Host–guest inclusion system of norathyriol with β-cyclodextrin and its derivatives: Preparation, characterization, and anticancer activity

Journal of Bioscience and Bioengineering VOL. 117 No. 6, 775e779, 2014 www.elsevier.com/locate/jbiosc

Hosteguest inclusion system of norathyriol with b-cyclodextrin and its derivatives: Preparation, characterization, and anticancer activity Bin Han,1 Bo Yang,1, * Xuemin Yang,1 Yulin Zhao,2 Xiali Liao,1 Chuanzhu Gao,1 Fen Wang,1 and Ruijian Jiang1 Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, PR China1 and Faculty of Chemical Engineering, Kunming University of Science and Technology, Kunming 650500, PR China2 Received 28 September 2013; accepted 2 December 2013 Available online 6 February 2014

The characterization, binding ability and inclusion complexation behavior of the inclusion complexes of norathyriol with b-cyclodextrin (b-CD) and its derivatives such as hydroxypropyl-b-cyclodextrin (HPbCD), sulfobutyl ether b-cyclodextrin (SBEbCD) and mono (6-ethylene-diamino-6-deoxy)-b-cyclodextrin (ENbCD) were investigated in both solution and solid state by means of femtosecond spectroscopy, 1H and 2D nuclear magnetic resonance, powder X-ray diffraction. The results showed that the aqueous solubility of the complexes was much higher than that of norathyriol. The cytotoxicity of complexes on human colon cancer cell lines HT-29, SW480, Lovo and HCT116 indicated that the antitumor activities of the complexes were better than that of norathyriol. This high antitumor activity, along with the satisfactory aqueous solubility of the complexes, will be potentially useful for their application on cancer chemotherapies. Ó 2013, The Society for Biotechnology, Japan. All rights reserved. [Key words: Norathyriol; b-Cyclodextrin; Inclusion complex; Binding ability; Characterization; Anticancer activity]

Norathyriol (1,3,6,7-tetrahydroxyxanthone, Fig. 1A), an activated metabolite of mangiferin (1,3,6,7-tetrahydroxyxanthone-C2-b-Dglucoside), was isolated by incubation with a bacterial mixture of human intestinal fences as described previously (1). Norathyriol is also a polyphenol and was reported to possess the following pharmacological activities such as anti-inflammatory (2), vasorelaxation (3) and antiplatelet activities (4). Li et al. (5) reported that norathyriol could suppresses UV-induced skin cancers by targeting ERKs. Recently, Bock et al. (6) found that the cleavage of mangiferin to norathyriol is followed by the absorption of norathyriol from the colon into the blood circulation, indicated that it may have a relation with the antioxidant and other effects of mangiferin. Norathyriol could also decreased serum uric acid and inhibited the activity of PTP1B (7). However, norathyriol has a low solubility and hydrophobicity in the solubilization test, which may limit the potent pharmaceutical application of norathyriol seriously. Therefore, in order to further the clinical application of norathyriol, the search for an efficient and nontoxic carrier for norathyriol has become more and more important. b-Cyclodextrin (bCD) might be an ideal carrier for norathyriol. It is well-known that bCD is truncated-cone polysaccharides mainly composed of seven D-glucose monomers linked by a-1,4-glucose bonds. It has a hydrophobic inner cavity and a hydrophilic outer surface, which can encapsulate model substrates to form hoste guest complexes or supramolecular species. It usually enhances drug solubility in aqueous solution and affects the chemical characteristics of the encapsulated drug. Because of its special

* Corresponding author. Tel.: þ86 13064281879; fax: þ86 871 65920637. E-mail addresses: [email protected], [email protected] (B. Yang).

hydrophobic cavity and the ability to improve chemical stability and physicochemical properties of drugs, bCD has been extensively investigated in hosteguest chemistry for construction of versatile supramolecular aggregations (8e10). Modified bCDs have also been extensively applied in supramolecular chemistry and medicinal chemistry (11). In previous study, the characterization, inclusion complexation behavior and binding ability of the inclusion complexes of mangiferin with bCD and its derivatives were investigated (12). In this paper, in order to improve the absolute bioavailability of norathyriol and prolong the duration of the norathyriol in the blood, we investigated the interaction of norathyriol with bCD and a series of modified bCDs such as mono-(6-ethylene-diamino-6-deoxy)-bcyclodextrin (ENbCD), 2-hydroxypropyl-b-cyclodextrin (HPbCD) and sulfobutyl ether b-cyclodextrin (SBEbCD) (Fig. 1B). We were particularly interested in exploring the solubilization effect of CDs on norathyriol and the enhancement in bioavailability of norathyriol/CD complexes compared to norathyriol. MATERIALS AND METHODS Materials Norathyriol (PC > 98%) was obtained from Kunming Pharmaceutical Corporation in Yunnan Province, PR China. bCD, HPbCD and SBEbCD were commercially available. ENbCD was synthesized according to references (13,14). Other reagents and chemicals were of analytical reagent grade and all the experiments were carried out using ultrapure water. Preparation of norathyriol/bCD, norathyriol/ENbCD, norathyriol/HPbCD and norathyriol/SBEbCD complexes Norathyriol (0.0530 mmol) and CDs (0.0125 mmol) were added to 10 mL of ultrapure water and the reagent was stirred for 5 days at room temperature in the dark. The filtrate was evaporated under reduced pressure to remove the solvent and dried in vacuo to give the norathyriol/CD complexes after the uncomplexed norathyriol was removed by filtration. Norathyriol/bCD

1389-1723/$ e see front matter Ó 2013, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2013.12.001

776

HAN ET AL.

A

B

J. BIOSCI. BIOENG., buffer solution (1.0 mL), and the varied amounts of CDs were added in order. The mixed solution was diluted to the mark with buffer and ultrasonically oscillated for 30 min at room temperature and the fluorescence spectra were measured at lex/ lem ¼ 380 nm/530 nm. 1 H NMR and 2D NMR All NMR experiments were carried out in D2O and tetramethylsilane (TMS) was used as a reference. Samples were dissolved in 99.98% 1 D2O and filtered before use. H NMR and 2D ROESY NMR spectra were acquired on a Bruker Avance DRX spectrometer at 500 MHz and 298 K.

Powder X-ray diffraction The powder X-ray diffraction (XRD) patterns were obtained using a D/Max-3B diffractometer with Cu-Ka radiation (k ¼ 1.5460  A, 40 kV, 100 mA) and at a scanning rate of 5 /min. Powder samples were mounted on a vitreous sample holder and scanned with a step size of 2q ¼ 0.02 between 2q ¼ 5 and 70. Solubilization test Excessive amounts of CDs and norathyriol were placed in 5 mL of water (approximately pH 7.0) respectively, sheltered from light and the mixture was stirred for 1.5 h at room temperature (25  2 C). The solution is filtered by a 0.45 mm cellulose acetate membrane and the filtrate was evaporated under reduced pressure to dryness, then the residue was dosed by appropriate weighing method.

C

In vitro cytotoxicity studies The cytotoxicity tests for mangiferin, norathyriol and norathyriol/CD complexes were evaluated in vitro for antiproliferative activity against human HCT116, Lovo, SW480 and HT-29 cell lines by the MTT cytotoxicity assay. The IC50 values that represented the concentration of a drug required for 50% reduction of cellular growth have been calculated. Cells were cultured at 5  105 cells/mL in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum at 37 C in a humidified atmosphere of 5% CO2 in air. Cells were seeded at 5  104 cells/mL and treated with the indicated amounts of the complex. The effects of the mangiferin, norathyriol and norathyriol/CD complexes were evaluated as cell survival after treatment. Cell viability was evaluated by a microculture tetrazolium reduction assay using 3-(4,5-dimethyltriazol-2-yl) 2,5diphenyltetrazolium bromide (MTT) (Sigma).

RESULTS AND DISCUSSION Stoichiometry Fig. 2 illustrates the Job plot for the norathyriol/ HPbCD system examined by femtosecond spectroscopy. The plot for HPbCD showed a maximum at a molar fraction of 0.5 in the concentration range, indicating the 1:1 inclusion complexation between host and guest. The same results were obtained from the complexes of norathyriol with bCD, SBEbCD and ENbCD, respectively.

FIG. 1. The structure of norathyriol (A) and CDs (B), the possible inclusion mode and typical NOESY correlations of the norathyriol/CDs inclusion complexes (C).

Spectral titration Quantitative investigation of the binding behavior of norathyriol with host CDs was examined in phosphate buffer solution by means of fluorescence spectroscopy. The complex stability constants (KS) were determined from the absorbance intensity change induced by adding the host molecule. The inclusion complexation of host (H) with guest (G) was expressed by Eq. 1 as the Job plot showed the 1:1 stoichiometry for the inclusion complexation of CDs with the guest molecule norathyriol.

complex: 1H nuclear magnetic resonance (NMR) (500 MHz, D2O): d 7.49 (s, 1H, H-8 of norathyriol), 7.13 (s, 1H, H-5 of norathyriol), 6.49 (s,1H, H-4 of norathyriol), 5.83 (s,1H, H-2 of norathyriol), 3.43e3.87 (m, 42H, H-2e6 of bCD), 4.93e4.99 (s, 7H, H-1 of bCD). Norathyriol/ENbCD complex: 1H NMR (500 MHz, D2O): d 7.55 (s, 1H, H-8 of norathyriol), 7.25 (s, 1H, H-5 of norathyriol), 6.49 (s, 1H, H-4 of norathyriol), 5.97 (s, 1H, H2 of norathyriol), 2.8e3.0 (m, 4H, CH2CH2 of ENbCD), 3.4e3.6 (m, 14H, H-2,4 of ENbCD), 3.7e3.9 (m, 28H, H-3,5,6 of ENbCD), 4.95 (s, 7H, H-1 of ENbCD). Norathyriol/ HPbCD complex: 1H NMR (500 MHz, D2O): d 7.43 (s,1H, H-8 of norathyriol), 6.86 (s,1H, H-5 of norathyriol), 6.53 (s, 1H, H-4 of norathyriol), 6.25 (s, 1H, H-2 of norathyriol), 3.30-4.15 (m, 100H, H-2e6 and CH2 and CH32,3,6 of HPbCD), 4.95e5.10 (s, 7H, H-1 of HPbCD). Norathyriol/SBEbCD complex: 1H NMR (500 MHz, D2O): d 7.77 (s, 1H, H-8 of norathyriol), 7.24 (s, 1H, H-5 of norathyriol), 6.50 (s, 1H, H-4 of norathyriol), 6.14 (s, 1H, H-2 of norathyriol), 4.95 (s, 7H, H-1 of SBEbCD), 3.34e3.50 (m, 6H, OeCH2CH2CH2CH2SO3Na of SBEbCD), 2.85 (m, 6H, OCH2CH2CH2CH2SO3Na of SBEbCD), 1.67 (m, 12H, OCH2CH2CH2CH2SO3Na of SBEbCD). Stoichiometry The stoichiometry for the inclusion complexation of norathyriol with CDs was determined by Job’s methods (15). The Job plots were determined with fluorescence spectra data obtained in a pH 8.0 buffer. The total molar concentration (i.e., the combined concentration of norathyriol and CDs) was kept constant (4.0  104 M), but the molar fraction of norathyriol (i.e., [MA]/ ([MA] þ [CD])) was varied from 0.1 to 0.9. Spectral titration The experimental procedure was carried out as follows: CDs solution (1.0  103 mol/L) were conducted in a pH 8.0 buffer. In a 10 mL colorimetric cylinder, norathyriol (2.0  105 mol/L), NaH2PO4-Na2HPO4 (pH 8.0)

FIG. 2. Job plot for the norathyriol/HPbCD system ([norathyriol] þ [HPbCD] ¼ 0.4 mM) in pH 8.0 buffer.

at

lem:

530

nm

VOL. 117, 2014

400

HOSTeGUEST INCLUSION SYSTEM OF NORATHYRIOL

TABLE 1. Stability constant (Ks) and Gibbs free energy change (DG) for inclusion complexations of host CD with norathyriol (pH 8.0) at 25 C.

250

(A)

(B)

200

Delta F

Fluorescence Intensity

CDs

300

150 100

bCD HPbCD SBEbCD ENbCD

obs calc

50

h 200

1

2

3

LogKs

DG (KJ/mol)

3.58 3.82 3.59 4.11

20.43 21.78 20.47 23.45

4

[G]0/mM

As illustrated in Fig. 3, with the stepwise addition of CDs, the absorbance intensity of norathyriol was increased. The complex stability constant for each hosteguest combination was obtained by using a nonlinear least-squares curve-fitting method (17). Fig. 3 shows the excellent fits between the calculated and experimental data. The plot of F as a function of [G]0 gave an excellent fit for each host examined, verifying the validity of the 1:1 complex stoichiometry as assumed above. The Ks values are reproducible within an error of 5% in the repeated measurements, The Ks values obtained are listed in Table 1, along with the free energy changes of complex formation (DG0) obtained upon addition of large excess of the host CDs.

a

100

0 500

600

Wavelength/nm

80

150

(C)

(D)

60

Delta F

Fluorescence Intensity

Ks (L/mol) 3802  20 6542  30 3859  25 12844  90

0 0

100

40

obs calc

20

i 0 0

1

2

3

4

[G]0/mM 50

777

a

0 500

600

Wavelength/nm FIG. 3. (A) Fluorescence emission spectra of norathyriol (2.0  103 mM) containing various concentrations of HPbCD (from a to h: 0.00, 0.02, 0.04, 0.08, 0.10, 0.20, 0.30, and 0.40 mM of HPbCD); emission at 530 nm. (B) Nonlinear least-squares curve-fitting analyses for the inclusion complexation of norathyriol and HPbCD. (C) Fluorescence emission spectra of norathyriol (2.0  103 mM) containing various concentrations of SBEbCD (from a to i: 0.00, 0.02, 0.04, 0.08, 0.12, 0.18, 0.25, 0.30 and 0.40 mM of SBEbCD); emission at 530 nm. (D) Nonlinear least-squares curve-fitting analyses for the inclusion complexation of norathyriol and SBEbCD. Ks

H þ G#H$G

(1)

The complex stability constants (Ks) were calculated for each hosteguest combination from the nonlinear squares fitting to Eq. 2: Ks ¼

½CD$norathyriol ½CD½norathyriol

¼





DF=Dε

½CD0  DF=Dε ½norathyriol0  DF=Dε

(2) 

Here [CD]0 and [norathyriol]0 refer to the total concentration of CD and norathyriol, respectively; Eq. 2 is achieved by Eq. 3:

DF ¼

Binding ability The size/shape-fit concept plays an important role in the formation of inclusion complexes of host CDs with guest molecules with various structures have been revealed by extensive studies. On the basis of this concept, several weak intermolecular forces such as van der Waals, ion-dipole, dipoleedipole, electrostatic, hydrophobic interactions and hydrogen bond are known to cooperatively contribute to inclusion complexation. bCD possesses a cyclic truncated cone cavity with a height of 0.79 nm, an inner diameter of 0.62 e 0.78 nm and a cavity volume of 0.262 nm3 (18). The stability of the complexes formed between CDs and norathyriol may dominated by the hosteguest size match. From Table 1, we can see that the binding constants for the complexation of norathyriol with bCD, HPbCD, SBEbCD and ENbCD were in the following order: ENbCD > HPbCD > SBEbCD > bCD. Modified derivatives gave a stronger Ks value than that of native bCD by comparing the enhancement effect of some kinds of CDs for norathyriol, which may possessed a more suited cavity size and can complex with the guest norathyriol better than native bCD. It was showed that the size-fit effect was the dominant controlling factor on the formation of inclusion complexes of norathyriol/CDs. 1

H NMR and 2D NMR analysis The 1H NMR spectra of norathyriol in the presence of host CDs was compared in order to explore the possible inclusion mode of norathyriol/CDs complexes (Fig. 4), where the 1H resonances of HPbCD, ENbCD and SBEbCD were assigned according to the reported method (19). When D2O is used as a solvent, norathyriol is transparent to 1H NMR under most conditions because of its poor water solubility. Assessment of the norathyriol/CDs complexes by 1H NMR clearly demonstrated the presence of the framework protons of the norathyriol molecule and implied a significant solubility increase of norathyriol/CDs compared with the native norathyriol. As illustrated in Fig. 4, norathyriol protons (3H) displayed chemical shifts at d 6.20e6.95 ppm and were distinct from the CD protons


 qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
2 ðDεÞ ½CD0 þ ½norathyriol0 þ 1=Ks  ðDεÞ2 ½CD0 þ ½norathyriol0 þ 1=Ks  4ðDεÞ2 ½CD0 ½norathyriol0

Here [CD]0 and [norathyriol]0 refer to the total concentration of CD and norathyriol, respectively; Dε is the proportionality coefficient which may be taken as a sensitivity factor for the absorbance intensity change (16).

2

(3)

(usually at d 3.0e5.0 ppm). The inclusion rates of norathyriol in the complexes were estimated by the 1H NMR spectrum and related to the stability constants calculated by the spectral titration. The inclusion rates of norathyriol in the complexes were

778

HAN ET AL.

J. BIOSCI. BIOENG.,

A H-3,5,6 of CD

H-2,4 of CD

H-1 of CD

a 8

7

6

5

4

3

H-3,5,6 of CD H-2,4 of CD

H-1 of CD

b 8

7

6

5

4

3

ppm

B a 8

b 8

7

6

H-4 of norathyriol H-8 of norathyriol H-2 of norathyriol H-5 of norathyriol

7 ppm

6

FIG. 4. (A) 1H NMR spectra of HPbCD in the absence and presence of norathyriol in D2O at 25  C. a: HPbCD; b: Norathyriol/HPbCD complex. (B) The enlarged NMR spectrum from approximately 6e8 ppm in the left box of panel A.

in the following order: EnbCD (83.6%) > HPbCD (25.1%) > SBEbCD (23.0%). It was observed that each inclusion rate was relevant to the stability constants calculated by the spectral titration (Table 1). As we know, 2D NMR spectroscopy could provides important information about the spatial proximity between host and guest molecules via observations of the intermolecular dipolar cross-

correlations. Two protons, which are closely located in space can produce an NOE cross-correlation between the relevant protons in ROESY or NOESY spectrum. The presence of NOE cross-peaks between protons of two species indicates spatial contacts within 0.4 nm (20). ROESY of the inclusion complex of norathyriol with CDs (Fig. 5) were obtained in order to further explore the inclusion mode, including a partial contour plot. The ROESY spectrum of the norathyriol/CDs complexes (Fig. 5) showed appreciable correlation of H-4 and H-5 protons of norathyriol with H-3 and H-5 protons of CDs, indicating that not only ring B and ring C, but also part of ring A of norathyriol is included in the CDs cavity. Furthermore, ROE correlation between the aromatic proton H-5 and H-8 of norathyriol was also observed. The possible inclusion mode of norathyriol with CDs as illustrated in Fig. 1C was deduced based on above-mentioned observations, together with the 1:1 (or 2:2) stoichiometry. XRD analysis The XRD patterns of norathyriol and SBEbCD as well as their physical mixtures and inclusion complexes are illustrated in Fig. 6. The physical mixture was prepared by grinding together a 1:1 M mixture of norathyriol and SBEbCD for 10 min in an agate mortar. As indicated in Fig. 6, norathyriol (Fig. 6B) was in a crystalline form. In contrast, the XRD of the norathyriol/SBEbCD inclusion complex (Fig. 6D) was amorphous and showed halo patterns, which was quite different from the superimposition of norathyriol and the norathyriol/SBEbCD physical mixtures, indicating the formation of the inclusion complex between CD and norathyriol. Solubilization The results showed that the water solubility of norathyriol/bCD, norathyriol/HPbCD, norathyriol/SBEbCD and norathyriol/ENbCD, compared with native norathyriol (0.36 mg/mL), was remarkably increased to approximately 21.7, 37.5, 29.4 and 47.2 mg/mL respectively. In the control experiment, a clear solution was obtained after dissolving the norathyriol/bCD (108.7 mg), norathyriol/HPbCD (191.2 mg), norathyriol/SBEbCD (214.6 mg) and norathyriol/ENbCD (201.3 mg) complexes, which was equivalent to 21.7, 37.5, 29.4, and 47.2 mg of norathyriol respectively, in 1 mL of water at room temperature. The results confirmed the accuracy of the obtained satisfactory water solubility of the norathyriol/CDs complexes, which would be beneficial for the pharmaceutical application of norathyriol.

FIG. 5. ROESY spectrum of norathyriol/ENbCD complex in D2O.

VOL. 117, 2014

HOSTeGUEST INCLUSION SYSTEM OF NORATHYRIOL

779

ACKNOWLEDGMENTS

3000 2000 1000 0

A 20

40

60

This work was supported by National Natural Science Foundation of China (NNSFC) (nos. 21062009 and 21362016), which is gratefully acknowledged.

Intensity/cps

8000

B

4000 0 6000 4000 2000 0 4000 3000 2000 1000 0

20

40

60

C 20

40

60

D 20

40

60

degrees FIG. 6. XRD patterns: (A) SBEbCD, (B) norathyriol, (C) norathyriol/SBEbCD physical mixture (1:1), (D) norathyriol/SBEbCD complex.

TABLE 2. IC50 values of mangiferin, norathyriol and norathyriol/CD complexes to four kinds of colon cancer cells. IC50 (mM)

Samples

Mangiferin Norathyriol Norathyriol/HPbCD Norathyriol/SBEbCD Norathyriol/ENbCD

HCT116

Lovo

SW480

HT-29

422 32.1 19.4 20.8 27.1

>474 72.2 21.8 22.4 31.6

464 21.6 19.7 19.2 46.3

390 22.9 15.8 18.1 46.3

In vitro cytotoxicity studies The norathyriol presented a satisfactory antiproliferative activity against HCT116, Lovo, SW480 and HT-29 cell lines, its IC50 values were 32.1, 72.2, 21.6 and 22.9 mM which were lower than that of mangiferin (422, >474, 464 and 390 mM respectively) (Table 2). In addition, the IC50 values of norathyriol in some norathyriol/CD complexes have also been calculated (Table 2). These results proved that the norathyriol in norathyriol/HPbCD and norathyriol/SBEbCD complexes presented an enhance antiproliferative activity against HCT116, Lovo, SW480 and HT-29 cell lines compared to native norathyriol. However, except for norathyriol/ENbCD when it against SW480 and HT-29 cell lines, the relative high complex stability constant (Ks) was the possible reason and it may be a resistance to the release of norathyriol from the complex. Concluding remarks The characterization, binding ability and inclusion complexation behavior of the inclusion complexes of norathyriol with bCD, HPbCD, SBEbCD and ENbCD were investigated in this paper and the results showed that bCD derivatives could significantly increase the water solubility and bioavailability of norathyriol. Considering the shortage in applications of norathyriol and the conveniently and environmentally friendly preparation of the norathyriol/CDs complex, these complexes should be regarded as a useful approach for the design of a novel formulation of norathyriol for medicine.

References 1. Sanugul, K., Akao, T., Li, Y., Kakiuchi, N., Nakamura, N., and Hattori, M.: Isolation of a human intestinal bacterium that transforms mangiferin to norathyriol and inducibility of the enzyme that cleaves a C-glucosyl bond, Biol. Pharm. Bull., 28, 1672e1678 (2005). 2. Lee, H.-Z., Lin, W.-C., Yeh, F.-T., Lin, C.-N., and Wu, C.-H.: Decreased protein kinase C activation mediates inhibitory effect of norathyriol on serotoninmediated endothelial permeability, Eur. J. Pharmacol., 353, 303e313 (1998). 3. Ko, F.-N., Lin, C.-N., Liou, S.-S., Huang, T.-F., and Teng, C.-M.: Vasorelaxation of rat thoracic aorta caused by norathyriol isolated from gentianaceae, Eur. J. Pharmacol., 192, 133e139 (1991). 4. Teng, C.-M., Lin, C.-N., Ko, F.-N., Lin, C.-K., and Huang, T.-F.: Novel inhibitory actions on platelet thromboxane and inositolphosphate formation by xanthones and their glycosides, Biochem. Pharmacol., 38, 3791e3795 (1989). 5. Li, J.-X., Malakhova, M., Mottamal, M., Reddy, K., Kurinov, I., Carper, A., Langfald, A., Oi, N., Kim, M.-K., Zhu, F., and other 4 authors: Norathyriol suppresses solar UV-induced skin cancer by targeting ERKs, Cancer Res., 72, 260e270 (2012). 6. Bock, C., Waldmann, K. H., and Ternes, W.: Mangiferin and hesperidin metabolites are absorbed from the gastrointestinal tract of pigs after oral ingestion of a Cyclopia genistoides (honeybush tea) extract, Nutr. Res., 28, 879e891 (2008). 7. Zhou, R.-G., Yang, Z.-X., Yang, J., Wang, J., Yang, B., and Zhang, W.: Total synthesis of norathyriol, Appl. Chem. Ind., 40, 1516e1517, 1521 (2011) (in Chinese). 8. Liu, Y. and Chen, Y.: Cooperative binding and multiple recognition by bridged bis(b-cyclodextrin)s with functional linkers, Acc. Chem. Res., 39, 681e691 (2006). 9. Loftsson, T. and Jävinen, T.: Cyclodextrins in ophthalmic drug delivery, Adv. Drug Deliv. Rev., 36, 59e79 (1999). 10. Villaverde, J., Maqueda, C., and Morillo, E.: Improvement of the desorption of the herbicide norflurazon from soils via complexation with b-cyclodextrin, J. Agric. Food Chem., 53, 5366e5372 (2005). 11. Zhang, Y.-C., Huang, L.-X., and Xu, Z.-K.: The properties, modification and application of cyclodextrin, Mod. Food. Sci. Technol., 24, 947e951 (2008) (in Chinese). 12. Yang, X.-M., Zhao, Y.-L., Chen, Y.-J., Liao, X.-L., Gao, C.-Z., Xiao, D., Qin, Q.-X., Yi, D., and Yang, B.: Hosteguest inclusion system of mangiferin with bcyclodextrin and its derivatives, Mater. Sci. Eng. C, 33, 2386e2391 (2013). 13. Singh, A. P., Cabrer, P. R., Alvarez-Parilla, E., Meijide, F., and Tato, J. V.: Complexation of 6-deoxy-6-(aminoethyl)amino-cyclodextrin with sodium cholate and sodium deoxycholate, J. Incl. Phenom. Macrocycl. Chem., 35, 335e348 (1999). 14. Potter, C. F., Russel, N. R., and McNamara, M.: Spectroscopic characterization of metallo-cyclodextrins for potential chiral separation of amino-acids and L/DDOPA, J. Incl. Phenom. Macrocycl. Chem., 56, 395e403 (2006). 15. Liu, Y., Chen, Y., Li, L., Zhang, H.-Y., Liu, S.-X., and Guan, X.-D.: Bridged bis(bcyclodextrin)s possessing coordinated metal center(s) and their inclusion complexation behavior with model substrates: enhanced molecular binding ability by multiple recognition, J. Org. Chem., 66, 8518e8527 (2001). 16. Liu, Y., Han, B.-Q., and Chen, Y.-T.: Molecular recognition and complexation thermodynamics of dye guest molecules by modified cyclodextrins and calixarenesulfonates, J. Phys. Chem., 106, 4678e4687 (2002). 17. Inoue, Y., Yamamoto, K., Wada, T., Everitt, S., Gao, X.-M., Hou, Z.-J., Tong, L.H., Jiang, S.-K., and Wu, H.-M.: Inclusion complexation of (cyclo)alkanes and (cyclo)alkanols with 6-O-modified cyclodextrins, J. Chem. Soc., Perkin Trans. 2, 1807e1816 (1998). 18. Ayala-Zavala, J. F., Del-Toro-Sánchez, L., Alvarez-Parrilla, E., and GonzálezAguilar, G. A.: High relative humidity in-package of fresh-cut fruits and vegetables: advantage or disadvantage considering microbiological problems and antimicrobial delivering systems? J. Food. Sci, 73, R41eR47 (2008). 19. Liu, Y., Chen, G.-S., Chen, Y., and Lin, J.: Inclusion complexes of azadirachtin with native and methylated cyclodextrins: solubilization and binding ability, Bioorg. Med. Chem., 13, 4037e4042 (2005). 20. Correia, I., Bezzenine, N., Ronzani, N., Platzer, N., Beloeil, J. C., and Doan, B. T.: Study of inclusion complexes of acridine with b- and (2,6-di-Omethyl)-b-cyclodextrin by use of solubility diagrams and NMR spectroscopy, J. Phys. Org. Chem., 15, 647e659 (2002).