Use of phenolic compounds for the preparation of a medicament for preventing or treating anti-hepatic fibrosis

Abstract

The application belongs to the field of traditional Chinese medicine biotechnology, and particularly relates to application of a phenolic compound in preparation of a medicine for preventing or treating anti-hepatic fibrosis. A pair of phenolic diastereoisomers (+)‑fresh bamboo juice phenol C and (-)‑fresh bamboo juice phenol C are separated from fresh bamboo juice, and it is found through anti-hepatic fibrosis activity screening test that (+)‑fresh bamboo juice phenol C has a significant inhibitory effect on human hepatic stellate cell LX-2, has a clinical application potential for anti-hepatic fibrosis treatment, and can be used in preparation of a medicine for preventing or treating anti-hepatic fibrosis, thereby providing a new basis for fresh bamboo juice pharmacological research.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine biotechnology, specifically relating to the application of a phenolic compound in the preparation of drugs for the prevention or treatment of liver fibrosis. Background Technology

[0002] Liver fibrosis is a process of chronic liver injury caused by factors such as alcohol consumption, metabolic disorders, and viruses; it is a reversible wound healing response. During persistent liver damage, chronic inflammation and the continuous accumulation of extracellular matrix (ECM) lead to the gradual replacement of liver parenchyma with scar tissue, ultimately resulting in cirrhosis. Many cell types, cytokines, and miRNAs are involved in the process of liver fibrosis and cirrhosis, such as Kupffer cells, hepatic stellate cells (HSCs), and platelet-derived growth factor. The development of liver fibrosis is closely related to the activation of HSCs. During the development of chronic liver injury, quiescent HSCs are activated and further differentiate into myofibroblasts, secreting large amounts of ECM. Therefore, inhibiting the activation of HSCs has become a research hotspot for improving liver fibrosis.

[0003] Fresh bamboo sap is a pale yellow, clear liquid extracted from the fresh stems of bamboo (a member of the Poaceae family) through roasting or dry distillation. It possesses properties such as clearing heat and resolving phlegm, relieving cough and asthma, and is primarily used to treat respiratory diseases. Its material basis is complex and diverse, mainly containing amino acids, sugars, phenols, alcohols, aldehydes, organic acids, phenylpropanoids and lignans, and inorganic elements. Recent studies have found that the pharmacological activity of fresh bamboo sap is closely related to phenolic compounds. While phenolic compounds generally possess chiral centers, traditional analytical methods can only detect their total amount and cannot distinguish diastereomers, thus hindering further development of their pharmacological properties. Furthermore, there are currently no reports on the anti-liver fibrosis effects of phenols in fresh bamboo sap. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides the application of phenolic compounds in the preparation of drugs for the prevention or treatment of liver fibrosis. These phenolic compounds are extracted from fresh bamboo sap and have significant pharmacological activity in inhibiting the proliferation of hepatic stellate cells, which can provide a theoretical basis for the treatment and application of phenolic components from fresh bamboo sap in the treatment of liver fibrosis.

[0005] The purpose of this invention is to provide the application of phenolic compounds in the preparation of drugs for the prevention or treatment of liver fibrosis, wherein the structure of the phenolic compound is shown in Formula I:

[0006] .

[0007] Furthermore, the phenolic compounds are extracted from fresh bamboo sap.

[0008] Specifically, the method for preparing the phenolic compound includes the following steps:

[0009] (1) Cut the bamboo into pieces, dry distill (temperature 120℃, pressure 0.7MPa), and concentrate under reduced pressure (temperature 60℃) to obtain an extract for later use;

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

[0011] (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 the 60% ethanol fraction of the macroporous resin.

[0012] (4) The 60% ethanol fraction was subjected to silica gel column chromatography with gradient elution using dichloromethane-methanol (volume ratio 40:1, 30:1, 20:1, 10:1, 5:1) as eluent. After thin-layer chromatography examination, similar components were combined to finally obtain six components A, B, C, D, E, and F.

[0013] (5) The C component was separated by a medium-pressure reversed-phase column and eluted with a methanol:water gradient (30%, 50%, 80%, 100%) to obtain four components C1 to C4.

[0014] (6) Pass component C4 through a gel and elute with methanol to obtain component C4.1;

[0015] (7) C4.1 was prepared by reverse-phase high-performance liquid chromatography with acetonitrile-water (22:78) as the mobile phase (7 mL / min) to obtain the compound;

[0016] (8) Dissolve the compound in methanol and perform 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 (20:80) as the mobile phase to obtain the phenolic diastereomers.

[0017] The phenolic diastereomer has chemical structural formulas as shown in Formula I and Formula II, wherein Formula I is the dextrorotatory isomer, named (+)-fresh bamboo sap phenol C, which is the phenolic compound of this invention, and Formula II is the levorotatory isomer, named (-)-fresh bamboo sap phenol C.

[0018]

[0019] Furthermore, the phenolic compound is used in combination with a pharmaceutically acceptable carrier.

[0020] Furthermore, the phenolic compound is a compound with the structure shown in Formula I or a pharmaceutically acceptable salt thereof as the active pharmaceutical ingredient.

[0021] Furthermore, the concentration of the active pharmaceutical ingredient is 0.01-100 μM.

[0022] Furthermore, the phenolic compounds have the activity of inhibiting the proliferation of hepatic stellate cells.

[0023] Advantages compared to existing technologies:

[0024] This invention isolates a pair of phenolic diastereomers, (+)-bamboo sap phenol C and (-)-bamboo sap phenol C, from fresh bamboo sap. Screening tests for anti-hepatic fibrosis activity revealed that (+)-bamboo sap phenol C significantly inhibited the proliferation of human hepatic stellate cells (LX-2), with the inhibition rate showing a dose-response relationship; the inhibitory effect increased with increasing drug concentration, indicating that (+)-bamboo sap phenol C possesses potential anti-hepatic fibrosis activity. In contrast, (-)-bamboo sap phenol C did not significantly inhibit the proliferation of these cells. Therefore, (+)-bamboo sap phenol C has clinical application potential in the treatment of anti-hepatic fibrosis and can be used in the preparation of drugs for the prevention or treatment of anti-hepatic fibrosis, providing new evidence for pharmacological research on fresh bamboo sap. Attached Figure Description

[0025] Figure 1 The chromatogram of the phenolic diastereomers of the present invention is shown, wherein the left peak is (+)-bamboo sap phenol C and the right peak is (-)-bamboo sap phenol C.

[0026] Figure 2 This is a high-resolution mass spectrum of the phenolic diastereomers of the present invention;

[0027] Figure 3 This is the UV spectrum of the phenolic diastereomers of the present invention;

[0028] Figure 4 The phenolic diastereomers of this invention 1 H NMR spectrum;

[0029] Figure 5 The phenolic diastereomers of this invention 13 C NMR spectrum;

[0030] Figure 6 The phenolic diastereomers of this invention 1 H- 1 H COSY spectrum;

[0031] Figure 7 This is the HMBC spectrum of the phenolic diastereomers of the present invention;

[0032] Figure 8 This is the HSQC spectrum of the phenolic diastereomers of the present invention;

[0033] Figure 9This is the NOESY spectrum of the phenolic diastereomers of the present invention;

[0034] Figure 10 The ECD spectrum of (+)-fresh bamboo sap phenol C, the diastereomer of the phenols of this invention;

[0035] Figure 11 This is the ECD spectrum of (-)-fresh bamboo sap phenol C, the diastereomer of the phenolic compound of the present invention. Detailed Implementation

[0036] The above-described technical features of the present invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. However, the present invention is not limited to these embodiments, nor are these embodiments limited in any way.

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the formulations involved in the following examples are all commercially available products that can be purchased from the market.

[0038] The bamboo used in this invention was collected from Tonggu County, Yichun City, Jiangxi Province, and is a species of bamboo belonging to the genus Phyllostachys in the family Poaceae. Phyllostachys edulis (Carr.) Fresh stems of H. de Lehaie.

[0039] The present invention will be further described in detail below with reference to the illustrations and embodiments:

[0040] Example 1: Preparation of phenolic diastereomers

[0041] A method for preparing phenolic diastereomers includes the following steps:

[0042] (1) Cut 10t of moso bamboo, dry distill at 120℃ and 0.7MPa, and concentrate under reduced pressure at 60℃ to obtain extract (96kg), for later use;

[0043] (2) Take 37.8 kg of extract and extract it three times with ethyl acetate and n-butanol (50 L each time). Concentrate to obtain ethyl acetate fraction (310 g) and n-butanol fraction (857 g).

[0044] (3) Pass 857g of n-butanol fraction through a D101 macroporous resin column and wash with 10%, 30%, 60% and 90% ethanol aqueous solutions respectively. Collect each component and concentrate under reduced pressure to obtain 60% ethanol fraction of macroporous resin (55g).

[0045] (4) 55g of 60% ethanol was subjected to silica gel column chromatography (100-200 mesh, 1000g, inner diameter 80mm, wet packing), with dichloromethane-methanol as the eluent gradient elution, wherein the volume ratio of dichloromethane-methanol was 40:1, 30:1, 20:1, 10:1, and 5:1 respectively. After thin-layer chromatography examination, similar components were combined to finally obtain six components A, B, C, D, E, and F.

[0046] (5) The C component (2.6g) was separated by medium-pressure reverse phase column elution and eluted with a methanol:water gradient (30%, 50%, 80%, 100%) to obtain four components C1 to C4.

[0047] (6) Pass fraction C4 (260 mg) through a Sephadex LH-20 gel and elute with methanol to obtain fraction C4.1;

[0048] (7) C4.1 (150 mg) was prepared by reverse-phase high-performance liquid chromatography using an XTerra prep MSC column. 18 The phenolic compound (7.8 mg) was prepared by using an acetonitrile-water (22:78) mobile phase in a column (300 mm × 19 mm, 10 μm) at a flow rate of 7 mL / min.

[0049] Example 2: Resolution and structural identification of phenolic diastereomers

[0050] 1. Splitting

[0051] The phenolic compounds obtained in Example 1 were dissolved in 1 mL of methanol and subjected to reversed-phase high-performance liquid chromatography (RP-HPLC) using a Phenomenex Lux Cellulose-4 (250 mm × 4.6 mm, 5 μm) chiral semi-preparative column with acetonitrile-water (20:80) as the mobile phase. Compounds I and II were obtained and named (+)-bamboo sap phenol C and (-)-bamboo sap phenol C, respectively. Their chromatograms are shown below. Figure 1 As shown, the left peak is (+)-bamboo sap phenol C, and the right peak is (-)-bamboo sap phenol C.

[0052]

[0053] 2. Structural Analysis

[0054] (±)-Fresh bamboo sap phenol C is a white powder, soluble in methanol; HRESIMS m / z was measured to be 299.1491 [M–H]–(calcd. forC) using Waters ACQUITY UPLC / Xevo G2 QTOF (HR-Q-TOF-MS: Waters Inc., USA) 15 H 23O6,299.1495), its molecular formula was determined to be C 15 H 24 O6. The high-resolution mass spectrum is shown below. Figure 2 As shown, other confirmatory maps are as follows: Figures 3 to 11 As shown, (±)-fresh bamboo sap phenol C 1 H-NMR, 13 The C-NMR data are shown in Table 1.

[0055] Table 1 (±)-Fresh bamboo sap phenol C 1 H-NMR, 13 C-NMR data

[0056]

[0057] a Recorded in CD3OD ( 1 H NMR 600 MHz, 13 C NMR 150 MHz).

[0058] 1 The 1H NMR spectrum showed signals for the 1, 3, 4, and 5 substituted hydrogens of the benzene ring: 6.63 (2H, s, H-2′, 6′); methoxy signal: 3.83 (6H, s, 3′, 5′-OCH3); and signals for the two oxymethimide hydrogens: 4.12 (1H, d, J = 6.4 Hz, H-3), 3.68 (1H, m, H-2); and one set of hydroxymethylene hydrogen signals: 3.69 (1H, m, H-1), 3.57 (1H, dd, J =12.0, 7.3 Hz, H-1); In addition, 1 1H NMR also yielded a set of signals for the hydrogen atoms of the hydroxyl group n-butyl: 3.34 (1H, m, H-1′′), 3.28 (1H, m, H-1′′), 1.52 (2H, m, H-2′′), 1.38 (2H, m, H-3′′), 0.89 (3H, t, J = 7.4 Hz, H-4′′). 13C10 NMR revealed 12 carbon signals, including four benzene ring carbon signals: 131.4 (C-1′), 106.1 (C-2′, 6′), 149.1 (C-3′, 5′), 136.1 (C-4′); four n-butyl carbon signals: 69.6 (C-1′′), 33.1 (C-2′′), 20.5 (C-3′′), 14.2 (C-4′′); one methoxy carbon signal: 56.7 (3′, 5′-OCH3); and three remaining carbon signals: 64.5 (C-1), 76.3 (C-2), 84.2 (C-3). Analysis of the above... 1 H NMR, 13 According to C NMR and HR-Q-TOF-MS data, this compound is a phenolic compound.

[0059] 1 In the H NMR spectrum, H-2 / H-3 shows a small coupling constant. J 2,3 = 6.4 Hz, therefore it is inferred that the relative configuration of this compound is erythrophoric; in the NOESY spectrum, there is a related signal of H-2 / H-3, further confirming the above inference. In the HBMC spectrum, δ H 4.12(1H, d, J = 6.4 Hz, H-3) and δ C The correlation at 69.6 (C-1′′) indicates that the oxy-n-butyl group is connected to C-3. δ H 3.83 (6H, s, 3′, 5′-OCH3) and δ C The correlation at 149.1 (C-3′, 5′) indicates that the methoxy group is attached to both the C-3′ and C-5′ positions. δ H 4.12 (d, J = 6.4 Hz, H-3) and δ C The correlations of 76.3 (C-2), 106.1 (C-2′, 6′), and 131.4 (C-1′) indicate that C-3 is connected to the C-1′ position of the benzene ring; therefore, the compound is identified as erythro-3-(4-hydroxy-3,5-dimethoxyphenyl)-3-n-butoxypropane-1,2-diol. Its main HMBC (H→C) correlation signals are shown below:

[0060] .

[0061] The CD chromatogram of the compound was determined by circular dichroism spectroscopy, and the results showed no obvious trend, suggesting that it might be a mixture. Chiral column analysis also confirmed that it was indeed a mixture.

[0062] (+)-Fresh bamboo sap phenol C is a white powder, soluble in methanol, and determined by a Perkin-Elmer 341 polarimeter (PERKINELMER, Inc., USA). +25° c 0.028, MeOH); ECD (MeOH) λ was determined using a JASCO J-815 circular dichroism spectrometer (JASCO Ltd., Japan). max (Δε) 223 (-10.86), 275(-8.18)nm. Its ECD shows a negative Cotton effect at 275nm, similar to that of forsythiayanoside D. Combined with its relative configuration being erythic, its absolute configuration is determined to be 2S, 3S.

[0063] (-)-Fresh bamboo sap phenol C is a white powder, soluble in methanol, and determined by a Perkin-Elmer 341 polarimeter (PERKINELMER, Inc., USA). -53° ( c 0.022, MeOH); ECD (MeOH) λ was determined using a JASCO J-815 circular dichroism spectrometer (JASCO Ltd., Japan). max (Δε) 224 (-9.02), 275 (6.33) nm. By virtue of the positive Cotton effect shown by its ECD at 275 nm, combined with its relative configuration at positions 2 and 3 being erythroform, its absolute configuration is opposite to that of (-)-bamboo sapol C, while the absolute configuration of (+)-bamboo sapol C is specified as 2R,3R.

[0064] Example 3: Study on anti-liver fibrosis activity

[0065] Test principle: The MTT method is used for measurement.

[0066] Mitochondria in living cells contain dehydrogenases associated with NAPP (nicotinamide adenine dinucleotide phosphate, coenzyme II). Succinate dehydrogenase can reduce exogenous yellow MTT (trade name thiazolyl blue, chemical name 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) to water-insoluble blue-purple crystals of formazan, which are then deposited in the cells. In dead cells, this enzyme disappears, and MTT is not reduced. After dissolving formazan in dimethyl sulfoxide (DMSO), the absorbance can be measured at 570 nm using a microplate reader; the optical density value is directly proportional to the number of living cells.

[0067] The cell line used was human liver stellate cell line LX-2.

[0068] Experimental methods: The proliferation of human hepatic stellate cells LX-2 was detected by MTT assay. Passaged human hepatic stellate cells LX-2 were cultured in DMEM medium (RNBK3466, Sigma-Aldrich, USA) with 3% fetal bovine serum at a concentration of 1×10⁶ cells / mL. 5 Cell suspension at a density of 100 μL / mL was seeded into 96-well culture plates. Once the cells reached near-monolayer size, the medium was replaced with DMEM containing different concentrations of the compound (3% serum). The following grouping experiments were performed, with 6 replicates per group. A blank solvent without the compound served as the negative control group, while different concentrations (0.01 μM, 0.1 μM, 1 μM, 10 μM, 100 μM) of the compound served as the sample groups. The 96-well culture plates were incubated at 37°C with 5% CO2 for 24 hours. Then, 20 μL of freshly prepared stock solution containing 0.5% MTT was added to each well, and the plates were incubated at 37°C for another 4 hours. The supernatant was then removed. 100 μL of DMSO was added to each well to dissolve intracellular crystals, and the plates were shaken for 5 minutes. The absorbance at 570 nm was measured using a microplate reader to reflect the number of viable cells. The calculation formula is as follows: Cell growth inhibition rate (%) = (Measurement value of control well - Measurement value of test well) / Measurement value of control well × 100%.

[0069] The test results are shown in Tables 2 and 3.

[0070] Table 2. Effects of (+)-Fresh bamboo sap phenol C at different concentrations on the proliferation of human hepatic stellate cells LX-2.

[0071]

[0072] Table 3. Effects of (-)-Fresh bamboo sap phenol C at different concentrations on the proliferation of human hepatic stellate cells LX-2

[0073]

[0074] The results in Tables 2 and 3 show that (+)-fresh bamboo sap phenol C has a significant inhibitory effect on human hepatic stellate cells LX-2. The inhibition rate is dose-dependent, and the inhibitory effect increases with increasing drug concentration, indicating that (+)-fresh bamboo sap phenol C has certain potential anti-hepatic fibrosis activity. However, (-)-fresh bamboo sap phenol C has no significant inhibitory effect on the proliferation of the above cells.

[0075] In summary, the phenolic compound (+)-fresh bamboo sap phenol C obtained in this invention has a significant inhibitory effect on the proliferation of human hepatic stellate cells, indicating its potential for clinical application in the treatment of liver fibrosis. It can be used in the preparation of drugs for the prevention or treatment of liver fibrosis, providing new evidence for the pharmacological research of fresh bamboo sap.

[0076] Finally, it should be emphasized that the above description 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 can have various changes and modifications. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The use of phenolic compounds in the preparation of drugs for the prevention or treatment of liver fibrosis, characterized in that, The structure of the phenolic compound is shown in Formula I: 。 2. The application according to claim 1, characterized in that, The phenolic compounds were extracted from fresh bamboo sap.

3. The application according to claim 1, characterized in that, The phenolic compound is used in combination with a pharmaceutically acceptable carrier.

4. The application according to claim 1, characterized in that, The phenolic compounds are compounds with the structure shown in Formula I or pharmaceutically acceptable salts thereof as the active pharmaceutical ingredient.

5. The application according to claim 4, characterized in that, The concentration of the active pharmaceutical ingredient is 0.01-100 μM.

6. The application according to claim 1, characterized in that, The phenolic compounds have the activity of inhibiting the proliferation of hepatic stellate cells.

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