TY - JOUR
AU - Farooq,, Muhammad
AB - Abstract Mosquitoes transmit serious diseases, which threaten humans and severely affect livestock. The half-lethal concentration (LC50) was calculated by log probit analysis. The LC50 and LC90 values of larvicidal activity of Cassia fistula Linn. hexane-methanol soluble fraction (HMSF) after 24 h of exposure were 21.04 and 34.68 µg/ml, respectively. The LC50 values after 24 h of exposure were 84.09 µg/ml and 108.08 µg/ml for chloroform–methanol soluble fraction (CMSF) and ethyl acetate-methanol soluble fraction (EMSF) respectively. The percent hatchability of eggs exposed to the hexane extract was 90 ± 5.0, 68.33 ± 7.6, 46.6 ± 11.5, 10 ± 0.0, and 0 ± 0.0% at 10, 20, 40, 60, and 80 ppm, respectively. The pupicidal activity of the hexane extract at 40 µg/ml was 0.0%. The LC50 value of adulticidal activity of the hexane extract was 12.8 mg/test tube. The biosafety of the hexane extract was assessed in nontarget organisms, i.e., zebrafish (Danio rerio) embryos and normal lung cells (BEAS-2B). The hexane extract of C. fistula was well tolerated by zebrafish embryos, and no mortality or toxicity was found in the embryos exposed to the highest tested concentration of 300 µg/ml. Similarly, all the concentrations tested against the normal lung cells (BEAS-2B) showed more than 95% survival. The gas chromatography–mass spectroscopy analysis identified 12 compounds, and 2-methyl hexanoic acid and 2-methyl butanoic acid were the major compounds identified in the hexane extract. The larvicidal activity of C. fistula extracts will help in the development of natural substitutes for vector management of mosquito populations. larvicidal, chromatography, zebrafish, lung cell Mosquitoes transmit many serious diseases, such as Zika virus infection, filariasis, West Nile virus disease, chikungunya, yellow fever, dengue, and malaria, which threaten humans and have severe effects on livestock (Elumalai et al. 2013). Mosquito species that belong to genera Culex, Aedes, and Anopheles are vectors for pathogens that contribute significantly to social suffering and poverty (Jiang et al. 2009). Culex pipiens is a hematophagous mosquito and a vector for many infectious diseases affecting humans (Vinogradova 2000). It is widely distributed in Europe, Africa, Asia, North America, South America, and Australia. In Saudi Arabia, the Cx. pipiens species is found in Al-Hofuf, Ash-Shu’bah, and in the eastern and southwestern regions of Saudi Arabia. It is a common mosquito species in the Riyadh district (Ahmed et al. 2011). It plays a critical role in the transmission of the Sindbis/Sindbis-like viruses (Togaviridae), Usutu virus (Flaviviridae), and West Nile virus (Flaviviridae). Furthermore, evidence shows that Cx. pipiens is refractory to Zika virus transmission (Aliota et al. 2016, Amraoui et al. 2016, Boccolini et al. 2016, Hart et al. 2017, Huang et al. 2016, Weger-Lucarelli et al. 2016, Heitmann et al. 2017, Kenney et al. 2017, Liu et al. 2017), while some other reports present conflicting information (Guo et al. 2016, Guedes et al. 2017). Synthetic insecticides have generally been questioned due to their harmful effects on humans and animals, increasing rates of resistance in mosquitoes, environmental sustainability, non-biodegradable nature, and cost of synthesis (Chowański et al. 2014). An alternative for the same is the use of secondary metabolites derived from plants as substitutes for synthetic insecticides. Plants synthesize a broad range of bioactive secondary metabolites (Bernhoft 2008), which are promising substitutes for synthetic insecticides used to control mosquito populations (Rahuman et al. 2009). Many household insecticide sprays contain pyrethrins, a class of plant-derived chemicals extracted from chrysanthemum flowers. However, insecticide-derived plants are not always safe, as they may cause life-threatening problems if inhaled. Frequent spraying of insecticides exposes people to various doses of these chemicals, mainly if it is done in an enclosed area; thus, there is a need to assess the toxicity of these insecticides on the lung tissue. Plant-derived insecticides are more promising than synthetic insecticides because of easier biodegradation, target specificity, low cost, and safety for humans, animals, and the environment (Rambabu et al. 2014). Approximately 2,000 plant species have insecticidal activity (Ghosh et al. 2012). Active compounds such as azadirachtin, isolated from neem, have been studied for their mosquitocidal potential (Isman 2006). Several other plant species, such as Tagetes minuta L. (Asterales: Asteraceae) (Perich et al. 1994), Curcuma domestica L. (Zingiberales: Zingiberaceae) (Ranaweera 1997), Piper longum L. (Piperales: Piperaceae) (Lee 2000), Piper nigrum L. (Piperales: Piperaceae) (Park et al. 2002), and Lantana camara L. (Lamiales: Verbenaceae) exhibit excellent larvicidal properties against mosquitoes (Hari and Mathew 2008). Cassia fistula Linn. of the family Leguminosae, is used to treat various illnesses, including ringworm and other skin infections (Rajan et al. 2001), diarrhea, fever, abdominal pain, and leprosy (Perry 1980). C. fistula possesses anti-inflammatory (Danish et al. 2011), hepatoprotective (Mukherjee et al. 1999), hypoglycemic (Bhakta et al. 1997), anticancer (Irshad et al. 2014), and antioxidant activities (Gupta et al. 2015). This study was designed to assess the larvicidal, ovicidal, pupicidal, and adulticidal activities of C. fistula fruit extract against Cx. pipiens under laboratory conditions. Furthermore, we aimed to characterize the chemical composition of the active extract. The resulting knowledge should be valuable as an aid to develop natural substitutes to improve vector management of mosquito populations that are resistant to synthetic chemical insecticides. Materials and Methods Plant Collection The fruits of C. fistula were purchased from a herbal shop in Riyadh, Kingdom of Saudi Arabia. The plant was authenticated in the Department of Botany, King Saud University, Riyadh, Saudi Arabia. A voucher specimen (KSU-BR-035) was deposited at the Bioproduct Research Chair, King Saud University, Riyadh, Saudi Arabia for future reference. Preparation of Extracts The pods of C. fistula were shade-dried for 7 d in a well-ventilated area at 30 ± 2°C. The pods of C. fistula were powdered using a commercial blender (Stardust, Japan). The finely ground pods (100 g) were placed in a cellulose thimble (28 × 100 mm - thickness 1.5 mm) and extracted using a Soxhlet apparatus (Korea) with 450 ml hexane. The same material was sequentially reextracted in ethyl acetate, chloroform, methanol, and water (450 ml). The extraction process was continued until the solvent color was clear. The extracts were evaporated using a rotary evaporator (Heidolph, Germany) at 150 rpm at a temperature of 45°C. The dried extracts of hexane, chloroform, and ethyl acetate were further fractionated by re-dissolving each fraction in methanol (300 ml). This extraction resulted in two fractions: methanol-soluble and methanol-insoluble fractions. The methanol-soluble fraction was called the hexane-methanol soluble fraction (HMSF), and the methanol-insoluble fraction was called the hexane-methanol insoluble fraction (HISF). The same procedure was repeated for the chloroform and ethyl acetate extracts as shown in Fig. 1. All extracts were filtered using Whatman No. 1 filter paper. The extracts were evaporated using a rotary evaporator at 150 rpm at a temperature of 45°C. The collected extracts were weighed and stored in glass vials at 4°C until further use. Fig. 1. Open in new tabDownload slide Procedure for the extraction of larvicidal active fraction from Cassia fistula fruit extract against Culex pipiens. The plant was extracted with Soxhelt apparatus using four different solvents of different polarity. The arrow indicates that the dry weight of each solvent obtained was further extracted with methanol to obtained two fraction of which one is soluble in methanol and the other is insoluble in methanol. Fig. 1. Open in new tabDownload slide Procedure for the extraction of larvicidal active fraction from Cassia fistula fruit extract against Culex pipiens. The plant was extracted with Soxhelt apparatus using four different solvents of different polarity. The arrow indicates that the dry weight of each solvent obtained was further extracted with methanol to obtained two fraction of which one is soluble in methanol and the other is insoluble in methanol. Mosquito Colonies Laboratory colonies of Cx. pipiens larvae were obtained from the Zoology Department, King Saud University, Riyadh, Saudi Arabia. Tap water was used for growing the larvae. The larvae were fed with fish feed (Tetra GmbH, Germany). The adults that emerged from the larvae were maintained in a wooden cage (25 × 35 × 6 cm) and fed with 10% sucrose (Loba Chemie, India) solution and mouse blood (Swiss albino). Eggs were collected after 2–3 d in a dish containing 100 ml of tap water. Mosquitoes were maintained under 12:12 (L:D) h photoperiod cycles at 28 ± 2°C. Larvicidal Bioassay Each test extract was individually added to 6-well plates (Corning Inc., NY) containing 6 ml of test solution. Initially, the third instar larvae were treated with different concentrations to assess the larvicidal activity range (between 0 and 100% mortality after 24 or 48 h of exposure). In total, three replicates of 20 larvae each were maintained at each concentration. The test extract (dissolved in methanol) was added to the wells, left to evaporate, and reconstituted in water. Methanol was added to the control wells to maintain solvent identity with the test solutions dissolved in methanol. All extracts that were insoluble in methanol were discarded due to poor solubility in water. Experiments were carried out at 28 ± 2°C. The number of dead larvae was counted and the mortality percentage was calculated (Al-Mekhlafi et al. 2018). Ovicidal Bioassay The egg rafts of Cx. pipiens were collected and treated with different concentrations of ethyl acetate fruit extract of C. fistula ranging from 10 to 60 ppm. The viability of the freshly laid eggs were assessed using stereomicroscope. About 30–40 viable eggs were collected for each concentration tested. After treatment, the eggs were moved to tap water cups for hatching assessment. Eggs were counted under a microscope. Each experiment was replicated three times. The hatching rate was assessed post-treatment (48 h) by the following formula (Su and Mulla 1999). %ofeggmortality=No.ofhatchedlarvaeTotalno.ofeggs Pupicidal Bioassay Mosquito pupae were used to observe the pupicidal activity of ethyl acetate fruit extract of C. fistula. Twenty freshly emerged pupae were kept in a 6-well plate containing 3 ml of tap water and the desired extract concentration (10, 20, 30, and 40 µg/ml) was prepared. At each tested concentration, three experimental replicates were evaluated, each containing 20 pupae. The control prepared as described above. The numbers of dead pupae were counted, and the percentage of mortality was calculated after 24 h of exposure. Adulticidal Bioassay The adulticidal bioassay was carried out using a 50 ml clean sterile glass test tube. The test tube was coated with different amounts of extracts (2.5, 5, 10, 15, 30, 60, 80, 100 µg) and allowed to dry (CDC 2018). Test tubes coated with solvent alone were used as controls. Three replicates were maintained per concentration. Using an automatic aspirator, 20 unfed female mosquitoes were released into each test tube. Mosquitoes were considered dead if they could no longer stand. All bioassays were carried out at 28 ± 2°C and relative humidity of 80 ± 2%. Mosquito mortality was determined 24 h post-exposure. The knocked-down mosquitoes were kept in a recovery jar providing with 10% sucrose solution to assess recovery/ mortality at 24-h period. Experiments were performed in triplicates with controls. LD50 values were determined using OriginPro 8.5 software. Embryo Toxicity and Teratogenicity Test Adult zebrafish were kept in Department of Zoology, King Saud University, and maintained as per the IAUAC and national guidelines for care and use of laboratory animals. Zebrafish embryos were obtained by natural pairwise breeding. Freshly fertilized eggs were harvested by syphoning, washed with distilled water, and transferred to sterile glass Petri dishes containing distilled water supplemented with 0.06% sea salt and 0.003% trypan blue. The embryos were screened under dissecting microscope and any unfertilized or developmentally abnormal eggs were removed. The extract was dissolved in methanol to make a stock concentration of 10 µg/ml. The required amount of final dilution was prepared in distilled water and 4 ml of this water was added to sterile 35-mm glass Petri dishes. Zebrafish embryos (25–30) were transferred by plastic Pasteur pipettes into 1 ml of distilled water thereby bringing the final volume to 5 ml in each 35 mm Petri dish. The embryos were transferred to an air incubator at a temperature of 29°C and incubated overnight. The mortality and embryonic development was monitored under a dissection microscope on the following day and thereafter every 24 h. The images were captured using Zeiss Observer D1 inverted microscope using Zeiss ZEN software. Cell Viability Bioassay BEAS-2B human lung cells (ATCC CCL-185) cells were obtained from the American Type Culture Collection (Manassas, VA). BEAS-2B cells were maintained in DMEM media supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) antibiotic/antimycotic cocktail (100 U/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B (Invitrogen, Carlsbad, CA) at 37°C and 5% CO2. The cells were treated with three different concentrations from 25 μg/ml to 100 μg/ml along with control (0.01% methanol). MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was used to assess the cell viability based on the conversion of MTT to formazan crystals in mitochondria by dehydrogenase enzyme. The absorbance (abs) was read in a multi-plate reader (Biochem Ltd, England) at 490 nm. The percent survival was calculated using the following formula: %survival=sampleabs/controlabs×100. Light Microscopy For untreated and treated BEAS-2B cells, the images were captured after 24 h using a phase acontrast microscope (Leica, Germany) to study the effect of the extracts on the morphology of the cells. Hoechst 33342 Staining BEAS-2B cells were treated with bioactive extract from the pods of C. fistula in 24-well plates for 24 h, and the cells were fixed and stained with the Hoechst 33342 (Life technology) at 37 °C for 15 min, in the dark. The cells were then washed three times with PBS and imaged using a fluorescence microscope (EVOS, ThermoFisher, Waltham, MA). This study was carried out to study the effect of the extracts on the nuclear morphology of the cells. Analysis of Hexane Extracts by Gas Chromatography–Mass Spectrometry Gas Chromatography–Mass Spectrometry (GC-MS) analysis of bioactive extract from the pods of C. fistula was performed using an Agilent GC-MS system (Agilent Technologies, Santa Clara, CA) equipped with an HP-88 capillary column (100 m in length × 250 μm in diameter × 0.20 μm film thickness). Helium (99.99%) was used as the carrier gas (flow rate 1 ml/min). Initially, the temperature was set at 50°C, then ramped at 5°C/min to 250°C. One microliter of the prepared extract was injected and subjected to full scan analysis (50–650 m/z). Relative quantities of phytocompounds were expressed as percentages based on peak areas in the chromatogram. Phytocompounds were identified using the National Institute Standard and Technology (NIST) library database contained in the GC-MS software. Statistical Analyses The data was analyzed using t-test and one-way analysis of variance (ANOVA). Significant differences between treatments were determined using Tukey’s tests (P ≤ 0.05). The LC50 and LC90 values were calculated using probit analysis (Finney 1971). Results The yields of dried material extracted using hexane, chloroform, ethyl acetate, methanol, and water are shown in Fig. 1. Water (101 g) extracted the largest quantity of plant material, whereas the lowest yield was obtained in the hexane extract (0.01 g), which was a yellow oil with an aromatic smell. Larvicidal Bioassay The larvicidal activities of HMSF, CMSF, and EMSF fruit extracts of C. fistula against third instar of Cx. pipiens are presented in Tables 1 and 2. The methanol and water extracts did not show activity. However, the best efficacy was exhibited by the HMSF extract of C. fistula against the larvae of Cx. pipiens. Mortality increased with the increasing concentrations of HMSF with LC50 and LC90 values 21.04 and 34.68 µg/ml, respectively after 24 h of exposure. CMSF and EMSF extracts induced mortality after 24 h of exposure with LC50 values of 84.0 and 108.0 µg/ml, respectively. No mortality was detected in the controls. The obtained results showed a significant (P < 0.05) increase in the ‘larvicidal’ activity as compared to the control Table 1. Larvicidal activities of Cassia fistula CMSF and EMSF of Cassia fistula fruit extract against third instar larvae of Culex pipiens Extracts . Mortality (%) . . Chloroform . Ethyl acetate . Concentration (ppm) 24 48 24 48 33 0 ± 00d 20 ± 5.77c 0 ± 00d 6.67 ± 3.33d 66 23.33 ± 3.33c 43.33 ± 3.33b 0 ± 00d 20 ± 00c 100 60 ± 5.77b 86.67 ± 3.33a 26.67 ± 3.33c 73.33 ± 3.33b 134 96.67 ± 3.33a 100 ± 00a 63.33 ± 3.33b 96.67 ± 3.33a 150 100 ± 00 - 100 ± 00a 100 ± 00a Control 0 ± 00d 3.33d 0 ± 00d 6.67 ± 3.33d F-value 225.12 225.12 225.12 225.12 P-value 0.00 0.00 0.00 0.00 LD50 (LCL-UCL) 84.09 (72.49–97.54) 56.69 (43.97–73.09) 108.08 (90.15–131.77) 82.89 (71.67–95.86) LD90 (LCL-UCL) 117.30 (102.12–136.07) 106.70 (82.76–137.57) 167.72 (138.73–202.77) 113.44 98.09–131.19)) Extracts . Mortality (%) . . Chloroform . Ethyl acetate . Concentration (ppm) 24 48 24 48 33 0 ± 00d 20 ± 5.77c 0 ± 00d 6.67 ± 3.33d 66 23.33 ± 3.33c 43.33 ± 3.33b 0 ± 00d 20 ± 00c 100 60 ± 5.77b 86.67 ± 3.33a 26.67 ± 3.33c 73.33 ± 3.33b 134 96.67 ± 3.33a 100 ± 00a 63.33 ± 3.33b 96.67 ± 3.33a 150 100 ± 00 - 100 ± 00a 100 ± 00a Control 0 ± 00d 3.33d 0 ± 00d 6.67 ± 3.33d F-value 225.12 225.12 225.12 225.12 P-value 0.00 0.00 0.00 0.00 LD50 (LCL-UCL) 84.09 (72.49–97.54) 56.69 (43.97–73.09) 108.08 (90.15–131.77) 82.89 (71.67–95.86) LD90 (LCL-UCL) 117.30 (102.12–136.07) 106.70 (82.76–137.57) 167.72 (138.73–202.77) 113.44 98.09–131.19)) Value represents mean ± SD of three replicates. Mortality of the larvae recorded after 24 and 48 h of exposure. Different letters in each column represent statistically significant differences at P < 0.05 using one-way ANOVA and followed by Tukey’s test. Control groups were treated with no extract. LC50, lethal concentration showing 50% mortality; LC90, lethal concentration showing 90% mortality; LCL, lower confidence limits; UCL, upper confidence limits. Open in new tab Table 1. Larvicidal activities of Cassia fistula CMSF and EMSF of Cassia fistula fruit extract against third instar larvae of Culex pipiens Extracts . Mortality (%) . . Chloroform . Ethyl acetate . Concentration (ppm) 24 48 24 48 33 0 ± 00d 20 ± 5.77c 0 ± 00d 6.67 ± 3.33d 66 23.33 ± 3.33c 43.33 ± 3.33b 0 ± 00d 20 ± 00c 100 60 ± 5.77b 86.67 ± 3.33a 26.67 ± 3.33c 73.33 ± 3.33b 134 96.67 ± 3.33a 100 ± 00a 63.33 ± 3.33b 96.67 ± 3.33a 150 100 ± 00 - 100 ± 00a 100 ± 00a Control 0 ± 00d 3.33d 0 ± 00d 6.67 ± 3.33d F-value 225.12 225.12 225.12 225.12 P-value 0.00 0.00 0.00 0.00 LD50 (LCL-UCL) 84.09 (72.49–97.54) 56.69 (43.97–73.09) 108.08 (90.15–131.77) 82.89 (71.67–95.86) LD90 (LCL-UCL) 117.30 (102.12–136.07) 106.70 (82.76–137.57) 167.72 (138.73–202.77) 113.44 98.09–131.19)) Extracts . Mortality (%) . . Chloroform . Ethyl acetate . Concentration (ppm) 24 48 24 48 33 0 ± 00d 20 ± 5.77c 0 ± 00d 6.67 ± 3.33d 66 23.33 ± 3.33c 43.33 ± 3.33b 0 ± 00d 20 ± 00c 100 60 ± 5.77b 86.67 ± 3.33a 26.67 ± 3.33c 73.33 ± 3.33b 134 96.67 ± 3.33a 100 ± 00a 63.33 ± 3.33b 96.67 ± 3.33a 150 100 ± 00 - 100 ± 00a 100 ± 00a Control 0 ± 00d 3.33d 0 ± 00d 6.67 ± 3.33d F-value 225.12 225.12 225.12 225.12 P-value 0.00 0.00 0.00 0.00 LD50 (LCL-UCL) 84.09 (72.49–97.54) 56.69 (43.97–73.09) 108.08 (90.15–131.77) 82.89 (71.67–95.86) LD90 (LCL-UCL) 117.30 (102.12–136.07) 106.70 (82.76–137.57) 167.72 (138.73–202.77) 113.44 98.09–131.19)) Value represents mean ± SD of three replicates. Mortality of the larvae recorded after 24 and 48 h of exposure. Different letters in each column represent statistically significant differences at P < 0.05 using one-way ANOVA and followed by Tukey’s test. Control groups were treated with no extract. LC50, lethal concentration showing 50% mortality; LC90, lethal concentration showing 90% mortality; LCL, lower confidence limits; UCL, upper confidence limits. Open in new tab Table 2. Larvicidal activities of Cassia fistula HMSF extract against third instar larvae of Culex pipiens Concentration (ppm) . Mortality (%) . . 24 . 48 . 5 0 ± 00d 13.33 ± 3.33d 15 30 ± 5.77c 33.33 ± 3.33c 25 43.33 ± 3.33b 60 ± 5.77b 35 93.33 ± 3.33a 100 ± 00a Control 00 ± 00d 3.33 ± 3.33d F-value 225.12 225.12 P-value 0.00 0.00 LD50 (LCL-UCL) 21.04 (17.19–25.76) 15.80 (9.47–26.35) LD90 (LCL-UCL) 34.68 (29.09–80.79) 33.86 (27.66–41.45) Concentration (ppm) . Mortality (%) . . 24 . 48 . 5 0 ± 00d 13.33 ± 3.33d 15 30 ± 5.77c 33.33 ± 3.33c 25 43.33 ± 3.33b 60 ± 5.77b 35 93.33 ± 3.33a 100 ± 00a Control 00 ± 00d 3.33 ± 3.33d F-value 225.12 225.12 P-value 0.00 0.00 LD50 (LCL-UCL) 21.04 (17.19–25.76) 15.80 (9.47–26.35) LD90 (LCL-UCL) 34.68 (29.09–80.79) 33.86 (27.66–41.45) Value represents mean ± SD of three replicates. Mortality of the larvae recorded after 24 and 48 h of exposure. Control groups were treated with no extract. Different letters in each column represent statistically significant differences at P < 0.05 using one-way ANOVA and followed by Tukey’s test. LC50, lethal concentration 50% mortality; LC90, lethal concentration 90% mortality; LCL, lower confidence limits; UCL, upper confidence limits. Open in new tab Table 2. Larvicidal activities of Cassia fistula HMSF extract against third instar larvae of Culex pipiens Concentration (ppm) . Mortality (%) . . 24 . 48 . 5 0 ± 00d 13.33 ± 3.33d 15 30 ± 5.77c 33.33 ± 3.33c 25 43.33 ± 3.33b 60 ± 5.77b 35 93.33 ± 3.33a 100 ± 00a Control 00 ± 00d 3.33 ± 3.33d F-value 225.12 225.12 P-value 0.00 0.00 LD50 (LCL-UCL) 21.04 (17.19–25.76) 15.80 (9.47–26.35) LD90 (LCL-UCL) 34.68 (29.09–80.79) 33.86 (27.66–41.45) Concentration (ppm) . Mortality (%) . . 24 . 48 . 5 0 ± 00d 13.33 ± 3.33d 15 30 ± 5.77c 33.33 ± 3.33c 25 43.33 ± 3.33b 60 ± 5.77b 35 93.33 ± 3.33a 100 ± 00a Control 00 ± 00d 3.33 ± 3.33d F-value 225.12 225.12 P-value 0.00 0.00 LD50 (LCL-UCL) 21.04 (17.19–25.76) 15.80 (9.47–26.35) LD90 (LCL-UCL) 34.68 (29.09–80.79) 33.86 (27.66–41.45) Value represents mean ± SD of three replicates. Mortality of the larvae recorded after 24 and 48 h of exposure. Control groups were treated with no extract. Different letters in each column represent statistically significant differences at P < 0.05 using one-way ANOVA and followed by Tukey’s test. LC50, lethal concentration 50% mortality; LC90, lethal concentration 90% mortality; LCL, lower confidence limits; UCL, upper confidence limits. Open in new tab The mean percentage of larvicidal activity at various concentrations was statistically significant as compared to the controls. Ovicidal Bioassay The ovicidal activity of HMSF of C. fistula was assessed by evaluating the hatchability of eggs. No ovicidal activity was observed in the control groups. At the highest concentration (80 ppm) of the HMSF extract, 100% ovicidal activity was reported against Cx. pipiens (Fig. 2A). Egg hatchability decreased with increasing concentrations of HMSF extract. The percent hatchability of Cx. pipiens was 90.0 ± 5.0, 68.3 ± 7.6, 46.6 ± 11.5, 10.0 ± 0.0, and 100.0 ± 0.0% at 10, 20, 40, 60, and 0 ppm of C. fistula extracts, respectively (ANOVA, F = 225.120 and P = 0.00). The obtained results showed a significant (P < 0.05) decrease in the hatchability as compared to the control. Fig. 2. Open in new tabDownload slide (A) Percentage hatchability, (B) Percentage pupicidal activity, and (C) Adulticidal activity of the ethyl acetate fruit extract of C. fistula against Culex pipiens using different concentrations after 24 h of exposure. Control groups were treated with no extract. Means followed by asterisks (**) are significantly different at P < 0.05 when compared to control using a t-test. Fig. 2. Open in new tabDownload slide (A) Percentage hatchability, (B) Percentage pupicidal activity, and (C) Adulticidal activity of the ethyl acetate fruit extract of C. fistula against Culex pipiens using different concentrations after 24 h of exposure. Control groups were treated with no extract. Means followed by asterisks (**) are significantly different at P < 0.05 when compared to control using a t-test. Pupicidal Bioassay HMSF exhibited pupicidal activity against Cx. pipiens (Fig. 2B). At concentrations of 40, 30, 20, and 10 µg/ml, the pupicidal mortality observed was 100, 43.3, 25.2, and 0.0%, respectively (ANOVA, F = 225.120 and P = 0.00). The LC50 value of the HMSF extract at 24 h was 31.2 µg/ml. The obtained results showed a significant (P < 0.05) increase in the pupicidal activity as compared to the control. Adulticidal Bioassay HMSF exhibited adulticidal activity against adult Cx. pipiens in a dose-dependent manner. HMSF induced 100% mortality in adult Cx. pipiens at 100 and 80 mg/test tube after 30 min of exposure. We observed that the mosquitoes did not recover even when moved to the recovery jar. The extract was lethal to the mosquitoes with an LD50 value of 12.8 mg/test tube (Fig. 2C). The obtained results showed a significant (P < 0.05) increase in the ‘dulticidal’ activity as compared to the control. Embryo Toxicity and Teratogenicity of HMSF The HMSF did not induce severe toxicity in treated zebrafish embryos. Zebrafish embryos were exposed to HMSF within concentration range of 100, 200, 600 µg/m for 72 h. The toxicity of fruit extract of C. fistula was judged by counting the number of dead embryos in each treatment and was recorded at 24, 48, and 72 h of post-treatment. As indicated by Fig. 3B, the treated embryos did not show significant level of mortality within indicated concentration range. However, zebrafish embryos which were treated with HMSF at ≥300 µg/ml, displayed mild level of developmental delay (Fig. 3A). The untreated embryos reached to ‘5 prim stage’ (embryonic developmental stage in zebrafish embryos at 24 h post-fertilization) after 24 h. However, on the other side, zebrafish embryos, which were treated, with 300 µg/ml of fruit extract of C. fistula were at 15-somite stage (20 h of post-fertilization). This mean that the treated embryos head around 4 h of developmental delay as compared to untreated control embryos. Fig. 3. Open in new tabDownload slide Developmental toxicity of fruit extract of C. fistula extract on zebrafish embryos. (A) Upper panel: Microphotograph of group of live zebrafish embryos either untreated (control) or treated with various concentration of fruit extract of C. fistula. The embryonic development of zebrafish embryos was un-affected when the embryos were exposed to the fruit extract of C. fistula within concentration range between 50 and 200 µg/ml and the embryonic development was very much similar to untreated control embryos, however, the fruit extract of C. fistula at concentration ≥ 300 µg/ml induced a mild level of developmental. The untreated embryos were at 5 prim stage (around 24 h post fertilization), while the development of zebrafish embryos which were treated with 300 µg/ml of fruit extract of C. fistula halted at n 15 somite stage, a stage which is attained at 20 h of post fertilization by zebrafish embryos. (B) Lower panel: line graph showing the percent mortality ratio of zebrafish embryos exposed to various concentration of fruit extract of C. fistula. Fig. 3. Open in new tabDownload slide Developmental toxicity of fruit extract of C. fistula extract on zebrafish embryos. (A) Upper panel: Microphotograph of group of live zebrafish embryos either untreated (control) or treated with various concentration of fruit extract of C. fistula. The embryonic development of zebrafish embryos was un-affected when the embryos were exposed to the fruit extract of C. fistula within concentration range between 50 and 200 µg/ml and the embryonic development was very much similar to untreated control embryos, however, the fruit extract of C. fistula at concentration ≥ 300 µg/ml induced a mild level of developmental. The untreated embryos were at 5 prim stage (around 24 h post fertilization), while the development of zebrafish embryos which were treated with 300 µg/ml of fruit extract of C. fistula halted at n 15 somite stage, a stage which is attained at 20 h of post fertilization by zebrafish embryos. (B) Lower panel: line graph showing the percent mortality ratio of zebrafish embryos exposed to various concentration of fruit extract of C. fistula. The fruit extract of C. fistula had effected the hatching process in treated zebrafish embryos. The embryos usually come out of their chorion and start swimming after 48 h of fertilization. The hatching ability of untreated embryos was not affected and >90% of untreated (control) embryos hatched around 48 h post-fertilization in this study, However, zebrafish embryos which were treated with fruit extract of C. fistula within concentration range of 100–600 µg/ml did not hatch at all at 48 hpf. Effect of C. fistula Fruit Extract on Lung Cell Growth MTT assay was carried out to examine the effect of fruit extract of C. fistula on cell growth. BEAS-2B cells were treated with different concentrations (100–25 µg/ml) of fruit extract of C. fistula for 24 h. As shown in Fig. 4A, the percentage survival of BEAS-2B cells treated with the fruit extract was similar to control. All the concentrations tested showed survival percentage >95%. This was further conformed by light and fluorescence microscopy, wherein the control and treated group, the cells retained their normal shape, adhered tightly to the flask, showed well-formed connections and radiations. Furthermore, no nuclear changes, such as chromatin condensation and DNA fragmentation were observed when cells were stained with Hoechst 33342 Staining (Fig. 4B). Fig. 4. Open in new tabDownload slide (A) Histogram showing the percentage of cell viability of Human lung normal cells (BEAS-2B) treated with different concentrations (100–25 µg/ml) of fruit extract of C. fistula for 24 h. The data represent the mean and standard deviation of three independent experiments. (B) Light and fluorescent micrographs of human lung cells BEAS-2B treated with 50 µg/ml fruit extract of C. fistula captured after 24 h. Photomicrograph of control (a) and treated (b) BEAS-2B cells captured with an inverted phase-contrast microscope. Fluorescent micrographs of Hoechst 33342 of control (c) and treated (d) BEAS-2B cell captured with fluorescence microscope. Fig. 4. Open in new tabDownload slide (A) Histogram showing the percentage of cell viability of Human lung normal cells (BEAS-2B) treated with different concentrations (100–25 µg/ml) of fruit extract of C. fistula for 24 h. The data represent the mean and standard deviation of three independent experiments. (B) Light and fluorescent micrographs of human lung cells BEAS-2B treated with 50 µg/ml fruit extract of C. fistula captured after 24 h. Photomicrograph of control (a) and treated (b) BEAS-2B cells captured with an inverted phase-contrast microscope. Fluorescent micrographs of Hoechst 33342 of control (c) and treated (d) BEAS-2B cell captured with fluorescence microscope. Analysis of HMSF Extract by GC-MS A GC-MS chromatogram of the HMSF extract of C. fistula revealed that it contained 12 phytocompounds. Comparing the mass spectra of the constituents with the NIST library, 12 phytocompounds were identified, which contributed to the larvicidal potential of C. fistula and are summarized in Table 3. Of the identified compounds, the most abundant compounds included 2-methyl hexanoic acid (49.81%) and 2-methyl butanoic acid (47.31%). Table 3. The chemical composition of the hexane fruit extract of Cassia fistula Peak No. . Name of compounds . Retention time (min) . Molecular formula . CAS NO . Peak area (%) . 1 1,1-oxybis (2-ethoxy) ethane 14.17 C8H18O3 000112-36-7 0.75 2 Boric acid, trimetheyl ester 14.88 C3H9BO3 000120-43-7 0.19 3 2-metyl butanoic acid 15.95 C5H10O2 000116-53-0 47.31 4 2-methyl hexanoic acid 16.15 C7H14O2 004536-23-6 49.81 5 8-methyl-hexahydro-pyrano(3,2-b)pyren-2-one 20.56 C7H12O2 1000190-63-9 0.27 6 2-cyclohexen-1-one, 2-methy1-5(1- methy1etheny1)-,(s) 21.18 C10H16O 002244-16-8 0.21 7 1-(1H-pyrrol-2-yl)-ethanone 23.96 C6H7NO 001072-83-9 0.19 8 6-Bromohexanoic acid, dodeec-9-ynyl ester 26.20 C17H 32O2 1000282-90-5 0.16 9 Ar-tumerone 27.36 C15H20O 1000292-71-0 0.17 10 Apiol 27.86 C12H14O4 000523-80-8 0.32 11 3,3,6-trimethyl 1-1,5-heptadiene-4-01 30.35 C10H16O 057590-19-9 0.20 12 1,2-Benzenedicarboxylic acid bis(2-methylpropyl) ester 33.27 C16H22O4 000084-69-5 0.41 Peak No. . Name of compounds . Retention time (min) . Molecular formula . CAS NO . Peak area (%) . 1 1,1-oxybis (2-ethoxy) ethane 14.17 C8H18O3 000112-36-7 0.75 2 Boric acid, trimetheyl ester 14.88 C3H9BO3 000120-43-7 0.19 3 2-metyl butanoic acid 15.95 C5H10O2 000116-53-0 47.31 4 2-methyl hexanoic acid 16.15 C7H14O2 004536-23-6 49.81 5 8-methyl-hexahydro-pyrano(3,2-b)pyren-2-one 20.56 C7H12O2 1000190-63-9 0.27 6 2-cyclohexen-1-one, 2-methy1-5(1- methy1etheny1)-,(s) 21.18 C10H16O 002244-16-8 0.21 7 1-(1H-pyrrol-2-yl)-ethanone 23.96 C6H7NO 001072-83-9 0.19 8 6-Bromohexanoic acid, dodeec-9-ynyl ester 26.20 C17H 32O2 1000282-90-5 0.16 9 Ar-tumerone 27.36 C15H20O 1000292-71-0 0.17 10 Apiol 27.86 C12H14O4 000523-80-8 0.32 11 3,3,6-trimethyl 1-1,5-heptadiene-4-01 30.35 C10H16O 057590-19-9 0.20 12 1,2-Benzenedicarboxylic acid bis(2-methylpropyl) ester 33.27 C16H22O4 000084-69-5 0.41 Open in new tab Table 3. The chemical composition of the hexane fruit extract of Cassia fistula Peak No. . Name of compounds . Retention time (min) . Molecular formula . CAS NO . Peak area (%) . 1 1,1-oxybis (2-ethoxy) ethane 14.17 C8H18O3 000112-36-7 0.75 2 Boric acid, trimetheyl ester 14.88 C3H9BO3 000120-43-7 0.19 3 2-metyl butanoic acid 15.95 C5H10O2 000116-53-0 47.31 4 2-methyl hexanoic acid 16.15 C7H14O2 004536-23-6 49.81 5 8-methyl-hexahydro-pyrano(3,2-b)pyren-2-one 20.56 C7H12O2 1000190-63-9 0.27 6 2-cyclohexen-1-one, 2-methy1-5(1- methy1etheny1)-,(s) 21.18 C10H16O 002244-16-8 0.21 7 1-(1H-pyrrol-2-yl)-ethanone 23.96 C6H7NO 001072-83-9 0.19 8 6-Bromohexanoic acid, dodeec-9-ynyl ester 26.20 C17H 32O2 1000282-90-5 0.16 9 Ar-tumerone 27.36 C15H20O 1000292-71-0 0.17 10 Apiol 27.86 C12H14O4 000523-80-8 0.32 11 3,3,6-trimethyl 1-1,5-heptadiene-4-01 30.35 C10H16O 057590-19-9 0.20 12 1,2-Benzenedicarboxylic acid bis(2-methylpropyl) ester 33.27 C16H22O4 000084-69-5 0.41 Peak No. . Name of compounds . Retention time (min) . Molecular formula . CAS NO . Peak area (%) . 1 1,1-oxybis (2-ethoxy) ethane 14.17 C8H18O3 000112-36-7 0.75 2 Boric acid, trimetheyl ester 14.88 C3H9BO3 000120-43-7 0.19 3 2-metyl butanoic acid 15.95 C5H10O2 000116-53-0 47.31 4 2-methyl hexanoic acid 16.15 C7H14O2 004536-23-6 49.81 5 8-methyl-hexahydro-pyrano(3,2-b)pyren-2-one 20.56 C7H12O2 1000190-63-9 0.27 6 2-cyclohexen-1-one, 2-methy1-5(1- methy1etheny1)-,(s) 21.18 C10H16O 002244-16-8 0.21 7 1-(1H-pyrrol-2-yl)-ethanone 23.96 C6H7NO 001072-83-9 0.19 8 6-Bromohexanoic acid, dodeec-9-ynyl ester 26.20 C17H 32O2 1000282-90-5 0.16 9 Ar-tumerone 27.36 C15H20O 1000292-71-0 0.17 10 Apiol 27.86 C12H14O4 000523-80-8 0.32 11 3,3,6-trimethyl 1-1,5-heptadiene-4-01 30.35 C10H16O 057590-19-9 0.20 12 1,2-Benzenedicarboxylic acid bis(2-methylpropyl) ester 33.27 C16H22O4 000084-69-5 0.41 Open in new tab Discussion Synthetic insecticides have been used globally to control insects of medical and agricultural importance. However, there is an urgent need to reduce the use of synthetic insecticides and develop alternatives to reduce the deleterious effects of these chemicals, such as the development of resistance in mosquitoes as well as harmful environmental and human health impacts (Tong and Bloomquist 2013, Jayaraman et al. 2015, Ramkumar et al. 2015). Botanical extracts are considered to be attractive alternatives in mosquito control management (Belmain et al. 2001, Isman et al. 2006, Mansour et al. 2012). In traditional practice, water and alcohol have been used as the major solvents for the extraction of secondary metabolites from plants. However, many potentially active compounds are not polar in nature, and therefore may not be extracted in these solvents, resulting in the inaccurate conclusion that a plant does not have bioactive properties (Sultana et al. 2009). Therefore, solvents of different polarity have been used in this study to extract secondary metabolites from plants. The findings of the current study showed that the C. fistula fruit extract is an efficient green insecticide against Cx. pipiens. HMSF of C. fistula exerted significant ovicidal, larvicidal, pupicidal, and adulticidal activity against Cx. pipiens. Controlling the eggs, larvae, and pupae in their aquatic habitat is more effective than targeting adult mosquitoes because eggs, larvae, and pupae are stationary targets (Puglisi et al. 2007). The larvicidal effect of leaf extracts of C. fistula was reported using different mosquitoes such as Anopheles stephensi (LC50, 17.97 µg/ml), Culex quinquefasciatus (LC50, 20.57 µg/ml) (Govindarajan et al. 2008), Culex tritaeniorhynchus (LC50, 136.59 µg/ml), Aedes albopictus (LC50, 118.64 µg/ml), Anopheles subpictus (LC50, 96.51 µg/ml), (Govindarajan et al. 2013) and Cx. quinquefasciatus (LC50, 203.492 µg/ml) (Ullah et al. 2018). The varied LC50 values are attributed to differences in concentration of the ingredients of plants, plant parts used, variation in season, and methods of drying (Sujatha et al. 1998, Tawatsin et al. 2006). In the present study, the LC50 value of the HMSF extract was calculated as 48.45 µg/ml. According to Komalamisra et al. (2005) and Kiran et al. (2006), compounds that exert larvicidal activity with LC50 below 100 or 50 µg/ml are significant larvicidal agents. In this study, the hexane extract of C. fistula met these criteria and hence, was considered a potential larvicide. The dose-dependent ovicidal, larvicidal, pupicidal, and adulticidal activities of C. fistula demonstrated promising potential against Cx. pipiens. However, the comparison of efficacy between C. fistula extracts with synthetic insecticides, such as permethrin, revealed that the latter had a stronger effect against Cx. pipiens (Mahyoub et al. 2016). These findings are in agreement with reports that plant-derived insecticides are typically less effective than synthetic insecticides (Mansour et al. 2000, Mohan et al. 2006, Mohan et al. 2010, Tong and Bloomquist 2013, Chansang et al. 2018). This could be attributed to complex mixtures of either active or inactive compounds in the plant extracts compared to a single active compound in synthetic insecticides. However, the complexity of naturally active compounds with different modes of action could be beneficial in either impeding the development of resistance in mosquito populations or enhancing bioactivity (Shaalan et al. 2005, Tak and Isman 2015). A toxicity test using the sensitive stage of zebrafish embryo is relatively a relatively easy and conclusive assay to perform, and reduces the cost and the period of the test (Wang et al. 2017). Embryo toxicity test is an alternative method to assess the developmental toxicity of molecules that can aid in avoid testing juveniles or adults (Embry et al. 2010, Jeffries et al. 2015) that might pose ethical issues. Evaluating the toxicity of HMSF on aquatic ecosystem as a nontarget organism is also necessary to assess the safety or the negative impact on fish population and their normal development (Pérez et al. 2013, Ku et al. 2015). Our results revealed that the extract was not toxic to the embryonic stages of zebrafish. This was evident from the normal morphological and development of embryonic stages of zebrafish. However, the laboratory conditions are mostly controlled and differ significantly from field conditions (Phyu et al. 2011, Yang et al. 2016). Therefore, field studies are required to evaluate the ecological risks of the extract. In nature, many plants exude volatile organic compounds into the atmosphere from flowers and fruits (Young et al. 1983, Muchalal et al. 1985, Effmert et al. 2005, Feng and Zhang 2017). Some of these volatile organic compounds may act as defensive compounds against insects and pathogens or as chemical signals in plant-microorganisms, plant-plant, and plant-animal interactions (Penuelas et al. 2001). The long pods of C. fistula are aromatic and smell unpleasant (Gupta et al. 2008). The reported adulticidal activity in this study (LC50, 12.8 µg/ml) is lower than the value reported in a study with methanolic leaf extracts of C. fistula against A. stephensi (LC50, 35.1 µg/ml) (Mehmood et al. 2014). The efficacy of C. fistula extracts in inducing the knockdown and 100% mortality after 30 min of adult mosquitoes still requires further investigation. This extract may have potential value as a substitute for other mosquitocidal agents, which could prove to be ineffective against the mosquitoes due to acquired resistance. In the current study, we assessed the toxicity of fruit extract of C. fistula on BEAS-2B normal lung cells in vitro. It was observed that the extract did not show marked toxicity in cell lines tested using light, and fluorescence microscopy, and MTT assay. Although the in vitro results cannot necessarily be extrapolated to the human body, the actual health risk should also be evaluated using other models. GC-MS is a precise spectroscopic tool used to identify secondary metabolites in plant extracts (Deshpande et al. 2013, Payum et al. 2016). The hexane extract of C. fistula was analyzed by GC-MS to identify compounds using the NIST library. GC-MS resolved separate compounds as a function of time and allowed for determination of relative concentration based on peak height. Our study revealed the presence of 12 volatile compounds. Several investigations have demonstrated the larvicidal activity of volatile compounds against mosquitoes (Elumalai et al. 2017, Afolabi et al. 2018). The compounds found in this extract are also found in the extract of other plants, such as Achillea tenuifolia (1,2-benzenedicarboxylic acid bis(2-methylpropyl) ester) (Manayi et al. 2014), Hiptage benghalensis (3,3,6-trimethyl 1-1,5-heptadiene-4-01) (Venkataramani et al. 2012), Curcuma longa (ar-tumerone) (Yue et al. 2012), Brucea javanica (ethanone, 1-(1H-pyrrol-2-yl)-) (Su et al. 2013), parsley (apiole) (Marín et al. 2016), Scapania verrucosa and Biophytum reinwardtii (2-methylhexanoic acid) (Guo et al. 2008, Sadasivan et al. 2014), Mangifera indica (2-methyl butanoic acid) (Pino et al. 2005), and Pinus roxburghii (boric acid, trimetheyl ester) (Kaushik et al. 2014). The larvicidal activity of these extracts can be attributed to the lipophilicity of these compounds. Lipophilic compounds can penetrate rapidly through the larval cuticle and disturb the composition of the cell membrane, resulting in cell death (Tabanca et al. 2015). The toxicity of these extracts against mosquito larvae provides a promising substitute for synthetic mosquitocides, which are fast losing efficacy. Ester derivatives, such as 2-methylhexanoic acid and hexadecanoic acid have been reported to attract insects and enhance oviposition activity (Knight et al. 1991, Tewari et al. 2015). Ar-turmerone has larvicidal activity with LC50 of 2.5 and 2.8 µg/ml, respectively against Aedis aegypti and Anopheles quadrimaculatus, respectively (Ali et al. 2015). Similarly, butanoic acid derivatives have also been reported to possess insecticidal activity (Miyamoto 1982). Conclusions To the best of our knowledge, the insecticidal activity of C. fistula fruit extract has not been previously reported. Our research findings revealed that the extracts of C. fistula are effective green insecticides against Cx. pipiens, causing high mortality at low concentrations, and thus providing an alternative to synthetic insecticides for management of mosquito population. This result was supported by the safety of the extract against nontarget lung cells (BEAS-2B) and zebrafish (Danio rerio) embryos. However, further studies are required to investigate the isolated active compounds to decipher their mechanisms of action and their effects on nontarget organisms. Acknowledgments We acknowledge the Deanship of Scientific Research at King Saud University for funding the work through the research group project No. RG-1439-030. Animal research ethics approval was obtained from King Saud University, with Approval No. KSU-RG-1439-030. References Cited Afolabi , O. J. , I. A. Simon-Oke , O. O. Elufisan , and M. O. Oniya . 2018 . 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TI - Target and Nontarget Toxicity of Cassia fistula Fruit Extract Against Culex pipiens (Diptera: Culicidae), Lung Cells (BEAS-2B) and Zebrafish (Danio rerio) Embryos
JF - Journal of Medical Entomology
DO - 10.1093/jme/tjz174
DA - 2020-02-27
UR - https://www.deepdyve.com/lp/oxford-university-press/target-and-nontarget-toxicity-of-cassia-fistula-fruit-extract-against-82mmc70vdg
SP - 493
VL - 57
IS - 2
DP - DeepDyve
ER -