Sida cordifolia L radix was collected in Karnataka, India (2012), and was donated by Pukka Herbs, Bristol. A voucher specimen was deposited in the DBN Economic Collections, Glasnevin Herbarium Dublin (DBN 06:201261). Plant roots were washed with isopropanol and water and lyophilised roots were homogenised to a fine powder using an IKA® A11 analytical mill (IKA® Werke GmbH & Co. KG, Staufen, Germany). Powdered roots were macerated successively in n-hexane, chloroform, methanol and double-distilled water (ddH2O) at room temperature (24h). N-hexane, chloroform and methanol extracts were concentrated by rotary evaporation (45°C). Aqueous extract was centrifuged at 4000g for 15min and then lyophilised and the subsequent extract underwent ethanol (abs.) precipitation (1:4 v/v) overnight. The resulting precipitate was centrifuged (1000g; 10min) and the supernatant discarded and the pellet (SQAP; 23.6%) was lyophilised.
Fractionation of SQAP
SQAP (2.0g; 50 mg/ml) was loaded onto a DEAE™ Sephadex A-50 column (GE Healthcare, Kent, UK) pre-equilibrated with ddH20, then eluted stepwise into low, medium, and high ionic strength solutions (0, 0.75, 2mol/l NaCl). After lyophilising, each of these were fractionated using 100 kDa molecular weight cut-off (MWCO) filters (Vivaspin, Sartorius, Gottingen, Germany) to obtain >100 kDa and <100 kDa fractions (SCAF0-5; supplementary data Table S1) where SCAF0 is the equivalent of SQAP. The protein content of SCAF fractions was determined (Bradford assay, Biorad, Hertfordshire, UK) and found to be negligible.
The Limulus Amebocyte Lysate (LAL) gel clot assay (Pyrosate; Associates of Cape Cod, Inc., Liverpool, UK) was performed to detect Gram-negative bacterial endotoxin contamination to a high degree of sensitivity (0.03 endotoxin units (EU)/ml). A glucan blocker was used to eliminate false positives due to activation of clotting enzyme by plant cellulose. A solution of lyophilised aqueous extract (100 ng/ml) in sterile certified endotoxin-free water (Sigma) was incubated with LAL reagent and the assay performed in accordance with the manufacturer’s instructions.
Immunological effects of SCAF fractions
Lymphocyte cell preparation
Spleens were aseptically removed from adult female Balb/C mice sacrificed by cervical dislocation and collected in 5ml RPMI-1640 medium (ThermoFisher Scientific™, Paisley, UK) containing 1% penicillin-streptomycin. Cells were collected into fresh RPMI medium using sterile 40 µM nylon cell strainers (BD Falcon, Oxford). Cell suspensions were centrifuged (300g; 10 min), the supernatant discarded, and erythrocytes depleted using a red blood cell lysis buffer (Red Blood Cell Lysing Buffer Hybri-Max, Sigma-Aldrich). Splenocytes were re-suspended in RPMI 1640-Glutamax medium containing 1% penicillin-streptomycin and 10% heat-inactivated foetal bovine sera (FBS). After cell counting (Countess® Automatic Cell Counter, Invitrogen) cells were seeded (2x105 cells/well) into 96-well plates (BD Falcon).
Splenocyte proliferation was measured by adding AlamarBlue (10% v/v; Invitrogen, ThermoFisher Scientific, Paisley, UK) to each well for 24h according to the manufacturer’s instructions, with optical density (OD; 570nm) determined using a microplate reader (Safire2, Tecan, Switzerland). SCAF0-SCAF5 diluted in RPMI medium (100µl; 10 ng/ml–1 mg/ml) were tested in triplicate with Concanavalin A (Con A; 5 µg/ml) (Sigma-Aldrich) as a positive control and supplemented media as a negative control. Results were expressed as the splenocyte proliferation index (OD treated cells divided by OD negative control cells).
Immunoglobulin (IgG) production was evaluated using an in-house sandwich enzyme-linked immunosorbent assay (ELISA). Splenocytes prepared as described above were incubated with extracts (48h), 96-well plates were centrifuged (300g; 10 min), supernatants collected by aspiration, and stored at -20°C until analysis. ELISA plates were coated overnight (4°C) with goat anti-mouse IgG capture antibody (Sigma Aldrich) diluted 1:1000 (10µg/ml; 75µl/well) in 0.1 M sodium bicarbonate buffer (pH 9.4). Wells were blotted then blocked with 1% non-fat dried milk (Marvel; 2h; 37°C). After washing, diluted cell supernatants were added (75µl; 2h; 37°C) to wells and incubated. Wells were washed with ELISA wash solution and 75µl of goat anti-mouse IgG secondary detector antibody (HRP-conjugated; 1:2000; Sigma-Aldrich) was added (1h; 37°C). Plates were again washed and tetramethylbenzidine (TMB) solution (Sigma-Aldrich) was added, after 10min H2SO4 (2.5M) was added and absorbance (450nm) measured.
Macrophage activation and phagocytosis
Murine macrophages from cell line RAW264.7 (European Collection of Cell Cultures (ECACC 91062702)) were cultured in Dulbecco’s modified Eagle’s medium (DMEM), containing 10% heat inactivated FBS and 1% penicillin-streptomycin. Cells were grown to confluence in 75cm2 culture flasks in the presence of 5% CO2 at 37°C. RAW 264.7 cells were cultured, seeded (1x104 cells/well), and exposed to SCAF fractions (24h; 37°C). Macrophage phagocytosis was assessed by the uptake of neutral red (NR) dye . Plates containing the cells had sterile homogenous NR solution (0.1% v/v) added to each well (6h). Contents were then discarded, adherent cells were washed twice with PBS, lysed (deionised water with 50% absolute ethanol and 1% glacial acetic acid), incubated at room temperature (2h), and optical densities measured (540nm). Lipopolysaccharide (LPS; 1.0µg/ml; Sigma-Aldrich) was used as a positive control and media as a negative control. Macrophage activation was determined by nitric oxide (NO) production as measured by the Griess assay where 50 µl of medium was transferred to a fresh 96-well microtiter plate before the assay (which included a sodium nitrite standard curve) was performed in accordance with the manufacturer’s instructions (ThermoFisher Scientific, Paisley).
Measurement of cytokine expression
RAW 264.7 cells (as described above) were seeded into 6-well cell culture plates (5x105 cells/well Corning® Costar, Sigma Aldrich) SCAF extracts were added to seeded wells (n=3), cells were incubated (37°C; 24h), then removed using a cell scraper. Triplicate samples were pooled, the total number of cells was determined, and diluted to a density of 3x105 cells/ml. Splenocytes were separately prepared at 1.5x06 cells/ml. Cells were centrifuged (300g; 15min), and RNA isolated from cell pellets (RNeasy Mini kit; Qiagen LTD, Manchester, UK). Purity of isolated RNA was assessed to ensure 260/280 ratio ≥ 2.0 (Nanodrop 2000c; Thermo Scientific, Loughborough, UK). Complementary DNA (cDNA) was synthesised from RNA samples (stored at -80°C) using the Transcriptor First-Strand cDNA synthesis kit (Roche Diagnostics Ltd, Sussex, UK) (n=3) according to the manufacturer’s instructions. RT-qPCR was performed on an Eppendorf Mastercycler (Eppendorf, Stevenage, UK) in accordance with the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines . Hydroxymethylbilane synthase (HPRT1) was used as the reference gene for normalisation. Primer sequences for the selected cytokines measured are shown in Table S2 along with post-amplification melting curves showing reaction specificity (Figure S1). RT-PCR products were resolved in 1.0% agarose gel electrophoresis. PCR reactions contained 100 ng cDNA and underwent 45 cycles of denaturation (94°C; 15s), annealing (58°C; 60s) and extension (72°C; 60s) using a LightCycler 480 system (Roche Diagnostics Ltd, Sussex, UK). The comparative cycle threshold (Ct) method was used to normalise the gene of interest to HPRT1 with treated samples (TRT) compared with untreated cells (CTL).
SCAF0 and SCAF5 (100 mg) were hydrolysed in trifluoracetic acid (105°C; 7h 10ml; 1M; TFA; Sigma-Aldrich) and lyophilised. Samples (2 mg) were derivatised by silylation reactions for 12h at room temperature with N,O-Bis(trimethylsilyl)trifluoroacetamide (500µl; BSTFA) with 1% trimethylchrosilane in 1 ml anhydrous pyridine. GC-MS analysis of silylated hydolysates was performed using gas chromatography (Agilent 7890A) interfaced with a mass selective detector (Agilent 5975C), with a ZB semi-volatiles column (30m x 0.25mm x 0.25 µM Zebron, Phenomenex Inc.) with helium as the carrier gas at a constant rate of 1 ml/min. The injector and MS source temperatures were set at 260°C and 230°C, respectively. The column temperature program consisted of injection at 80°C held for 1 min, with temperature increase of 15°C/min to 300°C, then held at 300°C for 15 min. The MS was operated in the electron impact mode with ionisation energy of 70 eV. The scan range was set from mass scan range was 50-550 Da. Injection volume was 1µl and the inlet had a split flow of 20 ml-1 (split ratio 20:1). Data was acquired and processed with the ChemStation software (Hewlett Packard) and monosaccharide identification was performed by comparison of retention time and mass spectra against known standards and/or the NIST mass spectral library (National Institute of Standards and Technology, USA).
Several Gram-positive and Gram-negative bacterial strains were selected for Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) assays. These were P. aeruginosa (ATCC 47085) (ATCC 27853) S. epidermis (NCTC 11964) S. aureus (NCTC 12493) (ATCC 43300) E. coli (ACTCC 11303) A. baumannii (NCTC 13304) and E. faecium (DZMZ 25390)). For the determination of MIC, the strains were tested against increasing concentrations of n-hexane (SCHEX), chloroform (SCCL), methanol (SCMEX) and aqueous (SCAQ) crude extracts using broth microdilution following the performance standards as recommended by the Clinical and Laboratory Standards Institute (CLSI) . Briefly, test strains were grown overnight at 37°C in Mueller Hinton Broth (MHB) with agitation and the organism suspension adjusted to the density of 0.5 McFarland standard. The test compounds were tested at concentration range from 0.003 – 8000µg/ml and the MIC determined as the lowest concentration (mmol) corresponding to absence of growth. Bactericidal activity was analysed by elucidation of the MBC where aliquots from each well showing no visible growth were plated onto a Mueller-Hinton agar plate. The agar plates were then incubated overnight at 37°C and checked for a 99.9% kill to determine MBC.
Isolation and characterisation of SC1 and SC2
Due to its potent antimicrobial activity the methanol extract (SCMEX: Table S3) was selected for further fractionation. SCMEX was dry loaded on to a 340g Biotage SNAP ultra-cartridge attached to a Biotage Isolera Spektra System, and flash chromatography was performed with an ethyl acetate: methanol gradient (95%:5% – 5%5:95%). Fractions were dried and screened for antimicrobial activity against methicillin-resistant Staph. aureus (MRSA (ATCC 43300)) by a modified thin-layer chromatography bioautogram overlay method described elsewhere . Bioautograms were developed by spraying with 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT; Invitrogen, ThermoFisher Scientific, Paisley, UK) in PBS (0.5mg/ml) and incubated for 30 min at 37°C. Clear white/yellow zones against a purple background on the plate indicated the presence of antimicrobial compounds. Fractions 16-31 exhibited antimicrobial activity and were combined and loaded onto a Sephadex LH20 column (40 x 2.5 cm pre-equilibrated with 5%:95% (ddH20: MeOH)) and eluted with solutions of increasing MeOH concentration. 7 fractions (SCMBu1-7) each 20ml in volume were obtained, but only fraction SCMBu5 retained antibacterial activity (Table S3). SCMBu5 underwent reversed-phase high performance chromatography (HPLC) using a W2690/5 HPLC system coupled with a W2996 photodiode array detector (Waters Corporation, Milford, USA) and using a Phenomenex Luna C18 (5u 100A 250 x 10 mm) column. Mobile phases A (water/acetonitrile 95%:5%) and B (acetonitrile/water 95%:5%) were prepared and a gradient elution programme (4.5 ml/min) of 0-5min (98%; 2%), then 5-20 min (50%:50%) was performed. Two chromatographic peaks referred to as SC1 and SC2 were subsequently collected, dried, prepared at 16mg/ml, filter sterilised and tested for antimicrobial activity. Molecular weights of SC1 and SC2 (Figures S2 & S3) were determined using Q-TOF LC/MS (Waters Xevo G2-S Q-TOF system) (Column; Acquity UPLC HSS T3 column 100Å, 1.8 µM, 2.1mm x 100mm). ESI parameters involved both negative and positive acquisition modes, a mass range 50-1200Da, a desolvation gas temperature of 450°C, desolvation gas flow of 14 L/min and the spray and cones voltages set at (1Kv and 30.0v, respectively). Structural verification studies involved Raman-IR spectroscopy (Thermo Scientific Nicolet iS5 FT-IR Spectrometer with Omnic Software™, Madison, Wisconsin, USA) and also 13C NMR and 1H NMR spectroscopy in CD3OD (400 MHz Bruker NMR, Billerica, MA, USA). The identities of SC1 and SC2 were confirmed by comparing chemical and spectroscopic properties reported against those in the literature.
Effect of fractions in Galleria mellonella
G. mellonella larvae (Livefoods Direct, Somerset, UK), were reared on an artificial diet at 25°C (dark). During experiments, larvae were kept in an incubator at 37°C in sterile Petri dishes (5 cm). Experimental groups consisted of 10 larvae (last instar) weighing 250-300 mg. In one series of experiments the ability of SCAF fractions to upregulate the number of haemocytes was determined. Sterile SCAF fractions of differing concentration were prepared in PBS and 20µl aliquots were injected into the larvae hemocoel through proleg (left) in dorsolateral region using a Terumo Myjector 1ml 29G (0.33 x 12 mm) needle. Larvae were subsequently incubated for 24h and haemolymph was removed, and haemocytes were enumerated using a haemocytometer. Secondly, the ability of pre-treatment of larvae with SCAF fractions to reduce bacterial load was investigated. Sterile SCAF0 or 5 fractions (20µl of 100 μg/ml or PBS negative control) were injected as described above and after overnight incubation, larvae from each group were inoculated with 20µl (2 x103 CFU MRSA; ATCC 43300) and maintained in an incubator at 37°C. At 48 and 72 h post-infection, larvae (n=7) from each group were removed and the bacterial load in haemolymph was determined by draining and diluting haemolymph. CFU/ml of haemolymph was determined by using the Miles and Misra Method to calculate CFU . In another series of experiments, the question of whether S. cordifolia methanol fractions SC1 and 2 could improve resistance of G. mellonella to MRSA infection was evaluated. A bacterial suspension of MRSA (ACTCC 4330) was prepared and larvae were inoculated as described above. . Six hours after infection, larvae were administered with either fraction (SC1 or SC2) and incubated (37°C; 48h), haemolymph was removed, and CFU/ml haemolymph were enumerated.
Results are expressed as mean ± standard error (SEM). Differences in means were evaluated by one-way analysis of variance (ANOVA) with a post hoc Tukey’s test. Analyses were performed using GraphPad Prism 8.0 software (GraphPad, California, USA).