MATERIALS AND METHODS

Solvents: The HPLC-MS grade acetonitrile were purchased from Merck Co., (Darmstadt, GER). High-purity deionized water was obtained from a Milli-Q water purification system (Millipore Bedford, MA, USA). The HPLC-grade formic acid used for preparation of the mobile phase were from Anaoua chemical supply (Houston, USA), methanol used for plant extraction was from ANPEL Scientific Instrument Co. (Shanghai, CHN).

Plant material and preparation of the extract: The roots of S. barbata were collected during June-July 2014 from Jiuzhaigou, Chengdu, China. The plant material was identified by Prof. Xun-Xhao. The roots were thoroughly washed with tap water, shade dried and then chopped into small pieces. Methanol (95%) was used for hot extraction which was carried out for 4 h using a soxhlet extraction apparatus. The extract was then concentrated under reduced pressure in a rotary evaporator at 40°C and was then kept in a refrigerator at 4°C prior to use.

Liquid chromatography-tandem mass spectrometry (LC-ESI-MSMS)/HPLC analysis: The HPLC analysis was carried out by aNexera HPLC system (Shimadzu, Japan) with a double-pump (LC-30AD), column oven (CTO 30A) and Autosampler (SIL-30AC). A Chromolith RP-18e column (4.6 mm ID, 50 mm length) (Merck) was used. The mobile phase was a gradient prepared from solvent A (0.1% aqueous formic acid) and solvent B (acetonitrile) and the conditions used for gradient elution were: 0-3 min, 5-18%B; 3-5 min, 18%B; 5-6 min, 18-45%B; 6-7 min, 45-100%B; 7-8 min, 100% B. The separation was conducted at 25 with a flow rate of 0.8 mL min^–1. The injection volume was 2 μL.

The LC-MS equipment (LC-MS QqQ-6410B Agilent Technologies) consisted of a chromatographic system (1260 In nity Agilent Technologies) coupled with an Agilent Triple Quad mass spectrometer tted with an ESI source. The MS conditions were the following: MS range 100-1200 Da, MSn spectra were obtained using both positive and negative modes, nebulizer gas 45 Psi, gas temperature 325°C, capillary voltage 4000 V.

Microorganism strains and culture media: Streptococcus pneumonia (ATCC 2730), Streptococcus pyogenes (ATCC 12344), methicillin-resistant Staphylococcus aureus (MRSA) (ATCC 43300), Bacillus subtilis (ATCC 6633), Pseudomonas aeruginosa (ATCC 25619) and Salmonella typhimurium (ATCC 10535) were used in the current study. The bacterialstrains were procured from the State Key Laboratory of Microbial Resources (SKLMR), the institute of microbiology, Chinese academy of Sciences, China. Bacterial strains were grown on nutrient agar plates at 37°C and maintained on nutrient agar slants. Cell suspension of microorganisms in 0.5% NaCl was adjusted at 0.5 Mcfarland to obtain approximately 10⁵ CFU mL^–1.

Preliminary screening for antimicrobial activity: The antimicrobial test was performed by disc diffusion assay as per NCCLS, 1997 (NCCLS., 1997). The nutrient agar plates containing an inoculum size of 10⁶ CFU mL^–1 on Saboraud glucose agar plates were used. Previously prepared extract impregnated disc (6 mm in diameter) at the concentrations of 200 μg mL^–1 for bacterial strains were placed aseptically on sensitivity plates with appropriate controls. Vancomycin (100 μg mL^–1) was used as standard antibacterial. Plates were incubated at 37°C for 30 h. Antibacterial activity wasrecorded by measuring the clean zone of growthinhibition on agar surface around the disc.

Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC): The MIC and MBC tests were carried out by the broth microdilution method (Yu et al., 2004). The extract was dissolved in sterilized physiological saline solution (0.9%) supplementedwith Tween-80 (Sigma) at final concentration of 0.8% (v/v). Serial doubling dilutions of the extract were prepared in a 96-well microtiter plate ranging from 10-0.125%. Each dilution (100 μL) was dispensed into the wells, then inoculated with (100 μL) of the bacterial suspension. The final concentration of each strain was adjusted to 10⁵-10⁶ CFU mL^–1. The concentration of the extract at which the bacteria show no visible growth is defined as Minimum Inhibitory Concentration (MIC) while, as the extract concentration at which the bacteria are killed is defined as Minimum Bactericidal Concentration (MBC). All experiments were performed in triplicate.

Reactive Oxygen Species (ROS) generation by the extract: The 2’, 7’-dichlorofluorescein diacetate (DCFDA) was used to detect the Reactive Oxygen Species (ROS) generation in these six bacterial strains induced by the methanol extract of S. barbata (Kye et al., 1999). The concentration of the extract used was 65 μg mL^–1 and the number of bacterial cellsused was adjusted to 10⁶ CFU mL^–1. After all cultures were incubated at 37°C for 4 h, they were centrifuged at 4°C for 20 min at 300×g and then each supernatant was treated with 50 μM DCFDA for 2 h. The ROS formed in the samples were detected using Fluorescence MultiDetection Reader (BIOTEK, USA) at 485 nm of fluorescence excitation wavelength and 528 nm of emission wavelength.

Scanning Electron Microscopy (SEM) evaluation: The SEM was performed as reported already in the literature (Yenugu et al., 2004; Agizzio et al., 2006). Salmonella typhimurium was grown in MH agar at 37°C was put in a saline solution comprising of 0.2% tween-80. Four different concentrations of the extract (0, 5, 25 and 75 μg mL^–1) were prepared and added into this suspension and was incubated at room temperature. After 24 h, the bacterial cells were centrifuged at 10,000xg for 10 min. The bacterial cells were then washed with 0.1 mol L^–1 tris-acetate buffer (pH 7.1), fixed in tris-acetate buffer containing 1.5% glutaraldehyde and then freeze-dried. Each of the bacterial culture was observed by SEM (Hitachi, Japan) at magnifications of 10,000x. The bacterial cell suspension in saline with no extract treatment served as a negative control.

Determination of the rate of killing of the tested bacterium using the S.B extract: This assay determining the rate of killing of the S. typhimurium was evaluated as per an already reported method with minor modifications (Odenholt et al., 2001). Tubes containing 10 mL of broth to which the extract had been added at concentrations of 1, 2 and 4 times the MIC were inoculated with a suspension of the test strain, giving a final bacterial count of approximately 5x10⁶ CFU mL^–1. The tubes were thereafter incubated at 37°C and the killing rate was determined over a period of 2 h. A 1.5 mL volume of each suspension was withdrawn at time intervals and transferred to 5 mL of nutrient broth recovery medium containing 5% “Tween 80” to neutralize the effect of the antimicrobial compound carry-overs from the test organisms. The suspension was then successively diluted and plated for viable counts. The plates were later incubated at 37°C for 48 h. The control plates contained the test microorganisms without the S.B extract. The remaining bacterial colonies were counted and compared with the counts of the culture control.

Determination of protein leakage in S. typhimurium bacterial cells: Twelve hour old bacterial cells were washed twice with saline by centrifugation at 12,000 rpm for 15 min followed by re-suspension in physiological saline. The bacterial cells were then treated with varying concentrations of the S. barbata extract relative to MIC (1x MIC, 2x MIC and 4x MIC) for 2 h. Each bacterial suspension treated with extract was centrifuged at 10,000 rpm and the supernatant obtained was evaluated for protein estimation using the Bradford assay (Bradford, 1976). The concentration of protein was estimated from the established standard curve obtained using Bovine Serum Albumin (BSA).

Statistical analysis: All the experiments were done in triplicate and the results were expressed as Mean±SD. Two-way analysis ANOVA was used for comparisons. Statistical significance was calculated at p<0.05.

RESULTS AND DISCUSSION

LC-ESI-MSMS analysis/HPLC analysis: The phytochemical analysis of the S. barbata root extract was carried out by LC-ESI-MS as well as HPLC-DAD techniques. The extract was run under both positive and negative ESI-MS conditions and it showed several major and minor ionic species. The total ion MS chromatogram is shown in Fig. 1, while as the identified compounds are shown in Fig. 2. Fragmentation of the major peaks was used for the identi cation of compounds. The identi cation of the chemical compounds was also carried out by comparing the molecular ion peaks along with the MS fragmentation pattern with those of the literature. The five chemical constituents identified were (1) Scutellarin, (2) Luteolin, (3) Apigenin, (4) 4’-Hydroxywogonin, (5) Luteolin-7-O-glucuronide (Fig. 2). These phytochemicals have already been reported previously in this medicinal plant (Yao et al., 2011).

Antibacterial activity of the S. barbata extract: The antibacterial activity of S. barbata extract was evaluated against a panel of six clinically important bacterial pathogens including Streptococcus pneumonia (ATCC 2730), Streptococcus pyogenes (ATCC 12344), methicillin-resistant Staphylococcus aureus (MRSA) (ATCC 43300), Bacillus subtilis ATCC 6633, Pseudomonas aeruginosa (ATCC 25619) and Salmonella typhimurium (ATCC 10535). Both the zones of inhibition as well as MIC/MBC values of the extract were calculated against these microbes using disc diffusion and microdilution assays respectively. The results of the disc diffusion assay (zones of inhibition) are shown in Fig. 3 while, as the results of the microdilution assay (MIC/MBC values) are shown in Table 1. As can be seen from Fig. 3, Salmonella typhimurium exhibited the maximum zone of inhibition at all extract concentrations and as such was most susceptible to the S. barbata extract treatment. Pseudomonas aeruginosa was the next most susceptible bacteria to the extract after S. typhimurium showing higher zones of inhibition at almost all extract doses. Streptococcus pneumonia, Streptococcus pyogenes, methicillin-resistant Staphylococcus aureus bacterial strains were relatively less susceptible to the S. barbata extract. The MIC/MBC values almost also displayed the same trend (Table 1) with S. typhimurium bacterial strain showing the lowest MIC and MBC values indicating that the extract has potent antibacterial effect against this pathogen. MRSA showed the highest value of MIC/MBC indicating that the extract has lesser effect on this bacterial pathogen.

Fig. 1:	LC-MS chromatogram of the methanol extract of S. barbata. The green numbers show peaks corresponding to compounds, (1) Scutellarin, (2) Luteolin, (3) Apigenin, (4) 4'-Hydroxywogonin and (5) Luteolin-7-O-glucuronide

Fig. 2:	Phytochemical constituents identified in the bioactive extract of Scutellaria barbata using HPLC/LC-MS analytical techniques

Fig. 3:	Effect of the different concentrations of the S. barbata extract on the zones of inhibition (antibacterial susceptibility) of the various bacterial strains

Table 1:	MIC and MBC values of the S. barbata extract as well as the standard antibacterial drug against a panel of bacterial strains

Generation of ROS in bacterial cells treated with different doses of S. barbata extract: There is a direct relation between Reactive Oxygen Species (ROS) generation and bacterialdeath. Various published reports demonstrated that plant extracts could lead to the generation of free radicals within bacterial and fungal cells which finally result in deleterious bacterial membrane damage.

Fig. 4:	Formation of ROS in S. pneumonia, S. pyogenes, MRSA, B. subtilis, P. aeruginosa and S. typhimurium bacterial cells exposed to different doses of the extract. Bacterial cultures were treated with the extract for 4 h

In the current study, ROS was measured in six bacterial cultures using DCFDA. After 4 h incubation, elevated levels of ROS were detectedand measured in the S. barbata extract treated bacterial group. As can be seen in Fig. 4, ROS generation was maximum in S. typhimurium at all extract concentrations. This result is consistent with the disc diffusion experiment as well as microdilution assay as both these experiments also showed that S. typhimurium is the most affected bacterial strain. The extract brings about generation of ROS in this bacteria more than other tested bacteria. The extract also induced variable ROS generation in other bacterial strains as can be seen in Fig. 4.

Morphological evaluation of S. typhimurium by scanning electron microscopy after extract treatment: The SEM results indicated that the S. barbata extract targets cell membrane of S. typhimurium bacteria. As can be seen in Fig. 5a-d, S. barbata extract induced potential morphological changes in S. typhimurium bacteria. As compared to the negative control (Fig. 5a, untreated bacterial cells) which showed normal cellular morphology, the S. barbata extract treated bacteria (Fig. 5b-d) showed lot of cellular damage especially at 75 μg mL^–1 concentration of extract (D). The membrane damage followed dose-dependent relation with the extract. This membrane damage in S. typhimurium is consistent with the Reactive Oxygen Species generation by the extract. The ROS generation can eventually lead to membrane damage which was demonstrated by SEM in this study.

Extent and rate of killing of S. typhimurium by the S.B extract: The extent and rate of killing of the S. typhimurium by the S.B extract at a concentration of 1x MIC, 2x MIC and 4x MIC is shown in Fig. 6. The percentage of bacterial cells killed within a 40 min contact time between the bacterial cells and the extract was around 24.7% which increased to 44.5% after 60 min time. After 80 min, about 95% of the cells were killed. This behaviour was also seen when the concentration of the S.B extract was doubled, that is 2x MIC. The concentration of the extract was again increased to 4x MIC and the percentage of bacterial cells killed after 40 min contact time was 64.2%. Thus the S.B extract showed a dose-dependent and time-dependent bactericidal effects on S. typhimurium bacterial cells.

Scutellaria barbata extract induced protein leakage in S. typhimurium microbes: Further, the bactericidal effects of the S. barbata extract were evaluated by observing its effect on protein leakage after bacterial cell membrane rupture. At a dose of 1x MIC and 40 min contact time of the bacterial cells with the extract, around 34.2 μg mL^–1 of protein leaked out of the S. typhimurium cells which increased to 38.1, 42.4, 56.2 and 58.1 μg mL^–1 after 60, 80, 100 and 120 min time periods respectively (Fig. 7). The same pattern of protein leakage was seen when the S. barbata extract concentration was increased to 2x MIC and then 4x MIC.

Fig. 5(a-d):	Electron micrographs of S. typhimurium exposed to different concentrations of the extract. The images were taken at (a) Magnification of 10,000x and S. typhimurium 12 h in saline, (b-d) Scanning electron micrographs of S. typhimurium treated for 12 h with 5, 25 and 75 μg mL–1 of the extract

Fig. 6:	The extent and the rate of killing of S. typhimurium by the S.B extract at concentrations of 1x MIC, 2x MIC, 4x MIC and the control. Each point represents the log10 of the mean survival of bacterial cells at a particular time interval in the presence of the extract

Fig. 7:	The effect of S. barbata extract on protein leakage from S. typhimurium bacterial cells at concentrations of 1x MIC, 2x MIC, 4x MIC and control. Each point characterizes the amount of protein leaked (μg mL–1) from the bacterial cells at a specific time interval in the presence of the extract

The protein leakage was much significant at 4x MIC concentration having values of 45.1, 57.3, 67.5, 71.3 and 77.3 μg mL^–1 after 40, 60, 80, 100 and 120 min contact of the bacterial cells with the extract, respectively.

DISCUSSION

There are approximately 20,000 plant species being used as ethno-medicines all over the world.However there has been an enormous increase in the multidrug resistant strains of clinically relevant bacterial pathogens which is posing a great challenge to the scientific community. This increase in multidrug resistance is believed to arise due to the indiscriminate use of antibiotics (Srividya et al., 2010; Martin and Yost, 2011; Bibi et al., 2011). Among the available treatments maximum drugs used to treat infectious diseases happen to be secondary metabolites isolated from natural resources. Importantly, these therapeutic secondary metabolites also work as prototypes or lead compounds for the derivation of novel antibiotics. One important feature of plant natural compounds is their hydrophobic nature which render them capable of partitioning the lipid bilayer of the membrane and thus cause disruption of the cell membrane and make them permeable making it vulnerable to the compound effects. Many plant derived compounds including phenols, quinones, terpenoids, flavones have been reported to possess antibacterial activity. Flavonoids and phenols exhibit effective antibacterial activity owing to their ability to form complexes with extra cellular and soluble proteins and to complex with bacterial cell walls leading to the death of the bacteria (Kuete, 2010; Cox et al., 2000; Carson et al., 2002; Cowan, 1999). More specifically flavonoids have been extensively studied and most of the studies have established their role as potential anti-microbial agents (Daglia, 2012; Cushnie and Lamb, 2005; Zhang et al., 2011).

Scutellaria barbata D. Don (Lamiaceae) is a perennial herb which is distributed throughout southern China. This herb is known in traditional Chinese Medicine as Ban-Zhi-Lian. It has been explored for the possible treatment of inflammation, tumor and gynecological diseases in China (Dai et al., 2008). Phytochemically, S. barbata is known to contain alkaloids, polyphenols and flavones (Jiangsu New Medical College, 1977; Qian, 1987). Scutellaria barbata is widely used as an antitumor agent in China. Ethanol extract of S. barbata has been reported to induce apoptosis in the lung cancer cells (A549) while as methylene chloride fraction of the plant has been shown to induce apoptosis in human leukemia cells through the mitochondrial signaling pathway (Yin et al., 2004; Cha et al., 2004). Flavonoids (apigenin and luteolin) were isolated from a 50% ethanolic extract of the plant as active constituents against methicillin-resistant Staphylococcus aureus. The E-1-(40-Hydroxyphenyl)-but-1-en-3-one was isolated from methanol extract of the plant and displayed strong cytotoxicity in K562 human leukaemia cell lines (Sato et al., 2000; Ducki et al., 1996).

S. barbata also contains essential oil with hexahydrofarnesylacetone, 3, 7, 11, 15-tetramethyl-2-hexadecen-1-ol, menthol and 1-octen-3-ol as main chemical constituents. The essential oil has also been reported to possess antibacterial activity against a wide-spectrum of bacterial strains including Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, methicillin-sensitive and methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus heamolyticus, Staphylococcus simulans, Enterococcus faecalis among others (Yu et al., 2004). Keeping in consideration the broad spectrum effects of S. barbata, we have evaluated the antibacterial activity and mode of action of the methanolic extract of the root of S. barbata along with phytochemical analysis of the extract to identify main chemical constituents. Antibacterial efficacy of the extract was measured by studying the zone of inhibition and MIC/MBC values of the extract against different bacterial strains using Disc diffusion and microdilution assays. Scanning Electron Microscopy (SEM) was employed to evaluate the morphological changes induced by the extract in cellular membrane of the bacteria. The extract inhibited growth inhibition of all tested bacteria with S. typhimurium being the most susceptible pathogen towards the extract followed by P. aeruginosa. The extract showed dose-dependent growth inhibitory effects. In addition it was found to induce potent ROS generation in all bacterial cultures with maximum peak in S. typhimurium. The SEM results revealed that the methanolic extract of S. barbata induces deleterious morphological changes in the bacterial cell membrane of S. typhimurium indicating that ROS generation can lead to membrane damage in bacterial cells. The percentage of bacterial cells killed within a 40 min contact time between the bacterial cells and the extract was around 24.7% which increased to 44.5% after 60 min time. After 80 min, about 95% of the cells were killed. This behaviour was evident when the extract concentrations was doubled, that is 2x MIC. Further the intracellular protein leakage was found among the the S. typhimurium bacterial cells when exposed to different doses of the extract indicating bacterial cell wall rupture by the extract and the biocidal effects of the extract on the cells, as evident by the killing rate of test results. Based on the previous studied on S. barbata regarding the potential of this herb as anti-inflammatory, anti-tumor, diuretic and gynecological diseases, it is evident that the herb can a potential lead against different bacterial strains that are clinically important.

CONCLUSION

In conclusion, this study demonstrates that the Scutellaria barbata methanol extract is a potent antimicrobial agent capable of inhibiting the growth of various clinically important microbes. The present study demonstrates that the antibacterial effect of the extract is mediated through membrane damage induced due to ROS generation by the extract followed by the leakage of protein molecules. The extract could be used as a safe and natural alternative to antibiotics with less incidence of showing multidrug resistance.