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    Home > Food News > Food Articles > Prof. Jinling Fan, Henan University of Science and Technology, et al.: Preparation and properties of photoglycyrrhizidine/cyclodextrin solid complex

    Prof. Jinling Fan, Henan University of Science and Technology, et al.: Preparation and properties of photoglycyrrhizidine/cyclodextrin solid complex

    • Last Update: 2022-11-04
    • Source: Internet
    • Author: User
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    Photoglycyrrhizin (GLD) is a unique hydrophobic isoflavone compound of licorice with a content of 0.
    1%~0.
    3%, which has various biological activities
    such as anti-pigmentation, antioxidant, anti-cell proliferation, anti-inflammatory, memory enhancement, anti-osteoporosis and antibacterial.
    GLD is poorly soluble in water (7 μg/mL, 25 °C), resulting in low dissolution, poor absorption and bioavailability in the gastrointestinal tract, and its application in related fields such as food and pharmaceuticals with water-soluble matrices is greatly limited
    .
    Therefore, improving the solubility of GLD in water is key to
    developing its potential application value.

    Cyclodextrin (CD) is a general term for a series of cyclic carbohydrates produced by amylose under the action of glucosyltransferase produced by bacillus, CD and its derivatives have a cylindrical structure composed of a hydrophilic outer surface and a relatively hydrophobic central cavity, this unique structural property enables CD to interact with a variety of compounds, especially hydrophobic compounds, through non-covalent forces (van der Waals forces, electrostatic interactions and hydrogen bonds), the latter is encapsulated in the cavity to form a host-guest complex, This improves the solubility of poorly soluble compounds, thereby improving their absorption and bioavailability
    in the body.


    Yao Peipei, Fan Jinling* and others from the School of Food and Biological Engineering, Henan University of Science and Technology, screened CDs suitable for GLD by molecular docking and phase solubility combination, and prepared solid clathrates.
    The effects of different drying methods and feeding ratios on the adduction rate, drug load and solubility of solid clathrates were investigated.
    Scanning electron microscopy (SEM), differential scanning calorimetry (DSC) method, Fourier transform infrared spectroscopy (FTIR) method and molecular docking technology were used to analyze the morphology of solid clathrates, the presence form of GLD, the interaction between GLD and 2-SBE-β-CD and spatial conformation.
    On this basis, the in vitro dissolution characteristics of GLD/2-SBE-β-CD solid clathrates and the inhibitory effect
    of GLD/2-SBE-β-CD solid clathrates on the proliferation of HepG-2 cells were further studied.


    1.
    Analysis of the cladding ability of CD and GLD and the stability of the clathrate


    Analysis of the results of molecular docking method screening CD


    The optimal combination of different types of CDs and GLD docking can be seen in Table 1
    .
    The optimal binding energy of α-CD and γ-CD is lower than that of β-CD, indicating that α-CD, γ-CD and GLD can form more stable complexes
    than β-CD 。 Among the derivatives of β-CD, the best binding energy of β-CD derivatives, triacetyl-β-CD and 6-quaternary-ammonium-β-CD, was higher than that of β-CD after substitution.
    The optimal binding energy of 2-SBE-β-CD, 6-HP-β-CD, 2,6-M-β-CD, 6-sulfate-β-CD, mono-6-amino-β-CD and 6-carboxymethyl-β-CD after substitution were lower than those of β-CD
    .
    Among them, the binding energy of 2-SBE-β-CD docking with GLD is significantly lower than that of other types of CD, which is -8.
    60 kcal/mol
    .
    These results show that the cavity size and type of substituent of CD are important factors affecting the ability of CD and its derivatives to envelop guests, and the stability of 2-SBE-β-CD and GLD encapsulation is the best
    .


    Analysis of the results of phase solubility screening of CDs


    α-CD, β-CD, γ-CD and three β-CD derivatives with less binding energy (6-HP-β-CD, 2,6-M-β-CD and 2-SBE-β-CD) were selected, and their encapsulation with
    GLD was further studied by phase solubility method.
    Among them, α-CD failed to form an clathrate with GLD, and the phase solubility curves of the other five CDs clad with GLD are shown in
    Figure 1.
    The correlation coefficient (R2) of all curve linear regression equations is greater than 0.
    98 (Table 2), that is, the phase solubility curve is AL type, indicating that CD and GLD are enveloped at a ratio of 1:1 substances
    .
    The K1:1 value is related to the stability of the clathrate, and the larger the K1:1 value, the better
    the stability of the clathrate formed by GLD and CD.

    It can be seen from Table 2 that the K1:1 of γ-CD and three β-CD derivatives are greater than β-CD, indicating that the encapsulation stability with GLD is better than that of β-CD.
    Among them, K1:1 of 2-SBE-β-CD and GLD is the largest, and the clathrate formed with GLD is the most stable
    .
    The ΔG values in GLD and different CD enveloping processes were negative, indicating that the enveloping process could be carried out spontaneously at 25 °C.
    The ΔG of γ-CD and three β-CD derivatives was lower than that of β-CD, and the ΔG of 2-SBE-β-CD and GLD was lower than that of other CDs, which coincided
    with the binding energy of the above molecules.
    It can be seen from the above results that the binding energy of 2-SBE-β-CD and GLD docking is the lowest and most stable through molecular docking simulation technology.
    Phase solubility experiments show that 2-SBE-β-CD has the best encapsulation effect on GLD.
    Therefore, 2-SBE-β-CD was selected for subsequent experiments
    .

    2.
    Analysis of the adclation rate, drug load and saturated solubility of GLD/2-SBE-β-CD solid clathrates


    Effect of preparation method on GLD/2-SBE-β-CD solid complex


    When the ratio of GLD to 2-SBE-β-CD is 1:1, the GLD/2-SBE-β-CD solid clathrate obtained by dry preparation and three wet preparation methods is shown in
    Figure 2.

    It can be seen from Table 3 that the inclusion rate and drug load of the clathrate obtained by the four preparation methods are not significant, the inclusion rate is 93.
    33%~97.
    68%, and the drug load is 11.
    19%~11.
    89%.
    The clathrates prepared by different drying methods can significantly improve the saturated solubility of GLD (7 μg/mL of GLD without encapsulation) because CD has a hydrophilic surface structure, and its cavity inner wall is composed of carbon chain bone structure, which exhibits hydrophobicity, so it can form clathrates with poorly soluble compounds through van der Waals force or hydrogen bonding, thereby improving the solubility
    of poorly soluble compounds 。 Except for the saturated solubility of the clathrate prepared by co-evaporation method that was significantly lower than that of the spray drying method, the saturated solubility of the clathrate prepared by the other three preparation methods (freeze drying method, spray drying method and kneading method) was not significantly different, all of which were greater than 83 mg/mL
    .


    Effect of feeding ratio on GLD/2-SBE-β-CD solid clathrates


    It can be seen from Table 4 that the ratio of GLD to 2-SBE-β-CD substances increased, the inclusion rate decreased and the drug load increased
    .
    When the ratio of GLD to 2-SBE-β-CD substance was 1.
    5:1, the inclusion rate was 86.
    09% and the drug load was 22.
    39%, compared with the volume ratio of GLD and 2-SBE-β-CD substances of 1:1 and 1:1.
    5, the inclusion rate decreased by 7.
    75% and 8.
    58%, and the drug load increased by 88.
    31% and 141.
    14%,
    respectively.

    The above results show that different preparation methods have no significant effect on the cladding rate and drug load of GLD/2-SBE-β-CD solid complexes, but have a certain effect
    on the water solubility of the clathrates.
    Appropriately increase the ratio of GLD to 2-SBE-β-CD substances, although the inclusion rate decreases to a certain extent, but can significantly increase the drug load
    .


    3.
    Characterization of GLD/2-SBE-β-CD solid clathrates


    SEM results analysis


    As shown in Figure 3, 2-SBE-β-CD is a spherical structure of different sizes, smooth surface and depression, which is consistent with the literature report.
    GLD has a columnar crystal structure with a clear outline; Both spherical 2-SBE-β-CD and columnar GLD can be observed in the GLD/2-SBE-β-CD physical mixture, indicating a simple mixture
    of the two 。 The morphological characteristics of GLD/2-SBE-β-CD clathrates are significantly different from physical mixtures, indicating that there is an interaction between host and guest molecules.
    The external morphology of the clathrate is quite different depending on the preparation method: the clathrate prepared by freeze-drying method has a sheet structure with sharp edges; The clathrates prepared by kneading and co-evaporation are irregular block structures.
    The clathrates prepared by spray drying method are spherical particles (less than 10 μm) with smooth surface and small particle size, and the particle size is significantly smaller than that prepared by freeze drying, kneading and co-evaporation
    .


    DSC results analysis


    As shown in Figure 4, GLD has a sharp melting peak at 233 °C, which is the melting point
    of the crystal.
    2-SBE-β-CD has two peaks, and a short and wide endothermic peak appears at 40~160 °C, which is the endothermic peak released by water in its cavity.
    There is an endothermic peak at 270 °C, which represents 2-SBE-β-CD decomposition, which is consistent with
    the results reported in the literature.
    The GLD/2-SBE-β-CD physical mixture had endothermic peaks at 233 °C and 270 °C, which was reflected in the simple superposition of GLD and 2-SBE-β-CD endothermic peaks, indicating that GLD still exhibited crystal characteristics
    in the physical mixture.
    There was no significant difference in the DSC diagram of GLD/2-SBE-β-CD solid clathrate prepared by freeze drying, spray drying, kneading and co-evaporation (the d curve in the figure was prepared and measured by freeze drying, and the rest was omitted).

    。 Compared with 2-SBE-β-CD, the peak intensity of the first peak of the GLD/2-SBE-β-CD solid clathrate is reduced and shifted, indicating that the insertion of GLD into the cavity causes the migration of water molecules, and the shift is speculated to be the formation of an interacting hydrogen bond between GLD and 2-SBE-β-CD.
    The second peak shifted to 276 °C, corresponding to the decomposition of 2-SBE-β-CD in GLD/2-SBE-β-CD solid complexes.
    The absence of melting peaks of GLD in the DSC curve of the clathrate indicates that the crystalline GLD transitions to an amorphous state
    due to the formation of GLD/2-SBE-β-CD solid clathrates.
    These results all indicate that 2-SBE-β-CD has encapsulated GLD in the cavity to form a solid clathrate
    .


    Analysis of FTIR results


    As shown in Figure 5, in the spectrum of GLD, a strong and wide peak appears at the 3 346 cm-1 wavenumber, which is the telescopic vibration of O—H; 1 518, 1 464 cm-1 wavenumber is the telescopic vibration of the aromatic ring C=C; The absorption peak
    of -CH3 at the wavenumber of 2 964 and 2 920 cm-1 is -CH3.
    In the spectrum of 2-SBE-β-CD, the characteristic band related to O—H telescopic vibration was exhibited before and after the 3 430 cm-1 wavenumber.
    2 935 cm-1 wavenumber is the telescopic vibration of C-H; 1 653 cm-1 wavenumber is the bending vibration of water molecules; 1 163, 1 039 cm-1 wavenumber is the telescopic vibration
    of C—O.
    In the spectra of the GLD/2-SBE-β-CD physical mixture, the characteristic absorption peaks of GLD and 2-SBE-β-CD still existed, indicating that the physical mixture was a simple superposition of host and guest molecules, and that there was no or only weak interaction
    between GLD and 2-SBE-β-CD.
    There was no significant difference in the FTIR plots of GLD/2-SBE-β-CD solid complexes prepared by different methods (the d curve in the figure was prepared and measured by freeze-drying method, and the rest was omitted).


    Analysis of GLD and 2-SBE-β-CD molecules


    As shown in Figure 6, GLD molecules are able to enter the cavity of 2-SBE-β-CD intact
    .
    GLD and 2-SBE-β-CD molecules form two hydrogen bonds: the oxygen of the C2 sulfonic acid group in two adjacent glucose units of 2-SBE-β-CD forms a hydrogen bond with the hydrogen at the C2 position and C3 position O—H on the B ring of the GLD molecule, respectively; The hydrogen bonding distances are 2.
    1 and 2.
    3 Å
    , respectively.
    It was shown that hydrogen bonding is one of
    the main forces of GLD interaction with 2-SBE-β-CD.


    4.
    GLD/2-SBE-β-CD solid clathrate analysis in simulated gastrointestinal fluid


    As can be seen from Fig.
    7, the cumulative dissolution rate of GLD and GLD/2-SBE-β-CD physical mixture is less than 10% in 1 h in simulated gastric juice at pH 1.
    2 and simulated intestinal fluid at pH 6.
    9.
    In contrast, the cumulative dissolution rate of GLD/2-SBE-β-CD complexes in simulated gastric juice and intestinal fluid was significantly improved, and the cumulative dissolution rate reached 87% and 83%
    in 1 h, respectively.
    The results show that GLD/2-SBE-β-CD solid clathrates can effectively increase the dissolution of GLD, which is caused
    by the hydrogen bonding interaction between GLD and CD and the decrease of the crystallinity of GLD in the clathrate.
    This indicates the potential of
    clathrates to improve GLD absorption.

    5.
    Inhibitory effect of GLD/2-SBE-β-CD solid clathrate on HepG-2 cell proliferation


    The result is shown
    in Figure 8.
    The GLD/DMSO group showed strong inhibition of HepG-2 cell proliferation with an IC50 of 37.
    68 μmol/L
    .
    The GLD/H2O group had a weak inhibitory effect on the proliferation of HepG-2 cells, and its IC50 was 162.
    29 μmol/L
    .
    The inhibition rate of HepG-2 cell proliferation in the 2-SBE-β-CD group was less than 2%, indicating that 2-SBE-β-CD is almost non-toxic
    .
    The IC50 value of GLD/2-SBE-β-CD group on HepG-2 cell proliferation inhibition was 74.
    69 μmol/L, which was significantly better than that of GLD/H2O group
    .

    Conclusion

    In addition to α-CD, β-CD, γ-CD, 6-HP-β-CD, 2,6-M-β-CD and 2-SBE-β-CD can increase the solubility of GLD to varying degrees, and the phase solubility curves of GLD are all AL type, indicating that the amount of substances formed with GLD is 1:1 clathrate.

    Among them, the ability of 2-SBE-β-CD to envelop GLD is better than that of other CDs and their derivatives.
    There was no significant difference in the cladding rate and drug load of GLD/2-SBE-β-CD solid clathrates prepared by different preparation methods, but the saturated solubility of clathrates was different
    .
    The ratio of GLD to 2-SBE-β-CD substances was appropriately increased, and although the inclusion rate decreased to a certain extent, the drug load could be significantly increased
    .
    When the ratio of GLD to 2-SBE-β-CD was 1.
    5∶1, the cladding rate and drug load of GLD/2-SBE-β-CD solid clathrates prepared by freeze-drying were 86.
    09% and 22.
    39%,
    respectively.
    When the ratio of GLD to 2-SBE-β-CD substance was 1:1, the saturated solubility of the clathrate prepared by freeze drying, spray drying and kneading was greater than 83 mg/mL
    .
    The morphology of GLD/2-SBE-β-CD clathrates obtained by different preparation methods was significantly different, and GLD existed as an amorphous amorphous structure in GLD/2-SBE-β-CD clathrates
    .
    GLD/2-SBE-β-CD complexes can effectively improve the dissolution of GLD in simulated gastric and intestinal fluids.
    The preparation method had no significant effect on the dissolution characteristics of GLD/2-SBE-β-CD complexes.
    At the same time, GLD/2-SBE-β-CD clathrate can also effectively ensure the anti-tumor proliferative activity
    of GLD.
    Therefore, GLD/2-SBE-β-CD clathrate is a promising form of GLD administration, which is worthy of in-depth research on pharmacokinetics and transmembrane transport mechanisms to better expand the application potential
    of GLD.


    About the corresponding author

    Professor Fan Jinling, School of Food Science and Engineering, Henan University of Science and Technology, doctoral supervisor, mainly engaged in the development and utilization of agricultural product processing by-products and natural product resources, natural product chemistry, bioavailability of functional active ingredients and other fields, presided over 1 general project of the National Natural Science Foundation of China, 1 project of the Natural Science Foundation of Henan Provincial Department of Education, and 4 horizontal cooperation projects of enterprises; As the first author or corresponding author, he has published more than 30 papers in Food Chemistry, Journal of Functional Foods, Journal of Analytical Chemistry, Applied Chemistry, Chinese Journal of Bioengineering and other journals, published 2 books, obtained 5 invention patents authorized by the state, presided over the completion of 2 provincial science and technology projects, and won 1 second prize of Henan Province Science and Technology Progress Award; Undertake the teaching tasks
    of "Principles of Food Engineering" for undergraduate students and "New Resources Science for Food" for graduate students.


    About the first author

    Yao Peipei, School of Food Science and Engineering, Henan University of Science and Technology, master candidate
    .
    In 2019, he graduated from Xuzhou Institute of Technology with a degree in engineering; In 2022, he graduated from Henan University of Science and Technology with a master's degree
    in engineering.
    His research direction is the utilization of functional food resources and quality control
    .
    1 published patent: a water-soluble photoglycyrrhizin clathrate and preparation method thereof
    .


    This article "Preparation and Properties of Photoglycyrrhizin/Cyclodextrin Solid Complex" is from Food Science, Vol.
    43, No.
    16, pp.
    9-18, 2022, authors: Yao Peipei, Fan Jinling, Li Defeng, Zhang Xiaoyu, Ren Guoyan, Du Lin
    .
    DOI:10.
    7506/spkx1002-6630-20210901-006
    。 Click to view information about
    the article.

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