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. Author manuscript; available in PMC: 2013 Feb 27.
Published in final edited form as: Phytochem Anal. 2009 Jul-Aug;20(4):298–306. doi: 10.1002/pca.1127

Comparison of the Phenolic Component Profiles of Skullcap (Scutellaria lateriflora) and Germander (Teucrium canadense and T. chamaedrys), a Potentially Hepatotoxic Adulterant

Long-Ze Lin a,*, James M Harnly a, Roy Upton b
PMCID: PMC3583524  NIHMSID: NIHMS322961  PMID: 19402188

Abstract

Introduction

Scutellaria lateriflora, commonly known as skullcap, is used as an ingredient in numerous herbal products. Unfortunately, it has occasionally been adulterated with Teucrium canadense or T. chamaedrys, commonly known as germander, which contains potentially hepatotoxic diterpenes. Chromatographic profiles of the phenolic components provide a means of distinguishing between these plants and enhancing public safety.

Objective

To develop a chromatographic method for the identification of Scutellaria lateriflora and two Teucrium species and to quantify the latter as adulterants.

Methodology

Samples were extracted with aqueous methanol and the extracts were analysed using a standardised LC-DAD-ESI/MS profiling method to obtain their phenolic profiles.

Results

Skullcap contained primarily flavonoids, while the major phenolic components of the two Teucrium species were the phenylethanoids, verbascoside and teucrioside. Using the phenylethanoids as markers, it was possible to clearly distinguish between the two genus and to determine 5% Teucrium mixed with Scutellaria using either ultraviolet absorption spectrometry or mass spectrometry in the total ion count mode. Using MS in the selective ion monitoring (SIM) mode, 1% Teucrium could be measured.

Conclusions

This study showed that chromatographic profiling was able to identify Scutellaria and Teucrium, separately and when mixed together.

Keywords: Scutellaria lateriflora, two Teucrium species, flavonoids, phenylethanoids, diterpenoids, LC-MS phenolic component profiles

Introduction

Skullcap (or mad dog skullcap), Scutellaria lateriflora L. (Lamiaceae) is a botanical medicine and supplement that is used as an ingredient in numerous herbal supplements that are available over the counter. S. lateriflora has a long history of use in Western herbal medicine as a nervine, to quiet and support the nervous system and to reduce anxiety, sleeplessness and various types of spasms (Rafinesque, 1830; King, 1866; Mills and Bone, 2000; Sarris, 2007; Wojcikowski et al., 2007). The single clinical trial testing its efficacy for relieving anxiety was based on a non-validated, subjective assessment scale (Wolfson and Hoffmann, 2003). Pre-clinical data have suggested pharmacological mechanisms (e.g. GABA and serotonin receptor modulation) that may be partially responsible for Scutellaria's putative effects (Awad et al., 2003; Gafner et al., 2003b; Xu et al., 2006).

S. lateriflora has been subjected to adulteration with various species of germander, Teucrium canadense L. and T. chamaedrys L. (Lamiaceae), which contain potentially hepatotoxic neoclerodane diterpenes such as teucrin (Fig. 1) (Bedir et al., 2003; Sundaresan et al., 2006; Rader et al., 2007). The hepatotoxicity of Teucrium arises from the bioactivation of teucrin A by cytochrome P450 to create reactive metabolites (Kouzi et al., 1994; Haouzi et al., 2000). The data suggest that oxidation of the furan ring is necessary for hepatotoxicity. The neoclerodane diterpenes in skullcap contain a tetrahydrofurofuran ring rather than the furan rings found in Teucrium (Bruno et al., 1998, 2002; Rosselli et al., 2004). The main phenolic components of T. canadense and T. chamaedrys are the phenylethanoid glycosides (Fig. 1; Sticher and Lahloub, 1982; Gafner et al., 2003a; Kadifkova-Panovska et al., 2005; Serrilli et al., 2007).

Figure 1.

Figure 1

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Structures of the phenylethanoids and diterpenoids of T. canadense and T. chamaedrys.

The purpose of this study was to develop analytical methodology that could be used to compare S. lateriflora and Teucrium using polyphenols and/or diterpenes. Phenolic profiles were acquired using high performance liquid chromatography with diode array and electrospray ionisation/mass spectrometric detection (LC-DAD-ESI/MS) (Lin and Harnly, 2007). Seventy phenolic components were identified in S. lateriflora and Teucrium spp. The chromatographic profiles showed clear differences between the two genus and the three species.

Experimental

Standards and chemicals

Luteolin (5,7,3′,4′-tetrahydroxyflavone), luteolin 7-O-glucoside, apigenin (5,7,4′-trihydroxyflavone), baicalein (5,6,7-trihydroxyflavone) and baicalin (baicalein 7-O-glucuronide) were purchased from Sigma Chemical Co. (Saint Louis, MO, USA). Wogonin (5,7-dihydroxy-8-methoxyflavone), scutellarein (5,6,7,4′-tetrhadroxyflavone), scutellarin (scutellarein 7-O-glucuronide), verbascoside and isoverbascoside were purchased from Chroma-Dex Inc. (Irvine, CA, USA). Luteolin 7-O-rutinoside, diosmetin (5,7,3′-trihydroxy-4′-methoxyflavone), diosmin (diosmetin 7-O-rutinoside) and chrysin (5,7-dihydroxyflavone) were purchased from Extrasynthese (Genay, Cedex, France).

Formic acid, hydrochloric acid (37%) and HPLC-grade solvents (acetonitrile and methanol) were purchased from VWR Scientific (Seattle, WA, USA). HPLC-grade water was prepared from distilled water using a Milli-Q system (Millipore Lab., Bedford, MA, USA).

Plant materials

Nine samples of the aerial parts of S. lateriflora (SL), four samples of Teucrium canadense (TCA) and seven samples of T. chamaedrys (TCH) were obtained from the American Herbal Pharmacopoeia (AHP). All the samples, except one, were dried plant materials. The exception was sample SL P120, which was in the form of a tincture prepared from 1.0 g of the plant materials extracted with 5.0 mL of 65% ethanol water. A collection of botanically auth-enticated and commercial materials was obtained. Vouchers for the authenticated materials and retention samples were deposited in the herbarium of the AHP.

Extraction

One hundred milligrams of dried ground sample was mixed with 5.0 mL of methanol–water (60:40, v/v) and sonicated for 60 min using an FS30H sonicator (40 kHz, 100 W) (Fisher Scientific, Pittsburgh, PA, USA) at room temperature. The slurry was centrifuged at 2500 rpm for 15 min (IEC Clinical Centrifuge, Damon/IEC Division, Needham, MA, USA). The supernatant was filtered through a 17 mm (0.45 μm) PVDF syringe filter (VWR Scientific, Seattle, WA, USA) and 50 μL of the extract was injected onto the LC column (Lin and Harnly, 2007).

The tincture was diluted 1:10 (v/v) with the aqueous methanol extraction solvent described above and filtered prior to injection. To avoid errors arising from unexpected degradation of the phenolic compounds, the LC determinations were completed within 24 h of the extraction.

Acid hydrolysed samples

Filtered sample extracts (0.50 mL) were mixed with concentrated HCl (37%, 0.10 mL), and heated in a covered tube at 85°C for 2 h. Then 0.40 mL of methanol was added to the mixture, and sonicated for 10 min. The solution was re-filtered prior to HPLC injection (Lin and Harnly, 2007).

LC-MS conditions

All samples were analysed by LC-DAD-ESI/MS. The instrument consisted of an Agilent 1100 HPLC (Agilent, Palo Alto, CA, USA), coupled sequentially with a DAD, and MS (MSD, SL mode). A Waters (Waters Corp., Milford, MA, USA) Symmetry column (C18, 5 μm, 250 × 4.6 mm) with a sentry guard column (Symmetry, C18, 5 μm, 3.9 × 20 mm) was used at flow rate of 1.0 mL/min. The column oven temperature was set at 25°C. The mobile phase consisted of A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile) and the gradient increased linearly from 10% B initially to 26% B (v/v) at 40 min, to 65% B at 70 min, and finally to 100% B at 71 min and held at 100% B until 75 min. The DAD was set at 280, 310, 330 and 350 nm to provide real-time traces of the chromatograms. The UV–VIS spectra from 190 to 650 nm were recorded for plant component identification. Mass spectra were simultaneously acquired in the positive and negative ionisation (PI and NI) modes at low and high fragmentation voltages (100 and 250 V) over the range of m/z 100–2000. MS data were available in the total ion count (TIC) and selective ion monitoring (SIM) mode (Lin and Harnly, 2007).

Detection of the diterpenoids of the two Teucrium plants (Sundaresan et al., 2006; Fig. 1) was accomplished in the SIM mode by monitoring m/z 329/327 (PI/NI) for teuflin and teucvidin (MW = 328), m/z 345/343 for teucrin A, teuflidin and isoteuflidin (MW = 344), m/z 377/375 for dihydroteugin (MW = 376) and m/z 391/389 for teucrin G (MW = 390). For Scutellaria lateriflora diterpenoids (Bruno et al., 1998), m/z 469/467 for scutelcyprol A (MW = 468), m/z 493/491 for scutellaterin A and scutecyprol A (MW = 492), m/z 535/533 for scutelaterin B (MW = 534), m/z 551/549 for ajugapitin (MW = 550) and m/z 553/551 for scutelaterin C (MW = 552) were monitored.

Results and Discussion

General characteristics of the phenolic components of Scutellaria and Teucrium

Figures 2–4 show the LC profiles of S. lateriflora, T. canadense and T. chamaedrys recorded at 350 nm. Tables 1–3 summarise the chromatographic data, i.e. retention times (tR), wavelength of maximum absorbance (λmax), protonated/deprotonated parent molecules ([M + H]+/[M−H]−), and major fragment ions (including aglycones, [A + H]+/[A−H]−), and the peak identification.

Figure 2.

Figure 2

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Chromatograms (350 nm) of 5 S. lateriflora samples: (A) SL 2322; (B) SL 2492; (C) SL 811; (D) SL 482; and (E) SL P120.

Figure 4.

Figure 4

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Chromatograms (350 nm) of 7 T. chamaedrys (TCH) samples: (A) TCH 2272; (B) TCH 2490; (C) TCH2311; (D) TCH 2708; and (E) TCH 137.

Table 1. Flavonoids of Scutellaria lateriflora.

Peak. no. tR (min) [M + H]+/[M–H]–(m/z) PI/NI aglycone, other ion (m/z) UV*λmax (nm) Identification
1 23.56 —/251 —/— 314 n.d.
2 23.31 479/477 303/301 282, 336 Pentahydroxyflavone 7-O-glucuronide
3 25.26 433/431 313/311 270, 332 Vitexina
4 28.85 465/463 289/287 286, 360sh Dihydroxyscutellarein 7-O-glucuronideb
5 29.37 463/461 287/285 282, 332 Scutellarein 7-O-glucuronideba
6 31.57 463/461 287/285 274, 336 Ikonnikosideb
7 33.64 463/461 287/285 n.d. Tetrahydroxyflavone 7-O-glucuronide
8 34.56 —/293 —/— 314 n.d.
9 36.37 447/445 271/269 270, 338 Apigenin 7-O-glucuronidea
10 38.44 477/475 301/299 272, 332 Trihydroxymethoxyflavone O-glucuronide
11 40.58 417/415 271/269 272, 314sh Baicalein 7-O-rhamnoside
12 42.77 447/445 271/269 278, 316 Baicalinba
13 46.03 477/475 301/299 274, 332 Lateriflorinb
14 46.71 449/447 273/271 242, 282, 364sh 5,6,7-Trihydroxyflavanone 7-O-glucuronideb
15 47.61 447/445 271/269 280, 360sh Hydroxymethoxyflavanone O-glucuronide
16 47.98 477/475 301/299 284, 350sh Hydroxyldimethoxyflavanone glucuronide
17 48.51 461/459 285/283 272, 312sh Oroxylin A 7-O-glucoronideb
18 49.73 507/505 331/329 278, 338sh Hydroxyl-trimethoxyflavanone glucuronide
19 50.28 461/459 285/283 274, 342sh Dimethoxy or flavanone glucoronide
20 50.83 463/461 287/285 288, 342sh Dihydrowogonin 7-O-glucuronide
21 51.70 491/489 315/313 272, 336sh Trimethoxy or flavanone glucuronide
22 52.24 345/343 —/— 282, 346sh Tetramethoxy or flavanone
23 54.74 271/269 —/— 276, 322 Bailcaleinba
24 55.22 715/713 271/269 n.d. n.d.
25 56.85 715/713 539/537 280, 318sh n.d.
26 58.55 287/285 —/— 292, 364sh Dihydroxymethoxyflavanone
27 61.54 285/283 —/— 268, 338 Wogoninba
28 62.56 315/313 —/— 272, 334 n.d.
29 63.08 285/283 —/— 270, 318 Oroxylin Ab
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a

Identified by comparison with a standard or reference compound.

b

Previously reported in the plant.

n.d., Not determined or unidentified.

Table 3. Verbascoside and teucrioside content in Teucrium.

Sample Verbascoside (% dry weight) Sample Teucrioside (% dry weight)
TCA2312a 0.23 TCH2490 0.45
TCA2312 0.69 TCH2311 0.88
TCA420 5.5 TCH2722 0.94
TCA407 9.6 TCH138 1.47
TCH137 1.56
TCH2490 2.02
TCH2708 2.30
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In general, peak identification was made by analysis of the recorded data listed in the tables. Both positive and provisional (isomers not specified) identification are provided for the main flavonoids. Positive identification was possible by comparison of data for specific peaks to data for standards or by comparison of the data in the tables with that reported in the literature (Harborne and Baxter, 1999; Gafner et al., 2003a, b; Bergeron et al., 2005). Confirmation of the identity of the aglycones was obtained by repeating the chromatographic analysis after hydrolysis of the samples.

The primary flavonoids found in Scutellaria were flavones and flavanones, which were easily distinguished by their UV spectra. For example, the UV spectra of the flavanones (e.g. dihydroxy-scutellarein 7-O-glucuronide, Table 1, peak 4) have a λmax around 286–290 nm (band II) with a shoulder between 340 and 360 nm (band I). This is different from the flavones (e.g. scutellarein 7-O-glucuroide, Table 1, peak 5), which have a λmax at 282 and 336 nm.

Glucuronides of the flavones and flavanones were the major flavonoid components of Scutellaria. The glucuronides were easily distinguished using in-source collision-induced dissociation with a high fragmentation voltage. The glucuronides contributed 176 amu to the molecular weight of the aglycones. In general, they behaved similarly to other glycosides. They were not, however, completely hydrolysed using the conditions normally used for hydrolysis of flavonoid glycosides (Lin and Harnly, 2007).

S. lateriflora samples

Figure 2 shows the chromatographic profiles of five samples of aerial parts of S. lateriflora (SL): (A) SL 2322, (B) SL 2492, (C) SL 811, (D) SL 482 and (E) SL P120 (an aqueous ethanol tincture). The first four [Fig. 2(A–D)] represent typical chromatograms of the extracts and agree well with four other samples (SL 1050, SL 1057, SL 2341 and SL 2491) that are not shown.

To date, 10 flavonoids have been positively identified in S. lateriflora (Gafner et al. 2003a, b). In this study, identification of dihydroxyscutellarein 7-O-glucuronide (Table 1, peak 4), scutellarein 7-O-glucuronide (scutellarin) (peak 5), ikonnikoside I (5,6,7,2′-tetrahydroxyflavone 7-O-glucuronide) (peak 6), baicalin (baicalein 7-glucuronide) (peak 12), lateriflorin (5,6,7-trihydroxy-2′-methoxyflavone 7-O-glucuronide) (peak 13), 5,6,7-trihydroxyflavanone 7-O-glucuronide (peak 14), oroxylin A 7-O-glucuronide (peak 17), baicalein (peak 23), wogonin (peak 27) and oroxylin (or oroxylin A, 5,7-dihydroxy-6-methoxyflavone) (peak 29) was based on previously reported data. Some of the flavonoids (peaks 3, 5, 12, 23 and 27) were identified by direct comparison with standards as indicated in Table 1. Vitexin (apigenin C8-glucoside, peak 3) has not been previously reported in this plant. Three previously reported flavonoids (scutellarein, 5,6,7,2′-tetrahydroxyflavone, and 5,6,7-trihydroxy-2′-methoxyflavone) were not detected in the extract at this concentration and injection volume.

All of the confirmed S. lateriflora samples [Fig. 2(A–D) and the five samples not shown] showed similarly characteristic profiles. In each, baicalin (peak 12) was the dominant flavonoid (greatest peak area) with scutellarein 7-O-glucuronide (peak 5), lateriflorin (peak 13), ikonnikoside (peak 6) and 5,6,7-trihydroxyflavanone 7-O-glucuronide (peak 14) also providing strong peaks.

Two Teucrium species

Figures 3 and 4 show the chromatographic profiles for T. canadense and T. chamaedrys samples, respectively. The data are summarised and identifications are provided in Table 2. The phenylethanoids, verbascoside and teucrioside, have been previously reported (Sticher and Lahloub, 1982; Gafner et al., 2003a; Serrilli et al., 2007) and were easily identified by their UV and MS data. The presence of verbascoside and its isomer isoverbascoside was confirmed by direct comparison to standards. Other phenylethanoid glycosides, cinnamic acid derivatives and flavonoids were identified as shown in Table 2.

Figure 3.

Figure 3

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Chromatograms (350 nm) of five T. canadense samples: (A) TCA 420; (B) TCA 407; (C) TCA 2312; (D) TCA 2312a.

Table 2. Phenolic compounds and diterpenoids of Teucrium canadense and T. chamaedrys.

Peak. no. tR (min) [M + H]+/[M–H]–(m/z) PI/NI aglycone, other ion (m/z) UV*λmax(nm) Identification
Teucrium canadense phenolic compounds (Fig. 3)
1 7.63 —/353 —/191,179,161,135 242,298sh,330 3-Caffeoylquinic acida
2 12.62 —/353,451 —/242 298sh,330 n.d.
3 18.52 —/639 —/— 242,298sh,330 Phenylethanoid glycoside
4 19.12 —/639 —/— 242,298sh,330 Phenylethanoid glycoside
5 25.73 —/653 —/— 242,298sh,330 Phenylethanoid glycoside
6 27.45 —/623 —/— 242,298sh,330 Verbascosidea
7 29.66 609/607 —/— 252,266,342 Flavone glucuronide
8 30.71 —/623 —/— 242,298sh,330 Isoverbascosidea
9 31.78 609/607 301/299 274,334 Trihydroxymethoxyflavone glycoside
10 32.59 639/637 331/329 268,330 Dimethoxytrihydroxyflavone glycoside
11 33.44 609/607 301/299 256,274,344 Trihydroxymethoxyflavone glycoside
12 36.36 447/445 271/269 268,338 Apigenin 7-O-glucuronidea
13 37.28 477/475 301/299 252,266,348 Trihydroxymethoxyflavone glucuronide
14 38.46 447/445 271/269 268,338 Trihydroxyflavone 7-glucuronide
15 40.26 637/635 285/283 268, 336 Dihydroxymethoxyflavone di-glucuronide
16 46.15 491/489 315/313 250,268,348 Dihydroxydimethoxyflavone glycoside
17 47.33 n.d. n.d. n.d. n.d.
18 50.16 461/459 285/283 266,344 Dihydroxymethoxyflavone 7-glucuronide
Teucrium canadense diterpenoids (Fig. 5)
1 60.89 329/— —/— — Teuflin
2 61.90 329/— —/— — Teucvidin
Teucrium chamaedrys phenolic compounds (Fig. 4)
1 11.23 —/353 —/191,179,161,135 216,298sh,330 5-Caffeoylquinic acida
2 14.92 —/179 —/— 216,298sh,326 Caffeic acida
3 17.76 —/771 —/— 216,298sh,330 Phenylethanoid glycoside
4 18.54 —/771 —/— 216,298sh,330 Phenylethanoid glycoside
5 23.19 611/609 287/285 252,268,342 Luteolin 7-O-diglucoside
6 24.16 787/785 287/285 252,268,342 Luteolin 7-O-glycoside
7 24.48 787/785 287/285 252,268,342 Luteolin 7-O-glycoside
8 25.21 581/579 287/285 252,268,342 Luteolin 7-O-pentosylglucoside
9 25.78 595/593 287/285 252,268,342 Luteolin 7-O-rutinoside
10 26.29 —/755 —/— 242,298sh,330 Teucreoside
11 27.37 —/755 —/— 242,298sh,330 Teucreoside isomer
12 27.69 —/623 —/— 242,298sh,330 Verbascosidea
13 29.85 757/755 287/285 252,268,342 Luteolin 7-O-glycoside
14 30.67 653/651 287/285 252,268,346 Luteolin 7-O-glycoside
15 32.20 771/769 301/299 282,330 Diosmetin 7-O-glycoside
16 33.27 609/607 301/299 252,268,342 Diosmetin 7-O-rutinoside
17 34.32 449/447 287/275 268,334 Tetrahydroxyflavone 7-O-glycoside
18 36.59 447/445 271/269 266,336 Apigenin 7-O-glucuronidea
19 37.80 653/651 287/285 276,304,328 Tetrahydroxyflavone glycoside
20 38.29 667/665 301/299 252,268,346 Diosmetin 7-O-glycoside
21 45.71 593/591 285/283 268,328 Dihydroxymethoxyflavone glycoside
22 47.43 287/275 —/— 268,334 Luteolina
23 49.19 667/665 301/299 278,306,328 Diosmetin 7-O-glycoside
24 52.50 n.d. n.d. 266,340 n.d.
25 53.78 331/329 —/— 256,272,346 Diosmetina
Teucrium chamaedrys diterpenoids (Fig. 5)
3 48.91 377/— —/— — Dihydroteugin
4 51.86 391/— —/— — Teucrin G
5 52.53 345/— —/— — Teucrin A, isoteuflidin, or teuflidin
1 60.89 329/— —/— — Teuflin
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a

Identified by comparison with a standard or reference compound.

n.d., Not determined or un-identified.

The four T. canadense samples (TCA 407, TCA 420, TCA 2312 and TCA 2312a) shown in Fig. 3 have similar phenolic profiles. For each, verbascoside (peak 6) was the largest peak, although the areas varied by as much as a factor of 42 (Table 3). The verbascoside content ranged from 0.23 to 9.66%, by dry weight, with TCA 407 having the highest verbascoside content and TCA 2312a the lowest.

Chromatographic profiles for five (TCH 137, TCH 2272, TCH 2311, TCH 2490 and TCH 2708) of the seven T. chamaedrys samples analysed are presented in Fig. 4. The two samples not shown (TCH 138 and TCH 2311b) had profiles identical to that of TCH 137. All contained teucrioside (peak 10) as their major phenolic compound with peak areas varying by a factor of 5. The teucrioside contents of the seven samples ranged from 0.45 to 2.30%, by dry weight (Table 3).

Verbascoside was used as a standard for the quantitative determination of teucrioside with a correction for the difference in the molecular weights since they are structurally identical except for the sugar at the R1 position. This difference, however, does not influence the absorption coefficient.

The profiles in Fig. 4 show that the same flavonoids appear in all the T. chamaedrys samples but the relative ratio of the individual flavonoid and total flavonoid content to that of teucrioside (based on peak area) varies considerably. TCH 2490 had the highest flavonoid content followed by TCH 2272. Taxonomically, however, the phenolic profiles show that these samples are very similar to each other and are distinguishable from T. canadense and the Scutellaria genus.

MS detection of diterpenoids in Teucrium and Scutellaria lateriflora

The terpenoids are only detectable by MS. This approach has been used previously for the determination of terpenoids in several plants, including the terpene lactones in Ginkgo biloba leaves and products (Lin et al., 2008). The structures of the seven diterpenoids reported in the literature (Sundaresan et al., 2006) for T. canadense and T. chamaedrys are shown in Fig. 1.

Figure 5 shows the characteristic chromatographic profiles, in the TIC and SIM modes, of two Teucrium samples (TCA 420 and TCH 2708) obtained with positive ionisation and a low fragmentation voltage (PI 100 V). The major diterpenoid peaks detected were teuflin (peak 1, tR 60.89 min, [M + H]+ = m/z 329), teucvidin (peak 2, tR 61.90 min, [M + H]+ = m/z 329), dihydroteugin (peak 3, tR 48.91 min, [M + H]+ = m/z 377), teucrin G (peak 4, tR 51.86 min, [M + H]+ = m/z 391) and teucrin A (peak 5, tR 52.53 min, [M + H]+ = m/z 345). Teucrin A may have co-eluted with its isomers (isoteuflidin and teuflidin, MW = 344). With MS detection, the isomers could not be differentiated.

Figure 5.

Figure 5

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Chromatograms of T. canadense and T. chamaedrys samples with MS detection: (A) TCA 420, PI TIC; (B) TCA 420, PI SIM; (C) TCH 2708, PI TIC; and (D) TCH 2708, PI SIM. Peaks 1 and 2 = teuflin (Mr 328) and teucvidin (Mr 328), peak 3 = dihydroteugin (Mr 376), peak 4 = teucrin G (Mr 390), peak 5 = teucrin A (Mr 344), isoteuflidin (Mr 344) and teuflidin (Mr 344).

Five diterpenoids have been reported in S. lateriflora (Bruno et al., 1998, 2002; Rosselli et al., 2004), but none of these compounds were detected in any of the authenticated S. lateriflora or Teucrium samples using TIC or SIM detection. However, in the SIM mode, a small peak was observed for ajugapatin (tR 66.42 min, [M + H]+ = m/z 551) in the S. galericulata sample (SG 2478b) (chromatogram not shown).

Estimation of Teucrium adulteration of Scutellaria lateriflora

The data presented above suggest that chromatographic profiling could be used for detecting adulteration of S. lateriflora by Teucrium. The phenylethanoid glycosides specific to T. canadense and T. chamaedrys, verbascoside (peak 6 in Fig. 3 and P-2 in Fig. 6) and teucrioside (peak 10 in Fig. 4 and P-1 in Fig. 6), respectively, have been previously proposed as possible markers for adulteration (Gafner et al., 2003a). While detection of these peaks in a chromatogram of S. lateriflora would definitely indicate adulteration, the level of adulteration would be difficult to determine. In this study, the verbascoside and teucrioside concentrations in the 11 plants tested varied by factors of 42 and 5, respectively (Table 3).

Figure 6.

Figure 6

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Chromatograms (350 nm) of extracts of S. lateriflora (SL 2322) containing: (A) 5% TCA 2312a; (B) 5% TCA 2312; (C) 1% TCA 407; (D) 5% TCH 2490; (E) 5% TCH 2311; and (F) 1% TCH 2708. Peak P-1 = teucrioside and P-2 = verbascoside, and the numbers for the UV peak area of peaks 1 in (A–C) are 37.28, 113.96 and 319.82, and those for peak 2 in (D–F) are 97.48, 131.10 and 154.82, respectively.

Figure 6 shows chromatographic traces (with UV detection at 350 nm) of S. lateriflora sample 2322 spiked with varying amounts of T. canadense (5% TCA 2312a, 5% TCA 2312 and 1% TCA 407) and T. chamaedrys (5% TCH 2490, 5% TCH 2311 and 1% TCH 2708). The final, diluted concentrations were 0.11, 0.034 and 0.097% (dry weight) for verbascoside and 0.022, 0.045 and 0.023% (dry weight) for teucrioside. In each case, the phenylethanoids were detectable. Similar results were obtained using MS detection in the TIC mode. For both detection systems, the detection limits (see Experimental section) for verbascoside and teucrioside were approximately 0.002%. With MS detection in the SIM mode, the detection limit was approximately 5 times lower, at 0.0004%.

Each of the remaining Scutellaria samples (SL 811, SL 842, SL 1050 and SL 2492) were spiked with either 1% (TCA 407, TCA 420, TCH 137, TCH 138, TCH 2272, TCH 2311b and TCH 2708) or 5% (TCA 2312a, TCA 2312, TCH 2490 and TCH 2311) of the Teucrium samples, depending on the phenylethanoid concentration. In each case, verbascoside and teucrioside were clearly detected using both UV and MS in the TIC mode (chromatograms not shown).

These preliminary results suggest that low levels of adulteration of S. lateriflora by Teucrium could be detected using chromatographic profiling and may be relevant for products consisting of powders. However, further analyses with more samples of varying concentrations of compounds and different degrees of admixtures would strengthen these findings and are necessary to establish a validated method.

Acknowledgments

This research is supported by the Agricultural Research Service of the US Department of Agriculture and an Interagency Agreement with the Office of Dietary Supplements of the National Institutes of Health.

Contract/grant sponsor: Agricultural Research Service of the US Department of Agriculture.

Contract/grant sponsor: Interagency Agreement with the Office of Dietary Supplements of the National Institutes of Health.

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