Encompassment of isoeugenol in 2-hydroxypropyl-β-cyclodextrin using ultrasonication: Characterization, antioxidant and antibacterial activities

Journal of Molecular Liquids 296 (2019) 111777

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Encompassment of isoeugenol in 2-hydroxypropyl-b-cyclodextrin using ultrasonication: Characterization, antioxidant and antibacterial activities Subramanian Siva a, Changzhu Li b, Haiying Cui a, **, Lin Lin a, * a b

School of Food and Biological Engineering, Jiangsu University, Zhenjiang, 212013, China Department of Bioresource, Hunan Academy of Forestry, Changsha, 410007, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 15 July 2019 Received in revised form 5 September 2019 Accepted 18 September 2019 Available online 8 October 2019

Isoeugenol is a natural dual antioxidant/prooxidant. In this research, the inclusion complex (IC) of isoeugenol with 2-hydroxypropyl-b-cyclodextrin (HPbCD) was prepared via the ultrasound (US) method. The US assisted isoeugenol/HPbCD-IC was investigated by various characterization techniques such as UV eVis absorption, fluorescence, powder X-ray diffraction, Fourier transform infrared spectroscopy, thermal gravimetric analysis and differential scanning calorimetry. Absorption, fluorescence and infrared studies indicated that the alkyl chain of isoeugenol was deeply included in the HPbCD cavity. Through determinations of phase solubility and water solubility studies, significant enhancement of isoeugenol water solubility was confirmed after IC formation with the HPbCD. Thermal data proved that the IC formation significantly enhanced the thermal stability of isoeugenol. Antioxidant test results indicated that isoeugenol/HPbCD-IC exhibited better antioxidant activity than free isoeugenol due to its solubility enhancement. Furthermore, the isoeugenol/HPbCD-IC showed higher antibacterial activity of 96 ± 0.2% and 97 ± 0.5% against Staphylococcus aureus and Escherichia coli bacteria, respectively. © 2019 Elsevier B.V. All rights reserved.

Keywords: Isoeugenol 2-Hydroxypropyl-b-cyclodextrin Phase solubility Antioxidant activity Antibacterial activity

1. Introduction Natural phenolic compounds are the most important components of essential oils (EOs) derived from various botanical sources and used in flavor, fragrance, food products and spiced beverages. These natural phenolic compounds have many biological activities such as antibacterial, anti-inflammatory, antifungal, antitumor activities, etc [1,2]. However, they have been gained particular interest of researchers because of their unique properties as antioxidants and free radical scavenging activity. Several studies in the literature have exemplified the effectiveness of using natural phenolic compounds in food products for preventing lipid oxidation [3]. The mechanism of antioxidant action of these phenolic compounds is different from each other due to various influencing factors. Lipid oxidation process can be retarded in the presence of natural phenolic antioxidants which provide hydrogen atom that effectively interrupts the free radical chain reaction [4]. Isoeugenol (2-methoxy-4-propenyl phenol), an isomer of eugenol, is a natural

* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (H. Cui), [email protected] (L. Lin). https://doi.org/10.1016/j.molliq.2019.111777 0167-7322/© 2019 Elsevier B.V. All rights reserved.

phenolic compound abundant in the EOs of numerous plants and is widely used to retard oxidation and extend shelf lives of food products [5]. Recently, isoeugenol is of interest for many researchers because of its dual antioxidant and/or prooxidant effects [6]. The biological activities of isoeugenol have been proposed to be due to free eOH group and a, b position of carbon‒carbon double bond in the alkyl chain and g position of a methyl group (Fig. 1A). Zhang and co-workers [7] have studied the antioxidant, protective effect against DNA damage and antibacterial activities of isoeugenol and eugenol. Interestingly, the results showed that isoeugenol exhibited higher biological activities than its isomer eugenol due to a carbon-carbon double bond nearer to the phenyl ring. Moreover, isoeugenol is a hydrophobic molecule (log P z 2.45) which exhibits poor absorption, like other naturally occurring phenolic compounds. a-, b- and g-cyclodextrins (CDs) are the most interesting cyclic oligomers composed of six, seven and eight D-glucopyranose units, respectively. These CDs exhibit internal hydrophobicity and external hydrophilicity because of their specific structures and abundant hydroxyl (‒OH) groups [8]. Primary hydroxyl groups are situated on the narrow rim and secondary hydroxyl groups are situated on the wider rim of the cavities. These arrangements of glucopyranose units present in CDs grant on them the possibility to

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Fig. 1. (A) The chemical structure of isoeugenol and HPbCD. (B) The schematic representation of ultrasound processed IC formation of isoeugenol and HPbCD.

serve as a host molecule. This CDs cavity has the ability to form inclusion complexes (ICs) with hydrophobic molecules such as EO compounds that are smaller than the cavity [9]. Non-covalent interactions such as hydrophobic interactions, van der Waals forces, electrostatic interactions and hydrogen bondings are the main driving forces for the formation of IC [10]. 2-hydroxypropyl-b-cyclodextrin (HPbCD) is a derivative of native bCD approved by FDA, with substitutions of the eOH groups by hydroxypropyl groups, resulting in a dramatic improvement of its solubility (~600 mg/mL) and complexation ability, than its parent bCD [11]. It is well tolerated in animals and humans because of its biocompatibility and non-toxicity. Consequently, HPbCD is extensively used as solubilizing agents in the food and pharmaceutical industries. In literature, a number of studies have been devoted to understanding the host/guest inclusion interactions of biologically active compounds with HPbCD [12,13]. It is also explained that how these non-covalent interactions alter their biological activities. In this respect, HPbCD-ICs of EOs and their compounds have been intensively studied with respect to their role as antioxidant, antibacterial, antifungal, etc. Specifically, the physicochemical studies targeting the interaction between EOs and components or HPbCD are of interest because of their potential applications in foods, pharmaceuticals, perfumes and cosmetics. It is also possible to observe an enhancement in the antioxidant and

antibacterial effects with the support of HPbCD [12e14]. Ultrasound (US) with the characteristics of acoustic cavitation (the formation, growth and implosive collapse of microbubbles) can generate high pressure and high temperature in liquid medium when irradiated with ultrasonic frequency. The US technology approach has been universally acknowledged to produce IC, nanoparticles and nanoemulsion from various kinds of materials. The exceptional advantages and characteristics of the US power and its assisted products have led to broad request in food, pharmaceutical and biomedical applications such as imaging and therapy [15e18]. Literature survey revealed few previous reports on the inclusion of isoeugenol into HPbCD. Kfoury et al. [19] investigated the inclusion in solution of isoeugenol with aCD, bCD and three bCD derivatives namely HPbCD, randomly methylated bCD (RAMEB) and a low methylated bCD (CRYSMEB) using the indirect UVeVis competition method (spectral displacement method) and static headspace-gas chromatography (SH-GC). Besides, Fourmentin et al. [20,21] reported that the aqueous solubility of isoeugenol with the effect of the CDs. The higher stability of isoeugenol upon encapsulation in aqueous solution was also determined; however, the solid IC of isoeugenol and HPbCD was not successfully prepared and characterized using analytical techniques. However, Higueras et al. reported that chitosan film incorporating HPbCD at a 1:1 wt ratio

S. Siva et al. / Journal of Molecular Liquids 296 (2019) 111777

are capable of sorbing selective amounts of isoeugenol [22]. To the best of our knowledge there is no report in literature about the IC formation of isoeugenol and HPbCD in solid state. This literature lacks prompted us to visit isoeugenol-HPbCD inclusion interaction and to acquire information on the solid isoeugenol/ HPbCD-IC. Therefore, the goal of the present work was to prepare the solid IC of isoeugenol and HPbCD by the US synthetic method and to investigate the structural and thermal properties by powder X-ray diffraction (PXRD), Fourier transform infrared spectroscopy (FT-IR), thermal gravimetric analysis (TGA) and differential scanning calorimetry (DSC). The UVeVis absorption and fluorescence spectroscopy were used for spectral characterization of isoeugenol with HPbCD in solution. Besides experimental measurements, semiempirical quantum mechanical calculations at PM3 (Parametric Model) level of theory was employed to accurately estimate complexation energies of the IC according to two proposed modes. In addition to the phase solubility, water solubility assessment was carried out to prove the solubility enhancement of isoeugenol through IC formation with HPbCD. The antioxidant activity of the solid HPbCD-IC was evaluated by measuring the clearance of DPPH radicals. Finally, the plate colony counting method was used to assess the antibacterial activities of the US assisted isoeugenol/ HPbCD-IC against gram-positive Staphylococcus aureus (S. aureus) and gram-negative Escherichia coli (E. coli) bacteria. 2. Materials and methods 2.1. Materials Isoeugenol (98%, MW z 164), HPbCD (98%, MW z 1375, degree of substitution z 0.6), ethanol and methanol were purchased from Sinopharm Chemical Reagent Co. Ltd., Shanghai, China. FT-IR grade potassium bromide (KBr, 99%) was received from Sigma-Aldrich Chemical Co., Mainland China. 2,2-diphenyl-1-picrylhydrazyl (DPPH, 99%) was obtained from Aladdin Industrial Corporation, Shanghai, China. The bacterial strains Staphylococcus aureus and Escherichia coli O157:H7 were supplied by China Center of Industrial Culture Collection, Beijing, China. Triple distilled water was used in preparing the sample solutions. 2.2. Preparation of solid isoeugenol/HPbCD-IC assisted by the US Isoeugenol/HPbCD-IC was prepared using ultrasound method according to our previous report [23]. Briefly, 0.04 M of isoeugenol was added dropwise to 0.04 M of HPbCD with distilled water:ethanol solvent mixture (4:1). The mixed solution was sonicated (Ymnl-1000Y ultrasonic device, horn tip end diameter ¼ 5 mm, Power ¼ 60 W) for 0.5 h with pulsed on and off for 1 s each. The powder of isoeugenol/HPbCD-IC was obtained by freeze-dried for 12 h. The obtained product was placed in a tightly sealed glass vial protected from light. 2.3. UVevis absorption and fluorescence spectroscopy A Shimadzu spectrophotometer (model 1650 PC) was used to carry out the UVeVis absorption measurements, using 1.0 cm quartz cells. A Horiba Jobin Yvon spectrofluorometer (model Fluoromax-4) was used to perform steady-state fluorescence measurements. The samples were excited at 265 nm and the emission wavelengths were scanned for 280e550 nm. The slit set was 5 nm for both excitation and emission. Fresh solutions of isoeugenol in HPbCD were prepared before measurements. The spectral measurement was performed by the incremental addition of HPbCD concentration (range from 0 to 10  103 M) to 0.2 mL of methanolic solutions containing isoeugenol (2  103 M) while keeping the isoeugenol concentration at 4  105 M during

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titration. The data were collected at room temperature (RT) only after solution equilibration achieved. The equilibrium of the solution was ensured by sonication for 15 min in an ultrasonic bath (Ymnl, model SBe5200DTD, Nanjing Immanuel Instrument Equipment Co. Ltd.). 2.4. Phase solubility study Phase solubility study was carried out according to the method in previous literature [23,24]. 10 mL of aqueous solutions of HPbCD ranging in concentration from 0 to 0.01 M were prepared. Next, excess amount of isoeugenol was added to the HPbCD solutions. Then, the mixed solution was sonicated for 30 min at RT. The flasks containing aqueous solutions were sealed and shaken continuously at 37  C in a thermostat shaker for 48 h. After reaching equilibrium, the suspensions were filtered through 0.22 mm membrane filter and appropriately diluted to find out the isoeugenol concentration. The absorbance was measured at lmax ~263 nm by using a UV-1801 spectrophotometer. The apparent stability constant (Ks) was calculated from the phase solubility diagram according to the following equation.

Ks ¼ slope=½interceptð1  slopeÞ

(1)

From the Ks, the change in Gibbs free energy (DG) upon complexation was calculated as follows [23]:

DG ¼  RT ln Ks

(2)

2.5. Characterizations of isoeugenol/HPbCD-IC (PXRD, FT-IR, TGA and DSC) Powder X-ray diffraction (PXRD) spectra were recorded on a X\’Pert PRO MPD diffraction spectrometer (Panalytical, Netherlands). Diffractograms were run over a 2q angle range of 5e50 at a scanning speed of 5 /min. FT-IR spectra of isoeugenol, HPbCD and isoeugenol/HPbCD-IC were measured over a frequency range of 400e4000 cm1 at a resolution of 4 cm1 using a Shimadzu IR Prestige-21 spectrometer. Samples were prepared using KBr pellet method. A thermogravimetric analyzer (STA 449C, NETZSCH Instruments, German) was used to examine thermal stability of the samples. The experimental atmosphere was nitrogen and the samples were heated from 25 to 500  C at a heating rate of 10  C/min. DSC thermograms of HPbCD and isoeugenol/HPbCD-IC were measured with a STA 449C analyzer (NETZSCH Instruments, German). The samples were heated from 25  C to 500  C at a rate of 10  C/min. All determinations were performed under a nitrogen atmosphere. 2.6. Computational studies The computational studies of isoeugenol/HPbCD-IC were carried out using the GAUSSIAN 03 program software package. The initial structures of isoeugenol and HPbCD were drawn by the Spartan 08 software. The HPbCD structure was built on the molecular structure of native bCD by adding six hydroxypropyl groups to the wider rim of glucopyranose units [24] and all structures were individually optimized by the semiempirical quantum mechanical method with PM3 level basis set. Next, the structures of ICs were constructed by introducing isoeugenol into the HPbCD cavity through the wider rim. Two possible inclusion modes were considered for isoeugenol/ HPbCD-IC. In the first mode namely A, the alkyl chain was oriented near at narrow rim of the HPbCD cavity. While in the second mode namely B, the aromatic ring was oriented near at narrow rim of the HPbCD cavity. For each system, energy minimization was performed, followed by optimization without imposing any restraints.

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The complexation energy (DEcomplex) of isoeugenol/HPbCD-IC was calculated using the following equation:

mean ± standard deviation.

DEcomplex ¼ EIC  ðEHPbCD þ Eisoeugenol Þ

3. Results and discussion

(3)

where EIC, EHPbCD and Eisoeugenol represent the total energy of IC, the free HPbCD and the free isoeugenol, respectively. 2.7. Evaluation of water solubility A UVeVis spectrophotometer was used to determine the solubility of isoeugenol and the HPbCD-IC in water [25]. A certain amount of isoeugenol and the isoeugenol/HPbCD-IC powder containing same amount of isoeugenol were placed in 15 mL glass vials. In the vials, 10 mL of distilled water was added and stirred overnight at RT. Then the solutions were filtered through 0.22 mm membrane filter and the appropriately diluted filtrates were taken for the analysis. A Shimadzu spectrophotometer was used to examine the sample in the wavelength range of 200e350 nm. 2.8. Antioxidant activity assay The antioxidant activity of the US assisted solid isoeugenol/ HPbCD-IC was studied using the DPPH radical scavenging assay. Fresh solution of DPPH (1.0  104 M) was prepared in methanol. Exactly, 6.4 mg of isoeugenol/HPbCD-IC was immersed in 4 mL of DPPH solution. Samples were shaken vigorously and left in the dark for 30 min. The absorbance of the resulting solution was measured at 517 nm with a UVeVis spectrophotometer. Next, the concentration dependent antioxidant tests of isoeugenol/HPbCD-IC were also performed at various IC concentrations. The sample was dissolved in methanolic solutions of DPPH to prepare different concentrations of 0.02, 0.05, 0.10, 0.20, 0.40, 0.80, 1.20 and 1.60 mg/mL. Then, the absorbance changes in the solutions were measured at 517 nm by a UVeVis spectrophotometer at 25  C after 30 min of incubation in the dark. The absorbance of DPPH was defined as 100% and the antioxidant activity (%) was determined by the following equation:

Antioxidant activityð%Þ ¼ ðAC  AS Þ = AC  100

(4)

where AC and AS represent the absorbance of control DPPH solution and the absorbance of DPPH solution with IC, respectively. The experiments were performed in triplicate and the results were given as mean ± standard deviation. 2.9. Antibacterial activity The antibacterial activity of the US assisted solid isoeugenol/ HPbCD-IC was tested against S. aureus and E. coli by the plate colony counting method. The bacterial cells of S. aureus and E. coli were grown for 26 h on a shaker at 100 rpm and 37  C. The inoculums were resuspended to provide a final density of ~105e106 colony forming units (CFU)/mL in PBS. Prepared isoeugenol/HPbCD-IC was immersed into the PBS encompassing bacteria in the concentration of 20 mg/mL. As a control experiment, the PBS containing the same concentration of bacteria was employed. The media were shaken at 100 rpm and 37  C for 26 h and 100 mL of diluted culture was spread on a nutrient agar (NA) plate. Then, the population of bacteria was counted. The percentage of antibacterial activity (%) was calculated by the following equation [26]:

Antioxidant activityð%Þ ¼ ðPC  PE Þ = PC  100

(5)

where, PE and PC are the population of experimental bacteria and the population of control bacteria, respectively. The experiments were performed in triplicate and the results are given as

3.1. UVevis absorption properties of isoeugenol/HPbCD-IC In earlier, Kfoury et al. [19] studied and compared the interaction between aCD, bCD, HPbCD, RAMEB and CRYSMEB, with isoeugenol in solution by the indirect UVeVis competition (spectral displacement) method using methyl orange as a competitor. Here, the inclusion interactions of isoeugenol in the absence and presence of increasing HPbCD concentrations were directly investigated by the UVeVis absorption spectroscopy. Fig. 2A presents the UVeVis absorption spectra of isoeugenol (4  105 M) in aqueous solutions containing different concentrations of HPbCD. The inset in Fig. 2A shows the variation of absorbance of isoeugenol with increasing concentration of HPbCD. In aqueous solution, isoeugenol displays the characteristic absorption bands at ~262 and 292 nm, corresponding to the transitions from S0 to S1. Upon addition of HPbCD, the absorbance was markedly enhanced at 262 nm with a small bathochromic shift of ~5 nm (267 nm). In addition, the absorption band at 292 nm was disappeared. These changes in the absorption spectra were due to the hydrophobic interaction between isoeugenol and HPbCD, which implied the formation of the IC [27,28]. However, a clear isosbestic point was observed at 218 nm, indicating the formation of an IC with 1:1 M ratio. The stoichiometry and association constant (Ka) for isoeugenol/ HPbCD-IC have been determined from the Benesi-Hildebrand double-reciprocal plot [27e30]. In order to determine the stoichiometry of the IC, the changes observed in the absorption spectra of isoeugenol in the presence of HPbCD were analyzed by using the Benesi-Hildebrand equation, assuming the IC formation with 1:1 M ratio between the guest and host molecules.

1=DA ¼ 1=Dε þ 1=Ka ½isoeugend0 Dε½HPbCD0

(6)

where DA is the difference between the absorbance of isoeugenol in the presence and absence of HPbCD and Dε is the difference between the molar absorption coefficient of isoeugenol and the IC. [isoeugenol]0 and [HPbCD]0 are the initial concentration of isoeugenol and HPbCD, respectively. Here, the reciprocals of the peak shift for the wavelength at the absorption maximum 1/(A ‒ A0) were calculated and plotted against the reciprocal of the concentration of host 1/[HPbCD] to produce the Benesi-Hildebrand plot (Fig. 2B). This plot gave a straight line with a linear correlation coefficient (r2 ¼ 0.985). In addition, the plot of 1/(A ‒ A0) as a function of 1/[HPbCD]2 for the formation of 1:2 ICs of isoeugenol and HPbCD has displayed a downward curve (data not shown). These results confirmed that the stoichiometric ratio of the IC is 1:1. From the straight line, the Ka for isoeugenol/HPbCD-IC was determined to be 214 ± 5 M1. The free energy change (DG) of the formed IC was determined according to equation (2) as 3.23 kcal mol1. The negative DG value of the IC suggested that the binding process was a spontaneous process. 3.2. Fluorescence emission properties of isoeugenol/HPbCD-IC To further inspect and quantify the host/guest-IC formation of HPbCD and isoeugenol in solution, the fluorescence emission spectra of isoeugenol in aqueous solution and with varying concentrations of HPbCD have been recorded (Fig. 2C). As evident from Fig. 2C, the HPbCD cavity effect on the fluorescence emission spectra of isoeugenol is more enunciated. Isoeugenol reveals a broad emission band in aqueous solution when excited at ~262 nm. The addition of HPbCD to the aqueous solution of isoeugenol leads to a very large enhancement in fluorescence intensity of isoeugenol as compared to that in pure water. However, the fluorescence

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Fig. 2. (A) UVeVis absorption and (C) fluorescence emission properties of isoeugenol in the absence and presence of increasing HPbCD concentrations. (C, D) BeH double reciprocal plot of 1/(A ‒ A0) and 1/(F  F0) versus 1/[HPbCD].

spectrum of isoeugenol in the presence of HPbCD displays a considerable bathochromic shift of about ~6 nm (347 nm). Such fluorescence enhancement of isoeugenol has been attributed to the restriction of its intramolecular torsional relaxation in the excited state [29,30]. This indicates that isoeugenol is present in a different micro environment in the presence of HPbCD. From the fluorescence data, the stoichiometry of the IC of isoeugenol in HPbCD and their corresponding Ka was also determined using the Benesi-Hildebrand relation as given by equation (7).

1=ðF  F0 Þ ¼ 1





F ’  F0 þ 1 Ka F ’  F0 ½HPbCD

(7)

where [HPbCD] represents the initial concentration of HPbCD. F0 is the fluorescence intensity of isoeugenol. F is the fluorescence intensity with a certain concentration of HPbCD and F’ is the limiting

intensity at higher HPbCD concentration. A good-linear relationship was obtained when 1/(F  F0) was plotted against 1/[HPbCD] (Fig. 2D), indicating that the stoichiometry of the isoeugenol/ HPbCD-IC was 1:1, which agrees well with the results obtained from UVeVis absorption studies. The estimated Ka was 402 ± 8 M1 and the DG value was 3.61 kcal mol1. 3.3. Phase solubility of the US assisted isoeugenol/HPbCD-IC Phase solubility study was performed to evaluate the complexation effect of HPbCD with water insoluble antioxidant isoeugenol and the stability constant (Ks) and also to determine the change of free energy (DG) involved in the IC formation. The phase solubility diagram was obtained by plotting the HPbCD concentration against the total solubility of isoeugenol (Fig. 3A). The phase

Fig. 3. (A) Phase solubility diagram of isoeugenol/HPbCD-IC at 37  C, (B) Powder XRD patterns of HPbCD and isoeugenol/HPbCD-IC, and (C) FT-IR spectra of HPbCD, isoeugenol and isoeugenol/HPbCD-IC.

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solubility diagram of isoeugenol with HPbCD at 37  C showed that the aqueous solubility of isoeugenol increased linearly as a function of HPbCD concentration, producing an AL-type of phase solubility diagram. Slope value for the solubility curve was lower than unity and correlation coefficient squared value (r2) was 0.990, confirming the formation of water soluble IC of isoeugenol and HPbCD with 1:1 M ratio. Kfoury and co-workers [19] studied the solubility and the apparent stability constant of isoeugenol with the two parent CDs and three bCD derivatives at 25  C. The solubility trend and the Ks value reported for the IC with HPbCD were very close to those found here. The calculated Ks value of the isoeugenol/HPbCD-IC was 410 ± 4 M1 and the DG value was 3.62 kcal mol1. These results confirmed the presence of strong interactions between the guest isoeugenol and the HPbCD cavity. Several authors previously showed that the aqueous solubility of other natural phenolic compounds such as trans-ferulic acid [31], caffeic acid [32] and quercetin [33] was significantly increased with the formation of HPbCD-ICs.

most of the characteristic vibrational bands observed for isoeugenol are undetectable in the isoeugenol/HPbCD-IC spectra (Fig. 3C). Although, the peaks corresponding to aromatic carbon‒ carbon double bond stretching of isoeugenol are found in the spectra of isoeugenol/HPbCD-IC at 1597 and 1514 cm1. The appearance of these peaks confirmed the formation of IC between the guest and host molecules. Furthermore, the increased intensity for the broad peak around 3396 cm1, indicating an increase in the number of hydroxyl groups, probably due to the IC formation of isoeugenol into the HPbCD cavity through the alkyl chain. It should be mentioned here that HPbCD was formed a more stable IC (1:1) with isoeugenol in solid because of better size-fit of the guest and the host. Analogous phenomenon was previously reported by Nuchuchua et al. [35] for eugenol within aCD, bCD and HPbCD cavities during IC formation. They found that the intensities of the peaks corresponding to aromatic carbon‒carbon double bond stretching of eugenol in HPbCD complex were much lesser than in other CD complexes. The order of decreasing the peak intensities

3.4. Structural characterization of the US assisted isoeugenol/ HPbCD-IC According to spectroscopic and phase solubility investigations, isoeugenol apparently forms an IC with HPbCD at a molar ratio of 1:1 in aqueous solution. A study by our research group reported that cuminaldehyde, a major constituent of cumin EO, forms an IC with HPbCD in solid state as a result of ultrasound treatment [23]. Generally, when the guest molecules are included in the CDs, their crystalline structures are altered and more number of amorphous structures are produced [34]. Therefore, PXRD was performed to investigate the structural features of isoeugenol/HPbCD-IC prepared by the ultrasound method. PXRD can give keen information about the IC formation between the host and guest molecules. It is also known that the diffraction pattern of the physical mixture would be a simple superposition of the patterns of the two components. The PXRD patterns for pure HPbCD and isoeugenol/ HPbCD-IC are shown in Fig. 3B. PXRD of isoeugenol was not performed because the physical state of isoeugenol was liquid under experimental temperature. The diffraction pattern of pure HPbCD showed a broad peak at 2q ~18.27, consistent with its amorphous character. In contrast, a unique diffraction pattern, which was different from that of HPbCD, was obtained for the isoeugenol/ HPbCD-IC. The appearance of a new diffraction peak at 2q ~32.23 indicates the formation of the IC between isoeugenol and HPbCD. A similar result has been observed by another researcher for the IC of benzyl isothiocyanate with HPbCD in the ultrasound synthetic process [15]. Furthermore, the less intensities and peak enlargement in the PXRD of the isoeugenol/HPbCD-IC revealed that the IC possesses more amorphous structures than pure HPbCD. The proposed structure of the formed IC is illustrated in Fig. 1B, where the alkyl chain is included inside the HPbCD cavity. The IC formation was further confirmed by observing the corresponding vibrational bands in the FT-IR spectra. The FT-IR spectra of isoeugenol, HPbCD and isoeugenol/HPbCD-IC are illustrated in Fig. 3C. The peak at 3402 cm1 in HPbCD corresponds to the hydrogen-bonded hydroxyl group while the peak at 1637 cm1 corresponds to the crystallized water band in the structure of the cavity. In addition, three sharp peaks at 1155, 1082 and 1038 cm1 are also found from HPbCD, which can be due to carbon‒oxygen stretching of the host molecule. In pure isoeugenol, the spectral response at 3501 cm1 represents the characteristic vibration of hydroxyl group and the two peaks at 1596 and 1509 cm1 are indicative of the carbon‒carbon double bond stretching vibrations of the aromatic ring. The absorption band between 750 and 1250 cm1 can also be assigned to carbon‒carbon double bond stretching vibrations arising from the isoeugenol. On the contrary,

Fig. 4. (A) TGA thermograms and (B) DSC curves of isoeugenol, HPbCD and isoeugenol/ HPbCD-IC. (C) Water solubility analysis of pure isoeugenol and solid isoeugenol/ HPbCD-IC having the same amount of isoeugenol.

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Fig. 5. (A) Coordinate systems to describe inclusion process of isoeugenol with HPbCD. The PM3 level of theory optimized structures for the most stable isoeugenol/HPbCD-IC (1:1) in both modes A (B) and B (C).

Table 1 The energetic features, HOMO and LUMO energy results of isoeugenol, HPbCD and the isoeugenol/HPbCD-ICs calculated by PM3 level of theory. Properties

Isoeugenol

HPbCD

Isoeugenol/HPbCD-IC Mode ‘A’

Mode ‘B’

E (kcal mol1) DE (kcal mol1) EHOMO (eV) ELUMO (eV) EHOMO e ELUMO (eV)

53.08

1713.28

8.63 0.16 8.47

10.65 1.38 12.03

1780.91 14.55 8.67 0.24 8.43

1778.38 12.02 8.79 0.32 8.48

was aCD < bCD < HPbCD. The above order suggested that the inclusion capability of CDs with the guest molecules. In other words, the HPbCD has stronger entrapping capacity than other CD cavities. 3.5. Thermal analysis of the US assisted isoeugenol/HPbCD-IC The results of PXRD and FT-IR spectroscopy suggested that isoeugenol and HPbCD formed an IC in solid state assisted by ultrasound. Thus, the thermal stability of isoeugenol/HPbCD-IC was examined using TGA and DSC. These investigations further supported the interaction between the guest and host molecules. TGA thermograms of isoeugenol, HPbCD and isoeugenol/HPbCD-IC are shown in Fig. 4A, as comparatively. The TGA thermogram of pristine HPbCD represents characteristic weight losses at two stages, as reported in the literature. The free isoeugenol started significant changes in the weight at about 140  C and maintained up to 290  C. Interestingly the isoeugenol/HPbCD-IC exhibited three stages of weight loss. The first stage weight loss below 100  C can be

attributed to the evaporation of water molecules that exist in the HPbCD cavity. The second stage weight loss around 110  Ce270  C can be related to the evaporation of encapsulated isoeugenol. At last, the third stage weight loss above 300  C can be attributed to the main thermal degradation of the HPbCD. Analysis of TGA thermograms revealed that the thermal onset for the decomposition of isoeugenol is shifted to a higher temperature [36,37]. This result indicates that the isoeugenol is encapsulated within the HPbCD cavity. Furthermore, the shift of decomposition of the guest component proved that the HPbCD cavity served as a thermal barrier and thus greatly increased the thermal stability of isoeugenol in the HPbCD-IC. In addition to TGA, DSC was applied in order to further reveal the thermal stability of ultrasound processed HPbCD-IC. Fig. 4B depicts the DSC curves of HPbCD and the isoeugenol/HPbCD-IC. The DSC thermogram of HPbCD displayed a broad endothermic peak around 89.9  C, which is associated with loss of water molecules [38]. Comparing with HPbCD curves, a distinctive endothermic peak is observed for the isoeugenol/HPbCD-IC. The water loss related peak is significantly shifted to a lower temperature (84.5  C). And the DSC peaks corresponding to the degradation of compounds at higher temperature are disappeared or significantly altered. Wang et al. [31] reported a similar observation of host molecule shifting its characteristic DSC peaks for the complexation of another phenolic compound, ferulic acid with HPbCD. These results indicated the formation of IC of isoeugenol and HPbCD with higher thermal stability and the resultant modification in thermal properties. 3.6. Water solubility of the US assisted isoeugenol/HPbCD-IC The absorbances of isoeugenol at ~263 nm and of the

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isoeugenol/HPbCD-IC at ~266 nm were measured to evaluate the water solubility [25]. Upon the IC formation, the solubility enhancement of insoluble antioxidant, isoeugenol, will lead to an increase of absorbance in the water medium. As shown in Fig. 4C, a significant absorbance enhancement was observed for the isoeugenol/HPbCD-IC containing the same amount of isoeugenol, while that of isoeugenol was lower. It proved that the formation of isoeugenol/HPbCD-IC by the ultrasonic synthetic method was a good method for producing the IC with privileged water solubility. This phenomenon also indicates that HPbCD is an efficient solubilizer of the hydrophobic EO compounds like isoeugenol. 3.7. Computational analysis of isoeugenol/HPbCD-IC Semiempirical quantum mechanical calculations were carried out for the isoeugenol/HPbCD-IC in the gas phase aiming to represent three-dimensional structures and intermolecular interactions [39,40]. As illustrated in Fig. 5A, isoeugenol exhibits better geometrical dimensions that are appropriate for forming ICs with the HPbCD cavity. The HPbCD cavity is accessible for isoeugenol through the inclusion of either its aromatic moiety or alkyl chain through either the wide or narrow rim of the HPbCD cavity. Although, it is important to highlight here that the inclusion processes resulted in marked changes for the wider rim inclusion compared with the narrow rim inclusion. For the IC of isoeugenol inside the HPbCD cavity, there are two possibilities: one is the alkyl chain oriented near at the narrow rim of the cavity (mode A) and other is the aromatic ring oriented near at the narrow rim of the cavity (mode B). The possible inclusion modes obtained by molecular modeling for isoeugenol complexed with HPbCD are displayed in Fig. 5. The corresponding energetic values obtained by PM3 method are given in Table 1. It can be understood from Fig. 5 that isoeugenol is wellfitted in the hydrophobic cavity of HPbCD. The calculated energies for isoeugenol/HPbCD-IC are negative (mode A ¼ 1778.38 kcal mol1 and mode B ¼ 1780.91 kcal mol1), which are lower than that of the isolated guest (53.08 kcal mol1) and host (1713.28 kcal mol1) molecules. This indicates that the IC formation process is thermodynamically favourable in both inclusion modes. Based on DE, the difference in the formation energy of IC (EIC) and the sum of the formation energies of the free guest (EIoseugenol) and host (EHPbCD) molecules, the relative stabilities of the two inclusion modes are compared. The DE values for mode A and mode B are 14.55 and 12.02 kcal mol1, respectively, which demonstrate that the isoeugenol forms stable ICs with the HPbCD. Further, mode A is more favourable than mode B by an energy difference of 2.53 kcal mol1. The PM3 calculation results pointed out the inclusion mode A as the preferred one, which is in agreement with the experimental results. The ICs are often stabilized by intermolecular interactions, such as hydrogen bondings. The green dotted lines in Fig. 5 show the intermolecular interactions between the HPbCD cavity and isoeugenol in the favourable configuration of each inclusion mode. In mode A, six intermolecular hydrogen bondings are detected between the isoeugenol and the inner cavity of HPbCD with a bond length range of 2.61e2.92 Å. Here the hydrogen bondings are defined as CeO/H, OeH/O and CeH/O. While in mode B, three intermolecular hydrogen bondings are formed. These findings indicated that intermolecular hydrogen bondings play a crucial role in the stability of ICs. This also explains why the DE for mode A is 2.53 kcal mol1 lower than that of mode B. To further quantify the stabilities of ICs, the EHOMO‒ELUMO energy gap, an important stability index, was calculated using the PM3 method (Table 1). The EHOMO‒ELUMO gap is slightly higher for mode A, which advocates that this complex is more stable than mode B. These results are in good agreement with the calculated IC

formation energies. Finally, the findings of computational studies are consistent with the results of spectroscopic studies and FT-IR analysis. 3.8. Antioxidant activity of the US assisted isoeugenol/HPbCD-IC It is well known that EOs extracted from natural plants exhibit a wide variety of biological properties including antioxidant, antimicrobial, anti-inflammatory, antitumor and antidiabetic properties. This is because of the presence of volatile compounds such as terpenes, terpenoids, phenolic and aliphatic compounds. Isoeugenol belongs to the class of natural phenolic compounds, encompassing a dual antioxidant/prooxidant properties. The result of Zhang et al. [7] indicated that isoeugenol exhibited higher antioxidant activity and greater protective effect against DNA damage than its isomer eugenol. The antioxidant activity of isoeugenol mainly came from the methoxyphenolic structure in which carbonecarbon double bond closer to the benzene ring. Therefore, in this study, the antioxidant activity of the US assisted isoeugenol/HPbCD-IC solid was determined using DPPH radical scavenging assay. Fig. 6A shows the photographs of the DPPH solution before and after the reaction with isoeugenol and isoeugenol/HPbCD-IC. Due to the poor water solubility of isoeugenol, the DPPH with its aqueous solution exhibits a quite low amount of antioxidant activity. Moreover, the color of the

Fig. 6. (A) Resulting DPPH solution photographs of solid isoeugenol/HPbCD-IC, (B) Sample concentration dependent antioxidant test graphs and (C) UVeVis absorption plots of antioxidant tests taking for the highest concentrations for solid isoeugenol/ HPbCD-IC (1.60 mg/mL).

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solution was still violet as shown in Fig. 6A. In contrast, the color of DPPH solution turns from purple to yellow at the end of the reaction with isoeugenol/HPbCD-IC and consequently the salient absorption band at 517 nm is disappeared (Fig. 6C). The antioxidant activity of isoeugenol and isoeugenol/HPbCD-IC was calculated according to equation (4) as of 86 ± 0.4% and 100 ± 0.6%, respectively. Based on the above results, it can be concluded that the antioxidant activity of isoeugenol was increased after IC formation with the HPbCD. Further, it is previously reported that the antioxidant properties of CD-ICs are closely related to their mode of binding. In this case, if the IC in mode B is formed, the antioxidant properties of isoeugenol might be quashed. But the results in the present analyses indicate that the antioxidant activity of isoeugenol is enhanced in HPbCD-IC. The similar results were also found for the ICs of trans-polydatin with natural bCD and its derivatives [41]. Therefore, we concluded that the alkyl chain of isoeugenol is deeply included and the phenyl ring with hydroxyl group of isoeugenol is located near to the wider rim of the HPbCD cavity. Here, the concentration dependent antioxidant tests of isoeugenol/HPbCD-IC were also performed at IC concentration range of 0.02e1.6 mg/mL. The percentage of antioxidant activity graphs of isoeugenol/HPbCD-IC depending on the concentration are displayed in Fig. 6B. The DPPH free radical scavenging rate is increased with the increasing concentrations of the IC. The free radical scavenging rate of isoeugenol/HPbCD-IC is appeared downward with increasing complex concentration. This result indicates a dose-effect relationship between the scavenging capacity of the IC and its concentrations [42]. The ability to scavenge free DPPH radicals reached 100% at the concentration of 1.6 mg/mL. The fifty percentage inhibition of isoeugenol/HPbCD-IC was 0.054 mg/mL.

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3.9. Antibacterial activity of the US assisted isoeugenol/HPbCD-IC The antibacterial activity of isoeugenol/HPbCD-IC (1:1) was tested against gram-positive S. aureus and gram-negative E. coli bacteria via the colony counting method (Fig. 7). Isoeugenol/ HPbCD-IC exhibited better antibacterial activity against both bacteria, owing to the higher stability and release of antibacterial agent at targeted microorganisms. The population of gram-positive S. aureus and gram-negative E. coli bacteria in the experimental group is decreased by 96 ± 0.2% and 97 ± 0.5% respectively, compared to the control group. These results may have occurred due to reversible interactions of isoeugenol in HPbCD-IC with bacterial membranes through a non-disruptive detergent like mechanism. This action destabilized the cytoplasmic membrane and the cell wall of pathogens to become permeable [43,44]. 4. Conclusions In summary, a natural antioxidant isoeugenol was successfully complexed with HPbCD in both solution and solid by the ultrasound method. The IC formation was characterized via UVeVis absorption spectroscopy, fluorescence spectroscopy, phase solubility, PXRD, FT-IR, TGA and DSC techniques. The solid state studies showed that the HPbCD-IC of isoeugenol had different physicochemical properties from uncomplexed isoeugenol. FT-IR and molecular modeling studies indicated that the alkyl chain of isoeugenol oriented near at narrow rim of the HPbCD cavity. Through determinations of phase solubility and water solubility test, significant enhancement of isoeugenol water solubility was confirmed after IC formation with the HPbCD. The IC formation also

Fig. 7. Typical photographs of colonies of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) before and after treatment with solid isoeugenol/HPbCD-IC.

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elevated the antioxidant and antibacterial activities of isoeugenol compared with those of free isoeugenol. In brief, the isoeugenol complexed with HPbCD could overcome the limitations of free EO component application in the food and pharmaceutical industries by increasing aqueous solubility, reducing volatility, enhancing radical scavenging activity and antimicrobial activity. Acknowledgements Financial support was provided by Hunan Science and Technology Major Project (Grant no. 2016NK1001-3), National Natural Science Foundation of China (Grant no. 31470594), Natural Science Foundation of Jiangsu Province (Grant no. BK20170070), Jiangsu Province Research Fund (Grant no. NY-013 and JNHB-131) and Jiangsu University Research Fund (Grant no. 11JDG050). References ~ oz, J. Ya n ~ ez-Ferna ndez, V. Fíla, Phenolic compounds recovered [1] R. 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