KR101461779B1 - Biomarker composition for predicting responder group to Korean black raspberry with DNA damage protecting activity in smokers - Google Patents

Abstract

The present invention relates to a biomarker composition for identifying a reaction group responsive to a bacterium having a DNA damage-inhibiting activity in a smoker, which comprises histamine, formate, lysine, tyrosine, A smoker containing one or more metabolites selected from the group consisting of phenylalanine, acetate, ornithine, glycine, and trimethylamine N-oxide (TMAO) A bacterium reaction group discriminating kit comprising a biomarker composition for identifying a bacterium responsive group and an antibody specifically binding to the metabolite. Also, the present invention provides a method for identifying a bacterium response group for a smoker, comprising the step of measuring the expression level of the metabolite from a test sample and comparing the expression level with a normal control sample.

Description

TECHNICAL FIELD [0001] The present invention relates to a biomarker composition for predicting response of a bacterium having a DNA damaging activity in a smoker,

The present invention relates to a biomarker composition for identifying a reaction group responsive to a bacterium having a DNA damage-inhibiting activity in a smoker.

Korean black raspberry (BR) is a fruit of a small strawberry tree ( Rubus coreanus Miquel) belonging to Rosaceae. Rubus was used as a folk remedy and functional food, and is well known to have antioxidant, anti-inflammatory, antibiotic, antiviral and immunomodulating activity. The therapeutic effect of the bokbunja has been investigated in various physiologically active phytochemicals including flavonoids, anthocyanins, tannins, triterpenoids, phenolic acids and organic acids phytochemical components. Due to the physiological activity of the bokbunja, the bokbunja are generally consumed as fresh fruits and also as fermented mainstream forms such as bokbunja wines. In addition, a variety of berry products such as fruit juice, tea, jam and dietary supplements are being developed in recent years.

Tobacco smoke contains a variety of carcinogens, including aromatic amines and polycyclic aromatic hydrocarbons, as well as high concentrations of oxidizing agents, which can cause oxidative damage as one of the many negative effects of smoking. Many studies have reported that supplementing antioxidants and foods such as almonds, grape juices, vitamins C and E can help prevent smoking-related diseases.

8-Hydroxy-2'-deoxyguanosine (8-OHdG) is known as a biomarker of oxidative DNA damage caused by free radicals. 8-OHdG levels in DNA increase in various disease conditions, including cancer, Alzheimer's and Lou Gehrig's. However, little is known about the level of urinary 8-OHdG in smokers and the effect of bramble ingestion on biological-system response.

Korean Patent No. 10-0585486 (registered May 26, 2006) discloses an antioxidative composition comprising a bokbunja extract or a compound derived therefrom, which is harmless to the human body and can replace conventional synthetic antioxidants. The present invention also provides an antioxidative composition comprising a methanol extract of brambles or an extract of brambled ethyl acetate as an active ingredient, but it is also possible to identify a reaction group responsive to brambles having a DNA damaging activity in a smoker of the present invention There is no mention of the biomarker composition for this purpose.

In the present invention, whether or not the 8-OHdG level of smokers is affected by the antioxidant activity of the bokbunja and whether there are individual differences are examined. It was also an object of the present invention to examine the effect of 4-week-old ruminant intake on urine 8-OHdG concentration and plasma metabolite profile of smokers. Bokbunja-ingested group was later divided into two groups based on urine 8-OHdG concentration level (responding group: more reduced individuals than placebo group, unreactive group: less decreased individuals than placebo group). Thereafter, in order to identify potential biomarkers that predict the efficacy of the brambles on urine 8-OHdG concentration, two groups (reacted and unreacted) samples before sample ingestion were analyzed by ¹ H NMR for metabolites Profiling was performed and the present invention was completed.

It is an object of the present invention to provide a pharmaceutical composition containing histamine, formate, lysine, tyrosine, phenylalanine, acetate, ornithine, glycine and trimethylamine N The present invention provides a biomarker composition for identifying a bacterium response group for a smoker comprising one or more metabolites selected from the group consisting of trimethylamine N-oxide (TMAO).

Another object of the present invention is to provide a bacterium reaction group discrimination kit comprising an antibody that specifically binds to the metabolite.

It is still another object of the present invention to provide a method for discriminating a bacterium response group to a smoker, comprising the step of measuring the expression level of the metabolite from a test sample and comparing with the normal control sample.

In order to achieve the above object, the present invention provides a pharmaceutical composition comprising histamine, formate, lysine, tyrosine, phenylalanine, acetate, ornithine, glycine, ) And a trimethylamine N-oxide (TMAO). The present invention also provides a biomarker composition for identifying a bacterium-responsive group for a smoker comprising one or two or more metabolites selected from the group consisting of trimethylamine N-oxide (TMAO) Specifically, the metabolite is glycine or trimethylamine N-oxide (TMAO).

More specifically, in the bacterium-responsive group, the decrease rate of the expression of 8-hydroxy-2'-deoxyguanosine (8-OHdG), which is an oxidative DNA damage marker, (placebo) control group.

The present invention also provides a bacterium response group discrimination kit for a smoker comprising an antibody specifically binding to the metabolite.

The determination kit of the present invention is produced by a conventional manufacturing method known to a person skilled in the art, and typically includes an antibody and a buffer, a stabilizer, an inactive protein and the like in lyophilized form. The antibody may be labeled with radionuclides, fluorescence, enzymes, and the like.

The present invention also relates to a method for detecting a metabolic syndrome comprising: measuring the metabolite expression level of the first or second aspect from a specimen sample; And comparing the expression level of the metabolite with a control sample to determine whether the bacterium is responsive to the bacterium. On the other hand, the sample is characterized by being blood or urine, but is not limited thereto.

Specifically, the method for measuring the level of metabolite expression is characterized by directly detecting the presence of the metabolite using NMR or HPLC, or using an antibody specific for the metabolite.

Immunoassay, which is widely known as an antigen-antibody reaction, is an enzyme-linked immunosorbent assay (ELISA, Coated Tube), an antibody-bound magnetic particle conjugated to a tube, followed by an antigen- Magnetic particle method which quantitatively quantifies enzyme reaction by reacting contaminants with each other, and latex particle method using antibody-bonded latex particles.

The present invention relates to a biomarker composition for identifying a reaction group responsive to a bacterium having a DNA damage-inhibiting activity in a smoker, and more particularly, to a biomarker composition for determining whether or not an 8-OHdG level of a smoker is affected by an antioxidant activity of a bacterium To determine whether these effects were individual differences, the effect of 4-week-old rats on urinary 8-OHdG concentration and plasma metabolite profiles was examined. As a result, potential biomarkers that could predict the efficacy of rubella on urine 8-OHdG concentrations could be found.

Fig. 1 shows a plasma sample ¹ H NMR spectrum (600 MHz) of a group of brambles in smokers. 1: isoleucine, 2: ethylmalonate, 3: valine, 4: lactate, 5: alanine, 6: lysine, 7: ornithine ornithine, 8: acetate, 9: proline, 10: glutamine, 11: pyruvate, 12: pyridoxine, 13: citrate, 14: N , N -dimethylglycine ( N , N -dimethylglycine) 15: creatinine 16: glucose 17: 1,3-dimethylurate 18: TMAO 19: Glycine, 20: guanidoacetate, 21: tyrosine, 22: histamine, 23: phenylalanine, 24: formate.
Figure 2 shows a PCA score plot derived from the ¹ H NMR spectrum of plasma samples in the 0 and 4-bore bacterium response group ( n = 7). The total number of principal components is three. ●: 0-bokbok responding group, △: 4-bokbok responding group
Figure 3 shows the relative mean values of metabolites contributing to the distribution of plasma samples 0 and 4 in the brambles intake group (* p < 0.05). The error bar represents the standard deviation ( n = 7). The following validation values for the compounds were obtained by validation analysis: formate, 0.973; Pyridoxine, 0.821; Tyrosine: 1.000; Acetate, 0.691; Histamine, 1.000; Glycine, 0.726; Ornithine, 0.902; Lysine, 0.820; Guanidoacetate, 0.984; Proline, 0.681; Ethylmalonate, 0.749; Isoleucine, 0.572; Phenylalanine, 1.000; N, N - dimethylglycine (N, N -dimethylglycine), 0.819 ; TMAO, 0.794; And valine, 0.620. a: 0-bokbok responders, b: 4-bokbok responders
Figure 4 shows PCA-derived score plots of plasma samples for the brambler response group and the brambler non-responsive group in the brambles intake group. The total number of principal components is three. ●: 0-bokbuk responding group, △: 0 bokbukjun non-responding group
Figure 5 shows the relative mean values of the metabolites contributing to the distribution of zero-bred and non-responsive plasma samples (* p < 0.05). The error bar represents the standard deviation ( n = 7). The following validation values for the compounds were obtained by validation analysis: glycine, 0.946; Histamine, 0.717; Formate, 0.671; Lysine, 0.743; Acetate, 0.668; Ornithine, 0.811; Phenylalanine, 0.708; Tyrosine, 0.569; And TMAO, 0.833.
a: 0, bokbunja response group, b: 0, bokbunja non-responsiveness group

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the present invention is not limited by these examples.

< Example  1> urine 8- OHdG  Measure level

1. Sample preparation of bokbunja

Bokbunja fruit was collected at harvest time from the farms in Goseong province, South Korea. Dr. Cho, Soo - Mook of the Rural Development Administration (Suwon, Korea), an expert on the collection and identification of natural products, collected and identified bokbunja samples. The surface of the fruit was immediately washed with distilled water, the sample was lyophilized and stored at -70 ° C. The test group received 30 g of lyophilized brambles daily for 4 weeks. Ingredients of the brambles intake group (BR) and the placebo were shown in Table 1. The color values of the berries of the fruit of the present invention were measured by a spectrophotometer (CM-3500d, Minolta, Osaka, Japan) equipped with a pulsed xenon arc lamp and the measured L ^* , a ^* (green degree and red degree) and b ^* (blue degree and yellow degree) were 39.95 ± 1.08, 24.71 ± 1.06 and 10.47 ± 0.82, respectively. The color value of berries fruit berries was used as a criterion for selecting samples. A control specimen (Voucher specimen) was placed at the College of Pharmacy, Chung-Ang University, Korea (CAUR 20090881P).

Free radical scavenging activity and total phenol content were measured by the methods reported previously (Process Biochemistry, 47 (1), 62-68). In the present invention, the free radical scavenging activity of the macromolecular sample is 29.1%, which is comparable to 29.6% as measured at 10 mg / L ascorbic acid. Meanwhile, the total phenol content was 74.02 mg gallic acid equivalent / g dry extract.

ingredient BR Placebo The freeze-dried brambles (g) 30 - Water (mL) 200 200 Carboxymethylcellulose (g) 0.5 1.5 Methyl-p-hydroxybenzoate (mg) 10 10 Sucrose (g) - 20 Starch (g) - 4 Red No. 40 (mg) - 10 Blue No. 1 (mg) - 0.7

2. Experimental group

38 healthy smokers, all boys aged between 20 and 30 years of age, were recruited from Chung-Ang Univ. And divided into two groups, bokbunjang (BR) or placebo. The researchers found that they smoked more than 10 cigarettes a day during the last four years. As shown in Table 2, the concentration of nicotine metabolites in the serum was measured to see if the tester continuously smoked for 4 weeks. Liver function tests measured ALP, AST and ALT levels in the plasma of each investigator and their values were in the normal range. Also, the tester was not taking any medication, limited to one cup of coffee or tea a day, and once a day of fruit or juice. The investigator did not take any functional food such as ginseng or vitamin supplements. All 38 participants agreed to the test. The protocol was reviewed and approved by the Institutional Review Board of the Central University (IACF 1-50).

Basic characteristics Placebo ( n = 19) BR ( n = 19) week 0 week 4 week 0 week 4 Age (yrs) 24.8 ± 2.9 24.5 ± 2.6 NM (ng / mL) 208.6 ± 113.1 217.8 ± 107.3 213.6 ± 157.9 206.8 ± 142.8 ALP (U / L) 62.4 ± 12.2 61.7 ± 12.0 63.9 ± 12.5 64.6 ± 12.5 AST (U / L) 22.6 ± 4.0 19.5 ± 4.9 20.4 ± 3.6 20.9 ± 7.9 ALT (U / L) 21.2 ± 9.8 18.8 ± 9.7 18.4 ± 7.2 16.8 ± 7.5 BMI (kg / m ² ) 23.0 ± 1.2 22.3 ± 1.3 22.5 ± 1.2 22.9 ± 1.6 pack-year (yrs) ^a 5.3 ± 2.1 5.6 ± 3.6

^a Pack-years = (cigarettes smoked / day x years smoked) / 20

NM, Nicotine metabolite; ALP, Alkaline phosphatase; AST, aspartate aminotransferase; ALT, alanine aminotransferase; And BMI, body-mass index,

3. Plasma and urine sample preparation, 8- OHdG  analysis

Plasma and urine samples were taken before and after the brambles or placebo and after 4 weeks of ingestion from all subjects. Blood was rapidly drawn from the tester after 12 hours, and the plasma was collected in a collection tube by centrifugation at 3,000 rpm for 15 minutes at 4 ° C in the blood. Urine samples were transferred to a conical tube (15 mL) containing 0.2% sodium azide and stored at -70 ° C until measurement.

Urinary 8-OHdG levels were measured using an 8-OHdG EIA kit (Cayman Chemical, MI, USA). Several controls such as sodium azide control, nonspecific binding control and blank control were used to minimize the background. The 8-OHdG EIA standard (300 ng / mL) in the kit was diluted from 10.3 pg / mL to 30 ng / mL to draw the standard curve. Briefly, acetylcholinesterase, 8-OHdG specific antibody and urine samples bound to 8-OHdG were added to the wells of a 96-well plate previously coated with mouse monoclonal antibody and incubated at 4 ° C for 18 hours Lt; / RTI &gt; The plate was washed to remove any unbound reagent, and then Ellman's reagent was used for the enzyme reaction. After 90 minutes of gentle shaking, the plates were measured at 420 nm and the 8-OHdG values of each sample were calculated according to the standard curve (R ² = 0.998).

Chemiluminescent immunoassay (CLIA) was performed using Immulite 2000 Nicotine Metabolite kit (Siemens, CA, USA) and Immulite 2000 immunoassay system (Siemens, CA, USA) for the analysis of plasma nicotine metabolism. It is a solid phase competitive chemiluminescent immunoassay with a calibration range of 10-500 ng / ml and an analytical sensitivity of 5 ng / ml.

4. Results

Table 3 shows the mean values of 8-OHdG before (0th week) and after (4th week) intake in the brambles and placebo groups, and the data of all the tester in both groups are shown in Table 4. Both groups showed a decrease in 8-OHdG levels after 4 weeks of ingestion. There was no significant difference in the BR group (25%) among the placebo group (12%) but no significant difference. In the present invention, the mean 8-OHdG concentration was also reduced by 12% in the placebo-fed group, which is presumed to be due to the placebo effect which is expected in the placebo-fed group by the tester of the raspberry. It has been reported that a placebo effect is exhibited not only in medicine but also in functional food when blind test and random extraction test are given to the tester.

Considering the placebo effect, the brambled diet group was divided into two groups: the brambles response group (BR-R group), in which the urine 8-OHdG level decreased more than 12% Non-responsive group (BR-NR group). The placebo-fed group was also divided into two groups: placebo-R group and placebo-NR group. The rats selected as bokbunja ( n = 12) and placebo ( n = 10) were shown in Table 4. Results: Twelve of the 19 brambles consumed brambles (63.2%) with a reduction of 8-OHdG of more than 12%, with 10 of the 19 placebo recipients receiving less than 12% of the 8-OHdG And 10.6% higher than the placebo group (52.6%).

As shown in Table 3, the reduction rate of urinary 8-OHdG levels in the bacterium-treated group was significantly higher than that of the total placebo group ( p <0.05). This indicates that the brambles have a beneficial effect in reducing the 8-OHdG level in the brambles response group compared to the mean reduction rate of urinary 8-OHdG levels in the total placebo-fed group.

The brambles are anthocyanins such as cyanidin 3-glucoside, cyanidin 3-rutinoside and cyanidin 3-sambubioside, It contains many phenolic compounds such as cinnamic acid, ferulic acid, protocatechuic acid and epicatechin. Cyanidin and phenolic compounds contained in the bokbunja may cause antioxidative effects in smokers.

BR (n = 19) Placebo (n = 19) NR (n = 7) R (n = 12) week 0
week 4
week 0 week 4 week 0 week 4 8-OHdG
(pg / mL) 1341.0
± 592.4 1961.1
± 481.6 3001.2
± 1373.0 1721.8
± 593.5 2068.1
± 862.0 1819.2
± 593.7 Decreamnet
(%) -75.8 36.6 ^a 12.0 ^b 24.8

Small letters showed statistical significance ( p <0.05).

R: Responder group, NR: Nonresponder group,

8-OHdG level (pg / mL)
No BR No Placebo week 0 week 4 week 0 week 4















1 ^a 4138.6 1585.3 One 1798.0 2442.9 2 ^a 3038.9 2306.2 2 1112.3 2073.1 3 ^a 2467.2 2021.8 3 2108.7 2567.3 4 1867.1 1697.2 4 1685.3 1662.1 5 ^a 1825.1 1574.9 5 ^b 2183.6 1896.2 6 ^a 4007.4 1399.2 6 ^b 3825.2 1926.2 7 ^a 3076.2 1595.9 7 ^b 2395.6 2090.8 8 652.9 1186.6 8 ^b 2769.4 1617.4 9 ^a 1505.5 1237.1 9 ^b 1988.9 1628.3 10 ^a 1896.2 1382.8 10 311.4 1168.6 11 1721.4 2651.5 11 1784.8 1957.1 12 910.7 1941.5 12 ^b 2467.2 1544.5 13 ^a 2800.5 2350.1 13 1065.7 1662.1 14 617.6 2306.2 14 ^b 3002.5 1270.8 15 ^a 2090.8 1798.0 15 1896.2 2090.8 16 1988.9 2223.1 16 ^b 1585.3 1134.2 17 ^a 6624.8 3076.2 17 ^b 3768.0 3002.5 18 ^a 3505.8 819.2 18 1825.1 2419.0 19 1628.3 1721.4 19 ^b 1721.4 408.5 Mean 2440.2
± 1435.9 1835.5
± 554.7 2068.1
± 862.0 1819.2
± 593.7 Decrement (%) 24.8 12.0

^a refers to the tester responding to ruminant intake (R group).

^b represents a tester in which urine 8-OHdG levels are reduced by more than 12% (placebo R group).

< Example  2> Plasma Metabolite NMR  Spectrum analysis

1. Plasma NMR  Measure

A frozen plasma sample (-70 ° C) was dissolved in a water bath at 40 ° C, 500ul of each plasma sample was transferred to a 10kDa filter, and centrifuged at 14,000xg for 15 minutes at 4 ° C. D ₂ O internal standard 0.05% 3- (trimethylsilyl) -propionic- 2,2,3,3- d 4 acid sodium salt buffer, a KH ₂ PO ₄ (1.232 g) in 100ml of D ₂ O containing the (TSP) Lt; / RTI &gt; The pH of the D ₂ O used for NMR measurements was adjusted to 6.0 with 520 ul of 1 N NaOD. NaCl (0.9 g) was dissolved in pH adjusted 10 mL D ₂ O containing KH ₂ PO ₄ and filled to 100 mL with water. 300 uL of 0.9% physiological saline was added to the 300 uL plasma supernatant and vortexed for a few seconds to mix thoroughly. The mixture was pipetted into a 5 mm NMR tube (Norell, Landisville, NJ). Samples were analyzed with an NMR spectrophotometer (Avance 600 FT-NMR, Bruker, Germany) at a temperature of 298 K, 600.13 MHz. For each plasma sample, ¹ H NMR and heteronuclear single quantum correlation (HSQC) analyzes were performed. ¹ H NMR spectra B ruker CPMG pulse sequence (Carr- Purcell-Meiboom-Gill T2 filter, cpmgpr1d; RD-90 ° - {τ-180 ° -τ} n-acquire) I obtained using, where τ is the spin Echo delay (τ = 100 μs, overall CPMG length = 40 ms). The CPMG pulse sequence produces a spectrum edited by the T2 relaxation time, which reduces broad-range resonance from high molecular weight compounds, which facilitates the observation of low molecular weight metabolites. A total of 128 transients with a spectral width of 10204.082 were collected at 64K data points. The acquisition time per scan of 3.21 seconds was used as a relaxation delay of 2 seconds. An exponential line broadening function of 0.30 Hz was applied to free induction decays before Fourier transform. The 2D ¹ H- ¹³ C HSQC spectrum was obtained with a 1.5 second relaxation delay, 8 scans and 8,012 Hz at F ₂ , and a spectrum width of 24,997 Hz at F ₁ .

Two-dimensional J- resolved ¹ H NMR spectra were recorded on a 600 MHz Varian spectrometer (VnmrJ 2.2, CA) at a temperature of 298 K. The spectrum was obtained using the following pulse sequence: [relaxation delay-90 ° -t ₁ -180 ° -t ₁ -acquire for time t ₂ ] where the relaxation delay was 1.4 seconds and t ₁ was incremented by an increasing delay. The data I obtained from the F ₂ using a, per incremental 16 FIDs collected from 4096 data points, which F ₂ at 6,009 Hz (chemincal shift axis), and F 30 Hz from the _first spectral width of the (spin-spin coupling constant axis) Were used. The data set was zero-filled with 384 data points and the spectrum was tilted 45 ° to provide orthogonality and then symmetric about the F ₁ axis. In subsequent scale calculations, the spectrum was represented by contour plots and skyline projections.

2. Statistical analysis

MestReNova (version 6.0.4, Mestrelab Research, Santiago de Compostela, Spain) was used to obtain the NMR spectrum, which was automatically encrypted in an ASCII file using AMIX (version 3.7, BrukerBioSpin, Billerica, . The ¹ H NMR portion of the spectrum from δ = 0.54 to δ = 10.00 was fragmented as a fraction with a width of 0.04 ppm, representing 230 integration sites in each NMR spectrum. 4.70-5.00 ppm was excluded to eliminate water resonance. ¹ H NMR signal analysis was performed by comparing chemical shifts and cleavage patterns in the Chenomx NMR suite software (version 5.1, Chenomx, Edmonton, Canada). Each spectral intensity data set is normalized to the total sum of the spectral components. The resulting data set was then converted to Microsoft Office Excel (version 2007, Microsoft, Redmond, WA, USA) format and multivariate analysis was performed on SIMCA-P + software (version 12.0, Umetrics, Umea Sweden) . All data were mean centered by scaling by unit deviation with preprocessing method and principal component analysis (PCA) was performed using SIMCA-P + software. The PCA model property measurements were expressed by the R ² X and Q ² parameters: R ² X represents the goodness of fit and Q ² represents the predictive verification power of the model. Significant differences in metabolite levels were detected by independent t-tests and subsequent corrections through Levene's test for the same variance using SPSS software (version 18, SPSS Inc., Chicago, IL, USA). Statistical significance was defined as p <0.05. Verification of the t-test was performed using Power and Precision software (version 4.1.0; Biostat, Englewood, NJ).

3. Results

FIG. 1 shows the positions of the respective peaks in a representative ¹ H NMR spectrum of plasma samples taken from the brambles. The chemical changes of standard compounds were compared and analyzed using Chenomx NMR Suite software. As shown in Table 5, the ¹ H NMR spectrum contains 24 designated metabolites: isoleucine, ethylmalonate, valine, lactate, alanine, lysine (lysine), ornithine (ornithine), acetate (acetate), proline (proline), glutamine (glutamine), pyruvate (pyruvate), pyridoxine (pyridoxine), a citrate (citrate), N, N -dimethylglycine (N , N - dimethylglycine), creatinine (creatinine), glucose (glucose), 1,3- dimethyl Wu rate (1,3-dimethylurate), trimethylamine N - oxide (trimethylamine N -oxide; TMAO), glycine (glycine) Guanidoacetate, tyrosine, histamine, phenylalanine, formate, and the like.

In order to identify the peaks identified in the 1D ¹ H-NMR spectrum, ¹ H- ¹ H 2D J -resolved and ¹ H- ¹³ C HSQC NMR analyzes were performed.

Compound Chemical shift, ppm (peak multiplicity, J value) Isoleucine 0.95 (t, J = 6.51), 1.03 (d, J = 6.90) Ethylmalonate 0.95 (t, J = 6.51), 1.68-1.74 (m) Valine 0.98 (d, J = 6.87), 1.03 (d, J = 6.90), 2.23-2.30 (m) Lactate 1.32 (d, J = 6.91), 4.11 (dd, J ₁ = 6.71, J ₂ = 13.06) Alanine 1.47 (d, J = 8.31) Lysine 1.68-1.74 (m), 1.85-1.93 (m) Ornithine 1.74-1.87 (m) Acetate 1.91 (s) Proline 1.95-2.09 (m), 2.30-2.36 (m) Glutamine 2.09-2.16 (m), 2.41-2.49 (m) Pyruvate 2.36 (s) Pyridoxine 2.42 (s), 7.42 (s) Citrate 2.52 (d, J = 15.92), 2.67 (d, J = 15.59) N , N- dimethylglycine 2.91 (s) Creatinine 3.03 (s), 4.05 (s) Glucose J = 8.90), 3.37-3.54 (m), 3.80-3.92 (m), 4.64 (d, J = 8.00), 5.22 (d, J = 4.07) 1,3-Dimethylurate 3.25 (s), 3.44 (s) TMAO 3.48 (s) Glycine 3.52 (s) Guanidoacetate 3.74 (s) Tyrosine 6.88 (d, J = 8.45), 7.18 (d, J = 8.41) Histamine 7.06 (s) Phenylalanine 7.32 (d, J = 7.80), 7.37 (t, J = 7.23), 7.42 (t, J = 7.63) Formate 8.44 (s)

PCA was performed using mean centered and Pareto-scaled NMR spectral data to identify outliers and confirm separation without priori classification. Outlier values are located outside the ellipse with 95% confidence in the PCA score plot. When PCA was applied to the spectral data of the bokbunja group ( n = 19), six outliers were excluded (five in the bokbunja reaction group and one in the bokbunja unresponsive group). As shown in FIG. 2, PCA was applied to the spectral data of the reaction group ( n = 7) in which the urine 8-OHdG level was decreased by 12% or more in order to investigate the effect of the brambles intake on the metabolic profile of the reaction group. In the PCA-derived score plot, the metabolite profile of the response group that ingested the 0 and 4-bolus rats was clearly distinguished ( R ² : 0.776, Q ² : 0.551).

As shown in Figure 3, 16 metabolites showed significant differences in the relevant values ( p < 0.05). In the group of bokbunja ingestion, isoleucine, pyridoxine, histamine, proline, lysine, tyrosine, phenylalanine, acetate, formate ), N , N -dimethylglycine ( N , N -dimethylglycine), ethylmalonate, valine, ornithine and TMAO, while guanidoacetate and Glycine was higher. In addition, the present inventors carried out a verification test for the independent t-test of the above 16 metabolites. Originally, the verification power analysis is used to determine the effective size of the test sample ( N ), with the appropriate verification power values. Generally, a verification power value of 0.8 or more indicates that the effective size is large. In order to calculate the effective number of samples, the analysis should be performed before proceeding with the present test, but the present inventors used a verification power analysis to statistically verify the results of this experiment from a small number of samples. Among these compounds, the relative values of histamine, guanidoacetate, glycine, formate and TMAO in smokers plasma were 1.000, 0.984, 0.726, 0.973 and 0.794, respectively It has changed greatly.

Histamine is known to stimulate the inflammatory response and regulate the immune response. Therefore, the decrease of histamine level by the consumption of bokbun is considered to be the anti - inflammatory effect of bokbunja in smokers. Also, in the present invention, guanidoacetate and glycine levels were increased in the plasma of the smokers of the smokers, indicating that the consumption of bokbun may improve the symptoms of heart disease of the smoker. On the other hand, the present inventors have found that formate and TMAO levels are reduced after 4 weeks of bramble ingestion. This suggests that consumption of brambles has a positive effect on the health of smokers in the light of previous reports.

In order to investigate the metabolic distinction between the bokbunja responding group and the bokbunja non-responding group at the 0th week, and to investigate the biomarker for predicting the effect of the bokbunja intake, Independent t-tests were performed. As shown in FIG. 4, the bractomic and non-bacterium non-responsive groups were clearly separated in the PCA-derived score plate ( R ² : 0.769, Q ² : 0.455). As shown in FIG. 5, in the present invention, the relative values of the nine metabolites were significantly different ( p <0.05): histamine, formate, lysine, tyrosine tyrosine, phenylalanine, acetate and ornithine were higher than those in the control group, while glycine and TMAO were higher in the response group.

In addition, the present inventors performed a verification test for the independent t-test of the above nine metabolites. Independent t-tests with a verified power score of> 0.8 are generally considered significant. The relative values of glycine and TMAO in smokers plasma were significantly different at the verifications of 0.946 and 0.833, respectively. Thus, the two compounds show potential as a potential biomarker for screening testers to determine the modulatory activity of the macromolecules on urine 8-OHdG numbers, which may be used as biomarkers for oxidative DNA damage by the free radicals of smokers .

The inventors have discovered that the methodology of the present invention may be applied to assess the bioactivity of the brambles as a natural or functional food in humans and to develop potential biomarkers for functionalized foods personalized for various symptoms.

Claims (7)

The compounds of formula (I) may be selected from the group consisting of histamine, formate, lysine, tyrosine, phenylalanine, acetate, ornithine, glycine and trimethylamine N- -oxidase &lt; / RTI &gt; (TMAO)). &lt; / RTI &gt; The biomarker composition according to claim 1, wherein the metabolite is glycine or trimethylamine N-oxide (TMAO). 2. The biomarker composition according to claim 1, wherein the metabolite is glycine or trimethylamine N-oxide (TMAO). The bacterium-responsive group of claim 1 or 2, wherein 8-hydroxy-2'-deoxyguanosine (8-OHdG), an oxidative DNA damage marker, Wherein the expression reduction rate of the bacterium is higher than that of the placebo control. delete Measuring the level of the metabolite of claim 1 or 2 from a sample of the sample; And
And comparing the expression level of the metabolite with a control sample to determine whether the bacterium is responsive to the bacterium. [6] The method of claim 5, wherein the metabolite expression level is measured by directly detecting the presence of the metabolite using NMR or HPLC. 6. The method of claim 5, wherein the specimen is blood or urine.

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