JP2006104213A - Food or drink used for lowering serum cholesterol and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new food or drink lowering serum cholesterol and originating from catechin. <P>SOLUTION: As a result of the examination of eight kinds of catechins for their serum cholesterol-lowering action, among ester type catechins, above all, non-epimers, (-)Cg and (-)GCg have been found to have more excellent serum cholesterol-lowering effect. On the basis of these findings, the food or drink containing (-)GCg, (-)Cg or their mixture as active ingredients has been proposed. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to food and drink used for lowering serum cholesterol and a method for producing the same.

  High levels of serum cholesterol not only induce hyperlipidemia, obesity, arteriosclerosis, thrombosis, but also become known to cause cerebral infarction, myocardial infarction, arteriosclerosis, etc. The total cholesterol level and the value of LDL-cholesterol, which is called bad, are now one of the health indicators that modern people pay most attention to.

  By the way, it is known that cholesterol contained in food is taken into a very small hydrophilic molecule called bile acid micelle by the action of bile acid secreted into the intestine and absorbed from the intestinal tract. Therefore, if the absorption of cholesterol can be inhibited in any process from the intake of food to the incorporation into bile acid micelles, an increase in cholesterol can be suppressed. From this point of view, various mechanisms of action have been reported and proposed for various substances, and among them, many reports have been made on tea extract components that are natural products.

  For example, Patent Document 1 discloses that (−) epicatechin (EC) contained in tea leaves has an effect of lowering blood cholesterol level, and Patent Document 2 discloses tea catechins, particularly (−) epigallo. Disclosed is a cholesterol-lowering agent containing catechin gallate (EGCg) as an active ingredient. Patent Document 3 discloses anti-high cholesterol containing (-) epigallocatechin gallate (EGCg) or (-) epicatechin gallate (ECg) as an active ingredient. Patent Document 4 discloses a cholesterol excretion promoter containing epicatechin gallate (ECg) as an active ingredient.

  In addition, Professor Keiichiro Muramatsu of Shizuoka University used rats as experimental animals and administered a high-fat, high-cholesterol diet or a cholesterol-free diet, in a tea catechin mixture (purity 91.2%, relative catechin content ( %); (−) EGCg: (−) EGC: (−) ECg: (−) EC = 58: 17: 15: 10) and (−) EGCg include a decrease in plasma total cholesterol concentration and LDL-cholesterol concentration. While an effect is recognized, it has been reported that there is no effect | action which lowers plasma HDL-cholesterol and LDL-cholesterol below a normal level (nonpatent literature 1).

  On the other hand, regarding the mechanism of action of the tea extract component, polyphenols contained in tea, red wine, etc. are a kind of anti-hypercholesterolemic effect. Polyphenols inhibit the formation of micelles of bile acids in the intestinal hepatic circulation. It has been suggested that expression occurs by interfering with reabsorption (Non-patent Document 2).

  In addition, the report that non-patent document 3 indicates that the absorption of cholesterol into the small intestinal mucosa is suppressed in a concentration-dependent manner due to the coexistence of tea catechin (EGCg), and the solubility of cholesterol in mixed micelles by EGCg and ECg. Report that this causes the suppression of cholesterol absorption from the intestinal tract (Non-patent Document 4). Furthermore, EGCg and ECg are very low concentrations of squalene epoxidase, which is one of the rate-limiting enzymes of cholesterol biosynthesis. There is a report (Non-patent Document 5) indicating that it is selectively inhibited.

Furthermore, with respect to the demonstration that tea catechins reduce cholesterol absorption using animal and bile acid micelle models, this effect is much higher for EGCg and ECg than for EGC and EC. There is also a description that considers that the hydrophobic region formed by the presence of the protein interacts with cholesterol and exhibits an excellent cholesterol absorption lowering effect (Non-patent Document 6).
JP 52-116473 A JP-A-60-156614 (Japanese Patent Publication No. 2-44449) JP-A-62-30711 JP-A-01-299224 (Registration No. 2812682) Functions of tea: Keiichiro Muramatsu, Academic Publishing Center, 2002, 124-126p Journal of Japanese Society of Nutrition and Food, vol. 39, no. 6, 495-500, 1986 T. Chisaka, H. Matsuda, Y. Kubomura et al .: Chem. Pharm. Bull., 36, 227 (1988) I. Ikeda, Y. Imasato, E. Sasaki et al .: Biochem. Biophys. Acta, 1127, 141 (1992) I. Abe, T. Seki, K. Umehara et al: Biochem. Biophys. Res. Commun., 268,767 (2000) Monthly “Tea” April 2001 issue, Tsutomu Nakayama, Faculty of Food and Nutrition Science, Shizuoka Prefectural University

  The present invention is intended to provide the serum cholesterol-lowering agent and the serum cholesterol-lowering food and drink according to the present invention based on new findings obtained as a result of further research on the serum cholesterol-lowering action of catechins.

  The inventor has (-) epicatechin (EC), (-) epigallocatechin (EGC), (-) epicatechin gallate (ECg) and (-) epigallocatechin gallate (EGCg), (-) catechin ( As a result of performing serum cholesterol lowering action test on all eight catechins of C), (−) gallocatechin (GC), (−) catechin gallate (Cg) and (−) gallocatechin gallate (GCg), ester type catechins (−) EGCg, (−) ECg, (−) GCg and (−) Cg were found to have an excellent serum cholesterol lowering action. Among these, (-) Cg and (-) GCg, which are non-epimers, have found a new finding that they exhibit a superior serum cholesterol lowering action than (-) ECg and (-) EGCg, which are epimers, and such findings The present invention has been conceived based on the above.

  The present invention relates to a serum cholesterol lowering agent comprising (−) GCg, (−) Cg or a mixture thereof as an active ingredient and food and drink used for lowering serum cholesterol (in the present invention, food and drink are collectively referred to as “food and beverage Therefore, foods and drinks include foods and beverages.

  (-) GCg and (-) Cg are epimers of (-) EGCg and (-) ECg, that is, diastereoisomers having a configuration in which one asymmetric carbon atom of (-) EGCg and (-) ECg is inverted. (All stereoisomers except enantiomers). These (-) GCg and (-) Cg are only slightly contained in natural products (for example, tea leaves), but (-) EGCg and (-) which are contained in large amounts in natural products (for example, tea leaves). ECg can be obtained to an industrially usable level by thermal isomerization.

  Conventionally, there have been almost no reports on the non-epimeric (−) GCg and (−) Cg pharmacological activities, particularly on the serum cholesterol lowering effect. Regarding the lowering action of serum total cholesterol and serum LDL-cholesterol, (−) Cg and (−) GCg which are non-epimers are more effective than (−) ECg and (−) EGCg which are epimers. The serum cholesterol-lowering agent and food and drink having a more excellent pharmacological effect than before by using these (-) GCg and (-) Cg as an active ingredient of a serum cholesterol-lowering agent or cholesterol-lowering food or drink. Could get.

The present invention also provides a method for producing a serum cholesterol lowering agent, which comprises a total of about 500 mg / L or more of ester-type catechins (-) EGCg, (-) ECg, (-) GCg, and (-) Cg. We propose a manufacturing method characterized in that a solution is prepared, heat-treated at about 80 ° C. or higher, and then concentrated and dried.
In addition, as a method for producing foods and drinks used to lower serum cholesterol, ester type catechins (-) EGCg, (-) ECg, (-) GCg and (-) Cg are contained in total of about 500 mg / L or more. We propose a production method characterized in that a catechin solution is prepared, heat-treated at about 80 ° C. or higher, concentrated and dried, and the resulting concentrated dried product is blended.
Moreover, as a manufacturing method of the drink used in order to reduce serum cholesterol, ester type catechins (-) EGCg, (-) ECg, (-) GCg, and (-) Cg are contained about 500-6000 mg / L in total. A catechin solution is prepared, and a manufacturing method characterized by heat-treating the catechin solution at about 80 ° C. or higher is proposed.
In any production method, a catechin solution containing a predetermined value or more of ester type catechins is prepared, and this is heat-treated to promote thermal isomerization of (−) EGCg and (−) ECg, and (−) GCg and (-) It has the feature which can raise content of Cg.

  In the present invention, “all catechins” means (−) EC, (−) EGC, (−) ECg and (−) EGCg, (−) C, (−) GC, (−) Cg and (−). It means eight catechins of GCg, and “ester type catechins” means four catechins of (−) EGCg, (−) ECg, (−) GCg and (−) Cg. “Epi form” means (−) EC, (−) EGC, (−) ECg and (−) EGCg, and “non-epi form” means (−) C, (−) GC, (− ) Cg and (-) GCg.

“Containing active ingredient as (−) GCg, (−) Cg or a mixture thereof” means that “(−) GCg or (−) Cg serum cholesterol lowering action is not inhibited, It means that other components, for example, (-) EC, (-) ECg, (-) EGCg and the like may be included.
In addition, a polymer of (−) GCg, a copolymer of (−) GCg and another catechin, a polymer of (−) Cg, or a copolymer of (−) Cg and another catechin is “( -) GCg, (-) Cg or a mixture thereof ".

  Next, an embodiment of the present invention will be described.

(Serum cholesterol lowering agent)
The serum cholesterol lowering agent of the present invention can be produced by blending (−) GCg, (−) Cg or a mixture thereof at a desired concentration. However, it is possible to appropriately mix other components or perform other treatments within a range not inhibiting the pharmacological activity of (−) GCg and (−) Cg.

(−) GCg and (−) Cg can be obtained by a conventionally known method or a method that will be known in the future.
(-) GCg and (-) Cg are scarcely present in natural plants including tea leaves. For example, "(-) EGCg, (-) ECg or a mixture thereof" is heated at about 80 ° C or higher. It can be obtained by promoting thermal isomerization (epimerization).
Therefore, “(−) GCg, (−) Cg or a mixture thereof” means “(−) EGCg or (−) ECg or a mixture thereof, or“ (−) such as tea extract or leachate ”, for example. The catechin solution containing EGCg and (−) ECg ”can be heated at about 80 ° C. or more to promote thermal isomerization of catechin, thereby increasing the content concentration of (−) GCg or (−) Cg, This heat-treated product can be obtained by separating and purifying (−) GCg, (−) Cg, a mixture thereof, or a mixture containing these in a high concentration.
At this time, it is preferable to heat-treat after preparing the catechin solution at pH 5-6. There is a possibility that catechin hardly undergoes thermal isomerization at pH 4.5 or less (Shinmatsu Shinichi et al .: Eclipse Journal, 39,178 (1992)).

The composition of the “catechin solution containing (−) EGCg and (−) ECg” is preferably prepared so as to contain a total of about 500 mg / L or more of ester type catechins.
As the separation / purification method, a conventionally known method or a method that will be known in the future can be employed. For example, the liquid to be treated (for example, tea extract) is subjected to reverse-phase HPLC using, for example, a water-acetonitrile-phosphoric acid mixture as a mobile phase, and gradient (-) GCg and (- -) Cg can be separated.

“(−) GCg, (−) Cg, or a mixture thereof” can be incorporated alone as an active ingredient of the present invention, but a substance that has been observed to lower serum cholesterol level in the future or in the future, For example, any of (-) EC, (-) ECg, (-) EGCg, or a mixture comprising a combination of two or more of these may be used as an active ingredient.
When compounded alone as an active ingredient of the present invention, for example, (−) GCg, (−) Cg or a mixture thereof is dissolved in purified water, physiological saline or the like to give a serum cholesterol lowering agent (for example, an oral administration agent, abdominal cavity, Internal administration agent etc.).

The serum cholesterol-lowering agent of the present invention can be used as an oral administration agent or a parenteral administration agent (intramuscular injection, intravenous injection, subcutaneous administration, rectal administration, transdermal administration, nasal administration, etc.). It is preferable to use a suitable formulation and dosage form.
Speaking of dosage forms, for example, for oral administration, it can be prepared in the form of solutions, tablets, powders, granules, dragees, capsules, suspensions, emulsions, pills, etc. It can be prepared in the form of injection, ampoule, rectal administration, oily suppository, water-soluble suppository and the like.
Regarding formulation (formulation), commonly used excipients, extenders, binders, wetting agents, disintegrants, surfactants, lubricants, dispersants, buffers, preservatives, solubilizers, It can be produced by a conventional method using a preservative, a flavoring agent, a soothing agent, a stabilizer and the like. Also, for example, lactose, fructose, glucose, starch, gelatin, magnesium carbonate, synthetic magnesium silicate, talc, magnesium stearate, methylcellulose, carboxymethylcellulose or salts thereof, gum arabic, polyethylene glycol, syrup, petrolatum, glycerin, ethanol, propylene Non-toxic additives such as glycol, citric acid, sodium chloride, sodium sulfite, and sodium phosphate can be blended.

The serum cholesterol lowering agent of the present invention includes pharmaceuticals, quasi-drugs, health foods / health drinks with specific pharmacological effects, foods for specified health / functional foods, food additives, other drugs for non-human animals, It can also be provided as a bait, a bait additive, and the like.
For example, by preparing as a quasi-drug and making it into a drinking form such as a bottled drink or a form such as a tablet, capsule, granule, etc., it can be made easier to ingest.

Examples of health foods, health drinks, foods for specified health use, and functional foods having a serum cholesterol lowering effect include (-) GCg, (-) Cg, or a mixture thereof, or a catechin solution containing this mixture, Choose from carbon dioxide, excipients (including granulating agents), diluents, or even sweeteners, flavors, flours, starches, sugars, fats and other proteins, carbohydrate ingredients, vitamins and minerals Or a currently known food or drink such as a sports drink, fruit drink, milk drink, tea drink, vegetable juice, milk drink, alcoholic drink, jelly, jelly drink, carbonated drink , Chewing gum, chocolate, candy, biscuits, snacks, bread, dairy products, fish paste products, livestock meat products, frozen desserts, dried foods, supplements, etc. It can be produced by pressing.
In particular, if a food or drink is prepared by adding (−) GCg, (−) Cg, or a mixture thereof, or a catechin solution containing this mixture to a food or drink containing a lot of fat, Since an increase can be suppressed, it can be provided as a low-cholesterol food or diet food or food.

(Serum cholesterol lowering food and drink)
Next, foods and drinks (ie, including serum cholesterol-lowering foods and beverages) having the serum cholesterol-lowering effect of the present invention, particularly tea-derived serum cholesterol-lowering beverages, will be described in detail.

As described above, (-) GCg and (-) Cg are hardly present in natural tea, but (-) EGCg, (-) ECg or a mixture thereof, which is contained in natural tea, is heated. It can be obtained by processing.
Therefore, a catechin solution containing (−) EGCg, (−) ECg, (−) GCg and (−) Cg in a predetermined amount or more (which may be included) is prepared and heat-treated. Can produce a tea-derived serum cholesterol-lowering beverage containing desired amounts of (-) GCg and (-) Cg.
For example, a tea extract obtained by extracting tea (containing (−) EGCg and (−) ECg) is added to water or the like to add (−) EGCg, (−) ECg, (−) GCg and (−) Cg. Of a desired composition containing a desired amount of (-) GCg and (-) Cg by preparing a catechin solution and heat-treating it. Serum cholesterol-lowering beverages can be produced.

  In this case, the tea that can be used as a raw material is not particularly limited as long as it contains (−) EGCg and (−) ECg. For example, fresh tea leaves, fermented tea such as black tea and puer tea, oolong tea, Extract one of semi-fermented teas such as baked tea, non-fermented teas such as green tea, kettle-roasted green tea, and roasted tea (single), or a mixture of two or more of these, or extract each A mixture of those obtained can be used.

  Tea extraction is performed by extracting tea with water, hot water or hot water, preferably 40 ° C to 100 ° C hot water, especially 90 ° C to 100 ° C hot water, or an organic solvent such as ethanol aqueous solution or ethanol harmless to the human body. To obtain a tea extract. Furthermore, this tea extract is purified in a direction to increase the content of catechin, especially (−) EGC and (−) ECg, by purification means such as solvent extraction method, resin adsorption method, filtration such as ultrafiltration and reverse osmosis filtration. To obtain a tea extract. During extraction, hydrochloric acid or the like may be added to increase the content of (−) EGC and (−) ECg, and extraction may be performed under acidic conditions.

  Commercially available tea extracts can also be used. For example, Teafuran 30A (trade name; manufactured by ITO EN) is a green tea extract obtained by subjecting green tea to hot water extraction treatment and drying this extract to a catechin concentration of about 30%. Teafuran 90S (trade name; ITO EN) Made by hot water extraction treatment of green tea with an adsorption column using water and low and high concentration alcohol and dried, the tea polyphenol concentration is about 85-99.5% Green tea extract. In addition, “Polyphenone” manufactured by Mitsui Norin Co., Ltd., “Sunphenon” manufactured by Taiyo Kagaku Co., Ltd., “Sun Oolong” manufactured by Suntory Ltd., etc. can be used as commercially available tea extracts.

  The composition of the “catechin solution” is preferably prepared so as to contain a total of about 500 mg / L or more of ester type catechins. If the concentration of ester-type catechins in the catechin solution is less than 500 mg / L, a concentration step is required on the way, which may increase production costs.

The catechin solution is preferably heat-treated at about 80 ° C. or higher. Looking at the test results, for example, 100 ° C. × 15 minutes heating, 115 ° C. × 20 minutes heating, 120 ° C. × 3-30 minutes heating, 123 ° C. × 10 minutes heating, 131 ° C. × 30 seconds heating, 133 ° C. × 45 seconds In any case, thermal isomerization of catechins has been observed.
If the heat treatment of the catechin solution is less than 80 ° C., the conversion of the ester-type catechins from the epi form to the non-epi form (thermal isomerization) does not easily occur, and the equilibrium shifts in the direction of the epi form, which is not preferable. .
Moreover, it is preferable to heat-process after preparing a catechin solution to pH 5-6. There is also a report that catechins hardly undergo thermal isomerization at pH 4.5 or less (Shinmatsu Shinichi et al .: Eclipse Journal, 39, 178 (1992)).

The serum cholesterol-lowering beverage of the present invention may be prepared by directly adding the catechin solution heat treatment solution obtained as described above or adding a green tea extract or other substances as it is to improve the flavor.
Incidentally, the serum cholesterol-lowering agent and the serum cholesterol-lowering food and drink of the present invention are prepared by further concentrating and drying the heat-treated liquid of the catechin solution obtained as described above, and blending the obtained solid substance. Can do. The concentration / drying step at this time can be performed by a normal concentration / drying method such as vacuum concentration or freeze-drying.

  The serum cholesterol-reduced food and drink obtained in this way is a health drink that can be consumed on a daily basis with peace of mind because the main ingredient uses ingredients obtained from natural products, especially tea that has been cited daily in large quantities. Can be provided.

  Next, the preferable composition of the serum cholesterol-lowering food and drink of the present invention will be described.

The serum cholesterol-lowering food / drink of the present invention comprises ester catechins (-) EGCg, (-) ECg, (-) GCg, and (-) Cg in total, which is about 70% by weight or more of the total content of catechins. It is preferable to contain and satisfy the following conditions (1) to (3).
(1) The total content of (−) GCg and (−) Cg is about 40 of the total content of (−) EGCg, (−) ECg, (−) GCg and (−) Cg, which are ester-type catechins. % By weight or more.
(2) The content of (−) ECg is about 20% by weight or less of the total content of (−) EGCg, (−) ECg, (−) GCg and (−) Cg which are ester type catechins.
(3) The content of (−) GCg is not less than the content of (−) ECg.

As will be described later, as a result of the study by the present inventor, it has been clarified that four types of ester catechins have a serum cholesterol lowering effect. However, only ester type catechins constitute catechins in foods that lower serum cholesterol. Attempting to do so makes it difficult to ingest due to lack of flavor. Therefore, when trying to improve the difficulty of ingestion by adding a green tea extract and setting the value of (−) EC in the food and drink as an index, the value is about twice that of a commercially available green tea beverage. The required addition amount of the green tea extract is about equal to the total catechin content of the food or drink at the maximum, and the total content of the four ester-type catechins in the serum cholesterol-lowering food and drink of the present invention in this case is It becomes about 70% by weight or more of the total content of all eight catechins. When a taste test was conducted by changing the content ratio of the ester-type catechins, it is preferable in terms of taste to prepare a beverage so that the total content of all catechins is about 70% by weight or more. confirmed.
Further, regarding the satisfactory conditions (1) to (3) of the ester-type catechins, the non-epi form of serum cholesterol reducing effect is superior to that of the epi form (which is effective as such). Therefore, it is preferable that the total content of non-epimers, particularly (−) GCg and (−) Cg, occupies at least about 40% by weight of the total content of ester-type catechins including epimers. is there. Regarding (2), since the serum cholesterol-reducing effect of ECg is the smallest among ester-type catechins, the content of (−) ECg is preferably about 20% by weight or less of the total content of ester-type catechins. It is. For (3), in order to compensate for the low ECg reduction effect with the most excellent GCg, it is preferable to set the content of (−) GCg to be equal to or more than the content of (−) ECg. It is.
The serum cholesterol-lowering food or drink of the present invention that satisfies the above conditions exhibits a serum cholesterol lowering effect equivalent to or higher than that of food or drink containing EGCg in the same amount as the total amount of the ester type catechins.

(Required intake and content)
The necessary intake of ester catechins in the serum cholesterol-lowering agent and the serum cholesterol-lowering food and drink of the present invention is considered to be about 300 to 2100 mg per day.
Although it varies depending on the method of use, it is preferable to take (−) GCg, (−) Cg or a mixture thereof in an amount of 120 to 2100 mg, preferably about 250 to 1500 mg per adult day in terms of dry weight.

Considering the above required intake, in the case of solid form serum cholesterol-lowering agents and serum cholesterol-lowering foods and drinks, it is preferable to contain about 0.1 to 100% by weight of ester-type catechins. ) GCg, (-) Cg or a mixture thereof is preferably blended in an amount of about 0.04 to 100% by weight, particularly about 0.1 to 40% by weight.
Less than about 0.1% by weight of ester-type catechins is not preferable because the amount of solids to be consumed may exceed about 2 kg per day.

On the other hand, in the case of a beverage form serum cholesterol-lowering agent and serum cholesterol-lowering food and drink, it is preferable to contain ester-type catechins at a concentration of about 500 to 6000 mg / L, and the beverage after the heat treatment is (−) GCg, (-) Cg or a mixture thereof is preferably contained at a concentration of about 200 to 6000 mg / L, particularly about 300 to 3000 mg / L.
An ester type catechin concentration of less than 500 mg / L is not preferable because the volume of a beverage to be ingested may exceed 4 L per day. Exceeding 6000 mg / L is not preferable because catechins have a high concentration and astringency is too strong. More preferably, the ester type catechin concentration is about 750 to 3750 mg / L.

<Test 1>
The ability of tea catechins to inhibit cholesterol absorption was measured, and the ability of isomerized catechins (non-epimeric catechins) to inhibit cholesterol absorption was compared.

  Among tea catechins, (-) EGCg, (-) ECg, (-) GCg and (-) Cg as ester type catechins, (-) EGC, (-) EC, (-) GC and (- ) The following tests were conducted using a total of 8 types of catechins of C as samples.

(Test method)
To a 15 mM phosphate buffer (pH 6.8), 0.5 mM cholesterol, 6.6 mM Na taurocholate, 0.6 mM egg yolk lecithin, 132 mM NaCl was added, and bile acid mixed micelles (hereinafter referred to as “bile acid” Micelle]).
These bile acid micelles were sealed with argon gas and kept at 37 ° C. for 24 hours to stabilize the micelles, and then dissolved in deionized water so that the above eight types of catechins had a final concentration of 1000 μM or 2000 μM, respectively. And incubated for 1 hour. At this time, the micelle solution became cloudy and precipitated in several catechin addition groups. In addition, only deionized water was added to the catechin-free group.
One hour after the start of the incubation, this solution was filtered through a 220 nm filter to obtain a clear micelle solution, and the cholesterol concentration in this solution was measured. The results are shown in FIG. 1 and FIG.

(result)
When FIG. 1 is seen, what added ester type catechin (-) EGCg, (-) ECg, (-) GCg, and (-) Cg to the bile acid micelle, the cholesterol concentration in a micelle is dependent on addition amount. Decreased. As for the effect, when added at 1000 μM, (−) EGCg, (−) GCg, and (−) Cg showed a strong lowering effect compared to (−) ECg (P <0.05).
Among the former three catechins, (−) GCg has the strongest lowering effect, significantly (P <0.05) lower than (−) EGCg, and then (−) Cg, (−) EGCg It was in order.
On the other hand, when FIG. 2 is seen, (-) EGC, (-) EC, (-) GC, and (-) Cg which are free catechin hardly reduced the cholesterol concentration in a bile acid micelle.

(Discussion)
As a result of the above, it has been clarified that the catechin gallate ester not only epi (epi) but also non-epi (isomer) precipitates cholesterol in the mixed bile acid micelle and desorbs it from the micelle. It was.
Furthermore, the cholesterol-lowering action of (−) GCg and (−) Cg, which are non-epimers (isomers), is stronger than (−) EGCg and (−) ECg, which are epimers (epi-forms). Can think.

<Test 2>
In order to examine the synergistic effect of the isomerized non-epimeric catechin mixture, using the same method as in Test 1, (-) GCg and (-) Cg-containing ester-type catechin mixture (beverage of Example 1), including these A comparative study of the cholesterol absorption inhibitory effect of the three ester-type catechin mixtures, EGCg (FIG. 3).

A bile acid mixed micelle was prepared in the same manner as in Test 1 from the beverage of Example 1 below as the ester-type catechin mixture containing (−) GCg and (−) Cg.
Further, as the ester type catechin mixture not containing (−) GCg and (−) Cg, Teafuran 90S (Itoen Green Tea Extract, see Table 1 for the composition) was used, and this example beverage was mixed with bile acid micelles as in Test 1. Was prepared.

As a result, it was found that any ester-type catechin mixture showed higher activity than EGCg. Moreover, in this test result, the difference of big activity was not recognized between the ester type catechin mixture which contains (-) GCg and (-) Cg, and the ester type catechin mixture which does not contain.
In addition, FIG. 3 attached to the application on February 10, 2003 showed that the ester-type catechin mixture not containing (−) GCg and (−) Cg showed higher activity. Since this drawing was found to be erroneous, it was replaced with the correct FIG.

<Test 3>
Teafuran 90S (Itoen catechin extract; see Table 1 for composition) was dissolved in warm water at 60 ° C to a concentration of 1450 mg / L, cooled to 35 ° C, vitamin C was added at 300 mg / L, then cyclodextrin was added. 1 wt%, green tea extract 0.2 wt% and baking soda about 300 mg / L were added, and finally adjusted to 1 kg with distilled water to obtain a catechin solution. At this time, 250 mg of polyphenol was contained per can.
After heating the catechin solution thus obtained to 95 ° C, in the case of cans, the cans are filled and subjected to heat treatment (retort sterilization) at 123 ° C for 10 minutes. Is heated to 95 ° C. and then subjected to heat treatment (UHT sterilization) at 133.5 ° C. for 45 seconds, filled into a PET bottle, and then the content of catechins in each step is measured. L) is shown in Table 2, and the isomerization rate (change rate%) of catechins is shown in Table 3.

Here, when the contribution rate regarding the cholesterol lowering action of (−) EGCg, (−) GCg, (−) ECg and (−) Cg in the beverage was determined, from Test 1, (−) EGCg, (−) GCg, Since (-) ECg and (-) Cg had residual cholesterol concentrations in micelles at 100 μM of 272, 244, 337, and 262, respectively, the precipitated cholesterol was different from the initial value of 425 μM by taking 153, 181, 88 163, and the cholesterol precipitation ability per 1 μM is 1.53, 1.81, 0.88, 1.63.
On the other hand, the catechin composition contained in 190 mL of the test beverage was determined as follows: (−) EGCg: (−) GCg: (−) ECg: (−) Cg = 51.3 mg (589.5 μM): 62.7 mg (720.5 μM) ): 19.0 mg (226.2 μM): 17.0 mg (202.4 μM) From the above cholesterol precipitation ability per 1 μM, the contribution rate regarding the cholesterol lowering effect of catechin contained in 190 mL of the test beverage is
(−) EGCg: (−) GCg: (−) ECg: (−) Cg = 33.0: 47.7: 7.3: 12.0 (%).

<Test 4: Human test>
1) Subjects This study was conducted on 42 subjects with a total serum cholesterol level of 200 mg / dL to 280 mg / dL and a mild high serum cholesterol level among men over 20 years of age who are living healthy. Extracted by doctors who do not participate directly.
Subject must be a person who has not undergone medical treatment at the hospital, patients taking lipid or hypotensive drugs such as hypolipidemic or antihypertensive drugs due to hospitalization or hospital visits, severe liver dysfunction, renal disorder, respiratory disorder, Those with endocrine and cardiovascular disorders were excluded from the subjects.
At the start of the test, 42 subjects were divided into 3 groups, Group A, Group B, and Group C, with 14 members in each group. There was no difference in blood pressure or total cholesterol levels in each group at the start.

2) Test Beverage In this test, 190 g of beverage was blended so that the amount of tea catechin was 150 mg per tea (250 mg as tea polyphenol: Theafranc 90S manufactured by ITO EN Co., Ltd.). Table 4 shows the tea catechin composition in the test beverage.
In addition to tea catechins, green tea extract containing no tea catechins, cyclodextrin, and vitamin C were added to the test beverages.
Moreover, the drink of the same component other than tea catechin was used as a placebo drink.

3) Study schedule The study was conducted in a double-blind manner. The test schedule is shown in FIG. All groups had blood tests at the beginning and end of each period.
Group A was ingested 3 control drinks per day, Group B was ingested 2 test drinks per day, and Group C was ingested 3 test drinks per day. Each group was fed with a meal.
In Group B, one control drink was ingested only at lunch time in order to match the total daily intake other than tea catechins with Group A and Group C.

4) Observation items Total cholesterol (TC), HDL-cholesterol (HDL-C), LDL-cholesterol (LDL-C), and neutral fat (TG) were measured as serum lipids for all subjects.
TC, HDL-C and TG were measured by an enzymatic method. About LDL-C, it computed by the formula (LDL-C = TC-HDL-C-TG / 5) of Friedewa1d.
In order to confirm the health condition, BUN, uric acid, total protein, ALP, GOT, GPT, LDH, γ-GTP, CPK, Na, K, Ca, Cl, Fe, UIBC, ferritin, HbA1c, hemoglobin, red blood cell count, White blood cell count, platelet count and hematocrit were measured. Furthermore, a dietary survey and an exercise amount survey were conducted for 3 days before each blood collection. For meals, subjects were asked to fill out a meal survey form, and nutritional calculations were performed in accordance with the “Fiveth Japanese Food Standard Composition Table”. Regarding the amount of exercise, a pedometer was distributed to all subjects, and the total number of steps per day was entered.
Blood was collected early in the morning on an empty stomach. Each blood collection day was examined by a doctor and observed for changes in physical condition.

5) Statistical analysis Each measured value is shown as an average value ± standard deviation. The significant difference test was performed using a SAS statistical analysis program with a risk rate of less than 5%.
As for the test of each measured value, when normality was recognized, a multiple comparison test (Dumet method) was performed. When normality was not recognized, the test was performed by the Steal method.

6) Results
1. Nutrition intake / exercise amount Table 5 shows the nutrient intake and exercise amount per day during the test period. The dietary survey / exercise survey were shown as average values for three days on weekdays before each blood collection day. The average value of intake energy throughout the whole was as low as 1638-1846 kcaL / day, but as a result of interviews by doctors, all subjects were employed, and as a eating habit, weekday mornings and lunches were very simple meals. It was also judged that there was a tendency to eat a lot with alcohol on holidays.
There was no significant difference in energy intake between groups. At 4 weeks from the start of intake, cholesterol intake in group C (106.1 ± 95.6 mg / d1) was significantly lower than that in group A (198.O ± 69.3 mg / d1). The amount of cholesterol intake was not immediately reflected in the serum cholesterol level, and in fact, no difference was observed in the serum lipid concentration of each group at this time point, so it was considered that there was no particular effect. There was no difference in nutrient intake and exercise volume among the other subjects.

2. Serum lipids Table 6 shows the transition of serum lipid-related test values during the test period. In group C (3 bottles / day), the TC value was 226.9 ± 20.2 mg / dL (at the start of ingestion) to 203.0 ± 22.7 mg / dL (after 8 weeks), which was significant (ρ <O.05 by Dunnet-test).
The TC value 236.O ± 19.1 mg / dL of group A (control beverage group) 8 weeks after the start was also significantly lower (ρ <O.01). Similarly, the LDL-C value was significantly decreased (ρ <O.01) from 141.6 ± 22.3 mg / dL (at the start of intake) to 116.9 ± 14.4 mg / dL (after 8 weeks) at the start of intake. Significant even for LDL-C value 147.3 ± 22.3 mg / d1 of group A (control beverage group) after week (ρ <0.01)
It was low.
In Group B (2 / day), the TC value changed from 236.O ± 25.8mg / d1 (at the start of intake) to 219.1 ± 21.Omg / d1 (after 8 weeks). The tendency was low (ρ <0.1) with respect to the TC value 236.O ± 19.1 mg / d1 of group A (control beverage group). On the other hand, there was no significant difference in the values of HDL-C, TG, and HbA1c in both groups B and C compared to the time of ingestion. In addition, no significant difference was observed with respect to Group A (control beverage group).

3. Blood components / minerals and other findings Table 7 shows changes in blood components and minerals of the subjects. During the intake period, significant changes were observed in each of the values of red blood cell count, white blood cell count, platelet count, hemoglobin, hematocrit, ferritin, and Fe, Ca, K, Cl, and Na in both groups B and C. I was not able to admit.
There was no significant difference between the groups.
After the follow-up period, a significant (ρ <0.01) increase in Na in group C (141.7 ± 1.3 mg / d1 → 143.4 ± 1.3 mg / d1) in group C compared to the beginning of ingestion, while in group B, significant (ρ <0.05) increase (4.7 ± O.2mg / d1 → 4.9 ± O.1mg / d1) was observed, but both were within the normal range, and the doctor judged that there was no medical problem.
During the test period, no significant changes were observed in γ-GTP, GOT, and GPT, and no findings that were considered to be adverse events were observed at the time of medical examination.

7) Discussion In this study, two or three drinks containing 250 mg of tea polyphenol per subject, 150 mg of tea catechin (EGCg51.3 mg, GCg62.7 mg, ECg19.Omg, Cg17.0 mg) per day for each subject. Was ingested for 8 weeks. Therefore, the daily intake is 500 mg for tea polyphenols in group B, 300 mg for tea catechins (EGCg102.6 mg, GCg125.4 mg, ECg38.Omg, Cg34.Omg), 750 mg for tea polyphenols in group C, 450 mg for tea catechins (EGCg153 .9mg, GCg188.1mg, ECg57.Omg, Cg51.Omg). The daily intake of 450 mg of tea catechin in Group C corresponds to the amount of tea catechin for 8-9 cups of normal green tea.
Ingested catechin forms an insoluble precipitate with dietary cholesterol, endogenous cholemeterol excreted in the intestinal tract, and cholesterol in bile acid micelles, thereby inhibiting cholesterol absorption from the small intestine, and bile acids and cholesterol in the stool Is excreted. In particular, ester-type catechins have higher ability to desorb cholesterol from bile acid micelles compared to other catechin species, so the effect of this beverage mainly containing ester-type catechins is due to inhibition of cholesterol absorption from the intestinal tract. It is assumed that there is.
In addition, tea catechin is known to change the intestinal bacterial flora, such as the growth of intestinal lactic acid bacteria. Dietary fiber is easily assimilated by lactic acid bacteria and the like, and as a result, the amount of propionic acid produced increases. Propionic acid produced has been reported to inhibit hepatic HMG-CoA synthase activity and suppress cholesterol synthesis. The contribution of the beginning is also conceivable. That is, during the tea catechin intake period, 1) inhibition of reuptake of endogenous cholesterol excreted in the intestinal tract, 2) inhibition of cholesterol synthesis due to the growth of lactic acid bacteria, and 3) bile acids suffered from intestinal circulation inhibition. Increased synthesis of cholesterol to bile acids is thought to reduce the endogenous cholesterol pool and the cholesterol saturation in the bile. After the end of tea catechin intake, the endogenous cholesterol pool recovers and bile acid synthesis decreases, and then excess endogenous cholesterol flows into the blood, leading to an increase in serum cholesterol levels. However, taking into account that it takes 4 weeks after the start of tea catechin intake until the decrease in serum cholesterol level becomes significant, it takes some time for the serum cholesterol level of the subject to return before the start of intake. It is inferred that there was a delay.
On the other hand, in this study, no decrease in serum mineral levels due to tea catechins was observed. In addition, no significant changes were observed in the values of serum iron, UIBC, hemoglobin, and hematocrit.
This result is consistent with the report by Record et al. That tea intake does not affect the absorption of trace metal elements. Therefore, although examination this time was 8 weeks, ingestion of the tea drink which mix | blended tea catechin can be ingested over a long term, without reducing each numerical value of a serum mineral.

  It has been reported that the affinity and stereochemical structure for lipid bilayers differ among gallate-type catechins (K. Kajiya et al .: Biosci. Biotech. Biochem. 2001, 65, pp. 2638- 2643). From this, it is considered that these differences influence the difference in the cholesterol lowering effect between ECg, EGCg, Cg and GCg.

<Test 5>
In this study, green tea catechins (epi form: (−) EC, (−) EGC, (−) ECg and (−) EGCg) and heat epimerized catechins (non-epi form) were used in thoracic cannulated rats. : (-) C, (-) GC, (-) Cg, and (-) GCg) were compared and examined for the effect of cholesterol on lymphatic absorption.

1) Materials Regarding catechin pure products, (-) EC, (-) EGC and (-) C are manufactured by Wako Pure Chemical Industries, Ltd. (-) GC, (-) Cg and (-) GCg are Nagara Chemical Industries, Ltd. (Nagara Science), (-) ECg and (-) EGCg were manufactured by Kurita Kogyo. The purity of these catechins (single substances) was 98% or more.
Further, ^4-14 C-cholesterol (55 mCi / mmoL) were purchased from Amersham Pharmacia Biotech, Inc..

Theafuran 90S manufactured by ITO EN was used as a mixture of green tea catechins. Thermally epimerized catechins (non-epimers) were prepared by heat-treating this catechin mixture at 120 ° C. for 5 minutes.
Table 8 shows the catechin composition of green tea catechins (epimer) and thermal epimerized catechins (non-epimer). From Table 8, it can be confirmed that the amount of Cg and GCg is increased in epimerized catechins (non-epimer) prepared from green tea catechins.

2) Test contents Eight-week-old SD rats were given a commercial Chinese-style meal (Chow Chow) for one week until surgery. The left thoracic lymphatic canal was pre-cannulated to the subarachnoid chyle of these rats.
A second intracorporeal catheter was placed inside the stomach for administration of the test emulsion. After the surgical procedure, the animals were placed in a restraining cage, and a solution containing 139 mM glucose and 85 mM NaCl was continuously injected into the stomach at a rate of 3.4 mL / h until the end of the experiment. The same solution was given as drinking water. The next morning, animals with a constant lymph flow rate were administered 3 mL of a ¹⁴ C-cholesterol-containing test emulsion with or without a catechin sample.
The test emulsion (3 mL) consists of 200 mg sodium taurocholic acid (complex bile acid), 50 mg fatty acid free bovine albumin fraction V, 200 mg triolein, 37 kBq ¹⁴ C-cholesterol and 25 Contains mg of cholesterol. The mixture was emulsified by sonication.

When administering tea catechins and heat epimerized catechins, 100 mg and 120 mg of these catechins were added to 3 mL of the emulsion, respectively. Since the content of catechins (the sum of ECg, EGCg, Cg, and GCg) decreased in the heat epimerized catechins, the catechin amount was adjusted to be the same level between the tea catechins and the epimerized catechins.
The molar ratio of total catechins to cholesterol in the lipid emulsion was about 2.5. Lymph was collected with an ice-cooled tube containing EDTA, and the radioactivity was measured.
All animal research is conducted at the Kyushu University Graduate School of Agricultural Science Animal Experiment Guidelines, the Act on the Protection and Management of Animals (Act No. 105 of October 1, 1973), and the breeding and storage of experimental animals. This was carried out in accordance with the “Standards for the Notification” (No. 6 Notification of the Prime Minister's Office on March 27, 1980).

FIG. 7 shows the changes over time in the absorption rate of cholesterol in the lymphatic vessels in each group administered with control, green tea catechins or heat epimerized catechins, in rats administered with a lipid emulsion inside the stomach. It was.
In addition, data are shown by the average value +/- standard error of 6 or 7 rats, and a, b, and c show that there is a significant difference between different characters (P <0.05).

(result)
Control group, green tea catechins ((−) EC, (−) EGC, (−) ECg and (−) EGCg) group and thermal epimerized catechins ((−) C, (−) GC, (−) Cg and The lymph flow rate was linear between the (−) GCg) groups and no difference was observed (175 ± 15 mL / 24h, 165 ± 16 mL / 24h and 154 ± 17 mL / 24h, respectively).
Administration of catechins effectively and significantly reduced cholesterol recovery to the lymph as compared to the control group during the 24-hour lymph collection (see FIG. 7).
Epimerized catechins were more effective and significant than tea catechins in reducing cholesterol absorption. The recovery rate of ¹⁴ C-cholesterol to the lymph was 40.5 ± 1.6%, 24.9 ± 3.0% and 15.2 ± 2.0% during the 24-hour lymph collection in the control, green tea catechin and heat epimerized catechin groups, respectively.

(Discussion)
From this test result, catechins before thermal epimerization of the heat epimerized catechins ((−) C, (−) GC, (−) Cg and (−) GCg)) increased during retort treatment, that is, green tea It has been found that plasma cholesterol concentrations are more effectively reduced than catechins ((−) EC, (−) EGC, (−) ECg and (−) EGCg).
It has also been demonstrated that plasma cholesterol levels are an independent risk factor for atherosclerosis (G. Rose and M. Shipley: Brit. Med. J. 1986, 293, pp. 306-307). ), When examined together with the results of this study, both green tea catechins and heat epimerized catechins are effective in preventing atherosclerosis by lowering plasma cholesterol and antioxidant activity, especially thermal epimerization Catechins are considered to be more effective than green tea catechins.
In addition, regarding the safety of heat epimerized catechins, tea drinks containing green tea catechins and heat epimerized catechins were given to humans for 8 to 20 weeks as total catechins to be 450 to 900 mg / day. No abnormality was reported in biochemical blood parameters (T. Nagao et al .: J. Oleo Sci. 2001, 50, pp. 717-728).

Example 1
Teafuran 90S (Itoen Green Tea Extract, see Table 1 for composition) was dissolved in warm water at 60 ° C to a concentration of 1450 mg / L, cooled to 35 ° C, vitamin C was added at 300 mg / L, then cyclodextrin 1% by weight of sodium bicarbonate and about 300 mg / L of baking soda were added to make 1 kg with distilled water to obtain a catechin solution. The obtained catechin solution was heated to 95 ° C., then filled into a can, and subjected to a heat treatment at 123 ° C. for 10 minutes to prepare a serum cholesterol-lowering beverage having the following composition.

Catechin 150mg
(-) GCg 62.7 mg
(−) EGCg 51.3 mg
(−) ECg 19.0 mg
(-) Cg 17.0 mg
Vitamin C 50mg
Cyclodextrin 500mg
Prepare the whole water to 190mL

  In this serum cholesterol-lowering beverage, all catechins contained are ester-type catechins (ie, 100 wt%), the concentration of ester-type catechins is 789 mg / L, and “(−) GCg + (− ) Cg ”is 53.1% by weight, and (−) ECg is 12.7% by weight.

In Test 1, it is the graph which compared the cholesterol absorption inhibitory effect of ester type catechin (EGCg, GCg, ECg, Cg) by showing the relationship between catechin addition amount and the cholesterol concentration in a micelle. In Test 1, it is the graph which compared the cholesterol absorption inhibitory effect of free catechin (EGC, GC, EC, C) by showing the relationship between the catechin addition amount and the cholesterol concentration in the micelle. In Test 2, it is the graph which compared the cholesterol absorption inhibitory effect of an isomerized catechin mixture, an ester type catechin mixture, and EGCg by showing the relationship between the catechin addition amount and the cholesterol concentration in the micelle. 6 is a graph showing a test schedule of Test 4. 5 is a graph showing changes with time in total cholesterol concentration in Test 4. 6 is a graph showing changes over time in LDL-cholesterol concentration in Test 4. In Test 5, a graph showing the change over time in the lymphatic absorption rate of cholesterol in each group administered with control, green tea catechins or heat epimerized catechins to rats administered with a lipid emulsion inside the stomach. It is.

Claims (5)

Ester catechins (-) EGCg, (-) ECg, (-) GCg, and (-) Cg are combined in a total amount of 70% by weight or more of the total content of catechins, and the following (1) to A food or drink used for lowering serum cholesterol, characterized by satisfying the condition of (3).
(1) The total content of (−) GCg and (−) Cg is 40 weights of the total content of (−) EGCg, (−) ECg, (−) GCg and (−) Cg, which are ester-type catechins % Or more.
(2) The content of (−) ECg is 20% by weight or less of the total content of (−) EGCg, (−) ECg, (−) GCg and (−) Cg which are ester type catechins.
(3) The content of (−) GCg is not less than the content of (−) ECg.   The food or drink according to claim 1, comprising 0.1 to 100% by weight of (-) EGCg, (-) ECg, (-) GCg and (-) Cg in total, which are ester-type catechins.   The food and drink according to claim 1, comprising a total of 500 to 6000 mg / L of (-) EGCg, (-) ECg, (-) GCg and (-) Cg which are ester-type catechins.   Ester-type catechins (-) EGCg, (-) ECg, (-) GCg, and (-) Cgchin solution containing 500 mg / L or more in total is prepared, and this is heat-treated at 80 ° C. or higher and then concentrated and dried. And the manufacturing method of the food / beverage used in order to reduce serum cholesterol characterized by mix | blending the obtained concentrated dried material.   Establishing a catechin solution containing a total of 500 to 6000 mg / L of ester-type catechins (-) EGCg, (-) ECg, (-) GCg and (-) Cg, and subjecting this to heat treatment at 80 ° C or higher. The manufacturing method of the drink used in order to reduce serum cholesterol characterized by the above-mentioned.

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