Flavonoid compound-chlorogenic acid co-amorphous substance and preparation method thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a flavonoid compound-chlorogenic acid co-amorphous substance and a preparation method thereof. After a flavonoid component with good miscibility with chlorogenic acid is screened out, a fusion quenching method or a ball milling method is adopted, so that the chlorogenic acid and a flavonoid compound are prepared into a flavonoid co-amorphous system. PXRD, DSC and FTIR results show that five flavonoid compounds and chlorogenic acid are successfully prepared into a co-amorphous system. Under a non-leakage tank condition, the five flavonoid compound-chlorogenic acid co-amorphous substance has remarkable dissolution / dissolution advantages compared with a single crystal medicine and a physical mixture thereof. Meanwhile, the formed co-amorphous substance is not recrystallized within 90 days under the conditions of 25 DEG C and 40 DEG C, and has excellent physical stability. In addition, through in-vitro antioxidant activity determination, the flavonoid compound co-amorphous substance shows good DPPH free radical removal capacity and has high antioxidant activity.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to a flavonoid compound-chlorogenic acid co-amorphous substance and a preparation method thereof. Background Art

[0002] Currently, most of the developed drugs and candidate drugs under development exhibit poor water solubility, which limits their oral absorption and therapeutic effects. Enhancing the water solubility of these candidate drugs has become a key issue for pharmaceutical enterprises and pharmaceutical companies to provide patients with high-quality, high-performance drug products. Amorphization is a common method to improve the solubility / dissolution rate of hydrophobic drugs by disordering the crystal lattice of hydrophobic drugs. In theory, disordered amorphous drugs have higher entropy, enthalpy and Gibbs free energy, and usually show better dissolution / dissolution performance than their crystalline drugs. However, the inherent risk of devitrification of amorphous drugs during preparation, storage and dissolution has seriously restricted their development, weakening or even completely losing the advantages of amorphous systems. Therefore, it is crucial to find effective strategies to stabilize amorphous systems.

[0003] Co-amorphous systems have attracted widespread attention in stabilizing amorphous drugs and improving the solubility / dissolution of hydrophobic drugs. Based on the functional groups in the active ingredient molecules that can form intermolecular forces, the drug molecules and suitable ligands (which can be pharmaceutical excipients or other drug molecules) are assembled into supramolecular complexes through non-covalent intermolecular forces (mostly hydrogen bonds) through the principles and methods of crystal engineering. Due to the molecular mixing or lattice rearrangement optimization of the drugs, they generally show excellent physical and chemical properties (such as higher solubility, stability and bioavailability, etc.).

[0004] Flavonoids are a large class of compounds that are widely present in nature, and their main sources are vegetables, fruits, herbs, grains, flowers and seeds. At the same time, flavonoids have a variety of pharmacological effects, such as antioxidant, antibacterial, anti-inflammatory, cardiovascular effects, anti-liver damage effects, etc. In recent years, many flavonoids with different activities have been discovered, such as luteolin has antiviral, antitumor, antibacterial and other effects; morin has antitumor and other effects; quercetin has a strong antioxidant effect. In addition, flavonoids are also important functional food additives, natural antioxidants, natural sweeteners, etc. However, due to their structural characteristics, they generally show low solubility / dissolution, and there is potential in vivo glucuronidase phase II metabolism, resulting in poor oral absorption, which limits their clinical application.

[0005] Chlorogenic acid (CA) is a phenylpropanoid compound produced by caffeic acid and quinic acid (1-hydroxyhexahydrogallic acid) during aerobic respiration in plants through the shikimic acid pathway. Its chemical structure is: Currently, chlorogenic acid exists as a food additive in foods and herbs (such as apples, coffee beans, tea, honeysuckle and licorice), and has great potential in the development of functional dietary foods, nutritional supplements, food materials, etc.

[0006] CN 102178675 A discloses a pharmaceutical composition prepared by combining quercetin, chlorogenic acid and pharmaceutically acceptable excipients, and the composition can be made into tablets or capsules. The composition is prepared by mixing quercetin, chlorogenic acid and a large amount of pharmaceutical excipients (lactose, corn starch, magnesium stearate, etc.), and the drug is still in a crystalline state. Summary of the invention

[0007] The present invention provides a flavonoid compound-chlorogenic acid co-amorphous substance and a preparation method thereof. To achieve the above-mentioned object, the present invention adopts the following technical scheme:

[0008] The flavonoid compound and chlorogenic acid co-amorphous product of the present invention is prepared by melt quenching method or ball milling method. First, 9 flavonoid compounds with good miscibility with chlorogenic acid are screened out by Hansen solubility calculation, including: genistein, quercetin, baicalein, kaempferol, chrysin, fisetin, apigenin, luteolin or daidzein.

[0009] The flavonoid compound is preferably: genistein, quercetin, baicalein, kaempferol or chrysin.

[0010] The flavonoids and chlorogenic acid are mixed in a molar ratio of 5:1 to 1:5; preferably, the molar ratio of flavonoids:chlorogenic acid = 2:1 to 1:2; most preferably, the molar ratio of flavonoids:chlorogenic acid = 1:1.

[0011] The method for preparing flavonoid compound-chlorogenic acid co-amorphous product by melt quenching method is as follows: the selected flavonoid compound and chlorogenic acid are ground and mixed according to different stoichiometric ratios, spread on an aluminum foil weighing dish, and placed in an oil bath to melt, and then the sample is immediately taken out and quenched in liquid nitrogen, vacuum dried and then ground to obtain the flavonoid compound-chlorogenic acid co-amorphous product.

[0012] The melting temperature of the melt quenching method is: 120 ~ 180 ° C, and the melting time is: 3 ~ 15 minutes.

[0013] The method for preparing flavonoid compound-chlorogenic acid co-amorphous product by ball milling is as follows: flavonoid compound and chlorogenic acid are placed in a ball milling jar, the ball milling frequency is 10-50 Hz, and the ball milling time is 60-360 min, so as to obtain flavonoid compound-chlorogenic acid co-amorphous product.

[0014] The flavonoid compound-chlorogenic acid co-amorphous substance of the present invention exhibits significantly improved water solubility compared to single crystalline drugs and physical mixtures thereof, and can achieve a high dissolution concentration and maintain a supersaturated state for a long time.

[0015] The flavonoid compound-chlorogenic acid co-amorphous substance of the present invention exhibits better physical stability than a single flavonoid amorphous substance.

[0016] Compared with a single flavonoid compound, the flavonoid-chlorogenic acid co-amorphous compound of the present invention exhibits a stronger ability to scavenge DPPH free radicals and has high antioxidant activity.

[0017] Beneficial effects of the present invention:

[0018] Based on the solubility / stability defects and potential metabolic inhibition of flavonoids, the present invention uses crystal engineering technology to design a supramolecular co-amorphous system by co-amorphizing flavonoids and the glucuronidase inhibitor chlorogenic acid to improve solubility / dissolution and enhance its physical stability and antioxidant activity, in order to achieve the purpose of improving the oral absorption and therapeutic effect of flavonoids, and provide new ideas for optimizing the drugability of flavonoids and other poorly soluble drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is the PLM diagram of the preliminary preparation products of various flavonoids and chlorogenic acid;

[0020] Figure 2 This is the PXRD pattern of the flavonoid compound-chlorogenic acid co-amorphous substance prepared in Example 1;

[0021] Figure 3 is the DSC graph of the flavonoid compound-chlorogenic acid co-amorphous substance prepared in Example 1;

[0022] Figure 4 is the FTIR graph of the flavonoid compound-chlorogenic acid co-amorphous substance prepared in Example 1;

[0023] Figure 5 It is a comparison diagram of the dissolution curves of genistein crystals, the physical mixture of genistein and chlorogenic acid prepared in Example 1, and the genistein-chlorogenic acid co-amorphous prepared in Example 1 under non-sink conditions;

[0024] Figure 6It is a comparison diagram of the dissolution curves of quercetin crystals, the physical mixture of quercetin and chlorogenic acid prepared in Example 1, and the quercetin-chlorogenic acid co-amorphous prepared in Example 1 under non-sink conditions;

[0025] Figure 7 It is a comparison diagram of the dissolution curves of baicalein crystals, the physical mixture of baicalein and chlorogenic acid prepared in Example 1, and the baicalein-chlorogenic acid co-amorphous substance prepared in Example 1 under non-sink conditions;

[0026] Figure 8 is a comparison diagram of the dissolution curves of kaempferol crystals, the physical mixture of kaempferol and chlorogenic acid prepared in Example 1, and the kaempferol-chlorogenic acid co-amorphous substance prepared in Example 1 under non-sink conditions;

[0027] Fig. 9 is a comparison diagram of the dissolution curves of chrysin crystals, the physical mixture of chrysin and chlorogenic acid prepared in Example 1, and the chrysin-chlorogenic acid co-amorphous prepared in Example 1 under non-sink conditions;

[0028] Fig.10 It is the PXRD pattern of stability comparison of genistein amorphous;

[0029] Fig.11 is the PXRD pattern of stability comparison of quercetin amorphous;

[0030] Fig.12 This is the PXRD pattern of the stability comparison of baicalein amorphous form;

[0031] Fig.13 This is the PXRD pattern of stability comparison of kaempferol amorphous;

[0032] Fig.14 This is the PXRD pattern of stability comparison of chrysin amorphous form;

[0033] Fig.15 is a PXRD diagram of stability comparison of genistein-chlorogenic acid co-amorphous prepared in Example 1;

[0034] Fig.16 is a PXRD diagram of stability comparison of quercetin-chlorogenic acid co-amorphous prepared in Example 1;

[0035] Fig.17 is a PXRD diagram for stability comparison of baicalein-chlorogenic acid co-amorphous substance prepared in Example 1;

[0036] Fig.18 is a PXRD diagram of stability comparison of kaempferol-chlorogenic acid co-amorphous material prepared in Example 1;

[0037] Fig.19is a PXRD diagram of stability comparison of chrysin-chlorogenic acid co-amorphous prepared in Example 1;

[0038] Fig. 20 is a graph showing the DPPH radical scavenging activity of genistein crystals and genistein-chlorogenic acid co-amorphous compound prepared in Example 1;

[0039] Fig.21 is a graph showing the DPPH radical scavenging activity of quercetin crystals and the quercetin-chlorogenic acid co-amorphous compound prepared in Example 1;

[0040] Fig. 22 It is a graph of DPPH radical scavenging activity of baicalein crystals and baicalein-chlorogenic acid co-amorphous prepared in Example 1;

[0041] Fig.23 is a graph showing the DPPH radical scavenging activity of kaempferol crystals and kaempferol-chlorogenic acid co-amorphous compound prepared in Example 1;

[0042] Fig.24 It is a graph showing the DPPH radical scavenging activity of chrysin crystals and chrysin-chlorogenic acid co-amorphous compound prepared in Example 1. DETAILED DESCRIPTION

[0043] The present invention is further described below in conjunction with test examples and embodiments.

[0044] Test Example 1: The Hansen solubility calculation of flavonoids-chlorogenic acid co-amorphous is performed as follows:

[0045] The Hansen solubility parameter (δ) was calculated by Molecular Modeling Pro software. When the solubility parameter difference (Δδ) of the two components was less than 7.0 MPa, they were considered to have good compatibility. From Table 1, it can be seen that 9 flavonoids (genistein, quercetin, baicalein, kaempferol, chrysin, fisetin, apigenin, luteolin, daidzein) have good miscibility with chlorogenic acid. However, myricetin, morin, proanthocyanidin, galangin, kauri biapigenin, etc. have poor miscibility with chlorogenic acid.

[0046] Table 1 Hansen solubility parameters (δ) of flavonoid crystals and chlorogenic acid crystals

[0047]

[0048] Test Example 2: The preparation product of flavonoid compound-chlorogenic acid was preliminarily analyzed by polarizing microscope, as follows:

[0049] Polarized light microscopy (PLM) was used to observe the crystal birefringence of the flavonoid compound-chlorogenic acid preparation product. The sample was placed on a glass slide, fixed and evenly dispersed with liquid paraffin, and observed under a magnification of 100 times.

[0050] According to the calculation of Hansen solubility parameters, the nine flavonoids have good miscibility with chlorogenic acid. The melt quenching method (specifically the same as in Example 1) was used to try to prepare a flavonoid-chlorogenic acid co-amorphous product. Through PLM observation, the preparation products of five flavonoids (genistein, quercetin, baicalein, kaempferol, chrysin) and chlorogenic acid showed no crystalline birefringence, indicating that an amorphous state can be formed ( Figure 1 ae).

[0051] Example 1

[0052] Weigh genistein (216.35 mg)-chlorogenic acid (283.65 mg), quercetin (230.17 mg)-chlorogenic acid (269.83 mg), baicalein (216.35 mg)-chlorogenic acid (283.65 mg), kaempferol (223.43 mg)-chlorogenic acid (276.57 mg), and chrysin (208.89 mg)-chlorogenic acid (291.11 mg) respectively, grind in a mortar and mix evenly for 5 minutes to obtain a physical mixture. Then pour the five physical mixtures into aluminum foil weighing dishes, spread them flat in an oil bath at 150°C for 5 minutes, and immediately take out the samples after complete melting and quench in liquid nitrogen for 2 minutes. Subsequently, vacuum dry and grind, and store at 4°C.

[0053] Test Example 3: Characterization of the flavonoid compound-chlorogenic acid co-amorphous substance of Example 1 is as follows:

[0054] 1. Powder X-ray Diffraction (PXRD)

[0055] Instrument: SmartLab9 X-ray diffractometer (Rigaku, Japan)

[0056] Target: Cu-Kα radiation

[0057] wavelength:

[0058] Tube voltage: 40KV

[0059] Tube current: 40mA

[0060] Step size: 0.02°

[0061] Scanning speed: 4° / min

[0062] Scanning range: 2θ, 5-40°

[0063] The crystal diffraction peaks of the genistein-chlorogenic acid combination, quercetin-chlorogenic acid combination, baicalein-chlorogenic acid combination, kaempferol-chlorogenic acid combination and chrysin-chlorogenic acid combination were detected respectively, and the corresponding PXRD patterns were as follows: Figure 2 shown.

[0064] like Figure 2 As shown, in Example 1, the crystal diffraction peaks of the genistein-chlorogenic acid combination, the quercetin-chlorogenic acid combination, the baicalein-chlorogenic acid combination, the kaempferol-chlorogenic acid combination and the chrysin-chlorogenic acid combination disappeared, showing that the product had diffuse diffraction rings with amorphous characteristics, indicating that it was in an amorphous state. In Examples 2 to 14, the genistein-chlorogenic acid combination, the quercetin-chlorogenic acid combination, the baicalein-chlorogenic acid combination, the kaempferol-chlorogenic acid combination and the chrysin-chlorogenic acid combination also showed similar PXRD patterns.

[0065] 2. Differential Scanning Calorimetry (DSC)

[0066] Instrument: HITACHIDSC 7020 differential scanning calorimeter (Hitachi Profile, Japan)

[0067] Range: 25-300℃

[0068] Heating rate: 10℃ / min

[0069] Nitrogen flow rate: 80mL / min

[0070] Genistein-chlorogenic acid co-amorphous, quercetin-chlorogenic acid co-amorphous, baicalein-chlorogenic acid co-amorphous, kaempferol-chlorogenic acid co-amorphous and chrysin-chlorogenic acid co-amorphous were detected respectively, and the corresponding DSC spectra were as follows: Figure 3 shown.

[0071] like Figure 3 As shown in Example 1, the DSC spectra of genistein-chlorogenic acid co-amorphous, quercetin-chlorogenic acid co-amorphous, baicalein-chlorogenic acid co-amorphous, kaempferol-chlorogenic acid co-amorphous and chrysin-chlorogenic acid co-amorphous showed that there was only one glass transition temperature (T g ), its T g The values ​​are 65.9°C, 106.6°C, 94.4°C, 98.3°C, and 96.2°C, respectively, further proving that it is a single-phase binary co-amorphous. In Examples 2 to 14, genistein-chlorogenic acid co-amorphous, quercetin-chlorogenic acid co-amorphous, baicalein-chlorogenic acid co-amorphous, kaempferol-chlorogenic acid co-amorphous, and chrysin-chlorogenic acid co-amorphous showed similar DSC spectra, showing a single glass transition temperature, and the glass transition temperature showed slightly different changes with the molar ratio.

[0072] 3. Infrared spectroscopy (FTIR)

[0073] Instrument: Thermo Scientific Nicolet iS50 Fourier transform infrared spectrometer (Thermo Fisher Scientific, America)

[0074] Range: 4000-400cm -1

[0075] Scan times: 32 times

[0076] Genistein-chlorogenic acid co-amorphous, quercetin-chlorogenic acid co-amorphous, baicalein-chlorogenic acid co-amorphous, kaempferol-chlorogenic acid co-amorphous and chrysin-chlorogenic acid co-amorphous were detected respectively, and the corresponding FTIR spectra were as follows Figure 4 shown.

[0077] like Figure 4 As shown in Example 1, the FTIR spectrum wave number (cm -1 ) are: 3405.11, 1777.86, 1733.15, 1611.27, 1514.13, 1443.90, 1348.05, 1251.19, 1177.26, 1077.55, 932.30, 833.80 and 599.70cm -1 .

[0078] FTIR spectrum of quercetin-chlorogenic acid co-amorphous compound (cm -1 ) are: 3404.84, 1773.39, 1651.71, 1607.95, 1560.78, 1514.90, 1443.77, 1316.05, 1247.98, 1200.46, 1148.12, 1081.29, 1034.96, 1005.65, 931.08, 882.10, 816.27, 635.82 and 597.49cm -1 .

[0079] FTIR spectrum of baicalein-chlorogenic acid co-amorphous compound (cm -1) are: 3412.12, 1778.10, 1717.13, 1659.06, 1616.28, 1512.42, 1470.29, 1449.74, 1370.32, 1284.72, 1161.89, 1079.97, 1034.47, 999.42, 977.44, 932.92, 915.62, 898.16, 850.70, 813.80, 770.59, 721.15, 685.66, 655.76, 641.19 and 614.27cm -1 .

[0080] FTIR spectrum of kaempferol-chlorogenic acid co-amorphous compound (cm -1 ) are: 3377.76, 2919.18, 2850.26, 1774.00, 1651.79, 1607.03, 1560.80, 1513.39, 1494.34, 1443.31, 1356.42, 1250.77, 1178.13, 1147.05, 1078.25, 1032.80, 976.57, 932.03, 881.03, 839.47, 815.47, 745.45, 674.66, 630.66, 611.20 and 505.97cm -1 .

[0081] FTIR spectrum of chrysin-chlorogenic acid co-amorphous compound (cm -1 ) are: 3404.80, 2717.71, 2632.47, 1775.23, 1654.63, 1612.68, 1577.92, 1555.39, 1500.07, 1449.73, 1357.19, 1273.16, 1168.79, 1077.67, 1033.28, 999.29, 977.66, 908.15, 842.92, 807.11, 782.17, 768.90, 746.51, 733.69, 711.47, 692.45, 642.17, 612.47, 558.99 and 430.66cm -1 .

[0082] In Examples 2 to 14, genistein-chlorogenic acid co-amorphous, quercetin-chlorogenic acid co-amorphous, baicalein-chlorogenic acid co-amorphous, kaempferol-chlorogenic acid co-amorphous and chrysin-chlorogenic acid co-amorphous showed the same or similar FTIR spectra.

[0083] Test Example 4: The apparent solubility test of the flavonoid compound-chlorogenic acid co-amorphous substance of Example 1 was conducted as follows:

[0084] Excess flavonoid crystals, flavonoid-chlorogenic acid physical mixture (molar ratio 1:1), and flavonoid-chlorogenic acid co-amorphous (molar ratio 1:1) were weighed and dissolved in 5 mL of pH 1.2 HCl buffer and pH 6.8 phosphate buffer solution, respectively, and placed in a 37°C air bath constant temperature oscillator and shaken for 24 h to achieve solubility equilibrium. Subsequently, the mixture was filtered through a filter membrane (0.22 mm) and diluted with an equal volume of methanol, and the filtrate was subjected to HPLC analysis. Each sample was measured in triplicate.

[0085] The HPLC conditions were as follows:

[0086] Instrument: Agilent 1260 High Performance Liquid Chromatograph

[0087] Chromatographic column: Ultimate XB-C18 (4.6mm×250mm, 5μm)

[0088] Mobile phase: acetonitrile-0.3% phosphoric acid water = 60:40 (V / V)

[0089] Flow rate: 1.0mL / min

[0090] From the apparent solubility measurement results in Table 2, the physical mixture and the crystalline drug showed similar apparent solubility in pH 1.2 HCl buffer and pH 6.8 phosphate buffer solution at 37°C. The solubility of the flavonoid-chlorogenic acid co-amorphous substance in pH 1.2 HCl buffer increased by 2.06 to 18.58 times and the solubility in pH 6.8 phosphate buffer solution increased by 2.02 to 36.25 times. Therefore, after the flavonoids and chlorogenic acid are prepared into a co-amorphous system, their molecules are disordered and have larger entropy, enthalpy and Gibbs free energy, thereby showing higher solubility compared with crystalline drugs. The solubility of the flavonoid-chlorogenic acid co-amorphous substance in Examples 2 and 8 showed similar significant improvements.

[0091] From Table 3, it can be seen that other flavonoids (daidzein, fisetin) and their products with chlorogenic acid did not show significant improvement in solubility in the two pH media.

[0092] Table 2 Solubility of flavonoid crystals, flavonoid-chlorogenic acid physical mixture and flavonoid-chlorogenic acid co-amorphous

[0093]

[0094]

[0095] Table 3 Solubility of other flavonoid crystals and flavonoid-chlorogenic acid preparation products (preparation method is the same as above, flavonoid-chlorogenic acid molar ratio is 1:1)

[0096]

[0097] Test Example 5: A non-sink dissolution test was performed on the flavonoid compound-chlorogenic acid co-amorphous substance in Example 1, as follows:

[0098] Take excess flavonoid crystals, flavonoid-chlorogenic acid physical mixture (molar ratio 1:1), flavonoid-chlorogenic acid co-amorphous (molar ratio 1:1). The dissolution test was carried out at 37°C and 100rpm. The dissolution media were HCl buffer (pH 1.2) and phosphate buffer solution (pH 6.8), respectively. Take 2ml of sample at the preset time point (immediately replenish the dissolution medium with the same temperature and volume), then filter with a filter membrane (0.22mm) and dilute with an equal volume of methanol, take the filtrate for HPLC analysis and record the peak area, and each sample is measured in parallel 3 times.

[0099] like Figures 5 to 9 As shown in Tables 4 and 6, in Example 1, there was no significant difference in the dissolution behavior between the flavonoid compound raw material and the physical mixture, while the dissolution of genistein-chlorogenic acid co-amorphous, quercetin-chlorogenic acid co-amorphous, baicalein-chlorogenic acid co-amorphous, kaempferol-chlorogenic acid co-amorphous and chrysin-chlorogenic acid co-amorphous was significantly increased compared to the corresponding flavonoid compounds and maintained at a supersaturated concentration for a long time. The dissolution behavior of the flavonoid compound-chlorogenic acid co-amorphous in Examples 2 and 8 was also significantly enhanced at different time points.

[0100] From Tables 5 and 7, it can be seen that the dissolution behavior of other flavonoid raw materials (daidzein, fisetin) and their preparation products with chlorogenic acid in pH 1.2 HCl buffer and pH 6.8 phosphate buffer solution is not significantly improved compared with the crystalline drug.

[0101] As shown in Table 4, at 12 h, under the condition of hydrochloric acid buffer solution of pH 1.2, the drug dissolution concentration of the genistein-chlorogenic acid co-amorphous compound in Example 1 (6616.30±1.05 μg / mL) was higher than that of the genistein bulk drug (3255.86±0.69 μg / mL), which was enhanced by 2.03 times; the drug dissolution concentration of the quercetin-chlorogenic acid co-amorphous compound in Example 1 (31.82±1.88 μg / mL) was higher than that of the quercetin bulk drug (2.10±0.10 μg / mL), which was enhanced by 15.15 times; the drug dissolution concentration of the baicalein-chlorogenic acid co-amorphous compound in Example 1 (19.55 ±0.99μg / mL) is higher than the baicalein raw material (5.69±1.17μg / mL), which is enhanced by 3.44 times; the drug dissolution concentration of the kaempferol-chlorogenic acid co-amorphous compound in Example 1 (10.14±0.55μg / mL) is higher than the kaempferol raw material (0.55±0.03μg / mL), which is enhanced by 18.44 times; the drug dissolution concentration of the chrysin-chlorogenic acid co-amorphous compound in Example 1 (2.77±0.06±1.05μg / mL) is enhanced by 15.39 times compared with the chrysin raw material (0.18±0.02μg / mL).

[0102] As shown in Table 6, at 12 h, under the conditions of pH 6.8 phosphate buffer, the drug dissolution concentration of the genistein-chlorogenic acid co-amorphous compound in Example 1 (4739.12±1.25 μg / mL) was higher than that of genistein (2354.07±2.80 μg / mL), which was enhanced by 2.01 times; the drug dissolution concentration of the quercetin-chlorogenic acid co-amorphous compound in Example 1 (25.80±1.17 μg / mL) was higher than that of quercetin (1.79±0.16 μg / mL), which was enhanced by 14.41 times; the drug dissolution concentration of the baicalein-chlorogenic acid co-amorphous compound in Example 1 ( 19.02±0.78μg / mL) is higher than baicalein (6.60±0.09μg / mL), which is enhanced by 2.88 times; the drug dissolution concentration of the kaempferol-chlorogenic acid co-amorphous compound in Example 1 (17.89±1.53μg / mL) is higher than kaempferol (0.33±0.13μg / mL), which is enhanced by 54.21 times; the drug dissolution concentration of the chrysin-chlorogenic acid co-amorphous compound in Example 1 (4.25±0.53μg / mL) is higher than chrysin (0.32±0.01μg / mL), which is enhanced by 13.28 times.

[0103] Table 4 Dissolution concentrations of flavonoid crystals, flavonoid-chlorogenic acid physical mixture and flavonoid-chlorogenic acid co-amorphous at different time points under pH 1.2 (μg / mL)

[0104]

[0105]

[0106] Table 5 Dissolution concentrations (μg / mL) of other flavonoid crystals and flavonoid-chlorogenic acid preparation products (preparation method is the same as above, flavonoid-chlorogenic acid molar ratio is 1:1) at different time points under pH 1.2

[0107]

[0108] Table 6 Dissolution concentrations of flavonoid crystals, flavonoid-chlorogenic acid physical mixtures and flavonoid-chlorogenic acid co-amorphous products at different time points under pH 6.8 (μg / mL)

[0109]

[0110]

[0111] Table 7 Dissolution concentrations (μg / mL) of other flavonoid crystals and flavonoid-chlorogenic acid preparation products (preparation method is the same as above, flavonoid-chlorogenic acid molar ratio is 1:1) at different time points under pH 6.8

[0112]

[0113] Test Example 6: A stability test was conducted on the flavonoid compound-chlorogenic acid co-amorphous substance in Example 1, as follows:

[0114] Genistein amorphous, quercetin amorphous, baicalein amorphous, kaempferol amorphous, chrysin amorphous, genistein-chlorogenic acid co-amorphous, quercetin-chlorogenic acid co-amorphous, baicalein-chlorogenic acid co-amorphous, kaempferol-chlorogenic acid co-amorphous and chrysin-chlorogenic acid co-amorphous were placed in a comprehensive drug stability test chamber at 25°C and 40°C for 3 months to observe their stability during storage. Powder X-ray diffraction (PXRD) was used to determine whether recrystallization occurred.

[0115] like Figures 10 to 14 As shown, genistein amorphous, quercetin amorphous, baicalein amorphous, kaempferol amorphous and chrysin amorphous were stored at 25°C and 40°C. The results showed that genistein amorphous, quercetin amorphous, baicalein amorphous and chrysin amorphous underwent crystal transformation within 14 days, and kaempferol amorphous underwent crystal transformation within 30 days.

[0116] like Figures 15 to 19As shown, the genistein-chlorogenic acid co-amorphous, quercetin-chlorogenic acid co-amorphous, baicalein-chlorogenic acid co-amorphous, kaempferol-chlorogenic acid co-amorphous and chrysin-chlorogenic acid co-amorphous in Example 1 were stored at 25°C and 40°C for 90 days, and no characteristic diffraction peaks appeared in the PXRD diffraction pattern. The results indicate that genistein-chlorogenic acid co-amorphous, quercetin-chlorogenic acid co-amorphous, baicalein-chlorogenic acid co-amorphous, kaempferol-chlorogenic acid co-amorphous and chrysin-chlorogenic acid have high physical stability.

[0117] Test Example 7: Antioxidant test was conducted on flavonoids and flavonoid-chlorogenic acid amorphous compound, as follows:

[0118] 500 μL of the serially diluted sample solution or extraction solvent (blank) was combined with 500 μL of DPPH solution, the mixture was shaken vigorously, and placed at 25°C for 30 min, and then the absorbance of the mixture was measured at 520 nm. Vitamin C (Vc) was selected as the positive control for each sample. The scavenging activity of DPPH free radicals was calculated as follows: Scavenging activity (%) = [(A0-A1) / A0] × 100%, where A0 and A1 are the absorbance of the blank and the flavonoid compound or its co-amorphous product.

[0119] like Fig. 20 As shown, in Example 1, at a concentration of 12.5 μg / mL, the scavenging DPPH free radical energy of genistein and genistein-chlorogenic acid co-amorphous compound (79.08%) was significantly stronger than that of genistein (6.93%), indicating that genistein-chlorogenic acid co-amorphous compound has strong antioxidant activity.

[0120] like Fig.21 As shown, at a concentration of 3.125 μg / mL, the DPPH radical scavenging ability of quercetin and quercetin-chlorogenic acid co-amorphous reached 65.28%, while that of quercetin only reached 44.63%, indicating that quercetin-chlorogenic acid co-amorphous exhibited strong antioxidant activity at low doses.

[0121] like Fig. 22 As shown, at low concentrations (6.25 μg / mL), the DPPH free radical scavenging ability of baicalin-chlorogenic acid co-amorphous reached 81.57%, and remained constant with the increase of dosage, indicating that the maximum DPPH scavenging activity was achieved, while baicalin only reached 44.46%, indicating that baicalin-chlorogenic acid co-amorphous exhibited strong antioxidant activity at low doses.

[0122] like Fig.23As shown, at the same concentration (6.25 μg / mL), the DPPH free radical scavenging ability of kaempferol and kaempferol-chlorogenic acid co-amorphous reached 72.61%, which is 2.20 times that of kaempferol, indicating that kaempferol-chlorogenic acid co-amorphous has strong antioxidant activity at low doses.

[0123] like Fig.24 As shown, chrysin and chrysin-chlorogenic acid co-amorphous showed significant antioxidant activity at a concentration of 12.5 μg / mL, with a scavenging activity of 79.24%, while chrysin was only 6.55%, indicating that chrysin-chlorogenic acid co-amorphous had significant antioxidant activity at low doses.

[0124] Example 2

[0125] Weigh genistein (138.04 mg)-chlorogenic acid (361.96 mg), quercetin (149.50 mg)-chlorogenic acid (350.50 mg), baicalein (138.04 mg)-chlorogenic acid (361.96 mg), kaempferol (143.86 mg)-chlorogenic acid (356.14 mg), and chrysin (132.02 mg)-chlorogenic acid (367.98 mg) respectively, grind in a mortar and mix evenly for 5 min. Pour into an aluminum foil weighing dish and spread flat in an oil bath at 150°C for 5 min to melt. After melting completely, take out the sample immediately and quench in liquid nitrogen for 2 min. Subsequently, vacuum dry and grind, and store at 4°C.

[0126] Example 3

[0127] Weigh genistein (302.01 mg)-chlorogenic acid (197.99 mg), quercetin (315.23 mg)-chlorogenic acid (184.77 mg), baicalein (302.02 mg)-chlorogenic acid (197.98 mg), kaempferol (308.85 mg)-chlorogenic acid (191.15 mg), and chrysin (294.67 mg)-chlorogenic acid (205.33 mg) respectively, grind in a mortar and mix evenly for 5 min. Pour into an aluminum foil weighing dish and spread flat in an oil bath at 150°C to melt for 5 min. After melting completely, take out the sample immediately and quench in liquid nitrogen for 2 min. Subsequently, vacuum dry and grind, and store at 4°C.

[0128] Example 4

[0129] Weigh genistein (216.35 mg)-chlorogenic acid (283.65 mg), quercetin (230.17 mg)-chlorogenic acid (269.83 mg), baicalein (216.35 mg)-chlorogenic acid (283.65 mg), kaempferol (223.43 mg)-chlorogenic acid (276.57 mg), chrysin (208.89 mg)-chlorogenic acid (291.11 mg) respectively, grind in a mortar and mix evenly for 5 min. Pour into an aluminum foil weighing dish and spread flat in an oil bath at 120°C to melt for 5 min. After melting completely, take out the sample immediately and quench in liquid nitrogen for 2 min. Subsequently, vacuum dry and grind, and store at 4°C.

[0130] Example 5

[0131] Weigh genistein (216.35 mg)-chlorogenic acid (283.65 mg), quercetin (230.17 mg)-chlorogenic acid (269.83 mg), baicalein (216.35 mg)-chlorogenic acid (283.65 mg), kaempferol (223.43 mg)-chlorogenic acid (276.57 mg), chrysin (208.89 mg)-chlorogenic acid (291.11 mg) respectively, grind in a mortar and mix evenly for 5 min. Pour into an aluminum foil weighing dish and spread flat in an oil bath at 180°C to melt for 5 min. After melting completely, take out the sample immediately and quench in liquid nitrogen for 2 min. Subsequently, vacuum dry and grind, and store at 4°C.

[0132] Example 6

[0133] Weigh genistein (216.35 mg)-chlorogenic acid (283.65 mg), quercetin (230.17 mg)-chlorogenic acid (269.83 mg), baicalein (216.35 mg)-chlorogenic acid (283.65 mg), kaempferol (223.43 mg)-chlorogenic acid (276.57 mg), chrysin (208.89 mg)-chlorogenic acid (291.11 mg) respectively, grind in a mortar and mix evenly for 5 min. Pour into an aluminum foil weighing dish and spread flat in a 150°C oil bath to melt for 3 min. After melting completely, take out the sample immediately and quench in liquid nitrogen for 2 min. Subsequently, vacuum dry and grind, and store at 4°C.

[0134] Example 7

[0135] Weigh genistein (216.35 mg)-chlorogenic acid (283.65 mg), quercetin (230.17 mg)-chlorogenic acid (269.83 mg), baicalein (216.35 mg)-chlorogenic acid (283.65 mg), kaempferol (223.43 mg)-chlorogenic acid (276.57 mg), and chrysin (208.89 mg)-chlorogenic acid (291.11 mg) respectively, grind in a mortar and mix evenly for 5 min. Pour into an aluminum foil weighing dish and spread flat in an oil bath at 150°C to melt for 15 min. After melting completely, take out the sample immediately and quench in liquid nitrogen for 2 min. Subsequently, vacuum dry and grind, and store at 4°C.

[0136] Example 8

[0137] Weigh genistein (216.35 mg)-chlorogenic acid (283.65 mg), quercetin (230.17 mg)-chlorogenic acid (269.83 mg), baicalein (216.35 mg)-chlorogenic acid (283.65 mg), kaempferol (223.43 mg)-chlorogenic acid (276.57 mg), chrysin (208.89 mg)-chlorogenic acid (291.11 mg) respectively, vortex for 10 min in a vortex instrument to mix evenly, place in a ball mill, add to a 250 mL agate ball mill, ball milling frequency of 30 Hz, grind 8 cycles, grind for 30 min each cycle, and cool for 10 min. After ball milling, place the sample in a vacuum drying oven at room temperature for 24 h, and seal the powder and store it at 4 ° C.

[0138] Example 9

[0139] Weigh genistein (138.04 mg)-chlorogenic acid (361.96 mg), quercetin (149.50 mg)-chlorogenic acid (350.50 mg), baicalein (138.04 mg)-chlorogenic acid (361.96 mg), kaempferol (143.86 mg)-chlorogenic acid (356.14 mg), chrysin (132.02 mg)-chlorogenic acid (367.98 mg) respectively, vortex for 10 min in a vortex instrument to mix evenly, place in a ball mill, add to a 250 mL agate ball mill, ball milling frequency of 30 Hz, grind 8 cycles, grind for 30 min each cycle, and cool for 10 min. After ball milling, place the sample in a vacuum drying oven at room temperature for 24 h, and seal the powder and store it at 4 ° C.

[0140] Example 10

[0141] Weigh genistein (302.01 mg)-chlorogenic acid (197.99 mg), quercetin (315.23 mg)-chlorogenic acid (184.77 mg), baicalein (302.02 mg)-chlorogenic acid (197.98 mg), kaempferol (308.85 mg)-chlorogenic acid (191.15 mg), chrysin (294.67 mg)-chlorogenic acid (205.33 mg), vortex for 10 min in a vortex instrument to mix evenly, place in a ball mill, add to a 250 mL agate ball mill, ball milling frequency of 30 Hz, grind 8 cycles, grind for 30 min each cycle, and cool for 10 min. After ball milling, place the sample in a vacuum drying oven at room temperature for 24 h, and seal the powder and store it at 4 ° C.

[0142] Embodiment 11

[0143] Weigh genistein (216.35 mg)-chlorogenic acid (283.65 mg), quercetin (230.17 mg)-chlorogenic acid (269.83 mg), baicalein (216.35 mg)-chlorogenic acid (283.65 mg), kaempferol (223.43 mg)-chlorogenic acid (276.57 mg), chrysin (208.89 mg)-chlorogenic acid (291.11 mg), vortex for 10 min in a vortex instrument to mix evenly, place in a ball mill, add to a 250 mL agate ball mill, ball milling frequency of 10 Hz, grind 8 cycles, grind for 30 min each cycle, and cool for 10 min. After ball milling, place the sample in a vacuum drying oven at room temperature for 24 h, and seal the powder and store it at 4 ° C.

[0144] Example 12

[0145] Weigh genistein (216.35 mg)-chlorogenic acid (283.65 mg), quercetin (230.17 mg)-chlorogenic acid (269.83 mg), baicalein (216.35 mg)-chlorogenic acid (283.65 mg), kaempferol (223.43 mg)-chlorogenic acid (276.57 mg), chrysin (208.89 mg)-chlorogenic acid (291.11 mg), vortex for 10 min in a vortex instrument to mix evenly, place in a ball mill, add to a 250 mL agate ball mill, ball milling frequency of 50 Hz, grind 8 cycles, each cycle grinding 30 min, cool 10 min. After ball milling, place the sample in a vacuum drying oven at room temperature for 24 h, and seal the powder and store it at 4 ° C.

[0146] Example 13

[0147] Weigh genistein (216.35 mg)-chlorogenic acid (283.65 mg), quercetin (230.17 mg)-chlorogenic acid (269.83 mg), baicalein (216.35 mg)-chlorogenic acid (283.65 mg), kaempferol (223.43 mg)-chlorogenic acid (276.57 mg), chrysin (208.89 mg)-chlorogenic acid (291.11 mg) respectively, vortex for 10 min in a vortex instrument to mix evenly, place in a ball mill, add to a 250 mL agate ball mill, ball milling frequency of 30 Hz, grind 2 cycles, grind 30 min each cycle, cool for 10 min. After ball milling, place the sample in a vacuum drying oven at room temperature for 24 h, and seal the powder and store it at 4 ° C.

[0148] Embodiment 14

[0149] Weigh genistein (216.35 mg)-chlorogenic acid (283.65 mg), quercetin (230.17 mg)-chlorogenic acid (269.83 mg), baicalein (216.35 mg)-chlorogenic acid (283.65 mg), kaempferol (223.43 mg)-chlorogenic acid (276.57 mg), chrysin (208.89 mg)-chlorogenic acid (291.11 mg) respectively, vortex for 10 min in a vortex instrument to mix evenly, place in a ball mill, add to a 250 mL agate ball mill, ball milling frequency of 30 Hz, grind 12 cycles, grind for 30 min each cycle, and cool for 10 min. After ball milling, place the sample in a vacuum drying oven at room temperature for 24 h, and seal the powder and store it at 4 ° C.

Claims

1. A flavonoid compound-chlorogenic acid co-amorphous substance, characterized in that: The co-amorphous material is composed of flavonoids and chlorogenic acid; wherein the flavonoids are genistein, quercetin, baicalein, kaempferol, chrysin, fisetin, apigenin, luteolin or daidzein.

2. The flavonoid compound-chlorogenic acid co-amorphous substance according to claim 1, characterized in that: The flavonoid compound is: genistein, quercetin, baicalein, kaempferol or chrysin.

3. The flavonoid compound-chlorogenic acid co-amorphous substance according to claim 1, characterized in that: The preparation method of the flavonoid compound-chlorogenic acid co-amorphous product is: weighing flavonoid compounds and chlorogenic acid and mixing them to form a physical mixture, and then using a melt quenching method or a ball milling method to prepare the flavonoid compound-chlorogenic acid co-amorphous product.

4. The flavonoid compound-chlorogenic acid co-amorphous substance according to claim 1, characterized in that: The molar ratio of the flavonoid compound to chlorogenic acid is 5:1 to 1:

5.

5. The flavonoid compound-chlorogenic acid co-amorphous substance according to claim 1, characterized in that: The molar ratio of the flavonoid compound to chlorogenic acid is 2:1 to 1:

2.

6. The flavonoid compound-chlorogenic acid co-amorphous substance according to claim 3, characterized in that: The melting temperature of the melt quenching method is 120-180° C., and the melting time is 3-15 minutes.

7. The flavonoid compound-chlorogenic acid co-amorphous substance according to claim 3, characterized in that: The ball milling frequency of the ball milling method is 10 to 50 Hz, and the ball milling time is 60 to 360 minutes.

8. A use of the flavonoid compound-chlorogenic acid co-amorphous substance according to claim 1, characterized in that: The flavonoid compound-chlorogenic acid co-amorphous substance is used for preparing antioxidant materials.

Images