WO2011162234A1 - Composition which is used for purpose of inhibiting biosynthesis of cholesterol - Google Patents
Composition which is used for purpose of inhibiting biosynthesis of cholesterol Download PDFInfo
- Publication number
- WO2011162234A1 WO2011162234A1 PCT/JP2011/064123 JP2011064123W WO2011162234A1 WO 2011162234 A1 WO2011162234 A1 WO 2011162234A1 JP 2011064123 W JP2011064123 W JP 2011064123W WO 2011162234 A1 WO2011162234 A1 WO 2011162234A1
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- Prior art keywords
- acid
- dolicoic
- cholesterol
- composition
- food
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Preparation or treatment thereof
- A23L2/52—Adding ingredients
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/06—Antihyperlipidemics
Definitions
- the present invention relates to a composition used to suppress biosynthesis of cholesterol containing dolicoic acid as an active ingredient, and its use as a medicine, food and drink, and reagent.
- statins represented by compactin are the “best-selling drugs in the world” and are used by 30 million people around the world every day, with sales of about 2 trillion yen. Since some statins may increase the risk of occurrence of side effects such as rhabdomyolysis, development of new drugs that are in vivo substances and have fewer side effects is expected (Non-patent Document 1).
- HMG-CoA reductase which is the rate-limiting enzyme of the mevalonate pathway, is an enzyme that generates MVA using HMG-CoA as a substrate.
- Compactin has an inhibitory action against HMG-CoA reductase to cause a decrease in cholesterol level. This inhibition is due to the fact that compactin has a structure very similar to that around the chiral 3-position carbon of HMG-CoA.
- Non-Patent Documents 3, 4, 9 Comparison of the forms of HMG and compactin in the crystal structure actually reported reveals that the structure around the 3-position carbon acts in the same state (Non-Patent Documents 3, 4, 9). ). In addition, when compactin or S- 2,3-DiGGA is allowed to act on human promyelocytic leukemia HL-60 cells, both compounds have been found to inhibit HMG-CoA reductase (Non-patent Document 5). ).
- the above-mentioned compound that inhibits HMG-CoA reductase is not a substance in the body (or has not been confirmed in vivo), and has the same activity as the above compound in order to develop a drug with few side effects. Identification of in-vivo substances has been desired.
- the present invention has been made in view of such a situation, and an object thereof is to identify an in-vivo substance having an activity of inhibiting cholesterol biosynthesis.
- a further object of the present invention is to provide a composition having the action of inhibiting the biosynthesis of cholesterol comprising the identified in vivo substance or a precursor thereof as an active ingredient.
- dolicoic acid which has been confirmed to exist in the human brain, is a compound having a structure around the 3-position carbon similar to S- 2,3-DiGGA (Non-Patent Documents 6 and 10).
- dolicoic acid is a compound obtained by oxidation of dolichol (Dolichol) in which a part of the cholesterol precursor FPP is metabolized.
- Dolichol dolichol
- the functions of dolicoic acid and dolichol in vivo have not yet been elucidated.
- there are some compounds having different side chains other than the structure around the 3-position carbon (Non-patent Document 7).
- dolicoic acid which is a substance in the body, in the biosynthesis of cholesterol.
- dolicoic acid exhibits a HL-60 cell growth inhibitory effect and induces morphological abnormalities due to a decrease in intracellular cholesterol.
- the present inventors analyzed the site of action of dolicoic acid in the mevalonate pathway involved in cholesterol biosynthesis, and found that dolicoic acid inhibits HMG-CoA reductase or a previous stage. Found to inhibit. Furthermore, from the analysis of the interaction between the HMG-CoA reductase substrate recognition site and dolicoic acid in silico, the structure around the 3rd carbon of dolicoic acid binds to the substrate recognition site of HMG-CoA reductase. It was strongly suggested that the effect was exerted.
- composition containing dolicoic acid or a precursor thereof is extremely useful as a pharmaceutical or food and drink for suppressing cholesterol biosynthesis and lowering blood cholesterol level.
- the present invention has been completed.
- a composition used for suppressing biosynthesis of cholesterol comprising dolicoic acid represented by the following chemical structural formula or a precursor thereof as an active ingredient.
- n represents a natural number, and “*” represents an asymmetric carbon atom.
- the composition according to (1) which is a pharmaceutical composition.
- the composition according to (2) which is used for preventing or treating a disease caused by an increase in blood cholesterol level.
- the composition according to (1) which is a food or drink.
- composition of the present invention By administering the composition of the present invention as a pharmaceutical or ingesting as a food or drink, the biosynthesis of cholesterol in cells can be suppressed, thereby reducing the blood cholesterol level in the living body. Therefore, the composition of the present invention is useful in the prevention and treatment of diseases caused by an increase in blood cholesterol level. Since dolicoic acid, which is an active ingredient of the composition of the present invention, is an in vivo substance, it is highly safe. In addition, the composition of the present invention can be easily taken by a healthy person on a daily basis as a food or drink such as a health food to improve blood cholesterol levels.
- the left of the figure (superposition of fatty oil droplets and nuclei) and the center of the figure (fatty oil droplets) are fluorescence micrographs, and the right is a phase contrast micrograph.
- the fatty oil droplets were stained with Nile red, and the nuclei were stained with Hoechst33342. It is a microscope picture which shows the continuation of FIG. 3A. It is the microscope picture which observed the form of the HL-60 cell 48 hours after each isoprenoid acid addition.
- the left of the figure (superposition of fatty oil droplets and nuclei) and the center of the figure (fatty oil droplets) are fluorescence micrographs, and the right is a phase contrast micrograph.
- the fatty oil droplets were stained with Nile red, and the nuclei were stained with Hoechst33342. It is a microscope picture which shows the continuation of FIG. 4A. It is a figure which shows the cholesterol biosynthesis mechanism (expectation) at the time of making a cholesterol inhibitor act in MVA coexistence. It is the microscope picture which made the compactin of various density
- the left side (superposition of fatty oil droplets and nuclei) is a fluorescence micrograph, and the right side is a phase contrast micrograph.
- the fatty oil droplets were stained with Nile red, and the nuclei were stained with Hoechst33342.
- the left side (superposition of fatty oil droplets and nuclei) is a fluorescence micrograph, and the right side is a phase contrast micrograph.
- the fatty oil droplets were stained with Nile red, and the nuclei were stained with Hoechst33342. It is a graph which shows the change of the total amount of cholesterol in 10 ml of cell liquids after each isoprenoid acid (10 micromol) addition. It is a graph which shows the change of the protein amount in 10 ml of cell liquids after each isoprenoid acid (10 micromol) addition. It is a graph which shows the change of the cholesterol total amount per protein amount after each isoprenoid acid (10 micromol) addition.
- FIG. 1 It is a figure which shows the structure of the substrate recognition site periphery (substrate S- 2,3-DiGGA prediction) of HMG-CoA reductase.
- the hydrophobic part where dolicoic acid may interact is surrounded by a frame.
- the present invention provides a composition used for suppressing cholesterol biosynthesis, containing dolicoic acid or a precursor thereof as an active ingredient.
- “suppressing the biosynthesis of cholesterol” means inhibiting the process of cholesterol biosynthesis by the mevalonate pathway.
- dolicoic acid which is an active ingredient of the composition of the present invention, competitively inhibits binding of the enzyme to HMG-CoA by binding to the substrate recognition site of HMG-CoA reductase. It is an action. This action suppresses intracellular biosynthesis of cholesterol.
- “Doricoic acid” in the present invention is a compound represented by the following chemical structural formula.
- n is a natural number, and the number is not particularly limited as long as the biosynthesis of cholesterol is suppressed.
- the number of n is typically 7-16 because the major dolicoic acid is C60 in archaea, C90-95 in rats, C95-100 in cattle and humans, and C105 up to penguins. .
- “*” Represents an asymmetric carbon atom.
- the “precursor of dolicoic acid” means a substance at a stage before dolicoic acid is generated in the metabolic process in the living body.
- the doliconic acid precursor is not particularly limited as long as it is a substance that can be metabolized to doliconic acid by administration to a living body and as a result can suppress the biosynthesis of cholesterol. Aldehyde derivatives), polyprenol (dehydrodricol), polyprenal (aldehyde derivative of polyprenol).
- composition of the present invention is in the form of a pharmaceutical composition used for suppressing biosynthesis of cholesterol, a food or drink (including animal feed), or a reagent used for research purposes (for example, in vitro or in vivo experiments). It can be.
- the function of mainly taking up cholesterol by hepatocytes is strengthened, thereby lowering the blood cholesterol level.
- the composition of the present invention has an action of lowering blood cholesterol level, diseases caused by increased blood cholesterol level, such as dyslipidemia, arteriosclerosis, myocardial infarction, cerebral hemorrhage, cerebral infarction, angina pectoris It is preferably used as a pharmaceutical composition administered for the prevention and treatment of gallstones, etc., and for the improvement of blood cholesterol level (including the prevention of the above-mentioned diseases), as a food and drink that is taken daily. be able to.
- diseases caused by increased blood cholesterol level such as dyslipidemia, arteriosclerosis, myocardial infarction, cerebral hemorrhage, cerebral infarction, angina pectoris
- It is preferably used as a pharmaceutical composition administered for the prevention and treatment of gallstones, etc., and for the improvement of blood cholesterol level (including the prevention of the above-mentioned diseases), as a food and drink that is taken daily. be able to.
- the composition in the present invention can be formulated by a known pharmaceutical method.
- a known pharmaceutical method for example, capsule, tablet, pill, liquid, powder, granule, fine granule, film coating, pellet, troche, sublingual, chewing agent, buccal, paste, syrup, suspension, To be used orally or parenterally as elixirs, emulsions, coatings, ointments, plasters, cataplasms, transdermal preparations, lotions, inhalants, aerosols, injections, suppositories, etc. Can do.
- the present invention is a composition comprising, as an active ingredient, dolicoic acid, which is an in vivo substance, and is advantageous in terms of high safety.
- carriers that are acceptable pharmacologically or as foods and beverages, specifically, sterile water and physiological saline, vegetable oils, solvents, bases, emulsifiers, suspensions, surfactants, stabilizers, Flavoring agent, fragrance, excipient, vehicle, preservative, binder, diluent, tonicity agent, soothing agent, extender, disintegrant, buffering agent, coating agent, lubricant, colorant, sweetness Can be suitably combined with an agent, a thickening agent, a flavoring agent, a solubilizing agent or other additives.
- composition of the present invention when used as a pharmaceutical composition, it may be used in combination with a known pharmaceutical composition used for the prevention or treatment of diseases caused by an increase in blood cholesterol level.
- the food or drink is, for example, a health food, a functional food, a food for specified health use, a dietary supplement, a food for a sick person, a food additive, or an animal feed.
- the food / beverage products of this invention can be ingested as a composition as described above, and can also be ingested as various food / beverage products.
- Specific examples of foods and drinks include edible oils, dressings, mayonnaise, margarine and other oils; juices, soft drinks, tea drinks, drinks, jelly drinks, functional drinks, and other beverages; beer, etc.
- Alcoholic beverages Carbohydrate-containing foods such as rice, noodles, breads and pasta; Noodle products such as fish ham, sausage and fish paste products; Retort products such as curry, sauce, and Chinese soup; Soups, milk, milk beverages, Dairy products such as ice cream, cheese, yogurt; fermented products such as miso, yogurt, fermented beverages, pickles; bean products; Western confectionery such as biscuits and cookies; Japanese confectionery such as buns and sheep cakes; , Jelly, pudding and other confectionery such as frozen confectionery; instant food such as instant soup and instant miso soup Microwave-safe food, and the like.
- health foods and drinks prepared in the form of powder, granules, tablets, capsules, liquid, paste or jelly are also included.
- composition of the present invention can be used for animals including humans, but is not particularly limited as animals other than humans, and may be used for various domestic animals, poultry, pets, laboratory animals, and the like. it can. Specific examples include but are not limited to pigs, cows, horses, sheep, goats, chickens, ducks, ostriches, ducks, dogs, cats, rabbits, hamsters, mice, rats, monkeys, and the like.
- the production of food and drink in the present invention can be performed by a production technique known in the technical field.
- one or more components effective for improving blood cholesterol level may be added.
- the dose or intake is appropriately selected according to the age, weight, symptom, health condition of the subject, type of composition (pharmaceutical, food and drink, etc.), etc.
- the for example, the dose or intake of the composition of the present invention per dose is generally 0.01 mg / kg body weight to 100 mg / kg body weight.
- the present invention also provides a method for improving (lowering) blood cholesterol level in a subject, characterized by administering or ingesting the composition of the present invention to the subject.
- the present invention also provides a method for preventing or treating a disease caused by an increase in blood cholesterol level in a subject, which comprises administering the composition of the present invention to the subject.
- the product of the composition of the present invention may be labeled with an indication that it is used to improve blood cholesterol levels.
- labeled product or instructions means that the product body, container, packaging, etc. are marked, or instructions, package inserts, promotional materials, or other printed materials that disclose product information. It means that the display is attached to.
- the indication that it is used to improve the blood cholesterol level can include information on the mechanism by which the blood cholesterol level is improved by administering or ingesting the composition of the present invention.
- Mechanisms include competitive inhibition of binding of HMG-CoA by inhibiting the process of cholesterol biosynthesis by the mevalonate pathway and binding to the substrate recognition site of HMG-CoA reductase. Information on obstructing.
- the display indicating that it is used to improve the blood cholesterol level can include information relating to use for the prevention or treatment of a disease caused by an increase in the blood cholesterol level.
- Example 1 Preparation of natural dolicoic acid Each bovine thyroid gland was purchased from Sendai City Food Monitoring Center (6-3-6 Ogimachi, Miyagino-ku, Sendai City, Miyagi Prefecture) for 100 yen each. PDC was purchased from Alfa Aesar. Normal phase silica gel plates (silica gel 60, F254), normal phase silica gel for column chromatography (silica gel 60), and reverse phase silica gel plates (HPTLC, RP18) were purchased from Merck. MeOH, CHCl 3 , hexane, benzene, ethyl acetate, and DMF were purchased from Wako Pure Chemical Industries, Ltd.
- KOH and HCl were purchased from Nacalai Tesque. Citronellic acid and citronellol were purchased from Sigma Aldrich.
- (1) Extraction of bovine thyroid-derived Dolichyl dolichoate Approximately 8 kg of bovine thyroid (300) was frozen in liquid nitrogen and sliced to a thickness of about 2 mm. All 50 sliced thyroid glands were placed in a grooved apple-shaped flask, and the lipid component containing water was extracted three times with MeOH: CHCl 3 (1: 2, v / v). The extract was filtered to remove meat pieces, and the solvent was removed with an evaporator. After the evaporator treatment, it was confirmed that the cholesterol component was precipitated.
- the obtained doliconic acid (C 100 ) was 5 mg (about 3.5 mol) or less.
- the fraction containing only dolichyl dolicate found in (1) was hydrolyzed with 15% KOH, neutralized with 4N HCl, and extracted three times with benzene.
- the Rf value (behavior) of dolicoic acid on normal phase TLC was lower than that of dolichol. This is due to the difference in the polarity of the carboxyl group rather than the hydroxyl group on normal phase TLC.
- the Rf value (behavior) in reverse phase TLC was the same as that of dolichol.
- Example 2 Preparation of chemical oxide dolicoic acid (1) Extraction of avian liver / heart-derived dolichol (chemically synthesized dolicoic acid) 1380 g of chicken liver / heart was added 1 times with 22.5% KOH (50% EtOH), 0.5% pyrogallol was added as an antioxidant, and saponification reaction was performed at 60 ° C. for 7 hours. Thereafter, the saponification solution containing dolichol was extracted three times with hexane, washed with distilled water to near pH 7, and dehydrated by being left overnight at room temperature with sodium sulfate. The saponified solution was concentrated and purified to dolichol only by column chromatography operation and preparative normal phase TLC.
- the dolicoic acid fraction fr.26-29 was collected.
- the obtained dolicoic acid (C 95 ) was 26.8 mg (20 ⁇ mol yield 26.3%), and an introduction experiment (Example 3 described later) on HL-60 cells was performed using this.
- the dolicoic acid prepared by normal phase TLC was confirmed.
- the Rf value of dolicoic acid was lower than that of dolicol, and it was confirmed that the compound was doliconic acid.
- dolicoic acid had a higher Rf value than dolichol. This is the same behavior described in the papers reported by Dorikyldrycoate. Although the paper is an HPLC detection, Dorikoin acid relative to C 18 has been reported to interact is lower than dolichols. Therefore, it was confirmed that the oxide obtained this time was dolicoic acid.
- the peak value of H of the —COOH group is expected to be around 11.00 ppm, but could not be confirmed in this analysis by 1 H-NMR.
- activity was shown in the methylation reaction with trimethylsilyldiazomethane for examining the presence or absence of a carboxylic acid group.
- CH 3 peak at the ⁇ -position was shifted to a lower magnetic field than in the case of dolichol ( ⁇ -CH 3 value 0.88 ppm), and 1 H-NMR analysis supported the production of dolicoic acid.
- Example 3 Examination of the action of isoprenoid acid having a chiral 3-position peripheral carbon structure on human promyelocytic leukemia HL-60 cells HL-60 cells were purchased from Tohoku University Aging Research Institute Medical Cell Resource Center. S- 2,3-DiGGA was prepared by Yasuhiro Suzuki (Non-Patent Document 5). The rac -phytanic acid prepared by Noriko Ishikawa was used (Non-patent Document 7). RPMI 1640 was purchased from Nippon Pharmaceutical, and NaHCO 3 was purchased from IRVINE SCIENTIFIC.
- Kanamycin, EtOH, Hoechst33342, and MVA were purchased from Wako Pure Chemical Industries, Ltd.
- penicillin G-streptomycin was purchased from GIBCO
- PBS was purchased from Takara.
- CO 2 was purchased from Tanuma Oxygen.
- Nile Red was purchased from Molecular Probes.
- FBS purchased BIOLOGICAL INDUSTRIES. MilliQ manufacturing equipment was purchased from MILLIPORE.
- T-flasks were purchased from Nacalai Tesque. Compactin was received from Dr. Akira Endo of Tokyo University of Agriculture and Technology.
- S -citronellic acid was purchased from Sigma Aldrich
- L -malic acid was purchased from Tokyo Chemical Industry Co., Ltd.
- the growth curve of HL-60 cells after addition of each isoprenoid acid is shown in FIG.
- EtOH was used as a background
- dolicoic acid after 5 days showed the same growth inhibitory effect as S- 2,3-DiGGA and compactin.
- L -malic acid and rac -phytanic acid showed similar effects.
- S -citronellyl acid and MVA did not show a very high inhibitory effect.
- a condition that is less than half of the value of the background (0.04% EtOH) on Day 5 is expressed as “Yes”, and other conditions are expressed as “None”.
- FIGS. 2 to 4 The morphology of HL-60 cells after addition of each isoprenoid acid is shown in FIGS. 2 to 4 as photomicrographs at 0, 24 and 48 hours after addition. Since no difference in conditions was observed at 0 hours after the addition, FIG. 2 shows compactin reported to cause morphological abnormalities with background EtOH.
- dolicoic acid showed the same effect as compactin on HL-60 cells.
- cell morphology observation morphological abnormalities were observed in the same manner as when compactin and S- 2,3-DiGGA were allowed to act only on the endogenous compound, dolicoic acid.
- Example 4 Recovery experiment of cholesterol biosynthesis by mevalonic acid From the results obtained in Example 3, dolicoic acid and S- 2,3-DiGGA can inhibit cholesterol by inhibiting HMG-CoA reductase. Expected. When HMG-CoA reductase or earlier is inhibited, it is considered that cholesterol biosynthesis is not inhibited by supplying MVA (FIG. 5). Therefore, the inhibitory action of each compound in the mevalonate pathway when dolicoic acid, S- 2,3-DiGGA and compactin were added to HL-60 cells in the presence of MVA was examined.
- Example 3 The experimental operation, reagents and equipment used in this example are as described in Example 3. However, 10 ⁇ M MVA was allowed to coexist in the presence of MVA. Moreover, what was prepared to 0.04% EtOH (same amount EtOH) was added in three types of concentration conditions of 10 ⁇ M, 5 ⁇ M and 1 ⁇ M. The cell morphology photograph shows one individual cell among the observed morphology (over 80%).
- dolicoic acid is shown (FIG. 8).
- dolicoic acid When dolicoic acid was allowed to act, no abnormal cell morphology was observed in the presence of MVA. Thus, it was strongly shown that dolicoic acid also inhibits HMG-CoA reductase or the previous step, like compactin. Moreover, dolicoic acid showed no effect at a low concentration (5 ⁇ M) compared to compactin and S- 2,3-DiGGA. A summary of the above results is shown in Table 2.
- Example 5 Cholesterol and protein analysis of HL-60 cells Cholesterol and protein analysis of HL-60 cells were performed as follows. Using 10 ml of cell solution of 1 ⁇ 10 6 cells / ml, dolicoic acid (10 ⁇ M), S- 2,3-DiGGA (10 ⁇ M) and compactin (10 ⁇ M) were allowed to act in the presence of 10% FBS. After the action, approximately 10 7 cells were collected by centrifugation (1,000 ⁇ g, 10 min), and suspended by adding PBS (phosphate-buffered saline). 300 ⁇ l of PBS was added to the cells collected by centrifugation again and suspended. Cells were sonicated and disrupted using a Branson sonicator (output 2, 50% duty for 2 minutes).
- PBS phosphate-buffered saline
- a cell growth curve is shown in FIG.
- dolicoic acid Compared to compactin, which has a gradual increase in cholesterol, dolicoic acid appears to be stagnant in the 3rd to 5th day, although the increase from the 1st to the 3rd day is dramatic. Observed. Considering increase / decrease in the number of cells, it was suggested that dolicoic acid acts more gently than compactin.
- dolicoic acid causes the structure around the 3-position carbon to act on the substrate recognition site for HMG-CoA reductase, and also interacts with the side chain at the large hydrophobic site shown in FIGS. It is considered that the inhibitory ability is shown. As a result, the amount of cholesterol in the cell is decreased, and it is considered that an abnormality was observed in the cell morphology due to the failure of the membrane structure.
- the composition of the present invention since the composition of the present invention has an action of suppressing the biosynthesis of cholesterol in cells, if it is used, the blood cholesterol level in the living body can be lowered. Moreover, since dolicoic acid, which is an active ingredient of the composition of the present invention, is an in vivo substance, it is highly safe. Therefore, the composition of the present invention is expected to be used as a pharmaceutical for the prevention or treatment of diseases caused by an increase in blood cholesterol, a health food for improving blood cholesterol level, and the like.
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Abstract
It has been found that dolichoic acid or a precursor thereof has cholesterol biosynthesis inhibitory activity and is extremely useful as a composition such as a pharmaceutical product, food or drink for lowering the blood cholesterol level.
Description
本発明は、ドリコイン酸を有効成分として含むコレステロールの生合成を抑制するために用いられる組成物、並びに、その医薬、飲食品、および試薬としての用途に関する。
The present invention relates to a composition used to suppress biosynthesis of cholesterol containing dolicoic acid as an active ingredient, and its use as a medicine, food and drink, and reagent.
生活習慣病の主要因は血中コレステロール値上昇による動脈硬化である。食の欧米化、運動不足そして生活ストレスなどによって悪玉コレステロールが血管壁に蓄積することで動脈硬化となることが知られている。現在、血中コレステロール値を低下させる対処法は、コンパクチン（Compactin）に代表されるスタチン類の投与が一般的に用いられている。スタチン類は世界中で毎日3000万人が服用し、売上高約2兆円の「世界で一番売れている薬」である。一部のスタチン類は横紋筋融解症などの副作用の発生リスクが高まることがあるため、生体内物質であり副作用のより少ない新薬の開発が期待されている（非特許文献１）。
The main cause of lifestyle-related diseases is arteriosclerosis due to elevated blood cholesterol levels. It is known that atherosclerosis is caused by the accumulation of bad cholesterol in the blood vessel wall due to Westernization of food, lack of exercise and life stress. At present, administration of statins represented by compactin is generally used as a countermeasure for lowering blood cholesterol level. Statins are the “best-selling drugs in the world” and are used by 30 million people around the world every day, with sales of about 2 trillion yen. Since some statins may increase the risk of occurrence of side effects such as rhabdomyolysis, development of new drugs that are in vivo substances and have fewer side effects is expected (Non-patent Document 1).
ヒトのコレステロール生合成は、メバロン酸経路で行われる。アセチルCoAがFPPに代謝され、FPPの構造においてαイソプレン側を頭部（Head）、ω末端側を尾部（Tail）としたとき、このFPPが「Head to Head」型に結合反応したスクアレン（Squalene）からコレステロールへと代謝される（非特許文献２）。
Human cholesterol biosynthesis is performed through the mevalonic acid pathway. When acetyl-CoA is metabolized to FPP and the α-isoprene side is the head and the ω-terminal side is the tail in the FPP structure, the squalene (Squalene) that FPP binds to the “Head to Head” type. ) To cholesterol (Non-patent Document 2).
メバロン酸経路の律速酵素であるHMG-CoA還元酵素は、HMG-CoAを基質としてMVAを生成する酵素である。コンパクチンはHMG-CoA還元酵素に対して拮抗的に阻害作用を示すことでコレステロール値の低下を引き起こす。この阻害はコンパクチンがHMG-CoAのキラルな3位炭素周辺に酷似した構造を持つことによる。
HMG-CoA reductase, which is the rate-limiting enzyme of the mevalonate pathway, is an enzyme that generates MVA using HMG-CoA as a substrate. Compactin has an inhibitory action against HMG-CoA reductase to cause a decrease in cholesterol level. This inhibition is due to the fact that compactin has a structure very similar to that around the chiral 3-position carbon of HMG-CoA.
実際に報告されている結晶構造中でのHMGとコンパクチンの形態を比較したところ、3位炭素周辺の構造が同じ状態で作用していることが判明している（非特許文献３、４、９）。また、ヒト前骨髄性白血病HL-60細胞に対してコンパクチンあるいはS-2,3-DiGGAを作用させると、両化合物ともにHMG-CoA還元酵素を阻害することが判明している（非特許文献５）。
Comparison of the forms of HMG and compactin in the crystal structure actually reported reveals that the structure around the 3-position carbon acts in the same state (Non-Patent Documents 3, 4, 9). ). In addition, when compactin or S- 2,3-DiGGA is allowed to act on human promyelocytic leukemia HL-60 cells, both compounds have been found to inhibit HMG-CoA reductase (Non-patent Document 5). ).
しかしながら、上記のHMG-CoA還元酵素を阻害する化合物は、生体内物質ではなく（あるいは生体内では未確認であり）、副作用の少ない医薬品などを開発するためにも、上記化合物と同様の活性を有する生体内物質の同定が望まれていた。
However, the above-mentioned compound that inhibits HMG-CoA reductase is not a substance in the body (or has not been confirmed in vivo), and has the same activity as the above compound in order to develop a drug with few side effects. Identification of in-vivo substances has been desired.
本発明は、このような状況に鑑みてなされたものであり、その目的は、コレステロールの生合成を阻害する活性を有する生体内物質を同定することにある。さらなる本発明の目的は、同定した生体内物質またはその前駆体を有効成分とする、コレステロールの生合成を阻害する作用を有する組成物を提供することにある。
The present invention has been made in view of such a situation, and an object thereof is to identify an in-vivo substance having an activity of inhibiting cholesterol biosynthesis. A further object of the present invention is to provide a composition having the action of inhibiting the biosynthesis of cholesterol comprising the identified in vivo substance or a precursor thereof as an active ingredient.
近年、ヒトの脳内で存在が確認されたドリコイン酸は、S-2,3-DiGGA同様の3位炭素周辺の構造を持つ化合物である（非特許文献６、１０）。予測される生合成経路においてドリコイン酸はコレステロールの前駆体FPPの一部が代謝されたドリコール（Dolichol）が酸化されることで得られる化合物である。しかしながら、生体内でのドリコイン酸及びドリコールの機能は、いまだ解明されていない。一方、ドリコイン酸の他に3位炭素周辺の構造以外の側鎖が異なる幾つかの化合物が存在する（非特許文献７）。
In recent years, dolicoic acid, which has been confirmed to exist in the human brain, is a compound having a structure around the 3-position carbon similar to S- 2,3-DiGGA (Non-Patent Documents 6 and 10). In the predicted biosynthetic pathway, dolicoic acid is a compound obtained by oxidation of dolichol (Dolichol) in which a part of the cholesterol precursor FPP is metabolized. However, the functions of dolicoic acid and dolichol in vivo have not yet been elucidated. On the other hand, in addition to dolicoic acid, there are some compounds having different side chains other than the structure around the 3-position carbon (Non-patent Document 7).
そこで、本発明者らは、生体内物質であるドリコイン酸を含む各種イソプレノイド酸の、コレステロール生合成における作用について鋭意検討を行った。その結果、本発明者らは、ドリコイン酸が、HL-60細胞の増殖抑制効果を示すとともに、細胞内コレステロールの減少による形態異常を誘導することを見出した。
Therefore, the present inventors have intensively studied the action of various isoprenoid acids including dolicoic acid, which is a substance in the body, in the biosynthesis of cholesterol. As a result, the present inventors have found that dolicoic acid exhibits a HL-60 cell growth inhibitory effect and induces morphological abnormalities due to a decrease in intracellular cholesterol.
さらに、本発明者らは、コレステロールの生合成に係るメバロン酸経路における、ドリコイン酸の作用点について解析を行ったところ、ドリコイン酸がHMG-CoA還元酵素を阻害するか、または、それ以前の段階を阻害することを見出した。さらに、インシリコにおけるHMG-CoA還元酵素の基質認識部位周辺とドリコイン酸との相互作用の解析から、ドリコイン酸の3位炭素周辺の構造がHMG-CoA還元酵素の基質認識部位に結合して、これら作用が発揮されていることが強く示唆された。
Furthermore, the present inventors analyzed the site of action of dolicoic acid in the mevalonate pathway involved in cholesterol biosynthesis, and found that dolicoic acid inhibits HMG-CoA reductase or a previous stage. Found to inhibit. Furthermore, from the analysis of the interaction between the HMG-CoA reductase substrate recognition site and dolicoic acid in silico, the structure around the 3rd carbon of dolicoic acid binds to the substrate recognition site of HMG-CoA reductase. It was strongly suggested that the effect was exerted.
以上から、本発明者らは、ドリコイン酸またはその前駆体を含む組成物が、コレステロールの生合成を抑制し、血中コレステロール値を低下させるための医薬や飲食品として極めて有用であることを見出し、本発明を完成するに至った。
From the above, the present inventors have found that a composition containing dolicoic acid or a precursor thereof is extremely useful as a pharmaceutical or food and drink for suppressing cholesterol biosynthesis and lowering blood cholesterol level. The present invention has been completed.
本発明は、より詳しくは、以下の発明を提供するものである。
（１） 下記化学構造式で表されるドリコイン酸またはその前駆体を有効成分として含んでなる、コレステロールの生合成を抑制するために用いられる組成物。 More specifically, the present invention provides the following inventions.
(1) A composition used for suppressing biosynthesis of cholesterol, comprising dolicoic acid represented by the following chemical structural formula or a precursor thereof as an active ingredient.
（１） 下記化学構造式で表されるドリコイン酸またはその前駆体を有効成分として含んでなる、コレステロールの生合成を抑制するために用いられる組成物。 More specifically, the present invention provides the following inventions.
(1) A composition used for suppressing biosynthesis of cholesterol, comprising dolicoic acid represented by the following chemical structural formula or a precursor thereof as an active ingredient.
（式中、ｎは、自然数を表す。また、「＊」は、不斉炭素原子を表す。）
（２） 医薬組成物である、（１）に記載の組成物。
（３） 血中コレステロール値の上昇に起因する疾患の予防または治療のために用いられる、（２）に記載の組成物。
（４） 飲食品である、（１）に記載の組成物。
（５） 血中コレステロール値を改善するために用いられる旨の表示を付した、（１）から（４）のいずれかに記載の組成物。 (In the formula, n represents a natural number, and “*” represents an asymmetric carbon atom.)
(2) The composition according to (1), which is a pharmaceutical composition.
(3) The composition according to (2), which is used for preventing or treating a disease caused by an increase in blood cholesterol level.
(4) The composition according to (1), which is a food or drink.
(5) The composition according to any one of (1) to (4), which is labeled to be used for improving blood cholesterol level.
（２） 医薬組成物である、（１）に記載の組成物。
（３） 血中コレステロール値の上昇に起因する疾患の予防または治療のために用いられる、（２）に記載の組成物。
（４） 飲食品である、（１）に記載の組成物。
（５） 血中コレステロール値を改善するために用いられる旨の表示を付した、（１）から（４）のいずれかに記載の組成物。 (In the formula, n represents a natural number, and “*” represents an asymmetric carbon atom.)
(2) The composition according to (1), which is a pharmaceutical composition.
(3) The composition according to (2), which is used for preventing or treating a disease caused by an increase in blood cholesterol level.
(4) The composition according to (1), which is a food or drink.
(5) The composition according to any one of (1) to (4), which is labeled to be used for improving blood cholesterol level.
本発明の組成物を医薬品として投与あるいは飲食品として摂取することによって、細胞におけるコレステロールの生合成を抑制し、これにより、生体における血中コレステロール値の低下を図ることができる。従って、本発明の組成物は、血中コレステロール値の上昇に起因する疾患の予防や治療において有用である。本発明の組成物の有効成分であるドリコイン酸は、生体内物質であるため、安全性が高い。また、本発明の組成物は、健康食品などの飲食品として、健常者が日常的に容易に摂取して、血中コレステロール値を改善することができる。
</ RTI> By administering the composition of the present invention as a pharmaceutical or ingesting as a food or drink, the biosynthesis of cholesterol in cells can be suppressed, thereby reducing the blood cholesterol level in the living body. Therefore, the composition of the present invention is useful in the prevention and treatment of diseases caused by an increase in blood cholesterol level. Since dolicoic acid, which is an active ingredient of the composition of the present invention, is an in vivo substance, it is highly safe. In addition, the composition of the present invention can be easily taken by a healthy person on a daily basis as a food or drink such as a health food to improve blood cholesterol levels.
本発明は、ドリコイン酸またはその前駆体を有効成分として含んでいる、コレステロールの生合成を抑制するために用いられる組成物を提供する。
The present invention provides a composition used for suppressing cholesterol biosynthesis, containing dolicoic acid or a precursor thereof as an active ingredient.
本発明において「コレステロールの生合成を抑制する」とは、メバロン酸経路によるコレステロールの生合成の過程を抑制することを意味する。本発明の組成物の有効成分であるドリコイン酸の典型的な作用は、HMG-CoA還元酵素の基質認識部位に結合することにより、該酵素がHMG-CoAに結合することを競合的に阻害する作用である。この作用により細胞内のコレステロールの生合成が抑制される。
In the present invention, “suppressing the biosynthesis of cholesterol” means inhibiting the process of cholesterol biosynthesis by the mevalonate pathway. The typical action of dolicoic acid, which is an active ingredient of the composition of the present invention, competitively inhibits binding of the enzyme to HMG-CoA by binding to the substrate recognition site of HMG-CoA reductase. It is an action. This action suppresses intracellular biosynthesis of cholesterol.
本発明における「ドリコイン酸」は、下記化学構造式であらわされる化合物である。
“Doricoic acid” in the present invention is a compound represented by the following chemical structural formula.
式中、nは自然数であり、コレステロールの生合成を抑制する限り、特に数に制限はない。例えば、主たるドリコイン酸が、古細菌ではC60、ラットではC90～95、ウシとヒトではC95～100、ペンギンでは最長C105であることから、nの数は、典型的には、7～16である。また、「＊」は、不斉炭素原子を表す。
In the formula, n is a natural number, and the number is not particularly limited as long as the biosynthesis of cholesterol is suppressed. For example, the number of n is typically 7-16 because the major dolicoic acid is C60 in archaea, C90-95 in rats, C95-100 in cattle and humans, and C105 up to penguins. . “*” Represents an asymmetric carbon atom.
本発明における「ドリコイン酸の前駆体」とは、生体内の代謝過程においてドリコイン酸が生成する前の段階の物質を意味する。ドリコイン酸の前駆体としては、生体への投与により、ドリコイン酸に代謝され、その結果、コレステロールの生合成を抑制しうる物質である限り、特に制限はないが、例えば、ドリコール、ドリカール（ドリコールのアルデヒド誘導体）、ポリプレノール（デヒドロドリコール）、ポリプレナール（ポリプレノールのアルデヒド誘導体）が挙げられる。
In the present invention, the “precursor of dolicoic acid” means a substance at a stage before dolicoic acid is generated in the metabolic process in the living body. The doliconic acid precursor is not particularly limited as long as it is a substance that can be metabolized to doliconic acid by administration to a living body and as a result can suppress the biosynthesis of cholesterol. Aldehyde derivatives), polyprenol (dehydrodricol), polyprenal (aldehyde derivative of polyprenol).
本発明の組成物は、コレステロールの生合成を抑制するために用いられる医薬組成物、飲食品（動物用飼料を含む）、あるいは研究目的（例えば、インビトロやインビボの実験）に用いられる試薬の形態であり得る。
The composition of the present invention is in the form of a pharmaceutical composition used for suppressing biosynthesis of cholesterol, a food or drink (including animal feed), or a reagent used for research purposes (for example, in vitro or in vivo experiments). It can be.
本発明の組成物の作用により、細胞内のコレステロールが減少した場合、主に肝細胞がコレステロールを取り込む働きを強め、これにより血中コレステロール値が低下する。
When the intracellular cholesterol is reduced by the action of the composition of the present invention, the function of mainly taking up cholesterol by hepatocytes is strengthened, thereby lowering the blood cholesterol level.
本発明の組成物は、血中コレステロール値を低下させる作用を有するため、血中コレステロール値の上昇に起因する疾患、例えば、脂質異常症、動脈硬化、心筋梗塞、脳出血、脳梗塞、狭心症、胆石などの予防や治療のために投与される医薬組成物として、また、血中コレステロール値の改善のために（上記疾患の予防を含む）、日常的に摂取される飲食品として好適に用いることができる。
Since the composition of the present invention has an action of lowering blood cholesterol level, diseases caused by increased blood cholesterol level, such as dyslipidemia, arteriosclerosis, myocardial infarction, cerebral hemorrhage, cerebral infarction, angina pectoris It is preferably used as a pharmaceutical composition administered for the prevention and treatment of gallstones, etc., and for the improvement of blood cholesterol level (including the prevention of the above-mentioned diseases), as a food and drink that is taken daily. be able to.
本発明における組成物は、公知の製剤学的方法により製剤化することができる。例えば、カプセル剤、錠剤、丸剤、液剤、散剤、顆粒剤、細粒剤、フィルムコーティング剤、ペレット剤、トローチ剤、舌下剤、咀嚼剤、バッカル剤、ペースト剤、シロップ剤、懸濁剤、エリキシル剤、乳剤、塗布剤、軟膏剤、硬膏剤、パップ剤、経皮吸収型製剤、ローション剤、吸引剤、エアゾール剤、注射剤、坐剤などとして、経口的または非経口的に使用することができる。本発明は、生体内物質であるドリコイン酸を有効成分とする組成物であり、安全性が高い点で有利である。
The composition in the present invention can be formulated by a known pharmaceutical method. For example, capsule, tablet, pill, liquid, powder, granule, fine granule, film coating, pellet, troche, sublingual, chewing agent, buccal, paste, syrup, suspension, To be used orally or parenterally as elixirs, emulsions, coatings, ointments, plasters, cataplasms, transdermal preparations, lotions, inhalants, aerosols, injections, suppositories, etc. Can do. The present invention is a composition comprising, as an active ingredient, dolicoic acid, which is an in vivo substance, and is advantageous in terms of high safety.
これら製剤化においては、薬理学上もしくは飲食品として許容される担体、具体的には、滅菌水や生理食塩水、植物油、溶剤、基剤、乳化剤、懸濁剤、界面活性剤、安定剤、香味剤、芳香剤、賦形剤、ベヒクル、防腐剤、結合剤、希釈剤、等張化剤、無痛化剤、増量剤、崩壊剤、緩衝剤、コーティング剤、滑沢剤、着色剤、甘味剤、粘稠剤、矯味矯臭剤、溶解補助剤あるいはその他の添加剤等と適宜組み合わせることができる。
In these preparations, carriers that are acceptable pharmacologically or as foods and beverages, specifically, sterile water and physiological saline, vegetable oils, solvents, bases, emulsifiers, suspensions, surfactants, stabilizers, Flavoring agent, fragrance, excipient, vehicle, preservative, binder, diluent, tonicity agent, soothing agent, extender, disintegrant, buffering agent, coating agent, lubricant, colorant, sweetness Can be suitably combined with an agent, a thickening agent, a flavoring agent, a solubilizing agent or other additives.
本発明の組成物を医薬組成物として用いる場合には、血中コレステロール値の上昇に起因する疾患の予防や治療に用いられる公知の医薬組成物と併用してもよい。
When the composition of the present invention is used as a pharmaceutical composition, it may be used in combination with a known pharmaceutical composition used for the prevention or treatment of diseases caused by an increase in blood cholesterol level.
本発明の組成物を飲食品として用いる場合、当該飲食品は、例えば、健康食品、機能性食品、特定保健用食品、栄養補助食品、病者用食品、食品添加物、あるいは動物用飼料であり得る。本発明の飲食品は、上記のような組成物として摂取することができる他、種々の飲食品として摂取することもできる。飲食品の具体例としては、食用油、ドレッシング、マヨネーズ、マーガリンなどの油分を含む製品；ジュース，清涼飲料水，茶飲料，ドリンク剤，ゼリー状飲料，機能性飲料等の各種飲料；ビール等のアルコール飲料；飯類，麺類，パン類およびパスタ類等の炭水化物含有食品；魚肉ハム，ソーセージ，水産練り製品等の練製品；カレー，あんかけ，中華スープ等のレトルト製品；スープ類、牛乳，乳飲料，アイスクリーム，チーズ，ヨーグルト等の乳製品；みそ，ヨーグルト，発酵飲料，漬け物等の発酵物；豆製品；ビスケット，クッキーなどの洋菓子類；饅頭や羊羹等の和菓子類，キャンディー類，ガム類，グミ，ゼリー，プリンなどの冷菓や氷菓などの各種菓子類；インスタントスープ，インスタントみそ汁等のインスタント食品や電子レンジ対応食品等が挙げられる。さらには、粉末、穎粒、錠剤、カプセル剤、液状、ペースト状またはゼリー状に調製された健康飲食品も挙げられる。
When the composition of the present invention is used as a food or drink, the food or drink is, for example, a health food, a functional food, a food for specified health use, a dietary supplement, a food for a sick person, a food additive, or an animal feed. obtain. The food / beverage products of this invention can be ingested as a composition as described above, and can also be ingested as various food / beverage products. Specific examples of foods and drinks include edible oils, dressings, mayonnaise, margarine and other oils; juices, soft drinks, tea drinks, drinks, jelly drinks, functional drinks, and other beverages; beer, etc. Alcoholic beverages; Carbohydrate-containing foods such as rice, noodles, breads and pasta; Noodle products such as fish ham, sausage and fish paste products; Retort products such as curry, sauce, and Chinese soup; Soups, milk, milk beverages, Dairy products such as ice cream, cheese, yogurt; fermented products such as miso, yogurt, fermented beverages, pickles; bean products; Western confectionery such as biscuits and cookies; Japanese confectionery such as buns and sheep cakes; , Jelly, pudding and other confectionery such as frozen confectionery; instant food such as instant soup and instant miso soup Microwave-safe food, and the like. Furthermore, health foods and drinks prepared in the form of powder, granules, tablets, capsules, liquid, paste or jelly are also included.
本発明の組成物は、ヒトを含む動物を対象として使用することができるが、ヒト以外の動物としては特に制限はなく、種々の家畜、家禽、ペット、実験用動物などを対象とすることができる。具体的には、ブタ、ウシ、ウマ、ヒツジ、ヤギ、ニワトリ、カモ、ダチョウ、アヒル、イヌ、ネコ、ウサギ、ハムスター、マウス、ラット、サルなどが挙げられるが、これらに制限されない。
The composition of the present invention can be used for animals including humans, but is not particularly limited as animals other than humans, and may be used for various domestic animals, poultry, pets, laboratory animals, and the like. it can. Specific examples include but are not limited to pigs, cows, horses, sheep, goats, chickens, ducks, ostriches, ducks, dogs, cats, rabbits, hamsters, mice, rats, monkeys, and the like.
本発明における飲食品の製造は、当該技術分野に公知の製造技術により実施することができる。当該飲食品においては、血中コレステロール値の改善に有効な1種もしくは2種以上の成分を添加してもよい。また、血中コレステロール値の改善以外の機能を発揮する他の成分あるいは他の機能性食品と組み合わせることによって、多機能性の飲食品としてもよい。
The production of food and drink in the present invention can be performed by a production technique known in the technical field. In the food and drink, one or more components effective for improving blood cholesterol level may be added. Moreover, it is good also as multifunctional food / beverage products by combining with the other component which exhibits functions other than the improvement of a blood cholesterol level, or another functional food.
本発明の組成物を投与または摂取する場合、その投与量または摂取量は、対象の年齢、体重、症状、健康状態、組成物の種類（医薬品、飲食品など）などに応じて、適宜選択される。例えば、1回当たりの本発明の組成物の投与量または摂取量は、一般に、0.01mg/kg体重～100mg/kg体重である。本発明は、このように、本発明の組成物を対象に投与もしくは摂取させることを特徴とする、対象における血中コレステロール値を改善する方法（低下させる方法）をも提供するものである。また、本発明の組成物を対象に投与することを特徴とする、対象における血中コレステロール値の上昇に起因する疾患の予防または治療の方法をも提供するものである。
When the composition of the present invention is administered or ingested, the dose or intake is appropriately selected according to the age, weight, symptom, health condition of the subject, type of composition (pharmaceutical, food and drink, etc.), etc. The For example, the dose or intake of the composition of the present invention per dose is generally 0.01 mg / kg body weight to 100 mg / kg body weight. Thus, the present invention also provides a method for improving (lowering) blood cholesterol level in a subject, characterized by administering or ingesting the composition of the present invention to the subject. The present invention also provides a method for preventing or treating a disease caused by an increase in blood cholesterol level in a subject, which comprises administering the composition of the present invention to the subject.
本発明の組成物の製品（医薬品、飲食品、試薬）またはその説明書は、血中コレステロール値を改善するために用いられる旨の表示を付したものであり得る。ここで「製品または説明書に表示を付した」とは、製品の本体、容器、包装などに表示を付したこと、あるいは製品の情報を開示する説明書、添付文書、宣伝物、その他の印刷物などに表示を付したことを意味する。血中コレステロール値を改善するために用いられる旨の表示においては、本発明の組成物を投与もしくは摂取することにより血中コレステロール値が改善される機序についての情報を含むことができる。機序としては、例えば、メバロン酸経路によるコレステロールの生合成の過程を抑制すること、HMG-CoA還元酵素の基質認識部位に結合することにより、該酵素がHMG-CoAに結合することを競合的に阻害すること、に関する情報が挙げられる。また、血中コレステロール値を改善するために用いられる旨の表示においては、血中コレステロール値の上昇に起因する疾患の予防または治療のために用いられること、に関する情報を含むことができる。
The product of the composition of the present invention (pharmaceutical product, food and drink, reagent) or its instructions may be labeled with an indication that it is used to improve blood cholesterol levels. Here, “labeled product or instructions” means that the product body, container, packaging, etc. are marked, or instructions, package inserts, promotional materials, or other printed materials that disclose product information. It means that the display is attached to. The indication that it is used to improve the blood cholesterol level can include information on the mechanism by which the blood cholesterol level is improved by administering or ingesting the composition of the present invention. Mechanisms include competitive inhibition of binding of HMG-CoA by inhibiting the process of cholesterol biosynthesis by the mevalonate pathway and binding to the substrate recognition site of HMG-CoA reductase. Information on obstructing. In addition, the display indicating that it is used to improve the blood cholesterol level can include information relating to use for the prevention or treatment of a disease caused by an increase in the blood cholesterol level.
以下、実施例及び比較例に基づいて本発明をより具体的に説明するが、本発明は以下の実施例に限定されるものではない。
Hereinafter, the present invention will be described more specifically based on examples and comparative examples, but the present invention is not limited to the following examples.
[実施例１] 天然ドリコイン酸の調製
ウシ甲状腺は仙台市食品監視センター(宮城県仙台市宮城野区扇町6-3-6)から1個100円で購入した。PDCはAlfa Aesarより購入した。順相シリカゲルプレート(シリカゲル60, F254)、カラムクロマトグラフィー用順相シリカゲル(シリカゲル60)、逆相シリカゲルプレート(HPTLC,RP18)はMerckより購入した。MeOH、CHCl3、ヘキサン、ベンゼン、酢酸エチル、DMFは和光純薬株式会社から購入した。KOH、HClはナカライテスクから購入した。シトロネリル酸及びシトロネロールはSigma Aldrichから購入した。
（１） ウシ甲状腺由来ドリキルドリコエート（Dolichyl dolichoate）の抽出
約8kgのウシ甲状腺(300個)を液体窒素で凍らせ、2mm程度の厚さにスライスした。スライスした甲状腺を溝付きリンゴ型フラスコに50個ごと入れMeOH：CHCl3(1:2,v/v)で水分を含む脂質成分を3回抽出した。抽出液はフィルターろ過することで肉片を取り除き、エバポレーターで溶媒を除去した。エバポレーター処理後はコレステロール成分が析出していることを確認した。 [Example 1] Preparation of natural dolicoic acid Each bovine thyroid gland was purchased from Sendai City Food Monitoring Center (6-3-6 Ogimachi, Miyagino-ku, Sendai City, Miyagi Prefecture) for 100 yen each. PDC was purchased from Alfa Aesar. Normal phase silica gel plates (silica gel 60, F254), normal phase silica gel for column chromatography (silica gel 60), and reverse phase silica gel plates (HPTLC, RP18) were purchased from Merck. MeOH, CHCl 3 , hexane, benzene, ethyl acetate, and DMF were purchased from Wako Pure Chemical Industries, Ltd. KOH and HCl were purchased from Nacalai Tesque. Citronellic acid and citronellol were purchased from Sigma Aldrich.
(1) Extraction of bovine thyroid-derived Dolichyl dolichoate Approximately 8 kg of bovine thyroid (300) was frozen in liquid nitrogen and sliced to a thickness of about 2 mm. All 50 sliced thyroid glands were placed in a grooved apple-shaped flask, and the lipid component containing water was extracted three times with MeOH: CHCl 3 (1: 2, v / v). The extract was filtered to remove meat pieces, and the solvent was removed with an evaporator. After the evaporator treatment, it was confirmed that the cholesterol component was precipitated.
ウシ甲状腺は仙台市食品監視センター(宮城県仙台市宮城野区扇町6-3-6)から1個100円で購入した。PDCはAlfa Aesarより購入した。順相シリカゲルプレート(シリカゲル60, F254)、カラムクロマトグラフィー用順相シリカゲル(シリカゲル60)、逆相シリカゲルプレート(HPTLC,RP18)はMerckより購入した。MeOH、CHCl3、ヘキサン、ベンゼン、酢酸エチル、DMFは和光純薬株式会社から購入した。KOH、HClはナカライテスクから購入した。シトロネリル酸及びシトロネロールはSigma Aldrichから購入した。
（１） ウシ甲状腺由来ドリキルドリコエート（Dolichyl dolichoate）の抽出
約8kgのウシ甲状腺(300個)を液体窒素で凍らせ、2mm程度の厚さにスライスした。スライスした甲状腺を溝付きリンゴ型フラスコに50個ごと入れMeOH：CHCl3(1:2,v/v)で水分を含む脂質成分を3回抽出した。抽出液はフィルターろ過することで肉片を取り除き、エバポレーターで溶媒を除去した。エバポレーター処理後はコレステロール成分が析出していることを確認した。 [Example 1] Preparation of natural dolicoic acid Each bovine thyroid gland was purchased from Sendai City Food Monitoring Center (6-3-6 Ogimachi, Miyagino-ku, Sendai City, Miyagi Prefecture) for 100 yen each. PDC was purchased from Alfa Aesar. Normal phase silica gel plates (silica gel 60, F254), normal phase silica gel for column chromatography (silica gel 60), and reverse phase silica gel plates (HPTLC, RP18) were purchased from Merck. MeOH, CHCl 3 , hexane, benzene, ethyl acetate, and DMF were purchased from Wako Pure Chemical Industries, Ltd. KOH and HCl were purchased from Nacalai Tesque. Citronellic acid and citronellol were purchased from Sigma Aldrich.
(1) Extraction of bovine thyroid-derived Dolichyl dolichoate Approximately 8 kg of bovine thyroid (300) was frozen in liquid nitrogen and sliced to a thickness of about 2 mm. All 50 sliced thyroid glands were placed in a grooved apple-shaped flask, and the lipid component containing water was extracted three times with MeOH: CHCl 3 (1: 2, v / v). The extract was filtered to remove meat pieces, and the solvent was removed with an evaporator. After the evaporator treatment, it was confirmed that the cholesterol component was precipitated.
析出物の主成分コレステロールはドリキルドリコエートよりも疎水性が低いため、Hexaneを完全に溶解するまで加え、低温でフィルターろ過することで物理的に析出成分を取り除いた。最終的に4℃でHexane 200mlに溶解した抽出物を濃縮しカラムクロマトグラフィー操作を行った。カラム条件は、シリカゲル60；170×30mm、溶媒；ベンゼン、流速；0.4ml/min、室温である。1フラクション4mlずつ回収した内、ドリキルドリコエートを含む画分はfr.56-74で回収した。
Since the main component cholesterol of the precipitate is less hydrophobic than dolichyl dolicoate, Hexane was added until completely dissolved, and the precipitate was physically removed by filtering at low temperature. Finally, the extract dissolved in 200 ml of Hexane at 4 ° C. was concentrated and subjected to column chromatography. Column conditions are silica gel 60; 170 × 30 mm, solvent; benzene, flow rate; 0.4 ml / min, room temperature. Among fractions 4 ml each, the fraction containing dolichyl dolicoate was collected at fr.56-74.
ドリキルドリコエートを含む画分を再度濃縮し、同条件で再度カラムクロマトグラフィー操作を行った。ドリキルドリコエート画分としてフラクション54～60本目を回収した。結果ドリキルドリコエート(C200)を51mg(19μmol)得た。
（２） ウシ甲状腺由来ドリコイン酸の精製(天然ドリコイン酸)
ドリキルドリコエート画分を回収後、エバポレーターで濃縮した。15% KOH(EtOH)で50℃の浴槽で加水分解し、4N HClで中和後、ベンゼンで3回抽出した。ドリコイン酸を含む加水分解物をカラムクロマトグラフィー操作で精製し、乾燥して計測した。結果、得られたドリコイン酸(C100)は5mg(約3.5mol)以下であった。
（３） ウシ甲状腺由来ドリコイン酸の解析(天然ドリコイン酸)
1次元目に順相TLC、2次元目に逆相TLCで展開する2次元TLC法を用いて、ドリキルドリコエートの加水分解で得られる物質を確認するとともに未だ報告の無いドリコイン酸の逆相TLC上での挙動(C18に対する挙動)を提唱すること目的とした。 The fraction containing dolichyl dolicoate was concentrated again, and column chromatography was performed again under the same conditions. The 54th to 60th fractions were collected as the driquil dolicoate fraction. Results 51 mg (19 μmol) of dolichyl dolicoate (C 200 ) was obtained.
(2) Purification of bovine thyroid-derived dolicoic acid (natural dolicoic acid)
After collecting the dolicyl dolicoate fraction, it was concentrated by an evaporator. Hydrolysis was performed with 15% KOH (EtOH) in a 50 ° C. bath, neutralized with 4N HCl, and extracted three times with benzene. The hydrolyzate containing dolicoic acid was purified by column chromatography, dried and counted. As a result, the obtained doliconic acid (C 100 ) was 5 mg (about 3.5 mol) or less.
(3) Analysis of bovine thyroid-derived dolicoic acid (natural dolicoic acid)
Using the two-dimensional TLC method developed in the normal phase TLC in the first dimension and the reverse phase TLC in the second dimension, the substance obtained by hydrolysis of dolichyl dolicoate was confirmed, and the reverse phase of doliconic acid that has not been reported yet The purpose was to propose the behavior on TLC (behavior for C18 ).
（２） ウシ甲状腺由来ドリコイン酸の精製(天然ドリコイン酸)
ドリキルドリコエート画分を回収後、エバポレーターで濃縮した。15% KOH(EtOH)で50℃の浴槽で加水分解し、4N HClで中和後、ベンゼンで3回抽出した。ドリコイン酸を含む加水分解物をカラムクロマトグラフィー操作で精製し、乾燥して計測した。結果、得られたドリコイン酸(C100)は5mg(約3.5mol)以下であった。
（３） ウシ甲状腺由来ドリコイン酸の解析(天然ドリコイン酸)
1次元目に順相TLC、2次元目に逆相TLCで展開する2次元TLC法を用いて、ドリキルドリコエートの加水分解で得られる物質を確認するとともに未だ報告の無いドリコイン酸の逆相TLC上での挙動(C18に対する挙動)を提唱すること目的とした。 The fraction containing dolichyl dolicoate was concentrated again, and column chromatography was performed again under the same conditions. The 54th to 60th fractions were collected as the driquil dolicoate fraction. Results 51 mg (19 μmol) of dolichyl dolicoate (C 200 ) was obtained.
(2) Purification of bovine thyroid-derived dolicoic acid (natural dolicoic acid)
After collecting the dolicyl dolicoate fraction, it was concentrated by an evaporator. Hydrolysis was performed with 15% KOH (EtOH) in a 50 ° C. bath, neutralized with 4N HCl, and extracted three times with benzene. The hydrolyzate containing dolicoic acid was purified by column chromatography, dried and counted. As a result, the obtained doliconic acid (C 100 ) was 5 mg (about 3.5 mol) or less.
(3) Analysis of bovine thyroid-derived dolicoic acid (natural dolicoic acid)
Using the two-dimensional TLC method developed in the normal phase TLC in the first dimension and the reverse phase TLC in the second dimension, the substance obtained by hydrolysis of dolichyl dolicoate was confirmed, and the reverse phase of doliconic acid that has not been reported yet The purpose was to propose the behavior on TLC (behavior for C18 ).
（１）において見出されたドリキルドリコエートのみの画分(fr.54)を15% KOHで加水分解、4N HClで中和後にベンゼンで3回抽出した。その結果、ドリコイン酸の順相TLC上でのRf値(挙動)はドリコールよりも低くかった。これは順相TLC上で水酸基よりもカルボキシル基の極性の違いに起因する。また、逆相TLCでのRf値(挙動)はドリコールと同じであった。
The fraction containing only dolichyl dolicate found in (1) (fr. 54) was hydrolyzed with 15% KOH, neutralized with 4N HCl, and extracted three times with benzene. As a result, the Rf value (behavior) of dolicoic acid on normal phase TLC was lower than that of dolichol. This is due to the difference in the polarity of the carboxyl group rather than the hydroxyl group on normal phase TLC. Moreover, the Rf value (behavior) in reverse phase TLC was the same as that of dolichol.
[実施例２] 化学的酸化物ドリコイン酸の調製
（１） トリ肝臓・心臓由来ドリコールの抽出(化学合成ドリコイン酸)
トリ肝臓・心臓1380gを22.5% KOH(50% EtOH)を1倍加え、0.5% ピロガロールを抗酸化剤として加え、60℃で7時間ケン化反応を行った。その後、ドリコールを含むケン化液をヘキサンで3回抽出し、蒸留水でpH7付近まで洗浄し、硫酸ナトリウムで一晩室温に放置することで脱水した。ケン化液は濃縮した後、カラムクロマトグラフィー操作及び分取用順相TLCでドリコールのみに精製した。カラム条件はシリカゲル60 700×130mm、トルエン/酢酸エチル(19:1,v/v)、0.8ml/min、1フラクション5ml回収、室温である。結果、ドリコール(主成分C95)を133.4mg(約100μmol)得た。カラムクロマトグラフィー操作後のドリコールを含む画分は分取用順相TLCで更に精製した。
（２） トリ肝臓・心臓由来ドリコールのPDC酸化(化学合成ドリコイン酸)
（１）で得られたドリコール(C95)の内100mg(76μmol)を用いて、PDC酸化反応を行った（非特許文献８）。50mlナス型フラスコにドリコールを入れ、窒素で溶媒を完全に飛ばした後、DMF 600μlに溶解したPDC 1.9g(5.1mmol、約70当量)を加え、塩化カルシウム管を接続し、室温で12時間反応させた。反応開始から1時間、2時間、3時間、4時間、12時間後の生成物を順相TLCで確認した。 [Example 2] Preparation of chemical oxide dolicoic acid (1) Extraction of avian liver / heart-derived dolichol (chemically synthesized dolicoic acid)
1380 g of chicken liver / heart was added 1 times with 22.5% KOH (50% EtOH), 0.5% pyrogallol was added as an antioxidant, and saponification reaction was performed at 60 ° C. for 7 hours. Thereafter, the saponification solution containing dolichol was extracted three times with hexane, washed with distilled water to nearpH 7, and dehydrated by being left overnight at room temperature with sodium sulfate. The saponified solution was concentrated and purified to dolichol only by column chromatography operation and preparative normal phase TLC. Column conditions are silica gel 60 700 × 130 mm, toluene / ethyl acetate (19: 1, v / v), 0.8 ml / min, 5 ml collection of 1 fraction, and room temperature. As a result, 133.4 mg (about 100 μmol) of dolichol (main component C 95 ) was obtained. The fraction containing dolichol after the column chromatography operation was further purified by preparative normal phase TLC.
(2) PDC oxidation of chicken liver / heart-derived dolichol (chemically synthesized dolicoic acid)
PDC oxidation reaction was performed using 100 mg (76 μmol) of the dolichol (C 95 ) obtained in (1) (Non-patent Document 8). After putting dolichol in a 50 ml eggplant-shaped flask and completely evaporating the solvent with nitrogen, add 1.9 g (5.1 mmol, about 70 equivalents) of PDC dissolved in 600 μl of DMF, connect a calcium chloride tube, and react at room temperature for 12 hours I let you. The products after 1 hour, 2 hours, 3 hours, 4 hours and 12 hours from the start of the reaction were confirmed by normal phase TLC.
（１） トリ肝臓・心臓由来ドリコールの抽出(化学合成ドリコイン酸)
トリ肝臓・心臓1380gを22.5% KOH(50% EtOH)を1倍加え、0.5% ピロガロールを抗酸化剤として加え、60℃で7時間ケン化反応を行った。その後、ドリコールを含むケン化液をヘキサンで3回抽出し、蒸留水でpH7付近まで洗浄し、硫酸ナトリウムで一晩室温に放置することで脱水した。ケン化液は濃縮した後、カラムクロマトグラフィー操作及び分取用順相TLCでドリコールのみに精製した。カラム条件はシリカゲル60 700×130mm、トルエン/酢酸エチル(19:1,v/v)、0.8ml/min、1フラクション5ml回収、室温である。結果、ドリコール(主成分C95)を133.4mg(約100μmol)得た。カラムクロマトグラフィー操作後のドリコールを含む画分は分取用順相TLCで更に精製した。
（２） トリ肝臓・心臓由来ドリコールのPDC酸化(化学合成ドリコイン酸)
（１）で得られたドリコール(C95)の内100mg(76μmol)を用いて、PDC酸化反応を行った（非特許文献８）。50mlナス型フラスコにドリコールを入れ、窒素で溶媒を完全に飛ばした後、DMF 600μlに溶解したPDC 1.9g(5.1mmol、約70当量)を加え、塩化カルシウム管を接続し、室温で12時間反応させた。反応開始から1時間、2時間、3時間、4時間、12時間後の生成物を順相TLCで確認した。 [Example 2] Preparation of chemical oxide dolicoic acid (1) Extraction of avian liver / heart-derived dolichol (chemically synthesized dolicoic acid)
1380 g of chicken liver / heart was added 1 times with 22.5% KOH (50% EtOH), 0.5% pyrogallol was added as an antioxidant, and saponification reaction was performed at 60 ° C. for 7 hours. Thereafter, the saponification solution containing dolichol was extracted three times with hexane, washed with distilled water to near
(2) PDC oxidation of chicken liver / heart-derived dolichol (chemically synthesized dolicoic acid)
PDC oxidation reaction was performed using 100 mg (76 μmol) of the dolichol (C 95 ) obtained in (1) (Non-patent Document 8). After putting dolichol in a 50 ml eggplant-shaped flask and completely evaporating the solvent with nitrogen, add 1.9 g (5.1 mmol, about 70 equivalents) of PDC dissolved in 600 μl of DMF, connect a calcium chloride tube, and react at room temperature for 12 hours I let you. The products after 1 hour, 2 hours, 3 hours, 4 hours and 12 hours from the start of the reaction were confirmed by normal phase TLC.
反応開始から12時間後、ドリコイン酸を含む反応液にpH2付近になるまでH2SO4 aqを加え、酢酸エチルで3回抽出した。抽出液にNa2SO4を加え、室温で1晩脱水を行った。後、フィルターろ過でNa2SO4を取り除き、濃縮した。ドリコイン酸を含む抽出液はカラムクロマトグラフィー操作でドリコイン酸のみに精製した。カラム条件はシリカゲル60 170×30mm、トルエン/酢酸エチル(9:1,v/v)、0.27ml/min、1フラクション5ml回収、室温である。
Twelve hours after the start of the reaction, H 2 SO 4 aq was added to the reaction solution containing doliconic acid until the pH reached about 2, and the mixture was extracted three times with ethyl acetate. Na 2 SO 4 was added to the extract and dehydrated overnight at room temperature. Thereafter, Na 2 SO 4 was removed by filtration and concentrated. The extract containing dolicoic acid was purified only to dolicoic acid by column chromatography. The column conditions are silica gel 60 170 × 30 mm, toluene / ethyl acetate (9: 1, v / v), 0.27 ml / min, 1 fraction 5 ml recovered, room temperature.
ドリコイン酸の画分fr.26-29を回収した。得られたドリコイン酸(C95)は26.8mg(20μmol 収率26.3%)あり、これを用いてHL-60細胞に対する導入実験(後述の実施例３)を行った。
（３） PDC酸化で得られたドリコイン酸の解析(化学合成ドリコイン酸)
実施例１（３）に記述したように、ドリコイン酸の逆相TLC上での挙動は未だ報告されていない。従って、化学的に調製したドリコイン酸を確認するとともに、逆相TLC上での挙動を調べた。また、1H-NMRによる解析も行った。 The dolicoic acid fraction fr.26-29 was collected. The obtained dolicoic acid (C 95 ) was 26.8 mg (20 μmol yield 26.3%), and an introduction experiment (Example 3 described later) on HL-60 cells was performed using this.
(3) Analysis of dolicoic acid obtained by PDC oxidation (chemically synthesized dolicoic acid)
As described in Example 1 (3), the behavior of dolicoic acid on reverse-phase TLC has not yet been reported. Therefore, the chemically prepared dolicoic acid was confirmed and the behavior on reverse phase TLC was investigated. Analysis by 1 H-NMR was also conducted.
（３） PDC酸化で得られたドリコイン酸の解析(化学合成ドリコイン酸)
実施例１（３）に記述したように、ドリコイン酸の逆相TLC上での挙動は未だ報告されていない。従って、化学的に調製したドリコイン酸を確認するとともに、逆相TLC上での挙動を調べた。また、1H-NMRによる解析も行った。 The dolicoic acid fraction fr.26-29 was collected. The obtained dolicoic acid (C 95 ) was 26.8 mg (20 μmol yield 26.3%), and an introduction experiment (Example 3 described later) on HL-60 cells was performed using this.
(3) Analysis of dolicoic acid obtained by PDC oxidation (chemically synthesized dolicoic acid)
As described in Example 1 (3), the behavior of dolicoic acid on reverse-phase TLC has not yet been reported. Therefore, the chemically prepared dolicoic acid was confirmed and the behavior on reverse phase TLC was investigated. Analysis by 1 H-NMR was also conducted.
順相TLCで調製したドリコイン酸を確認した。結果、天然物ドリコイン酸の2D-TLCの結果と同様に、ドリコイン酸のRf値はドリコールよりも低く、ドリコイン酸であることを確認した。
The dolicoic acid prepared by normal phase TLC was confirmed. As a result, similarly to the result of 2D-TLC of the natural product dolicoic acid, the Rf value of dolicoic acid was lower than that of dolicol, and it was confirmed that the compound was doliconic acid.
また、逆相TLC上での挙動を確認した結果、ドリコイン酸はドリコールよりもRf値が高かった。これはドリキルドリコエートが報告された論文に記述された挙動と同じである。論文ではHPLC検出であるが、C18に対してドリコイン酸はドリコールよりも相互作用が低いことが報告されている。従って、今回得られた酸化物がドリコイン酸であることを確認した。
Moreover, as a result of confirming the behavior on the reverse phase TLC, dolicoic acid had a higher Rf value than dolichol. This is the same behavior described in the papers reported by Dorikyldrycoate. Although the paper is an HPLC detection, Dorikoin acid relative to C 18 has been reported to interact is lower than dolichols. Therefore, it was confirmed that the oxide obtained this time was dolicoic acid.
-COOH基のHのピーク値は11.00ppm付近と予想されるが、1H-NMRによる本解析では確認する事が出来なかった。カルボン酸基存在の有無を調べる為のトリメチルシリルジアゾメタンによるメチル化反応では、活性を示した。また、α位のCH3のピークはドリコールの時(α-CH3値0.88ppm)よりも低磁場にシフトし、1H-NMR分析によりドリコイン酸の生成を支持できた。
The peak value of H of the —COOH group is expected to be around 11.00 ppm, but could not be confirmed in this analysis by 1 H-NMR. In the methylation reaction with trimethylsilyldiazomethane for examining the presence or absence of a carboxylic acid group, activity was shown. In addition, the CH 3 peak at the α-position was shifted to a lower magnetic field than in the case of dolichol (α-CH 3 value 0.88 ppm), and 1 H-NMR analysis supported the production of dolicoic acid.
[実施例３] キラルな3位炭素周辺構造を持つイソプレノイド酸のヒト前骨髄性白血病HL-60細胞に対する作用の検討
HL-60細胞は東北大学加齢研究所附属医用細胞資源センターより購入した。S-2,3-DiGGAは鈴木康裕修士が調製したものを用いた（非特許文献５）。rac-フィタン酸は石川乃梨子修士が調製したものを用いた（非特許文献７）。RPMI 1640は日本製薬より購入、NaHCO3はIRVINE SCIENTIFICより購入した。カナマイシン、EtOH、Hoechst33342、MVAは和光純薬工業株式会社より、ペニシリン G-ストレプトマイシンはGIBCOより、PBSはTakaraより購入した。CO2は田沼酸素より購入した。Nile RedはMolecular Probesより購入した。FBSはBIOLOGICAL INDUSTRIES社製を購入した。MilliQ製造装置はMILLIPOREより購入した。Tフラスコはナカライテスクより購入した。コンパクチンは東京農工大学の遠藤章博士から頂いた。S-シトロネリル酸はSigma Aldrichから購入し、L-リンゴ酸は東京化成工業株式会社から購入した。
（１）各イソプレノイド酸の細胞増殖への影響
保存されたHL-60細胞を37℃で溶かし、20倍量のRPMI1640(10% FBS)培地に加え、1200rpmで10分間遠心し、上清を除いた後に5mlのRPMI1640(10% FBS)培地に溶解し、50ml T-フラスコ内で37℃,5% CO2条件(CO2インキュベーター)で培養した。継代は2～3日ごとに2倍希釈した。 [Example 3] Examination of the action of isoprenoid acid having a chiral 3-position peripheral carbon structure on human promyelocytic leukemia HL-60 cells HL-60 cells were purchased from Tohoku University Aging Research Institute Medical Cell Resource Center. S- 2,3-DiGGA was prepared by Yasuhiro Suzuki (Non-Patent Document 5). The rac -phytanic acid prepared by Noriko Ishikawa was used (Non-patent Document 7). RPMI 1640 was purchased from Nippon Pharmaceutical, and NaHCO 3 was purchased from IRVINE SCIENTIFIC. Kanamycin, EtOH, Hoechst33342, and MVA were purchased from Wako Pure Chemical Industries, Ltd., penicillin G-streptomycin was purchased from GIBCO, and PBS was purchased from Takara. CO 2 was purchased from Tanuma Oxygen. Nile Red was purchased from Molecular Probes. FBS purchased BIOLOGICAL INDUSTRIES. MilliQ manufacturing equipment was purchased from MILLIPORE. T-flasks were purchased from Nacalai Tesque. Compactin was received from Dr. Akira Endo of Tokyo University of Agriculture and Technology. S -citronellic acid was purchased from Sigma Aldrich, and L -malic acid was purchased from Tokyo Chemical Industry Co., Ltd.
(1) Effects of each isoprenoid acid on cell growth Dissolved HL-60 cells at 37 ° C, added to 20 volumes of RPMI1640 (10% FBS) medium, centrifuged at 1200 rpm for 10 minutes, and removed the supernatant Thereafter, the resultant was dissolved in 5 ml of RPMI1640 (10% FBS) medium and cultured in a 50 ml T-flask at 37 ° C. under 5% CO 2 condition (CO 2 incubator). Passaging was diluted 2-fold every 2-3 days.
HL-60細胞は東北大学加齢研究所附属医用細胞資源センターより購入した。S-2,3-DiGGAは鈴木康裕修士が調製したものを用いた（非特許文献５）。rac-フィタン酸は石川乃梨子修士が調製したものを用いた（非特許文献７）。RPMI 1640は日本製薬より購入、NaHCO3はIRVINE SCIENTIFICより購入した。カナマイシン、EtOH、Hoechst33342、MVAは和光純薬工業株式会社より、ペニシリン G-ストレプトマイシンはGIBCOより、PBSはTakaraより購入した。CO2は田沼酸素より購入した。Nile RedはMolecular Probesより購入した。FBSはBIOLOGICAL INDUSTRIES社製を購入した。MilliQ製造装置はMILLIPOREより購入した。Tフラスコはナカライテスクより購入した。コンパクチンは東京農工大学の遠藤章博士から頂いた。S-シトロネリル酸はSigma Aldrichから購入し、L-リンゴ酸は東京化成工業株式会社から購入した。
（１）各イソプレノイド酸の細胞増殖への影響
保存されたHL-60細胞を37℃で溶かし、20倍量のRPMI1640(10% FBS)培地に加え、1200rpmで10分間遠心し、上清を除いた後に5mlのRPMI1640(10% FBS)培地に溶解し、50ml T-フラスコ内で37℃,5% CO2条件(CO2インキュベーター)で培養した。継代は2～3日ごとに2倍希釈した。 [Example 3] Examination of the action of isoprenoid acid having a chiral 3-position peripheral carbon structure on human promyelocytic leukemia HL-60 cells HL-60 cells were purchased from Tohoku University Aging Research Institute Medical Cell Resource Center. S- 2,3-DiGGA was prepared by Yasuhiro Suzuki (Non-Patent Document 5). The rac -phytanic acid prepared by Noriko Ishikawa was used (Non-patent Document 7). RPMI 1640 was purchased from Nippon Pharmaceutical, and NaHCO 3 was purchased from IRVINE SCIENTIFIC. Kanamycin, EtOH, Hoechst33342, and MVA were purchased from Wako Pure Chemical Industries, Ltd., penicillin G-streptomycin was purchased from GIBCO, and PBS was purchased from Takara. CO 2 was purchased from Tanuma Oxygen. Nile Red was purchased from Molecular Probes. FBS purchased BIOLOGICAL INDUSTRIES. MilliQ manufacturing equipment was purchased from MILLIPORE. T-flasks were purchased from Nacalai Tesque. Compactin was received from Dr. Akira Endo of Tokyo University of Agriculture and Technology. S -citronellic acid was purchased from Sigma Aldrich, and L -malic acid was purchased from Tokyo Chemical Industry Co., Ltd.
(1) Effects of each isoprenoid acid on cell growth Dissolved HL-60 cells at 37 ° C, added to 20 volumes of RPMI1640 (10% FBS) medium, centrifuged at 1200 rpm for 10 minutes, and removed the supernatant Thereafter, the resultant was dissolved in 5 ml of RPMI1640 (10% FBS) medium and cultured in a 50 ml T-flask at 37 ° C. under 5% CO 2 condition (CO 2 incubator). Passaging was diluted 2-fold every 2-3 days.
コンフルエント状態に達した細胞液を30mlずつファルコンチューブに入れ1200rpmで10分間遠心した後、上澄みを除き、1.5×105cells/mlの濃度になるように調製した。調製した細胞液は2.5mlずつ6穴プレートに分注した。分注した細胞液に対して終濃度10μMとなるように各イソプレノイド酸(0.04% EtOH)を添加した。但し、コンパクチンは終濃度1μMとなるように添加した。ドリコイン酸(C95で算出した)は分子量が他の化合物より10倍近く大きいため特に調製に気をつけた。24時間ごとに細胞数計測用25μlと形態観察用75μlをそれぞれ1.5mlエッペンチューブに入れた。
30 ml of the cell solution that reached a confluent state was placed in a falcon tube and centrifuged at 1200 rpm for 10 minutes, and then the supernatant was removed to prepare a concentration of 1.5 × 10 5 cells / ml. 2.5 ml of the prepared cell solution was dispensed into a 6-well plate. Each isoprenoid acid (0.04% EtOH) was added to the dispensed cell solution to a final concentration of 10 μM. However, compactin was added to a final concentration of 1 μM. Dorikoin acid (calculated at C 95) is molecular weight care especially prepared for nearly 10 times greater than the other compounds. Every 24 hours, 25 μl for counting cells and 75 μl for morphological observation were placed in a 1.5 ml Eppendorf tube.
時間経過ごとの細胞液25μlに対し0.4%トリパンブルーを1μl加え、5分間氷中で浸透させ、このうち3μlを血算盤に載せ、空気が入らないようにカバーグラスで固定し、倒立顕微鏡10×10倍で計測した。
Add 1 μl of 0.4% trypan blue to 25 μl of cell solution over time, and infiltrate in ice for 5 minutes. Place 3 μl of this on a pedometer and fix it with a cover glass to prevent air from entering. Measured 10 times.
各イソプレノイド酸添加後のHL-60細胞の増殖曲線を、図１に示した。EtOHをバックグラウンドとした時、5日後のドリコイン酸はS-2,3-DiGGA及びコンパクチンと同様の成長阻害効果を示した。そのほかにもL-リンゴ酸とrac-フィタン酸も同様に効果を示した。しかしS-シトロネリル酸とMVAに関しては、それほど高い抑制効果を示さなかった。なお、図１では、Day5でのバックグランド(0.04% EtOH)の数値の半分以下の条件を「あり」と表現し、それ以外は「なし」と表現している。
（２）各イソプレノイド酸の細胞形態への影響
時間経過ごとの細胞液75μlに対し1mM Nile redを1μMになるように加え、37℃で2時間浸透させた。この後、1200rpmで遠心し、上清を除き、1% グルタルアルデヒド20μlを加え、4℃で一晩固定した。細胞を固定した後、1200rpmで遠心し、上清を除き、沈殿をPBS(-)10μlで懸濁した。細胞浮遊液10μlに対し1mM Hoechst33342 2μlを加え、5μlをスライドグラスに取り、蛍光顕微鏡40×10倍で細胞形態を観察した。先ず、Hoechst33342染色を確認するために励起フィルター365nm(Band Pass)とバリアフィルター397nm(Long Pass)で観察した。次にNile red染色を観察するためにバリアフィルターを515-565nm(Long Pass)に切り替え観察した。最後に細胞の全体を確認するためにバリアフィルターを掛けずに位相差を観察した。細胞形態写真は観察された過半数(80%以上)に観られた形態のうち細胞1個体を示している。 The growth curve of HL-60 cells after addition of each isoprenoid acid is shown in FIG. When EtOH was used as a background, dolicoic acid after 5 days showed the same growth inhibitory effect as S- 2,3-DiGGA and compactin. In addition, L -malic acid and rac -phytanic acid showed similar effects. However, S -citronellyl acid and MVA did not show a very high inhibitory effect. In FIG. 1, a condition that is less than half of the value of the background (0.04% EtOH) onDay 5 is expressed as “Yes”, and other conditions are expressed as “None”.
(2) Effect of each isoprenoid acid oncell morphology 1 mM Nile red was added to 1 μM of 75 μl of cell solution every time and allowed to permeate at 37 ° C. for 2 hours. Thereafter, the mixture was centrifuged at 1200 rpm, the supernatant was removed, 20 μl of 1% glutaraldehyde was added, and the mixture was fixed at 4 ° C. overnight. After fixing the cells, the cells were centrifuged at 1200 rpm, the supernatant was removed, and the precipitate was suspended in 10 μl of PBS (−). 2 μl of 1 mM Hoechst33342 was added to 10 μl of the cell suspension, 5 μl was taken on a slide glass, and the cell morphology was observed with a fluorescence microscope 40 × 10 times. First, in order to confirm Hoechst33342 staining, observation was performed with an excitation filter 365 nm (Band Pass) and a barrier filter 397 nm (Long Pass). Next, in order to observe Nile red staining, the barrier filter was switched to 515-565 nm (Long Pass) and observed. Finally, in order to confirm the whole cell, the phase difference was observed without applying a barrier filter. The cell morphology photograph shows one individual cell among the observed morphology (over 80%).
（２）各イソプレノイド酸の細胞形態への影響
時間経過ごとの細胞液75μlに対し1mM Nile redを1μMになるように加え、37℃で2時間浸透させた。この後、1200rpmで遠心し、上清を除き、1% グルタルアルデヒド20μlを加え、4℃で一晩固定した。細胞を固定した後、1200rpmで遠心し、上清を除き、沈殿をPBS(-)10μlで懸濁した。細胞浮遊液10μlに対し1mM Hoechst33342 2μlを加え、5μlをスライドグラスに取り、蛍光顕微鏡40×10倍で細胞形態を観察した。先ず、Hoechst33342染色を確認するために励起フィルター365nm(Band Pass)とバリアフィルター397nm(Long Pass)で観察した。次にNile red染色を観察するためにバリアフィルターを515-565nm(Long Pass)に切り替え観察した。最後に細胞の全体を確認するためにバリアフィルターを掛けずに位相差を観察した。細胞形態写真は観察された過半数(80%以上)に観られた形態のうち細胞1個体を示している。 The growth curve of HL-60 cells after addition of each isoprenoid acid is shown in FIG. When EtOH was used as a background, dolicoic acid after 5 days showed the same growth inhibitory effect as S- 2,3-DiGGA and compactin. In addition, L -malic acid and rac -phytanic acid showed similar effects. However, S -citronellyl acid and MVA did not show a very high inhibitory effect. In FIG. 1, a condition that is less than half of the value of the background (0.04% EtOH) on
(2) Effect of each isoprenoid acid on
各イソプレノイド酸添加後のHL-60細胞の形態は添加後0時間、24時間及び48時間の顕微鏡写真を図２～４に示す。添加後0時間では条件の差が見られなかったことから、図２には、バックグランドのEtOHと形態異常を引き起こすことが報告されているコンパクチンを示した。
The morphology of HL-60 cells after addition of each isoprenoid acid is shown in FIGS. 2 to 4 as photomicrographs at 0, 24 and 48 hours after addition. Since no difference in conditions was observed at 0 hours after the addition, FIG. 2 shows compactin reported to cause morphological abnormalities with background EtOH.
48時間後に条件ごとの差が見られた。細胞形態に関しては予想通りドリコイン酸にS-2,3-DiGGA及びコンパクチンと同様の異常が観察された。油滴形成誘導に関しては全ての条件で存在が確認された。アポトーシス作用に関しては全ての条件で観られなかった。
After 48 hours, there was a difference for each condition. As expected, abnormalities similar to S- 2,3-DiGGA and compactin were observed in dolicoic acid as expected. The presence of oil droplet formation was confirmed under all conditions. Apoptosis was not observed under all conditions.
以上の結果のまとめを表１に示す。
A summary of the above results is shown in Table 1.
増殖抑制に関して、ドリコイン酸はHL-60細胞に対してコンパクチンと同等の効果を示した。細胞形態観察に関して、内在性化合物の内ドリコイン酸のみにコンパクチン及びS-2,3-DiGGAを作用させた時と同様に形態異常が観察された。
In terms of growth inhibition, dolicoic acid showed the same effect as compactin on HL-60 cells. Regarding cell morphology observation, morphological abnormalities were observed in the same manner as when compactin and S- 2,3-DiGGA were allowed to act only on the endogenous compound, dolicoic acid.
[実施例４] メバロン酸によるコレステロール生合成の回復実験
実施例３で得られた結果より、ドリコイン酸とS-2,3-DiGGAはHMG-CoA還元酵素を阻害することでコレステロール阻害することが予想された。HMG-CoA還元酵素もしくはそれ以前が阻害されている場合、コレステロール生合成はMVAを供給することで阻害されることは無いと考えられる（図５）。従って、HL-60細胞に対してMVA共存下でドリコイン酸、S-2,3-DiGGA及びコンパクチンを添加した場合の各化合物のメバロン酸経路内阻害作用を検討した。 [Example 4] Recovery experiment of cholesterol biosynthesis by mevalonic acid From the results obtained in Example 3, dolicoic acid and S- 2,3-DiGGA can inhibit cholesterol by inhibiting HMG-CoA reductase. Expected. When HMG-CoA reductase or earlier is inhibited, it is considered that cholesterol biosynthesis is not inhibited by supplying MVA (FIG. 5). Therefore, the inhibitory action of each compound in the mevalonate pathway when dolicoic acid, S- 2,3-DiGGA and compactin were added to HL-60 cells in the presence of MVA was examined.
実施例３で得られた結果より、ドリコイン酸とS-2,3-DiGGAはHMG-CoA還元酵素を阻害することでコレステロール阻害することが予想された。HMG-CoA還元酵素もしくはそれ以前が阻害されている場合、コレステロール生合成はMVAを供給することで阻害されることは無いと考えられる（図５）。従って、HL-60細胞に対してMVA共存下でドリコイン酸、S-2,3-DiGGA及びコンパクチンを添加した場合の各化合物のメバロン酸経路内阻害作用を検討した。 [Example 4] Recovery experiment of cholesterol biosynthesis by mevalonic acid From the results obtained in Example 3, dolicoic acid and S- 2,3-DiGGA can inhibit cholesterol by inhibiting HMG-CoA reductase. Expected. When HMG-CoA reductase or earlier is inhibited, it is considered that cholesterol biosynthesis is not inhibited by supplying MVA (FIG. 5). Therefore, the inhibitory action of each compound in the mevalonate pathway when dolicoic acid, S- 2,3-DiGGA and compactin were added to HL-60 cells in the presence of MVA was examined.
本実施例における実験操作、使用試薬・機器は、実施例３に記載した通りである。但し、MVA共存下条件ではMVAを10μM共存させた。また10μM、5μM及び1μMの3種類の濃度条件では0.04% EtOH(同量EtOH)に調製したものを添加した。細胞形態写真は観察された過半数(80%以上)に観られた形態のうち細胞1個体を示している。
The experimental operation, reagents and equipment used in this example are as described in Example 3. However, 10 μM MVA was allowed to coexist in the presence of MVA. Moreover, what was prepared to 0.04% EtOH (same amount EtOH) was added in three types of concentration conditions of 10 μM, 5 μM and 1 μM. The cell morphology photograph shows one individual cell among the observed morphology (over 80%).
S-2,3-DiGGAとコンパクチンを作用させた場合、MVA共存下での細胞形態の異常は見られなくなった(図６、７)。得られた結果より、実施例３で観察された細胞形態の異常はHMG-CoA還元酵素を阻害することで引き起こされることが再現的に示された。また、細胞形態の異常に関してはS-2,3-DiGGAとコンパクチンの濃度による差は見られなかった。
When S- 2,3-DiGGA and compactin were allowed to act, no abnormal cell morphology was observed in the presence of MVA (FIGS. 6 and 7). The obtained results reproducibly show that the abnormal cell morphology observed in Example 3 is caused by inhibiting HMG-CoA reductase. In addition, regarding the abnormal cell morphology, there was no difference due to the concentration of S- 2,3-DiGGA and compactin.
次にドリコイン酸の結果を示す(図８)。ドリコイン酸を作用させた場合、MVA共存下での細胞形態の異常は見られなくなった。これにより、ドリコイン酸もコンパクチンと同様にHMG-CoA還元酵素を阻害するまたはそれ以前の段階を阻害する事が強く示された。また、ドリコイン酸はコンパクチン及びS-2,3-DiGGAに比べ低濃度(5μM)では作用を示さなかった。以上の結果のまとめを表２に示す。
Next, the result of dolicoic acid is shown (FIG. 8). When dolicoic acid was allowed to act, no abnormal cell morphology was observed in the presence of MVA. Thus, it was strongly shown that dolicoic acid also inhibits HMG-CoA reductase or the previous step, like compactin. Moreover, dolicoic acid showed no effect at a low concentration (5 μM) compared to compactin and S- 2,3-DiGGA. A summary of the above results is shown in Table 2.
[実施例５] HL-60細胞のコレステロールとタンパク質分析
HL-60細胞のコレステロールとタンパク質分析は、次の通り行った。1×106cells/mlの細胞液10mlを用いて10%FBS存在下、ドリコイン酸(10μM)、S-2,3-DiGGA(10μM)、コンパクチン(10μM)を作用させた。作用後に凡そ107個の細胞を遠心分離法（1,000×g、10min）により集め、PBS(phosphate-buffered saline)を加えて懸濁した。再度遠心法により集めた細胞に対して300μlのPBSを加えて懸濁した。ブランソン製の超音波破砕機（出力２、50% dutyで２分間）を用いて細胞を超音波処理し破壊した。 [Example 5] Cholesterol and protein analysis of HL-60 cells Cholesterol and protein analysis of HL-60 cells were performed as follows. Using 10 ml of cell solution of 1 × 10 6 cells / ml, dolicoic acid (10 μM), S- 2,3-DiGGA (10 μM) and compactin (10 μM) were allowed to act in the presence of 10% FBS. After the action, approximately 10 7 cells were collected by centrifugation (1,000 × g, 10 min), and suspended by adding PBS (phosphate-buffered saline). 300 μl of PBS was added to the cells collected by centrifugation again and suspended. Cells were sonicated and disrupted using a Branson sonicator ( output 2, 50% duty for 2 minutes).
HL-60細胞のコレステロールとタンパク質分析は、次の通り行った。1×106cells/mlの細胞液10mlを用いて10%FBS存在下、ドリコイン酸(10μM)、S-2,3-DiGGA(10μM)、コンパクチン(10μM)を作用させた。作用後に凡そ107個の細胞を遠心分離法（1,000×g、10min）により集め、PBS(phosphate-buffered saline)を加えて懸濁した。再度遠心法により集めた細胞に対して300μlのPBSを加えて懸濁した。ブランソン製の超音波破砕機（出力２、50% dutyで２分間）を用いて細胞を超音波処理し破壊した。 [Example 5] Cholesterol and protein analysis of HL-60 cells Cholesterol and protein analysis of HL-60 cells were performed as follows. Using 10 ml of cell solution of 1 × 10 6 cells / ml, dolicoic acid (10 μM), S- 2,3-DiGGA (10 μM) and compactin (10 μM) were allowed to act in the presence of 10% FBS. After the action, approximately 10 7 cells were collected by centrifugation (1,000 × g, 10 min), and suspended by adding PBS (phosphate-buffered saline). 300 μl of PBS was added to the cells collected by centrifugation again and suspended. Cells were sonicated and disrupted using a Branson sonicator (
300μl液のうち250μlはコレステロール定量用に用いた。250μlをガラス試験管にとり1.5mlのクロロホルム：メタノール（2:1）液を加えて脂質を抽出した。上層の水溶液を除いた後、下層のクロロホルム液の濃縮乾燥を窒素ガスを用いて行った。得られた脂質に対して150μlのイソプロパノールを加えた。さらにコレステロール検出キットとしてのセロテック社製の酵素液Aを700μl加えて37℃で5分間インキュベーションした。更に酵素液Bを350μl加えて同様5分間インキュベーションした。吸光度を600nm波長で測定した。
Of the 300 μl solution, 250 μl was used for cholesterol quantification. 250 μl was placed in a glass test tube and 1.5 ml of chloroform: methanol (2: 1) solution was added to extract lipids. After removing the upper aqueous solution, the lower chloroform solution was concentrated and dried using nitrogen gas. 150 μl of isopropanol was added to the obtained lipid. Furthermore, 700 μl of Cellotech enzyme solution A as a cholesterol detection kit was added and incubated at 37 ° C. for 5 minutes. Furthermore, 350 μl of enzyme solution B was added and incubated for 5 minutes in the same manner. Absorbance was measured at 600 nm wavelength.
一方、300μl液のうち50μlはタンパク質定量用に用いた。バイオラド社製タンパク質アッセイ液を用い、800μlサンプル液に対して色素試薬200μlを加え室温で5分間反応させた。吸光度を595nm波長で測定した。
On the other hand, 50 μl of 300 μl solution was used for protein quantification. Using a protein assay solution manufactured by Bio-Rad, 200 μl of a dye reagent was added to an 800 μl sample solution and reacted at room temperature for 5 minutes. Absorbance was measured at a wavelength of 595 nm.
細胞液10ml中のコレステロール総量とタンパク質量の変化について、図９と１０に示す。また、タンパク質量当たりのコレステロール総量の変化について、図１１に示す。また、細胞増殖曲線を図１２に示す。
Changes in the total amount of cholesterol and the amount of protein in 10 ml of cell fluid are shown in FIGS. Moreover, it shows in FIG. 11 about the change of the cholesterol total amount per protein amount. A cell growth curve is shown in FIG.
徐々にコレステロール量が増加しているコンパクチンと比較して、ドリコイン酸は1日目から3日目の増加は劇的ではあるものの3日目から5日目での増加は停滞しているように観察された。細胞数の増減も考慮すると、ドリコイン酸は、コンパクチンと比べ、穏やかに作用している事が示唆された。
Compared to compactin, which has a gradual increase in cholesterol, dolicoic acid appears to be stagnant in the 3rd to 5th day, although the increase from the 1st to the 3rd day is dramatic. Observed. Considering increase / decrease in the number of cells, it was suggested that dolicoic acid acts more gently than compactin.
[参考例１]
実施例３及び４で得られた結果より、ドリコイン酸はコンパクチン及びS-2,3-DiGGAと同様にHL-60細胞に対して、増殖抑制効果と形態異常の作用を観察した。また、形態異常に関してドリコイン酸以外の内在性化合物では観られなかったことから、3位炭素周辺の構造のみならず、HMG-CoAとコンパクチンのように側鎖部分の疎水性がある位程度必要であることが示唆された(図１３)。ドリコイン酸は3位炭素周辺の構造と側鎖に疎水性を持つ(図１４)。また、HMG-CoA還元酵素の基質認識部位の周辺に目を向けた時、近辺に大きな疎水性箇所(図１５、１６)が存在することが分かった（非特許文献４）。 [Reference Example 1]
From the results obtained in Examples 3 and 4, dolicoic acid was observed to have a growth-suppressing effect and an abnormal morphological effect on HL-60 cells in the same manner as compactin and S- 2,3-DiGGA. In addition, since morphological abnormalities were not observed in endogenous compounds other than dolicoic acid, not only the structure around the 3-position carbon, but also the degree of hydrophobicity of the side chain portion like HMG-CoA and compactin is required. It was suggested that there was (FIG. 13). Doriconic acid has hydrophobicity in the structure and side chain around the 3-position carbon (FIG. 14). Moreover, it turned out that a large hydrophobic part (FIGS. 15 and 16) exists in the vicinity when the eyes are turned around the substrate recognition site of HMG-CoA reductase (Non-patent Document 4).
実施例３及び４で得られた結果より、ドリコイン酸はコンパクチン及びS-2,3-DiGGAと同様にHL-60細胞に対して、増殖抑制効果と形態異常の作用を観察した。また、形態異常に関してドリコイン酸以外の内在性化合物では観られなかったことから、3位炭素周辺の構造のみならず、HMG-CoAとコンパクチンのように側鎖部分の疎水性がある位程度必要であることが示唆された(図１３)。ドリコイン酸は3位炭素周辺の構造と側鎖に疎水性を持つ(図１４)。また、HMG-CoA還元酵素の基質認識部位の周辺に目を向けた時、近辺に大きな疎水性箇所(図１５、１６)が存在することが分かった（非特許文献４）。 [Reference Example 1]
From the results obtained in Examples 3 and 4, dolicoic acid was observed to have a growth-suppressing effect and an abnormal morphological effect on HL-60 cells in the same manner as compactin and S- 2,3-DiGGA. In addition, since morphological abnormalities were not observed in endogenous compounds other than dolicoic acid, not only the structure around the 3-position carbon, but also the degree of hydrophobicity of the side chain portion like HMG-CoA and compactin is required. It was suggested that there was (FIG. 13). Doriconic acid has hydrophobicity in the structure and side chain around the 3-position carbon (FIG. 14). Moreover, it turned out that a large hydrophobic part (FIGS. 15 and 16) exists in the vicinity when the eyes are turned around the substrate recognition site of HMG-CoA reductase (Non-patent Document 4).
この構造によってコンパクチンと同様にドリコイン酸はHMG-CoA還元酵素に対して基質認識部位に3位炭素周辺の構造を作用させ、かつ、図１５と１６に示す大きな疎水性部位に側鎖を相互作用させることで阻害能を示している事が考えられる。その結果として、細胞内のコレステロール量は減少し、膜の構成に障害が生じることで細胞形態に異常が観察されたと考えられる。
Due to this structure, as with compactin, dolicoic acid causes the structure around the 3-position carbon to act on the substrate recognition site for HMG-CoA reductase, and also interacts with the side chain at the large hydrophobic site shown in FIGS. It is considered that the inhibitory ability is shown. As a result, the amount of cholesterol in the cell is decreased, and it is considered that an abnormality was observed in the cell morphology due to the failure of the membrane structure.
以上説明したように、本発明の組成物は、細胞におけるコレステロールの生合成を抑制する作用を有するため、これを用いれば、生体における血中コレステロール値の低下を図ることができる。また、本発明の組成物の有効成分たるドリコイン酸は、生体内物質であるため、安全性が高い。従って、本発明の組成物は、血中コレステロールの上昇に起因する疾患の予防や治療のための医薬品や、血中コレステロール値の改善を図るための健康食品などとしての利用が期待される。
As described above, since the composition of the present invention has an action of suppressing the biosynthesis of cholesterol in cells, if it is used, the blood cholesterol level in the living body can be lowered. Moreover, since dolicoic acid, which is an active ingredient of the composition of the present invention, is an in vivo substance, it is highly safe. Therefore, the composition of the present invention is expected to be used as a pharmaceutical for the prevention or treatment of diseases caused by an increase in blood cholesterol, a health food for improving blood cholesterol level, and the like.
Claims (5)
- 下記化学構造式で表されるドリコイン酸またはその前駆体を有効成分として含んでなる、コレステロールの生合成を抑制するために用いられる組成物。
- 医薬組成物である、請求項１に記載の組成物。 The composition according to claim 1, which is a pharmaceutical composition.
- 血中コレステロール値の上昇に起因する疾患の予防または治療のために用いられる、請求項２に記載の組成物。 The composition according to claim 2, which is used for prevention or treatment of a disease caused by an increase in blood cholesterol level.
- 飲食品である、請求項１に記載の組成物。 The composition according to claim 1, which is a food or drink.
- 血中コレステロール値を改善するために用いられる旨の表示を付した、請求項１から４のいずれかに記載の組成物。 5. The composition according to any one of claims 1 to 4, which is labeled to be used for improving blood cholesterol level.
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