A phenolic compound, its preparation method and application

Abstract

The present invention belongs to the field of traditional Chinese medicine biotechnology, and specifically relates to a phenolic compound, its preparation method and application. The phenolic compound is a pair of diastereoisomers, where Formula I is the dextrorotatory form, named (+)-fresh bamboo juice phenol B, and Formula II is the levorotatory form, named (−)-fresh bamboo juice phenol B. The present invention successfully obtained a pair of diastereoisomeric phenolic compounds, (+)-fresh bamboo juice phenol B and (−)-fresh bamboo juice phenol B, from fresh bamboo juice. Through in vitro cell experiments, it was found that both of them have significant inhibitory effects on human gastric cancer cells, liver cancer cells, colorectal cancer cells, bladder cancer cells and lung cancer cells, and can be used in the preparation of drugs for preventing or treating tumors, showing broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of traditional Chinese medicine biotechnology, and particularly relates to a phenolic compound, a preparation method thereof and an application thereof. Background Art

[0002] Fresh bamboo juice is the liquid flowing out after the fresh stem is heated at high temperature. It is yellow to reddish-brown in color, slightly sweet in taste, and is used for treating symptoms such as excessive phlegm and cough due to lung heat, asthma and chest tightness, stiffness of the tongue due to stroke, excessive phlegm, and infantile convulsions due to phlegm-heat.

[0003] At present, the research on fresh bamboo juice mainly focuses on its pharmacological research, and the material basis of its medicinal efficacy is not clear. The chemical components of fresh bamboo juice are complex and diverse. Relevant research shows that fresh bamboo juice mainly contains chemical components such as amino acids, saccharides, phenols, alcohols, aldehydes, organic acids, phenylpropanoids and lignans, and inorganic elements, but most of them have not been confirmed as what compounds. In order to further improve the quality control standard of fresh bamboo juice and ensure the safety and effectiveness of the clinical application of fresh bamboo juice, it is necessary to conduct an in-depth analysis of its components. Summary of the Invention

[0004] Aiming at the problems existing in the prior art, the present invention provides a phenolic compound, a preparation method thereof and an application thereof. The phenolic compound is extracted from fresh bamboo juice, has a novel structure and pharmacological activity, and can provide a basis for the pharmacological research and safety of fresh bamboo juice.

[0005] The present invention provides a phenolic compound, which is a pair of diastereoisomers and has chemical structural formulas shown in Formula I and Formula II. Among them, Formula I is the dextrorotatory body, named (+)-fresh bamboo juice phenol B, and Formula II is the levorotatory body, named (-)-fresh bamboo juice phenol B.

[0006] 。

[0007] The present invention also provides a preparation method of the above phenolic compound, including the following steps:

[0008] (1) Cut the moso bamboo, carry out dry distillation, and concentrate under reduced pressure to obtain an extract, and set aside;

[0009] (2) Take an appropriate amount of the extract, extract it with ethyl acetate and n-butanol for 3 to 4 times respectively, and concentrate to obtain an ethyl acetate part and an n-butanol part;

[0010] (3) Pass the n-butanol part through a D101 macroporous resin column, wash it with 10%, 30%, 60%, and 90% ethanol aqueous solutions respectively, collect each component and concentrate it under reduced pressure to obtain a 60% ethanol part of the macroporous resin;

[0011] (4) Subject the 60% ethanol fraction to silica gel column chromatography, eluting with dichloromethane - methanol as the eluent in a gradient manner. After examination by thin - layer chromatography, combine the similar components to finally obtain six components A, B, C, D, E, and F;

[0012] (5) Subject fraction B to silica gel column chromatography, eluting with petroleum ether - ethyl acetate as the eluent in a gradient manner. After examination by thin - layer chromatography, combine the similar components to obtain 4 components B1 - B4;

[0013] (6) Subject fraction B4 to gel permeation chromatography, eluting with methanol to obtain 3 components B4.1 - B4.3;

[0014] (7) Subject B4.2 to preparative reverse - phase high - performance liquid chromatography to obtain phenolic compounds;

[0015] (8) Take the phenolic compounds and subject them to chiral separation by reverse - phase high - performance liquid chromatography to obtain phenolic compounds of formula Ⅰ and formula Ⅱ.

[0016] Further, in step (1) of the above technical solution, the temperature of dry distillation is 120 °C, the pressure of vacuum concentration is 0.7 MPa, and the temperature is 60 °C.

[0017] Further, in step (2) of the above technical solution, the mass - to - volume ratio of the extract to ethyl acetate or n - butanol is 1:1.2 - 1.5, preferably 1:1.32.

[0018] Further, in step (4) of the above technical solution, the pore size of the silica gel column chromatography is 100 - 200 mesh; when eluting in a gradient manner, the volume ratios of dichloromethane - methanol are 40:1, 30:1, 20:1, 10:1, and 5:1 in sequence.

[0019] Further, in step (5) of the above technical solution, when eluting in a gradient manner, the volume ratios of petroleum ether - ethyl acetate are 5:1, 4:1, 3:1, 2:1, 1:1, and 0:1 in sequence.

[0020] Further, in step (7) of the above technical solution, the preparative reverse - phase high - performance liquid chromatography uses acetonitrile - water with a volume ratio of 26:74 as the mobile phase, and the flow rate is 7 mL / min.

[0021] Further, in step (8) of the above technical solution, the reverse - phase high - performance liquid chromatography uses Phenomenex LuxCellulose - 4 as the chiral semi - preparative chromatographic column, and acetonitrile - water with a volume ratio of 38:62 as the mobile phase.

[0022] The present invention also provides a pharmaceutical composition comprising the above - mentioned phenolic compounds and a pharmaceutically acceptable carrier.

[0023] The present invention also provides an application of the above phenolic compound or pharmaceutical composition in the preparation of a drug for preventing or treating tumor diseases, and the tumors include human gastric cancer cells, human liver cancer cells, human colorectal cancer cells, human bladder cancer cells and human lung cancer cells.

[0024] Advantages over the prior art:

[0025] 1. The phenolic compound successfully obtained by the present invention uses fresh bamboo juice as a raw material, through dry distillation, vacuum concentration, extraction, macroporous resin column separation, two silica gel column chromatography separations, one gel filtration, reverse-phase preparative high-performance liquid chromatography, and finally reverse-phase high-performance liquid chromatography chiral resolution. The phenolic compound is a pair of diastereoisomer compounds (+)-fresh bamboo juice phenol B and (-)-fresh bamboo juice phenol B, and the preparation method is simple, rapid and efficient.

[0026] 2. In vitro cell experiments show that both (+)-fresh bamboo juice phenol B and (-)-fresh bamboo juice phenol B have significant inhibitory effects on human gastric cancer cells, liver cancer cells, colorectal cancer cells, bladder cancer cells and lung cancer cells, indicating that both have certain anti-tumor activities and can be used in the preparation of drugs for preventing or treating tumors; in comparison, the inhibitory effect of (-)-fresh bamboo juice phenol B on the proliferation of the above cells is stronger than that of (+)-fresh bamboo juice phenol B, indicating that the anti-tumor activity of (-)-fresh bamboo juice phenol B is stronger than that of (+)-fresh bamboo juice phenol B. Description of the drawings

[0027] Figure 1 It is the chromatogram of the diastereoisomers of the phenolic compound of the present invention, where the left peak is (+)-fresh bamboo juice phenol B and the right peak is (-)-fresh bamboo juice phenol B;

[0028] Figure 2 It is the high-resolution mass spectrum of the diastereoisomers of the phenolic compound of the present invention;

[0029] Figure 3 It is the UV spectrum of the diastereoisomers of the phenolic compound of the present invention;

[0030] Figure 4 It is the 1 H NMR spectrum of the diastereoisomers of the phenolic compound of the present invention;

[0031] Figure 5 It is the 13 C NMR spectrum of the diastereoisomers of the phenolic compound of the present invention;

[0032] Figure 6 It is the TOCSY spectrum of the diastereoisomers of the phenolic compound of the present invention;

[0033] Figure 7 It is the HMBC spectrum of the diastereoisomers of the phenolic compound of the present invention;

[0034] Figure 8 This is the HSQC spectrum of the diastereoisomers of the phenolic compounds of the present invention;

[0035] Figure 9 This is the NOESY spectrum of the diastereoisomers of the phenolic compounds of the present invention;

[0036] Figure 10 This is the ECD spectrum of (+)-phyllostachol B of the phenolic compounds of the present invention;

[0037] Figure 11 This is the ECD spectrum of (-)-phyllostachol B of the phenolic compounds of the present invention. Detailed implementation manners

[0038] Any of the above technical features of the present invention can be combined with the technical features specifically described hereinafter (such as in the examples) to form new or preferred technical solutions. However, the present invention is not limited to these examples, and these examples do not limit the present invention in any way.

[0039] Unless otherwise specified, the experimental methods in the following examples are all conventional methods. Unless otherwise specified, the preparations involved in the following examples are all ordinary commercially available products and can be obtained through market purchase.

[0040] The moso bamboo used in the present invention is collected from Tonggu County, Yichun City, Jiangxi Province, and is the fresh culm of Phyllostachys edulis (Carr.) H. de Lehaie of the genus Phyllostachys in the family Poaceae. Phyllostachys edulis (Carr.) of H. de Lehaie.

[0041] The present invention will be further described in detail below with reference to the drawings and examples:

[0042] Example 1: Preparation of phenolic compounds

[0043] The preparation method of phenolic compounds includes the following steps:

[0044] (1) Cut 10 t of moso bamboo, pyrolyze it at 120 °C, and concentrate it under reduced pressure at 60 °C and 0.7 MPa to obtain an extract (96 kg) for standby;

[0045] (2) Take 37.8 kg of the extract, extract it with ethyl acetate and n-butanol three times in sequence (50 L each time), and concentrate to obtain an ethyl acetate part (310 g) and an n-butanol part (857 g);

[0046] (3) Pass the 857 g of the n-butanol part through a D101 macroporous resin column, rinse it with 10%, 30%, 60%, and 90% ethanol aqueous solutions respectively, collect each component and concentrate it under reduced pressure to obtain a 60% ethanol part of the macroporous resin (55 g);

[0047] (4) 55 g of 60% ethanol was subjected to silica gel column chromatography (100 mesh - 200 mesh, 1000 g, inner diameter 80 mm, wet packing), and gradient elution was carried out with dichloromethane - methanol as the eluent. The volume ratios of dichloromethane - methanol were 40:1, 30:1, 20:1, 10:1, 5:1 in sequence. After inspection by thin layer chromatography, the components with similar properties were combined, and finally six components A, B, C, D, E, and F were obtained;

[0048] (5) Component B (1.3 g) was subjected to silica gel column chromatography (100 mesh - 200 mesh, 40 g, inner diameter 20 mm, wet packing), and gradient elution was carried out with petroleum ether - ethyl acetate as the eluent. The volume ratios of petroleum ether - ethyl acetate were 5:1, 4:1, 3:1, 2:1, 1:1, 0:1 in sequence. After inspection by thin layer chromatography, the components with similar properties were combined to obtain 4 components B1 - B4;

[0049] (6) Component B4 was passed through Sephadex LH - 20 gel and eluted with methanol to obtain 3 components B4.1, B4.2, and B4.3;

[0050] (7) Component B4.2 (100 mg) was subjected to preparative reverse - phase high - performance liquid chromatography. The chromatographic column was XTerra prep MSC 18 column (300 mm×19 mm, 10 μm), and elution was carried out with acetonitrile - water (26:74) as the mobile phase at a flow rate of 7 mL / min to obtain a phenolic compound (19 mg).

[0051] Example 2: Resolution and Structure Identification of Diastereoisomers of Phenolic Compounds

[0052] 1. Resolution

[0053] The phenolic compound obtained in Example 1 was taken and dissolved in 1 mL of methanol. After reverse - phase high - performance liquid chromatography using a Phenomenex Lux Cellulose - 4 (250 mm×4.6 mm, 5 μm) chiral semi - preparative chromatographic column and acetonitrile - water (38:62) as the mobile phase, compounds of formula I and formula II were obtained, which were named (+) - Phyllostachys nigra phenol B and (-) - Phyllostachys nigra phenol B respectively. Their chromatograms are as Figure 1 shown. The left peak is (+) - Phyllostachys nigra phenol B, and the right peak is (-) - Phyllostachys nigra phenol B.

[0054]

[0055] 2. Structure Analysis

[0056] (±)-Fresh bamboo extract phenol B is a yellow powder and soluble in methanol; HRESIMS was measured by Waters ACQUITY UPLC / Xevo G2 QTOF (HR-Q-TOF-MS: Waters Corporation, USA). m / z 405.1925 [M–H] – (calcd. for C 22 H 29 O7, 405.1913), and its molecular formula was determined to be C 22 H 30 O7. Among them, the high-resolution mass spectrum is as shown in Figure 2 Figure, and other confirmation spectra are as shown in Figures 3 to 11 Figure. The 1 H-NMR and 13 C-NMR nuclear magnetic data of (±)-fresh bamboo extract phenol B are shown in Table 1.

[0057] Table 1 1 H-NMR and 13 C-NMR nuclear magnetic data of (±)-fresh bamboo extract phenol B

[0058]

[0059] a [[ID=3④Recorded in CD3OD (1H NMR 600 MHz, 13 C NMR 150 MHz).

[0060] 1 In the 1H NMR spectrum, the signals of the substituted hydrogens at positions 1, 3, 4, and 5 of the benzene ring are given: 6.26 (2H, s, H-2′, 6′), and a set of ABX system proton signals: 6.63 (1H, d, J = 8.0 Hz, H-5′′), 6.55 (1H, dd, J = 8.0, 1.9 Hz, H-6′′), 6.53 (1H, d, J = 1.9 Hz, H-2′′); 4.38 (1H, d, J = 8.3 Hz, H-3), 2.98 (1H, ddd, J = 8.3, 7.3, 5.7 Hz, H-2) is the hydrogen signal of the methylene group adjacent to oxygen; 4.10 (1H, dd, J = 10.9, 5.⑦ Hz, H-1) and 3.91 (1H, dd, J= 10.9, 7.3 Hz, H-1) is the signal of the methylene hydrogen adjacent to oxygen; 3.71 (6H, s, 3′, 5′-OCH3) and 3.68 (3H, s, 3′′-OCH3) are the signals of three methoxy hydrogens; 3.34 (1H, m, H-1′′′), 3.29 (1H, m, H-1′′′), 1.55 (2H, m, H-2′′′), 1.42 (2H, m, H-3′′′) and 0.91 (3H, t, J = 7.4 Hz, H-4′′′) is the signal of the n-butyl hydrogen. 13 In the 13C NMR spectrum, ten aromatic ring carbon signals are shown: 131.8 (C-1′), 107.7 (C-2′, 6′), 148.7 (C-3′, 5′), 135.1 (C-4′), 133.4 (C-1′′), 112.2 (C-2′′), 148.4 (C-3′′), 146.7 (C-4′′), 115.4 (C-5′′), 121.3 (C-6′′); four n-butyl carbon signals: 69.4 (C-1′′′), 33.2 (C-2′′′), 20.6 (C-3′′′), 14.3 (C-4′′′); two methoxy carbon signals: 56.7 (3′, 5′-OCH3) and 56.6 (3′′-OCH3); and three other carbon signals: 64.8 (C-1), 56.3 (C-2), 85.7 (C-3). Analyzing the above 1 1H NMR, 13 13C NMR and HR-Q-TOF-MS data, it can be known that this compound is a phenolic compound.

[0061] In the HMBC spectrum, J 2,3 = 8.3 Hz, it can be inferred that the relative configuration of the compound is threo, and in the NOESY spectrum, 4.38 (1H, d, J = 8.3 Hz, H-3) has no correlation with 2.98 (1H, ddd, J = 8.3, 7.3, 5.7 Hz, H-2), further proving this inference. In the HMBC spectrum, δ H 4.38 (1H, d, J = 8.3 Hz, H-3) is correlated with δ C 69.4 (C-1′′′), 133.4 (C-1′′), indicating that C-3 is connected to the n-butyl group adjacent to oxygen and the benzene ring C-1′′ position; δ H2.98 (1H, ddd, J = 8.3, 7.3, 5.7 Hz, H-2) is correlated with δ C 131.8 (C-1′), indicating that C-2 is connected to the C-1′ position of the benzene ring. Therefore, the compound was identified as threo-2-(4-hydroxy-3,5-dimethoxyphenyl)-3-(4-hydroxy-3-methoxyphenyl)-3-butoxypropanol, and its main HMBC (H→C) correlation signals are shown as follows:

[0062] .

[0063] The CD spectrum of the compound was measured by a circular dichroism spectrometer, and the result did not show an obvious trend, suggesting that it might be a mixture. Analysis by a chiral column confirmed that it was indeed a mixture.

[0064] (+)-Phyllostachysin B is a yellow powder, soluble in methanol, and measured by a Perkin-Elmer 341 polarimeter (PerkinElmer, Inc., USA) +12° ( c 0.04, MeOH); ECD (MeOH) λ was measured by a JASCO J-815 circular dichroism spectrometer (JASCO Corporation, Japan) max (Δε) 242 (24.99), 282 (15.42) nm. The positive Cotton effects shown by its ECD at 242 nm and 282 nm are similar to those of eucophenolic D. Combining its relative configuration as threo, its absolute configuration was determined to be 2R, 3R.

[0065] (-)-Phyllostachysin B is a yellow powder, soluble in methanol, and measured by a Perkin-Elmer 341 polarimeter (PerkinElmer, Inc., USA) -16° ( c 0.04, MeOH); ECD (MeOH) λ was measured by a JASCO J-815 circular dichroism spectrometer (JASCO Corporation, Japan) max (Δε) 242 (-23.65), 282 (-12.91) nm. The negative Cotton effects shown by its ECD at 242 nm and 282 nm are similar to those of eucophenolic C. Combining its relative configuration as threo, its absolute configuration was determined to be 2S, 3S.

[0066] Example 3: Antitumor cell activity screening

[0067] Principle of MTT assay: There are dehydrogenases related to NAPP (nicotinamide adenine dinucleotide phosphate, coenzyme II) in the mitochondria of living cells. Succinate dehydrogenase can reduce exogenous yellow thiazolyl blue MTT (3-(4,5)-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to water-insoluble blue-violet crystalline formazan, which deposits in the cells. This enzyme disappears in dead cells, and MTT is not reduced. After dissolving formazan with dimethyl sulfoxide (DMSO), the absorbance can be detected at 570 nm and 630 nm using an enzyme-linked immunosorbent assay (ELISA) reader. The optical density value is proportional to the number of living cells.

[0068] The cell lines used were: SGC7901 (human gastric cancer cells), SMMC-7721 (human liver cancer cells), HCT116 (human colorectal cancer cells), BIU87 (human bladder cancer cells), and SPCA-1 (human lung cancer cells).

[0069] MTT assay method:

[0070] Take logarithmically growing cells, digest them, and thoroughly pipette them into a single-cell suspension. After counting, dilute them to 1×10 5 cells / mL, inoculate them into a 96-well culture plate, add 100 μL of cell suspension to each well, and place them in an incubator at 37°C and 5% CO2 for 24 hours. Then, discard the original culture medium. Design 4 concentration gradients for each sample, and then add 100 μL of medium containing each concentration gradient of the sample and paclitaxel (positive control) to the test wells. Each concentration has 6 parallel wells; add an equal volume of solvent to the control group. After culturing the 96-well culture plate in an incubator at 37°C, 5% CO2, and saturated humidity for 72 hours, add 20 μL of freshly prepared serum-free medium containing 5 mg / mL MTT to each well and continue to culture at 37°C for 4 hours. Then, remove the supernatant. Add 150 μL of DMSO to each well to dissolve the formazan precipitate, shake for 5 minutes, and measure the absorbance at 570 nm and 630 nm using an ELISA reader, which can reflect the number of living cells. The calculation formula is as follows: Tumor cell growth inhibition rate (%) = (measured value of control well - measured value of test well) / measured value of control well × 100%. The test results are shown in Tables 1 to 5.

[0071] Table 1 Detection results of the growth inhibition rate of phenolic compounds on gastric cancer cells (SGC7901) at different concentrations

[0072]

[0073] Table 2 Detection results of the growth inhibition rate of phenolic compounds on liver cancer cells (SMMC-7721) at different concentrations

[0074]

[0075] Table 3 Detection results of the growth inhibition rate of phenolic compounds on colorectal cancer cells (HCT116) at different concentrations

[0076]

[0077] Table 4 Detection results of the growth inhibition rate of phenolic compounds on bladder cancer cells (BIU87) at different concentrations

[0078]

[0079] Table 5 Detection results of the growth inhibition rate of phenolic compounds on lung cancer cells (SPCA-1) at different concentrations

[0080]

[0081] From the above detection results, it can be seen that (+)-phyllostachoside B and (-)-phyllostachoside B have significant inhibitory effects on human gastric cancer cells, liver cancer cells, colorectal cancer cells, bladder cancer cells and lung cancer cells. Moreover, the inhibition rate shows a certain dose-effect relationship with the drug concentration. As the drug concentration increases, the inhibitory effect increases, indicating that both have certain potential anti-tumor activities. In comparison, the inhibitory effect of (-)-phyllostachoside B on the proliferation of the above cells is stronger than that of (+)-phyllostachoside B, indicating that the anti-tumor activity of (-)-phyllostachoside B is stronger than that of (+)-phyllostachoside B.

[0082] In summary, the phenolic compounds obtained in this invention are a pair of diastereoisomers, and have significant inhibitory effects on human gastric cancer cells, liver cancer cells, colorectal cancer cells, bladder cancer cells and lung cancer cells, and all have anti-tumor activities. They can be used in the preparation of drugs for preventing or treating tumors and have broad application prospects.

[0083] Finally, it should be emphasized that the above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention should be included within the protection scope of the present invention.

Claims

1. Use of a phenolic compound in the preparation of a medicament for preventing or treating tumor diseases, characterized in that, The tumors are human colorectal cancer and human bladder cancer, and the phenolic compound has the chemical structural formula shown in Formula II: 。 2. Use of a pharmaceutical composition containing a phenolic compound in the preparation of a medicament for preventing or treating tumor diseases, characterized in that, The tumors are human colorectal cancer and human bladder cancer, and the phenolic compound has the chemical structural formula shown in Formula II: 。

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