KR102656187B1 - Pharmaceutical composition for preventing or treating tuberculosis disease comprising Agastache rugosa extract as effective component - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of tuberculosis containing a pear herb extract as an active ingredient. The pear herb extract alone significantly reduces the number of tuberculosis bacteria in mouse bone marrow-derived macrophages infected with tuberculosis bacteria, and isoniazid, an anti-tuberculosis drug ( When administered in combination with isoniazid, rifampicin, or bedaquiline, there is a synergistic effect on reducing tuberculosis bacteria, so pear extract, which is the active ingredient of the present invention; Pear extract and isoniazid mixture; Pear extract and rifampicin mixture; Or a mixture of pear extract and bedaquiline; can be usefully used as an anti-tuberculosis agent.

Description

Pharmaceutical composition for preventing or treating tuberculosis disease comprising Agastache rugosa extract as effective component}

The present invention relates to a composition for preventing, improving, or treating tuberculosis containing pear extract as an active ingredient.

Tuberculosis is an infectious disease caused by infection with Mycobacterium, especially Mycobacterium tuberculosis. It is estimated that one-third of the world's population is infected with Mycobacterium tuberculosis, and approximately 8 million new patients occur every year. Most infected people have no symptoms, but about 1/10 of them develop the disease, and if they do not receive appropriate treatment when they develop the disease, more than half of them will die.

In developed countries, the tuberculosis infection rate is less than 0.1% and the disease incidence rate is less than 25%. In Korea, the prevalence of active pulmonary tuberculosis (prevalence based on X-ray imaging) decreased from 5.1% in 1965 to 1.0% in 1995, the prevalence of positive bacteria decreased from 0.94% to 0.22%, and the annual risk of tuberculosis infection decreased from 5.3% to 0.5%. . The death rate per 100,000 people decreased from 10.4% in 1991 to 6.3 in 2001, but tuberculosis is still one of the top 10 causes of death.

The 6-month short-term treatment regimen involves prescribing the first-line anti-tuberculosis drugs, rifampicin, isoniazid, pyrazinamide, and ethambutol, in combination for 2 months, and then rifampin, isoniazid, and rifampin after 2 months. Three drugs of ethambutol are prescribed for 4 months. Streptomycin may be used instead of ethambutol during the initial 2 months of intensive treatment. In this case, maintenance treatment involves administering rifampin and isoniazid.

However, all of these antibiotics were invented at least 50 years ago, and the development of new treatments is not active. In addition, tuberculosis bacteria that are multi-drug resistant (MDR) and extensively drug resistant (XDR) are emerging, and the efficiency of conventional treatments is gradually decreasing. Therefore, research on new tuberculosis treatments or substances that can improve the sensitivity of existing tuberculosis treatments is required.

As prior art related to the treatment of tuberculosis, Korean Patent No. 1833048 discloses an anti-tuberculosis pharmaceutical composition containing cholinine and a method for manufacturing the same, and Korean Patent No. 0656969 discloses an anti-tuberculosis pharmaceutical composition containing crude Sancho extract as an active ingredient for the treatment of tuberculosis. Although pharmaceutical compositions and health functional foods have been disclosed, there has been no disclosure regarding a composition for preventing, improving, or treating tuberculosis containing the pear herb extract of the present invention as an active ingredient.

The present invention was developed in response to the above-mentioned needs, and provides a composition for preventing, improving or treating tuberculosis containing a pear extract as an active ingredient, and treating mouse bone marrow-derived macrophages infected with Mycobacterium tuberculosis with the pear extract alone. The present invention was completed by confirming that the number of Mycobacterium tuberculosis was reduced and that there was a synergistic effect on the reduction of Mycobacterium tuberculosis by combined treatment with anti-TB drugs such as isoniazid, rifampicin or bedaquiline.

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating tuberculosis containing pear extract as an active ingredient.

In addition, the present invention provides a health functional food composition for preventing or improving tuberculosis containing pear extract as an active ingredient.

Additionally, the present invention provides a feed additive for preventing or improving tuberculosis containing pear extract as an active ingredient.

In addition, the present invention provides a veterinary composition for preventing or treating tuberculosis containing pear extract as an active ingredient.

Additionally, the present invention provides a method of inhibiting the growth of tuberculosis bacteria by administering a pear herb extract alone or in combination with a pear herb extract and an anti-tuberculosis agent to animals other than humans.

The present invention relates to a pharmaceutical composition for the prevention or treatment of tuberculosis comprising a pear herb extract as an active ingredient, wherein the pear herb extract significantly reduces the number of tuberculosis bacteria in macrophages derived from the bone marrow of mice infected with tuberculosis bacteria, and the anti-tuberculosis drugs such as isoniazid, rifampicin, or When administered in combination with bedaquiline, there is a synergistic effect in reducing tuberculosis bacteria.

Figure 1 shows the results of confirming the inhibitory effect of pear extract on Mycobacterium tuberculosis in mouse bone marrow-derived macrophages infected with Mycobacterium tuberculosis (H37Rv). Rv is a group treated with Mycobacterium tuberculosis alone, Rv + RIF is a positive control group in which rifampicin, an anti-tuberculosis drug, was administered to macrophages derived from the bone marrow of mice infected with Mycobacterium tuberculosis, and Rv + Pear Choco is a group in which macrophages derived from the bone marrow of mice infected with Mycobacterium tuberculosis were treated with Pear Choco extract. . * means that the number of tuberculosis bacteria in the pear extract or rifampicin treated group was statistically significantly reduced compared to the untreated group, p<0.05.

In order to achieve the object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating tuberculosis containing pear extract as an active ingredient.

In one embodiment of the present invention, the composition of the present invention may further include one or more anti-tuberculosis agents selected from isoniazid, rifampicin, and bedaquiline in addition to the above active ingredients, preferably bedaquiline. It includes more, but is not limited to this.

The pear extract, which is an active ingredient of the present invention, has antibacterial activity against Mycobacterium tuberculosis H37Rv when used alone, and has a synergistic effect on antibacterial activity when administered in combination with an anti-tuberculosis agent.

In the present invention, the synergy effect is an effect obtained by multiple interactions of a single function. When the action of a substance is strengthened by the intervention of another substance, the two substances are said to have a synergistic effect (synergistic effect). means that a mixture cooperates to produce a total effect that is greater or more prolonged than the sum of the effects of the individual components taken independently.

The extraction solvent of the pear herb extract of the present invention may be water, a C ₁ to C ₄ lower alcohol, or a mixture thereof, preferably ethanol, but is not limited thereto.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, excipient, or diluent in addition to the above active ingredients.

In addition, it can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterilized injection solutions according to conventional methods, but is not limited thereto. .

Carriers, excipients, and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, and methyl. Examples include cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations contain the active ingredient plus at least one excipient such as starch, calcium carbonate, sucrose ( It is prepared by mixing sucrose, lactose, and gelatin. Additionally, in addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is.

Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, Witepsol, Macrogol, Tween 61, cacao, laurin, glycerogeratin, etc. can be used.

The appropriate dosage of the pharmaceutical composition according to the present invention is prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity. It can be. The concentration of the active ingredient included in the composition of the present invention can be determined considering the purpose of treatment, patient condition, required period, etc., and is not limited to a specific concentration range.

In addition, the present invention provides a health functional food composition for preventing or improving tuberculosis containing pear extract as an active ingredient.

The composition is preferably manufactured in a dosage form selected from powder, granule, pill, tablet, capsule, candy, syrup and beverage, but is not limited thereto.

When using the health functional food composition of the present invention as a food additive, the above-mentioned active ingredients can be added as is or used together with other foods or food ingredients, and can be used appropriately according to conventional methods. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use (prevention, health, or therapeutic treatment). Generally, when producing a food or beverage, the composition of the present invention is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, based on the raw materials. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

There are no special restrictions on the types of foods above. Examples of foods to which the above active ingredients can be added include meat, sausages, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, and drink preparations. , alcoholic beverages, vitamin complexes, etc., and includes all health functional foods in the conventional sense.

When the composition of the present invention is used as a health drink, it may contain various flavoring agents or natural carbohydrates as additional ingredients like conventional drinks. The above-mentioned natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as textrin and cyclotenstrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As a sweetener, natural sweeteners such as thaumatin and stevia extract or synthetic sweeteners such as saccharin and aspartame can be used. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g, per 100 g of the composition of the present invention.

The composition of the present invention contains various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal neutralizer, pH adjuster, stabilizer, preservative, glycerin, alcohol, carbonic acid. It may contain carbonating agents used in beverages.

In addition, the composition of the present invention may contain pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks. These ingredients can be used independently or in combination. Although the ratio of these additives is not very important, the composition of the present invention is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight.

Additionally, the present invention provides a feed additive for preventing or improving tuberculosis containing pear extract as an active ingredient.

The feed additive of the present invention corresponds to supplementary feed under the Feed Management Act. In the present invention, the term 'feed' may mean any natural or artificial diet, meal, etc., or a component of the meal, for or suitable for eating, ingestion, and digestion by animals. The type of feed is not particularly limited, and feed commonly used in the art can be used. Non-limiting examples of the feed include plant feeds such as grains, roots and fruits, food processing by-products, algae, fiber, pharmaceutical by-products, oils and fats, starches, cucurbits or grain by-products; Examples include animal feeds such as proteins, inorganic substances, fats and oils, minerals, oils and fats, single-cell proteins, zooplanktons or foods. These may be used alone or in combination of two or more types.

In addition, the present invention provides a veterinary composition for preventing or treating tuberculosis containing pear extract as an active ingredient.

The veterinary composition of the present invention may further include appropriate excipients and diluents according to conventional methods. Excipients and diluents that may be included in the veterinary composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate, cetanol, stearyl alcohol, liquid paraffin, sorbitan monostearate. , polysorbate 60, methylparaben, propylparaben, and mineral oil.

The veterinary composition according to the present invention may further include fillers, anti-aggregants, lubricants, wetting agents, spices, emulsifiers, preservatives, etc. The veterinary composition according to the present invention provides rapid and sustained release of the active ingredient after administration to an animal. or may be formulated using methods well known in the art to provide sustained release, including powders, granules, tablets, capsules, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, It may be in the form of a suppository, sterile injectable solution, or sterile topical medication.

The effective amount of the veterinary composition according to the present invention can be appropriately selected depending on the individual animal. Severity of the disease or condition, sensitivity to the active ingredient of the present invention depending on the individual's age, weight, health condition or gender, administration route, administration period, factors including other compositions mixed or used simultaneously with the composition, and other physiological or It can be determined based on factors well known in the veterinary field.

Additionally, the present invention provides a method of inhibiting the growth of tuberculosis bacteria by administering a pear herb extract alone or in combination with a pear herb extract and an anti-tuberculosis agent to animals other than humans.

In one embodiment of the present invention, the anti-tuberculosis agent may be one or more selected from isoniazid, rifampicin, and bedaquiline, preferably bedaquiline, but is not limited thereto.

Hereinafter, the present invention will be described in more detail using examples. These examples are only for illustrating the present invention in more detail, and it is obvious to those skilled in the art that the scope of the present invention is not limited thereto.

Preparation Example 1. Preparation of pear extract

12L of 70% (v/v) ethanol was added to 1 kg of aerial parts of pear herbaceous extract, extracted at 85°C for 3 hours, filtered, and the filtered extract was concentrated and dried under reduced pressure at 45°C to obtain 126.4g of pear herbaceous extract.

Example 1. Confirmation of the effect of reducing tuberculosis bacteria by treatment with pear extract alone in macrophages infected with tuberculosis bacteria

Macrophages (BMDM) extracted from the bone marrow of 6-week-old C57BL/6 female mice were counted and infected with pathogenic Mycobacterium tuberculosis H37Rv at an MOI of 1. After 3 hours, the medium was replaced with pear extract (200 μg/mL) or rifampicin (100 ng/mL; positive control), and cultured at 37°C and 5% CO ₂ for 48 hours. Afterwards, 500 ㎕ of sterilized tertiary distilled water was added to each well to collect intracellular tuberculosis bacteria and diluted 10-fold to a total of 100-fold. To measure live Mycobacterium tuberculosis within cells, 10㎕ each was dispensed into Mycobacterium tuberculosis selection medium (7H10 medium) containing ampicillin (25ng/ml) and cultured at 37°C for two weeks. Afterwards, the number of intracellular bacteria was measured through CFU analysis.

As a result, as shown in Figure 1, it was confirmed that the pear extract had an anti-tuberculosis effect.

Example 2. Confirmation of synergistic effect by combined administration of pear extract and anti-tuberculosis agent in macrophages infected with Mycobacterium tuberculosis

Macrophages (BMDM) extracted from the bone marrow of 6-week-old C57BL/6 female mice were counted and infected with pathogenic Mycobacterium tuberculosis H37Rv at an MOI of 1. 3 hours after infection, the medium was replaced with a medium containing pear extract or/and anti-tuberculosis drugs (isoniazid, rifampicin, or bedaquiline) at various concentrations.

After culturing for 24 hours, the number of viable tuberculosis bacteria in the cells was measured to confirm the minimal inhibitory concentration (MIC). MIC was defined as the lowest concentration that inhibits bacterial growth by more than 99%.

The FIC value for quantifying the interaction between the pear herb extract and the anti-tuberculosis agent was calculated using Equation 1 below, and the interaction according to the results is shown in Table 1.

As a result, as shown in Tables 2 to 4 below, when the pear extract was used alone, the minimum blocking concentration was 200 μg/mL, and when isoniazid was used alone, the minimum blocking concentration was 0.2 μg/mL, and rifampicin or When bedaquiline was used alone, the minimum inhibitory concentration was 0.1 μg/mL.

When pear herb extract and isoniazid were treated in combination, the minimum inhibitory concentration was 50 ㎍/mL for pear herb extract and 0.05 ㎍/mL for isoniazid (Table 2), and when pear herb extract and rifampicin were combined, the minimum inhibitory concentration was 50 ㎍ of pear herb extract. /mL, rifampicin was 0.05㎍/mL (Table 3), and when pear extract and bedaquiline were combined, the minimum inhibitory concentration was 50㎍/mL for pear extract and 0.005㎍/mL for bedaquiline (Table 4). Pear extract showed a synergistic effect when combined with anti-tuberculosis drugs, and especially showed the best synergy effect when combined with bedaquiline.

Therefore, when pear herb extract and an anti-tuberculosis agent are administered together, the synergistic effect between the two substances can enhance the anti-tuberculous activity of the pear herb extract and increase the drug sensitivity of the anti-tuberculous drug, specifically isoniazid, rifampicin or bedaquiline, to tuberculosis bacteria.

[Equation 1]

FIC value (ΣFIC _index )=FIC _B +FIC _A (A=pear extract, B=anti-tuberculosis drug)

FIC _RMP =MIC _{B combination} /MIC _{B alone}

FIC _A =MIC _{A combination} /MIC _{A alone}

FIC value effect ≤0.5 synergistic >0.5 to ≤0.75 partial synergy >0.76 to ≤1.0 additive >1.0 to ≤4.0 indifferent(noninteractive) >4.0 antagonistic

Isoniazid (INH)
+Pear herb extract MIC alone MIC combination FIC value effect INH
(㎍/mL) Pear herb extract
(㎍/mL) INH
(㎍/mL) Pear herb extract
(㎍/mL) H37Rv 0.2 200 0.05 50 0.75 partial synergy

Rifampicin (RIF)
+Pear herb extract MIC alone MIC combination FIC value effect RIF
(㎍/mL) Pear herb extract
(㎍/mL) RIF
(㎍/mL) Pear herb extract
(㎍/mL) H37Rv 0.1 200 0.05 50 0.75 partial synergy

Bedaquiline (BDQ)
+Pear herb extract MIC alone MIC combination FIC value effect BDQ
(㎍/mL) Pear herb extract
(㎍/mL) BDQ
(㎍/mL) Pear herb extract
(㎍/mL) H37Rv 0.1 200 0.005 50 0.3 synergy

Claims (10)

A pharmaceutical composition for preventing or treating tuberculosis containing pear extract as an active ingredient. The pharmaceutical composition for the prevention or treatment of tuberculosis according to claim 1, further comprising, in addition to the active ingredient, at least one anti-tuberculosis agent selected from isoniazid, rifampicin, and bedaquiline. The pharmaceutical composition for preventing or treating tuberculosis according to claim 1, wherein the active ingredient has antibacterial activity against Mycobacterium tuberculosis H37Rv. The pharmaceutical composition for preventing or treating tuberculosis according to claim 1, wherein the extraction solvent of the pear herb extract is water, a lower alcohol of C ₁ to C ₄ , or a mixture thereof. The pharmaceutical composition for preventing or treating tuberculosis according to claim 1, further comprising a pharmaceutically acceptable carrier, excipient, or diluent in addition to the active ingredient. A health functional food composition for preventing or improving tuberculosis containing pear extract as an active ingredient. A feed additive for preventing or improving tuberculosis containing pear extract as an active ingredient. A veterinary composition for preventing or treating tuberculosis containing pear extract as an active ingredient. A method of inhibiting the growth of tuberculosis bacteria by administering pear extract alone or in combination with pear extract and anti-tuberculosis drugs to animals other than humans. The method of claim 9, wherein the anti-tuberculosis agent is at least one selected from isoniazid, rifampicin, and bedaquiline.