Biological and phytochemical potential of Baccharis trimera (Less.) DC leaf extract on swine clinical isolates

Abstract Phytochemical studies have shown extracts obtained from the aerial parts of Baccharis trimera (Less.) DC has antioxidant and antimicrobial activities and the potential to treat some diseases. This study investigated the phenolic compounds, antioxidant and antimicrobial activity, and phytochemical potential of B. trimera leaf extract obtained by decoction on ATCC standard bacterial strains and 23 swine clinical isolates. Water was used as an extraction solvent based on the principles of green chemistry and at a low cost. The decoction process resulted in an extract rich in phenolic compounds and a high capacity for scavenging DPPH and ABTS radicals. Phytochemical analysis of aqueous extracts was performed using HPLC-DAD, and high concentrations of chlorogenic, ferulic, caffeic, and cinnamic phenolic acids were found. Antimicrobial activity was observed against gram-negative bacteria. B. trimera aqueous extract may be a promising low-cost agent for prophylactic treatment against swine enteropathogens and contribute to reducing production costs. Graphical Abstract


Introduction
new therapeutic approaches are promising alternatives in treating disease conditions, including using natural or plant-derived products, which have demonstrated efficiency in promoting health with fewer side effects when used correctly (Karimi et al. 2015).Baccharis trimera (Less.)dC, native to South america, has many pharmacological properties and has been used in treating diabetes mellitus and digestive and liver diseases, in addition to its anti-inflammatory, gastroprotective, hepatoprotective, antioxidant, hypotriglyceridemic, and hypoglycaemic effects (oliveira et al. 2010;Barbosa et al. 2020).
the composition of B. trimera extracts in biocompounds varies according to the extraction method, type of solvent, and part of the plant used.although organic solvents have high employability in secondary compound extraction processes, polar and non-toxic solvents such as water also show good results.Several studies have reported the efficiency of water use for extracting secondary compounds with biological activities (Rabelo et al. 2017;Sabir et al. 2017).
the use of water as a solvent to obtain extracts with bioactive properties has some advantages, such as lower toxicity and lower economic cost, compared to extraction with organic solvents.Focusing on a green and low-cost extraction process, aqueous extracts of B. trimera cultivated in southern Brazil were obtained and characterized for phenolic compounds, antioxidant activity, antimicrobial capacity, and phytochemical properties.
Some herbs with known effects related to the control of diarrhoea in humans and animals may have biological potential for use as additives and microbiota modulators (dadi et al. 2020).Bioactive compounds extracted from plants, used as phytobiotics, offer an important perspective in replacing commercial growth promoting formulations recommended today (Vidanarachchi et al. 2005), with a healthier approach that meets the demands of the consumer, in contrast to the few studies involving tests with microorganisms of veterinary origin, the present work seeks to elucidate the potential of the plant on pathogens of importance in pig farming (Salmonella and E. coli).

Phytochemicals and antioxidant ability
the B. trimera leaf extract obtained by extraction with boiling water showed high levels of total phenolic acids (288.2 ± 2.29 milligram of gallic acid equivalent per gram of extract (mg GaE/g)).Lower total phenolic contents (101 ± 4.1 mg GaE/g) were found by Sabir et al. Sabir et al. Sabir et al. (2017) in B. trimera leaf extract using a similar extraction process.phytochemical analysis (table 1) showed the presence of chlorogenic acid (9.189 mg 100 g −1 ), ferulic acid (2.138 mg 100 g −1 ), caffeic acid (0.13 mg 100 g −1 ), and cinnamic acid (0.009 mg 100 g −1 ) in the studied extract.Compounds such as gallic acid (45.8 mg g −1 ), rutin (15.1 mg g −1 ), and quercetin (5.1 mg g −1 ) were identified in extracts of B. trimera leaves obtained by the maceration method used by Sabir et al. (2017).Fifteen different compounds were found in the aqueous extract of B. trimera by Rabelo et al.
(2017), most of which were derived from caffeic and ferulic acids, in addition to the presence of three flavonoids.the high content of phenolic compounds in the extract of B. trimera leaves highlights the importance of this plant as a potential antioxidant source.
in the present study, the relationship between the extract dose and the standard required to obtain a 50% reduction in dppH radicals was 5.7 times greater than that of the standard (ascorbic acid), while this relationship was 14.6 times in a study by Sabir et al. (2017) with aqueous extract of the same species (iC 50 of the extract 415 µg mL −1 and iC 50 of the ascorbic acid standard 28.4 µg mL −1 ), demonstrating better results than those described in the literature.
the aqueous extract of B. trimera was also efficient in removing the aBtS radical, it removed approximately 80% of this radical at a concentration of 1,500 µg mL −1 .the relationship between the iC 50 values of the extract studied here and ascorbic acid used as a reference standard was 3.5 times.
phytochemical analyses of B. trimera leaf extracts have revealed the presence of phenols and flavonoids (Rabelo et al. 2017;Sabir et al. 2017). in this context, the ability to scavenge free radicals and the antioxidant potential of B. trimera extracts is possibly associated with the presence of simple phenol structures as well as polyphenolic substances.previous studies have shown a strong correlation between antioxidant activity and the presence of phenolic compounds in samples (Floegel et al. 2011).
the microbial inhibition curves of the american type Culture Collection (atCC) control bacterial strains showed the maintenance of the antimicrobial inhibitory effect of the extract.Whereas in the control assays, cell growth was maintained during the evaluation period (Figure 2).
Regarding the clinical isolates Escherichia coli 371, E. coli 348, Salmonella Choleraesuis 1542, and S. Senftenberg 25, B. trimera extract showed a differential inhibitory effect on microbial growth, highlighting the susceptibility of some strains to others.as expected, the growth of microorganisms cultivated without extract was maintained during the evaluation (Figure 3). the clinical isolate E. coli 371  showed the lowest sensitivity to the extract, with an inhibition curve profile similar to that of the control.in contrast, E. coli 348 and S. Senftenberg 25 were the most sensitive to the extract.
important antimicrobial activity of the aqueous extract of B. trimera against gram-negative pathogenic microorganisms clinically isolated from swine (E. coli and Salmonella spp.) was found in this study.it is worth mentioning that the antimicrobial activity of B. trimera extracts against gram-positive bacteria has been reported, but not against gram-negative bacteria.the work written by oliveira et al. (2005) showed antimicrobial activity only against the gram-positive bacterium.Similarly, aleixo et al. (2013) did not observe the antimicrobial activity of the hydroalcoholic extract of B. trimera against gram-negative microorganisms.
tincture of B. trimera leaf was effective in inhibiting gram-positive bacteria, however, it showed no activity against E. coli atCC 35218, contrary to what was found in this study, where the aqueous extract inhibited the growth of E. coli atCC strain and enterotoxigenic clinical isolates (da Silva et al. 2018).
the bacteriostatic effect of the extract was demonstrated by the spot-test method, in which it is possible to qualitatively evaluate the density of cells maintained in the stationary phase of cell growth, a characteristic of antimicrobials such as tetracyclines and macrolides (Guimarães et al. 2010).the profile of the inhibition curves reinforces the indication of the bacteriostatic effect observed in the spot test, whereas the control inoculum maintained the expected growth.

Experimental
the experimental session is described in supplementary material.

Conclusions
B. trimera extract obtained by decoction was rich in phenolic compounds, mainly phenolic acids such as chlorogenic, ferulic, caffeic, and cinnamic.these extracts showed a high ability to scavenge aBtS and dppH radicals and antimicrobial potential.the extract acted selectively with a more significant antimicrobial effect on gram-negative bacteria.it may be a promising and environmentally friendly, low-cost agent for prophylactic treatment against swine enteropathogens.

Figure 1 .
Figure 1.dPPh radical scavenging activity of the B. trimera aqueous extract (a) and ascorbic acid standard (B) aBts radical scavenging activity of the B. trimera aqueous extract (c) and ascorbic acid standard (d).

Figure 2 .
Figure 2. absorbance (nm) relative to cell growth of E. coli atcc 25922 (a) and S. typhimurium atcc 0028 (B) in the absence and presence of B. trimera extract at a concentration of 5 mg ml -1 and 10 mg ml -1 , respectively, incubated in an orbital shaker at a temperature of 37 °c at 150 rpm for 9 h.

Figure 3 .
Figure 3. absorbance (nm) related to cell growth of E. coli 371 (a) E. coli 371 (B), S. choleraesuis 1542 (c) and S. senftenberg 25 (d) in the absence and presence of B. trimera extract at a concentration of 20 mg ml -1 , incubated in an orbital shaker at a temperature of 37 °c at 150 rpm for 9 h.

Table 1 .
chromatographic parameters of compounds analysed by hPlc-dad.

Table 2 .
Minimum inhibitory concentration (MIc) of B. trimera extract on Salmonella enterica subspecies enterica and Escherichia coli serovars (clinical isolates and atcc strains) incubated at 37 °c for 16-20 h for growth.