A water-soluble phenolic acid, salviamarinic acid C, its preparation method and uses

Abstract

The present application relates to the technical field of medicine, in particular to a water-soluble phenolic acid salviamarinic acid C and a preparation method and application thereof, the present application proves that the level of Th17 cells and pro-inflammatory cytokines TNF-alpha, IL-4, IL-6, IL-17A of salviamarinic acid C in allergic asthma mice is significantly higher than that in normal mice, and the level of Treg cells and anti-inflammatory cytokines IL-35 is significantly lower, which can obviously alleviate the coughing and wheezing symptoms of asthma mice, reduce the IgE level in the serum of mice, reduce airway inflammation, and inhibit airway hyperresponsiveness and mast cell degranulation of mice.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, specifically to a water-soluble phenolic acid, salviamarinic acid C, its preparation method, and its uses. Background Technology

[0002] Asthma is a chronic inflammatory disease of the airways involving multiple cells and cellular components. Clinically, it is characterized by recurrent episodes of wheezing, shortness of breath, chest tightness, or cough. Data from the Global Initiative for Asthma (GINA) shows that asthma affects over 300 million people worldwide and is growing rapidly at a rate of 20%–25% per decade, making it one of the most common diseases globally.

[0003] Salvia miltiorrhiza (Salvia miltiorrhiza) Salviae Miltiorrhizae Radix et Rhizoma is a plant belonging to the genus Salvia in the family Lamiaceae. Salvia miltiorrhiza The dried roots and rhizomes of *Bunge* were first recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica) and listed as a superior herb. They are believed to have effects such as promoting blood circulation and regulating menstruation, removing blood stasis and relieving pain, cooling the blood and reducing swelling, and nourishing the blood and calming the mind. Yudanshen produced in Fangcheng, Nanyang, Henan Province, is characterized by its thick, purplish-red stems, high quality, and superior efficacy. It was praised as the "King of Danshen" by the renowned physician Zhang Zhongjing. In 2013, it obtained GAP certification, establishing a standardized GAP cultivation base in Henan. The cultivation area of ​​Yudanshen exceeds 100,000 mu (approximately 6,667 hectares), making it a well-known geographical indication brand. Summary of the Invention

[0004] The technical problem solved by this invention is to provide a water-soluble phenolic acid, salviamarinic acid C, its uses and preparation process, and to demonstrate its improvement on lung damage and inflammation in mice with allergic asthma, as well as its regulation of Th17 / Treg cells through in vitro and in vivo experiments.

[0005] Specifically, the present invention provides the first extraction and isolation of a novel water-soluble phenolic acid, salviamarinic acid C, with anti-asthmatic activity from *Salvia miltiorrhiza*, the structural formula of which is shown below:

[0006] This invention uses Nanyang Fangcheng Yudanshen as the research object and discovers for the first time that salviamarinic acid C has anti-asthmatic activity from Yudanshen, providing a basis for the development of anti-asthmatic drugs.

[0007] The second objective of this invention is to provide the use of the aforementioned novel compound. This invention isolates and extracts a novel water-soluble phenolic acid, salviamarinic acid C, from tanshinone extract. Its efficacy was evaluated using an ovalbumin (OVA)-induced asthmatic mouse model and a compound 48 / 80 (C48 / 80)-induced RBL-2H3 cell degranulation model. The results showed that salviamarinic acid C significantly alleviated cough and wheezing symptoms in asthmatic mice, reduced serum IgE levels, reduced airway inflammation, and inhibited airway hyperresponsiveness and mast cell degranulation in mice. Its effect was comparable to that of the positive control drug montelukast sodium, suggesting that salviamarinic acid C has a significant interventional effect on asthmatic mice.

[0008] Th17 and Treg cells are functionally antagonistic, working together to maintain immune homeostasis. IL-17A is a major effector secreted by Th17 cells, promoting the recruitment of various inflammatory cells to inflammatory areas and the production of various cytokines such as IL-4, IL-6, and TNF-α, thereby promoting inflammatory responses. IL-35 is an important factor discovered in recent years that mediates the immunosuppressive function of Treg cells. Furthermore, IL-6 is a key factor essential for Th17 cell differentiation; it interacts with TGF-β, promoting Th17 cell differentiation and inhibiting Treg cell proliferation, thus leading to a Th17 / Treg imbalance. This experiment investigated the regulation of the Th17 / Treg immune imbalance in allergic asthmatic mice by detecting the levels of Th17 and Treg cells in peripheral blood and lung tissue, as well as the levels of the above-mentioned related cytokines in alveolar lavage fluid and serum. The results showed that the levels of Th17 cells and pro-inflammatory cytokines TNF-α, IL-4, IL-6, and IL-17A were significantly increased in the asthmatic mouse model compared to normal mice, while the levels of Treg cells and the anti-inflammatory cytokine IL-35 were significantly decreased. Salvia marinic acid C intervention significantly improved the Th17 / Treg cell imbalance and related cytokine changes, suggesting that the intervention effect of salvia marinic acid C on asthma may be related to its regulation of the Th17 / Treg cell ratio and the expression of related cytokines in asthmatic mice.

[0009] A third objective of this invention is to provide a method for preparing the aforementioned novel compound I, comprising the following steps: (1) Take dried and pulverized Salvia miltiorrhiza, soak it in organic solvent for 10-36 hours, pulverize and filter, combine the filtrates and concentrate under reduced pressure to obtain extract; (2) The extract was dissolved in water and extracted in sequence with petroleum ether, dichloromethane, ethyl acetate and n-butanol to obtain the petroleum ether fraction, dichloromethane fraction, ethyl acetate fraction, n-butanol fraction and water fraction. (3) After concentrating and drying each fraction, dissolve the ethyl acetate fraction in methanol, mix with silica gel, elute, and perform thin-layer chromatography for identification; combine the fractions of dichloromethane and methanol, continue to run on a silica gel column, elute with a gradient of petroleum ether: acetone as the mobile phase, and perform thin-layer chromatography for identification; combine the fractions of petroleum ether and acetone, dissolve in methanol, run on an MCI column, elute with a gradient of methanol: water as the mobile phase, and perform thin-layer chromatography for identification; combine the fractions of methanol and water, dissolve in methanol, pass through a Toyopearl HW-40C column, elute with 60% methanol, and perform thin-layer chromatography for identification; (4) The fractions from step (3) were combined and separated by semi-preparative HPLC. The mobile phase was methanol:water and acetonitrile. The retention time was collected. R The fraction was concentrated and dried over a period of 18.9–21.0 min to obtain the compound salviamarinic acid C.

[0010] Further, the organic solvent in step (1) is one or more selected from haloalkanes, aromatic hydrocarbons, lipids and esters; preferably one or more selected from dichloromethane, 1,2-dichloroethane, trichloromethane, toluene, acetone and ethyl acetate. Further still, in step (1), dried and pulverized *Salvia miltiorrhiza* is soaked in 50% acetone at room temperature for 24 hours, and extracted three times using a flash extractor for 30 seconds each time. After filtration, the filtrates are combined and concentrated under reduced pressure to obtain an extract.

[0011] Further, step (2) specifically involves dissolving the extract from step (1) in water and extracting it six times each with petroleum ether, dichloromethane, ethyl acetate, and n-butanol to obtain the petroleum ether fraction, dichloromethane fraction, ethyl acetate fraction, n-butanol fraction, and water fraction. Each fraction is then concentrated, dried, and set aside for later use.

[0012] Further, step (3) specifically involves: dissolving the ethyl acetate fraction from step (2) in methanol, mixing it with 100-200 mesh silica gel, packing it onto a column with 200-300 mesh silica gel, eluting with a gradient of petroleum ether: dichloromethane and dichloromethane: methanol as the mobile phase at a flow rate of 10 ml / min, and using anisaldehyde-concentrated sulfuric acid thin-layer chromatography for identification. After elution for 3 days, the dichloromethane and methanol fractions are combined, dissolved in methanol, and then loaded onto a silica gel column again. The sample is mixed with 200-300 mesh silica gel, packed onto a column with 300-400 mesh silica gel, and eluted with a gradient of petroleum ether: acetone as the mobile phase at a flow rate of 5 ml / min. Anisaldehyde-concentrated sulfuric acid thin-layer chromatography is used for identification. The petroleum ether and acetone fractions are combined, dissolved in methanol, loaded onto an MCI column, eluted with a gradient of methanol: water as the mobile phase at a flow rate of 20 ml / min, and used anisaldehyde-concentrated sulfuric acid thin-layer chromatography for identification. The methanol-water fraction is combined, dissolved in methanol, and analyzed by Toyopearl. HW-40C column chromatography, eluted with 60% methanol, flow rate 0.5 ml / min, mobile phase volume 500 ml, and detected by anisaldehyde-concentrated sulfuric acid thin-layer chromatography.

[0013] Further, in step (3), the ratio of petroleum ether to dichloromethane is 100:0, 20:1, 10:1, 5:1, 2:1, and 0:1, respectively; the ratio of dichloromethane to methanol is 20:1, 10:1, 4:1, and 0:1, respectively; the ratio of petroleum ether to acetone is 50:1, 10:1, 5:1, 2:1, 1:1, and 0:1, respectively; the mobile phase is methanol and water gradient elution at a flow rate of 20 ml / min, and the ratios used are methanol:water = 40:60, 60:40, 80:20, and 100:0, respectively; the weight ratio of the sample to silica gel is 1:1.

[0014] Further, in step (4), the fractions from step (3) are combined and separated using semi-preparative HPLC. The chromatographic column is loaded with a mobile phase of methanol:water = 47:53 and acetonitrile:water = 32:68, both at a flow rate of 3 ml / min. The retention time is collected. R The fraction was concentrated and dried over a period of 18.9–21.0 min to obtain the compound salviamarinic acid C, in which the water contained 0.03% trifluoroacetic acid.

[0015] In a preferred embodiment of the present invention, the specific preparation process is as follows: 50 kg of dried and pulverized *Salvia miltiorrhiza* was soaked in 50% acetone at room temperature for 24 h, and then extracted three times using a flash extractor for 30 s each time. After filtration, the filtrates were combined and concentrated under reduced pressure to obtain 18.4 kg of extract. 18.4 kg of extract was dissolved in 16 L of water and then extracted six times each with 16 L of petroleum ether, dichloromethane, ethyl acetate, and n-butanol to obtain the petroleum ether fraction, dichloromethane fraction, ethyl acetate fraction, n-butanol fraction, and water fraction.

[0016] After concentrating and drying each fraction, dissolve ethyl acetate fraction A (239.0 g) in methanol and mix with 100-200 mesh silica gel. The sample to silica gel ratio is 1:1. Pack a column with 200-300 mesh silica gel and use petroleum ether:dichloromethane and dichloromethane:methanol as mobile phase gradient elution at a flow rate of 10 ml / 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, respectively. Check every 200 ml. The amount of each gradient mobile phase is determined by anisaldehyde-concentrated sulfuric acid thin-layer chromatography. Elution is completed in 3 days. Combine the fraction with dichloromethane:methanol = 10:1 and label it B2. After dissolving component B2 in methanol, it was loaded onto a silica gel column. The sample was mixed with 200-300 mesh silica gel at a ratio of 1:1. The column was packed with 300-400 mesh silica gel and eluted with a gradient of petroleum ether:acetone at a flow rate of 5 ml / min. The ratios used were 50:1, 10:1, 5:1, 2:1, 1:1, and 0:1, respectively. Thin-layer chromatography was performed using anisaldehyde-concentrated sulfuric acid. The fractions with a petroleum ether:acetone ratio of 1:1 were combined and designated as C3. Fraction C3 was dissolved in methanol and loaded onto an MCI column. Gradient elution was performed using a methanol:water mobile phase at a flow rate of 20 ml / min, with the following ratios: methanol:water = 40:60, 60:40, 80:20, and 100:0. Each 100 ml fraction was analyzed. The volume of each gradient mobile phase was determined by anisaldehyde-concentrated sulfuric acid thin-layer chromatography. The fraction with methanol:water = 60:40 was combined and labeled C3. Fraction C3 was then dissolved in methanol and eluted using a Toyopearl HW-40C column with 60% methanol at a flow rate of 0.5 ml / min. The mobile phase volume was 500 ml, and the fraction with anisaldehyde-concentrated sulfuric acid was analyzed by thin-layer chromatography. The fractions of 210-250 ml were combined and labeled D4. Component D4 was separated by semi-preparative HPLC using a YMC-Pack ODS-AA column (250×10mm, 5μm particle size, 12nm pore size). The mobile phases were methanol:water (trifluoroacetic acid content 0.03%) = 47:53 and acetonitrile:water (trifluoroacetic acid content 0.03%) = 32:68, with a flow rate of 3 mL / min for both phases. The retention time was t. R The fraction was concentrated and dried over a period of 18.9–21.0 min to obtain the compound salviamarinicacid C.

[0017] A third objective of this invention is to provide a pharmaceutical formulation selected from any one of injections, aerosols, sprays, capsules, tablets, granules, and suspensions. The aerosol is composed of water-soluble phenolic acid salviamarinic acid C, histidine, ethanol, lactose, polyethylene glycol 600, and a hydrofluoroalkane propellant, calculated by weight percentage. The water-soluble phenolic acid salviamarinic acid C accounts for 0.023-0.057% of the weight of the aerosol; the histidine accounts for 0.008-0.027%; the lactose accounts for 0.014-0.076%; the ethanol accounts for 3-8%; the polyethylene glycol 600 accounts for 0.014-0.024%; and the balance is a hydrofluoroalkane propellant.

[0018] Compared with the prior art, the technical advantages of the present invention are as follows: 1) To investigate the regulation of Th17 / Treg immune imbalance in allergic asthmatic mice by salvia marinic acid C. Results showed that Th17 cells and pro-inflammatory cytokines TNF-α, IL-4, IL-6, and IL-17A were present in the asthmatic mouse model. The levels of Th17 cells were significantly higher than those in normal mice, while the levels of Treg cells and the anti-inflammatory cytokine IL-35 were significantly lower. Salvia marinic acid C intervention significantly improved the imbalance of Th17 / Treg cells and the changes in related cytokines, suggesting that the intervention effect of salvia marinic acid C on asthma may be related to its regulation of the Th17 / Treg cell ratio and the expression of related cytokines in asthmatic mice.

[0019] Salviamarinic acid C was found to significantly alleviate cough and wheezing symptoms in asthmatic mice, reduce serum IgE levels, reduce airway inflammation, and inhibit airway hyperresponsiveness and mast cell degranulation. Its effects were comparable to those of the positive control drug montelukast sodium, suggesting that salviamarinic acid C has a significant intervention effect on asthmatic mice.

[0020] The aerosol prepared using the new compound achieved a spray area that met the basic requirements of the pharmacopoeia. This invention provides strong experimental evidence for elucidating the anti-asthmatic active ingredients of *Yudanshen* and also contributes to the complete analysis of the pharmacological components of *Yudanshen*. Attached Figure Description

[0021] Figure 1 This is the molecular structural formula of the compound of the present invention.

[0022] Figure 2The compound salviamarinic acid C 1 H-NMR (500 MHz, CD3OD).

[0023] Figure 3 The compound salviamarinic acid C 13 C-NMR (125 MHz, CD3OD).

[0024] Figure 4 DEPT 135 spectrum of compound salviamarinic acid C.

[0025] Figure 5 The compound salviamarinic acid C 1 H- 1 H COSY spectrum.

[0026] Figure 6 HSQC spectrum of compound salviamarinic acid C.

[0027] Figure 7 HMBC spectrum of compound salviamarinic acid C.

[0028] Figure 8 NOESY spectrum of compound salviamarinic acid C.

[0029] Figure 9 HR-ESI-MS spectrum of compound salviamarinic acid C.

[0030] Figure 10 UV spectrum of compound salviamarinic acid C.

[0031] Figure 11 IR spectrum of compound salviamarinic acid C.

[0032] Figure 12 Salvia marinic acid C improves the typical phenotype of OVA-induced allergic asthma, where A represents the number of coughs in mice; B represents the cough latency in mice; C represents tidal volume (TVb); D represents airway hyperresponsiveness (Penh); E represents apnea (Pau); F represents inspiratory time (Ti); G represents expiratory time (Te); H represents PGD2 levels in bronchoalveolar lavage fluid; I represents serum IgE levels (n=10); and J represents HE, Masson, and PAS staining of lung tissue. Yellow arrows indicate lesion sites. n=10, compared to the CON group, ##P <0.01; compared with the OVA group, * P <0.05, ** P <0.01. CON: Normal control group, OVA: Model group, Mon: Positive drug montelukast sodium group, S1-L: Low-dose (10mg / kg) salviamarinic acid C group, S1-H: High-dose (20mg / kg) salviamarinic acid C group.

[0033] Figure 13 Effects of salviamarinic acid C on inflammation in OVA-induced allergic asthmatic mice A: Serum IL-4 level; B: Serum IL-5 level; C: Serum IL-10 level; D: Serum IL-13 level; E: Serum TNF-α level; F: Serum IL-17A level; G: Serum GM-CSF level; H: Serum CXCL1 level; I: Serum CCL11 level; J: Lung tissue IL-4 level; K: Lung tissue IL-5 level; L: Lung tissue IL-10 level; M: Lung tissue IL-13 level; N: Lung tissue TNF-α level; O: Lung tissue IL-17A level; P: Lung tissue GM-CSF level; Q: Lung tissue CXCL1 level; R: Lung tissue CCL11 level. n=4, compared with the CON group, ## P <0.01; compared with the OVA group, * P <0.05, ** P <0.01. CON: Normal control group, OVA: Model group, Mon: Positive drug montelukast sodium group, S1-L: Low-dose (10mg / kg) salviamarinic acid C group, S1-H: High-dose (20mg / kg) salviamarinic acid C group.

[0034] Figure 14 Effects of salvia marinic acid C on Th17 / Treg ratio in peripheral blood of OVA-induced allergic asthmatic mice A: Flow cytometry of Treg cells in peripheral blood; B: Flow cytometry of Th7 cells in peripheral blood; C: Quantitative analysis of Treg cells in peripheral blood; D: Quantitative analysis of Th7 cells in peripheral blood. n=3, compared with the CON group, ## P <0.01; compared with the OVA group, * P<0.05, ** P <0.01. CON: Normal control group, OVA: Model group, Mon: Positive drug montelukast sodium group, S1-L: Low-dose (10mg / kg) salviamarinic acid C group, S1-H: High-dose (20mg / kg) salviamarinic acid C group.

[0035] Figure 15 Effects of salviamarinic acid C on Th17 / Treg ratio in lung tissue of OVA-induced allergic asthma mice A: Flow cytometry of Treg cells in lung tissue; B: Flow cytometry of Th7 cells in lung tissue; C: Quantitative mapping of Treg cells in lung tissue; D: Quantitative mapping of Th7 cells in lung tissue. n=3, compared with the CON group, # P < 0.05, ## P < 0.01; compared with the OVA group, * P < 0.05, ** P < 0.01. CON: Normal control group, OVA: Model group, Mon: Positive drug montelukast sodium group, S1-L: Low-dose (10 mg / kg) salviamarinic acid C group, S1-H: High-dose (20 mg / kg) salviamarinic acid C group.

[0036] Figure 16 Accelerated testing conditions were used to sample and test the droplet distribution of the aerosol at the end of months 0, 1, 2, 3, and 6. Detailed Implementation

[0037] The following non-limiting embodiments are provided to further illustrate the present invention.

[0038] I. Selection of Experimental Instruments and Consumables

[0039] Table 1 List of Main Instruments

[0040] 1.2 Experimental Reagents

[0041] 1.3 Experimental Materials Column chromatography packing materials: Diaion HP-20, MCI gel CHP-20 (Mitsubishi Chemical Corporation, Japan), Toyopearl HW-40C (TOSOH Corporation, Japan), thin layer chromatography silica gel (particle size range 10-40 mm) (Qingdao Ocean Chemical Plant), column chromatography silica gel H (100-200 mesh, 200-300 mesh, Qingdao Ocean Chemical).

[0042] Deployment System: (1) Petroleum ether: Ethyl acetate; (2) Dichloromethane: Methanol; (3) Ethyl acetate: Methanol; (4) Ethyl acetate: ethanol: water; Color developer: (1) Ferric chloride-potassium ferricyanide (1:1); (2) 1% anisaldehyde-concentrated sulfuric acid 1.4 Specific preparation method of the new water-soluble phenolic acid extract salviamarinic acid C Example 1: Method for extracting a new water-soluble phenolic acid, salviamarinic acid C, from Salvia miltiorrhiza. Take 50 kg of dried and pulverized *Salvia miltiorrhiza*, 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.

[0043] The extract was dissolved in 16L of water and then extracted six times each with 16L of petroleum ether, dichloromethane, ethyl acetate, and n-butanol to obtain the petroleum ether fraction, dichloromethane fraction, ethyl acetate fraction, n-butanol fraction, and water fraction.

[0044] After concentrating and drying each fraction, dissolve ethyl acetate fraction A (239.0 g) in methanol and mix with 100-200 mesh silica gel. The sample to silica gel ratio is 1:1. Pack a column with 200-300 mesh silica gel and use petroleum ether:dichloromethane and dichloromethane:methanol as mobile phase gradient elution at a flow rate of 10 ml / 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. Check every 200 ml. The amount of each gradient mobile phase is determined by anisaldehyde-concentrated sulfuric acid thin-layer chromatography. Elution is completed in 3 days. Combine the fraction with dichloromethane:methanol = 10:1 and label it B2. After dissolving component B2 in methanol, it was loaded onto a silica gel column and mixed with 200-300 mesh silica gel at a 1:1 ratio. The column was then packed with 300-400 mesh silica gel. A gradient elution was performed using petroleum ether:acetone as the mobile phase at a flow rate of 5 ml / min, with ratios of 50:1, 10:1, 5:1, 2:1, 1:1, and 0:1. Thin-layer chromatography with anisaldehyde-concentrated sulfuric acid was used for identification. The fraction with petroleum ether:acetone = 1:1 was combined and labeled C3. Component C3 was dissolved in methanol and loaded onto an MCI column. A gradient elution was performed using methanol:water as the mobile phase at a flow rate of 20 ml / min, with ratios of 40:60, 60:40, 80:20, and 100:0. Identification was performed every 100 ml. The amount of each gradient mobile phase was determined by thin-layer chromatography with anisaldehyde-concentrated sulfuric acid. The fraction with methanol:water = 60:40 was combined and labeled C3. Fraction C3 was dissolved in methanol and eluted with 60% methanol using a Toyopearl HW-40C column at a flow rate of 0.5 ml / min. The mobile phase volume was 500 ml. Identification was performed using anisaldehyde-concentrated sulfuric acid thin-layer chromatography. The fractions of 210-250 ml were combined and labeled D4. Fraction D4 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 phases were methanol:water (trifluoroacetic acid content 0.03%) = 47:53 and acetonitrile:water (trifluoroacetic acid content 0.03%) = 32:68, both at a flow rate of 3 ml / min. The retention time was collected. R The fraction was concentrated and dried over a period of 18.9–21.0 min to obtain the compound salviamarinicacid C. HPLC analysis showed it as a single peak.

[0045] HR-TOF-MS of water-soluble phenolic acid salviamarinic acid C yielded quasi-molecular ion peaks. m / z 755.5795 [M+Na] + (C) 37 H 32 O 16Na (calculated value 755.5791), its molecular formula is determined to be C. 37 H 32 O 16 ,UV (CH3OH) λ max nm: 205 (2.46), 254 (0.51); IR (CH3OH): ν max cm -1 :3363,2953,1732,1611,1520,1286,1198,1023.

[0046]

[0047] Figure 1 Structural formula of compound (I) NMR data of compound (I) salviamarinic acid C (in CD3OD)

[0048] Example 2: Method for extracting water-soluble phenolic acid salviamarinic acid C from Salvia miltiorrhiza 50 kg of dried and pulverized *Salvia miltiorrhiza* was soaked in 50% dichloromethane at room temperature for 10 h, and then extracted twice using a flash extractor for 50 s each time. After filtration, the filtrates were combined and concentrated under reduced pressure to obtain 17.2 kg of extract. The extract was dissolved in 16 L of water and extracted four times each with 16 L of petroleum ether, dichloromethane, ethyl acetate, and n-butanol to obtain the petroleum ether fraction, dichloromethane fraction, ethyl acetate fraction, n-butanol fraction, and water fraction. After concentrating and drying each fraction, 182.2 g of ethyl acetate fraction A was dissolved in methanol, and the rest of the process was the same as in Example 1. HPLC It is displayed as a single peak.

[0049] Example 3: Method for extracting water-soluble phenolic acid salviamarinic acid C from Salvia miltiorrhiza Take 50 kg of dried and pulverized *Salvia miltiorrhiza*, soak it in 50% ethyl acetate at room temperature for 36 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 17.5 kg of extract.

[0050] The extract was dissolved in 16L of water and then extracted five times each with 16L of petroleum ether, dichloromethane, ethyl acetate, and n-butanol to obtain the petroleum ether fraction, dichloromethane fraction, ethyl acetate fraction, n-butanol fraction, and water fraction.

[0051] After concentrating and drying each fraction, ethyl acetate fraction A (185.7g) was dissolved in methanol. Other steps were the same as in Example 1. HPLC showed a single peak.

[0052] II. Improvement of OVA-induced typical phenotype of allergic asthma by water-soluble phenolic acid salviamarinic acid C 2.1 Experimental animals Fifty SPF-grade BALB / c mice, female, weighing 18-22 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (license number: SCXK (Jing) 2021-0006). The experimental mice were housed in the standardized animal feeding room of Henan University of Chinese Medicine, with a relative humidity of 40%-60%, a temperature of (23±2) ℃, and free access to food and water under a 12 h light / 12 h dark cycle. The animal experiment was approved by the Experimental Animal Ethics Committee of Henan University of Chinese Medicine (approval number DWLL2018080003).

[0053] 2.2 Experimental drugs and reagents Montelukast sodium, purchased from MSD Pharmaceutical Co., Ltd. (positive control drug, product number: 14202007317); ovalbumin grade II (batch number: A5253), ovalbumin grade V (batch number: A5503), Compound 48 / 80 (batch number: C2313), mouse immunoglobulin E (IgE) (batch number: MM-0056M1), histamine (HIS) (batch number: MM-0548M1), leukotriene C4 (LTC4) (batch number: MM-0395M1), prostaglandin D2 (PGD2) (batch number: MM-43727M1), interleukin-4 (IL-4) (batch number: MM-0165M1), interleukin-5 (IL-5) (batch number: MM-0164M1), interleukin-10 (IL-10) (batch number: MM-0176M1), interleukin-13 (IL-13) (batch number: MM-0173M1), tumor necrosis factor- α (TNF- α ) (batch number: MM-0132M1), mast cell β tryptase ( βThe ELISA kits for MCT (batch number: MM-46274M1) and mMCP-1 (batch number: MM-45085M1) were purchased from Jiangsu Enzyme Immunoassay Biotechnology Co., Ltd. TSLP (catalog number: ab47943), IL-33 (catalog number: 66235-1-Ig), Anti-Mouse CD4 PE (catalog number: 2198944), Anti-Mouse IL-17A APC (catalog number: 2142931), Anti-Mouse CD4 FITC (catalog number: 11-0041-82), Anti-Mouse CD25 PE (catalog number: 12-0251-83), and Anti-Mouse Foxp3 APC (catalog number: 17-5773-82) were purchased from eBioscience, USA. Fetal bovine serum (FBS) was purchased from Hangzhou Sijiqing Pharmaceutical Co., Ltd. DMEM high glucose medium was purchased from Gibco Invitrogen. Ampicillin and streptomycin were purchased from Sigma-Aldrich. Thiazol blue (MTT) was purchased from Beijing Solarbio Science & Technology Co., Ltd., and dimethyl sulfoxide (DMSO) was purchased from Shanghai Maclean Biotechnology Co., Ltd.

[0054] 2.3 Experimental Apparatus 402A1 ultrasonic nebulizer (Jiangsu Yuyue Medical Equipment Co., Ltd.); YLS-8A multifunctional cough and asthma induction device (Jinan Yiyan Technology Development Co., Ltd.); BD FACS Aria III flow cytometer (BD Biosciences, USA); SORVALL ST16R Centrifuge5810R high-speed refrigerated centrifuge (Eppendorf, Germany); BioTek multifunctional microplate reader (Bio-Rad, USA); micropipette (Eppendorf, Germany).

[0055] 2.4 Animal Grouping Six- to seven-week-old female BALB / c mice were acclimatized for 3 days and then randomly divided into five groups: a normal control group (CON), a model group (OVA), a positive control group (Y), a low-dose salvia marinic acid C group (S1-L), and a high-dose salvia marinic acid C group (S1-H), with ten mice in each group. An allergic asthma mouse model was established using intraperitoneal injection of OVA and aluminum hydroxide gel, and OVA nebulization challenge. Except for the normal control group, the other experimental mice were sensitized by intraperitoneal injection of 0.2 mL of a suspension of chicken ovalbumin (OVA) and aluminum hydroxide (Al2(OH)3) (OVA 50 mg, Al2(OH)3 2 mg dissolved in 0.2 mL sterile saline) on days 0, 7, and 14. From days 21 to 27, they were challenged by nebulization with 1% OVA dissolved in saline (100 mg OVA dissolved in 10 mL saline), 30 min / time / day. The model group, positive control group, low-dose salviamarinic acid C group, and high-dose salviamarinic acid C group were administered equal volumes of physiological saline, montelukast sodium (2.6 mg / kg), salviamarinic acid C (10 mg / kg), and salviamarinic acid C (20 mg / kg), respectively, 30 min before challenge. The normal control group was sensitized by intraperitoneal injection of 0.2 mL of aluminum hydroxide (Al2(OH)3) suspension (Al2(OH)3 2 mg dissolved in 0.2 mL sterile physiological saline) per mouse on days 0, 7, and 14. From days 21 to 27, mice were challenged by nebulized physiological saline for 30 min, with an equal volume of physiological saline administered 30 min before challenge. Lung function was assessed and samples were collected 24 h after the last nebulization challenge.

[0056] 2.5 Cough and wheezing index testing Twenty-four hours after the last challenge, mice were placed in the aerosol chamber of a multifunctional cough-inducing and asthma-inducing device. Coughing was induced for 15 seconds with a 25% ammonia spray. The time from the start of the spray to the first cough, i.e., the cough latency period, was observed, and the number of coughs within 2 minutes was recorded. Similarly, asthma was induced by a spray of an equal mixture of 0.1% histamine phosphate and 2% acetylcholine chloride. The time from the start of the spray to the onset of shortness of breath and wheezing, i.e., the asthma-inducing latency period, was observed, and the number of wheezes within 2 minutes was recorded.

[0057] 2.6 Airway responsiveness testing Twenty-four hours after the last challenge, mice were sequentially placed into the FinePointe WBP plethysmography chamber, and baseline values ​​were adjusted with PBS. Baseline values ​​were recorded for approximately 2 minutes. Subsequently, each mouse was administered methacholine (Mech) via nebulization at escalating concentrations (0, 3.125 mg / mL, 6.25 mg / mL, 12.5 mg / mL, 25 mg / mL, and 50 mg / mL), with each dose nebulized for 30 seconds and continuous recording for 5 minutes. Changes in lung function and airway responsiveness (Penh) at different concentrations were measured.

[0058] 2.7 Collection of serum and bronchoalveolar lavage fluid samples After airway responsiveness testing, blood was collected by enucleation, centrifuged at 4000 rpm for 10 min, and serum samples were obtained and stored at -80℃ for later use. Following blood collection, endotracheal intubation was performed to collect bronchoalveolar lavage fluid (BALF). The entire lungs were lavaged three times with sterile saline, 0.5 mL per lavage. The BALF was centrifuged at 4000 rpm for 10 min, and the supernatant was collected and stored at -80℃ for later use.

[0059] 2.8 Pathological assessment of lung tissue Three mice were randomly selected from each group. The left lung and trachea that had not been irrigated were taken and fixed in 4% paraformaldehyde for 24 hours. H&E, PAS and Masson staining were performed respectively.

[0060] 2.9 Detection of relevant cytokine levels Following the kit instructions, the ELISA method was used to detect IgE, PGD2, HIS, and LTC4 in serum, as well as IL-4, IL-5, IL-10, IL-13, mMCP-1, and [other substances] in serum and BALF. β -Levels such as MCT.

[0061] 2.10 Flow cytometry detection of Th17 / Treg cell levels in mouse peripheral blood and lung tissue Peripheral blood was collected from mice via enucleation, and 100 μL of plasma was collected in flow cytometry tubes, labeled as Th17 and Treg cells, respectively. Th17 cells were labeled with PE-CD4 and APC-IL-17A antibodies, while Treg cells were labeled with FITC-CD4, PE-CD25, and APC-Foxp3 antibodies, respectively. Each tube was incubated with the corresponding external standard antibody in the dark for 30 min, followed by the addition of 1× erythrocyte lysis buffer and lysis for approximately 10 min until the liquid became clear. The cells were centrifuged at 300×g for 5 min, the supernatant was discarded, and the cells were resuspended in 2 mL of PBS. The washing process was repeated twice. Add 1 mL of cell permeabilization working solution to each tube to resuspend the cells and incubate at room temperature in the dark for 45 min. Add 2 mL of 1× cell permeabilization buffer to each tube, centrifuge at 500×g for 5 min, and discard the supernatant. Resuspend the cells in 100 μL of 1× cell permeabilization buffer, add the corresponding internal standard antibody (APC-Foxp3 antibody), and incubate in the dark for 30 min. Then, add 300 μL of PBS to each tube to resuspend the cells and perform analysis.

[0062] Three mice were randomly selected from each group. The lungs were quickly dissected and removed. Blood was rinsed off and the organs dried. A suitable amount of lung tissue was placed in a 1.5 mL centrifuge tube, minced, and digested with 1 mL of trypsin for 20-30 minutes until the cells became mucous. Serum was added to stop the digestion. The filtrate was collected and centrifuged at 1200 rpm for 5 minutes. After centrifugation, the supernatant was slowly discarded to obtain primary lung cells. The labeling and detection of Th17 and Treg cells in the primary lung cells were performed as described above.

[0063] 3. In vitro experiments 3.1 Detection of RBL-2H3 cell degranulation level The rat basophilic leukemia cell line (RBL-2H3) was purchased from the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. RBL-2H3 cells in logarithmic growth phase were cultured at 5 × 10⁻⁶ cells / cells. 4 Cells / mL were seeded in 24-well cell culture plates. After 24 h of culture, the cells were divided into a normal control group (CON), a model group (M, C48 / 80, 20 μg / mL), different concentrations of salvia marinic acid C groups (20 μg / mL C48 / 80 + 1 μM, 10 μM, 20 μM, 40 μM), and a total enzyme group (TE, 1% Triton X-100). After incubation at 37°C in a 5% CO2 incubator for 30 min, the cells were centrifuged, and the supernatant was used for measurement. β -Aminohexosidase ( β Release of Hex and histamine (HIS).

[0064] β-Hex release rate determination: Take 50 μL of cell supernatant and add 50 μL of 1 mmol / L... β -Aminohexosidase substrate solution, incubated at 37°C for 1 h, the reaction was terminated by adding 150 μL Na2CO3 / NaHCO3 stop solution, and the absorbance was measured at 405 nm using a microplate reader to calculate... β -Hex release rate. (Calculation formula:) 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% HIS release rate determination: The HIS release rate of RBL-2H3 cells was detected by ELISA, strictly following the kit instructions.

[0065] 3.2 Neutral Red Staining Cells from the supernatant were fixed with 4% formaldehyde (400 mL / well) and then stained with 0.5% neutral red (300 mL / 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.

[0066] 4. Statistical Methods

[0067] Experimental data are presented in the form of ` x ± s This indicates that SPSS 20.0 was used for one-way ANOVA statistical analysis. All statistical analysis results were presented using GraphPad Prism 8.0.

[0068] 5. Experimental Results 5.1 Improvement of the typical phenotype of OVA-induced allergic asthma by EB-A

[0069] Compared with normal mice, mice in the OVA group showed significantly increased airway responsiveness (Penh), coughing frequency, and PGD2 levels in alveolar lavage fluid, and a significantly shortened coughing latency. Salvia marinic acid C intervention significantly prevented these changes compared to the OVA group. Furthermore, the serum total IgE level in the OVA group was significantly higher than that in the normal group, while the serum total IgE level in the positive control group and the low- and high-dose salvia marinic acid C groups was significantly lower than that in the OVA group. P < 0.01). Furthermore, histopathological analysis of lung tissue showed significant inflammatory cell infiltration and collagen deposition in the lung tissue of mice with allergic asthma; these conditions were significantly improved after intervention with salviamarinic acid C, suggesting that salviamarinic acid C significantly improved the typical characteristics of OVA-induced allergic asthma. Results are shown in […]. Figure 12 .

[0070] 5.2 Effects of salviamarinic acid C on inflammation in OVA-induced allergic asthmatic mice The levels of various cytokines in serum and BALF were detected. Results showed that, compared with the CON group, the levels of IL-4, IL-5, IL-10, IL-13, TNF-α, IL-17A, GM-CSF, CXCLI, and CCL11 in the serum and BALF of mice in the OVA group were significantly upregulated. P < 0.01); Compared with the OVA group, the levels of IL-4, IL-5, IL-10, IL-13, TNF-α, IL-17A, GM-CSF, CXCLI, and CCL11 in the positive control group and the low- and high-dose salviamarinic acid C groups were significantly decreased ( P < 0.01 or P The result was <0.05%, suggesting that salviamarinic acid C can alleviate OVA-induced inflammation in mice with allergic asthma. See the results below. Figure 13 .

[0071] 5.3 Effects of salviamarinic acid C on the Th17 / Treg imbalance in peripheral blood and lung tissue of allergic asthmatic mice Th17 / Treg immune imbalance plays an important role in the pathogenesis of asthma, which can be detected by flow cytometry. Levels of Th17 and Treg cells in peripheral blood and lung tissue of mice with allergic asthma. Results showed that, compared with the CON group, the levels of Th17 and Treg cells in peripheral blood and lung tissue of mice in the OVA group were significantly increased. P <0.01); Compared with the OVA group, salviamarinic acid C significantly reduced the levels of Th17 and Treg cells in the peripheral blood and lung tissue of mice with allergic asthma ( P < 0.01 or P < 0.05). This indicates that salviamarinic acid C can improve the Th17 / Treg cell immune imbalance in allergic asthmatic mice. Results are shown in […]. Figure 14 and Figure 15 .

[0072] Aerosols containing salviamarinic acid C Example 1: An aerosol, the composition and preparation method of which are as follows:

[0073] Preparation method: Histidine and lactose are dissolved in 50% ethanol water, then water-soluble phenolic acid salviamarinic acid and polyethylene glycol 600 are added to dissolve them. The pH of the solution is adjusted to 4.5 by adding citrate-sodium citrate buffer. The propellant is then added under pressure and stirred. The solution is then filled into aerosol bottles under pressure to obtain the final product.

[0074] Example 2: An aerosol, the composition and preparation method of which are as follows:

[0075] The preparation process is the same as in Example 1.

[0076] Example 3: An aerosol, the composition and preparation method of which are as follows:

[0077] The preparation process is the same as in Example 1.

[0078] Comparative Example 1: An aerosol, the composition and preparation method of which are as follows:

[0079] The preparation process is the same as in Example 1.

[0080] Comparative Example 2: An aerosol, the composition and preparation method of which are as follows:

[0081] The preparation process is the same as in Example 1.

[0082] Comparative Example 3: An aerosol, the composition and preparation method of which are as follows:

[0083] The preparation process is the same as in Example 1.

[0084] Comparative Example 4: An aerosol, the composition and preparation method of which are as follows:

[0085] The preparation process is the same as in Example 1.

[0086] For details on droplet distribution detection of aerosols, please refer to [link / reference]. Figure 5 Simultaneously, an accelerated test was conducted for 6 months under the following conditions: temperature (40±2)℃ and relative humidity (75±5)%, lasting for 6 months. During the test, samples were taken at the end of months 0, 1, 2, 3, and 6 to examine the droplet distribution of the aerosol.

Claims

1. A method for preparing a water-soluble phenolic acid, salviamarinic acid C, characterized in that, Includes the following steps: (1) Take dried and pulverized Yu Danshen, soak it in 50% acetone at room temperature for 24 hours, and extract it three times with a flash extractor for 30 seconds each time. After filtration, combine the filtrates and concentrate under reduced pressure to obtain the extract. (2) Dissolve the extract from step (1) in water and extract it 6 times each with petroleum ether, dichloromethane, ethyl acetate and n-butanol to obtain the petroleum ether fraction, dichloromethane fraction, ethyl acetate fraction, n-butanol fraction and water fraction. Concentrate and dry each fraction for later use. (3) Dissolve the ethyl acetate fraction from step (2) in methanol, mix with 100-200 mesh silica gel, pack onto a 200-300 mesh silica gel column, and elute with a gradient of petroleum ether: dichloromethane and dichloromethane: methanol as the mobile phase at a flow rate of 10 ml / min. Use anisaldehyde-concentrated sulfuric acid thin-layer chromatography for identification. After elution for 3 days, combine the dichloromethane and methanol fractions, dissolve in methanol, and continue loading onto a silica gel column. Mix with 200-300 mesh silica gel, pack onto a 300-400 mesh silica gel column, and elute with a gradient of petroleum ether: acetone as the mobile phase at a flow rate of 5 ml / min. Use anisaldehyde-concentrated sulfuric acid thin-layer chromatography for identification. Combine the petroleum ether and acetone fractions, dissolve in methanol, load onto an MCI column, and elute with a gradient of methanol: water as the mobile phase at a flow rate of 20 ml / min. Use anisaldehyde-concentrated sulfuric acid thin-layer chromatography for identification. Combine the methanol-water fractions, dissolve in methanol, and pass through a Toyopearl filter. HW-40C column chromatography was performed using 60% methanol as the eluting agent at a flow rate of 0.5 ml / min and a mobile phase volume of 500 ml. Anisaldehyde-concentrated sulfuric acid thin-layer chromatography was used for detection. The ratios of petroleum ether to dichloromethane were 100:0, 20:1, 10:1, 5:1, 2:1, and 0:1; the ratios of dichloromethane to methanol were 20:1, 10:1, 4:1, and 0:1; and the ratios of petroleum ether to acetone were 50:1, 10:1, 5:1, 2:1, 1:1, and 0:

1. Methanol and water were used as the mobile phase for gradient elution at a flow rate of 20 ml / min, with ratios of methanol:water of 40:60, 60:40, 80:20, and 100:

0. The weight ratio of sample to silica gel was 1:

1. (4) The fractions from step (3) were combined and separated by semi-preparative HPLC. The chromatographic column was loaded with methanol:water = 47:53 and acetonitrile:water = 32:68 as the mobile phases, and the flow rate was 3 ml / min for both. The retention time was collected. R The fraction with a flow rate of 18.9~21.0 min was concentrated and dried to obtain the compound salviamarinic acid C, in which the aqueous phase contained 0.03% trifluoroacetic acid. The water-soluble phenolic acid salviamarinic acid C has the structural formula shown in Formula I: Ⅰ。

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