CN102293785B - Method for extracting secondary metabolites of insects or arthropods - Google Patents
Method for extracting secondary metabolites of insects or arthropods Download PDFInfo
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- CN102293785B CN102293785B CN2011102495700A CN201110249570A CN102293785B CN 102293785 B CN102293785 B CN 102293785B CN 2011102495700 A CN2011102495700 A CN 2011102495700A CN 201110249570 A CN201110249570 A CN 201110249570A CN 102293785 B CN102293785 B CN 102293785B
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- insecticide
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Abstract
The invention relates to a method for extracting secondary metabolites of insects or arthropods. The method comprises the following steps: immersing powder slag of insects of arthropods by dichloromethane for more than twice, wherein each immersion time is not less than 36 hours, then performing a solid-liquid separation; drying the obtained powder slag and immersing by a methanol aqueous solution for more than twice, wherein each immersion time is not less than 36 hours, then merging and immersing the obtained an alcohol extract and freeze drying to obtain an extract. The insect is selected from Formica aquilonia, Aspongopus chinensis Dallas, cockroach, mole cricket or maggot; the arthropod is selected from scorpion or spider. According to the invention, the obtained extract has different antibacterial degree to G+positive bacteria or G-negative bacteria, and is capable of separating and preparing a compound with bacteriostatic activity, the method for extracting secondary metabolites of insects or arthropods establishes a methodology foundation for chemical research of the insects which has important academic meaning and good application prospect.
Description
Technical field
The present invention relates to the extraction manufacture field of insecticide, be specifically related to the method for distilling of a kind of insecticide or arthropod secondary metabolites, it is extract obtained to G to adopt this method to extract
+Positive bacteria or G
-Negative bacterium has bacteriostasis in various degree, can be used to prepare the medicine with bacteriostatic activity.
Background technology
Insecticide is as most species on the earth; The species that quantity is maximum; The biological treasure-house of containing huge natural product resource beyond doubt, the record that Ancient Times in China just has many insecticides to be used as medicine, the record that all has insecticide to be used as medicine like traditional Chinese medical science monumental work Compendium of Material Medica and supplementary Amplifications of the Compendium of Materia Medica etc.According to not exclusively investigation; The kind of China insecticide has surpassed 100,000 kinds; Found to have medical value only 209 kinds, less than 2% of insecticide sum, and insects Chinese medicine commonly used is less than 20 kinds; The medical value that is to say many insecticides is still undiscovered, makes great efforts to excavate to have important scientific meaning and application prospect.
Medicinal insect resources is abundant, and a large amount of bioactive chemical compounds that has can be provided for the exploitation of new drug.The successful at present insects new drug of developing is the preparation of ferment cordyceps sinensis.Therefore in addition, Chinese medicine has uniqueness, rich experience in the application facet of insecticide, and medicinal insecticide is worth further research excavating source new drugs, great potential all being arranged aspect curing difficult and complicated cases.And because caste is various, the medical value of many insecticides is still undiscovered.Therefore insecticide is having potentiality aspect the developing new drug source, should make great efforts to excavate.
Insecticide is as important medicine resource and have the active lead compound of important physiological source; Countries such as Australia, Brazil, New Zealand, Canada and Britain are owing to enjoy unique ecological environment, and the research of insecticide natural product is in rank first always.The insecticide of China is studied at present, mainly concentrates on the secretions of insecticide, the utilization of metabolite and insect toxins, and in the application of bioactive macromolecule and the ecological research of insect chemistry.Biochemical active research only is confined to minority castes such as Mylabris, Cordyceps to the insect small-molecule chemical compound in early days.Medicinal insecticide still is limited in the compatibility of medicines in a prescription of Chinese herbal medicine, and what adopt so far in China is the traditional approach that Scorpio is used as medicine, and medicinal ingredient is unclear.Therefore the research of the natural product of insecticide (medicine) thing has crucial meaning.
The insecticide bacteriostatic active ingredients; Remove for many years in the research of the system of defense of insecticide, outside the application that relates to pesticide touches to some extent, and comparatively active as the insect antimicrobial peptide activity research in recent years; Insect antimicrobial peptide is the macromolecular compound research that insect bodies contains, and does not belong to the secondary metabolites category.Although further study antibacterial peptide structure and active relation and the mechanism of action report is arranged repeatly, compare, the also not nearly enough ideal of the antibacterial activity of insect antimicrobial peptide with traditional antibiotic.So just present Research analysis both at home and abroad shows, most of research work of relevant insecticide still just carries out in the discipline range of entomological taxonomy, chemical ecology at present, the inreal as yet relation of studying their biological activity and human diseases diligently of people.Up to now, do not see the report of achievement in research of other the micromolecular chemical compound of more similar insecticide secondary metabolites or insecticide natural product chemistry and original new drug as yet.
Summary of the invention
The object of the invention is to provide the method for distilling of a kind of insecticide or arthropod secondary metabolites; Adopt the extract obtained extractum of this method that G+ positive bacteria or G-negative bacterium are had bacteriostasis in various degree; Can be used to separate and prepare reactive compound, further find antibacterial or antimicrobial lead compound with bacteriostatic activity.
The method for distilling of a kind of insecticide or arthropod secondary metabolites; It comprises the steps: exsiccant insecticide or arthropod granulated slag are soaked twice above (soak and be advisable can flood granulated slag) with dichloromethane earlier; Each soak time is no less than 36h, then solid-liquid separation (merge gained lixiviating solution and concentrate drying and get liposoluble extract); The reuse methanol aqueous solution soaks more than twice after the gained granulated slag drying, and each soak time is no less than 36h, and merging gets extractum after soaking gained alcohol extract and lyophilization, and this extractum is water solubility extract.Two kinds of liposoluble extracts of above-mentioned this and water solubility extract are insecticide or the intravital secondary metabolites of arthropod.
Said insecticide is selected from Changbai Mountain Formica rufa, Aspongopus, Blatta seu periplaneta, Gryllotalpa or Chrysomyiame gacephala (Fab.) etc.; Said arthropod is selected from Scorpio or Aranea etc.
Concrete, this method for distilling is: exsiccant insecticide or arthropod granulated slag are soaked twice with dichloromethane earlier, soak 36-48h at every turn, then solid-liquid separation (merge gained lixiviating solution and concentrate drying and get liposoluble extract); The methanol aqueous solution of reuse mass concentration 70% soaks twice after the gained granulated slag drying, soaks 36-48h at every turn, merges 60 ℃ of placements of immersion gained alcohol extract Bing Yu – 24h postlyophilization and gets extractum, and this extractum is water solubility extract.
Adopt the inventive method gained insecticide extract extractum to G
+Positive bacteria or G
-Negative bacterium has bacteriostasis in various degree, can be used to separate the chemical compound that preparation has bacteriostatic activity, has established the methodology basis for the research of insect chemistry, has than important academic significance and good prospects for application.
The Changbai Mountain Formica rufa (
Tetramorium sp.) buy from Luoyang the big pharmacy of ten thousand states, be Hymenoptera Formicidae Myrmicinae.Both at home and abroad pharmacology and the immune function experiment to Formica rufa proves, Formica rufa has tangible antiinflammatory, relievings asthma, calmness, hepatoprotective, analgesic activity, and immune function of human body is had adaptation former state dual regulation.
Aspongopus (
Aspongopus chinensis Dallas) buy the big pharmacy of Zhang Zhongjing from Luoyang, i.e. Semiptera, Heteroptera, the melon of Pentatomiddae is deceived stinkbug.Function with cure mainly: regulating QI to relieve pain, warming middle-JIAO is supporing yang; Be used for the cold syndrome of the stomach distending pain, stomachache due to hyperactive liver-QI attacking the stomach, impotence due to deficiency of the kidney, soreness of waist and knee joint.
Scorpio (
Heterometrus spinifer) buy the big pharmacy of ten thousand states from Luoyang, belong to the dry body of Arthropoda Arachnoidea Buthidae animal Scorpio (Latin), main product in Henan, ground such as Shandong, Hebei.Scorpio has the effect of endogenous wind stopping relieving convulsion, dispersing pathogen accumulation, removing obstruction in the collateral to relieve pain, is used for the anxious intermittent infantile convulsion diseases such as wind, apoplexy facial paralysis, tetanic spasm, sore swollen toxin, scrofula tuberculosis, intractable migraine and general headache, rheumatic arthralgia of fainting.In recent years, along with domestic and international biologic pharmacological science, biochemistry and development of molecular biology, the application of Scorpio and charybdotoxin is constantly widened, become the treatment multiple difficult disease medicine and be applied to clinical.
Blatta seu periplaneta (
Blatta orientalis Linnaeus), available from Lao Yitang pharmacy, Bozhou, Anhui, formal name used at school is a cockroach, belong to the Insecta Blattaria (
Blattodea) Blattidae (
Blattidae); As the medicinal Shennong's Herbal that is stated from the earliest; Cure mainly " blood stasis disease heavily fortified point, cold and heat, removing mass gather, laryngopharynx numbness, endogenous cold loss of fecundity "; In the successive dynasties, important herbal ancient books and records were like " Newly Revised Canon of Materia Medica ", Compendium of Material Medica, supplementary Amplifications of the Compendium of Materia Medica, all record, also had modern age record old in many pharmacy monographs.Modern scientific research shows that the cockroach mucopolysaccharide has the effect of aspects such as anticancer, antiallergic, treatment gynaecopathia, hepatitis, hepatic injury.
Aranea (
Aranea ventricosa L. Koch), available from Lao Yitang pharmacy, Bozhou, Anhui, belong to Arthropoda (
Arthropoda), Arachnoidea (
Arachnida), Araneida (
AFaneoe).The geographical distribution of Aranea is very extensive, and adaptability is also very strong, has powerful natural evolution ability.Aranea all has a pair of poison gland, and it is terminal to be positioned at the head chelicera, and its opening often is positioned at chela pawl art end, can extremely survive in the rugged environment.
Gryllotalpa (Gryllotalpa africana Pal. De Beauvois) is that Herba indigoferae Pseudotinctoriae is used time-honored animal drugs, and available from Lao Yitang pharmacy, Bozhou, Anhui, Gryllotalpa is cold in nature, salty in the mouth; The National People's Congress, small intestinal, urinary bladder channel cures mainly various edema; Defecation is unfavorable, urine retention, miscellaneous diseases such as urinary system calculus.East Gryllotalpa Gryllotalpa orientalis Burmeister is the dispersed species of China; It also is the staple kind of Gryllotalpa medical material; The chemical constituent that it contains is except that aminoacid, trace element, and Wei in 2007, road intelligence etc. was therefrom got fatty acid and two big types of 13 kinds of chemical compounds of sterol and phenylacetic acids first.
Chrysomyiame gacephala (Fab.) (
Chrysomyia megacephala Fab.), the popular name maggot, available from Lao Yitang pharmacy, Bozhou, Anhui, the larva of general reference Calliphoridae insecticide.According to Chinese Medicine " book on Chinese herbal medicine is asked the source ", " book on Chinese herbal medicine is just read " record, this worm is cold in nature, after drying is ground into powder, supplies to put deposited external on the skin, can treat ecthyma, lip pustule.
The present invention compared with prior art has the following advantages and outstanding effect:
The present invention is from insecticide or arthropod source secondary metabolites; The reactive compound that research and development have bacteriostasis; Utilize intermiscibility biology " two sections solvent extraction methodes ": promptly extract the fat-soluble composition of insecticide earlier with methylene chloride, the reuse methanol aqueous solution extracts polar substances wherein.Can in the processing procedure of raw material, guarantee to obtain metabolite composition all in the insect bodies (the living metabolite of dimension complicated component, content in insect bodies is very low) so on the one hand; To guarantee also that on the other hand the BA of itself is constant in the leaching process, guarantee isolating reliability of raw material and accuracy, efficiently solve " bottleneck " problem of insecticide natural product research.The extract that employing the inventive method extracts is to G
+Positive bacteria or G
-Negative bacterium has bacteriostasis in various degree.
The bacteriostatic activity screening study of two sections solvent extraction methodes that the present invention implemented is suitable for all insecticides, arthropod and Amphibian.Through the worm source feature extraction thing that design obtains, prepare pure article chemical compound through further separating with bacteriostatic activity, can utilize Spectrum Analysis, electronic spraying and crystal diffraction technology to carry out structure then and identify and characterize; The worm source bacteriostatic activity noval chemical compound that screening and assessment obtains; Continuation is carried out the antibiotic efficacy determinations to it; Might find and optimize brand-new skeleton antibiotic medicine lead compound, lay the foundation, for the development of new antibiotic provides new way and new approaches for obtaining novel antibiotic medicine.The insecticide of the present invention's employing or arthropod breeding are fast in addition, quantity distributes extensively, is pathophorous media; They colonize in the dirty adverse circumstances, and adapting to goes forward side by side dissolves a cover and self secrete antibiotic ability and mechanism, can produce strong bacteriostasis.Lacking under new construction, the antibiotic present situation of novel targets at present; The research of the bacteriostatic active ingredients that insecticide and arthropod carry out based on two sections solvent extraction methodes of biocompatibility; Established the methodology basis for insecticide or arthropod chemistry research direction, had than important academic significance and good prospects for application.
The specific embodiment
Below through embodiment the present invention is done further explanation, but protection scope of the present invention is not limited thereto.Exsiccant insecticide or arthropod granulated slag get through following processing among each embodiment: get insecticide or arthropodan all 80 mesh sieves of pulverizing, promptly get after placing 60 ℃ of baking ovens to be dried to constant weight then.
Embodiment 1
The method for distilling of a kind of insecticide or arthropod secondary metabolites; It comprises the steps: exsiccant Changbai Mountain Formica rufa granulated slag (is soaked and is advisable can flood granulated slag with the dichloromethane immersion earlier; Immersion process can be auxiliary with ultrasound wave) twice (immersion for the first time finishes after-filtration; Granulated slag soaks with new dichloromethane once more), soak 48h at every turn; Filter then and obtain lixiviating solution (merge the gained lixiviating solution twice, concentrate drying gets CH
2Cl
2Extractum, this CH
2Cl
2Extractum is the fat-soluble metabolite of insecticide, sealing, put in the refrigerator preserve subsequent use) and granulated slag, the methanol aqueous solution of reuse mass concentration 70% soaks twice after the granulated slag drying, soaks 36h at every turn; Merge and soak 60 ℃ of gained alcohol extract Bing Yu – for twice and place the 24h postlyophilization and get extractum (this extractum is the insecticide water solublity metabolite that extracts gained, is designated as 70% methanol extractum in the table 1 and 2), it is subsequent use that preservation is put in the refrigerator in sealing.
Embodiment 2
With Aspongopus granulated slag replacement Changbai Mountain Formica rufa granulated slag, other is with embodiment 1.
Embodiment 3
With Blatta seu periplaneta granulated slag replacement Changbai Mountain Formica rufa granulated slag, other is with embodiment 1.
Embodiment 4
With Gryllotalpa granulated slag replacement Changbai Mountain Formica rufa granulated slag, other is with embodiment 1.
Embodiment 5
With Chrysomyiame gacephala (Fab.) granulated slag replacement Changbai Mountain Formica rufa granulated slag, other is with embodiment 1.
Embodiment 6
With Scorpio granulated slag replacement Changbai Mountain Formica rufa granulated slag, other is with embodiment 1.
Embodiment 7
With Aranea granulated slag replacement Changbai Mountain Formica rufa granulated slag, other is with embodiment 1.
The bacteriostatic test of above-mentioned each embodiment gained extractum (being the insecticide extract):
Get the extractum that each embodiment makes at last, use the methanol aqueous solution of mass concentration 70% to be made into the solution that concentration is 15 mg/mL and 30 mg/mL (gained CH in the leaching process respectively
2Cl
2Extractum is mixed with respective concentration with 70% methanol-oil and carries out the fungistatic effect contrast); With G-bacterium escherichia coli (E.coli), G+ bacterium bacillus subtilis (B. subtilis) as the bacterium source; Solvent 70% methanol-oil and 70% methanol aqueous solution are as negative control, and penicillin is as G+ bacterium positive control, and gentamycin sulfate is as G-bacterium negative control; Penicillin and gentamycin sulfate are all prepared 1 mg/mL and two concentration of 10 mg/mL, do biological activity test.Observe whether the generation of inhibition zone is arranged, and write down its inhibition zone size, the result sees table 1 and table 2 for details.
Bacteriostatic activity test: adopt flat band method that the insecticide extract is carried out the antibacterial activity screening and carries out antibacterial classification.
Experimental strain is selected the pathogen antibacterial gram positive bacteria that uses clinically for use: bacillus subtilis (B.Subtilis) and staphylococcus aureus (S.aureus Rosenbach); Gram negative bacteria: escherichia coli (E. coli) and bacillus pyocyaneus (P. Aeruginosa).
Positive control medicine: G
+Positive bacteria: penicillin (Penicillin G Sod); G
-Negative bacterium: gentamycin (Gentamicin).Corresponding two kinds of fat-soluble metabolite of extract (are CH
2Cl
2Extractum) and water solublity metabolite (i.e. 70% methanol extractum) use solvent 70% methanol-oil (oil is for commercially available vegetable oil, like Shandong flower Oleum Arachidis hypogaeae semen) and 70% methanol-water to estimate respectively as negative control.
Through investigating bacteriostatic test, the extract extractum of confirming insecticide that all are estimated or arthropod water solublity metabolite is to G
+Positive bacteria or G
-Negative bacterium has bacteriostasis in various degree, can be used to separate and prepare the chemical compound with bacteriostatic activity, has established the methodology basis for the chemical research of insecticide, has than important academic significance and good prospects for application.
Claims (2)
1. the method for distilling of insecticide or arthropod secondary metabolites is characterized in that, comprises the steps: exsiccant insecticide or arthropod granulated slag are soaked more than twice with dichloromethane earlier, and each soak time is no less than 36h, then solid-liquid separation; The reuse methanol aqueous solution soaks more than twice after the gained granulated slag drying, and each soak time is no less than 36h, and merging gets extractum after soaking gained alcohol extract and lyophilization; Said insecticide is selected from Changbai Mountain Formica rufa, Aspongopus, Blatta seu periplaneta, Gryllotalpa or Chrysomyiame gacephala (Fab.); Said arthropod is selected from Scorpio or Aranea.
2. the method for distilling of insecticide or arthropod secondary metabolites according to claim 1 is characterized in that, exsiccant insecticide or arthropod granulated slag are soaked twice with dichloromethane earlier, soaks 36-48h, then solid-liquid separation at every turn; The methanol aqueous solution of reuse mass concentration 70% soaks twice after the gained granulated slag drying, soaks 36-48h at every turn, merges 60 ℃ of placements of immersion gained alcohol extract Bing Yu – 24h postlyophilization and gets extractum.
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| CN103638058A (en) * | 2013-12-01 | 2014-03-19 | 大理学院 | Preparation of effective part of nephila spider and application of anti-gram medicine |
| CN104003923B (en) * | 2014-05-30 | 2016-06-15 | 河南科技大学 | The application of the extracting method of alkaloid compound and this compound in Gryllotalpa |
| CN108129538B (en) * | 2017-12-19 | 2020-06-16 | 河南科技大学 | Method for extracting antibacterial compound from scorpion |
| CN109170173A (en) * | 2018-10-17 | 2019-01-11 | 江门鑫肽生物蛋白有限公司 | A kind of insect metabolin additive and its preparation method and application |
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| CN1450169A (en) * | 2003-05-08 | 2003-10-22 | 福建师范大学 | Separation purification method for preparing sex-gene recombination spider dragline silk protein |
| CN1544593A (en) * | 2003-11-25 | 2004-11-10 | 中国人民解放军第二军医大学 | Cricket fatty oil and supercritical extracting process thereof |
| CN1559447A (en) * | 2004-02-18 | 2005-01-05 | 中国人民解放军军需大学 | Extracte of Aspongopus chinensis Dallas and Extraction method therefor medicinal use and preparn. thereof |
| CN1582970A (en) * | 2004-06-15 | 2005-02-23 | 上海澳隆科技发展有限公司 | Extraction of effective portion of gryllotalpidae for anti-cancers and its preparation |
| CN101156875A (en) * | 2007-04-25 | 2008-04-09 | 深圳信立泰药业股份有限公司 | Cockroach extractive for treating cardiovascular disease, preparation method and its composition |
-
2011
- 2011-08-29 CN CN2011102495700A patent/CN102293785B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1450169A (en) * | 2003-05-08 | 2003-10-22 | 福建师范大学 | Separation purification method for preparing sex-gene recombination spider dragline silk protein |
| CN1544593A (en) * | 2003-11-25 | 2004-11-10 | 中国人民解放军第二军医大学 | Cricket fatty oil and supercritical extracting process thereof |
| CN1559447A (en) * | 2004-02-18 | 2005-01-05 | 中国人民解放军军需大学 | Extracte of Aspongopus chinensis Dallas and Extraction method therefor medicinal use and preparn. thereof |
| CN1582970A (en) * | 2004-06-15 | 2005-02-23 | 上海澳隆科技发展有限公司 | Extraction of effective portion of gryllotalpidae for anti-cancers and its preparation |
| CN101156875A (en) * | 2007-04-25 | 2008-04-09 | 深圳信立泰药业股份有限公司 | Cockroach extractive for treating cardiovascular disease, preparation method and its composition |
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