JP2015128393A - Refined composition of post-fermented tea with enhanced lox-1 inhibitory activity - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a refined material of a post-fermented tea leaf extract with enhanced LOX-1 inhibitory activity, specifically a refined composition of post-fermented tea with reduced unpleasant odor derived from the post-fermented tea leaf and having very good flavor, and to provide a method for producing the same, and food and drink containing the refined composition of post-fermented tea.SOLUTION: Provided is a refined composition of post-fermented tea with enhanced LOX-1 inhibitory activity. A concentration of teabrownin in an aqueous solution, in which a concentration of a solid content of the refined material is 1% (w/v), is shown to be 1.3 or more in terms of absorbance at 380 nm. Also, in an aqueous solution having a concentration of a solid content of the refined material of 0.1% (w/v), a concentration of 1,2,3-trimethoxybenzene is 20 ng/mL or less, or a concentration of 1,2-dimethoxybenzene is 1 ng/mL or less.

Description

  The present invention relates to a composition for inhibiting oxidative LDL binding activity of a lectin-like oxidized low-density lipoprotein receptor (LOX-1) derived from an aqueous extract of post-fermented tea leaves obtained by processing tea belonging to the genus Camellia, and The present invention relates to a food composition containing the inhibitor composition.

Conventionally, it is widely known that green tea has the effect of reducing the risk of cardiovascular disease. Compared to Western countries, Japanese people have a high smoking rate, but epidemiological data that coronary artery disease is low, human tests to verify anti-arteriosclerosis of green tea (for example, Non-Patent Document 1) This is supported by a variety of in vitro tests.
In fact, in Japan, many green teas for improving health functions related to cardiovascular disease prevention are already on the market. Non-green tea beverages such as green tea to prevent cholesterol absorption, green tea with body fat accumulation suppression and combustion promotion effects, green tea beverages containing catechins at high concentrations, soft drinks containing catechins at high concentrations, etc. Has formed a large market, and it can be seen that social needs are high.

  Pu'er tea produced from the same Camellia plant is a post-fermented tea that includes a fermentation process by microorganisms in the production process, and is characterized by its unique flavor. Although there is no scientific demonstration, it has been prized for a long time as a "drug of healing that helps saliva swell, stops thirst, awakens alcohol, suppresses nausea, and helps digestion" (Non-patent Document 2). In recent years, effects such as fat absorption inhibitory effect (Non-Patent Document 3) and sugar absorption inhibitory action (Non-Patent Document 4) have been reported in animal experiments, and a slimming effect on humans is expected. On the other hand, it has been reported that an old-fashioned musty flavor, which is peculiar to Pu'er tea, is brought about by methoxylphenols represented by 1,2,3-trimethoxybenzene (TMB) (Non-patent Document 5). However, this unique flavor and color are not familiar to Japanese people and are not as popular as green tea, black tea and oolong tea. So far, although the improvement method (patent document 1) of the fermentation method of puer tea which improves unique flavor and hue, the improvement method (patent document 2) of the extraction method, etc. are proposed, these flavor improvement There is no report that the product has an effect of preventing or ameliorating cardiovascular disease.

  According to the recent demographic statistics of the Ministry of Health, Labor and Welfare of Japan, the number one death in Japan by disease is malignant neoplasm (tumor and cancer), second is heart disease, and third is cerebrovascular disease. The total number of deaths due to heart disease and cerebrovascular disease is about 26% of the total, which is almost equivalent to about 30%, which is the death rate of malignant neoplasms (2010 (2010)). As for coping with malignant neoplasm, early detection / early treatment is important considering the onset process, and clinical approach is desirable. On the other hand, heart diseases and cerebrovascular diseases are diseases that are located downstream of obesity and arteriosclerosis, which are chronic diseases caused by lifestyle-related diseases, and their treatment also lasts for a long time.

  Treatment methods for chronic diseases are usually coping with long-term medication and guidance on daily living habits, but from the characteristics of the disease, from foods that can be taken daily This is a process that allows for a preventive approach, and rather a method of dealing with such a process is desirable.

  Low density lipoprotein (LDL) is known as a risk factor for ischemic heart diseases including myocardial infarction. LDL is originally a lipid protein complex necessary for the transport of plasma lipids, but it contains a lot of unsaturated fatty acids that are susceptible to oxidation and is easily oxidatively modified. In recent years, it has been found that this oxidized LDL (oxidized LDL) causes a functional change of vascular endothelial cells and is an important factor responsible for pathophysiological activity.

  A lectin-like oxidized low-density lipoprotein receptor (LOX-1) was first isolated and identified from vascular endothelial cells by one of the inventors as a receptor that mediates the action of oxidized LDL on vascular endothelial cells. In subsequent studies, LOX-1 expression has been confirmed not only in endothelial cells, but also in macrophages, platelets, vascular smooth muscle cells, and the like (Non-Patent Documents 7 to 9).

  The expression of LOX-1 is increased due to diabetes, hypertension, hyperlipidemia and the like, and anti-LOX-1 antibody (anti-human LOX-1 mouse monoclonal antibody TS92) prepared by Tatsuya Sawamura is oxidized. By inhibiting the binding between LDL and LOX-1, the decrease in endothelium-dependent relaxation reaction by oxidized LDL is recovered, and further, the formation of infarct after myocardial infarction and the intimal thickening after balloon injury are suppressed. In addition, it has been suggested that LOX-1 is also involved in thrombus formation, endotoxin-induced inflammatory action, and the development of stroke, so that LOX-1 is at various levels in the development of cardiovascular disease. It is assumed that they are involved (Non-Patent Documents 7 and 10-15).

  For example, as an invention aimed at inhibiting the binding of a ligand to LOX-1, a pharmaceutical composition utilizing an antibody against oxidized LDL receptor, apple polyphenol, gallate catechin, etc. have been proposed by Tatsuya Sawamura (Patent Documents 3 to 5).

  However, as a result of investigations by the present inventors, apple polyphenol and gallate-type catechin have oxidized LDL and LOX-1 when the same serum-derived protein as the actual vascular system on which LOX-1 acts is present in the evaluation system. It was newly found that there is a problem that the activity of inhibiting the binding to the protein significantly decreases.

Japanese Patent No. 04583417 JP2013-9641A Japanese Patent No. 4497586 JP 2011-006326 A JP 2012-111747 A

J Am Coll Nutr. 2005 Oct; 24 (5): p.342-346 Yozo Tanimoto “Charm of Chinese Tea” Shibata Shoten Phytotherapy Research Volume 25, Issue 2, p.234-238, 2011 Numazu National College of Technology Research Report No. 46, pages 331-336 Food Chemistry Vol 130, Issue 4, 1 5 February 2012, p. 1074-1081 Sawamura T, et al. , Nature, 386: p. 73-77, (1997). Chen M, et al. , Biochem Biophys Res Commun, 282: p. 153-158 (2001). Kataoka H, et al. , Arterioscler Throm Vasc Biol, 21: pp. 955-960 (2001). Yoshida H, et al. , Biochem J, 334: p.9-13 (1998). Mehta JL, et al. , Cir Res 100 (11) p.1634-1642 (2007) Li et al. , J. et al. Pharmacol. Exp. Ther. 302: p. 601-605 (2002) Hinagata et al. , Cardiovasc Res, 69: p.263-271 (2006). Kakutani M. et al. , Et al. , Proc Natl Ac ad Sci USA. 97: p. 360-364 (2000) Honjo M. , Et al. Proc Natl Acad Sci USA. 100: p. 1274-1279 (2003) Inoue N. , Et al. , Clin Chem. 2010 Apr; 56 (4): pp. 550-558.

  As a result of conducting research on foods and the like that are more excellent in flavor and expected to have health functionality using tea extract, the present inventors have found that a component having a high cardiovascular disease prevention effect is a type of post-fermented tea. It was discovered for the first time that the tea extract had, and the present invention was completed for the first time by developing a technology capable of reducing the unpleasant odor derived from the post-fermented tea extract.

  Therefore, the present invention is a purified product of a post-fermented tea leaf extract with enhanced LOX-1 inhibitory activity, which has a very tasty post-fermented tea purified composition that reduces the unpleasant odor derived from the post-fermented tea leaf, and It aims at providing the manufacturing method and the food / beverage products containing the said post-fermented tea refinement | purification composition.

The gist of the present invention is as follows:
[1] A post-fermented tea purification composition having enhanced LOX-1 inhibitory activity,
The concentration of theabraunin in an aqueous solution in which the solid content of the purified product is 1% (w / v) is 1.3 or more at an absorbance of 380 nm, and
The 1,2,3-trimethoxybenzene concentration in an aqueous solution in which the solid content of the purified product is 0.1% (w / v) is 20 ng / mL or less, or the 1,2-dimethoxybenzene concentration is 1 ng / mL. A post-fermented tea refining composition, characterized in that:
[2] A method for producing a post-fermented tea purified composition containing a purified product of a post-fermented tea leaf extract having enhanced LOX-1 inhibitory activity,
Steps (a) to (c):
(A) a step of extracting fermented tea leaves with water or hot water after the step of immersing in lower alcohol or hydrous lower alcohol,
(B) a step of contacting the post-fermented tea leaf hot water extract or the post-fermented tea leaf extract obtained in the step (a) with a solid adsorbent to recover non-adsorbed components;
(C) The post-fermented tea leaf hot water extract or the post-fermented tea leaf extract obtained in the step (a) is treated with an ultrafiltration membrane having a fractional molecular weight of 1000 to 10,000, and passes through the ultrafiltration membrane. Recovering the components that do not
The method for producing a post-fermented tea purification composition according to [1], wherein the method comprises one or more steps selected from the group consisting of:
[3] A food or drink comprising the post-fermented tea purified composition according to [1],
About.

  In the present invention, “LOX-1 inhibitory activity” means an action that inhibits the binding of oxidized LDL to LOX-1 when evaluated by a method as described in Examples below. Further, “increased LOX-1 inhibitory activity” means that when LOX-1 inhibitory activity is evaluated by a method as described in Test Example 1 described later, the general method of brewing post-fermented tea is used. Furthermore, when the extract obtained by the post-fermentation tea leaf extraction method is used as a comparative product, this comparative product means that the action of inhibiting the binding of oxidized LDL to LOX-1 is enhanced.

The post-fermented tea purified composition of the present invention is expected to have excellent health functionality by significantly inhibiting the binding of oxidized LDL to LOX-1 in the vascular system, and suppresses unpleasant odors. It is very easy to ingest.
In addition, even when the post-fermented tea purified composition of the present invention is blended in foods and drinks, the unpleasant odor derived from the post-fermented tea leaves is remarkably suppressed, so that it is difficult to cause problems when ingested, and the obtained food and drink The health functionality of the product can be increased.

The left figure of FIG. 1 shows the photograph of the mesenteric artery for analysis of the lipid deposition area in the mesenteric artery of each extract in Test Example 2. Moreover, the right figure of FIG. 1 dyes the lipid deposition in each mesenteric artery of the rat ingesting each extract in Test Example 2 with Oil Red O, and the area of the lipid deposition site in the total area of the mesenteric artery The results of the average values obtained by testing one group at n = 12 and measuring the ratio are graphed.

  Hereinafter, the present invention will be described in detail.

The post-fermented tea purified composition of the present invention includes a purified product of post-fermented tea leaf extract having enhanced LOX-1 inhibitory activity,
The concentration of theabraunin in an aqueous solution in which the solid content of the purified product is 1% (w / v) is 1.3 or more at an absorbance of 380 nm, and
The 1,2,3-trimethoxybenzene concentration in an aqueous solution in which the solid content of the purified product is 0.1% (w / v) is 20 ng / mL or less, or the 1,2-dimethoxybenzene concentration is 1 ng / mL. It is characterized by the following.

  The post-fermented tea leaves used in the present invention are long-term ripened teas produced by fermenting the leaves of Camellia (genus Camellia) with microorganisms such as molds (genus Aspergillus, genus Rhizopus). In the classification of tea, it is called “post-fermented tea” and is widely used in China under the names such as “pu-er tea”, “black tea” and “general tea”. In Japan, Kochi Prefecture's "Soteishi Tea" and Toyama Prefecture's "Batabata Tea" are produced and consumed in small quantities. Any post-fermented tea leaves used in the present invention can be used as long as they are post-fermented with microorganisms as described above, but Pu'er tea is preferred from the viewpoint of easy availability.

  The purified product of the post-fermented tea leaf extract used in the present invention means a product obtained by further purifying the post-fermented tea leaf extract, and specifically, any product obtained by the method described later.

  As a method for measuring the degree of fermentation of post-fermented tea leaves, there is a method for measuring the theabrownin concentration. Tea brownin is a generic name for ethyl acetate in post-fermented tea leaf extract and water-soluble components that are not soluble in n-butanol. Surprisingly, tea brownin concentration correlates with LOX-1 inhibitory activity, and high tea brownin concentration The present inventors have shown for the first time in the art that these have high LOX-1 inhibitory activity. As a method for measuring the theabrownin concentration in the present invention, specifically, a purified product of post-fermented tea leaf extract is dissolved in water so that the solid content concentration becomes 1% (w / v), The treatment was carried out by the method shown in the Example, and the theabrownin concentration was represented by a value measured with an absorptiometer at an absorbance of 380 nm.

When the theabraunin concentration of the post-fermented tea leaf extract without the purification treatment is measured by the above method, the absorbance at 380 nm is 1.0 for normal hot water extraction and about 1.2 for pressure heating extraction. However, the post-fermented tea leaf extract obtained by these extraction methods is an extract containing many unpleasant flavors peculiar to post-fermented tea.
On the other hand, when the theabrownin concentration of the post-fermented tea purified composition of the present invention is measured by the above method, the absorbance at 380 nm is 1.3 or more, preferably 1.6 or more, more preferably 1.8 or more. is there. When the theabrownin concentration is less than 1.3, a sufficient LOX-1 inhibitory activity cannot be expected, and when it is less than 1.0, an unpleasant flavor stands out or almost no LOX-1 inhibitory activity is observed.

The purified product of the post-fermented tea leaf extract used in the post-fermented tea purified composition of the present invention is 1, 2, 3 in an aqueous solution having a solid content of 0.1% (w / v). -The trimethoxybenzene concentration is 20 ng / mL or less, or the 1,2-dimethoxybenzene concentration is 1 ng / mL or less.
In the present invention, attention is paid to 1,2,3-trimethoxybenzene and 1,2-dimethoxybenzene as the cause of the old-fashioned musty flavor, which is a unique flavor of post-fermented tea, and the respective contents are within the above ranges. By using the reduced purified product of post-fermented tea leaf extract, it is possible to obtain a post-fermented tea purified composition that significantly reduces the unpleasant odor peculiar to post-fermented tea.

  Specifically, the measurement of the concentration of 1,2,3-trimethoxybenzene and 1,2-dimethoxybenzene in the present invention was carried out using a purified product of post-fermented tea leaf extract having a solid content concentration of 0.1% (w / v) and dissolved in water, and this solution can be measured by a gas chromatography-mass spectrometer as shown in the following examples.

  As a purified product of post-fermented tea leaf extract used in the present invention, the concentration of 1,2,3-trimethoxybenzene is 20 ng / mL or less, or the concentration of 1,2-dimethoxybenzene is 1 ng / mL or less. If the concentration of 1,2,3-trimethoxybenzene is 20 ng / mL or less and the concentration of 1,2-dimethoxybenzene is 1 ng / mL or less, the unpleasant odor derived from post-fermented tea is more This is preferable because it is suppressed.

  The post-fermented tea refining composition of the present invention may be composed of a purified product of the post-fermented tea leaf extract.

The method for producing a post-fermented tea purification composition of the present invention includes the following steps (a) to (c):
(A) a step of extracting fermented tea leaves with water or hot water after the step of immersing in lower alcohol or hydrous lower alcohol,
(B) a step of contacting the post-fermented tea leaf hot water extract or the post-fermented tea leaf extract obtained in the step (a) with a solid adsorbent to recover non-adsorbed components;
(C) The post-fermented tea leaf hot water extract or the post-fermented tea leaf extract obtained in the step (a) is treated with an ultrafiltration membrane having a fractional molecular weight of 1000 to 10,000, and passes through the ultrafiltration membrane. Recovering the components that do not
One or more steps selected from the group consisting of:

In the step (a), first, post-fermented tea leaves are pretreated by immersing them in lower alcohol or hydrous lower alcohol. By this pretreatment, impurities adhering to the post-fermented tea leaves can be removed.
In general, the pretreatment using the lower alcohol or the hydrous lower alcohol is performed for about 10 minutes to one week under the temperature condition of the normal temperature to the pressurization and the normal temperature to the pressurization of the solvent in accordance with the lower alcohol to be used. Good. As the lower alcohol, a solvent that is usually used in accordance with the treatment step may be appropriately selected and used. For example, a primary alcohol such as methanol, ethanol, propanol, or butanol, or propylene glycol, 1,3-butylene glycol, etc. A polyhydric alcohol is mentioned. A primary alcohol is more preferable as the treatment solvent. The hydrous lower alcohol is a mixture of the lower alcohol and water. The ratio of the amount of water and the lower alcohol in the hydrous lower alcohol is such that the content of the lower alcohol is 50% (v / v) or more. There is no particular limitation.
Among them, when the residual organic solvent is not preferable, as in the case of adding the post-fermented tea purified composition of the present invention to food, it is preferable to use ethanol, hydrous ethanol or the like. It is most preferable to use 70 to 95% (v / v) as hydrous ethanol.
These lower alcohols or water-containing lower alcohols can be used alone as an extraction solvent, but can also be used in any combination of two or more.

  The pretreatment method in the step (a) is not particularly limited, and standing, stirring treatment, room temperature homogenization treatment, reflux extraction treatment, supercritical fluid extraction treatment, and the like can be used.

  Typical methods include the following. That is, 5 to 20 times the amount of lower alcohol or hydrous lower alcohol is added to post-fermented tea leaves, and the mixture is allowed to stand at normal pressure at room temperature for about one week, or near the boiling point of pressurized alcohol or hydrous lower alcohol or under pressurized conditions. After refluxing for about 30 to 120 minutes near the boiling point of the solvent used, filtration is performed, and the obtained tea leaves are air-dried or pressed to obtain post-extraction fermented tea leaves.

Next, the obtained post-fermented tea leaves for extraction are extracted with water or hot water. After the pretreatment as described above, the product obtained by extraction with water or hot water becomes a purified product from which impurities are removed rather than the extract of post-fermented tea leaves.
Either water or hot water may be used for the extraction, but extraction with hot water is preferred particularly in terms of extraction efficiency. The temperature of the hot water may be 80 to 130 ° C.

The amount of water or hot water used is preferably from 100 to 10,000 parts by weight, more preferably from 150 to 9,000 parts by weight, even more preferably from 200 to 5,000 parts by weight, based on 100 parts by weight of post-fermented tea leaves for extraction. .
When the amount of water or hot water used is 100 parts by weight or more, the LOX-1 inhibitory activity of the extract is increased, and when it is 10,000 parts by weight or less, solid-liquid separation after extraction is favorably performed. be able to.

  In extraction, water or hot water and post-fermented tea leaves for extraction may be placed in an extraction tank and heated, and stirred as necessary. What is necessary is just to adjust as heating temperature so that the temperature of the water in an extraction tank may be 80-130 degreeC. Moreover, although extraction time cannot be generally specified by the temperature at the time of extraction, from the viewpoint that the LOX-1 inhibitory activity of the extract becomes high and the amount of the extract can be secured, 0.1 to 5 hours are preferable, and more preferably 0. 2 to 4 hours are preferred.

  In order to promote extraction efficiency, steam can be sprayed directly on the fermented tea leaves after extraction with a pressure cooker, etc., or the jacket attached to the periphery of the pressure cooker can be heated and the temperature can be raised using this propagation heat. You may employ | adopt the method of heating while applying the method of making it easy, and using a retort.

A product obtained by drying a purified product of the post-fermented tea leaf extract obtained in the step (a) by a conventional method has an absorbance of 380 nm in an aqueous solution having a theabraunin concentration of 1% (w / v). .3 and more, and since the 1,2,3-trimethoxybenzene concentration is 20 ng / mL or less or the 1,2-dimethoxybenzene concentration is 1 ng / mL or less, the post-fermented tea purified composition is used as it is. Can be used as
However, usually, a solid-liquid separation process such as centrifugal separation or a foreign substance is removed by a filter press to obtain an extract, and the extract may be concentrated and used in the form of an extract. You may dry and use. The method for concentrating the extract is not particularly limited, and there are vacuum concentration, membrane concentration, freeze concentration, etc., and drying is performed by using a machine such as spray drying, freeze drying, kneader, and nauter mixer. Can do.

  Moreover, in the said (b) process or (c) process, the hot water extract of post-fermented tea leaves or the post-fermented tea leaf extract obtained by the said (a) process is used.

The hot-water extract of post-fermented tea leaves used in the step (b) or the step (c) refers to one extracted by immersing the post-fermented tea leaves in hot water.
As temperature conditions of hot water, what is necessary is just 80-130 degreeC.
The amount is preferably 100 to 10,000 parts by weight, more preferably 150 to 9,000 parts by weight, and still more preferably 200 to 5,000 parts by weight with respect to 100 parts by weight of the post-fermented tea leaves.
When the amount of water or hot water used is 100 parts by weight or more, the LOX-1 inhibitory activity of the extract is increased, and when it is 10,000 parts by weight or less, solid-liquid separation after extraction is favorably performed. be able to.

  As the extraction method, for example, a method of spraying steam directly onto post-fermented tea leaves with a pressure cooker or the like, a jacket attached around the pressure cooker is heated, and this propagation heat is used to easily raise the temperature. You may employ | adopt the method heated while pressing using a method and a retort.

In the step (b), the hot water extract of the post-fermented tea leaf or the post-fermented tea leaf extract obtained in the step (a) can be further purified using a solid adsorbent.
As the solid adsorbent, activated carbon, diatomaceous earth, silica gel, zeolite and the like can be used alone or in appropriate combination. For example, an adsorbent in which 66 parts by weight of diatomaceous earth is mixed with 34 parts by weight of activated carbon is packed in a column, and the hot water extract of the post-fermented tea leaves or the post-fermented tea leaf extract obtained in the step (a) is passed through. In this way, purification can be performed.

In the step (c), the post-fermented tea leaf hot water extract or the post-fermented tea leaf extract obtained in the step (a) is treated with an ultrafiltration membrane having a fractional molecular weight of 1000 to 10,000, Purification can proceed by collecting components that do not pass through the ultrafiltration membrane.
As the molecular weight cut-off of the ultrafiltration membrane is less than 1000, unpleasant components remain, a large amount of high molecular weight material that does not pass through the membrane remains, and it takes time for the solution to pass through the membrane. It is bad and not preferable. On the other hand, when the molecular weight cut off exceeds 10,000, the active ingredient also passes through the membrane and the recovery rate is deteriorated, which is not preferable for purification of the post-fermented tea leaf extract. The molecular weight cutoff of the ultrafiltration membrane is preferably 3,000 to 10,000.

  In the present invention, a purified product of post-fermented tea leaf extract may be produced using any of the steps (a), (b), and (c). For example, the step (a), (b) You may mix the refinement | purification material of the post-fermented tea leaf extract obtained using two or more processes among process, (c) processes.

  After using the purified product of post-fermented tea leaf extract obtained by the production method of the present invention as described above, after maintaining a high theabraunin concentration and enhancing the LOX-1 inhibitory activity without an unpleasant flavor A fermented tea refining composition can be obtained.

  The purified product of the post-fermented tea leaf extract obtained as described above has a stronger LOX-1 inhibitory activity than the post-fermented tea leaf extract that is a raw material, and the LOX-1 inhibitory action makes the heart. Since a vascular disease prevention and improvement effect can be achieved, the post-fermented tea purified composition with enhanced LOX-1 inhibitory activity of the present invention comprising the purified product of the post-fermented tea leaf extract as an active ingredient is a novel cardiovascular It is also useful as a disease prevention agent.

  In addition, since the safety of the post-fermented tea leaf extract used in the present invention has already been generally confirmed, the safety of the post-fermented tea purified composition with enhanced LOX-1 inhibitory activity of the present invention is also the same. It is thought that it is excellent.

  Since the post-fermented tea purified composition having enhanced LOX-11 inhibitory activity of the present invention is a refined composition with good flavor, it may be blended in food and drink. It does not specifically limit as food-drinks, For example, a drink, alcoholic beverage, jelly, confectionery, functional food, health food, health-oriented food, etc. are mentioned. In consideration of preservability, portability, ease of ingestion and the like, confectionery is preferable, and among confectionery, hard candy, soft candy, gummy candy, tablet, chewing gum and the like are preferable.

  In the case where the post-fermented tea purified composition with increased LOX-1 inhibitory activity of the present invention is added to food or drink, the content of the post-fermented tea purified composition with increased LOX-1 inhibitory activity in the food or drink is its physiological activity. Any amount can be used as long as the effect can be expected. Usually, it is preferable to appropriately determine the blending amount according to the form of the food or drink so that 10 to 10000 mg, more preferably 100 to 3000 mg can be ingested per day. For example, 5 to 50% by mass is preferable in the case of a solid food, and 0.01 to 10% by mass in the case of a liquid food such as a beverage. The preventive effect of cardiovascular disease induced by binding of oxidized LDL to LOX-1 by daily intake of foods and drinks containing the fermented tea purified composition after enhancing the LOX-1 inhibitory activity described above Be expected.

  Moreover, since the post-fermented tea purified composition having enhanced LOX-1 inhibitory activity obtained by the production method of the present invention is excellent in safety, not only for humans but also for non-human animals, for example, You may mix | blend with therapeutic agents or feed, such as a rat, a mouse | mouth, a guinea pig, a rabbit, a sheep, a pig, a cow, a horse, a cat, a dog, a monkey, a chimpanzee, a bird, an amphibian, a reptile. As feed, for example, livestock feed used for sheep, pigs, cattle, horses, chickens, etc., feed for small animals used for rabbits, rats, mice, etc., feed for seafood used for eel, Thailand, yellowtail, shrimp, etc., dogs, Pet foods used for cats, small birds, squirrels, etc. are listed.

  EXAMPLES Next, although this invention is demonstrated in detail based on an Example, this invention is not limited only to this Example.

(Test Example 1: Comparison of LOX-1 inhibitory activities of various tea extracts)
LOX-1 inhibitory activity for each hot water extract of green tea (non-fermented tea), black tea (strongly fermented tea) and puer tea (post-fermented tea) that are typical teas produced from Camellia plants The comparison was made as follows.

  Add 5 g each of green tea leaves (manufactured by Ujimori Tokusha), black tea leaves (manufactured by Kyoto Grain Systems), and puer tea leaves (manufactured by Kyoto Grain Systems) to 450 mL of distilled water, pressurize in an extraction tank at 120 ° C. for 20 minutes, After heating and hot water extraction, the extract extracted from the solid was vacuum dried to obtain a dried product of each extract.

The recombinant hLOX-1 protein used for measurement of LOX-1 inhibitory activity is the extracellular domain of human-derived LOX-1. Among the human LOX-1 cDNA (Genbank: NM002543), a region encoding the extracellular domain (ex-hLOX-1) (61st to 273rd nucleotide sequence) expressed and purified according to a conventional method was used. The recombinant hLOX-1 protein was confirmed to have the ability to bind to oxidized LDL, and was used as a test LOX-1 preparation by the following ELISA (Enzyme Linked Immunosorbent Assay).
Note that the oxidized LDL is obtained from Sawamura T, et al. It produced according to the method as described in Nature, 386: 73-77, (1997).

ELISA was performed using a maxi soap immunoplate (96 well type, manufactured by NUNC). The recombinant hLOX-1 protein purified as described above was adjusted to 5 μg / mL with phosphate buffered saline (PBS (−) buffer), and 50 μL was applied to each well. After allowing to stand overnight at 4 ° C., each well was washed twice with PBS (−) buffer at 400 μL × 2 times, and 300 μL of PBS (−) buffer containing 20% immunoblock was applied to each well. After standing at 25 ° C. for 2 hours, each well was washed with PBS (−) buffer at 400 μL × 2 times, and HamF12-HEPES buffer (HamF12 medium (manufactured by Gibco), 1% fetal bovine serum, 10 mM) 50 μL of each purified sample adjusted to 0.1 to 30 μg / mL with 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES) was applied to each well. After standing at 4 ° C. for 1 hour, each well was washed with PBS (−) buffer at 400 μL × 3 times, and oxidized LDL adjusted with HamF12-HEPES buffer so as to be 1 μg / mL was applied to each well. After standing at 4 ° C. for 1 hour, each well was washed with PBS (−) buffer at 400 μL × 3 times, and Anti-ApoB antibody (manufactured by Binding site) was added to 10 mM HEPES / 150 mM NaCl / 1% bovine serum albumin. An appropriate amount was diluted with (BSA), and 50 μL was applied to each well. After standing at room temperature for 1 hour, each well was washed with PBS (−) buffer 400 × L 3 times, and HRP-rabbit anti-sheep IgG antibody (manufactured by Invitrogen) was added to 10 mM HEPES / 150 mM NaCl / 1% BSA. A suitable amount was diluted with 50 μL and applied to each well. After standing at room temperature for 1 hour, each well was washed with 400 μL × 5 times with PBS (−) buffer, and 3,3 ′, 5,5′-tetramethylbenzidine (TMB) peroxidase-enzyme immunoassay ( 50 μL of EIA) -substrate-kit reagent (Bio-rad) was applied to each well. After an appropriate reaction time, 50 μL of 0.5 M sulfuric acid was applied to each well to stop the reaction. Finally, detection was performed at 450 nm, LOX-1 inhibitory activity (inhibition rate of oxidized LDL binding to LOX-1) was quantified, and IC ₅₀ (50% inhibitory concentration) was calculated and compared. In addition, IC _{50 under} test conditions in which fetal bovine serum was not included in the system when the inhibitor was added was also calculated and compared. Table 1 shows the results. Incidentally, IC ₅₀ in the table indicates that the number is low enough LOX-1 inhibiting activity high.

  As a result, each of green tea, black tea, and puer tea shows high activity when no serum-derived protein is contained in the inhibitor reaction (lower part of Table 1), but when serum-derived protein is present in the system (upper part of Table 1). ) In green tea and black tea, a decrease in inhibitory activity was observed. On the other hand, it became clear that the puer tea extract maintained high LOX-1 inhibitory activity even when serum-derived protein was present in the system, and the superiority of the puer tea extract in the presence of the serum-derived protein was clarified. It was.

(Test Example 2: Effectiveness test of Pu'er tea leaf extract)
Using the Puer tea leaf extract obtained in Test Example 1, the ability to inhibit lipid deposition in the mesenteric artery of spontaneous stroke model rats (SHRSP / Izm strain, male, 8 weeks old, manufactured by CLEA Japan, Inc.) was evaluated. did.

  A group of 12 model rats as a group, a 1% saline drinking group, a 1% saline drinking group containing 0.2% weight-concentrated puer tea hot water extract, 1% containing 0.2% weight apple polyphenol The test was conducted in 4 groups of a saline drinking water group and a 1% saline drinking water group containing a 0.2% weight concentration green tea hot water extract. All the groups were fed “High Fat Diet” (excluding α-tocopherol) manufactured by CLEA Japan, and these solid feed and saline were used in the form of free intake, and one week was the breeding period. In addition, experiments on lipid deposition of mesenteric arteries using spontaneous stroke model rats were carried out with the following modifications, with reference to a previous report (Japan Circuit Journal, Vol. 39, p601-609, 1975). It was.

  A model rat was orally administered with control saline or saline containing Puer tea leaf extract for 1 week, and then anesthetized with isoflurane, followed by laparotomy and thoracotomy. Serum was collected from the laparotomy / thoracotomy rat, and the mesenteric artery was removed together with the surrounding adipose tissue. After removal, the surrounding adipose tissue of the mesenteric artery was removed using tweezers in PBS buffer. The obtained mesenteric artery was washed and equilibrated with 60% isopropanol, and then the lipid deposited in the mesenteric artery was stained with Oil Red O staining solution (manufactured by Wako Pure Chemical Industries, Ltd.), and 60% isopropanol / 30 The decolorization operation was carried out while gradually decreasing the isopropanol concentration to% isopropanol / PBS buffer. Finally, the area of lipid deposits stained by Oil Red O staining was measured under a microscope.

  The serum total cholesterol concentration and HDL cholesterol concentration were measured from the collected serum using cholesterol E test Wako and HDL cholesterol E test Wako (manufactured by Wako Pure Chemical Industries, Ltd.). No significant difference was observed in blood total cholesterol concentration or HDL cholesterol concentration between rats ingested with saline containing apple polyphenol and green tea extract.

  Moreover, the measurement result (right figure of FIG. 1) of the lipid deposition area in the mesenteric artery obtained by Experiment 2 and the photograph (left figure of FIG. 1) of the analyzed actual mesenteric artery are shown. From this result, the lipid deposition area in the blood vessels stained with Oil Red O staining solution was significantly reduced in the Puer tea leaf extract ingestion group (indicated as “Puer” in the figure), and the average deposition area was It became smaller than apple polyphenol. That is, it is considered that Puer tea extract suppressed arteriosclerosis induced by a high fat diet. From this, it was clarified that the puer tea leaf extract has a significant effect on the prevention of arteriosclerosis, although the blood total cholesterol level does not change greatly.

  In addition, the weight gain rate of the rats administered with the Puer tea leaf extract in the above Examples was not changed compared with the rats not administered. In addition, when a rat administered with the Puer tea leaf extract was opened, no significant organ abnormality was observed. From the above, it was revealed that the Pu'er tea leaf extract is highly safe.

(Example 1: Production of purified product of Pu'er tea extract)
<Examination of immersion conditions with hydrous ethanol>
10 g of Pu'er tea leaves (manufactured by Kyoto Grain System) was added to 500 mL of 50, 60, 70, 80, 90, 100% (v / v) aqueous ethanol solution and refluxed for 2 hours. After refluxing, the Pu'er tea leaves were filtered with filter paper no. The filtrate was filtered through 101 (Advantech) to obtain a reflux extract (prototypes 1 to 6) as a filtrate. The tea leaves after reflux filtration were air-dried overnight, and the whole amount was added to 900 mL of distilled water, followed by pressure and heating extraction at 120 ° C. for 20 minutes in a pressure tank. Filtered at 101 to obtain a purified Puer tea leaf extract (prototypes 7 to 12).
The LOX-1 inhibitory activity of each extract and purified product was measured according to the procedure of Test Example 1, and the following formula was obtained from the obtained IC ₅₀ :
Relative activity ratio = comparative product IC ₅₀ / prototype IC ₅₀
Based on this, the relative activity ratio was determined. A sample having a higher inhibitory activity than a comparative product has a relative activity ratio of greater than 1, and a larger relative activity ratio indicates a higher inhibitory activity. As a comparative product, a puer tea leaf extract extracted by the method of Test Example 1 was used.

Further, the theabrownin concentration (TB value) of each extract purified product was measured by the following procedure. Prototypes 1 to 12 were dried using a centrifugal evaporator, each was dissolved in distilled water to a concentration of 10 mg / mL, and then vigorously shaken and mixed with an equal amount of n-butanol for 3 minutes to 2400 × g. Centrifuge at. The obtained lower layer water layer portion 80 μL, saturated aqueous oxalic acid solution 80 μL, distilled water 240 μL, 95% (v / v) ethanol 600 μL were mixed, and the absorbance at 380 nm was measured to obtain the TB value. As a comparative product, a puer tea leaf extract extracted by the method of Test Example 1 was used.
Table 2 shows the extraction conditions for each prototype, and Table 3 shows the relative activity ratio and TB value of each prototype and comparative product.

As a result, when compared with comparative products, LOX-1 inhibitory activity was particularly improved in prototypes 1, 2, and 11.
Since the LOX-1 inhibitory activity was particularly excellent for the above-mentioned prototypes 1, 2, and 11, the purified puer tea obtained by dissolving the comparative product and the prototypes 1, 2, and 11 in water at 0.5% weight concentration The flavors were compared by evaluation of 5 panelists. A score of 1 was given for those with no unpleasant odor, and a score of 0 was given for those with an unpleasant odor. Table 4 shows the total score of each evaluation product.
In addition, a total of 4 or more points shall be accepted.

As a result, the prototype 11 was a purified product having no unpleasant odor, good flavor, and increased LOX-1 inhibitory activity. On the other hand, although Prototypes 1 and 2 have an increase in LOX-1 inhibitory activity, they have an unpleasant odor and are significantly inferior to the comparative product, and are not suitable for the present invention.
Moreover, when the flavor of the Puer tea leaf extract refinement | purification thing in which the improvement of LOX-1 inhibitory activity was seen like prototypes 9, 11, and 12 was investigated similarly, all became a total score of 4 or more in evaluation. .
From the above results, the purified Puer tea leaf extract with improved LOX-1 inhibitory activity has a TB value of 1.3 or more, and the unpleasant odor is remarkably reduced and the flavor is good. On the other hand, it became clear that the purified product of Puer tea leaf extract having a TB value of less than 1.3 has an unpleasant flavor.

(Example 2: Production of purified product of Pu'er tea leaf extract)
20 g or 40 g of Pu'er tea leaves (manufactured by Kyoto Grain System Co., Ltd.) was added to 1000 mL of a 90% (v / v) aqueous ethanol solution and immersed for 1 week with stirring every 24 hours. After soaking, the Pu'er tea leaves were filtered with paper no. The mixture was filtered through 101 (Advantech Co., Ltd.), air-dried overnight, and 5 g of the obtained tea leaves were added to 450 mL of distilled water, followed by pressurization and heating at 120 ° C. for 20 minutes in a pressurized tank to perform hot water extraction. A prototype 13 was obtained from a 20 g soaked puer tea leaf, and a prototype 14 was obtained from a 40 g soaked puer tea leaf. The relative activity ratio and TB value of the LOX-1 inhibitory activity of the purified product of the obtained Puer tea leaf extract were measured in the same manner as in Example 1. The puer tea leaf extract extracted by the method of Test Example 1 was used as a comparative product. The results are shown in Table 5.

  As a result, both prototypes 13 and 14 showed LOX-1 inhibitory activity superior to that of the comparative product, and the TB value was 1.3 or more. In addition, a sensory test using the same panel as in Example 1 was performed. Both prototypes were refined products with excellent flavor because the total score of evaluation was 4 or more. That is, it was shown that a purified product of Pu'er tea leaf extract can be produced by a very simple method in which tea leaves are allowed to stand still in hydrous ethanol.

(Example 3: Production of purified product of Pu'er tea leaf extract)
100 g of Pu'er tea leaves (manufactured by Kyoto Grain System Co., Ltd.) were added to 900 mL distilled water, and extraction was performed by heating at 120 ° C. for 20 minutes. After collecting the extract, the extract was passed through powdered activated carbon to obtain prototype 15. Subsequently, the prototype 15 was subjected to an ultrafiltration membrane having a molecular weight cut-off of 10,000, ultrafiltration was performed, and components that did not pass through the filtration membrane were collected to obtain a prototype 16. The relative activity ratio and TB value of the LOX-1 inhibitory activity of the purified product of the obtained Puer tea leaf extract were measured in the same manner as in Example 1. The puer tea leaf extract extracted by the method of Test Example 1 was used as a comparative product. The results are shown in Table 6.

  As a result, both prototypes 15 and 16 showed LOX-1 inhibitory activity superior to that of the comparative product, and the TB value was 1.3 or more. In addition, a sensory test using the same panel as in Example 1 was performed. Both prototypes were refined products with excellent flavor because the total score of evaluation was 4 or more. That is, it was shown that the puer tea leaf extract purified product having a high TB value is a purified puer tea leaf extract having high LOX-1 inhibitory activity and excellent flavor.

(Example 4: Comparison of unpleasant odor of purified product of Pu'er tea leaf extract)
The TMB and 1,2-dimethoxybenzene (DMB) concentrations in the prototypes 11, 13, and 14 were measured, and the presence or absence of an unpleasant odor was compared. As comparative products, Puer tea extracted by the method of Test Example 1 (Comparative product 1) and Puer tea put in a teapot (Comparative product 2) were used. Comparative product 2 was obtained by putting 5 g of tea leaves into a teapot, adding 160 mL of hot water, rinsing the tea leaves, once discarding the hot water, and newly adding 200 mL of hot water and steaming for 3 minutes for extraction. Measurement of TMB and DMB was performed using gas chromatography-mass spectrometer (GC-MS) analysis. Equipment used is Rtx (registered trademark) -200MS (0.25 mmid × 30 m, film thickness 0.25 μm, manufactured by RESTEK), SPME fiber (50/30 μm, DVB / CAR / PDMS, manufactured by Spellco), GC-MS Used was JMS-Q1000GC K9 (manufactured by JEOL Ltd.). The SPME fiber was subjected to preheating (65 ° C., 1 minute), adsorption time (30 minutes), desorption time (10 minutes), and inlet temperature (250 ° C.). The GC conditions were as follows: temperature conditions (50 ° C. for 10 minutes → 320 ° C. up to 10 ° C./minute→320° C. for 10 minutes), split (ratio: 20), and carrier gas (helium).
Create a calibration curve using 1,2,3-trimethoxybenzene and 1,2-dimethoxybenzene (both manufactured by Sigma-Aldrich), unify the weight concentration of each prototype and comparative product to 0.1%, TMB and DMB concentrations contained in each were measured. The results are shown in Table 7.

As a result, it became clear that the TMB contained in the prototypes was less than 1/10 of the comparative products 1 and 2, and the DMB was also at a concentration below the detection limit, and the unpleasant odor of these prototypes was greatly improved. It became clear that
On the other hand, Comparative products 1 and 2 both strongly felt an unpleasant odor derived from Pu'er tea leaves.

(Example 5: Food containing Puer tea leaf extract purified product)
1 g of the solid material of the prototype 16 obtained in Example 3 was dissolved in 100 mL of ethanol in advance, and 500 g of paratinite (manufactured by Palatinit), 714 g of reduced maltose starch syrup (manufactured by Towa Kasei Kogyo Co., Ltd., Bx70) (500 g of solid content) ) Was mixed in a vacuum kettle and cooked to 155 ° C. under a vacuum degree of −600 mmHg. When this was put into a cooling plate and cooled to about 100 ° C., 15 g of citric acid, 1.1 mL of lemon flavor, and 1 mL of pigment were added and solidified after mixing to obtain a non-sugar hard candy. This non-sugar hard candy was easy to eat as a confectionery without the unpleasant odor peculiar to Pu'er tea.

Claims (3)

A post-fermented tea purification composition having enhanced LOX-1 inhibitory activity,
The concentration of theabraunin in an aqueous solution in which the solid content of the purified product is 1% (w / v) is 1.3 or more at an absorbance of 380 nm, and
The 1,2,3-trimethoxybenzene concentration in an aqueous solution in which the solid content of the purified product is 0.1% (w / v) is 20 ng / mL or less, or the 1,2-dimethoxybenzene concentration is 1 ng / mL. A post-fermented tea refining composition characterized in that: A method for producing a post-fermented tea purified composition comprising a purified product of a post-fermented tea leaf extract having enhanced LOX-1 inhibitory activity,
Steps (a) to (c):
(A) a step of extracting fermented tea leaves with water or hot water after the step of immersing in lower alcohol or hydrous lower alcohol,
(B) a step of contacting the post-fermented tea leaf hot water extract or the post-fermented tea leaf extract obtained in the step (a) with a solid adsorbent to recover non-adsorbed components;
(C) The post-fermented tea leaf hot water extract or the post-fermented tea leaf extract obtained in the step (a) is treated with an ultrafiltration membrane having a fractional molecular weight of 1000 to 10,000, and passes through the ultrafiltration membrane. Recovering the components that do not
The method for producing a post-fermented tea purified composition according to claim 1, comprising one or more steps selected from the group consisting of:   A food or drink comprising the post-fermented tea refining composition according to claim 1.