PHARMACEUTICAL COMPOSITION, PHARMACEUTICAL COMPOSITION FOR ANIMAL, AND FOOD COMPOSITION, EACH COMPRISING NATURAL SUBSTANCE EXTRACT, AND PREPARATION METHOD THEREFOR

Abstract

The present invention relates to a pharmaceutical composition, a pharmaceutical composition for animals, and a food composition, each composition comprising a Scutellaria baicalensis extract, a Liriope platyphylla extract, a Sophora flavescens extract, a Dictamnus root bark extract, and a Phellodendron amurense Ruprecht extract, and preparation methods therefor.

Claims

1. A composition comprising at least one extract selected from a group consisting of a Scutellaria baicalensis extract, a Liriope platyphylla extract, a Sophora flavescens extract, a Dictamnus root bark extract, and a Phellodendron bark extract.

2. The composition of claim 1, wherein the composition is used for the prevention, amelioration, or treatment of a skin related disease.

3. The composition of claim 1, wherein the composition comprises the Scutellaria baicalensis extract, the Liriope platyphylla extract, the Sophora flavescens extract, the Dictamnus root bark extract, and the Phellodendron bark extract.

4. The composition of claim 3, wherein the composition comprises 50 to 150 parts by weight of the Liriope platyphylla extract, 50 to 150 parts by weight of the Sophora flavescens extract, 50 to 150 parts by weight of the Dictamnus root bark extract, and 50 to 150 parts by weight of the Phellodendron bark extract, based on 100 parts by weight of the Scutellaria baicalensis extract.

5. The composition of claim 1, wherein a content of the extract in the composition is 0.5 v/v % to 5 v/v %.

6. The composition of claim 1, wherein the extract is obtained by extraction with at least one solvent selected from the group consisting of water, an organic solvent, and a mixture thereof.

7. The composition of claim 1, wherein the composition is a pharmaceutical composition.

8. The composition of claim 1, wherein the composition is a food composition.

9. A method for preventing or treating a skin-related disease, comprising a step of administering the composition of claim 1 to an individual in need thereof.

10. A method for preventing or treating a skin-related disease, comprising a step of causing an individual in need thereof to ingest the composition of claim 1.

11. The method of claim 9, wherein the skin-related disease is at least one disease selected from the group consisting of skin wrinkles, photoaging, melanosis, skin pigmentation, skin burns, skin inflammation, skin cancer, melasma, freckles, lentigines, nevus, psoriasis, atopic dermatitis, alopecia areata, vitiligo, athlete's foot, and dandruff.

12. A preparation method of a composition of claim 1, comprising a step of extracting at least one natural material selected from a group consisting of Scutellaria baicalensis, Liriope platyphylla, Sophora flavescens, Dictamnus root bark, and Phellodendron bark, independently from each other.

13. The preparation method of claim 12, wherein the natural material includes all of the Scutellaria baicalensis, the Liriope platyphylla, the Sophora flavescens, the Dictamnus root bark, and the Phellodendron bark, and the method further comprises a step of mixing, based on 100 parts by weight of the Scutellaria baicalensis extract, 50 to 150 parts by weight of the Liriope platyphylla extract, 50 to 150 parts by weight of the Sophora flavescens extract, 50 to 150 parts by weight of the Dictamnus root bark extract, and 50 to 150 parts by weight of the Phellodendron bark extract, to obtain a mixture.

14. The preparation method of claim 13, further comprising a step of drying the mixture.

15. The preparation method of claim 14, wherein the drying is spray drying.

16. The preparation method of claim 12, wherein the extraction is carried out at a temperature of 40 to 55° C., is carried out for 60 to 80 hours, or is carried out at a temperature of 40 to 55° C. for 60 to 80 hours.

17. The preparation method of claim 12, wherein the extraction is carried out with at least one solvent selected from the group consisting of water, an organic solvent, and a mixture thereof.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0113] FIG. 1 illustrates results showing an ability to inhibit reactive oxygen species of the composition according to an embodiment of the present invention.

[0114] FIG. 2 illustrates results showing an ability to inhibit NO synthesis of the composition according to an embodiment of the present invention.

[0115] FIG. 3 illustrates results showing an intracellular melanin production inhibition rate of the composition according to an embodiment of the present invention.

[0116] FIGS. 4A, 4B, 4C, and 4D illustrate results obtained by evaluating cytotoxicity in human-derived keratinocytes (HaCaT) of the composition according to an embodiment of the present invention.

[0117] FIG. 5 illustrates results obtained by evaluating NO production (NO assay) of the composition according to an embodiment of the present invention.

[0118] FIGS. 6A, 6B, 6C, 6D, and 6E illustrate results obtained by evaluating inhibition of expression of inflammatory cytokines of 1 mg/mL of the composition according to an embodiment of the present invention.

[0119] FIGS. 7A, 7B, 7C, 7D, and 7E illustrate results obtained by evaluating inhibition of expression of inflammatory cytokines of 3 mg/mL of the composition according to an embodiment of the present invention.

[0120] FIGS. 8A, 8B, 8C, 8D, and 8E illustrate results obtained by evaluating inhibited expression level of inflammatory cytokines depending on doses of the composition of Example 7-1.

[0121] FIGS. 9A, 9B, 9C, 9D, and 9E illustrate results obtained by evaluating inhibited expression level of inflammatory cytokines depending on doses of the composition of Example 7-3.

[0122] FIG. 10 illustrates a plan for evaluating anti-atopic efficacy of the composition according to an embodiment of the present invention.

[0123] FIG. 11 illustrates results obtained by measuring body weight in evaluation of the anti-atopic efficacy of the composition according to an embodiment of the present invention.

[0124] FIG. 12 illustrates clinical symptom results (photographs) in evaluation of the anti-atopic efficacy of the composition according to an embodiment of the present invention.

[0125] FIGS. 13A and 13B illustrate results (A) obtained by identifying skin irritation index and results (B) obtained by measuring ear thickness in evaluation of the anti-atopic efficacy of the composition according to an embodiment of the present invention.

[0126] FIG. 14 illustrates results obtained by measuring lymph node weight in evaluation of the anti-atopic efficacy of the composition according to an embodiment of the present invention.

DETAILED DESCRIPTION

[0127] Hereinafter, the present invention will be described in more detail by way of specific examples. However, the present invention is not limited to the following examples, and it will be apparent to those skilled in the art that various modifications or variations can be made within the spirit and scope of the present invention. Accordingly, the appended claims should be construed broadly and in a manner consistent with the spirit and scope of the present invention.

Example 1. Preparation of Herbal Medicinal Extracts

[0128] Each of Scutellaria baicalensis, Liriope platyphylla, Sophora flavescens, Dictamnus root bark, and Phellodendron bark was heated and stirred at a temperature of 50° C. for 72 hours in a mixture of butylene glycol and water. Then, filtration was performed under reduced pressure through a filter paper. The filtered extract was refrigerated at a temperature of 4° C. for 24 hours. Then, the extract was filtered again under reduced pressure to obtain an extract, which was refrigerated at a temperature of 4° C.

Example 2. Preparation of Composition Containing Extracts

[0129] The extracts prepared in Example 1 were weighed as shown in Table 1 and mixed. The mixture was stirred, filtered again under reduced pressure through a filter paper, concentrated, and freeze-dried to prepare a composition containing the extracts. A comparative example, in which a composition ratio of the respective extracts was different, was used as a control.

TABLE-US-00001 TABLE 1 Scutellaria Liriope Sophora Dictamnus Phellodendron baicalensis platyphylla flavescens root bark bark Example 2 100 g 100 g 100 g 100 g 100 g Comparative 100 g 100 g 200 g 100 g 100 g Example

Example 3. Evaluation of Antioxidant Activity with DCFH-DA Measurement

[0130] To measure intracellular reactive oxygen species (ROS), a fluorescence assay using DCFH-DA (2′,7′-dichlorofluorescein-diacetate) was performed. DCFH-DA is a non-polar molecule that can be easily introduced into cells. DCFH-DA is converted to fluorescent DCF in a case of being separated into DCFH, which is a non-fluorescent polar molecule, by an esterase, and then oxidized by intracellular reactive oxygen species. The DCFH-DA was measured with a fluorometer.

[0131] Specifically, human dermal fibroblasts (HDFn) were cultured in DMEM (supplemented with 10% FBS and containing 1% antibiotics) at a condition of 37° C. and 5% CO2. About 1×10.sup.5 cells per well were dispensed into each well of a 96-well plate. Then, treatment with the composition of Example 2 at 0, 0.5, 1, and 2 v/v % was performed, and culture was performed.

[0132] Then, 1 mM of H.sub.2O.sub.2, which is an oxidative stress-inducing substance, was added, and 20 μM of DCFH-DA (20 mM stock) was added. Then, the fluorescence reaction was analyzed with a spectrofluorometer (excitation 485 nm, emission 535 nm) at 37° C. for 90 minutes, and the results are illustrated in FIG. 1. Treatment with the composition of Comparative Example at 2 v/v % was also respectively performed in the same manner. Then, the fluorescence reaction was analyzed, and the results are illustrated in Table 2. From each result, the reactive oxygen species inhibition ability (%) was quantified by calculating % of fluorescence intensity of the experimental group relative to the untreated control (0 v/v %).

TABLE-US-00002 TABLE 2 Reactive oxygen species Item inhibition ability (%) Untreated control    0% Example 2-1 (2 v/v %)  46.7% Example 2-2 (3 v/v %) 65.30% Comparative Example (2 v/v %) 40.90%

[0133] As can be seen in FIG. 1, the antioxidant activity also increased as the concentration increased; and as can be seen in Table 2, it was identified that the highest ROS inhibitory effect was obtained at 3 v/v %. In particular, it was identified that the composition of Example 2 had an excellent effect as compared with the composition of Comparative Example, which contains twice the Sophora flavescens extract among the extracts, and thus had an excellent antioxidant effect.

Example 4. Evaluation of NO Synthesis Inhibition Ability

[0134] NO, which is a pathological secondary signaling substance secreted by many cells, regulates activity of COX and acts synergistically during inflammatory responses. NO produced from NOS induced by LPS in Raw264.7 cells was measured to identify an inflammatory response inhibitory effect.

[0135] Raw264.7 cells were subjected to treatment with the composition of Example 2 at 0, 0.5, 1, and 2 v/v %, and then the NO synthesis inhibition ability was evaluated using a NO quantification kit based on Expression 1. The control was treated only with LPS without adding a sample. The results are illustrated in FIG. 2.

NO synthesis inhibition ability (%)={1−(Slope of NO synthesis amount when sample is added)}/(NO synthesis amount when no sample is added)×100  [Expression 1]

[0136] As can be seen from FIG. 2, the composition of Example 2 according to the present invention had an increased anti-inflammatory effect as the concentration increased. Referring to Table 3, it was found that the composition of Example 2 according to the present invention had the highest anti-inflammatory effect at 3 v/v %.

[0137] Treatment with the composition of Comparative Example at 2 v/v % was performed to analyze its anti-inflammatory effect in the same manner as above, and the results are shown in Table 3.

TABLE-US-00003 TABLE 3 Item NO synthesis inhibition ability (%) Untreated control (LPS only)  0% Example 2-1 (2 v/v %) 69.3%.sup.  Example 2-2 (3 v/v %) 98% Comparative Example 65%

[0138] From the above results, it was identified that the composition of Example 2 had an excellent effect as compared with the composition of Comparative Example, which contains twice the Sophora flavescens extract among the extracts, and thus had an excellent anti-inflammatory effect.

Example 5. Evaluation of Whitening Efficacy Through Measurement of Intracellular Melanin Production

[0139] To identify a whitening effect of the extract, its effect on inhibition of melanin biosynthesis was measured using the mouse-derived melanocytes B16F1.

[0140] The B16F1 melanocytes were dispensed into each well and the cells were allowed to attach thereto. Then, treatment with the composition of Example 2 at 0, 0.5, 1, and 2 v/v and the composition of Comparative Example at 2 v/v % was performed, and incubation was performed. After incubation, the cells were detached with trypsin-EDTA. Subsequently, the number of cells was measured, and then the cells were collected by centrifugation. 1 N NaOH (10% DMSO) was added to the cell filtrate to extract melanin. The extracted melanin was dissolved, and the absorbance of melanin was measured with a microplate reader. Subsequently, melanin was quantified to measure a melanin production inhibition rate (%) of the sample. A melanin production inhibition rate (%) in the B16F1 melanocytes was calculated by Expression 2, and the results are shown in Table 4 and FIG. 3.

Melanin production inhibition rate (%)={(Melanin amount when no sample is added)−(Melanin amount when sample is added)}/(Melanin amount when no sample is added)×100  [Expression 2]

TABLE-US-00004 TABLE 4 Item Melanin production inhibition rate (%) Untreated control (LPS only) 0% Example 2-1 (2 v/v %) 14.5%   Comparative Example 0%

[0141] From the above results, it was identified that the composition of Example 2 inhibited melanin production in a concentration-dependent manner, and thus had whitening activity. In particular, from the viewpoint that the composition of Example 2 had a melanin production inhibition ability even though the composition of Comparative Example had no whitening effect, it was identified that the composition of the present invention had a specifically effective whitening effect.

Example 6. Efficacy Evaluation Depending on Extraction Conditions

[0142] To identify whether efficacy of the composition of the present invention varies depending on extraction methods, it was checked whether the respective compositions had antioxidant and anti-inflammatory effects in a case where extraction is performed using different extraction temperatures and times as shown in Table 5.

TABLE-US-00005 TABLE 5 Item Temperature Time Example 6-1 50° C. 72 hours Example 6-2 60° C. 96 hours Example 6-3 100° C.  1 and half hours

[0143] For evaluation of antioxidant and anti-inflammatory effects, experiments were conducted in the same manner as in Examples 3 and 4. The sample at 2 v/v % was used.

TABLE-US-00006 TABLE 6 Reactive oxygen species NO synthesis inhibition Item inhibition ability (%) ability (%) Untreated control    0%    0% Example 6-1   62% 74.10% Example 6-2 34.70% 61.10% Example 6-3 41.50%  40.8.%

[0144] The results are as shown in Table 6. It was identified that the active ingredients for antioxidant and anti-inflammatory properties were highly extracted under the extraction condition of 50° C. for 72 hours, thereby allowing excellent efficacy.

Example 7. Cytotoxicity Evaluation (MTT Assay)

[0145] As a colorimetric assay to evaluate metabolic activity of cells, this experiment was performed. To evaluate toxicity of a test substance, viability of the cells treated with the test substance was compared with cell viability of a control which was regarded as 100%. It was determined that the test substance was toxic in a case where the cell viability was 70% or lower.

[0146] Specifically, cytotoxicity evaluation was performed in human-derived keratinocytes (HaCaT). The composition was prepared in the same manner as in Example 2, except that as shown in Table 7, distilled water 100% or aqueous solution of ethanol (30% ethanol) was used in place of butylene glycol and water to compare differences between the solvents, and spray drying was performed in addition to freeze drying to compare differences between the drying methods. Then, the cells were subjected to treatment with each of the prepared compositions of Example 7 at doses of 0, 1, 3, 10, and 30 mg/mL, respectively.

TABLE-US-00007 TABLE 7 Item Solvent Drying method Remarks Example 7-1 Distilled water 100% Spray drying Suspended matters are present Example 7-2 Distilled water 100% Freeze drying — Example 7-3 Distilled water 70% + Spray drying Suspended Ethanol 30% matters are present Example 7-4 Distilled water 70% + Freeze drying Suspended Ethanol 30% matters are present

[0147] As a result, it was identified that Examples 7-1 to 7-4 were all non-cytotoxic at 10 mg/mL or lower (results of Example 7-1, Example 7-2, Example 7-3, and Example 7-4 are illustrated in FIG. 4A, FIG. 4B, FIG. 4C, and FIG. 4D, respectively).

Example 8. Evaluation of NO Production (NO Assay)

[0148] NO is a substance produced when cells are stimulated, and a concentration of NO released into culture medium was measured using a NaNO.sub.2 standard curve. NO production of LPS (0.1 μg/mL), which is a representative inflammation-inducing substance, is around 30 μM, and LPS was used as a control. Specifically, treatment with the respective compositions of Examples 7-1 to 7-4 at 0, 0.1, 0.3, 1, and 3 mg/mL, respectively, was performed in the presence of LPS (0.1 μg/mL).

[0149] As a result, it was identified that the composition of Example 7-2 had no significant effect on a decrease in NO production; the compositions of Examples 7-3 and 7-4 decreased NO production at 3 mg/mL or higher; and the composition of Example 7-1 decreased NO production at 1 mg/mL or higher (FIG. 5).

Example 9. Evaluation of Expression Level of Inflammatory Cytokines (RT-PCR)

[0150] (1) Evaluation of Inhibited Expression Level of Inflammatory Cytokines Caused by 1 mg/mL of Compositions of Examples 7-1 to 7-4

[0151] In a case where the cells are stimulated, the expression level of inflammatory cytokines (iNOS, COX-2, TNF-α, IL-6, IL-1β) increases. Thus, an attempt was made to determine whether the expression of inflammatory cytokines was inhibited in a case where treatment with each of the compositions of Examples 7-1 to 7-4 at 1 mg/mL was performed. Specifically, intracellular mRNA was extracted and the expression level of inflammatory cytokines was measured through RT-PCR.

[0152] As a result, overall, the composition of Example 7-1 had the highest inhibitory effect on expression of inflammatory cytokines, followed by then the composition of Example 7-3, followed by the composition of Example 7-4, and followed by the composition of Example 7-2 (FIGS. 6A-6E). Specifically, the two compositions having the highest inhibitory effect on expression of each inflammatory cytokine are shown in Table 8.

TABLE-US-00008 TABLE 8 Item iNOS COX-2 TNF-α IL-6 IL-1β Example 7-1 ◯ ◯ ◯ ◯ ◯ Example 7-2 — — — — — Example 7-3 — ◯ ◯ ◯ — Example 7-4 ◯ — — — ◯

[0153] (2) Evaluation of Inhibited Expression Level of Inflammatory Cytokines Caused by 3 mg/mL of Compositions of Examples 7-1 to 7-4

[0154] In addition, an attempt was made to determine whether the expression of inflammatory cytokines was inhibited in a case where treatment with each of the compositions of Examples 7-1 to 7-4 at 3 mg/mL was performed. Specifically, intracellular mRNA was extracted and the expression level of inflammatory cytokines was measured through RT-PCR.

[0155] As a result, overall, the composition of Example 7-1 had the highest inhibitory effect on expression of inflammatory cytokines, followed by then the composition of Example 7-3, followed by the composition of Example 7-2, and followed by the composition of Example 7-4 (FIGS. 7A-7E). Specifically, the two compositions having the highest inhibitory effect on expression of each inflammatory cytokine are shown in Table 9.

TABLE-US-00009 TABLE 9 Item iNOS COX-2 TNF-α IL-6 IL-1β Example 7-1 — ◯ ◯ — ◯ Example 7-2 ◯ — — ◯ — Example 7-3 ◯ ◯ — ◯ — Example 7-4 — — — — ◯

[0156] (3) Evaluation of Inhibited Expression Level of Inflammatory Cytokines Depending on Doses of Composition of Example 7-1

[0157] The composition of Example 7-1 had the highest inhibitory effect on expression of inflammatory cytokines. Thus, the expression level of inflammatory cytokines depending on doses of the composition of Example 7-1 was evaluated.

[0158] As a result, it was identified that the composition had a better inhibitory effect on expression of inflammatory cytokines (except TNF-α) as the dose increased; and most of the inflammatory cytokines (except TNF-α) were inhibited in a case where treatment with the composition at 3 mg/mL was performed (FIGS. 8A-8E).

[0159] (4) Evaluation of Inhibited Expression Level of Inflammatory Cytokines Depending on Doses of Composition of Example 7-3

[0160] The composition of Example 7-3 had the second-highest inhibitory effect on expression of inflammatory cytokines following the composition of Example 7-1. Thus, the expression level of inflammatory cytokines depending on doses of the composition of Example 7-3 was evaluated.

[0161] As a result, it was identified that the composition had a better inhibitory effect on expression of inflammatory cytokines (except TNF-α) as the dose increased; and most of the inflammatory cytokines (except TNF-α) were inhibited in a case where treatment with the composition at 3 mg/mL was performed (FIGS. 9A-9E).

Example 10. Evaluation of Anti-Atopic Efficacy

[0162] To evaluate in vivo anti-atopic efficacy, mice were prepared. The mice were acclimatized for 4 days, and then the hairs of the mice were shaved. One day after the hair removal, the mice were sensitized with 1% 2,4-dinitrochlorobenzene (DNCB). Two days after DNCB sensitization, the mice were sensitized once more with 1% DNCB. Starting from 3 days after the second DNCB sensitization, 0.6% DNCB was applied to the hair-removed area at two-day intervals for a total of 8 times. The next day after the second application of DNCB, the mice were orally administered with 500 mpk each of the compositions of Examples 7-1 to 7-4, and 1 mpk of dexamethasone and 1 mpk of 0.9% saline as controls, to evaluate their efficacy. To evaluate the results, evaluation of skin irritation (dermatitis score), measurement of ear thickness, measurement of body weight, and evaluation of clinical symptoms (photographs) were performed over a total of 6 times on the day of hair removal, the day of second DNCB sensitization, the day after the first DNCB application, and at intervals of 4 days starting from the second day after administration of each composition. The mice were sacrificed 13 days after administration of each composition. Subsequently, the lymph nodes of the sacrificed mice were weighed (Table 10 and FIG. 10).

TABLE-US-00010 TABLE 10 Substance Route of Number of Test groups Induction of atopy administered administration animals Normal — 0.9% saline 1 mpk Oral 8 Control Snsitization with 0.9% saline 1 mpk 8 Dexamethasone 1% DNCB + Dexamethasone 1 mpk 8 LK1 Application of 6% Example 7-1 500 mpk 8 LK2 DNCB Example 7-2 500 mpk 8 LK3 Example 7-3 500 mpk 8 LK4 Example 7-4 500 mpk 8

[0163] From the results of body weight measurement, it was found that the groups administered with the compositions of Examples 7-1 to 7-4 gained less weight than the control group (FIG. 11).

[0164] In addition, the clinical symptoms were directly checked by observing the affected area. As a result, it was visually found that for all groups administered with the compositions of Examples 7-1 to 7-4, the affected area was improved as compared with the control group; and in particular, it was found that for the group administered with the composition of Examples 7-3, the affected area was improved as compared with the control group (FIG. 12).

[0165] In addition, after oral administration for 2 weeks, the symptoms of atopic dermatitis were evaluated by skin irritation index. As a result, it was found that all groups administered with the compositions of Examples 7-1 to 7-4 had a lower index than the control group; and in particular, it was found that the composition of Example 7-3 had a lower skin irritation index than dexamethasone (FIG. 13A).

[0166] In addition, from the results of ear thickness measurement, it was found that for the groups administered with the compositions of Examples 7-1, 7-3, and 7-4, the ear thickness was significantly decreased (FIG. 13B).

[0167] In addition, the lymph node weight was measured after sacrifice of each mouse. As a result, it was found that for all groups administered with the compositions of Examples 7-1 to 7-4, the lymph node weight was decreased as compared with the control group (FIG. 14).

[0168] Overall, it was found that the composition of Example 7-3 exhibited the best effects.