CN113244296B - Medicinal volatile oil composition and preparation method and application thereof - Google Patents

Abstract

The invention discloses a medicinal volatile oil composition, a preparation method and application thereof, and belongs to the technical field of medicinal preparations. The raw materials of the medicinal volatile oil composition comprise the following components in parts by weight: 6-15 parts of rhizoma atractylodis volatile oil, 1-10 parts of wrinkled gianthyssop volatile oil, 6 parts of folium artemisiae argyi volatile oil, 1-15 parts of melaleuca alternifolia volatile oil, 2-6 parts of clove volatile oil and 2-6 parts of cinnamon volatile oil. The medicine volatile oil composition is prepared by mixing the 6 kinds of volatile oil according to the weight ratio, has scientific compatibility, has a synergistic effect, has obvious antibacterial, antiviral and anti-inflammatory effects, has no obvious toxic or side effect and medicine tolerance, and can provide a thought for solving the problem of the medicine resistance of antibiotics.

Description

Medicinal volatile oil composition and preparation method and application thereof

Technical Field

The invention relates to the technical field of pharmaceutical preparations, in particular to a pharmaceutical volatile oil composition, a preparation method and application thereof.

Background

Bacteria are the most abundant of all living things, and have a great influence on the health of human beings. It is estimated that the total number of bacterial cells in the human body and on the epidermis is about ten times the total number of human cells. The bacteria can spread diseases among normal human bodies through various modes, such as contact, digestive tract, respiratory tract, insect bite and the like, have strong infectivity and great harm to the society. Currently, the most effective antibacterial means is to use various chemically synthesized drugs such as antibiotics, sulfonamides, imidazoles, nitroimidazoles, quinolones, etc., but the biggest problem caused by using antibiotics is the problem of drug resistance.

The data of the last five years show that 25000 people die of the European Union each year due to antibiotic resistance, and the cost for treating diseases related to drug-resistant bacteria is up to 15 hundred million euros; the U.S. disease control center in 2017 estimates that 200 million people are infected with drug-resistant bacteria every year. Even though resistance is a natural biological phenomenon, resistance increases arise from two major factors: (1) incorrect use of antibiotics or use of ineffective methods to limit the use of antibiotics can lead to the development of new resistance; (2) global travel, food, animal, fertilizer, mud and other by-product businesses can increase the environmental spread of resistance. FDA reported in 2016 that medical antibiotics applied to food animals exceeded 50%. The data of Chinese academy of sciences show that in 16.2 ten thousand tons of antibiotics used in 2013 in China, 52 percent of the antibiotics for animals and 48 percent of the antibiotics for people are discharged into water and soil environments for more than 5 ten thousand tons of antibiotics in one year, and the abuse of the antibiotics is limited to be reluctant.

Chinese herbal medicine is a treasure in Chinese medicine, and Chinese people have a long history of using Chinese herbal medicine. Generally, Chinese herbal medicine bacteriostasis has the advantages of high safety, small toxic and side effect, difficult generation of drug resistance and the like.

The virus is a non-cellular life form, which is composed of a long chain of nucleic acids and a protein coat, and virus multiplication is performed only in living cells. Infectious diseases caused by viruses seriously threaten human health and public safety all the time, and serious patients can cause large-scale death due to viral diseases such as influenza, AIDS, measles, rubella, smallpox mumps, rubella, measles, chicken pox, respiratory virus infection, viral hepatitis, poliomyelitis, other enterovirus infection, epidemic encephalitis B, epidemic hemorrhagic fever and the like.

The most effective method at present is vaccine prevention and treatment aiming at viral diseases, but the virus has multiple virus species and multiple serotypes, certain difficulty is brought to the application of the vaccine, virus surface antigens can be frequently and constantly mutated, so that the originally effective virus vaccine can be quickly inactivated, and various drug-resistant strains also appear in anti-influenza drugs used at the first line of clinic at present. Moreover, the medicines mostly play a therapeutic role by inhibiting virus surface receptors, and have no obvious therapeutic effect on long-term autoimmune inflammation injury caused by viruses.

The traditional Chinese medicine has unique advantages in the aspect of antivirus, and firstly, part of the medicine can enhance the immune function of the organism and prevent viruses from entering cell tissues; secondly, many medicines have the effects of fever reduction, inflammation resistance and the like while resisting viruses, and have multiple effects on infection caused by viruses, such as shortening the fever time, controlling the diffusion of inflammation, promoting the absorption of the inflammation and the like, namely the medicines play roles in multiple ways and multiple directions; thirdly, the toxic and side effects are small, and normal tissue cells are generally rarely damaged.

The antiviral mechanism of the traditional Chinese medicine comprises: (1) direct antiviral effects include killing of the virus before it invades the cell, prevention of virus adsorption and penetration to the cell, inhibition of the virus self-replication process, and prevention of virus infection from infected cells to uninfected cells. (2) The indirect antiviral effect is that after the virus infects the organism, the virus must be parasitized in the organism cells to survive, so that the important role of mobilizing the immune defense system of the organism to play the antiviral effect is particularly important, which is in line with the basic thought of 'existence of vital qi and incapability of drying out of pathogenic factors' of the traditional Chinese medicine theory. Including promoting the development of immune organs, increasing the phagocytic capacity of phagocyte, enhancing the activity of natural killer cells, and enhancing the humoral immunity and cellular immunity of the body.

In recent years, with the continuous and extensive research on pharmacy, some active ingredients of traditional Chinese medicines with strong antiviral activity are proved to mainly comprise alkaloids, flavones, organic acids, volatile oil, polysaccharides and the like. Researches show that the volatile oil of hypericum, valerian, selfheal, elsholtzia, mulberry leaf, garlic, valerian, forsythia, cordate houttuynia, turmeric, patchouli, honeysuckle, siberian cocklebur and the like has obvious virus inhibiting and killing effects. The mechanism is mainly to utilize the characteristic that volatile oil penetrates cells and utilize chemical substances penetrating the cells to inhibit and eliminate intracellular viruses in the cells.

However, the existing problems are that the effect of the single traditional Chinese medicine volatile oil is single, for example, the documents of the atractylodes rhizome and the elsholtzia herb are mostly focused on antiviral activity, the tea tree oil, the cinnamon and the clove are mainly focused on antibacterial and anti-inflammatory activity, and although the single traditional Chinese medicine volatile oil is compounded, the medicine components are more diversified theoretically and can play the effect through multiple mechanisms, the realization of the effect needs to be established on the basis of scientific compatibility, and the existing compound volatile oil composition has poor comprehensive effects of sterilization, antivirus and anti-inflammatory due to unreasonable compatibility. Therefore, it is necessary to provide a pharmaceutical volatile oil composition with excellent comprehensive effects of sterilization, antivirus and anti-inflammation.

Disclosure of Invention

The invention aims to provide a medicinal volatile oil composition, a preparation method and application thereof, which are used for solving the problems in the prior art and realizing excellent comprehensive effects of sterilization, antivirus and anti-inflammation of the volatile oil.

In order to achieve the purpose, the invention provides the following scheme:

one of the technical schemes of the invention is to provide a medicinal volatile oil composition, which comprises the following raw materials in parts by weight:

6-15 parts of rhizoma atractylodis volatile oil, 1-10 parts of wrinkled gianthyssop volatile oil, 6 parts of folium artemisiae argyi volatile oil, 1-15 parts of melaleuca alternifolia volatile oil, 2-6 parts of clove volatile oil and 2-6 parts of cinnamon volatile oil.

Further, the raw materials comprise the following components in parts by weight: 15 parts of rhizoma atractylodis volatile oil, 10 parts of wrinkled gianthyssop volatile oil, 6 parts of folium artemisiae argyi volatile oil, 1 part of melaleuca alternifolia volatile oil, 2 parts of clove volatile oil and 2 parts of cinnamon volatile oil.

Further, the raw materials comprise the following components in parts by weight: the rhizoma atractylodis volatile oil is 6 parts, the agastache volatile oil is 6 parts, the folium artemisiae argyi volatile oil is 6 parts, the melaleuca alternifolia volatile oil is 6 parts, the clove volatile oil is 6 parts and the cinnamon volatile oil is 6 parts.

Further, the raw materials comprise the following components in parts by weight: the rhizoma atractylodis volatile oil is 10 parts, the agastache volatile oil is 1 part, the folium artemisiae argyi volatile oil is 6 parts, the melaleuca alternifolia volatile oil is 15 parts, the clove volatile oil is 2 parts and the cinnamon volatile oil is 2 parts.

Rhizoma atractylodis: is dried rhizome of Atractylodes lancea (Thunb.) or Atractylodes chinensis Koidz. The traditional Chinese medicine composition is mainly distributed in northeast, north China, Anhui, Jiangsu, Henan, Shaanxi, Hubei and other places of China, is a traditional Chinese medicine material, and has abundant medicinal resources. Cang Zhu is recorded in Shen nong Ben Cao Jing, listed as the superior. Warm in nature, bitter in taste, pungent and strong in flavor, enters spleen and stomach channels, and has the effects of dispelling wind and removing dampness, tonifying spleen and improving eyesight and the like. Modern researches show that the medicinal components of the rhizoma atractylodis are mainly volatile oil, and the main components in the volatile oil are atractylone, beta-cineol, atractylone, atractyl alcohol and the like, so that the rhizoma atractylodis has important pharmacological action. Pharmacological research shows that the rhizoma atractylodis has the effects of protecting the liver, resisting bacteria, viruses, inflammation and tumors, protecting the digestive system, the nervous system, the cardiovascular system and the like. The 75% ethanol extract of the Atractylodes lancea L can obviously inhibit the swelling of the feet of a mouse caused by carrageenan, has obvious inhibition effect on the increase of the permeability of capillary vessels in the abdominal cavity of the mouse caused by acetic acid, and shows good anti-inflammatory activity. The rhizoma atractylodis alcohol extract can obviously inhibit the expression of inflammatory cytokines such as TNF, IL-6, IL-8 and the like in human mast cells induced by phorbol myristate and calcium ion carriers and the activity of NF-kB, thereby slowing down the inflammatory reaction mediated by mast cells. In-vitro antibacterial tests show that the rhizoma atractylodis extract has different inhibitory activities on 15 fungi, has an eliminating effect on pseudomonas aeruginosa R plasmids, and can be used as a safe plasmid eliminating agent. Atractylone has inhibitory effect on H3N2, H5N1 influenza virus and influenza B virus, while atractylodin and rhizoma atractylodis water extract do not show antiviral activity.

Agastache rugosus: is the dry aerial part of the Benth. of Pogostemon cablin (Blanco) of the family Labiatae, originally recorded in foreign body journal of Dong Yang Fu. Originally produced in southeast Asia, the introduction and cultivation history of China is long, the introduction and cultivation history can be traced back to the Liang Dynasty or before, and the introduction and cultivation history is mainly distributed in Hainan province and Guangdong province. Herba Agastaches has effects of eliminating turbid pathogen with aromatics, stimulating appetite, relieving vomiting, relieving exterior syndrome and clearing summer-heat, and can be used for treating damp turbidity obstruction in middle energizer, cold-dampness retention of summer-heat, epigastric fullness emesis, abdominal pain vomiting and diarrhea, nasosinusitis headache, etc. More than 90 compounds have been found in patchouli, including many types of flavonoids, terpenes (sesquiterpenes, diterpenes, triterpenes), phenylpropanoids, steroids, alkaloids, etc. The patchouli volatile oil is considered as the main medicinal component of the patchouli and has biological activities of resisting pathogenic microorganisms, killing parasites, resisting inflammation, relieving pain, relieving fever, regulating immunity and the like. The main components are patchouli alcohol, patchouli ketone, patchouli alkene, alpha-guaiene, delta-guaiene, alpha-patchouliene, beta-patchouliene, etc. The patchouli leaf volatile oil can reduce the contents of prostaglandin E2 and NO in inflammatory tissues and serum, reduce the accumulation of lipid peroxidation product malondialdehyde in blood and shorten the inflammatory process; patchouli oil can inhibit acute inflammation mainly comprising vasodilatation, interstitial fluid exudation and edema and chronic inflammation mainly comprising tissue hyperplasia. A large number of experimental researches show that the patchouli has obvious inhibition effects on various fungi such as microsporum lanuginose, candida albicans, microsporum gypseum, cryptococcus neoformans and the like, and has inhibition effects on staphylococcus aureus, bacillus subtilis, pseudomonas aeruginosa, enteritis coccus and aerobacter. The in vitro antiviral experiment of patchouli alcohol shows that it can inhibit cytopathic effect caused by influenza virus, adenovirus and coxsackie virus, and the result shows that patchouli alcohol has the function of inhibiting the proliferation of the 3 respiratory tract viruses.

Folium artemisiae argyi: is a dry leaf of Artemisia argyi Levi. Et Vant. in Artemisia of Compositae, is widely distributed in China and has a long application history. It is pungent, bitter and warm in nature; has little toxicity; the traditional Chinese medicine composition enters liver, spleen and kidney channels, and is mainly used for treating diseases such as lower abdomen psychroalgia, congealing cold in channels and collaterals, infertility due to cold in uterus, threatened abortion, hematemesis, epistaxis, bloody purulent dysentery, skin itch and the like in clinic. Modern researches have shown that it has various pharmacological activities, including antiinflammatory, antibacterial, antiviral, antioxidant, antitumor and immunoregulatory effects. The folium Artemisiae Argyi chemical components mainly comprise volatile oil, flavonoids, tannins and polysaccharides, etc., wherein the volatile oil is the main active site separated from folium Artemisiae Argyi extract. The chemical components of the volatile oil are identified to be 180 compounds at present, wherein more than 90 compounds are common active components, mainly comprise ethers, alcohols, sesquiterpenes, esters, monoterpenes, ketones and aromatic compounds, and have the functions of broad-spectrum antibacterial property, antioxidation, anti-inflammation, anti-tumor, pain relief, asthma relief, immune regulation and the like. Researches find that the argy wormwood leaf volatile oil can relieve mouse ear swelling and rat foot swelling caused by xylene and inhibit capillary permeability increase. Its anti-inflammatory mechanism may be associated with dose-dependent inhibition of inflammatory mediators, such as Nitric Oxide (NO), Prostaglandins (PGE)2, Reactive Oxygen Species (ROS); meanwhile, the compound can inhibit mouse macrophage RAW264.7 from secreting cytokines, such as Tumor Necrosis Factor (TNF), interleukin (IL-6, IL-10), Interferon (IFN) -beta, the production of monocyte chemotactic protein (MCP-1), and inhibit the phosphorylation of Tyrosinase (TYR)701 in STAT1, TYR705 in STAT3 and upstream JAK2, and does not affect the related actions of JAK, STAT protein total level and the like. The volatile argyi leaf oil can be combined with the cell surface of a tested bacterium to destroy a cell wall structure so as to play a role in inhibiting bacteria, and researches show that the volatile argyi leaf oil has a good antibacterial effect on staphylococcus aureus, bacillus subtilis, listeria, escherichia coli, proteus, salmonella enteritidis, saccharomycetes, aspergillus niger and streptococcus. The bacteriostatic effect of the folium artemisiae argyi volatile oil extracted by the steam distillation method is obviously higher than that of the supercritical CO2 extraction method. The argyi leaf volatile oil also has a good inhibition effect on Respiratory Syncytial Virus (RSV), influenza virus (IFV) and Hepatitis B Virus (HBV).

Melaleuca alternifolia (Maiden & belche) Cheel) branches and leaves of Melaleuca alternifolia (Melaleuca alternifolia) belonging to Melaleuca of Myrtaceae, native to New southern Wales, Australia, and parts of New Zealand. In the 90 s of the 20 th century, Guangdong, Guangxi, Hainan, Fujian, Yunnan and the like in China were successfully introduced and formed a certain planting scale, and Qinzhou, 37013and Ning in Guangxi all had planting and processing bases, so that the resources are very rich. At present, the research on the chemical components of melaleuca alternifolia is mostly focused on the volatile essential oil components, the melaleuca alternifolia essential oil is also called tea tree oil, and the main components of the essential oil in branches, leaves, flowers and fruits are similar, and the essential oil mainly comprises terpinene-4-ol, 1, 8-cineole, gamma-terpinene, alpha-terpinene, iso-terpinolene and the like. The melaleuca alternifolia oil has various pharmacological activities of antibiosis, anti-inflammation, anti-tumor, antivirus, antioxidation and the like. The tea tree oil has good antibacterial effect, has the characteristics of wide antibacterial spectrum and strong antibacterial activity, and has remarkable antibacterial effect on staphylococcus aureus, staphylococcus epidermidis, escherichia coli and the like. The mechanism is that tea tree oil destroys the cell membrane of bacteria, resulting in the loss of electron density material in cells, thereby changing the cell morphology. Melaleuca alternifolia oil also has anti-influenza effects, and its antiviral mechanism may be related to its ability to prevent virus entry into host cells. The melaleuca alternifolia oil can inhibit influenza virus A/PR/8 subtype H1N1 by interfering virus uncoating, and the main components of the melaleuca alternifolia oil, namely terpinene-4-ol, terpinolene and alpha-terpinolene, play an important role. The tea tree oil shows good anti-inflammatory activity in vitro experiments, and plays a role in anti-inflammation by inhibiting the generation of peroxide and regulating the secretion of cytokines TNF-alpha, IL-1 beta, PEG2 and the like; further intensive research shows that the tea tree oil and the terpinen-4-ol as the main component inhibit the secretion of cytokines IL-1 beta, IL-6 and IL-10 in NF-kappa B, p38 and ERK/MAPK pathways, so as to produce effective protection effect on cells.

Clove: is a dried flower bud of clove Ewgewewia caryophylolata Thunb of Myrtaceae, the clove as a medicine is firstly seen in the famous medical records of the south-ward Qiliang pottery hong Jing, which is called as the chicken tongue incense in the book. It is pungent in flavor and warm in nature, and has effects of warming spleen, stomach, lung and kidney, lowering adverse qi, invigorating kidney and tonifying yang. The clove is native to Malaysia and Africa, and is cultivated in Guangxi, Guangdong and Hainan areas in recent times. The clove volatile oil is the main chemical component of clove and has the functions of resisting bacteria, diminishing inflammation, relieving pain and protecting digestive system. The clove volatile oil has more than 320 kinds of reported components, mainly comprises eugenol, eugenol acetate, beta-caryophyllene, alpha-pinene, acetyl eugenol, alpha-terpinene, alpha-caryophyllene and the like, and the biological activity of the clove volatile oil comprises antibiosis, antifungal, anti-inflammatory and drug resistance. The clove volatile oil has strong antibacterial effect on candida albicans, staphylococcus aureus, escherichia coli, salmonella, penicillium, aspergillus niger, hansenula and the like, and has good curative effect on bacterial infectious diseases such as acute bacillary dysentery, enteritis, upper respiratory tract infection, sore throat, acute and chronic tonsillitis and the like. The study shows that the clove volatile oil and the eugenol have the inhibiting effect on 8 helicobacter pylori, and the water extract obtained after the clove is extracted with the volatile oil has no bacteriostatic effect.

Cinnamon: cinnamon is the dry bark of Cinnamomum Cassia Presl of Lauraceae, and is listed as the top grade in Shen nong's herbal Jing. Cinnamon is native to srilanka and is mainly distributed in tropical and subtropical regions such as southeast asia. The method is mainly produced in Guangdong province, Guangxi Zhuang autonomous region, Hainan province, Yunnan province and the like in China. Pungent, sweet and big heat in nature, entering kidney, spleen, heart and liver meridians, and has the effects of tonifying fire, supporting yang, guiding fire to origin, dispelling cold, relieving pain, and warming and dredging channels and collaterals. Modern pharmacological studies show that cinnamon has biological activities of antibiosis, antioxidation, anti-inflammation, anticancer, blood sugar reduction and the like. Cinnamon contains volatile components such as cinnamaldehyde and non-volatile components such as polysaccharides, diterpenes, polyphenols, and flavones. In the volatile components of cinnamon, the content of cinnamaldehyde can reach more than 70%, and in addition, the cinnamon-rich cinnamon bark tea also contains substances with relatively high content such as cinnamyl acetate, coumarin and benzaldehyde. Researches show that the ethanol extract of cinnamon has stronger inhibition effect on escherichia coli, bacillus subtilis, staphylococcus aureus, aspergillus niger, penicillium and beer yeast, and possibly plays an antibacterial role by enhancing the sensitivity of drug-resistant strains to antibiotics. Cinnamon volatile oil is capable of reducing levels of cytokines TNF-alpha, IL-1, NO, PGE2, and COX-2 and iNOS expression in the rat plantar skin tissue induced by carrageenan. Cinnamaldehyde and its derivatives have similar anti-inflammatory activity and exert anti-inflammatory effects mainly by inhibiting the production of NO.

The preparation method of the rhizoma atractylodis volatile oil comprises the following steps:

taking a proper amount of rhizoma atractylodis medicinal material, crushing, sieving by a second sieve, adding 6 times of water, soaking for 1h, and extracting for 4h according to a method A under the item of a method for measuring 2204 volatile oil in the fourth part of 2015 edition of Chinese pharmacopoeia to obtain the rhizoma atractylodis volatile oil.

The preparation method of the clove volatile oil comprises the following steps:

taking a proper amount of clove medicinal material, crushing, sieving by a second sieve, adding 20 times of water, soaking for 1h, extracting for 6h by steam distillation, extracting clove oil from a collected liquid by adopting diethyl ether, drying by using anhydrous sodium sulfate, and volatilizing the diethyl ether at normal temperature to obtain the clove volatile oil.

The preparation method of melaleuca alternifolia volatile oil (tea tree oil) comprises the following steps:

shearing the melaleuca alternifolia branches and leaves into pieces with the length of about 40-60 mm by using scissors, and naturally evaporating the water on the surfaces of the melaleuca alternifolia branches and leaves to constant weight under the condition of room temperature. Extracting by steam distillation for 6h by volatile oil determination method A in appendix 2204 of Chinese pharmacopoeia, adding anhydrous magnesium sulfate to the obtained volatile oil to remove all water to obtain Melaleuca alternifolia volatile oil (tea tree oil).

The optimal process of the tea tree oil comprises the steps of soaking for 6 hours and extracting for 6 hours, wherein the ratio of materials to liquid is 1: 20.

The preparation method of the folium artemisiae argyi volatile oil comprises the following steps:

pulverizing folium Artemisiae Argyi, sieving with a second sieve, extracting volatile oil with a volatile oil extractor by a method A under the item of 2204 volatile oil determination method in the fourth part of Chinese pharmacopoeia of 2015 edition, and drying with anhydrous sodium sulfate to obtain folium Artemisiae Argyi volatile oil with special aroma.

The preparation method of the cinnamon volatile oil comprises the following steps:

pulverizing cortex Cinnamomi, sieving with a second sieve, soaking for 1 hr, adding 6 times of water, and steam distilling for 5 hr to obtain cortex Cinnamomi volatile oil.

The preparation method of the agastache volatile oil comprises the following steps:

weighing dried herba Agastaches aerial parts, pulverizing, sieving with 60 mesh sieve, extracting crude volatile oil by steam distillation, extracting crude volatile oil with diethyl ether for 3 times, drying the extractive solution with anhydrous sodium sulfate, and volatilizing diethyl ether at room temperature to obtain fragrant yellow herba Agastaches volatile oil.

The second technical scheme of the invention provides a preparation method of the medicinal volatile oil composition, which comprises the following steps:

mixing the raw materials according to the weight ratio to obtain the medicinal volatile oil composition.

The third technical scheme of the invention is to provide a pharmaceutical preparation, which comprises an effective amount of the pharmaceutical composition.

By "effective amount" according to the present invention is meant an amount of an agent according to the present invention which is required to exhibit a measurable therapeutic or prophylactic effect.

Further, a pharmaceutically acceptable carrier is included.

The carrier is one or more of diluent, lubricant, buffering agent, disintegrating agent, flavoring agent, surfactant or adhesive.

Various carriers known in the art can be widely used. For example, diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, and the like; wetting agents and binders such as water, glycerin, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethylcellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone and the like; disintegrating agents such as dried starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene sorbitol fatty acid ester, sodium dodecylsulfate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors such as sucrose, glyceryl tristearate, cacao butter, hydrogenated oil and the like; absorption accelerators such as quaternary ammonium salts, sodium lauryl sulfate and the like; lubricants, for example, talc, silica, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, and the like. Other carriers such as polyacrylic resin, liposome, and water soluble carrier such as PEG4000 and PEG6000, PVP, etc.

Colorants, preservatives, flavors, flavorants, sweeteners, or other materials may also be added to the compositions.

Further, the dosage forms of the pharmaceutical preparation include capsules, aerosols, sprays, powders, suspensions, emulsions, suppositories, ointments, creams, sustained-release preparations, controlled-release preparations, targeted preparations and microparticles.

The pharmaceutical composition or pharmaceutical formulation of the invention may be administered parenterally, transdermally, intranasally or intrarectally.

For topical administration, the compounds of the present invention may be prepared in the form of ointments and creams.

The third technical scheme of the invention is to provide the application of the medicinal volatile oil composition or the medicinal preparation in the aspects of bacteriostasis, disinfection and anti-inflammation.

The medicinal volatile oil composition and the medicinal preparation can also be used for disinfecting air, objects, human body surfaces and the like.

The invention discloses the following technical effects:

the invention firstly adopts a steam distillation method to extract the rhizoma atractylodis volatile oil, the agastache volatile oil, the folium artemisiae argyi volatile oil, the melaleuca alternifolia volatile oil, the clove volatile oil and the cinnamon volatile oil, and then the 6 kinds of volatile oils are mixed according to a certain proportion to prepare the medicine volatile oil composition.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1 shows that test agent A1 inhibits CPE produced by influenza virus;

FIG. 2 shows that test A2 inhibits CPE produced by influenza virus;

FIG. 3 shows that test A3 inhibits CPE production by influenza virus.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

The "parts" in the present invention are in parts by weight unless otherwise specified.

Unless otherwise stated, the following adopted medicinal materials all meet the regulation of the 1 st part of the pharmacopoeia of the people's republic of China, 2010 edition, and various reagents and pharmaceutic adjuvants are all commercial products and meet the relevant national standards.

First, experimental material of the embodiment of the invention

1. The Chinese herbal medicines used in the embodiment of the invention are as follows:

rhizoma atractylodis: the producing area: inner Mongolia; production batch number: 1806001, respectively; the production date is as follows: produced in 2018, 6 and 29 months; production enterprises: changda Chinese medicinal decoction pieces, Inc., Anguo city;

agastache rugosus: the producing area: the Guangdong; production batch number: 1906001, respectively; the production date is as follows: 6 months and 15 days in 2019; production enterprises: changda Chinese medicinal decoction pieces limited, Anguo city;

folium artemisiae argyi: the producing area: north of river; production batch number: 1907001, respectively; the production date is as follows: produced in 2019, 7 months and 17 days; production enterprises: changda Chinese medicinal decoction pieces, Inc., Anguo city;

melaleuca alternifolia: the producing area: guangxi; collected in Daizhong Daizhen in Guangxi province in 2019 in 2 months;

clove: the producing area: sichuan; production batch number: 1804001, respectively; the production date is as follows: 26 months 4 in 2018; production enterprises: changda Chinese medicinal decoction pieces, Inc., Anguo city;

cinnamon: the producing area: guangxi; production batch number: 1801001; the production date is as follows: 6 days 1 month 2018; production enterprises: changda Chinese medicinal decoction pieces Limited, Anguo city.

2. The experimental animals used in the present invention:

variety: a mouse; strain: ICR

Grade: SPF class (Specefic Pathologen Free, no specific Pathogen)

The source is as follows: purchased from beijing weitonglihua laboratory animals technology ltd, license No.: SCXK (Jing) 2016-: 110011201108760783.

sex: a female; weight: 20-22g

3. Animal house:

during the experiment, the animals were kept in animal houses of institute of traditional Chinese medicine of Chinese academy of sciences, and the license of experimental facilities SYXK (Jing) 2019-.

The facility management follows national standard GB 14925 of the people's republic of China and 2010 Experimental animal environment and facilities.

4. Feeding conditions are as follows:

artificial illumination is adopted for a 12-hour light and shade period, the environmental temperature is maintained at 20-26 ℃, the humidity is 40-70%, and the air exchange frequency per hour is more than or equal to 15 times. Animals were housed in polycarbonate mouse cages, each of which was housed with 5 homozygote mice from the same dose group. Clean animal cages and bedding were changed every 2 days.

5. Feed and its providing unit:

the SPF rat maintenance feed was provided by australian cooperative feed limited, beijing, and the feed manufacturing company examined the certification number: kyo feed (2014)06054, experimental animal production license number: SCXK (Jing) 2019-.

6. Drinking water: purified water, which can be freely ingested by animals, is replaced with water bottles and drinking water 3 times a week.

7. Strains, strains and cells:

(1) bacteria:

escherichia coli 8099. The generation number of the above strains is the 4 th generation. Taking a fresh culture (18-24h) of a nutrient agar culture medium slant of the strain, washing off lawn by using 5mL of 0.03mol/L Phosphate Buffer Solution (PBS) to enable the strain to be suspended uniformly, and then diluting the strain to a required concentration by using the PBS.

Salmonella, clinical isolate 104. And taking out the frozen salmonella strain from a refrigerator at-80 ℃, inoculating the salmonella strain into a TSB broth culture medium, and putting the culture medium in a shaker at 37 ℃ for enrichment culture for 16-18 h. The next day, enriched Salmonella were dipped with a sterile inoculating loop and streaked three times onto selective medium XLT4 plates. The colony morphology of the salmonella in an XLT4 culture medium is black in the middle, transparent halos are arranged around the salmonella colony, and single clones are selected and streaked on an XLT4 culture medium for 3-4 generations of purification.

Multiple drug resistant salmonella: clinical isolate 169. The culture and purification method is the same as above.

(2) Virus strain:

H1N1 Influenza (A/Weiss/43), purchased from China center for type culture Collection;

H1N1 Influenza (A/Virginia/ATCC2/2009), available from ATCC in the United states.

(3) Cell:

canine kidney epithelial cells (MDCK), purchased from the shanghai cell bank of the chinese academy. MDCK cells are routinely cultured for more than 30 generations in high-glucose DMEM medium containing 10% fetal calf serum.

Second, extraction process of volatile oil of each component

1. And (3) rhizoma atractylodis oil:

taking 10kg of rhizoma atractylodis medicinal material, crushing, sieving by a second sieve, adding 6 times of water, soaking for 1h, and extracting for 4h according to a method A under the item of a four-part 2204 volatile oil determination method in 2015 edition of Chinese pharmacopoeia to obtain 55mL of rhizoma atractylodis volatile oil.

2. Clove oil:

taking 1kg of clove medicinal material, crushing and screening the clove medicinal material by a second sieve, adding 20 times of water, soaking for 1h, extracting for 6h by steam distillation, extracting clove oil from collected liquid by adopting ether, drying by using anhydrous sodium sulfate, and volatilizing the ether at normal temperature to obtain 100mL of clove volatile oil.

3. Tea tree oil:

2kg of branches and leaves of Guangxi Traverse tea are cut into pieces with the length of about 40-60 mm by scissors, and the water on the surfaces of the pieces is naturally evaporated to constant weight under the condition of room temperature. Adding 20 times of water, soaking for 6h, performing steam distillation by using a volatile oil determination method A in appendix 2204 of Chinese pharmacopoeia to extract volatile oil, and adding anhydrous magnesium sulfate into the obtained tea tree oil to remove all water to obtain 46mL of tea tree oil.

4. Blumea oil:

crushing 5kg of folium artemisiae argyi, sieving the crushed folium artemisiae argyi by a second sieve, extracting volatile oil by a volatile oil extractor according to a method A under the item of a four-part 2204 volatile oil determination method of 'Chinese pharmacopoeia' of 2015 edition, and drying the volatile oil by anhydrous sodium sulfate to obtain 55mL of volatile oil with special aroma.

5. Cinnamon oil:

crushing 1kg of cinnamon, sieving with a second sieve, soaking for 1h, adding 6 times of water, and performing steam distillation for extraction for 5h to obtain 31mL of volatile oil.

6. Patchouli oil:

weighing 4kg of patchouli, crushing, sieving by a second sieve, extracting crude volatile oil by a steam distillation method, extracting the crude volatile oil by diethyl ether for 3 times, drying extract liquid by anhydrous sodium sulfate, and volatilizing the diethyl ether at normal temperature to obtain 40mL of yellow volatile oil with aromatic smell.

Thirdly, the volatile oil composition and each volatile oil component

Example 1

Taking 15mL of rhizoma atractylodis volatile oil, 10mL of wrinkled gianthyssop herb volatile oil, 6mL of folium artemisiae argyi volatile oil, 1mL of melaleuca alternifolia volatile oil (tea tree oil), 2mL of clove volatile oil and 2mL of cinnamon volatile oil, and fully mixing the 6 volatile oil extracts to prepare 36mL of volatile oil mixed liquid for later use as a test object A1 of an experimental part.

Example 2

Taking 6mL of rhizoma atractylodis volatile oil, 6mL of wrinkled gianthyssop herb volatile oil, 6mL of folium artemisiae argyi volatile oil, 6mL of cajeput volatile oil (tea tree oil), 6mL of clove volatile oil and 6mL of cinnamon volatile oil, and fully mixing the 6 volatile oil extracts to prepare 36mL of volatile oil mixed liquid for later use as a test object A2 of an experimental part.

Example 3

Taking 10mL of rhizoma atractylodis volatile oil, 1mL of wrinkled gianthyssop herb volatile oil, 6mL of folium artemisiae argyi volatile oil, 15mL of melaleuca alternifolia volatile oil (tea tree oil), 2mL of clove volatile oil and 2mL of cinnamon volatile oil, and fully mixing the 6 volatile oil extracts to prepare 36mL of volatile oil mixed liquid for later use as a test object A3 of an experimental part.

Example 4

The rhizoma atractylodis volatile oil stock solution is reserved and used as a test substance A4 of an experimental part.

Example 5

The stock solution of the volatile oil of Agastache rugosa is ready for use as the test substance A5 in the experimental part.

Example 6

The stock solution of the volatile oil of the artemisia leaf is reserved and used as a test object A6 of the experimental part.

Example 7

Melaleuca leucadendra volatile oil (tea tree oil) stock solution, which is used as a test substance A7 in the experimental part.

Example 8

Clove volatile oil stock solution is reserved as a test object A8 of the experimental part.

Example 9

Cinnamon volatile oil stock solution, which is ready for use, is used as a test substance A9 in the experimental part.

Fourthly, the medicine composition of the invention and the bacteriostatic action of each volatile oil component in the composition on salmonella

A. Experimental methods

1. Minimum Inhibitory Concentration (MIC)

The sensitivity of salmonella isolates to antibacterial drugs was determined using the CLSI recommended microbulette dilution method.

The recovered Salmonella 104 and 169 were inoculated into MH broth for enrichment, and the test was started after 10h culture and diluted to 5X 10 with MH broth⁵CFU/mL. The antibacterial drug was diluted with MH broth to the concentration to be tested. The bacterial solution and the drug working solution were added at a ratio of 1:1, and positive (MH broth containing the same number of colonies) and negative control (sterile MH broth) were set. In order to ensure the accuracy of the experiment,the antibacterial drug concentration is added from high to low, and Escherichia coli ATCC25922 is used as a quality control strain, and each drug is 3 in parallel. Quickly and lightly shaking by hand to ensure full mixing, and culturing at 37 ℃ for 16-18 h.

If the mixture is turbid, the bacteria will grow, otherwise the bacteria will not grow. And when the determination is carried out, the determination of the quality control bacteria is carried out, and then the determination of the salmonella isolate is carried out when the quality control bacteria is in the recommended range. The minimum concentration of each drug without bacterial growth is taken as the minimum inhibitory concentration of the salmonella isolate to the antibacterial drug. The specific criteria are shown in Table 1.

TABLE 1 concentration of antibacterial mother liquor and criteria for determination

2. Bacteriostasis rate

The bacteriostasis rate test of the tested substance to pathogenic bacteria adopts a plate counting method. Inoculating single colony of Salmonella (104) into MH broth for enrichment, culturing for 10h, and diluting with MH broth to 5 × 10 per tube before test⁵CFU/mL. Respectively adding the volatile oil test substance into each tube of bacterial liquid, placing the centrifugal tube in a shaking table at 37 ℃ for shake culture, and selecting 0h, 2h and 6h time points of shake culture to dilute each tube of bacterial liquid to 10^-3、10^-4. The above 10. mu.L of the bacterial solution was applied evenly to TSA plates, and positive control (MH broth containing the same colony number) and negative control (sterile MH broth) were set, 2 replicates for each drug. The plate is placed in an incubator at 37 ℃ for 24h, the result is counted, the average value of bacteria of 2 parallel plates is taken as the colony number of the drug at the time point, and the inhibition (sterilization) rate of each volatile oil is calculated by comparing the colony number of the positive plate.

The (bactericidal) inhibition rate (%) was (number of colonies on positive plate-number of colonies on administration group)/number of colonies on positive plate × 100%

B. Results of the experiment

1. Minimum Inhibitory Concentration (MIC)

1.1 minimum inhibitory concentration on clinical isolate 104 of Salmonella

Test results show that the quality control bacteria 25922 are controlled, the MIC of gentamicin is 0.5ug/mL, the MIC of gentamicin to 104 salmonella is 1ug/mL, the results all accord with the determination standard of CLSI (see table 1), and the results are shown in table 2. The bacteriostatic effect of the volatile oil of the atractylodes rhizome, the agastache rugosus, the artemisia leaf, the melaleuca alternifolia, the clove and the cinnamon on salmonella is as follows: clove, cinnamon, agastache rugosus and tea tree oil, 50% concentration of clove and cinnamon have the same bacteriostatic effect as 100% concentration of agastache rugosus and tea tree oil, but the atractylodes rhizome and the folium artemisiae argyi have no inhibitory effect on salmonella. The proportion of ageratum and tea tree oil in A1, A2 and A3 is respectively 30.6%, 33.3% and 44.4%, and the concentration of 3 Chinese medicine volatile oil compounds A1, A2 and A3 in different proportions is lower than the concentration (100%) of the two volatile oils, thus the same bacteriostasis can be achieved. The proportion of clove and cinnamon in A2 is 33.3%, and the clove and cinnamon have bacteriostatic action at a lower concentration, and the A2 still has a significant bacteriostatic effect at a lower concentration (25%) of a test substance, and shows a certain synergistic effect, and the results are shown in Table 3.

TABLE 2 minimum inhibitory concentration of gentamicin on Salmonella 104

Note: () The concentration of gentamicin is the inner.

TABLE 3 minimum inhibitory concentration test (%) -for Salmonella 104 for A1-A9

Note: "-" is negative, no colonies are seen; "+" is positive and there is bacterial growth.

1.2 minimum inhibitory concentration to multiple drug-resistant Salmonella clinical isolate 169

Test results show that the quality control bacteria 25922 are in control, the gentamicin MIC is 0.5ug/mL, and the determination standard of CLSI is met; gentamicin was still ineffective against Salmonella 169 at 64ug/mL, and was shown to be resistant according to the CLSI criteria, with the results shown in Table 4. The bacteriostatic effect of the volatile oil of the atractylodes rhizome, the agastache rugosus, the artemisia leaf, the melaleuca alternifolia, the clove and the cinnamon on salmonella is as follows: clove, cinnamon, wrinkled gianthyssop herb and tea tree oil are effective for multiple drug-resistant salmonella 169, while the atractylodes rhizome and the folium artemisiae argyi have no inhibition effect. The ratio of the effective 4 traditional Chinese medicine volatile oils in A1, A2 and A3 is 41.7%, 66.7% and 55.5%, respectively, and the concentration of the traditional Chinese medicine volatile oil compound A2 is lower than the concentration (100%) of the 4 volatile oils, respectively, so that the same antibacterial effect can be achieved, and the results are shown in Table 5.

TABLE 4 minimum inhibitory concentration of gentamicin against Salmonella 169

TABLE 5 minimum inhibitory concentration test (%) -for Salmonella 169 by A1-A9

2. Rate of inhibition of bacteria

The further bacteriostasis test is carried out on the agastache rugosus, melaleuca alternifolia, clove and cinnamon volatile oil with bacteriostasis and 3 Chinese medicine volatile oil compound A1, A2 and A3 which are mixed according to different proportions. The results show that the clove and the cinnamon have the bacteriostatic effect on the salmonella>Tea tree oil>Agastache rugosa, at the same action time (0h), same bacterial liquid concentration (10)^-3Diluted), the bacteriostasis rate of the clove and the cinnamon is 100 percent, and the tea tree oil is 88.5 percent; the concentration of the bacterial liquid is 10 times (10 times) lower than that of the bacterial liquid when the agastache rugosus acts for 6 hours^-4Dilution) was carried out, the bacteriostatic ratio was 80.0%, which was substantially consistent with the MIC test result. According to the percentage, A2 and A3 can play the same antibacterial (killing) effect at the concentration lower than that of the volatile oil used alone, the antibacterial (killing) effect time of A1, A2 and A3 is far shorter than the effect time (6h) of ageratum, and the quick sterilization effect is reflected. The results are shown in Table 6.

TABLE 6 test of the inhibitory rate of the test substances on Salmonella (%)

Fifthly, the pharmaceutical composition of the invention has bactericidal effect on escherichia coli

A. Test method

1. Neutralizer identification test

A PBS solution containing 0.5% of lecithin, 0.25% of glycine, 0.1% of sodium thiosulfate and 1% of Tween 80 is used as a neutralizer, samples A1, A2 and A3 are used as stock solutions, and a test is carried out according to a C3.1 neutralizer identification test in appendix C3 of GB 15979-. The test groups are: (1) the method comprises the following steps of (1) staining a bacteria specimen chip +5mL of PBS, (2) staining the bacteria specimen chip +5mL of neutralizer, (3) staining a photograph +5mL of neutralizer, (4) specimen chip +5mL of neutralizer + staining a photograph, (5) staining the bacteria photograph +5mL of PBS, (6) (7) and (8) are respectively the PBS, the neutralizer and the culture medium of the same batch. Samples A1, A2, A3 stock solutions were each allowed to act for 1 min. The experiment was repeated 3 times. 3 times of repeated experiments prove that the average growing colony numbers of the groups A1, A2 and A3 1 are 72, 0 and 0 CFU/plate respectively, and the average growing colony numbers of the groups 2 are 1.6 multiplied by 10 respectively³21, 15 CFU/plate, the average number of growing colonies of the 3 rd, 4 th and 5 th groups are similar, the error rates among the three groups are 3.33%, 3.14% and 4.86%, respectively, and the 6 th, 7 th and 8 th groups are aseptically grown. The results are shown in tables 7 to 9. And 3 repeated tests show that the neutralizer identification test meets the requirements, and the next bacteriostatic test can be carried out continuously.

TABLE 7A 1 neutralizer identification test results

Note: negative control was grown aseptically.

TABLE 8A 2 neutralizer identification test results

Note: negative control was grown aseptically.

TABLE 9A 3 neutralizer identification test results

Note: negative controls were grown aseptically.

B. Experiment for inhibiting bacteria

3 Chinese medicine volatile oil compound samples A1, A2 and A3 with better effect in a salmonella bacteriostasis experiment are selected for the following experiments. The test bacteria 24h slant culture was washed with PBS to prepare a bacterial suspension. The test piece (2X 3cm) and the control piece were each divided into 4 groups and placed in 4 sterilized petri dishes. Taking the bacterial suspension, respectively dripping 100uL on each sample to be tested and each control sample, uniformly coating, starting timing, acting for 2, 5, 10 and 20min, respectively putting the samples into test tubes containing 5mL of neutralizing agent by using sterile forceps, fully and uniformly mixing, properly diluting, then taking 2-3 dilutions, respectively sucking 0.5mL, placing in two plates, pouring 15mL of nutrient agar culture medium placed to 40-45 ℃ in advance, rotating the plates to be fully and uniformly, turning over the plates after agar solidification, culturing for 48h at 35 +/-2 ℃, and counting viable bacterial colonies. The test was repeated 3 times, and the sterilization rate% was calculated according to the following formula.

Percent bactericidal activity%

C. Results of the experiment

3 repeated experiments prove that the stock solutions of the samples A1, A2 and A3 act for 2min, the average sterilization rates of the stock solutions to escherichia coli are respectively 97.45%, 99.99% and 99.99%, and the average sterilization rates to the escherichia coli are respectively 98.86%, 99.99% and 99.99%, and the average sterilization rates to the escherichia coli are respectively 10min, 99.93%, 99.99% and 99.99%, and the average sterilization rates to the escherichia coli are respectively 20min, and the average sterilization rates to the escherichia coli are respectively more than 99.99%. The results are shown in Table 10.

TABLE 10 results of the bactericidal test of the pharmaceutical composition of the present invention against E.coli

Sixthly, the antiviral effect of the pharmaceutical composition on influenza virus

A. Test method

1) Cytotoxicity assays

Will be 1 × 10⁵Inoculating 100 mu L of MDCK cell suspension into a 96-well plate, and culturing for 24h in a cell culture box; diluting 50% A1, A2 and A3 (mother liquor) prepared by DMSO in a doubling ratio by using a cell maintenance solution, wherein the concentration is 1.0000%, 0.5000%, 0.2500%, 0.1250%, 0.0625%, 0.0313%, 0.0156%, 0.0078% and 0.0039%, and setting a blank control group; removing culture medium from 96-well plate, washing with sterile PBS for three times, removing excessive PBS during the last washing, and adding A1, A2, and A3 at different concentrations, 200 μ L per well, and 6 auxiliary wells per concentration; adding 200 μ L of cell maintenance solution into each well of the blank control group, and continuously culturing for 72 h; observing the cell state every day, after 72 hours, adding 20 mu L of MTT solution into each hole under the condition of keeping out of the sun, continuously incubating for 2-4 hours in an incubator, gently sucking out the culture solution, adding 150 mu L of DMSO into each hole, uniformly mixing for 10min on a shaking table, and then measuring the absorbance value at 490nm wavelength; the% cell viability was calculated. The median toxic concentration of the test substance (CC50) was further calculated by SPSS.

Cell viability ═ 100% (OD 490nm in A1, A2, A3 administration group/OD 490nm in normal group) × 100%

2) Determination of anti-influenza virus activity of test substances

Will be 1 × 10⁵Inoculating 100 mu L of cell suspension per well into a 96-well plate, and culturing for 24h in a cell culture box; taking out 96-well plate from incubator, removing culture medium in the plate, washing cells with prepared PBS, adding 100 μ L of 100TCID50 virus solution into each well for infection, adding virus-free virus adsorption solution into normal control group, and placing 96-well plate in incubatorIncubating for 2 h; diluting the mother liquor of the test substance by a virus maintenance solution in a multiple ratio, wherein the concentration is 0.1250%, 0.0625%, 0.0313%, 0.0156%, 0.0078%, 0.0039%, 0.0020% and 0.0010%; after adsorption, removing virus adsorption solution in the culture plate, washing with PBS twice, adding 200 μ L of diluent containing A1, A2 and A3 with different concentrations into each well, adding 200 μ L of virus maintenance solution into the virus group and the normal control group, repeating 6 wells each, observing pathological change state every day, and continuously culturing for 72 h; after 72h, adding 20 mu L of MTT solution into each hole under the condition of keeping out of the sun, incubating in an incubator for 2-4 h, removing the culture solution, adding 150 mu L of DMSO into each hole, and determining the OD value at 490 nm; the virus inhibition rate% was calculated. The half-effective concentration (EC50) and therapeutic index of the test substance were further calculated by SPSS.

The inhibition ratio of the virus (A1, A2, A3 administration group OD490 nm-virus group OD490 nm)/(normal group OD490 nm-virus group OD490 nm) × 100%

Therapeutic index SI ═ CC50/EC50

3) Test related to inactivation of influenza virus (TCID50)

MDCK cells were seeded in 96-well plates at 1X 10 per well⁴Culturing in a cell culture box for 24 hours; mixing 10 μ L of the mother liquor with 90 μ L of serum-free culture medium (dilution of the test sample is 20 times), sucking 50 μ L of the diluted test sample and equal volume of virus solution, mixing, standing at room temperature for 10min, mixing the serum-free culture medium and the virus solution as reference, and standing at room temperature for 10 min; diluting the reacted liquid with virus adsorbing liquid 10^-1、10^-2、10^-3、10^-4……10^-8Total 8 concentration gradients; taking the 96-well plate out of the cell incubator, removing the culture medium in the plate in an ultra-clean workbench, washing the cells with PBS (phosphate buffer solution) for three times at a rate of 100 mu L/well, adding the diluted virus solution into the 96-well plate, adding 6 wells per dilution and 100 mu L per well, adding 100 mu L of virus adsorption solution per well of a normal control group, and placing the 96-well plate in the incubator at 37 ℃ for adsorption for 2 hours; after 2h, removing virus adsorption solution, washing with PBS for three times, adding virus maintaining solution into 96-well plate, culturing for 72h, observing pathological change degree, and taking pathological change degree greater than 50% as pathological changeWells with less than 50% lesions were non-diseased wells and virus TCID50 was calculated according to Reed-Muench formula. The effect of the test article on the activity of influenza virus was examined by calculating the difference between TCID50 of the test article groups A1, A2, A3 and the control group.

B. Results of the experiment

The test substances A1, A2 and A3 all have certain antiviral activity on influenza virus Weiss strains, wherein the antiviral effect of A1 is superior to that of a positive drug ribavirin, the therapeutic index is 22.21, and the test substances also have certain antiviral effect on oseltamivir resistant strains, and the therapeutic index is 3.46. After the test substance is diluted 20 times and acts with the virus for 10min at room temperature, A1 can reduce the titer of the virus H1N1 Influenza (A/Weiss/43) by two gradients, and the virus TCID50 is 10 after A1 acts^-5Per 100. mu.L, whereas the virus control had a TCID50 of 10^-7And 100 mu L. The results are shown in Table 11 and FIGS. 1 to 3, in which the microscope magnifications of the photographs in FIGS. 1 to 3 are 200 times (20X 10).

FIG. 1 shows that test A1 inhibits CPE produced by influenza virus;

FIG. 2 shows that test A2 inhibits CPE produced by influenza virus;

FIG. 3 shows that test A3 inhibits CPE production by influenza virus.

TABLE 11 antiviral action of the pharmaceutical compositions of the invention against influenza viruses

Seventh, the invention pharmaceutical composition on xylene induced mouse ear swelling effect research

Test method

ICR mice, female. The skin on the back of the mice was depilated with depilatory cream in an area of 2X 3cm for about 1 minute each, after which the skin was thoroughly exposed by wiping with warm water. The next day after depilation, the hair were divided into normal control group, model group, positive hydrocortisone butyrate cream, A1, A2 and A3 groups, and 6 groups, 9-10 of them were used. The test substances are given to groups according to the corresponding dose of 1 time per day, the back skin of groups A1, A2 and A3 is smeared with about 0.2mL of 50% A1, A2 and A3 prepared by glycerol, and the administration area is about 2 multiplied by 3 cm; the hydrocortisone butyrate cream with the same area is smeared on the back skin of the positive medicine group by about 50mg, the glycerol with the same area is smeared on the normal control group and the model group by about 0.2 mL/patient, and the continuous administration is carried out for 6 days.

At 60min after the last administration, the two sides of the right ear of each group of mice except the normal control group are coated with 20uL dimethylbenzene to cause the auricle swelling of the mice, and the auricle of the right ear of the normal control group of mice is coated with the same volume of normal saline. 1h after xylene application (normal control group with normal saline), all animals were sacrificed by cervical dislocation. And cutting off double ears along the base line of the auricle, punching ear sheets at the symmetrical positions of the double ears by adopting a stainless steel puncher with the diameter of 6mm, weighing, and calculating swelling degree and the inhibition rate of ear swelling of each group of mice according to the following formulas.

Swelling degree (g) is right ear weight-left ear weight.

The swelling degree inhibition ratio (%) is (swelling degree model group-swelling degree drug group)/swelling degree model group x 100%

Statistical analysis

The test data of each group are expressed as mean +/-standard deviation, and the comparison between a normal control group and a model group and between the model group and each administration group is carried out by using student's TTEST, and P <0.05 is taken as a standard with statistical significance difference.

Test results

The A1, A2 and A3 groups all have a certain degree of inhibition effect on mouse auricle swelling caused by xylene, and the inhibition rates are 54.0%, 19.0% and 20.0%, wherein the ear swelling degree of the mice in the A1 group is statistically different from that of the mice in the model group (P is less than 0.05), and the ear swelling degree can be obviously reduced. The positive drug group also has obvious effect of relieving ear swelling caused by dimethylbenzene, and the inhibition rate is 52.1% (P is less than 0.05). The results are shown in Table 12.

TABLE 12 inhibition of ear swelling in mice by xylene of the pharmaceutical compositions of the present invention

Note: comparing with normal control group^###P<0.001; comparison with model group^*P<0.05。

The experiment shows that the research result of the bactericidal effect of the pharmaceutical composition on the non-drug-resistant clinical isolates, the drug-resistant clinical isolates and the escherichia coli of the salmonella shows that the pharmaceutical composition has definite bactericidal effect, and compared with 3 pharmaceutical compositions, the bactericidal effect A2 is better than A1 and A3, and the bactericidal effect is effective on the drug-resistant strains and is better than the single-use volatile oil.

A further anti-Influenza virus activity test on the 3 medicine combinations with good bacteriostatic effects shows that the anti-Influenza virus activity test of the 3 medicine combinations shows that the anti-Influenza virus activity of A1 is better than that of A2 and A3, and better than that of a positive medicine ribavirin, the titer of Influenza virus H1N1 Influenza (A/Weiss/43) can be obviously reduced, and meanwhile, the anti-Influenza virus activity test of the oseltamivir drug-resistant strain also has a certain anti-viral effect.

Further anti-inflammatory tests show that the pharmaceutical composition has an obvious inhibiting effect on mouse auricle swelling caused by xylene after being prophylactically administered for 6 days, and compared with 3 pharmaceutical compositions, the anti-inflammatory effect of A1 is equivalent to that of a positive hydrocortisone butyrate cream, and is better than that of A2 and A3, which indicates that the pharmaceutical composition can obviously inhibit swelling caused by acute inflammation.

The experimental result indicates that the pharmaceutical composition has obvious sterilization, antivirus and anti-inflammatory effects, low effect concentration, quick response, better effect than that of the volatile oil used singly and no obvious toxic or side effect.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Claims (2)

1. The medicinal volatile oil composition is characterized by comprising the following raw materials in parts by weight:

15 parts of rhizoma atractylodis volatile oil, 10 parts of wrinkled gianthyssop volatile oil, 6 parts of folium artemisiae argyi volatile oil, 1 part of melaleuca alternifolia volatile oil, 2 parts of clove volatile oil and 2 parts of cinnamon volatile oil;

the preparation method of the rhizoma atractylodis volatile oil comprises the following steps: pulverizing rhizoma Atractylodis, sieving with a second sieve, adding 6 times of water, soaking for 1h, and extracting according to method A of 2015 edition of Chinese pharmacopoeia in the fourth 2204 volatile oil determination method for 4h to obtain rhizoma Atractylodis volatile oil;

the preparation method of the clove volatile oil comprises the following steps: pulverizing flos Caryophylli, sieving with a second sieve, adding 20 times of water, soaking for 1 hr, extracting by steam distillation for 6 hr, extracting oleum Caryophylli from the collected liquid with diethyl ether, drying with anhydrous sodium sulfate, and volatilizing diethyl ether at room temperature to obtain flos Caryophylli volatile oil;

the preparation method of the melaleuca alternifolia volatile oil comprises the following steps: shearing branches and leaves of melaleuca alternifolia into branches and leaves with a length of about 40-60 mm by using scissors, naturally evaporating water on the surfaces of the branches and leaves to a constant weight at room temperature, performing steam distillation extraction for 6 hours by using a volatile oil determination method A in appendix 2204 of Chinese pharmacopoeia, and adding anhydrous magnesium sulfate into the obtained volatile oil to remove all water to obtain melaleuca alternifolia volatile oil;

the preparation method of the folium artemisiae argyi volatile oil comprises the following steps: pulverizing folium Artemisiae Argyi, sieving with a second sieve, extracting volatile oil with a volatile oil extractor by method A under the item of 2204 volatile oil determination method in the fourth part of Chinese pharmacopoeia 2015 edition, drying with anhydrous sodium sulfate to obtain folium Artemisiae Argyi volatile oil;

the preparation method of the cinnamon volatile oil comprises the following steps: pulverizing cortex Cinnamomi, sieving with a second sieve, soaking for 1 hr, adding 6 times of water, and steam distilling for 5 hr to obtain cortex Cinnamomi volatile oil;

the preparation method of the agastache volatile oil comprises the following steps: weighing dried herba Agastaches aerial parts, pulverizing, sieving with 60 mesh sieve, extracting crude volatile oil by steam distillation, extracting crude volatile oil with diethyl ether for 3 times, drying the extractive solution with anhydrous sodium sulfate, and volatilizing diethyl ether at room temperature to obtain herba Agastaches volatile oil;

the preparation method of the medicine volatile oil composition comprises the following steps:

mixing the raw materials according to the weight ratio to obtain the medicinal volatile oil composition;

the medicinal volatile oil composition is used for bacteriostasis, disinfection or anti-inflammation.

2. A pharmaceutical formulation comprising an effective amount of the pharmaceutical composition of claim 1; the pharmaceutical preparation is used for bacteriostasis, disinfection or anti-inflammation and comprises a pharmaceutically acceptable carrier;

the dosage forms of the pharmaceutical preparation comprise capsules, aerosols, sprays, powders, suspensions, emulsions, suppositories, ointments and creams.

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