Anti-bacterial and anti-fungal activity of xanthones obtained via semi-synthetic modification of α-mangostin from Garcinia mangostana

Article abstract of DOI:10.3390/molecules22020275

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 IE showed high anti-bacterial activity whereas II 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.

Full text of DOI:10.3390/molecules22020275

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.)
Department of Biology, Chemistry and Environmental Sciences, American University of Sharjah,
2
P.O. Box 26666 Sharjah, United Arab Emirates; iabuyousef@aus.edu (I.A.A.-Y.);
amajdalawieh@aus.edu (A.F.M.)
Institute of Sciences of Food Productions, CNR-ISPA, Lecce 73100, Italy
3
*
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 -mangostin. In the present
study, twelve synthetic analogs were obtained through semi-synthetic modification of -mangostin
α
α
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 [
is commercially used as dietary supplement for cancer patients [
contains high amounts of xanthones, such as -mangostin (Figure 1),
1
,
2
]. In addition, mangosteen with essential minerals
]. The pericarp of the fruit
-mangostin, -mangostin,
3
α
β
γ
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 [ ]. Natural compounds with antibacterial
5
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

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visible growth of a bacterium (bacteriostatic activity), whereas the minimum bactericidal concentration
(MBC) is the concentration that results in microbial death. Among the pathogens developing antibiotic
resistances, Pseudomonas aeruginosa and Staphylococcus aureus (S. aureus) are largely spread in hospitals
and healthcare units [9,10].
CH3
H3C
OH
O
O
CH3
O
CH3
H3C
OH
HO
Figure 1. Structure of α-mangostin.
In the literature, mangosteen fruit extracts were shown to contain different xanthones, identified
by HPLC analysis: -mangostin, -mangostin, -mangostin, 8-desoxygartanin and gartanin,
two isoprenylated xanthones, and 9-hydroxycalabaxanthone [11]. -Mangostin (3,6,8-trihydroxy-
α
β
γ
α
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
medicinal properties, such as anti-microbial activity against bacteria [
neuro-protective activity [19 22], lipase inhibition [23], and anti-inflammatory and anti-cancer
α
-mangostin from G. mangostana fruit possesses several
4,12–18], anti-oxidant and
–
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
possess anti-bacterial activity, especially against Gram-positive bacteria [12
that semi-synthetic analogs would be produced with enhanced anti-bacterial activity. In order to
increase the bioactivity and antibacterial properties of -mangostin, semi-synthetic modification
of the compound was performed by several authors, to lead to more active compounds [25 29],
α
- and non-
α
-mangostin xanthones have been shown to
–
18], it is most probable
α
–
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
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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 -mangostin and the purity
-mangostin ( ) using
α
was confirmed as 95% using HPLC. The structural characterization of the pure
α
I
1H-NMR spectra, 13C-NMR spectra, IR spectrum and high-resolution mass spectra is available in
Supplementary materials.

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2.2. Synthetic Modifications
Following the isolation,
core structure (Figure 2).
α
-mangostin was subjected to a series of chemical reactions to alter the
Figure 2. Reaction scheme leading to different derivatives.
The basic core structure xanthone (anthraquinone) was conserved intact while the functional
iso-prenyl and phenolic hydroxy groups were subjected to semi-synthetic modification. Twelve
different semi-synthetic derivatives were obtained (Table 1), each containing new key moieties that
were evaluated to detect the anti-microbial activity against various bacterial and fungal cultures.
(
I D), the alkylated product of α-mangostin with ethyl iodide, was found to be a di-alkylated
product. All other alkylations using the exact same reaction conditions reported in the table resulted in
tri-alkylated products. In the Ritter reaction, the addition of acetonitrile, followed by intra-molecular
addition of –OH to isoprene unit gave ether (or) pyran ring structures. Addition of acetonitrile is
followed by hydration to produce amide. This reaction is general for all the nitrile reaction products
studied (I A to I C). Compound ID, reaction of α-mangostin with ethyl iodide, produced mainly the
di-alkylated product, probably due to the possibility of ether cleavage of 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.
Compound
Information
Reaction of
α-Mangostin with
Molecular
Formula
S. No.
Structure
Mass
CH3
H3C
OH
O
O
CH3
α
-Mangostin
1.
-
C24H26O6
411 (M+1)+
O
(I)
CH3
H3C
OH
HO

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Table 1. Cont.
Compound
Information
Reaction of
α-Mangostin with
Molecular
Formula
S. No.
Structure
Mass
H3C
NHCOCH3
H3C
Ritter
product of
-mangostin
(I A)
OH
Acetonitrile in
Silica supported
sulphuric acid
O
O
2.
C26H31NO7
470 (M+1)+
O
α
CH3
H3C
H3C
OH
O
H3C
NHCOCH2CN
H3C
Ritter
product of
-mangostin
(I B)
OH
Malononitrile in
Silica supported
sulphuric acid
O
O
514
3.
4.
C27H30N2O7
O
α
(M+2+NH3)+
CH3
H3C
OH
O
H3C
H3C
NHCOCH2CH2CH3
H3C
Ritter
product of
-mangostin
(I C)
OH
Butyronitrile in
Silica supported
sulphuric acid
O
O
C28H25NO7
498 (M+1)+
O
CH3
α
H3C
H3C
OH
O
CH3
CH3
O
H3C
Ethyl Iodide
K2CO3/ACN
Alkylated
product of
O
O
467 (M+1)+
537 (M)+
CH3
C28H34O6
5.
6.
O
α
-mangostin
CH3
H3C
(I D)
M.W. 10 min
O
HO
CH3
H
3C
Bromopropane
K2CO3/ACN
CH3
H3
C
Alkylated
product of
O
O
CH3
C33H44O6
α
-mangostin
O
CH3
H3
H3
C
(I E)
M.W. 10 min
C
CH3
O
O
O
Cyclopentyl
bromide
CH3
H3C
Alkylated
product of
O
O
O
K2CO3/ACN
CH3
615 (M)+
C39H50O6
7.
8.
O
CH3
α
-mangostin
H3C
(I F)
M.W. 10 min
O
O
CH3
H3C
Propargyl bromide
K2CO3/ACN
Alkylated
product of
O
O
CH3
525 (M)+
O
C33H32O6
CH3
H3C
α
-mangostin
M.W. 10 min
(I G)
O
O
O
Benzyl bromide
K2CO3/ACN
CH3
H3
C
Alkylated
product of
O
O
CH3
681 (M)+
O
C45H44O6
9.
CH3
H
3C
α
-mangostin
(I H)
M.W. 10 min
O
O
O
Benzene sulphonyl
chloride
CH3
H3C
O
S
K2CO3/ACN
Alkylated
product of
O
O
O
O
CH3
O
O
831 (M)+
C42H38O12S3
CH3
10.
H3
C
α
-mangostin
O
S
O
(I I)
O
M.W. 10 min
S
O
O

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Table 1. Cont.
Compound
Information
Reaction of
α-Mangostin with
Molecular
Formula
S. No.
Structure
Mass
Chloroethyl
morpholine
O
hydrochloride
N
CH3
H3C
K2CO3/ACN
Alkylated
product of
O
O
O
CH3
750 (M)+
C42H59N3O9
11.
O
O
CH3
α
-mangostin
H3C
(I J)
O
M.W. 10 min
N
N
O
O
CH3
H3C
Acetic anhydride
K2CO3/ACN
CH3
O
O
O
CH3
Acylated
O
O
CH3
O
product of
536 (M)+
H3C
C26H28O7
12.
13.
α
-mangostin
M.W. 10 min
(I K)
O
O
H3C
H3C
O
CH3
H3C
Reduced
product of
OH
O
O
CH3
Palladium/Carbon
C25H30O6
415 (M+1)+
O
α
-mangostin
CH3
H3C
(I L)
OH
HO
2.3. Biological Assays
2.3.1. Anti-Bacterial Assay
The evaluation of the anti-bacterial potential for
examined against E. coli, Bacillus subtilis (B. subtilis), S. aureus, and P. aeruginosa, in accordance with an
experimental procedure. Two different concentrations (50 g/mL and 100 g/mL) of the -mangostin
α
-mangostin-based synthetic analogs was
µ
µ
α
and their synthetic analogs along with standard drug Ciprofloxacin were tested against the pathogens
and the results are given in Table 2. The zone of inhibition was determined in triplicates using the
diffusion technique, with values representing the average zone of inhibition.
By measuring the zone of inhibition (in mm), it is observed that all the derivatives of
exert moderate to high anti-bacterial activity. Compound (I E) showed maximum anti-bacterial activity
(up to 12 mm) at 100 g/mL concentration against all bacterial stains tested in comparison to the
other synthesized compounds. At low concentration of 50 g/mL, the acetyl derivative (I G) showed
α-mangostin
µ
µ
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
P. aeruginosa. Among these compounds, the acetyl (I K) and benzene sulphonyl (I I) derivatives of
-mangostin showed maximum anti-bacterial activity against the four bacterial strains tested. Most of
α-mangostin showed maximum inhibition against
α
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
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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
I A
I B
I C
I D
I E
5 ± 0.09
4 ± 0.18
6 ± 0.14
8 ± 0.11
7 ± 0.12
6 ± 0.13
6 ± 0.18
6 ± 0.20
5 ± 0.12
5 ± 0.08
4 ± 0.15
10 ± 0.14
9 ± 0.13
19 ± 0.11
8 ± 0.04
9 ± 0.16
10 ± 0.11
11 ± 0.08
10 ± 0.18
11 ± 0.22
9 ± 0.04
9 ± 0.12
9 ± 0.11
8 ± 0.15
7 ± 0.16
12 ± 0.16
11 ± 0.06
24 ± 0.05
7 ± 0.11
6 ± 0.15
6 ± 0.12
10 ± 0.06
4 ± 0.04
9 ± 0.15
5 ± 0.09
7 ± 0.05
6 ± 0.12
6 ± 0.04
5 ± 0.11
4 ± 0.10
5 ± 0.21
16 ± 0.07
9 ± 0.15
10 ± 0.11
9 ± 0.23
12 ± 0.04
10 ± 0.09
12 ± 0.16
11 ± 0.18
10 ± 0.11
9 ± 0.08
10 ± 0.20
8 ± 0.14
7 ± 0.14
8 ± 0.16
20 ± 0.14
5 ± 0.05
5 ± 0.12
5 ± 0.16
6 ± 0.18
5 ± 0.08
7 ± 0.22
7 ± 0.14
10 ± 0.21
8 ± 0.18
7 ± 0.11
4 ± 0.13
5 ± 0.08
5 ± 0.12
11 ± 0.15
9 ± 0.12
11 ± 0.22
8 ± 0.14
9 ± 0.06
7 ± 0.12
11 ± 0.18
11 ± 0.17
11 ± 0.19
10 ± 0.13
9 ± 0.15
7 ± 0.21
9 ± 0.08
8 ± 0.05
18 ± 0.06
6 ± 0.24
5 ± 0.11
4 ± 0.08
5 ± 0.16
9 ± 0.21
7 ± 0.05
5 ± 0.09
6 ± 0.14
8 ± 0.11
9 ± 0.19
3 ± 0.08
6 ± 0.22
6 ± 0.15
15 ± 0.12
10 ± 0.08
8 ± 0.16
8 ± 0.22
10 ± 0.13
9 ± 0.08
12 ± 0.03
9 ± 0.14
9 ± 0.18
11 ± 0.11
12 ± 0.06
6 ± 0.20
9 ± 0.06
8 ± 0.12
19 ± 0.16
8 ± 0.12
7 ± 0.11
4 ± 0.16
2 ± 0.16
4 ± 0.22
4 ± 0.08
3 ± 0.07
4 ± 0.12
9 ± 0.17
10 ± 0.14
11 ± 0.12
8 ± 0.20
7 ± 0.13
15 ± 0.16
10 ± 0.14
10 ± 0.06
6 ± 0.11
6 ± 0.13
6 ± 0.21
7 ± 0.17
5 ± 0.14
6 ± 0.19
13 ± 0.12
12 ± 0.09
13 ± 0.15
11 ± 0.18
10 ± 0.08
18 ± 0.04
4 ± 0.18
9 ± 0.11
3 ± 0.07
2 ± 0.18
7 ± 0.15
3 ± 0.04
4 ± 0.09
3 ± 0.16
7 ± 0.11
9 ± 0.08
6 ± 0.03
10 ± 0.15
9 ± 0.17
14 ± 0.13
7 ± 0.05
12 ± 0.02
5 ± 0.15
5 ± 0.14
9 ± 0.08
5 ± 0.05
7 ± 0.11
5 ± 0.19
10 ± 0.21
13 ± 0.11
8 ± 0.18
13 ± 0.18
12 ± 0.08
18 ± 0.08
I F
I G
I H
I I
I J
I K
I L
Std drug

Molecules 2017, 22, 275
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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 g/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 -mangostin, the acetyl (I K) and benzene sulphonyl
I I) derivatives at 100 g/mL concentration displayed maximum inhibition of 13 mm against
µ
α
(
µ
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 -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 g/mL) and two fungal pathogens (using Ketoconazol as a reference
α
µ
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
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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 1H-NMR spectra, 13C-NMR spectra,
and high-resolution mass spectra.
4.2. General Methods for Compound Analysis
The purity of the isolated
on analytical reversed phase develosil ODS column C18 (150 mm
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) (1H: 400 MHz, 13C: 100 MHz) and data recorded on a
α
-mangostin and the progress of the reaction was monitored by HPLC
4.6 mm, 0.5 m) using 0.02 M
×
µ
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
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and kept for 15 min for absorption. Six-millimeter Whatman No. 1 discs were pre-sterilized and
two concentrations (50 and 100 g/mL) of the test compounds in DMSO were applied to the sterile
disc papers. The standard drug Ciprofloxacin (50 and 100
µ
µ
g) 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
standard to determine the sensitivity of each fungal species.
µg) was used as a positive reference
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 1H-NMR spectra, 13C-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.
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©
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/).