Headspace Volatiles of Scutellaria baicalensis

Jeobp 12 (4) 2009 pp 435 - 442

435

ISSN 0972-060X

Headspace Volatiles of Scutellaria baicalensis Georgi Flowers Gary R. Takeoka 1*, David M. Rodriguez 2, Lan Dao 1 and Robert Patterson 2 1

Western Regional Research Center, U. S. Department of Agriculture, 800 Buchanan St., Albany, CA 94710, USA 2 Department of Biology, S. F. State University, 1600 Holloway Avenue, San Francisco, CA 94132, USA Received 20 March 2009; accepted in revised form 18 May 2009

Abstract: Volatile constituents of Baikal skullcap (Scutellaria baicalensis Georgi) flowers were isolated by solid-phase microextraction (SPME) and analyzed by GC and GC-MS. A total of 64 constituents was identified (constituting 57.1 - 89.9 % of the total area), 13 of which were tentatively identified. β-Caryophyllene (22.3 - 41.5 %), germacrene D (12.4 - 27.5 %), δ-cadinene (3.1 - 5.4 %), γ-muurolene (1.9 - 3.4 %), γ-cadinene (1.6 - 3.1 %), α-humulene (1.6 - 2.6 %), αcopaene (1.4 - 2.3 %), α-muurolene (1.0 - 2.6 %), bicyclogermacrene (1.1 - 2.1 %) and 3-octanone (0.9 - 3.0 %) were the major constituents of the flower volatiles. A small amount (0.5 %) of the uncommon volatile, 2-(methylamino)benzaldehyde was detected in one of the samples. Key Word Index: Scutellaria baicalensis, Lamiaceae, headspace volatiles, βcaryophyllene, germacrene D, 2-(methylamino)benzaldehyde, grape-like odor. Introduction: The genus Scutellaria L. (Lamiaceae) is widely distributed throughout the world and comprises about 300 species 1. They are pubescent herbs growing 20 - 60 cm tall with opposite, simple, entire, lanceolate and ciliate leaves, and flowers in a one-sided raceme 2. Scutellaria baicalensis Georgi is a perennial herb whose roots are used in traditional Chinese medicine (TCM) to clear heat, dry dampness and stop bleeding 3. S. baicalensis has been shown to inhibit the growth of human cancer cells in vitro and its inhibitory effect on these cells is probably mediated through inhibiting PGE2 production 4. The roots have also been used to treat bacterial and viral infections of the respiratory and gastrointestinal tract, reduce total cholesterol and decrease blood pressure 5. Twenty-nine flavonoids, eight flavone glycosides and two chalcones have been identified in S. baicalensis roots 3. S. baicalensis roots contain much higher concentrations of the flavones, baicalin (19335 vs. 1163 μg/g dry weight), baicalein (410 vs. 243 μg/g dry *Corresponding author (Gary R. Takeoka) E- mail: < [email protected] >

Gary R. Takeoka et al. / Jeobp 12 (4) 2009 pp 435 - 442

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weight), scutellarein (810 vs. 553 μg/g dry weight) and chrysin (526 vs. 51 μg/g dry weight) than the aerial parts 6. Rosselli et al.7 reported that S. rubicunda Hornem subsp. linnaeana (Caruel) Rech. oil contained mainly oxygenated monoterpenes (44.5 %) and sesquiterpenes (39.0 %) with the main constituents being β-caryophyllene (28.7 %), linalool (27.8 %), αterpineol (6.7 %), cayophyllene oxide (4.2 %), nerol (4.2 %), hexadecanoic acid (2.5 %) and α-cedrol (2.3 %). The oil contained smaller amounts of phenolic compounds (5.1 %) with 4-vinylguaiacol (3.1 %) being the main phenolic constituent. The composition of volatiles in the oil of various species of Scutellaria was recently reviewed 8. The essential oil of S. baicalensis roots has been reported to possess a medicinal odor as well as an aromatic floral odor with a sweet note 9. The major constituents of the essential oil were acetophenone, (E)-4-phenyl-3-buten-2-one, 1-phenyl-1,3-butanedione, palmitic acid and oleic acid 9. The flowers of S. baicalensis possess a Concord grape aroma. The aim of this study was to characterize the constituents responsible for its pleasant aroma. Experimental Scutellaria baicalensis flowers: Scutellaria baicalensis plants and seeds were obtained from Horizon Herbs (Williams, OR), Chinese Medicinal Plants (Petaluma, CA) and UC Berkeley Botanical Gardens (Berkeley, CA). The plants were grown in a greenhouse (Horizon Herbs and UC Berkeley Botanical Gardens) and outside (Chinese Medicinal Plants) at San Francisco State University. Voucher specimens were deposited at the Harry Thiers Herbarium, San Francisco State University. The purple flowers were picked in August 2006 and sampled on the same day. Solid-phase microextraction (SPME) of S. baicalensis flower volatiles: Whole flowers (15 - 18 flowers, no leaves or stems) were placed in a 240 mL screw top amber glass bottle sealed with a PTFE/silicone septum (22 mm diameter × 2.54 mm thickness). Bottles, screw caps and septa were purchased from Supelco (Bellefonte, PA). Manual SPME fiber holder and SPME fiber assembly (divinylbenzene/Carboxen/polydimethylsiloxane, DVB/CAR/PDMS, 50/30 μm coating, 2 cm length, StableFlex) were also obtained from Supelco. Prior to extraction, the fiber was heated in the injector port (230°C) of a HewlettPackard model 6890 gas chromatograph for 15 min. The SPME fiber was exposed in the upper space of the sealed bottle for 1 h (GC-FID and GC-MS) at room temperature. After sampling, the fiber was withdrawn and immediately introduced into the GC-MS or GCFID injector for desorption and analysis. Capillary gas chromatography: A Hewlett-Packard model 5890 Series II gas chromatograph equipped with a 60 m × 0.32 mm i.d. (df = 0.25 μm) DB-1 bonded-phase fused-silica capillary column (J&W Scientific, Folsom, CA) and a flame ionization detector (FID) were used for determination of Kováts indices and % area. The injector and detector temperatures were 180°C and 250°C, respectively. Narrow bore (0.75 mm i.d.) injector port liners were used. The oven temperature was programmed from 30°C (4 min. isothermal) to 200°C held for 15 min. at final temperature) at 2°C/min. The linear velocity of the helium


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carrier gas was 38 cm/s (30°C). Splitless injections were employed with a purge time of 1 min. Capillary gas chromatography-mass spectrometry: The system consisted of an Agilent 6890 gas chromatograph coupled to an Agilent 5973N quadrupole mass spectrometer (capillary direct interface). The quadrupole, transfer line and ion source temperatures were 130 °C, 200 °C and 170 °C, respectively. A 60 m × 0.25 mm i.d. (df = 0.25 μm) DB-1 bonded-phase fused-silica capillary column was used. Helium carrier gas was used at a column head pressure of 22 psi. Narrow bore (0.75 mm i.d.) injector port liners were used. The oven temperature was programmed from 30 °C (4 min isothermal) to 200 °C (held for 15 min at final temperature) at 2 °C/min. Splitless injections were employed with a purge time of 1 min. Mass spectra were generated at 70 eV at a scan range of m/z 35-350. Preliminary identifications were made using BenchTop/PBM (Palisade Corporation, Ithaca, NY) equipped with the Identification of Essential Oil Components by Gas Chromatography/ Mass Spectrometry, 4th Ed. library 10, and Agilent ChemStation equipped with The Wiley Registry of Mass Spectral Data, 8th Edition. MassFinder 3 (Dr. Hochmuth Scientific Consulting, Hamburg, Germany) was also used to identify constituents. Positive identifications were made by comparison of the compound’s Kováts index, I11 and mass spectrum of a reference standard. Results and discussion: Scutellaria baicalensis flowers from three sources were picked in full bloom. The flower sample from Horizon Herbs had a Concord grape odor and possessed the most pleasant odor of the three samples. The sample from UC Berkeley Botanical Gardens had a weaker Concord grape odor and also had an unpleasant sour aroma. The grape-like odor was much fainter in the sample from Chinese Medicinal Plants. This sample had the strongest sour odor and was the least pleasant of the three samples. Volatiles were isolated by SPME and analyzed by GC and GC-MS on the same day. Flower volatiles from Horizon Herbs (3.0 × 106 total area counts) and Chinese Medicinal Plants (2.3 × 106 total area counts) had similar total FID area counts and chromatographic patterns whereas the sample from UC Berkeley Botanical Gardens (4.5 × 105 total area counts) had much lower quantity of headspace volatiles and a different chromatographic profile than the other two samples. Volatile constituents of fresh S. baicalensis flowers were identified by comparison of the compound’s Kováts index, I11 and mass spectrum of a reference standard. A total of 64 constituents were identified (constituting 57.1 - 89.9 % of the total area), 13 of which were tentatively identified (Table 1). The % area values presented are preliminary and should only be considered as approximate since extraction efficiencies and response factors were not determined. 3-Octanone has been previously identified in alfalfa (Medicago sativa) flowers 12 and lavender (Lavandula angustifolia L.) flowers 13. (E)-4,8-dimethyl-1,3,7nonatriene is a tetra-norsesquiterpene 1 4,15,16, that is very widespread in scented plant species 17, 18. The rare natural product, 2-(methylamino)benzaldehyde was first identified in the flowers of Coryanthes mastersiana and C. picturata by Gerlach and Schill 19, 20. The highly diverse species C. mastersianum that belongs to the section Coryanthes has a floral


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scent dominated by 2-(methylamino)benzaldehyde which represents at least 80 % of the total volatiles 18. It is also the dominant headspace constituent of C. speciosa, C. kaiseriana, and C. picturata flowers. This compound has a peculiar dusty, leathery and sweet odor and is highly typical for species from the section Coryanthes of the genus Coryanthes 18. The related compound, 2-aminobenzaldehyde, has a tortilla aroma though it a weaker odorant with an odor threshold more than 50 times higher than 2-aminoacetophenone, the compound responsible for much of the characteristic odor of tortillas 21. Though 2-(methylamino)benzaldehyde possesses a grape-like odor, it was only present in one of the three samples and only at a level of 0.5 %. Its contribution to the grape-like odor of S. baicalensis flowers therefore seems unlikely. The flower scent contained large proportions of β-caryophyllene (22.3 - 41.5 %). High levels of β-caryophyllene (56.5 %) were found in the floral scent of S. californica 8. Similarly, large amounts (29.4 %, 22.5 %, 15.0 %, and 14.2 %, respectively) were reported in S. galericulata 22, S. churchilliana Fernald 23, S. pinnatifia A. Hamilt. ssp. alpina (Bornm.) Rech.24 and S. sieberi 25 oils. High percentages of germacrene D (12.4 - 27.5 %) were found in the floral scent. Ghannadi and Mehregan 24, Takeoka et al.8, and Lawrence 23 found large amounts of germacrene D (39.7 %, 6.9 % and 6.2 %, respectively) in S. albida ssp. albida oil, S. californica floral scent, and S. churchilliana Fernald oil. Sesquiterpene hydrocarbons comprised the majority of S. baicalensis flower volatiles (55.3 - 81.4 %) with additional abundant constituents being δ-cadinene (3.1 - 5.4 %), γmuurolene (1.9 - 3.4 %), γ-cadinene (1.6 - 3.1 %), α-humulene (1.6 - 2.6 %), α-copaene (1.4 - 2.3 %), α-muurolene (1.0 - 2.6 %), and bicyclogermacrene (1.1 - 2.1 %). These results are consistent with those of Ghannadi and Mehregan 24 and Takeoka et al 8, who reported that sesquiterpene hydrocarbons (83.1 % and 79.1 %, respectively) were the main constituents of S. pinnatifia A. Hamilt. ssp. alpina (Bornm.) Rech. oil and S. californica floral scent. Sesquiterpenes (78.3 %) were also the main constituents of S. lateriflora oil 26. In contrast, the oils of S. albida ssp. albida, S. barbata and S. rupestris ssp. adenotricha were much different as the former did not contain any sesquiterpene hydrocarbons 27 while the latter two only contained 2.9 % and 6.6 % sesquiterpene hydrocabons, respectively 2 5, 28 . There was a major unknown sesquiterpene hydrocarbon (19.1 % of the total headspace volatiles) in the flower sample from UC Berkeley Botanical Gardens. The compound had a Kováts retention index of 1302 on DB-1 and the following mass spectrum: 204 (100), 189 (64), 175 (59), 161 (21), 149 (25), 148 (38), 147 (30), 133 (32), 119 (44), 106 (34), 105 (46), 91 (47), 77 (16), 55 (10), 41 (15). The unknown comprised 1.7 % of the total headspace volatiles in the sample from Horizon Herbs but was not present in the sample from Chinese Medicinal Plants. Eleven other sesquiterpene hydrocarbons were present that we were unable to identify.

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Table 1. Percent composition of headspace constituents of Scutellaria baicalensis flowers.

IDB-1 Constituent

Ethyl acetate 2-Butanol 1-Penten-3-ol Methyl 2-methylpropanoate 3-Hydroxy-2-butanone (E)-2-Methyl-2-butenal Dimethyl disulfide 2-Butyl acetate Ethyl 2-methylpropanoate 2-Methylpropyl acetate 3-Hexanol Octane 2-Heptanone Hexanol 2-Methylbutyl acetate α-Thujene α-Pinene Sabinene 3-Octanone 3-Octanol (Z)-3-Hexenyl acetate δ-3-Carene p-Cymene 1,8-Cineole Limonene trans-β-Ocimene γ-Terpinene Terpinolene

exp.

601 592 672 675 684 720 726 750 751 764 784 800 872 860 868 925 931 963 968 983 989 1002 1009 1017 1019 1039 1048 1077 (3-(4-Methyl-3-pentenyl)furane) [perillene]1085 1-Octen-3-yl acetate 1095 (E)-4,8-Dimethyl-1,3,7-nonatriene 1104 Methyl octanoate 1106 3-Octyl acetate 1110 ((E)-Ectocarpene) 1142 Methyl salicylate 1168 2-(Methylamino)benzaldehyde 1232

A

% aread B

0.1 0.1 tr.f tr. tr. tr. tr. tr. tr. 3.0 1.2 tr. tr. tr. 0.1 0.1 0.1 tr. 1.7 tr. 0.1 0.5

0.3 0.2 0.4 0.9 -

ref.

601 596 671 674 674 715 722 750 751 764 784 800 872 860 869 922 929 964 969 980 986 1004 1010 1018 1020 1037 1048 1077 1090 1096 1104 1103 1110 1147 1166 1231

C 2.0 0.4 0.4 0.2 tr. 0.2 tr. tr. 0.2 0.1 0.5 0.4 0.3 2.7 0.8 tr. tr. tr. tr. 0.1 tr. tr. 0.1 tr. 0.1 -


442

table 1. (continued)

IDB-1 Constituent

exp.

ref. A

Geranial α-Cubebene α-Ylangene α-Copaene β-Bourbonene β-Cubebene (1,5-di-epi-β-Bourbonene) (Isocaryophyllene) β-Ylangene β-Caryophyllene (β-Copaene) Aromadendrene (Cadina-3,5-diene) α-Humulene (cis-Muurola-4(15), 5-diene) (7αH,10βH-Cadina-1(6),4-diene) γ-Muurolene Germacrene D (γ-Amorphene) Bicyclogermacrene α-Muurolene (δ-Amorphene) γ-Cadinene Calamenene δ-Cadinene (Zonarene) (Cadin-1,4-diene) (α-Cadinene) Total identified (%)

1240 1347 1369 1374 1381 1385 1385 1402 1409 1417 1424 1435 1440 1448 1456 1467 1470 1476 1485 1489 1492 1498 1504 1508 1514 1516 1523 1529

1241 1347 1370 1374 1382 1385 1390 1409 1415 1415 1430 1436 1448 1449 1462 1472 1469 1474 1492 1489 1492 1499 1505 1508 1514 1521 1523 1534

0.1 1.2 0.5 1.4 0.9 0.6 0.1 0.2 22.3 1.0 0.8 0.6 1.7 2.3 0.3 3.4 27.5 0.7 2.1 2.6 1.0 3.1 0.2 5.4 tr. 0.3 0.8 87.6

% aread B

C

1.0 0.2 2.3 0.5 0.4 23.1 0.8 0.6 0.4 1.6 1.1 0.2 2.0 12.4 0.4 1.2 1.0 0.6 1.6 0.1 3.2 0.2 0.4 57.1

0.9 0.3 1.5 6.4 0.6 0.1 41.5 1.1 0.6 0.3 2.6 1.6 0.2 1.9 13.0 0.4 1.1 1.4 0.5 1.6 0.1 3.1 tr. 0.2 0.4 89.9

A: S. baicalensis (Horizon Herbs) B: S. baicalensis (UC Berkeley Botanical Gardens) C: S. baicalensis (Chinese Medicinal Plants) d Peak area percentage of total FID area (assuming all response factors of 1). e Tentative identifications (in parentheses) assigned based on mass spectra and IDB-5 reported in MassFinder 3 (Dr. Hochmuth Scientific Consulting, Hamburg, Germany). f tr. indicates an area of less than 0.1%.