KR100542323B1 - Preparation method of compounds with apoptosis-inducing activity on cells from the Machilus thunbergii - Google Patents

Abstract

The present invention relates to a Hakku extract having an action of inducing apoptosis, and more particularly, to an apoptosis extract from a Hakku extract, and also less toxic to immune system diseases caused by abnormal increase and activation of inflammatory cells. The present invention relates to a method for separating lignans and lactone compounds which may be usefully used as a therapeutic agent and food additive for various intractable and chronic diseases including, and separating an active ingredient from pharmaceuticals and food additives and hawthorn containing the same.

Apoptosis, Lignan and Lactone Compounds, Prunus

Description

Preparation method of compounds with apoptosis-inducing activity on cells from the Machilus thunbergii}

Figure 1 shows the ¹ H-NMR of Tunberin A (Formula 1) of the present invention.

Figure 2 shows the ¹ H-NMR of the erythro- ortho birignan (Formula 2) of the present invention.

Figure 3 shows the ¹ H-NMR of the secoisolansipolide (Formula 3) of the present invention.

Figure 4 shows the effect of the lignan and lactone compounds on the DNA fragmentation phenomenon in the present invention.

 M: control group (molecular weight marker)

 1: Tunberin A 0 μM treatment

 2: 20 μM treatment of Tunberin A

 3: Tunberin A 40 μM Treatment

 4: Tunberin A 80 μM Treatment

 5: erythro-austrobi lignan 0 μM treatment

 6: Treatment with erythro-austrobi lignan 20 μM

 7: Treatment with erythro-austrobi lignan 40 μM

 8: Treatment with erythro-austrobi lignan 80 μM

 9: treatment with secoisolancipolide 0 μM

10: 20 μM treatment of secoisolansipolide

11: treatment with secoisolansipolide 40 μM

12: Seikoisolanxipolide 80 μM treatment

P: camptocecin treatment (positive control)

The present invention relates to a Hakku extract having an action of inducing apoptosis, and more particularly, to an apoptosis extract from a Hakku extract, and also less toxic to immune system diseases caused by abnormal increase and activation of inflammatory cells. The present invention relates to a method for separating lignans and lactone compounds which may be usefully used as a therapeutic agent and food additive for various intractable and chronic diseases including, and separating an active ingredient from pharmaceuticals and food additives and hawthorn containing the same.

The various kinds of cells that make up life can be killed through various pathways. Apoptosis, first referred to by Kerr et al. In 1972, is one of the physiological phenomena essential for the development of normal organs of multicellular organisms and for maintaining homeostasis of tissues, which are cells that respond to certain stimuli. It is a representative intracellular mechanism of action that occurs selectively at. Specifically, cells can cause planned death by various stresses, such as nutrient depletion, viruses, oxygen radicals or drugs that damage chromosomes, and such apoptosis may be due to the aggregation of chromosomes, blister formation, cell condensation, and apoptosis ( Specific morphological changes such as apoptotic body formation, activation of caspase enzyme systems, and observation of chromosomal fragments in the nucleus can be seen [John DR, Sten O, Boris Z, 2000, J. Structur. Biol. 129, 346-358; Takeuchi M, Hayakawa A, Takagi K, Hiramatsu K, Shimizu Y, Matsumoto S, Hiramatsu T, Ito Y, Kume H, Suzuki R, Yamaki K, 1999, Apoptosis 4, 461-468].

Apoptosis is known to not only perform a physiological function for maintaining tissue homeostasis, but also cause a proliferative disease of cells due to abnormal inhibition or promotion of apoptosis or various diseases due to the loss of the cells. In addition, one of the regulatory mechanisms of the immune response is to maintain homeostasis of lymphocyte tissues by apoptosis of lymphocytes, which are immune cells, and the process of killing target cells by lymphocytes is also caused by apoptosis.

In recent years, apoptosis plays an important role in maintaining tissue homeostasis along with cell proliferation. Thus, a new example has emerged that many diseases including immune diseases and tumors are caused by dysregulation of apoptosis. Therefore, in order to understand the pathogenesis of many diseases of the human body and to develop a treatment method accordingly, the understanding of the apoptosis control mechanism in each tissue of the human body has emerged as a big concern, and thus, cell death in the pharmaceutical industry field has high added value. Was injured.

Recently cyclosporine [Kitagaki et. al. BBRC, 1996, 222, 77-77] and SB203570 [Kankaanranta et. al, J. Pharmacol. Exp. Ther., 1999, 290, 621 to 628], and also reported that in natural products, bicarin or bicarin may be used as an anticancer agent as an apoptosis organic agent of cancer cells [PCT / JP93]. / 00614]. However, most of the compounds exhibiting apoptosis activity have reported apoptosis activity against cancer cell lines and are different from the apoptosis activity of inflammatory cells such as eosinophils.

Over the years, bronchial asthma is a clinical inflammation of the airways and eosinophils among various inflammatory cells have been known to play an important role in bronchial inflammation in asthma patients [Barnes PJ, 1992, J. Intern. Med. 231, 451-461; Busse WW, Nagata M, Sedgwich JB, Eur. Respir. J. 1996, 9 (Suppl 2), 132S-135S; Myra S, Laura M, John S, Christopher H, J. Immunol. 1992, 148 (11), 3543-3549; Gleich GJ, J. Allergy clin. Immunol . 1990, 85 (2), 422-36. Specifically, by-products of eosinophils may damage the epidermis, or may exhibit histotoxicity, such as edema and mucus in the airways of bronchial stenosis and asthma patients, and effectively controlling the excessive presence and activation of eosinophils is eosinophilic composition. It is known to be very important in treating diseases [Simon HU, Blaser K, 1995, Immun. Today 16, 53-55; Haslett C, 1997, British Med. Bull. 53, 669-683. Glucocorticoids or theophylline are known as therapeutic agents to remove these eosinophils and are currently used in the clinic to treat asthma. However, these have the disadvantage of showing various steroidal side effects such as metabolic abnormalities or cataracts in the long-term administration.

Therefore, there is an urgent need to develop a therapeutic agent that exhibits apoptosis activity of inflammatory cells and has fewer side effects.

On the other hand, Machilus thunbergii Sieb. Et Zucc., Lauraceae is an evergreen broad-leaved arborescent of the dicotyledonous plant of the genus Trifoliaceae , which is distributed in the coastal and foothills of southwestern Korea. Since the viscosity becomes stronger, it is used as a binder of the incense. Bark may also be used as a dye. In oriental medicine, bark is called epoch peel (피) and used for asthma and gastrointestinal diseases. Wood is made of furniture and marine materials. Flaxoids include flavonoids, lignans, neoolignans, and isoquinoline alkaloids. There are few studies of medicinal effects. No studies of apoptosis activity have been reported.

Accordingly, the present inventors, while studying a substance having an apoptosis effect, the lignan and lactone compounds obtained from the hawthorn tree show apoptosis activity, and also less toxic, including immune system diseases related to abnormal increase and activation of inflammatory cells The present invention has been completed by knowing that it can be used as a therapeutic agent and food additive for various refractory and chronic diseases.

Accordingly, it is an object of the present invention to provide a therapeutic agent and a food additive for various refractory and chronic diseases including immune system diseases containing hawthorn extract, lignan and lactone compounds, and derivatives thereof.

In addition, another object of the present invention is to provide a method for isolating a compound exhibiting apoptosis activity from hawthorn.

The present invention is characterized by the treatment and food additives of various refractory and chronic diseases, including immune system diseases, including hawthorn extract, lignan and lactone compounds, and derivatives thereof, as an active ingredient thereof.

In another aspect, the present invention is characterized by a method for separating lignans and lactone compounds exhibiting apoptosis activity from hawthorn trees.

Referring to the present invention in more detail as follows.

The present invention includes the use as a therapeutic agent and a food additive of various refractory and chronic diseases, including immune system diseases, including lignan and lactone compounds represented by the following Chemical Formulas 1 to 3, the active ingredients thereof, and lactone compounds and derivatives thereof.

More specifically, the present invention is a tunberin A (thunberin A) represented by the following formula (1), erythro-austrobailignan (erythro-austrobailignan) represented by the following formula (2), the secoisoransipolide represented by the following formula (3) It includes a therapeutic agent for a variety of refractory and chronic diseases, including immune system diseases containing secoisolancifolide) as an active ingredient.

The lignans and lactone compounds represented by the above formulas (1) to (3) may also be used as derivatives thereof, and derivatives of the lignans and lactone compounds that may be used in the present invention may be in the form of esters that are easily obtained by hydrolysis. Advantageously the ester containing an alkyl group of C ₁ ~C _4. However, the derivatives are not limited to the ester form.

The lignans and lactone compounds represented by Chemical Formulas 1 to 3 may be prepared by various methods, and specifically, may be obtained by chemical synthesis or extraction from plants.

In order to determine that the lignan and lactone compounds of the present invention show an activity against apoptosis, apoptosis induction of leucine HL-60 cells by fluorescence detection, apoptosis induction by caspase-3 activity assay, and Induction of apoptosis by DNA fragmentation assay was investigated. As a result, the lignan and lactone compounds of the present invention were shown to have apoptosis-inducing action, and no special cytotoxic side effects were observed, which may be more useful than conventional induction agents.

Because of this, lignan and lactone compounds can be used as a preventive and therapeutic agent for various refractory and chronic diseases, including immune system diseases.

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The lignan and lactone compounds and derivatives thereof of the present invention can be administered orally or parenterally during clinical administration and can be provided in the form of general pharmaceutical preparations.

The lignans and lactone compounds of the present invention and derivatives thereof may be administered in various oral or parenteral formulations during actual clinical administration, and when formulated, commonly used fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc. Prepared using diluents or excipients.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, sucrose or the like in lignan and lactone compounds and derivatives thereof. It is prepared by mixing lactose and gelatin. In addition to simple excipients, lubricants such as magnesium styrene talc are also used. Liquid preparations for oral administration include suspensions, solvents, emulsions, and syrups. In addition to the commonly used simple diluents, water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have.

Formulations for parenteral administration include sterile aqueous solutions, water-insoluble solvents, suspensions, emulsions, lyophilizers, suppositories. As the non-aqueous and suspending solvent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like can be used. As the base of the suppository, utopsol, macrogol, tween 61, cacao butter, laurin butter, glycerol gelatin and the like can be used.

The content in the preparation of the active ingredient according to the present invention can be appropriately selected depending on the absorbency, inactivation rate, excretion rate, age, sex and condition of the user in the body. In the case of the lignan and lactone compound of this invention, it is 0.01-100 mg / kg, Preferably it is 0.1-10 mg / kg, and can be administered 1-3 times a day.

The lignan and lactone compounds of the present invention did not show a toxicity change upon oral administration to 100 mg / kg, and the minimum lethal dose (LD ₅₀ ) of the oral administration was 100 mg / kg or more. Therefore, the lignan and lactone compounds of the present invention can be administered stably to a living body.

In addition, the present invention provides an immune system disease comprising the tunberin A represented by the formula (1), the erythro-ostrobirignan represented by the formula (2), the secoisolansipolide represented by the formula (3) or derivatives thereof as an active ingredient It can be used as a food additive for the treatment of various refractory and chronic diseases, including functional foods, dietary supplements or special nutritional products.

As used herein, the term "functional food" means a food product having improved functionality of a general food product by adding the lignan, a lactone compound and a derivative thereof to the general food product.

In the present specification, a 'health supplement' or 'special dietary supplement', as distinguished from a functional food, is a health food prepared by adding the lignan and lactone compound to a general food, or encapsulating, powdering, and suspension, and ingesting the same. If you mean to have a specific effect on health, but unlike the general medicine has the advantage that there is no side effect that can occur when using the drug as a raw material for long-term use.

The content of functional foods, dietary supplements and specialty health foods containing the lignans and lactone compounds and derivatives thereof according to the present invention can be varied according to the food groups used, the content of which is used in the pharmaceutical composition. Perform within the range of toxicities measured.

In addition, the present invention

1) extracting the powdered bark of Machilus thunbergii by dissolving it in methanol or a mixed solvent of methanol and water, concentrating under reduced pressure, and fractionating the mixture with hexane, ethyl acetate and chloroform to obtain a hexane fraction; And

2) fractionating the hexane fraction with a hexane-acetone mixture and separating the compounds of the following Chemical Formulas 1 to 3 with Rf values of 0.46, 0.3 and 0.5 on adsorption chromatography (elution solvent: hexane / ethyl acetate = 4/1). It includes a separation method consisting of steps.

Step 1) first extracts the stem bark of the pear tree successively several times using methanol to obtain a methanol extract and then concentrated under reduced pressure to obtain a pear tree methanol extract. Suspension was obtained by adding water to the methanol extract, and then fractionated into hexane, ethyl acetate and chloroform to obtain a fraction.

Step 2) extracts the hexane fraction of Lucemia HL-60 apoptosis from the fraction into lignan and lactone compounds by adsorption chromatography, which is commonly used by those skilled in the art. Any method may be used, preferably silica gel column chromatography. As the elution solvent used, a mixed solvent of hexane and acetone was used, and Rf values of 0.46, 0.3, and 0.5 on adsorption chromatography (elution solvent: hexane / ethyl acetate = 4/1) were used to obtain the compounds of Formulas 1 to 3, respectively. Separate.

Hereinafter, the present invention will be described in more detail based on the following examples, but the present invention is not limited thereto.

Example 1 Isolation of Lignan and Lactone Compounds from Stem Bark

Approximately 1.8 kg of dried stem bark pulverized pulverized bark tree pulverized to about 1 mm size was extracted four times with 12 L of methanol, and the extracts were collected and concentrated under reduced pressure to obtain 290 g of pear tree methanol extract. The obtained methanol extract was suspended in 1 L of distilled water, and then fractionated into hexane, ethyl acetate and chloroform, respectively.

Hexane fraction (35.3 g) was obtained by concentrating the hexane layer with Lucemia HL-60 apoptosis effect under reduced pressure, and then using silica gel column chromatography with elution solvent (hexane / acetone = 10/1 → 1/1). It was. The hexane layer was separated into four fractions, and a portion of the second fraction with Lucemia HL-60 apoptosis among the separated fractions was again subjected to silica gel column chromatography (eluent, hexane / ethyl acetate = 20/1 → 1). / 1) was separated into nine fractions. The third fraction (hexane / ethyl acetate = 4/1, Rf = 0.46) was again subjected to silica gel column chromatography (eluent, benzene / ethyl acetate = 50/1) to obtain the compound of formula 1 (tunberin A). The fifth fraction (hexane / ethyl acetate = 4/1, Rf = 0.3, 0.5) was again subjected to silica gel column chromatography (eluent, benzene / acetone = 70/1) to obtain the compound of Formula 2 ( Erythro-ostrobirignan) and a compound of formula 3 (secoisolansipolide) were obtained.

Example 2 Structural Analysis of Lignan and Lactone Compounds

(1) Tunberin A Compound of Formula 1

The Tunberin-A compound of Formula 1 obtained in Example 1 was well dissolved in methanol in the form of a white needle, but not in hexane.

In the infrared absorption spectrum, the peak at 3025 and 1220 cm ^-1 , and the absorption peak at 294 nm with chloroform as the solvent in the ultraviolet absorption spectrum.

Mass spectrometry showed a molecular weight of 340.

The structural formula is C ₂₀ H ₂₀ O ₅ as a result of measuring the number of hydrogen and carbon using high resolution mass spectrometry and nuclear magnetic resonance spectra. The hydrogen nuclear magnetic resonance spectrum analysis results are shown below. In addition, we determined the structure of the Thun suberic this by ¹³ C nuclear magnetic resonance spectrum and ¹ H- ¹ H COSY, HMQC, HMBC method of instrumental analysis.

¹ H NMR (300 MHz)

δ 6.98 (2H, d, J = 1.2, H-2, 2 ′), 6.81 (2H, d, J = 8.1, H-5, 5 ′), 6.90 (2H, dd, J = 8.1, 1.5, H -6, 6 '), 5.98 (2H, s, -OCH2O-) 4.43 (2H, d, J = 6.6, H-7, 7'), 2.27 (2H, m, H-8, 8 '), 1.02 (6H, doublet, J = 6.9, H-9, 9 ').

(2) erythro-ostrobirignan compound of formula (2)

The erythro-ostrobirignan compound of Formula 2 obtained in Example 1 is a yellow liquid form.

In the infrared absorption spectrum, peaks were shown at 3020, 2400, and 1220 cm ⁻¹ , and absorption peaks were shown at 302 and 274 nm with chlorform as a solvent in the ultraviolet absorption spectrum.

Mass spectrometry showed a molecular weight of 328. The structural formula was found to be C ₂₀ H ₂₄ O ₄ using mass spectrometry and nuclear magnetic resonance spectra. The hydrogen nuclear magnetic resonance spectrum analysis results are shown below. Also, the erythro by ¹³ C nuclear magnetic resonance spectrum and ¹ H- ¹ H instrumental analysis method of COSY - in Australia was determined by the structure of the lignans.

¹ H NMR (300 MHz)

δ 6.73 (1H, D, J = 8.1, H-5 '), 6.65 (1H, br s, H-2'), 6.60 (1H, d, J = 8.1, H-6 '), 6.59 (1H, d, J = 1.2, H-2), 6.83 (1H, d, J = 8.1, H-5), 6.63 (1H, d, J = 8.1, H-6), 5.92 (2H, s, -OCH2O- ), 3.87 (3H, s, -OCH3), 2.72 (2H, dd, J = 5.1, 13.5, H-7a, 7'a), 2.27 (2H, dd, J = 9.6, 13.5, H-7b, 7 'b), 1.73 (1H, m, H-8, 8'), 0.81 (6H, s, H-9, 9 ').

(3) Secoisolansipolide Compounds of Formula 3

The secoisolansipolide compound of Chemical Formula 3 obtained in Example 1 was a colorless liquid, well soluble in chloroform but not in water.

In the infrared absorption spectrum, peaks were found at 3400, 3025, 2925, 2400, 1780, 1680, 1500, and 1420 cm ⁻¹ , and absorption peaks at 258 nm with chloroform as the solvent in the ultraviolet absorption spectrum.

Mass spectrometry showed that the molecular weight was 284. The structural formula was found to be C ₁₆ H ₂₈ O ₄ using mass spectrometry and nuclear magnetic resonance spectra. The hydrogen nuclear magnetic resonance spectrum analysis results are shown below. In addition, the structure of the secoisolansipolide was determined by instrumental analysis of ¹³ C nuclear magnetic resonance spectra.

¹ H NMR (300 MHz)

δ 7.08 (1H, t, J = 7.8, H-6), 4.90 (1H, br s, H-3), 3.73 (3H, s, -OCH3), 2.35 (2H, q, J = 7.5, H- 7), 2.15 (3H, s, H-1), 1.51 (2H, quintet, J = 7.2, H-14), 1.26 (12H, br s, H-8-13), 0.88 (3H, t, J = 6.6, H-15).

Example 3: Induction of Apoptosis of Luchemia HL-60 Cells by Fluorescence Detection of Lignan and Lactone Compounds

In the present invention, the fluorescence detection is performed by double staining Annexin V-FITC / PI (fluoresce isothiocyanate- Annexin V / propidium iodide) on HL-60 cells, which are leucine cells, and together with necrosis and apoptosis of cells. Measured. The chromosome cleaved at the time of apoptosis binds to PI, and the phosphatidylserine (PS) exposed to the outside of the cell membrane binds to a fluorescent substance called Annexin V-FITC, thus the method of Annexin V-FITC kit (BD sciences). Cells were double stained and measured by fluorescence detector [Vermes, IC, Hannen, H., et al. 1995, J. Immunol. Meth. 184, 39-51. Fluorescence activity of the lignan and lactone compounds isolated in Example 1 was measured, and the results are expressed as the ratio (T / C) of the fluorescent activity of the experimental group to the control group, as shown in Table 1 below.

Effect of Lignan and Lactone Compounds on Fluorescent Activity division Concentration (micromolar) Fluorescence activity (T / C) Tunberin A (Formula 1) 50 2.54 100 3.49 Erythro-austroby lignan (Formula 2) 50 2.57 100 3.91 Secoisolansipolide (Formula 3) 50 2.88 100 5.05

As shown in Table 1, all three kinds of lignans and lactone compounds showed fluorescence activity, and among them, especially secoisolancipolide showed high activity at 100 μM.

Example 4 Induction of Apoptosis by Caspase-3 Activity Test of Lignan and Lactone Compounds

Apoptosis-inducing action of the samples in the present invention was confirmed by measuring the caspase-3 activity. At least 1 × 10 ⁵ (in 200 μl) of HL-60 cells were dispensed per well of a 96 well-plate and the samples were mixed at constant concentration and incubated for 16 hours in a 37 ° C. CO ₂ incubator. The supernatant was removed by centrifugation at 3000 rpm, washed once with PBS, and then mixed well by adding 15 µl of a lysis buffer, followed by standing at room temperature for 10 minutes. The cell lysate was taken and transferred to a fluorescence plate, respectively, and 100 µl of assay buffer and 10 µl of caspase-3 substrate (Ac-DEVD-AFC) were added and mixed at 37 ° C. The reaction was carried out for 1 hour, and the fluorescence activity of 400 nm and 505 nm was measured by LS50B fluorometer (perkin-Elmer Corp., Norwalk, CT) [MacFarlane, M., Kelvin, C., 1997, J. Cell Biol., 469-479]. The results are expressed as the ratio (T / C) of the fluorescent activity of the experimental group to the control group as shown in Table 2.

Effect of Lignan and Lactone Compounds on Caspase-3 Activity division Concentration (micromolar) Caspase-3 activity (T / C) Tunberin A (Formula 1) 100 0.36 ± 0.07 Erythro-austroby lignan (Formula 2) 100 3.04 ± 1.13 Secoisolansipolide (Formula 3) 100 0.17 ± 0.02

As shown in Table 2, the compounds of the formulas (1), (2) and (3) showed activity against caspase-3, and the activity of erythro-ostrobirignan was stronger than that of tumberin a and secoisolansipolide. appear.

Example 5 Induction of Apoptosis by DNA Segmentation Test of Lignan and Lactone Compounds

In the present invention, the DNA fragmentation of the specimens was mixed with 2 × 10 ⁶ (in 10 ml) HL-60 cells at 20, 40, 80 μM concentrations and cultured for 4 hours in a CO ₂ incubator at 37 ℃ Then, DNA was extracted using an apoptotic DNA ladder kit (Roche). The extracted DNA was subjected to electrophoresis on 1% agarose gel to confirm DNA fragmentation under UV light [Kojio et al., FEMS Immuno. and Med. Microbiol. 2000. 29, 275-281; Voytas, D., 1987. Current Protocols in Molecular Biology, Chapter 2. 5], and the results are as shown in FIG.

As shown in FIG. 4, lanes 1 to 4, 5 to 8, and 9 to 12 are tunberine A (Formula 1), erythro-austrobirignan (Formula 2), and secoisolansipolide (Formula 3). DNA fragmentation of the cells according to the concentration of).

Camptocecin, used as a positive control (P), is a known apoptosis inducer, and camptocecin has excellent activity, but has very strong cytotoxicity, and therefore can be used as a therapeutic agent such as inhibition of diarrhea hematopoiesis due to intestinal endothelial cell destruction. While there are serious side effects, the lignan and lactone compounds of the present invention, tunberin A, erythro-ostrobirignan and secoisolansipolide, have also been shown to induce excellent DNA fragmentation, and specific cytotoxic side effects It is not observed and appears to be safer to use.

Example 6: Acute Toxicity Test

Acute toxicity test was performed using 6-week-old specific pathogen-free (SPF) SD rats. Two animals per group were orally administered once at a dose of 100 mg / kg / ml of the three kinds of lactone compounds of the present invention. After administration of the test substance, mortality, clinical symptoms, and changes in body weight were observed, and hematological and hematological tests were performed.

As a result, no significant clinical symptoms or dead animals were noted in all animals treated with the test substance, and no toxic changes were observed in weight changes, blood tests, blood biochemical tests, and autopsy findings. As a result, all of the lactone compounds of the present invention did not show a toxicity change in rats up to 100 mg / kg / ml, respectively, and the minimum lethal dose (LD ₅₀ ) was determined to be a safe substance having 100 mg / kg.

Preparation Example 1 Preparation of Powder and Capsule

10 mg of lignan and lactone compound were mixed together with 14.8 mg of lactose, 3 mg of crystalline cellulose, and 0.2 mg of magnesium stearate. The mixture was filled into No. 5 gelatin capsules using a suitable apparatus.

The components of the powder and capsules are as follows.

Lignan and Lactone Compounds ·········· 10 mg

Lactose ... 14.8 mg

Crystalline Cellulose ... 3 mg

Magnesium Stearate 0.2 mg

Preparation Example 2 Preparation of Injection Solution

Injection was prepared by mixing 10 mg of lignan and lactone compound, 180 mg of mannitol, 26 mg of Na ₂ HPO ₄ .12H ₂ O and 2974 mg of distilled water. The solution was placed in a bottle and heated at 20 ° C. for 30 minutes for sterilization.

Lignan and Lactone Compounds 10 mg

Mannitol 180 mg

Na ₂ HPO ₄ · 12H ₂ O ············· 26

Distilled water ··················· 2974 mg

Preparation Example 3 Preparation of Beverage

0.1 g of lignan and lactone compound, powdered vitamin E, ferric nitrate, zinc oxide, nicotinic acid amide, vitamin A, vitamin B ₁ and vitamin B ₂ were prepared by mixing.

The components of the beverage are as follows.

Lignan and Lactone Compounds 0.1 g

15 g of vitamin C

Powdered vitamin E 7.5 g

Ferrous iron ···························· 19.75 g

Zinc Oxide ... 3.5 g

Nicotinic acid amide ... 3.5 g

0.2 g of vitamin A

Vitamin B ₁ .0.25 g

0.3 g of vitamin B ₂

·······························

Preparation Example 4 Preparation of Health Supplement

0.1 g of lignan and lactone compound, powdered vitamin E, ferric nitrate, zinc oxide, nicotinic acid amide, vitamin A, vitamin B ₁ and vitamin B ₂ were prepared by mixing.

The components of the beverage are as follows.

Lactone Compounds 0.1 g

15 g of vitamin C

7.5 g of powdered vitamin E ···················

5.75 g of ferric nitrate ·················

Zinc Oxide ······ 3.5 g

Nicotinic Acid Amide · ・ ・ ・ ・ ・ ・ ・ ・ 3.5 g

0.2 g of vitamin A

Vitamin B ₁ ······················ 0.25 g

Vitamin B ₂ 0.3 g 0.3 g

Starch ····················· 18.0 g

······························

As described above, the lignans and lactone compounds obtained from the pear tree of the present invention are excellent in apoptosis-inducing activity and various intractable properties including diseases related to abnormally proliferated inflammatory cells in vivo, namely, immune system diseases such as asthma and It can be used for the treatment or support for the treatment of chronic diseases, and because of its low toxicity, it can be very useful for the medicine or functional food industry.

Claims (5)

delete delete delete delete 1) extracting the powdered bark of Machilus thunbergii by dissolving it in methanol or a mixed solvent of methanol and water, concentrating under reduced pressure, and fractionating the mixture with hexane, ethyl acetate and chloroform to obtain a hexane fraction; And 2) The hexane fractions were fractionated with a hexane-acetone mixture and the Rf values were 0.46 and 0.5 on the adsorption chromatography (elution solvent: hexane / ethyl acetate = 4/1). Separation method of a compound represented by the following formula (1) or a compound represented by the following formula (3) from a hummus tree comprising the step of separating. [Formula 1]

[Formula 3]

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