JP2007326827A - Cyclooxygenase activity inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a highly safe extract exhibiting COX-inhibiting activities, preferably one exhibiting cyclooxygenase-inhibiting activities that strongly inhibits selectively cyclooxygenase-2 compared with cyclooxygenase-1, a cyclooxygenase activity inhibitor comprising the extract, and a composition comprising these. <P>SOLUTION: The extract is obtained by subjecting peach leaves to extraction with an organic solvent or an organic solvent aqueous solution and exhibits cyclooxygenase-inhibiting activities. The cyclooxygenase activity inhibitor comprises the extract. The composition comprises the extract or the cyclooxygenase activity inhibitor. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to an extract having cyclooxygenase inhibitory activity obtained from a peach leaf which is a natural product, a cyclooxygenase activity inhibitor, and a composition containing these.

  Cyclooxygenase (hereinafter referred to as COX) is a rate-limiting enzyme of the arachidonic acid cascade that produces chemical mediators such as prostaglandins and thromboxanes from arachidonic acid. There are two COX isozymes in the living body, which are called cyclooxygenase type 1 (COX-1) and cyclooxygenase type 2 (COX-2), respectively. COX-1 is constitutively expressed in many cells such as platelets, stomach, and vascular endothelial cells, and has effects such as platelet aggregation, gastric acid secretion inhibitory action, and gastric mucosa protection, and is involved in the protection of living organisms. COX-2 is an inducible enzyme whose production is induced by inflammatory stimuli or hormonal stimuli, and is thought to produce prostaglandins responsible for reactions such as fever, pain, and inflammation such as edema.

Non-steroidal anti-inflammatory agents known to date such as aspirin and indomethacin well inhibit both COX-1 and COX-2. This suppresses the biosynthesis of prostaglandins and is therefore effective for diseases involving prostaglandins. However, on the other hand, it is known that side effects such as peptic ulcer and dizziness occur because it inhibits not only COX-2 but also COX-1. For these reasons, it is desired to develop a highly safe COX activity inhibitor with fewer side effects, that is, a highly selective COX activity inhibitor for COX-2 (for example, Patent Document 1, Non-Patent Document). 1).
JP 2006-76910 A FitzGerald GA. Nature Reviews 2: 879-890, 2003.

  An object of the present invention is to provide a highly safe extract having a cyclooxygenase inhibitory activity having a COX inhibitory activity, preferably selectively selectively inhibiting COX-2 as compared with COX-1, and a cyclooxygenase activity inhibitor containing the extract And an object of the present invention is to provide a composition containing them.

From the viewpoint of safety, the present inventors conducted a search for COX inhibitors using an in vitro system for extracts of edible materials. As a result, peach leaves were found to have high COX inhibitory activity, and the present invention was completed.
That is, the gist of the present invention is as follows.
[1] An extract obtained by extracting peach leaves with an organic solvent or an organic solvent aqueous solution and containing COX inhibitory activity (hereinafter also referred to as COX inhibitory activity extract)
[2] The extract according to the above [1], which inhibits cyclooxygenase type 2 more strongly than cyclooxygenase type 1,
[3] A cyclooxygenase activity inhibitor comprising the extract according to [1] or [2],
[4] A composition comprising the extract according to [1] or [2] or the cyclooxygenase activity inhibitor according to [3].

The COX inhibitory activity extract of the present invention is highly safe because it is derived from food.
The COX activity inhibitor of the present invention exhibits selective inhibitory activity against COX-2, which has a strong inhibitory activity on COX-2, while the inhibitory activity of COX-1 is moderate. Therefore, it is expected that there are few side effects.

  Furthermore, the present invention can be applied to food compositions, external preparation compositions, pharmaceutical compositions, and quasi-drug compositions, and is based on COX activity inhibitory action, causing various diseases and functional declines associated with inflammation. Prevention and improvement effects are expected. The selectivity with respect to COX-2 here is a number in which the ratio (COX-1 / COX-2) of ICX values of COX-1 and COX-2 is 1.5 or more. In addition, IC50 value is a value measured by the method as described in the below-mentioned Example.

  The peach used in the present invention includes Prunus persica, which is a deciduous tree of the genus Rosaceae, which is native to northwestern China and is cultivated throughout Japan. The peach leaf used as an extract sample can be obtained by pulverizing the leaf with a mixer or the like. Peach leaves have long been used as bathing materials and tea materials.

  The extract sample used in the present invention may include branches and flowers in addition to peach leaves. These are pulverized with a mixer or the like. In addition, the sample is preferably dried for pulverization, but the dry state is not limited.

  The peach leaves used in the present invention contain amygdalin, which is a hydrocyanic acid glycoside, although the amount is small. Amygdalin itself is not toxic, but hydrogen cyanide is generated when it is decomposed by gastric acid of animals. Therefore, peach leaves are heated at 93 ° C. or higher for amygdalin decomposition temperature for about 10-15 minutes to decompose amygdalin. It is preferable to use peach leaves subjected to (for example, Merck Index (13th edition), page 100, item 601). In addition, it is necessary to be careful about ventilation.

  As the extraction solvent, an organic solvent or an organic solvent aqueous solution is used. Examples of the organic solvent include, but are not limited to, lower alcohols such as methanol, ethanol, propanol, and butanol, and esters such as ethyl acetate. Further, these organic solvents may be used alone or in combination of two or more kinds, or may be used as an organic solvent aqueous solution which is a mixed solvent of an organic solvent and water. From the viewpoint of safety, it is preferable to extract with ethanol or an aqueous ethanol solution. In addition, the ratio of each solvent in the case of mixing organic solvents or organic solvent and water is not particularly limited.

  As an extraction method, for example, the peach leaf sample and the extraction solvent are mixed, and the mixture is stirred for 1-5 hours at room temperature or refluxed at the boiling temperature of the extraction solvent for 1-5 hours, followed by filtration or centrifugation. A method of removing the sample residue from the extract by separation or the like and concentrating the extract by reduced pressure or ultrafiltration can be mentioned. Further, if necessary, the extraction solvent may be completely removed and dried by solidification or lyophilization.

  The COX inhibitory activity extract obtained as described above has COX inhibitory activity that selectively and strongly inhibits COX-2 as compared to COX-1. Here, the COX inhibitory activity is measured by the method described in Examples below.

  The extract derived from an oil layer (for example, ethyl acetate layer or 1-butanol layer) obtained by distributing the COX inhibitory activity extract with a low polarity organic solvent has COX inhibitory activity, and a highly polar solvent layer (for example, water The COX inhibitory activity was not observed in the extract derived from the layer). By partitioning with the ethyl acetate layer or 1-butanol layer, a more purified COX inhibitory activity extract can be obtained.

  The COX inhibitory activity extract of the present invention can be used as a COX activity inhibitor. Therefore, the present invention relates to a COX activity inhibitor comprising the COX inhibitory activity extract.

  Since the COX activity inhibitor contains the COX inhibitory activity extract as an active ingredient, it inhibits COX-2 more strongly than COX-1. The COX activity inhibitor may be mixed with other components other than the COX inhibitory activity extract as long as the COX inhibitory activity is not impaired. There is no limitation in particular as another component.

  The COX inhibitory activity extract or the COX activity inhibitor may be blended with various base materials to form a composition. The blending amount and the type of base material are not particularly limited, and may be set as appropriate. The base material is not particularly limited as long as it is a product used for foods, pharmaceuticals, quasi drugs, and the like, and examples of the oral administration base material include tablets, capsules, candy, gummi, and beverages. As a blending method for these various base materials, it can be produced by using known techniques in the fields of foods, pharmaceuticals, quasi drugs and the like.

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited thereto.

Example 1
100 ml of ethanol was added to 10.0 g of peach leaf powder and stirred at room temperature for 1 hour. The extract was filtered through filter paper, and the filtrate was concentrated with a rotary evaporator under reduced pressure. As a result, 0.82 g of extract was obtained from the 10.0 g sample. The extract was dissolved in dimethyl sulfoxide and used in the following experiments as a peach leaf extract adjusted to a concentration as described below.

(Test Example 1) COX Activity Inhibition Test A test was performed using Cayman chemical Cox Inhibitor Screening Assay (Cox Inhibitor Screening Assay, Catalog No. 560131).

(Sample solution)
As the sample, a peach leaf extract obtained in Example 1 was appropriately diluted with dimethyl sulfoxide so that the extract concentration was 10, 1.0, 0.1, 0.01 mg / ml. As a control, dimethyl sulfoxide containing no extract was used as a negative control. Moreover, what dissolved indomethacin (made by SIGMA), "NS-398" (made by CALBIOCHEM), and ibuprofen (made by Wako Pure Chemical Industries) in dimethylsulfoxide was used as a positive control.

(Method)
Arachidonic acid as a substrate was added to a COX solution as an enzyme solution to carry out a prostaglandin production reaction (hereinafter referred to as COX reaction). Each sample solution prepared at this time was added according to the test method of the kit, and the influence on the production amount of prostaglandins was examined. Prostaglandins produced by the COX reaction were quantified using an enzyme immunoassay (EIA method). At this time, the compound indomethacin used as a control is known as a compound that inhibits the activities of both COX-1 and COX-2. NS-398 is a compound that specifically inhibits COX-2 and ibuprofen is a compound that specifically inhibits COX-1 (for example, Timothy D. Warner. Et al. Proc. Natl. Acad. Sci. (1999)). 96: 7563-68). The enzyme activity inhibition rate was calculated from the prostaglandin production amount of each sample solution with respect to the control group. Using this result, the IC50 value (inhibitor amount showing 50% activity inhibition) of each inhibitor was determined. The results are shown in Table 1.

  From Table 1, the peach leaf extract which is the product of the present invention obtained in Example 1 showed inhibitory activity against COX-1 and COX-2. More specifically, similar to NS-398, COX-1 / COX-2 had a tendency to show a significantly large value. That is, a significantly stronger inhibitory effect was observed on COX-2 than on COX-1. Therefore, it is expected that the product of the present invention tends to specifically suppress COX-2 expressed during inflammation.

(Example 2)
210 ml of ethanol was added to 21.0 g of peach leaf powder and stirred at room temperature for 1 hour. The extract was filtered through filter paper, and the filtrate was concentrated with a rotary evaporator under reduced pressure. 1.04 g of extract was obtained from 21.0 g of sample.

  The above peach leaf extract was distributed using water and ethyl acetate. The obtained extract was dissolved in a mixed solution of ethyl acetate (40 ml) and water (40 ml) and allowed to stand, and then the upper layer was collected as an ethyl acetate fraction. This operation was repeated a total of 3 times to obtain about 120 ml of an ethyl acetate layer fraction. Further, this solution fraction was dried by a rotary evaporator to obtain 0.45 g of an ethyl acetate extract fraction. For the aqueous layer, 1-butanol was further added and distribution was performed in the same manner to obtain 0.42 g of an aqueous layer extraction fraction and 0.16 g of a butanol layer extraction fraction. Further, each extract fraction was dissolved in dimethyl sulfoxide so that the concentration was 1 mg / ml.

(Test Example 2)
In the same manner as in Test Example 1, the COX activity inhibition rate of the peach leaf fraction extract obtained in Example 2 was calculated. Regarding COX-1, 67.92% inhibition was observed in the ethyl acetate layer (AcOEt) and 61.48% inhibition in the butanol layer (BuOH), but the aqueous layer was comparable to the negative control. Similarly, for COX-2, 99.49% inhibition was observed in the ethyl acetate layer and 95.59% inhibition in the butanol layer, but inhibition in the aqueous layer was not observed (see FIG. 1).

(Test Example 3)
The peach leaf fraction extract obtained in Example 2 was adjusted with dimethyl sulfoxide so as to be 1.0, 0.1, 0.01 mg / ml. As a control, indomethacin dissolved in dimethyl sulfoxide was used.

  In the same manner as in Test Example 1, the COX activity inhibition rate of the peach leaf fraction extract was calculated. IC50 value of each inhibitor was calculated | required using this result. The results are shown in Table 2.

  From Table 2, the enzyme inhibitory activity comparable or more than the COX-2 inhibitory activity of indomethacin was seen in the extract obtained from the ethyl acetate layer fraction of the peach leaf extract. On the other hand, the extract obtained from the peach leaf butanol layer fraction showed about 1/30 of the inhibitory activity compared to the ethyl acetate layer fraction, but the aqueous layer fraction showed almost no activity. .

  The COX activity inhibitory extract of the present invention can be suitably used as a highly safe COX activity inhibitor, preferably a highly safe COX activity inhibitor that inhibits COX-2 more strongly than COX-1. .

FIG. 1 is a graph showing the results of an analysis of COX activity inhibition conducted in the same manner as in Test Example 1 using each extracted fraction of peach leaves by the partitioning operation of Example 2. The vertical axis shows the activity when no inhibitor is contained as 100, and the activity obtained when the compound or extract is added is subtracted from 100 as the inhibition rate (%).

Claims (4)

  An extract obtained by extraction with an organic solvent or an aqueous organic solvent solution of peach leaves and containing cyclooxygenase inhibitory activity.   The extract according to claim 1, which inhibits cyclooxygenase type 2 more strongly than cyclooxygenase type 1.   A cyclooxygenase activity inhibitor comprising the extract according to claim 1 or 2. A composition comprising the extract according to claim 1 or 2 or the cyclooxygenase activity inhibitor according to claim 3.

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