CN101461949B - Berberine cyclodextrin inclusion compound, preparation thereof and preparation method - Google Patents

Abstract

The invention relates to a hydrochloric acid berberine/hydroxide propyl-beta-cyclodextrin clathrate, a preparation thereof and a method for preparing the same. By a gradient temperature reduction sealing method, the hydrochloric acid berberine and the hydroxide propyl-beta-cyclodextrin between which the mol ratio is 1:0.2-10 are sealed so as to improve the water solubility and biological availability of the hydrochloric acid berberine; the clathrate has the advantages of high sealing rate, large medicine loading capability and high purity; and the clathrate can be prepared into a clinically acceptable formulation with quick curative effect. The invention also clarifies the pharmacological functions of the clathrate.

Description

A kind of berberine cyclodextrin inclusion compound, its preparation and preparation method

technical field

The present invention relates to a pharmaceutical preparation containing organic active ingredients and a preparation method thereof, in particular to a medicinal preparation of a six-membered ring heterocyclic system containing nitrogen as a ring heteroatom and a preparation method thereof.

Background technique

Berberine hydrochloride (berberine) is an isoquinoline alkaloid extracted from the rhizome of Coptis chinensis Franch. of Ranunculaceae or Phellodendron amurense Rupr. of Rutaceae. Molecular formula: C ₂₀ H ₁₈ ClNO _4. Molecular weight: 336.37, its molecular structure formula is:

The clinical application of berberine hydrochloride is mainly used to clear away heat and detoxify, and treat intestinal infection. However, it has been found in recent years that it also has the effects of treating arrhythmia, hypertension, hyperlipidemia, diabetes, depression and anti-tumor. Ehrlich ascites tumor, colon cancer cells, esophageal cancer cell lines, nasopharyngeal cancer cells, bladder cancer cells, malignant teratoma cells, etc. all have certain inhibitory and killing effects. However, berberine has a very bitter taste, poor solubility and low bioavailability, which greatly limit its clinical application.

Cyclodextrin is a cyclic oligosaccharide formed by 6-12 glucose molecules linked by enzymatic cyclization of starch. It has a hydrophobic inner cavity and a hydrophilic outer wall, so it can provide A hydrophobic binding site, its internal cavity is hydrophobic and its external surface is hydrophilic, so that it can form clathrates and molecular assembly systems with many organic and inorganic molecules based on van der Waals forces, hydrophobic interaction forces, and the matching between host and guest molecules. . The host-guest or supramolecular complexes formed by cyclodextrin and the active ingredients of the drug can significantly improve the solubility and stability of the drug, mask the bad smell of the drug, and reduce the stimulation of the drug. It is widely used in pharmaceuticals, food, cosmetics and other fields by improving drug bioavailability and regulating drug release.

Hydroxypropyl-β-cyclodextrin is a derivative of β-cyclodextrin. Compared with β-cyclodextrin, it has the advantages of better water solubility and higher safety. It is approved by the United States, India and other countries as an injection Used in pharmaceutical excipients. Moreover, hydroxypropyl-β-cyclodextrin has the effect of improving the fluidity of cell membrane lipid rafts and the inhibitor of multidrug resistance pumps (MDRs).

Many pharmaceutical workers make berberine into weak acid salts to improve its solubility; or make modern dosage forms such as nano-preparations and dripping pills to improve its absorption mechanism. However, the above methods have disadvantages. Converting berberine from hydrochloride to other weak acid salts, due to the restriction of chemical reaction balance and industrial crystallization technology, makes the yield low and greatly increases the cost of raw materials. The large-scale production process of nano-preparation is not stable, which restricts its industrial production, and it is still in the laboratory stage. The bitter taste of berberine is well known (coptis rhizome is used as a representative of bitter taste in many common sayings and allusions), so it is not wise at all to take berberine hydrochloride sublingually as a drop pill.

The current research is mainly to form inclusion complexes between berberine and β-cyclodextrin to improve the solubility and bioavailability of berberine (Li Zhiping, Ouyang Yuzhu, Zhang Aihua, Wu Yun, berberine β-cyclodextrin Preparation and antibacterial test of dextrin inclusion compound, Fine Chemical Intermediates, 2003, 6(33): 46-48; Yang Bo, Zhao Yulin, Xin Xiaoyan, Yang Minghui, Berberine hydrochloride-cyclodextrin in binary Interactions in inclusion systems, Chemical World, 2004, 6, 292-294). Although the formation of berberine/β-cyclodextrin inclusion complex can improve the solubility of berberine to a certain extent, but the encapsulation efficiency, drug loading and purity are low, the prepared berberine β-cyclodextrin inclusion complex The bioavailability of the drug is low, especially the bioavailability during oral administration is low, which affects the efficacy of the drug.

Sun Junmei et al. (Sun Junmei, Shen Hui, Luo Wen, He Gang, Study on antibacterial effect of berberine/β-cyclodextrin inclusion compound in Chinese medicine compatibility Journal of Chengdu University (Natural Science Edition) 2007, 26(4): 280 -282) The inclusion complex of berberine hydrochloride/β-cyclodextrin was prepared by ultrasonic method. After the berberine was completely dissolved in water, it was shaken in an ultrasonic oscillator for 2 hours, poured into a plate, and allowed to air-dry to crystallize naturally. The encapsulation efficiency, drug loading and purity of the clathrate prepared by the method are low, and the bioavailability of the clathrate is low, especially when it is administered orally, which affects the efficacy of the drug.

Contents of the invention

The primary purpose of the present invention is to provide a berberine hydrochloride/hydroxypropyl-β-cyclodextrin inclusion compound, its formulation and preparation method for the above problems. The prepared berberine hydrochloride/hydroxypropyl-β-cyclodextrin inclusion compound medicinal preparation has high solubility, high bioavailability and remarkable pharmacological effects.

In order to realize the purpose of the present invention, the present invention provides a kind of berberine hydrochloride/hydroxypropyl-β-cyclodextrin clathrate on the one hand, the molar ratio of berberine hydrochloride and hydroxypropyl-β-cyclodextrin 1:0.2-10, preferably 1:0.5-3, more preferably 1:0.5-2, most preferably 1:0.5-1.5.

Wherein, the berberine hydrochloride is anhydrous berberine hydrochloride without crystal water or berberine hydrochloride hydrate containing crystal water.

Another aspect of the present invention provides a preparation method of berberine hydrochloride/hydroxypropyl-β-cyclodextrin inclusion compound, comprising the following steps:

1) Heat the hydroxypropyl-β-cyclodextrin solution to 45-70°C, then add berberine hydrochloride while stirring to dissolve the berberine hydrochloride in the hydroxypropyl-β-cyclodextrin solution, continue Stir for 12-24 hours;

2) Gradient cooling of the berberine hydrochloride and hydroxypropyl-β-cyclodextrin solution to 20-30°C;

3) drying the cooled berberine hydrochloride/hydroxypropyl-β-cyclodextrin solution to obtain the clathrate finished product.

Wherein, the molar ratio of berberine hydrochloride and hydroxypropyl-β-cyclodextrin described in step 1) is 1: 0.2-10, preferably 1: 0.5-3, more preferably 1: 0.5-2 , the most preferred is 1:0.5-1.5; the stirring rate is 600-800rpm.

In particular, the solvent of the hydroxypropyl-β-cyclodextrin solution is selected from one of water, a buffer solution with a pH of 1 to 8, alcohol, dimethylsulfoxide, dimethylacetamide, and ketone, preferably water ; The heating temperature of the hydroxypropyl-β-cyclodextrin solution is preferably 45-60°C.

In particular, the mass ratio of the volume of the solvent to the hydroxypropyl-β-cyclodextrin is 100-1500:1, preferably 100-1000:1, more preferably 100-500:1, that is, hydroxypropyl-β-cyclodextrin - If the mass of cyclodextrin is 1 g, the volume of the solvent is 100-1500 mL or if the mass of hydroxypropyl-β-cyclodextrin is 1 kg, the volume of the solvent is 100-1500 L.

Wherein, the cooling rate of the gradient cooling described in step 2) is 3 to 20°C/h, preferably 3 to 10°C/h, more preferably 3 to 6°C/h; the drying described in step 3) is Freeze-drying, spray-drying or drying under reduced pressure, preferably freeze-drying and spray-drying.

In particular, during spray drying, the temperature of the inlet of the filtrate spray dryer is controlled to be 130-150°C, preferably 145°C; the temperature of the sample outlet is 70-80°C, preferably 75°C; The drying temperature is -40 to -80°C, preferably -45 to -50°C; the relative pressure is -0.05 to -0.08MPa, preferably -0.05 to -0.06MPa.

Another aspect of the present invention provides a method for preparing the above-mentioned berberine hydrochloride/hydroxypropyl-β-cyclodextrin inclusion compound, comprising the following steps:

1) Heat the hydroxypropyl-β-cyclodextrin solution to 45-70°C, then add berberine hydrochloride under the action of ultrasonic waves, and sonicate the berberine hydrochloride to dissolve in the hydroxypropyl-β-cyclodextrin in solution;

2) Gradient cooling of the berberine hydrochloride and hydroxypropyl-β-cyclodextrin solution to 20-30°C;

3) drying the cooled berberine hydrochloride and hydroxypropyl-β-cyclodextrin solutions to obtain the clathrate finished product.

Wherein, the molar ratio of berberine hydrochloride and hydroxypropyl-β-cyclodextrin described in step 1) is 1: 0.2-10, preferably 1: 0.5-3, more preferably 1: 0.5-2 , the most preferred is 1:0.5-1.5; the ultrasonic treatment time is 30-90 min, preferably 45-60 min.

In particular, the solvent of the hydroxypropyl-β-cyclodextrin solution is selected from one of water, a buffer solution with a pH of 1 to 8, alcohol, dimethylsulfoxide, dimethylacetamide, and ketone, preferably water ; The heating temperature of the hydroxypropyl-β-cyclodextrin solution is preferably 45-60°C.

In particular, the mass ratio of the volume of the solvent to the hydroxypropyl-β-cyclodextrin is 100-1500:1, preferably 100-1000:1, more preferably 100-500:1, that is, hydroxypropyl-β-cyclodextrin - If the mass of cyclodextrin is 1 g, the volume of the solvent is 100-1500 mL or if the mass of hydroxypropyl-β-cyclodextrin is 1 kg, the volume of the solvent is 100-1500 L.

Wherein, the cooling rate of the gradient cooling described in step 2) is 3 to 20°C/h, preferably 3 to 10°C/h, more preferably 3 to 6°C/h; the drying described in step 3) is Freeze-drying, spray-drying or drying under reduced pressure, preferably freeze-drying and spray-drying.

Another aspect of the present invention provides a berberine hydrochloride/hydroxypropyl-β-cyclodextrin inclusion complex prepared according to the above method.

Yet another aspect of the present invention provides a pharmaceutical preparation of the above-mentioned berberine hydrochloride/hydroxypropyl-β-cyclodextrin inclusion compound, wherein the inclusion compound of berberine hydrochloride/hydroxypropyl-β-cyclodextrin is mixed with The pharmaceutically acceptable auxiliary materials or carriers can be fully mixed to make any pharmaceutically acceptable dosage form.

Among them, the inclusion compound of berberine hydrochloride/hydroxypropyl-β-cyclodextrin and pharmaceutically acceptable auxiliary materials or carriers are made into tablets, capsules, granules, oral solutions, dropping pills, injections or cataplasms.

The advantages of the clathrate of berberine hydrochloride/hydroxypropyl-β-cyclodextrin of the present invention are as follows:

1. The inclusion compound of berberine hydrochloride/hydroxypropyl-β-cyclodextrin prepared by the present invention has high water solubility, high encapsulation efficiency, large drug loading capacity and high purity.

2. The preparation method of the clathrate of berberine hydrochloride/hydroxypropyl-β-cyclodextrin of the present invention is simple, and the ratio of raw materials is calculated according to the molar ratio. Berberine hydrochloride and hydroxypropyl-β-cyclodextrin The solution is subjected to gradient cooling to promote the inclusion of inclusion compounds, and the gradient cooling is beneficial to the process control in industrial production, so that the quality of the produced products is stable, and the encapsulation rate, drug loading and purity of the inclusion complex products are improved without increasing the temperature. Oral administration avoids the potential risk of secondary infection caused by the imbalance of intestinal flora, and provides a basic guarantee for the safety of clinical medication and the stability of drug efficacy.

3. The inclusion compound of berberine hydrochloride/hydroxypropyl-β-cyclodextrin of the present invention can be fully mixed with suitable pharmaceutical excipients to make any pharmaceutically acceptable dosage form, such as tablets, capsules, granules medicaments, oral solutions, pills, injections or cataplasms.

4. The pharmaceutical preparation of the inclusion compound of berberine hydrochloride/hydroxypropyl-β-cyclodextrin of the present invention has good water solubility, high intestinal absorption rate of the drug in vivo, fast absorption rate, large effective permeability coefficient, and biological The utilization is high, the drug can be promoted to exert its pharmacological effect rapidly, and the drug effect can be significantly improved.

Description of drawings

Figure 1 The infrared spectrum of berberine hydrochloride, hydroxypropyl-β-cyclodextrin, the berberine hydrochloride/hydroxypropyl-β-cyclodextrin inclusion compound prepared in Example 1 and Comparative Example 1

Figure 2 The X-ray powder diffraction pattern of berberine hydrochloride, hydroxypropyl-β-cyclodextrin, the berberine hydrochloride/hydroxypropyl-β-cyclodextrin inclusion compound prepared in Example 1 and Comparative Example 1

Figure 3 Differential scanning calorimetry of berberine hydrochloride, hydroxypropyl-β-cyclodextrin, berberine hydrochloride/hydroxypropyl-β-cyclodextrin inclusion compound prepared in Example 1 and Comparative Example 1 Analyze Spectrum

Specific examples:

The implementation of the technical solution of the present invention is described in detail below through the examples, but the present invention is not limited to the content contained in the following examples. The berberine hydrochloride used in the embodiments of the present invention is anhydrous berberine hydrochloride without crystal water, and the berberine hydrochloride containing crystal water is also applicable to the present invention.

Example 1:

1. Preparation of berberine hydrochloride/hydroxypropyl-β-cyclodextrin inclusion compound

1) Put 2g of hydroxypropyl-β-cyclodextrin in a beaker, add 1L of deionized water, and stir to dissolve;

2) After heating the hydroxypropyl-β-cyclodextrin solution to 60°C, slowly add 574 mg of berberine hydrochloride solution dissolved in a small amount of absolute ethanol to the hydroxypropyl-β-cyclodextrin while stirring In the solution, the stirring rate is 800rpm. After stirring for 12 hours, the temperature is gradually lowered at a rate of 5°C/h, and the temperature is slowly lowered to 25°C;

3) Filter the cooled solution with a 0.22 μm pore size microporous filter (to remove unincluded berberine hydrochloride);

The filtrate is placed in a spray dryer for spray drying. The inlet temperature of the filtrate spray dryer is 145° C., and the temperature of the sample outlet is 75° C. to obtain a white loose powder, namely berberine hydrochloride/hydroxypropyl-β- Cyclodextrin inclusion complex.

The content of berberine hydrochloride in the clathrate is determined by high performance liquid chromatography, and the encapsulation efficiency and drug loading capacity of the clathrate are detected, wherein:

Encapsulation efficiency=(the input amount of berberine hydrochloride quality/berberine hydrochloride in clathrate)×100%;

Drug loading=(mass of berberine hydrochloride in inclusion compound/total weight of inclusion compound)×100%.

Wherein: the high-performance liquid chromatography of berberine hydrochloride content measures chromatographic condition: chromatographic column is ThermoHypersil ODS-2 C18 post (250mm * 4.6mm, 5 μ m); Mobile phase is acetonitrile: water (containing 0.1% phosphoric acid and 0.05% Tris) ethylamine)=30:70; the flow rate was 1 mL/min; the column temperature was room temperature; the detection wavelength was 263 nm; the injection volume was 5 μL.

The enthalpy change ΔH of the clathrate was determined by differential scanning calorimetry.

The test results of the performance indexes of the prepared berberine hydrochloride/hydroxypropyl-β-cyclodextrin inclusion compound are shown in Table 1.

2. Preparation of Berberine Hydrochloride Hydroxypropyl-β-cyclodextrin Inclusion Complex Dispersible Tablets

Take an appropriate amount of hydroxypropyl-β-cyclodextrin inclusion compound of berberine hydrochloride, lactose, and sodium carboxymethylcellulose, and mix it with microcrystalline cellulose at the same time, then granulate with 75% ethanol, and dry at 60°C , plus an appropriate amount of magnesium stearate, after mixing evenly, compress into tablets according to the direct compression method to obtain dispersible tablets.

Example 2:

1. Preparation of berberine hydrochloride/hydroxypropyl-β-cyclodextrin inclusion compound

1) Put 1g of hydroxypropyl-β-cyclodextrin in a beaker, add 1L of deionized water, stir, and heat to 45°C to dissolve it;

2) After heating the hydroxypropyl-β-cyclodextrin solution to 45°C, slowly add 574 mg of berberine hydrochloride solution dissolved in a small amount of absolute ethanol to the hydroxypropyl-β-cyclodextrin while stirring In the solution, the stirring rate is 800rpm. After stirring for 24 hours, the temperature is gradually lowered at a rate of 3°C/h, and the temperature is slowly lowered to 20°C;

3) Filter the cooled solution with a 0.22 μm pore size microporous filter (to remove unincluded berberine hydrochloride);

4) Put the filtrate in a freeze dryer for freeze-drying under reduced pressure. The freeze-drying temperature is -45°C and the relative pressure is -0.06MPa to obtain a white and loose powder, namely berberine hydrochloride/hydroxypropyl-β-cyclo Dextrin inclusion complex.

The test results of the performance indexes of the prepared berberine hydrochloride/hydroxypropyl-β-cyclodextrin inclusion complex are shown in Table 1.

2. Preparation of berberine hydrochloride/hydroxypropyl-β-cyclodextrin inclusion complex capsules

Take an appropriate amount of berberine hydrochloride/hydroxypropyl-β-cyclodextrin inclusion compound, mix with an appropriate amount of auxiliary materials (lactose or starch), and fill capsules to obtain capsules.

Example 3:

1. Preparation of berberine hydrochloride/hydroxypropyl-β-cyclodextrin inclusion compound

1) Put 6g of hydroxypropyl-β-cyclodextrin in a beaker, add 2L of deionized water, and stir to dissolve;

2) After heating the hydroxypropyl-β-cyclodextrin solution to 50°C, slowly add 574 mg of berberine hydrochloride solution dissolved in a small amount of absolute ethanol to the hydroxypropyl-β-cyclodextrin while stirring In the solution, the stirring rate is 800rpm. After stirring for 12 hours, the temperature is gradually lowered at a rate of 6°C/h, and the temperature is slowly lowered to 25°C;

3) Filter the cooled solution with a 0.22 μm pore size microporous filter (to remove unincluded berberine hydrochloride);

4) Put the filtrate in a freeze dryer for freeze-drying under reduced pressure. The freeze-drying temperature is -45°C and the relative pressure is -0.06MPa to obtain a white and loose powder, namely berberine hydrochloride/hydroxypropyl-β-cyclo Dextrin inclusion complex.

The test results of the performance indexes of the prepared berberine hydrochloride/hydroxypropyl-β-cyclodextrin inclusion complex are shown in Table 1.

2. Preparation of berberine hydrochloride/hydroxypropyl-β-cyclodextrin inclusion compound cataplasm

Take an appropriate amount of berberine hydrochloride/hydroxypropyl-β-cyclodextrin inclusion compound to prepare an aqueous solution with a prescription concentration, mix it with the cataplasm matrix solution, stir it evenly with electromagnetic stirring, and apply it evenly on the non-woven fabric. Cover with a protective film and seal to obtain the poultice of the beta-cyclodextrin inclusion compound of berberine hydrochloride.

Example 4:

1. Preparation of berberine hydrochloride/hydroxypropyl-β-cyclodextrin inclusion compound

1) First add 4g of hydroxypropyl-β-cyclodextrin into a beaker containing 1L of deionized water, then place the beaker containing hydroxypropyl-β-cyclodextrin in an ultrasonic oscillator for ultrasonication, Make it dissolve while heating to 50°C;

2) After dissolving 574mg of berberine hydrochloride with a small amount of absolute ethanol, slowly add the berberine hydrochloride ethanol solution dropwise into the hydroxypropyl-β-cyclodextrin solution, and continue to sonicate to dissolve berberine hydrochloride in the hydroxyl Propyl-β-cyclodextrin solution, wherein the ultrasonic power of ultrasonic treatment is 300W, and the treatment time is 60min;

3) The ultrasonically treated solution is subjected to gradient cooling at a gradient cooling rate of 4.5°C/h, and slowly cooled to 30°C;

4) Filter the cooled solution with a 0.22 μm pore size microporous filter (to remove unincluded berberine hydrochloride);

5) The filtrate is placed in a freeze dryer for freeze-drying under reduced pressure. The freeze-drying temperature is -45°C and the relative pressure is -0.06MPa to obtain a white and loose powder, namely berberine hydrochloride/hydroxypropyl-β-cyclo Dextrin inclusion complex.

The test results of the performance indexes of the prepared berberine hydrochloride/hydroxypropyl-β-cyclodextrin inclusion complex are shown in Table 1.

2. Preparation of berberine hydrochloride/hydroxypropyl-β-cyclodextrin inclusion complex drop pills

1) Take PEG1000 and PEG4000 with a mass ratio of 1:1 as the drop pill base, take an appropriate amount of berberine hydrochloride/hydroxypropyl-β-cyclodextrin inclusion compound and mix them evenly with the drop pill base, and heat to 95°C;

2) Use simethicone as the coolant, adjust the temperature of the coolant to 5°C, drop the caliber to 3 mm, drop the speed to 50 drops/min, and drop the distance to 6 cm to obtain hydroxypropyl-β- Dropping pills of cyclodextrin inclusion complexes.

Example 5:

1. Preparation of berberine hydrochloride/hydroxypropyl-β-cyclodextrin inclusion compound

1) Put 20g of hydroxypropyl-β-cyclodextrin in a beaker, add 2L of deionized water, and stir to dissolve;

2) After heating the hydroxypropyl-β-dextrin solution to 45°C, slowly add 574 mg of berberine hydrochloride solution dissolved in a small amount of absolute ethanol dropwise to the hydroxypropyl-β-cyclodextrin solution while stirring , the stirring rate is 800rpm, after stirring for 12h, the gradient cooling is carried out, the gradient cooling rate is 3°C/h, and the temperature is slowly lowered to 20°C;

3) Filter the cooled solution with a 0.22 μm pore size microporous filter to remove unincluded berberine hydrochloride;

4) Put the filtrate in a freeze dryer for freeze-drying under reduced pressure. The freeze-drying temperature is -45°C and the relative pressure is -0.06MPa to obtain a white and loose powder, namely berberine hydrochloride/hydroxypropyl-β-cyclo Dextrin inclusion complex.

The test results of the performance indexes of the prepared berberine hydrochloride/hydroxypropyl-β-cyclodextrin inclusion complex are shown in Table 1.

2. Preparation of berberine hydrochloride/hydroxypropyl-β-cyclodextrin inclusion complex injection

Take an appropriate amount of hydroxypropyl-β-cyclodextrin inclusion compound of berberine hydrochloride, add an appropriate amount of mannitol after adding water for injection, adjust isotonicity, and then use an ultrafilter with a molecular weight cut-off of 5000 to remove pyrogens, and filter the filtrate with 0.22 um pore size microporous membrane filter to sterilize, subpackage, and freeze-dry to obtain freeze-dried powder for injection.

Comparative example 1

The preparation of the berberine hydrochloride/hydroxypropyl-β-cyclodextrin inclusion complex was the same as in Example 1 except that natural cooling at room temperature was adopted.

Comparative example 2

The preparation of the berberine hydrochloride/hydroxypropyl-β-cyclodextrin inclusion compound was the same as that in Example 2 except that natural cooling was adopted at room temperature.

Comparative example 3

The preparation of the berberine hydrochloride/hydroxypropyl-β-cyclodextrin inclusion complex was the same as in Example 3 except that natural cooling was adopted at room temperature.

Comparative example 4

The preparation of the berberine hydrochloride/hydroxypropyl-β-cyclodextrin inclusion complex was the same as in Example 4 except that natural cooling was adopted at room temperature.

Comparative example 5

The preparation of the berberine hydrochloride/hydroxypropyl-β-cyclodextrin inclusion compound was the same as in Example 5 except that natural cooling at room temperature was adopted.

Comparative example 6

Anhydrous berberine hydrochloride that does not contain crystal water is used as comparative example 6 of the present invention.

Table 1 Test results of inclusion compound performance indicators

Encapsulation rate (%) Drug loading(%) ΔH(J/g) Example 1 47.1 23.9 4.25 Example 2 30.5 18.6 5.25 Example 3 22.6 13.4 8.95 Example 4 34.3 36.5 3.85 Example 5 5.0 7.38 18.56 Comparative example 1 45.5 20.3 33.15 Comparative example 2 31.2 17.2 34.65 Comparative example 3 20.8 12.8 42.16 Comparative example 4 32.1 32.4 25.68 Comparative Example 5 0.08 6.65 50.24 Comparative example 6 / / 60.84

Detecting result shows: under the situation that berberine hydrochloride and hydroxypropyl-beta-cyclodextrin consumption are identical, the berberine hydrochloride/hydroxypropyl-beta-cyclodextrin clathrate prepared by the present invention is relatively compared with adopting room temperature The encapsulation efficiency of berberine hydrochloride/hydroxypropyl-β-cyclodextrin inclusion compound prepared under natural cooling treatment is high; the drug loading capacity is large; the purity is high, and the ΔH of differential scanning calorimetry (DSC) Low, which is beneficial to the preparation and production of pharmaceutical preparations and improves the bioavailability of drugs.

Test example 1 in vitro dissolution test

According to "Chinese Pharmacopoeia" 2005 edition dissolution test method 1, 200mL of simulated intestinal juice and simulated gastric juice were respectively used as the release medium, the temperature was 37±0.5°C, and the rotation speed was 50r/min. The berberine hydrochloride/hydroxypropyl-β-cyclodextrin inclusion compound prepared in Example 1 (approximately equivalent to 0.3 g of berberine hydrochloride) and Comparative Examples 1 and 6 (approximately equivalent to 0.3 g of berberine hydrochloride) g) Put it into a tumbler, place it in a dissolution vessel filled with 200mL of medium, take 1mL of samples at regular intervals (1mL of release medium is supplemented at the same time), filter with a 0.45μm microporous filter, and detect in the liquid phase after dilution.

The detection results of DE ₂₀ , DE ₆₀ , and DE ₁₂₀ of berberine hydrochloride and its inclusion complex in simulated intestinal juice are shown in Table 2, and the detection results of DE ₂₀ , DE ₆₀ , and DE ₁₂₀ in simulated gastric juice are shown in Table 3 .

DE ₂₀ , DE ₆₀ , and DE ₁₂₀ values represent the dissolution percentages of berberine hydrochloride within 20 minutes, 60 minutes, and 120 minutes, respectively.

Table 2 The dissolution rate of berberine hydrochloride and its clathrate in simulated intestinal fluid

DE ₂₀ (%) DE ₆₀ (%) DE ₁₂₀ (%) Example 1 80.16 ^*ab 95.66 ^*ab 96.04 ^*ab Comparative Example 1 73.82 91.37 92.54 Comparative example 6 37.53 59.65 65.6

*a Compared with the control example 6, the DE ₆₀ (%) and DE ₁₂₀ (%) of the embodiment 1 are all significantly improved (P＜0.01)

*b compares with control example 1, DE ₆₀ (%), DE ₁₂₀ (%) of embodiment 1 all have significant improvement (P＜0.05)

Table 3 The dissolution rate of berberine hydrochloride and its clathrate in simulated gastric juice

DE ₂₀ (%) DE ₆₀ (%) DE ₁₂₀ (%) Example 1 27.87 ^*ab 30.98 ^*ab 35.29 ^*ab Comparative Example 1 25.33 26.48 30.53 Comparative example 6 6.26 9.17 10.36

*a Compared with the control example 6, the DE ₆₀ (%) and DE ₁₂₀ (%) of the embodiment 1 are all significantly improved (P＜0.01)

*b compares with control example 1, DE ₆₀ (%), DE ₁₂₀ (%) of embodiment 1 all have significant improvement (P＜0.05)

Test result shows: in simulated gastric juice, the solubility of berberine hydrochloride is very low, and its stripping speed is also very slow (only stripping 9.17% in 60 minutes). After the berberine hydrochloride prepared by the invention is clathrated with hydroxypropyl-β-cyclodextrin, both the solubility and the dissolution rate are greatly improved, and 27.87% of it is dissolved within 20 minutes, and 30.98% of it is dissolved within 60 minutes. In the simulated intestinal fluid, the dissolution amount of pure berberine hydrochloride is 37.53% within 20 minutes, while the dissolution amount of berberine hydrochloride in the clathrate prepared by the present invention reaches 80.16%.

The clathrates prepared in other examples 2-5 also have the characteristics of high dissolution rate, which is beneficial to the absorption of drugs and helps to improve the bioavailability of drugs.

Test Example 2 Rat in vivo intestinal absorption test

The typical P-glycoprotein substrate berberine hydrochloride was selected as a model drug, and the in vivo intestinal absorption test of Sprague Dawley rats was carried out by using the rat in vivo one-way intestinal perfusion method.

12 Sprague Dawley rats were divided into 3 groups, 4 in each group: the berberine hydrochloride/hydroxypropyl-β-cyclodextrin inclusion complex prepared in Example 1 and Comparative Example 1 were respectively used as Example 1 group , control group 1, berberine hydrochloride as control group 6.

Before the experiment, the animals were acclimatized for at least 5 days and fasted for 14 hours (with free access to water). Rats were anesthetized with ether and injected intraperitoneally with 20% urethane solution (0.5mL·kg ^-1 ). After the painless reflex was confirmed, the abdomen was incised about 3 cm along the midline of the abdomen, and the jejunum segment (starting at 15 cm from the pylorus) was taken about 10 cm. Cannulae were intubated after incisions at the upper and lower ends, and ligated with thread. Cover the wound with absorbent cotton soaked in normal saline to moisturize, then gently rinse the intestinal contents with an appropriate amount of normal saline preheated to 37°C, then replace it with KR test solution at 37°C to balance for 10 minutes, and then use the test solution (containing phenol red 20 μg·mL ^-1 , concentration of berberine hydrochloride 20 μg·mL ^-1 ) for perfusion at a flow rate of 0.2 mL·min ^-1 , collected in a small bottle at the outlet, and quickly replaced the collection solution every 15 minutes vial. Finally, high performance liquid chromatography was used to detect the concentration of phenol red and berberine hydrochloride in the collected liquid at each time period, and the experiment lasted for 120 minutes. After the experiment, the animals were sacrificed, and the perfused intestines were removed to measure the length and inner diameter.

The net water flow rate (NWF), absorption rate constant (K _a ) and effective permeability coefficient (P _eff ) were calculated by the phenol red method.

According to the Grubbs method, the 8 K _a and P _eff values obtained in each group of data were selected for outlier; the "Independent-Samples t-test" analysis was performed on each group of data using SPSS11.5.

The average net water flow rate (NWF), average absorption rate constant (K _a ) and average effective permeability coefficient (P _eff ) of Example 1 group and Comparative example groups 1 and 6 are shown in Table 4.

Table 4 Intestinal absorption test results

Average clean water flow rate (μL·min ^-1 ·cm ^-1 ) Average absorption rate constant (×10 ^-2 min ^-1 ) Average effective permeability coefficient (×10 ^-3 min·cm ^-1 ) Example 1 49.32±4.76 2.87±0.31 ^*ab 2.55±0.12 ^*ab Comparative example 1 47.56±2.33 1.92±0.20 1.77±0.09 Comparative example 6 45.60±1.57 0.45±0.03 0.43±0.03

*a Compared with Comparative Example 6, the average absorption rate constant (K _a ) and average effective permeability coefficient (P _eff ) of Example 1 are significantly improved (P<0.01)

*b Compared with Comparative Example 1, the average absorption rate constant (K _a ) and the average effective permeability coefficient (P _eff ) of Example 1 are significantly improved (P<0.05)

The results showed that:

1) Significant difference (P＜0.05) in the _Ka value and _Peff value of the berberine hydrochloride of the control example group and the berberine hydrochloride/hydroxypropyl-β-cyclodextrin inclusion compound of the embodiment group , P<0.01).

2) The absorption rate constant and effective permeability coefficient of berberine hydrochloride in the berberine hydrochloride/hydroxypropyl-β-cyclodextrin inclusion compound of the embodiment group in the small intestine are significantly increased, and the average absorption rate constant reaches 2.87 ±0.31×10 ^-2 min ^-1 , which is 6.38 times of the average absorption rate constant of berberine hydrochloride and 1.49 times of the average absorption rate constant of the control group; the average effective permeability coefficient reaches 2.55±0.12×10 ^-3 min·cm ^-1 , which is 5.93 times of the average effective permeability coefficient of berberine hydrochloride and 1.44 times of the average effective permeability coefficient of the control group. It shows that the intestinal absorption rate and high efficiency of the clathrate prepared by the method of the present invention are high, and the bioavailability of the drug is high.

3) The berberine hydrochloride in the clathrate prepared in other examples 2-5 has a large absorption rate constant and a large effective permeability coefficient in the small intestine, and also has the characteristics of rapid intestinal absorption and high bioavailability of the drug.

Infrared spectrum test of test example 3 clathrate

Compress berberine hydrochloride, hydroxypropyl-β-cyclodextrin, the berberine hydrochloride/hydroxypropyl-β-cyclodextrin inclusion compound prepared in Example 1 and Comparative Example 1, and test the infrared spectrum. The infrared spectrogram is shown in Figure 1. Compared with the infrared spectrum of berberine hydrochloride and embodiment 1, the change of the material characteristic peak in the clathrate infrared spectrum obtained in embodiment 1 proves that the clathrate exists as a new compound, and have different characteristic peaks.

The characteristic peaks of the benzene ring in berberine hydrochloride: 1600.035cm ^-1 , 1505.883cm ^-1 , the characteristic peaks of the aryl ether: 1272.432cm ^-1 , which can be vaguely discerned in the infrared spectrum of Comparative Example 1, while that of Example 1 Significantly decreased in the infrared spectrum;

The characteristic peaks of the coupling effect of adjacent ether bonds in berberine hydrochloride: 1231.905cm ^-1 , 1104.853cm ^-1 , 1036.159cm ^-1 , can still be distinguished in the infrared spectrum of Comparative Example 1, while the infrared spectrum of Example 1 Significantly decreased or disappeared in the figure.

The changes of the material characteristic peaks in the infrared spectra of Examples 2-5 are basically the same as those in Example 1, which proves that the clathrate exists as a new compound and has different characteristic peaks.

X-ray powder diffraction identification test of test example 4 clathrate

The berberine hydrochloride/hydroxypropyl-β-cyclodextrin clathrate powder prepared in embodiment 1 and comparative example 1 is carried out X-ray diffraction, The X-ray powder diffraction pattern is shown in FIG. 2 . The diffraction angle of berberine hydrochloride can still be seen in the collection of collections of comparative example 1, and the diffraction diagram of clathrate in the collection of collections of embodiment 1 is obviously different from other two kinds of samples, and the intensity of diffraction peak and diffraction angle all take place significant change, little Most of the characteristic peaks of berberine also basically disappeared. It was proved that clathrates were formed and new substances were generated.

The intensity and the diffraction angle of the diffraction peak of the X-ray powder diffraction pattern of embodiment 2-5 are basically the same as the X-ray powder diffraction pattern of embodiment 1, and the diffraction of berberine hydrochloride still can be seen in the collection of illustrative plates of corresponding comparative example horn.

Differential scanning calorimetry analysis of test example 5 clathrate

The berberine hydrochloride/hydroxypropyl-β-cyclodextrin inclusion complex powder prepared in Example 1 and Comparative Example 1 was subjected to differential scanning calorimetry Analysis, the differential scanning calorimetry spectrum is shown in Figure 3. Berberine hydrochloride presents a sharp endothermic peak at 185.98°C. Hydroxypropyl-β-cyclodextrin has a broad endothermic peak at 102.06℃. In Comparative Example 1, a heating peak appeared at 117.48°C and 186.00°C, which were the melting point peaks of cyclodextrin and berberine hydrochloride respectively. alkali. Compared with Comparative Example 1 in Example 1, the peak area and ΔH in the heating curve of the berberine hydrochloride/hydroxypropyl-β-cyclodextrin inclusion compound all changed, and ΔH was only 4.25 J/g, indicating that the inclusion compound formed The purity of the complex is high, the inclusion complexation between berberine hydrochloride and hydroxypropyl-β-cyclodextrin is complete, and the inclusion efficiency is high.

The differential scanning calorimetric analysis results of the clathrates of Examples 2-5 are the same as the differential scanning calorimetric analysis results of Example 1.

The test results show that the purity of the clathrate prepared by the method of the present invention is obviously higher than that of the clathrate prepared by the conventional natural cooling method at room temperature.

Claims (7)

1. the preparation method of a berberine hydrochloride/hydroxypropyl-beta-cyclodextrin inclusion comprises the steps:

1) HP-solution is heated to 45～70 ℃, adds berberine hydrochloride then while stirring;

2) berberine hydrochloride and the HP-solution gradient that stirs is cooled to 20～30 ℃;

3) berberine hydrochloride and the HP-solution after will lowering the temperature carries out drying, makes the clathrate finished product;

Wherein, the mole proportioning of berberine hydrochloride described in the step 1) and HP-is 1: 0.2～10; The speed of gradient cooling step 2) is 3-6 ℃/h.

2. the preparation method of a berberine hydrochloride/hydroxypropyl-beta-cyclodextrin inclusion comprises the steps:

1) HP-solution is heated to 45～70 ℃, under the ultrasound wave effect, adds berberine hydrochloride then, supersound process is dissolved in the HP-solution berberine hydrochloride;

2) berberine hydrochloride and HP-solution gradient are cooled to 20～30 ℃;

3) berberine hydrochloride and the HP-solution after will lowering the temperature carries out drying, makes the clathrate finished product;

Wherein, the mole proportioning of berberine hydrochloride described in the step 1) and HP-is 1: 0.2～10; The speed of gradient cooling step 2) is 3-6 ℃/h.

3. preparation method as claimed in claim 1 or 2, the mole proportioning that it is characterized in that described berberine hydrochloride and HP-is 1: 0.5～3.

4. preparation method as claimed in claim 1 or 2 is characterized in that the solvent of described HP-solution is selected from a kind of in the buffer solution of water, pH 1～8, alcohol, dimethyl sulfoxide, dimethyl acetylamide, the ketone.

5. preparation method as claimed in claim 1 or 2 is characterized in that the drying described in the step 3) is lyophilization, spray drying or drying under reduced pressure.

6. berberine hydrochloride/hydroxypropyl-beta-cyclodextrin inclusion is according to being prepared from as claim 1-5 either party method.

7. pharmaceutical preparation as clathrate as described in the claim 6 is characterized in that the clathrate of berberine hydrochloride HP-and pharmaceutically acceptable auxiliaries or carrier fully are mixed and made into acceptable any dosage form on the pharmaceutics.

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