PHYTOTHERAPY RESEARCH

Phytother. Res. (2015)

Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/ptr.5362

In vivo Antimalarial Activity of -Mangostin and

the New Xanthone -Mangostin

Yulieth Upegui,1 Sara M. Robledo,1,2* Juan Fernando Gil Romero,3 Winston Quiones,3
Rosendo Archbold,3 Fernando Torres,3 Gustavo Escobar,3 Bibiana Nario3 and
Fernando Echeverri3*
1
PECET, Medical Research Institute, School of Medicine, University of Antioquia, Calle 67 No. 53-108, Medelln 050010, Colombia
2
Center for Development of Products against Tropical Diseases CIDEPRO, Calle 67 No. 53-108, Medelln 050010, Colombia
3
Institute of Chemistry, Faculty of Exact and Natural Sciences, University of Antioquia, Calle 67 No. 53-108, Medelln 050010,
Colombia

Based on the previously reported in vitro antiplasmodial activity of several xanthones from Garcinia mangostana,
two xanthones, -mangostin and a new compound, -mangostin, were isolated from mangosteen husk, and the
in vitro antiplasmodial and cytotoxic effects were determined. -Mangostin was more active against the resistant
Plasmodium falciparum chloroquine-resistant (FCR3) strain (IC50 = 0.2 0.01 M) than -mangostin
(IC50 = 121.2 1.0 M). Furthermore, the therapeutic response according to the administration route was evaluated
in a Plasmodium berghei malarial murine model. The greatest therapeutic response was obtained with intraperito-
neal administration; these xanthones reduced parasitemia by approximately 80% with a daily dose of 100 mg/kg
administered twice a day for 7 days of treatment. Neither compound was effective by oral administration. Noticeable
toxicological effects were not observed. In addition to the antimalarial effect of these xanthones isolated from G.
mangostana husk, the availability of larger amounts of husk raw material to purify the bioactive xanthones is advan-
tageous, permitting additional preclinical assays or chemical transformations to enhance the biological activity of
these substances. Copyright 2015 John Wiley & Sons, Ltd.
Keywords: Garcinia mangostana; xanthones; Plasmodium falciparum; Plasmodium berghei; antimalarial activity.

bottlenecks in the discovery and development of new

INTRODUCTION
Malaria is a parasitic endemic disease that is widely dis-
tributed worldwide and affects a large economically dis-
advantaged population living in rural areas of tropical
and subtropical countries (WHO, 2013a; Murray et al.,
2012). Conventional treatments include compounds
such as aminoquinolines, antifolates, artemisinin deriva-
tives, and the hydroxynaphthoquinone atovaquone. The
use of these compounds in affected populations presents
disadvantages related to their relatively high cost and
the development of resistant parasites (WHO, 2013b;
Toshihiro and Kazuyuki, 2012). Therefore, there is an
urgent need to discover and develop new and better
drugs. Unfortunately, the drug discovery and develop-
ment processes are not only expensive but also highly
time-consuming and complicated.
The search for novel and effective agents against the
Plasmodium parasite, which is the causative agent of
malaria, has led to the identification of thousands
of molecules present in nature; however, only a few of
these compounds have been further evaluated for their
pharmacological potential. One of the most important
* Correspondence to: Sara Robledo, Center for Development of Products
against Tropical Diseases CIDEPRO, Calle 67 No. 53108, Medelln
050010, Colombia; Fernando Echeverri, Institute of Chemistry, Faculty of
Exact and Natural Sciences, University of Antioquia, Calle 67 No. 53108,
Medelln 050010, Colombia.
E-mail: sara.robledo@udea.edu.co (Sara Robledo); feche@une.net.co (Fernando
Echeverri)
Copyright 2015 John Wiley & Sons, Ltd.
drugs from natural products is the availability of suffi-
cient plant material to obtain pure active molecules for
more advanced evaluation. This process includes assays
of the therapeutic response in an adequate animal
model and Quantitative Structure Activity Relation-
ships (QSAR) analysis of the natural products.
Xanthones isolated from Garcinia mangostana Linn.
G. mangostana Linn. (mangosteen, family Clusiaceae)
possesses a wide spectrum of pharmacological proper-
ties, including antioxidant, antitumor, antiallergic,
antiinflammatory, antibacterial, antifungal, and antiviral
activities (Enserink, 2007; Obolskiy et al., 2009; Balunas
et al., 2008; Michel et al., 2012). Several xanthones also ex-
hibit in vitro antiplasmodial activity, with IC50 < 0.8 g/mL
(Pedraza et al., 2008; Hay et al., 2004; Molinar et al., 2006;
Riscoe et al., 2005; Limei et al., 2007). However, this poten-
tial antiplasmodial activity must be confirmed by in vivo
studies in malaria animal models to determine the ade-
quate dose and scheme of administration and the cytotox-
icity and preliminary toxicology and pharmacokinetic
profiles. These compounds are primarily extracted from
the husk and latex. The husk is a by-product of the juice
and beverage industry and serves as a sufficient source of
raw materials to isolate xanthones in abundance for
demonstration of both their in vitro activity and their
antimalarial potential.
In this study, mangosteen husk was fractionated, and
two xanthones were isolated, the known -mangostin
and a new compound, -mangostin. The antiplasmodial
activity, cytotoxicity, and hemolytic potential of both
Received 03 February 2015
Revised 16 March 2015
Accepted 10 April 2015
 Y. UPEGUI ET AL.
xanthones were determined using in vitro assays, and the
therapeutic response was evaluated in a Plasmodium
berghei murine model for malaria (Ovenden et al., 2011).
MATERIALS AND METHODS
General experimental conditions. Chromatographic sepa-
rations were performed using a preparative high-
performance liquid chromatograph (prep-HPLC) (Gilson,
Villiers Le Bel, France) and a Supelco Ascentis C18 col-
umn (250 21.2 mm) (Bellefonte, PA, USA) with a linear
gradient beginning at 95% solvent A (H2O) (Merck,
Darmstadt, Germany) and decreasing to 40% A over a
period of 15 min before increasing to 50% A in 20 min,
followed by a gradient to 95% solvent B (MeOH)
(Merck) for 30 min, 95% B up to 35 min, and returning
to 95% A beginning at 38 min, with a flow rate of
7 mL/min and monitoring at = 254 nm. We performed ad-
ditional column chromatography using Sephadex LH-20
(Sigma-Aldrich, St. Louis, MO, USA).
Column chromatography was performed using silica
gel 60 (200300 mesh, Merck) and Sephadex LH-20
(Sigma-Aldrich). For thin-layer chromatography, silica
gel 60F254-impregnated aluminum sheets (0.25 mm,
Merck) were used. Compounds were detected under
ultraviolet light (254, 360 nm) after spraying with FeCl3
(3%) and heating at 110 C.
1
H, 13C, and 2D nuclear magnetic resonance (NMR)
spectra of the isolated compounds were recorded on a
Bruker DRX 300 spectrometer (Bruker Bio-Spin GmbH,
Rheinstetten, Germany) operating at 300 MHz for 1H-
NMR and 75 MHz for 13C-NMR, using a mixture of
CDCl3/acetone-d6 (Sigma-Aldrich) as the solvent, and
Tetramethylsilane (TMS) as an internal standard. Chemi-
cal shifts () are reported in ppm, and the coupling con-
stants (J) are reported in Hz. High-resolution mass
spectra were obtained using a Quadrupole-time-of-flight
(Q-Tof) mass spectrometer (Waters, Milford, MA, USA).
General extraction method. G. mangostana husks were
obtained from the local market in June 2013; approxi-
mately 350 g of pericarp was milled with 90% EtOH
and evaporated to dryness, yielding a deep yellow solid
(15 g). An aliquot (7 g) was extracted three times with
200 mL of n-hexane/CH2Cl2/MeOH (2:1:1) and evapo-
rated to yield 5 g, which was dissolved in the same sol-
vent mixture and separated using Sephadex LH-20
column chromatography (5 80 cm); 25 fractions of
100 mL were obtained, evaporated, and monitored by
thin-layer chromatography on silica gel using n-hexane/
EtOAc (3:1, v/v) or n-hexane/EtOAc (1:3, v/v). -
Mangostin 1 (3.2 g) and compound 2 (1.3 g) were purified
from fractions 1216 and 2022 as a deep yellow powder
and a gummy yellow solid, respectively.
In vitro cytotoxic activity. Cytotoxicity was assessed in
the promonocytic cell line U937 using the 3-(4,5-dimeth-
ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
(Sigma-Aldrich) assay as previously described (Robledo
et al., 2005; Taylor et al., 2011). The cells were adjusted
to a concentration of 1 105 cells/mL in 96-well plates
Copyright 2015 John Wiley & Sons, Ltd.
suspended in RPMI 1640 medium with 10% fetal bovine
serum and the corresponding concentration of com-
pound. Six double-serial concentrations for each com-
pound (from 200 to 6.25 g/mL) were evaluated. Cells
exposed to each concentration were incubated at 37 C
and 5% CO2 for 72 h. To determine the toxic effect,
20 L of MTT (0.5 mg/mL) was added to each well,
and the plates were subsequently incubated at 37 C
for 3 h. The amount of formazan produced by the grow-
ing cells was quantified at 570 nm using an Enzyme-
Linked Immunosorbent Assay (ELISA) microplate
reader (Bio-Rad Benchmark Microplate Reader Inc,
Hercules, CA, USA). Amphotericin B and doxorubicin
were used as the positive control, whereas untreated
cells (cultured in medium alone) were used as the nega-
tive control. All determinations were performed in trip-
licate in two isolated experiments. Cytotoxicity was
determined according to the percentages of viability
and cell growth inhibition obtained for each compound,
amphotericin B, or medium alone. Percentages of viabil-
ity were calculated using Equation (1), as follows: % via-
bility = (OD of treated cells)/(OD of control cells) 100,
where the Optical Density (OD) of the control cells cor-
responds to 100% viability. In turn, the percentage of cell
growth inhibition is calculated using Equation (2), as fol-
lows: % cell growth inhibition = 100 % viability. The re-
sults are expressed as the lethal concentration 50 (LC50)
calculated using the probit method (Finney, 1978).
In vitro hemolytic activity. The ability to induce hemoly-
sis was evaluated following the method described else-
where. In brief, 500 L of human red blood cells
(huRBCs), adjusted to 5% hematocrit in RPMI 1640
medium, were placed into each well of 24-well plate
and subsequently exposed against four different concen-
trations of compounds (200, 50, 12.5, and 3.125 g/mL).
Detection of free hemoglobin, after 48 h of incubation at
37 C, is the evidence that the compound induces
hemolysis. The measurement of the concentration of
free hemoglobin was performed by spectrophotometry
at 542 nm (Varioskan Flash Multimode Reader, Thermo
Scientific, Waltham, MA, USA), and intensity of color
(absorbance) was registered as optical densities. Deter-
minations were carried out by triplicate in at least two
independent experiments (Conceio et al., 2006).
In vitro antiplasmodial activity. The antiplasmodial
activity was determined using an asynchronous culture
of Plasmodium falciparum strain 3D7 (chloroquine-
sensitive) maintained under standard conditions as
described by Trager and Jensen (1976). The effect of the
compounds on the growth of the parasites was evaluated
using a fluorometric method by quantifying parasite
DNA using ethidium bromide staining (Insuasty et al.,
2015). Four serial concentrations in base 4 (from 200 to
6.25 g/mL) were evaluated. Briefly, the P. falciparum
cultures with total forms were adjusted from 1.5% to
2% parasitemia and 5% hematocrit in RPMI 1640
medium supplemented with 0+ human serum (total me-
dium). Subsequently, 500 L of a suspension of the para-
sites and 500 L of each concentration of the compound
were simultaneously placed in each well of a 24-well plate
for evaluation. Parasites exposed to each concentration
Phytother. Res. (2015)
 ANTIMALARIAL ACTIVITY OF A NEW XANTHONE

were incubated at 37 C in a N2 (90%), CO2 (5%), and O2 Table 1. Nuclear magnetic resonance data (300 and 75 MHz for
(5%) atmosphere for 48 h. The DNA was subsequently ex- 1
H and 13C, respectively) for -mangostin and -mangostin
tracted and purified using a lysis solution (10 mM Tris, pH
8.0, 5 mM EDTA, and 20 mM NaCl) and proteinase K CDCl3/acetone (1:2, v/v) d6, in ppm, J in Hz
(1 mg/mL). Briefly, parasites (1 mL) were centrifuged at
-Mangostin -Mangostin
800 g for 10 min. Then, supernatant was discarded, and pel-
let was resuspended in 200 L of lysis solution. After 1 h in- Atom H (HSQC) C H (HSQC) C
cubation at 65 C, DNA was precipitated with 300 L of
ethanol 90%. After being dried at room temperature, the 1 160.3 154.64
pellet was rehydrated with 200 L of TE solution (10 mM 2 110.2 109.55
TrisHCl and 1 mM EDTA pH 8.0). The DNA content 3 162.3 160.18
was determined using fluorometry (510 nm excitation and 4 6.31 (1H, s) 91.9 127.55
590 nm emission). Chloroquine diphosphate salt (Sigma- 4a 156.6 161.14
Aldrich) was used as a positive antiplasmodial activity 5 6.77 (1H, s) 101.6 6.60 (1H, s) 100.18
control, whereas unexposed parasites (cultured in me- 6 155.4 150.18
dium alone) were used as the negative control. 7 143.5 139.78
Antiplasmodial activity was determined by the inhibi- 8 137.2 127.55
tion of parasite growth according to the florescence 8a 111.0 111.44
emitted in arbitrary units using Equation (3) as follows: 9 181.8 182.12
% parasite growth = Arbitrary Fluorescent Units (AFU) 9a 102.6 103.09
exposed parasites/AFU control parasites 100. In turn, 10a 154.9 152.59
the percentage of inhibition was calculated using Equa- 11 3.20 (2H, d, 7 Hz) 26.0 3.20 (2H, d, 6.6 Hz) 25.50
tion (4) as follows: % inhibition = 100 % parasite 12 5.12 (1H, m) 122.7 5.12 (1H, m) 122.80
growth. The results are expressed as the effective con- 13 130.4 131.49
centration 50 (EC50) calculated using the probit 14 1.81 (3H, s) 25.1 1.62 (3H, s) 17.81
method (Finney, 1978). 15 1.68 (3H, s) 17.3 1.51 (3H, s) 25.64
16 4.00 (2H, d, 6 Hz) 21.2 4.0 (1H, d, 6.6 Hz) 21.14
17 5.25 (1H, m) 123.9 5.12 (1H, m) 122.40
18 130.6 131.28
In vivo antiplasmodial therapeutic response. The thera- 19 1.79 (3H, s) 16.9 1.51 (3H, s) 17.49
peutic efficacy was assessed in an established infection 20 1.66 (3H, s) 25.1 1.68 (3H, s) 25.55
using the Ranes test (Ryley and Peters, 1970). Briefly, 7-OMe 3.77 (3H, s) 61.5
BALB/c mice, 8 to 10 weeks old, were infected intraperito-
neal with 1 107 red blood cells infected with P. berghei HSQC, heteronuclear single quantum coherence.
ANKA. After infection was confirmed by a blood smear
stained with Giemsa, the mice were distributed into seven whose 1H-NMR and 13C-NMR are similar to previously
groups (six animals each), and treatment began using one reported values (Zarena and Udaya, 2011). The second
of (a) oral -mangostin 100 mg/kg/day for 7 days, (b) oral xanthone is a new compound containing several hy-
-mangostin 100 mg/kg/day for 7 days, (c) intraperitoneal droxyl groups in the xanthone moiety and two isoprene
-mangostin 50 mg/kg/day for 5 days, (d) intraperitoneal chains. This compound was named -mangostin 2. In
-mangostin 50 mg/kg/day for 7 days, (e) intraperitoneal - comparison with -mangostin, -mangostin possesses
mangostin 100 mg/kg twice daily for 7 days, (f) intraperito- only one aromatic proton and lacks the methoxy group
neal -mangostin 100 mg/kg twice daily for 7 days, and (g)
of -mangostin (Table 1).
oral chloroquine diphosphate salt 15 mg/kg/day for 4 days.
The structure of compound 2 was assigned based on
Parasitological and clinical follow-up was performed daily
mass spectrometry and 1D and 2D NMR as follows:
until 3 days post-treatment. At the end of the test, liver
m/z 412.1530 (calculated 412.1522) for C23H24O7. Two
function (serum Alanine aminotransferase (ALT) level)
isoprene groups were detected by signals at 1.51 (s,
and renal function (serum creatinine and Blood Urea Ni-
6H), 1.62 (s, 3H), and 1.68 (s, 3H) in addition to a pair
trogen (BUN) levels) were determined to evaluate the
of doublets at 3.20 and 4.00 for methylene groups, the
toxic effect of the treatment. Histopathological examina-
former at a peri position to a carbonyl group. Additionally,
tion of the liver, spleen, and kidneys was also performed.
two vinyl protons at 5.12 (m, 2H) and one aromatic
The therapeutic efficacy was calculated as the percentage
proton at 6.60 were observed. Although the position of
of chemosuppression in treated groups versus the un-
the aromatic proton was deduced through heteronuclear
treated control. The institutional ethical committee for ex-
multiple-bond correlations, it could initially be attributed
perimentation in animals approved all assays performed in
to H-5 because the aromatic proton exhibits a
the animal model (Act 77, 10 July 2012).
heteronuclear single quantum coherence correlation with
C-5 ( 100.18) that is similar to that observed for -
mangostin ( 101.73). Thus, the aromatic proton at 6.60
exhibited long-range correlations with quaternary carbon
RESULTS AND DISCUSSION atoms at 111.44 (C-8a), 139.78 (C-7), 150.18 (C-6),
and 152.59 (C-10a). Additionally, Cq at 111.44 also ex-
Characterization of compound 2 hibited 3J correlations with the methylene signal at 4.00
(H-16), which in turn exhibits several long-range correla-
Using a bioguided process, two compounds were obtained tions to quaternary carbon atoms at 127.55 (C-8),
from column chromatography using Sephadex LH-20. 122.40 (C-17), 131.28 (C-18), and 139.78 (C-7). There-
The first compound was identified as -mangostin 1, fore, the single aromatic proton must be attached to C-5.
Copyright 2015 John Wiley & Sons, Ltd. Phytother. Res. (2015)
 Y. UPEGUI ET AL.

Table 3. In vitro cytotoxicity and hemolytic activity (huRBC) of

extract, chromatographic fractions, and pure compounds from
Garcinia mangostana husk

ISb
Cytotoxicity Hemolysis
Product (U-937)a (huRBC)a 3D7 FCR3

Crude extractc 190.6 13.0 34.3 4.1 16.7 560.6

Fraction 1c 90.5 5.3 54.6 8.7 <4.5 47.6
Figure 1. Chemical structures of -mangostin 1 and the new com- -Mangostin 130.6 19.7 69.7 2.7 3.6 653.0
pound -mangostin 2, with some heteronuclear multiple-bond -Mangostin 262.6 40.2 39.0 3.2 21.1 <2.2
correlations.
Fraction 8c 41.6 8.6 53.2 3.7 37.8 416.0
Chloroquine 152.2 5.2 >200 3805.0 543.6
According to the molecular formula, the other aro- Amphotericin B 54.4 0.1 19.4 5.8 NA NA
matic ring must have three hydroxyl groups attached Doxorubicin 0.1 0.01 42.5 16.9 NA NA
in addition to the other isoprene chain. The protons of
the methylene group at 3.20 (H-11) exhibits 3J cou- huRBC, human red blood cell; IS, index of selectivity; NA, not
pling to signals at 160.18 (C-3) and 161.14 (C-4a); applicable.
a
therefore, the isoprene side chain is attached to C-4. Data represent the mean values SD of the lethal concentration
However, this carbon atom signal most likely overlaps 50 (LC50) in mM.
with the C-8 signal. Thus, -mangostin is 1,2,3,6,7-
b
IS = Cytotoxicity (LC50) in U937/IC50.
pentahydroxy-4,8-diisoprenylxanthone (Fig. 1).
c
LC50 is expressed as g/mL.

3D7 and FCR3 strains of -mangostin are similar to

In vitro antiplasmodial and cytotoxic activities
The crude ethanol extract of G. mangostana was
separated by column chromatography to obtain eight
fractions. After thin-layer chromatography analysis,
fractions containing same compounds were drawn to-
gether and then two fractions and two pure compounds
were obtained. All samples were analyzed in vitro
against asynchronous cultures of P. falciparum 3D7 and
FCR3 strains, which are chloroquine-sensitive and
chloroquine-resistant strains, respectively. Promising
chromatographic fractions and compounds were se-
lected as the products with IC50 values < 20 M (pure)
or <20 g/mL (extracts or fractions). As shown in
Table 2, almost all compounds were more active against
resistant than the sensitive P. falciparum strain. Fraction
1 and 8 were highly active against both P. falciparum
strains exhibiting IC50 2.0 M. Similar results were ob-
served for crude extract, although activity of this extract
on sensitive strain was nearly 10 M. Fraction 1 was
active against the resistant strain, but not against the
sensitive strain. Unexpectedly, strain FCR3 was more
sensitive than the 3D7 strain to -mangostin. Contrary,
-mangostin was active against the sensitive strain, but
not against the resistant strain. The IC50 values for the
those previously reported (Ruiz et al., 2011; Barea
et al., 2013; Fotie et al., 2003). It would be interesting
to determine which components of the fractions 1 and
8 might be responsible of activity in the resistant but
not the sensitive P. falciparum strain.
As shown in Table 3, the potential hemolysis on
huRBCs was higher than cytotoxicity on human U937
macrophages. The crude extract was less toxic than frac-
tions 1 and 8 to U937 cells (LC50 of 190.6 vs 90.5 and
41.6 g/mL, respectively), whereas -mangostin showed
less cytotoxicity than -mangostin (LC50 of 262.6 40.2
and 130.6 19.7 M, respectively). In contrast, all frac-
tions and compounds produced hemolysis of huRBCs.
Chloroquine did not exhibit toxicity on U937 cells nor
hemolytic activity on huRBCs. At contrary, amphotericin
B and doxorubicin were cytotoxic on U937 macrophages
and hemolytic on huRBCs.
The index of selectivity (IS) calculated for each prod-
uct after comparing the antiplasmodial activity against
the chloroquine-resistant and chloroquine-sensitive P.
falciparum strains and their cytotoxicity are summarized
in Table 3. All compounds except -mangostin exhibited
higher IS in the chloroquine-resistant P. falciparum
FCR3 strain. The best IS values were observed for -
mangostin.

Table 2. In vitro antiplasmodial activity of extracts and pure Antimalarial therapeutic response
compounds from Garcinia mangostana husk
The therapeutic response was determined at the chemo-
Product 3D7 FCR3
suppression level according to parasitemia. Thus, on
Crude extracta 11.40 1.8 0.34 0.07 our scale, the therapeutic response was high when
Fraction 1a >20.00 1.90 0.7
chemosuppression was >80%, moderate when between
-Mangostin 36.10 4.9 0.20 0.01
50% and 80%, and low when this value was <50%. In-
-Mangostin 12.40 1.0 >121.20 1.0
fected mice treated with -mangostin and -mangostin
Fraction 8a 1.10 0.3 0.10 0.0
orally administered at 100 mg/kg/day for 7 days exhib-
Chloroquine 0.04 0.03 0.28 0.09
ited chemosuppression of P. berghei parasitemia of
26.9% and 35.0% compared with the untreated control
The data represent the mean values SD of the inhibitory concentra- group (Table 4). However, when -mangostin or -
tion 50 (IC50) expressed as M against Plasmodium falciparum 3D7 mangostin was intraperitoneal administered once a
(chloroquine-sensitive) and FCR3 (chloroquine-resistant) strains. day at 50 mg/kg/day for 7 days, parasitemia decreased
a
IC50 is expressed as g/mL. by 45.8% and 54.3%, respectively. After treatment of
Copyright 2015 John Wiley & Sons, Ltd. Phytother. Res. (2015)
 ANTIMALARIAL ACTIVITY OF A NEW XANTHONE
Table 4. Therapeutic efficacy of xanthones from Garcinia mangostana
Product Dosea Schemeb
-Mangostin 100 1X-7 days
50 1X-5 days
100 2X-7 days
-Mangostin 100 1X-7 days
50 1X-7 days
100 2X-7 days
Chloroquine salt 15 1X-4 days
No treatment NA NA
The data are expressed as the mean SD of six animals per group of treatment compared with the control without treatment. NA, not
applicable.
a
mg/kg/day.
b
1X, once/day; 2X, twice/day.
c
Route of administration: O, oral; IP, intraperitoneal.
d
Data obtained on the first day post-treatment.
e
data obtained on the seventh day of treatment.
infected mice with 100 mg/kg/day -mangostin or -
mangostin, twice a day for 7 days, parasitemia decreased
by 80.7% and 79.9%, respectively (Table 4). The reduction
in parasitemia observed in this study is higher than previ-
ously reported values, in which the reduction of
parasitemia ranged from 15.1% to 70.5% following intra-
peritoneal administration. Because of similar levels of ac-
tivity for -mangostin and -mangostin at identical
concentrations, a mechanism of action based on the num-
ber of hydroxyl groups does not account for the activity
of these compounds.
The low in vivo activity of both xanthones when
orally administered is most likely related to the low bio-
availability due to reduced digestive absorption rates.
All animals exhibited the typical signs of the disease
such as weight loss, drowsiness, dehydration, dyspnea,
and goose bumps. The most critical symptom of the dis-
ease observed in the animals was respiratory distress
produced by multiple causes related to the infection, in-
cluding marked anemia, lung edema based on histopath-
ological analysis, and multi-organ failure. This symptom
was more severe in untreated animals and was further
confirmed by histopathological studies. Following treat-
ment, a greater reduction in these symptoms was
observed in the groups treated with the highest doses
of both xanthones and chloroquine. Hepatic disorders
were evident by the high levels of ALT and histologi-
cally by the congestion, cardiomegaly, and malarial pig-
ment deposits in the organ and were more pronounced
in the groups that were not able to control greater than
50% of the parasitemia. At the renal level, the creati-
nine level was within the normal range, although there
was a slight increase of BUN, most likely due to the
protein-rich diet. Histologically, atrophy was present in
addition to the presence and congestion of pigments,
findings that are consistent with the lysis of red blood
cells observed at the vascular level.
The antiplasmodial activity reported herein correlates
with that obtained in vitro in similar reports using the
same type of xanthones. Recently, Al-Massarani et al.
(2013) reported that -mangostin is active on schizonts
of P. falciparum that are resistant to chloroquine
(IC50 = 2.2 g/mL) but is also cytotoxic against human
MRC-5 fibroblasts (IC50 = 9.4 g/mL), yielding an IS of
3.4. In the present study, -mangostin was also active
Copyright 2015 John Wiley & Sons, Ltd.
Routec Parasitemiad (X SD) Chemosuppression n
O 25.2 8.5 26.9
IP 18.7 5.0 45.8
IP 6.6 4.5 80.7
O 22.4 6.9 35.0
IP 15.75 5.9e 54.3
IP 6.9 1.4 79.9
O 00 100
NA 34.5 5.3 NA
against total parasite forms (rings, trophozoites, and
schizonts) of chloroquine-resistant FCR3 P. falciparum
(IC50 = 0.2 M), while -mangostin did not show activity
against total forms of chloroquine-resistant P. falciparum
parasites (IC50 121.2 M). Moreover, -mangostin and
-mangostin were active against total parasite forms
(rings, trophozoites, and schizonts) of chloroquine-
sensitive 3D7 P. falciparum (IC50 = 36.1 and 12.4 M,
-mangostin and -mangostin, respectively). Both
compounds produced hemolysis in huRBCs, which are
the host cells for P. falciparum (LC50 = 69.7 mM and
39.0 M, -mangostin and -mangostin, respectively).
On the other hand, -mangostin showed antiplasmodial
activity at a concentration >121.2 M and hemolytic
activity at 39.0 M; thus, the antiplasmodial activity
reported is most likely to be due to the effect of the com-
pound on red blood cells rather than a direct effect on
the parasite. Nevertheless, in spite of the differences ob-
served in vitro, chemosuppression in mice infected with
P. berghei was similar for both compounds (Table 4).
Differences between the in vitro and in vivo data are
likely to be due to issues relating to absorption, distribu-
tion, metabolism, and excretion.
The high level of activity found in vitro for both G.
mangostana xanthones indicates that these compounds
may be considered promising molecules. Furthermore,
the influence of the administration scheme on the
antiplasmodial activity was confirmed by in vivo studies
in a P. berghei murine model. Suppression of the
parasitemia level was from 26.9% to 80.7% for -
mangostin and from 35.0% to 79.9% for -mangostin
depending on the dose, route of administration, and
therapeutic scheme. In contrast to the study by Al-
Massarani et al. (2013), the results reported herein
suggest that the biological activities of -mangostin and
the new metabolite -mangostin are differential and se-
lective. Thus, the antiplasmodial activity is higher than
the cytotoxic activity, yielding IS values of 3.6 and
653.0 for -mangostin (chloroquine-sensitive and
chloroquine-resistant P. falciparum strains, respectively)
and 21.1 and <2.2 for -mangostin (chloroquine-sensi-
tive and chloroquine-resistant P. falciparum strains,
respectively). However, a possible role of metabolism of
these compounds in the differential activity remains to
be understood. Although -mangostin and -mangostin
Phytother. Res. (2015)
 Y. UPEGUI ET AL.
were cytotoxic in vitro, the in vivo assays did not indicate
alterations in animal behavior or toxic symptoms. These
results confirm that in vitro cytotoxicity is not predictive
of in vivo toxicity.
Because of the extensive use of G. mangostana in the
nutraceutical food industry, the bioavailability of -
mangostin has been recently evaluated, indicating poor
bioavailability (Li et al., 2011; Gutirrez and Failla,
2013). This finding suggests that in addition to the route
of administration, the compound itself most likely re-
quires modification to obtain more effective therapeutic
levels necessary for antimalarial activity.
The search for new natural bioactive molecules re-
quires a considerable investment of time and money.
Moreover, the lack of raw material to purify bioactive
molecules in the amounts necessary for preclinical
and clinical bioassays is an additional limiting factor
to the further development of new and better drugs.
In this case, the raw material to obtain the
antiplasmodial xanthones -mangostin and -
mangostin is abundant, as husks are readily available
from the industrial waste of beverages of G.
mangostana that are sold in numerous countries as
energizing nutraceuticals and functional foods.
Additionally, both compounds are found in high
concentrations in the hulls; nearly 3.2 and 1.0 g of -
mangostin and -mangostin, respectively, can be
obtained from 15.0 g of raw extract, and their purifi-
cation is relatively simple using Sephadex LH-20 col-
umn chromatography. An additional advantage is the
apparently low toxicity of -mangostin, as indicated
by the LD50 values of certain chemically related
molecules (Marques et al., 2012) and various reports
of their biological activity and ethnomedical history
(Obolskiy et al., 2009; Balunas et al., 2008; Shan
et al., 2011).
REFERENCES
Al-Massarani SM, El Gamal AA, Al-Musayeib NM, et al. 2013.
Phytochemical, antimicrobial and antiprotozoal evaluation of
Garcinia mangostana pericarp and -mangostin, its major
xanthone derivative. Molecules 18: 10,59910,608.
Balunas MJ, Su B, Brueggemeier RW, et al. 2008. Xanthones from the
botanical dietary supplement mangosteen (Garcinia mangostana)
with aromatase inhibitory activity. J Nat Prod 71: 11611166.
Barea C, Pabn A, Prez S. 2013. New amide derivatives of
quinoxaline 1,4-di-N-oxide with leishmanicidal and
antiplasmodial activities. Molecules 18: 47184727.
Conceio K, Konno K, Richardson M, et al. 2006. Orpotrin: a
novel vasoconstrictor peptide from the venom of the Brazilian
Stingray Potamotrygon gr. orbignyi. Peptides 27: 30923099.
Enserink M. 2007. Malaria treatment: ACT two. Science 318:
560563.
Finney JD. 1978. Statistical Method in Biological Assay, 3rd edn.
Academic Press: London; 508.
Fotie J, Nkengfack AE, Rukunga G. 2003. In-vivo antimalarial ac-
tivity of some oxygenated xanthones. Ann Trop Med Parasitol
97: 683688.
Gutirrez F, Failla ML. 2013. Biological activities and bioavailability
of mangosteen xanthones: a critical review of the current
evidence. Nutrients 5: 31633183.
Han AR, Kim JA, Lantvit DD, et al. 2009. Cytotoxic xanthone
constituents of the stem bark of Garcinia mangostana (mango-
steen). J Nat Prod 72: 20282031.
Hay AE, Hlesbeux JJ, Duval O, et al. 2004. Antimalarial xan-
thones from Calophyllum caledonicum and Garcinia vieillardii.
Life Sci 75: 30773085.
Insuasty B, Ramrez J, Becerra D, et al. 2015. An efficient synthe-
sis of new caffeine-based chalcones, pyrazolines and
Copyright 2015 John Wiley & Sons, Ltd.
Finally, despite the high number of compounds iso-
lated from mangosteen (approximately 70), -mangostin
has not been reported (Obolskiy et al., 2009; Balunas
et al., 2008; Michel et al., 2012; Han et al., 2009). It may
be that this compound is unique to the Colombian eco-
type of G. mangostana.
In conclusion, we report in vitro antiplasmodial activ-
ity of -mangostin at previously reported levels and the
antiplasmodial activity of new xanthone -mangostin
isolated from G. mangostana. Moreover, promising
results were found against the chloroquine-resistant P.
falciparum FCR3 strain. In addition, the in vivo thera-
peutic antimalarial response was demonstrated for both
xanthones when evaluated using intraperitoneal admin-
istration; in contrast, -mangostin and -mangostin are
not orally absorbed, suggesting that their bioavailability
requires improvement through an appropriate formula-
tion. Finally, in this work, the utility of the industrial
waste of mangosteen husk as a raw material to produce
active pharmaceutical compounds for antimalarial drug
development is highlighted.
Acknowledgements
This work was sponsored by Colciencias-COLOMBIA (grant
111548925424) and CIDEPRO (CIIEs, Universidad de Antioquia).
Y.U. thanks Universidad de Antioquia for a grant in Sustainability
Program and Colciencias for the Young Research. The authors also
recognize A. Daza MDV for assistance with the bioassays in mice.
Conflict of Interest
The authors declare no conflict of interest.
pyrazolo[3,4-b][1,4]diazepines as potential antimalarial,
antitrypanosomal and antileishmanial agents. Eur J Med
Chem 93: 401413.
Li L, Brunner I, Han AR, et al. 2011. Pharmacokinetics of -
mangostin in rats after intravenous and oral application. Mol
Nutr Food Res 55: S67S74.
Limei Y, Mouming Z, Bao Y, et al. 2007. Phenolics from hull of
Garcinia mangostana fruit and their antioxidant activities.
Food Chem 104: 176181.
Marques ES, Silva S, Niero R, et al. 2012. Genotoxicity
assessment of Garcinia achachairu Rusby (Clusiaceae)
extract in mammalian cells in vivo. J Ethnopharmacol 142:
362366.
Michel T, Destandau E, Fougre L, et al. 2012. New hyphenated
CPCHPLCDADMS strategy for simultaneous isolation,
analysis and identification of phytochemicals: application to
xanthones from Garcinia mangostana. Anal Bioanal Chem
404: 29632972.
Molinar E, Gonzalez J, Ortega E, et al. 2006. Antiprotozoal activity
against Plasmodium falciparum and Trypanosoma cruzi of xan-
thones isolated from Chrysochlamys tenuis. Pharm Biol 44:
550553.
Murray CJ, Rosenfeld LC, Lim SS, et al. 2012. Global malaria mor-
tality between 1980 and 2010: a systematic analysis. Lancet
379: 413431.
Obolskiy D, Pischel I, Siriwatanametanon N, et al. 2009. Garcinia
mangostana L. A phytochemical and pharmacological review.
Phytother Res 23: 10471065.
Ovenden SP, Cobbe M, Kissell R, et al. 2011. Phenolic glycosides
with antimalarial activity from Grevillea Poorinda Queen. J
Nat Prod 74: 7478.
Phytother. Res. (2015)
 ANTIMALARIAL ACTIVITY OF A NEW XANTHONE
Pedraza J, Crdenas N, Orozco M, et al. 2008. Medicinal proper-
ties of mangosteen (Garcinia mangostana). Food Chem
Toxicol 46: 32273239.
Riscoe M, Kelly JX, Winter R. 2005. Xanthones as antimalarial
agents: discovery, mode of action, and optimization. Curr
Med Chem 12: 25392549.
Robledo S, Osorio E, Muoz D, et al. 2005. In vitro and in vivo
cytotoxicities and antileishmanial activities of thymol and
hemisynthetic derivates. Antimicrob Agents Chemother 49:
16521655.
Ruiz L, Ruiz L, Maco M. 2011. Plants used by native Amazonian
groups from the Nanay River (Peru) for the treatment of ma-
laria. J Ethnopharmacol 133: 917921.
Ryley JF, Peters W. 1970. The antimalarial activity of some quino-
lone esters. Ann Trop Med Parasitol 64: 209222.
Shan T, Ma Q, Guo K, et al. 2011. Xanthones from mangosteen ex-
tracts as natural chemopreventive agents: potential anticancer
drugs. Curr Mol Med 11: 666677.
Taylor VM, Cedeo DL, Muoz DL, et al. 2011. In vitro and in vivo
studies of the utility of dimethyl and diethyl carbaporphyrin
ketals in treatment of cutaneous leishmaniasis. Antimicrob
Agents Chemother 55: 47554764.
Copyright 2015 John Wiley & Sons, Ltd.
Toshihiro M, Kazuyuki T. 2012. Evolution of Plasmodium
falciparum drug resistance: implications for the development
and containment of artemisinin resistance. Jpn J Infect Dis
65: 465475.
Trager W, Jensen JB. 1976. Human malaria parasites in continu-
ous culture. Science 193: 673675.
World Health Organization. World malaria report 2013a. http://www.
who.int/malaria/publications/world_malaria_report_2013/en/
World Health Organization. World health statistics 2013b. http://
www.who.int/gho/publications/world_health_statistics/2013/en/
Zarena AS, Udaya SK. 2011. Xanthones enriched extracts from
mangosteen pericarp obtained by supercritical carbon dioxide
process. Sep Purif Technol 80: 172178.
SUPPORTING INFORMATION
Additional supporting information may be found in the
online version of this article at the publishers web-site.
Phytother. Res. (2015)