KR101327318B1 - Pharmaceutical composition for treating cancers comprising resveratrol derivatives isolated from the seeds of iris pseudacorus - Google Patents

Abstract

Yellow iris of the present invention ( Iris) pseudacorus seed extract and any one selected from the group consisting of irisferin A, irisferin B, irisferin C, and mixtures thereof, which are resveratrol compounds isolated and purified therefrom. The present invention relates to an anticancer composition containing an acceptable carrier, and the composition of the present invention can be used as a prophylactic and therapeutic agent for various malignancies such as lung cancer, ovarian cancer, melanoma, central nervous system cancer, colon cancer and leukemia.

Description

An anticancer composition comprising a resveratrol derivative compound isolated from a yellow iris seed, the present invention relates to a cancer drug composition.

The present invention has an anti-cancer effect, an organic solvent extract of yellow iris seeds and the active substance irisferin A (irisferin A), irisferin B (irisferin B), and irisferin C that are purified from the active ingredient It relates to an anticancer agent composition containing.

Yellow Iris pseudacorus) is a plant native to Europe and belongs to the lily and iris neck thereby relieve the symptoms of swelling in the body diuretic yellow roots and stems of iris may be effective in increasing abdominal distension and abdominal pain (primary Korea Prime Encyclopedia). In civilians, they are crushed on bruises.

The chemical composition of the yellow iris is a flavonoid, and quinones, triterpenoids, and the like have been reported ( Chem . Ind . 1975, 8, 349, and Liebigs Ann . Chem 1990, 563).

However, compounds such as irisferin A, irisferin B and irisferin C isolated from yellow iris seeds exhibit potent cell proliferation inhibitory effects against human cancer cell lines. It has not been reported yet that it is useful.

Primary color book of Korea Chem. Ind. 1975, 8, 349 Liebigs Ann. Chem 1990, 563

It is an object of the present invention to provide an anticancer composition comprising a compound isolated from a natural product.

In order to achieve the above object, the present invention is a yellow flower comprising a compound selected from the group consisting of iris perrin A, iris perrin B, iris perrin C, pharmaceutically acceptable salts and isomers thereof, and mixtures thereof It provides a pharmaceutical composition for the prevention and treatment of cancer containing iris seed extract as an active ingredient.

Since the composition of the present invention exhibits an excellent cell growth inhibitory effect on human-derived cancer cell lines, it can be usefully used for the prevention and treatment of various cancers.

Hereinafter, the present invention will be described in detail.

The pharmaceutical composition according to the present invention is a yellow iris seed extract comprising a compound selected from the group consisting of iris perrin A, iris perrin B, iris perrin C, pharmaceutically acceptable salts and isomers thereof, and mixtures thereof. Pharmaceutical composition for the prevention or treatment of cancer comprising as an active ingredient, A549 (human lung cancer cell line), SK-OV-3 (human ovarian cancer cell line), SK-MEL-2 (human melanoma cell line), XF498 ( It has an excellent cell proliferation inhibitory effect on cancer cell lines of human origin such as CNS cancer cell line) and HCT-15 (human colon cancer cell line).

Yellow iris seed extract according to the present invention can be prepared by solvent extraction of yellow iris seeds or dried products thereof. Specifically, 2 to 200 times the volume of the yellow iris seeds, preferably 10 to 30 times the organic solvent, is added to the dried and cut yellow iris seeds, and at 10 to 30 ° C. for 1 to 20 days, Preferably, yellow iris seed extract can be prepared by extracting for 5 to 10 days and filtration and concentration under reduced pressure. In this case, a solvent selected from the group consisting of methanol, aqueous methanol solution, ethanol, ethanol aqueous solution, butanol, dichloromethane, ethyl acetate and mixtures thereof may be used as the extraction solvent, and 80-100% methanol or aqueous methanol solution is preferable. .

The yellow iris seed extract thus obtained may be reextracted with butanol, dichloromethane or ethyl acetate, and more preferably reextracted with ethyl acetate. According to a preferred embodiment of the present invention, yellow iris seeds or dried products thereof are cooled for 7 days using 100% methanol, filtered, and the filtrate is concentrated under reduced pressure to obtain a methanol extract, and the methanol extract is suspended in distilled water, followed by the same amount. The yellow iris seed extract can be obtained by fractionating with dichloromethane and distilling off the water layer with the same amount of ethyl acetate and concentrating under reduced pressure.

In the present invention, from the yellow iris seed extract, a specific active ingredient exhibiting a cell proliferation inhibitory effect on a human-derived cancer cell line can be separated and purified by chromatography. The chromatography is preferably carried out one or more times of reverse phase or silica gel column chromatography. Dichloromethane / methanol (50: 1 (v / v)-1: 1 (v / v)) or methanol / water (20% MeOH to 100% MeOH) can be used as the mobile phase. At this time, the solvent used is eluted and separated by a gradient gradient elution method that is sequentially raised from non-polar to polar, or from polar to non-polar, and measures the effect of inhibiting cell proliferation on human-derived cancer cell lines of the collected isolates. Active fractions can be obtained.

Irisferrin A of Formula 1, Irisferin B of Formula 2, which exhibits a cell proliferation inhibitory effect on human cancer cell lines by purifying the active fraction showing the cell proliferation inhibitory effect on human-derived cancer cell lines again by chromatography Irisferrin C of 3 can be obtained. In addition, if necessary, iris perrin A, iris perrin B, and iris perrin C can be chemically synthesize | combined.

The pharmaceutically acceptable salts include, for example, hydrochloride, hydrobromide, sulfate, phosphate, acetate, malate, fumarate, lactate, tartrate, citrate, gluconate, besylate and camsylate, and the like. It is not limited to this.

Types of cancer that can be prevented or treated by the composition of the present invention include lung cancer, ovarian cancer, melanoma, central nervous system cancer, colon cancer, leukemia, and the like, preferably lung cancer, ovarian cancer, melanoma, central nervous system Cancer and colon cancer.

Yellow iris seed extract, the iris perrin A, iris perrin B, iris perrin C or mixtures thereof as an active ingredient is mixed with a suitable pharmaceutically acceptable carrier or excipient according to a conventional method or diluted with a diluent. Pharmaceutical compositions having the above functions can be prepared.

Examples of suitable carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose , Microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. The pharmaceutical composition may further include fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives and the like.

The pharmaceutical compositions of the present invention may be formulated using methods well known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. The formulations may be in the form of tablets, pills, powders, sachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powders and the like.

The pharmaceutical compositions of the invention can be administered via several routes including oral, transdermal, subcutaneous, intravenous or intramuscular. Typical daily dosages of the pharmaceutical compositions of the present invention are yellow iris seed extracts based on the active ingredient in the range of 10 to 100 mg / kg body weight, preferably 10 to 30 mg / kg body weight, and iris perrin A, iris perrin B, iris perrin C or mixtures thereof are in the range of 1 to 30 mg / kg body weight, preferably 1 to 10 mg / kg body weight, and can be administered once or in several divided doses. However, the actual dosage of the active ingredient should be determined in light of several relevant factors such as the route of administration, the age, sex and weight of the patient, and the severity of the disease, and therefore the dosage limits the scope of the invention in any aspect. It is not.

In addition, in the present invention, cancer prevention by inhibiting the growth of various cancer cells, including the yellow iris seed extract, or an active ingredient isolated therefrom, iris perrin A, iris perrin B, iris perrin C or a mixture thereof It provides a health promoting food or beverage composition that can be or alleviate.

Preferably, the cancer cells include lung cancer cells, ovarian cancer cells, melanoma cells, central nervous system cancer cells, and colon cancer cells.

The yellow iris seed extract of the present invention, the active ingredient or a mixture of the active ingredient may be added to exhibit the above effects, for example, various foods, beverages, snacks, confectionery, gums, ice cream , Tea bags, instant teas, granules, flavorings, vitamin complexes, and other health supplements, but are not limited thereto.

Yellow iris seed extract of the present invention, the active ingredient or a mixture of the active ingredient separated therefrom may be added to the raw material during food preparation or properly mixed with the cooked food to produce the above-mentioned food or beverage for health promotion, In this case, the content of the yellow iris seed extract, the active ingredient or a mixture of the active ingredient in the finally prepared food or drink may be in the range of 0.01 to 30% by weight, respectively.

The pharmaceutical composition, or health food or beverage of the present invention may further include other medicinal herbs or extracts thereof that are pharmaceutically acceptable to enhance or supplement the desired effect, and representative examples of such herbal medicines are pagoji, Happiness, green bean sprouts, lotus root meat and cloves. The herbal medicine may be used in an amount of 0.01 to 50% by weight based on the total weight of the composition.

Hereinafter, the present invention will be described in more detail based on the following examples. However, the following examples are not intended to limit the invention only.

Example

Example  1: Preparation of Yellow Iris Seed Extract and Isolation of Active Ingredients

10 kg of dried yellow iris seeds were cooled to 50 L of methanol at room temperature for 1 week, filtered and the filtrate was concentrated under reduced pressure to obtain 708.8 g of methanol extract. The methanol extract was suspended in 10 L of distilled water and extracted with the same amount of dichloromethane. The water layer was reextracted with the same amount of ethyl acetate and the extract was concentrated under reduced pressure to obtain 335 g of ethyl acetate reextract, and the remaining water layer was freeze-dried.

335 g of the ethyl acetate re-extract was mixed with dichloromethane / methanol as a mobile phase at a flow rate of 50 ml / min, and the mixing ratio of dichloromethane / methanol was 50: 1 (v / v) to 1: 1 (v). Silica gel column chromatography of concentration gradient elution method to raise sequentially to / v) was divided into a total of nine fractions (first fraction to ninth fraction). The second fraction was subjected to silica gel column chromatography again (dichloromethane / methanol 40: 1 (v / v)-1: 1 (v / v) as a mobile phase, flow rate 5 ml / min) to give irisperin A, Irisferin B and Irisferin C were isolated. The structure was confirmed by comparing the spectroscopic data of these ¹ H-NMR, ¹³ C-NMR, MASS and the literature.

< Irisferrin  A>

¹ H-NMR (800 MHz, MeOH- d4 ) δ: 0.88 (3H, t, J = 7.2 Hz), 1.05 (3H, m), 1.09 (3H, m), 1.19 (2H, m), 1.92 (2H , m), 2.00 (3H, m), 2.15 (1H, m), 2.26 (1H, m), 3.85 (3H, s), 4.33 (1H, d, J = 5.5 Hz), 5.29 (1H, m) , 5.29 (1H, m), 5.39 (1H, d, J = 5.5 Hz), 6.11 (1H, s), 6.13 (2H, d, J = 2.2 Hz), 6.18 (1H, t, J = 2.2 Hz) , 6.29 (1H, d, J = 16.4 Hz), 6.34 (1H, d, J = 16.4 Hz), 6.39 (1H, s), 6.59 (2H, d, J = 8.7 Hz), 6.77 (2H, d, J = 8.7 Hz), 6.85 (2H, d, J = 8.7 Hz), 7.17 (2H, d, J = 8.7 Hz)

¹³ C-NMR (200 MHz, MeOH- d4 ) δ: 134.1, 128.1, 116.3, 158.6, 116.3, 128.1, 94.7, 59.0, 147.6, 107.2, 160.3, 102.2, 160.3, 107.2, 130.1, 128.6, 116.4, 156.9, 116.4, 128.6, 134.8, 123.1, 136.5, 118.7, 162.8, 96.6, 160.4, 114.0, 184.0, 160.5, 108.5, 189.0, 114.4, 147.0, 57.1, 28.7, 28.6, 30.2, 30.4, 30.8, 30.6, 30.4, 31.0, 28.3, 131.0, 131.0, 28.3, 30.9, 30.1, 33.1, 23.9, 14.6

< Irisferrin  B>

¹ H-NMR (800 MHz, MeOH- d4 ) δ: 0.88 (3H, t, J = 7.2 Hz), 1.05 (3H, m), 1.09 (3H, m), 1.19 (2H, m), 1.92 (2H , m), 2.00 (3H, m), 2.15 (1H, m), 2.26 (1H, m), 3.85 (3H, s), 4.33 (1H, d, J = 5.5 Hz), 5.29 (1H, m) , 5.29 (1H, m), 5.39 (1H, d, J = 5.5 Hz), 6.11 (1H, s), 6.13 (2H, d, J = 2.2 Hz), 6.18 (1H, t, J = 2.2 Hz) , 6.29 (1H, d, J = 16.4 Hz), 6.34 (1H, d, J = 16.4 Hz), 6.39 (1H, s), 6.59 (2H, d, J = 8.7 Hz), 6.77 (2H, d, J = 8.7 Hz), 6.85 (2H, d, J = 8.7 Hz), 7.17 (2H, d, J = 8.7 Hz)

¹³ C-NMR (200 MHz, MeOH- d4 ) δ: 134.1, 128.1, 116.3, 158.6, 116.3, 128.1, 94.7, 59.0, 147.6, 107.2, 160.3, 102.2, 160.3, 107.2, 130.1, 128.6, 116.4, 156.9, 116.4, 128.6, 134.8, 123.1, 136.5, 118.7, 162.8, 96.6, 160.4, 114.0, 184.0, 160.5, 108.5, 189.0, 114.4, 147.0, 57.1, 28.7, 28.6, 30.2, 30.4, 30.6, 30.4, 28.3, 131.0, 131.0, 28.3, 30.9, 30.1, 33.1, 23.9, 14.6

< Irisferrin  C>

¹ H-NMR (800 MHz, MeOH- d4 ) δ: 0.87 (3H, t, J = 7.2 Hz), 1.09 (2H, m), 1.15 (2H, m), 1.17 (6H, m), 1.25 (2H , m), 1.30 (4H, m), 1.32 (4H, m), 1.96 (2H, m), 2.00 (2H, m), 2.26 (2H, m), 2.28 (2H, m), 3.84 (3H, s), 4.46 (1H, d, J = 5.6 Hz), 5.29 (1H, m), 5.31 (1H, m), 5.38 (1H, d, J = 5.6 Hz), 6.07 (1H, s), 6.11 ( 2H, d, J = 2.0 Hz), 6.14 (1H, t, J = 1.6 Hz), 6.32 (1H, d, J = 16.4 Hz), 6.37 (1H, d, J = 16.4 Hz), 6.41 (1H, s), 6.58 (2H, d, J = 8.8 Hz), 6.77 (2H, d, J = 8.0 Hz), 6.84 (2H, d, J = 8.0 Hz), 7.16 (2H, d, J = 8.8 Hz)

¹³ C-NMR (200 MHz, MeOH- d4 ) δ: 134.1, 128.1, 116.3, 158.6, 116.3, 128.1, 94.7, 59.0, 147.6, 107.2, 160.3, 102.2, 160.3, 107.2, 130.1, 128.6, 116.4, 156.9, 116.4, 128.6, 134.8, 123.1, 136.5, 118.7, 162.8, 96.6, 160.4, 114.0, 184.0, 160.5, 108.5, 189.0, 114.4, 147.0, 57.1, 28.7, 28.6, 30.2, 30.4, 30.8, 30.6, 30.4, 31.0, 28.3, 131.0, 131.0, 28.3, 30.9, 30.1, 33.1, 23.9, 14.6

Test Example  1: Yellow-flowered Iris Seed Extract and Human Derivatives On cancer cell lines  Cell proliferation inhibitory effect test

The effect of inhibiting cell proliferation on the human-derived cancer cell lines of methanol extract and ethyl acetate re-extract, iris perrin A, iris perrin B, and iris perrin C of yellow iris seed obtained in Example 1 was measured.

A549 (human lung cancer cell line), SK-OV-3 (human ovarian cancer cell line), SK-MEL-2 (human melanoma cell line), XF498 (central nervous system cancer cell line), and HCT-15 (human colon cancer cell line) The cancer cell line of human origin of the species was distributed from the US National Cancer Research Center (NCI) and used for subculture, and the inhibition of cancer cell proliferation is the most widely used method to measure the primary anticancer effect of drugs in the US NCI and various other testing institutions. It was measured according to the sulfrhodamine B (SRB) method (Skehan, P. et al., (1990) J. Natl. Cancer. Inst., 82, 1107).

1) Passage cancer cells were treated with trypsin-CDTA solution to separate them from the container attachment surface, and the number of cells per well was 5 × 10 ³ (A549, XF498), 1 × 10 ^{4 in} 96-well flat bottom microplates (Falcon), respectively. Dilutions were made so that (SK-MEL-2) and 2 × 10 ⁴ (SKOV-3) were dispensed. The cells were incubated in a CO ₂ incubator for 24 hours to allow the cells to anchor to the bottom of the well, and then the medium was removed by an inhaler, and the sample dissolved in the medium was put in each well by concentration, and the culture was continued for 48 hours. .

2) After 48 hours of incubation, aspirate the medium in each well, remove 10% of 10% trichloroacetic acid (TCA) solution in each well, and leave the cells at room temperature for 1 hour to fix the cells. Washing was done several times to completely remove excess TCA solution and to dry.

3) 100 μl of SRB staining solution (0.4% sulforhodamine in 1% acetic acid) was added to each well for 30 minutes, and the excess dye solution was repeatedly washed 5 to 6 times with 1% acetic acid and dried at room temperature. .

4) Add 100 μl of 10 mM Trisma base (unbuffered) solution to each well, and shake for 10 minutes with a titerplate shaker to elute the stained cells and microplate reader. Absorbance at 520 nm was measured using a microplate reader.

5) The cancer cell proliferation rate (% cell growth, inverse of anticancer activity) of the sample group into which the sample solution was added was calculated according to the following formula. That is, the number of cells (C), the number of cells (Tz) of zero time, and the number of cells (T) of the sample group, in which the same amount of medium was added instead of the sample solution, were converted from the absorbance values of each group, respectively. The cancer cell proliferation rate (% cell growth) of the sample group was calculated by Equation 1 for Tz> T and Equation 2 for Tz <T.

[Equation 1]

&Quot; (2) &quot;

6) Calculate the concentration (ED ₅₀ ) at which the sample inhibits the proliferation of the cancer cell by 50% using the data regression of the LOTUS program based on the cancer cell proliferation rate of the sample group measured at each concentration. It was.

As a result, 50% proliferation inhibitory concentration (ED ₅₀ ) for each cancer cell line obtained is shown in Table 1, and etoposide, which is widely used in clinical practice, was used as a control drug.

According to the method of the reference example, the methanol extract of the yellow iris seeds obtained in Example 1, the acetate re-extract of the yellow iris seeds, the inhibitory effect of the cancer cell proliferation of iris perrin A, iris perrin B, and iris perrin C 5, including A549 Species of human-derived cancer cell lines were tested for 50% proliferation inhibitory concentration (ED ₅₀ ) for each cancer cell line, and etoposide, which is widely used in clinical practice, was used as a reference drug.

As shown in Table 1, methanol extracts and ethyl acetate re-extracts of yellow iris seeds, and purified iris perrin A, iris perrin B, and iris perrin C all inhibited cell growth in a concentration-dependent manner against cancer cell lines of human origin. It can be expected to prevent and treat various cancers.

Formulation Example

For formulation, the extract of yellow iris seed obtained in Example 1 was used as it was or dried.

< Manufacturing example  1> Powder

The powders were prepared by mixing the following ingredients and then filling the airtight cloth according to a conventional powder preparation method:

Yellow Iris Seed Dry Extract

Or 2 g of active ingredient separated therefrom

Lactose 1 g

< Manufacturing example  2> tablets

The tablets were prepared by mixing the following ingredients and then tableting according to the conventional tablet preparation method:

Yellow Iris Seed Dry Extract

Or 100 mg of active ingredient separated therefrom

100 mg corn starch

Lactose 100 mg

2 mg of magnesium stearate

< Manufacturing example  3> Capsule

The capsules were prepared by mixing the following ingredients and filling the gelatin capsules according to a conventional capsule preparation method:

Yellow Iris Seed Dry Extract

Or 100 mg of active ingredient separated therefrom

100 mg corn starch

Lactose 100 mg

2 mg of magnesium stearate

< Manufacturing example  4> Injection

Injectables were prepared by dissolving the active ingredient in distilled water for injection and adjusting the pH to about 7.5 according to a conventional injection method, and then filling the 2 ml volume of the ampoule with sterilized distilled water and sterilizing the following remaining ingredients:

Yellow Iris Seed Dry Extract

Or 100 mg of active ingredient separated therefrom

Suitable amount of distilled water for injection

pH adjuster

< Manufacturing example  5> Wire

Brown rice, barley, glutinous rice, and yulmu were alphad by a known method, and then dried and roasted to make a powder having a particle size of 60 mesh. Black beans, black sesame seeds, and perilla were also steamed and dried in a known manner, and then ground to a powder having a particle size of 60 mesh.

The yellow iris seed seed extract of the present invention was concentrated under reduced pressure in a vacuum concentrator, and the dried product obtained by drying with a spray hot air dryer was pulverized with a particle size of 60 mesh to obtain a yellow iris seed extract dry powder.

Granules were prepared by combining the dry powder of the grains, seeds and yellow iris seed extract prepared above in the following proportions.

Cereals: Brown rice 30% by weight, barley 15% by weight, barley 20% by weight, glutinous rice 9% by weight,

Seeds: perilla 7% by weight, black beans 8% by weight, black sesame 7% by weight,

Yellow iris seed extract dry powder 3% by weight, ganoderma lucidum 0.5% by weight, turmeric 0.5% by weight

< Manufacturing example  6> Chewing gum

Chewing gum was prepared in a conventional manner by combining 20% by weight of gum base, 76.9% by weight of sugar, 1% by weight of perfume, and 2% by weight of water, and 0.1% by weight of yellow iris seed extract of the present invention.

< Manufacturing example  7> candy

Candy was prepared by the conventional method by combining 60% by weight of sugar, 39.8% by weight of starch syrup, and 0.1% by weight of perfume and 0.1% by weight of yellow iris seed extract of the present invention.

< Manufacturing example  8> Biscuits

Force 25.59% by weight, 22.22% by weight of gravity, 4.80% by weight, white salt 0.73% by weight, 0.78% by weight, palm shortening 11.78% by weight, ammonium 1.54% by weight, 0.17% by weight sodium bisulfite 0.16% by weight , Rice flour 1.45%, Vitamin B₁0.0001%, Vitamin B₂0.0001%, Milk flavor 0.04%, Water 20.6998%, Whole milk powder 1.16%, Substitute milk powder 0.29%, Monobasic calcium phosphate 0.03% , Biscuits were prepared by combining 0.29 wt% of spray salt and 7.27 wt% of spray oil with 1 wt% of yellow iris seed extract of the present invention.

< Manufacturing example  9> health drink

0.26% by weight of honey, 0.0002% by weight of thioctoamide, 0.0004% by weight of nicotinic acid, 0.0001% by weight of sodium riboflavinate, 0.0001% by weight of pyridoxine hydrochloride, 0.001% by weight of inositol, 0.002% by weight of orthoic acid and 98.7362% by weight of water Yellow iris seeds 1% by weight of blended to prepare a health drink in a conventional manner.

Preparation Example 10 Health Supplement

55% by weight of spirulina, 10% by weight of guar gum enzyme, 0.01% by weight of vitamin B ₁ hydrochloride, 0.01% by weight of vitamin B6 hydrochloride, 0.23% by weight of DL-methionine, 0.7% by weight of magnesium stearate, 22.2% by weight of lactose and 1.85% by weight of corn starch And blended 10% by weight of the yellow iris seed extract of the present invention to prepare a tablet-type health supplement food in a conventional manner.

Claims (8)

Containing as an active ingredient a compound selected from the group consisting of irisferin A, irisferin B, irisferin C, pharmaceutically acceptable salts thereof, and mixtures thereof A pharmaceutical composition for treating cancer selected from the group consisting of lung cancer, ovarian cancer, melanoma, central nervous system cancer, and colon cancer.
delete delete It contains a compound selected from the group consisting of iris perrin A, iris perrin B, iris perrin C, pharmaceutically acceptable salts thereof, and mixtures thereof, lung cancer cells, ovarian cancer cells, melanoma cells, central nervous system cancer cells Health foods having a cancer cell growth inhibitory effect selected from the group consisting of, and colon cancer cells.
5. The method of claim 4,
Health foods, characterized in that the food is in the form of a drink.
delete delete delete