A bis-ester type catechin compound and a preparation method thereof

Abstract

The application discloses a kind of double ester type catechins compound and preparation method, steps are as follows: tea is mixed with methanol aqueous solution, and tea extract is obtained by ultrasonic extraction;Tea extract is continuously carried out reverse phase liquid chromatography separation, and finally double ester type catechins compound is obtained by collecting specific eluent and the distillate of specific time period, which is obtained epigallocatechin-3,4'-di- O -gallate and gallocatechin-3,4'-di- O -gallate is first purified by separation, and double ester type catechins compound has high purity.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of compound extraction and preparation, and particularly relates to a diester-type catechin compound and a preparation method. BACKGROUND

[0002] Catechin is a kind of polyphenol compound widely existing in plants, and has a 2-phenyl benzopyran skeleton structure. Catechin is a kind of substance most abundant in tea leaves, and the content of catechin accounts for 12-14% of the dry weight of tea leaves.

[0003] In the process of plant secondary metabolism, catechin substances can form various catechin monomers, such as catechin (C), epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG), and epigallocatechin gallate (EGCG). These catechin compounds also exhibit various biological activities, including antioxidant, antitumor, anti-inflammatory, antibacterial, and antiviral activities. Hydroxylation and esterification are reactions easily occurring on the skeleton of catechin substances. In addition to the changes in the number of reactions and the reaction sites to produce isomers, catechin can also form polymers with different polymerization degrees, such as dimers and trimers. Therefore, catechin substances in tea leaves have various forms, but the structures of these compounds also have unstable characteristics. Under the action of external heating, catechin can be oxidized and degraded or isomerized to convert into corresponding stereoisomers.

[0004] Due to the similar structures of catechin compounds and the complex stereochemical properties, the thermal instability of diester-type catechin compounds is more prominent, which increases the difficulty of material basis research. The catechin compounds obtained by purifying tea leaves have relatively fixed types, and there are relatively few research reports. The extraction and preparation methods are also relatively single. SUMMARY

[0005] In view of the problems in the prior art, the application is designed to provide a diester-type catechin compound and a preparation method. The technical scheme is specifically as follows:

[0006] 1) mixing tea leaves with a methanol aqueous solution, ultrasonic extraction, and filtering to obtain a tea leaf extract;

[0007] 2) under room temperature, the tea extract is separated by reverse phase liquid chromatography through a packed column filled with packing material, and the fraction collected in 15-20 min is obtained as a first fraction, wherein 0.05% trifluoroacetic acid aqueous solution is used as mobile phase A, and methanol is used as mobile phase B; the gradient elution mode of the mobile phase is 8%-15% mobile phase B in 0-10 min, 15%-25% mobile phase B in 10-40 min, 25%-100% mobile phase B in 40-45 min, and 100% mobile phase B in 45-60 min; the flow rate is 80 mL / min, the injection volume is 125 mL, and the packing material is octadecyl bonded stationary phase;

[0008] The first fraction is pre-frozen in a refrigerator for 3-5 h, transferred to a vacuum freeze dryer, and subjected to freeze-drying under reduced pressure to remove the solvent, thereby obtaining a sample crude component;

[0009] 3) under room temperature, the sample crude component is separated by reverse phase liquid chromatography through a packed column filled with packing material, and the fraction collected in 19-21.5 min is obtained as a second fraction, wherein 0.05% trifluoroacetic acid aqueous solution is used as mobile phase A, and acetonitrile is used as mobile phase B; the gradient elution mode of the mobile phase is 15%-30% mobile phase B in 0-25 min, 30%-95% mobile phase B in 25-30 min, and 95% mobile phase B in 30-40 min; the flow rate is 80 mL / min, the injection volume is 20 mL, and the packing material is octadecyl bonded stationary phase;

[0010] The second fraction is pre-frozen in a refrigerator for 3-5 h, transferred to a vacuum freeze dryer, and subjected to freeze-drying under reduced pressure to remove the solvent, thereby obtaining a catechin fine component;

[0011] 4) under room temperature, the catechin fine component is separated by reverse phase liquid chromatography through a packed column filled with packing material, and the fractions collected in 27-30 min and 37-39 min are obtained as a third fraction and a fourth fraction, wherein 0.05% trifluoroacetic acid aqueous solution is used as mobile phase A, and methanol is used as mobile phase B; the gradient elution mode of the mobile phase is 25% mobile phase B in 0-20 min, 25%-35% mobile phase B in 20-30 min, and 35% mobile phase B in 30-45 min; the flow rate is 80 mL / min, the injection volume is 80 mL, and the packing material is a mixture of octadecyl bonded stationary phase and isopropanol;

[0012] The third and fourth fractions were pre-frozen in a refrigerator for 3-5 hours, then transferred to a vacuum freeze dryer and freeze-dried under reduced pressure to remove the solvent, yielding diester-type catechin compounds.

[0013] The diester-type catechin compound is epigallocatechin-3,4'-di- O -gallate or gallocatechin-3,4'-di- O -gallate.

[0014] Preferably, the tea is green tea, yellow tea, black tea, dark tea, white tea, or oolong tea.

[0015] Preferably, the concentration of the methanol-water solution used in the ultrasonic extraction of tea is 12%-15%.

[0016] Preferably, the mass-to-volume ratio of the tea leaves and the methanol aqueous solution is 1g:40-60L.

[0017] Preferably, the ultrasonic extraction time is 20-30 minutes. After ultrasonic extraction, the filtrate is collected by vacuum filtration and then filtered through a filter membrane to obtain tea extract.

[0018] Preferably, the pore size of the filter membrane is 0.45μm-0.5μm.

[0019] Preferably, the height of the filling column bed is 260 mm and the diameter is 50 mm.

[0020] Preferably, the temperature inside the refrigerator and the temperature inside the vacuum freeze dryer are both set to -80°C.

[0021] The diester-type catechin compound prepared by the above method has the following structural characteristics:

[0022] .

[0023] This invention utilizes ultrasonic extraction of tea leaves combined with multi-step continuous reversed-phase liquid chromatography separation and purification, and reduced-pressure freeze-drying technology. By employing specific chromatographic and gradient elution conditions, a previously unreported novel compound 1 (epigallocatechin-3,4'-di-) was extracted from tea leaves. O -gallate) and the monomeric compound 3 (gallocatechin-3,4'-di-) obtained for the first time through isolation and purification. O The high purity diester-type catechin compounds obtained by the preparation method (-gallate) can be applied in tea component research and the beverage, food industry, or biopharmaceutical industry, providing a certain material basis for tea quality control and commercial development. Attached Figure Description

[0024] Figure 1 It is a reverse-phase preparative chromatographic crude separation of tea extract;

[0025] Figure 2 This is the reversed-phase liquid-phase preparation spectrum of fraction F1;

[0026] Figure 3 This is the reversed-phase liquid-phase preparation spectrum of fraction F1-1;

[0027] Figure 4 This is the ultra-high performance liquid chromatography-high resolution mass spectrometry primary negative ion mode spectrum of compound 1;

[0028] Figure 5 This is the ultra-high performance liquid chromatography-high resolution mass spectrometry spectrum of compound 1 in secondary negative ion mode;

[0029] Figure 6 It is compound 1 1 H NMR spectrum;

[0030] Figure 7 It is compound 1 13 C NMR spectrum;

[0031] Figure 8 It is compound 1 1 H- 1 H COSY spectrum;

[0032] Figure 9 This is the HSQC spectrum of compound 1;

[0033] Figure 10 This is the HMBC spectrum of compound 1. Detailed Implementation

[0034] The present invention will be further described below with reference to the accompanying drawings to provide a better understanding of the technical solution. Example

[0035] Take 20g of green tea leaves and mix them with 1L of 15% methanol aqueous solution. Extract by ultrasonication for 30min, filter and collect the filtrate. Filter the filtrate through a 0.46μm pore size filter membrane to obtain tea extract.

[0036] At room temperature, the tea extract was separated by reversed-phase liquid chromatography using a packed column filled with packing material. The separation was based on the sample liquid phase chromatogram (e.g., ...). Figure 1The fraction was collected at the retention time shown in the figure. The fraction from 15 min to 20 min was collected to obtain the first fraction, which was labeled as fraction F1. In the reversed-phase liquid chromatography separation, 0.05% trifluoroacetic acid aqueous solution was used as mobile phase A and methanol was used as mobile phase B. The mobile phase gradient elution method was as follows: 0 min-10 min, mobile phase B accounted for 8%-15% of the volume and mobile phase A accounted for 85%-92% of the volume; 10 min-40 min, mobile phase B accounted for 15%-25% of the volume and mobile phase A accounted for 75%-85% of the volume; 40 min-45 min, mobile phase B accounted for 25%-100% of the volume and mobile phase A accounted for 0-75% of the volume; 45 min-60 min, mobile phase B accounted for 100% of the volume; the flow rate was 80 mL / min and the injection volume was 125 mL; the packing material was 320 g of octadecyl bonded stationary phase.

[0037] Fraction F1 was pre-frozen in a refrigerator at -80℃ for 4 hours, then transferred to a vacuum freeze dryer. The freezing temperature was set to -80℃, and the solvent was removed by freeze-drying under reduced pressure to obtain the crude sample components.

[0038] At room temperature, the crude components of the above sample were separated by reversed-phase liquid chromatography using a packed column filled with packing material. The chromatograms of the distillate fractions were prepared (e.g.,...). Figure 2 The fraction was collected based on the chromatographic peak shape shown. The elution peak in the 19-21.5 min range was collected to obtain the second fraction, which was labeled as fraction F1-1. During the reversed-phase liquid chromatography separation, 0.05% trifluoroacetic acid aqueous solution was used as mobile phase A, and acetonitrile was used as mobile phase B. The mobile phase gradient elution was as follows: 0 min-25 min, mobile phase B occupied 15%-30% of the volume, with the remainder being mobile phase A; 25 min-30 min, mobile phase B occupied 30%-95% of the volume, with the remainder being mobile phase A; 30 min-40 min, mobile phase B occupied 95% of the volume, with the remainder being mobile phase A. The flow rate was 80 mL / min, and the injection volume was 20 mL. The packing material was 320 g of octadecyl bonded stationary phase.

[0039] Fraction F1-1 was pre-frozen in a refrigerator at -80°C for 4 hours, then transferred to a vacuum freeze dryer. The freezing temperature was set to -80°C, and the solvent was removed by freeze-drying under reduced pressure to obtain the fine catechin fraction.

[0040] At room temperature, the above-mentioned fine catechin components were separated by reversed-phase liquid chromatography using a packed column packed with packing material. The fractions were analyzed using liquid chromatography chromatograms (e.g.,...). Figure 3Fractions were collected based on the chromatographic peaks shown. Elution peaks were collected at 27-30 min, 32-36 min, and 37-39 min, yielding the third, fifth, and fourth fractions, labeled F1-1-1, F1-1-2, and F1-1-3, respectively. The mobile phase A was 0.05% trifluoroacetic acid aqueous solution, and the mobile phase B was methanol. The gradient elution was as follows: 0-20 min, mobile phase B comprised 25% of the volume, and mobile phase A comprised 75%; 20-30 min, mobile phase B comprised 25%-35% of the volume, with the remainder being mobile phase A; 30-45 min, mobile phase B comprised 35% of the volume, and mobile phase A comprised 65%. The flow rate was 80 mL / min, and the injection volume was 80 mL. The packing material was 320 g of octadecyl bonded stationary phase packing.

[0041] Fractions F1-1-1 and F1-1-3 were pre-frozen in a refrigerator at -80°C for 4 hours, then transferred to a vacuum freeze dryer. The freezing temperature was set to -80°C, and the solvent was removed by freeze-drying under reduced pressure to obtain diester-type catechin compounds.

[0042] Fraction F1-1-2 was pre-frozen in a refrigerator at -80°C for 4 hours, then transferred to a vacuum freeze dryer. The freezing temperature was set to -80°C, and the solvent was removed by freeze-drying under reduced pressure to obtain monoester-type catechin compounds.

[0043] Among them, the diester-type catechin compound obtained by freeze-drying fraction F1-1-1 under reduced pressure is epigallocatechin-3,4'-di- O -gallate (a grayish-white powder, 93.2% purity, 18.6 mg by mass, designated as compound 1).

[0044] The diester-type catechin compound obtained by freeze-drying fraction F1-1-3 under reduced pressure is gallocatechin-3,4'-di- O -gallate (a grayish-white powder, 95.7% purity, 12.1 mg by weight, designated as compound 3). The monoester-type catechin compound obtained by freeze-drying fraction F1-1-2 is epicatechin-3- O -gallate (a white powder with a purity of 94.9% and a mass of 40.2 mg, designated as compound 2).

[0045] In this embodiment, the tea leaves may also be yellow tea, black tea, dark tea, white tea, or oolong tea.

[0046] Regarding the structural identification tests for compound 1: [The following methods were used] 1 H NMR,13 The structure of compound 1 prepared according to the method of this application was identified by C1NMR, two-dimensional nuclear magnetic resonance spectroscopy, and ultra-high performance liquid chromatography-tandem high-resolution mass spectrometry. Specific results are as follows: Figures 4-10 As shown.

[0047] Depend on Figure 4 and Figure 5 It can be seen that the HR-ESI-MS negative ion mode [MH] + m / z = 609.0886, molecular formula is C 29 H 22 O 15 The secondary negative ion mode fragments mainly include m / z = 457.0769, 305.0661, 169.0410.

[0048] Combination 1 H NMR spectrum ( Figure 6 ), 13 C NMR ( Figure 7 ), 1 H- 1 H COSY spectrum ( Figure 8 ), HSQC spectrum ( Figure 9 ) and HMBC spectrum ( Figure 10 ), and the analysis of each detection spectrum determined that compound 1 is epigallocatechin-3,4'-di- O -gallate. The NMR signal of compound 1 is assigned as follows:

[0049] 1 H NMR (600 MHz, DMSO- d 6) δ 9.55 (2H, s, 3', 5'-O) H ), 9.46 (1H, 5-O H ),9.42 (s, 1H), 9.42 (2H, s, 3'' , 5''-O H ), 9.34 (2H, s, 3''' , 5'''-O H ), 9.22(1H, s, 7-O H ), 7.07 (2H, s, H-2'', 6''), 6.90 (2H, s, H-2''', 6'''), 6.62(2H, s, H-2', 6'), 5.99 (1H, d, J 6,8= 2.5 Hz, H-6), 5.87 (1H, d, J 8,6 = 2.1Hz, H-8), 5.34 (1H, d, J 3,4 = 3.6 Hz, H-3), 5.05 (1H, s, H-2), 2.95 (1H, m, H-4α), 2.72 (1H, d, J 4α,β = 16.4 Hz, H-4β).

[0050] 13 C NMR (150 MHz, DMSO- d 6) δ 165.86 (C-10, C O), 164.31 (C-9, C O),157.10 (C-5), 156.98 (C-7), 155.75 (C-8a), 150.37 (C-3', C-5'), 146.03,145.92 (C-3'', 5'', 3''', 5'''), 139.05 , 139.00 (C-4'', 4'''), 136.26 (C-1'), 126.77 (C-4'), 119.66, 119.49 (C-1'',1'''), 109.75 (C-2'', C-6''), 109.20 (C-2''', C-6'''), 105.89 (C-2', C-6'), 97.64 (C-4a), 96.17 (C-6), 94.76 (C-8), 76.78 (C-2), 68.66 (C-3), 25.99 (C-4).

[0051] Regarding the structural identification tests for compound 2 mentioned above: [The following methods were used] 1 H NMR, 13 The structure of compound 2 was identified by C10 NMR. Compound 2 is epicatechin-3- O -gallate. The NMR signal of compound 2 is assigned as follows:

[0052] 1 H NMR (600 MHz, DMSO- d 6) δ 8.81-9.34 (7H, -O H ), 6.88 (1H, d,J 2’,6’ =1.9 Hz, H-2''), 6.83 (2H, s, H-2',6'), 6.75 (1H, dd, J 6’,5’ = 8.2, J 6’,2’ =1.8Hz, H-6''), 6.66 (1H, d, J 5’,6’ = 8.1 Hz, H-5''), 5.95 (1H, d, J 6,8 = 2.2 Hz, H-6), 5.84 (1H, d, J 8,6 = 2.2 Hz, H-8), 5.34 (1H, dt, J 3,4 = 4.3 Hz, J 3,2 = 1.9Hz, H-3), 5.03 (1H, s, H-2), 2.68-2.94 (2H, m, H-4).

[0053] 13 C NMR (150 MHz, DMSO- d 6) δ 165.65 (C-9), 157.02 (C-5), 156.95 (C-7), 156.07 (C-8a), 145.89 (C-3'', 5''), 145.20 (C-3', 4'), 139.01 (C-4''), 129.84(C-1'), 119.66 (C-5'), 118.01 (C-1''), 115.54 (C-6'), 114.73 (C-2'), 109.06 (C-2'', 6''), 97.71 (C-4a), 96.00 (C-6), 94.80 (C-8), 76.93 (C-2), 68.62 (C-3), 26.11 (C-4)

[0054] Regarding the structural identification tests for compound 3 mentioned above: samples were taken using... 1 H NMR, 13 The structure of compound 3 was identified by C10 NMR. The NMR signal of compound 3 is assigned as follows:

[0055] 1 H NMR (600 MHz, DMSO-d 6) δ 9.49 (s, 2H 3', 5'-O H ), 9.39 (d, J = 10.6Hz, 2H), 9.28 (s, 2H), 9.16 (d, J = 8.0 Hz, 1H), 7.06 (s, 2H), 6.87 (s, 2H), 6.58 (s, 2H), 5.97 (t, J = 2.3 Hz, 1H), 5.87 (d, J = 2.4 Hz, 1H), 5.38 (s,1H), 5.07 (d, J = 2.7 Hz, 1H), 3.03 – 2.89 (m, 1H), 2.71 (1H, d, J 4α,β = 15.8Hz, H-4β).

[0056] 13 C NMR (150 MHz, DMSO- d 6) δ 163.67 (C-10, C O), 162.20 (C-9, C O),154.97 (C-5), 154.86 (C-7), 153.69 (C-8a), 148.24 (C-3', C-5'), 143.90,143.79 (C-3'', 5'', 3''', 5'''), 136.97, 136.94 (C-4'', 4'''), 134.18 (C-1'),124.71 (C-4'), 117.55 (C-1'',1'''), 107.61 (C-2'', C-6''), 107.06 (C-2''', C-6'''), 103.77 (C-2', C-6'), 95.61 (C-4a), 94.02 (C-6), 92.65 (C-8), 74.62 (C-2), 66.35 (C-3), 23.96 (C-4).

[0057] Analysis of the structures of compounds 1 and 3 reveals that the compounds prepared by the method of this invention share the following common structural features:

[0058] ;

[0059] Among them, compound 1 obtained by this preparation method is a previously unreported catechin compound, and the configuration and substituents of compound 1 are 2. R ,3 R R1=G, R2=R3=R4=H, R5=OH; The monomeric compound 3, obtained for the first time through separation and purification, has the following configuration and substituents: 2. R ,3 S R1=G, R2=R3=R4=H, R5=OH, where the substituent G is:

[0060] .

Claims

1. A method for preparing diester-type catechin compounds, characterized in that, Includes the following steps: 1) Mix tea leaves with a methanol-water solution, extract using ultrasound, and filter to obtain a tea extract with a methanol-water concentration of 12-15%; 2) Under room temperature conditions, the tea extract from step 1) was separated by reversed-phase liquid chromatography and collected. The fraction in the 15-20 min range was collected to obtain the first fraction. The first fraction was pre-frozen in a refrigerator for 3-5 h and then transferred to a vacuum freeze dryer. The solvent was removed by freeze-drying under reduced pressure to obtain the crude sample component. Chromatographic separation conditions: 0.05% trifluoroacetic acid aqueous solution was used as mobile phase A, and methanol was used as mobile phase B; the mobile phase gradient elution mode was as follows: 0 min-10 min, 8%-15% mobile phase B; 10 min-40 min, 15%-25% mobile phase B; 40 min-45 min, 25%-100% mobile phase B; 45 min-60 min, 100% mobile phase B; the flow rate was 80 mL / min, and the injection volume was 125 mL; the packing material was octadecyl bonded stationary phase. 3) At room temperature, the crude components of the sample were separated by reversed-phase liquid chromatography. The fraction was collected according to the chromatographic peak shape of the liquid chromatogram. The elution peak in the 19-21.5 min range was collected to obtain the second fraction. The second fraction was pre-frozen in a refrigerator for 3-5 h, transferred to a vacuum freeze dryer, and lyophilized under reduced pressure to remove the solvent to obtain the fine catechin components. Chromatographic separation conditions: 0.05% trifluoroacetic acid aqueous solution was used as mobile phase A, and acetonitrile was used as mobile phase B; the mobile phase gradient elution mode was: 0 min-25 min, 15%-30% mobile phase B; 25 min-30 min, 30%-95% mobile phase B; 30 min-40 min, 95% mobile phase B; the flow rate was 80 mL / min, and the injection volume was 20 mL; the packing material was octadecyl bonded stationary phase. 4) At room temperature, the fine components of catechins were separated by reversed-phase liquid chromatography. Fractions were collected based on the peak shapes of the prepared chromatograms. Elution peaks were collected in the 27-30 min and 37-39 min intervals to obtain the third and fourth fractions, respectively. The third and fourth fractions were pre-frozen in a refrigerator for 3-5 hours, then transferred to a vacuum freeze dryer and lyophilized under reduced pressure to remove the solvent, yielding diester-type catechin compounds. The diester-type catechin compound is epigallocatechin-3,4'-di- O -gallate and gallocatechin-3,4'-di- O -gallate; Chromatographic separation conditions: 0.05% trifluoroacetic acid aqueous solution was used as mobile phase A, and methanol was used as mobile phase B; the mobile phase gradient elution mode was 0 min-20 min, 25% mobile phase B; 20 min-30 min, 25%-35% mobile phase B; 30 min-45 min, 35% mobile phase B; the flow rate was 80 mL / min, and the injection volume was 80 mL; the packing material was octadecyl bonded stationary phase.

2. The method for preparing a diester-type catechin compound as described in claim 1, characterized in that... In step 1), the ultrasonic extraction time is 20-30 minutes. After ultrasonic extraction, the filtrate is collected by vacuum filtration and then filtered through a filter membrane to obtain tea extract.

3. The method for preparing a diester-type catechin compound as described in claim 2, characterized in that... In step 1), the pore size of the filter membrane is 0.45μm-0.5μm.

4. The method for preparing a diester-type catechin compound as described in claim 1, characterized in that... The chromatographic column used for chromatographic separation has a height of 260 mm and a diameter of 50 mm.

5. The method for preparing a diester-type catechin compound as described in claim 1, characterized in that... The pre-freezing temperature of the refrigerator and the internal temperature of the vacuum freeze dryer are both set to -80℃.

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