Scutellaria Incana</Emphasis>

Acta Chromatographica 25(2013)3, 555–569 DOI: 10.1556/AChrom.25.2013.3.11 Comprehensive Profiling of Flavonoids in Scutellaria incana L. Using LC–Q-TOF–MS M. NURUL ISLAM1,2, F. DOWNEY1, AND C.K.-Y. NG1,* 1School of Biology and Environmental Science, University College Dublin, Belfield, Dublin 4, Ireland 2Present address: Department of Chemistry, University of Gothenburg, SE-412 96, Gothenburg, Sweden E-mail: [email protected] Summary. Scutellaria L. is a diverse genus of the Lamiaceae (Labiatae) family of over 300 herbaceous plants commonly known as skullcaps. Various species of Scutellaria are used as ethnobotanical herbs for the treatment of ailments like cancer, jaundice, cirrhosis, anxiety, and nervous disorders. Scutellaria incana L., commonly known as the Hoary skullcap, is a traditional medicinal plant used by native Americans as a sedative for nervousness or anxiety. S. incana metabolites were identified by comparing their high- performance liquid chromatography (HPLC) retention times and mass spectra with those of the corresponding authentic standards. Where standards were unavailable, the structures were characterized on the basis of their tandem mass spectrometry (MS/MS) spectra following collision-induced dissociation (CID) and the accurate masses of the corresponding deprotonated molecules [M–H]− (mass accuracy ± 5 ppm). A total of 40 flavonoids, including two phenolic glycosides, were identified from leaves, stems, and roots of S. incana. Differences in the flavonoid composition between leaves, stems, and roots in S. incana were observed although the flavonoid profile of S. incana is consistent with other Scutellaria species. Further work should focus on assessing the potential of S. incana as a source of these bioactive metabolites. Key Words: Scutellaria incana, flavonoids, phenolic glycosides, LC–MS Introduction Scutellaria L. is a diverse genus of the Lamiaceae (Labiatae) family of herbaceous plants commonly known as skullcaps. Scutellaria is widespread geo- graphically, and it has been estimated that there are over 300 species dis- tributed throughout the world. Various species of Scutellaria are used as ethnobotanical herbs for the treatment of ailments like cancer, jaundice, cirrhosis, anxiety, and nervous disorders [1]. Scutellaria baicalensis, commonly known as Baikal skullcap or Huang-qin, is widely used in Traditional Chi- nese Medicine (TCM). In addition to S. baicalensis, Scutellaria lateriflora, commonly known as the Blue skullcap, is widely used by native American herbalists as a sedative for treating nervous disorders, and recent studies have demonstrated that extracts of S. lateriflora exhibit anxiolytic properties [2]. 0231–2522 © 2013 Akadémiai Kiadó, Budapest Unauthenticated | Downloaded 09/26/21 11:55 PM UTC 556 M. Nurul Islam et al. The ethnobotanical properties of skullcaps have been attributed to the presence of bioactive phenolics and flavonoids like acteoside, scutellarin, scutellarein, baicalin, baicalein, wogonin, wogonoside, apigenin, chrysin, and oroxylin A. These compounds have demonstrated bioactivities in diverse cellular processes ranging from angiogenesis to apoptosis [3]. Readers are referred to Shang et al. [3] for a comprehensive review of the pharma- cological properties of bioactive metabolites from Scutellaria. Scutellaria incana L., commonly known as the Hoary skullcap, is a per- ennial herb native to North America. S. incana is used by native American herbalists as a sedative for the treatment of anxiety or nervousness [4]. We used high-performance liquid chromatography (HPLC) coupled with tandem mass spectrometry to profile and identify the flavonoid and phenolic composition of S. incana as a first step towards understanding its potential as a resource for pharmacologically active metabolites. Experimental Plants Seeds of S. incana L. (kindly provided by Royal Botanic Gardens, Kew, United Kingdom) were germinated, and plants were grown as previously described [5]. Briefly, seeds were surface-sterilized in 70% ethanol for 1 min followed by 15 min in 25% domestic bleach before rinsing with sterile de- ionized water. Seeds were then transferred to sterile Schenk–Hildebrandt medium supplemented with 300 mg L−1 casein, 30 g L−1 sucrose, 1 mg L−1 −1 GA3 and 8 g L plant cell-culture tested agar. The plant seeds were strati- fied for 6 days at 4°C before being transferred to a constant temperature room under constant illumination with a combination of red and blue LEDs at 20 µmol m−2 s−1. Seedlings were transferred to a compost/vermiculite (2:1) mix (Shamrock Multipurpose Compost, Shamrock Horticulture, United Kingdom) and grown in a greenhouse at 25°C under constant illumination at 200 μmol m−2 s−1. Chemicals and Reagents Baicalein (≥95%), baicalin (≥99%), norwogonin (≥95%), and acteoside (≥98%) were purchased from Extrasynthèse (France). Scutellarein (≥98%), scutellarin (≥98%), and wogonoside (≥98%) were supplied by Chengdu Biopurify Phytochemicals Ltd. (China). Apigenin (≥95%), apigenin glucoside (≥ 97%), and chrysin (≥96%) were purchased from Sigma-Aldrich (United Kingdom), and wogonin (≥95%) and oroxylin A (≥95%) were supplied by Shanghai Unauthenticated | Downloaded 09/26/21 11:55 PM UTC Flavonoids in Scutellaria incana L. 557 Chemical Technology Ltd. (China). Stock solutions of standard compounds were prepared in methanol at a concentration of 1 mg mL−1 and diluted with 70% methanol to obtain working solution for liquid chromatography– mass spectrometry (LC–MS) analysis. LC–MS grade and HPLC grade methanol, acetonitrile, formic acid, and water were purchased from Fluka (Germany). All other chemicals used in this study were of analytical grade unless otherwise noted. Sample Preparation for Analysis Leaf, stem, and root tissue from 3-month-old plants were harvested and flash frozen in liquid nitrogen before freeze-drying. Freeze-dried materials were ground into fine powder using a mortar and pestle. The ground fine plant materials were extracted with 70% methanol at a concentration of 10 mg mL−1 in a bath sonicator for 2 h. Plant debris were removed by cen- trifugation, and the extracts were diluted with 70% methanol to a final concentration of 5 mg mL−1. The samples were then filtered using 0.2 μm filter prior analysis, and 5 to 10 μL of filtered samples were analyzed by LC–MS. Liquid Chromatography–Mass Spectrometry (LC–MS) HPLC system consisted of binary pump, autosampler, column oven (1200 RRLC, Agilent Technologies, USA). The chromatographic separation of compounds in extract was achieved using a Gemini NX C18 (2.0 mm ID × 100 mm, particle size 3 μm, Phenomenex, Torrance, CA, USA) narrow bore analytical column. Column oven and autosampler temperature were maintained at 40°C and 4°C, respectively. The mobile phase consisted of 0.1% aqueous formic acid (solvent A) and acetonitrile containing 0.1% formic acid (solvent B). Elution was performed at a flow rate of 0.3 mL min−1 in a binary gradient mode. The initial elution condition was A–B (85:15, v/v) for 0.5 min, linearly changed to A–B (75:25, v/v) at 15 min, again gradient condition linearly changed to A–B (40:60, v/v) at 30 min, before being changed to A–B (30:70, v/v) at 33 min, returning to the initial condition at 35 min, followed by 8 min column re-equilibration. Total run time for sample analysis was 43 min. The HPLC system was coupled to an Agilent 6520 Q- TOF mass spectrometer equipped with a dual electrospray ion source. Elec- trospray ionization (ESI) was performed in the negative ion acquisition mode, with nitrogen used as the nebulizing agent. The gas temperature and flow rate were 360°C and 12 L min−1, respectively. The ESI needle voltage was adjusted to 3.5 kV, and fragmentor voltage was 150. Collision-induced dissociation (CID) of the analytes was accomplished using 18 to 35 V of en- Unauthenticated | Downloaded 09/26/21 11:55 PM UTC 558 M. Nurul Islam et al. ergy with high purity nitrogen as collision gas. Two reference ions (m/z 301.998139, m/z 601.978977, supplied by Agilent) were introduced to ESI source simultaneously with HPLC eluant for accurate mass measurement using in-built reference ions delivery system. MS and MS/MS data were collected with MassHunter Data Acquisition software, and MassHunter Qualitative Analysis (Agilent Technologies, USA) software was applied to identify molecular species. Molecular formulas of deprotonated molecules were generated in the MassHunter software using the Molecular Formula Generator algorithm. The algorithm has a built-in logic to eliminate chemi- cally implausible compositions. Only the common elements C, H, and O were considered in the generation of formulae, taking into account the chemical composition of flavonoids and phenolics. Results and Discussion Flavonoids can be analyzed by positive and negative electrospray ionization mode in mass spectrometry [5, 6]. However, negative ionization mode for ESI–MS has the added advantage for yielding extensive structural informa- tion, particularly for C-glycoside flavonoids via CID [7]. The compounds from S. incana extracts were identified by comparing their HPLC retention times and mass spectra with those of the corresponding authentic standards. Where standards were unavailable, the structures were characterized on the basis of their MS/MS spectra following CID and the accurate masses of the corresponding deprotonated molecules [M–H]− (mass accuracy ± 5 ppm). A total of 40 metabolites were identified from leaves, stems and roots of S. incana (Figs 1 and 2). The identity of the 40 metabolites

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