Cyclodextrin-Baicalein Inclusion Complex - MDPI

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May 27, 2016 - Center for Biotechnology Research in UBITA (CBRU), Institute for ..... Naidu, N.B.; Chowdary, K.; Murthy, K.; Satyanarayana, V.; Hayman, A.; ...
molecules Article

Characterization and Enhanced Antioxidant Activity of the Cysteinyl β-Cyclodextrin-Baicalein Inclusion Complex Hwanhee Kim 1 , Hu Yiluo 1 , Seyeon Park 2 , Jae Yung Lee 3 , Eunae Cho 4, * and Seunho Jung 1,4, * 1

2 3 4

*

Department of Bioscience and Biotechnology, Microbial Carbohydrate Resource Bank (MCRB), Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 05029, Korea; [email protected] (H.K.); [email protected] (H.Y.) Department of Applied Chemistry, Dongduk Women’s University, Seoul 136-714, Korea; [email protected] Department of Biological Science, Mokpo National University, Jeonnam 534-729, Korea; [email protected] Center for Biotechnology Research in UBITA (CBRU), Institute for Ubiquitous Information Technology and Applications (UBITA), Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 05029, Korea Correspondence: [email protected] (E.C.); [email protected] (S.J.); Tel.: +82-2-450-3520 (S.J. & E.C.)

Academic Editor: Bernard Martel Received: 4 May 2016; Accepted: 25 May 2016; Published: 27 May 2016

Abstract: Baicalein is a type of flavonoid isolated from the roots of a medicinal plant, Scutellaria baicalensis. Although it has attracted considerable attention due to its antiviral, anti-tumor, and anti-inflammatory activities, its limited aqueous solubility inhibits the clinical application of this flavonoid. The present study aimed to prepare and characterize a host-guest complex in an effort to improve the solubility and antioxidant activity of baicalein. The host molecule is a macrocyclic β-cyclodextrin (β-CD) functionalized with cysteine for a synergetic effect. The structure of the synthesized cysteinyl β-CD was analyzed using nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. The inclusion complex with baicalein was studied by UV-vis, NMR spectroscopy, scanning electron microscopy, and X-ray powder diffractometry. The formed cysteinyl β-CD/baicalein inclusion complex efficiently improved the solubility and antioxidant ability of baicalein. Therefore, we suggest that the present cysteinyl β-CD is a potential host molecule for inclusion complexation and for bioavailability augmentation. Keywords: cysteinyl β-cyclodextrin; baicalein; inclusion complex; antioxidant activity

1. Introduction In an inclusion complex system, macrocyclic host molecules are of great importance, as the cyclized and constrained conformation can provide the advantages of molecular selectivity and recognition [1]. The compound β-cyclodextrin (β-CD) is α-1,4-linked macrocyclic oligosaccharide containing seven glucose units. It can encapsulate hydrophobic compounds in its internal cavity [2]. Further, host-guest complexation can change the physico-chemical and biological characteristics of guest compounds. Due to its complex-forming ability, various applications are found in the pharmaceutical, food, and cosmetic industries [3]. To expand the applicability of β-CD, chemical derivatives such as hydroxypropyl and methyl β-CDs have been synthesized, and the inclusion complexing ability has been improved [4,5]. In addition, lipid derivatives of β-CD have been investigated as promising drug-carrier systems based on their self-assembled architecture [6,7]. However, few studies of amino acid or peptide derivatives have been reported to date [8,9]. Among them, cysteine modification is expected to provide Molecules 2016, 21, 703; doi:10.3390/molecules21060703

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been reported to date [8,9]. Among them, cysteine modification is expected to provide antioxidant effects through inactivation of hydroxyl radicals or through thethe support glutathione antioxidant effectsthe through the inactivation of hydroxyl radicals or through supportof of glutathione synthesis [10,11]. synthesis [10,11]. Baicalein (5,6,7-trihydroxyflavone) (5,6,7-trihydroxyflavone)isisanan active phytochemical isolated Baikal skullcap Baicalein active phytochemical isolated fromfrom Baikal skullcap root root extract. It is used against inflammatory diseases such as hepatitis, nephritis, bronchitis, asthma, extract. It is used against inflammatory diseases such as hepatitis, nephritis, bronchitis, asthma, and and atopic dermatitis In addition, antiviral [13], antibacterial [14],and andanti-cancer anti-canceractivities activities have have atopic dermatitis [12].[12]. In addition, antiviral [13], antibacterial [14], been found found [15]. [15]. The The beneficial beneficial effects effects on on human human health health are are also also closely closely related related to to the the antioxidant antioxidant been properties of baicalein because it has good hydrogen and electron donors, and the radical intermediates properties of baicalein because it has good hydrogen and electron donors, and the radical intermediates are stabilized stabilized by by possible possible resonance resonance forms forms [16]. [16]. Although Although interest interest in in baicalein baicalein with with regard regard to to its its are ˝ chemopreventive properties has arisen, its poor aqueous solubility (90 µg/mL, 25 °C) inhibits its chemopreventive properties has arisen, its poor aqueous solubility (90 µg/mL, 25 C) inhibits bioavailability. Thus, insolubility as a practical issue remains to be solved before additional clinical its bioavailability. Thus, insolubility as a practical issue remains to be solved before additional applications. clinical applications. Herein, we we aim aim to to synthesize synthesize cysteine-functionalized cysteine-functionalized β-CDs β-CDs for for inclusion inclusion complexation complexation with with Herein, baicalein. Inclusion with nuclear nuclear magnetic magnetic resonance resonance (NMR) (NMR) spectroscopy, spectroscopy, baicalein. Inclusion complexes complexes were were analyzed analyzed with scanning electron microscopy (SEM), and X-ray powder diffractometry (XRPD). The modified scanning electron microscopy (SEM), and X-ray powder diffractometry (XRPD). The modified properties of the complexed baicalein were also evaluated in terms of their aqueous solubility and properties of the complexed baicalein were also evaluated in terms of their aqueous solubility antioxidant activities. The The desired synergetic effect bybythe the and antioxidant activities. desired synergetic effect thebaicalein baicaleinguest guest compound compound and and the cysteine-conjugated host molecule was observed. cysteine-conjugated host molecule was observed. 2. Results 2. Results and and Discussion Discussion 2.1. 2.1. Structural Structural Analysis Analysis of of Mono-6-Cysteinyl-β-CD Mono-6-Cysteinyl-β-CD (Cysteinyl β-CD) β-CD) Cysteinyl Cysteinyl β-CD β-CD was prepared prepared as as described described in in the the Materials Materials and and Methods Methods (Scheme (Scheme 1). 1). After After purification, purification, the the structure structure of of the theresultant resultantproduct productwas wasanalyzed analyzedusing usingmatrix-assisted matrix-assistedlaser laserdesorption/ desorption/ ionization-time ionization-time of of flight flight (MALDI-TOF) (MALDI-TOF) mass mass spectrometry spectrometry and and NMR NMR spectroscopy. spectroscopy. Pseudo-molecular Pseudo-molecular + + + ion and[mono-6-cysteinyl [mono-6-cysteinylβ-CD β-CD−´HH+ +2Na] 2Na]) +were ) were observed ion peaks peaks ([mono-6-cysteinyl ([mono-6-cysteinyl β-CD β-CD + Na] and observed at at m/z = 1260.42 and 1282.40 (Figure The chemicalstructure structureisisshown shownin in Figure Figure 2a. Due to cysteinyl m/z = 1260.42 and 1282.40 (Figure 1).1). The chemical cysteinyl modification, between δ 5.00 and δδ 5.10. modification, the anomeric protons protons of β-CD were also separated separated between 5.10. In the the range range of of δδ 2.90 2.90 to to δδ 3.30, 3.30, upfield-shifted upfield-shifted methylene methylene protons protons on on the the C6 C6 position position of of glucose glucose were were detected detected with withthe the methylene methylenesignals signalsof of the the side side chain chain (Figure (Figure 2b). 2b). In In its its heteronuclear heteronuclear single single quantum quantum coherence coherence (HSQC) spectrum (Figure 2c), compared to C6 carbons (δ 62.47) with a free OH group, the substituted (HSQC) spectrum (Figure 2c), compared to C6 carbons (δ 62.47) with a free OH group, the substituted C6 C6′1 carbons carbons shifted significantly upfield to δ 36.48, 36.48, becoming becoming correlated correlated with with the the methylene methylene germinal germinal H6 Chiral carbons carbons of cysteine cysteine also appear at δ 56.38 with H6′1 signals. signals. Chiral with the the cross-peak cross-peak of of the the attached attached hydrogens Methylenecarbon carbonand andhydrogen hydrogenininthe thecysteine cysteineside sidechain chain were detected hydrogens (δ 3.73–3.95). Methylene were detected at at δ 35.71/3.17 and 35.71/2.98, respectively.The Theresults resultsof of these these structural structural analyses indicate that δ 35.71/3.17 and δ δ35.71/2.98, respectively. that cysteinyl cysteinyl β-CD β-CD was was successfully successfully synthesized. synthesized.

Figure 1. 1. MALDI MALDI TOF TOF mass mass spectrum spectrum of of cysteinyl cysteinylβ-CD. β-CD. Figure

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Figure 2. (a) Chemical structure of cysteinyl β-CD; (b) 11H-NMR spectra of β-CD (top) and cysteinyl Figure Figure 2. 2. (a) (a) Chemical Chemical structure structure of of cysteinyl cysteinyl β-CD; β-CD; (b) (b) 1H-NMR H-NMR spectra spectra of of β-CD β-CD (top) (top) and and cysteinyl cysteinyl β-CD (bottom); (c) HSQC spectrum of cysteinyl β-CD. Solvent: D2O. β-CD (bottom); (c) HSQC spectrum of cysteinyl β-CD. Solvent: D O. β-CD (bottom); (c) HSQC spectrum of cysteinyl β-CD. Solvent: D22O.

Scheme 1. Schematic illustration of synthesis of cysteinyl β-CD. Scheme 1. Schematic illustration of synthesis of cysteinyl β-CD. Scheme 1. Schematic illustration of synthesis of cysteinyl β-CD.

2.2. Phase Solubility Diagram 2.2. Phase Solubility Diagram 2.2. Phase Solubility Diagram The substituted cysteine may provide additional effects related to electrostatic interaction and The substituted cysteine may provide additional effects related to electrostatic interaction and hydrogen bonding sites as well as provide easily oxidized thiol groups. Together with the attached β-CD The substituted cysteine may additional effects related to electrostatic interaction and hydrogen bonding sites as well as easily oxidized thiol groups. Together with the attached β-CD cavity, cooperative recognition will leadoxidized to morethiol sophisticated complexation guest β-CD molecules. hydrogen bonding sites as well as easily groups. Together with thefor attached cavity, cavity, cooperative recognition will lead to more sophisticated complexation for guest molecules. In relation torecognition this, the inclusion capacity cysteinyl β-CD for baicalein investigated. phase cooperative will lead to moreofsophisticated complexation forwas guest molecules.The In relation In relation to this, the inclusion capacity of cysteinyl β-CD for baicalein was investigated. The phase solubility diagram is shown with for thatbaicalein of unmodified β-CD (Figure According to to this, the inclusion capacityinofcomparison cysteinyl β-CD was investigated. The3). phase solubility solubility diagram is shown in comparison with that of unmodified β-CD (Figure 3). According to Higuchi Connors [17], an AN-type for the cysteinyl is diagramand is shown in comparison with phase that ofdiagram unmodified β-CD (Figureβ-CD/baicalein 3). Accordingcomplex to Higuchi Higuchi and Connors [17], an AN-type phase diagram for the cysteinyl β-CD/baicalein complex is obtained. Although β-CD-induced changes in the β-CD/baicalein dielectric constant of theis aqueous and Connors [17], ancysteinyl AN -type phase diagram for the cysteinyl complex obtained. obtained. Although cysteinyl β-CD-induced changes in the dielectric constant of the aqueous complexation solventβ-CD-induced or self-association of the willconstant give rise negative deviation from Although cysteinyl changes in complexes the dielectric ofto the aqueous complexation complexation solvent or self-association of the complexes will give rise to negative deviation from linearity, curve indicates a soluble complex between cysteinyl β-CD and baicalein. solvent orthe self-association ofthe theformation complexesofwill give rise to negative deviation from linearity, the curve linearity, the curve indicates the formation of a soluble complex between cysteinyl β-CD and baicalein. The solubilizing efficiency is determined as the ratio between theand solubility of The the drug in the indicates the formation of a(SE) soluble complex between cysteinyl β-CD baicalein. solubilizing The solubilizing efficiency (SE) is determined as the ratio between the solubility of the drug in the presence a certain host concentration the drug alone in water. thethe solubilizing efficiency efficiencyof(SE) is determined as the ratioand between the solubility of the Here, drug in presence of a certain presence of a certain host concentration and the drug alone in water. Here, the solubilizing efficiency (SE of cysteinyl β-CD was much than that the of the original β-CD (SE 2.35) in the host9.21) concentration and the drug alone better in water. Here, solubilizing efficiency (SE 9.21) ofpresence cysteinyl (SE 9.21) of cysteinyl β-CD was much better than that of the original β-CD (SE 2.35) in the presence of 10 mmol β-CDs.better Afterthan attaching cysteine on β-CD, the(SE intrinsic solubility β-CDβ-CDs. could β-CD was much that of the original β-CD 2.35) inaqueous the presence of 10ofmmol of 10 mmol β-CDs. After attaching cysteine on β-CD, the intrinsic aqueous solubility of β-CD could be improved by cysteine about 100 (cysteinyl β-CD:aqueous >180 g/100 mL, 25of°C vs. β-CD: g/100 mL,by 25about °C). After attaching onfold β-CD, the intrinsic solubility β-CD could1.82 be improved be improved by about 100 fold (cysteinyl β-CD: >180 g/100 mL, 25 °C vs. β-CD:˝1.82 g/100 mL, 25 °C). Furthermore, the complexation with baicalein be more although also hasthe a 100 fold (cysteinyl β-CD: >180 g/100 mL, 25 ˝ Cmay vs. β-CD: 1.82effective, g/100 mL, 25 C). β-CD Furthermore, Furthermore, the complexation with baicalein may be more effective, although β-CD also has a capturable cavity. complexation with baicalein may be more effective, although β-CD also has a capturable cavity. capturable cavity.

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Figure 3. Phase solubility diagrams of baicalein with cysteinyl β-CD and β-CD in aqueous solution Figure 3. Phase solubility diagrams β-CDand andβ-CD β-CD aqueous solution Figure 3. Phase solubility diagramsofofbaicalein baicalein with with cysteinyl cysteinyl β-CD in in aqueous solution at 25 ˝ C. 25 °C. at 25at°C.

2.3. Studies 2.3. NMR Studies 2.3.NMR NMR Studies To non-covalent interactions level,NMR NMR spectroscopic analysis To examine non-covalent interactions at at the the molecular molecular level, spectroscopic analysis is isis Toexamine examine non-covalent interactions at the molecular level, NMR spectroscopic analysis 11H-NMR performed. Figure 4 showsthe the spectra, where the proton signals of baicalein are shifted in the performed. Figure 4 shows H-NMR spectra, where the proton signals of baicalein are shifted performed. Figure 4 shows the 1H-NMR spectra, where the proton signals of baicalein are shifted in the or downfield direction by the inclusion complex with cysteinyl β-CD. A downfield displacement in theupfield upfield or downfield by the complex inclusion complex with cysteinyl β-CD. A downfield upfield or downfield directiondirection by the inclusion with cysteinyl β-CD. A downfield displacement indicates an environment of electronegative atoms [18], and an upfield shift displacement is attributed displacement indicates an environment of electronegative atoms [18], and an upfield shift displacement indicates an environment of electronegative atoms [18], and an upfield shift displacement is attributed to the variation in the local polarity after insertion into a β-CD cavity [19]. The protons (2–6) in the istoattributed to the in the local polarity after insertion into a β-CD cavity [19]. (2–6) The protons the variation in variation the local polarity insertion into a β-CD the cavity [19]. The protons in the benzyl ring are shifted toward the after upfield direction, indicating incorporation in the cavity. For (2–6) in the benzyl ring are shifted toward the upfield direction, indicating the incorporation in the benzyl are8′ shifted the upfield direction, indicating the incorporation in the cavity. the ring 3′ and protons toward in the chromene moiety, a pronounced downfield-shifted and split pattern is For 1 and 81 protons in the chromene moiety, a pronounced downfield-shifted and split cavity. For the 3 the 3′ and 8′ protons in the chromene a pronounced and split pattern is observed in the presence of cysteinylmoiety, β-CD. This result placesdownfield-shifted stress on the chromene positioning pattern is observed in on thethe cysteinyl β-CD. This result places on the chromene observed inthe thecysteine presence ofpresence cysteinyl β-CD. Thistogether result places stress oninsertion. thestress chromene positioning toward primary of part of β-CD with the benzyl positioning toward the cysteine on the primary part of β-CD together with the benzyl insertion. toward the cysteine on the primary part of β-CD together with the benzyl insertion.

Figure 4. (a) 1H-NMR spectrum of baicalein (Solvent: D2O:MeOD, 70:30, v/v); (b) 1H-NMR spectrum of cysteinyl β-CD/baicalein complex (Solvent: D2O:MeOD, 70:30, v/v); (c) Enlarged spectrum of the aromatic region (top: baicalein, bottom: cysteinyl β-CD/baicalein complex). The inset shows the chemical baicalein.of baicalein (Solvent: D2O:MeOD, 70:30, v/v); (b) 1H-NMR Figure 4. (a)11structure H-NMR of spectrum spectrum 1

Figure 4. (a) H-NMR spectrum of baicalein (Solvent: D2 O:MeOD, 70:30, v/v); (b) H-NMR spectrum of cysteinyl β-CD/baicalein complex (Solvent: D2O:MeOD, 70:30, v/v); (c) Enlarged spectrum of the of cysteinyl β-CD/baicalein complex (Solvent: D2 O:MeOD, 70:30, v/v); (c) Enlarged spectrum of the aromatic region (top: baicalein, bottom: cysteinyl β-CD/baicalein complex). The inset shows the aromatic region (top: baicalein, bottom: cysteinyl β-CD/baicalein complex). The inset shows the chemical structure of baicalein. chemical structure of baicalein.

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In addition,two-dimensional two-dimensionalnuclear nuclear Overhauser effect (NOE) signals were detected in a In addition, Overhauser effect (NOE) signals were detected in a NOESY NOESY experiment. Given that two protons inproximity close proximity can induce an NOE cross-peak, experiment. Given that two protons locatedlocated in close can induce an NOE cross-peak, this this approach is useful for analyzing the intermolecular interaction of inclusion complexes [20]. approach is useful for analyzing the intermolecular interaction of inclusion complexes [20]. Both Both the β-CD cavity (H3,5) and cysteine protons NOEs benzyl-ring protons the β-CD cavity (H3,5) and cysteine protons (H71 )(H7′) shareshare NOEs withwith benzyl-ring protons (2–6)(2–6) and and chromene protons (Figure 5). Taken together, the of results these NMR spectroscopic chromene protons (31 ,81 )(3′,8′) (Figure 5). Taken together, the results theseof NMR spectroscopic analyses analyses suggest that the complex plausibleiscomplex is the A mode rather than(Figure the B mode (Figure The suggest that the plausible the A mode rather than the B mode 5c). The benzyl5c). moiety benzyl moiety into the β-CD cavity from the and narrow rim side, and the 5,6,7-trihydroxypenetrates into penetrates the β-CD cavity from the narrow rim side, the 5,6,7-trihydroxy-4H-chromene-4-one 4H-chromene-4-one is located near the cysteine residue. The proposed would be effective is located near the cysteine residue. The proposed structure would be structure effective for the cooperative for the cooperative complexation of baicalein complexation of baicalein and cysteinyl β-CD.and cysteinyl β-CD.

Figure5.5.(a) (a)NOESY NOESY spectrum spectrum of of cysteinyl cysteinyl β-CD/baicalein β-CD/baicaleincomplex complex(Solvent: (Solvent:DD (b)Enlarged Enlargedspectrum spectrum 2 O); Figure 2O); (b) of the area in the red rectangle; (c) The proposed model of the cysteinyl β-CD/baicalein complex. of the area in the red rectangle; (c) The proposed model of the cysteinyl β-CD/baicalein complex.

2.4. SEM SEM Analysis Analysis 2.4. SEM analysis analysis is is frequently frequently used used as as aa tool to visualize the surface morphological morphological changes changes in in SEM inclusion complexation complexation studies studies [21,22]. [21,22]. Figure Figure 66 shows shows the the SEM SEM images images of of baicalein, baicalein, cysteinyl cysteinyl β-CD, β-CD, inclusion the physical physicalmixture, mixture,and andthe theinclusion inclusion complex. Baicalein shows a rod shape of approximately 10 the complex. Baicalein shows a rod shape of approximately 10 µm µmlength, in length, whereas amorphous particles 2 µm size areobserved observedinincysteinyl cysteinylβ-CD. β-CD.The The physical physical in whereas amorphous particles 2 µm in in size are mixtures showed showed aa combined combined morphology morphology of baicalein and cysteinyl β-CD. β-CD. After After complexation, complexation, the the mixtures original morphologies morphologiesof of both both compounds compoundsdisappeared disappearedand andfibrous fibrous structures structureswere were newly newly observed. observed. original Thus, the the data obtained obtained from the SEM analysis suggest that the inclusion complex formation formation process process Thus, between baicalein and cysteinyl β-CD occurs in a solid state. between baicalein and β-CD occurs in a solid state.

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Figure 6. SEM photographs. (a) Baicalein; (b) Cysteinyl β-CD; (c) Physical mixture; (d) Cysteinyl β-CD/ Figure 6. SEM photographs. (a) Baicalein; (b) Cysteinyl β-CD; (c) Physical mixture; (d) Cysteinyl baicalein complex. (a) Baicalein; (b) Cysteinyl β-CD; (c) Physical mixture; (d) Cysteinyl β-CD/ Figure 6.inclusion SEMinclusion photographs. β-CD/baicalein complex. baicalein inclusion complex.

2.5. XRPD 2.5. XRPD 2.5. Further XRPD evidence for the cysteinyl β-CD/baicalein inclusion complex is provided by XRPD Further evidence the cysteinyl inclusion complex is provided by XRPD experiments. is for a suitable method β-CD/baicalein for the evaluationinclusion of inclusion complexation in powder or FurtherXRPD evidence for the cysteinyl β-CD/baicalein complex is provided by XRPD experiments. XRPD is a suitable method for the evaluation of inclusion complexation in powder microcrystalline states [23]. Figure 7 shows the disappearance of the sharp peak, indicating a lack experiments. XRPD is a suitable method for the evaluation of inclusion complexation in powderoforor microcrystalline states [23]. 7 7shows disappearance of the the sharp sharppeak, peak, indicating lack crystallinity with the formation of the inclusion In the diffractogram of theindicating physical mixture, microcrystalline states [23].Figure Figure showsthe thecomplex. disappearance of a alack ofof crystallinity with the formation ofof the complex. In the the diffractogram diffractogram the physical a crystallinity superimposed pattern of each compound is clearly displayed. Accordingly,ofof the formation ofmixture, the with the formation theinclusion inclusion complex. In the physical mixture, cysteinyl β-CD/baicalein inclusion complex is confirmed. a superimposed pattern of each compound is clearly displayed. Accordingly, the formation the a superimposed pattern of each compound is clearly displayed. Accordingly, the formation ofof the cysteinyl β-CD/baicalein cysteinyl β-CD/baicaleininclusion inclusioncomplex complex is confirmed. confirmed.

Figure 7. X-ray diffractograms. (a) Baicalein; (b) Cysteinyl β-CD; (c) Physical mixture; (d) Cysteinyl β-CD/baicalein inclusion complex.(a) Baicalein; (b) Cysteinyl β-CD; (c) Physical mixture; (d) Cysteinyl Figure 7. X-ray diffractograms. Figure 7. X-ray diffractograms. (a) Baicalein; (b) Cysteinyl β-CD; (c) Physical mixture; (d) Cysteinyl β-CD/baicaleininclusion inclusioncomplex. complex. β-CD/baicalein

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2.6. Antioxidant Effect 2.6. Antioxidant Effect Baicalein has been reported to scavenge reactive oxygen species which cause damage to lipids, Baicalein has been reported to scavenge reactive species which cause damage lipids, proteins, and DNA in cellular systems [24]. Based on oxygen the structure, one-step hydrogen atomtotransfer proteins, and DNA in cellular systems Based on thecan structure, one-step hydrogen atom[16]. transfer or an electron transfer followed by a [24]. proton transfer be the antioxidant mechanism The or an electron transfer followed by ainvestigated proton transfer the antioxidant mechanism The antioxidant effects of samples were basedcan on be a DPPH assay. Because DPPH [16]. is a stable antioxidant of samples were investigated baseddiscoloration on a DPPH assay. Because DPPH is activity. a stable free free radicaleffects generating a violet color, a progressive represents antioxidant The radical generating violet color,scavenging a progressive discoloration represents the antioxidant The amount amount of DPPH afree radical is estimated by observing decreaseactivity. in the absorbance at of DPPH scavengingactivity is estimated by cysteinyl observingβ-CD/baicalein the decrease in the absorbance at 518isnm [25]. 518 nm free [25].radical The antioxidant of the inclusion complex clearly The antioxidantfrom activity thefree cysteinyl β-CD/baicalein complex is clearly differentiated differentiated thatofof baicalein (Figure 8a). inclusion After efficient complexation, the limited from that of free baicalein (Figurewas 8a).highly After enhanced efficient complexation, antioxidant activity of baicalein together with the the limited aqueousantioxidant solubility. activity The few of baicalein was in highly enhanced together with the aqueous (DM solubility. negative in negative values the case of β-CD or 2,6-di-O-methyl-β-CD β-CD)The can few be due to the values enhanced the case of β-CD or 2,6-di-O-methyl-β-CD (DMthe β-CD) can be due DPPH to the enhanced from the solubility from the complex formation with violet-colored in water solubility [26]. Furthermore, complex withlittle the antioxidant violet-colored DPPHThe in water [26]. Furthermore, cysteinyl β-CD shows cysteinylformation β-CD shows activity. synergetic antioxidant activity may be affected little antioxidant activity. The synergetic antioxidant activity may affected by for thethe complexation by the complexation mode (Figure 5c). The trihydroxy groups, thebe specific sites antioxidant mode The trihydroxy groups, the specific sites for the antioxidant remain availablethis to effect,(Figure remain5c). available to scavenge radicals, and the sulfhydryl group of effect, cysteine can support scavenge andthe theβ-CD sulfhydryl of the cysteine can support activity. Given that activity the β-CD activity. radicals, Given that cavitygroup masks irrelevant benzylthis ring, the antioxidant of cavity masks the irrelevant benzyl theaantioxidant activity of baicalein be reduced, and, baicalein cannot be reduced, and, ring, rather, positive effect is considered by cannot baicalein solubilization. rather, a positive effect is byinvestigated baicalein solubilization. Recently, of baicalein Recently, the solubility of considered baicalein was with the addition of α-,the β-, solubility γ-CD, hydroxypropylwas investigated the addition of α-,DM β-, β-CD γ-CD,showed hydroxypropyl-β-CD (HP β-CD), and DM β-CD, β-CD (HP β-CD),with and DM β-CD, where the highest solubilizing effect [27]. Thus, the where DM β-CD showed highest effect [27]. the antioxidant activity of DM antioxidant activity of DMthe β-CD was solubilizing also compared, and thatThus, of cysteinyl β-CD showed three-fold β-CD also compared, and that of cysteinyl β-CD three-fold higher valuestreatment. comparedThe to higherwas values compared to the antioxidant effect ofshowed DM β-CD under an identical the antioxidant effect of DM spectra β-CD under an identical The corresponding absorption spectra corresponding absorption also support the treatment. antioxidant results (Figure 8b). Hence, we find also the antioxidant results (Figure 8b). Hence, we find that the synthesized β-CD is thatsupport the synthesized cysteinyl β-CD is effectively utilized to improve the solubilitycysteinyl and antioxidant effectively to improve the solubility and antioxidant activity of baicalein. activity of utilized baicalein.

Figure8.8. (a) (a)Consumption ConsumptionofofDPPH DPPHinin presence of baicalein, β-CDs complexes; Figure thethe presence of baicalein, β-CDs andand theirtheir complexes; (b) (b) Absorption spectra of baicalein in aqueous solution different β-CDs. Absorption spectra of baicalein in aqueous solution withwith different β-CDs.

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3. Materials and Methods 3.1. Chemicals The β-CD (97%), 1-(p-toluenesulfonyl)imidazole (98%), and baicalein (>99%) were obtained from Tokyo Chemical Industry Co., Ltd. Triethylamine (TEA), DM β-CD, L-cysteine (97%), 2,2-diphenyl-1picrylhydrazyl (DPPH), ethanol (>99.8%) were purchased from Sigma–Aldrich Chemicals Co. The used water was triply distilled. D2 O (99.96% at D) and CD3 OD (99.8% at D) were from Cambridge Isotope Laboratories, Inc. (Andover, MA, USA). 3.2. Synthesis of Cysteinyl β-CD The cysteinyl β-CD was synthesized from mono-6-O-p-toluenesulfonyl-β-CD (tosyl β-CD), the most important intermediate for mono-functionalization on the primary side of β-CD [28]. The tosyl β-CD was synthesized as described in our previous report [29]. The β-CD (5.0 g, 4.4 mmol) was dissolved in water by heating it to 60 ˝ C, and the cooled solution was mixed with 1-(p-toluenesulfonyl) imidazole (3.9 g, 17.7 mmol). After 6 h, a NaOH aqueous solution was added over a time of 20 min, and the unreacted precipitates were removed. Ammonium chloride (6.1 g, 115 mmol) was added to stop the reaction, and the mixture was concentrated to half of the original volume by blowing air. Tosyl β-CD began to precipitate, and the precipitates were washed and dried. Tosyl β-CD (921 mg, 0.7 mmol) and L-cysteine (251 mg, 2.1 mmol) were dissolved in a 40% (v/v) TEA aqueous solution (10 mL) at 85 ˝ C while stirring for two days under N2 . After 48 h, excess solvent was removed by evaporation under reduced pressure, and the concentrated sample was precipitated using ethanol (100 mL). The precipitate was collected, and the cysteinyl β-CD was purified using DEAE-Sephadex A-25 and a Bio-gel P-2 column. The lyophilized product was obtained at a 30% yield starting with β-CD. Mono-6-cysteinyl-β-CD. 1 H-NMR (500MHz, D2 O): δ 5.10–5.00 (m, 7H, H1), 3.95–3.73 (m, H3,6,71 ,5), 3.65–3.50 (m, H2,4), 3.29–3.23 (m, 1H, H61 a), 3.19–3.12 (m, 1H, H81 a), 3.08–3.01 (m, 1H, H61 b), 3.00–2.93 (m, 1H, H81 b); 13 C-NMR (500 MHz, D2 O): δ 175.26 (C91 ), 104.64 (C1,11 ), 86.49 (C41 ), 83.54 (C4), 75.60 (C3), 75.29 (C31 ), 74.63 (C51 ), 74.58 (C5), 74.50 (C2), 74.44 (C21 ), 62.47 (C6), 56.38 (C71 ), 36.48 (C61 ), 35.71 (C81 ); MALDI-TOF MS: 1260.42 [mono-6-cysteinyl β-CD + Na]+ . 3.3. MALDI-TOF Mass Spectrometry MALDI-TOF MS (Voyager- DETM STR Bio-Spectrometry, Applied Biosystems, Framingham, MA, USA) was carried out to obtain the mass spectrum of cysteinyl β-CD in the positive ion mode. The 2,5-Dihydroxybenzoic acid (DHB) was used as the matrix. 3.4. NMR Spectroscopy For the NMR spectroscopic analysis, a Bruker Avance 500 spectrometer (Bruker, Karlsruhe, Germany) was used to record the 1 H-NMR, 13 C-NMR, HSQC, and NOESY spectra. The NOESY spectra were recorded with 256/2048 complex data points using a pulse train to achieve a spin-lock field with a mixing time of 600 ms for the complex. 3.5. Phase Solubility Studies Baicalein was dissolved in methanol to obtain a 20 mM stock solution. In this case, 500 µL of the stock solution was added to an aqueous solution (500 µL) of β-CD and cysteinyl β-CD with various concentrations (0.0–10.0 mM) in capped vials. In each case, the mixture was magnetically stirred at 25 ˝ C and shielded from light to prevent the degradation of the baicalein. After 72 h, methanol was evaporated, lyophilized, and dissolved in 500 µL water. After filtering (PTFE 0.2 µm filter, ADVANTEC), the solubilized baicalein was evaluated at 270 nm using a spectrophotometer (UV 2450, Shimadzu Corporation, Shimadzu, Japan).

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3.6. Aqueous Solubility of β-CD and Cysteinyl β-CD The intrinsic solubility measurement was performed with some modification in the previous protocol [30]. The β-CD and cysteinyl β-CD were added to 250 µL of water, and the mixture was sonicated for 1 min and then stirred for 1 h at 25 ˝ C. After lyophilizing the soluble fraction, the mass was measured as a function of the volume. Because of increasing viscosity, a more accurate estimation of solubility was difficult. 3.7. Preparation of the Cysteinyl β-CD/Baicalein Complex Inclusion complexes of baicalein and cysteinyl β-CD were prepared using the suspension method [31]. Baicalein was dissolved in methanol and then added to an aqueous solution of an appropriate concentration of cysteinyl β-CD. The resulting mixture was stirred at 25 ˝ C for 72 h. After equilibration, methanol was evaporated and lyophilized. The dissolved sample was lyophilized and the sample was then dissolved in 500 µL water, after which it was filtered and freeze-dried. 3.8. SEM The surfaces morphology of the materials was examined using a field emission scanning electron microscope (JSM-6700F, JEOL, Ltd., Tokyo, Japan). The samples were precisely fixed on a brass stub using double-sided adhesive carbon tape, and were then made to be electrically conductive by sputter coating with a thin layer of gold in a vacuum. Images were taken at an accelerating voltage of 10 kV, and a magnitude level of 2300ˆ was used. 3.9. XRPD XPRD patterns for baicalein, cysteinyl β-CD, the physical mixtures and the inclusion complexes were traced employing high resolution X-ray diffractometer (Bruker D8 DISCOVER, Karlsruhe, Germany) at a scanning rate of 2˝ /min. The voltage/current used was 40 kV/40 mA and the angular range (2θ) covered was between 6˝ and 50˝ . 3.10. Antioxidant Activity The antioxidant activities of the samples are measured in terms of their radical scavenging ability (RSA), using the DPPH method [32]. A volume of 1 mL (ethanol:water, 50:50 v/v) of 50 µM DPPH was used. The reaction was started with an addition of 10 µL of each sample without and with a 2 mM β-CD concentration. The resulting mixtures were incubated in the dark at 25 ˝ C. After the system reached equilibrium at the end of 30 min, the absorbance levels of the solutions were measured by UV-Vis spectroscopy at 518 nm. The results were expressed as the percentage of DPPH eradication calculated according to the following equation: RSA “ p1 ´ AS {AB q ˆ 100

(1)

where the absorbance of the sample is AS and that of the blank sample is AB . 4. Conclusions In the present study, mono-6-cysteinyl-β-CD was successfully synthesized by reacting mono-6-O-p-toluenesulfonyl-β-CD with L-cysteine in water. The complexation characteristics of cysteinyl β-CD and baicalein were analyzed using UV-vis, NMR spectroscopy, SEM, and XRPD. The driving forces for the complexation are considered as non-covalent interactions such as hydrogen bonding, hydrophobic interaction, and van der Waals interactions between mono-6-cysteinyl-β-CD and baicalein. The results of NMR spectroscopic analyses show a complexation mode where the benzyl moiety penetrates into the β-CD cavity and the chromene part is located near the cysteine. This cooperative complexation can enhance the solubility and antioxidant activity of baicalein. Cysteinyl

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β-CD and this synergetic approach for inclusion complexation will be useful for the design and development of novel host molecules. Acknowledgments: This paper was supported by the KU Research Professor Program of Konkuk University. This research was also supported by the National Research Foundation of Korea, funded by the Korean Government (NRF-2015R1D1A1A01058686) and the Bio & Medical Technology Development Program of the NRF funded by the Korean government, MSIP (2015M3A9B8031831). SDG. Author Contributions: Seunho Jung conceived and designed the experiments; Hwanhee Kim and Hu Yiluo performed the experiments. Hwanhee Kim and Eunae Cho analyzed the data and wrote the paper. Jae Yung Lee contributed the reagents and the analysis tools. Seyeon Park supervised and provided consulting during the project. All authors reviewed the manuscript. Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds are Not available from the authors. © 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).