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MINI-REVIEW
Antimicrobial activity of resin acid derivatives
Sonia Savluchinske-Feio &Maria João Marcelo Curto &
Bárbara Gigante &J. Carlos Roseiro
Received: 24 March 2006 /Revised: 22 May 2006 /Accepted: 23 May 2006 / Published online: 5 August 2006
#Springer-Verlag 2006
Abstract The wide potential of resin acids as bioactive
agents gave rise to a growing effort in the search for new
applications of the natural forms and their derivatives. In
some of these compounds, the antimicrobial activity is
associated to the presence in the molecules of functional
groups such as the hydroxyl, aldehyde, and ketone or to
their cis or trans configurations. The resin acid family
covers a spectrum of antimicrobial activities against several
microorganisms, from bacteria to fungi, in which the mode
of action was studied by electron microscopy. The
morphological alterations are consistent with an unspecific
mode of action causing inhibition of the fungal growth or
damaging the fungal cells in parallel with a mechanism of
resistance based on the retention of the compound by the
lipid accumulation. The sterol composition of phytopatho-
genic fungi Botrytis cinerea and Lophodermium seditiosum
treated with methyl cis-7-oxo-deisopropyldehydroabietate
revealed the presence of ergosterol (M+ 396) and dihy-
droergosterol (M+ 398) in both cultures showing that this
compound did not interfere with the ergosterol metabolic
pathway of both fungi.
Keywords Resin acids .Antifungal .Antibacterial .
Natural products .Phytopathogenic .Dehydroabietic acid
Introduction
Conifers are subject to predation by a wide range of
herbivores and pathogens (over an uncommonly long
life span) with the most serious destruction worldwide
resulting from the infestation by tree-killing bark beetles
and their symbiotic fungal pathogens. The great success
of the conifers rests partly in the existence of complex
defense mechanisms that deter the pathogen aggression.
The principal chemical and physical defense of conifers
is made of the constitutive and inducible production of
oleoresin (often simply termed resin or pitch). The
viscous oleoresin secretion is composed of a complex
mixture of terpenoids, consisting of roughly equal parts
of volatile turpentine [mostly monoterpene (C
10
) olefins
with some sesquiterpenes (C
15
), including oxygenated
types] and rosin [diterpene (C
20
) resin acids] (Croteau and
Johnson 1985; Jonnessen and Stern 1978; Mutton 1962;
Norin 1972). Rosin, the distillation residue of pine resins, is
the main source of diterpene resin acids such as abietic (1),
levopimaric (2), palustric (3), neoabietic (4), and dehydroa-
bietic acid (5) along with other non-abietane compounds
(Carman and Marty 1970).
Accumulated resin is released upon tissue injury and/
or produced locally at the site of infestation, encasing
and destroying the beetle and associated fungal pathogen
(s) being expelled through the point of entry. This
process results not only in killing the attackers and
flushing the wound site but also moving the oleoresin to
the trunk surface where the turpentine evaporates to
permit the resin acids to form a physical barrier that
seals the wound (Croteau and Johnson 1985; Gijzen et al.
1993).
Conifer resins have long been studied for their industrial
importance and role in defense against herbivores and
Appl Microbiol Biotechnol (2006) 72:430–436
DOI 10.1007/s00253-006-0517-0
S. Savluchinske-Feio (*):M. J. M. Curto :B. Gigante
Departamento de Tecnologia de Indústrias Químicas,
Instituto Nacional de Engenharia, Tecnologia e Inovação,
Azinhaga dos Lameiros, 22,
1648-038 Lisboa, Portugal
e-mail: sonia.feio@ineti.pt
J. C. Roseiro
Departamento de Biotecnologia,
Instituto Nacional de Engenharia, Tecnologia e Inovação,
Azinhaga dos Lameiros, 22,
1648-038 Lisboa, Portugal
pathogens (Bohlmann and Croteau 1999; Phillips and
Croteau 1999; Trapp and Croteau 2001).
On the other hand, pine rosin, as a whole or its isolated
constituents, is used since immemorial times as a medicine
for infected wounds, boils, and pyodermas, as well as
rubefacients and vesicants in poultices and creams in
veterinary practice (San Feliciano et al. 1993). These
authors also reported the antiulcer, cardiovascular, allergen-
ic, and antiallergenic properties of these compounds.
More recently, it was shown that some diterpene resin
acids such as abietic acid (1) and isopimaric acid (6) have
growth-inhibiting effects on Ophiostoma ips, a conifer
pathogenic fungus (Kopper et al. 2005).
According to Soderberg et al. (1991), compounds 1and
5are responsible for the antibacterial activity against Gram-
positive bacteria. To increase the bioactivity of those acids,
some authors (Gigante et al. 2002) have investigated the
impact of their chemical modification on biological activity.
So, in this study, we review the recent work targeting
natural and higher nonnatural diterpenoids toward biological
activity.
1
CO2H
1
2
3
45
6
7
8
9
11
10
12
14
13
16
17
15
20
18
19
2
CO2H
3
CO2H
4
CO2H
5
CO2H
6
CO2H
7 R = O
8 R = OH, H
CO2H
R
9
OH
CO2H
10
OH
11
OH
HO2C
R
OH
HO
12 R = CO2CH3
13 R = CO2H
14 R = CH2OH
15 R = CH3
HO2C
OH
HO
16
Scheme 1 Resin acids
Appl Microbiol Biotechnol (2006) 72:430–436 431
Antimicrobial activity
In Table 1, a list with compounds and respective biological
activities is presented.
Naturally occurring diterpenoids with a dehydroabietane
skeleton (ketones, alcohols, and phenols) are often found
and isolated from plants and have been reported for their
bioactivity (Ulubelen et al. 2000; Mensah et al. 2000).
According to Smith et al. (2005), isopimaric acid (6)
extracted from immature cones of Pinus nigra inhibited the
growth of multidrug-resistant and methicillin-resistant
Staphylococcus aureus (MRSA) which are becoming
increasingly resistant to antibiotics.
Earlier, other studies were carried out with resin acids.
Oxidized resin acids such as 7-oxo-dehydroabietic acid (7),
7-hydroxy-dehydroabietic acid (8), and 13-hydroxy-podo-
carpa-8,11,13-trien-18-oic acid (9) have been shown to be
highly fungistatic and to inhibit in vitro both spore
germination and mycelial growth of Dothistroma pini,a
leaf pathogen of young Pinus radiata D. Don (Franich et al.
1983). Other studies on the antifungal activity of oxidized
resin acids (Henricks et al. 1979) showed that (7)
completely inhibited fungal growth, exhibiting significantly
greater activity than the common resin acids abietic (1),
levopimaric (2), palustric (3), and dehydroabietic acids (5).
Furthermore, (7) was found to be the major oxidized resin
CO2CH3
N
NR
5
R6
CO2CH3
R3
N
N
R4
H
17a R3 = R4 = H
17b R3 = Br; R4 = H
18a R3 = H; R4 = CH3
18b R3 = Br; R4 = CH3
19a R3 = H; R4 = CF3
19b R3 = Br; R4 = CF3
20 R3 = H; R4 = NHCO2CH3
21 R5 = R6 = CH3
22 R5 = R6 = C6H5
23 R5, R6
= C4H8
2´ 2´
3´
CO2CH3
NH
CO2CH3
Br
H
N
24
25
26 R = CO2CH3, R1= H2
27 R = CH2OH, R1= H2
28 R = CHO, R1= H2
29 R = CO2CH3, R1= O
R
R1
30 R = CO2H
31 R = CO2CH3
32 R = CH2OH
R
33
CO2CH3
O
34 R = CO2H, R1= H
35 R = CO2CH3, R1= H
36 R = CH2OH, R1= OH
37 R = CH2OH,
,
R1= H
38 R = CHO R1= H
R
39 R = CO2CH3
40 R = CHO
R
41
CH2OH
O
OH
H
R1
Scheme 1 (continued)
432 Appl Microbiol Biotechnol (2006) 72:430–436
acid in the reaction zone of spruce sapwood attacked by
Fomes annosus, a fungus responsible for one of the most
important diseases of conifers in the north temperate zone
of the world.
Fukui et al. (1978) extracted dehydroabietic acid (5),
ferruginol (10), and pisiferic acid (11) from the leaves and
twigs of Chamaecyparis pisifera and detected antibacterial
activity of these compounds. The lipid nature of (11)was
correlated with its antimicrobial activity; a greater lipophilicity
leads to a higher activity against Gram-positive bacteria,
whereas a lower degree of lipophilicity increases the activity
against Gram-negative bacteria (San Feliciano et al. 1993).
Other authors (Moujir and Gutierrez-Navajo 1996)
studied the structure–antimicrobial activity relationship of
abietane diterpenes extracted from Salvia mellifera, and
verified that the catechol group was essential for the
antimicrobial activity. According to Kobayashi et al.
(1988), the presence of carboxyl and hydroxyl functions
Table 1 Compounds and biological activities referred in the text
Compounds Biological activities Authors
Oxidized resin acids Antifungal Borglin (1947)
Dehydroabietylguanidines Antibacterial Muftic (1968,1970)
Pisiferic acid Antibacterial Fukui et al. (1978)
Ferruginol
Dehydroabietic acid
Oxidized dehydroabietic acid derivatives Antifungal Henricks et al. (1979)
Oxidized resin acids Antifungal Franich et al. (1983)
Oxidized pisiferic acid derivatives Antibacterial Kobayashi et al. (1988)
Abietane diterpenes Antibacterial Moujir and Gutierrez-Navajo (1996)
Dehydroabietic acid derivatives Antibacterial Savluchinske Feio et al. (1999)
Antifungal
Dehydroabietic acid Antifungal Vargas et al. (1999)
Oxidized dehydroabietic acid derivatives Antifungal Mensah et al. (2000)
Oxidized dehydroabietic acid derivatives Antibacterial Ulubelen et al. (2000)
Abietic acid derivatives Antiviral, anticancer Gigante et al. (2003)
Dehydroabietic acid derivatives Antiviral Fonseca et al. (2004)
Isopimaric acid Antibacterial Smith et al. (2005)
Table 2 Bioactivity of dehydroabietic acid derivatives against filamentous fungus (FF), yeasts, and bacteria
a,b,c
FF
d
Yeasts
e,f
Bacteria
e,g
RI (%) (μmol/ml) (μmol/ml
Compound T. mentagrophytes C. albicans 110 C. albicans 407 C. kruzei C. parapsilosis S. aureus
28 44 >70 >70 >70 >70 5.6
32 91 na na na na 6.5
36 100 na na na na 1.5
37 90 na na na na 6.5
38 n.t. 26.9 26.9 53.8 26.9 6.6
39 88 na na na na 27.7
40 39 12.7 12.7 25.4 6.3 3.9
41 100 na na na na 3.5
Amphotericin B 100 n.t. n.t. n.t. n.t. n.t.
5-Fluorocytosine n.t. 0.8 0.8 0.8 0.8 n.t.
Rifampicin n.t. n.t. n.t. n.t. n.t. 0.001
a
Antimicrobial activity of resin acid derivatives (Gigante et al. 2002)
b
No inhibition of growth was observed at 40 μmol/ml for any of the tested compounds against the Gram-negative bacteria, the P. aeruginosa and
S. marcescens, also tested
c
n.t. Not tested
d
Acetone as control
e
Values of MIC (μmol/ml)
f
na Not active below 80 μmol/ml
g
na Not active below 40 μmol/ml
Appl Microbiol Biotechnol (2006) 72:430–436 433
is the most important factor in the activity of pisiferic acid
(11) derivatives against Gram-positive bacteria.
Compound 5also showed antifungal activity against
phytopathogenic species and food contaminants, namely,
Geotrichum candidum (Vargas et al. 1999).
In recent years, many pathogens became a serious threat,
both in human and animal health, due to their resistance to
the known chemical control agents. This has occasioned a
growing effort in the search for new bioactive agents,
including natural products and their derivatives (Gigante et
al. 2002).
Having in account that the isolation of natural products
from plants or animals is a useful way for the discovery of
useful drugs, and that hemisynthesis or derivatization of
natural products can be a faster and economical approach in
the search for biologically active compounds, some authors
have been using dehydroabietic acid (5), the main compo-
nent of dismutated rosin, as a starting material for the
synthesis of industrially and physiologically important
products. Bactericidal and fungicidal activities of dehy-
droabietic acid abietylamines were long described in the
literature (Borglin 1947). The antibacterial activity of
dehydroabietylguanidine acetate against Gram-positive
and Gram-negative bacteria has been reported by Muftic
(1968,1970), who tested various abietyl derivatives,
including deisopropylated analogs. Muftic (1970) observed
that the isopropyl group was generally not essential for
antibacterial activity against Gram-positive and Gram-
negative bacteria, yet it was necessary for the inhibition of
mycobacteria.
Gigante et al. (2003) described the biological activities,
namely, antifungal, antitumoral, antimutagenic, antiviral,
and antiproliferative of catechols (12–15) prepared from 5
by a short and good yielding chemical process. Their
properties were compared with those of carnosic acid (16),
a naturally occurring catechol with an abietane skeleton and
known to possess potent antioxidant activity, as well as
anticancer and antiviral properties. From all those synthetic
catechols tested, compound 2showed the best activities
stronger than 16.
More recently, Fonseca et al. (2004) had also
explored the ability of dehydroabietic acid (5) derivatives
to act as synthetic precursors of heteroaromatic com-
pounds with potential biological activities, describing the
synthesis of a plethora of new heterocycles, such as
benzimidazoles (17–20), quinoxalines (21–23), and
indoles (24 and 25). Biological evaluation showed that
some of the compounds (19a,17b,23,and25)were
found to inhibit both varicella-zoster virus (VZV) and
cytomegalovirus (CMV) replication at a concentration
ca. 5- to 10-fold lower than the cytotoxic concentration
(MCC or CC50), when tested in human embryonic lung
cells. Those compounds can therefore be accredited with
some specificity in their anti-VZV and anti-CMV action.
The potencies of 17a,19a,17b,18b,23,24,and25
as anti-VZV agents were comparable to that of acyclovir,
while the potencies of 17a,19a,17b,23,and25 as anti-
CMV agents were comparable to that of ganciclovir.
Structure–activity relationship
The structure–activity relationship of some resin acid
derivatives (26,29,31,33,35,and39) was also studied,
and several exhibited antifungal and antibacterial activity
associated to the presence of functional groups (Savluchinske
Feio et al. 1999).
The oxidized test compounds (33 and 39)trans/cis
isomers were the most active, inhibiting the growth of
several filamentous fungi (Actinomucor harzii,Clado-
sporium cucumerinum,Mucor racemosus,Rhizopus
arrhizus,Rhizopus stolonifer,andSyncephalastrum race-
Fig. 1 Longitudinal and transverse section of young hyphae of untreat-
ed culture of Botrytis cinerea.NNucleus. Bar 1μm (Savluchinske Feio
et al. 2002)
Fig. 2 Longitudinal and transverse section of young hyphae from a
methyl cis-7-oxo-deisopropyldehydroabietate treated culture of Botry-
tis cinerea.MCell membrane, Llipid. Bar 1μm (Savluchinske Feio et
al. 2002)
434 Appl Microbiol Biotechnol (2006) 72:430–436
mosum) and Gram-positive bacterium, the S. aureus,but
did not inhibit the growth of Gram-negative bacteria, the
Escherichia coli and Klebsiella pneumoniae. However, in
combination, those two compounds (33 and 39) inhibited
the growth of those microorganisms, suggesting a
synergistic effect. The presence of the isopropyl group
(29) seemed to decrease the antibacterial activity of these
compounds. These findings are not in accordance with
those obtained by other authors (Fukui et al. 1978)who
attributed the antimicrobial activity of dehydroabietic acid
derivatives to the isopropyl group, but are in agreement
with the findings Savluchinske Feio et al. (1997)who
verified the antimicrobial activity of the deisopropylated
compounds 33 and 39.
Later on, in an enlarged study (Gigante et al. 2002), 15
resin acid derivatives (26–32 and 34–41) having different
A/B ring junction (natural, steroidal, or antipodal) with
carboxyl, ester, aldehyde, or alcohol functions at C-4 were
tested against some human pathogenic microorganisms,
Trichophyton mentagrophytes, several yeasts species, and
S. aureus. From the results showed, (Table 2), it is seen that
in general, the presence of the hydroxyl, the aldehyde, and
the ketone functions in the molecule at cis or trans
configurations was essential for the expression of antimi-
crobial activity. The aldehyde function showed to be
important for the antiyeast activity of these kinds of
compounds; 38 and 40 inhibited the growth of Candida
albicans 110, C. albicans 407, Candida kruzei,and
Candida parapsilosis. On the other side, the presence of a
hydroxyl in the aromatic ring (36) increased the antibacte-
rial activity against S. aureus as well as the antifungal
activity against T. mentagrophytes. The derivatives con-
taining the hydroxyl group (27,32,36,37, and 41) are
more active than the other oxidized compounds (28,29,30,
34,38, and 39), particularly on the growth inhibition of the
filamentous fungus. Compounds without the isopropyl
group (30–32 and 34–41) exhibited a stronger antifungal
activity than those containing this group in their structure
(27 and 28). No activity against Gram-negative bacteria, the
Pseudomonas aeruginosa and Serratia marcescens, was
observed.
Compounds 26,27,30,31,34, and 35 showed none or
weak activity toward the microorganisms tested.
Studies by transmission electron microscopy (TEM)
and mechanism of action
Some studies by TEM were performed on the phytopath-
ogenic fungi, the Botrytis cinerea and Lophodermium
seditiosum (Savluchinske Feio et al. 2002).
TEM photographs of B. cinerea and L. seditiosum
hyphae, on untreated cultures, showed a regular-shape
plasma membrane and a well-formed nuclear envelope
limiting the nucleus as well as the presence of normal
ultrastructures. No lipid accumulation in the hyphae apical
zone was observed. The treated hyphae with methyl cis-7-
oxo-deisopropyldehydroabietate (39) showed the presence of
several autophagic-like vacuoles, morphological alterations
in the lomasome, and lipid accumulation on the apical zone.
Some observations were also made on the spore germi-
nation of B. cinerea. Untreated cultures of young hyphae
showed a regular-shape plasma membrane and a well-formed
nucleus (Fig. 1). Cultures treated with 39 (Fig. 2)revealed
the presence of strongly stained lipid accumulations retained
by the vacuoles at the periphery of young hyphae.
The sterol composition of treated and untreated cultures
of B. cinerea and L. seditiosum determined by gas-
chromatography–mass-spectrometry (GC–MS) revealed
the presence of molecular ions and fragmentation patterns
characteristics of ergosterol (M+ 396) and of dihydroergos-
terol (M+ 398) in both cultures. Compound 39 does not
seem to interfere with the ergosterol metabolic pathway of
both fungi. The morphological alterations are consistent
with an unspecific mode of action for methyl cis-7-oxo-
deisopropyldehydroabietate (39) causing inhibition of the
fungal growth or damaging the fungal cells. TEM observa-
tions suggest a mechanism of resistance based on the
retention of the test compound by the lipid accumulation
(Savluchinske Feio et al. 2002).
The antifungal and antibacterial activities of resin acid
derivatives indicate that these compounds have future
applications in pharmacology and phyto-epidemiology
preventing infections in forests.
The structural effects observed in this type of com-
pounds is an important basis to select bioactive products to
obtain a higher activity with less environmental impact.
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