JP2008088187A - Lipid metabolism-improving agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a drug, a food and the like which can be taken in a large amount at one time, does not impose an daily ingestion burden, excels in safety, has a higher suppressing effect on increase in blood sugar level, prevents and improves hyperleptinemia and hyperinsulinemia, and improves lipid metabolism. <P>SOLUTION: An agent for suppressing the increase in blood sugar level comprises chlorogenic acids. An anti-obesity agent comprises chlorogenic acids of monocaffeoylquinic acid and dicaffeoylquinic acid at a weight ratio of 120/1 to 1/1. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a blood glucose level increase inhibitor, a hyperleptinemia preventive / ameliorating agent, a hyperinsulinemia preventive / ameliorating agent, a peroxisome proliferator activated receptor (PPAR) -dependent gene transcription activator. , Relating to fat metabolism promoters.

  The number of Japanese diabetic patients has been increasing in recent years and has become a major social problem. Type 2 diabetes, which accounts for more than 90% of diabetes, is combined with various degrees of decrease in insulin secretion from pancreatic β-cells and decreased insulin sensitivity (insulin resistance) in the skeletal muscle, liver and adipose tissues that are insulin target organs. It can be said that the insulin action is reduced and hyperglycemia occurs. Low insulin secretion is likely genetically regulated, and insulin resistance is associated with genetic predisposition as well as obesity caused by environmental factors such as overeating, high-fat diet, lack of exercise, and a lot of fat accumulated in the internal organs Visceral fat type obesity is considered to be greatly involved.

The presence of insulin resistance associated with obesity results in hyperinsulinemia as a compensation. The living body responds to the insulin resistance caused by obesity by oversecreting insulin, but when insulin resistance persists, the pancreatic β cells are exhausted, the insulin secretory ability gradually decreases, and progresses to a diabetic state. When the hyperglycemic state persists, glucose itself increases insulin secretion of pancreatic β cells and insulin resistance in the periphery, and glucose toxicity is exhibited. A vicious circle is formed here, and the degree of failure gets worse.
Therefore, it is considered that a drug that improves insulin resistance is extremely useful as a therapeutic drug for diabetes (Non-Patent Documents 1 to 4).

  On the other hand, leptin is a peptide hormone secreted from adipocytes, and it is becoming clear that it is deeply involved in appetite regulation and energy metabolism. In humans, generally obese people have higher blood leptin levels, which are positively correlated with body mass index (BMI) and body fat percentage. Therefore, the pathological condition of obesity in humans is not a leptin secretion deficiency but a decrease in leptin sensitivity (leptin resistance) in the center or periphery, which is thought to increase blood leptin concentration in a compensatory manner ( Non-Patent Documents 5 and 6).

PPAR (Peroxisome Proliferator Activated Receptor) is one of the nuclear receptors, and as a protein that mediates the action of increasing peroxisomes, which are organelles involved in lipolysis in 1990. It was identified and named Peroxisome Proliferator Activated Receptor α (peroxisome proliferator-activated receptor: PPARα) in the sense of a receptor that is activated by a peroxisome proliferator.
Subsequently, β / δ type and γ type were identified as isoform genes structurally similar to α type, and it is known that it consists of a total of three subtypes. Since there are two promoters in human and mouse γ-type genes, there are two types of proteins, PPARγ1 and PPARγ2, which differ in N-terminus due to differences in splicing. Each subtype of PPAR is activated in a ligand-dependent manner, and forms a heterodimer with RXR (Retinoid X Receptor) with 9-cis retinoic acid as a ligand, thereby responding to the PPAR response in the promoter region (transcriptional control region). It regulates the expression of various genes having sequences (PPAR responsive element; PPRE). In recent years, it has become clear that PPAR is involved in numerous physiological and pathological phenomena.
Among them, the function of PPARα is considered to be related to the maintenance of energy metabolism and homeostasis in a wide range such as fatty acid synthesis, transport and secretion, ATP production in fat consuming organs, and regulation of cell cycle. In particular, gene expression of enzymes important for fatty acid metabolism such as β-oxidation (Acyl-CoA oxidase, HMG-CoA synthase, Acyl-CoA synthase, Medium chain acyl dehydrogenase, Fatty acid binding protein, Lipoprotein lipase, etc.) It is becoming clear that there is a strong dependence.
In other words, it has become clear that a PPAR activator has an action of activating lipid metabolism in a living body. Activation of biological lipid metabolism is a useful action that leads to improvement of hyperlipidemia and anti-obesity.
In addition, PPARγ, especially PPARγ2, is expressed with relatively strong specificity in adipocytes, and has been shown to play a central role in adipocyte differentiation. (Non-patent documents 7 to 9)

Since the discovery that fibrate compounds, which are antihyperlipidemic agents, are agonists of PPARα, and that thiazolidine derivatives, known as antidiabetic agents, are agonists of PPARγ, the search for PPAR agonists following these compounds As a result, it is being developed as a drug for improving hyperlipidemia, insulin resistance, and diabetes.
And so far, as PPAR agonists, in addition to thiazolidine derivatives and fibrate compounds, fatty acids, leukotriene B4, indomethacin, ibuprofen, fenoprofen, 15-deoxy-Δ-12,14-PGJ2, etc. (Non-Patent Documents 10-14) Has been reported.
Biochemistry: 71, 11, pp1281-1298, 1999 Chemistry and Biology: Vol.37, No.2, pp120-125 Laboratory test: 42 vol.4, pp395-403, 1998 Mebio volume: Multiple risk factor syndrome 2, 1999 Diabetes & Metabolism, 23,16-24,1997 History of Separate Medicine: Adipocytes: 49-53, 199 Cells: 31 (6), 218-234, 1999 J. Lipid Res. 37,907-925, 1996 Curr.Opin.Lipidol. 10, 151-159, 1999 Cell.83,813-819,1995 J. Biol. Chem. 272 (6), 3406-3410, 1997 Proc.Natl.Acad.Sci.USA.94,4321-, 1997 J. Biol. Chem. 274 (10), 6718-6725, 1999 Mebio separate volume; Multiple Risk Factor Syndrome 2,88-96, 1999

  However, these compounds have problems such as side effects due to long-term intake. In view of the above situation, the search for antidiabetic and anti-insulin resistance drugs has been advanced, and so-called thiazolidine drugs such as troglitazone and pioglitazone have been developed and their effectiveness has been confirmed. On the other hand, serious liver disorders accompanied by death cases have been reported, and the development of drugs with excellent safety is desired.

  With regard to the anti-obesity effect of water-soluble substances, extracts from Kawara mugwort and porcupine and caffeic acid, chlorogenic acid, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, and chlorogenin contained therein Methyl acid has been reported to have an anti-obesity effect and an effect on lipid metabolism (Japanese Patent Laid-Open No. 61-40763). However, it is difficult to continuously use those derived from mugwort because of the relationship between taste and smell.

  The object of the present invention is that a large amount can be ingested at a time, it does not become a burden even if it is ingested on a daily basis, and is excellent in safety, has a higher blood sugar level increase inhibitory effect, a high leptinemia preventive / improving effect, The object is to provide drugs, foods and the like having the effect of preventing / ameliorating hyperinsulinemia and the effect of improving lipid metabolism.

  When the present inventors administered chlorogenic acids to C57B1 / 6J mice that develop obesity / type 2 diabetes in a diet-dependent manner, the present inventors suppressed obesity and suppressed blood glucose elevation and were effective in preventing and improving diabetes. Furthermore, it has been found that it has an inhibitory effect on the increase in blood insulin and leptin levels.

  Further, the present inventor has found that chlorogenic acids have a PPAR-dependent gene transcription activation action and a lipid metabolism-related gene transcription activation action, and an excellent lipid metabolism promoting effect can be obtained when used in combination with a specific substance.

The present invention provides a blood sugar level increase inhibitor comprising chlorogenic acids, a hyperleptinemia preventive / ameliorating agent, a hyperinsulinemia preventive / ameliorating agent, and a PPAR-dependent gene transcription activator.
The present invention also provides a lipid metabolism promoter comprising a chlorogenic acid having a weight ratio of monocaffeoylquinic acid / dicaffeoylquinic acid of 120/1 to 1/1.

  Furthermore, the present invention provides a lipid metabolism promoter comprising a chlorogenic acid and caffeine and having a chlorogenic acid / caffeine weight ratio of 4/1 to 2/5.

The chlorogenic acids of the present invention have an excellent PPAR-dependent gene transcription activation action, and in particular activate lipid metabolism by activating a gene having a PPAR responsive element (PPRE) in the transcriptional regulatory region of the gene. It has an effect on various lifestyle-related diseases.
In particular, taking chlorogenic acid can prevent and improve obesity, suppress or increase blood sugar, prevent and improve hyperleptinemia, and prevent and improve hyperinsulinemia. As a result, prevent and improve diabetes An effect is obtained.
Moreover, since chlorogenic acids have been taken as food on a daily basis for many years and have high safety, there is little concern about side effects even when taken as food or drink or drugs.

  Examples of the chlorogenic acids used in the present invention include 3-caffeoylquinic acid (neochlorogenic acid), 4-caffeoylquinic acid (cryptochlorogenic acid), 5-caffeoylquinic acid, and 3,4-dicaffeoylquinic acid. 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, 3-feruloylquinic acid, 4-feruloylquinic acid, 5-feruloylquinic acid, 3-ferloyl-4-caffeoyl And quinic acid.

  In addition, these substances extracted from other plants and derivatives thereof may be used. As the extract, caffeic acid dimethyl ether, 2-O-caffeoyl-arbutin, caffeoyl-carrelanine, 3-O -Caffe oil-Shikimic acid, caffeic acid docosanol ester, caffeic acid eicosanol ester, caffeic acid heneicosanol ester, caffeic acid tricosanol ester, caffeic acid tetracosanol ester, caffeic acid pentacosanol ester, cafe Acid hexacosanol ester, ferulic acid docosanol ester, ferulic acid eicosanol ester, ferulic acid heneicosanol ester, ferulic acid tricosanol ester, ferulic acid tetracosanol ester, ferulic acid pentacosanol ester, ferulic acid F Sakosa Nord ester, eicosyl - ferulic acid esters, Fukinoru acid, echinacoside (Echinacoside), 1,3- dicaffeoylquinic acid, Shikorikku acid (Cichoric acid), coniferyl alcohol, curcumin, lignans, lignins and the like.

Plant extracts containing these include, for example, coffee, apple, grape, onion, radish, lemon, cucumber, touki, pine, auren, turmeric, agi, sweet potato, sunflower leaves, sunflower seeds, morohaya, corn, Barley, wheat, rice and the like are preferable, and rice is particularly preferable. Here, rice means raw or dried products such as seeds of Oryza sativa LINNE. In particular, it may be extracted from plants containing a large amount of chlorogenic acid such as green coffee beans, southern leaves, and unripe apples. For example, warm ascorbic acid or citric acid from seeds of Coffea arabica LINNE A fresh coffee bean extract obtained by extraction with an acidic aqueous solution can be used instead of these.
Among the plant extracts, preferred are coffee, apple, grape, onion, radish, lemon, cucumber, toki, pine, auren, turmeric, agi, sweet potato, sunflower leaf, sunflower seeds, morohaya, corn, barley, Wheat, rice and the like are preferable, and rice is particularly preferable. Here, the rice includes raw or dried products such as seeds of Oryza sativa LINNE, and more preferably coffee, apples, and grapes.

Ferulic acid, which is a skeleton of chlorogenic acids, can be obtained by hydrolyzing and purifying ferulic acid ester obtained from the above step with sulfuric acid under heating under pressure, or by purifying bacteria (Pseudomonas), Syzygium aromaticum MERRILL et PERRY) It can be obtained by culturing with clove oil obtained by steam distillation from buds and leaves, or a culture solution containing eugenol obtained by purification from clove oil, and separating and purifying the culture solution. . When ferulic acid is prepared by chemical synthesis, for example, a method by a condensation reaction of vanillin and malonic acid can be mentioned (Journal of American Chemical Society, 74 , 5346, 1952). Although ferulic acid and the like have stereoisomers, any isomer can be used, and a mixture of isomers may be used.

The chlorogenic acids used in the present invention may be in the form of a salt such as sodium salt or potassium salt or a mixture thereof, in addition to those having a free carboxyl group.
Examples of the basic substance for salt formation of such salts include alkali metal hydroxides such as lithium hydroxide, sodium hydroxide and potassium hydroxide; alkaline earth metal such as magnesium hydroxide and calcium hydroxide. Hydroxides: inorganic bases such as ammonium hydroxide, basic amino acids such as arginine, lysine, histidine, ornithine; organic bases such as monoethanolamine, diethanolamine, and triethanolamine are used, especially alkali metals or alkaline earths Metal hydroxides are preferred.

  Furthermore, monocaffeoylquinic acid in chlorogenic acids when chlorogenic acids are used as an inhibitor of blood glucose level elevation, preventive / ameliorating hyperleptinemia, preventing / ameliorating hyperinsulinemia, or PPAR-dependent gene transcription activator The ratio by weight of monocaffeoylquinic acid / dicaffeoylquinic acid is preferably 120/1 to 1/1. Furthermore, it is preferably 100/1 to 2/1, particularly preferably 100/1 to 5/1. Monocaffeoylquinic acid includes 3-caffeoylquinic acid, 4-caffeoylquinic acid, 5-caffeoylquinic acid, and dicaffeoylquinic acid includes 3,4-dicaffeoylquinic acid, 3, 5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, 3-feruloylquinic acid, 4-feruloylquinic acid, 5-feruloylquinic acid, 3-feruloyl-4-caffeoylquinic acid Can be mentioned.

  Weight ratio of monocaffeoylquinic acid and dicaffeoylquinic acid content in chlorogenic acids Ingesting monocaffeoylquinic acid / dicaffeoylquinic acid in the range of 120/1 to 1/1 has an effect of promoting lipid metabolism. Excellent and preferred. The weight ratio of monocaffeoylquinic acid / dicaffeoylquinic acid is more preferably 25/1 to 1/1, and particularly preferably 25/1 to 2/1.

In addition, when chlorogenic acids are ingested in combination with caffeine at a certain ratio, an excellent lipid metabolism modification effect is obtained.
The weight ratio of the content of chlorogenic acids and caffeine is preferably 20/1 to 1/10, more preferably 10/1 to 2/5, and particularly preferably 4/1 to 2/5. In this case, the weight ratio of the content of monocaffeoylquinic acid and dicaffeoylquinic acid in the chlorogenic acids is such that monocaffeoylquinic acid / dicaffeoylquinic acid is 120/1 to 1/1, more preferably 100. / 1 to 2/1, particularly preferably 25/1 to 2/1.

  The chlorogenic acids of the present invention are formulated into an arbitrary form together with excipients and other additives. Examples of such excipients and additives include lactose, kaolin, sucrose, crystalline cellulose, corn starch, talc, agar, pectin, stearic acid, magnesium stearate, lecithin, sodium chloride and the like as solids. Examples of liquids include glycerin, peanut oil, polyvinyl pyrrolidone, olive oil, ethanol, benzyl alcohol, propylene glycol, and water.

The chlorogenic acid of the present invention and the composition containing the same can be administered or ingested to humans by any route such as oral, enteral, injection, transmucosal and transdermal, depending on the dosage form.
The amount varies depending on various factors such as age, weight, sex, administration method, etc., but in the case of oral administration, it is usually preferably 100 to 5000 mg per adult.
When used as a blood glucose level increase inhibitor, it is preferable to administer the range of 300 to 5000 mg, particularly 400 to 1000 mg, once a day to several times a day.
In addition, when used as an agent for preventing / ameliorating hyperleptinemia and an agent for preventing / ameliorating hyperinsulinemia, it should be administered in the range of 300 to 5000 mg, particularly 400 to 1000 mg, once a day to several times a day. Is preferred.
Even when used as a PPAR-dependent gene transcription activator, it is preferable to administer the range of 300 to 5000 mg, particularly 400 to 1000 mg, once a day to several times a day.
When used as a fat metabolism promoter, it is preferably administered in the range of 300 to 5000 mg, particularly 400 to 1000 mg, once a day to several times a day.

  Examples of the dosage form of the present invention include tablets, powders, granules, capsules, troches, syrups, emulsions, soft gelatin capsules, gels, pastes, injections, creams, gels, lotions, patches and the like. . Moreover, it can mix | blend suitably with various foodstuffs, such as a drink of various forms, snacks, dairy products, seasonings, starch processing products, and processed meat products. In particular, when used as a lipid metabolism promoter, it is preferably a container-packed beverage.

  The chlorogenic acids of the present invention are preferably 0.01 to 100% by weight, particularly preferably 0.05 to 95% by weight, and more preferably 0.1 to 0.1% by weight in the preparations because of ease of ingestion and administration. ~ 90% by weight.

Example 1
Efficacy test on obesity, blood glucose, blood insulin, blood leptin using C57BL / 6J mice, a model of diet-induced obesity / type 2 diabetes, 5 male mice each 7 weeks 7 C57BL / 6J mice Divide into groups and rear on each diet with the composition listed in Table 1. After 4 months, the body weight is measured, and after fasting for 12 hours, blood is collected from the abdominal aorta under ether anesthesia, and fasting serum glucose, insulin, and leptin levels are measured. The results are shown in Table 2.

In the second group, a significant increase in body weight was observed compared to the first group, whereas in the third group, the increase in body weight was less than that in the first group, and in comparison with the second group, Showed a low value. The chlorogenic acid of the present invention was excellent in anti-obesity effect.
In the second group, a significant increase in blood glucose level (glucose) was observed compared to the first group, whereas in the third group, the increase in blood glucose level relative to the first group was small, and the second group The blood glucose level was low for the group. The chlorogenic acid of the present invention was excellent in the effect of suppressing an increase in blood sugar level and was effective for diabetes.
Compared to the first group, the second group showed a significant increase in insulin level, whereas in the third group the insulin level increased less than the first group, and in the second group In contrast, the insulin level was low. The chlorogenic acid of the present invention was excellent in suppressing an increase in blood insulin level and was effective in insulin resistance.
Compared to the first group, the second group showed a significant increase in leptin levels, whereas in the third group the increase in leptin levels relative to the first group was small, and in the second group On the other hand, the leptin level was low. The chlorogenic acid of the present invention was excellent in suppressing the increase in blood leptin level and was also effective in leptin resistance.

Example 2
PPAR-dependent gene transcription activation test Intestinal epithelial cell line IEC-6 is seeded on a 12-well plate and cultured in DMEM (1% FCS) for 1 day. PPAR response element (underlined) (AACg TgACCTTTgTCCT ggTC AACg TgACCTTTgTCCT ggTC AACg TgACCTTTgTCCT ggTC) DNA chain, SV40 promoter gene, PPAR reporter plasmid containing firefly luciferase gene A control plasmid (TK-Luc: Promega) linked with a promoter gene was introduced at the same time using a transfection reagent (Superfect transfection reagent; QIAGEN) so as to be 0.5 μg / well each. Thereafter, the culture solution was replaced with a DMEM (-FCS) medium containing a test substance, and further cultured for 24 hours. After washing with PBS, cells were lysed using a dual luciferase assay system (Promega), a substrate solution containing luciferin was added to the lysate, and firefly and Renilla luciferase activities were measured with a luminometer. The PPAR activity ability was evaluated by measuring the transcriptional activity (luciferase activity 1) of a PPAR-dependent gene in this experimental system. The transcriptional activity of PPAR-dependent gene (luciferase activity 1) was defined as follows.

  PPAR-dependent gene transcriptional activity (luciferase activity 1) = (firefly luciferase activity by PPAR-Luc) / (renilla luciferase activity by TK-Luc)

  Table 3 shows the ability of each test substance to activate PPAR in IEC-6 cells. The PPAR-dependent transcription activity in the control is defined as 100, and the relative value is shown.

  Coffee extract containing chlorogen and chlorogenic acid was excellent in PPAR-dependent gene transcription activation.

Example 3
HMG-CoA synthase gene activation test A small intestinal epithelial cell line IEC-6 is seeded on a 12-well plate and cultured in DMEM (1% FCS) for 1 day. Rat HMG-CoA synthase gene transcription control region (-1081 / + 21), SV40 promoter gene, PPAR reporter plasmid containing firefly luciferase gene (HMGS-Luc), and thymidine kinase promoter gene upstream of the Renilla luciferase gene The control plasmid (TK-Luc: Promega) was introduced at the same time using a transfection reagent (Superfect transfection reagent; QIAGEN) so as to be 0.5 μg / well respectively. Thereafter, the culture solution was replaced with a DMEM (-FCS) medium containing a test substance, and further cultured for 24 hours. After washing with PBS, cells were lysed using a dual luciferase assay system (Promega), a substrate solution containing luciferin was added to the lysate, and firefly and Renilla luciferase activities were measured with a luminometer. Lipid metabolism gene activity was evaluated by measuring the transcriptional activity (luciferase activity 2) of HMG-CoA synthase in this experimental system. The gene transcription activity (luciferase activity 2) was defined as follows.

  Gene transcription activity (Luciferase activity 2) = (Firefly luciferase activity by HMGS-Luc) / (Renilla luciferase activity by TK-Luc)

  Table 4 shows the ability of each test substance to activate HMG-CoA synthase gene in IEC-6 cells. In addition, the transcriptional activity in control is set to 100 and the relative value is shown.

  Chlorogen was excellent in transcriptional activation of lipid metabolism enzyme gene.

Example 4
Anti-obesity effect test on body weight and visceral fat using diet-induced obese mice / chlorogenic acid + caffeine Seven-week old C57BL / 6J male mice were divided into 6 groups of 5 mice each, and the composition described in Table 5 was used. Breed on each diet. After one month, the body weight was measured, and the visceral fat mass (cold testicular fat, mesenteric fat, retroperitoneal fat and perirenal fat) was measured.

  In the second group, a significant increase in body weight and an increase in visceral fat mass were observed compared to the first group, whereas in the third group, the weight gain and total visceral fat mass were as compared to the second group. It showed a significant reduction. Furthermore, compared with the 3rd group, the 4th group added the caffeine rather than the chlorogenic acid alone, and the efficacy of the chlorogenic acid increased and the body weight gain inhibitory effect and the visceral fat mass increase inhibitory effect became strong.

Example 5
Anti-obesity effect test on body weight and visceral fat using diet-induced obese mice / anti-obesity effect test of chlorogenic acid alone and coffee bean extract 7-week-old C57BL / 6J male mice were divided into 4 groups of 5 each Divide and raise each diet with the composition described in Table 8. After 5 weeks, the body weight was measured and the visceral fat mass (cold testicular fat, mesenteric fat, retroperitoneal fat and perirenal fat) was measured.

  In the second group, a significant increase in body weight and increase in visceral fat mass was observed compared to the first group, whereas in the third group and the fourth group, the weight gain and total weight were increased compared to the second group. Visceral fat mass decreased.

Example 6
Capsules for prevention and improvement of lifestyle-related diseases
The following composition (400 mg) was encapsulated in an encapsulating agent.
Chlorogenic acid 68% by weight
Cornstarch 10
Cellulose 10
Tocopherol 2
Lactose 10

Example 7
Container-packed beverages were produced using coffee bean extract water by the following method, and monocaffeoylquinic acid, dicaffeoylquinic acid, chlorogenic acids and caffeine in the beverage were analyzed.
・ Analysis of monocaffeoylquinic acid and dicaffeoylquinic acid
HPLC (manufactured by Hitachi, Ltd.) was used. The model numbers of the unit units are as follows.
Plotter: D-2500
Detector: L-4200
Pump: L-7100
Auto sampler: L-7200
Column: lnertsil ODS-2, inner diameter 2.1 mm x length 250 mm
<Analysis conditions>
Sample injection volume: 10 μL
Flow rate: 0.3mL / min
Ultraviolet spectrophotometer detection wavelength: 325 nm
Eluent A: 0.05M acetic acid 3% acetonitrile solution Eluent B: 0.05M acetic acid 100% acetonitrile solution Concentration gradient time Eluent A Eluent B
0 minutes 100% 0%
5 minutes 100% 0%
20 minutes 80% 20%
35 minutes 80% 20%
45 minutes 0% 100%
60 minutes 0% 100%
70 minutes 100% 0%
120 minutes 100% 0%
<Retention time of monocaffeoylquinic acid and dicaffeoylquinic acid>
(Unit: minutes)
Monocaffeoylquinic acid: 19.7, 22.4, 23.5, total 3 points Dicaffeoylquinic acid: 32.2, 32.8, total 2 points The ratio of the mono-form and the di-form was calculated.

・ Analysis of chlorogenic acids Analytical instruments and analysis conditions are the same as those for mono- and di-forms.
<Retention time of chlorogenic acids>
(Unit: minutes)
Monocaffeoylquinic acid: 19.7, 22.4, 23.5 total 3 points Dicaffeoylquinic acid: 32.2, 32.8 total 2 points Ferlaquinic acid: 26.4, 27.1 total Two points Caffeoylquinic acid at the fifth position was used as the standard substance from the area% obtained here, and the weight% was obtained.

・ Caffeine analysis method and definition <Analytical equipment>
The same equipment was used for the analysis of mono and dicaffeoylquinic acid.
<Analysis conditions>
Sample injection volume: 10 μL
Flow rate: 1.0mL / min
Ultraviolet absorption spectrophotometer detection wavelength: 280 nm
Eluent A: 0.1M acetic acid aqueous solution Eluent B: 0.1M acetic acid acetonitrile solution Concentration gradient condition (% is volume%)
Time Eluent A Eluent B
0 minutes 97% 3%
5 minutes 97% 3%
37 minutes 80% 20%
43 minutes 80% 20%
43.5 minutes 0% 100%
48.5 minutes 0% 100%
49 minutes 97% 3%
62 minutes 97% 3%
<Retention time of caffeine>
(Unit: minutes)
Caffeine: 17.2 in total: 27.2% by weight was determined from the area% determined here using a standard substance.

Manufacture of packaged beverages:
(1) Production Example 1
200 g of extract was added to 200 g of coffee beans of Tanzania AA (Unicafe, L value 26) to obtain an extract. At this time, the Brix value of the extract was 9.95.
A packaged beverage having the composition shown in Table 11 was produced. In addition, after filling the beverage into a 190 mL steel can, the retort sterilization tester (manufactured by Nisaka Manufacturing Co., Ltd.) is used and kept at 123.5 ° C. for 25 minutes and subjected to heat treatment under the condition that F value (sterilization index) 43 is obtained. went. An F value of 3.1 is enough to kill botulinum.
(2) Production Example 2
After adding 2000 g of extract to 200 g of coffee beans of Tanzania AA (Unicafe, L value 26), only the last 200 g with 200 g added was collected to obtain an extract. At this time, the Brix value of the extract was 0.38.
A packaged beverage having the composition shown in Table 11 was produced. In addition, after filling the beverage into a 190 mL steel can, using a retort sterilization tester (manufactured by Nisaka Seisakusho), hold it at 121.5 ° C. for 10 minutes, and heat-treat it under the condition that F value (sterilization index) is 10. went. In addition, it becomes the level which can kill botulinum bacteria by F value 3.1.

  0.05M acetic acid was used to dilute the beverage concentration of the packaged beverage to 15% by weight after sterilization. Next, the mixture was centrifuged at 10,000 r / min for 5 minutes and centrifuged (Kubota Corporation KR-1500), and the supernatant was collected and analyzed by HPLC with an injection volume of 10 μL. The results are shown in Table 11.

  As a result of drinking the beverages of Production Examples 1 and 2, lipid metabolism was promoted with good taste.

Claims (11)

  An anti-obesity agent consisting of chlorogenic acids.   An antiobesity agent comprising a chlorogenic acid having a weight ratio of monocaffeoylquinic acid / dicaffeoylquinic acid of 120/1 to 1/1.   An anti-obesity agent comprising chlorogenic acids and caffeine, wherein the weight ratio of chlorogenic acids / caffeine is 4/1 to 2/5.   Visceral fat accumulation inhibitor consisting of chlorogenic acids.   A visceral fat accumulation inhibitor comprising a chlorogenic acid having a weight ratio of monocaffeoylquinic acid / dicaffeoylquinic acid of 120/1 to 1/1.   A visceral fat accumulation inhibitor consisting of chlorogenic acids and caffeine, wherein the weight ratio of chlorogenic acids / caffeine is 4/1 to 2/5.   Lipid metabolism promoter consisting of chlorogenic acids.   A lipid metabolism promoter comprising a chlorogenic acid having a weight ratio of monocaffeoylquinic acid / dicaffeoylquinic acid of 120/1 to 1/1.   A lipid metabolism promoter consisting of chlorogenic acids and caffeine, wherein the weight ratio of chlorogenic acids / caffeine is 4/1 to 2/5.   A packaged beverage containing the anti-obesity agent, visceral fat accumulation inhibitor, or lipid metabolism promoter according to any one of claims 1 to 9.   A peroxisome proliferator-activated receptor-dependent gene transcription activator comprising chlorogenic acids.