CN108124908B - Component C separated from sargassum henryi ethyl acetate extract, preparation method thereof and application of component C in marine fouling organism prevention and removal - Google Patents
Component C separated from sargassum henryi ethyl acetate extract, preparation method thereof and application of component C in marine fouling organism prevention and removal Download PDFInfo
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Abstract
The invention discloses a component C separated from an ethyl acetate extract of sargassum henryi, a preparation method thereof and application thereof in preventing and removing marine fouling organisms. The component C separated from the ethyl acetate extract of the sargassum henryi of the invention has obvious effect of inhibiting barnacle and mussel larva from attaching when being coated on a solid surface at low dose, thus being capable of being used for preparing marine fouling organism control agents. The component C is extracted from sargassum henryi, is a naturally-occurring organic compound, does not pollute the water environment and is enriched in organisms due to food chain transfer, is environment-friendly and high in safety, does not contain heavy metal elements such as copper and tin while effectively inhibiting the attachment of marine organisms, has good social benefits from the aspect of environmental protection, is wide in source, simple and convenient in separation and preparation approach, suitable for large-scale production, high in popularization potential and has good application prospects in marine fouling organism prevention and removal.
Description
The technical field is as follows:
the invention belongs to the field of natural products, and particularly relates to a component C separated from an ethyl acetate extract of sargassum henslowianum, a preparation method of the component C and application of the component C in marine fouling organism prevention and removal.
Background art:
marine fouling organisms refer to various organisms which are fixed or inhabit on underwater parts of ships and artificial facilities and have adverse effects on human economic activities, and the marine fouling organisms mainly have the harm of increasing resistance, reducing navigational speed and increasing fuel consumption; blocking a pipeline system, changing a metal corrosion process and initiating corrosion perforation; the dynamic load effect is increased, so that artificial facilities drift, are unbalanced and even overturn; compete for the attaching base and bait with the culture object, hinder the growth and development of the culture object and reduce the quality of aquatic products. Therefore, in the development of the marine economic industry, the control of marine fouling organisms is an essential part of the development of the marine economic industry.
Marine biofouling organisms consist of animals, plants and microorganisms, of which the more harmful and difficult to remove species are mainly limy hulls, sessile living stemless vines (barnacles) and bivalve mollusks (mussels and oysters). In tropical and subtropical coastal areas, balanus reticulatus is a typical representative of sessile tendrils and is also an absolute dominant species in fouling organism communities; perna viridis is a major bivalve mollusk in the east and south seas. Fouling organisms are generally divided into two life stages, namely a planktonic life stage from the development of larva out of an egg membrane until the surface of an object is explored intermittently to prepare for attachment; the larvae are selected to settle at the settlement position, and after the larvae are adhered to the surface of the adhering base and are transformed into larvae, the larvae are in the stage of fixation or adhering to the life. From the viewpoint of control, it is considered that the damage to human beings is caused after fixation or adhesion. If the adhesion of larvae can be effectively inhibited, the purpose of preventing and removing can be achieved. Therefore, the verification test of the invention adopts the balanus reticulates and the perna viridis as experimental objects, and the obtained research result has scientific reasonability and wide representativeness.
The traditional marine fouling organism control technology has the defects of high cost, short action time, poor target organism pertinence and the like, and even can generate the problem of environmental hazard, so the understanding of the natural chemical antifouling action mechanism of marine organisms can provide reference for developing pollution-free antifouling technologies, and the marine organisms themselves also can be important sources of novel antifouling agents. The content of the anti-adhesion active substances in marine animals and microorganisms is low, and the structure is relatively complex, so that the marine animals and microorganisms are inconvenient to deeply develop and widely apply. Therefore, the natural anti-fouling agent is searched from marine plants which are widely distributed and have large resource quantity, and the natural anti-fouling agent is not only a brand-new attempt, but also has important theoretical and practical significance.
Sargassum henryi (Sargassum hensellowianum c. agardh) belongs to phaeophyta, phaeophyceae, Fucales, Sargassaceae, and Sargassum, and is widely distributed on rock in low tide zone of coastal areas such as Fujian, Guangdong, and hong Kong. The algae is large in size, rich in resources, short in growth cycle, capable of being cultured in large scale and good in development and utilization prospect, and recently, people find that the algae contains bioactive components such as anti-tumor, anti-virus, blood fat reducing, anti-oxidation and free radical scavenging. However, there has not been any report on whether or not sargassum henryi contains an effective component for preventing the adhesion of larvae and the germination of spores of marine biofouling organisms and whether or not it can be applied to the field of marine biofouling organism control.
The invention content is as follows:
the invention aims to provide a component C separated from an ethyl acetate extract of sargassum henryi, a preparation method thereof and application thereof in preventing and removing marine fouling organisms.
The first purpose of the invention is to provide the application of sargassum henryi in preparing marine fouling organism control agents. Further relates to the application of the sargassum henryi ethyl acetate extract in preparing marine fouling organism control agents.
The second purpose of the invention is to provide a preparation method of component C separated from the ethyl acetate extract of sargassum henryi, which comprises the following steps:
extracting sargassum henslowianum with ethanol water solution, and concentrating the ethanol extract to obtain paste extract; sequentially extracting the paste extract with petroleum ether and ethyl acetate, and concentrating and drying ethyl acetate extract to obtain ethyl acetate extract; performing silica gel column chromatography on the ethyl acetate extract, performing gradient elution by using petroleum ether-acetone as an eluent according to the volume ratio of 100:0 to 50:1 to 20:1 to 10:1 to 5:1, and collecting petroleum ether: and (3) eluting a component Fr.3 with the acetone volume ratio of 5:1, performing gel column chromatography, eluting with an eluent with the chloroform-methanol volume ratio of 1:1, performing tracking detection by adopting Thin Layer Chromatography (TLC), and collecting a fraction on a thin layer plate which is eluted with petroleum ether: acetone is used as developing agent in the volume ratio of 5:1, and the Rf value is 0.65, thus obtaining component C of sargassum henryi ethyl acetate extract.
The ethanol water solution is preferably ethanol water solution with the volume fraction of 95%.
The third object of the present invention is to provide a separated fraction C of an ethyl acetate extract of sargassum henryi prepared according to the above preparation method of the separated fraction C of an ethyl acetate extract of sargassum henryi.
The component C of the ethyl acetate extract of sargassum henryi of the invention is coated at a coating amount of 10 mu g/cm2In the process, the attachment rate of barnacle larvae after 72 hours is 10.7 percent and is remarkably lower than 66.2 percent of a control group, which indicates that the component C separated from the sargassum henryi ethyl acetate extract has obvious inhibiting effect on the barnacle larvae. The coating amount of component C separated from ethyl acetate extract of Sargassum henryi is 10 μ g/cm2In time, the attachment rate of the mussel larvae after 72 hours is only 5.3 percent and is far lower than that of the control group, which shows that the component C separated from the ethyl acetate extract of the gulfweed can effectively inhibit the attachment of the mussel larvae. Therefore, the component C separated from the ethyl acetate extract of the sargassum henryi has good inhibition effect on larva attachment of the stemless cranberries and bivalves mollusks.
Therefore, the fourth object of the present invention is to provide the use of component C isolated from ethyl acetate extract of sargassum henryi for the prevention and treatment of marine biofouling organisms.
The marine fouling organisms are preferably stemless tendrils and bivalves mollusks.
The stemless tendrils are preferably balanus reticulatus, and the bivalves are preferably perna viridis.
Preferably, the component C is separated from the ethyl acetate extract of sargassum henryi and is coated on the solid surface in the amount of 10 mu g/cm2The above.
The fifth purpose of the invention is to provide the application of the component C separated from the ethyl acetate extract of sargassum henryi in preparing the marine fouling organism control agent.
The marine fouling organism control agent is preferably a barnacle larva control agent or a mussel larva control agent.
Compared with the prior art, the invention has the following beneficial effects:
the component C separated from the ethyl acetate extract of the sargassum henryi has obvious effect of inhibiting marine organism adhesion when being coated on a solid surface at a low dose, so that the ethyl acetate extract of the sargassum henryi can be used for preparing a marine fouling organism control agent. The component C of the sargassum henslowianum ethyl acetate extract is a naturally-occurring organic compound, does not pollute the water environment and cause the enrichment of the sargassum henslowianum in organisms through food chain transfer, is environment-friendly and high in safety, does not contain heavy metal elements such as copper and tin while effectively inhibiting the attachment of marine organisms, has good social benefits from the aspect of environmental protection, has wide sources of sargassum henslowianum, is simple and convenient in separation and preparation method of the component C of the sargassum henslowianum ethyl acetate extract, is suitable for large-scale production, has large popularization potential, and has good application prospect in preventing and removing marine fouling organisms.
The specific implementation mode is as follows:
the following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1:
the component C is extracted from sargassum henslowianum, and the separation and extraction steps comprise:
(1) extracting Sargassum henryi (Sargassum hensellowianum C. Agardh) with 95% ethanol water solution with volume fraction for 3 times, mixing extractive solutions to obtain ethanol extractive solution, vacuum rotary evaporating at 45 deg.C for concentration, and removing ethanol to obtain paste extract;
(2) adding water into the paste extract at a ratio of 6 ml per gram for re-suspending, extracting with isovolumetric petroleum ether for 3 times, respectively, discarding petroleum ether extract components, extracting the residual water layer with isovolumetric ethyl acetate for 3 times, combining ethyl acetate extracts, concentrating and drying to obtain ethyl acetate extract;
(3) subjecting the ethyl acetate extract to chromatographic separation in a normal pressure silica gel column (8000 g of silica gel, column length 180cm, column diameter 20cm), eluting with petroleum ether-acetone and chloroform-methanol at a petroleum ether-acetone volume ratio of 100:0, 50:1, 20:1, 10:1, 5:1, 3:1, 2:1, 1:1, 1:2, and chloroform-methanol at a chloroform-methanol volume ratio of 5:1, 3:1, 1:1, 0:100, performing tracking detection by Thin Layer Chromatography (TLC), combining similar components to obtain 25 components numbered as Fr.1-25;
(4) mixing petroleum ether: performing Sephadex LH-20 gel chromatography separation on a component Fr.3 eluted by an acetone volume ratio of 5:1, eluting by using chloroform to methanol volume ratio of 1:1 as an eluent, collecting eluted fractions at the speed of 8 min/tube, numbering according to the collection time sequence, numbering as 1-50, performing tracking detection by adopting Thin Layer Chromatography (TLC), wherein 32-36 tubes are the same component (petroleum ether to acetone volume ratio of 5:1 is used as a developing agent, and the Rf value is 0.65), and combining and concentrating to obtain a separated component C of the sargassum henslowianum ethyl acetate extract.
Example 2:
experimental groups: the component C of the ethyl acetate fraction of sargassum henryi was dissolved in a chloroform-methanol mixed solvent (chloroform-methanol mixed solvent is a mixture of chloroform and methanol at a volume ratio of 3: 1) to prepare an experimental solution having a concentration of 282.6. mu.g/ml. 1ml of 282.6. mu.g/ml of the test solution was added to a 6cm diameter dish and allowed to cover the bottom of the dish uniformly. After the solvent was completely volatilized, the amount of component C separated from the ethyl acetate extract of Sargassum henryi coated on the bottom of the petri dish was 10. mu.g/cm2. 13ml of seawater was then added to the petri dish.
Control group: adding 1ml chloroform-methanol mixed solvent (the chloroform-methanol mixed solvent is formed by mixing chloroform and methanol according to the volume ratio of 3: 1) to uniformly distribute the chloroform-methanol mixed solvent at the bottom of the culture dish, and adding 13ml seawater after the chloroform-methanol mixed solvent is completely volatilized.
Blank group: 13ml of seawater was added to the petri dish.
Each of the experimental, blank and control groups was provided with 4 replicates. 30 cyprids of balanus reticulates were added to each sample. Culturing in a constant temperature incubator at about 30 deg.C in dark environment. The observation was performed every 24 hours. The final attachment and death status of each group of larvae was statistically analyzed after 72 hours of culture, and the specific results are shown in table 1.
Table 1 lists the attachment and mortality rates of the cyprids of the experimental, control and blank groups. As can be seen, after 72 hours of incubation in the incubator, the rate of larval attachment was 69.1% for the blank groupThe attachment rate of the cyprids of the blank group and the control group has no significant difference (P) according to 66.2 percent of the control group>0.05), indicating that harmful substances which influence the activity of the cyprids cannot be left after the chloroform-methanol mixed solvent is volatilized. As for the experimental group in which the bottom of the culture dish was covered with the ethyl acetate extract of sargassum henryi to separate the component C, the attachment rate of the cyprids of the jinxing was 10.7%, which was smaller than that of the control group, and the difference was significant (P)<0.01), indicating that component C is separated at 10. mu.g/cm in the ethyl acetate extract of sargassum henryi2Can effectively inhibit the attachment of cyprids of balanus reticulatus under the dosage of the composition. In addition, the larval mortality rates of the blank group, the control group and the experimental group were all 0, indicating that the component C isolated from the ethyl acetate extract of sargassum henryi does not exert a poisoning effect on the larvae at this dose.
Table 1: attachment and death status of cyprids of balanus reticulatus 72 hours later
| Group of | Test dose | Adhesion Rate (%) | Mortality (%) |
| Blank group | － | 69.1 | 0 |
| Control group | － | 66.2 | 0 |
| Experimental group | 10μg/cm2 | 10.7 | 0 |
Example 3:
experimental groups: the component C of the ethyl acetate fraction of sargassum henryi was dissolved in a chloroform-methanol mixed solvent (chloroform-methanol mixed solvent is a mixture of chloroform and methanol at a volume ratio of 3: 1) to prepare an experimental solution having a concentration of 282.6. mu.g/ml. 1ml of 282.6. mu.g/ml of the test solution was added to a 6cm diameter dish and allowed to cover the bottom of the dish uniformly. After the solvent was completely volatilized, the amount of component C separated from the ethyl acetate extract of Sargassum henryi coated on the bottom of the petri dish was 10. mu.g/cm2Then 13ml of seawater was added to the petri dish.
Control group: adding 1ml chloroform-methanol mixed solvent (the chloroform-methanol mixed solvent is prepared by mixing chloroform and methanol at a volume ratio of 3: 1) to uniformly distribute the solvent at the bottom of the culture dish, and adding 13ml seawater when the solvent is completely volatilized.
Blank group: 13ml of seawater was added to the petri dish.
The experimental group, the blank group and the control group are all provided with 4 parallel samples, and about 30 perna viridis faceplate larvae are added into each sample. Culturing in dark environment in an incubator at a temperature of about 26 ℃. The observation was performed every 24 hours. Statistical analysis of the final attachment and death status of each group of larvae was performed after 72 hours of culture.
Table 2 lists the attachment and mortality rates of perna viridis larvae in the experimental, control and blank groups. It can be seen that after the culture in the constant temperature incubator for 72 hours, the attachment rate of the larvae of the blank group is about 55.6%, the attachment rate of the larvae of the control group is about 53.3%, and the attachment rates of the larvae of the dough plates of the blank group and the control group are not significantly different (P is greater than 0.05), which indicates that harmful substances which affect the activity of the larvae of the dough plates cannot be left after the chloroform-methanol mixed solvent is volatilized. The adhesion rate of larvae of the experimental group treated by the component C separated from the sargassum henryi ethyl acetate extract is only 5.3%, the difference is very obvious (P is less than 0.01) when the experimental group is far lower than that of the control group, and the fact that the component C separated from the sargassum henryi ethyl acetate extract can effectively inhibit the adhesion of larvae of perna viridis is shown. In addition, the larval mortality rates of the blank group, the control group and the experimental group were all 0, indicating that the component C isolated from the ethyl acetate extract of sargassum henryi does not exert a poisoning effect on the larvae at this dose.
Table 2: attachment and death of emerald green common mussel faceplates larvae after 72 hours
| Group of | Test dose | Percentage of adhesion% | The mortality rate is% |
| Blank group | － | 55.6 | 0 |
| Control group | － | 53.3 | 0 |
| Experimental group | 10μg/cm2 | 5.3 | 0 |
Claims (3)
1. The application of the component C separated from the ethyl acetate extract of sargassum henslowianum in preventing and controlling marine fouling organisms or preparing a marine fouling organism control agent is characterized in that the component C separated from the ethyl acetate extract of sargassum henslowianum is prepared by the following method: extracting sargassum henryi with 95% ethanol water solution by volume fraction, and concentrating the ethanol extract to obtain a paste extract; sequentially extracting the paste extract with petroleum ether and ethyl acetate, and concentrating and drying ethyl acetate extract to obtain ethyl acetate extract; and (3) performing silica gel column chromatography on the ethyl acetate extract, performing gradient elution by using petroleum ether-acetone as an eluent according to the volume ratio of 100:0 to 50:1 to 20:1 to 10:1 to 5:1, and collecting petroleum ether: and (3) eluting a component Fr.3 with the acetone volume ratio of 5:1, performing gel column chromatography, eluting with an eluent with the chloroform-methanol volume ratio of 1:1, performing tracking detection by adopting Thin Layer Chromatography (TLC), and collecting a fraction on a thin layer plate which is eluted with petroleum ether: acetone is used as a developing agent in a volume ratio of 5:1, the component with the Rf value of 0.65 is obtained, the component C of the sargassum henryi ethyl acetate extract is obtained, and the marine fouling organisms are stemless tendrils and bivalve mollusks.
2. The use according to claim 1, wherein said sessile tendrilis is balanus reticulatus and said bivalves are perna viridis.
3. The use according to claim 1 or 2, wherein said use comprises applying said component C of ethyl acetate extract of Sargassum henryi in an amount of 10 μ g/cm to a solid surface2The above.
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