Valorization of bioactive metabolites from Trichodesmium erythraeum bloom biomass for vector borne disease control, bactericidal and anticancer therapy

doi:10.21203/rs.3.rs-4788767/v1

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Valorization of bioactive metabolites from Trichodesmium erythraeum bloom biomass for vector borne disease control, bactericidal and anticancer therapy

https://doi.org/10.21203/rs.3.rs-4788767/v1

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In the marine environment, algae are regarded as globally distributed entity. The excessive accumulation of nutrients such as nitrogen or phosphorus causes an algal bloom, which is caused by the rapid growth of microscopic algae rather than macroscopic ones. Even though it has negative effects, its significance in terms of biological usage was limited to experimental perspective. In our study, we have utilized algal waste biomass of Trichodesmium erythraeum to valorize its natural metabolites for the vector control, bacterial inhibition and anticancer study. Among the solvents, the methanolic extract produced significant mosquitocidal action against A. stephensi, A. aegypti, and C. quinquefasciatus. The methanolic extract of T. erythraeum were found to inhibit the growth of clinical pathogens including B. subtilis, P. mirabilis, S. aureus, E. coli and K. pneumoniae. Further, the methanol extract mediated anticancer effect was assessed on non-small cell lung cancer cell lines. Moreover, the extract displayed no impact on non-target organism. FT-IR results suggested the incidence of alkanes (–C ≡ C–H: C–H stretch), nitrile (C ≡ N), (primary amines (N–H bend), aliphatic amines (C–N stretch), alkenes (= C–H bend) and nitro compounds (N–O asymmetric stretch). GCMS analysis listed the series of compounds such as Hexadecanoic acid, Bicyclo [10.1.0] tridec-1-ene, Glycidol stearate, Tricyclo [20.8.0.0 (7, 16)] triacontane, 1(22), 7 (16)-diepoxy- and Piperidine played vital role in vector control and bactericidal action. This study specifies on usage of algal metabolites and the obtained results imply that it might be useful for essential medicinal applications.

Cyanobacterium

Trichodesmium erythraeum

GC-MS

Mosquitocidal

Bactericidal

Anticancer

Mosquito-borne diseases continue to pose a serious threat to public health despite tremendous advancements in biomedical research worldwide, especially in tropical areas where effective control measures have not yet been fully implemented. This is demonstrated by the continued existence of several fatal mosquito-borne illnesses, including filariasis, chikungunya, dengue, malaria, and the Zika virus [1]. The World Health Organization (WHO) reported that there were 247 million cases of malaria worldwide in 2021, with an expected 619,000 fatalities from the disease [2]. Between 100 and 400 million cases of dengue fever are thought to occur each year, whereas 51 million cases of lymphatic filariasis are thought to have occurred worldwide in 2018. Notably, some mosquito species including Aedes aegypti for dengue, Anopheles stephensi for malaria, and Culex quinquefasciatus for lymphatic filariasis act as the main carriers of these diseases. Therefore, protecting human health, fostering environmental sustainability, and stopping the development of these diseases all depend on developing mosquito control methods. Mosquito control can be accomplished through three main methods: (i) direct targeting and killing of adult mosquitoes; (ii) repelling adult mosquitoes while they are feeding on blood; and (iii) larval control, the most extensively researched method that tries to lower adult mosquito populations by focusing on immature stages [3].

The development of innovative mosquito control techniques is required due to the growth of pesticide resistance in mosquito populations. These techniques ought to be economical, successful, safe for the environment, and have little effect on organisms that are not the intended targets [4]. This has sparked interest in researching natural insecticides that provide the fewest dangers to the health of people and the environment.

According to Brutyan et al. (2017) [5], research on microalgae, a varied collection of unicellular photosynthetic microorganisms with substantial ecological and commercial significance, is very promising. These quickly proliferating creatures are found in freshwater, marine environments, and damp rock surfaces. Notably, bioactive substances like steroids, phenolics, terpenoids, amino acids, and phlorotannin’s are abundant in microalgae. According to de Morais et al. (2015) [6], these substances have a broad spectrum of biological activity, including antimicrobial, fungicidal, antiviral, anticancer, and most importantly mosquitocidal qualities [7]. Many biological actions, including antioxidant, antifungal, antibacterial, antiviral, antialgal, antiprotozoal, anticancer, and anti-inflammatory properties, have been linked to cyanobacteria metabolites, according to a recent study [8]. This served as inspiration for this review, which emphasizes the potential of cyanobacteria in the search for antiviral and antibacterial drugs. Trichodesmium erythraeum is a filamentous, diazotrophic cyanobacterium that contributes significantly to the global nitrogen cycle, making it an important microalga in marine ecosystems [9]. This study demonstrates potential of T. erythraeum as anti-mosquitocidal, bactericidal and anticancer properties.

2.1 Collection of marine microalgae

A dense bloom of T. erythraeum was found and collected from the coastal waters of Tuticorin [Lat: 8° 46' 4.1952'' N Long: 78° 14' 9.2004'' E], east coast of India. Algal samples were captured from the surface water using a plankton net made of bolting silk (No. 25 µm mesh size with a round mouth diameter of 48 cm). The collected samples were then transported in an ice box to the laboratory and meticulously cleaned with Milli-Q water to eliminate all attached epiphytes. The microalgae identification was done based on the standard taxonomic monograph [10]. Subsequently, the algal biomass was centrifuged for 20 minutes at 3000 rpm, and the supernatant was decanted. The algal biomass was preserved at 4°C for future utilization.

2.2 Preparation of different extracts

The extraction was performed using cold percolation method. The biomass (1kg) were shade-dried at room temperature (RT) and then grounded. The powder (250g) was soaked with 750 ml (1:3 w/v) of different solvents such as ethanol, methanol, hexane and petroleum ether in an aspirator bottle for 72 h at room temperature with occasional shaking. Whatman paper (No. 1) filter paper was utilised to filter the extract using a Buchner funnel. Using a rotating evaporator set at 40ºC, the filtrate was condensed. Then, the yielded extracts were stored at 4ºC.

2.3 Collection of eggs and maintenance of vectors

Anopheles stephensi, Aedes aegypti, and Culex quinquefasciatus (hereafter As, Aa, and Cq) eggs were chosen for this study. Eggs of A. stephensi, A. aegypti, and C. quinquefasciatus were collected from the Vector Control Research Centre (VCRC), Puducherry, and transported to the laboratory. They were hatched in enamel trays filled with water, fed a mixture of dog biscuits and yeast until they reached the pupal stage. After pupation, pupae were transferred to plastic containers filled with water for adult emergence. Mosquitoes were maintained at 27 ± 2°C, 75–85% humidity, and a 14:10 light/dark photoperiod. A sugar solution was provided three days before blood-feeding.

2.4 Larvicidal bioassays

The larvicidal tests were done in accordance to Sharanappa et al. (2023) [11], which utilized cyanobacterial extracts as natural pesticides. The concentrations (25–125 µg/mL) of different extracts of T. erythraeum (methanol, hexane, petroleum ether and ethanol) was carried out with replications. A control group was maintained in dechlorinated water with 0.01% DMSO. Containers were kept in the laboratory under ambient conditions (28 ± 2°C, 12 hours light/dark cycle). Larvae were considered deceased if they showed no movement upon gentle brushing. Mortality was recorded after 24-hour exposure then, LD₅₀ and LD₉₀ values were calculated using Log probit analysis [12].

2.5 Adulticidal and Ovicidal activity

Adulticidal bioassays were conducted following the WHO guidelines [13]. T. erythraeum extract was diluted and applied to Whatman no. 1 filter papers (12×15 cm) tested at concentrations of 30–150 µg/mL. Control papers were treated with ethanol. Mosquito mortality was recorded after the recovery period and values were determined using Log probit analysis [12]. Ovicidal activity was assessed using the version of Su and Mulla's method [14] by expose to different concentrations (5–25 µg/mL) of T. erythraeum extract. Each experiment, including controls, was replicated six times (n = 6). Hatch rates were calculated 48 hours post-treatment using the formula: Percentage of egg mortality = (Number of hatched larvae / Total number of eggs) × 100.

2.6 Environmentally Friendly Toxicity Assay

Environmental toxicity was assessed using field-collected aquatic insects (Diplonychus indicus and Anisops bouvieri) and fish (Gambusia affinis). The impact of T. erythraeum methanol extract on these organisms was evaluated following a standardized protocol [15, 16]. The concentrations of the extract (300–1500 µg/mL) were tested, and mortality rates were observed after 48 hours and calculated using Log probit analysis [12].

2.7 Bactericidal activity

2.7.1Pathogenic bacterial strains

The bacterial strains Bacillus subtilis, Klebsiella pneumoniae, Proteus mirabilis, Escherichia coli, and Staphylococcus aureus were obtained from the Saveetha Institute of Medical and Technical Sciences, Saveetha University, Tamil Nadu, India.

2.7.2 Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) determination

Various concentrations, ranging from 15.6 µg/mL to 500 µg/mL, were prepared from the stock extract (10 mg/mL). After adding 0.5 mL of the test dilution to 2 mL of nutrient broth (NB), 500 µL of the standardized bacterial culture (CFU) was inoculated into each test tube. Nutrient broth alone was used as a control group. The samples were incubated for 24 hours at 37°C, and growth was measured using a UV spectrophotometer at 600 nm. Microdilutions were performed according to the standard method [17]. The minimum inhibitory concentration (MIC) is defined as the lowest concentration that exhibits 100% growth inhibition. The minimum bactericidal concentration (MBC) was identified as the lowest concentration at which no bacterial colonies were observed.

2.7.3 Antibacterial assay

Briefly, pure isolates of the microorganisms were subculture on their respective specific media for twenty-four hours at 37°C. A 100 µL aliquot of each test organism's inoculum was applied to Mueller-Hinton Agar plates. Discs were then impregnated with the extract at concentrations of 125 µg/mL, 250 µg/mL, and 500 µg/mL. After allowing the extract to diffuse for ten minutes, the plates were incubated for twenty-four hours at 37°C [18]. The diameter of inhibitory zones was measured in millimeters after 24 hours of incubation. Tetracycline was used as an antibiotic.

2.8 Cell lines and culture conditions

The NCCS (National Centre for Cell Science), located in Pune, India, provided the lung cancer cells and L132 (normal). The cells were cultured as monolayers in DMEM medium supplemented with 1% actinomycin and 10% fetal bovine serum. Cell lines were subculture and the system was kept at 37°C in a humidified environment.

2.8.1 Cytotoxicity valuation assay

Using 96-well plates, the MTT assay was used to assess the extract's effect on the survival of lung cell lines. Initially, 100 microliters of the cell suspension were mixed with each well having a concentration of 10,000 cells per well. The plates were incubated for 24 hours at 37°C with 5% CO2, and then different quantities of extract (12.5 to 400 µg/ml) were added. The cells were then cultured for a further twenty-four hours. Subsequently, each well received 20 µl of MTT reagent (5 mg/ml) produced in sterile PBS, and the mixtures were incubated for an additional 4 hours. The purple formazan crystals that had grown were then dissolved by adding 200 µl of DMSO to each well. Then using a Spectrophotometer, the absorbance was measured after 1 h at 570 nm and 620 nm [19].

2.9 Instrumentation

Nicolet 800 FTIR spectrometer-Nicolet, Madison, WI, USA, was utilized for FTIR spectroscopy. The biomolecules present in the T. erythraeum methanol extract were identified in the frequency 500 to 4000 cm − 1 [20]. GCMS study was performed using Agilent Technologies (6890 N) and JEOL GCMATE II equipment, including an autosampler and gas chromatography interfaced with mass spectrometry (GC-MS). A 624 ms capillary column (30 m × 0.32 mm × 1.8 m) operating in electron mode at 70 eV was utilized. Helium (99.999%) served as the carrier gas at a constant flow rate of 1.491 ml/min, with an injection volume of 1.0 ml. The injector temperature was maintained at 140°C, and the ion source temperature was set to 200°C. Mass spectra were recorded at 70 eV.

2.10 Statistical Analysis

The probit analysis tool and SPSS version 16.0, was used to analyze the data for one-way analysis of variance. The values were expressed as mean value ± SD.

3.1 Larvicidal activity

The mosquito larvicidal activities of various T. erythraeum extracts (hexane, methanol, petroleum ether, and ethanol) were analyzed for the concentrations ranged from 25 to 125 µg/mL against for the selected species (A. stephensi, A. aegypti, and C. quinquefasciatus) (Table 1–4). Increased concentrations of the extracts resulted in significantly higher larval mortality across all T. erythraeum extracts. The LC50 values for larval mortality were 44.22, 46.85, and 47.645 µg/mL for the hexane extract of T. erythraeum, while the methanol extract exhibited LC50 values of 38.62, 40.29, and 43.22 µg/mL against As, Aa, and Cq, respectively. Furthermore, the petroleum ether extract of T. erythraeum had LC50 values of 49.13, 50.82, and 54.82 µg/mL, while the ethanol extract showed LC50 values of 58.56, 64.56, and 76.25 µg/mL against to the selected species. The methanol extract showed prominent larvicidal activity, followed by hexane, petroleum ether, and ethanol, respectively. The LC₉₀ larvae mortality value was 106.77, 119.02, and 127.24 for methanol extract of T. erythraeum against As, Aa, and Cq, respectively. Thus, all the further studies, such as adulticidal activity, ovicidal activity, bio-toxicity analysis, and screening of phytocompounds, etc., were carried out only on the methanol extracts of T. erythraeum.

Table 1

Effect of *T. erythraeum* hexane extract against selected mosquito larvae
Organism	Concentration (µg/mL)	Morality of larvae (%)	LC₅₀ (µg/mL) (LCL-UCL)	LC₉₀ (µg/mL) (LCL-UCL)	Regression Equation	x² df = 3
An. stephensi	25	28.4 ± 0.4^a	44.22 (32.12–60.62)	127.17 (92.45–174.93)	Y = 2.8407x + 0.3282	0.531
	50	48.6 ± 1.2^b
	75	68.7 ± 1.4^c
	100	89.2 ± 2.6^d
	125	100.0 ± 0.0^e
Ae. aegypti	25	26.6 ± 0.8^a	46.85 (33.81–64.92)	140.26 (101.22-194.35)	Y = 2.7255x + 0.4485	0.537
	50	46.2 ± 0.6^b
	75	66.2 ± 2.2^c
	100	86.4 ± 1.6^d
	125	100.0 ± 0.0^e
Cx. quinquefasciatus	25	24.6 ± 1.2^a	47.645 (36.75–62.91)	121.39 (91.93–160.28)	Y = 3.3577x + 0.6215	0.547
	50	45.4 ± 0.8^b
	75	63.8 ± 1.6^c
	100	80.6 ± 2.2^d
	125	97.2 ± 1.4^e
Significant at p < 0.05; Control: Nil mortality; LC₅₀: Lethal concentration that kills 50% of the exposed larvae; LC₉₀: Concentration that kills 90% of the exposed larvae; LEL: Lower fiducial limit; UFL: Upper fiducial limit; x² : Chi-sq value; df: degrees of freedom.

Table 2

Effect of *T. erythraeum* methanol extract against selected mosquito larvae
Organism	Concentration (µg/mL)	Morality of larvae (%)	LC₅₀ (µg/mL) (LCL-UCL)	LC₉₀ (µg/mL) (LCL-UCL)	Regression Equation	x² df = 3
An. stephensi	25	32.8 ± 1.4^a	38.62 (28.07–53.15)	106.77 (77.601–146.92)	Y = 2.9511x + 0.3197	0.940
	50	57.6 ± 1.2^b
	75	74.8 ± 2.2^c
	100	92.5 ± 1.8^d
	125	100.0 ± 0.0^e
Ae. aegypti	25	30.5 ± 0.6^a	40.29 (29.68–55.84)	119.02 (85.89–164.95)	Y = 2806x + 0.4715	0.733
	50	54.2 ± 1.4^b
	75	72.5 ± 1.2^c
	100	89.6 ± 1.6^d
	125	100.0 ± 0.0^e
Cx. quinquefasciatus	25	27.8 ± 0.4^a	43.22 (31.80-61.27)	127.24 (94.04–181.18)	Y = 7402x + 0.4936	0.717
	50	50.6 ± 1.8^b
	75	70.4 ± 1.6^c
	100	86.8 ± 2.6^d
	125	100.0 ± 0.0^e
Significant at p < 0.05; Control: Nil mortality; LC₅₀: Lethal concentration that kills 50% of the exposed larvae; LC₉₀: Concentration that kills 90% of the exposed larvae; LEL: Lower fiducial limit; UFL: Upper fiducial limit; x² : Chi-square value; df: degrees of freedom.

Table 3

Effect of *T. erythraeum* petroleum ether extract against selected mosquito larvae
Organism	Concentration (µg/mL)	Morality of larvae (%)	LC₅₀ (µg/mL) (LCL-UCL)	LC₉₀ (µg/mL) (LCL-UCL)	Regression Equation	x² df = 3
An. stephensi	25	23.3 ± 1.2^a	49.13 (36.75–65.69)	130.877 (97.89 -174.97)	Y = 3.1372x – 0.2981	0.590
	50	46.2 ± 2.4^b
	75	62.6 ± 1.4^c
	100	78.2 ± 2.6^d
	125	95.4 ± 3.2^e
Ae. aegypti	25	22.4 ± 1.2^a	50.82 (38.50–69.77)	139.39 (103.55–187.63)	Y = 3.02223x + 0.1487	0.648
	50	45.2 ± 2.5^b
	75	60.4 ± 1.8^c
	100	76.6 ± 2.6^d
	125	94.0 ± 3.8^e
Cx. quinquefasciatus	25	20.8 ± 0.6^a	54.82 (40.77–75.29)	163.69 (119.19–224.82)	Y = 2.7558x + 0.2141
	50	42.8 ± 1.8^b
	75	58.8 ± 2.6^c
	100	70.6 ± 3.4^d
	125	90.2 ± 4.2^e
Significant at p < 0.05; Control: Nil mortality; LC₅₀: Lethal concentration that kills 50% of the exposed larvae; LC₉₀: Concentration that kills 90% of the exposed larvae; LEL: Lower fiducial limit; UFL: Upper fiducial limit; x² : Chi-square value; df: degrees of freedom.

Table 4

Effect of *T. erythraeum* ethanol extract against selected mosquito larvae
Organism	Concentration (µg/mL)	Morality of larvae (%)	LC₅₀ (µg/mL) (LCL-UCL)	LC₉₀ (µg/mL) (LCL-UCL)	Regression Equation	x² df = 3
An. stephensi	25	19.5 ± 1.2^a	58.56 (42.31–81.05)	180.16 (130.17-249.34)	Y = 2.6616x + 0.2982	0.778
	50	39.2 ± 2.6^b
	75	54.6 ± 1.8^c
	100	70.2 ± 4.2^d
	125	86.8 ± 3.6^e
Ae. aegypti	25	16.4 ± 0.8^a	64.56 (47.42–91.91)	203.39 (146.09–298.56)	Y = 2.5944x + 0.3072	0.872
	50	36.8 ± 1.5^b
	75	51.4 ± 2.4^c
	100	65.2 ± 2.2^d
	125	83.0 ± 1.6^e
Cx. quinquefasciatus	25	13.4 ± 0.4^a	76.25 (54.67-106.984)	244.79 (174.97–342.48)	Y = 2.5546x + 0.1912	0.674
	50	26.2 ± 1.2^b
	75	45.6 ± 2.8^c
	100	60.5 ± 3.1^d
	125	76.2 ± 2.6^e
Significant at p < 0.05; Control: Nil mortality; LC₅₀: Lethal concentration that kills 50% of the exposed larvae; LC₉₀: Concentration that kills 90% of the exposed larvae; LEL: Lower fiducial limit; UFL: Upper fiducial limit; x² : Chi-square value; df: degrees of freedom.

3.2 Adulticidal bioassay

The adult mortality of methanol extract of T. erythraeum was noticed at a concentration of 30, 60, 90, 120 and 150 µg/mL against chosen mosquitoes (Table 5). When the concentration of methanol extract of T. erythraeum increased, the adult mosquito population gradually declined, and significant mortality occurred between each dose. The adult mortality LC₅₀ values of methanol extract was 49.37, 52.49, and 55.80 µg/mL for As, Aa, and Cq, respectively. The highest concentration of 150 µg/mL of methanol extract killed the entire adult mosquito during the experimental period.

Table 5

Effect of *T. erythraeum* methanol extract against selected adult mosquito
Organism	Concentration (µg/mL)	Morality of larvae (%)	LC₅₀ (µg/mL) (LCL-UCL)	LC₉₀ (µg/mL) (LCL-UCL)	Regression Equation	x² df = 3
An. stephensi	30	28.2 ± 0.4^a	49.37 (36.55–69.47)	142.704 (103.51-196.72)	Y = 2.8512x + 0.1474	0.849
	60	55.4 ± 0.8^b
	90	72.8 ± 1.4^c
	120	88.6 ± 2.2^d
	150	100.0 ± 0.0^e
Ae. aegypti	30	27.6 ± 1.2^a	52.49 (37.72–73.05)	149.20 (107.20 -207.65)	Y = 2.717x + 0.3273	0.811
	60	52.8 ± 1.8^b
	90	70.5 ± 2.2^c
	120	86.4 ± 3.4^d
	150	100.0 ± 0.0^e
Cx. quinquefasciatus	30	25.4 ± 1.6^a	55.80 (40.12–77.60)	159.93 (115.01–222.39)	Y = 2.6922x + 0.2981	0.773
	60	49.6 ± 2.4^b
	90	68.6 ± 1.2^c
	120	84.2 ± 4.2^d
	150	100.0 ± 0.0^e
Significant at p < 0.05; Control: Nil mortality; LC₅₀: Lethal concentration that kills 50% of the exposed larvae; LC₉₀: Concentration that kills 90% of the exposed larvae; LEL: Lower fiducial limit; UFL: Upper fiducial limit; x² : Chi-square value; df: degrees of freedom.

3.3 Ovicidal bioassay

The selected mosquito eggs were treated at 5, 10, 15, 20 & 25 µg/mL of T. erythraeum methanol extract (Table 6). The hatchability rate was 40.6, 16.3, 5.4, 0, and 0 for Anopheles stephensi, meanwhile, 42.3, 19.5, 7.4, 0, and 0 for Aedes aegypti and 46.7, 23.3, 9.7, 2.8 and 0 for Culex. quinquefasciatus at different T. erythraeum methanol extract concentrations like 5, 10, 15, 20, and 25 µg/mL, respectively. The egg hatchability rate declined while increasing the concentration of methanol extract. Among the mosquito species, T. erythraeum methanol extract was highly susceptible to the tested mosquitoes.

Table 6. Ovicidal activity of T. erythraeum methanol extract against the selected mosquito

Concentration	Control	5 (µg/mL)	10 (µg/mL)	15 (µg/mL)	20 (µg/mL)	25 (µg/mL)
Mosquito species	*An. stephensi*
	100	40.6 ± 2.4^a	16.3 ± 1.6^b	6.4 ± 0.8^c	0.0 ± 0.0^d	0.0 ± 0.0^d
	*Ae. aegypti*
	100	42.3 ± 3.6^a	19.5 ± 1.3^b	7.8 ± 0.4^c	0.0 ± 0.0^d	0.0 ± 0.0^d
	*Cx. quinquefasciatus*
	100	46.7 ± 2.5^a	23.3 ± 1.4^b	9.7 ± 0.7^c	2.8 ± 0.2^d	0.0 ± 0.0^e

Significant at p<0.05; NH: No hatchability

3.4 The impact of T. erythraeum methanol extract on field-collected aquatic insects and fish

The environmentally friendly toxicity assay was performed to know the toxicity nature of T. erythraeum methanol extract at a concentration of 300, 600, 900, 1200, and 1500 µg/mL on aquatic insects such as Diplonychus indicus and Ani-sops bouvieri and the fish Gambusia affinis, respectively (Table 7). The mortality of selected aquatic organisms was notably increased when the concentration of T. erythraeum methanol extract increased. The aquatic insect's mortality LC₅₀ values were 799.16 and 791.32 µg/mL, while the fish Gambusia affinis mortality LC₅₀ value was 791.32 µg/mL when exposed to methanol extract of T. erythraeum respectively.

Table 7

The impact of *T. erythraeum* methanol extract against environment-friendly aquatic insects and fish
Eco-friendly Organism	Concentration (µg/mL)	Mortality (%) (48 h)	LC₅₀ (µg/mL) (LCL-UCL)	LC₉₀ (µg/mL) (LCL-UCL)	Regression Equation	x² df = 3
A. bouvieri	300	15.6 ± 1.2^a	799.16 (588.93 -1084.42)	2283.15 (1682.56-3098.12)	y-2.8505x-3.2344	0.490
	600	34.5 ± 2.8^b
	900	46.9 ± 1.6^c
	1200	62.6 ± 1.5^d
	1500	89.0 ± 3.3^e
D. indicus	300	13.8 ± 0.6^a	791.32 (591.43–1058.77)	2098.98 (1568.77 -2808.37)	y-3.016x – 3.7139	0.613
	600	30.7 ± 1.4^b
	900	52.4 ± 1.2^c
	1200	68.2 ± 0.8^d
	1500	86.8 ± 0.6^e
G. affinis	300	17.6 ± 1.2^a	745.30 (551.09–1007.94)	2004.69 (1482–2711.15)	y-2.8992x-3.2892	0.457
	600	37.2 ± 2.9^b
	900	48.5 ± 0.6^c
	1200	67.6 ± 1.6^d
	1500	90.8 ± 2.4^e
Significant at p < 0.05; Control: Nil mortality; LC₅₀: Lethal concentration that kills 50% of the exposed insects/fish; LC₉₀: Concentration that kills 90% of the exposed insects/fish; LEL: Lower fiducial limit; UFL: Upper fiducial limit; x²: Chi-square value; df: degrees of freedom.

3.5 Antibacterial activity

The antibacterial effect of different prepared solvent extracts was tested. The methanol-based algal extract showed more positive MIC and MBC results compared to other solvents on the tested pathogenic bacterial cultures (Tables 8 and 9). The results showed that the harmful bacterial strains were tested, with the highest inhibition observed against K. pneumoniae (18.2 mm), the lowest against S. aureus (10 mm), and moderate inhibition against the remaining pathogens (Fig. 2 & Table 10)

Table 8

Minimal Inhibitory Concentrations (MIC) of methanol extract from *T. erythraeum* against pathogenic bacterial strains
S. No	Name of the Strains	15.6 µg/mL	31.25 µg/mL	62.5 µg/mL	125.0 µg/mL	250.0 µg/mL	500.0 µg/mL	(+) ve C	(-) ve C
1.	E. coli	+++	++	++	*	––	––	––	+++
2.	B. subtilis	+++	++	++	*	––	––	––	+++
3.	K. pneumoniae	+++	+++	++	*	––	––	––	+++
4.	S. aureus	+++	+++	++	++	+	––	––	+++
5.	P. mirabilis	+++	++	++	*	––	––	––	+++
+++ depicts highly turbid; ++ depicts turbid; + indicates cloudiness; *MIC concentration; –– No growth, C- Control.

Table 9

Minimum Bactericidal Concentration (MBC) of methanol extract from *T. erythraeum* against pathogenic bacterial strains.
S. No	Name of the Strains	MBC (µg/mL)
1.	E. coli	250
2.	B. subtilis	250
3.	K. pneumoniae	250
4.	S. aureus	400
5.	P. mirabilis	250

Table 10

Antibacterial activity of methanol extract from *T. erythraeum* against pathogenic bacterial strains.
S. No	Name of the Strains		Zone of inhibition (mm)
		125 µg/mL	250 µg/mL	500 µg/mL	(+) ve C	(-) ve C
1.	Escherichia coli	7.6	11.5	17.2	18.4	––
2.	Klebsiella pneumoniae	8	12	18.2	17.8	––
3.	Bacillus subtilis	7.3	12.2	16.3	16.5	––
4.	Staphylococcus aureus	2	5	10	13.6	––
5.	Proteus mirabilis	4 2	10.4	15.3	17.1	––
mm – Millimetre; C – Control.

3.6 Anticancer potential of T. erythraeum

In the present study, methanol extract was tested against a lung cancer cell line (Fig. 3). A similar dosage was tested with normal L132 cells (data not shown). The cell proliferation of T. erythraeum methanolic extract on both cell lines is depicted in Fig. 3. For A549 cells, after a 24-hour incubation, the extract at a concentration of 25 µg/mL showed the least inhibition, while cells treated with 250 µg/mL demonstrated highest level (92%) and a toxicity value of 125.0 ± 5.0 µg/mL. In contrast, NCI-H460 cell lines showed 97% inhibition at a dose of 300 µg and an IC50 value of 175.0 ± 10.0 µg/mL after 24 hours. Our trials indicated that the percentage of viable cancer cells decreased as the extract treatment increased.

3.7 Functional compositions of T. erythraeum methanolic extract

Figure 4c displays the FTIR spectra analysis of T. erythraeum methanolic extract, showing 13 overall absorption peaks. The absorption peak at 3282.84 cm^− 1 indicates the presence of –C ≡ C–H: C–H stretch representing alkynes (terminal). Absorption peaks at 1625.99, 1519.91, 1448.54, 1394.53, 1230.58, and 1039.63 cm^− 1 are assigned for bends of N–H, and C–H, followed by the O-H, C–N stretch and a N–O asymmetric stretch, representing 1° amines, nitro compounds, alkanes, primary or secondary OH in-plane and aliphatic amines. Absorption peaks at 671.23, 596.00, 551.64, 443.63, and 416.62 cm^− 1 are assigned for = C–H bend, C–Cl stretch, C–Br stretch, and S-S stretch, respectively, representing alkenes, alkyl halides, alkyl halides and aryl disulfides.

3.8 Phytochemical compounds of T. erythraeum analysed in GC-MS

Figure 5 shows the GC-MS results of T. erythraeum methanolic extract. All the phytochemical compounds through the mass spectrum (MS) were identified as the known and unknown compounds listed in the NIST-08 library. GC-MS analysis of T. erythraeum methanolic extract showed the confirmation of Heneicosane, 9,12-Octadecadienoic acid (Z,Z)-, methyl est, 9-Octadecenoic acid, methyl ester, (E)-Methyl stearate, 9-Octadecenoic acid, 1,2,3-propanetriyl ester, OLEIC ACID, 3-HYDROXYPROPYL EST, HEXADECANOIC ACID, 2-HYDROXY-1, 6,9-OCTADECADIENOIC ACID, METHY, 9-Octadecenamide, (Z)-9,12-Octadecadienoic acid (Z,Z)-, 2,3-dihydr, Oleoyl chloride, BICYCLO[10.1.0] TRIDEC-1-ENE, 9-Octadecenoic acid, 1,2,3-propanetriyl ester, (R)-(-)-14-Methyl-8-hexadecyn-1-ol, Glycidol stearate, Methyl (Z)-5,11,14,17-eicosatetraenoate, Tricyclo[20.8.0.0(7,16)] triacontane, 1(22),7(Butyl 9,12,15-octadecatrienoate, Triarachine and Piperidine, 1-[5-(1,3-benzodioxol-5-yl)-1-ox as some of the significant bulk of phytochemical compounds. The major phytocompounds found in the T. erythraeum methanolic extract, respective retention time, molecular weight, and molecular formula are listed in Table 11.

Table 11

Key chemical constituents identified in methanol extract from *T. erythraeum* extract
Peak#	R. Time	Area	Area%	Compound Name
1.	27.526	1482967	2.47	Heneicosane
2.	38.468	217865	0.36	9,12-Octadecadienoic acid (Z,Z)-, methyl est
3.	38.620	568096	0.95	9-Octadecenoic acid, methyl ester, (E)-
4.	39.135	15537	0.03	Methyl stearate
5.	39.419	12428592	20.73	9-Octadecenoic acid, 1,2,3-propanetriyl ester
6.	39.630	261515	0.44	OLEIC ACID, 3-HYDROXYPROPYL EST
7.	41.969	2283871	3.81	HEXADECANOIC ACID, 2-HYDROXY-1,
8.	42.835	245494	0.41	6,9-OCTADECADIENOIC ACID, METHY
9.	42.942	482492	0.80	9-Octadecenamide, (Z)-
10.	43.828	342974	0.57	9,12-Octadecadienoic acid (Z,Z)-, 2,3-dihydr
11.	43.917	609768	1.02	Oleoyl chloride
12.	44.520	8785565	14.65	BICYCLO[10.1.0]TRIDEC-1-ENE
13.	44.603	17839905	29.76	9-Octadecenoic acid, 1,2,3-propanetriyl ester
14.	44.702	6571364	10.96	(R)-(-)-14-Methyl-8-hexadecyn-1-ol
15.	44.966	3627972	6.05	Glycidol stearate
16.	45.204	1077231	1.80	Methyl (Z)-5,11,14,17-eicosatetraenoate
17.	45.265	251463	0.42	Tricyclo[20.8.0.0(7,16)]triacontane, 1(22),7(
18.	45.837	1077312	1.80	Butyl 9,12,15-octadecatrienoate
19.	47.665	465700	0.78	Triarachine
20.	47.958	1316903	2.20	Piperidine, 1-[5-(1,3-benzodioxol-5-yl)-1-ox
		59952586	100.00

The application of chemical insecticides can lead to insecticide resistance and have adverse effects on the environment and public health. Therefore, there is an urgent need to expedite research into alternative, environmentally friendly strategies for mosquito control [21]. Consequently, this study aimed to assess the anti-mosquitocidal activity of various extracts of T. erythraeum against selected mosquito species such as As, Aa, and Cq. The findings indicate that the methanol extract of T. erythraeum exhibits insecticidal properties against mosquitoes.

Among the main methods used in mosquito control programmes are larvicides, which kill mosquito larvae in their breeding grounds before they have a chance to develop into adulthood and spread. Once mosquitoes reach adulthood and take to the air, they become challenging to track and eliminate. Therefore, the most effective approach to mosquito control is targeting them during the larval and immature stages (larvae and pupae), as these stages are particularly susceptible to control measures [22]. Naturally, certain algae species contain fatty acids and polyunsaturated fatty acids that exhibit significant toxicity to mosquito larvae [21, 23]. In the present investigation, the T. erythraeum methanol extract showed good larvicide activity against mosquito larvae, followed by hexane, petroleum ether, and ethanol. The larval mortality LC₅₀ values of methanol extract were 38.62, 40.29, and 43.22 µg/mL against As, Aa, and Cq, respectively. This result showed that T. erythraeum methanol extract might have a complex mixture of bioactive compounds, which could contribute jointly or independently to the mortality and delayed larvae growth. The current findings align with those of Sharanappa et al. 2023 [24], who reported that the cyanobacteria Nostoc muscorum exhibited the lowest LC50 values of 49.09 and 61.37 ppm against hexane extract on second-instar larvae of Spodoptera frugiperda while using the diet overlay method of bioassay. However, in our study, the methanol extract demonstrated significant larvicidal activity against selected mosquito larvae, likely due to the higher solubility of insecticidal bioactive compounds in the methanol extract compared to other extracts. Additionally, previous reports documented the larvicidal activity of microalgae such as Chlorella sp. and Chlorococcum sp. against the dengue vector [25]. The most frequently utilized tool in mosquito control is synthetic insecticides, which have many advantages like speedy action, easy application, etc. However, synthetic pesticides are non–non-biodegradable, and the continuous application of these insecticides leads to resistance in mosquitoes and affects non-target organisms [26]. The present study of adult mortality LC₅₀ values of T. erythraeum methanol extract were 49.37, 52.49, and 55.80 µg/mL for As, Aa, and Cq, respectively. The methanol extract of T. erythraeum may kill adult mosquitoes at low concentrations by inhibiting digestion or altering the behavior, growth, development, and cytotoxic nature. Further, reported studies elicited those microalgae like Chlorella sp. and Chlorococcum sp. showed good mosquitocidal activity against dengue vectors [25].

The egg stage remains one of the least understood phases of mosquito development, and comprehending it could lead to innovative approaches to mosquito control [27]. In our study, the hatchability rate of eggs significantly decreased (100%) upon treatment with the methanol extract, particularly at concentrations of 20 and 25 µg/mL. This finding indicates that the presence of secondary metabolites in T. erythraeum can disrupt embryonic development and hinder the survival of larvae within the egg. Previously, Golawska et al. (2014) demonstrated that bioactive compounds have the potential to disrupt embryo development, impair larval survival within the egg, or inhibit egg hatching [28].

Repeated vaccinations and applications of chemical mosquito control agents could result in adverse effects on the non-targeted organisms. This effect may result in the deterioration of the environmental ecosystem quality of the treated habitats [28]. To know the toxic impact of T. erythraeum methanol extract on non-target organisms, in the present study, aquatic insects like D. indicus and A. bouvieri and the fish G. Affinis were utilised. The aquatic insect mortality LC₅₀ values were 799.16 and 791.32 µg/mL for D. indicus and A. bouvieri. In contrast, the fish G. affinis mortality LC₅₀ value was 745.30 µg/mL when exposed to methanol extract of T. erythraeum, respectively. These results presented that the methanol extract showed less toxicity on non-target organisms, and LC₅₀ value ranged from 745 to 800 µg/mL for selected non-selected organisms, which is 14-fold higher than larvae mortality of LC₅₀ value for selected mosquitoes. Reports from Tufan-Cetin and Cetin [29] previously observed that both micro and macroalgae components exhibit significantly lower toxicity to non-target organisms compared to many pesticide groups. Additionally, they noted that the degradation processes of these components in the aquatic environment are relatively brief.

Previous reports on antibacterial study [30] stated that the ethanol extract of T. erythraeum demonstrated broad-spectrum antibacterial action against all tested pathogenic bacterial strains, in which Salmonella typhi and Staphylococcus aureus exhibited the largest zones of inhibition (15 mm and 12.33 mm) respectively. The extracts of methanol, hexane and chloroform showed significant inhibitory action (9.3–13.5 mm) against every pathogen, whereas the extract of acetone showed very little activity (9–12 mm). Extractions of Trichodesmium erythraeum in organic solvents (hexane and ethyl acetate) were screened in vitro against bacteria. Bacteria including B. subtilis (12 mm), S. aureus (12 mm), Enterococcus faecalis (16 mm), S. typhi (12 mm), P. aeruginosa (10 mm), P. vulgaris (14 mm), and Erwinia sp (9 mm) were all susceptible to the effects of hexane extract [31]. It is well known that cyanobacteria are abundant producers of secondary metabolites with a variety of structural variations and biological uses. Natural substances produced from freshwater and marine cyanobacteria have broad-spectrum antibacterial capabilities, albeit some may be hazardous [32]. Nevertheless, a clear connection between particular chemical groups of cyanobacterial metabolites and their bioactivities such as cytotoxicity, mortality, and antibacterial activity remains unclear. It was suggested that saturated fatty acids may interact with the cell membranes of bacteria, thereby upsetting physiological processes linked to antimicrobial defense and radical scavenging. Further research is necessary to fully comprehend the mechanisms underlying cyanobacterial metabolites and the structural characteristics that are associated with their bioactivity in order to produce novel lead molecules for the pharmaceutical industry [33].

The potential of marine cyanobacteria as a source of new anticancer drugs has attracted a lot of attention. Certain chemicals have been useful templates for the development of next-generation anticancer medicines, even beyond their proven cytotoxicity in tumor cell lines. Four cyanobacterial isolates, Nostoc punctiforme, Oscillatoria sp., Arthrospira platensis, and Leptolyngbya sp., exhibited dose-dependent cytotoxicity against the Hep-G2 human liver cancer cell line, with IC50 values ranging from 12.75 to 15.0 µg/mL [34]. This effect was observed within 48 hours of incubation across all tested cell lines, with the most pronounced reductions in cell viability seen in HT29 (colon cancer) and Hep-G2 cells (61.38 ± 3.26% and 62.78 ± 2.13%, respectively) [35]. These findings suggest varying degrees of antiproliferative and cytotoxic activity of cyanobacterial extracts against different cancer cell lines. Supporting this, extracts from Lyngbya carotinosum and Nodularia linckia displayed significant inhibitory effects on C6 glioma cells, with IC50 values of 112.69 and 121.48 µg/mL, respectively [36]. Furthermore, A549 lung cancer cells exhibited a dose-dependent response, with cell proliferation inhibited by 25–80% upon treatment with increasing concentrations (12.5 to 1000 µg/mL) of the cyanobacterial extracts [37]. The exact mechanisms behind the cytotoxicity of marine cyanobacterial chemicals are still mostly unknown. However, the research points to a possible function of apoptotic induction. Several apoptotic hallmarks such as cell cycle arrest, mitochondrial dysfunction, oxidative stress, modifications in the caspase cascade, changes in the expression levels of particular proteins, and disruptions in the dynamics of membrane sodium, have been observed in treated tumor cells, providing evidence in favor of this association [38].

Analysis by FTIR revealed the presence of alkanes, nitrile, primary amines, nitro compounds, primary or secondary OH in-plane, aliphatic amines, alkenes, alkyl halides, and aryl disulfides in methanol extract of T. erythraeum. These functional groups may play a vital role in the insecticide properties of T. erythraeum methanol extract. The fictional groups identified through the FTIR spectrum of the extract correlate with the compounds identified through GC-MS. GC-MS analysis helped retrieve many important organic volatile compounds of pharmaceutical importance in the present study. In T. erythraeum methanolic extract had Bicyclo [10.1.0] tridec-1-ene, 9-octadecenoic acid, 1,2,3-propanetriyl ester, (E, E, E)-, R)-(-)-14-methyl-8-hexadecyn-1-ol and glycidol stearate as some of the significant bulk of phytochemical compounds. The 9-octadecenoic is reported in other cyanobacteria like the methanol extract of Phormidium sp. [39] and Hypersaline Oscillatoria Salina [40], also Kalasariya et al. (2021) was reported 1,2,3-propanediol ester is presented in methanol extract of green algae Chaetomorpha crassa [41]. Further, the glycidol stearate was present in Mentha spicata leaves [42]. The present study identifies that the extract of cyanobacteria could be used to strengthen antibiotics and has potential future applications as an anticancer agent.

The present findings demonstrate that T. erythraeum methanol extract exhibits significant mosquitocidal properties against A. stephensi, A. aegypti, and C. quinquefasciatus. The LC50 and LC90 concentrations for larvicidal and adulticidal activity showed no adverse effects on aquatic organisms. Additionally, the extract displayed strong cytotoxic effects against lung cancer cells. These results provide substantial evidence that T. erythraeum methanol extract is both eco-friendly for aquatic organisms and toxic to lung cancer cells. Further research is needed to evaluate the in vivo effects of T. erythraeum methanol extract before it can be widely used in medicine, either alone or as carriers for other medications.

Author contribution

Surya- Conceptualization, Writing Original draft, Methodology, Data Curation, Supervision. Seema- Formal analysis, writing review and editing. Salmen and Aljawdah -Writing review and editing, Funding acquisition, Formal analysis, Data Curation. Sundaramanickam- Writing review and editing, Formal analysis.

Funding

The authors extend their appreciation to the Researchers Supporting Project number (RSP2024R385), King Saud University, Riyadh, Saudi Arabia for financial support. Acknowledgements

The authors would like to acknowledge the authorities of King Saud University, Saudi Arabia, and Saveetha University, Tamil Nadu, India, for providing necessary facilities.

Ethical approval

Not applicable.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

Data will be made available on request.

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Valorization of bioactive metabolites from Trichodesmium erythraeum bloom biomass for vector borne disease control, bactericidal and anticancer therapy

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1 Collection of marine microalgae

2.2 Preparation of different extracts

2.3 Collection of eggs and maintenance of vectors

2.4 Larvicidal bioassays

2.5 Adulticidal and Ovicidal activity

2.6 Environmentally Friendly Toxicity Assay

2.7 Bactericidal activity

2.7.1Pathogenic bacterial strains

2.7.2 Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) determination

2.7.3 Antibacterial assay

2.8 Cell lines and culture conditions

2.8.1 Cytotoxicity valuation assay

2.9 Instrumentation

2.10 Statistical Analysis

3. Results

3.1 Larvicidal activity

3.2 Adulticidal bioassay

3.3 Ovicidal bioassay

3.4 The impact of T. erythraeum methanol extract on field-collected aquatic insects and fish

3.5 Antibacterial activity

3.6 Anticancer potential of T. erythraeum

3.7 Functional compositions of T. erythraeum methanolic extract

3.8 Phytochemical compounds of T. erythraeum analysed in GC-MS

4. Discussion

Conclusion

Declarations

References

Status:

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