Use of pileostegia viburnoides extract in the preparation of a drug for preventing and treating pulmonary fibrosis

Abstract

This invention belongs to the field of pharmaceutical technology and discloses the application of *Hedyotis diffusa* extract in the preparation of drugs for the prevention and treatment of pulmonary fibrosis. The *Hedyotis diffusa* extract is an aqueous extract obtained by water extraction, filtration, combining the filtrates, and concentration under reduced pressure from the dried whole herb of *Hedyotis diffusa*. Experimental results show that the *Hedyotis diffusa* extract can improve bleomycin-induced pathological damage to rat lung tissue, reduce inflammatory cell infiltration and collagen deposition, decrease serum levels of inflammatory factors IL-6, IL-1β, and TNF-α, increase lung tissue SOD levels and decrease MDA levels, and downregulate the expression of pulmonary fibrosis-related proteins. This indicates that the *Hedyotis diffusa* extract has an ameliorative effect on pulmonary fibrosis and can be used to prepare drugs for the prevention and treatment of pulmonary fibrosis.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and in particular relates to the application of *Hypericum perforatum* extract in the preparation of drugs for the prevention and treatment of pulmonary fibrosis. Background Technology

[0002] Pulmonary fibrosis (PF) is a chronic, progressive interstitial lung disease characterized by abnormal proliferation of fibroblasts, excessive deposition of extracellular matrix, and destruction of lung tissue structure, ultimately leading to impaired gas exchange and decreased respiratory function. This disease is triggered by various factors, including environmental exposure, drug damage, and autoimmune abnormalities. Some patients experience a progressive disease course, and severe cases can lead to respiratory failure, resulting in a poor clinical prognosis. Currently, while existing chemical drugs used to treat PF can slow disease progression to some extent, they still suffer from limited efficacy, numerous adverse reactions, and insufficient long-term medication adherence. Therefore, developing novel drugs with a clear source, good safety profile, and anti-pulmonary fibrosis potential is of great significance.

[0003] In recent years, active ingredients derived from natural medicines and traditional Chinese medicine formulas have received widespread attention in the field of anti-pulmonary fibrosis. Existing studies have shown that some traditional Chinese medicines can intervene in the process of pulmonary fibrosis by regulating inflammatory responses, inhibiting fibroblast activation, and reducing extracellular matrix deposition. *Aristolochia crassifolia* is a plant belonging to the genus *Aristolochia* in the Rubiaceae family. Hedyotis scandens The dried whole herb of Roxb. has the effects of clearing heat and detoxifying, regulating the lungs and relieving cough, and is traditionally used to treat respiratory diseases such as pneumonia and bronchitis. Existing research on Roxb. mainly focuses on its chemical composition; its application in the prevention and treatment of pulmonary fibrosis lacks systematic research, and its related effects and mechanisms have not been clearly reported.

[0004] Therefore, it is necessary to study the role of *Hypericum perforatum* extract in pulmonary fibrosis in order to provide a natural source candidate for the preparation of drugs to prevent and treat pulmonary fibrosis. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art, explore the medicinal activity of *Hedyotis diffusa*, and provide the application of *Hedyotis diffusa* extract in the preparation of drugs for the prevention and treatment of pulmonary fibrosis, so as to solve the problems of limited selection of existing pulmonary fibrosis treatment drugs, many adverse reactions and insufficient candidate drugs from natural sources.

[0006] The technical solution of the present invention is as follows: Application of *Hypericum perforatum* extract in the preparation of drugs for the prevention and treatment of pulmonary fibrosis.

[0007] Preferably, the pulmonary fibrosis is drug-, inflammation-, or oxidative stress-induced pulmonary fibrosis.

[0008] More preferably, the pulmonary fibrosis is bleomycin-induced pulmonary fibrosis.

[0009] Preferably, the prevention and treatment of pulmonary fibrosis includes any one or more of the following: reducing collagen deposition in lung tissue, improving alveolar structure, and reducing the level of inflammatory factors.

[0010] Preferably, the extract of *Hypericum perforatum* is an aqueous extract of *Hypericum perforatum*.

[0011] Preferably, the concentration of the aqueous extract of *Hypericum perforatum* is 0.5~1.5 g / mL based on the amount of raw medicinal material.

[0012] Preferably, the method for preparing the aqueous extract of *Hypericum perforatum* includes: taking *Hypericum perforatum*, soaking it in water, reflux extraction, filtering, and concentrating the filtrate under reduced pressure to obtain the extract.

[0013] Preferably, the amount of water used for soaking is 10 to 20 times the mass of the creeping rhizome, and the soaking time is 1 to 4 hours.

[0014] Preferably, the reflux temperature is 95~105℃ and the reflux time is 20~60min.

[0015] Preferably, the number of extractions is 1 to 3.

[0016] Preferably, the content of *Houttuynia cordata* extract in the drug for preventing and treating pulmonary fibrosis is 1-100%.

[0017] Preferably, the drug for preventing and treating pulmonary fibrosis also includes a pharmaceutically acceptable carrier or excipient.

[0018] Technical effects of the present invention: 1. The extract of *Hypericum perforatum* of the present invention can be used to prevent and treat pulmonary fibrosis. Specifically, it improves its effect in preventing and treating pulmonary fibrosis by regulating inflammatory response, reducing collagen deposition in lung tissue, improving oxidative stress-related indicators in lung tissue, and downregulating the expression of fibrosis-related proteins in lung tissue.

[0019] 2. This invention validated the efficacy of *Hedyotis diffusa* extract in preventing and treating pulmonary fibrosis using a bleomycin-induced rat pulmonary fibrosis model. Experimental results showed that *Hedyotis diffusa* extract could improve pathological damage in rat lung tissue to varying degrees, reduce inflammatory cell infiltration and collagen deposition, decrease serum levels of inflammatory factors IL-6, IL-1β, and TNF-α, increase lung tissue SOD levels and decrease MDA levels, while downregulating the expression of pulmonary fibrosis-related proteins. These results indicate that *Hedyotis diffusa* extract has an ameliorative effect on pulmonary fibrosis and can serve as a natural source candidate for preparing drugs to prevent and treat pulmonary fibrosis. Attached Figure Description

[0020] Figure 1It is the pathological section diagram of rat lung tissue in Example 2; Figure 2 It is the result diagram of protein immunoblot analysis in Example 2. Specific implementation manners

[0021] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0022] Unless otherwise specified in the following embodiments, the implementation conditions are usually the conditions in conventional experiments, and the raw materials are all commercially available or prepared by conventional methods in the art.

[0023] Example 1 Preparation of Hedyotis scandens extract Take the dried whole herb of Hedyotis scandens (produced in Tengchong, Yunnan), after impurity removal and pulverization, place it in a round-bottom flask, add 15 times the amount of water equivalent to the mass of the crude drug, soak for 2 h, then heat to boiling and reflux for 30 min, filter, and collect the filtrate; repeat the above steps for the filter residue for another extraction; combine the two filtrates, and concentrate under reduced pressure using a rotary evaporator to obtain an extract (PJEC) with a concentration of 1 g / mL based on the crude drug.

[0024] Example 2 Improvement effect of Hedyotis scandens extract on bleomycin-induced pulmonary fibrosis in rats 1. Experimental materials (1) Experimental animals SPF-grade male SD rats, with a body weight of (200 - 210) g, purchased from Henan Specks Biotechnology Co., Ltd., license number SCXK (Henan) 2025 - 0005. The rats were housed in a standard animal room at a temperature of 20 - 25°C, a relative humidity of (55 ± 5)%, a 12 h / 12 h light-dark cycle, and free access to food and water. After 1 week of adaptive feeding, the experiment was started. This study was approved by the Wuhan Hualianke Animal Ethics Committee (Ethical Approval Number: HLK - 20250411 - 001).

[0025] (2) Main reagents <00​​​​​​Rats were randomly divided into a control group and a model group. All rats were anesthetized with intraperitoneal injection of aphthylamine (200 mg / kg) and fixed in a 45° supine position. The tongue was gently lifted and pulled downwards and outwards. The glottis was exposed by irradiating the larynx with an external cold light source. An intravenous cannula was inserted into the trachea, the guidewire was removed, and the tubing remained in the trachea. The rat was quickly laid flat, and an empty syringe containing a short column of saline solution was placed at the opening of the external cannula; successful cannulation was indicated by the observed sliding of the saline column. The rat plate was then placed at a 45° angle, and bleomycin sulfate solution (BLM, 5 mg / kg; 33.3 μL / 100g) was pipetted and evenly dripped into the trachea through the cannula. A small amount of air was then introduced to ensure no fluid residue remained. After confirming that all fluid had entered the lungs through the cannula, it was removed. The rat was then released from its restraints, kept upright, and its back was gently patted to promote even distribution of BLM in the lungs. Rats were returned to their cages, kept in a supine position with their heads elevated and feet lowered, and kept warm. The normal control group received intratracheal infusion of physiological saline (33.3 μL / 100 g) using the same method.

[0027] The rats in the model group were then randomly divided into five groups: Model group, low-dose group of *P. japonica* extract (PJEC-L, 1.575 g / kg, calculated as raw medicinal material, 0.5 times the clinical equivalent dose), medium-dose group of *P. japonica* extract (PJEC-M, 3.15 g / kg, calculated as raw medicinal material, clinical equivalent dose), high-dose group of *P. japonica* extract (PJEC-H, 6.3 g / kg, calculated as raw medicinal material, 2 times the clinical equivalent dose), and positive control drug prednisone (PNS, 5 mg / kg). Each intervention group was given different doses of PJEC and PNS by gavage (10 mL / kg) according to body weight. The control group and the model group were given an equal volume (10 mL / kg) of physiological saline by gavage once a day at the same time for 28 consecutive days.

[0028] (2) Sample collection Twenty-eight days after drug administration, blood was collected from the abdominal aorta, and serum was collected. The serum was centrifuged at 4000 r / min for 15 minutes, and the supernatant was retained and frozen at -80 °C. Rat lung tissue was collected and washed with physiological saline. The left lung was fixed with 4% paraformaldehyde fixative, embedded in paraffin, and sectioned for hematoxylin-eosin (H&E) and Masson staining. The remaining lung tissue was rapidly transferred to -80 °C for freezing and used in molecular biology experiments.

[0029] (3) Morphological analysis of lung tissue Fixed lung tissue was harvested, and after routine dehydration, clearing, paraffin embedding, and preparation of paraffin sections with a thickness of 4 μm, it was then subjected to H&E staining and Masson staining. Pathological changes and collagen fiber deposition in the lung tissue were observed under a light microscope. H&E staining was used to observe alveolar structure, alveolar septal thickness, and inflammatory cell infiltration; Masson staining was used to observe collagen fiber deposition in the lung tissue.

[0030] (4) Measurement of serum IL-6, IL-1β and TNF-α levels Serum samples were collected from rats in each group, and the levels of IL-6, IL-1β, and TNF-α in the serum were detected according to the ELISA kit instructions.

[0031] (5) Determination of SOD and MDA content in lung tissue Weigh an appropriate amount of lung tissue and test the SOD activity and MDA content in the lung tissue according to the instructions of the SOD and MDA kits.

[0032] (6) Western Blot (WB) A suitable amount of lung tissue was lysed using RIPA lysis buffer, centrifuged, and the supernatant was collected. Protein concentration was determined using a BCA protein quantification kit. Equal amounts of protein samples from each group were separated by SDS-PAGE electrophoresis and transferred to PVDF membranes. After blocking, primary antibodies against transforming growth factor-β1 (TGF-β1), phosphorylated Smad3 (p-Smad3), SMAD family member 3 (Smad3), α-smooth muscle actin (α-SMA), type I collagen (Collagen I), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were added, and the membranes were incubated overnight at 4 °C. After washing, the membranes were incubated with the corresponding HRP-labeled goat anti-rabbit secondary antibody. After development with ECL chemiluminescence buffer, images were acquired using a chemiluminescence imaging system, and grayscale analysis was performed using image analysis software. TGF-β1, Smad3, α-SMA, and Collagen I are expressed as their relative expression levels by the ratio of their gray values ​​to GAPDH, while p-Smad3 is expressed as its relative expression level by the ratio of its gray values ​​to those of p-Smad3 / Smad3.

[0033] 3. Experimental Results 1. Pathological observation of rat lung tissue like Figure 1As shown, the lung tissue structure of rats in the Control group was relatively intact, with clear alveolar outlines, thinner alveolar septa, and no obvious inflammatory cell infiltration or abnormal collagen fiber deposition. In the Model group, the lung tissue structure was significantly disordered, with thickened alveolar septa, some alveolar cavities narrowed, blocked, or fused, and numerous inflammatory cell infiltrations were observed. Masson staining revealed a significant increase in blue-stained collagen fibers, indicating that bleomycin treatment caused significant pulmonary fibrosis-related pathological changes in rats. Compared with the Model group, the pathological damage to lung tissue in the PJEC-L, PJEC-M, PJEC-H, and PNS groups all showed varying degrees of improvement, specifically in alveolar structure, reduced interstitial thickening, decreased inflammatory cell infiltration, and reduced collagen fiber deposition. The improvements were most significant in the PJEC-M, PJEC-H, and PNS groups. These results demonstrate that the extract of *Hypericum perforatum* described in this invention can improve bleomycin-induced pathological damage to rat lung tissue and reduce collagen deposition in lung tissue.

[0034] 2. Detection results of serum inflammatory factors IL-6, IL-1β, and TNF-α levels in rats Inflammation is a common pathological feature of pulmonary fibrosis. The levels of serum inflammatory factors IL-6, IL-1β, and TNF-α in rats were detected, and the results are shown in Table 1. Compared with the Control group, the serum levels of IL-6, IL-1β, and TNF-α in the Model group were significantly increased (P < 0.01), indicating that bleomycin treatment significantly increased the levels of serum inflammatory factors in rats. Compared with the Model group, the serum levels of IL-1β and TNF-α in the PJEC-L group were significantly decreased (P < 0.01, P < 0.05), and the IL-6 level showed a decreasing trend; the serum levels of IL-6, IL-1β, and TNF-α in the PJEC-M group were significantly decreased (P < 0.01); the serum levels of IL-6, IL-1β, and TNF-α in the PJEC-H group were significantly decreased (P < 0.01); and the serum levels of IL-6, IL-1β, and TNF-α in the PNS group were significantly decreased (P < 0.01). The above results indicate that the extract of *Hypericum perforatum* described in this invention can reduce the serum inflammatory factor levels in rats with pulmonary fibrosis to varying degrees.

[0035] Table 1. Serum IL-6, IL-1β, and TNF-α levels in each group of rats ( (n=8)

[0036] 3. Detection of SOD and MDA levels, markers of oxidative stress in rat lung tissue Oxidative stress plays a crucial role in the development and progression of pulmonary fibrosis. Excessive reactive oxygen species (ROS) can lead to decreased antioxidant enzyme activity and enhanced lipid peroxidation. Table 2 shows the results of detecting SOD and MDA levels, markers of oxidative stress, in rat lung tissue. Compared with the Control group, the Model group showed significantly decreased SOD activity (P < 0.01) and significantly increased MDA content (P < 0.01), indicating oxidative stress-related changes in rat lung tissue after bleomycin treatment. Compared with the Model group, the PJEC-L group showed no significant increase in SOD levels but a significant decrease in MDA content (P < 0.01); the PJEC-M group showed significantly increased SOD levels (P < 0.01) and significantly decreased MDA content (P < 0.05); the PJEC-H group showed significantly increased SOD levels (P < 0.01) and significantly decreased MDA content (P < 0.01); and the PNS group showed significantly increased SOD levels (P < 0.01) and significantly decreased MDA content (P < 0.01). The above results indicate that the extract of *Hypericum perforatum* described in this invention can improve oxidative stress-related indicators in the lung tissue of rats with pulmonary fibrosis to varying degrees.

[0037] Table 2. SOD and MDA levels in lung tissue of rats in each group ( (n=8)

[0038] 4. Western blot analysis (WB) The results of the Western blot analysis are shown in Table 3 and 4. Figure 2 As shown, compared with the Control group, the expression levels of TGF-β1, p-Smad3 / Smad3, α-SMA, and Collagen I proteins in the lung tissue of rats in the Model group were significantly increased (P < 0.01), indicating that the expression of fibrosis-related proteins in the lung tissue of the Model group was significantly upregulated. Compared with the Model group, the expression of the above proteins in the PJEC-L, PJEC-M, PJEC-H, and PNS groups were all decreased to varying degrees, with the decreases being more significant in the PJEC-M, PJEC-H, and PNS groups (P < 0.01). The results indicate that the extract of *Hypericum perforatum* described in this invention can downregulate the expression of fibrosis-related proteins in lung tissue, thereby improving pulmonary fibrosis-related changes.

[0039] Table 3. Relative expression levels of TGF-β1, α-SMA, p-Smad3 and Collagen I proteins in the lung tissue of rats in each group ( (n=3)

[0040] 5. Conclusion The above experimental results indicate that after bleomycin induction, the Model group rats exhibited significant pathological changes related to pulmonary fibrosis, manifested as disordered lung tissue structure, thickened alveolar septa, increased inflammatory cell infiltration, and increased collagen fiber deposition, accompanied by elevated serum inflammatory factor levels, abnormal oxidative stress-related indicators in lung tissue, and upregulated expression of fibrosis-related proteins. After intervention with *Hypericum perforatum* extract, the aforementioned pathological changes and related indicators in the PJEC-L, PJEC-M, and PJEC-H groups showed varying degrees of improvement, with the PJEC-M and PJEC-H groups showing more significant improvement. In conclusion, the *Hypericum perforatum* extract of this invention can reduce inflammatory cell infiltration and collagen deposition in lung tissue, improve oxidative stress-related indicators, and downregulate the expression of fibrosis-related proteins, thus possessing application value in the preparation of drugs for the prevention and treatment of pulmonary fibrosis.

[0041] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of *Hypericum perforatum* extract in the preparation of drugs for the prevention and treatment of pulmonary fibrosis, characterized in that: The extract of *Hypericum perforatum* is an aqueous extract of *Hypericum perforatum*.

2. The application according to claim 1, characterized in that: The pulmonary fibrosis mentioned is bleomycin-induced pulmonary fibrosis.

3. The application according to claim 1, characterized in that: The prevention and treatment of pulmonary fibrosis includes any one or more of the following: reducing collagen deposition in lung tissue, improving alveolar structure, and reducing the level of inflammatory factors.

4. The application according to claim 1, characterized in that: The concentration of the aqueous extract of the herb *Hedyotis diffusa* was 0.5–1.5 g / mL, calculated based on the crude drug amount.

5. The application according to claim 1, characterized in that: The preparation method of the aqueous extract of *Hypericum perforatum* includes: taking *Hypericum perforatum*, soaking it in water, reflux extraction, filtering, and concentrating the filtrate under reduced pressure to obtain the extract.

6. The application according to claim 5, characterized in that: The amount of water used for soaking should be 10 to 20 times the weight of the creeping ear fungus, and the soaking time should be 1 to 4 hours.

7. The application according to claim 5, characterized in that: The reflux temperature is 95~105℃, and the reflux time is 20~60min.

8. The application according to claim 5, characterized in that: The number of extractions is 1 to 3.

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