Aescin and mannitol eutectic crystal as well as preparation method and application thereof

Abstract

The invention relates to the technical field of eutectic crystals, in particular to an aesculin and mannitol eutectic crystal and a preparation method and application thereof.The XRD spectrum of the eutectic crystal at least has characteristic diffraction peaks at the positions where the angle 2 theta is 13.80 degrees, 17.41 degrees, 18.87 degrees, 19.99 degrees, 20.55 degrees, 21.46 degrees, 25.29 degrees, 27.15 degrees, 27.66 degrees, 28.30 degrees, 33.32 degrees, 34.13 degrees, 35.67 degrees, 36.33 degrees, 36.80 degrees and 44.06 degrees, and error tolerance of + / -0.2 degrees exists; characteristic endothermic peaks exist at 167.5 DEG C and 327 DEG C in a DSC spectrum, and error tolerance of + / -0.2 DEG C exists. The preparation method comprises the following steps: dissolving mannitol and escin in deionized water, heating and stirring, and carrying out high-temperature and high-pressure reaction, standing, crystallizing, filtering and drying to obtain the eutectic crystal which can be applied to cosmetics, health-care products, foods and medicines. The eutectic crystal provided by the invention has the advantages of no irritation, high safety, easy absorption and good stability.

Description

Technical Field

[0001] The present invention relates to the technical field of co-crystals, and in particular to a co-crystal of aescin and mannitol, and a preparation method and application thereof. Background Art

[0002] Aescin is a triterpenoid saponin extracted from the dried mature seeds of the Aesculus hippocastanum plant, and primarily includes aescin A, B, C, and D. Aescin is a pentacyclic triterpenoid saponin that can be extracted from the seeds, bark, and immature buds and leaves of the horse chestnut tree.

[0003] Aescin has multiple pharmacological effects:

[0004] Anti-inflammatory effect: It can inhibit the levels of inflammatory factors such as TNF-α and IL-1β, reduce the exudation of inflammatory tissues, and reduce tissue permeability;

[0005] Anti-exudative effect: reduces vascular permeability, inhibits capillary water exudation, and thus reduces edema;

[0006] Promote venous blood flow and lymphatic return: increase venous tension, accelerate venous blood flow, promote lymphatic return, and improve blood circulation and microcirculation;

[0007] Protect blood vessel walls: It has a protective effect on vascular endothelial cells and helps maintain the integrity of blood vessel walls;

[0008] Anti-aging and improve blood circulation of the skin around the eyes: It is rich in flavonoids, which have natural strong anti-aging and anti-wrinkle effects and can improve blood circulation of the skin around the eyes.

[0009] However, the safety, transdermal absorption and stability of aescin are poor, which affects its application in many fields. Summary of the Invention

[0010] In response to the problems of poor safety, transdermal absorption and stability of aescin in the prior art, the present invention proposes a co-crystal of aescin and mannitol, a preparation method and application thereof. The obtained co-crystal has the advantages of being non-irritating, highly safe, having good transdermal absorption and good stability.

[0011] Specifically, the present invention is achieved through the following technical scheme: an aescin and mannitol co-crystal, wherein the XRD pattern of the aescin and mannitol co-crystal has characteristic diffraction peaks at least at 2θ angles of 13.80°, 17.41°, 18.87°, 19.99°, 20.55°, 21.46°, 25.29°, 27.15°, 27.66°, 28.30°, 33.32°, 34.13°, 35.67°, 36.33°, 36.80°, and 44.06°, with an error tolerance of ±0.2°; and the DSC spectrum has characteristic endothermic peaks at 167.5°C and 327°C, with an error tolerance of ±0.2°.

[0012] As a preferred embodiment, the XRD pattern of the aescin and mannitol cocrystal also has characteristic diffraction peaks at 2θ angles of 11.11°, 15.22°, 28.76°, 30.70°, 31.65°, 39.74°, 40.97°, 44.82°, 48.46°, 49.24°, and 56.76°, with an error tolerance of ±0.2°; the grain sizes corresponding to the characteristic diffraction peaks are

[0013] As a preferred embodiment, the grain sizes corresponding to the characteristic diffraction peaks of 13.80°, 17.41°, 18.87°, 19.99°, 20.55°, 21.46°, 25.29°, 27.15°, 27.66°, 28.30°, 33.32°, 34.13°, 35.67°, 36.33°, 36.80°, and 44.06° are respectively

[0014] As a preferred embodiment, the infrared spectrum of the cocrystal of aescin and mannitol is at least 3279.98 cm -1 、2924.23cm -1 、2854.49cm -1 、1717.49cm -1 、1458.40cm -1 、1375.80cm -1 、1317.64cm -1 、1258.43cm -1 、1194.58cm -1 、1076.24cm -1 、1018.67cm -1 、949.92cm -1 、929.49cm -1 、877.83cm -1 、718.55cm-1 、621.12cm -1 There is a characteristic peak at ±0.2cm -1 error tolerance.

[0015] The present invention also provides a method for preparing a eutectic of aescin and mannitol, comprising the following steps:

[0016] S1. Weigh mannitol and deionized water according to the ratio, and stir to dissolve mannitol in the deionized water to form a mannitol solution;

[0017] S2. Weigh aescin and add it to the mannitol solution, stirring until the aescin is completely dissolved;

[0018] S3, continue heating and stirring the mixed solution in S2 for 1-2h;

[0019] S4, placing the solution in step S3 into a reactor and reacting for 0.5-1h;

[0020] S5, taking out the solution in S4 and letting it stand for crystallization;

[0021] S6. Filter the crystals in S5, dry them in a spray dryer, and grind them into fine powder.

[0022] As a preferred embodiment, the mass ratio of mannitol, aescin and deionized water is 2:1:7.

[0023] As a preferred solution, in step S3, stirring is performed in a heat-collecting constant-temperature heating magnetic stirrer at a temperature of 30 to 50° C. and a rotation speed of 150 to 250 r / min.

[0024] As a preferred solution, in step S4, the temperature is 100-120° C. and the pressure is 10-15 MPa.

[0025] The last aspect of the present invention also provides the use of aescin and mannitol co-crystal in cosmetics, food, health products and medicines.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] (1) Compared with the raw material aescin, the aescin and mannitol co-crystal prepared by the present invention has mild properties. The chicken embryo test results show that it is non-irritating to the skin, and the zebrafish test results show that it has high safety. In cosmetic applications, it reduces the occurrence of allergic symptoms such as skin redness, swelling, and itching;

[0028] (2) Compared with the raw material aescin, the aescin and mannitol co-crystal obtained by the present invention has improved transdermal absorption performance, so that it can penetrate into the skin more efficiently and be fully absorbed and utilized by human skin, thereby making the multiple effects of aescin such as anti-inflammatory and antioxidant better exerted;

[0029] (3) The stability of the co-crystal of aescin and mannitol prepared by the present invention is significantly improved compared to the raw material aescin. This property makes the co-crystal more reliable in the application of cosmetics, pharmaceuticals, health care products and other fields, and can maintain the effectiveness of the active ingredients for a long time, thereby extending the shelf life of the product. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is the XRD pattern of aescin, mannitol and aescin and mannitol cocrystal of the present invention;

[0031] Figure 2 is a DSC graph of the co-crystal of aescin and mannitol of the present invention;

[0032] Figure 3 is a DSC graph of aescin of the present invention;

[0033] Figure 4 It is the DSC diagram of mannitol of the present invention;

[0034] Figure 5 This is the infrared spectrum of the co-crystal of aescin and mannitol of the present invention;

[0035] Figure 6 This is a graph showing the irritation test of the aescin and mannitol co-crystal of the present invention (chicken embryo test results);

[0036] Figure 7 This is a test chart of the irritation of aescin of the present invention (chicken embryo test results);

[0037] Figure 8 This is a comparative data diagram of the transdermal absorption of aescin and mannitol cocrystal and aescin of the present invention;

[0038] Figure 9 This is a graph showing the stability test of the aescin and mannitol cocrystal and the aescin aqueous solution of the present invention. DETAILED DESCRIPTION

[0039] The following will be combined with the accompanying drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, rather than all the embodiments. The following description of at least one exemplary embodiment is actually only illustrative and is in no way intended to limit the present invention and its application or use. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0040] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0041] Unless otherwise specifically stated, the relative arrangement of the parts and steps, numerical expressions and numerical values ​​set forth in these embodiments do not limit the scope of the present invention. The technology, methods and equipment known to those of ordinary skill in the relevant art may not be discussed in detail, but in appropriate cases, the technology, methods and equipment should be considered as part of the authorization specification. In all examples shown and discussed here, any specific value should be interpreted as being merely exemplary, rather than as a limitation. Therefore, other examples of the exemplary embodiments may have different values. It should be noted that similar numbers and letters represent similar items in the following figures, and therefore, once an item is defined in one figure, it does not need to be further discussed in subsequent figures.

[0042] Example 1

[0043] A method for preparing a cocrystal of aescin and mannitol, comprising the following steps:

[0044] S1. Weigh mannitol and deionized water according to the ratio, and stir to dissolve mannitol in the deionized water to form a mannitol solution;

[0045] S2. Weigh aescin according to the ratio and add it to the mannitol solution, stirring until the aescin is completely dissolved;

[0046] S3, the mixed solution in S2 was placed in a heat-collecting constant temperature heating magnetic stirrer, and stirred at a temperature of 40°C and a speed of 200 r / min for 1.5 h;

[0047] S4, placing the solution in step S3 into a reactor and reacting for 45 min at a pressure of 13 MPa and a temperature of 110°C;

[0048] S5, taking out the solution in S4 and letting it stand for 24 hours for crystallization;

[0049] S6. Filter the crystals in S5, place them in a spray dryer for drying, and grind them to obtain a co-crystal powder of aescin and mannitol.

[0050] The mass ratio is shown in the following table:

[0051] Material Name Ratio Aescin 10％ Mannitol 20％ Deionized water 70％

[0052] Example 2

[0053] A method for preparing a cocrystal of aescin and mannitol, comprising the following steps:

[0054] S1. Weigh mannitol and deionized water according to the ratio, and stir to dissolve mannitol in the deionized water to form a mannitol solution;

[0055] S2. Weigh aescin according to the ratio and add it to the mannitol solution, stirring until the aescin is completely dissolved;

[0056] S3, put the mixed solution in S2 into a heat-collecting constant temperature heating magnetic stirrer, and stir at a temperature of 30°C and a speed of 150 r / min for 2 h;

[0057] S4, placing the solution in step S3 into a reactor and reacting for 60 min at a pressure of 10 MPa and a temperature of 100° C.;

[0058] S5, taking out the solution in S4 and letting it stand for 24 hours for crystallization;

[0059] S6. Filter the crystals in S5, place them in a spray dryer for drying, and grind them to obtain a co-crystal powder of aescin and mannitol.

[0060] The mass ratio is shown in the following table:

[0061] Material Name Ratio Aescin 10％ Mannitol 20％ Deionized water 70％

[0062] Example 3

[0063] A method for preparing a cocrystal of aescin and mannitol, comprising the following steps:

[0064] S1. Weigh mannitol and deionized water according to the ratio, and stir to dissolve mannitol in the deionized water to form a mannitol solution;

[0065] S2. Weigh aescin according to the ratio and add it to the mannitol solution, stirring until the aescin is completely dissolved;

[0066] S3, placing the mixed solution in S2 into a heat-collecting constant temperature heating magnetic stirrer, and stirring at a temperature of 50°C and a speed of 250 r / min for 1 h;

[0067] S4, placing the solution in step S3 into a reactor and reacting for 30 min at a pressure of 15 MPa and a temperature of 120°C;

[0068] S5, taking out the solution in S4 and letting it stand for 24 hours for crystallization;

[0069] S6. Filter the crystals in S5, place them in a spray dryer for drying, and grind them to obtain a co-crystal powder of aescin and mannitol.

[0070] The mass ratio is shown in the following table:

[0071] Material Name Ratio Aescin 10％ Mannitol 20％ Deionized water 70％

[0072] X-ray diffraction analysis was performed on the cocrystal of aescin and mannitol obtained in Example 1 and the raw materials. The specific test parameters were:

[0073] Voltage, current: 40kV, 40mA

[0074] Detector: DteX250(H)

[0075] Test range: 5-80°

[0076] Step size: 0.01°

[0077] Scanning speed: 10.00deg / min

[0078] Divergence slit: 10mm;

[0079] Get as Figure 1 The XRD pattern of the cocrystal of aescin and mannitol is shown in FIG. 2, which has characteristic diffraction peaks at 2θ angles of 13.80°, 17.41°, 18.87°, 19.99°, 20.55°, 21.46°, 25.29°, 27.15°, 27.66°, 28.30°, 33.32°, 34.13°, 35.67°, 36.33°, 36.80°, and 44.06°, with an error tolerance of ±0.2°; the grain sizes corresponding to the characteristic diffraction peaks are

[0080]

[0081] The XRD pattern of the cocrystal of aescin and mannitol also has characteristic diffraction peaks at 2θ angles of 11.11°, 15.22°, 28.76°, 30.70°, 31.65°, 39.74°, 40.97°, 44.82°, 48.46°, 49.24°, and 56.76°, with an error tolerance of ±0.2°; the grain sizes corresponding to each characteristic diffraction peak are

[0082]

[0083] The specific XRD data of the obtained aescin and mannitol co-crystal are shown in the following table:

[0084] Table 1 XRD data of cocrystal of aescin and mannitol

[0085]

[0086]

[0087] The DSC test was performed on the cocrystal of aescin and mannitol obtained in Example 1 and the raw materials. The test parameters were as follows: temperature 50-400°C, heating rate 10.0°C / min, Ar 20.0mL / min. The results are shown in FIG. Figure 2-4 The temperature corresponding to the peak in the figure is the temperature inside the high-temperature furnace of the instrument. Since the sample temperature is lower than the furnace temperature at the same time, the melting temperature of the sample is analyzed by software as follows:

[0088] 1) Aescin: Two broad melting peaks appeared, the first peak at 182.39°C and the second peak at 310.12°C;

[0089] 2) Mannitol: melting point is 169.19°C;

[0090] 3) Cocrystal of aescin and mannitol: Two melting peaks appeared, peak 1 at 167.5°C and peak 2 at 327°C. The melting peaks of the cocrystal were different from those of the two raw materials, indicating that the cocrystal was obtained.

[0091] The eutectic of aescin and mannitol obtained in Example 1 was subjected to infrared testing to obtain the following Figure 5 The infrared spectrum shown, Figure 5 The eutectic is shown at 3279.98 cm -1 、2924.23cm -1 、2854.49cm -1 、1717.49cm -1 、1458.40cm -1 、1375.80cm -1 、1317.64cm-1 、1258.43cm -1 、1194.58cm -1 、1076.24cm -1 、1018.67cm -1 、949.92cm -1 、929.49cm -1 、877.83cm -1 、718.55cm -1 、621.12cm -1 There is a characteristic peak at ±0.2cm -1 error tolerance.

[0092] Example 4 Efficacy Test (Sample 1# was prepared as in Example 1)

[0093] 1. Irritation test:

[0094] Test method: Chicken chorioallantoic membrane test for eye irritation / corrosion of cosmetics (SN / T 2329)

[0095] Determination of acute toxicity of water substances to freshwater fish (zebrafish) (GB / T13267-91)

[0096] Testing instruments: stereo microscope, fully automatic incubator

[0097] Sample Information:

[0098] serial number Sample name concentration 1# Cocrystal of aescin and mannitol 1.0％ 2# Aescin 1.0％

[0099] The results of the chicken embryo test are as follows:

[0100]

[0101]

[0102] The zebrafish test results are as follows:

[0103]

[0104] According to the above chicken embryo test results tabular data and Figure 6 and Figure 7 It can be seen that 1.0% aescin and mannitol cocrystal is non-irritating, and 1.0% aescin is mildly irritating; the zebrafish test results show that the safety of aescin and mannitol cocrystal is better than that of raw aescin.

[0105] 2. Transdermal absorption test

[0106] Sample Information:

[0107] serial number Sample name concentration 1# Cocrystal of aescin and mannitol 1.0％ 2# Aescin 1.0％

[0108] (1) Test method for release from the surface of miniature pig skin:

[0109] Pig skin model: Xiaoba fragrant pig skin

[0110] Supply solution: 1# aescin and mannitol eutectic solution, 2# aescin solution Experimental method:

[0111] Diffusion cell installation and skin condition testing:

[0112] Without adding receiving solution, use filter paper to absorb the moisture of the skin (weigh it) and place it on the top surface of the receiving pool with the skin stratum corneum facing upwards. Add water to the supply pool to check whether there is liquid infiltration into the receiving pool. If there is no infiltration, discard the physiological saline in the supply pool and wipe the water on the supply pool and skin surface dry;

[0113] The sample receiving solution was balanced by adding 5 mL of physiological saline solution (weighed) until it was in complete contact with the skin and no bubbles were expelled, and then placed in a water bath for 30 minutes to balance;

[0114] Add sample supply solution: Take 400 μL of 1# and 2# supply solution respectively and add them to the supply pool;

[0115] The diffusion cell temperature was 34°C and the stirring speed was 450 rpm.

[0116] Sampling test after 4 hours:

[0117] ① Supply liquid: weigh the total weight of the supply pool (place it in a 25mL beaker or centrifuge tube, the gross weight of the supply pool needs to be weighed in advance), rinse with 50% methanol and dilute to 10mL, shake it by ultrasonic for 20min, filter and ultrasonically degas;

[0118] ② Skin surface: Weigh the total weight (the skin weight needs to be weighed in advance), rinse with 50% methanol or wipe the surface with a cotton swab and dilute to 2 mL, shake well, filter and ultrasonically degas;

[0119] ③Inside the skin: mince the skin, add 2 mL of 50% methanol and sonicate for 20 minutes;

[0120] ④ Receiving solution: Take 1 mL of receiving solution, filter it, and then ultrasonically degas it for 1 minute before injection.

[0121] (2) Aescin B transdermal absorption test method:

[0122] Instrument: G3-Q TOF liquid chromatography-mass spectrometry;

[0123] Chromatographic column: ACQUITY UPLC HSS BEH C18 (2.1*50mm, 1.7μm);

[0124] Mobile phase: Phase A: 0.1% formic acid in water; Phase B: 0.1% formic acid in acetonitrile;

[0125] Elution method: gradient method; B phase ratio: 0-30min, 5%-95%; 30-35min, 95%; 35.5-40min, 5%;

[0126] Flow rate: 0.3 mL / min;

[0127] Mass spectrometry conditions:

[0128] Negative ion mode: capillary voltage 2.5 kV, cone voltage 40 V, source default voltage 80 V, source temperature 120 °C, desolvation temperature 450 °C, carrier gas flow rate 50 L / H, desolvation gas flow rate 900 L / H;

[0129] Positive ion mode: capillary voltage 2.5 kV, cone voltage 40 V, source default voltage 80 V, source temperature 120 °C, desolvation temperature 450 °C, carrier gas flow rate 50 L / H, desolvation gas flow rate 800 L / H.

[0130] Test results:

[0131]

[0132] From the above test results and Figure 8 It can be seen that using aescin b as the target and miniature pig skin as the model, it was found that the transdermal absorption performance of aescin and mannitol cocrystal was better than that of the raw material aescin.

[0133] 3. Stability test

[0134] Aescin content test method:

[0135] High performance liquid chromatograph: Waters E2695 high performance liquid chromatograph

[0136] Chromatographic column: Wondasil C18-WR (150*4.6mm, 5μm);

[0137] Mobile phase: Phase A: 0.2% phosphoric acid aqueous solution, Phase B: acetonitrile;

[0138] Elution method: gradient method;

[0139] Gradient program: different time proportions of acetonitrile in phase B: 0-6 min, 5%-10%; 6-20 min, 10%-20%; 20-30 min, 20%-34%; 30-45 min, 34%-45%; 46-56 min, 45%-100%; 56.5-60 min, 100%; 61 min-72 min, 5%;

[0140] Flow rate: 1.0 mL / min;

[0141] Detection wavelength: 220nm;

[0142] Analysis time: 72 minutes.

[0143] Test results:

[0144]

[0145] from Figure 9 As can be seen from the data in the above table, taking the aescin content as an indicator, the high temperature stability for 48 hours was examined and it was found that the stability of aescin and aescin cocrystal was better than that of aescin.

[0146] The above are preferred embodiments of the present invention. Those skilled in the art to which the present invention belongs can also change and modify the above embodiments. Therefore, the present invention is not limited to the above specific embodiments. Any obvious improvements, replacements or modifications made by those skilled in the art on the basis of the present invention fall within the scope of protection of the present invention.

Claims

1. A co-crystal of aescin and mannitol, characterized by: The XRD spectrum of the aescin and mannitol cocrystal has characteristic diffraction peaks at least at 2θ angles of 13.80°, 17.41°, 18.87°, 19.99°, 20.55°, 21.46°, 25.29°, 27.15°, 27.66°, 28.30°, 33.32°, 34.13°, 35.67°, 36.33°, 36.80°, and 44.06°, with an error tolerance of ±0.2°; the DSC spectrum has characteristic endothermic peaks at 167.5°C and 327°C, with an error tolerance of ±0.2°C.

2. The aescin and mannitol co-crystal according to claim 1, characterized in that: The XRD pattern of the aescin and mannitol cocrystal also has characteristic diffraction peaks at 2θ angles of 11.11°, 15.22°, 28.76°, 30.70°, 31.65°, 39.74°, 40.97°, 44.82°, 48.46°, 49.24°, and 56.76°, with an error tolerance of ±0.2°; The grain sizes corresponding to the characteristic diffraction peaks are 3. The aescin and mannitol co-crystal according to claim 1, characterized in that: The grain sizes corresponding to the characteristic diffraction peaks of 13.80°, 17.41°, 18.87°, 19.99°, 20.55°, 21.46°, 25.29°, 27.15°, 27.66°, 28.30°, 33.32°, 34.13°, 35.67°, 36.33°, 36.80°, and 44.06° are 4. The aescin and mannitol co-crystal according to claim 1, characterized in that: The infrared spectrum of the cocrystal of aescin and mannitol is at least 3279.98 cm -1 、2924.23cm -1 、2854.49cm -1 、1717.49cm -1 、1458.40cm -1 、1375.80cm -1 、1317.64cm -1 、1258.43cm -1 、1194.58cm -1 、1076.24cm -1 、1018.67cm -1 、949.92cm -1 、929.49cm -1 、877.83cm -1 、718.55cm -1 、621.12cm -1 There is a characteristic peak at ±0.2cm -1 error tolerance.

5. The method for preparing the co-crystal of aescin and mannitol according to any one of claims 1 to 4, characterized in that: Here are the steps: S1. Weigh mannitol and deionized water according to the ratio, and stir to dissolve mannitol in the deionized water to form a mannitol solution; S2. Weigh aescin according to the ratio and add it to the mannitol solution, stirring until the aescin is completely dissolved; S3, continue heating and stirring the mixed solution in S2 for 1-2h; S4, placing the solution in step S3 into a reactor and reacting for 0.5-1h; S5, taking out the solution in S4 and letting it stand for crystallization; S6. Filter the crystals in S5, dry them in a spray dryer, and grind them into fine powder.

6. The aescin and mannitol co-crystal and preparation method thereof according to claim 5, characterized in that: The mass ratio of mannitol, aescin and deionized water is 2:1:

7.

7. The method for preparing the co-crystal of aescin and mannitol according to claim 5, wherein: In step S3, stirring is performed in a heat-collecting constant-temperature heating magnetic stirrer at a temperature of 30 to 50° C. and a rotation speed of 150 to 250 r / min.

8. The method for preparing the co-crystal of aescin and mannitol according to claim 5, characterized in that: In step S4, the temperature is 100-120° C. and the pressure is 10-15 MPa.

9. Use of the aescin and mannitol co-crystal according to any one of claims 1 to 4 in cosmetics, foods, health products and medicines.

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