A traditional Chinese medicine composition for resisting new coronavirus

Abstract

The present invention discloses a traditional Chinese medicine composition for treating novel coronavirus pneumonia, which is used to treat pneumonia caused by novel coronavirus infection and relates to the field of traditional Chinese medicine. The traditional Chinese medicine for treating novel coronavirus pneumonia disclosed in the present invention includes gypsum, ephedra, ginger, poria, yam, scutellaria, roasted licorice, qinglongyi, dried lemon slices, atractylodes macrocephala, dandelion, immature bitter orange, and dried tangerine peel. The qinglongyi is the dried fleshy peel of the walnut plant Juglans mandshurica, which is dried and washed before use as medicine. In the prescription disclosed in the present invention, the two most critical herbs that can have an antiviral effect are qinglongyi and gypsum. Qinglongyi has a bitter and astringent taste, is neutral in nature, and enters the lung, spleen, stomach, and large intestine meridians; gypsum has a sweet and pungent taste, is extremely cold in nature, and enters the lung and stomach meridians. This traditional Chinese medicine formula can dispel cold and dampness, regulate qi and resolve phlegm, strengthen the spleen, and enhance immunity.

Description

Technical Field

[0001] The present invention belongs to the field of traditional Chinese medicine, and specifically relates to a traditional Chinese medicine composition for treating novel coronavirus pneumonia. Background Art

[0002] Epidemic disease is an abnormal life process caused by the disorder of homeostasis under the influence of certain pathogenic factors, which triggers a series of changes in metabolism, function, and structure, manifesting as abnormal symptoms, signs, and behavior. Disease is an abnormal life process caused by the disorder of homeostasis under certain conditions and after being damaged by pathogenic factors. It is a state of life caused by certain reasons. In this state, the morphology and function of the human body change to a certain extent, normal life activities are restricted or destroyed, and perceptible symptoms appear sooner or later. The outcome of this state can be recovery (return to normal) or long-term survival, or even death. COVID-19 is an acute infectious disease caused by the new coronavirus. It has an acute onset, rapid progression, and is highly contagious. The "Handbook of Diagnosis and Treatment of Novel Coronavirus Pneumonia in Traditional Chinese Medicine" classifies it as a "plague" or "epidemic disease" in traditional Chinese medicine (Wen Qing, Tian Kan, Lu Chao, Wang Shengming. The intervention of traditional Chinese medicine in the prevention and treatment of COVID-19 and its implications [J]. Journal of Nanjing Medical University, 2021, 21(2):149-153.).

[0003] Academician Tong Xiaolin, a Chinese medicine expert and chief researcher at the China Academy of Chinese Medical Sciences, defines COVID-19 as a "cold-dampness epidemic." The main organs invaded by this epidemic are the lungs and spleen. If the disease is located in the lungs, symptoms are similar to coughing, expectoration, wheezing, and shortness of breath. If the disease is located in the lungs, symptoms are similar to fatigue, loss of appetite, nausea, vomiting, and diarrhea. Clinical observations show that in the early stages of COVID-19, patients may have fever or no fever, or a low fever, accompanied by aversion to cold, headache, body pain, limb pain, fatigue, poor appetite, white and greasy tongue coating, and slippery pulse. This is a manifestation of dampness stagnating in the skin and suppressing Yang Qi. Some patients simply present with digestive tract symptoms such as diarrhea, loose stools, constipation, poor appetite, nausea, and vomiting, all of which are caused by the invasion of the gastrointestinal tract by cold-dampness evil and damage to the spleen and stomach (Tao Lingxia, Zhu Ying. Professor Zhu Ying's experience in treating COVID-19 with traditional Chinese medicine. Chinese Community Physician, 2020, 36(28): 94-95).

[0004] Western medications targeting the novel coronavirus are under development globally, but these can take up to 18 months or even longer. Drugs currently in clinical trials remain in the early stages of development due to toxicity and side effects. Furthermore, the effectiveness and safety of COVID-19 vaccines against variants are currently highly uncertain, and even if effective, they only target a single strain of the virus. Therefore, developing highly effective, safe, and short-term anti-COVID-19 medications is crucial to combating the epidemic. Traditional Chinese Medicine (TCM) is a treasure of the Chinese nation and has made significant contributions to China's health sector. For the prevention and treatment of epidemics, TCM possesses a comprehensive system of syndrome differentiation and treatment, encompassing a rich set of theoretical frameworks and extensive technical approaches. Furthermore, TCM offers several distinct advantages in preventing and treating epidemics, including proven efficacy, minimal adverse reactions, and flexible formulations. TCM has achieved remarkable success in preventing and treating major epidemics and has accumulated extensive practical experience.

[0005] The novel coronavirus variant, "Omicron," is currently spreading widely around the world. It is characterized by strong transmissibility, high pathogenicity, a short incubation period, and a rapid onset of disease, and is rapidly spreading worldwide. Due to the emergence of the Omicron variant, some scholars have defined "novel coronavirus pneumonia" as "novel coronavirus disease." This is because many patients infected with the novel coronavirus strain, "Omicron," do not exhibit symptoms of pneumonia, primarily experiencing upper respiratory tract infections, with most infections being mild or asymptomatic. However, it is a disease state, and patients generally experience a slow transition from "positive" to "negative" nucleic acid test results. A large number of studies have shown that the Omicron variant has super strong immune escape characteristics (Sun, C., Kang, YF., Liu, YT. et al. Parallel profiling of antigenicity alteration and immuneescape of SARS-CoV-2 Omicron and other variants. Signal Transduction and Targeted Therapy, 2022, 7, 42). After the nucleic acid test is positive, the time it takes for the nucleic acid test results of its infected patients to turn negative during treatment is significantly higher than that of the wild-type new coronavirus strain, as well as alpha, beta and delta strains. Recent authoritative research data indicates that the new subtypes of the Omicron variants, BA.2.12.1, BA.4, and BA.5, exhibit enhanced immune evasion capabilities and exhibit significant neutralization escape in the plasma of recovered Omicron BA.1 patients. This makes achieving herd immunity through Omicron infection extremely difficult (Cao, Y., Yisimayi, A., Jian, F. et al. BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection. Nature (2022). https: / / doi.org / 10.1038 / s41586-022-04980-y). Therefore, there is an urgent need to develop a drug to treat respiratory infections caused by Omicron variants and shorten the time it takes for patients to transition from a positive nucleic acid test result to a negative result.

[0006] Traditional Chinese medicine formulas have been tested for thousands of years and are already clinically effective. If they can be rapidly and scientifically tested, they could be applied to patients worldwide earlier and more rapidly than new drugs and vaccines, potentially saving countless lives. More importantly, the Chinese medicines mentioned in ancient prescriptions target more than just one viral infection; they can treat a wide range of infectious diseases, including the novel coronavirus, as well as new upper respiratory infections that may emerge globally in the future. Therefore, the mass production and clinical application of these formulas are of paramount importance. Summary of the Invention

[0007] The purpose of the present invention is to provide a Chinese medicine decoction composition for treating novel coronavirus pneumonia in response to the above technical problems.

[0008] The purpose of the present invention can be achieved by the following solutions:

[0009] The present invention provides a traditional Chinese medicine composition for treating novel coronavirus pneumonia, comprising gypsum, qinglongyi, and dried lemon slices.

[0010] Preferably, the traditional Chinese medicine composition comprises 5-25 parts of gypsum, 5-15 parts of Qinglongyi, and 6-18 parts of dried lemon slices.

[0011] Preferably, the traditional Chinese medicine composition further comprises ephedra, ginger, poria, yam, scutellaria, licorice, atractylodes, dandelion, immature bitter orange, and tangerine peel.

[0012] Preferably, the traditional Chinese medicine composition comprises the following raw materials in parts by weight: 5-25 parts of gypsum, 3-9 parts of ephedra, 3-9 parts of ginger, 10-20 parts of poria, 5-15 parts of yam, 3-9 parts of scutellaria, 3-9 parts of roasted licorice, 5-15 parts of qinglongyi, 6-18 parts of dried lemon slices, 5-15 parts of atractylodes, 3-9 parts of dandelion, 3-9 parts of immature bitter orange, and 3-9 parts of dried tangerine peel.

[0013] In some embodiments, the traditional Chinese medicine is composed of the following raw materials in parts by weight: 15 parts of gypsum, 6 parts of ephedra, 6 parts of ginger, 15 parts of poria, 12 parts of yam, 6 parts of scutellaria, 6 parts of roasted licorice, 5-15 parts of green dragon skin, 12 parts of dried lemon slices, 9 parts of white atractylodes, 6 parts of dandelion, 6 parts of immature bitter orange, and 6 parts of dried tangerine peel.

[0014] Preferably, the Qinglongyi is the dried fleshy peel of Juglans mandshurica, a plant of the genus Juglans, which is washed and used as medicine after drying.

[0015] The present invention also provides a use of Qinglongyi in preparing a traditional Chinese medicine composition for treating pneumonia caused by the new coronavirus. The Qinglongyi is prepared from the dried fleshy peel of Juglans mandshurica, a plant of the genus Walnut, which is washed and dried before use as medicine.

[0016] The present invention also provides a use of a traditional Chinese medicine composition comprising gypsum, qinglongyi, and dried lemon slices in preparing a preparation for treating pneumonia caused by the new coronavirus.

[0017] The present invention also provides a method for preparing a decoction of a traditional Chinese medicine composition, the method comprising the following steps:

[0018] S1. Crush the weighed gypsum, add water and simmer, and soak the remaining weighed medicinal materials in water during this period; the ingredients of the Chinese medicine composition are: 5-25 parts of gypsum, 3-9 parts of ephedra, 3-9 parts of ginger, 10-20 parts of poria, 5-15 parts of yam, 3-9 parts of scutellaria, 3-9 parts of roasted liquorice, 5-15 parts of Qinglongyi, 6-18 parts of dried lemon slices, 5-15 parts of white atractylodes, 3-9 parts of dandelion, 3-9 parts of immature bitter orange, and 3-9 parts of dried tangerine peel;

[0019] S2. After the gypsum is decocted, the medicinal materials and the soaking liquid are poured in, and the mixture is boiled over high heat, and then decocted over low heat; the decoction is filtered to remove the residue to obtain the medicinal liquid, i.e., the Chinese medicine composition decoction.

[0020] Preferably, in step S2, after filtering the medicinal liquid, the medicinal residue is boiled with water, decocted over a low fire, and filtered to obtain the medicinal liquid, which is repeated several times, and the obtained medicinal liquids are combined to form the Chinese medicine composition decoction. The number of repetitions is preferably 1-2 times.

[0021] Preferably, in step S2, the simmering time is 30-40 minutes.

[0022] In some embodiments, the preparation method specifically comprises the following steps: crushing the weighed raw gypsum, adding 300 ml of water, and simmering for 30 minutes. During this time, soaking the remaining 12 herbs in 500 ml of water; after the gypsum is decocted, pouring the herbs and the soaking liquid into a clay pot, bringing to a boil over high heat, and then simmering for 30-40 minutes. The decoction is filtered and placed in a glass or ceramic container. 500 ml of water is then added to the clay pot, the herbs are boiled again, and then simmered for 30 minutes. The two decoctions are combined and filtered to obtain the medicinal liquid of the traditional Chinese medicine formula.

[0023] The present invention provides a method for extracting juglone, which comprises the following steps:

[0024] The Qinglongyi slices were crushed, soaked in ethanol solution, and then ultrasonically extracted; the extract was filtered, the residue was further added with ethanol solution for reflux, the extract was filtered again, the two extracts were combined, and the extract was concentrated under reduced pressure to an extract without alcohol taste; the extract was dispersed in water and extracted with petroleum ether, chloroform, ethyl acetate, and n-butanol in sequence, and the extracts were concentrated and recovered.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The two most critical antiviral herbs in this invention are Qinglongyi (green dragon hairtail) and Gypsum Fibrosum. Qinglongyi is bitter and astringent, neutral in nature, and enters the lung, spleen, stomach, and large intestine meridians; Gypsum Fibrosum is sweet and pungent, extremely cold, and enters the lung and stomach meridians. This treatment formula, while taking into account the underlying conditions, can effectively improve the condition of confirmed COVID-19 patients with symptoms of asthma, chest tightness, and dyspnea, dispelling cold and dampness, regulating qi and resolving phlegm, strengthening the spleen, and boosting immunity. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Other features, objects and advantages of the present invention will become more apparent upon reading the detailed description of non-limiting embodiments with reference to the following drawings:

[0028] Figure 1 It is the analysis of juglone H NMR spectrum;

[0029] Figure 2 Schematic diagram of juglone significantly inhibiting the novel coronavirus at a concentration of 2 μM;

[0030] Figure 3 This is a schematic diagram showing that the decoction of Qinglongyi, lemon slices and gypsum significantly inhibits the new coronavirus at a concentration of 0.5%. DETAILED DESCRIPTION

[0031] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings and specific embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0032] The present invention discloses a traditional Chinese medicine containing Qinglongyi, which is used to treat pneumonia caused by novel coronavirus infection and relates to the field of traditional Chinese medicine. The traditional Chinese medicine disclosed in the present invention for treating novel coronavirus pneumonia includes gypsum, ephedra, ginger, Poria, Chinese yam, scutellaria, roasted licorice, Qinglongyi, dried lemon slices, Atractylodes macrocephala, dandelion, immature bitter orange, and tangerine peel. The Qinglongyi is made from the dried fleshy peel of Juglans mandshurica, a plant of the genus Walnut, which is washed and used as medicine after drying. The traditional Chinese medicine disclosed in the present invention is composed of the following raw materials by weight: 15 grams of gypsum, 6 grams of ephedra, 6 grams of ginger, 15 grams of Poria, 12 grams of Chinese yam, 6 grams of scutellaria, 6 grams of roasted licorice, 5-15 grams of Qinglongyi, 12 grams of dried lemon slices, 9 grams of Atractylodes macrocephala, 6 grams of dandelion, 6 grams of immature bitter orange, and 6 grams of tangerine peel. The decoction method for the traditional Chinese medicine is as follows: crush the weighed raw gypsum, add 300 ml of water, and simmer for 30 minutes. During this time, soak the remaining 12 herbs in 500 ml of water. After the raw gypsum is completely decocted, pour the herbs and the soaking liquid into a clay pot, bring to a boil over high heat, and simmer for another 30-40 minutes. The decoction is filtered and placed in a glass or ceramic container. 500 ml of water is added to the clay pot again, and the herbs are boiled again, followed by a simmer for 30 minutes. The two decoctions are combined and filtered to obtain a traditional Chinese medicine liquid, which is taken twice daily. In the prescription disclosed herein, the two most critical herbs that have antiviral effects are Qinglongyi and raw gypsum. Qinglongyi has a bitter and astringent taste, is neutral in nature, and enters the lung, spleen, stomach, and large intestine meridians; gypsum has a sweet and pungent taste, is extremely cold in nature, and enters the lung and stomach meridians. This Chinese medicine formula can dispel cold and dampness, regulate qi and resolve phlegm, strengthen the spleen and enhance immunity.

[0033] Example 1

[0034] This example involves the extraction and separation of juglone (I) from Qinglongyi medicinal material, and the molecular formula is as follows:

[0035] .

[0036] The following steps are involved:

[0037] Ten kilograms of Qinglongyi slices were crushed to a 20-30 mesh size. The pieces were then soaked in 10 volumes of 95% ethanol for 24 hours and then ultrasonically extracted for 30 minutes (at a frequency of 40 kHz). The extract was filtered, and the residue was further refluxed with 10 volumes of 95% ethanol for 2 hours. The extract was filtered again, the residue discarded, and the two extracts combined. The extracts were concentrated under reduced pressure until the extract was free of alcohol, with the temperature not exceeding 40°C. The extract was then dispersed in water and extracted sequentially with petroleum ether, chloroform, ethyl acetate, and n-butanol. The extracts were concentrated and recovered, all at a temperature not exceeding 40°C. The extracts were dried and weighed.

[0038] The ethyl acetate extraction part was taken to separate and analyze the effective components. The concentrated ethyl acetate extract (150.0 g) was repeatedly purified by silica gel column chromatography, with chloroform-methanol as the eluent for gradient elution. The same components were combined by thin layer chromatography (TLC) tracking and inspection. The chloroform-methanol (200:1) fraction was chromatographed on a silica gel column, and eluted with petroleum ether-acetone (200:1) to obtain 210 mg of orange-yellow needle-shaped crystals. The melting point of the compound is 158°C~159°C, it is insoluble in cold water, slightly soluble in hot water, and soluble in ethyl acetate, ethanol, ether and chloroform. The molecular weight obtained by LC-MS is 174.2. It was analyzed by nuclear magnetic resonance hydrogen spectrum (attached Figure 1 ), confirming that the compound is juglone (I), 1 H NMR (400 MHz, CDCl3): δ 11.88 (s, 1H, OH), 7.67 –7.55 (m, 2H), 7.26 (dd, J = 7.9, 1.7 Hz, 1H), 6.94 (s, 2H).

[0039] Example 2

[0040] This example relates to the determination of the inhibitory activity of juglone on the SARS-CoV-2 novel coronavirus 3CL protease, comprising the following steps:

[0041] The enzyme inhibitory activity of the compounds was determined using the fluorescence resonance energy transfer method reported in the literature (Jin et al. 2020. Structure of Mprofrom SARS-CoV-2 and discovery of its inhibitors. Nature, 582: 289–293). The catalytic activity and initial velocity of 3CL enzymes were determined by enzyme kinetics using the commercially available fluorescently labeled peptide MCA-AVLQSGFR-Lys(Dnp)-Lys-NH2 as a substrate (GL Biochem, Shanghai). The incubation system contained SARS-CoV-2 3CL protease (0.2 μM), the fluorescently labeled peptide (20 μM), and a series of test compound concentrations (0–10 μM). Fluorescence intensity was measured after 3 minutes of incubation using a microplate reader with excitation and detection wavelengths of 320 nm and 405 nm, respectively. The enzyme inhibition rate of the test compound at different concentrations was calculated based on the change in the initial velocity of substrate hydrolysis after the addition of the inhibitor. We determined the inhibitory activity of juglone, obtained by the extraction and separation method described in Example 1, against the novel coronavirus 3CL protease. In the enzyme inhibition activity assay, shikonin was used as a positive control. The results are shown in Table 1.

[0042] The results showed that the enzyme inhibition rate of the positive control shikonin at a concentration of 10 μM was 51.4%. At a concentration of 1 μM, the enzyme inhibition rate of juglone reached 99%, with the inhibitory activity being much higher than that of the positive control drug shikonin.

[0043] Table 1 Inhibitory activity of compounds against novel coronavirus 3CL enzyme

[0044]

[0045] Example 3

[0046] This example relates to the evaluation of the inhibitory effect of juglone on the binding of the S protein of the wild-type strain of the new coronavirus to the ACE receptor of the host cell.

[0047] Experimental principle:

[0048] First, human embryonic kidney cells (HEK293T) with high expression of the ACE2 receptor were established by transfecting a plasmid with an HIV lentiviral vector. Using an HIV lentiviral vector and the SARS-CoV-2 spike protein (S protein), the lentiviral envelope protein VSVG was replaced with the SARS-CoV-2 S protein. This was then co-transfected into 293T cells with a lentiviral packaging plasmid and a CMV-GFP-T2A-Luciferase plasmid to generate a SARS-CoV-2 S pseudovirus. This pseudovirus expresses the SARS-CoV-2 S protein on its surface and carries a GFP fluorescent reporter gene. Fluorescence intensity can be used to assess the activity of the pseudovirus in infecting cells. This pseudovirus lacks autonomous replication and is highly safe, making it suitable for drug screening against the SARS-CoV-2 receptor. Using a similar pseudovirus model, Li et al. determined the pseudovirus infection rate by measuring fluorescence intensity and identified drugs with anti-pseudovirus activity. The antiviral activity of this drug was also confirmed in a real virus infection model, indicating that the compound could be used to treat SARS-CoV-2 infection. The results shown by the compounds in the pseudovirus model are consistent with those in the real virus model (Li H, Cheng C, Li S, et al. Discovery and Structural Optimization of 3-O-β-Chacotriosyl Oleanane-type Triterpenoids as Potent Entry Inhibitors of SARS-CoV-2 Virus Infections[J]. European Journal of Medicinal Chemistry, 2021, 215:113242). This proves that the compounds screened using the pseudovirus model and their assay results can reflect the anti-real virus effect, and compounds that are effective against pseudoviruses can also be used to actually treat the new coronavirus.

[0049] Experimental methods:

[0050] HEK293T cells were used for pseudovirus S protein infection studies. HEK293T cells were trypsinized and plated at a density of 100,000 cells / mL. 100 μL was added to each well. After 24 hours, the supernatant was aspirated and the cells were administered with 92 μL of culture medium containing juglone (final concentration 2 μM) in dimethyl sulfoxide (DMSO) at a concentration of 0.5%. For the positive control, 92 μL of culture medium containing an equal volume of DMSO was added and mixed thoroughly. An equal volume of pseudovirus (8 μL per well after 20-fold dilution) was added to each well and mixed again. After 24 hours, the supernatant was aspirated and 200 μL of culture medium containing the same concentration of compound as in the original well was added. After an additional 48 hours, the cells were digested, diluted with phosphate-buffered saline (PBS), centrifuged at 1600 r / min for 10 minutes, and the supernatant was removed. Each sample was resuspended in 200 μL of PBS. FITC fluorescence was measured by flow cytometry to determine the proportion of positive cells. The inhibition rate was calculated according to the following formula: Inhibition rate (%) = (cell infection rate of the drug-free control group - cell infection rate of the drug-treated group) / cell infection rate of the drug-free control group × 100%.

[0051] After using the wild-type pseudovirus infection experiment of the new coronavirus, it was found that compared with the positive control group, the proportion of positive cells in the experimental group with added juglone decreased from 24.02% to 17.28%, and the virus inhibition rate was 28.06%.

[0052] Example 4

[0053] This example relates to an in vitro test of the activity of juglone against the novel coronavirus.

[0054] Experimental methods:

[0055] Vero E6 cells with good growth status were counted and seeded into 96-well plates at 3x104 cells per well.

[0056] The cells were cultured in a 37°C cell culture incubator for 24 hours to allow them to adhere. The cells were pretreated with juglone (2 μM) for 1 hour, and the supernatant was discarded. Subsequently, 2% DMEM medium containing juglone (2 μM) and 2019-nCoV virus (MOI parameter set to 0.01) was added and the cells were cultured for 2 hours. Subsequently, the supernatant containing the virus and juglone was discarded, the cells were washed twice with 1× PBS, and freshly prepared DMEM medium containing juglone (2 μM) was added and cultured for another 48 hours. After 48 hours, the cell culture supernatant was collected and trizol was added to remove the viral nucleic acid. Viral RNA was extracted from the supernatant culture medium using a kit (Qiagen, Germany), and the viral copy number (N protein) in the supernatant was detected by qRT-PCR analysis. A juglone test group (A) and a corresponding blank control group (the blank control group only added a solvent containing dissolved juglone, B) were set up in the test.

[0057] Experimental results:

[0058] The experimental results are as follows Figure 2 As shown in the results, juglone at a concentration of 2 μM can significantly inhibit the replication of the new coronavirus in VeroE6 cells, with a virus inhibition rate of more than 50%.

[0059] Example 5

[0060] This embodiment relates to the mechanism of action of Qinglongyi prescription in treating novel coronavirus pneumonia:

[0061] Qinglongyi is the dried fleshy peel of the Juglans mandshurica plant, a member of the Juglandaceae family. Qinglongyi has a neutral medicinal property, with a bitter and astringent taste. Its chemical components include polyphenols, terpenes, diarylheptanoids, flavonoids, and quinones. Quinones and flavonoids are the primary active ingredients. Traditional Chinese medicine theory and experience confirm that Qinglongyi has anti-inflammatory, analgesic, detoxifying, and swelling-reducing properties, and is used to treat stomach and abdominal pain, dysentery, carbuncles, sores, and stubborn ringworm. The "Fangmai Zhengzong" (Fangmai Zhengzong) records that Qinglongyi can treat persistent dysentery. The original text reads: "Take one liang of green walnut peel. Crush it, simmer in an iron pan, and then pound it again. Take three qian every morning with white soup." Qinglongyi can be taken orally as a decoction, with a typical daily dosage of 5 to 15 grams. For external use, a suitable amount of Qinglongyi mixed with sulfur can be applied to the affected area to treat vitiligo. Furthermore, there are no reports of toxic effects of Qinglongyi, nor have there been reports of serious adverse reactions when taken at the prescribed dosage (Yu Chuanlong, Huang Zhengming, Xiu Chengjuan, et al. Chinese Clinical Drug Dictionary: Chinese Herbal Medicine Pieces Volume. Beijing: China Medical Science and Technology Press, 2018). Modern pharmacological studies have shown that Qinglongyi has anti-inflammatory, analgesic, and anti-tumor effects. Juglone, the main naphthoquinone component in Qinglongyi, has strong inhibitory activity against the main protease of the novel coronavirus. It also blocks the viral S protein and inhibits its binding to the ACE2 receptor, demonstrating significant antiviral activity. The antiviral activity of juglone and other active ingredients underlies the anti-COVID-19 activity of Qinglongyi extract. Juglone binds to serum albumin at a rate of over 99.0%. When formulated into a nanoformulation, the compound exhibits a half-life of nearly 24 hours upon intravenous administration, demonstrating excellent pharmacokinetic properties and in vivo stability.

[0062] Example 6

[0063] This embodiment relates to an in vitro activity test of a decoction of Qinglongyi, lemon slices and gypsum against the true novel coronavirus.

[0064] Experimental methods:

[0065] Crush 15 grams of gypsum and add 300 milliliters of water. Simmer gently for 30 minutes. Meanwhile, soak 10 grams of Qinglongyi (green dragon hair) and 12 grams of dried lemon slices in 500 milliliters of water. Once the gypsum is cooked, pour the herbs and soaking liquid into a casserole, bring to a boil over high heat, and simmer for 30-40 minutes. Filter the decoction and set aside.

[0066] Vero E6 cells that are growing well were counted at 3x10 4 Each well was inoculated into a 96-well plate.

[0067] The cells were cultured in a 37°C cell culture incubator for 24 hours to allow them to adhere. The cells were pretreated with medium containing a decoction of medicinal herbs (0.5%, v / v) for 1 hour, and the supernatant was discarded. Subsequently, DMEM medium containing a decoction of Qinglongyi, lemon slices, and gypsum (0.5%, v / v) and the 2019-nCoV virus (MOI parameter set at 0.01) was added and the cells were cultured for 2 hours. The supernatant containing the virus and decoction was then discarded, the cells were washed twice with 1× PBS, and fresh DMEM medium containing the decoction (0.5%, v / v) was added for a further 48 hours. After 48 hours, the cell culture supernatant was collected and trizol was added to remove viral nucleic acid. Viral RNA was extracted from the supernatant using a kit (Qiagen, Germany), and the viral copy number (N protein) in the supernatant was determined by qRT-PCR analysis. A test group (C) and a corresponding blank control group (the blank control group only received sterile water, D) were set up in the test group.

[0068] Experimental results:

[0069] The experimental results are as follows Figure 3 As shown in the results, the decoction of Qinglongyi, lemon slices and gypsum at a concentration of 0.5% can significantly inhibit the replication of the new coronavirus in Vero E6 cells, with a virus inhibition rate of more than 60%.

[0070] Example 7

[0071] This embodiment relates to an in vitro activity test of a decoction of Qinglongyi, lemon slices and gypsum against the true novel coronavirus.

[0072] Experimental methods:

[0073] Crush 5 grams of raw gypsum and add 300 milliliters of water. Simmer gently for 30 minutes. Meanwhile, soak 5 grams of Qinglongyi (green dragon hair) and 6 grams of dried lemon slices in 500 milliliters of water. Once the gypsum is cooked, pour the herbs and soaking liquid into a casserole, bring to a boil over high heat, and simmer for 30-40 minutes. Filter the decoction and set aside.

[0074] Vero E6 cells that are growing well were counted at 3x10 4Each well was inoculated into a 96-well plate.

[0075] The cells were cultured in a 37°C cell culture incubator for 24 hours to allow them to adhere. The cells were pretreated with medium containing a decoction of medicinal herbs (0.5%, v / v) for 1 hour, and the supernatant was discarded. Subsequently, DMEM medium containing a decoction of Qinglongyi, lemon slices, and gypsum (0.5%, v / v) and the 2019-nCoV virus (MOI parameter set to 0.01) was added and the cells were cultured for 2 hours. The supernatant was then removed by aspiration to remove the virus and decoction, and the cells were washed twice with 1× PBS. Fresh DMEM medium containing the decoction (0.5%, v / v) was added and cultured for a further 48 hours. After 48 hours, the cell culture supernatant was collected and trizol was added to remove viral nucleic acid. Viral RNA was extracted from the supernatant using a kit (Qiagen, Germany), and the viral copy number (N protein) in the supernatant was determined by qRT-PCR analysis. A test group (C) and a corresponding blank control group (the blank control group only contained sterile water, D) were set up in the test.

[0076] Experimental results:

[0077] The experimental results showed that a decoction of 5 grams of Qinglongyi, 6 grams of lemon slices and 5 grams of gypsum at a concentration of 0.5% can significantly inhibit the replication of the new coronavirus in Vero E6 cells, with a virus inhibition rate of 52%.

[0078] Example 8

[0079] This embodiment relates to an in vitro activity test of a decoction of Qinglongyi, lemon slices and gypsum against the true novel coronavirus.

[0080] Experimental methods:

[0081] Crush 25g of gypsum and add 300ml of water. Simmer gently for 30 minutes. Meanwhile, soak 15g of Qinglongyi (green dragon hair) and 18g of dried lemon slices in 500ml of water. Once the gypsum is cooked, pour the herbs and soaking liquid into a casserole, bring to a boil over high heat, and simmer for 30-40 minutes. Filter the decoction and set aside.

[0082] Vero E6 cells that are growing well were counted at 3x10 4 Each well was inoculated into a 96-well plate.

[0083] The cells were cultured in a 37°C cell culture incubator for 24 hours to allow them to adhere. The cells were pretreated with medium containing a decoction of medicinal herbs (0.5%, v / v) for 1 hour, and the supernatant was discarded. Subsequently, DMEM medium containing a decoction of Qinglongyi, lemon slices, and gypsum (0.5%, v / v) and the 2019-nCoV virus (MOI parameter set to 0.01) was added and the cells were cultured for 2 hours. The supernatant was then removed by aspiration to remove the virus and decoction, and the cells were washed twice with 1× PBS. Fresh DMEM medium containing the decoction (0.5%, v / v) was added and cultured for a further 48 hours. After 48 hours, the cell culture supernatant was collected and trizol was added to remove viral nucleic acid. Viral RNA was extracted from the supernatant using a kit (Qiagen, Germany), and the viral copy number (N protein) in the supernatant was determined by qRT-PCR analysis. A test group (C) and a corresponding blank control group (the blank control group only contained sterile water, D) were set up in the test.

[0084] Experimental results:

[0085] The experimental results show that a decoction of 15 grams of Qinglongyi, 18 grams of lemon slices and 25 grams of gypsum at a concentration of 0.5% can significantly inhibit the replication of the new coronavirus in Vero E6 cells, with a virus inhibition rate of over 85%.

[0086] Example 9

[0087] In a specific embodiment of the present invention, a traditional Chinese medicine for treating the novel coronavirus is composed of the following ingredients by weight: 15 grams of gypsum, 6 grams of ephedra, 6 grams of ginger, 15 grams of poria, 12 grams of yam, 6 grams of scutellaria, 6 grams of roasted licorice, 10 grams of qinglongyi, 12 grams of dried lemon slices, 9 grams of atractylodes, 6 grams of dandelion, 6 grams of immature bitter orange, and 6 grams of dried tangerine peel. The decoction method of the traditional Chinese medicine is as follows: crush the weighed gypsum, add 300 milliliters of water and simmer for 30 minutes. During this time, soak the remaining 12 herbs in 500 milliliters of water. After the gypsum is decocted, pour the herbs and the soaking liquid into a casserole, boil over high heat, and then simmer for 30-40 minutes. Filter the decoction and place it in a glass or ceramic container. Add 500 milliliters of water to the casserole, boil the herbs again, and simmer for 30 minutes. The two decoctions are combined and filtered to obtain a Chinese medicine solution, which is taken twice a day.

[0088] Modern pharmacological research of the medicine in this embodiment:

[0089] As described in Example 6, a decoction of juglone, lemon slices, and gypsum exhibited significant anti-novel coronavirus activity. Polysaccharides from Ephedra sinica, administered orally at a dose of 400 mg / kg to mice with inflammation, significantly reduced the levels of inflammatory cytokines IL-8, IL-6, TNF-β, and MMP-9 in the lung tissue. In an inflammatory model, dandelion extract similarly reduced the expression of cytokines such as IL-1β, IL-6, and TNF-α.

[0090] A cytokine storm, including those containing IL-1, IL-8, IL-6, and TNF-α, is the primary cause of pathological damage in severe COVID-19 patients. After the novel coronavirus invades lung epithelial cells, the infected cells promote the release of numerous chemokines and cytokines, creating a positive feedback loop. Interleukins IL-6, IL-8, and tumor necrosis factor β (TNF-β) released by cells at the site of infection recruit lymphocytes and monocytes / macrophages to the site of infection, further releasing inflammatory factors. The cytokine storm releases large amounts of nitric oxide, further damaging blood vessels and causing septic shock. It also triggers tissue fibrosis in mechanically ventilated patients, contributing to lung damage in COVID-19 patients. Active ingredients in medicinal herbs such as ephedra and dandelion can significantly inhibit the expression of inflammatory cytokines, providing a protective effect at the site of inflammation.

[0091] Comparative Example 1

[0092] This comparative example involves an in vitro activity test of a decoction of Qinglongyi and lemon slices against the true novel coronavirus.

[0093] Experimental methods:

[0094] Soak 10g of Qinglongyi (green dragon hair) and 30g of dried lemon slices in 500ml of water for 30 minutes. Pour the herbs and the soaking liquid into a casserole, bring to a boil over high heat, and simmer for 30-40 minutes. Filter the decoction and set aside.

[0095] Vero E6 cells with good growth status were counted and seeded into 96-well plates at 3x104 cells per well.

[0096] The cells were cultured in a 37°C cell culture incubator for 24 hours to allow them to adhere. The cells were pretreated with medium containing a decoction of medicinal herbs (0.5%, v / v) for 1 hour, and the supernatant was discarded. Subsequently, DMEM medium containing a decoction of Qinglongyi and lemon slices (0.5%, v / v) and the 2019-nCoV virus (MOI parameter set to 0.01) was added and the cells were cultured for 2 hours. The supernatant was then removed by aspiration to remove the virus and decoction, and the cells were washed twice with 1× PBS. Fresh DMEM medium containing the decoction (0.5%, v / v) was added and cultured for a further 48 hours. After 48 hours, the cell culture supernatant was collected and trizol was added to remove viral nucleic acid. Viral RNA was extracted from the supernatant using a kit (Qiagen, Germany), and the viral copy number (N protein) in the supernatant was determined by qRT-PCR analysis. A test group (C) and a corresponding blank control group (the blank control group only contained sterile water, D) were set up in the test.

[0097] Experimental results:

[0098] The experimental results, shown in Table 2, show that a decoction of Qinglongyi and lemon slices at a concentration of 0.5% inhibited the replication of the novel coronavirus in Vero E6 cells, with a viral inhibition rate of 49%. The preparation and preparation method of this comparative example are essentially the same as those in Example 6, differing only in that gypsum is omitted from the medicinal formula, and 10 grams of Qinglongyi and 30 grams of dried lemon slices are used. At the same concentrations, the antiviral activity of the decoction in this comparative example was lower than that of the formula in Example 6 (15 grams of gypsum, 10 grams of Qinglongyi, and 12 grams of dried lemon slices).

[0099] Table 2 Antiviral activity of decoction preparations of different compositions against the novel coronavirus

[0100]

[0101] Comparative Example 2

[0102] This comparative example relates to an in vitro activity test of a decoction of gypsum and qinglongyi against the true novel coronavirus.

[0103] Experimental methods:

[0104] Crush 30 grams of raw gypsum and add 300 milliliters of water. Simmer for 30 minutes. During this time, soak 10 grams of Qinglongyi in 500 milliliters of water. After the gypsum is cooked, pour the herbs and the soaking liquid into a casserole, bring to a boil over high heat, and simmer for 30-40 minutes. Filter the decoction and set aside.

[0105] Vero E6 cells that are growing well were counted at 3x10 4 Each well was inoculated into a 96-well plate.

[0106] The cells were cultured in a 37°C cell culture incubator for 24 hours to allow them to adhere. The cells were pretreated with medium containing a decoction of the medicinal herbs (0.5%, v / v) for 1 hour, and the supernatant was discarded. Subsequently, a decoction of Qinglongyi and Gypsum (0.5%, v / v) and the 2019-nCoV virus (MOI parameter set to 0.01) in DMEM medium were added and the cells were cultured for 2 hours. The supernatant containing the virus and the decoction was then discarded, the cells were washed twice with 1× PBS, and fresh DMEM medium containing the decoction (0.5%, v / v) was added for a further 48 hours. After 48 hours, the cell culture supernatant was collected and trizol was added to remove viral nucleic acid. Viral RNA was extracted from the supernatant using a kit (Qiagen, Germany), and the viral copy number (N protein) in the supernatant was determined by qRT-PCR analysis. A test group (C) and a corresponding blank control group (the blank control group only contained sterile water, D) were set up in the test.

[0107] Experimental results:

[0108] The experimental results, shown in Table 2, show that a decoction of Qinglongyi and gypsum at a concentration of 0.5% inhibited the replication of the novel coronavirus in Vero E6 cells, with a viral inhibition rate of 45%. The preparation and preparation method of this comparative example are essentially the same as those in Example 6, differing only in that dried lemon slices were omitted and 10 grams of Qinglongyi and 30 grams of gypsum were used. At the same concentrations, the antiviral activity of the decoction in this comparative example was lower than that of the formulation in Example 6 (15 grams of gypsum, 10 grams of Qinglongyi, and 12 grams of dried lemon slices).

[0109] Comparative Example 3

[0110] The preparation method of this comparative example is basically the same as that of Example 6, except that gypsum is replaced with sodium sulfate, and the formula used is 15 grams of sodium sulfate, 10 grams of Qinglongyi, and 12 grams of dried lemon slices. The antiviral activity of the decoction is tested using the same test method as in Example 6.

[0111] The experimental results, shown in Table 2, show that a decoction containing a sodium sulfate formula at a concentration of 0.5% inhibited the replication of the novel coronavirus in Vero E6 cells, with a viral inhibition rate of 30%. The preparation and preparation method of this comparative example are essentially the same as those in Example 6, except that gypsum was replaced with sodium sulfate. The decoction used was 15 grams of sodium sulfate, 10 grams of qinglongyi, and 12 grams of dried lemon slices. At the same concentrations, the antiviral activity of the decoction in this comparative example was lower than that of the formula in Example 6 (15 grams of gypsum, 10 grams of qinglongyi, and 12 grams of dried lemon slices).

[0112] Comparative Example 4

[0113] The preparation and preparation method of this comparative example are essentially the same as those in Example 6, differing only in that lemon slices are replaced with astragalus slices. The formulation employed is composed of 15 g of gypsum, 10 g of qinglongyi, and 12 g of astragalus slices. The decoction was tested for antiviral activity using the same testing method as in Example 6.

[0114] The experimental results, shown in Table 2, show that a decoction containing the Astragalus root formula at a concentration of 0.5% inhibited the replication of the novel coronavirus in Vero E6 cells, with a viral inhibition rate of 37%. The preparation and preparation method of this comparative example are essentially the same as those in Example 6, except that dried lemon slices are replaced with Astragalus root slices. 15 grams of gypsum, 10 grams of Qinglongyi, and 12 grams of Astragalus root slices are used. At the same concentrations, the decoction of this comparative example exhibited lower antiviral activity than the formula described in Example 6 (15 grams of gypsum, 10 grams of Qinglongyi, and 12 grams of dried lemon slices).

[0115] Comparative Example 5

[0116] The preparation and preparation method of this comparative example are essentially the same as those in Example 6, differing only in that Qinglongyi is replaced with rhubarb. The formulation employed is composed of 15 grams of gypsum, 10 grams of rhubarb, and 12 grams of dried lemon slices. The decoction was tested for antiviral activity using the same testing method as in Example 6.

[0117] The experimental results are shown in Table 2. A decoction containing the rhubarb formula at a concentration of 0.5% inhibited the replication of the novel coronavirus in Vero E6 cells, with a viral inhibition rate of 11%. The preparation and preparation method of this comparative example are essentially the same as those in Example 6, except that the naphthoquinone-containing Qinglongyi (Qinglongyi) is replaced with anthraquinone-containing rhubarb (Qinglongyi), and 15 grams of gypsum, 10 grams of rhubarb, and 12 grams of dried lemon slices are used. At the same concentrations, the decoction of this comparative example exhibited lower antiviral activity than the formula described in Example 6 (15 grams of gypsum, 10 grams of Qinglongyi, and 12 grams of dried lemon slices).

[0118] The preferred embodiments of the present invention disclosed above are intended only to help illustrate the present invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the present invention to the specific embodiments described. Obviously, many modifications and variations are possible based on the content of this specification. These embodiments are selected and described in detail in this specification to better explain the principles and practical applications of the present invention, thereby enabling those skilled in the art to better understand and utilize the present invention. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A Chinese medicine composition for use against the new coronavirus, characterized in that: The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 5-25 parts of gypsum, 5-15 parts of qinglongyi, and 6-18 parts of dried lemon slices.

2. The Chinese medicine composition according to claim 1, characterized in that The Qinglongyi is the dried fleshy peel of the Juglans mandshurica plant of the genus Juglans, which is washed and used as medicine after drying.

3. Use of the traditional Chinese medicine composition according to claim 1 in preparing a preparation for resisting the new coronavirus.

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