JP2011256133A - Sr-b1 protein expression enhancer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel application of Xanthohumol related with a SR-B1 protein expression enhancer.SOLUTION: The SR-B1 protein expression enhancer is provided, which contains a compound represented by general formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient. In formula, Ris a hydrogen atom or a 1-5C alkyl group, and Ris a dimethyl allyl group or a geranyl group.

Description

  The present invention relates to an SR-B1 protein expression enhancer.

  Xanthohumol, a prenylflavonoid contained in hops, has been reported to have an action of suppressing the growth of cultured cells derived from breast cancer, colon cancer, and ovarian cancer (for example, Non-Patent Document 1). reference). Xanthohumol also has an action of suppressing the production of inflammatory interleukin (Patent Document 1), an action of suppressing the production of TNF-α (Patent Document 2), and an action of enhancing the production of adiponectin (Patent Document 3). It is known to have.

JP2008-120720A JP2008-106013 JP2007-262050

Food and Chemical Toxiology 37, 271-285, 1999

  An object of this invention is to provide the novel use of xanthohumol.

  The present inventors have obtained a novel finding that xanthohumol has an action of enhancing the expression of SR-B1 protein that functions as a HDL (high density lipoprotein) receptor in the liver and adrenal gland. The present invention is based on these novel findings.

That is, the present invention provides an SR-B1 protein expression enhancer containing a compound represented by the following general formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient. Here, in the general formula (1), R ¹ represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms, and R ² represents a dimethylallyl group or a geranyl group.

The compound represented by the general formula (1) is preferably a compound represented by the following formula (2), that is, xanthohumol.

  The SR-B1 protein expression enhancer of the present invention enhances the expression of SR-B1 protein by containing the compound represented by the above general formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient. be able to. The SR-B1 protein is a receptor protein that has a function of binding to HDL and selectively taking cholesteryl ester of HDL into cells in the liver or adrenal gland. HDL plays a central role in the so-called reverse cholesterol transport system that transfers extra cholesterol from peripheral tissues to the liver for catabolism or excretion. The SR-B1 protein expression enhancer of the present invention enhances the expression of SR-B1 protein and increases the uptake of cholesteryl ester from HDL cholesterol. This causes activation of the reverse cholesterol transfer system. Therefore, the SR-B1 protein expression enhancer of the present invention is used for the prevention or improvement of diseases that can be expected to be prevented or ameliorated by enhancing the expression of SR-B1 protein or activating the reverse cholesterol transfer system. Can do. The SR-B1 protein expression enhancer of the present invention can also be used in kits useful for studying the function of SR-B1 protein, studying the mechanism of cholesterol metabolism centering on the reverse cholesterol transfer system, and the like.

  Xanthohumol, which is one of the compounds represented by the general formula (1), is a component found in hops that have long been used as a raw material for food and drink such as beer, and its safety has also been established. . From this, it is thought that the SR-B1 protein expression enhancer of this invention is suitable for using as components, such as a pharmaceutical and food-drinks, for example.

  According to the present invention, a novel SR-B1 protein expression enhancer is provided.

It is a figure which shows the result of the western blot which analyzed SR-B1 protein expression in a human liver cancer origin cell line. It is a figure which shows the result of the Western blot which analyzed SR-B1 protein expression in a hamster. It is a graph which shows the blood total cholesterol level. It is a graph which shows a blood HDL cholesterol level. It is a graph which shows aortic deposition cholesterol value. It is a graph which shows the correlation with an aortic deposition cholesterol level and a blood HDL cholesterol level.

  Hereinafter, embodiments of the present invention will be described.

  In the present specification, the enhanced expression of SR-B1 protein means that the expression level of SR-B1 protein increases (the abundance increases) through enhanced production of SR-B1 protein or suppression of degradation.

The SR-B1 protein expression enhancer of the present invention contains a compound represented by the following general formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

In the general formula (1), R ¹ represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms, and R ² represents a dimethylallyl group or a geranyl group. Here, the alkyl group having 1 to 5 carbon atoms may be linear or branched. Examples of the alkyl group having 1 to 5 carbon atoms include methyl group, ethyl group, 1-methylethyl group (isopropyl group), 1,1-dimethylethyl group (tert-butyl group), propyl group, 1- Examples include methylpropyl group (sec-butyl group), 2-methylpropyl group (isobutyl group), 2,2-dimethylpropyl group (neopentyl group), butyl group, 3-methylbutyl group, and pentyl group.

As the compound represented by the general formula (1), for example, a compound in which R ¹ is a hydrogen atom or an alkyl group having 1 to 3 carbon atoms (methyl group, ethyl group, 1-methylethyl group, propyl group) is preferable. A compound in which R ¹ is a hydrogen atom, a methyl group or an ethyl group and R ² is a dimethylallyl group is particularly suitable. Xanthohumol (a compound represented by the following formula (2)), which is a hop-specific flavonoid, is one of particularly suitable compounds.

  The compound represented by the general formula (1) may be contained in the SR-B1 protein expression enhancer as a pharmaceutically acceptable salt. Such a pharmaceutically acceptable salt is formed by the compound represented by the general formula (1) and a base that forms a nontoxic salt. Specific examples include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as magnesium salt and calcium salt, ammonium salt and the like.

  The compound represented by the general formula (1) may be derived from a natural product (plant, microorganism, etc.) or artificially synthesized. Moreover, if there exists a commercially available thing, you may use it.

  For example, xanthohumol can be obtained by fractionating and purifying a hop extract. As the hop extract, for example, a commercially available hop extract may be used.

  When extracting from a hop, as a hop structure | tissue used for extraction, a spikelet is preferable. The hop may be subjected to treatments such as drying, freezing, processing, pulverization, and selection, and for example, hop pellets may be used.

  The variety of hops is not particularly limited, and may be any of existing varieties (for example, Czech Saats, German Haratau Magnum, German Haratau Tradition, German Perrelet). One kind may be used alone, or two or more kinds may be used in combination.

  Extraction from the hop can be performed, for example, by immersing the hop in a solvent and filtering it. As the solvent, for example, water, ethanol, and methanol are suitable. A solvent may be used individually by 1 type, or may be used in combination of 2 or more type. Ultrasonic treatment may be performed during the immersion. The obtained extract may be concentrated or dried by a known method (for example, concentration under reduced pressure, lyophilization), and further processing such as pulverization may be performed.

  Fractionation and purification of xanthohumol from the hop extract can be performed, for example, as follows. That is, first, an aqueous solution or aqueous suspension of a hop extract is distributed with hexane, and the obtained aqueous layer is distributed with ethyl acetate (pH 3). And the obtained organic layer is further distributed with saturated sodium hydrogencarbonate aqueous solution (pH 8-9), and the newly produced organic layer is collect | recovered. Next, this organic layer was subjected to silica gel column chromatography [diethyl ether / hexane (3: 7) → ethyl acetate / hexane (4: 6) → ethyl acetate / hexane (6: 4) → chloroform / methanol (5 : 5)] to fractionate the xanthohumol-eluted fraction. Finally, it is further purified using an ODS column or by recrystallization.

  The temperature during the above extraction, fractionation, and purification is preferably from 5 to 65 ° C, more preferably from 5 to 50 ° C, from the viewpoint of preventing isotoxol from isolating.

  Whether the obtained compound is xanthohumol can be confirmed by a known method (for example, mass spectrometry, elemental analysis, nuclear magnetic resonance spectroscopy, ultraviolet spectroscopy, infrared spectroscopy).

  The SR-B1 protein expression enhancer may contain only one type of the compound represented by the general formula (1) or a pharmaceutically acceptable salt thereof, or may contain two or more types. Moreover, you may further contain the other compound which has SR-B1 protein expression enhancement effect.

  The SR-B1 protein expression enhancer may be in the form of a solid (for example, powder), liquid (water-soluble or fat-soluble solution or suspension), paste, etc., and powders, granules, tablets, capsules Any dosage form such as an agent, a liquid, a suspension, an emulsion, an ointment, a plaster may be used. It can also take the form of a controlled release formulation. Furthermore, it may consist only of the compound represented by the general formula (1) or a pharmaceutically acceptable salt thereof.

  The above-mentioned various preparations include a compound represented by the general formula (1) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable additive (excipient, binder, lubricant, disintegrant, emulsifier). , Surfactants, bases, solubilizers, suspending agents, and the like).

  For example, the excipient includes lactose, sucrose, starch, dextrin and the like. Examples of the binder include polyvinyl alcohol, gum arabic, tragacanth, gelatin, hydroxypropylmethylcellulose, hydroxypropylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone and the like. Examples of the lubricant include magnesium stearate, calcium stearate, talc and the like. Examples of the disintegrant include crystalline cellulose, agar, gelatin, calcium carbonate, sodium bicarbonate, dextrin and the like. Examples of the emulsifier or surfactant include Tween 60, Tween 80, Span 80, and glyceryl monostearate. Examples of the base include cetostearyl alcohol, lanolin, polyethylene glycol, rice bran oil, fish oil (DHA, EPA, etc.), olive oil and the like. Examples of the solubilizer include polyethylene glycol, propylene glycol, sodium carbonate, sodium citrate, Tween 80 and the like. Examples of the suspending agent include Tween 60, Tween 80, Span 80, glyceryl monostearate, polyvinyl alcohol, polyvinyl pyrrolidone, methyl cellulose, hydroxymethyl cellulose, sodium alginate and the like.

  The SR-B1 protein expression enhancer can be used as a component of pharmaceuticals, foods and drinks (beverages, foods), food and drink additives, feeds, feed additives, and the like. For example, examples of the beverage include water, soft drinks, fruit juice beverages, milk beverages, alcoholic beverages, sports drinks, and nutritional drinks. Examples of foods include breads, noodles, rice, tofu, dairy products, soy sauce, miso, and confectionery. Moreover, it can also be used as a component of food for specified health use, food for special use, dietary supplement, health food, functional food, food for patients and the like.

  The SR-B1 protein expression enhancer may be ingested by humans or non-human mammals. The intake amount and the intake method can be appropriately determined according to the state, age, etc. of the individual. As a suitable intake method, for example, oral intake can be mentioned.

  The SR-B1 protein expression enhancer of the present invention has an action of enhancing the expression of SR-B1 protein both in vivo and in vitro. SR-B1 protein is a protein that acts as an HDL receptor and is involved in the so-called reverse cholesterol transport system. The SR-B1 protein expression enhancer of the present invention can be used for the prevention or improvement of diseases that can be prevented or ameliorated by enhancing the expression of SR-B1 protein or activating the reverse cholesterol transfer system. The SR-B1 protein expression enhancer of the present invention can also be used in kits used for studies such as functional analysis of SR-B1 protein, or studies on the mechanism of cholesterol metabolism centering on the reverse cholesterol transfer system. . The kit contains the SR-B1 protein expression enhancer of the present invention, and SR-B1 in the target sample by administration to the target sample (for example, cultured cells, mice, hamsters, rats, monkeys, humans, etc.). It can be suitably used for studies that enhance the expression of proteins and analyze their effects.

  Hereinafter, based on an Example, this invention is demonstrated more concretely. However, the present invention is not limited to the following examples.

[Example 1]
Enhanced expression of SR-B1 protein in human liver cancer cell lines (purification of xanthohumol)
The hop was immersed in methanol and sonicated to obtain a hop extract containing xanthohumol. This was purified by HPLC to prepare xanthohumol standard. This was serially diluted in 5% aqueous dimethyl sulfoxide (DMSO) solution to obtain a test sample having a predetermined concentration. HPLC was performed under the following conditions.
HPLC conditions:
Column: CAPCELL PAK C18 (φ1cm) (Shiseido)
Mobile phase: 77.5% CH3OH / 5% CH3COOH
Flow rate: 3 mL / min

(Cell culture)
The human liver cancer-derived cell line HepG2 (RIKEN Cell Bank, RCB1886) was cultured in a DMEM medium containing 10% FBS in a 5% CO ₂ incubator at 37 ° C. When it reached 100% confluence, xanthohumol (XN) was added to the medium so that the predetermined concentrations (0, 1.8, and 3.5 μM) were obtained, followed by further culturing for 3 days.

(Western blot analysis)
After culture, the medium was discarded and the cells were washed with PBS. Proteins were recovered from the cells using a cell protein recovery solution (PRO-PREP, Cosmo Bio) and subjected to Western blotting analysis. Western blot detection uses an anti-SR-B1 antibody (abcam, ab396) and an anti-β-actin antibody (abcam, ab6276) as the primary antibody, and an HRP-conjugated anti-rabbit IgG antibody (abcam) as the secondary antibody. This was performed by chemiluminescence (ECL) method using ab 6721).

(result)
The result of Western blotting is shown in FIG. The graph in FIG. 1 represents the value of SR-B1 emission intensity relative to the emission intensity of β-actin (Actb) as a relative value when CNT (XN concentration is 0 μM) is 1. In addition, a protein recovered from untreated HepG2 cells was used as a negative control (NC). In addition, the value of a graph represents the average value of the test result of 4 times performed independently, and an error bar represents the standard deviation.

  The cultured cells to which xanthohumol was added (XN 1.8 μM and 3.5 μM. In the graph, expressed as 1.8 and 3.5, respectively) were compared with CNT and NC to which xanthohumol was not added, The expression level of SR-B1 protein was significantly increased (FIG. 1).

[Example 2]
Increased expression of SR-B1 protein in high cholesterol diet model animals (grouping of test animals)
16 hamsters (5 weeks old, male, SPF Syrian hamsters, Japan SLC), weight and lipid parameters (total blood cholesterol level, triglyceride level and HDL cholesterol level) at the start of the test should not vary between groups Each group was divided into two groups of 8 animals (control group (CNT group), xanthohumol-administered group (XN group)). The hamster was used for the test at the age of 6 weeks after acclimation breeding for 1 week.

(Preparation of test feed)
The test feed (diet) was prepared to have the composition shown in Table 1 (low fat, high cholesterol). In Table 1, the unit of each component amount is g. Moreover, CRF-1 (Oriental Yeast Industry) was used as a feed during the acclimatization breeding period.

(Raising conditions)
After one week of acclimatization, each group of hamsters was allowed to freely feed and drink the test feed and tap water for 14 days. Throughout the acclimatization period and the subsequent test feed administration period, hamsters were individually housed (1 animal / cage) in a breeding apparatus (temperature 23 ± 5 ° C., relative humidity 55 ± 10%, Inocage, Oriental Giken).

(Xanthohumol administration)
Xanthohumol (85% purity xanthohumol powder, Hopsteiner) was dissolved in DMSO containing 2% (v / v) Tween 20 so that the dose was 5 mg / kg-body weight / day, and osmotic pressure A pump (ALZET® model 2006) was filled and embedded in the back of the hamster by surgery prior to the start of the study.

(Measurement of SR-B1 protein)
14 days after the start of test feed administration, for each group of hamsters, protein was collected from the liver tissue using a cellular protein collection solution (PRO-PREP, Cosmo Bio), and Western blotting analysis was performed in the same manner as in Example 1. went.

(result)
The result of the Western blot is shown in FIG. The graph in FIG. 2 represents the value of the luminescence intensity of SR-B1 relative to the luminescence intensity of β-actin (Actb) as a relative value when the CNT group is 1. Moreover, the value of a graph represents the value which averaged the measurement result about 8 animals in each group, and an error bar represents the standard deviation.

  In the hamster group (XN group) to which xanthohumol was administered, the expression level of SR-B1 protein was significantly increased compared to the CNT group (FIG. 2). From the results of Examples 1 and 2, it was confirmed that xanthohumol has an SR-B1 protein expression enhancing action.

Example 3
Reduction of blood HDL cholesterol and suppression of blood vessel cholesterol deposition For the hamster of Example 2 (CNT group and XN group), blood cholesterol and thoracic aortic arch cholesterol deposition were measured.

(Measurement of blood cholesterol)
At the start of the test and 7 days after the start of administration of the test feed, each group of hamsters was bled from the orbit using heparinized capillaries. In addition, 14 days after the start of test feed administration, heart blood was collected from each group of hamsters under diethyl ether anesthesia. The collected blood was centrifuged (5,000 × g, 5 minutes, room temperature) and separated from blood cells, and then subjected to cholesterol measurement. Total cholesterol level and HDL cholesterol level are measured using Cholesterol E-Test Wako (Wako Pure Chemical Industries, Code 437-1501), HDL-Cholesterol E-Test Wako (Wako Pure Chemical Industries, Code 431-52501). did. In addition, the precipitation operation of lipoproteins other than HDL at the time of measuring the HDL cholesterol value was performed by the polyethylene glycol precipitation method without using the precipitation reagent attached to the kit.

(Measurement of aortic deposited cholesterol)
14 days after the start of test feed administration, the hamsters in each group were thoracotomized under diethyl ether anesthesia and about 1 cm or less along the arched blood vessel from the end of the ascending aorta (about 3 mm from the heart (left ventricle)) (Corresponding to the aortic arch) was removed. This was washed with PBS and then homogenized in chloroform / methanol (2/1 [v / v]) to extract cholesterol. The residue was filtered and the filtrate was dissolved, and then dried under 5% cholic acid conjugation and subjected to cholesterol measurement. Cholesterol E-Test Wako (Wako Pure Chemical Industries, Ltd., Code 439-17501) was used for the measurement of cholesterol.

(Statistical analysis)
For each measurement item, Student's t-test was performed using statistical analysis software SPSS (ver. 13.0J).

(result)
The results are shown in FIGS. 3 to 5 show the measurement results of blood total cholesterol level, blood HDL cholesterol level and aortic deposition cholesterol level, respectively. The value in each graph represents the average value of the measurement results of 8 animals in each group, and the error bar represents the standard deviation. FIG. 6 is a plot of aortic deposition cholesterol levels and blood HDL cholesterol levels in individual test hamsters.

  There was no significant difference in the total blood cholesterol level between the CNT group and the XN group (FIG. 3). On the other hand, a decrease in the blood HDL cholesterol level of the XN group was observed with respect to the CNT group (FIG. 4). Decreased blood HDL cholesterol levels in the XN group correlated with increased SR-B1 protein expression levels, reflecting increased SR-B1 protein expression resulting in increased cholesteryl ester uptake from HDL cholesterol. It is considered a thing.

  On the other hand, a decrease in the aortic deposition cholesterol level in the XN group was observed with respect to the CNT group (FIG. 5). Further, a positive correlation tendency was observed between the aortic deposition cholesterol level and the blood HDL cholesterol level ( FIG. 6).

  In general, HDL cholesterol is called good cholesterol because it works to remove cholesterol adhering to blood vessels in the body, and it is thought that the higher the value in the blood, the lower the risk of arteriosclerosis etc. . On the other hand, in this example, although the blood HDL cholesterol level was reduced by xanthohumol, the aortic deposited cholesterol level was simultaneously reduced. This action of xanthohumol has much in common with the action of probucol, which is known as an arteriosclerosis inhibitor and a hyperlipidemia treatment agent (for example, Teramoto Minsei, Cholesterol-From basic to clinical, 2009) Life Science Publishing Co., Ltd., pages 197-201). Probucol is said to lower the blood HDL cholesterol level by activating the reverse cholesterol transfer system, and thereby exerts an anti-arteriosclerosis action and a hyperlipidemia treatment action. The action by xanthohumol confirmed in this example promotes the expression of SR-B1 protein, which is an HDL receptor, activates the reverse cholesterol transport system, and lowers the blood HDL cholesterol level. This suggests that xanthohumol and probucol have a similar mechanism of action.

  From the above examples, it was confirmed that xanthohumol has an action of enhancing the expression of SR-B1 protein. Furthermore, it was confirmed that xanthohumol has an action of lowering the blood HDL cholesterol level and an action of suppressing the deposition of cholesterol in blood vessels through the reduction of the blood HDL cholesterol level.

  The SR-B1 protein expression enhancer of the present invention can enhance the expression of SR-B1 protein, which is an HDL receptor, both in vivo and in vitro, and activates the reverse cholesterol transport system via this. To do. Therefore, it can be used for prevention or improvement of diseases that can be expected to be prevented or ameliorated by enhancing the expression of SR-B1 protein or activating the reverse cholesterol transfer system. Furthermore, since the SR-B1 protein expression enhancer of the present invention has an action of suppressing the deposition of cholesterol in blood vessels, it can also be used as a cholesterol deposition inhibitor. The SR-B1 protein expression enhancer of the present invention can also be used in kits used for studying the function of SR-B1 protein, studying the mechanism of cholesterol metabolism centering on the reverse cholesterol transfer system, and the like.

Claims (4)

An SR-B1 protein expression enhancer comprising a compound represented by the following general formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[In General Formula (1), R ¹ represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms, and R ² represents a dimethylallyl group or a geranyl group. ] The SR-B1 protein expression enhancer according to claim 1, wherein the compound represented by the general formula (1) is a compound represented by the following formula (2).
  A pharmaceutical comprising the SR-B1 protein expression enhancer according to claim 1 or 2.   Food-drinks containing the SR-B1 protein expression enhancer of Claim 1 or 2.