HERBAL COMPOSITION FOR PREVENTING OR TREATING BENIGN PROSTATIC HYPERPLASIA DISEASE

Abstract

The present invention relates to a composition for preventing or treating benign prostatic hyperplasia disease, the composition comprising, as active ingredients, extracts of Semen cuscutae, Semen nelumbinis, and Taraxacum platycarpum. When using the present invention, it is possible to effectively prevent or treat benign prostatic hyperplasia without side effects.

Claims

1-7. (canceled)

8. A method for alleviating or treating benign prostatic hyperplasia, comprising: administering to a subject in need thereof a composition comprising a composite extract from Semen cuscutae, Semen nelumbinis, and Taraxaci herba as an active ingredient.

9. The method of claim 8, wherein the Semen cuscutae, the Semen nelumbinis, and the Taraxaci herba are mixed at a formulation weight ratio (w/w) of 1-4:1-4:1-4.

10. The method of claim 8, wherein the extract is a water or ethanol extract.

11. The method of claim 10, wherein the ethanol is greater than 0% and less than or equal to 50% ethanol.

12. The method of claim 10, wherein the ethanol is 25% ethanol.

13. The method of claim 8, wherein the composition is a pharmaceutical composition.

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

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0045] FIG. 1 is a plot showing inhibitory effects of composite herbal extracts on testosterone-induced prostate epithelial cells (LNCaP).

[0046] FIG. 2 is a graph showing inhibitory effects of composite herbal extracts prepared with solutions of various ethanol concentrations on testosterone-induced prostate epithelial cells (LNCaP).

[0047] FIG. 3 is a graph showing inhibitory effects of the composite herbal extract and the single herbal extracts on testosterone-induced prostate epithelial cells (LNCaP).

[0048] FIG. 4 is a plot showing testosterone-induced benign prostatic hyperplasia rat models changing in body weight with treatment with the composite herbal extract or the single herbal extracts.

[0049] FIG. 5 is a graph showing inhibitory effects of the composite herbal extract and the single herbal extracts on prostate enlargement in benign prostatic hyperplasia-induced rat models.

[0050] FIG. 6 shows sizes of acinus in the prostate tissues excised from testosterone-induced benign prostatic hyperplasia rat models after treatment with the composite herbal extract and the single herbal extracts.

DETAILED DESCRIPTION

[0051] A better understanding of the present disclosure may be obtained through the following examples which are set forth to illustrate, but are not to be construed as limiting the present disclosure.

EXAMPLES

Preparation Example 1: Preparation of Composite Herbal Extracts by Various Formulation Ratios

[0052] Semen cuscutae, Semen nelumbinis, and Taraxaci herba were purchased from Humanherb Co. Ltd., which is an herb distributor in Korea, and washed and dried before use in experiments. The medicinal herbs Semen cuscutae, Semen nelumbinis, and Taraxaci herba were mixed at the weight ratios (w/w) indicated in Table 1, below, to form mixtures each weighing 240 g in total. Each of the mixtures was added with 10 volumes of a 25% ethanol solution and then well stirred at room temperature for 72 hours to conduct extraction. The extract thus formed was filtered, concentrated at 50-65° C. in a vacuum, and lyophilized to afford a total of seven composite herbal extract powders. Their yields are given in Table 1, below.

TABLE-US-00001 TABLE 1 Preparation Semen Semen Taraxaci Yield Ex. # Cuscutae nelumbinis Herba (%) 1-1 1 1 1 11.35 1-2 2 1 1 11.22 1-3 4 1 1 10.17 1-4 1 2 1 12.08 1-5 1 4 1 12.39 1-6 1 1 2 11.71 1-7 1 1 4 10.85

Preparation Example 2: Preparation of Composite Herbal Extracts by Various Extraction Times

[0053] Semen cuscutae, Semen nelumbinis, and Taraxaci herba were purchased from Humanherb Co. Ltd., which is an herb distributor in Korea, and washed and dried before use in experiments. The medicinal herbs Semen cuscutae, Semen nelumbinis, and Taraxaci herba were mixed at the weight ratio (w/w) of Preparation Example 1-6 to form a mixture weighing 240 g. The mixture was added with 10 volumes of a 25% ethanol solution and then well stirred at room temperature for 0.5, 3, 8, 24, or 72 hours to conduct extraction. The extract thus formed was filtered, concentrated at 50-65° C. in a vacuum, and lyophilized to afford a total of five composite herbal extract powders. Their yields are given in Table 2, below.

TABLE-US-00002 TABLE 2 Preparation Extraction Yield Ex. # time (hr) (%) Note 2-1 0.5 9.04 2-2 3 10.87 2-3 8 10.01 2-4 24 11.17 2-5 72 11.71 the same as in Preparation Example 1-6

Preparation Example 3: Preparation of Composite Herbal Extracts by Various Ethanol Concentrations

[0054] Semen cuscutae, Semen nelumbinis, and Taraxaci herba were purchased from Humanherb Co. Ltd., which is an herb distributor in Korea, and washed and dried before use in experiments. The medicinal herbs Semen cuscutae, Semen nelumbinis, and Taraxaci herba were mixed at the weight ratio (w/w) of Preparation Example 1-6 to form a mixture weighing 240 g. The mixture was added with 10 volumes of a 0, 10, 25, 50, 75, or 95% ethanol solution and then well stirred at room temperature for 72 hours to conduct extraction. The extract thus formed was filtered, concentrated at 50-65° C. in a vacuum, and lyophilized to afford a total of six composite herbal extract powders. Their yields are given in Table 3, below.

TABLE-US-00003 TABLE 3 Preparation Conc. of Ethanol Yield Ex. # solution (%) (%) Note 3-1 0 10.32 3-2 10 10.63 3-3 25 11.71 the same as in Preparation Example 1-6 or 2-5 3-4 50 12.28 3-5 75 8.81 3-6 95 5.16

Preparation Example 4: Preparation of Composite Herbal Extract Through Hot Water Extraction

[0055] Semen cuscutae, Semen nelumbinis, and Taraxaci herba were purchased from Humanherb Co. Ltd., which is an herb distributor in Korea, and washed and dried before use in experiments. The medicinal herbs Semen cuscutae, Semen nelumbinis, and Taraxaci herba were mixed in the respective amounts of 30 g, 30 g, and 60 g at the weight ratio (w/w) of Preparation Example 1-6 to form a mixture. The mixture was added with 10 volumes of distilled water and then well stirred at 95-100° C. for 3 hours to conduct extraction under reflux. The extract thus formed was filtered, concentrated at 50-65° C. in a vacuum, and lyophilized to afford a composite herbal extract powder. The yield was about 11.90%.

Comparative Example 1: Preparation of Extract from Single Herb

[0056] Semen cuscutae, Semen nelumbinis, and Taraxaci herba were purchased, washed, and dried for use in experiments. To 180 g of each of the herbs, 10 volumes of 25% ethanol solution were added, followed by stirring at room temperature for 72 hours to conduct extraction. The extract thus formed was filtered, concentrated at 50-65° C. in a vacuum, and lyophilized to afford a total of three single herbal extract powders. Their yields are given Table 4, below.

TABLE-US-00004 TABLE 4 Comparative Ex. # Herb Yield (%) 1-1 Semen Cuscutae 5.49 1-2 Semen nelumbinis 16.35 1-3 Taraxaci Herba 15.00

Experimental Example 1: Test of Inhibitory Activity of Composite Herbal Extract Against Testosterone-Induced Proliferation of Prostate Epithelial Cell Line

[0057] (Test Method)

[0058] LNCaP cells, which are the prostate epithelial cells derived from human prostate cancer, were seeded at a density of 1×10.sup.5 cells/well in 24-well plates containing an RPMI medium (Gibco U.S.A) and stabilized overnight. Then, the cells were cultured for 24 hours in a serum-free medium. After removal of the medium, the cells were incubated for 72 hours with 2 μM of testosterone (Tes) alone or in combination with 50, 100, or 200 μg/ml of the composite herbal extract of Preparation Example 1-6 or 4. Thereafter, growth of the cells was evaluated by MTT assay. Cell proliferation data were expressed as percentages of the cell counts relative to that of the Tes-treated group.

[0059] (Results)

[0060] When comparison was made of effects between the composite herbal extracts prepared with hot water (Preparation Example 4) and 25% ethanol solution (Preparation Example 1-6), the two extracts were both observed to inhibit prostate cell growth in a dose dependent manner (FIG. 1).

Experimental Example 2: Test of Inhibitory Activity of Composite Herbal Extract Prepared According to Various Ethanol Concentrations Against Testosterone-Induced Proliferation of Prostate Epithelial Cell Line

[0061] (Test Method)

[0062] LNCaP cells, which are the prostate epithelial cells derived from human prostate cancer, were seeded at a density of 1×10.sup.5 cells/well in 24-well plates containing an RPMI medium (Gibco, U.S.A) and stabilized overnight. Then, the cells were cultured for 24 hours in a serum-free medium. After removal of the medium, the cells were incubated for 72 hours with 2 μM of testosterone (Tes) alone or in combination with 200 μg/ml of each of the six composite herbal extracts of Preparation Example 3. Thereafter, growth of the cells was evaluated by MTT assay. Cell proliferation data were expressed as percentages of the cell counts relative to that of the Tes-treated group.

[0063] (Results)

[0064] Effects of the composite herbal extracts prepared according to various ethanol concentrations on testosterone-induced proliferation of prostate epithelial cells are given in Table 5, below.

TABLE-US-00005 TABLE 5 Cell growth % inhibition Substance (% of control) of cell growth NC (not treated) — — Tes (Testosterone) 100 — Testosterone + Preparation Ex. 3-1 45.91 54.09 Testosterone + Preparation Ex. 3-2 41.16 58.84 Testosterone + Preparation Ex. 3-3 34.59 65.41 Testosterone + Preparation Ex. 3-4 58.43 41.57 Testosterone + Preparation Ex. 3-5 74.00 26.00 Testosterone + Preparation Ex. 3-6 83.54 16.46

[0065] Comparison of effects of the composite herbal extracts according to various ethanol concentrations showed that all the cell groups treated with of the extracts prepared in Preparations Examples 3-1 to 3-6 were lower in cell growth rate than the Tes-treated group, demonstrating that the composite herbal extracts prepared at various ethanol concentrations have excellent inhibitory activity against the growth of prostate cells (FIG. 2).

Experimental Example 3: Comparison of Inhibitory Activity Against Testosterone-Induced Proliferation of Prostate Epithelial Cell Line Between Composite Herbal Extract and Individual Herbal Extract

[0066] (Experimental Method)

[0067] LNCaP cells, which are the prostate epithelial cells derived from human prostate cancer, were seeded at a density of 1×10.sup.5 cells/well in 24-well plates containing an RPMI medium (Gibco, U.S.A) and stabilized overnight. Then, the cells were cultured for 24 hours in a serum-free medium. After removal of the medium, the cells were incubated for 72 hours with 2 μM of testosterone (Tes) alone or in combination with 200 μg/ml of each of the six composite herbal extracts of Preparation Example 3 or each of the three single herbal extracts. Thereafter, growth of the cells was evaluated by MTT assay. Cell proliferation data were expressed as percentages of the cell counts relative to that of the Tes-treated group.

[0068] (Results)

[0069] Effects of the composite herbal extracts and the single herbal extracts on testosterone-induced proliferation of prostate epithelial cells are given in Table 6, below.

TABLE-US-00006 TABLE 6 200 μg/ml Cell growth % Inhibition rate (%) of cell growth NC — — Testosterone 100.0 — Preparation Ex. 1-6 44.9 55.1 Comparative Ex. 1-1 66.7 33.3 (Semen Cuscutae) Comparative Ex. 1-2 78.0 22.0 (Semen nelumbinis) Comparative Ex. 1-3 88.1 11.9 (Taraxaci Herba)

[0070] When treated with testosterone, the prostate epithelial cells were measured to grow by about 62.5%. Treatment with the composite herbal extract of Preparation Example 1-6 together with testosterone inhibited the cell growth by about 55.1%. In contrast, the single herbal extracts of Comparative Example 1-1 (Semen cuscutae), Comparative Example 1-2 (Semen nelumbinis), and Comparative Example 1-3 (Taraxaci herba) inhibited the growth of the cells by about 33.3%, 22.0%, and 11.9%, respectively. Thus, the composite herbal extract was observed to be far superior to the single herbal extracts in terms of growth inhibition at the same ethanol concentration (FIG. 3).

Experimental Example 4: Comparison of Inhibitory Activity Against Prostate Enlargement Between Single Herbal Extracts and Composite Herbal Extract in Testosterone-Induced Benign Prostatic Hyperplasia Rat Model

[0071] (Test Method)

[0072] After being acclimated for one week or longer, male SD rats 9 weeks old (weighing 350 g or less) were divided on the basis of weight into (1) a normal group, (2) a benign prostatic hyperplasia-induced and distilled water-administered group (negative control), (3) benign prostatic hyperplasia-induced and composite herbal extract-administered group (Comparative Example 1-6), (4) benign prostatic hyperplasia-induced and single Semen cuscutae herbal extract-administered group (Comparative Example 1-1), (5) benign prostatic hyperplasia-induced and single Semen nelumbinis herbal extract-administered group (Comparative Example 1-2), (6) benign prostatic hyperplasia-induced and single Taraxaci herba herbal extract-administered group (Comparative Example 1-3), and (7) benign prostatic hyperplasia-induced and finasteride-administered group (positive control). In order to induce benign prostatic hyperplasia, testosterone (dissolved in cotton seed oil) was subcutaneously injected at a dose of 3 mg/kg once/three days for a total of 10 times. For the control, the same amount of cotton seed oil was subcutaneously injected. The composite herbal extract of Preparation Example 1-6 and the single herbal extracts of Comparative Example 1 were each orally administered at a dose of 300 mg/kg once a day for 30 days. Finasteride (Sigma, dissolved in 0.2% Tween 80) was orally administered at a dose of 10 mg/kg once/day for 30 days. For the negative control, oral administration of distilled water alone was carried out. From the starting day of the test, the rats were weighed five times in total at regular intervals of one week. On the next day after the final administration and treatment, the rats were sacrificed with carbon dioxide gas. The prostate glands were excised and weighed. % Inhibition of prostate enlargement was expressed according to the following mathematical formula on the basis of the weight measurements.

% Inhibition of prostate enlargement=100−{(prostate weight of composite herbal extract group/single herbal extract or positive control group−prostate weight of control group)*100/(prostate weight of negative control−prostate weight of normal group)}  Mathematical Formula

[0073] In addition, the excised prostate tissue was fixed in 10% neutral buffered formalin and subjected to H&E (hematoxylin and eosin) staining. Slides of the stained tissues were observed and photographed with an optical microscope. From the images, sizes of the acini in the prostate glands were measured. The reduction in the size of acinus of each experimental group is expressed as a percentage relative to the size of acinus of the normal group.

[0074] (Results)

[0075] Benign prostatic hyperplasia was induced. The normal group steadily increased in body weight whereas the increase of body weight was suppressed in the groups treated with testosterone. However, significant differences in body weight were not observed among the negative control, the composite herbal extract group (Preparation Example 1-6), the single herbal extract groups (Comparative Examples 1-1, 1-2, and 1-3), and the positive group (FIG. 4). Effects of the composite herbal extract (Preparation Example 1-6), the single herbal extracts (Preparation Examples 1-1, 1-2, and 1-3), and the positive control on the testosterone-induced prostate enlargement are given in Table 7, below.

TABLE-US-00007 TABLE 7 Prostate weight/ % Inhibition body weight of prostate Experimental Group (g/kg) enlargement Normal 0.08 ± 0.01 — Negative control 2.26 ± 0.21 — Composite herbal extract 1.49 ± 0.26 35 Single Semen Cuscutae extract 1.82 ± 0.09 20 Single Semen nelumbinis extract 1.79 ± 0.20 21 Single Taraxaci Herba extract 2.14 ± 0.17 5 Positive control (Finasteride) 1.31 ± 0.10 43

[0076] The rats in which benign prostatic hyperplasia was induced by testosterone increased in prostate weight by about 30 fold compared to the normal group. However, the prostate weight in the composite herbal extract group was decreased by 35% compared to that in the testosterone-induced group (negative control), with a statistically significant difference. In contrast, the single herbal extract groups (Preparation Examples 1-1 (Semen cuscutae), 1-2 (Semen nelumbinis), and 1-3 (Taraxaci herba)) exhibited inhibitory effects on prostate enlargement by about 20%, 21%, and 5%, respectively, compared to the testosterone-induced group (negative control), but without a statistically significant difference (FIG. 5). The data indicate that the composite herbal extract exerts a far higher inhibitory activity against testosterone-induced prostate enlargement, compared to the single herbal extracts.

[0077] Effects of the composite herbal extract (Preparation Example 1-6), the single herbal extracts (Comparative Examples 1-1, 1-2, and 1-3), and the positive control on the size of acinus are summarized in Table 8, below.

TABLE-US-00008 TABLE 8 Reductive Size of effect on size Experimental Group acinus (%) of acinus (%) Normal 100 ± 30 — Negative control 481 ± 52 — Composite herbal extract 270 ± 48 44 Single Semen Cuscutae extract 375 ± 25 22 Single Semen nelumbinis extract 334 ± 18 30 Single Taraxaci Herba extract 482 ± 65 0 Positive control (Finasteride) 279 ± 10 42

[0078] The negative control increased in the size of acinus by 4.8 fold, compared to the normal group while the composite herbal extract exhibited the effect of reducing the size of acinus by 44%, with statistical significance. For the single herbal extracts (Comparative Examples 1-1, 1-2, and 1-3), the reduction rates were 22, 30, and 0%, respectively, which are far short from the effect of the composite herbal extract. Taken together, the data demonstrate that the composite herbal extract has a far higher inhibitory activity against testosterone-induced prostate enlargement, compared to the single herbal extracts (FIG. 6).