阅读说明：本技术 用于通过增强流动介导的扩张来改善内皮功能的组合物 (Compositions for improving endothelial function by enhancing flow-mediated dilation ) 是由 L·阿克蒂戈雷塔 R·贝尔-赖利德 于 2020-04-08 设计创作，主要内容包括：本发明涉及包含经酯酶处理的脱咖啡因的生咖啡提取物(HDGCE)的组合物,该组合物用于通过增强个体中流动介导的扩张来改善心血管终点诸如内皮功能的方法中,其中以100mg/天-400mg/天的量向所述个体施用有效剂量的HDGCE。(The present invention relates to compositions comprising esterase-treated decaffeinated green coffee extract (HDGCE) for use in a method of improving cardiovascular end-point such as endothelial function by enhancing flow-mediated dilation in an individual, wherein an effective dose of HDGCE is administered to the individual in an amount of 100 mg/day to 400 mg/day.)

1. A composition comprising an esterase-treated decaffeinated green coffee extract (HDGCE) for use in a method of improving cardiovascular flow and reducing the risk of cardiovascular disease in a healthy individual as measured by changes in flow-mediated dilation (FMD) measured in the individual, wherein an effective dose of the composition is administered to the individual in an amount of 100-400 mg/day.

2. The composition for use according to claim 1, wherein the composition is in the form of a functional food product.

3. A composition for use according to claims 1 and 2, wherein the composition comprises caffeic acid in a ratio of at least greater than 2: 1: ferulic acid.

4. A composition for use according to claim 3, wherein the composition has a ratio of caffeic acid to caffeic acid in the range of 3: 1 to 10: 1: ferulic acid.

5. Composition for use according to any one of claims 1 to 4, wherein the composition has a total content of caffeic acid and ferulic acid of between 30mg and 80mg and is a heat-treated composition.

6. Composition for use according to any one of claims 1 to 5, wherein the composition has a total content of caffeic acid and ferulic acid of between 35mg and 60mg and is a heat-treated composition.

7. The composition for use according to any one of claims 1 to 6, wherein the composition is a liquid beverage composition.

8. Composition for use according to any one of claims 1 to 7, wherein the individual is supplemented with at least 200kcal of a meal in addition to the administration of HDGCE.

9. The composition for use according to any one of claims 1 to 8, wherein the subject is a human.

10. The composition for use according to any one of claims 1 to 9, wherein the cardiovascular disease is endothelial dysfunction.

11. A process for preparing the composition according to claims 1 to 10, comprising the steps of:

(i) by mixing green coffee beans with water, steam, organic solvent, and supercritical CO₂And/or mixtures thereof to prepare a decaffeinated green coffee extract;

(ii) optionally drying the decaffeinated green coffee extract;

(iii) contacting the obtained decaffeinated green coffee extract with an esterase at a pH ranging from 4 to 7 and a temperature ranging from 20 ℃ to 50 ℃ for an incubation time ranging from 1 hour to 6 hours;

(iv) heating the enzyme-treated green coffee extract at a temperature in the range of 80 ℃ to 120 ℃ for 1 minute to 30 minutes to inactivate the enzymes and pasteurize the extract; and

(v) optionally drying said extract to obtain said esterase-treated decaffeinated green coffee extract (HDGCE).

12. The process of claim 11, wherein the drying step (ii) is spray drying or freeze drying.

13. The process according to claims 11 and 12, wherein the esterase is chlorogenic acid esterase dissolved in 1ml of water or buffer at a concentration ranging from 1U to 20U per 200mg of green coffee extract W/W.

Technical Field

The present invention relates to compositions comprising esterase-treated decaffeinated green coffee extract (HDGCE) for use in a method of improving cardiovascular end-point such as endothelial function by enhancing flow-mediated dilation in an individual, wherein an effective dose of HDGCE is administered to the individual in an amount of 100 mg/day to 400 mg/day.

Background

Chlorogenic acid is an ester formed between trans-cinnamic acid and quinic acid. Chlorogenic acids occur naturally in coffee and act primarily as mono-and diesters of quinic acid and phenolic groups (e.g., caffeine, ferulic, coumaric, methoxycinnamic) attached to different positions.

US 8,481,028 describes a method of obtaining hydrolysed chlorogenic acid using a microorganism and/or an enzyme capable of hydrolysing caffeoylquinic acids and diesters (e.g. 3-, 4-or 5-caffeoylquinic acids and diesters), and/or feruloylquinic acids and diesters (e.g. 3-, 4-or 5-feruloylquinic acids and diesters) to produce caffeic acid and ferulic acid, respectively.

Endothelium refers to the cells lining the inner surfaces of blood and lymph vessels, forming an interface between circulating blood or lymph in the lumen and the rest of the vessel wall. The endothelium slows many vascular functions and plays a key role in blood flow mechanisms and coagulation regulation. In a healthy artery that also has a healthy endothelium, the vessel can relax if the blood flow is increased. It has been observed that individuals with impaired endothelial function are more susceptible to cardiovascular disease (e.g., damage to the endothelium can result in atherosclerotic plaques, as well as cerebrovascular disease). Vascular dysfunction can be monitored by measuring flow-mediated dilation (FMD) in blood vessels. Mills et al (Clinical Nutrition 36 (2017)) 1520-1529 describe the improvement of human vascular function induced by modulation of coffee by chlorogenic acids and their metabolites.

In this article, the authors describe that 450mg and 900mg of pure 5-CQA showed no significant effect in 24 individuals.

Little research has been conducted on coffee and/or coffee components to assess markers of endothelial dysfunction. Interestingly, most of these studies have shown beneficial effects on vascular health. As demonstrated by Mills et al (Clinical Nutrition 36(2017)1520 and 1529), the observed effect was most pronounced at high chlorogenic acid coffee levels rather than low levels.

A recent study (Sanchez-Bridge et al Biofactors 42(3) (2016)259-67) details the absorption and metabolism of coffee chlorogenic acids and phenolic acids in humans after the administration of coffee with different roasting conditions. In addition, to assess the importance of intestinal absorption, unbaked coffee containing high levels of free phenolic acids after enzymatic hydrolysis was also used as a test beverage. Enzymatically hydrolyzed unbaked coffee is prepared by passing an unbaked coffee extract through a column containing beads with immobilized lactobacillus johnsonii (La1, NCC533) esterase.

It is an object of the present invention to improve the prior art and to provide a better new nutritional solution for improving cardiovascular endpoints such as endothelial function by enhancing flow-mediated dilation in an individual.

Disclosure of Invention

Thus, according to a first aspect of the invention, although this is not necessarily the broadest aspect of the invention, nor is it necessarily the only aspect of the invention, there is provided a method of improving vascular function and consequently cardiovascular and cerebrovascular disease, said method comprising administering to an individual an effective dose of an esterase-treated decaffeinated green coffee extract (HDGCE) composition in an amount of from 100 mg/day to 400 mg/day of hydrolysed green coffee extract.

In one embodiment, the composition comprises caffeic acid in a ratio of at least greater than 2: 1: ferulic acid. In another embodiment, the composition comprises caffeic acid in a ratio in the range of 3: 1 to 10: 1: ferulic acid.

The inventors have surprisingly found that consumption of a composition comprising HDGCE (173.1mg HDGCE dose) showed a significant increase in FMD at 1h (p-value 0.036) and 6h (p-value 0.017608) compared to placebo treatment. The increase in FMD at these time points was greater than 1% units of FMD. Improvement in endothelial dysfunction (1% flow-mediated dilation value) was associated with a 13% lower risk of cardiovascular events (Inaba et al, 2010, Int J Cardiovas Imaging 26: 621-640).

In one aspect, the invention relates to a composition comprising an esterase-treated decaffeinated green coffee extract (HDGCE) for use in a method of improving cardiovascular flow and reducing the risk of cardiovascular disease in a healthy individual, the risk of cardiovascular disease being measured by a change in flow-mediated dilation (FMD) measured in the individual, wherein an effective dose of the composition is administered to the individual in an amount of 100 mg/day to 400 mg/day.

In yet another aspect, the present invention relates to a method for preparing a composition comprising an esterase-treated decaffeinated coffee extract (HDGCE), the method comprising the steps of:

by mixing green coffee beans with water, steam, organic solvent, supercritical CO₂And/or mixtures thereof to prepare a decaffeinated green coffee extract;

-optionally drying, preferably spray drying or freeze drying, the decaffeinated green coffee extract;

-contacting the obtained decaffeinated green coffee extract with an esterase, preferably a chlorogenic acid esterase dissolved in 1ml of water or buffer, preferably in a concentration ranging from 1U to 20U/200mg of green coffee extract W/W, at a pH ranging from 4 to 7 and at a temperature ranging from 20 ℃ to 50 ℃, for an incubation time ranging from 1 hour to 6 hours;

-heating the above enzyme-treated green coffee extract at a temperature in the range of 80 ℃ to 120 ℃ for 1 min to 30 min to inactivate the enzymes and pasteurize the extract; and

-optionally drying the extract to obtain an esterase-treated decaffeinated green coffee extract (HDGCE).

Drawings

Figure 1 shows a purified esterase from lactobacillus johnsonii (l.johnsonii) having a Mw of about 34 KDa.

FIG. 2 shows the effect of pH on the esterase activity of Lactobacillus johnsonii (Lactobacillus johnsonii).

FIG. 3 shows the effect of temperature on Lactobacillus johnsonii esterase activity.

FIG. 4 shows the substrate specificity of Lactobacillus johnsonii esterase. 4-nitrophenylbutyrate (●); 4-Nitrobenzene acetate (. diamond-solid.); 4-nitrophenyl decanoate (tangle-solidup); 4-nitrophenyltetradecanoate (■); 4-Nitrobenzene dodecanoic acid ester

FIG. 5 shows CQA (●), FQA (■) and di-CQA of Lactobacillus johnsonii esterase (16.5U/ml) on decaffeinated green coffee extract (200g/L)Kinetic transformation of caffeic acid (. tangle-solidup.) and ferulic acid (. diamond-solid.) formation as a function of time. The reaction volume was 1 ml. Reactions were performed in duplicate.

Detailed Description

The term cardiovascular disease may include, for example, coronary artery disease (also known as coronary heart disease and ischemic heart disease), peripheral artery disease (e.g., peripheral endothelial dysfunction), renal artery stenosis and aortic aneurysm, formation of atherosclerotic plaques. There are also many cardiovascular diseases involving the heart, such as cardiomyopathy, hypertensive heart disease, heart failure, pulmonary heart disease, cardiac arrhythmias, endocarditis. The structures most commonly involved are heart valves, inflammatory cardiac hypertrophy, myocarditis, certain drugs, toxins and autoimmune disorders, eosinophilic myocarditis, valvular heart disease, congenital heart disease, and rheumatic heart disease. Preferably, the cardiovascular disease is selected from the group consisting of atherosclerotic plaque formation, coronary artery disease and endothelial dysfunction, preferably endothelial dysfunction. More preferably, the cardiovascular disease is endothelial dysfunction, preferably peripheral endothelial dysfunction.

Cerebrovascular disease includes a variety of medical conditions that affect the cerebral vasculature and cerebral circulation. Arteries that supply oxygen and nutrients to the brain are often damaged or deformed in these disorders. The most common manifestations of cerebrovascular disease are ischemic or minor strokes, sometimes hemorrhagic strokes. Hypertension (high blood pressure) is the most important risk factor for stroke and cerebrovascular disease, as it can alter the structure of blood vessels and lead to atherosclerosis. Atherosclerosis narrows the blood vessels in the brain, resulting in reduced brain perfusion. Other risk factors that lead to stroke include smoking and diabetes. Narrowed cerebral arteries can lead to ischemic stroke, but sustained elevated blood pressure can also lead to vessel tearing, resulting in hemorrhagic stroke. Preferably, the cerebrovascular disease is selected from ischemic stroke, mini-stroke and hemorrhagic stroke.

The term "functional food product" refers to a beverage or food composition comprising an HDGCE composition in an amount ranging from 100mg to 400mg of hydrolyzed green coffee extract. The food composition may be in the form of a powder, such as a chocolate or malt-based composition. The food composition may also be a snack, such as a cereal bar comprising HDGCE.

The term "esterase-treated" means that the decaffeinated green coffee extract is incubated with a purified chlorogenic acid esterase or a microorganism comprising such an esterase. Chlorogenic acid esterases are for example in US 8,481,028 or for example in Bel-Rhlid et al: biotransformation of caffeoyl quinic acids from green coffee extracts by Lactobacillus johnsonii NCC533 (2013): AMB express volume 3: page 28.

Esterases are hydrolytic enzymes that break down esters into acids and alcohols in a chemical reaction with water, known as hydrolysis. The incubation time may be 30 minutes to 6 hours at a temperature in the range of 20 ℃ to 50 ℃ related to the enzyme concentration, such that at least 80% of the total chlorogenic acids present in the decaffeinated green coffee extract are hydrolyzed. In one embodiment, the esterase is from lactobacillus johnsonii.

The term "decaffeinated green coffee extract" means by, for example, hot water or with organic solvents or supercritical CO well known to those skilled in the art₂Extracting the decaffeinated green coffee beans. The caffeine content is less than 5% W/W, and may be 2% to 3% W/W.

The term "amount of 100mg-400 mg/day" refers to 100mg-400mg dry weight of esterase-treated decaffeinated green coffee extract (HDGCE), which can be dissolved in, for example, water or incorporated into an edible food product such as a cereal, soluble coffee, chocolate or food supplement such as a capsule or tablet suitable for consumption by an individual. In one embodiment, an esterase-treated decaffeinated green coffee extract in an amount of 100mg to 400mg dry weight is dissolved in about 200ml of water.

The term "measurement of changes in flow-mediated dilation" refers to changes in Δ FMD values between an individual administered a placebo (an individual administered a composition without HDGCE) and an individual administered an active composition (a composition comprising 100 mg/day to 400 mg/day HDGCE). In one embodiment, the variation is at least 1% and the P value is less than 0.02.

In one embodiment, the HDGCE composition has a total content of caffeic acid and ferulic acid of between 30mg and 80mg, and the composition may be a heat-treated composition. In another embodiment, the HDGCE composition has a total content of caffeic acid and ferulic acid of between 35mg and 60mg, and the composition may be a heat-treated composition.

The composition used according to the invention may be in any suitable form, for example the composition may be in the form of a liquid composition, a beverage, such as a liquid drink, a shake, a nutritional composition or a liquid meal replacement.

An important approach to controlling food hygiene risks is to heat treat edible components that may carry food pathogens or spoilage organisms. Well-known examples of such heat treatments are pasteurization (e.g. heating the edible material to 70 ℃ for 2 minutes, or 75 ℃ for 26 seconds, or 80 ℃ for 5 seconds) and Ultra High Temperature (UHT) treatment (e.g. heating the edible material above 135 ℃ for at least 2 seconds).

The composition for use according to the invention may be administered in daily doses to provide HDGCE in an amount of 100mg to 400mg dry weight per individual per day. The dosage should ensure that the daily dose is sufficient to provide the desired effect to the individual at least during the intermediate period. In one embodiment, the subject is fasting when the composition is administered.

Another aspect of the invention is the non-therapeutic use of a composition comprising HDGCE in an amount of 100-400 mg/day for increasing flow-mediated dilation (FMD), wherein the amount of caffeic acid and ferulic acid is as follows: the ferulic acid ratio is at least greater than 2: 1, such as at least greater than 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1 or 10: 1.

In one embodiment, the individual is supplemented with at least 200kcal of a meal in addition to the administration of HDGCE.

In one embodiment, the subject is a human subject. In one embodiment, the subject is a healthy human subject.

Coffee extract

Many methods for preparing coffee extracts are known in the art, for example from EP 0916267. The coffee extract may be, for example, pure soluble coffee. Pure soluble coffee products are readily available and many methods for preparing pure soluble coffee products are known in the art, for example from EP 106930.

Another aspect of the invention provides a process for preparing an HDGCE composition comprising the steps of: (i) by mixing green coffee beans with water, steam, organic solvent, and supercritical CO₂And/or mixtures thereof to prepare a decaffeinated green coffee extract; (ii) optionally drying the decaffeinated green coffee extract; (iii) contacting the obtained decaffeinated green coffee extract with an esterase at a pH ranging from 4 to 7 and a temperature ranging from 20 ℃ to 50 ℃ for an incubation time ranging from 1 hour to 6 hours; (iv) heating the enzyme-treated green coffee extract at a temperature in the range of 80 ℃ to 120 ℃ for 1 minute to 30 minutes to inactivate the enzymes and pasteurize the extract; and (v) optionally drying the extract to obtain the esterase-treated decaffeinated green coffee extract (HDGCE). In one embodiment, the drying step (ii) is spray drying or freeze drying. In another embodiment, the esterase is preferably a chlorogenic acid esterase preferably dissolved in 1ml of water or buffer, preferably in a concentration ranging from 1U to 20U per 200mg of green coffee extract W/W.

Those skilled in the art will appreciate that they are free to incorporate all of the features of the invention disclosed herein. In particular, features described for therapeutic use of the composition may be combined with non-therapeutic use, and vice versa. In addition, features described with respect to different embodiments of the invention may be combined. Further advantages and features of the invention will be apparent from the figures and embodiments.

Examples

Example 1

Purification, characterization and cloning of esterase from Lactobacillus johnsonii (NCC533)

Esterase activity was identified in whole cells of Lactobacillus johnsonii. The enzyme was purified and characterized. The gene was annotated (LJ-1228, gene 11586011159347 reverse product ═ α/β hydrolase (see sequence below.) then overexpressed in food grade e.coli (e.coli) and linear gradient 1 to 0mol (NH) by HPLC (HIC column TSK gel phenyl-5 PW₄)₂SO₄NaPO of/L₄Solution (50mM) pH 7.0+1mM EDTA). The flow rate was 0.8 ml/min).

DNA sequence/nucleotide sequence

Atggagactacaattaaacgtgatggtctaaacttacatggtttacttgaaggaaccgataagattgaaaatgatacgattgctattttaatgcatggttttaaaggtgatttgggttatgatgacagcaagattttgtatgctctctctcactacttaaatgatcaaggcctcccaacaattcgttttgactttgatggatgcggaaaaagtgatggtaaatttgaagatatgactgtctatagcgaaatcctagatgggataaaaatattagattatgttcgtaatactgttaaggcaaaacatatctatttagtgggacactcccaaggtggagtagtagcgtcaatgctggctggatattatcgagatgttattgaaaaattggctttactctctcctgcagcaactcttaagtctgatgctttagatggagtttgtcagggtagtacttatgatccaacgcatatccctgaaactgtcaatgttagtggctttgaagtaggaggagcttactttagaacggctcaattattgcctatttatcaaacagcggaacattataatagggaaactttattgattcatggcttagcagataaagtcgtgtcacctaatgcttcaagaaaatttcatacacttttgcctaaaagtgagctccatttaattccagatgagggtcacatgtttaacggaaaaaatagacctgaagtattaaaattagttggtgagtttttaataaaataa

Amino acid sequence

Identification of the optimum pH of Lactobacillus johnsonii esterase

The optimum pH was determined by using glycine buffer, acetate buffer, Tris buffer, phosphate buffer and water. 5-CQA was used as substrate (50. mu. mol/ml). As we can see in fig. 2, the optimal pH of the enzyme is between 4.0 and 6.0. The reaction was carried out with purified esterase (0.01U/mg substrate) at 37 ℃ for 30 minutes.

Identification of optimal temperature for Lactobacillus johnsonii esterase

The optimal temperature was determined by using 5-CQA as substrate (50. mu. mol/ml) at different temperatures from 10 ℃ to 90 ℃. As we can see in FIG. 3, the optimal temperature of the enzyme is between 30 ℃ and 40 ℃. The reaction was carried out at pH 5.0 for 30 minutes with purified esterase (0.01U/mg substrate).

Substrate specificity

The substrate specificity of Lactobacillus johnsonii esterase was studied using different 4-nitrophenyl derivatives. As we can see in fig. 4, 4-nitrobenzene butyrate is the best substrate, whereas no conversion of 4-nitrobenzene dodecanoate is observed. The reaction was carried out at 37 ℃ for 10 minutes in sodium phosphate buffer pH 6.0. The substrate was used at a concentration of 0.2mM and the enzyme was used at 0.01U/mg substrate. Measurements were monitored every 30 seconds. The absorbance was set at 410 nm.

Decaffeinated green coffee extracts were treated with lactobacillus johnsonii esterase.

Reaction kinetics: laboratory scale test

A kinetic study of 200mg/ml Decaffeinated Green Coffee Extract (DGCE) was performed using different concentrations (1.65U/ml, 3.3U/ml, 4.95U/ml, 8.25U/ml and 16.25U/ml) of Lactobacillus johnsonii esterase. The reaction was carried out in a volume of 1ml at pH 4.5 and 37 ℃. The results are summarized in the table below. The concentrations of the different compounds are in mg/ml.

Time (h)	0	1	2	3	4
						Enzyme (U)	0	1.65	1.65	1.65	1.65
DGCE(mg/ml)	200	200	200	200	200
						CQA	52.11	21.51	18.05	16.18	15.14
FQA	11.03	9.89	9.09	9.56	8.3
						di-CQA	13.22	3.34	2.88	2.65	2.44
CA	0.49	19.82	21.51	22.35	22.08
						FA	0.21	3.76	4.11	4.45	4.36
Caffeine	3.20	3.13	3.09	3.10	3.00

Time (h)	0	1	2	3	4
						Enzyme (U)	0	3.3	3.3	3.3	3.3
DGCE(mg/ml)	200	200	200	200	200
						CQA	52.25	17.77	14.62	12.88	11.09
FQA	11.1	1.65	1.46	1.28	1.11
						di-CQA	13.29	2.41	2.14	2.08	1.83
CA	0.48	25.63	27.23	27.56	26.27
						FA	0.20	4.09	4.58	4.77	4.80
Caffeine	3.21	3.05	3.08	3.04	2.90

Time (h)	0	1	2	3	4
						Enzyme (U)	0	4.95	4.95	4.95	4.95
DGCE(mg/ml)	200	200	200	200	200
						CQA	50.85	15.88	12.86	10.93	10.04
FQA	10.74	1.66	1.29	1.07	0.57
						di-CQA	11.75	1.83	1.72	1.44	1.37
CA	0.49	26.42	27.77	27.82	27.65
						FA	0.21	4.30	4.79	4.94	5.19
Caffeine	3.12	3.03	3.04	2.94	2.98

Time (h)	0	1	2	3	4
						Enzyme (U)	0	8.25	8.25	8.25	8.25
DGCE(mg/ml)	200	200	200	200	200
						CQA	51.68	13.03	9.78	8.07	6.93
FQA	10.92	1.32	0.56	0.50	0.49
						di-CQA	13.48	1.53	1.17	0.95	0.90
CA	0.48	27.13	28.52	29.22	29.40
						FA	0.21	4.90	5.72	6.08	6.36
Caffeine	3.20	2.97	2.95	2.94	2.92

Time (h)	0	1	2	3	4
						Enzyme (U)	0	16.5	16.5	16.5	16.5
DGCE(mg/ml)	200	200	200	200	200
						CQA	51.46	8.90	6.34	4.87	3.91
FQA	10.93	0.54	0.48	0.44	0.42
						di-CQA	13.19	1,29	1.02	0.58	0.30
CA	0.48	27.26	28.79	29.19	29.90
						FA	0.21	4.90	5.72	6.08	6.36
Caffeine	3.18	2.80	2.83	2.79	2.82

Kinetics of reaction as a function of time

A kinetic study of decaffeinated green coffee extract (200mg/ml) was performed using Lactobacillus johnsonii esterase at a concentration of 16.25U/ml. The reaction was carried out in a reaction volume of 1ml at pH 4.5 and 37 ℃. Kinetics were carried out for 1, 2, 3 and 4 hours. The concentrations of the different compounds are in mg/ml.

Reaction kinetics as a function of enzyme concentration

The kinetics of decaffeinated green coffee extracts (200mg/ml) were studied using different concentrations of Lactobacillus johnsonii esterase (5. mu.l/ml, 10. mu.l/ml, 15. mu.l/ml, 25. mu.l/ml and 50. mu.l/ml, corresponding to 1.65U/ml, 3.3U/ml, 4.95U/ml, 8.25U/ml and 16.5U/ml, respectively). The reaction was carried out in a volume of 1ml at pH 4.5 and 37 deg.C (see FIG. 5). The reaction time was 4 hours. The concentrations of the different compounds are in mg/ml.

Pilot plant test for production

Decaffeinated green coffee extract treated with Lactobacillus johnsonii esterase

Decaffeinated green coffee extract (1.76Kg) was dissolved in water (8.8Kg) with stirring. The pH was then adjusted to pH 4.5 by the addition of hydrochloric acid (HCl, 0.36 Kg). To this solution was added 0.024Kg of enzyme (esterase from lactobacillus johnsonii) in two portions: 0.016Kg of enzyme was added at time T equal to 0h, and 0.008Kg of enzyme was added after 3h of reaction. The reaction was carried out at 37 ℃ for 6 hours. The mixture was then heated at 98 ℃ for 10 minutes to inactivate the enzyme. After centrifugation (2 min at 5000g) and filtration (0.45 μm), the mixture was freeze-dried and the resulting powder was used to prepare beverages for clinical studies.

UPLC analysis

The method for analyzing samples allows for quantitative determination of caffeic acid, chlorogenic acid isomers (5-CQA, 4-CQA, 3-CQA, 4-FQA, 5-FQA, 3, 4-diCQA, 3, 5-diCQA and 4, 5-diCQA) and caffeine in liquid coffee extracts and pure soluble coffee extracted from roast or green beans. The sample was then centrifuged (5000g) at 15 ℃ for 5 minutes. 100 μ L of the resulting supernatant was added to 900 μ L of methanol/water (80: 20) and filtered at 0.2 μm before analysis. The analysis was performed on a UPLC equipped with a pump, a degassing system, an injector with a sample loop of more than 5 μ Ι _ sample, photodiode array detectors (wavelength 325nm and 275nm) and appropriate data software. The separation of the molecules was carried out on an ACQUITY UPLC BEH Shield RP 18, 1.7 μm, 2.1X 100mm column (from Waters). Mobile phase a was 5% acetonitrile in water with 0.1% phosphoric acid and mobile phase B was 100% acetonitrile with 0.1% phosphoric acid. The flow rate was 0.4mL/min, the column temperature was 35 ℃ and the injection volume was 2. mu.L.

Clinical trial

Main object of

The primary objective of the experiment was to study the efficacy of improving endothelial function in healthy volunteers by oral administration of phenolic acid enriched hydrolyzed decaffeinated green coffee. Endothelial function was defined as the percentage change in internal diameter of the brachial artery from baseline during reactive hyperemia (% FMD).

Primary endpoint

The primary result is the mean change in% FMD from baseline (i.e., before dosing) at any time point after treatment. Response was calculated as the percent change in brachial artery diameter from baseline

Experimental design of HDGCE

This was a placebo-controlled, double-blind, randomized, single-center crossover trial of hydrolyzed green coffee extract in 20 healthy individuals. Individuals were randomly assigned to each group sequence. The trial was performed in one center and involved a 4 day (non-continuous) treatment period.

Statistical analysis

The primary analysis of the primary endpoint was the difference in the change in FMD from baseline mean at the peak between different doses of phenolic acid and control at each time point after baseline. A difference of 1% was considered clinically relevant and a p-value of less than 0.05 was considered statistically significant.

Description of the composition

Hydrolyzed green coffee extract containing phenolic acids and approximately 2% caffeine was used for the test. In this study, only one dose of phenolic acid-enriched coffee extract was administered to the individual per day. The decaffeinated green coffee extract, maltodextrin and flavors were wet blended and then freeze dried to ensure homogeneity of the final premix.

Placebo contained only maltodextrin and fragrance. The amounts of maltodextrin and flavour were similar between groups 4 to avoid any potential effect of these ingredients on the study results.

The following table describes the composition of the four investigated products after freeze-drying (considering 2% residual water after drying).

Table 1: hydrolyzed green coffee extract (HDGCE) composition

Immediately prior to administration, each dose of the study product or matching placebo was dissolved in 200mL of mineral water in opaque glass at room temperature, capped. In the morning, study products were administered under fasting conditions.

Treatment of an individual

23 individuals were screened, 3 of which were failed screening violating inclusion and/or exclusion criteria. All remaining 20 subjects participating in the study continued for all 4 visits.

Total analysis data set (called FAS or ITT for maintenance therapy)

All randomized individuals were included in the full analysis. The data set had 20 individuals in total. Of which only 42 individuals had data available for preliminary analysis.

Table 1: analyzing populations

Group of people	Total number of individuals	Individuals with complete baseline FMD data
			FAS/ITT	20	19
PP	14	14

Table 3: summary of statistical changes from baseline FMD

Results on endothelial dysfunction

Table 4: estimated difference and significance of dose 40 relative to placebo

Table 5: estimated difference and significance of dose 20 relative to placebo

Table 6: estimated difference and significance of dose 10 relative to placebo

Table 7: FMD iAUC summary statistics of HDGCE

P values relate to the comparison of iAUC for different doses to that of placebo. No dose was found to be statistically different from placebo and this was probably due to the very high variability observed.

We found that HDGCE doses of 173.1mg showed significant increases in FMD at 1 hour (p-value 0.036) and 6 hours (p-value 0.017608) compared to placebo treatment. The increase in FMD at these time points was greater than 1% units of FMD. Improvement in endothelial dysfunction (1% flow-mediated dilation value) was associated with a 13% lower risk of cardiovascular events (Inaba et al, (2010) Int J Cardiovas Imaging 26: 621-.

Conclusion

Statistical significance was observed between HDGCE 173.1mg (dose 40) and placebo at time points of 1 and 6 hours.