CN103299998A - Application of ginkgolic acid in killing of blue-green algae - Google Patents
Application of ginkgolic acid in killing of blue-green algae Download PDFInfo
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Abstract
The invention discloses application of ginkgolic acid in killing of a blue-green algae, application of an alcohol extract of ginkgo biloba sarcotesta in killing of the blue-green algae, a composition and application of the composition in killing of the blue-green algae. It is observed that the ginkgolic acid and a preparation containing the ginkgolic acid have strong activity of killing the blue-green algae from the embodiment of the invention. In addition, the ginkgolic acid is plant source algicide, is taken from the natural world and applied to the natural world, is low in toxicity and residue, and is an algicide which is friendly to the environment, and simple in using method; and the drug resistance is not easily generated.
Description
Technical field
The present invention relates to the application of ginkgolic acid in killing blue algae.
Background technology
In recent ten years, frequency economic loss more and more higher, that bring increasing (Zhao Yuhang, the Yang Hongsheng of some waters wawter bloom (blue-green algae) generation of China.A kind of research of Euglena wawter bloom in the fishpond.The aquatile journal, 1994,18 (2): 186-188), algal bloom has become the problem that has a strong impact on quality of water environment and water ecology safety, and not only great harm humans is healthy and other biological safety, also causes economically huge loss.According to the literature, the kind of these waters formation wawter blooms mainly contains microcystis kutz and the Aphanizomenon of Cyanophyta, wherein again with microcystic aeruginosa (the Heath R.L that has comparative advantage, Parker L.Photoperitation in isolated choloplasts kinetics and stoichiometry of fatty acid peroxidation.Arch Biophy, 1968,75 (2): 189-198).
Microcystic aeruginosa (Microcystic aeruginosa) is a kind of prokaryotic micro-organisms that extensively is present in aquatic ecosystem, belong to Cyanophyta (Cyanophyta), Chroococcaceae (Chroococcaceae), microcystis kutz (Microcystis Kutz) (Liu Wentao.Leek extract is to the research [D] of microcystic aeruginosa Water extract.Yangzhou University, 2009).Plant cell is many cells colony, and colony's glue is colourless by homogeneous, the cement shape that often becomes to disperse.The children plants the solid colony that body is ball-type or ellipsoid, after grow up to and be network-like hollow cystidium, utricule breaks and forms netted colloid colony subsequently.Cell dia 3-7 μ m presents blue-green, with particle or pseudo-cavity.Grow in the hydrostatic more, in spring and summer grow prolifically in season, often form wawter bloom.
At present, in order to control the growth of microcystic aeruginosa, carried out a large amount of research both at home and abroad, generally can adopt chemical method, change water law except algae, microorganism algal control and animal predation are except the methods such as algae (Tang Ping, Wu Guorong, Lu Changmei, girth virtue, Wei Jincheng.Eichhornia Crassipes Roots is on the impact [J] of grid algal structure and metabolism.ACTA Scientiae Circumstantiae, 2000,20 (3): 355-359).Wherein, changing water law cures the symptoms, not the disease, not only waste water resource, and cost is very high, and utilizes microorganism and animal control algae may have ecological risk and wayward, chemical method is method commonly used at present, its used medicine has multiple, but at present with the most use be copper sulphate, but this pharmacy effect is of short duration, and copper sulphate itself also is a kind of pollutant, should not repeatedly use.The drawbacks limit of these methods itself applying of they.In this case, received colleague's very big concern both at home and abroad with plant control body eutrophication, and the plant resource medicine since its get nature, use nature, low toxicity, low-residual, non-harmful characteristics and become the focus of Recent study.But these researchs lay particular emphasis on the interaction between water plants and the blue-green algae more, and are less for the research of terrestrial plant.
Ginkgo (Ginkgo biloba L.) is Ginkgoaceae Ginkgo plant, has another name called Gong Sunshu, maidenhair tree, is the rare rare tree of China.Ginkgo is precious medicinal plant, and its leaf, fruit and exosper etc. all have medicinal exploitation and be worth, and is called as " whole body all is precious living fossil ".A large amount of studies confirm that both at home and abroad, mainly contain multiple ginkgolic acid (Ginkgolic acids) in the gingko episperm, ginkgolic acid is the salicylic derivative of a class, side chain carbon number on its 6 can be 13 to 17, the pendant double bonds number can be 0 to 3, a homology mixture (Wang Jie, wish and establish one's virtue, Yu Biyu etc., extraction fromginkgoseed coat suppresses the research [J] of growth and antifeedant activity to cabbage caterpillar, Yangzhou University's journal (agricultural and life science version), 2002,23 (1): 72-75; H.Itokawa, N.Totsuka, K.Nakahara, et al.Antitumor principles from Ginkgo biloba L[J] .Chem.Pharm.Bull.1987,35:3016; Mohamed Yalpami, John H.P.Tyman.The phenolic acids of pistachio vera[J] .Phytochemistry, 1983,22 (10): 2263-2266; Maria Jose T.G.Gonzalez, Carlos J.C.Deoliveiveira, Jose O.Fernandes, et al.Further alkyl and alkenylphenols of knema laurina and knema austrosiameensis:location of the double bond in the alkenyl side chains[J] .Phytochemistry, 1996,43 (6): 1333-1337).
From gingko episperm, separated at present and identified ginkgoic acid (Wang Jie wishes and establishes one's virtue, Yu Biyu etc., extraction fromginkgoseed coat suppresses the research [J] of growth and antifeedant activity to cabbage caterpillar, Yangzhou University's journal (agricultural and life science version), 2002,23 (1): 72-75; Mohamed Yalpami, John H.P.Tyman.The phenolic acids of pistachio vera[J] .Phytochemistry, 1983,22 (10): 2263-2266; ), ginkgol (Maria Jose T.G.Gonzalez, Carlos J.C.Deoliveiveira, Jose O.Fernandes, et al.Further alkyl and alkenylphenols of knema laurina and knema austrosiameensis:location of the double bond in the alkenyl side chains[J] .Phytochemistry, 1996,43 (6): 1333-1337) with the ginkgo diphenol, structure is suc as formula shown in the I.The reports such as JaggyH contain 4 kinds of ginkgoic acid C in the gingko episperm
13:0, C
15:1, C
17:2And C
17:1And determine content and be respectively 0.19%, 3.10%, 0.03% and 0.22%, total ginkgoic acid content is 3.54% (Jaggy H, Koch E.Chemistry and biology of alkylphenols from Ginkgo biloba L.[J] .Pharmazie, 1997,52 (10): 735-738.).
Yet, a large amount of studies confirm that of external many scholars (Gayland F, Spencer, Larry W.Tjarks, Robert Kleiman.Alkyl and phenylalkyl anacardic acids from knema elegans seed oil[J] .Journal ofNatural products, 1980,43 (6): 724-730; Nigel J, Coates, Martin L.Gilpin, Mick N.Gwynn, et al.SB-202742, A Novel β-Lactamase inhibitor isolated from spondias mombin[J] .Journal of Natural products, 1994,57 (5): 654-657): the ginkgolic acid majority is that 5 kinds of compounds form, and is respectively C
13:0, C
15:0, C
15:1, C
17:1And C
17:2(Hidej Itokawa, Nobuo Totsuka, Keisuka Nakahara.Antitumor Principles from Ginkgo bilobaL[J] .Chem.Pharm.Bull, 1987,35 (7): 3016-3020), this with face upward blue or green grade of pomegranate and (face upward pomegranate green grass or young crops, Wu Xiangyang, Chen Jun.The content of ginkgoic acid [J] in the high effective liquid chromatography for measuring gingko episperm. 901) and (Li Hongqing, He Zhaofan, the Zhang Yongmin etc. such as Li Hongqing analytical chemistry, 2002,30 (8):.Gingko episperm alkyl phenol and alkyl constituents research [J]. Chinese herbal medicine, 2004,35 (1): research 18-20) is consistent.Facing upward the content that the blue or green RP-HPLC of employing of pomegranate method records ginkgolic acid in the gingko episperm is 5.46%, identifies 5 kinds of ginkgolic acid composition (C with LC-ESI-MS
13:0, C
15:0, C
15:1, C
17:1And C
17:2), wherein with ginkgolic acid (Ginkgolicacid, C
15:1) content is the highest, secondly is hydroginkgoic acid (Hydroginkgolieaeid, C
15:0), the inferior acid of gingko (Hydroginkgolinieacid, C
17:2).
C
13:0R＝(CH
2)
12CH
3
C
15:1R＝(CH
2)
7CH＝CH(CH
2)
5CH
3
C
17:2R＝(CH
2)
8CH＝CHCH
2CH＝CH(CH
2)
3CH
3
C
15:0R＝(CH
2)
14CH
3
C
17:1R＝(CH
2)
9CH＝CH(CH
2)
5CH
3
Studies show that, the multiple pharmacological activity of ginkgolic acid tool, present known clinical application has:
(1) bacteriostatic activity: the ginkgolic acid in the ginkgolic acid, hydroginkgoic acid etc. can suppress streptococcus, staphylococcus, anthrax bacteria etc.;
(2) antitumor and antiviral activity: the long-chain phenolic acid in the gingko episperm has antitumor activity, especially murine sarcoma S180 hamster V-79 cell is had stronger inhibitory action;
(3) be widely used in human diseases: domestic present large quantities of ginkgo agents listing, such as ginkgo oral liquid EBG-761 etc., be mainly used in human heart and brain blood disease, neurogenic disease etc., have relieving cough and asthma effect more than the separating Ginkgo phenolic acids extract;
(4) biological pesticide technical field: the ginkgolic acid compounds is in the purposes of killing on the Pomacea canaliculata, such as among the CN101911938A record (applicant: Yang Xiaoming, Ren Xiaofeng, Liu Weimin).Main application is for utilizing the ginkgolic acid compounds to kill the harmful mollusk of agricultural;
(5) biological pesticide technical field: ginkgolic acid has food refusal and toxic action to diamondback moth larvae;
(6) aquaculture field: phyteral external parasite preventing and treating agent for aquatic product animals and preparation method, such as among the CN1545890A record (applicant: Wang Gaoxue, Zhao Yunkui, Cheng Minglie, Dong Jun).Main application is for utilizing the ginkgolic acid compounds to kill the parasitic Dactylogyrus of fish.
Summary of the invention
The function that the purpose of this invention is to provide a kind of new killing blue algae is used, and particularly, is the method for utilizing the ginkgolic acid killing blue algae.
The invention provides the application of ginkgolic acid in killing blue algae.
The present invention also provides the application of alcohol extract in killing blue algae of gingko episperm.
In addition, the present invention also provides a kind of composition, it is characterized in that, said composition contains five kinds of compd A-E shown in general formula I,
R is-(CH in the compd A
2)
12CH
3R is-(CH in the compd B
2)
7CH=CH (CH
2)
5CH
3R is-(CH in the Compound C
2)
14CH
3R is-(CH in the Compound D
2)
9CH=CH (CH
2)
5CH
3R is-(CH in the compd E
2)
8CH=CHCH
2CH=CH (CH
2)
3CH
3Take the weight of said composition as benchmark, the total content of compd A-E surpasses 40 % by weight.In addition, also provide the application of above-mentioned composition in killing blue algae.
Microcystic aeruginosa is as the model organism of Cyanophyta, for its research conclusion whole Cyanophyta all had general directive significance, and therefore, the present inventor selects microcystic aeruginosa as research object.Particularly, the algicdal activity of the B-2 flow point group (being ginkgolic acid) in the alcohol extract of gingko episperm is measured, can be found out, this stream is grouped in the LC of the inhibition microcystic aeruginosa of the 3rd day and the 7th day
50Be respectively 4.25mg/L and 3.41mg/L.This explanation, ginkgolic acid has the extremely strong activity of killing microcystic aeruginosa, also namely has the activity of extremely strong killing blue algae.
In addition, ginkgolic acid is the plant resource algicide, gets the nature of naturally using, and low toxicity and low residue is difficult for producing drug resistance, is the simple algicide of a kind of environmental friendliness and using method.
Description of drawings
Accompanying drawing is used to provide a further understanding of the present invention, and consists of the part of specification, is used from explanation the present invention with following embodiment one, but is not construed as limiting the invention.In the accompanying drawings:
Fig. 1 is the HPLC chromatogram of B-2 flow point group sample in the alcohol extract of gingko episperm;
Fig. 2 is the HPLC chromatogram of ginkgolic acid standard items;
Fig. 3 is the liquid chromatography-mass spectrography figure of B-2 flow point group sample in the alcohol extract of gingko episperm, wherein, Fig. 3-the 1st, the TIC of B-2 flow point group sample, Fig. 3-the 2nd, (from left to right, the i.e. C of first chromatographic peak in the total ion current of B-2 flow point group sample
13:0) mass spectrogram, Fig. 3-the 3rd, (from left to right, the i.e. C of second chromatographic peak in the total ion current of B-2 flow point group sample
15:1) mass spectrogram, Fig. 3-the 4th, (from left to right, the i.e. C of the 3rd chromatographic peak in the total ion current of B-2 flow point group sample
17:2) mass spectrogram, Fig. 3-the 5th, (from left to right, the i.e. C of the 4th chromatographic peak in the total ion current of B-2 flow point group sample
15:0) mass spectrogram, Fig. 3-the 6th, (from left to right, the i.e. C of the 5th chromatographic peak in the total ion current of B-2 flow point group sample
17:1) mass spectrogram.
Embodiment
Below the specific embodiment of the present invention is elaborated.Should be understood that, embodiment described herein only is used for description and interpretation the present invention, is not limited to the present invention.
The invention provides the application of ginkgolic acid in killing blue algae, wherein, described ginkgolic acid has the structure shown in the formula I,
Wherein, R is-(CH
2)
12CH
3(be ginkgolic acid C
13:0) ,-(CH
2)
7CH=CH (CH
2)
5CH
3(be ginkgolic acid C
15:1) ,-(CH
2)
8CH=CHCH
2CH=CH (CH
2)
3CH
3(be ginkgolic acid C
17:2) ,-(CH
2)
14CH
3(be ginkgolic acid C
15:0) and-(CH
2)
9CH=CH (CH
2)
5CH
3(be ginkgolic acid C
17:1).That is, among the present invention, described " ginkgolic acid " is the mixture of above-mentioned five kinds of materials, and also, the present invention is identical with the definition of national standard " ginkgolic acid " to the definition of " ginkgolic acid ".The separately content of the not clear and definite wherein five kinds of compounds of national standard " ginkgolic acid ", and only refer to the mixture of these five kinds of compounds of from gingko episperm, extracting.
Although the ginkgolic acid among the present invention refers to the material identical with national standard, and preferably from gingko episperm, extract and obtain,, the present invention does not get rid of the synthetic ginkgolic acid that obtains.The method of described extraction has been as well known to those skilled in the art, as with the gingko episperm crushed material and alcohol mixture (wherein, the gingko episperm crushed material is 1 with the weight ratio of alcohol: 3-10) under refluxad process at least 1h, with the extract reduced pressure concentration that obtains, obtain the alcohol extract medicinal extract of gingko episperm, then gained medicinal extract is carried out multistage silica gel column chromatography, separate obtaining described ginkgolic acid.
Among the present invention, described " killing microcystic aeruginosa " or " kill algae " both can refer to also can refer to suppress the Growth and reproduction of frustule to the killing of existing frustule.
The present invention also provides the application of alcohol extract in killing blue algae of gingko episperm.
Among the present invention, the concept of described extract is known for those skilled in the art, refers to through physico chemistry and extracts separation process, and orientation is obtained and concentrated a certain or Multiple components, and does not change the structure of this composition and the product that forms.Proterties according to final products is different, and extract can be divided into vegetable oil, medicinal extract, powder, crystalline lens etc.
Among the present invention, the alcohol extract of described gingko episperm is preferably the alcohol extract medicinal extract of gingko episperm.
Described alcohol extract medicinal extract is the extract with the medicinal extract shape of alcohol extracting, and described alcohol can comprise for the various conventional alcohols material that extracts, for example methyl alcohol and/or ethanol.Described methyl alcohol and/or ethanol comprise anhydrous methyl alcohol and/or ethanol, also comprise methyl alcohol and/or the ethanol of technical grade, and wherein the purity of methyl alcohol and/or ethanol is higher than 97%.
There is no particular limitation for the kind of various ginkgolic acids in the alcohol extract of gingko episperm and content in the present invention, as long as contain ginkgolic acid.But take the weight of the alcohol extract of described gingko episperm as benchmark, the content of ginkgolic acid surpasses 40 % by weight in the alcohol extract of described gingko episperm, for example can be the 45-100 % by weight preferably.The alcohol extract that contains the gingko episperm of the ginkgolic acid in the preferable range has the better effect of killing blue algae.
The method that obtains the alcohol extract of above-mentioned gingko episperm can be the method for this area routine, for example can comprise, get the gingko episperm dry product, extract with alcohol reflux after pulverizing, then the concentrated fluid medicinal extract that obtains of pressurization use petroleum ether dissolution, removes residue, concentrated extract obtains the alcohol extract of described gingko episperm.
Wherein, the weight that the condition of described refluxing extraction comprises used alcohol for the 3-10 of the gingko episperm weight pulverized doubly, the time of refluxing extraction can for more than the 1h, be preferably 1.5-5 hour.The degree of petroleum ether dissolution extraction take the extract that obtains near colourless as standard, the weight of each benzinum that is used for dissolving can be 0.8-1.2 times of the gingko episperm weight pulverized, number of operations for example can be for 2-5 time.
The method of described extraction can also be included in before the petroleum ether dissolution, and then the solvent in the fluid medicinal extract that fully volatilizees adds alcohol and dissolve, and gets supernatant concentration, carries out afterwards the step of petroleum ether dissolution extraction again.The pure and mild alcohol for dissolving that is used for refluxing extraction can be the same or different, and is preferably identical.
The present invention also provides a kind of composition, it is characterized in that, said composition contains five kinds of compd A-E shown in general formula I,
R is-(CH in the compd A
2)
12CH
3R is-(CH in the compd B
2)
7CH=CH (CH
2)
5CH
3R is-(CH in the Compound C
2)
14CH
3R is-(CH in the Compound D
2)
9CH=CH (CH
2)
5CH
3R is-(CH in the compd E
2)
8CH=CHCH
2CH=CH (CH
2)
3CH
3Take the weight of said composition as benchmark, the total content of compd A-E surpasses 40 % by weight, for example can be the 45-100 % by weight.The present inventor finds can have the significant blue-green algae effect of killing as long as the total content of above-mentioned five kinds of compounds surpasses the composition of 40 % by weight.Therefore, above-mentioned composition of the present invention can be applicable to killing blue algae.
Described blue-green algae among the present invention is preferably microcystic aeruginosa (Microcystic aeruginosa).But those skilled in the art can recognize, because the typicalness of microcystic aeruginosa in Cyanophyta, described blue-green algae is not limited to microcystic aeruginosa.
The alcohol extract of ginkgolic acid of the present invention, gingko episperm and described composition can be made algicide, and for the purpose of convenience, preferably the alcohol extract with gingko episperm prepares algicide.Particularly, this algicide can contain alcohol extract, emulsifier and the organic solvent of gingko episperm, wherein, preferably, take the volume of this algicide as benchmark, the content of the alcohol extract of gingko episperm is 20-80 volume % in the described algicide, and the content of described emulsifier is 10-50 volume %, and the content of described organic solvent is 10-50 volume %; Further preferably, take the volume of this algicide as benchmark, the content of the alcohol extract of gingko episperm is 35-65 volume % in the described algicide, and the content of described emulsifier is 20-50 volume %, and the content of described organic solvent is 20-50 volume %.
The alcohol extract of described gingko episperm is identical with foregoing description, does not repeat them here.
Emulsifier is drug world surfactant-based material commonly used.Its effect is: when it is dispersed in dispersate surperficial, form film or electric double layer, can make disperse phase with electric charge, so just can stop the droplet of disperse phase to condense mutually, make the emulsion of formation more stable, be convenient to medicine and bring into play drug effect.
Emulsifier among the present invention can be the various emulsifier of this area routine, includes but not limited at least a in homogeneous mixture, 33#, 34#, NP-10, PNP-10, PF-690, BY-130 and the tween-120 of alkylphenol polyoxyethylene, 60 volume % calcium dodecyl benzene sulfonates and 10 volume % aliphatic acid polyethenoxy ethers of 30 volume %.
Among the present invention, described organic solvent also can consider solvability, toxicity and cost for the various organic solvents of routine, and preferably, described organic solvent is methyl alcohol and/or ethanol.
The method for preparing algicide of the present invention can be the method for preparing the biological medicament goods of routine, namely obtain at first according to the method described above the alcohol extract of gingko episperm, then alcohol extract and the organic solvent with gingko episperm mixes to fully dissolving, then in the solution that obtains, add emulsifier, fully stirring and evenly mixing namely makes described algicide.
Described algicide is applied to extremely that the concrete grammar of algae can comprise, adds this algicide in water body.
The addition of described algicide can be regulated as required; Generally preferably, take the volume of water as benchmark, the addition of described algicide is 1-10mL/m
3, be preferably 1-5mL/m
3
Below will describe the present invention by embodiment.In following examples:
Gingko episperm picks up from Taixing City, Jiangsu Province, crosses 40 mesh sieves after the solarization dry grinding.Microcystic aeruginosa (FACHB-905) is provided by aquatic institute of Chinese Academy of Sciences algae kind storehouse.
Ginkgolic acid standard items (be purchased from Nat'l Pharmaceutical ﹠ Biological Products Control Institute, wherein the total phenolic content of ginkgo is 99 % by weight); Used column chromatography silica gel (80-100 order, 200-300 order, 300-400 order) and thin plate silica gel (GF254 type, H type, G type) are all available from Qingdao Marine Chemical Co., Ltd.; It is pure that the organic solvents such as ethanol, methyl alcohol, benzinum, chloroform, ethyl acetate, acetic acid, dimethyl sulfoxide (DMSO) (DMSO), DMF (DMF) are domestic analysis; 10% sulfuric acid ethanol, FeCl
3The developer commonly used such as solution, iodine, BG-11 medium are the experiment autogamy.
Instrument comprises: ZF-2 type three is used ultraviolet device, Shanghai City An Ting Electronic Instruments Plant; Waters 2695 highly effective liquid phase chromatographic systems, Quattro Premier Micromass mass detector, Masslynx4.1 operation system of software; RE-52A rotary evaporator (Shanghai Yarong Biochemical Instrument Plant); BX41 light microscope: OLYMPUS; One of ALC-1100.2 percentage electronic balance, Beijing Sai Duolisi instrument system Co., Ltd; KQ-500E type ultrasonic cleaner.
The assay method of killing the activity of microcystic aeruginosa may further comprise the steps:
(1) medicine to be measured is dissolved with DMSO, is made into the liquid that initial concentration is 200mg/L, and with the liquid gradient dilution, make successively 200,100,50,25,12.5,6.25,3.125,1.0625,0.53125mg/L for reagent liquid, for subsequent use.
(2) for the preparation that tries algae liquid
With the renewed vaccination of well-grown algae liquid, test when selecting its exponential phase.Determine algae cell density by cell counting before the experiment.
(3) kill the microcystic aeruginosa experiment
Long-pending (frustule concentration is as 10 as benchmark take algae liquid
6Individual/as mL), will to be added in the microcystic aeruginosa algae liquid for the ratio of reagent liquid in 0.5 volume %, each concentration arranges 3 parallel group, and establishes the DMSO control group.After dosing the 3rd day and the 7th day added up respectively the amount of survival of frustule and taken the mean, and calculates algal control rate and corresponding LC under the different drug concentrations
50
1, experimental procedure
(1) extraction of test sample and initial gross separation
Under normal temperature condition, 20kg gingko episperm crushed material is extracted 3 times 75 ℃ of hot refluxs with anhydrous industrial alcohol (200L), each 2h merges extract, and reduced pressure concentration gets ethanol extract 6640g.And then with the ethanol extract of gained with isopyknic petroleum ether extraction 3-5 time, until extract is almost colourless, the reduced pressure concentration extract gets benzinum medicinal extract 3276g, extraction yield is 49.3 % by weight, residual residue is 3300g.
(2) separation of petroleum ether extract
(a) sample treatment
Get petroleum ether extract medicinal extract 100g, mix thoroughly with adding 100g silica gel after the methyl alcohol dissolving, put the upper evaporate to dryness of thermostat water bath (temperature 50 C), grind evenly.
(b) dress post and loading
The post of glass chromatography column directly is 100mm, and column length is 1200mm, column chromatography silica gel 2000g (the 200-300 order is pressed 1g sample 20g silica gel and calculated), chloroform wet method dress post, dry method loading.
(c) solvent system
Through thin-layer chromatography (TLC) prerun, determine that (1: 0,200: 1,100: 1,50: 1,25: 1,12.5: 1,5: 1,2: 1,1: 1 and methyl alcohol are washed post successively with chloroform-methanol, this ratio is volume ratio, the ratio of the mixing eluting solvent in the embodiment of the invention is volume ratio) as elution system, carry out gradient elution.
(d) flow point and algicdal activity detection are analyzed, merged to thin-layer chromatography (TLC)
Adopt silica GF254 Preparative TLC plate, the every flow of collecting is divided carry out the TLC analysis.Combined with fluorescent, sulfuric acid ethanol developer are 100 ℃ of colour developings, and each change of component in the test column chromatography is adjusted eluant, eluent polarity according to changing.Every 200mL collects a flow point, collects altogether 165 flow points.Adopt simultaneously TLC to check, merge similar flow point, vacuum and low temperature in vacuum drying chamber (50 ℃) drying, obtain altogether 5 flow point groups: A (1-15 flow point, 5.0g), B (the 16-62 flow point, 31.5g), C (the 63-105 flow point, 19.4g), D (106-135 flow point, 17.5g) and E (the 136-165 flow point, 13.2g).
Five components of A-E are carried out the algicdal activity test according to the method described above, record the B flow point and have algicdal activity.
(3) two stage column chromatography
Get the above-mentioned B flow point of 25.0g and be combined thing, mix thoroughly with adding 25.0g silica gel after the methyl alcohol dissolving, place the upper evaporate to dryness of thermostat water bath (temperature 50 C), grind evenly.Again carrying out silica gel column chromatography separates.
The post of glass chromatography column directly is 70mm, and column length is 1000mm, and column chromatography silica gel is 750g (the 300-400 order is pressed 1g sample 30g silica gel and calculated), benzinum wet method upper prop, dry method loading.
Elution system is petroleum ether-ethyl acetate (1: 0,200: 1,100: 1,99: 1,98: 2,97: 3,95: 5,90: 10,80: 20,2: 1 and methyl alcohol wash post), carries out gradient elution.
Analyze with thin-layer chromatography (TLC), combined with fluorescent, sulfuric acid ethanol developer are 100 ℃ of colour developings, and each change of component in the test column chromatography is adjusted eluant, eluent polarity according to changing.Every 100mL collects a flow point, collects altogether 126 flow points.
Each flow point of collecting is carried out TLC to be analyzed, merge similar flow point, vacuum and low temperature in the vacuum drying chamber (50 ℃) drying, obtain altogether 4 flow point group: B-1 (1-10 flow points, 1.5g), B-2 (11-40 flow point, 6.3g), B-3 (the 41-80 flow point, 4.8g), B-4 (the 80-126 flow point, 5.1g).
4 flow point groups are carried out the algicdal activity test according to the method described above, and wherein, B-2 flow point group has significant algicdal activity (specifically as described in Example 2).
(4) B-2 flow point group analysis and evaluation
(a) high performance liquid chromatography (HPLC) is analyzed
Accurately take by weighing ginkgolic acid standard items and B-2 flow point group sample 10.0mg, with the absolute methanol dissolving, surely be dissolved in the 10mL volumetric flask, be made into the solution that concentration is 1.0mg/mL, filter with 0.45 μ m Whatman disposable aspiration needle filter, carry out HPLC and analyze, measure content.Chromatographic column is Hyper ODS-2C18 (250mm * 10mm, 5 μ m) post, and it is 310nm that ultraviolet detects wavelength, and flow velocity is 1.0mL/min, and column temperature is 35 ℃, and mobile phase composition is CH
3OH-4%HAC (93.5: 6.5, V: V).
(b) liquid chromatography-mass spectrography (LC/ (-) ESI/MS) is analyzed
Chromatographic condition: Waters XTerra MS C18 chromatographic column (150 * 2.1mm, 5 μ m); Mobile phase: 85% methyl alcohol and 15% water (containing 0.1% formic acid in the water), flow velocity is 0.3mL/min, and column temperature is 30 ℃, and sample size is 10 μ L.
Mass spectrum condition: capillary voltage (ESI) 3.5kV, 110 ℃ of source temperatures, 300 ℃ of desolventizing temperature, taper hole air-flow 50L/h, desolventizing air-flow 650L/h.Electron spray ionisation anion mode.Sweep limits: 100-600 (m/z).
2, results and analysis
(1) HPLC analysis result
The HPLC analysis result shows, the HPLC chromatogram (as shown in Figure 1) of B-2 flow point group sample is basically identical with the HPLC chromatogram (as shown in Figure 2) of ginkgolic acid standard items, all contains 5 kinds of ginkgolic acids, and peak sequence is followed successively by C
13:0, C
15:1, C
17:2, C
15:0And C
17:1
(2) liquid chromatography-mass spectrography (LC/ (-) ESI/MS) identification
B-2 flow point group sample dissolution in hplc grade methanol, is adopted the ginkgolic acid in RPLC-electrospray ionization mass spectrometry (ES-MS) combination analysis evaluation sample.To shown in Fig. 3-6, can find out from liquid chromatogram and mass spectrogram: the molecular weight at five peaks is followed successively by 319.6,345.7,371.6,347.7 and 373.8 to ginkgolic acid liquid chromatography-mass spectrography figure such as Fig. 3-1, respectively with C
13:0, C
15:1, C
17:2, C
15:0And C
17:1Theoretical molecular is consistent.
Deducibility B-2 flow point group sample is ginkgolic acid thus.By HPLC the ginkgolic acid that separates is analyzed, with the peak area mean value substitution regression equation of gained, the total content of trying to achieve ginkgolic acid in the separator is 96.70 % by weight.
With B-2 flow point group sample (wherein, the content of ginkgolic acid is 96.70 % by weight) as described above method carry out algicdal activity and detect.
Added up at the 3rd day and the 7th day respectively and kill algae experimental result (representing with the algal control rate).The algal control experimental result that has shown the B-2 flow point group sample of variable concentrations in the table 1.
Table 1
According to the algal control rate of the 3rd day and the 7th day, calculate the LC of the 3rd day and the 7th day
50Be respectively 4.25mg/L and 3.41mg/L.
The above results explanation, B-2 flow point group sample, that is, ginkgolic acid has the excellent activity of killing microcystic aeruginosa.
Embodiment 3
Get gingko episperm dry product 10.0kg, be crushed to the 30-60 order.Extract with industrial methanol eddy, the concentrated fluid medicinal extract that obtains of pressurization behind the methyl alcohol that fully volatilizees, is used the industrial alcohol stirring and dissolving, gets supernatant, removes the impurity such as sediment sugar, starch.With petroleum ether dissolution extraction, remove residue behind the concentrated supernatant, concentrated extract is to extractum A, and is 40 % by weight with the content that the HPLC method among the embodiment 1 is measured ginkgolic acid in this extractum A.
After the methyl alcohol of the extractum A of 62.5 volumes and 25 volumes mixed, add the emulsifier of 12.5 volumes, strong agitation can obtain algicide A after evenly.During use, first with 1000 times water dilution algicide A, and then full pool spilling head, every cubic metre of volume with algicide A is 1-3mL.
Get gingko episperm dry product 10.0kg, be crushed to the 30-60 order.Extract with industrial methanol eddy, the concentrated fluid medicinal extract that obtains of pressurization behind the methyl alcohol that fully volatilizees, is used the industrial alcohol stirring and dissolving, gets supernatant, removes the impurity such as sediment sugar, starch.With petroleum ether dissolution extraction, remove residue behind the concentrated supernatant, concentrated extract is to medicinal extract B, and is 40 % by weight with the content that the HPLC method among the embodiment 1 is measured ginkgolic acid among this medicinal extract B.
After the dimethylbenzene of the medicinal extract B of 33.5 volumes and 6.5 volumes mixed, add the emulsifier of 10 volumes, strong agitation evenly after, add the water of 50 volumes, vigorous stirring becomes microemulsion to obtain algicide B.During use first with 1000 times water dilution algicide B, and then full pool spilling head, every cubic metre of volume with algicide B is 3-5mL.
Embodiment 5
Get gingko episperm dry product 10.0kg, be crushed to the 30-60 order.Extract with industrial methanol eddy, the concentrated fluid medicinal extract that obtains of pressurization behind the methyl alcohol that fully volatilizees, is used the industrial alcohol stirring and dissolving, gets supernatant, removes the impurity such as sediment sugar, starch.With petroleum ether dissolution extraction, remove residue behind the concentrated supernatant, concentrated extract is to medicinal extract C, and is 40 % by weight with the content that the HPLC method among the embodiment 1 is measured ginkgolic acid among this medicinal extract C.
After the ethanol of the medicinal extract C of 50 volumes and 30 volumes mixed, add the emulsifier of 20 volumes, strong agitation can obtain algicide C after evenly.During use first with 1000 times water dilution algicide C, and then full pool spilling head, every cubic metre of volume with algicide C is 3-4mL.
Use the above-mentioned algicide A for preparing to kill algae experiment, algicide concentration (ginkgolic acid the densimeter in algae liquid of algicide concentration wherein to contain) as shown in table 2.The frustule initial density is 10
6Individual/mL, the survival condition of the 7th day statistics frustule after the dosing.Table 2 shows the extremely algae experimental result of variable concentrations algicide.
Table 2
| Algicide concentration (mg/L) | 4.0 | 3.0 | 2.0 | 1.0 | 0.5 | 0.25 | CK |
| Cell number (10 6Individual/mL) | 2.7 | 3.55 | 4.3 | 5.1 | 6.15 | 7.35 | 8.9 |
| Kill algae rate (%) | 69.7 | 60.1 | 48.3 | 42.7 | 39.1 | 17.4 | 0 |
Can find out by embodiment 3, the algicide that utilizes the alcohol extract of gingko episperm to make has the activity of killing preferably microcystic aeruginosa, thereby has the preferably activity of killing blue algae.
In addition, also can carry out any combination between the various embodiment of the present invention, as long as it is without prejudice to thought of the present invention, it should be considered as content disclosed in this invention equally.
Claims (9)
1. the application of ginkgolic acid in the preferred microcystic aeruginosa of killing blue algae (Microcystic aeruginosa).
2. application according to claim 1, wherein, described ginkgolic acid extracts from gingko episperm and obtains.
3. the application of the alcohol extract of gingko episperm in the preferred microcystic aeruginosa of killing blue algae.
4. application according to claim 3, wherein, the alcohol extract of described gingko episperm is the alcohol extract medicinal extract of gingko episperm.
5. according to claim 3 or 4 described application, wherein, take the volume of the alcohol extract of described gingko episperm as benchmark, the content of ginkgolic acid surpasses 40 % by weight in the alcohol extract of described gingko episperm.
6. according to claim 3 or 4 described application, wherein, described alcohol extract is methanolic extract and/or ethanol extract.
7. a composition is characterized in that, said composition contains five kinds of compd A-E shown in general formula I,
R is-(CH in the compd A
2)
12CH
3R is-(CH in the compd B
2)
7CH=CH (CH
2)
5CH
3R is-(CH in the Compound C
2)
14CH
3R is-(CH in the Compound D
2)
9CH=CH (CH
2)
5CH
3R is-(CH in the compd E
2)
8CH=CHCH
2CH=CH (CH
2)
3CH
3Take the weight of said composition as benchmark, the total content of compd A-E surpasses 40 % by weight.
8. the application of composition claimed in claim 7 in killing blue algae.
9. application according to claim 8, wherein, described blue-green algae is microcystic aeruginosa.
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| CN105191993B (en) * | 2015-09-22 | 2017-12-08 | 安徽师范大学 | A kind of plant source algae-inhibiting agent and preparation method thereof |
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| CN113827626A (en) * | 2021-08-15 | 2021-12-24 | 江苏蓝星生物工程有限公司 | Method for extracting ginkgolic acid by using waste liquid in processing process of ginkgo leaf extract |
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