CN115057897B - Preparation method for preparing two glucopyranosides by using nematode culture - Google Patents
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- 241000244206 Nematoda Species 0.000 title claims abstract description 51
- 238000002360 preparation method Methods 0.000 title claims abstract description 22
- WYUFTYLVLQZQNH-JAJWTYFOSA-N Ethyl beta-D-glucopyranoside Chemical compound CCO[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O WYUFTYLVLQZQNH-JAJWTYFOSA-N 0.000 claims abstract description 23
- BZANQLIRVMZFOS-HOTMZDKISA-N (2r,3r,4s,5s,6r)-2-butoxy-6-(hydroxymethyl)oxane-3,4,5-triol Chemical compound CCCCO[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O BZANQLIRVMZFOS-HOTMZDKISA-N 0.000 claims abstract description 22
- WYUFTYLVLQZQNH-UHFFFAOYSA-N 1-Ethyl-D-galactoside Natural products CCOC1OC(CO)C(O)C(O)C1O WYUFTYLVLQZQNH-UHFFFAOYSA-N 0.000 claims abstract description 19
- BZANQLIRVMZFOS-UHFFFAOYSA-N n-butyl alpha-D-glucopyranoside Natural products CCCCOC1OC(CO)C(O)C(O)C1O BZANQLIRVMZFOS-UHFFFAOYSA-N 0.000 claims abstract description 19
- 239000000126 substance Substances 0.000 claims abstract description 9
- 238000000034 method Methods 0.000 claims abstract description 5
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 101
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 82
- 239000003208 petroleum Substances 0.000 claims description 41
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 33
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 14
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 12
- 239000001963 growth medium Substances 0.000 claims description 12
- 239000000741 silica gel Substances 0.000 claims description 12
- 229910002027 silica gel Inorganic materials 0.000 claims description 12
- 239000006228 supernatant Substances 0.000 claims description 10
- 239000000047 product Substances 0.000 claims description 9
- 239000000287 crude extract Substances 0.000 claims description 7
- 238000012258 culturing Methods 0.000 claims description 7
- 239000000499 gel Substances 0.000 claims description 7
- 238000002386 leaching Methods 0.000 claims description 7
- 238000010828 elution Methods 0.000 claims description 6
- 235000004535 Avena sterilis Nutrition 0.000 claims description 5
- 241001647031 Avena sterilis Species 0.000 claims description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 5
- 150000001875 compounds Chemical class 0.000 claims description 5
- 238000005406 washing Methods 0.000 claims description 4
- 238000005516 engineering process Methods 0.000 claims description 3
- 238000000605 extraction Methods 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 238000000926 separation method Methods 0.000 claims description 3
- 238000004809 thin layer chromatography Methods 0.000 claims description 3
- 238000000746 purification Methods 0.000 claims description 2
- 238000010898 silica gel chromatography Methods 0.000 claims description 2
- 238000002791 soaking Methods 0.000 claims description 2
- 230000001954 sterilising effect Effects 0.000 claims description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 11
- XLOMVQKBTHCTTD-UHFFFAOYSA-N Zinc monoxide Chemical compound [Zn]=O XLOMVQKBTHCTTD-UHFFFAOYSA-N 0.000 description 8
- 238000001228 spectrum Methods 0.000 description 8
- 238000004440 column chromatography Methods 0.000 description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 4
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 4
- 229910052799 carbon Inorganic materials 0.000 description 4
- 229910052739 hydrogen Inorganic materials 0.000 description 4
- 239000001257 hydrogen Substances 0.000 description 4
- HEGSGKPQLMEBJL-UHFFFAOYSA-N n-octyl beta-D-glucopyranoside Natural products CCCCCCCCOC1OC(CO)C(O)C(O)C1O HEGSGKPQLMEBJL-UHFFFAOYSA-N 0.000 description 4
- HEGSGKPQLMEBJL-RKQHYHRCSA-N octyl beta-D-glucopyranoside Chemical compound CCCCCCCCO[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O HEGSGKPQLMEBJL-RKQHYHRCSA-N 0.000 description 4
- 239000011787 zinc oxide Substances 0.000 description 4
- 238000001819 mass spectrum Methods 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- ZXSQEZNORDWBGZ-UHFFFAOYSA-N 1,3-dihydropyrrolo[2,3-b]pyridin-2-one Chemical compound C1=CN=C2NC(=O)CC2=C1 ZXSQEZNORDWBGZ-UHFFFAOYSA-N 0.000 description 2
- KBPLFHHGFOOTCA-UHFFFAOYSA-N 1-Octanol Chemical compound CCCCCCCCO KBPLFHHGFOOTCA-UHFFFAOYSA-N 0.000 description 2
- 239000000543 intermediate Substances 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 229910001958 silver carbonate Inorganic materials 0.000 description 2
- LKZMBDSASOBTPN-UHFFFAOYSA-L silver carbonate Substances [Ag].[O-]C([O-])=O LKZMBDSASOBTPN-UHFFFAOYSA-L 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- JHQAETOGKIPCPC-YXHKNWEZSA-N (2s,3s,4r,5r)-2,3,4-triacetyl-2,3,4,5,6-pentahydroxy-7-oxooctanoyl bromide Chemical compound CC(=O)C(O)[C@@H](O)[C@](O)(C(C)=O)[C@@](O)(C(C)=O)[C@](O)(C(C)=O)C(Br)=O JHQAETOGKIPCPC-YXHKNWEZSA-N 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Chemical class OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000006196 deacetylation Effects 0.000 description 1
- 238000003381 deacetylation reaction Methods 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- XELZGAJCZANUQH-UHFFFAOYSA-N methyl 1-acetylthieno[3,2-c]pyrazole-5-carboxylate Chemical compound CC(=O)N1N=CC2=C1C=C(C(=O)OC)S2 XELZGAJCZANUQH-UHFFFAOYSA-N 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 125000006239 protecting group Chemical group 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/02—Acyclic radicals, not substituted by cyclic structures
- C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
- C07H1/08—Separation; Purification from natural products
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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Abstract
The invention discloses a preparation method for preparing two glucopyranosides by using nematode culture, and relates to the technical field of glucopyranosides. According to the research of finding two glucopyranosides of ethyl-beta-D-glucopyranoside and butyl-beta-D-glucopyranoside in signal substances secreted by the nematode, the invention designs a brand new method for simultaneously obtaining two glucopyranosides of ethyl-beta-D-glucopyranoside and butyl-beta-D-glucopyranoside, and provides a new thought for the preparation process of the two glucopyranosides of ethyl-beta-D-glucopyranoside and butyl-beta-D-glucopyranoside.
Description
Technical Field
The invention relates to the technical field of glucopyranosides, in particular to a preparation method for preparing two glucopyranosides by using nematode culture.
Background
The ethyl-beta-D-glucopyranoside (wp-2) and the butyl-beta-D-glucopyranoside (wp-1) are commercially available common medical raw material intermediates, and the structures of the intermediates are respectively shown as formula 1 and formula 2:
at present, glucopyranoside compounds are mainly obtained by a chemical synthesis method, for example, the patent publication of the invention with the publication number of CN103159804B provides a preparation method of octyl-beta-D-glucopyranoside, which comprises the steps of mixing 2,3,4, 6-tetraacetyl bromo-glucose, octanol and zinc oxide, reacting to obtain 1-octyl-2, 3,4, 6-tetraacetyl-beta-D-glucopyranoside, and then performing deacetylation protecting group reaction to obtain the octyl-beta-D-glucopyranoside. Firstly, compared with silver carbonate diatomite, zinc oxide is low in price, so that the production cost of octyl-beta-D-glucopyranoside can be reduced; and secondly, zinc oxide is stable and is easy to obtain, so that the preparation method is simple to operate.
However, for the preparation of ethyl-beta-D-glucopyranoside (wp-2) and butyl-beta-D-glucopyranoside (wp-1), the yield of the preparation method is obviously inferior to that of octyl-beta-D-glucopyranoside, and zinc oxide is only cheaper than silver carbonate diatomite, so that the preparation of ethyl-beta-D-glucopyranoside (wp-2) and butyl-beta-D-glucopyranoside (wp-1) in the prior art needs to explore new preparation modes.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a preparation method for preparing two glucopyranosides by using nematode culture, which is used for simultaneously separating and obtaining two glucopyranosides of ethyl-beta-D-glucopyranoside (wp-2) and butyl-beta-D-glucopyranoside (wp-1) from nematode culture for the first time, and provides a brand new preparation method for solving the technical problem that the existing wp-1 and wp-2 are difficult to prepare.
The technical scheme adopted by the invention is as follows:
a preparation method for preparing two glucopyranosides by using nematode culture, which utilizes signal substances secreted by nematodes to extract two glucopyranosides, namely ethyl-beta-D-glucopyranoside and butyl-beta-D-glucopyranoside.
Preferably, the nematode is a holoophorate nematode.
More preferably, the preparation method provided by the invention specifically comprises the following steps:
(1) Biological culture: inoculating the nematodes into a sterile oat culture medium, and culturing for 8-15 d at 28 ℃;
(2) Crude extraction of the product: after the nematodes climb the bottle wall, adding sterile ddH into the oat culture medium 2 O, gently washing out nematodes, transferring to a separating funnel, discarding supernatant after the nematodes settle, repeating for 3-5 times to fully remove oat culture medium, and collecting nematodes; 3 volumes of sterile ddH were added to the nematodes 2 O, culturing at 20 ℃ and 180rpm for 6-10 h to secrete signal substances in the nematodes, centrifuging at 4000rpm for 10-30 min to remove nematode bodies and remaining supernatant, and standing upwardsAdding 3 times of absolute ethyl alcohol into the clear liquid for leaching, carrying out ultrasonic crushing halfway for 3 times each time of leaching for 48 hours, and concentrating by a rotary evaporator to obtain a crude extract;
(3) And (3) purifying a product: and separating and obtaining two glucopyranosides, namely ethyl-beta-D-glucopyranoside and butyl-beta-D-glucopyranoside by adopting a column chromatography technology.
Further, the preparation process of the sterile oat medium in the step (1) is as follows: 100g of oatmeal was weighed and 100mL of ddH was then added 2 O wets the oatmeal and is sterilized at 121℃until use.
Still further, in step (3), the product purification is specifically operated as: dissolving the crude extract with a small amount of methanol, and performing preliminary separation by a methanol gel column to obtain 4 components: A1-A4, mixing the component A2 with equal weight of silica gel, separating by silica gel column chromatography, eluting with ethyl acetate to obtain 4 components: a2-1, A2-2, A2-3 and A2-4, wherein the component A2-2 is mixed with equal weight of silica gel and then sequentially passes through a silica gel column, and step elution is sequentially carried out by using petroleum ether/acetone solution according to the step volume ratio of petroleum ether from more to less, so as to obtain two components: a2-2-1, A2-2-2; and (3) purifying the components A2-2-1 and A2-2-2 respectively by a methanol gel column to obtain the compound: ethyl-beta-D-glucopyranoside, butyl-beta-D-glucopyranoside.
Still further, the step volume ratio of the petroleum ether/acetone solution is set as follows: petroleum ether: acetone=50: 1. petroleum ether: acetone=40: 1. petroleum ether: acetone=30: 1. petroleum ether: acetone=20: 1. petroleum ether: acetone=10: 1. petroleum ether: acetone=8: 1. petroleum ether: acetone=5: 1. petroleum ether: acetone=4: 1. petroleum ether: acetone=3: 1. petroleum ether: acetone = 2:1, and petroleum ether: acetone = 2:1 to obtain two components A2-2-1 and A2-2-2 during elution.
In summary, compared with the prior art, the invention has the following advantages and beneficial effects:
1. according to the research of finding two glucopyranosides of ethyl-beta-D-glucopyranoside and butyl-beta-D-glucopyranoside in signal substances secreted by the nematode, a brand new method for simultaneously obtaining the two glucopyranosides of ethyl-beta-D-glucopyranoside and butyl-beta-D-glucopyranoside is designed, and a new thought is provided for the preparation process of the two glucopyranosides of ethyl-beta-D-glucopyranoside and butyl-beta-D-glucopyranoside;
2. the invention prepares two glucopyranosides of ethyl-beta-D-glucopyranoside and butyl-beta-D-glucopyranoside by biological culture, and has lower price and simpler preparation method on the used raw materials.
Drawings
FIG. 1 is a nuclear magnetic resonance carbon spectrum of wp-1 prepared in example 1;
FIG. 2 is a nuclear magnetic resonance hydrogen spectrum of wp-1 prepared in example 1;
FIG. 3 is an ultra-high resolution mass spectrum of wp-1 prepared in example 1;
FIG. 4 is a nuclear magnetic resonance carbon spectrum of wp-2 prepared in example 1;
FIG. 5 is a nuclear magnetic resonance hydrogen spectrum of wp-2 prepared in example 1;
FIG. 6 is a map of the ultra-high resolution mass of wp-2 prepared in example 1.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be further described in detail with reference to the accompanying drawings and embodiments. It should be understood that the particular embodiments described herein are illustrative only and are not intended to limit the invention, i.e., the embodiments described are merely some, but not all, of the embodiments of the invention.
The invention provides a preparation method for preparing two glucopyranosides by using nematode culture, which comprises the following steps:
(1) Biological culture: inoculating the full-tooth reviving nematode in a sterile oat culture medium, and culturing for 8-15 d at 28 ℃;
(2) Crude extraction of the product: after the nematodes climb the bottle wall, adding sterile ddH into the oat culture medium 2 O, gently washing out nematodes, transferring to a separating funnel, discarding supernatant after the nematodes settle, and repeating for 3-5 times to completely remove oat culture mediumCollecting nematodes; 3 volumes of sterile ddH were added to the nematodes 2 O, culturing at 20 ℃ and 180rpm for 6-10 hours to secrete signal substances in the nematodes, centrifuging at 4000rpm for 10-30 minutes to remove supernatant remained by the nematodes, adding 3 times of absolute ethyl alcohol into the supernatant for leaching, carrying out leaching for 48 hours each time, carrying out ultrasonic crushing midway, repeating for 3 times, and concentrating by a rotary evaporator to obtain a crude extract;
(3) And (3) purifying a product: and separating and obtaining two glucopyranosides, namely ethyl-beta-D-glucopyranoside and butyl-beta-D-glucopyranoside by adopting a column chromatography technology.
Example 1
The reagent used in this example was as follows:
oat medium: 100g oatmeal, 100mL ddH was added 2 O soaking oatmeal, and sterilizing at 121 ℃ for later use;
experimental nematodes: the whole-tooth reviving nematodes are cultured in oat medium.
Absolute ethanol (analytically pure, part of the sciences company, inc.) solvent for nematode extract; petroleum ether, ethyl acetate, acetone and methanol which are used for separating and purifying the compounds are all industrial pure solvents for heavy evaporation; the GF254 thin layer chromatography silica gel plate and 200-300 mesh column chromatography silica gel are used as ocean chemical plant of Qingdao in China, and the column chromatography methanol gel Sephadex LH-20 is a Pharmacia product; the nuclear magnetic resonance instrument is Bruker DRX-500 and Bruker Avance III-600NMR superconducting nuclear magnetic resonance instrument; the mass spectrometer was Finnigan LCQ-Advantage.
The test process comprises the following steps:
inoculating the whole-tooth reviving nematode in aseptic oat culture medium, culturing at 28deg.C for 8d-15d, adding aseptic ddH into oat culture medium after the nematode climbs the bottle wall 2 O, gently washing out nematodes, transferring to a separating funnel, discarding supernatant after the nematodes settle, repeating for 3-5 times to fully remove oat culture medium, and collecting nematodes; 3 volumes of sterile ddH were added 2 O, culturing at 20 ℃ for 7 hours at 180rpm to secrete signal substances in the nematodes, centrifuging at 4000rpm for 10 minutes to remove nematode bodies, adding 3 times of absolute ethyl alcohol into supernatant to carry out leaching, carrying out ultrasonic crushing halfway each time for 48 hours, repeating for 3 times, and concentrating by a rotary evaporator to obtain 3.5718g of crude extract.
The crude extract (3.5718 g) was dissolved with a small amount of methanol and subjected to preliminary separation by a methanol gel column to obtain 4 components A1 to A4. Component A2 (364.2 mg) was stirred with an equal weight of silica gel and then subjected to column chromatography on silica gel, followed by elution with ethyl acetate to give 4 components (A2-1, A2-2, A2-3, A2-4). Component A2-2 (37 mg) was stirred with an equal weight of silica gel and then passed through a silica gel column with petroleum ether (400 ml), petroleum ether: acetone=50: 1 (450 mL), petroleum ether: acetone=40: 1 (320 mL), petroleum ether: acetone=30: 1 (300 mL), petroleum ether: acetone=20: 1 (200 mL), petroleum ether: acetone=10: 1 (300 ml), petroleum ether: acetone=8: 1 (240 mL), petroleum ether: acetone=5: 1 (240 mL), petroleum ether: acetone=4: 1 (250 mL), petroleum ether: acetone=3: 1 (400 mL), petroleum ether: acetone = 2:1 (300 mL) in succession, in petroleum ether: acetone = 2:1 to obtain two components (A2-2-1, A2-2-2) during elution; components A2-2-1 and A2-2-2 were purified by passing them through methanol gel column, respectively, to obtain compounds wp-1 (10.5 mg) and wp-2 (6.3 mg).
The obtained compounds wp-1 and wp-2 are detected by Bruker DRX-500, bruker Avance III-600NMR superconducting nuclear magnetic resonance apparatus and mass spectrometer, and the patterns are respectively: the nuclear magnetic resonance carbon spectrum of wp-1 is shown in figure 1, the nuclear magnetic resonance hydrogen spectrum of wp-1 is shown in figure 2, the ultra-high resolution mass spectrum of wp-1 is shown in figure 3, the nuclear magnetic resonance carbon spectrum of wp-2 is shown in figure 4, the nuclear magnetic resonance hydrogen spectrum of wp-2 is shown in figure 5, and the ultra-high resolution mass spectrum of wp-2 is shown in figure 6. It was found that the obtained wp-1 and wp-2 were both butyl- β -D-glucopyranoside and ethyl- β -D-glucopyranoside.
The foregoing examples merely represent specific embodiments of the present application, which are described in more detail and are not to be construed as limiting the scope of the present application. It should be noted that, for those skilled in the art, several variations and modifications can be made without departing from the technical solution of the present application, which fall within the protection scope of the present application.
Claims (1)
1. A preparation method for preparing two glucopyranosides by using nematode culture is characterized in that the preparation method utilizes signal substances secreted by nematodes to extract two glucopyranosides, namely ethyl-beta-D-glucopyranoside and butyl-beta-D-glucopyranoside; the nematode is a full-tooth reviving nematode; the method specifically comprises the following steps:
(1) Biological culture: the nematodes are inoculated in a sterile oat culture medium, and are cultured for 8-15 d at the temperature of 28 ℃, and the preparation process of the sterile oat culture medium comprises the following steps: 100g of oatmeal was weighed and 100mL of ddH was then added 2 O soaking oatmeal, and sterilizing at 121 ℃ for later use;
(2) Crude extraction of the product: after the nematodes climb the bottle wall, adding sterile ddH into the oat culture medium 2 O, gently washing out nematodes, transferring to a separating funnel, discarding supernatant after the nematodes settle, repeating for 3-5 times to fully remove oat culture medium, and collecting nematodes; 3 volumes of sterile ddH were added to the nematodes 2 O, culturing at 20 ℃ and 180rpm for 6-10 hours to secrete signal substances in the nematodes, centrifuging at 4000rpm for 10-30 minutes to remove nematode bodies, retaining supernatant, adding 3 times of absolute ethyl alcohol into the supernatant for leaching, carrying out leaching for 48 hours each time, carrying out ultrasonic crushing midway, repeating for 3 times, and concentrating by a rotary evaporator to obtain a crude extract;
(3) And (3) purifying a product: separating and obtaining two glucopyranosides, namely ethyl-beta-D-glucopyranoside and butyl-beta-D-glucopyranoside by adopting thin layer chromatography and column chromatography technologies, wherein the product purification specifically comprises the following steps: dissolving the crude extract with a small amount of methanol, and performing preliminary separation by a methanol gel column to obtain 4 components: A1-A4, mixing the component A2 with equal weight of silica gel, separating by silica gel column chromatography, eluting with ethyl acetate to obtain 4 components: a2-1, A2-2, A2-3 and A2-4, wherein the component A2-2 is mixed with equal weight of silica gel and then sequentially passes through a silica gel column, and step elution is sequentially carried out by using petroleum ether/acetone solution according to the step volume ratio of petroleum ether from more to less, so as to obtain two components: a2-2-1, A2-2-2; and (3) purifying the components A2-2-1 and A2-2-2 respectively by a methanol gel column to obtain the compound: ethyl-beta-D-glucopyranoside, butyl-beta-D-glucopyranoside; the step volume ratio of the petroleum ether/acetone solution is sequentially set as follows: petroleum ether: acetone=50: 1. petroleum ether: acetone=40: 1. petroleum ether: acetone=30: 1. petroleum ether: acetone=20: 1. petroleum ether: acetone=10: 1. petroleum ether: acetone=8: 1. petroleum ether: acetone=5: 1. petroleum ether: acetone=4: 1. petroleum ether: acetone=3: 1. petroleum ether: acetone = 2:1, in petroleum ether: acetone = 2:1 to obtain two components A2-2-1 and A2-2-2 during elution.
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