A stable and high drug loading pharmaceutical for treating axial spondyloarthritis and a preparation method thereof

Abstract

This invention discloses a method for preparing a stable and high-drug-loading drug for treating axial spondyloarthritis, which is made from the following traditional Chinese medicine raw materials in parts by weight: 5-20 parts Eucommia ulmoides, 5-20 parts Angelica pubescens, 5-15 parts processed Aconitum carmichaelii, 5-20 parts Corydalis yanhusuo, 5-15 parts Paeonia lactiflora, 5-15 parts Pueraria lobata, and 3-10 parts Glycyrrhiza uralensis. The method is characterized by the following steps: 1) extracting Angelica pubescens, Eucommia ulmoides, and Corydalis yanhusuo with ethanol to obtain an alcohol extract; 2) extracting processed Aconitum carmichaelii, Glycyrrhiza uralensis, Paeonia lactiflora, and Pueraria lobata with water extraction and alcohol precipitation to obtain an aqueous extract. The extract obtained by the method of this invention has a more stable and comprehensive therapeutic effect, and the prepared traditional Chinese medicine preparation has a higher drug loading and good disintegration properties.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, specifically to a stable and high-drug-loading drug for treating axial spondyloarthritis and its preparation method. Background Technology

[0002] The predecessor of Jitongning tablets was a clinical preparation developed by the General Hospital of the Chinese People's Liberation Army, initially in capsule form. Now, it has been developed into tablets by Tasly Pharmaceutical Group using modern traditional Chinese medicine formulation methods. The formula consists of seven medicinal herbs: kudzu root, red peony root, licorice root, prepared aconite root, angelica root, eucommia bark, and corydalis rhizome. It has the effects of tonifying the liver and kidneys, dispelling wind and dampness, and promoting blood circulation. Clinically, it is mainly used to treat axial spondylitis and is currently undergoing phase III clinical trials. In its production process, alcohol precipitation is a crucial purification step that plays a vital role in ensuring the stability of the intermediate quality. Alcohol precipitation alters the solubility of the drug solution, thereby retaining the active ingredients and removing ineffective components, aiming to improve the safety and efficacy of the drug.

[0003] CN1301729C discloses a pharmaceutical preparation for treating ankylosing spondylitis, comprising a drug composition of seven herbs: Eucommia ulmoides, Angelica pubescens, processed Aconitum carmichaelii, Corydalis yanhusuo, Paeonia lactiflora, Pueraria lobata, and Glycyrrhiza uralensis. Six of the herbs are extracted with ethanol and concentrated to obtain an extract, which is then mixed with finely powdered Pueraria lobata to obtain the finished product. It is generally in capsule form, but the drug has poor stability, and the daily dosage is about 6g / day. Therefore, there is an urgent need to develop a pharmaceutical preparation for treating axial spondyloarthritis with high stability and high drug loading capacity. Summary of the Invention

[0004] To address the aforementioned issues, this invention optimizes the production process by using ethanol extraction of Angelica pubescens, Eucommia ulmoides, and Corydalis yanhusuo, and water extraction followed by alcohol precipitation of Aconitum carmichaelii, Glycyrrhiza uralensis, Paeonia lactiflora, and Pueraria lobata. This new process yields extracts with more stable and comprehensive medicinal effects. Furthermore, the herbal extracts are combined with microcrystalline cellulose, sodium carboxymethyl starch, and magnesium stearate, and then processed through fluidized bed granulation, tableting, and coating to obtain a finished formulation with better stability and higher drug loading compared to traditional capsules.

[0005] This invention claims protection for the following technical solutions:

[0006] A method for preparing a stable and high-drug-load drug for treating axial spondyloarthritis, comprising the following parts by weight of traditional Chinese medicine raw materials: 5-20 parts Eucommia ulmoides, 5-20 parts Angelica pubescens, 5-15 parts processed Aconitum carmichaelii, 5-20 parts Corydalis yanhusuo, 5-15 parts Paeonia lactiflora, 5-15 parts Pueraria lobata, and 3-10 parts Glycyrrhiza uralensis, characterized in that the preparation method comprises the following steps: 1) extracting Angelica pubescens, Eucommia ulmoides, and Corydalis yanhusuo with ethanol to obtain an alcohol extract; 2) extracting processed Aconitum carmichaelii, Glycyrrhiza uralensis, Paeonia lactiflora, and Pueraria lobata with water extraction and alcohol precipitation to obtain an aqueous extract.

[0007] The preparation of the aqueous extract in step 1 includes the following steps: processed aconite root, licorice root, red peony root, and kudzu root are placed in an extraction tank, water is added for extraction, the mixture is filtered, and the aqueous extract is concentrated to a relative density of 1.06-1.15; ethanol is added to a concentration of 65-75% for alcohol precipitation, the mixture is allowed to stand, filtered, and concentrated to obtain the aqueous extract. Preferably, the aqueous extract is concentrated to a relative density of 1.08-1.12.

[0008] The concentration temperature of the aqueous extract in step 1 is controlled below 80°C, and the concentration method is vacuum concentration.

[0009] The preparation of the aqueous extract in step 1 includes the following steps: Prepared Aconitum carmichaelii, licorice root, Paeonia lactiflora, and Pueraria lobata are placed in an extraction tank, decocted twice with water for 2 hours each time, filtered, and the aqueous extract is concentrated under reduced pressure to a relative density of 1.08-1.12; ethanol is added to a concentration of 68-73% for alcohol precipitation, the mixture is allowed to stand, filtered, and the supernatant is concentrated to a relative density of 1.25-1.30 to obtain the aqueous extract. Preferably, the alcohol precipitation step involves adding ethanol to a concentration of 70%.

[0010] The preparation of the alcohol extract in step 2 includes the following steps: Eucommia ulmoides, Angelica pubescens and Corydalis yanhusuo are put into an extraction tank, 65-85% ethanol is added for extraction, filtered, and the filtrate is concentrated under reduced pressure to a relative density of 1.25-1.30 to obtain the alcohol extract.

[0011] The preparation of the alcohol extract in step 1 includes the following steps: Eucommia ulmoides, Angelica pubescens, and Corydalis yanhusuo are placed in an extraction tank, and extracted twice with 6 times the amount of 74-76% ethanol, each time for 2 hours. The mixture is then filtered, and the filtrate is concentrated under reduced pressure to a relative density of 1.25-1.30. The extract is collected to obtain the alcohol extract. Preferably, the ethanol is 75% ethanol.

[0012] The present invention further provides a pharmaceutical composition prepared using the preparation method of the present invention, comprising a pharmaceutically active substance and a pharmaceutical carrier, wherein the pharmaceutically active substance is an alcohol extract and a water extract prepared according to the method described above.

[0013] The aqueous and alcoholic extracts of the active pharmaceutical ingredients constitute 50-75% of the pharmaceutical composition by weight, with the remainder being a drug carrier. Preferably, the drug carrier is selected from sodium carboxymethyl starch, magnesium stearate, and microcrystalline cellulose, and their weight percentages in the pharmaceutical composition are: sodium carboxymethyl starch 5%, magnesium stearate 0.5%, and microcrystalline cellulose 19.5-44.5%.

[0014] Most preferably, the weight percentages of each component in the pharmaceutical composition are: 65% for the aqueous extract and alcoholic extract of the active pharmaceutical ingredient, 5% for sodium carboxymethyl starch, 0.5% for magnesium stearate, and 29.5% for microcrystalline cellulose.

[0015] The present invention further provides a method for preparing the pharmaceutical composition of the present invention, characterized in that the aqueous extract and alcohol extract are taken, sodium carboxymethyl starch and microcrystalline cellulose are added internally, granulated, dried, and then sodium carboxymethyl starch and magnesium stearate are added externally, tableted, and coated with a film; wherein the weight ratio of internally added sodium carboxymethyl starch to externally added sodium carboxymethyl starch is 3:2.

[0016] The present invention has the following beneficial effects:

[0017] 1. Eucommia ulmoides, Angelica pubescens, Corydalis yanhusuo, and processed Aconitum carmichaelii were extracted with ethanol, while Paeonia lactiflora, Glycyrrhiza uralensis, and Pueraria lobata were extracted with water and precipitated with alcohol. The anti-inflammatory and analgesic effects of the extracts were relatively stable and comprehensive.

[0018] 2. The yield of the ethanol extract prepared from Eucommia ulmoides, Angelica pubescens, Corydalis yanhusuo, and processed Aconitum carmichaelii showed good parallelism with the transfer rate of osthol, and the process was stable and controllable.

[0019] 3. The preparation of water extract from processed Aconitum carmichaelii, licorice, Paeonia lactiflora and Pueraria lobata by water extraction and alcohol precipitation has good parallelism in terms of extract yield and paeoniflorin transfer rate, and the process is stable and controllable;

[0020] 4. The pharmaceutical composition tablets containing the above-mentioned aqueous extract and alcohol extract disclosed in this invention, after testing, have high drug loading, good appearance, hardness, disintegration and brittleness, and good stability compared to the spinal pain relief capsules. Attached Figure Description

[0021] Figure 1 The effect of concentration temperature of alcohol extract on paeoniflorin content;

[0022] Figure 2 The effect of concentration temperature of water extract on osthol content;

[0023] Figure 3 The properties of the tablets containing the composition of the present invention before and after accelerated stability testing (left: 0 months, right: 6 months).

[0024] Figure 4 Characteristics of Jitongning capsules before and after accelerated stability testing (left: 0 months, right: 6 months) Detailed Implementation

[0025] The present invention is further illustrated by the following examples, but these are not intended to limit the invention.

[0026] Unless otherwise specified, the reagents, methods and equipment used in this invention are conventional reagents, methods and equipment in this technical field.

[0027] Example 1: A method for preparing a drug for treating axial spinal arthritis.

[0028] Prescription: Eucommia ulmoides 600g, Angelica pubescens 600g, Corydalis yanhusuo 600g, processed Aconitum carmichaelii 400g, Paeonia lactiflora 400g, Pueraria lobata 400g, Glycyrrhiza uralensis 240g

[0029] Water extraction process: Licorice root, red peony root, kudzu root, and processed aconite root are added to an extraction tank with 10 times the amount of water and extracted for 2 hours, then filtered. The residue is then subjected to a second extraction with 10 times the amount of water added and extracted for 2 hours, then filtered and discarded. The filtrate is concentrated under reduced pressure to a relative density of 1.06–1.12 (60±1℃); ethanol is added to a concentration of 70%, and stirring is continued for 30 minutes, followed by standing for 12–24 hours; the clear liquid is collected and filtered; the ethanol is recovered from the filtrate, and the mixture is concentrated under reduced pressure to a relative density of 1.25–1.30 (60±1℃), then filtered and collected as a paste.

[0030] Alcohol extraction extract production process: Eucommia ulmoides, Angelica pubescens, and Corydalis yanhusuo are put into an extraction tank, 6 times the amount of 75% ethanol is added, and the mixture is extracted for 2 hours. After filtration, the residue is subjected to a second extraction, 6 times the amount of 75% ethanol is added, and the mixture is extracted for 2 hours. After filtration, the residue is discarded. The filtrate is concentrated under reduced pressure to a relative density of 1.25-1.30 (60±1℃), filtered, and the extract is collected.

[0031] Take the two extracts mentioned above, add 3% sodium carboxymethyl starch and an appropriate amount of microcrystalline cellulose to each prescription, granulate, dry, add 2% sodium carboxymethyl starch and 0.5% magnesium stearate to each prescription, make 1000 tablets, and coat them with a film.

[0032] Example 2: A method for preparing a drug for treating axial spinal arthritis.

[0033] Prescription: Eucommia ulmoides 500g, Angelica pubescens 500g, Corydalis yanhusuo 500g, processed Aconitum carmichaelii 500g, Paeonia lactiflora 500g, Pueraria lobata 500g, Glycyrrhiza uralensis 300g

[0034] Water extraction process: Licorice root, red peony root, kudzu root, and processed aconite root are added to an extraction tank with 10 times the amount of water and extracted for 2 hours, then filtered. The residue is then subjected to a second extraction with 10 times the amount of water added and extracted for 2 hours, then filtered and discarded. The filtrate is concentrated under reduced pressure to a relative density of 1.06–1.12 (60±1℃); ethanol is added to a concentration of 75%, and stirring is continued for 30 minutes, followed by standing for 12–24 hours; the clear liquid is collected and filtered; the ethanol is recovered from the filtrate, and the mixture is concentrated under reduced pressure to a relative density of 1.25–1.30 (60±1℃), then filtered and collected as a paste.

[0035] Alcohol extraction extract production process: Eucommia ulmoides, Angelica pubescens, and Corydalis yanhusuo are put into an extraction tank, 6 times the amount of 65% ethanol is added, and the mixture is extracted for 2 hours. After filtration, the residue is subjected to a second extraction, 6 times the amount of 65% ethanol is added, and the mixture is extracted for 2 hours. After filtration, the residue is discarded. The filtrate is concentrated under reduced pressure to a relative density of 1.25-1.30 (60±1℃), filtered, and the extract is collected.

[0036] Take the two extracts mentioned above, add 3% sodium carboxymethyl starch and an appropriate amount of microcrystalline cellulose to each prescription, granulate, dry, add 2% sodium carboxymethyl starch and 0.5% magnesium stearate to each prescription, make 1000 tablets, and coat them with a film.

[0037] Example 3: A method for preparing a drug for treating axial spinal arthritis.

[0038] Prescription: Eucommia ulmoides 600g, Angelica pubescens 600g, Corydalis yanhusuo 600g, processed Aconitum carmichaelii 450g, Paeonia lactiflora 450g, Pueraria lobata 450g, Glycyrrhiza uralensis 300g

[0039] Water extraction process: Licorice root, red peony root, kudzu root, and processed aconite root are added to an extraction tank with 10 times the amount of water and extracted for 2 hours, then filtered. The residue is then subjected to a second extraction with 10 times the amount of water added and extracted for 2 hours, then filtered and discarded. The filtrate is concentrated under reduced pressure to a relative density of 1.08–1.12 (60±1℃); ethanol is added to a concentration of 70%, and stirring is continued for 30 minutes, followed by standing for 12–24 hours; the clear liquid is collected and filtered; the ethanol is recovered from the filtrate, and the mixture is concentrated under reduced pressure to a relative density of 1.25–1.30 (60±1℃), then filtered and collected as a paste.

[0040] Alcohol extraction extract production process: Eucommia ulmoides, Angelica pubescens, and Corydalis yanhusuo are put into an extraction tank, 6 times the amount of 85% ethanol is added, and the mixture is extracted for 2 hours. After filtration, the residue is subjected to a second extraction, 6 times the amount of 85% ethanol is added, and the mixture is extracted for 2 hours. After filtration, the residue is discarded. The filtrate is concentrated under reduced pressure to a relative density of 1.25-1.30 (60±1℃), filtered, and the extract is collected.

[0041] Take the two extracts mentioned above, add 3% sodium carboxymethyl starch and an appropriate amount of microcrystalline cellulose to each prescription, granulate, dry, add 2% sodium carboxymethyl starch and 0.5% magnesium stearate to each prescription, make 1000 tablets, and coat them with a film.

[0042] Example 1: Determination of the process route

[0043] 1. Process route design

[0044] Based on the chemical composition of each herb in the formula, an extraction process route was designed, and the following four routes were selected for process screening.

[0045] Route 1: Ethanol extraction of Eucommia ulmoides, Angelica pubescens, Corydalis yanhusuo, and processed Aconitum carmichaelii; water extraction and alcohol precipitation of Paeonia lactiflora, Glycyrrhiza uralensis, and Pueraria lobata.

[0046] Design concept: The active ingredients in Eucommia ulmoides, Angelica pubescens, Corydalis yanhusuo, and processed Aconitum carmichaelii have good alcohol solubility, and ethanol can be used to extract them to the maximum extent. Therefore, ethanol extraction is adopted for these four herbs. Paeoniflorin in Paeonia lactiflora has good water solubility, and the main component of Pueraria lobata, puerarin, has good water and alcohol solubility and contains a large amount of starch. Water extraction and alcohol precipitation can effectively remove starch and retain the active ingredient puerarin. Therefore, Paeonia lactiflora, Pueraria lobata, and Glycyrrhiza uralensis are decocted in water and then subjected to alcohol precipitation.

[0047] Route 2: Ethanol extraction of Eucommia ulmoides, Angelica pubescens, and Corydalis yanhusuo; water extraction and alcohol precipitation of Aconitum carmichaelii, Glycyrrhiza uralensis, Paeonia lactiflora, and Pueraria lobata.

[0048] Design concept: The active ingredients in Eucommia ulmoides, Angelica pubescens, and Corydalis yanhusuo are highly soluble in alcohol, and ethanol can extract them to the maximum extent. Therefore, ethanol extraction is used for these three herbs. The processed Aconitum carmichaelii in the formula mainly contains aconitine alkaloids, which are both active and toxic components and must be controlled. Traditional Chinese medicine theory considers licorice... [25,26] Since peony root and aconite root can be decocted together to reduce the toxicity of aconite root, licorice root, red peony root, and processed aconite root are decocted together. Puerarin, the main component of kudzu root, has good water and alcohol solubility, and it contains a large amount of starch. Water extraction and alcohol precipitation can effectively remove starch components and retain the effective components of puerarin. Therefore, the process design involves water extraction and alcohol precipitation of four herbs: processed aconite root, kudzu root, red peony root, and licorice root.

[0049] Route 3: Kudzu root powder is used as medicine, while the other 6 ingredients are extracted with ethanol.

[0050] Design concept: The components of Eucommia ulmoides, Angelica pubescens, Corydalis yanhusuo, Paeonia lactiflora, processed Aconitum carmichaelii, and Glycyrrhiza uralensis have good water and alcohol solubility. Ethanol can extract their effective components to the maximum extent. Therefore, ethanol extraction technology was adopted for these six herbs. Kudzu root has a strong powdery nature. It was considered that kudzu root could be pulverized and used as raw powder in the medicine. This can replace some of the excipients, thereby reducing the amount of excipients used in the preparation.

[0051] Route 4: Water extract of the whole formula of Eucommia ulmoides, Angelica pubescens, Corydalis yanhusuo, processed Aconitum carmichaelii, Paeonia lactiflora, Pueraria lobata, and Glycyrrhiza uralensis.

[0052] Design concept: Following the actual clinical practice of clinical experience formulas, the whole formula is extracted by water.

[0053] 3.1. Process route sample preparation

[0054] Samples were prepared for process screening according to the above four process routes, and drug extracts were obtained respectively, and were denoted as: JTN-1 (equivalent to 5.23g crude drug / g powder), JTN-2 (equivalent to 6.11g crude drug / g powder), JTN-3 (equivalent to 3.77g crude drug / g powder), and JTN-4 (equivalent to 4.05g crude drug / g powder).

[0055] 3.2. Screening and Determination of Process Route

[0056] 3.2.1. Experimental Purpose

[0057] To investigate the effects of drug extract samples from different extraction processes on the number of writhing responses induced by acetic acid in mice and the effects on paw swelling in rats caused by carrageenan, so as to screen and determine the optimal extraction process route.

[0058] 3.2.2. Experimental Materials

[0059] Test drug: Drug extract (JTN), information on the equivalent amount of crude drug: JTN-1 (5.23 g of crude drug / g of powder), JTN-2 (6.11 g of crude drug / g of powder), JTN-3 (3.77 g of crude drug / g of powder), JTN-4 (4.05 g of crude drug / g of powder).

[0060] Tool drug: Glacial acetic acid: Tianjin Kaixin Chemical Industry Co., Ltd., batch number: 20090609. Dilute it with normal saline to a concentration of 0.67% before use.

[0061] Normal saline: Produced by Tianjin Jinlan Pharmaceutical Co., Ltd., batch number: 10080901.

[0062] Carrageenan suspension: Provided by Shenyang Pharmaceutical University, prepare it into a 1% concentration with distilled water on the day of use, and shake for 2 hours to form a suspension.

[0063] Materials and instruments: HSS-1B type constant temperature bath (also known as the constant temperature device for isolated intestinal tract and hot plate experiments, Chengdu Instrument Factory).

[0064] PL203 electronic balance: Mettler-Toledo Instruments (Shanghai) Co., Ltd.

[0065] T2000 electronic balance: American Double Jie Brothers (Group) Co., Ltd..

[0066] Experimental animals: ICR mice and SD rats, SPF grade, both male and female, purchased from Tianjin Shanchuanhong Experimental Animal Technology Co., Ltd., license number: SCXK(Jin)2009-0001. <00​​​​​​​​One hundred ICR mice, weighing 20-22g, were randomly divided into 10 groups of 10 mice each, with half males and half females. The drug extract groups were administered the drug extract once daily by gavage at the dosages shown in Table 2 for three consecutive days. The control group was administered the same volume of distilled water by gavage. Forty minutes after the last administration, each mouse was intraperitoneally injected with 0.2ml of 0.67% glacial acetic acid solution. The number of writhing movements per mouse within the second 10 minutes after acetic acid-induced pain was recorded using a counter. The average number of writhing movements in each treatment group was compared with the average number of writhing movements in the control group, and a statistical t-test was performed. The results (see Table 1) showed that all samples of the drug extract significantly reduced the number of writhing movements induced by acetic acid in mice, with JTN-2 and JTN-3 showing more significant analgesic effects. This indicates that the drug extract has a significant analgesic effect on chemically induced pain.

[0071] Table 1. Effects of acetic acid on the writhing response in mice (n=10)

[0072]

[0073] Compared with the control group, **P<0.01***P<0.001

[0074] 3.2.3.2. Effect of carrageenan on paw edema in rats

[0075] One hundred SD rats, weighing 200-220g, were randomly divided into 10 groups of 10 each, with half males and half females. The rats were administered the drugs as shown in Table 12-4, once daily for 4 consecutive days. The control group received the same volume of distilled water. Thirty minutes after the last administration, 0.05ml of 1% carrageenan paste was subcutaneously injected into the right hind paw pad of each rat to induce inflammation. The diameter of the right hind limb 0.5cm below the ankle joint was measured using a projector (8x magnification) before and at 1, 2, 3, 4, and 6 hours after inflammation. The difference in swelling between the pre- and post-inflammatory induction time points was used as the degree of swelling. The mean values ​​of each group were compared with the mean value of the control group, and a statistical t-test was performed. The results (see Table 2) showed that the drug extracts JTN-2 sample at doses of 3.2 and 0.8 g crude drug / kg and JTN-4 sample at doses of 3.2 g crude drug / kg could significantly inhibit carrageenan-induced paw edema in rats in this model, and had significant anti-inflammatory effects.

[0076] Table 2. Effects of carrageenan on paw edema in rats (n=10)

[0077]

[0078]

[0079] Compared with the control group, *P<0.05**P<0.01

[0080] 3.2.4. Experimental Conclusions

[0081] The results showed that: 1) all drug extracts could significantly reduce the number of writhing movements in mice induced by acetic acid, with JTN-2 and JTN-3 showing more significant analgesic effects; 2) the 3.2 and 0.8 g crude drug / kg dose groups of JTN-2 and the 3.2 g crude drug / kg dose of JTN-4 could significantly inhibit carrageenan-induced paw edema in rats, demonstrating significant anti-inflammatory effects.

[0082] Based on the above experimental results, the drug extracts of each sample have significant analgesic effects on writhing in mice and significant anti-inflammatory effects on carrageenan paw edema. Among the four samples, the JTN-2 sample has the most stable and comprehensive effects. Therefore, the process corresponding to the JTN-2 sample is selected as the optimal process route.

[0083] Experimental Example 2. Study on Extraction Process

[0084] The optimal process route, selected through pharmacodynamic comparative experiments, was used for process research. Specifically, Eucommia ulmoides, Angelica pubescens, and Corydalis yanhusuo were extracted with ethanol; Aconitum carmichaelii, Paeonia lactiflora, Pueraria lobata, and Glycyrrhiza uralensis were extracted with water and then precipitated with alcohol. For the alcohol extraction process, osthol from the principal herb Angelica pubescens was selected; and for the water extraction and alcohol precipitation process, paeoniflorin, which has a clear anti-inflammatory effect, was selected as the process evaluation indicator. The research data are as follows:

[0085] 1. Research on alcohol extraction process

[0086] This study selected osthol as the indicator for process evaluation. First, the ethanol extraction concentration was confirmed. Then, a three-factor, three-level orthogonal experiment was conducted to determine the extraction process and perform verification experiments. Through studies on concentration temperature, concentration specific gravity, etc., the ethanol extraction process was finally determined. Detailed research data are as follows:

[0087] 1.1. Selection of Indicator Components

[0088] Angelica pubescens is the principal ingredient in the formula. Modern pharmacological studies have shown that the main active ingredients in Angelica pubescens that have anti-inflammatory and analgesic effects are coumarins, among which osthol has the highest content. Therefore, osthol in Angelica pubescens was selected as the indicator ingredient for process investigation.

[0089] 1.2. Content Determination Method

[0090] The method for determining the content of osthol in Angelica pubescens under the entry for Angelica pubescens in the 2010 edition of the Pharmacopoeia of the People's Republic of China was optimized and converted into an UPLC method for determination.

[0091] The optimized method is as follows:

[0092] Using Thermo C 18A chromatographic column (100×4.6mm, 2.4μm) was used with acetonitrile-water (50:50) as the mobile phase (the ratio of the mobile phase can be finely adjusted according to the actual situation), the column temperature was 35℃, and the detection wavelength was 330nm.

[0093] 1.3. Extraction Process Research

[0094] 1.3.1. Confirmation of Ethanol Extraction Concentration

[0095] According to the proportions of the medicinal materials in the prescription, weigh out 30g of Eucommia ulmoides, 30g of Angelica pubescens, and 30g of Corydalis yanhusuo, and weigh out 5 portions in parallel. Add 8 times the amount of 55%, 65%, 75%, 85%, and 95% ethanol respectively, heat and reflux for 1 hour, filter, and record the volume of the extract.

[0096] Take an appropriate amount of the cooled extract, filter it through a 0.45 μm microporous membrane, inject the filtrate into a liquid chromatograph, determine and calculate the osthol extraction amount, see Table 3 for details.

[0097] Table 3 Confirmation of Ethanol Extraction Concentration

[0098] ethanol concentration 55％ 65％ 75％ 85％ 95％ Extraction volume (ml) 615 630 655 670 675 Osthol extract (mg) 169.9 171.1 183.8 168.1 158.1

[0099] As can be seen from Table 3, with the same amount of medicinal materials, the total amount of osthol extracted by 75% ethanol is the highest. Therefore, 75% ethanol was chosen as the extraction solvent.

[0100] 1.3.2. Orthogonal Experiment and Results

[0101] An orthogonal experimental design was used with 75% ethanol as the extraction solvent to investigate the three factors and three levels of solvent dosage, extraction time, and number of extractions. The orthogonal factor level table is shown in Table 4.

[0102] Table 4. Orthogonal Factor Level Table for Alcohol Extraction Conditions

[0103]

[0104] L9(3) is designed according to orthogonal principles. 4 An orthogonal array was used to arrange the experiment. For each experiment, 30g of Eucommia ulmoides, 30g of Angelica pubescens, and 30g of Corydalis yanhusuo were taken. Different amounts of 75% ethanol were added according to the experiment number. The extraction time and number of times were different. The extract was filtered and the volume of the extract was recorded. After the extract was filtered through a 0.45μm filter membrane, it was injected into a liquid chromatograph for determination. The extraction amount of osthol was calculated. The experimental results are shown in Table 5.

[0105] Table 5. Orthogonal Experiment Table for Alcohol Extraction Process

[0106]

[0107] Table 6. Analysis of Variance Table

[0108] Source of variance Sum of squared deviations Degrees of freedom F ratio F critical value Significance A 113.8 2 2.7 19.0 B 555.0 2 13.1 19.0 C 1947.0 2 45.9 19.0 * error 42.4 2

[0109] Results Analysis: Table 5 shows that the order of influence of each factor is C>B>A. Table 12-5 provides a direct analysis of the K values, indicating that the optimal extraction process is A3B2C2, i.e., adding 10 times the amount of 75% ethanol, extracting twice, each time for 2 hours. Table 6, the ANOVA table, shows that the number of extractions (factor C) has a significant impact on the extraction yield of osthol (P<0.05), while the solvent volume (factor A) and extraction time (factor B) have relatively small effects. Table 5 shows that the extraction yield of osthol is basically the same when extracting twice and three times. Furthermore, the K values ​​for solvent volume in Table 5 indicate that the solvent volume has a relatively small impact on the extraction yield of osthol. Considering the actual situation of large-scale production, and taking into account energy consumption and cost control, a solvent volume of 6 times the amount is preferable, i.e., A1B2C2. Therefore, the optimal extraction process is: Eucommia ulmoides, Angelica pubescens, and Corydalis yanhusuo added to 6 times the amount of 75% ethanol, extracted twice, each time for 2 hours.

[0110] 1.3.3. Verification Experiment

[0111] The experiment was conducted under the preferred extraction conditions A1B2C2 (i.e., Eucommia ulmoides, Angelica pubescens, and Corydalis yanhusuo were extracted twice with 6 times the amount of 75% ethanol for 2 hours each time). The experiment was repeated in triplicate. For each extraction, 30g of Eucommia ulmoides, 30g of Angelica pubescens, and 30g of Corydalis yanhusuo were weighed, and 540ml of 6 times the amount of 75% ethanol was added. The extraction time was 2 hours. After filtration, the residue was added to 540ml of 6 times the amount of 75% ethanol again and extracted for 2 hours. After filtration, the extracts were combined and the volumes were measured.

[0112] Take an appropriate amount of the cooled alcohol extract and pass it through a 0.45 μm organic microporous membrane. Collect the filtrate and inject it into a liquid chromatograph for analysis. Record the chromatogram and calculate the extraction amount of osthol based on the peak area. The experimental results are shown in Table 7.

[0113] Table 7. Results of the verification experiment

[0114]

[0115] As can be seen from the data in Table 7, the extraction yield of osthol in the three parallel experiments showed good parallelism and was basically consistent with the data from the orthogonal experiment. Therefore, the selected process is considered to be the optimal process, namely: Eucommia ulmoides, Angelica pubescens, and Corydalis yanhusuo are extracted twice with 6 times the amount of 75% ethanol, each time for 2 hours.

[0116] 1.4. Concentration Temperature Study

[0117] Accurately pipette 20 ml of the drug's ethanol extract into 25 ml volumetric flasks, making 9 aliquots. Place 8 aliquots separately in a water bath at 80℃ and 90℃ for 2, 4, 8, and 12 hours, respectively. Remove the aliquots, cool them, dilute to the mark with 75% ethanol, shake well, and filter through a 0.45 μm organic microporous membrane. Inject the filtrate into a liquid chromatograph for analysis, recording the chromatogram and the peak area of ​​osthol. The experimental results are shown in Table 8. Figure 1 .

[0118] Table 8 Results of Temperature Investigation in Alcohol Extraction and Concentration

[0119]

[0120] As can be seen from the data in Table 8, when the ethanol extract of the drug is kept in a water bath at 80℃ and 90℃, the peak area of ​​osthol tends to decrease with the extension of the holding time. Under the same holding time, the holding temperature has little effect on the content of osthol. However, in order to minimize the loss of drug components, it is more appropriate to control the concentration temperature of the ethanol extract below 80℃, and the concentration time should be shortened as much as possible during the production process.

[0121] Based on production experience, the extract has better properties when the specific gravity of the extract is generally between 1.25 and 1.30, which is conducive to production, storage and circulation of the extract. Therefore, the specific gravity of the extract from alcohol extraction is set at 1.25-1.30.

[0122] 1.5. Pilot-scale verification

[0123] The experiment was conducted according to the finalized extraction process: Eucommia ulmoides, Angelica pubescens, and Corydalis yanhusuo were extracted twice with 6 times the amount of 75% ethanol, each time for 2 hours. Four batches of pilot-scale ethanol extraction were carried out. The concentration temperature of the extract was controlled below 80℃, and the specific gravity of the ethanol extract was controlled between 1.25 and 1.30. The pilot-scale data are shown in Table 9.

[0124] Table 9. Pilot-scale Validation of Alcohol Extraction Process

[0125] batch number 201208079 201208080 201208083 201208085 Eucommia ulmoides (kg) 12.0 12.0 12.0 12.0 Angelica pubescens (kg) 12.0 12.0 12.0 12.0 Corydalis rhizome (kg) 12.0 12.0 12.0 12.0 Extract specific gravity (60℃) 1.260 1.257 1.271 1.250 Extract weight (kg) 8.05 7.93 8.25 8.75 Osthol content (%) 0.48％ 0.49％ 0.45％ 0.47％ Transfer rate (%) 55.5％ 5.8％ 53.3％ 59.1％ Dry yield (%) 14.4％ 14.5％ 15.6％ 15.5％

[0126] Note: The content of osthol in the medicinal herb Angelica pubescens is 0.58%.

[0127] As can be seen from the data in Table 9, when the concentration ratio of the alcohol extract is controlled at 1.25-1.30, the production process is smooth, the yield and the transfer rate of osthol are parallel, and the process is stable and controllable. Therefore, it is reasonable to control the recovery ratio of the alcohol extract at 1.25-1.30.

[0128] In summary, the alcohol extraction process is determined as follows: Eucommia ulmoides, Angelica pubescens, and Corydalis yanhusuo are heated and extracted twice with 75% ethanol. In actual industrial production, the ethanol content is not too precise, so 74-76% ethanol can be selected as the extraction solvent, each extraction lasting 2 hours. After filtration, the extract is concentrated under reduced pressure to a paste with a relative density of 1.25-1.30 (60±1℃).

[0129] 2. Research on water extraction and alcohol precipitation process

[0130] This study selected paeoniflorin as the indicator for process evaluation. An orthogonal experiment with three factors and three levels was conducted to determine the extraction process and perform verification experiments. Through studies on concentration temperature, alcohol precipitation concentration, and alcohol precipitation specific gravity, the water extraction and alcohol precipitation process was finally determined. Detailed research data are as follows:

[0131] 2.1. Selection of Indicator Components

[0132] The main component of red peony is paeoniflorin. Modern pharmacological studies have shown that paeoniflorin has anti-inflammatory and analgesic effects. The marketed product Pavlin capsules use paeoniflorin as the main component and are used to treat rheumatoid arthritis. Based on the indications of this product, paeoniflorin, which has anti-inflammatory and analgesic effects, was selected as the indicator component for investigation of the water extraction and alcohol precipitation process.

[0133] 2.2. Content determination method

[0134] Referring to the method for determining the content of paeoniflorin in the literature, it was converted into a UPLC analytical method, and the method was optimized to obtain an optimized method for determining the content of paeoniflorin.

[0135] The content determination method was used for the investigation of the water extraction and alcohol precipitation process.

[0136] 2.3. Extraction Process Research

[0137] 2.3.1. Orthogonal Experimental Design and Results

[0138] An orthogonal experimental design was used with water as the extraction solvent to investigate the three factors and three levels of orthogonal factors regarding solvent dosage, extraction time, and number of extractions. The orthogonal experimental factor level table is shown in Table 10.

[0139] Table 10. Orthogonal Factor Level Table for Water Extraction Conditions

[0140]

[0141]

[0142] L9(3) is designed according to orthogonal principles. 4An orthogonal array was used to arrange the experiment, and a total of 9 experiments were conducted. For each experiment, 20g of processed Aconitum carmichaelii, 20g of Paeonia lactiflora, 20g of Pueraria lobata, and 12g of Glycyrrhiza uralensis were used. According to the level corresponding to the experiment number, different multiples of water were added, and different extraction times and times were performed. The extract was filtered, and the volume of the extract was recorded. After the extract was cooled, it was filtered through a 0.45μm aqueous microporous membrane. The filtrate was injected into a liquid chromatograph for analysis, and the chromatogram was recorded. The extraction amount of paeoniflorin was calculated based on the peak area of ​​paeoniflorin. The experimental results are shown in Table 11.

[0143] Table 11 Orthogonal Experiment Table for Water Extraction Process

[0144]

[0145] Table 12 Analysis of Variance Table

[0146]

[0147]

[0148] Results Analysis: Table 11 shows that the order of influence of each factor is C>A>B. Intuitively, the optimal extraction process is A3B2C3, i.e., adding 10 times the amount of water, extracting twice, each time for 3 hours. Table 12, the ANOVA table, shows that the number of extractions (factor C), the solvent volume (factor A), and the extraction time (factor B) all have significant effects on the extraction yield of paeoniflorin (P<0.05). Furthermore, the K values ​​for 2 and 3 extractions in Table 11 are not significantly different. Considering the actual situation of large-scale production, and taking into account energy consumption and cost control, 2 extractions are preferable. Therefore, the optimal water extraction process is A3B2C2, i.e., adding 10 times the amount of water, extracting twice, each time for 2 hours.

[0149] 2.3.2. Verification Experiment

[0150] The experiment was conducted under the preferred extraction conditions A3B2C2, namely, 20g of prepared Aconitum carmichaelii, 20g of Paeonia lactiflora, 20g of Pueraria lobata, and 12g of Glycyrrhiza uralensis, with 10 times the amount of water (720ml), and the extraction time was 2 hours. After filtration, the residue was added to 10 times the amount of water (720ml), and the extraction was carried out for 2 hours. After filtration, the extracts were combined and the volume was measured.

[0151] Take an appropriate amount of the cooled extract, filter it through a 0.45 μm aqueous microporous membrane, collect the filtrate, and inject it into a liquid chromatograph for determination. Calculate the amount of paeoniflorin extracted. The experimental results are shown in Table 13.

[0152] Table 13 Verification Test Results

[0153]

[0154] 2.4. Concentration Temperature Study

[0155] Based on past production experience and literature reports, paeoniflorin is considered to have poor thermal stability, so we investigated the concentration temperature of the aqueous extract.

[0156] Eighteen portions of the drug's aqueous extract were transferred to 25 ml volumetric flasks. Sixteen portions were incubated in water baths at 80℃ and 90℃, respectively. Two portions were taken out at 2h, 4h, 8h, and 12h, cooled, and diluted to the mark with water. The solutions were then filtered through a 0.45 μm aqueous microporous membrane. The filtrate was injected into a liquid chromatograph for analysis, and the chromatogram and peak area of ​​paeoniflorin were recorded. The experimental results are shown in Table 14 and... Figure 2 .

[0157] Table 14 Data on water extraction and concentration temperature.

[0158]

[0159] As can be seen from the data in Table 14, when the aqueous extract of the drug was kept in a water bath at 80℃ and 90℃ for 12 hours, the peak area of ​​paeoniflorin changed little. This is inconsistent with the literature report that paeoniflorin has poor thermal stability. However, in order to reduce the loss of paeoniflorin components during the concentration process, we still controlled the concentration temperature of the aqueous extract below 80℃ and used vacuum concentration to shorten the concentration time as much as possible.

[0160] 2.5. Research on alcohol precipitation process

[0161] 2.5.1. Investigation of alcohol precipitation concentration

[0162] Based on production experience and literature reports, the alcohol precipitation effect is generally better when the concentrate before precipitation is controlled to be equivalent to 1-2g of crude drug per ml. Therefore, we conducted experiments according to the optimal extraction process, that is, adding 10 times the amount of water to processed Aconitum carmichaelii, Paeonia lactiflora, Pueraria lobata, and Glycyrrhiza uralensis, extracting twice, each time for 2 hours, and concentrating the concentrate to 1-2g of crude drug / ml.

[0163] Take 600g of processed Aconitum carmichaelii, 600g of Paeonia lactiflora, 600g of Pueraria lobata, and 360g of Glycyrrhiza uralensis. Add 10 times the amount of water and decoct twice, 2 hours each time. Filter and concentrate the filtrate under reduced pressure to obtain about 1900ml of concentrated water extract (1.14g crude drug / ml, relative density 1.085). Take 4 portions of concentrated water extract, 500g each (about 460ml), and precipitate them with alcohol to make the alcohol content 60%, 65%, 70%, and 75% respectively. After stirring evenly, let stand for more than 12 hours and measure the volume of the supernatant.

[0164] Water extract concentrate: Weigh approximately 1.2g of concentrate accurately, place it in a 20ml volumetric flask, add water to make up to volume, filter through a 0.45μm aqueous microporous membrane, and inject the filtrate into a liquid chromatograph for analysis;

[0165] Alcohol precipitation supernatant: Accurately pipette 5.0 ml of the alcohol precipitation supernatant into a 20 ml volumetric flask, dilute to the mark with 60% ethanol, shake well, filter through a 0.45 μm organic microporous membrane, and inject into the liquid chromatograph for determination. The results are shown in Table 15.

[0166] Table 15 Results of the experiment on alcohol precipitation concentration

[0167]

[0168]

[0169] *The specific gravity of the water extract concentrate is 1.085. Each alcohol precipitation sample weighs 500g, with a volume of approximately 460ml.

[0170] Table 15 shows that at an alcohol precipitation concentration of 60%, the interface between the supernatant and the precipitate is unclear. The precipitation effect is better when the concentration reaches 65% or higher, and the effect improves with increasing concentration. Looking at the total amount of paeoniflorin in the supernatant, a concentration of 60% results in significant paeoniflorin loss, while a concentration of 65% results in slightly greater loss. Concentrations of 70% and 75% show less paeoniflorin loss, and the transfer rate is not significantly different between the two concentrations. Therefore, 70% and 75% are both suitable concentrations. From an economic perspective, a concentration of 75% requires more ethanol, increasing production costs. Therefore, considering all factors, a concentration of approximately 70% is chosen.

[0171] 2.5.2. Investigation of the specific gravity of alcohol precipitation

[0172] The experiment was conducted at the preferred alcohol precipitation concentration of 70%, that is, the prepared Aconitum carmichaelii, Paeonia lactiflora, Pueraria lobata, and Glycyrrhiza uralensis were extracted twice with 10 times the amount of water, each time for 2 hours. The extracts were concentrated to be equivalent to about 1g, 1.5g, 2g and 3g of crude drug per ml, respectively, and then ethanol was added for alcohol precipitation to achieve an alcohol content of 70%.

[0173] Take 1200g of processed Aconitum carmichaelii, 1200g of Paeonia lactiflora, 1200g of Pueraria lobata, and 720g of Glycyrrhiza uralensis. Add 10 times the amount of water and decoct twice, 2 hours each time. Filter the decoction, combine the filtrates, and divide them into 4 equal portions. Concentrate each portion under reduced pressure to obtain 4 concentrated solutions (the relative densities of the 4 concentrated solutions are 1.06, 1.12, 1.15, and 1.19, respectively, which are equivalent to 0.98, 1.59, 2.23, and 2.92 g of crude drug / ml). Add 95% ethanol to each solution for alcohol precipitation to bring the alcohol content to 70%. After stirring evenly, let stand for more than 12 hours, take the supernatant, and measure the volume.

[0174] Alcohol precipitation supernatant: Accurately pipette 5.0 ml of the alcohol precipitation supernatant into a 20 ml volumetric flask, dilute to the mark with 70% ethanol, shake well, filter through a 0.45 μm organic microporous membrane, and inject the filtrate into the liquid chromatograph for determination. The results are shown in Table 12-16.

[0175] Table 16 Experimental data for alcohol precipitation specific gravity investigation

[0176]

[0177] As can be seen from Table 16, when the concentrate is concentrated to a relative density of 1.06-1.15 (60℃, each ml is equivalent to 0.98-2.23g of crude drug) and then subjected to alcohol precipitation, paeoniflorin can be well retained. Therefore, the concentration ratio of the concentrate is initially set as: relative density 1.06-1.15 (60℃).

[0178] 2.6. Verification of the water extraction and alcohol precipitation process

[0179] The experiment was conducted according to the finalized extraction and alcohol precipitation process: Prepared Aconitum carmichaelii, Paeonia lactiflora, Pueraria lobata, and Glycyrrhiza uralensis were extracted twice with 10 times their volume of water, each time for 2 hours. The extract was concentrated at a temperature below 80℃ until each ml was equivalent to approximately 1-1.6g of raw herbs. 95% ethanol was then added to achieve an alcohol content of 70%. The mixture was allowed to stand for at least 12 hours, and the supernatant was concentrated under reduced pressure. Based on past production experience, a specific gravity of 1.25-1.30 generally results in better extract properties and facilitates production, storage, and distribution. Therefore, the specific gravity of the water-extracted alcohol-precipitated extract was set at 1.25-1.30 in the verification experiment. Pilot-scale data are shown in Table 17.

[0180] Table 17 Verification Test of Water Extraction and Alcohol Precipitation

[0181] Serial Number 201208078 201208081 201208082 201208084 Processed aconite (kg) 8.0 8.0 8.0 8.0 Red peony root (kg) 8.0 8.0 8.0 8.0 Arrowroot (kg) 8.0 8.0 8.0 8.0 Licorice (kg) 4.8 4.8 4.8 4.8 Concentrated solution (g crude drug / ml) 1.32 1.58 1.56 1.09 Concentrate specific gravity (60℃) 1.100 1.095 1.120 1.087 Concentrate weight (kg) 24.0 20.0 20.7 28.8 Amount of ethanol added (kg) 49.5 41.4 42.0 60.1 Extract specific gravity (60℃) 1.271 1.280 1.293 1.268 Extract weight (kg) 3.01 2.23 2.36 3.06 Paeoniflorin content (%) 4.8％ 6.2％ 6.2％ 5.3％ Transfer rate (%) 45.2％ 43.2％ 45.7％ 50.7％ Dry yield (%) 6.5％ 5.1％ 5.3％ 6.4％

[0182] Note: The paeoniflorin content in red peony root is 4.0%.

[0183] As shown in Table 17, the concentrated solution before alcohol precipitation in the four batches of pilot-scale production was equivalent to 1.09-1.58 g crude drug / ml, with a corresponding specific gravity distribution of 1.087-1.120 (60±1℃). To facilitate production control, based on experimental investigation and the data from the four batches of pilot-scale production, the specific gravity of the water extract concentrate was determined to be 1.08-1.12 (60±1℃). Meanwhile, when the concentrated specific gravity of the water-extracted alcohol immersion extract was controlled at 1.25-1.30, the production process was smooth, the yield and the transfer rate of paeoniflorin showed good parallelism, and the process was stable and controllable. Therefore, the water extraction and alcohol precipitation process was determined to be reasonable overall.

[0184] In summary, the optimal water extraction and alcohol precipitation process is determined as follows: Prepared Aconitum carmichaelii, Paeonia lactiflora, Pueraria lobata, and Glycyrrhiza uralensis are decocted twice with water for 2 hours each time, filtered, and concentrated under reduced pressure to a relative density of 1.08-1.12 (60±1℃). Ethanol is added to bring the alcohol content to 70%. In actual production, the alcohol content during alcohol precipitation usually fluctuates between 68-73%. After standing for more than 12 hours, the supernatant is collected and concentrated under reduced pressure to obtain an extract with a relative density of 1.25-1.30 (60±1℃).

[0185] Experimental Example 3: Study on Formulation Process Conditions

[0186] 1. Dosage Form Selection

[0187] This product's formula originates from a hospital-prepared formulation from the Rheumatology Department of the General Hospital of the Chinese People's Liberation Army. The original dosage form was capsules. The kudzu root in the formula, after being pulverized, acts as an excipient. This results in a highly hygroscopic formulation, requiring strict control over environmental temperature and humidity, which is unfavorable for large-scale production and the distribution and sale of the drug. To improve product quality and patient suitability, a dosage form with good stability, portability, and ease of administration was developed. Based on the determined extraction process's yield range, the properties of the extract, and the patient population for this product's indications, micro-pellets, capsules, granules, and tablets were initially selected. Preliminary screening for formulation formability was conducted to investigate the maximum drug loading and daily dosage of different dosage forms. The results are shown in Table 18.

[0188] Table 18 Dosage Form Selection Results

[0189]

[0190] The results showed that, considering the four formulations, tablets have a larger drug loading capacity and a wider drug loading range. Compared with capsules and microgranules, they have the lowest single dose. Compared with granules, they are more conducive to improving patient medication adherence. Therefore, tablets were chosen for development.

[0191] 2. Investigation of the formulation and manufacturing process of unprocessed tablets

[0192] 2.1. Determination of Granulation Method

[0193] Tablets are typically made by directly compressing extracts or by granulating them before tableting. Common granulation methods include wet granulation, dry granulation, and fluidized bed granulation. Fluidized bed granulation offers advantages such as uniform particle size, good flowability and compressibility, time savings, and better compliance with GMP requirements. Furthermore, our company has a mature fluidized bed granulation production line, therefore we have chosen the fluidized bed granulation followed by tableting process.

[0194] 2.2. Selection of excipients

[0195] Dextrin, starch, and microcrystalline cellulose were used as candidate excipients, and fluidized bed granulation was performed at a drug loading of 65%. Granulation characteristics and particle size were examined. To ensure smooth tableting, based on experimental experience, 5% sodium carboxymethyl starch was added as a disintegrant and 0.5% magnesium stearate as a lubricant during tableting. The appearance, hardness, disintegration, and friability of the tablets were examined. The results are shown in Table 19.

[0196] Table 19 Results of the investigation of different proportions of auxiliary materials

[0197]

[0198] The results showed that formulations 1 and 3 were refined, with simple processes, good tablet appearance, and minimized the production difficulty of each step, thus improving production efficiency and making them most suitable for industrial production. However, formulation 1, using dextrin as a filler, had low tablet hardness after compression and was prone to cracking, making it unsuitable for subsequent processing requirements. Therefore, formulation 3, using microcrystalline cellulose as a filler excipient, was chosen as a dosage regulator to balance fluctuations in the powder extraction rate.

[0199] 2.3. Investigation on the Dosage of Disintegrant

[0200] Microcrystalline cellulose was used as a filler, and fluidized bed granulation was performed at a drug loading of 65%. Sodium carboxymethyl starch was added as a disintegrant at proportions of 3%, 5%, and 7%. 0.5% magnesium stearate was used as a lubricant for tableting. The disintegration and hardness of the tablets were investigated. The results are shown in 20.

[0201] Table 20 Results of the study on disintegrant dosage

[0202] Disintegrant ratio (%) Disintegration time (min) Hardness (N) 3 25 123-154 5 20 89-104 7 18 67-92

[0203] The results showed that when the amount of sodium carboxymethyl starch added as a disintegrant was above 5%, the tablets of the composition of the present invention had good disintegration effect. Therefore, it was determined that 5% sodium carboxymethyl starch should be added as a disintegrant.

[0204] 2.4. Investigation of internal and external disintegrant addition methods

[0205] When preparing tablets from drug-loaded granules, external disintegrants can promote disintegration between granules, while internal disintegrants can promote disintegration within the granules. Therefore, it is necessary to determine the appropriate ratio of external to internal disintegrant addition through investigation. Microcrystalline cellulose was used as a filler, and granulation was performed with a drug loading of 65%. Disintegrants were added at internal to external ratios of 2:3, 1:1, and 3:2, respectively. Tableting was performed using 0.5% magnesium stearate as a lubricant, and the tablet hardness, disintegration, and friability were investigated. The results are shown in Table 21.

[0206] Table 21 Results of the investigation on internal and external addition of disintegrants

[0207] Disintegrant (internal:external) addition ratio Disintegration (min) Hardness (N) Friability (%) 2:3 21 144-166 0.39 1:1 20 131-156 0.31 3:2 17 93-112 0.20

[0208] The results showed that the fragility and hardness of the tablets were optimal when the ratio of internal to external disintegrant addition was 3:2. Therefore, the optimal ratio of internal to external disintegrant addition was determined to be 3:2.

[0209] 2.5. Lubricant Dosage Assessment

[0210] Microcrystalline cellulose was used as an excipient, and fluidized bed granulation was performed at a drug loading of 65%. 5% sodium carboxymethyl starch was added as a disintegrant at an internal-external ratio of 3:2. Magnesium stearate (0.3%, 0.5%, and 0.7%) was added as a lubricant during tableting. The disintegration and hardness of the tablets were then investigated. Results are shown in Figure 22.

[0211] Table 22 Results of Lubricant Dosage Investigation

[0212] Lubricant ratio (%) Disintegration time (min) Hardness (N) Phenomenon description 0.3 21 46-67 Slight sticking and uneven surface 0.5 18 81-104 Tableting went smoothly, and the tablet surface was smooth. 0.7 17 123-154 Tableting went smoothly, and the tablet surface was smooth.

[0213] The results showed that tableting was smooth and the tablet surface was clean when the amount of magnesium stearate added was above 0.5%. Therefore, it was determined that 0.5% magnesium stearate should be added for tableting.

[0214] 2.6. Investigation of the range of fillers

[0215] Microcrystalline cellulose was used as an excipient, 5% sodium carboxymethyl starch was used as a disintegrant in an internal-external ratio of 3:2, and 0.5% magnesium stearate was used as a lubricant. The mixture was granulated by boiling and then compressed into tablets at drug loadings of 50%, 55%, 60%, 65%, 70%, 75%, and 80%. Granulation characteristics, tablet disintegration, and hardness were investigated. Results are shown in Figure 23.

[0216] Table 23 Results of the investigation on the range of fillers

[0217]

[0218]

[0219] The results showed that granulation and tableting were relatively smooth and the yield was normal when the drug loading was between 50% and 75%. Therefore, considering the fluctuation of the yield of multiple batches of raw herbs in the pilot test and the daily dosage of raw herbs, and taking into full consideration the patient's compliance, the design adopted a tablet specification of 0.65g / tablet (3 tablets twice a day). Each 1000 tablets is equivalent to 3240g of raw herbs. Each prescription contains 19.5g of sodium carboxymethyl starch, plus 13g of sodium carboxymethyl starch and 3.25g of magnesium stearate, to make 1000 tablets.

[0220] 2.7. Investigation of boiling granulation temperature

[0221] Microcrystalline cellulose and sodium carboxymethyl starch were used as substrates, and fluidized bed granulation was carried out at a drug loading of 65%. The extract was used as slurry to investigate fluidized bed granulation at different temperatures. The particle characteristics and yield were investigated, and the results are shown in Table 24.

[0222] Table 24 Results of Temperature Investigation of Fluidized Bed Granulation Materials

[0223]

[0224] The results showed that when the base material temperature was set at 60℃-80℃, the formulation proceeded smoothly, the material state was moderate, and it was suitable for fluidized bed granulation.

[0225] 3. Investigation of film coating formulation and process

[0226] Film coating is widely used in tablets and pills, especially for traditional Chinese medicine tablets with strong hygroscopicity and special odors, which shows its advantages. Film-coated tablets can effectively prevent moisture and mask the taste. Based on the above advantages, film coating is used to mask the taste and prevent moisture of plain tablets. The film coating process is studied as follows.

[0227] 3.1. Selection of Coating for Weight Gain

[0228] Bokolin's water-soluble moisture-proof film coating material was selected, with theoretical coating weight gains of 3%, 4%, and 5% of the tablet weight, respectively. The coating performance was investigated using a coating machine at 40°C, and the results are shown in Table 25.

[0229] Table 25 Results of the Investigation on the Amount of Coating Material Used

[0230] Coating material quantity 3％ 4％ 5％ Coating for weight gain 2.5％ 3.6％ 4.6％ Coating situation Cannot be completely covered Almost completely covered Fully coated, with clear coloring

[0231] The results showed that a coating material content of 5% was sufficient for complete coating. Therefore, the coating material content was chosen to be 5% of the weight of the uncoated tablets. Thus, the specifications of the coated tablets are: uncoated tablet size * (1 + theoretical weight gain) = 0.65g / tablet * (1 + 5%) = 0.68g / tablet.

[0232] 3.2. Coating Temperature Study

[0233] Take an appropriate amount of the uncoated tablets and coat them at 35℃, 40℃, 45℃ and 50℃ respectively. The coating conditions were examined and the results are shown in Table 26.

[0234] Table 26 Coating Temperature Investigation

[0235] Coating temperature 35℃ 40℃ 45℃ 50℃ Coating situation Severe adhesion of the film Well dried Well dried Severe chip adhesion and easy wear of the chip core.

[0236] The results showed that coating performance was good when the coating temperature was between 40℃ and 45℃. Therefore, a coating temperature of 40℃-45℃ was selected.

[0237] Experimental Example 4. Stability test of the composition of the present invention in tablets and spinal pain relief capsules.

[0238] This experiment uses a stability test chamber with accelerated placement and sample retention method for evaluation.

[0239] 1. Accelerated stability test conditions

[0240] The tablets (batch number 20230501, 0.68 g / tablet) and the capsules (batch number 20240601, 0.35 g / capsule) of the present invention prepared according to Experimental Example 3 were sealed in oral solid pharmaceutical high-density polyethylene bottles and placed at 40℃±2℃ and relative humidity 75%±5%. The results were measured periodically at 0, 1, 2, 3 and 6 months.

[0241] 2. Site Visit

[0242] Tablets: Properties, moisture content, disintegration time, diester alkaloids test, and content determination;

[0243] Capsules: Properties, moisture content, disintegration time, diester alkaloids test, and content determination;

[0244] The results are shown in Tables 27 and 28.

[0245] 3. Conclusion

[0246] The tablets containing the composition of this invention underwent an accelerated testing process over 6 months. Compared with the results at 0 months, all aspects of the tablets, including appearance, moisture content, disintegration time, diester alkaloids, and content determination, were within acceptable limits. The capsules containing Jitongning, after an accelerated testing process over 2 months, also showed acceptable levels of appearance, moisture content, disintegration time, diester alkaloids, and content determination compared to the results at 0 months. However, at 3 months, the moisture content exceeded the limit, and at 6 months, the appearance was unacceptable, with clumping of the contents. The contents of paeoniflorin and glycyrrhizic acid showed a significant decreasing trend at each time point. These results indicate that the tablets containing the composition of this invention are more stable in quality than the capsules containing Jitongning under the aforementioned testing conditions. Figure 3 and Figure 4 The images show the properties of the tablets and capsules of the composition of the present invention before and after accelerated stability testing.

[0247] Table 27 Accelerated stability study batch number of the tablet composition of the present invention: 20230501

[0248]

[0249]

[0250] Table 28 Accelerated Stability Study Batch Number for Jitongning Capsules: 20240601

[0251]

Claims

1. A method for preparing a stable and high-drug-loading medicament for treating axial spondylitis, comprising the following parts by weight of traditional Chinese medicine raw materials: 5-20 parts Eucommia ulmoides, 5-20 parts Angelica pubescens, 5-15 parts processed Aconitum carmichaelii, 5-20 parts Corydalis yanhusuo, 5-15 parts Paeonia lactiflora, 5-15 parts Pueraria lobata, and 3-10 parts Glycyrrhiza uralensis, characterized in that... The preparation method includes the following steps: 1) extracting Angelica pubescens, Eucommia ulmoides and Corydalis yanhusuo with ethanol to obtain an alcohol extract; 2) extracting Aconitum carmichaelii, Glycyrrhiza uralensis, Paeonia lactiflora and Pueraria lobata with water extraction and alcohol precipitation to obtain an aqueous extract.

2. The preparation method according to claim 1, characterized in that, The preparation of the aqueous extract includes the following steps: adding processed Aconitum carmichaelii, licorice, Paeonia lactiflora and Pueraria lobata into an extraction tank, adding water for extraction, filtering, concentrating the aqueous extract to a relative density of 1.06-1.15; adding ethanol to a concentration of 65-75% for alcohol precipitation, allowing to stand, filtering, and concentrating to a relative density of 1.25-1.30 to obtain the aqueous extract.

3. The preparation method according to claim 2, characterized in that, The concentration temperature of the aqueous extract is controlled below 80°C, and the concentration method is vacuum concentration.

4. The preparation method according to claim 2, characterized in that, The preparation of the aqueous extract includes the following steps: Prepared Aconitum carmichaelii, Glycyrrhiza uralensis, Paeonia lactiflora, and Pueraria lobata are placed in an extraction tank, and extracted twice with 10 times the amount of water, each time for 2 hours. After filtration, the aqueous extract is concentrated under reduced pressure to a relative density of 1.08-1.

12. Ethanol is added to a concentration of 68-73% for alcohol precipitation. After standing, the extract is filtered, and the supernatant is concentrated to a relative density of 1.25-1.30 to obtain the aqueous extract.

5. The preparation method according to claim 4, characterized in that, The alcohol precipitation step involves adding ethanol until the alcohol content reaches 70%.

6. The preparation method according to claim 1, characterized in that, The preparation of the alcohol extract includes the following steps: Eucommia ulmoides, Angelica pubescens and Corydalis yanhusuo are put into an extraction tank, 65-85% ethanol is added for extraction, filtered, and the filtrate is concentrated under reduced pressure to obtain an extract with a relative density of 1.25-1.30, thus obtaining the alcohol extract.

7. The preparation method according to claim 6, characterized in that, The preparation of the alcohol extract includes the following steps: Eucommia ulmoides, Angelica pubescens and Corydalis yanhusuo are put into an extraction tank, and 6 times the amount of 74-76% ethanol is added for extraction twice, each time for 2 hours. After filtration, the filtrate is concentrated under reduced pressure at below 80°C to a relative density of 1.25-1.30, and the extract is collected to obtain the alcohol extract.

8. The preparation method according to claim 7, characterized in that, The ethanol is 75% ethanol.

9. The pharmaceutical composition prepared according to the method of claim 1, characterized in that, It includes a pharmaceutically active substance and a pharmaceutical carrier, wherein the pharmaceutically active substance is an alcohol extract and a water extract prepared by the preparation method of claim 1.

10. The pharmaceutical composition according to claim 9, characterized in that, The water extract and alcohol extract of the active pharmaceutical ingredients account for 50-75% by weight in the pharmaceutical composition, with the remainder being a drug carrier.

11. The pharmaceutical composition according to claim 10, characterized in that, The drug carrier is selected from sodium carboxymethyl starch, magnesium stearate and microcrystalline cellulose, and their weight percentages in the drug composition are 5% sodium carboxymethyl starch, 0.5% magnesium stearate and 19.5-44.5% microcrystalline cellulose.

12. The pharmaceutical composition according to claim 11, characterized in that, The weight percentages of each component in the pharmaceutical composition are as follows: 65% for the aqueous and alcoholic extracts of the active pharmaceutical ingredient, 5% for sodium carboxymethyl starch, 0.5% for magnesium stearate, and 29.5% for microcrystalline cellulose.

13. The method for preparing the pharmaceutical composition according to claim 12, characterized in that, Take the water extract and alcohol extract, add sodium carboxymethyl starch and microcrystalline cellulose, granulate, dry, then add sodium carboxymethyl starch and magnesium stearate externally, compress into tablets, and coat with a film; wherein the weight ratio of the internally added sodium carboxymethyl starch to the externally added sodium carboxymethyl starch is 3:

2.

14. Use of the pharmaceutical composition of claims 9-12 in the preparation of a medicament for treating axial spondyloarthritis.

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