Application of solidago canadensis allelochemical in inhibition of heterosylvus rugosa

Abstract

The invention discloses an application of a solidago canadensis allelochemical in inhibition of heterosylvus rugosa, and belongs to the technical field of water pollution control. The solidago canadensis allelochemical comprises the following components: 30-45% of a flavonoid compound; 40-50% of an organic acid compound; 0.5 to 5 percent of terpenoids; and 10-25% of alkaloid. The solidago canadensis allelochemical can be used for treating and controlling the outbreak of the red-tide heterobending algae through allelopathy, has a remarkable inhibition effect on the growth of the red-tide heterobending algae, not only can be used for treating the red tide with the red-tide heterobending algae as a dominant species, but also can be used for turning the solidago canadensis into wealth, and has two purposes.

Description

Technical Field

[0001] The invention relates to the technical field of water pollution control, and in particular to application of Solidago canadensis allelopathic substances in inhibiting Heterosigma akashiwo Background Art

[0002] Solidago canadensis L., a perennial herbaceous plant of the Asteraceae family, is native to North America. It was introduced to Shanghai as a flower plant in 1935. After its introduction, it rapidly propagated and spread, resulting in reduced crop yields and quality degradation, destroying the local ecological balance and threatening local biodiversity. Turning Solidago canadensis L. into treasure can not only solve the harm of Solidago canadensis L., but also achieve efficient utilization of resources. Solidago canadensis L. can be used to extract antibacterial agents and natural pigments in addition to ornamental cut flowers, feed, etc. The Chinese patent document with publication number CN103800254A also discloses the application of Solidago canadensis L. in the preparation of whitening and anti-aging cosmetics. The Chinese patent document with publication number CN109122617A discloses a method for making grass carp fishing bait using Solidago canadensis L. In order to more effectively explore and make full use of the potential value of Solidago canadensis L., it is urgent to develop diversified application strategies.

[0003] In recent years, the excessive growth of algae has become one of the main challenges of aquatic ecological environment problems, especially in eutrophic water bodies. The rapid reproduction of algae not only affects the water quality, but also may cause algal blooms, produce a large amount of toxic substances, and endanger aquatic organisms and human health. Traditional algae control methods mainly rely on physical, chemical or biological means. However, these methods often have certain side effects. For example, the use of chemical agents may cause long-term pollution to the water ecology, and the effect of biological control measures may be unstable. Heterosigma akashiwo is a flagellate algae widely distributed in the world's coastal waters. It belongs to the plant kingdom and the genus Heterosigma. It is one of the most important red tide organisms and has formed red tides many times in many sea areas. It will go dormant at low temperatures. There are few algae inhibitors for Heterosigma akashiwo in the prior art. Therefore, it is urgent to develop inhibitors with excellent inhibitory effects on Heterosigma akashiwo.

[0004] Allelopathic substances have attracted widespread attention as a potential natural algae inhibitor. Different from traditional chemical agents, allelopathic substances are usually derived from plants, microorganisms or other natural substances, and have the advantages of being natural, green and low in toxicity. Allelopathic effect refers to the inhibition of the growth and development of other neighboring plants (including microorganisms and themselves) by plants through the secretion or release of chemical substances, thereby affecting the species diversity and stability in the ecosystem. As a plant with strong allelopathic effects, the allelopathic substances of Solidago canadensis in water bodies, especially the inhibitory effect on Heterosigma akashiwo, have not yet been fully developed and applied. Summary of the invention

[0005] In order to develop an inhibitor with excellent inhibitory effect on Heterosigma akashiwo, the present invention provides an application of Solidago canadensis allelopathic substances in inhibiting Heterosigma akashiwo, and natural allelopathic substances that can inhibit the growth of Heterosigma akashiwo are extracted from Solidago canadensis, which can not only control the red tide with Heterosigma akashiwo as the dominant species, but also turn Solidago canadensis into treasure, thus killing two birds with one stone.

[0006] The specific technical solutions adopted are as follows:

[0007] The application of allelopathic substances of Solidago canadensis in inhibiting Heterosigma akashiwo. The components of allelopathic substances of Solidago canadensis include:

[0008]

[0009] Furthermore, the flavonoid compounds include at least one of quercetin, luteolin, tamarind, astragaloside, psoralen, iris glycoside, kaempferol, catechin, and nobiletin; the organic acid compounds include at least one of caffeic acid, γ-aminobutyric acid, L-angesone, DL-phenylalanine, and cis-5,8,11,14,17-eicosapentaenoic acid; the terpene compounds include germacone; and the alkaloid compounds include trigonelline.

[0010] Canada solidago is an alien invasive species. The present invention uses the allelopathic substances extracted from Canada solidago as a growth inhibitor of Heterosigma akashiwo, and manages and controls the outbreak of Heterosigma akashiwo through allelopathic action, which can not only control the growth of Heterosigma akashiwo, but also turn Canada solidago from a pest into a treasure.

[0011] The present invention uses the allelopathic substance of Solidago canadensis to carry out an algae inhibition activity experiment on the dominant species of marine red tide, Heterosigma akashiwo, and finds that it has a significant inhibitory effect on the growth of Heterosigma akashiwo, especially at a higher concentration, the algae cells show a negative growth trend. The allelopathic substance is derived from natural plants, is easily degradable, has good ecological safety, and is suitable for use in natural water bodies, especially marine water bodies.

[0012] Specifically, the allelopathic substances of Solidago canadensis are extracted by the following method:

[0013] (1) Drying the Canada goosefoot to constant weight and then crushing it into 80-120 meshes;

[0014] (2) mixing the Canada solidago powder obtained in step (1) and an ethanol aqueous solution with a concentration of 70 to 85 wt %, and heating them in a water bath at 60 to 100° C. for 1 to 3 h, with a material-liquid mass volume ratio of 1 g:10 to 40 mL; after heating in a water bath, filtering the mixed solution and collecting the filtrate;

[0015] (3) removing the solvent from the filtrate to obtain an extract of the allelopathic substances in Solidago canadensis.

[0016] The extract obtained by the extraction method has a high content of active ingredients with inhibitory activity on the growth of Heterosigma akashiwo, has a high inhibitory activity on the growth of Heterosigma akashiwo, and the extraction method has a high extraction efficiency.

[0017] Furthermore, in step (1), at least one part of the Canada goosefoot is selected from leaves, stems and roots; and the Canada goosefoot is dried at 55-105° C. to a constant weight.

[0018] Too high or too low a mass percentage concentration of the ethanol aqueous solution will reduce the extraction efficiency. Preferably, in step (2), the mass percentage concentration of the ethanol aqueous solution is 70 to 80 wt%.

[0019] The mass volume ratio of the slurry to the liquid also has an important influence on the extraction rate of the allelopathic substances. Preferably, in step (2), the mass volume ratio of the slurry to the liquid is 1 g: 20-30 mL.

[0020] Appropriate water bath heating is conducive to the extraction of allelopathic substances in Canada goldenrod, but too high water bath temperature and too long water bath time are not conducive to the extraction of allelopathic substances. Too high water bath temperature will cause the allelopathic substances to be destroyed, and the volatilization of ethanol may also reduce the extraction rate of allelopathic substances; if the water bath time is too long, it will also increase the oxidation time of allelopathic substances in the air and reduce the extraction rate of active ingredients; preferably, the water bath heating temperature is 80-90°C, the water bath heating time is 1-2h; the number of water bath heating extractions is 1-3 times.

[0021] Furthermore, in step (2), the mixed solution is filtered using a 0.22-8 μm filter membrane and the filtrate is collected.

[0022] The present invention also provides the use of the allelopathic substance of Solidago canadensis in preventing and controlling red tide.

[0023] The present invention also provides a method for repairing a red tide water body, wherein the dominant species in the red tide water body is Heterosigma akashiwo, and the Solidago canadensis allelopathic substance is used and added to the red tide water body, so that the concentration of the Solidago canadensis allelopathic substance in the water body is greater than 0.05 g / L, preferably greater than or equal to 0.2 g / L, and most preferably is 0.25-2 g / L; when the concentration of the allelopathic substance in the water body increases, the growth inhibition effect on Heterosigma akashiwo shows an increasing trend; when the concentration of the allelopathic substance in the water body is 0.25-2 g / L, the growth of Heterosigma akashiwo can be completely inhibited in a short period of time, and the cost can be effectively controlled at the same time.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) The present invention uses natural plant allelopathic substances extracted from the invasive alien species Solidago canadensis as a new environmentally friendly growth inhibitor of Heterosigma akashiwo, and controls the outbreak of Heterosigma akashiwo through allelopathic action, thereby preventing and controlling the red tide caused by Heterosigma akashiwo. This can not only control the growth of Heterosigma akashiwo, but also turn Solidago canadensis from a pest into a treasure, thus killing two birds with one stone.

[0026] (2) The inhibition rate of Solidago canadensis allelopathic substances on Heterosigma akashiwo was very high, and the long-term inhibition effect was good at high concentrations, close to 100%;

[0027] (3) The allelopathic substances of Canada goldenrod are easily degradable, have no secondary pollution, are ecologically safe, and are green and environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the ultra high performance liquid chromatography (UHPLC) spectrum of the allelopathic substance extract prepared in Example 1.

[0029] Figure 2 This is a growth curve of the marine alga Heterosigma akashiwo when the water contains different concentrations of the allelochemicals extracted in Example 1.

[0030] Figure 3 The graph is a graph showing the inhibition rate of the marine alga Heterosigma akashiwo when the water contains different concentrations of the allelopathic substances extracted in Example 1.

[0031] Figure 4 This is a growth curve of the marine alga Heterosigma akashiwo when the water contains different concentrations of the allelochemicals extracted in Example 2.

[0032] Figure 5 The graph is a graph showing the inhibition rate of the marine alga Heterosigma akashiwo when the water contains different concentrations of the allelopathic substances extracted in Example 2. DETAILED DESCRIPTION

[0033] In order to make the purpose, features and advantages of the present invention more obvious and easy to understand, the following is a detailed description through specific embodiments. In the following description, many specific details are set forth in order to fully understand the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below. The technical features in each embodiment of the present invention can be combined accordingly without conflicting with each other.

[0034] The operating methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. The contents not described in detail in this specification belong to the prior art known to professionals in the field. The experimental materials used in the following examples, unless otherwise specified, can be purchased from conventional biochemical reagent companies.

[0035] The present invention explores the application of allelopathic substances extracted from Solidago canadensis in inhibiting Heterosigma akashiwo, in order to provide a green and environmentally friendly solution for the prevention and control of red tides with Heterosigma akashiwo as the dominant species.

[0036] Example 1

[0037] Firstly, the solidago canadensis (including stems and leaves) is placed at 105°C for 30 minutes to be sterilized, then dried at 55°C to a constant weight, crushed, and passed through 80 meshes to obtain solidago canadensis powder;

[0038] Weigh 60g of the above-mentioned Solidago canadensis powder and extract it with 70wt% ethanol aqueous solution, with a solid-liquid ratio of 1g:20mL. Mix 60g of Solidago canadensis powder with 1.2L 70wt% ethanol aqueous solution, place in a water bath at 80℃ for 2h, filter through a 0.45μm filter membrane, collect the filtrate, repeat the water bath heating extraction 3 times, and combine the Solidago canadensis extracts;

[0039] The solvent was removed from the Solidago canadensis extract by vacuum distillation using a rotary evaporator to obtain an extract; the extract was dissolved using 50% dimethyl sulfoxide, and dissolved in a 42°C water bath and with ultrasound to obtain a 1.0 g / mL allelopathic substance solution.

[0040] The extracted allelopathic substances were detected by UHPLC-Q-TOF MS. The UHPLC spectrum is shown in Figure 1 shown.

[0041] The UHPLC-Q-TOF MS instrument name and sample measurement parameters are as follows:

[0042] The samples were separated by Agilent 1290 Infinity LC ultra-high performance liquid chromatography system (UHPLC) HILIC column; column temperature 25°C; flow rate 0.5 mL / min; injection volume 2 μL; mobile phase composition A: water + 25 mM ammonium acetate + 25 mM ammonia, B: acetonitrile; gradient elution program is as follows: 0-0.5 min, 95% B; 0.5-7 min, B linearly changes from 95% to 65%; 7-8 min, B linearly changes from 65% to 40%; 8-9 min, B is maintained at 40%; 9-9.1 min, B linearly changes from 40% to 95%; 9.1-12 min, B is maintained at 95%; the samples were placed in the 4°C autosampler during the entire analysis process. In order to avoid the influence of fluctuations in instrument detection signals, the samples were analyzed continuously in a random order.

[0043] An AB Triple TOF 6600 mass spectrometer was used to collect the primary and secondary spectra of the samples.

[0044] The samples were separated by an Agilent 1290 Infinity LC ultra high performance liquid chromatography (UHPLC) system and analyzed by mass spectrometry using a Triple TOF 6600 mass spectrometer (AB SCIEX), using electrospray ionization (ESI) positive and negative ion modes for detection.

[0045] The ESI source setting parameters are as follows: nebulizer gas auxiliary heating gas 1 (Gas1): 60, auxiliary heating gas 2 (Gas2): 60, curtain gas (CUR): 30psi, ion source temperature: 600℃, spray voltage (ISVF) ±5500V (positive and negative modes); primary mass-to-charge ratio detection range: 60-1000Da, secondary product ion mass-to-charge ratio detection range: 25-1000Da, primary mass spectrometry scan accumulation time: 0.20s / spectra, secondary mass spectrometry scan accumulation time 0.05s / spectra; the secondary mass spectrum was obtained using the data-dependent acquisition mode (IDA), and the peak intensity value screening mode was used, declustering voltage (DP): ±60V (positive and negative modes), collision energy: 35±15eV, IDA settings are as follows: dynamic exclusion of isotope ion range: 4Da, 10 fragment spectra are collected for each scan.

[0046] The main components of allelopathic substances were analyzed and the results are shown in Table 1.

[0047] Table 1 Main components of allelopathic substances extracted from Solidago canadensis

[0048]

[0049] Example 2

[0050] First, the Canada solidago plants (including roots, stems, and leaves) were placed at 105° C. for 30 minutes to kill green, then dried at 55° C. to constant weight, crushed, and passed through 80 mesh.

[0051] Weigh 24g of the above-mentioned Solidago canadensis plant powder and extract it with 70wt% ethanol aqueous solution, with a solid-liquid ratio of 1g:30mL. Mix 24g of Solidago canadensis plant powder with 720mL of 70wt% ethanol aqueous solution, place in a water bath at 80℃ for 2h, filter through a 0.45μm membrane, and collect the filtrate as the Solidago canadensis extract.

[0052] The solvent was removed by vacuum distillation of the Solidago canadensis extract using a rotary evaporator to obtain an extract with a mass of 9.62 g. The extract was dissolved with 50% dimethyl sulfoxide, and dissolved in a 42°C water bath and with ultrasound to obtain a 1.0 g / mL allelopathic substance solution.

[0053] Example 3

[0054] The allelopathic substances of Solidago canadensis in Example 1 were diluted to 0.025, 0.05, 0.1, 0.2, 0.4, 0.8, and 1.0 g / mL using 50% dimethyl sulfoxide. Culture medium, algae species Heterosigma akashiwo in the logarithmic growth period, and allelopathic substance solution of Solidago canadensis were added to a 6-well plate, so that the concentrations of the experimental groups were 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, and 2 g / L, respectively. The experimental group with dimethyl sulfoxide was used as the solvent control group, and the culture system was 10 mL. The initial densities of Heterosigma akashiwo were 5×10 4 cells / mL. The culture medium used for Heterosigma akashiwo was f / 2 culture medium. The Heterosigma akashiwo algae were placed in a constant temperature and light incubator with the culture conditions of 25°C, 3000 Lux of light intensity, and 12:12 light-dark ratio. After activation inoculation twice, the algae growth cycle was adjusted to the logarithmic phase, counted, and set aside.

[0055] The algae cells were cultured for 7 days, and samples of the cultured algae cells were taken for testing every other day.

[0056] Calculation of algae inhibition rate:

[0057]

[0058] Among them C sample is the algae density of the experimental group, C control is the algae density of the control group.

[0059] The experimental results are as follows Figure 2 and Figure 3As shown. The results show that the allelopathic substances extracted from Solidago canadensis have a significant inhibitory effect on the marine algae Heterosigma akashiwo, and as the concentration increases, the inhibitory effect is strengthened and effective for a long time. Among them, the allelopathic substances extracted from Solidago canadensis have the strongest inhibitory effect on Heterosigma akashiwo. When the concentration of allelopathic substances exceeds 0.1g / L, it shows significant algae inhibition activity, especially in the late culture period, the inhibition rate is always close to 100%. And when the concentration of allelopathic substances exceeds 0.4g / L, the inhibition rate of Heterosigma akashiwo reaches 100%.

[0060] Example 4

[0061] The allelopathic substances of the whole plant of Solidago canadensis prepared in Example 2 were diluted to 0.125, 0.15, 0.2, 0.25, 0.5, and 0.1 g / mL using dimethyl sulfoxide solution. The tested algae species was Heterosigma akashiwo, and the other experimental steps and parameters were the same as in Example 3.

[0062] The experimental results are as follows Figure 4 and Figure 5 The results showed that under the action of the allelopathic substances of Solidago canadensis, the inhibition rate of algal cells was always very high; when the final concentration of the allelopathic substances was 0.25g / L, 0.3g / L, 0.4g / L, 0.5g / L, 1.0g / L and 2.0g / L, the allelopathic substances of the whole plant of Solidago canadensis had significant algae inhibition activity, and showed high inhibition effect in the early stage, and were effective for a long time, and the inhibition rate was always close to 100%; when the concentration was greater than or equal to 0.3g / L, the inhibition rate of Heterosigma akashiwo reached 100%.

[0063] The embodiments described above provide a detailed description of the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements or similar substitutions made within the scope of the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. The application of allelopathic substances of Solidago canadensis in inhibiting Heterosigma akashiwo, characterized in that: The components of allelopathic substances of Canada solidago include:

2. The use of the allelopathic substance of Solidago canadensis in inhibiting Heterosigma akashiwo according to claim 1, characterized in that: The flavonoid compounds include at least one of quercetin, luteolin, tamarind, astragaloside, psoralen, iris glycoside, kaempferol, catechin, and nobiletin; the organic acid compounds include at least one of caffeic acid, γ-aminobutyric acid, L-angesone, DL-phenylalanine, and cis-5,8,11,14,17-eicosapentaenoic acid; the terpene compounds include germacone; and the alkaloid compounds include trigonelline.

3. The use of the allelopathic substance of Solidago canadensis in inhibiting Heterosigma akashiwo according to claim 1, characterized in that: The allelopathic substance of Solidago canadensis is extracted by the following method: (1) Drying the Canada goosefoot to constant weight and then crushing it into 80-120 meshes; (2) mixing the Canada solidago powder obtained in step (1) and an ethanol aqueous solution with a concentration of 70 to 85 wt %, and heating them in a water bath at 60 to 100° C. for 1 to 3 h, with a material-liquid mass volume ratio of 1 g:10 to 40 mL; after heating in a water bath, filtering the mixed solution and collecting the filtrate; (3) removing the solvent from the filtrate to obtain the allelopathic extract of Solidago canadensis.

4. The use of the allelopathic substance of Solidago canadensis in inhibiting Heterosigma akashiwo according to claim 3, characterized in that: In step (1), at least one part of the Canada solidago is selected from leaves, stems and roots; and the Canada solidago is dried at 55-105° C. to a constant weight.

5. The use of the allelopathic substance of Solidago canadensis in inhibiting Heterosigma akashiwo according to claim 3, characterized in that: In step (2), the concentration of the ethanol aqueous solution is 70-80wt%; the mass volume ratio of the feed liquid is 1g:20-30mL.

6. The use of the allelopathic substance of Solidago canadensis in inhibiting Heterosigma akashiwo according to claim 3, characterized in that: In step (2), the water bath heating temperature is 80-90° C., the water bath heating time is 1-2 hours, and the water bath heating extraction times are 1-3 times.

7. The use of the allelopathic substance of Solidago canadensis in inhibiting Heterosigma akashiwo according to claim 3, characterized in that: In step (2), the mixed solution is filtered using a 0.22-8 μm filter membrane and the filtrate is collected.

8. Use of allelopathic substances of Solidago canadensis in preventing and controlling red tide, characterized in that: The components of allelopathic substances of Canada solidago include:

9. A method for repairing red tide water bodies, characterized in that: Utilize the allelopathic substances of Canada goldenrod, the components of the allelopathic substances of Canada goldenrod include:

10. The red tide water body restoration method according to claim 9, characterized in that: The dominant species in the red tide water body is Heterosigma akashiwo, and the allelopathic substance of Solidago canadensis is added into the red tide water body to make the concentration of the allelopathic substance of Solidago canadensis in the water body greater than 0.05 g / L.

Images