Tetrandrine ferulate and its application in preparing medicine for treating pulmonary fibrosis

Abstract

The invention discloses a tetrandrine ferulate and its application in the preparation of a medicament for treating pulmonary fibrosis. The pharmacological effects of tetrandrine and ferulic acid, as well as those after salification, are studied. It is found that both have an anti-pulmonary fibrosis effect, and the two can be used in combination to synergistically exert a more excellent anti-pulmonary fibrosis efficacy. At the same time, the solubility of tetrandrine can also be increased. This has not been reported in existing literature. The tetrandrine ferulate of the present invention has lower cytotoxicity, and the cell viability of normal human liver cells LO-2 and human embryonic fibroblasts MRC-5 is higher than that of tetrandrine, which alleviates the burden of liver and lungs to a certain extent and has good safety.

Description

Technical Field

[0001] The present invention relates to the technical field of medical drugs, and in particular to tetrandrine ferulate and application thereof in preparing a drug for treating pulmonary fibrosis. Background Art

[0002] Tetrandrine (Tet), also known as tetrandrine, is derived from the sun-dried roots of Stephania tetrandra S. Moore, a plant of the Menispermaceae family. It is a white solid that is easily soluble in ether, chloroform, and certain organic solvents. As one of the main active ingredients of Stephania tetrandra, it has many pharmacological effects. Its chemical structure is as follows:

[0003]

[0004] Tetrandrine is clinically used to treat a variety of diseases, including silicosis, liver fibrosis, and pulmonary fibrosis. However, its efficacy is limited by its high lipid solubility, poor water solubility, and low bioavailability.

[0005] Ferulic acid (FA) is an active ingredient in many traditional Chinese medicines, including Angelica sinensis and Chuanxiong rhizome. It has multiple biological effects, such as protecting the nervous and cardiovascular systems and anti-fibrosis. It also plays an important role in the treatment of fibrotic diseases, kidney disease, pulmonary hypertension, and brain diseases. Its chemical structure is as follows:

[0006]

[0007] In Chinese patent CN102898433A, a tetrandrine derivative, i.e., tetrandrine gallate, was designed and synthesized by using tetrandrine as a lead compound. The structure of tetrandrine was modified to prepare a new tetrandrine compound, i.e., tetrandrine gallate. Physical and chemical constant tests showed that the solubility of tetrandrine gallate was 43 times greater than that of tetrandrine. Pharmacological experiments showed that tetrandrine gallate had a significant inhibitory effect on the proliferation of tumor cell lines, and its effect was better than that of tetrandrine. It also showed significantly stronger antihypertensive, anti-inflammatory, and analgesic effects than tetrandrine, and its toxicity was lower than that of tetrandrine. This scheme mainly enhances the anti-cancer effect of tetrandrine by increasing its solubility.

[0008] In Chinese patent CN101352439A, a solution of a bio-organic acid (acidic acid, acetic acid, lactic acid, and gallic acid) is gradually added to tetrandrine until it is completely dissolved, followed by lyophilization to produce a pharmaceutical composition. This pharmaceutical composition transforms the crystalline state of tetrandrine into an amorphous state, significantly enhancing its water solubility and improving its bioavailability. However, research on improving its efficacy has not yet been conducted. Summary of the Invention

[0009] In view of the deficiencies in the prior art, the present invention aims to provide a tetrandrine ferulate and its application in the preparation of a medicament for treating pulmonary fibrosis.

[0010] In order to achieve the above object, the present invention adopts the following technical solutions:

[0011] A tetrandrine ferulate, whose chemical structural formula is shown in Formula 1:

[0012]

[0013] The present invention also provides a method for synthesizing the tetrandrine ferulate, the specific process of which is as follows:

[0014] Take tetrandrine and add anhydrous ethanol, and stir magnetically at 30°C to obtain an ethanol solution of tetrandrine; add anhydrous ethanol to ferulic acid and dissolve it to obtain an ethanol solution of ferulic acid, add the ethanol solution of ferulic acid to the ethanol solution of tetrandrine, stir magnetically until the solid is completely dissolved, and then a small amount of solid precipitates, and stir at 30°C for 24 hours; then control the water bath temperature at 70-73°C, concentrate under reduced pressure until solid precipitates obviously, stop concentrating, filter after 1 hour, and dry naturally to obtain tetrandrine ferulate solid.

[0015] Furthermore, the mass ratio of tetrandrine to ferulic acid is 15:11.

[0016] Furthermore, the tetrandrine ferulate can be used in the preparation of drugs for treating pulmonary fibrosis.

[0017] The beneficial effects of the present invention are:

[0018] (1) The present invention studied the pharmacological effects of tetrandrine and ferulic acid individually and after salt formation, and found that both have anti-pulmonary fibrosis effects, and the combination of the two can synergistically exert a better anti-pulmonary fibrosis efficacy, while also increasing the solubility of tetrandrine, which has not been reported in existing literature.

[0019] (2) The tetrandrine ferulate of the present invention has lower cytotoxicity and has higher cell viability on normal human liver cells LO-2 and human embryonic fibroblasts MRC-5 than tetrandrine, reduces the burden on the liver and lungs to a certain extent, and has better safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 HPLC spectrum of ferulic acid obtained in Example 3 of the present invention;

[0021] Figure 2 HPLC spectrum of tetrandrine obtained in Example 3 of the present invention;

[0022] Figure 3 HPLC spectrum of the tetrandrine ferulate obtained in Example 3 of the present invention;

[0023] Figure 4 This is a graph showing the effects of various agents obtained in Example 5 of the present invention on the body weight of rats with pulmonary fibrosis model;

[0024] Figure 5 This is a graph showing the effects of various agents obtained in Example 5 of the present invention on the lung function of rats with pulmonary fibrosis model;

[0025] Figure 6 This is a graph showing the effects of various agents obtained in Example 5 of the present invention on the lung coefficient of rats with pulmonary fibrosis model;

[0026] Figure 7 This is a graph showing the effects of various agents obtained in Example 5 of the present invention on lung imaging in rats with pulmonary fibrosis model;

[0027] Figure 8 HE staining of lung tissue of rats with pulmonary fibrosis model using each agent obtained in Example 5 of the present invention (HE200x);

[0028] Figure 9 This is a Masson staining image (Masson 200x) of lung tissue of rats with pulmonary fibrosis model treated with each agent obtained in Example 5 of the present invention;

[0029] Figure 10 Graph (630x) showing the effects of various agents obtained in Example 5 of the present invention on the expression of E-cad and N-cad in rats with pulmonary fibrosis model;

[0030] Figure 11 The figure shows the effects of various agents obtained in Example 5 of the present invention on the expression of α-SMA and Col-I in rats with pulmonary fibrosis model (200x);

[0031] Figure 12 This is a graph showing the effects of various agents obtained in Example 5 of the present invention on the levels of TNF-α and IL-6 in rats with pulmonary fibrosis model;

[0032] Figure 13 This is a graph showing the effects of ferulic acid, tetrandrine, and tetrandrine ferulate obtained in Example 6 of the present invention on MRC-5 cell viability;

[0033] Figure 14 This is a graph showing the effects of ferulic acid, tetrandrine, and tetrandrine ferulate obtained in Example 6 of the present invention on the viability of LO-2 cells. DETAILED DESCRIPTION

[0034] The present invention will be further described below in conjunction with the accompanying drawings. It should be noted that this embodiment is based on the technical solution and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to this embodiment.

[0035] Example 1

[0036] This embodiment provides a tetrandrine ferulate salt, the chemical structure of which is shown in Formula 1.

[0037]

[0038] Example 2

[0039] This embodiment provides a method for synthesizing the tetrandrine ferulate described in Example 1, and the specific process is as follows:

[0040] Place 0.3g of tetrandrine in a 50mL round-bottom flask, add 24mL of anhydrous ethanol, and magnetically stir at 30°C until the raw material does not dissolve, to obtain a tetrandrine ethanol solution. Weigh 0.22g of ferulic acid into a 25mL Erlenmeyer flask, add 3mL of anhydrous ethanol, and shake by hand to dissolve. Add this solution to the tetrandrine ethanol solution and magnetically stir for 3-4 minutes. After the solid is completely dissolved, a small amount of solid precipitates after 5 minutes. Stir at 30°C for 24 hours, then control the water bath temperature at 70-73°C and concentrate under reduced pressure until solid precipitation is apparent. Stop concentration, filter after 1 hour, and air dry to obtain 0.48g of a white solid, tetrandrine ferulate, with a melting point of 150-151°C.

[0041] The recrystallization process is as follows: 0.48 g of tetrandrine ferulate is placed in a 25 mL round-bottom flask, 5 mL of ethyl acetate is added, and the solid is completely dissolved in a water bath at 70°C. An appropriate amount of activated carbon is added and heated under reflux for 30 minutes. While hot, the liquid is filtered through diatomaceous earth, and the liquid is transferred to a conical flask to precipitate a white solid. The liquid is placed in a -20°C refrigerator overnight, filtered, and dried to obtain 0.43 g of a white solid with a yield of 89.58%.

[0042] The synthesis equation is shown below:

[0043]

[0044] The raw material formula and reaction parameters for synthesizing tetrandrine ferulate are shown in Table 1.

[0045] Table 1

[0046]

[0047] The test product results are as follows:

[0048] 1HNMR (CD3OD, 600MHz): δ7.55-7.44 (m, 3H), 7.15 (s, 2H), 7.09 (d, J=7.8Hz, 1H), 7.02 (d, J=8.4Hz, 2H) , 6.96 (m, 1H), 6.91 (m, 1H), 6.88-6.74 (m, 4H), 6.523 (s, 2H), 6.45 (d, J = 8.4Hz, 1H), 6.32 (d, J = 15.6Hz , 2H), 6.10 (s, 1H), 4.41 (m, 1H), 4.12 (d, J = 10.2Hz, 1H), 3.88 (s, 9H), 3.76 (S, 4H), 3.48 (q, J = 7.2Hz, 3H), 3.42 (s, 3H), 3.18 (s, 3H), 2.87 (S, 3H), 2.66 (d, J=15Hz, 1H), 2.52 (s, 3H), 1.18 (t, J=7.2Hz, 3H). 13 CNMR (CD3OD, 600MHz): δ177.22, 173.03, 154.19, 153.50, 150.09, 150.00, 148.69, 147.04, 1 43.90, 138.58, 132.58, 132.20, 130.99, 129.28, 126.00, 124.89, 123.23, 121.90, 121.83, 12 1.61, 120.17, 115.80, 114.83, 112.30, 112.17, 106.32, 72.86, 63.35, 63.19, 59.47, 55.29, 55.14, 54.88, 44.87, 44.66, 40.98, 39.90, 39.65, 43.10, 36.78, 22.20, 21.73, confirmed to be tetrandrine ferulate.

[0049] Example 3

[0050] This example is intended to test the HPLC spectrum of the tetrandrine ferulate synthesized in Example 1, and the specific method is as follows:

[0051] (1) Chromatographic conditions

[0052] Chromatographic column: YMC-pack pro C18 (4.6x150mm 5μm)

[0053] Mobile phase: acetonitrile-0.1% formic acid solution (13:87), as shown in Table 2

[0054] Table 2

[0055] Time (minutes) (Acetonitrile) A% (0.1% formic acid) B% 0 3 87 2 3 87 10 20 80 20 35 65 25 70 30 27 95 5 33 95 5 33.1 13 87 40 STOP

[0056] Column temperature: 35°C

[0057] Detection wavelength: 316nm and 280nm

[0058] Injection volume: 10 μL

[0059] (2) Content determination

[0060] Determination method: external standard method

[0061] Preparation of reference solution: Take 10 mg of ferulic acid and tetrandrine reference substances respectively, accurately weigh them, place them in a 10 mL volumetric flask, add methanol to dissolve them, and dilute to the scale.

[0062] Prepare the test solution: Accurately weigh approximately 10 mg each of ferulic acid, tetrandrine reference substance, and tetrandrine ferulate described in Example 1 into a 10 mL volumetric flask. Dissolve in methanol and dilute to the mark. Shake well, allow to stand, filter the supernatant, and obtain the filtrate.

[0063] (2) Experimental results

[0064] The HPLC spectrum of ferulic acid is as follows Figure 1 As shown, the HPLC spectrum of tetrandrine is as shown Figure 2 As shown, the HPLC spectrum of tetrandrine ferulate is as shown Figure 3 shown.

[0065] Example 4

[0066] This example aims to test the solubility of the tetrandrine ferulate described in Example 1.

[0067] (1) Experimental methods

[0068] According to the solubility determination method in the example of Part II of the 2020 edition of the "Pharmacopoeia of the People's Republic of China": take a sample, accurately weigh an appropriate amount, place it in a certain amount of solvent (water) (25±2℃), shake vigorously for 30s every 5 minutes, and observe the dissolution within 30 minutes.

[0069] (2) Experimental results

[0070] The experimental results are shown in Table 3. The experimental results show that the solubility of tetrandrine ferulate (Formula I) is significantly increased compared with that of tetrandrine.

[0071] Table 3

[0072] name Solubility Solubility increase factor Tetrandrine 8mg / 100mL ----- Tetrandrine ferulate 560mg / 100mL 70

[0073] Example 5

[0074] This example aims to test the effect of tetrandrine ferulate (Formula I) described in Example 1 in treating pulmonary fibrosis.

[0075] 1) Experimental methods

[0076] Sixty healthy adult Wistar rats, SPF grade, male, aged 8 to 10 weeks, weighing 200±20 g, were used. The rats were adaptively fed for 5 days and randomly divided into sham operation group (Sham group), bleomycin model group (BLM group), pirfenidone group (PFD group), tetrandrine group (Tet group), ferulic acid group (FA group) and tetrandrine ferulate group (Tet-FA group) according to their body weight, with 10 rats in each group.

[0077] A pulmonary fibrosis model was established by intratracheal injection of bleomycin (Louise, Organ, Barbara, et al. Structural and functional correlations in a large animal model of bleomycin-induced pulmonary fibrosis. [J]. Bmc Pulmonary Medicine, 2015. DOI: 10.1186 / s12890-015-0071-6). The procedure was as follows: 0.8% sodium anisobarbital was injected intraperitoneally. After anesthesia, the neck was disinfected with iodine. The skin was longitudinally incised, and the subcutaneous tissue and muscles of the neck were bluntly separated to expose the trachea. Bleomycin was then drawn up with a 1 mL syringe and rapidly injected into the trachea (5 mg kg -1 ) Then the rats were rotated upright so that the drug solution could reach the lungs evenly. Finally, the muscles and skin were sutured and the rats were fed normally after they woke up naturally. The rats in the sham group were injected with an equal amount of normal saline into the trachea, and the other groups were operated in the same way. Drug administration began 24 hours after modeling. The rats in the sham group and the BLM group were given the corresponding dose of normal saline by intraperitoneal injection, and the rats in the PFD group were given the corresponding dose of pirfenidone solution (50 mg·kg -1 ), and the rats in the Tet group were given the corresponding dose of tetrandrine solution (30 mg·kg -1 ), and the rats in the GA group were given corresponding doses of ferulic acid solution (30 mg·kg -1 ), the Tet-GA group was given the corresponding dose of tetrandrine ferulate solution (30 mg·kg -1 The rats were sacrificed on the 29th day.

[0078] Observe the following indicators:

[0079] (1) General status observation

[0080] The respiration, activity frequency, mental state, fur color, etc. of each group of rats were observed every day during the experiment. The body weight of the rats was weighed on the 28th day of modeling to observe the changes in the body weight of the rats.

[0081] (2) Pulmonary function test

[0082] A DSI / BUXCO airway resistance and pulmonary compliance monitoring system was used. After the instrument was calibrated and allowed to run for 10 minutes to reach a stable state, the rats were anesthetized and immobilized. The skin at the mid-cervical region was longitudinally incised, and the subcutaneous tissue and muscle were bluntly dissected to expose the trachea. A small incision was made in the trachea, and a flexible tube was inserted, secured with sutures, and the rats were transferred to the sealed chamber of the monitoring system. The airway in the chamber was connected to the endotracheal tube. Tidal volume, airway resistance, and dynamic pulmonary compliance were monitored and recorded in real time by a computer.

[0083] (3) Lung coefficient detection

[0084] After the pulmonary function test, blood was collected from the rat's abdominal aorta. The thorax was opened and the left and right lung tissues were removed. The lung tissues were wiped dry with filter paper to remove surface moisture and then weighed. The lung coefficient was calculated (lung coefficient = lung mass (mg) / body mass (g).

[0085] (4) Lung imaging examination

[0086] One hour after the last administration, the rats were anesthetized (0.8% sodium pentobarbital intraperitoneally), and the Micro CT scanning parameters were set as follows: tube voltage: 90 kV, tube current: 88 μA, imaging field of view: 72 ＊ After anesthesia, rats were placed in a prone position with their limbs spread out and head first. A chest CT scan was then performed, which took approximately 10 minutes. After completion, the images were reconstructed using SimpleViewer software.

[0087] (5) Pathological observation of lung tissue

[0088] The left lung was transferred to 4% paraformaldehyde solution and fixed at room temperature for 24 hours. The lung was then dehydrated, transparentized, immersed in wax, embedded, and sectioned. The lung was then stained with HE and Masson's staining. The pathological morphology was observed under an optical microscope.

[0089] (6) Detection of E-cad and N-cad, indicators of epithelial-mesenchymal transition in rat lung tissue

[0090] After dewaxing, the paraffin sections were subjected to antigen retrieval and blocked at room temperature for 60 min. The sections were incubated with the corresponding primary antibodies (N-cad, 1:200; E-cad, 1:200) at 4°C overnight, washed with PBS, and incubated at room temperature for 60 min after adding the corresponding secondary antibodies. The sections were washed again with PBS, counterstained with DAPI, and incubated at room temperature for another 30 min. After sealing, the sections were photographed using a laser confocal microscope.

[0091] (7) Detection of α-SMA and Col-I in rat lung tissue

[0092] Paraffin sections were dewaxed and antigen retrieval performed. Blocking was performed at room temperature for 30 minutes, followed by incubation with the corresponding primary antibodies (α-SMA, 1:100; Col-I, 1:100) overnight at 4°C. Sections were washed with PBS, and secondary antibodies were added and incubated at room temperature for 1 hour. Sections were washed again with PBS, counterstained with DAP1, incubated at room temperature in the dark, and mounted. Images were acquired using a fluorescence microscope using SlideViewer software.

[0093] (8) Detection of TNF-α and IL-6 levels in rat lung tissue

[0094] After the rat lung function test was completed, blood was collected from the abdominal aorta and placed in a centrifuge tube. The tube was kept at room temperature for 30 minutes and then centrifuged at 3000 r / min. -1 The cells were centrifuged for 5 minutes and the TNF-α and IL-6 enzyme-linked immunosorbent assay kits were used for determination.

[0095] (9) Data processing

[0096] The experimental data are expressed as mean ± standard deviation The data were expressed in the form of SPSS Statistics 25 statistical software, and one-way analysis of variance (One-Way-Anova) was used. P < 0.05 was considered to be statistically significant.

[0097] 2) Experimental results

[0098] (1) Effects of tetrandrine ferulate on the general condition of rats with pulmonary fibrosis model

[0099] The rats in the sham-operated group were observed to be in good spirits, active and agile, with lustrous fur, normal water and food intake, normal bowel movements, and continued weight gain. Following bleomycin modeling, the rats in the bleomycin model group experienced decreased food and water intake, decreased mental activity, dull yellow fur, arched backs, and dark purple discoloration of the lips, nails, and tail tips. Some rats experienced bleeding from the mouth and nose and difficulty breathing. Some rats in the pirfenidone, tetrandrine, ferulic acid, and tetrandrine ferulate groups developed arched backs and slight dark purple discoloration of the lips, nails, and tail tips, but their overall condition improved compared to the bleomycin model group.

[0100] In terms of body weight, the body weight of rats in the bleomycin model group was significantly lower than that in the sham operation group 28 days after modeling (P < 0.001). Compared with the bleomycin model group, the body weight of rats in the pirfenidone group, the tetrandrine group, the ferulic acid group and the tetrandrine ferulate group were all higher than those in the bleomycin model group (P < 0.001, P < 0.001, P < 0.001 and P < 0.001). Among them, the body weight of rats in the tetrandrine ferulate group was heavier than that in the other three groups. Figure 4 . 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.

[0101] (2) Effects of tetrandrine ferulate on lung function in rats with pulmonary fibrosis model

[0102] Compared with the sham operation group, the lung compliance and airway resistance of the rats in the bleomycin model group showed more obvious changes, 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 the rats in the pirfenidone, tetrandrine and tetrandrine ferulate groups was significantly reduced (P < 0.05, P < 0.05 and P < 0.01), while the lung compliance of the rats in the pirfenidone and tetrandrine ferulate groups was significantly increased (P < 0.001 and P < 0.001). Figure 5 .in, Figure 5 A corresponds to airway resistance function, and B corresponds to lung compliance function. Compared with the sham operation group ＊ P<0.05, ＊＊ P < 0.01, ＊＊＊ P<0.001, compared with the model group #P<0.05, ## P < 0.01, ### P<0.001.

[0103] (3) Effects of Tetrandrine Ferulate on the Lung Coefficient in Rats with Pulmonary Fibrosis

[0104] Compared with the sham operation group, the lung coefficient of rats in the bleomycin model group was significantly increased (P < 0.001). Compared with the bleomycin model group, the lung coefficient of rats in the pirfenidone group, the tetrandrine group, the ferulic acid group and the tetrandrine ferulate group was significantly decreased (P < 0.001, P < 0.001, P < 0.001 and P < 0.001). Among them, the lung coefficient of rats in the tetrandrine ferulate group was lower than that in the other three groups. Figure 6 . Figure 6 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.

[0105] (4) Effects of Tetrandrine Ferulate on Lung Imaging in Rats with Pulmonary Fibrosis

[0106] The lung CT scans of the sham-operated rats showed no abnormalities. The lung texture was clear, the transparency was uniform, there were no high or low dense shadows, no abnormal lines or flake shadows. The lung texture of the rats in the bleomycin model group was fuzzy, the transparency was uneven, there were dense shadows and honeycomb changes. Compared with the bleomycin model group, the above manifestations were milder in the pirfenidone group, the tetrandrine group, the ferulic acid group and the tetrandrine ferulate group. Figure 7 .

[0107] (5) Effects of Tetrandrine Ferulate on Lung Histopathology in Rats with Pulmonary Fibrosis

[0108] HE staining showed that there was no significant abnormality in the lung tissue structure of the rats in the sham operation group; the alveolar tissue structure of the rats in the bleomycin model group basically disappeared, the alveolar wall was diffusely thickened, pulmonary edema, diffuse infiltration of inflammatory cells (lymphocytes, neutrophils, plasma cells, etc.) in the interstitium, accumulation of foam cells, and significant pulmonary interstitial fibrosis; the alveolar wall thickening of the rats in the pirfenidone group was significantly reduced, with occasional inflammatory cell infiltration and hemorrhage; the focal alveolar edema of the rats in the ferulic acid group was observed. The rats in the tetrandrine group had mild thickening of the alveolar wall, focal infiltration of inflammatory cells, occasional eosinophilic exudate in the alveolar cavity, pulmonary edema, focal infiltration of inflammatory cells, significant pulmonary interstitial fibrosis and a small amount of hemorrhage; the rats in the tetrandrine ferulate group had mild thickening of the alveolar wall, focal infiltration of inflammatory cells, occasional eosinophilic exudate in the alveolar cavity, and improved edema compared with the model group; the rats in the tetrandrine ferulate group had focal infiltration of inflammatory cells in the lungs, a small amount of hemorrhage, and improved edema compared with the bleomycin model group. Figure 8 .

[0109] Masson staining showed that fibrous tissue was specifically stained blue. The lungs of rats in the bleomycin model group showed obvious fibrous tissue proliferation, mainly distributed in the alveolar interstitium around the damaged bronchi, indicating that the pulmonary fibrosis model was successfully replicated. Obvious fibrous tissue proliferation was also seen in the pirfenidone group, tetrandrine group, ferulic acid group and tetrandrine ferulate group, but the degree of lesions was alleviated compared with the bleomycin model group. Figure 9 .

[0110] (6) Effect of tetrandrine ferulate on the expression of E-cad and N-cad, the epithelial-mesenchymal transition indicators in lung tissue of rats with pulmonary fibrosis model

[0111] Compared with the sham operation group, the expression level of N-cad protein in the lung tissue of rats in the bleomycin model group was increased, while the expression level of E-cad protein was decreased; compared with the bleomycin model group, the expression level of N-cad protein was decreased in the pirfenidone group, the tetrandrine group, the ferulic acid group and the tetrandrine ferulate group, while the expression level of E-cad protein was increased. Among them, the tetrandrine ferulate group had a better reversal effect. Figure 10 .

[0112] (7) Effects of Tetrandrine Ferulate on the Expression of α-SMA and Col-1 in Lung Tissue of Rats with Pulmonary Fibrosis

[0113] Compared with the sham operation group, the expression levels of α-SMA and Col-1 in the lung tissue of rats in the bleomycin model group were significantly increased; compared with the bleomycin model group, the expression levels of α-SMA and Col-1 in the pirfenidone group, the tetrandrine group, the ferulic acid group and the tetrandrine ferulate group were decreased. Figure 11 .

[0114] (8) Effects of Tetrandrine Ferulate on TNF-α and IL-6 Contents in Lung Tissue of Rats with Pulmonary Fibrosis

[0115] Compared with the sham operation, the TNF-α and IL-6 levels of the rats in the bleomycin model group were significantly increased (P < 0.001, P < 0.001 and P < 0.001); compared with the bleomycin model group, the IL-6 level in the tetrandrine ferulate group was significantly decreased (P < 0.05); at the same time, the TNF-α levels in the pirfenidone group, ferulic acid group and tetrandrine ferulate group were significantly decreased (P < 0.01, P < 0.05 and P < 0.01), among which the tetrandrine ferulate group had a better effect than the ferulic acid group; the results are shown in Figure 12 . Figure 12 A corresponds to IL-6, B corresponds to TNF-α, compared with the sham operation group ＊ P<0.05, ＊＊ P < 0.01, ＊＊＊ P<0.001, compared with the model group # P<0.05, ## P < 0.01, ### P<0.001.

[0116] Example 6

[0117] This example aims to test the effect of tetrandrine ferulate on the viability of A549 and LO-2 cells.

[0118] 1) Experimental methods

[0119] MRC-5 cell culture: MRC-5 cells were cultured in MEM medium containing 10% FBS in a cell culture incubator at 37°C and 5% CO2. They were expanded to the third generation. Microscopic observation showed that the MRC-5 cells were spindle-shaped, with good overall growth and clear supernatant. Subsequent experiments could be performed when the cells were 70-80% confluent.

[0120] LO-2 cell culture: LO-2 cells were cultured in 1640 medium containing 10% FBS. The cells were placed in a cell culture incubator at 37°C and 5% CO2 and expanded to the third generation. Microscopic observation showed that the LO-2 cells were oval in morphology, with good overall cell growth and clear supernatant. Subsequent experiments could be performed when the cells were 70-80% confluent.

[0121] Cell viability assay: cells in logarithmic growth phase were selected and digested with trypsin to prepare cell suspension. 5 / mL -1Cells were seeded at a density of 100 μL / well in a 96-well cell culture plate. One day after seeding, the cell supernatant was aspirated and 100 μL / well of the dosing medium at various concentrations was added. 24 hours after drug addition, the supernatant was aspirated and 10 μL / well of CCK-8 and 10 μL / well of culture medium were added. The cells were incubated in a cell culture incubator at 37°C and 5% CO2 for 1 hour. The OD value was calculated using a CCK-8 plate reader at a wavelength of 450 nm. The OD value was calculated using the following formula.

[0122] Survival rate = (OD value of experimental well - OD value of control well) / (OD value of control well - OD value of blank well) × 100%

[0123] 2) Experimental results

[0124] The experimental results showed that different concentrations of drugs had an impact on the activity of MRC-5 cells after 24 hours of intervention. -1 At 50-200 μmol·L -1 At 50 μmol·L -1 There was a significant decrease (P<0.05), and the tetrandrine ferulate group was at 100μmol·L -1 The cell survival rate of the ferulic acid group was significantly decreased (P < 0.05). -1 The results showed that the cell activity of the tetrandrine ferulate group was higher than that of the tetrandrine group. Figure 13 As shown, Figure 13 In the medium, with 0 μmol·L -1 Group comparison ＊ P<0.05, ＊＊ P < 0.01, ＊＊＊ P<0.001

[0125] At the same time, the experiment further investigated the effects of different concentrations of drugs on the activity of LO-2 cells after 24 hours of intervention. -1 At the concentration of 50-200 μmol·L -1 At 50 μmol·L -1There was a significant decrease (P<0.05), and the tetrandrine ferulate group was at 100μmol·L -1 The cell survival rate of the ferulic acid group was significantly decreased (P < 0.05). -1 The results showed that the cell activity of the tetrandrine ferulate group was higher than that of the tetrandrine group. Figure 14 . Figure 14 In the medium, with 0 μmol·L -1 Group comparison ＊ P<0.05, ＊＊ P < 0.01, ＊＊＊ P<0.001.

[0126] Those skilled in the art can make various corresponding changes and modifications based on the above technical solutions and concepts, and all of these changes and modifications should be included in the scope of protection of the claims of the present invention.

Claims

1. A tetrandrine ferulate, characterized in that Its chemical structure is shown in Formula 1:

2. A method for synthesizing the tetrandrine ferulate according to claim 1, characterized in that: The specific process is: Take tetrandrine and add anhydrous ethanol, and stir magnetically at 30°C to obtain an ethanol solution of tetrandrine; add anhydrous ethanol to ferulic acid and dissolve it to obtain an ethanol solution of ferulic acid, add the ethanol solution of ferulic acid to the ethanol solution of tetrandrine, stir magnetically until the solid is completely dissolved, and then a small amount of solid precipitates, and stir at 30°C for 24 hours; then control the water bath temperature at 70-73°C, concentrate under reduced pressure until solid precipitates obviously, stop concentrating, filter after 1 hour, and dry naturally to obtain tetrandrine ferulate solid.

3. The synthesis method according to claim 2, characterized in that The mass ratio of tetrandrine to ferulic acid is 15:

11.

4. Use of the tetrandrine ferulate according to claim 1 in the preparation of a medicament for treating pulmonary fibrosis.

Images