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This is an author produced version of a paper published in Planta Medica
This paper has been peer-reviewed but does not include the final publisher proof- corrections or journal pagination. 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 Access to the published version may require subscription. Published with
permission from: Thieme
Gothenburg University Publications http://gup.ub.gu.se Research article:
Antiplasmodial Quinones from Pentas longiflora
and Pentas lanceolata
Planta Medica 2012, 78, 31-35
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ORIGINAL PAPER
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*
AFFILIATION
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, Sweden and the Swedish NMR Centre, University of Gothenburg, P.O. Box 465, SE-40530 Gothenburg, Sweden CORRESPONDENCE
* (AbiyYenesew) Tel/Fax: +254-02-4446138, E-mail: [email protected] (Mate Erdelyi)
Tel: +46-31-7869033, E-mail: [email protected] ABSTRACT The dichloromethane/methanol (1:1) extracts of the roots of Pentas longiflora and
Pentas lanceolata showed low micromolar (IC50 = 0.9-3µg/mL) in vitro antiplasmodial activity against chloroquine-resistant (W2) and chloroquine-sensitive (D6) strains of Plasmodium falciparum. 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
(7), 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 [1]. 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 [2]. 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 [3], there is an urgent need for novel antimalarial agents. Because of the high cost of the few still effective antimalarial drugs [4] traditional medicine remains an important source of treatment in developing countries. Pentas longiflora Oliver (Rubiaceae) is an important medicinal plant of Tropical East Africa [5]. In Kenya, a decoction of its roots mixed with milk is taken as a cure for malaria [6]. Although, its leaves have previously been tested for in vitro antimalarial activity; no attempts were made to isolate and identify the antiplasmodial constituents [7]. Pentas lanceolata (Forsk.) is mostly found in the highlands of Kenya and was reported to exhibit micromolar in vitro antiplasmodial activity against P. falciparum [8]. 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 and P. lanceolata. 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. Plant material: The roots of Pentas longiflora were collected from Nandi East district, Kenya
(Nandi Hills-Chebarus location) in August, 2009. The roots of Pentas lanceolata were collected 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 (1.1 kg) were
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 damnacanthol (10, 50 mg).
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 (each ca. 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 damnacanthol (10, 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, 40
Drugs: 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 Pentas lanceolata 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. [10]. Cytotoxicity assay: Experimental details are given in the supporting information.
5,6-Dihydroxydamnacanthol (11). 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 showed significant antiplasmodial activities (Table 1). From the root extract of P. longiflora the naphthoquinone derivatives pentalongin (1) [11, 12, 13], psychorubrin (2) [14] and mollugin (3) [15, 13] were
chromatographically isolated, identified and tested for antiplasmodial activities. The major constituents 1 and 2 showed good to moderate activities (IC50 < 1 µg/mL) whereas 3 has
marginal inhibition against the W2 chloroquine-resistant and D6 chloroquine-sensitive strains of P. falciparum (Table 1). Although these compounds were previously reported [11, 12] and studied for antibacterial [16], antifungal [17], and antiviral [18] 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. lanceolata resulted in the isolation of seven known anthraquinones (Figure 2), spectroscopically (NMR and MS) identified as tectoquinone (4) [15], rubiadin (5) [19], rubiadin-1-methyl ether (6)
[19], nordamnacanthal (7) [20], damnacanthal (8) [19], lucidin-ω-methyl ether (9) [26, 29], and
damnacanthol (10) [21]. Three of these (7, 9 and 10) are reported here for the first time from the
genus Pentas. In agreement with previous investigations on rubiadin-1-methyl ether (6),
damnacanthal (8), and lucidin-ω-methyl ether (9) [22], the anthraquinones isolated from the roots
of P. lanceolata showed moderate antiplasmodial activities (Table 1). The methanol extract yielded further amounts of 4, 5, 10 and a new compound 11 (Figure 3)
isolated as a red solid. The Q-TOF-MS spectrum provided the exact mass at m/z 317.0659 [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 [23]. 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 [24] substitution allowing its placement at C-1 rather than C-3. Hence, in similarity to previously identified anthraquinones of the Rubiaceae family [25], 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 was
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 (L. fil, Rubiaceae) [26]. An additional evidence for the biosynthetic route in the family Rubiaceae [25] yielding compound 11 is the presence of the 2-ethoxyl-derivative of 11, 2-ethoxymethyl-3,5,6-
trihydroxy-1-methoxyanthraquinone, in the extract of Putoria calibrica (L. fil) [27]. 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) [25], 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 [25] which is shown for example for morindone, as reported from the cell cultures of Morinda citrifolia [29] and for putorinoside A, isolated from Putoria calábrica [27]. As a consequence of the biosynthetic pathway, most, if not all, anthraquinones carry a carbon substituent at position 2 in ring A [25]. One of the rare exceptions from the above rule is 2-ethoxy-1-hydroxyanthraquinone isolated from Morinda citrifolia [30], 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 [25] the currently accepted nomenclature it is placed unambiguously at position 2 in ring A. Not following the above convention [31] may be perplexing in the evaluation of biosynthetic routes and bioactivities. Hence, the compounds anthraquinone [31] 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, the comparably high cytotoxicity (Table 1) of 1 and 2 makes their direct application as antimalarial
agents virtually impossible. The anthraquinones isolated from Pentas lanceolata, 4-11, 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. longiflora. 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 ACKNOWLEDGEMENT 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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Sample (purity in %)
Antiplasmodial activity
Selectivity
50* (µg/mL)
Pentas longiflora (root extract) Pentalongin (1, ≥ 98%)
Psychorubrin (2, ≥ 98%))
Mollugin (3, ≥ 95%)
Pentas lanceolata (root extract) Tectoquinone (4, ≥ 98%)
Rubiadin (5, ≥ 98%)
Rubiadin-1-methyl ether (6, ≥ 98%)
Nordamnacanthal (7, ≥ 99%)
Damnacanthal (8, ≥ 99%)
Lucidin-ω-methyl ether (9, ≥ 98%)
Damnacanthol (10, ≥ 98%)
5,6-Dihydroxydamnacanthol ﴾11, > 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
Table 2. NMR Spectroscopic Data (DMSO-d6) for 5,6-dihydroxydamnacanthol (11)
position δH (J in Hz)
HMBC (2J, 3J)
C-1a, 2, 3, 4a, 10 Figure 1. Compounds isolated from the roots of Pentas longiflora

Figure 2
. Structures of known compounds isolated from the roots of Pentas lanceolata
Figure 3: Structure of 5, 6-Dihydroxydamnacanthol (11)

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