A phenol diastereomer and its preparation method and application

Abstract

The present invention belongs to the field of traditional Chinese medicine biotechnology, and specifically relates to a phenolic diastereomer, a preparation method, and an application thereof. The phenolic diastereomer has the chemical structures of Formula I and Formula II, wherein Formula I is the dextrorotatory isomer, named (+)-fresh bamboo phenol D, and Formula II is the levorotatory isomer, named (-)-fresh bamboo phenol D. The present invention successfully obtains a pair of phenolic diastereomer compounds, (+)-fresh bamboo phenol D and (-)-fresh bamboo phenol D, from fresh bamboo phenol. Anti-neurotoxicity activity tests show that (+)-fresh bamboo phenol D has a significant protective effect against PC12 cell damage caused by glutamate. It can be used to prepare drugs for preventing or treating neurodegenerative diseases, and has broad application prospects.

Description

Technical Field

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

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

[0003] Recent studies have revealed that the pharmacological activity of fresh bamboo sap is closely related to phenolic compounds. However, current research on fresh bamboo sap focuses primarily on its pharmacological properties, leaving the specific structure of the active ingredients unclear. Fresh bamboo sap has a complex and diverse material base, primarily containing amino acids, sugars, phenols, alcohols, aldehydes, organic acids, phenylpropanoids and lignans, and inorganic elements. The relative content of phenols significantly influences its efficacy and safety. Phenolic compounds commonly contain chiral centers, but traditional analytical methods can only detect the total amount and cannot distinguish between diastereoisomers. The long-standing stagnation in analyzing the total amount of phenols in fresh bamboo sap can easily overlook the dual effects of chiral differences on efficacy or toxicity. Therefore, analyzing the isomers of the active ingredients in fresh bamboo sap can further improve the quality control standards of fresh bamboo sap and ensure its safety and efficacy in clinical applications. Summary of the Invention

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

[0005] The present invention provides a method for preparing phenol diastereomers, comprising the following steps:

[0006] (1) Cut the bamboo, dry distill, and concentrate under reduced pressure to obtain an extract, which is then set aside;

[0007] (2) Take an appropriate amount of extract, extract it with ethyl acetate and n-butanol 3 to 4 times respectively, and concentrate to obtain the ethyl acetate portion and the n-butanol portion;

[0008] (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. The fractions were collected and concentrated under reduced pressure to obtain a 60% ethanol fraction of the macroporous resin.

[0009] (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, similar components were combined to obtain six components A, B, C, D, E, and F.

[0010] (5) Group B was passed through a silica gel column with a gradient elution using petroleum ether-ethyl acetate as the eluent. After thin layer chromatography, similar components were combined to obtain four components B1 to B4;

[0011] (6) Component B4 was passed through gel and eluted with methanol to obtain three components B4.1 to B4.3;

[0012] (7) B4.1 was subjected to reverse phase preparative high performance liquid chromatography to obtain the compound;

[0013] (8) The compound was subjected to chiral separation by reverse-phase high performance liquid chromatography to obtain a pair of phenolic diastereomers.

[0014] The phenolic diastereoisomers have chemical structural formulas such as formula I and formula II, wherein formula I is a dextrorotatory isomer, named (+)-fresh bamboo phenol D, and formula II is a levorotatory isomer, named (-)-fresh bamboo phenol D.

[0015] .

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

[0017] Furthermore, in step (2) of the above technical solution, the mass volume ratio of the extract to ethyl acetate or n-butanol is 1:1.3-1.5, preferably 1:1.32.

[0018] 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.

[0019] 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.

[0020] Furthermore, in step (7) of the above technical solution, the reverse phase preparative high performance liquid chromatography uses acetonitrile-water in a volume ratio of 27:73 as the mobile phase, and the flow rate is 7 mL / min.

[0021] Furthermore, in step (8) of the above technical solution, the reversed-phase high performance liquid chromatography uses Phenomenex LuxCellulose-4 as a chiral semi-preparative column and acetonitrile-water with a volume ratio of 35:65 as the mobile phase.

[0022] The present invention also provides a phenol diastereomer prepared by the method.

[0023] The present invention also provides a phenolic compound having the chemical structural formula shown in Formula I above.

[0024] The present invention also provides a pharmaceutical composition comprising the above phenolic compound and a pharmaceutically acceptable carrier.

[0025] The present invention also provides a use of the above-mentioned phenolic compound or pharmaceutical composition in the preparation of a drug for preventing or treating neurodegenerative diseases, wherein the neurodegenerative diseases include Parkinson's disease and Alzheimer's disease.

[0026] Advantages compared to existing technologies:

[0027] 1. The present invention uses fresh bamboo sap as raw material, and obtains a compound through a series of steps such as dry distillation, reduced pressure concentration, extraction, macroporous resin column separation, two silica gel column chromatography separations, one-time gelation, and reverse phase preparative high performance liquid chromatography. Finally, chiral separation is performed by reverse phase high performance liquid chromatography to successfully obtain a pair of phenolic diastereomers (+)-fresh bamboo sap phenol D and (-)-fresh bamboo sap phenol D.

[0028] 2. Anti-neurotoxicity tests showed that (+)- and (-)-bamboo phenol D exhibited protective effects against glutamate-induced PC12 cell damage. Furthermore, the protective effect of (+)-bamboo phenol D increased with increasing drug concentration, while the protective effect of (-)-bamboo phenol D remained weak even at increasing concentrations. Therefore, (+)-bamboo phenol D may be used for the prevention or treatment of neurodegenerative diseases, providing new evidence for the pharmacological research of fresh bamboo phenol. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The figure is a chromatogram of the phenolic diastereomers of the present invention, wherein the left peak is (+)-fresh bamboo phenol D and the right peak is (-)-fresh bamboo phenol D;

[0030] Figure 2 is a high-resolution mass spectrum of the phenol diastereomers of the present invention;

[0031] Figure 3 is the UV spectrum of the phenol diastereomers of the present invention;

[0032] Figure 4 The phenol diastereomers of the present invention 1 H NMR spectrum;

[0033] Figure 5 The phenol diastereomers of the present invention 13 C NMR spectrum;

[0034] Figure 6 is the TOCSY spectrum of the phenol diastereomers of the present invention;

[0035] Figure 7 is the HMBC spectrum of the phenol diastereomers of the present invention;

[0036] Figure 8 HSQC spectra of phenol diastereomers of the present invention;

[0037] Figure 9 is the NOESY spectrum of the phenol diastereomers of the present invention;

[0038] Figure 10 The ECD spectrum of (+)-fresh bamboo phenol D, a phenolic diastereomer of the present invention;

[0039] Figure 11 The ECD spectrum of (-)-fresh bamboo phenol D, a phenolic diastereomer of the present invention. DETAILED DESCRIPTION

[0040] 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.

[0041] The experimental methods in the following examples are conventional methods unless otherwise specified. The preparations involved in the following examples are common commercial products and can be purchased from the market unless otherwise specified.

[0042] The bamboo used in the present invention is collected from Tonggu County, Yichun City, Jiangxi Province, and is a plant of the genus Phyllostachys in the Poaceae family. Phyllostachys edulis (Carr.) Fresh stems of H. de Lehaie.

[0043] The present invention is further described in detail below with reference to the accompanying drawings and examples:

[0044] Example 1: Preparation of phenolic diastereomers

[0045] The preparation method of phenol diastereomers comprises the following steps:

[0046] (1) 10 t of bamboo was cut and 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;

[0047] (2) Take 37.8 kg of extract and extract it with ethyl acetate and n-butanol three times (50 L each time), and concentrate to obtain ethyl acetate (310 g) and n-butanol (857 g);

[0048] (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 (55 g) of the macroporous resin.

[0049] (4) 55 g of the 60% ethanol fraction was chromatographed on a silica gel column (100-200 mesh, 1000 g, inner diameter 80 mm, wet packing) using 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, similar components were combined to obtain six components A, B, C, D, E, and F.

[0050] (5) Component B (1.3 g) was subjected to silica gel column chromatography (100-200 mesh, 40 g, inner diameter 20 mm, wet packing) 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-B4;

[0051] (6) Component B4 was passed through Sephadex LH-20 gel and eluted with methanol to obtain three components: B4.1, B4.2, and B4.3;

[0052] (7) B4.1 (120 mg) was subjected to reverse phase preparative HPLC using an XTerra prep MSC column. 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 phenolic compounds (20 mg).

[0053] Example 2: Separation and structural identification of phenolic diastereomers

[0054] 1. Split

[0055] The phenolic compound 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 (35:65) as the mobile phase to obtain compounds of Formula I and Formula II, which were named (+)-fresh bamboo phenol D and (-)-fresh bamboo phenol D, respectively. The chromatograms are shown in FIG. Figure 1 As shown, the left peak is (+)-fresh bamboo phenol D, and the right peak is (-)-fresh bamboo phenol D.

[0056]

[0057] 2. Structural analysis

[0058] (±)-Fresh bamboo phenol D was a yellow powder soluble in methanol. HRESIMS m / z 435.2040 [M–H]–(calcd. for C 23 H 31 O8, 435.2019), and its molecular formula was determined to be C 23 H 32 O8, where the high-resolution mass spectrum is as follows Figure 2 As shown, other confirmed spectra are as follows Figures 3 to 11 As shown, (±)-fresh bamboo phenol D 1 H-NMR, 13 The C-NMR data are shown in Table 1.

[0059] Table 1 (±)-Fresh bamboo phenol D 1 H-NMR, 13 C-NMR data

[0060]

[0061] a Recorded in CD3OD ( 1 H NMR 600 MHz, 13 C NMR 150 MHz).

[0062] 1 The signal fragments 6.28 (2H, s, H-2′′, 6′′) and 6.25 (2H, s, H-2′,6′) given in the H NMR spectrum indicate that there are two 1, 3, 4, or 5-substituted benzene rings in the structure of the compound; 4.35 (1H, d, J = 7.8 Hz, H-3) and 2.87 (1H, td, J = 7.8, 4.9 Hz, H-2) are two oxymethylene hydrogen signals; 3.83 (1H, dd, J = 10.2,4.8 Hz, H-1) and 3.75 (1H, m, H-1) are hydrogen signals of oxymethylene; 3.61 (6H, s, 3′, 5′-OCH3) and 3.60 (6H, s, 3′′, 5′′-OCH3) are four methoxy hydrogen signals; 3.22 (2H, m, H-1′′′), 1.46 (2H,m, H-2′′′), 1.35 (2H, m, H-3′′′) and 0.85 (3H, t,J = 7.4 Hz, H-4′′′) is the signal of the oxygen-linked n-butyl group. 13 C NMR showed 17 carbon signals, including 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); the remaining three carbon signals: 62.3 (C-1), 54.5 (C-2), 82.9 (C-3). 1 H NMR, 13 C NMR and HR-Q-TOF-MS data showed that the compound was a phenolic compound.

[0063] 1 In the H NMR spectrum, J 2,3 = 7.8Hz, it can be inferred that the relative configuration of the compound is threo. In addition, the NOESY spectrum does not show 4.35 (1H, d, J = 7.8 Hz, H-3) and 2.87 (1H, td, J = 7.8, 4.9 Hz, H-2) has related signals, which also proves that its relative configuration is Threu. In the HMBC spectrum, δ H 4.35 (1H, d, J = 7.8 Hz, H-3) and δ C 67.6 (C-1′′′), 130.9 (C-1′′) are correlated, indicating that C-3 is connected to the n-butyl oxygen group and the C-1′′ position of the benzene ring; δ H 2.87 (1H, td, J =7.8, 4.9 Hz, H-2) and δ C 130.7 (C-1′), indicating that C-2 is connected to the C-1′ position of the benzene ring. Therefore, the compound was identified as threo-2,3-bis-(3,5-dimethoxy-4-hydroxy)phenyl-3-butoxypropanol. Its main HMBC (H→C) related signals are shown below:

[0064] .

[0065] The CD spectrum of the compound measured by circular dichroism spectrometry showed no obvious trend, suggesting that it might be a mixture. Chiral column analysis also confirmed that it was indeed a mixture.

[0066] (+)-Fresh bamboo phenol D is a yellow powder, which is soluble in methanol and measured by Perkin-Elmer 341 polarimeter (PERKINELMER Co., Ltd., USA). +14° ( c 0.04, MeOH); ECD (MeOH) λ was measured by JASCO J-815 circular dichroism spectrometer (JASCO Co., Ltd., Japan) max (Δε) 252 (23.00), 277 (19.27) nm. Its ECD at 252 nm and 277 nm showed positive Cotton effects similar to those of eucophenolic D. Combined with its relative configuration being threo, its absolute configuration was determined to be 2R, 3R.

[0067] (-)-Fresh bamboo phenol D is a yellow powder, which is soluble in methanol and measured by Perkin-Elmer 341 polarimeter (PERKINELMER Co., Ltd., USA). -29° ( c 0.04, MeOH); ECD (MeOH) λ was measured by JASCO J-815 circular dichroism spectrometer (JASCO Co., Ltd., Japan) max (Δε) 246 (-27.97), 277 (-12.67) nm. The negative Cotton effect shown by its ECD at 246 nm and 277 nm is similar to that of eucophenolic C. Combined with its threo-relative configuration, its absolute configuration was determined to be 2S, 3S.

[0068] Example 3: Study on anti-neurotoxicity activity

[0069] Glutamic acid (Glu) is the primary free amino acid in the central nervous system and a crucial excitatory neurotransmitter. However, excessive release can damage cellular components, including mitochondria, leading to elevated levels of reactive oxygen species and metabolic disorders. Excessive accumulation of Glu can lead to neuronal degeneration and neurological diseases such as Parkinson's disease and Alzheimer's disease, both of which are associated with high concentrations of glutamate in the brain. This study aimed to establish a neuronal cell damage model by inducing Glu in rat adrenal pheochromocytoma cell lines, PC12. The study then investigated the protective effects of two novel compounds on Glu-induced neuronal damage, aiming to provide a theoretical basis for the treatment and application of fresh bamboo juice phenolics for neurodegenerative diseases.

[0070] Experimental method: PC12 cells (simulating neuronal cells) in logarithmic growth phase were taken, the original culture medium was discarded, and 5×10 3 10 cells / well were seeded into a 96-well plate in DMEM medium supplemented with 5% fetal bovine serum, 5% horse serum, penicillin (100 IU / mL), streptomycin (100 μg / mL), and L-glutamine (2 μM). The cells were cultured in a 37°C CO2 incubator for 24 hours. The monomeric compound was then added at final concentrations of 10 μM, 1 μM, and 0.1 μM. Glutamate was also added to the culture medium to a final concentration of 20 μM, and the cells were cultured for 24 hours. This constituted the sample group. In the glutamate model group, PC12 cells in the logarithmic growth phase were cultured with the original culture medium discarded and cultured in DMEM complete medium supplemented with 20 μM glutamate. In the blank control group, cells were cultured in DMEM supplemented with 5% fetal bovine serum and 5% horse serum for 24 hours.

[0071] After 24 hours of incubation, MTT (final concentration 0.5 mg / mL) was added to each well and incubated for an additional 4 hours. The supernatant was removed, and 150 μL of DMSO was added to each well. The cells were shaken on a microplate shaker for 5 minutes. The optical density at 570 nm was measured on a BIORAD 550 microplate reader. Cell viability was evaluated as relative protection rate, calculated as follows:

[0072] .

[0073] The test results are shown in Table 1.

[0074] Table 1 Detection results of the Glu-induced cell damage activity of PC12 cells at different concentrations of the compounds

[0075]

[0076] From the above test results, it can be seen that (+)-fresh bamboo phenol D and (-)-fresh bamboo phenol D both have a certain protective effect on PC12 cell damage caused by glutamate, but the protective effect of (-)-fresh bamboo phenol D on nerve cells remains weak as the drug concentration increases; while the protective effect of (+)-fresh bamboo phenol D on nerve cells gradually increases with the increase of drug concentration, indicating that (+)-fresh bamboo phenol D has a significant protective effect on nerve cells and can be used for the prevention or treatment of neurodegenerative diseases.

[0077] In summary, the phenolic diastereoisomer compound (±)-fresh bamboo phenol D obtained in the present invention, among which (+)-fresh bamboo phenol D has a significant protective effect on PC12 cell damage caused by glutamate, can be used to prepare drugs for preventing or treating neurodegenerative diseases, and has broad application prospects.

[0078] 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. Use of a phenolic compound in the preparation of a drug for preventing or treating neurodegenerative diseases, characterized in that: The phenolic compound has a chemical structure as shown in Formula I: 。 2. Use of a pharmaceutical composition comprising a phenolic compound in the preparation of a drug for preventing or treating neurodegenerative diseases, characterized in that: The neurodegenerative disease is Parkinson's disease or Alzheimer's disease, and the phenolic compound has a chemical structure as shown in Formula I: 。

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