CA3166282A1 - Use of pharmaceutical composition for preventing and treating novel coronavirus pneumonia - Google Patents

Abstract

Use of a pharmaceutical composition in the preparation of a medicament for preventing and treating novel coronavirus-infected pneumonia. The pharmaceutical composition is prepared from forsythia, honeysuckle, isatis root, Rheum officinale, Pogostemon cablin, Rhizoma Dryopteris Crassirhizomae, Rhodiola rosea, menthol, honey-fried Herba ephedra, fried Semen Armeniacae Amarae, Houttuynia cordate, liquorice and gypsum fibrosum. It is experimentally verified that the pharmaceutical composition has the effect of preventing and treating novel coronavirus-infected pneumonia.

Description

USE OF PHARMACEUTICAL COMPOSITION FOR PREVENTING AND TREATING
NOVEL CORONAVIRUS PNEUMONIA
[0001] This application claims the priority of Chinese Patent Application No.
202010079857.2 filed with the China National Intellectual Property Administration on February 4, 2020, Chinese Patent Application No. 202010094502.0 filed with the China National Intellectual Property Administration on February 16, 2020, Chinese Patent Application No.
202010105903.1 filed with the China National Intellectual Property Administration on February 21, 2020 and Chinese Patent Application No. 202010134508.6 filed with the China National Intellectual Property Administration on March 2, 2020, which are hereby incorporated by reference in entirety.
FIELD

[0002] The present disclosure relates to the use of a pharmaceutical composition in the manufacture of a medicament for preventing and treating pneumonia caused by novel coronavirus, and belongs to the field of medicine.
BACKGROUND

[0003] On January 12, 2020, the World Health Organization (WHO) officially named the novel coronavirus as "2019 novel coronavirus (2019-nCoV)". According to the research results published in the Lancet, sequencing and analyzing 2019-nCoV pathogen from patient samples revealed that the 2019-nCoV was genetically different from the virus causing severe acute respiratory syndrome (SARS) that was prevalent in 2003 and the virus causing middle east respiratory syndrome (MERS) discovered in 2012. Phylogenetic analysis showed that the novel coronavirus belongs to the subtype sarbecovirus of the genus betacoronavirus and genetically distant from the SARS virus.

[0004] It is reported that this coronavirus is an enveloped positive-sense single-stranded RNA
virus. The pathogen 2019-nCoV and its genome sequence have been identified, which has laid an important foundation for epidemic prevention work.

[0005] Novel coronavirus-infected pneumonia is also known as novel coronavirus pneumonia (NCP) or coronavirus disease 2019 (COVID-19). Its clinical manifestations typically include fever, fatigue, and dry cough. Few patients further have accompanying symptoms such as nasal obstruction, runny nose, sore throat and diarrhea. Severe patients may have breathing difficulties or die.

[0006] The disease is highly contagious. Some researchers have estimated the "basic reproductive number" (RO) of this epidemic as 2.2 by using a model.

[0007] Currently existing antiviral drugs such as Tamiflu (oseltamivir phosphate) do not have a good therapeutic effect on COVID-19. Oseltamivir is a neuraminidase inhibitor, which targets the neuraminidase component on the cell membrane of influenza A and B to exert corresponding effects. However, 2019-nCoV lacks neuraminidase, so Tamiflu is not effective in treating COVID-19 .

[0008] Clinical manifestations of COVID-19 disease mainly include fever, fatigue and dry cough, and a few patients have accompanying symptoms such as nasal obstruction, runny nose, sore throat and diarrhea. In severe cases, most patients develop dyspnea after a week, and severe patients rapidly develop acute respiratory distress syndrome, septic shock, refractory metabolic acidosis, or coagulopathy. Some patients only show low fever, mild fatigue, etc., without pneumonia, and mostly will recover after 1 week.

[0009] Laboratory tests founded that in the early stage of the COVID-19 disease, the total number of leukocytes is normal or decreases or the lymphocyte count decreases, some patients have increased liver enzymes, muscle enzymes or myoglobin, and most patients have increased C-reactive protein or erythrocyte sedimentation rate.

[0010] Early chest imaging founded that the patients show multiple small patchy opacities and interstitial changes, especially in the extrapulmonary zone, and then develop into multiple ground-glass opacities or infiltration shadows in both lungs, or lung consolidation in severe cases.

[0011] Sputum, throat swabs, lower respiratory tract secretions and other samples can be used to test for 2019-nCoV nucleic acid positive by real-time fluorescent RT-PCR.

[0012] Clinical classification of the C OVID-19 disease:

[0013] I. Common type, having fever or respiratory tract symptoms and showing pneumonia on imaging.

[0014] II. Severe type, meeting any of the following: 1. Respiratory distress, RR? 30 times/min;
oxygen saturation < 93% in resting state; partial pressure of oxygen in arterial blood (Pa02)/
fraction of inspired oxygen (Fi02) < 300 mmHg).

[0015] III. Critical type, showing respiratory failure and needing mechanical ventilation; having shock; or having other combined organ failure and needing ICU monitoring and treatment.

[0016] Treatment of the COVID-19 disease:

[0017] 1. General treatment: bed rest with vital signs and oxygen saturation under monitoring;
supportive and symptomatic treatment; ensuring heat and maintaining stable internal environment such as water, electrolyte and acid-base balance.

[0018] 2. Oxygen treatment: perform oxygen treatment immediately for patients with hypoxemia, to maintain oxygen saturation Sp02 > 90% in non-pregnant adult patients or Sp02 > 92-95% in pregnant patients.

[0019] 3. Antiviral treatment: try aerosol inhalation of alpha-interferon (5 million U each time for adults, addition of 2m1 of sterile water for injection, twice a day);
lopinavir/ritonavir (200mg/50mg, per capsule), 2 capsules each time, 2 times a day.

[0020] 4. Antibacterial drug treatment: avoid blind or inappropriate trial of antibiotics, especially combined use of broad-spectrum antibiotics.

[0021] 5. Other treatment measures: according to the degree of dyspnea and the development of chest imaging, use glucocorticoids for a short period as appropriate, at a dose of 1-2 mg/kg.d in the case of methylprednisolone, or at 100m1/d in the case of intravenously administrating Xuebijing twice a day; or use intestinal microecological regulators to maintain intestinal microecological balance.

[0022] However, there still lacks effective treatment or preventive medicine against the COVID-19, which has been raging around the world. Although some drugs been recommended worldwide to treat COVID-19, they are basically emergency or compassionate drugs, stilling having many deficiencies. For example, broad-spectrum antiviral drugs have no obvious activity against COVID-19, and many drugs for symptomatic treatment of viral pneumonia are also less effective.

Some other drugs may inhibit the virus but have obvious toxicity. Therefore, there is an urgent need for safe and effective drugs for the treatment of COVID-19.

[0023] The present disclosure is an improved invention based on the disclosure of the patent CN1194752C, the contents of which are incorporated herein in its entirety. The above- mentioned patent does not disclose the use of the pharmaceutical composition of this disclosure in the prevention and treatment of patients with novel coronavirus-infected pneumonia.
SUMMARY

[0024] The inventor unexpectedly found in research that the pharmaceutical composition of this disclosure can effectively prevent and treat novel coronavirus-infected pneumonia, and the pharmaceutical composition is prepared from the following materials in parts by weight:
forsythiae fructus 150-260 lonicerae japonicae flos 150-isatidis radix 150-260 rhei radix et rhizoma 30-55 pogostemonis herba 50-90 dryopteridis crassirhizomatis rhizoma 150-260 rhodiolae crenulatae radix et rhizoma 50-90 /-menthol 5-8 honey-fried ephedrae herba 50-90 stir-baked armeniacae semen amarum 50-90 houttuyniae herba 150-260 glycyrrhizae radix et rhizoma gypsum fibrosum 150-260.

[0025] On the basis of this discovery, in the first aspect, the present disclosure provides use of a pharmaceutical composition in the manufacture of a medicament for preventing and treating novel coronavirus-infected pneumonia, and the pharmaceutical composition is prepared from the following materials in parts by weight:
forsythiae fructus 150-260 lonicerae japonicae flos 150-isatidis radix 150-260 rhei radix et rhizoma 30-55 pogostemonis herba 50-90 dryopteridis crassirhizomatis rhizoma 150-260 rhodiolae crenulatae radix et rhizoma 50-90 1-menthol 5-8 honey-fried ephedrae herba 50-90 stir-baked armeniacae semen amarum 50-90 houttuyniae herba 150-260 glycynhizae radix et rhizoma gypsum fibrosum 150-260.

[0026] The pharmaceutical composition is preferably prepared from the following materials in parts by weight:
forsythiae fructus 150 lonicerae japonicae flos 260 isatidis radix 150 rhei radix et rhizoma 55 pogostemonis herba 50 dryopteridis crassirhizomatis rhizoma 260 rhodiolae crenulatae radix et rhizoma 50 1-menthol 8 honey-fried ephedrae herba 50 stir-baked armeniacae semen amarum houttuyniae herba 150 glycyrrhizae radix et rhizoma 90 gypsum fibrosum 150.

[0027] The pharmaceutical composition is also preferably prepared from the following materials in parts by weight:
forsythiae fructus 260 lonicerae japonicae flos 150 isatidis radix 260 rhei radix et rhizoma 30 pogostemonis herba 90 dryopteridis crassirhizomatis rhizoma 150 rhodiolae crenulatae radix et rhizoma 90 1-menthol 5 honey-fried ephedrae herba 90 stir-baked an-neniacae semen amarum houttuyniae herba 260 glycynhizae radix et rhizoma 50 gypsum fibrosum 260.

[0028] The pharmaceutical composition is also preferably prepared from the following materials in parts by weight:
forsythiae fructus 170 lonicerae japonicae flos 170 isatidis radix 170 rhei radix et rhizoma 34 pogostemonis herba 57 dryopteridis crassirhizomatis rhizoma 170 rhodiolae crenulatae radix et rhizoma 57 /-menthol 5 honey-fried ephedrae herba 57 stir-baked armeniacae semen amarum houttuyniae herba 170 glycynhizae radix et rhizoma 57 gypsum fibrosum 170.

[0029] The pharmaceutical composition is also preferably prepared from the following materials in parts by weight:
forsythiae fructus 255 lonicerae japonicae flos 255 isatidis radix 255 rhei radix et rhizoma 51 pogostemonis herba 85 dryopteridis crassirhizomatis rhizoma 255 rhodiolae crenulatae radix et rhizoma 85 /-menthol 7.5 honey-fried ephedrae herba 85 stir-baked anneniacae semen amarum houttuyniae herba 255 glycynhizae radix et rhizoma 85 gypsum fibrosum 255.

[0030] The active ingredients of the pharmaceutical composition of this disclosure may be prepared by:

[0031] step (1) weighing the materials according to weight ratio and performing selection to remove impurities;

[0032] step (2) chopping pogostemonis herba, extracting volatile oil from the pogostemonis herba with water having amount of 5-8 times of the pogostemonis herba for 4 h to collect the volatile oil and obtain an extract liquid, and filtering the extract liquid to discard residue and obtain filtrate;

[0033] step (3) performing extraction on a mixture of forsythiae fructus, honey-fried ephedrae herba, houttuyniae herba and rhei radix et rhizoma twice for 1-3 h each time by using 50-90%
ethanol in an amounts of 6-10 times of the mixture to obtain extract liquids, combining and filtering the extract liquids, distilling the filtrate to recover ethanol and obtain alcohol extract;

[0034] step (4) to a mixture of lonicerae japonicae flos, gypsum fibrosum, isatidis radix, dryoptelidis crassirhizomatis rhizoma, glycyrrhizae radix et rhizoma and rhodiolae crenulatae radix et rhizoma, adding water in an amount of 7-11 times of the mixture, heating to boil, adding stir-baked armeniacae semen amarum, boiling twice for 0.5-2.5 h each time to obtain extract liquids, combining and filtering the extract liquids to obtain filtrate, combining the obtained filtrate with the filtrate obtained after the extracting of volatile oil from the pogostemonis herba in step (2) to obtain a combined filtrate, concentrating the combined filtrate into a clear paste with a relative density of 1.10-1.15 measured at 60 C, adjusting the clear paste to an alcohol concentration of 70%
by adding ethanol, refrigerating, filtering, and distilling the filtrate to recover ethanol until no ethanol smell and obtain alcohol extract;

[0035] step (5) combining the clear paste obtained in step (4) with the alcohol extract obtained in step (3), concentrating the resultant into a clear paste with a relative density of 1.15-1.20 measured at 60 C, and drying to obtain dry paste powder; and

[0036] step (6) mixing the dry paste powder obtained in step (5), the volatile oil obtained in step (2) and /-menthol to prepare the active ingredients of the pharmaceutical composition.

[0037] The dosage form of the medicament of the present disclosure can be in a form selected from the group consisting of capsule, tablet, powder, granule, oral liquid, pill, tincture, syrup, suppository, gel, spray and injection.

[0038] To obtain the above dosage forms, it is preferable to add a pharmaceutically acceptable adjuvant during the preparation of these dosage forms, including fillers, disintegrants, lubricants, suspending agents, binders, sweeteners, flavoring agents, preservatives, bases and the like. The fillers include starch, pregelatinized starch, lactose, mannitol, chitin, microcrystalline cellulose, sucrose, and the like. The disintegrants include starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone, low-substituted hydroxypropyl cellulose, cross-linked sodium carboxymethyl cellulose and the like. The lubricants include magnesium stearate, sodium lauryl sulfate, talc, silicon dioxide and the like. The suspending agents include polyvinylpyrrolidone, microcrystalline cellulose, sucrose, agar, hydroxypropyl methylcellulose and the like. The binders include starch syrup, polyvinylpyrrolidone, hydroxypropyl methylcellulose and the like. The sweeteners include sodium saccharin, aspartame, sucrose, sodium cyclamate, glycyrrhetinic acid and the like. The flavoring agents include sweeteners and various flavors. The preservatives include parabens, benzoic acid, sodium benzoate, sorbic acid and salts thereof, benzalkonium bromide, chlorhexidine acetate, eucalyptus oil and the like. The bases include PEG6000, PEG4000, insect wax and the like.

[0039] The capsule may be prepared by a process comprising:

[0040] step (1) weighing the materials according to weight ratio and performing selection to remove impurities;

[0041] step (2) chopping pogostemonis herba, extracting volatile oil from the pogostemonis herba with water having amount of 5-8 times of the pogostemonis herba for 4 h to collect the volatile oil and obtain an extract liquid, and filtering the extract liquid to discard residue and obtain filtrate;

[0042] step (3) performing extraction on a mixture of forsythiae fructus, honey-fried ephedrae herba, houttuyniae herba and rhei radix et rhizoma twice for 1-3 h each time by using 50-90%
ethanol in an amounts of 6-10 times of the mixture to obtain extract liquids, combining and filtering the extract liquids, distilling the filtrate to recover ethanol and obtain alcohol extract;

[0043] step (4) to a mixture of lonicerae japonicae flos, gypsum fibrosum, isatidis radix, dryoptelidis crassirhizomatis rhizoma, glycyrrhizae radix et rhizoma and rhodiolae crenulatae radix et rhizoma, adding water in an amount of 7-11 times of the mixture, heating to boil, adding stir-baked armeniacae semen amarum, boiling twice for 0.5-2.5 h each time to obtain extract liquids, combining and filtering the extract liquids to obtain filtrate, combining the obtained filtrate with the filtrate obtained after the extracting of volatile oil from the pogostemonis herba in step (2) to obtain a combined filtrate, concentrating the combined filtrate into a clear paste with a relative density of 1.10-1.15 measured at 60 C, adjusting the clear paste to an alcohol concentration of 70%
by adding ethanol, refrigerating, filtering, and distilling the filtrate to recover ethanol until no ethanol smell and obtain alcohol extract;

[0044] step (5) combining the clear paste obtained in step (4) with the alcohol extract obtained in step (3), concentrating the resultant into a clear paste with a relative density of 1.15-1.20 measured at 60 C, and drying to obtain dry paste powder;

[0045] step (6) preparing the dry paste powder obtained in step (5) into particles with a suitable pharmaceutically acceptable adjuvant; and

[0046] step (7) dissolving /-menthol and the volatile oil obtained in step (2) into ethanol, spraying the resultant onto the particles obtained in step (6), sealing, mixing well, and packaging into the capsule.

[0047] The granule may be prepared by a process comprising:

[0048] step (1) weighing the materials according to weight ratio, performing selection to remove impurities, and chopping;

[0049] step (2) chopping pogostemonis herba, extracting volatile oil from the pogostemonis herba with water having amount of 5-8 times of the pogostemonis herba for 4 h to collect the volatile oil and obtain an extract liquid, and filtering the extract liquid to discard residue and obtain filtrate;

[0050] step (3) performing extraction on a mixture of forsythiae fructus, honey-fried ephedrae herba, houttuyniae herba and rhei radix et rhizoma twice for 1-3 h each time by using 50-90%
ethanol in an amounts of 6-10 times of the mixture to obtain extract liquids, combining and filtering the extract liquids, distilling the filtrate to recover ethanol and obtain alcohol extract;

[0051] step (4) to a mixture of lonicerae japonicae flos, gypsum fibrosum, isatidis radix, dryopteiidis crassirhizomatis rhizoma, glycyrrhizae radix et rhizoma and rhodiolae crenulatae radix et rhizoma, adding water in an amount of 7-11 times of the mixture, heating to boil, adding stir-baked armeniacae semen amarum, boiling twice for 0.5-2.5 h each time to obtain extract liquids, combining and filtering the extract liquids to obtain filtrate, combining the obtained filtrate with the filtrate obtained after the extracting of volatile oil from the pogostemonis herba in step (2) to obtain a combined filtrate, concentrating the combined filtrate into a clear paste with a relative density of 1.10-1.15 measured at 60 C, adjusting the clear paste to an alcohol concentration of 70%
by adding ethanol, refrigerating, filtering, and distilling the filtrate to recover ethanol until no ethanol smell and obtain alcohol extract;

[0052] step (5) combining the clear paste obtained in step (4) with the alcohol extract obtained in step (3), concentrating the resultant into a thick paste with a relative density of 1.15-1.20 measured at 60 C;

[0053] step (6) preparing the thick paste obtained in step (5) into particles with a suitable pharmaceutically acceptable adjuvant, and granulating the particles to obtain granulated particles;
and

[0054] step (7) dissolving /-menthol and the volatile oil obtained in step (2) into ethanol, spraying the resultant onto the particles obtained in step (6), sealing, mixing well, and bagging to obtain the granule.

[0055] The granule is preferably prepared by a process comprising:

[0056] step (1) weighing the materials according to weight ratio, performing selection to remove impurities, and chopping;

[0057] step (2) chopping pogostemonis herba, adding 6 times the amount of water to extract volatile oil for 4 h to collect the volatile oil and obtain an extract liquid, filtering the extract liquid to discard residue and obtain filtrate;

[0058] step (3) mixing forsythiae fructus, honey-fried ephedrae herba, houttuyniae herba and rhei radix et rhizoma, extracting twice with 8 times the amount of 50-90%
ethanol, the first time for 2 h and the second for 1.5 h, combining extracts and filtering, recovering the ethanol, and collecting filtrate;

[0059] step (4) mixing lonicerae japonicae flos, gypsum fibrosum, isatidis radix, dryopteridis crassirhizomatis rhizoma, glycyrrhizae radix et rhizoma and rhodiolae crenulatae radix et rhizoma, adding 9 times the amount of water to boil, adding stir-baked armeniacae semen amarum to boil 2 times, the first time for 1.5 h and the second for 1 h, combining extracts and filtering, combining the obtained filtrate with the water filtrate obtained after the extracting of volatile oil from the pogostemonis herba in step (2), concentrating into a clear paste with a relative density of 1.10-1.15 measured at 60 C, adding 95% ethanol to adjust to an alcohol concentration of 70%, refrigerating, filtering, and distilling the filtrate to recover ethanol until no ethanol smell and obtain alcohol extract filtrate;
- ici -

[0060] step (5) combining the clear paste obtained in step (4) with the alcohol extract obtained in step (3), concentrating the resultant into a thick paste with a relative density of 1.15-1.20 measured at 60 C;

[0061] step (6) preparing the thick paste obtained in step (5) into particles with a suitable pharmaceutically acceptable adjuvant, and granulating the particles to obtain granulated particles;
and

[0062] step (7) sieving out fine powder from the granulated particles obtained in step (6), dissolving /-menthol and the volatile oil obtained in step (2) into ethanol, spraying the resultant onto the fine powder and mixing well, mixing well with the granulated particles obtained in step (6), sealing for half an hour, and bagging to obtain the granule.

[0063] Other dosage forms of the medicament according to the present disclosure can be prepared from the materials weighed in proportion by conventional preparation methods. For example, according to the preparation process described in Fan Biting's "Pharmaceuticals of Traditional Chinese Medicine" (Shanghai Science Press, December 1997 1st edition), pharmaceutically acceptable conventional dosage forms can be made.

[0064] The present disclosure also provides use of the pharmaceutical composition in the manufacture of a medicament for relieving shortness of breath and/or dyspnea.

[0065] The present disclosure also provides use of the pharmaceutical composition in the manufacture of a medicament for relieving fever, cough or fatigue.

[0066] The present disclosure also provides use of the pharmaceutical composition in the manufacture of a medicament for relieving chest distress.

[0067] The present disclosure also provides use of the pharmaceutical composition in the manufacture of a medicament for relieving pulmonary moist rale.

[0068] The present disclosure also provides use of the pharmaceutical composition in the manufacture of a medicament for inhibiting cytokine elevation or chemokine elevation.

[0069] In the above uses, the cytokine or chemokine is preferably selected from the group consisting of INF-a, IL-6, CCL-2/MCP-1 and CXCL-1 MP- 1 0.

BRIEF DESCRIPTION OF DRAWINGS

[0070] FIG. 1 shows the inhibitory effect of the pharmaceutical composition of this disclosure that is the granules prepared in Example 4 (hereinafter referred to as LHQW or LH) on cytopathic effects caused by common coronavirus (HCoV-229E) and novel coronavirus (2019-nCoV) infections;

[0071] FIG. 2 is electron microscope images illustrating the inhibitory effect of LHQW on HCoV-229E- and 2019-nCoV-infected Vero E6 cells;

[0072] FIG. 3 shows the inhibitory effect of LHQW on mRNA expression of inflammatory factors caused by 2019-CoV infection of Huh-7 cells;

[0073] FIG. 4 shows the cytotoxicity of LHQW and Remdesivir on Vero E6 cells;

[0074] FIG. 5 shows the inhibitory effect of LHQW and Remdesivir on SARS-CoV-2 virus;

[0075] FIG. 6 shows the typical morphology of coronavirus observed under the electron microscope after LHQW and Remdesivir treatment; and

[0076] FIG. 7 shows the effect of LHQW in inhibiting SAR2-CoV-2-induced expression of cytokine and chemokine.
DETAILED DESCRIPTION

[0077] The following examples are intended to illustrate the preparation of the pharmaceutical compositions of this disclosure. It should be understood that they are intended to limit the scope of the present invention.
Examples Example 1

[0078] Formula:
forsythiae fructus 255 g lonicerae japonicae flos 255 g isatidis radix 255 g rhei radix et rhizoma 51 g pogostemonis herba 85 g dryopteridis crassirhizomatis rhizoma 255 g rhodiolae crenulatae radix et rhizoma 85 g /-menthol 7.5 g honey-fried ephedrae herba 85 g stir-baked armeniacae semen amarum 85 g houttuyniae herba 255 g glycyrrhizae radix et rhizoma 85 g gypsum fibrosum 255 g.

[0079] Preparation:

[0080] (1) The Chinese herbal materials were weighed according to the above formula and subjected to selection to remove impurities.

[0081] (2) The pogostemonis herba was chopped and subjected to extraction with 6 times the amount of water to extract volatile oil for 4 h and obtain an extract liquid.
The volatile oil was collected. The extract liquid was filtered to discard residue and obtain filtrate.

[0082] (3) The forsythiae fructus, honey-fried ephedrae herba, houttuyniae herba and rhei radix et rhizoma were subjected to extraction twice with 8 times the amount of 70%
ethanol for 2 h at the first time and for 1.5 h at the second time to obtain extract liquids. The extract liquids were combined and filtered. The filtrate was collected after ethanol was recovered from therein.

[0083] (4) To a mixture of the lonicerae japonicae flos, gypsum fibrosum, isatidis radix, dryopteridis crassirhizomatis rhizoma, glycyrrhizae radix et rhizoma and rhodiolae crenulatae radix et rhizome, water was added in an amount of 9 times of the mixture, and heated to boil. The stir-baked armeniacae semen amarum was added. The resulting mixture was boiled twice for 1.5 h at the first time and for 1 h at the second time to obtain extract liquids. The extract liquids were combined and filtered. The obtained filtrate was combined with the filtrate obtained after the extraction of volatile oil from pogostemonis herba in step (2), and concentrated into a clear paste with a relative density of 1.10-1.15 measured at 60 C. The clear paste was adjusted to an alcohol concentration of 70% by adding ethanol, refrigerated for 24 h, and filtered.
The filtrate was concentrated to obtain a clear paste, while ethanol was recovered from therein until no ethanol smell.

[0084] (5) The clear paste obtained in step (4) was combined with the alcohol extract liquid obtained in step (3). The resultant was concentrated into a clear paste with a relative density of 1.15-1.20 measured at 60 C. The clear paste was spray-dried to obtain dry paste powder.

[0085] (6) The drypaste powder obtained in step (5) was added with 142 g of starch, and prepared into to particles using 85% ethanol.

[0086] (7) The volatile oil obtained in step (2) and /-menthol were dissolved into ethanol, and sprayed onto the particles obtained in step (6). The resulting particles were sealed, mixed well, and encapsulated into 1000 capsules.
Example 2

[0087] Formula:
forsythiae fructus 260 g lonicerae japonicae flos 150 g isatidis radix 260 g rhei radix et rhizoma 30 g pogostemonis herb a 90 g dryopteridis crassirhizomatis rhizoma 150 g rhodiolae crenulatae radix et rhizoma 90 g /-menthol 5 g honey-fried ephedrae herba 90 g stir-baked armeniacae semen amarum 50 g houttuyniae herba 260 g glycyrrhizae radix et rhizoma 50 g gypsum fibrosum 260 g.

[0088] Preparation:

[0089] (1) The Chinese herbal materials were weighed according to the above formula and subjected to selection to remove impurities.

[0090] (2) The pogostemonis herba was chopped and subjected to extraction with 5 times the amount of water to extract volatile oil for 4 h and obtain an extract liquid.
The volatile oil was collected. The extract liquid was filtered to discard residue and obtain filtrate.

[0091] (3) The forsythiae fructus, honey-fried ephedrae herba, houttuyniae herba and rhei radix et rhizoma were subjected to extraction twice with 6 times the amount of 70%
ethanol for 1 h at the first time and for 2.5 h at the second time to obtain extract liquids. The extract liquids were combined and filtered. The filtrate was collected after ethanol was recovered from therein.

[0092] (4) To a mixture of the lonicerae japonicae flos, gypsum fibrosum, isatidis radix, dryopteridis crassirhizomatis rhizoma, glycyrrhizae radix et rhizoma and rhodiolae crenulatae radix et rhizome, water was added in an amount of 7 times of the mixture, and heated to boil. The stir-baked armeniacae semen amarum was added. The resulting mixture was boiled twice for 1.5 h at the first time and for 1 h at the second time to obtain extract liquids. The extract liquids were combined and filtered. The obtained filtrate was combined with the filtrate obtained after the extraction of volatile oil from pogostemonis herba in step (2), and concentrated into a clear paste with a relative density of 1.10-1.15 measured at 60 C. The clear paste was adjusted to an alcohol concentration of 70% by adding ethanol, refrigerated for 24 h, and filtered.
The filtrate was concentrated to obtain a clear paste, while ethanol was recovered from therein until no ethanol smell.

[0093] (5) The clear paste obtained in step (4) was combined with the alcohol extract liquid obtained in step (3). The resultant was concentrated into a clear paste with a relative density of 1.15-1.20 measured at 60 C. The clear paste was spray-dried to obtain dry paste powder.

[0094] (6) The dry paste powder obtained in step (5) was added with 151 g of starch, and prepared into to particles using 85% ethanol.

[0095] step (7) The volatile oil obtained in step (2) and /-menthol were dissolved into ethanol, and sprayed onto the particles obtained in step (6). The resulting particles were sealed, mixed well, and compressed into 935 tablets.
Example 3

[0096] Formula:
forsythiae fructus 185 g lonicerae japonicae flos 179 g isatidis radix 190 g rhei radix et rhizoma 36 g pogostemonis herb a 60 g dryopteridis crassirhizomatis rhizoma 170 g rhodiolae crenulatae radix et rhizoma 57 g /-menthol 5 g honey-fried ephedrae herba 60 g stir-baked armeniacae semen amarum 60 g houttuyniae herba 170 g glycyiThizae radix et rhizoma 60 g gypsum fibrosum 170g.

[0097] Preparation:

[0098] (1) The Chinese herbal materials were weighed according to the above formula and subjected to selection to remove impurities.

[0099] (2) The pogostemonis herba was chopped and subjected to extraction with 6 times the amount of water to extract volatile oil for 4 h and obtain an extract liquid.
The volatile oil was collected. The extract liquid was filtered to discard residue and obtain filtrate.

[0100] (3) The forsythiae fructus, honey-fried ephedrae herba, houttuyniae herba and rhei radix et rhizoma were subjected to extraction twice with 8 times the amount of 70%
ethanol for 2 h at the first time and for 1.5 h at the second time to obtain extract liquids. The extract liquids were combined and filtered. The filtrate was collected after ethanol was recovered from therein.

[0101] (4) To a mixture of the lonicerae japonicae flos, gypsum fibrosum, isatidis radix, dryoptelidis crassirhizomatis rhizoma, glycyrrhizae radix et rhizoma and rhodiolae crenulatae radix et rhizome, water was added in an amount of 9 times of the mixture, and heated to boil. The stir-baked armeniacae semen amarum was added. The resulting mixture was boiled twice for 1.5 h at the first time and for 1 h at the second time to obtain extract liquids. The extract liquids were combined and filtered. The obtained filtrate was combined with the filtrate obtained after the extraction of volatile oil from pogostemonis herba in step (2), and concentrated into a clear paste with a relative density of 1.10-1.15 measured at 60 C. The clear paste was adjusted to an alcohol concentration of 70% by adding 95% ethanol, refrigerated for 24 h, and filtered. The filtrate was concentrated to obtain a clear paste, while ethanol was recovered from therein until no ethanol smell.

[0102] (5) The clear paste obtained in step (4) was combined with the alcohol extract liquid obtained in step (3). The resultant was concentrated into a clear paste with a relative density of 1.25-1.30 measured at 60 C. The clear paste was spray-dried to obtain dry paste powder.

[0103] (6) The dry paste powder obtained in step (5), the volatile oil obtained in step (2) and /-menthol were prepared into 905 pills by a conventional method.
Example 4:

[0104] Formula of crude drugs:
forsythiae fructus 170 g lonicerae japonicae flos 170 g isatidis radix 170 g rhei radix et rhizoma 34 g pogostemonis herba 57 g dryopteridis crassirhizomatis rhizoma 170 g rhodiolae crenulatae radix et rhizoma 57 g /-menthol 5.0 g honey-fried ephedrae herba 57 g stir-baked armeniacae semen amarum 57 g houttuyniae herba 170 g glycyrrhizae radix et rhizoma 57 g gypsum fibrosum 170g.

[0105] Preparation:

[0106] 1. Extraction process:

[0107] (1) The Chinese herbal materials were weighed according to the above formula and subjected to selection to remove impurities, and chopped.

[0108] (2) The pogostemonis herba was subjected to extraction with 6 times the amount of water to extract volatile oil for 4 h and obtain an extract liquid. The volatile oil was collected with an oil yield of 0.33%. The extract liquid was filtered to discard residue and obtain filtrate which is water extract liquid.

[0109] (3) The forsythiae fructus, honey-fried ephedrae herba, houttuyniae herba and rhei radix et rhizoma were subjected to extraction twice with 8 times the amount of 70%
ethanol for 2 h at the first time and for 1.5 h at the second time to obtain extract liquids. The extract liquids were combined and filtered. The filtrate was combined. Ethanol was recovered from the filtrate until no ethanol smell.

[0110] (4) To a mixture of the lonicerae japonicae flos, gypsum fibrosum, isatidis radix, dryopteridis crassirhizomatis rhizoma, glycyrrhizae radix et rhizoma and rhodiolae crenulatae radix et rhizoma, water was added in an amount of 9 times of the mixture, and heated to boil. The stir-baked armeniacae semen amarum was added. The resulting mixture was boiled twice for 1.5 h at the first time and for 1 h at the second time to obtain extract liquids. The extract liquids were combined and filtered. The obtained filtrate was combined with the water solution after the extraction of volatile oil from pogostemonis herba in step (2), and concentrated into a clear paste with a relative density of 1.10-1.15 measured at 60 C. The clear paste was added 95% ethanol while being stirred, to adjust to an alcohol concentration of 70%, refrigerated for 24 h, and filtered.
The resultant was combined with the alcohol extract liquid obtained in step (3), and concentrated into a thick paste with a relative density of 1.15-1.20 measured at 60 C.

[0111] 2. Preparation process:

[0112] Preparation formula contained 335.5 g of the thick paste obtained in step (4), 5 g of /-menthol, 0.2 ml of the pogostemonis herba oil obtained in step (2), 342.5 g of icing sugar and 514.0 g of dextrin.

[0113] (5) Granulation: The icing sugar and the dextrin were mixed well according to the above formula, and prepared into soft materials using the thick paste obtained in step (4) as a binder. The resultant was pelletized with a 14-mesh sieve, dried at 60-65 C, and granulated with a 10-mesh sieve.

[0114] (6) Sub-packaging: An appropriate amount of fine powder was sieved out.
The /-menthol and pogostemonis herba volatile oil were dissolved into an appropriate amount of ethanol according to the formula, and sprayed into the fine powder sieved out in step (5). After being mixing well, the resulting fine powder was mixed well with the particles obtained in step (5). The resultant was sealed for half an hour, and packaged into bags. According to the above formula, 1000 g granules were prepared.
Experimental examples

[0115] The following in vitro studies and clinical trial studies were carried out to confirm the curative effect of the pharmaceutical composition of this disclosure in preventing and treating novel coronavirus induced-pneumonia. The granules prepared by the method in Example 4 (hereinafter referred to as LHQW or the drug of this disclosure or the pharmaceutical composition of this disclosure) was used.
Experimental example 1:

[0116] In vitro cell studies

[0117] The team of Professor Zifeng Yang from the State Key Laboratory of Respiratory Diseases of Guangzhou Medical University conducted a cell model study on the anti-new coronavirus and anti-inflammatory activities of LHQW, and found that LHQW had a good inhibitory effect on cytopathic effects caused by common coronavirus (HCoV-229E) and novel coronavirus (2019-nCoV) infections (FIG. 1), with IC50 of 338.74 and 414.02 gg/mL, and SI of 3.47 and 2.89, respectively (Table 1).
Table 1 In vitro inhibitory effect of LHQW on HCoV-229E and 2019-nCoV
LAW ( gem L
Coronavinis Cell Tc50 IC50 S
HOW-229E H uh- 7 1177.25142.17 338.7418.31 347 2019-neoV Vero E6 1197.75151.26 414.02114.18 2,89 TC50: Half-toxic concentration of the drug to cells IC50: half-maximal inhibitory concentration of the drug SI: Selection Index (TC50/IC50)

[0118] Transmission electron microscopy analysis showed that normal cells had intact morphology and were free of virus particle intracellularly and extracellularly; virus-infected cells contained a large number of virus particles in the cell membrane, cytoplasm and vesicles; and the LHQW-treated cells contained less virus particles than the virus-infected cells, and some viral particles were slightly deformed (see FIG. 2).

[0119] Huh-7 cells having been infected by 2019-CoV were added with different concentrations of LHQW. After 48 hours, cell mRNA was extracted, reverse transcribed into cDNA, and detected for the expression of inflammatory factors by RT-qPCR. The results showed that the two drugs significantly inhibited the mRNA expression of inflammatory factors such as INF-a, IL-6, CCL2/MCP-1 and CXCL-10/IP-10 in a dose-dependent manner (see FIG. 3).
Experimental example 2:

[0120] Clinical Research 1

[0121] Materials and methods

[0122] 1. Subject selection

[0123] Subjects who met the diagnostic criteria for novel coronavirus infected-pneumonia and were judged to be positive for 2019-nCoV by nucleic acid testing on specimens such as sputum, throat swabs and lower respiratory tract secretions, were selected from clinical cases.

[0124] Inclusion criteria: common-type inpatients, aged 18 years or older, meeting the diagnostic criteria for novel coronavirus infected-pneumonia, and with body temperature of >37.2 C.

[0125] Exclusion criteria: (1) patients with severe and critical novel coronavirus-infected pneumonia; (2) patients with acute respiratory diseases not caused by 2019-nCoV; (3) patients with any other chronic respiratory diseases, respiratory bacterial infections such as suppurative tonsillitis, acute tracheo-bronchitis, sinusitis, otitis media and other respiratory diseases that affect the evaluation of clinical trials; (4) patients with asthma requiring daily treatment, or underlying lung diseases such as severe pulmonary interstitial lesions and bronchiectasis confirmed by chest CT;
and (5) patients associated with serious primary immunodeficiency disease, acquired immunodeficiency syndrome, congenital respiratory malformation, congenital heart disease, abnormal lung development and other underlying diseases.

[0126] 2. Grouping method:

[0127] A. Test drug group: 21 common-type inpatients, aged 18 years or older, with body temperature of >37.2 C, and meeting the diagnostic criteria for novel coronavirus infected-pneumonia, were given the pharmaceutical composition of this disclosure. The treatment plan was conventional treatment (refer to the "Prevention and Control Program for New Coronavirus Infected Pneumonia" issued by the Chinese National Health Commission) plus the pharmaceutical composition of this disclosure at 1 sachet/time, 3 times/day. With age and body temperature used as covariates, propensity scores were calculated using a logistic regression model and matched at a 1 :1 ratio.

[0128] B. 21 patients were matched as the control group, and treated with conventional treatment plan, which was to give nutritional support treatment, symptomatic treatment, antiviral treatment and antibacterial drug treatment according to disease monitoring.

[0129] 3. Evaluation index: The rate of disappearance of main symptoms (fever, fatigue and cough), the time to fever disappearance and the rate of disappearance of other single symptoms were compared between the test drug group and the control group.

[0130] 4. Statistical method: Statistical analysis was performed using SAS 9.4 software. All statistical tests used two-sided test, and P value less than or equal to 0.05 were considered statistically significant for the differences tested. Descriptive analysis:
Enumeration data were expressed by the number of cases and constituent ratios, and measurement data were expressed by means and standard deviations. Quantitative data were compared by t test, and categorical data were analyzed using the chi-square test or the exact probability method.
II. Results

[0131] 1. Baseline data

[0132] A total of 42 patients with confirmed novel coronavirus infected-pneumonia who met the requirements were enrolled, including 16 males (76.2%) and 5 females (23.8%) with an average age of 57.1 14.0 years old in the experimental drug group, and 12 males (57.1%) and 9 females (42.9%) with an average age of 55.4 12.3 years old in the control group.
Baseline data comparison between the two groups, including the age, gender, and body temperature, blood pressure, heart rate, respiration, past medical history, and the time from onset to diagnosis, showed no statistically significant difference (P>0.05), indicating that the two group were comparable. See Table 2 for details.
Table 2 Baseline data of treatment group and control group Item Treatment group (N=21) Control group (N=21) P value Age (years) 57.1+14.0 62.4+12.3 0.202 Male 16(76.2%) 12(57.1%) 0.190 Body temperature 38.56+0.68 38.38+0.63 0.383 Systolic blood pressure 123.9+12.9 119.3+14.4 0.290 (mmHg) Diastolic blood pressure 75.3+10.2 72.4+9.8 0.361 (mmHg) Heart rate (beats/min) 88.5+10.8 88.4+11.6 0.978 Breathing (times/min) 20.0+2.3 19.8+1.3 0.687 Past medical history 10(47.6%) 10(47.6%) 1.000 Time from onset to 12.8+3.8 12.9+3.3 0.944 diagnosis (days)

[0133] 2. Comparison of the rate of disappearance of main symptoms between the two groups

[0134] (1) Baseline data: 21 cases in the experimental drug group, including 21 cases with fever (100%), 15 cases of cough (71.4%), and 12 cases with fatigue (57.1%); 21 cases in the control group, including 21 cases with fever (100%), 18 cases with cough (85.7%), and 13 cases with fatigue (61.9%); the baseline comparison between the groups had no statistically significant difference between (P>0.05). (2) Treatment results: In the experimental drug group, fever symptoms disappeared in 18 cases (85.7%) and cough symptoms disappeared in 7 cases (46.7%), which had statistically significant difference (P<0.05) as compared with these in the control group.
See Table 3 for details.
Table 3 Rate of disappearance of main symptoms in treatment group and control group Treatment group Control group Number of Number of Item N N cases with Disappearance cases with Disappearance P value symptom- rate symptom- rate disappeared disappeared Fever 21 18 85.7% 21 12 57.1%
0.040 Cough 15 7 46.7% 18 1 5.6% 0.012 Fatigue 12 5 41.7% 13 4 30.8% 0.688

[0135] 3. Comparison of the fever duration between the two groups

[0136] 21 cases in the experimental drug group had a fever duration of 4.6 3.2 d; 21 cases in the control group had a fever duration of 6.1 3.1 d. There was no statistically significant difference between the groups (P= 0.218).

[0137] 4. Comparison of the rate of disappearance of other symptoms between the two groups

[0138] In the treatment group, the rates of disappearance of expectoration and shortness of breath were 64.3% and 77.8% respectively, which had statistically significant difference (P<0.05) as compared with these in the control group. See Table 4 for details.
Table 4 Rate of disappearance of other symptoms in treatment group and control group Item Treatment group Control group P value Number of cases Number of cases Disappearance Disappearance N with symptom- N with symptom-rate rate disappeared disappeared 6 4 66.7% 7 2 28.6%
0.286 Muscle pain 14 9 64.3% 11 1 9.1%
0.012 Expectoration 3 1 33.3% 0 0 0 Stuffy nose 3 1 33.3% 0 0 0 Runny nose 3 1 33.3% 3 1 33.3%
1.000 Sore throat Shortness of 9 7 77.8% 5 0 0 0.021 breath 7 5 71.4% 9 2 22.2%
0.126 Chest distress 2 1 50.0% 2 1 50.0%
1.000 Dyspnea 4 2 50.0% 1 0 0 1.000 Headache 4 2 50.0% 3 2 66.7%
1.000 Nausea 4 3 75.0% 0 0 0 Vomiting 11 4 36.4% 12 2 16.7%
0.371 Anorexia 3 60.0% 3 2 66.7% 1.000 Diarrhea

[0139] During the research related to this disclosure, 42 confirmed patients with novel coronavirus-infected pneumonia with fever symptoms were enrolled, and they were accompanied by varying degrees of cough, fatigue, expectoration, muscle pain, shortness of breath, sore throat, 5 nausea, vomiting, anorexia or diarrhea. During the treatment, the inventor unexpectedly found that in the treatment group, the clinical symptoms such as fever, cough, expectoration and shortness of breath were significantly relieved during administration for the patients who additionally took the granules of Example 4 of the pharmaceutical composition of this disclosure, and the time to fever disappearance was shortened by an average of 1.5 days compared with that in the control group.
This finding indicates that the drug had clinical advantages in relieving fever symptoms, and also exhibited a positive trend in improving symptoms of fatigue, muscle pain, nasal obstruction, and headache.

[0140] The patients enrolled in this study were with mild to moderate new coronavirus infected-pneumonia, as considering that they can give better communication and feedback on the efficacy, rather than that severe patients cannot take the drug of this disclosure.
Experimental example 3:

[0141] Clinical Research 2

[0142] 1 Clinical data

[0143] 1.1 Diagnostic criteria

[0144] According to the "Diagnosis and Treatment Program for Novel Coronavirus Infected Pneumonia (Trial Version 4)" on the diagnostic criteria for suspected cases, a case who meets any one item of epidemiological histories of travel, residence, contacting or gathering, or meet any two of the clinical manifestations of fever and/or respiratory tract symptoms, total white blood cell counts showing normal/decreased/reduced lymphocyte count in the early onset stage, or NCP
imaging features, can be identified as a suspected case.

[0145] 1.2 Inclusion criteria

[0146] Inpatients aged 18 years or older who met the above-mentioned diagnostic criteria for suspected cases and had NCP imaging features.

[0147] 1.3 Exclusion Criteria

[0148] Patients diagnosed as severe and critical NCP; patients with bronchial asthma; patients with underlying lung diseases such as severe interstitial lung disease and bronchiectasis confirmed by chest X-ray computed tomography (CT); patients associated with serious immunodeficiency disease, congenital respiratory malformation, congenital heart disease, abnormal lung development or other underlying diseases.

[0149] 1.4 General data

[0150] A total of 101 suspected patients were collected, of which 38 patients who received conventional treatment were in the control group, and 63 patients who received conventional treatment combined with the drug of this disclosure (LHQW) were in the treatment group. Baseline data comparison between the two groups, including age, gender, body temperature, blood pressure, heart rate, respiration, past medical history, fever, fatigue, cough, routine treatment, and main laboratory test indicators showed no statistically significant difference (P>0.05), indicating that the test results were comparable. The general data of the patients in the treatment group and the control group are shown in Table 5.
Table 5 Comparison of general data between two groups Item Treatment Control group Statistics P

group (38 cases) t/x2 value (63 cases) Age (years) 59.1+16.56 60.2+17.01 -0.309 0.758 Male [cases (%)] 28(44.4) 18(47.4) 0.082 0.775 Body temperature ( C) 38.08+0.63 38.03+0.67 0.318 0.751 Heart rate [beats/min, (t+s)] 88.5+15.43 87.6+13.13 0.305 0.761 Breath [beats/min, (x s)] 19.4+2.03 18.8+1.65 1.315 0.192 Past medical history [cases 42(66.7) 28(73.7) 0.628 (%)]
--Hypertension 21(33.3) 11(29.0) 0.211 0.646 --Coronary heart disease 8(12.7) 3(7.9) 0.528 --Diabetes 5(7.9) 6(15.8) 0.323 --Cerebral infarction 10(15.9) 5(13.2) 0.138 0.710 Fever 60(95.2) 34(89.5) 0.421 Fatigue 40(63.5) 29(76.3) 1.801 0.180 Cough 54(85.7) 36(94.7) 0.201 Conventional treatment [cases (%)]
--Antibiotic 55(87.3) 34(89.5) 1.000 --Antiviral 54(85.7) 32(84.2) 0.042 0.837 --Antipyretic and analgesic 31(49.2) 17(47.4) 0.190 0.663 --Immunoglobulins 33(52.4) 19(50.0) 0.054 0.817 --Expectorant 54(85.7) 32(84.2) 0.042 0.837 --Asthma medicine 35(55.6) 22(57.9) 0.053 0.818 --Glucocorticoids 23(36.5) 13(34.2) 0.055 0.815 Blood leukocytes [109/L, (,-t 4.62+2.14 4.42+2.32 -0.441 0.330 +s)]
Neutrophils [%, (v+s)] 67.09+14.48 65.52+14.80 -0.524 0.301 Lymphocytes [%, (x+s)] 22.28+11.15 20.12+12.65 -0.896 0.186 C-reactive protein [mg/L, 51.50+46.05 50.41+45.27 -0.116 0.454 +s)]

[0151] 2. Methods

[0152] 2.1 Treatment methods

[0153] The two groups of patients were monitored for their condition, and given nutritional support treatment, symptomatic treatment, antiviral treatment, or antibacterial drug treatment. The main treatment drugs included: moxifloxacin hydrochloride and sodium chloride injection, 0.4 g, once a day, produced by Chengdu Zhengkang Pharmaceutical Co., Ltd., batch number 3419111102;
Ganciclovir Injection, 0.5 g, once a day, produced by Hubei Keyi Pharmaceutical Co., Ltd., batch number 191003; intravenous human immunoglobulin, 2.5 g, 1 time/day, produced by Guizhou Taibang Biological Products Co., Ltd., batch number 201906026; ambroxol hydrochloride injection, 30 mg, 2 times/day, produced by Tianjin Pharmaceutical Group Co., Ltd., batch number 1906116; doxofylline for injection, 0.2 g, once a day, produced by Ruiyang Pharmaceutical Co., Ltd., batch number 19082116; methylprednisolone sodium succinate for injection, 40 mg, once a day, produced by Liaoning Haisco Pharmaceutical Co., Ltd., batch number 20191027.

[0154] Control group: only given the above treatment

[0155] Treatment group: treated with the drug of this disclosure in combination with the above treatment, 6 g/sachet, 1 sachet each time, 3 times a day. The clinical data of patients treated for 10 days were collected.

[0156] 2.2 Observation indicators and methods

[0157] 2.2.1 Comparison of the disappearance of main symptoms in the two groups: rate of disappearance of (fever, fatigue and cough), time to fever disappearance, rate of disappearance of other single symptoms or signs (muscle pain, expectoration, nasal obstruction, runny nose, sore throat, shortness of breath, chest distress, dyspnea, headache, nausea, vomiting, anorexia, diarrhea, moist rale), and aggravation during treatment.

[0158] 2.2.2 Safety evaluation: blood routine, urine routine, stool routine, liver function and renal function were detected before and after treatment.

[0159] 2.3 Statistical methods

[0160] Statistical analysis was performed using SAS 9.4 software. All statistical tests used two-sided test. In descriptive analysis, enumeration data were expressed by the number of cases and constituent ratios, and measurement data were expressed by the mean standard deviation. The t test was used to compare the measurement data between groups, and the chi-square test or the exact probability method was used for the enumeration data. Fever duration was analyzed by survival analysis. The difference was statistically significant with P<0.05.

[0161] 3. Results

[0162] 3.1 Comparison of the rate of disappearance of main symptoms between the two groups of patients

[0163] Table 6 shows that compared with the control group, the disappearance of the symptoms of fever, cough or fatigue in the treatment group were significantly better than those in the control group (all P<0.05).

Table 6 Comparison of rate of disappearance of main symptoms between two groups of patients Fever Cough Fatigue Number Number Number of cases of cases of cases Grou Case with Disappear Case with Disappear Case with Disappear p numb sympto ance rate numb sympto ance rate numb sympto ance rate er m- (%) er m- (%) er m-(%) disappea disappea disappea red red red Treatm ent 60 52 86.7 54 30 55.6 40 33 82.5 group Control 34 23 67.7 36 11 30.6 29 17 58.6 group x2 - - 4.868 ¨ ¨ 5.443 ¨ ¨ 4.804 P 0.027 0.020 0.028

[0164] 3.2 Comparison of fever duration between the two groups of patients

[0165] There were 52 fever patients in the treatment group with a median fever duration of 6 days; and there were 23 fever patients in the control group with a median fever duration of 7 days.
There was no statistically significant difference between the two groups (P=0.171).

[0166] 3.3 Comparison of the rate of disappearance of other symptoms and signs between the two groups of patients

[0167] Table 7 shows that the rates of disappearance of shortness of breath and moist rale in the treatment group were 68.2% and 56.0%, respectively, which were significantly better than 20.0%
and 20.0% in the control group (z2 =9.817, 4.972), and the differences between the two groups were statistically significant. There was no statistically significant difference between the two groups in the rate of disappearance of other symptoms.
Table 7 Comparison of disappearance of other symptoms and signs between two groups of patients Treatment group (63 cases) Control group (38 cases) Number of Number of Disappear 2 Item Case cases with Disappearance Case cases with x P
ance rate number symptom- rate (%) number symptom-(% ) disappeared disappeared Muscle pain 9 7 77.8 7 4 57.1 0.596 Expectoration 42 24 57.1 23 11 47.8 0.519 0.471 Stuffy nose 3 2 66.7 4 3 75.0 1.000 Runny nose 6 5 83.3 3 3 100.0 -1.000 Sore throat 3 2 66.7 3 1 33.3 1.000 Shortness of . 9 817 22 15 68.2 20 4 20.0 0.002 breath Chest distress 24 17 70.8 19 12 63.2 0.285 0.594 Dyspnea 12 6 50.0 9 1 11.1 0.159 Headache 6 5 83.3 6 4 66.7 -1.000 Nausea 11 10 90.9 6 3 50.0 -0.099 Vomiting 4 3 75.0 3 1 33.3 0.486 Anorexia 48 36 75.0 30 19 63.3 1.209 0.272 Diarrhea 8 4 50.0 3 1 33.3 1.000 Moist rale 25 14 56.0 15 3 20.0 4.972 0.026

[0168] 3.4 Aggravation in the two groups of patients during treatment

[0169] Among 63 cases in the treatment group, condition of 4 cases (6.4%) deteriorated during the treatment. Among 38 cases in the control group, condition of 6 cases (15.8%) deteriorated during the treatment. There was no statistical difference between the two groups (P> 0.05).

[0170] 3.5 Safety analysis

[0171] During the treatment of the treatment group, no abnormality caused by the drug of this disclosure was found in the laboratory examinations of blood routine, liver and kidney function, and no adverse reaction of the drug of this disclosure was found, indicating good safety in clinical application of this drug.
Experimental example 4:

[0172] Clinical Research 3

[0173] 1. Subjects and methods

[0174] 1.1 Research subjects: Clinical data of patients were collected, wherein the patients met the common-type diagnostic criteria of the "Diagnosis and Treatment Program for Novel Coronavirus Infected Pneumonia (Trial Version 5)" and were judged to be NCP
positive by nucleic acid testing on specimens such as sputum, throat swabs and lower respiratory tract secretions. 54 patients were treated with the drug of this disclosure (LHQW) combined with conventional treatment (nutrition support treatment, symptomatic treatment, antiviral treatment and antibacterial drug treatment according to disease monitoring).

[0175] 1.2 Research methods: The clinical data of 54 patients with common-type NCP were analyzed, and retrospective analysis was performed on the rate of disappearance of main symptoms (fever, fatigue and cough) in days 3, 5, and 7, days to fever disappearance, and rate of disappearance of other symptoms and signs. Meanwhile, the absence of the main symptom was scored as 0 point and the presence as 1 point. The symptom score-reducing rate > 30% was regarded as effective treatment and < 30% as ineffective treatment. The treatment efficiency of all patients after 7 days of treatment was analyzed.

[0176] 2. Results

[0177] 2.1 General data

[0178] 54 inpatients, aged 25 to 95 years old with an average of (60.1 16.98) years old; Among them, 29 males (53.7%) and 25 females (46.3%); average body temperature (37.93 0.93) C, median body temperature 38.05 C; maximum body temperature before diagnosis (38.54 0.60) C;
average heart rate (87.9 11.80) beats/min, median 85.5 beats/min, maximum 112 beats/min;
average respiration (21.1 3.78) beats/min, median 20.0 times/min, maximum 30.0 times/min; 21 cases (38.9%) with a history of hypertension, 7 cases (13.0%) with a history of coronary heart disease, 10 cases (18.5%) with a history of diabetes, and 10 cases with a history of cerebral infarction (18.5%). Laboratory tests: among 54 cases, 31 cases (64.6%) with leukocytes within the normal range, 9 cases (18.8%) with leukocytes below the normal value, and 8 cases (16.7%) with leukocytes above the normal value; 25 cases (52.1%) with neutrophils within the normal range, and 23 cases (47.9%) with neutrophils above the normal value; 14 cases (29.2%) with lymphocytes within the normal range, and 34 cases (70.8%) with lymphocytes below the normal value; all cases with high-sensitivity C-reactive protein above the normal value (100%). The drug of this disclosure was administrated to the patients for an average day of 8.0 4.10 days, median 7.0 days, minimum 1.0 days, and maximum 16.0 days.
Table 8 General data of patients with common-type NCP
Item Result Item Result Age, cases (years) 54 (60.1+16.98) Neutrophils, cases (%) Male, cases (%) 29 (53.7%) --Normal 25 (52.1%) Body temperature, cases 54 (37.93+0.93) --Low 0 (0.0%) ( C) Heart rate, cases 54(87.9 11.80) --High 23 (47.9%) (beats/min) Breathing, cases 54 (21.1+3.78) Lymphocytes, cases (%) (times/min) Past medical history, --Normal 14 (29.2%) cases (%) --Hypertension 21(38.9%) --Low 34 (70.8%) --Coronary heart disease 7 (13.0%) --High 0 (0.0%) C-reactive protein, cases (.0/0) --Diabetes (%) --Cerebral infarction 10 (18.5%) --Normal 0 (0.0%) Blood leukocytes, cases --Low 0 (0.0c/
(%) o) --Normal 31(64.6%) --High 44(100%) Days of taking the drug of 9 (18.8%) 54(8.0 4.10) --Low this disclosure --High 8 (16.7%)

[0179] 2.2 Rate of disappearance of main symptoms (fever, fatigue and cough)

[0180] (1) Before treatment: Among the 54 patients, 40 patients (74.1%) were accompanied by fever, 37 patients (68.5%) were fatigued, and 30 patients (55.6%) had cough;
(2) After 3 days of 5 treatment, fever symptoms disappeared in 19 cases (disappearance rate 47.5%), fatigue symptoms disappeared in 13 cases (disappearance rate 35.1%), and cough symptoms disappeared in 6 cases (disappearance rate 20.0%); (3) After 5 days of treatment, fever symptoms disappeared in 25 cases (disappearance rate 62.5%), fatigue symptoms disappeared in 22 cases (disappearance rate 59.5%), cough symptoms disappeared in 15 cases (disappearance rate 50.0%); (4) After 7 days of treatment, 10 fever symptoms disappeared in 32 cases (disappearance rate 80.0%), fatigue symptoms disappeared in 28 cases (disappearance rate was 75.7%), and cough symptoms disappeared in 23 cases (disappearance rate was 76.7%). The results are shown in Table 9.
Table 9 Rate of disappearance of main symptoms in patients with common-type NCP
Before treatment 3 days after treatment 5 days after treatment 7 days after treatment Number of .
Case Disappeara Number of cases Disappeara Number of cases Disappeara Item Incide cases with N numbe nce rate with symptom- ace rate with symptom- nce rate nce symptom-(%) disappeared (%) disappeared (%) disappeared Fever 54 40 74.1% 19 47.5% 25 62.5% 32 80.0%
Fatigue 54 37 68.5% 13 35.1% 22 59.5% 28 75.7%
Cough 54 30 55.6% 6 20.0% 15 50.0% 23 76.7%

[0181] 2.3 Days of main symptom (fever, fatigue, cough) disappearance

[0182] (1) The average days of fever symptom disappearance were (3.6 2.14) days, the median days to disappearance were 3.0 days, the minimal days to disappearance were 1.0 days, and the maximal days to disappearance were 8.0 days; (2) The average days of fatigue symptom disappearance were (4.1 2.58) days, the median days to disappearance were 4.0 days, the minimal days to disappearance were 1.0 days, and the maximal days to disappearance were 12.0 days; (3) The average days of cough symptom disappearance were (5.3 2.63) days, the median days to disappearance were 5.0 days, the minimal days to disappearance were 1.0 days, and the maximal days to disappearance were 12.0 days.

[0183] 2.4 Rate of disappearance of other symptoms and signs

[0184] (1) Before treatment: Among the 54 patients, 13 patients (24.1%) were accompanied with chest distress, 8 patients (14.8%) with dyspnea, 10 patients (18.5%) with anorexia, and 19 patients (35.2%) with moist rale; (2) After 7 days of treatment: chest distress was in 2 cases and disappeared in 11 cases (disappearance rate 84.6%); dyspnea was in 0 cases and disappeared in 8 cases (disappearance rate 100%); anorexia was in 6 cases and disappeared in 4 cases (40.0%); moist rale was in 2 cases and disappeared in 17 cases (disappearance rate 89.5%). The results are shown in Table 10.
Table 10 Rate of disappearance of other symptoms and signs in patients with common-type NCP
Before treatment After treatment Number of cases Item N Case number Incidence with symptom-Disappearance rate (%) disappeared Chest 54 13 24.1% 11 84.6%
distress Dyspnea 54 8 14.8% 8 100%
Anorexia 54 10 18.5% 4 40.0%
Moist rale 54 19 35.2% 17 89.5%

[0185] 2.5 Treatment efficiency: Among 54 NCP patients, 23 cases (efficacy rate 46.9%) were effectively treated after 3 days of treatment, 34 cases (efficacy rate 69.4%) were effectively treated after 5 days of treatment, and 40 cases (efficacy rate 81.6%) were effectively treated after 7 days of treatment. The results are shown in Table 11.
Table 11 Efficacy rate of treatment in patients with common-type NCP
3 days after treatment 5 days after treatment 7 days after treatment Item N Case Case number Proportion Case number Proportion Proportion number Effective 54 23 46.9% 34 69.4% 40 81.6%
Ineffective 54 26 53.1% 15 30.6% 9 18.4%

[0186] 2.6 Safety Analysis: Analysis on the laboratory examinations of blood routine, liver and kidney function found that in the process of applying the drug of this disclosure in combination with conventional treatment, the drug of this disclosure caused no abnormality and no adverse reaction, indicating good safety in clinical application of this drug.
Experimental example 5:

[0187] 1. Subjects and methods

[0188] 1.1 Research subjects

[0189] Patients who were diagnosed as NCP positive by nucleic acid testing on specimens such as sputum, throat swabs and lower respiratory tract secretions, were enrolled.

[0190] 1.2 Inclusion criteria

[0191] Patients aged between 18 and 70 who met the common-type NCP diagnostic criteria of the "Diagnosis and Treatment Program for Novel Coronavirus Infected Pneumonia (Trial Version 5)" and had been hospitalized for more than 6 days.

[0192] 1.3 Exclusion criteria

[0193] (1) Severe and critical NCP patients; (2) patients with any other chronic respiratory diseases, respiratory bacterial infections such as purulent tonsillitis, acute tracheo-bronchitis, sinusitis, otitis media and other respiratory diseases that affect the evaluation of clinical trials; (3) patients with severe pulmonary interstitial disease, bronchiectasis, primary immunodeficiency disease, congenital respiratory malformation, congenital heart disease, lung dysplasia and other underlying diseases; (4) patients accompanied by severe liver disease (aspartate aminotransferase AST or alanine aminotransferase ALT exceeded 5 times of the upper limit of normal), or patients with severe renal insufficiency or undergoing continuous renal replacement therapy, hemodialysis, or peritoneal dialysis; (5) patient with multiple metastatic malignant tumors that cannot be resected, or with blood disease, cachexia, active bleeding, severe malnutrition, HIV, etc., or with severe neurological diseases or mental disorders.

[0194] 1.4 Grouping method

[0195] The patients who met the inclusion criteria were collected. Among them, 51 patients who had been treated with the drug of this disclosure for more than or equal to 5 days were enrolled in the statistics, to calculate their propensity scores by using a logistic regression model with age, body temperature and course of disease as covaiiates. In addition, another 51 patients were matched in a 1:1 ratio as a control group and received conventional treatment.

[0196] 1.5 Treatment method

[0197] The control group received conventional treatment including simple nutritional support treatment, symptomatic treatment, antiviral treatment and antibacterial drug treatment. The treatment group was treated with the drug of this disclosure (LHQW) in combination with the conventional treatment, at 6 g/sachet, 1 sachet each time, 3 times a day. The clinical data of the patients treated for 7 days were collected.

[0198] 1.6 Observation indicators

[0199] The clinical data of the two groups of patients were analyzed, including the disappearance rate, days to disappearance of main symptoms (fever, fatigue and cough), and the rate of disappearance of other symptoms and signs, efficacy rate for main symptoms, CT
improvement rate, clinical conversion-to-severe type rate, etc.

[0200] 1.7 Evaluation criteria

[0201] (1) Rate of disappearance of symptom: Rate of disappearance of symptom = number of cases with symptom disappearance after treatment / total number of cases x 100%; (2) Efficacy rate for main symptoms: the absence of the main symptoms (fever, fatigue and cough) was scored as 0 point and the presence as 1 point. Symptom score-reducing rate = (score before treatment -score after treatment) / score before treatment x 100%. The symptom score-reducing rate > 30%
was regarded as effective treatment and < 30% as ineffective treatment.
Efficacy rate of treatment = number of cases judged as effectively treated after treatment / total number of cases x 100%. (3) CT improvement rate = number of cases showing improvement in lung imaging (CT) after treatment / total number of cases x 100%; (4) Clinical conversion-to-severe type rate: according to the severe type diagnostic criteria of the "Diagnosis and Treatment Program for Novel Coronavirus Infected Pneumonia (Trial Version 5)[151 clinical conversion-to-severe type rate = number of cases from common type to severe type / total number of cases x 100%.

[0202] 1.8 Statistical methods

[0203] Statistical analysis was performed using SAS 9.4 software. All statistical tests used two-sided test. In descriptive analysis, enumeration data were expressed by the number of cases and constituent ratios, and measurement data were expressed by the mean standard deviation. The t test was used to compare the measurement data between groups, and the chi-square test or the exact probability method was used for the enumeration data. Fever duration was analyzed by survival analysis. P<0.05 represented that the difference was statistically significant.

[0204] 2. Results

[0205] 2.1 General data

[0206] A total of 102 confirmed common-type patients who met the requirements were collected, including 51 in the treatment group and 51 in the control group.

[0207] The treatment group included 26 males (51.0%) and 25 females (49.0%), who had an average age of 55.5 12.3 years old, average body temperature of 38.44 0.63 C, systolic blood pressure of 122.6 11.9 mmHg, diastolic blood pressure of 75.0 9.2 mmHg;
average heart rate of 89.0 11.6 beats/min, and average respiration of 19.8 2.4 beats /min.. There are 15 cases (29.4%) with a history of hypertension, 5 cases (9.8%) with a history of coronary heart disease, 4 cases (7.8%) with a history of diabetes, and 3 cases (5.9%) with a history of cerebral infarction.
Laboratory tests showed that 13 cases (25.5%) had decreased blood leukocytes, 20 cases (39.2%) had decreased lymphocytes, 40 cases (78.4%) had increased high-sensitivity C-reactive protein, 20 cases (39.2%) had increased erythrocyte sedimentation rate, and 16 cases (31.4%) had increased procalcitonin.

[0208] Among 51 cases in the control group, there were 27 males (52.9%) and 24 females (47.1%), with an average age of 55.8 11.6 years old, average body temperature of 38.33 0.64 C, systolic blood pressure of 127.0 16.3 mmHg, diastolic blood pressure of 74.8 10.3 mmHg, average heart rate of 88.7 13.4 beats/min, and average respiration of 20.0 2.8 beats/min. There are 17 cases (33.3%) with a history of hypertension, 2 cases (3.9%) with a history of coronary heart disease, 4 cases (7.8%) with a history of diabetes mellitus, and 6 cases (11.8%) with a history of cerebral infarction. Laboratory tests showed that 16 cases (31.4%) had decreased blood leukocytes, 24 cases (47.1%) had decreased lymphocytes, 42 cases (82.4%) had increased high-sensitivity C-reactive protein, 24 cases (47.1%) had increased erythrocyte sedimentation rate, and 17 cases (33.3%) had increased procalcitonin.

[0209] Baseline data comparison between the two groups, including the age, gender, and baseline data of body temperature, blood pressure, heart rate, respiration, past medical history, and laboratory test results showed no statistically significant difference (P>0.05), indicating that the two group were comparable. The results are shown in Table 12.
Table 12 Comparison of general data between two groups of patients Item Treatment group (N=51) Control group (N=51) Statistics t/x2 P value Age (years, :T s) 55.5 12.3 55.8 11.6 -0.127 0.550 Male (cases, %) 26(51.0%) 27(52.9%) 0.039 0.843 Body temperature ( C, .t s) 38.44 0.63 38.33 0.64 0.848 0.398 Systolic blood pressure 122.611.9 127.016.3 -1.561 0.122 (mmhg, .t s) Diastolic blood pressure 75.0 9.2 74.8 10.3 0.101 0.920 (mmhg, Tcs) Heart rate (beats/min, .T.. s) 89.0 11.6 88.7 13.4 0.126 0.900 Breathing (beats/min, fc s) 19.8 2.4 20.0 2.8 -0.527 0.599 Past medical history 27(52.9%) 26(51.0%) 0.039 0.843 (cases, %) --Hypertension 15(29.4%) 17(33.3%) 0.182 0.670 --Coronary heart disease 5(9.8%) 2(3.9%) 0.436 --Diabetes 4(7.8%) 4(7.8%) 1.000 --Cerebral infarction 3(5.9%) 6(11.8%) 0.487 Abnormal blood leukocytes 13(25.5%) 16(31.4%) 0.434 0.510 (4.) (cases, %) Abnormal lymphocyte (1) 20(39.2%) 24(47.1%) 0.639 0.424 (cases, %) Abnormal high-sensitivity C-reactive protein (f) 40(78.4%) 42(82.4%) 0.249 0.618 (cases, %) Abnormal erythrocyte sedimentation rate (f) 20(39.2%) 24(47.1%) 0.639 0.424 (cases, %) Abnormal procalcitonin (T) 16(31.4%) 17(33.3%) 0.045 0.832 (cases, %) Note: Male, past medical history, and laboratory test results were enumeration data, which were compared by chi-square test; the rest of the items were measurement data, which were compared by t test. 1 mmHgz,0.133 kPa.

[0210] 2.2 Comparison of rate of disappearance of main symptoms between the two groups of patients

[0211] (1) Baseline data: 51 cases in the treatment group, including 43 cases (84.3%) with fever, 31 cases (60.8%) with fatigue, and 37 cases (72.6%) with cough; 51 cases in the control group, including 41 cases (80.4%) with fever, 35 cases with fatigue, and 39 cases (76.5%) with cough.

There were no statistically significant differences between groups. (2) After 7 days of treatment, the fever symptoms disappeared in 36 cases (83.7%) in the treatment group, which was significantly better (x2=5.461, P=0.019) as compared with 25 cases (61.0%) in the control group;
the fatigue symptoms disappeared in 19 cases (61.3%) in the treatment group, which was significantly better (x2=4.813, P=0.028) as compared with 12 cases (34.3%) in the control group;
the cough symptoms disappeared in 23 cases (62.2 %) in the treatment group, which was significantly better (x2=5.243, P=0.022) as compared with 14 cases (35.9 %) in the control group.
The above differences between the groups were all statistically significant (P<0.05). The results are shown in Table 13.
Table 13 Comparison of rate of disappearance of main symptoms between two groups of patients Treatment group Control group Number of Number of Item N N cases with Disappearance cases with Disappearance Statistics x2 P value symptom- rate (%) symptom- rate (%) disappeared disappeared Fever 43 36 83.7% 41 25 61.0% 5.461 0.019 Fatigue 31 19 61.3% 35 12 34.3% 4.813 0.028 Cough 37 23 62.2% 39 14 35.9% 5.243 0.022

[0212] 2.3 Comparison of time of main symptom disappearance between two groups of patients

[0213] In the treatment group, there were 36 cases with fever disappearance at an average duration of 2.9 1.67 days, 19 cases with fatigue disappearance at an average duration of 3.5 1.50 days, and 23 cases with cough disappearance at an average duration of 3.9 1.98. In the control group, there were 25 cases with fever disappearance at an average duration of 3.9 1.29 days, 12 cases with fatigue disappearance at an average duration of 4.8 1.53 days, and 14 cases with cough disappearance at an average duration of 5.2 1.76 days. The time to disappearance of main symptom (fever, fatigue and cough) in the treatment group was shorter than that in the control group, and the difference between the groups was statistically significant (P<0.05). The results are shown in Table 14.
Table 14 Comparison of time of main symptom disappearance between two groups of patients Treatment group Control group Item Case Statistic t P value Result Case number Result number Fever 36 2.9+1.67 25 3.9+1.29 -2.453 0.017 Fatigue 19 3.5+1.50 12 4.8+1.53 -2.342 0.026 Cough 23 3.9+1.98 14 5.2+1.76 -2.083 0.045

[0214] 2.4 Comparison of efficacy rates for main symptoms between two groups of patients

[0215] After 7 days of treatment, main symptoms of 44 of the 51 patients in the treatment group were effectively treated at an efficacy rate of 86.3%, while main symptoms of 35 of the 51 patients in the control group were effectively treated at an efficacy rate of 68.6%.
The difference between the groups was statistically significant (P=0.033).

[0216] 2.5 Comparison of rate of disappearance of other symptoms and signs between two groups of patients

[0217] (1) Baseline data: among 51 cases in the treatment group, there were 9 cases with muscle pain (17.7%), 20 cases with expectoration (39.2%), 13 cases with shortness of breath (25.5%), 11 cases with chest distress (21.6%), 3 cases with dyspnea (5.9%), 7 cases with nausea (13.7%), 23 cases with anorexia (45.1%), and 22 cases with pulmonary moist rale (43.1%);
among 51 cases in the control group, there were 11 cases with muscle pain (21.6%), 19 cases with expectoration (37.3%), 14 cases with shortness of breath (27.5%), 19 cases with chest distress (37.3%), 7 cases with dyspnea (13.7%), 5 cases with nausea (9.8%), 26 cases with anorexia (51.0%), and 23 cases with pulmonary moist rale (45.1%); there was no statistically significant difference between the groups (P>0.05). (2) After 7 days of treatment, in the treatment group, the symptoms of expectoration, shortness of breath, chest distress, anorexia and moist rale disappeared respectively in 11 cases (55.0%), 8 cases (61.5%), 6 cases (54.6%), 8 cases (34.8%) and 10 cases (45.5%), and in the control group, while the mentioned symptoms disappeared respectively in 3 cases (15.8%), 2 cases (14.3%), 3 cases (15.8%), 2 cases (7.7%) and 3 cases (13.0%). The differences between the two groups were statistically significant (P<0.05). In the treatment group, muscle pain, dyspnea, and nausea disappeared respectively in 6 cases (66.7%), 2 cases (66.7%), and 4 cases (57.1%), while in the control group, the mentioned symptoms disappeared respectively in 2 cases (18.2%), 2 cases (28.6%) and 2 cases (40.0%), respectively. There was no statistical difference between the two groups (P>0.05). The results are shown in Table 15.
Table 15 Comparison of rate of disappearance of other symptoms and signs between treatment group and control group Treatment group Control group Number of cases . Number of cases .
Statistic Item Disappearance Disappearance P value N with symptom- N with symptom- x2 rate rate disappeared disappeared Muscle pain 9 6 66.7% 11 2 18.2% -0.065 Expectoration 20 11 55.0% 19 3 15.8%
6.510 0.011 Shortness of 13 8 61.5% 14 2 14.3% -0.018 breath Chest distress 11 6 54.6% 19 3 15.8% -0.042 Dyspnea 3 2 66.7% 7 2 28.6% -0.500 Nausea 7 4 57.1% 5 2 40.0% -1.000 Anorexia 23 8 34.8% 26 2 7.7% -0.032 Moist rale 22 10 45.5% 23 3 13.0%
5.750 0.016

[0218] 2.6 Analysis of conversion to severe type in two groups of patients during treatment

[0219] Among 51 cases in the treatment group, 4 cases (7.8%) became severe in the course of treatment; and among 51 cases in the control group, 11 cases (21.6%) became severe. The difference between the groups was statistically significant (P<0.05).

[0220] 2.7 Comparison of improvement rate of lung imaging (CT) between two groups

[0221] After 7 days of treatment, among 51 cases in the treatment group, 28 cases (54.9%) had improved lung CT, while among 51 cases in the control group, 23 cases (45.1%) had improved lung CT. There was no statistically significant difference between the groups (P>0.05).

[0222] The clinical research results showed that the administration of the pharmaceutical composition of this disclosure significantly improved the symptoms of the common-type patients in the early stage and the middle stage, enabled the patients to easily recover, and significantly reduced the patients transformed from middle stage to severe type, effectively avoiding the transformation to severe and critical types. Moreover, the combined administration of the pharmaceutical composition of this disclosure to critical-type patients stabilized their blood oxygen saturation, improved their dyspnea, promoted their inflammatory absorption of the lung, and avoided sequelae such as interstitial pneumonia and pulmonary fibrosis. The results show that the pharmaceutical composition of this disclosure can significantly improve fever, cough, fatigue, shortness of breath, pulmonary moist rale and other clinical symptoms and signs related to the disease. In addition, the proportion of severe cases in the treatment group also showed an obvious downward trend, indicating that the pharmaceutical composition of this disclosure had good clinical curative effect on suspected cases, such as helping to improve clinical symptoms, relieving disease severity and the like.
Experimental example 6

[0223] In vitro cell studies

[0224] 1. Materials and methods

[0225] 1.1 Cell lines and viruses

[0226] African green monkey kidney epithelial cells (Vero E6) and human hepatoma cell line Huh-7 were purchased from ATCC. Culture conditions: Dulbecco's modified Eagle's medium (DMEM, Gibco, USA) containing 10% fetal bovine serum (FBS). Culture temperature: 37 C. The novel coronavirus (SARS-CoV-2) was a clinical isolate from the First Affiliated Hospital of Guangzhou Medical University.

[0227] 1.2 Test sample

[0228] A. The pharmaceutical composition of this disclosure (LHQW, Example 4).
Preparation and storage of stock solution of the test sample: the test sample was dissolved in dimethyl sulfoxide (DMSO) and stored at -20 C. B. Remdesivir, provided by Professor Zhang Jiancun of Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, was dissolved in DMEM
medium.

[0229] 1.3 Cytotoxicity test

[0230] The cytotoxic effects of LHQW on Vero E6 cells and Huh-7 cells were evaluated by the MTT method. The original culture medium was discarded from the monolayer of Vero E6 cells and Huh-7 cells grown in a 96-well plate. The cells were washed with PBS and incubated with different concentrations of the test solution for 72 h. After 72 h, cells were stained with 0.5 mg/mL MTT
solution for 4 h. The supernatant was discarded, and the formed formazan crystals were dissolved in 200 ilL of dimethyl sulfoxide (DMSO). Absorbance at 570 nm was measured using a full-wavelength microplate reader (Thermo Fisher, USA).

[0231] 1.4 Cytopathic inhibition test

[0232] Vero E6 cells were seeded in a 96-well plate. After grew to monolayer cells, cells were inoculated with a coronavirus strain with a titer of 100 TCID50, and cultured at 37 C for 2 h. The culture medium was discarded. Cells were then added with different concentrations of LHQW
solution or positive control substance Remdesivir, and incubated for 72 h.
After 72 h, the infected Vero E6 cells showed 100% cytopathic effect under the microscope. The percentage of cytopathic effect in LHQW-treated cells was recorded. The half inhibitory concentration (IC50) of LHQW on virus-induced cytopathic effect was calculated.

[0233] 1.5 Plaque reduction assay

[0234] Vero E6 cells were cultured on a 6-well plate to a monolayer, and the supernatant was discarded. Cells were washed with PBS, and 50 PFU of SARS-CoV-2 was added to the plate and incubated for 2 h. The supernatant was discarded, and cells were added with basal medium containing 1.2% agarose (Invitrogen, USA) and different concentrations of LHQW
or positive control Remdesivir per ml and incubated at 37 C for 48 h. After that, cells were fixed with 4%
formalin for 20 min and stained with 1% crystal violet for 3 min. The plaque inhibition rate was calculated, and the half inhibitory concentration (IC50) of the drug on the virus was calculated.

[0235] 1.6 RT-qPCR method

[0236] The Huh-7 monolayer cells seeded in a 12-well plate were washed with PBS, and infected with coronavirus at a multiplicity of infection (MOD of 1 at 37 C for 2 h. The supernatant was discarded. The test substance was diluted to the desired concentration with medium containing 2%
FBS and added to a 12-well plate for 48 h of culture. The collected cells were used for RNA
isolation and qPCR detection. The primer and probe sequences used for the analysis are shown in Table 16. With GAPDH as an internal reference factor, the 2-AACt method was used to calculate the relative mRNA expression.

Table16 Primers for qRT-PCR used in this study Gene Primers and Probe Sequence (5'¨>3') IL-6 Forward CGGGAACGAAAGAGAAGCTC TA
Reverse CGCTTGTGGAGAAGGAGTTCA
Probe TCCCCTCCAGGAGCCCAGCT
MCP-1 Forward CAAGCAGAA.GTGGGTICAGGAT
Reverse AGTGAGTGTTCAAGTCTTCGGAGTT
Probe CATGGACCACCTGGACAAGCAAACC
TNF-a Forward AACATCCAACCTTCCCAAACG
Reverse GACCCTAAGCCCCCAATTCTC
Probe CCCCCTCCTTCAGACACCCTCAACC
IP-10 Forward GAAATTATTCCTGCAAGCCAATTT
Reverse TCACCCTTCTTTTTCAT-TGTAGCA
Probe TCCACGTGTTGAGATCA
GAPDH Forward GAAGGTGAAGGTCGGAGTC
Reverse GAAGATGGTGATGGGATTTC
Probe CAAGCTTCCCGTTCTCAGCC

[0237] 1.7 Electron Microscopic Analysis

[0238] Cells were fixed, dehydrated and embedded. The embedded paraffin block was cut into 70 nm ultrathin sections, stained with uranyl acetate and lead citrate, and then viewed under a JEM-1400 PLUS transmission electron microscope.

[0239] 2. Statistical analysis

[0240] Statistical analysis was performed using GraphPad Prism 7.0 software.
One-way analysis of variance (ANOVA) was used to compare the differences in the mRNA expression levels of pro-inflammatory cytokines among the groups. The test statistical significance level was set at p<0.05.

[0241] 3. Results

[0242] 3.1 In vitro antiviral activity of LHQW against SARS-CoV-2

[0243] Cell viability of Vero E6 cells treated with LHQW or remdesivir was determined by the MTT assay. At a concentration of 600 pg/mL, LHQW was significantly cytotoxic to VeroE6 cells (results are shown in FIG. 4A). The positive control Remdesivir was not cytotoxic to cells at a concentration of 50 RM (results are shown in FIG. 4B).

[0244] As shown in FIG. 5A, the inhibitory effect of LHQW on SARS-CoV-2 virus was determined by cytopathic effect inhibition assay and plaque reduction assay, with an IC50 value of 411.2 pg/mL (see FIGs. 5A and 5C). The IC50 value of Remdesivir against SARS-CoV-2 virus was 0.651 pM.

[0245] 48 h after virus infection of Vero E6 cells, virions were found in the cytoplasm, intracellular vesicles, endoplasmic reticulum, and cell membrane, and typical coronavirus morphology was observed under electron microscopy (see FIGs. 6B and 6F). After treatment with LHQW (600 pg), the viral expression decreased (see FIGs. 6C and 6G). More significantly, LHQW-treated cells altered virus morphology, as many oval-shaped virions were present on the cell surface (see FIG. 6H), which differ from the spherical, pointed virions in virus-infected cells (see FIGs. 6B and 6F).

[0246] 3.2 Inhibitory effect of LHQW on SARS-CoV-2-induced cytokine and chemokine expression

[0247] To determine the role of LHQW in inhibiting SAR2-CoV-2-induced cytokine and chemokine expression, mRNA expression levels of TNF-a, IL-6, CCL-2/MCP-1 and 10 were determined in LHQW-treated cells. The results showed that the expression of cytokines and chemokines increased after 48 h of virus infection, and LHQW had a significant inhibitory effect on these cytokines and chemokines, and the inhibitory effect was concentration-dependent.
The results are shown in FIG. 7.
Conclusion

[0248] The above research results show that the pharmaceutical composition LHQW of the present disclosure can exert its anti-novel coronavirus activity by inhibiting virus replication and reducing the release of cytokines from host cells.

Claims (16)

1. Use of a pharmaceutical composition in the manufacture of a medicament for preventing or treating novel coronavirus-infected pneumonia, wherein the pharmaceutical composition is prepared from the following materials in parts by weight:
forsythiae fructus 150-260 lonicerae japonicae flos 150-260 isatidis radix 150-260 rhei radix et rhizoma 30-55 pogostemonis herba 50-90 dryopteridis crassirhizomatis rhizoma 150-rhodiolae crenulatae radix et rhizoma 50-90 /-menthol 5-8 honey-fried ephedrae herba 50-90 stir-baked armeniacae semen amarum 50-90 houttuyniae herba 150-260 glycyrrhizae radix et rhizoma 50-90 gypsum fibrosum 150-260.

2. The use according to claim 1, wherein the pharmaceutical composition is prepared from the following materials in parts by weight:
forsythiae fructus 150 lonicerae japonicae flos 260 isatidis radix 150 rhei radix et rhizoma 55 pogostemonis herba 50 dryopteridis crassirhizomatis rhizoma 260 rhodiolae crenulatae radix et rhizoma 50 /-menthol 8 honey-fried ephedrae herba 50 stir-baked arrneniacae sernen amarum 90 houttuyniae herba 150 glycyrrhizae radix et rhizoma 90 gypsum fibrosum 150.

3. The use according to claim 1, wherein the pharmaceutical composition is prepared from the following materials in parts by weight:
forsythiae fructus 260 lonicerae japonicae flos 150 isatidis radix 260 rhei radix et rhizoma 30 pogostemonis herba 90 dryopteridis crassirhizomatis rhizoma 150 rhodiolae crenulatae radix et rhizoma 90 /-menthol 5 honey-fried ephedrae herba 90 stir-baked armeniacae semen amarum houttuyniae herba 260 glycyrrhizae radix et rhizoma 50 gypsum fibrosum 260.

4. The use according to claim 1, wherein the pharmaceutical composition is prepared from the following materials in parts by weight:
forsythiae fructus 170 lonicerae japonicae flos 170 isatidis radix 170 rhei radix et rhizoma 34 pogostemonis herba 57 dryopteridis crassirhizomatis rhizoma 170 rhodiolae crenulatae radix et rhizoma 57 /-menthol 5 honey-fried ephedrae herba 57 stir-baked armeniacae semen amarum houttuyniae herba 170 glycyrrhizae radix et rhizoma 57 gypsum fibrosum 170.

5. The use according to claim 1, wherein the pharmaceutical composition is prepared from the following materials in parts by weight:
forsythiae fructus 255 lonicerae japonicae flos 255 isatidis radix 255 rhei radix et rhizoma 51 pogostemonis herba 85 dryopteridis crassirhizomatis rhizoma 255 rhodiolae crenulatae radix et rhizoma 85 /-menthol 7.5 honey-fried ephedrae herba 85 stir-baked arrneniacae semen amarum 85 houttuyniae herba 255 glycyrrhizae radix et rhizoma 85 gypsum fibrosum 255.

6. The use according to any one of claims 1-5, wherein active ingredients of the pharmaceutical composition are prepared by:
step (1) weighing the materials according to weight ratio and performing selection to remove impurities;
step (2) chopping pogostemonis herba, extracting volatile oil from the pogostemonis herba with water having amount of 5-8 times of the pogostemonis herba for 4 h to collect the volatile oil and obtain an extract liquid, and filtering the extract liquid to discard residue and obtain filtrate;
step (3) performing extraction on a mixture of forsythiae fructus, honey-fried ephedrae herba, houttuyniae herba and rhei radix et rhizoma twice for 1-3 h each time by using 50-90% ethanol in an amounts of 6-10 times of the mixture to obtain extract liquids, combining and filtering the extract liquids, distilling the filtrate to recover ethanol and obtain alcohol extract;
step (4) To a mixture of the lonicerae japonicae flos, gypsum fibrosum, isatidis radix, dryopteridis crassirhizomatis rhizoma, glycyrrhizae radix et rhizoma and rhodiolae crenulatae radix et rhizoma, adding water in an amount of 7-11 times of the mixture, heating to boil, adding stir-baked armeniacae semen amarum, boiling twice for 0.5-2.5 h each time to obtain extract liquids, combining and filtering the extract liquids to obtain filtrate, combining the obtained filtrate with the filtrate obtained after the extracting of volatile oil from the pogostemonis herba in step (2) to obtain a combined filtrate, concentrating the combined filtrate into a clear paste with a relative density of 1.10-1.15 measured at 60 C, adjusting the clear paste to an alcohol concentration of 70%
by adding ethanol, refrigerating, filtering, and distilling the filtrate to recover ethanol until no ethanol smell and obtain alcohol extract;
step (5) combining the clear paste obtained in step (4) with the alcohol extract obtained in step (3), concentrating the resultant into a clear paste with a relative density of 1.15-1.20 measured at 60 C, and drying to obtain dry paste powder; and step (6) mixing the dry paste powder obtained in step (5), the volatile oil obtained in step (2) and /-menthol to prepare the active ingredients of the pharmaceutical composition.

7. The use according to any one of claims 1-5, wherein the medicament is in a form selected from the group consisting of capsule, tablet, powder, granule, oral liquid, pill, tincture, syrup, suppository, gel, spray and injection.

8. The use according to claim 7, wherein the capsule is prepared by a process comprising:
step (1) weighing the materials according to weight ratio and performing selection to remove impurities;
step (2) chopping pogostemonis herba, extracting volatile oil from the pogostemonis herba with water having amount of 5-8 times of the pogostemonis herba for 4 h to collect the volatile oil and obtain an extract liquid, filtering the extract liquid to discard residue and obtain filtrate;
step (3) performing extraction on a mixture of forsythiae fructus, honey-fried ephedrae herba, houttuyniae herba and rhei radix et rhizoma twice for 1-3 h each time by using 50-90% ethanol in an amount of 6-10 times amounts of the mixture to obtain extract liquids, combining and filtering the extract liquids, distilling the filtrate to recover ethanol and obtain alcohol extract;
step (4) to a mixture of lonicerae japonicae flos, gypsum fibrosum, isatidis radix, dryopteridis crassirhizomatis rhizoma, glycyrrhizae radix et rhizoma and rhodiolae crenulatae radix et rhizoma, adding water in an amount of 7-11 times of the mixture, heating to boil, adding stir-baked armeniacae semen amarum, boiling twice for 0.5-2.5 h each time to obtain extract liquids, combining and filtering the extract liquids to obtain filtrate, combining the obtained filtrate with the filtrate obtained after the extracting of volatile oil from the pogostemonis herba in step (2), concentrating the resultant into a clear paste with a relative density of 1.10-1.15 measured at 60 C, adjusting the clear paste to an alcohol concentration of 70% by adding ethanol, refrigerating, filtering, and distilling the filtrate to recover ethanol until no ethanol smell and obtain alcohol extract;
step (5) combining the clear paste obtained in step (4) with the alcohol extract obtained in step (3), concentrating the resultant into a clear paste with a relative density of 1.15-1.20 measured at 60 C, and drying to obtain dry paste powder;
step (6) preparing the dry paste powder obtained in step (5) into particles with a suitable pharmaceutically acceptable adjuvant; and step (7) dissolving /-menthol and the volatile oil obtained in step (2) into ethanol, spraying the resultant onto the particles obtained in step (6), sealing, mixing well, and packaging into the capsule.

9. The use according to claim 7, wherein the granule is prepared by a process comprising:
step (1) weighing the materials according to weight ratio, pefforming selection to remove impurities, and chopping;
step (2) chopping pogostemonis herba, extracting volatile oil from the pogostemonis herba with water having amount of 5-8 times of the pogostemonis herba for 4 h to collect the volatile oil and obtain an extract liquid, filtering the extract liquid to discard residue and obtain filtrate;
step (3) performing extraction on a mixture of forsythiae fructus, honey-fried ephedrae herba, houttuyniae herba and rhei radix et rhizoma twice for 1-3 h each time by using 50-90% ethanol in an amount of 6-10 times of the mixture to obtain extract liquids, combining and filtering the extract liquids, distilling the filtrate to recover ethanol and obtain alcohol extract;
step (4) to a mixture of lonicerae japonicae flos, gypsum fibrosum, isatidis radix, dryopteridis crassirhizomatis rhizoma, glycyrrhizae radix et rhizoma and rhodiolae crenulatae radix et rhizoma, adding water in an amount of 7-11 times of the mixture, heating to boil, adding stir-baked armeniacae semen amarum, boiling twice for 0.5-2.5 h each time to obtain extract liquids, combining and filtering the extract liquids to obtain filtrate, combining the obtained filtrate with the filtrate obtained after the extracting of volatile oil from the pogostemonis herba in step (2), concentrating the resultant into a clear paste with a relative density of 1.10-1.15 measured at 60 C, adjusting the clear paste to an alcohol concentration of 70% by adding ethanol, refrigerating, filtering, and distilling the filtrate to recover ethanol until no ethanol smell and obtain alcohol extract;
step (5) combining the clear paste obtained in step (4) with the alcohol extract obtained in step (3), concentrating the resultant into a clear paste with a relative density of 1.15-1.20 measured at 60 C, and drying to obtain dry paste powder;
step (6) preparing the dry paste powder obtained in step (5) into particles with a suitable pharmaceutically acceptable adjuvant; and step (7) dissolving /-menthol and the volatile oil obtained in step (2) into ethanol, spraying the resultant onto the particles obtained in step (6), sealing, mixing well, and bagging to obtain the granule.

10. The use according to claim 9, wherein the granule is prepared by a process comprising:
step (1) weighing the materials according to weight ratio, performing selection to remove impurities, and chopping;
step (2) chopping pogostemonis herba, extracting volatile oil from the pogostemonis herba with water having an amount of 6 times of the pogostemonis herba for 4 h, to collect the volatile oil with an oil yield of 0.33 0.05% and obtain an extract liquid, filtering the extract liquid to discard residue and obtain filtrate;
step (3) performing extraction on a mixture of forsythiae fructus, honey-fried ephedrae herba, houttuyniae herba and rhei radix et rhizoma twice for 2 h at the first time and for 1.5 h at the second time, with 50-90% ethanol having an amount of 8 times of the mixture, to obtain extract liquids, combining and filtering the extract liquids, recovering the ethanol, and collecting filtrate;
step (4) to a mixture of lonicerae japonicae flos, gypsum fibrosum, isatidis radix, dryopteridis crassirhizomatis rhizoma, glycyrrhizae radix et rhizoma and rhodiolae crenulatae radix et rhizoma, adding water in an amount of 9 times of the mixture, heating to boil, adding stir-baked armeniacae semen amarum, boil twice for 1.5 h at the first time and for 1 h at the second time to obtain extract liquids, combining and filtering the extract liquids to obtain filtrate, combining the obtained filtrate with the water extract obtained after the extracting of volatile oil from the pogostemonis herba in step (2), concentrating the resultant into a clear paste with a relative density of 1.10-1.15 measured at 60 C, adjusting the clear paste to an alcohol concentration of 70% by adding 95% ethanol, refrigerating, filtering, and distilling the filtrate to recover ethanol until no ethanol smell and obtain alcohol extract;
step (5) combining the clear paste obtained in step (4) with the alcohol extract obtained in step (3), concentrating the resultant into a thick paste with a relative density of 1.15-1.20 measured at 60 C;
step (6) preparing the thick paste obtained in step (5) into particles with a suitable pharmaceutically acceptable adjuvant, and granulating the particles to obtain granulated particles;
and step (7) sieving out fine powder from the granulated particles obtained in step (6), dissolving /-menthol and the volatile oil obtained in step (2) into ethanol, spraying the resultant onto the fine powder and mixing well, mixing well with the granulated particles obtained in step (6), sealing for half an hour, and bagging to obtain the granule.

11. The use according to any one of claims 1-5, wherein the medicament is used for improving shortness of breath and/or dyspnea.

12. The use according to any one of claims 1-5, wherein the medicament is used for improving fever, cough or fatigue.

13. The use according to any one of claims 1-5, wherein the medicament is used for improving chest distress.

14. The use according to any one of claims 1-5, wherein the medicament is used for improving pulmonary moist rale.

15. The use according to any one of claims 1-5, wherein the medicament is used for inhibiting cytokine elevation or chemokine elevation.

16. The use according to claim 15, wherein the cytokine or chemokine is selected from the group consisting of TNF-a, IL-6, CCL-2/MCP-1 and CXCL-10/I13-10.