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

Abstract

The application belongs to the technical field of medicine, and particularly relates to a flavonoid compound-green acid co-amorphous substance and a preparation method thereof. After screening flavonoid components with good miscibility with green acid, a flavonoid co-amorphous system is prepared from green acid and the flavonoid compound by using a melt quenching method or a ball milling method. PXRD, DSC and FTIR results show that five kinds of flavonoid compounds and green acid are successfully prepared into a co-amorphous system. Under non-leakage conditions, the five kinds of flavonoid compound-green acid co-amorphous substances have significant solubility / dissolution advantages compared with single crystal drugs and physical mixtures thereof. Meanwhile, under the conditions of 25 DEG C and 40 DEG C, the formed co-amorphous substances do not recrystallize within 90 days, and have excellent physical stability. In addition, through in-vitro antioxidant activity determination, the flavonoid compound co-amorphous substance shows good DPPH free radical scavenging ability and has strong antioxidant activity.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to a flavonoid compound-green acid co-amorphous substance and a preparation method thereof. BACKGROUND

[0002] At present, most of the developed drugs and the candidate drugs under development exhibit poor water solubility, which limits their oral absorption and therapeutic effect. Enhancing the water solubility of these candidate drugs has become a key problem for pharmaceutical companies and pharmaceutical companies to provide high-quality, high-performance drug products for patients. Amorphization is a common method for improving the solubility / solubility of hydrophobic drugs by disordering the crystal lattice of the hydrophobic drugs. In theory, amorphous drugs with disordered arrangement have higher entropy, enthalpy and Gibbs free energy, and generally exhibit better solubility / solubility than their crystal drugs. However, the inherent de-glassing risk of amorphous drugs during preparation, storage and dissolution process seriously restricts the development of amorphous drugs, and the advantages of amorphous system are weakened or even completely lost. Therefore, it is crucial to find effective strategies to stabilize the amorphous system.

[0003] Co-amorphous systems have attracted wide attention in stabilizing amorphous drugs and improving the solubility / solubility of hydrophobic drugs. According to the functional groups contained in the molecules of active ingredients, through the principle and method of crystal engineering, the drug molecules and suitable ligands (which can be pharmaceutical excipients or other drug molecules) are assembled into supramolecular complexes by non-covalent intermolecular forces (mainly hydrogen bonds). Due to the mixing of drug molecules at the molecular level or the rearrangement and optimization of the crystal lattice, the supramolecular complexes generally exhibit excellent physicochemical properties (such as higher solubility, stability and bioavailability, etc.).

[0004] Flavonoids are a large class of compounds widely existing in nature, mainly derived from vegetables, fruits, herbs, cereals, flowers and seeds. At the same time, flavonoids have various pharmacological effects, such as antioxidant, antibacterial, anti-inflammatory and cardiovascular effects, anti-liver injury effects, etc. In recent years, many flavonoids with different activities have been found, such as luteolin with antiviral, antitumor, antibacterial effects; morin with antitumor effects; quercetin with strong antioxidant effects. In addition, flavonoids are also important functional food additives, natural antioxidants, natural sweeteners, etc. However, due to their structural characteristics, they generally exhibit low solubility / solubility, and there is potential phase II metabolism of glucuronidase in vivo, which leads to poor oral absorption and limits their clinical application.

[0005] Chlorogenic acid (CA), a depside formed from caffeic acid and quinic acid (1-hydroxyhexahydrogallic acid), is a phenylpropanoid compound produced by plants during aerobic respiration via the shikimic acid pathway, and has the chemical structure of: At present, chlorogenic acid as a food additive exists in food 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 and the like.

[0006] CN 102178675 A discloses quercetin, chlorogenic acid and pharmaceutically acceptable adjuvants, which are prepared together to form a pharmaceutical composition, 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 adjuvants (lactose, corn starch, magnesium stearate, etc.), and the drug therein is still in a crystalline state. SUMMARY

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

[0008] The flavonoid-chlorogenic acid co-amorphous substance is prepared by a melt-quenching method or a ball milling method. First, nine flavonoids with good miscibility with chlorogenic acid are screened out through Hansen solubility calculation, including genistein, quercetin, baicalein, kaempferol, chrysin, fisetin, apigenin, luteolin or daidzein.

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

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

[0011] The method for preparing the flavonoid-chlorogenic acid co-amorphous substance by the melt-quenching method is as follows: the selected flavonoid and chlorogenic acid are ground and mixed in different stoichiometric ratios, laid flat on an aluminum foil weighing dish, and placed in an oil bath for melting, then the sample is immediately taken out and quenched in liquid nitrogen, ground after vacuum drying, and the flavonoid-chlorogenic acid co-amorphous substance is obtained.

[0012] The melting temperature of the melt-quenching method is 120-180℃, and the melting time is 3-15 min.

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

[0014] Compared with the single crystal drug and the physical mixture thereof, the flavonoid-chlorogenic acid co-amorphous substance has significantly improved water solubility, can reach a high dissolution concentration, and can maintain a supersaturated state for a long time.

[0015] Compared with the single flavonoid amorphous substance, the flavonoid-chlorogenic acid co-amorphous substance has better physical stability.

[0016] Compared with the single flavonoid compound, the flavonoid-chlorogenic acid co-amorphous substance has stronger DPPH free radical scavenging ability and high antioxidant activity.

[0017] The flavonoid-chlorogenic acid co-amorphous substance has the following beneficial effects:

[0018] Based on the solubility / stability defects and potential metabolic inhibition of the flavonoid compound, the supermolecular co-amorphous system is designed by co-amorphization of the flavonoid compound and the glucuronidase inhibitor chlorogenic acid by using the crystal engineering technology, so as to improve the solubility / dissolution, enhance the physical stability and the antioxidant activity, and achieve the purposes of improving the oral absorption and the curative effect of the flavonoid compound, and providing a new idea for the drug optimization of the flavonoid compound and other poorly soluble drugs. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is a PLM graph of the preliminary preparation product of the flavonoid compound and the chlorogenic acid;

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

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

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

[0023] Figure 5 is a dissolution curve comparison graph of the genistein crystal, the physical mixture of the genistein and the chlorogenic acid prepared in Example 1, and the genistein-chlorogenic acid co-amorphous substance prepared in Example 1 under the non-bath condition;

[0024] Figure 6is a plot of the dissolution profiles of quercetin crystals, a physical mixture of quercetin prepared in Example 1 and chlorogenic acid, and a co-amorphous of quercetin-chlorogenic acid prepared in Example 1 under non-puddle conditions;

[0025] Figure 7 is a plot of the dissolution profiles of baicalein crystals, a physical mixture of baicalein prepared in Example 1 and chlorogenic acid, and a co-amorphous of baicalein-chlorogenic acid prepared in Example 1 under non-puddle conditions;

[0026] Figure 8 is a plot of the dissolution profiles of kaempferol crystals, a physical mixture of kaempferol prepared in Example 1 and chlorogenic acid, and a co-amorphous of kaempferol-chlorogenic acid prepared in Example 1 under non-puddle conditions;

[0027] Figure 9 is a plot of the dissolution profiles of acacetin crystals, a physical mixture of acacetin prepared in Example 1 and chlorogenic acid, and a co-amorphous of acacetin-chlorogenic acid prepared in Example 1 under non-puddle conditions;

[0028] Figure 10 is a PXRD plot of genistein amorphous stability comparison;

[0029] Figure 11 is a PXRD plot of quercetin amorphous stability comparison;

[0030] Figure 12 is a PXRD plot of baicalein amorphous stability comparison;

[0031] Figure 13 is a PXRD plot of kaempferol amorphous stability comparison;

[0032] Figure 14 is a PXRD plot of acacetin amorphous stability comparison;

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

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

[0035] Figure 17 is a PXRD plot of baicalein-chlorogenic acid co-amorphous prepared in Example 1 stability comparison;

[0036] Figure 18 is a PXRD plot of kaempferol-chlorogenic acid co-amorphous prepared in Example 1 stability comparison;

[0037] Figure 19PXRD pattern of stability comparison of chrysin-greenophore co-amorphous prepared in Example 1;

[0038] Figure 20 Graph of DPPH radical scavenging activity of genistein crystal and genistein- greenophore co-amorphous prepared in Example 1;

[0039] Figure 21 Graph of DPPH radical scavenging activity of quercetin crystal and quercetin- greenophore co-amorphous prepared in Example 1;

[0040] Figure 22 Graph of DPPH radical scavenging activity of baicalein crystal and baicalein- greenophore co-amorphous prepared in Example 1;

[0041] Figure 23 Graph of DPPH radical scavenging activity of kaempferol crystal and kaempferol- greenophore co-amorphous prepared in Example 1;

[0042] Figure 24 Graph of DPPH radical scavenging activity of chrysin crystal and chrysin- greenophore co-amorphous prepared in Example 1. DETAILED DESCRIPTION

[0043] The present application is further illustrated by the following test examples and examples.

[0044] Test Example 1: Hansen solubility calculation of flavonoid-greenophore co-amorphous was carried out as follows:

[0045] Hansen solubility parameter (δ) calculation was carried out by Molecular Modeling Pro software, when the difference of solubility parameter (Δδ) of two components was less than 7.0 MPa, it was considered that they had good compatibility. As shown in Table 1, 9 kinds of flavonoids (genistein, quercetin, baicalein, kaempferol, chrysin, fisetin, apigenin, luteolin, daidzein) had good miscibility with greenophore. While the miscibility of myricetin, morin, procyanidine, galangal essence, khusimarin was poor.

[0046] Table 1 Hansen solubility parameter (δ) of flavonoid crystal and greenophore crystal

[0047]

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

[0049] The crystal birefringence of the prepared product of flavonoids-chlorogenic acid was observed by polarized light microscopy (PLM). The sample was placed on a glass slide, fixed with liquid paraffin and dispersed uniformly, and observed under 100 times magnification.

[0050] Through the calculation of Hansen solubility parameters, 9 kinds of flavonoids had good miscibility with chlorogenic acid. The melt-quenching method (specifically the same as Example 1) was used to try to prepare flavonoids-chlorogenic acid co-amorphous substances. Through PLM observation, the prepared products of 5 kinds of flavonoids (genistein, quercetin, baicalein, kaempferol, chrysin) and chlorogenic acid showed no crystal birefringence, indicating that amorphous form could be formed. Figure 1 a-e) in the middle.

[0051] Example 1

[0052] 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) were weighed respectively, ground in a mortar and mixed uniformly for 5 min to obtain physical mixtures. Then the five physical mixtures were poured into aluminum foil weighing dishes respectively, and laid flat in an oil bath at 150°C for melting for 5 min. After complete melting, the sample was immediately taken out and placed in liquid nitrogen for quenching for 2 min. Then, vacuum drying and grinding were carried out, and the sample was stored at 4°C.

[0053] Test Example 3: The flavonoids-chlorogenic acid co-amorphous substances of Example 1 were characterized 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] Scan speed: 4° / min

[0062] Scan range: 2θ, 5-40°

[0063] The crystal diffraction peaks of 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 are shown in Figure 2 .

[0064] As shown in Figure 2 , in Example 1, the crystal diffraction peaks of genistein-chlorogenic acid combination, quercetin-chlorogenic acid combination, baicalein-chlorogenic acid combination, kaempferol-chlorogenic acid combination and chrysin-chlorogenic acid combination disappeared, showing diffraction rings with amorphous characteristics, indicating that they were in amorphous state. The genistein-chlorogenic acid combination, quercetin-chlorogenic acid combination, baicalein-chlorogenic acid combination, kaempferol-chlorogenic acid combination and chrysin-chlorogenic acid combination in Examples 2-14 also showed similar PXRD patterns.

[0065] 2. Differential scanning calorimetry (DSC)

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

[0067] Range: 25-300℃

[0068] Heating rate: 10℃ / min

[0069] Nitrogen flow rate: 80 mL / min

[0070] The genistein-chlorogenic acid co-amorphous substance, quercetin-chlorogenic acid co-amorphous substance, baicalein-chlorogenic acid co-amorphous substance, kaempferol-chlorogenic acid co-amorphous substance and chrysin-chlorogenic acid co-amorphous substance were detected respectively, and the corresponding DSC patterns are shown in Figure 3 .

[0071] As shown in Figure 3 , in Example 1, the DSC patterns of genistein-chlorogenic acid co-amorphous substance, quercetin-chlorogenic acid co-amorphous substance, baicalein-chlorogenic acid co-amorphous substance, kaempferol-chlorogenic acid co-amorphous substance and chrysin-chlorogenic acid co-amorphous substance showed only one glass transition temperature (T g ), and the T g values were 65.9℃, 106.6℃, 94.4℃, 98.3℃, 96.2℃ respectively, further proving that they were single-phase binary co-amorphous substances. The genistein-chlorogenic acid co-amorphous substance, quercetin-chlorogenic acid co-amorphous substance, baicalein-chlorogenic acid co-amorphous substance, kaempferol-chlorogenic acid co-amorphous substance and chrysin-chlorogenic acid co-amorphous substance in Examples 2-14 showed similar DSC patterns, showing a single glass transition temperature, and the glass transition temperature changed slightly with the change of molar ratio.

[0072] 3. Infrared spectrum (FTIR)

[0073] Instrument: Thermo Scientific Nicolet iS50 Fourier Transform Infrared Spectrometer (Thermo Fisher Scientific, America)

[0074] Range: 4000-400 cm -1

[0075] Scan times: 32 times

[0076] The detection of genistein-chlorogenic acid co-amorphous substance, quercetin-chlorogenic acid co-amorphous substance, baicalein-chlorogenic acid co-amorphous substance, kaempferol-chlorogenic acid co-amorphous substance and chrysin-chlorogenic acid co-amorphous substance was detected respectively, and the corresponding FTIR spectrum was as shown in Figure 4 .

[0077] As shown in Figure 4 , in Example 1, the FTIR spectrum wave number (cm -1 ) of genistein-chlorogenic acid co-amorphous substance was: 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 599.70 cm -1 .

[0078] The FTIR spectrum wave number (cm -1 ) of quercetin-chlorogenic acid co-amorphous substance was: 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 597.49 cm -1 .

[0079] The FTIR spectrum wave number (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 614.27 cm -1 .

[0080] FTIR spectrum of quercetin-chlorogenic acid co-amorphate -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 505.97 cm -1 .

[0081] FTIR spectrum of chrysin-chlorogenic acid co-amorphate -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 430.66 cm -1 .

[0082] The genistein-chlorogenic acid co-amorphate, quercetin-chlorogenic acid co-amorphate, baicalein-chlorogenic acid co-amorphate, chrysin-chlorogenic acid co-amorphate, and apigenin-chlorogenic acid co-amorphate in Examples 2 to 14 exhibit the same or similar FTIR spectra.

[0083] Test Example 4: Apparent solubility test was performed on the flavonoid-chlorogenic acid co-amorphates of Example 1, as follows:

[0084] Excess of flavonoid crystal, flavonoid-chlorogenic acid physical mixture (molar ratio 1:1), flavonoid-chlorogenic acid co-amorphous (molar ratio 1:1) were weighed separately and dissolved in 5 mL of pH 1.2 HC1 buffer and pH 6.8 phosphate buffer solution, respectively, and placed in a 37°C air bath constant temperature shaker for 24 h to reach solubility equilibrium. Then, the filtrate was filtered (0.22 mm) and diluted with an equal volume of methanol, and the filtrate was analyzed by HPLC. Each sample was determined in triplicate.

[0085] The high performance liquid chromatography conditions are as follows:

[0086] Instrument: Agilent 1260 high performance liquid chromatograph

[0087] Column: Ultimate XB-C18 (4.6 mm x 250 mm, 5 μm)

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

[0089] Flow rate: 1.0 mL / min

[0090] From the apparent solubility determination results in Table 2, the physical mixture and the crystal drug exhibit similar apparent solubility in pH 1.2 HC1 buffer and pH 6.8 phosphate buffer solution at 37°C. The solubility of the flavonoid-chlorogenic acid co-amorphous is increased by 2.06-18.58 times in pH 1.2 HC1 buffer and 2.02-36.25 times in pH 6.8 phosphate buffer solution. Therefore, after the flavonoid and chlorogenic acid are prepared into a co-amorphous system, the molecules are arranged in disorder, have large entropy, enthalpy and Gibbs free energy, thereby exhibiting higher solubility compared with the crystal drug. The solubility of the flavonoid-chlorogenic acid co-amorphous in Examples 2 and 8 exhibits a similar significant increase.

[0091] From Table 3, the solubility of other flavonoids (daidzein, rhusinic acid) and their products with chlorogenic acid in the two pH media does not exhibit a significant improvement.

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

[0093]

[0094]

[0095] Table 3 solubility of other flavonoids, flavonoids-chlorogenic acid preparation products (preparation method as above, flavonoids-chlorogenic acid molar ratio 1:1)

[0096]

[0097] Test Example 5: non-puddle dissolution test of flavonoids-chlorogenic acid co-amorphous substance in Example 1 was carried out as follows:

[0098] Excess flavonoids crystal, flavonoids-chlorogenic acid physical mixture (molar ratio 1:1), flavonoids-chlorogenic acid co-amorphous substance (molar ratio 1:1) were taken. The dissolution test was carried out at 37°C, 100 rpm, and the dissolution medium was HCl buffer (pH 1.2) and phosphate buffer solution (pH 6.8) respectively. 2ml was sampled at the preset time point (the same temperature and volume of dissolution medium was immediately supplemented), then filtered (0.22mm) and diluted with equal volume of methanol, and the filtrate was analyzed by HPLC and the peak area was recorded, 3 replicates were determined for each sample.

[0099] As Figures 5-9 As shown in Table 4, Table 6, in Example 1, there was no significant difference in dissolution behavior between flavonoids drug substance and physical mixture, while the dissolution of genistein-chlorogenic acid co-amorphous substance, quercetin-chlorogenic acid co-amorphous substance, baicalein-chlorogenic acid co-amorphous substance, kaempferol-chlorogenic acid co-amorphous substance and chrysin-chlorogenic acid co-amorphous substance was significantly increased compared with the corresponding flavonoids and maintained at a high concentration for a long time. The dissolution behavior of flavonoids-chlorogenic acid co-amorphous substances in Examples 2 and 8 was also significantly enhanced at different time points.

[0100] From Table 5 and Table 7, the dissolution behavior of other flavonoids (daidzein, rhuskin) and their preparation products with chlorogenic acid in pH 1.2 HCl buffer and pH 6.8 phosphate buffer solution was not significantly improved compared with the crystal drug.

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

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

[0103] Table 4 Dissolution concentration (pg / mL) of flavonoid crystal, flavonoid-chlorogenic acid physical mixture and flavonoid-chlorogenic acid co-amorphous substance at different time points under pH 1.2 condition

[0104]

[0105]

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

[0107]

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

[0109]

[0110]

[0111] Table 7. Dissolution concentrations (μg / mL) of other flavonoid crystals and flavonoid-chlorogenic acid preparation products (preparation method as above, flavonoid-chlorogenic acid molar ratio 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 compound from Example 1, as detailed below:

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

[0115] like Figures 10-14 As shown, the amorphous products of genistein, quercetin, baicalin, kaempferol, and poplarin were stored at 25℃ and 40℃. The results showed that the amorphous products of genistein, quercetin, baicalin, and poplarin underwent crystallization after 14 days, while the amorphous product of kaempferol underwent crystallization after 30 days.

[0116] like Figures 15-19As shown in Table 1, the PXRD diffractograms of 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 showed no characteristic peaks at 25°C and 40°C after 90 days of storage, indicating that 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 had high physical stability.

[0117] Example 7: Antioxidant test of flavonoids, flavonoids-chlorogenic acid co-amorphous was carried out as follows:

[0118] 500 μL of serially diluted sample solution or extraction solvent (blank) was combined with 500 μL of DPPH solution, the mixture was mixed vigorously, and then 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 according to the following formula: scavenging activity (%) = [(A0-A1) / A0] x 100%, wherein A0 and A1 are the absorbance of the blank and flavonoids or co-amorphous thereof.

[0119] As shown in Table 2, the DPPH radical scavenging ability of genistein-chlorogenic acid co-amorphous was 79.08% at a concentration of 12.5 μg / mL, which was significantly stronger than that of genistein (6.93%), indicating that genistein-chlorogenic acid co-amorphous had strong antioxidant activity. Figure 20 As shown in Table 3, the DPPH radical scavenging ability of quercetin-chlorogenic acid co-amorphous was 65.28% at a concentration of 3.125 μg / mL, while that of quercetin was only 44.63%, indicating that quercetin-chlorogenic acid co-amorphous exhibited strong antioxidant activity at low doses.

[0120] Figure 21 As shown in Table 4, the DPPH radical scavenging ability of baicalein-chlorogenic acid co-amorphous was 81.57% at a low concentration (6.25 μg / mL), and remained constant as the dose increased, indicating that it reached the maximum DPPH scavenging activity, while that of baicalein was only 44.46%, indicating that baicalein-chlorogenic acid co-amorphous exhibited strong antioxidant activity at low doses.

[0121] As shown in Table 5, the DPPH radical scavenging ability of kaempferol-chlorogenic acid co-amorphous was 79.08% at a concentration of 12.5 μg / mL, which was significantly stronger than that of genistein (6.93%), indicating that genistein-chlorogenic acid co-amorphous had strong antioxidant activity. Figure 22 As shown in Table 6, the DPPH radical scavenging ability of chrysin-chlorogenic acid co-amorphous was 65.28% at a concentration of 3.125 μg / mL, while that of quercetin was only 44.63%, indicating that quercetin-chlorogenic acid co-amorphous exhibited strong antioxidant activity at low doses.

[0122] Figure 23 ​​As shown, at the same concentration (6.25 μg / mL), the DPPH radical scavenging ability of kaempferol- chlorogenic acid co-amorphous solid reached 72.61%, which was 2.20 times that of kaempferol, indicating that kaempferol-chlorogenic acid co-amorphous solid had strong antioxidant activity at a low dose.

[0123] As shown, at the same concentration (6.25 μg / mL), the DPPH radical scavenging ability of kaempferol- chlorogenic acid co-amorphous solid reached 72.61%, which was 2.20 times that of kaempferol, indicating that kaempferol-chlorogenic acid co-amorphous solid had strong antioxidant activity at a low dose. Figure 24

[0124] Example 2

[0125] Respectively, 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) were weighed into a mortar and ground and uniformly mixed for 5 min. They were poured into an aluminum foil weighing dish and laid flat in an oil bath at 150°C for 5 min of melting. After complete melting, the sample was immediately removed and quenched in liquid nitrogen for 2 min. Subsequently, vacuum drying and grinding were performed, and storage was performed at 4°C.

[0126] Example 3

[0127] Respectively, 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) were weighed into a mortar and ground and uniformly mixed for 5 min. They were poured into an aluminum foil weighing dish and laid flat in an oil bath at 150°C for 5 min of melting. After complete melting, the sample was immediately removed and quenched in liquid nitrogen for 2 min. Subsequently, vacuum drying and grinding were performed, and storage was performed at 4°C.

[0128] Example 4

[0129] ​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) were weighed separately, ground in a mortar and mixed homogeneously for 5 min. They were poured into an aluminium foil weighing dish and spread out on an oil bath at 120°C for 5 min. After complete melting, the samples were immediately removed and quenched in liquid nitrogen for 2 min. Subsequently, they were vacuum dried and ground and stored at 4°C.

[0130] Example 5

[0131] 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) were weighed separately, ground in a mortar and mixed homogeneously for 5 min. They were poured into an aluminium foil weighing dish and spread out on an oil bath at 180°C for 5 min. After complete melting, the samples were immediately removed and quenched in liquid nitrogen for 2 min. Subsequently, they were vacuum dried and ground and stored at 4°C.

[0132] Example 6

[0133] 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) were weighed separately, ground in a mortar and mixed homogeneously for 5 min. They were poured into an aluminium foil weighing dish and spread out on an oil bath at 150°C for 3 min. After complete melting, the samples were immediately removed and quenched in liquid nitrogen for 2 min. Subsequently, they were vacuum dried and ground and stored at 4°C.

[0134] Example 7

[0135] 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) were weighed separately, ground in a mortar and mixed homogeneously for 5 min. Transferred to an aluminium foil weighed dish and spread on an oil bath at 150 °C for 15 min. Immediately after complete melting, the sample was taken out and quenched in liquid nitrogen for 2 min. Subsequently, vacuum dried and ground and stored at 4 °C.

[0136] Example 8

[0137] 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) were weighed separately, vortexed for 10 min in a vortexer, placed in a ball mill and added to a 250 mL agate ball mill jar. The ball mill frequency was 30 Hz and the grinding was performed for 8 cycles of 30 min each, with 10 min cooling in between. The ball milled sample was dried in a vacuum oven at room temperature for 24 h and the powder was sealed and stored at 4 °C.

[0138] Example 9

[0139] 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) were weighed separately, vortexed for 10 min in a vortexer, placed in a ball mill and added to a 250 mL agate ball mill jar. The ball mill frequency was 30 Hz and the grinding was performed for 8 cycles of 30 min each, with 10 min cooling in between. The ball milled sample was dried in a vacuum oven at room temperature for 24 h and the powder was sealed and stored at 4 °C.

[0140] Example 10

[0141] 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) were weighed separately, vortexed for 10 min in a vortexer, placed in a ball mill, added to a 250 mL agate ball mill jar, and milled at a frequency of 30 Hz for 8 cycles, with each cycle being 30 min and a cooling period of 10 min. The milled samples were dried in a vacuum oven at room temperature for 24 h, sealed and stored at 4 °C.

[0142] Example 11

[0143] 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) were weighed separately, vortexed for 10 min in a vortexer, placed in a ball mill, added to a 250 mL agate ball mill jar, and milled at a frequency of 10 Hz for 8 cycles, with each cycle being 30 min and a cooling period of 10 min. The milled samples were dried in a vacuum oven at room temperature for 24 h, sealed and stored at 4 °C.

[0144] Example 12

[0145] 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) were weighed separately, vortexed for 10 min in a vortexer, placed in a ball mill, added to a 250 mL agate ball mill jar, and milled at a frequency of 50 Hz for 8 cycles, with each cycle being 30 min and a cooling period of 10 min. The milled samples were dried in a vacuum oven at room temperature for 24 h, sealed and stored at 4 °C.

[0146] Example 13

[0147] 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) were weighed separately, vortexed for 10 min in a vortexer, mixed uniformly, placed in a ball mill, added to a 250 mL agate ball mill jar, the ball milling frequency was 30 Hz, milled for 2 cycles, each cycle for 30 min, cooled for 10 min. The samples after ball milling were placed in a vacuum drying oven and dried at room temperature for 24 h, the powder was sealed and stored at 4°C.

[0148] Example 14

[0149] 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) were weighed separately, vortexed for 10 min in a vortexer, mixed uniformly, placed in a ball mill, added to a 250 mL agate ball mill jar, the ball milling frequency was 30 Hz, milled for 2 cycles, each cycle for 30 min, cooled for 10 min. The samples after ball milling were placed in a vacuum drying oven and dried at room temperature for 24 h, the powder was sealed and stored at 4°C.

Claims

1. A flavonoid-chlorogenic acid co-amorphate, characterized in that, The flavonoid is kaempferol, and the flavonoid-chlorogenic acid co-amorphous substance has no crystal diffraction peak detected in the range of 2θ = 5-40° by Cu-Kα radiation, and differential scanning calorimetry shows that the glass transition temperature thereof is 98.3℃; The flavonoid-chlorogenic acid co-amorphous substance is prepared by the following preparation method: a physical mixture is prepared by mixing the flavonoid and the chlorogenic acid at a molar ratio of 2:1-1:2, and then a melt quenching method or a ball milling method is used to prepare the flavonoid-chlorogenic acid co-amorphous substance; the melt quenching method has a melt temperature of 120-180℃ and a melt time of 3-15 min; The ball milling method has a ball milling frequency of 10-50 Hz and a ball milling time of 60-360 min.

2. Use of the flavonoid-chlorogenic acid co-amorphate according to claim 1, characterized in that, The flavonoid-chlorogenic acid co-amorphous substance is used for preparing an antioxidant material.

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