A method for inclusion of the ethanol extract of Ligusticum chuanxiong Hort.

Abstract

The invention discloses an inclusion method of the alcohol extract of Ligusticum chuanxiong, belonging to the technical field of medicines, and solves the problems of easy sticking to punches, pitting, and large dosage of excipients in the prior art. The inclusion method of the alcohol extract of Ligusticum chuanxiong of the invention comprises the following steps: under stirring conditions, an ethanol solution of the alcohol extract of Ligusticum chuanxiong is dropped into a saturated solution of hydroxypropyl-β-cyclodextrin for inclusion, refrigeration, filtration, and low-temperature drying to obtain an inclusion compound. The invention first uses hydroxypropyl-β-cyclodextrin to include the ethanol extract of Ligusticum chuanxiong, and the obtained inclusion compound has significantly accelerated dissolution in vitro. When used for preparing tablets, the dosage of excipients is small, there is no sticking to punches, the obtained tablets have good appearance, and all quality indexes meet the requirements. The method of the invention is simple, easy to operate, and has high inclusion rate and high yield, and is suitable for industrial production.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceuticals, and particularly relates to a method for inclusion of ethanol extract of Ligusticum chuanxiong Hort. Background Art

[0002] For liver disease patients who need to take medicine for a long time, whether the medicine is convenient to take directly affects their choice of therapeutic drugs. Commonly used oral solid preparations, such as tablets and capsules, have the advantages of mature preparation technology, high degree of mechanization and automation in production, stable quality, and convenient taking and carrying.

[0003] The applicant found in previous studies that the ethanol extract of Ligusticum chuanxiong Hort. has good hepatoprotective effects. Therefore, it is of great significance to prepare it into an oral preparation. However, the ethanol extract of Ligusticum chuanxiong Hort. is an alcohol-soluble substance with a viscous property. When preparing tablets, if it is directly used as the main drug for tabletting, problems such as sticking to the punch and pockmarked tablets are likely to occur. In the prior art, such substances are generally dispersed with diluent excipients, but there is a situation where the amount of excipients used is large, resulting in an increase in the dosage for patients, which seriously affects the compliance of patients taking medicine.

[0004] Therefore, providing a treatment method for the ethanol extract of Ligusticum chuanxiong Hort. so that the prepared preparation has good properties and the preparation process is easy to control with less excipient usage has become an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0005] The technical problem solved by the present invention is: to provide a method for inclusion of the ethanol extract of Ligusticum chuanxiong Hort., and solve the problems of sticking to the punch, pockmarked tablets and large amount of excipients in the prior art.

[0006] The technical solution adopted by the present invention is as follows:

[0007] A method for inclusion of the ethanol extract of Ligusticum chuanxiong Hort. disclosed by the present invention comprises the following steps: under stirring conditions, adding an ethanol solution of the ethanol extract of Ligusticum chuanxiong Hort. dropwise to a saturated solution of hydroxypropyl-β-cyclodextrin, carrying out inclusion, refrigerating, filtering, and drying at low temperature to obtain an inclusion compound.

[0008] The applicant found in previous studies that β-cyclodextrin has poor water solubility itself, while hydroxypropyl-β-cyclodextrin (HP-β-CD) as a new inclusion material can overcome the disadvantage of poor water solubility of β-cyclodextrin without changing the cavity of cyclodextrin. Therefore, the present invention uses HP-β-CD as the inclusion material of the inclusion compound to prepare the CX-E inclusion compound.

[0009] In the preferred embodiment of the present invention, the stirring speed is 100 - 300 r / min, preferably 300 r / min.

[0010] In a preferred embodiment of the present invention, the mass ratio of hydroxypropyl-β-cyclodextrin to the ethanol extract of Ligusticum chuanxiong Hort. is 1-20:1, preferably 5-15:1, and more preferably 10:1.

[0011] In some embodiments of the present invention, the inclusion temperature is 30-70 °C, preferably 60 °C.

[0012] In some embodiments of the present invention, the inclusion time is 0.5-2.5 hours, preferably 2 hours.

[0013] In some embodiments of the present invention, after the inclusion is completed, the solution is refrigerated at 2-6 °C for 12-24 hours.

[0014] In some embodiments of the present invention, after the inclusion is completed, a small amount of sodium chloride is added to the solution, and after dissolution is completed, it is refrigerated again.

[0015] In some embodiments of the present invention, after adding sodium chloride, the content of sodium chloride in the solution is 0.2-0.3 mg / ml.

[0016] The present invention unexpectedly finds that adding a small amount of sodium chloride to the solution before refrigeration can improve the yield of the inclusion complex.

[0017] In some embodiments of the present invention, the preparation method of the ethanol extract of Ligusticum chuanxiong Hort. is as follows: after adding water to Ligusticum chuanxiong Hort. medicinal materials and heating to extract volatile oil, filtering to obtain a water extract and medicinal residues; adding ethanol to the water extract for precipitation and filtering to obtain an ethanol precipitation solution; adding ethanol to the medicinal residues for extraction to obtain an ethanol extract; combining the ethanol precipitation solution and the ethanol extract, and evaporating the solvent to obtain the ethanol extract of Ligusticum chuanxiong Hort.

[0018] In some embodiments of the present invention, the concentration of the ethanol extract of Ligusticum chuanxiong Hort. in the ethanol solution of the ethanol extract of Ligusticum chuanxiong Hort. is 0.05-1 g / ml; preferably 0.1 g / ml.

[0019] CX-E described in the present invention is the ethanol extract of Ligusticum chuanxiong Hort.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention first uses hydroxypropyl-β-cyclodextrin to include the ethanol extract of Ligusticum chuanxiong Hort., and the obtained inclusion complex has significantly accelerated in vitro dissolution. When used for preparing tablets, the amount of excipients is small, there is no sticking to the punch, the obtained tablets have good appearance, and all quality indexes meet the requirements.

[0022] The method of the present invention is simple, easy to operate, and both the inclusion rate and the yield are relatively high, which is suitable for industrial production. Description of the Drawings

[0023] Appendix Figure 1Appearance comparison diagrams of the CX-E inclusion complex, CX-E extract paste, HP-β-CD, and physical mixture of HP-β-CD and CX-E extract paste in Test Example 1. Among them, a is the CX-E extract paste, b is HP-β-CD, c is the physical mixture of HP-β-CD and CX-E extract paste, and d is the CX-E inclusion complex;

[0024] Attached Figure 2 Optical microscope diagrams of the CX-E inclusion complex, HP-β-CD, and physical mixture of HP-β-CD and CX-E extract paste in Test Example 1; among them, a is HP-β-CD, b is the physical mixture of HP-β-CD and CX-E extract paste, and c is the CX-E inclusion complex;

[0025] Attached Figure 3 DSC analysis diagrams of the CX-E inclusion complex, CX-E, HP-β-CD, and physical mixture of HP-β-CD and CX-E in Test Example 1; among them, a is the physical mixture of HP-β-CD and CX-E extract paste, b is CX-E, c is HP-β-CD, and d is the CX-E inclusion complex;

[0026] Attached Figure 4 Infrared spectrum analysis diagrams of HP-β-CD, CX-E, physical mixture of HP-β-CD and CX-E, and CX-E inclusion complex in Test Example 1, where a is HP-β-CD, b is CX-E, c is the physical mixture of HP-β-CD and CX-E, and d is the CX-E inclusion complex;

[0027] Attached Figure 5 Cumulative dissolution curves of the CX-E inclusion complex, physical mixture of HP-β-CD and CX-E, and CX-E raw material drug in Test Example 1. Specific implementation mode

[0028] The preparation method of the ethanol extract of Ligusticum chuanxiong used in the examples of the present invention is as follows: Weigh 800 g of Ligusticum chuanxiong and put it into a 10000 mL round-bottomed flask, add 8 times the amount of water, and extract by steam distillation for 10 h. The water extract of Ligusticum chuanxiong is rotary-evaporated and concentrated to a crude drug content of 0.1 g / mL, and 95% ethanol is added under stirring until the ethanol content reaches 80%. Let it stand overnight for sedimentation, centrifuge at 4000 r / min for 5 min, separate the supernatant and the lower precipitate, and the supernatant is the ethanol precipitation solution. Take the residue of the cut crude drug and continue to extract it by heating under reflux with 5 times the amount of 70% ethanol to obtain an ethanol extract. Filter and combine the ethanol precipitation solution and the ethanol extract, recover ethanol under reduced pressure, and concentrate to 0.5 g / mL (calculated by crude drug content). Then purify the concentrated solution with HPD100 macroporous resin. First, elute with 1000 ml of pure water, and then elute with 1000 ml of ethanol. Collect the ethanol eluate, recover ethanol under reduced pressure, and evaporate it to dryness in an evaporating dish to obtain about 10 g of the ethanol extract of Ligusticum chuanxiong, denoted as CX-E.

[0029] In the embodiments of the present invention, the calculation formulas for the inclusion rate and the yield are as follows:

[0030]

[0031]

[0032] According to the applicant's previous component analysis of CX-E, it was determined that phenolic acid components are the main components in CX-E. Therefore, in the embodiments of the present invention, the method for measuring total phenolic acids was used to investigate the inclusion rate. The specific measurement method is as follows:

[0033] Preparation of the reference solution: Weigh an appropriate amount of ferulic acid reference substance accurately, dissolve it in ethanol, and prepare a ferulic acid solution with a concentration of 0.663 mg / mL. Dilute the reference solution to obtain reference solutions with different mass concentrations.

[0034] Preparation of the test solution: Weigh 8 mg of CX-E accurately, dissolve it in ethanol and make up the volume to 10 mL.

[0035] Establishment of the standard curve: Accurately pipette 0.5 mL of the diluted reference solutions with different concentrations respectively, add 4.5 mL of ethanol, then add 2 mL of 0.3% sodium dodecyl sulfate (SDS) solution and 2 mL of a uniformly mixed solution of 0.5% potassium ferricyanide - 1.0% ferric chloride (1:1) respectively, shake well, place in the dark for 5 min, add 0.1 mol / L hydrochloric acid solution to the scale, shake well, and measure the absorbance at a wavelength of 705 nm. Take the reference substance mass concentration as the abscissa and the absorbance as the ordinate, and calculate the regression equation.

[0036] Take the test solution, develop the color in the same way and measure the absorbance at 705 nm. Substitute the measured absorbance into the standard curve to calculate the total phenolic acid content in CX-E.

[0037] Example 1

[0038] This example discloses a screening test for the inclusion method. The ethanol extract of Ligusticum chuanxiong was included by the saturated aqueous solution method, the grinding method and the ultrasonic method respectively. Specifically:

[0039] 1. Saturated aqueous solution method: Add 60℃ pure water to HP-β-CD to prepare a saturated solution of HP-β-CD, weigh CX-E and dissolve CX-E with anhydrous ethanol to prepare a CX-E ethanol solution with a concentration of 0.1g / ml, wherein the weight ratio of HP-β-CD to CX-E is 10:1. At a speed of 300r / min, slowly drip the CX-E solution into the saturated aqueous solution of HP-β-CD and stir for 2h, then place it in a refrigerator for 24h, take it out, filter it, wash it with water, and then place it in a vacuum drying oven to dry it for 24h, grind it through an 80-mesh sieve to obtain a crude inclusion compound. Then wash the crude inclusion compound with anhydrous ethanol and dry it at low temperature to obtain a light yellow loose CX-E inclusion compound powder.

[0040] 2 Grinding method: Add HP-β-CD and an appropriate amount of pure water to a mortar and grind thoroughly, then slowly drip CX-E dissolved in anhydrous ethanol to obtain a CX-E ethanol solution with a concentration of 0.1g / ml, wherein the weight ratio of HP-β-CD to CX-E is 10:1. Grind into a paste and filter it, place it in a vacuum drying oven to dry for 24 hours, then wash it with water and place it in a vacuum drying oven to dry for 24 hours. Grind it through an 80-mesh sieve to obtain a crude inclusion compound. Wash the crude inclusion compound with anhydrous ethanol and dry it at low temperature to obtain a light yellow loose CX-E inclusion compound powder.

[0041] 3 Ultrasonic method: Add 60℃ pure water to HP-β-CD to prepare a saturated solution of HP-β-CD, place the saturated solution under 60℃ ultrasonic conditions, and then slowly drop CX-E dissolved in anhydrous ethanol to prepare a CX-E ethanol solution with a concentration of 0.1g / ml, wherein the feed weight ratio of HP-β-CD to CX-E is 10:1. After ultrasonication for 2h, refrigerate in a refrigerator for 24h, then filter and wash with water, then place it in a vacuum drying oven to dry for 24h, grind it through an 80-mesh sieve to obtain a crude inclusion compound. Then wash the crude inclusion compound with anhydrous ethanol and dry it at low temperature to obtain a light yellow loose CX-E inclusion compound powder.

[0042] The results are shown in the following table:

[0043] Table 1 Comparison results of inclusion methods

[0044]

[0045]

[0046] As shown in Table 1, the inclusion rate and yield of the CX-E inclusion compound prepared by the saturated aqueous solution method are the highest. Therefore, the saturated aqueous solution method is selected as the preparation method of the CX-E inclusion compound.

[0047] Test Example 1

[0048] The properties of the CX-E inclusion complex prepared by the saturated aqueous solution method according to Example 1 were investigated.

[0049] 1. The appearance of the CX-E inclusion complex is as shown in d in the appendix. In addition, in the appendix, Figure 1 a is the CX-E thick paste, B is HP-β-CD, and c is the physical mixture of HP-β-CD and the CX-E thick paste. As can be seen from the appendix Figure 1 Figure 1 , the CX-E inclusion complex sample after being included by HP-β-CD is a delicate and uniform light yellow powder. Compared with CX-E, the inclusion complex improves its viscous characteristics; the powder of the physical mixture of HP-β-CD and CX-E is uneven in thickness and color, and when it is used to prepare tablets, there will be problems such as pitted tablets and loose tablets, which is not conducive to the preparation of tablets. Therefore, it is necessary to use the inclusion complex prepared by the water-saturated solution method as an intermediate for tablets.

[0050] 2. The optical micrographs of HP-β-CD, the physical mixture of HP-β-CD and the CX-E thick paste, and the CX-E inclusion complex are as shown in the appendix Figure 2 As can be seen from the appendix Figure 2 , no physical mixture appears in the inclusion complex, and it can be clearly seen that a new phase has been formed.

[0051] 3. The DSC analysis diagrams of the CX-E thick paste, HP-β-CD, the physical mixture of HP-β-CD and the CX-E thick paste, and the CX-E inclusion complex are as shown in the appendix Figure 3 As can be seen from the Figure 3 analysis, both curve a of the physical mixture and curve b of CX-E have an exothermic peak 1 and 3 at about 230 °C, and this peak does not appear in the chuanxiong inclusion complex. Since the melting point of cyclodextrin is about 332.49 °C, it can be determined that the exothermic peak of peak 4 in curve c is the melting point peak of HP-β-CD. And there is a large exothermic peak in the chuanxiong inclusion complex at 310 - 350 °C in curve d, indicating that after cyclodextrin completely includes CX-E, its exothermic peak moves to the melting point of cyclodextrin. In addition, there is also an exothermic peak 2 of cyclodextrin in curve a of the physical mixture, indicating that the physical mixture only simply mixes CX-E and cyclodextrin, and the inclusion effect of this product is not a simple physical mixture.

[0052] 4. The infrared spectroscopy analysis diagrams of the CX-E thick paste, HP-β-CD, the physical mixture of HP-β-CD and the CX-E thick paste, and the CX-E inclusion complex are as shown in the appendix Figure 4 As can be seen from the appendix Figure 4 ​​It can be seen that the characteristic peak of the carbonyl group of CX-E at 1753.29 cm-1 has disappeared or significantly weakened after inclusion, while it still exists in the physical mixture, indicating that CX-E enters the cavity of HP-β-CD and its infrared vibration is restricted. The characteristic peak of the inclusion complex is similar to that of HP-β-CD, and the stretching vibration of the multi-associated hydroxyl group appears at 3388.93 cm-1; the overall infrared absorption characteristics of the physical mixture are similar to those of HP-β-CD. The absorption peak at 1751.36 cm-1 in the physical mixture is the characteristic peak of CX-E, indicating that its infrared spectrum is composed of the sum of the infrared spectrum of HP-β-CD with strong absorption and the infrared spectrum of CX-E with weak absorption.

[0053] 5. Determination results of the dissolution rate of the CX-E inclusion complex

[0054] According to the second method of the paddle method in General Chapter 0931 of the Fourth Part of the Chinese Pharmacopoeia (2020 Edition), take 6 portions each of CX-E 100 mg, the CX-E inclusion complex, and the CX-E / HP-β-CD physical mixture, accurately weigh them, and use 900 mL of pure water as the dissolution medium. Set the rotation speed to 100 r / min and the temperature to (37 ± 0.5) °C. At 2, 4, 8, 16, 30, and 60 min, respectively, draw 5 mL of the dissolution medium (while supplementing the same amount of medium at the same temperature). Filter the drawn medium, take the subsequent filtrate with the dissolution medium as the blank, determine the total phenolic acid content, calculate the dissolution rate, and draw the cumulative dissolution curve.

[0055] The results are as Figure 5 shown. The in vitro dissolution of the CX-E inclusion complex of the present invention is significantly accelerated. The cumulative dissolution amount of the inclusion complex at 60 min is about 2.6 times that of the CX-E raw material drug.

[0056] Example 2

[0057] This example discloses a screening test for the feeding amounts of HP-β-CD and CX-E. Compared with the preparation method of the saturated aqueous solution method in Example 1, the feeding mass ratio of HP-β-CD and CX-E is different, and the other conditions are the same. The results are shown in the following table:

[0058] Table 2 Investigation results of the feeding weight ratio

[0059] HP-β-CD: CX-E Inclusion rate (%) Yield rate (%) 1∶1 46.62 46.41 5∶1 53.95 52.19 10∶1 64.55 62.14 15∶1 60.98 59.58 20∶1 49.57 32.23

[0060] As can be seen from Table 2, when the feeding mass ratio of HP-β-CD and CX-E is 10:1, the inclusion rate and the recovery rate are the highest. Therefore, the feeding weight ratio of the inclusion complex is selected as 10:1.

[0061] Example 3

[0062] This example discloses an investigation experiment on the inclusion time. Compared with the preparation method of the saturated aqueous solution method in Example 1, the inclusion time is different, and the other conditions are the same. The results are shown in the following table:

[0063] Table 3 Investigation results of inclusion time

[0064]

[0065]

[0066] The results are shown in Table 3. When the inclusion time is 2.5 h, the yield is the largest. When the inclusion time is 2 h, the inclusion rate is relatively high. According to the comprehensive scoring results and considering the time cost issue, the inclusion time of the CX-E clathrate is therefore selected as 2 h.

[0067] Example 4

[0068] This example discloses an investigation experiment on the inclusion temperature. Compared with the preparation method of the saturated aqueous solution method in Example 1, the inclusion temperature is different, and the other conditions are the same. The results are shown in the following table:

[0069] Table 4 Investigation results of inclusion temperature

[0070] Inclusion temperature (°C) Inclusion rate (%) Yield rate (%) 30 22.49 20.19 40 41.05 42.82 50 58.14 60.22 60 70.05 69.98 70 68.37 65.21

[0071] The results are shown in Table 4. It can be seen from Table 4 that when the inclusion temperature is 60 °C and 70 °C, the inclusion rate and yield of the clathrate are both relatively large. However, it was found during the preparation process that when the temperature is too high, the sample is easily dried in the pot. Considering comprehensively, the inclusion temperature of the CX-E clathrate is selected as 60 °C.

[0072] Example 5

[0073] This example discloses an investigation experiment on the stirring speed. Compared with the preparation method of the saturated aqueous solution method in Example 1, the stirring speed is different, and the other conditions are the same. The results are shown in the following table:

[0074] Table 5 Investigation results of stirring speed

[0075] Stirring speed (r / min) Inclusion rate (%) Yield rate (%) 100 52.13 50.04 200 64.99 62.57 300 70.24 71.08

[0076] The results are shown in Table 5. It can be seen from Table 5 that the higher the stirring speed, the higher the inclusion rate and yield of the clathrate. Therefore, the stirring speed selected for the CX-E clathrate is 300 r / min.

[0077] Example 6

[0078] This example discloses a screening experiment on the amount of sodium chloride added. Compared with the preparation method of the saturated aqueous solution method in Example 1, after the inclusion is completed, sodium chloride is added and dissolved, and then refrigerated. The other conditions are the same. The results are shown in the following table:

[0079] Investigation Results of Sodium Chloride Dosage in Table 6

[0080] Sodium chloride concentration in inclusion solution (mg / ml) Inclusion rate (%) Yield rate (%) 0 75.81 76.95 0.1 76.37 78.29 0.2 76.65 83.53 0.3 76.09 84.09 0.5 75.52 82.14

[0081] As can be seen from the above table, after adding sodium chloride, it has no effect on the inclusion rate of the ethanol extract of Ligusticum chuanxiong, but can significantly improve the yield of the inclusion compound.

[0082] Example 7

[0083] Take the CX-E inclusion compound prepared by the saturated aqueous solution method in Example 1 to prepare tablets. The prescription is 61 wt.% of the inclusion compound, 38 wt.% of lactose. After mixing the powders evenly, wet granulation is carried out by adding 85% ethanol under a 20-mesh sieve, drying at low temperature, then adding 1 wt.% of magnesium stearate, and uniformly mixing and then tabletting to obtain the tablets.

[0084] It was found that the appearance, weight variation of tablets, friability, and disintegration time limit all met the requirements of the pharmacopoeia, and the hardness was moderate. There was no sticking phenomenon during the preparation process. It shows that using the inclusion method of the present invention for CX-E can obtain tablets with excellent quality.

[0085] The above embodiments are only one of the preferred embodiments of the present invention and should not be used to limit the protection scope of the present invention. Any modification or polishing that has no substantial meaning made on the main design concept and spirit of the present invention, as long as the technical problems solved are still the same as those of the present invention, should be included within the protection scope of the present invention.

Claims

1. A method for inclusion of the ethanol extract of Ligusticum chuanxiong Hort., characterized in that, It includes the following steps: Under stirring conditions, add the ethanol solution of the ethanol extract of Ligusticum chuanxiong to the saturated solution of hydroxypropyl-β-cyclodextrin for inclusion, refrigerate, filter, and dry at low temperature to obtain the inclusion complex; among them, after the inclusion is completed, add a small amount of sodium chloride to the solution, and after dissolution is completed, refrigerate again. After adding sodium chloride, the content of sodium chloride in the solution is 0.2 - 0.3 mg / ml.

2. The inclusion method according to claim 1, characterized in that, The mass ratio of hydroxypropyl-β-cyclodextrin to the ethanol extract of Ligusticum chuanxiong is 1 - 20:

1.

3. The inclusion method according to claim 1, characterized in that, The mass ratio of hydroxypropyl-β-cyclodextrin to the ethanol extract of Ligusticum chuanxiong is 5 - 15:

1.

4. The inclusion method according to claim 1, characterized in that, The mass ratio of hydroxypropyl-β-cyclodextrin to the ethanol extract of Ligusticum chuanxiong is 10:

1.

5. The inclusion method according to claim 1, characterized in that The inclusion temperature is 30 - 70 °C.

6. The inclusion method according to claim 1, characterized in that, The inclusion temperature is 60 °C.

7. The inclusion method according to claim 1, characterized in that The inclusion time is 0.5 - 2.5 hours.

8. The inclusion method according to claim 1, characterized in that, The inclusion time is 2 hours.

9. The inclusion method according to claim 1, characterized in that, After the inclusion is completed, place the solution at 2 - 6 °C and refrigerate for 12 - 24 hours.

10. The inclusion method according to any one of claims 1-9, characterized in that The preparation method of the ethanol extract of Ligusticum chuanxiong is as follows: After adding water to the Ligusticum chuanxiong medicinal materials and heating to extract the volatile oil, filter to obtain the water extract and the medicinal residues; Among them, add ethanol to the water extract for precipitation and filtration to obtain the ethanol precipitation solution; extract the medicinal residues with ethanol to obtain the ethanol extract; combine the ethanol precipitation solution and the ethanol extract, and evaporate the solvent to obtain the ethanol extract of Ligusticum chuanxiong.

11. The inclusion method according to claim 10, characterized in that, The concentration of the ethanol extract of Ligusticum chuanxiong in the ethanol solution of the ethanol extract of Ligusticum chuanxiong is 0.05 - 1 g / ml.

12. The inclusion method according to claim 10, wherein The concentration of the ethanol extract of Ligusticum chuanxiong in the ethanol solution of the ethanol extract of Ligusticum chuanxiong is 0.1 g / ml.

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