Use of phenolic compounds for the preparation of a medicament for the prevention or treatment of osteoporosis

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 osteoporosis. A pair of phenolic diastereoisomers (+)-fresh bamboo juice phenol E and (-)-fresh bamboo juice phenol E are separated from fresh bamboo juice, and it is found through anti-osteoporosis activity test that the ALP activity of (-)-fresh bamboo juice phenol E UMR106 cells has a promoting effect, has clinical application potential for treating osteoporosis, and can be used in preparation of a medicine for preventing or treating osteoporosis, thereby providing a new basis for fresh bamboo juice pharmacological research.

Description

TECHNICAL FIELD

[0001] 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 osteoporosis. BACKGROUND

[0002] Osteoporosis (OP) is a common progressive systemic bone disease characterized by reduced bone density with microstructure damage, increased bone fragility, and increased risk of fracture.

[0003] At present, bisphosphonates, calcitonin, estrogen and raloxifene are commonly used drugs for treating osteoporosis. However, the adverse reactions of these drugs seriously restrict their clinical application. For example, hormone-related therapies such as estrogen can maintain bone density, but increase the risk of cancer, thrombosis and heart disease; bisphosphonates can inhibit bone resorption and promote bone formation, but they can cause gastrointestinal problems such as esophageal inflammation and gastric ulcers. Therefore, more drugs with fewer adverse reactions need to be developed to prevent and treat OP. Traditional Chinese medicine has gradually become a hot spot in clinical research of OP due to its good overall therapeutic effect, fewer adverse reactions and suitability for long-term use.

[0004] Fresh bamboo juice is a light yellow and clear liquid extracted from fresh bamboo stems of Gramineae by fire roasting or dry distillation, and its components include amino acids, sugars, phenolic compounds, alcohols, aldehydes, organic acids, phenylpropanoids and lignans, inorganic elements, etc. Fresh bamboo juice has the effects of clearing heat and reducing phlegm, relieving cough and asthma, and is mainly used for the treatment of respiratory system diseases. In recent years, it has been found that the pharmacological activity of fresh bamboo juice is closely related to phenolic compounds. Phenolic compounds generally have chiral centers, and their pharmacological properties also differ significantly due to different isomers. Therefore, isomer analysis of phenolic compounds in fresh bamboo juice is expected to provide new basis for the study of the pharmacological activity of fresh bamboo juice. SUMMARY

[0005] In view of the problems in the prior art, the application provides application of a phenolic compound in preparation of a medicine for preventing or treating osteoporosis, which is extracted from fresh bamboo juice. Due to its special stereostructure, the phenolic compound has the pharmacological activity of promoting ALP activity of UMR106 cells, and can provide a theoretical basis for treatment and application of phenolic components in fresh bamboo juice to osteoporosis.

[0006] The application aims to provide application of a phenolic compound in preparation of a medicine for preventing or treating osteoporosis, and the phenolic compound has the structure as shown in Formula II.

[0007] .

[0008] Further, the phenolic compound is extracted from fresh bamboo juice.

[0009] Specifically, the preparation method of the phenolic compound comprises the following steps:

[0010] (1) cutting, dry distillation (temperature 120℃, pressure 0.7MPa), and vacuum concentration (temperature 60℃) of moso bamboo to obtain an extract, for standby;

[0011] (2) taking an appropriate amount of the extract, extracting with ethyl acetate and n-butanol for 3-4 times respectively, and concentrating to obtain an ethyl acetate part and an n-butanol part;

[0012] (3) subjecting the n-butanol part to D101 macroporous resin column, washing with 10%, 30%, 60%, and 90% ethanol aqueous solution respectively, collecting each component, and vacuum concentration to obtain a 60% ethanol part of macroporous resin;

[0013] (4) subjecting the 60% ethanol part to silica gel column chromatography, gradient eluting with dichloromethane-methanol (volume ratio 40:1, 30:1, 20:1, 10:1, 5:1) as an eluent, observing by thin layer chromatography, and combining similar components to finally obtain six components A, B, C, D, E, and F;

[0014] (5) subjecting the C component to medium-pressure reverse-phase column separation, gradient eluting with methanol: water (30%, 50%, 80%, 100%) to obtain four components C1-C4;

[0015] (6) subjecting the C2 component to gel, methanol elution, and then to silica gel column chromatography, gradient eluting with dichloromethane-methanol (40:1, 30:1, 20:1) as an eluent to obtain two components C2.1 and C2.2;

[0016] (7) subjecting C2.1 to reverse-phase preparative high performance liquid chromatography, preparing with acetonitrile-water (30:70) as a mobile phase (7mL / min) to obtain a compound;

[0017] (8) taking the compound, dissolving with methanol, and subjecting to reverse-phase high performance liquid chromatography with a Phenomenex Lux Cellulose-4 (250mm×4.6mm, 5μm) chiral semi-preparative chromatographic column, with acetonitrile-water (32:68) as a mobile phase to obtain a phenolic diastereoisomer.

[0018] The phenolic diastereoisomer has a chemical structural formula as formula I and formula II, wherein formula I is a dextrorotatory body, named as (+)-fresh bamboo juice phenol E, and formula II is a levorotatory body, named as (-)-fresh bamboo juice phenol E, which is the phenolic compound of the present application.

[0019]

[0020] Further, the medicine is a combination of the phenolic compound and a pharmaceutically acceptable carrier.

[0021] Further, the medicine takes the phenolic compound of formula II or a pharmaceutically acceptable salt thereof as the active ingredient.

[0022] Further, the concentration of the active ingredient is 0.01 μM-100 μM.

[0023] Further, the phenolic compound has the activity of promoting the enhancement of ALP of UMR106 cells.

[0024] Advantages over the prior art:

[0025] The present application separates a pair of phenolic diastereoisomers (+)-fresh bamboo sap phenol E and (-)-fresh bamboo sap phenol E from fresh bamboo sap. Anti-osteoporosis activity test shows that (-)-fresh bamboo sap phenol E has a significant promoting effect on the ALP of UMR106 cells, and the ALP activity of UMR106 cells is continuously increased with the increase of the concentration of the medicine, indicating that (-)-fresh bamboo sap phenol E has potential anti-osteoporosis activity; and (+)-fresh bamboo sap phenol E has no significant promoting effect on the ALP activity of UMR106 cells. Therefore, it is considered that (-)-fresh bamboo sap phenol E has the clinical application potential of anti-osteoporosis activity, and can be used for preparing medicines for preventing or treating osteoporosis, and provides a new basis for the pharmacological research of fresh bamboo sap. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 The chromatogram of the phenolic diastereoisomer of the present application, wherein the left peak is (+)-fresh bamboo sap phenol E and the right peak is (-)-fresh bamboo sap phenol E;

[0027] Figure 2 The high-resolution mass spectrum of the phenolic diastereoisomer of the present application;

[0028] Figure 3 The UV spectrum of the phenolic diastereoisomer of the present application;

[0029] Figure 4 The 1 H NMR spectrum of the phenolic diastereoisomer of the present application;

[0030] Figure 5 The 13 C NMR spectrum of the phenolic diastereoisomer of the present application;

[0031] Figure 6 The 1 H- 1 H COSY spectrum of the phenolic diastereoisomer of the present application;

[0032] Figure 7 The HMBC spectrum of the phenolic diastereoisomer of the present application;

[0033] Figure 8 HSQC spectrum of the phenolic diastereomer of the present application;

[0034] Figure 9 NOESY spectrum of the phenolic diastereomer of the present application;

[0035] Figure 10 ECD spectrum of (+)-neo-maoecol E of the phenolic diastereomer of the present application;

[0036] Figure 11 ECD spectrum of (-)-neo-maoecol E of the phenolic diastereomer of the present application. DETAILED DESCRIPTION

[0037] The above technical features of the present application and the technical features described in detail below (such as examples) can be combined with each other to form new or preferred technical solutions, but the present application is not limited to only these examples, and neither do these examples limit the present application in any way.

[0038] 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 unless otherwise specified, and can be purchased on the market.

[0039] The Phyllostachys pubescens used in the present application is collected from Tonggu County, Yichun City, Jiangxi Province, and is a plant of the genus Phyllostachys in the family Poaceae Phyllostachys edulis (Carr.) fresh stems of H. de Lehaie.

[0040] The present application will be further described in detail below in combination with examples:

[0041] Example 1: Preparation of the phenolic diastereomer

[0042] The preparation method of the phenolic diastereomer comprises the following steps:

[0043] (1) 10 t of Phyllostachys pubescens was cut and subjected to dry distillation at 120℃ and 0.7 MPa, and concentrated at 60℃ under reduced pressure to obtain an extract (96 kg) for standby;

[0044] (2) 37.8 kg of the extract was extracted with ethyl acetate and n-butanol for 3 times (50 L each time) in turn, and concentrated to obtain an ethyl acetate part (310 g) and an n-butanol part (857 g);

[0045] (3) The 857 g of the n-butanol part was subjected to D101 macroporous resin column, and eluted with 10%, 30%, 60%, and 90% ethanol aqueous solution respectively, and the components were collected and concentrated under reduced pressure to obtain a macroporous resin 60% ethanol part (55 g);

[0046] (4) The 55 g of the 60% ethanol fraction was subjected to silica gel column chromatography (100-200 mesh, 1000 g, 80 mm in diameter, wet column packing) using dichloromethane-methanol as the eluent in gradient elution, wherein the volume ratio of dichloromethane-methanol was 40:1, 30:1, 20:1, 10:1 and 5:1, respectively, and the similar fractions were combined by TLC detection, and finally six fractions A, B, C, D, E and F were obtained;

[0047] (5) The C fraction (2.6 g) was subjected to medium-pressure reverse-phase column separation using methanol:water (30%, 50%, 80%, 100%) in gradient elution, and four fractions C1-C4 were obtained;

[0048] (6) The C2 fraction (873 mg) was subjected to gel Sephadex LH-20 chromatography using methanol as the eluent, and then subjected to silica gel column chromatography using dichloromethane-methanol (40:1, 30:1, 20:1) as the eluent in gradient elution, and two fractions C2.1 and C2.2 were obtained;

[0049] (7) The C2.1 fraction (60 mg) was subjected to reverse-phase preparative high-performance liquid chromatography using an XTerra prep MS C 18 column (300 mm x 19 mm, 10 μm) and acetonitrile-water (30:70) as the mobile phase at a flow rate of 7 mL / min to prepare a phenolic compound (13.5 mg).

[0050] Example 2: Resolution and structural identification of phenolic diastereoisomers

[0051] 1. Resolution

[0052] 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 x 4.6 mm, 5 μm) chiral semi-preparative column and acetonitrile-water (32:68) as the mobile phase to obtain compound formula I and formula II, which were named as (+)-fresh bamboo sap extractive E and (-)-fresh bamboo sap extractive E, respectively. The chromatogram is shown in Figure 1 , wherein the left peak is (+)-fresh bamboo sap extractive E and the right peak is (-)-fresh bamboo sap extractive E.

[0053]

[0054] 2. Structural analysis

[0055] (±)-Fresh Zhileicheng Phenol E is a white powder, soluble in methanol; HRESIMS m / z 435.2014 [M-H]- (calcd. for C 23 H 31 O8,435.2019) was determined to be C 23 H 32 O8. The high resolution mass spectrum is shown in Figure 2 , and other confirmation spectrum is shown in Figures 3 to 11 , the 1 H-NMR, 13 C-NMR nuclear magnetic resonance data of (±)-Fresh Zhileicheng Phenol E are shown in Table 1.

[0056] Table 1 (±)-Fresh Zhileicheng Phenol E 1 H-NMR, 13 C-NMR nuclear magnetic resonance data

[0057]

[0058] a recorded in CD3OD 1 H NMR 600 MHz, 13 C NMR 150 MHz).

[0059] 1 In the H NMR spectrum, 6.38 (2H, s, H-2'', 6'') and 6.29 (2H, s, H-2', 6') indicate that the compound has two 1,3,4,5-substituted benzene rings; 4.57 (1H, d, J = 4.8 Hz, H-3) and 2.81 (1H, m, H-2) are two oxygen-containing methine hydrogen signals; 3.92 (1H, dd, J = 10.2, 6.6 Hz, H-1) and 3.73 (m, H-1) are oxygen-containing methylene hydrogen signals; 3.73 (6H, s, 3', 5'-OCH3) and 3.68 (6H, s, 3'', 5''-OCH3) are four methoxy hydrogen signals; 3.32 (1H, m, H-1''') and 3.26 (1H, m, H-1''') are oxygen-containing methylene hydrogen signals; 1.50 (2H, m, H-2'''), 1.36 (2H, m, H-3'''), 0.86 (3H, t, J = 7.2 Hz, H-4''') is an oxygen-containing n-butyl signal. 13The C10 NMR spectrum showed 17 carbon signals, including 8 benzene ring carbon signals: 131.3 (C-1′), 105.4 (C-2′, 6′), 148.8 (C-3′, 5′), 135.5 (C-4′), 133.1 (C-1′′), 108.4 (C-2′′, 6′′), 148.5 (C-3′′, 5′′), 135.3 (C-4′′); 4 n-butyl carbon signals: 69.4 (C-1′′′), 33.2 (C-2′′′), 20.6 (C-3′′′), 14.2 (C-4′′′); and 2 methoxy carbon signals: 56.7 (3′, 5′-OCH3), 56.5 (3′′, 5′′-OCH3); and three other carbon signals: 64.4 (C-1), 57.1 (C-2), and 83.0 (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.

[0060] 1 In the H NMR spectrum, H-2 / H-3 shows a small coupling constant. J 2,3 =4.8 Hz, therefore it is inferred that the relative configuration of this compound is erythropomorphic; in the NOESY spectrum, there are relevant signals at 4.57 (1H, d, J = 4.8 Hz, H-3) and 2.81 (1H, m, H-2), further confirming its erythropomorphic configuration. In the HMBC spectrum, δ H 4.57 (1H, d, J = 4.8 Hz, H-3) and δ C The correlation between 69.4 (C-1′′′) and 133.1 (C-1′′) indicates that C-3 is connected to the oxygen-bound n-butyl group and the C-1′′ position of the benzene ring; δ H 2.81 (1H, m, H-2) and δ C The correlation at 131.3 (C-1′) indicates that C-2 is connected to the C-1′ position of the benzene ring. Therefore, the compound is identified as erythro-2,3-bis-(3,5-dimethoxy-4-hydroxy)phenyl-3-butoxypropanol. Its main HMBC (H→C) correlation signals are shown below:

[0061] .

[0062] 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.

[0063] (+)-Fresh Zhili Phenol E is a white powder, soluble in methanol, determined by Perkin-Elmer 341 polarimeter (PERKINELMER, USA) +52° (0.034, MeOH); JASCO J-815 circular dichroism spectrometer (JASCO, Japan) ECD (MeOH) λ c 0.036, MeOH); JASCO J-815 circular dichroism spectrometer (JASCO, Japan) ECD (MeOH) λ max (Δε) 251 (-37.76), 282(4.05)nm. By comparing the optical rotation with Eucophenolic B, the optical rotation of the two is similar, combined with the relative configuration of the erythro type, so the absolute configuration of (+)-Fresh Zhili Phenol E is 2R, 3S.

[0064] (-)-Fresh Zhili Phenol E is a white powder, soluble in methanol, determined by Perkin-Elmer 341 polarimeter (PERKINELMER, USA) -47° (0.036, MeOH); JASCO J-815 circular dichroism spectrometer (JASCO, Japan) ECD (MeOH) λ c 0.036, MeOH); JASCO J-815 circular dichroism spectrometer (JASCO, Japan) ECD (MeOH) λ max (Δε) 251 (-37.76), 282(4.05)nm. By comparing the optical rotation with Eucophenolic B, the optical rotation of the two is similar, combined with the relative configuration of the erythro type, so the absolute configuration of (-)-Fresh Zhili Phenol E is 2S, 3R.

[0065] Among them, Eucophenolic A and Eucophenolic B come from the literature Yan J K, Shi X L, Donkor PO, et al. Two pairs of phenolic enantiomers from the leaves of Eucommia ulmoides Oliver [J]. Natural product research, 2019, 33(8): 1162-1168.

[0066] Example 3: Anti-osteoporosis activity research

[0067] UMR106 cells are a kind of rat osteosarcoma cells, which are often used to study the mechanism of various drugs affecting osteoporosis due to their osteoblast properties. Alkaline phosphatase (ALP) is an enzyme secreted by osteoblasts during differentiation, and its activity can be used to measure osteoblast differentiation. Based on this, this experiment aims to evaluate the anti-osteoporosis activity of phenolic compounds in fresh bamboo juice by investigating their promoting effect on the ALP activity of rat osteoblast-like UMR106 cells, and to provide a theoretical basis for the treatment and application of phenolic compounds in fresh bamboo juice for osteoporosis.

[0068] Test method: UMR106 cells were seeded in a 24-well plate at a density of 37500 cells / well and cultured in 5% FBS-DMEM for 3 days. Then the compound (±)-fresh bamboo juice phenol E was diluted in 1% FBS-DMEM to different concentrations and added to the 24-well plate for 1 day. After washing the cells once with ice PBS buffer, 200µL of lysis buffer (10mM Tris, 2mM MgCl, 1% Triton X-100) was added to each well, and the cells were frozen and thawed three times to ensure complete lysis. Take 40µL of cell lysate and 1000µL of ALP reaction reagent (10mM p-nitrophenyl phosphate, 0.1M Na2CO3, 1mM MgCl) and react at 37°C in the dark for 30 minutes, then add 500µ0.1M NaOH solution to terminate the reaction. The absorbance value was measured at 405nm, and the value measured with double distilled water was taken as the initial control value of the enzyme reaction (A 水 ), and the value measured for each concentration sample was A 样 . Another 40µL of cell lysate was diluted with 800µL of double distilled water to determine the protein content by Coomassie brilliant blue method; cells cultured in culture medium without adding compounds were used as negative control. The ALP activity was calculated as the change in alkaline phosphatase activity per minute per µg of protein at the specified time, i.e. ALP activity change = (A 样 -A 水 ) / min / µg protein. In order to make the results more intuitive and accurate, the final ALP activity was expressed as the ratio of ALP activity change of sample and negative control, i.e. ALP 样 / ALP 对 ×100%, where ALP 样 is the ALP activity change of different compound concentrations, and ALP 对 is the ALP activity change of the culture medium without adding compounds.

[0069] Principle of determination: Alkaline phosphatase in cells can hydrolyze p-nitrophenyl phosphate to p-nitrophenol under alkaline conditions, which has a maximum absorption at 405nm. According to the principle of enzyme kinetics, the rate of p-nitrophenol formation is proportional to the activity of alkaline phosphatase.

[0070] Preparation of protein standard curve: 0, 100, 200, 400, 800 μL of protein standard solution (0.1 mg / mL) was taken respectively, 200 μL of deionized water was added to each, and then an appropriate amount of coomassie brilliant blue reagent was added to make the final volume 1 mL. After standing at room temperature for 10 minutes, the absorbance value was measured at a wavelength of 595 nm. The absorbance (Y) / protein content (X) was plotted to draw the protein standard curve Y = 0.0063X + 0.1572 (R 2 =1.0000).

[0071] According to the above determination method, three parallel samples were prepared for each concentration of the test solution, and the average value and standard deviation were calculated. The detailed test results are shown in Tables 2 to 3.

[0072] Table 2 Effect of (+)-fresh bamboo phloquol E on ALP activity of UMR106 cells at different concentrations

[0073]

[0074] Table 3 Effect of (-)-fresh bamboo phloquol E on ALP activity of UMR106 cells at different concentrations

[0075]

[0076] From the test results in Tables 2 and 3, it can be seen that (-)-fresh bamboo phloquol E has a promoting effect on the ALP activity of UMR106 cells, and as the drug concentration increases, the ALP activity of UMR106 cells increases continuously, indicating that (-)-fresh bamboo phloquol E has a certain potential anti-osteoporosis activity; (+)-fresh bamboo phloquol E has no effect on the ALP activity of UMR106 cells, indicating that it has no promoting effect on the ALP activity of the above-mentioned cells.

[0077] In summary, the phenolic compound (-)-fresh bamboo phloquol E obtained by the present application has a significant promoting effect on the ALP activity of UMR106 cells, indicating that the (-)-fresh bamboo phloquol E has a clinical application potential for anti-osteoporosis activity, and can be used for preparing drugs for preventing or treating osteoporosis, and provides a new basis for the pharmacological research of fresh bamboo sap.

[0078] Finally, it should be emphasized that the above description is only the preferred embodiment of the present application and is not used to limit the present application. For those skilled in the art, the present application can have various changes and modifications, and any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. The use of phenolic compounds in the preparation of drugs for the prevention or treatment of osteoporosis, characterized in that, The phenolic compounds have the structure shown in Formula II: 。 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 drug is a combination of the phenolic compound and a pharmaceutically acceptable carrier.

4. The application according to claim 1, characterized in that, The drug uses a phenolic compound of formula II or a pharmaceutically acceptable salt thereof as its active pharmaceutical ingredient.

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

6. The application according to claim 1, characterized in that, The phenolic compounds have the activity of promoting ALP enhancement in UMR106 cells.

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