Abstract
Context: Parinari excelsa Sabine (Chrysobalanaceae) is an indigenous tree from West and Eastern Africa. This tree is used in Ivory Coast as an antimalaria remedy.
Objective: The in vitro antiplasmodial and antileishmanial activities of the stem bark, the leaf and the major compounds from the stem bark were investigated.
Materials and methods: The leaves and stem bark from P. excelsa were separately collected, air-dried and powdered. Two extracts (methylene chloride and methanol) were realized for both powders. Every extract was tested for its antiplasmodial and antileishmanial activities. Only the stem bark crude extracts were fractionated by column chromatography and their major components were analyzed by NMR, HRESIMS and IR methods. The compounds were tested for their antiplasmodial and antileishmanial activities.
Results: The comparison of the IC50 values of the crude extracts were in this ordrer: 3.41 (IC50 of PeBMc) <4.10 (IC50 of PeBMc) <4.42 (IC50 of PeLMe) against P. falciparum and 5.19 (IC50 of PeBMc) <12.32 (IC50 of PeBMe) <19.33 (IC50 of PeLMc) <32.37 (IC50 of PeLMe) against L. donovani. The stem bark crude extracts were the most active against both parasites. Their fractionation leaded to a new ventiloquinone, five triterpenes and one chlorogenic acid. All these compounds were isolated for the first time from P. excelsa. High activities were observed with (3β)-3-hydroxyolean-12-en-28-oic acid (IC50 = 8.2 µM) and 3β-hydroxyolean-5,12-dien-28-oic acid (IC50 = 7.7 µM) against L. donovani. With the antiplasmodial activity, the best activity was observed with 16β-hydroxylupane-1,20(29)-dien-3-one (IC50 = 28.3 µM).
Discussion and conclusion: These findings demonstrated that the constituents of P. excelsa stem bark have in vitro antiplasmodial and antileishmanial activities.
Introduction
Malaria is an important tropical disease which has the potential to affect nearly 40% of the world population and is responsible for 1–2 million deaths each year (CitationSnow et al. 2005). Human malaria is endemic to 90 countries and is caused by protozoan parasites of the genus Plasmodium, mainly Plasmodium falciparum. Development of resistance to mainstay drugs like chloroquine, and controlled use of new artemisinin analogs have created an urgent need to discover new antimalarial agents. Recently, the clinical use of artemisinin, a sesquiterpene lactone isolated from Artemisia annua L. (Asteraceae), for the treatment of malaria has prompted interest in the discovery of new pharmaceuticals of plant origin with antiplasmodial activity (CitationMuhammad et al. 2003). Another source of tropical disease is Leishmaniasis (Trypanosomatidae). It is caused by a number of species of protozoan parasites belonging to the genus Leishmania, and it is regarded as a major public health problem that affects around 12 million people in 80 countries and causes morbidity and mortality mainly in Africa, Asia, and Latin America (CitationRocha et al. 2005). Historically, the chemotherapy of Leishmaniasis has been based on the use of pentavalent antimonial drugs. Other medications, such as pentamidine and amphotericin B, have been used as alternative drugs. However, these medicines are not orally active, requiring long-term parenteral administration, not to mention that they lead to serious side effects (CitationRocha et al. 2005). Nature remains an ever evolving source for compounds of medicinal importance among which triterpenes (CitationMurata et al. 2008; CitationCamacho et al. 2000) and quinines (CitationEyong et al. 2008; CitationLaurent et al. 2006). Parinari excelsa Sabine (Chrysobalanaceae) is a woody species commonly found in forest of West Africa and Eastern Africa. It is known to be used traditionally for antidiarrhea (CitationNdiaye et al. 2008) and for anthelminthic remedies (Diehl et al. 2004). In Ivory Coast and in Tanzania, this plant is used for antimalarial remedies (CitationGessler et al. 1995; CitationKamanzi et al. 2004). It was thus decided to investigate the antiplasmodium falciparum K1 and antileishmania donovani activities of this specie and to isolate and characterize active compounds.
Material and methods
General experimental procedures
The fractionation and purification process were made using column chromatography and recrystallization methods. Silica gel 60 (230–400 mesh, Merck) and Sephadex LH20 were used as stationary phase. Analytical TLC (thin layer chromatography) was performed on percolated silica gel 60 F254 plates (Merck) and detection was achieved by spraying with sulfuric vanillin, followed by heating 5 min at 105°C. Infra-red (IR) spectra were recorded in MeOH on a JASCO 302-A spectrometer. NMR spectra were recorded on Bruker Avance DRX-400 spectrometer at 400 MHz (1H) and 100 MHz (13C); chemical shifts (δ) are in ppm rel. to Me4Si (internal standard). The electro-spray ionization mass spectroscopy (ESIMS) at 70ev by direct inlet probe, high resolution electro-spray ionization mass spectroscopy (HRESIMS) were detected on an Esquire 3000 plus ion trap mass spectrometer (Bruker Daltonics).
Plant material
The leaf and stem bark from P. excelsa were collected between July and August 2009, in its natural habitat in Ivory Coast, in the forest region near Abidjan (Southern Ivory Coast). Botanical determination was performed by Pr. L. Aké Assi (Centre National de Floristique, Université de Cocody, Abidjan). Voucher specimens (n°842) are deposited at the Herbarium of the Centre National de Floristique (CNF).
Preparation of crude extracts
Air-dried and powdered stem bark (2.3 kg) from P. excelsa was extracted (by solid-liquid extraction method) at room temperature with 8.5 L of cylohexane (2 × 24 h). The residue was air-dried and then extracted with 6.2 L of methylene chloride (reflux at 40°C, 4 × 24 h). The filtrates were taken to dryness under vacuum; 15.2 g of methylene chloride crude extract (PeBMc) were obtained. The residue was still air-dried and then, extracted the last time with 5.0 L of methanol (reflux at 64°C, 4 × 24 h). The filtrates were taken to dryness under vacuum; 12.7 g of methanol crude extract (PeBMe) were obtained. The same extraction protocol was used for the leaves. Air-dried and powdered leaves (1.3 kg), after extractions gave 15.2 g of methylene chloride crude extract (PeLMc) and 20.0 g of methanol crude extract (PeLMe). Tannins were removed from methanol crude extracts using Sephadex LH-20 exclusion chromatography, according to method described by CitationHoughton and Raman (1998). All crude extracts were stored at room temperature until testing.
Isolations and purifications
The antiplasmodial and antileishmanial assays were performed with on crude extracts (). The best activities were observed with PeBMc against L. donovani. (IC50 = 5.19 µg/mL) and P. falciparum. K1 (IC50 = 3.41 µg/mL). A good activity was also observed against P. falciparum with PeBMe (IC50 = 4.42 µg/mL). Therefore, only methylene chloride crude extract (PeBMc) and methanol crude extract (PeBMe) of the stem bark had been fractionated by column chromatography over silica gel. PeBMc (15.0 g) was fractionated a first time on silica gel column chromatography; column size was: diameter (d) = 3.5 cm and height (h) = 14 cm. Elution solvent was the mixture CH2Cl2/EtOAc (2:8 v/v) in increasing proportions to finish with EtOAc/MeOH (9:1 v/v). Six fractions were obtained according to their TLC profile. The first fraction was purified over silica gel column chromatography (column size: d = 1.2 cm, h = 7 cm) using the mixture CH2Cl2/EtOAc (7:3 v/v) as elution solvent. The compound 1 (22 mg) was obtained. Fractions 2–3 were also purified over Silica gel column chromatography (column size: d = 1.2 cm, h = 10 cm) using the mixture CH2Cl2/EtOAc (2:6 v/v) as elution solvent. Compounds 2 (25 mg) and 3 (18 mg) were isolated. Fractions 4–5 were also purified following the same method, column size was: d = 1.2 cm and h = 10 cm. The elution solvent was the mixture CH2Cl2/MeOH (9:1 v/v) and compounds 4 (15 mg) and 5 (12 mg) were obtained. Compounds 1 to 4 were purified by recrystallization in methanol. Crude extract PeBMe was fractionated by column chromatography over Silica gel (column size: d = 3.5 cm, h = 13 cm). Elution solvent was the mixture CH2Cl2/EtOAc (2:8 v/v) following a gradient system, to finish with EtOAc/MeOH (9:1 v/v). Two sub-fractions were grouped together according to their chromatography profile. The first fraction gave compound 6 (35 mg) by recrystallization in EtOAc (100%). The second sub-fraction was analyzed by HPLC inverse phase method. Elution solvent was the mixture: A (H2O with 0.1% TFA) and C (acetonitrile). The column size was C18. Major component was observed at the retention time (RT) 48.11 min and its absorption band was exhibited at the wavelengths maximum 229.0; 288.0 and 380 nm. The purification was future achieved by column chromatography over silica gel (column size: d = 1.2 cm, h = 5.4 cm) using the mixture EtOAc/MeOH (9:1). Compound 7 (26 mg) was obtained after purification by recrystallization in EtOAc (100%).
Table 1. In vitro antileishmanial and antiplasmodial activities and cytotoxicity of crude extracts and isolated compounds (1–7) from P. excelsa.
Table 2. 1H, 13C, COSY and HMBC NMR spectral data of 7.
Biological assays
Antimalarial assay
Quantitative assessment of antimalarial activity in vitro was determined by means of the microculture radioisotope technique based upon a method previously described by CitationDesjardins et al. (1979) and modified by CitationRidley et al. (1996). The assay uses the uptake of [3H]hypoxanthine by parasites as an indicator of viability. Continuous in vitro cultures of asexual erythrocytic stages of Plasmodium falciparum were maintained following the methods of CitationTrager and Jensen (1976). Plant extracts and isolated compounds were tested on K1 strain (multidrug pyrimethamine/chloroquine-resistant strain) (CitationThaithong & Beale, 1981). Initial concentration of plant extracts and isolated compounds was 30 µg/mL diluted with two-fold dilutions to make seven concentrations, the lowest being at 0.47 µg/mL. After 48 h incubation of the parasites with the extracts and compound at 37°C, [3H]hypoxanthine (Amersham, UK) was added to each well and the incubation was continued for another 24 h at the same temperature. The concentrations of the extract or the compound at which the parasite growth (= [3H]hypoxanthine uptake) was inhibited by 50% (IC50) was calculated by linear interpolation between the two drug concentrations above and below 50% (CitationHuber & Koella, 1993). Chloroquine was used as positive reference. The values given in are means of two independent assays; each assay was run in duplicate.
Antileishmanial assay
A transgenic cell line of Leishmania donovani promastigotes showing stable expression of luciferase was used as the test organism. Cells in 200 μL of growth medium (L-15 with 10% FCS) were plated at a density of 2 × 106 cells per mL in a clear 96-well microplate. Stock solutions of the standards and test compounds/extracts were prepared in DMSO. Culture medium without cells and the controls were incubated (at 26°C for 72 h) simultaneously, in duplicate, at six concentrations of the test compounds. An aliquot of 50 μL was transferred from each well to a fresh opaque/black microplate, and 40 μL of Steadyglo reagent was added to each well. The plates were read immediately in a Polar Star galaxy microplate luminometer. IC50 values were calculated from dose-response inhibition graphs. Miltefosine was tested as standard antileishmanial agents.
Cytotoxicity assay
Cytotoxicity assay of the plant extracts and isolated compounds was done following the method of CitationPage et al. (1993) with the modification of CitationAhmed et al. (1994). Cell line L6 (rat skeletal muscle myoblasts) were seeded in 96-well Costar microtiter plates at 2.2 × 105 cells/mL, 50 µL per well in MEM supplemented with 10% heat inactivated fetal bovine serum (FBS). A three-fold serial dilution ranging from 500 to 0.07 µg/mL of sample in test medium was added. Plates with a final volume of 100 µL per well were incubated at 37°C for 72 h in a humidified incubator containing 5% CO2. Alamar Blue was added as viability indicator according to CitationAhmed et al. (1994). After an additional 2 h of incubation, the plate was measured with a fluorescence scanner using an excitation wavelength of 536 nm and an emission wavelength of 588 nm (SpectraMax GeminiXS, Molecular Devices). IC50 values were calculated from the sigmoidal inhibition curve.
Results
Seven compounds were isolated from the stem bark of P. excelsa among which, five known triterpenes: 16β-hydroxylupane-1,20(29)-dien-3-one (1); (3β)-3-hydroxyolean-12-en-28-oic acid (2); 3β-hydroxy-olean-5,12-dien-28-oic acid (3); daucosterol (4) and 3-O-β-d-glucopyranosyl-stigmasta-5,11(12)-diene (5). One chlorogenic acid (6) was also isolated. Only compound 7 was new and, it was identified as 1-(7-allyl-5-(ethoxymethoxy)-8-methoxy-4,6-bis(methoxymethoxy)naphthalen-1-yl)ethanone. It was isolated as yellow amorphous, mp. 108–109°C. 1H NMR (400 MHz, CD3OD), δH(ppm): 7.56(1H, d, J = 8.0 Hz, H-2), 6.62 (1H, d, J = 8.0 Hz, H-3), 2.57 (3H, br s, H-12), 6.02 (4H, br s, H-13), 3.21 (2H, br s, H-14), 6.02 (2H, br s, H-15), 3.40 (2H, q, J = 7.0 Hz, H-16), 1.15 (3H, t, J = 7.0 Hz, H-17), 6.32 (2H, br s, H-18), 3.21 (2H, br s, H-19), 3.35 (2H, d, J = 6.0 Hz, H-20), 6.10 (1H, m, H-21), 4.93 (1H, dd, J = 2.1 and 14.6 Hz, H-22α) and 4.96 (1H, dd, J = 2.1 and 14.6 Hz, H-22β), 3.70 (3H, br s, H-23). 13C NMR (100 MHz, CD3OD), δC(ppm): 119.1 (C-1), 127.2 (C-2), 101.1 (C-3), 153.5 (C-4), 131.9 (C-5), 136.1 (C-6), 107.6 (C-7), 143.2 (C-8), 119.0 (C-9), 117.4 (C-10), 199.8 (C-11), 28.7 (C-12), 96.1 (C-13), 55.7 (C-14), 94.1 (C-15), 63.5 (C-16), 15.7 (C-17), 95.6 (C-18), 55.7 (C-19), 30.1 (C-20), 137.1 (C-21), 117.4 (C-23). IR (CHCl3); λ (cm−1): 1692 (CH3-C=O, ketone), 2840 (C-H of the methoxy CH3-O), 2865 and 2772 (C-H of methylenedioxy O-CH2-O), multiple bands 3090–3070 (CH=CH2). HREISMS m/z: 434.1878, (calcul. for C23H30O8. 434.1884). All these compounds were evaluated for their antileishmanial, antiplasmodial and cytotoxic activities. Compounds 2 and 3 showed the best antileishmanial activity with IC50 values of 7.7 and 8.2 µM, respectively. The IC50 values of compounds 1, 5 and 7 were 23.5, 171.5 and 152.7 µM, respectively. Two compounds, 4 and 6 were not active against this parasite. Concerning the antiplasmodial assay, only compound 1 presented a moderate activity (IC50 = 28.3 µM). The IC50 values of compounds 2, 3, 5, 6 and 7 were 69.9, 77.9, 145.0, 74.1 and 90.5 µM, respectively.
Discussion
Chemical structures of most of the isolated compounds were established by IR, HRESIMS, 1H (400 MHz) and 13C NMR (100 MHz) data analysis. The spectral data of all these compounds () were in agreement with the previously published data, thereby allowing for identification of 16β-hydroxylupane-1,20(29)-dien-3-one (1) (CitationWei et al. 2008), (3β)-3-hydroxyolean-12-en-28-oic acid (2) (CitationXiao-Qiang et al. 2008; Geraldo et al. 2008), 3β-hydroxy-olean-5,12-dien-28-oic acid (3) (CitationChoudhary et al. 2007; CitationZhang et al. 1993), daucosterol (4) (CitationDuarte et al. 2010; CitationSultana et al. 2010; CitationBayoumi et al. 2010), 3-O-β-d-glucopyranosyl-stigmasta-5,11(12)-diene (5) (CitationHussain et al. 2008) and chlorogenic acid (6) (CitationKusaura et al. 2010). The compound 7 was new. Its structure was established through HRESIMS, IR, 1H and 13C NMR, including 2D experiments (HMBC, HMQC and COSY). HRESIMS of 7 exhibited molecular ion peak at m/z: 434.18844 (C23H30O8). The IR spectrum of 7 showed absorption bands due to the ketone CH3-CO- at 1692 cm−1, C-H of the methyl ether (CH3-O) at 2840 cm−1 and C-H of methylenedioxy (O-CH2-O) at 2865 and 2772 cm−1. The multiple bands between 3090 and 3070 cm−1 was due to CH=CH2. The 1H NMR spectrum recorded in CD3OD, showed characteristic signals of two methyl groups at 1.15 ppm (3H, t, J = 7.0 Hz) and 2.57 ppm (3H, br s). Two methoxy groups were observed at 3.21 ppm (6H, br s) and one at 3.70 ppm (3H, br s). The quadruplet at 3.40 ppm (2H, q, J = 7.0 Hz) was for a methylene group (-CH2-) in the system -O-CH2-CH3. Singlet at 6.02 ppm (4H, br s) and 6.32 ppm (2H, br s) belonged to three systems -O-CH2-O-. The allylic system showed signals at 3.35 ppm (2H, d, J = 6.0 Hz), 4.92 ppm (1H, dd, J = 2.1 and 14.6 Hz), 4.96 ppm (1H, dd, J = 2.1 and 14.6 Hz) and 6.10 ppm (1H, m). The aromatic protons gave two doublets at 6.62 ppm (1H, J = 8.0 Hz) and 7.57 ppm (1H, d, J = 8.0 Hz). The 13C NMR spectrum exhibited characteristic chemical shifts of methyl groups at 15.7 ppm (CH3) and 28.7 ppm (CH3-CO). The methoxy groups were observed at 55.7 ppm (O-CH3). Signal at 63.5 ppm corresponded to the primary alcohol CH3-CH2-O-. The signals at 96.1 and 94.1 ppm belonged to the system -O-CH2-O-. The allylic system gave signals at 30.1 ppm (-CH2-), 117.4 ppm (=CH2) and 137.1 ppm (-CH=). The aromatic carbon gave signals at 119.1 ppm (C-1), 127.0 ppm (C-2), 101.1 ppm (C-3), 153.5 ppm (C-4), 131.9 ppm (C-5), 136.1 ppm (C-6), 107.5 ppm (C-7), 143.2 ppm (C-8), 119.0 ppm (C-9) and 117.4 ppm (C-10). A combination of 1H–1H COSY, HMQC and HMBC experiments allowed unambiguous assignments of all protons and carbons signals of 7. In 1H-1H COSY spectrum, correlation signals were observed between H-2 and H-3; H-16 and H-17; H-20 and H-21, and finally between H-21 and H-22. 13C-1H long-range correlation (2JC-H and 3JC-H) signals were observed between H-12(C-1, C-11), H-2(C-1, C-11, C-9, C-3 and C-4), H-13(C-14, C-4), H-16(C-15, C-17), H-18(C-6, C-19), H-20(C-7, C-21, C-22) and H-23(C-8). Combination of COSY, HMQC and HMBC analyses lead to deduction of chemical structure of 7. Consequently, this compound was unequivocally identified as a new ventiloquinone derivative: 1-(7-allyl-5-(ethoxymethoxy)-8-methoxy-4,6-bis(methoxymethoxy)naphthalen-1-yl)ethanone. All these compounds were isolated for the first time from P. excelsa. A wide variety of chemical compounds have been identified from Parinari genus, such as diterpenes and triterpenes (CitationUys et al. 2002), flavonoids, flavonols; flavanones and quinones (CitationBraca et al. 2005; CitationWerawattanachai et al. 2010). It is well known that the chemical composition of plant can change depending on several factors, including the collection site (CitationMissima et al. 2007). The isolation of triterpenes and quinones showed that our results are in agreement with those reported in the literature.
Figure 1. 16β-Hydroxylupane-1,20(29)-dien-3-one (1), (3-β)-3-hydroxyolean-12-en-28-oic acid (2), 3β-hydroxy-olean-5,12-dien-28-oic acid (3), Daucosterol (4), 3-O-β-d-glucopyranosyl-stigmasta-5,11(12)-diene (5), chlorogenic acid (6) and 1-(7-allyl-5-(ethoxymethoxy)-8-methoxy-4,6-bis(methoxymethoxy)naphthalen-1-yl)ethanone (7). Black arrow: HMB correlation (2JC-H and 3JC-H).
Regarding the in vitro antileishmanial assay, the crude extract PeBMc of P. excelsa exhibited an IC50 value of 5.2 µg/mL (). With the antiplasmodial assay, the same crude extract showed the best activity with an IC50 value of 3.41 µg/mL. Compounds 2 and 3 showed the best antileishmanial activity with IC50 value of 7.7 and 8.2 µM, respectively. Both compounds had very close antileishmanial activities and we can explain this by their almost identical structures. Only compound 1 presented a weak antiplasmodial activity (IC50 = 28.3 µM). This value was in the same range of those of some antiplasmodial triterpenes isolated from medicinal plants (CitationCamacho et al. 2000; CitationKirandeep et al. 2009). We noticed that the activity of the crude extract was better than that of the most active compounds from the plant. This would result from a concert action between two or several molecules. Their separation thus weakened their action. The new ventiloquinone derivative was not active against both Leishmania and Plasmodium parasites. The antiplasmodial and antileishmanial activities of all these compounds were evaluated for the first time here.
Conclusion
This study on P. excelsa leaded to the isolation and identification of triterpenes, chlorogenic acid and a new ventiloquinone derivative. Among these compounds, a new antiplasmodial and two new antileishmanial were identified. However, these activities were relatively moderated against both parasites, compared to those of crude extracts. Therefore, further studies are in progress to disclose other important biological effects of this medicinal plant.
Acknowledgments
We are thankful to Patrick Wehrung (Université de Strasbourg) for NMR and MS analyses data, Aké A. L (Jardin botanique de l’Université de Cocody-Abidjan) for plant identification.
Declaration of interest
The authors are grateful to AUF (Agence Universitaire de la Francophonie) for financial support.
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