CN103299998B - 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, the frequency economic loss that is more and more higher, that bring increasing (Zhao Yuhang, the Yang Hongsheng that occur of some waters wawter bloom (blue-green algae) of China.A kind of research of Euglena wawter bloom in fishpond.Aquatile journal, 1994,18 (2): 186-188), algal bloom has become the problem having a strong impact on quality of water environment and water ecology safety, not only great harm humans is healthy and other biological safety, also causes huge loss economically.According to the literature, the kind of these waters formation wawter bloom mainly contains microcystis kutz and the Aphanizomenon of Cyanophyta, wherein to have comparative advantage (Heath R.L with microcystic aeruginosa again, 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 being extensively 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, often becomes the cement shape of dispersion.Children's implant is the solid colony of ball-type or ellipsoid, after grow up to for network-like hollow cystidium, utricule breaks and forms netted colloid colony subsequently.Cell dia 3-7 μm, presents blue-green, with particle or pseudo-cavity.Be grown in 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, having carried out large quantifier elimination both at home and abroad, generally can adopt chemical method, changed 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, change water law to cure the symptoms, not the disease, not only waste water resource, and cost is very high, and utilize microorganism and animal to control algae may to there is ecological risk and wayward, chemical method is method comparatively conventional at present, its medicine used has multiple, but with the most use be at present copper sulphate, but this pharmacy effect is of short duration, and copper sulphate itself is also a kind of pollutant, should not repeatedly use.The shortcoming of these methods itself limits applying of they.In this case, control with plant the very big concern that body eutrophication has been subject to colleague both at home and abroad, and plant-based medicine due to its get nature, with nature, low toxicity, low-residual, non-harmful feature and become the focus of Recent study.But these study the interaction laid particular emphasis between water plants and blue-green algae more, and the research for terrestrial plant is less.
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 Development volue, is called as " whole body is all precious living fossil ".Large quantifier elimination confirms both at home and abroad, main containing multiple ginkgolic acid (Ginkgolic acids) in gingko episperm, ginkgolic acid is the salicylic derivative of a class, side chain carbon number on its 6 can be 13 to 17, pendant double bonds number can be 0 to 3, a homologous mixture (Wang Jie, wish and establish one's virtue, Yu Biyu etc., extraction fromginkgoseed coat is to the research [J] of cabbage caterpillar Developing restraint and antifeedant activity, 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).
Be separated from gingko episperm at present and identified ginkgoic acid (Wang Jie, wish and establish one's virtue, Yu Biyu etc., extraction fromginkgoseed coat is to the research [J] of cabbage caterpillar Developing restraint and antifeedant activity, 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) and ginkgo diphenol, structure is such as formula shown in I.The reports such as JaggyH, containing 4 kinds of ginkgoic acid C in 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.).
But, the large quantifier elimination of external many scholars confirms (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): ginkgolic acid majority is 5 kinds of compound compositions, 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 pomegranate green grass or young crops wait (face upward pomegranate green grass or young crops, Wu Xiangyang, Chen Jun.The content [J] of ginkgoic acid in high effective liquid chromatography for measuring gingko episperm. analytical chemistry, 2002,30 (8): 901) and (Li Hongqing, He Zhaofan, the Zhang Yongmin etc. such as Li Hongqing.Gingko episperm alkyl phenol and alkyl constituents research [J]. Chinese herbal medicine, 2004,35 (1): 18-20) research be consistent.Facing upward the blue or green content adopting RP-HPLC method to record ginkgolic acid in gingko episperm of pomegranate 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, is secondly hydroginkgoic acid (Hydroginkgolieaeid, C
15:0), the sub-acid of gingko (Hydroginkgolinieacid, C
17:2).
formula I
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
Research shows, the multiple pharmacological activity of ginkgolic acid tool, and current known clinical application has:
(1) bacteriostatic activity: the ginkgolic acid in ginkgolic acid, hydroginkgoic acid etc. can suppress streptococcus, staphylococcus, anthrax bacteria etc.;
(2) antitumor and antiviral activity: the long-chain phenolic acid in gingko episperm has antitumor activity, especially has stronger inhibitory action to S180 sarcoma hamster V-79 cell;
(3) human diseases is widely used in: domestic current large quantities of ginkgo agent listing, as ginkgo oral liquid EBG-761 etc., is mainly used in the heart and brain blood disease, neurogenic disease etc. of the mankind, has relieving cough and asthma effect more than separating Ginkgo phenolic acids extract;
(4) biological pesticide technical field: ginkgolic acid compounds is killing the purposes on Pomacea canaliculata, as described (applicant: Yang little Ming, Ren Xiaofeng, Liu Weimin) in CN101911938A.Main application kills the harmful mollusk of agricultural for utilizing ginkgolic acid compounds;
(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, as described (applicant: Wang Gaoxue, Zhao Yunkui, Cheng Minglie, Dong Jun) in CN1545890A.Main application kills the parasitic Dactylogyrus of fish for utilizing ginkgolic acid compounds.
Summary of the invention
The object of this invention is to provide a kind of function application of new killing blue algae, particularly, is the method utilizing 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 the alcohol extract of gingko episperm in killing blue algae.
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 as shown in general formula I,
formula I
In compd A, R is-(CH
2)
12cH
3; In compd B, R is-(CH
2)
7cH=CH (CH
2)
5cH
3; In Compound C, R is-(CH
2)
14cH
3; In Compound D, R is-(CH
2)
9cH=CH (CH
2)
5cH
3; In compd E, R is-(CH
2)
8cH=CHCH
2cH=CH (CH
2)
3cH
3; With the weight of said composition for benchmark, the total content of compd A-E is more than 40 % by weight.In addition, the application of above-mentioned composition in killing blue algae is additionally provided.
Microcystic aeruginosa is as the model organism of Cyanophyta, and the research conclusion for it all has general directive significance to whole Cyanophyta, and therefore, the present inventor selects microcystic aeruginosa as research object.Particularly, measure, can find out the algicdal activity of the B-2 flow point group (i.e. ginkgolic acid) in the alcohol extract of gingko episperm, this stream is grouped in the LC of the suppression microcystic aeruginosa of the 3rd day and the 7th day
50be respectively 4.25mg/L and 3.41mg/L.This illustrates, ginkgolic acid has the extremely strong activity killing microcystic aeruginosa, also namely has the activity of extremely strong killing blue algae.
In addition, ginkgolic acid is plant resource algicide, and that gets uses nature naturally, and low toxicity and low residue, not easily produces drug resistance, is a kind of environmental friendliness and the simple algicide of using method.
Accompanying drawing explanation
Accompanying drawing is used to provide a further understanding of the present invention, and forms a part for specification, is used from explanation the present invention, but is not construed as limiting the invention with embodiment one below.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. 3-1 is the TIC of B-2 flow point group sample, and Fig. 3-2 is first chromatographic peak in the total ion current of B-2 flow point group sample (from left to right, i.e. C
13:0) mass spectrogram, Fig. 3-3 is second chromatographic peak in the total ion current of B-2 flow point group sample (from left to right, i.e. C
15:1) mass spectrogram, Fig. 3-4 is the 3rd chromatographic peak in the total ion current of B-2 flow point group sample (from left to right, i.e. C
17:2) mass spectrogram, Fig. 3-5 is the 4th chromatographic peak in the total ion current of B-2 flow point group sample (from left to right, i.e. C
15:0) mass spectrogram, Fig. 3-6 is the 5th chromatographic peak in the total ion current of B-2 flow point group sample (from left to right, i.e. C
17:1) mass spectrogram.
Embodiment
Below the specific embodiment of the present invention is described in detail.Should be understood that, embodiment described herein, only for instruction and explanation of 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 formula I,
formula I
Wherein, R is-(CH
2)
12cH
3(i.e. ginkgolic acid C
13:0) ,-(CH
2)
7cH=CH (CH
2)
5cH
3(i.e. ginkgolic acid C
15:1) ,-(CH
2)
8cH=CHCH
2cH=CH (CH
2)
3cH
3(i.e. ginkgolic acid C
17:2) ,-(CH
2)
14cH
3(i.e. ginkgolic acid C
15:0) and-(CH
2)
9cH=CH (CH
2)
5cH
3(i.e. ginkgolic acid C
17:1).That is, in 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 respective 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 the five kinds of compounds extracted from gingko episperm.
Although the ginkgolic acid in the present invention refers to the material identical with national standard, and preferred extraction from gingko episperm obtains, the present invention does not get rid of the ginkgolic acid synthesizing and obtain.The method of described extraction is known to the skilled person, as by the mixture of gingko episperm crushed material and alcohol (wherein, the weight ratio of gingko episperm crushed material and alcohol is 1: 3-10) under reflux conditions process at least 1h, by the extract reduced pressure concentration obtained, obtain the alcohol extract medicinal extract of gingko episperm, then gained medicinal extract is carried out multistage silica gel column chromatography, be separated and obtain described ginkgolic acid.
In the present invention, described " killing microcystic aeruginosa " or " killing algae " both can refer to killing existing frustule, also can refer to the Growth and reproduction suppressing frustule.
The present invention also provides the application of the alcohol extract of gingko episperm in killing blue algae.
In the present invention, the concept of described extract is known for those skilled in the art, refers to through physico chemistry extraction and isolation process, directedly obtains and a certain or Multiple components that concentrates, and does not change the structure of this composition and the product that formed.Different according to the proterties of final products, extract can be divided into vegetable oil, medicinal extract, powder, crystalline lens etc.
In 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 of the medicinal extract shape with alcohol extracting, and described alcohol can comprise the various conventional alcohols material for extracting, such as methyl alcohol and/or ethanol.Described methyl alcohol and/or ethanol comprise anhydrous methyl alcohol and/or ethanol, and also comprise methyl alcohol and/or the ethanol of technical grade, wherein the purity of methyl alcohol and/or ethanol is higher than 97%.
For the kind of ginkgolic acid various in the alcohol extract of gingko episperm and content, there is no particular limitation, as long as containing ginkgolic acid in the present invention.But preferably, with the weight of the alcohol extract of described gingko episperm for benchmark, in the alcohol extract of described gingko episperm, the content of ginkgolic acid is more than 40 % by weight, such as, can be 45-100 % by weight.The alcohol extract of the gingko episperm containing the ginkgolic acid in preferable range has the effect of killing blue algae better.
The method obtaining the alcohol extract of above-mentioned gingko episperm can be the method for this area routine, such as can comprise, get gingko episperm dry product, extract with alcohol reflux after pulverizing, pressurization is concentrated obtains fluid medicinal extract, then uses petroleum ether dissolution, removing residue, concentrated extract, obtains the alcohol extract of described gingko episperm.
Wherein, the weight that the condition of described refluxing extraction comprises alcohol used be the 3-10 of the gingko episperm weight pulverized doubly, the time of refluxing extraction can be more than 1h, preferably 1.5-5 hour.The degree of petroleum ether dissolution extraction with the extract obtained close to colourless for standard, at every turn for the weight of benzinum of dissolving can for the 0.8-1.2 of gingko episperm weight that pulverizes doubly, number of operations can be such as 2-5 time.
The method of described extraction can also be included in before petroleum ether dissolution, and the solvent fully in volatilization fluid medicinal extract, then adds alcohol and dissolve, get supernatant concentration, carry out the step of petroleum ether dissolution extraction afterwards again.Can be the same or different for the alcohol of refluxing extraction and the alcohol for dissolving, 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 as shown in general formula I,
formula I
In compd A, R is-(CH
2)
12cH
3; In compd B, R is-(CH
2)
7cH=CH (CH
2)
5cH
3; In Compound C, R is-(CH
2)
14cH
3; In Compound D, R is-(CH
2)
9cH=CH (CH
2)
5cH
3; In compd E, R is-(CH
2)
8cH=CHCH
2cH=CH (CH
2)
3cH
3; With the weight of said composition for benchmark, the total content of compd A-E more than 40 % by weight, such as, can be 45-100 % by weight.The present inventor finds, as long as the composition of the total content of above-mentioned five kinds of compounds more than 40 % by weight can have kill blue-green algae effect significantly.Therefore, above-mentioned composition of the present invention can be applicable to killing blue algae.
Described blue-green algae in the present invention is preferably microcystic aeruginosa (Microcystic aeruginosa).But those skilled in the art can recognize, due to the typicalness of microcystic aeruginosa in Cyanophyta, described blue-green algae is not limited to microcystic aeruginosa.
Alcohol extract and the described composition of ginkgolic acid of the present invention, gingko episperm can make algicide, with conveniently, preferably use the alcohol extract of gingko episperm to prepare algicide.Particularly, this algicide can containing the alcohol extract of gingko episperm, emulsifier and organic solvent, wherein, preferably, with the volume of this algicide for benchmark, in described algicide, the content of the alcohol extract of gingko episperm is 20-80 volume %, and the content of described emulsifier is 10-50 volume %, and the content of described organic solvent is 10-50 volume %; Further preferably, with the volume of this algicide for benchmark, in described algicide, the content of the alcohol extract of gingko episperm is 35-65 volume %, 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 the surfactant-based material that drug world is conventional.Its effect is: when it is dispersed in dispersate surperficial, forms film or electric double layer, disperse phase can be made with electric charge, the droplet of disperse phase so just can be stoped to condense mutually, make the emulsion of formation more stable, is convenient to medicine performance drug effect.
Emulsifier in the present invention can be the various emulsifier of this area routine, includes but not limited at least one in the homogeneous mixture of the alkylphenol polyoxyethylene of 30 volume %, 60 volume % calcium dodecyl benzene sulfonates and 10 volume % aliphatic acid polyethenoxy ethers, 33#, 34#, NP-10, PNP-10, PF-690, BY-130 and tween-120.
In the present invention, described organic solvent also can be conventional various organic solvents, and consider solvability, toxicity and cost, preferably, described organic solvent is methyl alcohol and/or ethanol.
The method preparing algicide of the present invention can for the conventional method preparing biological medicament goods, namely the alcohol extract of gingko episperm is obtained first according to the method described above, then the alcohol extract of gingko episperm and organic solvent are mixed to and dissolve completely, then in the solution obtained, emulsifier is added, abundant stirring and evenly mixing, i.e. obtained described algicide.
Described algicide is applied to the concrete grammar killing algae and can comprises, and adds this algicide in water body.
The addition of described algicide can regulate as required; Generally preferably, with the volume of water for benchmark, the addition of described algicide is 1-10mL/m
3, be preferably 1-5mL/m
3.
Below will be described the present invention by embodiment.In following examples:
Gingko episperm picks up from Taixing City of Jiangsu Province, crosses 40 mesh sieves after shining 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 ginkgo total phenolics content is 99 % by weight); Column chromatography silica gel used (80-100 order, 200-300 order, 300-400 order) and thin plate silica gel (GF254 type, H type, G type) are all purchased 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 conventional developer such as solution, iodine, BG-11 medium are experiment autogamy.
Instrument comprises: ZF-2 type three ultraviolet device, Shanghai City An Ting Electronic Instruments Plant; Waters 2695 highly effective liquid phase chromatographic system, 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 killing the activity of microcystic aeruginosa comprises the following steps:
(1) candidate drug DMSO is dissolved, is made into the liquid that initial concentration is 200mg/L, and by liquid gradient dilution, make 200 successively, 100,50,25,12.5,6.25,3.125,1.0625,0.53125mg/L for reagent liquid, for subsequent use.
(2) for the preparation of examination algae liquid
By the renewed vaccination of well-grown algae liquid, select to test during its exponential phase.By cell counting determination algae cell density before experiment.
(3) microcystic aeruginosa experiment is killed
Amass as benchmark with algae liquid that (frustule concentration is for 10
6individual/mL), will be added in microcystic aeruginosa algae liquid in the ratio of 0.5 volume % for reagent liquid, each concentration arranges 3 parallel group, and establishes DMSO control group.Within the 3rd day after dosing and the 7th day, add up the amount of survival of frustule respectively and take the mean, calculating the algal control rate under different drug concentration and corresponding LC
50.
Embodiment 1
1, experimental procedure
(1) extraction of test sample and initial gross separation
Under normal temperature condition, by 20kg gingko episperm crushed material with anhydrous industrial alcohol (200L) 75 DEG C of circumfluence distillation 3 times, each 2h, merge extract, reduced pressure concentration obtains ethanol extract 6640g.And then by the ethanol extract of gained with isopyknic petroleum ether extraction 3-5 time, until extract is almost colourless, reduced pressure concentration extract obtains benzinum medicinal extract 3276g, and extraction yield is 49.3 % by weight, and residual residue is 3300g.
(2) separation of petroleum ether extract
(a) sample treatment
Get petroleum ether extract medicinal extract 100g, add 100g silica gel mix thoroughly with methyl alcohol after dissolving, put the upper evaporate to dryness of thermostat water bath (temperature 50 C), grinding evenly.
(b) dress post and loading
The post footpath of glass chromatography column is 100mm, and column length is 1200mm, column chromatography silica gel 2000g (200-300 order calculates by 1g sample 20g silica gel), chloroform wet method dress post, dry method loading.
(c) solvent system
Through thin-layer chromatography (TLC) prerun, determine with chloroform-methanol (1: 0,200: 1,100: 1,50: 1,25: 1,12.5: 1,5: 1,2: 1,1: 1 and methyl alcohol wash post successively, this ratio is volume ratio, the ratio of the mixing eluting solvent in the embodiment of the present invention is volume ratio) as elution system, carry out gradient elution.
D () thin-layer chromatography (TLC) is analyzed, merge flow point and algicdal activity detects
Adopt silica GF254 Preparative TLC plate, TLC analysis is carried out to each flow point collected.Combined with fluorescent, sulfuric acid ethanol developer are 100 DEG C of colour developings, and each change of component in test column chromatography, according to change adjustment eluant, eluent polarity.Every 200mL collects a flow point, collects 165 flow points altogether.Adopt TLC to check simultaneously, merge similar flow point, in vacuum drying chamber, vacuum and low temperature (50 DEG C) is dry, obtain 5 flow point groups altogether: A (1-15 flow point, 5.0g), B (16-62 flow point, 31.5g), C (63-105 flow point, 19.4g), D (106-135 flow point, 17.5g) with E (136-165 flow point, 13.2g).
A-E five components are carried out algicdal activity test according to the method described above, records B flow point and there is algicdal activity.
(3) level chromatography is separated
Get the above-mentioned B of 25.0g and flow packet combining thing, add 25.0g silica gel mix thoroughly with methyl alcohol after dissolving, be placed in the upper evaporate to dryness of thermostat water bath (temperature 50 C), grinding evenly.Again carry out silica gel column chromatography separation.
The post footpath of glass chromatography column is 70mm, and column length is 1000mm, and column chromatography silica gel is 750g (300-400 order calculates by 1g sample 30g silica gel), 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 methanol wash column post), carries out gradient elution.
Analyze with thin-layer chromatography (TLC), combined with fluorescent, sulfuric acid ethanol developer are 100 DEG C of colour developings, and each change of component in test column chromatography, according to change adjustment eluant, eluent polarity.Every 100mL collects a flow point, collects 126 flow points altogether.
TLC analysis is carried out to each flow point collected, merge similar flow point, in vacuum drying chamber, vacuum and low temperature (50 DEG C) is dry, obtain 4 flow point group: B-1 (1-10 flow points altogether, 1.5g), B-2 (11-40 flow point, 6.3g), B-3 (41-80 flow point, 4.8g), B-4 (80-126 flow point, 5.1g).
4 flow point groups are carried out 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 qualification
A () high performance liquid chromatography (HPLC) is analyzed
Accurately take ginkgolic acid standard items and B-2 flow point group sample 10.0mg, dissolve with absolute methanol, be surely dissolved in 10mL volumetric flask, be made into the solution that concentration is 1.0mg/mL, with 0.45 μm of Whatman disposable aspiration needle metre filter, carry out HPLC analysis, measure content.Chromatographic column is Hyper ODS-2C18 (250mm × 10mm, 5 μm) post, and UV detect wavelength is 310nm, and flow velocity is 1.0mL/min, and column temperature is 35 DEG C, and mobile phase consists of 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 μm); Mobile phase: 85% methyl alcohol and 15% water (containing 0.1% formic acid in water), flow velocity is 0.3mL/min, and column temperature is 30 DEG C, and sample size is 10 μ L.
Mass Spectrometry Conditions: capillary voltage (ESI) 3.5kV, source temperature 110 DEG C, desolventizing temperature 300 DEG C, taper hole air-flow 50L/h, desolventizing air-flow 650L/h.Electron spray ionisation negative-ion mode.Sweep limits: 100-600 (m/z).
2, results and analysis
(1) HPLC analysis result
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, and all containing 5 kinds of ginkgolic acids, 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
By B-2 flow point group sample dissolution in hplc grade methanol, adopt the ginkgolic acid in RPLC-electrospray ionization mass spectrometry (ES-MS) combination analysis qualification sample.Ginkgolic acid liquid chromatography-mass spectrography figure as shown in Fig. 3-1 to Fig. 3-6, as can be seen 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, 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.Analyzed the ginkgolic acid be separated by HPLC, the peak area mean value of gained is substituted into regression equation, and the total content of trying to achieve ginkgolic acid in separator is 96.70 % by weight.
Embodiment 2
By 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 detection.
Algae experimental result (representing with algal control rate) is killed respectively the 3rd day and the 7th day statistics.The algal control experimental result of the B-2 flow point group sample of variable concentrations is shown in 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 illustrates, B-2 flow point group sample, that is, ginkgolic acid has the excellent activity killing microcystic aeruginosa.
Embodiment 3
Get gingko episperm dry product 10.0kg, be crushed to 30-60 order.Extract with industrial methanol eddy, pressurization is concentrated obtains fluid medicinal extract, fully after volatilization methyl alcohol, uses industrial alcohol stirring and dissolving, gets supernatant, the impurity such as removing sediment sugar, starch.With petroleum ether dissolution extraction after concentrated supernatant, removing residue, concentrated extract to extractum A, and is 40 % by weight with the content that the HPLC method in embodiment 1 measures ginkgolic acid in this extractum A.
After the extractum A of 62.5 volumes being mixed with the methyl alcohol of 25 volumes, add the emulsifier of 12.5 volumes, after strong agitation is even, algicide A can be obtained.During use, first with the water dilution algicide A of 1000 times, and then full pool spilling head, every cubic metre of volume with algicide A is 1-3mL.
Embodiment 4
Get gingko episperm dry product 10.0kg, be crushed to 30-60 order.Extract with industrial methanol eddy, pressurization is concentrated obtains fluid medicinal extract, fully after volatilization methyl alcohol, uses industrial alcohol stirring and dissolving, gets supernatant, the impurity such as removing sediment sugar, starch.With petroleum ether dissolution extraction after concentrated supernatant, removing residue, concentrated extract to medicinal extract B, and is 40 % by weight with the content that the HPLC method in embodiment 1 measures ginkgolic acid in this medicinal extract B.
After being mixed with the dimethylbenzene of 6.5 volumes by the medicinal extract B of 33.5 volumes, add the emulsifier of 10 volumes, after strong agitation is even, add the water of 50 volumes, vigorous stirring becomes microemulsion to obtain algicide B.Water dilution algicide B first with 1000 times during use, 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 30-60 order.Extract with industrial methanol eddy, pressurization is concentrated obtains fluid medicinal extract, fully after volatilization methyl alcohol, uses industrial alcohol stirring and dissolving, gets supernatant, the impurity such as removing sediment sugar, starch.With petroleum ether dissolution extraction after concentrated supernatant, removing residue, concentrated extract to medicinal extract C, and is 40 % by weight with the content that the HPLC method in embodiment 1 measures ginkgolic acid in this medicinal extract C.
After being mixed with the ethanol of 30 volumes by the medicinal extract C of 50 volumes, add the emulsifier of 20 volumes, after strong agitation is even, algicide C can be obtained.Water dilution algicide C first with 1000 times during use, and then full pool spilling head, every cubic metre of volume with algicide C is 3-4mL.
Test case 1
The above-mentioned algicide A prepared is used to carry out killing algae experiment, algaecide concentration (algaecide concentration is with the densimeter of the ginkgolic acid wherein contained in algae liquid) as shown in table 2.Frustule initial density is 10
6individual/mL, the survival condition of the 7th day statistics frustule after dosing.What table 2 showed variable concentrations algicide kills algae experimental result.
Table 2
| Algaecide 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 be found out by embodiment 3, the algicide utilizing the alcohol extract of gingko episperm to obtain has the activity killing microcystic aeruginosa preferably, thus has the activity of good killing blue algae.
In addition, also can be combined between various different 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 (8)
1. the application of ginkgolic acid in killing blue algae.
2. application according to claim 1, wherein, described blue-green algae is microcystic aeruginosa (Microcystic aeruginosa).
3. application according to claim 1 and 2, wherein, described ginkgolic acid extracts and obtains from gingko episperm.
4. the application of the alcohol extract of gingko episperm in killing blue algae, it is characterized in that, with the volume of the alcohol extract of described gingko episperm for benchmark, in the alcohol extract of described gingko episperm, the content of ginkgolic acid is more than 40 % by weight, and described alcohol extract is methanolic extract and/or ethanol extract.
5. application according to claim 4, wherein, described blue-green algae is microcystic aeruginosa.
6. the application according to claim 4 or 5, wherein, the alcohol extract of described gingko episperm is the alcohol extract medicinal extract of gingko episperm.
7. the application of composition in killing blue algae, wherein, said composition contains five kinds of compd A-E as shown in general formula I,
In compd A, R is-(CH
2)
12cH
3; In compd B, R is-(CH
2)
7cH=CH (CH
2)
5cH
3; In Compound C, R is-(CH
2)
14cH
3; In Compound D, R is-(CH
2)
9cH=CH (CH
2)
5cH
3; In compd E, R is-(CH
2)
8cH=CHCH
2cH=CH (CH
2)
3cH
3; With the weight of said composition for benchmark, the total content of compd A-E is more than 40 % by weight.
8. application according to claim 7, wherein, described blue-green algae is microcystic aeruginosa.
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| CN103749550B (en) * | 2014-01-15 | 2015-09-09 | 东南大学 | A kind of method fully utilizing gingko episperm |
| CN104585167B (en) * | 2014-12-30 | 2016-09-28 | 南京林业大学 | The preparation of extraction fromginkgoseed coat nano-capsule |
| 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 |
| CN107711916A (en) * | 2017-09-11 | 2018-02-23 | 无锡市天智者生物科技有限公司 | A kind of natural Chinese medicinal herb for blue-green alga bloom of tagging and preparation method thereof |
| CN111109299A (en) * | 2019-12-24 | 2020-05-08 | 阙金龙 | Preparation method of algistat containing ginkgo endophyte |
| 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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