Two new monoterpene glycosides and trypanocidal terpenoids from Dracocephalum kotschyi

Article abstract of DOI:10.1248/cpb.52.1249

From the whole plant of Dracocephalum kotschyi BOISS., two new monoterpene glycosides (9, 10), together with seven known terpenoids and a phytosterol (1-8), were isolated. Their structures were determined to be limonen-10-al (1), geranial (2), neral (3), β-sitosterol (4), oleanolic acid (5), ursolic acid (6), p-mentha-8-en-1,2-diol (7), colosolic acid (8), limonen-10-ol 10-O-β-D-glucopyranoside (9), and limonen-10-ol 10-O-β-D- glucopyranosyl-(1→2)-β-D-glucopyranoside (10). Compounds 1 (3.1 μM), 2 (3.1 μM), 3 (3.1 μM), 5 (6.2 μM), 6 (6.2 μM), and 8 (6.2 μM) were effective against epimastigotes of Trypanosoma cruzi.

Full text of DOI:10.1248/cpb.52.1249

October 2004
Notes
Chem. Pharm. Bull. 52(10) 1249—1250 (2004)
1249
Two New Monoterpene Glycosides and Trypanocidal Terpenoids from
Dracocephalum kotschyi
Soodabeh SAEIDNIA,a Ahmad Reza GOHARI,a Nahoko UCHIYAMA,b Michiho ITO,b Gisho HONDA,*,b and
c
Fumiyuki KIUCHI
a Department of Pharmacy, Faculty of Pharmacy, Medical Sciences University of Mazandaran; Sari, P.O. Box 48175–861,
Iran: b Graduate School of Pharmaceutical Sciences, Kyoto University; 46–29 Yoshida-Shimoadachi, Sakyo-ku, Kyoto
606–8501, Japan: and c Tsukuba Medicinal Plant Research Station, National Institute of Health Sciences, Ministry of
Health, Labor and Welfare; Tsukuba, Ibaraki 305–0843, Japan. Received April 13, 2004; accepted June 28, 2004
From the whole plant of Dracocephalum kotschyi BOISS., two new monoterpene glycosides (9, 10), together
with seven known terpenoids and a phytosterol (1—8), were isolated. Their structures were determined to be
limonen-10-al (1), geranial (2), neral (3), b-sitosterol (4), oleanolic acid (5), ursolic acid (6), p-mentha-8-en-1,2-
diol (7), colosolic acid (8), limonen-10-ol 10-O-b-D-glucopyranoside (9), and limonen-10-ol 10-O-b-D-glucopyra-
nosyl-(1Æ2)-b-D-glucopyranoside (10). Compounds 1 (3.1 mM), 2 (3.1 mM), 3 (3.1 mM), 5 (6.2 mM), 6 (6.2 mM), and 8
(6.2 mM) were effective against epimastigotes of Trypanosoma cruzi.
Key words Dracocephalum kotschyi; monoterpene glucoside; trypanocidal activity
The genus Dracocephalum (Labiatae) is widely distributed phy (CC) and Lobar (Si-60 and RP-8) CC to give compounds
in Iran. One of the important endemic species, Draco- 1 (400 mg), 2 (10 mg), 3 (7 mg), 4 (18 mg), 5 (34 mg), 6
cephalum kotschyi BOISS., is found in many parts of the Al- (177 mg), 7 (9 mg), and 8 (4 mg). After washing with hexane
borz Mountains.1) This plant is used in herbal medicine as a and EtOAc, the MeOH extract was separated by ODS,
remedy for stomach disorders. Its usefulness for headache, Sephadex LH-20, and Lobar RP-8 CC to afford compounds 9
congestion, and liver disorders has also been described in (17 mg) and 10 (9 mg).
traditional medicine documents.2) However, no phytochemi-
Compounds 2—8 were identified as geranial (2),4) neral
cal work has been reported on this plant. Recently, some (3),4) b-sitosterol (4),5) oleanolic acid (5),6) ursolic acid (6),7)
diterpenes have been isolated from Dracocephalum komarovi p-mentha-8-en-1,2-diol (7),8) and colosolic acid (8)9) by com-
with strong trypanocidal activity against epimastigotes of parison of their spectral data with those reported. Compound
Trypanosoma cruzi, the causative agent of American try- 1 was identified by the analysis of its NMR spectra as limo-
panosomiasis.3) In this paper, we report the isolation and nen-10-al (1). Although this is a known compound, its NMR
identification of two new monoterpene glycosides, limonen- data were not available in the literature. Therefore 1 was
10-ol 10-O-b-D-glucopyranoside (9) and limonen-10-ol treated with NaBH4 to give limonen-10-ol (1a), of which the
10-O-b-D-glucopyranosyl-(1Æ2)-b-D-glucopyranoside (10), structure was confirmed by NMR data.
together with eight known terpenoids (1—8) and describe the
Compound 9 was obtained as a colorless gum. The molec-
trypanocidal activity of the isolated compounds.
Table 1. 13C-NMR of Limonene-10-ol Derivatives
Results and Discussion
No.
1a)
1aa)
9b)
10b)
Dried whole plants of D. kotschyi collected in Iran were
successively extracted with EtOAc and MeOH. The EtOAc
extract was fractionated using silica gel column chromatog-
raphy using hexane–EtOAc and MeOH as eluents to give 7
fractions (fr. A—G). The fractions showed strong in vitro try-
panocidal activity against epimastigotes of T. cruzi (fr. C—F)
were further separated using silica gel column chromatogra-
1
2
3
4
5
6
7
8
9
10
1ꢀ
2ꢀ
3ꢀ
4ꢀ
5ꢀ
6ꢀ
1ꢁ
2ꢁ
3ꢁ
4ꢁ
5ꢁ
6ꢁ
133.8
120.0
30.6
31.5
29.9
27.6
23.4
154.7
132.9
194.6
133.8
120.4
31.3
36.9
30.5
28.2
23.4
153.6
107.8
65.2
133.4
121.1
31.3
36.8
30.5
28.2
23.5
150.8
109.8
71.1
133.5
121.1
31.4
37.0
30.6
28.2
23.5
150.5
109.3
71.0
102.2
83.8
103.9
75.2
78.6c)
71.7
78.1
71.6
78.7c)
62.8
78.5
62.5c)
106.3
76.7
78.1
71.3
78.5
62.7c)
a) In CDCl3; b) in pyridine-d5; c) interchangeable in each column.
Fig. 1
* To whom correspondence should be addressed. e-mail: ghonda@pharm.kyoto-u.ac.jp
© 2004 Pharmaceutical Society of Japan

1250
Vol. 52, No. 10
after CC with hexane–EtOAc (3 : 2). Compound 7 (9 mg) was yielded from
fraction E5 (1.6 g) by silica gel CC (benzene–EtOAc, 3 : 1) and then Lobar
RP-8 (60% MeOH). Fraction F (250 mg) was subjected to silica CC
(hexane–EtOAc, 3 : 2) to give compound 8 (4 mg).
ular formula C16H26O6 was revealed by high-resolution FAB-
1
MS. The H- and 13C-NMR spectra indicated it to be a
monoterpene glycoside. The 13C-NMR data (Table 1) of the
aglycon part were very similar to those of limonen-10-ol
(1a), except for the chemical shift of C-10, which showed a
glycosylation shift (ꢂ5.9 ppm compared to corresponding
carbon of 1a). The sugar part was concluded to be b-D-glu-
cose from the 13C-NMR chemical shifts and the coupling
constant of the anomeric proton (Jꢃ7.6 Hz).10) Thus the
structure was concluded to be limonen-10-ol 10-O-b-D-glu-
copyranoside.
Compound 10 was obtained as a colorless powder. The
presence of the limonen-10-ol part and two glucoses was
concluded from the 1H- and 13C-NMR spectra (Table 1). This
was in accordance with the molecular formula C22H35O11, as
revealed by HR-FAB-MS. One glucose unit was located at
C-10 of the limonen-10-ol unit, and the other was located at
C-2ꢀ of the first glucose unit based on the HMBC correla-
tions and glycosylation shifts (C-10, ꢂ5.8 ppm compared to
corresponding carbon of 1a; C-2ꢀ, ꢂ8.6 ppm compared with
the corresponding carbon of 9). The configurations of the
anomeric protons were determined to be b from their cou-
pling constants (Jꢃ7.9, 7.6 Hz).10) Thus the structure of 10
was concluded to be limonen-10-ol 10-O-b-D-glucopyra-
nosyl-(1Æ2)-b-D-glucopyranoside.
Trypanocidal activities of the isolated compounds against
epimastigotes of T. cruzi were tested and the minimum lethal
concentrations (MLC) were determined. Limonen-10-al (1,
MLCꢃ3.1 mM), geranial (2, 3.1 mM), neral (3, 3.1 mM),
oleanolic acid (5, 6.2 mM), ursolic acid (6, 6.2 mM), and
colosolic acid (8, 6.2 mM) showed trypanocidal activity. Al-
though limonen-10-al (1) showed strong activity, limonen-
10-ol (1a), p-mentha-8-en-1,2-diol (7) and limonen-10-ol
glucosides (9, 10) were not active even at 400 mM. b-Sitos-
terol (4) also did not show any activity at this concentration.
The MeOH extract (20 g) was washed with hexane and EtOAc to give a
residue (18 g). The residue (6 g) was subjected to reverse-phase silica gel
(ODS) with aqueous MeOH (30%, 50%, 80%) to give six fractions (M1—
M6). Fractions M5 (130 mg) and M4 (268 mg), after chromatographic sepa-
ration with Sephadex LH-20 (MeOH) and then Lobar RP-8 (60% MeOH),
gave compounds 9 (17 mg) and 10 (9 mg), respectively.
Limonen-10-al (1): Colorless oil, [a]D25 ꢂ88.0° (cꢃ0.03, CHCl3). 1H-
NMR (CDCl3): dH 1.52 (1H, m, H-5a), 1.66 (3H, s, H-7), 1.78 (1H, m, H-
5b), 1.89 (2H, m, H-6), 1.93 (1H, m, H-3a), 2.17 (1H, m, H-3b), 2.71 (1H,
m, H-4), 5.41 (1H, br s, H-2), 5.99 (1H, s, H-9a), 6.25 (1H, s, H-9b), 9.54
(1H, s, H-10); 13C-NMR (CDCl3): Table 1.
Limonen-10-ol 10-O-b-D-Glucopyranoside (9): Colorless gum; [a]D24
ꢄ7.6° (cꢃ0.26, MeOH); IR (KBr) nmax 3344, 2916, 2356, 1588, 1076,
1041 cmꢄ1; positive-ion HR-FAB-MS: m/z 337.1632 [MꢂNa]ꢂ; Calcd for
C16H26O6Na, 337.1628. 1H-NMR (pyridine-d5): dH 1.40 (1H, m, H-5a), 1.54
(3H, s, H-7), 1.75 (1H, m, H-5b), 1.81 (2H, m, H-6), 1.82 (1H, m, H-3a),
2.14 (1H, m, H-3b), 2.29 (1H, m, H-4), 3.97 (1H, m, H-5ꢀ), 4.08 (1H, m, H-
2ꢀ), 4.26 (2H, m, H-3ꢀ, 4ꢀ), 4.32 (1H, d, Jꢃ12.9 Hz, H-10a), 4.39 (1H, dd,
Jꢃ11.9, 5.5 Hz, H-6ꢀa), 4.57 (1H, dd, Jꢃ11.8, 2.2 Hz, H-6ꢀb), 4.62 (1H, d,
Jꢃ12.9 Hz, H-10b), 4.92 (1H, d, Jꢃ7.6 Hz, H-1ꢀ), 4.98 (1H, br s, H-9a),
5.32 (1H, br s, H-2), 5.38 (1H, br s, H-9b); 13C-NMR (pyridine-d5): Table 1.
Limonen-10-ol
10-O-b-D-Glucopyranosyl-(1Æ2)-b-D-glucopyranoside
(10): Colorless powder; [a]D24 ꢄ8.3° (cꢃ0.03, MeOH); IR (KBr) nmax 3348,
2920, 2360, 1600, 1442, 1380, 1257, 1076 cmꢄ1; Negative-ion HR-FAB-
MS: m/z 475.2178 [MꢄH]ꢄ; Calcd for C22H35O11, 475.2179. 1H-NMR
(pyridine-d5): dH 1.39 (1H, m, H-5a), 1.56 (3H, s, H-7), 1.73 (1H, m, H-5b),
1.78 (2H, m, H-6), 1.81 (1H, m, H-3a), 2.10 (1H, m, H-3b), 2.22 (1H, m, H-
4), 3.90 (2H, m, H-5ꢀ, 5ꢁ), 4.09 (1H, m, H-2ꢁ), 4.19 (1H, m, H-2ꢀ), 4.21 (1H,
m, H-4ꢁ), 4.23 (1H, m, H-3ꢁ), 4.26 (1H, d, Jꢃ13.4 Hz, H-10a), 4.29 (1H, m,
H-4ꢀ), 4.31 (1H, m, H-3ꢀ), 4.34 (1H, m, H-6ꢀa), 4.39 (1H, dd, Jꢃ11.4,
4.4 Hz, H-6ꢁa), 4.46 (1H, br d, Jꢃ11 Hz, H-6ꢀb), 4.46 (1H, dd, Jꢃ11.4,
2.3 Hz, H-6ꢁb), 4.57 (1H, d, Jꢃ13.4 Hz, H-10b), 4.94 (1H, d, Jꢃ7.6 Hz, H-
1ꢀ), 4.99 (1H, br s, H-9a), 5.32 (1H, br s, H-2), 5.38 (1H, d, Jꢃ7.9 Hz, H-1ꢁ),
5.53 (1H, br s, H-9b); 13C-NMR (pyridine-d5): Table 1.
NaBH4 Reduction of 1 Compound 1 (50 mg) in MeOH (5 ml) was
treated with NaBH4 at room temperature for 10 min. The solution was con-
centrated under reduced pressure and the residue was purified by silica gel
column chromatography (hexane : EtOAc, 3 : 1) to give 1a (10 mg).
Limonen-10-ol (1a): Colorless oil; [a]D24 ꢂ101.2° (cꢃ0.16, MeOH); H-
1
NMR (CDCl3): dH 1.65 (3H, s, H-7), 4.14 (2H, s, H-10), 4.91 (1H, s, H-9a),
5.05 (1H, s, H-9b), 5.40 (1H, br s, H-3); 13C-NMR (CDCl3): Table 1.
Trypanocidal Assay The trypanocidal assay was performed as de-
Experimental
General Procedures Optical rotations were recorded on a HITACHI U-
3210 or a JASCO P-1020 polarimeter. IR spectra were recorded with a Shi-
madzu FT-IR-8700 spectrometer. Mass spectra were measured on a JEOL
scribed previously.11) Gentian violet is used as
a positive control
(MLCꢃ6.2 mM).
JMS-HX/HX110A spectrometer. H- and 13C-NMR spectra were measured
1
on a JEOL JNM-LA500 (500 MHz for 1H and 125 MHz for 13C) spectrome-
ter with tetramethylsilane as an internal standard, and chemical shifts are
given in d. Silica gel 60F254 precoated plates (Merck) were used for TLC.
The spots were detected by spraying anisaldehyde–H2SO4 reagent followed
by heating.
Acknowledgments Two of the authors (A. R. G. and S. S.) are grateful
to the Ministry of Health and Medical Education of Iran for financial sup-
port.
References
Plant Material D. kotschyi BOISS. was collected on the Tochal Moun-
tain, Alborz, north of Tehran, Iran, at 3200 m above sea level, in July 2002.
A voucher specimen (ESM-4577) was deposited at the Experimental Station
of Medicinal Plants, Kyoto University Graduate School of Pharmaceutical
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Extraction and Isolation Dried whole plants of D. kotschyi (1 kg) were
cut into small pieces and successively extracted with EtOAc and MeOH at
room temperature overnight to obtain EtOAc (50 g) and MeOH (143 g) ex-
tracts. The EtOAc extract (15 g) was subjected to silica gel CC with
hexane–EtOAc (19 : 1, 0 : 1) and MeOH as eluent to give seven fractions
(A—G). Fraction C was compound 1 (400 mg). Fraction D (800 mg) was
fractionated by a silica gel CC with hexane–CHCl3 (1 : 1) to afford 4 parts
(D1—D4). Fraction D3 (190 mg) was chromatographed twice with
hexane–EtOAc (19 : 1) on a Lobar Si-60 column to give compounds 2
(10 mg) and 3 (7 mg). Fraction E (8 g) was subjected to silica gel CC
(hexane–EtOAc, 65 : 35) to obtain eight fractions (E1—E8). From fraction
E2 (1.3 g), compound 4 (18 mg) was obtained by silica gel CC with
hexane–EtOAc (19 : 1), hexane : acetone (8 : 2) and then CHCl3–EtOAc
(9 : 1). Fraction E3 (600 mg) was submitted to silica gel CC with
hexane–EtOAc (3 : 1) and then CHCl3–EtOAc (19 : 1) to give compound 5
(34 mg). Compound 6 (177 mg) was obtained from fraction E4 (746 mg)
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