Phytochemical, Antimicrobial and Antiprotozoal Evaluation of Garcinia Mangostana Pericarp and α-Mangostin, Its Major Xanthone Derivative

Five xanthone derivatives and one flavanol were isolated from the dichloromethane extract of Garcinia mangostana. Dichloromethane, ethyl acetate extract and the major xanthone (α-mangostin) were evaluated in vitro against erythrocytic schizonts of Plasmodium falciparum, intracellular amastigotes of Leishmania infantum and Trypanosoma cruzi and free trypomastigotes of T. brucei. The major constituent α-mangostin was also checked for antimicrobial potential against Candida albicans, Escherichia coli, Pseudomonas aeruginosa, Bacillius subtilis, Staphylococcus aureus, Mycobacterium smegmatis, M. cheleneoi, M. xenopi and M. intracellulare. Activity against P. falciparum (IC50 2.7 μg/mL) and T. brucei (IC50 0.5 μg/mL) were observed for the dichloromethane extract, however, with only moderate selectivity was seen based on a parallel cytotoxicity evaluation on MRC-5 cells (IC50 9.4 μg/mL). The ethyl acetate extract was inactive (IC50 > 30 µg/mL). The major constituent α-mangostin showed rather high cytotoxicity (IC50 7.5 µM) and a broad but non-selective antiprotozoal and antimicrobial activity profile. This in vitro study endorses that the antiprotozoal and antimicrobial potential of prenylated xanthones is non-conclusive in view of the low level of selectivity.


Introduction
The genus Garcinia (Guttiferae, syn. Clusiaceae) contains well-known fruit trees with about 35 genera and up to 800 species of which the fruits of many are edible and serve as a substitute for tamarinds in curries [1]. Garcinia mangostana Linn., known as mangosteen, is cultivated in the tropical rainforest of Southeast Asian nations like Indonesia, Malaysia, Sri Lanka, Philippines and Thailand where traditional medicine uses the pericarp for the treatment of abdominal pain, diarrhea, cystitis, eczema, dysentery, wound suppuration and chronic ulcers [2,3]. In vitro and in vivo laboratory studies have demonstrated that extracts of G. mangostana have very diverse pharmacological activities including anti-inflammatory, cytotoxic, antioxidant, antitumoral, immunomodulatory, neuroprotective, anti-allergic, antibacterial and antiviral properties [4][5][6][7]. Phytochemical investigation of the pericarp of G. mangostana revealed the presence of prenylated xanthones, benzophenones, bioflavonoids and triterpenes [8][9][10]. Over 68 xanthone-type constituents were reported [11], of which the prenylated cage-type is particularly encouraging for further biological and chemical studies. The most studied xanthones are the α-, β-, and γ-mangostins, garcinone E, 8-deoxygartanin and gartanin [7,12].
The present study evaluated the in vitro antileishmanial, antiplasmodial and antitrypanosomal potential of the dichloromethane and ethyl acetate extracts of G. mangostana, as well as the isolation and characterization of its xanthone constituents.

In Vitro Antiprotozoal and Antimicrobial Activity
The dichloromethane and ethyl acetate extracts of G. mangostana were evaluated in an integrated in vitro screen for their antiplasmodial, antileishmanial and antitrypanosomal potential (Table 3). While the ethyl acetate extract showed no antiprotozoal activity at all, a pronounced inhibitory effect (IC 50 ) was obtained with the dichloromethane extract against Plasmodium falciparum (IC 50 2.7 µg/mL) and Trypanosoma brucei (IC 50 0.5 µg/mL), but only with acceptable selectivity (SI) for T. brucei (SI 18.8). Some side activity was also noted against T. cruzi and Leishmania infantum (IC 50 7.6 and 7.5 µg/mL), but with low selectivity.
The major constituent α-mangostin was also checked for antimicrobial potential against Candida albicans, Escherichia coli, Pseudomonas aeruginosa, Bacillius subtilis, Staphylococcus aureus, Mycobacterium smegmatis, M. cheleneoi, M. xenopi and M. intracellulare (Table 4). Although inhibitory activity could be indicated against B. subtilis and S. aureus (MIC 1.6 and 3.2 µg/mL) and the Mycobacterium species (MIC 1.5 µg/mL), selectivity was quite low in view of the observed cytotoxicity on MRC-5 cells (IC 50 7.5 µM) ( Table 3). No activity at all was found against C. albicans, E. coli and P. aeruginosa (IC 50 >200 µg/mL).  To the best of our knowledge, no data exist in the literature regarding the antiprotozoal activity and potential significance of G. mangostana as a source of antitrypanosomal and antiplasmodial compounds. G. parvifolia (Miq) has been used as a herbal remedy to treat malaria [21] and α-mangostin was found active against P. falciparum with IC 50 values of 5.1 and 17 µM [22,23]. In our study, α-mangostin was found slightly more potent (IC 50 2.2 µM), but also cytotoxic to MRC-5 cells (IC 50 7.5 µM), hence suggesting a non-specific inhibition. The latter also explains the observed activity against L. infantum, T. brucei and T. cruzi, with IC 50 values between 8.0 and 9.0 µM ( Table 3). Another illustration of non-selectivity are several studies quoting the antimicrobial potential of G. mangostana extract [24,25]. However, the observed IC 50 values may still justify the claimed (topical) uses of G. mangostana to treat infections in the traditional medicine.
This study clearly illustrates that interpretation of the antiprotozoal and antimicrobial potential of prenylated xanthones proves to be far from easy in view of the low level of selectivity. Available data in literature must be interpreted with great caution, particularly when parallel cytotoxicity data are not available. One route of further research on xanthones could be through structural modification with the sole option to maximize efficacy and reduce toxicity, e.g., non-selectivity.

General
The UV and IR spectra were recorded on Hitachi-UV-3200 and JASCO 320-A spectrometers. The 1 H-, 13 C-NMR and 2D-NMR spectra were recorded on a Bruker AMX-500 spectrometer with tetramethylsilane (TMS) as internal standard. Chemical shifts are given in ppm (δ) relative to tetramethylsilane internal standard and scalar coupling constants (J) are reported in Hertz. FAB and HRFABMS (neg. ion mode, matrix: glycerol) were registered on a JEOL JMS-HX110 mass spectrometer. Thin layer chromatography (TLC) was performed on precoated silica gel F254 plates (E. Merck, Darmstadt, Germany); detection was done at 254 nm and by spraying with p-anisaldehyde/H 2 SO 4 reagent. All chemicals were purchased from Sigma Chemical Company (St. Louis, MO, USA).

Plant Material
The fruits of G. mangostana Linn. were purchased from a local market at Riyadh city in 2009.

Reference Drugs
For the different tests, appropriate reference drugs were used as positive control: tamoxifen for MRC-5, chloroquine for P. falciparum, miltefosine for L. infantum, benznidazole for T. cruzi and suramin for T. brucei. All reference drugs were either obtained from the fine chemical supplier Sigma-Aldrich (Taufkirchen, Germany; tamoxifen, suramin) or from WHO-TDR (Geneva, Switzerland; chloroquine, miltefosine, benznidazole).

Biological Assays
The integrated panel of microbial screens and standard screening methodologies were adopted as previously described [26]. All assays were performed in triplicate at the Laboratory of Microbiology, Parasitology and Hygiene at the University of Antwerp (Antwerp, Belgium). Extracts were tested at five concentrations (64, 16, 4, 1 and 0.25 μg/mL) to establish a full dose-titration and determination of the IC 50 (inhibitory concentration 50%). The final in-test concentration of DMSO did not exceed 0.5%, which is known not to interfere with the different assays [26]. The selectivity of activity was assessed by simultaneous cytotoxicity evaluation on the MRC-5 fibroblast cell line. The criterion for activity was an IC 50 <10 μg/mL and a selectivity index (SI) of >4.

Antileishmanial Activity
L. infantum MHOM/MA (BE)/67 amastigotes were collected from the spleen of an infected donor hamster and used to infect primary peritoneal mouse macrophages. To determine in vitro antileishmanial activity, 3 × 10 4 macrophages were seeded in each well of a 96-well plate. After 2 days outgrowth, 5 × 10 5 amastigotes/well, were added and incubated for 2 h at 37 °C. Pre-diluted plant extracts were subsequently added and the plates were further incubated for 5 days at 37 °C and 5% CO 2 . Parasite burdens (mean number of amastigotes/macrophage) were microscopically assessed on 500 cells after Giemsa staining of the testplates, and expressed as a percentage of the blank controls without plant extract.

Antitrypanosomal Activity
Trypanosoma brucei Squib-427 strain (suramin-sensitive) was cultured at 37 °C and 5% CO 2 in Hirumi-9 medium, supplemented with 10% fetal calf serum (FCS) [28]. About 1.5 × 10 4 trypomastigotes/well were added to each well and parasite growth was assessed after 72 h at 37 °C by adding resazurin [29]. For Chagas disease, T. cruzi Tulahuen CL2 (benznidazole-sensitive, LacZ-reporter strain) [30] was maintained on MRC-5 cells in minimal essential medium (MEM) supplemented with 20 mM L-glutamine, 16.5 mM sodium hydrogen carbonate and 5% FCS. In the assay, 4 × 10 3 MRC-5 cells and 4 × 10 4 parasites were added to each well. After incubation at 37 °C for 7 days, parasite growth was assessed by adding the substrate chlorophenol red α-D-galactopyranoside. The color reaction was read at 540 nm after 4 h and absorbance values were expressed as a percentage of the blank controls.

Antimicrobial Activity
Samples were tested for antimicrobial activity according to the Clinical Laboratory Standard Institution using American type of Culture Collection (ATCC) standard [31].
3.6.5. Cytotoxicity Assay MRC-5 SV 2 cells were cultivated in MEM, supplemented with L-glutamine (20 mM), 16.5 mM sodium hydrogen carbonate and 5% FCS. For the assay, 10 4 MRC-5 cells/well were seeded onto the test plates containing the pre-diluted sample and incubated at 37 °C and 5% CO 2 for 72 h. Cell viability was assessed fluorimetrically after 4 h of addition of resazurin. Fluorescence was measured (excitation 550 nm, emission 590 nm) and the results were expressed as % reduction in cell viability compared to control [26].

Conclusions
Interpretation of the antiprotozoal and antimicrobial potential of prenylated xanthones proves to non-conclusive in view of the low level of selectivity. One route of further research on this subject could be through structural modification with the sole option to maximize efficacy and avoid non-selectivity.