New Flavonoid Glycosides from Elsholtzia rugulosa Hemsl

Elsholtzia rugulosa Hemsl. is known in China as a local herbal tea, medicinal herb and honey plant. Chemical examination of E. rugulosa led to the isolation of two new flavonoid glycosides, apigenin 4'-O-α-D-glucopyranoside (1) and 5,7,3',4'-tetrahydroxy-5'-C-prenylflavone-7-O-β-D-glucopyranoside (2), together with nine known flavonoids. Their structures were elucidated on the basis of spectroscopic evidence.


Introduction
Elsholtzia rugulosa Hemsl. (Lamicaeae), which is distributed in the Yunnan, Sichuan and Guizhou provinces of China, is known as a local herbal tea, medicinal herb and honey plant [1]. In these regions, the title plant is also widely used by local people in the treatment of colds, headaches, coughs, pharyngitis and fever [2]. Several flavonoids, maltol glycosides and cyanogenic glycosides have been isolated from E. rugulosa [3,4]. The antiviral activities of these flavonoids were also reported [4]. As a part of our systematical investigation of Chinese tea and herbal tea plants, and in the search for biologically active flavonoids from plants sources, a detailed study on ethanol extracts of E. rugulosa was carried out [5,6,7]. This led to the isolation of two new flavonoids glycosides, apigenin 4'-O-α-D-glucopyranoside (1) and 5,7,3',4'-tetrahydroxy-5'-C-prenylflavone 7-O-β-D-glucopyranoside (2), together with nine known flavonoids 3-11. Herein, we present the details of this study.
Compound 1 was obtained as a yellow amorphous powder, and had a molecular formula C 21 H 20 O 10 , derived from its negative HR-FAB-MS (m/z 431.1280 [M-H] -) and 13 C-NMR spectrum. Comparison of the NMR data with those of apigenin [10], and the further 2D-NMR spectral data allow elucidation the structure of compound 1 as shown in Figure 1. The UV spectrum exhibited absorption maxima at 265 nm (band II) and 331 nm (band I), that are characteristic flavone skeleton bands. The IR spectrum of 1 indicated the presence of hydroxyl (3,376 cm -1 ) and carbonyl functions (1,640 cm -1 ). The occurrence of a flavone skeleton in the molecule could be easily deduced from the 1 H-NMR spectrum, in which compound 1 showed the signals for an exchangeable proton at δ 12.95 (1H, s), A 2 B 2 -type aromatic protons at δ 7.94 (d, H2', 6') and 6.92 (d, H3', 5') on B-ring, two doublets at δ 6.43 (d, H6) and 6.82 (d, H8) on A-ring, together with an olefinic proton at δ 6.86 (s, H3) on a flavone C-ring. In addition, the 1 H-NMR also exhibited signals due to one α-glucopyranosyl unit [δ 5.42 (d, J = 3.7 Hz, H1")]. The J value (3.7 Hz) of the anomeric proton indicated the α-configuration of the glucose moiety [16]. This was supported by the IR spectrum showing a strong band at 770, 780 cm -1 , probably due to one glucosyl unit, and the enzymatic hydrolysis displaying the Rf values consistent with those of a standard sample of D-glucose, as well as anomeric carbon signal δ 99.9 (C(1')) of α-D-glucosyl group observed, in accord with those of literature values [17,18]. The 13 C-NMR spectrum of 1 exhibited 21 carbons whose aglycon chemical shift were in good agreement with those of apigenin and the sugar moiety chemical shifts were in good agreement with those of α-D-glucosyl moiety [18]. The attachment of the glucopyranosyl moiety was deduced to be at C-4′ according to glycosylation rule. The conclusion was further confirmed by the HMBC spectrum in which the anomeric proton of the glucopyranosyl moiety at δ 5.42 (d, H1'') showed long range correlation with C(4') (δ 161.1). Therefore, the structure of 1 was determined to be apigenin 4'-O-α-D-glucopyranoside.
Compound 2 was obtained as a pale yellow amorphous powder. The molecular formula C 26 H 28 O 11 was derived by negative ion HR-FAB-MS (m/z: 515.1913 [M-H] -) in combination with the presence of 26 carbon signals in its 13 C-NMR spectrum, and the further 2D-NMR spectral data allow to elucidate the structure of compound 2 as shown in Figure 1.

Plant Material
The aerial parts of E. rugulosa were collected from Yunnan Province, China. The voucher specimen (No. 0215159) was deposited in the KUN Herbarium of Kunming Institute of Botany, Chinese Academy of Sciences.

Extraction and Isolation
Dried plant material (400 g) of E. rugulosa was refluxed four times with ethanol (4.0 L) for 3 h. After removal of the organic solvent under reduced pressure, the aqueous solution afforded precipitates, which were removed by filtration, and the filtrate was partitioned with ethyl ether to yield ethyl ether and aqueous fractions. The aqueous fraction was concentrated to a small volume (120 mL) and applied to a Dianion HP 2MGL column, eluting with H 2 O-MeOH (1:0-0:1) to afford five fractions (fr. [1][2][3][4][5]

Enzymatic hydrolysis of compounds 1 and 2
An aqueous solution of 1 (3 mg) and maltase (1 mg) was incubated at 37 °C for 80 h. The solution was extracted with CHCl 3 and aglycone produced was identified as apigenin by comparison with compound 6 on silica gel TLC using CHCl 3 -MeOH-H 2 O (8:2:0.2), R f = 0.68. The aqueous layer was concentrated to a residue, which was dissolved by water and examined for identification of the component sugar, and D-glucose was identified by direct comparison on silica gel TLC with an authentic sample, using CHCl 3 -MeOH-H 2 O (7:3:0.5). R f = 0.23.
A solution of 2 (2 mg) in H 2 O (1 mL) were treated with crude cellulase (7 mg) at 37 °C for 60 h. The reaction mixture was diluted with H 2 O (2 mL), and extracted with CHCl 3 (3 mL × 2). The aqueous layer was concentrated to a residue, which was dissolved by water and examined for identification of the component sugar, and D-glucose was identified by direct comparison on silica gel TLC with an authentic sample, using CHCl 3 -MeOH-H 2 O (7:3:0.5). R f = 0.23.