Phenolic diastereoisomer as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of traditional Chinese medicine biology, and particularly relates to a phenolic diastereoisomer and a preparation method and application thereof.The phenolic diastereoisomer has the chemical structural formula shown in the formula I and the formula II, the formula I is dextroisomer and is named as (+)-fresh bamboo juice phenol D, and the formula II is levoisomer and is named as (-)-fresh bamboo juice phenol D. A pair of phenolic diastereoisomer compounds (+)-fresh bamboo juice phenol D and (-)-fresh bamboo juice phenol D are successfully obtained from fresh bamboo juice, and an anti-neurotoxicity activity test shows that (+)-fresh bamboo juice phenol D has a remarkable protection effect on PC12 cell injury caused by glutamic acid and can be used for preparing medicines for preventing or treating neurodegenerative diseases; wide application prospects are realized.

Description

Technical Field

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

[0002] Fresh bamboo juice is a pale yellow clear liquid extracted from the fresh bamboo stems of Gramineae plants by roasting or dry distillation, and has the effects of clearing heat and resolving phlegm, relieving cough and asthma, etc., and is widely used in the treatment of respiratory diseases.

[0003] In recent years, studies have found that its pharmacological activity is closely related to phenolic compounds. However, the current research on fresh bamboo juice mainly focuses on its pharmacological research, and the specific structure of its active pharmaceutical ingredients is not clear. The material basis of fresh bamboo juice is complex and diverse, mainly containing amino acids, sugars, phenols, alcohols, aldehydes, organic acids, phenylpropanoids and lignans, inorganic elements and other components, among which the relative content of phenols significantly affects the efficacy and safety. Phenolic compounds generally have chiral centers, but traditional analytical methods can only detect the total amount and cannot distinguish diastereoisomers. And staying at the total analysis of phenols in fresh bamboo juice for a long time is likely to ignore the dual effects of chiral differences on efficacy or toxicity. Therefore, analyzing the isomers of the active ingredients in fresh bamboo juice can further improve the quality control standard of fresh bamboo juice and ensure the safety and effectiveness of the clinical application of fresh bamboo juice. Summary of the Invention

[0004] Aiming at the problems existing in the prior art, the present invention provides a phenolic diastereoisomer, a preparation method thereof, and an application thereof. The phenolic diastereoisomer is extracted from fresh bamboo juice, and its dextrorotatory form has significant pharmacological activity against glutamate, which can provide a new basis for the pharmacological research of fresh bamboo juice.

[0005] The present invention provides a preparation method of a phenolic diastereoisomer, comprising the following steps: (1) Cutting bamboo culms, subjecting them to dry distillation, and concentrating under reduced pressure to obtain an extract, for later use; (2) Taking an appropriate amount of the extract, extracting it 3 - 4 times with ethyl acetate and n-butanol respectively, and concentrating to obtain an ethyl acetate part and an n-butanol part; (3) Passing the n-butanol part through a D101 macroporous resin column, flushing it with 10%, 30%, 60%, and 90% ethanol aqueous solutions respectively, collecting each component and concentrating under reduced pressure to obtain a 60% ethanol part of the macroporous resin; (4) Subjecting the 60% ethanol part to silica gel column chromatography, eluting it with dichloromethane - methanol as an eluent by gradient, examining it by thin layer chromatography, and combining similar components to finally obtain six components A, B, C, D, E, and F; (5) Passing the B group through a silica gel column, eluting it with petroleum ether - ethyl acetate as an eluent by gradient, examining it by thin layer chromatography, and combining similar components to obtain 4 components B1 - B4; (6) Subject component B4 to gel filtration and elute with methanol to obtain three components B4.1 to B4.3; (7) Subject B4.1 to preparative reverse-phase high performance liquid chromatography to obtain a compound; (8) Take the compound and subject it to chiral resolution by reverse-phase high performance liquid chromatography to obtain a pair of phenolic diastereoisomers.

[0006] These phenolic diastereoisomers have chemical structural formulas as shown in Formula I and Formula II, wherein Formula I is the dextrorotatory form, named (+)-phyllostachys nigra phenolic D, and Formula II is the levorotatory form, named (-)-phyllostachys nigra phenolic D.

[0007] 。

[0008] 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.

[0009] Further, in step (2) of the above technical solution, the mass-volume ratio of the extract to ethyl acetate or n-butanol is 1:1.3 - 1.5, preferably 1:1.32.

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

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

[0012] Further, in step (7) of the above technical solution, the reverse-phase preparative high performance liquid chromatography uses acetonitrile - water with a volume ratio of 27:73 as the mobile phase, and the flow rate is 7 mL / min.

[0013] 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 35:65 as the mobile phase.

[0014] The present invention also provides a phenolic diastereoisomer prepared by the above method.

[0015] The present invention also provides a phenolic compound having the chemical structural formula as shown in Formula I above.

[0016] The present invention also provides a pharmaceutical composition comprising the above phenolic compound and a pharmaceutically acceptable carrier.

[0017] The present invention also provides an application of the above-mentioned phenolic compound or pharmaceutical composition in the preparation of a medicament for preventing or treating neurodegenerative diseases, which include Parkinson's disease and Alzheimer's disease.

[0018] Advantages over the prior art: 1. The present invention uses fresh bamboo juice as a raw material, and through a series of steps such as dry distillation, vacuum concentration, extraction, macroporous resin column separation, two silica gel column chromatography separations, one gel filtration, and reverse-phase preparative high-performance liquid chromatography, compounds are obtained. Finally, through chiral separation by reverse-phase high-performance liquid chromatography, a pair of phenolic diastereoisomers, (+)-fresh bamboo juice phenol D and (-)-fresh bamboo juice phenol D, are successfully obtained.

[0019] 2. The anti-neurotoxicity activity test shows that (+)-fresh bamboo juice phenol D and (-)-fresh bamboo juice phenol D have a certain protective effect on the damage of PC12 cells caused by glutamate. At the same time, as the drug concentration increases, the protective effect of (+)-fresh bamboo juice phenol D on nerve cells increases, while the protective effect of (-)-fresh bamboo juice phenol D on nerve cells remains weak even when the concentration is increased. Therefore, (+)-fresh bamboo juice phenol D can be used for the prevention or treatment of neurodegenerative diseases, providing a new basis for the pharmacological research of fresh bamboo juice. Description of the drawings

[0020] Figure 1 It is the chromatogram of the phenolic diastereoisomers of the present invention, where the left peak is (+)-fresh bamboo juice phenol D and the right peak is (-)-fresh bamboo juice phenol D; Figure 2 It is the high-resolution mass spectrum of the phenolic diastereoisomers of the present invention; Figure 3 It is the UV spectrum of the phenolic diastereoisomers of the present invention; Figure 4 It is the 1 1H NMR spectrum of the phenolic diastereoisomers of the present invention; Figure 5 It is the 13 13C NMR spectrum of the phenolic diastereoisomers of the present invention; Figure 6 It is the TOCSY spectrum of the phenolic diastereoisomers of the present invention; Figure 7 It is the HMBC spectrum of the phenolic diastereoisomers of the present invention; Figure 8 It is the HSQC spectrum of the phenolic diastereoisomers of the present invention; Figure 9 It is the NOESY spectrum of the phenolic diastereoisomers of the present invention; Figure 10 It is the ECD spectrum of (+)-fresh bamboo juice phenol D of the phenolic diastereoisomers of the present invention; Figure 11 This is the ECD spectrum of (-)-fresh bamboo extract phenol D, a phenolic diastereoisomer of the present invention. Specific embodiments

[0021] 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.

[0022] 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.

[0023] The moso bamboo used in the present invention is collected from Tonggu County, Yichun City, Jiangxi Province, and is the fresh culm of Phyllostachys heterocycla (Carr.) Mitford cv. Pubescens Mazel ex H. de Lehaie. Phyllostachys edulis (Carr.) The fresh culm of H. de Lehaie.

[0024] The present invention will be further described in detail below with reference to the drawings and examples: Example 1: Preparation of phenolic diastereoisomers The preparation method of phenolic diastereoisomers includes the following steps: (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; (2) Take 37.8 kg of the extract, extract it with ethyl acetate and n-butanol successively three times (50 L each time), and concentrate to obtain an ethyl acetate part (310 g) and an n-butanol part (857 g); (3) Pass the 857 g of 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 (55 g); (4) Pass the 55 g of the 60% ethanol part through silica gel column chromatography (100 mesh - 200 mesh, 1000 g, inner diameter 80 mm, wet packing), and elute it with dichloromethane - methanol as the eluent in a gradient manner, where the volume ratio of dichloromethane - methanol is 40:1, 30:1, 20:1, 10:1, 5:1 in sequence. After inspection by thin layer chromatography, combine the similar components to finally obtain six components A, B, C, D, E, and F; (5) Pass the B component (1.3 g) through silica gel column chromatography (100 mesh - 200 mesh, 40 g, inner diameter 20 mm, wet packing), and elute it with petroleum ether - ethyl acetate as the eluent in a gradient manner, where the volume ratio of petroleum ether - ethyl acetate is 5:1, 4:1, 3:1, 2:1, 1:1, 0:1 in sequence. After inspection by thin layer chromatography, combine the similar components to obtain 4 components B1 - B4; (6) Subject the B4 component to Sephadex LH-20 gel filtration and elute with methanol to obtain three components: B4.1, B4.2, and B4.3. (7) Subject B4.1 (120 mg) to reversed-phase preparative high-performance liquid chromatography using an XTerra prep MSC 18 column (300 mm × 19 mm, 10 μm) with acetonitrile-water (27:73) as the mobile phase at a flow rate of 7 mL / min to obtain a phenolic compound (20 mg).

[0025] Example 2: Resolution and Structure Identification of Phenolic Diastereoisomers 1. Resolution Take the phenolic compound obtained in Example 1, dissolve it in 1 mL of methanol, and subject it to reversed-phase high-performance liquid chromatography using a Phenomenex Lux Cellulose-4 (250 mm × 4.6 mm, 5 μm) chiral semi-preparative column with acetonitrile-water (35:65) as the mobile phase to obtain Compounds I and II, which are named (+)-fresh bamboo extract phenol D and (-)-fresh bamboo extract phenol D, respectively. Their chromatograms are as Figure 1 shown. The left peak is (+)-fresh bamboo extract phenol D, and the right peak is (-)-fresh bamboo extract phenol D.

[0026]

[0027] 2. Structure Analysis (±)-Fresh bamboo extract phenol D is a yellow powder and is soluble in methanol. The HRESIMS m / z 435.2040 [M–H]– (calcd. for C 23 H 31 O8, 435.2019) was measured by Waters ACQUITY UPLC / Xevo G2 QTOF (HR-Q-TOF-MS: Waters Corporation, USA), determining its molecular formula to be C 23 H 32 O8. The high-resolution mass spectrum is as Figure 2 shown, and other confirmation spectra are as Figures 3 to 11 shown. The 1H-NMR and 1 13C-NMR data of (±)-fresh bamboo extract phenol D are shown in Table 1. 13 The 13C-NMR data of (±)-fresh bamboo extract phenol D are shown in Table 1.

[0028] Table 1 1H-NMR and 1 13C-NMR Data of (±)-Fresh Bamboo Extract Phenol D 13 The 13C-NMR data of (±)-fresh bamboo extract phenol D

[0029] aRecorded in CD3OD ( 1 H NMR 600 MHz, 13 C NMR 150 MHz). 1 The signal fragments 6.28 (2H, s, H-2′′, 6′′) and 6.25 (2H, s, H-2′,6′) given in the 1H NMR spectrum indicate the presence of two 1,3,4,5-substituted benzene rings in the structure of the compound; 4.35 (1H, d, J J = 7.8 Hz, H-3) and 2.87 (1H, td, J J = 7.8, 4.9 Hz, H-2) are the signals of two oxygenated methine hydrogens; 3.83 (1H, dd, J J = 10.2,4.8 Hz, H-1) and 3.75 (1H, m, H-1) are the signals of oxygenated methylene hydrogens; 3.61 (6H, s, 3′, 5′-OCH3) and 3.60 (6H, s, 3′′, 5′′-OCH3) are the signals of 4 methoxy hydrogens; 3.22 (2H, m, H-1′′′), 1.46 (2H,m, H-2′′′), 1.35 (2H, m, H-3′′′) and 0.85 (3H, t, J J = 7.4 Hz, H-4′′′) are the signals of oxygenated n-butyl. 13 13C NMR shows 17 carbon signals, among which there are 8 benzene ring carbon signals: 130.7 (C-1′), 106.9 (C-2′, 6′), 147.3 (C-3′, 5′), 133.8 (C-4′), 130.9 (C-1′′), 104.9 (C-2′′, 6′′), 147.2 (C-3′′,5′′), 134.3 (C-4′′); 4 n-butyl carbon signals: 67.6 (C-1′′′), 31.6 (C-2′′′), 19.1 (C-3′′′), 13.8 (C-4′′′); 2 methoxy carbon signals: 55.9 (3′, 5′-OCH3), 55.8 (3′′, 5′′-OCH3); and the remaining 3 carbon signals: 62.3 (C-1), 54.5 (C-2), 82.9 (C-3). Analyzing the above 1 1H NMR, 13 13C NMR and HR-Q-TOF-MS data, it can be known that the compound is a phenolic compound.

[0030] 1 In the 1H NMR spectrum, J 2,3= 7.8 Hz, it can be inferred that the relative configuration of the compound is threo. Moreover, in the NOESY spectrum, no correlation signals were observed between 4.35 (1H, d, J = 7.8 Hz, H-3) and 2.87 (1H, td, J = 7.8, 4.9 Hz, H-2), which also proves that its relative configuration is threo. In the HMBC spectrum, δ H 4.35 (1H, d, J = 7.8 Hz, H-3) was correlated with δ C 67.6 (C-1′′′) and 130.9 (C-1′′), indicating that C-3 is connected to the oxygen-bearing n-butyl group and the benzene ring C-1′′ position; δ H 2.87 (1H, td, J =7.8, 4.9 Hz, H-2) was correlated with δ C 130.7 (C-1′), indicating that C-2 is connected to the benzene ring C-1′ position. Therefore, the compound was identified as threo-2,3-bis-(3,5-dimethoxy-4-hydroxy)phenyl-3-butoxypropanol. The main correlation signals of its HMBC (H→C) are shown as follows: .

[0031] 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 also confirmed that it was indeed a mixture.

[0032] (+)-Sinocalamus phenolic D is a yellow powder, soluble in methanol, measured by a Perkin-Elmer 341 polarimeter (PerkinElmer, Inc., USA) +14° ( c 0.04, MeOH); the ECD (MeOH) λ was measured by a JASCO J-815 circular dichroism spectrometer (JASCO Corporation, Japan) max (Δε) 252 (23.00), 277(19.27) nm. The positive Cotton effects shown by its ECD at 252 nm and 277 nm are similar to those of eucophenolic D. Combining its relative configuration as threo, its absolute configuration was determined to be 2R, 3R.

[0033] (-)-Sinocalamus phenolic D is a yellow powder, soluble in methanol, measured by a Perkin-Elmer 341 polarimeter (PerkinElmer, Inc., USA) -29° ( c0.04, MeOH); ECD (MeOH) λ was determined using a JASCO J-815 circular dichroism spectrometer (JASCO Corporation, Japan). max (Δε) 246 (-27.97), 277 (-12.67) nm. The negative Cotton effects shown by its ECD at 246 nm and 277 nm are similar to those of eucophenolic C. Considering its relative configuration is threo, its absolute configuration was determined to be 2S, 3S.

[0034] Example 3: Activity study on anti-neurotoxicity Glutamic acid (Glu) is the main free amino acid in the central nervous system and an important excitatory neurotransmitter. However, when released in excess, it can damage cellular components including mitochondria, increase the content of reactive oxygen species or cause metabolic disorders. The massive accumulation of Glu can lead to the degeneration of nerve cells and cause neurological diseases. For example, Parkinson's disease and Alzheimer's disease are both related to the accumulation of high concentrations of glutamate in the brain. Based on this, this experiment intended to establish a nerve cell damage model by inducing rat adrenal pheochromocytoma cell line PC12 cells with Glu, and study the protective effects of two new compounds on the damaged nerve cells after Glu injury, aiming to provide a theoretical basis for the treatment and application of phenolic components in fresh bamboo juice for neurodegenerative diseases.

[0035] Experimental method: PC12 cells (simulating neuron cells) in the logarithmic growth phase were taken, and the original culture medium was discarded. They were inoculated into 96-well plates containing DMEM medium with 5% fetal bovine serum, 5% horse serum, penicillin (100 IU / mL), streptomycin (100 μg / mL) and L-glutamine (2 μM) at a density of 5×10 3 / tube, and cultured in a 37°C CO2 incubator for 24 hours. Then, monomeric compounds with final concentrations of 10 μM, 1 μM and 0.1 μM were added, and glutamate was added to the medium to a final concentration of 20 μM, and cultured for 24 hours, which was the sample group. In the glutamate model group, PC12 cells in the logarithmic growth phase were taken, and the original culture medium was discarded, and they were cultured with DMEM complete medium containing 20 μM glutamate. In the blank control group, they were cultured with DMEM medium containing 5% fetal bovine serum and 5% horse serum, and all were cultured for 24 hours.

[0036] After culturing for 24 hours in each group, MTT (final concentration 0.5 mg / mL) was added to each well and cultured for another 4 hours. The supernatant was removed, 150 μL DMSO was added to each well, and it was shaken on a micro shaker for 5 minutes. The optical density value at 570 nm was measured on a BIORAD 550 microplate reader. The cell viability was evaluated by the relative protection rate, and the calculation method was as follows: .

[0037] The test results are shown in Table 1.

[0038] Table 1 Detection results of the activities of PC12 cells damaged by Glu with the compound at different concentrations

[0039] From the above detection results, it can be seen that both (+)-phyllostachol D and (-)-phyllostachol D have a certain protective effect on the damage of PC12 cells caused by glutamate. However, the protective effect of (-)-phyllostachol D on nerve cells remains weak with the increase of the drug concentration; while the protective effect of (+)-phyllostachol D on nerve cells gradually increases with the increase of the drug concentration, indicating that (+)-phyllostachol D has a significant protective effect on nerve cells and can be used for the prevention or treatment of neurodegenerative diseases.

[0040] In summary, for the phenolic diastereoisomer compound (±)-phyllostachol D obtained in the present invention, among which (+)-phyllostachol D has a significant protective effect on the damage of PC12 cells caused by glutamate and can be used in the preparation of drugs for the prevention or treatment of neurodegenerative diseases, having broad application prospects.

[0041] 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 method for preparing phenolic diastereoisomers, characterized in that, It includes the following steps: (1) Cut, carbonize, and concentrate under reduced pressure the moso bamboo to obtain an extract for standby; (2) Take an appropriate amount of the extract, extract it 3 - 4 times with ethyl acetate and n-butanol respectively, and concentrate to obtain the ethyl acetate part and the n-butanol part; (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; (5) Pass group B through a silica gel column, 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; (6) Pass component B4 through a gel and elute it with methanol to obtain 3 components B4.1 - B4.3; (7) Subject B4.1 to preparative reversed-phase high-performance liquid chromatography to obtain a compound; (8) Take the compound, perform chiral resolution by reversed-phase high-performance liquid chromatography to obtain a pair of phenolic diastereoisomers with chemical structural formulas as shown in Formula I and Formula II: 。 2. The preparation method of the phenolic compound according to claim 1, wherein In step (1), the carbonization temperature is 120 °C, the pressure for concentration under reduced pressure is 0.7 MPa, and the temperature is 60 °C.

3. The preparation method of the phenolic compound according to claim 1, characterized in that, In step (2), for each extraction, the mass-volume ratio of the extract to ethyl acetate or n-butanol is 1:1.3 - 1.

5.

4. The method for preparing a phenolic compound according to claim 1, characterized in that, In step (4), 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.

5. The method for preparing a phenolic compound according to claim 1, characterized in that, In step (5), 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.

6. The preparation method of the phenolic compound according to claim 1, characterized in that, In step (7), the preparative reversed-phase high-performance liquid chromatography uses acetonitrile - water with a volume ratio of 27:73 as the mobile phase, and the flow rate is 7 mL / min.

7. The preparation method of the phenolic compound according to claim 1, wherein In step (8), the reversed-phase high-performance liquid chromatography uses Phenomenex Lux Cellulose-4 as the chiral semi-preparative chromatographic column, and acetonitrile - water with a volume ratio of 35:65 as the mobile phase.

8. A phenolic compound, characterized in that, Prepared by the preparation method according to any one of claims 1 - 7, and having the chemical structural formula as shown in Formula I.

9. A pharmaceutical composition, characterized in that, It contains the phenolic compound as described in claim 10. Use of a phenolic compound as described in claim 8 or a pharmaceutical composition as described in claim 9 in the preparation of a medicament for preventing or treating neurodegenerative diseases, characterized in that, ​

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