Application of allelochemicals from Solidago virgaurea in inhibiting Heterosigma akashiwo

Abstract

The present invention discloses the application of allelochemicals from Solidago virgaurea in inhibiting Heterosigma akashiwo, belonging to the technical field of water pollution control. The components of the allelochemicals from Solidago virgaurea include: 30-45% flavonoid compounds; 40-50% organic acid compounds; 0.5-5% terpene compounds; 10-25% alkaloids. The allelochemicals from Solidago virgaurea can control the outbreak of Heterosigma akashiwo through allelopathy, and have a significant inhibitory effect on the growth of Heterosigma akashiwo. It can not only control the red tide with Heterosigma akashiwo as the dominant species, but also turn Solidago virgaurea from waste into treasure, killing two birds with one stone.

Description

Technical Field

[0001] The present invention relates to the technical field of water pollution control, and particularly relates to the application of allelochemicals from Solidago canadensis in inhibiting Heterosigma akashiwo. Background Art

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

[0003] In recent years, the overgrowth of algae has become one of the main challenges in the water ecological environment problems. Especially in eutrophic waters, the rapid reproduction of algae not only affects water quality, but may also lead to the phenomenon of water blooms, producing a large amount of toxic substances, endangering 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 ecosystem, and the effects of biological control measures may be unstable. Heterosigma akashiwo is a flagellate alga widely distributed in the coastal waters of the world. It belongs to the plant kingdom and the genus Heterosigma, and is one of the main red tide organisms. It has formed red tides many times in multiple sea areas and will go dormant at low temperatures. There are few algicides for Heterosigma akashiwo in the prior art. Therefore, it is urgent to develop an inhibitor with excellent inhibitory effect on Heterosigma akashiwo.

[0004] Allelochemicals, as a potential natural algal inhibitor, have attracted extensive attention. Different from traditional chemical agents, allelochemicals usually originate from plants, microorganisms or other natural substances, and have the advantages of being natural, green and low-toxic. Allelopathy refers to the phenomenon that plants inhibit the growth and development of neighboring plants (including microorganisms and themselves) by secreting or releasing chemical substances, thereby affecting the species diversity and stability in the ecosystem. Solidago virgaurea, as a plant with strong allelopathic effects, the allelochemicals in its water body, especially the inhibitory effect on Heterosigma akashiwo, have not 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 the application of allelochemicals from Solidago virgaurea in inhibiting Heterosigma akashiwo. Extracting natural allelochemicals that can inhibit the growth of Heterosigma akashiwo from Solidago virgaurea can not only control the red tide dominated by Heterosigma akashiwo, but also turn Solidago virgaurea from waste to treasure, killing two birds with one stone.

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

[0007] The application of allelochemicals from Solidago virgaurea in inhibiting Heterosigma akashiwo, and the components of the allelochemicals from Solidago virgaurea include:

[0008]

[0009] Further, the flavonoid compounds include at least one of quercetin, luteolin, tamarixetin, astragalin, psoralidin, iridin, kaempferol, catechin, nobiletin; the organic acid compounds include at least one of caffeic acid, γ-aminobutyric acid, L-carnosine, DL-phenylalanine, cis-5,8,11,14,17-eicosapentaenoic acid; the terpene compounds include germacrone; the alkaloid compounds include trigonelline.

[0010] Solidago virgaurea belongs to an alien invasive species. The present invention uses the allelochemicals extracted from Solidago virgaurea as a growth inhibitor of Heterosigma akashiwo, and controls the outbreak of Heterosigma akashiwo through allelopathy, which can not only control the growth of Heterosigma akashiwo, but also turn Solidago virgaurea from a harmful thing into a useful resource.

[0011] The present invention conducts an experiment on the algicidal activity of allelochemicals from Solidago virgaurea against Heterosigma akashiwo, the dominant species of marine red tide, and finds that it has a significant inhibitory effect on the growth of Heterosigma akashiwo. Especially at higher concentrations, the algal cells show a negative growth trend. This allelochemical 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 allelochemicals of Solidago virgaurea are obtained by the following method:

[0013] (1) Dry Solidago virgaurea to constant weight and then crush it to 80 - 120 mesh;

[0014] (2) Mix the Solidago virgaurea powder obtained in step (1) evenly with an ethanol aqueous solution with a concentration of 70 - 85 wt%, heat it in a water bath at 60 - 100 °C for 1 - 3 h, and the mass - to - volume ratio of the material to the liquid is 1 g:10 - 40 mL; after water - bath heating, filter the mixture and collect the filtrate;

[0015] (3) Remove the solvent from the filtrate to obtain the extract of the allelochemicals in Solidago virgaurea.

[0016] The content of the active ingredient with inhibitory activity against the growth of Heterosigma akashiwo in the extract obtained by the above extraction method is relatively high, the inhibitory activity against the growth of Heterosigma akashiwo is relatively high, and the extraction efficiency of the above extraction method is relatively high.

[0017] Further, in step (1), at least one part of leaves, stems, and roots of Solidago virgaurea is selected; Solidago virgaurea is dried to constant weight at 55 - 105 °C.

[0018] If the mass percentage concentration of the ethanol aqueous solution is too high or too low, the extraction efficiency will be reduced. Preferably, in step (2), the mass percentage concentration of the ethanol aqueous solution is 70 - 80 wt%.

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

[0020] Appropriate water - bath heating helps the leaching of allelochemicals in Solidago virgaurea, but too high a water - bath temperature and too long a water - bath time are adverse to the leaching of allelochemicals. Too high a water - bath temperature will cause the allelochemicals to be damaged, and the volatilization of ethanol may also reduce the extraction rate of allelochemicals; if the water - bath time is too long, it will also increase the oxidation time of allelochemicals 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 - 2 h; the number of water - bath heating extractions is 1 - 3 times.

[0021] Further, in step (2), filter the mixture with a filter membrane of 0.22 - 8 μm and collect the filtrate.

[0022] The present invention also provides the use of the allelochemicals of Solidago virgaurea in preventing and controlling red tides.

[0023] The present invention also provides a method for restoring red tide water bodies. The dominant species in the red tide water bodies is Heterosigma akashiwo. By using the allelopathic substances of Solidago canadensis, the allelopathic substances of Solidago canadensis are added to the red tide water bodies, so that the concentration of the allelopathic substances of Solidago canadensis in the water bodies > 0.05 g / L, preferably ≥ 0.2 g / L, and most preferably 0.25 - 2 g / L; when the concentration of the allelopathic substances in the water bodies increases, the growth inhibition effect on Heterosigma akashiwo shows an increasing trend; when the concentration of the allelopathic substances in the water bodies is 0.25 - 2 g / L, the growth of Heterosigma akashiwo can be completely inhibited in the short term, and the cost can be effectively controlled at the same time.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] (1) The present invention uses the natural plant allelopathic substances extracted from the alien invasive species Solidago canadensis as a new type of environmentally friendly growth inhibitor for Heterosigma akashiwo, and controls the outbreak of Heterosigma akashiwo through allelopathy to prevent the red tide caused by it. It can not only control the growth of Heterosigma akashiwo, but also turn the harm of Solidago canadensis into a treasure, killing two birds with one stone;

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

[0027] (3) The allelopathic substances of Solidago canadensis are easily degradable, have no secondary pollution, have good ecological safety, and are green and environmentally friendly. Description of the Drawings

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

[0029] Figure 2 It is the growth curve of the marine alga Heterosigma akashiwo when the water bodies contain different concentrations of the allelopathic substances extracted in Example 1.

[0030] Figure 3 It is the inhibition rate curve of the marine alga Heterosigma akashiwo when the water bodies contain different concentrations of the allelopathic substances extracted in Example 1.

[0031] Figure 4 It is the growth curve of the marine alga Heterosigma akashiwo when the water bodies contain different concentrations of the allelopathic substances extracted in Example 2.

[0032] Figure 5 It is the inhibition rate curve of the marine alga Heterosigma akashiwo when the water bodies contain different concentrations of the allelopathic substances extracted in Example 2. Detailed Embodiments

[0033] To make the objectives, features, and advantages of the present invention more apparent and understandable, the following provides a detailed description through specific embodiments. A number of specific details are set forth in the following description in order to provide a thorough understanding of 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 departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below. The technical features in various embodiments of the present invention can be combined accordingly without conflict.

[0034] For the operating methods of the following embodiments without specific conditions noted, they are generally in accordance with conventional conditions or the conditions recommended by the manufacturer. The content not described in detail in this specification belongs to the prior art well-known to those skilled in the art. The experimental materials used in the following embodiments, unless otherwise specified, can be obtained from conventional biochemical reagent companies.

[0035] The present invention explores the application of allelochemicals extracted from Solidago virgaurea in inhibiting Heterosigma akashiwo, with the expectation of providing a green and environmentally friendly solution for the prevention and control of red tides dominated by Heterosigma akashiwo.

[0036] Example 1

[0037] First, Solidago virgaurea (including stems and leaves) was blanched at 105°C for 30 minutes, then dried at 55°C to constant weight, pulverized, and passed through a 80-mesh sieve to obtain Solidago virgaurea powder.

[0038] 60 g of the above-mentioned Solidago virgaurea powder was weighed and extracted with an ethanol aqueous solution with a mass percentage concentration of 70 wt%, and the solid-liquid ratio was 1 g:20 mL. 60 g of Solidago virgaurea powder was mixed with 1.2 L of 70 wt% ethanol aqueous solution, heated in a water bath at 80°C for 2 h, filtered through a 0.45-μm filter membrane, and the filtrate was collected. The water bath heating extraction was repeated 3 times, and the Solidago virgaurea extract was combined.

[0039] The Solidago virgaurea extract was subjected to vacuum distillation using a rotary evaporator to remove the solvent, obtaining an extract; the extract was dissolved with 50% dimethyl sulfoxide, and dissolved in a water bath at 42°C with ultrasonic assistance to obtain a 1.0 g / mL allelochemical solution.

[0040] The extracted allelochemicals were detected by UHPLC-Q-TOF MS, and the UHPLC chromatogram is as Figure 1 shown.

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

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

[0043] The AB Triple TOF 6600 mass spectrometer was used to collect the first-level and second-level spectra of the samples.

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

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

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

[0047] Table 1 Main components of allelochemicals extracted from Solidago virgaurea

[0048]

[0049] Example 2

[0050] First, place the Solidago virgaurea plant (including roots, stems, and leaves) in an oven at 105 °C for 30 minutes for fixation, then place it in an oven at 55 °C for drying to a constant weight, crush it, and pass it through a 80-mesh sieve.

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

[0052] Use a rotary evaporator to perform vacuum distillation on the Solidago virgaurea extract to remove the solvent, obtaining an extract with a mass of 9.62 g. Dissolve the extract with 50% dimethyl sulfoxide, and dissolve it in a water bath at 42 °C with ultrasonic assistance to obtain an allelochemical solution with a concentration of 1.0 g / mL.

[0053] Example 3

[0054] Dilute the allelochemicals of Solidago virgaurea in Example 1 with 50% dimethyl sulfoxide to 0.025, 0.05, 0.1, 0.2, 0.4, 0.8, and 1.0 g / mL. Add the culture medium, the algal species Heterosigma akashiwo in the logarithmic growth phase, and the Solidago virgaurea allelochemical solution to a 6-well plate, so that the concentrations of the experimental groups are 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, and 2 g / L respectively. The experimental group with added dimethyl sulfoxide is used as the solvent control group, and the culture system is 10 mL. The initial density of the Heterosigma akashiwo algal species is 5×10 4 cells / mL. The culture medium used for Heterosigma akashiwo is f / 2 medium. Place the Heterosigma akashiwo algal species in a constant-temperature light incubator, and the culture conditions are 25 °C, a light intensity of 3000 Lux, and a light-dark ratio of 12:12. After activation and inoculation twice, adjust the algal growth cycle to the logarithmic phase, count the cells, and set aside for use.

[0055] Cultivate the algal cells for 7 days, and sample and detect the cultured algal cells every other day.

[0056] Calculation of algal inhibition rate:

[0057]

[0058] where C sample is the algal density of the experimental group, and C control is the algal density of the control group.

[0059] The experimental results are as shown in Figure 2 and Figure 3As shown in the figure. The results show that the allelochemicals extracted from Solidago virgaurea have a significant inhibitory effect on Heterosigma akashiwo, and with the increase of concentration, the inhibitory effect is enhanced and effective in the long term. Among them, the allelochemicals extracted from Solidago virgaurea have the strongest inhibitory effect on Heterosigma akashiwo. When the concentration of allelochemicals exceeds 0.1 g / L, significant algicidal activity is exhibited, especially in the later stage of cultivation, and the inhibition rate is always close to 100%. And when the concentration of allelochemicals exceeds 0.4 g / L, the inhibition rate on Heterosigma akashiwo reaches 100%.

[0060] Example 4

[0061] The whole-plant allelochemicals of Solidago virgaurea prepared in Example 2 were diluted to 0.125, 0.15, 0.2, 0.25, 0.5, and 0.1 g / mL with dimethyl sulfoxide solution. The test algal species was Heterosigma akashiwo, and other experimental steps and parameters were the same as those in Example 3.

[0062] The experimental results are as Figure 4 and Figure 5 shown in the figure. The results show that under the action of the allelochemicals of Solidago virgaurea, the inhibition rate of algal cells is always very high; when the final concentrations of the allelochemicals are 0.25 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 1.0 g / L, and 2.0 g / L, the whole-plant allelochemicals of Solidago virgaurea have significant algicidal activity, and show a high inhibition effect in the initial stage, are effective in the long term, and the inhibition rate is always close to 100%; when the concentration is greater than or equal to 0.3 g / L, the inhibition rate on Heterosigma akashiwo reaches 100%.

[0063] The above-described embodiments have described the technical solutions of the present invention in detail. It should be understood that the above are only specific embodiments of the present invention and are not used to limit the present invention. Any modifications, supplements, or substitutions in a similar manner 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: Flavonoids 30-45%; Organic acid compounds 40-50%; Terpenoids 0.5% to 5%; Alkaloids 10-25%; The allelopathic substance of Solidago canadensis is extracted by the following method: (1) Dry the Canada goosefoot to constant weight and then crush it into 80-120 mesh; (2) The Canada solidago powder obtained in step (1) and an ethanol aqueous solution with a concentration of 70 to 85 wt% are mixed evenly, and heated 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, the mixed solution is filtered and the filtrate is collected; (3) removing the solvent from the filtrate to obtain a Solidago canadensis allelopathic substance extract; When the allelopathic substances of Solidago canadensis inhibit Heterosigma akashiwo, the concentration of the allelopathic substances of Solidago canadensis in the water body is greater than 0.05g / L.

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: 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 to 105° C. to a constant weight.

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

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

6. The use of the allelopathic substance of Solidago canadensis in inhibiting Heterosigma akashiwo according to claim 1, characterized in that: In step (2), the mixed solution is filtered through a 0.22-8 μm filter membrane and the filtrate is collected.

7. 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: Flavonoids 30-45%; Organic acid compounds 40-50%; Terpenoids 0.5% to 5%; Alkaloids 10-25%; The dominant species in the red tide water body is Heterosigma akashiwo, and the allelopathic substance of Solidago canadensis is added to the red tide water body, so that the concentration of the allelopathic substance of Solidago canadensis in the water body is greater than 0.05 g / L; The allelopathic substance of Solidago canadensis is extracted by the following method: (1) Dry the Canada goosefoot to constant weight and then crush it into 80-120 mesh; (2) The Canada solidago powder obtained in step (1) and an ethanol aqueous solution with a concentration of 70 to 85 wt% are mixed evenly, and heated 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, the mixed solution is filtered and the filtrate is collected; (3) The solvent in the filtrate is removed to obtain the allelopathic extract of Solidago canadensis.

Images