A pair of diastereoisomers in fresh bamboo juice, and preparation method and application thereof

Abstract

The present invention belongs to the field of traditional Chinese medicine biotechnology, and specifically relates to a pair of diastereoisomers in fresh bamboo juice, their preparation methods and applications. In the diastereoisomers, formula I is the dextrorotatory form, named (+)-fresh bamboo juice phenol A, and formula II is the levorotatory form, named (−)-fresh bamboo juice phenol A. The present invention has successfully obtained a pair of diastereoisomer compounds, (+)-fresh bamboo juice phenol A and (−)-fresh bamboo juice phenol A, from fresh bamboo juice. Through in vitro cell experiments, it is found that (+)-fresh bamboo juice phenol A has 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, with broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of traditional Chinese medicine biotechnology, and particularly relates to a pair of diastereoisomers in fresh bamboo juice, a preparation method thereof, and an application thereof. Background Art

[0002] Fresh bamboo juice is a liquid flowing out from the fresh stems of plants of the Gramineae family ( Gramineae ), Bambusa glaucescens ( Phyllostachys glauca McClure), Phyllostachys nuda ( Phyllostachys nuda McClure) and their congeners after being heated at high temperature. It is yellow to reddish-brown in color, slightly sweet in taste, and has the efficacy of clearing away heat and resolving phlegm. It is used for treating excessive phlegm in cough due to lung heat, asthma with chest distress, stiffness of the tongue in stroke, infantile convulsion due to phlegm-heat, etc.

[0003] Fresh bamboo juice was first recorded as "bamboo juice" in "Shennong Ben Cao Jing", and later was called "light bamboo juice" and "bamboo juice". It was only called "fresh bamboo juice" in modern times. The name mainly varies due to the types of bamboo used in the preparation and the different processing techniques. In ancient times, light bamboo, bitter bamboo, violet bamboo, etc. were mostly used for the preparation of fresh bamboo juice. By the 1977 edition of "Chinese Pharmacopoeia", the types of bamboo used for the preparation of fresh bamboo juice were more extensive, including Bambusa glaucescens, Phyllostachys nuda and their congeners. Its functions and indications are complex and diverse. From the "maintaining wind spasm, opening the mind, making the body light and strengthening qi" recorded in "Shennong Ben Cao Jing", to the "great heat in the chest, improving eyesight, and promoting the flow of the nine orifices" mentioned in subsequent documents, and then to the "clearing away heat and resolving phlegm, used for excessive phlegm in cough due to lung heat, asthma with chest distress, stiffness of the tongue in stroke, profuse phlegm, infantile convulsion due to phlegm-heat" recorded in the 1977 edition of "Chinese Pharmacopoeia", the records of its efficacy and application have become increasingly complete.

[0004] However, at present, the pharmacodynamic material basis of fresh bamboo juice is not clear, and the current standard for quality control is low. Therefore, it is urgent to conduct a more in-depth study on the active ingredients to further improve the quality control standard and ensure the safety and effectiveness of the clinical application of fresh bamboo juice. Relevant research shows that fresh bamboo juice mainly contains chemical components such as amino acids, saccharides, phenols, alcohols, aldehydes, organic acids, phenylpropanoids and lignans, inorganic elements, etc. However, the exact components and structures of many of them are not clear, and there are even fewer reports on diastereoisomers. Summary of the Invention

[0005] Aiming at the problems existing in the prior art, the present invention provides a pair of diastereoisomers in fresh bamboo juice, a preparation method thereof, and an application thereof. This pair of diastereoisomers has a novel structure and pharmacological activity, providing a basis for the pharmacological research and safety of fresh bamboo juice.

[0006] The present invention provides a pair of diastereoisomers, and the chemical structural formulas of the diastereoisomers are shown as Formula I and Formula II. Among them, Formula I is the dextrorotatory body, named (+)-fresh bamboo phenol A, and Formula II is the levorotatory body, named (-)-fresh bamboo phenol A:

[0007] 。

[0008] The present invention also provides a method for preparing the above diastereomers, comprising the following steps:

[0009] (1) Cut moso bamboo, carry out dry distillation, and concentrate under reduced pressure to obtain an extract, and reserve it for later use;

[0010] (2) Take an appropriate amount of the extract, extract it 3 - 4 times successively with ethyl acetate and n-butanol, and concentrate to obtain an ethyl acetate part and an n-butanol part;

[0011] (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 under reduced pressure to obtain the 60% ethanol part of the macroporous resin;

[0012] (4) Subject the 60% ethanol part to silica gel column chromatography, elute it with dichloromethane - methanol as the eluent by gradient elution, check it by thin layer chromatography, and combine the similar components to finally obtain six components A, B, C, D, E, and F;

[0013] (5) Subject component B to silica gel column chromatography, elute it with petroleum ether - ethyl acetate as the eluent by gradient elution, check it by thin layer chromatography, and combine the similar components to obtain 4 components B1 - B4;

[0014] (6) Subject component B2 to preparative reverse-phase high performance liquid chromatography to obtain diastereomers;

[0015] (7) Take phenolic compounds and obtain diastereomers of formula I and formula II after chiral separation by reverse-phase high performance liquid chromatography.

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

[0017] Further, in step (2) of the above technical solution, the mass - volume ratio of the extract to ethyl acetate or n-butanol is 1:1 - 1.4, preferably 1:1.3.

[0018] Further, in step (4) of the above technical solution, the pore size of the silica gel column chromatography is 100 - 200 mesh; when gradient eluting, 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 gradient eluting, 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 (6) of the above technical solution, the reverse-phase preparative high performance liquid chromatography uses acetonitrile-water with a volume ratio of 24:76 as the mobile phase, and the flow rate is 10 mL / min.

[0021] Further, in step (7) of the above technical solution, the reverse-phase high performance liquid chromatography uses Phenomenex LuxCellulose-4 (250 mm × 4.6 mm, 5 μm) as the chiral semi-preparative chromatographic column, and acetonitrile-water with a volume ratio of 24:76 is used as the mobile phase.

[0022] The present invention also provides a pharmaceutical composition comprising the above compound of formula I and a pharmaceutically acceptable carrier.

[0023] The present invention also provides an application of the above compound of formula I or the 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 present invention uses fresh bamboo juice as a raw material, and through dry distillation, vacuum concentration, extraction, macroporous resin column separation, two silica gel column chromatography separations, reverse-phase preparative high performance liquid chromatography separation, and finally reverse-phase high performance liquid chromatography chiral resolution, a pair of diastereoisomer compounds (+)-fresh bamboo juice phenol A and (-)-fresh bamboo juice phenol A are successfully obtained. The preparation method is simple and rapid.

[0026] 2. In vitro cell experiments show that (+)-fresh bamboo juice phenol A has significant inhibitory effects on human gastric cancer cells, liver cancer cells, colorectal cancer cells, bladder cancer cells and lung cancer cells, indicating that it has anti-tumor activity and can be used in the preparation of drugs for preventing or treating tumors, and has broad application prospects. Description of the drawings

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

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

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

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

[0031] Figure 5 It is the 1313C NMR spectrum;

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

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

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

[0035] Figure 9 is the ECD spectrum of (+)-phyllostachol A of the diastereoisomers of the present invention;

[0036] Figure 10 is the ECD spectrum of (-)-phyllostachol A of the diastereoisomers of the present invention. Detailed implementation manners

[0037] Any of the above technical features of the present invention can be combined with the technical features specifically described below (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.

[0038] 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.

[0039] The moso bamboo of the present invention is collected from Tonggu County, Yichun City, Jiangxi Province, and is identified as Phyllostachys heterocycla (Carr.) Mitford var. pubescens Mazel ex H. de Lehaie by senior experimentalist Wu Ziping of Tonggu Weisheng Industrial Co., Ltd. Phyllostachys edulis (Carr.) Fresh culms of H. de Lehaie.

[0040] The present invention will be further described in detail below with reference to the drawings and examples:

[0041] Example 1: Preparation of diastereoisomers

[0042] The preparation method of diastereoisomers includes the following steps:

[0043] (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;

[0044] (2) Take 37.8 kg of the extract, extract it 3 times (50 L each time) with ethyl acetate and n-butanol in sequence, and concentrate to obtain an ethyl acetate part (310 g) and an n-butanol part (857 g);

[0045] (3) The 857 g of n-butanol fraction was passed through a D101 macroporous resin column and rinsed with 10%, 30%, 60%, and 90% ethanol aqueous solutions respectively. Each fraction was collected and concentrated under reduced pressure to obtain the 60% ethanol fraction of the macroporous resin (55 g).

[0046] (4) The 55 g of the 60% ethanol fraction was subjected to silica gel column chromatography (100 mesh - 200 mesh, 1000 g, inner diameter 80 mm, wet packing), and eluted with dichloromethane - methanol as the eluent in a gradient manner, where the volume ratios of dichloromethane - methanol were 40:1, 30:1, 20:1, 10:1, and 5:1 in sequence. After inspection by thin layer chromatography, the similar fractions were combined to finally obtain six fractions A, B, C, D, E, and F.

[0047] (5) The fraction B (1.3 g) was passed through silica gel column chromatography and eluted with petroleum ether - ethyl acetate as the eluent in a gradient manner, where the volume ratios of petroleum ether - ethyl acetate were 5:1, 4:1, 3:1, 2:1, 1:1, and 0:1 in sequence. After inspection by thin layer chromatography, the similar fractions were combined to obtain 4 fractions B1 - B4.

[0048] (6) The fraction B2 (60 mg) was prepared by reverse-phase preparative high performance liquid chromatography (using acetonitrile - water (24:76) as the mobile phase and a flow rate of 10 mL / min) to obtain the diastereoisomers (5 mg).

[0049] Example 2: Resolution and Structure Identification of Diastereoisomers

[0050] 1. Resolution

[0051] Take the diastereoisomers obtained in Example 1, add 1 mL of methanol to dissolve them, and perform reverse-phase high performance liquid chromatography using a Phenomenex Lux Cellulose - 4 (250 mm × 4.6 mm, 5 μm) chiral semi-preparative chromatographic column with acetonitrile - water (24:76) as the mobile phase to obtain Compound I and Compound II, which were named (+)-Fresh Bamboo Extract Phenol A and (-)-Fresh Bamboo Extract Phenol A respectively. Their chromatograms are as Figure 1 shown.

[0052] 2. Structure Analysis

[0053] (±)-Fresh Bamboo Extract Phenol A is a yellow powder and is soluble in methanol; HRESIMS was measured by Waters ACQUITY UPLC / Xevo G2 Q TOF (HR - Q - TOF - MS: Waters Corporation, USA) m / z 269.1399 [M–H] – (calcd. forC 14 H 23 O5, 269.1389), and its molecular formula was determined to be C 14 H24 O5, where the high-resolution mass spectrum is as Figure 2 shown, and the other confirmation spectra are as Figures 3 to 10 shown. The 1 1H-NMR, 13 13C-NMR nuclear magnetic data of (±)-phyllostachoside A are shown in Table 1.

[0054] Table 1 The 1 1H-NMR, 13 13C-NMR nuclear magnetic data of (±)-phyllostachoside A

[0055]

[0056] a Recorded in DMSO( 1 1H NMR 600 MHz, 13 13C NMR 150 MHz).

[0057] 1 The 1H NMR spectrum ( Figure 4 ) gives the substitution hydrogen signals of benzene ring 1, 3, 4, 5: 6.52 (2H, s, H-2′, 6′); the tertiary hydrogen signal: 4.14 (dd, J J = 7.8, 4.8 Hz, 1H, H-1); the methoxy signal: 3.73 (6H, s, 3′, 5′-OCH3); and a set of methylene hydrogen signals: 3.48 (1H, dd, J J = 11.4, 7.8 Hz, H-2) and 3.35 (1H, dd, J = 11.4, 4.8 Hz, H-2). In addition, 1 1H NMR also gives a set of characteristic signals of the oxygen-linked n-butyl group: 3.27 (2H, m, H-1′′), 1.47 (2H, m, H-2′′), 1.32 (2H, m, H-3′′) and 0.84 (3H, t, J J = 7.4 Hz, H-4′′). 13 The 13C NMR spectrum ( Figure 5), fourteen carbon signals were shown, among which 130.4 (C-1′), 104.0 (C-2′, 6′), 147.8 (C-3′, 5′), 134.7 (C-4′) were benzene ring carbon signals; 83.1 (C-1) was a tertiary carbon signal; 66.2 (C-2) was a methylene carbon signal; the carbon spectrum data also showed the characteristic carbon signals of n-butyl: 67.8 (C-1′′), 31.6 (C-2′′), 18.9 (C-3′′) and 13.8 (C-4′′) and the benzene ring substituted methoxy carbon signal 55.9 (3′, 5′-OCH3). Analyzing the above 1 H NMR, 13 C NMR and HR-Q-TOF-MS data, it can be known that this compound is a phenolic compound.

[0058] In the HMBC spectrum ( Figure 7 ), δ H 3.73 (6H, s, 3′, 5′-OCH3) was correlated with δ C 147.8 (C-3′, 5′), indicating that the methoxy group was connected to C-3′ and C-5′ positions; δ H 4.14 (dd, J = 7.8, 4.8 Hz, H-1) was correlated with δ C 66.2 (C-2), 104.0 (C-2′, 6′), 130.4 (C-1′), indicating that C-1 was connected to the benzene ring C-1′ position; δ H 3.48 (dd, J = 11.3,7.6 Hz, Ha-2), 3.35 (dd, J = 11.3, 4.4 Hz, Hb-2) were simultaneously correlated with δ C 67.8 (C-1′′), indicating that C-2 was connected to the oxygen-linked n-butyl group. Thus, the compound was identified as 1-hydroxy-2-butoxy-1-(4-hydroxy-3,5-dimethoxyphenyl)-ethane, and its main HMBC (H→C) correlation signals are shown as follows:

[0059] .

[0060] By measuring the ECD spectrum of the compound with a circular dichroism spectrometer, no obvious trend was shown, and it was speculated that it might be a mixture. Analyzed by a chiral column, it was confirmed that it was indeed a mixture.

[0061] (+)-Fresh Bamboo Extract Phenol A, yellow powder, soluble in methanol, measured with a Perkin-Elmer 341 polarimeter (PerkinElmer Inc., USA) -73° (c 0.022, MeOH); ECD (MeOH) was measured on a JASCO J-815 circular dichroism spectrometer (JASCO Corporation, Japan). λ max (Δε) 213 (-11.03), 272 (3.14) nm ( Figure 9 ) By comparing the specific rotation with that of (2R)-amino-2-phenylpropanoic acid, the specific rotations of the two were similar. Therefore, the absolute configuration of (+)-phyllostachol A was assigned as R.

[0062] (-)-Phyllostachol A is a yellow powder, soluble in methanol. The specific rotation was measured on a Perkin-Elmer 341 polarimeter (PerkinElmer, Inc., USA). +32° (c 0.02, MeOH); ECD (MeOH) was measured on a JASCO J-815 circular dichroism spectrometer (JASCO Corporation, Japan). λ max (Δε) 218 (-8.77), 272 (-3.52) nm ( Figure 10 ) By comparing the specific rotation with that of (S)-Methoxy-(3,5-dimethoxy-4-hydroxyphenyl)ethanediol, the specific rotations of the two were similar. Therefore, the absolute configuration of (-)-phyllostachol A was assigned as S.

[0063] Example 3: Screening for anti-tumor cell activity

[0064] Test principle: MTT method: There is a dehydrogenase 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 and deposit it 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 with an enzyme-linked immunosorbent assay reader. The optical density value is proportional to the number of living cells.

[0065] 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).

[0066] Test method:

[0067] MTT method: Take logarithmically growing cells, after digestion, 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 culture 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 samples at each concentration gradient and paclitaxel (positive control) to the test wells, with 6 parallel wells for each concentration; 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 on an enzyme-linked immunosorbent assay (ELISA) reader, which can reflect the number of viable 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.

[0068] Table 1 Detection results of the growth inhibition rate of the compound on gastric cancer cells (SGC7901) at different concentrations

[0069]

[0070] Table 2 Detection results of the growth inhibition rate of the compound on liver cancer cells (SMMC-7721) at different concentrations

[0071]

[0072] Table 3 Detection results of the growth inhibition rate of the compound on colorectal cancer cells (HCT116) at different concentrations

[0073]

[0074] Table 4 Detection results of the growth inhibition rate of the compound on bladder cancer cells (BIU87) at different concentrations

[0075]

[0076] Table 5 Detection results of the growth inhibition rate of the compound on lung cancer cells (SPCA-1) at different concentrations

[0077]

[0078] As can be seen from the above test results, (+)-phyllolitorin A has significant inhibitory effects on human gastric cancer cells, liver cancer cells, colorectal cancer cells, bladder cancer cells and lung cancer cells. The inhibition rate shows a certain dose-effect relationship with the drug concentration. As the drug concentration increases, the inhibitory effect increases, indicating that (+)-phyllolitorin A has certain potential anti-tumor activity; while (-)-phyllolitorin A has no inhibitory effect on the proliferation of the above cells.

[0079] 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 shall be included within the protection scope of the present invention.

Claims

1. A compound, characterized in that, The said compound has a structure shown in Formula I: 。 2. A method for preparing diastereoisomers, characterized in that, It includes the following steps: (1) Cut moso bamboo, carry out dry distillation, and concentrate under reduced pressure to obtain an extract for standby. Among them, the temperature of dry distillation is 120 °C, the pressure of concentration under reduced pressure is 0.7 MPa, and the temperature is 60 °C; (2) Take an appropriate amount of the extract, extract it 3 - 4 times successively with ethyl acetate and n-butanol, and concentrate to obtain an ethyl acetate part and an n-butanol part. Among them, the mass-volume ratio of the extract to ethyl acetate or n-butanol is 1:1 - 1.4; (3) Pass 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 under reduced pressure to obtain the 60% ethanol part of the macroporous resin; (4) Subject the 60% ethanol part to silica gel column chromatography, elute it with dichloromethane - methanol as the eluent in a gradient manner, check it by thin-layer chromatography, and combine the similar components to finally obtain six components A, B, C, D, E, and F. Among them, when eluting in a gradient manner, the volume ratio of dichloromethane - methanol is 40:1, 30:1, 20:1, 10:1, 5:1 in sequence; (5) Subject component B to silica gel column chromatography, elute it with petroleum ether - ethyl acetate as the eluent in a gradient manner, check it by thin-layer chromatography, and combine the similar components to obtain 4 components B1 - B4. Among them, when eluting in a gradient manner, the volume ratio of petroleum ether - ethyl acetate is 5:1, 4:1, 3:1, 2:1, 1:1, 0:1 in sequence; (6) Subject component B2 to preparative reverse-phase high performance liquid chromatography to obtain diastereoisomers. Among them, the preparative reverse-phase high performance liquid chromatography uses acetonitrile - water with a volume ratio of 24:76 as the mobile phase, and the flow rate is 10 mL / min; (7) Diastereomers I and II were obtained after chiral resolution of phenolic compounds by reversed-phase high performance liquid chromatography. The structural formulas are shown below. Among them, Phenomenex Lux Cellulose-4 was used as the chiral semi-preparative chromatographic column for reversed-phase high performance liquid chromatography, and acetonitrile-water with a volume ratio of 24:76 was used as the mobile phase.

3. The preparation method of a diastereoisomer according to claim 2, characterized in that, In step (4), the pore size of the said silica gel column chromatography is 100 mesh - 200 mesh.

4. A pharmaceutical composition, characterized in that, It contains the compound of Formula I as described in Claim 1 and a pharmaceutically acceptable carrier.

5. Use of a compound of formula I as claimed in claim 1 or a pharmaceutical composition as claimed in claim 4 in the preparation of a medicament for preventing or treating tumor diseases, characterized in that, The said tumors are human gastric cancer cells, human liver cancer cells, human colorectal cancer cells, human bladder cancer cells, and human lung cancer cells.

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