WO2014019486A1

WO2014019486A1 - New application of patchouli oil

Info

Publication number: WO2014019486A1
Application number: PCT/CN2013/080302
Authority: WO
Inventors: 彭成; 魏晓露; 万峰; 林大胜
Original assignee: 成都华神集团股份有限公司; 成都中医药大学
Priority date: 2012-07-30
Filing date: 2013-07-29
Publication date: 2014-02-06
Also published as: CN103393752A; CN103191183A

Abstract

Application of patchouli oil in preparing antiviral drugs, health food, food, cosmetics, disinfectant, or everyday chemicals, and an antiviral drug, health food, food, cosmetic, disinfectant, or everyday chemical, characterized by being a preparation prepared by using patchouli oil as an active ingredient, added with auxiliary materials or auxiliary ingredients acceptable in drugs, health food, food, cosmetics, disinfectant, or everyday chemicals.

Description

New use of musk oil

The present invention relates to new uses of musk oil, and in particular to the use of musk oil in the preparation of antiviral drugs, health foods, foods, cosmetics, disinfectants or daily chemicals.

Background technique

Infectious diseases caused by viruses have always been a serious threat to human life and health. According to the statistics,

More than 80% of infectious diseases are caused by viruses. Viral diseases have been involved in various fields of clinical medicine. At present, viruses and diseases caused by them have become the focus of many disciplines. The most common diseases of viral infections are infectious diseases caused by respiratory viruses and enteroviruses.

Respiratory virus is a large group of viruses that can invade the respiratory tract and cause local lesions in the respiratory tract, or the respiratory tract is the portal of the respiratory tract. It is the main pathogen causing acute respiratory infection. At present, there are more than 10 kinds of viruses known to cause acute respiratory infections, including influenza virus, parainfluenza virus, adenovirus, herpes simplex virus, respiratory syncytial virus and the like. According to statistics, more than 90% of acute respiratory infections are caused by viruses, especially respiratory infections are common and frequently-occurring diseases in the clinic. In severe cases, bronchiolitis and pneumonia can cause acute respiratory distress and heart failure, and even lead to death. Respiratory viruses are susceptible to variability, which increases their pathogenicity or triggers new diseases. Among them, influenza is the main pathogenic virus causing influenza in acute respiratory infections. The influenza is characterized by strong contagiousness, rapid spread, and repeated susceptibility to the population. According to the latest epidemic report issued by the World Health Organization, the H1 Influenza A has caused more than 10,000 deaths worldwide, and its activities in Central Asia and South Asia have continued to strengthen. China is a flu-prone area. Influenza epidemics or local outbreaks occur almost every year, causing more than 100 million people to suffer from the flu. The social and economic burden caused by the flu-to-hospital treatment of more than 500,000 people is difficult to count. The harm of adenovirus can not be ignored. AdV (AdVovirus) is a common virus in children with respiratory tract infection. The most common disease is pharyngeal tonsillitis, followed by pneumonia, gastroenteritis, bronchitis and otitis media. Conjunctivitis, encephalitis, keratitis, enteritis, etc., the mortality rate is very high.

Coxsaekieviurs belongs to the group Picomaviridea and Enterroviurs, which can be divided into group A and group B according to their different pathogenic ability to suckling rats. Coxsackievimsgroup B (CVB) can cause epidemic chest pain, aseptic encephalitis, meningoencephalitis and pericarditis. In particular, CVB3 is the main pathogen of viral myocarditis, which can cause focal necrosis of myocardial tissue. With pathological changes such as inflammatory cell infiltration and cardiomyocyte lysis, persistent viral infection can lead to dilated cardiomyopathy, which poses a great threat to humans. Since the 1990s, researchers have discovered a number of new antiviral drugs. More than 20 antiviral drugs have been used in clinical practice, but the exact efficacy is still controversial, and antiviral chemical synthesis drugs have long been After application, the virus is easily resistant to it, and it has become a thorny problem in the treatment of viral diseases. Drug ribazole, which is now routinely used to control viral infections

(ribavirin) inhibits viral replication by inhibiting phosphonium nucleus dehydrogenase, and is effective in early administration. However, there is bone marrow cytotoxicity when ribavirin is administered intravenously. Therefore, it is best to use only a small amount of aerosol to be administered through the respiratory tract. However, leukopenia often occurs in clinical applications. When the dose is too large, it can cause headache, irreversible anemia, elevated serum bilirubin, etc. Side effects, teratogenic reports in animal experiments. Therefore, the research on antiviral drugs has important practical significance. It is imperative to find an antiviral drug with high efficiency, safety and few side effects.

China's traditional Chinese medicine resources are extremely rich, with a long history of application, and accumulated a lot of information and experience in long-term practice. The efficacy of traditional Chinese medicine is certain, the side effects are small, the source of medicine is abundant and the price is low. In the theory of traditional Chinese medicine, it is proposed that there are single-drug drugs such as honeysuckle, Daqingye, Atractylodes, and Astragalus, and compound drugs such as Banlangen granules can exert advantages in antiviral. In vitro experiments and clinical practice have shown that most viruses are sensitive to Chinese herbal medicines, such as respiratory viruses (influenza virus, measles virus, mumps virus), enteroviruses (poliovirus, rotavirus, coxsackie virus), coal The use of traditional Chinese medicines such as viruses (epidemic JEV, encephalitis virus) can achieve better results. The general pharmacological action principle is to reduce the viral infection, prevent the virus-induced pathological changes, regulate the immune function, improve the pulmonary circulation, reduce and eliminate the inflammatory exudate of the diseased part, and reduce or heal the symptoms of the viral infectious disease. Chinese medicine anti-virus has potential advantages and broad prospects.

Patchouli is a long-lived genus of the Labiatae (Pogostemon cablin (Blanco) Benth.), also known as the fragrant incense, native to tropical Asia such as the Philippines, and is now widely distributed in China, India, Japan, Indonesia, Malaysia, Madagascar, Brazil, Paraguay and Russia. At present, Guangleixiang is cultivated in Guangzhou, Zhaoqing, Zhanjiang, Hainan, Guangxi, Sichuan and other provinces (regions:). It is one of the “Ten Great South Medicines”, and its medicinal materials are divided into brands according to the place of origin (Guangzhou Production:), Zhixiang (Zhaoqing:), Zhanxiang (Zhanjiang:) and Nanxiang (Hainan:) 4 kinds. It has a mild taste, mild warmth, and belongs to the spleen, stomach, lungs, and is used as a whole herb. It has the effects of aroma, turbidity, appetizing and vomiting, and release of heat. Commonly used in wet turbidity, vomiting, vomiting, stagnation, chest tightness, cold and dampness, abdominal pain, vomiting and diarrhea, nasal headache, exogenous cold. The volatile oil of patchouli has anti-inflammatory, analgesic and antibacterial activities.

Musk is a musk plant of the genus Agastache rugosa (Fisch. Et Mey.) O. Ktze. The aboveground part contains volatile oil and has the same effect as patchouli. Modern pharmacology shows that patchouli has anti-pathogenic microorganisms, anti-inflammatory, antipyretic and analgesic effects. It is the main component of common Chinese medicines for treating influenza and colds, such as Huoxiang Zhengqishui and anti-virus oral liquid, but its efficacy The material basis is still unclear and there is no report on its anti-influenza activity in vivo. The volatile oil of musk has an antibacterial effect.

Summary of the invention The object of the present invention is to provide a new use of musk oil, and a medicament, a health food, a food, a cosmetic, a disinfectant or a daily chemical which uses musk oil as an active ingredient.

Use of the musk oil of the present invention in the preparation of an antiviral drug, a health food, a food, a cosmetic, a disinfectant or a daily chemical.

Wherein the virus is an influenza virus, CVB-3 and/or an adenovirus.

The medicine, health food, food, cosmetics, disinfectant or daily chemical is a medicine, health food, food, cosmetics, disinfectant or daily chemical for preventing, treating or assisting in the treatment of viral infection.

The medicament, health food, food, cosmetic, disinfectant or daily chemical product is a preventive, therapeutic or adjuvant treatment for respiratory infectious diseases, pneumonia, otitis media, viral myocarditis, conjunctivitis, encephalitis, keratitis, hepatitis or/and Enteritis drugs, health foods, foods, cosmetics, disinfectants or daily chemicals.

The respiratory infectious disease is rhinitis, pharyngitis, tonsillitis, laryngitis, bronchitis or/and the sesame oil is derived from patchouli Pogostemon c<a*/ «(Blanco)Benth. or Musk Agastache rugosa Fisc .Et Mey.) O.Ktze. Extracted volatile oil.

The musk oil derived from the patchouli pogiwfe iw o^/ «(Blanco)Benth. has a content of not less than 40% (w/w) and a musk ketone content of not less than 20% (w /w) o

The musk oil is prepared as follows: Take the musk Pogostemon

Ca «(Blanco)Benth. or Musk Agiwtoc e gova (Fisch.Et Mey.) O.Ktze. The whole grass, powdered into coarse powder, added with water, soaked, extracted by steam distillation, that is.

The antiviral medicine, health food, food, cosmetics, disinfectant or daily chemical product of the invention is characterized in that the musk oil is used as an active ingredient, and the medicine, health food, food, cosmetics, disinfectant or antimony product is acceptable. Preparation of excipients or auxiliary ingredients.

The preparations are liquid preparations, gas preparations, solid preparations, and semisolid preparations.

The content of the musk oil in the preparation is 0.1% to 100% (w/w) o

The musk oil of the invention has antiviral action, in particular, has significant inhibitory effects on influenza virus, CVB-3 and/or adenovirus, and has therapeutic effects on acute respiratory infections caused by viruses and other inflammations, wherein, for viral myocarditis, Pneumonia is effective and has good clinical application prospects.

It is apparent that various other modifications, substitutions and changes can be made in the form of the above-described embodiments of the present invention without departing from the spirit and scope of the invention.

The above content of the present invention will be further described in detail below by way of specific embodiments of the embodiments. However, the scope of the above-mentioned subject matter of the present invention should not be construed as being limited to the following examples. Any technique implemented based on the above description of the present invention is within the scope of the present invention. detailed description

Example 1 Preparation of Musk Oil of the Present Invention

Take the patchouli ί^ί« £^ο« ^/ «(Β1&η( ))Βεηώ. whole grass, crushed through a 20-40 mesh sieve, add ₁₀ - ₁₄ times the weight of distilled water, soak for 1-5 hours, then The steam distillation method is used for 2-6 hours to obtain the musk oil of the present invention, which is also known as patchouli oil, wherein it contains penaeyl alcohol (C ₁₅ H ₂₆ C is not less than 40% w/w, and musk ketone is not allowed. Less than 20% w/w.

Example 2 Preparation of Musk Oil of the Present Invention

Take Agastache rugosa (¥isc i.Et Mey.) O.Ktze. whole grass, crushed through 20-40 mesh sieve, add 10-14 times weight of distilled water, soak for 1-5 hours, then use steam distillation The extracting of the musk oil of the present invention is carried out for 2-6 hours.

The musk oil of the present invention can also be extracted according to the pharmacopoeia (2005 edition:) Appendix XD volatile oil assay, and can also be extracted by prior art techniques such as organic solvent extraction, supercritical CO ₂ extraction, or by purchasing a commercially available product. Example 3 Preparation of Mouthwash of the Invention

The musk oil prepared in Example 1 or Example 2 was uniformly dispersed with propylene glycol, Tween-60, distilled water, glycerin, surfactant, and isopropanol, filtered, and the filtrate was taken and packaged to obtain a mouthwash. Example 4 Preparation of the Cosmetic of the Invention

Take the sesame oil prepared in Example 1 or Example 2, with appropriate amount of lanolin, glyceryl monostearate, petrolatum, leucovorin, stearic acid, liquid paraffin, benzoic acid, triethanolamine, propylene glycol, distilled water, Tween - 60, partial sodium sulfite, flavor, preservatives, brain peptide, sodium citrate, cetyl alcohol, evenly mixed, cooled storage, that is, cosmetics. Example 5 Preparation of Disinfectant Water of the Invention

The appropriate amount of the musk oil prepared in Example 1 or Example 2 was mixed with Tween-60, sodium carboxymethylcellulose, polyethylene glycol 200, and distilled water to obtain a disinfecting water. Example 6 Preparation of the daily chemical product of the invention - cleaning agent

The eucalyptus oil prepared in Example 1 or Example 2 is prepared by adding an appropriate amount of a carboxylic acid polymer, a nonionic surfactant, an anionic surfactant, an enzyme, an enzyme stabilizer, a foam control agent, Tween-80, and water. . Example 7 Preparation of Daily Chemicals of the Invention - Hand Sanitizer

Take the musk oil prepared in Example 1 or Example 2, with glycerin, sodium benzoate, sodium chloride, go Ionic water, essence, triethanolamine, glyceryl monostearate, hydrogenated lanolin, stearic acid, ethyl paraben, and white spirit. After evenly dispersed, the hand sanitizer is obtained. Example 8 Food of the invention

Take the musk oil and bayberry prepared in Example 1 or Example 2, add appropriate amount of orange peel powder, cinnamon powder, granulated sugar, clove powder, licorice powder, cumin powder, salt and alum to prepare the bayberry candied fruit.

After taking the red bayberry, put a layer of about 20cm thick bayberry, then put a layer of mixed salt, alum, and Baiqiu Li alcohol, and immediately compact. Later, the same mixture of bayberry and salt alum, Baiqiu Li alcohol was placed in the same phase, and then pressed into a bayberry. Take the sucrose, spice powder, orange peel powder, cinnamon powder, male clove powder, licorice powder, cumin powder, then soak, hang, mix, and package, then get the bayberry honey. Example 9 Health food of the present invention

Take the sesame oil prepared in Example 1 or Example 2, add appropriate amount of sodium carboxymethyl cellulose, and the appropriate amount of withered leaves, with two-pot greening machine, and the cockroach, Amomum villosum L., arboreal, bergamot, Daqingye fresh tea, chrysanthemum, cassia seed, honeysuckle, atractylodes, hawthorn, white peony, mulberry leaf, schisandra, cumin, yam are mixed evenly to prepare liver tea. Example 10 Preparation of Drug Granules of the Invention

The musk oil prepared in Example 1 or Example 2 was added to an appropriate amount of starch and dextrin to prepare granules.

The beneficial effects of the present invention are demonstrated by pharmacodynamic tests as follows:

Experimental Example 1 In vitro antiviral experiment of musk oil of the present invention

1, materials and instruments

1.1 cell strain and virus strain

1.1.1 cell line:

MDCK cells (canine kidney cells), Hep-2 cells (human laryngeal carcinoma epithelial cells), and HeLa cells (cervical cancer cells) were purchased from the China National Institute for the Control of Pharmaceutical and Biological Products.

1.1.2 virus strain:

Influenza A virus is selected from A/PR/8/34 strain, serotype is H1N1 (hereinafter referred to as influenza virus), which is derived from National Influenza Center; Coxsackie virus is selected from group B type 3 (hereinafter referred to as CVB-3), source From the Sichuan Provincial Clinical Examination Center; adenovirus uses the common type 3 (Ad-3), fed by the Gansu Provincial Center for Disease Control Gift.

1.2 drugs and control drugs

Test drug: Inventive drug: Patchouli oil prepared in Example 1.

Positive drug: Ribavirin injection: The specification is 100mg/ml, produced by Tianjin Pharmaceutical Co., Ltd. Jiaozuo Co., Ltd. Approval number: National Pharmaceutical Standard H19992467, Production batch number: 10110521; Acyclovir for injection: The specification is 0.25g/bottle, produced by Tianjin Pharmaceutical Co., Ltd. Approval number: National Pharmaceutical Standard H20034034, Production Lot No.: 10122403.

1.3 medium, reagents and consumables

RPMI-1640 medium (GibcoTM, Lot 1313945); DMEM medium (GibcoTM, Lot 1345538); Fetal bovine serum (HyClone Biochemicals (Beijing) Co., Ltd., batch number NVM0347); Trypsin (Amresco) Penicillin G sodium, streptomycin sterile cell culture flasks and 96-well cell culture plates (Coming, USA). 1, 5, 10ml disposable straw, 200μ1, 1ml - secondary shot (Jiangsu Haimen Bio Consumables Co., Ltd.)

1.4 main instruments

Water-proof constant temperature C0 ₂ incubator (model MCO-15AC, Sanyo, Japan);

OLYMPUS inverted microscope (model CKX41, Olympus, Japan);

Micropipette (produced by GILSON, France);

Water-proof electric heating constant temperature incubator (Model GSV-DA-1, Huangshi Medical Equipment Factory, Hubei Province); Horizontal centrifuge (Model LXJ-II, Shanghai Medical Analytical Instrument Factory);

Electronic balance (model JA-2603, Shanghai Tianping Instrument Factory).

2, test method

2.1 Pretreatment of drugs and positive controls

The inventive drug patchouli oil was firstly dissolved in a white liquid preparation with 0.5% Tween-80, and its initial concentration was 20% (the density of patchouli oil was 1.012 g/ml, and the concentration was 202.4 mg/ml). The white liquid was diluted 1/10 with 0.5% Tween-80 before use, and filtered for sterilization.

The positive control drug ribavirin injection for influenza virus and Coxsackie virus and the positive control drug for adenovirus injection were diluted 1/10 with sterile water (ie 10 mg/ml) with acyclovir. Take a backup.

2.2 Determination of 50% tissue cell infection dose

Influenza virus and adenovirus were diluted with 3% fetal bovine serum DMEM and Coxsackie virus B3 with 3% fetal bovine serum RPMI-1640 to different concentrations of virus solution.

Influenza virus was inoculated into MDCK cells, Coxsackie virus B3 strain was inoculated into HeLa cells, adenovirus was inoculated into Hep-2 cells, and cultured for 7 days at 37 ° C in 5% CO ₂ incubator to observe cytopathic lesions such as rounded cells and shedding of infected cells ( Cytopathic effect, CPE), to determine the 50% tissue culture infective dose (TCID50) caused by each virus. 2.3 anti-virus experiment

Add 0.1ml of 100xTCID50 influenza virus solution to MDCK monolayer cells in 96-well culture plate, add 0.1ml of 100xTCID50 CVB-3 virus solution to HeLa monolayer cells, and add 100xTCID50 Ad-3 virus solution to Hep-2 monolayer cells. O.lml, adsorbed at 37 ° C, 5% C0 _{2 for} 2 hours, aspirate the virus solution, add diluted drug of the invention and positive control drug, and make 3 duplicate wells per concentration. The experiment also set up an infection control that was not administered with an equal amount of virus and a normal cell control without virus infection. Each plate was further cultured at 37 ° C, 5% 0 _{2 for} 5 days, and CPE was observed under a microscope every day.

3. Experimental results and calculation of pharmacodynamic indicators

3.1 TCID _{5 of} each virus. value

CPE appeared about 3 days after influenza virus inoculation of MDCK cells, CPE appeared about 5 days after Coxsackievirus B3 inoculation of HeLa cells and adenovirus Ad-3 inoculated with Hep-2 cells. The TCID50 titer of influenza virus is 10" ⁴ , and the TCID50 titer of coxsackievirus B3 and adenovirus Ad-3 is ^10-12 .

3.3 Antiviral experimental effect and pharmacodynamic index calculation of the invention drug

3.3.1 Experimental results

The experimental results are shown in Table 1:

Table 1 Antiviral experiment results of the invention drugs (n=3)

Percentage of MDCK cell lesions (%) Percentage of HeLa cell lesions (%) Percentage of Hep-2 cell lesions (%)

(mg/ml) invention drug ribavirin invention drug ribavirin invention drug acyclovir

1.25 0 —— 0 0

0.625 0 —— 0 0

0.312 0 —— 0 0

0.156 25 —— 25 25

0.078 50 —— 50 25

0.039 100 —— 100 50

0.020 100 —— 100 100

0.092 25 25 - 25

0.088 50 25 —— 25

0.084 75 50 —— 50

0.08 100 50 —— 75

0.078 100 75 —— 100

0.075 100 100 - 100

0.072 100 100 —— 100

Cell control cells grow well, no cell lesions (0)

Virus control cells all have lesions (100)

As can be seen from Table 1, the present invention is a musk oil against influenza virus, coxsackievirus B3 strain and adenosis The experiment demonstrates that the musk oil of the present invention is excellent in inhibiting influenza A virus, CVB-3 and adenovirus. Experimental Example 2 In vivo antiviral experiment of musk oil of the present invention

1, materials and instruments

1.1 virus strain

Influenza A virus is selected from A/PR/8/34 strain, serotype is H1N1 (hereinafter referred to as influenza virus), which is derived from National Influenza Center; Coxsackie virus is selected from group B type 3 (hereinafter referred to as CVB-3), source From the Sichuan Provincial Clinical Examination Center; the adenovirus uses the common type 3 (Ad-3), which is donated by the Gansu Provincial Center for Disease Control.

1.2 drugs and control drugs

Test drug: Inventive drug: Patchouli oil prepared in Example 1.

1.3 Animals: Mouse Bab/c male, weighing 10 ± 2 g, male and female

Mouse Bab/c male, weighing 18 ± 2g, male and female

1.4 medium, reagents and consumables

RPMI-1640 medium (GibcoTM, Lot 1313945); DMEM medium (GibcoTM, Lot 1345538); Fetal bovine serum (HyClone Biochemicals (Beijing) Co., Ltd., batch number NVM0347); Trypsin (Amresco) Penicillin G sodium, streptomycin sterile cell culture flasks and 96-well cell culture plates (Coming, USA). 1, 5, 10ml disposable straw, 200 μ 1 , 1ml - secondary shot (Jiangsu Haimen Bio Consumables Co., Ltd.)

Ether (analytically pure, Chengdu Kelon Chemical Reagent Factory, batch number: 20111215); 0.9% physiological salt water (Chengdu Kelun Pharmaceutical Co., Ltd., batch number: H120115)

Lactate Dehydrogenase (LDH) Assay Kit, Nanjing Institute of Bioengineering, Batch No.: 20121013; Superoxide Dismutase (SOD) Assay Kit, Nanjing Institute of Bioengineering, Batch No.: 20121012; Malondialdehyde (MDA) Determination kit, Nanjing Institute of Bioengineering, batch number: 20121010; CK-MB (creatine kinase isoenzyme), TNF-α (tumor activating factor alpha) assay kit, RD, USA, batch number: 201210 1.4 main instruments

OLYMPUS inverted microscope (model CKX41, Olympus, Japan);

Micropipette (produced by GILSON, France);

Electronic balance (Model JA-2603, Shanghai Tianping Instrument Factory);

Microplate reader, Thermo Scientific, USA, Model: Varioskan Flash.

2, test method

2.1 Pretreatment of drugs and positive controls

The inventive drug musk oil was first dissolved in 0.5% Tween-80 as a white liquid preparation with an initial concentration of 20% (musk oil density was 1.012 g/ml, concentration 202.4 mg/ml). The white liquid was diluted 1/10 with 0.5% Tween-80 before use, and filtered for sterilization.

2.2 In vivo antiviral experiment of the invention drug

2.2.1 Establishment of a pathogenic model of virus-infected animals

Forty Bab/c mice, SPF grade, weighing 10 ± 2 g, were randomly divided into 4 groups, 10 in each group. After the injection site was routinely disinfected, 0.3 ml of CVB3 virus solution of the original doubling, 1/2 and 1/4 was intraperitoneally injected into the group, and the normal control group was intraperitoneally injected with 0.3 ml of physiological saline.

Another Bab/c mice, SPF grade, weighing 18 ± 2 g, were randomized into groups of 10 animals each. According to the group, first mild anesthesia with ether, then nasal infection of the original dou, 1/2 and 1/4 of the influenza virus solution or adenosis virus lml, the normal control group with lml saline solution nasal drops.

The animal room keeps the air fresh, the relative humidity is 60%, the temperature is 22 ± 2 °C, the feed is supplemented regularly, the water is changed, the litter is replaced every day, the morbidity and mortality of the mice are observed, and recorded to determine whether the CVB3, flu is successfully established. Virus and adenovirus infection in animal pathogenic models.

2.2.2 In vivo antiviral pathogenic model test of the invention drug

2.2.2.1 In vivo drug model test of antiviral myocarditis

140 Bab/c mice, weighing 10 ± 2 g, were randomly divided into 7 groups, 20 in each group. to mark.

The clinical injection dosage and LD _{5 of} patchouli oil. Based on the safety dose range, the drug treatment groups of high, medium, low and ultra-low concentrations were established, ie 1.25% (0.127g/kg), 0.95% (0.096g/kg), 0.625 of the original drug concentration. % (0.063g/kg) and 0.48% (0.049g/kg). At the same time, normal control group, CVB3 virus model control group and ribavirin positive drug control group (positive drug) were set up. The concentration was 0.15 g/kg).

The injection site of the mice was routinely sterilized. Except for the normal control group, the other groups were intraperitoneally injected with the CVB3 virus solution required for the viral model, and the normal control group was intraperitoneally injected with normal saline, and the injection amount was the same as that of the other groups. After 24 hours, the intraperitoneal injection was started, and the injection amount was 0.1 ml/10 g once a day. The animal room kept the air fresh, the relative humidity was 60%, the temperature was 22 ± 2 °C, and the feed, water, and litter were replenished regularly every day. After 7 days of continuous treatment, the drug was stopped for the 15th day. The signs and deaths of the mice were observed daily, and the body weights were weighed and recorded.

On the 5th, 10th, and 15th day, 4 animals were randomly sacrificed on the 5th, 10th, and 15th day. Blood was collected from the eyelids, and the heart, liver, lungs, and kidneys were removed. After removing the surface blood with filter paper, Weigh and calculate the body rate of heart, liver, lung and kidney. Then, 100 mg of each organ was taken and fully padded with 2 ml of RPMI-1640 cell maintenance solution. After the tissue homogenate was filtered and sterilized by microfiltration membrane, it was repeatedly frozen and thawed three times, centrifuged at 3000 rPm for 30 min, and 0.1 ml of the supernatant was inoculated into a 96-well plate which had grown into a single layer of HeLa cells, and each sample was made to be 3 complexes. After incubating at 37 °C, 5% CO 2 for 2 h, discard the liquid in the well. After washing with PBS for 3 times, add 1640 cell maintenance solution, and observe the cell growth and the corresponding CPE level at 37 ° C, 5% CO 2 culture for 72 h-120 ho. Those who did not appear CPE were blindly transmitted 3 times without CPE and were no virus infection. The blood collected from the eyelids was placed at 4 ° C overnight, centrifuged at 3000 rpm for 20 min, and serum was taken for serum biochemical indicators.

2.2.2.2 In vivo anti-pneumonia model test of the invention drug

140 Bab/c mice, weighing 18 ± 2 g, were randomly divided into groups of 20 each. to mark. Based on the clinical dose of patchouli oil, the drug treatment groups of high, medium, low and ultra-low concentrations were established in the safe dose range, ie 10% of the original concentration of the drug ( 1.27g/kg). 5% (0.508g/k _g ), 2.5% (0.254g/kg) and 1.25% (0.127g/kg) _o set normal control group, influenza virus model control group, adenovirus model control group, ribavi Lin-positive drug control group and acyclovir-positive drug control group.

In addition to the normal control group, the model group was given nasal infections to the mice according to the amount of influenza virus and adenovirus virus required for successful modeling. The normal control group was given saline nasal drops, and the amount of nasal drops was the same as that of other groups. . After 24 hours, the intragastric administration was started, and the amount of gastric perfusion was 0.3 ml/10 g once a day.

The animal room kept fresh air, relative humidity 60%, temperature 22 ± 2 °C, daily feeding, water, and replacement of litter. After 7 days of continuous treatment, the drug was stopped for the 15th day. The signs and deaths of the mice were observed daily, and the body weights were weighed and recorded.

On the 5th, 10th, and 15th day, 4 anatomists were randomly sacrificed from the animals on the 5th, 10th, and 15th day. The lung lesions were observed by the naked eye. After removing the whole lung filter paper, the surface blood was removed. Quantity, calculate the lung rate. The murine lungs are then inoculated with corresponding sensitive cells to determine if the lung disease is caused by infection with the test virus.

3. Test results 3.1 Establishment of a virus model in mice

The original doubling, 1/2 and 1/4 of CVB3 virus solution was intraperitoneally injected into mice, 0.3 ml/mouse, and it was observed that the hair of the mice was shiny before the injection of the virus, and the activity, diet, and urine were normal. After the injection of the drug, compared with the normal control group, the mice in the original doubling virus group began to decrease in activity on the 2nd to 3rd day, the diet decreased, the weight was reduced, the hair was tarnished, and curls appeared, 6~7 The days of death, all died on the 8th day, the heart was found to be significantly smaller than the normal group of mice, while the milky white spot-like, strip-like fat-like lesions were seen under the epicardium. In the 1/2 original concentration group and the 1/4 original concentration group, the mice also showed decreased activity, decreased diet and weight loss on the 2nd to 3rd day, but increased activity on the 5th day, the diet increased, and the status gradually increased. Reverted to normal group mouse status. Explain that the establishment of a model of viral myocarditis requires intraperitoneal injection of mice with original CVB3 virus solution, 0.3 ml / only.

The original doubling virus, 1/2 and 1/4 of influenza virus solution and adenovirus solution were intranasally inoculated into mice, 1 ml/mouse. It was observed that the model mice were smaller than the normal control mice. The rats began to die on the day of the nose, and all died on the fourth day. The 1/2 original concentration group showed decreased activity on the 2nd to 3rd day, the diet decreased, the hair lost luster, and curls appeared. Death occurred in ~6 days, and all died on the 7th day. The lung lesions were obvious after dissection. The 1/4 original concentration group showed discomfort on the second day, but the state improved on the third to fourth days. The influenza virus and adenovirus required to establish a pneumonia model are all 1/2 original concentration group, 1 ml/only.

3. 2 antiviral effect of the invention drug in vivo

3. 2.1 Effect of the invention drug on a mouse model of viral myocarditis

3. 2.1.1 General situation impact

The experimental results are shown in Table 2:

Table 2 Body weight and death of each experimental group

The normal group of mice performed normally, and by the end of the observation, the mortality rate was 0%; At the end of the modeling, the mice in the model group, the positive group and the inventive drug group showed weight loss to varying degrees, with poor skin, decreased activity, decreased food intake, and increased urination.

The mice in the model group began to die on the 6th to 7th day. At the end of the observation, the mortality rate was as high as 90%. The symptoms of the positive drug group and the inventive drug group were all reduced, the activity amount, the food intake increased, and the body weight began to rise:

Among them, the mortality rate of the positive group was 40%; the mortality rate of the invention drug was 70%; the mortality rate of the invention drug was 1.2%; the mortality rate of the invention drug was 0.6%; 40% of the invention drug; The rate is 65%.

The therapeutic effect of the musk oil of the present invention on viral myocarditis increases first and then decreases with the increase of the dosage, and the effect is optimal at the dosage of 0.096 g/kg.

When the dosage of patchouli oil of the invention is 0.063~0.096g/kg, the therapeutic effect is better than that of the positive drug (0.15kg/g), indicating that the medicament of the invention has excellent therapeutic effect on viral myocarditis.

3. 2.1.2 Effect of the invention drug on the body rate of heart, lung and kidney in mice

The experimental results are shown in Table 3:

Table 3 Body weight and heart, lung and kidney liver body rate results of mice in each experimental group ( _n = 4)

The cardiopulmonary kidney of the normal group was red and shiny;

In the model group, the heart color was narrowed and contracted, and the epicardium showed milky white spots, cord-like fat-like lung enlargement, and liver and kidney did not change;

In the positive group and the concentration group of the inventive drug, the color of the heart is reduced to a gradual recovery of color, and the heart is outside. Membrane milk white dot-like, strip-like fat-like reduction trend, lung swelling is reduced.

The patchouli oil of the invention can alleviate various organ diseases, can effectively reduce the color of the heart and gradually return to normal, further illustrating that the patchouli oil of the invention can alleviate viral myocarditis.

3. 2.1.3 Effect of the invention drug on serum biochemical parameters in mice

The sera of mice immunized with virus for 5d, 10d and 14d were tested for serum biochemical indicators. Compared with the model group, LDH, CK-MB and TNF-α levels and MDA content were significantly lower, and SOD activity was significantly higher (PO.01). Compared with the model group, the levels of LDH, CK-MB and TNF-a and MDA content in the positive group and the inventive drug group were decreased to some extent, and the SOD activity was increased to different degrees (P<0.01). Compared with the normal group, the levels of LDH, CK-MB and TNF-a and MDA content increased with different degrees, and SOD activity decreased to different degrees (P<0.05). The levels of LDH and CK-MB in the normal group did not change much with time, SOD activity increased slightly, MDA content and TNF-a level decreased significantly. The LDH and CK-MB levels in the model group increased with time. Significantly decreased, SOD activity, MDA content and TNF-a levels increased significantly; LDH and CK-MB levels in the positive group, 0.95% and 0.625% concentration groups decreased with time, SOD activity increased, MDA content and The level of TNF-a was significantly decreased, and the index changes to the normal level; the levels of LDH, CK-MB, MDA and TNF-a decreased with time in the 1.25% and 0.48% concentrations, and SOD activity increased. The indicator changes tend to be at the model group level. The results of serum biochemical indicators in each experimental group are shown in Table 4.

Table 4 Serum biochemical parameters of different experimental groups in different experimental groups (n=4)

2* .21 93*" •56* ^# .75*".3*" .53**

CK-MB 3.50±0.06* 4.78±0.98 4.03±0.69* ^# 4.11±0.46* ^# 3.97±0.67* 4.35±0.29* ^# 4.64±0.61 *

SOD 10.91±0.82* 7.73±0.14 10.25±0.67*# 9.17±0.29* ^# 10.01±0.37* ^# 9.83±0.65* ^# 10.64±0.76* ^#

MDA 18.33±1.38* 29.45±3.2 20.11±1.56* ^# 24.13±1.98* ^# 22.33±1.62* ^# 18.96±3.95* ^# 26.10±2.21* ^#

TNF-a 26.28±2.65* 64.24士4.83 44.18±5.92* ^# 31.47±5.83* 48.85±5.29* ^# 37.25士1.74* ^# 56.51士2.43*#

(* Compared with the model groups, P <0.01, ^# and normal group, PO.05)

As can be seen from Table 4, the inventive drug can significantly reduce the LDH, CK-MB and TNF-a water and MDA content, and increase the SOD activity. Compared with the model group, the difference is significant (ρο.οι ), and the progress proves that the invention is wide. Therapeutic effect of musk oil on viral myocarditis.

3. 2.2 Effect of the invention drug on the mouse model of pneumonia

3. 2.2.1 General situation impact

After 2 to 3 days of virus infection, the mice in the virus group showed weight loss to varying degrees, accompanied by hair loss, decreased activity, decreased feeding, and increased urination. The mice in the model group began to die 12 days after the 6th to 7th days. 2 mice remained until the end of the observation; the weight loss of the mice in the 4 concentration groups of the invention drug was significantly higher than that in the model. The mice in the ribavirin, acyclovir-positive group and the inventive drug concentration group died at 5-6 days, positive. The mice in the group began to gradually improve in 7-8 days (ie, 1 day after stopping the drug), the active food intake increased, and the body weight began to rise. However, some of the mice in the concentration group of the invention drug had some changes.

3. 2.2.2 Effect of the invention drug on lung index in mice

The results of the invention of drugs for the treatment of pneumonia are shown in Tables 5 and 6.

Table 5 Drug anti-influenza effect in vivo Body weight and lung index comparison ( _n = 4)

It can be seen from Table 5 that when the concentration of the drug of the invention is more than 5%, the lung index can be effectively improved, indicating that the inventive drug can effectively treat pneumonia caused by influenza virus.

Table 6 Drug anti-adenovirus effect in vivo Body weight and lung index comparison ( _n = 4)

Body weight 25.6 0.5 15.0 0.5 22.6 1.8 14.5 1.5 15, 8 0.5 17.3 1.5 15.5 1.2 Lung index 0.8102 0.0246 1.9024 0.1461 1.2134±0.0121 1.8135 0.0233 1.7673 0.0214 1.7554 0.0826 1.8159±0.0217 As can be seen from Table 6, the drug of the invention can effectively improve the lung index, It is indicated that the invention drug can effectively treat pneumonia caused by adenovirus.

In summary, musk oil has an antiviral effect and is well resistant to influenza virus, CVB-3 and adenovirus, and can treat respiratory tract infections, pneumonia, otitis media, viral myocarditis, conjunctivitis, encephalitis, keratitis, Hepatitis and enteritis, among which, the curative effect on viral myocarditis and pneumonia is exact, the pharmacological effect is strong, and the market application prospect is good.

Claims

claims

1. The use of Patchouli oil in the preparation of antiviral drugs, health food, food, cosmetics, disinfectants or daily chemical products.

2. The use according to claim 1, characterized in that: the virus is influenza virus, CVB-3 and/or adenovirus.

3. The use according to claim 1, characterized in that: the medicine, health food, food, cosmetics, disinfectant or daily chemical products are medicines, health food, food, cosmetics for preventing, treating or assisting in the treatment of viral infection. , disinfectants or daily chemical products.

4. The use according to claim 3, characterized in that: the medicine, health food, food, cosmetics, disinfectant or daily chemical products are used to prevent, treat or assist in the treatment of respiratory infectious diseases, pneumonia, otitis media, viral Drugs, health foods, food, cosmetics, disinfectants or daily chemical products for myocarditis, conjunctivitis, encephalitis, keratitis, hepatitis or/and enteritis.

5. The method according to claim 4, characterized in that: the respiratory tract infectious disease is rhinitis, pharyngitis, tonsillitis, laryngitis, tracheitis or/and bronchitis.

6. The use according to any one of claims 1 to 5, characterized in that: the Patchouli oil is derived from Patchouli Pogostemon ca / «(Blanco) Benth. or Patchouli Agiwtoc e

Volatile oil extracted from go∞(Fisch.Et Mey.)O.Ktze.

7. The use according to claim 6, characterized in that: the Patchouli oil derived from Pogostemon ca/(Blanco) Benth. has a Patchouli alcohol content of not less than 40% (w/w) ), the patchoulione content is not less than 20% (w/w

8. The use according to any one of claims 1 to 7, characterized in that: the Patchouli oil is prepared according to the following method: Take Patchouli Pogostemon o^/«(Blanco) Benth. or Patchouli AgiMtoc £ ? The whole plant of go∞ (Fisch. Et Mey.) O. Ktze. is crushed into coarse powder, added with water, soaked, and extracted by steam distillation.

9. An antiviral drug, health food, food, cosmetics, disinfectant or daily chemical product, characterized in that: it uses Patchouli oil as the active ingredient, plus drugs, health food, food, cosmetics, disinfectants or Preparations prepared from excipients or auxiliary ingredients acceptable in daily chemical products.

10. The medicine, health food, food, cosmetics, disinfectant or chemical product according to claim 9, characterized in that: the preparation is a liquid preparation, a gas preparation, a solid preparation, or a semi-solid preparation.

11. The medicine, health food, food, cosmetics, disinfectant or daily chemical products according to claim 10, characterized in that: the content of Patchouli oil in the preparation is 0.1%~100% (w/w