Lamiaceae) and Their Biological Activityjphp 1336 1..13 Received November 26, 2010 3 Accepted June 29, 2011 DOI a B 4 10.1111/J.2042-7158.2011.01336.X Nilufar Z

Research Paper

JPP 2011, ••: ••–•• © 2011 The Authors 1 JPP © 2011 Royal Flavonoids in Scutellaria immaculata and S. ramosissima Pharmaceutical Society 2 (Lamiaceae) and their biological activityjphp_1336 1..13 Received November 26, 2010 3 Accepted June 29, 2011 DOI a b 4 10.1111/j.2042-7158.2011.01336.x Nilufar Z. Mamadalieva , Florian Herrmann , 5 ISSN 0022-3573 Mahmoud Z. El-Readib,c, Ahmad Tahranib, Razan Hamoudb, 6 Dilfuza R. Egamberdievaa, Shahnoz S. Azimovaa and Michael Winkb

7 aInstitute of the Chemistry of Plant Substances AS RUz, Tashkent, Uzbekistan, bInstitute of Pharmacy and 8 Molecular Biotechnology, Heidelberg University, Germany and cBiochemistry department, Faculty of 9 Pharmacy, Al-Azhar University, Assiut, Egypt 10 11 Abstract

12 Objectives The aim of this study was to investigate the flavonoid composition of Scutel- 13 laria immaculata and S. ramosissima (Lamiaceae) and the in-vitro biological activity of 14 their extracts and flavonoids. 15 Methods The flavonoid composition of S. immaculata (Si) and S. ramosissima (Sr) were 16 analysed using LC-MS. Antimicrobial activity was studied in vitro against a range of 17 bacteria and fungi using diffusion and microdilution methods. Anti-trypanosomal and cell 18 proliferation inhibitory activity of the extracts and flavonoids was assessed using MTT. The 19 antioxidant activity of the flavonoids and extracts were evaluated using DPPH* test. 20 Key findings LC-MS investigation of Si and Sr plants allowed the identification, for the 21 first time, of an additional 9 and 16 flavonoids, respectively. The methanol, chloroform and 22 water extracts from these plants and six flavonoids (scutellarin, chrysin, apigenin, apigenin- 23 7-O-glucoside, cynaroside and pinocembrine) exhibited significant inhibition of cell growth 24 against HeLa, HepG-2 and MCF-7 cells. The chloroform extract of Sr showed potent 25 cytotoxic effects with IC50 (drug concentration which resulted in a 50% reduction in 26 cell viability) values of 9.25 Ϯ 1.07 mg/ml, 12.83 Ϯ 1.49 mg/ml and 17.29 Ϯ 1.27 mg/ml, 27 respectively. The highest anti-trypanosomal effect against T. b. brucei was shown by the 28 chloroform extract of Sr with an IC50 (drug concentration which resulted in a 50% inhibi- 29 tion of the biological activity) of 61 mg/ml. The pure flavonoids showed an IC50 range 30 between 3 and 29 mm, with cynaroside as the most active compound with an IC50 value of 31 3.961 Ϯ 0.133 mm. The chloroform extract of Sr has potent antimicrobial activity against 32 Streptococcus pyogenes (minimum inhibitory concentration, MIC = 0.03 mg/ml). Pinocem- 33 brine exhibited a strong activity against the all bacteria except Escherichia coli and yeasts. 34 Water extracts of Sr and Si exhibited potent antioxidant activity with IC50 values of 35 5.62 Ϯ 0.51 mg/ml and 3.48 Ϯ 0.02 mg/ml, respectively. Scutellarin exerted stronger anti- 36 oxidant activity than other flavonoids. 37 Conclusions This is the first study reporting an in-vitro biological investigation for Si and 38 Sr. Especially the chloroform extract of Sr showed potent anticancer and antimicrobial 39 activity. Cynaroside had a highly selective and strong cytotoxicity against T. b. brucei while 40 showing only mild effects against cancer cells. 41 Keywords antimicrobial; antioxidant; anti-trypanosomal; cytotoxicity; LC-MS; 42 S. immaculata; S. ramosissima 22 43 44 Introduction

45 The genus Scutellaria (Lamiaceae) comprises over 360 species, many of which are medici- 46 nally important. Essential oils, ﬂavonoids, phenylethanoid glycosides, iridoid glycosides, 47 Correspondence: Michael Wink, diterpenes, triterpenoids, alkaloids, phytosterols, polysaccharides and other secondary com- 48 Institute of Pharmacy and pounds have been isolated from Scutellaria species.[1] Some Scutellaria species are used to 49 Molecular Biotechnology, treat neurological disorders, cancer, inﬂammatory diseases and viral and bacterial infections. Heidelberg University, Im 50 Scutellaria baicalensis S. barbata Neuenheimer Feld 364, 69120 Extracts from and inhibited cancer cell proliferation and [2] 51 Heidelberg, Germany. showed anti-tumour effects. Previous studies demonstrated anti-trypanosomal effects of 52 E-mail: [email protected] S. baicalensis.[3,4]

1 2 Journal of Pharmacy and Pharmacology 2011; ••: ••–••

1 The pharmacological effects of Scutellaria species are (Ն95%) were purchased from Sigma Aldrich (Sternheim, 59 2 attributed particularly to flavonoids, which are abundant in Germany), chrysin (Ն97%) and apigenin-7-O-glucoside were 60 3 this genus. More than 60 flavonoids have been described in from Roth (Karlsruhe, Germany). Cynaroside and pinocem- 61 4 S. baicalensis and the most commonly studied flavonoids brine (Ն95%) were obtained from the Institute of the Chem- 62 5 from this plant include baicalein, baicalin, wogonin and istry of Plant Substances, Tashkent, Uzbekistan. 63 6 wogonoside. Flavonoids of Scutellaria have received scien- 7 tific attention as modifiers of inflammatory processes, as well Preparation of crude plant extracts 64 8 as for their antiviral, anti-retroviral, anti-tumour and anti- The plant material (root or aerial parts) was air-dried at room 65 9 bacterial properties.[1] temperature before grinding it to a fine powder with a Waring 66 10 These compounds show almost no or minor toxicity to blender. After grinding, 100 g of plant material was extracted 67 11 normal epithelial and normal peripheral blood and myeloid with 500 ml solvent (methanol, chloroform or water). Extrac- 68 12 cells. The anti-tumour functions of these flavones are largely tion with each solvent was carried out for one day. The solvent 69 13 due to their ability to scavenge oxygen radicals, attenuate was evaporated in a rotary vacuum evaporator at 40°C. Yields 70 14 NF-kappaB activity, suppress COX-2 gene expression, inhibit were calculated as %. The extracts were then kept in a refrig- 71 15 several genes important for regulation of the cell cycle, and erator until further use. 72 16 to prevent viral infections.[5] Baicalein and baicalin were 17 shown to protect several types of tissue against damage from HPLC analysis of flavonoids 73 18 reactive oxygen species (ROS)[6] and these flavonoids are The final concentration of all samples was 20 mgin1ml 74 19 reported to be largely responsible for the antimicrobial methanol. The HPLC system (Merck-Hitachi L-6200A) 75 20 effects.[7,8] Baicalein has also been shown to inhibit HIV-1 consisted of a Rheodyne injector (20 ml loop). Separation 76 [9] 21 reverse transcriptase. was carried out using a RP-C18e LichroCART 250–4, 5 mm 77 22 In Uzbekistan alone, 32 Scutellaria species have been column (Darmstadt, Germany). The mobile phase consisted of 33 78 23 found and some of them are considered as medicinal plants.[10] A: water HPLC grade with 0.5% formic acid, B: acetonitrile. 79 24 These plants are used in Uzbek traditional medicine to The gradient program was as follows: for methanol and chlo- 80 25 treat epilepsy, inflammation, allergies, chorea, nervous roform fractions, from 0% to 50% B in 50 min then to 100% 81 26 tension states and high blood pressure.[11] S. immaculata in 5 min; for water fraction, from 0% to 25% in 50 min then 82 27 Nevski ex Juz. and S. ramosissima M. Pop. are perennial to 100% in 5 min. 83 28 shrubs that grow in Northern Tien Shan, Pamirs and Altay 29 mountains (Central Asia). Chemical analysis of these plants Mass-spectrometry 84 30 indicate the presence of flavonoids.[12–16] Although, there are A Quattro II system from VG with an ESI interface was used 85 31 widespread reports about the anti-tumour, antibacterial and in a negative ion mode under the following conditions: drying 86

32 antioxidant effects of some species of this genus, in-vitro and nebulizing gas, N2; capillary temperature, 120°C; capil- 87 33 biological studies have not yet been conducted on S. immacu- lary voltage, 3.00 kV; lens voltage, 0.5 kV; cone voltage 30 V; 88 34 lata and S. ramosissima. full scan mode in mass range m/z 200–1000. 89 35 This study aimed to analyse the flavonoid composition 36 of the methanol, chloroform and water extracts of the aerial Cytotoxicity assay 90 37 parts (Srap), roots of S. ramosissima (Srr), and aerial parts of Cell cultures 91 38 S. immaculata (Siap) using LC-MS. In addition in-vitro bio- HeLa (cervical cancer), HepG-2 (hepatic cancer) and MCF-7 92 39 logical properties (cytotoxic, antioxidant, anti-trypanosomal (breast cancer) cell lines were maintained in DMEM complete 93 40 and antimicrobial activity) of the extracts and flavonoids were media (l-glutamine supplemented with 10% heat-inactivated 94 41 evaluated in detail. fetal bovine serum, 100 U/ml penicillin and 100 mg/ml strep- 95 96 42 tomycin) in addition to 10 mm non-essential amino acids. Bloodstream forms of T. b. brucei TC221 cells (causative 44 97 43 Materials and Methods organism of the epidemic nagana) were grown in BALTZ 98 [17] 99 44 Plant material medium supplemented with 20% inactivated fetal bovine serum (FBS) and 0.001% b-mercaptoethanol. Cells were 100 45 The aerial parts and roots of S. ramosissima (Accession no. grown at 37°C in a humidified atmosphere of 5% CO2. All 101 46 20088052) and aerial parts of S. immaculata (Accession no. experiments were performed with cells in the logarithmic 102 47 20088045) employed in this investigation were collected from growth phase. 103 48 Tashkent and Namangan region of Uzbekistan, respectively, at 104 49 the flowering stage during the summer of 2008. Plants were Cytotoxicity and cell proliferation assay 105 50 identified in the Department of Herbal Plants (Institute of The cytotoxicity of extracts and isolated compounds was 106 51 the Chemistry of Plant Substances, Uzbekistan) by Dr O.A. determined in triplicate using the 3-(4,5-dimethylthiazol-2- 107 52 Nigmatullaev and voucher specimens were deposited at this yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability 108 53 Department. assay.[18] The extracts and flavonoids were dissolved in dim- 109 54 ethyl sulfoxide (DMSO) and further serially diluted with the 110 55 Chemicals medium in two-fold fashion into ten different concentra- 111 56 Media and supplements for cell cultures, doxorubicin tions so as to attain final concentrations ranging from 0.977 112 57 (Ն98%) and quercetin (Ն98%) were obtained from Gibco to 500 mg/ml for extracts and from 0.977 to 500 mm for 113 58 (Invitrogen, Karlsruhe, Germany). Scutellarin and apigenin individual substances, in 96-well plates. A sample (100 ml) 114 •• Nilufar Z. Mamadalieva et al. 3

51 5 containing medium was dispensed into each well in. The 6 mm were cut off and delivered with 40 ml of each extract 58 2 concentration of the solvent, DMSO, did not exceed 0.05% in (16 mg/ml) and of each pure substance (1 mm). Ampicillin 59 3 the medium that contained the highest concentration of extract (1 mg/ml), vancomycin (1 mg/ml), nystatin (1 mg/ml) and 60 4 or compound tested. Wells containing the solvent and wells DMSO were used as controls. The diameters of the growth 61 5 without the solvent were included in the experiment. Cells inhibition zones were measured in triplicate after incubation 62 6 (2 ¥ 104 cells/well of exponentially growing cells of each indi- at 37°C for 24 h (bacteria) or 48 h (yeast). 63 7 vidual HeLa, HepG-2, MCF-7, T. b. brucei, TC221 cells) 64 8 were seeded in a 96-well plate (Greiner Labortechnik), the Determination of minimum inhibitory 65 9 cells were cultivated for 24 h and then incubated with various concentration and minimum 66 10 concentrations of the serially diluted tested samples at 37°C microbicidal concentration 67 11 for 24 h and then with 0.5 mg/ml MTT for 4 h. The formed Micro-dilution method was used to determine MIC as 68 12 formazan crystals were dissolved in 100 ml DMSO. The described by NCCLS.[19] Plant extracts and compounds were 69 13 absorbance was detected at 570 nm with a Tecan Safire II first dissolved in DMSO 5% to 8 mg/ml and then were diluted 70 14 Reader. The cell viability rate (%) of three independent two fold with MHB (bacteria) and SDB (fungi) in 96-well 71 15 experiments was calculated by the following formula: plates to obtain a range of concentrations (4–0.007 mg/ml). 72 6 16 The bacterial and fungal suspensions of 1 ¥ 10 CFU/ml were 73 Cell viability rate() %= ( OD of treated cells then added and the plates were incubated at 37°C for 24 h 74 × (1) 75 17 OD of control ceells) 100% (bacteria) or at 25°C for 48 h (fungi). The concentration in the first well that showed no visible turbidity matching with a 76 negative control was defined as the MIC. Three microlitres 77 18 The T. b. brucei TC221 viability results were additionally from each clear well (with no visible turbidity) were inocu- 78 19 confirmed by counting cells under a light microscope. Dimi- lated in appropriate agar media and incubated under the 79 20 nazene, suramin and doxorubicin were used as positive appropriate conditions. The minimum microbicidal concen- 80 21 controls. tration (MMC) was determined as the concentration that 81 22 23 Antimicrobial activity showed no growth on agar after incubation. Each test was 82 performed in triplicate. 83 24 Test microorganisms 25 The antimicrobial activity was evaluated using standard Antioxidant activity 84 26 strains: the Gram-positive bacteria Streptococcus pyogenes The antioxidant and radical scavenging activity of flavonoids 85 27 ATCC 12344 and methicillin-resistant Staphylococcus aureus and extracts was evaluated according to a previous described 86 28 NTCC 10442; the Gram-negative bacteria Escherichia coli method using diphenyl picryl hydrazyl (DPPH*).[20] Equal 87 29 ATCC 25922 and Pseudomonas aeruginosa ATCC 27853; volumes of sample solutions containing 0.02–10 mg/ml of the 88 30 and the fungi Candida albicans ATCC 90028 and Candida tested samples and 0.2 mm methanolic solution of DPPH* 89 31 glabrata ATCC MYA 2950. All microorganism cultures were were pipetted into 96-well plates. The absorbance was mea- 90 32 supplied by Medical Microbiology Laboratory, Hygiene Insti- sured against a blank at 517 nm using a Tecan Safire II Reader 91 33 tute, University of Heidelberg, Germany. after incubation in the dark for 30 min at room temperature 92 34 compared with DPPH* control after background subtraction. 93 35 Culture media Quercetin was used as a positive control. The percent inhibi- 94 36 Columbia medium with 5% sheep blood (BD) was used for tion was calculated from three different experiments using the 95 37 bacteria sub-culturing and minimum bactericidal concentra- following equation: 96 38 tion (MBC) determination. Mueller-Hinton Broth (MHB) 39 (Fluka) was used for bacterial inocula dilution and minimum 97 =− × 40 inhibitory concentration (MIC) determination. All bacterial RSA() % [(Abs517control Abs 517sample )/Abs 517control ] 100 (2) 98 41 cultures were incubated at 37°C for 24 h. CHROM agar

42 Candida (BD) was used for sub-culturing fungi and MBC where RSA = radical scavenging activity; Abs517 = absorption 99 43 determination. Sabouraud dextrose broth (SDB) (Oxid) was at 517 nm 100 44 used for fungal inocula dilution and MIC determination. All 45 fungal cultures were incubated at 25°C for 48 h. Statistical analysis 101 46 All experiments were carried out three times unless mentioned 102 47 Inoculum preparation in the procedure. Continuous variables were presented as 103 48 One or two colonies from an 18–24 h agar plate were sus- mean Ϯ SD. The IC50 was determined as the drug concentra- 104 49 pended in saline to a turbidity matching 0.5 McFarland tion that resulted in a 50% reduction in cell viability or inhibi- 105 50 ª1 ¥ 108 colony-forming units (CFU)/ml, then 100 ml of this tion of the biological activity. IC50 values were calculated 106 51 suspension was diluted to 1 : 100 with 900 ml broth to obtain using a four parameter logistic curve (SigmaPlot 11.0) and all 107 52 1 ¥ 106 CFU/ml.[19] the data were statistically evaluated using Student’s t-test or the 108 53 Kruskal–Wallis test (GraphPad Prism 5.01; GraphPad Soft- 109 54 Diffusion method ware, Inc., San Diego, USA) followed by Dunn’s post-hoc 110 55 Columbia medium with 5% sheep blood and CHROM agar multiple comparison test when the significance value was 111 56 Candida were inoculated with 1 ¥ 106 CFU/ml of bacterial <0.05 using the same significance level. The criterion for 112 57 and fungal suspensions, respectively. Wells with diameter of statistical significance was generally taken as P < 0.05. 113 4 Journal of Pharmacy and Pharmacology 2011; ••: ••–••

1 Results wogonin (25), chrysin (26) and 5,2′-dihydroxy-6,7,8- 61 trimethoxyflavone (27) (Tables 1 and 2 and Figure 1). Fla- 62 2 Identification of flavonoids vonoids 1–7, 9–14 and 16–27 from aerial parts and 3–8, 63 3 The flavonoids from S. immaculata and S. ramosissima were 10–16 and 18–27 from roots were identified in S. ramosissima 64 4 identified by comparison of their LC-MS spectra with pub- for the first time (Figures 2–4). Flavonoids 14, 21 and 25 had 65 5 lished data or were confirmed by comparison with authentic been isolated before from the aerial and roots from this 66 [12–14,16] 6 6 6 samples.[21,22] (Figures 2–4). The LC-MS investigation of plant. 67 7 S. ramosissima allowed the identification of the following In this study we have recorded flavonoids 1, 8, 9, 11–14, 68 8 additional flavonoids: chrysin-6-arabinosyl-8-C-glucoside 16, 18–20, 21–23, 25 and 26 from aerial parts of S. immacu- 69 9 (4), isorhamnetin-7-O-rhamnosyl-glucoside (5), rhamnetin-7- lata for the first time (Tables 1 and 2, and Figures 2–4). The 70 10 O-rhamnosyl-glucoside (6), scutellarin (8), baicalin (9), occurrence of flavonoids 14, 25, and 26 in S. immaculata is in 71 11 5,7,2′,5′-tetrahydroxy-8,6′-dimethoxyflavone (10), oroxylin agreement with the previously reported flavonoid profile.[15,16] 72 12 A-7-O-glucoside (11), 5,6,7-trihydroxyflavanone 73 13 (dihydroxybaicalein)-7-O-glucuronide (12), norwogonin-7- Cytotoxic and anti-trypanosomal activity 74 14 O-glucuronide (13), chrysin-7-O-glucuronide (14), oroxylin The anti-proliferative activity of nine extracts from aerial 75 15 A-7-O-glucuronide (15), wogonin-7-O-glucuronide (16), nor- parts and roots of S. ramosissima and from aerial parts of 76 16 wogonin (21), 5,7,3-trihydroxy-4′-methoxyflavone (22), S. immaculata, including six isolated flavonoids scutellarin 77 17 baicalein (23), 5,7,4′-trihydroxy-8-methoxyflavone (24), (8), chrysin (26), apigenin (28), apigenin-7-O-glucoside (29), 78 18 19 Table 1 Identification of flavonoids in Scutellaria species by LC-MS

- 20 Peak [M-H] m/z Compound Reference

21 1 345 Unknown – 22 2 431 Unknown – 23 3 341 Unknown – 24 4 547 Chrysin-6-arabinosyl-8-C-glucoside 17 25 5 623 Isorhamnetin-7-O-rha-glu 16 26 6 623 Rhamnetin-7-O-rha-glu 16 27 7 637 Unknown – 28 8 461 Scutellarin – 29 9 445 Baicalin 17 30 10 345 5,7,2′,5′-Tetrahydroxy-8,6′-dimethoxy flavone 17 31 11 445 Oroxylin A-7-O-glucoside 17 32 12 447 5,6,7-Trihydroxy flavanone(dihydroxybaicalein)-7-O-glucuronide 17 33 13 445 Norwogonin-7-O-glucuronide 17 34 14 + 15 429 + 459 Chrysin-7-O-glucuronide + oroxylin A-7-O-glucuronide 17 35 16 459 Wogonin-7-O-glucuronide 17 36 17 431 Unknown – 37 18 329 Unknown – 38 19 327 Unknown – 39 20 359 Unknown – 40 21 + 22 269 + 299 Norwogonin + 5,7,3-trihydroxy-4′-methoxyflavone 16 41 23 + 24 269 + 299 Baicalein +5,7,4′-trihydroxy-8-methoxyflavone 16 42 25 283 Wogonin 17 43 26 253 5,7-Dihydroxyflavone (chrysin) 17 44 27 343 5,2′-Dihydroxy-6,7,8-trimethoxyflavone 17 45 46 47 Table 2 Flavonoids in S. immaculata and S. ramosissima

48 Plant material Extracts (yield, %) Flavonoids

49 S. ramosissima (aerial parts) CHCl3 (3.5%) 10, 11, 14, 18–27 50 MeOH (7.2%) 1–4, 6, 9, 11–14, 16, 17, 19–22, 24–27

51 H2O (4.5%) 3–7, 11–14, 16, 22 52 S. ramosissima (roots) CHCl3 (2.3%) 10, 11, 14, 18–27 53 MeOH (11.8%) 3–6, 8–13, 15–27

54 H2O (5.3%) 3–7, 11–14, 16, 18, 20, 22, 23, 26, 27 55 S. immaculata (aerial parts) CHCl3 (2.1%) 8, 14, 18–20, 25, 26 56 MeOH (8.1%) 1, 8, 9, 11–14, 16, 18–23, 26

57 H2O (3.9%) 8, 11, 13, 14 58 59 Flavonoid numbers as in Table 1. %, Yield of the each extract was calculated on the basis of air-dried weight of the plant material (aerial part or roots). 60 •• Nilufar Z. Mamadalieva et al. 5

R6 COOH

R5 O R7 O O HO O R4 OH R O 3 O OH HO OH OH O OH O R8 R2 R1 OH O 4-6, 8-11, 13-16, 21-30 12 31

Substance R1 R2 R3 R4 R5 R6 R7 R8 4 Chrysin-6-arabinosyl-8-C-glucoside H Ara H Glu H H H H

5 Isorhamnetin-7-O-rha-glu OH H Rha-Glu H H OCH3 OH H 6 Rhamnetin-7-O-rha-glu Rha-Glu H OCH3 H H OH OH H 8 Scutellarin H OH Glu acid H H H OH H 9 Baicalin H OH Glu acid H H H H H

10 5,7,2`,5`-Tetrahydroxy-8,6`- HHHOCH3 OCH3 OH H OH dimethoxy flavones

11 Oroxylin A-7-O-glucoside H OCH3 Glu H H H H H 13 Norwogonin-7-O-glucuronide H H Glu acid OH H H H H 14 Chrysin-7-O-glucuronide H H Glu acid H H H H H

15 Oroxylin A-7-O-glucuronide H OCH3 Glu acid H H H H H 16 Wogonin-7-O-glucuronide H H Glu acid OCH3 HHHH 21 Norwogonin H H H OH H H H H

22 5,7,3-Trihydroxy-4`-methoxyflavone OH H H H H H OCH3 H 23 Baicalein H OH H H H H H H

24 5,7,4`-Trihydroxy-8-methoxyflavone H H H OCH3 HHOHH 25 Wogonin H H H OCH3 HHHH 26 Chrysin H H H H H H H H

27 5,2`-Dihydroxy-6,7,8- H OCH3 OCH3 OCH3 OH H H H trimethoxyflavone 28 Apigenin H HH H H H OH H 29 Apigenin-7-O-glucoside H H Glu H H H OH H 30 Cynaroside H H Glu H H OH OH H

1 Figure 1 Chemical structures of flavonoids studied. 2 3 cynaroside (30) and pinocembrine (31), was determined in between 17 and 64 mm, while the IC50 value of chrysin in 24 4 HeLa, HepG-2, MCF-7 cells and against T. b. brucei. The T. b. brucei was 11 mm and of apigenin 8 mm. The resulting 25 5 IC50 values are presented in Table 3. selectivity index for chrysin ranges between 1 and 2 and for 26 6 SrapC, SrrC and SiapC substantially inhibited the prolif- apigenin between 2 and 7. Cynaroside was remarkably differ- 27 7 eration of cancer cells. The IC50 values of SrapC in all three ent from all tested flavonoids with IC50 values between 149 28 8 human cancer cell lines were in the range 9–17 mg/ml, those and 184 mm in the three cancer cell lines and an IC50 value 29 9 of SrrC were between 9 and 30 mg/ml and those of SiapC were of 4.9 mm in T. b. brucei. The selectivity index thus ranges 30 10 between 13 and 31 mg/ml, with HepG-2 as the most sensitive between 30 and 37. 31 11 and MCF-7 as the least sensitive cell line. The IC50 values 12 against T. b. brucei were in the range 0.6–0.8 mg/ml. This Antimicrobial activity 32 13 results in a selectivity index for T. b. brucei in relation to the A total of six microbial strains (two Gram-positive, two 33 14 cancer cell lines in the range 13–50 (15–50 for SrapC, 13–24 Gram-negative bacteria, two fungi) were used in this investi- 34 15 for SrrC and 17–39 for SiapC). The highest selectivity index gation. Methanol, chloroform and water extracts of Siap, 35 16 was obtained for T. b. brucei and MCF-7 cells and the lowest Srap, Srr and flavonoids 8, 26 and 28–31 were tested at 36 17 index was between T. b. brucei and HepG-2. various concentrations, ranging from 4 to 0.007 mg/ml. MIC 37 18 Chrysin and apigenin showed the strongest cytotoxicity and MMC values are reported in Table 4. 38 19 of all tested flavonoids in all three human cancer cell lines and All chloroform extracts showed strong growth inhibition 39 20 in T. b. brucei, while cynaroside was highly selective for against Streptococcus pyogenes. The SiapC exhibited moder- 40 21 T. b. brucei with only minor toxicity in all three human cancer ate antimicrobial activity (MIC = 1 mg/ml) compared with 41 22 cell lines. The IC50 values of chrysin in all three human SrapC and SrrC, which exhibited stronger antimicrobial 42 23 cancer cell lines range between 13 and 22 mm and of apigenin activity (MIC = 0.06 and 0.03 mg/ml, respectively) against 43 31 30 29 28 27 26 25 24 23 22 21 20 19 18 17 16 15 14 13 12 10 11 9 8 7 6 5 4 3 2 1 oouii ( Doxorubicin .immaculata S. h aasonaemeans are shown data The Sample 2 Figure 6 al 3 Table oouii ( Doxorubicin ( Diminazene ramosissima S. ramosissima S. pgnn(28) Apigenin control) (positive (mg/ml) Suramin Apigenin-7- hyi (26) Chrysin yaoie( Cynaroside ioebie( Pinocembrine ctlai (8) Scutellarin 100 081209_002 Sm(SG,10×1) 100 081209_004 Sm(SG,10×1) 100 081209_003 Sm(SG,10×1) MeOH H CHCl H H MeOH MeOH CHCl CHCl .050 00 50 00 50 00 50 00 50 00 55.00 50.00 45.00 40.00 35.00 30.00 25.00 20.00 15.00 10.00 5.00 0.00 .050 00 50 00 50 00 50 00 50 00 55.00 50.00 45.00 40.00 35.00 30.00 55.00 25.00 50.00 20.00 45.00 15.00 40.00 10.00 35.00 5.00 30.00 25.00 0.00 20.00 15.00 10.00 5.00 0.00 0 % 0 % 0 % 2 2 2 O O O (c) (b) (a) ora fPamc n hraooy21;•:••–•• ••: 2011; Pharmacology and Pharmacy of Journal 3 3 3 nityaooa n nipoieaieatvt of activity anti-proliferative and Anti-trypanosomal fmtao xrcs (a) extracts. methanol of (-) LC-MS guoie(29) O-glucoside 30) /l pstv control) (positive mg/ml) pstv control) (positive m) /l pstv control) (positive mg/ml) ara parts) (aerial 31) (roots) parts) (aerial Ϯ Dotie rmtreidpnetexperiments. independent three from obtained SD 1 1 2 3 3 .immaculata S. 29.692 36.999 27.864 11.431 1.785 0.723 0.879 1.698 7.950 3.645 1.822 3.857 8.670 0.084 4.724 4.939 0.611 .b brucei b. T. IC50 4 4 5 – – Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ (m) 6 .3 23.181 0.130 1.373 .2 9.219 0.127 20.552 0.329 0.689 2.656 0.990 .1 170.663 0.012 1.094 1.574 0.091 .2 1317.177 0.129 .7 137.832 0.575 0.399 .0 148.990 0.106 .1 109.825 1.017 .2 127.826 1.123 6 .immaculata S. 8 8 eilprs (b) parts. aerial 9 9 9 10 11 11 11 12 12 12 13 13 13 and 62.898 57.197 86.365 32.086 66.573 74.571 33.306 22.808 14 14 .ramosissima S. 15 1.84 1.07 16 16 .ramosissima S. 16 HeLa 17 17 Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ 18 18 19 19 0.19 .4 9.04 1.543 2.860 .3 12.83 0.934 13.86 1.597 3.613 4.104 0.829 3.211 6.232 2.512 0.11 4.323 9.411 .8 291.61 4.981 3.793 273151.64 12.773 015150.97 10.165 7.221 19 20 20 20 C0(mg/ml) IC50 21 21 21+22 22 22 eilprs (c) parts. aerial xrcsadioae flavonoids isolated and extracts 24 23 23+24 42.81 40.29 66.57 32.06 52.59 62.91 64.56 13.98 94.60 0.67 0.39 HepG-2 .ramosissima S. – – Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ 25 0.07 .430.41 1.04 3.88 .917.29 1.49 31.52 1.15 3.44 4.70 2.28 4.20 6.27 0.04 5.72 34 151.64 23.46 1.09 41 184.13 14.19 51 171.63 15.19 5.16 25 26 26 26 27 27 roots. 31.84 62.53 54.30 93.19 44.05 53.01 69.73 17.25 22.73 0.48 0.28 MCF-7 Scan ES- Scan ES- Scan ES- – – Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ 3.68e6 2.75e6 1.59e6 3.35 0.04 0.37 0.47 1.27 1.32 0.22 5.10 3.25 3.88 5.93 0.02 1.33 14.87 2.48 17.37 11.78 Time BPI BPI BPI •• Nilufar Z. Mamadalieva et al. 7

091209_003 Sm (SG, 10×1) Scan ES- 26 100 BPI 3.50e6 (a)

% 18 20 14 25 8 19

0 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 × 091209_002 Sm (SG, 10 1) 20 Scan ES- 100 BPI 21+22 6.08e6 (b)

26 27 % 18 23+24 19 25 10 11 14 0 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 × 091209_001 Sm (SG, 10 1) 20 Scan ES- 100 BPI 26 6.15e6 (c) 21+22 27

% 19 23+24 18 25 10 11 14 0 Time 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00

1 Figure 3 LC-MS (-) of chloroform extracts. (a) S. immaculata aerial parts. (b) S. ramosissima roots. (c) S. ramosissima aerial parts.

3 S. pyogenes. SiapM, SrapM and SrrM exhibited signifi- Antioxidant activity 29 4 cant antimicrobial activity against Staphylococcus aureus, The free radical scavenging activity was determined using 30 5 Escherichia coli, Pseudomonas aeruginosa and S. pyogenes. DPPH* assay. The DPPH* scavenging capacity of the extracts 31 > 76 7 All plant extracts showed weak inhibition (MIC 3 mg/ml) and flavonoids were compared with the known antioxidative 32 7 against P. aeruginosa; only SrapC (MIC = 1 mg/ml) was substance quercetin. The DPPH* radical-scavenging activity 33 8 found to have moderate activity. Data in Table 4 indicate a of the extracts and flavonoids studied are shown in Table 5. 34 9 weak activity against Candida albicans only for SrrC and All plants extracts possessed significant DPPH* radical 35 > 10 SrrM (MIC 4 mg/ml), while the other extracts were inactive scavenging activity. The most active plant extract was SiapW 36 11 against this strain. Candida glabrata was resistant to the (IC50 = 3.48 Ϯ 0.02 mg/ml) compared with the most active 37 12 SiapM, SrrM and SrapW, while the other extracts showed a single compound scutellarin (4.77 Ϯ 0.53 mg/ml). 38 > 13 weak inhibition (MIC 2 mg/ml) against this yeast. 39 14 The antimicrobial activity of flavonoids (inhibition zones, Discussion 40 15 MIC and MMC values) is shown in Table 4. Apigenin-7-O- 16 glucoside (29) exhibited a strong activity against all the bac- Scutellaria species are characterized by their richness in fla- 41 817 8 teria and yeasts (MIC = 0.25–0.5 mm). Cynaroside (30)was vonoids. Malikov and Yuldashev reported 208 phenolic com- 42 18 active against S. aureus, E. coli, P. aeruginosa and S. pyo- pounds from Scutellaria baicalensis, which has the highest 43 919 9 genes with MIC = 0.5 mm and had no activity on fungal number of known phenolic compounds (>60) and represents 44 20 strains. Pinocembrine (31) showed potent antimicrobial activ- the best studied species in the genus.[15] 45 21 ity against S. pyogenes, S aureus, C. albicans and C. glabrata S. immaculata has been studied previously and 17 46 1022 10 (MIC = 0.25 mm). Apigenin (28), was inactive against flavonoids (chrysin-7-O-glucuronide, scutellarein-7-O- 47 23 S. aureus, P. aeruginosa and C. albicans and proved to be the glucoside, apigenin-7-O-glucoside, baicalein-7-O-glucoside, 48 24 most active substance against S. pyogenes, E. coli and C. gla- norwogonin-7-O-glucoside, oroxyloside, wogonoside, 49 1125 11 brata (MIC = 0.25–0.5 mm). Scutellarin (8) showed strong immaculoside, 5,2′-dihydroxy-6,7,6′-trimethoxyflavan- 50 26 growth inhibition against S. pyogenes, E. coli, C. albicans and one, 5,2′-dihydroxy-6,7,8,6′-tetramethoxyflavanone, chrysin, 51 27 C. glabrata. Only chrysin (26) was inactive against any of the wogonin, apigenin, isoscutellarein, scutellarein, cosmo- 52 28 bacterial pathogens at the concentrations tested. siin (apigenin-7-O-b-d-glucopyranoside), and wogonin- 53 8 Journal of Pharmacy and Pharmacology 2011; ••: ••–••

101209_001 Sm (SG, 10×1) Scan ES- 8 100 BPI 3.26e6 (a) 14

% 11 13

0 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 101209_003 Sm (SG, 10×1) Scan ES- 11 100 BPI 14 20 1.28e6 (b) 13 16 5 22

% * 3 6 27 26 4 18 7 23 *=12 0 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 101209_002 Sm (SG, 10×1) Scan ES- 11 100 BPI 14 1.22e6 (c) 16 5 13

% * 3 6 4 7 22 *=12

0 Time 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00

1 Figure 4 LC-MS (-) of water extracts. (a) S. immaculata aerial parts. (b) S. ramosissima roots. (c) S. ramosissima aerial parts.

3 7-O-b-d-glucopyranoside) were identified;[15,16] and from ion bonding. As a result the conformation of proteins are 28 4 S. ramosissima, chrysin 7-O-b-d-glucuronide, 2(S)-2′,5,7- disturbed and in consequence their biological activity is 29 5 trihydroxyflavanone 7-O-(Me b-d-glucopyranosiduronate), altered.[31] These physicochemical properties can explain the 12 30 6 2(S)-2′,5,7-trihydroxyflavanone 7-O-(Et b-d-glucopyranosi- wide range of activities of polyphenols. Flavonoids are well 31 7 duronate), 5,2′-dihydroxy-7-O-b-d-glucopyranosylflavone, known for their wide range of biological activity. Recent 32 8 rivularin, 5,2′-dihydroxy-7-O-b-d-glucopyranosylflavanone, studies regarding the cytotoxic activity of the flavonoids of 33 9 oroxylin A, wogonin, norwogonin, 5,2′,6′-trihydroxy-6,7,8- Scutellaria indicate that some flavonoids, like baicalein,[32,33] 34 10 trimethoxyflavone, and 5,6-dihydroxy-7,8-dimethoxyfla- wogonin[34] and luteolin,[35] possess substantial anti-cancer 35 11 vone.[12,13,23] However, flavonoids have not been determined activity. For example, baicalein showed substantial toxicity 36 12 fully in S. immaculata and S. ramosissima. In this investiga- to P-glycoprotein or MRP1-expressing multidrug-resistant 37 13 tion evidence for the presence of an additional 19 and 12 cells.[36] The anti-tumour effects of these flavonoids may be 38 14 flavonoids from S. immaculata and S. ramosissima, respec- due to their ability to inhibit several genes important for 39 15 tively, is provided (Tables 1 and 2). Our data confirm that the regulation of the cancer cell cycle, and to prevent viral 40 16 Scutellaria is characterized by a substantial accumulation and infections.[5] 41 17 a broad structural diversity of flavonoids. Their biological The cytotoxicity of chloroform extracts was superior to 42 18 properties vary considerably with only minor modifications in that of the methanol and water extracts. Especially, the SrrC 43 19 their structure. The number and specific positions of phenolic showed a high toxicity level in all cell lines (Table 3). Chrysin 44 20 hydroxyl groups and the nature of the substitutions deter- (26) and norwogonin (21) might be responsible for the toxic- 45 21 mine whether flavonoids function as strong antioxidative,[24–26] ity since they are the main compounds of the chloroform 46 22 anti-inflammatory, anti-proliferative or enzyme-modulating extracts while the methanol and water extracts mainly contain 47 23 agents.[27–30] The phenolic hydroxyl groups of flavonoids can scutellarin (8) and oroxylin A-7-O-glucoside (11). Chrysin 48 24 dissociate to negatively charged phenolate ions under physi- (26) was the most cytotoxic single substance tested with a 49 25 ological conditions. Therefore, flavonoids can interact with cytotoxicity ranging between 11 mm in T. b. brucei and 22 mm 50 26 several proteins (including transporters, enzymes and tran- in HeLa cells, which makes it 12 times less toxic than the 51 27 scription factors) by binding to them via hydrogen bridges and positive control doxorubicin (1.84 mm). 52 ••

1 Table 4 Minimum inhibitory concentrations (MIC) and minimum microbicidal concentrations (MMC) of the ﬂavonoids, S. immaculata and S. ramosissima plant extracts against different pathogens using 2 the broth micro-dilution method

3 Sample Extract G+ Staphylococcus aureus G+ Streptococcus pyogenes G- Escherichia coli G-Pseudomonas aeruginosa Yeast Candida albicans Yeast Candida glabrata 4 MRSA ATCC 10442 ATCC 12344 ATCC 25922 ATCC 27853 ATCC 90028 ATCC MYA 2950 5 I.z. MIC MMC I.z. MIC MMC I.z. MIC MMC I.z. MIC MMC I.z. MIC MMC I.z. MIC MMC 6 (mm) (mg/ml) (mg/ml) (mm) (mg/ml) (mg/ml) (mm) (mg/ml) (mg/ml) (mm) (mg/ml) (mg/ml) (mm) (mg/ml) (mg/ml) (mm) (mg/ml) (mg/ml)

7 S. immaculata MeOH 5 Ϯ 0.5 2 4 5 Ϯ 12 44Ϯ 0.8 2 4 4 Ϯ 0.63 4 NANANANANANA

98 (aerial parts) CHCl3 6 Ϯ 11 47Ϯ 0.5 1 2 – 4 >45Ϯ 0.2 3 4 NA NA NA – 4 >4

10 H2O5Ϯ 12 45Ϯ 0.2 2 5 4 Ϯ 0.5 2 4 4 Ϯ 13 4 NANANA5Ϯ 12 4 11 S. ramosissima MeOH 4 Ϯ 0.8 2 4 5 Ϯ 1.5 1 3 4 Ϯ 0.6 2 4 4 Ϯ 0.5 4 5 NA NA NA 4 Ϯ 0.5 >4 >4

1312 (aerial parts) CHCl3 8 Ϯ 10.547Ϯ 2 0.06 0.5 NA NA NA 6 Ϯ 0.8 1 4 NA NA NA 5 Ϯ 0.3 2 4

14 H2O4Ϯ 0.6 2 4 5 Ϯ 0.2 4 4 – 4 >44Ϯ 0.8 4 >4 NANANANANANA 15 S. ramosissima MeOH 5 1 5 5 Ϯ 0.2 1 4 4 Ϯ 0.5 2 >44Ϯ 0.0 3 4 5 >4 >4NANANA

1716 (roots) CHCl3 8 Ϯ 10.548Ϯ 1 0.03 0.5 NA NA NA 5 Ϯ 0.5 1 4 5 Ϯ 0.2 >4 >4–4>4

18 H2O5Ϯ 11 24Ϯ 12 5 – 4 >44Ϯ 0.2 3 4 NA NA NA – 4 >4 19 Apigenin (28)NANANA4Ϯ 0.5 0.5 >0.5 – 0.5 >0.5NANANANANANA5Ϯ 1 0.25 0.5 20 Apigenin-7-O-glucoside (29)– 0.5 >0.5 5 0.5 >0.5 – 0.5 0.5 – 0.5 >0.5 5 Ϯ 1 >0.5 >0.5 5 Ϯ 1 0.25 0.5 21 Chrysin (26) NANANANANANANANANANANANANANANANANANA 16 22 Cynaroside (30)–0.5>0.5 – 0.5 >0.5 – 0.5 >0.5 – 0.5 >0.5NANANANANANA 23 Pinocembrine (31) – 0.25 1 – 0.25 1 NA NA NA – 0.5 >0.5 4 Ϯ 1 0.25 0.25 5 0.25 0.25 24 Scutellarin (8)NANANA4Ϯ 0.3 0.5 >0.5 – 0.5 >0.5 NA NA NA 6 Ϯ 0.5 >0.5 >0.5 5 Ϯ 0.6 0.25 0.5 25 Ampicillin – 25 >25 25 Ϯ 1 0.05 0.1 5 Ϯ 112.525NANANANTNTNTNTNTNT iua .Mamadalieva Z. Nilufar 26 Vancomycin 10 Ϯ 0.2 0.8 12.5 15 Ϯ 10.10.4NANANANANANANTNTNTNTNTNT 27 Nystatin NT NT NT NT NT NT NT NT NT NT NT NT 10 Ϯ 1.2 0.2 0.4 12 Ϯ 1 0.2 0.2 28 29 NA, not active; NT, not tested. 30 17 tal. et 9 10 Journal of Pharmacy and Pharmacology 2011; ••: ••–••

1 Table 5 Antioxidant activity of pure isolated of the flavonoids, S. im- promising candidates for lead structures. SrrC was the most 62 2 maculata and S. ramosissima plant extracts and isolated flavonoids using active against Staphylococcus aureus and S. pyogenes, fol- 63 3 the DPPH* radical scavenging assay lowed by SrapC and SiapC (Table 4). 64 The pure flavonoids showed a wide spectrum of activity 65 4 Sample IC50 (mg/ml) against both yeast and bacteria (Table 4). The strong activity 66 5 S. immaculata (aerial parts) of apigenin (28), apigenin-7-O-glucoside (29), pinocembrine 67 6 MeOH 6.41 Ϯ 0.62 (31) and scutellarin (8) against yeast is promising. All of 68 Ϯ 7 CHCl3 30.09 3.21 them have the R7 hydroxyl group, which might play an im- 69 Ϯ 8 H2O 3.48 0.02 portant role in their activity. Apigenin-7-O-glucoside (29)is 70 9 S. ramosissima (aerial parts) highly active against all strains of bacteria and yeast. Its 71 10 MeOH 9.62 Ϯ 0.98 higher activity compared with its aglycon results from the 72 11 CHCl3 13.86 Ϯ 1.43 higher bioavailability due to the sugar chain. 73 12 H2O 5.62 Ϯ 0.51 13 S. ramosissima (roots) S. aureus, responsible for skin infections, proved sensi- 74 14 MeOH 10.77 Ϯ 1.12 tive to apigenin-7-O-glucoside (29), cynaroside (30) and 75

15 CHCl3 12.88 Ϯ 1.50 pinocembrine (31). In this context, cynaroside (30) seems to 76 16 H2O 5.81 Ϯ 0.53 have highly selective properties since it is selectively toxic 77 17 Apigenin (28) 206.17 Ϯ 18.12 for bacteria and T. b. brucei, while yeast and cancer cells 78 18 18 18 Apigenin-7-O-glucoside (29) 286.54 Ϯ 25.96 are less sensitive. Further research should be conducted to 79 Ϯ 19 Chrysin (26) 308.27 28.34 explain this selectivity. Chrysin (26) is another exceptional 80 20 Cynaroside (30) 13.90 Ϯ 1.46 flavonoid since it is not toxic for bacteria and yeast while 81 21 Pinocembrine (31) 413.01 Ϯ 35.21 being the most toxic flavonoid for T. b. brucei and cancer 82 22 Scutellarin (8) 4.77 Ϯ 0.53 23 Quercetin (positive control) 3.37 Ϯ 0.77 cells. We suggest that the selectivity of flavonoids on their 83 24 target is much higher than previously expected and thus 84 25 19 19 The data are represented as mean Ϯ SD. make flavonoids very interesting lead structures for the 85 26 development of new drugs. 86 27 Our studies established that these extracts contain various 87 28 As was previously shown, flavonoids are potential lead phenolic compounds, such as baicalin, baicalein and wogonin 88 29 structures for the discovery of new trypanocidal drugs.[37] (Table 1), which are known for their antioxidative activ- 89 30 The activity of flavonoids is closely linked to their structure ity.[39,40] The water and methanol extracts showed a high anti- 90 31 and the number of phenolic OH groups.[38] The flavonoids oxidant activity, 3–5 mg/ml, for all plants. This range is 91 32 with a high number of phenolic OH groups were especially similar to that of the two most active pure compounds, cynaro- 92 33 active in all human cancer cell lines and in T. b. brucei side (30) and scutellarin (8) (4 and 13 mg/ml, respectively), 93 34 (Table 3). Chrysin (26) and apigenin (28) were most potent while the other flavonoids are around 100 times less active. 94 35 in both human cancer cell lines and in T. b. brucei, while Minor modifications in the molecules were again responsible 95 36 cynaroside (30) was highly selective with mild cytotoxicity for strong variations in their activity. 96 37 in human cancer cell lines (IC50 values 149–184 mm)but 38 showing strong cytotoxicity in T. b. brucei (4.9 mm). The 97 39 selectivity index between 30 and 37 seems promising for a Conclusions 98 40 potential anti-trypanosomal use of cynaroside (30). Suramin, 41 still used as a standard drug against trypanosomiasis with Our results highlight that two Scutellaria species are charac- 99 42 a selectivity index of 280 and a toxicity of 4.7 mg/ml in terized by a substantial accumulation and a broad structu- 100 43 T. b. brucei, is known to cause severe side effects. Thus, new ral diversity of flavonoids. The cytotoxicity of chloroform 101 44 lead structures for the treatment of trypanosomiasis are extracts was more potent than other extracts. Especially the 102 45 urgently required. chloroform extract of Srr showed a high level of toxicity in all 103 46 Comparison of the different flavonoid molecules shows cell lines. Chrysin was the most cytotoxic single substance 104 47 that the R6 hydroxyl group of cynaroside (30) is missing in all tested against T. b. brucei and in HeLa cells, and was 12 times 105 48 inactive flavonoids. We suggest that this R6 hydroxyl group less toxic than doxorubicin. Especially cynaroside showed 106 49 forms a crucial part in the activity against T. b. brucei and strong cytotoxicity in T. b. brucei. Among the tested plant 107 50 explains the high selectivity between cancer cells and chloroform extracts, Srr is a potential source of novel anti- 108 51 T. b. brucei while all other flavonoids are less selective. It is microbial components because of a stronger bactericidal 109 52 probable that the activity of the extracts is caused not by only effect on clinically isolated microorganisms, particularly on 110 53 a single flavonoid but rather several ones working together methicillin-resistant S. aureus (MRSA). Also pure flavonoids 111 54 synergistically. Further research should be conducted to are in the same range of the tested antibiotics, thus making 112 55 confirm the structural dependency of the effect of flavonoids them promising candidates for lead structures. The water 113 56 on T. b. brucei. extracts showed high antioxidant activity for all plants, with 114 57 Compared with antibiotics (positive control), the broth scutellarin as the most antioxidant substance. Our findings 115 58 micro-dilution method showed that all plant extracts have suggest that chloroform extracts and flavonoids of S. imma- 116 59 antimicrobial properties with an inhibition zone (I.z.), ranging culata and S. ramosissima are potentially useful for the 117 60 from 4 to 8 mm. The MIC values of the pure compounds are development of therapeutic treatments of microbial MRSA 118 61 in the same range as the tested antibiotics, thus making them infections, trypanosomiasis and cancer. 119 •• Nilufar Z. Mamadalieva et al. 11

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AUTHOR QUERY FORM

Dear Author, During the preparation of your manuscript for publication, the questions listed below have arisen. Please attend to these matters and return this form with your proof. Many thanks for your assistance.

Query Query Remark References q1 AUTHOR: Please supply a short title of up to 40 characters that will be used as the running head. q2 AUTHOR: please reduce the number of key words to a maximum of five q3 AUTHOR: add name of manufacturer (e.g. Merck, Darmstadt, Germany) q4 AUTHOR: give name in full here (Trypanosoma?) q5 AUTHOR: is some text missing here? q6 WILEY-BLACKWELL: unusual to cite figures out of numerical order – is renumbering necessary? q7 AUTHOR: would it be more accurate to say ‘Most’ plant extracts (since the text goes on to say that SrapC has moderate activity)? q8 AUTHOR: units don’t match those used in Table 4 (mg/ml) q9 AUTHOR: units don’t match those used in Table 4 (mg/ml) q10 AUTHOR: units don’t match those used in Table 4 (mg/ml) q11 AUTHOR: units don’t match those used in Table 4 (mg/ml) q12 AUTHOR: Please note that the references have been reordered from 31 onwards. q13 AUTHOR: References 10 and 11: please cofirm if the book titles, the city locations and the names of publishers for these references are correct. q14 AUTHOR: added ‘O’ – please check q15 AUTHOR: add definition of IC50 to table footnote q16 AUTHOR: added ‘O’ – please check q17 AUTHOR: explain ‘–‘ and ‘I.z.’ in footnote q18 AUTHOR ‘O’ added – please check q19 AUTHOR: please add number of values used to calculate mean Ϯ sd; add definition of IC50 to footnote

USING e-ANNOTATION TOOLS FOR ELECTRONIC PROOF CORRECTION

Required software to e-Annotate PDFs: Adobe Acrobat Professional or Adobe Reader (version 8.0 or above). (Note that this document uses screenshots from Adobe Reader X) The latest version of Acrobat Reader can be downloaded for free at: http://get.adobe.com/reader/

Once you have Acrobat Reader open on your computer, click on the Comment tab at the right of the toolbar:

This will open up a panel down the right side of the document. The majority of tools you will use for annotating your proof will be in the Annotations section, pictured opposite. We’ve picked out some of these tools below:

1. Replace (Ins) Tool – for replacing text. 2. Strikethrough (Del) Tool – for deleting text.

Strikes a line through text and opens up a text Strikes a red line through text that is to be

box where replacement text can be entered. deleted.

How to use it How to use it  Highlight a word or sentence.  Highlight a word or sentence.  Click on the Replace (Ins) icon in the Annotations  Click on the Strikethrough (Del) icon in the section. Annotations section.  Type the replacement text into the blue box that appears.

3. Add note to text Tool – for highlighting a section 4. Add sticky note Tool – for making notes at to be changed to bold or italic. specific points in the text.

Highlights text in yellow and opens up a text Marks a point in the proof where a comment box where comments can be entered. needs to be highlighted.

How to use it How to use it

 Highlight the relevant section of text.  Click on the Add sticky note icon in the

 Click on the Add note to text icon in the Annotations section.

Annotations section.  Click at the point in the proof where the comment

 Type instruction on what should be changed should be inserted. regarding the text into the yellow box that  Type the comment into the yellow box that appears. appears.

USING e-ANNOTATION TOOLS FOR ELECTRONIC PROOF CORRECTION

5. Attach File Tool – for inserting large amounts of 6. Add stamp Tool – for approving a proof if no

text or replacement figures. corrections are required.

Inserts an icon linking to the attached file in the Inserts a selected stamp onto an appropriate

appropriate pace in the text. place in the proof.

How to use it How to use it  Click on the Attach File icon in the Annotations  Click on the Add stamp icon in the Annotations section. section.  Click on the proof to where you’d like the attached  Select the stamp you want to use. (The Approved file to be linked. stamp is usually available directly in the menu that appears).  Select the file to be attached from your computer or network.  Click on the proof where you’d like the stamp to

 Select the colour and type of icon that will appear appear. (Where a proof is to be approved as it is,

in the proof. Click OK. this would normally be on the first page).

7. Drawing Markups Tools – for drawing shapes, lines and freeform

annotations on proofs and commenting on these marks.

Allows shapes, lines and freeform annotations to be drawn on proofs and for comment to be made on these marks..

How to use it

 Click on one of the shapes in the Drawing

Markups section.

 Click on the proof at the relevant point and draw the selected shape with the cursor.

 To add a comment to the drawn shape,

move the cursor over the shape until an

arrowhead appears.

 Double click on the shape and type any

text in the red box that appears.

For further information on how to annotate proofs, click on the Help menu to reveal a list of further options:

MARKED PROOF Please correct and return this set Please use the proof correction marks shown below for all alterations and corrections. If you wish to return your proof by fax you should ensure that all amendments are written clearly in dark ink and are made well within the page margins.

Instruction to printer Textual mark Marginal mark Leave unchanged under matter to remain Insert in text the matter New matter followed by indicated in the margin or Delete through single character, rule or underline or or through all characters to be deleted Substitute character or substitute part of one or through letter or new character or more word(s) through characters new characters Change to italics under matter to be changed Change to capitals under matter to be changed Change to small capitals under matter to be changed Change to bold type under matter to be changed Change to bold italic under matter to be changed Change to lower case Encircle matter to be changed Change italic to upright type (As above) Change bold to non-bold type (As above) or Insert ‘superior’ character through character or under character where required e.g. or

Insert ‘inferior’ character (As above) over character e.g. Insert full stop (As above) Insert comma (As above) or and/or Insert single quotation marks (As above) or

or and/or Insert double quotation marks (As above) or Insert hyphen (As above) Start new paragraph No new paragraph Transpose Close up linking characters

Insert or substitute space through character or between characters or words where required

Reduce space between between characters or characters or words words affected