COMPOSITION FOR TREATING BENIGN PROSTATE HYPERPLASIA AND FUNCTIONAL HEALTH FOOD COMPRISING AUCKLANDIAE RADIX EXTRACT AND COSTUNOLIDE AS ACTIVE INGREDIENTS

Abstract

The present invention relates to a composition for treating benign prostatic hyperplasia and a health functional food, containing an extract of Aucklandiae Radix and costunolide as active ingredients.

Claims

1. A composition containing an Aucklandiae Radix extract as an active ingredient for treatment and prevention of benign prostatic hyperplasia.

2. The composition of claim 1, wherein the Aucklandiae Radix extract is obtained using any one of ethanol, methanol, and water as a solvent.

3. A composition containing costunolide as an active ingredient for treatment and prevention of benign prostatic hyperplasia.

4. A health functional food containing the ingredient of any one of claims 1 to 3 for alleviation of benign prostatic hyperplasia.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1 shows the evaluation results of NO production rate and antiinflammatory effect according to the treatment with a 100% methanol extract of Aucklandiae Radix, a 30% ethanol extract of Aucklandiae Radix, a 70% ethanol extract of Aucklandiae Radix, and costunolide.

[0024] FIGS. 2A-2C show the measurement results of levels of inflammatory cytokines (IL-1, IL-6, TNF-) according to the treatment with a 30% ethanol extract of Aucklandiae Radix, a 70% ethanol extract of Aucklandiae Radix, and costunolide.

[0025] FIG. 3 shows benign prostatic hyperplasia alleviation effect according to the treatment with a 100% methanol extract of Aucklandiae Radix, a 30% ethanol extract of Aucklandiae Radix, a 70% ethanol extract of Aucklandiae Radix, and costunolide.

[0026] FIGS. 4 and 5 show the results of prostate pathology biopsy according to the Aucklandiae Radix extracts and costunolide.

MODE FOR CARRYING OUT THE INVENTION

[0027] Hereinafter, the present invention will be described in detail with reference to examples. The examples are provided merely to help with the understanding of the present invention, and the scope of the present invention is not limited to the examples.

[0028] <1. Manufacture of Aucklandiae Radix Extracts>

[0029] Aucklandiae Radix used in the present tests was purchased from Hunjin Pharmacy.

[0030] The purchased Aucklandiae Radix was subjected to extraction a total of twice using a reflux extractor (COSMOS660/80L, Misung Scientific. co. Ltd., Yangju, Kyunggido, Korea), and then the extracts were used in mixture. A solvent was added at 10 times the weight of the sample at the first extraction and added at 7 times the weight of the sample at the second extraction, followed by heat extraction for 3 hours each time of extraction.

[0031] The extracts were filtered (Whatman No 2, Maidstone, England), lyophilized (PVTFD10R, Ilsinb iobase, Dounducheon, Gyeonggido, Korea), milled (Cyclotec 1093 Sample Mill, Tecator Co., Sweden), and then stored frozen at 20 C. before use.

[0032] As the solvent, 100% methanol, 70% ethanol, or 30% ethanol was used, and the extraction conditions were identical therefor.

[0033] <2. HPLC Analysis of Aucklandiae Radix Extract>

[0034] The Aucklandiae Radix extracts were weighed 10 mg, respectively, and shaken to dissolve in methanol for 30 minutes or longer. The undissolved components were removed through a membrane filter (0.45 m), and only the clear solution was subjected to HPLC analysis in the above conditions to obtain chromatograms, and the obtained results were summarized into a table (Table 1). The approximate concentrations were calculated by inserting each peak area from the chromatograms into the calibration equation, and the results verified that compared with the 30% ethanol extract, the 70% ethanol extract contained costunolide by about 1.8-fold and dehydrocostus lactone by about 1.6-fold.

TABLE-US-00001 TABLE 1 Concentration (ug/mL) EtOH extract Costunolide Dehydrocostuslactone a) 30% extract 80.0 (100%) 106.4 (100%) b) 70% extract 145.4 (182%) 165.1 (155%)

[0035] <3. Evaluation of Antiinflammatory Effect of Aucklandiae Radix Extracts and Costunolide>

[0036] 1) Culture of Macrophages

[0037] RAW 264.7 cells, which were macrophages used in the present tests, were available from American Type Culture Collection (ATCC). These cells were cultured in CO.sub.2 incubator (Formascientific, Inc.) using DMEM containing 10% fetal bovine serum (FBS). The differentiation of cells was observed through microscopic observation, and then the cells were used in the tests.

[0038] 2) Evaluation of Nitric Oxide (NO) Change and Antiinflammatory Effect

[0039] To induce the activation of macrophages, RAW 264.7 cells were treated with 200 ng/ml lipopolysaccharide (LPS) while simultaneously treated with various concentrations (g/ml) of a 100% methanol extract of Aucklandiae Radix (expressed by MeOH 100%), a 30% ethanol extract of Aucklandiae Radix (expressed by EtOH 30%), and a 70% ethanol extract of Aucklandiae Radix (expressed by EtOH 70%), and costunolide, and then the cells were cultured in a 37 C., 5% CO.sub.2 incubator for 18 hours. After 18 hours, 100 l of the supernatant was transferred to a new plate for each group, and 100 l of a solution, in which Griess reagent A (2% sulfanilamide in 5% phosphoric acid) and Greiss reagent B (0.2% naphthylethylenediamine dihydrochloride) were mixed at a rate of 1:1, was added thereto. Thereafter, the absorbance was measured at 540 nm.

[0040] As a test result, the amount of NO was increased in the cell only treatment groups as the control groups rather than the LPS alone treatment groups, and the amount of NO was dose-dependent and was significantly decreased in all the groups treated with the Aucklandiae Radix extracts and costunolide (FIG. 1). It can be verified from these results that the Aucklandiae Radix extracts and costunolide had an anti-inflammatory effect of preventing tissue damage by suppressing excessive inflammation of cells.

[0041] 3) Evaluation of Inflammatory Cytokine Changes and Antiinflammatory Effect

[0042] RAW 264.7 cells were treated with 200 ng/ml LPS while simultaneously treated with various concentrations (g/ml) of Aucklandiae Radix extracts (EtOH 30% and 70%) and costunolide, and then the cells were cultured in a 37 C., 5% CO.sub.2 incubator for 18 hours. After 18 hours, ELISA analysis kits (eBioScience 88-7013, 88-7064 USA, BD 555268 USA) were used to check the levels of the inflammatory cytokines IL-1, IL-6, and TNF- in the supernatant of each group.

[0043] FIGS. 2A-2C show the measurement results through the ELISA analysis kit of the levels of the inflammatory cytokines IL-1 (FIG. 2A), IL-6 (FIG. 2B), and TNF- (FIG. 2C) in the cultures of macrophages activated by LPS. When compared with the cells treated with only LPS, the cells treated with the Aucklandiae Radix extracts and costunolide showed decreases in the secretion levels of the inflammatory cytokines, indicating that the Aucklandiae Radix extracts and costunolide had an antiinflammatory effect.

[0044] <4. Evaluation of Effect of Aucklandiae Radix Extract on Alleviation of Benign Prostatic Hyperplasia>

[0045] 1) Construction of Benign Prostatic Hyperplasia Animal Models

[0046] After 6-week-old male Wistar rats (Samtako, Korea) were obtained and acclimated for one week, six animals constituted each test group when each animal weighed 340-350 g.

[0047] As for the test configuration, the animals were divided into groups without testicle removal and groups undergoing 4-hour recovery after testicle removal, and each group was subcutaneously injected with 5 mg/kg testosterone to induce benign prostatic hyperplasia for 8 weeks (BPH induced group). Test groups were constructed by oral administration of Aucklandiae Radix extracts and costunolide at different concentrations (mg/kg) once a day, seven times a week, for 8 weeks. Positive control groups were used by oral administration of finasteride, which has been used as a medicine for benign prostatic hyperplasia.

[0048] 2) Changes in Prostate Weight and Volume

[0049] After the completion of the test, the rats of all the test groups were euthanized, and then prostate tissue and main organs were extracted, and weights and volumes thereof were measured using an electronic scale for animal weights and a caliper (mm.sup.3) (Table 2).

[0050] The results verified that compared with the normal control groups, the BPH induced groups showed significantly increased weights and volumes, indicating the introduction of benign prostatic hyperplasia.

[0051] The prostate weights and volumes of the Aucklandiae Radix extract administration groups were all decreased to similar values to those of finasteride (BPH medicine) administration groups (Fina), and especially, the costunolide administration groups (0.075 g/ml) showed a significant reduction effect compared with the finasteride administration groups (FIG. 3).

TABLE-US-00002 TABLE 2 [weight: g, volume: mm.sup.3] Administered Body Prostate Ratio Prostate Liver Spleen material Test group weight (a) weight (b) (b/a .Math. 100) volume weight weight MeOH control 391 0.97 0.25 2,353 0.71 BPH 384 1.82 0.48 6,145 0.64 7.5 mg/kg 377 1.37 0.36 3,530 0.65 Fina 379 1.23 0.29 2,322 0.60 EtOH 30 control 489 1.09 0.22 2,952 11.28 0.72 BPH 414 1.87 0.45 6,079 9.57 0.62 3.75 mg/kg 439 1.57 0.36 4,820 9.55 0.67 7.5 mg/kg 419 1.57 0.38 4,864 10.14 0.66 15 mg/kg 411 1.47 0.36 4,443 9.86 0.72 Fina 415 1.42 0.34 4,255 9.46 0.70 EtOH 70 control 489 1.09 0.22 2,952 11.28 0.72 BPH 414 1.87 0.45 6,079 9.57 0.62 3.75 mg/kg 420 1.44 0.34 4,322 10.07 0.65 7.5 mg/kg 420 1.46 0.35 4,394 9.33 0.62 Fina 415 1.42 0.34 4,255 9.46 0.70 Costunolide control 489 1.09 0.22 2,952 11.28 0.72 BPH 414 1.87 0.45 6,079 9.57 0.62 0.075 mg/kg 433 1.40 0.32 4,126 10.72 0.62 Fina 415 1.42 0.34 4,255 9.46 0.70

[0052] 3) Measurement of Hepatotoxicity and Kidney Function Change

[0053] For analysis of biochemical markers in serum, blood was obtained through the abdominal vein from rats of all the groups at necropsy. The blood was coagulated for about 30 minutes, and then centrifuged for 5 minutes at 10,000 rpm to separate serum. After serum separation, a biochemistry analyzer (AU480, Beckman Coulter, USA) was used to check liver function (AST, ALP), lipoprotein (total cholesterol (T-CHO)), HDL cholesterol(HDL-C), LDL cholesterol (LDL-C)), and kidney function (creatine) levels (Table 3). The results verified that all the groups administered with the extracts and costunolide showed no significant changes, indicating no hepatoxicity and kidney toxicity.

TABLE-US-00003 TABLE 3 Administered AST ALP T-CHO HDL LDL CRE material Test group (U/L) (U/L) (mg/dL) (mg/dL) (mg/dL) (mg/dL) MeOH control 77.20 32.60 59.20 19.40 16.00 0.40 BPH 107.60 43.80 60.20 19.60 14.40 0.30 7.5 mg/kg 105.80 51.40 67.60 21.60 17.20 0.28 Fina 88.20 37.20 59.00 19.20 13.20 0.30 EtOH 30 control 96.00 91.50 93.33 60.00 12.67 0.37 BPH 125.17 85.67 88.17 56.17 13.50 0.35 3.75 mg/kg 89.00 71.33 66.50 43.00 10.67 0.30 7.5 mg/kg 121.67 86.33 79.50 50.00 12.67 0.35 15 mg/kg 145.00 70.83 86.33 55.00 13.00 0.32 Fina 80.67 81.50 70.83 43.83 13.67 0.27 EtOH 70 control 96.00 91.50 93.33 60.00 12.67 0.37 BPH 125.17 85.67 88.17 56.17 13.50 0.35 3.75 mg/kg 90.33 67.67 73.00 47.17 12.50 0.28 7.5 mg/kg 70.50 64.33 71.83 47.67 12.00 0.30 Fina 80.67 81.50 70.83 43.83 13.67 0.27 Costunolide control 96.00 91.50 93.33 60.00 12.67 0.37 BPH 125.17 85.67 88.17 56.17 13.50 0.35 0.075 mg/kg 78.33 61.17 79.67 51.50 13.17 0.27 Fina 80.67 81.50 70.83 43.83 13.67 0.27

[0054] 4) Prostate Pathology Biopsy

[0055] After the completion of the test, the prostate tissue extracted from each group was fixed with 10% neutral formalin and stored. The tissue of each group was processed into paraffin blocks, and slides for microscopic inspection of tissue were manufactured and stained with hematoxylin & eosin (H&E). The microscopic inspection of tissue was conducted through a microscope (Olympus, Tokyo, Japan) at a magnification of 200.

[0056] It can be seen that compared with the normal control groups, the benign prostatic hyperplasia induced groups showed atrophy and hypertrophy of epithelial cells of prostate vesicles and overall thickening of the basement membrane. The test groups administered with Aucklandiae Radix extracts and costunolide showed alveolar glands maintained in a round shape without atrophy similar to the normal control group, and showed significantly reduced endothelial thicknesses compared with the benign prostatic hyperplasia induced group (FIGS. 4 and 5).

[0057] <5. Evaluation of Toxicity of Aucklandiae Radix Extracts>

[0058] For investigation of a safety area of the Aucklandiae Radix extracts, the acute toxicity test and MTD toxicity test were carried out.

[0059] For the acute toxicity test, 7-week-old healthy male and female SD rats were divided into one normal control group and two drug administration groups (two groups orally administered with the Aucklandiae Radix extract at a single dose of 1,000 mg and 2,000 mg per kg of body weight), and five animals were taken for each group, and then tested. As a test result, there were no significant differences in body weight, feed intake, and the like between the normal control group and the drug administration groups, and no toxicity-related clinical signs were observed (Table 4: Acute toxicity test results).

TABLE-US-00004 TABLE 4 Male Female B.W. (g) C.S.* B.W. (g) C.S.* Group N Day 1 Day 7 Day 14 Day 14 Day 1 Day 7 Day 14 Day 14 Control 5 210 9 240 13 283 18 No sign 178 11 193 8 216 11 No sign 1000 mg/kg 5 208 10 247 10 285 12 No sign 177 10 195 10 214 15 No sign 2000 mg/kg 5 207 11 242 13 285 16 No sign 175 9 193 6 208 9 No sign Body weight, feed intake, and clinical signs: no significant differences between normal control group and Aucklandiae Radix extract administration groups C.S.*: Clinical sign

[0060] For the MTD toxicity test, 7-week-old healthy male and female SD rats were divided into two medicine administration groups (two groups orally administered with 30% and 70% ethanol extracts of the Aucklandiae Radix extract for 5 days), and three animals were taken for each group, and then tested. As a test result, there were no significant differences in body weight, feed intake, and the like between the medicine administration groups, and no toxicity-related signs were observed in clinical testing and necropsy (Table 5: MTD toxicity test results).

TABLE-US-00005 TABLE 5 Male B.W. (g) C.S.* N.F.** Group (mg/kg/day) N Day 1 Day 3 Day 5 Day 1-5 Day 1-5 KSB30% EtOH 1000 5 days 3 222 7 232 7 248 6 No change No change KSB70% EtOH 1000 5 days 3 220 4 231 4 244 2 No change No change Body weight, feed intake, and clinical signs: no significant differences between two groups. C.S.*: Clinical Sign, NF**: Necropsy Findings

[0061] It can be seen from the above results that Aucklandiae Radix extracts can alleviate the thickened prostate by increasing immune-related antiinflammatory responses and reducing prostate weight and volume without general toxicity.