Ligustrazine ferulate as well as preparation method and application thereof

Abstract

The invention discloses ligustrazine ferulate and a preparation method and application thereof, and belongs to the technical field of pharmacy, the preparation method comprises the following steps: dissolving ligustrazine in methanol to obtain a methanol solution of the ligustrazine, weighing the ligustrazine, adding the ligustrazine into a bottle, magnetically stirring at 28 DEG C for 24 hours, controlling the water bath temperature at 50 DEG C, and performing vacuum concentration and suction filtration to obtain the ligustrazine ferulate. The ligustrazine ferulate provided by the invention is higher in solubility, and can play a better anti-pulmonary fibrosis drug effect.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmacy, and in particular to ferulic acid ligustrazine, a preparation method thereof and an application thereof. Background Art

[0002] Pulmonary fibrosis is a progressive interstitial pneumonia, which is characterized by alveolar structure remodeling, limited ventilation function, and continuous aggravation of pulmonary interstitial fibrosis, and ultimately can lead to respiratory failure with extremely poor prognosis.

[0003] Clinically, appropriate drugs are usually selected for treatment according to the specific symptoms and conditions of patients. Pirfenidone and nintedanib are commonly used anti-fibrotic drugs in clinical practice. However, the exact mechanism of action of pirfenidone is not yet clear, and it cannot reverse the already formed fibrosis. At the same time, the therapeutic anti-inflammatory effect of nintedanib is weaker than the prophylactic anti-inflammatory effect. At the same time, glucocorticoids and N-acetylcysteine are also widely used in clinical treatment. However, long-term use of glucocorticoids will cause obesity, damage to the digestive system, and a certain inhibitory effect on the body's immune system. Therefore, it is often used as an adjuvant treatment drug and cannot be the first choice for long-term treatment of pulmonary fibrosis. The clinical effect of N-acetylcysteine remains to be studied, and the improvement of symptoms is not obvious.

[0004] Ligustrazine (Lig), as shown in formula I, is an alkaloid with various pharmacological activities, mainly derived from the dry rhizome of the Umbelliferae plant Ligusticum chuanxiong Hort. It has the effects of antiplatelet aggregation, dilation of small arteries, improvement of microcirculation, promoting blood circulation and removing blood stasis, etc. Ligustrazine is mainly used in clinical treatment of occlusive vascular diseases, cerebral thrombosis, vasculitis, coronary heart disease, angina pectoris and other diseases. The disadvantages of ligustrazine, such as being easily soluble in hot water and petroleum ether, soluble in chloroform and dilute hydrochloric acid, slightly soluble in ether, insoluble in cold water, and low bioavailability, affect the exertion of its efficacy.

[0005]

[0006] Ferulic acid (FA) as shown in Formula II is mainly derived from various plants, such as the rhizomes of Ferula assafoetida and Ligusticum chuanxiong in the Umbelliferae family, the whole herb of Lycopodium selago in the Lycopodiaceae family, the whole herb of Equisetum hyemale in the Equisetaceae family, etc. Ferulic acid has the effects of antiplatelet aggregation, inhibiting the release of platelet 5-hydroxytryptamine, inhibiting the production of platelet thromboxane A2, enhancing prostaglandin activity, analgesia, and relieving vasospasm; it is the basic raw material for the production of drugs for treating cardiovascular and cerebrovascular diseases and leukopenia, such as Xinxuankang and Limai capsules. Ferulic acid has poor stability in aqueous solution and is sensitive to light, pH value, and temperature. Under sunlight irradiation, its content will drop sharply within three days; as the temperature rises, its degradation rate will also increase significantly; and under high pH conditions, it will accelerate degradation and produce colored products. This means that products containing ferulic acid require special storage conditions, such as using dark or light-proof packaging and placing them in a place protected from direct sunlight; in the high-temperature season, refrigeration may even be required to maintain its activity.

[0007] Summary of the Invention

[0008] Based on this, the present invention provides a ligustrazine ferulate, its preparation method and application. The ligustrazine ferulate of the present invention can exert a better anti-pulmonary fibrosis pharmacodynamic effect and has higher solubility. The content of the present invention is as follows:

[0009] The first object of the present invention is to provide a ligustrazine ferulate. The technical point is that the molecular formula of the ligustrazine ferulate is C 28 H 29 O 8 N 2 , and the melting point of the ligustrazine ferulate is 152 °C - 154 °C, and the structural formula is as shown in Formula III:

[0010]

[0011] The second object of the present invention is to provide a preparation method of ligustrazine ferulate. The technical point is that it includes the following steps

[0012] Step 1: Place ligustrazine at the bottom of a round-bottom flask, and then add methanol to dissolve the ligustrazine to obtain a methanol solution of ligustrazine;

[0013] Step 2: Weigh ferulic acid and place it in the methanol solution of ligustrazine in Step 1 and dissolve it until the liquid turns pink;

[0014] Step 3: Stir the liquid in Step 2 at 28 °C for 24 h, concentrate it in a water bath at 50 °C, add absolute ethanol and filter to obtain the crystals of crude ligustrazine ferulate, and place the crystals of crude ligustrazine ferulate in a petri dish and dry them naturally;

[0015] Step 4: Recrystallize the crude tetramethylpyrazine ferulate obtained by drying in Step 3.

[0016] To better implement the above technical solution, in Step 1 of the preparation method of tetramethylpyrazine ferulate of the present invention, the mass-volume ratio of tetramethylpyrazine to methanol is 1 (g): 20 (mL).

[0017] To better implement the above technical solution, in Step 2 of the preparation method of tetramethylpyrazine ferulate of the present invention, the molar ratio of ferulic acid to tetramethylpyrazine is 23:11.

[0018] The third object of the present invention is to provide an application of tetramethylpyrazine ferulate, and the technical point lies in the application of tetramethylpyrazine ferulate in drugs for treating pulmonary fibrosis.

[0019] To better implement the above technical solution, in the application of tetramethylpyrazine ferulate in drugs for treating pulmonary fibrosis of the present invention, the tetramethylpyrazine ferulate is 30 mg·kg -1 .

[0020] Compared with the prior art, a tetramethylpyrazine ferulate, its preparation method and application of the present invention can achieve the following beneficial effects:

[0021] The tetramethylpyrazine ferulate of the present invention can exert a better anti-pulmonary fibrosis pharmacological effect and has higher solubility at the same time. Description of the Drawings

[0022] The drawings described herein are used to provide a further understanding of the present invention, form a part of the present invention, and the schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention. In the drawings:

[0023] Figure 1 It is the HPLC chart for synthesizing tetramethylpyrazine ferulate;

[0024] Figure 2 It is the mass spectrometry chart for synthesizing tetramethylpyrazine ferulate;

[0025] Figure 3 It is the result chart of the influence of tetramethylpyrazine ferulate on the body weight of rats with pulmonary fibrosis model;

[0026] Figure 4 It is the result chart of the influence of tetramethylpyrazine ferulate on the lung coefficient of rats with pulmonary fibrosis model;

[0027] Figure 5 It is the result chart of the influence of tetramethylpyrazine ferulate on the lung function of rats with pulmonary fibrosis model (A airway resistance result; B dynamic lung compliance result);

[0028] Figure 6 Figure of H&E staining of lung tissue of rats with pulmonary fibrosis model by ligustrazine ferulate (H&E 200×);

[0029] Figure 7 Figure of Masson staining of lung tissue of rats with pulmonary fibrosis model by ligustrazine ferulate (Masson 200×);

[0030] Figure 8 Figure of the effects of ligustrazine ferulate on the contents of TNF-α and IL-1β in the serum of rats with pulmonary fibrosis model (A Results of TNF-α content; B Results of IL-1β content);

[0031] Figure 9 Figure of the effects of ligustrazine ferulate on the contents of HYP and FN in the lung tissue of rats with pulmonary fibrosis model (A Results of HYP content; B Results of FN content);

[0032] Figure 10 Figure of the effects of ligustrazine ferulate on the expressions of α-SMA and Col-Ⅰ in rats with pulmonary fibrosis model (200×);

[0033] Figure 11 Figure of the effects of ligustrazine ferulate on the translational levels of α-SMA and Col-Ⅰ proteins in rats with pulmonary fibrosis model (A Western blot lane diagram; B Ratio of α-SMA / β-actin gray value; C Ratio of Col-Ⅰ / β-actin gray value). Detailed implementation manners

[0034] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with specific embodiments of the present invention and the corresponding drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0035] The technical solutions provided by each embodiment of the present invention will be described in detail below in conjunction with the drawings.

[0036] Example 1 - Preparation of ligustrazine ferulate

[0037] Take 1.6 g of ligustrazine and put it into a 100 mL round-bottom flask. Add 32 mL of methanol to dissolve it. Weigh 4.64 g of ferulic acid and put it into the above flask. Stir magnetically for a while until it is completely dissolved. The liquid turns pink. Stir at 28 °C for 24 h. Control the water bath temperature at 50 °C and concentrate it under reduced pressure to dryness. Add 10 mL of anhydrous ether and stir for a while. The solid becomes loose. Filter by suction, wash it with 10 mL of anhydrous ether, and place it in a petri dish to dry naturally to obtain 5.68 g of ligustrazine ferulate. Its melting point is 152 - 154 °C.

[0038] The recrystallization process is as follows: Put 5.68 g of ligustrazine ferulate into a 25 mL round-bottom flask, add 10 mL of ethyl acetate, heat it in a water bath to 50 °C until the solid is completely dissolved, add an appropriate amount of activated carbon, heat under reflux for 30 min, filter it through diatomaceous earth while it is hot, transfer the liquid to a triangular flask, white solid precipitates, place it in a -20 °C refrigerator overnight, filter by suction and dry to obtain 4.97 g of white solid, and the yield is 87.5%.

[0039] Its synthesis equation is shown as follows:

[0040]

[0042] The raw material formula and reaction parameters for synthesizing ligustrazine ferulate are shown in Table 1.

[0043] Table 1

[0044] Ligustrazine Ferulic acid Polar solvent Reaction temperature Reaction time 1.60 g, 11 mmol 4.64 g, 23 mmol Methanol, 32 mL 28℃ 24h

[0045] The test results of the product are as follows:

[0046] 1 HNMR(CD 3 OD, 600 MHz): δ7.59 (d, J = 18.0 Hz, 2H), 7.17 (d, J = 6.0 Hz, 2H), 7.05 (dd, J1 = 8.4 Hz, J2 = 1.8 Hz, 2H), 6.80 (d, J = 8.4 Hz, 2H), 6.30 (s, J = 16.2 Hz, 2H), 3.89 (s, 6H), 2.45 (s, 12H). 13 CNMR(CD 3 OD, 600 MHz): δ169.56, 149.11, 148.39, 147.97, 145.43, 144.50, 126.39, 122.58, 115.07, 114.52, 110.28, 55.04, 19.49.

[0047] HPLC spectrum and mass spectrum of ligustrazine ferulate prepared in Test Example 1 - Example 1

[0048] The specific method is as follows:

[0049] 1) Experimental conditions

[0050] (1) Chromatographic conditions

[0051] Chromatographic column: Waters ACQUITYTM Premier HSS T3 (2.1 mm × 100 mm, 1.8 μm); column temperature: 35 °C; mobile phase and gradient elution conditions are shown in Table 2, injection volume: 10 μL; flow rate: 0.3 mL·min -1 .

[0052] Table 2 Mobile phase gradient elution conditions

[0053]

[0054] (2) Mass spectrometry conditions

[0055] Electrospray ionization source (ESI) was used, with positive and negative ion scanning modes. Under the positive ion mode, the capillary temperature was 350 °C, the capillary voltage was 35 V, the spray voltage was 3.5 kV, the sheath gas (N2) flow rate was 40 psi (1 psi ≈ 6.895 kPa), and the auxiliary gas (N2) flow rate was 10 psi; under the negative ion mode, the capillary voltage was -35 V, the spray voltage was -3.5 kV, the sheath gas (N2) flow rate was 35 psi, and the auxiliary gas (N2) flow rate was 10 psi. The first-order mass spectrometry of the sample was performed in the FT mode for full scan (resolution: 30,000, scan range m / z (50 - 2000)), and the second-order and third-order mass spectrometry used data-dependent scan.

[0056] 2) Experimental results

[0057] The HPLC chromatogram of ferulic acid ligustrazine ( Figure 1 ) showed that the sample contained 2 main components and multiple trace components. The product of the main peak was collected for further mass spectrometry detection. The mass spectrometry analysis results showed that the m / z value of the main component in the sample was 521.0938, which was basically consistent with the theoretical molecular weight (M - H - ) 521.0938 (molecular formula: C 28 H 29 O 8 N 2 ) of ferulic acid ligustrazine, indicating that the main component in the sample was ferulic acid ligustrazine, and other components were by-products ( Figure 2 ).

[0058] Test Example 2 - Solubility test of ferulic acid ligustrazine described in Example 1

[0059] According to the solubility determination method in the second part of the Pharmacopoeia of the People's Republic of China (2020 Edition): Take the sample, accurately weigh an appropriate amount, place it in a certain amount of solvent (water) at (25 ± 2°C), shake vigorously for 30 s every 5 min, and observe the dissolution situation within 30 min. The experimental results are shown in Table 3. The experimental results show that the solubility of ferulic acid ligustrazine (Formula I) has increased significantly compared with ligustrazine.

[0060] Table 3 Solubility Results of Ferulic Acid Ligustrazine

[0061] Name Solubility Solubility increase multiple Ligustrazine 5.09 mg / 100 mL ----- Ligustrazine ferulate 230 mg / 100 mL 45.18

[0062] Application of Ferulic Acid Ligustrazine Prepared in Example 2 - Example 1 in the Treatment of Pulmonary Fibrosis

[0063] This example aims to study the application of ferulic acid ligustrazine prepared in Example 1 in the treatment of pulmonary fibrosis.

[0064] 1) Experimental Method

[0065] 60 healthy adult Wistar rats, SPF grade, male, 8 - 10 weeks old, weighing 200 ± 20 g. The rats were adaptively fed for 5 d and then randomly divided into sham operation group (Sham group), bleomycin model group (BLM group), pirfenidone group (PFD group), ligustrazine group (Lig group), ferulic acid group (FA group), and ferulic acid ligustrazine group (Fig - FA group), with 10 rats in each group.

[0066] According to the method of intratracheal injection of bleomycin in the paper "Structural and functional correlations in a large animal model of bleomycin - induced pulmonary fibrosis", a pulmonary fibrosis model was established: Intraperitoneal injection of 0.8% pentobarbital sodium. After anesthesia, the neck was disinfected with iodophor, the skin was longitudinally incised, the subcutaneous tissue and muscle of the neck were bluntly separated to expose the trachea. A 1 mL syringe was used to aspirate bleomycin and quickly injected into the trachea (5 mg·kg -1 ) Then the rat was rotated upright to ensure that the drug solution evenly reached the lungs. Finally, the muscle and skin were sutured, and the rats were routinely fed after natural awakening. The rats in the sham operation group were intratracheally injected with an equal amount of normal saline, and the operations of the other groups were the same. Administration started 24 h after modeling. The rats in the sham operation group and the bleomycin group were orally given the corresponding dose of normal saline, the rats in the PFD group were given the corresponding dose of pirfenidone solution (50 mg·kg -1 ) by gavage, and the rats in the Fig group were given the corresponding dose of ligustrazine (30 mg·kg -1), The rats in the FA group were given the corresponding dose of ferulic acid (30 mg·kg -1 ), The rats in the Fig-FA group were given the corresponding dose of ligustrazine ferulate (30 mg·kg -1 ), for 28 consecutive days. The rats were sacrificed on the 29th day.

[0067] The following indicators were observed:

[0068] (1) General condition observation

[0069] During the experiment, the respiratory condition, activity frequency, mental state, fur color, etc. of the rats in each group were observed every day. On the 28th day of modeling, the body weight of the rats was measured, and the change in body weight was observed.

[0070] (2) Pulmonary function detection

[0071] Using the DSI / BUXCO airway resistance and lung compliance monitoring system, after the instrument was calibrated and run for 10 min to reach a stable state, the rats were anesthetized, fixed, and the skin in the middle of the neck was longitudinally incised. The subcutaneous tissue and muscle were bluntly separated to expose the trachea. A small incision was made in the trachea, a soft tube was inserted, tied with a suture, and the rat was transferred to the airtight cavity of the detection system. The air path of the cavity was connected to the tracheal intubation. The airway resistance and dynamic lung compliance of the rats were monitored and recorded in real time through a computer.

[0072] (3) Lung coefficient detection

[0073] After the pulmonary function measurement, blood was taken from the abdominal aorta of the rats, the chest cavity was opened, and the left and right lung tissues were dissected. After drying the moisture on the lung tissue surface with filter paper, it was weighed, and the lung coefficient was calculated. Lung coefficient = lung mass (mg) / body mass (g).

[0074] (4) Histopathological observation of lung tissue

[0075] The left lung was transferred to 4% paraformaldehyde solution and fixed at room temperature for 24 h, then dehydrated, cleared, infiltrated with wax, embedded and sectioned, and then stained with H&E and Masson. The pathological morphology was observed under an optical microscope.

[0076] (5) Detection of HYP and FN contents in rat lung tissue

[0077] The right lung was cut into small pieces about 1 cm 3 left and right and placed in a cryopreservation tube and stored in a -80°C refrigerator. When measuring, the right lung tissue was taken out from the -80°C refrigerator, 100 mg of lung tissue was weighed, cut into pieces and put into a grinding tube with steel beads, and 1 mL of pre-cooled protein lysate was added to the grinding tube. The homogenizer was used for 4000 r·min -1 After homogenization, it was lysed on ice for 30 min and centrifuged at 14000 r·min at 4°C-1 After centrifuging for 10 min and extracting the supernatant, the protein content was determined by the BCA method, and then the detection was carried out according to the instructions of the rat HYP and FN enzyme-linked immunosorbent assay kits.

[0078] (6) Detection of TNF-α and IL-1β contents in rat serum

[0079] Blood was collected and placed in a centrifuge tube, left standing at room temperature for 30 min, and centrifuged at 3000 r·min -1 After centrifuging for 5 min, the supernatant was taken, and the determination was carried out according to the instructions of the rat TNF-α and IL-1β enzyme-linked immunosorbent assay kits.

[0080] (7) Detection of α-SMA and Col-I in rat lung tissue

[0081] The paraffin sections were dewaxed and then antigen repaired, blocked at room temperature for 30 min, and then the sections were incubated overnight at 4 °C with the corresponding primary antibodies (α-SMA, 1:100; Col-I, 1:100). The sections were washed with PBS, secondary antibody was added and incubated at room temperature for 1 h. The sections were washed again with PBS, counterstained with DAPI, incubated at room temperature in the dark, mounted, photographed under a fluorescence microscope, and image acquisition was performed using SlideViewer software.

[0082] And the tissue supernatant in (5) was taken, after protein quantification by the BCA method, loading buffer was added for denaturation (100 °C, 5 min), 50 μg of protein was separated by 8% SDS-PAGE electrophoresis, transferred to a membrane, blocked at room temperature for 1 h (5% skim milk powder), and then incubated with α-SMA (1:1000), Col-Ⅰ (1:1000) and β-actin (1:2500) respectively, washed with TBST 3 times, incubated with secondary antibody (1:2000), developed with ECL, the band pictures were collected by a gel imaging system, and finally the gray value was calculated using ImageJ software, and the relative expression level of the protein to be detected was expressed as the gray value ratio of the target protein / β-actin.

[0083] (8) Data processing

[0084] The experimental data were expressed in the form of mean ± standard deviation and statistically analyzed using SPSS Statistics 25 statistical software for each group of data. One-way analysis of variance (One-Way-Anova) was used, and P < 0.05 indicated significant differences in data statistics.

[0085] 2) Experimental results

[0086] (1) Effects of ferulic acid ligustrazine on the general state of rats with pulmonary fibrosis model

[0087] It was observed that the rats in the sham operation group had smooth hair color during the experiment, normal food intake and water intake, were lively, and their body weights maintained normal growth. The rats in the model group had yellow and dull hair, purple claws, lips and tail tips, were in low spirits, and some rats showed symptoms of respiratory distress, asthma and dyspnea in the later stage of the experiment, accompanied by reduced appetite. Some rats in the pirfenidone group, ligustrazine group, ferulic acid group and ligustrazine ferulate group showed arched backs, slightly purple and dull lips, claws and tail tips, and slightly decreased food and water intake, and their overall condition was better than that of the model group.

[0088] In terms of body weight, the body weights of the rats in the bleomycin model group were significantly lower than those in the sham operation group at 28 days after modeling (P<0.001); compared with the bleomycin model group, the body weights of the rats in the pirfenidone group, ligustrazine group, ferulic acid group and ligustrazine ferulate group were higher than those of the rats in the bleomycin model group (P<0.001, P<0.001, P<0.001 and P<0.001), and among them, the body weight of the rats in the ligustrazine ferulate group was higher than the other three groups. The results are shown in Figure 3 (compared with the sham operation group * P<0.05, ** P<0.01, *** P<0.001, compared with the bleomycin model group # P<0.05,

[0089] ## P<0.01, ### P<0.001).

[0090] (2) Effects of ligustrazine ferulate on the lung coefficient of rats with pulmonary fibrosis model

[0091] Compared with the sham operation group, the lung coefficient of the rats in the bleomycin model group was significantly increased (P<0.001). Compared with the bleomycin model group, the lung coefficients of the rats in the pirfenidone group, ligustrazine group, ferulic acid group and ligustrazine ferulate group were significantly decreased (P<0.001, P<0.001, P<0.001 and P<0.001), and among them, the lung coefficient of the rats in the ligustrazine ferulate group was lower than the other three groups. The results are shown in Figure 4 (compared with the sham operation group * P<0.05, ** P<0.01, *** P<0.001, compared with the bleomycin model group # P<0.05, ## P<0.01, ### P<0.001)

[0092] (3) Effects of ligustrazine ferulate on the lung function of rats with pulmonary fibrosis model

[0093] Compared with the sham operation group, obvious changes occurred in the lung compliance and airway resistance of rats in the bleomycin model group, mainly manifested as a significant increase in airway resistance (P<0.001) and a significant decrease in dynamic lung compliance (P<0.001); compared with the bleomycin model group, the airway resistance of rats in the pirfenidone, ligustrazine, ferulic acid, and ligustrazine ferulate groups was significantly decreased (P<0.001, P<0.01, and P<0.001), and at the same time, the lung compliance of rats in the ligustrazine ferulate group was significantly increased (P<0.001). The results are shown in Figure 5 -B (compared with the sham operation group * P<0.05, ** P<0.01, *** P<0.001, compared with the bleomycin model group # P<0.05, ## P<0.01, ### P<0.001).

[0094] (4) Effects of ligustrazine ferulate on the lung histopathology of rats with pulmonary fibrosis model

[0095] As shown in the H&E staining results in Figure 6, the lung structure of rats in the sham operation group was intact, the alveolar ducts and alveoli were normal in morphology, the small bronchi and terminal bronchioles had clear structures and were evenly distributed. Compared with the sham operation group, the rats in the model group had basically lost the original alveolar tissue structure, the remaining alveolar structures were dilated, the alveolar walls were significantly thickened, and there was diffuse infiltration of inflammatory cells (lymphocytes, neutrophils, plasma cells, etc.) in the interstitium, and foam cells were seen to accumulate. Compared with the model group, pirfenidone, ligustrazine, ferulic acid, and ligustrazine ferulate groups all showed thickening of the lung interstitium and reduced inflammatory response; in the ligustrazine ferulate group, alveolar dilation and fibroplasia were occasionally seen, and a small amount of lymphocyte infiltration and focal alveolar hemorrhage were seen in the lung interstitium.

[0096] Figure 7 It can be seen from the Masson staining shown in Figure 6 that the fibrous tissue was specifically stained blue. Obvious fibrous tissue hyperplasia occurred in the lungs of rats in the bleomycin model group, mainly distributed in the alveolar interstitium around the damaged bronchi, indicating that the pulmonary fibrosis model was successfully replicated; the fibrous tissue hyperplasia in the pirfenidone, ligustrazine, ferulic acid, and ligustrazine ferulate groups was mainly mild and moderate, and was significantly less than that in the model group. Generally speaking, the degree of lesions in the ligustrazine ferulate group was alleviated compared with the model group, and the ligustrazine ferulate group had a better effect.

[0097] (5) Effects of ligustrazine ferulate on the contents of serum TNF-α and IL-1β in rats with pulmonary fibrosis model

[0098] Compared with the sham operation group, the contents of TNF-α and IL-1β in the serum of rats in the bleomycin model group were significantly increased (P < 0.001 and P < 0.01); in terms of TNF-α, compared with the bleomycin model group, the contents of TNF-α in the rats in the pirfenidone, ligustrazine, ferulic acid, and ligustrazine ferulate groups were all significantly decreased (P < 0.01, P < 0.001, P < 0.001, and P < 0.001); in terms of IL-1β, compared with the bleomycin model group, the contents of IL-1β in the rats in the pirfenidone and ligustrazine ferulate groups were both significantly decreased (P < 0.01 and P < 0.05), and the results are shown in Figure 8 -B (compared with the sham operation group * P < 0.05, ** P < 0.01, *** P < 0.001, compared with the bleomycin model group # P < 0.05, ## P < 0.01, ### P < 0.001).

[0099] (6) Effects of ligustrazine ferulate on the contents of HYP and FN in the lung tissues of rats with pulmonary fibrosis model Compared with the sham operation group, the contents of HYP and FN in the lung tissues of rats in the bleomycin model group were significantly increased (P < 0.001 and P < 0.001); in terms of HYP, compared with the bleomycin model group, the contents of HYP in the rats in the pirfenidone, ligustrazine, ferulic acid, and ligustrazine ferulate groups were all significantly decreased (P < 0.01, P < 0.01, P < 0.001, and P < 0.001); in terms of FN, compared with the bleomycin model group, the contents of FN in the rats in the ferulic acid and ligustrazine ferulate groups were both significantly decreased (P < 0.01 and P < 0.01), and the results are shown in Figure 9 -B (compared with the sham operation group * P < 0.05, ** P < 0.01, *** P < 0.001, compared with the bleomycin model group # P < 0.05, ## P < 0.01, ### P < 0.001).

[0100] (7) Effects of ligustrazine ferulate on the expression levels of α-SMA and Col-I in the lung tissues of rats with pulmonary fibrosis model

[0101] Compared with the sham operation group, the expression levels of α-SMA and Col-I in the lung tissues of rats in the bleomycin model group were significantly increased; compared with the bleomycin model group, the expression levels of α-SMA and Col-I in the pirfenidone, ligustrazine, ferulic acid, and ligustrazine ferulate groups were decreased, and the results are shown in Figure 10 。

[0102] Meanwhile, the Western blot results showed that, compared with the sham operation group, the protein translation levels of α-SMA and Col-I in the lung tissues of rats in the bleomycin model group were significantly increased (P<0.01 and P<0.01), and the protein translation levels of α-SMA and Col-I in the pirfenidone and ligustrazine ferulate group were significantly down-regulated (P<0.01, P<0.05 and P<0.01, P<0.05). The results are shown in Figure 11 A-B-C (compared with the sham operation group * P<0.05, ** P<0.01, *** P<0.001, compared with the bleomycin model group # P<0.05, ## P<0.01, ### P<0.001).

[0103] The above are only the embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, various modifications and changes can be made to the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the scope of the claims of the present invention.

Claims

1. A ligustrazine ferulate, characterized in that: The molecular formula of the ligustrazine ferulate is C 28 H 29 O8N2, the melting point of ligustrazine ferulate is 152°C-154°C, and the structural formula is shown in Formula III:

2. A method for preparing ligustrazine ferulate, characterized in that: The following steps are included Step 1, placing ligustrazine at the bottom of a round-bottom flask, and then adding methanol to dissolve the ligustrazine to obtain a methanol solution of ligustrazine; Step 2, weighing ferulic acid and placing it in the methanol solution of ligustrazine in step 1 to dissolve it until the liquid turns pink; Step 3, stirring the liquid in step 2 at 28°C for 24h, concentrating in a water bath at 50°C, adding anhydrous ethanol and filtering to obtain crude ligustrazine ferulate crystals, and placing the crude ligustrazine ferulate crystals in a watch glass to dry naturally; Step 4: recrystallize the crude ligustrazine ferulate obtained by drying in step 3.

3. The method for preparing ligustrazine ferulate according to claim 2, characterized in that: The mass-to-volume ratio of ligustrazine to methanol in the step 1 is 1 (g):20 (mL).

4. The method for preparing ligustrazine ferulate according to claim 1, characterized in that: In the step 2, the molar ratio of ferulic acid to ligustrazine is 23:

11.

5. An application of ligustrazine ferulate, characterized in that: The application of ligustrazine ferulate in medicine for treating pulmonary fibrosis.

6. The use of ligustrazine ferulate according to claim 5, characterized in that: The ligustrazine ferulate is 30 mg kg -1 .

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