Use of reishi spore oil for preventing and treating pulmonary nodules

Abstract

Disclosed is use of reishi spore oil in the preparation of a drug for preventing and treating nodular diseases. Animal experiments show that reishi spore oil has the effect of significantly inhibiting the formation and development of benign granulomatous nodules in the lungs of mice, and also possesses good safety. The reishi spore oil can be used for preparing a drug for preventing and treating the formation and development of pulmonary nodules. The reishi spore oil comprises one or more components of triglyceride, ergosterol, and vitamin E; and the reishi polysaccharide ingredient is not detected.

Description

An application of Ganoderma lucidum spore oil in the prevention and treatment of pulmonary nodules Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the application of Ganoderma lucidum spore oil in the prevention and treatment of pulmonary nodules. Background Technology

[0002] Reishi mushroom is a precious treasure in traditional Chinese medicine, often referred to as the "immortal herb." It is used to treat restlessness, insomnia, palpitations, cough and asthma due to lung deficiency, shortness of breath due to weakness, and loss of appetite. Reishi spores are extremely small spores ejected from the cap of the mushroom during its mature growth. They are the reproductive cells of Reishi and contain all of its genetically active substances. Modern pharmacological research shows that Reishi spores have effects such as enhancing immunity, lowering blood sugar and lipids, anti-tumor activity, anti-hypoxia ability, and scavenging free radicals. Reishi spore oil is a fat-soluble active substance obtained from Reishi spores through cell wall disruption and supercritical carbon dioxide extraction. Studies have shown that it has effects such as enhancing immunity and protecting the liver. Current research generally considers the main effective components of Reishi to be polysaccharides and triterpenoids. Research on the various effects of Reishi has focused more on polysaccharides and triterpenoids. Research on other components in spore oil, such as the triglycerides which constitute the largest proportion, and their efficacy analysis are currently less reported.

[0003] In recent years, with the widespread application of multi-slice computed tomography (CT) scans and the extensive implementation of lung cancer screening, the detection rate of pulmonary nodules has significantly increased globally. An article published in the authoritative journal *JAMA*, through observation of patients with pulmonary nodules, found that at least 95% of pulmonary nodules are benign and do not present the possibility of malignancy. Most patients with pulmonary nodules recover spontaneously and have a good prognosis, but one-third of them will progress to chronic disease, eventually leading to pulmonary fibrosis, pulmonary hypertension, respiratory failure, and even death. Given the current prevalence of respiratory infections caused by viruses and mycoplasma, the prevention and treatment of pulmonary nodules has become a focus of media and public attention in recent years. Clinically, for slowly progressing or unchanging benign pulmonary nodules, follow-up observation is the primary approach, and guidelines lack recommendations for effective prevention and treatment drugs. Currently, clinical treatment options for pulmonary sarcoidosis are limited, with glucocorticoids being the first-line treatment. However, long-term, high-dose use has serious adverse reactions, and relapse is common after discontinuation. Therefore, the search for safe and effective treatments for pulmonary nodules remains necessary. Summary of the Invention

[0004] The purpose of this invention is to provide an application of Ganoderma lucidum spore oil in the preparation of drugs for preventing and treating the development of pulmonary nodules. The prevention and treatment of pulmonary nodules has become a focus of media and public attention in recent years, but guidelines still lack recommendations for effective preventative and therapeutic drugs. Based on the above reasons and needs, Ganoderma lucidum spore oil has been found to possess novel properties in preventing and treating the development of pulmonary nodules, thus providing a new application of Ganoderma lucidum spore oil as a drug, namely, its application in the preparation of drugs for the prevention or treatment of pulmonary nodules.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0006] An application of Ganoderma lucidum spore oil in the preparation of a formulation for the prevention and treatment of sarcoidosis.

[0007] Animal model experiments have shown that Ganoderma lucidum spore oil has a significant inhibitory effect on the occurrence and development of benign granulomatous nodules in the lungs of mice, and has good safety. Therefore, it can be used to prepare drugs for the prevention and treatment of lung nodules.

[0008] Furthermore, current experimental modeling methods are not entirely targeted at the lungs; they may also have similar effects on preventing and treating sarcoidosis in other organs besides the lungs, which can be further developed and utilized.

[0009] Preferably, the sarcoidosis includes pulmonary nodules.

[0010] Preferably, the sarcoidosis includes benign granulomatous nodules.

[0011] Preferably, the causes of sarcoidosis include one or more of microorganisms and their related derivatives, and exogenous particles.

[0012] Preferably, the microorganisms include one or more of bacteria, viruses, and mycoplasma; the exogenous particles include one or more of carbon nanoparticles, metal particles, and smoke.

[0013] Preferably, the Ganoderma lucidum spore oil includes one or more components selected from triglycerides, ergosterol, and vitamin E.

[0014] Preferably, the Ganoderma lucidum spore oil contains >60% triglycerides, 0.1% to 0.5% ergosterol, 0.5% to 4% vitamin E, and no Ganoderma lucidum polysaccharides were detected.

[0015] Preferably, the content of triglyceride components in the Ganoderma lucidum spore oil is >90%.

[0016] Preferably, the vitamin E content in the Ganoderma lucidum spore oil is 2% by mass.

[0017] More preferably, no Ganoderma triterpenoids were detected in the Ganoderma lucidum spore oil.

[0018] A formulation obtained through the above-described application.

[0019] Preferably, the formulation comprises a drug; the drug further comprises a pharmaceutically acceptable carrier.

[0020] Preferably, the drug comprises one of the following: tablets, capsules, lozenges, granules, powders, pills, powders, ointments, pills, suspensions, solutions, emulsions, injections, suppositories, creams, sprays, drops, or patches.

[0021] Compared with the prior art, implementing the present invention has the following beneficial effects:

[0022] It has been reported that sarcoidosis is characterized by CD4+ T cell-mediated excessive immunity, therefore corticosteroids are still used as first-line treatment in clinical practice. Ganoderma lucidum spore oil is generally considered to have immune-enhancing effects, and multiple studies have reported that it can upregulate the ratio of CD4+ T cells in peripheral blood lymphocytes, thereby enhancing the body's immune capacity. However, this invention unexpectedly discovered that Ganoderma lucidum spore oil with a triglyceride content >60%, ergosterol content of 0.1%–0.5%, and vitamin E content of 0.5%–4% has a significant effect in preventing and treating pulmonary nodules. It can significantly inhibit the occurrence and development of pulmonary granulomas in mice with benign granulomatous pulmonary nodules, without significantly affecting the weight or overall health of the mice, and no obvious adverse reactions were observed, suggesting good safety. The Ganoderma lucidum spore oil of this invention has the beneficial effect of being used to prepare drugs for the prevention and treatment of pulmonary nodules. Attached Figure Description

[0023] Figure 1 shows the HPLC component detection results of Ganoderma lucidum spore oil in an embodiment of the present invention;

[0024] Figure 2 shows the TLC polysaccharide detection results of Ganoderma lucidum spore oil in an embodiment of the present invention;

[0025] Figure 3 shows the results of Micro-CT tomographic scan of mouse lungs in an embodiment of the present invention;

[0026] Figure 4 is a diagram of the pathological examination results of mouse lung tissue in an embodiment of the present invention. Detailed Implementation

[0027] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments are not intended to limit the present invention. Modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and substance of the present invention are all within the scope of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0028] Example 1

[0029] Preparation and component analysis of Ganoderma lucidum spore oil

[0030] The method for preparing Ganoderma lucidum spore oil used in this invention is as follows:

[0031] Ganoderma lucidum spore powder was subjected to cell wall disruption, followed by supercritical CO2 extraction to obtain crude Ganoderma lucidum spore oil. An appropriate amount of silica gel was dissolved in a petroleum ether-ethyl acetate mixture (v / v) of 8.5–9.5:0.5–1.5, and packed into a column using a wet packing method. The column diameter to height ratio was 1:10–20, and the mass ratio of crude Ganoderma lucidum spore oil to silica gel was 1:10–40. The crude Ganoderma lucidum spore oil was loaded onto the elution column and eluted with the above petroleum ether-ethyl acetate mixture under normal pressure. The eluent was collected, combined, concentrated under reduced pressure to remove the solvent, and vacuum dried. Vitamin E was added to achieve a content of 2% by mass to obtain the Ganoderma lucidum spore oil. Through separation and purification, a large amount of impurities and water were removed, resulting in a clear and transparent Ganoderma lucidum spore oil with stable component content.

[0032] The contents of ergosterol and triglycerides in Ganoderma lucidum spore oil were determined by high performance liquid chromatography (HPLC), as shown in Figure 1. Peak 1 is ergosterol with a content of 0.37%, and peaks 2-7 are different triglyceride components, with a total content of 93.29%. No peaks representing triterpenoid components were found.

[0033] Thin-layer chromatography (TLC) was used to detect whether Ganoderma lucidum spore oil contained polysaccharides, with starch and glucose selected as positive controls.

[0034] 1. Take 10g of Ganoderma lucidum spore oil, add 5ml of water to a separatory funnel, shake thoroughly, and allow to stand for separation. Separate the aqueous layer and discard the oil layer. Concentrate the aqueous layer under reduced pressure to 2ml, add 6ml of strong acid, shake quickly, and place in a water bath at 85℃ for 30min. Remove and let cool to room temperature before use.

[0035] 2. Take 10g of starch and prepare a 10% starch solution. Take 2ml of the solution, add 6ml of strong acid, shake quickly and mix well. Place in a water bath at 85℃ for 30 minutes. Remove and let cool to room temperature before use.

[0036] 3. Take 10 mg of anhydrous glucose and prepare a glucose solution containing 1 mg per ml. Take 2 ml of the solution, add 6 ml of strong acid, shake quickly, and place in a water bath at 85°C for 30 minutes. Remove and let cool to room temperature before use.

[0037] Five μl of each of the solutions reacted with the three strong acids was spotted onto the same silica gel thin-layer plate. Methanol was used as the developing solvent. After development, the plates were dried at 105°C and the spots were observed, as shown in Figure 2. In the positive control group, spots representing monosaccharides appeared after hydrolysis of the starch and glucose solutions, while no spots were observed at the corresponding positions in the Ganoderma lucidum spore oil. This indicates that no sugar components (including polysaccharides, disaccharides, and monosaccharides) were detected in the Ganoderma lucidum spore oil.

[0038] Example 2

[0039] Pharmacological study of Ganoderma lucidum spore oil on mice with benign granulomatous pulmonary nodules

[0040] 1. Laboratory animals

[0041] SPF grade C57BL / 6J mice (male, 6-8 weeks old) were housed in an SPF-grade animal room with 12h light / 12h darkness, temperature 22±2℃, and humidity 50±5%.

[0042] 2. Experimental Materials

[0043] Freund's incomplete adjuvant, Mycobacterium tuberculosis superoxide dismutase A (SodA), and agarose beads were used for model establishment.

[0044] Ganoderma lucidum spore oil: The Ganoderma lucidum spore oil obtained in Example 1 was used.

[0045] 3. Experimental Methods

[0046] (1) Establishment and administration of a mouse model of benign granulomatous pulmonary nodules

[0047] Mice were randomly divided into four groups: a control group, a model group, a low-dose Ganoderma lucidum spore oil group, and a high-dose Ganoderma lucidum spore oil group, with 12 mice in each group. Drug administration began 30 days before model establishment. The control and model groups were given soybean oil as a solvent, while the Ganoderma lucidum spore oil group was administered Ganoderma lucidum spore oil by gavage. The low-dose and high-dose groups received 0.3 g / kg and 1.2 g / kg of Ganoderma lucidum spore oil, respectively. The administration volume for each group was 5 mL / kg, once daily for 51 consecutive days, and changes in body weight were recorded.

[0048] On day 31, the model group and Ganoderma lucidum spore oil group were subcutaneously injected with an emulsion containing SodA and Freund's incomplete adjuvant, while the control group was subcutaneously injected with Freund's incomplete adjuvant emulsion. Drug administration continued simultaneously. On day 44, the model group and Ganoderma lucidum spore oil group were injected via tail vein with agarose beads conjugated with SodA, while the control group was injected with PBS. Drug administration continued until day 51.

[0049] (2) Micro-CT imaging of mouse lungs

[0050] Before sampling, mice in each group were anesthetized with isoflurane gas, and their breathing was observed. After several minutes, the mice's breathing became steady, and they were placed in a small animal Micro-CT scanner with a breathing mask on. The instrument parameters were adjusted, the chamber was set, the camera was adjusted, and the mice's breathing and body position were observed. After ensuring the position of the mice's chest cavity and their breathing status, a tomographic scan was performed to detect changes in the mice's lung imaging.

[0051] Micro-CT statistics: The lungs of each mouse were kept under the same window level, and the CT values ​​of the same size area were selected for each window level of each mouse and then analyzed.

[0052] (3) Pathological examination of mouse lung tissue

[0053] Fixation and embedding: Lung tissue was fixed with 4% paraformaldehyde, washed and trimmed, and the tissue was trimmed to create a smooth cut surface. It was dehydrated with 50%, 70%, 80%, 95%, and 100% ethanol solutions, and then cleared by immersion in xylene I and II respectively. After that, it was cleared by immersion in 60℃ wax I and II respectively, and finally embedded in paraffin.

[0054] Section staining: The embedded tissue was cut into paraffin sections with a thickness of approximately 4 μm. The sections were dewaxed by immersing them in clearing agent I, clearing agent II, and clearing agent III, respectively. They were then soaked in alcohol solutions of 100%, 90%, 80%, and 70% for 5 min each. After washing with water, the sections were stained with hematoxylin for 3 min, differentiated with 0.5% hydrochloric acid ethanol for a few seconds, washed with tap water for 15 min for blue inversion, and stained with eosin for 2 min. The sections were then dehydrated and cleared by immersing them in 95% ethanol I, 95% ethanol II, 100% ethanol I, 100% ethanol II, clearing agent I, clearing agent II, and clearing agent III, respectively. Finally, the sections were mounted with neutral resin.

[0055] Granuloma area statistics: Microscopic observation and photography were performed, and ImageJ software was used to statistically analyze the granuloma area of ​​the lung pathology images.

[0056] 4. Experimental Results

[0057] During the experiment, the animals in all groups were in good general condition. The results of animal weight measurements are shown in Table 1. After SodA injection into the mice to establish the model, the mice's weight decreased to some extent one day after modeling and then gradually recovered. Compared with the control group, the model group had a lower weight, but the difference was not significant. Ganoderma lucidum spore oil did not significantly improve the weight loss in the benign granulomatous pulmonary nodule model mice. There was no significant difference in the weight of the animals in each group at the time of sampling.

[0058] Table 1. Animal weight test results (g)

[0059] The effects of Ganoderma lucidum spore oil on lung imaging in mice with granulomatous pulmonary nodules are shown in Figure 3. Observation of lung imaging results revealed that, compared with the control group, the model group showed gray ground-glass opacities in different lung positions; the gray ground-glass opacities were reduced in the low- and high-dose Ganoderma lucidum spore oil groups. CT value is the corresponding value of the attenuation coefficient of each tissue in a CT image to X-rays. X-ray scanning of the mouse lungs resulted in different CT values ​​due to the varying absorption capacity of different lung tissue components for X-rays. Mice with benign granulomatous pulmonary nodules had higher CT values ​​compared to normal mice. The statistical results of CT values ​​for each group are shown in Table 2. Compared with the control group, the CT value of the model group was significantly increased (P<0.01); compared with the model group, the CT values ​​of the low- and high-dose Ganoderma lucidum spore oil groups were significantly decreased (P<0.05 or P<0.01), indicating statistical significance. This suggests that low- and high-dose Ganoderma lucidum spore oil has an ameliorative effect on granulomatous pulmonary nodules.

[0060] Table 2 Statistical results of Micro-CT scans of mouse lungs Note: Compared with the control group, **P<0.01; compared with the model group, # P<0.05, ## P<0.01

[0061] The effects of Ganoderma lucidum spore oil on the pathological changes of the lungs in mice with granulomatous pulmonary nodules are shown in Figure 4. In the control group, the alveolar tissue was intact and without abnormalities. In the model group, multiple granulomas were observed around agarose beads in the lung tissue, with the center of the granulomas composed of epithelioid cells and surrounded by numerous lymphocytes. Compared with the model group, the area of ​​granulomas in the lungs of mice in the Ganoderma lucidum spore oil group was improved to some extent, with only a thin layer of epithelioid cells surrounding the naked beads and fewer surrounding lymphocyte infiltrations. The results of the granuloma area statistics are shown in Table 3. Compared with the model group, the area of ​​granulomas in the lungs of the low-dose Ganoderma lucidum spore oil group was significantly reduced (P<0.05), and the difference was statistically significant. The area of ​​granulomas in the high-dose Ganoderma lucidum spore oil group also showed a certain degree of reduction, suggesting that low and high doses of Ganoderma lucidum spore oil have an ameliorative effect on granulomatous pulmonary nodules.

[0062] Table 3. Statistical results of granuloma area in mouse lung tissue. Note: Compared with the model group, *P<0.05

[0063] The above studies, through the establishment of a mouse model of granulomatous pulmonary nodules and the use of techniques such as Micro-CT imaging and pathological analysis, showed that Ganoderma lucidum spore oil with triglyceride content >60%, ergosterol content 0.1%–0.5%, and vitamin E content 0.5%–4% can alleviate the progression of pulmonary nodules by reducing the formation and growth of granulomas in mice with granulomatous pulmonary nodules.

[0064] In summary, this invention, through experiments and research on Ganoderma lucidum spore oil with a triglyceride content >60%, ergosterol content of 0.1%–0.5%, and vitamin E content of 0.5%–4%, found that Ganoderma lucidum spore oil has the effect of preventing and treating the occurrence and development of pulmonary nodules. It can significantly reduce the growth of granulomas in mice with granulomatous pulmonary nodules, without significantly affecting the weight and overall health of the mice, suggesting that Ganoderma lucidum spore oil has good safety. The Ganoderma lucidum spore oil of this invention has the beneficial effect of being used to prepare drugs for the prevention or treatment of pulmonary nodules.

[0065] The above-disclosed embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. Therefore, any equivalent variations made in accordance with the claims of the present invention are still within the scope of the present invention.

Claims

1. An application of Ganoderma lucidum spore oil, characterized in that, Used to prepare formulations for the prevention and treatment of sarcoidosis.

2. The application as described in claim 1, characterized in that, The sarcoidosis includes pulmonary nodules.

3. The application as described in claim 1, characterized in that, The sarcoidosis includes benign granulomatous nodules.

4. The application as described in claim 1, characterized in that, The causes of sarcoidosis include one or more of microorganisms and their related derivatives, and exogenous particles.

5. The application as described in claim 4, characterized in that, The microorganisms include one or more of bacteria, viruses, and mycoplasma; the exogenous particles include one or more of carbon nanoparticles, metal particles, and smoke.

6. The application as described in claim 1, characterized in that, The Ganoderma lucidum spore oil contains one or more components of triglycerides, ergosterol, and vitamin E.

7. The application as described in claim 1, characterized in that, The Ganoderma lucidum spore oil contained >60% triglycerides, 0.1%–0.5% ergosterol, 0.5%–4% vitamin E, and no Ganoderma lucidum polysaccharides were detected.

8. A formulation obtained by the application as described in claim 1.

9. The formulation as described in claim 8, characterized in that, The formulation includes a drug; the drug further contains a pharmaceutically acceptable carrier.

10. The formulation of claim 9, characterized in that, The drug includes one of the following: tablets, capsules, lozenges, granules, powders, pills, powders, ointments, pills, suspensions, solutions, emulsions, injections, suppositories, creams, sprays, drops, or patches.

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