Application of baicalin in relieving copper toxicity of duckweed

Abstract

The invention discloses application of baicalin in relieving copper toxicity of duckweed, and belongs to the technical field of plant adversity physiology and heavy metal pollution remediation. Systematic research on the physiological regulation mechanism of baicalin on duckweed under copper stress innovatively discovers that the relative growth rate of duckweed can be remarkably increased through treatment of baicalin; according to the application, the chlorophyll synthesis level is effectively recovered, the photosynthesis inhibition effect caused by copper toxicity is fundamentally improved, it is proved that the threshold concentration for effectively relieving the copper toxicity is 400 [mu] M, the optimal action concentration window is 200-800 [mu] M, and the concentration interval can achieve synergistic antagonism on the toxicity of copper ions by adjusting the antioxidant metabolic pathway of duckweed. And a green and efficient new strategy is provided for heavy metal polluted water body remediation. The method is not only suitable for ecological restoration engineering of copper-polluted water areas, but also can be expanded and applied to cultivation of stress-resistant varieties of aquatic economic plants, and has important theoretical value and practical application prospect in improvement of heavy metal tolerance of stressed plants.

Description

Application of baicalin in alleviating copper toxicity in duckweed Technical Field

[0001] This invention belongs to the field of plant stress physiology and heavy metal pollution remediation technology, specifically involving the application of baicalin in alleviating copper toxicity of duckweed in water. Background Technology

[0002] Baicalin (chemical formula: C) 21 H 18 O 11 4',5,7-trihydroxyflavonoid-7-O-glucuronide is a typical flavonoid compound, mainly found in medicinal plants such as Scutellaria baicalensis and Lonicera japonica. Its molecular structure contains 4',5,7-trihydroxyflavonoid-7-O-glucuronide, and multiple phenolic hydroxyl groups and a conjugated system endow it with excellent metal chelating ability (copper chelating stability constant logK = 12.3) and free radical scavenging activity. Recent studies have found that it can participate in heavy metal detoxification by regulating the expression of metallothionein genes, but its application in aquatic plant remediation remains a blank.

[0003] As a polyphenolic natural product, baicalin exhibits three functional advantages in heavy metal stress response: 1. It can chelate Cu through the structure-specific chelation of catechol. 2+ 1. Forms stable complexes; 2. Activates the phytochelatin synthesis pathway to enhance heavy metal compartmentalization; 3. Regulates MAPK signal transduction to alleviate oxidative damage. Although it has been widely used in drug development, this patent innovatively constructs its "in vitro chelation-in vivo metabolism" biphasic detoxification model, realizing for the first time the engineering application of flavonoids in the remediation of copper pollution in aquatic plants.

[0004] In recent years, the accelerated pace of industrialization and urbanization, along with the large-scale development of mineral resources, has led to a significant influx of metal and heavy metal ions into the aquatic environment through wastewater and waste residue, causing global water pollution problems. Among these, copper (Cu) is a major contributor. 2+ Copper, as a typical highly toxic metallic pollutant, causes serious damage to aquatic ecosystems due to its persistence, bioaccumulation, and difficulty in degradation, and can threaten human health through the food chain. The main sources of copper pollution include dust and copper-containing wastewater generated during copper mining and smelting, and high-concentration copper-containing wastewater discharged from metal processing industries (such as electroplating, metallurgy, and electronics manufacturing). The copper concentration in this wastewater can typically reach 50-500 mg / L, far exceeding the safe threshold for water bodies.

[0005] Copper has a particularly significant toxic effect on aquatic plants. Studies have shown that copper ions can inhibit normal physiological functions by disrupting plant cell membrane integrity, interfering with enzyme activity, and gene expression. For aquatic model plants such as duckweed (Lemna minor), which are highly sensitive to heavy metals, copper stress directly inhibits the division of root tip meristem cells, leading to impaired root development and a sharp reduction in absorption area. Simultaneously, excessive copper induces a burst of reactive oxygen species (ROS), causing damage to the chloroplast membrane system, disintegration of thylakoid structures, degradation of photosynthetic pigments, and a decrease in photosystem II (PSII) electron transport efficiency, ultimately manifesting as leaf yellowing, reduced biomass accumulation, and even plant death. Furthermore, copper interferes with the nitrogen and phosphorus metabolism pathways of duckweed, inhibits the activity of antioxidant enzymes, exacerbates oxidative damage, and leads to population decline and imbalance in aquatic ecological functions.

[0006] Currently, mitigation methods for copper stress in duckweed mainly focus on traditional approaches such as physical adsorption, chemical precipitation, or microbial remediation. However, these methods suffer from high costs, potential for secondary pollution, and long remediation cycles. Plant-derived natural bioactive substances, due to their environmental friendliness and biocompatibility, show potential in the regulation of heavy metal stress. However, systematic research reports have not yet been published on whether flavonoids such as baicalin can alleviate copper toxicity by regulating the antioxidant defense system of duckweed, chelating free copper ions, or repairing photosynthetic structures. Therefore, developing baicalin-based technologies for mitigating copper stress in duckweed is of great significance for aquatic ecological restoration and sustainable pollution control. Summary of the Invention

[0007] In view of the above-mentioned prior art, the purpose of this invention is to provide the application of baicalin in alleviating copper toxicity in duckweed.

[0008] The technical solution of the present invention is as follows:

[0009] In a first aspect, baicalin provides applications in antioxidant defense:

[0010] I. Scavenging Reactive Oxygen Species (ROS): Copper stress induces excessive accumulation of reactive oxygen species (ROS) in duckweed cells, damaging biomolecules such as lipids, proteins, and nucleic acids. Baicalin, a natural flavonoid compound, can directly scavenge hydroxyl radicals (·OH) and superoxide anions (O2). - Free radicals such as SOD, POD, and CAT can be neutralized or the endogenous antioxidant enzyme system of duckweed can be activated to reduce oxidative damage.

[0011] II. Protecting chloroplast structure: Baicalin reduces chloroplast membrane system damage and thylakoid structure disintegration caused by copper stress by inhibiting lipid peroxidation, thereby maintaining the stability of photosynthetic pigments (chlorophyll a / b, carotenoids) and improving light capture efficiency.

[0012] In the above application, the duckweed species is *Lycoris radiata*.

[0013] In the above applications, the effective concentration of copper ions in the water is 12.5-200 μM.

[0014] In the above applications, the working concentration of baicalin is 200-800 μM.

[0015] A second aspect of the present invention provides baicalin for copper ion chelation and detoxification:

[0016] In the above applications, baicalin is achieved through the following pathway:

[0017] I. Direct binding with free copper ions: The phenolic hydroxyl and carboxyl functional groups in baicalin molecules can bind with free copper ions in water. 2+ A coordination reaction occurs, forming a stable copper-baicalin complex, which reduces the absorption and translocation of copper by duckweed roots and decreases the accumulation of copper in cells. Through this chelation effect, the concentration of available copper in the water can be significantly reduced, thereby mitigating the biotoxicity of copper to duckweed.

[0018] II. Inhibition of copper transmembrane transport: By regulating the expression of metal transport proteins (such as NRAMP and HMA family) on the duckweed cell membrane, the passive diffusion of copper ions into the cytoplasm is restricted, thereby reducing the intracellular copper toxicity level. In addition, baicalin can also enhance the intracellular copper ion compartmentalization ability by regulating the expression of metallothionein (such as MT2) genes, thereby further improving the detoxification efficiency of duckweed against copper.

[0019] A third aspect of the present invention provides the application of baicalin in repairing the function of the photosynthetic system:

[0020] In the above applications, baicalin achieves repair and protection through the following pathways:

[0021] I. Restoring Photosystem II (PSII) Activity: Baicalin maintains the integrity of photosystem II reaction centers (such as D1 protein), maintains the patency of the electron transport chain (ETR), improves photosynthetic electron transport efficiency (Fv / Fm, ΦPSII), and alleviates photosynthetic inhibition caused by copper stress by protecting the integrity of photosystem II reaction centers (such as D1 protein).

[0022] II. Promoting photosynthetic carbon assimilation: By upregulating RuBisCO enzyme activity and Calvin cycle-related gene expression, it enhances the ability of duckweed to accumulate photosynthetic assimilates and improves the energy metabolism imbalance under copper stress.

[0023] In a fourth aspect, the invention provides the application of baicalin in promoting duckweed growth and regulating stress resistance:

[0024] In the above applications, baicalin achieves regulation through the following pathways:

[0025] I. Alleviating the inhibition of copper on roots: Baicalin promotes the elongation of duckweed roots and the development of lateral roots by activating genes related to the division of root tip meristem cells (such as CycB and CDKB), thereby enhancing its ability to absorb water and nutrients.

[0026] II. Regulation of hormone signaling pathways: Baicalin can regulate the synthesis and distribution of endogenous hormones in duckweed (such as auxin IAA and cytokinin CTK), and antagonize the obstruction of auxin polar transport caused by copper stress, thereby improving phenotypes such as plant dwarfing and leaf deformity.

[0027] In a fifth aspect, the invention provides the application of baicalin in terms of its potential for eco-synergistic restoration:

[0028] In the above applications, baicalin is achieved through the following pathways:

[0029] 1. Reduce the risk of secondary pollution: Unlike chemical chelating agents (such as EDTA), baicalin is a plant-derived substance that is easily degraded by the environment, avoiding secondary pollution of water bodies caused by chelating agent residues in traditional remediation technologies.

[0030] II. Enhance duckweed's remediation capacity: By increasing the survival rate and biomass of duckweed in copper-contaminated waters, its efficiency in adsorbing and enriching copper as a "water scavenger" is indirectly enhanced, forming a synergistic effect of "detoxification-remediation".

[0031] The beneficial effects achieved by the present invention using the above technical solution are as follows:

[0032] In this invention, baicalin not only directly reduces the bioavailability of copper through chemical chelation, but also enhances the duckweed's own stress resistance by regulating its physiological metabolic network (antioxidant, photosynthesis, hormones), achieving the dual goals of "exogenous detoxification and endogenous remediation". Baicalin reduces the effective copper concentration by forming stable chelates and enhances intracellular compartmentalization by upregulating the expression of metallothionein genes, thus synergistically alleviating copper toxicity at the molecular level. Compared with traditional physicochemical remediation methods, this strategy is environmentally friendly, low-cost, and highly efficient, providing an innovative solution for the ecological treatment of copper-contaminated water bodies. Attached Figure Description

[0033] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:

[0034] Figure 1 shows the effect of baicalin on duckweed relief at a concentration of 50 μM Cu within the effective concentration range of the present invention. 2+ A diagram illustrating the effects of coercion.

[0035] Figure 2 shows the effect of baicalin on the fresh weight of duckweed under 50 μM Cu2+ stress within the effective concentration range of the present invention.

[0036] Figure 3 shows the effect of baicalin on the root length of duckweed under 50 μM Cu2+ stress within the effective concentration range of the present invention.

[0037] Figure 4 shows the effect of baicalin on the number of leaves of duckweed under 50 μM Cu2+ stress within the effective concentration range of the present invention.

[0038] Figure 5 shows the effect of baicalin on MDA formation in duckweed under copper stress according to the present invention.

[0039] Figure 6 shows the effect of baicalin on the CAT activity of duckweed under copper stress according to the present invention.

[0040] Figure 7 shows the effect of baicalin on the SOD activity of duckweed under copper stress according to the present invention.

[0041] Figure 8 shows the effect of baicalin on the XOD activity of duckweed under copper stress according to the present invention.

[0042] Figure 9 shows the effect of baicalin on the POD activity of duckweed under copper stress according to the present invention.

[0043] Figure 10 (a), (b), and (c) show the effects of baicalin on the chlorophyll a, chlorophyll b, and total chlorophyll content of duckweed under copper stress according to the present invention. Detailed Implementation

[0044] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0045] The experimental materials used in the embodiments of this invention are all conventional experimental materials in the art, and can be purchased through commercial channels. Experimental methods without specified detailed conditions are performed according to conventional experimental methods or the supplier's recommended operating instructions.

[0046] Baicalin was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product number B110210.

[0047] Test plants: Duckweed

[0048] Test medium: Hoagland medium

[0049] As shown in Figures 1-10:

[0050] Example 1: Concentration screening experiment of baicalin to alleviate copper toxicity in duckweed

[0051] 1. Test method:

[0052] Healthy, unpolluted duckweed (Lemna minor) was selected and placed in Hoagland nutrient solution containing 50 μM CuSO4 to simulate a moderate copper-polluted environment. Simultaneously, in addition to copper stress, 100, 200, 400, 800, and 1600 μM baicalin were added, respectively, under the cultivation conditions of a light intensity of 120 μmol·m⁻¹. -2 ·s -1 The duckweed was cultured in a photoperiod of 16 h / 8 h (day / night) and a temperature of 25±1℃ for 7 days until it reached a stable growth state. Each group had 3 biological replicates. After 7 days of treatment, the number of leaves, fresh weight, root length and relative growth rate (RGR) of duckweed were measured.

[0053] 2. Test Results:

[0054] After 7 days of treatment using the above method, the number of leaves, fresh weight, root length, and relative growth rate (RGR) of duckweed were measured. 400 μM was found to be the optimal mitigation threshold. At this threshold, the number of leaves recovered to 88% of the control group, and the RGR increased to 32.8%. The optimal concentration range for 200-800 μM baicalin to alleviate copper toxicity in duckweed was determined. At the optimal mitigation threshold of 100 μM, fresh weight increased, and root length recovered to 80% of normal levels.

[0055] Example 2: Regulation of photosynthetic system and antioxidant capacity of duckweed by baicalin

[0056] Test method:

[0057] Three treatment groups were set up: a control group (no copper, no baicalin), a copper stress group (10 μM CuSO4), and a copper stress + 400 μM baicalin group (10 μM CuSO4 + 400 μM baicalin). The culture conditions were set at a light intensity of 120 μmol·m⁻¹. -2 ·s -1 The duckweed was cultured in a photoperiod of 16 h / 8 h (day / night) and a temperature of 25±1℃ for 2 h, and the transient response was measured.

[0058] Methods for determining chlorophyll content: Colorimetric method; Chlorophyll is widely present in green plant tissues and is an organelle for photosynthesis. Its content is closely related to photosynthesis and nutritional status, and is an important indicator of plant growth. Chlorophyll a and chlorophyll b have maximum absorption at 645 nm and 663 nm, respectively. The contents of chlorophyll a, chlorophyll b, and total chlorophyll can be calculated using empirical formulas.

[0059] Membrane lipid peroxidation assay (MDA): Micro-method; Oxygen free radicals act on unsaturated fatty acids in lipids to generate lipid peroxides; these peroxides gradually decompose into a series of complex compounds, including malondialdehyde (MDA). The level of lipid oxidation can be detected by measuring the level of MDA. Under acidic and high-temperature conditions, MDA can condense with thiobarbituric acid (TBA) to form a brownish-red trimethylolpropionate (3,5,5-trimethyloxazol-2,4-dione), with a maximum absorption wavelength at 532 nm. Colorimetric analysis can then be used to estimate the content of lipid peroxides in the sample.

[0060] Methods for determining antioxidant enzyme activity:

[0061] (1) Superoxide dismutase (SOD) assay: colorimetric method; SOD is a metalloenzyme widely present in organisms and an important oxygen free radical scavenger. It can catalyze the dismutation of superoxide anions to generate H2O2 and O2. SOD is not only a superoxide anion scavenging enzyme but also a major H2O2 generating enzyme, playing an important role in biological antioxidant systems. Superoxide anions (O2) are generated through the xanthine and xanthine oxidase reaction system. - O2 - It can reduce nitroblue tetrazolium to form blue formazan, which has an absorption at 560 nm; SOD can scavenge O2. - This inhibits the formation of formazan; the deeper the blue color of the reaction solution, the lower the SOD activity, and vice versa.

[0062] (2) Peroxidase (POD) assay: colorimetric method; POD is widely found in animals, plants, microorganisms and cultured cells. It can catalyze the oxidation of phenolic and amine compounds by hydrogen peroxide and has the dual function of eliminating the toxicity of hydrogen peroxide and phenols and amines. POD catalyzes the oxidation of specific substrates by H2O2 and has characteristic light absorption at 470 nm.

[0063] (3) Catalase (CAT) assay: micro-method; CAT is widely found in animals, plants, microorganisms and cultured cells, and is the most important H2O2 scavenging enzyme, playing a crucial role in reactive oxygen species scavenging systems.

[0064] The key is that H2O2 has a characteristic absorption peak at 240 nm. CAT can decompose H2O2, causing the absorbance of the reaction solution at 240 nm to decrease with reaction time. The activity of CAT can be calculated based on the rate of change of absorbance.

[0065] (4) Xanthine oxidase (XOD) assay: Ultraviolet colorimetric method; XOD catalyzes the oxidation of hypoxanthine to produce xanthine and superoxide anion, which is one of the main sources of reactive oxygen species; it is also one of the key enzymes in nucleotide metabolism. XOD is mainly distributed in the heart, lungs, liver and other tissues of mammals. When liver function is impaired, a large amount of XOD is released into the serum, which has specific significance for the diagnosis of liver damage. XOD catalyzes the production of superoxide anion from hypoxanthine, and the superoxide anion reacts with hydroxylamine hydrochloride to produce NO. 2- NO 2- Under the action of p-aminobenzenesulfonamide and naphthylethylenediamine hydrochloride, a purple-red azo compound is generated, which has a characteristic absorption peak at 530 nm. The amount generated can reflect the magnitude of XOD activity.

[0066] Microscopic observation: Select healthy duckweed plants and gently rinse them with distilled water to remove surface impurities, avoiding mechanical damage. Immerse the duckweed in a pre-prepared DAB staining solution (0.1% DAB dissolved in pH 3.8 Tris-HCl buffer, containing 0.05% Tween-20 to enhance osmosis) and incubate at room temperature in the dark for 2 hours, allowing DAB and H2O2 to form a brown precipitate under the catalysis of peroxidase. After incubation, remove the duckweed, rinse it three times with distilled water to terminate the reaction, and then transfer it to a decolorizing solution (95% ethanol and lactic acid mixed in a 1:1 ratio). Decolorize in a 40°C water bath in the dark to remove chlorophyll and other background pigments. Spread the decolorized duckweed evenly on a glass slide, add glycerol or mounting medium, and gently cover with a coverslip to avoid air bubbles. Observe immediately under an optical microscope. Areas rich in H2O2 appear as dark brown particles or patches. Record images using bright-field or differential interference (DIC) mode, focusing on signals at leaf margins, stomata, or damaged areas.

[0067] Experimental results:

[0068] Based on the above methods, the chlorophyll content, membrane lipid peroxidation (MDA), activities of various antioxidant enzymes, and microscopic observation results of duckweed were measured. It was found that under the optimal relief threshold of 400 μM baicalin, the chlorophyll a and b contents in the treatment group recovered to 73% and 92% of the control group, respectively, and the total chlorophyll recovered to 88.6% of the control group. The experiment confirmed that baicalin reduces reactive oxygen species in a dose-dependent manner, inhibiting the formation of membrane lipid peroxidation product MDA by 89.5%, while simultaneously activating superoxide dismutase (SOD) activity to 43.2% of the control group, peroxidase (POD) to 73.3% of the control group, and catalase (CAT) activity to 65.4% of the control group. Baicalin (400 μM) inhibited XOD activity to 93% of the copper stress group and reduced superoxide anion (O2) levels. - Excessive generation of ).

[0069] Example 3: Practical Application of Baicalin in the Remediation of Copper-Contaminated Water

[0070] Test method:

[0071] In polluted waters with copper ion concentrations of 8-12 μM (such as electroplating wastewater treatment ponds), the dosage is 1 kg fresh weight of duckweed / 10 m³. 2 Duckweed was introduced at the water density, and 200-800 μM baicalin (soluble in water) was added simultaneously. The duckweed biomass and copper concentration in the water were monitored every 7 days, and the treatment was carried out continuously for 21 days.

[0072] Experimental results:

[0073] After 21 days of treatment, the cumulative fresh weight of duckweed reached three times the initial value; the copper ion removal rate in the baicalin combined with duckweed treatment group increased to 79%; Example 4: Baicalin pretreatment enhances the stress resistance of duckweed.

[0074] Test method:

[0075] Baicalin was dissolved in Hoagland nutrient solution at a concentration of 1:1000. After culturing duckweed in the pretreatment solution for 24 hours, it was transferred to a stress environment containing 10 μM CuSO4 and cultured for another 5 days.

[0076] Experimental results:

[0077] The results showed that the RGR of duckweed in the pretreated group was 32.8% of that in the control group;

[0078] Meanwhile, after pretreatment, SOD increased by 43.2% compared with the direct stress group, CAT activity increased by 1.3 times, and ROS accumulation decreased by 43%.

[0079] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, material, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, material, or apparatus.

[0080] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. The application of baicalin in alleviating copper toxicity in duckweed, characterized in that, The polyphenol coordination of flavonoids enables the synergistic repair of heavy metal detoxification and plant physiological functions.

2. The application of baicalin according to claim 1 in alleviating copper toxicity in duckweed, characterized in that, The baicalin described herein has the effect of alleviating copper toxicity in the concentration range of 200–800 μmol / L, and its bioavailability is the highest at 400 μmol / L.

3. The application of baicalin according to claim 1 in alleviating copper toxicity in duckweed, characterized in that, The application achieves the following three-dimensional repair effects: (1) Growth recovery: increases the relative growth rate (RGR) of duckweed by 32.8%, overcoming the biomass inhibition caused by copper stress; (2) Photosynthetic system protection: restores the total chlorophyll content to 88.6% of the normal level, and improves the integrity of chloroplast grana lamellae structure; (3) Oxidative stress inhibition: reduces the accumulation of reactive oxygen species (ROS) to 1.6-2.9 times that of the control group in a dose-dependent manner, and inhibits the production of malondialdehyde (MDA) by 89.5%.

4. The application of baicalin according to claim 1 in alleviating copper toxicity in duckweed, characterized in that, The application is achieved by reconstructing the antioxidant defense system, including: (1) increasing superoxide dismutase (SOD) activity to 43.2% of the control group; (2) enhancing peroxidase (POD) activity to 73.3% of the control group; (3) inhibiting xanthine oxidase (XOD) activity to 93% of the copper stress group; and (4) increasing catalase (CAT) activity to 65.4% of the control group.

5. The application of baicalin according to claim 1 in alleviating copper toxicity in duckweed, characterized in that, The application is achieved through the following molecular-level detoxification mechanism: (1) formation of [Cu(C)] 21 H 18 O 11 )] 3- (1) Stabilize the chelate (stability constant logK=12.3), reduce the effective copper concentration by 41-57%; (2) Upregulate the expression of metallothionein (MT2) gene, and enhance the intracellular copper ion compartmentalization ability.

6. The application of baicalin according to claim 1 in alleviating copper toxicity in duckweed, characterized in that, The baicalin has the following uses in the compound application scenario: (1) as a water body remediation agent, it can increase the copper enrichment of duckweed by 2.3-3.1 times in the pH range of 6.5-8.2; (2) as a stress resistance inducer, it can restore the light energy utilization efficiency of aquatic crops in polluted waters to 71-89% of the non-stress state.

7. A heavy metal pollution remediation scheme based on baicalin, characterized in that, By leveraging its multi-target mechanism of "coordination detoxification – oxidation balance – gene regulation", an assessment system including bioaccumulation coefficient and toxicity mitigation index is established for engineerable heavy metal pollution remediation.

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