KR20090086817A - Liver toxicity disorder composition comprising an extract from the seed of opuntia ficus-indica var. saboten and compounds isolated therefrom - Google Patents

Abstract

A composition for preventing and treating hepatotoxic diseases, which contains Opuntia ficus-indica var. saboten seed extract or compound isolated from the extract is provided to have excellent liver protection effect and liver recovery effect in generation of hepatotoxicity. A composition for preventing and treating hepatotoxicity comprises an alcohol extract of Opuntia ficus-indica var. saboten seed as an active ingredient. A method for producing the alcohol extract of Opuntia ficus-indica var. saboten seed comprises: a step of adding 0.1-10 ‘± of alcohol solvent per 1kg of seed powder and performing reflux at 60-100‹C for two to six hours; and a step of filtering and compressing. The method further comprises a step of adding 1-5L of ethylacetate(EtOAc) to have fraction.

Description

LIFE TOXICITY DISORDER COMPOSITION COMPRISING AN EXTRACT FROM THE SEED OF OPUNTIA FICUS-INDICA VAR. SABOTEN AND COMPOUNDS ISOLATED THEREFROM}

The present invention relates to a composition for the prevention and treatment of hepatotoxic diseases, which contains a seed extract of palm cactus fruit or a compound isolated therefrom.

The liver is one of the most active organs in the human body, and is located between the digestive system and the systemic circulation system in the human body, and functions to protect the whole body from external in vitro substances. Once in vivo, the in vitro material is passed through the liver, so the liver is more at risk of being exposed to many toxic substances in addition to nutrients, which is more likely to be damaged. The liver is an organ with excellent regenerative capacity, and if there is a slight damage, the liver recovers to a normal level.However, if the damage persists, part of the liver tissue is completely destroyed and liver function is reduced. . In addition, our bodies are constantly exposed to pollutants and toxic substances due to industrialization, and our liver is constantly suffering from detoxification.In addition to substances that cause hepatotoxicity, mental stress, heavy drinking, smoking, etc. can increase liver damage. The body's defense and detoxification can cause abnormalities in the immune system, causing other diseases. Liver diseases are classified into viral liver disease, alcoholic liver disease, drug-toxic liver disease, fatty liver, autoimmune liver disease, metabolic liver disease and other liver diseases depending on the cause of the disease. Liver disease is not detected in the early stage of the disease, so it is found only to proceed quite a bit, but it is the cause of death not only in Korea but also in the world, but there is no effective treatment and diagnosis. Despite the severity of liver disease, there is no effective treatment method for liver disease. Therefore, there is an urgent need to develop drugs to treat and prevent liver damage while maintaining the structure and function of liver tissue.

Hepatoprotective agents extracted from natural plants and applied in clinical practice to date are isomers such as silybin, silydiamine and silycristine extracted from a plant called Silybum marianum . There is a silymarin formulation consisting of. As such, despite numerous efforts to develop liver disease therapeutics from natural products, only a handful of examples are currently in use or clinical trials. Thus, the present inventors investigated the liver protective activity using the seeds of palm cactus for the purpose of searching for a substance having a hepatoprotective effect and using it in food and medicine. It is known by the present inventors that the stem of the palm cactus has antioxidant activity, and flavonoid glycosides isolated from the palm cactus stem have been reported to effectively exhibit antioxidant activity [Saleem M. et. al., 2006, Phytochemistry 67: 1390; Lee EH et. al., 2003, Arch. Pharm. Res. 26: 1018; Dok-Go H. et. al., 2003, Brain Res. 965: 130. However, there are no reports on the hepatoprotective activity of palm cactus seeds.

Opuntia ficus-indica var.saboten Makino , which has been used in folklore for burns, edema, indigestion, boils and bronchial asthma, is a perennial plant belonging to the Cactaceae family native to the southern United States [Lee Young-no, 1998] , Korea Plant Book Pakhaksa p150]. Currently, it is being cultivated as a special crop in Jeju Island, and it is developed and marketed as a beauty product such as health supplement food and soap. Ethanol extracts of the fruit and stem of palm cactus have been known to have effects such as gastric mucosal protection, hypoglycemic action, and immune enhancing effect by analgesic and anti-inflammatory action. Recently, the inventors have found that the alcohol extract of palm cactus stem has antioxidant and neuroprotective effects, and has been applied for a patent application (Korean Patent Registration No. 523562). In addition, the radical scavenging activity of palm cactus, tyrosinase inhibitory activity involved in melanin biosynthesis, antiallergic activity [Lee Nam Ho et al. 2000, Journal of Pharmacy 44: 613].

However, there is no report on the hepatoprotective activity of the alcoholic extracts of palm cactus fruit seeds and the compounds isolated therefrom.

Thus, the inventors of the present invention, while studying a liver protective material for a variety of plants, it was confirmed that the material showing the liver protective effect in the cactus fruit of the palm. The alcohol extracts of palm cactus fruit seeds and the compounds obtained by separating and identifying the components contained in the extracts (Formulas 1 to 8) protect cytotoxicity after inducing toxicity of HepG2 cells, human liver cancer cell lines with t-BHP. As a result of searching for the effect, the present invention was found to have an excellent effect on the protection of the liver, and completed the present invention by confirming that it is a useful material for liver diseases such as hepatitis, fatty liver, cirrhosis and hepatotoxicity.

Accordingly, an object of the present invention is to provide a composition for preventing or treating hepatotoxic disease, which contains a seed extract of palm cactus fruit or compounds isolated therefrom (Formula 1 to 8) as an active ingredient.

The present invention is characterized by a composition for preventing or treating hepatotoxic disease, which contains a seed extract of palm cactus fruit or compounds isolated therefrom (Formulas 1 to 8) as an active ingredient.

The present invention is an alcohol extract of palm cactus fruit seeds, the compounds purified from the alcohol extract is purified and hepatotoxicity when the hepatotoxicity is induced by toxic substances such as t-BHP is excellent in the ability to protect the liver cells to prevent and treat liver disease You can expect an excellent effect.

The present invention will be described in more detail as follows.

The present invention relates to a composition for the prevention and treatment of hepatotoxic diseases, which contains extracts having a hepatoprotective effect obtained by extracting seeds of palm cactus with alcohol and compounds isolated therefrom (Equations 1 to 8) as an active ingredient.

Compounds of Formulas 1 to 8 of the present invention can be obtained by the following extraction and separation methods.

Remove the pulp from the palm cactus, wash it cleanly, crush the dried seeds with a grinder, degrease it with about 1 to 5 times hexane (n-Hexane), and then lower alcohols with 1 to 6 carbon atoms or 5 to 30%. Add to the alcohol solvent of 1 to 2 liters / kg into the extraction device and the ultrasonic extraction or reflux extraction at 60 ~ 100 ℃ for 1 to 24 hours. The alcohol solvent was filtered through a filter paper, and the resulting solution was concentrated and dried using a thickener to obtain a lower alcohol extract. The alcohol fraction was added to water, and then dichloromethane, ethyl acetate (EtOAc) and butanol (BuOH) were sequentially distributed. To prepare a dichloromethane fraction, ethyl acetate fraction, butanol fraction, the remaining water fraction. After obtaining the crude fraction of the obtained ethyl acetate fraction using Sephadex, the compounds of Chemical Formulas 1 to 8 may be obtained by using silica gel, reverse phase silica gel, and Sephadex.

Since the activity was the strongest in the ethyl acetate fraction, we tried to identify the structure by separating the component showing the liver protective effect in this fraction. Ethyl acetate fractions of palm cactus fruit seeds were separated using Sephadex, reversed phase column chromatography (RP-18), and the like. As a result, Americanol A (Formula 1), Isoaminol A (Formula 2), and iso Americanin A (Formula 3), 9′-O-methylisoaminol A (Formula 4), Americanin D (Formula 5), 3,3′-Bismethylmethylnorresinol (Formula 6), Princepin ( Formula 7), 8 compounds of iso-prinepin (Formula 8) could be separated.

In particular, these compounds were first isolated from palm cactus by the inventors.

Alcohol extracts obtained through the above process and the compounds purified from ethyl acetate fractions and ethyl acetate fractions fractionated therefrom induce toxicity of HepG2 cells, human liver cancer cell lines with t-BHP, and search for cytotoxic effects As a result, it was found that the hepatoprotective effect was excellent in the ethyl acetate fraction (Table 1), and the pure compounds showed an excellent effect on the hepatoprotective effect (Table 2). 9'-O-methylisoaminol A (Formula 4) of the isolated compound effectively inhibited the damage of hepatocytes, and its effect was superior to the cytotoxic protective effect than silicide or quercetin used as a reference drug. Americanol A (Formula 1), isoaminol A (Formula 2), isoaminine A (Formula 3), 9′-O-methylisoaminol A (Formula 4), americanin D (Formula 5), 3 Ethyl acetate fractions from 3'-bis-methyl-pineoresinol (Formula 6), Prispine (Formula 7), and isoprinesepin (Formula 8) from the seeds of palm cactus were expressed in hepG2 cells, a liver cancer cell line. BHP has been shown to be an active ingredient that plays an important role in suppressing the damage of hepatocytes induced by toxicity. Therefore, the present invention provides Americanol A (Formula 1), isoaminol A (Formula 2), isoaminine A (Formula 3), 9'-O-methylisoaminol A (Formula 4), Americanin D (Formula 4) 5), 3,3′-Bismethyl pinorresinol (Formula 6), Princepin (Formula 7), Isoprinsepin (Formula 8), mixtures thereof, and their large amounts of Opuntia ficus-indica var sabote n) The alcohol extracts or ethyl acetate fractions of fruit seeds are effective in the prevention and treatment of hepatotoxic diseases due to their good liver protection. That is, the extract and the compounds isolated therefrom prevent hepatotoxic diseases, specifically, drug liver damage, viral liver damage, hepatitis, liver cirrhosis, liver cancer, hepatic coma due to the protective effect of hepatotoxicity in HepG2 cells. And there is a therapeutic effect.

The pharmaceutical compositions of the present invention can be used to prepare pharmaceutical formulations according to conventional methods. In the preparation of the formulation, it is preferred that the active ingredient is mixed with the carrier, diluted with the carrier, or enclosed in a carrier in the form of a capsule, assay or other container. Accordingly, the pharmaceutical compositions of the present invention may be formulated in oral dosage forms such as tablets, pills, powders, assays, elixirs, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules. It may also be formulated in the form of a solution or suspension as an injectable formulation, or may be formulated in the form of an ointment, cream, gel or lotion as a transdermal formulation.

Examples of suitable carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, Propylhydroxybenzoate, talc, magnesium stearate and mineral oil. The formulation may further comprise fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives and the like. 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.

When administering the alcohol extract or ethyl acetate fraction of the present invention for clinical purposes, the total daily dose to be administered to an adult in a single dose or in a separate dose ranges from about 200 mg to 20 g, preferably 500 mg to 10 g. While sufficient, some diseases may require a higher or lower daily dosage. In addition, specific dosage levels for a particular patient may vary depending on the particular compound to be used, weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion, drug mixing and the severity of the disease.

In addition, when the pharmaceutical composition of the present invention is clinically administered to obtain a desired therapeutic effect on hepatotoxic disease, the alcoholic extract of palm cactus, and the compounds isolated therefrom, may be selected from one or more components of known hepatotoxic diseases. It may be mixed and administered at the same time.

Hereinafter, the present invention will be described in detail with reference to Examples, but the technical scope of the present invention is not limited to the following Examples.

Preparation Example 1 Preparation of Palm Cactus Fruit Extract, Separation and Purification of Active Ingredient

The pulp was removed from the palm cactus (Jeonnyeoncho) from Jeju-do, washed with water, and then pulverized with 9.44 kg of well-dried seeds, which were then degreased with n -hexane (12.5 L) to remove the fatty components of the seeds. The degreased seed powder was dried and then refluxed with MeOH (2.5 L) at 80 ° C. for 4 hours using a reflux apparatus. After cooling to room temperature, the solution filtered with filter paper was dried using a vacuum concentrator to obtain 224.6 g of methanol extract. This methanol extract was suspended in distilled water (1.0 L), followed by dichloromethane (CH ₂ Cl ₂ , 0.5 L × 3), ethyl acetate (EtOAc, 0.5 L × 3) and butanol (BuOH, 0.5 L × 3). The dichloromethane fraction, ethyl acetate fraction, butanol fraction, and the remaining water (H ₂ O) fraction were prepared.

Production Example  2: Palm cactus  Ethyl Acetate of Fruit Seeds From fractions  Isolation of Hepatocellular Protective Components

24.7 g of ethyl acetate fractions fractionated from alcohol extracts of palm cactus fruit seeds were divided into 14 subfractions by Sepadex LH-20 column chromatography using methanol as a developing solvent. Small fraction E9 (1.42 g) was subjected to column chromatography using silica gel. As the developing solvent, 14 small fractions were obtained using CH ₂ Cl ₂ : MeOH (5: 1) by increasing the amount of methanol from a mixed solvent of CH ₂ Cl ₂ : MeOH (11: 1). Small fraction E9d (34.0 mg) was separated using 40% CH ₃ CN as a developing solvent using preparative reversed-phase silica gel TLC to obtain 24.7 mg of isoaminein A (Formula 3). Small fraction E9f (153.7 mg) was subjected to column chromatography using reversed phase silica gel. The solvent used was obtained using a developing solvent of 40% acetonitrile (CH ₃ CN) to obtain 95.2 mg of 3,3'-bismethylmethyl pyrecinole (Formula 6).

Small fraction E10 (963.0 mg) was subjected to column chromatography using silica gel. Small fraction E10i (227.1 mg) obtained using a mixed solvent of CH ₂ Cl ₂ : MeOH (12: 1) as the developing solvent was separated using preparative HPLC (LiChrosorb RP-18 250-10, 7 μm). It was. Gradually increasing the amount of methanol from 35% MeOH and flowing it at a flow rate of 3.4 ml / min using 50% MeOH yielded 43.6 mg of Princecepin (Formula 7) and 65.7 mg of Isoprine Sepin (Formula 8), respectively. Could. Small fraction E7 (1.75 g) was subjected to column chromatography using reverse phase silica gel. The developing solvent was divided into 18 subfractions using 25% CH ₃ CN. Small fraction E7h (176.5 mg) was subjected to column chromatography using silica gel. CH ₂ Cl ₂ : MeOH (11: 1) was used as a developing solvent to obtain 73.3 mg of 3,3′-bismethylmethylpyrecinole (formula 6).

Small fraction E7i (133.7 mg) was subjected to column chromatography using silica gel (developing solvent-CH ₂ Cl ₂ : MeOH / 13: 1) and reversed phase silica gel (developing solvent-55% MeOH) to 4.9 mg of Americanin D ( Formula 5) was obtained purely.

Small fraction E7l (447.5 mg) was subjected to column chromatography using silica gel (CH ₂ Cl ₂ : MeOH / 13: 1) and preparative HPLC (LiChrosorb RP-18 250-10, 7 μm). The developing solvent was a gradient system of 30-70% CH ₃ CN, and the solvent was developed at a flow rate of 3.1 ml / min to form 11.7 mg of Americanol A (Formula 1) and 21.2 mg of isoaminol A. 17.5 mg of 9'-O-methylisoaminol A (Formula 4) were obtained, respectively, and 44.5 mg of isoaminine A (Formula 3) was further obtained.

Preparation Example 3 Structure Determination of Components Isolated from Ethyl Acetate Fraction of Palm Cactus Fruit Seeds

Physical properties and ¹ H-NMR (400 MHz) and ¹³ C-NMR (100 MHz) spectra of the components obtained in Preparation Example 2 were measured.

The chemical shift of each peak was expressed as a relative value of the chemical shift value (3.3 ppm, 49.8 ppm) of methanol as a measurement solvent, and the results are shown in Tables 1 to 3 below.

Compound 1 ( Americol A) : colorless compound, Mp 129 ° C., [α] ²² _D + 1.50 ° ( c 0.54, MeOH)

Positive-ion mode (ES-MS): m / z = 353 [M + Na] ⁺

[Formula 1]

Compound 2 ( isoaminol A): colorless compound, Mp 103-105 ° C., [α] ²² _D + 1.44 ° ( c 0.98, MeOH), ES-MS (positive-ion mode): m / z = 353 [ M + Na] ⁺

[Formula 2]

Compound 3 ( isoamine A): colorless compound, Mp 140 ° C., [α] ²² _D + 0.19 ° ( c 2.13, MeOH), ES-MS (positive-ion mode): m / z = 351 [M + Na] ⁺

[Formula 3]

Compound 4 (9'- O - methylisobutyl America play A): colorless _{^{compound, [α] 22 D + 2.57}} ° (c 0.70, MeOH), ES-MS (positive-ion mode): m / z = 367 [ M + Na] ⁺

[Formula 4]

Compound 5 ( American D): Colorless compound, Mp 162 ° C. (dec.), [Α] ²² _D + 2.76 ° ( c 0.15, MeOH), ES-MS (positive-ion mode): m / z = 351 [M + Na] ⁺

[Formula 5]

Compound 6 (3,3-bis-methyl-de Pinoresinol ): light yellow compound, [α] ²² _D + 0 ° ( c 0.44, MeOH), ES-MS (positive-ion mode): m / z = 353 [M + Na] ⁺

[Formula 6]

Compound 7 ( Princepine ): colorless compound, Mp 117 ° C, [α] ²² _D -1.61 ° ( c 1.74, MeOH)

Positive-ion mode (ES-MS): m / z = 517 [M + Na] ⁺

[Formula 7]

Compound 8 ( isoprincepine ): colorless compound, Mp 136-137 ° C., [α] ²² _D + 0.46 ° ( c 2.62, MeOH) ES-MS (positive-ion mode): m / z = 517 [M + Na] ⁺

[Formula 8]

As a result, in the present invention, the structure isolated from the ethyl acetate fraction of palm cactus fruit seeds is known Americanol A ( 1 ), isoaminol A ( 2 ), isoaminine A ( 3 ), 9′- O -methyl. Isomericol A ( 4 ) [Waibel R. et. al., 2003, Phytochemistry 62: 805, Americanin D ( 5 ) [Woo, WS et. al., 1980, Tetrahedron Lett . 21: 4258], 3,3'-bisdemethyl pinoresinol ( 6 ), princeepin ( 7 ), isoprinsepine ( 8 ) [Waibel R. et. al., 2003, Phytochemistry 62: 805].

Example 1: t -Induced hepatocyte damage repair by BHP

The alcohol extract extracted from the seed of palm cactus and the purely purified compound from ethyl acetate were dissolved in DMSO (dimethyl sulfoxide) to make a 10 mg / ml stock solution, which was diluted with medium and used at an appropriate concentration. It was. HepG2 cells, which are human hepatocytes, were stabilized by dispensing 2 × 10 ⁴ cells / well in 96-well plates and incubating for 24 hours. The cultured cells were treated with concentrations of palm cactus alcohol extracts and the compounds isolated therefrom for 2 hours, and treated with 200 μM of t- BHP to induce toxicity for 3 hours. MTT assay [Hansen MB et. al., 1989, J. Immunol . Methods 119: 119] were used.

After incubating the cells with MTT for 4 hours, the reagents were removed, and the resulting formazan was dissolved by adding DMSO to each well, and the absorbance was measured at 570 nm using a microplate reader. . Cell viability of the toxic treatment group and the sample treatment group was expressed as a percentage of the control group, with the cell survival rate of the control group without t-BHP and the sample being 100%. The results of three repeated experiments are shown in Tables 4 and 5 below. Respectively.

(continue)

As shown in Table 4, when the HepG2 cells were treated with 200 μM t-BHP, it was found that the cell viability was lowered to 47.84% compared to the control group, and the cytotoxicity of the solvent extracts extracted from the seeds of palm cactus fruit. It can be seen that by the concentration-dependent inhibition. In particular, the ethyl acetate fraction is much better than the silybin used as a control drug, and shows a protective effect similar to quercetin, which is well known as an antioxidant.

This suggests that the solvent extracts extracted from the seeds of palm cactus fruit have superior protection against hepatocellular damage due to toxicity.

(continue)

As shown in Table 5, HepG2 cells treated with 200 μM t-BHP lowered the cell viability was increased concentration-dependently by the compound 1 ~ 8 purified from the ethyl acetate fraction of the palm cactus fruit seeds. In particular, 9'-O-methylisoaminol A (Compound 4) shows a much better protective effect than the silybin used as a reference drug, and is superior to the protective effect of quercetin, which is known as an antioxidant. Therefore, it can be seen that the excellent hepatoprotective effect of the ethyl acetate fraction fractionated from the alcoholic extracts of the seeds of palm cactus fruit originated from these compounds, and these compounds were found to be useful as hepatocellular protective agents due to toxicity.

Example 2: Hepatocellular Necrosis Inhibition Effect by t-BHP

When the cells are necrosed by cytotoxicity, lactate dehydrogenase (LDH) is released, and its activity was measured as an indicator of cell necrosis.

HepG2 cells, which are human hepatocytes, were stabilized by dispensing 2 × 10 ⁴ cells / well in 96-well plates and incubating for 24 hours. The cultured cells were treated with concentrations of alcohol extracts and purely separated compounds extracted from the seeds of palm cactus fruits and incubated for 2 hours, and treated with 200 μM of t- BHP to induce toxicity for 3 hours. The amount of LDH released from the supernatant of the cultured cells was measured using a kit (Roche) [Chen Q. et. al., 1997, J. Biol . Chem . , 272: 14532. The results are shown in Table 6 and Table 7 after three repeated experiments.

(continue)

As shown in Table 6, when the 200 μM t-BHP treatment HepG2 cells, the amount of LDH outflow due to cell damage was increased to 56.07% compared to 11.20% of the control group. However, when the solvent extracts extracted from the seeds of palm cactus were treated, it was found that cell necrosis was suppressed and the amount of LHD leakage decreased in a concentration dependent manner. In particular, the ethyl acetate fraction showed a much better cell death suppression effect than the silicide used as a reference drug, and showed similar efficacy to the inhibitory effect of quercetin, which is well known as an antioxidant.

It was found that the solvent extracts extracted from the seeds of palm cactus fruit were excellent in inhibiting hepatocellular necrosis by toxicity.

(continue)

As shown in Table 7 above, HepG2 cells were treated with 200 μM t-BHP to increase the amount of LDH outflow-dependently reduced by the compounds 1 to 8 separated from the ethyl acetate fractions of the palm cactus fruit seeds. Could know. In particular, 9′-O-methylisoaminol A (Compound 4) exhibited much better cell death suppression effect than silybin used as a reference drug, and showed superior efficacy to the necrosis suppression effect of quercetin, which is well known as an antioxidant. have. Therefore, it could be seen that the superior cell necrosis inhibitory effect of ethyl acetate fractions extracted from the alcohol extracts of the seeds of palm cactus fruit was due to these compounds, and these compounds were found to be useful in inhibiting hepatocellular necrosis caused by toxicity. .

Claims (6)

A composition for preventing and treating hepatotoxic diseases, which contains a seed alcohol extract of Opuntia ficus-indica var.saboten fruit as an active ingredient. According to claim 1, wherein the alcohol extract of the palm cactus fruit seeds 1) Extract the powder of palm cactus fruit seeds by adding 0.1-10 l of alcohol solvent per kg and refluxing at 60-100 ° C. for 2-6 hours, 2) the extract obtained by filtration and evaporation under reduced pressure; Composition obtained by extracting with ethyl acetate (EtOAc) 1 to 5 L from the extract of 2). The composition of claim 2, wherein the alcohol solvent of 1) is selected from the group consisting of lower alcohols having 1 to 4 carbon atoms and at least 50% aqueous alcohol solution. The composition for the prevention and treatment of hepatotoxic diseases containing at least one selected from the compounds represented by the following formula (1) as an active ingredient. [Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

[Formula 7]

[Formula 8]

5. A composition according to claim 4, wherein the compound is isolated from the seed alcohol extract of Opuntia ficus-indica var.saboten fruit. 5. The composition according to claim 1 or 4, wherein the hepatotoxic disease is drug liver damage, viral liver damage, hepatitis, liver cirrhosis, liver cancer or hepatic coma.