Antimicrobial flavonoids isolated from Indian medicinal plant Scutellaria oblonga inhibit biofilms formed by common food pathogens

Abstract Scutellaria oblonga Benth., a hitherto phytochemically unexplored Indian medicinal folklore plant was extracted with acetone and subjected to chromatography to yield nine flavonoids, for the first time from this plant. Antimicrobial assays were performed against 11 foodborne pathogens, and three molecules (Techtochrysin, Negletein and Quercitin-3-glucoside) depicted significant activity. These molecules were assessed for their rate of antibacterial action using time–kill curves which depicted complete inhibition of most of the bacteria within 12–16 h. The significant biofilm-reducing capability exhibited by these three molecules formed a significant finding of the current study. In most of the experiments, a 90–95% reduction in biofilms was observed. Thus, flavonoids as natural molecules from S. oblonga could be further researched to be used as potent antimicrobial and antibiofilm agents.


Introduction
Food in general or food products form an important component of human life. However, as other biotic stuffs, it is also prone to spoilage and deterioration which requires vigilance (Kazemi 2015). The consumption of such unsafe foods causes foodborne diseases left to be cured under the potency of commercial antibiotics in today's world. However, of late, ABSTRACT Scutellaria oblonga Benth., a hitherto phytochemically unexplored Indian medicinal folklore plant was extracted with acetone and subjected to chromatography to yield nine flavonoids, for the first time from this plant. Antimicrobial assays were performed against 11 foodborne pathogens, and three molecules (Techtochrysin, Negletein and Quercitin-3-glucoside) depicted significant activity. These molecules were assessed for their rate of antibacterial action using time-kill curves which depicted complete inhibition of most of the bacteria within 12-16 h. The significant biofilm-reducing capability exhibited by these three molecules formed a significant finding of the current study. In most of the experiments, a 90-95% reduction in biofilms was observed. Thus, flavonoids as natural molecules from S. oblonga could be further researched to be used as potent antimicrobial and antibiofilm agents. food pathogens have become highly resistant and effect strategies (e.g. Biofilms, quorum sensing, etc.) to evade antimicrobial invasion of antibiotics (Walsh & Fanning 2008). This creates an urgent need to discover and establish new potent antimicrobial molecules without compromising safety. Since decades, polyphenols (especially flavonoids) from plants have been acting as potent antimicrobial molecules. The complex structural diversity of plant-derived molecules makes the microbes difficult to evade their antimicrobial mechanisms. Also, plant molecules are green and safe for prolonged use in management of diseases. Scutellaria is a genus which is known for its species containing enormous amount of flavonoids (Mamadalieva et al. 2013;Bhat et al. 2014;Li et al. 2014;Wang et al. 2014;Wozniak et al. 2014). Its phytochemical constituents have been reported for various bioactivities ranging from antimicrobial, anti-inflammatory to anticancer (Shang et al. 2010;Kim et al. 2014). One of its species, Scutellaria oblonga Benth. is a tropical shrub found widely in Nilgiris of Tamilnadu and forests of Kerala, India . It is known as Kattutulsi in Tamil and is used in traditional folk medicine to treat infections . To our best of knowledge, there are no previous reports on phytochemical analysis, isolation and characterisation of chemical constituents from S. oblonga. In the current study, we report isolation of nine flavonoids for the first time from leaves of S. oblonga. We also report the antimicrobial activity, time-kill analysis of potent antimicrobial flavonoids and antibiofilm potencies of active flavonoids against food borne pathogens.

Results and discussion
Totally nine flavonoids ( Figure S1) were isolated and characterised (Figures S2-S4) from leaves of S. oblonga using acetone extraction and subsequent column chromatography using silica gel (Satyan et al. 2011;Sivasubramanian et al. 2013). Based on the acetone extraction, the total flavonoids yield was 942 mg/kg of dried leaf material. Although, acetone extract did not show considerable antimicrobial activity (>512 μg/ml) against all of the pathogens, all the individual compounds remarkably depicted considerable potency in inhibiting food pathogens (Table 1). Minimum inhibitory concentrations (MICs) of techtochrysin, negletein and quercitin-3-glucoside depicted significant antimicrobial action and were advanced for To assess the cidal action of these potent antimicrobial flavonoids, minimum bactericidal concentrations of these flavonoids were also observed. Techtochrysin, negletein and quercitin-3-glucoside as anticipated (from their MICs), depicted significant (p < 0.001) killing action against food pathogens compared to negative control. For e.g. Quercitin-3-glucoside killed 99.9% cells of B. subtilis, E. faecalis and S. dysenteriae at 24 μg/ml.
To achieve better understanding about rate of action of tested compounds, time-kill analysis was performed (Subramaniam et al. 2014) and the observations are depicted ( Figure S5). Relatively, negletein took much time (16-20 h) to kill P. aeruginosa, S. enterica, K. pneumonia and S. dysenteriae spp. compared to techtochrysin and quercitin-3-glucoside. The remaining pathogens were killed within 6-10 h. Similarly, for techtochrysin, considerable resistive patterns were observed with K. pneumonia (16 h) and moderate resistance with P. aeruginosa (10 h). The remaining pathogens were killed within 6 h. Quercitin-3-glucoside had to struggle significantly to kill E. coli and K. pneumonia (16 h), while it killed all other pathogens within 6 h except S. enterica (10 h).
Hence, to further assess the antibiofilm activities of these three potent molecules, experiments were done using four biofilm-forming bacteria (Figure 1). Observations depict that quercitin-3-glucoside showed gratifying biofilm inhibition at its MIC, reducing nearly 92-98% biofilms in all the tested pathogens. This observation was further followed by negletein which depicted 83.4, 88, 72.3 and 87.9% biofilm reduction against S. aureus, B. subtilis, P. aeruginosa and E. coli, respectively. Techtochrysin however showed relatively less but still significant (p < 0.001) biofilm inhibition of 88.9, 73.5 and 75.5% against B. subtilis, S. aureus and E. coli compared to negative control. However, it showed only moderate inhibition (66.7%) against P. aeruginosa. Similar reports were previously documented for naringenin and quercitin which depicted antibiofilm activity against food pathogens E. coli O157:H7 and Vibrio harveyi (Vikram et al. 2010).

Conclusion
In conclusion, totally nine flavonoids were isolated from leaves of S. oblonga for the first time and their structures were elucidated using spectral analysis. Antimicrobial assays with all the tested flavonoids depicted significant to moderate activity (MIC and MBC) against various food pathogens that may cause serious food-related diseases. To further obtain insights about potent flavonoids, techtochrysin, negletein and quercitin-3-glucoside were assessed with time-kill curve analysis and the results obtained indicate killing most of the bacteria within 12 h and rest within 24 h. Also, potent antibiofilm activity exhibited by these three flavonoids forms a significant result obtained in the current study. Four biofilm-forming pathogens were tested and molecules depicted significant reduction in biofilms in most of the experiments. Thus, S. oblonga could be used as a source of potent bioactive flavonoids and these molecules could be further researched for their application as antibiofilm agents especially against food pathogens.

Supplementary material
Supplementary material relating to this article is available online, alongside Figures S1-S5.