CN1288163C - Preparation process for compound of proto steroid soap oside and application thereof - Google Patents
Preparation process for compound of proto steroid soap oside and application thereof Download PDFInfo
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- CN1288163C CN1288163C CN 200410015473 CN200410015473A CN1288163C CN 1288163 C CN1288163 C CN 1288163C CN 200410015473 CN200410015473 CN 200410015473 CN 200410015473 A CN200410015473 A CN 200410015473A CN 1288163 C CN1288163 C CN 1288163C
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Abstract
The present invention provides two steroid sapogenin compounds which belongs to the derivative of spirostane type steroids replaced by polyhydroxy. The steroid sapogenin has obvious antibacterial activity in vivo and in vitro for peronophythora litchii chen, and can be used as a germicidal agent for preventing and curing peronophythora litchii chen.
Description
Technical field
The present invention relates to two kinds of sapogenin compounds and application thereof.In particular to the preparation of two kinds of steroid sapogeniness and as sterilant in the application of peronophythora litchi (Peronophythoralitchii Chen) aspect preventing and treating.
Background technology
Peronophythora litchi is the disease that is caused by lower fungi (Peronophythora litchii Chen), can cause that the tender tip of lichee is withered, fallen flowers, shedding, decayed fruit, is the main disease that has a strong impact on the lichee yield and quality.At present, the control peronophythora litchi remains based on the chemosynthesis medicament on producing---the many Mils of thunder (Ridomil), and medicament kind is more single, and Shang Weijian has botanical fungicide to be applied to the report of this sick control.
Summary of the invention
Purpose of the present invention provides the active two kinds of sapogenin compounds of plant-sourced that can suppress lichee frost vaccine.
Another object of the present invention provides the preparation method of this compound.
Of the present invention also have a purpose that the application of this active compound in the peronophythora litchi control is provided.
Two compounds provided by the invention are the spirostane type steroid derivative that poly-hydroxy replaces.First compound (abbreviation Compound I), its name is called 1 β, 2 β, 3 β, 4 β, 5 β, 7 α-hexahydroxy-spiral shell steroid-25 (27)-alkene-6-ketone (1 β, 2 β, 3 β, 4 β, 5 β, 7 α-hexahydroxyspirost--25 (27)-en-6-one), its structure is suc as formula I; Second compound (being called for short Compound I I), its name is called spiral shell steroid-25 (27)-alkene-1 β, 2 β, 3 β, 4 β, 5 β, 6 β, 7 α-seven alcohol (spirost-25 (27)-ene-1 β, 2 β, 3 β, 4 β, 5 β, 6 β, 7 α-heptol), its structure is suc as formula II.The spectroscopic data of two compounds is seen embodiment six.
Formula I
Formula II
Compound I and Compound I I all are to be raw material with liliaceous plant Tupistra chinensis Bak. (Tupistrachinensis Bak.) rhizome, separate obtaining by methods such as methyl alcohol immersion, organic solvent branch, silica gel column chromatographies.Its concrete preparation process of separating is: (1) volume ratio by weight adds 3～5 times of Tupistra chinensis Bak. crushed materials heavy methyl alcohol or ethanol in the Tupistra chinensis Bak. crushed material, under refluxad extract, extracting temperature is 60～80 ℃, extraction time is 4～8 hours, or volume ratio adds 3～8 times of Tupistra chinensis Bak. crushed materials heavy methyl alcohol or ethanol in the Tupistra chinensis Bak. crushed material by weight, lixiviate got methyl alcohol or ethanol extraction more than 24 hours; (2) methyl alcohol or ethanol extraction, branch's extraction is more than 3 times successively with 5～10 chloroform and ethyl acetate for volume ratio by weight, and each extraction liquid gets corresponding extract through concentrate drying; (3) chloroform and acetic acid ethyl ester extract are merged, obtain Compound I with silica gel (200～300 order) column chromatography, reverse phase silica gel (RP-18) column chromatography and gel (SephadexLH-20) column chromatography for separation.
Two compounds of the present invention have the activity of anti-peronophythora litchi.With the spore germination method peronophythora litchi biological activity tracking and testing that exsomatizes is proved: concentration (EC in effective inhibition that Compound I is sprouted the peronophythora litchi sporocyst
50) be 100.28 μ g/ml, Compound I I is to concentration (EC in effective inhibition of peronophythora litchi sporocyst sprouting
50) be 124.37 μ g/ml; With inoculation method the live body antibacterial tests that two compounds carry out peronophythora litchi is proved: concentration is Compound I and the Compound I I soup of 500 μ g/mL, method with spraying, immersion or coating can be prevented and treated litchi skill peronospora tabacina, and lichee (kind is " a Huaihe River branch ") peronospora tabacina is had tangible prevention effect.
Two compounds of the present invention can be used as effective constituent separately, combination or composite with the sterilant (thunderous many Mils) of control peronophythora litchi, be used for the control of peronophythora litchi.Plant milk extract, branch's extract and separated part that use contains steroid saponin compound of the present invention also can.
Embodiment
Below in conjunction with example essence of the present invention is described, but content of the present invention is not limited thereto.
Embodiment one: the thick extraction of compound with separate
Tupistra chinensis Bak. rhizome dry powder 3kg, with 95%, 9L methyl alcohol soaks repeatedly till leach liquor is almost colourless, methyl alcohol vat liquor concentrating under reduced pressure gets brown paste 283g, with this enriched material 1500mL sherwood oil, the branch's extraction successively of chloroform and ethyl acetate, concentrating under reduced pressure gets each branch's extract, ethyl acetate extract and chloroform extract are merged 15.5g, last silica gel (100-200 order, 300g) post, carry out the gradient gradient elution with chloroform and methyl alcohol mixed liquor, chloroform and methanol mixed ratio, volume ratio is 9: 1 and 8: 1, is that a cut receives by 400mL, and with the spore germination method to the peronophythora litchi biological activity tracking and testing that exsomatizes, 22-26 cut 1.78g and 27-37 cut 1.32g two active portion.
Embodiment two: the extraction of Compound I with separate
With embodiment one described 22-26 cut, last silica gel (200-300 order, 60g) post, carry out wash-out with chloroform and methyl alcohol, chloroform and methyl alcohol volume ratio are 8: 1.5, by 50mL is that cut is accepted, and with the spore germination method to the peronophythora litchi biological activity tracking and testing that exsomatizes, must 9-12 cut 0.37g active portion.With reverse phase silica gel (RP-18) post on this active portion, with methyl alcohol and water mixed liquid wash-out, methyl alcohol and water volume ratio are 8: 2, are that a cut receives by 10mL, and with the spore germination method to the peronophythora litchi biological activity tracking and testing that exsomatizes, 11-15 cut 0.11g active portion.With gel on this active portion (Sephadex LH-20) post, use the tetrahydrofuran (THF) wash-out, be that a cut receives by 2mL, get 5-7 cut 32mg, be Compound I.
Embodiment three: the extraction of compound ii with separate
With reverse phase silica gel post (RP-18) on the embodiment one described 27-37 cut, with methyl alcohol and water mixed liquid wash-out, methyl alcohol and water volume ratio 8: 2, by 10mL is that a cut receives, and with the spore germination method to the peronophythora litchi biological activity tracking and testing that exsomatizes, get 17-30 cut 852mg active portion, be Compound I I.
Embodiment four: Compound I and Compound I I measure the isolated activity of peronophythora litchi sporocyst germination inhibitor
With Compound I and compound, being made into concentration is the storage liquid of 1000 μ g/mL earlier.Be taken under 25 ℃ and the 98%RH condition again and cultivate, cell age is 5 days a peronophythora litchi bacterial classification, uses aseptic water washing, makes the different concns sporangia suspension.Be made into concentration 50 with the storage liquid of Compound I and Compound I I respectively with the sporangia suspension of different concns, 100,150, the pastille bacterium liquid of 200,250 μ g/ml, the concentration of pastille bacterium liquid are following 20 sporocysts in every visual field, pastille bacterium liquid is shaken up, place under 25 ℃ and the 98%RH condition and cultivated 6 hours, establish blank simultaneously, the pastille bacterium of getting then after the cultivation is added up germination rate or inhibiting rate under 10 * 40 power microscopes.Data statistic analysis gets: Compound I is to concentration (EC in effective inhibition of peronophythora litchi sporocyst sprouting
50) be 100.28 μ g/ml, Compound I I is to concentration (EC in effective inhibition of peronophythora litchi sporocyst sprouting
50) be 124.37 μ g/ml.Result such as accompanying drawing 1.
Fig. 1 is the inhibition that Compound I and Compound I I sprout the peronophythora litchi sporocyst
Compound I and compound ii are to the live body bacteria inhibition assay of peronophythora litchi
Selected lichee (kind is the Huaihe River branch) is washed fruit with surface disinfectant first, then wash down liquid, airing with running water, then dip Compound I and the Compound I I liquid (compound method is with embodiment four) that concentration is 500 μ g/mL with writing brush, evenly coat litchi point place, airing, dip white phytophthora sporangia suspension (2 * 10 with writing brush again5Individual/as mL), evenly to coat dispenser place, individual packing and after cultivating 3 days under 25 ℃ and the 98%RH condition, sick, the strong fruit of statistics is calculated infection rate. Result of the test such as table 1:
Table 1: Compound I and Compound I I are to the antibacterial as a result * of the live body of peronophythora litchi
| Process | Concentration (μ g/mL) | Sick fruit (individual) | Strong fruit (individual) | Susceptible rate (%) |
| Contrast | 79 | 11 | 87.8 a | |
| Compound I | 500 | 15 | 75 | 16.7b |
| Compound I I | 500 | 17 | 73 | 18.9 b |
*The same person of following word parent phase is illustrated on 5% level difference not significantly (DMRT method).
The Structural Identification of steroid saponin
Compound I: be white powder, molecular formula is C27H
40O
9 Proton nmr spectra (400MHz, tritium is for pyridine): δ 4.32 (1H, br s, H-1), (4.40 1H, br s, H-2), 4.74 (1H, br s, H-3), 5.81 (1H, d, J=3.2Hz, H-4), 4.54 (1H, br s, H-7), 4.56 (1H, m, H-16), (0.87 3H, s, H-18), 1.45 (3H, s, H-19), 1.06 (3H, d, J=6.8Hz, H-21), 4.01 (1H, d, J=12.0Hz, H-26eq), 4.41 (1H, d, J=12.0Hz, H-26ax), 4.76 (1H, br s, H-27a), 4.79 (1H, br s, H-27b): carbon-13 nmr spectra (100MHz, tritium is for pyridine): see Table 2.
Compound I I: be white powder, molecular formula is C27H
42O
9 Proton nmr spectra (400MHz, tritium is for pyridine): δ 4.34 (1H, br s, H-1), (4.31 1H, br s, H-2), 4.74 (1H, d, J=3.0Hz, H-3), 5.31 (1H, d, J=3.0Hz, H-4), 5.00 (1H, d, J=2.8Hz, H-6), 4.48 (1H, br s, H-7), 2.61 (1H, br t, J=11.6 Hz, H-8), 4.56 (1H, dd, J=14.4,7.6Hz, H-16), 0.93 (3H, s, H-18), 1.98 (3H, s, H-19), 1.06 (3H, d, J=6.8Hz, H-21), 3.98 (1H, d, J=12.0Hz, H-26eq), 4.40 (1H, d, J=12.0Hz, H-26ax), 4.75 (1H, br s, H-27a), (4.78 1H, br s, H-27b): carbon-13 nmr spectra (100MHz, tritium is for pyridine): see Table 2.
Table 2: the carbon-13 nmr spectra data of Compound I and Compound I I
| Carbon | Compound I | Compound I I | Carbon | Compound I | Compound I I |
| 1 2 3 4 5 6 7 8 9 10 11 12 13 14 | 76.5 67.9 75.5 71.2 86.3 211.1 75.2 40.9 38.0 50.2 22.0 39.4 40.7 49.3 | 79.7 67.2 75.6 69.8 78.2 73.6 71.8 34.6 37.6 40.4 21.4 39.8 40.4 50.1 | 15 16 17 18 19 20 21 22 23 24 25 26 27 | 31.5 81.3 62.9 16.3 13.0 42.1 15.0 109.5 33.2 29.0 144.4 65.1 108.8 | 32.0 81.3 63.1 16.4 15.6 42.1 15.0 109.5 33.2 29.0 144.5 65.0 108.7 |
Claims (1)
1, a kind of sapogenin compound 1 β, 2 β, 3 β, 4 β, 5 β, the preparation method of 7 α-hexahydroxy-spiral shell steroid-25 (27)-alkene-6-ketone is characterized in that with the Tupistra chinensis Bak. being raw material, and raw material after crushed, volume ratio adds the methyl alcohol or the ethanol of 3～5 times of volumes in the Tupistra chinensis Bak. crushed material by weight, under refluxad extract, extracting temperature is 60～80 ℃, and extraction time is 4～8 hours, or volume ratio by weight, in the methyl alcohol or the ethanol of 3～8 times of volumes of Tupistra chinensis Bak. crushed material adding, lixiviate got methyl alcohol or ethanol extraction more than 24 hours; Methyl alcohol or ethanol extraction, by its weightmeasurement ratio, with 5～10 times chloroforms and ethyl acetate branch's extraction successively more than 3 times, each extraction liquid through concentrate extract accordingly; Chloroform and acetic acid ethyl ester extract are merged, separate obtaining described sapogenin compound 1 β, 2 β with 200～300 order silica gel column chromatographies, RP-18 reversed-phase silica gel column chromatography and SephadexLH-20 gel filtration chromatography, 3 β, 4 β, 5 β, 7 α-hexahydroxy-spiral shell steroid-25 (27)-alkene-6-ketone.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410015473 CN1288163C (en) | 2004-02-27 | 2004-02-27 | Preparation process for compound of proto steroid soap oside and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410015473 CN1288163C (en) | 2004-02-27 | 2004-02-27 | Preparation process for compound of proto steroid soap oside and application thereof |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2006100849907A Division CN100420380C (en) | 2004-02-27 | 2004-02-27 | Use of sapogenin compound as bactericidal agent |
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| CN1560070A CN1560070A (en) | 2005-01-05 |
| CN1288163C true CN1288163C (en) | 2006-12-06 |
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| CN 200410015473 Expired - Fee Related CN1288163C (en) | 2004-02-27 | 2004-02-27 | Preparation process for compound of proto steroid soap oside and application thereof |
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Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105494378B (en) * | 2015-12-29 | 2017-12-29 | 中国科学院华南植物园 | Application of 6 benayl aminopurines in anti-Peronophythora Litchii medicine is prepared |
| CN105859825B (en) * | 2016-05-06 | 2017-09-29 | 广东药科大学 | One class spirostanol saponins and its application |
| CN105924494B (en) * | 2016-05-06 | 2017-12-12 | 广东药科大学 | A kind of furostanol saponin class compound and its application |
| CN111848707B (en) * | 2020-09-01 | 2021-08-17 | 中国林业科学研究院林产化学工业研究所 | Steroid compound, preparation method and application thereof |
| CN112209986B (en) * | 2020-09-21 | 2022-11-22 | 西北农林科技大学 | Steroid compound, preparation method and application |
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