Metabolomics (2011) 7:446–453 DOI 10.1007/s11306-010-0269-9
ORIGINAL ARTICLE
Comparative analysis of bioactive phytochemicals from Scutellaria baicalensis, Scutellaria lateriflora, Scutellaria racemosa, Scutellaria tomentosa and Scutellaria wrightii by LC-DAD-MS M. Nurul Islam • Frances Downey Carl K. Y. Ng
•
Received: 25 August 2010 / Accepted: 18 December 2010 / Published online: 25 December 2010 Ó Springer Science+Business Media, LLC 2010
Abstract Scutellaria is a geographically widespread and diverse genus of the Lamiaceae family of herbaceous plants commonly known as skullcaps. Scutellaria is used widely as an ethnobotanical herb for the treatment of various ailments ranging from cancers, cirrhosis, jaundice, hepatitis, anxiety and nervous disorders. We used (1) reverse-phase liquid chromatography coupled to a diode array detector (LC-DAD), and (2) multiple reaction monitoring (MRM) using mass spectrometry (LC-MS/MS) to quantify the levels of acteoside, scutellarin, scetellarein, baicalin, baicalein, wogonin, wogonoside, apigenin, chrysin, and oroxylin A in aqueous methanolic extracts of roots, shoots and leaves of S. baicalensis, S. lateriflora, S. racemosa, S. tomentosa and S. wrightii. Our results indicate that both methods (LC-DAD and LC-MS/MS) were robust for the detection of the 10 analytes from Scutellaria extracts although greater sensitivities were achieved using LC-MS/ MS in MRM mode. MRM enabled the detection of low levels of analytes which were otherwise undetected using LC-DAD. The baicalin content of S. wrightii roots were 5-fold higher than the commonly used S. baicalensis. Additionally, we also showed that leaves of both S. wrightii and S. tomentosa are good sources of scutellarin compared to S. baicalensis. Our data clearly demonstrated that previously uncharacterized species, S. wrightii and S. tomentosa are both good sources of flavonoids, particularly scutellarin, baicalin, wogonin and baicalein. Electronic supplementary material The online version of this article (doi:10.1007/s11306-010-0269-9) contains supplementary material, which is available to authorized users. M. Nurul Islam F. Downey C. K. Y. Ng (&) School of Biology and Environmental Science, University College Dublin, Belfield, Dublin 4, Ireland e-mail:
[email protected]
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Keywords Scutellaria Flavonoid Phenolics LC-DAD-MS
1 Introduction Scutellaria is a geographically widespread and diverse genus of the Lamiaceae (Labiatae) family of herbaceous plants commonly known as skullcaps. It has been estimated that there are over 300 species distributed throughout the world. Scutellaria is used widely as an ethnobotanical herb for the treatment of various ailments ranging from cancers, cirrhosis, jaundice, hepatitis, anxiety and nervous disorders (Cole et al. 2007). Scutellaria baicalensis, commonly known as Baikal skullcap or Huang-qin is one of the most commonly prescribed herbs in Traditional Chinese Medicine (TCM). In addition to the use of S. baicalensis in TCM, Scutellaria lateriflora, commonly known as the Blue skullcap (or Hoodwort, Virginian skullcap or mad-dog skullcap) is widely used by native American herbalists as a sedative and for treating nervous disorders. Extracts of S. lateriflora has recently been shown to exhibit anxiolytic properties (Awad et al. 2003). More recently, Cole et al. (2008) have analysed the content of 4 flavonoids (baicalin, baicalein, scutellarin and wogonin) from S. racemosa, the South American skullcap and showed that S. racemosa contained significantly higher levels of wogonin compared to S. baicalensis and S. lateriflora. The interest in S. racemosa stems from the observation that S. racemosa exhibits neuro-protective activity (cited in Cole et al. 2008). The widespread use of Scutellaria as an ethnobotanical herb by diverse communities of indigenous peoples suggests that diverse species of Scutellaria may contain common active phytochemicals responsible for their
LC-DAD-MS analysis of phytochemicals from Scutellaria
medicinal properties (Cole et al. 2007, 2008). In order to validate this, it is important to expand on the number of Scutellaria species and the number of bioactive metabolites examined. In this study, we examined the phenolic and flavonoid content in Scutellaria wrightii (Wright’s skullcap) and S. tomentosa, in addition to previously studied species, S. baicalensis, S. lateriflora and S. racemosa. Ten bioactive metabolites were examined from aqueous methanolic extracts obtained from roots, stems and leaves, and they include acteoside, scutellarin, scutellarein, baicalin, baicalein, wogonin, wogonoside, apigenin, chrysin, and oroxylin A. These metabolites were chosen for their demonstrated bioactivity in diverse cellular processes like apoptosis and angiogenesis. For example, acteoside has been shown to inhibit human promyelocytic HL-60 leukemia cell proliferation via inducing cell cycle arrest at G0/G1 phase and differentiation into monocyte (Lee et al. 2007), oroxylin A has been shown to induce apoptosis in cancer cells via p53 and therefore is a potential candidate for cancer therapy (Mu et al. 2009), and baicalin has been shown to induce apoptosis in human mucoepidermoid carcinoma Mc3 cells in vitro and in vivo (Xu et al. 2010). Chrysin has been shown to suppress IL-6-induced angiogenesis via down-regulation of JAK/STAT3 and VEGF in vitro and in vivo (Lin et al. 2010), and wogonoside can inhibit lipopolysaccharide-induced angiogenesis in vitro and in vivo via toll-like receptor 4 signal transduction (Chen et al. 2009). Readers are referred Shang et al. (2010) for an extensive review of the pharmacological properties of these bioactive metabolites. The aim of this study is to compare, using HPLC coupled with diode array detection and tandem mass spectrometry, the bioactive phytochemical content of roots, stems, and leaves of Scutellaria baicalensis, S. lateriflora, S. racemosa, S. tomentosa and S. wrightii with respect to the levels of acteoside, scutellarin, scutellarein, baicalin, baicalein, wogonin, wogonoside, apigenin, chrysin, and oroxylin A. More specifically, this study aims to determine if previously uncharacterized S. tomentosa and S. wrightii can be potentially good sources of bioactive phytochemicals.
2 Materials and methods 2.1 Scutellaria seeds germination and plant growth Seeds of S. lateriflora, S. racemosa, S. tomentosa, S. wrightii (kindly provided by Royal Botanic Gardens, Kew, United Kingdom), and S. baicalensis (kindly provided by Dr. Graham Wilson, University College Dublin) were surface-sterilized in 70% ethanol for 1 min followed by 15 min in 25% domestic bleach before rinsing with sterile deionized water. Seeds were then transferred to
447
sterile Schenk-Hildebrandt medium supplemented with 300 mg l-1 casein, 30 g l-1 sucrose, 1 mg l-1 GA3 and 8 g l-1 plant cell-culture tested agar. The plated seeds were stratified 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 lmol 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 lmol m-2 s-1. 2.2 Extraction of phenolic and flavonoids Leaf, stem and root tissue from 3-month old plants were harvested and flash frozen in liquid N2 before freeze-drying. Freeze-dried materials were ground into fine powder using a mortar and pestle. To determine the content of bioactive components, the ground 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 centrifugation and the extracts were diluted with 70% methanol to a final concentration of 2 mg ml-1. The samples were then filtered using a 0.2 lm filter prior to LC-DAD analysis. Samples were suitably diluted for LCMS/MS analysis considering the sensitivity of mass spectrometry. An aliquot of the diluted sample (100 ll) was spiked with an internal standard (digoxin, 10 ll of 200 lg ml-1) and 5 ll of sample was subjected to LC-MS/ MS analysis. 2.3 Chemicals and reagents Baicalein (C95%), baicalin (C99%), and acteoside (C98%) were purchased from Extrasynthe`se (France). Scutellarein (C98%), scutellarin (C98%), wogonoside (C98%) were supplied by Chengdu Biopurify Phytochemicals Ltd. (China). Apigenin (C95%) and chrysin (C96%) were purchased from Sigma-Aldrich (United Kingdom), and wogonin (C95%) and oroxylin A (C95%) were supplied by Shanghai Zhanshu Chemical Technology Ltd. (China). HPLC-grade and LC-MS-grade water, acetonitrile and methanol were purchased from Fisher Scientific, UK. All other chemicals used were of analytical grade unless otherwise specified. 2.4 Standard solutions Standard stock solutions (1 mg ml-1) of acteoside, scutellarin, scutellarein, baicalin, wogonoside, apigenin, baicalein, wogonin, chrysin and oroxylin A were obtained by dissolving the compounds in methanol. The standard solutions were serially diluted with 70% methanol to obtain
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working standard solutions of various concentrations. Calibration curves were constructed using ten calibration standard mixture solutions ranging in concentration from 1 to 80 lg ml-1 for acteoside, scutellarin, baicalin and wogonoside. The calibration range for apigenin, wogonin, chrysin and oroxylin A was 0.25–20 lg ml-1, 0.5–40 lg ml-1 for scutellarein, and 0.5–20 lg ml-1 for baicalein.
2.5 Quantitative analysis of bioactive metabolites by HPLC-DAD Fifteen ll of each extract were injected into the HPLC system for quantitative analysis. Chromatography was performed with a HPLC system consisting of binary pump, autosampler, column oven and DAD detector (1200 RRLC, Agilent Technologies, USA). The chromatographic separation of compounds was achieved using a Luna C18 (4.6 mm I.D. 9 150 mm, particle size 3 lm, Phenomenex, Torrance, CA, USA) analytical column. Column oven and autosampler temperatures were maintained at 35°C and 4°C, respectively. The mobile phase consists of 0.1% aqueous formic acid (solvent A) and organic modifier (acetonitrile:methanol, 50:50, containing 0.1% formic acid, solvent B). Elution was performed at a flow rate of 1.0 ml min-1 in a binary gradient mode. The initial elution condition was A–B (80:20, v/v) for 3 min, linearly changed to A–B (50:50, v/v) at 20 min and maintained in this condition for up to 30 min, before being changed to A–B (30:70, v/v) at 36 min, returning to initial condition at 37 min, followed by 6 min column reequilibration. Total run time for sample analysis was 43 min and the absorbance was measured at 280 nm with a diode array detector (Agilent Technologies, Germany). The chromatographic peaks of the analytes in extracts were identified by comparing their retention time and UV spectra with those of the reference standards and further confirmed by spiking samples with the reference compounds. Data acquisition and analysis was achieved using MassHunter software (Agilent Technologies, USA). Calibration curves for acteoside, scutellarin, scutellarein, baicalin, wogonoside, apigenin, baicalin, wogonin, chrysin and oroxylin A were generated by plotting peak area against concentration by least-squares regression analysis. Each calibration curve was obtained using 7 different analyte concentrations. The linear correlation coefficient (r2) for all calibration curves was [0.99 and intercept was close to zero, indicating good linearity in the concentration range (Supplementary Table 1). Additionally, the analytical method was robust as indicated by the relative lack of intra- and inter-day variations in the levels measured (Supplementary Table 2).
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2.6 Quantitative analysis of bioactive metabolites by LC-MS/MS The HPLC system was coupled to an Agilent 6460 triplequadrupole mass spectrometer (Agilent Technologies, USA) equipped with a Jet Stream ion source. Electrospray ionization (ESI) was performed in the positive acquisition mode, with nitrogen used as the nebulising agent. The gas temperature and flow rate was 350°C and 8 l min-1, respectively and the sheath gas temperature and flow rate was 325°C and 10 l min-1, respectively. The ESI needle voltage was adjusted to 3.5 kV and the optimum fragmentor voltage and collision energy were determined by analysis of reference compounds in selected ion and product ion scanning mode. The product ion spectra and the postulated fragmentation patterns of acteoside, scutellarin, scutellarein, baicalin, wogonoside, apigenin, baicalein, wogonin, chrysin and oroxylin A are shown in Supplementary Fig. 1. Multiple-reaction monitoring (MRM) detection was applied using nitrogen as the collision gas with a dwell time of 75 ms for each transition of the protonated molecules (Supplementary Table 3). Data acquisition and analysis was controlled by MassHunter software (Agilent Technologies, USA). Separate optimized chromatographic protocol was applied for LC-MS/MS analysis of samples. Elution of analytes was performed using a flow rate of 0.3 ml min-1 in a binary gradient mode on narrow bore analytical column Gemini C18 (2.00 mm I.D. 9 150 mm, particle size 3 lm, Phenomenex, Torrance, CA, USA) and column oven and autosampler temperature was maintained at 35°C and 4°C, respectively. The mobile phase consists of 0.1% formic acid (solvent A) and acetonitrile with 0.1% formic acid (Solvent) B. The initial elution condition was A–B (75:25, v/v), linearly changed to A–B (40:60, v/v) at 3.5, again change to (60:40, v/v) at 6 min and maintained this condition up to 10 min, again gradient was changed to A–B (20:80, v/v) at 14 min returning to initial condition at 15 min, followed by 7 min column re-equilibration. The calibration curves were obtained using 10 calibration standard mixture solutions at concentration ranging from 25 to 1000 ng ml-1 for baicalein, chrysin and apigenin, 12.5–1000 ng ml-1 for wogonin and oroxylin A, 200–10,000 ng ml-1 for acteoside, 100–10,000 ng ml-1 for scutellarin, baicalin and wogonoside, and 25–2000 ng ml-1 for scutellarein. 100 ll of standard solution at each concentration was spiked with 10 ll of internal standard (digoxin, 200 lg ml-1). The volume of standard solution injected into LC-MS/MS system was 5 ll. The calibration curves for LC-MS/MS analysis were generated by plotting the peak area ratios for the analytes from the internal standard vs. the concentration. The linear correlation coefficient (r2) for all calibration curves was [0.99 and intercept was closer to zero, indicating good linearity in the concentration range
LC-DAD-MS analysis of phytochemicals from Scutellaria
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(Supplementary Table 4). Additionally, the analytical method was robust as indicated by the relative lack of intraand inter-day variations in the levels measured (Supplementary Table 5).
3 Results and discussion 3.1 Optimization of chromatographic separation and mass spectrometric analysis Flavonoids and phenolics (Fig. 1) were analyzed by reverse-phase liquid chromatography due to the polar nature of these compounds. The rapid and simultaneous determination of bioactive compounds in different Scutellaria species in a single HPLC run in isocratic elution mode was considered difficult due to variations in the physicochemical properties of the analytes. As such, a gradient elution method was developed to ensure that all constituents could be well separated and quantified in one analysis run. In the course of the experiments, various mixtures of water, methanol and acetonitrile were used as mobile phase, but poor peak shape and tailing of analytes were observed (data not shown). The addition of formic acid to the mobile phase to minimize the ionization of
(A)
phenol group of flavonoid compounds resulted in good resolution, as well as satisfactory peak symmetry and shape. The optimized mobile phase was selected as 0.1% formic acid as aqueous phase and 0.1% formic acid in mixture of methanol, acetonitrile (50:50, v/v) as organic modifier. Selection of gradient condition is also critical for analysis of multi-component plant extracts. In the current study, gradient program was initiated with solvent A and solvent B in the ratio of 80:20, resulting good peak shape but acteoside peak was merged with other peaks present in samples. Therefore, an isocratic hold for 3 min in the ratio of (A:B, 80:20, v/v) was necessary to overcome the overlapping phenomenon. Representative LC-DAD chromatograms of samples (exemplified using extracts from leaf, stem and root of S. tomentosa) and standard mixture show that all compounds were completely separated in an entire analytical run (Fig. 2). Chromatograms of samples analyzed using LC-MS/MS demonstrated good separation of compounds (Supplementary Fig. 2).
R1 R4 R3
O
R2 OH R1 = OH R1 = OH R1 = H R1 = H R1 = OH R1 = H R1 = H R1 = H R1 = H
R2 = OH R2 = OH R2 = OH R2 = H R2 = H R2 = OH R2 = H R2 = H R2 = 0CH3
O
R3 = O-Glucuronic acid R3 = OH R3 = O-Glucuronic acid R3 = O-Glucuronic acid R3 = OH R3 = OH R3 = OH R3 = OH R3 = OH
R4 = H R4 = H R4 = H R4 = 0CH3 R4 = H R4 = H R4 = 0CH3 R4 = H R4 = H
Scutellarin Scutellarein Baicalin Wogonoside Apigenin Baicalein Wogonin Chrysin Oroxylin A
OH
(B)
O
O HO O OH
O
OH
O
O
OH
OH
HO
HO
OH
Fig. 1 Chemical structures of flavonoids (a) and acteoside (b) from Scutellaria
Fig. 2 Representative LC–DAD chromatograms of standard compounds (a), and aqueous methanolic extracts of S. tomentosa leaves (b), stems (c), and roots (d). (1) acteoside, (2) scutellarin, (3) scutellarein, (4) baicalin, (5) wogonoside, (6) apigenin, (7) baicalein, (8) wogonin, (9) chrysin and (10) oroxylin A
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3.2 Comparative analyses of metabolites from Scutellaria species Using LC-DAD, we observed the presence of all 10 analytes in S. baicalensis and S. tomentosa (Table 1). Roots of S. baicalensis contained high levels of baicalin (25.41 ± 0.10 lg mg-1) relative to stems (7.66 ± 0.01 lg mg-1) and leaves (4.03 ± 0.10 lg mg-1) (Table 1), in agreement with previous studies showing that baicalin is predominantly found in roots of S. baicalensis (Horvath et al. 2005; Makino et al. 2008; Parajuli et al. 2009). Direct comparisons of the levels quantified in this study with previous reports are difficult due to differences in the manner in which the levels were presented. For example, baicalin levels were expressed as lg mg-1 extract in Parajuli et al. (2009) as opposed to lg mg-1 dry weight of plant material in the present study. The levels of baicalin observed in S. baicalensis in this study (25.41 ± 0.10 lg mg-1 dry weight of plant material) are similar to levels (19.33 ± 0.72 lg mg-1 dry weight of plant material) reported by Zgo´rka and Hajnos (2003). However, the levels of baicalin reported in this study and by Zgo´rka and Hajnos (2003) differed by 4- to 5-fold to the levels reported by Xie et al. (2002). Such differences may be due to differences in growth conditions although it is difficult to be certain as exact growth conditions were not reported in some previous reports (Xie et al. 2002; Zgo´rka and Hajnos 2003). Nevertheless, the observation that baicalin is predominantly found in the roots compared to leaves and shoots (in agreement with previous reports) lends credence to our observations not only of S. baicalensis, but also of the other species reported in this study. High levels of baicalin were also observed in roots of S. wrightii compared to stems and leaves (Table 1). Interestingly, the levels of baicalin in roots were about 5-fold higher in S. wrightii compared to S. baicalensis (Table 1). In S. racemosa, comparable levels of baicalin were observed between leaves, stems and roots as opposed to the situation in S. tomentosa where baicalin was observed to be higher in roots (17.30 ± 0.22 lg mg-1) followed by stems (10.63 ± 0.26 lg mg-1) and then leaves (1.05 ± 0.03 lg mg-1) (Table 1). Very low levels of baicalin were detected in leaves and roots of S. lateriflora (Table 1), in contrast to Parajuli et al. (2009) who reported comparable levels of baicalin between roots of S. baicalensis and S. lateriflora. Interestingly, our data suggest that baicalein were found to be present in higher levels in S. tomentosa (4.70 ± 0.19 lg mg-1) and S. wrightii (4.99 ± 0.30 lg mg-1) compared to S. baicalensis, S. racemosa and S. lateriflora. The highest levels of scutellarin were found in leaves of S. wrightii (51.50 ± 0.59 lg mg-1), about 2-fold higher than the levels of scutellarin observed in leaves of S. tomentosa (24.29 ± 0.09 lg mg-1) and stems of S. wrightii
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(21.05 ± 0.09 lg mg-1) (Table 1). In general, highest levels of scutellarin were found in leaves in S. lateriflora, S. racemosa, S. tomentosa, and S. wrightii with the notable exception of S. baicalensis, where the highest levels of scutellarin were observed in stems (Table 1). Relatively low and comparable levels of scutellarin were found in roots of all species examined, suggesting that the stems and leaves are better sources of scutellarin compared to roots. Comparable levels of wogonoside were detected in the roots of S. baicalensis (12.63 ± 0.14 lg mg-1) and S. wrightii (11.90 ± 0.09 lg mg-1) (Table 1). In contrast, the levels of wogonoside was lower by 2-fold (5.68 ± 0.04 lg mg-1) in roots of S. tomentosa (Table 1). The highest levels of wogonin were found in roots of S. tomentosa (10.15 ± 0.18 lg mg-1) followed by roots of S. baicalensis (6.71 ± 0.14 lg mg-1), roots of S. wrightii (4.23 ± 0.05 lg mg-1), and roots of S. lateriflora (2.04 ± 0.06 lg mg-1) (Table 1). Scutellaria tomentosa appears to be a good source of chrysin with roots of S. tomentosa showing the highest levels of chrysin (3.89 ± 0.09 lg mg-1) compared to all the other species examined (Table 1). All species examined showed relatively low levels of oroxylin A with the exception of S. racemosa where oroxylin A was observed to be predominantly found in leaves and stems. Comparable levels of acteoside were detected in roots of S. baicalensis (2.35 ± 0.05 lg mg-1) and S. tomentosa (2.18 ± 0.06 lg mg-1) (Table 1). Low levels of scutellarein were observed in leaves, stems and roots of S. baicalensis whereas scutellarein were not detected in all other species except in roots of S. tomentosa (Table 1). Apigenin were notably absent in S. lateriflora and S. racemosa and only low levels were detected in S. baicalensis, S. tomentosa, and S. wrightii (Table 1). To increase the sensitivity of detection, we use mass spectrometry with multiple reaction monitoring (MRM) to quantify the levels of the various analytes in Scutellaria extracts. LC-MS/MS in MRM mode is a selective and specific technique for simultaneous quantitation of a number of compounds without the need for chromatographic separation except for isomers with the same precursor/ product ion transitions. LC conditions were optimized using narrow bore column, water/acetonitrile with 0.1% formic acid as mobile phase with a short run time. Isomeric compounds, apigenin (m/z 271/153), baicalein (m/z 271/123), wogonin (m/z 285/270), and oroxylin A (m/z 285/270) were chromatographically separated. Results obtained using MRM (Table 2) were largely in agreement with those obtained using LC-DAD (Table 1). Due to the increased sensitivity afforded by MRM, we were able to detect the presence of scutellarein in S. wrightii and S. lateriflora (Table 2), which we were unable to with LC-DAD (Table 1).
0.54 ± 0.02
–
Stem
Root
0.79 ± 0.04
Values (lg mg
0.98 ± 0.02
21.05 ± 0.09 –
–
–
0.35 ± 0.01
–
–
–
–
–
–
–
–
0.31 ± 0.01
0.25 ± 0.00
0.26 ± 0.01
Scutellarein
dry weight) are means ± SD, n = 3
0.89 ± 0.04
Root
-1
0.51 ± 0.02
0.91 ± 0.01
Leaf
Stem
51.50 ± 0.59
0.93 ± 0.02
2.18 ± 0.06
Root
S. wrightii
24.29 ± 0.09
12.42 ± 0.35
0.52 ± 0.03
1.49 ± 0.06
Leaf
0.51 ± 0.01
Root 0.59 ± 0.07 S. tomentosa
Stem
1.74 ± 0.03
0.83 ± 0.07
1.25 ± 0.03
Leaf
6.53 ± 0.14
–
Stem
S. racemosa
–
Leaf
9.22 ± 0.43
0.92 ± 0.04
2.35 ± 0.05
Root
S. lateriflora
8.64 ± 0.09
1.27 ± 0.13
1.34 ± 0.04
Leaf
5.97 ± 0.23
Scutellarin
Stem
S. baicalensis
Acteoside
122.14 ± 1.42
0.92 ± 0.02
0.51 ± 0.07
17.30 ± 0.22
10.63 ± 0.26
1.05 ± 0.03
11.11 ± 0.24
10.59 ± 0.11
15.21 ± 0.11
0.52 ± 0.02
–
0.51 ± 0.01
25.41 ± 0.10
7.66 ± 0.01
4.03 ± 0.10
Baicalin
11.90 ± 0.09
0.51 ± 0.01
–
5.68 ± 0.04
1.08 ± 0.05
–
2.04 ± 0.04
1.58 ± 0.09
–
–
–
–
12.63 ± 0.14
5.61 ± 0.07
7.90 ± 0.10
Wogonoside
Table 1 Quantitative LC-DAD analysis of bioactive analytes from different species of Scutellaria
–
–
0.37 ± 0.02
–
0.67 ± 0.05
0.53 ± 0.03
–
–
–
–
–
–
0.25 ± 0.04
–
Apigenin
4.99 ± 0.30
–
–
4.70 ± 0.19
–
–
0.53 ± 0.05
–
–
0.61 ± 0.04
–
1.04 ± 0.14
–
–
Baicalein
4.23 ± 0.05
0.17 ± 0.01
–
10.15 ± 0.18
0.34 ± 0.03
–
0.63 ± 0.06
0.26 ± 0.01
–
2.04 ± 0.06
–
6.71 ± 0.14
0.52 ± 0.02
0.62 ± 0.01
Wogonin
–
0.14 ± 0.02
–
1.30 ± 0.20
3.89 ± 0.09
1.01 ± 0.03
–
0.20 ± 0.00
0.32 ± 0.03
–
–
–
0.22 ± 0.02
1.60 ± 0.06
–
Chrysin
0.35 ± 0.01
0.13 ± 0.01
–
0.59 ± 0.01
0.21 ± 0.01
–
0.34 ± 0.03
5.86 ± 0.15
12.42 ± 0.18
0.22 ± 0.02
–
0.61 ± 0.02
–
–
Oroxylin A
LC-DAD-MS analysis of phytochemicals from Scutellaria 451
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0.63 ± 0.02 –
Stem Root
0.62 ± 0.03
Root
Values (lg mg
1.04 ± 0.01
22.66 ± 0.54 0.03 ± 0.00
0.05 ± 0.00
0.04 ± 0.00
0.34 ± 0.02
0.03 ± 0.00
0.03 ± 0.00
–
–
–
– 0.03 ± 0.00
0.03 ± 0.00
0.28 ± 0.01
0.27 ± 0.00
0.21 ± 0.00
Scutellarein
dry weight) are means ± SD, n = 3
0.88 ± 0.02
Root
-1
0.40 ± 0.01
0.87 ± 0.01
Leaf
Stem
49.07 ± 0.58
1.01 ± 0.01
2.28 ± 0.04
S. wrightii
Root
13.22 ± 0.25
0.38 ± 0.02
1.34 ± 0.03
Leaf
Stem
23.79 ± 0.52
0.49 ± 0.01
1.38 ± 0.01
S. tomentosa
1.85 ± 0.02
0.71 ± 0.04
Stem
6.55 ± 0.11
0.98 ± 0.00 0.02 ± 0.00
Leaf
S. racemosa
–
Leaf
8.84 ± 0.04
1.01 ± 0.01
2.21 ± 0.14
Root
S. lateriflora
9.32 ± 0.13
1.07 ± 0.06
1.26 ± 0.13
Leaf
5.84 ± 0.16
Scutellarin
Stem
S. baicalensis
Acteoside
117.45 ± 0.26
1.04 ± 0.03
0.58 ± 0.01
12.42 ± 0.34
10.25 ± 0.25
14.11 ± 0.03
11.17 ± 0.28
9.65 ± 0.14
13.26 ± 0.21
0.03 ± 0.00 0.49 ± 0.01
0.42 ± 0.00
23.82 ± 0.70
8.00 ± 0.21
4.17 ± 0.22
Baicalin
11.71 ± 0.04
0.46 ± 0.00
–
5.58 ± 0.08
0.93 ± 0.03
–
1.94 ± 0.01
1.77 ± 0.02
–
– –
–
11.37 ± 0.08
5.87 ± 0.06
7.82 ± 0.22
Wogonoside
Table 2 Quantitative LC-MS/MS analysis of bioactive analytes from different species of Scutellaria
–
0.06 ± 0.00
0.39 ± 0.00
0.02 ± 0.00
0.55 ± 0.01
0.41 ± 0.00
–
–
0.02 ± 0.00
– –
–
–
0.27 ± 0.00
–
Apigenin
4.71 ± 0.05
0.02 ± 0.00
0.02 ± 0.00
4.86 ± 0.26
0.03 ± 0.00
–
0.48 ± 0.02
0.03 ± 0.00
0.02 ± 0.00
– 0.57 ± 0.03
–
0.89 ± 0.01
0.04 ± 0.00
0.03 ± 0.00
Baicalein
3.97 ± 0.03
0.14 ± 0.00
0.03 ± 0.00
8.50 ± 0.11
0.42 ± 0.02
0.06 ± 0.00
0.71 ± 0.01
0.28 ± 0.01
0.09 ± 0.01
0.02 ± 0.00 1.88 ± 0.03
0.02 ± 0.00
5.84 ± 0.02
0.56 ± 0.01
0.61 ± 0.03
Wogonin
0.03 ± 0.00
0.16 ± 0.00
0.05 ± 0.00
1.46 ± 0.06
3.59 ± 0.08
0.91 ± 0.02
0.06 ± 0.00
0.17 ± 0.00
0.29 ± 0.00
– 0.04 ± 0.00
0.02 ± 0.00
0.18 ± 0.00
1.59 ± 0.01
–
Chrysin
0.40 ± 0.01
0.14 ± 0.01
–
0.58 ± 0.01
0.17 ± 0.00
0.03 ± 0.00
0.31 ± 0.00
4.38 ± 0.07
10.97 ± 0.18
– 0.15 ± 0.00
–
0.57 ± 0.00
0.10 ± 0.00
0.04 ± 0.00
Oroxylin A
452 M. Nurul Islam et al.
LC-DAD-MS analysis of phytochemicals from Scutellaria
453
4 Conclusion
References
Overall, our results indicate that both methods (LC-DAD and LC-MS/MS) were robust for the detection of the 10 analytes from Scutellaria extracts although greater sensitivities were achieved using LC-MS/MS in MRM mode. MRM enabled the detection of low levels of analytes which were otherwise undetected using LC-DAD. Additionally, LC-MS/MS provided greater specificity, structural information and shorter analytical time compared to LC-DAD. The baicalin content of S. wrightii roots was 5-fold higher than the commonly used S. baicalensis. Additionally, we also showed that leaves of both S. wrightii and S. tomentosa are good sources of scutellarin compared to S. baicalensis. In Japan, roots of skullcaps species (S. baicalensis and S. lateriflora) are classified as ‘raw materials exclusively used as pharmaceuticals’, whereas aerial parts (stems and leaves) are classified as ‘non-pharmaceutical’ (Makino et al. 2008). Considering the widespread use of skullcaps in herbal remedies and dietary supplements, our data demonstrating the presence of flavonoids in aerial parts of skullcaps will have important implications for the classification of roots and aerial tissues as ‘exclusively use for pharmaceuticals’ and ‘non-pharmaceuticals’, respectively. When the results of this study are placed in the context of previous studies of different Scutellaria species, large variations in the quantified levels of metabolites were immediately obvious. Such variations can be attributed in part to prevailing growth conditions. Standardization of growth conditions would facilitate comparisons between studies. This can be achieved through the use of in vitro culture of Scutellaria explants where growth conditions can be tightly regulated. Work by the Murch and Saxena laboratories (Murch et al. 2004; Alan et al. 2007; Cole et al. 2008) have contributed much towards establishing conditions important for successful in vitro culture of Scutellaria. Our data clearly demonstrated that previously uncharacterized species, S. wrightii and S. tomentosa are both good sources of flavonoids, particularly scutellarin, baicalin, wogonin and baicalein. Future work should focus on establishing in vitro conditions for the axenic cultures of S. wrightii and S. tomentosa to facilitate experiments for establishing elite germplasm (via in vitro manipulation for selection of natural and induced mutations, and genetic manipulation) as useful sources bioactive metabolites.
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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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