Cytotoxycity and antiplasmodial activity of phenolic derivatives from Albizia zygia (DC.) J.F. Macbr. (Mimosaceae)

Background The proliferation and resistance of microorganisms area serious threat against humankind and the search for new therapeutics is needed. The present report describes the antiplasmodial and anticancer activities of samples isolated from the methanol extract of Albizia zygia (Mimosaseae). Material The plant extract was prepared by maceration in methanol. Standard chromatographic, HPLC and spectroscopic methods were used to isolate and identify six compounds (1–6). The acetylated derivatives (7–10) were prepared by modifying 2-O-β-D-glucopyranosyl-4-hydroxyphenylacetic acid and quercetin 3-O-α-L-rhamnopyranoside, previously isolated from A. zygia (Mimosaceae). A two-fold serial micro-dilution method was used to determine the IC50s against five tumor cell lines and Plasmodium falciparum. Results In general, compounds showed moderate activity against the human pancreatic carcinoma cell line MiaPaca-2 (10 < IC50 < 20 μM) and weak activity against other tumor cell lines such as lung (A-549), hepatocarcinoma (HepG2) and human breast adenocarcinoma (MCF-7and A2058) (IC50 > 20 μM). Additionally, the two semi-synthetic derivatives of quercetin 3-O-α-L-rhamnopyranoside exhibited significant activity against P. falciparum with IC50 of 7.47 ± 0.25 μM for compound 9 and 6.77 ± 0.25 μM for compound 10, higher than that of their natural precursor (IC50 25.1 ± 0.25 μM). Conclusion The results of this study clearly suggest that, the appropriate introduction of acetyl groups into some flavonoids could lead to more useful derivatives for the development of an antiplasmodial agent.


Background
Albizia is a large genus belonging to the Mimosaceae plant family. It comprises at least 150 species mostly trees and shrubs native to tropical and subtropical regions of Asia and Africa [1]. In traditional medicine, the roots bark of Albizia zygia are used against cough, while its stem bark is used as a purgative, antiseptic, aphrodisiac, to treat gastritis, fever, conjunctivitis, as well as to fight worms and overcome female sterility [2,3]. The methanol extract of its stem bark has been reported to exhibit strong activity against P. falciparum K1 strain and Trypanosoma brucei rhodesiense [4][5][6]. The genus Albizia is phytochemically known as a source of saponin compounds with a large number of sugar moieties [3,7,8]. Despite this predisposition to produce saponins, previous works have also reported flavonoids, alkaloids and tannins [9][10][11]. Thus, we carried out and reported herein the fractionation and purification of methanol extract of A. zygia followed by the acetylation of the two most abundant isolated compounds obtained, 2-O-β-D-glucopyranosyl-4hydroxyphenylacetic acid and quercetin 3-O-α-L-rhamnopyranoside. The cytotoxic and antiplasmodial activities of compounds are also reported.

General experimental procedures
Column chromatography were proceeded with Silica gel 60 F 254 (70-230; Merck; Darmstadt, Germany). TLC developed on precoated silica gel Kieselgel 60 F 254 plates (0.25 mm thick) and compounds were detected by spraying with 50% H 2 SO 4 on it before being heated at 100°C. Semi-preparative and preparative HPLC was performed using a Gilson FX-281322H2 High Performance Liquid Chromatography coupled to a DAD detector and an automatic fraction collector. ASunfire C18 column (10 μm, 10 × 250 mm) and (5 μm, 10 × 150 mm) were used in these separations. (+)-ESITOF-MS was performed as previously described [12]. We recorded NMR spectra on a Bruker Avance III spectrometer, equipped with a 1.7 mm TCI microcryoprobe, (500.0 and 125.0 MHz for 1 H and 13 C NMR, respectively). The chemical shifts are given in part per million (ppm) using the signal of the residual solvent as internal reference. The coupling constant (J) are in Hertz.

Plant material
The leaves of Albizia zygia (DC) J.F. Macbr were collected on the slopes of the cliff of Santchou, West Region of Cameroon in March 2013. It is a public and well known wild. Thus, access and collection of samples do not require any permission according to the legislation of Cameroon. These leaves were identified at the National Herbarium of Cameroun (NHC) by comparison to a voucher specimen under the number N°43,969 HNC.

Extraction and isolation
Dried leaves of A. zygia were ground to a fine powder (0.77 Kg) and macerated with methanol (5 L) for 24 h (repeated 3 times) at room temperature. After filtration and removal of the solvent in vacuo, a crude extract of 42.0 g was obtained. The extract was subjected to silica gel column chromatography (CC) eluting with gradient of n-hexane-EtOAc and then EtOAc-MeOH to afford four major fractions (A-D). Fraction A was not further investigated, it contains mostly fatty material and fraction B (3.2 g) was separated by column chromatography over silica gel with a (5-30%) of n-hexane-EtOAc to give quercetin (6) (27.0 mg). Fraction C (12.6 g) was separated by column chromatography over silica gel using gradient (5-50%) of CH 2 Cl 2 -MeOH to give a mixture of compounds 2 and 3 (97.3 mg). Fraction D (20.8 g) was subjected to silica gel column chromatography eluted with gradient (5-40%) of EtOAc-MeOH to give phaseoloidin (1) (335.6 mg) and a mixture of 4 and 5 (9.8 mg). Further purification of the two above mentioned mixtures by semi-preparative HPLC eluted with a gradient of acetonitrile-water from 5 to 100% as mobile phase, afforded quercetin 3-O-α-L-rhamnopyranoside (2) (44.4 mg) and kampherol 3-O-α-L-rhamnopyranoside (3) (13.7 mg) from the first mixture, and quercetin 3,4′-di-O-α-L-rhamnopyranoside (4) (1.6 mg) and kaempferol 3, 4′-di-O-α-L-rhamnopyranoside (5) (1.1 mg) from the second one.
Anticancer assays: Five tumor cell lines (MiaPaca-2 (CRL-1420), a carcinoma pancreatic from 65 years adult; Hep G2 (HB-80665), a perpetual cell line which was derived from the liver tissue of a 15-year-old Caucasian American male with a well-differentiated hepatocellular carcinoma; A549 (CCL-185), a carcinoma lung from 58-year-old Caucasian made; A2058 (CRL-11147), Human skin melanoma from a 43 years Caucasian adult derived from lymph node and MCF-7 (HTB-22), a breast adenocarcinoma from 69 years woman) were obtained from ATCC. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) colorimetric assay, which measures mitochondrial metabolic activity, was employed to estimate the amount of living cells. According to the huge amount of celles to be plated, SelecT (TAP Biosystems, Royston, UK), a cell culture robotic system was used to process ten thousand cells per well (for 72 h assay). Cells were seeded at a concentration of 1× 104 cells/well in 200 μl culture medium and incubated at 37°C in 5% CO 2 . After 24 h, the automated liquid-handling system Biomek FX (Beckman Coulter, Pasadena, CA, USA) was used to replace the medium with a final volume of 200 μL and 1 μL of compound (dilution 1/200) and to add controls to the plates and which were then be incubated for 72 h. The test compounds were examined in triplicate with serial two-fold dilutions. After incubation, MTT solution was prepared at 5 mg/mL in PBS 1X and then diluted at 0.5 mg/mL in MEM without phenol red. The sample solution in wells was removed and 100 μL of MTT dye was added to each well. The plates were gently shaken and incubated for 3 h at 37°C in 5% CO 2 incubator. The supernatant was removed and 100 μL of DMSO 100% was added. The plates were gently shaken to solubilize theoriginated formazan and absorbance at 570 nm was read in a Victor2 Wallac spectrofluorometer (PerkinElmer, Waltham, MA, USA). IC 50 values were calculated as the concentration that decreases 50% of the cell viability using Genedata Screener software (Genedata AG, Basel, Switzerland). Curve fitting followed the Smart Fit strategy with Hill model selection.

Results
The methanol extract of the leaves of A. zygia was purified over silica gel, Sephadex LH-20 column chromatography and HPLC to afford six phenolic compounds (1-6); two of them were subjected to acetylation to give four new semi-synthetic compounds. The structures of the isolated compounds were determined by spectroscopic and spectrometric data and comparison with those of similar reported compounds. Both, naturally occurring and semi-synthetically prepared metabolites were screened for their antiplasmodial and cytotoxic properties.

Chemical transformation
The starting materials, 2-O-β-D-glucopyranosyl-4hydroxyphenylacetic acid and quercetin 3-O-α-L-rhamnopyranoside, isolated from the leaves of A. zygia, were subjected to acetylation by reacting with acetic anhydride in pyridine, followed by semi-preparative HPLC purification. The structures of the semi-synthetic derivatives 7-10 ( Fig. 2) were determined on the basis of their NMR and HRESI-MS data and comparison with those of compounds 1 and 2.
Compound 7 was obtained as colorless oil with a molecular formula of C 22   Aliphatic hydroxyl groups, like those of the sugar moiety, are more reactive than those of the phenol groups [17,18]. In addition to these signals common to compound 2, the spectrum also showed three methyl groups at δ H 1.99 (s, 3H), 2.02 (s, 3H) and 2.13 (s, 3H) corresponding to three acetyl groups. The HMBC spectrum revealed that these methyls were located on the sugar moiety.
Compound 10 was obtained as yellow amorphous powder. Its molecular formula, C 29 H 28 O 15  In addition to signals corresponding to the three acetyl groups already observed in compound 9 at δ H 1.98 (s, 3H), 2.02 (s, 3H) and 2.13 (s, 3H), the spectrum showed an additional methyl group attributable to an aromatic acetyl group at δ H 2.37 (s, 3H) linked to C-7. One can noticed the deshielding of signals from carbons C-8 and C-6 compared to their homolog compounds 9 and 2. The fact that only the hydroxyl at C-7 was acetylated can be explained also by the chelation observed between the hydroxyl group at C-5 and the carbonyl at C-4 and between the two hydroxyl groups at C-3′ and C-4′, which will make the latter hydroxyl groups less reactive than the OH-7. Appropriate NMR and MS spectra are provide as supplementary material (Additional file 1: fig. S1 -fig. S14).

Antiplasmodial activity
The natural compounds isolated from the leaves of A. zygia as well as their semi-synthetic derivatives were tested against Plasmodium falciparum (Table 1) using a microdilution method in liquid medium as previously described [13]. The two semi-synthetic derivatives of quercetin 3-O-α-L-rhamnopyranoside exhibited significant activity against P. falciparum with IC 50 values of 7.5 ± 0.25 μM for compound 9 and 6.8 ± 0.25 μM for compound 10. However, the natural precursor of these two semi-synthetic derivatives showed a weak activity (IC 50 25.1 ± 0.25 μM), similar to that of kaempferol 3-O-

Discussion
The genus Albizia is so far a source of natural occurring saponins and phenolics [3,7,8,20,21]. In our study, no saponins were isolated but phenolic compounds were obtained. Chemical composition of plants can differ from one species to another in a group of plants. That can be due to the ecological region where plants are growing. However, this experiment allowed us to confirm once more that Albizia genus continues to be a source of polar compounds as our phenolics were glycosylated. This study aimed also at identifying how acetylation of phenolic compounds can interfere with the antiplasmodial and anticancer activities by comparing IC 50 values of precursors to those of semi-synthetic compounds. The results indicate that acetylated derivatives display in general a better activity than their natural precursors.
The antiplasmodial activities of the isolated compounds were 19-100.0 μM and that of acetylated derivatives were 6.8-100.0 μM against Plamodium falciparum strain 3D7. Derivatives 9 (7.5 μM) and 10 (6.8 μM) scored the highest in vitro activity among the compounds tested. Several flavonoids have been reported to exert a moderate antiplasmodial activity in a number of different P. falciparum strains [22][23][24]. As a result, we present herein a difference in activity of high hydroxylated flavonoids compared to their acetylated derivatives. This result is interesting insofar that acetylation reaction is easy to achieve in laboratories and flavonoids are very common in plants. Thus, the appropriate introduction of acetyl groups into flavonoids may lead to more useful derivatives for the development of an antiplasmodial agent. In fact, the two acetylated compounds 9 and 10 were over 3 times more active than their natural precursor quercetin 3-O-α-L-rhamnopyranoside (2). However, the absence of activity of phaseolidin (1) and its corresponding derivatives 7 and 8 highlighted that hydroxyl groups are not related to the absence of activity of compound 1 on the protozoal P. falciparum. This is the first report of the antiplasmodial activity of the 2-O-β-D-glucopyranosyl-4-hydroxyphenylacetic acid and quercetin 3-O-α-L-rhamnopyranoside derivatives.
On the other hand and according to the screening program of the National Cancer Institute, USA, a compound is generally considered to have in vitro cytotoxic activity if it exhibits an IC 50 ≤ 4.0 mg/mL or 10.0 μM, following its incubation for 48 and 72 h with cancer cells [19]. In the present report, IC 50 values equal or around this threshold (10.0 μM) were obtained with compounds 10 (10.8 and 12.2 μM against MCF-7and A2050 respectively) and 7 (10.0 μM against Miapaca-2). In general, as shown in Table 2, the lowest IC 50 were obtained with the semisynthetic derivatives (IC 50 10.0-64.9 μM) compared to the parent compounds (IC 50 16.8-121.2 μM). The current result is in the same line with those previously described in the literature which shows that flavonoids have good anticancer properties [25,26]. All the compounds isolated and described in this report could be said to be generally non-cytotoxic when compared to the standard drug Doxorubicin which showed an IC 50 ≈ 0.0 μM. However, the theoretical more effectivity and safety of our compounds was calculated. Compound 10 presented a better safety capability (SI = 9.57) compared to its counterpart compound 9 (SI = 3.03). For the others, the toxicity of the drugs was not far enough from the antiplasmodial effects (SI < 3) to guarantee their useness. The toxicity of the flavonoids could be said to be related to the hydroxyl group at C-7.

Conclusion
The objective of this study was to highlight the effect of structure transformation through acetylation of phenolic compounds over anticancer and antiplasmodial activities. The results clearly suggest that, the appropriate introduction of acetyl groups into flavonoids may lead to more useful derivatives for the development of antiplasmodial and anticancer agents.