CN103636679B - Method for preparing phenolic acid and alkaloid from enteromorpha through separation and purification and application thereof - Google Patents
Method for preparing phenolic acid and alkaloid from enteromorpha through separation and purification and application thereof Download PDFInfo
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Abstract
The invention relates to a method for preparing phenolic acids and alkaloids from enteromorpha through separation and purification. The method comprises the steps of adding absolute methanol into enteromorpha dry powder to prepare a dried substance, and adding distilled water to prepare supernate; adding ethyl acetate after the pH of the supernate is adjusted and carrying out multiple extraction to prepare components A, B, C and D; eluting a methanol solution with the component A by a column, carrying out sample application of methanol solutions with B, C and D on silicon gel GF254, combining and collecting strips at the same Rf, dissolving in acetone, preparing nine components through centrifugation and vacuum concentration, and carrying out sample application of three in the nine components on the silicon gel GF254 by a scoring method for purifying to prepare eight components. The fourteen components are alkaloids and phenolic acids. The alkaloids and phenolic acids can be used for inhibiting the growth of karenia mikimotoi or skeletonema costatum. The method provided by the invention is high in maneuverability. The prepared phenolic acids and alkaloids are high in purity, and the breakthrough of preparing the phenolic acids and the alkaloids from the enteromorpha through extraction is realized. The various phenolic acids and alkaloids, prepared by the method, have alga inhibition activity, and actual application value.
Description
Technical field
The invention belongs to marine biochemical engineering field, be specifically related to one separation and purification from Enteromorpha and prepare phenolic acid and alkaloidal method.The invention still further relates to the obtained phenolic acid of the method and alkaloidal purposes.
Background technology
Because halobiontic living environment is different from terrestrial life, be in high salt, high pressure, in the environment such as anoxic, for seeking survival and competing living space, a lot of marine organisms metabolism produces chemical substance and the secondary metabolites (Secondary metabolites) of some structure uniquenesses, phenolic acid (phenolic acid, and alkaloid (Marine alkaloids PA), MA) be exactly important composition composition wherein, this also creates the biologic activity of phenolic acid and alkaloid uniqueness, mainly comprise antitumor action, next has antibacterial, antiviral, anti-oxidant, the effects such as anti-inflammatory, good curative effect is had to mankind's various diseases.In addition, its mechanism of action has diversity and unique feature, this is that the halobiontic phenolic acid of exploitation and alkaloidal medical value provide good foundation and wide prospect, in the future have more ocean phenolic acid and alkaloid is found, and exploitation becomes the medicine such as antitumor, antibacterial and antiviral.
Enteromorpha (
enteromorpha prolifera) classification belongs to Chlorophyta, Ulvaceae, Enteromorpha, being commonly called as " green laver ", " Ulva lactuca L ", is important economical alga, is distributed widely in the Shi Zhaozhong of river mouth and middle tide band, how is used as food, feed and fertilizer etc. by people.China's wild Enteromorpha resource is very abundant, only in the annual output of ALONG COASTAL FUJIAN just at 100,000 more than t, all can occur throughout the year in aquaculture sea area.Compared with other marine algas, Enteromorpha growth and breeding is fast, and need not purchase seedling, be a kind of cheap marine biomass resource.In recent years due to seawater nutrient laden, Enteromorpha occurs that explosion type grows, and causes " green tide ", causes Oceanic disasters.Long-term solution and the basic point of Enteromorpha control to the resource exploitation of Enteromorpha, at present, less to the research range of Enteromorpha comprehensive utilization, low side exploitation mainly concentrates on the exploitation of biomass fertilizers and biomass energy, high-end utilization mainly around food and additive thereof, several aspects such as feed, medicine and Enteromorpha bioactivator.
Summary of the invention
Technical problem to be solved by this invention is for the deficiencies in the prior art, provide a kind of newly prepare phenolic acid and alkaloidal method from the separation and purification Enteromorpha, the method is workable, obtained phenolic acid and alkaloid purity high.
Another technical problem to be solved by this invention there is provided phenolic acid obtained by said method and alkaloidal purposes.
Technical problem to be solved by this invention is realized by following technical scheme.The present invention is that phenolic acid and alkaloidal method are prepared in one separation and purification from Enteromorpha, and be characterized in, its step is as follows:
(1) Enteromorpha dry powder is added absolute methanol, normal temperature, dark lower immersion, obtain leaching liquor after filtration; Evaporated under reduced pressure, obtains evaporate to dryness thing; Evaporate to dryness thing adds distilled water, fully vibrates, hold over night at 4 DEG C, and centrifugal, filtration, removes precipitation, obtain supernatant;
(2) supernatant is adjusted pH to 11, add extraction into ethyl acetate, gained upper strata evaporated under reduced pressure, obtain component A, regulate gained lower floor pH to 7, extraction into ethyl acetate, gained upper strata evaporated under reduced pressure, obtains B component, regulate gained lower floor pH to 2 again, extraction into ethyl acetate, gained the upper and lower are evaporated under reduced pressure respectively, obtains component C and component D; A, B, C, D 4 kinds of components are dissolved in absolute methanol respectively, obtained A, B, C, D 4 kinds of methanol solutions;
(3) methanol solution of component A is loaded on Sephadex LH-20 gel filtration chromatography, with 30%, 50%, 70%, 90% and 100% methanol solution for eluting solvent; Detect through thin-layer chromatography, merge and collect eluent, reduced pressure concentration, respectively under 30-50%, 70% and 100% methanol solution wash-out, collects 3 kinds of components, is designated as component AA, component AB and component AC; By component AA, and B component, C, D methanol solution point sample in silica G F
254on, carry out thin-layer chromatography detection; Take volume ratio as the chloroform/acetone/formic acid of 15:3:2 be solvent, after expansion, observing under ultraviolet 254 nm, there are 4 spots in component AA,
r fbe 0.375,0.688,0.775 and 0.825; There are 2 spots in the methanol solution of B component,
r fbe 0.602 and 0.851; There is 1 spot in the methanol solution of component C,
r fbe 0.843; Employing volume ratio is the chloroform/methanol of 8:1 is solvent, and under ultraviolet 254 nm, 2 spots appear in the methanol solution of component D,
r fbe respectively 0.333 and 0.575; On this basis, with method of scoring repeatedly point sample, merge collection identical
r fplace's band, is again dissolved in acetone, through centrifugal and reduced pressure concentration, is prepared into 9 kinds of components, is designated as component AA successively
1, component AA
2, component AA
3, component AA
4, B component
1, B component
2, component C
1, component D
1with component D
2; Wherein, component AA
2, component AA
4with component C
1again with method of scoring point sample in silica G F
254on carry out purifying, be that the chloroform/acetone/formic acid of 18:1:3 is solvent with volume ratio, be prepared into 8 kinds of components, be designated as component AA successively
21, component AA
22, component AA
23, component AA
41, component AA
42, component AA
43, component AA
44with component C
11;
(4) detect through compound qualitative detection, length scanning and thin-layer chromatography, in above-mentioned 14 kinds of components, component D
1with component D
2be alkaloid, all the other components are phenolic acid; Evaporated under reduced pressure, to obtain final product respectively.
The phenolic acid that above-described the inventive method is obtained or alkaloid, can be used for suppressing the growth of the triumphant human relations algae of Michaelis or Skeletonema Costatum.
Compared with prior art, the inventive method is workable, obtained phenolic acid and alkaloid purity high, achieve and extract obtained phenolic acid and alkaloidal breakthrough from waterside platform.The multiple phenolic acid that the method is obtained and alkaloid have marine alga algistatic activity, have actual application value.
Embodiment
Below further describe concrete technical scheme of the present invention, so that those skilled in the art understands the present invention further, and do not form the restriction to its right.
Embodiment 1, phenolic acid and alkaloidal method are prepared in one separation and purification from Enteromorpha, and its step is as follows:
(1) Enteromorpha dry powder is added absolute methanol, normal temperature, dark lower immersion, obtain leaching liquor after filtration; Evaporated under reduced pressure, obtains evaporate to dryness thing; Evaporate to dryness thing adds distilled water, fully vibrates, hold over night at 4 DEG C, and centrifugal, filtration, removes precipitation, obtain supernatant;
(2) supernatant is adjusted pH to 11, add extraction into ethyl acetate, gained upper strata evaporated under reduced pressure, obtain component A, regulate gained lower floor pH to 7, extraction into ethyl acetate, gained upper strata evaporated under reduced pressure, obtains B component, regulate gained lower floor pH to 2 again, extraction into ethyl acetate, gained the upper and lower are evaporated under reduced pressure respectively, obtains component C and component D; A, B, C, D 4 kinds of components are dissolved in absolute methanol respectively, obtained A, B, C, D 4 kinds of methanol solutions;
(3) methanol solution of component A is loaded on Sephadex LH-20 gel filtration chromatography, with 30%, 50%, 70%, 90% and 100% methanol solution for eluting solvent; Detect through thin-layer chromatography, merge and collect eluent, reduced pressure concentration, respectively under 30-50%, 70% and 100% methanol solution wash-out, collects 3 kinds of components, is designated as component AA, component AB and component AC; By component AA, and B component, C, D methanol solution point sample in silica G F
254on, carry out thin-layer chromatography detection; Take volume ratio as the chloroform/acetone/formic acid of 15:3:2 be solvent, after expansion, observing under ultraviolet 254 nm, there are 4 spots in component AA,
r fbe 0.375,0.688,0.775 and 0.825; There are 2 spots in the methanol solution of B component,
r fbe 0.602 and 0.851; There is 1 spot in the methanol solution of component C,
r fbe 0.843; Employing volume ratio is the chloroform/methanol of 8:1 is solvent, and under ultraviolet 254 nm, 2 spots appear in the methanol solution of component D,
r fbe respectively 0.333 and 0.575; On this basis, with method of scoring repeatedly point sample, merge collection identical
r fplace's band, is again dissolved in acetone, through centrifugal and reduced pressure concentration, is prepared into 9 kinds of components, is designated as component AA successively
1, component AA
2, component AA
3, component AA
4, B component
1, B component
2, component C
1, component D
1with component D
2; Wherein, component AA
2, component AA
4with component C
1again with method of scoring point sample in silica G F
254on carry out purifying, be that the chloroform/acetone/formic acid of 18:1:3 is solvent with volume ratio, be prepared into 8 kinds of components, be designated as component AA successively
21, component AA
22, component AA
23, component AA
41, component AA
42, component AA
43, component AA
44with component C
11;
(4) detect through compound qualitative detection, length scanning and thin-layer chromatography, in above-mentioned 14 kinds of components, component D
1with component D
2be alkaloid, all the other components are phenolic acid; Evaporated under reduced pressure, to obtain final product respectively.
Embodiment 2, from Enteromorpha, phenolic acid and alkaloidal methods experiment are prepared in separation and purification:
Enteromorpha dry product is ground into 0.3 mm powder, adds absolute methanol, under normal temperature, dark, soak 3-4d.Repeatedly extract after 3-4 time, merge leaching liquor, through centrifugal, filtration and evaporated under reduced pressure, be prepared into evaporate to dryness thing; This evaporate to dryness thing adds appropriate distilled water, fully vibrates, hold over night at 4 DEG C, and centrifugal, filtration, removes precipitation, retains supernatant.With 2-5 mol/L sodium hydroxide, supernatant pH is adjusted to 11, adds extraction into ethyl acetate 3-4 time.Upper strata evaporated under reduced pressure, obtains component A.Regulate lower floor pH to 7, extraction into ethyl acetate 3-4 time, upper strata evaporated under reduced pressure, is prepared into B component.Again lower floor pH is adjusted to 2, extraction into ethyl acetate 3-4 time, the upper and lower are evaporated under reduced pressure respectively, obtains component C and component D.Above-mentioned 4 kinds of components are dissolved in absolute methanol respectively, are prepared into 4 kinds of methanol solutions.Component solution A loads on Sephadex LH-20 gel filtration chromatography, with 30%, 50%, 70%, 90% and 100% methanol solution for eluting solvent.Detect through thin-layer chromatography, merge and collect eluent, reduced pressure concentration, respectively under 30-50%, 70% and 100% methanol solution wash-out, collects 3 kinds of components, is designated as component AA, component AB and component AC.In these 3 kinds of components, because component AB and component AC is considerably less, therefore, in subsequent separation process, only purified components AA.By component AA solution and B component, component C and component solution D point sample in silica G F
254on, front 3 kinds of components are with chloroform/acetone/formic acid (15:3:2) for solvent, and component D adopts chloroform/methanol (8:1) to be solvent.After expansion, observe under ultraviolet 254 nm.There are 4 spots in component AA,
r fbe 0.375,0.688,0.775 and 0.825; There are 2 spots in B component,
r fbe 0.602 and 0.851; There is 1 spot in component C,
r fbe 0.843; There are 2 spots in component D,
r fbe respectively 0.333 and 0.575.On this basis, with method of scoring repeatedly point sample, merge collection identical
r fplace's band, is again dissolved in acetone, through centrifugal and reduced pressure concentration, is prepared into 9 kinds of components, is designated as component AA successively
1, component AA
2, component AA
3, component AA
4, B component
1, B component
2, component C
1, component D
1with component D
2; Wherein, component AA
2, component AA
4with component C
1again with method of scoring point sample in silica G F
254on carry out purifying, with chloroform/acetone/formic acid (18:1:3) for solvent, be prepared into 8 kinds of components, be designated as component AA successively
21, component AA
22, component AA
23, component AA
41, component AA
42, component AA
43, component AA
44with component C
11.Get the concentrate of 0.1 mL, 14 kinds of components, after solvent volatilization, add 1 mol/L glacial acetic acid solution 1 mL, abundant vibration, add the potassium ferricyanide-ferric trichloride developer (0.6 % potassium ferricyanide solution and 0.9 % liquor ferri trichloridi mix by 0.9:1.0 before using, matching while using), observe color reaction, find component AA
1, component AA
21, component AA
22, component AA
23, component AA
3, component AA
41, component AA
42, component AA
43, component AA
44, B component
1, B component
2with component C
11presenting dirty green, is the positive reaction of phenolic acid; Length scanning shows, and these 12 kinds of components have characteristic absorption peak within the scope of 260-330 nm, are the characteristic absorption peak of phenolic acid; 0.1 mL concentrate of 12 kinds of components, after solvent volatilization, adds bismuth potassium iodide solution 1 mL, fully vibrates, only component D
1with component D
2occurring yellow mercury oxide, is alkaloidal positive reaction.By these 14 kinds of component solution point samples in silica G F
254on, respectively in chloroform/methanol, launch under 3 kinds of solvents such as cyclohexane/ethyl acetate and n-butanol/acetic acid/water, find that these 14 kinds of components all present single spot; Simultaneously, adopt universal developer (10% sulfuric acid solution and iodine) and exclusive developer (potassium ferricyanide-liquor ferri trichloridi or bismuth potassium iodide solution), find under these 3 kinds of developers, these 14 kinds of components present single spot equally, show that purity reaches thin layer pure.Finally, algistatic activity detects and shows, these 14 kinds of components all can suppress the growth of the triumphant human relations algae of Michaelis and Skeletonema Costatum.Evaporated under reduced pressure, is prepared into 14 kinds of algistatic activity materials.
Embodiment 3, from Enteromorpha, phenolic acid and alkaloidal methods experiment are prepared in separation and purification:
Take Enteromorpha dry powder 480 g, be dissolved in 700 mL absolute methanols, under room temperature dark, soak 3d.After extracting 3 times, merge extract, through centrifugal, filter and evaporated under reduced pressure, be prepared into 14.8 g evaporate to dryness things.Add 20 mL distilled water in this evaporate to dryness thing, fully vibrate, hold over night at 4 DEG C, centrifugal, filtration, removes precipitation, retains supernatant.With 2 mol/L sodium hydroxide, supernatant pH is adjusted to 11, adds extraction into ethyl acetate 3 times.Evaporated under reduced pressure at 40 DEG C, upper strata, obtains blackish green evaporate to dryness thing 1.782 g(component A).Regulate lower floor pH to 7, extraction into ethyl acetate 3 times, evaporated under reduced pressure at 40 DEG C, upper strata, is prepared into green evaporate to dryness thing 0.271 g(B component).Again lower floor pH is adjusted to 2, extraction into ethyl acetate 3 times, the upper and lower, respectively 40 DEG C of evaporated under reduced pressure, obtain brown color evaporate to dryness thing 0.225 g(component C) and brown color evaporate to dryness thing 5.108 g(component D).Above-mentioned 4 kinds of components are dissolved in absolute methanol respectively, are prepared into 4 kinds of methanol solutions.Component solution A loads on Sephadex LH-20 gel filtration chromatography, with 30%, 50%, 70%, 90% and 100% methanol solution for eluting solvent.Detect through thin-layer chromatography, merge and collect eluent, reduced pressure concentration, respectively under 30-50%, 70% and 100% methanol solution wash-out, collects 3 kinds of components, is designated as component AA, component AB and component AC.In these 3 kinds of components, because component AB and component AC is considerably less, therefore, in subsequent separation process, only purified components AA.By component AA solution and B component, component C and component solution D point sample in silica G F
254on, front 3 kinds of components are with chloroform/acetone/formic acid (15:3:2) for solvent, and component D adopts chloroform/methanol (8:1) to be solvent.After expansion, observe under ultraviolet 254 nm.There are 4 spots in component AA,
r fbe 0.375,0.688,0.775 and 0.825; There are 2 spots in B component,
r fbe 0.602 and 0.851; There is 1 spot in component C,
r fbe 0.843; There are 2 spots in component D,
r fbe respectively 0.333 and 0.575.On this basis, with method of scoring repeatedly point sample, merge collection identical
r fplace's band, is again dissolved in acetone, through centrifugal and reduced pressure concentration, is prepared into 9 kinds of components, is designated as component AA successively
1, component AA
2, component AA
3, component AA
4, B component
1, B component
2, component C
1, component D
1with component D
2; Wherein, component AA
2, component AA
4with component C
1again with method of scoring point sample in silica G F
254on carry out purifying, with chloroform/acetone/formic acid (18:1:3) for solvent, be prepared into 8 kinds of components, be designated as component AA successively
21, component AA
22, component AA
23, component AA
41, component AA
42, component AA
43, component AA
44with component C
11.Get the concentrate of 0.1 mL, 14 kinds of components, after solvent volatilization, add 1 mol/L glacial acetic acid solution 1 mL, abundant vibration, add the potassium ferricyanide-ferric trichloride developer (0.6 % potassium ferricyanide solution and 0.9 % liquor ferri trichloridi mix by 0.9:1.0 before using, matching while using), observe color reaction, find component AA
1, component AA
21, component AA
22, component AA
23, component AA
3, component AA
41, component AA
42, component AA
43, component AA
44, B component
1, B component
2with component C
11presenting dirty green, is the positive reaction of phenolic acid; Length scanning shows, and these 12 kinds of components have characteristic absorption peak within the scope of 260-330 nm, are the characteristic absorption peak of phenolic acid; 0.1 mL concentrate of 12 kinds of components, after solvent volatilization, adds bismuth potassium iodide solution 1 mL, fully vibrates, only component D
1with component D
2occurring yellow mercury oxide, is alkaloidal positive reaction.By these 14 kinds of component solution point samples in silica G F
254on, respectively in chloroform/methanol, launch under 3 kinds of solvents such as cyclohexane/ethyl acetate and n-butanol/acetic acid/water, find that these 14 kinds of components all present single spot; Simultaneously, adopt universal developer (10% sulfuric acid solution and iodine) and exclusive developer (potassium ferricyanide-liquor ferri trichloridi or bismuth potassium iodide solution), find under these 3 kinds of developers, these 14 kinds of components present single spot equally, show that purity reaches thin layer pure.Finally, algistatic activity detects and shows, these 14 kinds of components all can suppress the growth of the triumphant human relations algae of Michaelis and Skeletonema Costatum.Evaporated under reduced pressure, is prepared into 14 kinds of algistatic activity materials, AA
1yellow oil 0.014 g, AA
21yellow powder 0.004 g, AA
22yellow crystalline powder 0.005 g, AA
23yellow powder 0.002 g, AA
3yellow crystal 0.040 g, AA
41yellow needles 0.004 g, AA
42pale yellow powder 0.005 g, AA
43pale yellow powder 0.004 g, AA
44pale yellow crystals 0.002 g, B
1yellow particle thing 0.018 g, B
2yellow oil 0.008 g, C
11pale yellow powder 0.002 g, D
1yellow oil 0.041 g and D
2yellow powder 0.052 g.
Embodiment 4, from Enteromorpha, phenolic acid and alkaloidal methods experiment are prepared in separation and purification:
Take Enteromorpha dry powder 800 g, be dissolved in 1500 mL absolute methanols, under room temperature dark, soak 4d.After extracting 4 times, merge extract, through centrifugal, filter and evaporated under reduced pressure, be prepared into 18.872 g evaporate to dryness things.Add 50 mL distilled water in this evaporate to dryness thing, fully vibrate, hold over night at 4 DEG C, centrifugal, filtration, removes precipitation, retains supernatant.With 3 mol/L sodium hydroxide, supernatant pH is adjusted to 11, adds extraction into ethyl acetate 4 times.Evaporated under reduced pressure at 40 DEG C, upper strata, obtains blackish green evaporate to dryness thing 2.651 g(component A).Regulate lower floor pH to 7, extraction into ethyl acetate 4 times, evaporated under reduced pressure at 40 DEG C, upper strata, is prepared into green evaporate to dryness thing 0.402 g(B component).Again lower floor pH is adjusted to 2, extraction into ethyl acetate 4 times, the upper and lower, respectively 40 DEG C of evaporated under reduced pressure, obtain brown color evaporate to dryness thing 0.327 g(component C) and brown color evaporate to dryness thing 10.167 g(component D).Above-mentioned 4 kinds of components are dissolved in absolute methanol respectively, are prepared into 4 kinds of methanol solutions.Component solution A loads on Sephadex LH-20 gel filtration chromatography, with 30%, 50%, 70%, 90% and 100% methanol solution for eluting solvent.Detect through thin-layer chromatography, merge and collect eluent, reduced pressure concentration, respectively under 30-50%, 70% and 100% methanol solution wash-out, collects 3 kinds of components, is designated as component AA, component AB and component AC.In these 3 kinds of components, because component AB and component AC is considerably less, therefore, in subsequent separation process, only purified components AA.By component AA solution and B component, component C and component solution D point sample in silica G F
254on, front 3 kinds of components are with chloroform/acetone/formic acid (15:3:2) for solvent, and component D adopts chloroform/methanol (8:1) to be solvent.After expansion, observe under ultraviolet 254 nm.There are 4 spots in component AA,
r fbe 0.375,0.688,0.775 and 0.825; There are 2 spots in B component,
r fbe 0.602 and 0.851; There is 1 spot in component C,
r fbe 0.843; There are 2 spots in component D,
r fbe respectively 0.333 and 0.575.On this basis, with method of scoring repeatedly point sample, merge collection identical
r fplace's band, is again dissolved in acetone, through centrifugal and reduced pressure concentration, is prepared into 9 kinds of components, is designated as component AA successively
1, component AA
2, component AA
3, component AA
4, B component
1, B component
2, component C
1, component D
1with component D
2; Wherein, component AA
2, component AA
4with component C
1again with method of scoring point sample in silica G F
254on carry out purifying, with chloroform/acetone/formic acid (18:1:3) for solvent, be prepared into 8 kinds of components, be designated as component AA successively
21, component AA
22, component AA
23, component AA
41, component AA
42, component AA
43, component AA
44with component C
11.Get the concentrate of 0.1 mL, 14 kinds of components, after solvent volatilization, add 1 mol/L glacial acetic acid solution 1 mL, abundant vibration, add the potassium ferricyanide-ferric trichloride developer (0.6 % potassium ferricyanide solution and 0.9 % liquor ferri trichloridi mix by 0.9:1.0 before using, matching while using), observe color reaction, find component AA
1, component AA
21, component AA
22, component AA
23, component AA
3, component AA
41, component AA
42, component AA
43, component AA
44, B component
1, B component
2with component C
11presenting dirty green, is the positive reaction of phenolic acid; Length scanning shows, and these 12 kinds of components have characteristic absorption peak within the scope of 260-330 nm, are the characteristic absorption peak of phenolic acid; 0.1 mL concentrate of 12 kinds of components, after solvent volatilization, adds bismuth potassium iodide solution 1 mL, fully vibrates, only component D
1with component D
2occurring yellow mercury oxide, is alkaloidal positive reaction.By these 14 kinds of component solution point samples in silica G F
254on, respectively in chloroform/methanol, launch under 3 kinds of solvents such as cyclohexane/ethyl acetate and n-butanol/acetic acid/water, find that these 14 kinds of components all present single spot; Simultaneously, adopt universal developer (10% sulfuric acid solution and iodine) and exclusive developer (potassium ferricyanide-liquor ferri trichloridi or bismuth potassium iodide solution), find under these 3 kinds of developers, these 14 kinds of components present single spot equally, show that purity reaches thin layer pure.Finally, algistatic activity detects and shows, these 14 kinds of components all can suppress the growth of the triumphant human relations algae of Michaelis and Skeletonema Costatum.Evaporated under reduced pressure, is prepared into 14 kinds of algistatic activity materials, AA
1yellow oil 0.032 g, AA
21yellow powder 0.009 g, AA
22yellow crystalline powder 0.008 g, AA
23yellow powder 0.004 g, AA
3yellow crystal 0.078 g, AA
41yellow needles 0.008 g, AA
42pale yellow powder 0.012 g, AA
43pale yellow powder 0.007 g, AA
44pale yellow crystals 0.005 g, B
1yellow particle thing 0.028 g, B
2yellow oil 0.011 g, C11 pale yellow powder 0.004 g, D1 yellow oil 0.079 g and D
2yellow powder 0.095 g.
Embodiment 5, from Enteromorpha, phenolic acid and alkaloidal methods experiment are prepared in separation and purification:
Take Enteromorpha dry powder 1200 g, be dissolved in 3500 mL absolute methanols, under room temperature, dark, soak 4d.After extracting 4 times, merge extract, through centrifugal, filter and evaporated under reduced pressure, be prepared into 30.072 g evaporate to dryness things.Add 100 mL distilled water in this evaporate to dryness thing, fully vibrate, hold over night at 4 DEG C, centrifugal, filtration, removes precipitation, retains supernatant.With 5 mol/L sodium hydroxide, supernatant pH is adjusted to 11, adds extraction into ethyl acetate 4 times.Evaporated under reduced pressure at 40 DEG C, upper strata, obtains blackish green evaporate to dryness thing 4.012 g(component A).Regulate lower floor pH to 7, extraction into ethyl acetate 4 times, evaporated under reduced pressure at 40 DEG C, upper strata, is prepared into green evaporate to dryness thing 0.584 g(B component).Again lower floor pH is adjusted to 2, extraction into ethyl acetate 4 times, the upper and lower, respectively 40 DEG C of evaporated under reduced pressure, obtain brown color evaporate to dryness thing 0.516 g(component C) and brown color evaporate to dryness thing 16.128 g(component D).Above-mentioned 4 kinds of components are dissolved in absolute methanol respectively, are prepared into 4 kinds of methanol solutions.Component solution A loads on Sephadex LH-20 gel filtration chromatography, with 30%, 50%, 70%, 90% and 100% methanol solution for eluting solvent.Detect through thin-layer chromatography, merge and collect eluent, reduced pressure concentration, respectively under 30-50%, 70% and 100% methanol solution wash-out, collects 3 kinds of components, is designated as component AA, component AB and component AC.In these 3 kinds of components, because component AB and component AC is considerably less, therefore, in subsequent separation process, only purified components AA.By component AA solution and B component, component C and component solution D point sample in silica G F
254on, carry out thin-layer chromatography detection.With chloroform/acetone/formic acid (15:3:2) for solvent, after expansion, observing under ultraviolet 254 nm, there are 4 spots in component AA,
r fbe 0.375,0.688,0.775 and 0.825; There are 2 spots in B component,
r fbe 0.602 and 0.851; There is 1 spot in component C,
r fbe 0.843.Adopt chloroform/methanol (8:1) to be solvent, under ultraviolet 254 nm, there are 2 spots in component D,
r fbe respectively 0.333 and 0.575.On this basis, with method of scoring repeatedly point sample, merge collection identical
r fplace's band, is again dissolved in acetone, through centrifugal and reduced pressure concentration, is prepared into 9 kinds of components, is designated as component AA successively
1, component AA
2, component AA
3, component AA
4, B component
1, B component
2, component C
1, component D
1with component D
2; Wherein, component AA
2, component AA
4with component C
1again with method of scoring point sample in silica G F
254on carry out purifying, with chloroform/acetone/formic acid (18:1:3) for solvent, be prepared into 8 kinds of components, be designated as component AA successively
21, component AA
22, component AA
23, component AA
41, component AA
42, component AA
43, component AA
44with component C
11.Get the concentrate of 0.1 mL, 14 kinds of components, after solvent volatilization, add 1 mol/L glacial acetic acid solution 1 mL, abundant vibration, add the potassium ferricyanide-ferric trichloride developer (0.6 % potassium ferricyanide solution and 0.9 % liquor ferri trichloridi mix by 0.9:1.0 before using, matching while using), observe color reaction, find component AA
1, component AA
21, component AA
22, component AA
23, component AA
3, component AA
41, component AA
42, component AA
43, component AA
44, B component
1, B component
2with component C
11presenting dirty green, is the positive reaction of phenolic acid; Length scanning shows, and these 12 kinds of components have characteristic absorption peak within the scope of 260-330 nm, are the characteristic absorption peak of phenolic acid; 0.1 mL concentrate of 12 kinds of components, after solvent volatilization, adds bismuth potassium iodide solution 1 mL, fully vibrates, only component D
1with component D
2occurring yellow mercury oxide, is alkaloidal positive reaction.By these 14 kinds of component solution point samples in silica G F
254on, respectively in chloroform/methanol, launch under 3 kinds of solvents such as cyclohexane/ethyl acetate and n-butanol/acetic acid/water, find that these 14 kinds of components all present single spot; Simultaneously, adopt universal developer (10% sulfuric acid solution and iodine) and exclusive developer (potassium ferricyanide-liquor ferri trichloridi or bismuth potassium iodide solution), find under these 3 kinds of developers, these 14 kinds of components present single spot equally, show that purity reaches thin layer pure.Finally, algistatic activity detects and shows, these 14 kinds of components all can suppress the growth of the triumphant human relations algae of Michaelis and Skeletonema Costatum.Evaporated under reduced pressure, is prepared into 14 kinds of algistatic activity materials, AA
1yellow oil 0.041 g, AA
21yellow powder 0.011 g, AA
22yellow crystalline powder 0.010 g, AA
23yellow powder 0.005 g, AA
3yellow crystal 0.092 g, AA
41yellow needles 0.010 g, AA
42pale yellow powder 0.015 g, AA
43pale yellow powder 0.008 g, AA
44pale yellow crystals 0.006 g, B
1yellow particle thing 0.043 g, B
2yellow oil 0.021 g, C
11pale yellow powder 0.006 g, D
1yellow oil 0.096 g and D
2yellow powder 0.105 g.
The experiment of algal control circle is adopted to carry out algistatic activity detection.At several diameter be 1.5 cm circular filter paper sheet on, drip 5 μ L component acetone to be measured concentrates and acetone (as a control group) respectively, after acetone volatilizees completely, filter paper is positioned on the solid culture medium of the Skeletonema Costatum of inoculation exponential phase, be placed in GXZ-260B intelligent illumination incubator to cultivate, temperature (26 ± 1) DEG C, intensity of illumination 62 μm of ol m
-2s
-1, Light To Dark Ratio is 12:12.After 2d, the growth of observation filter paper and around tested micro-algae, finds that 14 kinds of components to be measured all reveal comparatively significantly inhibitory action to the growth table of Skeletonema Costatum.By measuring algal control circle, the algal control circle of these 14 kinds of components to be measured is all more than 1.0 cm.
Claims (2)
1. from Enteromorpha, phenolic acid and an alkaloidal method are prepared in separation and purification, it is characterized in that, its step is as follows:
(1) Enteromorpha dry powder is added absolute methanol, normal temperature, dark lower immersion, obtain leaching liquor after filtration; Evaporated under reduced pressure, obtains evaporate to dryness thing; Evaporate to dryness thing adds distilled water, fully vibrates, hold over night at 4 DEG C, and centrifugal, filtration, removes precipitation, obtain supernatant;
(2) supernatant is adjusted pH to 11, add extraction into ethyl acetate, gained upper strata evaporated under reduced pressure, obtain component A, regulate gained lower floor pH to 7, extraction into ethyl acetate, gained upper strata evaporated under reduced pressure, obtains B component, regulate gained lower floor pH to 2 again, extraction into ethyl acetate, gained the upper and lower are evaporated under reduced pressure respectively, obtains component C and component D; A, B, C, D4 kind component is dissolved in absolute methanol respectively, obtained A, B, C, D4 kind methanol solution;
(3) methanol solution of component A is loaded on Sephadex LH-20 gel filtration chromatography, with 30%, 50%, 70%, 90% and 100% methanol solution for eluting solvent; Detect through thin-layer chromatography, merge and collect eluent, reduced pressure concentration, respectively under 30-50%, 70% and 100% methanol solution wash-out, collects 3 kinds of components, is designated as component AA, component AB and component AC; By component AA, and B component, C, D methanol solution point sample in silica G F
254on, carry out thin-layer chromatography detection, be that the chloroform/acetone/formic acid of 15:3:2 is solvent with volume ratio, after expansion, observe under ultraviolet 254nm, there are 4 spots in component AA, R
fbeing 0.375,0.688,0.775 and 0.825, there are 2 spots, R in the methanol solution of B component
fbe 0.602 and 0.851, component C methanol solution there is 1 spot, R
fbe 0.843; Employing volume ratio is the chloroform/methanol of 8:1 is solvent, and under ultraviolet 254nm, R appears 2 spots, in the methanol solution of component D
fbe respectively 0.333 and 0.575; On this basis, with method of scoring repeatedly point sample, merge and collect identical R
fplace's band, is again dissolved in acetone, through centrifugal and reduced pressure concentration, is prepared into 9 kinds of components, is designated as component AA successively
1, component AA
2, component AA
3, component AA
4, B component
1, B component
2, component C
1, component D
1with component D
2; Wherein, component AA
2, component AA
4with component C
1again with method of scoring point sample in silica G F
254on carry out purifying, be that the chloroform/acetone/formic acid of 18:1:3 is solvent with volume ratio, be prepared into 8 kinds of components, be designated as component AA successively
21, component AA
22, component AA
23, component AA
41, component AA
42, component AA
43, component AA
44with component C
11;
(4) detect through compound qualitative detection, length scanning and thin-layer chromatography, said components AA
1, component AA
21, component AA
22, component AA
23, component AA
3, component AA
41, component AA
42, component AA
43, component AA
44, B component
1, B component
2, component C
11, component D
1with component D
2in 14 kinds of components: component D
1with component D
2be alkaloid, all the other components are phenolic acid; Evaporated under reduced pressure, to obtain final product respectively.
2. the phenolic acid that obtains of method or the purposes of alkaloid in the growth suppressing the triumphant human relations algae of Michaelis or Skeletonema Costatum as claimed in claim 1.
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| CN1850630A (en) * | 2006-05-26 | 2006-10-25 | 中国海洋大学 | Method for preparing enteromorpha extract for inhibiting red tide algae and its use |
| CN101485343A (en) * | 2009-03-02 | 2009-07-22 | 中国海洋大学生物工程开发有限公司 | Extraction technology of active ingredients of enteromorpha and preparation method of enteromorpha seaweed fertilizer |
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| CN1850630A (en) * | 2006-05-26 | 2006-10-25 | 中国海洋大学 | Method for preparing enteromorpha extract for inhibiting red tide algae and its use |
| CN101485343A (en) * | 2009-03-02 | 2009-07-22 | 中国海洋大学生物工程开发有限公司 | Extraction technology of active ingredients of enteromorpha and preparation method of enteromorpha seaweed fertilizer |
Non-Patent Citations (2)
| Title |
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| "浒苔对4种赤潮微藻生长的影响";孙颖颖等;《淮海工学院学报》;20100915;第19卷(第3期);第75-78页 * |
| "浒苔提取物对4种赤潮微藻生长的抑制作用";孙颖颖等;《环境科学》;20100615;第31卷(第6期);1663-1669页 * |
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