CN1321642A - Cynanchun komarovii extract and its application - Google Patents
Cynanchun komarovii extract and its application Download PDFInfo
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- CN1321642A CN1321642A CN 00106234 CN00106234A CN1321642A CN 1321642 A CN1321642 A CN 1321642A CN 00106234 CN00106234 CN 00106234 CN 00106234 A CN00106234 A CN 00106234A CN 1321642 A CN1321642 A CN 1321642A
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Abstract
The present invention relates to a crude extract of Cynanchum komarovii Al.Iljinski and its activity for resisting plant virus. The active component resisting virus of tobacco mosaic disease in the ethyl alcohol extract of above-ground portion of the Cynanchum komarovii Al.Iljinski is phenanthronidolizidine alkaloid, in which 7-demethoxytylophorine (antofine) is its main active component. As compared with existent extensively-used preparation for resisting tobacco mosaic disease, its activity can be raised by 1-2 orders of magnitude. Said invention can be extensively used for controlling viral disease of plant.
Description
The invention belongs to the natural anti-plant viral disease preparation in the biocontrol agent.
Biocontrol agents such as sterilant, sterilant and weedicide have all been obtained development rapidly in recent years, and the disease pest and weed in many agriculture productions has all obtained control effectively.Yet because the singularity of structure of plant virus and physiology, biochemical aspect, the human at present preparation that does not also find efficient, safe anti-plant viral disease, thereby plant virus is also just more and more big to the harm of agricultural and the loss that caused, therefore at present to the very urgent (Zhu Shuifang of needs of the preparation of efficient, safe anti-plant viral disease, world agriculture, 1989,1,40-42; Sunsun Kubo plants the disease newspaper day, and 1990,65,481-487).
The existing people of Niuxin Pozicao (Cynanchum komanovii Al.Iljinski) has carried out fitochemical studies, and isolates corresponding alkaloid (Fang Shengding, Zhang Rui, Chen Yan etc., Botany Gazette, 1989,31 (2), 934-938; 1991,33 (11), 870-875), but do not report that this plant has the anti-plant viral disease activity, did not more mention the activity of the Antiphytoviral of 7-demethoxytylophorine.
The purpose of this invention is to provide a kind of Cynanchun komarovii extract and application thereof, the present invention is the preparation of efficient, a safe anti-plant viral disease, to tobacco mosaic virus (TMV) (Tobacco Mosaic Virus, TMV) stronger inhibition activity is arranged, luxuriant and rich with fragrance parallel indoles Li Xiding Alkaloid (phenanthroindolizidine alkaloids) is its reactive site.
The present invention is the ethanol extraction that picks up from the over-ground part of the husky Niuxin Pozicao (Cynanchum komanovii Al.Iljinski) of giving birth to of West Inner Mongolia, its main component is following alkaloid compound, and 7-demethoxytylophorine (antofine) is its main effectively activeconstituents:
X=H, OCH
3: Y=O, electron pair; The 7-demethoxytylophorine
The CA accession number of 7-demethoxytylophorine is 054781-87-2.(Ralph?C.Bick?and?WanneeSinchai：The?Alkaloids?XIX，Academic?Press，Inc.1981.Chapter?3，Phenanthroindolizidine?and?Phenanthroquinolizidine?Alkaloids，pp191)
The invention process method is described below:
With over-ground part natural air drying, the chopping of the Niuxin Pozicao of full-bloom stage harvesting, extract No. three to four times ethanol extract that must black behind the extracting solution precipitation with heavily steaming industrial alcohol.According to a conventional method it is divided into neutrality, acidity and alkaline position then.Biological activity determination shows to have only alkaline position that tangible above-mentioned biological activity is arranged.Compound 1,2,3 are isolated through Saphadex LH-20 gel column, silicagel column in the alkalescence position.After recrystallization is further purified, carry out physical constant and spectroscopic data and measure, determine its structure at last.
1,7-demethoxytylophorine 2, N-oxidation 7-demethoxytylophorine 3, tylophorine
antofine???????????????????N-oxide?antofine????????????????tylophorine
Wherein 1 content maximum allows to carry out more careful biological activity determination,
The present invention has very high resisting tobacco mosaic virus (TMV) activity, reach 70～90% at the withered spot inhibiting rate of 500ppm concentration, surpass contrast medicine virus of A, virazole, DADHT and DHT (DADHT and DHT are abroad commercialization plant virus controlling medicine), the prevention effect of potato virus Y also is better than DADHT.Further adopt the biological activity tracking to determine that reactive site is an alkaloid, this alkaloid (total alkali) reaches 70～80% at the withered spot inhibiting rate of 50ppm concentration.The further separation of this alkaloid (total alkali) and structure, biological activity are identified, the proof active constituent is the 7-demethoxytylophorine, inhibiting rate to tobacco mosaic disease under the 1ppm concentration still reaches 60%, exceeds 1～2 order of magnitude than the activity of at present the most frequently used resisting tobacco mosaic disease preparation.This active substance that clearly the present invention found will can be used as the preparation of an anti-plant viral disease efficiently, particularly resisting tobacco mosaic disease.Moreover, involved in the present invention to the crude extract of Niuxin Pozicao and the preparation that the total alkaloids separated all can be used as anti-plant viral disease, particularly tobacco mosaic disease.
The present invention finds that the ethanol extract total alkaloids of over-ground part of Niuxin Pozicao to the viroses of plant, particularly has the obvious suppression effect to tobacco mosaic disease; Alkaloid position in this plant extraction liquid is the main position of control of plant virosis; Main active constituent is luxuriant and rich with fragrance parallel indoles Li Xiding Alkaloid in this alkaloid position; Main active constituent 1 is 7-demethoxytylophorine (antofine) in this alkaloid; The 7-demethoxytylophorine particularly has very strong restraining effect to tobacco mosaic disease to the viroses of plant;
Substantive distinguishing features that the present invention gives prominence to and unusual effect can be embodied from following embodiment, but they are not that the present invention is imposed any restrictions.
Embodiment:
Over-ground part natural air drying, the chopping of the Niuxin Pozicao that the West Inner Mongolia sand of full-bloom stage harvesting is living, getting 10kg leaches three times with the industrial alcohol that heavily steams (cover vegetable material and be degree) normal temperature, united extraction liquid, vacuum precipitation (40 ℃) gets 1185g black thickness medicinal extract.This medicinal extract is 70% (1000ppm) to the inhibiting rate of tobacco mosaic virus disease.
Aqueous citric acid solution stirring and dissolving with the medicinal extract 150g and the 500mL 5% of gained, with 250mL ethyl acetate extraction three times, the sodium hydroxide solution extraction of united extraction liquid and usefulness 250mL 5% three times, tell organic phase, use anhydrous sodium sulfate drying, the vacuum precipitation, the dope 35g of black, be neutral position; Merge sodium hydroxide extraction liquid, the hcl acidifying with 10% is used the 250mL ethyl acetate extraction three times again behind pH=3～4, the combined ethyl acetate extracting solution, and anhydrous sodium sulfate drying, the vacuum precipitation gets acid sites 21g.The lemon aqueous acid is after suction filtration is removed insoluble impurities, and the ammoniacal liquor with 25% neutralizes behind the pH=10, extracts three times with trichloromethane 250mL, and united extraction liquid is used anhydrous sodium sulfate drying, and the vacuum precipitation gets the 0.8g alkaline matter, promptly alkaline position.Biological activity determination shows to have only alkaline position that obvious suppression plant virus activity is arranged.
The alkaline matter of gained at first carries out gel chromatography (Sephadex LH-20, CHCl
3: CH
3OH=1: 1) to remove the material that contains carbohydrate.Carry out silica gel column chromatography (Shandong marine chemical industry factory rapid column chromatography silica gel then, 150g, elutriant are trichloromethane: methyl alcohol: the lower floor of water tertiary mixture, 10: 1: 1,5: 1: 1,5: 2: 1), with the alkaloid (developer is a Dragendorf reagent) in the thin-layer chromatography detection elutriant, the fraction that will contain same substance merges, precipitation, obtain compound 145mg, 216mg, 38mg with trichloromethane and methanol mixture recrystallization then.
1: lurid crystal, m.p.205～208 ℃, [α]
D 25=-195 °.MS: direct injection, M
+363 (79%), 294 (100%).
1H NMR,
13C NMR is all consistent with known compound 7-demethoxytylophorine (antofine).
2: colourless acicular crystal, m.p.197～198 ℃, [α]
D 25=-65 °.MS: direct injection, M+379 (3%), 363 (25%), 294 (100%).
1H NMR,
13C NMR is all consistent with N-oxidation 7-demethoxytylophorine.
3: pale yellow crystals, m.p.289-291 ℃, MS:393 (35%), 324 (100%).
Compound 1,2 and 3 part nuclear magnetic data see Table 1.
Table 1 compound 1,2 and 3 nuclear magnetic data (CDCl
3, TMS=0ppm)
| Compound | ?????????????1 | ????????????????2 | ????????3 | ||
| The position | ????δ ???? 13C | ????δ 1H(J＝Hz) | ????δ ???? 13C | ????δ 1H ????(J＝Hz) | ????δ 1H ????(J＝Hz) |
| ????1 | ????1 ????03.6 | ????7.24(s) | ????1 ????03.6 | ????7.16(s) | ????7.34(s) |
| ????2 | ????1 ????49.1 | ????1 ????49.4 | |||
| ????3 | ????1 ????48.1 | ????1 ????48.8 | |||
| ????4 | ????1 ????03.6 | ????7.84(s) | ????1 ????04.0 | ????7.77(s) | ????7.84(s) |
| ????4a | ????1 ????23.3 | ????1 ????20.9 | |||
| ????4b | ????1 ????29.9 | ????1 ????30.4 | |||
| ????5 | ????1 ????04.3 | ????7.84(s) | ????1 ????04.7 | ????7.77(s) | ????7.84(s) |
| ????6 | ????1 ????57.3 | ????1 ????57.7 | |||
| ????7 | ????11 ????4.7 | ????7.15(dd， ????J＝8.8Hz) ????J＝2.0Hz | ????11 ????5.0 | ????7.07(dd， ????J＝8.8Hz) ????J＝1.5Hz | |
| ????8 | ????1 ????25.1 | ????7.33(d， ????J＝8.8Hz) | ????1 ????23.6 | ????7.48(d， ????J＝8.8Hz) | ????7.16(s) |
| ????8a | ????1 ????25.1 | ????1 ????23.4 | |||
| ????8b | ????1 ????26.7 | ????1 ????23.8 | |||
| ????9 | ????5 ????3.3 | ????H β4.05(d， ????J＝15Hz) ????H α3.65(d， ????J＝15Hz) | ????6 ????9.54 | ????H β5.09(d， ????J＝15Hz) ????H α4.47(d， ????J＝15Hz) | ????H β4.63(d， ????J＝15Hz) ????H α3.70(d， ????J＝15Hz) |
| ????11 | ????5 ????5.6 | ????H β3.26(m) ????H α2.45(m) | ????6 ????6.05 | ????H β3.39(m) ????H α3.28(m) | ????H β2.54(m) ????H α3.47(m) |
| ????12 | ????2 ????1.3 | ????1 ????9.91 | ????1.90-2.22(m) | ????1.9-2.1(m) | |
| ????13 | ????3 ????0.9 | ????1.74-1.80(m) ????1.97-2.15(m) | ????2 ????7.07 | ????2.30-2.47(m) | ????1.78(m)， ????2.32(m) |
| ????13a | ????5 ????9.9 | ????2.45(m) | ????6 ????9.22 | ????2.98(m) | ????2.55(m) |
| ????14 | ????3 ????3.1 | ????H β3.44(m) ????H α2.89(m) | ????2 ????7.33 | ????H α3.34(m) ????H β3.10(m) | ????H β2.90(m) ????H α3.38(m) |
| ????14a | ????1 ????25.5 | ????1 ????23.7 | |||
| ????14b | ????1 ????26.7 | ????1 ????26.0 | |||
| ????- ????OCH 3(2) | ????5 ????5.8 | ????4.07(s) | ????5 ????5.90 | ????3.98(s) | ????4.05(s) |
| ????- ????OCH 3(3) | ????5 ????5.6 | ????4.10(s) | ????5 ????5.90 | ????4.05(s) | ????4.11(s) |
| ????- ????OCH 3(6) | ????5 ????5.5 | ????3.98(s) | ????5 ????5.42 | ????3.95(s) | ????4.11(s) |
| ????- ????OCH 3(7) | ????4.05(s) | ||||
[6]Fumiko?Abe，Yukiko?Inase，Tatsuo?Yamauchi，et?al.，Phytochemistry，1995，
39(3)，695～699
Give birth to the survey method:
1, half leaf method.
1), tobacco mosaic virus (TMV) TMV (tobacco mosaic virus) for the prelibation source:; 2), marmor upsilon PVY (Potato Virus Y).
Test plant: three lives cigarette (Nicotiana tabacum cv.Samsum, the host of TMV system is used to breed malicious source); Coral west cigarette (N.Tabacum cv.Xanthi-NC, TMV dead leaf host are used for antiviral biological activity determination).
Virus inoculation: with the juice inoculation method that rubs.Get the sick leaf of three lives cigarette of fresh typical TMV virus symptom, and the adding phosphate buffer solution (0.01M, pH7.2), grind in mortar, the butt writing brush that boiled with boiling water dips in taking juice then, inoculates on the western tobacco leaf face of the coral that spreads silicon carbide, and get express developed with clear water, leave standstill moments later and move again.
Sample preparation: TMV inoculation was cut the inoculation blade after 1.5～2 hours, was cut into two along the blade master pulse and equated half leaf, and a half vane soaks leaf with different compounds to be handled, and second half blade is dipped in clear water and compares, and " Invest, Then Investigate " was added up half leaf withered spot number in 72 hours.Suppressing percentage calculates by following formula:
The mixture that is extracted and the biological activity test of isolated compound the results are shown in Table 2.
Table 2 activity of resisting tobacco mosaic virus
| Test compound | Concentration ppm | Withered spot inhibiting rate % |
| ????1 | ????1 | ????60 |
| ????1 | ????10 | ????65 |
| ????1 | ????50 | ????80 |
| ????2 | ????500 | ????40 |
| Total alkali | ????50 | ????70～80 |
| Crude extract | ????500 | ????70～90 |
| Virus of A | ????500 | ????40～50 |
| Virazole | ????500 | ????40～50 |
| ????DADHT * | ????500 | ????50 |
| ????DHT ** | ????500 | ????50 |
*DADHT:1,5-diacetyl-2,4-dioxy six hydrogen-1,3,5-triazines
*DHT:2,4-dioxy six hydrogen-1,3,5-triazines
2, plant method:
Utilize the plant method to test crude extract and the control TMV of DADHT and the activity of PVY of Niuxin Pozicao, method is as follows:
A inoculates earlier and afterwards sprays medicine, the same half leaf method of virus inoculation, and the TMV inoculation sprayed the organic compound of different concns after 1.5～2 hours, and other establishes the clear water contrast, 72 hours " Invest, Then Investigate " statistics blade withered spot numbers;
B inoculates behind the spray medicine earlier, sprays compound earlier after 24 hours, inoculation TMV virus (the same half leaf method of method), and other establishes clear water and compares, 72 hours " Invest, Then Investigate " statistics blade withered spot numbers.
Test-results sees Table 3.
Table 3 crude extract results from pot experiment test, 500ppm
Claims (4)
1. Cynanchun komarovii extract, its main component is following alkaloid compound:
X=H, OCH
3Y=O, electron pair;
It is characterized in that the preparation method is as follows: with over-ground part natural air drying, the chopping of the Niuxin Pozicao of full-bloom stage harvesting, extract three to four times, obtain ethanol extract, ethanol extract that must black behind the extracting solution precipitation with heavily steaming industrial alcohol.
2. the application of the said Cynanchun komarovii extract of claim 1 is characterized in that it is used for the inhibition of the viroses of plant.
3. according to the application of the said Cynanchun komarovii extract of claim 2, it is characterized in that it is used for the inhibition of tobacco mosaic virus (TMV) TMV, marmor upsilon PVY.
4. according to the application of claim 2,3 said Cynanchun komarovii extracts, the effective active composition that it is characterized in that it is the 7-demethoxytylophorine, and working concentration is 1-1000ppm.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB001062344A CN1156475C (en) | 2000-04-28 | 2000-04-28 | Cynanchun Komarovii extract and its application |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB001062344A CN1156475C (en) | 2000-04-28 | 2000-04-28 | Cynanchun Komarovii extract and its application |
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| Publication Number | Publication Date |
|---|---|
| CN1321642A true CN1321642A (en) | 2001-11-14 |
| CN1156475C CN1156475C (en) | 2004-07-07 |
Family
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB001062344A Expired - Lifetime CN1156475C (en) | 2000-04-28 | 2000-04-28 | Cynanchun Komarovii extract and its application |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1310592C (en) * | 2005-09-13 | 2007-04-18 | 西北大学 | Pollution-less botanical pesticide for fish |
| CN101189968B (en) * | 2006-11-23 | 2011-06-01 | 南开大学 | Phenanthroindolizidine and phenanthroquinolizidine derivatives and applications of salts in pesticides |
| WO2012146194A1 (en) * | 2011-04-29 | 2012-11-01 | Industrial Technology Research Institute | Extracts of cynanchum sp. and active ingredients contained therein in use of arthritis treatment |
| CN103446211A (en) * | 2013-09-24 | 2013-12-18 | 兰州理工大学 | Cynanchum komarovii total alkaloid and preparing method and application thereof |
| WO2021208711A1 (en) * | 2020-04-17 | 2021-10-21 | 南开大学 | Application of tylophora alkaloid or salt thereof in resisting coronavirus |
-
2000
- 2000-04-28 CN CNB001062344A patent/CN1156475C/en not_active Expired - Lifetime
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1310592C (en) * | 2005-09-13 | 2007-04-18 | 西北大学 | Pollution-less botanical pesticide for fish |
| CN101189968B (en) * | 2006-11-23 | 2011-06-01 | 南开大学 | Phenanthroindolizidine and phenanthroquinolizidine derivatives and applications of salts in pesticides |
| WO2012146194A1 (en) * | 2011-04-29 | 2012-11-01 | Industrial Technology Research Institute | Extracts of cynanchum sp. and active ingredients contained therein in use of arthritis treatment |
| GB2498677A (en) * | 2011-04-29 | 2013-07-24 | Ind Tech Res Inst | Extracts of cynanchum SP.and active ingredients contained therein in use of arthritis treatment |
| JP2014512400A (en) * | 2011-04-29 | 2014-05-22 | インダストリアル テクノロジー リサーチ インスティテュート | Kinancum plant extract for the treatment of arthritis and active ingredients contained therein |
| US9549958B2 (en) | 2011-04-29 | 2017-01-24 | Industrial Technology Research Institute | Extracts of Cynanchum sp. and active ingredients contained therein in use of arthritis treatment |
| CN103446211A (en) * | 2013-09-24 | 2013-12-18 | 兰州理工大学 | Cynanchum komarovii total alkaloid and preparing method and application thereof |
| CN103446211B (en) * | 2013-09-24 | 2016-02-10 | 兰州理工大学 | A kind of Cynanchum Komarovii total alkaloids and its preparation method and application |
| WO2021208711A1 (en) * | 2020-04-17 | 2021-10-21 | 南开大学 | Application of tylophora alkaloid or salt thereof in resisting coronavirus |
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| Publication number | Publication date |
|---|---|
| CN1156475C (en) | 2004-07-07 |
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Granted publication date: 20040707 |