KR100830236B1 - The anti-prostate cancer effect of platycodon grandiflorum extracts - Google Patents

Abstract

A hot water extract of Platycodon grandiflorum is provided to inhibit histone acetyl transferase and show excellent anti-cancer effect on androgen receptor mediated prostatic carcinoma cell lines, thereby being usefully used as a medicine and a health food. A composition or a health food for preventing and improving prostatic carcinoma comprises an extract of Platycodon grandiflorum, which is prepared by extracting roots of the Platycodon grandiflorum with hot water having a temperature of 90-100 deg.C and then fractionating the extract using various organic solvents such as hexane, chloroform and ethyl acetate, as an effective ingredient.

Description

Composition for preventing and treating prostate cancer containing bellflower extract {The anti-prostate cancer effect of Platycodon grandiflorum extracts}

1 is a diagram showing the specific inhibitory effect of HAT protein on various plant extracts.

Figure 2 is a chart showing the transcriptional activity inhibiting activity of bellflower extract in prostate cancer cell line.

Figure 3 is a photograph showing the mRNA expression inhibitory effect of the androgen receptor regulatory gene of bellflower extract in prostate cancer cell line.

4 is a chart showing cell viability of prostate cancer of the Platycodon grandiflorum extract in various cancer cell lines.

The present invention relates to a composition for preventing and treating prostate cancer containing bellflower extract, and more particularly, the extract of hot water extract of Platycodon grandiflorum inhibits histone acetyltransferase of uric acid and androgen receptor-mediated prostate, a male hormone. The present invention relates to a new medicinal use of bellflower extract which can be usefully used as a material for medicines and health foods by showing superior anticancer effects in cancer cell lines.

Prostate cancer is the highest incidence of hormone receptor mediated adult cancer in the United States, and the number of cancers in Korea is increasing rapidly. Androgen (androgen) is essential for the development and maintenance of the reproductive system and the growth of the prostate gland, as well as regulating the activity of ligand-dependent hormone receptors belonging to the steroid hormone receptor superfamily. Deeply involved in the development and progression of human resources [Park, SK] et al. Prostate. 66 (12): 1285-91 (2006); Taplin M.E. et al. J Cell Biochem. 15; 91 (3): 483-90, 2004; Chen, L. et al. Mol Cancer Ther. 4 (9): 1311-9, 2005; Yoon, H.G. et al. Mol Endocrinol. 20 (5): 1048-60, 2006].

Histones are tightly bound to DNA with genetic information to form nucleosomes in the nucleus, and post-translational modifications of these DNA-binding proteins can regulate gene expression and signaling. The acetylation of histones during this transformation is largely regulated by two enzymes, histone acetyltransferase (HAT) and histone deacetylase (HDAC), which are transcription factors and signal transduction parameters. It can also regulate the activity of the same nonhistone protein. HAT proteins increase the acetylation of hormone receptors, and acetylated hormone receptors overexpress each target protein, causing cancer growth and development. As a result, inhibition of hyperacetylation in hormone receptor-related cancers is recognized as a molecular target for the search for new cancer inhibitors. In particular, hyperacetylation of androgen receptors and histones is associated with the development and proliferation of androgen receptor-independent prostate cancer. Relevant and apoptosis suppression mechanisms have been reported in vitro in cancer cell lines [Kang, J. et al. Biochem Pharmacol. 69 (8): 1205-13, 2005; Stimson, L. et al. Mol Cancer Ther. 4 (10): 1521-32, 2005; Balasubramanya, K. et al. J Biol Chem. 279 (49): 51163-71, 2004; Druesne, N. et al. Carcinogenesis. 25 (7): 1227-36, 2004; Fu, M. et al. Biochem Pharmacol. 68 (6): 1199-208, 2004].

In order to control such acetylation, HDAC activity inhibitors have been released so far mainly using HDAC protein as a target protein for cancer therapy. Relatively limited HAT inhibitors compared to these various HDAC inhibitors have been published. Inhibitors studied to date include synthetic peptide-CoA conjugates, isothiazolone, polyprenylated benzophenone, curcumin, and anacardic acid [Kang, J. et al. Biochem Pharmacol. 69 (8): 1205-13, 2005; Inche, AG Drug Discov Today. 11 (3-4): 97-109, 2006; Lau, OD et al. Mol Cell. 5 (3): 589-95, 2000; Debes, JD et al. Cancer Res. 63 (22): 7638-40, 2003; Sun, Y. et al, FEBS Lett. 580 (18): 4353-6, 2006]. Recently, studies on plant-derived inhibitors that are easier to secure safety than the synthetic compounds such as peptide-CoA conjugate or isothiazolone have been actively conducted. Curcumin in turmeric mentioned above, anacardic acid from cashew nuts, and benzophenone in Garcinia indica are examples of plant-derived HAT inhibitors. The securing of such HAT inhibitory activity-containing plant extracts is constantly needed for the development of new anticancer drugs.

The bellflower ( Platycodon grandiflorum ) is a perennial herb that grows in the grassland of Sanya, native to Korea, northeastern China and Japan. In addition, it is a perennial herb belonging to the campanula, the roots are thick, and the stems are single or pedigerous, 40 to 100 cm high. The leaves are regenerated or resilient, long egg-shaped or oval with almost no petioles. Flowers bloom in June-August, flower color is blue purple, bell-shaped and blooms one by one at the end of stem or branch. The white flowering system is called the white bellflower, and the root is used for medicine or food for keratinized patients. The root of bellflower has been used for edible vegetable or medicinal, and the root is washed with water, dried or peeled, dried and dried. The root contains 2% triterpenoid saponin and 0.03% sterol [Saeki, T. et al., Planta Med. 65: 428 (1937). Other saccharides include inulin and platinum codinin (polysaccharides composed of 10 molecules of fructose). It is known that the main efficacy of bellflower in oriental medicine is good for the symptoms such as sore throat, nausea, difficulty urination, diarrhea and post-heavy, which are closely related to cough, phlegm and lung disorders [Lee, KJ et al., Food Chem. Toxicol. 40: 517 (2002), Han, SB et al., Int. Immunopharmacol. 1: 1969 (2001), Choi, CY et al., Cancer Lett. 166: 17 (2001). The inhibitory effect of the extract of Bellflower extract on cancer cell proliferation [Lee, Ji-Young et al., Vol. 30 (1), 13-21, 1998], etc., has been described, and it is only demonstrated for lymphocytes and solid cancers. It has not been reported as an inhibitor of HAT activity for the prevention of prostate cancer.

Accordingly, the present inventors have ensured safety in developing new food materials required for the development of functional foods, food drugs, and medicines for inhibiting hormone receptor-related cancer as described above, and for a long time in order to reduce the risk of the substance in clinical trials. While conducting a search for HAT activity inhibitory ability on 500 kinds of food and medicinal plants that have been used, the present invention was confirmed by confirming high HAT inhibitory activity in water extract of bellflower, which has long been common in Korea, and confirmed in vitro . It became.

Accordingly, an object of the present invention is to provide a composition for the prevention and treatment of prostate cancer, which contains Platycodon grandiflorum extract as an active ingredient.

In addition, the present invention has another object to provide a health food for preventing and improving prostate cancer containing the bellflower ( Platycodon grandiflorum ) extract as an active ingredient.

The present invention is characterized by the composition for the prevention and treatment of prostate cancer containing bellflower ( Platycodon grandiflorum ) extract as an active ingredient.

In the present invention, the extract is characterized in that the hot water extract of bellflower root.

In the present invention, the extract may be further fractionated with various organic solvents such as nucleic acid, chloroform, ethyl acetate after extracting the bellflower root with water at 90 ~ 100 ℃, may be further purified by various chromatography after organic solvent fractionation have.

In the present invention, the HAT activity inhibitor is characterized in that it is used for the prevention and treatment of prostate cancer which is a hormone receptor-related cancer. The mechanism of action of HAT and its anticancer activity, which are closely related to prostate cancer, are well known in the art [Stimson, L. et al. Mol Cancer Ther. 4 (10): 1521-32, 2005; Balasubramanyam, K. et al. J Biol Chem. 279 (49): 51163-71, 2004], the plant extract of the present invention will be highly useful as a prophylactic and therapeutic agent for prostate cancer by inhibiting HAT activity. In addition, the present invention is a natural material isolated from the roots of bellflower, which is an edible plant, has no side effects and is very safe compared to conventional organic synthetic drugs.

In order to achieve another object of the present invention, the present invention provides a health food for the prevention and improvement of prostate cancer, which is a hormone receptor mediated cancer containing the HAT activity inhibitor according to the present invention as an active ingredient.

In order to achieve another object of the present invention, the present invention provides a composition for the prevention and treatment of prostate cancer, which is a hormone receptor mediated cancer containing the HAT activity inhibitor according to the present invention as an active ingredient.

Hereinafter, the present invention configured as described above will be described in more detail HAT activity inhibitory substance and its in vitro prostate cancer inhibitory activity from bellflower roots.

In the present invention, plant-derived extracts with inhibitory activity of HAT, which is involved in the growth and development of hormone-mediated cancers, are extracted from HeLa cell line, a human cancer cell line, among extracts extracted from 500 food and medicinal plants. The LNCaP cell line, a human prostate cancer cell line, was used to examine the molecular mechanism of inhibition of growth and transcriptional activity and inhibition of mRNA expression of androgen receptor regulatory genes in vitro . The present invention was completed by obtaining an extract.

In the present invention, the bellflower extract includes all of the extracts, fractions and purified products obtained in each step of the extraction, fractionation and purification treatment, diluents or concentrates thereof, or dried products thereof.

Bellflower extract, which is a raw material of the present invention, may be formulated in the form of granules, tablets, capsules, or drinks by conventional methods known in the art. Moreover, in order to make storage and handling easy, you may add the carrier used for normal formulation, such as dextrin and cyclodextrin, and other arbitrary preparations. In addition, auxiliary raw materials or additives conventionally added to foods or drugs are not particularly limited, for example, glucose, fructose, sucrose, maltose, sorbitol, stevioside, loxoside, corn syrup, lactose, citric acid, tartaric acid, malic acid , Succinic acid, lactic acid, L-ascorbic acid, d1-α-tocopherol, sodium elisolvinate, glycerin, propylene glycol, glycerin fatty acid ester, polyglycerin fatty acid ester, sucrose fatty acid ester, sorbitan fatty acid ester, gum arabic , Casein, gelatin, pectin, agar, vitamin Bs, nicotinic acid amide, calcium panthenate, amino acids, calcium salts, pigments, flavors, preservatives and the like.

In addition, the present invention includes a health food containing the bellflower extract as an active ingredient.

Health food is a food prepared by adding bellflower extract to food materials, such as beverages, teas, spices, gums, confectionery, or encapsulated, powdered, or suspensions. However, unlike general medicine, there is no side effect that can occur when taking long-term use of the drug as a food raw material. Since the health food of this invention obtained in this way can be ingested routinely, high anticancer and cancer prevention effect can be expected, and it is very useful.

The amount of bellflower extract in such a health food can not be defined uniformly depending on the type of health food, but may be added within a range that does not impair the original taste of the food, usually 0.01 to 50 % By weight, preferably in the range of 0.1 to 20% by weight. In the case of food in the form of granules, tablets or capsules, it is usually added in the range of 0.1 to 100% by weight, preferably 5 to 100% by weight.

In addition, the bellflower extract, which is an active ingredient of the inhibitory activity of HAT of the present invention, is appropriately administered so that the daily intake amount of adult is 1 to 3000 mg. In addition, the dosage can be appropriately increased or decreased depending on age, symptoms, and the like.

Next will be described in more detail with reference to the embodiment of the present invention configured as described above. These examples are only for illustrating the present invention in detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

Preparation Example 1 Preparation of Bellflower Hot Water Extract

In order to confirm the HAT inhibitory activity of the bellflower water extract selected from more than 500 kinds of food and medicinal biological resources was carried out as follows.

The root portion of the bellflower was blanched at 100 ° C. for 5 minutes to extract reflux by adding 5 times the amount of distilled water to the sample. The process was repeated three times, and the extract was concentrated under reduced pressure and dried to obtain a hot water extract. Got it.

Example 1: Confirmation of HAT inhibitory activity against the bellflower hot water extract

HeLa cells, a human cervical cancer cell line, were grown to full confluence on 150 mm plates, harvested, and then pelleted by centrifugation at 1000 rpm for 3 minutes. 20 mL of cold buffer A (10 mM pH 7.9 Hepes, 1.5 mM MgCl ₂ , 10 mM KCl, 0.5 mM DTT, 0.2 mM PMSF) was added to the collected pellets, and the mixture was left on ice for 10 minutes, followed by 5 minutes at 4 ° C and 3000 rpm. Centrifugation was performed, pellets were collected again, and 6 mL of the buffer A as described above was homogenized. The prepared cell homogenate was centrifuged at 4 ° C. and 12500 rpm for 30 minutes to collect pellets, and buffer C (20 mM pH 7.9 Hepes, 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl ₂ , 0.2 mM EDTA, 0.5 mM DTT, 4 ml of 0.2 mM PMSF) was added, resuspended, and allowed to stand on ice for 30 minutes, followed by centrifugation at 4 ° C and 12500 rpm for 30 minutes. Finally, the supernatant obtained by centrifuging the supernatant at 4 ° C and 30000 rpm for 1 hour was collected and used for the experiment. In order to measure the inhibitory activity of HAT, 50 ㎍ of the nuclear extract prepared was measured for absorbance at 440 nm using a HAT activity colorimetric assay kit [Biovision, CA, USA].

As a result of the above method, 31% of inhibitory activity was confirmed in the bellflower when the plant extract of Table 1 was treated at a concentration of 0.1 mg / ml. The following Table 1 is a chart showing the inhibitory effect of the plant extract when measuring the HAT activity. Here, HAT activity inhibition capacity (%) was converted according to the following equation (1).

Ac: absorbance at distilled water treatment

Ab: absorbance of blank

As: absorbance of sample treatment

Example 2 Measurement of Various HAT Protein Activities Using IP (Immunoprecipitation)

In order to measure the activity of various HAT proteins such as P300, PCAF, and CBP and examine the inhibition specificity, HeLa nuclear extract (50 μg) prepared in the same manner as in Example 1 was pre-treated at 4 ° C. for 2 hours. After clearing, Protein A / G agarose bead (10 μl, Santa Cruz, CA, USA) and 2 μl of the antibody of each HAT protein were added and washed overnight, followed by eluted product. HAT assay was performed according to the method of 1 to compare specific inhibitory activity. As a result, it was confirmed that the bellflower hot water extract has an inhibitory effect on CBP (25%) and P300 (30%) (FIG. 1). It is expected that the hot water extract of bellflower will have an enzymatic inhibitory effect on CBP and P300 among several HAT proteins, and thus the androgen receptor activity inhibitory effect will be excellent.

Example 3: Confirmation of transcriptional activity inhibitory activity of bellflower hot water extract in prostate cancer cell line

LNCaP cell line, a prostate cancer cell line, was cultured in RPMI1640 medium containing 10% (w / v) fetal bovine serum for 3 days, and LNCaP cell line containing 10% CS-FCS (charcoal-stripped FCS) [Life Technologies] After incubation for 2 days again with red-free RPMI1640 medium, transfection was performed with a vector for the Luciferase reporter assay (pGL3-PSA) including a PSA regulatory promoter site. After 24 hours of incubation, the synthetic androgen R1881 was treated with 1 nM, and again after 6 hours, the bellflower hydrothermal extract was treated, and reporter analysis was performed using a lysate of cells after 16 hours. Bellflower hot water extract was confirmed to inhibit transcription to a level similar to the control (Fig. 2).

Example 4 Inhibition of mRNA Expression of Androgen Receptor Regulator Gene of Bellflower Hot Water Extract in Prostate Cancer Cell Lines

The LNCaP cell line was cultured in RPMI1640 medium containing 10% (w / v) fetal bovine serum for 3 days and the LNCaP cell line was exchanged with phenol red-free RPMI1640 medium containing 10% charcoal-stripped FCS (CS-FCS) [Life Technologies]. After incubation for 3 days, the synthetic androgen R1881 at a concentration of 1 nM. After 6 hours, 16 hours later, total RNA was extracted by RT or PCR treatment. Related target gene primers are as follows:

NKX 3-1:

5'-AGCCGCTCACGTCCTTCCTCATCC-3 '(SEQ ID NO: 1)

5'-GGGGCCCGGTGCTCAGCTCGTCGTTCT-3 '(SEQ ID NO: 2)

TSC22:

5'-ATTTTTCTCTATTAGTTCTTTGATTTG-3 '(SEQ ID NO: 3)

5'-GACTTGATAATAGCTCCTCTGGT-3 '(SEQ ID NO: 4)

PSA:

5'-GCCCACCCAGGAGCCAGCACT-3 '(SEQ ID NO: 5)

5'-GGCCCCCAGAATCACCCGAGCAG-3 '(SEQ ID NO: 6)

GAPDH:

5'-CGCGGGGCTCTCCAGAACATCATCC-3 '(SEQ ID NO: 7)

5'-CTCCGACGCCTGCTTCACCACCTTCTT-3 '(SEQ ID NO: 8)

RT-PCR was performed with the above primers, and it was confirmed that each androgen receptor regulatory gene was inhibited (FIG. 3). The mRNA level of each gene was significantly increased by the treatment of synthetic androgen R1881, and the expression was reduced to the control (R1881 untreated group) level by treatment of bellflower hot water extract (100 ㎍ / ml). By obtaining the androgen receptor regulatory genes of Bellflower hot water extract confirmed the inhibitory effect.

Example 5 Inhibitory Activity of Growth and Proliferation in Prostate Cancer Cell Lines

To examine the activity of Bellflower hot-water extracts on the growth and proliferation of prostate cancer, 1 x 10 ³ cervical cancer cell line (HeLa), breast cancer cell line (MCF-7), esophageal cancer cell line (HCE4) and prostate cancer cell line (LNCaP), respectively. , 1.0 x 10 ⁴ , After seeding in 96 well-plate at 1.0 x 10 ⁴ and 1.5 x 10 ⁴ cells / well, and incubating at 37 ° C for 18 hours, the bellflower hydrothermal extract was treated with the final concentration of 200 ㎍ / mL, and after 48 hours, MTT [3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide: 1 mg / ml] was added and reacted at 37 ° C. for 2 hours. The formed formazan was absorbed at 570 nm and reference 630 nm. It was confirmed by measurement. According to the above method, the bellflower hot water extract showed 44% cell viability at a concentration of 200 ㎍ / mL, which was superior to esophageal cancer (85.2%), cervical cancer (99.1%) and breast cancer (91.7%) cell lines. The anticancer effect could be confirmed (FIG. 4).

Formulation Example 1 Preparation of Tablet

The following formulation tablets (based on 200 parts by weight of total composition) were prepared by a conventional tablet press.

20 parts by weight of powder of bellflower extract

Dextrin 72 parts by weight

80 parts by weight per minute

8 parts by weight of glycerin fatty acid ester

Mixing and tableting of raw materials were easy, and refined refinement | purification was obtained.

Formulation Example 2: Preparation of Capsule

By the usual method, the capsule which has the following compositions was manufactured. In addition, No. 1 hard gelatin capsule was used for the capsule.

Composition in 1 Capsule (200 mg)

5 mg of powder of bellflower extract

Cornstarch 60.0 mg

Lactose 100.0 mg

Calcium Lactate 10.0 mg

Hydroxypropylcellulose (HPC-L) 10.0 mg

Formulation Example 3 Preparation of Granules

By conventional methods, instant tea granules of the following formulations were prepared by fluid bed granulator (based on a total composition of 970 parts by weight).

20 parts by weight of powder of bellflower extract

40 parts by weight of oligosaccharide

50 parts by weight of citric acid

50 parts by weight of sugar

Dextrin 810 parts by weight

Mixing of the raw materials and granulation with a fluidized bed granulator were easy, and a fine grain of instant tea was obtained.

Formulation Example 4 Preparation of Injection

Injection solution containing 10 mg of Bellflower hot water extract was prepared by the following method.

1 g of bellflower hot water extract, 0.6 g of sodium chloride and 0.1 g of ascorbic acid were dissolved in distilled water to make 100 ml. The solution was bottled and sterilized by heating at 20 ° C. for 30 minutes.

Formulation Example 5: Beverage Preparation

After dissolving 500 mg of Bellflower hot-water extract according to the present invention in an appropriate amount of water, vitamin C as an auxiliary component, citric acid, sodium citrate, oligosaccharides as a copulating agent is added, and an appropriate amount of sodium benzoate is added as a preservative, followed by the addition of water. To 100 ml to prepare a beverage composition. At this time, taurine, myo-inositol, folic acid, pantothenic acid, etc. may be added alone or together.

As described above, the bellflower extract according to the present invention has an excellent effect of inhibiting the HAT activity, and thus can be used in medicines or health foods for the prevention and treatment of hormone receptor mediated prostate cancer using the same as an active ingredient.

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Claims (2)

A composition for preventing and treating prostate cancer, comprising bellflower ( Platycodon grandiflorum ) extract as an active ingredient. Healthy food for preventing and improving prostate cancer, comprising bellflower ( Platycodon grandiflorum ) extract as an active ingredient.

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