A novel water-soluble phenolic acid salviamarinic acid A extracted from Salvia miltiorrhiza Bunge var. przewalskii Maxim. and its preparation method and application

Abstract

A new water-soluble phenolic acid salviamarinic acid A extracted from Salvia miltiorrhiza Bunge var. yuchensis, was extracted by tissue disruption with 50% aqueous acetone. A new water-soluble phenolic acid compound, namely salviamarinic acid A, was isolated and identified from the ethyl acetate fraction of Salvia miltiorrhiza Bunge var. yuchensis. An in vitro model of pulmonary fibrosis was constructed by TGF-β1-induced human normal lung epithelial cells BEAS-2B to screen its activity. The experimental results showed that salviamarinic acid A could significantly increase cell viability, cell index, cell motility and E-cadherin expression; and significantly reduce the expression of TGF-β1, α-SMA, and CollagenⅠ, with significant anti-pulmonary fibrosis activity, and significant economic and social benefits.

Description

Technical Field

[0001] The present invention relates to the field of medicine, in particular to a new water-soluble phenolic acid salviamarinic acid A extracted from Salvia miltiorrhiza var. yudanshen, a preparation method thereof and applications thereof. Background Art

[0002] Salviae Miltiorrhizae Radix et Rhizoma is the dried root and rhizome of the plant Salvia miltiorrhiza Bunge of the Labiatae family, and has the effects of promoting blood circulation to regulate menstruation, removing stasis to relieve pain, cooling blood to resolve carbuncles, clearing the heart to remove vexation, nourishing blood and tranquilizing the mind, etc. Since ancient times, there has been a saying that a single Salvia miltiorrhiza is as effective as the four drugs for nourishing blood. Fangcheng County in Nanyang, Henan Province is located on the dividing line between the Tongbai Mountains and the Funiu Mountains, the Huaihe River Basin and the Yangtze River Basin, and the north subtropical zone and the south warm temperate zone. It has a special geographical location and unique climate. The Salvia miltiorrhiza var. yudanshen produced there has the characteristics of thick roots, purple-red color, good quality and excellent curative effect. It is called the best of Salvia miltiorrhiza by Zhang Zhongjing, the medical sage. In 2013, it passed the GAP certification and a standardized GAP planting base for Salvia miltiorrhiza var. yudanshen in Henan was established. The planting scale of Salvia miltiorrhiza var. yudanshen exceeds 100,000 mu, and it is a famous geographical brand. However, at present, the literature reports mainly focus on the germplasm cultivation of Salvia miltiorrhiza var. yudanshen, and there are few literature reports on the chemical composition research of Salvia miltiorrhiza var. yudanshen. Many of its chemical components have not been discovered, and the experimental study on the pharmacodynamic material basis needs to be further carried out.

[0003] Pulmonary fibrosis is a complex pathological process, mainly characterized by severe damage to lung epithelial cells, excessive repair of damage caused by the proliferation of (myo)fibrotic cells and abnormal collagen deposition. Currently, except for lung transplantation, even treatment drugs mainly based on pirfenidone and nintedanib can only delay the decline rate of patients' lung function and cannot reverse the process of pulmonary fibrosis. TGF-β1 is currently recognized as the most potent profibrotic cytokine, playing a key role in interstitial lung fibrosis diseases and participating in processes such as the proliferation, activation, and differentiation of lung fibroblasts into myofibroblasts. The epithelial-mesenchymal transition (EMT) process is an important source of fibroblasts. During the occurrence of EMT, the synthesis and degradation of collagen will be imbalanced. If the expression of E-cadherin is inhibited, the contact connection structure between epithelial cells will be damaged, promoting epithelial cells to undergo EMT. The decrease of E-cadherin is the core link of the whole EMT. CollagenΙ is one of the main components of the extracellular matrix and mesenchyme, playing a role in maintaining the stability of the cytoskeleton by regulating the migration of collagen. The increase of CollagenΙ can induce the occurrence of tissue fibrosis; α-SMA is the main marker protein of EMT. It is found that almost all fibroblasts and fibrous tissues express α-SMA. α-SMA participates in the fibrosis of the body and cells while regulating interstitialization and is a marker of the production of myofibroblasts. The TGF-β1-induced lung epithelial cell injury model is a commonly used in vitro model of pulmonary fibrosis.

[0004] In recent years, traditional Chinese medicine has made certain progress in the treatment of pulmonary fibrosis. Due to its significant efficacy, low side effects, and the fact that traditional Chinese medicine exerts its pharmacological effects through multiple pathways, multiple targets, and multiple components, it has gradually shown advantages. Salvia miltiorrhiza has the effects of removing blood stasis and relieving pain, cooling blood and dissipating carbuncles, and clearing the heart and relieving vexation. And clinical studies have shown that Salvia miltiorrhiza contains a variety of chemical components and has a wide range of biological activities. Its traditional pharmacology mainly focuses on dilating blood vessels, promoting blood circulation to remove blood stasis, and improving microcirculation. However, there are few research reports on its treatment of pulmonary fibrosis. So far, there has been no relevant public report. Summary of the Invention

[0005] In view of the above situation, to overcome the defects of the prior art, the purpose of the present invention is to provide a new water-soluble phenolic acid salviamarinic acid A extracted from Salvia miltiorrhiza Bunge var. przewalskii Maxim. and its preparation method and application, which can effectively solve the problems that there is less research on the chemical components and pharmacodynamic material basis of Salvia miltiorrhiza Bunge var. przewalskii Maxim., and the pharmacodynamic material basis and pharmacological action mechanism for the treatment of pulmonary fibrosis are not yet clear.

[0006] To achieve the above purpose, the technical solution solved by the present invention is a new water-soluble phenolic acid salviamarinic acid A extracted from Salvia miltiorrhiza Bunge var. przewalskii Maxim., and its molecular structural formula is:

[0007]

[0008] The preparation method of the novel water-soluble phenolic acid salviamarinic acid A extracted from Salvia miltiorrhiza var. yudanshen comprises the following steps:

[0009] (1) Take 50 kg of dried and pulverized Salvia miltiorrhiza var. yudanshen, impregnate it with 50% acetone at room temperature for 24 h, and use a flash extractor to perform tissue disruption extraction 3 times, 30 s each time. Filter, and after combining the filtrates, concentrate under reduced pressure to obtain 18.4 kg of extract paste;

[0010] (2) Dissolve the extract paste in 16 L of water, and extract it 6 times successively with 16 L of petroleum ether, dichloromethane, ethyl acetate, and n-butanol each to obtain a petroleum ether fraction, a dichloromethane fraction, an ethyl acetate fraction, an n-butanol fraction, and an aqueous fraction;

[0011] (3) After concentrating and drying each fraction, take 239.0 g of the ethyl acetate fraction A and dissolve it in methanol. Mix the sample with silica gel in a ratio of 1:1 (sample to silica gel). Pack a column with 200 - 300 mesh silica gel, and use petroleum ether:dichloromethane and dichloromethane:methanol as mobile phases for gradient elution at a flow rate of 10 ml / min. The ratios used are successively petroleum ether:dichloromethane = 100:0, 20:1, 10:1, 5:1, 2:1, 0:1 and dichloromethane:methanol = 20:1, 10:1, 4:1, 0:1. Detect once every 200 ml, and judge the amount of each gradient mobile phase by anisaldehyde - sulfuric acid thin-layer detection. Finish elution in 3 d, combine the fractions with dichloromethane:methanol = 4:1, and label it as B6; After dissolving component B6 in methanol, continue to load it onto a silica gel column. Mix the sample with silica gel in a ratio of 1:1 (sample to silica gel). Pack a column with 200 - 300 mesh silica gel, and use ethyl acetate:methanol as the mobile phase for gradient elution at a flow rate of 6 ml / min. The ratios used are successively 16:1, 8:1, 4:1, 2:1, 1:1, 0:1. Perform anisaldehyde - sulfuric acid thin-layer detection, and combine the fractions with ethyl acetate:methanol = 2:1, and record it as C5; Dissolve component C5 in methanol, perform column chromatography on a Sephadex LH-20 column, elute with 70% methanol at a flow rate of 0.6 ml / min, and the amount of the mobile phase is 400 ml. Perform anisaldehyde - sulfuric acid thin-layer detection, and combine the fractions of 200 - 230 ml and label it as F3; Separate component F3 by semi-preparative HPLC. Load it onto a YMC-Pack ODS-AA chromatographic column with a specification model of: 250×10 mm, particle size 5 μm, pore size 12 nm. The mobile phase is methanol:water = 67:33, and the flow rate is 3 ml / min. Collect the fractions with a retention time t R = 18.9 - 21.0 min, concentrate and dry to obtain the compound salviamarinic acid A.

[0012] Use of the novel water-soluble phenolic acid salviamarinic acid A extracted from Salvia miltiorrhiza Bunge var. przewalskii Maxim. prepared by the said method in the preparation of anti-pulmonary fibrosis drugs.

[0013] The raw materials of the present invention are abundant, the preparation method is easy to operate, the obtained product has good quality and good use effect, and can significantly increase cell viability, cell index, cell motility and E-cadherin expression; and significantly reduce TGF-β1, α-SMA, CollagenⅠ expression, with significant economic and social benefits. Description of the Drawings

[0014] Figure 1 It is the structural formula of the compound salviamarinic acid A of the present invention.

[0015] Figure 2 It is of the compound salviamarinic acid A of the present invention 1 H-NMR(500MHz,CD 3 OD).

[0016] Figure 3 The 13 C-NMR(125MHz,CD 3 OD).

[0017] Figure 4 DEPT 135 spectrum of the compound salviamarinic acid A.

[0018] Figure 5 The 1 H- 1 H COSY spectrum of the compound salviamarinic acid A.

[0019] Figure 6 HSQC spectrum of the compound salviamarinic acid A.

[0020] Figure 7 HMBC spectrum of the compound salviamarinic acid A.

[0021] Figure 8 HR-ESI-MS spectrum of the compound salviamarinic acid A.

[0022] Figure 9 UV spectrum of the compound salviamarinic acid A.

[0023] Figure 10IR spectrum of compound salviamarinic acid A

[0024] Figure 11 It is the influence diagram of the compound salviamarinic acid A of the present invention on the in vitro model of pulmonary fibrosis of human normal lung epithelial cells BEAS - 2B induced by TGF - β1; wherein, A: After 24 h of administration treatment, MTT was used to detect cell viability; B: The cell index of cells after 72 h of administration treatment was observed by a label - free real - time monitoring system; C and D: The cell migration distance of cells after 48 h of administration treatment was observed by a label - free real - time monitoring system; E: Fluorescence imaging diagrams of E - cadherin, TGF - β1, α - SMA, and CollagenⅠ; F: Quantification of TGF - β1; G: Quantification of α - SMA; H: Quantification of E - cadherin; I: Quantification of CollagenⅠ. n = 4. Compared with the CON group, ## P < 0.01; compared with the M group, * P < 0.05, ** P < 0.01. CON: Normal control group; TGF - β1: Model group; 5, 10 μM are different dose groups of salviamarinic acid A. Detailed implementation manners

[0025] The following combines examples and specific situations to elaborate in detail on the specific implementation manners of the present invention.

[0026] In the specific implementation of the present invention, it can be given by the following examples.

[0027] Example 1

[0028] A preparation method of a new water - soluble phenolic acid salviamarinic acid A extracted from Salvia miltiorrhiza Bge. var. przewalskii Maxim. includes the following steps:

[0029] (1) Take 50 kg of Salvia miltiorrhiza Bge. var. przewalskii Maxim. and dry - crush it, soak it with 50% acetone at room temperature for 24 h, and use a flash extractor to perform tissue - crushing extraction 3 times, 30 s each time. Filter, and after combining the filtrates, concentrate under reduced pressure to obtain 18.4 kg of extract.

[0030] (2) Dissolve the extract with 16 L of water, and extract it 6 times successively with 16 L of petroleum ether, dichloromethane, ethyl acetate, and n - butanol each to obtain a petroleum ether fraction, a dichloromethane fraction, an ethyl acetate fraction, an n - butanol fraction, and a water fraction.

[0031] (3) After concentrating and drying each part, take part A (239.0 g) of ethyl acetate fraction, dissolve it with methanol, mix the sample with silica gel at a ratio of 1:1 (sample to silica gel) using silica gel of 100 - 200 mesh, pack a column with silica gel of 200 - 300 mesh, and perform gradient elution with petroleum ether:dichloromethane and dichloromethane:methanol as the mobile phases at a flow rate of 10 ml / min. The ratios used are successively petroleum ether:dichloromethane = 100:0, 20:1, 10:1, 5:1, 2:1, 0:1 and dichloromethane:methanol = 20:1, 10:1, 4:1, 0:1. Detect once every 200 ml, and judge the usage amount of each gradient mobile phase by anisaldehyde - sulfuric acid thin layer detection. After 3 days of elution, combine the fractions with dichloromethane:methanol = 4:1 and label it as B6; after dissolving fraction B6 with methanol, continue to load it onto a silica gel column, mix the sample with silica gel at a ratio of 1:1 (sample to silica gel) using silica gel of 100 - 200 mesh, pack a column with silica gel of 200 - 300 mesh, perform gradient elution with ethyl acetate:methanol as the mobile phase at a flow rate of 6 ml / min. The ratios used are successively 16:1, 8:1, 4:1, 2:1, 1:1, 0:1. Detect by anisaldehyde - sulfuric acid thin layer, and combine the fractions with ethyl acetate:methanol = 2:1 and record it as C5; dissolve fraction C5 with methanol, perform column chromatography on a Sephadex LH - 20 column, elute with 70% methanol at a flow rate of 0.6 ml / min, with a mobile phase usage amount of 400 ml, detect by anisaldehyde - sulfuric acid thin layer, and combine the fractions of 200 - 230 ml and label it as F3; separate fraction F3 by semi - preparative HPLC, load it onto a YMC - Pack ODS - AA chromatographic column with a specification model of: 250×10 mm, particle size 5 μm, pore size 12 nm, with the mobile phase of methanol:water (the content of trifluoroacetic acid is three ten - thousandths) = 67:33, flow rate 3 ml / min, collect the fractions with retention time t R = 18.9 - 21.0 min, concentrate and dry to obtain the compound salviamarinic acid A.

[0032] The compound salviamarinic acid A prepared by the present invention can significantly increase cell viability, cell index, cell motility and E - cadherin expression; and significantly reduce the expression of TGF - β1, α - SMA, Collagen Ⅰ. The relevant experimental data are as follows:

[0033] I. Instruments and Reagents

[0034] 1.1 Experimental Instruments

[0035] Table 1 List of Main Instruments

[0036]

[0037]

[0038] 1.2 Experimental Reagents

[0039] Table 2 List of Main Reagents

[0040]

[0041]

[0042] 1.3 Experimental Materials

[0043] Salvia miltiorrhiza var. yudanshen was harvested in September 2020 from Fangcheng County, Nanyang City, Henan Province. It was identified by Professor Chen Suiqing and Professor Dong Chengming of Henan University of Chinese Medicine as the dried roots and rhizomes of the Labiatae plant Salvia miltiorrhiza Bunge. The specimen (20200901B) is stored in the Research Laboratory of Chinese Medicine Chemistry, Henan University of Chinese Medicine. The column chromatography packing materials Diaion HP-20 and MCIgel CHP-20 were purchased from Mitsubishi Chemical Corporation, Japan. Toyopearl HW-40C was purchased from TOSOH Corporation, Japan. Sephadex LH-20 was purchased from Pharmacia Biotech. The thin-layer chromatography silica gel (particle range 10 - 40 μm) and column chromatography silica gel H (100 - 200 mesh, 200 - 300 mesh) were purchased from Qingdao Ocean Chemical Industry.

[0044] II. Extraction and Isolation

[0045] According to the preparation method of Example 1 above, extraction and separation were carried out to obtain the compound salviamarinic acid A.

[0046] III. Structure Identification

[0047] Colorless waxy solid (CH 3 OH). HR-ESI-MS gave the quasi-molecular ion peak m / z 553.2550 [M+H] + , (calcd. For C 28 H 25 O 12 553.2554), determining its molecular formula to be C 28 H 24 O 12 ; UV (MeOH) λ max : 207(0.33), 288(1.23); IR (KBr) ν max cm -1 : 3342, 2953, 1736, 1609, 1509, 1447, 1286, 1031 cm -1 , the specific spectrum is shown in Figure 2-10 , the NMR data (in CD 3 OD) are shown in Table 3 below, and its structural formula is shown in Figure 1As shown:

[0048] Table 3 NMR data of compound salviamarinic acid A (in CD 3 OD)

[0049]

[0050]

[0051] IV. Activity Screening

[0052] 4.1 Detection of cell viability by MTT method

[0053] BEAS-2B cells were cultured in DMEM medium containing 10% fetal bovine serum and penicillin-streptomycin solution, and placed in an incubator at 37°C and 5% CO2. Logarithmic growth phase BEAS-2B cells were selected for the experiment. Fresh medium was changed every 2 days, and subcultured at a ratio of 1:3 - 1:4. Cell cryopreservation solution: 90% FBS + 10% DMSO. Logarithmic growth phase BEAS-2B cells were seeded at 5000 cells / well in a 96-well plate. After 24 hours of adhesion, BEAS-2B cells were divided into a normal control group (CON), a model group (TGF-β1, 1 ng / mL), and a salviamarinic acid A group (1, 2.5, 5, 10, 20, 40 μM + TGF-β1, 1 ng / mL). After culturing for 24 hours, 20 μL of a medium solution containing 5 mg / mL MTT was added to each well, and the culture was continued for 4 hours. The culture medium was carefully aspirated, and 150 mL of DMSO was added to each well and shaken for 10 minutes to completely dissolve it. The absorbance value (OD) of each well was measured at 490 nm with an enzyme-linked immunosorbent assay (ELISA) reader, and the average OD value and cell viability were calculated.

[0054] 4.2 Detection of cell index and cell motility by a cell label-free real-time monitoring system

[0055] Logarithmic growth phase BEAS-2B cells were seeded at 5000 cells in a 16-well plate (CIMPlate16, ACEA, Belgium). After 24 hours, they were divided into a normal control group (CON), a model group (M, TGF-β1 1 ng / mL), and a salviamarinic acid A group (5, 10 μM + TGF-β1 1 ng / mL), and the culture was continued for 72 hours. The cell index and cell motility were detected by a cell real-time label-free system (xCELLigence system).

[0056] 4.3 Detection of the expression of collagen deposition-related proteins by a high-content imaging analysis system

[0057] BEAS-2B cells in logarithmic growth phase were seeded in 96-well plates at a density of 5000 cells per well. After 24 hours, the cells were divided into a normal control group (CON), a model group (M, TGF-β1 1 ng / mL), and a salviamarinic acid A group (5, 10 μM + TGF-β1 1 ng / mL). After 24 hours of drug treatment, the supernatant was aspirated, fixed with formaldehyde, permeabilized with Triton, blocked with bovine serum albumin for 1.5 hours, and then incubated overnight with primary antibodies against TGF-β1, α-SMA, E-Cadherin, and Collagen Ι [all diluted 1:1000 in bovine serum albumin (BSA)]. Secondary antibodies (diluted 1:1000 in BSA) were added and incubated for 1 hour, followed by washing 4 times with PBST for 5 minutes each time and 1 time with PBS for 5 minutes. The results were scanned using a high-content imaging analysis system, and the fluorescence values were processed and statistically analyzed as protein expression levels.

[0058] V. Results and Discussion

[0059] In this invention, a TGF-β1-induced in vitro model of pulmonary fibrosis in human normal lung epithelial cells BEAS-2B was used to screen the activity of salviamarinic acid A. The results are as Figure 11 shown, indicating that salviamarinic acid A can significantly increase cell viability, cell index, cell motility, and E-cadherin expression (P < 0.05 or P < 0.01); and significantly reduce the expression of TGF-β1, α-SMA, and Collagen Ι (P < 0.01), suggesting that it can improve collagen deposition and has significant anti-pulmonary fibrosis activity.

[0060] In summary, the raw materials of this invention are abundant, and the preparation method is easy to operate. A new water-soluble phenolic acid compound, salviamarinic acid A, was isolated and identified from the ethyl acetate fraction of Salvia miltiorrhiza Bunge var. przewalskii Maxim. for the first time. A TGF-β1-induced in vitro model of pulmonary fibrosis in human normal lung epithelial cells BEAS-2B was used to screen its activity. The experimental results showed that salviamarinic acid A can significantly increase cell viability, cell index, cell motility, and E-cadherin expression; and significantly reduce the expression of TGF-β1, α-SMA, and Collagen Ι, suggesting that it may play an anti-pulmonary fibrosis role by improving collagen deposition and has significant anti-pulmonary fibrosis activity, which is very conducive to popularization and application and has significant social and economic benefits.

[0061] It should be noted that the above are only the preferred embodiments of the present invention, and there is no limitation to the present invention in any form. Any person skilled in the art can, within the scope of the technical solution of the present invention, make changes or modifications to equivalent embodiments of equivalent changes by using the technical content disclosed above, and all fall within the protection scope of the present invention.

Claims

1. A water-soluble phenolic acid salviamarinic acid A extracted from Salvia miltiorrhiza Bge. var. przewalskii Maxim., characterized in that, its molecular structural formula is: 。 2. A preparation method of the water-soluble phenolic acid salviamarinic acid A extracted from Salvia miltiorrhiza Bge. var. przewalskii Maxim. according to claim 1, characterized in that, it comprises the following steps: (1) Take 50 kg of Salvia miltiorrhiza Bge. var. przewalskii Maxim., dry and crush it, impregnate it with 50% acetone at room temperature for 24 h, and use a flash extractor to perform tissue disruption extraction 3 times, 30 s each time, filter, and concentrate the filtrate under reduced pressure to obtain 18.4 kg of extract; (2) Dissolve the extract in 16 L of water, and extract it 6 times successively with 16 L of petroleum ether, dichloromethane, ethyl acetate, and n-butanol each to obtain a petroleum ether fraction, a dichloromethane fraction, an ethyl acetate fraction, an n-butanol fraction, and an aqueous fraction; (3) After concentrating and drying each part, 239.0 g of the ethyl acetate fraction A was dissolved in methanol, mixed with silica gel of 100 - 200 mesh, and the ratio of sample to silica gel was 1:

1. The column was packed with silica gel of 200 - 300 mesh, and gradient elution was carried out with petroleum ether:dichloromethane and dichloromethane:methanol as the mobile phase at a flow rate of 10 ml / min. The ratios used were successively petroleum ether:dichloromethane = 100:0, 20:1, 10:1, 5:1, 2:1, 0:1 and dichloromethane:methanol = 20:1, 10:1, 4:1, 0:

1. Detection was carried out every 200 ml, and the amount of each gradient mobile phase was judged by anisaldehyde - sulfuric acid thin layer detection. Elution was completed in 3 days. The fractions with dichloromethane:methanol = 4:1 were combined and labeled as B6; after dissolving fraction B6 in methanol, it was continued to be loaded onto a silica gel column, mixed with silica gel of 100 - 200 mesh, and the ratio of sample to silica gel was 1:

1. The column was packed with silica gel of 200 - 300 mesh, and gradient elution was carried out with ethyl acetate:methanol as the mobile phase at a flow rate of 6 ml / min. The ratios used were successively 16:1, 8:1, 4:1, 2:1, 1:1, 0:

1. Detection was carried out by anisaldehyde - sulfuric acid thin layer, and the fractions with ethyl acetate:methanol = 2:1 were combined and recorded as C5; fraction C5 was dissolved in methanol and separated by Sephadex LH - 20 column chromatography, eluted with 70% methanol at a flow rate of 0.6 ml / min, and the amount of the mobile phase was 400 ml. Detection was carried out by anisaldehyde - sulfuric acid thin layer, and the fractions of 200 - 230 ml were combined and labeled as F3; fraction F3 was separated by semi - preparative HPLC, loaded onto a YMC - Pack ODS - AA chromatographic column with a specification model of: 250×10 mm, particle size 5 μm, pore size 12 nm, the mobile phase was methanol:water = 67:33, and the flow rate was 3 ml / min. The fractions with retention time t R = 18.9 - 21.0 min were collected, concentrated and dried to obtain the compound salviamarinic acid A.

3. Use of the water-soluble phenolic acid salviamarinic acid A extracted from Salvia miltiorrhiza Bge. var. przewalskii Maxim. according to claim 1 in the preparation of anti-pulmonary fibrosis drugs.

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