Preparation,Characterization and Antioxidant Activity of Inclusion Complex of Bakuchiol with Hydroxypropyl-β-cyclodextrin

DENG Yunxia LIU Minyan WANG Lu BIAN Yonggang SHAO Zhiyu

College of Chemistry,Chemical Engineering and Biotechnology,Donghua University,Shanghai 201620,China

Abstract:Bakuchiol isolated from Psoralea corylifolia is a naturally occurring prenylated phenolic monoterpene with a variety of bioactivities. The aim of this study was to improve the water solubility and thermal stability of bakuchiol through complexing it with hydroxypropyl-β-cyclodextrin(HP-β-CD). The bakuchiol/HP-β-CD inclusion complex’s behavior and characterization were investigated by ultraviolet-visible (UV-vis) spectroscopy,Fourier transform infrared spectroscopy (FT-IR),thermogravimetric analysis (TGA),X-ray diffraction(XRD),1H nuclear magnetic resonance(NMR),and two-dimensional (2D) NMR. The obtained results indicated the formation of 1∶1 inclusion complex for bakuchiol with HP-β-CD. Water solubility of bakuchiol was significantly improved by complexation with HP-β-CD as demonstrated by phase solubility studies. The encapsulation of bakuchiol was confirmed by UV-vis,FT-IR,and XRD. The thermal stability was effectively enhanced by TGA and derivative thermogravimetry(DTG) analysis. In vitro antioxidant activity showed that bakuchiol/HP-β-CD inclusion complex had a little higher antioxidant ability than free bakuchiol. Moreover,we got the possible inclusion mode for the bakuchiol/HP-β-CD inclusion complex through NMR analysis. These results suggest that the inclusion complex can be a potentially useful approach in the design of novel formulations of bakuchiol for medical applications.

Key words:bakuchiol;hydroxypropyl-β-cyclodextrin(HP-β-CD);nclusion complex;characterization

Introduction

Bakuchiol is a naturally occurring prenylated phenolic monoterpene with a chemical formula C18H24O，which is one of the main active components ofPsoraleacorylifoliaL.(Leguminosae) that has been widely used in China to treat various diseases such as leucoderma and other skin diseases,cardiovascular diseases,nephritis,osteoporosis,and cancer[1-2].It also has been isolated fromOtholobiumpubescens[3]andPsoraleaglandulosaL.[4],Ulmusdavidianavar.japonica[5].As a bioactive component,bakuchiol has been reported to possess a range of significant pharmacological activities including antimicrobial,antioxidant,antiinflammatory,anti-influenza viral activity,antidepressant,anti-metastasis activity hepatoprotective,reduction of bone loss，estrogenic activities[6-10]and protective effects in the heart,liver,skin and other organs[11].However,bakuchiol has been shown to be poor water solubility and is easy to be oxidized or decomposed when exposed to the air,light,or heat,which restricts its medical applications.The Pharmacokinetic studies reported that bakuchiol possessed poor absorption and low bioavailability probably mainly due to its low water solubility[12-13].In order to protect the bakuchiol from denaturating and improve its water solubility bioavailability,encapsulation may be one of the options.

Cyclodextrins (CDs) are cyclic oligosaccharides with 6(α-CD),7(β-CD) or 8(γ-CD) glucose residues consisting of (α-1,4)-linked α-D-glucopyranose units,which are derived from the enzymatic degradation of starch.They have a relatively hydrophilic external surface and a hydrophobic interior cavity allowing them to include hydrophobic guests[14].The physical,chemical and biological properties of many natural bioactive molecules can be improved after their encapsulation within CD.Moreover,CDs have a high level of biocompatibility and are approved by Food and Drug Administration( FDA).Hydroxypropyl-β-CD (HP-β-CD)is a hydroxyalkylated β-CD derivative that exhibits better water solubility,safe cytotoxicity and satisfactory inclusion ability,which makes bioactive molecules suitable for oral and parental application[15].However,there is little study about the inclusion complex of bakuchiol with HP-β-CD.

In our study,the bakuchiol extracted fromPsoraleacorylifoliaL.was included by HP-β-CD,the characterizations of the complex were investigated through the analysis of ultraviolet-visible (UV-vis),Fourier transform infrared spectroscopy(FT-IR),thermogravimetric analysis(TGA),X-ray diffraction(XRD),1H nuclear magnetic resonance(NMR),nuclear overhauser effect spectroscopy(NOESY),and the phase solubility,solubility and antioxidant activity were also evaluated.

1 Experiments

1.1 Materials

HP-β-CD (average molecular weight = 1 460) was purchased from Tokyo Chemical Industry Co.,Ltd.，Japan and used as received.1,1-Diphenyl-2-picrylhydrazyl (DPPH) was purchased from Wako Pure Chemical Industries,Ltd.(Japan),Methanol was of high-performance liquid chromatography(HPLC) grade.And other reagents and chemicals were of analytical reagent grade.All experiments were carried out by ultrapure or deionised water.

The seeds ofPsoraleaglandulosaL.were purchased in Anhui Province,China,in Jan.2013,and identified by Associate Prof.CAO Zhangjun,Donghua University,Shanghai.A voucher specimen (Ps-2013-1) was deposited in the College of Chemistry,Chemical Engineering and Biotechnology,Donghua University,Shanghai.

1.2 HPLC analysis

Analytical HPLC were performed on a Waters 600E with a Waters 2489 UV-vis detector and a SunFireTM-C18 column (4.6 mm×150 mm,5 μm),methanol/water (90/10 by volume) was used as the mobile phase and the eluent was detected by an UV-vis detector at a wavelength of 260 nm.The injection volume was 20 μL and the flow rate was 1.0 mL/min.

1.3 Preparation of bakuchiol

The dried seeds ofPsoraleacorylifoliaL.(1 kg) were ground and extracted with 95% EtOH (2.5 L) at room temperature for three times to give 263 g extracts,and which were suspended in water and then partitioned with petroleum ether and chloroform successively to give petroleum ether soluble fraction (7.2 g) and chloroform soluble fraction (115 g),respectively.Parts of the chloroform fraction were chromatographed on a silica gel column (200-300 mesh) eluted successively with petroleum ether-acetone (100∶1 to 10∶1) to obtain compound1(1.1 g) which was identified as bakuchiol by NMR analysis.

1.4 Preparation of the inclusion complex

The inclusion complex of bakuchiol with HP-β-CD was prepared by the freeze-drying method.Briefly,the bakuchiol was dissolved in trace acetone and slowly dropped into aqueous solution of HP-β-CD under magnetic stirring at a molar ratio of 1∶1 at room temperature,and the mixture protected from light was stirred continuously for another 4 h.Subsequently,the undissolved bakuchiol solids were removed by filtration (pore diameter,0.45 μm),and the solution was lyophilized in a freeze dryer and stored at 4 ℃ for further tests.

The physical mixture was prepared through mixing bakuchiol and HP-β-CD together (molar ratio,1∶1) in an agate mortar until a homogeneous mixture was obtained.

1.5 Phase solubility study

The phase solubility diagram was performed according to a modification of the method of Higuchi and Connors[16].Briefly,excess of bakuchiol was added into vials containing increasing concentrations of HP-β-CD (0.5-20.0 mmol/L).Vials protected from light with aluminum foil were agitated.When equilibrium had been reached (about after for 4 h at 25 ℃),the resulting solution was filtered over 0.45 μm millipore membrane and appropriately diluted for HPLC analysis at 260 nm.All experiments were performed in triplicate.The apparent stability constantsK1∶1were calculated from the linear fit of the curve according to the following equation.

whereSslopeis the value found in the linear regression andS0is the aqueous solubility of the drug in the absence of HP-β-CD determined by using HPLC.

1.6 Solubility study

A solubility study was carried out according to a previously reported method[17].In brief,excess amounts of the bakuchiol and bakuchiol/HP-β-CD inclusion complex were added into 4 mL deionised water in order to ensure the solution reaching saturation;then,the suspension was stirred continuously protecting from light at 25 ℃ for 24 h.After equilibrium was attained,the solution was filtered by the 0.45 μm millipore membrane to remove the excess drug.Then the filtrate was injected into HPLC and the concentration of bakuchiol was determined by the standard curve.All tests were performed in triplicate.

1.7 Characterization

1.7.1UV-visspectroscopy

The UV-vis spectra of bakuchiol and the inclusion complex were recorded on a UNIC UV-2100 spectrophotometer (United Products &Instruments,Inc.,USA)by scanning from 800 nm to 200 nm.

1.7.2FT-IRanalysis

FT-IR spectra of bakuchiol,HP-β-CD,the physical mixture and the inclusion complex were measured using the KBr disk method on a Nicolet 6 700 FT-IR spectrometer (Thermo Fisher Scientific,America) with 4 cm-1resolution and a scanning range of 4 000 cm-1and 400 cm-1.

1.7.3Thermalanalysis

The thermal behavior of the empty HP-β-CD,the free bakuchiol,the physical mixture and the inclusion complex were studied by TGA using a NETZSCH TG 209 F1(NETZSCH Proven Excellence,Germany).Samples (4-5 mg) were sealed in an aluminum pan and heated from 25 ℃ to 600 ℃ at a rate of 10 ℃/min under nitrogen environment.

1.7.4XRDanalysis

XRD patterns were recorded by using a D/max-2550 PC ( Rigaku,Japan) powder diffractometer using Cu- Kα radiation with 200 mA,40 kV,and a scanning rate of 5 (°)/min.Powder samples were mounted on a sample holder and scanned in the 2θangle range from 5° to 50° with a step size of 2θ= 0.02°.

1.7.5NMRspectroscopy

1H NMR and NOESY spectra of bakuchiol,HP-β-CD and their inclusion complex were conducted on a Bruker AV-500 spectrophotometer(Bruker,Karlsruhe,Germany) at 300 K in DMSO-d6or D2O,respectively.Tetramethylsilane (TMS) was used as an internal standard.Chemical shifts (δ) were indirectly referenced to TMS.Samples were equilibrated for at least 24 h before measurement.

1.8 In vitro antioxidant activity

Antioxidant activity of bakuchiol and the inclusion complex were evaluated in vitro against free DPPH radicals according to a previous method with some modifications[18].A methanol solution of DPPH (0.1 mmol/L) was prepared.Then,100 μL of this solution were added to 100 μL of sample solutions (bakuchiol alone or inclusion complex with same contents dissolved in methanol).Mixtures were vortexed vigorously and incubated at room temperature for 30 min in dark.The absorbance was measured with a Multiskan spectrum microplate spectrophotometer at 517 nm in the presence of the inclusion complex or bakuchiol solution.Butylated hydroxyanisole(BHA) was applied as the positive control.Blank sample was prepared by mixing 100 μL of methanol with 100 μL of DPPH solution.All preparations and assays were performed in triplicate.The absorbance of the sample was compared with the absorbance of the control.The percentage scavenging activity A was calculated as

A= (Acontrol-Asample)/Acontrol×100%,

whereAcontrolwas the absorbance of the DPPH solution without the inclusion complex or bakuchiol solution,andAsamplewas the absorbance of the DPPH solution with the inclusion complex or bakuchiol solution.

2 Results and Discussion

2.1 Phase solubility analysis

The solubility method is useful for studying inclusion complex of poorly soluble drugs with CDs in water.Figure 1 shows the phase solubility diagram of bakuchiol in the presence of various HP-β-CD concentrations at 25 ℃.The aqueous solubility of bakuchiol increased linearly with increasing HP-β-CD concentration within the studied concentration range.The solubility curve having a slope of 0.218 6 with a correlation coefficient squared values of 0.985 5 (R2>0.98) was regarded as a straight line and could be classified as ALtype according to Higuchi and Connors[16].And it was assumed that the solubility increase was due to the formation of 1∶1 complex[16-19],suggesting the formation of a complex characterized by having a greater solubility than that of bakuciol alone.TheS0is the aqueous solubility of bakuchiol and the slope is the value found in the linear regression.The apparent stability constantK1∶1was calculated as 6 994 M-1，which indicated that the interactions between bakuchiol and HP-β-CD were very strong.

Fig.1 Phase solubility diagram of bakuchiol/HP-β-CD system

2.2 Solubility of bakuchiol

The water solubility of bakuchiol and complex were evaluated by preparation of its saturation solution.The results revealed that the solubility of bakuchiol was only about 10.24 μg/mL in water.In contrast,the water solubility of bakuchiol/HP-β-CD inclusion complex remarkably increased to approximately 1.72 mg/mL,which was over 160-fold higher than the solubility of bakuchiol with the absence of HP-β-CD.These results revealed that HP-β-CD notably improved the water solubility of bakuchiol by the formation of bakuchiol/HP-β-CD inclusion complex.

2.3 Characterization of bakuchiol/HP-β-CD inclusion complex

2.3.1UV-visspectraanalysis

The UV-vis spectrum is an important tool to investigate the influence of the complexation on the absorption of bakuchiol.The maximum absorption wavelength of bakuchiol was at 260 nm,while the inclusion complex’s wavelength was at 247 nm (shown in Fig.2),the observed blue shift might be due to the perturbation of the chromophore electrons of the drug due to the inclusion into the CD cavity.These results might suggest the possibility of the complex formation between bakuchiol and HP-β-CD.

Fig.2 UV-vis spectra of (a)bakuchiol (0.05 mg/mL) and(b) bakuchiol/HP-β-CD (1 mg/mL)

2.3.2FT-IRanalysis

Fig.3 FT-IR spectra of (a) bakuchiol ;(b) HP-β-CD;(c) physical mixture;(d) bakuchiol/HP-β-CD inclusion complex

2.3.3TGAandDTGanalyses

The thermal stability of the encapsulated bakuchiol was assessed by TGA and DTG analyses,and compared with the free molecule and the empty HP-β-CD,which were shown in Figs.4-5.The thermal decomposition of HP-β-CD existed two weight loss steps at 29-90 ℃ and 282-390 ℃.The first stage was due to the evaporation of water binding to HP-β-CD and the second one was associated with the decomposition of HP-β-CD.Bakuchiol quickly lost mass from 142 ℃ to 250 ℃ as shown in Fig.4.The physical mixture apparently contained only the free species,which followed by the decomposition of both compounds,and represented by a continuous mass loss in the TG curve.The inclusion complex also showed two weight loss steps at 29-114 ℃ and 274-364 ℃,which was similar to HP-β-CD due to the influence of HP-β-CD,and reduced bakuchiol’s decomposition.Moreover,the decomposition peak (shown Fig.5) of inclusion complex was 339 ℃,which was much higher than that of pure bakuchiol of 244 ℃ and a little lower than pure HP-β-CD of 344 ℃.This observation indicated that the formation of the bakuchiol/HP-β-CD inclusion complex retarded the weight loss of bakuchiol during heating,and then bakuchiol/HP-β-CD inclusion complex was more stable than bakuchiol.These findings were consistent with the literature,in which the thermal stability of guest molecules was improved when they were included with HP-β-CD[20].

Fig.4 Thermal stability analyses on TGA of (a) bakuchiol;(b) physical mixture;(c) bakuchiol/HP-β-CD inclusion complex;(d) HP-β-CD

Fig.5 Thermal stability analyses on DTG of (a) bakuchiol;(b) physical mixture;(c) bakuchiol/HP-β-CD inclusion complex;(d) HP-β-CD

2.3.4XRDanalysis

The powder XRD patterns of HP-β-CD and their inclusion complex are illustrated in Fig.6.HP-β-CD showed an amorphous state that lacked crystalline peaks,the bakuchiol/HP-β-CD inclusion complex also noted an amorphous halo pattern from the diffractogram.These results further proved that bakuchiol had been incorporated into the cavity of HP-β-CD and presented as the amorphous or disordered structure.As bakuchiol is not solid at room temperature,the corresponding XRD patterns can not be obtained.However,through comparative analysis of existing data,we consider that bakuchiol/HP-β-CD inclusion complex was confirmed.

Fig.6 Powder XRD of (a) HP-β-CD and (b) bakuchiol/HP-β-CD inclusion complex

2.3.5NMRanalysis

NMR spectroscopy is an important tool to prove the formation of the inclusion complex and study the inclusion mode.The majority of bakuchiol protons displayed chemical shifts at δ1-2 or 5-8 and distincted from those of the HP-β-CD protons.It is well known that if a guest molecule is incorporated into the CDs cavity,the hydrogen atoms located in the interior of the cavity (C-3-H and C-5-H),with H-3 closer to the wider rim and H-5 on the opposite,will be considerably shielded by the guest molecule and show a significant upfield shift,whereas the hydrogen atoms on the outer surface (C-2-H,C-4-H and C-6-H) will be unaffected or show only a marginal upfield shift[21].After inclusion complexation with bakuchiol,the H-3 proton of HP-β-CD shifted 0.012 and the H-5 proton of HP-β-CD shifted 0.055 ( shown in Table 1).And the H-12 proton of bakuchiol shifted 0.094,H-18 proton of bakuchiol shifted 0.041 (shown in Table 2).These results may indicate that the bakuchiol should be entered in the HP-β-CD cavity from the narrow side.

Table 1 1H chemical shifts (δ) corresponding to HP-β-CD proton in the free and complexed state in D2O

Table 2 1H chemical shifts (δ) corresponding to bakuchiol in the free and complexed state in DMSO-d6

2D NMR provides the more direct evidence for the spatial proximity between host and guest atoms following the observation of intermolecular dipolar cross-correlations.Two protons closely located in space can produce a Nuclear Overhauser Effect (NOE) cross-correlation in NOESY.In the NOESY of bakuchiol/HP-β-CD inclusion complex (shown in Fig.7),strong NOE cross peaks were observed between H-5 (red front in Fig.7) of HP-β-CD and H-12 of bakuchiol,H-3 (red front in Fig.7) of HP-β-CD and H-17,H-18 of bakuchiol,which indicated that the 14,15 methyls of bakuchiol were near the narrow bottom and the vinyl at position of C-9 got closer to the wide bottom.

Fig.7 Parts of 2D NOESY spectrum of bakuchiol/HP-β-CD inclusion complex in DMSO-d6

2.4 Antioxidant activity analysis

The DPPH radical scavenging assay is extensively used to quantify antioxidant activity of natural compounds,with the advantages of relatively simple,highly reproducible and comparable with other methods[22].As can be seen in Fig.8,the rate of DPPH radical scavenging increased with the increase of bakuchiol or bakuchiol/HP-β-CD inclusion complex concentration,which was significantly lower than the positive control BHA.Phenolic compounds with free hydroxyls are considered to possess more or less antioxidative activity,since phenoxy radicals are readily formed by donation of phenolic hydrogens,meanwhile,the DPPH radical scavenging ability of an antioxidant is believed to be closely associated with its hydrogen-donating ability[23].It should be noted that the bakuchiol/HP-β-CD inclusion complex had the little higher antioxidant ability than free bakuchiol to scavenge DPPH free radical under the same bakuchiol concentration,since the HP-β-CD had no antioxidant activity.It might be due to the formation of hydrogen bonds between bakuchiol and HP-β-CD,which resulted in the formation of a stable phenoxyl radical,and then improved the hydrogen-donating ability of guest molecules.A similar phenomenon was also observed in canthaxanthin /HP-β-CD complex studies[24].

Fig.8 DPPH radical scavenging activities of BHA,bakuchiol,bakuchiol/HP-β-CD and HP-β-CD

3 Conclusions

We successfully prepared the water-soluble inclusion complex with bakuchiol and HP-β-CD in the molar ratio of 1∶1 and determined that the resulting bakuchiol/HP-β-CD inclusion complex exhibited different physicochemical characteristics in contrast with free bakuchiol.In addition,the inclusion complex effectively enhanced the water solubility and thermal stability,and kept antioxidant ability of bakuchiol through the formation of inclusion complex.We got the possible inclusion mode of the bakuchiol/HP-β-CD inclusion complex.This inclusion complex can be regarded as an important and useful approach in the design of novel formulations of bakuchiol for medical applications.