CN103636679A - 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 Yu Haiyang Biochemical Engineering field, be specifically related to a kind of from Enteromorpha separation and purification prepare phenolic acid and alkaloidal method.The invention still further relates to phenolic acid and alkaloidal purposes that the method makes.
Background technology
Because halobiontic living environment is different from terrestrial life, in high salt, high pressure, in the environment such as anoxic, for seeking survival and competing living space, the chemical substance that a lot of marine organisms metabolism produce some structure uniquenesses is secondary metabolites (Secondary metabolites), phenolic acid (phenolic acid, PA) and alkaloid (Marine alkaloids, MA) be exactly important composition composition wherein, this has also brought up 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, mankind's various diseases is had to good curative effect.In addition, its mechanism of action has diversity and unique feature, this provides good foundation and wide prospect for developing halobiontic phenolic acid and alkaloidal medical value, future has more ocean phenolic acid and alkaloid is found, and exploitation becomes the medicines such as antitumor, antibacterial and antiviral.
Enteromorpha (
enteromorpha prolifera) belong to Chlorophyta in classification, Ulvaceae, Enteromorpha, is 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 by people as food, feed and fertilizer etc.The wild Enteromorpha resource of China is very abundant, only in the annual output of ALONG COASTAL FUJIAN just more than 100,000 t, cultivation all can occur in marine site throughout the year.Compare with other marine algas, Enteromorpha growth and breeding is fast, need not purchase seedling, is a kind of cheap marine biomass resource.Due to seawater nutrient laden, there is explosion type growth in Enteromorpha, causes " green tide ", causes Oceanic disasters in recent years.It is long-term solution and the basic point of Enteromorpha control that the resource of Enteromorpha is developed, at present, research range to Enteromorpha comprehensive utilization is less, low side exploitation mainly concentrates on the exploitation of biomass fertilizers and biomass energy, high-end utilization is 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, provides a kind of new separation and purification from Enteromorpha to prepare phenolic acid and alkaloidal method, and the method is workable, and the phenolic acid making and alkaloid purity are high.
Another technical problem to be solved by this invention has been to provide the prepared phenolic acid of said method and alkaloidal purposes.
Technical problem to be solved by this invention is to realize by following technical scheme.The present invention be a kind of from Enteromorpha separation and purification prepare phenolic acid and alkaloidal method, be characterized in, its step is as follows:
(1) Enteromorpha dry powder is added to absolute methanol, normal temperature, dark lower immersion, obtain leaching liquor after filtering; Evaporated under reduced pressure, obtains evaporate to dryness thing; Evaporate to dryness thing adds distilled water, fully vibration, and standing over night at 4 ℃, centrifugal, filter, remove precipitation, obtain supernatant;
(2) supernatant is adjusted to pH to 11, add ethyl acetate extraction, gained upper strata evaporated under reduced pressure, obtains component A, regulate the pH to 7 of gained lower floor, ethyl acetate extraction, gained upper strata evaporated under reduced pressure, obtains B component, regulate again the pH to 2 of gained lower floor, ethyl acetate extraction, gained the upper and lower are evaporated under reduced pressure respectively, obtains component C and component D; A, B, C, 4 kinds of components of D are dissolved in respectively in absolute methanol, make A, B, C, 4 kinds of methanol solutions of D;
(3) methanol solution of component A is loaded on Sephadex LH-20 gel filtration chromatography, 30%, 50%, 70%, 90% and 100% methanol solution of take is eluting solvent; Through thin-layer chromatography, detect, merge and collect eluent, reduced pressure concentration, under 30-50%, 70% and 100% methanol solution wash-out, collects 3 kinds of components respectively, is designated as component AA, component AB and component AC; By component AA, and the methanol solution point sample of B component, C, D is in silica G F
254upper, carry out thin-layer chromatography detection; Chloroform/acetone/formic acid that the volume ratio of take is 15:3:2 is solvent, after expansion, under ultraviolet 254 nm, observes, and 4 spots appear 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; Adopting the chloroform/methanol that volume ratio is 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 point sample repeatedly, merge collect identical
r fplace's band, is dissolved in acetone again, through centrifugal and reduced pressure concentration, is prepared into 9 kinds of components, is designated as successively component AA
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, chloroform/acetone/formic acid that the volume ratio of take is 18:1:3 is solvent, is prepared into 8 kinds of components, is designated as successively component AA
21, component AA
22, component AA
23, component AA
41, component AA
42, component AA
43, component AA
44with component C
11;
(4) through compound qualitative detection, length scanning and thin-layer chromatography, detect, 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, obtains respectively.
The phenolic acid that above-described the inventive method makes 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, and the phenolic acid making and alkaloid purity are high, has realized from waterside platform and has extracted and make phenolic acid and alkaloidal breakthrough.Multiple phenolic acid and alkaloid that the method makes 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, a kind of from Enteromorpha separation and purification prepare phenolic acid and alkaloidal method, its step is as follows:
(1) Enteromorpha dry powder is added to absolute methanol, normal temperature, dark lower immersion, obtain leaching liquor after filtering; Evaporated under reduced pressure, obtains evaporate to dryness thing; Evaporate to dryness thing adds distilled water, fully vibration, and standing over night at 4 ℃, centrifugal, filter, remove precipitation, obtain supernatant;
(2) supernatant is adjusted to pH to 11, add ethyl acetate extraction, gained upper strata evaporated under reduced pressure, obtains component A, regulate the pH to 7 of gained lower floor, ethyl acetate extraction, gained upper strata evaporated under reduced pressure, obtains B component, regulate again the pH to 2 of gained lower floor, ethyl acetate extraction, gained the upper and lower are evaporated under reduced pressure respectively, obtains component C and component D; A, B, C, 4 kinds of components of D are dissolved in respectively in absolute methanol, make A, B, C, 4 kinds of methanol solutions of D;
(3) methanol solution of component A is loaded on Sephadex LH-20 gel filtration chromatography, 30%, 50%, 70%, 90% and 100% methanol solution of take is eluting solvent; Through thin-layer chromatography, detect, merge and collect eluent, reduced pressure concentration, under 30-50%, 70% and 100% methanol solution wash-out, collects 3 kinds of components respectively, is designated as component AA, component AB and component AC; By component AA, and the methanol solution point sample of B component, C, D is in silica G F
254upper, carry out thin-layer chromatography detection; Chloroform/acetone/formic acid that the volume ratio of take is 15:3:2 is solvent, after expansion, under ultraviolet 254 nm, observes, and 4 spots appear 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; Adopting the chloroform/methanol that volume ratio is 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 point sample repeatedly, merge collect identical
r fplace's band, is dissolved in acetone again, through centrifugal and reduced pressure concentration, is prepared into 9 kinds of components, is designated as successively component AA
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, chloroform/acetone/formic acid that the volume ratio of take is 18:1:3 is solvent, is prepared into 8 kinds of components, is designated as successively component AA
21, component AA
22, component AA
23, component AA
41, component AA
42, component AA
43, component AA
44with component C
11;
(4) through compound qualitative detection, length scanning and thin-layer chromatography, detect, 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, obtains respectively.
Embodiment 2, and 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, soaks 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 vibration, and standing over night at 4 ℃, centrifugal, filter, remove precipitation, retain supernatant.With 2-5 mol/L sodium hydroxide, supernatant pH is adjusted to 11, adds ethyl acetate extraction 3-4 time.Upper strata evaporated under reduced pressure, obtains component A.Regulate the pH to 7 of lower floor, ethyl acetate extraction 3-4 time, upper strata evaporated under reduced pressure, is prepared into B component.The pH of Zai Jiang lower floor is adjusted to 2, ethyl acetate extraction 3-4 time, and 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 respectively in absolute methanol, are prepared into 4 kinds of methanol solutions.Component A solution loads on Sephadex LH-20 gel filtration chromatography, and 30%, 50%, 70%, 90% and 100% methanol solution of take is eluting solvent.Through thin-layer chromatography, detect, merge and collect eluent, reduced pressure concentration, under 30-50%, 70% and 100% methanol solution wash-out, collects 3 kinds of components respectively, is designated as component AA, component AB and component AC.In these 3 kinds of components, due to component AB and component AC considerably less, therefore, in subsequent separation process, purified components AA only.By component AA solution and B component, component C and component D solution point sample in silica G F
254upper, it be solvent that front 3 kinds of components be take chloroform/acetone/formic acid (15:3:2), and component D employing chloroform/methanol (8:1) is solvent.After expansion, under ultraviolet 254 nm, observe.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 point sample repeatedly, merge collect identical
r fplace's band, is dissolved in acetone again, through centrifugal and reduced pressure concentration, is prepared into 9 kinds of components, is designated as successively component AA
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, the chloroform/acetone/formic acid (18:1:3) of take is solvent, is prepared into 8 kinds of components, is designated as successively component AA
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 14 kinds of components of 0.1 mL, after solvent evaporates, add 1 mol/L glacial acetic acid solution 1 mL, fully vibration, add the potassium ferricyanide-ferric trichloride developer (0.6 % potassium ferricyanide solution and 0.9 % liquor ferri trichloridi mix matching while using by 0.9:1.0 before 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 demonstration, 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 evaporates, adds bismuth potassium iodide solution 1 mL, fully vibration, 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
254upper, respectively in chloroform/methanol, under 3 kinds of solvents such as cyclohexane/ethyl acetate and n-butanol/acetic acid/water, launch, 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), discovery is under these 3 kinds of developers, and these 14 kinds of components present single spot equally, show that purity has reached 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, and 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.Extract after 3 times, merge extract, through centrifugal, filtration and evaporated under reduced pressure, be prepared into 14.8 g evaporate to dryness things.In this evaporate to dryness thing, add 20 mL distilled water, fully vibration, standing over night at 4 ℃, centrifugal, filter, remove precipitation, retain supernatant.With 2 mol/L sodium hydroxide, supernatant pH is adjusted to 11, adds ethyl acetate extraction 3 times.Evaporated under reduced pressure at 40 ℃, upper strata, obtains blackish green evaporate to dryness thing 1.782 g(component A).Regulate the pH to 7 of lower floor, ethyl acetate extraction 3 times, evaporated under reduced pressure at 40 ℃, upper strata, is prepared into green evaporate to dryness thing 0.271 g(B component).The pH of Zai Jiang lower floor is adjusted to 2, ethyl acetate extraction 3 times, the upper and lower, respectively 40 ℃ 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 respectively in absolute methanol, are prepared into 4 kinds of methanol solutions.Component A solution loads on Sephadex LH-20 gel filtration chromatography, and 30%, 50%, 70%, 90% and 100% methanol solution of take is eluting solvent.Through thin-layer chromatography, detect, merge and collect eluent, reduced pressure concentration, under 30-50%, 70% and 100% methanol solution wash-out, collects 3 kinds of components respectively, is designated as component AA, component AB and component AC.In these 3 kinds of components, due to component AB and component AC considerably less, therefore, in subsequent separation process, purified components AA only.By component AA solution and B component, component C and component D solution point sample in silica G F
254upper, it be solvent that front 3 kinds of components be take chloroform/acetone/formic acid (15:3:2), and component D employing chloroform/methanol (8:1) is solvent.After expansion, under ultraviolet 254 nm, observe.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 point sample repeatedly, merge collect identical
r fplace's band, is dissolved in acetone again, through centrifugal and reduced pressure concentration, is prepared into 9 kinds of components, is designated as successively component AA
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, the chloroform/acetone/formic acid (18:1:3) of take is solvent, is prepared into 8 kinds of components, is designated as successively component AA
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 14 kinds of components of 0.1 mL, after solvent evaporates, add 1 mol/L glacial acetic acid solution 1 mL, fully vibration, add the potassium ferricyanide-ferric trichloride developer (0.6 % potassium ferricyanide solution and 0.9 % liquor ferri trichloridi mix matching while using by 0.9:1.0 before 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 demonstration, 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 evaporates, adds bismuth potassium iodide solution 1 mL, fully vibration, 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
254upper, respectively in chloroform/methanol, under 3 kinds of solvents such as cyclohexane/ethyl acetate and n-butanol/acetic acid/water, launch, 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), discovery is under these 3 kinds of developers, and these 14 kinds of components present single spot equally, show that purity has reached 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 acicular crystal 0.004 g, AA
42pale yellow powder 0.005 g, AA
43pale yellow powder 0.004 g, AA
44faint yellow crystallization 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, and 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.Extract after 4 times, merge extract, through centrifugal, filtration and evaporated under reduced pressure, be prepared into 18.872 g evaporate to dryness things.In this evaporate to dryness thing, add 50 mL distilled water, fully vibration, standing over night at 4 ℃, centrifugal, filter, remove precipitation, retain supernatant.With 3 mol/L sodium hydroxide, supernatant pH is adjusted to 11, adds ethyl acetate extraction 4 times.Evaporated under reduced pressure at 40 ℃, upper strata, obtains blackish green evaporate to dryness thing 2.651 g(component A).Regulate the pH to 7 of lower floor, ethyl acetate extraction 4 times, evaporated under reduced pressure at 40 ℃, upper strata, is prepared into green evaporate to dryness thing 0.402 g(B component).The pH of Zai Jiang lower floor is adjusted to 2, ethyl acetate extraction 4 times, the upper and lower, respectively 40 ℃ 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 respectively in absolute methanol, are prepared into 4 kinds of methanol solutions.Component A solution loads on Sephadex LH-20 gel filtration chromatography, and 30%, 50%, 70%, 90% and 100% methanol solution of take is eluting solvent.Through thin-layer chromatography, detect, merge and collect eluent, reduced pressure concentration, under 30-50%, 70% and 100% methanol solution wash-out, collects 3 kinds of components respectively, is designated as component AA, component AB and component AC.In these 3 kinds of components, due to component AB and component AC considerably less, therefore, in subsequent separation process, purified components AA only.By component AA solution and B component, component C and component D solution point sample in silica G F
254upper, it be solvent that front 3 kinds of components be take chloroform/acetone/formic acid (15:3:2), and component D employing chloroform/methanol (8:1) is solvent.After expansion, under ultraviolet 254 nm, observe.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 point sample repeatedly, merge collect identical
r fplace's band, is dissolved in acetone again, through centrifugal and reduced pressure concentration, is prepared into 9 kinds of components, is designated as successively component AA
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, the chloroform/acetone/formic acid (18:1:3) of take is solvent, is prepared into 8 kinds of components, is designated as successively component AA
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 14 kinds of components of 0.1 mL, after solvent evaporates, add 1 mol/L glacial acetic acid solution 1 mL, fully vibration, add the potassium ferricyanide-ferric trichloride developer (0.6 % potassium ferricyanide solution and 0.9 % liquor ferri trichloridi mix matching while using by 0.9:1.0 before 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 demonstration, 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 evaporates, adds bismuth potassium iodide solution 1 mL, fully vibration, 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
254upper, respectively in chloroform/methanol, under 3 kinds of solvents such as cyclohexane/ethyl acetate and n-butanol/acetic acid/water, launch, 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), discovery is under these 3 kinds of developers, and these 14 kinds of components present single spot equally, show that purity has reached 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 acicular crystal 0.008 g, AA
42pale yellow powder 0.012 g, AA
43pale yellow powder 0.007 g, AA
44faint yellow crystallization 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, and 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, room temperature, the dark lower 4d that soaks.Extract after 4 times, merge extract, through centrifugal, filtration and evaporated under reduced pressure, be prepared into 30.072 g evaporate to dryness things.In this evaporate to dryness thing, add 100 mL distilled water, fully vibration, standing over night at 4 ℃, centrifugal, filter, remove precipitation, retain supernatant.With 5 mol/L sodium hydroxide, supernatant pH is adjusted to 11, adds ethyl acetate extraction 4 times.Evaporated under reduced pressure at 40 ℃, upper strata, obtains blackish green evaporate to dryness thing 4.012 g(component A).Regulate the pH to 7 of lower floor, ethyl acetate extraction 4 times, evaporated under reduced pressure at 40 ℃, upper strata, is prepared into green evaporate to dryness thing 0.584 g(B component).The pH of Zai Jiang lower floor is adjusted to 2, ethyl acetate extraction 4 times, the upper and lower, respectively 40 ℃ 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 respectively in absolute methanol, are prepared into 4 kinds of methanol solutions.Component A solution loads on Sephadex LH-20 gel filtration chromatography, and 30%, 50%, 70%, 90% and 100% methanol solution of take is eluting solvent.Through thin-layer chromatography, detect, merge and collect eluent, reduced pressure concentration, under 30-50%, 70% and 100% methanol solution wash-out, collects 3 kinds of components respectively, is designated as component AA, component AB and component AC.In these 3 kinds of components, due to component AB and component AC considerably less, therefore, in subsequent separation process, purified components AA only.By component AA solution and B component, component C and component D solution point sample in silica G F
254upper, carry out thin-layer chromatography detection.Chloroform/acetone/the formic acid (15:3:2) of take is solvent, after expansion, under ultraviolet 254 nm, observes, and 4 spots appear 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.Adopting chloroform/methanol (8:1) is solvent, and under ultraviolet 254 nm, 2 spots appear in component D,
r fbe respectively 0.333 and 0.575.On this basis, with method of scoring point sample repeatedly, merge collect identical
r fplace's band, is dissolved in acetone again, through centrifugal and reduced pressure concentration, is prepared into 9 kinds of components, is designated as successively component AA
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, the chloroform/acetone/formic acid (18:1:3) of take is solvent, is prepared into 8 kinds of components, is designated as successively component AA
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 14 kinds of components of 0.1 mL, after solvent evaporates, add 1 mol/L glacial acetic acid solution 1 mL, fully vibration, add the potassium ferricyanide-ferric trichloride developer (0.6 % potassium ferricyanide solution and 0.9 % liquor ferri trichloridi mix matching while using by 0.9:1.0 before 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 demonstration, 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 evaporates, adds bismuth potassium iodide solution 1 mL, fully vibration, 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
254upper, respectively in chloroform/methanol, under 3 kinds of solvents such as cyclohexane/ethyl acetate and n-butanol/acetic acid/water, launch, 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), discovery is under these 3 kinds of developers, and these 14 kinds of components present single spot equally, show that purity has reached 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 acicular crystal 0.010 g, AA
42pale yellow powder 0.015 g, AA
43pale yellow powder 0.008 g, AA
44faint yellow crystallization 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.
Adopt the experiment of algal control circle to carry out algistatic activity detection.At several diameters, be on the circular filter paper sheet of 1.5 cm, drip respectively 5 μ L component acetone to be measured concentrates and acetone (as a control group), after acetone volatilizees completely, filter paper is positioned on the solid culture medium of the Skeletonema Costatum of inoculating exponential phase, being placed in GXZ-260B intelligent illumination incubator cultivates, temperature (26 ± 1) ℃, intensity of illumination 62 μ mol m
-2s
-1, Light To Dark Ratio is 12:12.After 2d, observe filter paper and the growth of tested micro-algae around, find 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 over 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 to absolute methanol, normal temperature, dark lower immersion, obtain leaching liquor after filtering; Evaporated under reduced pressure, obtains evaporate to dryness thing; Evaporate to dryness thing adds distilled water, fully vibration, and standing over night at 4 ℃, centrifugal, filter, remove precipitation, obtain supernatant;
(2) supernatant is adjusted to pH to 11, add ethyl acetate extraction, gained upper strata evaporated under reduced pressure, obtains component A, regulate the pH to 7 of gained lower floor, ethyl acetate extraction, gained upper strata evaporated under reduced pressure, obtains B component, regulate again the pH to 2 of gained lower floor, ethyl acetate extraction, gained the upper and lower are evaporated under reduced pressure respectively, obtains component C and component D; A, B, C, 4 kinds of components of D are dissolved in respectively in absolute methanol, make A, B, C, 4 kinds of methanol solutions of D;
(3) methanol solution of component A is loaded on Sephadex LH-20 gel filtration chromatography, 30%, 50%, 70%, 90% and 100% methanol solution of take is eluting solvent; Through thin-layer chromatography, detect, merge and collect eluent, reduced pressure concentration, under 30-50%, 70% and 100% methanol solution wash-out, collects 3 kinds of components respectively, is designated as component AA, component AB and component AC; By component AA, and the methanol solution point sample of B component, C, D is in silica G F
254upper, carry out thin-layer chromatography detection; Chloroform/acetone/formic acid that the volume ratio of take is 15:3:2 is solvent, after expansion, under ultraviolet 254 nm, observes, and 4 spots appear 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; Adopting the chloroform/methanol that volume ratio is 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 point sample repeatedly, merge collect identical
r fplace's band, is dissolved in acetone again, through centrifugal and reduced pressure concentration, is prepared into 9 kinds of components, is designated as successively component AA
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, chloroform/acetone/formic acid that the volume ratio of take is 18:1:3 is solvent, is prepared into 8 kinds of components, is designated as successively component AA
21, component AA
22, component AA
23, component AA
41, component AA
42, component AA
43, component AA
44with component C
11;
(4) through compound qualitative detection, length scanning and thin-layer chromatography, detect, 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, obtains respectively.
2. the phenolic acid that method makes as claimed in claim 1 or the alkaloid purposes in the growth that suppresses the triumphant human relations algae of Michaelis or Skeletonema Costatum.
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| CN106561722A (en) * | 2016-11-08 | 2017-04-19 | 淮海工学院 | Algal inhibition application of undaria pinnatifida and separation and purification method of undaria pinnatifida algal inhibition active substance |
| CN106561722B (en) * | 2016-11-08 | 2019-05-10 | 淮海工学院 | The algal control purposes of thallus laminariae and the isolation and purification method of thallus laminariae algistatic activity substance |
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