AU2020101225A4

AU2020101225A4 - Preparation Method of Curcumin-Cyclodextrin Supramolecular Inclusion Compound

Info

Publication number: AU2020101225A4
Application number: AU2020101225A
Authority: AU
Inventors: Xiaohuang Cao; Jianping Chen; Hai Liu; Ying Liu; Xiaoming QIN; Suhua SHEN; Saiyi ZHONG
Original assignee: Guangdong Ocean University
Current assignee: Guangdong Ocean University
Priority date: 2020-07-02
Filing date: 2020-07-02
Publication date: 2020-08-06
Anticipated expiration: 2028-07-02

Abstract

of Description The invention provides a preparation method of curcumin-cyclodextrin supramolecular inclusion compound, which comprises the following steps: Si. Under alkaline conditions, carrying out polymerization reaction on p-cyclodextrin and epichlorohydrin to generate cyclodextrin polymer; S2. Mixing cyclodextrin polymer and curcumin, and taking water as a solvent to obtain a water-soluble curcumin supramolecular inclusion compound. The cyclodextrin polymer provided by the invention has good water solubility and can wrap more curcumin, and is a good embedding material. The curcumin/cyclodextrin polymer supramolecular inclusion compound prepared by the invention is proved to be formed by DSC, FT-IR and XRD identification, and has better water solubility compared with the traditional curcumin/cyclodextrin inclusion compound. Meanwhile, the invention adopts HPLC as a method for detecting curcumin content, and has the advantages of strong specificity, high sensitivity, good repeatability, convenient operation and the like. The preparation method is simple, the obtained inclusion compound yield and inclusion rate meet the requirements of production and clinical application, and have better application prospect. curcumin 3427 cyclodextrin polymer 1510 physical mixture 1035 2922 4000 3600 3200 2800 2400 2000 1600 1200 800 400 d (cm1) Figure 3 8.78 17.13 7.83 15.79 13.76 inclusluSon CommplX03 5 10 15 20 25 30 35 40 45 50 55 Figure 4 2/2

Description

curcumin

3427 cyclodextrin polymer 1510

physical mixture

1035

2922

4000 3600 3200 2800 2400 2000 1600 1200 800 400 d (cm1 )

Figure 3

inclusluSon CommplX03 8.78 17.13

7.83 15.79 13.76

10 15 20 25 30 35 40 45 50 55

Figure 4

2/2

Description

Preparation Method of Curcumin-Cyclodextrin Supramolecular Inclusion

Compound

Technical Field The invention belongs to the technical field of traditional Chinese medicine preparations, and particularly relates to a preparation method of curcumin-cyclodextrin supramolecular inclusion compound.

Technical Background Curcumin is a natural flavonoid extracted from the rhizome of Curcuma longa L. of Zingiberaceae. Studies have shown that curcumin has many effects such as anti-oxidation, anti-inflammation and anti-aging. In recent years, a large number of experimental facts have proved that curcumin has inhibitory effect on a variety of tumor cells, and has been repeatedly confirmed in many animal experiments. Currently, curcumin has been listed as the third generation of tumor therapeutic drugs by the National Cancer Institute of the United States. However, curcumin is hardly soluble in water and is not convenient for injection, so oral administration is currently widely used in zoology experimental research and clinical experimental research of curcumin. However, curcumin is not easy to absorb after oral administration, and it is easily degraded in digestive tract. Its bioavailability is extremely low, and it is difficult to volatilize its anticancer and anti-cancer effects in clinical and food industries. It can be seen that this insoluble characteristic of curcumin greatly limits its application value in food and medicine. Therefore, it is particularly important to try to increase the water solubility of curcumin so as to change the route of administration and improve its bioavailability.

Cyclodextrin is a cyclic oligosaccharide compound produced by the interaction between cyclodextrin glucose transferase produced by Bacillus basophilus and starch. Cyclodextrins have many homologs, which can be divided into a-cyclodextrin, p-cyclodextrin and y-cyclodextrin according to their structures. Among them, P -cyclodextrin is composed of glucose with 7 chair configurations. Its outer end has good hydrophilicity, its inner part has hydrophobicity, and it has the properties similar to surfactant. It provides favorable conditions for the preparation of inclusion compound, thus it is widely used in thefield of traditional Chinese medicine. Studies have shown that cyclodextrin inclusion of curcumin can improve its water solubility by about 100 times and its anti-inflammatory and anti-angiogenic activities have been significantly improved. Moreover, the inclusion compound formed by P -cyclodextrin and curcumin can effectively induce leukemia cell apoptosis. Meanwhile, Chauhan et al also found that the inclusion compound formed by P -cyclodextrin and curcumin has better anti-tumor effect than curcumin alone. At the same time, cyclodextrin, as an anticancer drug carrier, has the advantages of increasing affinity with cancer cells, enabling the drug to have specificity of anticancer pharmacological activity, enhancing its targeting effect, selectively killing cancer cells, remarkably improving the inhibiting and killing effect of the drug on drug-resistant tumor cells, overcoming drug resistance, improving curative effect, reducing administration dose and reducing toxic and side effects. However, considering the problem of low water solubility if cyclodextrin is directly used as drug carrier. Therefore, there is an urgent need for a preparation method and material with high water solubility to overcome the above shortcomings.

Summary of the Invention The invention aims to provide a preparation method of curcumin-cyclodextrin supramolecular inclusion compound.

Based on the theory of supramolecular chemistry, the invention adopts polymerization reaction to synthesize cyclodextrin into cyclodextrin polymer with good water solubility as a drug carrier to prepare curcumin supramolecular inclusion compound, which provides new technical means and scientific basis for the application and development of curcumin in anti-tumor clinical drugs.

The above object of the present invention is achieved by the following scheme:

A preparation method of curcumin-cyclodextrin supramolecular inclusion compound comprises the following steps:

Si. Under alkaline conditions, p-cyclodextrin and epichlorohydrin are polymerized to generate cyclodextrin polymer;

S2. Mixing cyclodextrin polymer with curcumin, and using water as solvent to obtain water-soluble curcumin supramolecular inclusion compound.

Preferably, the mass volume ratio of p-cyclodextrin to epichlorohydrin is (2-3): 1.

Preferably, the mixing mass ratio of cyclodextrin polymer and curcumin is (3-5): 1.

More preferably, the mass ratio of cyclodextrin polymer to curcumin is 4: 1.

Preferably, the alkaline condition in Si is to add sodium hydroxide and dissolve it with water, wherein the mass fraction of sodium hydroxide is 30%-35%.

Preferably, the polymerization time is 20 to 30 hours.

Preferably, cyclodextrin polymer is obtained after polymerization reaction through ultrasonic, dialysis, filtration and drying.

Compared with the prior art, the invention has the following beneficial effects:

The cyclodextrin polymer provided by the invention has good water solubility, can wrap more curcumin, and is a good embedding material. The curcumin/cyclodextrin polymer supramolecular inclusion compound prepared by the invention is proved to be formed by DSC, FT-IR and XRD identification, and has better water solubility compared with the traditional curcumin/cyclodextrin inclusion compound. Meanwhile, the invention adopts HPLC as a method for detecting curcumin content, and has the advantages of strong specificity, high sensitivity, good repeatability, convenient operation and the like. Moreover, the preparation method is simple, the obtained inclusion compound yield and inclusion rate meet the requirements of production and clinical application, and have better application prospect.

Brief Description of Drawings Fig. 1 is an infrared spectrum of cyclodextrin polymer (a) and -cyclodextrin (b).

Fig. 2 is a differential scanning calorimeter of curcumin, cyclodextrin polymer, physical mixture of the two and inclusion compound.

Fig. 3 is an infrared spectrum of curcumin, cyclodextrin polymer, physical mixture of the two and inclusion compound.

Fig. 4 is an XRD spectrum of curcumin, cyclodextrin polymer, physical mixture of the two and inclusion compound.

Detailed Description of the Preferred Embodiments The present invention will be further described below with reference to specific examples and drawings, but the specific examples do not limit the present invention in any way. The culture medium, reagents, etc. described in the following examples are available through purchase by those of ordinary skill in the Art ..

Embodiment 1: Preparation of Curcumin Supramolecular Inclusion Compound

1. Preparation of Cyclodextrin Polymer

Weigh 15.76 g NaOH into a three-necked flask, add 32 mL H2 0, and stir to dissolve. Weigh 20 g of p-cyclodextrin again, add it into a three-necked flask, stir until it is completely dissolved, slowly add 9.64 mL of epoxy chloropropane (EPC) at °C, stir for 24 h, and cool to room temperature. Pour the reaction solution into a conical flask and perform ultrasound for 5 min. Pour the solution into a dialysis bag (first wet and clean with water, then wash the pan twice with steam room water), put it into a large beaker containing steam smoke water for dialysis, change the steam feed water every hour at the beginning, then change the water for extended time, and dialyze to neutrality. Filter that solution in the dialysis bag, filtering out insoluble substance, filtering with cellulose membrane of 0.45 [m, performing rotary steaming aft filtering, steaming to viscous state, adding anhydrous ethanol to separate out white solid, filtering, and vacuum drying to obtain cyclodextrin polymer. The infrared spectrum identification results are shown in Figure 1 below. Comparing the spectra of a and b, it can be seen that there is a strong and wide absorption peak near 3388cm-1

, which belongs to -OH stretching vibration absorption peak. A C-H stretching vibration absorption peak appears near 2926 cm-1 , which belongs to CH2 antisymmetric stretching vibration peak. An in-plane rocking vibration peak of -CH2 appeared near 750 cm- 1. However, there is no characteristic absorption peak with strong C-Cl bond at 650-800 cm-1 and no characteristic absorption peak with epoxy group at 1328 cm-1 in epichlorohydrin, which indicates that epichlorohydrin is successfully crosslinked on cyclodextrin.

2. Preparation of Curcumin Supramolecular Inclusion Compound

Curcumin and cyclodextrin polymer are mixed evenly according to the mass ratio of 1:4, added into a mortar for grinding, poured into a conical flask after grinding evenly, added with 50 mL of water, and subjected to ultrasound for 5 minutes. After the conical flask is placed on a magnetic stirrer and stirred for 2 days, the inclusion compound solution is filtered to remove undissolved curcumin, and then the solution is subjected to rotary evaporation and vacuum drying to obtain the water-soluble curcumin supramolecular inclusion compound.

3. Identification of Curcumin Supramolecular Inclusion Compound 3.1 DSC

Test conditions: N2 atmosphere, flow rate of 30 m/min, aluminum crucible, heating rate of 10.0°C/min, scanning range of 80-220°C. Curcumin, p-cyclodextrin polymer, physical mixture of the two (consistent with the feeding ratio) and inclusion compound are taken and tested under the synchronous thermal analyzer according to the above conditions. The experimental results are shown in fig. 2. As can be seen from fig. 2, curcumin has an endothermic peak at 185.7°C, which is its melting peak. However, cyclodextrin polymer has no melting peak in the scanning range, which indicates that its stability is relatively good. However, the melting peak of curcumin appeared in the mixture, indicating that curcumin was not included. However, the inclusion compound showed a new endothermic peak at 181.9°C, and the melting peak of curcumin in the inclusion compound was significantly weakened, indicating that the inclusion compound had been formed. 3.2 FI-TR

Test conditions: curcumin, j-cyclodextrin polymer, their physical mixture (consistent with the feeding ratio) and inclusion compound are mixed with KBr, ground into powder in agate mortar and mixed evenly, tabletted, and respectively measured under Fourier infrared spectrometer under the same conditions, with scanning wave number ranging from 4000 cm- 1. The experimental results are shown in fig. 3. As can be seen fromfig. 3, curcumin has a -OH stretching vibration absorption peak at 3427 cm- 1, and 1510cm-1 belongs to a C=O stretching vibration characteristic peak. Cyclodextrin polymer has a strong absorption peak at 3390 cm-1

, which is the -OH stretching vibration absorption peak. The strong absorption peak at 2920 cm-1 belongs to the anti-symmetric stretching vibration absorption peak of -CH2. The strong absorption peak at 1035cm-1 belongs to the stretching vibration absorption peak of C-O-C. The characteristic peaks of curcumin and cyclodextrin polymers can be seen from the chromatogram of the mixture, indicating that the peak shape of the mixture is the superposition of the two peak shapes. However, it can be seen from the peak shape of the inclusion compound that the characteristic peak of curcumin disappears or weakens in the inclusion compound, which proves that curcumin and cyclodextrin polymer form an inclusion compound. 3.3 XRD

Test conditions: Cu-Ka radiation, LynxExe array detector, tube voltage 40Kv, tube current 40mA, scanning step length 0.02°, scanning speed 17.7s/ step, scanning range 5-55°. Curcumin, P -cyclodextrin polymer, physical mixture of the two (same as the feeding ratio) and inclusion compound are taken and measured in X- ray powder diffractometer according to the above conditions. The experimental results are shown in fig. 4. As can be seen from fig. 4, curcumin has specific crystallization diffraction peaks at 20 = 7.83°, 8.78°, 11.99°, 13.76°, 15.79°, 17.13°, 21.01°, indicating that curcumin is a crystalline substance. However, cyclodextrin polymer has two broad diffraction peaks at 20 of 10°-15° and 15°-22.5°, indicating that cyclodextrin polymer is an amorphous powder. The characteristic peak of curcumin can be seen from the diffraction peak shape of the mixture, which indicates that it is not included, and the peak shape of the mixture is the superposition of the peak shapes of curcumin and cyclodextrin polymer. However, it can be seen from the peak shape of the inclusion compound that the diffraction peak of curcumin disappears or weakens in the inclusion compound, which proves that curcumin and cyclodextrin polymer form an inclusion compound.

4. Determination of Curcumin by High Performance Liquid Chromatography

4.1 Chromatographic Conditions

The chromatographic column is Scienhome Kromasil C18 column (150mmx4.6mm, 5um), room temperature (25-30°C), mobile phase is methanol -1% citric acid (70:30), volume flow rate is 1 mL/min, detection wavelength is 425 nm, and injection volume is 10 pL.

4.2 Investigation of Linear Relation

Accurately weigh 12.25 mg of curcumin reference substance, dissolve it with methanol and fix it in a 100 mL volumetric flask to obtain mother liquor with a mass concentration of 122.5 [g/ml. Remove 0.2, 0.4, 0.6, 0.8, 1.6, 2.4, 3.2, 5.0 mL from the mother liquor respectively and place them in a 10 mL volumetric flask. The mobile phase is constant volume to scale to obtain a series of reference substance solutions with mass concentrations of 2.45, 4.90, 9.80, 19.60, 29.40, 39.20, 49.00 g/ml respectively. Take 10 1 of the above reference substance solution and inject it into a high performance liquid chromatograph for determination. Taking the concentration of the curcumin reference substance as the abscissa and the peak area of the curcumin reference substance as the ordinate, a linear regression equation is obtained: Y=102211X-256580, r=0.9940, indicating that the peak area of curcumin has a good linear relationship in the range of concentration of 2.45-49.00 [g/ml.

4.3 Preparation of Test Solution

Weigh an appropriate amount of inclusion compound sample (about equivalent to curcumin 5mg), place it in a brown volumetric flask, add distilled water to dissolve it, add 95% ethanol to constant volume to scale, vibrate and mix well, filter through a 0.45 m microporous filter membrane, and dilute the filtrate to obtain the final product.

4.4 Precision Test

Accurately suck 10 L of the same inclusion compound test sample solution, and continuously inject 5 samples. Determine the peak area of curcumin, and calculate its RSD to be 0.49%.

4.5 Reproducibility Test

Accurately weigh 5 samples of the same inclusion compound, prepare 5 sample solutions according to the method of 2.3, inject samples, measure the peak area of curcumin, and calculate its RSD to be 0.37%.

4.6 Sample Recovery Rate Test

Five inclusion compound samples containing 107.2 mg/g of curcumin were taken and weighed precisely. 100mg of curcumin reference substance was added respectively to prepare the test solution. The sample was injected and the peak area of curcumin was determined. The calculated recovery rate was 106.2% with RSD of 1.36%.

4.7 Sample Determination

Inject 10 L of test solution into high performance liquid chromatography, measure, and calculate the content of curcumin in the inclusion compound according to linear equation. Calculate the inclusion rate of the inclusion compound according to the following formula.

Inclusion rate = curcumin content in inclusion compound/curcumin input amount x100%

Yield = inclusion compound yield/ (cyclodextrin polymer input + curcumin input) x 100%

According to the calculation, the inclusion rate of the inclusion compound is 34.5%, and the yield is 63.5%.

Claims

1. A preparation method of curcumin-cyclodextrin supramolecular inclusion compound is characterized by comprising the following steps:

2. The preparation method according to claim 1, characterized in that the mass volume ratio of p-cyclodextrin to epichlorohydrin is (2-3): 1.

3. The preparation method according to claim 1, characterized in that the mixing mass ratio of cyclodextrin polymer and curcumin is (3-5): 1.

4. The preparation method according to claim 3, characterized in that the mixing mass ratio of cyclodextrin polymer and curcumin is 4: 1.

5. The preparation method according to claim 1, characterized in that the alkaline condition in S Iis to add sodium hydroxide and dissolve with water, wherein the mass fraction of sodium hydroxide is 30%-35%.

6. The preparation method according to claim 1, wherein the polymerization time is 20 to 30 hours.

7. The preparation method according to claim 1, characterized in that the cyclodextrin polymer is obtained after ultrasonic, dialysis, filtration and drying after the polymerization reaction.