Antipromastigote and cytotoxic activities of some chemical constituents of Hypericum lanceolatum Lam. (Guttifereae)

Phytochemical investigation of the ethanol extract of the stem bark of Hypericum lanceolatum Lam. (Guttifereae) afforded eight known compounds including 2,2ʹ,5,6ʹ-tetrahydroxybenzophenone ( 1 ), 5-hydroxy-3-methoxyxanthone ( 2 ), 3-hydroxy-5-methoxyxanthone ( 3 ), betulinic acid ( 4 ), hydroquinone ( 5 ) 6,7-dihydroxy-1,3-dimethoxyxanthone ( 6 ), calophyllumin A ( 7 ), and 1,3,5,6-tetrahydroxy-4-prenylxanthone ( 8 ). Compound 1 was submitted to acetylation reaction to give 5-acetoxy-2,2ʹ,6ʹ-trihydroxybenzophenone ( 9 ), a new hemisynthetic derivative. Compounds 5 and 8 were isolated for the first time from this plant. The structures were established by extensive analysis of their mass spectrometry and nuclear magnetic resonance (NMR) spectroscopic data and comparison with those from the literature. The isolated compounds ( 1 , 2 , 4 , 5 , and 8 ) and the derivative of benzophenone ( 9 ) were tested for their antipromastigote and cytotoxic activities against visceral leishmaniasis parasite Leishmania donovani and macrophage RAW 264.7 cell line, respectively. Compound 9 was the most active with an IC 50 value of 6.1 µg/ml, while compounds 1 , 2 , 4, and 8 were moderately active with IC 50 values ranging from 11.4 to 34.8 µg/ml against L. donovani and were not cytotoxic except compound 5 that was very toxic and not active. The findings of the present study suggested that compounds 1 , 2 , 4 , and 8 could be considered as a potential source of therapeutic medicine for the treatment of leishmaniasis.


INTRODUCTION
Leishmaniasis is a noncontagious, infectious-parasitic disease, caused by protist parasites of the genus Leishmania and transmitted by the bite of a female phlebotomine sand fly.These neglected diseases are considered as a serious health concern, which is occurring in Africa, Asia, Southern Europe, and Latin America (WHO, 2016).According to the World Health Organization records, around 12 million people are infected with leishmaniasis, and 350 million people are considered at risk of acquiring an infection (WHO, 2016).The disease is spreading at an alarming rate with an estimated 2 million new cases occurring annually (WHO, 2016).Leishmaniasis can be categorized into different forms such as mucocutaneous leishmaniasis (MCL), visceral leishmaniasis, and cutaneous leishmaniasis (CL) (Alves et al., 2003).Reported studies revealed about 0.5 and 1.5 million cases of visceral and CL, respectively (Ejazi and Ali, 2013;Shah et al., 2014).Chemotherapy remains the mainstay for the control of leishmaniasis, as effective vaccines have not been developed (Den Boer et al., 2011;Kedzierski, 2011).Available drugs based on pentavalent antimonial as sodium stibugluconate (Pentostam) and meglumine antimoniate (Glucantime) are the current treatment (Rodriguez et al., 2015).However, these limited synthetic drugs require long-term treatments are of high toxicity with severe side effects, high cost, and face severe resistance associated with therapeutic failures (Duthie et al., 2012;Essid et al., 2015).This situation underlines the urgent need to search for new agents for the treatment of leishmaniasis.In recent years, the use of medicinal plants as an alternative for leishmaniasis treatment is quite common in endemic areas (Bahmani et al., 2015).

General experimental procedures
Mass spectral data [Electron impact ionization mass spectroscopy (EIMS)] were recorded on a Finnigan MAT-95 spectrometer (70 eV) with perfluorkerosine as reference substance for high-resolution electrospray ionization time-of flight mass spectrometry (HR-ESI-TOF-MS) (Japan).Melting points of the isolated compounds were determined using an Electrothermal IA9000 Series digital melting-point apparatus (Bibby scientific, Great Britain) and were uncorrected.Ultraviolet and visible spectra were recorded in MeOH at 25°C using a Kontron Uvikon spectrophotometer.The infrared (IR) spectra were measured on a Perkin Elmer 1750 FTIR spectrometer.The nuclear magnetic resonance (NMR) spectra were measured on Bruker 300, 500, and 600 MHz NMR Avance II spectrometers equipped with cryoprobe, with TMS as an internal reference.Chemical shifts were recorded in δ (ppm), and the coupling constants (J) are in hertz (Hz).Silica gel 60 F 254 Merck;Darmstadt,Germany) was used for column chromatography.Precoated silica gel Kieselgel 60 F 254 plates (0.25 mm thick) were used for thin-layer chromatography (TLC), and spots detected by spraying with 50% sulfuric acid (H 2 SO 4 ) followed by heating at 100°C.All solvents were distilled before use.

Sample collection
The stem bark of H. lanceolatum was collected on the Mount Bamboutos flanks (Western Region, Cameroon) in May 2011.The sample identification was confirmed by Mr. Victor Nana, a botanist at the Cameroon National Herbarium in Yaounde, where a voucher specimen was deposited (Voucher No 32356/HNC).

Extraction and isolation
The dried stem barks of Hypericum lanceolatum (2.0 Kg) were extracted with ethanol (EtOH) (10 l) for three days at room temperature.The resulting extract was then concentrated with the aid of rotavapor to yield 50 g of crude extract.

In vitro antipromastigote assay
In vitro antileishmanial activity of compounds was investigated against Leishmania donovani promastigote using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide/ phenazinemethosulfate (MTS/PMS, Promega) colorimetric assay (Wong et al., 2014).Briefly, stationary-phase promastigotes were seeded into 96-well flat-bottomed microtiter plates at 1.10 7 parasites per well, in a final volume of 100 μl medium, and were incubated at 28°C with a series of concentrations of compounds, which dissolved in dimethyl sulfoxide and ranging from 12.5 to 200 μg/ml, in triplicate.After 72 hours of incubation, the plates were examined under an inverted microscope to assure the growth of the controls and sterile conditions and 10 μl of MTS/PMS was added to each well of the microtiter plate.The plate was then incubated at 28°C for color development.After 4 hours of incubation, the optical density (OD) values were read at 490 nm using an automated microtiter plate reader (TECAN Infinite M200 Pro Plate Reader, Austria).Amphotericin B was used as a positive control, while DMSO was used as a negative control.The experiments were conducted in triplicate.The percent growth inhibition was calculated from the absorbance relative to the negative control, and 50% cell cytotoxicity (IC 50 ) values were determined.

Cytotoxicity assay
A toxicological assessment was carried out against RAW cell lines using the resazurin assay and has been used to determine the selectivity indices of active compounds as described by Süzgeç-Selçuk et al. (2011).From sub-confluent cultures in 75 cm 2 culture flasks, they were trypsinized, counted, suspended in respective medium, and then seeded into triplicate wells of a 96-well plate (100 μl per well) at concentrations of 1.10 5 cells per ml and incubated.Cells were allowed to attach overnight and then treated in triplicate with 10 μl per well of 5-fold serially diluted individual compound (3.2-2,000 μg/ml) in the culture medium and incubated for 48 hours.After incubation, 10 μl of 2.5 mM of resazurin solution was added to each well and then incubated for 4 hours at 37°C.Fluorescence signal was measured using the microplate reader at the excitation and emission wavelengths of 530 nm and 590 nm, respectively.Curcumin was used as a positive control.Experiments were conducted in triplicate.The percent growth inhibition was calculated from the absorbance relative to the negative control, and the concentration of extract that inhibited 50% of cells (CC 50 values) was determined.The selectivity index (SI) ratio (CC 50 for macrophages/IC 50 for amastigotes) was used to compare the toxicity of the compounds against the macrophages and their activity against the parasites.

Statistical analysis
Results were expressed as mean ± standard deviation (SD).The IC 50 and CC 50 values were calculated fitting the data as a non-linear regression using a dose-response inhibitory model, in the GraphPad Prism 7.0 program.

Chemical modification of compound 1
2,2ʹ,5,6ʹ-tetrahydroxybenzophenone (1) (20 mg) was dissolved in pyridine (3 ml) and acetic anhydride (3 ml) and stirred at room temperature for 24 hours.Ten milliliters of water were added to the mixture and stirred for 30 minutes.Extraction with CH 2 Cl 2 and purification over a silica gel column with n-hexane-AcOEt (7:3) as solvent gave 5-acetoxy-2,2ʹ,6ʹtrihydroxybenzophenone (9) (5 mg) (Kopa et al., 2014).Compound 9 was obtained as yellow crystals having a melting point of 246°C-247°C.Its IR spectrum exhibited strong absorptions at 1,610 cm− 1 (OH) and 1,757 cm− 1 corresponding to typical vibration bands of a conjugated carbonyl and an ester carbonyl, respectively.Its molecular formula was deduced as C 32 H 38 O 8 from the rigorous analysis of its 1D and 2D NMR data jointly with its EIMS spectra, which did not show the peak of the molecular ion but that of a fragment ion at m/z 270 [M-H 2 O] + corresponding to the loss of a molecule of H 2 O. Another important fragment ion was observed at m/z 228 [M-COMe-H 2 O+H] + .The comparison of the molecular weight of this compound with that of 2,2ʹ,5,6ʹtetrahydroxybenzophenone (1) thus reveals a difference in mass of m/z 43 corresponding to the molar mass of an acetyl unit.1D and 2D NMR data of compound 9 are almost identical to those of its precursor 2,2ʹ,5,6ʹ-tetrahydroxybenzophenone (1).The main differences are observed on the 1 H NMR spectrum of compound 9 by the disappearance of the signal of a hydroxyl group in favor of a characteristic methyl signal of an acetyl group at δ H 2.36 (3H, s).This is confirmed by the appearance on the 13 C NMR spectrum of compound 9 of two additional signals at δ C 181.7 and δ C 21.0, respectively, corresponding to carbonyl and methyl of an acetyl group.Moreover, when comparing the data from the 13 C NMR spectra of the two compounds, we can clearly see that the chemical shifts of carbons C-5 and C-4 have undergone a decrease of δ C − 5.2 and an increase of δ C + 4.9, respectively, and the chemical shift of the H-4 proton evolved toward the weak fields (δ H + 0.63).This can only be explained by the presence of the C-5 acetate group, and the fact that the H-4 proton is strongly deshielded by the proximity of the carbonyl group of the acetate moiety.This position of MeO-group at C-5 instead of C-2 or C-2ʹ is confirmed by the HMBC correlation of the methyl protons of the acetyl group with the carbon at C-5 (δ H 146.7).
According to the literature, these different observations allowed us to identify this compound as 5-acetoxy-2,2ʹ6ʹtrihydroxybenzophenone (9), which is a semisynthetic derivative characterized here for the first time.
Based on the molecular framework of isolated xanthones, the relationship between the in vitro antileishmanial activity and the chemical structure were examined with respect to different functional group therein.Structural comparison between 5-hydroxy-3-methoxyxanthone (2) and 1,3,5,6-tetrahydroxy-4prenylxanthone (8) revealed the presence of three hydroxyl groups and one prenyl in compound 8, which are absent in compound 2 that contains only one hydroxyl group and one methoxy group, which are absent in compound 8. Azebaze et al. (2008) showed that the prenyl group at position 4 could increase the activity of xanthones.However, the presence of the hydroxyl group in both molecules had an important effect on their antileishmanial activity of compound 8.
Betulinic acid (4), a naturally occurring lupane-type triterpene found in many plant species, which has been reported to anti-HIV-1, antibacterial, antifungal, antiplasmodial, and anti-inflammatory activities (Yogeeswari and Sriram 2005), and has also been reported to inhibit growth of cancer cells, without affecting normal cells (Einzhammer and Xu 2004).Chan-Bacab et al. (2003) have been reported that betulinic acid (4) has the leishmanicidal activity.These results, therefore, confirm the antileishmanial potential of this molecule.
Hydroquinone (5) was previously isolated from the ingredient of many plant-derived products, and an important metabolite of benzene is found to possess strong antiproliferative activity against promastigote forms on Leishmania major and Leishmania tropica in vitro.
2,2ʹ5,6ʹ-tetrahydroxybenzophenone (1) was previously isolated from the stem bark of H. lanceolatum and was not showed any activity against Plasmodium falciparum.However, it was not cytotoxic on LLC-MK2 monkey kidney epithelial cells as with on macrophage RAW 264.7 cells line.Acetylation reaction of this compounds gave 5-acetoxy-2,2ʹ,6ʹ-trihydroxybenzophenone (9).The structural comparison of compound (1) and compound (9) revealed only the replacement of the hydroxyl group by acetyl group at position C-5.Although the change had an important effect on antileishmanial activity (IC 50 16.2 to 6.1 µg/ml), our results showed that the product of acetylation, 5-acetoxy-2,2ʹ,6ʹtrihydroxybenzophenone (9) was about 10 times more active than its precursor 1.The acetyl group presents at position C-5 is likely to be responsible for most potential antileishmanial activity.Moreover, we also noted that the cytotoxicity increases.The observed differences in bioactivity of the two compounds may only due to the corresponding change in cytotoxicity (CC 50 ˃ 200-130 µg/ml).To the best of authors' knowledge, antileishmanial activities of all tested compound have never been reported except for betulinic acid (4).
The cytotoxicity of all these compounds was also assessed (Table 1) on macrophages Raw 264.7 cells line in culture.The results of this assay indicated the selective toxicity by most of the compounds.However, 2,2ʹ,5,6ʹ-tetrahydroxybenzophenone (1) showed no signs of cytotoxicity on macrophages (CC 50 value of > 200 μg/ml), while compound 5-acetoxy-2,2ʹ,6ʹ-trihydroxybenzophenone (9) was slightly cytotoxic with a SI of 21.4.Additionally, the less active compound, hydroquinone (5) was cytotoxic at substantially lower concentration (CC 50 0.1132 µg/ml) than its antileishmanial concentration (IC 50 107.9µg/ml).All the other compounds showed higher CC 50 values compared to their IC 50 values.

ACKNOWLEDGEMENT
Authors are grateful to the pharmacognosy team of the University of Liege for their contribution to analysis of some of the compounds.The authors are also grateful to Bill and Melinda Gates Foundation through the postdoctoral fellowship training program in Infectious Diseases at Noguchi Memorial Institute for Medical Research, Ghana (Global Health Grant number OPP52155), for the financial support to carry out biological activities.

FINANCIAL SUPPORT
None.

CONFLICT OF INTERESTS
Authors declare that they do not have any competing interests.

Table 1 .
Antipromastigote and cytotoxic activities of isolated compounds from the ethanol extract of the stem bark of H. lanceolatum.