CN113332400B - Antiviral traditional Chinese medicine volatile oil composition and application thereof - Google Patents
Antiviral traditional Chinese medicine volatile oil composition and application thereof Download PDFInfo
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- CN113332400B CN113332400B CN202110539405.2A CN202110539405A CN113332400B CN 113332400 B CN113332400 B CN 113332400B CN 202110539405 A CN202110539405 A CN 202110539405A CN 113332400 B CN113332400 B CN 113332400B
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/35—Extraction with lipophilic solvents, e.g. Hexane or petrol ether
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses an antiviral traditional Chinese medicine volatile oil composition and application thereof. The antiviral traditional Chinese medicine volatile oil composition oil provided by the invention has the characteristics of high safety, strong drug effect, long acting time and the like, and is remarkable in that the antiviral traditional Chinese medicine volatile oil composition provided by the invention is not easy to generate drug resistance.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicines, and in particular relates to an antiviral traditional Chinese medicine volatile oil composition and application thereof.
Background
Influenza is an acute respiratory infectious disease caused by influenza virus, and is high in spring and autumn. Because the influenza virus gene is easy to be mutated, the virus gene recombination process is complex and unpredictable, and the influenza virus gene is easy to be mutated into a new influenza virus variant strain. In addition, avian Influenza Virus (AIV) frequently infects humans directly from birds across species, and even once has caused a pandemic worldwide, posing a huge potential for human health and public health security. Human influenza is mainly caused by influenza a and influenza b viruses. Influenza a viruses often have antigenic variation and can be further classified into subtypes such as H1N1, H3N2, H5N1, and H7N 9. H1N1 is an RNA virus belonging to the family Orthomyxoviridae. Its hosts are birds and some mammals. Among them, influenza a virus is susceptible to variation in antigenicity, which causes a worldwide pandemic many times. For example, in a pandemic of 1918 to 1919, at least 2000 to 4000 million people die worldwide from influenza.
Most of the existing medicines for treating viruses are synthetic medicines, such as acyclovir, erythromycin and the like, although the effect is considerable, the bioavailability is low, the metabolism period of the medicines is long, the toxicity of the medicines is high, the medicines can relapse, and most importantly, the medicines are easy to generate medicine resistance. The traditional Chinese medicine can effectively treat various diseases caused by viruses from the two aspects of treatment based on syndrome differentiation and holism concept, has small toxic and side effects, low recurrence rate and good tolerance of patients, and the traditional Chinese medicine volatile oil is an important effective component of the traditional Chinese medicine, but the research on the antivirus of the traditional Chinese medicine volatile oil is rarely reported.
Disclosure of Invention
The invention aims to provide an antiviral traditional Chinese medicine volatile oil composition and application thereof, so as to solve the technical problems. In order to realize the purpose, the technical scheme of the invention is as follows:
in a first aspect, the antiviral traditional Chinese medicine volatile oil composition provided by the invention comprises the following raw materials in parts by weight: 5 to 10 parts of argyi leaf volatile oil, 5 to 10 parts of rhizoma atractylodis volatile oil, 5 to 10 parts of elsholtzia volatile oil, 5 to 10 parts of tea tree volatile oil, 5 to 10 parts of rhizoma kaempferiae volatile oil, 5 to 10 parts of clove volatile oil, 5 to 10 parts of rosemary volatile oil, 5 to 10 parts of origanum vulgaris volatile oil, 5 to 10 parts of juniper wood volatile oil, 5 to 10 parts of cajeput volatile oil, 10 to 20 parts of forsythia volatile oil and 10 to 20 parts of wild chrysanthemum volatile oil.
Preferably, the antiviral traditional Chinese medicine volatile oil composition comprises the following raw materials in parts by weight: 7.5 parts of folium artemisiae argyi volatile oil, 7.5 parts of rhizoma atractylodis volatile oil, 7.5 parts of herba elsholtziae volatile oil, 7.5 parts of tea tree volatile oil, 6 parts of rhizoma kaempferiae volatile oil, 6 parts of clove volatile oil, 6 parts of rosemary volatile oil, 6 parts of origanum vulgaris volatile oil, 8 parts of juniper volatile oil, 8 parts of cajeput volatile oil, 15 parts of fructus forsythiae volatile oil and 15 parts of wild chrysanthemum volatile oil.
Preferably, the folium artemisiae argyi volatile oil, the rhizoma atractylodis volatile oil, the herba elsholtziae volatile oil, the tea tree volatile oil, the rhizoma kaempferiae volatile oil, the clove volatile oil, the rosemary volatile oil, the oregano volatile oil, the juniper volatile oil, the cajeput volatile oil, the fructus forsythiae volatile oil and the wild chrysanthemum volatile oil are respectively extracted by an ultrasonic-assisted steam distillation method.
Further preferably, the ultrasound-assisted steam distillation method comprises the following steps:
(1) Pulverizing and sieving the raw materials to obtain coarse powder;
(2) Fully and uniformly mixing the obtained coarse powder with water, and performing ultrasonic oscillation extraction to obtain a coarse extract;
(3) Putting the crude extract into a steam distillation device, and extracting volatile oil by adopting a steam distillation method; stopping heating, cooling, and collecting distillate;
(4) Extracting volatile oil from the distillate by using an organic solvent, removing water and the organic solvent to obtain the volatile oil.
More preferably, the weight ratio of the coarse powder to the water in the step (2) is 1.
Still more preferably, the conditions for ultrasonic oscillation extraction in step (2) are as follows: the power is 200-400W, the temperature is 50-60 ℃, and the time is 1-3 hours.
Still more preferably, the time for extracting the water vapor in the step (3) is 2 to 10 hours.
Still more preferably, the organic solvent in step (4) is selected from one or more of n-butanol, diethyl ether, petroleum ether, ethanol, ethyl acetate, acetone and n-hexane.
In a second aspect, the invention provides an application of the antiviral traditional Chinese medicine volatile oil composition in preparation of an antiviral medicine.
Preferably, the antiviral drug is a dosage form prepared by a pharmaceutically acceptable carrier and the antiviral traditional Chinese medicine volatile oil composition.
Further preferably, the pharmaceutically acceptable carrier is selected from one or more of excipients, binders, fillers, lubricants, antioxidants, preservatives and thickeners.
Further preferably, the formulation is selected from any one of nasal drops, gels, pastes, patches, liniments, tinctures, lotions, plastics and emplastrums.
Preferably, the antiviral drug is any one of anti-influenza virus drugs and anti-avian influenza virus drugs.
Compared with the prior art, the invention has the beneficial effects that:
compared with the synthetic medicines on the market, the antiviral traditional Chinese medicine volatile oil composition provided by the invention has the characteristics of high safety, strong drug effect, long acting time and the like, and is worthy of notice that the antiviral traditional Chinese medicine volatile oil composition provided by the invention is not easy to generate drug resistance.
The Chinese medicinal volatile oil composition provided by the invention takes twelve Chinese medicinal volatile oils as raw materials, the twelve volatile oils have different effective components, can realize synergistic effect after compatibility and have synergistic effect on virus resistance, wherein,
wild chrysanthemum: compositae plant, whose main components include luteolin-7-O-beta-D-glucoside, apigenin-7-O-beta-D-glucoside, diosmetin-7-O-beta-D-glucoside, and linarin, and can resist various virus-induced diseases such as influenza virus, so that flos Chrysanthemi Indici volatile oil has good antiviral effect;
fructus forsythiae: the main components of the trifoliaceae plant comprise forsythoside A, 4-methyl hydroxycinnamate, benzyl acetate, 4-methyl hydroxyphenylacetate, 1, 3-cyclohexanedione, forsythin, rutin and the like, so that the extracted forsythia volatile oil has good antiviral effect;
folium artemisiae argyi: the essential oil of Compositae plant comprises monoterpene components such as eucalyptol, camphor and borneol, and sesquiterpene components such as bicyclic sesquiterpene beta-caryophyllene, and flavonoids mainly including eupatilin and jaceosidin, which have good antiviral and antiinflammatory effects;
rhizoma atractylodis: the effective components of Compositae plant mainly include atractylodin, atractylol and beta-eucalyptol. The sesquiterpene compound in the rhizoma atractylodis has good anti-inflammatory, antiviral, liver-protecting and antibacterial effects;
herba Moslae: the main components of the volatile oil of labiate plants comprise flavonoids, glycosides, coumarins and the like, wherein the elsholtzia aqueous extract is proved to promote the production of IL-2 and gamma interferon (IFN-gamma) in serum and has the function of resisting influenza A virus H1N 1;
tea plant: theaceae plant, whose essential oil comprises p-cymene, terpinen-4-ol, terpinolene, 1, 8-cineole, alpha-pinene, gamma-terpinene, etc., can effectively treat influenza virus, etc.;
and (3) rhizoma kaempferiae: the Zingiberaceae plant has abundant volatile oil, and mainly contains p-methoxy ethyl cinnamate, kaempferol, kaempferide, quercetin, ferulic acid, etc., and rhizoma Kaempferiae has anti-tumor, antiinflammatory, and antioxidant effects;
clove: the essential oil of the plant of the family Oleaceae mainly contains eugenol, and can effectively resist tobacco mosaic virus and novel coronavirus;
rosemary: labiatae plants, which mainly comprise phenolic acid compounds, such as rosmarinic acid, rosmarinic phenol, carnosic acid, carnosol, caffeic acid, ursolic acid and the like, are mainly used in anti-inflammatory and antibacterial drugs, and volatile oil of the labiatae plants mainly comprises limonene, camphor, alpha-pinene, beta-pinene, camphene, terpineol and the like and also has good antibacterial and anti-inflammatory effects;
and (3) oregano: the volatile oil of the labiate plant mainly comprises carvacrol, thymol, rosmarinic acid, 3, 4-dihydroxy benzoic acid, 2, 5-dihydroxy benzoic acid and other substances, and can be clinically used for treating viruses such as respiratory syncytial virus and novel coronavirus and the like;
chinese juniper: the cypress family plant is the combined name of the red juniper and the hinoki, and the hinoki leaves are used as the medicine, and have the effects of cooling blood and stopping bleeding, dispelling wind and regulating dampness, and cooling and astringing;
cajeput (root of cajeput) of cajeput: myrtaceae plant mainly contains volatile oil, and its components include terpinen-4-ol, 1, 8-cineole, gamma-terpinen, alpha-terpinene, terpinolene, etc., and the volatile oil has good antibacterial activity, antiviral activity, antiinflammatory activity, and antitumor effect.
Detailed Description
The following further describes embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
Examples 1 to 8
Pulverizing and sieving dried folium Artemisiae Argyi to obtain coarse powder; fully and uniformly mixing the obtained coarse powder with water according to the weight ratio of 1; placing the obtained crude extract in a steam distillation device, heating to boil, keeping a slightly boiling state for continuous extraction for 8 hours, stopping heating, cooling, collecting distillate, extracting by using ethanol, wherein the volume ratio of the ethanol to the distillate is 1.
Pulverizing dried rhizoma Atractylodis to obtain coarse powder; fully and uniformly mixing the obtained coarse powder with water according to the weight of 1; placing the obtained crude extract in a steam distillation device, heating to boil, keeping a slightly boiling state for continuous extraction for 4 hours, stopping heating, cooling, collecting distillate, extracting by using ethanol, wherein the volume ratio of the ethanol to the distillate is 2.
Pulverizing dried herba Moslae whole plant, and sieving to obtain coarse powder; fully and uniformly mixing the obtained coarse powder with water according to the weight of 1; placing the obtained crude extract in a steam distillation device, heating to boil, keeping a slightly boiling state for continuous extraction for 8 hours, stopping heating, cooling, collecting distillate, extracting with ethanol, wherein the volume ratio of the ethanol to the distillate is 1. Crushing and sieving dried tea leaves to obtain coarse powder; mixing the obtained coarse powder with water according to the weight of 1, performing ultrasonic oscillation extraction for 3 hours under the conditions of power of 300W and temperature of 60 ℃ to obtain a crude extract, placing the obtained crude extract in a steam distillation device, heating to boil, keeping a slightly boiling state, continuously extracting for 5 hours, stopping heating, cooling, collecting distillate, extracting with ethanol, wherein the volume ratio of the ethanol to the distillate is 1.
Pulverizing and sieving dried rhizoma Kaempferiae to obtain coarse powder; fully and uniformly mixing the obtained coarse powder with water according to the weight of 1; placing the obtained crude extract in a steam distillation device, heating to boil, keeping a slightly boiling state for continuous extraction for 5 hours, stopping heating, cooling, collecting distillate, extracting with ethanol, wherein the volume ratio of ethanol to distillate is 1, taking an oil phase, adding anhydrous sodium sulfate 0.02 times of the weight of the oil phase, standing overnight, removing water, and recovering ethanol from the rest liquid under reduced pressure at 40 ℃ and vacuum degree of-0.05 Mpa until no obvious distillate flows out, thereby obtaining the tea tree volatile oil.
Pulverizing dried flos Caryophylli, and sieving to obtain coarse powder; fully and uniformly mixing the obtained coarse powder with water according to the weight of 1; placing the crude extract in a steam distillation device, heating to boil, keeping the micro-boiling state for continuous extraction for 6 hours, stopping heating, cooling, collecting distillate, extracting with ethanol, collecting oil phase, recovering ethanol under reduced pressure at 50 deg.C and vacuum degree of-0.03 Mpa until no obvious distillate flows out to obtain crude volatile oil, adding petroleum ether into the crude volatile oil for extraction, collecting oil phase, adding anhydrous sodium sulfate 0.02 times the weight of the crude volatile oil, standing overnight, removing water, recovering petroleum ether from the residual liquid under reduced pressure at 50 deg.C and vacuum degree of-0.03 Mpa until no obvious distillate flows out to obtain flos Caryophylli volatile oil.
Pulverizing dried herba Rosmarini officinalis, and sieving to obtain coarse powder; fully and uniformly mixing the obtained coarse powder with water according to the weight ratio of 1; placing the obtained crude extract in a steam distillation device, heating to boil, keeping a slightly boiling state for continuous extraction for 6 hours, stopping heating, cooling, collecting distillate, extracting by using ethanol, wherein the volume ratio of the ethanol to the distillate is 1.
Pulverizing dried origanum vulgare, and sieving to obtain coarse powder; fully and uniformly mixing the obtained coarse powder with water according to the weight ratio of 1; placing the obtained crude extract in a steam distillation device, heating to boil, keeping a slightly boiling state, continuously extracting for 6 hours, stopping heating, cooling, collecting distillate, extracting with diethyl ether for 2 times, wherein the volume of diethyl ether used in each extraction is the same as that of the distillate, combining the extract solutions, adding 0.02 times of anhydrous sodium sulfate into the extract solution, standing overnight, removing water, and recovering diethyl ether from the rest liquid under reduced pressure at 35 deg.C and vacuum degree of-0.05 Mpa until no obvious fraction flows out to obtain volatile oil of origanum vulgaris.
Pulverizing dried Chinese juniper and sieving to obtain coarse powder; fully and uniformly mixing the obtained coarse powder with water according to the weight of 1; placing the crude extract in a steam distillation device, heating to boil, keeping slightly boiling state, continuously extracting for 10 hr, stopping heating, cooling, collecting distillate, extracting with petroleum ether for 3 times, wherein the volume of petroleum ether used in each extraction is the same as that of distillate, mixing extractive solutions, adding 0.02 times of anhydrous sodium sulfate into extractive solution, standing overnight, removing water, and recovering petroleum ether under reduced pressure at 45 deg.C and vacuum degree of-0.06 Mpa to obtain volatile oil of Sabina chinensis.
Pulverizing dried Melaleuca alternifolia L, and sieving to obtain coarse powder; fully and uniformly mixing the obtained coarse powder with water according to the weight of 1; placing the obtained crude extract in a steam distillation device, heating to boil, keeping a slightly boiling state for continuous extraction for 10 hours, stopping heating, cooling, collecting distillate, extracting for 2 times by using ethanol, wherein the volume of the ethanol used in each extraction is the same as that of the distillate, combining the extract solutions, recovering the ethanol under reduced pressure at 45 ℃ and under the vacuum degree of-0.03 Mpa until no obvious distillate flows out to obtain a crude volatile oil product, adding ethyl acetate into the crude volatile oil product for extraction, wherein the volume ratio of the ethyl acetate to the crude volatile oil product is 1.
Pulverizing dried fructus forsythiae, and sieving to obtain coarse powder; fully and uniformly mixing the obtained coarse powder with water according to the weight ratio of 1; placing the obtained crude extract in a steam distillation device, heating to boil, keeping a slightly boiling state, continuously extracting for 8 hours, stopping heating, cooling, collecting distillate, extracting with ethanol for 3 times, wherein the volume of ethanol used in each extraction is the same as that of the distillate, combining the extract solutions, adding 0.02 time of anhydrous sodium sulfate into the extract solution, standing overnight, removing water, and recovering organic solvent from the rest liquid under reduced pressure at 50 ℃ and vacuum degree of-0.03 Mpa until no obvious fraction flows out to obtain fructus forsythiae volatile oil.
Pulverizing and sieving dried flos Chrysanthemi Indici to obtain coarse powder; fully and uniformly mixing the obtained coarse powder with water according to the weight of 1; placing the crude extract in a steam distillation device, heating to boil, keeping slightly boiling state, continuously extracting for 6 hours, stopping heating, cooling, collecting distillate, extracting with ethanol for 3 times, wherein the volume of ethanol used in each extraction is the same as that of the distillate, mixing the extractive solutions, adding 0.02 times of anhydrous sodium sulfate into the extractive solution, standing overnight, removing water, and recovering organic solvent under reduced pressure at 50 deg.C and vacuum degree of-0.03 Mpa until no significant fraction flows out to obtain flos Chrysanthemi Indici volatile oil.
Weighing the volatile oil raw materials in the following table weight parts, and uniformly mixing the volatile oil raw materials.
Research on effect of traditional Chinese medicine volatile oil composition on treatment of mice infected by influenza virus H1N1/FM1 strain
1. Experimental materials
1.1 animals and groups
50 SPF-grade ICR mice with half male and female, 9-12 weeks old and 13-15g weight are selected and purchased from the scientific and technical center of laboratory animals of Jiangxi university of traditional Chinese medicine [ quality certification number: SCXK 2018-0003]. And (3) feeding the clean animal house, wherein the unified illumination time is 06-18, and the constant temperature and humidity are adopted for freely drinking water and feeding solid feed. Adapted feeding was used for the experiment after 3 d. The method is divided into the following 5 groups according to a random number table method, and each group comprises 10 pieces:
normal control group
Model control group
Chinese medicinal volatile oil composition with large, medium and small dosage groups
1.2 Experimental drugs
The Chinese medicinal volatile oil composition was prepared according to the method of example 1.
1.3 Primary reagents
Trizol Reagent is purchased from Ambion by life technology (batch No. 257403), influenza Virus AReal Time RT-PCR Kit is purchased from Shanghai river Biotech GmbH (batch No. NOP 20200101), ethyl ether is purchased from Shanghai Hongsheng Fine chemical Co., ltd (batch No. 20181009), mouse alpha interferon (IFN-alpha) enzyme linked immunoassay is purchased from Shanghai enzyme linked Biotech Co., ltd (batch No. NO 03/2019), mouse MDA enzyme linked immunoassay is purchased from Shanghai enzyme linked Biotech Co., ltd (batch No. NO 07/2020), mouse tumor necrosis factor alpha (TNF-alpha) enzyme linked immunoassay is purchased from U.S. bio-technology (batch No. NO 546999).
1.4 Main Instrument
<xnotran> A2 (: thermo MSC 1.8), A2 (: thermo MSC 1.2) Thermo , A2 (: nuaire NU-425-400E) Nuaire, (: YP1002 MAX100 g) , (: ltem-AR 1140MAX110 g) Ohaus Corp.Brock.WJ.USA, (: AL 204) - ( ) , real-Time PCR Instrument (: quantStudio 5) Appliedbiosystems, (: LX-300) Kylin-Bell Lab Instruments, (: QL-901) , ( Eppendorf-5810R) eppendorf, (: AR 1140) Ohaus Corp.Brock.NJ.USA, ( Enspire) PerkinElmer, (: SONIC RUPTOR 400) MONI homogenizer company, (: fastprepTM-24) MP Biomedicals, IVC (: ZJ-4) . </xnotran>
2. Experimental methods
2.1 model building
Except for the normal control group, the mice were lightly anesthetized with ether and infected nasally with 15 drops of LD50 influenza virus (H1N 1/FM1 strain) of 35. Mu.l each. The administration is started on the day of infection, and the large, medium and small dose groups are administered by nasal drip at each time, the administration amount of each time is shown in table 1, 1 time per day, and 4 days continuously, and a normal control group and a model control group are subjected to nasal drip by distilled water under the same conditions. Dissections were done after weighing on day 5. Weighing lung, and calculating lung index and lung index inhibition rate. The results were statistically processed using an interclass comparison t-test, where the formula is as follows:
lung index (%) = [ lung wet weight (g)/body weight (g) ] × 100%;
lung index inhibition (%) = [ model control lung weight (g) -administration group lung weight (g) ]/[ model control lung weight (g) -normal control lung weight (g) ] × 100%.
2.2 Effect on the nucleic acid viral load of Lung tissue
Dissecting a mouse, subpackaging lung tissues and storing in a low-temperature refrigerator at minus 80 ℃ for storage, taking out from the low-temperature refrigerator at minus 80 ℃ during detection, placing in a cracking tube containing a cracking medium, adding 1ml of TRIzol Reagent, screwing a tube cover, placing cracking light on a vibration cracking instrument, taking down after vibration cracking for 20s, incubating at room temperature for 10min, incubating at 4 ℃,12000rpm, and centrifuging for 10min; transferring the clarified supernatant into a new 1.5ml centrifuge tube; adding 0.2ml chloroform, covering the tube cap tightly, shaking the centrifuge tube for 15s with force, and incubating at room temperature for 2-3min until liquid is layered; centrifuging at 12000rpm at 4 deg.C for 15min; carefully transferring the transparent supernatant into a new 1.5ml centrifuge tube, adding 0.5ml isopropanol, mixing uniformly, and incubating at room temperature for 30min; centrifuging at 12000rpm at 4 deg.C for 10min; the supernatant was discarded and the pellet was washed gently with 1ml of 75% ethanol (the white pellet was floated gently); centrifuging at 7500rpm for 5min at 4 deg.C; sucking up the supernatant, and temporarily drying the RNA precipitate for 5-10min; dissolving the precipitate with 30 μ l DEPC water, and storing at-80 deg.C in refrigerator.
Treatment of the samples before detection: each sample was diluted 1000 times and tested on the machine.
And (3) treating the nucleic acid of the reference substance: DEPC-H 2 O as a negative control. The positive control was diluted 10, 100, 1000 fold gradient.
Preparing a reagent: taking n multiplied by 18 mu l IFVA nucleic acid fluorescence PCR detection mixed solution, n multiplied by 1 mu l internal reference substance, and n multiplied by 1 mu l RT-PCR enzyme (n is the number of reaction tubes), shaking and mixing evenly for a plurality of seconds, and centrifuging at 3000rpm for a plurality of seconds.
Sample adding: and (3) placing 20 mu l of the mixed solution into a PCR tube, then respectively adding 5 mu l of each of the sample nucleic acid extracting solution, the DEPC-H2O and the positive control into the PCR tube, tightly covering the tube cover, centrifuging for several seconds to place all the liquid at the bottom, and immediately carrying out PCR amplification reaction.
The cycle parameters are set as: x10 min at 45 ℃; x 15min at 95 ℃; circulating for 40 times according to 95 ℃ multiplied by 15sec → 60 ℃ multiplied by 60 sec; single-point fluorescence detection was performed at 60 ℃ in a 25. Mu.l reaction system.
2.3 Effect on inflammatory factors in Lung tissue (Elisa method)
After the lung tissue of the mouse is weighed, the lung tissue of the mouse is collected and stored at-4 ℃. After 50mg of lung tissue was weighed and 500. Mu.L of physiological saline was added, the tissue was homogenized using an ultrasonic cell disruptor and centrifuged at 1000g at 4 ℃ for 10 minutes using a low-temperature high-speed centrifuge. And sucking the supernatant, subpackaging, and storing in a refrigerator at-80 ℃ for later use. When in detection, the operation is carried out according to the instruction of the kit, and the absorbance of the microplate reader at 450nm is used for detecting each index.
2.5 statistical methods
Statistical analysis was performed using SPSS 22.0 software, and comparison between groups was performed using a control t-test and one-way analysis of variance to
Indicates that the Dunnet test was performed for multiple comparisons of differences between groups using independent sample t-test or one-way analysis of variance (ANOVA), as P<0.05 indicates that the statistical difference is statistically significant.
3 results of the experiment
TABLE 1 therapeutic Effect on influenza Virus H1N1/FM1 Strain infected mouse model of pneumonia
Note: comparison with Normal group ## P<0.01; compared with the model control group, ** P<0.01
TABLE 2 Effect of Chinese medicinal volatile oil compositions on pulmonary tissue viral load of H1N1 influenza A Virus-infected mice
Note: compared with the normal control group, ## P<0.01; compared with the model control group, * P<0.05， ** P<0.01。
TABLE 3 Effect of Chinese medicinal volatile oil compositions on the content of inflammatory factors in Lung tissue of H1N1 influenza A Virus-infected mice (N = 6)
Note: compared with the normal control group, # P<0.05， ## P<0.01; compared with the model group of epidemic virus attacking lung, * P<0.05， ** P<0.01。
4 conclusion
Table 1 the results show that: after the influenza A H1N1 virus FM1 strain is adopted to infect the mice, the pulmonary index of the mice is obviously increased, and the mice have a dominant difference (P < 0.01) compared with a normal control group; after the traditional Chinese medicine volatile oil composition is applied to the high-dose and medium-dose groups for 4 days on the infection day, the lung indexes of the high-dose and medium-dose groups are obviously reduced, and the lung indexes are obviously different from those of a model control group (P < 0.01). The inhibition rates were 45.18% and 64.04%, respectively.
Table 2 the results show: no influenza A H1N1 virus nucleic acid expression exists in lung tissues of animals in a normal control group; the lung tissue of the mouse in the model control group has obvious nucleic acid expression, and has obvious difference compared with the normal group (P < 0.01); the three dosage groups of the traditional Chinese medicine volatile oil composition can obviously reduce the expression level of virus nucleic acid in lung tissues, and have obvious difference (P < 0.01) compared with a model group.
Table 3 the results show that: the contents of inflammatory factors MDA and TNF-a in the lung tissues of mice in a model control group are both obviously increased, and compared with a normal control group, the contents of the inflammatory factors MDA and TNF-a are obviously different (P is less than 0.01), the contents of the inflammatory factors IFN-alpha in the lung tissues of the mice are obviously reduced, and compared with the normal control group, the contents of the inflammatory factors IFN-alpha are obviously different (P is less than 0.05); the small-dose group of the traditional Chinese medicine volatile oil composition can obviously reduce the MDA content in lung tissues, and has significant difference (P < 0.05) compared with a model control group, and the three dose groups of the traditional Chinese medicine volatile oil composition can obviously reduce the TNF-alpha content in lung tissues, and has significant difference (P < 0.01) compared with the model control group.
(II) protection effect of traditional Chinese medicine volatile oil composition on mouse death caused by influenza virus H1N1/FM1 strain
1. Experimental Material
1.1 animals and groups
80 SPF-grade ICR mice, half of each sex, 9-12 weeks old and 13-15g of body weight, are selected and purchased from the scientific and technical center of laboratory animals of the university of traditional Chinese medicine in Jiangxi (quality certification number: SCXK 2018-0003]. And (3) feeding the clean animal house, wherein the unified illumination time is 06-18, and the constant temperature and humidity are adopted for freely drinking water and feeding solid feed. Adapted feeding was used for the experiment after 3 d. The method is divided into the following 4 groups according to a random number table method, and each group comprises 20 pieces:
model control group
Chinese medicinal volatile oil composition with large, medium and small dosages
1.2 Experimental drugs
The traditional Chinese medicine volatile oil composition is prepared according to the method of example 1.
1.3 Primary reagents
Trizol Reagent is purchased from Ambion by life technology (batch No. 257403), influenza Virus AReal Time RT-PCR Kit is purchased from Shanghai river Biotech GmbH (batch No. NOP 20200101), ethyl ether is purchased from Shanghai Hongsheng Fine chemical Co., ltd (batch No. 20181009), mouse alpha interferon (IFN-alpha) enzyme linked immunoassay is purchased from Shanghai enzyme linked Biotech Co., ltd (batch No. NO 03/2019), mouse MDA enzyme linked immunoassay is purchased from Shanghai enzyme linked Biotech Co., ltd (batch No. NO 07/2020), mouse tumor necrosis factor alpha (TNF-alpha) enzyme linked immunoassay is purchased from U.S. bio-technology (batch No. NO 546999).
1.4 Main Instrument
<xnotran> A2 (: thermo MSC 1.8), A2 (: thermo MSC 1.2) Thermo , A2 (: nuaire NU-425-400E) Nuaire, (: YP1002 MAX100 g) , (: ltem-AR 1140MAX110 g) Ohaus Corp.Brock.WJ.USA, (: AL 204) - ( ) , real-Time PCR Instrument (: quantStudio 5) Appliedbiosystems, (: LX-300) Kylin-Bell Lab Instruments, (: QL-901) , ( Eppendorf-5810R) eppendorf, (: AR 1140) Ohaus Corp.Brock.NJ.USA, ( Enspire) PerkinElmer, (: SONIC RUPTOR 400) MONI homogenizer company, (: fastprepTM-24) MP Biomedicals, IVC (: ZJ-4) . </xnotran>
2. Experimental methods
2.1 determination of viral infection amount: mu.l of influenza virus H1N1/FM1 strain solution is inoculated into a culture solution bottle containing 10ml, the bottle is placed into an incubator and cultured for 24 hours at 37 ℃, and then the virus titer is determined. Then diluting the virus liquid by 10 times with normal saline, and using 6 concentration gradients for infecting mice, wherein each concentration is 10, observing the death condition of the animals within 2 weeks after infection, and taking the concentration with the death rate of 90-95% as the infection concentration of the animals in the formal test.
2.2 groups of mice were lightly anaesthetized with ether and infected nasally with 2 drops of LD50 influenza virus (strain FM 1), 30. Mu.l each. Administration was started on the day of infection, and the large, medium and small dose groups were administered at the doses shown in table 4 1 time per day for 5 consecutive days, and the model control group was nasally dropped with distilled water under the same conditions. The animals were observed for mortality within 2 weeks after infection and mortality, mortality protection, mean survival days and life extension were calculated. Results were statistically processed using the interclass comparison chi-square test and t-test. The calculation formula is as follows:
mortality (%) = [ number of deaths/total number of animals ] × 100%;
mortality protection (%) = [ (mortality of model control group-mortality of administration group)/mortality of model control group ] × 100%;
life extension rate (%) = [ (days-survival of drug administration group-days-survival of model control group)/days-survival of model control group ] × 100%.
2.3 statistical methods
Statistical analysis was performed using SPSS 22.0 software, and comparison between groups was performed using a control t-test and one-way analysis of variance to
Indicates that the Dunnet test was performed for multiple comparisons of differences between groups using independent sample t-test or one-way analysis of variance (ANOVA), as P<0.05 indicates that the statistical difference is statistically significant.
3 results of the experiment
TABLE 4 mortality protection against influenza virus H1N1/FM1 strain infected mouse model of pneumonia
Note: p <0.01, P <0.05 compared to model control group
Discussion 4
In 2 weeks after mice are infected by the influenza A H1N1 virus FM1 strain, the mortality of animals in a model control group is 95 percent, and the average survival days are 8.15 days; the traditional Chinese medicine volatile oil composition is administered on the same day of infection, and the mortality rate of mice in a large-dose group of the traditional Chinese medicine volatile oil composition is remarkably reduced for 5 consecutive days, and is remarkably different from that in a model group (P is less than 0.05); the survival days of mice can be prolonged by three dosage groups of the traditional Chinese medicine volatile oil composition, and compared with a model control group, the survival days of the mice have dominant differences (P <0.05, P < -0.01) and the life prolonging rate reaches 30.06 percent, 23.31 percent and 15.34 percent.
(III) protection effect of traditional Chinese medicine volatile oil composition on mouse death caused by influenza virus H1N1/PR8 strain
1. Experimental Material
1.1 animals and groups
50 SPF-grade ICR mice with half male and female, 9-12 weeks old and 13-15g weight are selected and purchased from the scientific and technological center of laboratory animals of pharmaceutical university in Jiangxi (quality certification number: SCXK 2018-0003]. And (3) feeding the clean animal house, wherein the unified illumination time is 06-18, and the constant temperature and humidity are adopted for freely drinking water and feeding solid feed. Adapted feeding was used for the experiment after 3 d. The method is divided into the following 5 groups according to a random number table method, and each group comprises 10 pieces:
normal control group
Model control group
Chinese medicinal volatile oil composition with large, medium and small dosages
1.2 Experimental drugs
The Chinese medicinal volatile oil composition was prepared according to the method of example 1.
1.3 Primary reagents
Trizol Reagent is purchased from Ambion by life technology (batch No. 257403), influenza Virus AReal Time RT-PCR Kit is purchased from Shanghai river Biotech GmbH (batch No. NOP 20200101), ethyl ether is purchased from Shanghai Hongsheng Fine chemical Co., ltd (batch No. 20181009), mouse alpha interferon (IFN-alpha) enzyme linked immunoassay is purchased from Shanghai enzyme linked Biotech Co., ltd (batch No. NO 03/2019), mouse MDA enzyme linked immunoassay is purchased from Shanghai enzyme linked Biotech Co., ltd (batch No. NO 07/2020), mouse tumor necrosis factor alpha (TNF-alpha) enzyme linked immunoassay is purchased from U.S. bio-technology (batch No. NO 546999).
1.4 Main Instrument
<xnotran> A2 (: thermo MSC 1.8), A2 (: thermo MSC 1.2) Thermo , A2 (: nuaire NU-425-400E) Nuaire, (: YP1002 MAX100 g) , (: ltem-AR 1140MAX110 g) Ohaus Corp.Brock.WJ.USA, (: AL 204) - ( ) , real-Time PCR Instrument (: quantStudio 5) Appliedbiosystems, (: LX-300) Kylin-Bell Lab Instruments, (: QL-901) , ( Eppendorf-5810R) eppendorf, (: AR 1140) Ohaus Corp.Brock.NJ.USA, ( Enspire) PerkinElmer, (: SONIC RUPTOR 400) MONI homogenizer company, (: fastprepTM-24) MP Biomedicals, IVC (: ZJ-4) . </xnotran>
2. Experimental methods
2.1 except for the normal control group, mice were lightly anesthetized with ether and infected with 15 drops of LD50 influenza virus (H1N 1/PR8 strain) in the nose, 35. Mu.l each. Administration was started on the day of infection, and the large, medium and small dose groups were nasally administered in the dose of table 5 each time, 1 time per day for 4 consecutive days, and the normal control group and the model control group were nasally administered with distilled water under the same conditions. Dissections were done after weighing on day 5. Weighing lung, and calculating lung index and lung index inhibition rate. Results were statistically processed using the comparative t-test between groups. The calculation formula is as follows:
lung index (%) = [ lung wet weight (g)/body weight (g) ] × 100%;
lung index inhibition (%) = [ model control lung weight (g) -administration group lung weight (g) ]/[ model control lung weight (g) -normal control lung weight (g) ] × 100%.
2.2 statistical methods
Statistical analysis was performed using SPSS 22.0 software, and comparison between groups was performed using a control t-test and one-way analysis of variance to
Indicates that the Dunnet test was performed for multiple comparisons of differences between groups using independent sample t-test or one-way analysis of variance (ANOVA), as P<0.05 indicates that the statistical difference is statistically significant.
3 results of the experiment
TABLE 5 therapeutic Effect on influenza Virus H1N1/PR8 strains in the pneumonia model of mice
Note: comparison with Normal group ## P<0.01; p compared to model control group<0.05， ** P<0.01
4 discussion of
After the influenza virus H1N1/PR8 is adopted to infect a mouse, the lung index of the mouse is obviously increased, and the lung index has obvious difference (P < 0.01) compared with a normal control group; after the traditional Chinese medicine volatile oil composition is applied for treatment for 4 days on the infection day, the lung index of a large-dose group of the traditional Chinese medicine volatile oil composition is obviously reduced, and compared with a model control group, the lung index of the large-dose group of the traditional Chinese medicine volatile oil composition has a dominant difference (P < 0.05), and the lung index inhibition rate is 36.59%.
Comparative example 1
To further illustrate the beneficial effects of the present invention, the present comparative example prepared the traditional Chinese medicine volatile oil composition by the similar method as in example 1, and the present comparative example only differs from example 1 in that: in this comparative example, the distillate was not further extracted, but the collected distillate was used as a volatile oil raw material.
Comparative example 2
To further illustrate the beneficial effects of the present invention, the present comparative example prepared the traditional Chinese medicine volatile oil composition by the similar method as in example 1, and the present comparative example only differs from example 1 in that: in this comparative example, after the distillate of each of the Chinese medicinal materials was prepared in example 1, ethanol was used as an extractant, and the volume ratio of ethanol to the distillate was 1.
(IV) therapeutic action of each composition on pneumonia model of mice infected by different influenza viruses
In order to further illustrate the beneficial effects of the present invention, the test method of example 1 was followed to test the efficacy of the Chinese medicinal volatile oil compositions obtained in examples 2 to 9 and comparative examples 1 to 2, and the test results are shown in tables 6 to 8 below.
TABLE 6 therapeutic Effect of the compositions on pneumonia model of influenza virus H1N1/FM1 strain-infected mice
Note: comparison with Normal group ## P<0.01; compared with the model control group, ** P<0.01
TABLE 7 mortality protection against influenza virus H1N1/FM1 strain infected mouse model of pneumonia
Note: p <0.01, P <0.05 compared to model control group
TABLE 8 therapeutic Effect on influenza Virus H1N1/PR8 strains in the pneumonia model of mice
Note: comparison with Normal group ## P<0.01; p compared to model control group<0.05， ** P<0.01
The results in tables 6 to 8 show that: compared with examples 6-8 and comparative examples 1-2, the traditional Chinese medicine volatile oil composition in examples 1-5 has good treatment effect on influenza virus H1N1/FM1 strain-infected mouse pneumonia models, influenza virus H1N1/FM1 strain-infected mouse pneumonia models and influenza virus H1N1/PR8 strain-infected mouse pneumonia models, wherein the comprehensive treatment effect of example 1 on pneumonia infected by three viruses is optimal.
By analyzing the treatment data of the embodiment 1 and the embodiments 6 to 8, the embodiment 1 of the invention adopts a plurality of traditional Chinese medicine volatile oil components, the effective components of the components are different, the components have synergy, and the traditional Chinese medicine volatile oil component can play an obvious role in treating pneumonia infected by three viruses. By analyzing the treatment data of the embodiment 1 and the comparative examples 1-2, the volatile oil component obtained by the specific process and the specific organic solvent extraction in the embodiment 1 of the invention has higher purity and more effective component content under the same dosage, and the treatment effect on various diseases infected by human coronavirus is obviously better than that of the medicine obtained in the comparative example 3.
The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in the embodiments without departing from the principles and spirit of the invention, and these embodiments are still within the scope of the invention.
Claims (3)
1. The anti-influenza virus traditional Chinese medicine volatile oil composition is characterized by comprising the following raw materials in parts by weight:
7.5 parts of folium artemisiae argyi volatile oil, 7.5 parts of rhizoma atractylodis volatile oil, 7.5 parts of herba elsholtziae volatile oil, 7.5 parts of tea tree volatile oil, 6 parts of rhizoma kaempferiae volatile oil, 6 parts of clove volatile oil, 6 parts of rosemary volatile oil, 6 parts of origanum vulgaris volatile oil, 8 parts of juniper volatile oil, 8 parts of cajeput volatile oil, 15 parts of fructus forsythiae volatile oil and 15 parts of wild chrysanthemum flower volatile oil.
2. The use of the anti-influenza virus traditional Chinese medicine volatile oil composition of claim 1 in the preparation of anti-influenza virus drugs.
3. The application of the anti-influenza-virus traditional Chinese medicine volatile oil composition in preparation of an anti-influenza-virus medicine according to claim 2, wherein the anti-influenza-virus medicine is a dosage form prepared from a pharmaceutically acceptable carrier and the anti-influenza-virus traditional Chinese medicine volatile oil composition.
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