AN ORAL PHARMACEUTICAL COMPOSITION COMPRISING AN EXTRACT OF COMBINED HERBS COMPRISING LONGANAE ARILLUS FOR THE TREATMENT OR ALLEVIATION OF INFLAMMATORY DISEASE AND THE USE THEREOF

Abstract

The present invention relates to an oral pharmaceutical composition comprising a combined herb extract of Longanae Arillus, Ligustici Tenuissimi Rhizoma and Polygalae radix, the present inventors demonstrated that the anti-inflammatory/anti-rheumatic effects of inventive combined composition is potent by accomplishing in vitro experiments such as the inhibitory test on the expression of cytokines involved in inflammation (RPLPO, TSLP, GM-CSF and IL-1beta) (Experimental Example 1); Cell viability test on HT-29 and THP-1 cell (in vitro Experimental Example 2); Anti-inflammatory activity in THP-1 cell (in vitro, Experimental Example 3); inhibitory effect on autophagy activity (in vitro, Experimental Example 4.) as well as in vivo experiments such as inhibitory effect on arthritis sing by arthritis-induced rat animal model (in vivo, Experimental Example 5), therefore, it is confirmed that inventive combined extract is very useful in the alleviation or treatment of inflammatory disease and arthritis disease as a form of oral pharmaceutical composition.

Claims

1. An oral pharmaceutical composition comprising a combined herb extract of Longanae Arillus, Ligustici Tenuissimi Rhizoma and Polygalae radix, as an active ingredient for preventing and treating inflammatory diseases selected from group of pruritus caused by dermatitis, atopic dermatitis, conjunctivitis, periodontitis, rhinitis, middle ear infection, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, gout, rheumatoid fever, lupus, fibromyalgia, tendinitis, tenosynovitis Peritendinitis, myositis hepatitis, cystitis, nephritis, Sjogren's syndrome, chronic inflammation and acute inflammation.

2. The oral pharmaceutical composition according to claim 1, wherein said “combined herb extract” is (a) combined herb extract of Longanae Arillus, Ligustici Tenuissimi Rhizoma and Polygalae radix with the mixed ratio based on the dried weight of Longanae Arillus, Ligustici Tenuissimi Rhizoma and Polygalae radix (w/w) ranging from 0.01-100:0.01-100:0.01-100 weight part (w/w); or (b) the combination of each extract of Longanae Arillus, Ligustici Tenuissimi Rhizoma and Polygalae radix with the mixed ratio based on the dried weight of Longanae Arillus, Ligustici Tenuissimi Rhizoma and Polygalae radix (w/w) ranging from 0.01-100:0.01-100:0.01-100 weight part (w/w).

3. The oral pharmaceutical composition according to claim 1, wherein said extract is extracted with at least one solvent selected from water methanol, ethanol, propanol, butanol, acetone, ethyl acetate, chloroform, hexane, butyleneglycol, propyleneglycol or glycerin.

4. A health functional food comprising a combined herb extract of Longanae Arillus, Ligustici Tenuissimi Rhizoma and Polygalae radix, as an active ingredient to prevent or improve inflammatory diseases selected from group of pruritus caused by dermatitis, atopic dermatitis, conjunctivitis, periodontitis, rhinitis, middle ear infection, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, gout, rheumatoid fever, lupus, fibromyalgia, tendinitis, tenosynovitis Peritendinitis, myositis hepatitis, cystitis, nephritis, Sjogren's syndrome, chronic inflammation and acute inflammation.

5. The health functional food according to claim 4, wherein said health functional food is provided as powder, granule, tablet, capsule, pill, suspension, emulsion, syrup, tea bag, leached tea, or beverage type.

6. (canceled)

7. A method of treating or alleviating inflammatory diseases selected from group of pruritus caused by dermatitis, atopic dermatitis, conjunctivitis, periodontitis, rhinitis, middle ear infection, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, gout, rheumatoid fever, lupus, fibromyalgia, tendinitis, tenosynovitis Peritendinitis, myositis hepatitis, cystitis, nephritis, Sjogren's syndrome, chronic inflammation and acute inflammation in a mammal comprising orally administering to said mammal an effective amount of the combined herb extract of Longanae Arillus, Ligustici Tenuissimi Rhizoma and Polygalae radix and pharmaceutically acceptable carrier thereof.

8. (canceled)

Description

BEST MODE FOR CARRYING OUT THE INVENTION

[0053] It will be apparent to those skilled in the art that various modifications and variations can be made in the compositions, use and preparations of the present invention without departing from the spirit or scope of the invention.

[0054] The present invention is more specifically explained by the following examples. However, it should be understood that the present invention is not limited to these examples in any manner.

EXAMPLES

[0055] The following Examples and Experimental Examples are intended to further illustrate the present invention without limiting its scope.

Example 1. The Preparation of Inventive Combined Extract (1)

[0056] 20 g of dried Longanae Arillus (Buyoung Yakup Co., Ltd.), 20 g of dried Ligustici Tenuissimi Rhizoma (Buyoung Yakup Co. Ltd.) and 20 g of dried Polygalae radix(Buyoung Yakup Co. Ltd.) were cut into small pieces, mixed with 6 fold volume (v/w) of 20% ethanol in water and the mixture was subjected to reflux extraction at 90±5° C. for 3 days. After filtration of the extract through filter paper (pore size, less than 10 μm) to remove the debris, the remaining debris was further extracted two times with 4 fold volume (v/w) of 20% ethanol in water and the extract was filtered with filter paper (pore size, less than 10 μm).

[0057] The collected extract was mixed with together and concentrated under vaccuo (16-21 Brix) to afford concentrated extract. The concentrated extract was dried with freeze drying process and pulverized (less than 50 mesh) to obtain 20.5 g (powder as dried basis, yield 33.4%) of inventive combined extract (1) (designated as “WIN-1001X” hereinafter)

Example 2-6. The Preparation of Inventive Combined Extract (2)-(6)

[0058] Excepting adopting different combined ratio as well as different solvents disclosed in Example 1, all the procedure was identical with those in Example 1 to obtain various inventive combined extract of Longanae Arillus (LA), Ligustici Tenuissimi Rhizoma (LT) and Polygalae radix (PR) i.e., inventive combined extract (2) to inventive combined extract (6) of the present invention, which are used as a test samples in following experiment.

TABLE-US-00001 TABLE 1 various kinds of combined extract Sample weight (g) Extract Final Example LA* PR* LT* solvent* name weight yield Example 2 10 5 50 10% WIN-1002X 16.6 g 25.6% EtOH Example 3 20 50 5 Water WIN-1003X 24.7 g 32.9% Example 4 10 80 20 70% WIN-1004X 32.3 g 29.4% BuOH Example 5 5 50 20 50% WIN-1005X 21.5 g 28.7% EtOH Example 6 30 10 2 hexane WIN-1006X 12.6 g 30.1% *Longanae Arillus (LA), Ligustici Tenuissimi Rhizoma (LT), Polygalae radix (PR)

Experimental Example 1. Inhibitory Effect on Cytokine Expression (In Vitro)

[0059] In order to determine the anti-inflammatory activity of inventive extract, following inhibition test of cytokine expression using HaCaT cell was performed according to the procedure disclosed in the literature (Jeong et al., 2019, J. Invest. Dermatol., May; 139 (5): pp 1098-1109).

[0060] HaCaT cell (human epithelial keratinocyte cell, 300493, CLS) was inoculated into DMEM medium containing 10% Fetal bovine serum, 100 units/ml of penicillin, 100 μg/ml of streptomycin (D6429, Sigma-Aldrich Co. Ltd) and was incubated in the incubator (HERA cell 150i, Thermo Fisher Scientific Co. Ltd.) maintaining optimum humidity (85-95%) and 5% CO.sub.2 atmosphere.

[0061] For performing gene expression test, the incubated cells were transferred to 12 wells and 50 ng/ml of TNF alpha (RC214-12, Biobasic Co. Ltd) was treated therewith for 1 hour to induce inflammatory response. Dexamethasone (200 nM, positive control, “DEX”, D4902, Sigma-Aldrich Co. Ltd.) and distilled water (negative control, “DIW”) were used as comparative controls.

[0062] 1 hour after inducing the inflammation, 1 μg/ml of inventive extract prepared in Examples was treated with identical medium and subjected to incubation for 1 hour. After the incubation, RNA (FATRR-001, Favorgen) was extracted from the cell and cDNA was synthesized from the RNA using by cDNA synthesis kit (RRO36A, TAKARA). The polymerization reaction was performed using by the synthesized cDNA and Sybrgreen kit (RT500M, Enzynomics) and then Real-time-PCR was performed using by primers for various cytokines involved in skin inflammation (RPLPO, TSLP, GM-CSF and IL-1beta) as disclosed in Table 2.

TABLE-US-00002 TABLE 2 The used primers in RT-PCR method human* direction sequence Sequence I.D RPLP0 forward 5′-AGC CCA GAA CAC TGG TCT 1 C-3′ reverse 5′-ACT CAG GAT TTC AAT GGT 2 GCC-3′ TSLP forward 5′-TAT GAG TGG GAC CAA AAG 3 TAC CG-3′ reverse 5′-GGG ATT GAA GGT TAG GCT 4 CTG G-3′ GM- forward 5′-TCC TGA ACC TGA GTA GAG 5 CSF ACA C-3′ reverse 5′-TGC TGC TTG TAG TGG CTG 6 G-3′ IL-1β forward 5′-CTC CAG GGA CAG GAT ATG 7 GA-3′ reverse 5′-TCT TTC AAC ACG CAG GAC 8 AG-3′ * abbreviation-RPLP0 (Ribosomal Protein Lateral Stalk Subunit P0); TSLP (thymic stromal lymphopoietin); GM(Granulocyte-macrophage)-CSF (colony stimulating factor); IL (interleukin)

[0063] As can be seen in Table 3 showing quantitative result of the RT-PCR, the test sample group treated with the inventive extract, sharply inhibited the expressed level of various cytokine involved in inflammation comparing with negative control group treated with distilled water (DIW) and it has been confirmed that the inhibitory activity of the test sample on the expression of various cytokine involved in inflammation is equivalent to that of positive control group treated with dexamethasone (DEX).

[0064] Accordingly, it has been confirmed that the various kind of inventive combined extract prepared in Examples 1-6 have potent inhibitory effect on inflammation.

TABLE-US-00003 TABLE 3 Inhibition effect on cytokine expression TNFα TNFα TNFα TNFα — TNFα WIN- WIN- WIN- WIN- TNFα — DIW 1001X 1002X 1003X 1005X Dex TSLP 1 132.4692 47.43735 60.85783 48.9323 55.34286 52.49334 0.462769 26.91228 9.645089 24.95619 19.85678 26.59252 11.2336 GM-CSF 1 4.473627 1.982161 2.069408 2.384771 1.917569 1.935997 0.111 0.817826 0.889233 0.326074 0.871501 0.599711 0.581338 IL-1β 1 4.152715 1.407169 1.437399 2.064964 1.662578 1.080503 0.483565 1.087056 0.394622 0.1926 0.620225 0.193175 0.413136

Experimental Example 2. Cell Viability Test on HT-29 and THP-1 Cell (In Vitro)

[0065] In order to confirm the cytotoxicity of inventive extract on HT-29 cell and THP-1 cell, following cell viability using HT-29 cell and THP-1 cell was according to the previous known procedure in the art.

[0066] 2-1. Procedure

[0067] HT-29 cell (human colon epithelial cell, Korean Cell Line Bank, Korean Cell Line Research Foundation, 101, Daehak-ro, Jongno-gu, Seoul, 03080, Korea) was inoculated into DMEM medium containing 10% Fetal bovine serum and 1% penicillin-streptomycin solution and THP-1 cell (human monocyte cell, Korean Cell Line Bank, Korean Cell Line Research Foundation, 101, Daehak-ro, Jongno-gu, Seoul, 03080, Korea) was inoculated into RPMI medium containing 10% Fetal bovine serum and 1% penicillin-streptomycin solution to incubate.

[0068] 25, 50, 100, 250, 500 and 1000n/mL of test samples were added to HT-29 cell and THP-1 cell. 24 hours after the incubation, CCK-8 (cell counting kit-8, Dojindo Molecular Technologies, Inc.) was added thereto and the absorbance (optical density) was determined at 450 nm to determine cell viability.

[0069] 2-2. Test Result

[0070] As can be seen in Table 4, it has been confirmed that the cell viability of test sample group treated with the inventive extract (25-1000 μg/mL) in HT-29 cell showed similar to that of negative control group treated with only medium and that in THP-1 cell showed 90.5% and 26.5% at the concentration of 500 and 1000 μg/mL, respectively, which showed some different with that of negative control group and less than 500 μg/mL of test sample was applied to following test.

TABLE-US-00004 TABLE 4 cell viability test result WIN1001X μg/mL HT-29 cell THP-1 cell 0 100% 100% 25 100.5 ± 0.6% 102.2 ± 1.1% 50 103.3 ± 3.2% 103.7 ± 1.5% 100 105.0 ± 1.4% 104.4 ± 1.6% 250 109.2 ± 3.1% 101.1 ± 0.6% 500 105.9 ± 3.3%  90.5 ± 0.8% 1000  90.3 ± 0.1%  26.5 ± 5.8%

Experimental Example 3. Anti-Inflammatory Activity in THP-1 Cell (In Vitro)

[0071] In order to determine the anti-inflammatory activity of inventive extract, following inhibition test of the level of pro-inflammatory cytokines using THP-1 cell was performed according to the previous known procedure in the art.

[0072] 3-1. Determination on the Level of IL-1Beta

[0073] 3-1-1. Test Procedure

[0074] 10, 20, 50, 100, and 500 μg/mL lipopolysaccharide (LPS) was added to human monocyte cell lines (THP-1 cell Korean Cell Line Bank, Korean Cell Line Research Foundation, 101, Daehak-ro, Jongno-gu, Seoul, 03080, Korea) to prepare an inflammatory model.

[0075] 24 hours after LPS treatment, the level of IL-1beta, an inflammatory cytokine, in the collected cell supernatant solution was measured.

[0076] In addition, 10, 25, 50 and 100 μg/mL of test samples were treated in THP-1 cells for four hours and LPS was treated therewith to confirm the anti-inflammatory effect of the test sample.

[0077] 24 hours after LPS treatment, the level of IL-1beta, a pro-inflammatory cytokine, was measured using ELISA reader (IL-1beta/IL-1F2 Duo set ELISA, R&D Systems).

[0078] 3-1-2. Test Result

[0079] As can be seen in Table 5, it has been confirmed that the test sample prepared in Examples sharply reduced the level of IL-1beta which had been increased with the dose-dependently increased LPS.

[0080] Accordingly, it has been confirmed that the inventive combined extract prepared in Examples has potent inhibitory effect on inflammatory response.

TABLE-US-00005 TABLE 5 Inhibition effect on IL-lbeta (pro-inflammatory cytokine) IL-1β level (μg/ mL) WIN1001X LPS 20 LPS 50 LPS 100 LPS 500 μg/mL μg/mL μg/mL μg/mL μg/mL 0  117.7 ± 14.0 262.8 ± 9.0 284.2 ± 9.0  319.5 ± 10.4 10  71.1 ± 6.5 214.5 ± 5.5 242.1 ± 4.0 312.2 ± 9.3 25  84.9 ± 7.6 186.5 ± 6.9 213.2 ± 6.2 285.1 ± 9.3 50   68.0 ± 12.4 158.4 ± 5.3 186.4 ± 3.2  245.2 ± 11.0 100 133.4 ± 8.6  198.9 ± 10.3 233.9 ± 6.0 311.1 ± 8.4

[0081] 3-2. Determination on the Level of IL-10

[0082] 3-2-1. Test Procedure

[0083] 10, 20, 50, 100, and 500 μg/mL of lipopolysaccharide (LPS) was added to human monocyte cell lines (THP-1 cell Korean Cell Line Bank, Korean Cell Line Research Foundation, 101, Daehak-ro, Jongno-gu, Seoul, 03080, Korea) to prepare an inflammatory model.

[0084] 24 hours after LPS treatment, the level of IL-10, an inflammatory cytokine, in the collected cell supernatant solution was measured.

[0085] In addition, 10, 25, 50 and 100 μg/mL of test samples were treated in THP-1 cells for four hours and LPS was treated therewith to confirm the anti-inflammatory effect of the test sample.

[0086] 24 hours after LPS treatment, the level of IL-10, a pro-inflammatory cytokine, was measured using ELISA reader (IL-10, Duo set ELISA, R&D Systems).

[0087] 3-2-2. Test Result

[0088] As can be seen in Table 6, it has been confirmed that the test sample prepared in Examples sharply reduced the level of IL-10 which had been increased with the dose-dependently increased LPS.

[0089] Accordingly, it has been confirmed that the inventive combined extract prepared in Examples has potent inhibitory effect on inflammatory response.

TABLE-US-00006 TABLE 6 Inhibition effect on IL-10 (pro-inflammatory cytokine) WIN1001X IL-10 level (pg/mL) μg/mL LPS 50 μg/mL LPS 100 μg/mL LPS 500 μg/mL 0 5.3 ± 1.0 12.0 ± 1.8 14.2 ± 1.1 10 6.2 ± 1.6  7.3 ± 1.7 15.6 ± 1.7 25 10.6 ± 2.1   6.7 ± 1.8 19.4 ± 1.8 50 5.2 ± 1.6 14.0 ± 1.6 24.5 ± 1.0 100 45.5 ± 2.5  67.3 ± 4.3 87.0 ± 4.8

Experimental Example 4. Inhibitory Effect on Autophagy Activity (In Vitro)

[0090] In order to determine the effect of inventive extract on the expression of Inflammatory factor in immune cell, following test using THP-1 cell was performed according to the previous known procedure in the art.

[0091] 4-1. Test Procedure

[0092] In order to confirm the correlation between ant-inflammatory activity and autophagy pathway, 10, 20, 50, 100, and 500 μg/mL lipopolysaccharide (LPS) was added to human monocyte cell lines (THP-1 cell Korean Cell Line Bank, Korean Cell Line Research Foundation, 101, Daehak-ro, Jongno-gu, Seoul, 03080, Korea) to prepare an inflammatory model.

[0093] After the treatment of 10, 25, 50 and 100 μg/mL of test samples, 20 and 100 μg/mL of lipopolysaccharide (LPS) was added thereto to confirm the expression of Beclin 1 and LC3B (autophagy marker) through immuno-blotting test using anti-Beclin 1 antibody (Abcam) and LC3B-antibody (Cell Signaling), respectively.

[0094] The expressed level of Beclin 1 and LC3B was quantified by scanning the resulting photo-sensitized film with ChemiDoc™MPImagingSystem (Biorad).

[0095] 4-2. Test Result

[0096] As can be seen in Table 7, it has been confirmed that the test group treated with test samples and 100 μg/mL of lipopolysaccharide (LPS), showed the dose-dependently increasing effect on the expression of LC3B-1 and Beclin1.

TABLE-US-00007 TABLE 7 effect on the expression of Beclin 1 and LC3B LPS 20 ug/mL LPS 100 ug/mL WIN1001X ratio ratio ratio ratio μg/mL Beclin/β-actin LC3/β-actin Beclin/β-actin LC3/β-actin 0 0.67 1.18 0.81 1.35 10 0.76 0.86 0.63 1.59 25 0.37 1.05 0.82 2.49 50 0.41 2.55 1.39 2.28 100 0.25 3.47 1.10 4.57

Experimental Example 5. Inhibitory Effect on Arthritis (In Vivo)

[0097] To confirm the inhibitory effect of inventive extract on arthritis, the animal model test using by arthritis-induced rat animal model, was performed according to the previous known procedure in the art.

[0098] 5-1. Test Procedure

[0099] In order to evaluate the efficacy of test sample on MIA(Monosodium iodoacetate)-induced osteoarthritis rat model, following test was performed at “Joint and Immune disease T2B center(chief MD. PARK, Sung-whan) located in “The Catholic University of Korea, Seoul ST. Mary's Hospital, Seoul).

[0100] 5-1-1. Test Protocol

[0101] 5-1-1-1. Animal Model Test Procedure

[0102] (1) Test animals: Rats (Male Wista rats, Central Lab. Animal, Seoul, Korea, 7 to 8 weeks aged, 200 to 250 g) were bred in the well-controlled breeding room in polysulfone cage (2-3 mice per cage) maintaining the temperature of 21±2° C. and relative humidity of 50±20% with the light cycle of day/dark (08:00˜20:00) at the interval of 12 hours and acclimated to the surround environment

[0103] Test method: 3 mg/kg of Monosodium iodoacetate (MIA, 12512, Sigma, Poole, UK) was dissolved in the injection saline to re reach to 60 mg/ml concentration on the day of the experiment (day 0). After dividing each groups, the experimental animals were placed in the anesthesia chamber and anesthetized with diethyl ether. 50 μL of MIA (3 mg/body) was injected into the right knee joint through the infrapatellar ligament using a 1cc syringe (26.5 gauge) in order to inducing osteoarthritis

[0104] (2) Test sample: the inventive extract prepared in Examples

[0105] (3) Positive control: Celecoxib® (Hanlim Pharmaceutical Company, Seoul, KOREA)

[0106] (4) Treatment route: oral administration 3 days after inducing osteoarthritis (once a day)

[0107] (5) The establishment of test groups: See Table 8.

[0108] 5-1-1-2. Administration Route and Test Period

[0109] (1) Administration Route

[0110] After inducing osteoarthritis with MIA, test sample treatment groups (G2, G3) were prepared by homogenizing the test samples with vehicle (saline) according to the prescribed dose and orally administrating 1 mL of test sample once a day.

[0111] The negative control group (G1) was treated with only vehicle and orally administrated once a day according to the sample schedule as the test substance administration.

[0112] The positive control group (G4) was homogenized to the vehicle according to the prescribed dose and orally administered once a day.

[0113] (2) Test Period

[0114] 24 rats were divided into 4 groups per 6 rats, and the test samples and positive control substance were orally administered at a specified time.

TABLE-US-00008 TABLE 8 The establishment of test groups Group Drug administration Dose n G1 MIA + vehicle (saline) — 6 G2 MIA + WIN1001X (saline) 100 mg/kg 6 G3 MIA + WIN1001X (saline) 150 mg/kg 6 G4 MIA + Celecoxib (0.5% CMC)  30 mg/kg 6 negative control group(G1), test sample group(G2, G3), positive control group (G4), n = 24

[0115] 5-2. Evaluation Contents

[0116] 5-2-1. Determination of Pain Threshold

[0117] 5-2-1-1. Determination Method of Pain Threshold

[0118] The pain threshold (nociceptive latency, threshold) was determined by using dynamic plantar aesthesiometer (Ugo Basile, 37400, Comerio, Italy), a device that gradually increases the force on the feet of Male Wistarats over a certain period of time, according to a von Freestyle evaluation method to measure pain thresholds ((Kwon J Y et al., Sci Rep. 2018 Sep. 14; 8(1):13832)

[0119] Prior to measurement, the animal was placed in an acrylic box with a wire mesh bench and acclimated for five minutes.

[0120] The pain threshold was determined by measuring the weight showing paw withdrawal behavior which animal withdraws its feet by applying a slow force of 0 to 50 g over 10 seconds using metal filaments in the center of each animal's right hind foot.

[0121] To avoid tissue damage, the cut-off threshold was set to 50 g, and the measurement time was set to the day before the inducing day of osteoarthritis by the treatment of MIA to calculate base line value in the normal control group (G1), test sample groups (G2, G3) and positive control group (G4) and the same value was obtained in the base line (day 3).

[0122] The same value was measured before the treatment of test samples (day 3) and the value was measured once a week at a certain time by administering the test samples in the articular cavity of rats.

[0123] The test result of pain threshold was calculated according to (a) paw withdrawal latencies (sec) and (b) paw withdrawal threshold (g).

[0124] 5-2-1-2. Test Result of Determination of Pain Threshold

[0125] As the test result of the pain measurement, it has been confirmed that the test sample group treated with 100 and 150 mg/kg of test samples showed potent inhibitory effect on pain comparing with negative control group treated with vehicle, dose-dependently manner. (Table 9-10)

TABLE-US-00009 TABLE 9 The inhibitory effect on pain of each group according to time lapse (paw withdrawal latencies, sec) MIA WIN1001X WIN1001X Celecoxib inducement vehicle (s) 100 mg/kg (s) 150 mg/kg (s) 30 mg/kg (s) Before MIA 16.8 ± 0.8  16.2 ± 0.6 16.5 ± 0.6 16.0 ± 1.5 treatment 3 days after 9.4 ± 1.2  8.0 ± 1.1  9.7 ± 1.1  9.2 ± 2.1 MIA treatment 6 days after 8.6 ± 1.0 11.0 ± 1.3 11.2 ± 1.3  9.9 ± 2.2 MIA treatment 11 days after 9.1 ± 1.4 12.4 ± 2.0 11.4 ± 1.8 10.2 ± 0.9 MIA treatment 13 days after 9.0 ± 1.6 11.4 ± 1.0 12.1 ± 1.5 10.4 ± 0.9 MIA treatment 17 days after 9.5 ± 0.4 11.1 ± 0.4 13.1 ± 1.4 10.9 ± 1.8 MIA treatment 21 days after 9.0 ± 1.6 12.8 ± 1.6 13.5 ± 1.6 11.9 ± 1.5 MIA treatment 25 days after 9.0 ± 0.5 13.3 ± 0.5 13.9 ± 2.0 11.6 ± 1.0 MIA treatment

TABLE-US-00010 TABLE 10 The inhibitory effect on pain of each group according to forced weight (paw withdrawal threshold, g). Celecoxib MIA WIN1001X WIN1001X 30 inducement vehicle (g) 100 mg/kg (g) 150 mg/kg (g) mg/kg (g) Before MIA 33.6 ± 1.7 32.5 ± 1.2 33.0 ± 1.2 33.8 ± 2.9 treatment 3 days after 19.0 ± 2.4 16.2 ± 2.1 19.7 ± 2.2 18.6 ± 4.1 MIA treatment 6 days after 17.4 ± 1.8 22.3 ± 2.7 22.6 ± 2.5 19.9 ± 4.3 MIA treatment 11 days after 18.5 ± 2.8 25.0 ± 4.0 23.1 ± 3.6 20.7 ± 1.7 MIA treatment 13 days after 18.3 ± 3.2 23.0 ± 2.0 24.5 ± 2.9 21.0 ± 1.6 MIA treatment 17 days after 19.2 ± 0.8 22.5 ± 0.9 26.3 ± 2.7 22.0 ± 3.4 MIA treatment 21 days after 18.3 ± 3.1 25.7 ± 3.3 27.3 ± 3.2 24.0 ± 2.9 MIA treatment 25 days after 18.2 ± 1.1 26.7 ± 0.9 27.9 ± 4.0 23.4 ± 1.9 MIA treatment

[0126] 5-2-2. Test on the Determination of Weight Bearing

[0127] 5-2-2-1. Determination of Weight Bearing

[0128] In order to measure the change in weight (or weight distribution) between normal hind legs (left) and arthritis-induced hind legs (right), the load of both hind feet was determined using testing apparatus (Model 600, IITC, USA) according to the method described in the literature (Kwon J Y et al., Sci Rep. 2018 Sep. 14; 8(1):13832).

[0129] The test animal shall be correctly located in the holder and fixed so that both feet can be stepped symmetrically, since the load can be varied depending on the position of the animal's foot. The particular person performs the fixation to reduce the possible error to the maximum extent.

[0130] When each animal was correctly located in the holder, the machine was operated and the weight bearing of each group was measured twice for 5 seconds per measurement.

[0131] The mean value for each test result was digitized into the weight bearing(g) of each foot.

[0132] After obtaining a baseline value before MIA treatment (Day 0), the weight bearing in the normal control group (G1), test sample groups (G2, G3) and positive control group (G4), was determined at a certain time twice a week, 3 days before the treatment of test sample (day 3).

[0133] The test results of the weight load measurement were transformed into each weight bearing ratio according to below Math 1.

Weight ratio (%)={weight of right hind limb/(weight of right hind limb+weight of left hind limb)}×100  [Math.1]

[0134] 5-2-2-2. Test Result of Weight Bearing

[0135] As can be seen in Table 11, it has been confirmed that the test sample group potently improved the balance capability of both limbs comparing with the negative control group.

TABLE-US-00011 TABLE 11 effect on the weight bearing Celecoxib MIA WIN1001X WIN1001X 30 inducement vehicle (%) 100 mg/kg (%) 150 mg/kg (%) mg/kg (%) Before MIA 50.6 ± 1.2 49.6 ± 0.8 50.3 ± 1.3 49.3 ± 0.9 inducement 3 days 36.9 ± 1.8 37.9 ± 2.7 35.2 ± 2.4 34.7 ± 4.0 after MIA inducement 6 days 35.5 ± 1.5 38.3 ± 1.7 38.6 ± 1.3 38.5 ± 1.8 after MIA inducement 11 days 33.8 ± 2.9 41.0 ± 2.5 39.1 ± 3.0 40.0 ± 3.2 after MIA inducement 13 days 32.3 ± 2.8 43.6 ± 1.4 44.2 ± 1.5 44.5 ± 0.7 after MIA inducement 17 days 32.2 ± 1.7 42.9 ± 1.2 42.5 ± 1.3 43.9 ± 1.3 after MIA inducement 21 days 31.6 ± 2.0 40.6 ± 1.2 41.0 ± 1.5 39.7 ± 3.7 after MIA inducement 25 days 33.3 ± 1.8 42.3 ± 2.3 42.0 ± 1.4 41.7 ± 1.9 after MIA inducement

[0136] 5-2-3. Inhibitory Test on Bone Injury (Histological Analysis, Micro-CT)

[0137] In order to determine the inhibitory effect of inventive extract on bone injury, following histological analysis was performed according to the known method disclosed in the literature (Kwon J Y et al., Sci. Rep. 2018 Sep. 14; 8(1):13832).

[0138] 5-2-3-1. Inhibitory Effect on Bone Injury Caused by Osteoarthritis (Histological Analysis, Micro-CT)

[0139] In order to determine the inhibitory effect of inventive extract on bone injury caused by osteoarthritis, the MIA and test sample were administered to the right knee joint of sacrificed rats (male Wistar rats) and fixed with formalin.

[0140] The degree of bone injury around femur area of the knee joint in each group consisting of 3 rats, was determined using X-ray source (70 kV, 142 uA, AI 0.5 mm filter, rotation step 0.6°) and animal scanner (SKYSCAN1272 ex-vivo micro-CT, Bruker micro CT, Belgium) to perform micro-CT photography at 15 μm of Pixel resolution including section formation (NRecon), section rotation (Data Viewer), data Analysis (CTAN), Volume rendering generation (CTVox), and surface rendering (CTAN+CTVol).

[0141] 5-2-3-2. Test Result of Histological Analysis (Micro-CT)

[0142] As can be seen in Table 12, it has been confirmed that the test sample group treated with 100 and 150 mg/kg of test samples showed potent inhibitory effect on bone injury comparing with negative control group (vehicle group) through the test result of Micro-CT photography.

TABLE-US-00012 TABLE 12 inhibitory effect on bone injury (Micro-CT photography) WIN1001X WIN1001X Celecoxib vehicle 100 mg/kg 150 mg/kg 30 mg/kg Bone surface 45.7 ± 8.9 59.3 ± 2.9 56.7 ± 2.5 57.5 ± 0.5 (%)

[0143] 5-2-4. Inhibitory Test on Bone Injury (Histopathological Analysis)

[0144] In order to determine the inhibitory effect of inventive extract on bone injury, following histopathological analysis was performed according to the known method disclosed in the literature (Kwon J Y et al., Sci Rep. 2018 Sep. 14; 8(1):13832).

[0145] 5-2-3-1. Inhibitory Effect on Bone Injury Caused by Osteoarthritis (Histopathological Analysis)

[0146] In order to determine the inhibitory effect of inventive extract on bone injury caused by osteoarthritis, the MIA and test sample were administered to the right knee joint of sacrificed rats (male Wistar rats) and the right knee joint of the rat fixed with formalin was sliced to stain with Safranin O.

[0147] The histopathological analysis was performed by photographing the femur area of knee joint (×200 fold). The test result was calculated and evaluated according to the known methods including Total Mankin score (Bulstra S K et al., 1989, Clin Orthop Relat Res: 294-302.) and OARSI score (Pritzker K. P. H. et al., 2006, Osteoarthritis Cartilage 14:13-29).

[0148] 5-2-3-2. Test Result of Histopathological Analysis

[0149] As can be seen in Table 13 (total Mankin score method) and Table 14 (OARSI score), it has been confirmed that the test sample group treated with 100 and 150 mg/kg of test samples showed potent inhibitory effect on bone injury comparing with negative control group (vehicle group) through the test result of histopathological analysis

TABLE-US-00013 TABLE 13 inhibitory effect on bone injury (total Mankin score method) WIN1001X WIN1001X Celecoxib vehicle 100 mg/kg 150 mg/kg 30 mg/kg Total Mankin 7.5 ± 0.8 2.8 ± 0.6 4.7 ± 0.7 4 ± 0.6 score

TABLE-US-00014 TABLE 14 inhibitory effect on bone injury (OARSI score method) WIN1001X WIN1001X Celecoxib vehicle 100 mg/kg 150 mg/kg 30 mg/kg OARSI score 4 ± 0.4 1.9 ± 0.4 2.0 ± 0.2 2.4 ± 0.4

[0150] Statistics Analysis

[0151] All data were expressed in mean and standard deviation (mean±SD), and statistical significance verification was determined to be significant (P<0.05), using two-way ANOVA, one-way ANOVA, in GraphPad PRISM Version 5.0 (USA) analysis program.

MODE FOR THE INVENTION

[0152] Hereinafter, the formulating methods and kinds of excipients will be described, but the present invention is not limited to them. The representative preparation examples were described as follows.

[0153] Preparation of Injection

[0154] WIN-1001X extract: 100 mg

[0155] Sodium metabisulfite: 3.0 mg

[0156] Methyl paraben: 0.8 mg

[0157] Propyl paraben: 0.1 mg

[0158] Distilled water for injection: optimum amount

[0159] Injection preparation was prepared by dissolving active component, controlling pH to about 7.5 and then filling all the components in 2e ample and sterilizing by conventional injection preparation method.

[0160] Preparation of Powder

[0161] WIN-1002X extract: 500 mg

[0162] Corn Starch: 100 mg

[0163] Lactose: 100 mg

[0164] Talc: 10 mg

[0165] Powder preparation was prepared by mixing above components and filling sealed package.

[0166] Preparation of Tablet

[0167] WIN-1003X extract 200 mg

[0168] Corn Starch 100 mg

[0169] Lactose 100 mg

[0170] Magnesium stearate optimum amount

[0171] Tablet preparation was prepared by mixing above components and entabletting.

[0172] Preparation of Capsule

[0173] WIN-1004X extract: 100 mg

[0174] Lactose: 50 mg

[0175] Corn starch: 50 mg

[0176] Talc: 2 mg

[0177] Magnesium stearate optimum amount

[0178] Tablet preparation was prepared by mixing above components and filling gelatin capsule by conventional gelatin preparation method.

[0179] Preparation of Liquid

[0180] WIN-1005X extract: 1000 mg

[0181] Sugar: 20 g

[0182] Polysaccharide: 20 g

[0183] Lemon flavor: 20 g

[0184] Liquid preparation was prepared by dissolving active component, and then filling all the components in 1000 ml ample and sterilizing by conventional liquid preparation method.

[0185] Preparation of Health Food

[0186] WIN-1001X extract: 1000 mg

[0187] Vitamin mixture: optimum amount

[0188] Vitamin A acetate: 70 g

[0189] Vitamin E: 1.0 mg

[0190] Vitamin B.sub.10: 13 mg

[0191] Vitamin B.sub.2: 0.15 mg

[0192] Vitamin B6: 0.5 mg

[0193] Vitamin B1: 20.2 g

[0194] Vitamin C: 10 mg

[0195] Biotin: 10 g

[0196] Amide nicotinic acid: 1.7 mg

[0197] Folic acid: 50 g

[0198] Calcium pantothenic acid: 0.5 mg

[0199] Mineral mixture: optimum amount

[0200] Ferrous sulfate: 1.75 mg

[0201] Zinc oxide: 0.82 mg

[0202] Magnesium carbonate: 25.3 mg

[0203] Monopotassium phosphate: 15 mg

[0204] Dicalcium phosphate: 55 mg

[0205] Potassium citrate: 90 mg

[0206] Calcium carbonate: 100 mg

[0207] Magnesium chloride: 24.8 mg

[0208] The above mentioned vitamin and mineral mixture may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the present invention.

[0209] Preparation of Health Beverage

[0210] WIN-1002X extract: 1000 mg

[0211] Citric acid: 1000 mg

[0212] Oligosaccharide: 100 g

[0213] Apricot concentration: 2 g

[0214] Taurine: 1 g

[0215] Distilled water: 900 ml

[0216] Health beverage preparation was prepared by dissolving active component, mixing, stirred at 85° C. for 1 hour, filtered and then filling all the components in 1000 ml ample and sterilizing by conventional health beverage preparation method.

[0217] The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the present invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

INDUSTRIAL APPLICABILITY

[0218] As described in the present invention, the present invention provides an oral pharmaceutical composition comprising a combined herb extract of Longanae Arillus, Ligustici Tenuissimi Rhizoma and Polygalae radix, the present inventors demonstrated that the anti-inflammatory/anti-rheumatic effects of inventive combined composition is potent by accomplishing in vitro experiments such as the inhibitory test on the expression of cytokines involved in inflammation (RPLPO, TSLP, GM-CSF and IL-1beta) (Experimental Example 1); Cell viability test on HT-29 and THP-1 cell (in vitro Experimental Example 2); Anti-inflammatory activity in THP-1 cell (in vitro, Experimental Example 3); inhibitory effect on autophagy activity (in vitro, Experimental Example 4.) as well as in vivo experiments such as inhibitory effect on arthritis using by arthritis-induced rat animal model (in vivo, Experimental Example 5), therefore, it is confirmed that inventive combined extract is very useful in the alleviation or treatment of inflammatory disease and arthritis disease as a form of oral pharmaceutical composition.