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Citation for the published paper:
Endale, Milkyas; Alao, John Patrick; Akala, H. M.; Rono, N. K.; Eyase, F. L.;
Derese, S.; Ndakala, A.; Mbugua, M.; Walsh, D. S.; Sunnerhagen, Per;
Erdelyi, Mate; Yenesew, Abiy
Antiplasmodial Quinones from Pentas longiflora and Pentas lanceolata
Planta Medica, 78 ( 1 ) s. 31-35
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Gothenburg University Publications
Antiplasmodial Quinones from Pentas longiflora and Pentas lanceolata
Planta Medica 2012
, 31-35 The publisher's copyright information is available on:
The publisher's version of the article is available on:
Antiplasmodial Quinones from Pentas longiflora
and Pentas lanceolata
Milkyas Endale, 1 John Patrick Alao, 2 Hoseah M. Akala, 3 Nelson K. Rono, 1 Fredrick L.
Eyase,3 Solomon Derese,1 Albert Ndakala, 1 Martin Mbugua, 1 Douglas S. Walsh, 3 Per
Sunnerhagen, 2 Mate Erdelyi, 4* Abiy Yenesew1*
1Department of Chemistry, University of Nairobi, P.O. Box 30197-00100, Nairobi, Kenya.
2Department of Cell- and Molecular Biology, University of Gothenburg, SE-405 30 Gothenburg,
Sweden. 3United States Army Medical Research Unit-Kenya, MRU 64109, APO, AE 09831-
4109, USA. 4Department of Chemistry, University of Gothenburg, SE-412 96 Gothenburg,
and the Swedish NMR Centre, University of Gothenburg, P.O. Box 465, SE-40530
* (AbiyYenesew) Tel/Fax: +254-02-4446138, E-mail: [email protected]
. (Mate Erdelyi)
Tel: +46-31-7869033, E-mail: [email protected]
The dichloromethane/methanol (1:1) extracts of the roots of Pentas longiflora
showed low micromolar (IC50 = 0.9-3µg/mL) in vitro
against chloroquine-resistant (W2) and chloroquine-sensitive (D6) strains of Plasmodium
. Chromatographic separation of the extract of Pentas longiflora
led to the isolation of
the pyranonaphthoquinones pentalongin (1
) and psychorubrin (2
) with IC50 values below 1
µg/mL and the naphthalene derivative mollugin (3
) which showed marginal activity. Similar
treatment of Pentas lanceolata
led to the isolation of eight anthraquinones (4-11
, IC50 = 5-
31µg/mL) of which one is new (5,6-dihydroxydamnacanthol, 11
) while three - nordamnacanthal
), lucidin-ω-methyl ether (9
and damnacanthol (10
) - are reported here for the first time from
the genus Pentas
. The compounds were identified by NMR and mass spectroscopic techniques.
KEYWORDS Pentas longiflora
, Pentas lanceolata
, Rubiaceae, anthraquinone, 5,6-
dihydroxydamnacanthol, pyranonaphthoquinone, malaria
According to the estimates of the World Health Organization almost one million deaths are
caused by malaria each year in Africa alone, of which most are children under the age of five .
In addition, this mosquito-borne disease has a serious economic impact due to loss of
commercial and labor outputs predominantly in countries with tropical and subtropical climates.
Over 300, 000, 000 people worldwide are infected, and each year, nearly one third of these
exhibit acute manifestations of the disease . While awaiting the development of a malaria
vaccine, millions of lives are still dependent upon treatment with chemotherapeutic agents. Since
most of the available drugs are becoming increasingly ineffective due to the rapid emergence of
resistant Plasmodium falciparum
strains , there is an urgent need for novel antimalarial
agents. Because of the high cost of the few still effective antimalarial drugs  traditional
medicine remains an important source of treatment in developing countries.
Oliver (Rubiaceae) is an important medicinal plant of Tropical East
Africa . In Kenya, a decoction of its roots mixed with milk is taken as a cure for malaria .
Although, its leaves have previously been tested for in vitro
antimalarial activity; no attempts
were made to isolate and identify the antiplasmodial constituents . Pentas lanceolata
is mostly found in the highlands of Kenya and was reported to exhibit micromolar in vitro
antiplasmodial activity against P. falciparum
. Although extracts of these plants have been
assayed against a range of diseases [8, 9], their constituents have not been investigated for
antiplasmodial activity. Motivated by the traditional uses and the preliminary screening reports
[7, 8, 9], we performed isolation, characterization and antiplasmodial investigation of
naphthoquinones and anthraquinones found in the extracts of the roots of P. longiflora
MATERIALS AND METHODS
General experimental procedures:
Column chromatography was performed on oxalic acid
impregnated silica gel [the silica gel was deactivated by mixing 2 kg of silica gel 60 (70-230
mesh) with 3% oxalic acid (30 g in 1L water) and allowed to stand for 30 min, filtered and dried
in an oven (100oC) for 45 min]. TLC was done using silica gel 60 F254 (Merck) precoated plates
NMR analyses were carried out on Varian 800, 600, 500 and 200 MHz spectrometers. Structural
assignment was performed based on gCOSY, gTOCSY, gNOESY, gHSQC, gHMBC, and
gH2BC spectra. ESI LC-MS was performed on a Perkin Elmer PE SCIEX API 150 EX
instrument equipped with a Turbolon spray ion source and a Gemini 5 mm C18 110 Å HPLC
column using a water-acetonitrile gradient (80:20 to 20:80). High-resolution mass spectral
analysis (Q-TOF-MS) was performed at Stenhagen Analyslab AB, Gothenburg, Sweden.
Compound purity was determined by NMR and HPLC. Analytical HPLC was run on a Hewlett
Packard Series 1050 HPLC using the Software Chromulan (Pikron Ltd), a Gemini 5 mm
C18 110 Å HPLC column and methanol-water mixture as eluent.
The roots of Pentas longiflora
were collected from Nandi East district, Kenya
(Nandi Hills-Chebarus location) in August, 2009. The roots of Pentas lanceolata
from Ngong forest in December, 2009. The plant materials were identified by Mr Patrick Chalo
Mutiso, School of Biological Sciences, University of Nairobi. Specimens are deposited at the
Herbarium, School of Biological Sciences, University of Nairobi under voucher numbers MEA
2009/001 (Pentas longiflora
) and MEA 2009/002 (Pentas lanceolata
Extraction and isolation:
The dried and grounded roots of Pentas longiflora
extracted by cold percolation with CH2Cl2:MeOH (1:1) three times for 24 hrs in each case. The
extract was concentrated using a rotary evaporator to yield a brownish crude extract (50 g, 4.54
%). A 35 g portion of the crude extract was subjected to column chromatography (80 cm length
and 80 mm diameter column size, 350 g oxalic acid impregnated silica gel) with increasing
gradient of acetone in n-hexane. Two hundred fractions (each ca.
200 mL) were collected.
Fractions 15-17 (2% acetone in n-hexane) were purified by Sephadex LH-20 (eluent
CH2Cl2:MeOH; 1:1) to give mollugin (3,
34 mg). Fractions 18-25 (3% acetone in n-hexane) were
purified by column chromatography on oxalic acid impregnated silica gel (eluent, 2% acetone in
n-hexane) to give pentalongin (1
, 40 mg). Fractions 90-112 (20% acetone in n-hexane) were
combined and purified by Sephadex LH-20 (eluent, CH2Cl2/MeOH; 1:1) to give psychorubrin (2
The ground roots (1.4 kg) of Pentas lanceolata
were extracted with CH2Cl2:MeOH (1:1) and
then with methanol three times for 24 hrs in each case. The extracts were concentrated using a
rotary evaporator to yield a brownish crude extract (57 g, 4.8%) and (100 g, 7.1%), respectively.
A 54 g portion of the crude CH2Cl2:MeOH (1:1) extract was subjected to column
chromatography (80 cm length and 80 mm diameter, 420 g oxalic acid impregnated silica gel)
with increasing gradient of ethyl acetate in n-hexane. A total of 550 fractions (each 200 mL)
were collected. Fractions 10-13 (2% ethyl acetate in n-hexane) were combined and purified on
Sephadex LH-20 (eluent, CH2Cl2:MeOH; 1:1) to give tectoquinone (4
, 40 mg). Fractions 15-25
(eluent, 3% ethyl acetate in n-hexane) were combined and purified by Sephadex LH-20 (eluent,
CH2Cl2:MeOH; 1:1) to give rubiadin (5
, 680 mg) and rubiadin-1-methyl ether (6
, 50 mg).
Fractions 30-35 (5% ethyl acetate in n-hexane as eluent) were combined and purified by column
chromatography to give damnacanthal (8,
320 mg). Fractions 53-65 (7% ethyl acetate in n-
hexane) were combined and purified on Sephadex LH-20 with CH2Cl2:MeOH; (1:1) as eluent to
give nordamnacanthal (7
, 20 mg). Fractions 131-135 (18% ethyl acetate in n-hexane) were
combined and purified using column chromatography on oxalic acid impregnated silica gel
(increasing gradient of ethyl acetate in n-hexane) to give lucidin-ω-methyl ether (9
, 50 mg).
Fractions 400-405 (50% ethyl acetate in n-hexane) were combined and purified by MPLC
(increasing gradient of ethyl acetate in n-hexane as eluent, flow rate of 30 mL/min) to give
The methanol extract (70 g) was subjected to column chromatography on oxalic acid
impregnated silica gel (80 cm length and 80 mm diameter, 500 g oxalic acid impregnated silica
gel) eluting with increasing gradient of methanol in dichloromethane. A total of 100 fractions
200 mL) were collected. Fractions 5-11 (100% dichloromethane) were combined and
purified on Sephadex LH-20 (eluent, CH2Cl2:MeOH; 1:1) to give rubiadin (5,
20 mg) and
rubiadin-1-methyl ether (6,
18 mg). Fractions 21-25 (eluent, 1% of methanol in CH2Cl2) were
combined and further purified on Sephadex LH-20 (eluent, CH2Cl2:MeOH; 1:1) to give
15 mg). Fractions 87-90 (5% MeOH in CH2Cl2) were combined and purified
using Sephadex LH-20 (eluent, CH2Cl2/MeOH; 1:1) to give 5,6-dihydroxydamnacanthol (11,
The reference antimalarial drugs, chloroquine and mefloquine having well-documented
IC50 values were tested alongside test samples pyranonaphthoquinones and a naphthalene
derivative isolated from the roots of Pentas longiflora;
anthraquinones isolated from the roots of
as described above.
Drug susceptibility testing:
Two laboratory clones of Plasmodium falciparum,
the Sierra Leone
D6 chloroquine-sensitive and the Indochina W2 chloroquine-resistant were maintained in
continuous culture to attain replication robustness prior to assays. Drug susceptibility was tested
by the Malaria SYBR Green I-based in vitro
assay technique described in Juma et al
Experimental details are given in the supporting information.
Red solid. UV (CH3OH) max 218, 274, 308, 424, nm. 1H
NMR (Table 2). 13C NMR (Table 2) HRMS (ESI): m/z
= 317.0659 [M+H]+, calcd. 317.06558.
RESULTS AND DISCUSSION
In our hands, the root extracts of P. longiflora
and P. lanceolata
antiplasmodial activities (Table 1). From the root extract of P. longiflora
derivatives pentalongin (1
) [11, 12, 13], psychorubrin (2
)  and mollugin (3
) [15, 13] were
chromatographically isolated, identified and tested for antiplasmodial activities. The major
showed good to moderate activities (IC50 < 1 µg/mL) whereas 3
marginal inhibition against the W2 chloroquine-resistant and D6 chloroquine-sensitive strains of
(Table 1). Although these compounds were previously reported [11, 12] and
studied for antibacterial , antifungal , and antiviral  properties, their antiplasmodial
activities are reported here for the first time.
Chromatographic separation of the dichloromethane/methanol (1:1) extract of the roots of P.
resulted in the isolation of seven known anthraquinones (Figure 2), spectroscopically
(NMR and MS) identified as tectoquinone (4
) , rubiadin (5
) , rubiadin-1-methyl ether (6
, nordamnacanthal (7
) , damnacanthal (8
) , lucidin-ω-methyl ether (9
) [26, 29], and
) . Three of these (7, 9
) are reported here for the first time from the
. In agreement with previous investigations on rubiadin-1-methyl ether (6
), and lucidin-ω-methyl ether (9
) , the anthraquinones isolated from the roots
of P. lanceolata
showed moderate antiplasmodial activities (Table 1).
The methanol extract yielded further amounts of 4
and a new compound 11
isolated as a red solid. The Q-TOF-MS spectrum provided the exact mass at m/z
[M+H]+, suggesting a molecular formula of C16H12O7. The UV-VIS absorption maxima at 218,
274, 308 and 424 nm suggests a 9,10-anthraquinone skeleton . Its 1H NMR spectrum (Table
2) revealed an aromatic singlet, a pair of ortho-
coupled aromatic protons, a methoxy and an
oxymethylene substituent as well as three solvent accessible and one chelated (H 12.40)
hydroxyl groups. Two carbonyl functionalities were indicated by 13C-NMR. HMBC correlation
of the methoxy protons with C-1, the oxymethylene protons with C-1, C-2 and C-3 (Table 2) are
consistent with the methoxy, oxymethylene, and a hydroxyl substitution in ring A. The high
chemical shift of the methoxyl group (C 62.4 ppm) is indicative of di-ortho
allowing its placement at C-1 rather than C-3. Hence, in similarity to previously identified
anthraquinones of the Rubiaceae family , ring A of 11
is oxygenated at C-1 and C-3 and has
the oxymethylene at C-2. The aromatic singlet at H 7.55 ppm (H-4) showed HMBC correlation
with the C-10 carbonyl (C 189.2 ppm), indicating their peri
position. The high chemical shift of
this carbonyl is indicative of a peri
hydroxyl group at C-5, which is further confirmed by the
HMBC correlation of the aromatic doublet at H 7.57 ppm (H-8) to the carbonyl at C 178.7 ppm
(C-9), but not with the one at C 188.5 ppm (C-10). These three bond heteronuclear correlations
confirm the dihydroxy-substitution at C-5 and C-6 in ring C. Therefore, compound 11
characterized as 3,5,6-trihydroxy-1-methoxy-2-hydroxymethyl-9,10-anthraquinone (Figure 3) for
which the trivial name 5,6-dihydroxydamnacanthol is proposed. Our assignation is in good
agreement with that of the recently reported and closely-related 2-hydroxymethyl-1-methoxy-
3,5,6-trihydroxyanthraquinone-3-O--glycopyranoside, isolated from Putoria calabrica
Rubiaceae) . An additional evidence for the biosynthetic route in the family Rubiaceae 
yielding compound 11
is the presence of the 2-ethoxyl-derivative of 11
trihydroxy-1-methoxyanthraquinone, in the extract of Putoria calibrica
(L. fil) .
Based on the biosynthesis of anthraquinones of the Rubiaceae, most of these compounds are
substituted with hydroxyl, methoxyl and/or methyl groups in ring A (Figure 2) , and some
carry additional hydroxyl or alkoxyl groups in ring C, mainly at positions 5 and 6 [25, 28]. These
latter oxygen atoms are introduced at a late stage of the biogenesis  which is shown for
example for morindone, as reported from the cell cultures of Morinda citrifolia
 and for
putorinoside A, isolated from Putoria calábrica
. As a consequence of the biosynthetic
pathway, most, if not all, anthraquinones carry a carbon substituent at position 2 in ring A .
One of the rare exceptions from the above rule is 2-ethoxy-1-hydroxyanthraquinone isolated
from Morinda citrifolia
, a compound lacking carbon (CH2, CHO, CH, etc) substitution at C-
2. We would like to emphasize that if carbon substitution is present in an anthraquinone derived
from the family Rubiaceae, based on biogenetics  the currently accepted nomenclature it is
placed unambiguously at position 2 in ring A. Not following the above convention  may be
perplexing in the evaluation of biosynthetic routes and bioactivities. Hence, the compounds
anthraquinone  should be correctly named as 5,6-dimethoxy-2-methyl-9,10-anthraquinone
and 6-hydroxy-5-methoxy-2-methyl-9,10-anthraquinone. Since complete and correctly assigned
spectroscopic characterization was not available for several anthraquinones described here,
detailed MS, and 1H and 13C NMR analysis (based on homo- and heteronuclear correlation
spectra providing unambiguous assignment) is reported (Supporting information).
Despite their promising activity against the W2 and D6 strains of Plasmodium falciparum
comparably high cytotoxicity (Table 1) of 1
makes their direct application as antimalarial
agents virtually impossible. The anthraquinones isolated from Pentas lanceolata
, show low
cytotoxicity indicating the safer applicability of the anthraquinone containing indigenous
decoction of P. lanceolata
as compared to that of the pyranonaphtoquinone containing P.
In conclusion, the pyranonaphthoquinones and some of the anthraquinones isolated from the
roots of P. lanceolata
and P. longiflora
showed good to moderate antiplasmodial activities
against the W2 and D6 strains of Plasmodium falciparum
, and an overall low cytotoxicity for
anthraquinones. Careful analysis of their structure-activity relationship followed by rational
synthetic modifications has potential for identifying more applicable agents in the fight against
M. Endale is thankful to the German Academic Exchange service
(DAAD) and the Natural Products Research Network for Eastern and Central Africa
(NAPRECA) for a Ph.D. Scholarship. J.P. Alao and P. Sunnerhagen are thankful to the Chemical
Biology Platform at the University of Gothenburg. M. Erdelyi is thankful for the financial
support of the Swedish Research Council (VR2007-4407) and the Royal Society of Arts and
Sciences in Göteborg.
Herewith we declare the absence of any conflict of interest, financial or personal, for all authors.
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Table 1. In vitro
antiplasmodial activity and cytotoxicity of crude extracts and pure compounds
Sample (purity in %)
, ≥ 98%)
, ≥ 98%))
, ≥ 95%)
, ≥ 98%)
, ≥ 98%)
Rubiadin-1-methyl ether (6
, ≥ 98%)
, ≥ 99%)
, ≥ 99%)
Lucidin-ω-methyl ether (9
, ≥ 98%)
, ≥ 98%)
, > 99%﴿
* Data are the mean of at least 3 independent experiments. §
The mean value of at least 6
independent experiments are given; 95% confidence interval and dose-response curves are
presented in the supporting information
NMR Spectroscopic Data (DMSO-d6) for 5,6-dihydroxydamnacanthol (11
position δH (J in Hz)
C-1a, 2, 3, 4a, 10
Compounds isolated from the roots of Pentas longiflora
. Structures of known compounds isolated from the roots of Pentas lanceolata
Structure of 5, 6-Dihydroxydamnacanthol (11
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