Accepted Manuscript Title: Inclusion complex of barbigerone with hydroxypropyl--cyclodextrin: Preparation and in vitro evaluation Author: Neng Qiu Xia Cheng Guangcheng Wang Wenwen Wang Jiaolin Wen Yongkui Zhang Hang Song Liang Ma Yuquan Wei Aihua Peng Lijuan ChenThese authors contributed equally to this work. PII: DOI: Reference:
S0144-8617(13)00927-2 http://dx.doi.org/doi:10.1016/j.carbpol.2013.09.035 CARP 8124
To appear in: Received date: Revised date: Accepted date:
25-5-2013 1-9-2013 14-9-2013
Please cite this article as: Qiu, N., Cheng, X., Wang, G., Wang, W., Wen, J., Zhang, Y., Song, H., Ma, L., Wei, Y., Peng, A., & Chen, L., Inclusion complex of barbigerone with hydroxypropyl--cyclodextrin: Preparation and in vitro evaluation, Carbohydrate Polymers (2013), http://dx.doi.org/10.1016/j.carbpol.2013.09.035 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.
Highlights (for review)
Research highligts Barbigerone complexes with HP-β-CD were prepared by a simple coevaporation method. The bar-HP-β-CD complexes were characterized by UV-vis, FT-IR, 1HNMR, XRD
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and SEM. The water solubility of barbigerone was remarkably increased by the solubilizing
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effects of HP-β-CD.
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The bar- HP-β-CD complexes still maintain good anti-tumor activities in vitro.
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*Manuscript
Inclusion complex of barbigerone with
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hydroxypropyl-β-cyclodextrin: Preparation and in vitro
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evaluation
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Neng Qiua,b,c,1, Xia Chenga,1, Guangcheng Wanga,Wenwen Wanga, Jiaolin Wena,
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Yongkui Zhang b, Hang Song b ,Liang Maa, Yuquan Weia, Aihua Penga, Lijuan Chena,
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a State Key laboratory of Biotherapy, West China Hospital, West China Medical
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School, Sichuan University, Chengdu, 610041, PR China.
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b Department of Bio-pharmaceutical Engineering, School of Chemical Engineering,
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Sichuan University, Chengdu, 610065, PR China.
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c College of Materials and Chemistry & Chemical Engineering, Chengdu University
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of Technology, Chengdu, 610059, PR China.
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Abstract
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These authors contributed equally to this work.
The aim of this study was to improve the water solubility of barbigerone by
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complexing it with hydroxypropyl-β-cyclodextrin (HP-β-CD). The inclusion
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complexation behavior, characterization and interactions of barbigerone with
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HP-β-CD were investigated in both solution and the solid state by means of UV/VIS),
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1
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formation of barbigerone-HP-β-CD (bar-HP-β-CD) inclusion complex, and the
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bar-HP-β-CD inclusion compounds exhibited different spectroscopic features and
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properties from barbigerone. The results demonstrated that the water solubility of
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H NMR, FT-IR, PXRD, SEM. All the characterization information demonstrated the
Corresponding author. Li-Juan Chen,Tel.: +86 28 85164063; fax: +86 28 85164060. E-mail:
[email protected]. 1
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barbigerone was notably increased in the presence of HP-β-CD. Furthermore,
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preliminary in vitro cytotoxicity assay showed that bar-HP-β-CD still maintain the
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anticancer activity of barbigerone. These results suggest that HP-β-CD will be
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potentially useful in the delivery of water-insoluble anticancer agents such as
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barbigerone.
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Keywords:
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Solubility; Characterization; Anti-cancer.
Inclusion
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Hydroxypropyl-β-cyclodextrin;
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Barbigerone;
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1. Introduction
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Barbigerone(2’,4’,5’-trimethoxy-6’’,6’’-dimethylpyrano(2’’,3’’:7,8) (Fig. 1 a), a
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naturally occurring isoflavone, was first isolated from the seeds of Tephrosia
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barbigerain 1979 (Villain., 1980) and could be found in other plants(Wangensteen,
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Alamgir, Rajia, Samuelsen, &Malterud, 2005; Yenesew, Midiwo, & Waterma,1998).
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It has been reported to have diverse pharmaceutical properties, including antioxidant,
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15-lipoxygenase inhibitory activity (Wangensteen et al., 2006), and anti-plasmodial
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activity against the malarial parasite Plasmodium falciparum (Yenesew et al., 2003).
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Our previous studies has shown that barbigerone can inhibit murine lung cancer cell
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proliferation by inducing apoptosis via mitochondrial apoptotic pathway and by
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decreasing phosphorylated p42/44 MAPK and Akt (Li et al., 2009). In the present
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study, barbigerone was found to effectively suppress tumor angiogenesis and tumor
43
growth in vitro and in vivo. Further studies indicated that barbigeronge inhibited
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tumor angiogenesis and tumor growth through VEGFR2 signaling pathways (Li,
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Wang, Ye, Peng, & Chen, 2012). Moreover, it exhibited less toxicity to non-cancer
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cells than tumor cells (Li et al., 2009). However, the aqueous solubility of barbigerone
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is almost nil, making it difficult for deep research. Therefore, it is important to
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introduce effective method to enhance the solubility of barbigerone.
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Cyclodextrins, a family of cyclic amylose-derived oligomers with a hydrophilic outer
50
surface and a lipophilic central cavity, are well known for its abilities to form
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inclusion complex with various guest molecules (Davis, & Brewster, 2004). Druing
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the past decades, cyclodextrins has been successfully used as comlexing agents to
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enhance the solubility, stability and bioavailabilty of drug molecules (Uekama,
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Hirayama, &Irie, 1998; Loftsson, &Duchêne, 2007). The α-, β-, and γ-cyclodextrin
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are the most common cyclodextrins used as formulation vehicles consisting of six,
56
seven, and eight D-(+)-glucopyranose units attached by α-1,4 linkage (Loftsson,
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&Brewster, 1996). β-cyclodextrin is the most useful and the lowest price, however, its
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solubility in water is relatively low (approximately 2%) and the toxicity of
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β-cyclodextrin limit its further application in pharmaceutical formulations (Davis, &
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Brewster, 2004; Irie, &Uekama). 2-hydroxylpropyl-β-cyclodextrin (HP-β-CD, Fig. 1b)
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is an alternative to β-cyclodextrin having a higher aqueous solubility (above 60%) and
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may be slightly more toxicologically benign (Gould, &Scott, 2005). Recently,
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HP-β-CD has been widely used to improve the solubility of poorly water-soluble
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drugs (Ma et al., 2012; Liu, Qiu, Gao, &Jin, 2006). However, to the best of our
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knowledge, no scientific study about the effect of cyclodextrins on enhancing
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solubility and dissolution rate of barbigerone has been reported.
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In the present study, with the aim to increase the aqueous solubility of barbigerone,
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the complex of barbigerone with HP-β-CD was prepared by a simple coevaporation
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method. UV/Vis spectroscopy was measured to evaluate the formation of
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bar-HP-β-CD complex in aqueous solution. Drug/HP-β-CD interactions in solution
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were investigated by phase solubility analysis and the interactions in the solid state
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were characterized by 1HNMR, FT-IR, XRD and SEM, aiming to verify the formation
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of inclusion complex. Finally, the inhibitory effects on cell growth of bar-HP-β-CD
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evaluated to prove the retaining bioactivity of free barbigerone.
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2. Materials and methods
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2.1 Materials Barbigerone (≥98% in purity) was obtained from Millettia pachycarpa Benth,
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separated and purified in our laboratory, as reported previously (Ye et al., 2010).
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HP-β-CD was obtained as a gift from Shijiazhuang Pharmaceutical Group Co., Ltd.
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(Shijiazhuang, China). 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium
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bromide (MTT) and Dimethyl sulfoxide (DMSO) was purchased from Sigma
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(Sigma-Aldrich Inc.). Methanol [high-performance liquid chromatography (HPLC)
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grade] was purchased from Fisher Chemical (Loughborough, UK). All other reagents
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and solvents, which are analytic grade, were purchased from Ke-Long Chemical
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Reagent Factory (Chengdu, China) and used without further purification.
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2.2 High-performance liquid chromatography analysis
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Reverse-phase high performance liquid chromatography (HPLC, Waters 2695
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separation module with a Waters 2998 photodiode array detector) was used to
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determine the concentration of barbigerone. Chromatographic analysis was performed
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on a C18 analytical column (Sunfire, 4.6 mm×150 mm) with methanol/ water (80/20,
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v/v %) as the mobile phase and UV detection at 264 nm. The injection volume was
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10μl and the flow rate was 1.0ml/min.
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2.3 Phase solubility study
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The effects of HP-β-CD on the solubility of barbigerone were investigated
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according to the method established by Higuchi and Conors (Higuchi, & Connors, 5
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1965). Excess amounts of barbigerone were added into 2ml of aqueous HP-β-CD
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solutions with different concentrations (ranging from 0~20 mM). The obtained
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suspensions were shaken at 37°C in a thermostatically controlled shaking air bath
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(100rpm). After 48h, aliquots were withdrawn, filtered through a 0.45μm Millipore
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filtration to remove the excess barbigerone. The amount of barbigerone was then
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measured by the above-mentioned HPLC method. All experiments were carried out in
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triplicate.
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2.4 Preparation of bar-HP-β-CD complex
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A simple coevaporation method was used to prepare bar-HP-β-CD complex. Briefly, 150mg HP-β-CD was completely dissolved in dehydrated alcohol.
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solution was kept at 50°C with moderate stirring for 30min to let it freely swell. Then
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barbigerone (5mg) was added into the system and stirred for 1h. Then the organic
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solvent was evaporated under reduced pressure in a rotary evaporator at 50°C and
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dried in vacuum to produce the bar-HP-β-CD complexes. The inclusion complex can
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be redissolved in distilled water or 5% glucose solution to obtain a clear solution for
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further use.
The
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Physical mixtures: A physical mixture of barbigerone and HP-β-CD (1:1, molar
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ratio) were prepared by thoroughly mixing the two components in an agate mortar for
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10 min.
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2.5 Solubility study
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Solubility studies were carried out according to the method reported previously (Hu, 6
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Zhang, Song, Gu, &Hu, 2012). In brief, excess amounts of barbigerone were added
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into 2ml of water, then the suspensions were kept strirring at 25°C for 48h. After
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equilibrium attainment, samples were withdrawn and filtered through a 0.22μm
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Millipore filtration to remove the excess barbigerone. The filtrate was centrifuged and
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injected into HPLC apparatus for analysis. The concentration of barbigerone was then
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calculated against a concentration-peak area calibration curve. The solubility of
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bar-HP-β-CD complex in water was tested in the similar procedure (Ma et al., 2012).
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Briefly, an excess amount of bar-HP-β-CD complexes were added to 2ml water and
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the mixture were kept stirring for 1h at 25°C. After removing the insoluble substances,
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the filtrate was then injected into HPLC and the concentration of barbigerone was
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then determined by the standard curve of barbigerone.
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2.6 Characterization
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2.6.1 UV spectroscopy measurement
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Complex formation between barbigerone and HP-β-CD was studied using the
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spectral shift method. Given the poor water solubility of barbigerone, a water/ethanol
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(V: V= 3:1) solution was used in the spectral measurements. The concentration of
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barbigerone was kept constant at 5μg/ml while the HP-β-CD concentration were
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increased from 1 to 15mmol/L. The mixtures were shaken for 1h before the UV-vis
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spectrum was recorded on a Lambda 35 UV spectrophotometer (Perkin Elmer, United
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States) equipped with a conventional 1cm path (1cm×1cm×4cm) quartz cell at 25 °C.
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The measurements were done in the wavelength range from 200 to 500 nm.
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2.6.2 1HNMR spectroscopy
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HNMR spectra were obtained on an Ascend 400 MHz NMR Spectrometer
(Bruker, Switzerland). HP-β-CD, bar-HP-β-CD complexes and barbigerone were
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dissolved in D2O, D2O (or DMSO-d6) and DMSO-d6 respectively. Chemical shifts
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were reported in ppm with tetramethylsilane (TMS) as the internal standard.
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2.6.3 Fourier-transform infrared spectroscopy (FT-IR)
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FT-IR spectra were recorded on the Fourier transform infrared spectrometer (Nicolet
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6700, Thermo Fisher Scientific, Waltham, MA) according to the KBr disc technique.
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Samples were prepared as KBr disks with 1 mg of complex in 100 mg of KBr. The
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FTIR measurements were performed in the scanning range of 4000-400 cm-1 at
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ambient temperature.
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2.6.4 Powder X-ray diffractometry (XRD)
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X-ray powder diffraction patterns were performed on a Philips X'Pert Pro
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diffractometer using Ni-filtered and Cu Kα radiation (45kV, 35mA).The scanning rate
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employed was 0.15 °/min in a diffraction angle (2θ) range of 3°-40°.
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2.6.5 Scanning electron microscopy (SEM)
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Scanning electron microscopy (SEM) was used to evaluate the morphology of
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barbigerone, HP-β-CD, bar-HP-β-CD inclusion complex and the physical mixture. All
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samples were made electrically conductive by coating with a thin layer of gold before
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being examined using a JEOL JSM-7500F scanning electron microscope operated at
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5.0 kV.
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2.7 Cell culture
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The human colon cancer cell line HCT116 and human liver cancer cell line 8
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HepG2 were purchased from the American Type Culture Collection (Manassas,
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Virginia). HCT116 and HepG2 cells were cultured in Dulbecco’s Modified Eagle
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Medium (DMEM, GIBICO) supplemented with 10% (v/v) heat-inactivated fetal calf
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serum, 100 units ml-1 penicillin and 100 units ml-1 streptomycin, and maintained at
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37°C, 95% relative humidity, under 5% CO2.
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2.8 In vitro bioactivity study
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The in vitro cytotoxicity of bar-HP-β-CD complex on HCT116 and HepG2 cell
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lines was evaluated by MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium
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bromide) test, and free barbigerone was used as control. Bar-HP-β-CD was dissolved
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in sterilized water and free barbigerone were dissolved in DMSO. Exponential
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growing cells were seeded in a 96-well plate at a seeding density of 4.0~5.0×10-4 per
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well, and cultured for 24 h. After that, harvested cells were exposed to bar-HP-β-CD
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or free barbigerone (diluted with DMEM, the final concentration of DMSO was less
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than 0.1%) at the indicated dosages. For each condition, three replicates were
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performed, six wells were left as blank controls. Cells were then incubated at 37°C in
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a humidity atmosphere for 48h and 20μl MTT (5 mg/ml, dissolved in saline) was
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added to each well. After further incubation for 3h at 37°C, the incubated medium
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was removed and 150μl of DMSO were added into each well to dissolve the formazan
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crystals. The absorbance was measured at 570 nm by a Spectramax M5 Microtiter
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Plate Luminometer (Molecular Devices, USA). Cell viability was given as the ratio of
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the absorbance of treated cells to that of blank controls.
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3 Results and Discussion
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3.1 Phase solubility studies
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The phase solubility behavior of barbigerone in HP-β-CD solution was investigated at
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37 °C and presented in Fig. 2A. The phase solubility study is a useful method for
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studying drug/cyclodextrin interactions because it provides not only the solubilizing
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ability of CDs but also the stability constant (Ks) of the inclusion complexes by
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analyzing the solubility curves (Higuchi, & Connors, 1965). From the diagram, it
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could be observed that aqueous solubility of barbigerone increased linearly as the
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concentration of HP-β-CD increased, over the entire concentration range studied. The
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solubility curve has a slope of 0.0247 with a correlation coefficient squared values of
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0.9951 (r2>0.990) was regarded as a straight line and can be classified as A L type.
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Since the slope is less than one, it was assumed that the solubility increase is due to
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the formation of 1:1 complex (Higuchi, & Connors, 1965). The apparent 1:1 stability
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constants (Kc) of bar-HP-β-CD complexes was calculated from the slope of the linear
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plot of the diagram according to the equation Kc=slope/ S 0 (1-slope),where S0 is the
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solubility of pure barbigerone at the same temperature in water. The value of Kc was
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calculated to be 20,659 M-1. The high Kc value indicates that the inclusion complex
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formed between barbigerone and HP-β-CD are quite stable.
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3.2 Preparation of bar-HP-β-CD complex
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In this study, a simple coevaporation method was used to prepare bar-HP-β-CD
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inclusion complex. Given the poor water solubility of barbigerone, absolute ethanol
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was used in the preparation. Barbigerone and swollen HP-β-CD were together
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dissolved in dehydrated ethanol instead of water, the bar-HP-β-CD inclusion complex
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was totally formed by self-association after stirring for only 1h at 50°C, while the
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complexation time in previous method needed at least 24h and most of the drug still
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not embedded into HP-β-CD. During the experiment, the maximum mass ratio of
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barbigerone/HP-β-CD could reach up to 1:30 (molar ratio: 1:7.9), when the amount of
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HP-β-CD increased, the amount of complexed barbigerone might also increase. The
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inclusion complex had a very good water solubility and can be directly dissolved in
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distilled water or 5% Glucose solution. Moreover, the aqueous solution of
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bar-HP-β-CD complex was quite stable and can be kept at 4°C for at least 12 weeks.
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3.2 Solubility of barbigerone
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The water solubility of barbigerone and complex were evaluated by preparation its
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saturation solution. An excess amount of barbigerone (or bar-HP-β-CD complex) was
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added into 2ml distilled water and kept moderate stirring at 25°C for 48h. The
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insoluble substance was removed by filtration before the concentration of the filtrates
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were determined using HPLC. Although barbigerone has excellent solubility in
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organic solvents such as chloroform, dichloromethane and ethanol, the solubility is
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only about 0.5μg/ml in water. In contrast, the water solubility of bar-HP-β-CD
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inclusion complex was remarkably increased to approximately 6.83 mg/ml. A clear
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solution can be obtained after dissolving the bar-HP-β-CD complex in water with a
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final concentration of barbigerone at 6mg/ml. These results revealed that HP-β-CD
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notably improved the water solubility of barbigerone by the formation of
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bar-HP-β-CD inclusion complex and the water soluble bar-HP-β-CD complex will be
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beneficial to the medical utilization of this compound.
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3.4 Characterization of bar-HP-β-CD complex
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3.4.1 UV spectroscopy UV-visible spectroscopy is an important tool to investigate the influence of the
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complexation on the absorption of barbigerone. The measurements were carried out in
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water/ethanol (V: V= 3:1) mixed solution owing to the rather low water solubility of
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barbigerone. As illustrated in Fig. 2B, two typical absorption peaks of barbigerone in
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the absence of HP-β-CD were observed at 264nm and 231nm, the spectrum of bar-
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HP-β-CD was very similar to those of barbigerone. It was noted that the absorbance
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intensity of barbigerone at 264nm gradually decreased with increasing concentrations
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of HP-β-CD from 1 to 15 mmol/L. As the HP-β-CD concentration increased, a
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slightly red shift of absorption peak was observed in the curve of bar-HP-β-CD
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complex. These changes might be partly attributed to the shielding of chromophore
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groups in barbigerone molecule due to the inclusion into the HP-β-CD cavity (Chow,
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&Karara, 1986; Liu, Qiu, Gao, &Jin, 2006). These results might suggest the
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possibility of the complex formation between barbigerone and HP-β-CD.
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3.4.2 1HNMR spectroscopy
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The 1HNMR of HP-β-CD inclusion complex and bar-HP-β-CD (in D2O) were
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shown in Fig. 3a and 3b. In the spectra of HP-β-CD, the signals for H-1, H-2, H-3,
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H-4, H-5 and H-6 located at 4.988, 3.531, 3.929, 3.499, 3.770 and 3.770, respectively
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(Table 1), were corresponding to the previous literature (Ma et al., 2012). In the
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spectrum for bar-HP-β-CD inclusion complex, appreciable chemical shift changes
248
were observed with respect to the spectra for the free HP-β-CD. The chemical shifts 12
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of HP-β-CD protons in the absence and presence of barbigerone were listed in Table 1
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(Δδ=δbar-HP-β-CD complex -δHP-β-CD). From Table 1 we could see the signals of H-1, H-2
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and H-4 protons on the outer surface of HP-β-CD changed only slightly with Δδ
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values of -0.003, -0.010 and -0.003 respectively. While significant chemical shift
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changes were exhibited by H-5 and H-3 protons in the inner surface of HP-β-CD with
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an up-field shift of 0.042 and 0.017 ppm, respectively. It is noteworthy that the
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chemical shift variation for H-3 was smaller than H-5 after resulting inclusion
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complex. Since both H-3 and H-5 protons from each sugar unit are located in the
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internal cavity of HP-β-CD, and H-3 protons are near the wide side of the cavity
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while H-5 protons near the narrow side (Bernini, 2004), we can propose from the
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1
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penetrate inside the cavity from the narrow side.
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To further explore the possible inclusion mode of the bar-HP-β-CD, we compared the
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1
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and 3d). Owing to its poor water solubility, DMSO-d6 was used as a solvent. As
264
illustrated in Fig. 3c and 3d, the majority signals of barbigerone appeared at
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5.9~8.3ppm, which were distinct from the HP-β-CD protons (3.2~5.2ppm). Most of
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the signals of barbigerone protons were emerged in the spectra of bar-HP-β-CD
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complex. However, the signals of barbigerone were much weaker than that of
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HP-β-CD, this phenomenon may be partly attributed to the low percentage of
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barbigerone in the inclusion complex. As expected, HP-β-CD-induced shift changes
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were also observed for barbigerone protons’ signals between the free and complexed
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HNMR spectrum of barbigerone in the absence and presence of HP-β-CD (Fig. 3c
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state (Table 1). From table 1, we can see the most significant HP-β-CD-induced
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variations were observed in H-2, H-3’ and H-6’ protons, which were attributed to the
273
aromatic protons (Fronza, Mele, Redenti, &Ventura, 1996) (Fig. 1a) from D ring of
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barbigerone. However, inclusion complexation with HP-β-CD had a negligible effect
275
on the δ values of H-5, H-6, H-4’’ and -CH3 protons, which were assigned to the
276
protons from A and B ring in barbigerone.
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Based on these 1HNMR results, we deduced the phenyl group (D ring) of
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barbigerone deeply inserted into the cavity of HP-β-CD and the possible inclusion
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modes for the bar-HP-β-CD complex was illustrated in Fig. 4.
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3.4.3 Fourier-transform infrared spectroscopy (FT-IR)
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The FT-IR spectra of barbigerone, HP-β-CD, bar-HP-β-CD inclusion complex and
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the physical mixture were shown in Fig. 5A. The FT-IR spectrum of barbigerone
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showed strong adsorption bands in the range of 1640~1510cm-1, 1460~1110cm-1, and
284
980~660cm-1 (Fig. 5A(a)). The sharp absorption band appeared at 1641cm-1
285
corresponding to the carbonyl (C=O) stretching vibration. The intense absorption was
286
found at 1210 cm-1 attributed to the C-O stretching vibration. The vibration of -CH3
287
could be found in the region 2980~2830 cm-1. The FT-IR spectra of pure HP-β-CD
288
(Fig. 5A(b)) illustrated intense broad absorption bands at 3600~3000 cm-1, which
289
corresponded to the free stretching vibration. The vibration of –CH3 and –CH could
290
be found at 2970 cm-1. The large band appeared in the region 1150~1030 cm-1 could
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be ascribed to the C-O stretching vibration.
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For the inclusion complex, the spectra was very similar to that of HP-β-CD (Fig.
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5A(c)). the characteristic C=O stretching band of barbigerone at 1641 cm -1 and the
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-CH3 stretching band in the region 2980~2830 cm-1 were masked by the intense band
295
corresponding to the –OH bending vibration of HP-β-CD. This effect was expected
296
due to the low barbigerone to HP-β-CD molar ratio (about 1:7.8), which resulted in
297
the inclusion complexes containing less than 4% by weight. However, we can find the
298
low intensity band with a leftward shift at 1569 cm-1, 1515cm-1 and 1207 cm-1, which
299
indicates the presence of barbigerone in the inclusion complex. In addition, no
300
additional peaks were found in the spectrum of inclusion complex, suggesting the
301
absence of any chemical reactions between HP-β-CD and barbigerone. However, the
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spectrum of physical mixture correspond to the simple superposition of the spectra of
303
the individual component (Fig. 5A(d)). Some characteristic absorption peaks of
304
barbigerone were easy to be observed, indicating the natural structure of barbigerone
305
still existed without any interactions with HP-β-CD.
306
3.4.4 XRD analysis
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293
X-ray powder diffractometry is a useful method for the detection of CD
308
complexation in powder or microcrystalline states. Fig. 5B showed the X-ray
309
diffraction patterns of barbigerone, HP-β-CD, bar-HP-β-CD complex and physical
310
mixture. The powder diffraction pattern of barbigerone displayed several sharp peaks
311
at diffraction angels (2θ) of 9.8, 16.9, 17.1, 21.5, 24.0 (Fig. 5B(a)) suggesting that the
312
compound existed as crystalline form. On the other hand, HP-β-CD was amorphous
313
lacking crystalline peaks (Fig. 5B(b)). These observations is consistent with the
314
previous reports (Hu et al., 2012; Yang et al., 2012). In the case of the physical
Ac
307
15
Page 16 of 30
mixture, the diffraction pattern (Fig. 5B(d)) was simply the superposition of the two
316
patterns of the crystalline barbigerone and the amorphous HP-β-CD. In contrast, the
317
XRD of their inclusion complex (Fig. 5B(c)) exhibited the amorphous halo pattern,
318
which was quite different from a superposition of the XRD of HP-β-CD and the
319
barbigerone. The spectrogram of bar-HP-β-CD complex was similar to that of the
320
amorphous HP-β-CD and the characteristic crystalline peaks of barbigerone was
321
disappeared, indicating some new complex compounds were formed and existed in
322
amorphous state.
323
3.4.5 Scanning electron microscopy (SEM)
an
us
cr
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315
Scanning electron microscopy (SEM) is a qualitative method used to visualize the
325
surface structure of raw materials or the prepared products. The SEM images of
326
barbigerone, HP-β-CD, bar- HP-β-CD inclusion complex and their physical mixtures
327
were illustrated in Fig. 6. Pure barbigerone existed in needle-like crystal with many
328
different size (Fig. 6a), whereas HP-β-CD was observed as spherical particles with
329
cavity structures (Fig. 6b). In the physical mixture, the characteristic HP-β-CD
330
microspheres, which were mixed with barbigerone crystals or adhered to their surface,
331
were clearly observed (Fig. 6d). However, the bar-HP-β-CD inclusion complex
332
appeared in the form of irregular blocky particles (Fig. 6c) in which the original
333
morphology of both components disappeared, which confirmed the formation of the
334
inclusion complex of barbigerone and HP-β-CD.
335
3.5 In vitro bioactivity of bar-HP-β-CD complex
336
In this study, MTT assays were used to evaluate the bioactivity of bar-HP-β-CD
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Page 17 of 30
complex. The MTT assay relies on the mitochondrial activity of cells and reflects
338
their metabolic activity. The effects of bar-HP-β-CD complex and free barbigerone on
339
HCT116 and HepG2 cells’ viability were illustrated in Fig. 7. After incubation for 48h,
340
both bar-HP-β-CD complex and free barbigerone decreased the viability of HCT116
341
cells in a dose-dependent manner (Fig. 7a), and bar-HP-β-CD complex showed
342
slightly higher cytotoxicity than free barbigerone. Both the inclusion complex and
343
free barbigerone exhibited significant cytotoxicity in HepG2 cells at various
344
concentrations (Fig. 7b). These results indicated that the cell cytotoxicity of
345
bar-HP-β-CD was close to free barbigerone and inclusion complexation with
346
HP-β-CD retained the bioactivity.
347
5. Conclusion
348
In this study, barbigerone was successfully complexed with HP-β-CD to form an
349
inclusion complex by a simple coevaporation method. The results of UV-VIS,
350
1
351
complex. The stability constant of the inclusion complexes was found to be 20,659
352
M-1. The water solubility of barbigerone was remarkably increased by complexation
353
with HP-β-CD. Further in vitro studies showed that bar-HP-β-CD inclusion complex
354
retained the anti-cancer activity of barbigeorne. This inclusion should be regarded as a
355
promising strategy in the delivery of water-insoluble anticancer agents such as
356
barbigerone.
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HNMR, FT-IR, XRD and SEM all proved the formation of bar-HP-β-CD inclusion
357 358
Acknowledgements 17
Page 18 of 30
This work was supported by the National Key Technologies R&D Program of China
360
(2012ZX09103101-009)
361
References
362
Bernini, A., Spiga, O., Ciutti, A., Scarselli, M., Bottoni, G., Mascagni, P., & Niccolai, N. (2004).
363
NMR studies of the inclusion complex between beta-cyclodextrin and paroxetine. European
364
Journal of Pharmaceutical Sciences, 22, 445-450.
cr
ip t
359
Chow, D.D., & Karara, A.H. (1986). Characterization, dissolution and bioavail, ability in rats of
366
ibuprofen-β-cyclodextrin complex system. International Journal of Pharmaceutics, 28,
367
95-101.
369
Davis, M.E., & Brewster, M.E. (2004). Cyclodextrin, based pharmaceutics: past, present and
an
368
us
365
future. Nature Reviews Drug Discovery, 3, 1023-1035.
Fronza, G., Mele, A., Redenti, E., & Ventura, P. (1996). 1 H NMR and Molecular Modeling Study
371
on the Inclusion Complex â-Cyclodextrin-Indomethacin. The Journal of Organic Chemistry,
372
61, 909-914.
ed
374
Gould, S., Scott, R.C., (2005). 2-Hydroxypropyl-beta-cyclodextrin (HP-beta-CD): a toxicology review. Food and Chemical Toxicology, 43, 1451-1459.
ce pt
373
M
370
Higuchi, T., & Connors, K. A. (1965). Phase solubility techniques. New York: Wiley Interscience.
376
Hu. L., Zhang, H., Song, W., Gu, D., &Hu, Q. (2012). Investigation of inclusion complex of
377
cilnidipine with hydroxypropyl-β-cyclodextrin. Carbohydrate Polymers, 90, 1719-1724.
378
Irie. T., & Uekama, K. (1997). Pharmaceutical applications of cyclodextrins. III. Toxicological
379
Ac
375
issues and safety evaluation. Journal of Pharmaceutical Sciences, 86,147- 162.
380
Li, X., Wang, X., Ye, H., Peng, A., &Chen, L. (2012). Barbigerone, an isoflavone, inhibits tumor
381
angiogenesis and human non-small-cell lung cancer xenografts growth through VEGFR2
382
signaling pathways. Cancer Chemotherapy and Pharmacology, 70, 425-437.
383
Li, Z.G., Zhao, Y.L., Wu, X., Ye, H.Y., Peng, A., Cao, Z.X., Mao, Y.Q., Zheng,Y.Z., Jiang, P.D.,
384
Zhao, X., Chen, L.J., &Wei, Y.Q. (2009). Barbigerone, a natural isoflavone, induces
385
apoptosis in murine lung, cancer cells via the mitochondrial apoptotic pathway. Cellular
386
Physiology and Biochemistry, 24, 95-104. 18
Page 19 of 30
387
Liu, J., Qiu, L., Gao, J., &Jin, Y. (2006). Preparation, characterization and in vivo evaluation of
388
formulation of baicalein with hydroxypropyl-beta-cyclodextrin. International Journal of
389
Pharmaceutics, 312, 137-143.
391 392 393
Loftsson, T., & Brewster, M.E. (1996). Pharmaceutical applications of cyclodextrins. 1. Drug solubilization and stabilization. Journal of Pharmaceutical Sciences, 85, 1017-1025. Loftsson, T., & Duchêne, D. (2007). Cyclodextrins and their pharmaceutical applications.
ip t
390
International Journal of Pharmaceutics, 329, 1-11.
Ma, S.X., Chen, W., Yang, X.D., Zhang, N., Wang, S.J., Liu, L., & Yang, L.J. (2012).
395
Alpinetin/hydroxypropyl-β-cyclodextrin host, guest system: preparation, characterization,
396
inclusion mode, solubilization and stability. Journal of Pharmaceutical and Biomedical Analysi,
397
67-68, 193-200.
401 402 403
us
an
400
Reviews, 98, 2045-2076.
Villain, C. (1980). Barbigerone, a new pyranoisoflavone from seeds of tephrosia barbigera. Phytochemistry, 19, 988-989.
M
399
Uekama, K., Hirayama, F., & Irie, T. (1998). Cyclodextrin Drug Carrier Systems. Chemical
Wangensteen, H., Alamgir, M., Rajia, S., Samuelsen, A.B., & Malterud, K.E. (2005). Rotenoids
ed
398
cr
394
and isoflavones from Sarcolobus globosus. Planta Medica, 71, 754-758. Wangensteen, H., Miron, A., Alamgir, M., Rajia, S., Samuelsen, A.B., & Malterud, K.E. (2006).
405
Antioxidant and 15, lipoxygenase inhibitory activity of rotenoids, isoflavones and phenolic
406
glycosides from Sarcolobus globosus. Fitoterapia, 77, 290-295.
ce pt
404
Yang, R., Chen, J.B., Dai, X.Y., Huang, R., Xiao, C.F., Gao, Z.Y., Yang, B., Yang, L.J., Yan, S.J.,
408
Zhang, H.B., Qing, C., & Lin, J. (2012). Inclusion complex of GA, 13315 with cyclodextrins:
409
Preparation, characterization, inclusion mode and properties. Carbohydrate Polymers, 89,
410
89-97.
Ac
407
411
Ye, H., Zhong, S., Li, Y., Tang, M., Peng, A., Hu, J., Shi, J., He, S., Wu, W., & Chen, L. (2010).
412
Enrichment and isolation of barbigerone from Millettia pachycarpa Benth. using high, speed
413
counter, current chromatography and preparative HPLC. Journal of Separation Science, 2010,
414
33, 1010-1017.
415
Yenesew, A., Derese, S., Midiwo, J.O., Oketch., Rabah, H.A., Lisgarten, J., Palmer, R.,
416
Heydenreich, M., Peter, M.G., Akala, H., Wangui, J., Liyala, P., &Waters, N.C. (2003). 19
Page 20 of 30
417
Anti-plasmodial activities and X, ray crystal structures of rotenoids from Millettia usaramensis
418
subspecies usaramensis. Phytochemistry , 64, 773-779.
419 420
Yenesew, A., Midiwo, J.O., & Waterma, P.G. (1998). Rotenoids, isoflavones and chalcones from the stem bark of Millettia Usaramensis subspecies usaramensis. Phytochemistry, 47, 295-300.
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Figures and Tables
425
Fig. 1. The chemical structures of barbigerone and HP-β-CD.
426
Fig. 2. (A): Phase solubility diagram of bar-HP-β-CD system. The HP-β-CD concentration was in
427
the range of 0~20 mM. Each point is the mean of three determinations. (B): The effect of
428
HP-β-CD concentration on the UV-VIS spectra of barbigerone (5μg/ml) in aqueous solution; the
429
HP-β-CD concentration was (a) 0mM; (b) 1mM; (c) 2mM; (d) 5mM; (e) 15mM, respectively.
430
Fig. 3. 1HNMR spectra..(a) bar-HP-β-CD inclusion complex and (b) HP-β-CD in D2O; 1HNMR
431
spectra of barbigerone (c) and (d) bar-HP-β-CD inclusion complex in DMSO-d6.
432
Fig. 4. Possible inclusion mode of bar-HP-β-CD complex.
433
Fig. 5. (A) FT-IR spectra of (a) barbigerone, (b) HP-β-CD, (c) bar-HP-β-CD inclusion complex, (d)
434
barbigerone and HP-β-CD physical mixture(1:1, molar ratio); (B) XRD patterns of (a)
435
barbigerone, (b) HP-β-CD, (c) bar-HP-β-CD inclusion complex, (d) barbigerone and HP-β-CD
436
physical mixture.(1:1, molar ratio)
437
Fig. 6. Scanning electron microphotographs: (a) barbigerone, (b) HP-β-CD, (c) bar-HP-β-CD
438
inclusion complex, (d) barbigerone and HP-β-CD physical mixture (1:1, molar ratio).
439
Fig. 7. Cell viability of bar-HP-β-CD inclusion complexes and free barbigerone on cancer cells.(a)
440
human colon cancer cells (HCT116) and (b) human liver cancer cell (HepG2; data were given as
441
mean ±SD, n=3)
444 445
cr
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443
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442
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446 447 448 449 450 451 21
Page 22 of 30
Table(1)
1
Table 1. Chemical shift (δ, ppm) change values relating to the signals of HP-β-CD and
2
barbigerone in the free and the complexed states. HP-β-CD
bar-HP-β-CD complex
Δδ
H-1
4.988
4.985
-0.003
H-2
3.531
3.541
-0.010
H-3
3.929
3.912
H-4
3.499
3.495
H-5
3.770
3.728
H-6
3.770
3.754
Me
1.045
1.041
protons
6.773
H-6’
6.875
H-5
cr us
-0.003
6.872
-0.003
7.839
7.838
-0.001
H-6
6.918
6.919
0.001
H-4’’
6.793
6.793
0.000
1.466
-0.001
ce pt
5
-0.004
1.467
Ac
4
-0.016
6.770
-CH3 3
-0.042
-0.004
M
H-3’
-0.003
8.241
ed
8.245
-0.017
an
barbigerone H-2
ip t
HP-β-CD
1
Page 23 of 30
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Figure(1)
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Figure(2)
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Figure(3)
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Figure(4)
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Figure(5)
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Ac ce p
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Figure(6)
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Ac ce p
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Figure(7)
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