CN102451228A - Preparation method and application of antibacterial and antiviral elsholtzia densa benth volatile oil - Google Patents
Preparation method and application of antibacterial and antiviral elsholtzia densa benth volatile oil Download PDFInfo
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- CN102451228A CN102451228A CN2010105195686A CN201010519568A CN102451228A CN 102451228 A CN102451228 A CN 102451228A CN 2010105195686 A CN2010105195686 A CN 2010105195686A CN 201010519568 A CN201010519568 A CN 201010519568A CN 102451228 A CN102451228 A CN 102451228A
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Abstract
The invention relates to elsholtzia densa benth volatile oil with antibacterial, antiviral and cholinesterase inhibition effect, and its application in the preparation of products with antibacterial, antiviral and cholinesterase inhibition effect. Elsholtzia densa benth volatile oil is extracted by steam distillation or supercritical technology, and is analyzed by gas chromatography-mass spectrometry; 41 components are identified, and the total content accounts for 84% of the volatile oil. The elsholtzia densa benth volatile oil has inhibition effect on aureus staphylococcus, pseudomonas aeruginosa, escherichia coli, candida albicans, influenza A virus, and cholinesterase. The elsholtzia densa benth volatile oil is applicable to industries of food, health products, medicine, cosmetics and the like, and can be used to prepare various products with antibacterial, antiviral and cholinesterase inhibition effect.
Description
Technical field
The invention belongs to medical technical field, be specifically related to that Elsholtzia densa. volatile oil is antibiotic, antiviral and cholinesterase inhibition and preparation method thereof and in the purposes of food, pharmaceutical preparations.
Technical background
At present because abuse of antibiotics causes the continuous rising of drug resistance to become one of common difficult problem of paying close attention in the whole world.Especially the resistant rate of staphylococcus aureus has reached 90%, and the intestinal microbial population that while antibiotics causes is disorderly, also increases the weight of primary infection, from natural product, seeks anti-infectives, and realistic meaning is arranged.
Elsholtzia densa. Elsholtzia densa Benth. is a Labiatae Herba Moslae platymiscium, is distributed in provinces and regions such as Yunnan Province of China, Hebei, Shanxi, Shaanxi, Gansu, Qinghai, Sichuan, Tibet, Xinjiang.For Herba Moslae usefulness, holding concurrently to be used for dermatosis outward in Tibet.At present to the existing report of composition of the volatile oil of Elsholtzia densa., for example Zhang Ji etc. extracts Elsholtzia densa. volatile oil with steam distillation and through the gas chromatography/mass spectrometry technology it is analyzed, and identifies and has confirmed 84 kinds of chemical compounds wherein; The 88.34%. main chemical compositions that its content accounts for full oil is germacrene (18.83%), D-limonene (11.17%), 2; 5,5-three or four bases-1,3; 6-heptantriene (6.30%), 6-methylene-dicyclo [3; 1,0], hexane (5.90%), oxidation caryophyllene (3.94%), caryophyllene (3.36%), 4-carbon-3,5-dimethyl hexamethylene-1-alkene (2.88%), α-3-cyclohexene-1-alcohol (2.06%).Pharmacological research to Elsholtzia densa. volatile oil does not appear in the newspapers.
The present invention relates to have the Elsholtzia densa. volatile oil of antibacterial, antiviral and choline esterase inhibition and had the application in antibiotic, antiviral and the treatment alzheimer disease product, filled up the blank that Elsholtzia densa. is used in this field in preparation.
Summary of the invention
1. the method for preparing of Elsholtzia densa. of the present invention
(1) steam distillation: take by weighing Elsholtzia densa. medical material 500g, pulverize, join in the alembic; Add entry 8-12 and doubly measure, volatile oil extractor is installed, vapor distillation 3-5 hour; From volatile oil extractor is heavy,, tells and reply a part, put into-10～-15 ℃ of refrigerator and cooled and freeze and spend the night in the aqueous volatile oil separatory funnel; The water intaking deicing obtains light yellow volatile oil, yield 0.8-2%.
(2) take by weighing Elsholtzia densa. 1kg, pulverize, place the 5L supercritical extraction reactor.Open extraction kettle and separating still heating apparatus, open compressor pump after waiting to reach design temperature, make extraction kettle pressure remain 20.0MPa, temperature is 50 ℃, with the C02 flow velocity cycling extraction of 40kg/h.The separating still pressure and temperature is respectively 10.0MPa and 30 ℃, and cycling extraction 3 hours obtains yellow oil, and peat-reek is arranged, and extraction ratio is 1.5-4%.
2. Elsholtzia densa. chemical composition of volatile oil of the present invention
Gained Elsholtzia densa. volatile oil is analyzed through GC-MS, and gas chromatography-mass spectrography technology gained mass spectrum has been confirmed the chemical composition of part in the Elsholtzia densa. volatile oil through computer mass spectrometric data library searching.GC conditions: quartz capillary column VarianVF-5MS CP8944,30m * 0.25mm, thickness 0.25 μ m.Heating schedule: since 60 ℃, be raised to 250 ℃ with 5 ℃/min, be raised to 280 ℃ with 15 ℃/min again, carrier gas is He, post flow 1.0mL/min, 270 ℃ of injector temperatures.Mass spectrum condition: EI power supply; Ionization voltage 70eV; 230 ℃ of ion source temperatures; Sweep limits 30～550aum; Sample size 1 μ l, split ratio: 22: 1.The Analysis and Identification structure shows that Elsholtzia densa. volatile oil main chemical compositions is patchouli alcohol (13.82%), 2, and 4-diethyl acrylic-1-methyl isophthalic acid-vinyl cyclohexane (9.04%), decahydro-4a-methyl isophthalic acid-methylene-7-(1-first and second bases)-naphthalene alkene (8.97%), 1,2,3; 3a, 4,5,6,7-octahydro-1,4-dimethyl-7-(1-first and second bases)-azulene (6.37%), 1,2; 3,4,5,6,7,8-octahydro 1,4-dimethyl-7 (1-first and second bases)-azulene (5.92%); The different propylene valeric acid of 5-epoxy (5.21%), Patchoulene (3.96%), ylangene (3.90%), 1,2,3,3a; 4,5,6, the 7-octahydro-. α, α, 3,8-tetramethyl-5-azulene methanol (3.27%); Caryophyllene (2.15%), 2,6,6-trimethyl-1-epoxy hexene-1-acetaldehyde (2.12%), 1,2,3; 5,6,7,8,8a-octahydro-1,8a-dimethyl-7-(1-first and second bases)-naphthalene alkene (2.02%) etc., total content accounts for 84.59% of full oil.
Through experiment showed, Xinjiang Elsholtzia densa. volatile oil, have broad-spectrum antibacterial, antivirus action, Elsholtzia densa. volatile oil is to antibacterials such as staphylococcus, escherichia coli genus, Rhodopseudomonass; The Candida fungus all has certain inhibition growth effect.Especially the effect that common bacteria pathogen staphylococcus aureus is had very strong inhibition growth has the effect of stronger inhibition growth to Candida albicans.Therefore, Xinjiang Elsholtzia densa. volatile oil can be applicable to industries such as food, medicine, health product, cosmetics, prepares the various forms of products with bacteriostasis.
The specific embodiment:
Embodiment 1 Xinjiang Elsholtzia densa. volatile oil antibacterium effect
Strain and culture medium
Experimental strain is provided by pharmacological evaluation chamber, Chinese Academy of Medical Sciences Beijing Union Medical College Institute of Medical Plants.Tabulate as follows:
Belong to strain
Staphylococcus staphylococcus aureus ATCC25923 (Staphylococcus aureus)
Escherichia coli belongs to escherichia coli ATCC25922 (Escherichia coli)
Antibacterial bacillus pyocyaneus ATCC10104 (Pseudomonas aeruginosa) such as Rhodopseudomonas
Culture medium is used in experiment: nutrient agar, nutrient broth medium are all available from Nat'l Pharmaceutical & Biological Products Control Institute.
2 experimental techniques
2.1 the mensuration of minimal inhibitory concentration MIC adopts Microdilution plate method
The test bacterial concentration is 105cfu/ml.Good bacterium liquid is added in the 96 porocyte culture plates every hole 100 μ l to draw dilution.Elsholtzia densa. volatile oil is dissolved in earlier among the DMSO, and the reuse nutrient broth medium is diluted to certain concentration (initial concentration 100mg/ml is used for gram negative bacteria, and initial concentration 80 μ g/ml are used for gram positive bacteria) with Elsholtzia densa. volatile oil.For gram negative bacteria escherichia coli and bacillus pyocyaneus, contrast dilution 7 holes make each hole Chinese medicine concentration be respectively 100mg/ml, 50mg/ml, 25mg/ml, 12.5mg/ml, 6.25mg/ml, 3.125mg/ml, 1.56mg/ml successively.For the gram positive bacteria staphylococcus aureus, contrast dilution 7 holes make each hole Chinese medicine concentration be respectively 80 μ g/ml, 40g/ml, 20 μ g/ml, 10 μ g/ml, 5 μ g/ml, 2.5 μ g/ml, 1.25 μ g/ml successively.Last not dosing of hole (only adding culture medium and antibacterial) adds dilution bacterium liquid 100 μ l, is the bacterial growth control wells.Stay the string hole not add antibacterial (only adding culture medium and medicine) and make the medicine control wells, positive control is a penicillin.Place the 1min that vibrates on the agitator, make in the hole behind the abundant mixing of solution, microwell plate is added a cover and is placed in the square enamel tray that is covered with wet gauze, in 37 ℃ of incubators, hatches 24 hours, observes the contained lowest drug concentration in no bacterial growth hole and is minimal inhibitory concentration.
3 result of the tests
Table 2 Elsholtzia densa. volatile oil minimal inhibitory concentration (MIC) (mg/ml of unit)
Pathogen MIC
Staphylococcus aureus 0.005
Bacillus pyocyaneus 25
Escherichia coli 50
The external antifungic action of embodiment 2 Elsholtzia densa. volatile oil
Strain and culture medium
Experimental strain is provided by pharmacological evaluation chamber, Chinese Academy of Medical Sciences Beijing Union Medical College Institute of Medical Plants.Tabulate as follows:
Belong to strain
Candida Candida albicans (Candida albicans)
Culture medium is used in experiment: husky fort agar culture medium, husky fort fluid medium is all available from Nat'l Pharmaceutical & Biological Products Control Institute.
2 experimental techniques
2.1 the mensuration of minimal inhibitory concentration MIC adopts Microdilution plate method
Experimental technique is with embodiment 1,
Experimental result is following:
Table 3: Elsholtzia densa. volatile oil minimal inhibitory concentration (MIC) (mg/ml of unit)
Pathogen MIC
Candida albicans 2.5
Experimental result shows: Elsholtzia densa. volatile oil has broad-spectrum antibacterial action.Antibacterial, fungus are responsive to Elsholtzia densa. volatile oil, to gram positive bacteria: staphylococcus aureus; Gram negative bacteria: escherichia coli, bacillus pyocyaneus; The fungus Candida albicans all has the effect that suppresses growth.Have important practice significance and using value.Minimal inhibitory concentration MIC scope is 0.005-50mg/ml.
Embodiment 3 Elsholtzia densa. volatile oil influenza first type virus activities
Test philosophy: with MDCK (Testis et Pentis Canis) cell is virus host, and working sample suppresses virus and causes cytopathy degree (CPE).
Test material and method:
1. Strain: influenza first type virus (15/90), influenza B virus (13/97) is cultivated at the chick embryo allantois intracavity and to be gone down to posterity (2008.11)-80 ℃ of preservations.
2. sample treatment: sample is dissolved in DMSO, and the reuse culture fluid is made into suitable initial concentration, does 3 times of dilutions with culture fluid, each 8 dilution factor.
3. positive control drug: virazole (RBV), Xinxiang pharmaceutcal corporation, Ltd (lot number 20080301).
4. method of testing: mdck cell is inoculated 96 well culture plates, puts 5%CO2, and 37C cultivated 24 hours.Mdck cell adds influenza first type virus (15/90) 10-5, influenza B virus (13/97) 1/210-2 respectively, and 37 ℃ of absorption hypsokinesis in 2 hours venom of preventing or cure a disease adds different dilution factor medicines respectively.If virus control and cell contrast, 37C cultivated 40 hours, observed result, and record CPE calculates each sample resisiting influenza virus half-inhibition concentration (IC50).
Test result:
Annotate:
(1) "-" expression sample does not have influenza first type virus activity at maximal non-toxic dosage in the table.
(2) TC
50: the poisonous concentration of medicine half; IC
50: medicine is to viral half-inhibition concentration; SI: selection index, SI=TC
50/ IC
50
Embodiment 4 Elsholtzia densa. volatile oil choline esterase inhibitions
1 material and reagent
Acetylcholinesterase derives from C57 strain normal male Mus, available from Beijing Military Medical Science Institute animal center.Acetylthiocholine iodide, tacrine, DTNB (the two nitrobenzoic acids of 5,5 '-two sulfur) be all available from Sigma company, 0.01M PH7.2-7.4PBS PBS (Beijing Suo Laibao biotechnology company), sodium lauryl sulphate (SDS, Sigma company); Galanthamine hydrobromide, other chemical reagent of donepezil hydrochloride (Chinese medicine biological products assay institute) are analytical pure (Beijing Chemical Plant).Herba Moslae volatile oil is according to the preparation of pharmacopeia volatile oil extraction method.
2 instrument and equipments
MQX200 type microwell plate scanning ELIASA (U.S. BiO-TEK company), ten thousand/electronic balance (FA-1104); KQ5200B type ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.); SSW type micro computer electric heating thermostatic water bath (Shanghai Boxun Industrial Co., Ltd.).
3 methods
3.1 the processing of cerebral tissue
Get the full brain of normal mouse, use glass homogenizer homogenate behind the PBS of 9 times of amounts of adding, 4 ℃ centrifugal, 10000r/min, 10min.Get supernatant ,-20 ℃ frozen subsequent use.
3.2 the foundation of the high-throughput screening method of acetylcholine esterase active
Adopt the Ellman method of improvement
[6]: add 32 μ L enzyme liquid, 160 μ L inhibitor (the matched group inhibitor is 1 ‰ DMSO) and 400 μ L developer DTNB (blank control group adds 400 μ LSDS cessation reactions after adding DTNB) in the reaction system; Moisturizing is to 1.2ml; Hatch 5min for 37 ℃; Add substrate A TCH (acetylthiocholine iodide) 37 ℃ and hatch 15min, add 400 μ L SDS cessation reactions, measure absorbance at the 412nm place with ELIASA.As positive control, and ask the IC50 value with tacrine.
ACHE inhibition (%)={ 1-(A
Sample-A
Blank)/(A
Contrast-A
Blank) * 100
4 results
Herba Moslae volatile oil is active suitable with the positive drug galanthamine hydrobromide to the inhibition of Acetylcholinesterase.
Through above practical implementation instance 1-4, broad-spectrum antiseptic, antiviral and acetylcholine esterase effect that Elsholtzia densa. volatile oil has are described, can in food, health product, cosmetics and the medicine of preparation bacteriostasis, use.Such as Elsholtzia densa. volatile oil staphylococcus aureus is had very strong inhibitory action, can be prepared as various forms of medicines, for clinical serious day by day antibiotics resistance plays an important role.Elsholtzia densa. volatile oil has stronger inhibitory action to Candida albicans, can be prepared as the health product and the medicine of various forms of preventions and treatment sexually transmitted disease (STD), has important practice significance and application prospect.
Claims (7)
1. Elsholtzia densa. extractive of volatile oil is characterized in that this volatile oil has antibiotic, antiviral and choline esterase inhibition.
2. Elsholtzia densa. extractive of volatile oil according to claim 1 is characterized in that this extract is to extract from the Elsholtzia densa. herb.
3. according to claim 1 and 2 described Elsholtzia densa. extracts, it is characterized in that it can being vapor distillation, also can be CO
2Supercritical extraction obtains.
4. Elsholtzia densa. volatile oil bacteriostasis according to claim 1 is characterized in that the bacterium that suppresses is gold-coloured staphylococci, bacillus pyocyaneus, escherichia coli, Candida albicans.
5. Elsholtzia densa. volatile oil antivirus action according to claim 1 is characterized in that influenza first type virus.
6. Elsholtzia densa. volatile oil according to claim 1 suppresses the acetylcholine esterase effect, it is characterized in that the cholinesterase activity that suppresses can be used for the treatment of alzheimer disease.
7. according to the described Elsholtzia densa. extractive of volatile oil of claim 1 to 6, it is characterized in that this extract can use in preparation has food, health product and the medicine of antibiotic, antiviral, the effect of treatment alzheimer disease.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103599178A (en) * | 2013-11-27 | 2014-02-26 | 王丽君 | Preparation method of bacteriostatic herba elsholtziae oil |
| CN113173928A (en) * | 2021-04-25 | 2021-07-27 | 武汉国粹医药科技有限公司 | Terpenoid, preparation method and application thereof, and antibacterial agent |
| CN113773907A (en) * | 2021-07-27 | 2021-12-10 | 吉林化工学院 | Preparation of elsholtzia volatile oil at different parts and detection of chemical components |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103599178A (en) * | 2013-11-27 | 2014-02-26 | 王丽君 | Preparation method of bacteriostatic herba elsholtziae oil |
| CN113173928A (en) * | 2021-04-25 | 2021-07-27 | 武汉国粹医药科技有限公司 | Terpenoid, preparation method and application thereof, and antibacterial agent |
| CN113173928B (en) * | 2021-04-25 | 2022-05-27 | 武汉国粹医药科技有限公司 | Terpenoid, preparation method and application thereof and antibacterial agent |
| CN113773907A (en) * | 2021-07-27 | 2021-12-10 | 吉林化工学院 | Preparation of elsholtzia volatile oil at different parts and detection of chemical components |
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Application publication date: 20120516 |