A new water-soluble phenolic acid salviamarinic acid B extracted from radix salviae miltiorrhizae and preparation method and application thereof

Abstract

The application discloses a new water-soluble phenolic acid salviamarinic acid B extracted from radix salviae miltiorrhizae, adopts a C48 / 80 induced RBL-2H3 cell degranulation in vitro model to simulate the degranulation phenomenon of mast cells in vitro, and first separates and identifies a new water-soluble phenolic acid compound (salviamarinic acid B) from the ethyl acetate part of radix salviae miltiorrhizae, which can inhibit the release of beta-Hex, reduce the levels of mMCP-1, beta-MCT, HIS and LTC4, significantly improve the degranulation phenomenon of RBL-2H3 cells, has significant anti-asthma activity, and has significant social and economic benefits.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, and in particular to a novel water-soluble phenolic acid, salviamarinic acid B, extracted from *Salvia miltiorrhiza*, its preparation method, and its application. Background Technology

[0002] Danshen (Salviae Miltiorrhizae Radix et Rhizoma) is a traditional Chinese medicine widely used in clinical practice. It is the dried root and rhizome of *Salvia miltiorrhiza* Bunge, a plant belonging to the Lamiaceae family. First recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica), it is listed as a superior-grade herb. It possesses various medicinal properties, including promoting blood circulation and regulating menstruation, removing blood stasis and relieving pain, cooling the blood and reducing swelling, clearing the heart and relieving irritability, nourishing blood and calming the mind. Throughout Chinese history, physicians have placed great emphasis on the relationship between the quality and origin of medicinal materials. Promoting the production and use of authentic medicinal materials is an effective method used by Chinese physicians to control the quality of medicinal materials and ensure clinical efficacy. Yu Danshen produced in Fangcheng, Nanyang, Henan Province, is characterized by its thick, purplish-red stems, high quality, and superior efficacy. It was praised as the premier Danshen by the renowned physician Zhang Zhongjing and received GAP certification in 2013. A standardized GAP cultivation base for Yu Danshen has been established in Henan, with a planting area exceeding 100,000 mu (approximately 6,667 hectares), making it a well-known geographical brand. However, current literature reports mostly focus on the germplasm culture of *Codonopsis pilosula*, while research on the chemical composition and pharmacological material basis of *Codonopsis pilosula* is relatively limited.

[0003] Asthma is a chronic inflammatory airway disease that seriously threatens public health. Its clinical manifestations include recurrent episodes of shortness of breath, wheezing, cough, and / or chest tightness, accompanied by varying degrees of airflow limitation, airway hyperresponsiveness, and airway remodeling. Asthma can be classified into allergic, non-allergic, occupational, aspirin-exacerbated respiratory disease, potentially fatal, exercise-induced, and cough-variant asthma. Allergic asthma is the most common type, accounting for approximately 80% of persistent asthma cases. Currently, the clinical treatment goal for asthma patients is primarily to control recurrent attacks; there is no effective cure. Mast cells are the core effector cells involved in allergic asthma. When the body comes into contact with allergens, the high-affinity IgE receptors (FcεRⅠ) on the surface of mast cells rapidly cross-link, leading to mast cell activation and the release of a large number of allergy mediators, such as β-aminohexosidase (β-Hex), histamine (HIS), leukotriene C4 (LTC4), mast cell β-trypsin (β-MCT), and mast cell protease (mMCP-1), which play a key role in the pathogenesis of asthma.

[0004] Traditional Chinese medicine (TCM) has a long history of treating asthma. Similar records of asthma can be found in the *Huangdi Neijing* (Yellow Emperor's Inner Classic), which includes symptoms such as wheezing and shortness of breath. Modern TCM often categorizes asthma under the terms "asthmatic disease" or "asthmatic syndrome." TCM, with its holistic regulatory advantages, has a definite and stable effect in preventing and treating asthma recurrence and exacerbation, with fewer adverse reactions. The use of Danshen (Salvia miltiorrhiza) in the treatment of lung diseases has been studied for some time. Zhang Xichun, a representative figure of the school of thought integrating TCM and Western medicine, proposed in *Medical Records of Integrating Chinese and Western Medicine* that TCM formulas with Danshen can treat lung abscesses, among other conditions. Danlong Oral Liquid, the first TCM preparation using Danshen as the principal ingredient for treating asthma, was the first new TCM drug approved for marketing in my country in 2017, indicating that Danshen has important efficacy in treating asthma. However, the pharmacodynamic material basis and pharmacological mechanism of Danshen in treating asthma are still unclear, and no public reports have been published to date. Summary of the Invention

[0005] In view of the above situation and to overcome the shortcomings of the existing technology, the purpose of this invention is to provide a new water-soluble phenolic acid, salviamarinic acid B, extracted from *Salvia miltiorrhiza*, its preparation method, and its application. This invention can effectively solve the problem that there is limited research on the chemical composition and pharmacological material basis of *Salvia miltiorrhiza*, and that the pharmacological material basis and mechanism of action for treating asthma are still unclear.

[0006] To achieve the above objectives, the technical solution provided by this invention is a novel water-soluble phenolic acid, salviamarinic acid B, extracted from *Salvia miltiorrhiza*, with the following molecular structural formula:

[0007]

[0008] The method for preparing the novel water-soluble phenolic acid salviamarinic acid B extracted from *Salvia miltiorrhiza* includes the following steps:

[0009] (1) Take 50 kg of dried and pulverized Yudanshen, soak it in 50% acetone at room temperature for 24 h, and extract it three times with a flash extractor for 30 s each time. After filtration, combine the filtrates and concentrate under reduced pressure to obtain 18.4 kg of extract.

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

[0011] (3) After concentrating and drying each fraction, dissolve 239.0g of ethyl acetate fraction A in methanol, mix with 100-200 mesh silica gel, and use a 1:1 ratio of sample to silica gel. Pack a column with 200-300 mesh silica gel and use a gradient elution with petroleum ether:dichloromethane and dichloromethane:methanol as mobile phases at a flow rate of 10ml / min. The ratios used are 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, and are checked every 200ml. The amount of each gradient mobile phase is calculated as anisaldehyde-concentrated sulfur. Acid thin-layer chromatography was used to identify the fraction. After elution for 3 days, the fraction containing dichloromethane:methanol (4:1) was combined and labeled B6. Fraction B6 was dissolved in methanol and then loaded onto a silica gel column. The sample was mixed with 100-200 mesh silica gel (sample to silica gel ratio 1:1) and packed into 200-300 mesh silica gel. Gradient elution was performed using ethyl acetate:methanol as the mobile phase at a flow rate of 6 ml / min, with ratios of 16:1, 8:1, 4:1, 2:1, 1:1, and 0:1. Anisaldehyde-concentrated sulfuric acid thin-layer chromatography was used for identification. The fraction containing ethyl acetate:methanol (4:1) was combined and labeled C4. Fraction C4 was dissolved in methanol and analyzed using Sephadex. LH-20 column chromatography was performed, eluting with 75% methanol at a flow rate of 0.6 mL / min and a mobile phase volume of 400 mL. Anisaldehyde-concentrated sulfuric acid thin-layer chromatography was used for identification, and the fractions of 180-210 mL were combined and labeled F4. Fraction F4 was separated by semi-preparative HPLC using a YMC-Pack ODS-AA column (250 × 10 mm, 5 μm particle size, 12 nm pore size). The mobile phase was methanol:water (trifluoroacetic acid content 0.03%) = 72:28, at a flow rate of 2.5 mL / min, and the retention time was [not specified]. R The fraction with a flow rate of 16.5–18.0 min was concentrated and dried to obtain the compound salviamarinic acid B.

[0012] The application of the novel water-soluble phenolic acid salviamarinic acid B extracted from *Salvia miltiorrhiza* prepared by the method in the preparation of anti-asthmatic drugs.

[0013] The raw materials of this invention are abundant, the preparation method is easy to operate, the product obtained is of good quality and has good effects. It can inhibit the release of β-Hex, reduce the levels of mMCP-1, β-MCT, HIS and LTC4, significantly improve the degranulation phenomenon of RBL-2H3 cells, and has significant anti-asthmatic activity, with significant economic and social benefits. Attached Figure Description

[0014] Figure 1 This is the structural formula of the compound salviamarinic acid B of this invention.

[0015] Figure 2It is the compound salviamarinic acid B of this invention. 1 H-NMR (500MHz, CD3OD).

[0016] Figure 3 It is the compound salviamarinic acid B of this invention. 13 C-NMR (125MHz, CD3OD).

[0017] Figure 4 This is the DEPT 135 spectrum of the compound salviamarinic acid B of this invention.

[0018] Figure 5 It is the compound salviamarinic acid B of this invention. 1 H- 1 H COSY spectrum.

[0019] Figure 6 This is the HSQC spectrum of the compound salviamarinic acid B of this invention.

[0020] Figure 7 This is the HMBC spectrum of the compound salviamarinic acid B of this invention.

[0021] Figure 8 This is the HR-ESI-MS spectrum of the compound salviamarinic acid B of this invention.

[0022] Figure 9 This is the UV spectrum of the compound salviamarinic acid B of this invention.

[0023] Figure 10 This is the IR spectrum of the compound salviamarinic acid B of this invention.

[0024] Figure 11 This is a graph showing the effect of the compound salviamarinic acid B of this invention on the in vitro intervention of C48 / 80-induced degranulation of RBL-2H3 cells. In the graph, A: β-Hex release rate of RBL-2H3 cells; B: HIS release rate of RBL-2H3 cells; C: Neutral red staining; D: LTC4 release rate of RBL-2H3 cells; E: mMCP-1 release rate of RBL-2H3 cells; F: β-MCT release rate of RBL-2H3 cells. n=6. Compared with group NC, ## P<0.01; compared with group M, * P<0.05, **P<0.01. CON: normal control group; M: model group; 5, 10 μM were different doses of salviamarinic acid B. Detailed Implementation

[0025] The specific implementation of the present invention will be described in detail below with reference to examples and specific circumstances.

[0026] The present invention can be described in the following embodiments.

[0027] Example 1

[0028] This invention discloses a method for preparing a novel water-soluble phenolic acid, salviamarinic acid B, extracted from *Salvia miltiorrhiza*, comprising the following steps:

[0029] (1) Take 50 kg of dried and pulverized Yudanshen, soak it in 50% acetone at room temperature for 24 h, and extract it three times with a flash extractor for 30 s each time. After filtration, combine the filtrates and concentrate under reduced pressure to obtain 18.4 kg of extract.

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

[0031] (3) After concentrating and drying each fraction, dissolve 239.0g of ethyl acetate fraction A in methanol, mix with 100-200 mesh silica gel, and use a 1:1 ratio of sample to silica gel. Pack a column with 200-300 mesh silica gel and use a gradient elution with petroleum ether:dichloromethane and dichloromethane:methanol as mobile phases at a flow rate of 10ml / min. The ratios used are 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, and are checked every 200ml. The amount of each gradient mobile phase is calculated as anisaldehyde-concentrated sulfur. Acid thin-layer chromatography was used to identify the fraction. After elution for 3 days, the fraction containing dichloromethane:methanol (4:1) was combined and labeled B6. Fraction B6 was dissolved in methanol and then loaded onto a silica gel column. The sample was mixed with 100-200 mesh silica gel (sample to silica gel ratio 1:1) and packed into 200-300 mesh silica gel. Gradient elution was performed using ethyl acetate:methanol as the mobile phase at a flow rate of 6 ml / min, with ratios of 16:1, 8:1, 4:1, 2:1, 1:1, and 0:1. Anisaldehyde-concentrated sulfuric acid thin-layer chromatography was used for identification. The fraction containing ethyl acetate:methanol (4:1) was combined and labeled C4. Fraction C4 was dissolved in methanol and analyzed using Sephadex. LH-20 column chromatography was performed, eluting with 75% methanol at a flow rate of 0.6 mL / min and a mobile phase volume of 400 mL. Anisaldehyde-concentrated sulfuric acid thin-layer chromatography was used for identification, and the fractions of 180-210 mL were combined and labeled F4. Fraction F4 was separated by semi-preparative HPLC using a YMC-Pack ODS-AA column (250 × 10 mm, 5 μm particle size, 12 nm pore size). The mobile phase was methanol:water (trifluoroacetic acid content 0.03%) = 72:28, at a flow rate of 2.5 mL / min, and the retention time was [not specified]. R The fraction with a flow rate of 16.5–18.0 min was concentrated and dried to obtain the compound salviamarinic acid B.

[0032] The compound salviamarinic acid B prepared in this invention can inhibit the release of β-aminohexosidase, reduce the levels of mast cell protease 1 (mMCP-1), β-trypsin (β-MCT), histamine (HIS), and leukotriene C4 (LTC4), and significantly improve the degranulation phenomenon of RBL-2H3 cells. Relevant experimental data are as follows:

[0033] I. Instruments and Reagents

[0034] 1.1 Experimental Apparatus

[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] The *Salvia miltiorrhiza* Bunge specimen 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 Traditional Chinese Medicine as the dried root and rhizome of the Lamiaceae family. The specimen (20200901B) is stored in the Laboratory of Traditional Chinese Medicine Chemistry, Henan University of Traditional Chinese Medicine. The column chromatography packing materials Diaion HP-20 and MCIgel CHP-20 were purchased from Mitsubishi Chemical Corporation, Japan; Toyopearl HW-40C from TOSOH Corporation, Japan; and Sephadex LH-20 from Parmacia Biotech. Thin-layer chromatography silica gel (particle size range 10–40 μm) and column chromatography silica gel H (100–200 mesh, 200–300 mesh) were purchased from Qingdao Haiyang Chemical.

[0044] II. Extraction and Separation

[0045] The compound salviamarinic acid B was extracted and separated according to the preparation method of Example 1 above.

[0046] III. Structural Identification

[0047] Colorless waxy solid (CH3OH). HR-ESI-MS yielded a quasi-molecular ion peak at m / z 797.5080 [M+Na]. + ,(calcd.For C 40 H 38 O 16 Na797.5077), its molecular formula was determined to be C 40 H 38 O 16 ;UV(MeOH)λ max :208(3.45), 254(1.10); IR(KBr)ν max cm -1 :3363,2950,1733,1609,1282,1199,1030cm -1 See the detailed diagrams. Figure 2-10 The NMR data (in CD3OD) are shown in Table 3 below, and its structural formula is shown in [Table 3]. Figure 1 As shown:

[0048] Table 3. NMR data (in CD3OD) for compound salviamarinic acid B.

[0049]

[0050]

[0051] IV. Activity Screening

[0052] 4.1 Detection of β-Hex release rate in C48 / 80-induced RBL-2H3 cells using substrate chromogenic assay

[0053] RBL-2H3 cells were cultured at 37°C in a 5% CO2 incubator until the logarithmic growth phase, and then cultured at a cell density of 2 x 10⁻⁶ cells / year. 4 Cells / mL were seeded in 24-well plates. After 24 hours, the cells were divided into a normal (NC) group, a model group (M, C48 / 80, 20 μg / mL), different concentrations of salvia marinic acid B groups (20 μg / mL C48 / 80 + 1 μM, 2.5 μM, 5 μM, 10 μM, 20 μM, 40 μM), and a total enzyme group (1% Trinton X-100). The drugs and C48 / 80 solutions were prepared using Trussin reagent. Five blank wells (containing no cells, only Trussin reagent) were also included. After 30 minutes of stimulation, 50 μL of cell supernatant was collected, and 50 μL of 1 mmol / L β-aminohexosidase substrate solution was added. The mixture was incubated at 37°C for 1 hour, and the reaction was terminated by adding 150 μL of Na2CO3 / NaHCO3 stop solution. The absorbance was measured at 405 nm using a microplate reader, and the β-Hex release rate was calculated. The calculation formula is as follows:

[0054] β-Hex release rate = (OD value of experimental group supernatant - OD value of blank well supernatant) / (OD value of total enzyme supernatant - OD value of blank well supernatant) × 100%

[0055] 4.2 Detection of mast cell activation and degranulation markers using ELISA

[0056] Take the supernatant from step 4.1 and use ELISA to detect the levels of mast cell proteinase 1 (mMCP-1), β-trypsin (β-MCT), histamine (HIS), and leukotriene C4 (LTC4). Follow the instructions carefully in the ELISA kit manual.

[0057] 4.3 Neutral red staining to observe morphological changes in RBL-2H3 cells during C48 / 80-induced degranulation.

[0058] Cells from section 4.1, after the supernatant was collected, were fixed with 4% formaldehyde (400 μL / well), and then stained with 0.5% neutral red (300 μL / well) at room temperature for 10 min. The staining solution was discarded, and the cells were washed twice with PBS before being observed and photographed under an inverted microscope.

[0059] V. Results and Discussion

[0060] This invention uses a C48 / 80-induced RBL-2H3 cell degranulation in vitro model to simulate the degranulation phenomenon of mast cells in vitro, and screens for the activity of salviamarinic acid B. The results are as follows: Figure 11 As shown, compared with the normal group, after treating cells with 20 μg / mLC48 / 80 solution for 30 min, the morphology of RBL-2H3 cells changed from spindle to round, and the release rates of β-Hex, HIS, mMCP-1, β-MCT, and LTC4 were significantly increased (P<0.01). Administration of salviamarinic acid B could significantly reverse this phenomenon (P<0.05 or P<0.01), suggesting that salviamarinic acid B can inhibit mast cell activation and degranulation, and has significant anti-asthmatic activity.

[0061] In summary, this invention utilizes abundant raw materials and employs an easy-to-operate preparation method. For the first time, a novel water-soluble phenolic acid compound, salviamarinic acid B, was isolated and identified from the ethyl acetate fraction of *Salvia miltiorrhiza*. This compound inhibits β-Hex release, reduces mMCP-1, β-MCT, HIS, and LTC4 levels, and significantly improves RBL-2H3 cell degranulation. This suggests that it exerts an anti-asthmatic effect by inhibiting mast cell degranulation, demonstrating significant anti-asthmatic activity. This is highly beneficial for widespread application and offers significant social and economic benefits.

[0062] It should be noted that the above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Any person skilled in the art who can make changes or modifications to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention shall fall within the protection scope of the present invention.

Claims

1. A method for preparing a novel water-soluble phenolic acid, salviamarinic acid B, extracted from *Salvia miltiorrhiza*, characterized in that... The molecular structure of the novel water-soluble phenolic acid salviamarinic acid B is as follows: ； The preparation method is as follows: (1) Take 50 kg of dried and pulverized Yudanshen, soak it in 50% acetone at room temperature for 24 h, and extract it three times with a flash extractor for 30 s each time. After filtration, combine the filtrates and concentrate under reduced pressure to obtain 18.4 kg of extract. (2) Dissolve the extract in 16L of water, and extract it 6 times each with 16L of petroleum ether, dichloromethane, ethyl acetate and n-butanol to obtain petroleum ether fraction, dichloromethane fraction, ethyl acetate fraction, n-butanol fraction and water fraction. (3) After concentrating and drying each fraction, take 239.0g of ethyl acetate fraction A, dissolve it in methanol, mix it with 100-200 mesh silica gel, and use a 1:1 ratio of sample to silica gel. Pack the column with 200-300 mesh silica gel and use petroleum ether:dichloromethane and dichloromethane:methanol as the mobile phase gradient elution at a flow rate of 10ml / min. The ratios used are petroleum ether:dichloromethane = 100:0, 20:1, 10:1, 5:1, 2:1, 0:1 and dichloromethane:methanol respectively. The mobile phase ratios were 20:1, 10:1, 4:1, and 0:1, with checks performed every 200 ml. The volume of each gradient mobile phase was determined by anisaldehyde-concentrated sulfuric acid thin-layer chromatography. Elution was completed after 3 days. The fraction with dichloromethane:methanol = 4:1 was combined and labeled B6. After dissolving fraction B6 in methanol, it was loaded onto a silica gel column. The sample was mixed with 100-200 mesh silica gel, with a sample-to-silica gel ratio of 1:

1. The column was packed with 200-300 mesh silica gel, and gradient elution was performed using ethyl acetate:methanol as the mobile phase at a flow rate of 6. The flow rates were measured at 16:1, 8:1, 4:1, 2:1, 1:1, and 0:1, with ratios of 16:1, 8:1, 4:1, 2:1, 1:1, and 0:1, respectively. Anisaldehyde was identified by TLC with concentrated sulfuric acid. The fractions of ethyl acetate:methanol = 4:1 were combined and labeled C4. Fraction C4 was dissolved in methanol and eluted with 75% methanol on a Sephadex LH-20 column at a flow rate of 0.6 ml / min. The mobile phase volume was 400 ml. Anisaldehyde-concentrated sulfuric acid was used for TLC, and the fractions of 180-210 ml were combined and labeled F4. Fraction F4 was separated by semi-preparative HPLC using a YMC-Pack ODS-AA column (250×10 mm, 5 μm particle size, 12 nm pore size). The mobile phase was methanol:water = 72:28, the flow rate was 2.5 ml / min, and the retention time was collected. R The fraction was concentrated and dried over a period of 16.5 to 18.0 minutes to obtain the compound salviamarinic acid B.

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