MUSHROOM-CONTAINING CHINESE MEDICINE COMPOUND COMPOSITION FOR KELOID SCAR TISSUE AND APPLICATION THEREOF

Abstract

The present invention provides a mushroom-containing Chinese medicine compound composition for treating keloid scar tissue, the composition including: brown strain of Flammulina velutipes extract, Artemisinin, Matrine, Triptolide, Tetramethylpyrazine, Tetrandrine, Curcumin, Resveratrol, EGCG, Quercetin and Asiaticoside. Said mushroom-containing Chinese medicine compound composition is used to inhibit the proliferation of keloid fibroblast, and to treat, inhibit or reduce the symptoms of keloid fibroblast.

Claims

1. A mushroom-containing Chinese medicine complex composition for keloid scar tissue, the mushroom-containing Chinese medicine complex composition comprising: 2 parts by weight of brown strain of Flammulina velutipes extract; 1 part by weight of Artemisinin; 1 part by weight of Matrine; 2 parts by weight of Triptolide; 2 parts by weight of Tetramethylpyrazine; 16 parts by weight of Tetrandrine; 4 parts by weight of Curcumin; 16 parts by weight of Resveratrol; 1 part by weight of Epigallocatechin gallate (EGCG); 2 parts by weight of Quercetin; and 2 parts by weight of Asiaticoside.

2. The mushroom-containing Chinese medicine complex composition of claim 1, further comprising: 1 part by weight of Tremella fuciformis extract.

3. The mushroom-containing Chinese medicine complex composition of claim 1, wherein the mushroom-containing Chinese medicine complex composition is configured such that a proliferation of keloid fibroblasts is inhibited and an apoptosis of scar tissue cells is promoted, so that hypertrophic scars of keloid scars are treated, inhibited or reduced.

4. The mushroom-containing Chinese medicine complex composition of claim 2, wherein the mushroom-containing Chinese medicine complex composition is configured such that a proliferation of keloid fibroblasts is inhibited and an apoptosis of scar tissue cells is promoted, so that hypertrophic scars of keloid scars are treated, inhibited or reduced.

5. The mushroom-containing Chinese medicine complex composition of claim 1, wherein the mushroom-containing Chinese medicine complex composition is prepared in a form of ointment, colloid, gel, solution, emulsion, patch or spray.

6. The mushroom-containing Chinese medicine complex composition of claim 2, wherein the mushroom-containing Chinese medicine complex composition is prepared in a form of ointment, colloid, gel, solution, emulsion, patch or spray.

7. A use of the mushroom-containing Chinese medicine complex composition according to claim 1 for preparing a drug that promotes apoptosis of scar tissue, produces tissue reconstitution, and reduces scar proliferation.

8. A use of the mushroom-containing Chinese medicine complex composition according to claim 2 for preparing a drug that promotes apoptosis of scar tissue, produces tissue reconstitution, and reduces scar proliferation.

9. A use of the mushroom-containing Chinese medicine complex composition according to claim 1 for preparing a drug for treating, inhibiting or reducing keloid scars.

10. A use of the mushroom-containing Chinese medicine complex composition according to claim 2 for preparing a drug for treating, inhibiting or reducing keloid scars.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIGS. 1(A) to 1(C) are photographs showing the effect of a first example using a mushroom-containing Chinese medicine complex composition according to one embodiment of the present invention.

[0018] FIGS. 2(A) to 2(D) are photographs showing the effect of a second example using the mushroom-containing Chinese medicine complex composition according to one embodiment of the present invention.

[0019] FIG. 3 is a graph showing the effect of different doses (3.125, 6.25, 12.5, 50, and 100 μM) of 10 Chinese medicine ingredients according to the present invention on KFS cell growth.

[0020] FIG. 4 is a graph showing the regulation of KFS apoptosis-related proteins of the 10 ingredients of Chinese medicine according to the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Ointment Preparation Process of the Present Invention

[0022] When calculated by the dry weight ratio of a material, the ointment includes: 1 part by weight of Tremella fuciformis extract; 2 parts by weight of brown strain of Flammulina velutipes extract; 1 part by weight of Artemisinin; 1 part by weight of Matrine; 2 parts by weight of Triptolide; 2 parts by weight of Tetramethylpyrazine; 16 parts by weight of Tetrandrine; 4 parts by weight of Curcumin; 16 parts by weight of Resveratrol; 1 part by weight of Epigallocatechin gallate (EGCG); 2 parts by weight of Quercetin; and 2 parts by weight of Asiaticoside.

[0023] After extracting the mushroom extract with hot water at 100° C. (weight ratio 1:1), the above 10 Chinese herbal ingredients are added and mixed with an ointment base, thereby preparing an ointment formulation.

[0024] Results on Example Implementation

[0025] The example is provided as follows, in which the ointment is directly applied to the affected area. As shown in FIG. 1(A), the first example of a keloid scar is a scar having been more than 10 years, and has problems that affect the quality of life, such as pain, itching, and foreign body sensation. Medication and surgical treatment have been conducted, but failed to diminish the scar. Instead, a new scar on the right side in FIG. 1(A) has derived from the original scar on the left side, which is a typical feature of keloid scars.

[0026] After 1 month of treatment using the ointment prepared according to the present invention, as shown in FIG. 1(B), the flexibility, size, thickness, area, and symptom such as pain, itchiness, and foreign body sensation were significantly improved. As shown in FIG. 1(C), more than 90% of the scar area disappeared within 3 months of use.

[0027] The second example as shown in FIG. 2(A) is an example of a typical bruise scar. The scar was rarely cured because the wound scabs and peels off repeatedly for 2 weeks. Moreover, disinfection of the affected area with iodine causes pigmentation. After one week of treatment using the ointment prepared according to the present invention, the lesion was cured but the scar still remained as shown in FIG. 2(B); After 3 weeks of use, it can be clearly seen that more than 90% of the scar is removed as shown in FIG. 2(C); After 4 weeks of use, it can be seen that pigmentation is remarkably removed as shown in FIG. 2(D).

[0028] Experimental Method and Results of the Chinese Medicine Ingredients of the Present Invention in Keloid Cells

[0029] Materials

[0030] Fetal bovine serum (FBS), penicillin G, Dulbecco's modified eagle medium (DMEM), and streptomycin prepared by Invitrogen (Carlsbad, Calif., USA). Pyruvic acid and non-essential amino acids prepared by Biological Industries (Kibbutz Beit Haemek, Israel). Primary antibody prepared by Santa Cruz Biotechnology (Santa Cruz, Calif., USA) against procaspase 3/8/9, cleaved caspase 3/8/9, cleaved PARP, cell pigment c, bax, bcl-2 and β-actin. Secondary antibody prepared by Santa Cruz Biotechnology coupled to horseradish peroxidase (HRP). Other reagents prepared by Sigma-Aldrich (St. Louis, Mo., USA).

[0031] Preparation of Test Formulations

[0032] The combination of 10 Chinese medicine ingredients in this experiment is shown in Table 1 below, and used as an in vitro study.

TABLE-US-00001 TABLE 1 Mixed dose No. 1 2 3 4 5 6 7 10 Arte- Ma- Tri- Tetram- Te- Cur- Re- 9 Asia- Ingre- mis- tri- pto- ethyl- tran- cu- svera- 8 Quer- tico- dients inin ne lide pyrazine drin min trol EGCG cetin side uM 3.125 3.125 6.25 6.25 50 12.5 50 3.125 6.25 6.25 Ratio 1 1 2 2 16 4 16 1 2 1

[0033] Experiment Result

[0034] Cell Culture

[0035] KFS cells were obtained from the Biosource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (Hsinchu, Taiwan). KFS cells were cultured in Dulbecco's modified eagle medium (DMEM) containing 10% FBS, 1.5 g/L sodium bicarbonate, 4 mM L-glutamine, 4.5 g/L glucose, 100 units/mL penicillin G, and 100 μg/mL streptomycin sulfate. All cells were incubated in a humidified environment with 5% CO2 and 37° C. The medium was refreshed every 2 days.

[0036] Cell Metabolic Activity Assay (MTT Assay)

[0037] KFS cells (2×10.sup.4 cells/well) were seeded in 96-well plates overnight. KFS cells were exposed to different concentrations of a test drug in 100 μL of medium (for example, combination of 10 Chinese medicine ingredients). After 24 hours treatment, 10 μL of 5 mg/mL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was added to each well. After incubation for 4 hours, cells were washed twice with 1×PBS. Next, 200 μL of dimethyl sulfoxide (DMSO) was added to each well. An absorbance value at 570 nm was determined for each well by using 650 nm as a reference wavelength. Compared with a control group (untreated with reagents), the absorbance may be correlated with the percentage of active cells. The cell metabolic activity rate is calculated as follows: Cell metabolic activity (%)=OD (treatment)/OD (control)×100%.

[0038] The results are shown in FIG. 3. The combination of 10 Chinese medicine ingredients did not affect the cell metabolic activity of KFS cells. FIG. 3 shows the effect of different doses of 10 compounds (3.125, 6.25, 12.5, 50 and 100 μM) on KFS cell growth. That is, after 24 hours of treatment, all IC50 values of the 10 traditional Chinese medicine components were >100 μM.

[0039] Western Blot Analysis

[0040] The obtained cells were cleaved with a PRO-PREP protein extraction reagent kit (iNtRON Biotechnology, Gyeonggi-do, Korea), and centrifuge with 10,000 rpm for 30 minutes at 4° C. A supernatant was incubated in 6× loading buffer containing 0.35 M Tris-HCl (pH 6.8), 10% SDS, 30% glycerol, 0.12% bromophenol blue, and 6% β-mercaptoethanol at 95° C. for 5 minutes. About 50 μg of total protein was isolated through 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then, blotted onto a polyvinylidene fluoride (PVDF) membrane (NEF1002001PK; PerkinElmer, Boston, Mass., USA). A non-specific binding of the blotting membrane was blocked using 5% skim milk dissolved in 1× Tris buffered saline (TBS) and 0.2% Tween20 (0.2% TBST). The membrane was incubated overnight at 4° C. with primary antibody (in 0.2% TBST). Then, secondary antibody (in 0.2% TBST) was applied at 4° C. for 3 hours. The predicted protein bands may be visualized by using an ECL reaction (Amersham, Arlington Height, Ill., USA). Luminescence signals were acquired using a Fujifilm LAS-4000 system (SanLeandro, Calif., USA). A band intensity was analyzed using Multi Gauge software (Fujifilm). The band intensity of individual protein was normalized to that of β-actin, and expressed as fold change compared to the control group (untreated with reagent).

[0041] FIG. 4 shows the regulation of KFS apoptosis-related protein by the combination of 10 Chinese medicine ingredients. The 10 Chinese medicine ingredients were administered to KFS cells via the combination of 0.25×, 0.5× and 1.0×.

CONCLUSION

[0042] According to the cell test method, after dissolving the 10 Chinese medicine ingredients (1. Artemisinin, 2. Matrine, 3. Triptolide, 4. Tetramethylpyrazine, 5. Tetrandrine, 6. Curcumin, 7. Resveratrol, 8. EGCG, 9. Quercetin, 10. Asiaticoside) in an appropriate solution through the method described above, the effects on the proliferation of keloid fibroblasts and the expression of apoptosis proteins were tested. It is confirmed that Tetrandrine, Curcumin, and Resveratrol thereamong inhibit the growth of keloid fibroblasts when the cell concentration is 6.25 to 100 micromolar concentration.

[0043] Next, the 10 Chinese medicine ingredients (1. Artemisinin, 2. Matrine, 3. Triptolide, 4. Tetramethylpyrazine, 5. Tetrandrine, 6. Curcumin, 7. Resveratrol, 8. EGCG, 9. Quercetin, 10. Asiaticoside) were mixed in the following ratio, so as to be acted on keloid fibroblasts at a concentration ratio of 0.25 times to 1 times. It was found that the protein expression of the anti-apoptosis protein Bcl2 may be reduced. For the caspase protease family that produces apoptosis, the combination of the 10 Chinese medicine ingredients may reduce the expression of procaspase 9 in the endogenous pathway of apoptosis and the procaspase 8 protein in the exogenous pathway of apoptosis. The response to increases in cleaved caspase 9 and cleaved caspase 8 signifies that the response pathway for apoptosis of keloid fibroblasts is enhanced.

[0044] The above examples are merely some embodiments of the mushroom-containing Chinese medicine complex composition for keloid scar tissue of the present invention, and are not intended to limit the scope of the invention. Simple equivalent changes and modifications of the spirit or relative components according to the embodiments disclosed in the claims and the detailed description for the Chinese medicine complex composition of the present invention will be understood as covered within the scope of the invention.