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Cytotoxic Activity of Diterpenoids Isolated from Salvia hypargeia

Authors:
  • Bezmialem Vakif University, Faculty of Pharmacy

Abstract and Figures

Ten diterpenoids [6a-hydroxysalvinolone (1), 6a-hydroxytaxodone (2), aethiopinone (3), microstegiol (4), ferruginol (5), saprorthoquinone (6), 11,12-dioxoabieta-8,13-diene (7), taxodione (8), hypargenin A (9), hypargenin D (10)] and three triterpenoids [lupeol 3-acetate, d-oleanol 3-acetate and ß-sitosterol] were isolated from the roots of Salvia hypargeia, a plant endemic to Turkey. The crude extract and compounds 1-2, 5-10 were tested against a panel of human cancer cell lines [human breast cancer (BC 1), human lung cancer (LU 2), human colon cancer (COL 2), human epidermoidal carcinoma in mouth (KB), vinblastine-resistant KB-VI, hormone-dependent human prostate cancer (LNCaP)] as well as P388 and ASK cells in culture. The crude extract was active in all of the test systems, except KB. Isolates 1 and 8 were also found to mediate a generalized cytotoxic response.
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ABSTRACT
Ten diterpenoids [6a-hydroxysalvinolone (1), 6-
hydroxytaxodone (2), aethiopinone (3), microstegiol
(4), ferruginol (5), saprorthoquinone (6), 11,12-dioxo-
abieta-8,13-diene (7), taxodione (8), hypargenin A (9),
hypargenin D (10)] and three triterpenoids [lupeol 3-
acetate, -oleanol 3-acetate and ß-sitosterol] were iso-
lated from the roots of Salvia hypargeia, a plant
endemic to Turkey. The crude extract and compounds
1-2, 5-10 were tested against a panel of human cancer
cell lines [human breast cancer (BC 1), human lung
cancer (LU 2), human colon cancer (COL 2), human
epidermoidal carcinoma in mouth (KB), vinblastine-
resistant KB-VI, hormone-dependent human prostate
cancer (LNCaP)] as well as P388 and ASK cells in
culture. The crude extract was active in all of the test
systems, except KB. Isolates 1and 8were also found
to mediate a generalized cytotoxic response.
INTRODUCTION
Salvia species (Labiatae) have been used as medicinal
plants throughout the world. They possess antifungal
and antibacterial activities (Gao et al., 1979) and are
used against tuberculosis (Dobrynin et al., 1976), as
anticancer agents and for the treatment of heart disease
(Ginda et al., 1988). In Turkish folk medicine, Salvia
species have several uses, such as antiseptic, antibacte-
rial, diuretic, hemostatic, spasmolitic, carminative and
wound healing (Baytop, 1984). Phytochemical research
revealed that the principal secondary metabolites in the
roots of Salvia species are found to be diterpenoids
which are responsible for biological activities.
In a previous study with the roots of the endemic
plant Salvia hypargeia Fisch. et Mey., collected from
eastern Turkey (Sivas), six new diterpenoids, hypar-
genins A-F (Ulubelen et al., 1988) were isolated. Later,
a NOE experiment showed that hypargenin C was tax-
odione (8) (unpublished data). Recently, we collected
the same plant from a different location, the Adana dis-
trict (southern Turkey), and studied its roots. Only three
of the previously isolated compounds were found to be
present: hypargenin A (9), hypargenin D (10) and tax-
odione (8). However, seven other diterpenes, previ-
ously obtained from other Salvia species, were isolated
from this plant for the first time. These were, 6-
hydroxysalvinolone (1) (Topcu et al., 1996), 6-
hydroxytaxodone (2) (Kupchan et al., 1968),
aethiopinone (3) (Boya et al., 1981), microstegiol (4)
(Ulubelen et al., 1992), ferruginol (5) (Cambie et al.,
1971), saprorthoquinone (6) (Lin et al., 1989), and
11,12-dioxo-abieta-8,13-diene (7) (Ulubelen et al.,
1995). In addition, -sitosterol (Swift, 1952), lupeol 3-
acetate (Ames et al., 1951) and -oleanol 3-acetate
(Djerassi & Lippman, 1955) were obtained. The diter-
penoids were either abietane (1, 2, 5, 7-10) or
rearranged abietane (3, 4, 6) types.
As reported herein, the crude plant extract and all
diterpenoids (except 3and 4) were tested against a
panel of cultured cancer cells comprised of human
breast cancer (BC 1), human lung cancer (LU1), human
colon cancer (COL 2), human epidermoidal carcinoma
in mouth (KB), vinblastine-resistant KB-VI, hormone-
dependent human prostate cancer (LNCaP), as well as
Keywords: Salvia hypargeia Fisch. et Mey., Lamiaceae, diter-
penoids, triterpenoids, cytotoxic activity.
Address correspondence to: A. Ulubelen, Faculty of Phar-
macy, University of Istanbul, 34452 Istanbul, Turkey.
CYTOTOXIC ACTIVITY OF DITERPENOIDS ISOLATED FROM SALVIA
HYPARGEIA
Ayhan Ulubelen1, GülaçtıTopçu1, Hee-Byung Chai2and John M. Pezzuto2
1Faculty of Pharmacy, University of Istanbul, 34452 Istanbul, Turkey,Tubitak, Marmara Research Center,
Department of Chemistry, P.O. 21, TR-41470, Gebze, Kocaeli, Turkey
2Program for Collaborative Research in the Pharmaceutical Sciences, College of Pharmacy,
University of Illinois, Chicago, Illinois, 60612–7231, USA
Pharmaceutical Biology 1388-0209/99/3702-0148$15.00
1999, Vol. 37, No. 2, pp. 148–151 © Swets & Zeitlinger
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P388 and ASK cells in culture. The crude extract was
found to be active in all the systems, except for KB.
Taxodione (8) was highly active in all systems, and the
other tested compounds showed activities in select cell
cultures.
MATERIALS AND METHODS
General Experimental Procedures
The spectra were recorded with the following instru-
ments. IR: Perkin-Elmer 980 in CHCl3; NMR: Bruker
CYTOTOXIC ACTIVITY OF DITERPENOIDS 149
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AC 200 L, 200 MHz (for 1H) and 50.32 MHz (for 13C)
in CDCl3; HRMS: ZabSpec; Optical rotation: Optical
Act. Ltd. AA-5 polarimeter; Separations: Chroma-
totron Model 7924 T; TLC: on Kieselgel 60 F254 (E.
Merck) precoated plates; CC: on silica gel 60 and
Sephadex LH-20 (Fluka).
Plant Material
The roots of Salvia hypargeia were collected from the
highland area of Adana (Tekir-Burucek) southern
Turkey, at an altitude of 1850 m. The plant was identi-
fied by Prof. Dr. Kerim Alpinar (Istanbul), and a
voucher is deposited in the herbarium of the Faculty of
Pharmacy, University of Istanbul (ISTE 68316).
Extraction and Isolation
Dried and powdered roots (1 kg) of S. hypargeia were
extracted with acetone in a Soxhlet apparatus. The fil-
tered extract was evaporated to dryness in vacuo, and
58 g of a residue were obtained. The residue was
roughly separated by column chromatography (5 70
cm) using 70–200 mesh silica gel, that was eluted with
petroleum ether, a gradient of ethyl acetate-ethanol, and
ethanol up to 100%. Fractions (1–28), (36–43),
(44–68), (69–73) and (74–87) were combined. The first
combined fraction (1–28), having only oily material,
was discarded, and the remaining combined fractions
were separated on a Chromatotron apparatus using sil-
ica gel rotors and eluting with petroleum ether and
chloroform, followed by ethanol. The compounds were
collected from the rotors under UV light (254 nm) and,
when necessary, purified on preparative TLC plates.
The amounts were: 1, 15 mg; 2, 13 mg; 3, 5 mg; 4, 8
mg; 5, 20 mg; 6, 25 mg; 7, 25 mg; 8, 18 mg; 9, 10 mg;
10, 10 mg. Optical rotations []D(recorded in CHCl3
at 22°C) are as follows: 1, 170° (c, 1.0); 2, 0° (c,
0.4); 3, 3° (c, 0.27); 4, 41.5° (c, 0.3); 5, 22° (c,
2.0); 6, 0° (c, 0.4); 7, 42.5° (c, 0.52); 8, 11° (c, 3.0);
9, 0° (c, 0.2); 10, 0° (c, 0.3).
Cytotoxic Evaluation
All compounds were evaluated for cytotoxic potential,
except 3and 4, the latter of which was reported active
against P388 (ED50 3.0 g) in a previous study
(Ulubelen et al., 1992). The evaluation procedures have
been described previously (Likhitwitayawuid et al.,
1993).
RESULTS AND DISCUSSION
As shown in Table 1, the crude extract obtained from
Salvia hypargeia was active in all cell lines tested
except KB. The ED50 values of the diterpenoid isolates
are also given in Table 1. Taxodione (8), previously
shown to mediate antitumor activity by (Kupchan et al.,
1968) in the Walker intramuscular carcinosarcoma 256
model, was the most active substance tested. 6-
Hydroxytaxodone (2), although structurally similar to
8, did not exhibit similar activity. Compounds 2and 5
showed weak but selective activity against colon cancer
cells (Col 2) and human prostate cancer cells (LNCaP).
Compounds 1and 6mediated generalized responses.
Other compounds were either not active (7) or medi-
ated weak responses in one or two systems. The activ-
ity of 1and 8was not negated in the multidrug-resistant
KB-VI cell culture system, indicating they were not
subject to efflux by P-glycoprotein which is overex-
pressed in this cell line. None of the tested compounds
mediated a stronger cytotoxic response with KB-VI
150 A. ULUBELEN ET AL.
Table 1. Cytotoxic activity of compounds 1, 2, 5-10, and ellipticinea
Compound BC1 LU1 COL2 KB KB-VI LNCaP P388
14.7 4.2 10.1 9.7 5.6 4.0 >5
2>20 >20 9.0 >20 >20 12.9 >5
5>20 >20 9.7 >20 >20 >20 >5
69.2 16.4 3.3 >20 9.1 >20 2.3
7>20 >20 >20 >20 >20 >20 >5
81.2 5.1 0.7 3.4 4.1 0.7 0.3
9>20 >20 >20 >20 >20 >20 >5
10 12.6 >20 12.3 >20 >20 >20 >5
Ellipticineb0.2 0.02 0.3 0.04 0.3 0.8 0.1
aData are given as ED50 values in g/ml, BC1, human breast cancer; COL2, human colon cancer; LU1, human lung cancer; KB, originally
derived from human nasopharyngeal cancer; KB-VI, multidrug-resistant KB; LNCaP, human prostate cancer; P388, mouse lymphocytic
leukemia.
bEllipticine was used as a positive control. For data obtained with additional positive control test compounds, see references (Likhitwitayawuid
et al., 1993 and Pisha et al., 1995).
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cells in the presence of vinblastine (1 g/ml), nor were
they antimitotic as judged by the ASK test system (data
not shown). The cytotoxic responses of compounds 1,
6, and 8could be due to the extensive conjugation affil-
iated with these structures. This concept should be
tested with model compounds in the future.
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... Ferruginol has also been found in angiosperms and has been isolated from the roots o f several species o f Salvia (Lamiaceae) (Kabouche et al., 2005;Nakanishi et al., 1983;Tan et al., 2002;Ulubelen et al., 2000Ulubelen et al., , 1999aUlubelen et al., , 1999b; Coleus barbatus (Lamiaceae) (Kelecom 1984) and also from Harpagophytum procumbens (Pedaliaceae), known as Devil's Claw (Clarkson et al., 2003a). ...
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Salvia macrosiphon Boiss. is an aromatic perennial herb belonging to the family Lamiaceae. Phytochemical studies and biological activities of this plant have been rarely documented in the literature. The current study aimed to investigate antibacterial and cytotoxic activity of different fractions of aerial parts of S. macrosiphon. Also, we tried to isolate and identify cytotoxic compounds from the plant. In this respect, the hydroalcoholic extract of the corresponding parts of the plant was fractionated into four fractions. Then, antibacterial and cytotoxic activity of each fraction were examined. It was found that the chloroform fraction had a good antibacterial activity against gram-positive and gram-negative bacteria. The most potent cytotoxicity was also obtained by the n-hexane fraction comparing with etoposide as the reference drug which was selected for the study and characterization of secondary metabolites. Accordingly, 13-epi manoyl oxide (1), 6α-hydroxy-13-epimanoyl oxide (2), 5-hydroxy-7,4'-dimethoxyflavone (3), and β-sitosterol (4) were isolated and evaluated for their cytotoxic activity. Among them, compound 1 revealed significant cytotoxicity against A549, MCF-7, and MDA-MB-231. It merits mentioning that it showed high selectivity index ratio regarding the low cytotoxic effects on Human Dermal Fibroblast which can be considered as a promising anticancer candidate.
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Transformed root cultures of Salvia species are rich in abietane-type diterpenoids. These compounds have various biological activities, such as antimicrobial, cytotoxic, antitumor, anti-inflammatory as well as an ability to induce apoptotic process anti- and inhibit acetyl- and butyrylcholinesterase. Some of them also possess cardioactive properties. Due to many pharmacological activities of abietane diterpenoids, scientists are actively searching for new valuable sources of biomass and its secondary metabolite. Bioactive secondary metabolites can be obtained from field plants. However, in vitro plant cultures, including transformed root cultures, may be an interesting source of these phytocompounds. Genetically modified roots (hairy roots) of Salvia species are obtained by transformation with Agrobacterium rhizogenes strains. Transformed roots have many advantages, such as the possibility of growth without exogenous phytohormones, rapid unlimited growth, genetic stability even after several years of cultivation, and often the possibility of biosynthesis of valuable secondary metabolites in higher amounts than in intact plants. Some of them are able to biosynthesize new compounds, including diterpenes, which have not been detected in the mother plant. Salvia sp. hairy roots can often be grown on a larger scale in bioreactors. After optimizing the culture conditions and/or using elicitors, they are able to biosynthesize abietane diterpenoids in quantities significantly exceeding not only mother plants but also those grown in flasks. The above properties of this kind of plant cultures make them an interesting experimental model in studies aimed at increasing the productivity of biologically active diterpenes.
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