KR101478487B1 - Composition for treating prostate disease comprisng extract and hydrolysable Tannin from CLEYERA JAPONICA - Google Patents

Abstract

The present invention relates to a Cleyera japonica extract or hydrolysable tannin isolated therefrom and a composition including the same for treating prostate diseases. More specifically, the present invention relates to a compound represented by chemical formula 1 or a pharmaceutically acceptable salt thereof, and a composition including the same as an active ingredient for treating prostate diseases.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for treating a prostate disease, which comprises a tiba tree leaf extract or a hydrolysable tannin separated therefrom.

The present invention relates to a composition for treating a prostate disease, which comprises a tsukuba leaf extract or a hydrolysable tannin isolated therefrom, and more particularly, to a composition for treating a prostate disease comprising a compound represented by the formula (I) or a pharmaceutically acceptable salt thereof, To a composition for treating a prostate disease.

Cryptomeria japonica (Cleyera japonica) is an evergreen small arboreal tree that lives in the southern coast of Korea, Jeju Island, Japan, China, and Taiwan. The height is less than 10m, white flowers gradually turn yellow in May-June, and black berries ripen in October. The shape of the winter snow is called a skein. They are mainly planted for ornamental purposes, and wood is used for construction, equipment and chambr. Especially in Japan, it is called a sacred tree in the sense of a tree planted between the gods and human beings, and is used as a sacred sacrifice to be used in ceremonies and weddings. Recent studies have reported that simple acid species and flavonoids have been isolated from the branches, and active studies only report antioxidant efficacy.

On the other hand, diseases that occur in the prostate gland can be divided into prostate, prostate hyperplasia, and prostate cancer. Prostatitis involves acute / chronic inflammatory diseases of the prostate gland and is known to occur more than once in a lifetime in more than 50% of adult men. The cause of prostatitis has not been clarified yet, but infection, stress, and autoimmunity are presumed to be pathogenic. Antibiotics are used only for bacterial prostatitis, but no standard therapy has yet been established for non-bacterial prostatitis.

Prostatic hyperplasia is a benign tumor that develops in the extrathoracic prostate, and prostate cancer is a malignant tumor that develops in the outside. Prostatic hyperplasia is a typical male adult disease, and its prevalence is more than 40% of the male population over 65 years old. Male hormone imbalance due to senescence of the testes is known to be the most potent cause, but high fat diet, obesity, chronic inflammation and autoimmune are also considered as etiologies. 5α-reductase inhibitors and α-blockers have been used to treat benign prostatic hyperplasia. Prostate cancer is the most prevalent cancer in Western men. In Korea, the prevalence rate has increased rapidly by 13.2% per year for the last 10 years. The etiology of prostate cancer is known to be genetic predisposition, such as race, family history, and age. Recently, high - fat diet, obesity, and smoking have been estimated to be important factors for the development of prostate cancer. Chemotherapy regimens such as GnRH agonist / antagonist hormones and taxol (docetaxel, carbazitaxel, etc.) have been used as standard therapy in clinical practice.

Although various antibiotics are used for the treatment of these prostatitis, they are effective only in bacterial prostatitis. In the case of non-bacterial prostatitis, recurrences are common and often develop chronic. In the case of enlarged prostate, 5α-reductase inhibitors reduce the size of the prostate, and α-blockers are used to improve the lower urinary tract symptoms. However, although the 5α-reductase inhibitors have been used for a relatively long period of time, the symptoms may be improved, but the side effects such as loss of sexual desire and sexual dysfunction may occur. It improves symptoms of lower urinary tract, but it is not only worry about side effects such as dizziness, hypotension, etc., but it is hard to expect fundamental therapeutic effect. Prostate cancer is initially treated with hormone therapy, but there are serious side effects of male sexual deprivation due to chemical castration and the nature of the disease necessarily recurs into hormone refractory cancer within 6 months - 2 years. At this time, chemotherapeutic agents such as taxol are administered, but it is known that prostate cancer does not contribute to life extension of the patient.

Accordingly, development of natural materials effective for the treatment of prostate diseases without side effects has been the subject of major researches, and studies have been conducted (Korean Patent Laid-Open No. 10-2012-0021281) .

DISCLOSURE OF THE INVENTION The present invention has been made in order to solve the problems in the prior art as described above. The present inventors have made efforts to discover active compounds effective for treating prostate diseases from natural products, and found that dimeric ellagitannin containing valoneoyl group And the compounds were found to have free radical and superoxide scavenging activity, nitric oxide (NO) production inhibiting activity, and prostate tumor cell proliferation inhibiting activity, and on the basis thereof, the present invention was completed.

Accordingly, it is an object of the present invention to provide a compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof.

[Chemical Formula 1]

It is another object of the present invention to provide a pharmaceutical composition for preventing or treating prostate disease, which comprises the compound or a pharmaceutically acceptable salt thereof as an active ingredient.

It is still another object of the present invention to provide a health functional food composition for preventing or ameliorating a prostate disease, which comprises the compound or a pharmaceutically acceptable salt thereof as an active ingredient.

It is still another object of the present invention to provide a pharmaceutical composition for preventing or treating prostate disease, which comprises an extract of Tibetan Leaf as an active ingredient.

It is still another object of the present invention to provide a health functional food composition for preventing or ameliorating a prostate disease, which comprises a tsukuba leaf extract as an active ingredient.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

The present invention provides a compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof.

[Chemical Formula 1]

The present invention provides a pharmaceutical composition for preventing or treating prostate disease, comprising the compound or a pharmaceutically acceptable salt thereof as an active ingredient.

In one embodiment of the present invention, the compound is characterized in that it is isolated from a bibuli leaf extract.

In another embodiment of the present invention, the prostate disease is prostate cancer, hypertrophy of the prostate, prostatitis, prostatitis or prostatitis.

In another embodiment of the present invention, the composition is characterized by having a nitric oxide (NO) production inhibiting activity.

The present invention provides a health functional food composition for preventing or ameliorating a prostate disease, comprising the compound or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention provides a pharmaceutical composition for preventing or treating prostate disease, which comprises an extract of Aspergillus oryzae as an active ingredient.

In an embodiment of the present invention, the prostate disease is prostate cancer, hypertrophy of the prostate, prostatitis, prostatitis or prostatitis.

In another embodiment of the present invention, the composition is characterized by having nitric oxide (NO) production inhibiting activity.

The present invention provides a health functional food composition for preventing or ameliorating a prostate disease, comprising the compound or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention provides a method of treating a prostate disease comprising the step of administering the compound or composition to a subject.

The present invention provides the use of the compound or composition for treating a prostate disease.

Camptothin A is superior to DPPH radical and superoxide scavenging activity and nitric oxide (NO) production inhibitory activity, and is also useful as a hormone-independent prostate tumor cell, PC- 3 is effectively reduced through apoptosis, and it is expected that it can be usefully used as an active ingredient of a pharmaceutical composition or functional food composition for preventing, ameliorating or treating prostate disease.

Fig. 1 shows the structure of Compound 1 isolated from a tsukuba leaf extract.
FIG. 2 shows the morphological changes of PC-3 cells using electron microscopy of the extract of Compound No. 1 isolated from the leaves of T. ssp.
FIG. 3 shows the results of observing PC-3 cell apoptosis patterns through Annexin_V / PI dual staining of Compound 1 isolated from a biscuit leaf extract.
FIG. 4 shows the results of observation of expression and translocation of apoptosis-related pro / anti-apoptotic protein by western blot and immunofluorescence staining of Compound 1 isolated from a biscuit leaf extract.
FIG. 5 shows the results of western blotting and immunofluorescence staining of Compound 1 isolated from the leaves of T. bovis leaf and the expression of apoptosis-related caspase protein, PARP, cytochrome c and translocation.

Hereinafter, the present invention will be described in detail.

A compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof.

[Chemical Formula 1]

More specifically, the compound represented by Formula 1 is camptothin A, which is a dimeric ellagitannin compound isolated and identified from Cleyera japonica extract.

As salts, acid addition salts formed by pharmaceutically acceptable free acids are useful. Acid addition salts include those derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, and aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates, Dioleate, aromatic acid, aliphatic and aromatic sulfonic acids. Such pharmaceutically innocuous salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate chloride, bromide, Butyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, succinate, maleic anhydride, maleic anhydride, , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, Methoxybenzoate, phthalate, terephthalate, benzene sulfonate, toluene sulfonate, chlorobenzene sulfide Propyl sulphonate, naphthalene-1-yne, xylenesulfonate, phenylsulfate, phenylbutyrate, citrate, lactate,? -Hydroxybutyrate, glycolate, maleate, Sulfonate, naphthalene-2-sulfonate or mandelate.

The acid addition salt according to the present invention can be obtained by a conventional method, for example, by dissolving the compound represented by the formula (1) in an excess amount of an acid aqueous solution, and then mixing the salt with a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile ≪ / RTI > It may also be prepared by evaporating a solvent or excess acid in this mixture and then drying or by suction filtration of the precipitated salt.

In addition, the base may be used to make a pharmaceutically acceptable metal salt. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or an alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt. The corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable salt (for example, silver nitrate).

The present invention provides a pharmaceutical composition for preventing or treating prostate disease, comprising the compound or a pharmaceutically acceptable salt thereof as an active ingredient. Prostate diseases that can be prevented or treated by the pharmaceutical composition of the present invention include, but are not limited to, prostate cancer, hypertrophy of the prostate, prostatitis, prostatitis, and prostatitis.

It is apparent to those skilled in the art that the compound represented by Chemical Formula (1) of the present invention has the same activity as a chemical compound synthesized from a natural product.

In the present invention, the compound represented by the above formula (1) can be obtained by a conventional method for separating and purifying a single compound from the extract. For example, filtration using silica gel or celite gel column, size-exclusion chromatography using liquid column chromatography, ion-exchange chromatography, partition chromatography, affinity chromatography affinity chromatography or a combination of these chromatographies.

The single compounds isolated from the extracts can be used in various fields such as MALDI-TOF MS, electron ionization mass spectroscopy (EI MS), chemical ionization mass spectroscopy (CI), fast atom bombardment ionization (FABI) And can be finally identified using mass spectrometry such as infrared spectroscopy (IR Sectrum), nuclear magnetic resonance (NMR), or circular dichroism (CD).

The compound of the present invention represented by the above formula (1) can be prepared as a pharmaceutically acceptable salt or solvate according to a conventional method in the art.

As used herein, the term "prophylactic " means any act that inhibits prostate disease or delays its development by administration of the pharmaceutical composition according to the present invention.

As used herein, the term "treatment" refers to any action that improves or alters the symptoms of prostate disease by administration of the pharmaceutical composition of the present invention.

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the formulation and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dosage level is determined depending on the type of disease, severity, , Sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including co-administered drugs, and other factors well known in the medical arts. The pharmaceutical composition according to the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the pharmaceutical composition according to the present invention may vary depending on the age, sex, condition, body weight, absorbency, inactivation rate, excretion rate, type of disease, May be administered in an amount of 0.001 to 150 mg, preferably 0.01 to 100 mg, per 1 kg of body weight per day, every other day, or one to three times per day. However, the dosage may be varied depending on the route of administration, the severity of obesity, sex, weight, age, etc. Therefore, the dosage is not limited to the scope of the present invention by any means.

The pharmaceutical composition of the present invention may be formulated into a unit dosage form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

In Example 1 of the present invention, the compound represented by the formula (1), more specifically, the camptothin A compound was isolated and identified (see Examples 1 and 2), and their free radical and excess oxide scavenging activity, NO (nitric oxide) production inhibitory activity and inhibitory effect on prostate tumor cell proliferation, the compounds showed superior or equivalent free radical and superoxide scavenging activity, NO (nitric oxide) production inhibitory activity and inhibitory effect on prostate tumor cell proliferation than the control group, It was confirmed that apoptosis of tumor cells was efficiently induced (see Examples 3 to 8).

Accordingly, the present invention provides a method for treating a prostate disease, comprising the step of administering the pharmaceutical composition to a subject. The term "individual" as used herein refers to a subject in need of treatment for a disease, and more specifically refers to a human or non-human primate, mouse, rat, dog, cat, It means mammals.

The present invention also provides a health functional food composition for preventing or ameliorating a prostate disease comprising the compound or a pharmaceutically acceptable salt thereof as an active ingredient.

The term "improvement" as used in the present invention means all actions that at least reduce the degree of symptom associated with the condition being treated. Herein, the functional food composition may be used simultaneously with or separately from the agent for treatment before or after the onset of the disease for the prevention or improvement of the prostate disease.

There is no particular limitation on the kind of the food. Examples of foods to which the active ingredient can be added include dairy products including dairy products, meat, sausage, bread, biscuits, rice cakes, chocolates, snacks, confectionery, pizza, ramen noodles, other noodles, gums, ice cream, , Beverages, alcoholic beverages and vitamin complexes, dairy products, and dairy products, all of which include health functional foods in a conventional sense.

In the health functional food composition of the present invention, the active ingredient may be added directly to the food or may be used together with other food or food ingredients, and may be appropriately used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (for prevention or improvement). In general, the composition of the present invention is added in an amount of not more than 15% by weight, preferably not more than 10% by weight based on the raw material, in the production of food or beverage. However, in the case of long-term consumption intended for health and hygiene purposes or for health control purposes, the amount may be less than the above range.

The health functional food composition of the present invention has no particular limitation on the other ingredients other than the above-mentioned active ingredients as essential ingredients in the indicated ratios and may contain various flavors or natural carbohydrates as additional ingredients such as ordinary drinks . Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above . The ratio of the above-mentioned natural carbohydrate can be appropriately determined by a person skilled in the art.

In addition to the above, the health functional food composition of the present invention can be used as a nutritional supplement, a vitamin, a mineral (electrolyte), a flavoring agent such as a synthetic flavoring agent and a natural flavoring agent, a coloring agent and a thickening agent (cheese, chocolate, Alginic acid and its salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks and the like. These components may be used independently or in combination. The ratios of these additives can also be appropriately selected by those skilled in the art.

The present invention provides a pharmaceutical composition for preventing or treating prostate disease, which comprises an extract of Aspergillus oryzae as an active ingredient.

The present invention also provides a health functional food composition for preventing or ameliorating a prostate disease, which comprises a tsukuba leaf extract as an active ingredient.

In the present invention, the tsukiba leaf extract can be extracted using a conventional solvent known in the art for extracting an extract from a natural product, that is, under ordinary temperature and pressure conditions. For example, in the present invention, the woody plant extract of water is selected from the group consisting of water, an anhydrous or a lower alcohol having 1-4 carbons (methanol, ethanol, propanol, butanol), acetone, ethyl acetate, butyl acetate, dichloromethane (CH ₂ Cl ₂ ) But are not limited to, solvents selected from the group consisting of chloroform, hexane and 1,3-butylene glycol. In addition, the method of extracting the extract from the white mold can be extracted through various methods such as hot water extraction, cold extraction, reflux extraction, and ultrasonic extraction, but is not limited thereto.

The present invention also includes fractions obtained by further purifying and extracting the extract of the present invention. The additional separation is preferably carried out by performing column chromatography, more preferably by size-exclusion chromatography using liquid column chromatography, ion-exchange chromatography, partition chromatography, affinity chromatography but are not limited to, affinity chromatography or a combination of these chromatography.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

Example  One. A skeptical tree  From the extract dimeric ellagitannins  Compound separation

A cobbler japonica ) leaves were extracted with 80% acetone three times at room temperature, filtered and concentrated under reduced pressure to obtain 430 g of the extract. The extract was then suspended in H ₂ O and then filtered through Celite to separate the water soluble layer (282.43 g) and the water insoluble layer (127.67 g) The water-soluble layer (282.43 g) was divided into 8 fractions by Sephadex LH-20 column chromatography (0 → 100% MeOH, gradient system). These fractions showed excellent antioxidant activity. 7 (27.25 g) and 8 (57.43 g) were subjected to YMC-gel ODS-A (40% → 100% MeOH, gradient system) and Sephadex LH-20 column chromatography (20% → 100% MeOH, gradient system) , Compound 1 (5.5 g) was obtained through repeated purification using MCI-gel CHP-20P (0 → 100% MeOH, gradient system).

Example  2. Identification of compound structure

¹ H and ¹³ C-NMR spectra of the compound 1 obtained in Example 1 were measured, and the results are as follows.

(1) Compound 1: Camptothin A (1) (Amorphous yellowish white powder)

¹ H-NMR (600 MHz, Acetone- d ₆ + D ₂ O): δ _H 7.03-7.09 (each s, val-H _C H-each of four form), 6.95, 6.92, 6.91, 6.91 (2H, each s, gal-H of each four form), 7.02, 7.01, 6.97, 6.67, 6.67, 6.64, 6.64 ( each s, HHDP-H of each four form), 6.64, 6.63, 6.62, 6.62 (each s, val-H A (each s, HHDP-H of each four form), 6.24, 6.24, 6.21, 6.21 (each s, val-H _B of each four form), 5.78, 5.77 (each t, J = 10.2 Hz, H-3 L of α - α, α - β form), 5.46, 5.41 (each t, J = 10.2 Hz, H-3 L of β - α, β - β form ), 5.36, 5.35 (each t, J = 10.2 Hz, H-3 R of α - α, β - α form), 5.26, 5.24 (each t, J = 3.6 Hz, H _{-1 R of α - α, β} - α form), 5.24-5.22 (each t, J = 3.6 Hz, H-3 R of α - β, β - β form), 5.22, 5.20 (each t, J = 3.6 Hz, H-1 _L of, α - α , α - β form), 5.26-5.20, 5.23-5.18 (complicated peaks, H-6a _L and H-6a _R (each dd, J = 7.8, 9.6 Hz, H-2 _L of each four form), 5.09-5.00 (complicated peaks, H-4 _L of each four form), 4.93-4.83 (each t, J = 10.2 Hz , H-4 R of each four form), 4.85, 4.71 (each d, J = 7.8 Hz, H-1 R of α - β, β - β form), 4.85, 4.73, 4.58, 4.51 (each m, H -5 L of each four form), 4.48, 4.44 (each d, J = 7.8 Hz, H-1 L of β - α, β - β form), 4.38, 4.21, 4.16, 4.05 (each m, H-5 R of each four form), 3.86-3.71 (each dd, J = 1.2, 12.0 Hz, H-6b L and H-6b _R of each four form), 3.58, 3.53 (each dd, J = 7.8, 9.0 Hz, H-2 _R of each four form).

^{13 C-NMR (150 MHz,} Acetone- d 6 + D 2 O): δ C 168.0-166.9 (HHDPC-7, HHDPC-7 ', valC-7, valC-7', valC-7 "of each four form ), 166.2-163.9 (gal C-7, gal C-7 'of each four form), 145.7-146.6 (val C- val C-6, val C-6 ', val C-5, gal C-5', HHDP C-4, HHDP C-4 ', HHDP C- of each four form), 142.5-142.2 (val C-5 "of each four form), 139.5-139.0 (val C-3 ", val C- val C-5 ', HHDP C-5, HHPD C-5' of each four form), 125.6 (val C- Of each four form), 120.5-119.7 (gal C-1, gal C-1 'of each four form), -124.9 (val C-2, val C-2', HHDP C-2, HHDP C- (Val C-1, val C-1, HHDP C-1, HHDP C-1 'of each four form), 109.4-109.0 val C-6 ", gal C-2, gal C-2 ', gal C-6, gal C-6'of each four form), 107.2-106.7 (HHDP C-3, HHDP C- -3 of each four form), 105.1-104.3 (val C-3 'of each four f orm), 97.7-95.3 (C-1 L of β - α form, C-1 L of β - β form, C-1 R of α - β form, C-1 R of β - β form), 93.0- _{90.0 (C-1 L of α} - β form, C-1 L of α - α form, C-1 R of β - α form, C-1 R of α - α form), 75.0-73.4 (C-2 _{L of β - α form, C} -2 L of β - β form, C-2 L of α - β form, C-2 L of α - α form, C-3 L of β - β form, C-3 of β-form α _L), 73.2-71.2 _(C-R 3 of α - β form, _C-R 3 of β - β form, C-3 _L of α - β form, C-3 _L of α-α form), 71.2-70.2 (C-3 R of β - α form, C-3 R of α - α form, C-2 R of α - β form, C-2 R of β - β form, C-2 _{R of β - α form, C} -2 R of α - α form, C-4 L of β - α form, C-4 L of β - β form, C-4 R of α - β form, C-4 _{R of β - β form, C} -4 L of α - β form, C-4 L of α - α form, C-4 R of β - α form, C-4 R of α - α form), 66.3- _{66.1 (C-5 L of β} - α form, C-5 L of β - β form, C-5 R of α - β form, C-5 R of β - β form, C-5 L of α - β form, C-5 _L of α - α form, C-5 _R of β - α form, C-5 _R of α - α form), 63.4-6 _{2.7 (C-6 L of β} - α form, C-6 L of β - β form, C-6 R of α - β form, C-6 R of β - β form, C-6 L of α - β form, C-6 _L of α - α form, C-6 _R of β - α form, C-6 _R of α - α form).

Based on the above results, the structure of the compound 1 obtained in Example 1 is shown in Fig.

Example  3. DPPH Radical Scarce  Measure

3-1. Experimental Method

20 μl of each sample (12.5-100 ug / ml of Camellia sinensis leaf extract, 1.5625-100 μM of camptothin A) was added to 180 μl of DPPH (200 mM) solution for 30 minutes and the absorbance was measured at 518 nm. The DPPH radical scavenging activity of each sample was determined by [1- (sampleO.D.-blankO.D.) / (ControlO.D.-blankO.D.)] X 100.

3-2. A skeptical tree  Of leaf extract Radical Scatters  Confirm

Bijjugi leaf extract IC ₅₀ values showed excellent DPPH radical scavenging activity by about 20㎍ / ㎖ (see Table 1).

3-3. camptothin  Of A Radical Scatters  Confirm

Example Compound 1 obtained in the camptothin A 1 showed excellent DPPH radical scavenging activity, in particular, IC ₅₀ values were excellent DPPH radical scavenging activity as from about 3.82 uM to about 5 times that of vitamin C 17.51 uM (see Table 2).

Example  4. Superoxide Radical Scarce  Measure

4-1. Experimental Method

20 μl of each concentration sample (12.5 μl of bismuth-tree leaf extract, 12.5 μl of each sample) was added to 160 μl of a mixture solution containing phosphate buffer (50 mM), EDTA (0.05 mM), NBT (1 mM), and hypoxanthine 20 μl of xanthine oxidase (1.2 U / μl) was added thereto, and the mixture was allowed to stand at 37 ° C for 10 minutes, and the absorbance at 590 nm was measured. The superoxide radical scavenging activity of each sample was determined by [1- (sampleO.D.-blankO.D.) / (ControlO.D.-blankO.D.)] X 100.

4-2. A skeptical tree  Of leaf extract Radical Scatters  Confirm

The IC ₅₀ value of the extract from the leaves of Bismuthia japonica was 39.10 ㎍ / ㎖, which showed about twice the superoxide radical scavenging ability (see Table 1) when compared to vitamin C 78.30 ㎍ / ㎖.

4-3. camptothin  Of A Radical Scatters  Confirm

The IC ₅₀ value of compound 1 camptothin A obtained in Example 1 was 2.40 uM and showed superoxide radical scavenging ability (see Table 2) 20 times or more as compared to 44.48 uM vitamin C.

The IC ₅₀ values of the DPPH radical scavenging activity and the superoxide radical scavenging activity measured on the leaves of the sickle leaves are shown in Table 1 below.

sample

DPPH radical scavenging ability (占 퐂 / ml)
Superoxide radacal scavenging ability (占 퐂 / ml)
Tibetan leaf extract

20.93 ± 1.18
39.10 ± 1.00
Vitamin C

5.99 ± 0.16
78.30 ± 2.24

Table 2 shows the IC ₅₀ values of DPPH radical scavenging activity and superoxide radical scavenging activity measured for camptothin A, which is Compound 1 obtained in Example 1 of the present invention.

sample

DPPH radical scavenging ability ([mu] M)
Superoxide radar scavenging ability ([mu] M)
Camptothin

3.82 + 0.14
2.40 ± 0.05
Vitamin C

17.51 ± 0.31
44.48 ± 3.12

From the results of the above experiment, it was found that the compound of the formula (I) isolated from the extract and the compound of the present invention has excellent DPPH radical scavenging ability and superoxide radical scavenging ability.

Example  5. NO ( nitric oxide ) Production inhibition efficacy evaluation

5-1. Experimental Method

In order to evaluate the nitric oxide (NO) production inhibitory effect of Compound 1 isolated and identified through Examples 1 and 2, the IC ₅₀ value of NO production inhibitory activity was measured as follows. In RAW 264.7 macrophage cells, LPS (lipopolysaccharide) was used to express NO synthase enzyme and the amount of NO produced was measured by Griess method. More specifically, RAW 264.7 macrophage cells were cultured in DMEM at a concentration of 5 × 10 ⁴ per 1 ml of medium, diluted to 1 × 10 ⁴ per 1 ml, added to 96 wells, and incubated for 2 hours, 20 μl of each sample (12.5-100 ug / ml of bismuth-tree leaf extract, 3.125-100 μM of camptothin A) containing 20 μl of LPS (1 μg / ml) and L-NMMA (N-Methylarginine) After incubation for 20 hours, the amount of NO produced in the culture was quantitated using a Griess reagent.

The NO production inhibitory activity IC ₅₀ value of each sample was determined as [1- (sample OD-blank OD) / (control OD-blank OD)] X 100.

5-2. NO  ( nitric oxide ) Production inhibition

The inhibitory nitric oxide (NO) production IC ₅₀ values of Compound 1, which was isolated from the leaves of the present invention, are shown in Table 3 below.

sample

NO production inhibitory activity (占 퐂 / ml)
NO production inhibitory activity ([mu] M)
Tibetan leaf extract

41.96 ± 1.50
-
Camptothin

-
3.94 ± 0.06
L-NMMA

12.5>
12.5>

From the results of the above experiment, it was confirmed that the compound of the present invention has an anti-inflammatory effect, and camptothin A has an excellent anti-inflammatory activity.

Example  6. Evaluation of cell proliferation inhibitory activity

6-1. Experimental Method

In order to evaluate the ability of Compound 1 isolated and identified in Examples 1 and 2 to inhibit prostate tumor cell proliferation, the following experiment was conducted.

That is, the cell proliferation inhibitory activity in PC-3 cells, which are prostate tumor cells by the respective test substances (bismuth-tree leaf extract and Compound 1), was measured by MTT [3- (4,5-dimethylthiazol-2- -diphenyltetrazolium bromide] assay. More specifically, PC-3 was added to a 24-well plate at a density of 10 ⁵ / well and cultured for 48 hours to allow cells to adhere. Then, each test material (12.5-100 ug / ml of Camellia sinensis leaf extract, ~ 100 uM). After 48 hours, the morphological changes were observed using an electron microscope. After removing the culture medium, PBS containing 0.5 mg / ml MTT was added, followed by reaction for 4 hours. Finally, MTT-formazan DMSO and absorbance was measured at 540 nm. The tumor cell proliferation inhibition IC ₅₀ value of each sample was determined by [(sampleO.D.-blankO.D.) / (ControlO.D .-- blankO.D.)] X 100.

6-2. Identification of cell proliferation inhibitory activity

The IC ₅₀ values of the hibiscus leaf extract and Compound 1 isolated therefrom are shown in Table 4 below.

sample
Prostate cancer proliferation inhibiting efficacy
(占 퐂 / ml)
Prostate cancer proliferation inhibiting efficacy
(μM)
Tibetan leaf extract
41.96 ± 1.50

-
Camptothin

-
3.94 ± 0.06

From the results of the above experiment, it was found that the compound of the present invention and the compound 1 isolated therefrom have excellent prostate cancer proliferation inhibitory activity.

Example  7. PC -3 cell Using Flow cytometry

7-1. Experimental Method

Flow cytometry was performed using Annexin V / PI double staining to determine cell proliferation inhibition in PC-3 cells. Cells treated with 48h of each test substance were washed with PBS and treated with trypsin. Annexin V-FITC and PI dissolved in binding buffer were sequentially reacted for 15 minutes each time, and flowcytometry (BD-LSR II, San Jose, USA).

7-2. Of flow cytometry  result

As shown in FIG. 3, Compound 1 strongly inhibited proliferation of PC-3 cells, which are hormone-independent prostate tumor cells, and it was confirmed that apoptosis was effectively induced.

Example  8. Cells apoptosis  relation protein  Expression pattern evaluation

8-1. Experimental Method

Western blot and immunofluorescence staining were performed to evaluate the proteins involved in the apoptosis of Compound 1 in PC-3 cells. More specifically, cells treated with Compound 1 at 0, 24, 36, and 48 h were washed with PBS, harvested by treatment with trypsin, and then subjected to western blotting to obtain pro-apoptotic protein (Bak, Bax) (Bcl-2, Bcl-xL), cytochrome c, cleaved caspase-3, cleaved caspase-9 and cleaved PARP.

8-2. protein  Expression pattern

As shown in Fig. 4 and Fig. 5, Bak expression was increased, bax expression was decreased, and mitochondrial expression was increased. Expression of anti-apoptotic proteins, Bcl-2 and Bcl-xL, was decreased, cytochrome c expression was increased, but mitochondrial expression was decreased. In addition, the expression of cleaved caspase-3, cleaved caspase-9 and cleaved PARP was increased.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (10)

A compound represented by the following formula (1):
[Chemical Formula 1]

A pharmaceutical composition for preventing or treating prostate cancer, which comprises the compound of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
3. The composition according to claim 2, wherein the compound is isolated from a hibiscus leaf extract.
delete 3. The composition of claim 2, wherein the composition has nitric oxide (NO) production inhibitory activity.
A health functional food composition for preventing or improving prostate cancer, which comprises the compound of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
A pharmaceutical composition for prevention or treatment of prostate cancer, which comprises an extract of Aspergillus oryzae as an active ingredient.
delete 8. The composition of claim 7, wherein the composition has nitric oxide (NO) production inhibitory activity.
A health functional food composition for prevention or amelioration of prostate cancer, which comprises an extract of Aspergillus oryzae as an active ingredient.

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