CN102696638A - Sweroside pharmaceutical preparation and application thereof in prevention of tobacco mosaic virus - Google Patents
Sweroside pharmaceutical preparation and application thereof in prevention of tobacco mosaic virus Download PDFInfo
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- CN102696638A CN102696638A CN2012101155763A CN201210115576A CN102696638A CN 102696638 A CN102696638 A CN 102696638A CN 2012101155763 A CN2012101155763 A CN 2012101155763A CN 201210115576 A CN201210115576 A CN 201210115576A CN 102696638 A CN102696638 A CN 102696638A
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Abstract
The invention discloses a sweroside pharmaceutical preparation and application thereof in prevention of a tobacco mosaic virus. The sweroside pharmaceutical preparation comprises 0.1-99% of sweroside and the balance pharmaceutic adjuvant and/excipient which is/are acceptable in pharmacology. The sweroside used in the sweroside pharmaceutical preparation can be separated and prepared from swertia cincta burk, the method is simple and the extraction rate is high. The sweroside pharmaceutical preparation has wide application prospects in prevention of the tobacco mosaic virus. The sweroside is separated and obtained from the swertia cincta burk through a modern separation technology. An experiment shows that the sweroside can significantly inhibit the tobacco mosaic virus (TMV) infection activity and serves as a novel plant virus resistance activity ingredient to be used for preventing a TMV disease, and the inhibition effect of the sweroside pharmaceutical preparation is better than that of other sold TMV resistant plant preparations (such as ningnanmycin). The sweroside pharmaceutical preparation is a pure biological agent, is harmless to people and animals, has a significant effect on TMV disease prevention and can be used in production of organic tobaccos.
Description
Technical field
The invention belongs to medicinal plants and applied technical field, be specifically related to a kind of sweroside pharmaceutical preparation, from surrounded swertia herb (
Swertia cinctaBurk) method of extraction sweroside and the application of pharmaceutical preparation in preparation prevention and control tobacco mosaic virus disease and anti-virus formulation.
Background technology
Tobacco mosaic virus (
Tobacco mosaic virus, be the pathogene of tobacco mosaic etc. TMV), belong to the Viraceae Tobamovirus of wearing armor (
Tobamovirus).The host of TMV is extensive, can infect 300 various plants of 30 sections, on crop, endangers seriously at grains such as tobacco, tomatoes, and the loss that the mosaic disease that the whole world is only caused by TMV every year causes just surpasses 100,000,000 dollars, causes enormous economic loss to agricultural production.Yet because TMV has not complete immune metabolic system as animal of obligate parasitism property and plant to the host, therefore, plant is in case just be in the state that is endangered by virus infections throughout one's life.These factors have brought very big difficulty all for the research and development of the Antiphytoviral medicament of high selectivity.In present agricultural production, the chemical pesticide control tobacco mosaic virus disease is used the most extensive, but the chemical control effect is limited, and is prone to cause residual the exceeding standard of farming, therefore presses for the development of new anti-virus formulation.
Plant has been synthesized the abundant secondary metabolite with antimicrobial acivity of type during evolution, and they can optionally resist the infringement of the pathogen that comprises TMV.Owing to be the antiviral substance of plant generation itself, the plant resource viral inhibitors is little or do not have toxicity, mechanism of action uniqueness and aboundresources to the toxicity of host own.Therefore, screening novel plant source antivirus preparation is significant.
The process that virus endangers the plant generation can be divided into infecting duplicates in host, the body and three parts of symptom expression.The harm that therefore, all can alleviate the viroses of plant to the inhibition of any part wherein.Though some anti-plant virus agent can activate the host and produce system's resistance the not effect of these three parts in addition.Simultaneously, the antiviral activity of many active substances performance possibly be the result that concurs of one or more mechanism wherein.Inventor's early-stage Study shows that a lot of plant resource viral inhibitors are the systemic acquired resistances through activated plant, thereby enhancement of plant is to the resistance of virus.
Gentianaceae Swertia plant (
Swertia) mainly originate in ground such as Yunnan, Guizhou, Sichuan, be southwest minority autonomic drug source commonly used.This platymiscium has 170 kinds, and China has 80 kinds approximately, mainly is distributed in the southwest, and only Yunnan just has 38 kinds.This genus medicinal plant has effects such as clearing heat secreting bile, cardiac stimulant, relaxing liver and strengthening stomach, detoxifcation mostly, is used to treat diseases such as hepatitis, icteric hepatitis, liver and gall, but does not have this platymiscium of bibliographical information to have the effect of Antiphytoviral as yet.
Summary of the invention
First purpose of the present invention is to provide a kind of sweroside pharmaceutical preparation; Second purpose is to provide a kind of method of extracting sweroside; The 3rd purpose is to provide the formulation of sweroside pharmaceutical preparation; The 4th purpose is to provide the application of sweroside in the sick preparation of preparation resisting tobacco mosaic virus.
First purpose of the present invention is achieved in that described sweroside pharmaceutical preparation contains sweroside 0.1 ~ 99%, and all the other are pharmaceutic adjuvant acceptable and/or excipient on the pharmacology.
Second purpose of the present invention is achieved in that with the surrounded swertia herb to be raw material, and extracts the sweroside compound by the technology that comprises the following steps:
A, get the surrounded swertia herb that dries in the shade and pulverize, with 75 ~ 85% methanol extraction 2 ~ 3 times, each 1 ~ 3h;
B, merging extract, filter cleaner, merging filtrate, concentrating under reduced pressure gets the medicinal extract of relative density 1.2 ~ 1.3 then;
The water of C, 1 ~ 3 times of volume of medicinal extract adding is stirred to and is suspension, with concentration 95 ~ 100% dichloromethane solutions extraction 2 ~ 3 times, each 15 ~ 25min;
D, combining extraction liquid are crossed the decolouring of MCI post, use the methanol-eluted fractions of concentration 70 ~ 80% afterwards, collect eluent;
E, use the macroporous resin column desugar again, and, collect eluent, concentrate to such an extent that contain the medicinal extract of 70 ~ 80% methyl alcohol with the eluant solution of methanol (V/V) 0 ~ 100:100 ~ 0;
F, last HPLC separate, and with 70 ~ 80% methanol aqueous solution wash-outs, carry out purifying with semi-preparative column at last, obtain the sweroside compound.
The 3rd purpose of the present invention be achieved in that by prescription requirement ratio with the sweroside compound with to nontoxic pharmaceutically acceptable auxiliaries of humans and animals and/or mixed with excipients, be mixed with aqua, tablet, granule or microemulsion.
The 4th purpose of the present invention is achieved in that the application of described sweroside pharmaceutical preparation in preparation control tobacco mosaic virus disease preparation.
The present invention use modern separation technology from surrounded swertia herb (
Swertia cincta Burk) obtain a kind of iridoid---sweroside middle the separation.Experiment proof sweroside has the activity that significant inhibition TMV infects; Can be used as the active component of novel anti plant virus, can be used for preparing the pharmaceutical preparation of preventing and treating tobacco mosaic virus disease, it suppresses effect and is better than commercial anti tobacco mosaic plant medical agent; Like medicines such as Ningnanmycins; Pharmaceutical preparation of the present invention is not only nontoxic, harmless to people and animals, safe and reliable, and the control tobacco mosaic virus disease is had significant effect.
Description of drawings
Fig. 1 detects the content block diagram of virus for the TAS-ELISA method.Among Fig. 1: the negative contrast of CK-, the positive contrast of CK+, positive greater than positive control, negative less than 2 times of negative controls.Ningnanmycin is the marketed drugs contrast, and sweroside-1,5,10,15 and 20 representes that respectively the concentration of sweroside is 1 μ g/ml, 5 μ g/ml, 10 μ g/ml, 15 μ g/ml and 20 μ g/ml.
Fig. 2 is an effect contrast photo of the present invention.Wherein left side figure is The compounds of this invention and control group effect comparison; Right figure is existing compound and the contrast of control group effect of drugs.
Embodiment
Below in conjunction with embodiment the present invention is further described, but never in any form the present invention is limited, any change or improvement based on training centre of the present invention is done all belong to protection scope of the present invention.
Sweroside pharmaceutical preparation of the present invention is: contain sweroside 0.1 ~ 99%, all the other are pharmaceutic adjuvant acceptable and/or excipient on the pharmacology.
As preferred implementation:
Contain sweroside 0.5 ~ 90%, all the other are pharmaceutic adjuvant acceptable and/or excipient on the pharmacology.
Contain sweroside 1 ~ 50%, all the other are pharmaceutic adjuvant acceptable and/or excipient on the pharmacology.
Contain sweroside 5 ~ 20%, all the other are pharmaceutic adjuvant acceptable and/or excipient on the pharmacology.
The sweroside that is used to prepare above-mentioned sweroside pharmaceutical preparation of the present invention, its preparation method is: be raw material with the surrounded swertia herb, and extract the sweroside compound by the technology that comprises the following steps:
A, get the surrounded swertia herb that dries in the shade and pulverize, with 75 ~ 85% methanol extraction 2 ~ 3 times, each 1 ~ 3h;
B, merging extract, filter cleaner, merging filtrate, concentrating under reduced pressure gets the medicinal extract of relative density 1.2 ~ 1.3 then;
The water of C, 1 ~ 3 times of volume of medicinal extract adding is stirred to and is suspension, with concentration 95 ~ 100% dichloromethane solutions extraction 2 ~ 3 times, each 15 ~ 25min;
D, combining extraction liquid are crossed the decolouring of MCI post, use the methanol-eluted fractions of concentration 70 ~ 80% afterwards, collect eluent;
E, use the macroporous resin column desugar again, and, collect eluent, concentrate to such an extent that contain the medicinal extract of 70 ~ 80% methyl alcohol with the eluant solution of methanol (V/V) 0 ~ 100:100 ~ 0;
F, last HPLC separate, and with 70 ~ 80% methanol aqueous solution wash-outs, carry out purifying with semi-preparative column at last, obtain the sweroside compound.
As preferred implementation:
Described macroporous resin column is any one among D900, D130, D101, D201 or the D301.
Described semi-preparative column is any one in the uncommon semi-preparative column of Ai Jieer semi-preparative column, HPLC semi-preparative column or Japanese aestheticism.Described Ai Jieer semi-preparative column carries out purifying, adopts methanol/water solution to carry out wash-out, obtains the sweroside compound.
The formulation of sweroside pharmaceutical preparation of the present invention is: with the sweroside compound and to nontoxic pharmaceutically acceptable auxiliaries of humans and animals and/or mixed with excipients, be mixed with aqua, tablet, granule or microemulsion by prescription requirement ratio.
Described pharmaceutical carrier or excipient are one or more solids, semisolid and liquid diluent, filler and pharmaceutical preparation assistant agent.Adopt pharmacy and the generally acknowledged method of field of food be prepared into various formulations, like liquid preparation (injection, supensoid agent, emulsion, solution, syrup etc.), solid pharmaceutical preparation (tablet, capsule, granule, electuary etc.), spray, aerosol etc.Auxiliary substance, stabilizing agent be can contain in the above-mentioned preparation, humectant and other additives commonly used helped, like starch, albumen powder, pectin, sucrose, dextrin, dolomol, mannitol, neopelex etc.Above-mentioned preparation can be processed according to the conventional preparation technology of various preparations.
The application of described sweroside pharmaceutical preparation in preparation control tobacco mosaic virus disease preparation, wherein the sweroside solution concentration is 1 ~ 20 μ g/ml.
Embodiment 1
Get the surrounded swertia herb that dries in the shade and pulverize, with 75% methanol extraction 2 times, 1h at every turn; Merge extract, filter cleaner, merging filtrate, concentrating under reduced pressure gets the medicinal extract of relative density 1.2 ~ 1.3 then; The water of 1 times of volume of medicinal extract adding is stirred to and is suspension, with concentration 98% dichloromethane solution extraction 2 times, and each 15min; Combining extraction liquid is crossed the MCI decolouring, uses the methanol-eluted fractions of concentration 75% afterwards, collects eluent; Use the desugar of D900 macroporous resin column again,, collect eluent with the eluant solution of methanol (V/V) 10:90; Concentrate medicinal extract, last HPLC separates, the methanol aqueous solution wash-out with 70%, the Ai Jieer semi-preparative column carries out purifying at last, the methanol aqueous solution wash-out with 70% obtains the sweroside compound.
Embodiment 2
Get the surrounded swertia herb that dries in the shade and pulverize, with 85% methanol extraction 3 times, 3h at every turn; Merge extract, filter cleaner, merging filtrate, concentrating under reduced pressure gets the medicinal extract of relative density 1.2 ~ 1.3 then; The water of 3 times of volumes of medicinal extract adding is stirred to and is suspension, with concentration 95% dichloromethane solution extraction 3 times, and each 25min; Combining extraction liquid is crossed the decolouring of MCI post, uses the methanol-eluted fractions of concentration 70% afterwards, collects eluent; Use the desugar of D130 macroporous resin column again,, collect eluent with the eluant solution of methanol (V/V) 30:70; Concentrate medicinal extract, last HPLC separates, 80% methanol aqueous solution wash-out, the HPLC semi-preparative column carries out purifying at last, the methanol aqueous solution wash-out with 80% obtains the sweroside compound.
Embodiment 3
Get the surrounded swertia herb that dries in the shade and pulverize, with 80% methanol extraction 3 times, 2h at every turn; Merge extract, filter cleaner, merging filtrate, concentrating under reduced pressure gets the medicinal extract of relative density 1.2 ~ 1.3 then; The water of 2 times of volumes of medicinal extract adding is stirred to and is suspension, with concentration 100% dichloromethane solution extraction 3 times, and each 20min; Combining extraction liquid is crossed the decolouring of MCI post, uses the methanol-eluted fractions of concentration 80% afterwards, collects eluent; Use the desugar of D101 macroporous resin column again,, collect eluent with the eluant solution of methanol (V/V) 70:30; Concentrate medicinal extract, last HPLC post separates, 75% methanol aqueous solution wash-out, last Japanese aestheticism is wished semi-preparative column and is carried out purifying, the methanol aqueous solution wash-out with 75% obtains the sweroside compound.
Embodiment 4
Adopt the desugar of D201 macroporous resin column, all the other are with embodiment 1.
Embodiment 5
Adopt the desugar of D301 macroporous resin column, all the other are with embodiment 2.
Embodiment 6
The preparation of the sweroside aqueous solution of 1 μ g/ml.Take by weighing the sweroside compound 2 μ g that separate to purify and be dissolved in a small amount of DMSO solution, fully mix, treat that it fully dissolves after, the distilled water that adds surplus is processed aqua to final volume 2ml, promptly gets the sweroside aqueous solution of 1 μ g/ml.Get final product according to the desired concn dilution during use.
Embodiment 7
The preparation of the sweroside aqueous solution of 20 μ g/ml.Take by weighing the sweroside compound 20 μ g that separate to purify and be dissolved in a small amount of DMSO solution, fully mix, treat that it fully dissolves after, the distilled water that adds surplus is processed aqua to final volume 1ml, promptly gets the sweroside aqueous solution of 20 μ g/ml.Get final product according to the desired concn dilution during use.
Embodiment 8
The preparation of the 100 μ g/ml sweroside aqueous solution.Take by weighing the sweroside compound 100 μ g that separate to purify and be dissolved in a small amount of DMSO solution, fully mix, treat that it fully dissolves after, the distilled water that adds surplus is processed aqua to final volume 1ml, promptly gets the sweroside aqueous solution of 100 μ g/ml.Get final product according to the desired concn dilution during use.
Embodiment 9
Preparation 1g sweroside pulvis takes by weighing the 10mg sweroside, adds a little DMSO and makes it abundant dissolving; And then add 0.1g bovine serum albumin white powder, and add the abundant stirring and evenly mixing of water, add 20% starch then as excipient, fully mixing grinds after the drying and processes pulvis.During use Dilution for powder to desired concn is got final product.
Embodiment 10
Preparation 1g sweroside pulvis takes by weighing the 0.95g sweroside, adds a little DMSO and makes it abundant dissolving; And then add 0.01g bovine serum albumin white powder, and add the abundant stirring and evenly mixing of water, add surplus starch then as excipient, fully mixing grinds after the drying and processes pulvis.During use Dilution for powder to desired concn is got final product.
Embodiment 11
Preparation 1g sweroside granule takes by weighing the 0.15g sweroside, adds a little DMSO and makes it abundant dissolving; And then add 0.5g bovine serum albumin white powder, and add the abundant stirring and evenly mixing of water, add 15% starch and 15% pectin then as excipient, fully mixing becomes granule after granulation and the drying.During use granule being diluted to desired concn gets final product.
Embodiment 12
Preparation 1g sweroside granule takes by weighing the 0.001g sweroside, adds a little DMSO and makes it abundant dissolving; And then add 0.5g bovine serum albumin white powder, and add the abundant stirring and evenly mixing of water, add 20% starch and 20% pectin then as excipient, fully mixing becomes granule after granulation and the drying.During use granule being diluted to desired concn gets final product.
Embodiment 13
Preparation 1g sweroside emulsion takes by weighing the 0.99g sweroside, adds a little DMSO and makes it abundant dissolving; Add 10% neopelex then as emulsifier, fully mixing becomes emulsion.During use emulsion being diluted to desired concn gets final product.
Embodiment 14
Preparation 1g sweroside tablet takes by weighing the 0.4g sweroside, adds a little DMSO and makes it abundant dissolving; And then add 0.1g wheat gluten (powder), and add the abundant stirring and evenly mixing of water, add 40% starch then as excipient, fully mixing is suppressed into tablet after the drying.During use tablet being diluted to desired concn gets final product.
Embodiment 15
The TMV formulations prepared from solutions of 10 μ g/ml.Draw the TMV mother liquor 1ml of the 100 μ g/ml that prepared, the phosphate buffer that adds surplus promptly gets the TMV solution of 10 μ g/ml to final volume 10ml.
Embodiment 16
The inhibitory action test that sweroside just infects TMV.Adopt the withered spot method of half leaf to Nicotiana glutinosa artificial infection TMV.Choose healthy and well-grown Nicotiana glutinosa, place a night in the darkroom.Every cigarette seedling is selected 3 identical blades of horizontal level.The right half part of each blade is evenly sprayed the sweroside solution 100 μ l of 10 μ g/ml, and left-half is sprayed the distilled water that 100 μ l contain identical DMSO concentration, the sprinkling amount all soak with the blade face and not following be as the criterion.Behind 2 h with the TMV 200 μ l of the even frictional inoculation 10 μ g/ml of whole blade.With 1000 times of liquid of 8% Ningnanmycin aqua is contrast.Each handles 3 strain cigarettes totally 10 tobacco leaves.Add up the withered spot number order of left and right sides blade after 5 days respectively and calculate withered spot inhibiting rate according to the withered spot number on the blade.The inhibiting rate that compound infects TMV=[(positive control withered spot number-compound treatment withered spot number)/positive control withered spot number] * 100%.
Experimental result: table 1 for the compound sweroside when 10 μ g/ml concentration and the rejection ratio that on Nicotiana glutinosa, TMV just infected of control drug Ningnanmycin.Can know that by the result compound sweroside is 48.5% to the average inhibiting rate of TMV, and Ningnanmycin has only 35% to the inhibiting rate of TMV.Therefore, sweroside inhibition effect that TMV is just infected is better than marketed drugs " Ningnanmycin ".
The comparison of the inhibiting rate that table 1 sweroside and Ningnanmycin just infect TMV on Nicotiana glutinosa
Embodiment 17
The sweroside solution of variable concentrations is to the value-added influence test of TMV.Choose healthy and well-grown system host's tobacco bred K326, place a night in the darkroom.Sweroside solution with variable concentrations evenly is sprayed on the blade face, the sprinkling amount all soak with the blade face and not following be as the criterion.Behind the 2h with the TMV 200 μ l of the even frictional inoculation 10 μ g/ml of whole blade.With 1000 times of liquid of 8% Ningnanmycin aqua is contrast.Inoculate after 3 days and detect the viral propagation situation of TMV with TAS-ELISA.
Experimental result: along with the increase of sweroside concentration, the content of virion reduces gradually.When sweroside concentration was 10 μ g/ml, it obviously was superior to the marketed drugs Ningnanmycin to the inhibition effect that TMV duplicates in the host.When the concentration of sweroside reaches 15 μ g/ml, detected exist (Fig. 1) less than virus.
Embodiment 18
Sweroside forms the influence test of symptom on the cigarette seedling to TMV.Choose healthy and well-grown withered spot host Nicotiana glutinosa, place a night in the darkroom.Evenly be sprayed on right half blade face, blade face with the sweroside solution of 15 μ g/ml, left half foliage spray clear water compares, the sprinkling amount all soak with the blade face and not following be as the criterion.Behind the 2h with the TMV 200 μ l of the even frictional inoculation 10 μ g/ml of whole blade.With 1000 times of liquid of 8% Ningnanmycin aqua is contrast.The symptom of observing the cigarette seedling on the 5th day behind the inoculation TMV.
Experimental result: as shown in Figure 2, with inoculating TMV behind the 15 μ g/ml sweroside solution-treated cigarette seedlings, there is not withered spot symptom to occur after 5 days on the cigarette seedling basically.The withered spot number order significantly is less than positive control, also obviously is less than the cigarette seedling that Ningnanmycin is handled.Show that sweroside can suppress or delay the performance of TMV symptom on tobacco, it suppresses effect and is better than the marketed drugs Ningnanmycin.
Claims (10)
1. a sweroside pharmaceutical preparation is characterized in that containing sweroside 0.1 ~ 99%, and all the other are pharmaceutic adjuvant acceptable and/or excipient on the pharmacology.
2. sweroside pharmaceutical preparation as claimed in claim 1 is characterized in that containing sweroside 0.5 ~ 90%, and all the other are pharmaceutic adjuvant acceptable and/or excipient on the pharmacology.
3. according to claim 1 or claim 2 sweroside pharmaceutical preparation is characterized in that containing sweroside 1 ~ 50%, and all the other are pharmaceutic adjuvant acceptable and/or excipient on the pharmacology.
4. according to claim 1 or claim 2 sweroside pharmaceutical preparation is characterized in that containing sweroside 5 ~ 20%, and all the other are pharmaceutic adjuvant acceptable and/or excipient on the pharmacology.
5. a method for distilling that extracts any described sweroside of claim 1 ~ 4 is characterized in that with the surrounded swertia herb being raw material, and extracts the sweroside compound by the technology that comprises the following steps:
A, get the surrounded swertia herb that dries in the shade and pulverize, with 75 ~ 85% methanol extraction 2 ~ 3 times, each 1 ~ 3h;
B, merging extract, filter cleaner, merging filtrate, concentrating under reduced pressure gets the medicinal extract of relative density 1.2 ~ 1.3 then;
The water of C, 1 ~ 3 times of volume of medicinal extract adding is stirred to and is suspension, with concentration 95 ~ 100% dichloromethane solutions extraction 2 ~ 3 times, each 15 ~ 25min;
D, combining extraction liquid are crossed the decolouring of MCI post, use the methanol-eluted fractions of concentration 70 ~ 80% afterwards, collect eluent;
E, use the macroporous resin column desugar again, and, collect eluent, concentrate to such an extent that contain the medicinal extract of 70 ~ 80% methyl alcohol with the eluant solution of methanol (V/V) 0 ~ 100:100 ~ 0;
F, last HPLC separate, and with 70 ~ 80% methanol aqueous solution wash-outs, carry out purifying with semi-preparative column at last, obtain the sweroside compound.
6. the method for extraction sweroside as claimed in claim 5 is characterized in that described macroporous resin column is any one among D900, D130, D101, D201 or the D301.
7. the method for extraction sweroside as claimed in claim 5 is characterized in that described semi-preparative column is any one in the uncommon semi-preparative column of Ai Jieer semi-preparative column, HPLC semi-preparative column or Japanese aestheticism.
8. the formulation of the described any sweroside pharmaceutical preparation of claim 1 ~ 4; It is characterized in that by prescription requirement ratio with the sweroside compound with to nontoxic pharmaceutically acceptable auxiliaries of humans and animals and/or mixed with excipients, be mixed with aqua, tablet, granule or microemulsion.
9. the application of the described any sweroside pharmaceutical preparation of claim 1 ~ 4 in preparation control tobacco mosaic virus disease preparation.
10. sweroside pharmaceutical preparation as claimed in claim 9 is 1 ~ 20 μ g/ml in the sweroside solution concentration that preparation prevents and treats in the tobacco mosaic application.
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| CN107573392A (en) * | 2017-10-16 | 2018-01-12 | 南开大学 | The Genipin derivative of a kind of glycosyl substitution and its preparation and application |
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| CN102696638B (en) | 2013-10-16 |
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