Use of the sesquiterpene lactone loliolide for the preparation of a medicament for the treatment of viral pneumonia

Abstract

This invention belongs to the field of traditional Chinese medicine pharmaceutical technology, specifically relating to the use of loliolide, a sesquiterpene compound, in the treatment of viral pneumonia. The invention isolates and extracts loliolide from *Lysimachia christinae*. Whole-animal experiments have confirmed that this compound has a good therapeutic effect on H1N1 influenza A virus-induced viral pneumonia in mice, significantly improving body weight, lung index, lung inflammation, viral titer, and pathological changes in virus-infected mice. Loliolide can be further used to prepare new drugs for the treatment of viral pneumonia.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology and relates to a new pharmaceutical use of the sesquiterpene compound loliolide, specifically the use of the sesquiterpene chemical loliolide in the preparation of drugs for treating viral pneumonia. Background Technology

[0002] Existing technologies disclose that viral pneumonia is caused by upper respiratory tract viral infection that spreads downwards, leading to lung inflammation and, in severe cases, impaired lung gas exchange. Influenza viruses first invade the upper respiratory tract mucosa, replicate, and release viral particles and toxins, causing infection. During the course of the disease, immune cells accumulate and activate, and alveolar epithelial cells and vascular endothelial cells undergo apoptosis, releasing large amounts of inflammatory mediators such as tumor necrosis factor (TNF-α), leukotrienes, IL-1, IL-6, and IL-8 (Burk M, El-Kersh K, Saad M, et al. Viral infection in community-acquired pneumonia: A systematic review and meta-analysis[J]. European Respiratory Review, 2016, 25(140):178-188.). Due to the rapid and massive replication of the virus, the body's immune system is overactivated, further leading to immune dysregulation and, in severe cases, a cytokine storm. An overactive immune system can induce alveolar epithelial cell death, exacerbate epithelial cell permeability, cause vascular leakage and pulmonary edema, clinically manifested as non-cardiac pulmonary edema and refractory hypoxemia, and even ARDS (Channappanavar R, Perlman S. Pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology[J]. Seminars in Immunopathology,2017,39(5):529-539).

[0003] The formula for Qingdai and Mabo (a type of Chinese medicine) originates from *Shi Jinmo's Paired Herbs*, written by the renowned modern Chinese medicine expert Shi Jinmo. In his clinical prescriptions, Shi Jinmo used Mabo and Qingdai together, with Qingdai as the principal herb and Mabo as the assistant herb. Mabo clears heat and detoxifies, promotes lung function, and benefits the throat; Qingdai cools the blood and stops bleeding. Mabo is neutral in nature and has a particularly strong dispersing effect, while Qingdai is bitter and cold in nature and has a stronger heat-clearing effect. When used together, they reach the upper burner to reduce swelling and pain, clear heat, and benefit the throat. It can be used for acute pharyngitis, chronic pharyngitis, and tonsillitis (Shi Jinmo's Paired Herbs, [M] Beijing: People's Medical Publishing House, 2002). Modern pharmacological studies have shown that the aqueous extract of Indigofera tinctoria has anticomplement, antioxidant, and antiviral activities in vitro, and also significantly improves lung damage in mice with viral pneumonia (Tu Peng. Study on the heat-clearing and detoxifying material basis of Indigofera tinctoria and its protective effect on mice with viral acute lung injury: [D] Shanghai: Fudan University, 2018). However, the pharmacodynamic material basis of Indigofera tinctoria in treating viral pneumonia has not been reported.

[0004] Based on the current state of the technology, the inventors of this application intend to provide a new pharmaceutical use for the sesquiterpene compound loliolide, specifically involving the use of the sesquiterpene chemical loliolide in the preparation of drugs for treating viral pneumonia. Summary of the Invention

[0005] The purpose of this invention is to provide new pharmaceutical uses for the sesquiterpene compound loliolide based on the existing technology and current status, specifically involving the use of the sesquiterpene chemical loliolide in the preparation of drugs for treating viral pneumonia.

[0006] This invention proposes the use of a loliolide, a sesquiterpene compound, in a drug for treating viral pneumonia. The structural formula of the loliolide is as follows:

[0007]

[0008] In this invention, the preparation method of the sesquiterpene compound loliolide is as follows: Indigofera tinctoria and Lycoperdon perlatum are taken at a mass ratio of 8:3. Lycoperdon perlatum is chopped and extracted by boiling at 78°C with 10 times its volume of pure water. The aqueous extract is concentrated. Then, it is extracted with ethyl acetate to obtain ethyl acetate extract (EA). The obtained ethyl acetate extract is enriched and separated by silica gel column, ODS reversed-phase column and gel column. Combined with UPLC-MS / MS tracking, the compound loliolide is finally obtained by semi-preparative HPLC separation.

[0009] This invention experimentally confirms that the sesquiterpene compound loliolide is the active ingredient in the treatment of viral pneumonia using the formula Indigofera tinctoria and Lycoperdon perlatum. This invention isolates and prepares loliolide from Indigofera tinctoria and Lycoperdon perlatum, and whole-animal model experiments have confirmed its significant therapeutic effect on viral pneumonia, making it suitable for the development of drugs for viral pneumonia.

[0010] In this invention, loliolide was used in an in vivo experiment to treat H1N1 influenza virus-induced viral pneumonia in mice:

[0011] (1) Lung Index

[0012] On the fourth day after viral infection, the animal was weighed, blood was collected, the lungs were completely dissected and removed, the entire lung lobe was placed on filter paper, and after the filter paper was dried, it was weighed. This was taken as the wet weight of the lung. The lung index was calculated as follows: lung index = wet weight of lung (mg) / body weight (g).

[0013] (2) Measurement of total lung protein

[0014] After reserving a portion of the entire lung for pathological sections, the remaining lung lobes were rinsed with physiological saline, then homogenized with an appropriate amount of ice-bathed PBS. The lung homogenate was aliquoted, with 100 μL of lung homogenate added to an appropriate amount of PBS. The homogenate was then centrifuged at low temperature and high speed, and the supernatant was harvested and aliquoted. Protein content was determined using the BCA method. A standard curve was constructed using bovine serum albumin as the standard protein solution. After adding the sample to be tested to the BCA working solution, the absorbance (A) value was read at 562 nm using a multi-functional automated microplate reader (Thermo, Bio-Tek).

[0015] (3) Measurement of IL-6, TNF-α, and IL-10 in mouse lung homogenate

[0016] Mouse lung homogenate was centrifuged at high speed to collect the supernatant, which was then aliquoted and frozen. The supernatant was analyzed using ELISA according to the IL-6, TNF-α, and IL-10 assay kit instructions.

[0017] (4) Neuraminidase activity test

[0018] Add 70 μL of neuraminidase detection buffer to each well of a 96-well fluorescent microplate, and add 10 μL of neuraminidase sample to each well. Then add 10 μL of Milli-Q water to each well to make a total volume of 90 μL. Before detection, add 10 μL of neuraminidase fluorescent substrate to each well and mix for 1 minute. Incubate at 37°C for half an hour for fluorescence detection. Wavelength settings: 322 nm excitation wavelength, 450 nm emission wavelength.

[0019] (5) Detection of lung and nucleoprotein expression levels

[0020] Lung tissue slides were subjected to dewaxing, antigen retrieval, antibody binding (primary antibody: Anti-virus nucleoprotein), and counterstaining steps to obtain immunohistochemical sample slides. The replication status of the lung virus in each group was observed under a microscope.

[0021] (6) Detection of pathological changes in the lungs

[0022] The pathological changes in the lungs of mice with viral pneumonia were observed and photographed using HE staining.

[0023] Experiments have confirmed that loliolide has a significant therapeutic effect on acute lung injury induced by influenza virus H1N1; loliolide can significantly reduce the lung index in mice, improve lung pathological damage, inhibit lung inflammation, and reduce the viral titer in the lungs.

[0024] The loliolide described in this invention can be used to prepare drugs for the prevention and treatment of viral pneumonia.

[0025] The beneficial effects of this invention are as follows: loliolide was isolated and prepared from indigo and puffball, and whole animal model experiments confirmed that it has a significant therapeutic effect on viral pneumonia and can be used to prepare drugs for viral pneumonia. Attached Figure Description

[0026] Figure 1 The effect of loliolide on body weight in mice infected with H1N1 virus.

[0027] Figure 2 The effect of loliolide on the lung index of H1N1 virus-infected mice was shown in the figures: **P<0.01, ***P<0.001, vs. Model Control.

[0028] Figure 3 The effect of loliolide on IL-6, TNF-α and IL-10 in the lungs of mice infected with H1N1 virus was shown. Where: A is IL-6, B is TNF-α and C is IL-10, *P<0.05, **P<0.01, ***P<0.001, vs Model Control.

[0029] Figure 4 The effect of loliolide on neuraminidase in the lungs of mice infected with H1N1 virus was shown in the figure. ***P<0.001, vs. Model Control.

[0030] Figure 5 The effect of loliolide on the expression of viral nucleoprotein in the lungs of mice infected with H1N1 virus.

[0031] Figure 6 The effect of loliolide on lung pathological changes in mice infected with H1N1 virus. Detailed Implementation

[0032] The present invention will be further illustrated by the following examples.

[0033] Example 1: Isolation and preparation of loliolide

[0034] 29 kg and 11 kg of *Lycoperdon perlatum* herb were taken separately and mixed in an 8:3 ratio. The peeled *Lycoperdon perlatum* was chopped and extracted by boiling in 10 times its volume of pure water at 78℃. Then, it was extracted with ethyl acetate, and the extracts were combined to obtain ethyl acetate extract (EA). The obtained extract was enriched and separated using silica gel column chromatography, ODS reversed-phase column chromatography, and gel column chromatography. With UPLC-MS / MS monitoring, the compound loliolide (30 mg) was finally prepared by semi-preparative HPLC.

[0035] Example 2: Protective effect of loliolide against influenza virus-induced viral pneumonia in mice

[0036] Thirty male BALB / c mice (14-15g) were randomly divided into 5 groups (A, B, C, D, E) according to body weight: Group A was the normal control group, Group B was the H1N1 virus model group, Group C was the EA group (5mg / kg), Group D was the loliolide group (5mg / kg), and Group E was the drug oseltamivir group (20mg / kg), with 6 mice in each group. All animals were anesthetized with isoflurane gas and infected with 3LD via intranasal instillation. 50 30 μL of H1N1 virus solution was used, with 30 μL of DMEM medium administered intranasally to group A, and H1N1 diluted solution used to infect groups B, C, D, and E. Two hours after infection, mice in groups C and D were administered EA and loliolide via gavage, respectively, at a dose of 5 mg / kg. Group E was given the positive control drug oseltamivir at a dose of 20 mg / kg. Administered once daily for four consecutive days, with daily mouse weight recorded. Ninety-six hours after viral infection, mice were weighed, and blood was collected by enucleation. The entire lung was carefully removed, the blood was dried with filter paper, and the weight was recorded. The left lung was carefully removed and placed in 10% formalin for pathological evaluation; the right lung was rinsed thoroughly with physiological saline and stored at -80°C for the measurement of inflammatory factors, neuraminidase activity, and other indicators.

[0037] (1) Effect of loliolide on body weight of mice infected with H1N1 virus

[0038] Body weight change is a macroscopic indicator for assessing the protective effect of drugs against viral pneumonia in mice. Daily body weight of the mice was recorded, and body weight changes were plotted. Results are as follows: Figure 1As shown: Compared with the normal group, the mice in the model group had a significantly lower body weight; compared with the model group, the mice treated with loliolide had a slightly higher body weight.

[0039] (2) Effects of loliolide on lung index in mice infected with H1N1 virus

[0040] The lung index reflects the degree of edema in the lungs of virus-infected mice; a higher value indicates a more severe degree of lung damage. Results are as follows: Figure 2 As shown, compared with the normal group, the lung index of mice in the model group was significantly increased (P<0.001); after administration of loliolide (5mg / kg), the lung index of mice was significantly decreased (P<0.001).

[0041] (3) Effects of loliolide on lung inflammation in mice infected with H1N1 virus

[0042] Results of lung inflammatory factor detection showed that, compared with the normal group, the release levels of IL-6 and TNF-α in the lung homogenate of the model group mice were significantly increased (both P<0.001), and the release level of IL-10 was significantly decreased (P<0.001); after drug administration, the levels of IL-6 and TNF-α in the lung homogenate of the loliolide group were significantly decreased (IL-6: P<0.01; TNF-α: P<0.05), and the release level of IL-10 was significantly increased (P<0.05) (e.g. Figure 3 (As shown).

[0043] (4) The effect of loliolide on viral titers in the lungs of H1N1 virus-infected mice. The results of the test on neuraminidase activity in the lungs of virus-infected mice showed (e.g.) Figure 4 As shown in the figure, compared with the normal group, the neuraminidase activity in the lungs of mice infected with influenza virus was significantly increased (P<0.001); after administration, the neuraminidase activity in the loliolide group was significantly decreased (P<0.001). Similarly, the immunohistochemical results of H1N1 viral nucleoprotein also showed that the expression of viral nucleoprotein was enhanced after mice were infected with influenza virus, indicating an increase in viral cytoplasm in the lungs; compared with the model group, the expression level of viral nucleoprotein decreased after loliolide administration (e.g., ...). Figure 5 (As shown).

[0044] (5) Effects of loliolide on lung pathological changes in mice infected with H1N1 virus

[0045] Pathological examination results showed that in the normal group, the alveolar contours were clear, the structure was intact, there was no bleeding, and there was virtually no inflammation; in the model group, pathological sections showed significantly thickened alveolar walls, alveolar atrophy and deformation, a large accumulation of immune cells, and severe inflammation. The loliolide group significantly improved lung pathological damage, with clearer alveolar contours, relatively intact structure, reduced bleeding, and significantly alleviated inflammatory symptoms. Figure 6 As shown.

[0046] In summary, loliolide has a significant therapeutic effect on H1N1 influenza virus-induced viral pneumonia in mice; it alleviates weight loss in mice, significantly reduces lung index, lung neuraminidase activity, viral nucleoprotein (NP) protein expression level, inhibits lung inflammation, reduces lymphocyte infiltration, and alleviates lung injury.

[0047] The loliolide described in this invention can be further used to prepare drugs for treating viral pneumonia.

Claims

1. The use of the sesquiterpene compound loliolide in the preparation of a drug for treating H1N1 influenza virus pneumonia, wherein the structural formula of the sesquiterpene compound loliolide is as follows: 。 2. Use according to claim 1, characterized in that, The loliolide, a sesquiterpene compound, was prepared by the following method: Two medicinal materials, Indigofera tinctoria and Lycoperdon perlatum, were taken at a mass ratio of 8:

3. Lycoperdon perlatum was chopped and extracted by boiling in 10 times its volume of pure water at 78℃. The aqueous extract was concentrated and then extracted with ethyl acetate to obtain ethyl acetate extract. The obtained ethyl acetate extract was enriched and separated by silica gel column, ODS reversed-phase column and gel column. Combined with UPLC-MS / MS tracking, the compound loliolide was finally obtained by semi-preparative HPLC separation.

3. Use according to claim 1, characterized in that, The sesquiterpene compound loliolide significantly reduced the lung index in mice infected with H1N1 influenza virus.

4. Use according to claim 1, characterized in that, The sesquiterpene compound loliolide improved lung pathological damage in mice infected with H1N1 influenza virus.

5. Use according to claim 1, characterized in that, The sesquiterpene compound loliolide inhibits lung inflammation in mice infected with H1N1 influenza virus and reduces the viral titer in the lungs of mice infected with H1N1 influenza virus.

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