Constituents and inhibitory effect on human pathogenic bacteria of the roots of scutellaria baicalensis

Vietnam Journal of Science and Technology 57 (1) (2019) 7-14 Doi:10.15625/2525-2518/57/1/12758 CONSTITUENTS AND INHIBITORY EFFECT ON HUMAN PATHOGENIC BACTERIA OF THE ROOTS OF SCUTELLARIA BAICALENSIS  Le Duc Long 1 , Nguyen Huu Tung 2 , Nguyen Thi Thom 3 , Gyung Ja Choi 4 , Vu Dinh Hoang 3 , Le Dang Quang 1, * 1 R&D Center of Bioactive Compounds, Vietnam Institute of Industrial Chemistry, 22 Pham Ngu Lao, Hoan Kiem, Ha Noi 2 VNU School of Medicine and Pharmacy, Vietnam National University, 144 Xuan Thuy, Cau Giay, Ha Noi 3 Hanoi University of Science and Technology, 1 Dai Co Viet, Hai Ba Trung, Ha Noi 4 Center for Eco-friendly New Materials, Korea Research Institute of Chemical Technology, 141 Gajeong-Ro, Yuseong-Gu, Daejeon, 305-600, Republic of Korea * Email: ledangquang2011@gmail.com Received: 23 July 2018; Accepted for publication: 15 November 2018 Abstract. Methanol extract of the roots of Scutellaria baicalensis effectively inhibited the bacterial growth of human pathogenic bacteria Staphylococcus aureus ATCC 6538, Bacillus cereus ATCC 21768 and Bacillus subtilis ATCC 6633 at MICs of 2,000 µg/mL. Hexane, ethyl acetate and aqueous residues were prepared by successively partitioning the methanol extract with hexane and ethyl acetate. Among them, only ethyl acetate layer showed antibiotic effect; whereas hexane and aqueous layers were inactive against tested bacteria. The ethyl acetate residue was fractionated by silica gel column chromatography to afford three flavonoids and an oligosaccharide. Their chemical structures were elucidated as wogonin (SB1), baicalein (SB4), baicalin (SB5) and tetrasaccharide (SB10) on the basis of the analysis of NMR and MS spectroscopic data. The isolates were evaluated for in vitro inhibitory effect against human pathogenic bacteria using micro dilution bioassay method. Baicalein (SB4) showed a broad- spectrum inhibition against various human pathogenic bacteria. In particular, it was found to potently inhibit S. aureus ATCC 6538 and B. cereus ATCC 21768 with MICs of 9.5 and 38 µg/mL, respectively. The study results demonstrated antibiotic effect of the extracts from the roots of S. baicalensis and characterization of compounds isolated from the plant materials. Keywords: activity, flavonoid, isolation, Scutellaria baicalensis. Classification numbers: 1.2.1.  Presented at the Conference: “The 6th National Workshop on Research and Development of Natural Products” Le Duc Long et al. 8 1. INTRODUCTION The genus Scutellaria consists of over 350 species worldwide and has been used by many cultures to treat a variety of medical conditions, including anxiety, nervous disorders, liver disease and cancers [1]. S. baicalensis is widely grown in Vietnam where root decoction has traditionally been used in medicine as a cancer treatment, remedy for inflammation and atherosclerosis [1, 2]. The flavonoids such as baicalein, wogonin, and baicalin are the major compounds in S. baicalensis. In recent years, the flavonoids isolated from the roots of S. baicalensis have demonstrated therapeutic potential for anti-inflammatory, anti-HIV, anti-human herpes virus type 6 (HHV-6), antioxidant, neuroprotective and anti-cancer properties [3]. The antibiotic activity of the roots of S. baicalensis and its constitutents are also considered because this plant has a wide range of uses in traditional medicine. The aim of this study, therefore, is to isolate and identify metabolites from the roots of S. baicalensis and to test for their antimicrobial activity against the growth of human pathogenic bacteria. 2. MATERIALS AND METHODS 2.1. Bacterial strains and culture conditions The extracts and the isolated compounds were tested for their antibacterial activity against Gram-positive bacteria such as Bacillus cereus ATCC 21768, Bacillus subtilis ATCC 6633, and Staphylococcus aureus ATCC 6538 which were obtained from the ATTC (Manassas, VA, USA) and Gram-negative bacteria such as Escherichia coli (American Type Culture Collection) ATCC 25922, Burkhoderia cepacia ATCC 25416, Enterobacter cloacae ATCC 13047 and Pseudomonas aeruginosa ATCC 9027. The bacterial strains were activated on nutrient agar (NA), then, transferred in nutrient broth (NB) at 37 °C for 24 h. The bacterial suspensions were diluted with sterile saline to get a turbidity equivalent to that of the 0.5 McFarland standard. 2.2. Plant materials Scutellaria baicalensis Georgi was purchased in Ninh Hiep folk medicine market in August 2016 and identified by Mr. Nghiem Duc Trong from the Department of Botany, Hanoi University of Pharmacy. Materials were crushed to powdery sizes of from 0.1 to 3 mm. The root powder (moisture content 7-10 %) was kept in dry environment at R&D Center of Bioactive Compounds, Vietnam Institute of Industrial Chemistry prior to extraction experiments. 2.3. Isolation of constituents from Scutellaria baicalensis The roots of S. baicalensis (2 kg) were crushed and extracted three times with methanol at 65 o C for 8 h and then filtrated and evaporated under reduced pressure to yield 390 g crude methanol extract. Then, the crude methanol extract was dissolved with 1.5 L of distilled water and partitioned with hexane and ethyl acetate (1.5 L; 3 times for each), consecutively. Each organic layer was evaporated under reduced pressure to yield hexane soluble residue (41.5 g) and ethyl acetate soluble residue (237.6 g). The hexane and ethyl acetate residue were tested by thin layer chromatography; the presence of flavonoids and oligosaccharides was observed in the ethyl acetate residue. The ethyl acetate residue (20 g) was subjected to column chromatography on silica gel (400 g silica gel 60Å (40–63 μm), 4.5 × 70 cm) which was eluted with the gradient solvent system of Constitutents and inhibitory effect on human pathogenic bacteria. 9 hexane/ethyl acetate (10:0; 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, 0:10, v/v) and finally washed out with the mixture of ethyl acetate and methanol in the ratio of 8:2 (v/v). In the results 7 fractions F1-F7 were yielded. Fraction 2 was fractionated on a Sephadex LH-20 column (3 g, 2.0 × 70 cm) eluted with methanol and the selected fractions were crystallized with hexane to give crystals of SB1 (842.7 mg). Fraction 4 (2.986 g) was applied on a silica gel column (60 g silica gel 60 Å (40–63 μm), 3.0 × 40 cm), eluted with hexane/ethyl acetate (7:3; 6:4, 5:5, v/v, 240 mL each and fraction volume of 50 mL) to yield two pure compounds SB4 (46.7 mg) and SB5 (387 mg). Fraction 6 (3 g) was dissolved with 150 mL of methanol and mixed with 4.5 g of silica gel 60 Å (40–63 μm). Then, the mixture was fractionated by a silica gel column chromatography (90 g silica gel 60Å (40–63 μm), 3.4 × 50 cm), eluted with a gradient system of ethyl acetate/methanol (8:2, 75:25, 7:3, 65:35, 6:4, 55:45, 5:5, 45:55, 4:6, 45:65, v:v) and then washed out with methanol:water in the ratio of 40:1 (v/v) to produce four fractions, F61 to F64. Fraction F61 was separated on a Sephadex LH-20 column (2 g, 2.0 × 50 cm) eluted with methanol to yield SB10 (468 mg). 2.4. Structural characterization of the isolated compounds SB1: Wogonin ESI-MS m/z 284.07 [M+H] + ; 1 H-NMR ((CD3)2CO, 500 MHz): 6.328 (1H, s, H-3), 6.817 (1H, s, H-6), 8.122 (1H, dd, J=7 Hz, J= 1.5 Hz, H-2’), 7.642 (2H, m, J= 8,5 Hz, H-3’, H-5’), 7.646 (1H, m, H-4’), 8.122 (1H, dd, J=5Hz, J=3,5 Hz, H-6’); 13C-NMR ((CD3)2CO, 125 MHz): 164.64 (C-2), 106.25 (C-3), 183.45 (C-4), 150.93 (C-5), 158.46 (C-8), 158.03 (C-7), 99.99 (C- 6), 130.26 (C-9), 105.62 (C-10), 132.50 (C-1’), 127.80 (C-2’, C-6’), 128.93 (C-3’, C-5’), 132.96 (C-4’). SB4: Baicalein ESI-MS m/z 293.2 [M+Na] + , m/z 271.2 [M+H] + ; 1 H-NMR ((CD3)2CO, 500 MHz): 6.76 (1H, s, H-3), 12.76 (1H, s, 5-OH), 6.68 (1H, s, H-8), 8.07 (2H, dd, J= 1Hz, J= 7.5 Hz, H-2’, H- 6’), 7.58 (2H, m, H-3’, H-5’), 7.6 (1H, m, H-4’); 13C-NMR ((CD3)2CO-d6, 125 MHz): 164.75 (C-2), 105.86 (C-3), 183.52 (C-4), 147.87 (C-5), 153.82 (C-6), 151.70 (C-7), 94.9 (C-8), 130.09 (C-9), 105.59 (C-10), 132.63 (C-1’), 127.31 (C-2’, C-6’), 129.89 (C-3’, C-5’), 132.69 (C-4’). SB5: Baicalin ESI-MS m/z 447,1 [M+H] + ; 1 H-NMR (DMSO-d6, 500 MHz): 7.06 (1H, s, H-3), 7.01 (1H, s, H-8), 8.07 (2H, m, H-2’, H-6’), 7.61 (2H, m, H-3’), 7.63 (1H, m, H-4’), 12.60 (1H, s, 5-OH), 8.69 (1H, s, 6-OH); 13 C-NMR (DMSO-d6, 125 MHz): 163.55 (C-2), 106.14 (C-3), 182.56 (C-4), 146.78 (C-5), 130.62 (C-6), 151.28 (C-7), 93.74 (C-8), 149.21 (C-9), 104.76 (C-10), 130.85 (C- 1’), 126.39 (C-2’, C-6’),129.18 (C-3’, C-5’), 132.06 (C-4’), 99.93 (C-1’’), 72.81 (C-2’’), 75.25 (C-3’’), 71.33 (C-4’’), 75.51 (C-5’’), 170.08 (C-6’’). SB10: Tetrasaccharide ( (O-α-D-glucopyranosyl-(1→2)-O-β-D-fructofuranosyl-(1→6)-O- α-D-glucopyranosyl-(1→2)-O-β-D-fructofuranoside). ESI-MS m/z 689.1 [M+Na] + ; 1 H-NMR (CD3OD, 500 MHz): 5.156 (1H, s, H-1), 3.737 (1H, s, H-2), 3.799 (1H, t, H-3), 3.519 (1H, t, H-4),4.626 (1H, t, H-5), 3.628 (1H, t, H-6), 3.705 (1H, s, H-7), 3.985 (1H, s, H-9),3.767 (1H, s, H-10), 4.530 (1H, t, H-11), 3.417 (1H, m, H-12), 5.148 (1H, s, H-1’), 3.726 (1H, s, H-2’), 3.777 (1H, t, H-3’), 3.501 (1H, t, H-4’), 3.598 (1H, t, H-6’), 3.690 (1H, s, H-7’), 3.888 (1H, s, H-9’),3.761 (1H, t, H-10’), 4.514 (1H, t, H-11’), 3.410 (1H, m, H-12’); 13C-NMR (CD3OD, 125 MHz): 93.80 (C-1), 72.87 (C-2), 76.59 (C-3), 69.26 (C-4), 77.88 (C-5), 62.69 (C-6), 65.76 (C-7), 99.15 (C-8),77.85 (C-9), 76.59 (C-10), 83.03 (C-11), Le Duc Long et al. 10 62.52 (C-12), 98.00 (C-1’), 71.53 (C-2’), 76.11 (C-3’), 71.09 (C-4’), 77.81 (C-5’), 64.45 (C-6’), 64.97 (C-7’), 103.05 (C-8’), 77.36 (C-9’), 76.11 (C-10’), 83.03 (C-11’), 62.55 (C-12’). Figure 1. The 1 H-NMR and 13 C-NMR spectra of baicalein (SB4). NMR spectra were recorded of an acetone-d6 solution of SB4 on a a Bruker AMX-500 (500 MHz). Chemical shifts are given in parts per million (ppm) downfield from tetramethylsilane. 2.5. Instrumental analyses Nuclear magnetic resonance (NMR) data of the isolated compounds were recorded on a Bruker AMX-500 (500 MHz) spectrometer (Bruker Analytische Messtechnik GmbH, Rheinstetten, Germany) at 500 MHz for 1 H-NMR spectra and 125 MHz for 13 C-NMR spectra (Fig. 1). Tetramethylsilane (TMS) is using as an internal standard. The electrospray ionization mass spectra (ESI-MS) of the isolated compounds were recorded on an MSD1100 single- quadruple mass spectrometer equipped with an electrospray ionizer (Hewlett-Packard Co., Palo Alto, CA, USA). Chemical structures of the isolates were determined by NMR data analyses and comparison with those of the previously reported in literature. 2.6. In vitro antibacterial assay against human pathogenic bacteria All bacterial strains were grown on nutrient agar (NA) and nutrient broth (NB) at 37°C for 24 h and then suspended in sterile saline at a density equivalent to that of the 0.5 McFarland standards. Bacterial suspensions with a concentration of 10 5 cfu/mL were used for in vitro antibacterial activity test. In vitro antibacterial assay was conducted in 96-well microtiter plates on the basis of a modification of broth micro-dilution method [4]. The tested materials were dissolved in dimethyl sulfoxide (DMSO) to get the stock solutions with dilution factor of 100. The working concentrations of the extracts and isolated compounds ranged from 9.5 to 2,000 μg/ml. DMSO (2 %) was used as the negative control, at which it did not affect the bacterial growth. Chloramphenicol (Sigma-Aldrich, USA) was used as a positive control against all the bacteria. The assay was repeated twice with two replicates for each sample against the individual bacterial species at all the test concentrations. The minimum inhibitory concentration (MIC, µg/mL) was Constitutents and inhibitory effect on human pathogenic bacteria. 11 determined as the lowest concentration that completely inhibited the growth of the bacteria which were incubated at 37°C for 1 day and 2 days. The optical density (OD) was measured spectro-photometrically at 600 nm. The growth inhibition for each dilution was determined using the formula: Percent inhibition (%) = 100 × [1 – OD of treated well/ OD of negative control well], where OD is the optical density of each well 24h after incubation; values of OD of negative control well and OD of treated well were corrected with OD of its blank wells corresponding to each concentration. 3. RESULTS AND DISCUSSION 3.1. Isolation and characterization of metabolites from the roots of S. baicalensis Compounds SB1, SB4 and SB5 were detected to be flavonoids which produced dark green color with FeCl3 and yellow color with KOH/ethanol reagents. Their 13 C-NMR spectra data displayed that all of the compounds have 15 carbons in flavonoid skeleton which consists of two phenyl rings (A and B-rings) and heterocyclic ring (C-ring). In the A-ring of each structure, a carbonyl group at C-4 was presented in a range of δ 183.45-183.56 ppm; two oxygenated aromatic carbons at C-5 and C-7in SB1 did from δ 150.62 to 158.03 ppm. The heterocyclic ring revealed the presence of phenolic group in a range of δ 7.06-8.69 ppm in 1H-NMR spectra of the three compounds. The 1 H-NMR spectrum of SB1 displayed a signal at δ 6.328 ppm that is expected for an olefinic proton and appeared as a singlet. A signal δ 6.817 ppm also appeared as a singlet, which is assigned as a proton at C-6 (δ 99.99 ppm) in the A-ring. Two sets of protons in the B-ring (H-2’, H-6’ and H-3’, H-5’) are symmetrical in each pair. Its 13C-NMR spectral data showed that the δ 158.46 ppm represented at C-8 position of A-ring connected to a methoxy group. By the 1 H-NMR and 13 C-NMR spectra data, SB1 was determined to be wogonin by comparison with NMR spectral data from previously published literatures [5]. SB4 was deduced to be baicalein based on the analysis of 1 H- and 13 C-NMR spectra (Figure 1). In the structure of bacalein, aromatic oxygenated carbons δ 147.87 (C-5), 153.82 (C-6) and 151.70 (C-7) in A-ring are connected to hydroxyl groups may be corresponding in antibacterial activity (Figure 2) [6]. Structure of compound SB5 is the similar to that of compound SB1. The 13 C-NMR spectrum of SB5 showed 19 signals arising from one flavone skeleton including 15 carbons (C-2 to C-10 and C-1’ to C-6’). 1H-NMR and 13C-NMR spectra of SB5 revealed a hexanoic pattern having an acid group (δ 71.33, 72.81, 75.25, 75.51, 99.93, 170.08 ppm), whose chemical shift values were in good agreement with those of β-D-glucuronic acid. Based on symmetry considerations, two carbon atoms of the B-ring were chemically equivalent; hence, only 13 signals from the 15 carbons were observed. Instead of a hexose, a hexanoic acid was present: the strongly deshielded resonance from the carbon atom at δ 170.08 ppm is the result of an acid functional group. The NMR data obtained for compound SB5 are consistent with that of baicalin [7]. Compound SB10 was obtained as white colorless crystals. The 13 C-NMR and JMOD spectra showed twenty-four carbons in the range from δ 62.55 to 103.05 ppm, which is characteristic of a tetrasaccharide corresponding to the carbons bonded with oxygen at position of those in glucose and fructose. Its 1 H-NMR spectrum showed anomeric proton signals and symmetry in pairs. Two signals at δ 5.156 (1H, s, H-1) and 5.148 (1H, s, H-1’) were determined to be two protons anomeric in two glucose moieties. Two signals of anomeric carbons at 93.80 and 98.00 ppm together with two quaternary oxygenate carbons at δ 99.15 and 103.05 suggested Le Duc Long et al. 12 that this compound comprised two glucose molecules and two fructose molecules. Compared with the previously reported data in the literature, compound SB10 was identified as O-α-D- glucopyranosyl-(1→2)-O-β-D-fructofuranosyl-(1→6)-O-α-D-glucopyranosyl-(1→2)-O-β-D- fructofuranoside (Figure 3), an analog of sucrosyl-(1→2)- β- isomaltulose [8]. Figure 2. Chemical structure of compounds isolated from the roots of S. baicalensis. 3.2. Antibacterial activity of layers and isolated compounds from the roots of S. baicalensis In the study, the methanol extract and the two compounds as SB1 and SB4 were selected for testing against seven human pathogenic bacteria. MICs values (μg/mL) at 1 day and 2 days after incubation were detected by visualizing with naked eye and listed in Table 1. SB1 was not active against all bacterial strains at the range of tested concentrations. Methanol extract of S. baicalensis inhibited the bacterial growth of S. aureus ATCC 6538, B. cereus ATCC 21768 and B. subtilis ATCC 6633 with the same MICs of 2,000 µg/mL. SB4 was found to potently inhibit bacterial strains such as S. aureus ATCC 6538 and B. cereus ATCC 21768 with MICs of 9.5 and 38 µg/mL, respectively. Table 1. MIC values of the methanol extract and isolated compounds from S. baicalensis against human pathogenic bacterial strains. Bacterial strains MIC (μg/mL)a SB1 SB4 Methanol extract Chl S. aureus ATCC 6538 - 9.5 b (19) c 2,000 (2,000) 40 (40) B. cereus ATCC 21768 - 38 (75) 2,000 (2,000) 40 (40) B. subtilis ATCC 21768 - 1,000 (2,000) 2,000 (2,000) 40 (40) B. cepacia ATCC 25416 - 1,000 (>2,000) - 20 (20) aMIC: minimum inhibition concentration (μg/mL) is the lowest concentration of antimicrobial agent that completely inhibits growth of the organism in microdilution wells as detected by the naked eye. b MIC values at 1 day after incubation. c (MIC) values at 2 days after incubation. (-) no inhibition. SB1: wogonin; SB4: baicalein. Chl: chloramphenicol. Constitutents and inhibitory effect on human pathogenic bacteria. 13 Figure 3. Fig 3.1: Effects of baicalein (SB4) and methanol extract (ME) of Scutellaria baicalensis on the bacterial growth of Bacillus cereus ATCC 21768 at 1 day (1 DAT) and 2 days (2 DAT) after incubation. Fig. 3.2. In vitro dose-response of baicalein for antibacterial activity against B. cereus ATCC 21768. (A) Dose-effect and (B) Log of dose-effect curves. Table 2. Calculated inhibitory concentrations IC50 and IC90 of baicalein causing 50 % and 90 % inhibition against the bacterial growth of Bacillus cereus ATCC 21768. a Inhibition values were calculated on the basis of Probit analysis of dose-response data expressed in Fig. 3.2. b 95 % confidence interval. Besides, it significantly affected the bacterial growth of B. subtills ATCC 6633 and B. cepacia ATCC 25416 strains at 1,000 µg/mL. The effects of SB4 and methanol extract on the bacterial growth of B. cereus ATCC 21768 were further tested by broth micro-dilution method in 96-well places and their inhibition rates were presented in Figure 3. Baicalein (SB4) showed a dose-response activity against B. cereus ATCC 21768 (Figure 3.2); it effectively inhibited the bacterial growth with low IC50 and IC90 values in a range of 25.8 to 109.7 µg/mL (Table 2). The difference between the antibacterial efficacy of SB1 and SB4 against B. cereus ATCC 21768 may be due to a different pattern of hydroxyl and methoxy groups at positions C-5, C-6, C-7 and C-8 in A-ring of each compound. Three bacterial strains E. coli ATCC 25922, P. aeruginosa ATCC 9027 and E. cloacae ATCC 13047 were resistant to all of the test materials (data not shown). In addition, our results of the potent antibacterial activity against B. cereus ATCC 21768, notably, the inhibition of baicalein against the bacterial growth of S. aureus ATCC 6538 is highly paid attention by natural product chemists and be evident by certain studies in synergistic effects of baicalein with other antibiotics such asciprofloxacin, oxacillin and vancomycin [10]. 4. CONCLUSIONS The present study exhibited that methanol extract of S. baicalensis had an inhibition for bacterial growth of S. aureus ATCC 6538, B. cereus ATCC 21768 and B. subtilis ATCC 6633. Conc. (µg/mL) 1 DAT 2 DAT IC50 25.8 a (11.0- 60.5) b 32.7 (5.6 - 56.8) IC90 78.6 (40.4-152.9) 109.7 (55.3 -164.0) 0 20 40 60 80 100 9.5 19 38 75 150 300 600 In h ib it io n r at e (% ) Concentration (µg/mL) SB4-2 DAT SB4-1 DAT ME-2 DAT ME-1 DAT Fig. 3.1 Fig. 3.2 Le Duc Long et al. 14 Its major constituents were identified as wogonin (SB1), baicalein (SB4), baicalin (SB5) and a tetrasaccharide (SB10). Of them, SB4 strongly inhibited bacterial strains such as S. aureus ATCC 6538, B. cereus ATCC 21768 and B. subtilis ATCC 6633. Our study suggested that S. baicalensis is a good plant resource containing potent antibacterial substances for developing botanical drugs to treat human bacterial pathogens. Acknowledgements. 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