CN102219687A - Elephantopus scaber extract as well as preparation method and applications thereof in preparing antiviral drugs - Google Patents
Elephantopus scaber extract as well as preparation method and applications thereof in preparing antiviral drugs Download PDFInfo
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Abstract
The invention discloses an elephantopus scaber extract as well as preparation method and applications thereof. The preparation method for the elephantopus scaber extract comprises the steps of extracting entirely-dried elephantopus scaber or roots with ethanol or water, and then purifying through macroporous absorption resin columns to obtain the extract, wherein activate ingredients are caffeoylquinic acid substances. In the invention, the elephantopus scaber extract with anti-respiratory virus effect is extracted and enriched from herb-elephantopus scaber widely applied in civil society at Guangdong Province, China for the first time, and can be used for preparing drugs or health-care products for preventing respiratory syncytial virus, parainfluenza 3-type virus or influenza A virus. The invention not only provides a certain scientific proof for civil application of the elephantopus scaber, but also provides new resources for developing novel and safety and efficient anti-respiratory virus drugs.
Description
Technical field
The invention belongs to the field of Chinese medicines, particularly a kind of extract that from Chinese medicine Elephantopus scaber L. (Elephantopus scaber), obtains and preparation method thereof and the purposes in the preparation antiviral.
Background technology
Elephantopus scaber L. (Elephantopus scaber) is that composite family (Compositae) Elephantopus scaber L. belongs to (Elephantopus) plant, also be Scabrous Elephantfoot Herb, Scabrous Elephantfoot Herb, God in charge of the Earth's English, mainly be distributed in the south China and the southwest of China, the effect of tool heat-clearing, cool blood, dampness removing, this herbal medicine is widely used in that China Guangdong is among the people, be used for the treatment of flu, Whooping cough, yellow subcutaneous ulcer, pharyngitis, conjunctivitis, ephritis, eczema and worm snakebite etc., and include in version " Chinese pharmacopoeia in 1977.Main chemical ingredients has sesquiterpenoids, flavonoid and caffeoyl quinic acid compounds in the Elephantopus scaber L..Modern pharmacological research shows, effects such as it is antiviral, antibacterial, antitumor and analgesic that Elephantopus scaber L. has, wherein, there is the crude extract of research report Elephantopus scaber L. to have anti respiratory syncytial virus (RSV) activity preferably, but, do not study report to the antiviral activity position of Elephantopus scaber L. and with activeconstituents.Result for retrieval at State Intellectual Property Office's coordinate indexing and CNKI, Scifinder Scholar shows, do not find with the coffee mesitoyl quinine acid material as Elephantopus scaber L. extract of effective constituent and preparation method thereof with at the patent and the document of the purposes aspect the preventing respiratory viruses.
As everyone knows, respiratory virus infection is one of communicable disease of serious harm human health, and wherein influenza virus, parainfluenza virus and respiratory syncytial virus are the main pathogens of respiratory virus infection.Because virus is endotrophic microorganism, duplicating with many biological processes of host cell of it is closely related, and therefore, searching can optionally suppress the viral antiviral that does not influence the host cell function again certain difficulty; In addition, the factors such as resistance of the easy variability of virus antigen and antiviral cause difficulty also for the research and development of antiviral.Therefore, FDA approval at present is used for clinical antiviral drug than antimicrobial drug much less, but also has problems such as toxic side effect and resistance.Therefore, need badly and seek and exploitation safety, novel antiviral medicine efficiently.
Summary of the invention
In order to solve the shortcoming and defect that above-mentioned prior art exists, primary and foremost purpose of the present invention is to provide a kind of Elephantopus scaber L. extract.
Another object of the present invention is to provide above-mentioned Elephantopus scaber L. preparation method of extract.
A further object of the present invention is to provide the purposes of above-mentioned Elephantopus scaber L. extract in the preparation antiviral.
Purpose of the present invention is achieved through the following technical solutions: a kind of Elephantopus scaber L. preparation method of extract comprises following operation steps:
(1) with Elephantopus scaber L. herb or root dry and pulverize after, be that 0～100% ethanolic soln extracts with mass percent concentration, united extraction liquid filters, being evaporated to does not have alcohol and distinguishes the flavor of, and obtains crude extract medicinal extract; Or, adopt water extraction and alcohol precipitation method to extract with Elephantopus scaber L. herb or root drying with after pulverizing, and extracting liquid filtering, concentrated, obtain crude extract medicinal extract;
(2) crude extract medicinal extract is become crude extract with dissolved in distilled water, obtain the Elephantopus scaber L. extract through purification with macroreticular resin.
Step (1) is described to be that the number of times that 0～100% ethanolic soln extracts is 1～3 time with mass percent concentration, extracts 1～3 hour at every turn.
Step (2) is described, and to obtain the Elephantopus scaber L. extract through purification with macroreticular resin be the macroporous adsorptive resins of earlier crude extract being flowed through, with distilled water wash-out pillar, follow the tracks of with thin-layer chromatography (TLC) analysis, on silica gel thin-layer plate, launch the FeCl that mass percent concentration 5% is sprayed in the back until effluent liquid
3Ethanolic soln shows blueness or black-and-blue spot; Use the ethanolic soln wash-out pillar of mass percent concentration 50～60% again, follow the tracks of, on silica gel thin-layer plate, launch the FeCl that mass percent concentration 5% is sprayed in the back until effluent liquid with thin-layer chromatography (TLC) analysis
3Ethanolic soln does not develop the color; Collect the effluent liquid of ethanolic soln wash-out pillar, reclaim solvent, concentrate, drying obtains the Elephantopus scaber L. extract.
The described Elephantopus scaber L. of step (1) is a composite family Elephantopus scaber L. platymiscium.
The described water extraction and alcohol precipitation method of step (1) is to extract by the method for pharmaceutical industry routine.
A kind of Elephantopus scaber L. extract that obtains according to method for preparing, comprise following activeconstituents: 3,5-O-dicaffeoylquinic acid, 3,4-O-dicaffeoylquinic acid, 4,5-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid methyl esters, 3,4-O-dicaffeoylquinic acid methyl esters, 4,5-O-dicaffeoylquinic acid methyl esters and 5-O-caffeoylquinic acids.
Dry product weight with the Elephantopus scaber L. extract is calculated, quality percentage composition＞50% of total phenol in the Elephantopus scaber L. extract.
The purposes of above-mentioned Elephantopus scaber L. extract in preparation antiviral or healthcare products.
Described virus be respiratory syncytial virus (RSV), parainfluenza 3 C-type virus Cs (PIV 3) or influenza A virus (FluA, H1N1).
Described antiviral contains the Elephantopus scaber L. extract and the pharmaceutically acceptable carrier for the treatment of significant quantity.
The relative prior art of the present invention has following advantage and beneficial effect:
(1) first from the widely used herbal medicine Elephantopus scaber L. among the people of Guangdong, extract and enrichment have an Elephantopus scaber L. extract of preventing respiratory viruses effect, antiviral experiment shows: this extract has the activity of very strong anti respiratory syncytial virus (RSV) and parainfluenza 3 C-type virus Cs (PIV 3), and cytotoxicity is little, so selectivity index (SI value, the SI=CC of its anti-RSV and PIV 3
50/ IC
50) be higher than the positive control drug virazole; In addition, this extract also has the activity of certain anti-influenza A virus, the SI value of its anti-influenza A virus and virazole quite.
(2) clear and definite this Elephantopus scaber L. extract mainly is made up of the good coffee mesitoyl quinine acid material of activity.
(3) the present invention has been not only for the application among the people of Elephantopus scaber L. provides certain scientific basis, and for develop safety, novel anti respiratory tract disease cytotoxic drug provides new resources efficiently.
Embodiment
The present invention is described in further detail below in conjunction with embodiment, but embodiments of the present invention are not limited thereto.
Embodiment 1:
Getting the composite family Elephantopus scaber L. drafts a document the medicine Elephantopus scaber L. and (picks up from Guangdong, identify by the Guangzhou medicinal material senior engineer officer of the company wheat ball that shakes) dry root 200g, pulverize, be 95% aqueous ethanolic solution refluxing extraction 2 times with the 2000mL mass percent concentration respectively, each 1 hour, filter merging filtrate, being evaporated to does not have the alcohol flavor, obtains crude extract medicinal extract;
Take by weighing macroporous adsorbent resin (D101) 500g, adding distil water 800mL stirs, and the water flotation process is removed resin monomer or fragment, at room temperature place 24h then, after treating the abundant swelling of resin, resin is drained, again with ethanol wet method dress post, flow cleaning pillar to the effluent liquid and the volume ratio of water with ethanol is the muddiness that is not white in color when mixing at 1: 5, do not have the alcoholic acid taste with the abundant drip washing pillar of distilled water to effluent liquid then, place the balance pillar that spends the night, obtain macroporous resin column.
With above-mentioned crude extract medicinal extract 500mL dissolved in distilled water, filter, filtrate is slowly flow through in the macroporous resin column that above-mentioned processing and balance cross, continue to wash pillar with distilled water, elution process is analyzed with TLC and is followed the tracks of, and the water elution process is launched the FeCl of back, sprinkling mass percent concentration 5% on silica gel thin-layer plate until effluent liquid
3Ethanolic soln shows blueness or black-and-blue spot promptly stops.Then use the ethanolic soln wash-out pillar of mass percent concentration 50～60% again, the same analysis with TLC of effluent liquid followed the tracks of, and launches the FeCl of back, sprinkling mass percent concentration 5% on silica gel thin-layer plate until effluent liquid
3Ethanolic soln does not develop the color, and collects effluent liquid, reclaims solvent, and concentrated, lyophilize obtains Elephantopus scaber L. extract 1.Calculate quality percentage composition＞50% of total phenol in this Elephantopus scaber L. extract with extract dry product weight.
Embodiment 2:
The dry root of 200g Elephantopus scaber L. is pulverized, with the distilled water refluxing extraction of 2000mL 3 hours, filter, filtrate concentrates with Rotary Evaporators, ethanolic soln to the ethanol content that under agitation adds mass percent concentration 95% is a mass percent concentration 80%, leaves standstill the leaching supernatant liquor 24 hours, decompression recycling ethanol must obtain crude extract medicinal extract to there not being the alcohol flavor.Crude extract medicinal extract gets Elephantopus scaber L. extract 2 through the macroporous adsorptive resins purifying, and purification process is consistent with embodiment 1.Calculate quality percentage composition＞50% of total phenol in the Elephantopus scaber L. extract with extract dry product weight.
Embodiment 3:
The dry root of 200g Elephantopus scaber L. is pulverized, and the distilled water with 2000mL refluxes 3 times respectively, and each 1～3 hour, filter, merging filtrate gets crude extract medicinal extract.Crude extract medicinal extract gets Elephantopus scaber L. extract 3 through the macroporous adsorptive resins purifying, and purification process is consistent with embodiment 1.Calculate quality percentage composition＞50% of total phenol in the Elephantopus scaber L. extract with extract dry product weight.
Embodiment 4: the total phenol content of Elephantopus scaber L. extract is measured (colorimetry):
In the 10mL volumetric flask, add the acetone-water solution 8ml of determinand (Elephantopus scaber L. extract of the present invention), add Folin-Ciocalteau reagent 0.5mL then, shake up, placed 1 minute.Adding mass percent concentration again is 20% metabisulfite solution 1.5mL, shakes up, and places 1 hour.Under 750nm, measure optical density.Simultaneously in another volumetric flask, add acetone-water, blank by the same step colour developing conduct.
In addition, with 3,5-O-dicaffeoylquinic acid (self-control, the HPLC analytical results shows its purity＞95%) is done standard model bioassay standard curve.The standard substance that will be dried to constant weight in moisture eliminator are made into series concentration, by the same method, measure optical density at 750nm, make typical curve.
The measurement result demonstration, in the Elephantopus scaber L. extract, with 3,5-O-dicaffeoylquinic acid meter, quality percentage composition＞50% of total phenol in the Elephantopus scaber L. extract.
Embodiment 5: the extraction of coffeic acyl quininic acid derivative in the Elephantopus scaber L. extract 1, separation and structure are surveyed and are identified
With the Elephantopus scaber L. extract 1 of embodiment 1 gained through silica gel column chromatography, chloroform-methanol solvent system wash-out repeatedly, again through Sephadex LH-20 gel filtration chromatography separation repeatedly, chloroform-methanol and methanol-water solvent system wash-out, and then through preparation HPLC purifying, methyl alcohol-trifluoracetic acid solvent elution, obtain 7 coffee mesitoyl quinine acid compounds, their chemical structure adopts the method for spectroscopic analysis to identify that data are as follows:
Compound 1: yellow powder, the smoked khaki color that shows of thin-layer chromatography iodine, it is black-and-blue that iron trichloride shows, and is shown with phenolic hydroxyl group and exists; ESI-MSm/z:515[M-H]
-, 1031[2M-H]
- 1H-NMR (500MHz, CD
3OD) δ ppm:7.56 (1H, d, J=16.0Hz, H-7 '), 7.52 (1H, d, J=16.0Hz, H-7 "), 7.01 (2H, d; J=2.0Hz, H-2 ', H-2 "), 6.91 (2H, dd, J=7.0,2.0Hz, H-6 ', H-6 "), 6.73 (2H; d, J=7.0Hz, H-5 ', H-5 "), 6.30 (1H, d, J=16.0Hz, H-8 '), 6.21 (1H, d, J=16.0Hz, H-8 "), 5.38 (1H, m, H-5); 5.32 (1H, m, H-3), 3.91 (1H; dd, J=7.5,3.0Hz, H-4); 2.09-2.27 (4H, m, H-2, H-6).
13C-NMR (125MHz, CD
3OD) δ ppm:74.8 (C-1), 37.6 (C-2), 72.6 (C-3), 70.9 (C-4), 72.1 (C-5), 36.0 (C-6), 177.5 (C-7), 123.1 (C-1 ', C-1 "); 116.5 (C-2 ', C-2 "), 147.2 (C-3 ', C-3 "); 149.6 (C-4 ', C-4 "), 115.6 (C-5 ', C-5 "); 116.5 (C-6 ', C-6 "), 146.8 (C-7 ', C-7 "); 115.2 (C-8 ', C-8 "), 168.4 (C-9 '), 168.9 (C-9 ").
Compound 2: pale yellow powder, the smoked khaki color that shows of thin-layer chromatography iodine, it is black-and-blue that iron trichloride shows, and is shown with phenolic hydroxyl group and exists; ESI-MSm/z:515[M-H]
-, 1031[2M-H]
- 1H-NMR (500MHz, CD
3OD) δ ppm:7.53 (1H, d, J=16.5Hz, H-7 '), 7.46 (1H, d, J=16.0Hz, H-7 "), 6.96 (H, d, J=2.0Hz, H-2 '); 6.94 (1H, d, J=1.5Hz, H-2 "), 6.87 (2H, dd, J=6.5,2.0Hz, H-6 ', H-6 "), 6.70 (1H, d; J=8.5Hz, H-5 '), 6.68 (1H, d, J=8.0Hz, H-5 "), (6.23 1H, d, J=16.5Hz, H-8 '), 6.13 (1H, d, J=16.0Hz, H-8 "), 5.57 (1H, m; H-3), 5.06 (1H, dd, J=9.0,2.5Hz; H-4), 4.32 (1H, dt, J=3.0,2.5Hz; H-5), 2.05-2.23 (4H, m, H-2, H-6).
13C-NMR (125MHz, CD
3OD) δ ppm:75.9 (C-1), 39.4 (C-2), 69.0 (C-3), 75.8 (C-4), 69.4 (C-5), 38.3 (C-6), 177.0 (C-7), 127.7 (C-1 ', C-1 "); 115.2 (C-2 ', C-2 "), 146.8 (C-3 ', C-3 "); 149.7 (C-4 ', C-4 "), 116.5 (C-5 ', C-5 "); 123.1 (C-6 ', C-6 "), 147.7 (C-7 ', C-7 "); 114.8 (C-8 ', C-8 "), 168.3 (C-9 '), 168.6 (C-9 ").
Compound 3: pale yellow powder, the smoked khaki color that shows of thin-layer chromatography iodine, it is black-and-blue that iron trichloride shows, and is shown with phenolic hydroxyl group and exists; ESI-MSm/z:515[M-H]
-, 1031[2M-H]
- 1H-NMR (500MHz, CD
3OD) δ ppm:7.64 (1H, d, J=16.0Hz, H-7 '), 7.53 (1H, d, J=16.0Hz, H-7 "), 7.10 (H, d, J=2.0Hz, H-2 '); 7.08 (1H, d, J=2.0Hz, H-2 "), 6.97 (2H, dd, J=6.5,2.0Hz, H-6 ', H-6 "), 6.76 (1H, d; J=8.5Hz, H-5 '), 6.72 (1H, d, J=8.0Hz, H-5 "), (6.30 1H, d, J=16.0Hz, H-8 '), 6.23 (1H, d, J=16.0Hz, H-8 "), 5.72 (1H, m; H-5), 5.15 (1H, dd, J=9.0,3.0Hz; H-4), 4.36 (1H, dt, J=3.0,2.5Hz; H-3), 2.04-2.31 (4H, m, H-2, H-6).
13C-NMR (125MHz, CD
3OD) δ ppm:77.5 (C-1), 40.7 (C-2), 69.5 (C-3), 77.1 (C-4), 70.7 (C-5), 38.8 (C-6), 177.0 (C-7), 127.7 (C-1 ', C-1 "); 115.1 (C-2 ', C-2 "), 146.7 (C-3 ', C-3 "); 149.6 (C-4 ', C-4 "), 116.5 (C-5 ', C-5 "); 123.1 (C-6 ', C-6 "), 147.5 (C-7 ', C-7 "); 114.9 (C-8 ', C-8 "), 168.5 (C-9 ', C-9 ").
Compound 4: yellow powder, the smoked khaki color that shows of thin-layer chromatography iodine, it is black-and-blue that iron trichloride shows, and is shown with phenolic hydroxyl group and exists; ESI-MSm/z:529[M-H]
-, 1059[2M-H]
-UV λ
Max(MeOH): 251 (sh), 300 (sh), 331;
1H-NMR (500MHz, CD
3OD) δ ppm:5.25 (1H, m, H-3), 3.92 (1H, dd, J=6.5,3.0Hz, H-4), 5.35 (1H, m, H-5), 2.10-2.29 (4H, m, H-2, H-6), 7.01 (1H, d, J=2.5Hz, H-2 "), 7.02 (1H, d, J=2.5Hz, H-2 '); 6.73 (2H, d, J=8.0Hz, H-5 ', H-5 "), 6.91 (2H, dd, J=8.0,2.5Hz, H-6 ', H-6 "), 7.50 (1H, d, J=16.0Hz, H-7 '); 7.57 (1H, d, J=16.0Hz, H-7 "), 6.17 (1H, d, J=16.0Hz, H-8 '), 6.29 (1H, d, J=16.0Hz, H-8 "), 3.64 (3H, s, OCH
3).
13C-NMR (125MHz, CD
3OD) δ ppm:74.6 (C-1), 36.7 (C-2), 69.7 (C-3), 72.0 (C-4), 72.2 (C-5), 35.7 (C-6), 175.6 (C-7), 127.6 (C-1 '), 127.9 (C-1 "); 115.2 (C-2 '), 115.2 (C-2 "), 146.9 (C-3 '), 146.9 (C-3 "), 149.6 (C-4 '), 149.8 (C-4 "), 116.5 (C-5 '), 116.5 (C-5 "), 123.0 (C-6 '), 123.1 (C-6 "), 147.1 (C-7 '), 147.4 (C-7 ") 114.9 (C-8 '), 115.5 (C-8 "), 167.9 (C-9 '), 168.8 (C-9 "), 53.0 (OCH3).
Compound 5: yellow powder, the smoked khaki color that shows of thin-layer chromatography iodine, it is black-and-blue that iron trichloride shows, and is shown with phenolic hydroxyl group and exists; ESI-MS m/z:529[M-H]
-, 1059[2M-H]
-UV λ
Max(MeOH): 251 (sh), 300 (sh), 332;
1H-NMR (500MHz, CD
3OD) δ ppm:5.37 (1H, m, H-3), 4.98 (1H, dd, J=7.5,3.0Hz, H-4), 4.35 (1H, m, H-5), 2.09-2.19 (4H, m, H-2, H-6), 6.88 (1H, d, J=2.5Hz, H-2 '), 6.89 (1H, d, J=2.5Hz, H-2 "); 6.63 (2H, d, J=8.0Hz, H-5 ', H-5 "), 6.78 (2H, dd, J=8.0,2.5Hz, H-6 ', H-6 "), 7.51 (1H, d, J=16.0Hz, H-7 "), (7.37 1H, d, J=16.0Hz, H-7 '), 6.17 (1H, d, J=16.0Hz, H-8 '), 6.09 (1H, d, J=16.0Hz, H-8 "), 3.63 (3H, s, OCH
3).
Compound 6: yellow powder, the smoked khaki color that shows of thin-layer chromatography iodine, it is black-and-blue that iron trichloride shows, and is shown with phenolic hydroxyl group and exists; ESI-MSm/z:529[M-H]
-, 1059[2M-H]
-UV λ
Max(MeOH): 250 (sh), 300 (sh), 332;
1H-NMR (500MHz, CD
3OD) δ ppm:4.37 (1H, m, H-3), 5.10 (1H, dd, J=8.0,3.1Hz, H-4), 5.49 (1H, m, H-5), 2.07-2.21 (4H, m, H-2, H-6), 6.86 (1H, d, J=2.5Hz, H-2 '), 6.89 (1H, d, J=2.5Hz, H-2 "), 6.73 (2H, d, J=8.0Hz, H-5 '; H-5 "), 6.79 (2H, dd, J=8.0,2.5Hz, H-6 ' and H-6 "), 7.61 (1H, d, J=16.0Hz, H-7 '); 7.52 (1H, d, J=16.0Hz, H-7 "), 6.25 (1H, d, J=16.0Hz, H-8 '), 6.16 (1H, d, J=16.0Hz, H-8 "), 3.70 (3H, s, OCH
3).
Compound 7: pale yellow powder, the smoked khaki color that shows of thin-layer chromatography iodine, it is black-and-blue that iron trichloride shows, and is shown with phenolic hydroxyl group and exists; ESI-MS m/z:354;
1H-NMR (500MHz, CD
3OD) δ ppm:7.56 (1H, d, J=16.0Hz, H-β), 7.00 (1H, d, J=2.0Hz, H-2 '), 6.92 (1H, dd, J=8.5,2.0Hz, H-6 '), 6.73 (1H, d, J=8.5Hz, H-5 '), 6.31 (1H, d, J=16.0Hz, H-α), 5.32 (1H, m, H-5), 4.29 (1H, m, H-4), 3.67 (1H, m, H-3).
13C-NMR (125MHz, CD
3OD): 75.6 (C-1), 38.1 (C-2), 74.9 (C-3), 73.0 (C-4), 69.0 (C-5), 41.7 (C-6), 147.0 (C-α), 115.1 (C-β), 128.1 (C-1; ), 116.5 (C-2 '), 146.9 (C-3 '), 149.7 (C-4 '), 115.8 (C-5 '), 123.0 (C-6 '), 177.8 (C-7), 169.0 (C-8).
The structure of compound 1-7 is respectively 3,5-O-dicaffeoylquinic acid (1), 3,4-O-dicaffeoylquinic acid (2), 4,5-O-dicaffeoylquinic acid (3), 3,5-O-dicaffeoylquinic acid methyl esters (4), 3,4-O-dicaffeoylquinic acid methyl esters (5), 4,5-O-dicaffeoylquinic acid methyl esters (6) and 5-O-caffeoylquinic acids (7), except that compound 1 and 3, other four coffeic acyl quininic acid derivatives separate from Elephantopus scaber L. first and obtain.Their structure is as shown below:
Compound 1:R
1=R
3=Caffeoyl group, R
2=R
4=H
Compound 2:R
1=R
2=Caffeoyl group, R
3=R
4=H
Compound 3:R
2=R
3=Caffeoyl group, R
1=R
4=H
Compound 4:R
1=R
3=Caffeoyl group, R
2=H, R
4=CH
3
Compound 5:R
1=R
2=Caffeoyl group, R
3=H, R
4=CH
3
Compound 6:R
2=R
3=Caffeoyl group, R
1=H, R
4=CH
3
Compound 7:R
3=Caffeoyl group, R
1=R
2=R
4=H
Embodiment 6: the extraction of coffeic acyl quininic acid derivative in the Elephantopus scaber L. extract 2, separation and structure are surveyed and are identified
With the Elephantopus scaber L. extract of embodiment 2 gained through silica gel column chromatography, chloroform-methanol solvent system wash-out repeatedly, again through Sephadex LH-20 gel filtration chromatography separation repeatedly, methanol-water solvent system wash-out, and then through preparation HPLC purifying, methyl alcohol-trifluoracetic acid solvent elution, obtain 7 coffee mesitoyl quinine acid compounds, their chemical structure adopts the method for spectroscopic analysis to identify that the result is consistent with embodiment 5 gained compound 1-7.
Embodiment 7: the extraction of coffeic acyl quininic acid derivative in the Elephantopus scaber L. extract 3, separation and structure are surveyed and are identified
With the Elephantopus scaber L. extract 3 of embodiment 3 gained through silica gel column chromatography, chloroform-methanol solvent system wash-out repeatedly, again through Sephadex LH-20 gel filtration chromatography separation repeatedly, methanol-water solvent system wash-out, and then through preparation HPLC purifying, methyl alcohol-trifluoracetic acid solvent elution, obtain 7 coffee mesitoyl quinine acid compounds, their chemical structure adopts the method for spectroscopic analysis to identify that the result is consistent with embodiment 5 gained compound 1-7.
Embodiment 8: the preventing respiratory viruses determination of activity of Elephantopus scaber L. and coffic acid derivative wherein
Cell, virus and experiment material: select respiratory syncytial virus (RSV) for use, host cell is laryngeal cancer cell (Hep-2); Selecting parainfluenza 3 C-type virus Cs (PIV 3), host cell for use is that (FluA, H1N1), host cell is a dog kidney passage cell (MDCK) for Hep-2 and influenza A virus.
Positive control drug is a virazole; Cell is grown in the MEM substratum that mass percent concentration is 10% calf serum (FBS); Keeping liquid is the MEM substratum of 1%FBS.
The preparation of sample solution: embodiment 1 gained Elephantopus scaber L. extract 1, embodiment 2 gained Elephantopus scaber L. extracts 2, embodiment 3 gained Elephantopus scaber L. extracts 3, coffeic acyl quininic acid derivative and virazole usefulness are kept the sample solution that the liquid partition is not made into 200 μ g/mL and 100 μ g/mL.
Cytotoxicity with tetrazolium salts (MTT) colorimetric method for determining compound: Hep-2 cell or mdck cell are cultivated in 96 well culture plates, Deng monolayer cell grow good after, add with keeping liquid and dilute good sample (concentration is 200.0～3.1 μ g/mL),, cultivated 3 days in the 5%CO2 incubator at 37 ℃.Add 10 μ lMTT solution (5mg/mL disposes with buffered soln), continue to cultivate 4 hours.The sucking-off sample solution adds methyl-sulphoxide, under the room temperature, 96 orifice plates is placed microvoid orifice plate vibrator vibration 10 minutes.Measure the OD value in each hole with microplate reader, the measurement wavelength is 570nm, and reference wavelength is 630nm, the half lethal toxicity concentration (CC of calculation sample pair cell
50).Establish 4 equalizing ports for every group, every group of experiment repeats 3 times.Calculation result, the curve that draws is obtained half toxic concentration (CC
50).
Inhibition degree (cytopathic effect reduction assay) by observation sample pair cell pathology effect is measured antiviral activity: Hep-2 cell or mdck cell are cultivated in 96 well culture plates, Deng monolayer cell grow good after, add with keeping liquid and dilute 100 good sesquialters and count infective dose (100TCID
50) viral liquid, add again with keeping liquid and dilute good series concentration sample solution (concentration is 100～0.4 μ g/mL).At 37 ℃, cultivated in the 5%CO2 incubator 3～4 days.Every day observation of cell pathology effect (CPE) under inverted microscope degree, and record: the no CPE of-expression; + expression 0～25% cell has CPE; 2+ represents that 25～50% cells have CPE; 3+ represents that 50～70% cells have CPE; 4+ represents that 75～100% cells have CPE.Estimate half-inhibition concentration (IC at last
50).Selectivity index (SI)=CC
50/ IC
50Experimental result sees Table 1-3:
The anti respiratory syncytial virus activity of table 1 Elephantopus scaber L. extract and coffic acid derivative
* the host cell of respiratory syncytial virus (RSV) is Hep-2, CC
50Be half toxic concentration, IC
50Be half-inhibition concentration, the SI value is a selectivity index, by CC
50/ IC
50Calculate and obtain.
The anti-parainfluenza virus cytotoxic activity of table 2 Elephantopus scaber L. extract and coffic acid derivative
* the host cell of parainfluenza 3 C-type virus Cs (PIV 3) is Hep-2, CC
50Be half toxic concentration, IC
50Be half-inhibition concentration, the SI value is a selectivity index, by CC
50/ IC
50Calculate and obtain.
Table 3 Elephantopus scaber L. extract and coffic acid derivative anti-influenza virus activity
* the host cell of influenza A H1N1 virus (Flu A H1N1) is MDCK, CC
50Be half toxic concentration, IC
50Be half-inhibition concentration, the SI value is a selectivity index, by CC
50/ IC
50Calculate and obtain, "-" is illustrated in to measure in the concentration range does not have activity.
From the data of table the 1-3 as seen: all greater than 200 μ g/mL, its cytotoxicity is more much lower than Western medicine virazole to the half toxic concentration of Hep-2 and mdck cell for (1) Elephantopus scaber L. extract.(2) Elephantopus scaber L. extract three kinds of Respiroviruses that this experiment is adopted all have vitro inhibition activity in various degree.The Elephantopus scaber L. extract is best to the inhibition activity of respiratory syncytial virus (RSV), IC
50Be worth suitable with positive control drug, the SI value of their SI value serious offense virazole; The Elephantopus scaber L. extract also has better inhibited activity to parainfluenza 3 C-type virus Cs (PIV3), and their SI value is also greater than the SI value of virazole; The Elephantopus scaber L. extract has certain restraining effect to influenza A virus (Flu AH1N1), and less because of toxicity, the SI value of their SI value and virazole is suitable.(3) 7 coffeic acyl quininic acid derivatives separating from the Elephantopus scaber L. extract have good in vitro to suppress active, also stronger to the vitro inhibition activity of PIV3 to RSV; 3 dicaffeoylquinic acid methyl esters have certain inhibition activity to Flu A H1N1, and wherein 3, the activity of 5-O-dicaffeoylquinic acid methyl esters vitro inhibition Flu A H1N1 is better than virazole.
Above-mentioned test-results shows that the Elephantopus scaber L. extract that extract, purifying obtains have preventing respiratory viruses activity preferably, and toxicity is lower than Western medicine from the widely used herbal medicine Elephantopus scaber L. among the people of Guangdong; Contain active very strong serial coffeic acyl quininic acid derivative in the Elephantopus scaber L. extract.
The foregoing description is a preferred implementation of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (10)
1. Elephantopus scaber L. preparation method of extract is characterized in that comprising following operation steps:
(1) with Elephantopus scaber L. herb or root dry and pulverize after, be that 0～100% ethanolic soln extracts with mass percent concentration, united extraction liquid filters, being evaporated to does not have alcohol and distinguishes the flavor of, and obtains crude extract medicinal extract; Or, adopt water extraction and alcohol precipitation method to extract with Elephantopus scaber L. herb or root drying with after pulverizing, and extracting liquid filtering, concentrated, obtain crude extract medicinal extract;
(2) crude extract medicinal extract is become crude extract with dissolved in distilled water, obtain the Elephantopus scaber L. extract through purification with macroreticular resin.
2. a kind of Elephantopus scaber L. preparation method of extract according to claim 1 is characterized in that: step (1) is described to be that the number of times that 0～100% ethanolic soln extracts is 1～3 time with mass percent concentration, extracts 1～3 hour at every turn.
3. a kind of Elephantopus scaber L. preparation method of extract according to claim 1 is characterized in that: the described water extraction and alcohol precipitation method of step (1) is to extract by the method for pharmaceutical industry routine.
4. a kind of Elephantopus scaber L. preparation method of extract according to claim 1, it is characterized in that: step (2) is described, and to obtain the Elephantopus scaber L. extract through purification with macroreticular resin be the macroporous adsorptive resins of earlier crude extract being flowed through, with distilled water wash-out pillar, on silica gel thin-layer plate, launch the FeCl that mass percent concentration 5% is sprayed in the back until effluent liquid
3Ethanolic soln shows blueness or black-and-blue spot; Use the ethanolic soln wash-out pillar of mass percent concentration 50～60% again, on silica gel thin-layer plate, launch the FeCl that mass percent concentration 5% is sprayed in the back until effluent liquid
3Ethanolic soln does not develop the color; Collect the effluent liquid of ethanolic soln wash-out pillar, reclaim solvent, concentrate, drying obtains the Elephantopus scaber L. extract.
5. a kind of Elephantopus scaber L. preparation method of extract according to claim 1 is characterized in that: the described Elephantopus scaber L. of step (1) is a composite family Elephantopus scaber L. platymiscium.
6. Elephantopus scaber L. extract that method according to claim 1 prepares, it is characterized in that: this extract comprises following activeconstituents: 3,5-O-dicaffeoylquinic acid, 3,4-O-dicaffeoylquinic acid, 4,5-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid methyl esters, 3,4-O-dicaffeoylquinic acid methyl esters, 4,5-O-dicaffeoylquinic acid methyl esters and 5-O-caffeoylquinic acids.
7. Elephantopus scaber L. extract according to claim 6 is characterized in that: the dry product weight with the Elephantopus scaber L. extract is calculated, quality percentage composition＞50% of total phenol in the Elephantopus scaber L. extract.
8. the purposes of Elephantopus scaber L. extract according to claim 6 in preparation antiviral or healthcare products.
9. purposes according to claim 8 is characterized in that: described virus is respiratory syncytial virus, parainfluenza 3 C-type virus Cs or influenza A virus.
10. purposes according to claim 8 is characterized in that: described antiviral contains the Elephantopus scaber L. extract and the pharmaceutically acceptable carrier for the treatment of significant quantity.
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| CN117069685A (en) * | 2023-10-13 | 2023-11-17 | 暨南大学 | Cinnamoyl alkyl polyketone compound, preparation method and application thereof |
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