A kind of fuzi decoction granules with toxicity removed and medicinal effect retained and preparation method thereof

Abstract

The present invention provides a Fuzi decoction granule with detoxification and retained efficacy and a preparation method thereof, belonging to the technical field of traditional Chinese medicine preparation. The preparation method of the Fuzi decoction granule of the present invention comprises: soaking Fuzi and decocting Fuzi in advance for 60 to 120 minutes, decocting Fuzi with soaked Poria, ginseng, bran-fried Atractylodes macrocephala, white peony root, red peony root and cinnamon twig for 30 to 60 minutes, and filtering the filtrate to obtain the Fuzi decoction granule after concentrating and granulating. The Fuzi decoction granule obtained by the preparation method of the present invention can remove toxicity and retain efficacy on osteoarthritis.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine preparation, and in particular relates to a fuzi decoction granule with toxicity removed and medicinal effect retained, and a preparation method thereof. Background Art

[0002] Knee osteoarthritis (KOA) is the joint degenerative disease with the highest clinical incidence and the second leading cause of disability, which seriously affects people's quality of life. KOA is more common in middle-aged and elderly people and is related to genetic and physical factors; trauma, inflammation, cumulative strain or congenital diseases can also cause KOA. With the aging of the population and the aggravation of obesity in my country, the incidence of KOA has been increasing year by year. KOA can cause joint pain, deformity and activity dysfunction, thereby increasing the incidence of cardiovascular events and all-cause mortality. The all-cause mortality rate increases by nearly 1 times with KOA.

[0003] Drug therapy is an important part of the treatment of KOA. The Consensus of Chinese Medicine Experts on Diagnosis and Treatment of Knee Osteoarthritis proposes that the main TCM syndrome of KOA is cold-dampness and arthralgia, commonly known as "old cold legs". Fuzi Decoction comes from Treatise on Febrile Diseases. It is mainly used to treat patients with yang deficiency, cold-dampness and internal invasion, and pain in the body joints. It is a classic prescription for warming yang and dispersing cold, suitable for the treatment of "old cold legs" KOA. In the prescription, Fuzi replenishes fire and invigorates the original yang, warms the meridians and dispels cold; ginseng greatly replenishes the original qi and generates qi and blood; Poria strengthens the spleen and penetrates dampness, and ginseng and Fuzi replenish qi and warm yang, while Atractylodes macrocephala dries dampness and moves the spleen, dispels cold and dampness; and Paeonia lactiflora harmonizes the Ying and stops pain. The whole prescription works together to warm the yang and move the spleen, dispel dampness and stop pain. However, Fuzi in the prescription is highly toxic, and clinical application not only produces toxic side effects, but may also be fatal.

[0004] The toxic component of aconite is aconitine, which is soluble in water and ethanol and is extremely toxic. Oral administration of 0.2 mg of aconitine can cause poisoning reactions in humans, and 3 to 5 mg can be fatal, so it is listed as a low-grade product in the Shennong's Herbal Classic. The "toxicity" and "effect" of aconite coexist. It has both toxicity and therapeutic effects. Improper use can cause toxic side effects, while correct use often produces rapid and efficient effects and can treat many critical and severe diseases. Therefore, some doctors call it a tiger and wolf drug and dare not use it. Therefore, there are always potential risks in the clinical application of aconite, and the toxicity of aconite is the primary problem to be solved in clinical practice.

[0005] First decoction is the main method of reducing the toxicity of Radix Aconiti Lateralis Preparata. Under large doses, it is often decocted for half an hour and tasted. After half an hour, it is not numb in the mouth, and then it is decocted and taken with other medicines. However, the above-mentioned detoxification method still cannot guarantee the safety of the clinical application of Radix Aconiti Lateralis Preparata, and no real non-toxic Radix Aconiti Lateralis Preparata has been developed. The reason is that the clinical correlation between the toxicity, dosage, and processing method of Radix Aconiti Lateralis Preparata is unclear and the unified standard of processing of Radix Aconiti Lateralis Preparata is lacking. The present invention is just the application development achievement obtained by studying the above-mentioned problems, and has a good application prospect. Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide a Fuzi decoction granule with toxicity removed and efficacy retained and a preparation method thereof, which can retain the efficacy of Fuzi decoction while removing the toxicity of Fuzi.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The invention provides a method for preparing a granule of aconite decoction with toxicity removed and efficacy retained, comprising: (1) soaking aconite for 25 to 35 minutes and decocting for 60 to 120 minutes; (2) mixing and soaking tuckahoe, ginseng, stir-fried atractylodes macrocephala with bran, white peony root, red peony root and cassia twig to obtain soaked Chinese medicine; (3) adding the soaked Chinese medicine in step (2) into the decoction container in step (1), decocting together with aconite for 30 to 60 minutes, filtering and obtaining a filtrate; and (4) concentrating the filtrate in step (3) to obtain a paste, mixing the paste with maltodextrin, spray drying and granulating to obtain the granule.

[0009] Preferably, the mass ratio of the aconite root, Poria cocos, ginseng, stir-fried Atractylodes macrocephala with bran, white peony root, red peony root and cinnamon twig is 15:9:6:12:15:15:12.

[0010] Preferably, the mass ratio of the aconite root to water in step (1) is 1:8-12.

[0011] Preferably, the mass ratio of the total mass of the Poria cocos, ginseng, stir-fried Atractylodes macrocephala, white peony root, red peony root, and cinnamon twig to water in step (2) is 1:8-12.

[0012] Preferably, the soaking time in step (2) is 25 to 35 minutes.

[0013] Preferably, the relative density of the paste is 1.02 to 1.12.

[0014] Preferably, the mass ratio of the paste to maltodextrin is 1:0.3.

[0015] Preferably, the spray drying temperature is 165-190°C.

[0016] Preferably, the particle size of the granules is 10 to 16 meshes.

[0017] The present invention also provides the Fuzi decoction granules obtained by the above preparation method, wherein the Fuzi decoction granules contain benzoylhypaconitine≥43.35μg / mg, benzoylmesaconitine≥268.52μg / mg, benzoylaconitine≥28.75μg / mg, hypoaconitine≤5.63μg / mg, mesaconitine≤0.05μg / mg, and do not contain aconitine.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention provides a method for preparing a fuzi decoction granules with detoxification and retained efficacy, comprising: soaking aconite in advance and decocting for 60 to 120 minutes, then decocting with soaked tuckahoe, ginseng, bran-fried atractylodes, white peony root, red peony root and cassia twig for 30 to 60 minutes, filtering the filtrate, concentrating and granulating to obtain the aconite decoction granules. The aconite decoction granules obtained by the preparation method of the present invention can remove toxicity and retain efficacy on osteoarthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The effect of aconite decoction at different times on the body weight of mice (g, x±s); FZ-30 compared with the normal control group, *P<0.05, **P<0.01; FZ-60 compared with the normal control group, #P<0.05, ##P<0.01;

[0021] Figure 2 The effects of each treatment on blood biochemistry of mice for 2 weeks (g, x±s); FZ-30 compared with the normal control group, *P<0.05, **P<0.01;

[0022] Figure 3 The effects of each treatment on blood biochemistry of mice for 4 weeks (g, x±s); FZ-30 compared with the normal control group, *P<0.05, **P<0.01;

[0023] Figure 4 Statistical analysis and histopathological observation of the effects of non-long-term decoction of Fuzi and long-term decoction on pain in MIA-induced OA rat model; A: HE staining, SO staining; Scale bar = 100 μm; B: Mankins score for histopathological observation; C: Mechanical pain at 0 and 4 weeks; ##P<0.01 compared with the normal group; **P<0.01 compared with the model group;

[0024] Figure 5 Immunohistochemical analysis of the effects of non-long-fried Aconite Decoction and long-fried Aconite Decoction on the expression of Col2 and MMP13 in rat cartilage; A: Immunohistochemical staining of Col2 and MMP13 in cartilage, scale bar = 100μm; B: Quantitative examination of the percentage of Col2 positive area and the percentage of MMP13 positive cells; ##P<0.01 compared with the normal group; *P<0.05 compared with the model group; **P<0.01 compared with the model group. DETAILED DESCRIPTION

[0025] The invention provides a method for preparing a toxicity-removing and efficacy-retaining aconite decoction granules, comprising: (1) soaking aconite for 25 to 35 minutes and decocting for 60 to 120 minutes; (2) mixing and soaking tuckahoe, ginseng, bran-fried atractylodes macrocephala, white peony root, red peony root and cassia twig to obtain soaked Chinese medicine; (3) adding the soaked Chinese medicine in step (2) into the decoction container in step (1), decocting together with aconite for 30 to 60 minutes, filtering and obtaining a filtrate; and (4) concentrating the filtrate in step (3) to obtain a paste, mixing the paste with maltodextrin, spray drying and granulating to obtain the granules.

[0026] The aconite root of the present invention is a processed aconite root, preferably including black shunpian, white fupian, yellow fupian, salt fupian, light fupian, etc. The aconite root decoction time in step (1) is preferably 90 minutes, and the decoction time in step (3) is preferably 30 minutes. The decoction time of the present invention can remove the toxicity in the aconite root and retain the drug effect on osteoarthritis.

[0027] In the present invention, the mass ratio of the aconite, Poria, ginseng, stir-fried Atractylodes macrocephala, white peony root, red peony root and cinnamon twig is preferably 15:9:6:12:15:15:12; the mass ratio of the aconite to water in step (1) is 1:8-12, more preferably 1:10; the mass ratio of the total mass of the Poria, ginseng, stir-fried Atractylodes macrocephala, white peony root, red peony root and cinnamon twig to water in step (1) is preferably 1:8-12, more preferably 1:10; the soaking time in step (2) is preferably 25-35 min; the relative density of the paste is preferably 1.02-1.12, more preferably 1.08.

[0028] The mass ratio of the paste to maltodextrin of the present invention is preferably 1:0.3; the spray drying temperature is preferably 165-190°C, more preferably 180°C; the particle size of the granules is preferably 10-16 mesh, more preferably 14 mesh; and packaging is preferably included after the granulation.

[0029] The present invention has no special limitation on the source of the raw materials, and conventional commercial products in the art may be used.

[0030] The present invention also provides the Fuzi decoction granules obtained by the above preparation method, wherein the Fuzi decoction granules contain benzoylhypaconitine≥43.35μg / mg, benzoylmesaconitine≥268.52μg / mg, benzoylaconitine≥28.75μg / mg, hypoaconitine≤5.63μg / mg, mesaconitine≤0.05μg / mg, and do not contain aconitine.

[0031] The mechanical equipment used in the process of preparing Fuzitang granules of the present invention is not particularly limited, and conventional equipment in the art can be used. In the present invention, the equipment used for concentration is a small extraction and concentration unit, model 100L; the equipment used for drying is a centrifugal spray dryer, model ZPG-5, the equipment used for mixing is a three-dimensional motion mixer, model SYH-1000, the equipment used for granulation is a dry granulator, model LGJ80, and the equipment used for packaging is an automatic granule packaging machine, model DXDK40VI.

[0032] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0033] Example 1

[0034] (1) Soak 15g of Aconite (Heishunpian) in 10 times the amount of water for 30 minutes, then boil for 90 minutes;

[0035] (2) Mix 9 g of Poria cocos, 6 g of ginseng, 12 g of bran-fried Atractylodes macrocephala, 15 g of white peony root, 15 g of red peony root, and 12 g of cinnamon twig, add 10 times of water and soak for 30 min, filter, and obtain the soaked Chinese medicine;

[0036] (3) adding the Chinese medicine soaked in step (2) into the decoction container of step (1) and decocting together with aconite for 30 minutes, filtering to obtain a filtrate;

[0037] (4) The filtrate of step (3) was concentrated to a paste with a relative density of 1.08, mixed with 25.2 g of maltodextrin, and spray-dried at an inlet temperature of 165-190° C. and an outlet temperature of 70-90° C. to obtain a spray powder, which was sieved through a 14-mesh sieve to form granules, and packaged at 10 g per bag.

[0038] Example 2

[0039] (1) Soak 15g of Aconite (Heishunpian) in 8 times the amount of water for 35 minutes, then boil for 60 minutes;

[0040] (2) Mix 9 g of Poria cocos, 6 g of ginseng, 12 g of bran-fried Atractylodes macrocephala, 15 g of white peony root, 15 g of red peony root, and 12 g of cinnamon twig, add 8 times the amount of water and soak for 35 min, filter, and obtain the soaked Chinese medicine;

[0041] (3) adding the Chinese medicine soaked in step (2) into the decoction container of step (1) and continuing to decoct together with the aconite for 60 minutes, filtering to obtain a filtrate;

[0042] (4) The filtrate of step (3) was concentrated to a paste with a relative density of 1.02, mixed with 25.2 g of maltodextrin, and spray-dried at an inlet temperature of 165-190° C. and an outlet temperature of 70-90° C. to obtain a spray powder, which was sieved through a 14-mesh sieve to form granules, and packaged at 10 g per bag.

[0043] Example 3

[0044] (1) Soak 15g of Aconite (Heishunpian) in 12 times the amount of water for 25 minutes, then boil for 120 minutes;

[0045] (2) 9 g of Poria cocos, 6 g of ginseng, 12 g of bran-fried Atractylodes macrocephala, 15 g of white peony root, 15 g of red peony root, and 12 g of cinnamon twig were mixed, soaked in 8 times water for 25 min, and filtered to obtain the soaked Chinese medicine;

[0046] (3) adding the Chinese medicine soaked in step (2) into the decoction container of step (1) and continuing to decoct with aconite for 30 minutes, filtering to obtain a filtrate;

[0047] (4) The filtrate of step (3) was concentrated to a paste with a relative density of 1.12, mixed with 25.2 g of maltodextrin, and spray-dried at an inlet temperature of 165-190° C. and an outlet temperature of 70-90° C. to obtain a spray powder, which was sieved through a 14-mesh sieve to form granules, and packaged at 10 g per bag.

[0048] Comparative Example 1

[0049] The specific implementation is the same as that of Example 1, except that the boiling time in step (1) is 0 min, and the boiling time in step (3) is 30 min.

[0050] Comparative Example 2

[0051] The specific implementation is the same as that of Example 1, except that the boiling time in step (1) is 30 minutes, and the boiling time in step (3) is 30 minutes.

[0052] Comparative Example 3

[0053] (1) Mix 15 g of aconite root (Heishun tablets), 9 g of Poria cocos, 6 g of ginseng, 12 g of bran-fried Atractylodes macrocephala, 15 g of white peony root, 15 g of red peony root, and 12 g of cinnamon twig, add 10 times the amount of water, soak for 30 min, decoct for 30 min, and filter to obtain a filtrate;

[0054] (2) The filtrate of step (2) was concentrated to a paste with a relative density of 1.08, mixed with 25.2 g of maltodextrin, and spray-dried at an inlet temperature of 165-190° C. and an outlet temperature of 70-90° C. to obtain a spray powder, which was sieved through a 14-mesh sieve to form granules, and packaged at 10 g per bag.

[0055] Experimental Example 1

[0056] The contents of three toxic alkaloids and three functional alkaloids in the Fuzi Decoction granules of Example 1 and Comparative Example 3 were determined.

[0057] The detection method is as follows:

[0058] 1. Preparation of reference solution

[0059] Accurately weigh functional aconitine (benzoylhypaconitine, benzoylmesaconitine, benzoylaconitine) and toxic aconitine (hypaconitine, mesaconitine, aconitine) standards, add dichloromethane to make up to volume, and shake well to dissolve as a reference mixed solution.

[0060] 2. Preparation of test solution

[0061] Take 18 mL of the aqueous solution of the Fuzi Decoction granules of Example 1 and Comparative Example 3 (configured into a solution with a concentration of 2.0 g / ml), respectively, extract with dichloromethane and chloroform, respectively, 3 times, 18 mL / time, combine the dichloromethane and chloroform layer extracts, recover to dryness under reduced pressure at 55°C, add dichloromethane to make up to 3 mL, and filter through a 0.45 μm microporous filter membrane to obtain the test solution.

[0062] 3. UPLC-MS / MS analysis

[0063] The chromatographic conditions were as follows:

[0064] Chromatographic column: BEH C18 2.1mm*100mm, 1.7μm; mobile phase A: 5mM ammonium formate aqueous solution, mobile phase B: acetonitrile; flow rate: 1.0mL / min, column temperature 30℃, injection volume 5μL.

[0065] The content of the active ingredient in the reference solution was determined, and a standard curve was prepared. According to the standard curve, the contents of the six components in the Fuzi Decoction Granules of Example 1 and Comparative Example 3 were calculated. The specific results are shown in Table 1.

[0066] Table 1 Contents of three toxic alkaloids and three functional alkaloids in Fuzi Decoction Granules

[0067]

[0068] It can be seen from Table 1 that, compared with Comparative Example 3, the Fuzi Decoction Granules prepared by the method of Example 1 of the present invention can significantly increase the content of functional aconitine and reduce the content of toxic aconitine.

[0069] Experimental Example 2

[0070] Acute toxicity test in ICR mice

[0071] 1. Experimental animals: 40 ICR mice, half male and half female, weighing 25-30 g, provided by the Animal Center of Zhejiang Chinese Medical University.

[0072] 2. Aconite Granule Dissolving Solution:

[0073] Take 50g of the aconite granules (1g of granules is equal to 3g of crude drug) of Example 1 and Comparative Examples 1-2 respectively, add water to dissolve, and then make the volume to 150ml to obtain a crude drug concentration of 1g / ml.

[0074] 3. Grouping and medication:

[0075] Take 40 clean-grade ICR mice and randomly group them according to body weight: normal control group, Fuzi decoction granules (Fuzi decoction 30min) group (Comparative Example 1), Fuzi decoction granules (Fuzi decoction 60min) group (Comparative Example 2), Fuzi decoction granules (Fuzi decoction 120min) group (Example 1), 10 mice in each group, half male and half female. Before the experiment, mice were fasted but not water-restricted for 12h. Each group of mice was administered with the maximum oral gavage dose (0.02ml / g / day), once a day for 28 consecutive days, and the normal control group was given distilled water every day.

[0076] 4. Daily weighing of mice and observation of their condition after intragastric administration:

[0077] (1) Observation

[0078] During the administration period (28 days), the weight changes of mice in each group were observed every day (for specific results, see Figure 1 ), diet, appearance, behavior, secretions, excretions, death and poisoning reactions (symptoms, severity, onset time, duration, and whether it is reversible).

[0079] Depend on Figure 1 It can be seen that compared with the normal control group, the body weight of mice in each medication group was significantly decreased (P < 0.05); by comparing the body weight difference, it can be seen that the toxicity of Fuzi decoction granules with different Fuzi decoction times to mice was significantly different (P < 0.05), indicating that as the decoction time is delayed, the toxicity of Fuzi decoction granules to mice decreases.

[0080] The mice in the normal control group were active, walking back and forth in the cage, and their food and water intake were normal; the mice in the Fuzi Decoction Granules (Fuzi decocted in water for 30 minutes) group showed rapid breathing, restlessness, and then closed their eyes, with cloudy and dull eyeballs, depression, shortness of breath, low body temperature, laziness, and poor coordination; there was no significant change in the mice treated with Fuzi Decoction Granules (Fuzi decocted in water for 60 minutes) and Fuzi Decoction Granules (Fuzi decocted in water for 120 minutes), and their activity level was reduced.

[0081] (2) Blood test

[0082] After the 28-day observation period, blood was collected from the surviving mice in each group, and the blood samples were placed horizontally for 2 hours, centrifuged at 3000r / min for 10 minutes, and the supernatant was obtained to obtain mouse serum. The following indicators in the serum were detected using relevant instruments and equipment: alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea nitrogen (BUN), uric acid (UA), creatinine (CREA), and lactate dehydrogenase (LDH). Specific results are shown in Tables 2-3 and Figure 2 .

[0083] Table 2 Results of leukocyte and classification examination of mice fed with Aconite for 28 days at different decoction times

[0084]

[0085] ** indicates significant difference

[0086] Table 3 Results of red blood cell and platelet examination in mice fed with Aconite for 28 days at different decoction times

[0087]

[0088] As shown in Tables 2 and 3, compared with the normal control group, the white blood cells, lymphocytes, and neutrophils in the blood of mice in the group of decocting Fuzi for 30 minutes were increased and the difference was statistically significant (P < 0.05), indicating that Fuzi Decoction Granules (decocting Fuzi for 30 minutes) can cause partial inflammation in mice. Compared with the normal control group, the lymphocytes and neutrophils in the groups of Fuzi Decoction Granules (decocting Fuzi for 60 minutes) and Fuzi Decoction Granules (decocting Fuzi for 120 minutes) showed abnormal changes to varying degrees (P < 0.05). The total number of red blood cells and platelets in the groups of Fuzi Decoction Granules (decocting Fuzi for 30 minutes) and Fuzi Decoction Granules (decocting Fuzi for 60 minutes) also showed abnormal changes (P < 0.05), and the above indicators in the group of Fuzi Decoction Granules (decocting Fuzi for 120 minutes) did not change at all (P > 0.05).

[0089] Depend on Figure 2 and Figure 3 It can be seen that compared with the normal control group, the ALT, GOT, CK and LDH levels in the blood of mice in the Fuzi Decoction Granules (Fuzi decoction for 30 minutes) group were increased and the difference was statistically significant (P < 0.05), indicating that Fuzi Decoction Granules (Fuzi decoction for 30 minutes) has certain toxicity to the liver and heart. However, there was no significant difference in ALT, AST, CK and LDH levels in the Fuzi Decoction Granules (Fuzi decoction for 60 minutes) group compared with the normal control group (P > 0.05).

[0090] The above results indicate that the safety of the Fuzi Decoction Granules (Fuzi decocted in water for 120 min) group is better than that of other groups.

[0091] Experimental Example 3

[0092] Therapeutic effect of Fuzitang granules on osteoarthritis rats

[0093] 1. Grouping

[0094] According to the random number table method, 40 SD rats were randomly divided into 4 groups (10 rats in each group): a sham operation group, a model group, a non-long-decoction Fuzi Decoction granule group (Comparative Example 3) and a long-decoction Fuzi Decoction granule group (Example 1).

[0095] 2. Modeling

[0096] Except for the sham operation group, the other groups were modeled by intra-articular injection of iodoacetic acid. First, 3% sodium pentobarbital (0.15 ml / 100 g) was injected intraperitoneally for anesthesia. After the rats were anesthetized, the hair of the knee joint was shaved, and the rats were fixed and flexed after disinfection with iodine tincture. 50 μL of iodoacetic acid (20 mg / ml) was injected into the knee joint cavity using a microsyringe to obtain osteoarthritis (OA) model rats.

[0097] 3. Treatment

[0098] After modeling, the rats in the non-long-term fried Fuzi decoction granules group / long-term fried Fuzi decoction granules group were gavaged with 5.4 g / kg / d, and the sham operation group and the model group were gavaged with the same mass of normal saline; the treatment lasted for 30 days.

[0099] 4. Statistical analysis and histopathological observation of pain in rat OA model

[0100] Pain behavioral testing: Mechanical hypersensitivity was measured using an "up-down" test method with a series of von Frey filaments (ranging from 0.6-26 g, UGO Basile, Italy). Briefly, the von Frey filament was pressed vertically against the mid-metatarsal surface of the hind paw with enough force to bend it, and the 50% PWT was calculated.

[0101] Sampling: After the experiment, the rats were anesthetized by intraperitoneal injection of sodium pentobarbital (3% concentration, 0.15ml / 100g), and blood was collected from the heart using a 10ml syringe. Then, scissors were used to remove the muscles near the knee joint, and the knee joint was removed using bone-breaking forceps, and fixed in a 4% paraformaldehyde solution for 3 days. After 3 days of fixation, the paraformaldehyde on the surface of the joint sample was rinsed off with running water, and then the sample was placed in an embedding box, marked, and placed in an EDTA solution for decalcification. The EDTA decalcification solution was replaced every day, and decalcification continued for about 2 months. When the bone tissue in the joint became soft, the sample was placed under running water and rinsed overnight. The sample was then placed in a dehydrator for dehydration, and finally embedded in paraffin. Each sample was cut into 3μm thick slices using a microtome for subsequent staining.

[0102] Staining: First, bake the tissue slices in a 60℃ oven overnight to prevent them from falling off; then dewax the slices in 3 cylinders of xylene, 10 minutes in each cylinder; then rehydrate with gradient alcohol (alcohol concentrations are 100%, 100%, 95%, 95%, and 70%), 5 minutes in each cylinder; finally, place in pure water and let stand for 3 minutes. HE staining steps are: dewaxing and rehydration; hematoxylin for 2 minutes, pure water rinse for 3 minutes / time, three times in total; 1% hydrochloric acid alcohol differentiation for 2 to 3 seconds, pure water rinse for 3 minutes / time, three times in total; 0.5% ammonia water anti-blue for 10 seconds, pure water rinse for 3 minutes / time, three times in total; eosin for 1 minute, pure water rinse for 3 minutes / time, three times in total; soak in 95% ethanol, 100% ethanol, xylene I, xylene II, and xylene III for 1 minute each, dehydrate and make transparent, and seal with neutral resin. The steps of SO staining are as follows: dewaxing and rehydration; fast green staining for 5 minutes, rinsing with pure water for 3 minutes each time, for a total of three times; differentiation with 1% acetic acid for 10 seconds, rinsing with pure water for 3 minutes each time, for a total of three times; safranin O staining for 1 minute, rinsing with pure water for 3 minutes each time, for a total of three times; differentiation with 95% ethanol for a few seconds, rinsing with pure water for 3 minutes each time, for a total of three times; drying in an oven at 37°C and then placing in xylene to make it transparent, and sealing with neutral resin.

[0103] Statistical analysis and histopathological observation of pain are shown in Figure 4 .

[0104] Depend on Figure 4 It can be seen that histopathological staining showed obvious cartilage degeneration, chondrocyte reduction and apoptosis, massive destruction of collagen, matrix disorder, and pain behavior test results showed that OA rats had more sensitive pain response, all of which showed a typical OA phenotype. Compared with the normal group, the number of chondrocytes and the content of glycosaminoglycans (an important component of cartilage matrix) in the model group were significantly reduced, and the Mankin's score was also significantly increased (P<0.01)( Figure 4 B). In addition, the mechanical pain threshold (reflected by MWT) of the model group was significantly decreased (compared with NC, P < 0.01).

[0105] 5. Immunohistochemistry

[0106] The steps are as follows: dewax and rehydrate the joint tissue sections of rats in each group, wash with PBS (1×) for 3 min / time, three times in total; perform antigen repair with sodium citrate solution in a 60°C oven for 4 h; take out the dye jar and let it warm up at room temperature for 30 min, wash with PBS (1×) for 3 min / time, three times in total; put the sections into 0.1% Triton solution to punch the tissue, wash with PBS (1×) for 3 min / time, three times in total; carefully wipe off the PBS around the tissue, draw a circle around the tissue with a water-blocking pen to fix the range of subsequent reagents; carefully wipe off the PBS around the tissue, add an appropriate amount of endogenous peroxidase blocker, incubate at room temperature for 10 min, wash with PBS (1×) for 3 min / time, three times in total; add 100 μL of primary antibody (Col2, MMP13, 1:100) diluted with PBS to each section, incubate at 4°C Overnight, wash with PBS (1×) for 3 minutes / time, three times in total; carefully wipe off the PBS around the tissue, add about 50μl of secondary antibody (enhanced enzyme-labeled goat anti-rabbit / anti-mouse IgG polymer), incubate at room temperature for 20 minutes, and wash with PBS (1×) for 3 minutes / time, three times in total; wipe off the PBS around the tissue, add an appropriate amount of freshly prepared DAB color developing solution, incubate at room temperature for 5-8 minutes, and wash with PBS (1×) for 3 minutes / time, three times in total; hematoxylin for 2 minutes, rinse with pure water for 3 minutes / time, three times in total; differentiate with 1% hydrochloric acid alcohol for 2-3 seconds, rinse with pure water for 3 minutes / time, three times in total; reverse blue with 0.5% ammonia water for 10 seconds, rinse with pure water for 3 minutes / time, three times in total; soak in 95% ethanol, 100% ethanol, xylene I, xylene II, and xylene III for 1 minute each, dehydrate and make transparent, and seal with neutral resin. After sealing, the sections were placed on a drying rack and ventilated in a fume hood for several hours. After the neutral resin solidified and the xylene evaporated, photos were taken under an optical microscope. The expression of MMP13 was quantified by counting the number of positive cells, and the expression of Col2 was quantified by calculating the positive area. The ratio of the antigen-positive area / cell volume in the selected area to the total area / total cell volume was used as the final statistical indicator.

[0107] Immunohistochemistry results are shown in Figure 5 .

[0108] Depend on Figure 5It can be seen that, in contrast, rats treated with Fuzi decoction showed a significant increase in the number of chondrocytes, a decrease in apoptotic chondrocytes, and improved the integrity of articular cartilage structure and the synthesis of glycosaminoglycans, which alleviated the pain sensitization of OA rats, and the effect of long-fried Fuzi decoction was better than that of non-long-fried Fuzi decoction. These results were also shown by Mankin's score. The Mankin's score of the model group rats was significantly higher than that of the normal group; while the Mankin's score of the Fuzi decoction group was significantly lower than that of the model group, and the effect of long-fried Fuzi decoction was better than that of non-long-fried Fuzi decoction. The results of immunohistochemistry showed that the main component of cartilage matrix (Col2) and cartilage matrix decomposition marker (MMP13) in the model group had abnormal changes, and after Fuzi decoction treatment, the expression of these markers was significantly restored to normal levels, and the effect of long-fried Fuzi decoction was better than that of non-long-fried Fuzi decoction. The results showed that Fuzi decoction had a successful therapeutic effect on OA rats, and the effect of long-decocted Fuzi decoction was better than that of non-long-decocted Fuzi decoction. This protective effect may be achieved by inhibiting the catabolism of chondrocytes, chondrocyte hypertrophy and promoting the anabolism of chondrocytes.

[0109] In summary, the technical solution of the present invention for preparing Fuzi decoction granules by long-term decoction of Fuzi has lower toxicity and better effect in treating osteoarthritis than non-long-term decoction Fuzi decoction granules.

[0110] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a Fuzi decoction granules for treating osteoarthritis, removing toxicity and retaining efficacy, It is characterized in that include: (1) Soak the aconite root for 25-35 minutes and boil it for 90-120 minutes; (2) Mix and soak Poria cocos, ginseng, bran-fried Atractylodes macrocephala, white peony root, red peony root, and cinnamon twig to obtain soaked Chinese medicine; (3) adding the Chinese medicine soaked in step (2) into the decoction container of step (1) and decocting together with aconite for 30 to 60 minutes, filtering to obtain a filtrate; (4) concentrating the filtrate from step (3) to obtain a paste, mixing it with maltodextrin, spray drying it, and granulating it to obtain the granules; The mass ratio of the aconite root, poria, ginseng, bran-fried atractylodes macrocephala, white peony root, red peony root, and cinnamon twig is 15:9:6:12:15:15:12; The mass ratio of the aconite root to water in step (1) is 1:8-12; The mass ratio of the total mass of the Poria cocos, ginseng, fried Atractylodes macrocephala, white peony root, red peony root, and cinnamon twig to water in step (2) is 1:8-12; The soaking time in step (2) is 25 to 35 minutes.

2. The preparation method according to claim 1, It is characterized in that The relative density of the paste is 1.02-1.

12.

3. The preparation method according to claim 1, It is characterized in that The mass ratio of the paste to maltodextrin is 1:0.

3.

4. The preparation method according to claim 1, It is characterized in that The spray drying temperature is 165-190°C.

5. The preparation method according to claim 1, It is characterized in that The particle size of the granules is 10-16 meshes.

6. The Fuzi Decoction granules obtained by the preparation method according to any one of claims 1 to 5, It is characterized in that The Fuzi Decoction granules contain benzoylhypaconitine≥43.35 μg / mg, benzoylmesaconitine≥268.52 μg / mg, benzoylaconitine≥28.75 μg / mg, hypoaconitine≤5.63 μg / mg, mesaconitine≤0.05 μg / mg, and do not contain aconitine.

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