Extracts of Sida cordifolia contain polysaccharides possessing immunomodulatory activity and rosmarinic acid compounds with antibacterial activity

Background The overuse of antibiotics has led to increased antimicrobial resistance, but plant-derived biological response modifiers represent a potential alternative to these drugs. This investigation examined the immunomodulatory and antibacterial activities of Sida cordifolia (used in ethnomedicinal systems to treat infectious disease). Methods Successive extractions were performed from the roots of these plants in hexane, chloroform, methanol and water. Immunomodulatory activity was determined in a series of experiments measuring the responses of splenocytes, macrophages and an in vivo model of innate immunity (Galleria mellonella). Antibacterial activity was assessed by determining minimum inhibitory/bactericidal concentrations (MIC/MBCs) for various Gram-positive and Gram-negative bacterial strains. Results Immunomodulatory activity was confined to the aqueous extract, and further fractionation and biochemical analysis yielded a highly potent polysaccharide-enriched fraction (SCAF5). SCAF5 is a complex mixture of different polysaccharides with multiple immunomodulatory effects including immune cell proliferation, antibody secretion, phagocytosis, nitric oxide production, and increased expression of pro-inflammatory cytokines. Furthermore, Galleria mellonella pre-treated with SCAF5 produced more haemocytes and were more resistant (P < 0.001) to infection with methicillin-resistant Staphylococcus aureus (MRSA) with a 98% reduction in bacterial load in pre-treated larvae compared to the negative control. The antibacterial activity of Sida cordifolia was confined to the methanolic fraction. Extensive fractionation identified two compounds, rosmarinic acid and its 4-O-β-d-glucoside derivative, which had potent activity against Gram-positive antibiotic-resistant bacteria, including MRSA. Conclusions Sida cordifolia counters bacterial infections through a dual mechanism, and immunomodulatory polysaccharides from this plant should be isolated and characterised to realise their potential as anti-infective agents. Such properties could be developed as an antibiotic alternative (1) in the clinic and (2) alternative growth promoter for the agri-food industry. Supplementary Information The online version contains supplementary material available at 10.1186/s12906-022-03502-7.


Background
Antibiotics derived from fungi or bacteria (or synthetic derivatives) are one of the most important discoveries of the last century and are widely used for effectively treating human and animal disease [1]. These compounds also serve as prophylactic growth promoters in the domesticated livestock industry by reducing subclinical disease burden and/or reducing the number of metabolites produced by microbes which may act to depress growth [2]. However, due to their widespread overuse in medicine and the agri-food industry, antibiotic resistance among bacterial pathogens has emerged as one of the greatest threats to global public health [3,4]. According to the World Health Organization (WHO), the global systematic misuse and overuse of these drugs in human medicine and food production has created an alarming level of antimicrobial resistance which could render antibiotic therapy redundant [5].
As antibiotic resistance exposes humans to potentially fatal infectious diseases, one approach has been a shift in focus to discovery of alternatives to these drugs. It is estimated that 25-50% of all medical drugs on the market were originally derived from plants, which have been shown to produce a plethora of antimicrobial secondary metabolites to protect themselves [6]. Furthermore, phytochemicals exhibiting antibacterial activity against antibiotic-resistant pathogenic bacteria have been extensively described elsewhere [6,7]. This includes the polyphenol rosmarinic acid, isolated and identified in the present study, which has previously been shown to inhibit the growth of Staphylococcus aureus and prevent it from forming biofilms [8]. One of the most promising plant-derived alternatives to prophylactic antimicrobials since the emergence of widespread antibiotic resistance is the use of phytochemical immunomodulators such as exogenously-derived Biological Response Modifiers (BRMs) [9]. Immunomodulators are compounds that interact with the host immune system and up-or downregulate specific immune responses. BRMs are chemically diverse and include a range of phytochemicals such as lectins, saponins and polysaccharides [10]. BRMs are postulated to instigate immunomodulatory responses by triggering one or more of the diverse range of pattern recognition receptors (PRRs) expressed by immune cells such as macrophages [9,10]. Mammals and plants possess PRRs which sense infection by recognising signals called pathogen-associated molecular patterns (PAMPs) unique to each invading microorganism. Upon detection of a PAMP (e.g. Gram-negative bacteria lipopolysaccharide (LPS)), the PRR (in this example, a macrophageassociated Toll-like receptor 4 (TLR4)) triggers an array of antimicrobial innate and adaptive immune responses that eliminate the potential threat. Relatively few BRMs have been discovered thus far and there is merit in screening plant materials used to treat infectious disease. Polysaccharides extracted from certain shrubs and medicinal plants are known to trigger various immunological responses [11]. For example, a polysaccharide isolated from Lycium barbarum stimulates splenocyte proliferation and macrophages activation [12,13]. In murine macrophage RAW 264.7 cells polysaccharides extracted from Ganoderma sinese promoted phagocytosis, release of NO and release cytokines such as IL-1α, IL-6, IL-10 and TNF-α [14].
For the current study Sida cordifolia L., a perennial subshrub in the Malvea tribe of the subfamily malvoideae (Malvaceae), was investigated. In India, the Sida genus is represented by circa 19 species of Sida of which the most widespread is Sida cordifolia [15]. The medicinal use of Sida in India can be traced back over 2000 years to the Charaka Samhita, where the roots were mixed with milk or honey ('vyādhikshamatva') to enhance immunity and in the treatment of ailments suggestive of infective disease [16,17]. Similarly, in parts of Central America where it is known as Chichibe, S. rhombifolia was found to be used by the Mopan Maya of Belize and Guatemala to enhance immunity, and as an anti-infective agent for the treatment of wounds [18]. In Africa, species of Sida were used to treat infective diseases and ailments normally associated with the immune system including malarial fever, and it is currently used as an antimicrobial and anti-inflammatory agent [19,20]. The widespread, international use of this species in different ethnomedical systems, as an anti-inflammatory, anti-pyretic, anti-infective and wound healing agent, and as an antidote to snake venom suggests this plant possesses immunomodulating and antimicrobial compounds.
Here, we investigated the in vitro and in vivo immunomodulating and antibacterial properties of Sida cordifolia and characterised some of its bioactive components. Galleria mellonella larvae were used as an invertebrate in vivo model as (i) it's a commonly used in vivo invertebrate infection model to study the efficacy of antimicrobial drugs [21], and (ii) its innate Keywords: Antibiotic resistance, Antibiotic alternative, Feed supplement, Sida cordifolia, Immunomodulation, Immunostimulant, Plant-derived biological response modifiers, Antimicrobial, Galleria mellonella, Polysaccharides, Rosmarinic acid immune system is similar to vertebrates, and, (iii) infection models have been shown to produce comparable data to animal studies [22]. An enriched aqueous fraction (referred to as SCAF5) possesses immunomodulatory activity. The fraction appears to be a complex mixture of polysaccharides, and their observed in vitro immunomodulating activities were verified in vivo in an established insect model of innate immunity. The plant also contained potent antibacterial activity which resided in the methanol fraction and had activity against antibiotic-resistant Gram-positive bacteria. Two antibacterial compounds were isolated and identified as rosmarinic acid and its derivative rosmarinic acid 4-O-β-d-glucoside.

Extraction
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 (ddH 2 O) at room temperature (24 h). N-hexane, chloroform and methanol extracts were concentrated by rotary evaporation (45 °C). Aqueous extract was centrifuged at 4000 g for 15 min and then lyophilised and the subsequent extract underwent ethanol (abs.) precipitation (1:4 v/v) overnight. The resulting precipitate was centrifuged (1000 g; 10 min) and the supernatant discarded, and the pellet was lyophilised.

Endotoxin contamination
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.

Lymphocyte proliferation
Splenocyte proliferation was measured by adding AlamarBlue (10% v/v; Invitrogen, ThermoFisher Scientific, Paisley, UK) to each well for 24 h according to the manufacturer's instructions, with optical density (OD; 570 nm) determined using a microplate reader (Safire2, Tecan, Switzerland). Concanavalin A (Con A) is a lectin which binds to mannose-containing receptors and is well characterised activator of murine lymphocytes [23].. SCAF0-SCAF5 diluted in RPMI medium (100 μl; 10 ng/ml-1 mg/ml) were tested in triplicate with (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).

Antibody secretion
Immunoglobulin (IgG) production was evaluated using an in-house sandwich enzyme-linked immunosorbent assay (ELISA). Splenocytes prepared as described above

Macrophage activation and phagocytosis
The murine macrophage cell line RAW264.7 (European Collection of Cell Cultures ECACC 91062702) was cultured in Dulbecco's modified Eagle's medium (DMEM), containing 10% heat inactivated FBS and 1% penicillinstreptomycin. Cells were grown to confluence in 75cm 2 culture flasks in the presence of 5% CO 2 at 37 °C. RAW 264.7 cells were cultured, seeded (1 × 10 4 cells/well), and exposed to SCAF fractions (24 h; 37 °C). Macrophage phagocytosis was assessed by the uptake of neutral red (NR) dye [24]. Sterile homogenous NR solution (0.1% v/v) was added to cells in each well (6 h). 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 (2 h), and optical densities measured (540 nm). Lipopolysaccharide (LPS; 1.0 μg/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 [25] using a sodium nitrite standard curve which was performed in accordance with the manufacturer's instructions (Ther-moFisher Scientific, Paisley).

Measurement of cytokine expression
RAW 264.7 cells (as described above) were seeded into 6-well cell culture plates (5 × 10 5 cells/well Corning ® Costar, Sigma Aldrich). SCAF extracts were added to seeded wells (n = 3), cells were incubated (37 °C; 24 h), then removed using a cell scraper. Triplicate samples were pooled, the total number of cells was determined, and diluted to a density of 3 × 10 5 cells/ ml. Splenocytes were separately prepared at 1.  [26]. 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 (Fig. 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; 15 s), 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).

Monosaccharide composition
SCAF0 and SCAF5 (100 mg) were hydrolysed in trifluoracetic acid (105 °C; 7 h 10 ml; 1 M; TFA; Sigma-Aldrich) and lyophilised. Samples (2 mg) were derivatised by silylation reactions for 12 h at room temperature with N,O-Bis (trimethylsilyl) trifluoroacetamide (500 μl; 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 (30 m × 0.25 mm × 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 1 μl 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). . The strains used in this study are type strains from several culture collections used for routine screening assays whose antimicrobial susceptibility profiles are well characterised [27][28][29].
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) [30]. 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 -8000 μg/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.

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 20 μl aliquots were injected into the larvae hemocoel through proleg (left) in dorsolateral region using a Terumo Myjector 1 ml 29G (0.33 × 12 mm) needle [30]. Larvae were subsequently incubated for 24 h 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 (20 μl of 100 μg/ml or PBS negative control) were injected as described above and after overnight incubation, larvae from each group were inoculated with 20 μl (2 × 10 3 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 [31]. 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; 48 h), haemolymph was removed, and CFU/ ml haemolymph were enumerated.

Data analysis
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).

Chemical and immunomodulatory properties of SCAF fractions Chemical properties
The protein content of SCAF fractions was negligible indicating that polysaccharide was the major component. Expressed as a percentage, the dry weight protein content of SCAF0, 1, 2, 3, 4 and 5 were 0.8, 1.0, 2.1, 0.1, 0 and 0%, respectively. No endotoxin contamination was detected for any of the SCAF fractions even at lowest detection limit of the assay (0.03 EU/ml).

Immunomodulatory responses in RAW 264.7 cells
Only the aqueous extract (SCAQ) significantly increased macrophage proliferation and nitric oxide (NO) production (Fig. 1). The largest increase in phagocytosis in SCAF-treated cells (Fig. 3A) was observed with 100 μg/ml SCAF5 (3.6-fold compared to negative control: P < 0.001) which was comparable to the LPS positive control (3.54fold). This increase remained significantly different to the untreated cells at 10 μg/ml (2.4-fold; P < 0.001) but no phagocytic activity was observed at lower doses.

Cytokine expression
Cytokine gene expression in splenocytes and RAW 264.7 cells treated in vitro with SCAF5 as determined by RT-qPCR are shown in Figs. 4A and B. SCAF5 upregulated the expression of a wider range of proinflammatory cytokines in the mixed population of primary splenic lymphocytes (TNF-α, IFN-β, IFN-γ, and IL1-β and IL-6) than in the macrophages (IL1-β and IL-6). The most marked increase in SCAF5-induced cytokine expression levels was observed with IL-6 where a 4.2fold increase (splenocytes) and 3.3-fold (macrophages) was observed compared with untreated controls. An approximate 2-fold increase in TNF-α, IFN-β, IFNγ, and IL1-β was elicited in splenocytes whereas only IL1-β (~ 2.5-fold) was elevated to a similar magnitude in macrophages. SCAF5 also elicited a 2.1-fold increase in macrophage-associated iNOS expression in RAW264.7 cells which presumably leads to the increased NO generation. IL-10 and IL-12p35 levels were unaffected following SCAF5 treatment of either the splenic lymphocytes or macrophages.

In vitro antibacterial activity of SC1 and SC2
The initial methanol extract possessed no antibacterial activity against Gram-negative bacteria E. coli (ATCC 11303) and P. aeruginosa (PAO1) but exhibited bacteriostatic activity against Gram-positive organisms with MIC values ranging from 0.5-2.0 mg/ml ( Table 1) (Table S3). SCMEXBu5 exhibited no activity against Gram-negative organisms (data not shown). After further fraction and purification by HPLC two peaks with identical UV λ max profiles (Fig. 5A) were isolated (SC1 & SC2). SC1 and SC2 both exhibited similar antibacterial potency (MIC and MBC) against an array of Gram-positive bacteria (Table 2). Both compounds exhibited the greatest potency against the antibiotic-resistant MRSA strains (1.25 mmol MIC/MBC) and to a lesser extent against MRSE (5.0-20 mmol MIC/MBC). SC1 and SC2 possessed no bioactivity against Gram-negative microorganisms (data not shown).

In vivo antibacterial activity of SC1 and SC2
Bioautography by means of agar overlay visibly showed the in vitro antibacterial activities of SC1 and SC2 against MRSA (ATCC 43300; Fig. 5B). The in vivo antibacterial activity against this antibiotic-resistant strain was analysed by injecting Galleria larvae with either SC1 or SC2 (50 or 100 μM dose in PBS) 24 h before challenge with MRSA (Fig. 5C). Injection of larvae with SC2 (100 μM) 24 h prior to MRSA challenge led to a markedly lower bacterial load (1243-fold decrease; 2.1 × 10 2 CFU/ml) compared to the PBS negative control (2.66 × 10 5 CFU/ ml). In fact, bacterial growth of the antibiotic-resistant Fig. 2 Immunomodulating activity of S. cordifolia ion exchange and size fractionated aqueous fractions (SCAF) on lymphocytes (splenocytes) in vitro. A Lymphoproliferation in response to SCAF0-5 treatment was measured using Alamar blue and results were expressed as proliferation index (OD treated cells divided by OD negative control cells). B Antibody secretion (IgG) following incubation with SCAF0-5 was measured by ELISA and results were expressed as optical density at 450 nm. Concanavalin A (Con A) was used as a positive control at a pre-determined optimal final concentration of 5 μg/ml and complete media was used as a negative non-proliferative control (0 μg/ml). All statistical analysis was carried out using a one-way ANOVA with Tukey's Multiple Comparison Test to compare differences between samples and negative controls (*p < 0.05, **p < 0.01, ***p < 0.001, n = 3). Groups: SCAF 0 (crude polysaccharide fraction of S. cordifolia); SCAF 1: Low ionic strength < 100 kDa; SCAF 2: medium ionic strength and between 10 and 100 kDa; SCAF 3: medium ionic strength and > 100 kDa; SCAF 4 high ionic strength and between 10 kDa-100 kDa; SCAF 5: high ionic strength and < 100 kDa  (1.05 × 10 5 CFU/ml) decrease in bacteria counts, respectively, compared to the negative control.

Discussion
The dual immunomodulatory and antimicrobial activity of S. cordifolia revealed by this study indicates its potential usefulness for treating and/or preventing bacterial infection. Such properties could be developed as (i) an alternative approach to clinically proscribed antibiotics, but equally, (ii) a ground root extract could be developed as a feed additive for the agri-food industry. This would result in harnessing the ability of polysaccharides to nonspecifically stimulate the immune system in combination with the well documented ability of rosmarinic acid to inhibit bacterial growth. The replacement of antibiotics in livestock could slow, and potentially reverse, the emergence of antibiotic resistance in a matter of years as has been demonstrated by the ban of vancomycin antibiotics in the Netherlands [33]. Companion animals are also an emerging source of concern in the field of antibiotic resistance, with several microbiological threats from pets, including MRSA, having been identified as potential threats to humans [9,10]. Our initial in vitro screening of crude root extracts (see Fig. 1) found only the aqueous fraction (SCAQ) possessed immunomodulating activity as measured by lymphocyte stimulation (cell proliferation and increased antibody secretion) and macrophages activation (enhanced phagocytosis and NO production). Ethanol precipitation of the aqueous extraction resulted in the isolation of palebrown precipitate (SCAF0) also with immunomodulatory activity. Biochemical analysis (Molisch's test; data not shown) indicated the presence of carbohydrates. Further fractionation (based on ionic strength and molecular size filtration) of this carbohydrate-enriched extract produced five fractions (SCAF1-5). A further round of in vitro immunological screening identified that the fraction with highest ionic strength and largest molecular size (> 100 kDa), SCAF5, had the most potent immunomodulatory activity. SCAF5 (100 μg/ml) elicited potent lymphoproliferative activity equivalent to the positive control lectin mitogen, and the bioactivity of this fraction was further confirmed by the marked increases in antibody levels in treated splenocytes as well as macrophage activation. Given that protein and potential immunomodulating LPS contaminants were undetectable in the bioactive fraction, the immunomodulatory activity shown here can be attributed solely to plant polysaccharides. Hydrolysis and GC-MS analysis determined the monosaccharide composition of polysaccharides present in SCAF0 and SCAF5. The profiles obtained demonstrate that these fractions remain impure and contain a complex mixture of different polysaccharides. It is likely that key components of the plant cell wall, such as rhamnogalacturonan-I (RG-I), homogalacturonan (HG), xylogalacturonan (XGA), rhamnogalacturonan-II (RG-II) and other polysaccharides are present and each of these could potentially contribute to the overall immunomodulatory effects observed. Indeed, it has been demonstrated elsewhere that some of these polysaccharide structures from certain plant roots have immunological activity [34]. For example, a high-molecular weight arabinogalactan branched RG-I, isolated from the roots of Vernonia kotschyana, has shown T-cell-independent induction of B cell proliferation and induces the chemotaxis of human macrophages, T-cells, and natural killer (NK) cells [35]. Similarly, arabinogalactan-containing RG-I domains from hot water solublised pectic fractions from the roots of Panax ginseng were effective in causing lymphocyte proliferation [9]. Much work remains to be done to understand the specific motifs within these complex cell wall polysaccharides responsible for these observed immunological properties. Since the immunomodulatory effects observed here are quite diverse it is unclear if these are the results of a single polysaccharide, or if multiple polysaccharides are acting in synergy. To elucidate this, it will be necessary to fragment, purify, structurally characterise and test individual molecules from within the SCAF5 fraction. The reason why only certain plant species are rich in immunological activity is also unclear, but an improved understanding of the precise polysaccharide structures involved should also help elucidate this.
Proinflammatory cytokines (e.g. IFN-γ and TNF -α ) are produced by macrophages which increase expression of inducible NO synthase (iNOS), resulting in enhanced NO production [36]. SCAF5 stimulated NO production (See figure on next page.) Fig. 3 Immunomodulating activity of S. cordifolia ion exchange and size fractionated aqueous fractions (SCAF) on a macrophage cell line (RAW264.7) in vitro. A Macrophage phagocytic activity in response to SCAF0-5 treatment was measured using the Neutral Red phagocytosis assay and results were expressed as the optical density at 540 nm. B Production of nitric oxide indirectly by measuring NO 2− production in macrophages was achieved using the Griess reagent assay and expressed as μM. Lipopolysaccharide (LPS) was used a positive control at a pre-determined optimal concentration (1.0 μg/ml) and complete media was used as a negative control. All statistical analysis was carried out using a one-way ANOVA with Tukey's Multiple Comparison Test to compare differences between samples and negative controls (*p < 0.05, **p < 0.01, ***p < 0.001, n = 3). Groups are SCAF 0 (crude polysaccharide fraction of S. cordifolia); SCAF 1: Low ionic strength < 100 kDa; SCAF 2: medium ionic strength and between 10 and 100 kDa; SCAF 3: medium ionic strength and > 100 kDa; SCAF 4 high ionic strength and between 10 kDa-100 kDa; SCAF 5: high ionic strength and < 100 kDa in macrophages, and these results were corroborated by RT-qPCR analysis which showed a two-fold increase in iNOS mRNA expression (Fig. 4). Increases in the expression of iNOS and SCAF5-induced NO observed in this study are indicative of a T H 1-mediated response [37]. There are several T H cell subsets classified based on their unique cytokine profiles and functions [38],with the T H 1-mediated response contributing towards microbicidal activity (i.e., NO production and phagocytosis), which are highly effective in clearing intracellular pathogens (e.g., bacteria and viruses) compared with the T H 2 phenotype which is implicated in less desirable hypersensitivity reactions [39]. Further evidence for the activation of a T H 1 microbicidal response was observed with the 2-fold increase in transcripts for T H 1-associated cytokines interferon (IFN-γ) and tumour necrosis factor (TNF-α). A further indication that SCAF5 promotes an antimicrobial T H 1-mediated immune response is evidenced by a lack of expression of the T H 2-associated cytokine IL-10 in both cell types. The most marked increase in cytokine expression in macrophages and splenocytes was that of IL-6. This pro-inflammatory cytokine is secreted by T cells and activated macrophages following activation by microbial PAMPs and is critical for defence against a number of intracellular pathogens [40]. IL-6 induces antibody production and activation of macrophages and T-cells which corresponds to our experimental observations. An approximate doubling in the levels of expression of IL-1β (splenocytes and macrophages) and IFN-β (splenocytes) was also observed with SCAF5.
In vivo immunomodulation was confirmed in a novel model of innate immunity using the insect larvae from Galleria mellonella (G. mellonella). These larvae are an inexpensive and ethically favourable alternative to animal anti-microbial infection models due to its susceptibility to infection, larger size facilitating experimental manipulation, short life cycle and ability to grow at physiological temperatures (37 °C) [21,41]. Intriguingly insects possess a highly effective immune system containing humoral and cellular components with many similarities to the vertebrate innate immune system. For example, their cellular component is composed of phagocytes (haemocytes) which possess the capacity to detect highly conserved antimicrobial pathogen associated molecular patterns (PAMPs) [42]. The haemocytes of G. mellonella are analogous to vertebrate immune cells such as macrophages because they are capable of phagocytosis, and they express pathogen recognition receptors (PRR) for detecting pathogens. The immunomodulatory activity of SCAF5 observed in vitro was reproduced in vivo following administration of either SCAF fraction (SCAF0 or 5) in this primitive arthropod model, and SCAF5 was again found to exhibit the most potent activity as determined by a marked increase in the number of haemocytes in haemolymph. The proliferation of haemocytes was almost equivalent in magnitude to the response elicited by the pre-optimised LPS positive control. Subsequently larvae pre-treated with SCAF5 were extremely resistant (P < 0.001) to infection with methicillin-resistant Staphylococcus aureus (MRSA) with a 98% reduction in bacterial load compared to the negative control.
Ion exchange and size fractionation indicates that SCAF5 possesses a high ionic strength and a molecular size greater than 100 kDa, which was confirmed by electrophoretic studies which indicated the size of the bioactive component within this fraction ranged from 100 kDa-150 kDa (data not shown). SCAF2 and SCAF4 with molecular weights ranging from 10 to 100 kDa exhibited more moderate activity profiles and  suggesting that the size of the bioactive component may be an important determinant for the immunomodulatory activity. GC-MS analysis of SCAF5 confirmed the bioactive component consisted of polysaccharides and that the type of monomer constituents were similar, but that the ratio of sugars differed. SCAF5 contained higher levels of both mannose and glucuronic and uronic acid and this may explain the need for a high ionic strength solution to elute SCAF5. The increased proportion of mannose in SCAF5 may suggest the immunomodulatory activity is mediated via the mannose PRR found on immune cells [43]. Alternatively, glucuronic acid is a functional group of the commercial adjuvant QS-21 which essential for its immunogenic function [44]. Preliminary in vitro antimicrobial susceptibility screening assays indicated that the crude methanol fraction contained compounds of interest. Fractionation by flash chromatography resulted in the isolation of the bioactive active fraction SCMEXBu5 and subsequent HPLC fractionation elucidated two peaks (SC1 & SC2) with identical UV λ max profiles. Bioautography by means of agar overlay confirmed both SC1 and SC2 exhibited antibacterial activities against methicillin-resistant S. aureus (MRSA) and the source of the antimicrobial activity was subsequently identified as rosmarinic acid 4-O-β-d-glucoside (SC1) and rosmarinic acid (SC2). The activity was specific to Gram-positive bacteria including antibiotic resistant strains of Staphylococcus aureus and Staphylococcus epidermidis and the observed selectivity for Grampositive bacteria suggests the mechanism of action involves targeting the cell wall. The bactericidal and bacteriostatic activity of SC2 reported in this study are equivalent in concentration range (mg/ml) as other antimicrobial agents tested against the same panel of reference strains [27].
The antimicrobial properties of rosmarinic acid and its derivatives has been described previously [8] and these studies also confirm our observed specificity for Gram-positive bacteria. No mechanism of action has yet been elucidated although it has been suggested that rosmarinic acid targets cell surface virulence factors unique to Gram-positive bacteria which mediate the initial hostbacteria interactions [45,46]. Interestingly, both in vitro and in vivo studies have provided extensive evidence that rosmarinic acid is a potent and effective anti-tumour agent [47][48][49] and studies are currently underway to elucidate its putative anti-cancer properties with crude and fractionated S. cordifolia extracts. Antimicrobial activity was demonstrated to also be effective in reducing in vivo microbial load in Galleria larvae which were infected with MRSA. Subsequent administration of the highest concentration of SC2 (100 μM) decreased in vivo MRSA load by 99.9% (a 1243-fold decrease in CFU/ml) in haemolymph and a complete elimination of bacteria was observed in 5 out of 7 larvae.

Conclusion
S. cordifolia exhibits dual antimicrobial activities in that it stimulates the immune system via a T H 1-mediated antimicrobial response, priming a host in readiness for a potential infection as demonstrated with the G. mellonella in vivo model. The presence of compounds with direct antimicrobial activity (against Gram-positive bacteria including MRSA) is effective in limiting the extent of an infection in a host already infected. A more detailed characterisation of the polysaccharides involved will enable progress to be made in respect to fully understanding the properties of this extract and its potential applications. Nonetheless, the fact that rosmarinic acid is also reported to have antiviral activity indicates that there is potential for using S. cordifolia crude extracts as an inexpensive animal feed supplement to prevent infectious disease and reduce the use of antibiotic growth promoters in livestock and companion animals.