CN111088291B - Secondary metabolite of nematophagous fungus isolate and crude extract and medical use thereof - Google Patents
Secondary metabolite of nematophagous fungus isolate and crude extract and medical use thereof Download PDFInfo
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Abstract
The invention provides a secondary metabolite of a nematophagous fungus isolate, a crude extract and medical application thereof, which are prepared according to nematophagous fungiDuddingtonia flagransThe characteristics of the secondary metabolite of the isolate F088 and crude extracts (comprising a water phase and an organic phase) thereof are that after the secondary metabolite and crude extracts (comprising a water phase and an organic phase) thereof are diluted by water or an organic solvent, the effects of resisting escherichia coli, chicken coccidiosis and breast cancer and the like are respectively observed through in-vitro cell culture, animal experiments and the like, and meanwhile, the working concentration of the secondary metabolite and crude extracts (comprising a water phase and an organic phase) and other drugs playing roles is provided, so that new candidate drugs are provided for resisting escherichia coli, chicken coccidiosis and breast cancer and the like.
Description
The technical field is as follows:
the invention provides a nematophagous fungusDuddingtonia flagransThe extraction and separation preparation method of the secondary metabolite of the isolate F088 and the crude extract thereof, and the antibacterial, anti-insect and anti-tumor effects of the secondary metabolite and the crude extract (including aqueous phase and organic phase) thereof and the application thereof in the fields of medicine and veterinary medicine, belong to the field of biological pharmacy.
Background art:
nematophagous fungi are mainly classified as nematophagous fungi according to their insecticidal mechanism: (Nematode– trapping fungi) Opportunistic fungi (A), (B)Opportunistic fungi) Toxic producing fungi: (Toxin–producing Fungi) And endoparasitic fungi: (Endoparasitic fungi). The nematode-trapping fungi are fungi which are specialized by vegetative hyphae to form various trapping structures so as to trap nematodes and widely exist in the nature. If nematodes are present in the environment, the bacteria can kill the nematodes by producing typical predation structures. Wherein the nematode-trapping fungi areDuddingtonia flagrans （For shortD. flagrans，Now called as：Arthrobtrys flagrans）Is the most potential strain internationally acknowledged at present for preventing and treating animal parasitic nematodes, because the strain can produce a large amount of chlamydospores with double-layer thick walls and resist adverse environments such as animal gastrointestinal tracts and the like without losing activity. A great deal of research on the in-vivo and in-vitro nematicidal performance of the strain is carried out, and the strain is considered to have better effect. However, at present, no nematophagous fungi are available at home and abroadD. flagransThe secondary metabolite is researched, and comprises the extraction, separation and biological functions of the secondary metabolite and a crude extract (comprising a water phase and an organic phase) thereof. Because the bacteria are fermentedThe secondary metabolite produced in the process is complex in composition, and the effective components thereof are not clear. Thus, it is clear thatD. flagransThe fermentation secondary metabolite and the crude extract (including water phase and organic phase) thereof have important effective components and biological functions. At present, no relevant report exists at home and abroad.
The invention content is as follows:
the invention aims to provide a nematophagous fungusDuddingtonia flagransExtraction, separation and preparation methods of a isolate F088 secondary metabolite and a crude extract (comprising an aqueous phase and an organic phase) thereof.
The invention also provides the antibacterial, anti-insect and anti-tumor effects of the secondary metabolite and the crude extract (comprising water phase and organic phase) thereof, and the application thereof in the fields of medicine and veterinary medicine, and the secondary metabolite and the crude extract are suitable for industrial production.
The invention relates to a nematophagous fungusDuddingtonia flagransAn isolate secondary metabolite characterized as being produced by the method comprising:
storing nematophagous fungi in test tube inclined plane at 4 deg.CDuddingtonia flagransSeparating strain F088, cutting agar block containing mycelium, inoculating to the center of 2% corn flour agar culture medium, and culturing in constant temperature incubator at 30 deg.C for 5 days to activate strain; then activatedDuddingtonia flagransInoculating the isolate F088 into a liquid culture medium, placing the isolate in a constant-temperature shaking incubator, and culturing for 10 d at 30 ℃; filtering with sterilized gauze, centrifuging to remove mycelium, and obtaining fermentation product, i.e. secondary metabolite.
The invention relates to a nematophagous fungusDuddingtonia flagransThe organic phase and the water phase of the secondary metabolite of the isolate are characterized in that:
fungus capable of feeding nematodeDuddingtonia flagransFully and uniformly mixing the secondary metabolite of the isolate with equal amount of ethyl acetate, standing overnight, and layering to obtain an upper organic phase and a lower water phase;
respectively collecting an organic phase and a water phase; placing the organic phase at 4 deg.C, and extracting the water phase for the second time; after extraction overnight, placing the separated organic phase in a refrigerator at 4 ℃, and extracting the rest aqueous phase for the third time; the organic phases obtained three times after the extraction were collected by rotary evaporation: continuously evaporating at 70 deg.C for 30 min; the aqueous phase obtained after three extractions was placed in a new distillation flask and the rotary evaporation was continued: continuously evaporating at 90 deg.C for 55min to finish evaporation; collecting the obtained organic phase and water phase at 80 deg.C, and drying to obtain crude extracts of secondary metabolite in organic phase and water phase.
The nematophagous fungi of the inventionDuddingtonia flagransThe secondary metabolite of the isolate and the crude extract thereof are used in the traditional Chinese medicine for preparing the anti-breast cancer medicine.
The nematophagous fungi of the inventionDuddingtonia flagransThe secondary metabolite of the isolate and the crude extract thereof have medical application in preparing anti-Escherichia coli medicaments.
The nematophagous fungi of the inventionDuddingtonia flagransThe secondary metabolite of the isolate is used for preparing anti-coccidiosis drugs for animals.
The invention has the positive effects that: according to nematophagous fungiDuddingtonia flagransThe characteristics of the secondary metabolite of the isolate F088 and crude extracts (comprising a water phase and an organic phase) thereof are that after the secondary metabolite and crude extracts (comprising a water phase and an organic phase) thereof are diluted by water or an organic solvent, the effects of resisting escherichia coli, chicken coccidiosis and breast cancer and the like are respectively observed through in-vitro cell culture, animal experiments and the like, and meanwhile, the working concentration of the secondary metabolite and crude extracts (comprising a water phase and an organic phase) and other drugs playing roles is provided, so that new candidate drugs are provided for resisting escherichia coli, chicken coccidiosis and breast cancer and the like.
Drawings
FIG. 1: nematophagous fungi of different dilution timesDuddingtonia flagransExtract (secondary metabolite, beta-glucan),
Aqueous phase, organic phase) on the viability of Mda-mb231 cells;
FIG. 2: nematophagous fungiDuddingtonia flagransThe water phase stock solution of the extract and the coliform inhibiting conditions of different dilution times.
Detailed Description
The following examples are intended to further illustrate, but not limit, the present invention. It will be appreciated by those skilled in the art that changes in this embodiment may be made without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims.
Example 1
Storing nematophagous fungi in test tube inclined plane at 4 deg.CDuddingtonia flagransIsolate F088, a 5X 5 mm agar block containing hyphae was cut, inoculated into the center of 2% CMA (corn flour agar medium), and cultured in a constant temperature incubator at 30 ℃ for 5 days (activated strain), and the culture was terminated. Then activatedDuddingtonia flagransThe isolate F088 was inoculated in a liquid medium and cultured in a constant temperature shaking incubator at 30 ℃ and 200 r/min for 10 days. Filtering with 4 layers of sterilized gauze, centrifuging to remove mycelium to obtain fermentation product, and obtaining nematophagous fungiDuddingtonia flagransA secondary metabolite of isolate F088.
Fermentation product (secondary metabolite) traits: the fermentation liquor is dark brown, is accompanied by mycelia with different lengths, and has relatively transparent liquid and no special odor.
Example 2
After mixing the secondary metabolite 80 mL obtained in example 1 and an equal amount of ethyl acetate (80 mL) thoroughly, the mixture was added to a 250 mL separatory funnel and turned upside down several tens of times to mix the two thoroughly; placing the separating funnel filled with the secondary metabolite and the ethyl acetate in a super-clean workbench, and standing overnight; after extraction overnight, the separating funnel is divided into an upper layer and a lower layer, the upper layer is an organic phase, and the lower layer is an aqueous phase. Respectively and slowly collecting the organic phase and the aqueous phase in a 100mL beaker; the collected organic phase was placed in a refrigerator at 4 ℃ and the aqueous phase was subjected to a second extraction. After extraction overnight, the organic phase obtained by separation was placed in a refrigerator at 4 ℃ and the remaining aqueous phase was subjected to a third extraction. Collecting the organic phase obtained in three times in a distillation flask, simultaneously connecting a rotary evaporator and a vacuum pump, continuously evaporating at 70 ℃ for 30min at 100 r/min, and ending evaporation when the bottom of the flask is less than about 2mL of liquid; and (3) placing the water phase obtained after the three-time extraction in a new distillation flask, simultaneously connecting a rotary evaporator and a vacuum pump, continuously evaporating at 90 ℃ for 55min at 100 r/min, and ending evaporation when the bottom of the flask is less than about 2mL of liquid.
Collecting the obtained organic phase and water phase in small plates (before operation, marking and weighing the two plates respectively), drying in a drying oven at 80 deg.C, and weighing together with the plates after the water phase and the organic phase in the plates are dried; the weight of the crude extract was obtained (as a result, organic phase dry weight: 19.4 mg, aqueous phase dry weight: 904.1 mg).
The following pharmacological tests confirm the nematophagous fungiDuddingtonia flagransThe secondary metabolite of the isolate F088 and the crude extract (including aqueous phase and organic phase) thereof have the effects of resisting bacteria, insects, tumors and the like, and can be applied as medicaments for resisting bacteria, insects, tumors and the like.
Test example 1
The secondary metabolite and the crude extract (including water phase and organic phase) thereof of the invention are used for resisting breast cancer test
1 method
Detection of nematophagous fungi by MTT methodDuddingtonia flagransThe effect of the extracts (secondary metabolite, aqueous phase and organic phase) on the proliferation of breast cancer cells (Mda-mb 231). Mda-mb231 cells were grown to logarithmic phase first, harvested by digestion, counted at 5X 103Per well was seeded in 96 well cell culture plates with 100. mu.l DMEM medium per well. Place 96-well cell culture plates at 37 ℃ in 5% CO2Culturing for 24-48 h in an incubator, completely sucking the culture medium after the cells adhere to the wall, and adding the nematophagous fungi diluted by times (5, 10, 20, 40, 80, 160 and 320)Duddingtonia flagransThe extract (secondary metabolite, aqueous phase, organic phase) of (a) was simultaneously set with 5 replicates per gradient for a non-medicated cell control, a media control and a secondary metabolite diluent control. After 24 h of action, 10. mu.l of CCK-8 solution was added to each well at 37 ℃ with 5% CO2Culturing for 1h in an incubator, detecting the absorbance OD450nm by an enzyme-labeling instrument, and calculating the cell survival rate according to the following formula:
cell survival rate (%) = (experimental OD value-diluent control OD value)/(cell control OD value-culture medium control OD value) × 100%
2 results
CCK-8 detection results show that nematophagous fungiDuddingtonia flagransAfter the extracts (secondary metabolite, aqueous phase, organic phase) of (1) were allowed to act on Mda-mb231 cells for 24 hours, the cell survival rates (%) of the secondary metabolite, aqueous phase and organic phase were respectively as follows: 30.18 +/-1.32, 20.06 +/-1.09 and 30.18 +/-0.81, and the lower the dilution factor, the lower the cell survival rate, which shows that the nematophagous fungiDuddingtonia flagransThe extract (secondary metabolite, water phase, organic phase) has obvious inhibition effect on the growth of Mda-mb231 cells, and the inhibition effect is against nematophagous fungiDuddingtonia flagransThe concentration of the extract (secondary metabolite, aqueous phase, organic phase) was increased and enhanced (Table 1).
TABLE 1 Effect of bacterial extracts of different dilution factor of the present invention on the survival (%) of Mda-mb231 cells
And (4) conclusion: the invention has obvious inhibition effect on the growth of Mda-mb231 cells, and the inhibition effect is against nematophagous fungiDuddingtonia flagransThe concentration of the extract (secondary metabolite, aqueous phase, organic phase) was increased and enhanced (Table 1).
Test example 2
The secondary metabolite of the invention is tested against chicken Eimeria tenella
1 method
30 18-day-old chickens were randomly divided into 5 groups of 6 chickens. A test group 1 (secondary metabolite is given on the same day of insect attack), a test group 2 (nematode-feeding fungus is given three days after insect attack), a test group 3 (secondary metabolite is given three days after insect attack), a red control group and a white control group are set. 2 x 10 to4An individual Eimeria tenella sporulated oocyst was inoculated into 18 day old chickens. Feces were collected from day 7 post inoculation for the entire day for each group of chickens until the end of day 8. The feces collected every day were stirred well and diluted to 1000mL, 10 of which was aspirated with a pipettePlacing the solution in a small beaker, reacting for 1h by using 1mL of sodium hypochlorite solution, and adding saturated saline solution to reach the constant volume of 60 mL. Filtering with a 60-mesh copper sieve, collecting filtrate, counting oocysts by using a Macmester method, calculating the ovulation amount of each group of chickens, and recording the ovulation amounts respectively. ACI determination was performed by survival, relative rate of weight gain (%), relative oocyst yield and cecal lesion score.
Results
The test groups 2 and 3 die one each on the fifth day of insect attack, the control group dies one on the sixth day of insect attack, the test group 3 dies one on the seventh day of insect attack, and the test group 3 dies two on the eighth day of insect attack. The ACI test results show that: the ACI of the secondary metabolite given by the attack insects on the same day, the nematode-feeding fungus given by the attack insects three days later and the secondary metabolite given by the attack insects three days later are respectively: 197.9, 142.9, and 118.45, indicating that: the relative rate of gain of worms to the secondary metabolite group was higher on the same day with the highest ACI (see table 2).
TABLE 2 Eimeria tenella resistance test
And (4) conclusion: nematophagous fungiDuddingtonia flagransThe secondary metabolite has good effect of resisting chicken Eimeria tenella, and ACI reaches 197.9.
Test example 3
The secondary metabolite and the crude extract (including water phase and organic phase) thereof of the invention inhibit the test of Escherichia coli
3.1 method
3.1.1 Escherichia coli Strain activation and bacterial liquid preparation
Coating and inoculating the frozen and preserved escherichia coli DH5a to an LB solid culture medium, and culturing for 12h at 37 ℃; selecting a single colony, inoculating the single colony to a liquid culture medium, carrying out shake cultivation at 37 ℃ and 200 r/min overnight, and keeping the bacterial liquid for later use.
Preparation of the culture Medium
First, 100ml of LB solid medium was prepared, and 20ml of the medium was poured into a sterilized petri dish and allowed to stand horizontally for coagulation. Then, 200. mu.l of Escherichia coli DH5a bacterial solution was inoculated into the culture medium, and the resulting mixture was uniformly spread on a sterilized loop. And (4) uniformly drilling 6 holes on the culture dish uniformly coated with the bacterial liquid by using a gun head.
Nematophagous fungiDuddingtonia flagransDiluting the crude extract (including secondary metabolite, water phase, organic phase) with medicinal liquid
3.1.3.1 aqueous liquid medicine
A96-well plate was used, and 100. mu.l of 20% DMSO was added to each of the 2 nd to 6 th wells in line A. Respectively adding 100 μ l of the extracted aqueous phase liquid medicine into the 1 st and 2 nd holes of the A row, uniformly mixing, taking 100 μ l of diluent from the 2 nd hole to the 3 rd hole, and sequentially carrying out dilution by times according to the method until reaching the 6 th hole.
3.1.3.2 organic phase and Secondary metabolite liquid medicine
Diluting the organic phase and the metabolite liquid medicine according to the operation method of 3.1.3.1.
3.1.3.3 bacteriostatic test
And sequentially adding 100 mu l of aqueous-phase liquid medicine diluted by different times into each hole of the culture dish, sealing by a sealing film, and marking. Similarly, the culture dish is perforated according to the above operation, and the diluted organic phase liquid medicine and the secondary metabolite liquid medicine are respectively added. Another plate was punched and 100. mu.l of 20% DMSO was added as a control. And (5) placing the culture dish in a 37 ℃ incubator for culturing for 12h, and observing the bacteriostatic effect.
Test results
A distinct antibacterial ring was observed in the petri dish with the addition of the aqueous liquid drug. Bacteriostatic rings can be observed around the aqueous-phase liquid medicine stock solution and the liquid medicines diluted by 2 times, 4 times and 8 times, and the bacteriostatic rings are less obvious along with the increase of the dilution times of the liquid medicines. This phenomenon indicates that the higher the concentration of the aqueous drug solution, the better the inhibition of E.coli (see FIG. 2). However, no antibacterial ring was observed in the culture dishes containing the organic phase liquid medicine, the metabolite liquid medicine and DMSO, indicating that the organic phase liquid medicine and the secondary metabolite have no inhibitory effect on Escherichia coli.
And (4) conclusion: nematophagous fungiDuddingtonia flagransThe aqueous crude extract has strong Escherichia coli inhibiting effect.
Claims (5)
1. A secondary metabolite of an isolate of the nematophagous fungus Duddingtonia flagrans, characterized in that it is prepared by:
cutting an isolated strain F088 of nematophagous fungus Duddingtonia flagrans preserved in a test tube inclined plane at 4 ℃, inoculating an agar block containing hyphae into the center of a 2% corn flour agar culture medium, and placing the agar block in a constant temperature incubator for culturing an activated strain for 5 days at 30 ℃; then inoculating the activated Duddingtonia flagrans isolate F088 into a liquid culture medium, placing the liquid culture medium in a constant-temperature shaking incubator, and culturing for 10 d at 30 ℃; filtering with sterilized gauze, centrifuging to remove mycelium, and obtaining fermentation product, namely: a secondary metabolite.
2. An organic phase and aqueous phase crude extract of a secondary metabolite of an isolate of nematophagous fungi Duddingtonia flagrans, characterized in that:
fully and uniformly mixing the secondary metabolite obtained in the claim 1 with ethyl acetate with the same volume, standing overnight for layering to obtain an upper organic phase and a lower aqueous phase; respectively collecting an organic phase and a water phase; placing the organic phase at 4 deg.C, and extracting the water phase for the second time; after extraction overnight, placing the separated organic phase at 4 ℃, and performing third extraction on the rest aqueous phase; the organic phases obtained three times after the extraction were collected by rotary evaporation: continuously evaporating at 70 deg.C for 30 min; the aqueous phase obtained after three extractions was placed in a new distillation flask and the rotary evaporation was continued: continuously evaporating at 90 deg.C for 55min, and ending evaporation; and (3) respectively collecting the organic phase and the water phase obtained in the step (a) at 80 ℃ and drying to respectively obtain the crude extracts of the organic phase and the water phase of the secondary metabolite.
3. The use of the secondary metabolite of the nematode-feeding fungus Duddingtonia flagrans isolate of claim 1 or the organic phase and aqueous phase crude extract of claim 2 in the preparation of anti-breast cancer drugs for traditional Chinese medicine.
4. The medical use of the aqueous crude extract of the secondary metabolite of the isolate of the nematophagous fungus Duddingtonia flagrans of claim 2 in the preparation of anti-e.
5. The nematophagous fungus of claim 1Duddingtonia flagransThe secondary metabolite of the isolate is used for preparing the veterinary use of the anti-coccidiosis drugs.
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| 利用食线虫真菌防治植物寄生线虫的研究进展;莫明和 等;《大理学院学报》;20091231;第8卷(第12期);第1-9页 * |
| 捕食线虫性真菌Duddingtonia flagrans胞外蛋白质的产生及其杀虫作用的研究;王瑞 等;《中国兽医科学》;20140430;第44卷(第4期);第387-393页 * |
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