CN114712341A - Application of carnosic acid in preparing medicine for preventing and treating influenza - Google Patents

Application of carnosic acid in preparing medicine for preventing and treating influenza Download PDF

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CN114712341A
CN114712341A CN202210397264.XA CN202210397264A CN114712341A CN 114712341 A CN114712341 A CN 114712341A CN 202210397264 A CN202210397264 A CN 202210397264A CN 114712341 A CN114712341 A CN 114712341A
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influenza
carnosic acid
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陈建新
彭海英
刘雅红
亓文宝
武力
罗伟
胡海波
邱电
何应敏
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Hunan Zhizhiyuan Agriculture And Animal Husbandry Co ltd
South China Agricultural University
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Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of carnosic acid in preparation of a medicine for preventing and treating influenza. A large amount of experimental data prove that carnosic acid has a remarkable antiviral effect on influenza viruses at a cellular level, and animal experiments prove that carnosic acid has a remarkable protective effect on mice infected with influenza viruses and the survival rate of the mice is improved. The carnosic acid can be extracted from plants, has wide sources and high safety, and is very suitable for being prepared into a medicament for preventing and treating influenza virus.

Description

Application of carnosic acid in preparation of medicine for preventing and treating influenza
Technical Field
The invention belongs to the technical field of biological medicines. More particularly, relates to the application of carnosic acid in preparing medicaments for preventing and treating influenza.
Background
Influenza, referred to as Influenza for short, is an acute respiratory infectious disease caused by Influenza Virus (IVs), and Influenza is classified into pandemic Influenza and seasonal Influenza according to the size of the spread range. Influenza is a zoonosis, has a wide host range, and can be infected in both birds and mammals, wherein waterfowls are the main hosts of influenza viruses. Influenza viruses that can infect birds are collectively referred to as avian influenza viruses, wherein all avian influenza viruses belong to the influenza a virus. Generally, the avian influenza virus only infects a natural host, and two subtypes of H5N1 and H7N9 of the avian influenza also have the potential of infecting human beings and causing human diseases, wherein the H5N1 subtype influenza virus belongs to highly pathogenic avian influenza virus, and causes great harm to both birds and human beings.
Among the traditional methods of prevention and treatment, vaccination is one of the most effective strategies for preventing influenza. However, for some high risk groups such as the old, children and the like, effective immunity is difficult to generate by vaccination; on the other hand, due to the high variability of influenza viruses, effective vaccines are constantly being updated for the prevention of infection by influenza viruses, and a period of at least six months is usually required from the identification of a strain to the development of a corresponding vaccine. Therefore, the anti-influenza virus medicine is still the main prevention and treatment method for preventing and treating influenza diseases at present and has a vital function.
Drugs approved by FDA for clinical treatment of influenza mainly include M2 ion channel inhibitors (amantadine and rimantadine), Neuraminidase (NA) inhibitors (peramivir, oseltamivir, and zanamivir), and recently japanese viral polymerase inhibitors fapirovir and Cap-dependent endonuclease inhibitors bazalovir, which are developed in japan, in limited varieties. However, researches show that influenza epidemic strains are easy to have drug resistance due to long-term single drug administration, for example, amantadine and rimantadine which are used in the beginning of 1970 are used for a long time, the current influenza strains are completely resistant, and the WHO does not recommend amantadine and rimantadine as seasonal influenza virus treatment drugs; in recent years, there have been increasing reports of oseltamivir resistance (Tani N, Kawai N, Chong Y, et al. Susceptibility of epidemic viruses to neuroamidinate inhibitors and treatment-epidemic resistance in the Japanese 2019-20 influenza search [ J ]. Journal of Infection,2022,84(2): 151-. Therefore, the development of new drugs against influenza virus is imminent.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defect and the defect that the existing common anti-influenza virus has drug resistance, and provides the application of carnosic acid in preparing the drugs for preventing and treating influenza.
The above purpose of the invention is realized by the following technical scheme:
carnosic Acid (CA) belongs to a phenol diterpene compound, and the molecular formula is C20H28O4Molecular weight is 332.43, and the product is light yellow solid, slightly soluble in water, and easily soluble in organic solvent such as ethanol. Carnosic acid is widely distributed in rosemary, sage and clary sage and has antioxidant, antibacterial, antiinflammatory, antitumor, neuroprotective and obesity inhibiting biological activities.
The inventor extracts high-purity carnosic acid from rosemary air-dried leaves, and determines that the content of the carnosic acid is more than 98% by adopting a high performance liquid chromatography. Indirect immunofluorescence microscopic analysis, virus titer determination and other methods find that carnosic acid has obvious antiviral effect on low-pathogenicity influenza viruses such as high-pathogenicity influenza viruses H5N1 and H1N1 in human lung cancer epithelial cells (A549), and animal experiments confirm that carnosic acid has obvious protective effect on mice infected with the high-pathogenicity influenza viruses H5N 1. The carnosic acid is proved to have obvious anti-influenza virus effect.
Accordingly, the present invention claims the use of carnosic acid in the preparation of a medicament for the prevention or treatment of influenza, said carnosic acid having the structure of formula I:
Figure BDA0003599525290000021
wherein the carnosic acid is commercially available or extracted from a plant rich in carnosic acid.
Carnosic acid may be prepared by the following method:
s1, pulverizing the dry leaves of rosemary by a pulverizer, sieving the pulverized leaves (20 meshes), and heating and refluxing the pulverized leaves of rosemary by using an ethanol solution with the volume fraction of 60-80% at 50 ℃ for two times, wherein each time lasts for 2 hours; collecting filtrate, and distilling under reduced pressure to remove solvent to obtain extract;
s2, weighing H1020 resin, filling the H1020 resin into a column by a wet method, and treating the resin in the column by a conventional method; uniformly dispersing the extract obtained in the step S1 by using 40-60% ethanol solution, adding the extract into a resin column to adsorb carnosic acid, wherein the adsorption temperature is 25 ℃, and the saturation adsorption time is 3 hours; desorbing by using 85-95% ethanol solution as a desorbent; recovering the desorption solution, and removing the solvent by reduced pressure distillation to obtain a crude carnosic acid product;
s3, purifying the crude carnosic acid obtained in the step S2 by silica gel column chromatography, wherein the granularity of silica gel for column chromatography is 83-165 mu m, and the eluent is ethyl acetate-n-hexane 4:6(v/v) with the diameter-height ratio of 1: 10; and (4) carrying out post-treatment on the eluent to obtain carnosic acid.
Further, the influenza is caused by an influenza virus which is an influenza a virus, an influenza b virus, an influenza c virus, or an influenza d virus.
Preferably, the influenza virus is an influenza a virus.
Preferably, the influenza a virus is an influenza a virus of H1N1, H5N1, H3N2, H7N1, H7N2, H7N3, H7N7, H7N9, H9N2 or H10N 8. More preferably, the influenza a virus is an influenza a virus of H1N1, H5N1 or H3N 2.
Furthermore, the medicine takes carnosic acid or pharmaceutically acceptable salt thereof as an effective component.
Further, the virus inhibitory concentration of carnosic acid on cells is 5 μ M or more. Preferably, the virus inhibitory concentration of carnosic acid on cells is 5-20. mu.M.
The use of carnosic acid as claimed in the present invention for the preparation of a medicament for the prevention and treatment of influenza, which medicament, in addition to being beneficial for the treatment of humans, can be applied in the veterinary medicine field for the treatment of pets, animals of the introduced species and animals in farms, including avians, mammals, rodents and the like.
Furthermore, the virus inhibitory dose of carnosic acid on mice is 10-40 mg/kg/d. The corresponding dosage conversion can be carried out according to different application objects (such as human beings or animals).
Furthermore, the medicine also contains pharmaceutically acceptable auxiliary materials.
Further, the dosage form of the medicament is oral preparation, injection or inhalant. Preferably, the oral preparation includes tablets, capsules, granules, powders, oral liquids, and the like.
The invention has the following beneficial effects:
the invention provides a new application of carnosic acid in preparing a medicament for preventing and treating influenza, which is proved by a large amount of experimental data that carnosic acid has a remarkable antiviral effect on influenza viruses at a cellular level, and animal experiments prove that carnosic acid has a remarkable protective effect on mice infected with influenza viruses and the survival rate of the mice is improved. The carnosic acid can be extracted from plants, has wide sources and high safety, and is very suitable for being prepared into a medicament for preventing and treating influenza virus.
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FIG. 1 shows the cytotoxicity assay results of carnosic acid on A549 cells in example 1.
FIG. 2 is a fluorescence diagram of the inhibition effect of different concentrations of carnosic acid on the expression of highly pathogenic influenza virus H5N1NP protein in A549 cells analyzed by the indirect immunofluorescence method in example 2.
Fig. 3 is a statistical chart of the data in example 3, which analyzed the inhibition of highly pathogenic influenza virus H5N1 replication by different concentrations of carnosic acid in a549 cells by end point dilution.
FIG. 4 is a graphic representation of the statistical data of example 4, which was analyzed by end-point dilution of different concentrations of carnosic acid in A549 cells for inhibition of replication of influenza virus H1N1pdm 09.
Figure 5 is a statistical plot of the data from example 5 analyzed by end point dilution of different concentrations of carnosic acid for inhibition of replication of influenza virus H3N2 in a549 cells.
FIG. 6 is a data set of the protective effects of carnosic acid on mice infected with the highly pathogenic influenza virus H5N1 in example 6.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
The carnosic acid adopted in the embodiment of the invention is prepared by the following method:
s1, pulverizing the dry leaves of rosemary by a pulverizer, sieving by a sieve (20 meshes), and heating and refluxing the pulverized leaves of rosemary by using 70 percent ethanol solution with volume fraction at 50 ℃ for two times, wherein each time lasts for 2 hours; collecting the filtrate, and distilling under reduced pressure to remove the solvent to obtain an extract, wherein the yield is 18-23%, and the content of carnosic acid in the extract is 11-15%;
s2, weighing H1020 resin, filling the H1020 resin into a column by a wet method, and treating the resin in the column by a conventional method; dispersing the extract obtained in the step S1 uniformly by using 50% ethanol solution, adding the extract into a resin column to adsorb carnosic acid, wherein the adsorption temperature is 25 ℃, and the saturation adsorption time is 3 hours; desorbing with 90% ethanol solution as desorbent; recovering the desorption solution, and removing the solvent by reduced pressure distillation to obtain a crude carnosic acid product with the carnosic acid content of 70-78%;
s3, purifying the carnosic acid crude product obtained in the step S2 by adopting a silica gel column chromatography, wherein the granularity of silica gel for column chromatography is 83-165 mu m, and the eluent is ethyl acetate-n-hexane 4:6(v/v) and the diameter-height ratio is 1: 10; the eluent is post-treated to obtain carnosic acid with the purity of more than 98 percent.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1 cytotoxicity assay results of carnosic acid on A549 cells
1. The experimental method comprises the following steps:
when A549 cells in a 96-well plate grow to about 90% of a monolayer in an adherent manner, the supernatant is aspirated and discarded, each well is washed by PBS, a compound to be tested which is diluted twice by times by DMEM culture solution is added, a blank control group is set, and each group is provided with 6 repeated wells with 100 mu L/well. And placing the mixture in a cell incubator to incubate for 24h, 48h or 72h respectively. The liquid in each well was aspirated and discarded, washed twice with PBS, and added with 0.5mg/mL of a tetramethylazodicarbonamide (MTT) solution at 100. mu.L/well under dark conditions, and incubated for 4 hours at 37 ℃ in an incubator under dark conditions. The liquid in each well was aspirated, and dimethyl sulfoxide (DMSO) was added at 150. mu.L/well, followed by shaking in the dark for 10 min. Detecting OD value of cells with wavelength of 570nm by using a full-wavelength enzyme-labeled detector, calculating relative activity of the cells, and calculating the obtained data by using GraphPadprism8.0 to obtain CC of each compound to be detected50The value is obtained.
2. The experimental results are as follows:
experimental results referring to FIG. 1, it can be seen that CC is observed when carnosic acid acts on A549 cells for 48h50The product has a purity of 37.89 mu M and good safety.
Example 2 analysis of inhibitory Effect of different concentrations of carnosic acid on the expression of highly pathogenic influenza virus H5N1NP protein in A549 cells by Indirect immunofluorescence method
1. The experimental method comprises the following steps:
when the human alveolar epithelial carcinoma cells (A549) in the 96-well plate grow to about 90% of a monolayer in an adherent manner, the supernatant is aspirated and discarded, each well is washed with PBS, and DMEM culture solution is added to dilute the solution to 100TCID50100 mu L/well of influenza virus solution, 37 ℃ and 5% CO2Culturing in a cell incubator for 1h, and setting a blank control group. After 1h of infection, the liquid in each well was aspirated, carnosic acid with concentrations of 5. mu.M, 10. mu.M and 20. mu.M diluted in DMEM medium was added, a virus control and a positive drug control were set, and the incubation was continued in an incubator for 48 h. Terminating the culture after 48h and discardingWashing the supernatant with PBS for 3 times, adding 120 μ L of paraformaldehyde with mass concentration of 4% into each well, and fixing for 15 min; washing with PBS for 3 times, adding 50 μ L Triton X-100 with mass concentration of 0.25%, and standing at room temperature for 10 min; adding BSA (bovine serum albumin) with the mass concentration of 5% to block the hybrid protein, and blocking for 1h at room temperature; washing with PBS for 3 times, adding murine influenza NP protein monoclonal antibody (diluted 1: 500), and incubating overnight at 4 deg.C; washing with PBS 3 times, adding secondary antibody (Alexa) in dark
Figure BDA0003599525290000051
488-labeled anti-mouse IgG, 1: 1000) incubating for 1h at 37 ℃ in the dark; washing with PBS for three times in dark condition, staining cell nucleus with 300nM DAPI, incubating at room temperature for 5min, and washing with PBS for two times; and observing under a fluorescence microscope and photographing and recording.
2. The experimental results are as follows:
the experimental result is shown in fig. 2, and it can be seen from the figure that the medicinal carnosic acid has a significant inhibition effect on the synthesis of NP protein of influenza virus H5N1 in A549 cells within the concentration range of 5-20 μ M, and shows a good dose-effect relationship.
Example 3 analysis of the inhibitory effect of different concentrations of carnosic acid on the replication of highly pathogenic influenza virus H5N1 in A549 cells by end-point dilution
1. The experimental method comprises the following steps:
the steps of infecting A549 cells with influenza virus H5N1 and adding drugs are the same as in example 1, the culture is stopped after incubation in an incubator for 48H, the cell plate is repeatedly frozen and thawed three times at-80 ℃ and 4 ℃ to fully lyse the cells, so that the virus in the cells is completely released to cell supernatant, and then the supernatant of each well is collected. The freeze-thawed samples were placed on ice and diluted using an end point dilution (Ramakrishnan M A. determination of 50% end point using a single template for use [ J ]]World journal of virology,2016,5(2):85), diluting each group of cell samples by 10 times with a DMEM culture solution, taking out 96-well plates with MDCK cells growing adherently to about 90% of a single layer, washing with PBS once, inoculating the sample diluent, 100 μ L/well, 6 repeat wells, placing in a cell incubator for incubation for 2 h; washing away the influenza virus which does not adsorb cells by PBS, replacing a blank DMEM culture solution, culturing at 100 mu L/hole continuously; recording after 48hIn the case of cytopathic effect (CPE), TCID of each group of samples was calculated by the Reed-Muench method50The value is obtained.
2. The experimental results are as follows:
results of the experiment are shown in fig. 3, where "+" indicates a significant difference (P <0.05), "+" indicates a very significant difference (P <0.01) and "+" indicates a very significant difference (P <0.01) compared to the virus-infected control group.
As can be seen from the figure, the medicine carnosic acid has obvious inhibition effect on replication of influenza virus H5N1 progeny virus in A549 cells within the concentration range of 5-20 mu M, and shows good dose-effect relationship. Particularly, when the concentration of the carnosic acid is in the range of 10-20 mu M, the carnosic acid shows a very significant difference on the replication of the progeny virus of the influenza virus H5N1 in A549 cells.
Example 4 analysis of the inhibitory Effect of different concentrations of carnosic acid on replication of influenza virus H1N1pdm 09 in A549 cells by end-point dilution
1. The experimental method comprises the following steps:
when A549 cells in a 96-well plate grow to about 90% of a monolayer adherent thereto, the supernatant is aspirated and washed with PBS, and DMEM culture solution is added to dilute the solution to 100TCID50H1N1 influenza virus solution (100 μ L/well, 37 deg.C, 5% CO)2Culturing in a cell incubator for 2h, and setting a blank control group. After 2h of infection, the liquid in each well was aspirated, carnosic acid at concentrations of 5. mu.M, 10. mu.M and 20. mu.M diluted in DMEM medium was added, a virus control and a positive drug control were set, and the incubation was continued in an incubator for 48 h. After 48h, the culture is terminated, the cell plate is repeatedly frozen and thawed three times at-80 ℃ and 4 ℃ to fully lyse the cells, so that the viruses in the cells are completely released to cell supernatants, and then the supernatants of all the wells are collected. Placing the freeze-thawed sample on ice, adopting an end point dilution method, diluting each group of cell samples by 10 times by using a DMEM culture solution, taking out 96-well plates with MDCK cells growing in an adherent manner to about 90% of a monolayer, washing the 96-well plates once by using PBS, inoculating the sample diluent, inoculating 100 mu L/well and 6 repeated wells, and placing the inoculated sample diluent in a cell culture box for incubation for 2 hours; washing away the influenza virus which does not adsorb cells by PBS, replacing blank DMEM culture solution, culturing at 100 mu L/hole continuously; the CPE status was recorded after 48h,calculating TCID of each group of samples by a Reed-Muench method50The value is obtained.
2. The experimental results are as follows:
results of the experiment referring to fig. 4, "+" indicates significant difference (P <0.05) and "+" indicates very significant difference (P <0.01) compared to the virus-infected control group.
As can be seen from the figure, the medicine carnosic acid has obvious inhibition effect on generation of progeny viruses of influenza virus H1N1pdm 09 in A549 cells within the concentration range of 10-20 mu M, and shows good dose-effect relationship. In particular, when the concentration of carnosic acid was 20 μ M, the inhibitory effect on progeny virus of influenza virus H1N1pdm 09 in a549 cells was very significant.
Example 5 analysis of the inhibitory Effect of different concentrations of carnosic acid on replication of influenza virus H3N2 in A549 cells by end-point dilution
1. The experimental method comprises the following steps:
the experimental procedure of reference example 4 was followed, except that the virus infected with a549 cells was replaced with influenza virus H3N2, and other parameters and procedures were as in reference example 4.
When A549 cells in a 96-well plate grow to about 90% of a monolayer adherent thereto, the supernatant is aspirated and washed with PBS, and DMEM culture solution is added to dilute the solution to 100TCID50H3N2 influenza virus liquid (100 μ L/well, 37 deg.C, 5% CO)2Culturing in a cell incubator for 2h, and setting a blank control group. After 2h of infection, the liquid in each well was aspirated, carnosic acid at concentrations of 5. mu.M, 10. mu.M and 20. mu.M diluted in DMEM medium was added, a virus control and a positive drug control were set, and the incubation was continued in an incubator for 48 h. After 48h, the culture is terminated, the cell plate is repeatedly frozen and thawed three times at-80 ℃ and 4 ℃ to fully lyse the cells, so that the viruses in the cells are completely released to cell supernatants, and then the supernatants of all the wells are collected. Placing the freeze-thawed sample on ice, adopting an end point dilution method, diluting each group of cell samples by 10 times by using a DMEM culture solution, taking out 96-well plates with MDCK cells growing in an adherent manner to about 90% of a monolayer, washing the 96-well plates once by using PBS, inoculating the sample diluent, inoculating 100 mu L/well and 6 repeated wells, and placing the inoculated sample diluent in a cell culture box for incubation for 2 hours; PBS washed off unadsorbed finesChanging the blank DMEM culture solution for the influenza virus of the cells, culturing the influenza virus of the cells at 100 mu L/hole; recording CPE condition after 48h, and calculating TCID of each group of samples by using Reed-Muench method50The value is obtained.
2. The experimental results are as follows:
results of the experiment see fig. 5, where "×" indicates that the difference is very significant (P <0.001) compared to the virus-infected control group.
It can be seen from the figure that the medicine carnosic acid 20 mu M of the invention has extremely obvious inhibition effect on generation of progeny virus of influenza virus H3N2 in A549 cells.
Example 6 protective Effect of carnosic acid on mice infected with highly pathogenic influenza Virus H5N1
1. The experimental method comprises the following steps:
SPF-grade BALB/c male mice 8 weeks old were housed in a P3 laboratory under sterile conditions. Mice were randomly divided into placebo, virus control and carnosic acid treated groups, 10 mice per group. After mice are anesthetized by isoflurane, influenza virus H5N1(5 LD) is instilled in nasal cavity5030 μ L), and the blank control group was instilled with 30 μ L of PBS through the nasal cavity. Mice were inoculated with influenza virus and administered 0.2mL per mouse by gavage. Once daily for 7 consecutive days. Wherein, carnosic acid is dispersed in 0.5% sodium carboxymethylcellulose water solution, the setting dosage is 10mg/kg/d, 20mg/kg/d and 40mg/kg/d, and the virus control group and the blank control group are used for mice to be gavaged with 0.5% sodium carboxymethylcellulose water solution with the same volume. Mice were observed for morbidity and their mortality was recorded for 14 consecutive days.
2. The experimental results are as follows:
results of the experiment referring to fig. 6, it can be seen that 9 mice in the virus control group died 8 days after infection and 1 mouse survived by the end of the experiment. Whereas the carnosic acid groups 10mg/kg/d, 20mg/kg/d and 40mg/kg/d survived 3, 7 and 5 respectively at the end of the experiment. Three doses of carnosic acid were shown to have protective effects on H5N1 infected mice, with the 20mg/kg/d carnosic acid group having the best protective effect and the survival rate of the mice being 70%.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims (10)

1. Use of carnosic acid in the manufacture of a medicament for the prevention or treatment of influenza, wherein the carnosic acid has the structure of formula I:
Figure FDA0003599525280000011
2. the use according to claim 1, wherein the influenza is caused by an influenza virus which is an influenza a virus, an influenza b virus, an influenza c virus or an influenza d virus.
3. Use according to claim 2, wherein the influenza virus is an influenza a virus.
4. Use according to claim 3, characterized in that the influenza A virus is an influenza A virus of H1N1, H5N1, H3N2, H7N1, H7N2, H7N3, H7N7, H7N9, H9N2 or H10N 8.
5. The use according to claim 4, wherein the influenza A virus is an influenza A virus of H1N1, H5N1 or H3N 2.
6. The use according to any one of claims 1 to 5, wherein the medicament comprises carnosic acid or a pharmaceutically acceptable salt thereof as an active ingredient.
7. The use of claim 6, wherein the cellular viral inhibitory concentration of carnosic acid is 5 μ M or greater.
8. The use of claim 6, wherein the amount of viral suppression of carnosic acid in mice is 10-40 mg/kg/d.
9. The use according to claim 6, wherein the medicament further comprises a pharmaceutically acceptable excipient.
10. The use according to claim 6, wherein the medicament is in the form of an oral preparation, an injection preparation or an inhalation preparation.
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111202766A (en) * 2020-01-19 2020-05-29 昆明医科大学 Application of rosemary extract in antiviral pneumonia medicine

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111202766A (en) * 2020-01-19 2020-05-29 昆明医科大学 Application of rosemary extract in antiviral pneumonia medicine

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DALIA ELEBEEDY ET AL: "In vitro and computational insights revealing the potential inhibitory effect of Tanshinone IIA against influenza A virus", 《COMPUTERS IN BIOLOGY AND MEDICINE》 *
DALIA ELEBEEDY ET AL: "In vitro and computational insights revealing the potential inhibitory effect of Tanshinone IIA against influenza A virus", 《COMPUTERS IN BIOLOGY AND MEDICINE》, no. 141, 17 December 2021 (2021-12-17), pages 1 - 14 *

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