Preparation and application of luteolin nanocrystal

Abstract

The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to a luteolin nanocrystal as well as a preparation method and application thereof. According to the luteolin nanocrystal, firstly, the particle size and PDI are taken as indexes, the prescription composition and the preparation process are subjected to single-factor investigation, then optimization and verification are carried out in combination with star point effect surface test design, the nanocrystal is prepared by adopting a micro medium grinding method, and then the solid nanocrystal is prepared through freeze drying. The defects that traditional nanocrystalline particles are prone to aggregation or overall sedimentation, inconvenient to store and carry and the like are overcome, the stability of the preparation is improved, the medication compliance of patients is improved, and a basis is provided for clinical research.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical preparations, and in particular to a luteolin nanocrystal and a preparation method and application thereof. Background Art

[0002] Luteolin (Luteolin, Lut), also known as yellow flavonoids and yellow spirit, has a chemical name of 3',4',5,7-tetrahydroxyflavone. It is a natural flavonoid compound, named after the genus Oleaceae, Oleaceae. Lut is widely present in many traditional Chinese medicines and fruits and vegetables, and mostly exists in the form of glycosides in vegetables such as celery and green peppers.

[0003] This product is yellow needle-shaped crystalline powder, with the molecular formula of C 15 H 10 O6, molecular weight is 286.23, melting point is 330℃, and it is weakly acidic due to the presence of more phenolic hydroxyl groups. Lut belongs to BCS class II drugs, which is highly lipid soluble, resulting in low solubility and bioavailability in vivo, which limits its clinical application.

[0004] Nanocrystals (NC), also known as nanosuspensions (NS), are a nanoscale liquid colloidal dispersion system formed by a drug being highly dispersed in a medium in a crystalline or amorphous state under the action of a small amount of stabilizer. The particle size is usually in the range of 200~800 nm. Traditional preparation methods to improve the solubility of poorly soluble drugs have shortcomings, such as poor safety due to the large amount of excipients, low drug loading, or the requirement of drugs with specific physical and chemical properties. NC can not only significantly improve the solubility of poorly soluble drugs by reducing the particle size and improve their bioavailability, but also reduce the potential toxicity caused by the amount of excipients. It has the advantages of simple prescription, high drug loading, wide application range and easy large-scale production. In addition, if the particle size is controlled or the group is modified, the drug can reach specific tissues or parts to achieve a targeted effect. NC is a thermodynamically unstable system, and the drug particles are at the nano level with a huge specific surface area and surface energy. They are prone to Ostwald ripening, which leads to irreversible aggregation and sedimentation, and poor stability. At the same time, liquid drugs are also inconvenient to carry and store.

[0005] Therefore, there is an urgent need to seek a luteolin solid nanocrystal with good stability and simple preparation method. Summary of the invention

[0006] The present invention aims to solve at least one of the technical problems existing in the prior art. For this purpose, the present invention provides a luteolin nanocrystal, a preparation method thereof, and an application thereof. The solid luteolin nanocrystal provided by the present invention can improve the solubility and dissolution rate of luteolin, has a good drug solubilization effect, and realizes the solidification of liquid drugs at the same time.

[0007] The present invention also provides a preparation method for the above-mentioned luteolin nanocrystal.

[0008] The present invention also provides an application of the above-mentioned luteolin nanocrystal.

[0009] A first aspect of the present invention provides a luteolin nanocrystal, comprising the following components: Stabilizer: 10%-50% luteolin; 5 mL of distilled water; 10 mL of grinding medium; 3% of lyoprotectant.

[0010] According to the embodiments of the first aspect of the present invention, it has at least the following beneficial effects: The stabilizer described in the present invention, such as sodium dodecyl sulfate, soy lecithin, hydroxypropyl methylcellulose, poloxamer 188 and 407, polyvinylpyrrolidone K30, vitamin E polyethylene glycol succinate, and tween-80, obtains the luteolin nanocrystal described in the present invention by limiting the addition ratio of the stabilizer, so that the NC is not prone to interparticle aggregation or Ostwald ripening.

[0011] The luteolin nanocrystal described in the present invention combines the advantages of high drug loading of liquid nanocrystals and good stability of solid preparations, has a good drug solubilization effect, can increase the probability of the drug being absorbed by the gastrointestinal tract in the dissolved state, improve the oral bioavailability and oral efficacy of luteolin, and thus improve the drug effect; moreover, the solid nanocrystal has good stability and low toxicity, realizes the solidification of liquid drugs, and solves the problems of inconvenient transportation, storage, and administration of liquid preparations.

[0012] According to some embodiments of the present invention, the lyoprotectant is at least one of PEG-4000, β-cyclodextrin, dextran-70, sodium citrate, mannitol, sucrose, and lactose.

[0013] Preferably, the lyoprotectant is sucrose.

[0014] According to some embodiments of the present invention, the stabilizer is at least one of sodium dodecyl sulfate, soy lecithin, hydroxypropyl methylcellulose, poloxamer 188 and 407, polyvinylpyrrolidone K30, vitamin E polyethylene glycol succinate, and tween-80.

[0015] The second aspect of the present invention provides a method for preparing the above-mentioned luteolin nanocrystals, comprising the following steps: S1. Mix the stabilizer and luteolin in proportion, and use a grinding medium to prepare Lut-NC by the micro-medium grinding method; S2. Mix the mixture obtained in step S1 with a freeze-drying protectant, and adopt the freeze-drying technology to solidify Lut-NC to obtain solid luteolin nanocrystals.

[0016] According to the embodiments of the second aspect of the present invention, it has at least the following beneficial effects: The preparation method provided by the present invention has simple steps, convenient and controllable operation, good repeatability, and good social and economic benefits.

[0017] According to some embodiments of the present invention, the ratio of drug to excipient is 5:1.

[0018] According to some embodiments of the present invention, the grinding medium is directly 0.2 - 0.4 mm.

[0019] According to some embodiments of the present invention, the mixing and stirring speed is 700 r / min.

[0020] According to some embodiments of the present invention, the grinding time is 12 h.

[0021] Among them, the particle size will first decrease to a certain extent and then increase with the prolongation of the grinding time. The reason is that although the increase in grinding time provides continuous energy, making the impact effect of the drug and the medium better and better, the excessive energy generated for a long time and the gradually increasing temperature inside the system will cause the smaller particles to redissolve or aggregate.

[0022] The third aspect of the present invention provides the application of the above-mentioned valsartan solid self-microemulsifying drug delivery system as a pharmaceutical preparation.

[0023] According to the embodiments of the third aspect of the present invention, it has at least the following beneficial effects: The present invention uses luteolin nanocrystals as an intermediate of pharmaceutical preparations, and can prepare pharmaceutical preparations such as tablets, capsules and granules. The obtained pharmaceutical preparations can improve the probability of the drug being absorbed by the gastrointestinal tract in the dissolved state, improve the efficacy of luteolin, and provide more choices for oral dosage forms.

[0024] According to some embodiments of the present invention, the dosage form of the pharmaceutical preparation includes at least one of tablets, pellets, powders, capsules and granules.

[0025] Other features and advantages of the present invention will be described in the subsequent specification, and part of them will become obvious from the specification or be understood by implementing the present invention. Brief Description of the Drawings

[0026] The above and / or additional aspects and advantages of the present invention will become apparent and be readily understood from the description of the embodiments in conjunction with the following drawings, wherein: Figure 1 It is a graph for investigating the addition amount of luteolin API in the present invention; Figure 2 It is a graph for investigating the drug-excipient ratio in the present invention; Figure 3 It is a graph for investigating the diameter of the grinding medium in the present invention; Figure 4 It is a graph for investigating the dosage of the grinding medium in the present invention; Figure 5 It is a graph for investigating the rotation speed in the present invention; Figure 6 It is a graph for investigating the grinding time in the present invention; Figure 7 It is the two-dimensional contour map and three-dimensional solid map of the response surface model Y1 of the central composite design - central point method in the present invention; Figure 8 It is the two-dimensional contour map and three-dimensional solid map of the response surface model Y2 of the central composite design - central point method in the present invention; Figure 9 It is the appearance state diagram of Lut-SNC (B), reconstituted Lut-SNC (C) and Lut-NC (A) before lyophilization in the present invention; Figure 10 It is the SEM image of luteolin solid nanocrystals SME in step (2) of Example 1 of the present invention; Figure 11 It is the DSC SEM image of luteolin API, PVP K30, sucrose, PM and luteolin solid nanocrystals in the present invention; Figure 12 It is the PXRD SEM image of luteolin API, PVP K30, sucrose, PM and luteolin solid nanocrystals in the present invention; Figure 13 It is the FT-IR SEM image of luteolin API, PVP K30, sucrose, PM and luteolin solid nanocrystals in the present invention; Figure 14 It is the dissolution curve of luteolin API, PM and luteolin solid nanocrystals in pH 1.2 HCl in the present invention; Figure 15 It is the dissolution curve of luteolin API, PM and luteolin solid nanocrystals in pH 6.8 PBS in the present invention; Figure 16 It is the plasma drug-time curve (0 - 24h) of luteolin API, PM and luteolin solid nanocrystals in the present invention; Figure 17This is the mean plasma concentration-time curve of luteolin capsules, PM capsules, and luteolin solid nanocrystal capsules of the present invention in rats. Embodiment

[0027] The embodiments of the present invention will be described in detail below. The same or similar reference numerals throughout the embodiments represent the same or similar elements or elements having the same or similar functions. The embodiments described below are exemplary and are only used to explain the present invention and should not be construed as limiting the present invention.

[0028] In the description of the present invention, if the first, second, etc. are described, it is only for the purpose of distinguishing technical features and should not be construed as indicating or implying relative importance or implicitly indicating the quantity of the indicated technical features or implicitly indicating the sequence relationship of the indicated technical features.

[0029] In the description of the present invention, it should be understood that the orientation or positional relationship indicated by terms such as up and down, etc., is based on the orientation or positional relationship shown in the embodiments, and is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as limiting the present invention.

[0030] The terms "preferably", "more preferably", etc. in the present invention refer to embodiments of the present invention that can provide certain beneficial effects in certain cases. However, in the same or other cases, other embodiments may also be preferred. In addition, the description of one or more preferred embodiments does not imply that other embodiments are not available, nor is it intended to exclude other embodiments from the scope of the present invention.

[0031] When a numerical range is disclosed herein, the above range is considered continuous and includes the minimum and maximum values of the range, as well as each value between such minimum and maximum values. Further, when the range refers to integers, it includes each integer between the minimum and maximum values of the range. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein.

[0032] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of the present invention.

[0033] The reagents, methods, and equipment used in the present invention are all conventional reagents, methods, and equipment in this technical field, unless otherwise specified.

[0034] Instruments and reagents used in the present invention: Instruments: Thermostatic heating magnetic stirrer with heat collection: DF-101S, Yuhua Instrument Co., Ltd., Gongyi City; Analytical balance: T6328B, Shanghai Tianping Instrument Factory; Electronic analytical balance: SMD200-2, Ohaus International Trade (Shanghai) Co., Ltd.; Ultra-microelectronic balance type electronic analytical balance: AG245, Mettler Toledo AG, Switzerland; Digital display tachometer: DLY-2301, Delixi Group Co., Ltd.; Particle size analyzer: Nano ZS90, Malvern Instruments Ltd., UK; Freeze dryer: FD-1A-50, Beijing Boyikang Experimental Instrument Co., Ltd.; Reagents: Luteolin (purity 98%, batch number P201005), Beijing Solarbio Science & Technology Co., Ltd.; Zirconia beads (0.2 - 0.4, 0.4 - 0.6, 0.6 - 0.8 mm), Hunan Ruichen Ceramics Co., Ltd.; Hydroxypropyl methylcellulose E30 (HPMC E30), Anhui Shanhe Pharmaceutical Excipients Co., Ltd.; Polyethylene glycol copolymer of polyvinyl alcohol (Kollicoat®IR), BASF SE, Germany; Polyvinylpyrrolidone K30 (PVP K30), Anhui Shanhe Pharmaceutical Excipients Co., Ltd.; Sodium carboxymethyl cellulose (CMC-Na), Tianjin Damao Chemical Reagent Factory; Tween-80 (Tween-80), Tianjin Comio Chemical Reagent Co., Ltd.; Vitamin E polyethylene glycol succinate (TPGS), Wuhan Guobangda Pharmaceutical Chemical Co., Ltd.; Poloxamer 188 (P188), BASF SE, Germany; Sodium dodecyl sulfate (SDS), Tianjin Damao Chemical Reagent Factory; Soybean lecithin (SPC), Shandong Yousuo Chemical Technology Co., Ltd.; Sucrose, Tianjin Bodi Chemical Co., Ltd.; Sodium citrate, Tianjin Hengxing Chemical Reagent Manufacturing Co., Ltd.; Lactose, Anhui Shanhe Pharmaceutical Excipients Co., Ltd.; Mannitol, Shanghai Pharmaceutical Excipients Factory; Polyethylene Glycol 4000 (PEG 4000), Tianjin Damao Chemical Reagent Factory; Dextran-70, Shanghai Macklin Biochemical Co., Ltd.; Absolute Ethanol (Analytical Reagent), Shandong Yuwang Chemical Reagent Co., Ltd.; Example

[0035] This example provides a luteolin solid nanocrystal, and its preparation method includes the following steps: (1) Weigh accurately 20 mg of polyvinylpyrrolidone K30 and 100 mg of luteolin into a 50 mL vial, add 5 mL of distilled water. After it is completely dispersed, add an appropriate amount of grinding medium (select zirconia beads), place it on a magnetic stirrer, and grind at 700 r / min for 12 h. Separate the upper layer liquid to obtain luteolin nanocrystals.

[0036] (2) Take an appropriate amount of luteolin nanocrystals and place them in a petri dish. Add 3% sucrose freeze-drying protectant to it, gently shake to dissolve it. After the above sample system is mixed evenly, place it in a -20 °C refrigerator for pre-freezing for 24 h, take it out and freeze-dry for 24 h to obtain luteolin solid nanocrystals. Example

[0037] This example provides a luteolin solid nanocrystal, and its preparation method includes the following steps: (1) Weigh accurately 20 mg of polyvinylpyrrolidone K30 and 100 mg of luteolin into a 50 mL vial, add 5 mL of distilled water. After it is completely dispersed, add an appropriate amount of grinding medium (select zirconia beads), place it on a magnetic stirrer, and grind at 300 r / min for 12 h. Separate the upper layer liquid to obtain luteolin nanocrystals.

[0038] (2) Take an appropriate amount of luteolin nanocrystals and place them in a petri dish. Add 3% sucrose freeze-drying protectant to it, gently shake to dissolve it. After the above sample system is mixed evenly, place it in a -20 °C refrigerator for pre-freezing for 24 h, take it out and freeze-dry for 24 h to obtain luteolin solid nanocrystals. Example

[0039] This example provides a luteolin solid nanocrystal, and its preparation method includes the following steps: (1) Weigh accurately 20 mg of polyvinylpyrrolidone K30 and 100 mg of luteolin into a 50 mL vial, add 5 mL of distilled water. After it is completely dispersed, add an appropriate amount of grinding medium (select zirconia beads), place it on a magnetic stirrer, and grind at 500 r / min for 12 h. Separate the upper layer liquid to obtain luteolin nanocrystals.

[0040] (2) Weigh an appropriate amount of luteolin nanocrystals and place them in a petri dish. Add 3% sucrose cryoprotectant to it, gently shake to dissolve it. After the above sample system is mixed evenly, place it in a -20 °C refrigerator for pre-freezing for 24 h, take it out and freeze-dry for 24 h to obtain solid luteolin nanocrystals. Example

[0041] This example provides a solid luteolin nanocrystal, and its preparation method includes the following steps: (1) Weigh 20 mg of polyvinylpyrrolidone K30 and 100 mg of luteolin precisely into a 50 mL vial, add 5 mL of distilled water. After it is completely dispersed, add an appropriate amount of grinding medium (select zirconia beads), place it on a magnetic stirrer, and grind at 900 r / min for 12 h. Separate the upper liquid to obtain luteolin nanocrystals.

[0042] (2) Weigh an appropriate amount of luteolin nanocrystals and place them in a petri dish. Add 3% sucrose cryoprotectant to it, gently shake to dissolve it. After the above sample system is mixed evenly, place it in a -20 °C refrigerator for pre-freezing for 24 h, take it out and freeze-dry for 24 h to obtain solid luteolin nanocrystals. Example

[0043] This example provides a solid luteolin nanocrystal, and its preparation method includes the following steps: (1) Weigh 20 mg of polyvinylpyrrolidone K30 and 100 mg of luteolin precisely into a 50 mL vial, add 5 mL of distilled water. After it is completely dispersed, add an appropriate amount of grinding medium (select zirconia beads), place it on a magnetic stirrer, and grind at 1100 r / min for 12 h. Separate the upper liquid to obtain luteolin nanocrystals.

[0044] (2) Weigh an appropriate amount of luteolin nanocrystals and place them in a petri dish. Add 3% sucrose cryoprotectant to it, gently shake to dissolve it. After the above sample system is mixed evenly, place it in a -20 °C refrigerator for pre-freezing for 24 h, take it out and freeze-dry for 24 h to obtain solid luteolin nanocrystals. Example

[0045] This example provides a solid luteolin nanocrystal, and its preparation method includes the following steps: (1) Weigh 20 mg of polyvinylpyrrolidone K30 and 100 mg of luteolin precisely into a 50 mL vial, add 5 mL of distilled water. After it is completely dispersed, add an appropriate amount of grinding medium (select zirconia beads), place it on a magnetic stirrer, and grind at 700 r / min for 3 h. Separate the upper liquid to obtain luteolin nanocrystals.

[0046] (2) Weigh an appropriate amount of luteolin nanocrystals and place them in a petri dish. Add 3% sucrose freeze-drying protectant to it and gently shake to dissolve it. After the above sample system is mixed evenly, place it in a -20 °C refrigerator for pre-freezing for 24 h, take it out and freeze-dry for 24 h to obtain luteolin solid nanocrystals. Example

[0047] This example provides a kind of luteolin solid nanocrystals, and its preparation method includes the following steps: (1) Weigh 20 mg of polyvinylpyrrolidone K30 and 100 mg of luteolin precisely in a 50 mL vial, add 5 mL of distilled water. After it is completely dispersed, add an appropriate amount of grinding medium (select zirconia beads), place it on a magnetic stirrer, and grind at 700 r / min for 6 h. Separate the upper liquid to obtain luteolin nanocrystals.

[0048] (2) Weigh an appropriate amount of luteolin nanocrystals and place them in a petri dish. Add 3% sucrose freeze-drying protectant to it and gently shake to dissolve it. After the above sample system is mixed evenly, place it in a -20 °C refrigerator for pre-freezing for 24 h, take it out and freeze-dry for 24 h to obtain luteolin solid nanocrystals. Example

[0049] This example provides a kind of luteolin solid nanocrystals, and its preparation method includes the following steps: (1) Weigh 20 mg of polyvinylpyrrolidone K30 and 100 mg of luteolin precisely in a 50 mL vial, add 5 mL of distilled water. After it is completely dispersed, add an appropriate amount of grinding medium (select zirconia beads), place it on a magnetic stirrer, and grind at 700 r / min for 9 h. Separate the upper liquid to obtain luteolin nanocrystals.

[0050] (2) Weigh an appropriate amount of luteolin nanocrystals and place them in a petri dish. Add 3% sucrose freeze-drying protectant to it and gently shake to dissolve it. After the above sample system is mixed evenly, place it in a -20 °C refrigerator for pre-freezing for 24 h, take it out and freeze-dry for 24 h to obtain luteolin solid nanocrystals. Example

[0051] This example provides a kind of luteolin solid nanocrystals, and its preparation method includes the following steps: (1) Weigh 20 mg of polyvinylpyrrolidone K30 and 100 mg of luteolin precisely in a 50 mL vial, add 5 mL of distilled water. After it is completely dispersed, add an appropriate amount of grinding medium (select zirconia beads), place it on a magnetic stirrer, and grind at 700 r / min for 15 h. Separate the upper liquid to obtain luteolin nanocrystals.

[0052] (2) Weigh an appropriate amount of luteolin nanocrystals and place them in a petri dish. Add 3% sucrose freeze-drying protectant to it and gently shake to dissolve. After the above sample system is mixed evenly, place it in a -20 °C refrigerator for pre-freezing for 24 h, take it out and freeze-dry for 24 h to obtain luteolin solid nanocrystals. Example

[0053] This example provides a kind of luteolin solid nanocrystals, and its preparation method includes the following steps: (1) Weigh accurately 20 mg of polyvinylpyrrolidone K30 and 100 mg of luteolin into a 50 mL vial, add 5 mL of distilled water. After it is completely dispersed, add an appropriate amount of grinding medium (select zirconia beads), place it on a magnetic stirrer, and grind at 700 r / min for 18 h. Separate the upper layer liquid to obtain luteolin nanocrystals.

[0054] (2) Weigh an appropriate amount of luteolin nanocrystals and place them in a petri dish. Add 3% sucrose freeze-drying protectant to it and gently shake to dissolve. After the above sample system is mixed evenly, place it in a -20 °C refrigerator for pre-freezing for 24 h, take it out and freeze-dry for 24 h to obtain luteolin solid nanocrystals. Example

[0055] This example provides a kind of luteolin solid nanocrystals, and its preparation method includes the following steps: (1) Weigh accurately 20 mg of polyvinylpyrrolidone K30 and 100 mg of luteolin into a 50 mL vial, add 5 mL of distilled water. After it is completely dispersed, add an appropriate amount of grinding medium (select zirconia beads), place it on a magnetic stirrer, and grind at 700 r / min for 18 h. Separate the upper layer liquid to obtain luteolin nanocrystals.

[0056] (2) Weigh an appropriate amount of luteolin nanocrystals and place them in a petri dish. Add 3% sucrose freeze-drying protectant to it and gently shake to dissolve. After the above sample system is mixed evenly, place it in a -20 °C refrigerator for pre-freezing for 24 h, take it out and freeze-dry for 24 h to obtain luteolin solid nanocrystals. Example

[0057] This example provides a kind of luteolin solid nanocrystals, and its preparation method includes the following steps: (1) Weigh accurately 20 mg of polyvinylpyrrolidone K30 and 100 mg of luteolin into a 50 mL vial, add 5 mL of distilled water. After it is completely dispersed, add an appropriate amount of grinding medium (select zirconia beads), place it on a magnetic stirrer, and grind at 700 r / min for 21 h. Separate the upper layer liquid to obtain luteolin nanocrystals.

[0058] (2) Weigh an appropriate amount of luteolin nanocrystals and place them in a petri dish. Add 3% sucrose freeze-drying protectant to it, gently shake to dissolve it. After the above sample system is mixed evenly, place it in a -20 °C refrigerator for pre-freezing for 24 h, take it out and freeze-dry for 24 h to obtain solid luteolin nanocrystals. Example

[0059] This example provides a solid luteolin nanocrystal, and its preparation method includes the following steps: (1) Weigh 20 mg of polyvinylpyrrolidone K30 and 100 mg of luteolin precisely into a 50 mL vial, add 5 mL of distilled water. After it is completely dispersed, add an appropriate amount of grinding medium (select zirconia beads), place it on a magnetic stirrer, and grind at 700 r / min for 24 h. Separate the upper layer of liquid to obtain luteolin nanocrystals.

[0060] (2) Weigh an appropriate amount of luteolin nanocrystals and place them in a petri dish. Add 3% sucrose freeze-drying protectant to it, gently shake to dissolve it. After the above sample system is mixed evenly, place it in a -20 °C refrigerator for pre-freezing for 24 h, take it out and freeze-dry for 24 h to obtain solid luteolin nanocrystals.

[0061] Test Example 1 1.1 Investigation of the type of stabilizer Some studies have shown that NCs with particle sizes in the range of 100 - 400 nm have slower particle growth and aggregation rates, which can greatly avoid recrystallization or aggregation after particle dissolution; the smaller the PDI value, the more uniform its particle size distribution, and usually PDI < 0.3 is sufficient. After comprehensive consideration, PVP K30 was selected as the stabilizer, as shown in Table 1 specifically.

[0062] Table 1 Effects of different stabilizers on particle size and PDI

[0063] 1.2 Investigation of the drug addition amount From Figure 1 it can be seen that when 25 mg and 100 mg of the drug are added, the obtained Lut-NC has smaller particle sizes and PDI. To make the preparation have a higher drug content, 100 mg of the drug was selected.

[0064] 1.3 Investigation of the drug-excipient ratio From Figure 2It can be seen that when the drug-excipient ratio decreases from high to low, the particle size and PDI of Lut-NC show a "U"-shaped change trend. The particle size and PDI results of NC obtained when the drug-excipient ratio is either too high or too low are not ideal. This is because when the excipient content is low, the whole system cannot achieve a good stabilization effect. When the excipient content is too high, a "bridging" effect will occur, increasing the contact and collision opportunities between drug particles instead, making aggregation more likely and it is also difficult to stabilize the preparation. After comprehensive consideration, the drug-excipient ratio of 5:1 was selected.

[0065] 1.4 Investigation of the diameter of grinding media Fix the prescription composition and other preparation conditions. Select zirconia beads with three different diameter ranges of 0.2 - 0.4, 0.4 - 0.6, and 0.6 - 0.8 mm as the grinding media. Prepare Lut-NC according to the operation under item "S1", and investigate the influence of the diameter of the grinding media on the particle size and PDI of NC. Prepare 6 groups of samples in parallel, and each group of samples is measured 3 times repeatedly.

[0066] It can be seen from Figure 3 that when zirconia beads with a diameter range of 0.2 - 0.4 mm are selected to prepare Lut-NC, it has appropriate particle size and PDI. At the same time, the separation degree between the drug and the grinding media is relatively high, and drug loss is not likely to occur.

[0067] 1.5 Investigation of the amount of grinding media Prepare Lut-NC according to the operation under item "S1", and set the amounts of grinding media to 5, 7.5, 10, 12.5, and 15 mL respectively. Measure the particle size and PDI of the obtained NC to screen the amount of grinding media. Prepare 6 groups of samples in parallel, and each group of samples is measured 3 times repeatedly.

[0068] It can be seen from Figure 4 the results that when the amount of grinding media gradually increases, the particle size and PDI of Lut-NC generally show a trend of first decreasing and then increasing. This is because when the amount of grinding media is small, the contact between drug particles and between drug particles and the grinding media is incomplete, and they cannot collide sufficiently. When the amount of grinding media is too large, the impact force between particles and the grinding media will weaken at the same rotation speed, and the effect is not good. Combining the data in the following table, the amount of grinding media of 10 mL was selected.

[0069] 1.6 Investigation of the rotation speed Prepare Lut-NC according to the operation under item "S1", and set the grinding speeds to 300, 500, 700, 900, and 1100 r·min -1 , and here the grinding time is set to 2 h separately to avoid the poor discrimination of each group of data due to long-term grinding. The remaining preparation processes remain unchanged, and investigate the influence of different grinding speeds on the particle size and PDI of NC. Prepare 6 groups of samples in parallel, and each group of samples is measured 3 times repeatedly.

[0070] When the rotational speed is low, the entire internal system of the grinding chamber is not sufficient to provide enough kinetic energy to fully pulverize the drug particles. Therefore, the particle size and PDI are relatively large. When the rotational speed gradually increases, the internal energy of the system continuously increases, and the impact and pulverization effect on the particles is improved. However, when the rotational speed reaches a certain value, the grinding medium will adhere to the wall of the grinding chamber and move in a circular motion together with the drug, resulting in insufficient collision and shear force. At the same time, the medium will also be consumed. Combined with the following Figure 5 Determine the rotational speed to be 700 r·min -1 。

[0071] 1.7 Investigation of grinding time Prepare Lut-NC according to the operation under "S1", and set the grinding times to be 3, 6, 9, 12, 15, 18, 21, and 24 h in sequence. Determine the grinding time by measuring the particle size and PDI. Prepare 6 groups of samples in parallel, and measure each group 3 times repeatedly.

[0072] Figure 6 The results show that the particle size of the particles will first decrease to a certain extent and then increase with the extension of the grinding time. The reason for the analysis is that although the increase in the grinding time provides continuous energy, making the impact effect between the drug and the medium better and better, the excessive energy generated for a long time and the gradually increasing temperature inside the system will cause the smaller particles to redissolve or aggregate. Therefore, the grinding time is selected to be 12 h.

[0073] 1.8 Optimization by central composite design Although the particle size and PDI of Lut-NC prepared according to the optimal results after single-factor investigation already meet the requirements, it is hoped that the process conditions such as the grinding time can be optimized. Therefore, the following factors are selected as independent variables: the amount of grinding medium (A), the grinding time (B), and the rotational speed (C). Based on the results of single-factor investigation, a central composite design experiment with 3 factors and 5 levels is carried out with the particle size (Y1) and PDI (Y2) as evaluation criteria. Prepare Lut-NC with different prescription processes according to the given central composite design scheme, record the particle size and PDI, and perform fitting of the mathematical model and Anova data analysis on them.

[0074] The coded values and actual operation values of each independent variable in the central composite design are shown in Table 2, and the experimental results of each group are shown in Table 3.

[0075] Table 2 Factors and levels of central composite design

[0076] Table 3 Central composite design and results

[0077] After fitting with Design-Expert 10.0.7 statistical software, the equation is obtained as follows: Y1 = 988.95 - 72.14*A - 29.82*B - 0.97*C + 0.24*AB + 0.02*AC - 0.01*BC + 2.67*A2 + 1.29*B2 + 0.0006*C2 (R2 = 0.9911, P<0.0001) Y2 = 2.24 - 0.15*A - 0.11*B - 0.002*C + 0.001*AB + 0.00002*AC + (4.9327E-19)*BC + 0.006*A2 + 0.004*B2 + (1.08485E-06)*C2 (R2 = 0.9363, P<0.0001) As can be seen from the above equations, the mathematical model after fitting is a quadratic polynomial fitting model, and the R2 of both equations is greater than 0.9, that is, they have good correlations. Subsequently, Anova data analysis was performed on them, and the results are shown in Tables 4 and 5.

[0078] As can be seen from the following two tables, for the models established for particle size (Y1, nm) and PDI (Y2), P<0.001, indicating that the variances of the established models are extremely significant and can be used for subsequent analysis and prediction; the lack-of-fit term P>0.05 is not significant, indicating that the prediction accuracies of the above two models are relatively high, and the influence of other unknown factors can be ignored.

[0079] Table 4 Results of variance analysis of Y1

[0080] Table 5 Results of variance analysis of Y2

[0081] Two-dimensional contour plots and three-dimensional solid plots of Y1 and Y2 were respectively drawn through Design-Expert 10.0.7 software, and the results are shown in Figure 7 and Figure 8 .

[0082] According to the results given by the software, the optimal process conditions were determined as follows: the dosage of grinding medium was 10 mL, the grinding time was 12 h, and the rotation speed was 700 r·min -1 . Three batches of Lut-NC were prepared in parallel according to the above process, and the particle size and PDI were measured to verify the predicted formulation.

[0083] As can be seen from the results in Table 6, the difference between the measured values and the predicted values is not significant, and RE<5%, indicating that the model has good predictability, high accuracy, and good credibility.

[0084] Table 6 Predicted values and observed values of the optimized formulation

[0085] 1.9 Stability investigation As can be seen from Table 7, the particle size of Lut-NC changed by about 10 nm within 14 days, the increase in PDI was slightly larger, and a little precipitation occurred, but it could be redispersed by gentle shaking. On the 20th day, it was obvious to the naked eye that the amount of precipitation increased, and it was difficult to redisperse even by shaking, indicating that Lut-NC was no longer stable.

[0086] Table 7 Stability results of Lut-NC

[0087] Note: - indicates that the particle size and PDI could not be measured because precipitation occurred in Lut-NC and it was difficult to disperse. The above results indicate that Lut-NC has good stability within 14 days, but unstable phenomena such as precipitation and difficulty in dispersion will occur if stored continuously. In addition, liquid preparations are also inconvenient in terms of carrying and transportation. Therefore, Lut-NC was solidified to enhance its stability and facilitate subsequent taking and carrying.

[0088] Screening of types and dosages of lyoprotectants in Test Example 2 (I) Investigation of types of lyoprotectants According to the different action principles of lyoprotectants, high molecular polymers, salts, polyhydric alcohols, etc. were selected for experiments. In this study, PEG-4000, β-cyclodextrin, dextran-70, sodium citrate, mannitol, sucrose, and lactose were selected as lyoprotectants. Lut-SNC was prepared according to the operation under "S1", and 6 groups were prepared in parallel, with each group of samples measured 3 times repeatedly. The appearance and morphology of the lyophilized powder, the ease of reconstruction, the particle size and PDI after reconstruction were used as indicators to evaluate the quality of the lyophilization effect.

[0089] When sucrose was selected as the lyoprotectant, the obtained Lut-SNC was in a loose and porous sponge-like shape without caking, and was easy to reconstruct. There was no obvious difference in the appearance morphology compared with that before lyophilization. As can be seen from Table 8, the particle size increased by about 20 nm, and the change in PDI was small. Therefore, sucrose was selected as the lyoprotectant.

[0090] Table 8 Effects of different cryoprotectants on particle size and PDI (X±S, n = 6)

[0091] (II) Investigation of dosages of lyoprotectants Lut-SNC was prepared according to the operation under "S1", and 0.1%, 0.5%, 1.0%, 3.0%, 5.0% (w / v) of sucrose were added in turn. 6 groups were prepared in parallel, with each group of samples measured 3 times repeatedly. The evaluation indexes were the same as those in item "2.4.1".

[0092] AsFigure 9 As shown, when 3.0% sucrose was added, the appearance of the obtained Lut-SNC showed little difference before and after freeze-drying, and it was easy to reconstruct. At the same time, the particle size and PDI changed slightly. Therefore, the amount of sucrose used as the lyoprotectant was selected to be 3.0% (w / v).

[0093] Test Example 3 Morphology and Particle Size Distribution of Luteolin Solid Nanocrystals (I) Appearance Observation Prepare 3 batches of Lut-SNC in parallel according to the optimal formulation and process conditions, and observe their appearance with the naked eye.

[0094] From Figure 10 it can be seen that Lut-SNC (B) is a yellow, loose, porous sponge-like solid, which can be easily twisted into powder. The powder state is loose and delicate, and it is extremely easy to redissolve. The appearance state of Lut-SNC (C) after redissolution has no obvious difference from that of Lut-NC (A) before freeze-drying.

[0095] (II) SEM Observation Take appropriate amounts of PVP K30, sucrose, Lut, and Lut-SNC powders and stick them on the conductive tape of the lead plate. Under vacuum conditions, sputter gold treatment is carried out with a current of 30 mA, and then the microscopic morphological characteristics of the above samples are observed under an electron microscope and photographed for recording.

[0096] As Figure 11 shown, from the SEM results, it can be seen that the original Lut powder mostly appears as irregular long columnar crystals, and the size belongs to the micron level; Lut-SNC mostly appears as short and thick rod-like shapes, and the crystallization characteristics are extremely unclear. Occasionally, crystallization debris can be seen, indicating that the drug particles have transformed into the amorphous state. The overall size is relatively small, mostly between 100 and 200 nm, which is consistent with the Malvern particle size measurement results; PVP K30 appears spherical under the electron microscope, with slightly concave surfaces; sucrose appears as giant cubic shapes under the electron microscope. Due to the large size of sucrose, different scales are selected for photographing and recording to facilitate the observation of its microscopic structure.

[0097] (III) Redispersibility The particle size of the redissolved Lut-SNC was measured to be (120.95 ± 3.54) nm, the PDI was (0.207 ± 0.062), and the Zeta potential was (-35.95 ± 1.04) mV. Comparing with the particle size, PDI, and Zeta potential before freeze-drying, the specific data are shown in Table 9. From the data in Table 9, it can be known that the particle size increased by about 20 nm after freeze-drying; the PDI decreased slightly. It is speculated that this may be due to the aggregation of too small particles after freeze-drying, resulting in an increase in the overall particle size, so the particle size distribution is more uniform. It may also be due to the differences in Lut-SNC between different batches; the Zeta potential value changed little, indicating good redispersibility.

[0098] Comparison of particle size, PDI and Zeta potential before and after lyophilization (X±S, n=3) (4) Differential scanning calorimetry (DSC) DSC mainly determines whether the crystal form of a sample has changed by comparing the intensity of the endothermic / exothermic peaks of different samples, or whether a peak appears at the same position of the same sample after being treated by different means. It can be Figure 12 seen that the Lut API has a characteristic endothermic peak at 320 °C, PVP K30 has a broad endothermic peak at about 125 °C, and sucrose has two characteristic peaks at about 200 °C and 230 °C. All three characteristic peaks can be seen in the PM group, but compared with the pure substances, the peak intensity is slightly weakened and the peak position is slightly changed. The characteristic peak of Lut disappears in Lut-SNC, and only the peaks of the two excipients can be observed. The experimental results indicate that the crystal state of the drug may have changed during the formulation process.

[0099] (5) Powder X-ray diffraction analysis (PXRD) When analyzing the PXRD pattern, the crystal structure and changes of the sample are mainly identified and analyzed according to the position and intensity of the diffraction peaks such as sharp peaks (crystalline form) and diffuse peaks (amorphous form) in the pattern.

[0100] Analysis Figure 13 shows that: the Lut API has sharp diffraction peaks at 7.48°, 13.62°, 15.08° and 27.00°, indicating that it is a crystalline substance; sucrose shows diffraction peaks at 12.96° and 25.24°, indicating that it also exists in a crystalline form; PVP K30 shows two broad diffuse peaks at 11.76° and 20.26°, indicating that PVP K30 belongs to an amorphous substance; the peak shape of PM shows the combination of the diffraction peaks of Lut and sucrose but with reduced intensity, and no diffuse peak appears. It is speculated that this may be because the content of PVP K30 is low; compared with PM and Lut, the overall peak shape of Lut-SNC shows an upward bulge, indicating that its crystallinity has been greatly reduced after grinding and has changed to an amorphous state, but there are small sharp peaks at 13.62°, 15.08° and 27.00°, indicating that there is still a very small part existing in a weak crystalline state.

[0101] PXRD can also be combined with DSC to make a judgment on the sample to verify whether its existence form is crystal or non-crystal. However, the experimental results show that the judgments of the two on the crystal form of Lut-SNC are slightly different. It is speculated that the reason why all the characteristic peaks of Lut disappear in the DSC pattern is that the sensitivity of DSC is low and it cannot detect Lut existing in a weak crystalline form.

[0102] (6) Fourier transform infrared spectroscopy (FT-IR) FT-IR is often used to analyze whether there are interactions between the components of a sample, or whether the characteristic chemical structures in the sample change during the preparation process. In the spectrum, this is manifested as the displacement, disappearance, increase or decrease in intensity, or the appearance of new peaks of absorption peaks.

[0103] Observation Figure 14 It can be seen that the characteristic absorption peaks of Lut raw material medicine are mainly located at 3365.25 cm−1 (O-H stretching vibration peak), 1655.78 cm−1 (C=O stretching vibration peak), 1619.99 cm−1 (benzene ring skeleton stretching vibration peak), 1371.61 cm−1 (O-H stretching vibration peak), 1166.60 cm−1 (C-O-C stretching vibration peak), and 1050.38 cm−1 (ortho-disubstituted phenolic hydroxyl stretching vibration peak); the characteristic peaks of PVP K30 appear at 3437.43 cm−1 (O-H stretching vibration peak), 1658.14 cm−1 (C=O stretching vibration peak), and 1289.37 cm−1 (C-N stretching vibration peak); the characteristic absorption peaks of sucrose mainly include 3563.05 cm−1, 3387.27 cm−1, 3333.32 cm−1 (O-H stretching vibration peak), and 1238.99 cm−1 (C-O-C stretching vibration peak). The characteristic peaks of the Lut-SNC group are roughly the same as those of the PM group, showing the superposition of the characteristic peaks of Lut and the excipients, indicating that its original active structure has not changed. However, the peak shape between 3200 and 3500 cm−1 becomes flatter, and the peak intensity decreases. It is speculated that there may be a hydrogen bond interaction between Lut and the excipients.

[0104] Test Example 4 Determination of the dissolution, solubility, and equilibrium solubility of luteolin solid nanocrystals (I) Content determination After determination, the drug loading of Lut-SNC was (80.27 ± 0.28)%.

[0105] (II) Dissolution determination From Figure 15 and 16 it can be seen that the cumulative release degrees of Lut in pH 1.2 HCl and pH 6.8 PBS are both relatively low; there is no significant difference in the dissolution of PM and Lut; Lut-SNC can release more than 80% within 5 minutes in pH 6.8 PBS, and the cumulative release within 2 hours is 93.97%, which is nearly 3 times higher than that of Lut. Although the cumulative release in pH 1.2 HCl solution is less than 40%, it is also about 2 times higher than that of Lut. The results show that the nanocrystal technology can significantly improve the solubility of Lut.

[0106] (III) Determination of equilibrium solubility As can be seen from Table 10, compared with the raw drug, the equilibrium solubility of Lut-SNC in different media was significantly improved.

[0107] Table 10 Equilibrium solubility of Lut and Lut-SNC in different pH media (n = 5)

[0108] Pharmacokinetic experiment of Lut-SNC in rats in Test Example 5 In this test example, the pharmacokinetic experiment of Lut-SNC capsules prepared in Example 1 was carried out in rats. The specific steps are as follows: (1) Preparation of test drugs Raw drug capsule group: The raw drug was suspended in 0.5% CMC-Na solution and magnetically stirred for 10 min to obtain a Lut suspension; PM capsule group: The raw drug and all excipients were accurately weighed according to the prescription amount. After treatment, they were suspended in 0.5% CMC-Na solution and magnetically stirred for 10 min to obtain a PM suspension; Lut-SNC capsule group: The raw drug and excipients were accurately weighed according to the prescription amount and then prepared into capsules. The capsule contents were suspended in 0.5% CMC-Na solution and magnetically stirred for 10 min to obtain a Lut-SNC suspension.

[0109] (2) Administration plan and blood sampling: Fifteen healthy SD rats with a body weight of (200 ± 20) g were randomly divided into 3 groups with 5 rats in each group. They were fasted for 12 h and allowed free access to water. Lut, PM and Lut-SNC capsule suspensions were administered according to Lut 9 mg. Samples were taken at 0.083, 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 12, 24 h after administration in the Lut and PM groups; an additional sampling time point of 0.167 h was added in the Lut-SNC capsule group. 0.5 mL of blood was taken from the orbital cavity in each case and drained into a centrifuge tube infiltrated with 1% heparin through a capillary glass tube. After gently shaking, it was centrifuged at 5000 r·min -1 for 10 min. After separating the upper plasma layer, the operation was carried out according to the method under "2.1.3", and HPLC was used for sample injection and determination to calculate the blood drug concentration and pharmacokinetic parameters.

[0110] Tables 11, 12 and 13 represent the in vivo blood drug concentration determination results of the Lut raw drug, PM and Lut-SNC capsule groups, respectively. The in vivo blood drug concentration-time curves are as Figure 17 shown.

[0111] Table 11 Blood drug concentration after single administration of Lut capsules (n = 5)

[0112] Note: ND indicates undetectable. Table 12 Plasma drug concentration after single-dose administration of PM capsules (n = 5)

[0113] Table 13 Plasma drug concentration after single-dose administration of Lut-SNC capsules (n = 5)

[0114] The relevant pharmacokinetic parameters were calculated and analyzed using DAS 2.0 and SPSS software. The detailed results are shown in the following table. Among them, Tables 14, 15, and 16 represent the in vivo pharmacokinetic data of the Lut, PM, and Lut-SNC capsule groups, respectively.

[0115] Table 14 Pharmacokinetic parameters of Lut capsules after single-dose administration

[0116] Table 15 Pharmacokinetic parameters of PM capsules after single-dose administration

[0117] Table 16 Pharmacokinetic parameters of Lut-SNC capsules after single-dose administration

[0118] In the bioequivalence trial, the main parameters analyzed were AUC, C max , and T max . Among them, AUC and C max followed a normal distribution after logarithmic transformation, so multivariate analysis of variance was used for significance testing. However, T max is a non-continuous parameter that follows a single-parameter Poisson distribution and does not have additivity, so the rank sum test of non-parametric method was used. For AUC, when the 90% confidence interval (CI) falls between 80% and 125%, it indicates bioequivalence between the two. For C max , when the CI falls between 70% and 143%, it indicates bioequivalence between the two. However, at this time, it still cannot prove complete bioequivalence between the two, and further two one-sided T-tests need to be used for verification. Conversely, if the confidence intervals of each parameter are not within the above range, it directly proves bioinequivalence between the two. max For C

[0119] The C max values of rats after oral administration of Lut capsules, PM capsules, and Lut-SNC capsule groups alone were (3.43 ± 0.55), (4.08 ± 0.96), and (16.95 ± 4.78) μg·mL -1 respectively. The C max values of the three groups were logarithmically transformed and then analyzed by analysis of variance.

[0120] Table 17 ANOVA of Lut capsules, PM capsules and Lut-SNC capsules C max

[0121] Table 18 Results of homogeneous subsets of Lut capsules, PM capsules and Lut-snc capsules regarding C max

[0122] As can be seen from the results in Table 17, P < 0.01, indicating significant differences among the three; regarding the homogeneous subsets, it can be understood that numerous data are divided into different subsets (automatically completed by statistical software), and the values in the same subset have no differences, while there are significant differences among different subsets. In Table 18, the three dosing groups are divided into 2 subsets, indicating that there is no significant difference in C between the Lut and PM capsule groups max , and there are significant differences between the Lut-SNC capsule group and the two groups. This proves that the nanocrystal technology can improve the solubility of Lut and its release and absorption processes in the gastrointestinal tract.

[0123] The average T of rats orally administered Lut alone and the PM group max were (0.90 ± 0.14) h and (0.70 ± 0.11) h respectively, and the average T of the Lut-SNC capsule group max was (0.30 ± 0.11) h. According to the characteristics of T max , an independent two-sample rank sum test was performed.

[0124] Table 19 Comparison of t results of Lut capsules, PM capsules and Lut-SNC capsules max

[0125] As can be seen from the results in Table 19, there are significant differences in T among the three groups of Lut, PM and Lut-SNC capsules max . It is speculated that the reason for the significant difference in T between the Lut capsule group and the PM capsule group may be that the PM group contains more water-soluble excipients, so T max is slightly shortened; the T of the Lut-SNC capsule group max is significantly shortened, proving that after Lut is prepared into Lut-SNC capsules, the drug will be rapidly released in vivo, which is consistent with the in vitro release results. max

[0126] The AUC of the API and the PM group were (23.07 ± 1.45) and (18.37 ± 5.94) μg·mL -1 ·h -1 ​​​​, the AUC of the Lut-SNC capsule group was (62.10 ± 14.87) μg·mL -1 ·h -1 . Similarly, after logarithmic transformation of the AUC, analysis of variance was performed.

[0127] Table 20 Analysis of variance of AUC among Lut capsules, PM capsules and Lut-SNC capsules 0→∞

[0128] Table 21 Results of the homogeneous subsets of AUC among Lut capsules, PM capsules and Lut-SNC capsules 0→∞

[0129] The results in Table 20 showed that P < 0.01, indicating significant differences among the three; from the results in Table 21, it was known that there was no significant difference in AUC between the Lut and PM capsule groups, and there were significant differences between the Lut-SNC capsule group and the two. The results indicated that when Lut was prepared into Lut-SNC capsules, its in vivo absorption after oral administration was significantly improved.

[0130] Relative bioavailability refers to the rate and extent to which the same drug is absorbed into the body and participates in the circulation in different dosage forms. Generally, the relative bioavailability Fr(%) of the test preparation after single-dose oral administration is calculated as the ratio of the test preparation (AUC 0→t , T) to the reference preparation (AUC 0→t, R), and the calculation formula is as follows: Fr = AUC 0→t ,T / AUC 0→t ,R × 100% (Eq. 5-1) Where T and R represent the test preparation and the reference preparation, respectively.

[0131] The calculation results of the relative bioavailability of the Lut-SNC capsule group compared with the Lut and PM capsule groups are as follows: Fr(PM capsules / Lutcapsules) = AUC 0→24 , PM capsules / AUC 0→24 , Lutcapsules × 100% = 85.56% Fr(Lut-SNC capsules / Lutcapsules) = AUC 0→24 ,Lut-SNC capsules / AUC 0→24 ,Lutcapsules × 100% = 288.91% ​​Fr(Lut-SNC capsules / PMcapsules)=AUC 0→24 ,Lut-SNC capsules / AUC 0→24 ,PMcapsules×100%=337.67% As can be seen from the results, when Lut raw material drug was used as the reference, the relative bioavailability of the PM group was 85.56%, which was between 80% and 125%, indicating that there was no significant difference between the two. When Lut raw material drug and PM were used as references respectively, the relative bioavailabilities of the Lut-SNC capsule group were 288.91% and 337.67% respectively, both greater than 125%, indicating that there were significant differences between the Lut-SNC capsule and the two, that is, they were bioinequivalent, indicating that the nanocrystal technology could significantly improve the oral bioavailability of Lut.

[0132] Based on the above analysis results of pharmacokinetic parameters, the T of the Lut-SNC capsule group max was significantly shortened, probably because its dissolution rate and cumulative dissolution degree in the first 10 min were relatively high, and the C max and AUC values increased significantly. The relative bioavailability was 2.89 times higher than that of Lut and 3.88 times higher than that of PM. It was speculated that this might be because the particle size was smaller, the dissolution rate increased, and it was more likely to adhere to the gastrointestinal mucosa. In addition, the nano-drug would accumulate in the Peyer's patches and then enter the blood circulation through the lymphatic circulation, thus changing the absorption pathway and making it absorb more efficiently.

[0133] In this in vivo pharmacokinetic experiment, although it could prove that the self-made Lut-SNC capsules had certain advantages, due to the limited conditions of experimental animals, beagle dogs could not be used for pharmacokinetic experiments and evaluations, and only rat experiments were carried out. At the same time, because rats could not swallow the capsules, their contents were taken for gavage. In addition, there was no marketed preparation for comparison, and only the raw material drug was used as the reference and the PM group was added for auxiliary proof. The above content was the deficiency of this study, and it was hoped that subsequent experiments could be supplemented and completed when there was an opportunity.

[0134] In summary, the present invention improves the solubility of Lut raw material drug by preparing Lut-SNC capsules, thereby improving its oral bioavailability. First, a methodology for the determination of the content and dissolution of Lut was established. Secondly, the optimal formulation of Lut-NC was obtained through single-factor investigation and central composite design. Subsequently, Lut-SNC was prepared by freeze-drying. The solid-phase characterization, content and in vitro dissolution of Lut-SNC were determined. Finally, an in vivo pharmacokinetic experiment on rats was carried out.

[0135] Lut-NC was prepared by micro-media grinding method. With particle size and PDI as indicators, the prescription composition and preparation process were investigated by single factor, and optimized and verified by central composite design. The optimal prescription of Lut-NC: Lut:PVP K30 = 5:1, zirconia beads with a size of 0.2 - 0.4 mm were selected as grinding media (dosage 10 mL), grinding for 12 h, rotation speed 700 r·min -1 . Lut-SNC was obtained by freeze-drying, and the optimal prescription: Lut-NC + 3% sucrose.

[0136] Solid-phase characterization of Lut-SNC was carried out: The results of Malvern and SEM showed that the particle size of Lut-SNC belonged to the nanometer level, the average particle size was (120.95 ± 3.54) nm, the PDI value was (0.207 ± 0.062), and the Zeta potential was (-35.95 ± 1.04) mV; The results of DSC and PXRD showed that the crystallization intensity of Lut was greatly weakened, but there was still a small part existing in a weaker crystalline state; The FT-IR results showed that the original structure of Lut in Lut-SNC did not change, and there might be hydrogen bond interactions. The drug loading of Lut-SNC was (80.27 ± 0.28)%; The results of in vitro dissolution experiments showed that Lut-SNC could greatly improve the solubility of Lut.

[0137] An in vivo analytical detection method for plasma samples containing Lut was established, and this method was accurate and reliable. The results of pharmacokinetic experiments in rats showed that the oral bioavailability of Lut raw material drug was low, and the improvement of oral bioavailability in the Lut-SNC capsule group was very significant. When Lut and PM groups were used as references respectively, Fr were 288.91% and 337.67% respectively.

[0138] The above has described the embodiments of the present invention in detail, but the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those of ordinary skill in the art, various changes can be made without departing from the gist of the present invention. In addition, without conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other.

Claims

1. A luteolin solid nanocrystal, characterized in that, It comprises the following components: Stabilizer: luteolin 10%-50%; Distilled water 5 mL; Grinding medium 10 mL; Lyoprotectant 3%.

2. The luteolin solid nanocrystal according to claim 1, characterized in that, The stabilizer is at least one of sodium dodecyl sulfate, soy lecithin, hydroxypropyl methylcellulose, poloxamer 188 and 407, polyvinylpyrrolidone K30, vitamin E polyethylene glycol succinate, and tween-80.

3. The luteolin solid nanocrystal according to claim 1, wherein One of its drug-excipient ratios is 50:1, 25:1, 10:1, 5:1, 2:1, and 1:

1.

4. The luteolin solid nanocrystal according to claim 1, characterized in that, The diameter of the grinding medium is one of three types: 0.2-0.4, 0.4-0.6, 0.6-0.8 mm.

5. The luteolin solid nanocrystal according to claim 1, wherein The lyoprotectant is at least one of PEG-4000, β-cyclodextrin, dextran-70, sodium citrate, mannitol, sucrose, and lactose.

6. A preparation method of the valsartan solid self - microemulsifying drug delivery system according to any one of claims 1 to 5, characterized in that, The preparation method comprises the following steps: S1. The stabilizer and luteolin are proportionally mixed, and Lut-NC is prepared by using a grinding medium and a micro-medium grinding method. S2. The mixture obtained in step S1 is mixed with the lyoprotectant, and Lut-NC is solidified by freeze-drying technology to obtain luteolin solid nanocrystals.

7. The preparation method according to claim 6, characterized in that, In step S1, the drug-excipient ratio is 5:1; preferably, in step S1, the grinding speed is 700 r / min; preferably, in steps S1 and S2, the grinding time is 12 h.

8. Use of the luteolin solid nanocrystals according to any one of claims 1-5 or the luteolin solid nanocrystals obtained by the preparation method according to any one of claims 6-7 as a pharmaceutical preparation.

9. The application according to claim 9, wherein The dosage form of the pharmaceutical preparation includes at least one of tablets, pellets, powders, capsules, and granules.

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