KR100659771B1 - Isolation of velutin and betulinic acid isolated from the organic extract of Korean mistletoe and its anti-tumor activity - Google Patents

Abstract

The present invention relates to an active compound berutin (= velutin; 5,4'-dihydroxy-7,3'-) having the structural formula shown in FIG. 2 isolated from Korean mistletoe Viscum album coloratum belonging to a parasitic plant. Dimethoxyflavone) and betulinic acid (betulinic acid) and the method of preparing the compound comprising the separation and purification of water or ethanol extraction and silica gel chromatography from the parasitic plants and the anticancer activity of these compounds, the compound May be used as a functional food, food additive or pharmaceutical material having antitumor activity.

Korean Mistletoe, Water Extract, Organic Extract, Berutin, Beturinic Acid, Anticancer Activity, Functional Samples, Food Additives, Medical Ingredients

Description

Purification of anticancer compound berutin and beturin acid from Korean mistletoe extract and preparation method of the extract {Isolation of velutin and betulinic acid isolated from the organic extract of Korean mistletoe and its anti-tumor activity}             

1 is a process for purifying berutin and beturinic acid from the organic extract of Korean mistletoe through a biochemical method

2 is a chemical structural formula of berutin and beturin acid

Figure 3 is the chemical structure of berutin and beturin acid isolated from Korean mistletoe

4 is the nuclear nuclear magnetic resonance spectrum of berutin

5 is the mass spectrum of berutin

6 is a hydrogen nuclear magnetic resonance spectrum of beturin acid

Figure 7 is the carbon nuclear magnetic resonance spectrum of beturin acid

8 is the DEPT spectrum of beturin acid

Figure 9 is the action of activating intracellular caspase-3 enzyme in the process of induction of apoptosis of tumor cells by Korean mistletoe-derived berutin and beturin acid

    A parasitic plant, Biscum Alboom Columtoum, belongs to the mistletoe family and is called Mistletoe by Korean name, and the name of the herb is leafy, grainy, parasitic, or northern herbaceous.北 桑寄生), etc. (Introduction to Pharmaceutical Botanical Sciences, Korean Learning Materials, 1982, p. 137, and Korea Plant Resources, Seoul National University Press, 1996, Vol. 1, 140). This plant grows native to Korea, including Odaesan, Jirisan, and Sobaeksan, and has been used in the private sector sometimes for low back pain, high blood pressure, gynecological diseases, pain relief, sedatives, etc., but it has not been actively used as a functional food or medicine.

Existing inventions related to the compounds contained in Korean mistletoe include lectins, but the compounds disclosed in the above patents are chemical glycoproteins and compounds having the skeleton of the triterpene invented in this patent (beturin acid) and flavones. It is a compound having completely different physicochemical properties from a compound having a skeleton of (berutin). In addition, the structural and physicochemical properties of berutin and beturin acid of the present invention have already been found in other plants, but these substances are components of mistletoe and have a strong cytotoxicity against cancer cells. It was first discovered by the inventors to be by activation.

The biological characteristics of Korean mistletoe are as follows.

  Habitat: Parasitic species on oak, hackberry, alder, chestnut and birch trees in the seas 50 ~ 1,100 m above sea level throughout Korea

  Characteristics: An evergreen parasitic shrub that grows in a nest and has a diameter of 1 m. The leaves are college, lanceolate, 3-6 cm long, 6-12 mm wide, and narrower toward the bottom.

The present inventors have tried to separate the substance having the above activity from the biscum alboom colorolatum as described above, the separation, purification, chemical structure determination of the compound having anticancer activity from the dichloromethane extract of the plant collected in Korea and The present invention has been completed by establishing a method for extracting a pharmaceutical and food composition containing a compound having the above activity.

The active compound of the present invention can be obtained by extracting, separating and purifying Korean mistletoe from the outpost, and the extraction, purification and separation are performed as follows, for example.

First, the mistletoe from Korea was collected to remove foreign substances, and then the plants were treated with hot water, dichloromethane, or ethanol, preferably 95% ethanol for 2 hours to 24 hours, preferably 5 hours at room temperature to 100 ° C, preferably 95 ° C. After extraction, the extract is lyophilized, distilled, or distilled under reduced pressure, or dried by spray drying, and then extracted with hot water extract, ethanol, or 95% ethanol. Partition extraction is performed by adding methane, and the components soluble in dichloromethane are collected to obtain a dried product after distillation, distillation under reduced pressure, or drying by spray drying.

The dried product was divided into three to ten, preferably ten fractions using a mixed solvent of silica gel and ethyl benzene as mobile phase, and the fraction showing activity among them was again divided into silica gel and ethyl benzene-ethyl acetate as mobile phase. The solvent is divided into two to eight, preferably two fractions. Among the fractions showing anticancer activity, silica gel (a mixed solution of benzene-ethyl acetate as a mobile phase) and high-performance liquid chromatography (reverse phase silica gel as a fixed phase, water-methanol as a mobile phase, or water-aceto) until a pure substance is obtained. Nitrile-acetic acid, preferably a mixed solution of water-methanol-acetic acid) to obtain the anticancer active materials berutin and beturin acid.

The compounds separated by the above method were analyzed by analytical methods such as NMR (nuclear magnetic resonance analysis) and MS (mass spectrometry) to confirm the physicochemical properties and chemical structures of the compounds. .

The compounds of the present invention exhibit excellent inhibitory activity in the proliferation inhibitory activity assay against cancer cells. Accordingly, the present invention provides a pharmaceutical or food composition comprising the anticancer active compound as an active ingredient. The physicochemical properties of the purified compound berutin and beturin acid exhibiting this activity are as follows and the structural formula is shown in FIG.

Berutin and beturin acids of the present invention have the physicochemical properties as described above and have anticancer activity and can be found not only in mistletoe but also in other plants and fungi, and thus the preparation method for the compound of the present invention is only barely It is not limited to how to use flesh.

A) berutin

  1) Properties of substance Yellow powder, or yellow needle crystal 2) Molecular weight (Molecular) 314 (C₁₇H₄₈O₃The mass spectrum is shown in FIG. 3) color reaction FeCl₃Bisexual, H₂SO₄4) Hydrogen and Carbon Nuclear Magnetic Resonance (DMSO)d                                                 ₆ Hydrogen nuclear magnetic resonance spectra of tetramethylsilane (TMS) as a standard in the solvent are shown in FIG. 3 and carbon nuclear magnetic resonance spectra are shown in FIG. 5) Soluble Soluble in DMSO, methanol, ethanol and poorly soluble in chloroform, benzene, hexane, water.

B) mountains of beturin

  1) Properties of the substance White powder, or white crystals 2) Molecular weight (Molecular) 456 (C₃₀H₁₄O₆3) Color reaction FeCl₃Voice to, H₂SO₄4) Hydrogen and Carbon Nuclear Magnetic Resonance on CDCl₃ Hydrogen nuclear magnetic resonance spectra of tetramethylsilane (TMS) as a standard in the solvent are shown in FIG. 6, and the carbon nuclear magnetic resonance spectra are shown in FIG. 5) Soluble Soluble in chloroform, benzene and poorly soluble in water, methanol.

Such pharmaceutical, or food, compositions may comprise pharmaceutically or food acceptable excipients, carriers, diluents, etc. together with the active compound and may be formulated according to known methods using readily available ingredients. Can be. In preparation of the formulation, the active ingredient or extract containing the active ingredient may be mixed with the carrier, diluted with the carrier, or contained in a carrier in the form of a capsule, sachet, paper or other container. If the carrier serves as a diluent, it may be a solid, semisolid, or liquid substance which acts as a vehicle, excipient or medium for the active ingredient. Thus, the preparation may be in the form of tablets, pills, dispersants, granules, elixirs, suspensions, emulsifiers, solutions, syrups, aerosols, soft and hard gelatin capsules, sterile injectable solutions, sterile packaged powders, ointments, or biscuits, in the form of sticks Confectionary, bread, candy, beverages and the like.

Examples of suitable carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, water , Sugar syrup, methylcellulose, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate and mineral oil. The composition may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, and the like, in addition to the above components. Compositions of the present invention can be formulated using techniques known in the art to release the active ingredient rapidly, continuously or delayed after administration to a patient.

The pharmaceutical or food composition of the present invention may be administered orally or parenterally, and the active compound is effective for a wide range of administration. For example, daily dosages of active compounds typically range from about 0.1 to 20 mg per kg of body weight and range from 100 mg to 10 g as water or ethanol extract when used as a functional food. However, the amount of the compound to be administered will be determined by the physician or pharmacist in light of the relevant conditions, including the condition of the disease to be treated, the route of administration chosen, the age, weight and response of the individual patient, and the severity of the disease. It is not intended to limit the scope of the invention.

The present invention discloses a method for preparing berutin and beturin acid having anticancer activity purified from Viscum album coloratum (Korean name Mistletoe) and its activity as a pharmaceutical and food composition comprising the same. . More specifically, the berutin and beturin compounds having anticancer activity from Korean mistletoes, which are parasitic on oak, alder, elm, etc., by hydrothermal extraction, ethanol extraction, solubility difference, silica gel and high-performance liquid chromatography It relates to a method for preparing the compound comprising separating and purifying the compound, and to a pharmaceutical and food composition comprising the compound.

Hereinafter, the present invention will be described in detail with reference to Examples, but the following Examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention.

Example 1 Preparation of Extracts Containing Anticancer Active Material Berutin and Beturin Acid from Korean Mistletoe

Fresh Korean mistletoe, or 1 kg of its dry matter, was finely chopped and placed in 10 liters of 95% ethanol, and extracted three times in a water bath at 95 ° C. for 3 hours. The extract was filtered under reduced pressure, or lyophilized, and dispersed in an appropriate amount of water. The mixture was extracted three times with 10 liters of dichloromethane, washed with saturated brine, dehydrated with anhydrous sodium sulfate, and filtered. Concentration under reduced pressure afforded 38.9 g of a dichloromethane soluble extract containing anticancer active ingredients, berutin and beturin acid (FIG. 1).

Example 2 Preparation of Berutin and Beturinic Acid

38.9 g of the dichloromethane soluble component obtained in Example 1 was eluted with silica gel column chromatography (benzene: ethyl acetate = 100: 1 and eluted with a slope of the solvent to be 10: 1 as shown in FIG. 1). . Divided into eleven fractions from I to XI, among which fr. Purification was again attempted for the VIII portion.

First of all fr. For VIII, silica gel column chromatography (benzene: ethyl acetate = 10: 1 to 1: 1 was eluted with a concentration gradient, Merck Art. No. 9385, 1.8 x 44 cm) to obtain fr. Divide into two fractions of VIII-I and VIII-II and fr. Fast Liquid Column Chromatography for VIII-I [Column; Μ-Bondapak C-18 (7.8 × 300 mm) from Merck, mobile phase; 100% methanol, flow rate; 0.6 ml / min, detection; Refractive index photometer] to give 12 mg of beturinic acid.

Meanwhile fr. VIII-II was subjected to silica gel column chromatography (benzene: ethyl acetate = 5: 1, Art. No. 9385 from Merck, 1.8 x 44 cm), and then recrystallized in a methanol-chloroform mixed solvent to give a yellow needle-like compound berutin 4 The mg was purified purely to determine the chemical structure (Figure 2). In addition, through the hydrogen nuclear magnetic resonance spectrum (FIG. 4), the carbon nuclear magnetic resonance spectrum (FIG. 4) and the mass spectrum (FIG. 5) for beturin, the betulinic acid has a hydrogen nuclear magnetic resonance spectrum (FIG. 6). The structure was confirmed by the carbon nuclear magnetic resonance spectrum (FIG. 7) and the DEPT spectrum (FIG. 8).

Example 3 Cytotoxicity of Cancerous Cells to Active Ingredients

The cytotoxic activity of cancer cells was measured for NIH3T3 cancer cell lines. In other words, add 50 µl of the solution containing 2.5 × 10 ⁴ cancer cells per well to a 96-well plate, and add 50 µl of EMEM-5% FBS solution containing appropriate amounts of berutin and beturin acid to it for 36 hours at 37 ° C. After culturing for a while, the cell inhibitory activity of the cells was expressed in% using a Cell Counting Kit (manufactured by Dojindo, Japan). As a result, as shown in Table 1, berutin and beturin acid inhibited the growth of cancer cells by 50% at 75 and 18.75μg / ml concentration, respectively.

Table 1. Inhibitory Effects of Berutin and Beturin Acids on NIH3T3 Cancer Cells

Compound IC ₅₀ (μg / ml) ^{* 1} VD-2 (velutin) 75.00 VD-6 (betulinic acid) 18.75

^{* 1} 50% cancer cell growth effective concentration

On the other hand, as a result of examining the morphological aspects of cancer cell injury activity by these substances, it was confirmed that the cell death by apoptosis, we have already isolated from Korean mistletoe to investigate the activity of intracellular caspase-3 related to apoptosis induction. The enzyme activity was compared with VD-3 having cancer cell apoptosis-inducing activity. As a result, it was found that the treatment of berutin (VD-2) and beturin acid (VD-6) increased the enzymatic activity of caspase-3 when treated with cancer cells (FIG. 9). These results indicate that beroutine and beturin acid induce apoptosis from cancer cells through the activation of caspase-3 enzyme.

As described above, the present invention relates to the anticancer activity of Korean mistletoe-derived berutin and beturin acid, and the cancer cell apoptosis-inducing activity according to the present invention is caused by Korean mistletoe organic extract. The present invention relates to belutin and beturin acid from Korean mistletoe, and an organic extract containing these substances, and provides a method for isolating anticancer substances contained in Korean mistletoe and preparing an extract fraction thereof. In addition, the present invention revealed that the activation of caspase-3 enzyme is involved in the action mechanism of cancer cell apoptosis induced by beturin and berutin acid. These substances can be used as functional foods or food additives as compounds that can induce apoptosis of cancer cells, and in particular, as pharmaceutical compositions that can be applied as anticancer agents and as an adjuvant of anticancer therapy against cancer, It can be used as a usable injection.

Claims (5)

After extracting Korean mistletoe with 95% ethanol at 95 ° C. for 5 hours, 5 times dichloromethane was added to the extract, and the extract was partitioned. The components soluble in dichloromethane were collected to obtain a dried product. Silica gel was fractionated using a mixed solvent of benzene-ethyl acetate, followed by the active fraction, and fractionated using a mixed solvent of benzene-ethyl acetate as a kagel and a mobile phase and purely separated from the fraction showing anticancer activity. Separation and purification method of berutin or betulinic acid, characterized in that and performing high performance liquid chromatography delete delete Korean Mistletoe Extract Containing Berutin or Betulinic Acid Separated and Purified by the Method of Claim 1 Anticancer pharmaceutical composition characterized in that it contains Berrutin or Betulinic acid purified from Korean mistletoe as an active ingredient

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