Pair of diastereoisomers in fresh bamboo juice as well as preparation method and application of diastereoisomers

Abstract

The invention belongs to the technical field of traditional Chinese medicine biology, and particularly relates to a pair of diastereoisomers in fresh bamboo juice as well as a preparation method and application of the diastereoisomers, in the diastereoisomers, a formula I is a dextroisomer and is named as (+)-fresh bamboo juice phenol A, and a formula II is a levoisomer and is named as (-)-fresh bamboo juice phenol A. According to the invention, a pair of diastereoisomer compounds (+)-fresh bamboo juice phenol A and (-)-fresh bamboo juice phenol A are successfully obtained from fresh bamboo juice, and an in-vitro cell test shows that the (+)-fresh bamboo juice phenol A has a remarkable inhibition effect on gastric cancer cells, liver cancer cells, colorectal cancer cells, bladder cancer cells and lung cancer cells of a human body; the compound can be used for preparing medicines for preventing or treating tumors, and has a wide application prospect.

Description

Technical Field

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

[0002] Fresh bamboo sap is a member of the Poaceae family ( Gramineae )Plant Powder Green Bamboo( Phyllostachys glauca McClure), Jingzhu ( Phyllostachysnuda McClure) and its relatives, the fresh stems are heated to high temperature and the liquid that flows out is yellow to reddish brown in color and slightly sweet in taste. It has the effect of clearing away heat and resolving phlegm. It is used for lung heat, cough with phlegm, shortness of breath and chest tightness, tongue stiffness in stroke, phlegm-heat convulsion in children, etc.

[0003] Fresh bamboo juice was first recorded as "bamboo juice" in Shennong's Herbal Classic, and later it was called "light bamboo juice" and "bamboo juice". It was not until modern times that it was called "fresh bamboo juice". The name mainly varies due to the different types of bamboo used in the preparation and the processing technology. In ancient times, light bamboo, bitter bamboo, and violet bamboo were mostly used to prepare fresh bamboo juice. In the 1977 edition of the Chinese Pharmacopoeia, the types of bamboo used to prepare fresh bamboo juice were recorded to be more extensive, including pink-green bamboo, pure bamboo and bamboos of the same genus. Its functions and indications are complex and diverse, from the "Treat wind-induced phlegm, clear the mind, lighten the body and benefit the qi" recorded in "Shennong Bencao Jing", to the "severe heat in the chest, brighten the eyes, and clear the nine orifices" mentioned in later documents, to the "clearing heat and resolving phlegm, used for lung heat, cough with excessive phlegm, asthma and chest tightness, stiff tongue due to stroke, excessive phlegm, and phlegm-heat and convulsions in children" recorded in the 1977 edition of "Chinese Pharmacopoeia", the records of its efficacy and application are becoming increasingly complete.

[0004] However, the material basis of the efficacy of fresh bamboo sap is not clear at present, and the current standard quality control standard is low. Therefore, it is urgent to conduct a more in-depth study of the effective ingredients to further improve the quality control standards and ensure the safety and effectiveness of the clinical application of fresh bamboo sap. Relevant studies have shown that fresh bamboo sap mainly contains chemical components such as amino acids, sugars, phenols, alcohols, aldehydes, organic acids, phenylpropanoids and lignans, and inorganic elements, but many of them are not clear about their exact composition and structure, and reports on diastereoisomers are rare. Summary of the invention

[0005] In view of the problems existing in the prior art, the present invention provides a pair of diastereoisomers in fresh bamboo sap and a preparation method and application thereof. The pair of diastereoisomers has a novel structure and pharmacological activity, providing a basis for the pharmacological research and safety of fresh bamboo sap.

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

[0007] The present invention also provides a method for preparing the above diastereomers, comprising the following steps: (1) Cutting the bamboo, dry distilling, and concentrating under reduced pressure to obtain an extract for later use; (2) Take an appropriate amount of extract, extract it with ethyl acetate and n-butanol for 3 to 4 times in sequence, and concentrate it to obtain the ethyl acetate part and the n-butanol part; (3) The n-butanol fraction was passed through a D101 macroporous resin column and washed with 10%, 30%, 60%, and 90% ethanol aqueous solutions, respectively. Each component was collected and concentrated under reduced pressure to obtain a 60% ethanol fraction of the macroporous resin; (4) The 60% ethanol fraction was subjected to silica gel column chromatography with a gradient elution of dichloromethane-methanol as the eluent. After thin layer chromatography inspection, similar components were combined to finally obtain six components A, B, C, D, E, and F; (5) Component B was subjected to silica gel column chromatography with petroleum ether-ethyl acetate as the eluent for gradient elution. After thin layer chromatography, similar components were combined to obtain four components B1 to B4; (6) Preparing component B2 by reverse phase preparative HPLC to obtain diastereomers; (7) The phenolic compound is subjected to chiral separation by reverse phase high performance liquid chromatography to obtain diastereoisomers of formula I and formula II.

[0008] Furthermore, in step (1) of the above technical solution, the temperature of the carbonization is 120°C, the pressure of the reduced pressure concentration is 0.7 MPa, and the temperature is 60°C.

[0009] Furthermore, 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.

[0010] Furthermore, in step (4) of the above technical solution, the pore size of the silica gel column chromatography is 100-200 mesh; during gradient elution, the volume ratio of dichloromethane to methanol is 40:1, 30:1, 20:1, 10:1, and 5:1, respectively.

[0011] Furthermore, in step (5) of the above technical solution, during gradient elution, the volume ratios of petroleum ether to ethyl acetate are 5:1, 4:1, 3:1, 2:1, 1:1, and 0:1, respectively.

[0012] Furthermore, in step (6) of the above technical solution, the reverse phase preparative high performance liquid chromatography uses acetonitrile-water in a volume ratio of 24:76 as the mobile phase, and the flow rate is 10 mL / min.

[0013] Furthermore, in step (7) of the above technical solution, the reversed-phase high performance liquid chromatography uses a Phenomenex LuxCellulose-4 (250 mm×4.6 mm, 5 μm) chiral semi-preparative chromatography column and acetonitrile-water with a volume ratio of 24:76 as the mobile phase.

[0014] The present invention also provides a pharmaceutical composition comprising the compound of formula I and a pharmaceutically acceptable carrier.

[0015] The present invention also provides an application of the above formula I or pharmaceutical composition in the preparation of drugs for preventing or treating tumor diseases, wherein the tumors include human gastric cancer cells, human liver cancer cells, human colorectal cancer cells, human bladder cancer cells and human lung cancer cells.

[0016] Advantages compared to the prior art: 1. The present invention uses fresh bamboo sap as a raw material, and successfully obtains a pair of diastereoisomer compounds (+)-fresh bamboo sap phenol A and (-)-fresh bamboo sap phenol A through dry distillation, reduced pressure concentration, extraction, macroporous resin column separation, two silica gel column chromatography separations, reverse phase preparative high performance liquid separation, and finally reverse phase high performance liquid chromatography chiral separation. The preparation method is simple and fast to operate.

[0017] 2. In vitro cell tests show that (+)-bamboo phenol A has a significant inhibitory effect 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 to prepare drugs for preventing or treating tumors, with broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is a chromatogram of the diastereoisomers of the present invention, wherein the left peak is (+)-fresh bamboo phenol A, and the right peak is (-)-fresh bamboo phenol A; Figure 2 is a high-resolution mass spectrum of the diastereomers of the present invention; Figure 3 is the UV spectrum of the diastereomers of the present invention; Figure 4 The diastereomers of the present invention are 1 H NMR spectrum; Figure 5 The diastereomers of the present invention are 13 C NMR spectrum; Figure 6 is the TOCSY spectrum of the diastereomer of the present invention; Figure 7 is the HMBC spectrum of the diastereomers of the present invention; Figure 8 is the HSQC spectrum of the diastereomers of the present invention; Fig. 9 The ECD spectrum of the diastereoisomer (+)-fresh bamboo phenol A of the present invention; Fig.10 The ECD spectrum of the diastereoisomer (-)-fresh bamboo phenol A of the present invention is shown in FIG. DETAILED DESCRIPTION

[0019] The above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form new or preferred technical solutions, but the present invention is not limited to these embodiments, and these embodiments do not limit the present invention in any way.

[0020] The experimental methods in the following examples are conventional methods unless otherwise specified. The preparations involved in the following examples are common commercial products unless otherwise specified and can be purchased from the market.

[0021] The bamboo of the present invention was collected from Tonggu County, Yichun City, Jiangxi Province, and was identified as a plant of the genus Phyllostachys in the Gramineae family by Wu Ziping, a senior experimenter of Tonggu County Weisheng Industrial Co., Ltd. Phyllostachys edulis (Carr.) Fresh stems of H. de Lehaie.

[0022] The present invention is further described in detail below in conjunction with the drawings and embodiments: Example 1: Preparation of diastereomers The method for preparing diastereomers comprises the following steps: (1) 10 t of bamboo was cut, dry distilled at 120°C, and concentrated under reduced pressure at 60°C and 0.7 MPa to obtain an extract (96 kg), which was set aside; (2) Take 37.8 kg of the extract, extract it with ethyl acetate and n-butanol three times (50 L each time), and concentrate it to obtain the ethyl acetate part (310 g) and the n-butanol part (857 g); (3) 857 g of the n-butanol fraction was passed through a D101 macroporous resin column and washed with 10%, 30%, 60%, and 90% ethanol aqueous solutions, respectively. The fractions were collected and concentrated under reduced pressure to obtain a 60% ethanol fraction of the macroporous resin (55 g); (4) 55 g of the 60% ethanol fraction was subjected to silica gel column chromatography (100-200 mesh, 1000 g, inner diameter 80 mm, wet column packing) with dichloromethane-methanol as the eluent for gradient elution, wherein the volume ratio of dichloromethane to methanol was 40:1, 30:1, 20:1, 10:1, and 5:1, respectively. After thin layer chromatography inspection, similar components were combined to finally obtain six components A, B, C, D, E, and F; (5) Component B (1.3 g) was subjected to silica gel column chromatography using petroleum ether-ethyl acetate as the eluent for gradient elution, wherein the volume ratio of petroleum ether-ethyl acetate was 5:1, 4:1, 3:1, 2:1, 1:1, and 0:1, respectively. After thin layer chromatography, similar components were combined to obtain four components B1 to B4; (6) Component B2 (60 mg) was prepared by reverse phase preparative HPLC (acetonitrile-water (24:76) as the mobile phase, flow rate of 10 mL / min) to obtain diastereomers (5 mg).

[0023] Example 2: Separation and structural identification of diastereomers 1. Split The diastereoisomer obtained in Example 1 was dissolved in 1 mL of methanol and subjected to reverse phase high performance liquid chromatography using a Phenomenex Lux Cellulose-4 (250 mm × 4.6 mm, 5 μm) chiral semi-preparative column and acetonitrile-water (24:76) as the mobile phase to obtain compounds of formula I and formula II, which were named (+)-fresh bamboo phenol A and (-)-fresh bamboo phenol A, respectively. The chromatograms are shown in FIG. Figure 1 shown.

[0024] 2. Structural analysis (±)-Fresh bamboo juice ether A is a yellow powder, soluble in methanol; HRESIMS was measured by Waters ACQUITY UPLC / Xevo G2 Q TOF (HR-Q-TOF-MS: Waters Co., Ltd., 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 H 24 O5, where the high-resolution mass spectrum is as follows Figure 2 As shown, other confirmed spectra are as follows Figures 3 to 10 As shown, (±)-Fresh bamboo phenol A 1 H-NMR, 13 The C-NMR data are shown in Table 1.

[0025] Table 1 (±)-Fresh bamboo extract phenol A 1 H-NMR, 13 C-NMR data

[0026] a Recorded in DMSO( 1 H NMR 600 MHz, 13C NMR 150 MHz). 1 H NMR spectrum ( Figure 4 ) gives the substituted hydrogen signals of benzene rings 1, 3, 4, and 5: 6.52 (2H, s, H-2′, 6′); tertiary hydrogen signal: 4.14 (dd, J = 7.8, 4.8 Hz, 1H, H-1); methoxy signal: 3.73 (6H, s, 3′, 5′-OCH3); and a group of methylene hydrogen signals: 3.48 (1H, dd, J = 11.4, 7.8 Hz, H-2) and 3.35 (1H, dd,J = 11.4, 4.8 Hz, H-2). In addition, 1 H NMR also gave a set of characteristic signals of n-butyl oxygen: 3.27 (2H, m, H-1′′), 1.47 (2H, m, H-2′′), 1.32 (2H, m, H-3′′) and 0.84 (3H, t, J = 7.4 Hz, H-4′′). 13 C NMR spectrum ( Figure 5 ), 14 carbon signals are displayed, of which 130.4 (C-1′), 104.0 (C-2′, 6′), 147.8 (C-3′, 5′), 134.7 (C-4′) are benzene ring carbon signals; 83.1 (C-1) is a tertiary carbon signal; 66.2 (C-2) is a methylene carbon signal; the carbon spectrum data also shows 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). Analysis of the above 1 H NMR, 13 CNMR and HR-Q-TOF-MS data show that the compound is a phenolic compound.

[0027] In the HMBC spectrum ( Figure 7 ), δ H 3.73 (6H, s, 3′, 5′-OCH3) and δ C 147.8 (C-3′, 5′), indicating that the methoxy group is connected to the C-3′ and C-5′ positions; δ H 4.14 (dd, J = 7.8, 4.8 Hz, H-1) and δ C66.2 (C-2), 104.0 (C-2′, 6′), 130.4 (C-1′), indicating that C-1 is connected to the C-1′ position of the benzene ring; δ H 3.48 (dd, J = 11.3,7.6 Hz, Ha-2), 3.35 (dd, J = 11.3, 4.4 Hz, Hb-2) and δ C 67.8 (C-1′′) are also correlated, indicating that C-2 is connected to the oxygen-n-butyl group. Therefore, the compound was identified as 1-hydroxy-2-butoxy-1-(4-hydroxy-3,5-dimethoxyphenyl)-ethane, and its HMBC (H→C) main related signals are as follows: .

[0028] The ECD spectrum of the compound measured by circular dichroism spectrometer showed no obvious trend, and it was speculated that it might be a mixture. Chiral column analysis confirmed that it was indeed a mixture.

[0029] (+)-Fresh bamboo phenol A is yellow powder, dissolved in methanol, and measured by Perkin-Elmer 341 polarimeter (Perkin-Elmer Co., Ltd., USA). -73° (c 0.022, MeOH); JASCO J-815 circular dichroism spectrometer (JASCO Co., Ltd., Japan) was used to measure ECD (MeOH) λ max (Δε)213(-11.03), 272(3.14)nm( Fig. 9 ). By comparing the optical rotation with (2R)-amino-2-phenylpropanoic acid [(2R)-amino-2-phenylpropanoic acidl], the optical rotations of the two are similar, so the absolute configuration of (+)-fresh bamboo phenol A is designated as R.

[0030] (-)-Fresh bamboo phenol A is yellow powder, dissolved in methanol, and measured by Perkin-Elmer 341 polarimeter (PERKINELMER Co., Ltd., USA). +32° (c 0.02, MeOH); JASCO J-815 circular dichroism spectrometer (JASCO Co., Ltd., Japan) was used to measure ECD (MeOH) λ max (Δε)218(-8.77),272(-3.52)nm( Fig.10). By comparing the optical rotation with (S)-Methoxy-(3,5-dimethoxy-4-hydroxyphenyl)ethanediol, the optical rotations of the two were similar, so the absolute configuration of (-)-fresh bamboo phenol A was designated as S.

[0031] Example 3: Anti-tumor cell activity screening Test principle: MTT method: 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-purple 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 570nm and 630nm using a microplate reader. The optical density value is proportional to the number of living cells.

[0032] 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).

[0033] Test method: MTT method: Take logarithmically growing cells, digest them, and fully blow them into a single cell suspension. After counting, dilute them into 1×10 5 / mL, inoculated in a 96-well culture plate, 100μL of cell suspension was added to each well, and the cells were placed in an incubator at 37℃ and 5% CO2 for 24 hours, and the original culture medium was discarded. Four concentration gradients were designed for each sample, and then 100μL of culture medium containing samples of each concentration gradient and paclitaxel (positive control) was added to the test wells, with 6 parallel wells for each concentration; an equal volume of solvent was added to the control group. After the 96-well culture plate was placed in a saturated humidity incubator at 37℃ and 5% CO2 for 72 hours, 20μL of freshly prepared serum-free culture medium containing 5mg / mL MTT was added to each well, and the supernatant was removed after further culture at 37℃ for 4 hours. 150μL of DMSO was added to each well to dissolve the Formazan precipitate, shaken for 5 minutes, and the absorbance at 570nm and 630nm was measured on an enzyme reader to reflect the number of living cells. The calculation formula is as follows: Tumor cell growth inhibition rate (%) = (control well measurement value-test well measurement value) / control well measurement value × 100%. The test results are shown in Tables 1 to 5.

[0034] Table 1 Detection results of the growth inhibition rate of gastric cancer cells (SGC7901) at different concentrations

[0035] Table 2 Detection results of the growth inhibition rate of liver cancer cells (SMMC-7721) at different concentrations of the compounds

[0036] Table 3 Detection results of the growth inhibition rate of colorectal cancer cells (HCT116) at different concentrations

[0037] Table 4 Detection results of the inhibition rate of the compounds on the growth of bladder cancer cells (BIU87) at different concentrations

[0038] Table 5 Detection results of the growth inhibition rate of lung cancer cells (SPCA-1) at different concentrations of the compounds

[0039] From the above test results, it can be seen that (+)-fresh bamboo phenol A has a significant inhibitory effect 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 (+)-fresh bamboo phenol A has a certain potential anti-tumor activity; while (-)-fresh bamboo phenol A has no inhibitory effect on the proliferation of the above cells.

[0040] Finally, it should be emphasized that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various changes and modifications. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A pair of diastereoisomers, characterized in that The chemical structural formulas of the diastereomers are shown in Formula I and Formula II: 。 2. A method for preparing diastereomers according to claim 1, characterized in that: The following steps are involved: (1) Cutting the bamboo, dry distilling, and concentrating under reduced pressure to obtain an extract for later use; (2) Take an appropriate amount of extract, extract it with ethyl acetate and n-butanol for 3 to 4 times in sequence, and concentrate it to obtain the ethyl acetate part and the n-butanol part; (3) The n-butanol fraction was passed through a D101 macroporous resin column and washed with 10%, 30%, 60%, and 90% ethanol aqueous solutions, respectively. Each component was collected and concentrated under reduced pressure to obtain a 60% ethanol fraction of the macroporous resin; (4) The 60% ethanol fraction was subjected to silica gel column chromatography with a gradient elution of dichloromethane-methanol as the eluent. After thin layer chromatography inspection, similar components were combined to finally obtain six components A, B, C, D, E, and F; (5) Component B was subjected to silica gel column chromatography with petroleum ether-ethyl acetate as the eluent for gradient elution. After thin layer chromatography, similar components were combined to obtain four components B1 to B4; (6) Preparing component B2 by reverse phase preparative HPLC to obtain diastereomers; (7) The phenolic compound is subjected to chiral separation by reverse phase high performance liquid chromatography to obtain diastereoisomers of formula I and formula II.

3. The method for preparing a diastereomer according to claim 2, characterized in that: In step (1), the temperature of carbonization is 120°C, the pressure of reduced pressure concentration is 0.7 MPa, and the temperature is 60°C.

4. The method for preparing a diastereomer according to claim 2, characterized in that: In step (2), the mass volume ratio of the extract to ethyl acetate or n-butanol is 1:1-1.

4.

5. The method for preparing a diastereomer according to claim 2, characterized in that: In step (4), the pore size of the silica gel column chromatography is 100-200 mesh; during gradient elution, the volume ratio of dichloromethane to methanol is 40:1, 30:1, 20:1, 10:1, and 5:1, respectively.

6. The method for preparing a diastereomer according to claim 2, characterized in that: In step (5), during gradient elution, the volume ratios of petroleum ether to ethyl acetate are 5:1, 4:1, 3:1, 2:1, 1:1, and 0:1, respectively.

7. The method for preparing a diastereomer according to claim 2, characterized in that: In step (6), the reverse phase preparative HPLC uses acetonitrile-water in a volume ratio of 24:76 as the mobile phase at a flow rate of 10 mL / min.

8. The method for preparing a diastereomer according to claim 2, characterized in that: In step (7), the reverse phase high performance liquid chromatography uses Phenomenex Lux Cellulose-4 as a chiral semi-preparative column and acetonitrile-water with a volume ratio of 24:76 as the mobile phase.

9. A pharmaceutical composition, characterized in that The diastereomer comprises the diastereomer of formula I as claimed in claim 1 and a pharmaceutically acceptable carrier.

10. Use of the diastereoisomer of formula I as claimed in claim 1 or the pharmaceutical composition as claimed in claim 9 in the preparation of a drug for preventing or treating tumor diseases, characterized in that: The tumors include 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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