molecules

Article
Anti-Bacterial and Anti-Fungal Activity of Xanthones
Obtained via Semi-Synthetic Modification of
α-Mangostin from Garcinia mangostana
Srinivasan Narasimhan 1 , Shanmugam Maheshwaran 1 , Imad A. Abu-Yousef 2 ,
Amin F. Majdalawieh 2 , Janarthanam Rethavathi 1 , Prince Edwin Das 1 and Palmiro Poltronieri 3, *
1 Asthagiri Herbal Research Foundation, 162A, Perungudi Industrial Estate, Perungudi,
Chennai 600096, India; asthagiri.herbal@gmail.com (S.N.); mahesh7605@yahoo.co.in (S.M.);
rethavathij@gmail.com (J.R.); prince.ahrf@gmail.com (P.E.D.)
2 Department of Biology, Chemistry and Environmental Sciences, American University of Sharjah,
P.O. Box 26666 Sharjah, United Arab Emirates; iabuyousef@aus.edu (I.A.A.-Y.);
amajdalawieh@aus.edu (A.F.M.)
3 Institute of Sciences of Food Productions, CNR-ISPA, Lecce 73100, Italy
* Correspondence: palmiro.poltronieri@ispa.cnr.it; Tel.: +39-083-242-2609; Fax: +39-083-242-2620

Academic Editors: Daniela Barlocco and Fiorella Meneghetti

Received: 14 December 2016; Accepted: 8 February 2017; Published: 12 February 2017

Abstract: The microbial contamination in food packaging has been a major concern that has paved the
way to search for novel, natural anti-microbial agents, such as modified α-mangostin. In the present
study, twelve synthetic analogs were obtained through semi-synthetic modification of α-mangostin
by Ritter reaction, reduction by palladium-carbon (Pd-C), alkylation, and acetylation. The evaluation
of the anti-microbial potential of the synthetic analogs showed higher bactericidal activity than
the parent molecule. The anti-microbial studies proved that I E showed high anti-bacterial activity
whereas I I showed the highest anti-fungal activity. Due to their microbicidal potential, modified
α-mangostin derivatives could be utilized as active anti-microbial agents in materials for the
biomedical and food industry.

Keywords: α-mangostin; anti-bacterial; anti-fungal; packaging; textiles; biomedical device;

semi-synthetic modification

1. Introduction
The fruit of Garcinia mangostana Linn. (mangosteen), of the family Guttiferae, has been used in Asian
traditional medicines for the treatment of skin infections, wounds, diarrhea, dysentery, suppuration,
leucorrhea, chronic ulcers, and gonorrhea [1,2]. In addition, mangosteen with essential minerals
is commercially used as dietary supplement for cancer patients [3]. The pericarp of the fruit
contains high amounts of xanthones, such as α-mangostin (Figure 1), β-mangostin, γ-mangostin,
etc., and considerable amounts of other bioactive compounds, such as terpenes, anthocyanins, tannins,
flavonoids and polyphenols [4].
Xanthones are naturally-occurring compounds with a distinct chemical structure, known as tricyclic
aromatic system, with known antibacterial properties [5]. Natural compounds with antibacterial
properties may be applied to treat local infections [5–7], wounds and lesions difficult to heal,
circumventing antibiotic resistant pathogens with multidrug resistance (MDR) genes, or may be
combined with antibiotics to increase their effect. Therefore, studies on bacteria inhibition in vitro or
in vivo have been performed on a wide array of natural compounds and peptides [8]. In microbiology,
the minimum inhibitory concentration (MIC) is the lowest concentration of a chemical that prevents

Molecules 2017, 22, 275; doi:10.3390/molecules22020275 www.mdpi.com/journal/molecules

1. Introduction
The fruit of Garcinia mangostana Linn. (mangosteen), of the family Guttiferae, has been used in
Molecules 2017, 22, 275 2 of 13
Asian traditional medicines for the treatment of skin infections, wounds, diarrhea, dysentery,
suppuration, leucorrhea, chronic ulcers, and gonorrhea [1,2]. In addition, mangosteen with essential
minerals
visible is commercially
growth used
of a bacterium as dietary supplement
(bacteriostatic for cancer
activity), whereas thepatients
minimum [3].bactericidal
The pericarp of the fruit
concentration
contains
(MBC) high
is the amounts of that
concentration xanthones,
results insuch as α-mangostin
microbial death. Among(Figure 1), β-mangostin,
the pathogens γ-mangostin,
developing antibiotic
etc., and considerable amounts of other bioactive compounds, such as terpenes, anthocyanins,
resistances, Pseudomonas aeruginosa and Staphylococcus aureus (S. aureus) are largely spread in hospitals
tannins,
and flavonoids
healthcare unitsand polyphenols [4].
[9,10].

H3C CH3

OH O
CH3
O
H3C CH3

HO O OH

Figure 1. Structure of α-mangostin.

Molecules 2017, 22, 275; doi:10.3390/molecules22020275 www.mdpi.com/journal/molecules
In the literature, mangosteen fruit extracts were shown to contain different xanthones, identified
by HPLC analysis: α-mangostin, β-mangostin, γ-mangostin, 8-desoxygartanin and gartanin,
two isoprenylated xanthones, and 9-hydroxycalabaxanthone [11]. α-Mangostin (3,6,8-trihydroxy-
2-methoxy-1,7-bis(3-methyl but-2-enyl)xanthen-9-one) is a compound purified as a yellow crystalline
solid, with molecular mass 410.45 g/mol, having a xanthone core structure. It is prepared by heating
of phenyl salicylate (salol). It is used in preparation of xanthydrol, which is used for the determination
of urea levels in the blood.
Recent reports have revealed that α-mangostin from G. mangostana fruit possesses several
medicinal properties, such as anti-microbial activity against bacteria [4,12–18], anti-oxidant and
neuro-protective activity [19–22], lipase inhibition [23], and anti-inflammatory and anti-cancer
properties [24]. Biologically active molecules from medicinal plants are utilized as therapeutic agents,
but most of the secondary metabolites do not exhibit optimum efficacy. This is due to the lack of
specificity and the absence of biologically active functional groups. Thus, by elucidating the structure
of the active compound and the pharmacophores, the functional groups are considered as essential
for the bioactivity of a compound. Since α- and non-α-mangostin xanthones have been shown to
possess anti-bacterial activity, especially against Gram-positive bacteria [12–18], it is most probable
that semi-synthetic analogs would be produced with enhanced anti-bacterial activity. In order to
increase the bioactivity and antibacterial properties of α-mangostin, semi-synthetic modification
of the compound was performed by several authors, to lead to more active compounds [25–29],
with no excessive toxicity. Alongside their anti-microbial properties, the synthetic analogs possess
wound healing and anti-inflammatory activity and, hence, they could be exploited in the treatment of
skin infections.
Following the discovery of the medicinal properties of the synthetic analogs, it is suggested
that these analogs possess better therapeutic value than the parent molecule and are potential drug
candidates for application as antimicrobials. It has been envisaged that mangosteen xanthones may be
applied in several fields and industries, such as textiles, fabrics, and polymers for medical devices and
biomaterials for applications in biomedicine [30–32], in biomaterials for oral hygiene and prevention
of dental caries [30], in materials preventing biofilm formation [31], and wrapping foil polymers for
food packaging [32]. However, it should be demonstrated that these new compounds are non-toxic
and safe, and the derived materials do not release them too fast, possibly being covalently linked,
to maintain the bioactivity for longer periods. In addition, pathogens are able to survive on steel
surfaces and in pipelines where food products are processed, establishing biofilms. Therefore, it is
of utmost importance to find new treatments of surfaces and processing lines in the food industry
in order to eliminate bacterial contaminations. Several approaches have been proposed to release of
active ingredients to the surface and kill the micro-organisms. For example, Poverenov and colleagues
prepared numerous active anti-microbial surfaces on the basis of polymers, cellulose, and glass, with
Molecules 2017, 22, 275 3 of 13

potent inhibition against Bacillus cereus (B. cereus), Alicyclobacillus acidoterrestris (A. acidoterrestris),
Escherichia coli (E. coli), and Pseudomonas aeruginosa (P. aeruginosa) [12–16]. Similar research on
anti-microbial food-contact materials were developed based on curcumin [33–35], polyphenols and
natural compounds [36–38], essential oils to control pest pathogens [39], active-passive modified
atmosphere for microbial control [40,41], and various polymeric-based anti-microbial films [28–32].
Using nanotechnological approaches, new materials based on the antimicrobial property of silver
nanoparticles have been studied and produced [42–45].
The perishable foods market is in the need of anti-microbial materials due to economic
losses caused by bacterial and fungal growth on foods throughout the entire food supply chain.
Such anti-microbial materials should extend the shelf-life of the product on the market shelves up
to the consumer table. One challenge is to find methods for improved treatment (i.e., modified
atmosphere, type of film, packages composed by various active materials) and application of effective,
safe anti-bacterial and anti-fungal compounds [46–52]. These methods may ensure the safety of foods
and alleviate the economic losses due to food deterioration. It is envisaged that new anti-microbial
compounds could be incorporated in food packaging and films to improve the shelf-life of ready-to-eat
foods and packaged fresh products.
In parallel to these studies, other recent reports described similar antibacterial properties in
xanthones such as mangiferin from edible plants, as well as from other medicinal plants [53,54].
In the field of polymer preparation, electro-spinning techniques have been applied to
G. mangostana extracts, with good results in formation of polylactic acid fiber mats to be used in
wound dressing [31]. Electro-spinning allows the deposition of small and medium sized molecules
on the surface of a forming polymer [31]. In addition, during the spinning and deposition of the
bioactive compounds, eventual solvents present as residues of extraction or purification steps may be
evaporated, reducing the possibility of contamination of material being in contact with food products.
Furthermore, other deposition techniques may be applied with higher performance and improved
purity of xanthones to become part of the polymer. Considering a covalent linkage of the bioactive
core inside the polymer, a preliminary study examined functionalized polyxanthones in the form of
poly-azoxanthone esters (PAXA), through polycondensation, and showed their applicability in food
packaging and in pharmaceutical industry [32].
In search for new anti-bacterial agents we performed semi-synthetic modification of α-mangostin
using Ritter reaction, reduction by palladium-carbon (Pd-C), alkylation and acetylation to improve
the bioactivity of the base compound. In this study, we describe the selective enrichment of
α-mangostin (demonstrated by the NMR peaks and HPLC graphs) its semi-synthetic modification,
the products generated, their chemical structure, and the inhibition activity against four pathogens,
two Gram-positive and two Gram-negative bacteria, and two fungi, evaluated as diameter or halo
of growth inhibition. Herein, we studied the anti-microbial activity of α-mangostin and its synthetic
analogs and confirmed the development of new anti-microbial xanthones with higher antibacterial and
antifungal activity. The candidate molecules with higher bioactivity may be applied in the composition
of antimicrobial textiles and polymers that could find applications in biomedical devices and in
food packaging.

2. Results

2.1. α-Mangostin Isolation and Purification

α-Mangostin was isolated from the dried fruits of G. mangostana using ethyl acetate and
yielded 26 g (26%) of dried ethyl acetate crude extract. The crude extract subjected to column
chromatography with ethyl acetate and hexane yielded 5–6 g of pure α-mangostin and the purity
was confirmed as 95% using HPLC. The structural characterization of the pure α-mangostin (I) using
1 H-NMR spectra, 13 C-NMR spectra, IR spectrum and high-resolution mass spectra is available in

Supplementary materials.
Molecules 2017, 22, 275 4 of 13

2.2. Synthetic Modifications

Following the isolation, α-mangostin was subjected to a series of chemical reactions to alter the
core
Molecules 2017, 22,(Figure
structure 275 2). 4 of 14

Molecules 2017, 22, 275 4 of 14

Figure 2. Reaction scheme leading to different derivatives.

Figure 2. Reaction scheme leading to different derivatives.

The basic core structure xanthone (anthraquinone) was conserved intact while the functional
The basic
iso-prenyl andcore structure
phenolic xanthone
hydroxy groups (anthraquinone)
were subjected was to conserved
semi-syntheticintactmodification.
while the functionalTwelve
Figure 2. Reaction scheme leading to different derivatives.
iso-prenyl and phenolic hydroxy groups were subjected to semi-synthetic
different semi-synthetic derivatives were obtained (Table 1), each containing new key moieties that modification. Twelve
different semi-synthetic
were evaluatedThe basicto detect derivatives were
the anti-microbial
core structure xanthone obtained
activity (Table
(anthraquinone) was 1),
against each containing
various
conserved bacterial
intact while new
and key moieties
thefungal cultures.that
functional
were (Ievaluated
iso-prenyl to detect
and phenolic
D), the alkylated the anti-microbial
hydroxy
product of groups activity against
were subjected
α-mangostin with ethyl various bacterial
to semi-synthetic and
iodide, wasmodification. fungal
found to beTwelve cultures.
a di-alkylated
(I D), the
different alkylated
semi-synthetic product of
derivatives α-mangostin with
were obtained (Table ethyl
1), eachiodide, was
containing
product. All other alkylations using the exact same reaction conditions reported in the table found
new to be
key moieties a di-alkylated
that
resulted
product.wereAllevaluated
other to detect theusing
alkylations anti-microbial
the activity
exact same against
reaction various bacterial
conditions and fungal
reported in cultures.
the table resulted by
in
in tri-alkylated products. In the Ritter reaction, the addition of acetonitrile,
(I D), the alkylated product of α-mangostin with ethyl iodide, was found to be a di-alkylated
followed
tri-alkylated
intra-molecular products. In of the–OH
Rittertoreaction, the addition of acetonitrile, followed by intra-molecular
product. Alladdition
other alkylations using isoprene unit
the exact same gave ether
reaction (or) pyran
conditions reportedring structures.
in the table resultedAddition of
addition
acetonitrileof –OH to
is followed isoprene unit
by hydration gave ether
to produce(or) pyran ring structures. Addition of acetonitrile is
in tri-alkylated products. In the Ritter reaction,amide. This reaction
the addition is general
of acetonitrile, for
followed allbythe nitrile
followed
reaction by hydration
intra-molecular
products studied to produce
addition (I of A
–OH amide.
to to This
isoprene
I C). reaction
unit gave ID,
Compound is general
etherreaction
(or) pyran for
ofringall the nitrile reaction
structures. Addition
α-mangostin with ethyl products
of iodide,
studied
produced mainly the di-alkylated product, probably due to the possibility of ether cleavagethe
(I A to
acetonitrile I C).
is Compound
followed by ID,
hydrationreaction
to of
produceα-mangostin
amide. This with
reactionethyl
is iodide,
general forproduced
all the mainly
nitrile of
reactionproduct,
di-alkylated products probably
studied (I A to ItoC). theCompound ID,ofreaction of α-mangostin with ethyl iodide, during
reaction product HI during due the reaction. possibility
All other ether cleavage
alkylation of reaction
reactions product
carried HI out with
the produced
reaction. mainly
All otherthe di-alkylated
alkylation product,
reactions probably
carried out due to alkyl-bromide
the possibility ofyielded
ether cleavage of
alkyl-bromide yielded only tri-alkylated products (I E with
to I J).
reaction product HI during the reaction. All other alkylation reactions carried out with
only tri-alkylated
products (I E to I J).
alkyl-bromide yielded only tri-alkylated products (I E to I J).
Table 1. α-Mangostin and its synthetic derivatives.
Table
Table1.1.α-Mangostin and
α-Mangostin and itsits synthetic
synthetic derivatives.
derivatives.
Reaction of
Compound Reaction of
Molecular
S. No. Compound
Compound Structure α-Mangostin
Reaction of Molecular
Molecular Mass
S. No.Information
S. No. Structure
Structure α-Mangostin Formula
Mass Mass
Information
Information withwith Formula
α-Mangostin Formula
CH3 with
H C
H3C3 CH3

OH
OH O
O
α-Mangostin
α-Mangostin
α-Mangostin CHCH
3 3
1. - +
24 C26
24H 626O(M+1) 411 (M+1)+
1. 1. O O - - C24HC
26O6H O411 6 411+ (M+1)
(I) (I)(I) CH3CH3
H3CH3C

HO O OH
HO O OH
H 3C
H3C H3CNHCOCH 3
Ritter H3 C NHCOCH 3
OH O Acetonitrile in
Ritter
product of
2. OH O O Acetonitrile in
Silica supported C26H31NO7 470 (M+1)+
α-mangostin
product of CH3
2. sulphuric
Silicaacid
supported C26H31NO7 470 (M+1)+
(I A)
α-mangostin H3C O
CH3
O OH sulphuric acid
(I A) H 3 C H 3C O

O O OH
H 3C
 reaction product HI during the reaction. All other alkylation reactions carried out with
alkyl-bromide yielded only tri-alkylated products (I E to I J).

Table 1. α-Mangostin and its synthetic derivatives.

Reaction of
Molecules Compound Molecular
No. 22, 275
S. 2017, Structure α-Mangostin Mass 5 of 13
Information Formula
with
H3 C CH3

OH O
Table 1. Cont.
α-Mangostin CH3
1. O - C24H26O6 411 (M+1)+
(I) CH3
H3 C
Compound Reaction of Molecular
S. No. Structure Mass
Information HO OH α-Mangostin with Formula
O
Molecules 2017, 22, 275 H 3C 5 of 14
Molecules 2017, 22, 275 H3 C NHCOCH 3 5 of 14
Ritter
Ritter
OH O Acetonitrile
Acetonitrile in in
product
productof of Table 1. Cont.
2. 2. Molecules
Molecules 2017, 22,
2017, 22, 275
275 O
Table 1. Cont. Silica supported
Silica supported C26HC3126
NO H731 NO of470
55 of 714 470+(M+1)+
14 (M+1)
α-mangostin
α-mangostin CH3
sulphuric acidacid
sulphuric
(I(IA)
Molecules
A) 22, 275 H3C
2017, Reaction of
Compound Reaction of Molecular 5 of 14
S. No. Compound O Structure
O Table OH α-Mangostin Molecular Mass
S. No. Information H 3C StructureTable 1.
1. Cont.
Cont. α-Mangostin Formula Mass
Information with Formula
Table
H3C 1. Cont. with
Reaction
Reaction of
of
Compound
Molecules 2017, 22, 275 Reaction of Molecular 5 of 14
S. No.
S. No. Compound StructureH3CH3C NHCOCH2CN α-Mangostin
Structure α-Mangostin Molecular Mass
Mass
Information
Information
Ritter
Ritter H3 C NHCOCH2CN Reaction of
Malononitrile Formula
Formula
Compound OH with
with Molecular
S. No. Ritter O
StructureTable
H33C
C 1. Cont.
Malononitrile
Malononitrile
α-Mangostin in Mass
product
product of
of
Information OH O H 3
in Silica Formula 514 514
3. 3. product of
α-mangostin H33C
H C O
NHCOCH
NHCOCH CN
2CN
Silica
with supported
in Silica
supported
C27H30N OH
C227 7
30 N(M+2+NH
2 O7514 3)+ +
3. α-mangostinRitter 3
H 3C O CH3
2
2
Malononitrile C27H30N2O7 (M+2+NH 3)
Ritter
α-mangostin OH
OH O H C CH3 sulphuric
Malononitrile
Reaction of
supported acid (M+2+NH3) +
(I(Iproduct
S. No. B)B)Compound
product of H3C
of
O
Structure 3 NHCOCH2CN sulphuric
in
in Silica
α-Mangostin
Silica
acid
Molecular
Mass 514
514
3.
3. (I B)Ritter H C
Information OOH sulphuric
Malononitrile acid C27
Formula
C H30
27H N2O7
30N22O77
α-mangostin H3 C
α-mangostin O OH O O CH33
CH
with
supported
supported
27 30 (M+2+NH333))+++
(M+2+NH
product of H3 C O O H3HC3C OH 3 in Silica 514
3. (I B)
(I B) 3 C
H sulphuric acid
sulphuric acid C27H30N2O7
α-mangostin H333C H3 C O
NHCOCHCH2CN supported (M+2+NH3)+
Ritter O H3CH3C NHCOCH OH 23CH 2CHMalononitrile
OH 3
Ritter(Iproduct
B) H3C
H C O OH O
O Butyronitrile in
of H333C
O H3 C NHCOCH 2CH 2CH 3 sulphuric acid
in Silica 514
3. Ritter of
Ritter OH H33C
C Butyronitrile
product α-mangostin H3C O OH
O
O OH
H33C
H
C
3 OOH
NHCOCH CH
CH22CH
NHCOCH CH33 supported
2CH
SilicaC27Hin
Butyronitrile
30N2O7
inC28H
(M+2+NH3)+
4. product
product ofof(I B)
Ritter
Ritter 3
H 3C O 2
3 2
2 3 Silica
Butyronitrile
Butyronitrile in
in
25NO7 498 (M+1)+
4. 4. α-mangostin CH3 sulphuric supported
acid
Silica supportedC28H25C NO 498 (M+1)+
α-mangostin
product ofof
H3 C OH
OH O H C
O O
NHCOCH 2CH CH 3 supported
Silica 287H25 NO 7 (M+1)
498+
α-mangostin
4. product
(I C)Ritter H3C H3C O
3
O OH CH 3 2 Silicasulphuric
Butyronitrile in acid C
C H25 NO77 + 498 (M+1)
(M+1)++
4. α-mangostin OH
O
O sulphuric
supported acid
28H
28
acid 28 25 7
25NO 498
of H3C
(IC)
C)
(Iα-mangostin O H 3C CH33
OHCH sulphuric
supported
product H 3C O O 3 Silica
4. (I C) H33C
C)Ritter H H C H3 C OOH 2CH 2CH 3
NHCOCH sulphuric acid C28H25NO7 498 (M+1)+
(I
α-mangostin 3C
O O sulphuric
Butyronitrile acid
in Iodide
OH CH
3
CH H3COH CH3 3 supported
Ethyl
H33CC O
O OH3 O
O
(Iproduct
C) of HH C CH3 O H3C CH3 SilicaEthyl
sulphuric Iodide
acid
4.
Alkylated
33
CH33 H33C CH33
COOHCH
CH EthylK2Iodide
CO 3/ACN
C28H25NO7 498 (M+1)+
α-mangostinH 3C O CH
O3 O H 3 3 3 Ethyl
supported Ethyl
Iodide
K2CO3/ACN Iodide
Alkylated
Alkylated O K CO /ACN
product of(I C)
Alkylated HCH
3C 3 O3 CH3 K 2CO
sulphuric 3/ACN
acid
Alkylated CH O H3C Ethyl
2
2 Iodide
3
3
467 (M+1)+
5. product
product of
of CHC3
H3CH O O
O O O
O OH O
K2CO3/ACN
K2 CO3 /ACN C28H34O6
5. 5. α-mangostin
product
product
Alkylated
5.α-mangostin
of
of H C CH 3
3
3 CH3O O
CH3
M.W. 10 min C C28 H34
28H
CO28H34O C628 H34
467 O467
(M+1) 467+
6 +++ (M+1) (M+1)+
O CHCH3 Ethyl Iodide 34O66 467 (M+1)
5. α-mangostin 3 CH
O3 H3C 28 34
6
α-mangostin O
(I D)
α-mangostin
product of H3C H C
H333C CH 3 CH333 K2COM.W.
M.W.
M.W.
/ACN10 10
min
10 min
min
5. (I(ID)D)(I Alkylated
D) O OO
3
M.W. 10 min C28H34O6 467 (M+1)+
(I D)
α-mangostin
product of H3C CH3 HO O
O CH3 CH3
HO O O CH M.W. 10 min C H O 467 (M+1)+
5. (I D) HO
HO
H 3C O
O
O OO CHCH3 3 28 34 6
M.W. Bromopropane
α-mangostin 3
H3 C HH H 3C CH3CH3 3
10 min
HO
H3C
3CC
CH O CH3 Bromopropane
Bromopropane
Bromopropane
(I D) 3
3 O HH333C
H CC CH
CH 3 3
K2Bromopropane
CO3/ACN
Alkylated
Alkylated H 3C
3 3
3
Alkylated
Alkylated
Alkylated HO O O CH K CO
K22CO
Bromopropane
2 /ACN
K2333CO 3/ACN
/ACN
product of OO H3C CH3 3
product of CH3 H3CO O
O O K2 CO3 /ACN
6. 6.
product of O
O H 3C Bromopropane
C33H44CO336H44O O537 (M)537
product of
Alkylated CH33 K2CO3/ACN +
6. 6. product
6. α-mangostin of CHCH
3 3
O
CH3
O
O
CH3 C33
33H44
CO3366H44O63353744(M)537
44 6+ C6 537
H (M)++
(M)+ (M)+
α-mangostin
α-mangostin
Alkylated
product of H3C
α-mangostin O O OCH
CH K2CO3/ACN
6. α-mangostin H C
H333C
CH3
O CH
33
3 3
M.W.
M.W.
M.W.
10 min C33H44O6
10 min
10 min 537 (M)+
(Iα-mangostin
E)E) ofH3C O
6.(I(I (Iproduct
(I E)
E) CH3 O
CH
M.W. 10 min
M.W. C10
33Hmin
44O6 537 (M)+
E) H CH C
H33C
α-mangostin3 H C3 OO O O 3
CHCH
CH 3 3 M.W. 10 min
(I E) O O O CH3
O 3
H 3C 3 H 3 C O O
3 M.W. 10 min
CH 3
(I E) H 3C O O CH3
O O CHCHCH Cyclopentyl
Cyclopentyl
Cyclopentyl
H 3C
O O HH33C
H CCO CH 33 3 3 Cyclopentyl
O 33 3 Cyclopentyl
Cyclopentyl
CH 3
HHC
3
3C CH
CH 3
bromide
Cyclopentyl
Cyclopentylbromide
bromide
H 3C 3 bromide
bromide
Alkylated O
OO O
O Kbromide
K CO
222CO /ACN
/ACN
Alkylated
Alkylated
Alkylated CH CH33
CH O bromide K2333CO 3/ACN
Alkylated
product
Alkylated of 3 3 OOO O
O O K2CO K32/ACN
CO 3/ACN
7. productproduct
product of of
Alkylated
of CHCH 3CH
3 3
O O OCH
CH33
K2CO 3/ACN K2 CO 3 /ACN
C39 H50 O6 615 (M)+++
7. 7. 7. α-mangostin
product
product of
product
α-mangostin of of
H
H
H
C 3
3
3
C
C OO O CH 3 3 C 39H
39 50CO3966H50O
50 C639615
H50(M)
O615
6 (M)+615 (M)+
7. α-mangostin
7.α-mangostin
7. 3
H HC C CHCH
3 CH
3 3
C39HC 396H50O
50O 615
C639H 6615 (M) 615
(M)
50O
+ +
(M)+
α-mangostin
(I F)
(I
α-mangostin F)
α-mangostin H 3 C 3 3 M.W. 10
M.W. 10 min
min
(I(IF)F) (I F)(I F) O
O O O
O M.W. 10 min
(I F) O OO O
OO O
M.W. 10
M.W. 10M.W.
minmin 10 min
O
O
O M.W. 10 min
O O O

H3C
H C CH3
CH Propargyl
Propargyl bromide
Propargyl
Propargyl
HH3
3C3C CH3 33 Propargyl
HH
3C CHCH Propargyl
Propargyl
3C 3 3 bromide
bromide
bromide
Alkylated OO
O O H 3C CH3 K COPropargyl
bromide K 2 CO3 /ACN
bromide
/ACN
Alkylated
Alkylated
Alkylated CHCH
CH 3 3
O
O K223CO
K2CO 2 333/ACN
/ACN
productAlkylated
product
of
product of of
of
product CH3
3
3 OO O
O O
K2CO bromide
K23/ACN
CO3/ACN
8. 8. Alkylated
8. 8. product of H CCH 3 O O
OO CH
CHCH 3 K2CO3/ACN C633
C
C33H32O 33HH32 O6 525 C
32O66
33 32
525
33525
(M) (M)
H32(M)
+ O6+ +
+ 525 (M) +
Alkylated
α-mangostin
α-mangostinα-mangostin
α-mangostin H
H333CCH C O 3 3 3
8. product of H 3C
3
3 OCH3 C33H32O6 525 (M)+
8. (I of
G)(I G)
α-mangostin
product O CHM.W. M.W. 10 min C33H32O6 525 (M)+
8. (I G)(I G)
α-mangostin
(I G)
H 3C
OO O OO
O
10 min
M.W.
3 M.W. 10
10 min
min
CH3 M.W. 10 min C33H32O6 525 (M)+
α-mangostin H 3C O O
O
(I G) O O O M.W. 10 min
(I G) O O O M.W. 10 min
O O O Benzyl
Benzyl bromide
Benzyl bromide
bromide
Benzyl
KBenzyl
2CO3/ACN
bromide
H3C CH3 K222CO
CObromide
/ACN
H33C
C CH33
CH K K3332/ACN
CO3 /ACN
H K2Benzyl
CO3/ACN bromide
Alkylated H33C CH33
O O Benzyl bromide
Molecules 2017,
product of 22, 275
Alkylated CH3 K2CO3/ACN 6 of 14
O
9. CH3
CH
O
O
O
O H3C O CH3 K2CO3/ACN C45 H44 O6 681 (M)+
Alkylated
product of O CH3
CH
9. Alkylated
α-mangostin
Alkylated H3CCH333 H3C O
O
3 C45H44O6 681 (M)+
α-mangostin
product
product of
of H33C
C CH3
O CH
9.
9. (I H)
product of
(I H) H
H3 C O
O O Table 1. Cont.
O CH333 M.W. 10 min
M.W. C45
10 minCC H44
45H O6
44O66
45 44
681 (M)
681 (M)+++
α-mangostin
9. Alkylated
α-mangostin 3 CH3 O O 45H44O6 681 (M)+
α-mangostin O M.W. 10
10 min
min
Alkylated
(I H)
(I H) CH3 O OO
O
M.W.
M.W. 10 min
product (Iof
H) O O
O O O CH3 Reaction of
H C O O
9. product Compound
of 3 CH3α-Mangostin Molecular C45H44O6 681 (M)+
9. α-mangostin
S. No. H3C Structure CMass
45H44O6 681 (M)+
Information Formula
α-mangostin
(I H) O with M.W. 10 min
O O Benzene sulphonyl
M.W. 10 min
(I H) O O O Benzene sulphonyl
chloride chloride
H 3C CH 3
O K2CO3/ACN
S K2 CO3 /ACN
Alkylated O O O
Alkylated CH 3
product of O
10. product of CH3 C H O12 S3 831 (M)+
10.
α-mangostin H 3C C42H38O12S3 83142
(M)+38
α-mangostin
O O
(I I) (I I) O O O M.W. 10 min
M.W. 10 min
S S
O O

O Chloroethyl
N
morpholine
H3 C CH3 hydrochloride
K2CO3/ACN
O O
Alkylated CH3
product of O
H3C CH3
11. C42H59N3O9 750 (M)+
α-mangostin
(I J) O O
O M.W. 10 min
N N

O O
 S. No. Structure α-Mangostin Mass
Information Formula
Table 1. Cont. with
Benzene sulphonyl
Reactionchloride
of
Compound H
H33C
C CH
CH33 Molecular
S. No. O
O Structure K22CO33/ACN
α-Mangostin Mass
Information SS Formula
with
O
O O
O O
O Benzene sulphonyl
Alkylated CH
CH33
Molecules 2017, 22, 275 O
O CH chloride 6 of 13
product of H 3C CH 3 CH33 K2CO3/ACN
10. H33C
H C O C42
42H38
38O12
12S33 831 (M)++
α-mangostin S
O
OCH O O O O
O
(I I)
Alkylated 3O
O O O
O M.W. 10 min
SS O O SS
product of H 3O CH3 OO
10. O
C Table 1. Cont. C42H38O12S3 831 (M)+
α-mangostin
O O
(I I) O O O M.W. 10 min
S S
O O
Compound Reaction of Molecular
S. No. Structure Mass
Information O
O α-Mangostin with Formula
Chloroethyl
N
Chloroethyl
morpholine
N
O H
H33C
C CH
CH33 morpholine
Chloroethyl
hydrochloride
N
morpholine
Khydrochloride
22CO33/ACN
O
O H3 C CH3 hydrochloride
O
O
Alkylated CH
CH33 K2 CO3 /ACN
Alkylated O
K2CO3/ACN
product of O O O CH
11. product of
Alkylated H33C
H C CH3 CH33 C42
42H59
59N33O99
H59 N3 O750 (M)++
11.11. α-mangostin
product of O
CH3
C42 9 750 (M)+
α-mangostin
11. H 3 C C42H59N3O9 750 (M)+
(I(IJ)
J)
α-mangostin OO O
O
O
O
M.W. 10 min
(I J) O O
N
N O N
N M.W. 10M.W.
min 10 min
N N

O
O O O
O
O

CH
CH
CH33 H3H C CHCH
HC33C 3CH33 Acetic
AceticAcetic anhydride
anhydride
anhydride
K2COK 3/ACN
OO
O
OO
O OO K22CO 33/ACN
2 CO3 /ACN
CH CH3 O
Acylated Acylated
Acylated CH33 O
product of H3 C O
CH3
O
product
12.
product ofof H C CH
CH33 C26H28O7 536 (M)+
12.12. α-mangostinH33C 26C
C26 H26 H7728 O7 536 (M)
28O
28 536
++ (M)
+
α-mangostin
α-mangostin O M.W. 10 min
(I K) O O
O M.W.
M.W.1010
minmin
(I(IK)
K) O
O O
O O
H3 C H 3C O
O
H
H33C
C H
H33C
C O
O
O
O
H3C CH3

OH H
H33C
C CH
CH33
Reduced O
CH3
product of O 415
Reduced
13.
Reduced H3C OH
OH O CH3 Palladium/Carbon C25H30O6
α-mangostin
CH O (M+1)+
product CH33
productof(I
ofL) 415 (M+1)+
415
13.13.
α-mangostin HO O OH O
O CH
CH33
Palladium/Carbon C2525C
Palladium/Carbon H25 H O6
30O6630
30
α-mangostin H
H33C
C (M+1)++
(I(IL)
L)
HO
HO O OH
OH
O
2.3. Biological Assays

2.3.1. Anti-Bacterial Assay

2.3. Biological Assays
2.3. Biological Assays
The evaluation of the anti-bacterial potential for α-mangostin-based synthetic analogs was
examined against E. coli, Bacillus subtilis (B. subtilis), S. aureus, and P. aeruginosa, in accordance with
2.3.1. Anti-Bacterial
2.3.1. Anti-Bacterial AssayAssay
an experimental procedure. Two different concentrations (50 μg/mL and 100 μg/mL) of the
α-mangostin
The evaluationand their synthetic analogs along with standard drug Ciprofloxacin were tested against
The evaluation
the pathogens
ofandof
the the anti-bacterial
anti-bacterial
theBacillus
results subtilis
are given
potential
potential for α-mangostin-based
forzone
α-mangostin-based synthetic analogs was
synthetic analogs was
examined against E. coli, (B.insubtilis),
Table 2. S.The
aureus, of inhibition
and was determined
P. aeruginosa, in
in accordance with
examined triplicatesE.
against coli,
using theBacillus
diffusion subtilis
technique,(B.
withsubtilis), S. aureus,
values representing theand
averageP. aeruginosa, in
zone of inhibition. accordance with an
an experimental procedure. Two different concentrations (50 μg/mL and 100 μg/mL) of the
By measuring the zone of inhibition (in mm), it is observed that all the derivatives of
experimental
α-mangostinprocedure.
and exert Two
their moderate different
synthetictoanalogs concentrations
along withactivity. (50
standard µg/mL and
drug Ciprofloxacin100 µg/mL) of the α-mangostin
α-mangostin high anti-bacterial Compound (I E) showedwere tested against
maximum
and their
the synthetic
pathogens analogs
and
anti-bacterial activity along
the (up to 12with
results are atstandard
mm) given drug
in Table
100 μg/mL 2. Ciprofloxacin
The zone
concentration were tested
againstofallinhibition
bacterial against
wastested
stains determined
in the in
pathogens
and thetriplicates
results using the
are given
comparison to thediffusion
in Table
other technique,
2. The
synthesized with
zonevalues
compounds. representing
ofAtinhibition wasthedetermined
low concentration average
of zonethe
50 μg/mL, of inhibition.
inacetyl
triplicates using the
By measuring
diffusion technique, with thevalues
zone of inhibition (in
representing themm),
averageit is zone
observed that all the derivatives of
of inhibition.
α-mangostin exert moderate to high anti-bacterial activity. Compound (I E) showed maximum
By measuring the zone of inhibition (in mm), it is observed that all the derivatives of α-mangostin
anti-bacterial activity (up to 12 mm) at 100 μg/mL concentration against all bacterial stains tested in
exert moderate
comparisontotohigh the anti-bacterial
other synthesized activity. Compound
compounds. At low(I concentration
E) showed maximum of 50 μg/mL, anti-bacterial
the acetyl activity
(up to 12 mm) at 100 µg/mL concentration against all bacterial stains tested in comparison to the
other synthesized compounds. At low concentration of 50 µg/mL, the acetyl derivative (I G) showed
maximum inhibition against E. coli. The butyl derivative (I C) showed maximum inhibition against
B. subtilis. The propenyl derivative (I G) showed maximum inhibition against S. aureus. The ethyl
(I D) and benzene sulphonyl (I I) derivatives of α-mangostin showed maximum inhibition against
P. aeruginosa. Among these compounds, the acetyl (I K) and benzene sulphonyl (I I) derivatives of
α-mangostin showed maximum anti-bacterial activity against the four bacterial strains tested. Most of
the derivatives showed better anti-bacterial activity against Gram-positive bacteria B. subtilis and
S. aureus followed by the Gram-negative bacteria P. aeruginosa and E. coli. The agar disk-diffusion
method may not be appropriate method to determine the minimum inhibitory concentration (MIC),
as it is impossible to quantify the amount of the antimicrobial agent diffused into the agar medium.
Since only two concentrations (50 and 100 µg/mL) were tested, at this time we could not calculate
the appropriate MIC value. Further experiments are required to evaluate the effective microbicidal
concentrations, such as MBC tests.
Molecules 2017, 22, 275 7 of 13

Table 2. Anti-microbial and anti-fungal activity of the α-mangostin and their synthetic analogs.

Zone of Inhibition in mm
S. No.
E. coli B. subtilis S. aureus P. aeruginosa C. albicans A. niger
Conc. (µg/mL) 50 100 50 100 50 100 50 100 50 100 50 100
I 5 ± 0.09 8 ± 0.04 7 ± 0.11 9 ± 0.15 5 ± 0.05 9 ± 0.12 6 ± 0.24 10 ± 0.08 8 ± 0.12 10 ± 0.14 4 ± 0.18 7 ± 0.05
IA 4 ± 0.18 9 ± 0.16 6 ± 0.15 10 ± 0.11 5 ± 0.12 11 ± 0.22 5 ± 0.11 8 ± 0.16 7 ± 0.11 10 ± 0.06 9 ± 0.11 12 ± 0.02
IB 6 ± 0.14 10 ± 0.11 6 ± 0.12 9 ± 0.23 5 ± 0.16 8 ± 0.14 4 ± 0.08 8 ± 0.22 4 ± 0.16 6 ± 0.11 3 ± 0.07 5 ± 0.15
IC 8 ± 0.11 11 ± 0.08 10 ± 0.06 12 ± 0.04 6 ± 0.18 9 ± 0.06 5 ± 0.16 10 ± 0.13 2 ± 0.16 6 ± 0.13 2 ± 0.18 5 ± 0.14
ID 7 ± 0.12 10 ± 0.18 4 ± 0.04 10 ± 0.09 5 ± 0.08 7 ± 0.12 9 ± 0.21 9 ± 0.08 4 ± 0.22 6 ± 0.21 7 ± 0.15 9 ± 0.08
IE 6 ± 0.13 11 ± 0.22 9 ± 0.15 12 ± 0.16 7 ± 0.22 11 ± 0.18 7 ± 0.05 12 ± 0.03 4 ± 0.08 7 ± 0.17 3 ± 0.04 5 ± 0.05
IF 6 ± 0.18 9 ± 0.04 5 ± 0.09 11 ± 0.18 7 ± 0.14 11 ± 0.17 5 ± 0.09 9 ± 0.14 3 ± 0.07 5 ± 0.14 4 ± 0.09 7 ± 0.11
IG 6 ± 0.20 9 ± 0.12 7 ± 0.05 10 ± 0.11 10 ± 0.21 11 ± 0.19 6 ± 0.14 9 ± 0.18 4 ± 0.12 6 ± 0.19 3 ± 0.16 5 ± 0.19
IH 5 ± 0.12 9 ± 0.11 6 ± 0.12 9 ± 0.08 8 ± 0.18 10 ± 0.13 8 ± 0.11 11 ± 0.11 9 ± 0.17 13 ± 0.12 7 ± 0.11 10 ± 0.21
II 5 ± 0.08 8 ± 0.15 6 ± 0.04 10 ± 0.20 7 ± 0.11 9 ± 0.15 9 ± 0.19 12 ± 0.06 10 ± 0.14 12 ± 0.09 9 ± 0.08 13 ± 0.11
IJ 4 ± 0.15 7 ± 0.16 5 ± 0.11 8 ± 0.14 4 ± 0.13 7 ± 0.21 3 ± 0.08 6 ± 0.20 11 ± 0.12 13 ± 0.15 6 ± 0.03 8 ± 0.18
IK 10 ± 0.14 12 ± 0.16 4 ± 0.10 7 ± 0.14 5 ± 0.08 9 ± 0.08 6 ± 0.22 9 ± 0.06 8 ± 0.20 11 ± 0.18 10 ± 0.15 13 ± 0.18
IL 9 ± 0.13 11 ± 0.06 5 ± 0.21 8 ± 0.16 5 ± 0.12 8 ± 0.05 6 ± 0.15 8 ± 0.12 7 ± 0.13 10 ± 0.08 9 ± 0.17 12 ± 0.08
Std drug 19 ± 0.11 24 ± 0.05 16 ± 0.07 20 ± 0.14 11 ± 0.15 18 ± 0.06 15 ± 0.12 19 ± 0.16 15 ± 0.16 18 ± 0.04 14 ± 0.13 18 ± 0.08
Molecules 2017, 22, 275 8 of 13

2.3.2. Anti-Fungal Assay

The first compound evaluated was the natural product α-mangostin, which was compared
against the synthetic analogs to prove that the analogs had better efficacy than the parent molecule.
Two compounds, I H and I J, at 100 µg/mL concentration, showed maximum anti-fungal activity
against Candida albicans, with a 13 mm inhibition halo, in respect to the other tested compounds.
In addition, among all derivatives of α-mangostin, the acetyl (I K) and benzene sulphonyl
(I I) derivatives at 100 µg/mL concentration displayed maximum inhibition of 13 mm against
Aspergillus niger (Table 2). The zone of inhibition was determined in triplicates using the diffusion
technique, with values representing the average zone of inhibition. The alkylated product of
α-mangostin (I I) showed the most significant activity against fungal strains, with 12 mm inhibition of
C. albicans and 13 mm inhibition of A. niger. The xanthonoid skeleton with benzene sulphonyl moiety
showed enhanced anti-fungal activity for α-mangostin-based derivatives.
The cytotoxicity studies were carried out in silico using a software to individuate reactive groups
as from databases on chemical compounds. The in silico toxicity was predicted using the Toxtree
implementation of the modified Cramer rules and Verhaar scheme. The software Toxtree v.2.6.13
(IdeaConsult Ltd., Sofia, Bulgaria), along with Cramer rules and Verhaar scheme, were run to study
the toxicity of organic molecules [55–59]. The module was developed for the Long-Range Research
Initiative (LRI) and European Chemical Industry Council (CEFIC) CEFIC-LRI project AMBIT for a new
open software tool. A typical evaluation is given for one of the products. The study showed all the
molecules to be non-toxic. However this has to be validated by in vitro or in vivo studies which are
beyond the scope of the present study. Indeed such studies will form part of an in depth evaluation of
potential molecules. The in silico studies for compounds I A–C are attached as separate file named
“Mangostin and derivatives IA-IC in silico studies”. An example of results of cytotoxicity studies is
presented in the Supplementary file S2, “I A, I B, I C α-mangostin derivatives in silico toxicity test”.

3. Discussion
In this study the semi-synthetically modified α-mangostin-derived compounds were shown to
exhibit bioactive properties and antimicrobial activity greater than the parent natural molecule when
tested against various microbial and fungal cultures. Due to their anti-microbial properties, they can
be utilized as wound healers and in the treatment of skin infections, and in packaging materials for the
extension of food shelf life. We have shown the semi-synthetic modification of α-mangostin using the
Ritter reaction reduction by palladium-carbon (Pd-C), alkylation, and acetylation.
Studies are ongoing in the field of materials for packaging, to extend the shelf-life of food products.
The biomedical industry is also interested in a safe way for the containment of biofilm formation, and
production of devices with potential to avoid bacterial growth. Textiles, polymer films, and wrapping
materials are manufactured by electro-spinning and layer-by-layer deposition, the first one better
suited to eliminate residual solvents. The perishable foods represent a loss due to fungal and bacterial
growth, requiring short times from the shelves to the table. Thus, improvement in treatments and
use of anti-fungal films may ensure higher safety standard for human health in addition to decrease
of economic losses at retailer shops and during food supply chain. It is expected that new materials
and films, based on available and novel, semi-synthetic antimicrobial compounds, after assessment
of safety for humans or animals, grade of release of antimicrobials during the time, may find their
application according to the industry needs.
α-Mangostin and derived semi-synthesis products have been shown in several studies to have the
potential to be exploited as antimicrobial agents. In this study, twelve α-mangostin derivatives were
analyzed for inhibition of two Gram-positive and two Gram-negative bacteria (using Ciprofloxacin as
a reference drug at 50 and 100 µg/mL) and two fungal pathogens (using Ketoconazol as a reference
drug at 50 and 100 µg/mL) to determine the sensitivity of each bacterial species.
Looking for derivatives with improved activity in respect to the original compound α-mangostin
xanthone. Starting from this preliminary characterization, it will be possible to test these compounds
Molecules 2017, 22, 275 9 of 13

in vitro and in vivo, on animal models, to check the safety of the compounds, and to evaluate the
inhibition of bacterial growth, such as a bactericide activity on infected wounds. The antimicrobial
compounds, such as I E, have been shown to have inhibitory activity and need to be studied further
for applications in healthcare.

4. Materials and Methods

4.1. Isolation and Purification of α-Mangostin from Mangoosteen Fruit

All of the chemicals and reagents were purchased from either Sigma-Aldrich (St. Louis, MO,
USA) or Merck (Darmstadt, Germany) chemicals. The extraction of α-mangostin from the dried fruits
of G. mangostana was carried out with Soxhlet extraction methodology. A known weight (100 g) of
the fruit hulls was used, and extracted twice with 300 mL of ethyl acetate. The extract was filtered
through a Whatman filter paper No.1 (Brentford, UK) by suction. The filtrate was concentrated under
reduced pressure to obtain the ethyl acetate crude extract (26 g). Then 20 g of ethyl acetate crude extract
was subjected to TLC column chromatography using ethyl acetate and hexane (1:1) as the eluent.
The fractions containing α-mangostin were pooled and evaporated to obtain a solid of 95% purity by
HPLC (High Performance Liquid Chromatography). The obtained solid fraction was used as starting
material for semi-synthetic modification and for microbial studies. The compound was repeatedly
recrystallized using benzene for structural characterization using 1 H-NMR spectra, 13 C-NMR spectra,
and high-resolution mass spectra.

4.2. General Methods for Compound Analysis

The purity of the isolated α-mangostin and the progress of the reaction was monitored by HPLC
on analytical reversed phase develosil ODS column C18 (150 mm × 4.6 mm, 0.5 µm) using 0.02 M
potassium dihydrogen phosphate in water and a acetonitrile 50:50 ratio as the mobile phase, with
1.0 mL/min flow rate for 30 min, and a UV detector wavelength of 254 nm. The main product was
analyzed by nuclear magnetic resonance (NMR) (1 H: 400 MHz, 13 C: 100 MHz) and data recorded on a
Bruker instrument (Billerica, MA, USA), with chemical shifts expressed in δ ppm. NMR spectra were
obtained in MeOD with tetramethylsilane (TMS) as a reference compound. Mass was determined
using a Shimadzu analyzer (Columbia, MD, USA).

4.3. Anti-Microbial Activity Assay

The study of anti-microbial activity of the synthetic analogs was performed by measuring the
diameter of the inhibition halo from the outer surface of the disc, expressed in millimeters. The zone
of inhibition of the α-mangostin was compared with its synthetic analogs to determine the rate of
inhibition. The zone of inhibition was determined in triplicates using the diffusion technique, with
values representing the average zone of inhibition. The nutrient broth medium (50 mL) was prepared
and sterilized in autoclave at 121 ◦ C for 15 min. Gram-positive bacteria (B. subtilis and S. aureus) and
Gram-negative bacteria (E. coli and P. aeruginosa) were inoculated in tubes of nutrient broth, whereas
the fungal cultures (C. albicans and A. niger) were inoculated in tubes of potato dextrose agar, and
incubated at 37 ◦ C for 24 h; then the suspension was centrifuged at 8000× g for 5 min, the pellet was
suspended in double-distilled water, and the cell density was standardized spectrophotometrically
(A610 nm). All of the microbial cultures were adjusted to 0.5 McFarland standards, which is visually
comparable to a microbial suspension of approximately 1.5 × 108 cfu/mL.

4.3.1. Anti-Bacterial Assay

The bacterial species, chosen as representatives of spoilage and pathogenic species commonly
found contaminating surfaces and workplaces, were used to evaluate the anti-bacterial activities
of the synthetic compounds. The bacteria were maintained on Muller Hilton broth media at 37 ◦ C.
Then, Muller Hilton agar plates were swabbed with 100 µL inocula of the test microorganisms
Molecules 2017, 22, 275 10 of 13

and kept for 15 min for absorption. Six-millimeter Whatman No. 1 discs were pre-sterilized and
two concentrations (50 and 100 µg/mL) of the test compounds in DMSO were applied to the sterile
disc papers. The standard drug Ciprofloxacin (50 and 100 µg) was used as a positive reference standard
to determine the sensitivity of each bacterial species. Then the plates were inoculated at 37 ◦ C for 24 h.

4.3.2. Anti-Fungal Assay

All of the synthesized compounds were screened for anti-fungal activity by disc diffusion method.
PDA medium was autoclaved at 121 ◦ C for 15 min and poured into each Petri plate and the solidified
media plates were swabbed with 0.2 mL of fungal cultures. Six-millimeter Whatman No. 1 discs
were loaded with the test compounds in DMSO at 50 and 100 µg/mL concentration. Then the plates
were inoculated at 28 ◦ C for 72 h. The diameter of the clear zone around the well was measured and
expressed in millimeters. Standard drug Ketoconazole (50 and 100 µg) was used as a positive reference
standard to determine the sensitivity of each fungal species.

5. Conclusions
Among the semi-synthetic derivatives of α-mangostin, I E showed higher anti-bacterial activity
whereas I I showed the most significant anti-fungal activity. These compounds pave the way
to the synthesis of non-toxic compounds with better efficacy compared to the natural product
for anti-microbial treatment. It is envisaged that these new anti-microbial compounds could be
incorporated in packaging materials, textiles, biomedical devices, and film polymers, to improve the
microbiological safety of surfaces in contact with bacteria.

Supplementary Materials: The following are available online. Figure S1: title Molecules Narasimhan Poltronieri
supplementary.doc in Supplementary.zip. Supplementary materials include 1 H-NMR spectra, 13 C-NMR spectra,
and high-resolution mass spectra for compounds I, I A–I L. Supplementary materials can be accessed from the
PhD thesis “Studies on isolation and the semi-synthetic modification of bio active molecules from G. mangostana
and Embelia ribes” submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy
by S. Maheshwaran, under the guidance of Dr. S. Narasimhan at the University of Madras (April 2013).
Supplementary word file S2: “I A, I B, I C α-mangostin derivatives in silico toxicity test”.
Acknowledgments: We declare no private funding for this work. P.P. was suportedc by the Italian Ministry for
Innovation: PON02_00186_3417512 S.I.Mi.S.A. Innovative instruments for the improvement of food safety.
Author Contributions: N.S. designed the experiments and supervised their execution. S.M. performed the
chemical modifications. I.A.A.Y., A.F.M. and J.R. performed the structure analyses. P.E.D. performed data analyses.
P.P. designed and supervised the microbiological assays done by S.M. All authors contributed to manuscript
writing and preparation.
Conflicts of Interest: The authors declare no conflicts of interest.

References
1. Martin, F.W. Durian and mangosteen. In Tropical and Subtropical Fruits: Composition, Properties and Uses;
Nagy, S., Shaw, P.E., Eds.; The AVI Publishing Company, Inc.: Westport, CT, USA, 1980; pp. 407–414.
2. Kanchanapoom, K.; Kanchanapoom, M. Mangosteen. In Tropical and Subtropical Fruits; Shaw, P.E., Chan, H.T.,
Nagy, S., Eds.; Agscience Inc.: Auburndale, FL, USA, 1998; pp. 191–215.
3. Suksamrarn, S.; Komutiban, O.; Ratananukul, P.; Chimnoi, N.; Lartpornmatulee, N.; Suksamrarn, A.
Cytotoxic prenylated xanthones from the young fruit of Garcinia mangostana. Chem. Pharm. Bull. 2006, 54,
301–305. [CrossRef] [PubMed]
4. Pedraza-Chaverri, J.; Cárdenas-Rodríguez, N.; Orozco-Ibarra, M.; Pérez-Rojas, J.M. Medicinal properties of
mangosteen (Garcinia mangostana). Food Chem. Toxicol. 2008, 46, 3227–3239. [CrossRef] [PubMed]
5. Iinuma, M.; Tosa, H.; Tanaka, T.; Asai, F.; Kobayashi, A.; Shimano, R.; Miyauchi, K.-I. Antibacterial activity of
xanthones from guttiferaeous plants against methicillin-resistant Staphylococcus aureus. J. Pharm. Pharmacol.
1996, 48, 861–865. [CrossRef] [PubMed]
Molecules 2017, 22, 275 11 of 13

6. Duangsrisai, S.; Choowongkomon, K.; Bessa, L.; Costa, P.; Amat, N.; Kijjoa, A. Antibacterial and EGFR-tyrosine
kinase inhibitory activities of polyhydroxylated xanthones from Garcinia succifolia. Molecules 2014, 19,
19923–19934. [CrossRef] [PubMed]
7. Chomnawang, M.T.; Surassmo, S.; Nukoolkarn, V.S.; Gritsanapan, W. Antimicrobial effects of Thai medicinal
plants against acne-inducing bacteria. J. Ethnopharmacol. 2005, 101, 330–333. [CrossRef] [PubMed]
8. Lebedeva, A.A.; Zakharchenko, N.S.; Trubnikova, E.V.; Medvedeva, O.A.; Kuznetsova, T.V.; Masgutova, G.A.;
Zylkova, M.V.; Buryanov, Y.I.; Belous, A.S. Bactericide and immunomodulating properties of transgenic
Kalanchoe pinnata synergize with antimicrobial peptide cecropin P1 in vivo. J. Immunol. Res. 2017, in press.
9. Miguel-de Abreu, P.; Farias, P.G.; Paliva, G.S.; Almeida, A.M.; Morals, P.V. Persistence of microbial
communities including Pseudomonas aeruginosa in a hospital environment: A potential health hazard.
BMC Microbiol. 2014, 14, 118.
10. Jain, A.; Agarwal, A.; Verma, R.K. Cefoxitin Disc diffusion test for detection of methicillin-resistant
Staphylococci. J. Med. Microbiol. 2008, 57, 957–961. [CrossRef] [PubMed]
11. Walker, E.B. HPLC analysis of selected xanthones in mangosteen fruit. J. Separ. Sci. 2007, 30, 1229–1234.
[CrossRef] [PubMed]
12. Mohamed, G.A.; Ibrahim, S.R.M.; Shaaban, M.I.A.; Ross, S.A. Mangostan axanthones I and II, new xanthones
from the pericarp of Garcinia mangostana. Phytotherapia 2014, 98, 215–221.
13. Sakagami, Y.; Iinuma, M.; Piyasena, K.G.; Dharmaratne, H.R. Antibacterial activity of alpha-mangostin
against vancomycin resistant Enterococci (VRE) and synergism with antibiotics. Phytomedicine 2005, 12,
203–208. [CrossRef] [PubMed]
14. Phitaktim, S.; Chomnawang, M.; Sirichaiwetchakoon, K.; Dunkhunthod, B.; Hobbs, G.; Eumkeb, G.
Synergism and the mechanism of action of the combination of α-mangostin isolated from
Garcinia mangostana L. and oxacillin against an oxacillin-resistant Staphylococcus saprophyticus. BMC Microbiol.
2016, 16, 195. [CrossRef] [PubMed]
15. Ragasa, C.Y.; Crisostomo, C.J.J.; Garcia, K.D.C.; Shen, C. Antimicrobial xanthones from Garcinia mangostana L.
Philipp. Sci. 2010, 47, 63–75.
16. Alsultan, Q.M.N.; Sijam, K.; Rashid, T.S.; Ahmad, K.B. GC-MS Analysis and antibacterial activity of
mangosteen leaf extracts against plant pathogenic bacteria. Am. J. Plant Sci. 2016, 7, 1013–1020. [CrossRef]
17. Tatiya-Aphiradee, N.; Chatuphonprasert, W.; Jarukamjorn, K. In vivo antibacterial activity of
Garcinia mangostana pericarp extract against methicillin-resistant Staphylococcus aureus in a mouse superficial
skin infection model. Pharm. Biol. 2016, 54, 2606–2615. [CrossRef] [PubMed]
18. Dharmaratne, H.R.W.; Sakagami, Y.; Piyasena, K.G.P.; Thevanesam, V. Antibacterial activity of xanthones
from Garcinia mangostana (L.) and their structure-activity relationship studies. Nat. Prod. Res. 2013, 27,
938–941. [CrossRef] [PubMed]
19. Weecharangsan, W.; Opanasopit, P.; Sukma, M.; Ngawhirunpat, T.; Sotanaphun, U.; Siripong, P. Antioxidative
and neuroprotective activities of extracts from the fruit hull of mangosteen (Garcinia mangostana Linn.).
Med. Princ. Pract. 2006, 15, 281–287. [CrossRef] [PubMed]
20. Guzmán-Beltrán, S.; Orozco-Ibarra, M.; González-Cuahutencos, O.; Victoria-Mares, S.; Merchand-Reyes, G.;
Medina-Campos, O.N.; Pedraza-Chaverri, J. Neuroprotective effect and reactive oxygen species scavenging
capacity of mangosteen pericarp extract in cultured neurons. Curr. Top. Nutr. Res. 2008, 6, 149–158.
21. Márquez-Valadez, B.; Lugo-Huitrón, R.; Valdivia-Cerda, V.; Miranda-Ramírez, L.R.; Pérez-De La Cruz, V.;
González-Cuahutencos, O.; Rivero-Cruz, I.; Mata, R.; Santamaría, R.; Pedraza-Chaverrí, J. The natural
xanthone α-mangostin reduces oxidative damage in rat brain tissue. Nutr. Neurosci. 2009, 12, 35–42.
[CrossRef] [PubMed]
22. Pedraza-Chaverrí, J.; Reyes-Fermín, L.M.; Nolasco-Amaya, E.G.; Ibarra, M.O.; Medina-Campos, O.N.;
González-Cuahutencos, O.; Rivero-Cruz, I.; Mata, R. ROS scavenging capacity and neuroprotective effect
of α-mangostin against 3-nitropropionic acid in cerebellar granule neurons. Exp. Toxicol. Pathol. 2009, 61,
491–501. [CrossRef] [PubMed]
23. Chae, H.-S.; Kim, E.-Y.; Han, L.; Kim, N.-R.; Lam, B.; Paik, J.H.; Yoon, K.D.; Choi, Y.H.; Chin, Y.-W. Xanthones
with pancreatic lipase inhibitory activity from the pericarps of Garcinia mangostana L. (Guttiferae). Eur. J.
Lipid Sci. Technol. 2016, 118, 1416–1421. [CrossRef]
Molecules 2017, 22, 275 12 of 13

24. Balunas, M.J.; Su, B.; Brueggemeier, R.W.; Kinghorn, A.D. Xanthones from the botanical dietary supplement
mangosteen (Garcinia mangostana) with aromatase inhibitory activity. J. Nat. Prod. 2008, 71, 1161–1166.
[CrossRef] [PubMed]
25. Lin, C.N.; Chung, M.I.; Liou, S.J.; Lee, T.H.; Wang, J.P. Synthesis and anti-inflammatory effects of xanthone
derivatives. J. Pharm. Pharmacol. 1996, 48, 532–538. [CrossRef] [PubMed]
26. Chin, Y.W.; Kinghorn, A.D. Structural characterization, biological effects, and synthetic studies on xanthones
from mangosteen (Garcinia mangostana), a popular botanical dietary supplement. Mini. Rev. Org. Chem. 2008,
5, 355–364. [CrossRef] [PubMed]
27. Fei, X.; Jo, M.; Lee, B.; Han, S.; Lee, K.; Jung, J.; Seo, S.; Kwak, Y. Synthesis of xanthone derivatives based
on α-mangostin and their biological evaluation for anti-cancer agents. Bioorg. Med. Chem. Lett. 2014, 24,
2062–2065. [CrossRef] [PubMed]
28. Morelli, C.F.; Biagiotti, M.; Pappalardo, V.M.; Rabuffetti, M.; Speranza, G. Chemistry of α-mangostin. Studies
on the semisynthesis of minor xanthones from Garcinia mangostana. Nat. Product Res. 2015, 29, 750–755.
[CrossRef] [PubMed]
29. Zou, H.; Koh, J.; Li, J.; Qiu, S.; Aung, T.T.; Lin, H.; Lakshminarayanan, R.; Dai, X.; Tang, C.; Lim, F.H.; et al.
Design and synthesis of amphiphilic xanthone-based, membrane-targeting antimicrobials with improved
membrane selectivity. J. Med. Chem. 2013, 56, 2359–2373. [CrossRef] [PubMed]
30. Samprasit, W.; Rojanarata, T.; Akkaramongkolporn, P.; Ngawhirunpat, T.; Kaomongkolgit, R.; Opanasopit, P.
Fabrication and in vitro/in vivo performance of mucoadhesive electrospun nanofiber mats containing
α-mangostin. AAPS Pharm. Sci. Tech. 2015, 16, 1140–1152. [CrossRef] [PubMed]
31. Suwantong, O.; Pankongadisak, P.; Deachathai, S.; Supaphol, P. Electrospun poly(L-lactic acid) fiber mats
containing crude Garcinia mangostana extracts for use as wound dressings. Polym. Bull. 2014, 71, 925–949.
[CrossRef]
32. Lakouraj, M.M.; Rahpaima, G.; Mohseni, M. Synthesis, characterization, metal sorption, and biological
activities of organosoluble and thermally stable azoxanthone-based polyester. Polym. Adv. Technol. 2015, 26,
234–244. [CrossRef]
33. Vimala, K.; Yallapu, M.M.; Varaprasad, K.; Reddy, N.N.; Ravindra, S.; Naidu, N.S.; Raju, K.M. Fabrication
of curcumin encapsulated chitosan-PVA silver nanocomposite films for improved antimicrobial activity.
J. Biomater. Nanobiotechnol. 2011, 2, 55–64. [CrossRef]
34. Mun, S.H.; Joung, D.K.; Kim, Y.S.; Kang, O.H.; Kim, S.B.; Seo, Y.S.; Kwon, D.Y. Synergistic antibacterial effect
of curcumin against methicillin-resistant Staphylococcus aureus. Phytomedicine 2013, 20, 714–718. [CrossRef]
[PubMed]
35. Shlar, I.; Poverenov, E.; Vinokur, Y.; Horev, B.; Droby, S.; Rodov, V. High throughput screening of
nanoparticle-stabilizing ligands: Application to preparing antimicrobial curcumin nanoparticles by
antisolvent precipitation. Nano-Micro Lett. 2015, 7, 68–79. [CrossRef]
36. Dogra, N.; Choudhary, R.; Kohli, P.; Haddock, J.D.; Makwana, S.; Horev, B.; Vinokur, Y.; Droby, S.; Rodov, V.
Polydiacetylene nanovesicles as carriers of natural phenylpropanoids for creating antimicrobial food-contact
surfaces. J. Agric. Food Chem. 2015, 63, 2557–2565. [CrossRef] [PubMed]
37. Ravichandran, M.; Hettiarachchy, N.S.; Ganesh, V.; Ricke, S.C.; Singh, S. Enhancement of antimicrobial
activities of naturally occurring phenolic compounds by nanoscale delivery against Listeria monocytogenes,
Escherichia coli O157:H7 and Salmonella typhimurium in broth and chicken meat system. J. Food Saf. 2011, 31,
462–471. [CrossRef]
38. Fitzgerald, D.J.; Stratford, M.; Gasson, M.J.; Ueckert, J.; Bos, A.; Narbad, A. Mode of antimicrobial action
of vanillin against Escherichia coli, Lactobacillus plantarum and Listeria innocua. J. Appl. Microbiol. 2004, 97,
104–113. [CrossRef]
39. Rodov, V.; Nafussi, B.; Ben-Yehoshua, S. Essential oil components as potential means to control postharvest
pathogens of citrus fruit. Fresh Prod. 2011, 5, 43–50.
40. Horev, B.; Sela, S.; Vinokur, Y.; Gorbatsevich, E.; Pinto, R.; Rodov, V. The effects of active and passive modified
atmosphere packaging on the survival of Salmonella enterica serotype Typhimurium on washed romaine
lettuce leaves. Food Res. Int. 2012, 45, 1129–1132. [CrossRef]
41. Izumi, H.; Rodov, V.; Bai, J.; Wendakoon, S. Physiology and quality of fresh-cut produce in CA/MA storage.
In Fresh-Cut Fruit and Vegetables: Physiology, Technology and Safety, 1st ed.; Pareek, S., Ed.; CRC Press:
Boca Raton, FL, USA, 2016; pp. 271–318.
Molecules 2017, 22, 275 13 of 13

42. Fadida, T.; Kroupitski, Y.; Peiper, U.M.; Bendikov, T.; Sela, S.; Poverenov, E. Air-ozonolysis to generate
contact active antimicrobial surfaces: Activation of polyethylene and polystyrene followed by covalent graft
of quaternary ammonium salts. Colloids Surf. B Biointerfaces 2014, 122, 294–300. [CrossRef] [PubMed]
43. Reidy, B.; Haase, A.; Luch, A.; Dawson, K.A.; Lynch, I. Mechanisms of silver nanoparticle release,
transformation and toxicity: A critical review of current knowledge and recommendations for future
studies and applications. Materials 2013, 6, 2295–2350. [CrossRef]
44. Malka, E.; Perelshtein, I.; Lipovsky, A.; Shalom, Y.; Naparstek, L.; Perkas, N.; Patick, T.; Lubart, R.; Nitzan, Y.;
Banin, E.; et al. Eradication of multi-drug resistant bacteria by a novel Zn-doped CuO nanocomposite. Small
2013, 9, 4069–4076. [CrossRef] [PubMed]
45. Meridor, D.; Gedanken, A. Preparation of enzyme nanoparticles and studying the catalytic activity of
the immobilized nanoparticles on polyethylene films. Ultrason. Sonochem. 2013, 20, 425–431. [CrossRef]
[PubMed]
46. Poverenov, E.; Danino, S.; Horev, B.; Granit, R.; Vinokur, Y.; Rodov, V. Layer-by-Layer electrostatic deposition
of edible coating on fresh cut melon model: Anticipated and unexpected effects of alginate-chitosan
combination. Food Bioprocess Technol. 2014, 7, 1424–1432. [CrossRef]
47. Poverenov, E.; Rutenberg, R.; Danino, S.; Horev, B.; Rodov, V. Gelatin-chitosan composite films and edible
coatings to enhance the quality of food products: Layer-by-Layer vs. blended formulations. Food Bioprocess
Technol. 2014, 7, 3319–3327. [CrossRef]
48. Poverenov, E.; Shemesh, M.; Gulino, A.; Cristaldi, D.A.; Zakin, V.; Yefremov, T.; Granit, R. Durable contact
active antimicrobial materials formed by a one-step covalent modification of polyvinyl alcohol, cellulose
and glass surfaces. Colloids Surf. B Biointerfaces 2013, 112, 356–361. [CrossRef] [PubMed]
49. Chung, D.; Papadakis, S.E.; Yam, K.L. Evaluation of a polymer coating containing triclosan as the
antimicrobial layer for packaging materials. Int. J. Food Sci. Technol. 2003, 38, 165–169. [CrossRef]
50. Bastarrachea, L.; Dhawan, S.; Sablani, S.S. Engineering properties of polymeric based antimicrobial films for
food packaging: A review. Food Eng. Rev. 2011, 3, 79–93. [CrossRef]
51. Kurt, P.; Wood, L.; Ohman, D.E.; Wynne, K.J. Highly effective contact antimicrobial surfaces via polymer
surface modifiers. Langmuir 2007, 23, 4719–4723. [CrossRef] [PubMed]
52. Zhang, F.; Kang, E.T.; Neoh, K.G.; Wang, P.; Tan, K.L. Surface modification of stainless steel by grafting of
poly(ethylene glycol) for reduction in protein adsorption. Biomaterials 2001, 22, 1541–1548. [CrossRef]
53. El-Seedi, H.R.; El-Ghorab, D.M.H.; El-Barbary, M.A.; Zayed, M.F.; Goransson, U.; Larsson, S.; Veerporte, R.
Naturally occurring xanthones; latest investigations: Isolation, structure elucidation and chemosystematic
significance. Curr. Med. Chem. 2009, 16, 2581–2626. [CrossRef] [PubMed]
54. Jyoshna; Khare, P.; Shanker, K. Mangiferin: A review of sources and interventions for biological activities.
BioFactors 2016, 42, 504–514. [CrossRef] [PubMed]
55. Cramer, G.M.; Ford, R.A.; Hall, R.L. Estimation of Toxic Hazard—A Decision Tree Approach. J. Cosmet.
Toxicol. 1978, 16, 255–276. [CrossRef]
56. Patlewicz, G.; Jeliazkova, N.; Safford, R.J.; Worth, A.P.; Aleksiev, B. An evaluation of the implementation
of the Cramer classification scheme in the Toxtree software. SAR QSAR Environ. Res. 2008, 19, 495–524.
[CrossRef] [PubMed]
57. Lapenna, S.; Worth, A. Analysis of the Cramer Classification Scheme for Oral Systemic Toxicity-Implications for Its
Implementation in Toxtree; EUR 24898 EN; Publications Office of the European Union: Luxembourg, 2011.
58. Verhaar, H.J.M.; van Leeuwen, C.J.; Hermens, J.L.M. Classifying environmental pollutants. 1. Structure-activity
relationships for prediction of aquatic toxicity. Chemosphere 1992, 25, 471–491. [CrossRef]
59. Verhaar, H.J.M.; Solbe, J.; Speksnijder, J.; van Leeuwen, C.J.; Hermens, J.L.M. Classifying environmental
pollutants: Part 3. External validation of the classification system. Chemosphere 2000, 40, 875–883. [CrossRef]

Sample Availability: Samples of the compounds I and I A to I L are available from the authors.

© 2017 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).