Pharmaceutical Biology

ISSN: 1388-0209 (Print) 1744-5116 (Online) Journal homepage: https://www.tandfonline.com/loi/iphb20

Chemical composition and antibacterial activity of

a hexane extract of Tunisian caprifig latex from the
unripe fruit of Ficus carica

Houda Lazreg-Aref, Massoud Mars, Abdelwaheb Fekih, Mahjoub Aouni &

Khaled Said

To cite this article: Houda Lazreg-Aref, Massoud Mars, Abdelwaheb Fekih, Mahjoub Aouni &
Khaled Said (2012) Chemical composition and antibacterial activity of a hexane extract of Tunisian
caprifig latex from the unripe fruit of Ficus�carica, Pharmaceutical Biology, 50:4, 407-412, DOI:
10.3109/13880209.2011.608192

To link to this article: https://doi.org/10.3109/13880209.2011.608192

Published online: 02 Dec 2011.

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Pharmaceutical Biology, 2012; 50(4): 407–412
© 2012 Informa Healthcare USA, Inc.
ISSN 1388-0209 print/ISSN 1744-5116 online
DOI: 10.3109/13880209.2011.608192

Research Article

Chemical composition and antibacterial activity

of a hexane extract of Tunisian caprifig latex from the
unripe fruit of Ficus carica
Houda Lazreg-Aref1, Massoud Mars3, Abdelwaheb Fekih4, Mahjoub Aouni2, and Khaled Said1
1
Laboratoire de Génétique, Biodiversité et Valorisation des Bio ressources (UR 03ES09), Institut Supérieur de
Biotechnologie, Monastir, Tunisie, 2Laboratoire des Maladies Transmissibles et Substances Biologiquement Actives,
Faculté de Pharmacie, Monastir, Tunisie, 3Laboratoire U.R. Agro biodiversité, Département des sciences Horticoles
Institut Supérieur Agronomique, and 4Laboratoire de Chimie, Faculté de Médecine Dentaire, Monastir, Tunisie

Abstract
Context: The plant kingdom has become a target in the search for new drugs and biologically active lead compounds.
The common Jrani Tunisian caprifig Ficus carica L. (Moraceae) is one of the large number of plant species that are used
in folklore medicine yet to be investigated for the treatment of many diseases, including those of infectious nature.
Objective: Hexane extract of the Tunisian common Jrani caprifig latex was assayed for antibacterial activity against
several Gram-positive and Gram-negative bacteria. Chemical composition of the extract was also investigated.
Materials and methods: The hexane extract was obtained from Tunisian Jrani caprifig latex by maceration, and then
analyzed by gas chromatography–mass spectrometry. The extract was tested in vitro for antibacterial activity by the
disc diffusion method and minimal inhibitory concentration (MIC) was also determined for all the test cultures.
Results: Thirty-six compounds of the extract were identified, 90.56% of the total area of peaks were coumarins. A
strong bactericidal effect was demonstrated. The most sensitive bacteria were Staphylococcus saprophyticus clinical
isolate, and Staphylococcus aureus ATCC 25923, with a MIC of 19 µg/mL.
Discussion and conclusion: These findings demonstrate an effective in vitro antibacterial activity of the hexane extract
of caprifig latex.
Keywords:  Medicinal plant, coumarin, Gram-positive bacteria, Gram-negative bacteria

Introduction Ballow, 2000). The screening of plant extracts and plant

Renewed attention in recent decades to alternative
medicines and natural therapies has stimulated a new
wave of research interest in traditional practices (Braun
& Cohen, 2007). The increased prevalence of antibiot-
ic-resistant bacteria emerging from the extensive use
of antibiotics may render the current antimicrobial
agents insufficient to control at least some bacterial
infections. Antimicrobial resistance among key micro-
bial pathogens continues to grow at an alarming rate
worldwide. Resistance among strains of Staphylococcus
aureus, Pseudomaunas ssp., Streptococcus ssp., and
Enterobacteriaceae has been described (Bhavnani &
products for antimicrobial activity has shown that
higher plants represent a potential source of new anti-
infective agents (Arias et  al., 2004), and only 5–15%
have been studied for a potential therapeutic value
(Kinghorn, 1992). Caprifig Ficus carica L. (Moraceae) a
fig tree which produces both male and female flowers
and is used to fertilize the female trees of the species.
Unlike common figs, the caprifig produces three crops
of synconia. These are known by their Italian terms, pro-
fichi, mammoni, and mamme. Fresh leaves and syconia
of caprifigs in Tunisian folk medicine are used by agri-
culturalists as an additive in chicken alimentation for

Address for Correspondence:  Houda Lazreg Aref, Laboratoire de Génétique, Biodiversité et Valorisation des Bio ressources (UR 03ES09),
Institut Supérieur de Biotechnologie, 5000 Monastir, Tunisie. Tel: +216 97 654 133. E-mail: ibrahimhoudarf@yahoo.fr
(Received 20 October 2010; revised 14 June 2011; accepted 23 July 2011)

407
408  H. Lazreg-Aref et al.
prevention against bacteria and virus infections; their
high amount of latex should be an effective source of
protection.
Lazreg Aref et al. (2010) have investigated the antimi-
crobial activity of F. carica latex extracts of Bidhi Bither
variety (Saint-Pedro type) against some resistant bacte-
ria. This study investigated the antibacterial activity of the
hexane extract of caprifig latex and defined its chemical
composition using gas chromatography–mass spectrom-
etry (GC-MS) technique.
Materials and methods
Plant material
The caprifig Jrani latex was collected from unripe ined-
ible fig fruit from Mesjed Aissa agricultural field located
in the central cost of Tunisia in June 2010. The latex was
held in ice during the period of collection. The identifi-
cation of this variety was established by Prof. Massoud
Mars, professor of arboriculture at the high school of hor-
ticulture of Chott Meriam Sousse, Tunisia, Department
of Agriculture and Arboriculture, and the code collection
of the tree is JR1.
Plant extract
The fresh latex was macerated in 1 L of hexane (Merck,
Germany) for 10 days then filtered and evaporated under
reduced pressure to give a gummy extract.
GC-MS analysis
The analysis of Jrani caprifig latex hexane extract was
performed on a GC-MS HP model 1909S-433 inert MSD
(Agilent Technologies, J&W Scientific Products, Palo
Alto, CA, USA), equipped with an Agilent Technologies
capillary DB-5MS column (30 m in length; 0.25 mm i.d.;
0.25 mm film thickness), and coupled to a mass selec-
tive detector (MSD1909S-433, ionization voltage 70 eV;
all Agilent, Santa Clara, CA, USA). The carrier gas was
He and was used at 1 mL/min flow rate. The oven tem-
perature program was as follows: 2 min at 150°C ramped
from 150°C to 240°C at 10°C/min and 1 min at 240°C then
ramped from 240°C to 280°C at 5°C/min and 15 min at
280°C. The chromatograph was equipped with a split/
splitless injector used in the split mode. The split ratio was
1:100. Bis-(trimethylsilyl)-acetamide (BSTFA) (100 mL)
was added to 100 mL of extract. The control of the GC-MS
system and the data peak processing were carried out by
means of MSDCHEM software. Identification of compo-
nents was assigned by matching their mass spectra with
Wiley and NIST library data and standards of the main
components.
Microorganisms
A collection of 10 test microorganisms including Gram-
positive and Gram-negative bacterial strains was used.
The groups included five organisms of American Type
Culture Collection (ATCC): Pseudomonas aeruginosa
ATCC 27950, Pseudomonas aeruginosa ATCC 2783,
 Pharmaceutical Biology
Staphylococcus aureus ATCC 25923, Escherichia coli
ATCC 25922, Enterococcus faecalis ATCC 29212, and five
clinical organisms were obtained from the laboratory of
microbiology, Faculty of Pharmacy, Monastir, Tunisia:
Staphylococcus epidermidis, Staphylococcus saprophyti-
cus, Staphylococcus aureus, Enterobacter cloacae, and
Escherichia coli. All the test cultures were stored at +4°C
on Muller-Hinton Agar (MH) (Biorad), subcultured every
2 weeks, and checked for purity.
Antimicrobial activity
Disc diffusion assay
The conventional disc diffusion method (Murray
et  al., 1995) was employed for the initial assessment
of antibacterial potential of the extract. The dried Jrani
(caprifig) extract was dissolved in dimethyl-sulfoxide
(DMSO; Merck) and then in sterile water, to reach a
final concentration of 20 mg/mL, and sterilized by fil-
tration by 0.45 µm Millipore filters. The media used was
Muller-Hinton Agar (Biorad). The discs (6 mm in diam-
eter) were impregnated with 10 µL of the extract (200
µg/disc) at a concentration of 20 mg/mL and placed on
the inoculated Agar (for the preparation of the inocula,
colonies of bacteria were suspended in Mueller-Hinton
(MH) Broth (Biorad); the suspensions contained 108
CFU/mL of bacteria). Negative controls were pre-
pared using the DMSO solvent employed to dissolve
the latex extract. Oxacillin (5 µg/disc; Gibco), tetra-
cyclin (30 µg/disc; Gibco), and erythromycin (15 µg/
disc; Gibco) served as positive reference standards
to determine the sensitivity of bacterial stains tested.
The inoculated plates were incubated at 37°C for 24 h.
The growth inhibition was assessed as the diameter (in
mm) of the zone of inhibited microbial growth. Each
assay in this experiment was performed in triplicate.
Micro-dilution assay
The minimal inhibitory concentration (MIC) values were
also studied for the microorganisms that were deter-
mined as sensitive to the extract in disc diffusion assay.
The inocula were prepared in broth cultures, and suspen-
sions were adjusted to 0.5 Mc Farland standard turbid-
ity. Extract dissolved in 10% DMSO was first diluted to
the highest concentration (2.5 mg/mL) to be tested, and
then serial two-fold dilutions were made in a concen-
tration range from 2.5 to 0.0195 mg/mL in 10 mL sterile
test tubes containing sterile water. The final concentra-
tion of DMSO in tubes was 0.78%. The MIC values of the
extract against bacterial strains were determined based
on a micro-well dilution method according to NCCLS
[National Committee for Clinical Laboratory Standards
(NCCLS), 2008] as described with slight modifications as
follows.
The 96-well plates were prepared by dispensing into
each well 95 µL of MH Broth and 5 µL of the inocula.
Extract (100 µL) initially prepared at a concentra-
tion of 2.5 mg/mL was added into the first well. Then,
100 µL from its serial dilution was transferred into six

consecutive wells. The last well, containing 195 µL of MH
Broth without compound and 5 µL of the inoculum on
each strip, was used as negative control. The final volume
in each well was 200 µL. Tetracycline (Gibco®) at the
concentration range of 400–0.39 µg/mL was prepared
in sterile water and used as standard drug for positive
control, and the DMSO was maintained as negative con-
trol. The plates were incubated at 37°C for 24 h, and the
MIC was determined from the lowest concentration of
the compound to inhibit the growth of microorganisms.
Inhibition of proliferation was assessed by optical den-
sity measurements (625 nm). These experiments were
replicated three times.
Results
The average percentage of individual compounds of
Jrani caprifig (Ficus carica) latex extract is presented in
Antibacterial activity of caprifig latex extract  409
Table 1. GC-MS analysis resulted in the identification of
36 ­compounds representing 96.12% of the total extract.
Among the identified compounds, three were sesquit-
erpens (2.83%), two were triterpens (0.46%), two were
oxygenated triterpens (0.74%), only one monoterpene
[bornanone-3. (1.04)], 14 were coumarins (90.56%) and
six were alcans (0.34%). Furthermore, the most abundant
compounds (>8%) of extract were lanosta-8 (13.17%), urs-
12-en-24-oic acid (21.52%), aristolone (15.63%), olean-12-
en-3-ol, acetate (23.47%), and maragenin I acetate (8.78%).
Table  2 represents the chemical formula and molecular
weight of some major components of the extract.
Figure 1 shows the GS-MS chromatogram of ana-
lyzed hexane extract of latex. Antimicrobial activity of
extract was tested against five ATCC cultures and five
clinical bacterial isolates. Table 3 shows diameters of
inhibition zones of latex extracts and Table 4 shows the
MIC values.

Table 1.  Chemical composition of hexane extract obtained by GC-MS.

Retention time (min) Area (%) Constituent Wiley library reference no. CAS no. Qual
4.70 0.06 Germacrene-D 121792 023986-74-5 98
5.04 0.01 Delta-Cadinene 121465 000483-76-1 97
7.03 0.33 2, 6, 10-Dodecatrien-1-ol 148283 004602-84-0 83
8.88 0.05 Dodecan-2-on 93122 006175-49-1 72
9.09 0.38 Hexadecanoic acid 213894 000112-39-0 98
10.73 0.33 Cis-Linoleic acid methyl ester 243137 000112-63-0 99
11.69 0.08 Pentadecane (CAS) 134011 000629-62-9 96
11.88 0.04 1, E-8, Z-10-Hexadecatriene 145418 080625-33-8 87
12.76 0.03 Tricosane (CAS) 275685 000638-67-5 96
13.90 0.13 Triacontane 34922 000638-68-6 91
14.51 0.02 8-Dimethoxynaphthalene 120716 105372-17-6 90
15.76 0.51 bis (2-Ethylhexyl) phthalate 326908 000117-81-7 91
16.30 0.05 Hexacosane 311168 000630-01-3 94
16.72 0.06 7-Pentadecyne 128099 022089-89-0 64
17.55 0.13 Heptacosane 32679 000593-49-7 95
18.79 0.10 Octacosane (CAS) 329269 000630-02-4 96
19.23 0.33 Squalene 337959 007683-64-9 93
20.05 0.13 Nanocosane (CAS) 337002 000630-03-5 97
20.61 0.37 Oxirane 345735 007200-26-2 89
21.73 0.29 1-Ethyl-3-acetyl-5 182541 112482-88-9 86
22.51 0.72 4-Methoxycarbonyl-2, 6-diphenylpyridine 237065 069209-39-8 74
22.98 1.04 Bornanone-3 49880 013854-85-8 70
23.38 0.19 1-Ethyl-3-acetyl-5 182541 112482-88-9 78
23.54 0.15 1, 6, 10, 14, 18, 22-Tetracosahexaen-3-ol 345737 054159-46-5 64
23.67 1.02 [3.2] Metacyclophane-10-ene 145464 121733-15-1 91
24.79 0.77 5-HYDROXY-6 236998 063955-63-5 78
27.96 0.98 9, 19-Cyclolanost-24-en-3-ol 345634 000469-38-5 93
28.96 0.94 beta-Amyrin 345611 000559-70-6 83
29.47 13.17 Lanosta-8 360770 002671-68-3 96
30.20 0.24 6-aza-B-Homo-5αcholestane 345497 066233-39-4 92
30.25 0.37 Lupeol 345599 000545-47-1 87
32.33 21.52 Urs-12-en-24-oic acid 360707 020475-86-9 91
32.75 15.63 Aristolone 141923 006831-17-0 86
32.95 23.47 Olean-12-en-3-ol, acetate 360750 001616-93-9 93
33.38 8.78 Maragenin I acetate 360711 071545-16-9 89
33.62 2.32 α-Amyrenyl acetate 360746 000863-76-3 64
Total terpene: 4.81%; Total coumarin: 90.56%; Qual, Quality.

© 2012 Informa Healthcare USA, Inc.

410  H. Lazreg-Aref et al.

Table 2.  Chemical formula and molecular weight of major component of Jrani hexane latex extract.
Name CAS Molecular formula Molecular weight
Aristolone 006831-17-0 218.33908

Lanosta-8 002671-68-3 468.76368

Olean-12-en-3-ol, acetate 001616-93-9 468. 76368

Urs-12-en-24-oic acid 020475-86-9 468.36

alpha.-Amyrenyl acetate 000863-76-3 468.76368

Bornanone-3 013854-85-8 152.23644

The highest inhibition zones recorded were 28, 28, and
26 mm, against S. saprophyticus, S. aureus ATCC25923,
and S. epidermidis, respectively, whereas the lowest
zones observed were 14, 14, and 15 mm against P. aerugi-
nosa ATCC27950, P. aeruginosa ATCC2783, and E. faeca-
lis ATCC29212, respectively.
The MIC values ranged from 19 to 312 µg/mL indi-
cated that the extract was an effective antimicrobial
agent. The antibacterial activity of bioactive compound
produced by hexane extract is comparable with tetra-
cycline as standard antibiotic. Interestingly, the hexane
extract from caprifig latex had MIC values of 19 µg/mL
against S. aureus ATCC 25923 and 39 µg/mL against
S. epidermidis, which was approximately two and three
times lower than that of tetracycline (MIC 25 and 100 µg/
mL), respectively (Table 4). This extract could be good
candidates for further studies of its antibacterial bioac-
tive compounds.
Discussion
The phytochemical analysis reveals that the aqueous
extract of ripe dried fruit of F. carica contains alka-
loids, flavonoids, coumarins, saponins, and terpenes
(Teixeira et  al., 2006; Vaya & Mahmood, 2006). Some
phenolic compounds with reported pharmacological
properties have already been isolated from fig leaves,
namely, furanocoumarins like psoralen and bergapten;
flavoloids like rutin, quercetin, and luteolin; pheno-
lic acids like ferrulic acid; and also phytosterols like
toraxasterol (Ross & Kasum, 2002; Vaya & Mahmood,
2006). Coumarins constitute an important class of

 Pharmaceutical Biology
 Antibacterial activity of caprifig latex extract  411

Figure 1.  GS-MS chromatogram of analyzed hexane extract of Jrani caprifig latex.

Table 3.  Antimicrobial activity of the extract in Agar diffusion assay.

Growth inhibition zone diametera (mm)
Hexane Erythromycin Oxacillin Tetracycline DMSO
Strains extract (15 µg/disc) (5 µg/disc) (30 UI) (10%)
Pseudomonas aeruginosa ATCC 27950 14 – – 9 –
Pseudomonas aeruginosa ATCC 2783 14 – – 14 –
Staphylococcus aureus ATCC 25923 28 22 28 22 –
Escherichia coli ATCC 25922 22 – – 24 –
Enterococcus faecalis ATCC 29212 15 16 10 22 –
Staphylococcus epidermidis 26 – – – –
Staphylococcus saprophyticus 28 21 27 32 –
Staphylococcus aureus 18 24 24 34 –
Enterobacter cloacae 19 – – 21 –
Escherichia coli 20 – – 16 –
–, no activity.
a
Inhibition zones including diameter of the paper disc (6 mm).

Table 4.  Minimum inhibitory concentration (µg/mL) of the

2000). Free 6-OH in coumarin nucleus has been found
extract. to be important for antifungal activity, the free hydroxyl
MIC (µg/mL) group at position 7 is important for antibacterial activity
Hexane (Sardari et al., 1999). Interestingly, coumarins have also
Strains extract Tetracycline inhibitory effect on DNA gyrase which may be linked to
Pseudomonas aeruginosa ATCC 27950 312 100 the anti-HIV (human immunodeficiency virus) activity
Pseudomonas aeruginosa ATCC 2783 312 100 (Matern et al., 1999).
Staphylococcus aureus ATCC 25923   19   25 In the present study, we describe the antibacterial
Escherichia coli ATCC 25922   39   10 activity for the first time of caprifig (F. carica) latex
Enterococcus faecalis ATCC 29212 312   25 extract which showed a strong activity against S. sap-
Staphylococcus epidermidis   39 100 rophyticus, S. aureus, E. coli, S. epidermidis, E. cloacae,
Staphylococcus saprophyticus   19    1 E. faecalis, and P. aeruginosa (MIC, 19 to 312 µg/mL)
Staphylococcus aureus   78    1 that can be due to the presence of coumarins with
Enterobacter cloacae   78   25 90.56% of the total area.
Escherichia coli   78   10

phytochemicals. Antimicrobial activities of the hexane

extract of latex containing mostly coumarins were eval-
uated. There are reports on efficacies of pure coumarins
against Gram-positive and Gram-negative bacteria as
well as fungi (Kayser & Kolodziej, 1997; Bisignano et al.,
© 2012 Informa Healthcare USA, Inc.
Conclusion
Based on the results of this study, it can be concluded that
caprifig latex is an agent which is used as folklore medi-
cine and could be a source for finding new antibacterial
agents in order to treat and control infections.
412  H. Lazreg-Aref et al.
Acknowledgments
The authors are grateful to Prof. Ben Ouada Hafed
(Directeur de l’Institut Supérieur des Sciences Appliquées
et de Technologie de Mahdia) and Prof. Rachid Chamli
(Laboratoire de Pharmacognosie et phytothérapie,
Faculté de pharmacie 5000 Monastir Tunisie) for provid-
ing facilities for this work.
Declaration of interest
The authors report no conflicts of interest. The authors alone
are responsible for the content and writing of the paper.
References
Arias ME, Gomez JD, Cudmani NM, Vattuone MA, Isla MI. (2004).
Antibacterial activity of ethanolic and aqueous extracts of Acacia
aroma Gill. ex Hook et Arn. Life Sci, 75, 191–202.
Bhavnani SM, Ballow CH. (2000). New agents for Gram-positive
bacteria. Curr Opin Microbiol, 3, 528–534.
Bisignano G, Sanogo R, Marino A, Aquino R, D’Angelo V, Germanò
MP, De Pasquale R, Pizza C. (2000). Antimicrobial activity of
Mitracarpus scaber extract and isolated constituents. Lett Appl
Microbiol, 30, 105–108.
Braun L, Cohen M. (2007). Herbs and Natural Supplements: An Evidence-
based Guide, second ed. Elsevier, Australia, ISBN: 072953796X, p. 791.
Kayser O, Kolodziej H. (1997). Antibacterial activity of extracts and
constituents of Pelargonium sidoides and Pelargonium reniforme.
Planta Med, 63, 508–510.
 Pharmaceutical Biology
Kinghorn AD. (1992). Plants as sources of medicinally and
pharmaceutically important compounds. In: Nigg HN, Seigler
D, (eds.), Phytochemical Resources for Medicine and Agriculture.
New York. Plenum Press, pp. 75–95.
Lazreg Aref H, Bel Hadj Salah K, Chaumont JP, Fekih A, Aouni M, Said
K. (2010). In vitro antimicrobial activity of four Ficus carica latex
fractions against resistant human pathogens. Pak J Pharm Sci, 23,
53–58.
Matern U, Lüer P, Kreusch D. (1999). Biosynthesis of coumarins.
In: Barton D, Nakanishi K, Meth-Cohn O, Sankawa U (eds.):
Comprehensive Natural Products Chemistry, Vol. 1, Polyketides
and Other Secondary Metabolites Including Fatty Acids and Their
Derivatives. Elsevier Science Ltd., Oxford, UK, pp. 623–637.
Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolke RH. (1995).
Manuel of Clinical Microbiology. 6th Edn. Washington. DC, ASM,
p. 300.
NCCLS. (2008). Performance Standards for Antimicrobial Susceptibility
Testing; Ninth Informational Supplement. NCCLS document
M100-S9. National Committee for Clinical Laboratory Standards,
Wayne, PA, pp. 120–126.
Ross JA, Kasum CM. (2002). Dietary flavonoids: Bioavailability,
metabolic effects, and safety. Annu Rev Nutr, 22, 19–34.
Sardari S, Mori Y, Horita K, Micetich RG, Nishibe S,
Daneshtalab M. (1999). Synthesis and antifungal activity of
coumarins and angular furanocoumarins. Bioorg Med Chem, 7,
1933–1940.
Teixeira DM, Patão RF, Coelho AV, da Costa CT. (2006). Comparison
between sample disruption methods and solid-liquid extraction
(SLE) to extract phenolic compounds from Ficus carica leaves.
J Chromatogr a, 1103, 22–28.
Vaya J, Mahmood S. (2006). Flavonoid content in leaf extracts of the
fig (Ficus carica L.), carob (Ceratonia siliqua L.) and pistachio
(Pistacia lentiscus L.). Biofactors, 28, 169–175.