Anti-coronavirus applications of elsholtzia volatile oils

Abstract

The application belongs to the field of traditional Chinese medicine, and provides an antiviral application of volatile oil of Mosla chinensis Maxim. The volatile oil of Mosla chinensis Maxim. has the characteristics of high safety, strong efficacy and convenient administration, can inhibit two kinds of proteins 3CLpro and PLP, and can reduce acute lung injury caused by N protein. The volatile oil of Mosla chinensis Maxim. can be prepared into various application forms and used in antiviral products, and has great market potential.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine, specifically relating to the application of Elsholtzia zedoaria volatile oil in the fight against coronavirus. Background Technology

[0002] The information disclosed in this background section is intended to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] In recent years, respiratory infectious diseases have seriously endangered people's health and brought about significant public safety issues, especially the novel coronavirus of 2019. Coronaviruses are a common family of RNA viruses in nature, widely distributed and prone to mutation. For a long time, coronaviruses have been considered important pathogens causing respiratory and gastrointestinal diseases in mammals and birds. They are infectious to humans, causing diseases such as colds and pneumonia. The Coronaviridae family can be classified into four genera: α, β, γ, and δ, of which seven species pose a risk of infection to humans. HCoV-229E, HCoV-HKU1, HCoV-NL63, and HCoV-OC43 are widely distributed in the human population; they generally have low virulence and cause milder, flu-like symptoms after infection. The other three (SARS-CoV, MERS-CoV, and SARS-CoV-2) are highly dangerous, characterized by high infectivity, high mortality, and widespread prevalence. They can cause severe respiratory illnesses, and severe cases can lead to lung failure and even death.

[0004] Over the past two decades, there have been three major coronavirus outbreaks: SARS-CoV in 2002, MERS in 2012, and SARS-CoV-2 in 2019. SARS-CoV and MERS-CoV can cause symptoms such as fever, dry cough, headache, difficulty breathing, lower respiratory tract infection, lymphopenia, and diarrhea, and in severe cases, even death. However, they are less infectious and have shorter incubation periods than SARS-CoV-2. According to the World Health Organization, as of 2024, SARS-CoV-2 had caused more than 770 million confirmed cases globally, leading to a severe public health crisis and widespread health damage. Its typical clinical symptoms include fever, dry cough, fatigue, and shortness of breath; severe cases can develop into acute respiratory distress syndrome (ARDS), acute lung injury, septic shock, and even death.

[0005] The SARS coronavirus primarily encodes two proteases, including 3C-like protease (3CLpro) and papain-like cysteine ​​protease (PLpro). These two enzymes play crucial roles in viral replication and infection mechanisms; therefore, inhibiting them can effectively suppress the SARS coronavirus and are important targets for the discovery of anti-coronavirus drugs.

[0006] Searching for potential antiviral drugs or lead compounds with low toxicity from natural plants is one of the important directions in antiviral research. Elsholtzia ciliata is a plant belonging to the Lamiaceae family. Mosla chinensis Maxim) or Jiangxiangru ( Mosla chinensis The dried aerial parts of *Elsholtzia ciliata* (also known as 'Jiangxiangru') are a traditional Chinese medicine used for both medicinal and edible purposes. *Elsholtzia ciliata* is warm in nature and pungent in taste, possessing diaphoretic and exterior-releasing properties, as well as the functions of resolving dampness and harmonizing the middle jiao (digestive system). Clinically, it is often used to treat summer-heat colds, chills and fever, headache without sweating, abdominal pain, vomiting, and diarrhea. CN111494456A discloses the application of *Elsholtzia ciliata* water extract in the preparation of anti-novel coronavirus drugs. However, *Elsholtzia ciliata* and *Elsholtzia jiangnanensis* are mainly produced in Guangdong, Guangxi, Fujian, Hunan, Zhejiang, and Jiangxi provinces, while the northern variety is *Elsholtzia ciliata*. There are currently no research reports on the anti-coronavirus effects of *Elsholtzia ciliata* volatile oil. Summary of the Invention

[0007] This invention provides an application of Elsholtzia volatile oil in the fight against coronavirus.

[0008] To achieve the above objectives, the present invention adopts the following technical solution.

[0009] Application of Elsholtzia volatile oil in the preparation of anti-coronavirus drugs.

[0010] The coronavirus mentioned is human coronavirus (HCoV).

[0011] The application is as an inhibitor of 3CL and PLP proteins or to alleviate acute lung injury.

[0012] The volatile oil of *Elsholtzia ciliata* is *Elsholtzia ciliata* (*Elsholtzia ciliata*). Elsholtzia stauntoni The dried aerial parts of Benth were extracted by steam distillation.

[0013] The main components of the Elsholtzia volatile oil are trans-caryophyllene, α-caryophyllene, palmitoleic acid, caryophyllene oxide, α-pinene, Δ-juniperne, and iso-orangene epoxide, with the content of the main components being greater than 90 wt%; and the content of trans-caryophyllene is 65 wt%-70 wt%, and the content of α-caryophyllene is 8 wt%-12 wt%.

[0014] The present invention has the following advantages:

[0015] This invention provides the application of Elsholtzia zedoaria volatile oil in the fight against coronaviruses. Elsholtzia zedoaria volatile oil is characterized by high safety, strong efficacy, and convenient administration. It inhibits both 3CLpro and PLP proteins and can reduce acute lung injury induced by N protein. Elsholtzia zedoaria volatile oil can be prepared into various application forms for use in antiviral products, and has enormous market potential. Attached Figure Description

[0016] Figure 1 Total ion chromatogram of Elsholtzia ciliata oil;

[0017] Figure 2 The fitted graph shows the inhibition rates of different concentrations of Elsholtzia oil on PLP (A) and 3CLpro (B).

[0018] Figure 3 The fitted graph shows the inhibition rate of different concentrations of Elsholtzia ciliata oil against HCoV-229E.

[0019] Figure 4 The expression levels of inflammatory factor mRNAs under different treatments;

[0020] Figure 5 Lung function in mice under different treatments;

[0021] Figure 6 The expression levels of inflammatory factor mRNA in lung tissue of mice under different treatments. Detailed Implementation

[0022] The present invention will be further described below with reference to the embodiments and accompanying drawings, but the present invention is not limited to the following embodiments.

[0023] Example 1: Inhibition of 3C-like protease (3CL) and papain-like protease (PLP) by Elsholtzia ciliata oil

[0024] 1. Preparation of 3CLpro and PLP proteins

[0025] According to the article "Inhibitory Effects of Six Traditional Chinese Medicines on the Activity of SARS-CoV-2 3CLpro and PLP" (2023), recombinant plasmids pGEX-6P-1-SARS-CoV-2-3CL and pET-32a(+)-SARS-CoV-2-PLP were obtained. After transforming the recombinant plasmids into BL21 competent cells, recombinant Escherichia coli was obtained to express the two proteins. The recombinant bacteria were cultured and induced by IPTG. The cell lysate was purified by HisTrap HP affinity chromatography to obtain the SARS-CoV-2 3CL and PLP proteins.

[0026] 2. Drug and component analysis

[0027] The oil of *Elsholtzia ciliata* (purity: 98%, Chinese Pharmacopoeia 2020 edition 2204 volatile oil determination method A) was purchased from Jiangxi Hengcheng Natural Fragrance Oil Co., Ltd. (batch number: 20220620) and extracted by steam distillation of *Elsholtzia ciliata*.

[0028] (1) Sample processing

[0029] Take 10 μL of Elsholtzia oil and place it in a 2 mL EP tube. Add 990 μL of ethyl acetate and sonicate to mix. Use a 1 mL disposable syringe to draw the solution and pass it through a 0.45 μm microporous organic filter membrane into a sample vial. This is the sample solution to be tested.

[0030] (2) Analysis conditions

[0031] The chromatographic column was an HP-5MS column (30 m × 0.25 mm × 0.25 μm); the carrier gas was high-purity helium, the flow rate was 1 mL / min, the injection was split, the split ratio was 50:1, the injection volume was 1.0 μL, and the temperature was programmed (initial 70℃, rate 10℃ / min to 100℃, 3℃ / min to 160℃, 5℃ / min to 190℃, hold for 1 min).

[0032] Mass spectrometry conditions: EI ion source, ion source temperature 230℃, ionization energy 70 eV, mass scan range m / z 12-550.

[0033] (3) Results

[0034] Total ion chromatogram as follows Figure 1 As shown. The percentage of each integral chromatographic peak to the total peak area was calculated using the area normalization method. Then, the mass spectrometry data of the components of Elsholtzia ciliata oil obtained by GC-MS were analyzed. All data were analyzed using NIST 2.3 spectral library search and automatic deconvolution technology.

[0035] Table 1. Identification results of the main chemical components of Elsholtzia oil

[0036]

[0037] Fifty-one components were isolated from Elsholtzia ciliata oil, and 26 of them were identified (see Table 1), accounting for 98% of the total peak area. Among them, trans-caryophyllene, α-caryophyllene, palmitoleic acid, caryophyllene oxide, α-pinene, Δ-juniperene, and iso-orangeene epoxide all had contents greater than 1%, making them the main components and accounting for 92.24% of the total peak area.

[0038] 2. The IC of Elsholtzia ciliata oil on 3CLpro and PLP 50

[0039] Elsholtzia oil was diluted with DMSO starting at 100 μg / mL to prepare a series of solutions. Selenium ibex or GRL0617 was diluted with DMSO starting at 1,000 μmol / L and 50,000 μmol / L to prepare a series of solutions. The inhibition rates and IC50 values ​​against 3CLpro and PLP were determined according to the following method. 50 :

[0040] In 96-well plates with a clear bottom, 40 μL of 3CLpro or PLP (final concentration 100 nmol / L) was mixed with 10 μL of Elsholtzia oil, 10 μL of selenium ibex (final concentration 10 μL / well), or 10 μL of GRL0617 (final concentration 10 μL / well). After incubation at room temperature for 30 min, 50 μL of 3CLpro or PLP fluorescent substrate (final concentration 25 nmol / L or 30 nmol / L) was added. Fluorescence intensity was immediately detected using a multi-mode microplate reader. The detection conditions were: gain 100, room temperature, excitation light 336 nm (3CLpro), 360 nm (PLP), emission light 490 nm, detection interval 30 s, and total detection time 60 min. The drug inhibition rate was calculated based on the increase in fluorescence intensity of different drugs after 1 h of reaction, using the formula:

[0041] Inhibition rate = ×100%;

[0042] The IC50 values ​​for 3CLpro and PLP were fitted based on the inhibition rates at different concentrations. 50 .

[0043] The fitted curve is as follows Figure 2 As shown, the IC50 of Elsholtzia ciliata oil on 3CLpro 50 The concentration was (45.05 ± 3.78) μg / mL; the IC50 of ebuselenium against 3CLpro was... 50 The concentration was (29.34 ± 1.65) μg / L; the IC50 of Elsholtzia oil against PLP was (29.34 ± 1.65) μg / L. 50 The concentration was (8.002 ± 1.395) μg / mL; the IC50 of GRL0617 against PLP was... 50 The concentration was (1.26 ± 0.0402) mg / L. These results indicate that Elsholtzia oil inhibits both 3CLpro and PLP, with a stronger inhibitory effect on PLP.

[0044] Example 2: In vitro anti-HCoV-229E effect of Elsholtzia ciliata oil

[0045] Commercially available HCoV-229E was used for experiments. The cells were dissolved in DMSO to their maximum solubility concentration, and then diluted twice with cell maintenance medium to obtain test solutions containing six volatile components of the traditional Chinese medicine, including those at maximum solubility. Huh-7 cells (1.5 × 10⁻⁶) were seeded in 24-well plates.5 Each well was incubated at 37°C with 5% CO2 for 24 h, and the culture medium was discarded. 500 µL of the test solution was added to each well, and after incubation for 4 h, the culture medium was discarded. Then, 500 µL of HCoV-229E virus dilution buffer (MOI=0.1, diluted with the test solution) was added to each well, and after incubation for another 1 h, the culture medium was discarded. 500 µL of the test solution was added to each well. A virus control well without drug treatment was also included. After incubation at 34°C with 5% CO2 for 48 h, the cells were subjected to three freeze-thaw cycles and then centrifuged using a low-temperature high-speed centrifuge. The viral supernatant was stored at -80°C.

[0046] The TCID of the collected supernatant was determined using the CCK8 method and the Reed-Muench method. 50 According to TCID 50 The efficacy, i.e., the inhibition rate of Elsholtzia ciliata oil against HCoV-229E, was calculated using the following formula:

[0047] Inhibition rate = ×100%;

[0048] Starting from a maximum final concentration of 50 μg / mL, six 2-fold dilutions were performed. Each concentration of the drug was tested according to the above method, and the results were fitted to obtain the IC50. 50 .

[0049] The fitting graph of Elsholtzia ciliata oil to HCoV-229E is as follows: Figure 3 As shown: IC 50 It was 1.602 ± 0.597 μg / mL.

[0050] Example 3: In vitro anti-N protein lesions and inflammation effects of Elsholtzia ciliata oil

[0051] 1. Obtaining the N protein

[0052] Based on the plasmid pET-32a(+)-N-protein provided by Professor Tao Shengze of Shanghai Jiao Tong University, a recombinant plasmid pET28A-SARS-CoV-2-N for SARS-CoV-2 N protein was designed. After transformation into BL21 competent cells, the plasmid was induced, purified, and endotoxin removed, then aliquoted and stored. Before the experiment, polymyxin B (250 μg / mL) was added and incubated in a 37℃ biochemical incubator for 1 h to ensure that there was no endotoxin present to interfere with the experiment.

[0053] 2. Protective effect of Elsholtzia oil against N-protein-induced cytopathic effects

[0054] The human lung epithelial cell line BEAS-2B (B2B) was cultured according to the standard procedure in the manufacturer's instructions and then used for experiments.

[0055] B2B cells were seeded into 96-well plates at a concentration of 1.6×10 5 cells / mL and placed back into the cell incubator at 37 °C and 5% CO2 for culture; 6 h after seeding, the original culture medium was discarded, rinsed with PBS, and then cultured in serum-free DMEM overnight for starvation; groups of elsholtzia oil (dissolved in DMSO at the cell level, final concentration 0.1 μg / mL), blank control group, and model group (N protein + 0.1% DMSO) were set up, and OD 450 was detected. The protection rate was calculated according to the following formula:

[0056] Protection rate = × 100%.

[0057] The protection rate of elsholtzia oil against the cytopathic effect caused by N protein was 31.61 ± 3.347.

[0058] 3. Effect of elsholtzia oil on cell inflammation caused by N protein

[0059] B2B cells were seeded into 96-well plates at a concentration of 1.6×10 5 cells / mL and placed back into the cell incubator at 37 °C and 5% CO2 for continued culture; 6 h after seeding, the original culture medium was discarded, rinsed with PBS, and then cultured in serum-free DMEM overnight for starvation; groups of elsholtzia oil (final concentration 0.1 μg / mL), blank control group, and model group were set up, and the mRNA expression levels of inflammatory factors IL-6, IL-8, IL-18, IL-1β, and TNF-α were detected.

[0060] The results were as Figure 4 shown: elsholtzia oil had a tendency to downregulate the secretion of cell inflammatory factors caused by N protein.

[0061] Example 4 Effect of elsholtzia oil on acute lung injury in mice caused by N protein

[0062] SPF-grade C57BL / 6JNifdc male mice, 4 weeks old, weighing 17 - 19 g, batch number 110011231110791025, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. [SCXK (Beijing) 2022 - 0006]. During the feeding period, the mice had free access to food and water, the light was on for 12 h in a cycle, the room temperature was maintained at 22 - 25 °C, and the humidity was 40% - 60%. They were randomly divided into three groups, with 3 mice in each group: normal group, elsholtzia oil group, and model group.

[0063] After intraperitoneal injection of 150 μL of 1% sodium pentobarbital for anesthesia, the treatment group received 25 μL of 1% Elsholtzia oil via nebulization daily, while the model and normal groups received 25 μL of normal saline via nebulization. Pre-treatment was administered for 3 days. On the third day, 50 μL (60 μg) of N protein stimulation was infused into both the treatment and model groups via intravenous drip. Lung function was assessed immediately 24 hours after N protein stimulation. Animals were euthanized, and lungs, spleen, and thymus were harvested, weighed, and organ indices were calculated. Lung tissue was preserved for analysis of the relative mRNA expression of inflammatory factors IL-6, IL-1β, IFN-γ, and TNF-α.

[0064] Lung function in mice, such as Figure 5 As shown, after N protein stimulation, respiratory rate, tidal volume, minute ventilation, cumulative volume, maximum expiratory flow rate, maximum inspiratory flow rate, relative time, and Penh were significantly upregulated, while inspiratory time and expiratory time were significantly downregulated. Treatment with Elsholtzia oil can significantly adjust the respiratory function of mice, restoring it to a level indistinguishable from that of the normal group.

[0065] After dissecting the mice, there were no significant differences in the indices of various organs.

[0066] Expression of inflammatory factor mRNA in lung tissue, such as Figure 6 As shown, Elsholtzia oil can downregulate the relative expression of four inflammatory factors: IL-6, IL-1β, IFN-γ, and TNF-α.

[0067] The above results indicate that Elsholtzia oil can combat N protein-induced acute lung injury in mice.

Claims

1. The use of volatile oil of Mosla chinensis Maxim. in the preparation of a drug against coronavirus, characterized in that, The volatile oil of *Elsholtzia ciliata* is *Elsholtzia ciliata* (*Elshol Elsholtzia stauntoni The dried aerial parts of Benth were extracted by steam distillation. The main components of the Elsholtzia volatile oil are trans-caryophyllene, α-caryophyllene, palmitoleic acid, caryophyllene oxide, α-pinene, Δ-juniperne, and iso-cisperene epoxide, with the content of the main components being greater than 90 wt%; and the content of trans-caryophyllene is 65 wt%-70 wt%, and the content of α-caryophyllene is 8 wt%-12 wt%. The coronavirus in question is HCoV.

2. The application of the Elsholtzia volatile oil according to claim 1 in the preparation of drugs for the prevention and treatment of acute lung injury.

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