CN101336940B - Thalassia hemprichii extract and preparation method and use thereof - Google Patents
Thalassia hemprichii extract and preparation method and use thereof Download PDFInfo
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- CN101336940B CN101336940B CN200810030161XA CN200810030161A CN101336940B CN 101336940 B CN101336940 B CN 101336940B CN 200810030161X A CN200810030161X A CN 200810030161XA CN 200810030161 A CN200810030161 A CN 200810030161A CN 101336940 B CN101336940 B CN 101336940B
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Abstract
The invention relates to a Thalassia hemprichii extract, and a preparation method and an application thereof. The preparation method comprises soaking Thalassia hemprichii with an organic solvent to obtain a crude extract, performing sequential liquid extraction of the crude extract with chloroform or ethyl acetate and n-butanol, collecting water layer, performing macroporous resin normal pressure column chromatography, eluting with a water-methanol solvent system or a water-ethanol solvent system, collecting methanol or ethanol eluate, rinsing with methanol, and collecting component indissoluble in methanol and having strong ultraviolet absorption at ultraviolet wavelength of 254nm to obtain the product. The inventive Thalassia hemprichii extract has strong anti-herpes simplex virus (HSV-1) activity, in vitro anti-CVB3 virus activity, insect antifeedant activity, and strong bryozoan (Bugula neritina) larvae attachment inhibitory activity; and can be used for preparing antiviral drugs, biological pesticides and ocean green anti-fouling agents.
Description
Technical field
The present invention relates to a kind of Old Taylor algae (Thalassia hemprichii) extract, also relate in addition this preparation method of extract with and the preparation antiviral, the application in biological pesticide and the marine antifoulant.
Background technology
Old Taylor algae (also claiming safe Lay grass) belongs to Hydrocharctaceae Old Taylor Trentepohlia, be the sociales in the sea grass bed in waters, China South Sea, but its economic worth also is not developed at present.
Summary of the invention
The objective of the invention is to from the Old Taylor algae, extract compound, thereby realize the economic worth of Old Taylor algae with pharmaceutical value.
We pass through organic solvent extraction, normal pressure column chromatography for separation technology etc., from the Old Taylor algae, separate and obtain the extract that a kind of principal constituent is the flavonoid glycoside compound, this compound has antiviral, the effect that insect antifeedant is active and anti-marine fouling organism larva adheres to, thereby can be applied to prepare antiviral, and the green stain control agent in biological pesticide and ocean, thus realized purpose of the present invention.
Thalassia hemprichii extract of the present invention is characterized in that containing the flavonoid glycoside compound and is prepared by following step:
(1) the Old Taylor algae is at room temperature soaked with organic solvent, is collected extracting solution, concentrate crude extract;
(2) above-mentioned crude extract is outstanding water-soluble, with chloroform or ethyl acetate, propyl carbinol extraction successively respectively, collect water layer and concentrate;
(3) above-mentioned spissated water layer is carried out macroporous resin normal pressure column chromatography, with water-methanol solvent system or water-ethanol solvent system is elutriant, carried out gradient elution in 100: 0 to 0: 100 from volume ratio, collect component, concentrate and obtain thick component with methyl alcohol or ethanol elution;
(4) with above-mentioned thick component washed with methanol, be insoluble to methyl alcohol after the collection flushing, and the component of strong uv-absorbing is arranged under ultraviolet light wavelength 254nm, obtain Thalassia hemprichii extract.
The described organic solvent of step (1) can be methyl alcohol, ethanol, methanol-water mixed solvent or ethanol-water mixed solvent etc., and described immersion can be 3 times, and each 5 days, described concentrating can be adopted conventional method such as concentrating under reduced pressure.
The described extraction of step (2) can be each 3～4 times, and described concentrating can be adopted conventional method such as concentrating under reduced pressure.
Described the concentrating of step (3) can be adopted conventional method such as concentrating under reduced pressure.
Thalassia hemprichii extract of the present invention is yellow amorphous powder, under ultraviolet light wavelength 254nm strong uv-absorbing is arranged, its proton nmr spectra [
1H NMR (500MHz, DMSO)] show below data: δ
H7.50～7.82ppm (a plurality of aromatic nucleus hydrogen), 6.10～6.80ppm (a plurality of aromatic nucleus hydrogen), 4.71～5.17ppm (a plurality of sugared terminal hydrogen), 3.20～3.82ppm (a plurality of sugared methynes and methylene radical hydrogen), its
1H NMR spectrum is similar to flavonoid glycoside compound the luteolin3 '-O-methyl-7-glucuronide that is separated to from the Old Taylor grass, and (document sees reference: Stochmal A, Placente S, Pizza C, et al.JAgric Food Chem, 2001,49:753-758.), just many fignal centers, in conjunction with flavonoid compound is this research fact of principal character chemical ingredients of Old Taylor algae, so infer that the main chemical compositions of this extract is a flavonoid glycoside compound.
The preparation method of Thalassia hemprichii extract of the present invention and optimal conditions thereof are with above-mentioned.
Experimental results show that Thalassia hemprichii extract of the present invention has very strong anti-herpes simplex virus HSV-I virus activity, its IC50 value is about 6.25 μ g/mL, also has external anti-CVB3 virus activity, the pathology that when concentration is 100 μ g/mL, can suppress CVB3 virus over half, compound of the present invention in addition has insect antifeedant activity preferably, food refusal rate to prodenia litura 2 instar larvaes when concentration is 500 μ g/mL is 93%, cell lethality to prodenia litura SL cell when concentration is 100 μ g/mL is 91%, in addition, compound of the present invention also has the activity (EC that very strong inhibition multicell grass tongue worm larva adheres to
50Be 0.005 μ g/mL).
Thalassia hemprichii extract of the present invention can be used to prepare antiviral, can be used as biological pesticide and marine antifoulant.
Embodiment
Following examples are to further specify of the present invention, are not limitations of the present invention.
Embodiment 1:
Old Taylor algae (Thalassia hemprichii) with air-dry dry weight 4Kg is a raw material, and the about 35L soaking at room temperature of ethanol organic solvent 3 times of chopping back each 5 days, get crude extract with the extracting solution concentrating under reduced pressure.With crude extract (70g) outstanding water-soluble (1L), extract each successively 3 times with chloroform (600mL) and propyl carbinol (500mL), collect water layer and concentrating under reduced pressure, water layer 23g after concentrating is carried out macroporous resin (D101 macroporous adsorbent resin) normal pressure post (normal pressure glass column, diameter 9cm, long 1.8m) chromatography, with the water-methanol solvent systems is eluent, carried out gradient elution in 100: 0 to 0: 100 from volume ratio, collect the component with methanol-eluted fractions, concentrating under reduced pressure obtains thick component.Thick component is used washed with methanol, can not be dissolved in methyl alcohol after the collection flushing, and the component of strong uv-absorbing is arranged under ultraviolet light wavelength 254nm, obtaining Thalassia hemprichii extract of the present invention.The proton nmr spectra of this extract [
1H NMR (500MHz, DMSO)] show below data: δ
H7.50～7.82ppm (a plurality of aromatic nucleus hydrogen), 6.10～6.80ppm (a plurality of aromatic nucleus hydrogen), 4.71～5.17ppm (a plurality of sugared terminal hydrogen), 3.20～3.82ppm (a plurality of sugared methynes and methylene radical hydrogen), its
1H NMR spectrum to from the Old Taylor algae, be separated to flavonoid glycoside compound luteolin3 '-O-methyl-7-glucuronide is similar, just many fignal centers, in conjunction with flavonoid compound is this research fact of principal character chemical ingredients of Old Taylor algae, so infer that the main chemical compositions of this extract is a flavonoid glycoside compound.
Embodiment 2:
Collect logarithmic phase vero cell, be inoculated in 96 well culture plates, every porocyte number is 1.0 * 10
5/ 100 μ L place 5%, C0
2Incubator is cultivated.Next day, every hole added the solution 50 μ L of the Thalassia hemprichii extract of the good embodiment 1 of dilution with growth media sucking-off in the micropore, added HSV-I virus immediately and used liquid 50 μ L, established the normal cell contrast simultaneously, virus control, 37 ℃, 5%CO
2Incubator is cultivated.Observation cytopathic effect (CPE), every day the observation of cell growing way, with-,+, ++, +++, ++ ++ expression cytopathy situation.The result shows that compound of the present invention is 25ug/ml to the maximal non-toxic concentration of vero cell, and it suppresses the IC of herpes simplex virus I-type pathology
50Value is 6.25ug/mL.
Embodiment 3:
Collect logarithmic phase HepG2 cell, be inoculated in 96 well culture plates, every porocyte number is 1.0 * 10
5/ 100 μ L place 5%, CO
2Incubator is cultivated.Next day, every hole added the solution 50 μ L of the Old Taylor grass extract of the good embodiment 1 of dilution, adds CVB3 virus immediately and uses liquid 50 μ L, establishes the normal cell contrast simultaneously, virus control, 37 ℃, 5%C0 with growth media sucking-off in the micropore
2Incubator is cultivated.Observation cytopathic effect (CPE), every day the observation of cell growing way, with-,+, ++, +++, ++ ++ expression cytopathy situation.The result shows that compound of the present invention is 200 μ g/mL to the Cytotoxic maximal non-toxic concentration of hela, can suppress the pathology of virus over half when concentration is 100 μ g/mL.
Embodiment 4:
The product of embodiment 1 is made into 500 μ g/mL acetone solns (the indissoluble thing has added several methyl-sulphoxides), and with concentration be 10 μ g/mL nimbin in contrast.(Φ=1.5cm) distinguish immersion test liquid and contrast liquid 1 second puts into culture dish after drying, and 3 in every ware inserts 2 prodenia litura 2 instar larvaes with cassava leaves.Every sample preparation is established 5 repetitions.Handle 48 hours " Invest, Then Investigate "s and get the food leaf area, and change fresh cabbage leaves and feed, the death condition of examination worm in 6 days after the recording processing.The result shows that compound of the present invention food refusal rate to prodenia litura 2 instar larvaes when concentration is 500 μ g/mL is 93%.
Embodiment 5:
With prodenia litura SL cell is object, with mtt assay the product of embodiment 1 has been carried out the cell virulence test.The concentration of the product of embodiment 1 is 100 μ g/mL (being 100ppm), and solvent is DMSO.The result shows that compound of the present invention cell lethality to prodenia litura SL cell when concentration is 100 μ g/mL is 91%.
Embodiment 6:
The Thalassia hemprichii extract that embodiment 1 is obtained is dissolved in methyl-sulphoxide (DMSO) as specimen, add the seawater (FSW) that filters (0.22 μ m) through autoclave then and be made into different concentration (μ g/mL O.05,0.2 μ g/mL, 1 μ g/mL, 10 μ g/mL and, 50 μ g/mL).Add the specimen that 20 larvas that move about and 1mL prepare in each orifice plate of polystyrene 24 aperture plate, each sample is done 4 parallel holes, with being added with the FSW seawater of DMSO as the negative control thing.24 aperture plate that to be furnished with specimen are put in the incubator of 28C and place after 1 hour the observation experiment result.Calculate by microscope: 1) attached to the larva number on the wooden partition, 2) not attached to the larva number on the wooden partition, 3) in the dust larva number.Calculate the per-cent that accounts for total larva number of experiment usefulness attached to the larva number on the wooden partition, it suppresses the half effective inhibition concentration EC that larva adheres to by EPAPROBIT ANALYSIS PROGRAM Version 1.5 computed in software again
50The result shows, the half effective inhibition concentration EC that compound of the present invention adheres to multicell grass tongue worm larva
50Be 0.005 μ g/mL.
Claims (7)
1. Thalassia hemprichii extract is characterized in that containing the flavonoid glycoside compound and is prepared by following step:
(1) Old Taylor algae (Thalassia hemprichii) is at room temperature soaked with organic solvent, is collected extracting solution, concentrate crude extract;
(2) above-mentioned crude extract is outstanding water-soluble, with chloroform or ethyl acetate, propyl carbinol extraction successively respectively, collect water layer and concentrate;
(3) above-mentioned spissated water layer is carried out macroporous resin normal pressure column chromatography, with water-methanol solvent system or water-ethanol solvent system is elutriant, carried out gradient elution in 100: 0 to 0: 100 from volume ratio, collect component, concentrate and obtain thick component with methyl alcohol or ethanol elution;
(4) with above-mentioned thick component washed with methanol, be insoluble to methyl alcohol after the collection flushing, and the component of strong uv-absorbing is arranged under ultraviolet light wavelength 254nm, obtain Thalassia hemprichii extract.
2. a kind of Thalassia hemprichii extract according to claim 1, it is characterized in that the described organic solvent of step (1) is methyl alcohol, ethanol, methanol-water mixed solvent or ethanol-water mixed solvent, described immersion is 3 times, each 5 days, described concentrating is concentrating under reduced pressure, the described extraction of step (2) is each 3～4 times, and described concentrating is concentrating under reduced pressure, and described the concentrating of step (3) is concentrating under reduced pressure.
3. the preparation method of a Thalassia hemprichii extract, its feature comprises the steps:
(1) the Old Taylor algae is at room temperature soaked with organic solvent, is collected extracting solution, concentrate crude extract;
(2) above-mentioned crude extract is outstanding water-soluble, with chloroform or ethyl acetate, propyl carbinol extraction successively respectively, collect water layer and concentrate;
(3) above-mentioned spissated water layer is carried out macroporous resin normal pressure column chromatography, with water-methanol solvent system or water-ethanol solvent system is elutriant, carried out gradient elution in 100: 0 to 0: 100 from volume ratio, collect component, concentrate and obtain thick component with methyl alcohol or ethanol elution;
(4) with above-mentioned thick component washed with methanol, be insoluble to methyl alcohol after the collection flushing, and the component of strong uv-absorbing is arranged under ultraviolet light wavelength 254nm, obtain product.
4. the preparation method of a kind of Thalassia hemprichii extract according to claim 3, the described organic solvent of its characterization step (1) is methyl alcohol, ethanol, methanol-water solvent or ethanol-water mixed solvent, described immersion is 3 times, each 5 days, described concentrating is concentrating under reduced pressure, the described extraction of step (2) is each 3～4 times, and described concentrating is concentrating under reduced pressure, and described the concentrating of step (3) is concentrating under reduced pressure.
5. the application of the described Thalassia hemprichii extract of claim 1 in preparation anti-herpes simplex virus I-form medicine.
6. the described Thalassia hemprichii extract of claim 1 is as the application of killing the biological pesticide of prodenia litura.
7. the described Thalassia hemprichii extract of claim 1 is as the application of marine antifoulant.
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| CN101336940B true CN101336940B (en) | 2010-11-03 |
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| CN101564050B (en) * | 2009-05-23 | 2013-06-19 | 中国船舶重工集团公司第七二五研究所 | Method for preparing Ulva pertusa Kjellm extract for marine antifoulant |
| CN102091086B (en) * | 2011-02-21 | 2012-03-28 | 中国科学院南海海洋研究所 | Application of sulfated flavonoid glycoside in preparation of herpes simplex virus resistant drug |
| CN103054131B (en) * | 2011-09-08 | 2014-10-01 | 浙江海洋学院 | Preparation method of high-purity Caulerpa racemesa polyphenol and application thereof to fresh-keeping of shrimp |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6395534B1 (en) * | 1999-03-31 | 2002-05-28 | Council Of Scientific And Industrial Research | White rot-lignin-modifying fungus Flavodon flavus and a process for removing dye from dye containing water or soil using the fungus |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6395534B1 (en) * | 1999-03-31 | 2002-05-28 | Council Of Scientific And Industrial Research | White rot-lignin-modifying fungus Flavodon flavus and a process for removing dye from dye containing water or soil using the fungus |
Non-Patent Citations (1)
| Title |
|---|
| 李盛英等.国外海洋抗肿瘤活性物质的研究与开发.《海洋通报》.2003,(第02期),76-82. * |
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