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Transmission blocking effects of neem (Azadirachta indica) seed kernel limonoids on Plasmodium berghei early sporogonic development
Fitoterapia 114 (2016) 122–126
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Fitoterapia journal homepage: www.elsevier.com/locate/fitote
Transmission blocking effects of neem (Azadirachta indica) seed kernel limonoids on Plasmodium berghei early sporogonic development Sofia Tapanelli a, Giuseppina Chianese b, Leonardo Lucantoni c, Rakiswendé Serge Yerbanga d, Annette Habluetzel a,⁎, Orazio Taglialatela-Scafati b,⁎ a
School of Pharmacy, University of Camerino, Piazza dei Costanti, 62032 Camerino, MC, Italy Department of Pharmacy, University of Naples Federico II, Via Montesano 49, 80131 Naples, Italy Discovery Biology, Eskitis Institute for Drug Discovery, Griffith University, Nathan, 4111, Queensland, Australia d Institut de Recherche en Sciences de la Santé, 01BP545 Bobo Dioulasso, Burkina Faso b c
a r t i c l e
i n f o
Article history: Received 15 June 2016 Received in revised form 8 September 2016 Accepted 14 September 2016 Available online 15 September 2016 Keywords: Azadirachta indica Neem Malaria Transmission blocking Deacetylnimbin
a b s t r a c t Azadirachta indica, known as neem tree and traditionally called “nature's drug store” makes part of several African pharmacopeias and is widely used for the preparation of homemade remedies and commercial preparations against various illnesses, including malaria. Employing a bio-guided fractionation approach, molecules obtained from A. indica ripe and green fruit kernels were tested for activity against early sporogonic stages of Plasmodium berghei, the parasite stages that develop in the mosquito mid gut after an infective blood meal. The limonoid deacetylnimbin (3) was identified as one the most active compounds of the extract, with a considerably higher activity compared to that of the close analogue nimbin (2). Pure deacetylnimbin (3) appeared to interfere with transmissible Plasmodium stages at a similar potency as azadirachtin A. Considering its higher thermal and chemical stability, deacetylnimbin could represent a suitable alternative to azadirachtin A for the preparation of transmission blocking antimalarials. © 2016 Published by Elsevier B.V.
1. Introduction Prompt, diagnosis-based treatment with an effective drug is crucial for the management of patients affected with malaria. Although significant progress has been achieved in rolling back this mosquito born parasitic disease over the last decade, many sub-Saharan countries still face major challenges to assure universal access to antimalarial treatment. This explains the still unacceptably high death toll of malaria: in 2015 the number of malaria deaths in children aged under five globally amounted about 300,000, 90% of which occurring in Africa and most cases being due to infection with Plasmodium falciparum [1]. Noteworthy, the two historically most important antimalarial drugs derive from plants, namely quinine from Cinchona tree and artemisinin from Qinghao (Artemisia annua L, Asteraceae). Both these plants have been used for centuries for the management of malarial fevers by the indigenous populations of South America and China, respectively. Since the beginning of the XXI century, artemisinin-based combination therapy (ACT) is recommended as first line treatment for the management of uncomplicated malaria [1]. Its wide employment has undoubtedly contributed to the reduction of malaria morbidity and mortality, and recent figures by ⁎ Corresponding authors. E-mail addresses: [email protected] (A. Habluetzel), [email protected] (O. Taglialatela-Scafati).
http://dx.doi.org/10.1016/j.fitote.2016.09.008 0367-326X/© 2016 Published by Elsevier B.V.
WHO indicate that control efforts (treatment of patients and vector control by insecticide-treated bed nets) have allowed to avert globally about 6.2 million deaths in the period from 2001 to 2015 [1]. However, ACTs impact only to a limited extent on gametocytes, the transmission stage of the parasite. Gametocytes reach peak densities in the blood stream 6 to 10 days after the onset of a clinical malaria episode [2] and persist in the blood circulation for prolonged periods, remaining able to infect hematophagous Anopheles vectors, thus perpetuating transmission. Primaquine is the drug of choice to eliminate mature gametocytes, but the drug can cause hemolysis in individuals with glucose 6-phosphate dehydrogenase (G6PD) deficiency and, therefore, has limited clinical applicability [3]. Current antimalarial drug research efforts focus on the discovery of new curative antimalarials, given the threat of emergence and spread of P. falciparum parasite strains resistant to ACTs [4]. However, growing attention is put on the discovery of transmission blocking molecules able to reduce the diffusion of the disease by hitting mature Plasmodium gametocytes in the human host and/or early sporogonic stages developing in the mosquito mid-gut. Ideally, the next generation antimalarial drug candidates should be effective against asexual and transmissible stages. In addition, if targeting the early sporogonic stages, they should have a prolonged half-life, matching the persistence of gametocytes in the patients' blood circulation for 1 to 3 weeks after a malaria attack [5], thus blocking transmission in the mosquitoes.
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Azadirachta indica A. Juss. (Meliaceae), well known as neem tree and renowned as medicinal plant, may represent a treasure trove for the discovery of new antimalarials. The plant, acknowledged for its multiple therapeutic properties, has been declared “the tree of the XXI century” by the United Nations [6]. A large number of secondary metabolites have been purified from different parts of neem, with a large predominance of terpenoids. Fruits and leaves are rich in limonoids [7], stem bark in diterpenoids, while the root bark has been found to produce both tri- and diterpenoids [8]. In particular, seed kernels have been recognized as a prolific source of limonoids and their chemical composition has been reported to change according to the maturation stage [9]. Azadirachtin A (1) and its analogues, nimbin (2) and salannin (3) are abundantly present in the unripe green fruit kernels, while azadirone (4) (Fig. 1), azadiradione and epoxyazadiradione are present in higher concentrations in the ripe kernels. Various neem parts (leaves, stem bark, roots and seeds) have been used traditionally in Ayurvedic medicine and are still widely employed in India and African countries for the preparation of antimalarial remedies [10]. Validation studies conducted with leaf and bark neem extracts to confirm activity against erythrocyte stages produced contrasting results [11]. On the other hand, gametocytocidal activity was demonstrated in seed kernel and leaf preparations [12]. Parasite stages developing in the mosquito vector have been found to be possible targets for neem constituents. Investigation on pure neem metabolites revealed that gedunin and azadirone (4) inhibit the development of the malaria parasite blood stages in vitro [13] and that azadirachtin A (1) interferes with the parasite transmission from human to the mosquito host blocking the development of the motile male malarial gamete in vitro [14,15]. The commercial product NeemAzal®, derived from seed kernel and rich in azadirachtin A (1), has been recently found to completely block parasite transmission [16]. The present study was aimed at investigating in detail the antimalarial potential of neem fruit limonoids, selecting in particular those able to interfere with early sporogonic development of the parasite. Given the previously reported differences in their chemical compositions, we analyzed seed kernel extracts derived from green as well as ripe fruits, characterizing fractions and pure molecules for activity against early sporogonic stages of Plasmodium berghei by using an in vitro ookinete development assay (ODA). 2. Experimental 2.1. General experimental procedures
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All the solvents used for extraction, medium pressure liquid chromatography (MPLC), HPLC and the chemicals for biological tests were purchased from Sigma-Aldrich (Milan, Italy). All solvents were of analytical grade and used as supplied. Ultrapure water was obtained from a MilliQ plus system (Millipore, Bedford, MA). 1H (500 MHz) and 13C (125 MHz) NMR spectra were measured on a Varian INOVA spectrometer. Chemical shifts were referenced to the residual solvent signal (CDCl3: δH 7.26, δC 77.0). Low- and high-resolution ESI-MS spectra were performed on a LTQ OrbitrapXL (Thermo Scientific) mass spectrometer. MPLC was performed on a Büchi apparatus using a silica gel (230–400 mesh) column; HPLC were achieved on a Knauer apparatus equipped with a refractive index detector. Synergi™ (4 μm, Polar-RP 80 Å, 250 × 4.6 mm, Phenomenex) column was used, with elution with MeOH/H2O mixtures and 0.8 ml/min as flow rate. 2.2. Plant material Azadirachta indica ripe and green fruits were collected near Farakoba, in Burkina Faso in May 2014 by R. S. Y. and Dr. Pascal Dipama of the Institut de Recherche en Sciences de la Santé (IRSS), BoboDioulasso. The plant was identified by Dr. Tahita Paulette (Institut de l'Environnement et de Recherches Agricole, Center of Protection des Végétaux) and deposited at the Unit of “Parcelle expérimentale de IRSS Bobo Dioulasso”, voucher number A. indica RF052014 and A indica GF052014 for Azadrirachta indica ripe and green fruits, respectively. 2.3. Extraction and isolation A. indica ripe and green fruits were dried in the shade, epicarp and mesocarp were removed and seed kernels grinded to obtain a fine powder. Ripe (RF: 135 g) and green (GF: 135 g) seed kernel powder were repeatedly extracted with MeOH (1.5 L × 3) at room temperature for 24 h and then concentrated under vacuum to obtain crude methanol extracts (RF: 26 g; GF: 24 g). The obtained materials were then partitioned between H2O and EtOAc to yield organic extract (RF: 12.0 g; GF: 14.6 g). EtOAc extracts were subjected to chromatography over silica column (230–400 mesh) eluting with a solvent gradient of increasing polarity from n-hexane to EtOAc. Fractions eluted with n-hexane/EtOAc 7:3 afforded a mixture of limonoids (240 mg) including only traces of nimbin (3) (2.0 mg) from GF EtOAc extract, while 98.8 mg of nimbin were obtained from RF. Fractions eluted with n-hexane/EtOAc 6:4 were further purified by RP-HPLC (MeOH/H2O 9:1) to obtain deacetylnimbin (5) from both extracts (323.0 mg from GF and
426.0 mg from RF). Fractions eluted with n-hexane/EtOAc 55:45 Fig. 1. Chemical structure of neem limonoids.
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contained pure salannin (451 mg from GF and 376 mg from RF). Lastly, the fractions eluted with increasing polarity from n-hexane/EtOAc 5:5 to 2:8 gave a mixture of azadirachtin A (1) and analogues (ca. 1.0 g of azadirachtins from both extracts). 2.4. Animals, parasite and ookinete development assay Two to four weeks old female BALB/c mice (20 ± 3 g) were used for the experiments. Mice were reared and maintained in the animal breeding facilities of the University of Camerino (Italy) and experimental protocols were performed in accordance with the Italian Legislative Decree on the “Use and protection of laboratory animals” (D. Lgs. 116 of 10/27/ 92). The rodent malaria parasite Plasmodium berghei PbCTRPp.GFP [17] was used in these studies. The parasite was maintained in the laboratory of parasitology at the University of Camerino by mouse to mouse acyclic and mouse to mosquito to mouse cyclic passages. To avoid repeated acyclic passages, which could compromise the parasite continued ability to transmit to mosquitoes [18], mice were infected for each experiment with PbCTRPp.GFP thawed from glass hematocrit capillaries kept in liquid nitrogen storage. To assess in vitro transmission blocking (TB) activity of fractions and compounds targeted to early sporogonic development, the ookinete development assay (ODA) was employed as described in [19]. Briefly, for each experiment 3 donor mice were inoculated i.p. with 50–75 μl of PbCTRPp.GFP infected blood, and four days after inoculation blood from mice with parasitemia around 2% was collected and 107 parasitized red blood cells i.p. inoculated into phenylhydrazine (200 μl, 1.2 mg/ml) pre-treated BALB/c mice (n = 6). On day 4 post infection, Giemsa stained blood smears were prepared from experimental mice. Mice with parasitemia around 5% and with mature gametocytes were assessed for microgamete exflagellation. A drop of blood was taken from the mouse tail tip and diluted in exflagellation medium [19]; ookinete medium was prepared by adjusting exflagellation medium to pH 7.4 and by adding 20% heat inactivated fetal bovine serum and 1% penicillin (10.000 U)/streptomycin (10 mg/ml) before use. Blood samples diluted in exflagellation medium (1:25) were placed in handmade coverslip glass slide chambers and incubated for 20 min at 19 °
C. Numbers of microgamete-extruding gametocytes, visible as vibrating “exflagellation centers” were counted under the microscope (400 ×). Mice showing more than 3 exflagellation centers per 1000 red blood cells were selected as blood donors for the ookinete development assay (ODA). Fractions (n = 28 GF; n = 23 RF) containing mixture of limonoids and pure molecules were dissolved in DMSO to obtain stock solutions at 30 mg/ml. Ookinete medium was dispensed to the wells of 96-wells microplates and samples of the pre-diluted fractions and molecules were added to each well to obtain the desired final concentration in a total volume of 100 μl, at a final DMSO concentration of 0.1% (0.1% DMSO as control). Infected blood collected from donor mice was added to the wells at a 1:20 dilution (corresponding to a hematocrit of 1–2%) and mixed swiftly. The plate was immediately transferred to 19 °C and incubated for 22–24 h. To assess the development of early sporogonic stages, each well content was mixed, then aliquots were diluted 1:50 in PBS (pH 7.4) in a new plate and GFP-expressing zygotes and ookinetes were counted at the fluorescent microscope (FITC fluorescent filter, 400× magnification). All samples were independently tested in triplicate wells using blood from at least 2 mice on different plates.
3. Results and discussion Seed kernel powders from ripe (RF) and unripe (GF) neem fruits were exhaustively extracted with MeOH and the obtained materials were then partitioned between H2O and EtOAc to yield 12.0 g of organic extract from RF and 14.6 g from GF. The EtOAc extracts from GF and RF were then subjected to MPLC chromatography over silica gel to afford nimbin (2) (much more abundant in ripe fruits), salannin (3), azadirachtin A (1) (both present in the same amounts in GF and RF) and deacetylnimbin (5) (present in large amounts in both extracts). These compounds were identified on the basis of a comparison of their spectral data with those present in the literature [20–22]. The very similar composition found for ripe and green fruits (with the exception of nimbin content) contrasts with previous reports indicating a marked change in the secondary metabolite content depending on the maturation stage [9].
Fig. 2. Inhibitory activity of A. indica fruit fractions on early sporogonic development at a test concentration of 50 μg/ml; A) Effects of fractions from green fruits (GF); B) Effects of fractions from ripe fruits (RF); FA = fatty acids.
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Fig. 3. Impact of fraction GF 12 on ookinete maturation. A) In wells treated with GF_12 at 50 μg/ml, ESS developed to round zygotes as in DMSO control wells (grey bars) whereas maturation to elongated ookinetes was impaired (black bars); B) Giemsa stained EES after 22–24 h in vitro culture (1000× magnification); a) mature, banana-shaped ookinete from DMSO 0.1% control well; b) zygote form from a GF_12 treated well.
Screening of fractions obtained from A. indica GF and RF seed kernels at 50 μg/ml revealed that some of them could inhibit early sporogonic development in vitro (Fig. 2). Parasitized blood incubated with azadirachtin(s)-containing fractions from green fruits (GF_19 to GF_23) induced a reduction of about 40%–70% of early sporogonic stage (ESS) development. These data appeared in line with our earlier results, which showed that pure azadirachtin A (1) was able to block ESS development almost completely at 50 μg/ml and reduced ESS by 50% at 17 μM (CI95:15–19 μM) [16]. Fractions from ripe fruits containing nimbin (2), tested at 50 μg/ml, reduced ESS development by about 40% (CI95: 34, 5–45, 5), while a complete block of development was observed with ripe fruit fraction RF_15 rich in deacetylnimbin (5). On the other hand, salannin (3), reported in the literature to possess moderate activity against Plasmodium falciparum asexual blood stages in vitro [23], did not reveal any effect on ESS. Also no activity on ESS was observed with either ripe or green fruit extract fractions containing fatty acids and glycerides (GF_1 to GF_9 and RF_1 to RF_7, data not shown). Interestingly, ookinete development assay (ODA) screening at 50 μg/ ml revealed a marked reduction of total ESS numbers by RF_15 (from ripe fruit) but not by GF_12 (from green fruit) (Fig. 2), although the two fractions appeared to contain similar amounts of deacetylnimbin (5). In order to clarify this issue, stage specific effects on the parasite undergoing ESS development were examined in detail. Microscope observations of GF-12 treated wells revealed zygote formation, but few zygotes underwent further development to form banana shaped ookinetes (Fig. 3). Differential counts showed that after 22–24 h of incubation with 50 μg/ml GF_12 only 35% (SD ± 10) of parasites fully developed into banana-shaped ookinetes, compared to 80% (SD ± 9) in the DMSO control wells (*P = 0.049). Pure deacetylnimbin (5), obtained after HPLC purification from both fractions GF_12 and RF_15, was tested in the ODA at 50 μg/ml (100 μM) and was found to completely inhibit ESS development confirming the sporontocidal activity of the molecule. The initial discrepant activities of fractions GF_12 and RF_15 might be therefore attributed to the presence of minor constituents in GF_12 which, interacting with deacetylnimbin (5), cause an arrest/delay in zygote development, rather than killing zygotes or interfering with earlier developmental processes. Testing the pure compound deacetylnimbin (5) in the ODA at various concentrations, the molecule displayed a dose-dependent inhibitory activity which reached complete inhibition at 100 μM and faded to negligible activity below 0.7 μM. In the range of 6–25 μM an inhibitory activity of about 50% (40% - 60%) was observed (Fig. 4). The dose-
dependency of the antiplasmodial effect of deacetylnimbin (3) points to a specific activity against the ESS. The pattern of antimalarial transmission blocking activity determined for the main limonoids present in neem fruits has been summarized in Fig. 5. The structural similarity within these compounds and their different activities allow the drawing of preliminary structure-activity relationships. The presence of an α,β unsaturated ketone in ring A seems to be not directly related to the biological activity, since this functionality is present in deacetylnimbin but absent in azadirachtin A. Similarly, azadirachtin A and salannin exhibited very different bioactivities although they share practically the same structure of rings A/B. Interestingly, both the active limonoids show the presence of a free\\OH group at ring B and the key role of this group at that position is clearly evidenced by the poor activity of nimbin, which differs only for the acetylation of that group. Interestingly, in 1987 Yamasaki and Klocke had found that the growth-inhibitory activity of azadirachtin A against the major agricultural insect pest Heliothis uirescens was strictly dependent from the presence of free hydroxyl groups on the structure, which must also include a lipophilic moiety (possibly for transport phenomena) [24]. Studies aimed at assessing the cytotoxicity of neem tree limonoids using N1E-115 neuroblastoma (mouse), 143B.TK- osteosarcoma (human) and Sf9 (insect) cultured cell lines had already assessed the
Fig. 4. In vitro inhibitory activity of deacetylnimbin (5) on ESS development of P. berghei CTRPgfp at different concentrations. Columns represent the mean % inhibition at a given concentration from tests performed in triplicate wells on at least 2 plates with blood from different mice and bars depict the ±SD.
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Fig. 5. Transmission blocking activity of the main limonoids of neem fruits.
lack of toxicity of deacetylnimbin (5), nimbin (2) and azadirachtin (1) at concentrations up to 200 μM [25]. Evaluation of nimbin and deacetylnimbin on leukemia (HL60), lung (A549), stomach (AZ521), and breast (SK-BR-3) cancer cell lines revealed that nimbin (2) was low to nontoxic for any of these cell lines, while deacetylnimbin (5) had a selective activity on the leukemia cancer cell line (IC50 of 18 ± 3 μM) worthy of further investigation [26]. In summary, our investigation has revealed that extracts of neem seed kernels, at different maturation stages, contain large amounts of deacetylnimbin (5), a limonoid endowed with a considerable transmission blocking activity on Plasmodium berghei early sporogonic development. Since deacetylnimbin shows a better thermal and light stability compared to the somewhat unstable azadirachtin A, further studies are encouraged to explore its biological properties and mechanism(s) of action in greater detail. Hopefully, once the activity will be confirmed also on Plasmodium falciparum parasites, pure deacetylnimbin, deacetylnimbin-enriched neem kernel extracts, or even simplified synthetic analogues, could fulfill the need for transmission blocking agents to be included in combination treatments aimed not only at curing malaria patients but also at preventing the diffusion of this deadly infection. Acknowledgments We thank Prof. Robert Sinden from Imperial College, London, for kindly providing Plasmodium berghei (PbCTRPp.GFP). References [1] World Health Organization, www.who.int (Accessed on May 22, 2016). [2] K.I. Barnes, N.J. White, Population biology and antimalarial resistance: the transmission of antimalarial drug resistance in Plasmodium falciparum, Acta Trop. 94 (2005) 230–240. [3] R.E. Howes, K.E. Battle, A.W. Satyagraha, J.K. Baird, S.I. Hay, G6PD deficiency: global distribution, genetic variants and primaquine therapy, Adv. Parasitol. 81 (2013) 133–201. [4] A. Mbengue, S. Bhattacharjee, T. Pandharkar, H. Liu, G. Estiu, R.V. Stahelin, S.S. Rizk, D.L. Njimoh, Y. Ryan, K. Chotivanich, C. Nguon, M. Ghorbal, J.-J. Lopez-Rubio, M. Pfrender, S. Emrich, N. Mohandas, A.M. Dondorp, O. Wiest, K. Haldar, A molecular mechanism of artemisinin resistance in Plasmodium falciparum malaria, Nature 520 (2015) 683–687. [5] K. Stepniewska, R.N. Price, C.J. Sutherland, C.J. Drakeley, L. von Seidlein, F. Nosten, N.J. White, Plasmodium falciparum gametocyte dynamics in areas of different malaria endemicity, Malar. J. 7 (2008) e249. [6] United Nations Environment Programme Neem: the UN's tree of the 21st Century. Nairobi: United Nations Environment Programme, http://www.unep.org/wed/ tree-a-day/neem.asp2012. [7] G. Chianese, S.R. Yerbanga, L. Lucantoni, A. Habluetzel, N. Basilico, D. Taramelli, E. Fattorusso, O. Taglialatela-Scafati, Antiplasmodial triterpenoids from the fruits of neem, Azadirachta indica, J. Nat. Prod. 73 (2010) 1448–1452.
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Fitoterapia journal homepage: www.elsevier.com/locate/fitote
Transmission blocking effects of neem (Azadirachta indica) seed kernel limonoids on Plasmodium berghei early sporogonic development Sofia Tapanelli a, Giuseppina Chianese b, Leonardo Lucantoni c, Rakiswendé Serge Yerbanga d, Annette Habluetzel a,⁎, Orazio Taglialatela-Scafati b,⁎ a
School of Pharmacy, University of Camerino, Piazza dei Costanti, 62032 Camerino, MC, Italy Department of Pharmacy, University of Naples Federico II, Via Montesano 49, 80131 Naples, Italy Discovery Biology, Eskitis Institute for Drug Discovery, Griffith University, Nathan, 4111, Queensland, Australia d Institut de Recherche en Sciences de la Santé, 01BP545 Bobo Dioulasso, Burkina Faso b c
a r t i c l e
i n f o
Article history: Received 15 June 2016 Received in revised form 8 September 2016 Accepted 14 September 2016 Available online 15 September 2016 Keywords: Azadirachta indica Neem Malaria Transmission blocking Deacetylnimbin
a b s t r a c t Azadirachta indica, known as neem tree and traditionally called “nature's drug store” makes part of several African pharmacopeias and is widely used for the preparation of homemade remedies and commercial preparations against various illnesses, including malaria. Employing a bio-guided fractionation approach, molecules obtained from A. indica ripe and green fruit kernels were tested for activity against early sporogonic stages of Plasmodium berghei, the parasite stages that develop in the mosquito mid gut after an infective blood meal. The limonoid deacetylnimbin (3) was identified as one the most active compounds of the extract, with a considerably higher activity compared to that of the close analogue nimbin (2). Pure deacetylnimbin (3) appeared to interfere with transmissible Plasmodium stages at a similar potency as azadirachtin A. Considering its higher thermal and chemical stability, deacetylnimbin could represent a suitable alternative to azadirachtin A for the preparation of transmission blocking antimalarials. © 2016 Published by Elsevier B.V.
1. Introduction Prompt, diagnosis-based treatment with an effective drug is crucial for the management of patients affected with malaria. Although significant progress has been achieved in rolling back this mosquito born parasitic disease over the last decade, many sub-Saharan countries still face major challenges to assure universal access to antimalarial treatment. This explains the still unacceptably high death toll of malaria: in 2015 the number of malaria deaths in children aged under five globally amounted about 300,000, 90% of which occurring in Africa and most cases being due to infection with Plasmodium falciparum [1]. Noteworthy, the two historically most important antimalarial drugs derive from plants, namely quinine from Cinchona tree and artemisinin from Qinghao (Artemisia annua L, Asteraceae). Both these plants have been used for centuries for the management of malarial fevers by the indigenous populations of South America and China, respectively. Since the beginning of the XXI century, artemisinin-based combination therapy (ACT) is recommended as first line treatment for the management of uncomplicated malaria [1]. Its wide employment has undoubtedly contributed to the reduction of malaria morbidity and mortality, and recent figures by ⁎ Corresponding authors. E-mail addresses: [email protected] (A. Habluetzel), [email protected] (O. Taglialatela-Scafati).
http://dx.doi.org/10.1016/j.fitote.2016.09.008 0367-326X/© 2016 Published by Elsevier B.V.
WHO indicate that control efforts (treatment of patients and vector control by insecticide-treated bed nets) have allowed to avert globally about 6.2 million deaths in the period from 2001 to 2015 [1]. However, ACTs impact only to a limited extent on gametocytes, the transmission stage of the parasite. Gametocytes reach peak densities in the blood stream 6 to 10 days after the onset of a clinical malaria episode [2] and persist in the blood circulation for prolonged periods, remaining able to infect hematophagous Anopheles vectors, thus perpetuating transmission. Primaquine is the drug of choice to eliminate mature gametocytes, but the drug can cause hemolysis in individuals with glucose 6-phosphate dehydrogenase (G6PD) deficiency and, therefore, has limited clinical applicability [3]. Current antimalarial drug research efforts focus on the discovery of new curative antimalarials, given the threat of emergence and spread of P. falciparum parasite strains resistant to ACTs [4]. However, growing attention is put on the discovery of transmission blocking molecules able to reduce the diffusion of the disease by hitting mature Plasmodium gametocytes in the human host and/or early sporogonic stages developing in the mosquito mid-gut. Ideally, the next generation antimalarial drug candidates should be effective against asexual and transmissible stages. In addition, if targeting the early sporogonic stages, they should have a prolonged half-life, matching the persistence of gametocytes in the patients' blood circulation for 1 to 3 weeks after a malaria attack [5], thus blocking transmission in the mosquitoes.
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Azadirachta indica A. Juss. (Meliaceae), well known as neem tree and renowned as medicinal plant, may represent a treasure trove for the discovery of new antimalarials. The plant, acknowledged for its multiple therapeutic properties, has been declared “the tree of the XXI century” by the United Nations [6]. A large number of secondary metabolites have been purified from different parts of neem, with a large predominance of terpenoids. Fruits and leaves are rich in limonoids [7], stem bark in diterpenoids, while the root bark has been found to produce both tri- and diterpenoids [8]. In particular, seed kernels have been recognized as a prolific source of limonoids and their chemical composition has been reported to change according to the maturation stage [9]. Azadirachtin A (1) and its analogues, nimbin (2) and salannin (3) are abundantly present in the unripe green fruit kernels, while azadirone (4) (Fig. 1), azadiradione and epoxyazadiradione are present in higher concentrations in the ripe kernels. Various neem parts (leaves, stem bark, roots and seeds) have been used traditionally in Ayurvedic medicine and are still widely employed in India and African countries for the preparation of antimalarial remedies [10]. Validation studies conducted with leaf and bark neem extracts to confirm activity against erythrocyte stages produced contrasting results [11]. On the other hand, gametocytocidal activity was demonstrated in seed kernel and leaf preparations [12]. Parasite stages developing in the mosquito vector have been found to be possible targets for neem constituents. Investigation on pure neem metabolites revealed that gedunin and azadirone (4) inhibit the development of the malaria parasite blood stages in vitro [13] and that azadirachtin A (1) interferes with the parasite transmission from human to the mosquito host blocking the development of the motile male malarial gamete in vitro [14,15]. The commercial product NeemAzal®, derived from seed kernel and rich in azadirachtin A (1), has been recently found to completely block parasite transmission [16]. The present study was aimed at investigating in detail the antimalarial potential of neem fruit limonoids, selecting in particular those able to interfere with early sporogonic development of the parasite. Given the previously reported differences in their chemical compositions, we analyzed seed kernel extracts derived from green as well as ripe fruits, characterizing fractions and pure molecules for activity against early sporogonic stages of Plasmodium berghei by using an in vitro ookinete development assay (ODA). 2. Experimental 2.1. General experimental procedures
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All the solvents used for extraction, medium pressure liquid chromatography (MPLC), HPLC and the chemicals for biological tests were purchased from Sigma-Aldrich (Milan, Italy). All solvents were of analytical grade and used as supplied. Ultrapure water was obtained from a MilliQ plus system (Millipore, Bedford, MA). 1H (500 MHz) and 13C (125 MHz) NMR spectra were measured on a Varian INOVA spectrometer. Chemical shifts were referenced to the residual solvent signal (CDCl3: δH 7.26, δC 77.0). Low- and high-resolution ESI-MS spectra were performed on a LTQ OrbitrapXL (Thermo Scientific) mass spectrometer. MPLC was performed on a Büchi apparatus using a silica gel (230–400 mesh) column; HPLC were achieved on a Knauer apparatus equipped with a refractive index detector. Synergi™ (4 μm, Polar-RP 80 Å, 250 × 4.6 mm, Phenomenex) column was used, with elution with MeOH/H2O mixtures and 0.8 ml/min as flow rate. 2.2. Plant material Azadirachta indica ripe and green fruits were collected near Farakoba, in Burkina Faso in May 2014 by R. S. Y. and Dr. Pascal Dipama of the Institut de Recherche en Sciences de la Santé (IRSS), BoboDioulasso. The plant was identified by Dr. Tahita Paulette (Institut de l'Environnement et de Recherches Agricole, Center of Protection des Végétaux) and deposited at the Unit of “Parcelle expérimentale de IRSS Bobo Dioulasso”, voucher number A. indica RF052014 and A indica GF052014 for Azadrirachta indica ripe and green fruits, respectively. 2.3. Extraction and isolation A. indica ripe and green fruits were dried in the shade, epicarp and mesocarp were removed and seed kernels grinded to obtain a fine powder. Ripe (RF: 135 g) and green (GF: 135 g) seed kernel powder were repeatedly extracted with MeOH (1.5 L × 3) at room temperature for 24 h and then concentrated under vacuum to obtain crude methanol extracts (RF: 26 g; GF: 24 g). The obtained materials were then partitioned between H2O and EtOAc to yield organic extract (RF: 12.0 g; GF: 14.6 g). EtOAc extracts were subjected to chromatography over silica column (230–400 mesh) eluting with a solvent gradient of increasing polarity from n-hexane to EtOAc. Fractions eluted with n-hexane/EtOAc 7:3 afforded a mixture of limonoids (240 mg) including only traces of nimbin (3) (2.0 mg) from GF EtOAc extract, while 98.8 mg of nimbin were obtained from RF. Fractions eluted with n-hexane/EtOAc 6:4 were further purified by RP-HPLC (MeOH/H2O 9:1) to obtain deacetylnimbin (5) from both extracts (323.0 mg from GF and
426.0 mg from RF). Fractions eluted with n-hexane/EtOAc 55:45 Fig. 1. Chemical structure of neem limonoids.
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contained pure salannin (451 mg from GF and 376 mg from RF). Lastly, the fractions eluted with increasing polarity from n-hexane/EtOAc 5:5 to 2:8 gave a mixture of azadirachtin A (1) and analogues (ca. 1.0 g of azadirachtins from both extracts). 2.4. Animals, parasite and ookinete development assay Two to four weeks old female BALB/c mice (20 ± 3 g) were used for the experiments. Mice were reared and maintained in the animal breeding facilities of the University of Camerino (Italy) and experimental protocols were performed in accordance with the Italian Legislative Decree on the “Use and protection of laboratory animals” (D. Lgs. 116 of 10/27/ 92). The rodent malaria parasite Plasmodium berghei PbCTRPp.GFP [17] was used in these studies. The parasite was maintained in the laboratory of parasitology at the University of Camerino by mouse to mouse acyclic and mouse to mosquito to mouse cyclic passages. To avoid repeated acyclic passages, which could compromise the parasite continued ability to transmit to mosquitoes [18], mice were infected for each experiment with PbCTRPp.GFP thawed from glass hematocrit capillaries kept in liquid nitrogen storage. To assess in vitro transmission blocking (TB) activity of fractions and compounds targeted to early sporogonic development, the ookinete development assay (ODA) was employed as described in [19]. Briefly, for each experiment 3 donor mice were inoculated i.p. with 50–75 μl of PbCTRPp.GFP infected blood, and four days after inoculation blood from mice with parasitemia around 2% was collected and 107 parasitized red blood cells i.p. inoculated into phenylhydrazine (200 μl, 1.2 mg/ml) pre-treated BALB/c mice (n = 6). On day 4 post infection, Giemsa stained blood smears were prepared from experimental mice. Mice with parasitemia around 5% and with mature gametocytes were assessed for microgamete exflagellation. A drop of blood was taken from the mouse tail tip and diluted in exflagellation medium [19]; ookinete medium was prepared by adjusting exflagellation medium to pH 7.4 and by adding 20% heat inactivated fetal bovine serum and 1% penicillin (10.000 U)/streptomycin (10 mg/ml) before use. Blood samples diluted in exflagellation medium (1:25) were placed in handmade coverslip glass slide chambers and incubated for 20 min at 19 °
C. Numbers of microgamete-extruding gametocytes, visible as vibrating “exflagellation centers” were counted under the microscope (400 ×). Mice showing more than 3 exflagellation centers per 1000 red blood cells were selected as blood donors for the ookinete development assay (ODA). Fractions (n = 28 GF; n = 23 RF) containing mixture of limonoids and pure molecules were dissolved in DMSO to obtain stock solutions at 30 mg/ml. Ookinete medium was dispensed to the wells of 96-wells microplates and samples of the pre-diluted fractions and molecules were added to each well to obtain the desired final concentration in a total volume of 100 μl, at a final DMSO concentration of 0.1% (0.1% DMSO as control). Infected blood collected from donor mice was added to the wells at a 1:20 dilution (corresponding to a hematocrit of 1–2%) and mixed swiftly. The plate was immediately transferred to 19 °C and incubated for 22–24 h. To assess the development of early sporogonic stages, each well content was mixed, then aliquots were diluted 1:50 in PBS (pH 7.4) in a new plate and GFP-expressing zygotes and ookinetes were counted at the fluorescent microscope (FITC fluorescent filter, 400× magnification). All samples were independently tested in triplicate wells using blood from at least 2 mice on different plates.
3. Results and discussion Seed kernel powders from ripe (RF) and unripe (GF) neem fruits were exhaustively extracted with MeOH and the obtained materials were then partitioned between H2O and EtOAc to yield 12.0 g of organic extract from RF and 14.6 g from GF. The EtOAc extracts from GF and RF were then subjected to MPLC chromatography over silica gel to afford nimbin (2) (much more abundant in ripe fruits), salannin (3), azadirachtin A (1) (both present in the same amounts in GF and RF) and deacetylnimbin (5) (present in large amounts in both extracts). These compounds were identified on the basis of a comparison of their spectral data with those present in the literature [20–22]. The very similar composition found for ripe and green fruits (with the exception of nimbin content) contrasts with previous reports indicating a marked change in the secondary metabolite content depending on the maturation stage [9].
Fig. 2. Inhibitory activity of A. indica fruit fractions on early sporogonic development at a test concentration of 50 μg/ml; A) Effects of fractions from green fruits (GF); B) Effects of fractions from ripe fruits (RF); FA = fatty acids.
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Fig. 3. Impact of fraction GF 12 on ookinete maturation. A) In wells treated with GF_12 at 50 μg/ml, ESS developed to round zygotes as in DMSO control wells (grey bars) whereas maturation to elongated ookinetes was impaired (black bars); B) Giemsa stained EES after 22–24 h in vitro culture (1000× magnification); a) mature, banana-shaped ookinete from DMSO 0.1% control well; b) zygote form from a GF_12 treated well.
Screening of fractions obtained from A. indica GF and RF seed kernels at 50 μg/ml revealed that some of them could inhibit early sporogonic development in vitro (Fig. 2). Parasitized blood incubated with azadirachtin(s)-containing fractions from green fruits (GF_19 to GF_23) induced a reduction of about 40%–70% of early sporogonic stage (ESS) development. These data appeared in line with our earlier results, which showed that pure azadirachtin A (1) was able to block ESS development almost completely at 50 μg/ml and reduced ESS by 50% at 17 μM (CI95:15–19 μM) [16]. Fractions from ripe fruits containing nimbin (2), tested at 50 μg/ml, reduced ESS development by about 40% (CI95: 34, 5–45, 5), while a complete block of development was observed with ripe fruit fraction RF_15 rich in deacetylnimbin (5). On the other hand, salannin (3), reported in the literature to possess moderate activity against Plasmodium falciparum asexual blood stages in vitro [23], did not reveal any effect on ESS. Also no activity on ESS was observed with either ripe or green fruit extract fractions containing fatty acids and glycerides (GF_1 to GF_9 and RF_1 to RF_7, data not shown). Interestingly, ookinete development assay (ODA) screening at 50 μg/ ml revealed a marked reduction of total ESS numbers by RF_15 (from ripe fruit) but not by GF_12 (from green fruit) (Fig. 2), although the two fractions appeared to contain similar amounts of deacetylnimbin (5). In order to clarify this issue, stage specific effects on the parasite undergoing ESS development were examined in detail. Microscope observations of GF-12 treated wells revealed zygote formation, but few zygotes underwent further development to form banana shaped ookinetes (Fig. 3). Differential counts showed that after 22–24 h of incubation with 50 μg/ml GF_12 only 35% (SD ± 10) of parasites fully developed into banana-shaped ookinetes, compared to 80% (SD ± 9) in the DMSO control wells (*P = 0.049). Pure deacetylnimbin (5), obtained after HPLC purification from both fractions GF_12 and RF_15, was tested in the ODA at 50 μg/ml (100 μM) and was found to completely inhibit ESS development confirming the sporontocidal activity of the molecule. The initial discrepant activities of fractions GF_12 and RF_15 might be therefore attributed to the presence of minor constituents in GF_12 which, interacting with deacetylnimbin (5), cause an arrest/delay in zygote development, rather than killing zygotes or interfering with earlier developmental processes. Testing the pure compound deacetylnimbin (5) in the ODA at various concentrations, the molecule displayed a dose-dependent inhibitory activity which reached complete inhibition at 100 μM and faded to negligible activity below 0.7 μM. In the range of 6–25 μM an inhibitory activity of about 50% (40% - 60%) was observed (Fig. 4). The dose-
dependency of the antiplasmodial effect of deacetylnimbin (3) points to a specific activity against the ESS. The pattern of antimalarial transmission blocking activity determined for the main limonoids present in neem fruits has been summarized in Fig. 5. The structural similarity within these compounds and their different activities allow the drawing of preliminary structure-activity relationships. The presence of an α,β unsaturated ketone in ring A seems to be not directly related to the biological activity, since this functionality is present in deacetylnimbin but absent in azadirachtin A. Similarly, azadirachtin A and salannin exhibited very different bioactivities although they share practically the same structure of rings A/B. Interestingly, both the active limonoids show the presence of a free\\OH group at ring B and the key role of this group at that position is clearly evidenced by the poor activity of nimbin, which differs only for the acetylation of that group. Interestingly, in 1987 Yamasaki and Klocke had found that the growth-inhibitory activity of azadirachtin A against the major agricultural insect pest Heliothis uirescens was strictly dependent from the presence of free hydroxyl groups on the structure, which must also include a lipophilic moiety (possibly for transport phenomena) [24]. Studies aimed at assessing the cytotoxicity of neem tree limonoids using N1E-115 neuroblastoma (mouse), 143B.TK- osteosarcoma (human) and Sf9 (insect) cultured cell lines had already assessed the
Fig. 4. In vitro inhibitory activity of deacetylnimbin (5) on ESS development of P. berghei CTRPgfp at different concentrations. Columns represent the mean % inhibition at a given concentration from tests performed in triplicate wells on at least 2 plates with blood from different mice and bars depict the ±SD.
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Fig. 5. Transmission blocking activity of the main limonoids of neem fruits.
lack of toxicity of deacetylnimbin (5), nimbin (2) and azadirachtin (1) at concentrations up to 200 μM [25]. Evaluation of nimbin and deacetylnimbin on leukemia (HL60), lung (A549), stomach (AZ521), and breast (SK-BR-3) cancer cell lines revealed that nimbin (2) was low to nontoxic for any of these cell lines, while deacetylnimbin (5) had a selective activity on the leukemia cancer cell line (IC50 of 18 ± 3 μM) worthy of further investigation [26]. In summary, our investigation has revealed that extracts of neem seed kernels, at different maturation stages, contain large amounts of deacetylnimbin (5), a limonoid endowed with a considerable transmission blocking activity on Plasmodium berghei early sporogonic development. Since deacetylnimbin shows a better thermal and light stability compared to the somewhat unstable azadirachtin A, further studies are encouraged to explore its biological properties and mechanism(s) of action in greater detail. Hopefully, once the activity will be confirmed also on Plasmodium falciparum parasites, pure deacetylnimbin, deacetylnimbin-enriched neem kernel extracts, or even simplified synthetic analogues, could fulfill the need for transmission blocking agents to be included in combination treatments aimed not only at curing malaria patients but also at preventing the diffusion of this deadly infection. Acknowledgments We thank Prof. Robert Sinden from Imperial College, London, for kindly providing Plasmodium berghei (PbCTRPp.GFP). References [1] World Health Organization, www.who.int (Accessed on May 22, 2016). [2] K.I. Barnes, N.J. White, Population biology and antimalarial resistance: the transmission of antimalarial drug resistance in Plasmodium falciparum, Acta Trop. 94 (2005) 230–240. [3] R.E. Howes, K.E. Battle, A.W. Satyagraha, J.K. Baird, S.I. Hay, G6PD deficiency: global distribution, genetic variants and primaquine therapy, Adv. Parasitol. 81 (2013) 133–201. [4] A. Mbengue, S. Bhattacharjee, T. Pandharkar, H. Liu, G. Estiu, R.V. Stahelin, S.S. Rizk, D.L. Njimoh, Y. Ryan, K. Chotivanich, C. Nguon, M. Ghorbal, J.-J. Lopez-Rubio, M. Pfrender, S. Emrich, N. Mohandas, A.M. Dondorp, O. Wiest, K. Haldar, A molecular mechanism of artemisinin resistance in Plasmodium falciparum malaria, Nature 520 (2015) 683–687. [5] K. Stepniewska, R.N. Price, C.J. Sutherland, C.J. Drakeley, L. von Seidlein, F. Nosten, N.J. White, Plasmodium falciparum gametocyte dynamics in areas of different malaria endemicity, Malar. J. 7 (2008) e249. [6] United Nations Environment Programme Neem: the UN's tree of the 21st Century. Nairobi: United Nations Environment Programme, http://www.unep.org/wed/ tree-a-day/neem.asp2012. [7] G. Chianese, S.R. Yerbanga, L. Lucantoni, A. Habluetzel, N. Basilico, D. Taramelli, E. Fattorusso, O. Taglialatela-Scafati, Antiplasmodial triterpenoids from the fruits of neem, Azadirachta indica, J. Nat. Prod. 73 (2010) 1448–1452.
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