High-efficiency formation mechanism of mangiferin/γ-cyclodextrin complex

Abstract

Abstract: Cyclodextrins (CDs) are widely applied to improve the aqueous solubility and stability of nutraceuticals with the advantages of high safety and low cost. In this study, the binding capacities of CDs (α-CD, β-CD, maltosyl-β-CD, hydroxypropyl-β-CD and γ-CD) with mangiferin were compared by the phase solubility method and isothermal titration calorimetry. γ-CD could completely encapsulate mangiferin at the molar ratio of 1:1, which coincided with the molecular docking and ONIOM calculation results that γ-CD had the lowest interaction energy with mangiferin. The independent gradient model (IGM) visualized the intermolecular non-covalent binding and electrostatic potential around mangiferin during interaction. By complexing with γ-CD, mangiferin was molecularly dispersed in the matrix of γ-CD with its aromatic ring in the hydrophobic cavity. Compared with pure mangiferin, the mangiferin/γ-CD complex exhibited the stronger chemical antioxidant activities. The obtained result can promote the application of mangiferin and γ-CD in foods.

Introduction

Mangiferin, a common polyphenol, is widely found in mango and other medicinal and edible plants, which possesses the strong resistance to acid and enzyme hydrolysis (Gómez-Zaleta et al., 2006; Prabhu et al., 2006). In recent years, its nutritional and therapeutic value in the pharmaceutical and food industry has been widely recognized. It has many heath-promotion properties, such as antiviral, immunoregulatory, anticancer, anti-inflammatory, antibacterial, and antioxidant activities (Andreu et al., 2005; Carvalho et al., 2007; Ferreira et al., 2010). Especially being used with other herbs, mangiferin can significantly improve the biological activities (Jyotshna et al., 2016). Moreover, the molecular structure of mangiferin partly conforms to Lipinski's rule of drug properties. It has the potential to be a natural medicine or nutraceutical. However, the low aqueous solubility and poor stability of mangiferin limit its application in foods and medicines (Khurana et al., 2017). Although a lot of work has been carried out to improve its water solubility and stability, such as tablets and dropping pills, the effect is not prospective (Liu et al., 2001; Potter et al., 2006). Forming the complex with cyclodextrins (CDs) is a feasible strategy (You et al., 2018). Cyclodextrin is a kind of cyclic oligosaccharide with the unique hydrophobic cavity and external hydrophilic structure, which is widely applied in analytical chemistry, organic synthesis and medicine. It can encapsulate hydrophobic molecules to enhance their aqueous solubility, thermostability and bioavailability, and prevent the release of undesirable odor (Deshaware et al., 2018; Li et al., 2019).

At present, the commonly used CDs mainly include α-cyclodextrin (α-CD), β-cyclodextrin (β-CD), (2-Hydroxypropyl)-β-cyclodextrin (HP-β-CD), 6-O-α-maltosyl-β-cyclodextrin (M-β-CD), γ-cyclodextrin (γ-CD), which generally possess low chemical activity and high safety. The addition of CD rarely causes adverse effects in foods, drugs and cosmetics. Some studies about the complexes of mangiferin had been performed. It was reported that the solubility in water and antioxidant activity of mangiferin could be improved by complexing with β-CD (Ferreira et al., 2013; Huang et al., 2011; Zhang et al., 2010). But to the best of our knowledge, these studies mainly focus on the physicochemical characterization of the complex of mangiferin and β-CD or HP-β-CD.

The traditional instrumental characterization mainly reflects the intermolecular mechanism by investigating the changes of spectral signal, which usually fail to clarify the underlying mechanism. The development of theoretical calculation method, such as quantum chemistry and molecular dynamics, provides a powerful tool for further mechanism research. Cheng et al. (2018) evaluated the conformational stability of amylose-linoleic acid complex in water by using molecular dynamics simulation. The molecular docking method also could be used to clarify the molecular recognition of folic acid subunits with CDs (Ceborska et al., 2018). The recently developed independent gradient model (IGM) could visualize and quantify intramolecular and intermolecular interactions (Lefebvre et al., 2017).

In view of this, the binding capacities of α-CD, β-CD, M-β-CD, HP-β-CD and γ-CD for mangiferin were systematically compared by using phase solubility method and isothermal titration calorimetry (ITC). The corresponding interaction mechanism was investigated by the comprehensive use of molecular docking, ONIOM calculation and IGM analysis. On this basis, the complex of mangiferin/γ-CD was prepared and characterized, whose antioxidant were also evaluated.

Materials and Methods

Reagents    Mangiferin (purity 95%), potassium bromide (spectrophotometric grade), α-CD, β-CD, HP-β-CD, M-β-CD, γ-CD and deuterium oxide (D2O, 99%) were from Aladdin (Shanghai, China). Acetonitrile (HPLC grade) was the product of TEDIA (Fairfield, OH). Both 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) were purchased from Sigma-Aldrich Chemical, Inc. (St. Louis, MO, USA).

Phase solubility study    The phase solubility measurement was performed based on the method of Liu et al. (2016). Mangiferin (60 mg) was added to 5 mL of CD aqueous solution at different concentrations (0–10 mM). The mixture was shaken at 30 °C for 72 h, and filtered. Then, the content of mangiferin in the supernatant was determined by an Agilent 1260 HPLC (Santa Clara, CA) with the injection volume of 10 µL. The mobile phase consisted of 60% acetonitrile and 40% water. The separation was performed on a Tnature C18 column (4.6 × 250 mm, 5 µm particle size) at 30 °C with the flow rate of 0.8 mL/min. The concentration of mangiferin was determined by using the external standard method at the detection wavelength of 370 nm. The curve of the solubility of mangiferin vs. the concentration of the CD was fitted linearly. The corresponding slope and intercept values of the regression line were recorded. Then, the apparent stability constant (K_s) could be obtained based on the following Higuchi-Connors equation:   

ITC method    The ITC measurements were performed on an ITC Standard Volume instrument (TA Instruments-Waters LLC., New Castle, DE) according to the previous method (Zhang et al., 2017). Before the measurement, mangiferin and CD were solved in the ultrapure water and degassed for 15 min, respectively. Then, 1.3 mL of 0.3 mM mangiferin solution was loaded into the sample cell with the same amount of water as the blank. Afterwards, 30 portions of the 8 µL CD solution (10 mM) were gradually injected into the sample cell at 30 °C with the interval time of 300 s. In order to eliminate the thermal effect caused by dilution, the titration of the CD solution into the ultrapure water was also carried out at the same condition. The thermal change during the titration was recorded and processed by the TA Nanoanalyze 3.3 software.

Determination of interaction modes    The molecular docking was carried out to obtain the binding mode of mangiferin and CD by using the AutoDock 4.2 software (Hou et al., 2013; Morris et al., 2009). The 3D structure of mangiferin was from Pubchem¹⁾ and optimized by the PM6-D3H4 method of MOPAC 2016 software (Stewart, 2007). Both the conformations of β-CD and γ-CD were from RCSB protein data bank (http://www.rcsb.org/). The 3D structure of M-β-CD was set up by introducing one α-maltosyl group to the hydroxyl group at C6 of β-CD, and also optimized by the PM6-D3H4 method. Then, the optimum binding modes of mangiferin and CDs were determined by the Lamarckian genetic algorithm (LGA) method. The points of the grid box in all directions were set at 40 with the grid spacing of 0.375 Å. In order to evaluate the binding capacity between mangiferin and CD, the interaction energies of the mangiferin-CD complexes obtained by molecular docking was further calculated by ONIOM method of Gaussian 09 (Chung et al., 2015). For the total energy of the complex (E_oniom), the full system (complex) was optimized by the PM3 method (a low level of theory) while the inner layer (mangiferin) was optimized by both PM3 and B3LYP/6-31G(d) (a high level of theory). The energies of mangiferin (E_comp^[Mg]) and CD (E_comp^[CD]) in the optimized complex were also calculated by using B3LYP/6-31G(d) and PM3, respectively. Then, the interaction energy (INE_[Mg-CD]) could be obtained by using the following equation (Li et al., 2019):   

IGM method    In this study, the IGM analysis of the optimized complex was performed to investigate the interaction between mangiferin and CD. The PDB file containing the conformation of the complex was imported into the Multiwfn 3.6 (dev) software (Lu and Chen, 2012). Mangiferin and CD were regarded as fragments. Then, the complex was covered by the high-quality grid. The results of the density gradient of atoms (δ_g), the electron density (p) and the sign of the second Hessian eigenvalue in Laplacians (sign(λ₂)) at each grid point were calculated based on pro-molecular density (Lefebvre et al., 2017). The obtained δ_g^inter could be further decomposed into δ_g^inter and δ_g^intra, which corresponded to the interfragment and intrafragment interactions.The interfragment interaction could be visualized by VMD 1.9.3 (Humphrey et al., 1996). The electrostatic potential on the molecular surface could be also exhibited by Gaussian 09 and VMD 1.9.3.

Preparation of the complex and physical mixture of mangiferin and γ-CD    Mangiferin (2 mM) and CD (2 mM) were mixed in 50 mL of the ultrapure water, shaken at 30 °C for 72 h and centrifuged at 3 000 rpm (618×g) for 15 min. The obtained supernatant was lyophilized by an Alpha 1–2 freezing drier (Christ, Germany) and collected as the complex of mangiferin and γ-CD. Mangiferin and CD were also mixed at the molar ratio of 1:1 in the mortar, and collected as the physical mixture of mangiferin and γ-CD.

Characterization of the mangiferin/γ-CD complex    In order to investigate the physicochemical properties of the mangiferin/γ-CD complex, the UV, IR, XRD and NMR spectra of mangiferin, γ-CD, their physical mixture and complex were compared. The UV spectra of the aqueous solution of the samples were obtained on a PERSEE TU1810PC UV spectrophotometer (Beijing, China) with the scanning range of 220–400 nm. Their FT-IR spectra (400–4 000 cm⁻¹) were recorded on a TENSOR 27 spectrophotometer (Bruker, Ettlingen, Germany) by the KBr method. For XRD analysis, their diffraction patterns in the range of 5–70^o were measured with a Bruker D8 Advance X-ray diffractometer (Ettlingen, Germany) using Cu Ka radiation (wavelength = 1.54056 A°). Both the ¹H-NMR spectra of mangiferin and its complex in D2O were also collected by a 400 MHz Avance spectrometer (Bruker, Ettlingen, Germany) at 25 °C.

Chemical antioxidant assay    Both the DPPH and ABTS radical scavenging activities of mangiferin and its complex with γ-CD were evaluated according to the previous report (Geng et al., 2016). For the DPPH assay, 2 mL of the ethanol solution of the sample containing 0–16 µg mangiferin/mL were mixed with 2 mL of the ethanol solution of DPPH (2×10⁻⁴ mol/L). The mixture was incubated in the dark at 25 °C for 30 min. Then, its absorbance at 517 nm (Abs_sample) was read by a UNICO 7200 spectrophotometer (Shanghai, China). The absorbance of control (Abs_control) with ethanol instead of sample was also measured. The DPPH radical scavenging activity of the sample could be calculated based on Equation 3:   

For the ABTS assay, 200 mL ABTS (7 mM) and 3.52 mL potassium persulfate (2.45 mM) were mixed, kept in the dark at room temperature for 12 h, and collected as the ABTS test solution. Before test, the ABTS test solution was diluted until its absorbance of 0.70 ± 0.02 at 734 nm. 0.15 mL of the sample aqueous solution at different concentrations (0–60 µg mangiferin/mL) and 2.85 mL of the diluted ABTS test solution were mixed and incubated at room temperature for 10 min. Then, the absorbance of mixture (Abs_sample) at 734 nm was determined by a UNICO 7200 spectrophotometer (Shanghai, China). The absorbance of control (Abs_control) with water instead of sample was also measured. The ABTS radical scavenging activity of the sample could be calculated based on Equation 4:   

Statistical analysis    The results were expressed as mean ± SD (n=3). The statistical comparison was based on the Student's t test. The p value of < 0.05 was considered to be significant.

Results and Discussion

Phase solubility analysis    The phase solubility analysis was widely applied to evaluate the binding capacities of guest molecules with CDs. Fig. 1 showed the phase solubility results of mangiferin with different CDs. α-CD failed to encapsulate mangiferin.β-CD (K_s, 573 M⁻¹), HP-β-CD (K_s, 881 M⁻¹) and M-β-CD (K_s, 465 M⁻¹) possessed the similar binding capacities. But γ-CD (K_s, 2701 M⁻¹) exhibited the highest binding capacity with mangiferin. The concentration of dissolved mangiferin increased in the linear manner with the addition amount of γ-CD (r² = 0.9964), which suggested that γ-CD could completely encapsulate mangiferin at the molar ratio of 1:1. The hydrophobicity and volume of guest molecules are the important factors affecting the inclusion effect of CDs. It could be concluded that mangiferin could better match the cavity of γ-CD in hydrophobicity and molecular volume. So it could continuously expel water molecules from the cavity, and enter into the cavity. As a result, the aqueous solubility of mangiferin was significantly improved by γ-CD. For other used CDs, their cavity sizes were inferior to that of γ-CD, which led to their poor performance.

Fig. 1.

Phase-solubility diagrams of the inclusion complexes formed by mangiferin and different CDs

ITC analysis    ITC measurement can directly obtain the binding constant and thermodynamic parameters between host and guest molecules (Bouchemal and Mazzaferro, 2012). From Table 1 and Fig. 2, it was found that the binding constant (K_a) of γ-CD with mangiferin was significantly superior to those of other CDs, and for α-CD, the heat flow induced by its interaction with mangiferin was not observed, which coincided with the phase solubility results. All the binding-site number (n) was near 1, indicating that mangiferin interacted with CDs at the molar ratio of 1:1. The negative ΔG and ΔH suggested that the binding of mangiferin with those CDs (β-CD, M-β-CD, HP-β-CD and γ-CD) was a spontaneous process, in which the heat was released. The lowest ΔG (the change of free energy) and ΔH (the change of enthalpy) values were also found by γ-CD, confirming that it had the highest binding capacity with mangiferin.

Table 1. Thermodynamics Parameters of mangiferin and CDs from ITC Study

CD	γ-CD	HP-β-CD	β-CD	6-O-α-D-β-CD	α-CD
Ka (M−1)	1.746×104	1.499×103	9.56×102	9.11×102	--
n	0.849	0.738	0.960	0.900	--
ΔH(kJ/mol)	−33.5	−18.98	−15.86	−8.615	--
Kd (M)	5.728×10−5	6.670×10−4	1.046×10−3	1.098×10−3	--
ΔS(J/mol·K)	−29.29	−1.795	4.738	28.24	--
ΔG(kJ/mol)	−24.62	−18.44	−17.30	−17.16	--

Fig. 2.

ITC results of mangiferin and CDs

Interaction modes and energies    In the above study, α-CD failed to bind mangiferin. Although HP-β-CD could encapsulate mangiferin, it was actually a mixture of the products with different degrees and substitution sites. As a result, when we investigated the interaction mechanism by using the theoretical method, we didn't take α-CD and HP-β-CD into account. The molecular docking is a simple and convenient method to study the binding patterns between host and guest molecules (Anighoro and Bajorath, 2019), which can provide initial conformation for subsequent high level calculations (molecular dynamics, quantum chemistry). The initial binding modes of mangiferin and the CDs (γ-CD, β-CD and M-β-CD) were obtained by the molecular docking method (Fig. 3 A1, A2 and A3). All the binding modes suggested that mangiferin entered the cavities of these CDs. Then, these binding modes was further optimized by the ONIOM calculation method, which could provide the more accurate interaction modes and energies. The ONIOM calculation processes were exhibited in Fig. 4. The optimized complexes had the more reasonable conformations (Fig. 3 B1, B2 and B3). For example, the glucosyl group of mangiferin extended out of the CD cavity, which could interacted better with water molecules.γ-CD, β-CD and M-β- CD interacted with mangiferin by 3, 4 and 3 hydrogen bonds, respectively. The interaction energy of mangiferin with γ-CD (−71.08 kJ/mol) were lower than those with β-CD (−32.23 kJ/mol) and M-β-CD (−41.69 kJ/mol), which convincingly explained the outstanding performance of γ-CD in the phase solubility and ITC assays.

Fig. 3.

Molecular docking and ONIOM calculation results (binding mode and interaction energy) of mangiferin with CDs (A1, A2 and A3 are the molecular docking results of γ-CD, β-CD and M-β-CD, respectively; B1, B2 and B3 are the ONIOM calculation results of γ-CD, β-CD and M-β-CD, respectively)

Fig. 4.

ONIOM calculation process for the complexes of mangiferin and different CDs

IGM analysis    The IGM method is the newest development of molecular simulation (Lefebvre et al., 2017). It can quantify and visualize the interactions between and within segments of a system by calculating the functions of δ_g^inter, δ_g^intra and sign(λ₂)p. The scatter diagrams of δ_g^inter and δ_g^intra vs. sign(λ₂)p for γ-CD, β-CD and M-β-CD were shown in Fig. 5 (A1, A2 and A3), respectively. The black and red scatters represented δ_g^intra and δ_g^inter, respectively. For all the CDs, a red scatter peak at the sign(λ₂)p of −0.04 could be observed, which confirmed the presence of hydrogen bonds between mangiferin and CD (Lefebvre et al., 2017). By using VMD program, the colour-filled δ_g^inter isosurfaces for the mangiferin/CD complexes could be visualized (Fig. 5 B1, B2 and B3). These surfaces were located in the cavities between CDs and mangiferin, and consisted of the blue and green zones, which represented hydrogen bonding and van der Waals force, respectively. It could be concluded that hydrogen bonding and van der Waals force played the important role in maintaining these complexes. In Fig. 5 (C1, C2 and C3), the electrostatic interactions between mangiferin and CDs were also exhibited. The electrostatic potential value of the red region was positive, indicating that this region was easier to accept electrons, or more nucleophilic than other regions. The blue region had the negative electrostatic potential value, which meant that this region was more likely to provide electrons, and more electrophilic than other regions. For the complexes, the electrostatic potential of mangiferin could almost match that of CD. The hydrogen bonding formed at the areas of overlap to maintain the complexes.

Fig. 5.

IGM and electrostatic potential results of the interaction between mangiferin and CDs (A1, A2 and A3 are the scatter diagrams of δ_g^inter and δ_g^intra vs. sign(λ2)p for the complexes of mangiferin with γ-CD, β-CD and M-β-CD, respectively; B1, B2 and B3 are the colour-filled δ_g^inter isosurfaces for the complexes of mangiferin with γ-CD, β-CD and M-β-CD, respectively; C1, C2, C3 are the electrostatic potential on the molecular surface for the complexes of mangiferin with γ-CD,

The complex of mangiferin and γ-CD    For its high binding performance, γ-CD was selected to prepare the complex with mangiferin. By using the traditional lyophilization method, the mangiferin/γ-CD complex was prepared, whose mangiferin content was determined as 15.3 ± 0.2% by HPLC. The UV, IR and XRD spectra of mangiferin, γ-CD, their physical mixture and complex were measured. For the absence of unsaturated bonds, no characteristic UV absorbance peak was found by γ-CD. But for mangiferin, its physical mixture and complex had the same characteristic UV absorbance peaks at 240, 255, 320 and 366 nm, suggesting that mangiferin and γ-CD interacted by non- covalent bonds (Fig. 6). In the IR analysis (Figure 7), mangiferin exhibited the characteristic absorption bands of hydroxyl group (3 365 cm⁻¹), C=O group (1 650 cm⁻¹), aromatic nucleus (1 496, 1 566 and 1 588 cm⁻¹), C-H and C-O stretching vibration (1 096 and 1 074 cm⁻¹). γ-CD showed the main absorption bands at 3 365 cm⁻¹ (for O-H stretching vibration), 2 938 cm⁻¹ (for C-H stretching vibration), 1 650 (O-H bending vibration), and 1 158 and 1 051 cm⁻¹ (for C-H and C-O stretching vibration). For their physical mixture, the spectral superposition effect of mangiferin and γ-CD could be found. That is, the spectra of mangiferin and γ-CD simply overlapped, and there was no change in the characteristic peaks. But the FT-IR spectrum of the complex mainly reflected the characteristics of γ-CD, in which the intensities of the characteristic absorption bands of mangiferin between 1 500–1 600 cm⁻¹ decreased, suggesting that the aromatic rings of mangiferin entered the cavity of γ-CD. In the XRD analysis (Figure 8), mangiferin and γ-CD exhibited the numerous sharp peaks, consistent with their crystalline nature. Their characteristics peaks could be also found in the XRD pattern of the physical mixture. But the XRD pattern of the complex showed three broad peaks, confirming its amorphous character. It could be concluded that during the formation of the complex, mangiferin was molecularly dispersed in the matrix of γ-CD, which led to the loss of their crystalline features. The comparison of the NMR chemical shifts of protons on the aromatic rings of mangiferin in the absence and presence of γ-CD was shown in Table 2. The significant upfield shifts of H6, H10 and H13 were observed, which was due to the shielding effect of γ-CD on the embedded mangiferin. Compared with H10 and H13, H6 could be closer to the center of the cavity for its lager shift value (Li et al., 2019). Based on UV, IR, XRD and NMR result, it could be concluded that mangiferin was entrapped inside the cavity of γ-CD, and the complexation was maintained by non-covalent bonds, which coincided with the theoretical calculation results.

Fig. 6.

UV spectra of mangiferin, γ-CD, their physical mixture and complex

Fig. 7.

IR spectra of mangiferin, γ-CD, their physical mixture and complex

Fig. 8.

XRD spectra of mangiferin, γ-CD, their physical mixture and complex

Table 2. NMR Chemical shifts of the protons on the aromatic rings of mangiferin before and after complexation with γ-CD

	H	δFree (ppm)	δComplex (ppm)	Δδ (ppm)
	6	6.88	6.26	−0.62
	10	6.94	6.47	−0.47
	13	7.40	7.03	−0.37

Chemical antioxidant activities    The authoritative DPPH and ABTS radical scavenging assays were applied to evaluate the effect of complexation on the chemical antioxidant activities of mangiferin (Fig. 9). The results of two chemical antioxidant assays were displayed as IC₅₀ values. In the DPPH assay, the IC50 values of mangiferin, mangiferin/γ-CD complex and tertiary butylhydroquinone (TBHQ) were 11.07 µg/mL, 8.02 µg/mL (mangiferin equivalent) and 5.53 µg/mL, respectively. As for ABTS assay, the mangiferin/γ-CD complex exhibited the highest ABTS radical scavenging activity (27.27 µg/mL, mangiferin equivalent) followed by mangiferin (38.90 µg/mL) and TBHQ (44.16 µg/mL). Meanwhile, considering that γ-CD has poor antioxidant activity, we can infer that the antioxidant activity of mangiferin could be improved by complexation. The similar phenomena had also been reported in the previous studies (Cheng et al., 2018; Li et al., 2019). It could be due to the influence of the formation of complex on the electron distribution of mangiferin, which then affected the ability of phenolic hydroxyl group to gain and lose electrons. At the same time, the phenolic hydroxyl group entered the cavity after complexation, avoiding the oxidation of air and making it more stable (Abdelmalek et al., 2016; Li et al., 2019). The antioxidant activity of the obtained mangiferin/γ-CD complex was comparable to that of TBHQ. It has the potential application in foods and medicines as the natural antioxidant.

Fig. 9.

DPPH and ABTS radical scavenging activities of mangiferin, the mangiferin/γ-CD complex, γ-CD and TBHQ

Conclusions

In this study, the binding capacities of the CDs (α-CD, β-CD, HP-β-CD, M-β-CD and γ-CD) with mangiferin were compared by using the phase solubility and ITC methods. γ-CD exhibited the highest binding capacity, which attributed to its lowest interaction energy with mangiferin based on the analysis of molecular docking, ONIOM calculation and IGM visualization. By complexing with γ-CD, the antioxidant activity of mangiferin could be significantly improved. The obtained results in this study can prompt the application of mangiferin as nutraceuticals. The proposed method of combining molecular docking, ONIOM calculation and IGM visualization provides a convenient way to investigate the interaction between polyphenols and CDs.

Acknowledgements    This work was supported by National Natural Science Foundation of China (No.32072180 and 31771941), Natural Science Foundation of Henan Province of China (No. 212300410005) and Key Scientific Research Project of Colleges and Universities in Henan Province of China (20zx016).

Conflict of interest    There are no conflicts of interest to declare.

References

•  Abdelmalek,  L.,  Fatiha,  M.,  Leila,  N.,  Mouna,  C.,  Nora,  M., and  Djameleddine,  K. (2016). Computational study of inclusion complex formation between carvacrol and β-cyclodextrin in vacuum and in water: Charge transfer, electronic transitions and NBO analysis. J. Mol. Liq., 224, 62-71.
•  Andreu,  G. P.,  Delgado,  R.,  Velho,  J. A.,  Curti,  C., and  Vercesi,  A. E. (2005). Iron complexing activity of mangiferin, a naturally occurring glucosylxanthone, inhibits mitochondrial lipid peroxidation induced by Fe2⁺-citrate. Eur. J. Pharmacol., 513, 47-55.
•  Anighoro,  A., and  Bajorath,  J. (2016). Three-dimensional similarity in molecular docking: prioritizing ligand poses on the basis of experimental binding modes. J Chem. Inf. Model., 56, 580-587.
•  Bouchemal,  K., and  Mazzaferro,  S. (2012). How to conduct and interpret ITC experiments accurately for cyclodextrin-guest interactions. Drug Discov. Today, 17, 623-629.
•  Carvalho,  A. C. S.,  Guedes,  M. M.,  Souza,  A. L. D.,  Trevisan,  M. T. S., and  Rao,  V. S. N. (2007). Gastroprotective effect of mangiferin, a xanthonoid from Mangifera indica, against gastric injury induced by ethanol and indomethacin in rodents. Planta Med., 73, 1372-1376.
•  Ceborska,  M.,  Kedra-Krolik,  K.,  Kowalska,  A. A., and  Kozbial,  M. (2018). Comparative study of molecular recognition of folic acid subunits with cyclodextrins. Carbohyd. Polym., 184, 47-56.
•  Cheng,  L.,  Feng,  T.,  Zhang,  B.,  Zhu,  X.,  Hamaker,  B.,  Zhang,  H., and  Campanella,  O. (2018). A molecular dynamics simulation study on the conformational stability of amylose-linoleic acid complex in water. Carbohyd. Polym., 81, 242-252.
•  Chung,  L. W.,  Sameera,  W. M. C.,  Ramozzi,  R.,  Page,  A. J.,  Hatanaka,  M.,  Petrova,  G. P.,  Harris,  T. V.,  Li,  X.,  Ke,  Z., and  Liu,  F. (2015). The ONIOM method and its applications. Chem. Rev., 115, 5678-5796.
•  Deshaware,  S.,  Gupta,  S.,  Singhal,  R. S.,  Joshi,  M., and  Variyar,  P. S. (2018). Debittering of bitter gourd juice usingβ-cyclodextrin: Mechanism and effect on antidiabetic potential. Food Chem., 262, 78-85.
•  Ferreira,  F. D.,  Valentim,  I. B.,  Ramones,  E. L. C.,  Trevisan,  M. T. S.,  Olea-Azar,  C.,  Perez-Cruz,  F.,  de Abreu,  F. C., and  Goulart,  M. O. F. (2013). Antioxidant activity of the mangiferin inclusion complex with β-cyclodextrin. LWT-Food Sci.Technol., 51, 129-134.
•  Ferreira,  F. D. R.,  Silva,  E. G. D.,  Leo,  L. P. M. D.,  Calvo,  E. J., and  Abreu,  F. C. D. (2010). Electrochemical investigations into host-guest interactions of a natural antioxidant compound with β-cyclodextrin. Electrochim. Acta, 56, 797-803.
•  Geng,  S.,  Chen,  Y. S.,  Abbasi,  A. M.,  Ma,  H. J.,  Mo,  H. Z., and  Liu,  B. G. (2016). Tannin fraction from Ampelopsis grossedentata leaves tea (Tengcha) as an antioxidant and α-glucosidase inhibitory nutraceutical. Int. J. Food Sci. Tech., 51, 2692-2700.
•  Gómez-Zaleta,  B.,  Ramírez-Silva,  M. T.,  Gutiérrez,  A.,  González-Vergara,  E.,  Guizado-Rodríguez,  M., and  As-Hernández,  A. R. (2006). UV/vis, ¹H, and 1³CNMR spectroscopic studies to determine mangiferin pK_a values. Spectrochim. Acta A, 64, 1002-1009.
•  Hou,  X.,  Du,  J.,  Zhang,  J.,  Du,  L.,  Fang,  H., and  Li,  M. (2013). How to improve docking accuracy of AutoDock4.2: A case study using different electrostatic potentials. J. Chem. Inf. Model., 53, 188-200.
•  Huang,  L.,  He,  J.,  Lu,  R.,  Ge,  X., and  Guo,  J. (2011). Investigation on a host-guest inclusion system by β-cyclodextrin derivative and its analytical application. Bioorg. Med. Chem. Lett., 21, 1113-1117.
•  Humphrey,  W.,  Dalke,  A., and  Schulten,  K. (1996). VMD: Visual molecular dynamics. J. Mol. Graph. Model., 14, 33-38.
•  Jyotshna,  Khare,  P., and  Shanker,  K. (2016). Mangiferin: A review of sources and interventions for biological activities. BioFactors, 42, 504-514.
•  Khurana,  R. K.,  Bansal,  A. K.,  Beg,  S.,  Burrow,  A. J.,  Katare,  O. P.,  Singh,  K. K., and  Singh,  B. (2017). Enhancing biopharmaceutical attributes of phospholipid complex-loaded nanostructured lipidic carriers of mangiferin: Systematic development, characterization and evaluation. Int. J. Pharmaceut., 518, 289-306.
•  Lefebvre,  C.,  Rubez,  G.,  Khartabil,  H.,  Boisson,  J. C.,  Contrerasgarcía,  J., and  Hénon,  E. (2017). Accurately extracting the signature of intermolecular interactions present in the NCI plot of the reduced density gradient versus electron density. Phys. Chem. Chem. Phys., 19, 17928-17936.
•  Li,  J.,  Geng,  S.,  Wang,  Y.,  Lv,  Y.,  Wang,  H.,  Liu,  B., and  Liang,  G. (2019). The interaction mechanism of oligopeptides containing aromatic rings with β-cyclodextrin and its derivatives. Food Chem., 286, 441-448.
•  Liu,  B. G.,  Zeng,  J.,  Chen,  C.,  Liu,  Y. L.,  Ma,  H. J.,  Mo,  H. Z., and  Liang,  G. Z. (2016). Interaction of cinnamic acid derivatives with beta-cyclodextrin in water: Experimental and molecular modeling studies. Food Chem., 194, 1156-1163.
•  Liu,  Y.,  Chen,  Y.,  Li,  L.,  Zhang,  H.Y.,  Liu,  S.X., and  Guan,  X. D. (2001). Bridged Bis(β-cyclodextrin)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, 8518-8527.
•  Lu,  T., and  Chen,  F. (2012). Multiwfn: A multifunctional wavefunction analyzer. J. Comput. Chem., 33, 580-592.
•  Morris,  G. M.,  Huey,  R.,  Lindstrom,  W.,  Sanner,  M. F., and  Olson,  A. J. (2009). AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility. J. Comput. Chem., 30, 2785-2791.
•  Potter,  C. F.,  Russell,  N. R., and  McNamara,  M. (2006). Spectroscopic characterisation of metallo-cyclodextrins for potential chiral separation of amino acids and L/D-DOPA. J. Incl. Phenom. Macro., 56, 395-403.
•  Prabhu,  S.,  Jainu,  M.,  Sabitha,  K., and  Devi,  C. S. (2006). Role of mangiferin on biochemical alterations and antioxidant status in isoproterenol-induced myocardial infarction in rats. J. Ethnopharmacol., 107, 126-133.
•  Stewart,  J. J. P. (2007). Optimization of parameters for semiempirical methods V: Modification of NDDO approximations and application to 70 elements. J. Mol. Model., 13, 1173-1213.
•  You,  G. J.,  Sun,  L. L.,  Cao,  X. X.,  Li,  H. H.,  Wang,  M.,  Liu,  Y. N., and  Ren,  X. L. (2018). Comprehensive evaluation of solubilization of flavonoids by various cyclodextrins using high performance liquid chromatography and chemometry. Lwt-Food Sci. Technol., 94, 172-177.
•  Zhang,  C. L.,  Liu,  J. C.,  Yang,  W. B.,  Chen,  D. L., and  Jiao,  Z. G. (2017). Experimental and molecular docking investigations on the inclusion mechanism of the complex of phloridzin and hydroxypropyl-β-cyclodextrin. Food Chem., 215, 124-128.
•  Zhang,  H.,  Hou,  Y.,  Liu,  Y.,  Yu,  X.,  Li,  B., and  Cui,  H. (2010). Determination of mangiferin in rat eyes and pharmacokinetic study in plasma after oral administration of mangiferin-hydroxypropyl-β-cyclodextrin inclusion. J. Ocul. Pharmacol. Th., 26, 319-324.
• i) https://pubchem.ncbi.nlm.nih.gov (Sep. 16, 2004