KR20110037502A - Composition for treating or preventing benign prostate hyperplasia containing ascorbate - Google Patents

Abstract

PURPOSE: A composition for preventing or treating prostatic hypertrophy containing ascorbate is provided to suppress growth of prostate grand. CONSTITUTION: A composition for preventing or treating prostatic hypertrophy contains ascorbate as an active ingredient. Ascorbate has a form of vitamin C, ascorbyl ester, ascoarbyl palmitate, ascorbyl phosphate ester, mineral ascorbate, dehydroascorbate, or vitamin C metabolite. The composition is provided as a health supplementary food, functional food, or food additive. A drug for treating micturition disorder due to prostatic hyperplasia contains the composition.

Description

Composition for treating or preventing benign prostate hyperplasia containing ascorbate}

The present invention relates to the field of treatment or prevention of prostate-related diseases, and more particularly, to a composition for treating or preventing prostatic hyperplasia comprising ascorbate as an active ingredient.

Benign prostatic hyperplasia (BPH) is one of the most common benign tumors in men after middle age. The main triggers for the development of enlarged prostate are age and male hormones. As the prostate grows, the urethra is compressed and resistance to urine flow increases, leading to prostate disease. Histological changes in prostate hypertrophy begin at age 35 and occur in 60% of men in their 60s and 90% in their 80s, among which 50% of patients complain of various symptoms of urination disorder due to prostate hyperplasia. Therapies include medical and surgical methods, the former including drugs such as alpha1-adrenergic receptor blockers or 5-alpha-reductase inhibitors, the latter being an open prostatectomy and a transesophageal prostatectomy that resections the enlarged prostate. Can be mentioned.

Ascorbate, which contains vitamin C, is one of the most easily encountered vitamins in almost every diet. Vitamin C deficiency causes scurvy, protects the body from oxidative stress as an antioxidant and also acts as a cofactor for many important enzyme reactions (Padayatty S, et al. (2003) "Vitamin C as an Antioxidant: evaluation of its role in disease prevention ". J Am Coll Nutr 22 (1): 18-5; Stanner SA, et al. (2004)" A review of the epidemiological evidence for the antioxidant hypothesis ". Public Health Nutr 7 (3): 407 -2).

However, despite much interest and research on prostatic hyperplasia, there is no known association between vitamin C-containing ascorbate and its prophylactic and therapeutic effects.

The present application is a new use of ascorbate, to provide a composition for treating or preventing prostatic hyperplasia comprising ascorbate.

The present application provides a composition for treating or preventing prostatic hyperplasia comprising ascorbate as an active ingredient.

The present application also relates to the ascorbate in the form of vitamin C, ascorbyl ester, ascorbyl palmitate, ascorbyl phosphate ester, mineral ascorbate, dehydroascollate or vitamin C metabolites, or mixtures thereof Phosphorus, a composition is provided.

The application also provides a composition wherein the mineral ascorbate is calcium ascorbate, magnesium ascorbate, zinc ascorbate, sodium ascorbate or potassium ascorbate.

The application also provides a composition, wherein the ascorbate inhibits the expression of HIF-1α.

The present application also provides a composition wherein the composition is provided as a dietary supplement, a functional food or a food additive.

The present application also provides an agent for improving urination disorder due to enlarged prostate, comprising the composition of the present application.

In one embodiment the present invention relates to a composition for the treatment or prevention of enlarged prostate, including ascorbate.

Ascorbate herein may be in any suitable form (derivative). For example, vitamin C, L-ascorbic acid, L-3-ketoserehexuronic acid lactone, ascorbic acid, L- L-xylo-ascorbic acid, L-threo-hex-2-enoic acid gamma lactone, ascorbyl palmitate, ascorbyl ester, ascorbyl phosphate ester, mineral ascorbate, dehydroascorbate (also called oxidized vitamin C), vitamin C metabolite or derivatives thereof , Analogs and functional equivalents. Ascolville phosphate esters include, but are not limited to, mono, di and trisodium phosphate, magnesium phosphate and calcium phosphate. Ascorbate of the present invention may exist in one or more different forms.

Mineral ascorbate is a compound of mineral and vitamin C, including but not limited to calcium ascorbate, magnesium ascorbate, zinc ascorbate, sodium ascorbate and potassium ascorbate.

Ascorbic acid is the name of vitamin C as defined by the IUPAC-IUB Division on Biochemical Names, and is also called L-ascolbic acid, L-xyllo-ascolbic acid, and L-threo-hex-2-enoic acid gamma lactone. Pure vitamin C has the formula C ₆ H ₈ O ₆ and has a molecular weight of 176.13 Da. There are four kinds of stereoisomers, namely L-ascorbic acid and D-araboascolbic acid, which are known to be active in vitamin C, and L-araboascolbic acid and D-xylloascolbic acid, and as long as they have vitamin C activity. Such isomers also fall within the scope of the present invention. In one embodiment of the invention the composition comprises vitamin C. In another embodiment, it includes mineral ascorbate.

The pharmacophores of vitamin C are ascorbate ions, and therefore all forms of compounds and derivatives thereof containing such ascorbate ions are included within the scope of the present invention.

Ascorbate included in the composition herein may be one or more derived. For example, they may be natural or synthetic, and they may be mixed. Natural origin includes, for example, fruits or vegetables, and ascorbate-rich fruits include, for example, cantaloupe, honeydew, kiwi, mango, orange, tangerine, papaya, strawberry, watermelon, and the like. Examples of vegetables rich in ascorbate include peppers, potatoes, broccoli, bean sprouts, cabbage, sweet potatoes, and tomatoes. In one embodiment the compositions or nutraceuticals of the present disclosure may comprise such natural-derived vegetables and / or fruits or extracts / concentrates thereof.

Ascorbate comprising vitamin C, which falls within the scope of this application, may be commercially available, synthetic or extracted from natural products, and such methods are known. Vitamin C can be purchased, for example, from BASF / Takeda, DSM and Merck, Sigma-Aldrich. Extraction / synthesis methods of vitamin C are described, for example, in US Pat. Hsieh H-J et al., (2005) "Synthesis of mixed esters of ascorbic acid using methyl esters of palm and soybean oils". Prep Biochem Biotechnol 35 (2): 113-118; And Hancock RD et al. (2002) "Biotechnological approaches for L-ascorbic acid". Reference may be made to Trends Biotechnol 20 (7) 299-305 et al.

In one embodiment of the present disclosure, ascorbate may be included in the composition at about 0.1% to 99.9% by weight. For example, about 0.1%, about 0.2%, about 0.5%, about 1% about 2%, about 5%, about 7%, about 10%, about 12%, about 15%, about 18%, about 20%, About 22%, about 24%, about 25%, about 27%, about 30%, about 32%, about 35%, about 37%, about 40%, about 42%, about 45%, about 47%, about 50 %, About 52%, about 55%, about 57%, about 60%, about 62%, about 65%, about 67%, about 70%, about 72%, about 75%, about 77%, about 80%, About 82%, about 85%, about 87%, about 90%, about 92%, about 95%, about 97%, about 98%, about 99%, about 99.5%, or about 99.9% (all above weight%) It includes. In another embodiment, ascorbate is included in at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at most 99.9%.

The composition of the present invention has a proliferation inhibitory effect on Benign Prostatic Hyperplasia (BPH). Prostatic hyperplasia refers to the phenomenon of enlargement of the prostate gland for various reasons, and the detailed cause is unknown, but it is speculated that the prostate hypertrophy is closely related to the change of endocrine hormone, a testosterone including testosterone. The enlarged prostate gland has various urination disorders such as nocturia, frequent urination, delayed onset of urination, residual urine, weakness of urination, as well as complications such as acute and chronic urinary tract, urinary tract infections and hematuria, bladder stones, kidney failure, etc. cause. Compositions comprising ascorbate herein can inhibit prostate growth as well as inhibit the growth of prostate, resulting in a reduction in enlarged prostate tissue, thereby preventing or treating such urination disorders and complications thereof. Thus, in another aspect, the present application also provides a urination disorder improving agent comprising the composition of the present application.

Both prostatic hyperplasia and prostate cancer can lead to tissue hypoxia due to uncontrolled sustained growth of cells, which leads to angiogenesis. This angiogenesis is promoted by Vascular Endothelial Growth Factor (VEGF), which is regulated by the transcription factor HIF-1. HIF-1 is a protein composed of α and β monomers and is known as a protein that plays an important role in the survival of cells in the hypoxic state, HIF-1 α monomers by HIF-1-α prolyl hydroxylase (PHD) Regulated in response to hypoxia (Li et al., "Vitamin C supplementation prevents testosteron-induced hyperplasia of rat prostate by down-regulating HIF-1α". J of Nutritional Biochemistry (2009), in press, first published online 2009 26 August www.ncbi.nlm.nih.gov). HIF-1 is overexpressed in the prostate tissue of patients with prostatic hyperplasia (Park SE et al., “HIF-1alpha promotes survival of prostate cells at a high zinc environment.” Prostate. 2007 Oct 1; 67 (14): 1514- 23).

In one embodiment herein, prostatic hyperplasia is testosterone (17beta-hydroxy-4-androsten-3-one), dehydrotestosterone (17beta-hydroxy-5alfa-androsten-3one), 5a-Dihydrotestosterone (5a-DHT), andro It is caused by male hormones including 4-androsten-3, 17-dione, dehydroepiandrosterone (DHA), and the like (3beta-hydroxy-5-androsten-17-one). In another embodiment, prostatic hyperplasia is caused by testosterone.

Although not intended to be limited to this theory, vitamin C included in the composition of the present disclosure inhibits the proliferative effect of the prostate through down-regulation of HIF-1α. Downregulation of HIF-1α is dependent on PHD, which downregulates transcription of about 80 genes, including VEGF. In one embodiment of the invention, ascorbate, in particular a composition comprising vitamin C, inhibits HIF-1α expression. The HIF-1α is derived from mammals including humans. In another embodiment of the invention, ascorbate, in particular a composition comprising vitamin C, inhibits the expression of target genes of VEGF and HIF.

The composition of the present application may further contain at least one active ingredient exhibiting the same or similar function in addition to the active ingredient or a compound which maintains / increases the stability, solubility and / or absorption of ascorbate.

In addition, the composition of the present application may be used alone or in combination with methods using surgery, drug treatment and biological response modifiers for the treatment or prevention of prostate hyperplasia.

The composition of the present invention may be prepared in the form of a pharmaceutical composition or health food for the treatment or prevention of enlarged prostate.

For the preparation of a pharmaceutical composition, in addition to the active ingredient described above, it may be prepared by including one or more pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, and one or more of these components, as needed. And other conventional additives such as buffers and bacteriostatic agents can be added.

In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable formulations, pills, capsules, granules, or tablets such as aqueous solutions, suspensions, emulsions, and the like, and may act specifically on target organs. Target organ specific antibodies or other ligands may be used in combination with the carriers so as to be used. Furthermore, it may be suitably formulated according to each disease or component by a suitable method in the art or using a method disclosed in Remington's Pharmaceutical Science (Recent Edition, Mack Publishing Company, Easton PA). have.

The composition of the present invention is not particularly limited thereto, but may be administered parenterally (for example, intravenously, subcutaneously, intraperitoneally, or topically) or orally, depending on the desired method. Parenteral administration is preferable, and administration by intravenous injection is more preferable. Dosage varies widely depending on the patient's weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion and severity of disease. Typically the dosage unit comprises, for example, about 0.1 mg to 1000 mg but does not exclude ranges below and above this range. The daily dose may be about 1 μg to 10 g, and may be administered once to several times a day.

Various ascorbate containing vitamin C contained in the composition of the present invention as a substance proved to be stable and administered to rats by intravenous injection as a result of toxicity test, 50% lethal dose (LD ₅₀ ) by the toxicity test It is believed to be a safe substance, each about 10 g / kg.

The composition of the present invention not only provides a prophylactic or therapeutic effect of superior acid prostate hyperplasia, but also has no toxicity and side effects due to the drug, so that it can be used safely even for long-term administration. Thus, the composition of the present invention can be used as a major, subsidiary and food additive of food.

In this regard, the composition of the present invention may be provided as a dietary supplement, functional food, food additives and the like. The dietary supplement, functional food, food additives have a function for preventing, treating or improving symptoms of enlarged prostate.

As used herein, the term "food" means a natural product or processed product containing one or more nutrients, and preferably means that it can be directly eaten through a certain processing process, By the meaning, it is intended to include all foods, food additives, functional foods and beverages.

As used herein, the term "functional food" refers to a biological defense rhythm of a food group or a food composition that has added value to the food by using physical, biochemical, or biotechnological techniques to act and express the function of the food for a specific purpose. It means a food processed and designed to fully express the bioregulatory function related to regulation, disease prevention and recovery to the living body, and particularly includes "health functional food".

As used herein, the term "health food" is a food manufactured and processed using raw materials or ingredients having a useful function for the human body, and is useful for health uses such as nutrient control or physiological action on the structure and function of the human body. Refers to foods that are manufactured and processed by extracting, concentrating, refining, or mixing certain ingredients as raw materials or ingredients contained in food raw materials for the purpose of health supplement to obtain effect.

In particular, the functional food or dietary supplement in the present invention means a food having a prostatic hypertrophy treatment effect that can improve or relieve the symptoms of prostatic hyperplasia compared to before ingesting the food, the present invention belongs to If you have a general knowledge in the Korean medical association, you can refer to the data presented by the Korean Medical Association to determine the exact criteria of the symptoms of prostatic hyperplasia, and can compare with this to determine the degree of improvement.

The functional foods and dietary supplements may further include food acceptable food additives, and may further include appropriate carriers, excipients and diluents commonly used in the manufacture of functional foods.

Examples of the food to which the composition of the present invention can be added include various foods, beverages, gums, teas, vitamin complexes, and functional foods. In addition, the food in the present invention, special nutrition products, processed meat products, fish meat products, tofu, jelly, noodles (eg, ramen, noodles, etc.), health supplements, seasoned foods (eg, soy sauce, miso, gochujang, mixed soy sauce) Etc.), sauces, sweets (e.g. snacks), dairy products (e.g. fermented milk, cheese, etc.), other processed foods, kimchi, pickles (various kimchi, pickles, etc.), beverages (e.g. fruits, vegetable drinks, soy milk, Fermented beverages, etc.), natural seasonings (eg, ramen soup, etc.), but is not limited thereto. The food, beverage or food additive may be prepared by a conventional production method.

In the present invention, the drink is a generic term for drinking to quench thirst or to enjoy the taste and is intended to include a functional drink. The beverage is not particularly limited to other ingredients other than including the present composition as an active ingredient as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates, etc. as additional ingredients, as in the usual beverage. Examples of such natural carbohydrates include monosaccharides such as glucose, fructose and other disaccharides such as maltose, sucrose and the like and polysaccharides such as dextrin, cyclodextrin and the like, and Sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. The proportion of natural carbohydrates is generally about 1 to 20 g, preferably 5 to 12 g per 100 ml of the composition of the present invention. It may contain.

Functional beverage in the present invention is a biological defense rhythm control, disease prevention and the like having a beverage group or a beverage composition that has added value to the beverage by using physical, biochemical, biotechnological techniques, etc. to function and express the function of the beverage to a specific purpose It means a beverage that is designed and processed to fully express the gymnastic function related to recovery, and preferably means a beverage for improving the symptoms of prostatic hyperplasia.

The functional beverage is not particularly limited to other ingredients except ascorbate containing vitamin C as an essential ingredient in the ratio indicated, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. have. Examples of such natural carbohydrates include monosaccharides such as glucose, fructose and other disaccharides such as maltose, sucrose and the like and polysaccharides such as dextrin, cyclodextrin and the like, and xylitol Sugar alcohols such as sorbitol and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tautin, stevia extract (e.g., Rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of said natural carbohydrates is generally about 1-20 g, preferably 5-12 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. These components can be used independently or in combination. The proportion of such additives is not so critical, but may be selected in the range of 0.001 to 20 parts by weight per 100 parts by weight.

The composition or ascorbate of the present invention may be added as it is, or used with other foods or food ingredients, and may be appropriately used according to conventional methods. The mixed amount of the active ingredient may be suitably determined depending on the purpose of use (prevention, health or therapeutic treatment). Generally, in the preparation of food or beverage, the composition or ascorbate of the present invention is 99.9% by weight, 90% by weight, 80% by weight, 70% by weight, 60% by weight, 50% by weight, 40 It is added in an amount of up to 30% by weight, up to 20% by weight, up to 15% by weight or up to 10% by weight. However, in the case of long-term intake for health and hygiene or health control, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety. .

Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

The present invention may be practiced using conventional techniques within the skill of those skilled in the art of cell biology, cell culture, molecular biology, gene transformation techniques, microbiology, DNA recombination techniques, immunology, unless otherwise noted. See also the following books and literature for a more detailed description of general techniques. For general methods of molecular biology and biochemistry, see Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. Eds., John Wiley & Sons 1999); DNA Cloning, Volumes I and II (Glover ed., 1985); Oligonucleotide Synthesis (Gait ed., 1984); Nucleic Acid Hybridization (Hames and Higgins eds. 1984); Transcription And Translation (Hames and Higgins eds. 1984); Culture Of Animal Cells (Freshney and Alan, Liss, Inc., 1987); Gene Transfer Vectors for Mammalian Cells (Miller and Calos, eds.); Current Protocols in Molecular Biology and Short Protocols in Molecular Biology, 3rd Edition (Ausubel et al., Eds.); And Recombinant DNA Methodology (Wu, ed., Academic Press). See also Gene Transfer Vectors For Mammalian Cells (Miller and Calos, eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al., Eds.); Immobilized Cells And Enzymes (IRL Press, 1986); Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (Weir and Blackwell, eds., 1986); Protein Methods (Bollag et al., John Wiley & Sons 1996); Non-viral Vectors for Gene Therapy (Wagner et al. Eds., Academic Press 1999); Viral Vectors (Kaplitt & Loewy eds., Academic Press 1995); Immunology Methods Manual (Lefkovits ed., Academic Press 1997); And Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998). For general techniques regarding cell culture and medium, see Large Scale Mammalian Cell Culture (Hu et al., Curr. Opin. Biotechnol. 8 (1997), 148); Serum-free Media (Kitano, Biotechnology 17 (1991), 73); Large Scale Mammalian Cell Culture (Curr. Opin. Biotechnol. 2 (1991), 375); And Suspension Culture of Mammalian Cells (Birch et al., Bioprocess Technol. 19 (1990), 251).

In addition, the experiments described below are basically described in Li et al, Journal of Nutritional Biochemistry (2009), in press, on August 26, 2009, www.ncbi.nlm.nih.gov). Since it was performed on the basis of what is described in the cited references, reference may be made to the article for more detailed experimental methods other than those described below.

Example  1: Testosterone-induced of vitamin C HIF -1α expression suppression

Testosterone sensitive LNCaP cells (human prostate cancer cells) were obtained from ECACC (London, UK) and RPMI-1640 medium supplemented with 10% fetal bovine serum, L-glutamine (2 mM), HEPES (10 mM), penicillin and streptomycin (Invitrogen) was incubated at 37 ℃ 5% CO ₂ . For this experiment, LNCaP cells were incubated for 24 hours in a serum-free medium for 24 hours, followed by testosterone (Sigma-Aldrich). Add 1 or 10 μM and incubate further for 24 hours. To see the effects of vitamin C, PBS (Phosphate Buffered Saline, pH 7.4, negative control) or vitamin C (final concentrations 10, 50 and 100 μM, Sigma-Aldrich) were treated with 2 μM of testosterone. Incubated for an additional 8 hours. Then, protein extracts were prepared from the cells, and Western blot was performed. First, the extract was subjected to electrophoresis on 8% SDS / polyacrylamide gel, and then transferred to Immunobilon-P membrane (Millipore) as recommended by the manufacturer, and TTBS ((Tris-buffered Saline with 5% skim milk) Blocked for 1 hour in tween-20, 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.01% Tween) The membrane was then anti-HIF-1α (1: 2000 dilution, direct preparation (Chun YS et al). ,, Biochem Pharmacol 2001: 61: 947-54)), anti-ARNT (1: 3000 dilution), antitubulin (1: 3000 dilution) or anti-VEGF (1: 3000 dilution) (the antibodies are Santa Cruz Biotechnology ), And incubated overnight at 4 ° C. As a secondary antibody for color development, diluting the horseradish peroxidase-conjugated anti-rabbit or anti mouse serum (GE Healthcare Co., Ltd.) at 1: 5000 was performed as described above. The reaction was carried out for 1 hour in the blocking solution.The antibody complex was prepared by using the Enhanced Chemiluminescence Plus Kit (GE Healthcare) as instructed by the manufacturer. By color development.

Experiments using vitamin C in the same manner as described above were performed using RWPE-1 (non-tumoral human prostate epithelial cells, ATCC) cells. However, testosterone was treated for 48 hours using 5 μM, and then PBS or vitamin C (treated at 25 μM or 100 μM final concentration) was added, and the nuclear extract was used for western blot. RWPE-1 cells were cultured in keratinocyte serum-free medium containing 5 ng / ml human recombinant EGF and 0.05 mg / ml cerebellar pituitary extract (Invitrogen).

The results are described in FIG. 1 and show that vitamin C inhibits HIF-1α expression induced by testosterone. As shown in FIG. 1A, HIF-1α increased expression by testosterone, while ARNT (HIF-1β) and tubulin remained unchanged. However, as shown in FIGS. 1B and 1C, this expression-induced phenomenon disappeared in both cell types when treated with vitamin C, indicating that vitamin C inhibits the expression of HIF-1α. On the other hand, there was no effect on the expression of tubulin and lamin B used as a control.

To test the effects of testosterone, HIF-1α, and vitamin C on the expression of VEGF, the following experiments were conducted.

LNCaP cells were transfected with VEGF promoter-luciferase plasmid and β-gal plasmid (Dr. Huang, University of Utah) using Lipofectamine (Invitrogen) as recommended by the manufacturer, and then cultured in serum-free medium for 24 hours. . Then 2 μM testosterone was incubated for 24 hours in the presence or absence of 25 μM or 100 μM of vitamin C. Extracts were prepared from the cells and luciferase activity was measured on Lumat LB9507 luminometer (Berthold), and this value was divided by β-gal activity and corrected for transfection efficiency. Total RNA was isolated from the cells using Trizol (Invitorgen) as recommended by the manufacturer and then reversed against VEGF, HIF-1α and Actin (control) using reverse transcriptase polymerase chain reaction (RT-PCR) and autoradiography. Quantitative experiments in Li et al, ibid It was performed as described in.

The results are described in Figures 1 D and E. Bars in the graph are mean ± standard deviations obtained from 9 experiments and represent * P <0.05 vs untreated control, #P <0.05 vs testosterone treated groups. As shown in FIG. 1D, the promoter activity of VEGF by testosterone was decreased by treatment of vitamin C. As shown in Figure 1E, VEGF mRNA expression was induced by testosterone as well as by the activity of the VEGF promoter, but decreased to control levels by vitamin C.

These results show that vitamin C decreases the expression and transcription of HIF-1α by increasing the expression of VEGF by testosterone.

Example  2: induced by testosterone by vitamin C Prostate  Proliferation inhibition

LNCaP cells were cultured in the presence of testosterone (5 μM final concentration once treatment) or testosterone and vitamin C (500 μM, twice daily, for a total of 5 days) as described in Example 1, followed by trypan blue assay (Trypan Blue The viable cell number was measured by an exclusion assay. The cell pellet was suspended in PBS, mixed 1: 1 with 0.4% trypan blue dye (Sigma-Aldrich), incubated at room temperature for 3 minutes, and then stained cells (dead cells), unstained. The number of cells (living cells) was measured respectively.

The results are in FIG. 2, which shows that vitamin C inhibits cell proliferation induced by testosterone. In the figure, C represents an untreated control group, T represents a testosterone treated group, and T + VC represents a testosterone and vitamin C treated group. FIG. 2A shows the cells on the 5th day of culture under an optical microscope. The cells treated with testosterone (central picture) showed no change in cell morphology compared to the untreated control (left picture), but the number of cells increased significantly. It can be seen that the density is high, and this increase in cell number is suppressed by the treatment of vitamin C (picture on the right). Figure 2B is a result of measuring the number of live cells by trypan blue analysis, compared with the testosterone treated cells compared to the non-treated group increased by 31%, when treated with vitamin C 44% compared to the testosterone treated group There was a decrease in cell number. This proves that vitamin C has an anti-proliferative effect on prostate cells.

Example  3: caused by vitamin C HIF -1α inhibitory PHD2  Dependencies

LNCaP and RWPE-1 cells were transfected with 50 nM of control nucleic acid (Universal Control RNA, Invitrogen) or PHD2 siRNA using Lipofecamine RNAiMAX (Invitrogen) as recommended by the manufacturer, then treated with 2 μM of testosterone for 24 hours, followed by vitamin Treatment with C 100 μM for 8 hours. SiRNA targeting PHD2 corresponds to 885-905 coding sequence of human PHD2 (NM_022051) and was synthesized by Invitrogen. PHD2 is known to be responsible for the degradation of HIF-1α. Each total cell extract (LNCaP) and nuclear extract (RWPE-1) was prepared from the treated cells and Western blot was performed in the manner described in Example 1. In the case of using DMOG (dimethyloxalylglycine, Frontier Scientific), the same as above, but after the testosterone treatment 1MM of DMOG for 1 hour was added vitamin C.

The results are in FIG. 3 and show that vitamin C inhibits HIF-1α expression in a PHD2 dependent manner. As shown in FIG. 3A, vitamin C inhibited the synergistic effect of testosterone-induced HIF-1α expression, which was offset by removal of PHD2 by siRNA. The siRNA treatment effect can be seen from the decrease in the amount of PHD2 protein expression. In addition, as shown in 3B, when the enzyme activity of PHD2 was inhibited using its inhibitor, DMOG, HIF-1α expression inhibited by vitamin C was increased again. These results indicate that vitamin C destabilizes HIF-1α through activation of PHD2.

Example  4: Inhibition / prevention of prostate growth caused by testosterone by vitamin C

Three-week old 300-350 g rats (Sprague-Dawley rat, Orient, Korea) were used for animal experiments. Rats were allowed to feed and water freely during the day, keeping the temperature at 20-25 ° C and 40-45% humidity. In addition, the light-dark cycle in the cage was adjusted to repeat day and night every 12 hours. Corn oil was subcutaneously injected into rats (n = 5) used as a control group (C) for the experiment, and testosterone (10 mg / kg / day) subcutaneously to BPH group (n = 5) (T) rats. (n = 5) (T + VC) Rats were intraperitoneally injected with vitamin C (100 mg / kg / day) in addition to testosterone. Dosing period was 4 weeks. One day after the final administration, the prostate was extracted, weighed, and used for immunohistochemistry. To monitor the progress of BPH, blood was collected from the tail during dosing and the prostate specific antigen (PSA) was measured using the Enzyme Linked ImmunoSorbant Assay (ELISA) system (USCN Life Science and Technology, USA) system as recommended by the manufacturer.

The extracted prostate was fixed in formalin and embedded in paraffin. Continuous sections were cut out to a thickness of 6 μM. The sections were then removed from the paraffin using a series of different concentrations of alcohol on slide glass (twice for 10 minutes each at 100%, 95%, and 80% ethanol), rehydrated in distilled water, and then Hematoxalin and Eosin (H & E). , Sigma-Aldrich) dye was used to stain the tissue as recommended by the manufacturer and observed under a magnification of x100 under an optical microscope. The area of the epidermal layer was analyzed using ImageJ 1.36B image analysis software (NIH, USA).

The results are in Figure 4, showing that vitamin C delays the growth of testosterone induced epidermal cells. 4A shows PSA concentrations in plasma. The dots represent the results and the horizontal lines represent the mean values, and the values of the C, T and T + VC groups were 257 ± 77.7, 843.8 ± 307.8 and 474.4 ± 192.6 (unit pg / ml), respectively. Serum PSA concentrations increased rapidly after testosterone treatment, and this increase was eliminated by vitamin C treatment. Figure 4B is a measure of the weight of the extracted prostate, the values of the C, T and T + VC group was 470 ± 110, 904 ± 138, and 804 ± 127 (unit mg), respectively. The testosterone treatment increased the size of the prostate gland, which slowed significantly in the vitamin C group. 4C shows the epidermal cell layer (red area), stromal (white part) and lumen (central space) constituting the prostate tissue. The epidermal tissue and stroma of the testosterone treated group were enlarged compared to the control group, and in the vitamin C treated group, this hypertrophy, in particular, the proliferation of epidermal cells was significantly reduced to the control level. Figure 4D is a measure of the area of the epidermis, which is consistent with the results of Figure 4C. Each individual data and average (horizontal line) are shown in the graph.

Example  5: induced by testosterone by vitamin C HIF -1α and VEGF  Suppress expression

The paraffin block prepared in Example 4 was also deparaffinized / rehydrated as in Example 4, and then heated for 5 minutes with microwave in sodium citrate (pH 6.0) to increase antigen exposure. Block the nonspecific site for 1 hour in PBS (pH 7.4) solution containing 2.5% bovine serum albumin and 2% normal rabbit serum, and then add anti-HIF-1α (Santa Cruz, 1: 100 dilution) in the blocking buffer. And incubated overnight at 4 ° C. No antibody was added to the negative control. It was then developed in response to horseradishperoxidase conjugated antibody (1: 200 dilution, Santa Cruz), ABC kit (Vector, USA) and DAB kit (Dako Cytomation, Denmark) as recommended by the manufacturer.

The concentration of VEGF was homogenized from the prostate gland extracted from each group, and then 50 μg of protein was analyzed by anti-VEGF antibody (Santa Cruz, 1: 3000 dilution) Western blotting as described in Example 1, and VEGF and tubulin (control). ) Images were analyzed using ImageJ 1.36B image analysis software (NIH, USA).

The results are in FIG. 5 and show that vitamin C inhibits the expression of HIF-1α and VEGF induced by testosterone. FIG. 5A was determined to be HIF-1α positive (arrow) when having twice the value (density staining, Adobe Photoshop Elements 2.0) compared to the background value as a result of immunohistostaining. The number of positive cells per 100 cells was measured and shown in the graph (lower right), and the horizontal line is the average value. The number of HIF-1α positive cells per 100 cells of C, T and T + VC is 8.3 ± 1.2, 19.1 ± 5.4, 11.9 ± 3.1 (unit positive cells / 100 cells), respectively. Increased HIF-1α positive cell numbers in the testosterone treated group (T) showed a decrease in control levels by treatment of vitamin C. FIG. 5B is Western blot results for VEGF (left) and VEGF / tubulin ratio is shown graphically (right). The concentration of VEGF was significantly increased in the testosterone treated group (T), but this phenomenon disappeared with the addition of vitamin C (T + C). These results demonstrate that vitamin C significantly reduces the expression of HIF-1α and VEGF in vivo.

Example  6: In the rat  Inhibition / treatment of enlarged prostate with vitamin C

Proliferation Cell Nuclear Antigen (PCNA) expression, a cell proliferation marker, was examined by tissue immunostaining as described in Example 5 using an anti-PCNA antibody (Santa Cruz, 1: 100 dilution).

For apoptosis analysis, TUNNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick and labelling) staining was performed using the ApopTag in situ apoptosis detection kit (Oncor, USA) as recommended by the manufacturer. In summary, the extracted treated prostate sections of Example 4 were reacted at 37 ° C. for 2 hours in a solution containing TdT (Terminal deoxynucleotidyl transferase) enzyme. The reaction was then stopped using the Stop / Wash buffer and reacted with antidigoxygenin peroxidase followed by 5 min in diaminobenzidine and 0.01% H ₂ O ₂ . Then counter staining with hematoxylin was observed under an optical microscope.

The results are in FIG. 6 and show that vitamin C interferes with the proliferation and survival of epidermal cells in vivo. FIG. 6A was determined to be PCNA positive (arrow) when the tissue had five times greater than the background value (density staining, Adobe Photoshop Elements 2.0) as a result of immunohistostaining for PCNA. The number of positive cells per 100 cells was measured and shown in the graph (lower right), and the horizontal line is the average value. The number of PCNA positive cells per 100 cells of C, T and T + VC is 1.6 ± 0.7, 6.0 ± 1.1 and 2.9 ± 0.6 (unit positive cells / 100 cells), respectively. Compared to the control group, the significantly increased PCNA positive cell number in the testosterone treated group (T) was shown to decrease to the control level by the treatment of vitamin C. FIG. 6B shows TUNNEL positive (arrow) cell counts per 100 cells as a result of TUNNEL staining, and is shown as a graph (lower right), and horizontal lines are average values. The number of PCNA positive cells per 100 cells of C, T and T + VC is 4.9 ± 3.1, 0.4 ± 0.1, 6.9 ± 1.0 (unit positive cells / 100 cells), respectively. Compared with the control group, apoptosis was significantly decreased in the testosterone treated group (T), and the number of cells (TUNNEL positive cells) killed by the treatment of vitamin C was increased. These results indicate the antiproliferative effect and apoptosis promoting effect of vitamin C.

Example  7: LPS By processing In the rat Enlarged prostate  Development and its inhibition by vitamin C

Three-week old 300-350 g rats (Sprague-Dawley rat, Orient, Korea) were used for animal experiments. Rats were allowed to feed and water freely during the day, keeping the temperature at 20-25 ° C and 40-45% humidity. In addition, the light-dark cycle in the cage was adjusted to repeat day and night every 12 hours. Corn oil was subcutaneously injected into rats (n = 5) used as the control group (C) for the experiment, and LPS (lipopolysaccharide) (100 μg / kg / day) was applied to the BPH group (n = 5) (LPS) rats. Sigma-Aldrich) was injected intraperitoneally, and VC group (n = 5) (LPS + VC) rats were intraperitoneally injected with vitamin C (100 mg / kg / day) plus LPS. Dosing period was 4 weeks. One day after the final administration, the prostate was removed, weighed, and used for immunohistochemistry as described in Example 4. The results are in FIGS. 7 and 8.

As shown in Figure 7 when administered with LPS the weight of the prostate is about 1.6 times compared to the control group This is a result showing that prostate hyperplasia can be caused by LPS.

As shown in FIG. 8, it is shown that vitamin C delays the growth of epidermal cells induced by LPS. 8 shows the epidermal cell layer (red area), stromal (white area) and lumen (central space) constituting the prostate tissue. The epidermal tissue and stroma of the LPS-treated group were very enlarged compared to the control group, and the vitamin C-treated group showed that the hypertrophy, in particular, the proliferation of epidermal cells was significantly reduced to the control level.

Example  8: caused by vitamin C LPS  Induced by HIF -1α expression suppression

The rats of Example 7 were made paraffin blocks as described in Examples 4 and 5 and sections were obtained from which immunohistochemistry for HIF-1α expression was performed. The results are in FIG.

As shown in FIG. 9, the expression of HIF-1α was significantly increased when LPS was treated compared to the control group, and this increase in expression was significantly reduced by the treatment of vitamin C.

Example  9: LPS  Processed In the rat  Inhibition / treatment of enlarged prostate with vitamin C

Tissue immunoassay was carried out to express the cell proliferation marker PCNA (Proliferation Cell Nuclear Antigen) as described in Example 6 using an anti-PCNA antibody (Santa Cruz, 1: 100 dilution) against the prostate extracted from the rat of Example 7. It was examined by staining. The results are in FIG. 10 and show that vitamin C interferes with the proliferation and survival of epidermal cells in vivo.

FIG. 10 was determined to be PCNA positive (brown portion) (FIG. 10A) when the tissue had five times greater value (density staining, Adobe Photoshop Elements 2.0) than the background value as a result of immunohistostaining for PCNA. The number of positive cells per 100 cells was measured and shown in FIG. 10B. The number of PCNA positive cells per 100 cells of C, LPS, LPS + VC 2.2, 7, 3.6 (unit positive cells / 100 cells). Compared with the control group, the significantly increased PCNA positive cell number in the LPS-treated group was shown to decrease to the control level by the treatment of vitamin C. These results are consistent with the vitamin C antiproliferative effect on prostatic hypertrophy by testosterone treatment.

Statistical processing

All data were analyzed using Microsoft Excel 2006. Statistical differences were examined using the Mann-Whitney U test, and both were compared with a two-sided test. The results are expressed as mean and standard deviation, and were judged to be significant when the P value was <.05.

The bars in FIGS. 4-6 and 8-10 are 50 μm.

The composition of the present invention comprising ascorbate containing the vitamin C may be administered in the following formulations, and the following formulation examples are merely illustrative of the present invention, thereby not limiting the content of the present invention.

Formulation example  1. Preparation of Tablets

Vitamin C 200mg

Lactose 100mg

Starch 100mg

Magnesium stearate proper amount

Tablets were prepared by mixing and tableting the above components according to a conventional tablet production method.

Formulation example  2. Preparation of Capsule

 Vitamin C 100mg

Lactose 50mg

Starch 50mg

Talc 2mg

Magnesium stearate proper amount

According to a conventional capsule preparation method, the above ingredients were mixed and filled into gelatin capsules to prepare capsules.

Formulation example  3. Liquid  Produce

Vitamin C 1000mg

20 mg of sugar

Isomerized sugar 20mg

Lemon flavor

Purified water was added to make a total of 1000 ml. According to the conventional method for preparing a liquid, the above components were mixed, and then filled into a brown bottle and sterilized to prepare a liquid.

Formulation example  4. Preparation of injections

 Vitamin C 100mg

Sodium metabisulfite 3.0mg

Methylparaben 0.8mg

Propylparaben 0.1mg

Appropriate sterile distilled water for injection

The above components were mixed and prepared in a conventional manner so that the final volume was 2 ml, and then filled into 2 ml ampoules and sterilized to prepare an injection.

Formulation example  5. Manufacture of health drinks

 Vitamin C 1000mg

Citric Acid 1000mg

Oligosaccharide 100g

Mesyl Concentrate 2g

1 g of taurine

Add 900 ml of purified water

After mixing the above components in accordance with a conventional healthy beverage production method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2L container, sealed sterilized and then refrigerated and stored in the present invention Used to prepare healthy beverage compositions.

Formulation example  6. Manufacture of health food

 Vitamin C 1000mg

Vitamin mixture proper amount

Vitamin A Acetate 70mg

Vitamin E 1.0mg

Vitamin B1 0.13mg

Vitamin B2 0.15mg

Vitamin B6 0.5mg

Vitamin B12 0.2mg

Biotin 10mg

Nicotinic Acid 1.7mg

Folic acid 50mg

Calcium Pantothenate 0.5mg

Vitamin E 1.0mg

Mineral mixture

Ferrous Sulfate 1.75mg

Zinc Oxide 0.82mg

Magnesium Carbonate 25.3mg

15 mg potassium monophosphate

Dicalcium Phosphate 55mg

Potassium Citrate 90mg

Calcium Carbonate 100mg

Magnesium chloride 24.8mg

The composition ratio of the above-mentioned vitamin and mineral mixtures is composed of relatively suitable ingredients suitable for health foods in a preferred manufacturing example, but the compounding ratios may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method. Health foods of granule formulations were prepared.

1 is a result showing that the increase in the expression of HIF-1α in prostate cells by testosterone is inhibited by vitamin C, in association with the expression and transcription of VEGF is also inhibited.

2 is a result showing that the proliferation of prostate cells by testosterone is inhibited by vitamin C.

3 is a Western blot showing that HIF-1α inhibition by vitamin C is PHD2 dependent.

4 is a PSA concentration and prostate tissue staining showing that vitamin C inhibits the growth of testosterone-induced epidermal cells in vivo.

FIG. 5 shows immunohistostaining and western blot results showing that vitamin C inhibits testosterone-induced HIF-1α and VEGF expression in vivo.

FIG. 6 is an immunohistostaining and apoptosis assay showing that vitamin C inhibits prostate cell proliferation and survival by testosterone in vivo.

7 is a result showing that the prostate weight is increased by the LPS in the animal model using the rat shows that prostate hypertrophy may be caused by the LPS.

FIG. 8 is a tissue staining result showing that proliferation of LPS-induced prostatic epidermal cells is suppressed by administration of vitamin C in a rat animal model.

9 is an immunohistostaining result showing that HIF-1a increase is inhibited by vitamin C in LPS-induced prostatic epidermal cells in vivo.

10 is an immunohistostaining result showing that vitamin C inhibits prostate epithelial cell proliferation and survival induced by LPS in vivo.

Claims (7)

Prostatic hyperplasia treatment or prevention composition comprising ascorbate as an active ingredient. The method of claim 1, wherein the ascorbate is in the form of vitamin C, ascorbyl ester, ascorbyl palmitate, ascorbyl phosphate ester, mineral ascorbate, dehydroascollate or vitamin C metabolite, or A composition thereof. The composition of claim 2, wherein the mineral ascorbate is calcium ascorbate, magnesium ascorbate, zinc ascorbate, sodium ascorbate or potassium ascorbate. The composition of claim 1, wherein the ascorbate inhibits the expression of HIF-1α. The composition according to any one of claims 1 to 4, wherein the composition is provided as a dietary supplement, a functional food or a food additive. The composition of claim 5, wherein the dietary supplement, the functional food, and the food additive are for preventing or improving symptoms of prostatic hyperplasia. An agent for improving urination disorder due to enlarged prostate, comprising the composition according to any one of claims 1 to 4.

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