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
Total Page：16
File Type：pdf, Size：1020Kb
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.