TWI417091B - Anti-obesity agents and pharmaceuticals, foods or cosmetics that have a fat accumulation inhibitory effect - Google Patents

Description

Anti-obesity agent and pharmaceutical, food or cosmetic with fat accumulation inhibition

The present invention relates to an anti-obesity agent and a pharmaceutical, food or cosmetic having a lip accumulation inhibiting action.

In recent years, the proportion of obesity has been increasing along with the changes in the European and American lifestyles or lifestyles. Since the energy absorbed by the food and drink exceeds the energy consumed by exercise or the like, the excess energy generated is accumulated in the body with fat, and this state continues to cause obesity. Obesity is highly correlated with the onset of lifestyle-related diseases such as hypertension, diabetes, and arteriosclerosis. From the point of view of prevention and treatment of such diseases, prevention or elimination of obesity is extremely important. Moreover, prevention or elimination of obesity is extremely important not only from a medical point of view, but also from a cosmetic point of view. In such a social context, various modes of action are known as anti-obesity agents and pharmaceuticals, foods or cosmetics having anti-obesity effects.

Anthraquinone derivatives which exhibit lipase activation (for example, refer to Patent Document 1) and isoquinolines having lipolysis are known as substances related to the mechanism of decomposition of fat. Anisoquinoline alkaloid derivatives (for example, refer to Patent Document 2). In addition, an anti-obesity agent containing a saponin compound containing Ilex paraguariensis as an active ingredient is known as a substance having a lipase-inhibiting effect of a free fatty acid (see, for example, Patent Document 3). A substance in the blood neutral neutral fat reducing agent characterized by a cycloartane-type triterpene or a glycoside thereof, which is caused by a decrease in neutral fat and a cholesterol lowering effect (for example, a reference patent) Document 4). An anthocyanin is known to act through the endocrine factor adiponectin according to the action mechanism of the adipocyte-specific mechanism of action. (cyanidin) compound as an active ingredient of an adiponectin expression promoter (for example, refer to Patent Document 5) and an anti-obesity agent containing an anthocyanin 3-glucoside as an active ingredient (for example) Refer to Patent Document 6). An anti-obesity effect is suppressed by an increase in the number of fat cells, and fat accumulation containing biabolol oxide-A-β-glucoside contained in chamomile is known. Inhibitor (for example, refer to Patent Document 7). A fat metabolism-improving composition containing arctiin and/or arctigenin is known as a substance which inhibits fat accumulation and inhibits the effect of fat cell differentiation (for example, refer to Patent Document 8). . Further, an external preparation having a fat accumulation inhibitory action for inhibiting the action of adipocyte differentiation is known to contain a fat accumulation inhibitor containing an omega-3 polyunsaturated fatty acid as an active ingredient (for example, reference) Patent Document 9).

On the other hand, the white crane ganoderma lucidum (hereinafter also referred to as the white crane ganoderma lucidum), which is also known as "the novel compound A to be described later", according to the present invention, is also known as Rhinacanthus nasutus (L). .) Kurz) is an evergreen shrub belonging to the genus Ganoderma lucidum, which is believed to be native to the Deccan Plateau in southern India. It is known to have insect repellent, anti-inflammatory and antibacterial to skin fungi. The action (for example, refer to Non-Patent Document 1) is mainly used as a Chinese medicine in China, Taiwan, and the like, and has recently been used as a Chinese medicine in Japan. Others, disclosed in the previous application filed by the applicant: Ganoderma lucidum has the ability to remove active oxygen (for example, refer to Patent Document 10); has an effect of promoting excretion (for example, refer to Patent Document 11); has an antiallergic action (for example, refer to Patent Document 12), and has an antitumor effect (for example, refer to Patent Document 13) .

[Patent Document 1] Japanese Patent Laid-Open Publication No. 2006-347952

[Patent Document 2] Japanese Patent Laid-Open Publication No. 2008-308446

[Patent Document 3] Japanese Patent Laid-Open Publication No. 2009-196902

[Patent Document 4] Japanese Patent Laid-Open Publication No. 2006-290882

[Patent Document 5] International Publication No. WO2004/078741

[Patent Document 6] Japanese Patent Laid-Open Publication No. 2003-252766

[Patent Document 7] Japanese Patent Laid-Open Publication No. 2006-213648

[Patent Document 8] Japanese Patent Laid-Open Publication No. 2008-297209

[Patent Document 9] Japanese Patent No. 3607062

[Patent Document 10] Japanese Patent Publication No. 9-143091

[Patent Document 11] Japanese Patent Laid-Open No. 9-169662

[Patent Document 12] Japanese Patent Laid-Open Publication No. 2001-10964

[Patent Document 13] Japanese Patent Laid-Open Publication No. 2002-53481

[Non-Patent Document 1] Original Color Makino and Han Herb Large Illustration, 492 pages, Beilong Pavilion, 1988

However, the white crane ganoderma lucidum plant body, the white crane ganoderma lucidum extract, or the "white crane ganoderma lucidum D" or the "new compound A described later" contained in the white crane ganoderma lucidum plant body or the white crane ganoderma lucidum extract have fat accumulation inhibition. The role (inhibition of adipocyte differentiation, anti-metabolic syndrome) has not been known.

Therefore, an object of the present invention is to provide an "anti-obesity agent" which has no side effects, is safe and has an excellent fat accumulation inhibitory action, and "a pharmaceutical or food containing the anti-obesity agent and having an excellent fat accumulation inhibiting action" Cosmetics."

As a result of searching for a compound having a fat accumulation inhibitory effect, the present inventors have found that the white crane Ganoderma lucidum D and the novel compound A described later have excellent fat accumulation inhibitory action (inhibition of differentiation of the precursor white fat cells). ), the completion of the present invention has been achieved. In order to solve the above problems, the present invention consists of the following matters.

[1] A use of the white crane ganoderma lucidum D represented by the following formula (1) for preparing an anti-obesity agent,

[2] A use of the white crane Ganoderma lucidum D represented by the following formula (1) for preparing an anti-obesity agent having a fat accumulation inhibiting action, [Chemical Formula]

[3] The use of the above-mentioned [1] or [2] white crane ganoderma lucidum D for the preparation of a pharmaceutical, food or cosmetic having a fat accumulation inhibiting action.

Further, the anti-obesity agent of the above [2] may also be referred to as a fat accumulation inhibitor.

According to the present invention, it is also known that the test examples described later provide an "anti-obesity agent which has no side effects, is safe and has an excellent fat accumulation inhibitory action", and "includes the anti-obesity agent and has a fat accumulation inhibiting action. Pharmaceutical, food or cosmetic products."

The white crane Ganoderma lucidum D and/or the novel compound A used in the present invention can be obtained by extracting and purifying the process using Ganoderma lucidum as raw material, and can also be obtained from other plant bodies. Further, in the present invention, white crane ganoderma lucidum D and/or novel compound A obtained by synthesis can also be used. Further, it is also possible to use a white crane ganoderma lucidum or an extract of a plant body containing a white crane ganoderma lucidum D and/or a novel compound A, a crude purified product or a dried matter of a plant body or a paste of a plant body.

When it is extracted and purified from a plant body such as Ganoderma lucidum, it is used in the present invention. When the white crane Ganoderma lucidum D and/or the novel compound A, any extraction and purification process generally used in the industry can be used. After collecting the leaves, stems, roots, flowers, etc. of the raw materials in an appropriate period, they are left untouched or handed over to a drying process which is usually ventilated and dried, and used as an extraction raw material. When extracting the white crane Ganoderma lucidum D and/or the novel compound A from the above-mentioned dried plant body, it can be carried out by referring to a well-known method (for example, refer to "Fight Medicine, Vol. 16, pp. 929-934, 2009").

That is, after the raw material is pulverized or finely cut, extraction is carried out using a solvent, and the solvent may be an alcohol such as water, ethanol, methanol or isopropanol; a ketone such as acetone or methyl ethyl ketone; methyl acetate or ethyl acetate; Esters such as esters; oleophilic solvents such as hexane or chloroform are used singly or as a mixed solvent. The extraction temperature is usually from 0 to 100 ° C, preferably from 5 to 50 ° C. The extraction time is about 1 hour to 10 days, and the amount of the solvent is usually 1 to 30 times by weight, preferably 5 to 10 times by weight, per dry raw material. The extraction operation can be carried out by stirring or by immersion. The extraction operation can be repeated 2 to 3 times as needed. The purification method from the extract obtained by the above operation through filtration or centrifugation to remove the insoluble residue, or the extract of the plant from the juice of the white crane Ganoderma lucidum D and the novel compound A is well known. The separation and purification method of the crude drug may be any method, but the two-phase solvent distribution method, the countercurrent distribution method, the column chromatography method, and the preparative high performance liquid method may be used alone or in combination (preparative high performance liquid) Chromatography and so on. For example, the two-phase solvent distribution method may be exemplified by the above-mentioned extract, which is passed through n-hexane, chloroform, methyl ethyl ketone, ethyl acetate, A method of dispersing a solvent such as methyl acetate or the like with water, a method of recovering a target compound to a solvent phase, and the like. The column chromatography method may be exemplified by ion exchange column chromatography; a method using a positive phase system or a reverse phase silica gel as a carrier; and adsorption column chromatography using DIAION HP-20 or the like. The method may be a gel filtration method using a modified polydextrose gel such as Sephadex LH-20 or the like as a carrier, or may be used singly or in combination or repeatedly. The high-performance liquid chromatography method includes a method of using a column of a reverse phase system such as octadecyl silica, and a method of using a column of a normal phase system such as tantalum or the like. .

The administration route of the anti-obesity agent of the present invention is not particularly limited, and examples thereof include enteral administration such as oral administration and intrarectal administration, mucosal administration by nasal administration, intravenous administration, and the like. Injection administration such as subcutaneous administration or the like. The dosage form of the anti-obesity agent of the present invention may be in the form of a preparation suitable for the administration method, and examples thereof include a tablet, a powder, a fine granule, a granule, a capsule, a powder, a pill, and a lozenge ( A solid agent such as a solution; a solution, a suspension, an emulsion, a syrup, an injection, or the like; a gel-form preparation or the like. The pure product, the purified product, the crude purified product, etc. of the white crane Ganoderma lucidum D and/or the novel compound A may be administered as they are, or may be administered together with a pharmacologically acceptable excipient. When the excipient is a monosaccharide, a disaccharide, a polysaccharide, an inorganic salt, a fat or oil, distilled water or the like, it can be used as a preparation. Additives such as a binder, a lubricant, a dispersing agent, a suspending agent, an emulsifier, a diluent, a buffer, an antioxidant, a bacterial inhibitor, or the like may also be used in the formulation.

The effective dose of Ganoderma lucidum 醌 D and/or novel compound A varies depending on the route of administration, the form of the form, the symptoms of the disease, the age of the subject, and the like, and is usually 0.1 to 1000 mg, preferably 0.5 to 300 mg per day for an adult. More preferably, it is 1~100mg. The content of the white crane Ganoderma lucidum D and/or the novel compound A in the oral anti-obesity agent of the present invention can be set to be most suitable for each administration form according to the form of the preparation, the effective administration amount, and the amount of the preparation as the preparation. The active ingredient content of the formulation.

The form of the food may be in the form of tea, or the dried product of the plant body containing the white crane Ganoderma lucidum D and/or the novel compound A, or the pure product of the white crane Ganoderma lucidum D and/or the novel compound A, A partially purified product of the compound, a crude extract of the compound derived from a plant containing the compound, a plant paste containing the compound, a food product containing the dried plant matter of the compound, and the like.

Tea can also be used alone or in combination with other tea ingredients. If other tea ingredients are green tea, oolong tea, Pu'er tea, black tea, sencha, brown rice tea, eucommia tea, persimmon leaf tea, mulberry leaf tea, etc., which can usually be used as tea, any tea raw material can be used.

When a plant extract is to be obtained, any method can be used if it is used for extraction by ordinary water, extraction by ethanol or aqueous ethanol, and the like. A crude extract or a partially purified product of a plant body derived from the white crane Ganoderma lucidum D and/or the novel compound A can be obtained by a usual method.

The form of the food having the fat accumulation inhibiting action of the present invention, in addition to tea, is a health drink, jelly, biscuit, lozenge, pill, soft gel A capsule, a hard capsule, a powder, a fine granule, a granule, and the like can be usually used as a form of food, and any form can be used. As the auxiliary raw material, an additive such as an excipient, a binder, a lubricant, a dispersing agent, a suspending agent, an emulsifier, a diluent, a buffer, an antioxidant, a bacterial inhibitor or the like can also be used.

The effective intake amount of the white crane Ganoderma lucidum D and/or the novel compound A according to the food having a fat accumulation inhibiting action according to the present invention varies depending on the form of ingestion, the state of health of the subject, the age of the subject, and the like, and is usually 0.001 per day for an adult. ~100 mg, preferably 0.01 to 10 mg, more preferably 0.1 to 1 mg.

The content of the white crane Ganoderma lucidum D and/or the novel compound A in the food having the fat accumulation inhibiting action of the present invention varies depending on the form of the food, and is usually 0.0001 to 1% by weight, preferably 0.001 to 0.5% by weight, more preferably It is 0.01 to 0.1 wt%.

Examples of the form of the external pharmaceutical or cosmetic of the present invention are not particularly limited.

Examples of the form of the external pharmaceuticals include ointments, ointments, cataplasms, tapes, and external preparations. The pharmaceutical product of the present invention, the white crane Ganoderma lucidum D and/or the novel compound A may contain other pharmaceutical ingredients as needed, and a binder, a dispersing agent, a suspending agent, an emulsifier, a diluent, a buffer, an antioxidant may also be used. Additives such as bacterial inhibitors.

The form of the cosmetic may be any form which can be used as an external preparation or a cosmetic preparation, such as a lotion, a cosmetic liquid, an emulsion, a cream, a gel, a pack, a cosmetic powder, a facial cleanser, and a bathing agent. . The above cosmetic preparation is in addition to the pure product of the white crane Ganoderma lucidum D and/or the novel compound A, a partially purified product of the compound, a crude extract of the compound derived from the plant, or In addition to the essential components such as the plant body containing the compound, the components to be blended in the cosmetic preparation may be contained as needed. Examples of the compounding component include solid oil, semi-solid oil, liquid oil, low molecular weight moisturizer, polymer moisturizer, fat-soluble moisturizer, emollient, surface active agent, preservative, and anti-corrosion agent. An antioxidant, a pH adjuster, ethanol, water, and the like.

The effective dose of the white crane Ganoderma lucidum D and/or the novel compound A is different depending on the symptoms of the subject, the age of the subject, and the like, and is usually 0.001 to 100 mg, preferably 0.01 to 10 mg per day for the adult. Good is 0.1~1mg.

The content of the white crane Ganoderma lucidum D and/or the novel compound A in the pharmaceutical or cosmetic having the fat accumulation inhibiting effect of the present invention is usually 0.0001 to 1% by weight, preferably 0.001 to 0.5% by weight, preferably more preferably 0.001 to 0.5% by weight. It is 0.01 to 0.1 wt%.

The following are the following examples of the fractionation, isolation, and fat accumulation inhibition of the white crane Ganoderma lucidum D and the novel compound A, and the white crane Ganoderma lucidum C as a comparative example, test examples and examples of toxicity, and examples. Although the invention is illustrated in more detail, the invention is not limited by such examples.

1. Division and single departure of Baihe Ganoderma lucidum D and novel compound A and white crane Ganoderma lucidum C

1-1, Baihe Ganoderma Lucidum D

(1), the division and single separation of the white crane Ganoderma lucidum

The division and separation of the white crane Ganoderma lucidum D are in accordance with the flow shown in Figure 1. Row. That is, using 25 L of 90% (v/v) ethanol, the dried roots (5 Kg) of Rhinocanthus nastus (L.) Kurz were subjected to a total of three extractions at room temperature for 24 hours, and the materials were combined. The mixture was concentrated to give a dry solid (407 g).

Subsequently, the suspension was dissolved in 7 L of 90% (v/v) methanol, and then partitioned three times with an equal amount of hexane, and then a 90% (v/v) methanol phase was concentrated and concentrated under reduced pressure. Purified water was added to the reduced pressure concentrate to fill 5 L, and transferred to a separatory funnel for 3 times of two-phase solvent partitioning with 5 L of chloroform. Next, the chloroform obtained by this operation was combined to obtain one dry matter, and 69.3 g of a dry solid was obtained.

69.0 g of the mixture was subjected to a gel column chromatography (80 mm φ × 150 mm, manufactured by Kanto Chemical Co., Ltd.) using hexane/ethyl acetate as a solvent. That is, after washing the ruthenium column with 3 bed volume (BV: Bed Volume) of the solvent hexane/ethyl acetate (9:1), the solvent was dissolved in 1 BV hexane/ethyl acetate (8:2). The dissolution was carried out to obtain a fraction A (dry solid 4.71 g).

Next, Sephadex LH-20 column chromatography (20 mm φ × 200 mm, manufactured by Pharmacia Co., Ltd.) using methanol as a solvent was carried out for Part A. That is, after washing the Sephadex LH-20 column with 1.5 BV of methanol, it was eluted with 0.5 BV of methanol to obtain a portion A-2 (1.34 g).

Further, Part A-2 was divided by Flash ODS column chromatography (20 mm φ × 150 mm, manufactured by Wako Pure Chemical Industries, Ltd.) using water/methanol as a solvent. That is, the Flash ODS tube is used in 180 ml of 50% (v/v) methanol. After the column was washed, it was gradually eluted with 60% (v/v) methanol and 70% (v/v) methanol, and dissolved in 80% (v/v) methanol to obtain a fraction A-2- containing the objective compound. 2 (dry solid 407 mg).

Further, preparative high performance liquid chromatography (ODS column, 20 mm φ×250 mm, manufactured by Nomura Chemical Co., Ltd., mobile phase: 72% (v/v) acetonitrile/water/0.1% (v/v) formic acid, detection: A portion of A-2-2 (dry solid 407 mg) was purified by a 254 nm UV monitor to obtain a white crane Ganoderma lucidum D (dry solids 18.8 mg).

The ¹ H NMR spectrum was obtained by nuclear magnetic resonance spectroscopy for the white crane Ganoderma lucidum D, which was divided and separated by the above method, and the following peaks were observed, and the literature (Journal of Natural Products) The values in Volume 59, pages 808 to 811, and 1996) are roughly the same.

δ 1.06 (H-, 6H, s), 2.74 (H-, 2H, s), 4.03 (H-, 2H, s), 5.97 (H-, 2H, s), 6.70 (H-, 1H, d) , 7.38 (H-, 1H, s), 7.56 (H-, 1H, d), 7.63 (H-, 1H, t), 7.67 (H-, 1H, t), 8.02 (H-, 2H, d) .

Further, in the above, high performance liquid chromatography was carried out using a Waters 515 system and a Waters 600 system (all manufactured by Japan Waters Co., Ltd.). Moreover, general-purpose experimental instruments and experimental apparatus were used for the ruthenium column chromatography, the Sephadex LH-20 column chromatography, and the Flash ODS chromatography.

1-2, novel compound A

(1), the division and single departure of the novel compound A

The division and separation of the novel compound A are in accordance with the flow shown in Figure 1. Row. That is, using 25 L of 90% (v/v) ethanol, the dried roots (5 Kg) of Rhinocanthus nastus (L.) Kurz were subjected to a total of three extractions at room temperature for 24 hours, and the materials were combined. The mixture was concentrated to give a dry solid (407 g).

Subsequently, the suspension was dissolved in 7 L of 90% (v/v) methanol, and then partitioned three times with an equal amount of hexane, and then a 90% (v/v) methanol phase was concentrated and concentrated under reduced pressure. Purified water was added to the reduced pressure concentrate to fill 5 L, and transferred to a separatory funnel to carry out three-phase solvent distribution three times with chloroform. Next, the chloroform obtained by this operation was combined to obtain one dry matter, and 69.3 g of a dry solid was obtained.

69.0 g of the mixture was subjected to a gel column chromatography (80 mm φ × 150 mm, manufactured by Kanto Chemical Co., Ltd.) using hexane/ethyl acetate as a solvent. That is, in a solvent solution of 3BV in hexane/ethyl acetate (9:1), 2BV in hexane/ethyl acetate (8:2), 2BV in hexane/ethyl acetate (7: 3) After washing the ruthenium rubber column in this order, it was eluted with 2BV of a solvent solvent hexane/ethyl acetate (6:4) to obtain a part C (dry solid 3.73 g).

Next, Sephadex LH-20 column chromatography (20 mm φ × 200 mm, manufactured by Pharmacia Co., Ltd.) using methanol as a solvent was carried out for Part C. That is, after washing the column with 2 BV of methanol, it was eluted with 1 BV of methanol to obtain a part F (dry solid 369 mg).

Further, Part F (dry solid 369 mg) was subjected to Flash ODS column chromatography (20 mm φ × 150 mm, manufactured by Nomura Chemical Co., Ltd.) using water/methanol as a solvent. That is, after washing the Flash ODS column with 180 ml of 50% (v/v) methanol, the steps were sequentially followed by 60% (v/v) methanol, 70% (v/v). After washing with methanol, it was dissolved in 80% (v/v) methanol to obtain a portion G (dry solids: 75.6 mg) containing the compound.

Further preparative high performance liquid chromatography (ODS column, 20mm φ × 250mm, manufactured by Nomura Chemical Co., mobile phase: 45% (v / v) acetonitrile, detection: 254nm UV monitor) on part of G (dry solid) Purification of 75.6 mg) gave the dried solid (9.8 mg) of Compound A.

(2) Structural analysis of novel compound A

Structural analysis of novel compound A was carried out using high resolution mass spectrometry (HRFABMS) and nuclear magnetic resonance spectroscopy ( ¹ H NMR, ¹³ C NMR). The results are shown below.

(2-1) Results obtained by high-resolution mass spectrometry (HRFABMS)

In the high-resolution mass spectrometry (HRFABMS), "m/z 272.1039 [M] ⁺ (calcd. 272.1049 △ 0.9 mmu)." was observed, and the molecular formula was found to be "C ₁₆ H ₁₆ O ₄ ". Further, in the low-resolution mass spectrometry (LRFABMS), "m/z 272." was observed.

(2-2) Results obtained by nuclear magnetic resonance spectroscopy ( ¹ H NMR)

In the nuclear magnetic resonance spectroscopy ( ¹ H NMR), the following peaks were observed.

" ¹ H NMR (CDCl _3, 500 MHz): δ 3.69 (H-14, J = 11.2 Hz, 1H, d), 3.75 (H-14, J = 11.2 Hz, 1H, d), 3.95 (8-OMe, 3H, s), 4.22 (H-2, J = 11.7 Hz, 1H, d), 4.31 (H-2, J = 11.7 Hz, 1H, d), 5.93 (H-4, J = 11.7 Hz, 1H, d), 6.43 (H-5, J = 11.7 Hz, 1H, d), 6.51 (H-7, 1H, s), 7.47 (H-10, J = 1.5, 6.7, 7.5 Hz, 1H, m), 7.50 (H-11, J=1.5, 6.7, 7.5 Hz, 1H, m), 8.14 (H-9, J=1.5, 7.5 Hz, 1H, d), 8.24 (H-12, J=1.5, 7.5 Hz) , 1H, d)."

(2-3) Results obtained by nuclear magnetic resonance spectroscopy ( ¹³ C NMR)

In the nuclear magnetic resonance spectroscopy ( ¹³ C NMR), the following peaks were observed.

" ¹³ C NMR (CDCl _3, 125 MHz): δ 55.7 (8-OMe), 65.9 (C-14), 74.4 (C-2), 74.9 (C-3), 106.8 (C-7), 119.1 (C -6), 121.6 (C-9), 122.7 (C-12), 126.0 (C-12a), 126.3 (C-10), 126.7 (C-11), 127.6 (C-8a), 130.1 (C- 5), 132.1 (C-4), 148.9 (C-13), 150.6 (C-8)."

Further, in the above, the high-resolution mass spectrometer was a JEOL JMS SX-102 mass spectrometer (manufactured by JEOL Ltd.). Moreover, nuclear magnetic resonance spectroscopy apparatus ⁽¹ H NMR and ¹³ C NMR) using JEOL JNM-GSX500 means nuclear magnetic resonance spectroscopy (JEOL Co., Ltd.).

From the above results, HMQC spectrum and HMBC spectrum, the novel compound A is a novel compound represented by the above formula (2).

2. Test example of fat accumulation inhibition

The test for inhibiting the accumulation of fat was carried out by measuring the fat accumulation inhibitory activity against the precursor white fat cells derived from SD rats (Sprague-Dawley rat).

(1) Test method

Precursor white fat cells (purchased from TAKARA BIO Co., Ltd., Japan) from SD rats were subjected to subculture once, and then stored in a liquid nitrogen container and stored for subsequent experiments. The frozen cells were melted in a warm water at 37 ° C according to a usual method, and suspended in a minimal medium at a cell concentration of 1 × 10 ⁵ cells/ml [ascorbic acid, biotin, pantothenic acid) Acid), triiodothyronine, octanoic acid, fetal bovine serum, penicillin (50U/ml), streptomycin (50μg/ml) added to high Glucose DMEM (Dulbecco's Modified Eagle's Medium) medium (manufactured by Lonza Co., Ltd.), each disseminate 1 ml in a 24-well culture plate (1 cm ² /well, NUNC) at 37 ° C, 5% carbonic acid gas ( The culture is carried out in the presence of carbonic acid gas). The basic medium was aspirated at a time point when the cells proliferated to 90% to 95% confluent, and the differentiation medium was added [insulin (10 μg/ml), dexamethasone (2.5 μM), 3-isobutyl- 3-isobutyl-1-methylxanthine (0.5 mM) was added to the minimal medium], and further culture was continued for 48 hours at 37 ° C in the presence of 5% carbonic acid gas. After 48 hours of culture in the differentiation medium, the differentiation medium was aspirated, washed twice with calcium-free, magnesium-PBS (Dulbecco's Phosphate-Buffered Saline) solution (hereinafter PBS), and then added to the white crane containing Ganoderma lucidum.醌D or novel compound A (all dissolved in DMSO (dimethyl sulfoxide: dimethyl sulfoxide), the final DMSO concentration in the medium = 0.2% (v / v), in addition, by adding only 1 / 500 volume of DMSO The cell culture medium was used as a control medium (maintenance medium) [10 μg/ml insulin was added to the minimal medium], and further cultured at 37 ° C, 5% carbonation gas for 6 days.

After the culture for 6 days, the medium was aspirated, washed twice with PBS, and 200 μl of Mildform (manufactured by Wako Pure Chemical Industries, Ltd.) was added to each well, and the cells were fixed by standing at 4 ° C for 2 hours. Next, Mildform was aspirated and washed once with 60% (v/v) isopropanol. Then, 100 μl of the prepared oil red O reagent was added to each well [2/3 amount of ultrapure water was added to dissolve the oil red O in isopropanol at a concentration of 0.5% (w/v). The stock solution was allowed to stand at room temperature for 15 minutes, filtered through a membrane filter (0.45 μm) and prepared, and allowed to stand at room temperature for 15 minutes. After standing for 15 minutes, the cells were washed twice with 60% (v/v) isopropanol, and after drying the medium, 100 μl of isopropanol was added to each well and shaken to elute the oil red O pigment. 80 μl of the pigment solution was collected in a 96-well microplate, and the absorbance at 540 nm was measured using an immuno reader (manufactured by Dainippon Pharmaceutical Co., Ltd.). The relative accumulation value of the neutral fat was calculated from the absorbance.

(2) Test results

Table 1 shows the relative values of the "neutral fat accumulation value of the precursor white fat cells" of the white crane Ganoderma lucidum D and the novel compound A and the white crane Ganoderma lucidum C as a comparative example.

As shown in Table 1, it was confirmed that the white crane Ganoderma lucidum D and the novel compound A exhibited excellent fat accumulation inhibition at a low concentration of 10 μg/ml (excellent neutral fat accumulation inhibitory activity against the precursor white fat cells). .

3. Toxicity test cases

The toxicity test was carried out by measuring the proliferation inhibitory activity against the precursor white fat cells derived from SD rats.

(1) Test method

The precursor white fat cells (purchased from TAKARA BIO Co., Ltd., Japan) from SD rats were subcultured once, and then stored in a liquid nitrogen container and stored for subsequent experiments. The frozen cells were melted in a warm water at 37 ° C according to a usual method, using a minimal medium [ascorbic acid, biotin, pantothenic acid, triiodothyronine, caprylic acid, fetal bovine serum, penicillin (50 U/ml), streptomycin ( 50 μg/ml) was added to high glucose DMEM medium (manufactured by Lonza Co., Ltd.), and dispersed in a 96-well culture plate (NUNC) at a cell concentration of 1 × 10 ⁴ cells/100 μl, and carried out in the presence of 5% carbonation gas at 37 ° C for 48 hours. to cultivate. After 48 hours of culture, the white crane Ganoderma lucidum D or the novel compound A (all dissolved in DMSO, the final DMSO concentration in the medium = 0.1% (v/v) was added, in addition, by adding only 1/1000 volume of DMSO. The cell culture medium was used as a control group, and further cultured in the presence of 5% carbonation gas at 37 ° C for 48 hours.

The degree of cell proliferation was determined by using (MTT: thiazole blue) [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Nacalai Tesque] ( Method of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Nacalai Tesque) [Refer to Tokyo Chemical Co., Ltd., New Biochemistry Experiment Lecture 12 Molecular Immunology I Immune Cells, Cellular media (cytokine) pp. 358-359]. That is, after the culture of each compound added to the toxicity test object for 48 hours, the medium was changed, and 10 μl of the MTT solution [5 mg/ml was dissolved in 90 μl of the culture solution of each well of a 96-well culture plate (0.33 cm ² /well). After the solution of calcium-free, magnesium-PBS (Dulbecco's Phosphate-Buffered Saline), it was filtered through a membrane filter (0.22 μm), and shaken to homogenize it at 37 ° C in 5% carbonic acid gas. Incubate for 4 hours. After incubation for 4 hours, 10% (w/v) SDS-50% (v/v) N,N-dimethylformamide-0.005N hydrochloric acid solution 100 μl was added to each well at 37 ° C. After standing for 18 hours in the presence of 5% carbonic acid gas, the absorbance at 590 nm was measured using an immunoreader (manufactured by Dainippon Pharmaceutical Co., Ltd.) at 750 nm as an index of survival rate.

(2) Test results

The "survival rate of the precursor white fat cells" showing the white crane Ganoderma lucidum D and the novel compound A and the white crane Ganoderma lucidum C as a comparative example are shown in Table 2.

As shown in Table 2, it was confirmed that the white crane Ganoderma lucidum D and the novel compound A showed low toxicity (low proliferation inhibitory activity against the precursor white fat cells).

[Examples] Example 1. Preparation of a tablet

The white crane Ganoderma lucidum D, which is divided and separated according to the method described in "1-1, White Crane Ganoderma lucidum D (1), White Crane Ganoderma lucidum 醌 D, and separate), is produced by the following prescription. Lozenges (500 mg per spindle).

(modulation method)

Ganoderma lucidum D (0.2 g), dried corn starch (2 g), talc (1.8 g), and calcium stearate (0.2 g) were added to lactose (95.8 g) for mixing. Next, a tablet was produced by a usual method using a single stroke tablet press.

Example 2, production of hard capsules

The novel compound A which was divided and isolated according to the method described in "1-2, Novel Compound A (1), Novel Compound A Division, Separation" described above was used to prepare a hard capsule by the following prescription. (360 mg per capsule).

(modulation method)

Lactose (220 g) and corn starch (110 g) were added to and mixed with the novel compound A (5 g), and an aqueous solution of hydroxypropylcellulose (25 g) was added thereto and kneading was carried out. Next, pellets were produced by a usual method using an extrusion granulator. A hard capsule is prepared by filling the granules into a gelatin hard capsule.

Example 3, production of soft capsules

The white crane Ganoderma lucidum D, which is divided and separated according to the method described in "1-1, White Crane Ganoderma lucidum D (1), White Crane Ganoderma lucidum 醌 D, and separate), is produced by the following prescription. Soft capsules (170 mg per capsule).

(modulation method)

White crane Ganoderma lucidum D (0.5 g) was added to soybean oil (169.5 g) and mixed. Then, by using a rotary soybean type automatic molding machine, as usual Fill the soft capsule with a method to make a soft capsule.

Example 4, preparation of pills

The white crane Ganoderma lucidum D, which is divided and separated according to the method described in "1-1, White Crane Ganoderma lucidum D (1), White Crane Ganoderma lucidum 醌 D, and separate), is produced by the following prescription. Pills (100 mg each).

(modulation method)

The raw materials were mixed by the above mixing, water was added in an appropriate amount, and a homogenous kneaded product was produced by a kneader, and the obtained kneaded product was rolled, pelletized by a pelletizer, and dried to prepare a pellet.

Example 5, production of powder

Using the method described in "1-1, White Crane Ganoderma lucidum D (1), White Crane Ganoderma lucidum 醌 D division, single separation", the white crane Ganoderma lucidum 醌 D is divided and separated by the following prescription. The usual method is to make powder (1000mg per pack).

Example 6, the production of jelly

Using the method described in "1-1, White Crane Ganoderma lucidum D (1), White Crane Ganoderma lucidum 醌 D division, single separation", the white crane Ganoderma lucidum 醌 D is divided and separated by the following prescription. Common methods are used to make jelly (100g).

(Modulation method).

The above ingredients were mixed and heated to 90 °C. After confirming the dissolution of the gelatin, it was filled in a container and cooled. Jelly is made by curing gelatin.

Example 7, production of ointment

Using the method described in "1-1, White Crane Ganoderma lucidum D (1), White Crane Ganoderma lucidum 醌 D division, single separation", the white crane Ganoderma lucidum 醌 D is divided and separated by the following prescription. A common method is used to make an ointment (100 g).

(oil phase component)

(aqueous phase component)

(modulation method)

The oil phase component and the water phase component were separately heated to 80 ° C and made uniform, and the aqueous phase was added to the oil phase while stirring, and the mixture was emulsified and cooled to prepare an ointment.

Example 8 Production of a tape agent

The novel compound A which was divided and isolated according to the method described in "1-2, Novel Compound A (1), Novel Compound A, and Separation" described above was prepared by the usual method by the following prescription. Tape agent (100g).

(adhesive solvent)

(medicinal ingredients)

Novel Compound A 0.1g

(modulation method)

The adhesive solvent and the medicinal ingredient are uniformly dispersed, and the medicinal ingredient and the percutaneous absorption enhancer are added to the adhesive solvent, and the composition is stirred at room temperature. The composition was stretched on a polyester film subjected to a silicone treatment, dried and cooled at 120 ° C, and then the adhesive layer was transferred to a polyethylene film to prepare a tape. Agent.

Example 9, Modulation of slimming lotion

Using the method described in "1-1, White Crane Ganoderma lucidum D (1), White Crane Ganoderma lucidum 醌 D division, single separation", the white crane Ganoderma lucidum 醌 D is divided and separated by the following prescription. A commonly used method for making slimming lotion (100g).

(oil phase composition)

(water phase composition)

(modulation method)

The oil phase component and the water phase component were uniformly dissolved, and the oil phase was added to the water phase while stirring to prepare a lotion.

Example 10: Production of massage gel for slimming

The novel compound A which was divided and isolated according to the method described in "1-2, Novel Compound A (1), Novel Compound A, and Separation" described above was prepared by the usual method by the following prescription. Massage gel (100g) for slimming.

(oil phase composition)

(water phase composition)

Dipropylene glycol 5.0g

(modulation method)

The oil phase component and the water phase component were separately heated to 80 ° C and made uniform, and the aqueous phase was added to the oil phase while stirring, and the mixture was emulsified and cooled to prepare a massage gel.

Figure 1 is a flow chart of the division of the white crane ganoderma lucidum root, the single white crane Ganoderma lucidum D and the novel compound.

Claims (3)

A use of the white crane ganoderma lucidum D represented by the following formula (1) for the preparation of an anti-obesity agent, A use of the white crane ganoderma lucidum D represented by the following formula (1) for preparing an anti-obesity agent having a fat accumulation inhibiting action, The use of the white crane ganoderma lucidum D in the first or second aspect of the patent application is for the preparation of a pharmaceutical, food or cosmetic having a fat accumulation inhibiting effect.