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 herba- ceous 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 eth- nobotanical herbs for the treatment of ailments like cancer, jaundice, cirrho- sis, 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 proper- ties [2].

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The ethnobotanical properties of skullcaps have been attributed to the presence of bioactive phenolics and flavonoids like acteoside, , , , , , wogonoside, , , and . These compounds have demonstrated bioactivities in di- verse 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 tan- dem 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%), (≥95%), and acteoside (≥98%) were purchased from Extrasynthèse (France). Scutellarein (≥98%), scutel- larin (≥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

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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 con- centration 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% for- mic 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 stan- dards. 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 and their corresponding LC retention times and CID spectral data are shown in Table I.

R R 4 4' 5 R3 2'

R2O O 7 6'

R6 R1 5

OH O

Fig. 1.

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Compound R1 R2 R3 R4 R5 R6 1 Ara H Glu H OH H 2 Glu H Glu H H H 3 Glu H Ara H OH H 4 H H Glu H OH H 5 Ara H Glu H H H 6 Glu H H H OH H 8 Glu H Ara H H H 10 OH GluA H H OH H 12 H H Glu H H H 13 H Glu H H OH H 14 H GluA H H OH H 15 OCH3 GluA H OH H H 16 OH Glu H H H H 17 OH H H H OH H 18 OH GluA H H H H 19 OCH3 GluA OH H H H 20 OH GluA OCH3 H H H 21 H GluA OH H H H 22 H H OCH3 H OH H 23 H GluA H H H H 24 OCH3 GluA H H H H 25 O-GluA H H H H H 27 H GluA OCH3 H H H 28 OCH3 CH3 H O-Glu H OH 29 H H OH H H H 30 OCH3 H H OH H H 31 H H H H OH H 32 OCH3 GluA OCH3 H H H 33 OH H H H H H 34 H CH3 OH OH H H 35 H CH3 OCH3 OH H OCH3 36 H H OCH3 H H H 37 OCH3 CH3 OCH3 OH H H 38 H H H H H H 39 OCH3 H H H H H 40 OCH3 CH3 OH H H H

R 4' 4 R3 2'

R2O O 7 6'

R1 5

OH O Fig. 1.

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Compound R1 R2 R3 R4 9 OH GluA H OH 26 H GluA OCH3 H

OH

R2O O O OH R O HO 1 O O OH

CH3 OH

HO

Compound R1 R2 7 Caffeoyl H 11 H Caffeoyl

HO O

C Caffeoyl = HO

Fig. 1. Chemical structures of the 40 metabolites identified in S. incana L. Compound names are listed in Table I

Flavonoids C-Glycosides

In ESI–MS/MS spectra, ions of [M–H-60]−, [M–H-90]−, [M–H-120]− are con- sidered as characteristic of C-glycoside flavonoids under low collision en- ergy. The isomeric compounds 1 and 3 in CID spectra of the [M–H]− ions at m/z 563 showed fragment ions at m/z 503 [M−H-60]−, 473 [M–H-90]−, 443 [M– H-120]−, 383 [M–H-180]−, and 353 [M–H-210]− at retention times of 2.5 min and 3.0 min which correspond to characteristic ions of C-glycosides (Table I). In contrast to O-glycosides, C-glycosides do not exhibit abundant aglycone ions. The MS/MS spectra for these compounds are in good agreement with previously characterized apigenin-6-C-arabinosyl-8-C-glucoside and api- genin-6-C-glucosyl-8-C-arabinoside in S. baicalensis [7, 8]. A similar ap- proach was used for identification of compound 2 as chrysin-6, 8-di-C- glucoside at a retention time of 2.8 min (Table I). The MS/MS spectra at m/z 577 exhibited 517 [M–H-60]−, 487 [M–H-90]−, 457 [M–H-120]−, 367 [M–H- 180]−, and 337 [M–H-240]− which suggested the presence of di-C-glucosyl [7]. In negative mode, ESI analysis of compounds 4 and 6 yielded

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DOI: 10.1556/AChrom.25.2013.3.11 Flavonoids in Table I. LC–MS characterization of flavonoids and phenolic glycosides in aqueous methanolic extract of Scutellaria incana

Chemical Obsd, Calcd, Error m/z Of fragment ions and relative RT Peak Compound Tissuea formula m/z m/z (ppm) abundances (min)

503.1(4%), 473.1(11%), 443.1(13%), Scutellaria incana 1 Apigenin-6-C-ara-8-C-glu C26H28O14 563.1408 563.1406 −0.3 383.1(79%), 353.1(100%), 2.5 L, S, R 325.1(14%), 297.1(16%) 517.1(5%), 487.1(13%), 457.1(30%), 2 Chrysin-6,8-di-C-glu C27H30O14 577.1570 577.1563 −1.25 367.1(54%), 337.1(100%), 2.8 L, S, R 309.1(11%), 281.1(12%)

503.1(2%), 473.1(11%), 443.1(10%), L 3 Apigenin-6-C-glu-8-C-ara C26H28O14 563.1408 563.1399 1.29 383.1(68%), 353.1(100%), 3.0 L, S, R 325.1(18%), 297.1(15%) 341.1(11%), 311.1(100%), 4 C H O 431.0985 431.0984 −0.30 4.1 L, S 21 20 10 283.1(29%) 487.1(7%), 457.1(8%), 367.1(80%), 5 Chrysin-6-C-ara-8-C-glu C26H28O13 547.1455 547.1457 0.39 337.1(100%), 309.1(21%), 4.2 L, S, R 281.1(21%) Unauthenticated |Downloaded 09/26/21 11:55 PMUTC 341.1(43%), 311.1(100%), 6 Isovitexin C H O 431.0977 431.0984 1.55 4.7 L, S 21 20 10 283.1(31%) 461.2(14%), 315.2(5%), 179.0(5%), 7 Acteosideb CH O 623.1972 623.1981 1.51 5.1 L, S, R 29 36 15 161.0(100%), 135.0(7%) 487.1(2%), 457.1(18%), 367.1(66%), 8 Chrysin-6-C-glu-8-C-ara C26H28O14 547.1456 547.1457 0.21 337.1(100%), 309.1(17%), 5.3 L, S, R 281.1(21%) 9 Dihydroscutellarin C21H20O12 463.0885 463.0882 −0.65 287.1(100%), 269.0(5%) 6.4 S 10 Scutellarinb C21H18O12 461.0724 461.0725 0.32 285.0(100%) 6.5 L, S, R 461.2(21%), 315.2(5%), 179.0(4%), 11 Isoacteoside C H O 623.1973 623.1981 1.35 6.8 L, S, R 29 36 15 161.0(100%), 135.0(5%) 12 Chrysin-8-C-glucoside C21H20O9 415.1044 415.1035 −2.27 325.1(7%), 295.1(100%), 267.1(33) 7.2 L, S, R 561

0231–2522 © 2013 Akadémiai Kiadó, Budapest 562 M. Nurul Islam et al. 562 Table I. (continued) M. Nurul Islam et a Islam et M.Nurul Chemical Obsd, Calcd, Error m/z Of fragment ions and relative RT Peak Compound Tissuea formula m/z m/z (ppm) abundances (min)

13 Apigenin-7-glucosideb C21H20O10 431.0968 431.0984 3.63 269.0(55%) 8.4 L, S, R 14 Apigenin-7-glucuronide C21H18O11 445.0778 445.0776 −0.37 269.0(100%) 9.4 L, S, R 5,7,2′-Trihydroxy-6- 15 methoxyflavone-7- C22H20O12 475.0880 475.0882 0.42 299.1(100%), 284.0(41%) 10.4 L, S, R glucuronide 16 Baicalein-7-O-glucoside C21H20O10 431.0977 431.0984 1.55 269.0(100%) 11.2 L, S, R 17 Scutellareinb C15H10O6 285.0402 285.0405 0.91 267.0(6%), 239.0(20%), 229.0(6%) 11.4 L 18 Baicalinb C21H18O11 445.0774 445.0776 0.53 269.0(100%) 12.4 L, S, R 5,7,8-Trihydroxy-6- 19 methoxyflavone-7- C22H20O12 475.0887 475.0882 −1.05 299.1(100%), 284.0(36%) 12.7 L, S, R glucuronide 5,6,7-Trihydroxy-8- 20 methoxyflavone-7- C22H20O12 475.0879 475.0882 0.63 299.1(100%), 284.0(33%) 13.4 L, S, R glucuronide

Unauthenticated |Downloaded 09/26/21 11:55 PMUTC 21 Norwogonin-7-glucuronide C21H18O11 445.0779 445.0776 −0.59 269.0(100%) 15.4 L, S, R 284.0(100%), 256.0(6%), 255.0(5%), 22 4′-Hydroxylwogonin C16H12O6 299.0573 299.0561 −3.96 15.6 L, S, R 228.0(17%), 212.0(20%) 23 Chrysin-7-glucuronide C21H18O10 429.0833 429.0827 −1.35 253.1(100%), 175.0(7%), 113.0(49%) 15.8 L, S, R 24 Oroxylin A-7-glucuronide C22H20O11 459.0930 459.0933 0.62 283.1(100%), 268.0(23%) 16.4 L, S, R 25 Baicalein-6-glucuronide C21H18O11 445.0781 445.0776 269.0(100%) 17.3 R, S Dihydrowogonin 7- 26 C H O 461.1087 461.1087 0.51 285.1(100%), 270.0(20%) 17.4 R, S glucuronide 22 20 11 27 Wogonosideb C22H20O11 459.0931 459.0933 0.40 283.1(100%), 268.0(32%) 17.7 L, S, R 5,2′6′-Trihydroxy-6,7- 28 dimethoxyflavone 2′- C23H24O12 491.1194 491.1195 0.2 329.1(100%), 314.0(10%), 299.0(7%) 17.9 R, S glucoside 29 Norwogoninb C15H10O5 269.0452 269.0455 1.20 241.0(10%), 225.0(10%), 197.0(60%), 18.8 L, S, R l.

Flavonoids in Scutellaria incana L. 563 Table I. (continued) Flavonoids in

Chemical Obsd, Calcd, Error m/z Of fragment ions and relative RT Peak Compound Tissuea formula m/z m/z (ppm) abundances (min)

5,7,2′-Trihydroxy-6- 284.0(100%), 256.0(10%), Scutellaria incana 30 C H O 299.0560 299.0561 0.37 18.8 R, S methoxyflavone 16 12 6 255.0(6%), 228.0(12%), 212.0(22%) 31 Apigeninb C15H10O5 269.0446 269.0455 3.51 225.0(3%), 151.0(18%), 117.0(100%) 19.1 L, S 5,7-Dihydroxy-6,8- 313.1(100%), 298.1(17%), 32 dimethoxyflavone-7-O- C H O 489.1039 489.1038 −0.1 19.7 R 23 22 12 283.0(12%) glucuronide 251.0 (22%), 241.0(38%), L. 33 Baicaleinb C15H10O5 269.0446 269.0455 3.14 (223.0(60%), 213.0(22%), 20.3 S, R 195.0(90%) 5,8,2′-Trihydroxy-7- 284.0(100%), 256.0(4%), 255.0(5%), 34 C H O 299.0559 299.0561 0.71 20.9 R methoxyflavone 16 12 6 228.0(9%), 212.0(11%) 5,2′-Dihydroxy-7,8,6′- 328.1(11%), 313.0(100%), 35 C H O 343.0826 343.0823 −0.8 23.3 R trimethoxyflavone 18 16 7 298.0(8%), 285.0(34%), 270.0(10%)

b 268.0(100%), 240.0(6%),

Unauthenticated |Downloaded 09/26/21 11:55 PMUTC 36 Wogonin CH O 283.0620 283.0612 −2.83 24.5 L, S, R 16 12 5 239.0(11%), 163.0(21%)

5,2′-Dihydroxy-6,7,8- 328.1(15%), 313.0(32%), 37 C H O 343.0826 343.0823 −1.96 24.8 R trimethoxyflavone 18 16 7 298.0(100%), 285.0(9%), 270.0(7%) 225.0(5%), 209.1(15%), 181.1(9%), 38 Chrysinb CH O 253.0509 253.0506 −1.05 24.9 L, S, R 15 10 4 165(10%), 143(100%), 268.0(100%), 240.0(4%), 239.0(9%), 39 Oroxylin Ab CH O 283.0611 283.0612 0.34 25.3 R 16 12 5 163.0(10%) 5,8-Dihydroxy-6,7- 40 C H O 313.0708 313.0718 3.06 298.0(6%), 283.0(100%), 255.0(25) 25.6 R dimethoxyflavone 17 14 6

aL = leaf, S = stem, R = root

bCompared with standard 563

564 M. Nurul Islam et al. ion at m/z 431. CID spectra showed the same fragmentation pattern com- prising m/z 341 [M–H-90]− and 311 [M–H-120]− which are identical to spec- tra of previously published data and as such are identified as vitexin and is- ovitexin from Oolong tea leaves [9]. Vitexin and isovitexin are ubiquitous compounds found in plant kingdom including Scutellaria species [10]. CID revealed that the [M–H]− ion at m/z 547 eluting at 4.2 min and 5.3 min was chrysin-6-C-arabinosyl-8-C-glucoside and chrysin-6-C-glucosyl-8-C-arabi- noside. The CID spectra for these compounds are in good agreement with published spectra [7, 11]. Consistent with previous studies, compound 12 showed a deprotonated molecule at m/z 415 and upon CID fragmentation gave an [M–H-90]− ion at 325 and a [M–H-120]− at m/z 295, suggesting the presence of a C-glycoside in this compound. On the basis of the MS/MS and accurate mass data, compound 12 was identified as chrysin-8-C-glucoside, previously identified in Scutellaria species [11].

Flavonoid O-glycosides

The presence of O-glucoside and O-glucuronide in flavonoids could be re- vealed by MS/MS experiment in product ion scan mode indicating neutral loss of 162 and 176, respectively, from precursor ions (Table I). Unlike C- glycosides, O-glycosides generated the most intense aglycone ion after loss of sugar molecule under low collision energy. Compound 10 was identified as scutellarin by comparative analysis of mass spectra and retention time with the authentic standard (Table I). MS/MS spectra of [M–H]− at m/z 461 exhibited a single ion at m/z 285, indicating the presence of glucuronic acid. A similar experimental approach was used for identifying compound 9 as dihydroscutellarin which has been shown to be present in S. baicalensis [10, 12]. MS/MS spectra of compound 9 revealed that [M–H]− ion at m/z 463 gave a strong signal of aglycone at m/z 287. Compound 18 was identified as baicalin by comparing the retention time and mass spectra with the authen- tic standard. The deprotonated molecule at m/z 445 yielded the most abun- dant aglycone ion at m/z 269, releasing one glucuronic acid molecule which confirmed the identity of compound. Compounds 14, 21, and 25 which showed different LC retention times (9.4 min, 15.4 min, and 17.3 min) yielded MS/MS spectra similar to the baicalin standard under CID (Table I), indicating that they are isomeric compounds. On the basis of previously published work [10, 13] which showed that apigenin-O-7-glucuronide eluted before baicalin whereas Norwogonin-7-O-glucuronide and baicalein- 6-O-glucuronide eluted later than baicalin in reverse phase liquid chroma- tography, compounds 14, 21, and 25 are plausibly identified as apigenin-O- 7-glucuronide, norwogonin-7-O-glucuronide and baicalein-6-O-glucuro-

Unauthenticated | Downloaded 09/26/21 11:55 PM UTC Flavonoids in Scutellaria incana L. 565 nide. Compounds 13 and 16 both showed [M–H]− at m/z 431, and retention times of 8.4 min and 11.2 min, respectively. Upon fragmentation by CID, both compounds generated ion [M–H-162]− at m/z 269. On the basis of their retention time and mass spectra, compound 13 is identified as apigenin-7- glucoside by comparison with the authentic standard and compound 16 tentatively identified as baicalein-7-glucoside [13]. Compounds 24 and 27 yielded [M–H]− ions at m/z 459 and similar MS/MS spectra following CID, indicating that they are isomeric pairs. Compound 27 was identified as wogonoside by comparison with the refer- ence standard. The [M–H-176]− ion at m/z 283 and the [M–H-176-15]− ion at m/z 268 indicated the presence of glucuronic acid and methyl group. By ex- amining the available data from published literature and the differences in their retention times, compound 24 was plausibly identified as oroxylin A- 7-O-glucuronide [7, 11, 13]. MS/MS analysis of compound 26 of m/z 461 produced a major ion at m/z 285 and a minor ion at m/z 270, indicating the presence of glucuronic acid and a methyl residue. This compound was ten- tatively identified as dihydrowogonin-7-glucuronide as reported previously in Scutellaria [10]. Compound 23 gave a [M–H]− ion at m/z 429 and a reten- tion time of 15.8 min, and subsequent CID yielded an ion at m/z 253, corre- sponding to the neutral loss of glucuronic acid. The compound was tenta- tively identified as chrysin-7-glucuronide based on accurate mass, MS/MS spectra, and previous reports [1, 13]. The three isomeric compounds 15, 19, and 20 that eluted at retention times 10.4, 12.7, and 13.4 min exhibited [M–H]− at m/z 475 and accurate mass 475.0882 ± 1.5 ppm. Upon CID, these compounds produced ions at m/z 299 and m/z 284, indicating the presence of a methyl and three hy- droxyl groups in these glucuronide compounds. These compounds were plausibly identified on the basis of their accurate mass and MS/MS spectra as 5,7,2′-trihydroxy-6-methoxyflavone-7-glucuronide (compound 15), 5,7,8- trihydroxy-6-methoxyflavone-7-glucuronide (compound 19), and 5,6,7- trihydroxy-8-methoxyflavone-7-glucuronide (compound 20) [13]. Compound 28 was identified as 5,2′6′-trihydroxy-6,7-dimethoxyflavone 2′-glucoside which was previously characterized and identified from S. bai- calensis [14]. MS/MS spectra revealed the product ions at m/z 329, 314, and 299, suggesting the presence of glucose and two methyl moieties. Com- pound 32 showed [M–H]− at m/z 489 and its CID spectra generated ions of m/z 313, 298, and 283, indicating the neutral loss of glucuronic acid and the presence of two methyl groups. Therefore, the structure of the compound was tentatively assigned as 5,7-dihydroxy-6,8-dimethoxyflavone-7-O- glucuronide [13].

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LC–MS analyses of compounds 7 and 11 generated deprotonated mole- cules [M–H]− at m/z 623 with retention times of 5.1 min and 6.8 min, and their accurate mass measurements (calculated m/z 623.1981, observed m/z 623.1972 ± 1.51 ppm) suggest that these two compounds are likely to be isomers. Further investigation following CID of the m/z 623 ion of com- pounds 7 and 11 generated 5 main fragment ions at m/z 461, 315, 179, 161, and 135. The ion at m/z 461 corresponds to neutral loss of the caffeoyl moi- ety as [M–H-162]− from the parent ion. The ion at m/z 315 resulted from the loss of caffeoyl moiety and rhamnose moiety (M–H-162-146]− from the par- ent ion. Similarly, the ion at m/z 179 corresponds to loss of aglycone and di- saccharide (glucose and rhamnose). The m/z 161 ion arises from similar fragmentation pathway with a further loss of one molecule of water. Com- pound 7 was identified as acteoside by comparison with the retention time and mass spectra of the authentic standard, and compound 11 with similar MS/MS spectra is identified as isoacteoside.

Flavonoids Aglycones

ESI–MS analysis of flavonoids exhibited some diagnostic features such as loss of H2O (18), CO (28), CO2 (44), C2H2O (42). Methoxylated flavonoids − − were found to produce [M–H–CH3] , [M–H–CH3–CO] , and [M–H–CH3– HCO]− fragment ions which are characteristic fragmentation pattern of de- protonated molecules under CID. Compound 17 was assigned as scutel- larein by comparison with the authentic standard. Compound 17 appears to be found exclusively in leaf tissue in S. incana (Table I). CID spectra of [M–H]− at m/z 285 gave 267, 239, and 229 which were derived from loss of H2O, CO, and 2CO from precursor ion. Compounds 29, 31, and 33 displayed ion at m/z 269 with similar accu- rate mass. These compounds were identified as norwogonin, apigenin, and baicalein by direct comparison of their retention times and MS/MS spectra with those authentic standards. Compound 38 was identified as chrysin by comparing its retention time and MS/MS spectra with the reference stan- dard. MS/MS spectra gave a series of product ions at m/z 225, 209, 181, 165, − − − and 143 corresponding to [M–H–CO] , [M–H–CO2] , [M–H–CO2–CO] , [M– − − H–2CO2] , and [M–H–C3O2–C2H2O] . The product-ion spectra and accurate mass of the two isomeric com- pounds (36 and 39) at m/z 283 are very similar. The predominant ion at m/z − 268 [M–H–CH3] is likely to result from the formation of a stable ion. In ad- dition, these compounds exhibited concurrent loss of CO (28) or HCO (29), resulting in minor ions at m/z 240 and 239, respectively. The retention times and MS/MS spectra of compounds 36 and 39 obtained in this experiment

Unauthenticated | Downloaded 09/26/21 11:55 PM UTC Flavonoids in Scutellaria incana L. 567 are similar to the reference compounds of wogonin and oroxylin A, respec- tively. There are three isomers, 22, 30, and 34, which yielded similar accurate masses, and the identical ion [M–H]− at m/z 299. Additionally, their MS/MS spectra showed a [M–H–CH3]− at m/z 284 as the base peak. The ions at m/z 256, 255, 228 and 212 correspond to successive loss of CO, HCO, 2CO, and C2O3. These data, together with previously published reports, enabled the identification of these compounds as 4′-hydroxywogonin, 5,7,2′-trihydroxy- 6-methoxyflavone, and 5,8,2′-trihydroxy-7-methoxyflavone, respectively [15, 16]. Compounds 35 and 37, with similar accurate masses, produced the [M–H]− at m/z 343, suggesting that they are isomeric pairs. Analyses of their CID spectra suggest that both compounds contained three methyl groups (ions of m/z 328, 313, and 297 generated from parent ions). The ions at m/z − − 285 and 270 correspond to [M–H–2CH3–CO] and [M–H–3CH3–CO] . Based on the MS/MS data, the structure of the compounds was suggested as 5,2′- dihydroxy-7,8,6′-trimethoxyflavone and 5,2′-dihydroxy-6,7,8-trimethoxyfla- vone, previously known compounds in S. baicalensis [7]. Compound 40 yielded a double loss of methyl group at m/z 298 and 283 from the [M–H]− ions of the m/z 313 in CID spectra. The ion at m/z 255 indi- − cated the loss of CO as [M–H–2CH3–CO] from parent ion. In agreement with previous reports, the compound was assigned as 5,8-dihydroxy-6,7- dimethoxyflavone [15].

Conclusion

In summary, a total of 40 flavonoids, including 2 phenolic glycosides were successfully identified by LC–MS in crude extracts of leaf, stem and root tis- sues of S. incana. There are differences in the flavonoid composition be- tween leaves, stems, and roots (Fig. 2 and Table I). Some flavonoids (com- pounds 32, 34, 35, 37, 39, and 40) are detected exclusively in roots, whereas dihydroscutellarin (compound 9) was found only in stems and scutellarein (compound 17) only in leaves. The majority of the compounds identified are found in leaves, stems, and roots (Fig. 2 and Table I). The flavonoid profile of S. incana is consistent with other Scutellaria species. Further work should fo- cus on assessing the potential of S. incana as a source of these bioactive me- tabolites.

Unauthenticated | Downloaded 09/26/21 11:55 PM UTC 568 M. Nurul Islam et al.

13 x10 21 0.9 0.8 0.7

0.6 22+23 Leaf 0.5 6 12 0.4 0.3 1 2 18 0.2 14 17 3 9 10 20 21 24 4+5 38 0.1 16

7+8 15 11 19 27 29 31 36 0 x10 2 1 13 0.9 0.8 0.7 0.6 Stem 0.5 0.4 7+8

0.3 21+22 27 0.2 19 24 36 10 25+26 1 2 16 18 23

4+5 11 14 0.1 3 6 12 15 20 28 38 29+30 31 33 0 2 x10 1 13 0.9 25+26 25+26 0.8 Root 0.7 0.6 21+22 0.5 36+37 27 0.4 28 0.3 5 18 0.2 19 24

1 2 7+8 40 11 23 35 38 0.1 10 16 20 34 3 12 14 15 29+30 3233 39 0 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 Counts (%) vs. Acquisition Time (min)

Fig. 2. Total ion chromatogram (TIC) of methanolic extracts from leaf, stem and root of S. incana L. Identities of peaks are listed in Table I

Acknowledgments

This work is supported by Science Foundation Ireland Research Frontiers Programme Grants (06/SFI/RFP/GEN034 and 08/SFI/RFP/EOB1087) and a Science Foundation Ireland Equipment Grant (06/SFI/RFP/GEN034ES) to C.K.-Y.N.

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