CN109662984B - Application of laggera pterodonta and rosemary extract in medicine for resisting type II dengue virus - Google Patents
Application of laggera pterodonta and rosemary extract in medicine for resisting type II dengue virus Download PDFInfo
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- CN109662984B CN109662984B CN201910116941.4A CN201910116941A CN109662984B CN 109662984 B CN109662984 B CN 109662984B CN 201910116941 A CN201910116941 A CN 201910116941A CN 109662984 B CN109662984 B CN 109662984B
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- pterodonta
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- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Abstract
The invention relates to application of laggera pterodonta and rosemary extracts in a medicine for resisting type II dengue virus. The pterodonta foetida extract, the rosemary extract and the composition thereof carry out in-vitro neutralization and proliferation inhibition detection on samples of the pterodonta foetida extract, the rosemary extract and the composition thereof through a series of experiments such as cytopathy, Real Time RT-PCR and the like, and the active ingredients of the pterodonta foetida extract, the rosemary extract and the composition thereof have the activity of resisting the II-type dengue virus, and the substances have the prospect of being developed into single-component and compound medicines for resisting the II-type dengue virus.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to application of laggera pterodonta and rosemary extracts in a medicine for resisting II-type dengue virus.
Technical Field
Laggera Pterodonta (DC.) Benth is a medicinal plant of Laggera of Compositae, is called herba leonuri, smelly leaves, Laggera Pterodonta and the like, is distributed in Yunnan, Sichuan, Tibet and other places, is mainly produced in Yunnan province in China, and has rich medicine sources. The experiences of using laggera pterodonta in Yunnan folk are largely collected in Yunnan Chinese herbal medicine, Kunming folk herbal medicine and Yunnan herbal medicine, and the like, and the medicine has good effects of resisting bacteria and diminishing inflammation, clearing heat and removing toxicity, and is widely used for treating diseases such as cold, sphagitis, bronchitis, malaria and the like. The compounds that have been identified from this genus of plants are mainly eudesmane-type sesquiterpenes and flavonols.
Rosemary has tranquilizing, refreshing and dyspepsia relieving effects. The medicinal liquor is smashed and soaked in boiled water for drinking for 2-3 times in 1 day, and can play a role in calming and inducing diuresis. It can also be used for treating insomnia, palpitation, headache, and dyspepsia. Can be used for treating trauma and arthritis by external application. It also has effects in strengthening heart, promoting metabolism, and promoting blood circulation of peripheral blood vessel.
The discovery of natural antiviral drugs from plants is an important approach for developing antiviral drugs. The laggera pterodonta medicinal material has abundant resources, is developed into antibacterial medicaments for treating sore throat and cough due to lung heat, and has no systematic basic research and application research on the virus effect. At present, no literature report of the application of the pterodonta and the rosemary extract in resisting the type II dengue virus exists.
Disclosure of Invention
The invention aims to provide application of laggera pterodonta and rosemary extracts in resisting type II dengue virus and an extraction method of the laggera pterodonta extracts.
The invention enriches and extracts the active pharmaceutical ingredients of laggera pterodonta and rosemary, cultures II type dengue virus in vitro, researches the antiviral action of the active pharmaceutical ingredients of laggera pterodonta, develops and utilizes the advantages and characteristics of Chinese traditional medicine, deeply researches the pharmacological mechanism of the active ingredients, further searches compounds with biological activity, and develops the application of the laggera pterodonta extract which has definite curative effect, little side effect, economy and effectiveness and can be widely applied to clinical resistance to the II type dengue virus. Also, a composition against type II dengue virus comprising the above ingredients is provided. The technical scheme of the invention is as follows:
the invention relates to an application of laggera pterodonta extract in a medicine for resisting II-type dengue virus.
The invention relates to application of rosemary extract and a composition thereof in medicaments for resisting II-type dengue virus.
The invention relates to a composition for resisting type II dengue virus, which comprises the following raw materials in parts by weight: 8-12 g of laggera pterodonta extract, 4-8 g of rosemary extract, 4-8 g of radix bupleuri and 4-8 g of forsythia.
Further, the active ingredients of the laggera pterodonta extract comprise the following compounds: 3, 4-di-O-caffeoylquinic acid, 3, 5-di-O-caffeoylquinic acid, 4 ', 5-dihydroxy-6, 7-dimethoxyflavone, 5, 6-dihydroxy-3, 4 ', 7-trimethoxyflavone, 4 ', 5-dihydroxy-3, 6, 7-trimethoxyflavone, 5-hydroxy-3, 3 ', 4 ', 6, 7-pentamethoxyflavone, 4 ', 5-dihydroxy-3, 3 ', 6, 7-tetramethoxyflavone, 3 ', 5-dihydroxy-3, 4 ', 6, 7-tetramethoxyflavone.
Further, the medicine is tablet, capsule, injection, aerosol, powder, pill, mixture, granule, medicinal granules, dripping pill, paste, oral liquid, or controlled release, sustained release, nanometer preparation.
The invention relates to a preparation method of laggera pterodonta extract, which comprises the following steps:
(1) adding an extracting agent into a laggera pterodonta medicinal material, and performing cold soaking or percolation, reflux heating and ultrasonic extraction to recover a solvent to obtain a primary extract;
(2) adding 2-3 times of water by weight of the primary extract into the primary extract, standing for 2-3 hours, concentrating the water solution under reduced pressure, adding 2-3 times of absolute ethyl alcohol by weight of the concentrate, and standing for 4-5 hours; repeatedly removing water insoluble substance with alcohol solution, or directly recovering solvent to dry to obtain laggera pterodonta extract;
or, loading the primary extract on a macroporous resin column or an active carbon column, washing with water, removing water-soluble impurities, eluting with 20-95% ethanol, collecting ethanol eluate, and recovering solvent to dry to obtain laggera pterodonta extract;
or passing the primary extract solution through polyamide column or sephadex column, eluting with water and 95% ethanol in sequence, collecting ethanol eluate, and recovering solvent to dry to obtain laggera pterodonta extract.
Further, in the step (1), the extractant is one of ethanol, methanol, acetone and ethyl acetate, and the dosage of the extractant is 10 times of the mass of the laggera pterodonta medicinal material.
As another embodiment, the preparation method of laggera pterodonta extract according to the present invention comprises the following steps of performing supercritical extraction on laggera pterodonta medicinal materials: the extraction pressure is 25Mpa, the extraction time is 3h, and the temperature is 45 ℃.
As another embodiment, the present invention relates to a method for preparing pterodonta foetida extract, comprising heating pterodonta foetida in water bath under conventional conditions, and extracting volatile oily liquid, i.e. volatile oil, with a volatile oil extractor.
Further, rosemary extracts include carnosic acid; the composition comprises acetyl carnosic acid, carnosic acid and carnosine. The rosemary extract is obtained by separating rosemary material by conventional method.
Compared with the prior art, the invention has the following beneficial effects:
the pterodonta foetida extract, the rosemary extract and the composition thereof carry out in-vitro neutralization and proliferation inhibition detection on samples of the pterodonta foetida extract, the rosemary extract and the composition thereof through a series of experiments such as cytopathy, Real Time RT-PCR and the like, and the active ingredients of the pterodonta foetida extract, the rosemary extract and the composition thereof have the activity of resisting the II-type dengue virus, and the substances have the prospect of being developed into single-component and compound medicines for resisting the II-type dengue virus.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. Those skilled in the art will recognize that the specific techniques or conditions, not specified in the examples, are according to the techniques or conditions described in the literature of the art or according to the product specification. The reagents or instruments used are conventional products which can be obtained by purchase, and are not indicated by manufacturers.
Example 1
In the composition for resisting the type II dengue virus, the laggera pterodonta extract is combined with bupleurum, forsythia, rosemary and medicinal excipients to form a medicinal composition. The weight parts of each component are as follows: 8 g of laggera pterodonta extract, 4 g of rosemary extract, 4 g of radix bupleuri, 4 g of fructus forsythiae and necessary medicinal excipient (the dosage can be prepared according to the prior art).
The method for extracting the laggera pterodonta extract comprises the following steps:
adding laggera pterodonta into one of ethanol, methanol, acetone or ethyl acetate which is 10 times of the laggera pterodonta medicinal material by weight, and performing cold soaking or percolation, reflux heating and ultrasonic extraction by using the conventional method to obtain an initial extract.
The primary extract is treated by the following method: adding water 2 times the weight of the primary extract, standing for 2 hr, filtering to remove insoluble chlorophyll and resin, concentrating the water solution under reduced pressure, adding 2 times the weight of anhydrous ethanol, standing for 4 hr, and filtering to remove ethanol insoluble substance; repeatedly removing water insoluble substance with alcohol solution, or directly recovering solvent to dry to obtain laggera pterodonta extract; or subjecting the primary extract to macroporous resin column or activated carbon column, washing with water, removing water soluble impurities, eluting with 20-95% ethanol, collecting ethanol eluate, and recovering solvent to dry to obtain laggera pterodonta extract; or passing the primary extract solution through polyamide column or sephadex column, eluting with water and 95% ethanol in sequence, collecting ethanol eluate, and recovering solvent to dry to obtain herba laggerae pterodonta extract. Extracting other materials with ethanol under reflux according to the ratio, and adding.
The active ingredients of the laggera pterodonta extract comprise the following compounds: 3, 4-di-O-caffeoylquinic acid, 3, 5-di-O-caffeoylquinic acid, 4 ', 5-dihydroxy-6, 7-dimethoxyflavone, 5, 6-dihydroxy-3, 4 ', 7-trimethoxyflavone, 4 ', 5-dihydroxy-3, 6, 7-trimethoxyflavone, 5-hydroxy-3, 3 ', 4 ', 6, 7-pentamethoxyflavone, 4 ', 5-dihydroxy-3, 3 ', 6, 7-tetramethoxyflavone, 3 ', 5-dihydroxy-3, 4 ', 6, 7-tetramethoxyflavone.
The above compound can be isolated by the following method:
separating the pterodonta foetida extract obtained by the method by using a silica gel column chromatography, and performing gradient elution by using petroleum ether-ethyl acetate (10-0: 1) to obtain a component of 1-70; fractions 10 were chromatographed on C18 column eluting with a methanol-0.1% formic acid gradient and the other fractions were separated on silica gel eluting with chloroform to give the above compounds.
Adding medicinal excipient into the above herba laggerae extract according to the prior art method, and making into tablet, capsule, injection, aerosol, powder, pill, mixture, granule, dripping pill, unguent, oral liquid, or controlled release, sustained release, or nanometer preparation.
The rosemary extract is obtained by separating rosemary material by conventional method. The rosemary A61 extract included carnosic acid, the carnosic acid content being 20%.
Example 2
In the composition for resisting the type II dengue virus, the laggera pterodonta extract is combined with bupleurum, forsythia, rosemary and medicinal excipients to form a medicinal composition. The weight parts of each component are as follows: 12 g of laggera pterodonta extract, 8 g of rosemary extract, 6 g of bupleurum, 6 g of forsythia and necessary medicinal excipient (the dosage can be prepared according to the prior art).
In the embodiment, the laggera pterodonta medicinal material is extracted by a supercritical method according to the following conditions: the extraction pressure is 25Mpa, the extraction time is 3h, and the temperature is 45 ℃.
Example 3
In the composition for resisting the type II dengue virus, the laggera pterodonta extract is combined with bupleurum, forsythia, rosemary and medicinal excipients to form a medicinal composition. The weight parts of each component are as follows: 10 g of laggera pterodonta extract, 6 g of rosemary extract, 8 g of bupleurum, 5 g of forsythia and necessary medicinal excipient (the dosage can be prepared according to the prior art). The rest is the same as in example 1.
Example 4
In the embodiment, after the laggera pterodonta medicinal material is heated in a water bath under the conventional condition, a volatile oily liquid is obtained by extracting with a volatile oil extractor, namely the volatile oil.
Example 5
In this example, the composition of rosemary extract includes acetyl carnosic acid, carnosic acid and carnosine. The synthesis method of acetyl carnosic acid, carnosic acid and carnosol comprises the following steps:
A. synthesis of acetyl carnosic acid
The formula of acetyl carnosic acid is as follows:
dissolving crude carnosic acid in anhydrous pyridine solution, adding 12.0eq acetic anhydride, stirring under nitrogen protection for 20h in a wet and dark place, performing TLC point plate tracking until carnosic acid raw material point disappears, stopping stirring, pouring the reaction solution into ice bath distilled water (50mL), stirring for 30min, extracting with ethyl acetate (3X 50mL), and extracting the organic layer with 5% HCl solution (3X 50mL) and saturated NaHCO respectively 3 The solution (3X 50mL), saturated NaCl solution (3X 50 mL). Using anhydrous Na for organic house 2 SO 4 Drying for 1h, filtering, concentrating at 45 deg.C under reduced pressure and vacuum to obtain oily substance, and recrystallizing with petroleum ether and ethyl acetate to obtain white needle crystal.
B. Synthesis of carnoside
The structural formula of the carnoside is as follows:
acetyl extractionCarnosic acid was dissolved in anhydrous THF and 19.0eq of SOCl was slowly added dropwise with stirring 2 Slowly heating the reaction temperature from room temperature to 65 ℃ under the protection of nitrogen, refluxing for 10h, tracking by a TLC point plate, and stopping stirring until the acetyl carnosic acid raw material point disappears. Concentrating the reaction liquid to be dry in a vacuum decompression device, adding anhydrous THF in ice bath to dissolve the reaction liquid, and introducing NH into the reaction liquid under the protection of nitrogen 3 After reaction for 3h, stirring was stopped, the reaction mixture was poured into 50mL ice-bath distilled water, stirred for 10min, extracted with ethyl acetate (3X 50mL), and anhydrous Na was added to the organic layer 2 SO 4 Drying for 1h, filtering, concentrating at 45 deg.C under reduced pressure and vacuum to obtain oily substance, and recrystallizing with petroleum ether and ethyl acetate to obtain white powdery solid.
C. Synthesis of cyanogen sage
The structural formula of the carnosic acid is as follows:
dissolving carnoside in anhydrous DCM, slowly adding 30.0eq of SOCl dropwise with stirring 2 The mixture is stirred and reacted for 5 hours at room temperature, and the stirring is stopped after TLC point plate tracking till the point of the raw material of the carnoside disappears. 10mL of distilled water was added to the reaction flask, the mixture was stirred for 10min, the mixture was extracted with DCM (3X 25mL), and the organic layer was placed over anhydrous Na 2 SO 4 Drying for 1h, filtering, concentrating at 45 deg.C under reduced pressure and vacuum to obtain oily substance, and recrystallizing with petroleum ether and ethyl acetate to obtain white needle crystal.
D. Synthesis of carnosic acid
The structural formula of carnosol is as follows:
dissolving acetyl carnosic acid in anhydrous THF, slowly adding 30.0eq SOCl dropwise under stirring 2 Slowly heating the reaction temperature from room temperature to 65 ℃ under the protection of nitrogen, refluxing for 10h, and tracking by TLC (thin layer chromatography) spot plate until the acetyl carnosic acid raw material spot disappearsThe stirring was stopped. Concentrating the reaction liquid to dryness in a vacuum decompression device, adding anhydrous THF in ice bath to dissolve the reaction liquid, and introducing CH into the reaction liquid under the protection of nitrogen 3 NH 2 After 4h of reaction, stirring was stopped, the reaction mixture was poured into 50mL of ice-bath distilled water, stirred for 10min, extracted with ethyl acetate (3X 50mL), and the organic layer was added with anhydrous Na 2 SO 4 Drying for 1h, filtering, concentrating at 45 deg.C under reduced pressure and vacuum to obtain oily substance, and gradient eluting with petroleum ether/ethyl acetate by silica gel column chromatography to obtain white powdery solid.
The following are experimental studies of laggera pterodonta extract, rosemary extract, and compositions thereof against type ii dengue virus.
Separation culture and titer determination of I and II type dengue viruses
1. Culture of C6/36 cells:
RPMI-1640 medium and MEM medium were mixed in a 1:2 mixing, adding 10% fetal calf serum (SIJIQING, ZHEJIANGTANGHANGZHUANGWU, Biotech Co., Ltd.), and adding penicillin and streptomycin conventionally. The culture condition is 28 +/-2 ℃ water-proof constant temperature incubator, and the growth condition of the cells is regularly observed.
2. Amplification of type ii dengue virus standard:
1) preparing virus adsorption solution: the RPMI-1640 medium and the MEM medium were mixed at a ratio of 1:2 to prepare the desired amount.
2) Removing culture medium from C6/36 cells grown to a monolayer, washing the cell surface with PBS 3 times, adding virus adsorption solution containing type II dengue virus standard strain, placing at 37 deg.C for adsorption for 1.5h, and shaking 1 time every 20 minutes to make the virus solution act on the cell surface uniformly.
3) Preparing a maintenance liquid: RPMI-1640 medium and MEM medium were mixed at a ratio of 1:2, and 5% fetal calf serum (SIJIQING, Zhejiang Hangzhou Biotechnology Ltd.) was added with penicillin and streptomycin.
4) The adsorbed culture flasks were supplemented with a maintenance solution, cultured at 37 ℃ and harvested when the cytopathic effect (CPE) reached 75% or more. The supernatant was collected by centrifugation at 4000rpm for 10min and dispensed into 1.5mL centrifuge tubes and stored at-70 ℃.
3. And (3) determining the titer of the type II dengue virus standard strain:
1) virus gradient dilution:
adding 100 μ l virus solution into EP tube, adding 900 μ l culture medium to 1mL, and mixing to obtain 10% -1 Diluting; get 10 -1 Adding 100 μ l of diluted virus solution into new EP tube, adding 900 μ l of culture medium, and mixing to obtain 10 -2 Diluting; get 10 -2 Adding 100 μ l of diluted virus solution into new EP tube, and adding 900 μ l of culture medium to obtain 10 -3 Dilution of 10 in this order -11 。
2) The virus solution was added in a gradient to a 96-well plate at 100. mu.l/well with a blank BHK-21 cell control (NC).
TABLE 1 dengue virus type II Standard Strain titre assay
3) Calculating the virus titer: after 12 days, the 96-well plate was removed and the number of lesion wells was counted. 8 wells are completely diseased under 10-1 to 10-4 dilution, 2 wells are diseased under 10-5 dilution, and no disease is found under 10-6 dilution or the following dilution.
The titer of this batch of type II dengue virus standard strain was 5.6X 10 as calculated by the Karber method 5 TCID 50/mL. TCID 50: 50% of the infected tissue cells.
Secondly, processing traditional Chinese medicine samples
1. Twelve traditional Chinese medicine samples are provided, provided by Kunming medical university, and are dissolved in 20 microliter of acetone for experiment and diluted into different working concentrations by cell maintenance solution, and the positive control medicine is ribavirin injection (specification: 1 mL: 100mg, manufacturer: Henan Tian Fangyao pharmaceutical Co., Ltd., batch No. 120621650), and Tamiflu capsules (75 mg/capsule, manufacturer: Basel Haofume-Ross Co., Ltd., production batch No. B1364). Twelve traditional Chinese medicine samples were as follows:
a: carnosic acid (commercially available product). B: acetyl carnosic acid (synthesized as in the examples). C: carnosic acid (synthesized as described in the examples). D: carnosidine (synthesized as described in the examples). E: laggera pterodonta volatile oil (prepared according to the examples). F: laggera pterodonta extract fraction 10 (prepared according to the examples) was isolated on silica gel. G: laggera pterodonta extract silica gel the fraction of stage 70 (prepared according to the examples). H: pterodonta supercritical extract (prepared according to the method of the example). I: ilicic acid (commercially available product). J: laggerac acid (a commercially available product). K: rosemary a61 extract (containing 20% carnosic acid) (prepared by the example method). L: pterodontolis glycol (commercially available product).
The above medicines include herba Laggera extract, herba Laggera product, herba Rosmarini officinalis extract and their composition.
2. Dissolving the traditional Chinese medicines:
scraping appropriate amount of Chinese medicinal sample into EP tube with dead weight removed, weighing, adding acetone or maintenance solution for dissolving, and diluting with maintenance solution to initial mother liquor concentration, as shown in Table 2.
TABLE 2 drug dissolution table
3. Dilution of pharmaceutical products
The 14 drugs were diluted to two concentrations of 50. mu.g/ml and 100. mu.g/ml with maintenance solution and the required amounts were prepared in the proportions shown in Table 3.
TABLE 3 dilution of drugs
Thirdly, coarse screening of traditional Chinese medicine with neutralizing and killing effect and proliferation inhibiting effect on II-type dengue virus
1. The traditional Chinese medicine has the effects of neutralizing and killing the II type dengue virus:
the experimental process comprises the following steps:
1) the culture medium in the cell culture flask was removed, and fresh C6/36 cell culture medium was added to prepare a cell suspension, which was inoculated into a 96-well cell culture plate at 200. mu.l/well. The sealing film is wound and sealed, and the mixture is cultured in an incubator at 28 ℃.
2) After the cells in the 96-well plate grow to form a single layer, the culture solution is discarded, and the maintenance solution is added.
3) Diluting the medicine: diluting 14 drugs with maintenance solution to two concentrations of 50 μ g/ml and 100 μ g/ml
4) Mixing 10 μ l of 50 μ g/ml and 100 μ g/ml medicinal liquid with 3 μ l of virus liquid for 1min, inoculating 96-well plate with full monolayer C6/36 cells, setting virus control, normal cell control, drug control without virus, acetone control and acetone control without virus, setting three multiple wells for each sample, adding maintenance liquid to total system 200 μ l, and making final drug concentration at 2.5 μ g/ml and 5 μ g/ml. Sealing the plate with sealing film at 37 deg.C and 5% CO 2 Incubate in incubator, observe under microscope every day and record the degree of cytopathic effect (CPE). CPE records were according to the general standard: -is a cell-free lesion; + is lesion in 25% of cells; + is 50% of cells with lesions; + + + is 75% cells with lesions; + + + + + is 100% of cells with lesions.
As a result: in the neutralization experiment, the medicine without virus (the final concentration is 5 mug/ml) has no obvious influence on the growth of C6/36 cells (see table 4), which shows that the medicine per se has no toxic and side effect on the cells at the concentration; within 48 hours after inoculation, all the medicaments with the final concentration of 2.5 mu g/ml and 5 mu g/ml have certain virus neutralization effect; at 72 hours post-inoculation, drugs A (2014122501), B (2014122502), E (LPB-01), J (DEOA) and K (MDXA61) neutralized DENV-II at a final concentration of 2.5. mu.g/ml, and drugs A (2014122501), B (2014122502), E (LPB-01), F (LPB-10) and G (LPB-70) neutralized DENV-II at a final concentration of 5. mu.g/ml (see Table 5).
TABLE 4 drug control lesion results without added virus
TABLE 5 neutralization of experimental cytopathic results
2. The traditional Chinese medicine has the inhibition effect on the proliferation of the type II dengue virus:
the experimental process comprises the following steps:
1) after the cells in the 96-well plate overgrow with a monolayer, the supernatant was discarded, 3. mu.l of virus solution was inoculated, and the solution was adsorbed at 37 ℃ for 2 hours.
2) Changing different concentrations of the drug-containing maintenance solution to make the final concentration of the drug be 2.5 μ g/ml and 5 μ g/ml, setting the total system at 200 μ l, setting virus control, normal cell control, acetone control, repeating the above three holes for each sample, sealing with sealing film, standing at 37 deg.C, and 5% CO 2 The cells were incubated, microscopically observed day by day and the cytopathic effect (CPE) was recorded, see table 6.
As a result: in the growth inhibition test, when drugs D (2014122505), F (LPB-10), G (LPB-70), H (LPB-SFE), I (LSL-Q), Daffy and ribavirin with a final concentration of 5 mu G/ml are used 24H after inoculation, the proliferation of DENV-II is inhibited.
At 48H after inoculation, when the final concentration of the medicine is 2.5 mu G/ml, the medicines D (2014122505), G (LPB-70), H (LPB-SFE), I (LSL-Q), J (DEOA), L (PDDO), Daphne and ribavirin have the inhibition effect on the proliferation of DENV-II; when the final concentration of the medicine is 5 mug/ml, the medicines A (2014122501), B (2014122502), F (LPB-10), G (LPB-70), I (LSL-Q), J (DEOA), Daphne and ribavirin have the inhibition effect on the proliferation of DENV-II,
at 72h after inoculation, when the final concentration of the medicine is 2.5 mu g/ml, the medicines J (DEOA), L (PDDO) and ribavirin have certain inhibition effect on the proliferation of DENV-II; when the final concentration of the drugs is 5 mu g/ml, the drugs A (2014122501), B (2014122502), E (LPB-01), F (LPB-10) and J (DEOA) have an inhibition effect on the proliferation of DENV-II, and the cytopathic effect of other drug groups has no significant difference from a virus positive control, which indicates that other drugs have no inhibition effect on the proliferation of DENV-II.
TABLE 6 proliferation inhibition cytopathic results
3. Coarse screening of traditional Chinese medicine samples and small results:
1) the medicine without the addition of the type II dengue virus (the final concentration is 5 mu g/ml) has no obvious influence on the growth of the C6/36 cells, and the cells have no pathological changes, which shows that the medicine per se has no toxic and side effects on the C6/36 cells under the concentration.
2) Neutralization and killing effects of the medicine on the type II dengue virus: within 48 hours after the inoculation, all the drug experimental groups with final concentrations of 2.5 mu g/ml and 5 mu g/ml are used, the degree of C6/36 cytopathic effect is lower than that of the virus control group, and all the drugs with final concentrations of 2.5 mu g/ml and 5 mu g/ml have the effect of neutralizing the virus; at 72 hours post-inoculation, virus positive control wells were completely diseased with drugs A (2014122501), B (2014122502), E (LPB-01), J (DEOA), and K (MDXA61) at a final concentration of 2.5 μ G/ml, with C6/36 cytopathic levels lower than those of the virus control group, indicating that they have a neutralizing effect on the virus, and with drugs A (2014122501), B (2014122502), E (LPB-01), F (LPB-10), and G (LPB-70) at a final concentration of 5 μ G/ml, with C6/36 cytopathic levels lower than those of the virus control group, indicating that they have a neutralizing effect on the virus.
3) Proliferation inhibition of type ii dengue virus by drugs: in the growth inhibition test, drugs D (2014122505), F (LPB-10), G (LPB-70), H (LPB-SFE), I (LSL-Q), Daffy and ribavirin with the final concentration of 5 mu G/ml are used 24H after inoculation, and the degree of C6/36 cytopathic effect of the drugs D (2014122505), F (LPB-SFE), I (LSL-Q), Daffy and ribavirin is lower than that of a virus control group, which indicates that the drugs D (2014122505), F (LPB-10), G (LPB-70), H (LPB-SFE), I (LSL-Q), Daffy and ribavirin have the effect of inhibiting the virus proliferation.
At 48H after inoculation, when the final concentration of the medicines is 2.5 mu G/ml, the C6/36 cytopathic degree of the experimental group using the medicines D (2014122505), G (LPB-70), H (LPB-SFE), I (LSL-Q), J (DEOA), L (PDDO), Daphne and ribavirin is lower than that of the virus control group, which indicates that the medicines have the inhibiting effect on the proliferation of the virus; when the final concentration of the drugs is 5 mu G/ml, the C6/36 cytopathic degree of the experimental group using the drugs A (2014122501), B (2014122502), F (LPB-10), G (LPB-70), I (LSL-Q), J (DEOA), Duffy and ribavirin is lower than that of the virus control group, which indicates that the drugs have an inhibiting effect on the proliferation of viruses.
At 72h after inoculation, the virus positive control holes are completely diseased, when the final concentration of the medicine is 2.5 mu g/ml, the degree of C6/36 cytopathy of the experimental group using the medicines J (DEOA), L and ribavirin is lower than that of the virus control group, which indicates that the experimental group has an inhibiting effect on the proliferation of the virus; when the final concentration of the drugs is 5 mu g/ml, the C6/36 cytopathic degree of the experimental group using the drugs A (2014122501), B (2014122502), E (LPB-01), F (LPB-10) and J (DEOA) is lower than that of the virus control group, which indicates that the drugs have an inhibiting effect on the proliferation of viruses.
Fourth, research of neutralization and killing effect and proliferation inhibition effect of Chinese medicine on type II dengue virus
1. Collection of type II dengue Virus supernatant
The operation flow of the neutralization experiment and the proliferation inhibition experiment is the same as the above, the initial concentration of the medicine is 100 mug/ml, the acetone control with the addition of virus, the virus positive control and the normal cell control are arranged, each sample is respectively provided with 3 multiple holes, the cytopathic degree is observed under a microscope day by day, the supernatant is collected every 24 hours, 4000rpm is carried out, centrifugation is carried out for 8min, and the supernatant is lightly sucked and frozen at-70 ℃.
2. Extraction of RNA from type II dengue virus
Viral RNA extraction kit from Qiagen (Qiagen, Inc.)
Viral RNA Mini Kit), the procedure was as follows:
1) mu.l AVE and 310. mu.g Carrier RNA were mixed well and dispensed into PCR tubes at 17. mu.l/tube and stored at-20 ℃ (repeated freeze-thaw was avoided as much as possible <5 times).
2) [ 560. mu.l AVL + 5.6. mu.l (AVE + Carrier RNA) ]/each sample, mixed well and ready for use.
3) [ 560. mu.l AVL + 5.6. mu.l (AVE + Carrier RNA) ] + 140. mu.l dengue virus solution, mixed and shaken for 15 s.
4) Incubate at room temperature for 10 min.
5) And (5) carrying out short-time centrifugation to remove water drops remained on the wall of the centrifugal tube.
6) Add 560. mu.l ethanol and mix well by shaking for 15 s.
7) And (4) removing liquid remained on the wall of the centrifugal pipe by short-time centrifugation.
8) And taking 630 mu l of sample from the mixed solution in the previous step, adding the sample into a dry column (a 2mL centrifuge tube), centrifuging for 1min at 6000-8000 g, and transferring the column into a new centrifuge tube.
9) Repeat step 8).
10) Add 500. mu.l AW1, 6000g and centrifuge for 1min, transferring the column to a new centrifuge tube.
11) Add 500. mu.l AW2, centrifuge for 3min at 14000g, transfer the column to a fresh centrifuge tube, centrifuge for 2min at 14000 g.
12) The column was placed in a 1.5mL EP tube, 60. mu.l RNase-free water was added, allowed to stand at room temperature for 5min, and centrifuged at 6000g for 1min at-70 ℃.
3. Relative quantification of dengue II RNA in neutralization and proliferation inhibition assays by Real time PCR
The experimental process comprises the following steps:
1) reverse transcription of viral RNA into cDNA
Reverse transcription was performed using the RevertAID First Strand cDNA Synthesis Kit from Fermentas, Inc., and the reverse transcription system is shown in Table 6.
Mu.l of RNA, 1. mu.l of gDNase and 2. mu.l of 5 XgDNA buffer are mixed uniformly, heated at 42 ℃ for 4min, heated at 85 ℃ for 5s, mixed uniformly with a mixture of (1. mu.l of RT Enzyme, 1. mu.l of primer mix, 4. mu.l of 5 Xreaction buffer, 4. mu.l of RNase Free water), heated at 37 ℃ for 40min and heated at 4 ℃ for 20min to obtain the required cDNA.
TABLE 6 RT reaction systems (20. mu.l)
2) Relative quantification of initial template amount by Real time PCR
Real time PCR primers: forward primer: GCAGGGATACTGAAGAGATGGG and Reverse primer: TGGTTCTCCGTTACGTGTGG, and the target fragment is 186bp in length.
Firstly, calculating the number of the required subsystems in the experiment, and specifically preparing the system according to the proportion. The system is divided into a plurality of parts with loss, generally 0.5 to 1 part of the system is added, and the system is prepared as shown in Table 7.
TABLE 7 qPCR System formulation
② mixing evenly, adding 1 mul cDNA template, subpackaging to eight-connected calandria according to the plate arrangement mode, the total is 20 mul/tube.
After the sample is added, the cover of the eight-row tubes is covered, and the sequence is marked on the edge of the uppermost edge of the eight-row tubes (the mark cannot be written on the cover of the reaction tube, the transparent fluorescence acquisition area in the middle of the tube is prevented from being touched by a naked hand, each hole is ensured to be tightly covered, and otherwise, the repeatability or the possibility of melting curve peak drift is influenced).
Thirdly, each eight-connected pipe is placed on an eight-connected pipe centrifugal machine for centrifuging for a moment, and liquid is prevented from remaining on the pipe wall.
Fourthly, Real time PCR reaction process:
a:95.0℃for 3:00
b:95.0℃for 0:10
c:55.0℃for 0:20
+Plate Read
d:72.0℃for 0:30
Plate Read
e:GOTO b，39more times
f:Melt Curve：65.0℃to 95.0℃：Increment 0.5℃for 0:05
+Plate Read
real time PCR plate description: since the variety of the drugs used in the study is large, the drugs involve a neutralization killing test and a proliferation inhibition test, the samples are sampled at 3 time points (24h, 48h and 72h), 3 multiple wells are required to be respectively arranged when the samples are subjected to qPCR, and the detection of all the samples cannot be completed in 1 96-well PCR instrument at one time, so that the drug experiments are arranged in 4 plates: drugs B, C, D and E row one, F, G, H and acetone row one, A, J, K and L row one, and I, Duffy and ribavirin row one, all provided DENV-II positive control wells.
4. Real Time PCR results
1) Real Time PCR experimental data statistics
The Ct value for each sample is automatically output by the instrument.
Calculating the delta Ct value of the sample: delta Ct-mean Ct value of drug treated samples-mean Ct value of virus positive control samples.
Proliferation ratio of DENV-II in C6/36 cells in drug-treated versus virus-positive control: sample/control group 2 -△Ct When the drug inhibits the amplification of DENV-II (1-2) -△Ct )×100％。
The specific data statistics are as follows:
(ii) neutralization assay Real Time PCR data and calculations, see Table 8.
TABLE 8 neutralization assay Real Time PCR data and calculation
② proliferation inhibition test Real Time PCR data and calculation, see table 9.
TABLE 9 proliferation inhibition assay Real Time PCR data and calculation
2) Summary of Real Time PCR detection results
For the neutralization test:
through Real Time PCR detection, after the medicine is taken for 24 hours, all the medicines do not generate obvious inhibition effect on the type II dengue virus; after 48 hours of administration, the cells using drugs A (2014122501), B (2014122502), G (LPB-70), I (LSL-Q), J (DEOA), K (MDXA61), L (PDDO), Duffy and ribavirin all showed a decrease in virus expansion rate of 60.6%, 95.0%, 54.0%, 73.6%, 70.1%, 71.6%, 53.2%, 88.3% and 76.7% respectively compared with the positive control cells; after 72H, the drugs B (2014122502), F (LPB-10), G (LPB-70) and H were administered
The virus amplification of the (LPB-SEE) and the I (LSL-Q) cells is reduced compared with that of the positive control cells, and the amplification rates are respectively reduced by 66.3%, 67.3%, 74.5%, 50.2% and 61.8%.
②, for the proliferation inhibition test:
the dengue virus type II solution used was 3. mu.l, the final drug concentration was 5. mu.g/ml. Through Real Time PCR detection, after the medicine is taken for 24 hours, the virus amplification amount of the cells using the medicine D (2014122505), the tamiflu and the ribavirin is reduced compared with that of positive control cells, and the amplification rates are respectively reduced by 52.38%, 78.9% and 67.6%; after 48 hours of administration, the virus amplification amount of the cells using the medicines B (2014122502), E (LPB-01), I (LSL-Q), Duffy and ribavirin is reduced compared with that of positive control cells, and the amplification rates are respectively reduced by 52.1%, 56.1%, 53.2%, 73.9% and 72.9%; after 72h of administration, the rate of viral expansion was 54.5% less in cells using drug B (2014122502) than in positive control cells.
Fifthly, experimental results:
12 traditional Chinese medicine samples, and the neutralization and proliferation inhibition effects of the tamiflu and the ribavirin on the II-type dengue virus are detected in vitro through a series of experiments of cytopathic effect and Real Time RT-PCR.
The results of coarse screening of traditional Chinese medicine samples obtained by observing the pathological condition of C6/36 cells under a microscope show that:
1) when the final concentration of the medicine is 5 mu g/ml, the medicine without adding the type II dengue virus has no obvious influence on the growth of C6/36 cells, and the cells have no pathological changes, which shows that the medicine has no toxic or side effect on C6/36 cells under the concentration.
2) Neutralization and killing effects of the medicine on the type II dengue virus: within 48 hours after the inoculation, all the drug experimental groups with final concentrations of 2.5 mu g/ml and 5 mu g/ml are used, the degree of C6/36 cytopathic effect is lower than that of the virus control group, and all the drugs with final concentrations of 2.5 mu g/ml and 5 mu g/ml have the effect of neutralizing the virus; at 72 hours post-inoculation, virus positive control wells were completely diseased with drugs A (2014122501), B (2014122502), E (LPB-01), J (DEOA), and K (MDXA61) at a final concentration of 2.5 μ G/ml, with C6/36 cytopathic levels lower than those of the virus control group, indicating that they have a neutralizing effect on the virus, and with drugs A (2014122501), B (2014122502), E (LPB-01), F (LPB-10), and G (LPB-70) at a final concentration of 5 μ G/ml, with C6/36 cytopathic levels lower than those of the virus control group, indicating that they have a neutralizing effect on the virus.
3) Proliferation inhibition of the drug against type ii dengue virus: in the growth inhibition test, drugs D (2014122505), F (LPB-10), G (LPB-70), H (LPB-SFE), I (LSL-Q), Daffy and ribavirin with the final concentration of 5 mu G/ml are used 24H after inoculation, and the degree of C6/36 cytopathic effect of the drugs D (2014122505), F (LPB-SFE), I (LSL-Q), Daffy and ribavirin is lower than that of a virus control group, which indicates that the drugs D (2014122505), F (LPB-10), G (LPB-70), H (LPB-SFE), I (LSL-Q), Daffy and ribavirin have the effect of inhibiting the virus proliferation.
At 48H after inoculation, when the final concentration of the medicines is 2.5 mu G/ml, the experimental group using the medicines D (2014122505), G (LPB-70), H (LPB-SFE), I (LSL-Q), J (DEOA), L (PDDO), Daphne and ribavirin has lower C6/36 cytopathic degree than the virus control group, which indicates that the medicines have the effect of inhibiting the proliferation of viruses; when the final concentration of the drugs is 5 mu G/ml, the C6/36 cytopathic degree of the experimental group using the drugs A (2014122501), B (2014122502), F (LPB-10), G (LPB-70), I (LSL-Q), J (DEOA), Duffy and ribavirin is lower than that of the virus control group, which indicates that the drugs have an inhibiting effect on the proliferation of viruses.
At 72h after inoculation, the virus positive control holes are completely diseased, when the final concentration of the medicine is 2.5 mu g/ml, the experimental group using the medicines J (DEOA), L and ribavirin has lower C6/36 cytopathic degree than the virus control group, which indicates that the medicines have an inhibiting effect on the proliferation of the virus; when the final concentration of the drugs is 5 mu g/ml, the C6/36 cytopathic degree of the experimental group using the drugs A (2014122501), B (2014122502), E (LPB-01), F (LPB-10) and J (DEOA) is lower than that of the virus control group, which indicates that the drugs have an inhibiting effect on the proliferation of viruses.
Further extracting type II dengue virus RNA in each group of experimental supernatants, performing Real Time RT-PCR with type II dengue virus specific primers to compare the initial template amount of each sample, and the results show that:
1) for the neutralization test: after the medicine is taken for 24 hours, all the medicines have no obvious inhibition effect on the type II dengue virus; after 48 hours of administration, the cells using drugs A (2014122501), B (2014122502), G (LPB-70), I (LSL-Q), J (DEOA), K (MDXA61), L (PDDO), Duffy and ribavirin all showed a decrease in virus expansion rate of 60.6%, 95.0%, 54.0%, 73.6%, 70.1%, 71.6%, 53.2%, 88.3% and 76.7% respectively compared with the positive control cells; after 72 hours of administration, the cells using drugs B (2014122502), F (LPB-10), G (LPB-70), H (LPB-SEE) and I (LSL-Q) all showed a decrease in virus amplification as compared to the positive control cells, with 66.3%, 67.3%, 74.5%, 50.2% and 61.8% reduction in amplification rate, respectively.
2) For the proliferation inhibition assay: the dengue virus type II solution used was 3. mu.l, the final drug concentration was 5. mu.g/ml. After the medicine is taken for 24 hours, the virus amplification amount of the cells using the medicine D (2014122505), the tamiflu and the ribavirin is reduced compared with that of positive control cells, and the amplification rates are respectively reduced by 52.38%, 78.9% and 67.6%; after 48 hours of administration, the virus amplification of the cells using the medicines B (2014122502), E (LPB-01), I (LSL-Q), Duffy and ribavirin is reduced compared with that of positive control cells, and the amplification rates are respectively reduced by 52.1%, 56.1%, 53.2%, 73.9% and 72.9%; after 72 hours of administration, the virus amplification rate of the cells using the drug B (2014122502) is reduced by 54.5 percent compared with that of positive control cells, which shows that the cells have certain effects of resisting the II type dengue virus and protecting the cells in vitro.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (6)
1. The application of the laggera pterodonta extract in preparing the anti-II type dengue virus medicine is characterized in that: the preparation method of the laggera pterodonta extract comprises the following steps:
(1) adding an extracting agent into a laggera pterodonta medicinal material, and performing cold soaking or percolation, reflux heating and ultrasonic extraction to recover a solvent to obtain a primary extract; the extractant is one of ethanol, methanol, acetone and ethyl acetate, and the dosage of the extractant is 10 times of the mass of the laggera pterodonta medicinal material;
(2) adding 2-3 times of water by weight of the primary extract into the primary extract, standing for 2-3 hours, concentrating the water solution under reduced pressure, adding 2-3 times of absolute ethyl alcohol by weight of the concentrate, and standing for 4-5 hours; repeatedly removing water insoluble substance with the alcoholic solution, or directly recovering solvent to dry to obtain Laggera pterodonta extract;
or, washing the primary extract with macroporous resin column or activated carbon column, removing water soluble impurities, eluting with 20% -95% ethanol, collecting ethanol eluate, and recovering solvent to dry to obtain laggera pterodonta extract;
or passing the primary extract solution through polyamide column or sephadex column, eluting with water and 95% ethanol in sequence, collecting ethanol eluate, and recovering solvent to dry to obtain laggera pterodonta extract.
2. The application of the laggera pterodonta extract in preparing the medicine for resisting the II-type dengue virus is characterized in that: the pterodonta foetida medicinal material is extracted by a supercritical method according to the following conditions: the extraction pressure is 25Mpa, the extraction time is 3h, and the temperature is 45 ℃.
3. The application of the laggera pterodonta extract in preparing the medicine for resisting the II-type dengue virus is characterized in that: heating the laggera pterodonta medicinal material in water bath under conventional conditions, and extracting volatile oily liquid by using a volatile oil extractor to obtain volatile oil serving as a laggera pterodonta extract.
4. A composition for use against dengue type ii virus, comprising: the traditional Chinese medicine composition is prepared from the following raw material medicines in parts by weight:
8-12 g of laggera pterodonta extract, 4-8 g of rosemary extract or composition, 4-8 g of bupleurum and 4-8 g of forsythia;
the laggera pterodonta extract is prepared by the preparation method in the application of any one of claims 1 to 3, and the active ingredients comprise the following compounds: 3, 4-di-O-caffeoylquinic acid, 3, 5-di-O-caffeoylquinic acid, 4 ', 5-dihydroxy-6, 7-dimethoxyflavone, 5, 6-dihydroxy-3, 4 ', 7-trimethoxyflavone, 4 ', 5-dihydroxy-3, 6, 7-trimethoxyflavone, 5-hydroxy-3, 3 ', 4 ', 6, 7-pentamethoxyflavone, 4 ', 5-dihydroxy-3, 3 ', 6, 7-tetramethoxyflavone, 3 ', 5-dihydroxy-3, 4 ', 6, 7-tetramethoxyflavone; the rosemary extract is obtained by separating rosemary medicinal materials by a conventional method; the rosemary composition is acetyl carnosic acid, carnosic acid and carnosic acid methylamine;
the synthesis method of acetyl carnosic acid, carnosic acid and carnosol comprises the following steps:
A. synthesis of acetyl carnosic acid
Dissolving crude carnosic acid in anhydrous pyridine solution, adding 12.0eq acetic anhydride, stirring under nitrogen protection for 20h in a wet and dark place, tracking by TLC point plate until carnosic acid raw material point disappears, stopping stirring, pouring the reaction solution into ice bath distilled water, stirring for 30min, extracting with ethyl acetate, and respectively using 5% HCl solution and saturated NaHCO solution as organic layers 3 Washing the solution and a saturated NaCl solution; using anhydrous Na for organic house 2 SO 4 Drying for 1h, filtering, concentrating at 45 deg.C under reduced pressure in vacuum device to obtain oily substance, and recrystallizing with petroleum ether and ethyl acetate to obtain white needle crystal;
B. synthesis of cyanogen of salvia officinalis
Dissolving carnoside in anhydrous DCM, slowly adding 30.0eq of SOCl2 dropwise with stirring, reacting for 5h with stirring at room temperature, performing TLC spot plate tracking until the carnoside raw material spot disappears, and stopping stirring; adding 10mL distilled water into the reaction flask, stirring for 10min, extracting with DCM, adding anhydrous Na into the organic layer 2 SO 4 Drying for 1h, filtering, concentrating at 45 deg.C under reduced pressure in vacuum device to obtain oily substance, and recrystallizing with petroleum ether and ethyl acetate to obtain white needle crystal;
C. synthesis of carnosol
Dissolving acetyl carnosic acid in anhydrous THF, and slowly adding 30.0eq SOCl dropwise under stirring 2 Slowly heating the reaction temperature from room temperature to 65 ℃ under the protection of nitrogen, refluxing for 10 hours, tracking by a TLC point plate, and stopping stirring until the raw material point of the acetyl carnosic acid disappears;
concentrating the reaction liquid to dryness in a vacuum decompression device, adding anhydrous THF in ice bath to dissolve the reaction liquid, and introducing CH into the reaction liquid under the protection of nitrogen 3 NH 2 After reacting for 4h, stopping stirring, pouring the reaction solution into 50mL of ice-bath distilled water, stirring for 10min, extracting with ethyl acetate, and adding anhydrous Na into an organic layer 2 SO 4 Drying for 1h, filtering, concentrating at 45 deg.C under reduced pressure in vacuum to obtain oily substance, and performing silica gel column chromatography with petroleum ether/ethyl acetate gradientElution afforded a white powdery solid.
5. The composition of claim 4, wherein: the composition can be made into tablet, capsule, injection, aerosol, powder, pill, mixture, granule, dripping pill, unguent, or oral liquid.
6. The composition of claim 4, wherein: the dosage form of the composition is controlled release, sustained release and nano preparation.
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