Plant collection and chromatography of the extract
The use of plant material in the study complies with relevant institutional, national, and international guidelines and legislation. The present study complies with the IUCN Policy Statement on Research Involving Species at Risk of Extinction and the Convention on the Trade in Endangered Species of Wild Fauna and Flora, and is not in the red list of IUCN. The aerial (leaves and stem) parts of C. juncea were collected at Wokha, Nagaland, India, circa March 2018. The plant was authenticated by Dr. Ravichandran N, Centre for Advanced Research in Indian System of Medicine (CARISM), SASTRA Deemed to be University, Thanjavur, India. A voucher specimen (CARISM 00147) was deposited in the herbarium of CARISM, SASTRA Deemed to be University, Thanjavur, India. The plant parts (1 kg) were dried at ambient temperatures, pulverized to a powder, and percolated with MeOH. After filtration, the extract was concentrated in a rotary evaporator and further lyophilized to obtain a residue, CJM (45 g).
CJM (20 g) was taken for column chromatography with silica gel (100–200 mesh) packed in a glass column (1 cm in diameter, 15 cm in length) and eluted with CHCl3-MeOH (2,8,15,25 and 30%), and MeOH successively. Two fractions eluted at 25% and 30% CHCl3-MeOH were pooled together and subsequently run in Sephadex LH-20 chromatography with an elution system of 30% CHCl3-MeOH.
Synthesis of biogenic silver and copper nanoparticles
Optimization for the synthesis of C. juncea extract capped AgNPs / Kaempferitrin@AgNPs
The effects of various influencing factors were considered for the synthesis of biogenic AgNPs. Initially two of the factors were kept constant and the desired factor was varied at different levels. (i) C. juncea extracts (2, 3, 4 mg/mL)/ Kaempferitrin (0.5, 1 and 1.5 mg/mL) (ii) Silver nitrate (SN) molarity: 1, 2 and 3 mM, (iii) Sunlight exposure time (hrs or min). OD was noted using UV–Vis spectroscopy at a regular time interval of 30, 60 and 120 min in the case of extract and 60, 90 and 120 min for Kaempferitrin (Fig. S1). The as-prepared, optimized biogenic AgNPs were characterized and continued for further studies.
Optimization for the synthesis of C. juncea extract capped CuNPs/Kaempferitrin@CuNPs
For CuNPs synthesis, i) C. juncea extracts (2, 3, 4 mg/mL)/ Kaempferitrin (0.5, 0.75 and 1 mg/mL) ii) Cupric acetate monohydrate (CAM) molarity: 1, 2 and 3 mM iii) Reducing agents (RA) combination: Ammonium hydroxide (NH4OH) and Hydrazine hydrate (H6N2O) were used at different volume as RA1; 5 and 10µL/mL, RA2; 10 and 20 µL/mL and RA3; 15 and 30 µL/mL respectively (Fig. S1). Synthesized CuNPs by the optimization were characterized and retained for further studies.
Characterization of nanoparticles functionalized with secondary metabolites
UV–visible spectroscopy was employed for analysis of bio-reduction of silver nitrate/Cupric acetate monohydrate to AgNPs/CuNPs using (Lambda 25, PerkinElmer, Waltham, MA) by monitoring the spectra from 200 to 800 nm. X-ray diffraction (XRD) patterns were recorded using Rigaku Ultima III diffractometer (Rigaku, Tokyo, Japan) with Cu-Ka radiation in the 2-h range from 10 to 80 kV. The dispersed AgNPs/CuNPs were taken in polystyrene cuvette and subjected to the particle size analysis and zeta potential, done with Laser Diffractometry coupled ZetaSizer Nano-series (ZS-90 Red, Malvern Instruments, Malvern, England). The surface morphology of AgNPs/CuNPs was analyzed using the Field Emission Transmission Electron Microscope (FE-TEM 2701F, JEOL, Tokyo, Japan).
Stability studies for AgNPs and CuNPs
To analyze the stability and agglomeration of synthesized nanoparticles (KF@AgNPs and KF@CuNPs), the nanoparticles synthesized at optimized conditions were prepared in bulk volume and stored in a dark place, with all NPs of equal volumes. Each sample was analyzed by UV–visible spectrum (200–800 nm) at various time points, followed by Zeta potential and Zeta sizer analysis for confirmation.
Antimicrobial studies
Bacterial strains
Methicillin resistant Staphylococcus aureus ATCC43300 was employed in the present study. The strain was maintained as 15% glycerol stocks at -80 oC. The strains were subcultured from a glycerol stock onto LBA/TSA plates for regular use. The isolated MRSA colony was inoculated into LB broth.
Screening for antibacterial activity
Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) was evaluated for bacteriostatic and bactericidal effect of CJE@AgNPs/CuNPs and KF@AgNPs/CuNPs, as reported earlier using micro broth two fold dilution method was used23,24.
Time kill kinetic assay curve
Time kill assay was performed to evaluate bactericidal potential of biogenic AgNPs/CuNPs. Briefly, overnight culture of MRSA was diluted to 0.05 OD in sterile broth and cells were allowed to grow till 0.1 OD (106 cells), measuring the optical density at 595 nm. At this time point, treatment was initiated with 1X MBC of AgNPs/CuNPs. Samples were retrieved at different time points (0, 1, 2, 3, 4, 5 & 24 h), serially diluted and plated onto Nutrient agar plates and plate counts were determined for both untreated and treated samples25.
Alizarin Red-Q assay
The amount of copper ions that are released from CuNPs in the absence and presence of MRSA is quantified as copper ions complexed with Alizarin red at fixed absorbance of 510 nm26. Overnight culture was diluted to 0.3 OD and were treated with CuNPs at their 1X MIC. Copper ions treated sterile media and media without copper ions were maintained as controls. Pelleted cells treated with CuNPs are resuspended in 1 mL of 0.2 M sodium acetate buffer pH 5.0. Alizarin Red-Q (200 µl) was added to samples and absorbance at 510 nm of these samples was recorded at different time intervals.
Studies on biofilm inhibition
Crystal violet (CV) staining
MRSA overnight grown culture was diluted to 0.05 OD and the bacteria were allowed to attach onto the surface of sterilized glass slides in a sterile petri plate. After allowing the cells to attach on glass surface for 5–10 min, the slides were gently immersed into sterile Brain Heart Infusion (BHI) broth with or without KF@AgNPs and KF@CuNPs and allowed to incubate for 24 h. Post incubation, the slide was carefully taken out using forceps and the unbounded planktonic cells were removed by sterile PBS wash thrice and were air dried. The slides were stained using CV (0.1%) for 20-25 min. Excess CV dye was washed away using double distilled H2O and stained slides were dried for 40 min in an oven. Biofilm formed on the slides were imaged using (Nikon Eclipse Ni-U, Japan) optical microscopy27.
Fluorescent live/dead imaging
For fluorescence live/dead imaging of biofilm, bacteria were allowed to attach on the surface of the sterile glass slides placed within Petri plates, the slides were immersed using sterile BHI broth. Different groups were allocated untreated and KF@AgNPs and KF@CuNPs treated independently. Untreated groups were maintained as control. After 24 h of incubation, the slides were washed gently with sterile PBS and stained using a mixture of Fluorescein diacetate and propidium iodide @ 1:1 ratio. The image was captured using a Nikon fluorescence microscope (Nikon Eclipse Ni-U, Japan)28.
SEM imaging
For SEM imaging studies, overnight culture of MRSA was diluted to an OD of 0.05 and allowed to attach on the surface of sterile cover glass, placed within microtiter plate (24 well), The coverglass was immersed in sterile BHI broth and kept for 24 h incubation, sterile PBS was used to wash the unbound cells and 2% glutaraldehyde was introduced as fixative. Further, washed with sterile PBS and air-dried. The biofilms, which were formed on the surface of the cover glass, were subjected to dehydration using a series of ethanol wash (50%-100%) for each 10 min. Subsequently, the biofilms were air-dried and sputter coated with platinum, image was captured by FE- SEM, JEOL 6701F (Tokyo, Japan)29.
Cell membrane permeability assay
Mid-log phase culture of MRSA was centrifuged and the pelleted cells were washed with sterile PBS twice and then re-suspended with PBS of equal volume. KF@AgNPs and KF@CuNPs and CTAB 0.2 mmol/l along with propidium iodide 30 µmol/1 were added and incubated at 37 °C for 2 h. Fluorescence intensity of PI was measured by using (Jasco FP-8500, Jasco, Tokyo, Japan) spectrofluorimeter. Permeability index was determined by calculating the fluorescence ratio between cells without and treated with CTAB. The fluorescence normalization was carried out by subtracting fluorescence of treated cells with untreated cells30.
BATH assay
Bacterial cell surface hydrophobicity was evaluated by determining the ability of cells to partition from the aqueous phase to Hexadecane. To determine the changes in surface hydrophobicity when treated with KF@AgNPs and KF@CuNPs hexadecane-aqueous partition method as reported earlier31 was adopted. After vortexing the mixture containing MRSA cells, hexadecane and PBS, final absorbance is taken at OD530 from the aqueous phase.
Reactive Oxygen species (ROS) assay
Production of ROS after treatment with Sub-MIC of KF@AgNPs and KF@CuNPs in MRSA were determined using fluorophore Dichloro-dihydro-fluorescein diacetate (DCFH-DA) which gets reduced to dichlorofluorescein in a ROS mediated formation, which was quantified using a fluorescence spectrophotometer (JASCO FP-8500, JASCO, Tokyo, Japan) at Ex 485 nm and Em 538 nm32.
Studies on in vivo model system
Ethical Statement
The study was performed in compliance with all relevant ethical laws and guidelines in India. The CPCSEA guidelines for laboratory animal facilities (Central Act 26 of 1982) in India were adhered to in all the in vivo experiments. The experimental protocols were approved by the Institutional Animal Ethics Committee (CPCSEA-493/SASTRA/IAEC/RPP) of SASTRA Deemed University, India . Reporting of all experimental procedures complied with recommendations in ARRIVE guidelines.
Zebrafish toxicity study design
Adult zebrafish (Danio rerio) irrespective of sex, aged 2 months, measuring 4 to 5 cm in length, weighing approx. 300 mg, were purchased from a local aquarium in Thanjavur, Tamil Nadu, and India. Animal acclimatization was carried out following established protocols33. To check the effect of CuNPs on liver enzyme profiles of zebrafish, 5 fish each were exposed to the 1X MIC of the MNPs/Standard drug for 48 h. At the end of exposure (48 h), fish were anesthetized with ms-222 and euthanized by decapitation. The liver tissues were pooled from fish within each group and homogenized in an ice-cold buffer (Tris–HCl, 0.1 M, pH 7.4). The homogenate was centrifuged (10,000 × g, 10 min, 4 °C) and the supernatant was used for all analyses in triplicates. Using the supernatant liver, α- and β-carboxylesterase enzyme activities were estimated34, acetylcholinesterase (AChE) activity was measured by DNTB (5,5′-dithio-bis-[2-nitrobenzoic acid]) degradation35 Protein was estimated by the Lowry method36.
Histological study on zebrafish liver.
All fish from the treated and untreated group (n = 5) were euthanized in ice cold water at the end of the experiment before fixing in buffered 10% formalin solution for 24 h and later paraffin wax was used to embed. Sections of 5 μm thickness were cut and the thin sections of the tissue were stained by both eosin and hematoxylin for histological examination of the liver37.
Effect of KF@AgNPs and KF@CuNPs on in-vivo zebrafish infection model
Overnight grown MRSA was diluted to 0.05 OD with sterile media and 10 µl of culture was delivered through an intramuscular injection using a 3/10-cc U-100 insulin syringe with a 0.5-in-long, 29-gauge needle. The fish were grouped (n = 5) as infected treated and infected untreated. After 3 h post-infection, 10 µl of KF@AgNPs and KF@CuNPs at 1X MIC were delivered near the site of infection, grouped as treated38. The fishes were anesthetized with ms-222 after 24 h and euthanized by decapitation and the muscle tissues were collected by dissection, homogenized in sterile PBS; plated onto LB agar plates (serially diluted) to determine the microbial load in respective treatments.
Statistical analysis
All experiments were performed thrice and data are presented as the mean ± standard deviation (SD). One-way analysis of variance (ANOVA), followed by a post hoc multiple comparisons (Tukey test) was done for grouped data analysis. Values were considered statistically significant at p < 0.05.
