New flavonoid glycosides from Elsholtzia rugulosa Hemsl.

Article abstract of DOI:10.3390/molecules14104190

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

Full text of DOI:10.3390/molecules14104190

Molecules 2009, 14, 4190-4196; doi:10.3390/molecules14104190
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molecules
ISSN 1420-3049
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Article
New Flavonoid Glycosides from Elsholtzia rugulosa Hemsl.
Gaimei She 1, Zhiqin Guo 1, Haining Lv 1 and Dongmei She 2,*
1
School of Chinese Pharmacy, Beijing University of Chinese Medicine, Beijing 100102, China;
E-Mails: shegaimei@126.com (G.S.); medicine_guo@163.com (Z.G.);
seagullning@163.com (H.L.)
2
The Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100049,
China
* Author to whom correspondence should be addressed; E-Mails: dongmeishe@yahoo.com.cn;
Tel.: +86-10-84738628; Fax: +86-10-84738611.
Received: 21 September 2009; in revised form: 12 October 2009 / Accepted: 20 October 2009 /
Published: 20 October 2009
Abstract: 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.
Keywords: Elsholtzia rugulos; flavonoid glycosides; apigenin 4'-O-α-D-glucopyranoside;
5,7,3',4'-tetrahydroxy-5'-C-prenylflavone-7-O-β-D-glucopyranoside
1. 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-

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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.
2. Results and Discussion
Repeated column chromatography (CC) of the chlorophyll-free fraction of an ethanol extract of E.
rugulosa on Dianion HP 2MGL, Sephadex LH-20, MCI-gel CHP-20P, and silica gel, resulted in the
isolation of 11 compounds, of which nine known flavonoids were identified as luteolin (3) [8] luteolin
7-O-β-D-glucoside (4) [8], luteolin 3'-O-β-D-glucuronide-6''-methylester (5) [9], apigenin (6) [10],
apigenin 7-O-β-D-glucoside (7) [11], quercetin 3-O-β-D-glucuronide-6''-methylester (8) [12],
kaempferol (9) [13], 3',4',5,7-tetrahydroxy-8-prenyl-flavone (10) [14] and 7,4-dimethylkaempferol
(11) [15], respectively, by direct comparison with authentic samples or comparison of the
spectroscopic data with reported literature values. Among them, compounds 7-10 were isolated for the
first time from E. rugulosa. The two new compounds were identified as apigenin 4'-O-α-D-
glucopyranoside (1) and 5,7,3',4'-tetrahydroxy-5'-C-prenylflavone 7-O-β-D-glucopyranoside (2), and
their structures were elucidated as follows.
Compound 1 was obtained as a yellow amorphous powder, and had a molecular formula C21H20O10,
13
derived from its negative HR-FAB-MS (m/z 431.1280 [M-H]-) and 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.
Figure 1. Structures of compounds 1 and 2.
HO
HO
OH
O
OH
HO
HO
HO
HO
O
OH
O
O
O
O
OH
HO
O
OH
O
OH
2
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 1H-NMR spectrum, in which compound 1 showed the signals for an
exchangeable proton at δ 12.95 (1H, s), A2B2-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 1H-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

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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
13
literature values [17,18]. The 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 C26H28O11
was derived by negative ion HR-FAB-MS (m/z: 515.1913 [M-H]-) in combination with the presence of
13
26 carbon signals in its C-NMR spectrum, and the further 2D-NMR spectral data allow to elucidate
the structure of compound 2 as shown in Figure 1.
The signals at δ 6.78 (s, H3) ascribable to C3- proton on a flavone C-ring, and two aromatic proton
signals at δ 6.61 (d, H6) and 6.48 (d, H8) due to H-6, 8 on A-ring protons, respectively, two broad
singlet signals at high field in the aromatic region [δ 7.40 (d, H2'), 6.95 (d, H6')] on B-ring were
observed in 1H-NMR spectrum, which suggested the occurrence in the molecule of a flavone skeleton
with a tetra-substituted B-ring. In addition, one glucopyranosyl unit [δ(H) 5.08 (d, H1"), δ(C) 101.9
1
C(1'')] was evident in the H- and 13 C-NMR of 2. On enzymatic hydrolysis, compound 2 liberated D-
glucose and the J value (8.1 Hz) of the anomeric proton concluded the β-configuration of the D-
glucose moiety. The HMBC correlations of glucosyl H-1'' [δ 5.08 (d, H1'')] in 2 with the C(7) (δ 166.7)
confirmed that the location of glucopyranosyl groups were at C-7 in 2. These NMR features were
resembled to those of luteolin 7-O-β-D-glucoside (4) [8], except for the existence of an additional set
of signals arising from a prenyl group in 2. Characteristic signals of prenyl group were observed at δ
1.68 (s, H5'''), 1.62 (s, H4'''), 3.62 (m, H1'''), 5.16 (br. t, H2'''), confirming that 2 was a prenylated
flavone glycoside [20]. The downfield chemical shift of C(5') of 1 at δ 127.1 indicated that the
additional prenyl group was linked at the C(5') position, which was further confirmed by the HMBC
correlations of H1''' (δ 3.62) of prenyl unit with the carbon at δ 127.1 C(5')) of the flavone glycoside
(Figure 2). On the basis of the above evidence, the structure of 2 was elucidated as 5,7,3',4'–
tetrahydroxy-5'-C-prenylflavone 7-O-β-D- glucopyranoside.
Figure 2. Key HMBC correlations of 2.
OH
OH
HO
HO
O
O
C
O
HO
OH
OH O
H
HMBC

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3. Experimental
3.1. General
Column chromatography (CC) was performed on Dianion HP 2MGL (Mitsuishi Chemical Co.),
Sephadex LH-20 (Pharmacia Fine Chemical Co. Ltd.), MCI-gel CHP20P (Mitsubishi Chemical Co.)
and silica gel (Qingdao Haiyang Chemical Co.). TLC was carried on silica gel G precoated plates
(Qingdao Haiyang Chemical Co.) with CHCl3-MeOH-H2O (9:1:0.1 or 7:3:0.5). The spots were
detected by spraying with 10% H2SO4 ethanol solution, followed by heating. UV spectra were
obtained on a UV 210A Shimadzu spectrometer (Shimadzu, Kyoto, Japan). IR spectra were recorded
1
on a Shimadzu IR-450 spectrometer as KBr pellets. H- and 13C-NMR, HSQC and HMBC spectra
were recorded with Bruker AM-400 and DRX-500 spectrometers operating at 500 and 400 MHz for
1H, and 125 and 100 MHz for 13C, respectively. FABMS and HRFABMS were recorded on an
AutoSpec 3000 spectrometer (VG, Manchester, UK) with glycerol as the matrix.
3.2. 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.
3.3. 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 H2O-MeOH (1:0-0:1) to afford five fractions
(fr. 1-5). Fr. 2 (0.4 g) was subjected to CC on silica gel (CHCl3-MeOH-H2O, 9:1:0.1-7:3:0.5),
Sephadex LH-20 and MCI-gel CHP-20P, eluting with H2O-MeOH (1:0-0:1) to afford compounds 2 (6
mg) and 5 (15 mg). Fr. 3 (1.7 g) was subjected to CC on silica gel (CHCl3-MeOH-H2O, 9:1:0.1-
7:3:0.5), Sephadex LH-20 (H2O-MeOH, 1:0-0:1) and MCI-gel CHP-20P (H2O-MeOH, 1:0-0:1) to
afford 1 (8 mg), 3 (34 mg), 4 (59 mg) and 11 (16 mg). Repeated CC on Sephadex LH-20 and MCI-gel
CHP-20P, eluting with H2O-MeOH (1:0-0:1), respectively, gave 7 (9 mg), 8 (10 mg) and 9 (50 mg)
from Fr. 4 (1.7 g), and 6 (22 mg) and 10 (24 mg) from Fr. 5 (0.7 g).
Compound 1: Yellow amorphous powder. UV-Visible λmax (nm) MeOH: 265, 296, 331; IR (KBr, cm-1):
3.376, 1.640, 1.613, 1.508, 1.055, 780, 770; 1H NMR (500 MHz, DMSO-d6): 7.94 (d, J = 8.6 Hz, H2',
H6'), 6.92 (d, J = 8.6 Hz, H3', H5'), 6.86 (s, H3), 6.82 (d, J = 1.6 Hz, H8), 6.43 (d, J = 1.6 Hz, H6),
5.42 (d, J = 3.7 Hz, H1''), 5.10 (d, J = 3.9, 12.5 Hz, Ha6''), 5.05 (dd, J = 3.9, 12.5 Hz, Hb6''), 4.63 (m,
13
H2''), 3.71 (m, H4''), 3.17-3.58 (m, H3'', H5'') ppm; C-NMR (125 MHz, DMSO-d6): 181.9 (s, C-4),
162.9 (s, C-2), 161.4 (s, C-5), 161.1 (s, C-4'), 156.9 (s, C-9), 128.6 (d, C-2', C-6'), 121.0 (s, C-1'),

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116.0 (d, C-3', 5'), 105.4 (s, C-10), 103.1 (d, C-3), 99.9 (d, C-1''), 99.5 (d, C-6), 94.8 (d, C-8), 77.2 (d,
C-3''), 76.3 (d, C-5''), 73.1 (d, C-2''), 69.5 (d, C-4''), 60.6 (t, C-6''); HR-FAB-MS (neg.): 431.1280 [M-
H]- (calcd. for C21H19O10 431.1102).
Compound 2: Yellow amorphous powder. UV-Visible λmax (nm) MeOH: 256, 267, 346; IR (KBr, cm-
1
1): 3,450, 2,920, 1,650, 1,573, 1,515, 990-600; H-NMR (500 MHz, MeOH+DMSO-d6): 7.40 (d, J =
1.9 Hz, H2'), 6.95 (d, J = 1.9 Hz, H6'), 6.78 (s, H3), 6.61 (d, J = 1.8 Hz, H6), 6.48 (d, J = 1.8 Hz, H8),
5.16 (br t, J = 6.7 Hz, H2'''), 5.08 (d, J = 8.1 Hz, H1''), 3.78-4.40 (m, H2'', H3'', H4'', H5''), 3.62 (m,
H1'''), 1.68 (s, H5'''), 1.62 (s, H4''') ppm; 13C-NMR (125 MHz, MeOH+DMSO-d6): 183.7 (s, C-4),
166.7 (s, C-7), 162.1 (s, C-5), 158.9 (s, C-9), 151.1 (s, C-4'), 147.3 (s, C-3'), 130.1 (s, C-3'''), 123.7 (s,
C-1'), 127.1 (s, C-5'), 122.1 (s, C-2'''), 117.6 (d, C-6'), 114.6 (d, C-2'), 107.1 (s, C-10), 104.5 (s, C-3),
101.9 (d, C-1''), 101.0 (d, C-6), 96.1 (d, C-8), 78.5 (d, C-3''), 77.9 (d, C-5''), 74.7 (d, C-2''), 71.2 (d, C-
4''), 62.9 (t, C-6''), 28.4 (t, C-1'''), 25.6 (q, C-5'''), 17.8 (q, C-4'''); HR-FAB-MS (neg.): 515.1913 [M-H]-
(calcd for C26H27O11 515. 1657).
3.4. 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 CHCl3 and aglycone produced was identified as apigenin by comparison with
compound 6 on silica gel TLC using CHCl3-MeOH-H2O (8:2:0.2), Rf = 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 CHCl3-MeOH-H2O (7:3:0.5). Rf = 0.23.
A solution of 2 (2 mg) in H2O (1 mL) were treated with crude cellulase (7 mg) at 37 °C for 60 h.
The reaction mixture was diluted with H2O (2 mL), and extracted with CHCl3 (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 CHCl3-MeOH-H2O (7:3:0.5). Rf = 0.23.
4. Conclusions
A detailed phytochemical investigation on 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 compounds (3-11). Among them, compounds 7-10
were isolated for the first time from E. rugulosa.
Acknowledgements
The authors are grateful to the staffs of the analytical group at State Key Laboratory of
Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy
of Sciences, for measuring the spectral data.

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References and Notes
1. Wu, C.Y. Flora of China; Science Press: Beijing, China, 1988; Vol. 66, p. 308.
2. Jiangshu New College of Medicine. The Dictionary of Chinese Medicine; Shanghai Press of
Science and Technology: Shanghai, China, 1985; p. 2132.
3. Liu, A.L.; Liu, B.; Qin, H.L.; Lee, S.M.; Wang, Y.T.; Du, G.H. Anti-influenza virus activities of
flavonoids from the medicinal plant Elsholtzia rugulosa. Planta Med. 2008, 74, 847–850.
4. Li, H.Z.; Nakashima, T.; Tanaka, T.; Zhang, Y.J.; Yang, C.R. Two new maltol glycosides and
cyanogenic glycosides from Elsholtzia rugulosa Hemsl. J. Nat. Med. 2008, 62, 75–79.
5. He, Z.D.; Liu, Y.Q.; Yang, C.R. Glycosides from Ligustrum purpurascens. Acta Bot. Yunnanica
1992, 14, 328–336.
6. Ouyang, M.A.; Wang, H.Q.; Liu, Y.Q.; Yang, C.R. Triterpenoid saponins from the leaves of Ilex
latifolia. Phytochemistry 1997, 45, 1501–1506.
7. She, G.M.; Wang, D.; Zeng, S.F.; Zhang, Y.J.; Chang, C.R. New antioxidative phenylethanoids
and sugar esters from Ku-Ding tea (the leaves of Ligustrum purpurascens). J. Food Sci. 2008, 73,
476–482.
8. Chen, H.Y.; Zhou, C.X.; Lou, Y.J.; Duan, Z.H.; Zhao, Y. Chemical constituents from Elsholtzia
blanda. Zhongguo Zhong Yao Za Zhi 2005, 30, 1589–1591.
9. Ma, J.Y.; Wang, Z.T.; Xu, L.S.; Xu, G.J. A sesquiterpene lactone glucoside from Ixeris
denticulata f. pinnatipartita. Phytochemistry 1999, 50, 113–115.
10. Shen, C.C.; Chang, Y.S.; Ho, L.K. Nuclear magnetic resonance studies of 5, 7-dihydroxy
flavonoids. Phytochemistry 1993, 34, 843–845.
11. Jiang, L.; Yao, Q.Q.; Xie, Y.Y. Study on chemical constituents of Sonchus arvensis L. Food Drug
2009, 11, 27–29.
12. Zhang, R.L.; Sun, X.C.; Li, W.X.; Wu, L.J.; Huang, J.; Sun, B.H. Isolation and identification of
chemical constituents of Polygonum perfoliatum L. J. Shenyang Pharm. Univ. 2008, 25, 105–107.
13. Zhou, Z.H.; Yang, C.R. Chemical constituents of crude green tea, the material of Pu-er tea in
Yunnan. Acta Bot. Yunnanica 2000, 22, 343–350.
14. Yang, L.; Che, Q.M.; Bi, C.; Sun, Q.S. Flavonoid compounds in solid wastes of Radix
Glycyrrhizae. Chin. Tradit. Herb. Drugs. 2007, 38, 671–673.
15. Zhao, Y.; Lin, Q.C.; Zhao, Y.; Chen, Y.G. Studies on the constituents from the herb of Elshotzia
rugulosa. Zhongguo Zhong Yao Za Zhi 2004, 29, 1144–1146.
16. Wang, X.K. Natural Medicinal Chemistry; People's Medical Publishing House: Beijing, China,
1988; p. 218.
17. Mathela, D.K.; Pant, A.K.; Mathela, C.S. A pyrone glycoside from Erigeron karwinskyanus.
Phytochemistry 1984, 23, 2090.
18. Gao, Y.M.; Wang, M.Z.; Wang, J.P.; Zhao, Q.; Qin, H.Y.; Mu, H J.; Guan, G.J. Chemical
constituents from Lonicera japonica. Chin. Tradit. Herb. Drugs. 1995, 26, 568–569.
13
19. Markham, K.R.; Ternai, B.; Stanly, R.; Geiger, H.; Mabry, T.J. C-NMR studies of flavonoids-
III. Tetrahedron 1978, 34, 1389–1397.

Molecules 2009, 14
4196
20. Bohlmann F.; Abraham, W.R. Neus Prenylflavone aus Helichrysum hypocephalum.
Phytochemistry 1979, 18, 1851–1853.
Sample Availability: Samples of the compounds are available from the authors.
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