Collection and processing of plant
The present study majorly concentrates upon the use of Suaeda maritima in treating fish bacterial infections. This plant was collected from Gilakaladindi mangrove fields located in Machilipatnam (16°0' N latitude and 81°10' E longitude, Andhra Pradesh, India). Identification and confirmation of the collected plant was done at Botanical Survey of India (BSI).The collected leaves were cleaned thoroughly under tap water to remove dirt and rinsed using double distilled water followed by cutting of leaves into small pieces. The plant material is shade dried, pulverized and the resultant powder was stored in an air tight container.
Chemicals
Hexane, Diethyl ether, ethanol, ascorbic acid, sodium hydroxide, ferric chloride, chloroform, acetic anhydride, sulphuric acid, glacial acetic acid, methanol, formic acid, acetonitrile and Potato Dextrose Agar (PDA) media used in the present study were procured from Merck.
Extraction and Phytochemical screening
Four different solvents viz., hexane, diethyl ether, ethanol and distilled water have been used for extracting the bioactive compounds present in the leaves of S. maritima. The plant sample and the solvents are mixed in 1: 10 (w/v) ratio in a Soxhlet apparatus for 6 h at suitable temperatures. The extracts were filtered through Whatman No. 1 filter paper and concentrated using rotary evaporator (Divya et al. 2020). The concentrated crude extracts were used for screening various phytochemicals such astannins, alkaloids, flavonoids, saponins, steroids, terpenoids, phenols, proteins, glycosides, anthraquinones, phytosterols and reducing sugars using standard protocols (Harborne 1984).
Antibacterial activity
Antibacterial activity of all the four extracts against Staphylococcus aureus (MTCC737), Klebsiella pneumonia (MTCC 3384), Bacillus subtilis (MTCC 441) and Pseudomonas aeruginosa, (MTCC 1688) was determined through agar well diffusion method. 100 µl of bacterial cultures (12 h) were inoculated on to the petri plate containing solidified medium and dried for 5 min. In order to perform well diffusion method, equidistant wells, each with 6 mm diameter were cut in the agar with the help of a cork-borer. Further, 40 μl of each extract was loaded into the wells and amikacin was used as a positive control. The plates were incubated for 24 h at 37 °C to find out their antibacterial efficacy of the extract. The antibacterial activity was assessed by measuring the diameter of zone of inhibition around the well containing S. maritima leaf extract (Guntur et al. 2018).
Antioxidant activity
Conversely, the antioxidant activity of S. maritima leaf extract was determined using DPPH radical scavenging assay and FRAP assay.
DPPH radical scavenging activity
For carrying out DPPH (2,2-diphenyl-1-picryl-hydrazyl) radical absorbance assay, the reaction mixture (5 ml) was preparedwith DPPH solution at different concentrations of extract (31.25, 62.5, 125, 250, 500 μg/ml). The mixture was incubation in the dark for 30 min and absorbance was measured at 517 nm. Ascorbic acid was used as standard and blank was prepared using methanol (Shaheena et al. 2019). Further, DPPH radical scavenging activity was calculated by the following equation:
Where A0 is the absorbance of the control reaction and A1 is the absorbance of the sample of the tested extracts.
Similarly, the IC50 value was calculated by interpolation of linear regression analysis. Percentage of free radical activity was plotted against concentration of antioxidant substance so as to obtain the IC50 value.
Ferric reducing antioxidant power (FRAP) assay
The reagent was prepared in 300mM acetate buffer by adding 10 mM 2,4,6-tri (2-pyridyl-s-triazine) (TPTZ) solution in 40 mM HCl and 20 mM FeCl3 solution in proportion of 10:1:1 (v/v), respectively. All the four extracts and standard ascorbic acid at varying concentrations (15.62, 31.25, 62.5, 125, 250 and 500 μl/ml) are added to FRAP reagent and incubated at 37 °C for 15 min. The absorbance was measured at 593 nm and the results were recorded as μg of ascorbic acid equivalents (AAE) per ml (Benzie and Strain 1999).
Purification and characterization of S. maritima leaf extract
Purification of sample by Column chromatography
Active extracts of S. maritima (20g) was subjected to column chromatography on silica gel of 100 – 200 mesh size (Merck) packed in a cylindrical glass column. Sample was passed in the pre-packed column and eluted with mixture of hexane: chloroform as mobile phase to vary polarity to separate and collect fractions. Extract was fractionated via gradient separation of mobile phase and each elute was carefully collected and labelled for further analysis. All the fractions were pooled based on similarity in colour and tested for antibacterial and antioxidant activity. Based on activity specific fractions were further purified using preparative TLC.
Preparative Thin Layer Chromatography (TLC)
Considering the results obtained from antibacterial and antioxidant activity, active fraction of S. maritima leaf obtained from column chromatography was subjected to TLC for separation of active compounds. Preparative TLC plates were prepared by homogeneous mixing of 30 g of silica gel and small amount of calcium sulfate in 60 ml of distilled water and uniformly spreading it on glass plates (20 x 20 cm) with 250 μ thickness. The plates were kept undisturbed for 10 min at room temperature followed by 1 h at 105 °C in hot air oven and then placed in a desiccator for 2 h. TLC of S. maritima active fraction was performed in the lidded tank comprising Butanol: Acetone: Water (12:6:3) as solvent system after spotting the sample on prepared silica gel plate. The solvent in the tank was maintained up to 1 cm beneath the origin. Because of the capillary action, the solvent travels on the plate along with the sample and various components of the sample gets separated. Formation of the bands was observed under the UV light. The procedure was followed until good resolution was noticed and each band was carefully scrapped and assessed for their antibacterial and antioxidant activity.
Characterization of phytochemicals by Mass spectroscopy
Crude and partially purified extracts of S. maritima were loaded onto Agilent 1100 LC/MS System with separate Chemstation Rev.A.09.01 (1206) software. Each extract (20 μL) was injected along with the mobile phase (formic acid (0.1 %) in water (50 %) and acetonitrile (50 %)) at a flow rate of 0.5 mL/min. The electrospray ionization was set in negative ionization mode in 60-200 V and capillary voltage at 4000 V. Nitrogen is used as nebulising gas at 350 °C and 30 psi with flow rate of 8-10 L/min (Shivani et al. 2020).
In vivo studies
Experimental design
Fingerlings of Catla catla weighing 10±0.9 g and length of 8±0.5 cm were procured from local hatchery and screened for any pathogenic infections. Fish fingerlings were acclimatized in the aerated laboratory tubs contained 40 L of freshwater (temperature 28 ± 2 oC and 12:12 L: D period) 10 days prior to experimentation. The fishes were fed twice in a day with commercial food and 30 % of water was changed daily to reduce ammonia toxicity. Various water parameters such as temperature: 26.49±0.13oC, pH: 7.94±0.04, electrical conductivity: 392.22±0.62 S/cm, total dissolved solids: 279.33±0.69 mg/L, dissolved oxygen: 6.44±0.05 mg/L, total alkalinity: 204±7.30 mg/L as CaCO3, total hardness: 180.44±3.74 mg/L as CaCO3, orthophosphate: 0.03±0.001 mg/L, ammonia-nitrogen: 1.66±0.21 mg/L and nitrate-nitrogen: 0.21±0.03 mg/L were maintained during experimental period.
After acclimatization, fish fingerlings were randomly divided into 5 groups of nine each in triplicate and classified as group 1 (control), group 2 (negative control), group 3 (positive control), group 4 (crude extract) and group 5 (partially purified extract). Fish fingerlings in all the groups were starved for 24 h before initiating challenge studies and later fed with pellet feed containing 1 ml of Pseudomonas aureginosa suspension (103 CFU). Group 1 was fed with regular feed and feeding was carried in a similar fashion after 12 h of interval. After 24 h, treatment for group 3, 4 and 5 was initiated with amoxicillin, crude extract of S. maritima (25 mg/g of body weight) and partially purified extract of S. maritima (10 mg/g of body weight) respectively along with feed twice a day for 5 days. The dosage of crude and partially purified extracts was determined on the basis of pilot study (data not shown). The remaining feed was siphoned out before the next feeding. Fingerlings were examined cautiously for pathological lesions, behavioural changes and mortality throughout the experimentation period.
Confirmation of P. aeruginosa
Bacteria from experimental moribund fish fingerlings was isolated and inoculated on an agar plate by spread plate technique to satisfy the Koch’s postulates. The isolated bacteria were identified morphologically through Gram's staining and biochemically through catalase, indole, methyl red, citrate utilization and gelatin hydrolysis tests.
Catalase activity (CAT) and Superoxide dismutase activity (SOD)
Liver was carefully dissected from all the groups followed by homogenization in Tris-HCl buffer (15mM, pH 7.4) and centrifuged at 2000xg for 10 min for measurement of antioxidant enzyme levels. The supernatant was used for estimation of protein (Bradford 1976), CAT (Aebi 1984) and SOD (McCord and Fridovich 1969) activity. Catalase activity was measured at 240 nm to note the consumption of H2O2/min in the presence of sample. SOD activity was measured by mixing the sample with EDTA and pyrogallol and observed for increase in absorbance at 420 nm. Both the activities were expressed as units per mg of protein.
Statistical analyses
All the experiments in the present study were performed in triplicates and the results were expressed as mean ± standard deviation. The statistical analysis was performed using IBM SPSS statistics for windows, 20.0 software (IBM Corp., Armonk, N.Y., USA).