Skip to main content
NCBI home page
Plant Direct logoLink to Plant Direct
. 2021 Dec 20;5(12):e372. doi: 10.1002/pld3.372

Metabolite profiling reveals organ‐specific flavone accumulation in Scutellaria and identifies a scutellarin isomer isoscutellarein 8‐O‐β‐glucuronopyranoside

Bryce C Askey 1, Dake Liu 2, Garret M Rubin 2, Andrew R Kunik 1, Yeong Hun Song 1, Yousong Ding 2,✉, Jeongim Kim 1,3,✉
PMCID: PMC8689113  PMID: 34977451

Abstract

Scutellaria is a genus of plants containing multiple species with well‐documented medicinal effects. S. baicalensis and S. barbata are among the best‐studied Scutellaria species, and previous works have established flavones to be the primary source of their bioactivity. Recent genomic and biochemical studies with S. baicalensis and S. barbata have advanced our understanding of flavone biosynthesis in Scutellaria. However, as over several hundreds of Scutellaria species occur throughout the world, flavone biosynthesis in most species remains poorly understood. In this study, we analyzed organ‐specific flavone profiles of seven Scutellaria species, including S. baicalensis , S. barbata , and two species native to the Americas ( S. wrightii to Texas and S. racemosa to Central and South America). We found that the roots of almost all these species produce only 4′‐deoxyflavones, while 4′‐hydroxyflavones are accumulated exclusively in their aerial parts. On the other hand, S. racemosa and S. wrightii also accumulated high levels of 4′‐deoxyflavones in their aerial parts, different with the flavone profiles of S. baicalensis and S. barbata . Furthermore, our metabolomics and NMR study identified the accumulation of isoscutellarein 8‐O‐β‐glucuronopyranoside, a rare 4′‐hydroxyflavone, in the stems and leaves of several Scutellaria species including S. baicalensis and S. barbata , but not in S. racemosa and S. wrightii . Distinctive organ‐specific metabolite profiles among Scutellaria species indicate the selectivity and diverse physiological roles of flavones.

1. INTRODUCTION

Medicinal plants have been used in the traditional medicines of indigenous populations for thousands of years. Due to this widespread usage, modern research techniques are being applied to identify the compounds responsible for these medicinal properties and to characterize their modes of action (Shang et al., 2010). A negative consequence of increased attention to and demand for medicinal plants is the endangerment of native plant populations resulting from overharvesting (Cole et al., 2007). Therefore, development of biotechnology‐based mass production systems for these medicinal compounds is desirable. Development of effective biotechnology for chemical production requires an understanding of the biosynthesis of the compounds of interest. In this work, we analyze the levels of flavones in various organs of multiple species from the Scutellaria genus to better understand flavone biosynthesis in Scutellaria.

Part of the mint family Lamiaceae, Scutellaria is a genus of plants containing several hundred species with well‐documented medicinal effects. Extracts from the aerial parts of S. barbata are commonly applied in Eastern medicines to treat swelling, inflammation, and cancer (Tao & Balunas, 2016). These activities, especially its anticancer effects, have drawn significant attention to S. barbata, and early phase clinical trials of aqueous extracts have demonstrated its selective cytotoxicity towards breast cancer cells (Chen et al., 2012). In addition, S. barbata extracts have exhibited remarkable activity towards multi‐drug resistant strains of bacteria (Tsai et al., 2018). S. baicalensis is another species extensively applied in Eastern medicines, with extracts of its roots being prescribed to treat diarrhea, dysentery, hypertension, inflammation, and a variety of other diseases (Zhao, Tang, et al., 2019). Numerous clinical studies have demonstrated the neuroprotective, antibacterial, antitumor, antioxidant, and other beneficial health effects of these extracts (Saralamma et al., 2017; Tao et al., 2018; Zhu et al., 2016).

One class of bioactive compounds in Scutellaria is flavones (Karimov & Botirov, 2017; Zhao, Tang, et al., 2019). Scutellaria species produce two classes of flavones: 4′‐hydroxyflavones and 4′‐deoxyflavones (Figures 1 and S1). 4´‐Hydroxyflavones, including apigenin and its derivatives, are relatively common across the plant kingdom, whereas 4′‐deoxyflavones, which include chrysin and its derivatives, are relatively rare outside of Scutellaria (Kato et al., 1992; Rao et al., 2002; Rao et al., 2009). Recent works in S. baicalensis and S. barbata have identified multiple enzymes responsible for flavone biosynthesis in Scutellaria and have described the differential activity of specific enzymes towards either 4′‐hydroxyflavones or 4′‐deoxyflavones (Zhao et al., 2016, 2018; Zhao, Yang, et al., 2019). The enzyme selectivity leads to an organ‐specific pattern of flavone accumulation. In this pattern, 4′‐hydroxyflavones accumulate at higher concentrations in the aerial parts of the plant than in the roots, and the roots contain higher concentrations of 4′‐deoxyflavones as compared to the aerial parts (Tao & Balunas, 2016; Xu et al., 2020).

FIGURE 1.

FIGURE 1

Open in a new tab

Proposed 4′‐hydroxyflavone and 4′‐deoxyflavone pathway. Structures of glycosylated flavones are not shown to save space but are included in Figure S1. Enzyme names in blue are specific isoforms that have been identified in S. baicalensis , and enzyme names in black are general names. Flavones that were quantified have names in bold and are numbered to match the labeling of Figure 2. Enzymes are phenylalanine ammonia lyase (SbPAL), cinnamate 4‐hydroxylase (SbC4H), cinnamate‐CoA ligase (SbCLL‐7), 4‐coumarate CoA ligase (SbCLL‐1), chalcone synthase (SbCHS‐1), pinocembrin‐chalcone synthase (SbCHS‐2), chalcone isomerase (SbCHI), flavone synthase I (SbFNSI), flavone synthase II (SbFNSII), flavone 6‐hydroxylase (SBF6H), flavone 8‐hydroxylase (SbF8H), and 8‐O‐methyl transferase (Sb8‐OMT)

Flavone profiles of S. baicalensis and S. barbata have been described, and their reference genomes have been established to further support the biosynthetic studies of flavones. However, due to the large number of uncharacterized species in the genus, it is unknown if the overall flavone pathway and the organ‐specific accumulation patterns of S. baicalensis and S. barbata are well conserved across the genus. In this work, we aimed to expand the current knowledge of flavone diversity in Scutellaria by analyzing metabolite profiles of seven species. These species included two well‐studied species, S. baicalensis and S. barbata, and two species native to warm climates, S. racemosa and S. wrightii. Furthermore, we selected three other Scutellaria species widely distributed in Europe, Asia, and North America, including S. altissima, S. tournefortii, and S. parvula (Hasaninejad et al., 2009; Shang et al., 2010; Sutter et al., 2011), respectively. During this analysis, we unexpectedly identified a 4′‐hydroxyflavone which has not been included in the recent biosynthetic studies of S. baicalensis. We elucidated the structure of this 4′‐hydroxyflavone and quantified its level in the seven species. Our results revealed diversity in site and type of flavone accumulated across the species we selected.

2. MATERIALS AND METHODS

2.1. Plant growth conditions

Plants of seven Scutellaria species were grown from seed at the University of Florida (Gainesville, Florida, USA) in indoor, climate‐controlled conditions at 21–23°C. Fluorescent lighting of intensity 140 μE m−2 s−1 was applied in a 16 h light/8 h dark cycle. Plants were watered every 5–8 days, and root, stem, and leaf tissue samples were collected in biological triplicate 6–8 weeks after germination. Seeds of all species except for those of S. racemosa and S. wrightii were obtained from retailers (S. altissima, S. baicalensis, and S. tournefortii from Plant World Seeds and S. barbata and S. parvula from Prairie Moon Nursery). To collect seeds of S. racemosa, mature plants were taken from a field in Hattiesburg, Mississippi, USA, and grown in indoor, climate‐controlled conditions at the University of Florida until seeds were ready to harvest. Seeds of S. wrightii were collected directly from mature plants grown in outdoor greenhouse conditions at Far South Wholesale Nursery (Austin, Texas, USA). Herbarium vouchers of all species were submitted to the University of Florida Herbarium, and voucher numbers are provided in Table S3.

2.2. Flavone extraction and quantification

With High Performance Liquid Chromatography (HPLC), 15 flavones were quantified from root, stem, and leaf tissue samples of plants. The flavones quantified included seven 4′‐hydroxyflavones, which were apigenin, apigenin‐7‐glucuronide (apigenin 7‐G), scutellarein, scutellarin, hispidulin, hispiduloside, and isoscutellarein‐8‐glucuronide (isoscutellarein 8‐G). The remaining eight flavones were 4′‐deoxyflavones, which were chrysin, chrysin‐7‐glucuronide (chrysin 7‐G), baicalein, baicalin, oroxylin A, oroxyloside, wogonin, and wogonoside. The fresh weight of each tissue sample was determined with an analytical balance immediately after harvesting. Samples were frozen in liquid nitrogen and stored in −80°C until further analysis. An extraction solution of 50% HPLC grade methanol was added to ground samples so that the following ratio was achieved: 30 mg tissue/ml solvent. Samples were then sonicated for 1 h at room temperature. Following sonication, the extraction solution was withdrawn and further diluted with additional 50% methanol to achieve a final ratio of 30 mg tissue/ml solvent. To remove any remaining particulate, extractions were centrifuged at 15,000 rpm for 5 min and syringe filtered with a filter having a pore size of .45 μm.

Flavones were quantified in this final extraction with a Thermo Scientific (Massachusetts, USA) UltiMate 3000 HPLC system. Flavones were separated with an Acclaim RSLC 120 C18 column (2.2 μm, 3.0 × 100 mm) and eluted by a mixture of .1% formic acid (A) and 100% acetonitrile (B). Following an 8‐min equilibration with 5% B prior to injection, the following gradient was applied: 2 min, 25% B; 2 to 6 min, 25% B; 9 min, 50% B; 9 to 11 min, 50% B; 15 min, 95% B; and 15 to 23 min, 95% B. A flowrate of .5 ml/min was used and the column oven temperature set to 40°C. Peak areas were measured at wavelength 276 nm. Calibration mixes of .1, .5, 1, 5, 10, 25, 50, and 100 ppm were prepared with all 14 flavone standards except isoscutellarein 8‐G and used to convert peak areas to concentrations in ppm. Chemical standards used to prepare calibration mixes were purchased from ChemFaces (Wuhan, China) or MilliporeSigma (Massachusetts, USA) and dissolved in dimethylsulfoxide to generate stocks of 1,000, 2000, or 4,000 ppm. These stocks were then diluted with 50% methanol and mixed to generate calibrations mixes of the varying concentrations. With the peak areas of these calibration mixes and the molecular weight of each metabolite, flavone concentrations in μmol/g fresh weight were calculated. For relative concentration of isoscutellarein 8‐G, only peak areas are reported.

Peaks in tissue sample chromatograms were identified based on their retention time and UV spectra (Figure S2) as compared with standards in the calibration mixes. Statistical testing was conducted by comparing the level of a flavone in the organ of a species to the level of the same flavone in the same organ of S. baicalensis. An unpaired Student's t test with a significance cutoff of .05 was applied to identify values as statistically significant.

2.3. LC‐HRMS

LC‐HRMS and HRMS/MS experiments were conducted on Thermo Scientific™ Q Exactive Focus mass spectrometer with Dionex™ Ultimate™ RSLC 3000 uHPLC system, equipped with H‐ESI II probe on Ion Max API Source. Water with .1% formic acid (A) and acetonitrile with .1% formic acid (B) were used as the mobile phases to separate analytes on an Agilent Poroshell 120 EC‐C18 column (2.7 μm, 3.0 × 50 mm). A typical LC program with a .5 ml/min flow rate included 10% B for 2 min, 10–95% B in 8.5 min, 95% B for 2.5 min, 95 to 10% B in .5 min, and re‐equilibration in 2% B for 2 min. The eluents from the first 2 min and last 3 min were diverted to a waste bottle by a diverting valve. MS1 signals were acquired under the Full MS positive ion mode covering a mass range of m/z 150–2,000, with a resolution at 35,000 and an AGC target at 1e6. Fragmentation was obtained using MS2 discovery and Parallel Reaction Monitoring (PRM) mode using an inclusion list of calculated parental ions. Precursor ions were selected in the orbitrap typically with an isolation width of 3.0 m/z and fragmented in the HCD cell with step‐wise collision energies (CE) of 20, 25, and 30. For some ions, the isolation width was 2.0 m/z and step‐wise CE of 15, 20, and 25 were used.

2.4. NMR analysis

For the NMR analysis, 1.6 mg of compound was dissolved in 40 μl DMSO‐d6. 1D and 2D spectra were recorded in a 1.7 mm TCI CryoProbe on a Bruker Avance Neo‐600 Console system (Magnex 14.1 T/54 mm AS Magnet) at Advanced Magnetic Resonance Imaging and Spectroscopy facility, McKnight Brain Institute, the University of Florida. Spectroscopy data were collected and processed using Topspin 4.1.3 software.

2.5. Chemical shifts

1 H NMR (600 MHz, DMSO‐d6): δH 12.82 (OH, br, s, 5), 1.34 (OH, br, s, 4′), 8.07 (2H, d, J = 8.66 Hz, 2′, 6′), 6.93 (2H, d, J = 8.66 Hz, 3′, 5′), 6.83 (1H, s, 3), 6.29 (1H, s, 6), 5.44 (OH, br, s, 7), 4.82 (1H, J = 7.91 Hz, 1″), 3.82 (1H, d, J = 9.65 Hz, 5″), 3.51 (1H, t, J = 9.26 Hz, 4″), 3.49 (1H, t, J = 8.55 Hz, 2″), 3.35 (1H, t, J = 9.03 Hz, 3″). 13 C NMR (151 MHz, DMSO‐d6): δc 181.72 (C‐4), 169.96 (C‐6″), 163.86 (C‐2), 161.12 (C‐4′), 157.23 (C‐7), 156.90 (C‐5), 149.19 (C‐9), 128.85 (C‐2′, C‐6′), 125.11 (C‐8), 120.96 (C‐1′), 115.96 (C‐3′, C‐5′), 106.25 (C‐1″), 103.35 (C‐10), 102.33 (C‐3), 98.86 (C‐6), 76.00 (C‐5″), 75.20 (C‐3″), 73.69 (C‐2″), 71.41 (C‐4″).

3. RESULTS

3.1. Organ‐specific flavone diversity across seven Scutellaria species

We selected seven species of Scutellaria for organ‐specific flavone profiling with High Performance Liquid Chromatography (HPLC). These species included S. altissima, S. baicalensis, S. barbata, S. parvula, S. racemosa, S. tournefortii, and S. wrightii. S. baicalensis and S. barbata have been used in East Asian medicines for thousand years. S. racemosa is native to South and Central America (Krings & Neal, 2001), and S. wrightii occurs in southwestern regions of North America, such as Texas (Nelson & Goetze, 2010). S. altissima, S. tournefortii, and S. parvula are widely distributed in Europe, Asia, and North America (Hasaninejad et al., 2009; Shang et al., 2010; Sutter et al., 2011), but their flavone profiles have not been studied extensively. We grew plants of each species from seed in climate‐controlled conditions and harvested tissue samples from the roots, stems, and leaves of mature plants in biological triplicate. We then quantified concentrations of six 4′‐hydroxyflavones (1; apigenin, 2; apigenin 7‐glucuronide (apigenin 7‐G), 3; scutellarein, 4; scutellarin, 5; hispidulin, 6; hispiduloside) and eight 4′‐deoxyflavones (7; chrysin, 8; chrysin 7‐glucuronide (chrysin 7‐G), 9; baicalein, 10; baicalin, 11;oroxylin A, 12; oroxyloside, 13; wogonin, 14; wogonoside) in these samples (Figure 2; Table 1). S. baicalensis is one of the most studied and medicinally applied species, and we selected it as a reference for statistical testing in this work.

FIGURE 2.

FIGURE 2

Open in a new tab

Metabolite data collected from the (a) leaves, (b) stems, and (c) roots of seven Scutellaria species via High Performance Liquid Chromatography (HPLC). Samples were taken in biological triplicate, and the average concentration of each metabolite calculated. Metabolites are numbered to match their order of occurrence in the flavone pathway, shown in Figure 1

TABLE 1.

Organ‐specific flavone concentrations collected from seven Scutellaria species via HPLC

Species Organ Apigenin Apigenin 7‐G Scutellarein Scutellarin Isoscutellarein 8‐G a Hispidulin Hispiduloside
S. baicalensis Leaves .19 ± .03 .13 ± .01 .27 ± .14 .10 ± .10 .10 ± .05 n.d. n.d.
S. baicalensis Stems .12 ± .12 .22 ± .01 1.04 ± .43 1.76 ± .28 2.17 ± .60 .03 ± .02 n.d.
S. baicalensis Roots n.d. n.d. n.d. n.d. n.d. n.d. n.d.
S. altissima Leaves n.d. * .28 ± .15 .65 ± .28 4.17 ± 1.22 * n.d. n.d. n.d.
S. altissima Stems n.d. n.d. * n.d. * 1.11 ± .13 * .26 ± .01 * n.d. n.d.
S. altissima Roots n.d. n.d. n.d. n.d. n.d. n.d. n.d.
S. barbata Leaves n.d. * .01 ± .01 * 4.59 ± .33 * 1.51 ± .70 * 2.34 ± .49 * .10 ± .02 * n.d.
S. barbata Stems n.d. .11 ± .02 * 1.90 ± .23 1.13 ± .25 3.89 ± .60 * .09 ± .01 * n.d.
S. barbata Roots n.d. n.d. n.d. n.d. n.d. n.d. n.d.
S. parvula Leaves .11 ± .01 * n.d. * .22 ± .04 n.d. n.d. .13 ± .01 * n.d.
S. parvula Stems .02 ± .02 n.d. * .11 ± .01 * .16 ± .08 * n.d. .07 ± .00 * n.d.
S. parvula Roots .06 ± .03 * n.d. n.d. .82 ± .19 * n.d. .10 ± .03 * n.d.
S. racemosa Leaves n.d. * n.d. * n.d. * 1.20 ± .35 * n.d. .63 ± .18 * n.d.
S. racemosa Stems n.d. n.d. * n.d. * .94 ± .33 n.d. .25 ± .06 * .06 ± .06
S. racemosa Roots n.d. n.d. n.d. n.d. n.d. n.d. n.d.
S. tournefortii Leaves n.d. * .28 ± .07 * n.d. * 2.98 ± .65 * .18 ± .03 n.d. n.d.
S. tournefortii Stems n.d. n.d. * n.d. * 1.47 ± .46 .90 ± .24 * n.d. n.d.
S. tournefortii Roots n.d. n.d. n.d. n.d. n.d. n.d. n.d.
S. wrightii Leaves n.d. * n.d. * n.d. * .13 ± .09 n.d. .02 ± .00 * n.d.
S. wrightii Stems n.d. .03 ± .01 * n.d. * 2.10 ± .25 n.d. .63 ± .08 * n.d.
S. wrightii Roots n.d. n.d. n.d. n.d. n.d. n.d. n.d.
Species Organ Chrysin Chrysin 7‐G Baicalein Baicalin Oroxylin A Oroxyloside Wogonin Wogonoside
S. baicalensis Leaves 4.84 ± .53 1.45 ± .24 .19 ± .08 .20 ± .11 n.d. n.d. n.d. n.d.
S. baicalensis Stems .18 ± .04 .09 ± .01 .06 ± .03 .87 ± .87 n.d. n.d. .11 ± .11 .02 ± .02
S. baicalensis Roots n.d. .30 ± .01 .20 ± .03 32.81 ± 2.22 .23 ± .13 .87 ± .87 3.49 ± .18 6.43 ± .43
S. altissima Leaves .75 ± .07 * 2.62 ± .18 * .70 ± .18 * n.d. n.d. n.d. n.d. n.d.
S. altissima Stems .04 ± .02 * .35 ± .05 * .15 ± .05 .47 ± .38 n.d. .06 ± .06 .34 ± .26 1.26 ± .51 *
S. altissima Roots n.d. n.d. * .07 ± .00 * 5.07 ± .66 * .14 ± .02 .64 ± .07 2.90 ± .21 * 2.30 ± .26 *
S. barbata Leaves .03 ± .00 * n.d. * n.d. * n.d. n.d. n.d. n.d. n.d.
S. barbata Stems n.d. * n.d. * n.d. n.d. n.d. n.d. .03 ± .02 n.d.
S. barbata Roots n.d. n.d. * 1.49 ± .04 * 2.59 ± .49 * .08 ± .01 .34 ± .18 3.88 ± .34 1.77 ± .30 *
S. parvula Leaves 25.34 ± .72 * .79 ± .09 * .22 ± .03 .07 ± .03 .59 ± .11 * n.d. .07 ± .07 .06 ± .02 *
S. parvula Stems 5.37 ± .41 * .91 ± .31 * .34 ± .14 * .17 ± .08 .37 ± .03 * .03 ± .03 1.24 ± .09 * .10 ± .05
S. parvula Roots .40 ± .08 * .18 ± .04 * .12 ± .03 5.35 ± .94 * 1.86 ± .31 * 2.18 ± .33 5.62 ± 1.10 n.d. *
S. racemosa Leaves .12 ± .01 * n.d. * .34 ± .29 7.59 ± 1.95 * 14.39 ± 2.58 * 29.39 ± 8.00 * .02 ± .02 n.d.
S. racemosa Stems .03 ± .03 * n.d. * .24 ± .22 6.36 ± 2.13 * 5.09 ± 1.09 * 15.17 ± 4.14 * .44 ± .03 * .45 ± .19 *
S. racemosa Roots n.d. n.d. * .08 ± .02 * 6.98 ± 1.20 * .44 ± .11 2.02 ± .38 1.82 ± .06 * 1.60 ± .25 *
S. tournefortii Leaves .49 ± .02 * 4.69 ± .97 * .53 ± .13 * n.d. n.d. n.d. n.d. .06 ± .06
S. tournefortii Stems .01 ± .01 * .15 ± .04 .28 ± .07 * .08 ± .04 n.d. .06 ± .03 .13 ± .05 2.43 ± .66 *
S. tournefortii Roots n.d. .08 ± .04 * .13 ± .06 .93 ± .08 * .08 ± .01 1.24 ± .14 2.33 ± .20 * 7.61 ± .15 *
S. wrightii Leaves .82 ± .17 * .13 ± .09 * .78 ± .18 * 1.21 ± .63 .10 ± .02 * .16 ± .05 * n.d. n.d.
S. wrightii Stems .99 ± .37 * 2.23 ± .11 * 3.10 ± 1.31 * 29.90 ± .92 * 2.24 ± .43 * 14.87 ± .26 * .62 ± .07 * .40 ± .11 *
S. wrightii Roots n.d. n.d. * 18.10 ± 1.37 * 43.99 ± 9.53 1.17 ± .38 * 4.22 ± 1.17 * 3.13 ± .53 3.09 ± .87 *
Open in a new tab

Note: Units for all flavones are μmol/g fresh weight, except for isoscutellarein 8‐G, which has units of peak area. Data are presented as mean ± standard error, as calculated from samples taken in biological triplicate.

Abbreviations: HPLC, High Performance Liquid Chromatography; n.d., not detected.

*

p value < .05 relative to S. baicalensis for the same organ and flavone.

a

Data have units of peak area.

Our root‐specific flavone profiling indicated that the 4′‐deoxyflavone pathway appears to be very well‐conserved across all seven species. We detected at least six of eight tested 4′‐deoxyflavones in the root of each species (Figure 2c; Table 1). Interestingly, although chrysin is proposed to serve as a precursor for all other 4′‐deoxyflavones, we found it only in the root of S. parvula, and its glycosylated form, chrysin 7‐G, in the roots of three species, S. baicalensis, S. parvula, and S. tournefortii. On the other hand, we observed the accumulation of baicalein, baicalin, oroxylin A, and oroxyloside in the roots of all seven species (Figure 2c; Table 1). S. wrightii was the only species to accumulate a significantly greater amount (p < .05, Student's t test) of baicalin and oroxyloside in its roots than S. baicalensis. We also detected significantly elevated (p < .05) levels of baicalein and oroxylin A in the roots of S. wrightii as compared to S. baicalensis (Figures 2c and 3; Table 1). Chrysin can also be converted to wogonin and wogonoside through the reaction of SbF8H and Sb8‐OM (Figure 1). We detected both wogonin and wogonoside in the roots of all species except for S. parvula, where we did not detect wogonoside. S. tournefortii was the only species to accumulate a significantly greater (p < .05) amount of wogonoside in its roots than S. baicalensis, and no species accumulated a significantly greater amount of wogonin (Figure 2c; Table 1). Finally, the absence of 4′‐hydroxyflavones in the roots of all but one species (S. parvula) indicates their specificity to the aerial organs of the plant in most species we selected (Figure 2c).

FIGURE 3.

FIGURE 3

Open in a new tab

Organ‐specific (a) oroxylin A and (b) oroxyloside concentrations in seven Scutellaria species, as determined via High Performance Liquid Chromatography (HPLC). Concentrations were averaged from tissue samples taken from three biological replicates, and error bars represent standard error. Asterisk (*) indicates p value < .05 relative to S. baicalensis for the same organ and flavone

Aerial tissue‐specific flavone profiles of the selected species were much more varied than root‐specific profiles (Figure 2a,b). First, of the six 4′‐hydroxyflavones we selected, we detected two to four in the leaves of all species analyzed and one to five in their stems. We were unable to detect hispidulin, or its glucoside, hispiduloside, in the leaves or stems of two species, S. altissima and S. tournefortii. Hispiduloside was particularly rare, and out of all tissue samples taken, we only detected it in the stems of S. racemosa (Figure 2a,b; Table 1). Although these more advanced steps in the biosynthetic pathway may not be well conserved, the accumulation of scutellarin in the aerial tissues of all seven species indicates at least partial retention of 4′‐hydroxyflavone biosynthesis in these species (Figure 2a,b). Apigenin is a biosynthetic precursor of all other selected 4′‐hydroxyflavones (Figure 1). Interestingly, it was scarcely accumulated, as we detected apigenin in the aerial tissues of only two species, S. baicalensis and S. parvula. This pattern is analogous to the scarce accumulation of chrysin in our root tissue samples.

In addition to 4′‐hydroxyflavones, we observed that several species accumulate one to eight of the selected 4′‐deoxyflavones in their aerial parts. Remarkably, the leaves of all seven species accumulated chrysin, and except for S. barbata, the stems of all species also accumulated chrysin. This widespread accumulation of chrysin in the aerial parts is strikingly different from its scarcity in the roots (Figure 2). Of note, S. parvula, S. racemosa, and S. wrightii accumulated significantly higher (p < .05) concentrations of oroxylin A in their stems and leaves as compared to S. baicalensis (Figure 3; Table 1). S. racemosa and S. wrightii also accumulated significantly higher (p < .05) concentrations of oroxyloside in their stems and leaves than S. baicalensis (Figure 3; Table 1). Overall, our detection of chrysin in the leaves of all species analyzed and baicalein in stems and leaves of most species suggests that specificity of 4′‐deoxyflavones to roots is less than that of 4′‐hydroxyflavones to aerial tissues.

3.2. The structural elucidation of a new scutellarin isomer

During our metabolite analysis, we detected multiple new metabolites which we were unable to unambiguously assign their identities. Of these unknown metabolites, one drew our interest because of its pattern of accumulation across the tissue samples we collected (Figure 4). In our HPLC chromatograms, we detected the peak corresponding to this metabolite in the aerial parts of S. baicalensis and S. barbata, but not in S. racemosa. The peak was absent in root chromatograms collected from all seven species. The aerial specificity of this unknown metabolite led us to hypothesize that it was a 4′‐hydroxyflavone. To elucidate its structure, we analyzed the unknown metabolite from our S. barbata leaf extracts by the liquid chromatography‐high resolution mass spectrometry (LC‐HRMS). Interestingly, its molecular weight was identical to scutellarin ([M + H]+ m/z 463.0866, calculated for C21H19O12 +, 463.0871), but they were eluted with different retention times (t m = 6.28 min for scutellarin vs. 6.94 min for the unknown compound) (Figure 5a). Furthermore, they gave rise to the same major MS/MS fragment, suggesting them to be two isomers (Figure S3).

FIGURE 4.

FIGURE 4

Open in a new tab

Comparison of chromatograms collected via HPLC from S. barbata , S. baicalensis , and S. racemosa stems. Time interval displayed was selected to center the unknown peak in the chromatograms

FIGURE 5.

FIGURE 5

Open in a new tab

Characterization of a new scutellarin isomer. (a) Standard scutellarin and isolated compound were eluted with different retention times in LC‐HRMS analysis. (b) Key NMR correlations of isolated compound. 1D‐NOESY and1D‐ROESY correlations are represented by red and green two‐way arrows, respectively (left). A three‐bond HMBC correlation from H‐1″ to C‐8 (right)

To further elucidate the structure of this compound, we performed 1D and 2D NMR analysis (Figures S4–S6). Comparison of its 1H and 13C chemical shifts to those of scutellarin allowed the assignment of d‐glucuronide (C1″ to 6″), 1,4‐disubstituted benzene ring (C1′ to 6′), and the carbons on the flavone ring (Jiang et al., 2016) (Figure 5b; Table S1). Based on the 1H chemical shift and coupling constant of the anomer proton H‐1″ (J = 7.86 Hz), the glucuronyl moiety was determined to be on the β configuration (Ko et al., 2018). Compared with scutellarin, the aromatic proton at δH 6.99 (1H, s) was initially assigned to H‐8 of the flavone. However, according to 1D‐selective nuclear overhauser effect spectroscopies (NOESY, resonance frequency at 6.28 or 12.81 ppm) and an 1D‐selective rotating frame overhauser enhancement spectroscopy (ROESY, resonance frequency at 12.81 ppm), OH‐5 correlates with H‐3 (δ = 6.83), and a proton at δ = 6.29 (Figure 5b), leading to the assignment of this proton at position 6 (δ = 6.29 ppm). This assignment was further supported by the weak NOE effects of H‐6 with OH‐5 and H‐1″ on the glucuronyl moiety, which further indicated the proximity of the glucuronyl moiety at position 7 or 8. H‐1″ also showed weak NOE effects with H2′ and H6′ on the 1,4‐disubstituted benzene ring, suggesting the potential configuration of the compound, where the glucuronyl moiety could be close to the aromatic system. According to an HMBC correlation from H1″ to C8 of the flavone, we then definitely assigned the glucuronyl moiety at position 8 (Figure 5b). Collectively, our 1D and 2D NMR analysis revealed the isolated compound as isoscutellarein 8‐O‐β‐glucuronopyranoside (isoscutellarein 8‐G). Comparison with the reported 1H and 13C chemical shifts of this compound (Billeter et al., 1991) confirmed this structural determination (Table S2).

After confirming the identity of this unknown metabolite as isoscutellarein 8‐G, we then quantified its relative abundance in all organ‐specific tissue samples we collected (Figure 6; Table 1). Isoscutellarein 8‐G was accumulated only in the aerial parts of all species, matching the pattern which we had previously observed for 4′‐hydroxyflavones including scutellarin. We detected isoscutellarein 8‐G in the stems of S. altissima, and in the stems and leaves of S. baicalensis, S. barbata, and S. tournefortii. S. barbata accumulated the greatest overall concentration of isoscutellarein 8‐G in its stems and leaves at levels significantly greater (p < .05) than that of S. baicalensis. In contrast, S. parvula, S. racemosa, and S. wrightii accumulated no isoscutellarein 8‐G in their aerial parts. It is noteworthy that these three species accumulated oroxylin A and its glycoside in their aerial parts (Figure 3).

FIGURE 6.

FIGURE 6

Open in a new tab

Organ‐specific isoscutellarein 8‐glucuronide peak areas in seven Scutellaria species, as determined via High Performance Liquid Chromatography (HPLC). Peak areas were averaged from tissue samples taken from three biological replicates, and error bars represent standard error. Asterisk (*) indicates p value < .05 relative to S. baicalensis for the same organ and flavone

4. DISCUSSION

From our analysis of organ‐specific flavone diversity, we determined profiles for S. baicalensis and S. barbata, which matched closely with previous publications (Xu et al., 2020; Zhao et al., 2016). In these flavone profiles, high concentrations of 4′‐deoxyflavones accumulated in the roots, and much lower concentrations of 4′‐deoxyflavones and 4′‐hydroxyflavones accumulated in the stems and leaves (Figure 2; Table 1). As described by Zhao et al. (2016), the root‐favored accumulation of 4′‐deoxyflavones by S. baicalensis is due to root‐specific overexpression of several enzyme isoforms with activity exclusively, or near exclusively in 4′‐deoxyflavone biosynthesis (Zhao et al., 2016). In contrast to the pattern we observed in S. baicalensis and S. barbata, we identified that S. racemosa and S. wrightii accumulated higher concentrations of 4′‐deoxyflavones in their aerial parts as compared to their roots (Figure 2; Table 1). Also, all seven species accumulated chrysin and/or chrysin 7‐glucuronide in their leaves (Figure 2a; Table 1). This suggests that the expression of 4′‐deoxyflavone enzyme isoforms is not perfectly root‐specific and some enzymes having activities towards 4′‐deoxyflavone precursors such as SbCLL‐7 and SbCHS‐2 may be active in both roots and aerial parts at least under our growth conditions. It is also possible that some fraction of 4′‐deoxyflavones are synthesized in the roots and then transported to the aerial parts. The fact that 4′‐hydroxyflavones were not detected in roots of most species indicates the selectivity of enzymes towards either 4′‐deoxyflavones or 4′‐hydroxyflavones (or their respective precursors), as well as organ‐specific regulation of biosynthetic gene expression.

We found that S. racemosa accumulates the highest concentrations of oroxylin A, and its 7‐glucuronide, oroxyloside, in its leaves, among all organs of all species (Figure 3; Table 1). S. wrightii also accumulated notable amounts of oroxylin A and oroxyloside in its stem, but not in its leaves. Oroxylin A is a 4′‐deoxyflavone which has been demonstrated to exhibit memory enhancement and neuroprotective effects in rat models (Jeon et al., 2011, 2012). The most likely route for oroxylin A biosynthesis is the methylation of baicalein at its 6‐OH group (Figure 1) (Elkin et al., 2018). Although previous works have identified a variety of O‐methyltransferases (OMTs) in plants, the enzymes with high specificity for the 6‐OH group in flavonoids are rare, as this reaction is biochemically unfavorable (Zhang et al., 2016). Work in sweet basil (Ocimum basilicum), a species also in the Lamiaceae family with Scutellaria, identified a methyltransferase capable of specific methylation of the 6‐OH group of scutellarein (Berim et al., 2012). Scutellarein is a 4′‐hydroxyflavone identical in structure to baicalein apart from its 4′‐OH group. To ensure the proper orientation of its substrate, and thus its regioselectivity, the O. basilicum OMT uses a threonine residue to form a hydrogen bond with the 4′‐OH group of scutellarein. However, as baicalein has no 4′‐OH group, it would be impossible for a regioselective OMT in S. racemosa or S. wrightii to rely on this interaction during the methylation of baicalein. Research by Zhang et al. (2016) in a liverwort species (Plagiochasma appendiculatum) identified a methyltransferase (PaF6OMT) that is capable of methylation of the 6‐OH group in baicalein. As this OMT has not yet been structurally characterized, how it achieves its specificity remains unknown. Future work in S. racemosa and S. wrightii should be directed towards characterizing its biosynthesis of oroxylin A, with specific attention paid to the potential specialization of OMTs in the pathway. Overall, S. racemosa and S. wrightii are promising targets for biotechnology improvement due to the significant bioactive effects of oroxylin A and oroxyloside. Considering that both species occur in warm area (Texas and South America) (Krings & Neal, 2001; Nelson & Goetze, 2010), accumulation of oroxylin A and oroxyloside in these species may indicate the physiological relevance of oroxylin A and oroxyloside in these species.

Isoscutellarein 8‐G was first detected in the liverwort species Marchantia berteroana (Markham & Porter, 1975). Following this initial report, Miyaichi, Imoto, et al. (1988) and Miyaichi, Kizu, et al. (1988) detected the flavone in the aerial parts of S. indica and S. baicalensis. Aside from these works, few other studies have reported isoscutellarein 8‐G in Scutellaria, though several have detected its aglycone and 7‐O‐glycosylated forms (Karimov & Botirov, 2017). This rarity in detection may be a result of its low abundance relative to other glycosylated flavones in Scutellaria. A potential reason for this low abundance is its unique glycosylation at the 8‐O position. Flavone 7‐O glycosylation is more common in Scutellaria due to the presence of a hydroxyl group at the 7‐O position in all flavones synthesized via the core flavone pathway (Figure 1). On the other hand, 8‐O glycosylation first requires the activity of an 8‐hydroxylase to add the free hydroxyl group to which the carbohydrate will be attached. As the purpose of glycosylation is typically to increase the stability of the flavone for long term storage (Slámová et al., 2018), it is possible that 8‐O glycosylation provides slightly greater stability as compared to 7‐O glycosylation. Therefore, it would be preferable to glycosylate isoscutellarein at the 8‐O position, even though a free hydroxyl group is also present at the 7‐O position. Several species may have evolved regioselective glycosyltransferase enzymes for this purpose. Researchers working with a glycosyltransferase from Bacillus cereus demonstrated that a single amino acid substitution could alter the primary site of quercetin glycosylation with high specificity (Chiu et al., 2016). Perhaps, a similar mutation occurs in several Scutellaria species to allow the biosynthesis of isoscutellarein 8‐G. Alternatively, it is possible that the glycosyltransferase enzymes of these species which accumulate isoscutellarein 8‐G have less strict regioselectivity, and are capable of glycosylation at both 7‐G and 8‐G positions. Quantification of isoscutellarein 7‐G alongside isoscutellarein 8‐G would provide valuable insight regarding these theories. Based on current understanding of flavone biosynthesis, we propose a possible route of isoscutellarein and isoscutellarein 8‐G production from apigenin (Figure S7). Further organ‐specific transcriptome study is required to identify enzymes responsible for of isoscutellarein and isoscutellarein 8‐G production.

Our quantification of isoscutellarein 8‐G across the seven Scutellaria species we analyzed revealed an intriguing pattern. Isoscutellarein 8‐G was entirely absent in the species of S. parvula, S. racemosa, and S. wrightii, all of which accumulate high concentrations of 4′‐deoxyflavones such as oroxylin A and oroxyloside in their aerial parts. This specific example is representative of a broader pattern—species with high accumulation of 4′‐deoxyflavones in their aerial parts accumulated low concentrations of 4′‐hydroxyflavones. This substitution of 4′‐hydroxyflavones with 4′‐deoxyflavones potentially indicates an evolution to utilize 4′‐deoxyflavones to fulfill the physiological roles which 4′‐hydroxyflavones do in other species. Works in species outside of Scutellaria have demonstrated the anti‐herbivory effects of several 4′‐hydroxyflavones we quantified here (Gallon et al., 2019; Sosa et al., 2004). However, little is known about the physiological role that 4′‐deoxyflavones play in plants. Further research should be devoted to exploring the role of 4′‐deoxyflavones in plant growth and stress response to better understand the evolutionary advantage their biosynthesis and accumulation offers.

AUTHOR CONTRIBUTIONS

B.C.A., Y.D., and J.K. designed the research project; B.C.A., D.L., G.M.R, and Y.S. performed the experiments and analyzed the data; B.C.A., Y.D., and J.K. wrote the manuscript.

CONFLICT OF INTEREST

The authors declare no competing interest.

Supporting information

Figure S1. Proposed 4′‐hydroxyflavone and 4′‐deoxyflavone pathway with structures of glycosylated flavones included. Enzyme names in blue are specific isoforms that have been identified in Styloscolex baicalensis , and enzyme names in black are general names. Flavones that were quantified have names in bold and are numbered to match the labeling of Figure 2.

Figure. S2. UV spectra of (a) apigenin, (b) apigenin 7‐G, (c) scutellarein, (d) scutellarin, (e) hispidulin, (f) hispiduloside, (g) chrysin, (h) chrysin 7‐G, (i) baicalein, (j) baicalin, (k) oroxylin A, (l) oroxyloside, (m) wogonin, and (n) wogonoside, and (p) isoscutellarein 8‐G. All spectra collected from standards except for isoscutellarein 8‐G, which was collected from isolated fraction sent for further analysis.

Figure S3. MS (a), and MS/MS (b) spectra of standard scutellarin and isolated compound.

Figure S4. 1H NMR spectrum of isolated compound (600 MHz, DMSO‐d6). Water signals were suppressed by presaturation.

Figure S5. 13C NMR spectrum of isolated compound (151 MHz, DMSO‐d6).

Figure S6. 2D NMR spectra of isolated compound. (a) 1H‐1H COSY; (b) HSQC; (c) HMBC; (d) 2D‐NOESY, in which positive and negative contours are highlighted in gray and red, respectively.

Figure S7. Proposed pathway for biosynthesis of isoscutellarein 8‐glucuronide in Scutellaria.

Table S1. Comparison of 1H (600 MHz, DMSO‐d6) chemical shifts of unknown compound with those previously published for scutellarin1.

Table S2. Comparison of 1H (600 MHz, DMSO‐d6) and 13C (151 MHz, DMSO‐d6) chemical shifts of unknown compound with those previously published for isoscutellarein 8‐O‐β‐glucuronopyranoside2. Table S3. Voucher information for the species used in this study.

Click here for additional data file. (1.1MB, docx)

ACKNOWLEDGMENTS

This work was supported by the United States Department of Agriculture (USDA)‐National Institute of Food and Agriculture Hatch project (005681), a startup fund from the Horticultural Sciences Department and Institute of Food and Agricultural Sciences at the University of Florida to J. K, and by National Institutes of Health (NIH, R35 GM128742) to Y.D. NMR studies were performed in the McKnight Brain Institute at the National High Magnetic Field Laboratory's AMRIS Facility, which is supported by the National Science Foundation Cooperative Agreement No. DMR‐1644779, the State of Florida, and an NIH award, S10RR031637. We thank John B. Nelson at A.C. Moore Herbarium and the late William Mark Whitten at the UF for collecting S. racemosa in the field. We also thank Dr. Sangtae Kim for the discussion and Dr. Swathi Nadakuduti for scutellarin standard.

Askey, B. C. , Liu, D. , Rubin, G. M. , Kunik, A. R. , Song, Y. H. , Ding, Y. , & Kim, J. (2021). Metabolite profiling reveals organ‐specific flavone accumulation in Scutellaria and identifies a scutellarin isomer isoscutellarein 8‐O‐β‐glucuronopyranoside. Plant Direct, 5(12), e372. 10.1002/pld3.372

Contributor Information

Yousong Ding, Email: yding@cop.ufl.edu.

Jeongim Kim, Email: jkim6@ufl.edu.

REFERENCES

  1. Berim, A. , Hyatt, D. C. , & Gang, D. R. (2012). A set of regioselective O‐methyltransferases gives rise to the complex pattern of Methoxylated flavones in sweet basil. Plant Physiology, 160, 1052–1069. 10.1104/pp.112.204164 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Billeter, M. , Meier, B. , & Sticher, O. (1991). 8‐hydroxyflavonoid glucuronides from Malva sylvestris. Phytochemistry, 30, 987–990. 10.1016/0031-9422(91)85292-8 [DOI] [Google Scholar]
  3. Chen, V. , Staub, R. E. , Fong, S. , Tagliaferri, M. , Cohen, I. , & Shtivelman, E. (2012). Bezielle selectively targets mitochondria of cancer cells to inhibit glycolysis and OXPHOS. PLoS ONE, 7, e30300. 10.1371/journal.pone.0030300 [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chiu, H.‐H. , Hsieh, Y.‐C. , Chen, Y.‐H. , Wang, H.‐Y. , Lu, C.‐Y. , Chen, C.‐J. , & Li, Y.‐K. (2016). Three important amino acids control the regioselectivity of flavonoid glucosidation in glycosyltransferase‐1 from Bacillus cereus. Applied Microbiology and Biotechnology, 100, 8411–8424. 10.1007/s00253-016-7536-2 [DOI] [PubMed] [Google Scholar]
  5. Cole, I. B. , Saxena, P. K. , & Murch, S. J. (2007). Medicinal biotechnology in the genus scutellaria. Vitro Cellular & Developmental Biology ‐ Plant, 43, 318–327. 10.1007/s11627-007-9055-4 [DOI] [Google Scholar]
  6. Elkin, Y. N. , Kulesh, N. I. , Stepanova, A. Y. , Solovieva, A. I. , Kargin, V. M. , & Manyakhin, A. Y. (2018). Methylated flavones of the hairy root culture Scutellaria baicalensis. Journal of Plant Physiology, 231, 277–280. 10.1016/j.jplph.2018.10.009 [DOI] [PubMed] [Google Scholar]
  7. Gallon, M. E. , Silva‐Junior, E. A. , Amaral, J. G. , Lopes, N. P. , & Gobbo‐Neto, L. (2019). Natural products diversity in plant‐insect interaction between Tithonia diversifolia (Asteraceae) and Chlosyne lacinia (Nymphalidae). Molecules, 24, 3118. 10.3390/molecules24173118 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hasaninejad, M. , Jamzad, Z. , & Yousefi, M. (2009). Nutlet micro‐morphology in Scutellaria L.(Lamiaceae) in Iran. Iranian Journal of Botany, 15, 227–239. [Google Scholar]
  9. Jeon, S. J. , Bak, H. , Seo, J. , Han, S. M. , Lee, S. H. , Han, S.‐H. , Kwon, K. J. , Ryu, J. H. , Cheong, J. H. , Ko, K. H. , Yang, S. I. , Choi, J. W. , Park, S. H. , & Shin, C. Y. (2012). Oroxylin a induces BDNF expression on cortical neurons through adenosine A2A receptor stimulation: A possible role in neuroprotection. Biomolecules & Therapeutics (Seoul), 20, 27–35. 10.4062/biomolther.2012.20.1.027 [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Jeon, S. J. , Rhee, S. Y. , Seo, J. E. , Bak, H. R. , Lee, S. H. , Ryu, J. H. , Cheong, J. H. , Shin, C. Y. , Kim, G.‐H. , Lee, Y. S. , & Ko, K. H. (2011). Oroxylin a increases BDNF production by activation of MAPK–CREB pathway in rat primary cortical neuronal culture. Neuroscience Research, 69, 214–222. 10.1016/j.neures.2010.11.008 [DOI] [PubMed] [Google Scholar]
  11. Jiang, Z. , Yang, J. , Jiao, Y. , Li, W. , Chai, X. , Zhang, L. , Jiang, M. , & Wang, Y. (2016). Determination of scutellarin in breviscapine preparations using quantitative proton nuclear magnetic resonance spectroscopy. Journal of Food and Drug Analysis, 24, 392–398. 10.1016/j.jfda.2015.12.004 [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Karimov, A. M. , & Botirov, E. K. (2017). Structural diversity and state of knowledge of flavonoids of the Scutellaria L. Russian Journal of Bioorganic Chemistry, 43, 691–711. 10.1134/S1068162017070068 [DOI] [Google Scholar]
  13. Kato, M. J. , Yoshida, M. , & Gottlieb, O. R. (1992). Flavones and lignans in flowers, fruits and seedlings of Virola venosa. Phytochemistry, 31, 283–287. 10.1016/0031-9422(91)83055-P [DOI] [Google Scholar]
  14. Ko, J.‐H. , Nam, Y. H. , Joo, S.‐W. , Kim, H.‐G. , Lee, Y.‐G. , Kang, T. H. , & Baek, N.‐I. (2018). Flavonoid 8‐O‐glucuronides from the aerial parts of Malva verticillata and their recovery effects on alloxan‐induced pancreatic islets in zebrafish. Molecules, 23, 833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Krings, A. , & Neal, J. C. (2001). South American skullcap (Scutellaria racemosa: lamiaceae) in the southeastern United States. SIDA Contributions to Botany, 19, 1171–1179. [Google Scholar]
  16. Markham, K. R. , & Porter, L. J. (1975). Isoscutellarein and hypolaetin 8‐glucuronides from the liverwort Marchantia berteroana. Phytochemistry, 14, 1093–1097. 10.1016/0031-9422(75)85194-6 [DOI] [Google Scholar]
  17. Miyaichi, Y. , Imoto, Y. , Saida, H. , & Tomimori, T. (1988). Studies on the constituents of Scutellaria species X: On the flavonoid constituents of the leaves of Scutellaria baicalensis GEORGI. Japanese Journal of Pharmacognosy, 42, 216–219. [Google Scholar]
  18. Miyaichi, Y. , Kizu, H. , Tomimori, T. , & Lin, C.‐C. (1988). Studies on the constituents of Scutellaria species XI: On the flavonoid constituents of the aerial parts of Scutellaria indica L. Chemical & Pharmaceutical Bulletin (Tokyo), 37, 794–797. 10.1248/cpb.37.794 [DOI] [Google Scholar]
  19. Nelson, A. D. , & Goetze, J. R. (2010). Hydroballochory in two Texas species of Skullcap (Scutellaria drummodii, S. wrightii; Lamiaceae). Texas Journal of Science, 62, 263‐270. [Google Scholar]
  20. Rao, V. M. , Damu, G. L. V. , Sudhakar, D. , & Rao, C. V. (2009). Two new bio‐active flavones from Grangea maderaspatana (Artemisia maderaspatana). Asian Journal of Chemistry, 21, 1552–1558. [Google Scholar]
  21. Rao, Y. K. , Reddy, M. V. B. , Rao, C. V. , Gunasekar, D. , Blond, A. , Caux, C. , & Bodo, B. (2002). Two new 5‐deoxyflavones from Albizia odoratissima. Chemical & Pharmaceutical Bulletin (Tokyo), 50, 1271–1272. 10.1248/cpb.50.1271 [DOI] [PubMed] [Google Scholar]
  22. Saralamma, V. V. G. , Lee, H. J. , Hong, G. E. , Park, H. S. , Yumnam, S. , Raha, S. , Lee, W. S. , Kim, E. H. , Sung, N. J. , Lee, S. J. , et al. (2017). Korean Scutellaria baicalensis Georgi flavonoid extract induces mitochondrially mediated apoptosis in human gastric cancer AGS cells. Oncology Letters, 14, 607–614. 10.3892/ol.2017.6184 [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Shang, X. , He, X. , He, X. , Li, M. , Zhang, R. , Fan, P. , Zhang, Q. , & Jia, Z. (2010). The genus Scutellaria an ethnopharmacological and phytochemical review. Journal of Ethnopharmacology, 128, 279–313. 10.1016/j.jep.2010.01.006 [DOI] [PubMed] [Google Scholar]
  24. Slámová, K. , Kapešová, J. , & Valentová, K. (2018). “Sweet flavonoids”: Glycosidase‐catalyzed modifications. International Journal of Molecular Sciences, 19, 2126. 10.3390/ijms19072126 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sosa, T. , Chaves, N. , Alias, J. C. , Escudero, J. C. , Henao, F. , & Gutiérrez‐Merino, C. (2004). Inhibition of mouth skeletal muscle relaxation by flavonoids of Cistus ladanifer L.: A plant defense mechanism against herbivores. Journal of Chemical Ecology, 30, 1087–1101. 10.1023/B:JOEC.0000030265.45127.08 [DOI] [PubMed] [Google Scholar]
  26. Sutter, R. D. , Govus, T. E. , Smyth, R. L. , Nordman, C. , Pyne, M. , & Hogan, T. (2011). Monitoring change in a central US calcareous glade: Resampling transects established in 1993. Natural Areas Journal, 31, 163–172. 10.3375/043.031.0209 [DOI] [Google Scholar]
  27. Tao, G. , & Balunas, M. J. (2016). Current therapeutic role and medicinal potential of Scutellaria barbata in traditional Chinese medicine and Western research. Journal of Ethnopharmacology, 182, 170–180. 10.1016/j.jep.2016.02.012 [DOI] [PubMed] [Google Scholar]
  28. Tao, Y. , Zhan, S. , Wang, Y. , Zhou, G. , Liang, H. , Chen, X. , & Shen, H. (2018). Baicalin, the major component of traditional Chinese medicine Scutellaria baicalensis induces colon cancer cell apoptosis through inhibition of oncomiRNAs. Scientific Reports, 8, 14477. 10.1038/s41598-018-32734-2 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Tsai, C.‐C. , Lin, C.‐S. , Hsu, C.‐R. , Chang, C.‐M. , Chang, I.‐W. , Lin, L.‐W. , Hung, C.‐H. , & Wang, J.‐L. (2018). Using the Chinese herb Scutellaria barbata against extensively drug‐resistant Acinetobacter baumannii infections: In vitro and in vivo studies. BMC Complementary and Alternative Medicine, 18, 96. 10.1186/s12906-018-2151-7 [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Xu, Z. , Gao, R. , Pu, X. , Xu, R. , Wang, J. , Zheng, S. , Zeng, Y. , Chen, J. , He, C. , & Song, J. (2020). Comparative genome analysis of Scutellaria baicalensis and Scutellaria barbata reveals the evolution of active flavonoid biosynthesis. Genomics, Proteomics & Bioinformatics, 18, 230–240. 10.1016/j.gpb.2020.06.002 [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Zhang, Y.‐Y. , Xu, R.‐X. , Gao, S. , & Cheng, A.‐X. (2016). Enzymatic production of oroxylin A and hispidulin using a liverwort flavone 6‐O‐methyltransferase. FEBS Letters, 590, 2619–2628. 10.1002/1873-3468.12312 [DOI] [PubMed] [Google Scholar]
  32. Zhao, Q. , Cui, M.‐Y. , Levsh, O. , Yang, D. , Liu, J. , Li, J. , Hill, L. , Yang, L. , Hu, Y. , Weng, J.‐K. , Chen, X. Y. , & Martin, C. (2018). Two CYP82D enzymes function as flavone hydroxylases in the biosynthesis of root‐specific 4′‐deoxyflavones in Scutellaria baicalensis. Molecular Plant, 11, 135–148. 10.1016/j.molp.2017.08.009 [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Zhao, Q. , Yang, J. , Cui, M.‐Y. , Liu, J. , Fang, Y. , Yan, M. , Qiu, W. , Shang, H. , Xu, Z. , Yidiresi, R. , Weng, J. K. , Pluskal, T. , Vigouroux, M. , Steuernagel, B. , Wei, Y. , Yang, L. , Hu, Y. , Chen, X. Y. , & Martin, C. (2019). The reference genome sequence of Scutellaria baicalensis provides insights into the evolution of Wogonin biosynthesis. Molecular Plant, 12, 935–950. 10.1016/j.molp.2019.04.002 [DOI] [PubMed] [Google Scholar]
  34. Zhao, Q. , Zhang, Y. , Wang, G. , Hill, L. , Weng, J.‐K. , Chen, X.‐Y. , Xue, H. , & Martin, C. (2016). A specialized flavone biosynthetic pathway has evolved in the medicinal plant, Scutellaria baicalensis. Science Advances, 2, e1501780. 10.1126/sciadv.1501780 [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Zhao, T. , Tang, H. , Xie, L. , Zheng, Y. , Ma, Z. , Sun, Q. , & Li, X. (2019). Scutellaria baicalensis Georgi. (Lamiaceae): A review of its traditional uses, botany, phytochemistry, pharmacology and toxicology. The Journal of Pharmacy and Pharmacology, 71, 1353–1369. 10.1111/jphp.13129 [DOI] [PubMed] [Google Scholar]
  36. Zhu, D. , Wang, S. , Lawless, J. , He, J. , & Zheng, Z. (2016). Dose dependent dual effect of Baicalin and herb Huang Qin extract on angiogenesis. PLoS ONE, 11, e0167125. 10.1371/journal.pone.0167125 [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1. Proposed 4′‐hydroxyflavone and 4′‐deoxyflavone pathway with structures of glycosylated flavones included. Enzyme names in blue are specific isoforms that have been identified in Styloscolex baicalensis , and enzyme names in black are general names. Flavones that were quantified have names in bold and are numbered to match the labeling of Figure 2.

Figure. S2. UV spectra of (a) apigenin, (b) apigenin 7‐G, (c) scutellarein, (d) scutellarin, (e) hispidulin, (f) hispiduloside, (g) chrysin, (h) chrysin 7‐G, (i) baicalein, (j) baicalin, (k) oroxylin A, (l) oroxyloside, (m) wogonin, and (n) wogonoside, and (p) isoscutellarein 8‐G. All spectra collected from standards except for isoscutellarein 8‐G, which was collected from isolated fraction sent for further analysis.

Figure S3. MS (a), and MS/MS (b) spectra of standard scutellarin and isolated compound.

Figure S4. 1H NMR spectrum of isolated compound (600 MHz, DMSO‐d6). Water signals were suppressed by presaturation.

Figure S5. 13C NMR spectrum of isolated compound (151 MHz, DMSO‐d6).

Figure S6. 2D NMR spectra of isolated compound. (a) 1H‐1H COSY; (b) HSQC; (c) HMBC; (d) 2D‐NOESY, in which positive and negative contours are highlighted in gray and red, respectively.

Figure S7. Proposed pathway for biosynthesis of isoscutellarein 8‐glucuronide in Scutellaria.

Table S1. Comparison of 1H (600 MHz, DMSO‐d6) chemical shifts of unknown compound with those previously published for scutellarin1.

Table S2. Comparison of 1H (600 MHz, DMSO‐d6) and 13C (151 MHz, DMSO‐d6) chemical shifts of unknown compound with those previously published for isoscutellarein 8‐O‐β‐glucuronopyranoside2. Table S3. Voucher information for the species used in this study.

Click here for additional data file. (1.1MB, docx)

Articles from Plant Direct are provided here courtesy of Wiley

RESOURCES