Equations 8 [44].
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From the optimized molecular structures by PM3 method, three and five hydrogen
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bonds between brazilin and HPβCD molecules were found in inclusion Complex 1
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and Complex 2, respectively (Figure 11). At the same time, the hydrogen bonds
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lengths of two complexes were 2.604–2.682 Å and 2.555–2.992 Å, respectively. It is
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obviously observed that the hydrogen bonds in Complex 2 were looser than Complex
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1. This phenomenon revealed why the binding energy of inclusion Complex 1 was
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lower than that of the Complex 2.
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3.8. Solubilization
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The water solubility of the brazilin/HPβCD complex was evaluated via the
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preparation of its saturated solution [45]. An excess amount of complex was added in
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ultrapure water (2 mL, ca. pH 6.0), and the solution was stirred for 24 h at room
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temperature in the dark. After removing the insoluble complex using filtration, the
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absorbance of the filtrate was carried out at 287 nm in UV and the residue is dosed
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using the standard curve of brazilin. The study suggested that the water solubility of
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the brazilin/HPβCD complex, compared with that of native brazilin (ca. 63.9 mg/mL),
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was improved to approximately 103.5 mg/mL via the solubilizing effects of HPβCD.
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In the control experiment, a clear solution was obtained after dissolving the
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in 1 mL of water at room temperature. The results indicated the reliability of the
327
enhanced suitable water solubility of the brazilin/HPβCD complex.
328
3.9. Stability in biological environments
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To assess the stability of brazilin/HPβCD in biological environments, we
330
investigated the absorbance changes of brazilin and brazilin/HPβCD in different
331
buffer solutions, such as the simulated gastric acid (ca. pH 1.5) [46]. The solid brazilin
332
and brazilin/HPβCD complex were rapidly dissolved in the buffer solution, and the
333
absorbance was carried out at 287 nm in UV at room temperature in an interval of 25
334
min. Figure 12 showed the relative absorbance A/A0 (A is the absorbance at the
335
recording time and A0 is the original absorbance) of brazilin or brazilin/HPβCD
336
complex at pH 1.5 with an interval of 25 min. The relative absorbance of free brazilin
337
changed to 1.05% at the first 2 h (Figure 10a), however, the relative absorbance of
338
brazilin/HPβCD varied only 0.17% (Figure 10b), and the relative absorbance of free
339
brazilin changed with a fast speed after 2 h. These studies suggested that
340
brazilin/HPβCD was more stable than free brazilin in simulated biological
341
environments.
343
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4. Conclusions
344
The preparation, characterization, inclusion mode and molecular modelling of the
345
host–guest system of brazilin/HPβCD inclusion complex were studied for the first
346
time. The formation of the brazilin/HPβCD inclusion complex were verified by means
16
ACCEPTED MANUSCRIPT of XRD, DSC, SEM, 1H and 2D NMR and UV–vis spectroscopy. Binding energy and
348
ONIOM2 energy calculations demonstrated the most stable inclusion model of
349
brazilin/HPβCD complex. The results showed that HPβCD could improve not only
350
the thermal stability but also the stability in biological environments of brazilin.
351
Considering the limitation of utilization of brazilin, as well as the simple and
352
environmentally-friendly preparation of the brazilin/HPβCD complex, this inclusion
353
complexation should be considered as a significant step in the development of a new
354
formulation of brazilin for the application as food, dye or drug.
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355 356 357
Notes
The authors declare no competing financial interest.
359
Acknowledgment
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This work was supported by NSFC (21762051, 21562048 and 21662043), the
361
Program for the Changjiang Scholars and Innovative Research Team in University
362
(IRT17R94), Donglu Scholar & Excellent Young Talents of Yunnan University (X.D.
363
Yang), Innovative Team of Yunnan Minzu University (L.J. Yang) and Key
364
Laboratory of Resource Clean Conversion in Ethnic Regions of Yunnan.
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ACCEPTED MANUSCRIPT Figure captions
507
Figure 1. The structures of brazilin and HPβCD.
508
Figure 2. The Job’s plot for the brazilin/HPβCD system ([brazilin]+[HPβCD] =
509
5.6×10-5 M) at 287 nm in an ultrapure water/ethanol (V: V = 4:1, pH = 3.0).
510
Figure 3. UV-vis spectral changes in brazilin (0.058 mM) upon addition of HPβCD
511
(0–4.00 mM, from a-g) in an ultrapure water/ethanol (V: V = 4:1, pH = 3.0) mixed
512
solution, and the nonlinear least-squares analysis (inset) of the differential intensity
513
(∆A at 287nm) to calculate the complex stability constant (Ks).
514
Figure 4. 1H NMR spectra of HPβCD in the absence and presence brazilin of in D2O
515
at 25 ºC, respectively. (a) β-CD, and (b) brazilin/HPβCD complex (asterisk highlights
516
the water peak).
517
Figure 5. ROESY spectrum of the brazilin/HPβCD complex in D2O.
518
Figure 6. Possible inclusion mode and key NOESY (↔) correlations of the
519
brazilin/HPβCD complex.
520
Figure 7. (A) XRD patterns of (a) brazilin, (b) HPβCD, and (c) brazilin/HPβCD
521
inclusion complex. (B) DSC thermograms of (a) brazilin, (b) HPβCD, and (c)
522
brazilin/HPβCD inclusion complex.
523
Figure 8. Scanning electron microphotographs: (a) HPβCD, (b) brazilin, (c) brazilin
524
and HPβCD physical mixture (1:1 molar ratio), and (d) brazilin/HPβCD inclusion
525
complex.
526
Figure 9. Two possible models of brazilin in HPβCD.
527
Figure 10. Binding energies of the inclusion complexation of brazilin into HPβCD at
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ACCEPTED MANUSCRIPT 528
different positions (Z) and models: model A and model B.
529
Figure 11.
530
B-2) at PM3 level of theory. The hydrogen bonds are shown as a dotted.
531
Figure 12. The relative absorbance A/A0 (A is the absorbance at the recording time
532
and A0 is the original absorbance) of (a) brazilin at pH 1.5, (b) brazilin/HPβCD
533
complex at pH 1.5.
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The optimized structure of Complex 1 (A-1, A-2) and Complex 2 (B-1,
26
ACCEPTED MANUSCRIPT 534
Table 1. The stability constant (Ks and logKs) and Gibbs free energy change (−∆Gº)
535
for the inclusion complexation of HPβCD with brazilin in an ultrapure water/ethanol
536
(V:V = 4:1, pH 3.0) mixed solution. Host
Ks/M-1
log Ks
−∆Gº/kJ·mol-1
brazilin
HPβCD
1932
3.29
18.76
537
SC
538 539
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540 541 542
547 548 549 550
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Guest
551 552 553 554 27
ACCEPTED MANUSCRIPT Table 2. The chemical shifts (δ) of the HPβCD and brazilin/HPβCD complex.
H-1 H-2 H-3 H-4 H-5 H-6 H-Me
d dd dd dd m dd s
δ (ppm) HPβCD
brazilin/HPβCD complex
4.99 3.54 3.87 3.49 3.77 3.77 1.03
4.93 3.49 3.98 3.47 3.80 3.78 1.07
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555
SC
556 557
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558 559 560
564 565 566 567 568
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569 570 571 572 28
ACCEPTED MANUSCRIPT 573
Table 3. Energetic features, dipolemoments and thermodynamic parameters
574
calculations for brazilin, HPβCD and their two complexes inclusion models by in
575
vacuum at PM3 level.
576 577
HPβCD
1.60 -----18.68 -----19.27 ------19.76 -----130.91 ------
12.56 ------565.97 ------565.38 ------749.77 -----618.44 ------
a
584 585 586
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583
AC C
582
TE D
579
581
12.47 -1.68 -559.15 -11.86 -558.56 -12.45 -758.72 10.80 671.35 -78.00
Complex 2 17.79 3.638 -558.90 -11.61 -558.39 -12.28 -761.77 7.759 682.13 -67.21
Dipole moment in Debye. bBinding energy changes. cEnthalpy changes. dGibbs energy changes. e Entropy changes. Acomplex =A ‒ (Abrazilin ‒ ACD), A=Dipole, E, H, G, S.
578
580
Complex 1
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Dipole (D) ∆Dipolea (D) E (kcal/mol) ∆Eb (kcal/mol) H (kcal/mol) ∆Hc (kcal/mol) G (kcal/mol) ∆Gd (kcal/mol) S (Cal/mol-Kelvin) ∆Se (Cal/mol-Kelvin)
brazilin
SC
a
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587 588 589 590 29
ACCEPTED MANUSCRIPT 591
Table 4. Relative energies of the Complex 1 and Complex 2 calculated at ONIOM2 in
592
vacuum (kcal/mol). Complex 2
-625657.99 -621962.13
-625656.45 -621961.01
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EONIOM2 (B3LYP/6-31G* : PM3) EONIOM2 (HF/6-31G* : PM3)
Complex 1
593 594
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595 596
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603 604 605 606 607
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608 609 610 611 30
ACCEPTED MANUSCRIPT OR1
6 4'
6'
A HO
612
3'
2'
B 1'
9
4
D 4a
3
OH
8
O H-5
R3O
OR2
8a
CO
1
O 7
Figure 1. The structures of brazilin and HPβCD.
SC
615 616
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617 618 619
624 625 626 627
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H-3
HPβCD R1=R3=H, R2=CH2CH(CH3)OH
Brazilin
613 614
H-3
2
H-5
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HO
OH
7
5
H
5'
628 629 630 631 31
ACCEPTED MANUSCRIPT 632
636 637 638
0.04
0.02
0.00 0.0
0.2
0.4
0.6
0.8
1.0
R=[BRA]/[BRA]+[HPβCD]
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635
0.06
SC
634
0.08
Relative Absorbance
633
Figure 2. The Job’s plot for the brazilin/HPβCD system ([brazilin]+[HPβCD] =
640
5.6×10-5 M) at 287 nm in an ultrapure water/ethanol (V: V = 4:1, pH = 3.0).
M AN U
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641 642
646 647 648 649 650
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651 652 653
32
ACCEPTED MANUSCRIPT 0.60
g a
A0-A
0.014
0.45
Esp Calcd
0.007
0.30
0.000 0
1
2
3
4
[HP-β-CD]/mmol*L-1 0.15
0.00
250
300
λ/nm
350
400
SC
654
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Absorbance
0.021
655
Figure 3. UV-vis spectral changes in brazilin (0.058 mM) upon addition of HPβCD
657
(0–4.00 mM, from a-g) in an ultrapure water/ethanol (V: V = 4:1, pH = 3.0) mixed
658
solution, and the nonlinear least-squares analysis (inset) of the differential intensity
659
(∆A at 287nm) to calculate the complex stability constant (Ks).
663 664 665 666
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ACCEPTED MANUSCRIPT 671 H -5,6
＊
672 673
H-3
a
H-1
675
＊
b
676 7.5
7.0
6. 5
6 .0
5.5
5. 0
4 .5
4.0
3. 5
3 .0
2.5
1 .5
1.0
0. 5
pp m
SC
677
2. 0
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Figure 4. 1H NMR spectra of HPβCD in the absence and presence brazilin of in D2O
679
at 25 ºC, respectively. (a) β-CD, and (b) brazilin/HPβCD complex (asterisk highlights
680
the water peak).
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685 686 687 688 689
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690 691 692
34
ACCEPTED MANUSCRIPT YUNNAN UNIVER. AV.DRX500 YLJ-425 in D2O 13060904 Roesy H-5'
H-5 of Brazilin
H-9 of Brazilin
H-8
ppm 0.5 1.0 1.5 2.0
H-5 and H-6 of HPβCD
B
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2.5 3.0 3.5
B
4.0
A
H-3 of HPβCD
4.5 5.0
7.5
7.0
6.5
6.0
694
698 699 700 701 702
SC 4.0
3.5
3.0
2.5
703 704 705 706 35
6.0 6.5 7.0 7.5
2.0
Figure 5. ROESY spectrum of the brazilin/HPβCD complex in D2O.
TE D
697
4.5
EP
696
5.0
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5.5
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5.5
1.5
1.0
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ACCEPTED MANUSCRIPT
H-3 HO O
H
8
5
A
B
C
D HO H-5
OH
9
OH 5' H Brazilin
H H
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H-5
H-3
707
Brazilin/HPβCD complex
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Figure 6. Possible inclusion mode and key NOESY (↔) correlations of the
710
brazilin/HPβCD complex.
711 712
716 717 718 719 720
EP
715
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721 722 723
36
1000 500
B
a
0
b
500
b
o 83 C
exo
250
c
20
a
o 1 0 8 .5 C DSC/(w/g)
A intensity[cps]
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o 360 C o 267 C
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20
30
40
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2θ[degree.]
100
c
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4 00
o
T e m p e ra tu re / C
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Figure 7. (A) XRD patterns of (a) brazilin, (b) HPβCD, and (c) brazilin/HPβCD
727
inclusion complex. (B) DSC thermograms of (a) brazilin, (b) HPβCD, and (c)
728
brazilin/HPβCD inclusion complex.
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Figure 8. Scanning electron microphotographs: (a) HPβCD, (b) brazilin, (c) brazilin
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and HPβCD physical mixture (1:1 molar ratio), and (d) brazilin/HPβCD inclusion
746
complex.
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Figure 9. Two possible models of brazilin in HPβCD.
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model A model B
0 -2 -4 -6 -8 -10 -12 -4
-2
0
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Z coordinate (Ao)
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Binding Energy (kcal/mol)
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Figure 10. Binding energies of the inclusion complexation of brazilin into HPβCD at
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different positions (Z) and models: model A and model B.
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Figure 11.
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B-2) at PM3 level of theory. The hydrogen bonds are shown as a dotted.
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The optimized structure of Complex 1 (A-1, A-2) and Complex 2 (B-1,
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A/A0/%
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a b 98
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Time(min)
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Figure 12. The relative absorbance A/A0 (A is the absorbance at the recording time
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and A0 is the original absorbance) of (a) brazilin at pH 1.5, (b) brazilin/HPβCD
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complex at pH 1.5.
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ACCEPTED MANUSCRIPT 1
Highlights
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● A novel brazilin/HPβCD inclusion complex was prepared.
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● The inclusion behavior and characterization of brazilin with HPβCD were
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investigated.
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● The molecular modelling demonstrated the most stable inclusion model.
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● The brazilin/CD complex is more stable than free brazilin.
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● The brazilin/CD complex will be potentially useful for its dye and medical
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application.
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S0143-7208(17)32012-0
DOI:
10.1016/j.dyepig.2017.12.010
Reference:
DYPI 6416
To appear in:
Dyes and Pigments
Received Date: 24 September 2017 Revised Date:
3 December 2017
Accepted Date: 4 December 2017
Please cite this article as: Yang L-J, Chang Q, Zhou S-Y, Yang Y-H, Xia F-T, Chen W, Li M, Yang X-D, Host–guest interaction between brazilin and hydroxypropyl-β-cyclodextrin: Preparation, inclusion mode, molecular modelling and characterization, Dyes and Pigments (2018), doi: 10.1016/ j.dyepig.2017.12.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Graphical Abstract
H-3 HO
HP¦ÂCD
O H
8
D HO H-5
OH
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B
C
5
H H
OH 5' H Brazilin
Brazilin/HPβCD Complex
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H-5
Su-Mu
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Host–guest interaction between brazilin and
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hydroxypropyl-β-cyclodextrin: Preparation, inclusion mode,
3
molecular modelling and characterization
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Li-Juan Yang a,*, Qing Chang a, Shu-Ya Zhou a, Yun-Han Yang a, Fu-Ting Xia a,
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Wen Chen b, Minyan Li c, Xiao-Dong Yang b,*
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Functional Materials of Yunnan, Yunnan Minzu University, Kunming 650500, P. R.
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China
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School of Chemistry & Environment, Engineering Research Center of Biopolymer
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b
Key Laboratory of Medicinal Chemistry for Natural Resources, Ministry of Education, School of Chemical Science and Technology, Yunnan University,
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Kunming, 650091, P. R. China
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Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104-6323, United States
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c
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* Corresponding author. E-mail addresses: [email protected] (L.J Yang), [email protected] (X.D. Yang)
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ABSTRACT: Brazilin, the major constituent from the heartwood of Caesalpinia sappan, has been
24
widely used as a natural red color dye or food ingredient and drug. However, the
25
application of brazilin is greatly restricted by its low stability. In this study, the
26
preparation, characterization, inclusion mode, stability and solubility of brazilin with
27
hydroxypropyl-β-cyclodextrin (HPβCD) were studied for the first time in both the
28
solid state and solution by means of XRD, DSC, SEM, 1H and 2D NMR and UV–vis
29
spectroscopy. The molecular modelling demonstrated the most stable inclusion model
30
of brazilin/HPβCD complex. The results illustrated that the stability of brazilin were
31
improved in the inclusion complex with HPβCD. This satisfactory characterization
32
and stability of the brazilin/HPβCD complex should be possibly useful for its
33
utilization as dye, food or drug.
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Keywords:
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Brazilin; Hydroxypropyl-β-cyclodextrin; Inclusion mode; Molecular modelling;
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Characterization
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1. Introduction Caesalpinia sappan L. (Chinese name Su-Mu) is a plant that has been used as a
46
natural red color dye for textile dyes, inks, paints, varnish tints and wood stains, as
47
well as a traditional ingredient of food in Southeast and East Asia [1]. The heartwood
48
of C. sappan has been used as an analgesic and anti-inflammatory drugs in Chinese
49
folk medicine [2]. A lot of research has verified that its heartwood contains abundant
50
homoisoflavones and several phenolics and chalcones. Among these homoisoflavones,
51
brazilin (7,11b-dihydrobenz[b]indeno-[1,2-d]pyran-3,6a,9,10(6H)-tetrol, Figure 1) is
52
regarded to be one of the most important active constituents due to its many
53
pharmacological activities [3]. Scientific studies have demonstrated that brazilin
54
has a wide range of biological activity including antioxidant, antibacterial, anticancer,
55
anti-inflammatory, hypoglycemic, vasorelaxant and hepatoprotective properties
56
[2,4–9]. In dye and food industry, brazilin has been widely used as a natural colorant
57
and preservative agent [1]. However, the application of brazilin as a natural dye or
58
food ingredient and drug is largely restricted by its low stability [1,10]. Brazilin is
59
easily oxidized by air and light to produce brazilein [11]. Although much effort has
60
been made to improve its stability by introducing some lyophillization or
61
nanodispersion techniques [12-13], it is still not possible to sufficiently enhance the
62
stability, which prevents its usage for applications in dye and food. As a result, it is
63
significant to find an efficient carrier for brazilin with the aim of its further
64
application.
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At the same time, cyclodextrins (CDs) are truncated-cone polysaccharides chiefly
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ACCEPTED MANUSCRIPT comprised of 6 to 8 D-glucose monomers connected by α-1,4-glucosidic bonds. CDs
67
possess a hydrophobic interior cavity and a hydrophilic exterior surface, which can
68
encapsulate model molecules to construct supramolecular complexes [14–16]. These
69
properties enable them to be effectively used as substrate carriers and generally
70
improve poor stability and aqueous solubility of the encapsulated drug
71
in medical industry [17–22]. The similar results have been confirmed in the dye and
72
pigment industry [23–30]. For example, CDs not only increased the stability of
73
natural colorant laccaic acid A, but also improved its water solubility and dyeing
74
effect [23]. The thermal stability of dope-dyed polyurethanes were improved by using
75
disperse orange 31 as the dye and β-CD as the additive [25]. Hydroxypropyl-β-
76
cyclodextrin (HPβCD, Figure 1) is a hydroxyalkylated β-CD derivative, which has
77
quite high water solubility, low toxicity and suitable inclusion capacity [31,32].
78
Recently, our group reported that the host–guest complexes of CDs with natural
79
products such as hesperetin, pinocembrin, naringenin, crassicauline A and nimbin,
80
remarkably improved the water solubility and stability of the products [33–37]. As a
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continuation of our research on natural products/CD inclusion complexes, a
82
host–guest system of homoisoflavones (brazilin) with HPβCD was investigated. To
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the best of our knowledge, so far no scientific research on the inclusion behavior of
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brazilin/CD complexes has been reported.
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In the present study, we investigated firstly the preparation and characterization of
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brazilin/HPβCD host–guest system, as well as the inclusion mode and molecular
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modelling of the supramolecular complex, which will present a valuable method for
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getting new natural brazilin-based products of dye, food or drug.
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2. Materials and methods
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2.1. Materials
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Brazilin (FW = 286, PC>98%) was isolated from heartwood of Caesalpinia sappan
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L. in Yunnan Province, P.R. China. HPβCD (average FW = 1380) was obtained from
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Sigma-Aldrich Co. LLC. All reagents used were A. R. grade. All aqueous solutions
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were prepared using ultrapure water.
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2.2. Preparation of brazilin/HPβCD inclusion complex
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Brazilin (0.03 mM, 8.6 mg) and HPβCD (0.01 mM, 13.8 mg) were completely
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dissolved in a mixed solution of ultrapure water/ethanol (ca. 10 ml, V:V = 4:1), and
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the mixture was stirred at room temperature for 5 days in the dark. After evaporating
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the ethanol from the mixture, the uncomplexed brazilin was removed by filtration.
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The filtrate was evaporated under reduced pressure to remove the solvent and dried in
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vacuum to obtain the brazilin/HPβCD complex (yield 80%).
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2.3. Preparation of brazilin and HPβCD physical mixture
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The physical mixture was prepared by grinding together a mixture of brazilin and
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HPβCD (in 1:1molar ratio) in an agate mortar for 5 min.
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2.4. Job's plot
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The stoichiometry of inclusion complex was determined by the continuous
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variation Job's method [38]. The Job's plot was processed by the UV-Vis Spectra data
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recorded in an ultrapure water/ethanol (V:V = 4:1, pH 3.0) mixed solution. The total
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ACCEPTED MANUSCRIPT molar concentration of brazilin and HPβCD was held at 5.6×10-5 M and the molar
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fraction of brazilin ([brazilin]/([brazilin]+[HPβCD])) changed from 0.1 to 0.9. The
112
absorbance was run on at 287 nm in UV at 37 oC. And the absorption of each brazilin
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solution in the presence and absence of HPβCD were also carried out in the same
114
condition.
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2.5. Determination by UV Spectra
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Absorption spectra measurements were recorded on an Agilent UV 8453 using a
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conventional 1cm (1cm×1cm×4cm) quartz cell in a thermostated compartment. The
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concentration of brazilin was held constant at 0.058 mM. A suitable amount of
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HPβCD was added with the final concentrations varied from 0 to 4.000 mM (0, 0.231,
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0.471, 0.960, 1.372, 1.960, 4.000 mM). The absorption spectra measurement was
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carried out after 1 h. Given the poor water solubility of brazilin, an ultrapure
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water/ethanol (V:V = 4:1) solution was used in the spectral measurements.
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2.6. 1H and 2D NMR
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H NMR and 2D NMR experiments for HPβCD and brazilin/HPβCD complex were
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recorded on a Bruker Avance DRX500 spectrometer at 298 K. Tetramethylsilane
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(TMS) was used as a standard. All NMR experiments were conducted in D2O.
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2.7. Powder X-ray diffraction (XRD)
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XRD patterns were performed by a D/Max-3B diffractometer with Cu Kα radiation
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(100 mA, 40 kV) and a scanning rate of 5°/min. Samples were sett on a vitreous
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sample holder and scanned with a range of 2θ = 0.02° between 2θ = 3° and 50°.
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2.8. Thermal analyses
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ACCEPTED MANUSCRIPT The differential scanning calorimetry (DSC) measurements were carried out with a
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2960 SDT V3.0F instrument or NETZSCH STA 449F3 with a heating rate of 10
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ºC/min from room temperature to 400 ºC in a nitrogen atmosphere.
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2.9. Scanning electron microphotographs (SEM)
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SEM pictures were run on a FEL QUANTA 200. The samples were fixed on metal
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stubs with double-sided adhesive tapes. The photographs were taken at an excitation
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voltage of 15, 20 or 30kV and a magnification of 1080, 1200, 1400 or 2000×.
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2.10. Solubilization test
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An excess amount of complex was added in ultrapure water (2 mL, ca. pH 6.0), and
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the solution was stirred for 24 h at room temperature in the dark. The mixture is
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filtered on a 0.45 µm cellulose acetate membrane. The absorbance of the filtrate was
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measured at 287 nm in UV and the residue was dosed by the standard curve of
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brazilin.
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2.11. Molecular Modelling
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Molecular modelling calculations were performed using Gaussian 03 program
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(Gaussian Inc., Wallingford, USA) [39]. The initial geometry of brazilin and HPβCD
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were constructed with Chembio3D ultra (Version 10.0, Cambridge Soft.com., USA)
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and then fully optimized by PM3 method without any symmetry constraint. The
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glycosidic oxygen atoms of HPβCD were placed onto the XY plane; their center was
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defined as the origin of the coordinate system. Then, the brazilin molecule was placed
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on the Z-axis, Which approaches and passes through the HPβCD cavity from +5 Å to
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-5 Å with a stepwise 1 Å. For each step, the geometry of the complex is fully
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possible models were investigated. In order to improve the accuracy of the results, the
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ONIOM2 (B3LYP/6-31G*:PM3) and (HF/6-31G*:PM3)] methods were used to
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calculated the most stable structure of the two models. All quantum theory
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calculations were performed at 1 atm and 298.15 K in vacuum. The structure with
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lowest heat energy at all positions was obtained as the optimal complex structure and
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then established in PyMol.
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3. Results and discussion
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3.1. Stoichiometry (Job's plot)
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host-guest molecules [31]. A Job's plot was used to confirm the stoichiometry of
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brazilin and HPβCD using the UV–vis spectroscopy. The method of continuous
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variation to generate Job's plots was used by preparing different mixtures of brazilin
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covering the whole range of molar fractions of HPβCD but keeping constant the total
169
concentration of the solutions. In Figure 2, x-coordinate is the mole fraction of
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brazilin, and y-coordinate is the relative absorbance intensity of brazilin in the
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presence and absence of HPβCD. The stoichiometric ratio of inclusion compound can
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be judged from the highest point of the curve. If the abscissa corresponding to the
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highest point of the curve is 0.5, the stoichiometric ratio of the inclusion compound is
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1:1; if the highest point is 0.67 or 0.75, the corresponding stoichiometric ratio is 1:2 or
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1:3. In the Figure 2, the relative absorbance intensity was the largest when the value
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of the mole fraction of brazilin was 0.5, which clearly suggested the 1:1 inclusion
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stoichiometry of the brazilin/HPβCD complex. 3.2. Spectral titration
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Quantitative study of the inclusion complexation behavior of HPβCD with brazilin
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was performed in an ultrapure water/ethanol (V:V = 4:1) (v:v = 4:1) solution using a
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spectrophotometric titration method because of the quite low water solubility of
182
brazilin. As shown in Figure 3, the absorbance intensity of brazilin increased steadily
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with the stepwise addition of HPβCD. In the control experiment, the pH of the
184
solution did not vary obviously during the experimental process. These results
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suggested that the inclusion behavior rely mostly on the individual structural features
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of HPβCD and brazilin. Assuming a 1:1 stoichiometry for the brazilin/HPβCD
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inclusion complex, the inclusion complexation of brazilin with HPβCD could be
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expressed as Equation 1. Then, the stability constant (Ks) can be calculated from
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Equation 2, in which [Brazilin/CD], [brazilin], [CD], [Brazilin]0 and [CD]0 were the
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equilibrium concentration of the brazilin/HPβCD inclusion complex, the equilibrium
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concentration of brazilin, the equilibrium concentration of HPβCD, the original
192
concentration of brazilin, and the original concentration of HPβCD, respectively, and
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∆ε was the differential molar extinction coefficient of brazilin in the absence and
194
presence of HPβCD. Based on Lambert-Beer Law, the concentration of the
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brazilin/HPβCD complex was equal to ∆A/∆ε (Equation 2). We can obtain Equation 3
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from Equation 2. At last, Ks can be got from the investigation of the sequential vary of
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absorption (∆A) at HPβCD concentrations, by a non-linear least squares method
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Equation 1
Brazilin / CD
[Brazilin / CD] ∆Α / ∆ε = [Brazilin ][CD] ([Brazilin ]0 − ∆Α / ∆ε )([CD]0 − ∆Α / ∆ε )
∆Α = {∆ε ([Brazilin
]0 + [CD]0 + 1/ Ks) ±
∆ε 2 ([Brazilin
]0 + [CD]0 + 1/ Ks)2 − 4∆ε 2 [Brazilin ]0 + [CD]0 } / 2 Equation 3
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Equation 2
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From a nonlinear least squares curve-fitting method [40], the stability constant (Ks) could be derived. Figure 3 (inset) displayed a representative curve-fitting plot for the
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titration of brazilin with HPβCD, which showed the good fit between the calculated
203
and measured data and the 1:1 stoichiometry of the brazilin/HPβCD inclusion
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complex. In repeated experiment, the Ks value can be reproducible within a 5%
205
margin of error. The Ks and −∆Gº (Gibbs free energy change) of the brazilin/HPβCD
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inclusion complex were shown in Table 1.
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3.3. 1H and 2D NMR analysis
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To investigate the possible inclusion mode of the brazilin/HPβCD complex, the 1H
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NMR spectra of brazilin in the absence and presence of HPβCD were compared
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(Figure 4). Due to its poor water solubility, brazilin is transparent to 1H NMR under
211
most conditions when D2O is used as a solvent. Investigation of the brazilin complex
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by 1H NMR obviously confirmed the presence of the protons of the brazilin, in
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accordance with the great solubilization. As shown in Figure 4, the most of brazilin
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protons exhibited chemical shifts at δ 6.0–7.5 ppm, which were different from the
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HPβCD protons (usually at δ 1.0–5.0 ppm). By contrastive analysis of the integration
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area of brazilin protons with that of the HPβCD’s H-1 protons, we calculated the
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inclusion stoichiometry of the brazilin/HPβCD complex, that is, 1:1 for the brazilin
218
and HPβCD. To further explore the inclusion mode, the chemical shifts of HPβCD protons in
220
the absence and presence of brazilin were investigated (Table 2). Inclusion
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complexation with brazilin had an insignificant effect on the δ values of the H-4, H-5
222
and H-6 protons of HPβCD (∆δ = 0.01–0.03 ppm). On the contrary, those values of
223
the H-2 and H-3 protons showed relatively weak but significant changes (∆δ =
224
0.05–0.11 ppm). It is noteworthy that the H-3 protons shifted largely (∼0.11 ppm),
225
but the H-5 protons changed relatively weak shifts (∼0.03 ppm) after inclusion
226
complex formation. Since both H-3 and H-5 protons are located in the interior of
227
HPβCD cavity, and H-3 protons are near the wide side of cavity while H-5 protons
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near the narrow side, this phenomena may suggest that brazilin should enter the
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HPβCD cavity from the wide side of HPβCD cavity (near H-3 protons).
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Two-dimensional (2D) NMR spectroscopy provides important information about
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the spatial proximity between host and guest atoms by observation of intermolecular
232
dipolar cross-correlations. Two protons closely located in space can produce a nuclear
233
overhauser effect (NOE) crosscorrelation in NOESY or ROESY. To get additional
234
conformational information, 2D ROESY spectra of the inclusion complex of brazilin
235
with CDs were carried out. The ROESY spectrum of the brazilin/HPβCD complex
236
(Figure 5) displayed significant correlations of the H-5/H-8 protons (D ring) and H-5'
237
proton (A ring) of brazilin with the H-5/H-6 and H-3 protons of HPβCD (peaks A), as
238
well as key correlations between the H-9 protons (B ring) of brazilin and the H-3
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protons of HPβCD (peaks B). These phenomenons suggested that brazilin should
240
penetrate the wide side of HPβCD cavity from the D ring of brazilin. Based on these observations, together with the 1:1 stoichiometry deduced by the
242
experiments of NMR spectra, Job's plot and UV–vis spectral titration, the possible
243
inclusion mode of brazilin with HPβCD was illustrated in Figure 6.
244
3.4. XRD analysis
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The lack of crystallinity is an evidence for the formation of inclusion complex. The
246
XRD patterns of brazilin, HPβCD and their inclusion complex were exhibited in
247
Figure 7A. As shown in Figure 7A, the XRD patterns of brazilin was in crystalline
248
form (Figure 7A(a)), wihch displayed sharp and highly intense peak at 8.15°, 13.03°,
249
16.06°, 16.46°, 17.66°, 18.34°, 18.58°, 19.10°,19.84°, 22.03° and 24.42°, while
250
HPβCD was amorphous (Figure 7A(b)). It was noteworthy that XRD pattern of the
251
brazilin/HPβCD complex (Figure 7A(c)) did not presented the crystalline pattern of
252
brazilin, in which the sharp diffraction peaks at 21.01°-32.66° of HPβCD partly
253
disappeared, the peaks shape are widened and the intensity of the peaks are
254
significantly reduced. The results further suggested that brazilin had been included
255
into the HPβCD and presented as the amorphous structure. In other words, the
256
brazilin/HPβCD inclusion complex has been formed.
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3.5. Thermal analysis
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The DSC themogram presented the thermal properties of the brazilin/HPβCD
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complex. As exhibited in Figure 7B, the DSC curve of brazilin showed an
260
endothermic peak at 108.5 0C (Figure 7B(a)). On the contrary, the DSC curve of
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ACCEPTED MANUSCRIPT HPβCD displayed a board endothermic band, between 60 and 160 oC, which had a
262
maximum at 83 oC (Figure 7B(b)), suggesting a dehydration process. Nevertheless,
263
the DSC curve of the brazilin/HPβCD complex showed an endothermic peak at about
264
267 0C (Figure 7B(c)). These phenomenons further validated the formation of
265
brazilin/HPβCD complex, and the thermal stability of brazilin in inclusion complex
266
was improved obviously compared with free brazilin, which will be helpful for the
267
application of brazilin.
268
3.6. SEM analysis
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The SEM photographs of HPβCD, brazilin, their physical mixture and inclusion
270
complex were exhibited in Figure 8. Typical SEM photograph of HPβCD showed
271
amorphous spherical particles with cavity structures (Figure 8a) and free brazilin
272
showed irregularly shaped crystal particles (Figure 8b). Then, the physical mixture of
273
brazilin and HPβCD showed a comparable morphology with free compounds (Figure
274
8c). In contrast, the brazilin/HPβCD inclusion complex displayed a compact and
275
homogeneous plate-like structures and was rather distinct from the sizes and shapes of
276
free brazilin and HPβCD (Figure 8d), which verified the formation of the
277
brazilin/HPβCD complex.
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3.7. Molecular Modelling
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Molecular modelling calculations were carried out to further elaborate the
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complexation mechanism of brazilin and HPβCD. There are two models for brazilian
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entering HPβCD cavity (Figure 9). Their molecular structures were established using
282
quantum theory calculation to accurately exemplify the formation of the
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ACCEPTED MANUSCRIPT brazilin/HPβCD inclusion complexes. The binding energy curves plot of model A and
284
model B computed using PM3 method is shown in Figure 10. Semi-empirical PM3
285
method is widely used to study the inclusion pattern of drugs and cyclodextrins
286
[41-43]. The energy minimized complex of model A and model B is named Complex
287
1 and Complex 2, respectively. The binding energy changes (∆E), enthalpy changes
288
(∆H), Gibbs free energy changes (∆G) and entropy changes (∆S) of the
289
brazilin/HPβCD inclusion complexes were calculated from Equations 4-7. Energetic
290
features, dipolemoments and thermodynamic parameters energy calculations of
291
Complex 1 and Complex 2 were listed in Table 3. Generally, the more negative ∆E is,
292
the stronger the interaction is between brazilin and HPβCD molecules. As a result, the
293
two stable patterns for two possible brazilin/HPβCD inclusion models were in the
294
following order: Complex 1 > Complex 2 (Figure 10). Negative values of the ∆H and
295
∆E show that the preparation of brazilin/HPβCD complex was exothermic processes.
296
Meanwhile, Negative values of the ∆H and ∆S suggested that these processes were
297
enthalpy-entropy cooperative processes.
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In order to improve the accuracy of the PM3 method, Complex 1 and Complex 2
300
were calculated by ONIOM2 [(B3LYP/6-31G*:PM3) and (HF/6-31G*:PM3)]
301
methods, and relative energies of the complexes calculated at ONIOM2 in vacuum
302
were shown in Table 4. The total optimized energies of Complex 1 were -625657.99
14
ACCEPTED MANUSCRIPT and -621962.13 kcal/mol. They were all less than energies of the Complex 2
304
(-625656.45 and -621961.01 kcal/mol). These results follow the same trend as the
305
PM3 method optimizations. In other word, the Complex 1 is more stable than
306
Complex 2. The ONIOM2 energy is described as Equations 8 [44].
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307
From the optimized molecular structures by PM3 method, three and five hydrogen
309
bonds between brazilin and HPβCD molecules were found in inclusion Complex 1
310
and Complex 2, respectively (Figure 11). At the same time, the hydrogen bonds
311
lengths of two complexes were 2.604–2.682 Å and 2.555–2.992 Å, respectively. It is
312
obviously observed that the hydrogen bonds in Complex 2 were looser than Complex
313
1. This phenomenon revealed why the binding energy of inclusion Complex 1 was
314
lower than that of the Complex 2.
315
3.8. Solubilization
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The water solubility of the brazilin/HPβCD complex was evaluated via the
317
preparation of its saturated solution [45]. An excess amount of complex was added in
318
ultrapure water (2 mL, ca. pH 6.0), and the solution was stirred for 24 h at room
319
temperature in the dark. After removing the insoluble complex using filtration, the
320
absorbance of the filtrate was carried out at 287 nm in UV and the residue is dosed
321
using the standard curve of brazilin. The study suggested that the water solubility of
322
the brazilin/HPβCD complex, compared with that of native brazilin (ca. 63.9 mg/mL),
323
was improved to approximately 103.5 mg/mL via the solubilizing effects of HPβCD.
324
In the control experiment, a clear solution was obtained after dissolving the
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ACCEPTED MANUSCRIPT HPβCD/brazilin (125.0 mg) complex, which was equivalent to 103.5 mg of brazilin,
326
in 1 mL of water at room temperature. The results indicated the reliability of the
327
enhanced suitable water solubility of the brazilin/HPβCD complex.
328
3.9. Stability in biological environments
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To assess the stability of brazilin/HPβCD in biological environments, we
330
investigated the absorbance changes of brazilin and brazilin/HPβCD in different
331
buffer solutions, such as the simulated gastric acid (ca. pH 1.5) [46]. The solid brazilin
332
and brazilin/HPβCD complex were rapidly dissolved in the buffer solution, and the
333
absorbance was carried out at 287 nm in UV at room temperature in an interval of 25
334
min. Figure 12 showed the relative absorbance A/A0 (A is the absorbance at the
335
recording time and A0 is the original absorbance) of brazilin or brazilin/HPβCD
336
complex at pH 1.5 with an interval of 25 min. The relative absorbance of free brazilin
337
changed to 1.05% at the first 2 h (Figure 10a), however, the relative absorbance of
338
brazilin/HPβCD varied only 0.17% (Figure 10b), and the relative absorbance of free
339
brazilin changed with a fast speed after 2 h. These studies suggested that
340
brazilin/HPβCD was more stable than free brazilin in simulated biological
341
environments.
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4. Conclusions
344
The preparation, characterization, inclusion mode and molecular modelling of the
345
host–guest system of brazilin/HPβCD inclusion complex were studied for the first
346
time. The formation of the brazilin/HPβCD inclusion complex were verified by means
16
ACCEPTED MANUSCRIPT of XRD, DSC, SEM, 1H and 2D NMR and UV–vis spectroscopy. Binding energy and
348
ONIOM2 energy calculations demonstrated the most stable inclusion model of
349
brazilin/HPβCD complex. The results showed that HPβCD could improve not only
350
the thermal stability but also the stability in biological environments of brazilin.
351
Considering the limitation of utilization of brazilin, as well as the simple and
352
environmentally-friendly preparation of the brazilin/HPβCD complex, this inclusion
353
complexation should be considered as a significant step in the development of a new
354
formulation of brazilin for the application as food, dye or drug.
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Notes
The authors declare no competing financial interest.
359
Acknowledgment
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This work was supported by NSFC (21762051, 21562048 and 21662043), the
361
Program for the Changjiang Scholars and Innovative Research Team in University
362
(IRT17R94), Donglu Scholar & Excellent Young Talents of Yunnan University (X.D.
363
Yang), Innovative Team of Yunnan Minzu University (L.J. Yang) and Key
364
Laboratory of Resource Clean Conversion in Ethnic Regions of Yunnan.
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inclusion mode, solubilization and stability. Carbohydr Polym 2011;83:1321–8.
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ACCEPTED MANUSCRIPT Figure captions
507
Figure 1. The structures of brazilin and HPβCD.
508
Figure 2. The Job’s plot for the brazilin/HPβCD system ([brazilin]+[HPβCD] =
509
5.6×10-5 M) at 287 nm in an ultrapure water/ethanol (V: V = 4:1, pH = 3.0).
510
Figure 3. UV-vis spectral changes in brazilin (0.058 mM) upon addition of HPβCD
511
(0–4.00 mM, from a-g) in an ultrapure water/ethanol (V: V = 4:1, pH = 3.0) mixed
512
solution, and the nonlinear least-squares analysis (inset) of the differential intensity
513
(∆A at 287nm) to calculate the complex stability constant (Ks).
514
Figure 4. 1H NMR spectra of HPβCD in the absence and presence brazilin of in D2O
515
at 25 ºC, respectively. (a) β-CD, and (b) brazilin/HPβCD complex (asterisk highlights
516
the water peak).
517
Figure 5. ROESY spectrum of the brazilin/HPβCD complex in D2O.
518
Figure 6. Possible inclusion mode and key NOESY (↔) correlations of the
519
brazilin/HPβCD complex.
520
Figure 7. (A) XRD patterns of (a) brazilin, (b) HPβCD, and (c) brazilin/HPβCD
521
inclusion complex. (B) DSC thermograms of (a) brazilin, (b) HPβCD, and (c)
522
brazilin/HPβCD inclusion complex.
523
Figure 8. Scanning electron microphotographs: (a) HPβCD, (b) brazilin, (c) brazilin
524
and HPβCD physical mixture (1:1 molar ratio), and (d) brazilin/HPβCD inclusion
525
complex.
526
Figure 9. Two possible models of brazilin in HPβCD.
527
Figure 10. Binding energies of the inclusion complexation of brazilin into HPβCD at
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ACCEPTED MANUSCRIPT 528
different positions (Z) and models: model A and model B.
529
Figure 11.
530
B-2) at PM3 level of theory. The hydrogen bonds are shown as a dotted.
531
Figure 12. The relative absorbance A/A0 (A is the absorbance at the recording time
532
and A0 is the original absorbance) of (a) brazilin at pH 1.5, (b) brazilin/HPβCD
533
complex at pH 1.5.
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The optimized structure of Complex 1 (A-1, A-2) and Complex 2 (B-1,
26
ACCEPTED MANUSCRIPT 534
Table 1. The stability constant (Ks and logKs) and Gibbs free energy change (−∆Gº)
535
for the inclusion complexation of HPβCD with brazilin in an ultrapure water/ethanol
536
(V:V = 4:1, pH 3.0) mixed solution. Host
Ks/M-1
log Ks
−∆Gº/kJ·mol-1
brazilin
HPβCD
1932
3.29
18.76
537
SC
538 539
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540 541 542
547 548 549 550
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ACCEPTED MANUSCRIPT Table 2. The chemical shifts (δ) of the HPβCD and brazilin/HPβCD complex.
H-1 H-2 H-3 H-4 H-5 H-6 H-Me
d dd dd dd m dd s
δ (ppm) HPβCD
brazilin/HPβCD complex
4.99 3.54 3.87 3.49 3.77 3.77 1.03
4.93 3.49 3.98 3.47 3.80 3.78 1.07
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555
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556 557
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564 565 566 567 568
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ACCEPTED MANUSCRIPT 573
Table 3. Energetic features, dipolemoments and thermodynamic parameters
574
calculations for brazilin, HPβCD and their two complexes inclusion models by in
575
vacuum at PM3 level.
576 577
HPβCD
1.60 -----18.68 -----19.27 ------19.76 -----130.91 ------
12.56 ------565.97 ------565.38 ------749.77 -----618.44 ------
a
584 585 586
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583
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582
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579
581
12.47 -1.68 -559.15 -11.86 -558.56 -12.45 -758.72 10.80 671.35 -78.00
Complex 2 17.79 3.638 -558.90 -11.61 -558.39 -12.28 -761.77 7.759 682.13 -67.21
Dipole moment in Debye. bBinding energy changes. cEnthalpy changes. dGibbs energy changes. e Entropy changes. Acomplex =A ‒ (Abrazilin ‒ ACD), A=Dipole, E, H, G, S.
578
580
Complex 1
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Dipole (D) ∆Dipolea (D) E (kcal/mol) ∆Eb (kcal/mol) H (kcal/mol) ∆Hc (kcal/mol) G (kcal/mol) ∆Gd (kcal/mol) S (Cal/mol-Kelvin) ∆Se (Cal/mol-Kelvin)
brazilin
SC
a
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587 588 589 590 29
ACCEPTED MANUSCRIPT 591
Table 4. Relative energies of the Complex 1 and Complex 2 calculated at ONIOM2 in
592
vacuum (kcal/mol). Complex 2
-625657.99 -621962.13
-625656.45 -621961.01
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EONIOM2 (B3LYP/6-31G* : PM3) EONIOM2 (HF/6-31G* : PM3)
Complex 1
593 594
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595 596
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603 604 605 606 607
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608 609 610 611 30
ACCEPTED MANUSCRIPT OR1
6 4'
6'
A HO
612
3'
2'
B 1'
9
4
D 4a
3
OH
8
O H-5
R3O
OR2
8a
CO
1
O 7
Figure 1. The structures of brazilin and HPβCD.
SC
615 616
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617 618 619
624 625 626 627
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H-3
HPβCD R1=R3=H, R2=CH2CH(CH3)OH
Brazilin
613 614
H-3
2
H-5
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HO
OH
7
5
H
5'
628 629 630 631 31
ACCEPTED MANUSCRIPT 632
636 637 638
0.04
0.02
0.00 0.0
0.2
0.4
0.6
0.8
1.0
R=[BRA]/[BRA]+[HPβCD]
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635
0.06
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634
0.08
Relative Absorbance
633
Figure 2. The Job’s plot for the brazilin/HPβCD system ([brazilin]+[HPβCD] =
640
5.6×10-5 M) at 287 nm in an ultrapure water/ethanol (V: V = 4:1, pH = 3.0).
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646 647 648 649 650
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651 652 653
32
ACCEPTED MANUSCRIPT 0.60
g a
A0-A
0.014
0.45
Esp Calcd
0.007
0.30
0.000 0
1
2
3
4
[HP-β-CD]/mmol*L-1 0.15
0.00
250
300
λ/nm
350
400
SC
654
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Absorbance
0.021
655
Figure 3. UV-vis spectral changes in brazilin (0.058 mM) upon addition of HPβCD
657
(0–4.00 mM, from a-g) in an ultrapure water/ethanol (V: V = 4:1, pH = 3.0) mixed
658
solution, and the nonlinear least-squares analysis (inset) of the differential intensity
659
(∆A at 287nm) to calculate the complex stability constant (Ks).
663 664 665 666
TE D
662
EP
661
AC C
660
M AN U
656
667 668 669 670 33
ACCEPTED MANUSCRIPT 671 H -5,6
＊
672 673
H-3
a
H-1
675
＊
b
676 7.5
7.0
6. 5
6 .0
5.5
5. 0
4 .5
4.0
3. 5
3 .0
2.5
1 .5
1.0
0. 5
pp m
SC
677
2. 0
RI PT
674
Figure 4. 1H NMR spectra of HPβCD in the absence and presence brazilin of in D2O
679
at 25 ºC, respectively. (a) β-CD, and (b) brazilin/HPβCD complex (asterisk highlights
680
the water peak).
M AN U
678
681
685 686 687 688 689
EP
684
AC C
683
TE D
682
690 691 692
34
ACCEPTED MANUSCRIPT YUNNAN UNIVER. AV.DRX500 YLJ-425 in D2O 13060904 Roesy H-5'
H-5 of Brazilin
H-9 of Brazilin
H-8
ppm 0.5 1.0 1.5 2.0
H-5 and H-6 of HPβCD
B
RI PT
2.5 3.0 3.5
B
4.0
A
H-3 of HPβCD
4.5 5.0
7.5
7.0
6.5
6.0
694
698 699 700 701 702
SC 4.0
3.5
3.0
2.5
703 704 705 706 35
6.0 6.5 7.0 7.5
2.0
Figure 5. ROESY spectrum of the brazilin/HPβCD complex in D2O.
TE D
697
4.5
EP
696
5.0
AC C
695
5.5
M AN U
A
693
5.5
1.5
1.0
0.5
ppm
ACCEPTED MANUSCRIPT
H-3 HO O
H
8
5
A
B
C
D HO H-5
OH
9
OH 5' H Brazilin
H H
RI PT
HPβCD
H-5
H-3
707
Brazilin/HPβCD complex
SC
708
Figure 6. Possible inclusion mode and key NOESY (↔) correlations of the
710
brazilin/HPβCD complex.
711 712
716 717 718 719 720
EP
715
AC C
714
TE D
713
M AN U
709
721 722 723
36
1000 500
B
a
0
b
500
b
o 83 C
exo
250
c
20
a
o 1 0 8 .5 C DSC/(w/g)
A intensity[cps]
ACCEPTED MANUSCRIPT
o 360 C o 267 C
0
10
724
20
30
40
50
2θ[degree.]
100
c
RI PT
10
2 00
300
4 00
o
T e m p e ra tu re / C
725
Figure 7. (A) XRD patterns of (a) brazilin, (b) HPβCD, and (c) brazilin/HPβCD
727
inclusion complex. (B) DSC thermograms of (a) brazilin, (b) HPβCD, and (c)
728
brazilin/HPβCD inclusion complex.
729 730
734 735 736 737 738
EP
733
AC C
732
TE D
731
M AN U
SC
726
739 740 741
37
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
742 743
Figure 8. Scanning electron microphotographs: (a) HPβCD, (b) brazilin, (c) brazilin
745
and HPβCD physical mixture (1:1 molar ratio), and (d) brazilin/HPβCD inclusion
746
complex.
749 750 751 752
EP
748
AC C
747
TE D
744
753 754 755
38
ACCEPTED MANUSCRIPT 756 757 758 759
RI PT
760 761 762
SC
763 764
766
M AN U
765
Figure 9. Two possible models of brazilin in HPβCD.
767
771 772 773 774 775
EP
770
AC C
769
TE D
768
776 777 778
39
ACCEPTED MANUSCRIPT
4
model A model B
0 -2 -4 -6 -8 -10 -12 -4
-2
0
2
Z coordinate (Ao)
779
6
M AN U
780
4
SC
-6
RI PT
Binding Energy (kcal/mol)
2
781
Figure 10. Binding energies of the inclusion complexation of brazilin into HPβCD at
782
different positions (Z) and models: model A and model B.
786 787 788 789 790
EP
785
AC C
784
TE D
783
791 792 793 794 40
ACCEPTED MANUSCRIPT 795 796 797
RI PT
798 799 800
SC
801
M AN U
802 803 804 805
TE D
806
Figure 11.
808
B-2) at PM3 level of theory. The hydrogen bonds are shown as a dotted.
810 811 812 813
AC C
809
The optimized structure of Complex 1 (A-1, A-2) and Complex 2 (B-1,
EP
807
814 815
41
ACCEPTED MANUSCRIPT 102
A/A0/%
101
100
a b 98
0
25
50
75
100
125
150
175
200
225
Time(min)
816
RI PT
99
Figure 12. The relative absorbance A/A0 (A is the absorbance at the recording time
818
and A0 is the original absorbance) of (a) brazilin at pH 1.5, (b) brazilin/HPβCD
819
complex at pH 1.5.
AC C
EP
TE D
M AN U
SC
817
42
ACCEPTED MANUSCRIPT 1
Highlights
2
● A novel brazilin/HPβCD inclusion complex was prepared.
4
● The inclusion behavior and characterization of brazilin with HPβCD were
5
investigated.
6
● The molecular modelling demonstrated the most stable inclusion model.
7
● The brazilin/CD complex is more stable than free brazilin.
8
● The brazilin/CD complex will be potentially useful for its dye and medical
9
application.
AC C
EP
TE D
M AN U
SC
RI PT
3