Ecotoxicological effects of total flavonoids in Cirsium japonicum DC on Microcystis aeruginosa Open Access

Flavonoids are phenolic secondary metabolites, consisting of two benzene rings linked by an intermediate 3-carbon atom, which can be grouped into the C6-C3-C6 on structure (Sulaiman & Balachandran 2012; Ponte et al. 2021). Various types of natural flavonoids distributed in vegetative and reproductive organs of plants have many kinds of effects. In the field of medicine, total flavonoids have biological activity against free radicals (Peng et al. 2019; Li et al. 2021) which have been used in the therapy of diseases such as diabetes (Zou et al. 2021), tumours and cancer (Ganai et al. 2021; Song et al. 2021). In addition, antimicrobial studies have shown that total flavonoids in herbaceous plants not only inhibited fungal growth but also reduced bacterial activity, such as total flavonoids in Eryngium billardieri F. Delaroche showed antibacterial activity to Penicillium chrysogenum and Aspergillus niger (Daneshzadeh et al. 2020), and in Potentilla kleiniana, Mosla chinensis Maxim. Jiangxiangru caused the death of Pseudomonas aeruginosa (Cloutier et al. 2021; Tao et al. 2022), Bacillus subtilis and Staphylococcus aureus (Li et al. 2015) respectively. Except for higher plants (Mazila Ramli et al. 2021), macro-eukaryotes algae containing secondary metabolites as experimental material with antioxidant properties also have been reported. Bhuyar et al. (2021) revealed that marine macroalgae including red (Kappaphycus alvarezii), green (Kappaphycus striatus) and brown (Padina gymnospora) scavenged radical DPPH and ABTS by polysaccharides, and Myriophyllum brasiliense total flavonoids inhibited the growth and reproduction of M. aeruginosa (Saito et al. 1989). Total flavonoids is one of the most widely studied for secondary metabolites in plants because of its strong antioxidant effect (Perez-Vizcaino & Fraga 2018; Ramli et al. 2021). The results of previous studies have shown that total barley straw flavonoids interfered with the second electron receptor (QB) of M. aeruginosa, interrupted electron transfer in the reaction centre of photosynthetic system II, and led to a decrease in photosynthetic rate, morphological changes and cell membrane damage in algal cells (Feng et al. 2013; Huang et al. 2015). As total flavonoids can affect the photosynthetic physiology and cell structure of M. aeruginosa, it has become an urgent task for researchers to search for plants with a abundance of total flavonoids which are widely distributed, and to improve polluted water by using them.

C. japonicum DC is a perennial herbaceous plant of the genus Cirsium in the Compositae family and is distributed in China, Japan, Korea and other countries (Tian et al. 2021). It is a common Chinese herbal medicine, which has the effects of stopping bleeding and removing blood stasis (Jang et al. 2020). C. japonicum DC is widely cultivated in China as a Chinese herbal medicine with high yield and low cost. Usually, after drying, the whole plant or root of C. japonicum DC is ground into powder as medicine (Ye et al. 2022), but resistance researches using its seeds are not widely available. In recent years, studies have revealed high content of flavonoids in the seeds, and the method of extracting flavonoids from the seeds is simple and easy, with high extraction efficiency and good stability, and it is suitable for large-scale industrial production (Park et al. 2020). At present, there are many researches on the antibacterial effect of total flavonoids from C. japonicum DC, which showed damage to hyphae of Fusarium oxysporum f. sp. melonis (Liu et al. 2020) and inhibition of growth of Pyricularia grisea and Ceratocystis fimbriata (Yoon et al. 2011; Wei et al. 2013). However, few studies are available from the literature on the inhibition of microalgae by the total flavonoids of C. japonicum DC. Therefore, using seeds of this plant as materials to study the inhibition effect on microcystis has certain innovation and practical significance.

In recent years, eutrophication and outbreaks of cyanobacterial blooms in lakes have led to large-scale water pollution in different regions of the world (Ayele & Atlabachew 2021; Rumyantsev et al. 2021; Viso-Vázquez et al. 2021). Excessive reproduction of cyanobacteria is the main reason for algal blooms (Plaas & Paerl 2020; Papadimitriou et al. 2022). M. aeruginosa is one of the most common dominant algae species in cyanobacteria (Srisuksomwong & Pekkoh 2020; Xie et al. 2021), and its special cell structure, such as pseudo-empty cells and glial sheath, enables the cyanobacteria to move freely in the vertical direction, grow fast and release algal toxins, which ensure the dominant position of M. aeruginosa in many aquatic microorganisms (Dyer & Needoba 2020; Yan et al. 2020). Therefore, M. aeruginosa is selected as the target algae species to explore a way of bloom control. This experiment uses the total flavonoids of C. japonicum DC to study its inhibitory effect on the growth and physiology of M. aeruginosa, which can provide a theoretical reference for the prevention and control of water blooms.

A large number of granular, intact seeds of C. japonicum DC (purchased from Bozhou City, China) were selected and placed in an oven (DHG-9075A, Changzhou Haibo Instrument Equipment Co., Ltd China) at 60 °C for 12 h. After drying, seeds were ground into powder. The method of extraction by Wang et al. (2018) was modified slightly as follows: 5 g of powder was taken and added with 100 mL 70% ethanol. After ultrasonic treatment at 40 °C for 30 min, the extraction solution was then filtered and concentrated, and an appropriate amount of 30% ethanol was added for rotary evaporation (60 °C). The obtained product was dissolved and the volume adjusted to 50 mL with deionised water, which was the total flavonoids solution of C. japonicum DC. The total flavonoids content (TFC) was detected by NaNO₂-Al (NO₃)₃-NaOH colorimetric method with Rutin as standard (Huang et al. 2018).

Algal culture: M. aeruginosa purchased from freshwater Algae Species Bank, Chinese Academy of Sciences (FACHB 1328) were activated and cultured by BG11 medium in an artificial climate box (QHX-250BS-III, Shanghai Xinmiao Medical Machinery Manufacturing Co., LTD. China) at 25 ± 1 °C under a light intensity of 2800 lux and a light-dark cycle ratio of 12:12 h.

Experimental treatment: 40 mL algal solution with a concentration of 60 × 10⁶ cells/mL was added to each conical flask. Six treatments were set up: CK (0 mg/L), 10, 20, 30, 40, and 50 mg/L total flavonoids of C. japonicum DC. Total volume of treated algal fluid was up to 50 mL in each bottle, and incubated for 9 d with the same conditions as above and repeated three times.

The basic procedure for sample preparation was to fix M. aeruginosa with 5 mL of 2.5% glutaraldehyde for 4 h, cleaning with buffered phosphate. Then the samples were dehydrated with 30–100% ethanol and natural drying (Jayakumar et al. 2021; Wang et al. 2021). Eventually, M. aeruginosa cell morphology was characterised by scanning electron microscopy (Zeiss, Gemini SEM 300, Germany).

The linear equation of algal density and absorbance was obtained by hematocyte counting (Czarny et al. 2021) and spectrophotometry (Orr & Jones 1998): Y = 23.832X–0.2335, r² = 0.9995 (Y is the density of algal cells, the unit is 10⁶ cells/mL, and X is the absorbance value at 680 nm). The absorbance of algal liquid was measured by ultraviolet-visible spectrophotometer (T6 New Century, Beijing Purkinje General Instrument Co., Ltd. China), and the algal density was calculated by continuous detection for 9 days.

Concentrations of chlorophyll a, carotenoid, phycocyanin (PC) and allophycocyanin (APC) were measured spectrophotometrically (Tazart et al. 2019). Four mL of M. aeruginosa was centrifuged at 8000 rpm for 10 min. Alga pellets were mixed with 4 mL of ethanol. The mixture was then allowed to stand overnight at 4 °C. It was then centrifuged at 8000 rpm for 10 min. The supernatant was analysed by reading absorbance at 470, 649 and 665 nm (Zhang et al. 2019, 2021). Chlorophyll a and carotenoid content were calculated using the formulas: Chl a (mg/L) = 13.95 × OD₆₆₅–6.88 × OD₆₄₉; Carot (mg/L) = (1000 OD₄₇₀–2.05Chl a)/245.

Five mL of M. aeruginosa was centrifuged at 10,000 rpm for 10 min. The pellet was mixed with 5 mL of PBS. The liquid was placed in an ultra-low temperature refrigerator, dissolved at 8 h intervals at room temperature, away from light, and frozen/thawed three times. The pellets were then centrifuged at 12,000 rpm for 10 min. The supernatant was analysed by reading absorbance at 620 and 650 nm (Xu et al. 2021). The content was calculated using the following formula: PC (mg/L) = (OD₆₂₀–0.7 × OD₆₅₀)/7.38.

Five mL of M. aeruginosa solution was centrifuged at 10,000 rpm for 10 min to obtain a precipitate of seaweed cells, floating in 95% ethanol. The algal cells were then crushed using an ultrasonic cell crusher (SCIENTZ, JY92-II, China) and centrifuged at 7000 rpm for 15 min at 4 °C. Finally, the supernatant was zeroed with 95% ethanol and the photometric values were measured at 615 and 652 nm (Jayakumar et al. 2021). APC content was calculated as follows: APC (mg/L) = (OD₆₅₂–0.208 × OD₆₁₅)/5.09.

Activity of superoxide anion (O₂^−.) induced by total flavonoids were determined using the reagent kit (Nanjing Jiancheng Bioengineering Co., Ltd, China) after algal cells were broken (Changzhou Putian, FK-A(JJ-2), China) and measurement of Malondialdehyde (MDA) was referred and modified (Wu et al. 2013; Zheng et al. 2021). The algal solution treated were centrifuged (4 °C, 10,000 r/min, 10 min), 0.5 g of algal cells were taken, adding 6 mL trichloroacetic acid (TCA) solution and a small amount of quartz sand, ground in an ice bath, centrifuged (4000 r/min, 10 min). Then, the supernatant and thiobarbituric acid (TBA) were mixed with 2 mL each, followed by a boiling water bath 15 min and centrifugated (4000 r/min, 10 min) after rapid cooling. Zeroed with 2 mL of deionised water, the supernatant was taken to determine the absorbance at 532 and 450 nm. The content of the MDA-TBA reaction product was calculated according to the formula: MDA (mol/L) =6.45 × 10⁻⁶ × OD₅₃₂–0.56 × 10⁻⁶ × OD₄₅₀.

Superoxide dismutase (SOD) and scorbate oxidase (APX) play an important role in the metabolization of reactive oxygen species (ROS) which leads to MDA production. Therefore, the activities of SOD and APX can reflect stress resistance. SOD and APX activities were measured by inferring the methods of Rong et al. (2018) and Wu et al. (2020) respectively. The case in detail was as follows: 2.85 mL of reaction mixture including 50 mmol/L Tris-HCl buffer (pH 7.8), 0.1 mmol/L EDTA, 0.1 mmol/L nitroblue tetrazolium (NBT), and 13.37 mmol/L methionine. The reaction was set out by adding 100 μL 0.1 mmol/L riboflavin and 50 μL enzyme extraction containing 50 mmol/L Tris-HCl buffer, 1 mmol/L EDTA, 1 mmol/L MgCl₂, and 5 mmol/L MgCl₂. Then OD₅₆₀ was measured immediately after 15 min illumination with light intensity of 2000 lux. Tris-HCl buffer was used instead of the enzyme extraction solution under 2000 lux light for 15 min as a control. The unit of SOD enzyme activity is defined as the amount of enzyme required to inhibit the NBT reduction by 50%. Reaction mixture of 2.9 mL (composed by 50 mmol/L Tris-HCl buffer (pH 7.0), 0.1 mmol/LEDTA and 0.1 mmol/L H₂O₂) detecting APX was added by 50 μL enzyme extraction the same as SOD. After shaking and water bath (25 °C, 5 min), 50 μL 30 mmol/L ASA was added correspondingly. APX activity was calculated according to the decrease of OD₂₉₀ measured every 10 s.

The data obtained from the experiments were subjected to data statistics and one-way analysis of variance (ANOVA) using Excel 2019 and SPSS 25.0 software, and the means ± standard deviation was obtained. Multiple comparisons of the different treatments were made by Duncan's method, and the results of data processing were plotted using Origin 2017.

In this sdudy, a calibration curve between absorbance for triplicate reading and mass concentration of Rutin were generated (Y = 0.3357X + 0.0035, R² = 0.9994). Concentration of total flavonoids solution was obtained (815 ± 2.6 mg/L) by referencing standard curve and TFC of C. japonicum DC seeds was 8.15 ± 0.026 mg/g dry extract.

Cells of M. aeruginosa from the six treatment groups showed different morphologies after 9 days (Figure 1). The cells of the control emerged with a smooth surface, round and full shape, while the appearance of algal cells under 10–50 mg/L total flavonoids treatment showed different depression and wrinkles, a positive correlation between cytomorphosis and total flavonoids concentration of C. japonicum DC was found and the cells of M. aeruginosa treated with 50 mg/L total flavonoids had obvious cracks and reduced integrity. The results showed that the high concentration of total flavonoids in C. japonicum DC caused damage to cell structure and mass mortality.

The density of M. aeruginosa cells in the control was the highest as the treatment time increased (Figure 2), 10 mg/L treatment group appeared with faintish cell division at the same time. However, the cells density of 20–50 mg/L treatments decreased and showed the negative proliferation of algal cells with increasing of total flavonoids concentration. On the eighth day, density of M. aeruginosa cells in 50 mg/L decreased in part to 30 ×10⁶ cells/mL, inhibition percentage of cells reproduction by total flavonoids of C. japonicum DC was 12.5, 29.8, 42.5, and 58.8% in 20–50 mg/L treatments, respectively, after 9 days. Density change displayed that the higher the treatment concentration, the more significant hold-up effect on growth.

During the process, contents of Chlorophyll a and carotenoid in M. aeruginosa treated by 10–50 mg/L total flavonoids of C. japonicum DC all declined (Figure 3(a) and 3(b)) compared with the control, but there were no significant differences between treatment groups on the first day (p > 0.05). However, two pigment concentrations of 10–50 mg/L treatments were significantly different (p < 0.05) after 5 days, which mostly reduced by 14.2% (14.1%), 26.6% (27.7%), and 44.4% (45.2%), respectively, on the ninth day at 50 mg/L total flavonoids treatment.

As shown in Figure 4(a), the change trend of PC content under 10–50 mg/L treatments was same as chlorophyll a and carotenoid on the ninth day, and they decreased significantly (P < 0.05) compared to the control. The PC content in 50 mg/L total flavonoids treatment was the lowest (0.238 mg/L), indicating that high concentration total flavonoids may severely disrupt the light trapping system of the algal cells. In terms of changes in the content of APC, it was positively correlated with the total flavonoids concentration in the range of 10–40 mg/L (Figure 4(b)). However, at 50 mg/L treatment, the APC content significantly decreased.

Figure 5(a) showed changes of O₂^−. activity when M. aeruginosa were exposed to 10–50 mg/L total flavonoids. Compared with the control, O₂^−. activity in four treatment groups (20–50 mg/L) significantly increased on the ninth day. The change trend of O₂^−. activity showed positive correlation with treatment concentration, which was consistent with the MDA content (Figure 5(b)).

SOD and APX activities of M. aeruginosa treated with 10–50 mg/L total flavonoids were higher than those of the control on the ninth day. Although SOD activities of five treatments all exceeded the control, there were no significant differences during treatments of 20–40 mg/L of total flavonoids (Figure 6(a)). However, it increased dramatically at 50 mg/L. At the same time, APX activities increased with elevating total flavonoids concentration and was significantly different (p < 0.05) among 10–30 mg/L treatments. The value of APX reached maximum at 50 mg/L (Figure 6(b)), which indicated that the stress of total flavonoids of C. japonicum DC on M. aeruginosa was serious.

To date, some research concerning content and antioxidant activity of total flavonoids in higher plants has been reported. The results showed that TFC of methanolic root extract of Arisaema jacquemontii was 35.5 ± 2.2 mg/g (Baba & Malik 2015) and hydro peel extract of Passifora edulis was 8.364 ± 0.002 mg/g (Ramli et al. 2020), following ethanol seed extract of C. japonicum DC in the current experiment was 8.15 ± 0.026 mg/g. The difference above probably related to species, sampling organ and extraction agent under the premise of using the same standard (Ramli et al. 2020). Although TFC in seeds of C. japonicum DC is significantly lower than that of Arisaema jacquemontii root (located at 1900–4500 meters above sea level), C. japonicum DC is an ideal plant for studying the effects of total flavonoids because of its strong fecundity, wide distribution and easy seed availability. Moreover, the total flavonoids activity varies in different plants. Meng et al. (2015) and Xing & Sun (2020) have found that certain concentrations of total flavonoids of Ailanthus altissima and Sophora japonica effectively inhibited the growth of M. aeruginosa: 200 mg/L total flavonoids of A. altissima on the fifth day and 11.86 mg/L total flavonoids of S. japonica on the seventh day had the best inhibitory effect, whereas the algal densities of the treatment with the best inhibitory effect of total flavonoids of A. altissima and total flavonoids of S. japonica were always greater than the initial concentration of M. aeruginosa. In contrast, this experiment showed that algal cells density of the 20–50 mg/L treatments were lower than the initial on the second day, and the algal density was reduced by about half under treatment of 50 mg/L total flavonoids of C. japonicum DC on the eighth day. Therefore, it is assumed that the total flavonoids of the herb C. japonicum DC have higher activity in inhibiting growth of M. aeruginosa. In addition, Zhao et al. (2020) demonstrated that the flavonoids in Aloe vera caused changes in cell permeability, with an overall rupture of the cell membrane and massive exudation of inclusions, correspondingly inhibiting the proliferation of M. aeruginosa. In a similar manner, M. aeruginosa in this experiment showed an increase in cell surface depression with increasing concentrations of the total flavonoids of C. japonicum DC and a dramatic decrease in algal density, with a minimum value of 24.7 × 10⁶ cells/mL. This may be due to the effects of total flavonoids of C. japonicum DC in disrupting cell morphology and inhibiting algal cell division, resulting in partial cell death (Xu et al. 2021). The inhibitory effect of total flavonoids of C. japonicum DC on the growth of M. aeruginosa cells showed positive correlation.

Chlorophyll a and carotenoid are the photosynthetic pigments of algal cells, and reduced cellular integrity can directly or indirectly affect chlorophyll a and carotenoid contents, hinder photosynthesis and lead to a decrease in the ability to carry out photochemical reactions and maintain cellular metabolism of M. aeruginosa (Deng et al. 2020; Yang et al. 2021). The effects of total flavonoids extracted from herbal plants on photosynthetic pigments of M. aeruginosa have been studied by predecessors. Tazart et al. (2019) confirmed that the chlorophyll a and carotenoid contents of M. aeruginosa were significantly lower than the control after 6 days of exposure to 201.8 and 106.5 mg/L total flavonoids of Ranunculus aquatilis and Nasturtium officinale respectively. The same phenomenon was observed in M. aeruginosa treated for 8 days with 25.83 and 37.61 mg/L total flavonoids of Thymus satureioides Coss. and Artemisia herba alba L (Tebaa et al. 2018). As for the present study, the chlorophyll a and carotenoid contents of M. aeruginosa treated with 10–50 mg/L total flavonoids of C. japonicum DC showed a significant decrease compared with the control (P < 0.05) after 5 days of treatment which expressed serious damage to pigments. Moreover, PC is an important light-trapping protein that supports photosynthesis in M. aeruginosa (Liang et al. 2020). When M. aeruginosa were treated by 50 mg/L of total flavonoids of Aloe vera (L.) Burm, PC content all increased during the processing (Zhao et al. 2020). However, the opposite result appeared in this experiment after 9 days of treatment with 10–50 mg/L total flavonoids of C. japonicum DC. The serious toxic effect of total flavonoids from C. japonicum DC on M. aeruginosa may be the reason for the difference.

O₂^−. as the preliminary reactive oxygen species (ROS) in cells derived from pseudoring electron transport in the photosynthetic chain. The oxidation of ROS will be enhanced once the O₂^–. increases (Wang et al. 2019), which damages cell membranes by accumulation of MDA coming from membrane lipid peroxidation (Wu et al. 2021). The experiments of Mecina et al. (2019) found that the highest O₂^−. concentration in M. aeruginosa was characterized on the sixth day with 500 mg/L flavonoids isolated from Tridax procumbens, meanwhile MDA content increased. In the present study, O₂^−. activity in M. aeruginosa cells exposed to total flavonoids of C. japonicum DC strengthened with the increase of treatment concentration, and the increase of MDA content accordingly, suggesting that the total flavonoids of C. japonicum DC induced the production of ROS and was responsible for the inhibition on the growth of M. aeruginosa.

SOD is the only enzyme with oxygen free radical as substrate in nature, which plays an important role in ROS metabolism and can quench superoxide anion (O₂^−.). It is the most important enzyme for scavenging O₂^−. in the organisms. Moreover, when the activity of APX is significantly increased, the production rate of O₂^−.decreased significantly and lipid peroxidation weakened (Zhang et al. 2016). Therefore, SOD and APX activity can reflect the resistance to adversity, indicating the severity of the environmental stress (Tian et al. 2018). Indeed, the SOD and APX activities of M. aeruginosa were higher than those of the control under the treatment of 10–50 mg/L total flavonoids, and the activities of SOD and APX were the strongest at 50 mg/L total flavonoids treatment in the current research, showing different trends with secondary metabolites of berberine from golden thread (Zhang et al. 2011). APC has certain antioxidant capacities and maintains algal cell activity and accelerates its own metabolic rate under adverse conditions (Wu et al. 2013). However, the APC content decreased in the largest dose of total flavonoids in the present experiment. The reason is that increasing O₂^−. activity not only caused severe membrane lipid peroxidation of M. aeruginosa (Figure 5(b)) but also damaged photosynthetic proteins (Figure 4(a) and 4(b)). This suggests that APC might be an important site of action of total flavonoids of C. japonicum DC in M.aeruginosa. The M. aeruginosa cells have a certain ability to stabilize the internal environment by increasing SOD and APX activities. However, M.aeruginosa did not remove all O₂^−. and MDA with the increasing dose and cell growth was seriously effected.

The growth of M. aeruginosa was inhibited by 10–50 mg/L total flavonoids of C. japonicum DC, and showed a concentration-time effect, with the lowest density of algae treated at 50 mg/L total flavonoids. The addition of different concentrations of total flavonoids of C. japonicum DC disrupted the synthesis of photosynthetic pigments and PC in M. aeruginosa, blocking the capture and absorption of light by the cells. Furthermore, although the SOD and APX activities of M. aeruginosa increased under the stress of total flavonoids of C. japonicum DC, the antioxidant activity was weakened by the increase of O₂^−., MDA and the decrease of APC content, which broke the intracellular equilibrium and then caused oxidative damage and cell death. The total flavonoids from C. japonicum DC are more effective in inhibiting M. aeruginosa. It is worth noting that C. japonicum DC seeds, with low cost and high flavonoids content, are a new source of effective and inexpensive biological algal inhibitors.

This work was supported by the Key Laboratory of Bioresource and Environmental Biotechnology of Anhui Higher Education Institutes, Huainan Normal University, and was given financial assistance by the Projects of Education Department of Anhui Province (KJ2020A0649). The useful comments of anonymous reviewers are also acknowledged.

The authors declare that there are no conflicts of interest.

All relevant data are included in the paper or its Supplementary Information.