CN116744908A

CN116744908A - Pharmaceutical composition comprising high levels of active principles of natural origin

Info

Publication number: CN116744908A
Application number: CN202180091670.7A
Authority: CN
Inventors: 康珉硕; 郑润锡; 李寿敏; 金度润; 文昇官; 郑多喜; 郑银河; 金廷勋; 朴如瞋; 柳宪昇; 崔源宰; 宋光镐
Original assignee: SK Chemicals Co Ltd
Current assignee: SK Chemicals Co Ltd
Priority date: 2020-11-24
Filing date: 2021-11-24
Publication date: 2023-09-12
Also published as: KR102618628B1; KR20230153973A; KR20220071956A; WO2022114795A1

Abstract

One aspect of the present disclosure relates to a preparation containing crude drug extracts of clematis root, trichosanthes root and prunella spike as active ingredients at a high content, which are useful for the treatment or improvement of arthritis and the like. The formulation according to one aspect of the present disclosure contains an active ingredient in a high content while also being small in size, is excellent in convenience of taking medicine, and has a release pattern and releasability that exhibit excellent therapeutic effects on arthritis even if taken 2 times for 1 day. Another aspect of the present disclosure relates to a formulation comprising 280-320mg of crude drug extracts of clematis root, trichosanthes root and prunella spike, which is taken 2 times a day, and which has a remarkable therapeutic effect on arthritis due to a specific release pattern.

Description

Pharmaceutical composition comprising high levels of active principles of natural origin

Technical Field

The present application claims priority based on korean patent application No. 10-2020-0159042 filed on 24 months 11 in 2020, the entire contents of the specification and the drawings of which are incorporated herein by reference.

The present disclosure relates to a preparation containing crude drug extracts of radix Clematidis, radix Trichosanthis, and Spica Prunellae. In particular, the present disclosure relates to a formulation that can be taken 2 times a day, contains high levels, and also has a release profile that exhibits significant efficacy.

Background

Osteoarthritis is the most common arthritis in adults known as degenerative arthritis. With the development of the advanced society, osteoarthritis patients are rapidly increasing, which not only causes the reduction of the personal life quality of the patients, but also causes socioeconomic loss.

Arthritis (arthritis) is a disease in which inflammation occurs at joints, and is very various. The most common arthritis is degenerative arthritis (osteoarthritis), and in addition to this, there are rheumatoid arthritis (rheumatoid arthritis), gout, psoriatic arthritis, and the like. The cause of the disease varies among arthritis, but the phenomenon of cartilage damage is the same.

Degenerative arthritis (osteoarthritis) is a representative chronic joint disease that occurs most frequently in arthritis. Degenerative arthritis is a phenomenon in which cartilage tissue surrounding the end of bones gradually wears out, causing inflammation and severe pain in joints, and subchondral bones are abnormally hardened. Therefore, cartilage that has a buffering effect on friction caused by joint movement is worn away, and severe pain and dyskinesia occur when the joint is moved.

Although there are many studies for controlling degenerative arthritis, fundamental therapies based on the pathological causes of the Disease have not been developed, and up to now, in addition to surgical methods, drug therapies for anti-inflammation and pain relief, such as nonsteroidal anti-inflammatory drugs (NSAIDs), disease-modifying osteoarthritis therapeutic agents (Disease-modifying osteoarthritis drug, DMOAD), and the like, have been mainly developed. In addition, although chondroprotective agents such as hyaluronic acid, glucosamine, and chondroitin have been developed and commercially available, their therapeutic effects have not been established in the fields of chondroprotection, regeneration induction, and the like.

On the other hand, complex crude drug extracts of Clematis chinensis, trichosanthis radix and Prunellae Spica are known to have anti-inflammatory, analgesic, joint protecting and immunosuppressive effects, and are widely used as therapeutic agents for osteoarthritis and rheumatoid arthritis.

Although tablets containing such crude drug extracts in high amounts can be produced, there is a possibility that the side effects caused by the increase may occur when the amount is simply increased in order to improve the efficacy. In addition, the preparation containing the compound crude drug extract of radix Clematidis, radix Trichosanthis and Spica Prunellae, which is commercially available at present, is administered 3 times a day, and the administration convenience is low.

Therefore, in order to prevent side effects caused by the high content formulation and to improve convenience of administration, a formulation having delayed release may be considered, but the results confirmed by the present inventors through experiments show unexpected problems in using a complex crude drug extract formulation of radix Clematidis, radix Trichosanthis and Spica Prunellae.

Disclosure of Invention

Accordingly, the present disclosure solves the problem of providing a preparation, particularly a tablet, which contains extracts of radix Clematidis, radix Trichosanthis and Spica Prunellae as active ingredients in a high content, and which is small in size and convenient to take, has a release pattern that can exhibit a preferable drug effect, and can be taken 2 times a day.

In order to solve the above problems, an aspect of the present application provides a preparation, particularly a tablet, comprising 280 to 320mg of crude drug extracts of Clematis chinensis, trichosanthis radix and Prunellae Spica as an active ingredient, wherein the content of the active ingredient is 40 to 90% by weight (preferably 50 to 90% by weight, more preferably 60 to 90% by weight) relative to the total weight of the pre-coated tablet or the pre-empty capsule mixture, and ethyl cellulose, an ethyl methacrylate copolymer or a mixture thereof is contained to regulate release.

The present inventors manufactured a preparation containing crude drug extracts of Clematis chinensis, trichosanthis radix and Prunellae Spica in high content for 2 times per 1 day, but the results of the study showed unexpected problems. In particular, while various binders may be used to delay or modulate release, this presents a problem in that it is difficult to completely dissolve all the extracts contained therein. In further studies, relatively more excipients, disintegrants are used to reduce the relative content of the extract, also to achieve delayed release and good release. However, the size of 1 granule of the preparation to be taken in this case becomes too large, and the taking is inconvenient. That is, due to the special physical properties of the crude drug extracts of radix Clematidis, radix Trichosanthis and Prunellae Spica, it is not easy to contain high contents of the crude drug extracts of radix Clematidis, radix Trichosanthis and Prunellae Spica at high weight% relative to the weight of the preparation and to achieve both good release pattern and high release property.

The present inventors have confirmed that such contradictory problems can be eliminated by using ethylcellulose, an ethyl methacrylate copolymer or a mixture thereof as a binder, thereby completing one aspect of the present application.

As crude drug extracts of clematis root, trichosanthes root and prunella spike of the present application, extract of "clematis root, trichosanthes root, prunella spike 30% ethanol extraction (40→1)" in a powder state having hygroscopicity of yellow brown-brown may be used, and such crude drug extracts of clematis root, trichosanthes root and prunella spike may be crude drug extracts manufactured according to the method described in korean patent No. 0180567, no. 0483707 (WO 2002-094301 A1) or the like, for example.

Of the above 2 binder components of the present application, ethylcellulose is more preferable, and thus, a preferred embodiment of the present application provides a preparation, particularly a tablet, characterized in that the preparation contains 280-320mg of crude drug extracts of radix Clematidis, radix Trichosanthis and Spica Prunellae as an active ingredient, the content of which is 40 to 90 wt% (preferably 50 to 90 wt%, more preferably 60 to 90 wt%) relative to the total weight of the tablet before coating, and ethylcellulose is contained to regulate release.

As such ethylcellulose, the following ethylcellulose solution can be used: a5% by weight solution was prepared with a toluene/ethanol 80:20 (volume ratio) mixed solvent, having a viscosity of 3 to 22mPa.s when the viscosity was measured at normal temperature.

The present inventors developed a preparation comprising 280-320mg of crude drug extracts of Clematis chinensis, trichosanthis radix and Prunellae Spica, which is administered 2 times a day. For this reason, formulations having various release patterns (rates) were manufactured and evaluated, and as described below, in the case of having a specific release pattern, the therapeutic effect on arthritis (for example, osteoarthritis (degenerative arthritis), rheumatoid arthritis, etc.) was remarkably remarkable. In general, for a formulation to be administered 2 times per 1 day, the sustained release properties are generally adjusted so as to release the active ingredient for a long period of time, similarly to examples 4 to 4 described later. However, very particularly, as shown in fig. 4 to 10, for the purpose of treating arthritis by crude drug extracts of clematis root, trichosanthes root and prunella spike, the effect of treating arthritis is improved as compared with a preparation taken 3 times a day only to a degree that the release of the active ingredient is slightly delayed (see fig. 4).

That is, an aspect of the present disclosure provides a dissolution pattern showing a preferred arthritis therapeutic effect in a formulation comprising 280-320mg of crude drug extracts of Clematis chinensis, trichosanthis radix and Prunellae Spica and taken 2 times for 1 day. In the dissolution pattern of the present application, which can achieve an excellent therapeutic effect on arthritis, when the dissolution test is performed under the condition of 900ml of water medium by the paddle (pad) test method at 37.+ -. 0.5 ℃ and 50rpm, the release of rosmarinic acid is 45 minutes 40 to 70% (more preferably 45 to 65%, still more preferably 45 to 60%), 90 minutes 75% or more (preferably 80% or more), and 120 minutes 80% or more (preferably 85% or more).

If it is stated that this mode of release can be achieved, other additives than the binders or delayed release agents mentioned above or described below can be used. That is, the scope of the present application is not limited to the types of the specific additives described above or described later, as long as the conditions can achieve the specific release pattern according to the present disclosure. The specific additives described above or below are only used to achieve a more preferred aspect of the present disclosure.

Specifically, another aspect of the present application provides a preparation for treating arthritis, in particular, a tablet, comprising 280 to 320mg of "30% ethanol extract (40→1) of Clematis chinensis and Prunella vulgaris" as an active ingredient, which is administered 2 times per 1 day, wherein when a dissolution test is performed under the condition of 900ml of water medium by a paddle (pad) test method at 37.+ -. 0.5 ℃ and 50rpm, the release of Rosmarinic Acid (Rosmarinic Acid) is 45 minutes to 70%, 90 minutes to 75% (more preferably 45 minutes to 40 minutes to 70%, 90 minutes to 75% to 120 minutes to 80%) or more). Wherein, the content of the extract of the above radix Clematidis, radix Trichosanthis, prunellae Spica 30% ethanol extraction (40→1) is preferably 40 to 90 wt% (preferably 50 to 90 wt%, more preferably 60 to 90 wt%) relative to the total weight of the mixture before filling of the tablet or empty capsule before coating.

In a preferred embodiment of the present application, the preparation for treating arthritis is a tablet or capsule comprising 280 to 320mg of crude drug extracts of Clematis chinensis, trichosanthis radix and Prunellae Spica as active ingredients, and is administered 2 times per 1 day, and when the dissolution test is performed under the condition of 900ml of water by a paddle (pad) test method at 37.+ -. 0.5 ℃ and a speed of 50rpm, the release of rosmarinic acid is 45 to 65% for 45 minutes, 80% or more for 90 minutes, 85% or more for 120 minutes (more preferably 45 to 60% for 45 minutes, 80% or more for 90 minutes, and 85% or more for 120 minutes). Wherein, the content of the extract of the above-mentioned "radix Clematidis, radix Trichosanthis, spica Prunellae 30% ethanol extraction (40→1)" is preferably 40 to 90% by weight (preferably 50 to 90% by weight, more preferably 60 to 90% by weight) relative to the total weight of the pre-coated tablet or capsule filling excluding the weight of empty capsules.

Preferably, a preferred embodiment of the present application provides a tablet comprising 280 to 320mg of "30% ethanol extract (40→1) of radix Clematidis trichosanthes kirilowii and Prunellae Spica" extract as an active ingredient, wherein the content of the active ingredient is 40 to 90% by weight (preferably 50 to 90% by weight, more preferably 60 to 90% by weight) based on the total weight of the tablet before coating, and the release of rosmarinic acid is 45 minutes to 70%, 90 minutes to 75% (more preferably 45 minutes to 70%, 90 minutes to 75%, 120 minutes to 80%, more preferably 45 minutes to 65%, 90 minutes to 80%, 120 minutes to 85% or more) when the tablet is taken 2 times per 1 day by a paddle (pad) test method at 37±0.5 ℃ and 50rpm under a condition of 900ml medium of water, and the release adjustment is achieved by the inclusion of ethylcellulose, an ethyl methacrylate copolymer or a mixture thereof (more preferably ethylcellulose).

The present application provides a tablet comprising 280 to 320mg of crude drug extracts of Clematis chinensis, trichosanthes kirilowii Maxim and Prunella vulgaris as active ingredients, wherein the content of the active ingredients is 40 to 90% by weight (preferably 50 to 90% by weight, more preferably 60 to 90% by weight) based on the total weight of the tablet before coating, and the release of rosmarinic acid is adjusted by ethyl cellulose when the tablet is taken 2 times per 1 day under conditions of 900ml of water by a paddle test method at 37.+ -. 0.5 ℃ and a speed of 50rpm, wherein the release of rosmarinic acid is 45 minutes to 70%, 90 minutes 75% or more, 120 minutes 80% or more (more preferably 45 minutes to 65%, 90 minutes 80% or more, 120 minutes 85% or more). The content of the above ethylcellulose is preferably 0.1 to 10% by weight, more preferably 0.4 to 5% by weight, and still more preferably 0.8 to 3% by weight, relative to the total weight of the pre-coated tablet.

In one embodiment of the present application, the tablet may further optionally contain a pharmaceutically acceptable excipient, an adsorbent, a disintegrant, a lubricant, and the like.

In one aspect of the present application, as the release regulator, various substances such as polyvinylpyrrolidone, ethylcellulose, methacrylic acid ethyl ester copolymer, hydroxypropyl methylcellulose, hydroxypropyl cellulose, and acrylic acid copolymer can be used. However, in view of achieving good releasability while reducing the size of the tablet, ethylcellulose, ethyl methacrylate copolymer or a mixture thereof is preferable. The release-modifying substance is preferably 0.1 to 10% by weight, more preferably 0.4 to 5% by weight, still more preferably 0.8 to 3% by weight, relative to the total weight of the pre-coated tablet.

As the excipient, microcrystalline cellulose, lactose, mannitol, etc. can be used, and the content of such excipient is preferably 5 to 40% by weight, more preferably 10 to 35% by weight, still more preferably 15 to 30% by weight, relative to the weight of the tablet before coating.

As the above-mentioned adsorbent, light anhydrous silicic acid (including hydrophobic light anhydrous silicic acid), aluminum magnesium silicate, and the like can be used, and the content of such adsorbent is preferably 0.5 to 9% by weight, more preferably 1 to 7% by weight, still more preferably 2 to 5% by weight, relative to the weight of the tablet before coating.

As the above-mentioned disintegrants, croscarmellose sodium, sodium carboxymethyl starch, crospovidone, low-substituted hydroxypropylcellulose, and the like can be used. However, in terms of achieving good releasability while reducing the size of the tablet, croscarmellose sodium, low-substituted hydroxypropylcellulose, or a mixture thereof is preferable. When croscarmellose sodium, low-substituted hydroxypropylcellulose or a mixture thereof is used, good releasability is achieved even under the condition that the content of the active ingredient becomes high. As the disintegrant, even if it is croscarmellose sodium or low substituted hydroxypropylcellulose, for the purpose of the present application, croscarmellose sodium is more particularly preferred. Such disintegrants are preferably present in an amount of 1 to 10% by weight, more preferably 1 to 8% by weight, and even more preferably 2 to 5% by weight, relative to the weight of the pre-coated tablet.

As the lubricant, stearyl fumarate, magnesium stearate, stearic acid, talc, etc. can be used, and the content of such lubricant is preferably 0.1 to 4% by weight, more preferably 0.2 to 2% by weight, relative to the weight of the pre-coated tablet.

In one embodiment of the present application, the tablets according to the present application may also be coated for various purposes of protection from external impact, appearance, regulation of release, etc. For example, the tablet may be coated with a coating composition comprising a film former, a plasticizer, a releasing agent, etc., and may be coated in an amount of 1 to 15% by weight, preferably 3 to 10% by weight of the coating substance based on the total weight of the tablet before coating. As such a film coating composition (article), various coating material articles sold by Colorcon corporation, which contain polyvinyl alcohol as a basic coating substance, can be used.

A preferred embodiment of the present application provides a tablet comprising 280 to 320mg of crude drug extracts of Clematis chinensis, trichosanthis radix and Prunellae Spica as active ingredients, wherein the content of the active ingredients is 40 to 90% by weight (preferably 50 to 90% by weight, more preferably 60 to 90% by weight) based on the total weight of the tablet before coating, and the tablet is administered 2 times per 1 day, and when the dissolution test is carried out under the condition of 900ml of water medium by a paddle (pad) test at 37.+ -. 0.5 ℃ and 50rpm, the release of rosmarinic acid is 45 minutes to 70%, 90 minutes to 75% or more, 120 minutes to 80% or more (more preferably 45 minutes to 65%, 90 minutes to 80% or more, 120 minutes to 85% or more), and the release is adjusted by ethyl cellulose, and the tablet comprises croscarmellose sodium as a disintegrant.

The above-mentioned preparation according to the present application can be produced into tablets, capsules, etc. by wet granulation using a solvent such as water, ethanol, isopropyl alcohol, etc. or dry granulation using an extrusion force, and then mixing the granules with a disintegrating agent, a lubricant, etc. and then mixing the components. The above formulation according to the present application can be manufactured into tablets by direct compression without a granule manufacturing process. However, in terms of maintaining the release pattern for achieving one of the objects of the present application without deviation, a wet granulation tablet may be preferable.

Accordingly, one embodiment of the present application provides a method for producing a tablet, comprising the steps of: making granule from crude drug extracts of radix Clematidis, radix Trichosanthis and Prunellae Spica, adsorbent, binder, optional excipient, optional disintegrating agent, etc.; a step of mixing the above-produced granules with pharmaceutically acceptable additives such as a lubricant and a disintegrant, and tabletting the mixture to produce tablets; and optionally a step of coating the above-mentioned tablets.

One aspect of the present disclosure provides a preparation containing a high content of crude drug extracts of radix Clematidis, radix Trichosanthis, and Spica Prunellae, which can be administered 2 times a day. The formulation according to the present disclosure has a specific release pattern, and shows excellent therapeutic effects on arthritis even if taken 2 times for 1 day. In addition, the preparation according to one aspect of the present disclosure contains crude drug extracts of radix Clematidis, radix Trichosanthis, and Spica Prunellae in high content, and is small in size and excellent in convenience of administration.

Drawings

The following drawings attached to the present specification serve to further understand the technical idea of the present application together with the above-described aspects of the application, and therefore, the present application is not to be construed as being limited to only the matters described in such drawings.

FIG. 1 is a graph showing the dissolution pattern in a pH1.2 medium according to the kind of binder.

FIG. 2 is a graph showing dissolution patterns in a pH6.8 medium according to ethylcellulose viscosity.

Fig. 3 is a graph showing dissolution patterns in water according to examples and comparative examples of the present application.

Fig. 4 is a graph showing dissolution patterns of a formulation having a plurality of dissolution patterns in water.

Fig. 5 is a result of evaluating the effect of a formulation with multiple dissolution patterns on gait after performing Meniscectomy and anterior cruciate ligament resection.

Fig. 6 is a result of evaluating the effect of a formulation having various dissolution patterns on arthritis, which is a bipedal balance test (Incapacitance test).

FIG. 7 is the results of ELISA assays to evaluate the effect of formulations with various dissolution patterns on cytokine production in arthritis.

FIG. 8 is the results of an immunohistochemical staining analysis to evaluate the effect of a formulation with multiple dissolution patterns on the biomarker in arthritis.

Fig. 9 is a result of evaluating the effect of a formulation having a plurality of dissolution patterns by an osteoarthritis score (OARSI score) through a histopathological analysis.

FIG. 10 shows the results of immunohistochemical analysis, i.e., hematoxylin-Eosin (H & E) and Safranin O (Safranin-O) staining of Joint surface (Joint surface), cartilage (cartilage) and synovium (synovium) after the end of the experiment.

Detailed Description

In the following, examples and the like are described in detail to facilitate understanding of the present application. However, the embodiment according to the present application may be modified into various forms, and the scope of the present application is not to be interpreted as being limited to the following embodiments. The embodiments of the present application are provided to more fully illustrate the application to those skilled in the art.

Example 1: dissolution pattern validation using multiple binders

Tablets containing 30% ethanol extract (40.fwdarw.1) of radix Clematidis, radix Trichosanthis, and Spica Prunellae were prepared by using the contents and components shown in Table 1. Mixing the extract, crospovidone, hydrophobic light anhydrous silicic acid and binder. Then adding magnesium stearate, mixing and tabletting.

TABLE 1

Polyvinylpyrrolidone: kollidon K30, viscosity of about 5.5-8.5 cps (20 ℃,10% w/v)

Ethyl methacrylate copolymer: eudragit L100-55 with a viscosity of about 50-200 cps

Hydroxypropyl methylcellulose: metolose, viscosity of about 100cps (20 ℃/2% w/v)

Hydroxypropyl cellulose: nisso HPC-L with a viscosity of about 6-10 cps (20 ℃/2% w/v)

Acrylic acid copolymer: carbomer 971, viscosity about 4000-11000 (0.5% w/w)

Experimental example 1: evaluation of elution according to the type of binding agent

The dissolution test of the tablets manufactured in example 1 above was performed using the korean pharmacopoeia 12 th edition dissolution test method. The stirring was carried out at a stirring speed of 50rpm and a dissolution temperature of 37.0.+ -. 0.5 ℃ using a paddle method. The dissolution was carried out in pH 1.2.900 ml. To improve the stability of the test solution, the assay was performed after pretreatment with acetonitrile. The dissolution test results are shown in table 2 below and fig. 1. The dissolution rate was measured as the time-dependent content of rosmarinic acid as the active ingredient of the crude drug extract. The rosmarinic acid content was determined by the same method as described in document J.Sep.Sci.2015,0,1-8 ("Quality evaluation of Salvia miltiorrhiza Bge.by ultra high performance liquid chromatography with photodiode array detection and chemical fingerprinting coupled with chemometric analysis").

TABLE 2

As shown in Table 2 above, examples 1-1, 1-4, 1-6 exhibited large dissolution retardation effects, and examples 1-2 and 1-3 exhibited similar dissolution retardation effects. The dissolution retardation effect of examples 1 to 5 was small. However, in the other examples except examples 1-2 and 1-3, even if the release for a very sufficient time was evaluated, it was confirmed that the elution amount was less than 80%, and it was found that the release was not practically usable. This is presumably because the combination of physical properties of the specific extract and physical properties peculiar to the polymer used in the present application is considered to be related to, for example, a function of preventing gelation of the crude drug extract, but the present application is not limited to such a theoretical mechanism.

The ethyl methacrylate copolymer used in examples 1-3 has a problem in that it shows a preferable release pattern in an acidic medium, but the release pattern is different in a different pH medium, and thus it is not easy to adjust the release, and as a result, it is slightly less desirable than the ethylcellulose of examples 1-2.

The inventors have conducted various evaluations, and it is considered that the dissolution pattern shown in examples 1-2 or 1-3 in the medium of pH1.2 is a preferable dissolution pattern of a preparation containing 300mg of a tablet taken 2 times, and therefore, in such a layer, the binders other than examples 1-2 and 1-3 have a problem of too delayed release.

As a result, surprisingly, as a release regulating component of a formulation comprising 300mg of crude drug extract of clematis root, trichosanthes root and prunella spike and taken 2 times a day, ethylcellulose and ethyl methacrylate copolymer, more preferably ethylcellulose, are preferable as compared with HPMC and the like which are generally used.

Example 2: delayed dissolution mode validation using ethylcellulose

A tablet containing 30% ethanol extract (40.fwdarw.1) of radix Clematidis, radix Trichosanthis, and Spica Prunellae was prepared by using the contents and components shown in Table 3 below. First, ethyl cellulose was dissolved in isopropyl alcohol to prepare a binding solution, and then an extract and hydrophobic light anhydrous silicic acid were added to the binding solution to prepare wet particles. The wet granules produced above were mixed with crospovidone and magnesium stearate, and tableted.

TABLE 3

Component (Unit: mg)	Example 2-1	Example 2-2	Examples 2 to 3	Examples 2 to 4
					Crude drug extract	300	300	300	300
Hydrophobic light anhydrous silicic acid	15	15	15	15
					Ethyl cellulose 4cps	20
Ethyl cellulose 7cps		20
					Ethylcellulose 10cps			20
Ethylcellulose 20cps				20
					Crosslinked povidone	15	15	15	15
Magnesium stearate	2	2	2	2
					Total weight of tablet	352	352	352	352

Experimental example 2: evaluation of dissolution in a Medium at pH6.8 according to ethylcellulose viscosity

The dissolution of examples 2-1 to 2-4 described above was evaluated by the dissolution test method of korean pharmacopoeia version 12. The stirring was carried out at a stirring speed of 50rpm and a dissolution temperature of 37.0.+ -. 0.5 ℃ using a paddle method. The dissolution was carried out in pH 6.8.900 ml. To improve the stability of the test solution, the assay was performed after pretreatment with acetonitrile. The results of the dissolution test of rosmarinic acid are shown in table 4 and fig. 2 below.

TABLE 4

Time (minutes)	Example 2-1	Example 2-2	Examples 2 to 3	Examples 2 to 4
					5	12	10	9	6
10	13	12	14	11
					15	17	17	17	16
30	27	25	25	25
					45	34	33	32	33
60	43	39	39	39
					90	65	60	52	56
120	92	85	72	78
					180	96	95	94	92
240	96	95	95	93

As shown in table 4 above, the formulation of the present application using ethylcellulose showed a good release pattern even at ph6.8, and could show a release of nearly 100% even at ph 6.8. Further, from examples 2-1 to 2-4, which are modes in which dissolution is slower and slower, it was confirmed that this is consistent with the viscosity of ethylcellulose becoming higher.

Example 3 and comparative example 1: evaluation of dissolution property based on various components

Tablets were produced according to the contents and components described in table 5 below. Example 3 crude drug extracts of radix Clematidis, radix Trichosanthis and Spica Prunellae, hydrophobic light anhydrous silicic acid and ethyl cellulose were prepared by wet granulation. Microcrystalline cellulose, croscarmellose sodium, and stearyl fumarate were mixed in the wet granules produced as described above, and the mixture was tableted. The core thus produced is coated with a coating material. Comparative example 1 crude drug extracts of radix Clematidis, radix Trichosanthis and Spica Prunellae, light anhydrous silicic acid, corn starch, microcrystalline cellulose and sodium carboxymethyl starch were prepared by dry mixing. Magnesium stearate was mixed with the above-produced mixture, followed by tabletting. The core thus produced is coated with a coating material.

TABLE 5

Component (Unit: mg)	Example 3	Comparative example 1
			Crude drug extract	300	200
Microcrystalline cellulose	95	113
			Corn starch		50
Light anhydrous silicic acid		10
			Hydrophobic light anhydrous silicic acid	10
Ethyl cellulose 4cps	8
			Croscarmellose sodium	13
Sodium carboxymethyl starch		25
			Magnesium stearate		2
Stearyl fumarate	2
			Coating material	28	30
Total weight of tablet	456	430

Experimental example 3: dissolution evaluation of example 3 and comparative example 1

The dissolution patterns of example 3, comparative example 1 and comparative example 2 described above were evaluated by the korean pharmacopoeia 12-edition dissolution test method. The stirring was carried out at a stirring speed of 50rpm and a dissolution temperature of 37.0.+ -. 0.5 ℃ using a paddle method. The dissolution was carried out in 900ml of purified water. To improve the stability of the assay, the assay was performed after pretreatment with acetonitrile and pH1.2 buffer. The results of the dissolution test of rosmarinic acid are shown in table 6 and fig. 3 below.

TABLE 6

Time (minutes)	Example 3	Comparative example 1
			5	5	6
10	14	24
			15	20	39
30	40	67
			45	57	81
60	71	82
			90	88	84
120	94	85
			180	98	86
240	99	86

As shown in table 6 above, the formulation according to the application shows a good release pattern even in water. The inventors of the present application conducted various evaluations and found that when the dissolution medium was water, the dissolution mode (for example, 45 minutes 40 to 70%, 90 minutes 75% or more, and more preferably 120 minutes 85% or more) as shown in example 3 was preferable, but it was not easy to realize the dissolution mode as described above. For example, the dissolution results of comparative example 1 show that although the use of excipients and disintegrants in large amounts reduced the content of the extract and increased the initial dissolution rate, the final dissolution rate was not increased.

Example 4: drug efficacy evaluation according to dissolution pattern

After tablets of 30% ethanol extracts (40.fwdarw.1) of radix Clematidis, radix Trichosanthis, and Spica Prunellae having various elution patterns were prepared according to the formulations shown in Table 7 below, the effect of the elution pattern on the drug efficacy was evaluated. 200mg quick-release tablet, 300mg delayed-release tablet and 300mg sustained-release tablet were produced. First, a binding agent is dissolved in water, ethanol, isopropanol, or a suitable solvent among these solvents to prepare a binding solution. Then, the extract, light anhydrous silicic acid, excipient, and the like are added to the binding liquid, thereby producing wet particles. The wet granules produced as described above were mixed with a disintegrant and a lubricant, and tableted. Film coating is then carried out by conventional methods.

TABLE 7

Experimental example 4-1: dissolution evaluation

Dissolution of 200mg immediate release tablet, 300mg delayed release tablet and 300mg sustained release tablet was evaluated by the same method as in experimental example 3. That is, the stirring was carried out at a stirring speed of 50rpm and a dissolution temperature of 37.0.+ -. 0.5 ℃ using a paddle method. The dissolution was carried out in 900ml of purified water. The dissolution results of rosmarinic acid are summarized in table 8 below and shown in fig. 4.

TABLE 8

Time/dissolution% (n=6)	Example 4-1	Example 4-2	Examples 4 to 3	Examples 4 to 4
					0	0	0	0	0
5	3	6	3	2
					10	24	21	9	5
15	45	36	15	7
					30	85	71	31	12
45	98	93	49	16
					60	100	102	66	20
90	102	106	88	29
					120	104	106	95	38
180	105	105	100	57
					240	106	106	103	71
360	106	105	105	89

The time when the average dissolution rate reached 80% was observed, and it was confirmed that "200mg immediate release tablet" was 30 minutes, "300mg immediate release tablet" was 45 minutes, "300mg delayed release tablet" was 90 minutes, and "300mg sustained release tablet" was 360 minutes. Therefore, each dosage form is judged to have the characteristics of a quick release tablet, a delayed release tablet and a slow release tablet respectively.

Experimental example 4-2: evaluation of therapeutic Effect of osteoarthritis

Experimental animals, test substances and test groups

Osteoarthritis treatment efficacy was evaluated using Beagle dog (Beagle dog) Meniscectomy (Meniscectomy) and anterior cruciate ligament resection (ACLT) models. As test substances, 200mg immediate release tablets, 300mg delayed release tablets and 300mg sustained release tablets manufactured in example 4 were used. As a positive control, celecoxib preparation (manufactured (lot) No. EF6274, capsule) from the company of the Buddha was used. As a negative control group (blank), tablets composed of the formulation shown in table 9 below were used.

TABLE 9

Component (Unit: mg)	Blank (placebo)
		Microcrystalline cellulose PH102 (excipient)	319
Corn starch (excipient)	47
		Sodium carboxymethyl starch (disintegrant)	9
Magnesium stearate (Lubricant)	5
		Film coating	29
Total weight of tablet	409

The composition of the test groups is shown in table 10 below.

TABLE 10

Beagle dog (Beagle dog) (Xi An Dile general biomedical limited (Xi' an Dilepu Biology & Medicine co., ltd)) was orally administered for 8 weeks according to the number of administrations of table 9 described above. Specifically, after the animal opens the mouth in a state of being naturally placed in the rearing box, the test substance is placed on the inner side of the tongue and the mouth is closed, and then the animal swallows by gently touching the throat. About 10mL of water was injected with a syringe after swallowing was confirmed.

Meniscectomy (Meniscectomy) and anterior cruciate ligament resection

Prior to surgery, hair around the knees on both sides of the animal was removed with scissors (clippers). Animals were anesthetized and the portions to be dissected were extensively sterilized with Povidone (Povidone) and 70% alcohol (alcohol). Then, the skin of the right knee was incised. The surrounding tissue is blunt disjunct (exposing) the articular surface of the thigh end. A defect window is formed on the inner joint surface, and the anterior cruciate ligament is resected. Wound closure was performed using 4-0 nylon. For the left knee, only anterior cruciate ligament resections were performed.

After 1 week of Meniscectomy (Meniscectomy), the animals were subjected to 1/day, 7 days of manual exercise (30 minutes/day).

Viewing and inspection items

(1) Gait evaluation

Gait evaluation was performed to evaluate the efficacy of the formulation manufactured in example 4 above on an animal model of osteoarthritis. The test substance was administered 2 times/week before and after administration. Gait evaluation was performed based on the evaluation criteria shown in table 11, and images were captured by a digital camera.

TABLE 11

Score of	Parameters (parameters)
		0	No apparent lameness
1	Intermittent, slight weight-loaded lameness, slightly altered gait
		2	Moderate body weight load lameness-apparent lameness, obvious gait change
3	Severe weight load lameness-just "touch toe"
		4	Non-load-bearing

The results are shown in FIG. 5. As shown in fig. 5, the result of 300mg of the delayed release sheet G6 is most preferable. In particular, only 300mg delayed release tablet G6 having a specific dissolution pattern showed good results compared to 300mg immediate release tablet G5 and 300mg sustained release tablet G7, which is a very particular result.

(2) Bipedal balance test (Incapacitance test)

The effect of the dosage form manufactured in example 4 on the weight load of an animal model of osteoarthritis was confirmed. Hindlimb weight was measured using a bipedal balance tester (Incapacitance tester) (1029-S, linton instrumentation, USA). In this trial, for osteoarthritis induced beagle dogs, the right hindlimb, on which both anterior and medial meniscectomy were performed, presented more severe pain than the left hindlimb, on which only anterior cruciate ligament resection was performed, and thus relied on left hindlimb to stand, thus determining that the weight load of the right hindlimb was relatively lighter than that of the left hindlimb. In the bipedal balance test, the weights (g) of the feet on both sides were measured without touching the instrument sensor on the abdomen of the beagle, and the body weight load ratio (%) was calculated by the method of the following expression 1 using the measured weights of the feet. The weight load is a force supported by the feet and the weight of the feet is balanced in a normal state, and therefore the weight load (%) of one foot is shown to be 50%, but the more severe the pain caused by the osteoarthritis induction, the lower the weight load (%) of the leg after the osteoarthritis induction.

[ calculation formula 1]

Weight load ratio (%) = [ weight of osteoarthritis-induced hind leg/(weight of both hind legs) ]×100

The post-limb heavy load distribution rate level was measured 1 time per week and 8 weeks before and after the administration of the test substance using a bipedal balance tester (Incapacitance tester). The results are shown in FIG. 6.

As shown in fig. 6, the result of 300mg of the delayed release sheet G6 is most preferable. In particular, only 300mg delayed release tablet G6 having a specific dissolution pattern showed good results compared to 300mg immediate release tablet G5 and 300mg sustained release tablet G7, which is a very particular result.

(3) ELISA assay

To confirm the activity of biomarkers associated with arthritis development, three cytokines (Cytokine) were analyzed for IL-1 beta, MMP-3 and TNF-alpha as biomarkers in joint fluid using joint fluid collected on the day of necropsy. The assay utilized a commercially available ELISA kit (kit). The results are shown in FIG. 7.

One of the powerful mechanisms for inducing osteoarthritis is the increased production of pro-inflammatory cytokines (pro-inflammatory cytokine) such as TNF- α, IL-1 β, IL-6, and the like, and the increased secretion of MMPs such as collagenase (collagenase) and stromelysin (stromelysin), thereby inducing damage to articular cartilage. That is, it is known that when osteoarthritis is developed, the expression of MMP-3, MMP-9, MMP-13, etc. is increased, and the increase of MMP causes damage to collagen matrix (collage matrix) constituting cartilage, thereby deteriorating degenerative arthritis. Furthermore, the following facts are ascertained: in the breakdown of aggrecan (aglecan) and type II collagen (type II collagen), which are representative chondrocyte extracellular matrices, ADAMTS5 and MMP13 play a decisive role in the development of degenerative arthritis.

There are various causes of the way arthritis progresses. One of the important reasons is related to the activity of macrotage (macrophages). When macrophages (macrotage) present in synovial fluid are activated by PAMPs, DAMPs and inflammatory cells (inframammomers), polarization is M1 macrophages (macrotage) which exert an effect associated with inflammation. Such M1-macrophages (macrocage) secrete inflammatory cytokines (cytokine) (e.g., IL-1B, IL-6, TNF- α), growth factors, MMPs (ex: MMP1, MMP2, MMP3, MMP 13) and TIMP, which, as a result, cause inflammation, cartilage breakdown and osteophyte formation (osteophyte formation). Such cytokines (cytokine), MMPs and secreted proteins maintain macrophage (macrocage) activity of M1 type in an autocrine (autocrine) form, and M1-macrophage (macrocage) promotes the breakdown of extracellular matrix (ECM) components by altering signaling pathways of chondrocytes including TGF- β, JNK, akt, NF- κb and β -catenin signaling. The ECM thus degraded, by acting as a DAMP, stimulates macrophage activation and polarization (polarization) to M1-macrophages again, leading to repetition of inflammation and cartilage degradation.

The levels (levels) of the major cytokines (cytokine) (IL-1. Beta., TNF-. Alpha.) and MMPs (MMP 3) secreted in macrophages (macrocage) responsible for such arthritis were confirmed by ELISA examination, and as a result, the levels (levels) of IL-1. Beta. In G3, G4, G5, G6 and G7 were significantly reduced compared to the induced group G2, and for the levels (levels) of TNF-. Alpha. The levels (levels) were significantly reduced in the test groups G3, G4, G5 and G6 compared to the induced group G2. For MMP3 levels (levels) in joint fluid, the test groups G3, G4, G5, G6 and G7 were significantly reduced compared to the induction group G2. As shown in fig. 7, the result of 300mg of the delayed release sheet G6 is most preferable. In particular, only 300mg delayed release tablet G6 having a specific dissolution pattern showed good results compared to 300mg immediate release tablet G5 and 300mg sustained release tablet G7, which is a very particular result. From such results, it is considered that the levels (levels) of inflammatory cytokines (cytokine) and MMP3 are reduced, and the ELISA-based examination is helpful for the alleviation of arthritis.

(4) Immunohistochemical staining

In addition, in order to confirm the blocking effect of MMP-13, a biomarker involved in the occurrence of arthritis, and the production effect of type II Collagen (Collagen type II), analyses of type II Collagen (Collagen type II) and MMP-13 were performed with an immunohistochemical staining (IHC) marker.

On the day of necropsy, joint fluid was collected, and then, after euthanizing the animals, visual observation of the defect was performed and photographed. Defective parts were extracted and fixed in 10% neutral buffered formalin solution. The collected joint fluid was stored in an ultralow temperature freezer set at-70 ℃ or lower until analysis. The fixed tissue is subjected to conventional tissue treatment processes such as deashing, trimming, dehydrating, paraffin embedding, stripping and cutting to prepare a sample for histopathological examination. Then, hematoxylin-Eosin (H & E), safranin O (Safranin-O) and immunohistochemical staining (Collagen type II) and MMP-13) were performed, staining was performed, histopathological changes were observed using an optical microscope (Olympus BX53, japan), and analysis of immunohistochemical staining was performed using an Image analyzer (Zen 2.3blue edition,Carl Zeiss,Germany).

In the case of immunohistochemical staining, MMP13 (R & D System, MAB 511-500), collagen II (Collagen II) (abcan, ab 34712) were used as primary antibodies, in the case of MMP13, DAKO, envision + System-HRP labelled polymer anti-mouse (K4001) was used as secondary antibodies, and in the case of Collagen II DAKO, envision + System-HRP labelled polymer anti-rabit (K4003) was used as secondary antibodies. Then, after staining with DAB, contrast staining was performed with Hematoxylin (Hematoxylin).

The results are shown in fig. 8.

As shown in fig. 8, 300mg of the delayed release sheet G6 showed good results. In particular, only 300mg delayed release tablet G6 having a specific dissolution pattern showed good results compared to 300mg immediate release tablet G5 and 300mg sustained release tablet G7, which is a very particular result.

(5) Histopathological analysis experiment

In the OARSI scoring (score) project after H & E staining, cartilage structure (Cartilage structure) and chondrocyte (chondryte) levels in the articular surface were confirmed, and synovial lining (synovial lining), inflammatory cell infiltration (inflammatory cell infiltration), and synovial hyperplasia (Synovial hyperplasia) levels in the articular cavity were confirmed. Proteoglycan (proteolycan) levels in the articular surface were confirmed in the OARSI score (score) project after the Safranin-O staining. The corresponding evaluation method was scored with reference to the OARSI histopathological initiative-dog Osteoarthritis histological evaluation recommendation (The OARSI histopathology initiative-recommendations for histological assessments of Osteoarthritis in the dog) (ostoarthritis cartilage.2010oct;18suppl 3: s 66-79.).

The results are shown in fig. 9 and 10. As shown in fig. 9, the 300mg delayed release sheet G6 showed good results. In particular, only 300mg delayed release tablet G6 having a specific dissolution pattern showed good results compared to 300mg immediate release tablet G5 and 300mg sustained release tablet G7, which is a very particular result.

In addition, as shown in FIG. 10, as a result of dyeing with Safranin O, normal cartilage tissue was destroyed by induction and the damage level of proteoglycan tissue was increased in the osteoarthritis induction group. In contrast, for the dosing group, the stained proteoglycan tissues were distributed largely around the synovium. In H & E staining, it was confirmed that the osteoarthritis induced group showed deeper cracking and erosion of the articular surface compared to the cartilage tissue of the normal group, and that the level of damage to such articular surface was reduced in the administered group. It was confirmed that the osteoarthritis induced group had increased injury due to excessive infiltration of periarticular synovial cells, increased villi levels, and a relatively decreased infiltration of such cartilage and bone in the administered group, as compared to the normal group of articular synovial tissue. Furthermore, 300mg of the delayed release tablet G6 showed good results. In particular, only 300mg delayed release tablet G6 having a specific dissolution pattern showed good results compared to 300mg immediate release tablet G5 and 300mg sustained release tablet G7, which is a very particular result.

Claims

1. A preparation for treating arthritis is tablet or capsule containing 280-320mg crude drug extract of radix Clematidis, radix Trichosanthis and Prunellae Spica as effective components, and is administered for 2 times in 1 day,

when the dissolution test was performed under the condition of 900ml of water medium by the paddle test method at 37.+ -. 0.5 ℃ and 50rpm, the release of rosmarinic acid was 45 minutes 40-70%, 90 minutes 75% or more and 120 minutes 80% or more.

2. The formulation according to claim 1, wherein the content of the crude drug extracts of the active ingredients, i.e., clematis root, trichosanthes root and prunella spike, is 40 to 90% by weight with respect to the total weight of the pre-coated tablet or capsule filling excluding the weight of empty capsules.

3. The formulation of claim 1, wherein the formulation is a tablet.

4. A formulation according to any one of claims 1 to 3, wherein the formulation releases rosmarinic acid 45-65% in 45 minutes, 80% in 90 minutes and 85% in 120 minutes when subjected to dissolution test in 900ml of water medium by a paddle test at 37±0.5 ℃ and 50 rpm.

5. A formulation according to any one of claims 1 to 3, wherein the formulation comprises ethylcellulose, ethyl methacrylate copolymer or mixtures thereof to modulate release.

6. The formulation of claim 5, wherein the formulation comprises ethylcellulose to modulate release.

7. A formulation characterized by comprising 280-320mg of crude drug extract of clematis root, trichosanthes root and prunella spike as active ingredient, the content of the active ingredient being 40 to 90% by weight relative to the total weight of the mixture before filling of tablet or empty capsule before coating, and ethyl cellulose, ethyl methacrylate copolymer or their mixture being contained for the modified release.

8. The formulation of claim 7, wherein the formulation comprises ethylcellulose.

9. The formulation according to claim 7 or 8, wherein the content of active ingredient of the formulation is 50 to 90% by weight relative to the total weight of the pre-coated tablet or pre-empty capsule fill mixture.

10. The formulation of claim 7 or 8, wherein the formulation is a tablet.