CN113533786A

CN113533786A - Method for researching anti-pollution action mechanism of diindole alkaloid on Nitzschia closterium based on electron microscopic imaging technology

Info

Publication number: CN113533786A
Application number: CN202110093693.3A
Authority: CN
Inventors: 王开玲; 宗鹏; 徐颖; 窦峥嵘
Original assignee: Dali University
Current assignee: Dali University
Priority date: 2021-01-24
Filing date: 2021-01-24
Publication date: 2021-10-22

Abstract

The invention relates to a method for researching an anti-pollution action mechanism of diindole alkaloid on Nitzschia closterium based on an electron microscopic imaging technology. In particular to the first proposal of the algae inhibiting activity (EC) of a compound DIM-Ph-1' -OH on the fouling organism Nitzschia closterium₅₀And EC₉₀The value is 0.35 and 1.0 mu g/mL respectively, the NOEC is observed to be 0.05 mu g/mL for 96 h), and the compound can enhance the roughness of the cell wall of the Nitzschia closterium and weaken the mechanical strength of the Nitzschia closterium under the concentration of 0.35-2.0 mu g/mL, so that the variety and the number of adsorption groups of the cell wall surface to trace metals are reduced, thereby inhibiting the growth of algae cells, and simultaneously, Ca in the algae cells is caused to increase²⁺Efflux and cell wall pair Ca²⁺The adsorption capacity of the cell is weakened, so that a certain amount of calcium ions are lacked in the cell, the synthesis of the cell wall is hindered, and the algae inhibiting activity is exerted.

Description

Method for researching anti-pollution action mechanism of diindole alkaloid on Nitzschia closterium based on electron microscopic imaging technology

Technical Field

The invention belongs to the field of research on an action mechanism of bis-indole alkaloid for inhibiting growth of Nitzschia closterium, and particularly relates to a research method for an anti-pollution action mechanism of bis-indole alkaloid on Nitzschia closterium based on an electron microscopy imaging technology.

Background

Nitzschia Closterium (NC) belongs to the phylum Diatomae, the class of Euptectidae, the order of Dictyotales, the family of Calystegiaceae. Nitzschia closterium belongs to red tide algae species, has records of red tide outbreak in China, and is one of main algae species in marine fouling diatom. The research on the influence mechanism of the antifouling agent on the adhesion of the antifouling agent is particularly important for developing a novel environment-friendly antifouling agent. The invention provides a method for researching an anti-pollution action mechanism of diindole alkaloid on Nitzschia closterium based on an Atomic Force Microscope (AFM) and assisted by a Non-invasive Micro-test Technology (NMT).

Disclosure of Invention

The invention provides an algistat, which is characterized in that the algistat takes a compound 4- (di (1H-indol-3-yl) methyl) phenol (DIM-Ph-1' -OH) as an active ingredient.

Another embodiment of the present invention provides an algistat characterized in that it preferably contains 0.35-2.0 μ g/mL of DIM-Ph-1' -OH; further preferably, DIM-Ph-1' -OH is contained in an amount of 0.35, 0.5, 1.0 or 2.0. mu.g/mL.

Another embodiment of the invention provides the use of DIM-Ph-1' -OH in the preparation of an algistat, in particular for inhibiting the growth of Nitzschia closterium. The algistat is prepared by increasing roughness of cell wall of Nitzschia closterium and weakening mechanical strength thereof to weaken adsorption capacity of cell wall surface and simultaneously cause Ca in algae cell²⁺The cell wall synthesis is blocked by efflux, and the algae inhibiting activity is exerted.

The algistat preferably inhibits the growth of Nitzschia closterium, and particularly, DIM-Ph-1' -OH can increase the roughness of the cell wall of the Nitzschia closterium and weaken the mechanical strength of the cell wall of the Nitzschia closterium under the concentration of 0.35-2.0 mu g/mL (preferably 0.35, 0.5, 1.0 and 2.0 mu g/mL), so that the types and the number of adsorption groups of the surface of the Nitzschia closterium on trace metals are reduced, and the reproduction of the alga cells is inhibited; at the same time, cause Ca in algal cells²⁺Efflux, cell wall to Ca²⁺The adsorption capacity of the cell is weakened, so that a certain amount of calcium ions are lacked in the cell, the synthesis of the cell wall is hindered, and the algae inhibiting activity is exerted.

Drawings

FIG. 1 is a graph showing the effect of different concentrations (μ g/mL) of DIM-Ph-1' -OH on the growth of Nitzschia closterium cells.

FIG. 2 is a graph of the results of AFM experiments with different mass concentrations (μ g/mL) of DIM-Ph-1' -OH treatment of Nitzschia closterium: (A) the roughness of the cell wall of the algae observed by AFM, wherein A, B, C, D, E is the global map of single algae cell of CON-7 (blank control group), CON-S (DMSO solvent control group), INH-L (0.05 μ g/mL), INH-M (0.35 μ g/mL) and INH-H (1.0 μ g/mL), a, b, c, d and e are the local topography maps of A, B, C, D, E, and F, G, H, I, J is the local enlargement maps of a, b, c, d and e; (B) surface roughness statistics of the atomic force measurements of the groups CON-7, CON-S, INH-L, INH-M and INH-H; (C) young's modulus statistics were measured for groups CON-7, CON-S, INH-L, INH-M, and INH-H atomic forces. Statistics of the data were analyzed using SPSS, p x < 0.01.

FIG. 3 is a graph showing the results of NMT experiments on algal cells after 4 days of DIM-Ph-1' -OH treatment of Nitzschia closterium at different mass concentrations (μ g/mL): (A) algal cell H⁺Average flow velocity and direction; (B) algal cell Ca²⁺Average flow velocity and direction. Statistics of the data were analyzed using SPSS, p<0.05,p**<0.01。

Detailed Description

4.1 Experimental materials

4.1.1 Medium

f/2 culture medium

4.1.2 Primary reagents

Mecline: 4- (di (1H-indol-3-yl) methyl) phenol (DIM-Ph-1' -OH), CAS No.: 151358-47-3.

Mecline: cell culture grade Dimethyl sulfoxide (DMSO)

4.1.3 Main Instrument

Main experimental instrument

4.1.4 sources of algal species

Nitzschia closterium (Nitzschia closterium) is from the university of Xiamen Algae species Bank (Center for Collections of Marine Algae, CCMA).

4.2 Experimental methods

4.2.1 culture of Nitzschia closterium

Preparing f/2 culture medium with sterilized seawater, inoculating 10%, and performing transfer culture every 5 days. The pH of the culture medium was 7.0, and the salinity of the seawater was 3.3%. Placing the transferred Nitzschia closterium in a light culture box for amplification culture at 23 deg.C under the intensity of light of 60 μmol/(m)²S), light-to-dark ratio 14 h: 10h, gently shake manually 2 times daily.

4.2.2 Compound DIM-Ph-1' -OH algal inhibitory Activity test on Nitzschia closterium

Nitzschia closterium which is cultured for 4 days and is in logarithmic growth phase is taken as experimental algae. According to the results of the preliminary experiments, the final concentrations of DIM-Ph-1' -OH in the exposure experiment were 0.05, 0.50, 1.0 and 2.0. mu.g/mL, the blank control group (CON-7) was an algal solution without any reagent added thereto, and the solvent control group (CON-S) was an algal solution containing 2.0. mu.g/mL of DMSO reagent. DIM-Ph-1' -OH mother liquor and algae liquor with different concentration gradients are respectively added into a 150mL conical flask, and the initial density of the Nitzschia closterium cells is adjusted to be 5 multiplied by 10⁵cells/mL, final volume of solution 50mL, each concentration set for 3 biological replicates. Each group was cultured in a light incubator by shaking gently by hand 2 times a day s, and 200. mu.L of algal solution was counted by a flow cytometer every 24 hours for 7 days. Statistical data, plotting growth curves and calculating the half maximum Effect Concentration (EC) of 96h₅₀) 90% maximum Effect Concentration (EC)₉₀) And observed to give a maximum no-effect concentration (NOEC) of 96 h.

4.2.3 determination of algal cell surface morphology, roughness and surface mechanics by Atomic Force Microscopy (AFM)

Nitzschia closterium cells cultured in 4d log phase in DIM-Ph-1' -OH exposure were used for the experiments. Dripping 50 μ L of each group of algae cells on a clean cover glass with proper size, fixing the cover glass on a round iron sheet with double faced adhesive tape, and standing at room temperature for 40-60min to achieve the effect of algae cell sedimentation. Soaking algae cells in high-purity water for 30s each time for 5 times. The algae cells were dried in a common moisture-proof cabinet for 48h before AFM experiments.

The surface topography pictures of the cells were obtained by using an automatic scanning mode (Scan Asyst Air mode) in an AFM gas phase environment and selecting the corresponding probe (tip radius: 2-12nm, modulus of elasticity 0.4N/m, Bruker). AFM model L01F4C8 multifunctional atomic force microscope (BRUKER), scanning rate of all AFM modes in the experiment is set to be 2Hz, room temperature is 25 ℃, and indoor relative humidity is 50%. Meanwhile, the Young modulus of each group of Nitzschia closterium cell walls is measured by using the mechanical mode of AFM, so that the mechanical strength of the siliceous cell walls and the possible influence of the siliceous cell walls on the surface properties, particularly the surface adsorption capacity, are quantitatively analyzed. Young modulus of the cell surface of the algal cells is measured in Force Volume mode, and a probe (tip radius: 8nm, elastic coefficient 20-80N/m, Bruker) of type RTESP is selected.

4.2.4 measurement of cell surface ion flux Using non-invasive microassay technique (NMT)

Determination of H in the different groups of experiments Using non-invasive micrometering technical Equipment (NMT Physiolyzer, Yange, USA)⁺And Ca²⁺Real-time rate of in and out of Nitzschia closterium cells, i.e. H⁺And Ca²⁺Flow rate. 1 plastic petri dish was prepared and one piece of the adhesive slide was treated with polylysine. The adhesive slide was placed on the bottom of the culture dish, and 100. mu.L of Nitzschia closterium cells in logarithmic growth phase (transfer culture 4d) were dropped on the adhesive slide and allowed to stand for 10min to allow the algal cells to completely adhere. A quantity of test solution was aspirated and slowly added along the edge of the petri dish while gently pressing the edge of the adhesive slide with forceps to prevent the adhesive slide from floating. Also, care should be taken to add the test solution in an amount that ensures that it will not over adhere to the slide. The waste solution in the dish was aspirated, 5-10 mL of fresh test solution was added, left for 30min, and then the test was started. On-displayLook for a single well-growing, morphologically normal algal cell under the microscope, and convert H⁺Or Ca²⁺The flow sensor was placed about 10 μm above the center of the cell surface of the algal cells to begin the test. Each sample was tested for 10min and each experimental group tested 6 algal cells as biological replicates. H⁺And Ca²⁺Flow rate data were read directly from imFluxes V2.0 software (YoungerUSA LLC, Amherst, MA 01002, USA) in mol cm^-2·s^-1The efflux values are negative and the absorption values are negative.

4.3 results and discussion

4.3.1 algal inhibitory Activity of the Compound DIM-Ph-1' -OH

DIM-Ph-1' -OH (2.0, 1.0, 0.5 and 0.05. mu.g/mL) at different concentrations stressed Nitzschia closterium growth, as shown in FIG. 1, at a concentration of 2. mu.g/mL, no increase in Nitzschia closterium cell number was obtained within 7 days, the level of algal cells at the time of initial addition was maintained, and the proliferation was completely inhibited; when the concentration is 1 mu g/mL, the number of the algae cells is increased very slowly within 72h, and the increase rate is increased after 72h, which shows that DIM-Ph-1' -OH at the concentration can effectively inhibit the growth of Nitzschia closterium cells within a certain time range; when the concentration is 0.5 mug/mL, the increase of the number of the algae cells is inhibited to a certain degree compared with the blank control group CON-7 and the solvent control group CON-S, but the inhibition degree is less than 2.0 and 1.0 mug/mL; at a concentration of 0.05. mu.g/mL, there was no significant difference in algal cell number and growth rate compared to the blank control and solvent control. In addition, there was no significant difference in algal cell growth in the solvent control group compared to the blank control group. Similar results were obtained in triplicate. The EC of the growth of the Nitzschia closterium cells stressed by DIM-Ph-1' -OH at 96h is obtained by statistical software₅₀A value of 0.35. mu.g/mL, EC₉₀The value was 1.0. mu.g/mL, and NOEC of 0.05. mu.g/mL was observed for 96 h.

According to the result of the algae inhibiting activity experiment, the conditions of an experimental group for measuring the roughness and surface mechanical data of the cell surface by an Atomic Force Microscope (AFM) and measuring the ion current of the cell surface by a non-damage micrometering technology (NMT) are determined. I.e. the experiments were divided into five groups: (1) the CON-7 is connected with the controller,i.e. blank control, no compound and other additions; (2) CON-S, a solvent control, added DMSO only (final concentration 2.0. mu.g/mL); (3) INH-L, a low concentration (0.05. mu.g/mL,) treatment group that is also the no visible effect concentration (NOEC) of DIM on Nitzschia closterium; (4) INH-M, middle concentration (0.35. mu.g/mL) treatment group, half maximal Effect Concentration (EC) of DIM-Ph-1' -OH on Nitzschia closterium₅₀) (ii) a (5) INH-H, a high concentration (1.0. mu.g/mL) treated group, DIM-Ph-1' -OH inhibited growth of Nitzschia closterium by about 90% (EC)₉₀)。

4.3.2 algal cell surface morphology, roughness and surface force variation based on Atomic Force Microscope (AFM) measurements

As shown in FIG. 2, the roughness of the cell walls of algae in the INH-M group was significantly higher than those of the CON-7, CON-S, INH-L and INH-H groups, while there was no significant difference in the roughness of the cell walls between the CON-7, CON-S, INH-L and INH-H groups. Research shows that the cell wall plays an important role in the process of absorbing and utilizing trace metals in the normal growth process of the algae cells, and the change of the roughness of the cell wall can change the number of surface adsorption groups, thereby possibly inhibiting the reproduction of the algae cells. The research is preliminarily determined according to the results of measuring the surface morphology and the roughness of the Nitzschia closterium cells under the stress of DIM-Ph-1 '-OH by AFM (atomic force microscopy) (see figure 2A and B), and the DIM-Ph-1' -OH is very likely to reduce the number of adsorption groups on the surface of the Nitzschia closterium cells to trace metals by changing the roughness of the cell walls of the Nitzschia closterium cells, thereby interfering the normal physiological activity of the algae cells to exert the algae inhibiting activity. Meanwhile, the Young modulus of each group of Nitzschia closterium cell walls is measured by using the mechanical mode of AFM, so that the mechanical strength of the siliceous cell walls and the possible influence of the siliceous cell walls on the surface properties, particularly the surface adsorption capacity, are quantitatively analyzed. As shown in FIG. 2C, the Young 'S modulus of INH-M and INH-H groups with significant algae inhibitory activity was significantly lower than that of CON-7, CON-S and INH-L, so that the compound DIM-Ph-1' -OH can affect the surface adsorption capacity of Nitzschia closterium cell wall by weakening its mechanical strength, thereby inhibiting the growth of algae cells.

4.3.3 DIM-Ph-1' -algal cell surface H under OH stress based on in situ non-invasive micrometering technique (NMT) measurements⁺And Ca²⁺Variation of ion flow

As shown in FIG. 3, except for the INH-H group H⁺、Ca²⁺The ion flow directions of the groups are all outflow, the other 4 groups CON-7, CON-S and INH-L and INH-M are all inflow, and the average flow velocity changes are different. At H⁺In the test results, the rate of influx in INH-L and INH-M in the experimental group with the addition of DIM-Ph-1' -OH compound decreased with increasing compound concentration compared to CON-7, with INH-H even appearing to be efflux (FIG. 3A). Excess cation absorption is via H in the external solution⁺Is balanced and is associated with the accumulation of cations and internally synthesized organic acid anions in the vacuoles of the cells. Ca on algal cell surface²⁺The NMT test results of (1) showed that the rates of influx of INH-L and INH-M in the experimental groups with DIM-Ph-1' -OH added were increased as the concentration of the compound was increased, and the INH-H group showed Ca, as compared with CON-7²⁺Outer row (fig. 3B). Calcium ions are reported to be closely related to many physiological activities of algae, such as stabilizing cell membranes, stabilizing cell walls, and participating in intracellular signaling as a second messenger, and adhesion of diatoms also requires the participation of calcium^[1,2]. Thus, DIM-Ph-1' -OH at high concentrations causes Ca on the cell surface of Nitzschia closterium²⁺Efflux, cell wall to Ca²⁺The decrease of the adsorption capacity of the compound and the lack of a certain amount of calcium ions in the cells cause the obstruction of the synthesis of the cell wall, which is probably one of the reasons that the compound inhibits the growth and adhesion of the nitzschia closterium.

4.4 conclusion

EC when compound DIM-Ph-1' -OH stresses Nitzschia closterium for 96h₅₀A value of 0.35. mu.g/mL, EC₉₀The value is 1.0 mu g/mL, and the NOEC of 0.05 mu g/mL is observed for 96h, which indicates that DIM-Ph-1' -OH has high-efficiency algae inhibiting activity and further confirms the broad-spectrum anti-pollution activity of the compound.

The method is characterized in that the surface appearance, roughness and surface force change of the nitzschia closterium cells are measured by an Atomic Force Microscope (AFM), and DIM-Ph-1' -OH can further influence the integrity of cell walls by increasing the roughness of the cell walls, so that the types and the number of adsorption groups of the surfaces of the nitzschia closterium cells on trace metals are reduced, normal physiological activities of the nitzschia closterium cells can not be carried out, and the growth and the propagation of the nitzschia closterium cells are fully inhibited. Meanwhile, DIM-Ph-1' -OH can also influence the surface adsorption capacity of the Nitzschia closterium cell wall by weakening the mechanical strength of the Nitzschia closterium cell wall, thereby inhibiting the growth rate of the algal cells.

Determination of algal cell surface H under DIM-Ph-1' -OH stress by non-invasive micrometering technique (NMT)⁺And Ca²⁺The change of ion current can be obtained, and the high concentration group treatment of the algae cells can cause the algae cells to absorb excessive cations to cause the algae cells to need to discharge H⁺Equilibrium is reached and may be associated with the accumulation of cations and internally synthesized organic acid anions in the vacuole of the cell. DIM-Ph-1' -OH causes Ca at high concentrations²⁺Efflux, cell wall to Ca²⁺The weakening of the adsorption capacity of the cell and the lack of a certain amount of calcium ions in the cell can cause the obstruction of the synthesis of the cell wall, which is probably an important action mechanism of DIM-Ph-1' -OH for exerting the algae inhibiting activity.

The research reports the algae inhibiting activity (EC) of the anti-pollution diindole alkaloid compound DIM-Ph-1' -OH on the fouling organism Nitzschia closterium for the first time₅₀A value of 0.35. mu.g/mL, EC₉₀The value is 1.0 mu g/mL, the NOEC of 96h is observed to be 0.05 mu g/mL), and the anti-fouling algae action mechanism of the compound is preliminarily researched by an atomic force microscopic imaging technology and a non-damage micrometering technology, so that the broad-spectrum anti-fouling activity of the compound is verified, and a data support is provided for the subsequent development of the compound as a commodity anti-fouling agent.

Reference to the literature

[1]Wigglesworth Cooksey B,Cooksey K E.Can diatoms sense surfaces？:State of our knowledge[J].Biofouling,1992,5(3):227-238.

[2]Vardi A,Formiggini F,Casotti R,et al.A stress surveillance system based on calcium and nitric oxide in marine diatoms[J].Plos Biology,2006,4(3):e60。

Claims

1. An algistat characterized by comprising 4- (di (1H-indol-3-yl) methyl) phenol (DIM-Ph-1' -OH) as an active ingredient.

2. The algistat according to claim 1, characterized in that it preferably contains DIM-Ph-1' "-OH in an amount of 0.35-2.0 μ g/mL.

3. The algistat according to claim 2, characterized in that it further preferably contains DIM-Ph-1' "-OH at 0.35, 0.5, 1.0, 2.0 μ g/mL.

The application of DIM-Ph-1' -OH in preparing an algistat.

5. Use according to claim 4, in particular for the preparation of a medicament for inhibiting the growth of Nitzschia closterium.

6. The use as claimed in any one of claims 4 to 5, wherein the algistat inhibits the proliferation of algal cells by increasing the roughness and weakening the mechanical strength of algal cell walls to reduce the kinds and number of adsorption groups of trace metals on the cell wall surface and interfere with the normal physiological activities of algae.

7. Use according to any one of claims 4 to 5, characterized in that the algistat is produced by causing intracellular Ca in algae cells²⁺The cell wall synthesis is blocked by efflux, and the algae inhibiting activity is exerted.