1 SUPPLEMENTARY MATERIAL Composition of the essential oils of three Uzbek Scutellaria species (Lamiaceae) and their antioxidant activities Nilufar Zokirjonovna Mamadalieva*a, Farukh Sharopovb, Prabodh Satyalc, Shahnoz Sadikovna Azimovaa, and Michael Winkb a Institute of the Chemistry of Plant Substances, Mirzo Ulugbek Str 77, Tashkent 100170, Uzbekistan b Heidelberg University, Institute of Pharmacy and Molecular Biotechnology, Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany c Department of Chemistry, University of Alabama in Huntsville, Huntsville, AL 35899, USA The chemical composition of the essential oils obtained from aerial parts of Scutellaria immaculata Nevski ex Juz., S. ramosissima M. Pop. and S. schachristanica Juz. (Lamiaceae) growing wild in Uzbekistan, were analyzed by GC and GC-MS. The main constituent of the essential oils from S. immaculata were acetophenone (30.39%), eugenol (20.61%), thymol (10.04%) and linalool (6.92%), whereas that of S. schachristanica produced acetophenone (34.74%), linalool (26.98%), eugenol (20.67%). The S. ramosissima oil is dominated by germacrene D (23.96%), β-caryophyllene (11.09%), linalool (9.63%) and hexadecanoic acid (8.34%). The essential oils of Scutellaria species exhibited weaker antioxidant effects in DPPH, ABTS and FRAP assays. In FRAP assay, only eugenol exhibited a substantial reducing power IC50=2476.92±15.8 (mM Fe (II)/g). Keywords: Flora of Uzbekistan, Scutellaria, GC-MS, essential oils, antioxidant activity 2 Experimental Plant material. The aerial parts (stems, leaves, flowers) of S. immaculata, S. ramosissima and S. schachristanica employed in this investigation were collected in the flowering stage (May-July 2014) from Namangan, Tashkent and Jizzakh regions of Uzbekistan, respectively and dried in shadow. The authenticated voucher specimens were kept in the Department of Herbal Plants (Institute of the Chemistry of Plant Substances, Uzbekistan) [S. immaculata (accession no. 20088045), S. ramosissima (accession no. 20088052) and S. schachristanica (accession no. 20141424)]. Isolation of essential oils. The air-dried aerial parts (moistury content was 10-12% w.b.) of the S. immaculata, S. ramosissima and S. schachristanica (200 g each) were hydrodistilled for 2 h, using a Clevenger-type apparatus (yields of 0.2%, 0.12% and 0.09%, respectively). As only small amounts of essential oil were present, the oils were trapped in dichloromethane, which were dried over anhydrous sodium sulphate and left over night in a hood to remove the dichloromethane solvent till the weights were fixed stored at -4°C until use. Gas chromatography – mass spectrometry (GC-MS) analysis. The essential oils were analyzed by GC-MS using a Shimadzu GCMS-QP2010 Ultra operated in the EI mode [(electron energy = 70eV), scan range = 3.0 scans/sec], and GC-MS solution software. GC-MS analyses were performed on ZB-5 (Phenomenex, USA) fused silica capillary columns (30 m × 0.32 mm (i.d.), film thickness: 0.25 μm). The carrier gas was helium with a column head pressure 80 psi and flow rate of 1.37 ml/min. Injector temperature was 250°C and the ion source temperature was 200°C, increase in temperature rate 2°C/min to 260°C. The GC oven temperature program was programmed for 50°C initial temperature, increase in the rate 2°C/min to 260°C. A 5% w/v solution of the sample in CH2Cl2 was prepared and 0.1 µL was injected in splitting mode (30:1). Identification of the oil components was based on their retention indices determined by reference to a homologous series of n-alkanes (Kovats RI), and by comparison of their mass spectral fragmentation patterns with those reported in the literature (Adams 2007) and stored in the MS library (NIST 11, WILEY 10, FFNSC version 1.2). The values provided are averages from three analyses. Relative standard deviations from these replicate injections were all small (less than 2%). Antioxidant activity. The antioxidant activity of the essential oils was evaluated by 2,2-diphenyl1-picrylhydrazyl (DPPH), 2,2'-azinobis-[3-ethylbenzthiazoline-6-sulfonic acid] (ABTS) and ferric reducing antioxidant power (FRAP) assays. DPPH, ABTS and FRAP assays were analyzed as described earlier by us (Mamadalieva et al. 2011; Sharopov et al. 2015). All 3 experiments were carried out three times unless mentioned in the procedure. Continuous variables were presented as mean ± SD. The IC50 was determined as the drug concentration that resulted in a 50% reduction or inhibition of the biological activity. Statistical significance was assessed using a one way analysis of variance (ANOVA) followed by a multiple comparison test (Tukey’s post hoc test), where p<0.05 was considered significant. References Adams RP. 2007. Identification of Essential Oil Components by Gas Chromatography /Mass Spectrometry. 4th edn., Allured Publishing Corporation, Carol Stream, IL, p. 804. Mamadalieva NZ, Herrmann F, El-Readi MZ, Tahrani A, Hamoud R, Egamberdieva D, Azimova SS, Wink M. 2011. Flavonoids in Scutellaria immaculata and S. ramosissima and their biological activities. J Pharm Pharmacol. 63:1346–1357. Sharopov F S, Wink M, Setzer WN. 2015. Radical scavenging and antioxidant activities of essential oil components-an experimental and computational investigation. Nat Prod Comm. 10: 153–156. 4 Table S1. Qualitative and quantitative composition (% v/v) of volatile compounds from Uzbek Scutellaria species Compound KIa KIb Composition, % S. S. S. imma- ramosissima schachri- culata Method of identification stanica Prenol 774 765 -- -- 0.05 Hexanal 798 797 -- 0.39 -- 837 832 -- -- 0.13 MS, RI Pent-3-en-1-ol 845 849 -- -- 0.17 MS, RI E-Hexen-1-ol 860 863 0.26 -- -- MS, RI 2-Heptanone 887 889 0.11 -- 0.07 MS, RI Styrene 892 893 0.23 -- -- MS, RI Isobutyl isobutyrate 912 908 -- -- 0.07 MS, RI Benzaldehyde 960 952 2.49 -- 0.13 MS, RI, AU 1-Octen-3-one 975 972 1.02 -- -- MS, RI, AU 1-Octen-3-ol 978 972 2.89 2.08 3.73 MS, RI, AU Octan-3-one 983 979 0.65 -- 0.55 MS, RI, AU Acetylcyclohexane 985 -- -- 0.23 MS, RI 3-Octanol 996 994 0.43 -- 0.27 MS, RI, AU 1-Acetylcyclohexene 1025 1023 -- -- 0.25 MS, RI Limonene 1028 1024 -- 1.14 -- MS, RI, AU 1,8-Cineole 1032 1026 2.25 -- -- MS, RI, AU (Z)-β-Ocimene 1038 1032 -- 0.47 -- MS, RI, AU Benzeneacetaldehyde 1042 1036 0.21 -- -- MS, RI Acetophenone 1058 1059 30.39 4.67 34.74 MS, RI Octanol 1069 1063 -- -- 1.56 MS, RI, AU o-Guaiacol 1083 1087 -- -- 1.19 MS, RI Linalool oxide (Furanoid) 1086 1084 -- -- MS, RI MS, RI, AU 3-Methylcyclo pentanone 0.05 MS, RI 5 Linalool 1099 1095 6.92 9.63 26.98 MS, RI, AU Phenylethyl alcohol 1111 1106 1.99 -- 0.55 MS, RI β-Cyclocitral 1123 1222 -- 0.25 -- MS, RI Camphor 1147 1141 0.76 -- -- MS, RI, AU 2-(1Z)Propenylphenol 1152 -- -- n-Nonanol 1171 1165 0.37 -- -- MS, RI Borneol 1172 1165 0.74 -- -- MS, RI, AU Isopinocamphone 1176 1172 0.40 -- -- MS, RI Terpinen-4-ol 1180 1177 -- 0.06 -- MS, RI, AU 2-Methoxy-p-cresol 1186 1188 0.29 -- 1.89 α-Terpineol 1189 1182 -- 1.39 -- MS, RI, AU γ-Terpineol 1194 1199 1.05 -- 3.57 MS, RI, AU Decanal 1206 1201 -- 0.70 -- cis-Piperitone epoxide 1249 1257 -- -- p-Anisaldehyde 1254 1247 -- -- 0.05 MS, RI, AU Piperitone 1254 1249 -- -- 0.10 MS, RI, AU Thymol 1289 1289 10.04 3.01 0.30 MS, RI, AU Carvacrol 1297 1298 1.62 -- -- MS, RI, AU 4-Vinyl guaiacol 1308 1315 2.50 2.42 1.64 MS, RI Eugenol 1350 1356 20.61 5.29 20.67 MS, RI, AU α-Cubebene 1352 1345 -- 0.46 -- MS, RI trans-Benzalacetone 1356 1330 1.91 -- -- MS, RI Nonalactone 1358 1358 0.15 -- -- MS, RI β-Bourbonene 1386 1387 -- 1.08 -- MS, RI Vanillin 1392 1393 0.19 -- -- MS, RI, AU α-Caryophyllene 1403 1408 1.46 -- -- MS, RI β-Caryophyllene 1416 1417 1.56 11.09 0.10 MS, RI, AU Aromadendrene 1438 1439 -- 0.80 -- MS, RI, AU Geranyl acetone 1446 1453 0.19 -- 0.04 α-Humulene 1455 1452 0.38 2.03 -- 0.09 0.12 MS, RI MS, RI MS, RI MS, RI MS, RI MS, RI, AU 6 Apocynin (acetovanillone) 1478 1480 0.38 -- -- MS, RI Germacrene D 1478 1484 -- 23.96 -- MS, RI, AU Ar-Curcumene 1480 1483 0.35 -- -- MS, RI α-Muurolene 1500 1500 -- 0.58 -- MS, RI γ-Cadinene 1515 1522 -- 1.96 -- MS, RI δ-Cadinene 1526 1537 -- 1.53 -- MS, RI Spathulenol 1580 1577 0.46 3.17 -- MS, RI, AU Caryophyllene oxide 1581 1582 1.95 5.90 0.44 MS, RI, AU Humulene epoxide II 1608 1608 0.30 -- -- MS, RI 10-epi-γ- Eudesmol 1621 1622 0.11 -- -- MS, RI α-Cadinol 1654 1652 0.50 1.94 -- MS, RI Phytone 1840 1838 0.81 -- 0.22 MS, RI Hexadecanoic acid 1958 1959 -- 8.34 -- MS, RI (Z)-9-Octadecanoic acid 2116 2097 -- 1.92 -- MS, RI Heptacosane 2700 2700 -- 1.12 -- MS, RI Nonacosane 2900 2900 -- 1.65 -- MS, RI Monoterpene hydrocarbons 0.76 1.86 0.00 Oxygenated monoterpenoids 43.82 19.38 51.83 Sesquiterpene hydrocarbons 3.75 43.49 0.10 Oxygenated sesquiterpenoids 3.32 11.01 0.44 Benzenoid compounds 40.58 7.09 40.28 Fatty acid and hydrocarbon derivatives 5.73 16.2 6.47 Others 0.96 0 0.83 Total identified 98.92 99.03 99.95 38 29 30 Identified compounds a - Retention induces on ZB-5 fused silica capillary column; b- Reported Retention induces on DB-5 fused silica capillary column (Adams). The values provided are averages from three analyses. Relative standard deviations from these replicate injections were all small (less than 2%). 7 Table S2. Antioxidant activity of the essential oils and major compounds of Scutellaria species DPPH ABTS Sample FRAP mM FeSO4/1 g IC50, µg/ml IC50, µg/ml sample S. immaculata 31.5±1.8 37.8±0.9 720.19±4.8 S. ramosissima 82.8±3.1 93.6±0.8 837.23±3.2 S. schachristanica 57.6±2.7 66.6±1.2 779.64±8.6 Eugenol 0.50±0.007 1.20±0.01 2476.92±15.8 Thymol 0.50±0.008 1.71±0.01 974.09±22.5 Linalool 1914.40±12.5 8821.00±24.9 283.33±3.2 Ascorbic acid 0.30±0.005 7.20±0.22 3327.45±21.4 8 Figure S1. Free radical scavenging of S. immaculata essential oil by the DPPH assay (IC50 = 31.5 µg/ml) Figure S2. Free radical scavenging of S. immaculata essential oil by the ABTS assay (IC50 = 37.8 µg/ml)