Monoterpene glucosides from Origanum syriacum

Article abstract of DOI:10.1016/S0031-9422(01)00386-7

From the aerial parts of Origanum syriacum, three new monoterpene glucosides thymoquinol 2,5-O-β-diglucopyranoside (3), carvacrol 2-O-β-glucopyranosyl-(1→2)-β-glucopyranoside (4) and p-menth-1-ene-3,4-diol 4-O-β-glucopyranoside (5) together with two known

Full text of DOI:10.1016/S0031-9422(01)00386-7

Phytochemistry 58 (2001) 1149–1152
www.elsevier.com/locate/phytochem
Monoterpene glucosides from Origanum syriacum
a,
a
a
b
b
b
M.S. Kamel *, M.H. Assaf , H.A. Hasanean , K. Ohtani , R. Kasai , K. Yamasaki
aDepartment of Pharmacognosy, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt
bInstitute of Pharmaceutical Sciences, Hiroshima University School of Medicine, 1-2-3 Kasumi, Minami-Ku, Hiroshima 734, Japan
Received 19 June 2001; received in revised form 14 August 2001
Abstract
From the aerial parts of Origanum syriacum, three new monoterpene glucosides thymoquinol 2,5-O-b-diglucopyranoside (3),
carvacrol 2-O-b-glucopyranosyl-(1!2)-b-glucopyranoside (4) and p-menth-1-ene-3,4-diol 4-O-b-glucopyranoside (5) together with
two known monoterpene glucosides thymoquinol 2-O-b-glucopyranoside (1), thymoquinol 5-O-b-glucopyranoside (2) have been
isolated. The structures of the isolated compounds were verified by means of MS and NMR spectral analyses. # 2001Elsevier
Science Ltd. All rights reserved.
Keywords: Origanum syriacum; Lamiaceae; Thymoquinol glucosides; Carvacrol glucoside
1
. Introduction
was subjected to a column chromatography of Diaion
HP-20. The methanol eluate was repeatedly chromato-
graphed on columns of silica gel and then by MPLC and
HPLC to afford 5 glycosides.
Origanum syriacum L. (Lamiaceae) is an aromatic,
herbaceous and perennial plant growing wild in the Sinai
Desert of Egypt (Tackholm, 1974). The essential oils and
the constituents of many Origanum species have been
intensively studied (Sendra et al., 1980; Ravid et al., 1983;
Akgul et al., 1987; Harvala et al., 1987; Halim et al., 1991;
Afsharypuor et al., 1997). In folk medicine, Origanum
species are used as powerful disinfectants, flavouring
agents, in perfumes and in scenting soaps (Gunther,
The structure of compound 1 was determined as thy-
moquinol 2-O-b-glucopyranoside while that of 2 was
identified as thymoquinol 5-O-b-glucopyranoside pre-
viously isolated from Schisandrae chinensis (Yahara et
al., 1993). The enzymatic hydrolysis of compounds 1 and
2 produced thymoquinol as aglycone (1a). In general,
this is the first report for isolation of compounds 1 and
2 from family lamiaceae. The molecular formula of
compound 3 was deduced as C H O from HR FAB-
1
949; Chiej, 1984; Kotb, 1985). It was reported that
thymol and carvacrol represent the major constituents of
the essential oils of Origanum species (Sarer et al., 1982).
From Egyptian majorana (Origanum vulgare), arbutin
and methyl arbutin have been already isolated and
quantitatively estimated (Assaf et al., 1987). The present
study deals with isolation and assignment of three new
and two known monoterpene glucosides from the aerial
parts of Origanum syriacum L.
2
2
34 12
MS spectrometry (see Experimental). The enzymatic
hydrolysis of 3 gave also the aglycone 1a. 13C NMR
spectrum (Table 1) and DEPT experiment of 3 displayed
the presence of two unsubstituted b-glucopyranosyl
0
0
units from the signals at ꢀ 104.0 (C-1, 1 ); 75.1, 75.0 (C-
0
0
0
2, 2 ); 78.6 (C-3, 3 ); 71.3, 71.4 (C-4, 4 ); 78.7 (C-5, 5 )
0
and 62.4 (C-6, 6 ) (Bradbury et al., 1984) together with
10 carbon signals for the aglycone as follows: 3 methyl
2
. Results and discussion
groups (ꢀ 16.4, 23.3 and 23.2), 3 methines (ꢀ 119.1, 114.9
and 26.8) and 4 quaternary carbons (ꢀ 152.3, 150.9, 137.0
1
3
The ethanolic extract of the aerial parts of O. syr-
iacum was deffated with CH Cl and the aqueous layer
and 126.2). Comparison of the C NMR data of 3 with
those of 1, 2 and 1a (Table 1) revealed that compound 3
is a bisdesmoside and its aglycone is 2,5-disubstituted
2
2
1
thymoquinol. From H NMR spectrum of 3, the two
singlets at ꢀ 7.60 and 7.50 were assigned for H-3 and H-
6, respectively (Metwally et al., 1986). The one proton
*
Corresponding author. Tel.: +20-088-332-196; fax: +20-088-
32-776.
E-mail address: mkamel@mailcity.com (M.S. Kamel).
3
0
031-9422/01/$ - see front matter # 2001Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(01)00386-7

1150
M.S. Kamel et al. / Phytochemistry 58 (2001) 1149–1152
multiplet at ꢀ 3.80 together with two methyl doublets at
1.26 and 1.21 were obvious for H-8, 9 and 10, respec-
Table 1
13C NMR spectral data of compounds 1–5 (100 MHz,C
ꢀ
5
5
D N)
tively and the methyl singlet at ꢀ 2.40 was assigned for
H-7 (Laswell et al., 1977). The b-configuration of the
glucopyranosyl units was deduced from the coupling
constants (6.8, 7.3 Hz) of the anomeric proton doublets
1ae
C
1
2
3
4
5
1
2
3
4
5
6
7
8
9
137.2
148.7
120.1
122.8
152.0
112.9
16.4
133.8
148.3
118.4
122.6
149.6
113.7
16.4
133.6
150.5
117.6
126.1
151.9
115.9
16.2
137.0
150.9
119.1
126.2
152.3
114.9
16.4
124.9
156.4
119.8
147.9
112.8
130.7
16.3
143.4
123.4
72.8
79.8
33.1
25.2
16.9
34.1
1
at ꢀ 5.46 and 5.47, respectively in the H NMR spectrum
of 3. The negative FAB-MS spectrum of 3 exhibited
M+ at m/z 489 [M–H] as well as significant peaks at
ꢀ
ꢀ
ꢀ
m/z 327 [M–H-glucose] and 165 [glucose] . Conse-
quently the structure of compound 3 was assigned as
thymoquinol 2,5-O-b-diglucopyranoside.
26.4
23.5a
23.3a
27.4
23.2
27.3
22.9a
22.8a
26.8
23.3a
23.2a
34.2
24.3a
24.0a
21.4a
21.2a
1
0
23.2
Glc
1
104.5
75.175.0
78.3b
71.4
104.3
104.0
100.1
83.4
78.1a
71.1b
78.3a
62.2c
97.9
75.3
78.1b
71.9
78.5b
62.7
b
2
75.1
3
78.2b
71.3
78.4b
78.6c
71.3d
78.7c
62.4
4
5
78.6b
6
62.4
62.3
Glc
0
0
1
104.0
75.0b
78.6c
71.4d
78.7c
62.4
106.5
76.8
78.6a
71.3b
78.7a
62.4c
0
0
0
0
0
2
3
4
5
6
a, b, c, d
e
Assignments may be interchangeable in each column.
Data published for the first time for thymoquinol.
were similar to those reported for carvacrol (Zechmeis-
ter, 1979). The downfield shift of C-2 (ꢀ 83.4) of the
inner glucopyranosyl residue indicated its substitution
with the terminal glucopyranosyl unit at this position
(Bradbury et al., 1984). The negative FAB–MS spectrum
of 4 exhibited M at m/z 473 [M–H] as well as sig-
+
ꢀ
ꢀ
nificant peaks at m/z 311 [M–H-glucose] and 149 [M–
ꢀ
H–2 glucose] . Consequently the structure of compound
4
was assigned as carvacrol 2-O-b-glucopyranosyl-(1!2)-
The molecular formula of compound 4 was deduced
b-glucopyranoside.
The molecular formula of compound 5 was deduced
as C H O from HR FAB–MS spectrometry (see
2
2
34 11
Experimental). 13C NMR spectrum (Table 1) and
DEPT experiment of 4 displayed the presence of one
unsubstituted b-glucopyranosyl unit from the signals at
as C H O from HR FAB-MS spectrometry (see
1
6
28
7
Experimental). 13C NMR spectrum (Table 1) and
DEPT experiment of 5 displayed the presence of one b-
glucopyranosyl unit from the signals at ꢀ 97.9 (C-1),
75.3 (C-2), 78.1(C-3), 7 1. 9 (C-4) 78.5 (5) and 62.7 (C-6)
(Bradbury et al., 1984) together with 10 carbon signals
for the aglycone as follows: 3 methyl groups (ꢀ 16.9,
21.4 and 21.2), 2 methylene groups (ꢀ 33.1and 25.2), 3
methines (ꢀ 123.4, 72.8 and 34.1) and 2 quaternary car-
bons (ꢀ 143.4 and 79.8). The carbon signals at ꢀ 72.8 and
79.8 indicated the attachment of two hydroxyl groups to
these carbons (Breitmaier et al., 1987). On the other
0 0 0 0
ꢀ
106.5 (C-1 ), 76.8 (C-2 ), 78.6 (C-3 ), 71.3 (C-4 ) 78.7
0
0
(
C-5 ) and 62.4 (C-6 ), one substituted b-glucopyranosyl
unit from the signals at ꢀ 100.1 (C-1), 83.4 (C-2), 78.1
C-3), 71.1 (C-4), 78.3 (C-5) and 62.2 (C-6) (Bradbury et
(
al., 1984) together with 10 carbon signals for the agly-
cone as follows: 3 methyl groups (ꢀ 16.3, 24.3 and 24.0),
4
methines (ꢀ 130.7, 119.8, 112.8 and 34.2) and 3 qua-
1
ternary carbons (ꢀ 156.4, 147.9 and 124.9). H NMR
spectrum of 4 revealed the presence of one methyl singlet
1
(ꢀ 2.5) and two methyl doublets (ꢀ 1.15 and 1.16)
hand, H NMR spectrum of 5 revealed the presence of
assignable for Me-7, 9 and 10, respectively. Moreover,
the two doublet signals at ꢀ 5.5 and 5.6 (J=7.6 and 7.2
Hz, respectively) deduced the b-configuration of the
one methyl singlet (ꢀ 1.5) and two methyl doublets (ꢀ
0.87) assignable for Me-7, 9 and 10 respectively. The
broad singlets at ꢀ 5.5 and 4.7 were assigned for H-2 and
3 respectively proving the attachment of the hydroxyl
1
3
glucopyranosyl units. C NMR signals of the aglycone

M.S. Kamel et al. / Phytochemistry 58 (2001) 1149–1152
1151
groups to C-3 and C-4 of the aglycone together with the
3.2. Extraction and isolation of compounds (1–5)
location of the double bond between C-1and 2. Fur-
thermore, comparison of 13C NMR spectrum of 5 with
p-menth-1-ene-4-ol confirmed the location of the
hydroxyl group at C-3 of 5 from its downfield shift to ꢀ
The air-dried powdered aerial parts of O. syriacum (2
kg) were extracted with 70% EtOH. The dried ethanolic
extract (450 g) was suspended in H O and deffated with
2
7
2.8 (Zechmeister, 1979). Moreover, the one proton
CH Cl . The aq. fr. was applied to a column of Diaion
2
2
1
multiplet signal at ꢀ 3.9 in the H NMR spectrum of 5
was interpreted for H-8 while the four protons multiplet
at ꢀ 1.8 – 2.2 was assigned for two methylene groups (H-
HP-20 and eluted with H O, MeOH and acetone succes-
2
sively. The MeOH eluate (30 g) was chromatographed
by silica gel CC using CH Cl –MeOH–H O (85:15:1)
2
2
2
5
and 6). The doublet signal at ꢀ 5.16 (J=8 Hz) deduced
and finally with (70:30:3) to give 5 fractions. Fraction 2
(500 mg) was separated by MPLC on RP-18 using 65%
MeOH and HPLC using ODS column and solvent I to
afford compounds 1 (white powder, 90 mg), 2 (white
powder, 40 mg) and 5 (white powder, 15 mg). Fraction
3 (350 mg) was subjected to HPLC using first ODS col-
umn with solvent II and then Polyamine column with
solvent III to give compounds 3 (white powder, 60 mg)
and 4 (white powder, 40 mg).
the b-configuration of the glucopyranosyl unit. The
attachment of the b-glucosyl moiety to C-4 of the agly-
cone was based on the downfield shift of this carbon (ꢀ
7
9.8) in addition to the upfield shift of the anomeric
carbon of the glucosyl unit (ꢀ 97.9) that is characteristic
for the attachment of the glucosyl moiety to a tertiary
alcohol (Zhou et al., 1981; Mohamed et al., 1994). In
general, the absolute configuration of the chiral center
C-3 could not be determined because of the location of
both a double bond and a quaternary chiral center (C-4)
vicinal to it. The negative FAB-MS spectrum of 5
3.2.1. Compound (1)
Thymoquinol 2-O-b-glucopyranoside, HR FAB-MS
exhibited M+ at m/z 331[M–H] as well as a significant
ꢀ
(negative) m/z: 327.1469 [M–H]
ꢀ
C H O (req.
16 23 7
ꢀ
327.1444). 13C NMR (C D N, Table 1). H NMR
1
peak at m/z 169 [M–H-glucose] . Consequently the
structure of compound 5 was assigned as p-menth-1-
ene-3, 4-diol 4-O-b-glucopyranoside.
5
5
(C D N): ꢀ 7.50 (1H, s, H-3), 7.10 (1H, s, H-6), 5.40 (1H,
5 5
d, J=6.8 Hz, H-1Glc), 3.82 ( 1H , m, H-8), 2.40 (3H, s,
Me-7), 1.21 and 1.20 (each 3H, d, J=6.1and 6.6 Hz,
respectively, Me-9 and 10).
3
. Experimental
3
.2.2. Compound (1a)
1H and 13C NMR (TMS as int. standard): 400 MHz
Thymoquinol, 13C NMR (C D N, Table 1). H NMR
1
5
5
and 100 MHz respectively were recorded on a Jeol JNM
a-400 spectrometer. FAB MS spectra were taken on a Jeol
JMS-SX 102 spectrometer by direct inlet method at an
ionizing voltage of 70 eV MPLC: RP-18 column (20 mm
i.d. ꢁ40 cm); flow rate of mobile phase 3 ml/min.
HPLC: D-ODS-5 and polyamine columns (each 20 mm
i.d. ꢁ25 cm, YMC) with a Toyo Soda high speed chro-
matograph HLC-803 D pump and a Tosoh refraction
index (RI-8) detector; flow rate of mobile phase 6 ml/min,
injection vol. 0.8–1.0 ml. On the ODS column, 60%
MeOH (I) and 55% MeOH (II) were used while 90%
MeCN (III) was used on the polyamine column. CC:
Kieselgel 60 (70–230 mesh, Merck) and Diaion HP 20
(C D N): ꢀ 7.20 (1H, s, H-3), 7.10 (1H, s, H-6), 3.70
5 5
(1H, m, H-8), 2.40 (3H, s, Me-7), 1.30 (6H, d, J=7.1
Hz, Me-9 and 10).
3.2.3. Compound (2)
Thymoquinol 5-O-b-glucopyranoside, HR FAB-MS
ꢀ
(negative) m/z: 327.1419 [M–H]
C H O (req.
16 23 7
327.1444). 13C NMR (C D N, Table 1). H NMR
(C D N): ꢀ 7.60 (1H, s, H-3), 7.0 (1H, s, H-6), 5.40 (1H,
1
5
5
5
5
d, J=6.4 Hz, H-1Glc), 3.60 ( 1H , m, H-8), 2.40 (3H, s,
Me-7), 1.32 (6H, d, J=6.8 Hz, Me-9 and 10).
3.2.4. Compound (3)
25
Thymoquinol 2,5-O-b-diglucopyranoside, [ꢁ]D ꢀ12.5ꢂ
(Mitsubishi). TLC: silica gel 60 precoated plates F-254 and
HPTLC using RP-18 precoated plates, F-254 s (Merck).
(MeOH; c 1.20). HR FAB–MS (negative) m/z: 489.1955
M–H] C H O (req. 489.1972). 13C NMR (C D N,
ꢀ
22 33 12 5 5
[
1
3
.1. Plant material
Table 1). H NMR (C D N): ꢀ 7.60 (1H, s, H-3), 7.50
5 5
(
1H, s, H-6), 5.47 (1H, d, J=7.3 Hz, H-1Glc), 5.46 ( 1H ,
0
The aerial parts (stems and leaves) of O. syriacum L.
Lamiaceae) were collected from Saint Kathrine, Sinai
d, J=6.8 Hz, H-1 Glc), 3.80 (1H, m, H-8), 2.40 (3H, s, Me-
7), 1.26 and 1.21 (each 3H, d, J=7.1Hz, Me-9 and 10 ).
(
desert, Egypt in May 1997. The plant was identified by
Dr. Salah El-Naggar, Department of Botany, Faculty of
Science, Assiut University, Assiut, Egypt. A voucher spe-
cimen is deposited at the Herbarium of Department of
Pharmacognosy, Faculty of Pharmacy, Assiut University,
Assiut, Egypt.
3.2.5. Compound (4)
Carvacrol 2-O-b-glucopyranosyl-(1!2)-b-glucopyr-
2
5
ꢂ
anoside, [ꢁ]D ꢀ25.1 (MeOH; c 1.60). HR FAB–MS
ꢀ
(negative) m/z: 473.2058 [M–H] C H O
2
(req.
2
33 11
1
473.2023). 13C NMR (C D N, Table 1). H NMR
5
5

1152
M.S. Kamel et al. / Phytochemistry 58 (2001) 1149–1152
(
C D N): ꢀ 7.40 (1H, bs, H-3), 7.04 (1H, d, J=8 Hz, H-
5
), 6.76 (1H, bd, J=7.6 Hz, H-5), 5.60 (1H, d, J=7.2
0
Assaf, M.H., Ali, A.A., Makboul, A.M., Beck, J.P., Anton, R., 1987.
Preliminary study of phenolic glycosides from Origanum majorana,
quantitative estimation of arbutin, cytotoxic activity of hydro-
quinone. Planta Medica 53 (4), 343–345.
5
6
Hz, H-1Glc), 5.50 ( 1H , d, J=7.6 Hz, H-1 Glc), 2.80
(
1H, m, H-8), 2.50 (3H, s, Me-7), 1.16 and 1.15 (each
H, d, J=6.8 Hz, Me-9 and 10).
.2.6. Compound (5)
Bradbury, H., Jenkins, J., 1984. Determination of the structures of
trisaccharides by 13C-NMR spectroscopy. Carbohydr. Res. 126,
3
1
25–156.
Breitmaier, E., Voelter, W., 1987. Carbon-13 NMR spectroscopy, 3rd.
Edition. Verlagsgesellschaft mbH: Weinheim, Germany, D-6940.
Chiej, R., 1984. The Macdonald Encyclopedia of Medicinal Plants.
Macdonald Co. Ltd, London, pp. 212–297.
3
2
5
p-Menth-1-ene-3,4-diol 4-O-b-glucopyranoside, [ꢁ]
D
o
ꢀ
18.9 (MeOH; c 1.50). HR FAB–MS (negative) m/z:
31.1758 [M–H] C H O (req. 331.1757). 13C NMR
C D N, Table 1). H NMR (C D N): ꢀ 5.50 (1H, bs,
5 5 5 5
ꢀ
3
16 27 7
Gunther, E., 1949. The Essential Oils, Vol. 3, 535. Van Nostrand Co,
New York.
1
(
H-2), 5.16 (1H, d, J=8.0 Hz, H-1Glc), 4.70 ( 1H , bs, H-
3
Halim, A.F., Mashaly, M.M., Zaghloul, A.M., Abdel-Fattah, H., De
Potter, H.L., 1991. Chemical constituents of the essential oils of
Origanum syriacum and Stachys aegyptiaca. International Journal of
Pharmacognosy 29 (1), 1–5.
), 3.90 (1H, m, H-8), 1.80–2.20 (4H, m, H-5 and 6),
.50 (3H, s, H-7), 0.87 (6H, d, J=6.8 Hz, Me-9 and 10).
1
Harvala, C., Menounos, P., Argyriadou, N., 1987. Essential oil from
Origanum dictamnus. Planta Medica 53 (4), 107–109.
Kotb, F., 1985. Medicinal plants in Libya, 632, Arab Encyclopedia
House.
3
.2.7. Enzymatic hydrolysis of compound 1
Compound 1 (20 mg) was dissolved in 0.5 ml MeOH
and a solution of b-glucosidase (100 mg) in 20 ml water
was added. The mixture was extracted with diethyl ether
Laswell, W, Hufford, C., 1977. Aromatic constituents from Uvaria
chamae. Phytochemistry 16, 1439–1441.
ꢂ
(
3 times ꢁ20 ml) after stirring at 37 C for 2 days. The
Metwally, M., Dawidar, A., Metwally, S., 1986. A new thymol deri-
vative from Pulicaria undulata. Chemical Pharmaceutical Bulletin 34
combined ethereal extracts were evaporated and the
residue was purified by HPLC using 95% MeCN on
polyamine column to afford the aglycone 1a (6 mg).
Compounds 2 and 3 have been subjected to same pro-
cedure as 1 to afford the aglycone 1a.
(
Mohamed, K., Ohtani, K., Kasai, R., Yamasaki, K., 1994. Dolabellane
1), 378–379.
diterpene glucosides from Chrozophora obliqua. Phytochemistry 37,
4
95–500.
Ravid, U., Putievsky, E., 1983. Essential oils of Majorana syriaca,
Coridothymus capitatus and Satureja thymbra plants growing wild in
Israel. Planta Medica 49, 248–249.
Acknowledgements
Sarer, E., Schefler, J., Baerheim, S., 1982. Monoterpenes of the essen-
tial oil of Origanum majorana. Planta Medica 46, 236–239.
Sendra, J.M., Cunat, P., 1980. Volatile constituents of Spanish Origa-
num (Coriandothymus capitatis) essential oil. Phytochemistry 19, 89–
92 and 1513–1517.
The authors are grateful to the Japan Society for
Promotion of Science (JSPS) and a Grant-in-Aid, Min-
istry of Education and Culture, Japan for the financial
support of this work. Also, we would like to thank the
Research Center of Molecular Medicine of the Hiroshima
University School of Medicine, Japan, for NMR mea-
surements.
Tackholm, V., 1974. Students Flora of Egypt, 2nd edn., 562. Coop-
erative Printing Co, Beirut.
Yahara, S., Sakamoto, C., Nohara, T., Niiho, Y., Nakajima, Y., Ito,
H., 1993. Thymoquinol 5-O-beta-d-glucopyranoside, kaempferol 3-
O-beta-rutinoside and protocatechuic acid from fruit of Scisandrae
chinensis. Shoyakugaku zassi 47 (4), 420–422.
Zechmeister, L., 1979. Progress in the Chemistry of Organic Natural
Products, Vol. 36, 24. Springer-Verlag, Wien, New York.
Zhou, J., Wu, M., Taniyasu, S., Besso, H., Tanaka, O., Saruwatari,
Y., Fuwa, T., 1981. Dammarane saponins of Sanchi-ginseng, root
of Panax notoginseng (Burk.) F. H. Chen (Araliaceae): structures of
new saponins, notoginsenosides-R1and -R2, and identification of
ginsenosides-Rg2 and Rh-1. Chemical Pharmaceutical Bulletin 29,
2844–2850.
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