Ginkgo biloba endopleura extract, preparation method thereof and application of ginkgo biloba endopleura extract in preparation of anti-pulmonary fibrosis drugs
By preparing ginkgo seed peel extract, the problem of lack of safe and effective anti-pulmonary fibrosis drugs in the prior art was solved, and the significant improvement effect on pulmonary fibrosis was achieved, and a variety of dosage forms were provided for treatment options.
Patent Information
- Application Number
- CN202510728440.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-03
- Publication Date
- 2025-08-08
AI Technical Summary
The lack of safe and effective anti-pulmonary fibrosis drugs in the prior art leads to limited treatment options for patients with pulmonary fibrosis, and large side effects, and waste of resources.
Ginkgo seed peel extract is prepared, and the ginkgo seed peel powder is obtained by methanol extraction, centrifugation, concentration and lyophilization. It is used to prepare anti-pulmonary fibrosis drugs, including powders, tablets, granules, capsules, solutions, emulsions and suspensions.
Ginkgo Intragranular Skin Extract significantly improves the pathological symptoms of pulmonary fibrosis in mice, reduces pulmonary edema and inflammatory factors, reduces inflammation and collagen deposition in lung tissues, and provides a safe and effective anti-pulmonary fibrosis treatment plan.
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Figure CN120437186A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of biomedicine, and in particular to a ginkgo endocarp extract, a preparation method thereof, and application thereof in the preparation of anti-pulmonary fibrosis drugs. Background Art
[0002] Pulmonary fibrosis (PF) is an interstitial lung disease characterized by excessive proliferation and activation of myofibroblasts (MFBs) and abnormal accumulation of the extracellular matrix (ECM) in lung tissue lesions. It results from repeated damage and abnormal repair of lung tissue. It is a major cause of disability and mortality in various lung diseases and a necessary stage in the progression of many lung diseases to lung cancer. Clinically, patients often experience symptoms such as dyspnea, dry cough, and hypoxemia, and in the late stages, respiratory failure often leads to death. PF is also a common complication of various autoimmune diseases, such as systemic sclerosis, rheumatoid arthritis, systemic lupus erythematosus, polymyositis, and dermatomyositis, and is one of the main factors affecting the poor prognosis of these diseases.
[0003] Idiopathic pulmonary fibrosis (IPF) is a pulmonary fibrosis (PF) disease of unknown etiology with the highest clinical mortality rate, a median survival of less than three years and a five-year survival rate of less than 30%. FDA-approved drugs such as pirfenidone and nintedanib only mitigate the progression of PF, lacking a curative effect and associated with significant side effects. Consequently, current clinical treatments for PF primarily rely on steroids, immunosuppressants, and cytotoxic drugs, supplemented with oxygen therapy and pulmonary rehabilitation. These treatments are associated with significant side effects, high costs, and inconsistent long-term efficacy, placing a significant burden on society and patients' families. Lung transplantation is the ultimate lifeline for patients with advanced PF, but limited lung organ resources and high treatment costs make it difficult for patients to obtain the treatment. The shortage of effective anti-PF drugs has become a significant challenge, and the development of safe and effective anti-PF drugs is of great practical significance and clinical value.
[0004] Ginkgo biloba, a living fossil of the plant kingdom, has high medicinal value. Its endocarp is used to treat lung diseases such as phlegm-related asthma and excessive phlegm. However, during the preparation and consumption of ginkgo biloba, its endocarp is discarded as waste, resulting in a wasteful use of resources. To date, there are no reports on the pharmacological activity of the endocarp. Therefore, there is an urgent need to develop a method for utilizing the endocarp of ginkgo biloba. Summary of the Invention
[0005] The purpose of the present invention is to provide a Ginkgo endocarp extract, a preparation method thereof, and an application thereof in the preparation of an anti-pulmonary fibrosis drug, so as to solve the problems existing in the above-mentioned prior art. The Ginkgo endocarp extract provided by the present invention has a significant improvement effect on various pathological symptoms of pulmonary fibrosis in mice and can effectively improve pulmonary fibrosis.
[0006] To achieve the above object, the present invention provides the following solutions:
[0007] The present invention provides a method for preparing a ginkgo endocarp extract, comprising the following steps:
[0008] Taking ginkgo seeds, removing the outer seed coat, drying, collecting the inner seed coat, freeze-drying, and grinding to obtain ginkgo inner seed coat powder;
[0009] The ginkgo endocarp powder is mixed with a methanol aqueous solution, subjected to vortex extraction, centrifuged, and the supernatant is collected, concentrated, and freeze-dried to obtain the ginkgo endocarp extract.
[0010] Preferably, the volume fraction of methanol in the methanol aqueous solution is 70%.
[0011] Preferably, the mass volume ratio of the ginkgo inner seed coat powder to the methanol aqueous solution is 1 g:8 mL.
[0012] Preferably, the vortex extraction is as follows: vortexing once every 30 minutes, each vortexing lasting 30 seconds, and vortexing 6 times in total.
[0013] The present invention also provides a ginkgo endocarp extract obtained according to the preparation method.
[0014] The present invention also provides a use of the Ginkgo endocarp extract in preparing a medicine for preventing and / or treating pulmonary fibrosis.
[0015] Preferably, the pulmonary fibrosis is bleomycin-induced pulmonary fibrosis.
[0016] The present invention also provides a medicine for preventing and / or treating pulmonary fibrosis, comprising the ginkgo endocarp extract.
[0017] Preferably, a pharmaceutically acceptable excipient is also included.
[0018] Preferably, the dosage form of the drug includes powder, tablet, granule, capsule, solution, emulsion and suspension.
[0019] The present invention discloses the following technical effects:
[0020] The present invention prepares a Ginkgo biloba endocarp extract and conducts a Ginkgo biloba endocarp extract administration experiment using a classic mouse pulmonary fibrosis model induced by bleomycin. The experimental results demonstrate that the Ginkgo biloba endocarp extract provided by the present invention can improve pulmonary fibrosis by ameliorating alveolar collapse in mice, reducing pulmonary edema, inflammatory factors, and the degree of inflammatory factors, alleviating pulmonary tissue edema, inflammation, and collagen deposition. This invention provides technical support for the development of Ginkgo biloba endocarp extract as an anti-pulmonary fibrosis drug and also lays a foundation for the development and utilization of Ginkgo biloba endocarp resources. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.
[0022] Figure 1 The total ion current of Ginkgo biloba seed coat extract (GBE) in positive / negative ion mode; A is negative ion mode; B is positive ion mode;
[0023] Figure 2 Figure 2 is the effect of GBE on histopathological lesions, lung index and inflammatory factors in each group; A is H&E staining of lung sections in different experimental groups; B is lung index in different experimental groups; CE is the expression level of inflammatory factors TNF-α (C), IL-6 (D), and IL-1β (E) in BALF of different experimental groups; Control is the control group, BLM is the bleomycin model group, GBE (L) is the low-dose GBE group, GBE (M) is the medium-dose GBE group, GBE (H) is the high-dose GBE group, and Pirfenidone is the positive drug group; compared with the control group, ##P<0.01; compared with the bleomycin model group, *P<0.05, **P<0.01;
[0024] Figure 3Figure 3 is the result of the effect of GBE on collagen deposition, PF marker cytokines and proteins; A is a representative image of Masson's trichrome staining of lung sections; B is the hydroxyproline content of each experimental group; C is the expression of TGF-β1 in BALF of each experimental group; D is the immunofluorescence staining result of the relative quantitative expression analysis of α-sma; E is the statistical result of the relative quantitative expression analysis of α-sma in lung tissue; Control is the control group, BLM is the bleomycin model group, GBE(L) is the low-dose GBE group, GBE(M) is the medium-dose GBE group, GBE(H) is the high-dose GBE group, and Pirfenidone is the positive drug group; compared with the control group, ##P<0.01; compared with the bleomycin model group, *P<0.05, **P<0.01. DETAILED DESCRIPTION
[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0026] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.
[0027] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.
[0028] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.
[0029] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.
[0030] Example 1
[0031] 1. Preparation of Ginkgo Biloba Seed Coat Extract
[0032] 1.1 Preparation of Ginkgo Biloba Seed Coat Powder
[0033] Ginkgo seeds were collected on the campus of Linyi University in mid-November 2023. The outer seed coat was removed to obtain ginkgo nuts, which were placed in a constant temperature oven at 45°C and dried for 96 h. The inner seed coat was collected by manual shelling and placed in a freeze dryer for vacuum freeze drying for 36 h (-50°C-6 h, -30°C-6 h, -10°C-6 h, 0°C-6 h, 20°C-6 h, 30°C-6 h), and ground into powder using a grinder (MM 400, Retsch) (30 Hz, 1.5 min) to obtain ginkgo inner seed coat powder.
[0034] 1.2 Extraction method
[0035] Accurately weigh 5 g of Ginkgo biloba inner seed coat powder and vortex extract it with 40 mL of 70% methanol-water solution pre-cooled at -20°C. The vortexing procedure was as follows: vortex once every 30 minutes, each time for 30 seconds, for a total of 6 times.
[0036] After centrifugation at 12000 rpm for 3 min, the supernatant was aspirated and concentrated to a paste using a rotary evaporator (40°C, -1 MPa); the mixture was freeze-dried in vacuo for 36 h (-50°C for 6 h, -30°C for 6 h, -10°C for 6 h, 0°C for 6 h, 20°C for 6 h, and 30°C for 6 h) to obtain a freeze-dried powder;
[0037] Take 1 g of freeze-dried powder, add 100 mL of 1.5% sodium carboxymethyl cellulose solution, and vortex mix thoroughly (vortex once every 5 minutes, 30 seconds each time, vortex 3 times) to obtain a suspension of Ginkgo biloba seed coat extract with a concentration of 1 g / 100 mL. This suspension is used as the mother liquor for subsequent in vivo anti-pulmonary fibrosis activity studies in animals, and the mother liquor is stored at 4°C for future use.
[0038] 2. Mass Spectrometry Total Ion Chromatography Analysis of Ginkgo Biloba Seed Coat Extract
[0039] 2.1 Liquid phase conditions mainly include:
[0040] (1) Chromatographic column: Agilent SB-C 18 1.8μm, 2.1mm*100mm;
[0041] (2) Mobile phase: Phase A is ultrapure water (added with 0.1% formic acid), phase B is acetonitrile (added with 0.1% formic acid);
[0042] (3) Elution gradient: at 0.00 min, the B phase ratio was 5%, which increased linearly to 95% within 9.00 min and maintained at 95% for 1 min. From 10.00 to 11.10 min, the B phase ratio was reduced to 5% and equilibrated at 5% for 14 min.
[0043] (4) Flow rate: 0.35 mL / min; column temperature: 40°C; injection volume: 2 μL.
[0044] 2.2 Mass spectrometry conditions mainly include:
[0045] The electrospray ionization (ESI) source temperature was 500°C; the ion spray voltage (IS) was 5500 V (positive ion mode) / -4500 V (negative ion mode); the ion source gas I (GSI), gas II (GSII), and curtain gas (CUR) were set to 50, 60, and 25 psi, respectively, and the collision-induced ionization parameter was set to high.
[0046] 2.3 Mass spectrometry total ion current analysis
[0047] like Figure 1 As shown in Figures A and B, the present invention constructs a fingerprint of the Ginkgo biloba inner seed coat extract based on liquid chromatography-mass spectrometry analysis through mass spectrometry positive and negative ion total ion current analysis, thereby providing a fingerprint to ensure the preparation of a stable and repeatable Ginkgo biloba inner seed coat extract.
[0048] The main chemical components of the Ginkgo endocarp were summarized based on mass spectrometry results. The results showed that, in order of quantity, the components found in the Ginkgo endocarp are flavonoids and their glycosides, lipids, phenolic acids, alkaloids, nucleotides / amino acids, and their derivatives. Among them, lipids and alkaloids are rarely reported in Ginkgo leaves and ginkgo nuts, and are therefore specific components of the Ginkgo endocarp that warrant attention.
[0049] Example 2 Study on the Anti-pulmonary Fibrosis Effect of Ginkgo Biloba Seed Coat Extract
[0050] 1. Animal Modeling
[0051] The experimental mice (C57BL / 6 mice, 24-26 g, 7-8 weeks) were randomly divided into a control group, a bleomycin (BLM) model group, a GBE (Ginkgo biloba seed coat extract) low-dose group (50 mg / kg), a GBE medium-dose group (100 mg / kg), a GBE high-dose group (150 mg / kg) and a positive drug group (pirfenidone 100 mg / kg), with 10 mice in each group.
[0052] Mice were anesthetized with an intraperitoneal injection of 5 mL / kg chloral hydrate (5%), followed by an intratracheal instillation of 10 mg / kg BLM. A control group of mice was injected with an equal volume of normal saline. Seven days later, mice in the control group and the BLM-induced pulmonary fibrosis group were treated with an equal volume of normal saline once daily.
[0053] Mice in the GBE low-dose, GBE medium-dose, and GBE high-dose groups were gavaged with 50, 100, and 150 mg / kg body weight of GBE, respectively. During the experiment, the stock solution prepared in Example 1 was vortexed (3 times every 5 minutes, 30 seconds each time) before each administration. The solution was then diluted with 1.5% sodium carboxymethylcellulose solution and administered by gavage. The drug was prepared freshly and used immediately.
[0054] The mice in the positive drug group were given 100 mg / kg body weight of pirfenidone by gavage and all mice were killed after 28 days.
[0055] The lung index, lung tissue and hydroxyproline content in lung homogenate of mice were examined and measured, and the changes in the lungs of mice were counted and analyzed.
[0056] 2. Experimental Results
[0057] 2.1GBE improves histopathological damage and inflammation in pulmonary fibrosis
[0058] Alveoli and interstitial structures are typical foci of pulmonary fibrosis. Histopathological sections can effectively reflect the severity of pulmonary fibrosis. Figure 2 As shown in center A, the control group showed thickened and narrowed alveolar septa, intact alveolar spaces, and normal distribution of interalveolar reticulin. However, the BLM group showed destructive alveolar collapse, with inflammatory cells and myofibroblasts widely distributed within the dense interstitial tissue, and significantly increased septal thickening. GBE at a dose of 50 mg / kg had no significant effect on the pathological features of alveolar collapse, dense interstitial infiltration, and thickened alveolar septa, whereas GBE at doses of 100 and 150 mg / kg significantly improved these features. These results directly reflect the therapeutic effect of GBE on pulmonary fibrosis.
[0059] The lung index can reflect the degree of lung tissue damage and edema. Figure 2 Middle B shows that the lung index of the bleomycin model group was significantly higher than that of the control group. 50 mg / kg of GBE had no significant effect on improving histopathology, but 100 and 150 mg / kg of GBE significantly reduced the lung index in a dose-dependent manner.
[0060] Inflammation is a common pathological feature of pulmonary fibrosis. Figure 2As shown in Figures C, D, and E, the bleomycin model group showed significant increases in the three inflammatory cytokines TNF-α, IL-6, and IL-1β compared to the control group. GBE at doses of 100 and 150 mg / kg significantly reduced inflammatory cytokine levels in a dose-dependent manner. GBE at 150 mg / kg had the same inhibitory effect on these three inflammatory factors as pirfenidone. GBE at 50 mg / kg had no significant effect on the infiltration of inflammatory cytokines. These results indicate that GBE significantly reduces the infiltration of inflammatory cytokines during PF.
[0061] 2.2GBE improves collagen deposition in pulmonary fibrosis and inhibits TGF-β and α-SMA
[0062] Extensive collagen deposition is the most typical feature of pulmonary fibrosis. Figure 3 As shown in Figures A and B, compared with the control group, mice in the bleomycin model group showed excessive collagen deposition in the alveoli and interstitial spaces of the lung tissue (blue staining). Treatment with 100 and 150 mg / kg of GBE significantly reduced collagen deposition. This result was confirmed by quantitative analysis of hydroxyproline, a major amino acid in collagen. GBE at 50 mg / kg had no significant inhibitory effect on hydroxyproline. Furthermore, the inhibitory effect of GBE at 150 mg / kg on hydroxyproline was similar to that of the positive drug group (pirfenidone 100 mg / kg). These results demonstrate that GBE significantly reduces collagen deposition in the lung tissue of PF mice.
[0063] TGF-β1 and α-SMA are representative markers of PF, such as Figure 3 As shown in Figures C, D, and E, TGF-β1 and α-SMA levels were significantly higher in the bleomycin model group than in the control group. Treatment with 100 and 150 mg / kg of GBE significantly reduced TGF-β1 and α-SMA expression in a dose-dependent manner. However, 50 mg / kg of GBE had no significant inhibitory effect on TGF-β1 expression. However, 50 mg / kg of GBE significantly inhibited α-SMA expression. The inhibitory effect of 150 mg / kg of GBE on TGF-β1 and α-SMA was similar to that of the positive drug group (pirfenidone 100 mg / kg).
[0064] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.
Claims
1. A method for preparing a Ginkgo biloba seed coat extract, characterized in that: The following steps are involved: Taking ginkgo seeds, removing the outer seed coat, drying, collecting the inner seed coat, freeze-drying, and grinding to obtain ginkgo inner seed coat powder; The ginkgo endocarp powder is mixed with a methanol aqueous solution, subjected to vortex extraction, centrifuged, and the supernatant is collected, concentrated, and freeze-dried to obtain the ginkgo endocarp extract.
2. The preparation method according to claim 1, wherein The volume fraction of methanol in the methanol aqueous solution is 70%.
3. The preparation method according to claim 1, wherein The mass volume ratio of the ginkgo inner seed coat powder to the methanol aqueous solution is 1g:8mL.
4. The preparation method according to claim 1, wherein The vortex extraction was performed as follows: vortexing once every 30 minutes, each vortexing lasting 30 seconds, for a total of 6 times.
5. A Ginkgo biloba endocarp extract obtained according to the preparation method according to any one of claims 1 to 4.
6. Use of the Ginkgo endocarp extract according to claim 5 in the preparation of a medicament for preventing and / or treating pulmonary fibrosis.
7. The use according to claim 6, characterized in that The pulmonary fibrosis is bleomycin-induced pulmonary fibrosis.
8. A drug for preventing and / or treating pulmonary fibrosis, characterized in that: The invention comprises the ginkgo endocarp extract according to claim 5.
9. The drug according to claim 8, wherein Pharmaceutically acceptable excipients are also included.
10. The drug according to claim 8, wherein The dosage forms of the drug include powders, tablets, granules, capsules, solutions, emulsions and suspensions.