CN114931592A - Application of scutellaria baicalensis extract in preparation of medicine for preventing and/or treating respiratory virus infection - Google Patents

Application of scutellaria baicalensis extract in preparation of medicine for preventing and/or treating respiratory virus infection Download PDF

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CN114931592A
CN114931592A CN202210756842.4A CN202210756842A CN114931592A CN 114931592 A CN114931592 A CN 114931592A CN 202210756842 A CN202210756842 A CN 202210756842A CN 114931592 A CN114931592 A CN 114931592A
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enzymolysis
extract
scutellaria baicalensis
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coronavirus
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刘志华
刘璐
冯遇
姜丹
陈路晓
王玉洁
刘瑾如
王丽
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Guangxi Wuhe Boao Pharmaceutical Co ltd
Beijing Wuhebao Pharmaceutical Co ltd
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Abstract

The invention discloses an application of a scutellaria baicalensis extract in preparing a medicament for preventing and/or treating respiratory virus infection. The respiratory viral infection is caused by at least one of the following viruses: coronavirus, influenza virus. The Scutellariae radix extract contains baicalein and oroxylin A, and further contains wogonin. The Scutellariae radix extract can be obtained by enzymolysis-alcohol extraction. The specific extraction method comprises the following steps: adding water into Scutellariae radix, performing enzymolysis at specific temperature, and drying to constant weight; weighing the radix Scutellariae enzymolysis powder, adding anhydrous ethanol, extracting, filtering, mixing filtrates, concentrating under reduced pressure, and vacuum drying to obtain baicalein extract.

Description

Application of scutellaria baicalensis extract in preparation of medicine for preventing and/or treating respiratory virus infection
Technical Field
The invention belongs to the field of medicines, and particularly relates to application of a scutellaria baicalensis extract in preparation of a medicine for preventing and/or treating respiratory virus infection.
Background
The acute respiratory tract infection caused by virus includes viral upper respiratory tract infection and viral lower respiratory tract infection, the former is manifested by common cold, acute pharyngitis, tonsillitis and laryngitis, and the latter is manifested by trachitis, bronchitis and pneumonia. The viral respiratory tract infection has high morbidity, people are generally infected easily, children, old people, malnutrition and patients suffering from chronic diseases are more susceptible, and the diseases are more frequently encountered in winter and spring. Among them, influenza virus and coronavirus are currently attracting much attention due to their strong infectivity and high lethality rate.
Coronaviruses are widely found in nature, and natural hosts thereof include domestic animals, birds, rats, wild mammals and the like, particularly flying mammalian bats, which are natural storage hosts of various coronaviruses and are also sources of infection of viral spread, transmission and epidemic diseases of animals or humans. Studies have shown that human coronavirus infection can cause respiratory diseases, such as bronchitis, bronchiolitis and pneumonia, Severe Acute Respiratory Syndrome (SARS) in winter to 2003 in 2002, and respiratory syndrome in middle east in 2012 to 2014 are all caused by coronavirus infection. The new coronavirus (2019-nCoV) in 2019 is a new coronavirus which is newly discovered after SARS coronavirus and MERS coronavirus and has high pathogenicity in human, and is a protozoon causing new pneumonia outbreak. Coronavirus is highly infectious and is generally susceptible to people. Due to the sudden outbreak of virus and the strong variability, no suitable antiviral drugs are available for effective treatment at present.
Influenza virus is an RNA virus that causes influenza in humans and animals, causes acute upper respiratory infection, is rapidly transmitted through the air, and is frequently pandemic periodically around the world. Influenza virus infection causes host cells to denature, necrose and even shed, resulting in mucosal congestion, edema and increased secretions, which can cause nasal obstruction, runny nose, sore throat, dry cough and other symptoms of upper respiratory tract infection, and when the virus spreads to the lower respiratory tract, bronchiolitis and interstitial pneumonia may be caused. Influenza often causes secondary infection because influenza virus infection reduces the ability of airway mucosal epithelial cells to clear and adhere foreign bodies, thus greatly reducing the ability of a human body to resist respiratory infection, and secondary pneumonia caused by influenza is one of the main causes of death due to influenza death.
Viral infections also induce interferon expression and cellular immune opsonization, which results in several autoimmune responses, including high fever, headache, calf muscle and general muscle pain, and the toxic-like products of viral metabolism and products released by cellular necrosis, which are also responsible for and aggravate these responses. Therefore, the research and development of antiviral drugs for resisting the infection and transmission of viruses and treating and relieving symptoms such as related pneumonia are urgent.
The traditional Chinese medicine has the advantages of low drug resistance, few side effects, few adverse reactions and the like, has the comprehensive effects of inhibiting virus replication, regulating the immune function, improving blood circulation, relieving fever and pain, resisting bacteria and diminishing inflammation and the like, and has unique advantages and wide development prospect in the aspect of preventing and treating upper respiratory tract virus infection. At present, most of single Chinese medicines with direct antiviral effect are heat-clearing and detoxifying traditional Chinese medicines such as fructus forsythiae, honeysuckle, wild chrysanthemum flower, coptis chinensis, scutellaria baicalensis, folium isatidis, houttuynia cordata, radix bupleuri, great burdock achene and the like.
Among complex components of traditional Chinese medicines, flavonoids are active components which are clearly researched at present, and have the effects of resisting tumors, oxidation, inflammation, bacteria, viruses and the like. Researches show that the flavonoids not only can directly kill viruses and prevent the viruses from being absorbed, penetrated, copied and transfected, but also can enhance the immunity of the organism, prevent the viruses from causing cytopathic effect, improve clinical symptoms and stimulate all immune defense systems of the organism to play an antiviral role.
Baicalein and baicalin are main flavonoid substances in scutellaria baicalensis, and in recent years, the baicalein serving as aglycone has better pharmacological activities of inhibiting inflammatory reaction, resisting viruses and removing free radicals than the baicalin. However, because the content of baicalein in the medicinal materials is low, most of the existing literature researches are concentrated on baicalin or extracts taking baicalin as a main component, and the researches on baicalein and other effective components are less; the related virus models are mostly HIV, respiratory syncytial virus, dengue fever virus, H1N1 influenza virus, cytomegalovirus, encephalitis virus, herpes simplex virus and the like.
Disclosure of Invention
The invention aims to provide application of a scutellaria baicalensis extract in preparing a medicament for preventing and/or treating respiratory virus infection.
The respiratory viral infection according to the present invention is caused by at least one of the following viruses: coronavirus, influenza virus.
The coronavirus can be human coronavirus HCOV-OC 43; the influenza virus may specifically be influenza virus H3N2 (influenza virus a/hanfang/359/95).
The scutellaria baicalensis extract contains baicalein, oroxylin A and wogonin. Furthermore, the scutellaria baicalensis extract does not contain baicalin.
Further, the scutellaria baicalensis extract is obtained by extracting through an enzymolysis-alcohol extraction method.
The specific extraction method comprises the following steps: adding water into Scutellariae radix, performing enzymolysis at specific temperature, and drying to constant weight to obtain Scutellariae radix enzymolysis medicinal powder; weighing Scutellariae radix enzymolysis powder, adding anhydrous alcohol or ethanol water solution, extracting, filtering, mixing filtrates, concentrating under reduced pressure, and vacuum drying to obtain baicalein extract.
Wherein the mass ratio of the scutellaria baicalensis medicinal material to water is 1: (5-6), preferably 1: 5.
the temperature of the enzymolysis is 40 ℃, the time is 12-24 hours, preferably 24 hours, the pH value is 6-7, preferably 6.
The mass ratio of the scutellaria enzymolysis medicinal powder to absolute ethyl alcohol or an ethanol water solution is 1: (10-12), preferably 1: 10. The volume fraction of ethanol in the ethanol water solution is 50-100%, specifically 50%, 60%, 70%, 80%, 95%.
The extraction may be reflux extraction. The reflux extraction can be carried out for 1 time, and the extraction time can be 2 h.
By adopting an enzymolysis-alcohol extraction method and controlling parameters such as enzymolysis temperature, enzymolysis time, ethanol adding amount and the like, the active component extract with single baicalein content can be obtained.
The enzymolysis refers to enzymolysis by using endogenous enzyme-beta-D-glucuronidase contained in the scutellaria baicalensis medicinal material.
A further preferred extraction method is as follows:
taking a proper amount of scutellaria baicalensis, adding 5 times of water, adjusting the pH to 6, performing enzymolysis at 40 ℃ for 24 hours, and drying to constant weight; weighing Scutellariae radix enzymolysis powder, adding 10 times of anhydrous alcohol, reflux extracting for 2 hr, extracting for 1 time, filtering, concentrating the filtrate under reduced pressure, and vacuum drying at 60 deg.C to obtain Scutellariae radix extract.
The method also comprises the step of further separating the scutellaria baicalensis extract:
filling pretreated macroporous resin LX-38 into a resin column, wherein the ratio of the diameter to the height of the filled column is 1: and 8, repeatedly washing the column with deionized water to fully compress the resin in the column, and when sampling, putting the deionized water on the upper part of the column to be flush with the surface of the resin and then starting sampling. Taking 100mg of scutellaria baicalensis extract, wherein the sample loading concentration is 1mg/mL, adsorbing and loading samples at a certain speed, removing impurities and eluting by using 0.8BV of 30% ethanol when the liquid level of the sample loading is flush with the resin surface, then eluting by using 4BV of 50% ethanol, 4BV of 60% ethanol and 3BV of 100% ethanol, collecting 100% ethanol eluent, spin-drying the solvent, and carrying out vacuum drying to obtain the component C.
Alternatively, the Scutellariae radix extract can be obtained by acid hydrolysis.
The specific extraction method comprises the following steps: decocting a scutellaria baicalensis medicinal material in water, concentrating decoction, adjusting the pH value to 1.0-2.0 by acid, keeping the temperature at 80 ℃ for 30min, standing overnight, and removing supernatant to obtain a precipitate; adding water into the precipitate, uniformly dispersing, adding alkali to adjust the pH to be 7.0, adding an equal volume fraction of 85% ethanol aqueous solution, stirring to dissolve, filtering, removing the precipitate, adding acid into the filtrate to adjust the pH to be 1.0-2.0, keeping the temperature at 60 ℃ for 30min, standing, and removing the supernatant to obtain the precipitate; washing the precipitate with water and alcohol in sequence, and drying under reduced pressure to obtain yellow powder; adding 5-6 times of concentrated sulfuric acid into the yellow powder, stirring, dripping 4-5 times of distilled water, standing for 15min, stirring the solution, pouring into water, precipitating yellow precipitate, filtering to obtain precipitate, washing with water to neutrality, and vacuum drying under reduced pressure to obtain Scutellariae radix extract.
The decocting with water is carried out at least 1 time, preferably twice.
Decocting the medicinal materials in water every time, wherein the mass ratio of the medicinal materials of the scutellaria baicalensis to the water is 1: the time for decoction can be 2 hours 10.
The medicine provided by the invention comprises the scutellaria baicalensis extract and a pharmaceutically acceptable carrier or auxiliary material.
Further, the medicine also comprises pharmaceutically acceptable antiviral medicines or other related active substances.
The dosage form of the medicine comprises an oral preparation, an injection preparation, a transdermal administration preparation, a mucosa administration preparation, a lung inhalation administration preparation or an intestinal administration preparation;
the dosage form of the medicament comprises: drop, oral liquid, tablet, capsule, granule, pellicle, gel, powder, emulsion, self-emulsifying preparation (such as self-microemulsion), dripping pill, suppository, aerosol, spray, powder spray, patch, solution, ointment or cream.
The medicament of the present invention may be administered by any known administration method.
The dosage of the drug of the present invention to be administered may vary widely depending on the nature and severity of the disease to be prevented or treated, the individual condition of the patient or animal, the route and dosage form of administration, and the like. The above-described dosage may be administered in one dosage unit or divided into several dosage units, depending on the clinical experience of the physician and the regimen comprising the use of other therapeutic means.
The medicine can be taken alone or combined with other therapeutic medicines or symptomatic medicines. When the medicament of the invention has synergistic effect with other therapeutic medicaments, the dosage of the medicament is adjusted according to actual conditions.
Compared with the prior art, the invention has the following beneficial technical effects:
the invention extracts and separates the prepared scutellaria root extract which takes baicalein as the main component, has good inhibiting effect on coronavirus of specific virus strain, further separates and purifies the scutellaria root extract, and the prepared extract which takes wogonin as the main component also has good inhibiting effect on coronavirus and influenza virus of specific virus strain.
Drawings
FIG. 1 is an HPLC chart of a mixed control solution of baicalin, baicalein, wogonin, oroxylin A and Scutellariae radix extracts 1 to 4, wherein A is an HPLC chart of the mixed control solution, B is an HPLC chart of extract 1, C is an HPLC chart of extract 2, D is an HPLC chart of extract 3, and E is an HPLC chart of extract 4 (wherein 1 is baicalin, 2 is baicalein, 3 is wogonin, and 4 is oroxylin A);
FIG. 2 is an HPLC chart of a mixed control solution of baicalein, wogonin, oroxylin A and fraction B, C, wherein A is an HPLC chart of the mixed control solution, B is an HPLC chart of fraction B, and C is an HPLC chart of fraction C (wherein 1 is baicalein, 2 is wogonin, and 3 is oroxylin A);
FIG. 3 is the results for component C against influenza virus protein;
FIG. 4 is a graph of component C anti-influenza mRNA results;
FIG. 5 shows the results for component C anti-coronavirus protein;
FIG. 6 shows the results for component C anti-coronavirus mRNA.
Detailed Description
The present invention will be further illustrated with reference to the following specific examples, but the present invention is not limited to the following examples. The method is a conventional method unless otherwise specified. The starting materials are commercially available from the open literature unless otherwise specified.
Example 1 preparation of Scutellaria baicalensis Georgi extract by enzymolysis-alcohol extraction method 1-2
The preparation process flow of the enzymolysis-alcohol extraction method comprises the following steps: taking a scutellaria baicalensis medicinal material, adding a certain amount of water, carrying out enzymolysis (carrying out enzymolysis by endogenous enzyme-beta-D-glucuronidase) at a certain temperature, and drying to constant weight; weighing the enzymolysis medicinal powder, adding a certain amount of anhydrous ethanol, reflux-extracting, filtering, concentrating the filtrate under reduced pressure, and vacuum-drying at 60 deg.C to obtain Scutellariae radix extract.
By adopting an enzymolysis-alcohol extraction method and through research, the range of an enzymolysis solvent (water) is set to be 4, 5, 6, 7 and 8 times, the range of enzymolysis temperature is set to be 40 ℃, 50 ℃, 60 ℃ and 70 ℃, the range of enzymolysis time is set to be 4h, 6h, 8h, 12h and 24h, the range of enzymolysis pH is set to be 3, 4, 5, 6 and 7, reflux extraction is carried out for 2h in 10 times of 100% ethanol, extraction is carried out for 1 time, and the sum of the contents of baicalein, wogonin and oroxylin in the obtained extract is used as an index to investigate the reaction parameters.
The influence of the addition amount of the enzymolysis solvent (water) on the sum of the contents of baicalein, wogonin and oroxylin is examined under the conditions that the enzymolysis time is 24h, the enzymolysis pH is 5 and the enzymolysis temperature is 40 ℃, and the table 1 shows.
TABLE 1
Figure BDA0003722836370000051
As can be seen from table 1, when the amount of the enzymolysis solvent is 5 times, the sum of the contents of baicalein, wogonin and oroxylin in the obtained extract is relatively high.
The influence of the enzymolysis temperature on the sum of the contents of baicalein, wogonin and oroxylin is examined under the conditions of 5 times of enzymolysis solvent (water), 24h of enzymolysis time and 5 of enzymolysis pH, and is shown in Table 2.
TABLE 2
Figure BDA0003722836370000052
As can be seen from Table 2, the sum of the contents of baicalein, wogonin and oroxylin A in the extract is relatively high at 40 ℃.
The influence of the enzymolysis time on the sum of the contents of baicalein, wogonin and oroxylin is examined under the conditions of 5 times of enzymolysis solvent (water), 40 ℃ of enzymolysis temperature and 5 ℃ of enzymolysis pH, and is shown in Table 3.
TABLE 3
Figure BDA0003722836370000053
Figure BDA0003722836370000061
As shown in Table 3, the total content of baicalein, wogonin and oroxylin in the extract is relatively high when the enzymolysis time is 24 hours.
The influence of the enzymolysis pH on the sum of the contents of baicalein, wogonin and oroxylin is examined under the conditions of 5 times of enzymolysis solvent (water), 40 ℃ of enzymolysis temperature and 24 hours of enzymolysis time, and is shown in Table 4.
TABLE 4
Figure BDA0003722836370000062
As shown in Table 4, when the pH of the enzymatic hydrolysis is 6, the sum of the contents of baicalein, wogonin and oroxylin in the extract is relatively high.
In summary, as can be seen from tables 1-4, the amount of the added enzymolysis solvent is 5 times, the enzymolysis temperature is 40 ℃, the enzymolysis time is 24 hours, and the enzymolysis pH is 6, so that the sum of the contents of baicalein, wogonin and oroxylin in the obtained extract is relatively high.
Based on the above reference index, the ethanol concentration is temporarily determined to be 100%, the reflux extraction time is 2h, the reflux extraction frequency is 1, the influence of the amount of the added ethanol on the sum of the contents of the baicalein, the wogonin and the oroxylin in the extract is examined, and the result is shown in table 5.
TABLE 5
Figure BDA0003722836370000063
As can be seen from Table 5, when the amount of ethanol is 10 times, the sum of the contents of baicalein, wogonin and oroxylin in the extract is relatively high.
Under the conditions of an enzymolysis solvent, enzymolysis temperature, enzymolysis time and enzymolysis pH value unchanged, 10 times of ethanol is selected, the concentration of the ethanol is 100%, and the reflux extraction time is 2 hours, the influence of the reflux extraction times on the sum of the contents of baicalein, wogonin and oroxylin in the extract is examined, and the result is shown in table 6.
TABLE 6
Figure BDA0003722836370000064
As can be seen from Table 6, when the number of reflux extractions is 1, the sum of the contents of baicalein, wogonin and oroxylin in the extract is relatively high.
Under the conditions of an enzymolysis solvent, enzymolysis temperature, enzymolysis time and enzymolysis pH value unchanged, 10 times of ethanol is selected, the concentration of the ethanol is 100%, and the reflux extraction times are 1, the influence of the reflux extraction time on the sum of the contents of baicalein, wogonin and oroxylin in the extract is examined, and the result is shown in table 7.
TABLE 7
Figure BDA0003722836370000071
As can be seen from Table 7, when the reflux extraction time is 2 hours, the sum of the contents of baicalein, wogonin and oroxylin in the extract is relatively high.
Under the conditions of an enzymolysis solvent, enzymolysis temperature, enzymolysis time and enzymolysis pH value unchanged, 10 times of ethanol is selected, the reflux extraction frequency is 1 time, and the reflux extraction time is 2 hours, the influence of the ethanol concentration on the sum of the contents of baicalein, wogonin and oroxylin in the extract is examined, and the result is shown in Table 8.
TABLE 8
Figure BDA0003722836370000072
As can be seen from Table 8, the sum of the contents of baicalein, wogonin and oroxylin in the extract was relatively high at an ethanol concentration of 100%.
In summary, as can be seen from tables 1 to 8, when the scutellaria baicalensis is extracted by the enzymolysis-alcohol extraction method, the sum of the contents of baicalein, wogonin and oroxylin is used as an index, the selected extraction conditions are that the addition amount of an enzymolysis solvent is 5 times, the enzymolysis temperature is 40 ℃, the enzymolysis time is 24 hours, the enzymolysis pH is 6, the amount of ethanol is 10 times, the ethanol concentration is 100%, the reflux extraction times is 1, and the reflux extraction time is 2 hours.
Extracting with different ethanol concentrations under the conditions of enzymolysis solvent times, enzymolysis temperature, enzymolysis time, enzymolysis pH value, ethanol times, reflux extraction times and reflux extraction time, to obtain two extracts with the highest and the lowest contents of baicalein, wogonin and oroxylin, which are Scutellariae radix extract 1 and Scutellariae radix extract 2 respectively.
The preparation method of the scutellaria baicalensis extract 1 comprises the following steps: taking a proper amount of scutellaria baicalensis, adding 5 times of water, adjusting the pH value to 6, carrying out enzymolysis at 40 ℃ for 24 hours, and drying to constant weight; weighing Scutellariae radix enzymolysis powder, adding 10 times of anhydrous alcohol, reflux extracting for 2 hr, extracting for 1 time, filtering, concentrating the filtrate under reduced pressure, and vacuum drying at 60 deg.C to obtain Scutellariae radix extract 1.
The preparation method of the scutellaria baicalensis extract 2 comprises the following steps: taking a proper amount of scutellaria baicalensis, adding 5 times of water, adjusting the pH value to 6, carrying out enzymolysis at 40 ℃ for 24 hours, and drying to constant weight; weighing Scutellariae radix enzymolysis powder, adding 10 times of 50% ethanol, reflux extracting for 2 hr, extracting for 1 time, filtering, concentrating the filtrate under reduced pressure, and vacuum drying at 60 deg.C to obtain Scutellariae radix extract 2.
Example 2 preparation of Scutellaria baicalensis Georgi extract by acid hydrolysis 3-4
The preparation process flow of the acid hydrolysis method comprises the following steps: decocting a scutellaria baicalensis medicinal material in water, concentrating decoction, adjusting the pH value to 1.0-2.0 by using 2mol/L hydrochloric acid, preserving heat at 80 ℃ for 30min, standing overnight, and removing supernatant to obtain a precipitate; adding water into the precipitate, uniformly dispersing, adding 40% sodium hydroxide to adjust the pH value to 7.0, adding equal amount of 85% ethanol, stirring to dissolve, filtering, removing the precipitate, adding 2moL/L hydrochloric acid into the filtrate to adjust the pH value to 1.0-2.0, keeping the temperature at 60 ℃ for 30min, standing, and removing the supernatant to obtain a precipitate; washing the precipitate with water and alcohol in sequence, and drying under reduced pressure to obtain yellow powder; adding appropriate amount of the yellow powder into appropriate amount of concentrated sulfuric acid, stirring with glass rod, dripping distilled water, standing for 15min, stirring the solution, pouring into water to separate out yellow precipitate, filtering to obtain precipitate, washing with water to neutrality, and vacuum drying under reduced pressure to obtain Scutellariae radix extract.
By adopting an acid hydrolysis method and controlling and inspecting the dosage of concentrated sulfuric acid, the scutellaria baicalensis extracts with the baicalein content of more than 80 percent are obtained, namely scutellaria baicalensis extract 3 and scutellaria baicalensis extract 4 respectively.
The preparation method of the scutellaria baicalensis extract 3 comprises the following steps: decocting scutellaria baicalensis medicinal materials for 2 hours by adding 10 times of water, decocting twice, combining the two decoctions, concentrating, adjusting the pH value to 1.0-2.0 by using 2mol/L hydrochloric acid, preserving heat for 30min at 80 ℃, standing overnight, and removing supernatant to obtain a precipitate; adding water into the precipitate, uniformly dispersing, adding 40% sodium hydroxide to adjust the pH value to 7.0, adding equal amount of 85% ethanol, stirring to dissolve, filtering, removing the precipitate, adding 2moL/L hydrochloric acid into the filtrate to adjust the pH value to 1.0-2.0, keeping the temperature at 60 ℃ for 30min, standing, and removing the supernatant to obtain a precipitate; washing the precipitate with water and alcohol in sequence, and drying under reduced pressure to obtain yellow powder; adding 5 times of concentrated sulfuric acid into the yellow powder, stirring with a glass rod, adding 4 times of distilled water dropwise, standing for 15min, stirring the solution, pouring into water, precipitating yellow precipitate, filtering to obtain precipitate, washing with water to neutrality, and vacuum drying under reduced pressure to obtain Scutellariae radix extract 3.
The preparation method of the scutellaria baicalensis extract 4 comprises the following steps: decocting scutellaria baicalensis medicinal materials for 2 hours by adding 10 times of water, decocting twice, combining the two decoctions, concentrating, adjusting the pH value to 1.0-2.0 by using 2mol/L hydrochloric acid, preserving heat for 30min at 80 ℃, standing overnight, and removing supernatant to obtain a precipitate; adding water into the precipitate, uniformly dispersing, adding 40% sodium hydroxide to adjust the pH value to 7.0, adding equal amount of 85% ethanol, stirring to dissolve, performing suction filtration, removing the precipitate, adding 2moL/L hydrochloric acid into the filtrate to adjust the pH value to 1.0-2.0, performing heat preservation at 60 ℃ for 30min, standing, and removing the supernatant to obtain a precipitate; washing the precipitate with water and alcohol in sequence, and drying under reduced pressure to obtain yellow powder; adding 6 times of concentrated sulfuric acid into appropriate amount of the yellow powder, stirring with glass rod, adding 5 times of distilled water, standing for 15min, stirring the solution, pouring into water, precipitating yellow precipitate, filtering, washing the precipitate with water to neutrality, and vacuum drying under reduced pressure to obtain Scutellariae radix extract 4.
Example 3 measurement of Scutellaria Baicalensis Georgi extract content
1. Preparation of test solution
Precisely weighing about 15mg of the extract, placing the extract into a 25mL measuring flask, adding a 95% ethanol solution containing 0.02% VC, ultrasonically dissolving, fixing the volume to the scale, and shaking up. Precisely transferring 1mL of the solution into a 25mL measuring flask, diluting to scale with a 95% ethanol solution containing 002% VC, shaking, filtering, and collecting the filtrate to obtain the sample solution.
2. Preparation of control solutions
In addition, a proper amount of reference substance is precisely weighed, and a mixed reference substance solution containing 19.23 mu g of baicalin, 19.66 mu g of baicalein, 7.98 mu g of wogonin and 5.14 mu g of oroxylin A per milliliter is prepared by the same method. Measuring 10 μ L of sample solution and reference solution, injecting sample under 3-degree chromatographic condition, recording the peak area of each chromatogram, and calculating the content of each component according to external standard method.
3. Chromatographic conditions
Using high performance liquid chromatography, a Diamonsil C18 column (4.6 mm. times.250 mm, 5 μm), methanol-0.05% aqueous phosphoric acid (65:35, v/v) was eluted at mobile phase isocratic, flow rate: 1mL/min, column temperature: 25 ℃, sample introduction: 10 μ L, detection wavelength: 275 nm.
4. As a result, the
Chromatogram of the reference substance and Scutellariae radix extract 1-4 are shown in FIG. 1, and content detection results are shown in Table 9.
TABLE 9 content measurement results of extract 1-4 samples
Figure BDA0003722836370000091
Note: "/" indicates that the component is not included.
As can be seen from FIG. 1, none of the extracts 1 to 4 contained baicalin.
Example 4 Scutellaria Baicalensis Georgi extract 1-4 antiviral efficacy test study
1. Anti-coronavirus HCoV-OC43 activity screen
1.1 test principle
MRC-5 (human embryonic lung fibroblast) cells are taken as virus hosts, and the degree of cytopathic effect (CPE) caused by virus inhibition of the samples is measured.
1.2 test materials and methods
(1) Virus strain: the coronavirus HCoV-OC43 was stored at-80 ℃.
(2) Sample treatment: the samples were prepared into stock solutions with DMSO just before use, and then diluted 3-fold with culture medium, 8 dilutions each.
(3) Positive control drug: ribavirin (RBV), Hubei Tian Yao pharmaceutical Co., Ltd. (batch No. 31712252).
(4) The test method comprises the following steps: MRC-5 cells were seeded in 96-well culture plates and infected with coronavirus 10 after 24 hours -2 Adding maintenance solution containing samples with different dilutions and positive control drug, setting cell control hole and virus control hole, observing cytopathic degree (CPE) of each group when the virus control group has pathological Changes (CPE) of 4+, and calculating half Inhibitory Concentration (IC) of the sample to virus by Reed-Muench 50 )。
1.3 results
The results are shown in Table 10. The result shows that compared with antiviral medicament ribavirin positive medicament, the scutellaria baicalensis extract 1-4 shows good anti-coronavirus activity, wherein the anti-coronavirus HCoV-OC43 activity of the extract 1 is strongest, and the effect of anti-coronavirus is better when the content of baicalein is not higher. The applicant then isolated extract 1, see example 5.
TABLE 10 test results of 1-4 anti-coronavirus HCoV-OC43 test of Scutellaria baicalensis Georgi extract
Figure BDA0003722836370000101
Note: (1) TC (tungsten carbide) 50 : half toxic concentration of drug; IC (integrated circuit) 50 : the median inhibitory concentration of the drug on the virus; and (3) SI: i.e. selection index, whose value is equal to TC 50 /IC 50 Theoretically, to determine the safety index of the drug effect, the index SI is selected>1, indicates that the drug is effective and safe, and the greater the SI value, the safer the drug.
2. Anti-coronavirus HCoV-229E activity screening
2.1 test principle
H460 cells were used as virus hosts, and the extent of cytopathic effect (CPE) of the sample on the virus-induced cells was determined.
2.2 test materials and methods
(1) Virus strain: the coronavirus HCoV-229E was stored at-80 ℃.
(2) Sample treatment: the samples were prepared into stock solutions with DMSO just before use, and then diluted 3-fold with culture medium, 8 dilutions each.
(3) Positive control drug: ribavirin (RBV), Hubei Tian Yao pharmaceutical Co., Ltd. (batch No. 31712252).
(4) The test method comprises the following steps: inoculating H460 cells to a 96-well culture plate, and placing 5% CO 2 Culturing at 35 ℃. Infection with coronavirus 10 after 24 hours -3 Simultaneously adding maintenance solution containing samples with different dilutions and positive control drug, setting cell control hole and virus control hole, and setting 5% CO 2 Culturing at 35 ℃. Observing the pathological change degree (CPE) of each group of cells when the pathological change degree (CPE) of the virus control group reaches 4+, and respectively calculating the half Toxic Concentration (TC) of the sample to the cells by using a Reed-Muench method 50 ) And half maximal Inhibitory Concentration (IC) against virus 50 )。
2.3 results
The results are shown in Table 11. The results showed that Scutellariae radix extract 1 showed no activity against coronavirus HCoV-229E.
TABLE 11 Scutellaria baicalensis Georgi extract 1 test results against coronavirus HCoV-229E
Figure BDA0003722836370000111
Note: (1) TC (tungsten carbide) 50 : half toxic concentration of drug; IC (integrated circuit) 50 : the median inhibitory concentration of the drug on the virus; and (3) SI: selecting an index, SI ═ TC 50 /IC 50 。
Example 5 preparation of component B and component C
Filling pretreated macroporous resin LX-38 into a resin column, wherein the ratio of the diameter to the height of the filled column is 1: and 8, repeatedly washing the column with deionized water to fully compress the resin in the column, and loading the deionized water on the upper part of the column until the deionized water is flush with the surface of the resin. Taking the scutellaria baicalensis extract 1(100mg) prepared in example 1, wherein the sample loading concentration is 1mg/mL, adsorbing and loading the sample at a certain speed, when the liquid level of the sample loading is flush with the resin surface, removing impurities and eluting by using 0.8BV of 30% ethanol, then eluting by using 4BV of 50% ethanol, 4BV of 60% ethanol and 3BV of 100% ethanol, respectively collecting 60% ethanol and 100% ethanol eluates, spin-drying the solvent, and performing vacuum drying to obtain a component B (60% ethanol eluent) and a component C (100% ethanol eluent).
Example 6 determination of the content of component B and component C
1. Preparation of test solution
Accurately weighing 10mg of the sample, placing the sample in a 100mL measuring flask, completely dissolving the sample by absolute ethyl alcohol, fixing the volume to a scale, and shaking up to obtain a stock solution of the test solution. Precisely transferring 0.5mL of the stock solution into a 50mL measuring flask, and diluting with anhydrous ethanol to a constant volume to obtain the final product.
2. Preparation of control solution
Weighing appropriate amount of reference substance, adding anhydrous alcohol to obtain mixed reference substance solution containing baicalein 30 μ g, wogonin 8 μ g, and oroxylin A4 μ g per ml.
3. Chromatographic conditions
The same as in example 2, 3 chromatographic conditions.
4. Results
The chromatogram of the mixed control solution and component B, C is shown in FIG. 2, and the content measurement results are shown in Table 12.
TABLE 12 determination of the content of component B, C
Figure BDA0003722836370000121
Example 7 component B, C screening for anti-coronavirus HCoV-OC43 Activity
1 principle of testing
H460 cells were used as virus hosts, and the extent of cytopathic effect (CPE) of the samples due to the inhibition of the virus was determined.
2 test materials and methods
(1) Virus strain: the coronavirus HCoV-OC43 was stored at-80 ℃.
(2) Sample treatment: the samples were prepared into stock solutions with DMSO just before use, and then diluted 3-fold with culture medium, 8 dilutions each.
(3) Positive control drug: ribavirin (RBV), Hubei Tian Yao pharmaceutical Co., Ltd. (batch No. 31712252).
(4) The test method comprises the following steps: inoculating H460 cells into 96-well culture plate, and placing 5% CO 2 Culturing at 35 ℃. Infection with coronavirus 10 after 24 hours -3 Simultaneously adding maintenance solution containing samples with different dilutions and positive control drug, setting cell control hole and virus control hole, and setting 5% CO 2 Culturing at 35 ℃. Observing the pathological change degree (CPE) of each group when the pathological change degree (CPE) of the virus control group reaches 4+, and respectively calculating the half Toxic Concentration (TC) of the sample to the cells by using a Reed-Muench method 50 ) And half maximal Inhibitory Concentration (IC) against the virus 50 )。
3 results
The results are shown in Table 13. The results show that the component C has certain activity against coronavirus HCoV-OC43, and the rest of the tested medicines do not show activity.
TABLE 13 component B, C test results against coronavirus HCoV-OC43
Figure BDA0003722836370000122
Note: (1) in the table "-" indicates that the sample had no anti-viral activity at the maximum non-toxic dose.
(2)TC 50 : half toxic concentration of drug; IC (integrated circuit) 50 : the median inhibitory concentration of the drug on the virus; and (3) SI: i.e. selection index, whose value is equal to TC 50 /IC 50， In order to judge the safety index of the drug effect, theoretically, the index SI is selected>1, indicates that the drug is effective and safe, and the greater the SI value, the safer the drug. Comparative example 1, component B, C screening for anti-coronavirus HCoV-229E Activity
1 principle of testing
H460 cells were used as virus hosts, and the extent of cytopathic effect (CPE) of the sample on the virus-induced cells was determined.
2 test materials and methods
(1) Virus strain: the coronavirus HCoV-229E was stored at-80 ℃.
(2) Sample treatment: the samples were prepared into stock solutions with DMSO just before use, and then diluted 3-fold with culture medium, 8 dilutions each.
(3) Positive control drug: ribavirin (RBV), Hubei Tian Yao pharmaceutical Co., Ltd. (batch No. 31712252).
(4) The test method comprises the following steps: inoculating H460 cells into 96-well culture plate, and placing 5% CO 2 Culturing at 35 ℃. Infection with coronavirus 10 after 24 hours -3 Simultaneously adding maintenance solution containing samples with different dilutions and positive control drug, setting cell control hole and virus control hole, and setting 5% CO 2 Culturing at 35 ℃. Observing the pathological change degree (CPE) of each group of cells when the pathological change degree (CPE) of the virus control group reaches 4+, and respectively calculating the half Toxic Concentration (TC) of the sample to the cells by using a Reed-Muench method 50 ) And half maximal Inhibitory Concentration (IC) against virus 50 )。
3 results
The results are shown in Table 14. The results showed that neither fraction B, C showed activity against coronavirus HCoV-229E.
TABLE 14 component B, C test results against coronavirus HCoV-229E
Figure BDA0003722836370000131
Note: TC (tungsten carbide) 50 : half toxic concentration of drug; IC (integrated circuit) 50 : the median inhibitory concentration of the drug on the virus; and (3) SI: i.e. selection index, whose value is equal to TC 50 /IC 50， In order to judge the safety index of the drug effect, theoretically, the index SI is selected>1, indicates that the drug is effective and safe, the greater the SI value the safer the drug.
Example 8 component C protein and mRNA assays against influenza and coronavirus
1 anti-influenza virus protein and mRNA assay
1.1Western blot method for detecting protein expression
MDCK cells (5X 10/ml) 5 ) Inoculating to 12-well plate, culturing overnight, washing with PBS 1 time, and culturing with influenza virus strain A/Hanfang/359/95 (H3N2)1/210 -4 Infecting cells for 2h, and adding liquid medicine to be detected and positive control medicine with different concentrations 2h after infection. And extracting total cell protein after culturing for 24h, and carrying out Western blot detection. BETA-Actin primary antibody (1:1000) and M2 primary antibody (1:400) were incubated at 4 ℃ overnight, and after washing with Tris-HCl buffer saline (TBST) for 3 times, horseradish peroxidase-labeled secondary antibody was incubated for 1h, and after washing with TBST for 3 times, Enhanced Chemiluminescence (ECL) developer (Thermo Co.) was added, and development was performed with a gel imager.
1.2 fluorescent quantitation method for detecting protein mRNA expression
MDCK cells (5X 10/ml) 5 ) Inoculated in a 24-well plate for overnight culture, washed 1 time with PBS and then inoculated with influenza virus strain A/Hanfang/359/95 (H3N2)1/210 -4 Infecting cells for 2h, and adding liquid medicine to be detected and positive control medicine with different concentrations 2h after infection. After 24h of culture, total RNA in cells was extracted using RNeasy Mini Kit, and the mRNA expression level of influenza virus M2 was measured by fluorescence quantification.
1.3 results
The results are shown in FIGS. 3 and 4. The results show that component C dose-dependently inhibited protein M2 expression and M2 mRNA levels of seasonal influenza virus strain a/hanfang/359/95 (H3N 2). In conclusion, component C has a good anti-influenza virus effect.
2 anti-coronavirus HCOV-OC43 protein and mRNA results
2.1 detection of protein expression by Western blot
H460 cells were plated on a 12-well plate at 1.5X 105/mL, incubated overnight at 37 ℃ and infected with HCoV-OC43 at an MOI of 0.005, followed by addition of gradient drug, incubated at 35 ℃ for 48hr to extract protein, and the NP protein expression level was determined using western blot.
2.2 fluorescent quantitation for protein mRNA expression
H460 cells were 1.5X 10 5 Spreading in 12-well plate per mL, culturing at 37 deg.C overnight, infecting HCoV-OC43 with MOI of 0.005 while adding gradient concentration medicine, culturing at 35 deg.C for 24hr, extracting RNA, and detecting NP protein mRNA expression level by qPCR.
2.3 results
The results are shown in FIGS. 5 and 6. The results show that the component C can inhibit HCoV-OC43 protein level and mRNA level when the concentration is 20 mug/mL, 10 mug/mL and 5 mug/mL, and the inhibition rate can reach more than 70%. In conclusion, component C has a very good anti-coronavirus effect.

Claims (7)

1. The application of the scutellaria baicalensis extract in preparing the medicines for preventing and/or treating respiratory virus infection;
the scutellaria baicalensis extract is extracted according to a method comprising the following steps: adding water into Scutellariae radix, performing enzymolysis at specific temperature, and drying to constant weight to obtain Scutellariae radix enzymolysis medicinal powder; weighing Scutellariae radix enzymolysis powder, adding anhydrous ethanol, extracting, filtering, mixing filtrates, concentrating under reduced pressure, and vacuum drying to obtain baicalein extract.
2. Use according to claim 1, characterized in that: the respiratory viral infection is caused by at least one of the following viruses: coronavirus, influenza virus.
3. Use according to claim 2, characterized in that: the coronavirus is human coronavirus HCOV-OC 43; the influenza virus is influenza virus H3N 2.
4. Use according to any one of claims 1-3, characterized in that:
the mass ratio of the scutellaria baicalensis medicinal material to water is 1: (5-6);
the temperature of the enzymolysis is 40 ℃, the time is 12-24 hours, and the pH value is 6-7;
the mass ratio of the scutellaria enzymolysis medicinal powder to the absolute ethyl alcohol is 1: (10-12);
the extraction is reflux extraction; the reflux extraction frequency is 1 time, and the extraction time is 2 h.
5. Use according to claim 4, characterized in that: the scutellaria baicalensis extract is extracted according to a method comprising the following steps: taking a proper amount of scutellaria baicalensis, adding 5 times of water, adjusting the pH value to 6, carrying out enzymolysis at 40 ℃ for 24 hours, and drying to constant weight; weighing the radix Scutellariae enzymolysis powder, adding 10 times of anhydrous alcohol, reflux extracting for 2 hr for 1 time, filtering, concentrating the filtrate under reduced pressure, and vacuum drying at 60 deg.C to obtain radix Scutellariae extract.
6. Use according to claim 5, characterized in that: the method also comprises the step of separating and extracting the obtained scutellaria baicalensis extract, and specifically comprises the following steps: filling pretreated macroporous resin LX-38 into a resin column, wherein the ratio of the diameter to the height of the filled column is 1: and 8, taking 100mg of the scutellaria baicalensis extract, wherein the sample concentration is 1mg/mL, when the liquid level of the sample is flush with the resin surface, removing impurities and eluting by using 0.8BV of 30% ethanol, eluting by using 4BV of 50% ethanol, 4BV of 60% ethanol and 3BV of 100% ethanol, collecting 100% ethanol eluent, spin-drying the solvent, and performing vacuum drying to obtain the component C.
7. Use according to any one of claims 1 to 6, characterized in that: the medicament further comprises a pharmaceutically acceptable carrier;
or the medicament also comprises a pharmaceutically acceptable antiviral medicament or other related active substances.
CN202210756842.4A 2022-06-30 2022-06-30 Application of scutellaria baicalensis extract in preparation of medicine for preventing and/or treating respiratory virus infection Pending CN114931592A (en)

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