Ent-trachyloban-19-oic acid isolated from Iostephane heterophylla as a promising antibacterial agent against Streptococcus mutans biofilms

Article abstract of DOI:10.1016/j.fitote.2011.12.022

From the roots of Iostephane heterophylla, six known compounds, namely, ent-trachyloban-19-oic acid (1), the mixture of ent-kaur-16-en-19-oic acid (2) and ent-beyer-15-en-19-oic acid (3), xanthorrhizol (4), 16α-hydroxy-ent- kaurane (5) and 16α-hydroxy-ent

Full text of DOI:10.1016/j.fitote.2011.12.022

Fitoterapia 83 (2012) 527–531
Contents lists available at SciVerse ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
Ent-trachyloban-19-oic acid isolated from Iostephane heterophylla as a
promising antibacterial agent against Streptococcus mutans biofilms
Dulce M. Hernández a,1, Gloria Díaz-Ruiz b, Blanca E. Rivero-Cruz a, Robert A. Bye c,
María Isabel Aguilar a, J. Fausto Rivero-Cruz a,
⁎
a
Departamento de Farmacia, Universidad Nacional Autónoma de México, Ciudad Universitaria, 04510 D.F., Mexico
Departamento de Alimentos y Biotecnología, Facultad de Química, Universidad Nacional Autónoma de México, Ciudad Universitaria, 04510 D.F., Mexico
Instituto de Biología, Universidad Nacional Autónoma de México, Ciudad Universitaria, 04510 D.F., Mexico
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 October 2011
Accepted in revised form 18 December 2011
Available online 9 January 2012
From the roots of Iostephane heterophylla, six known compounds, namely, ent-trachyloban-19-
oic acid (1), the mixture of ent-kaur-16-en-19-oic acid (2) and ent-beyer-15-en-19-oic acid
(3), xanthorrhizol (4), 16α-hydroxy-ent-kaurane (5) and 16α-hydroxy-ent-kaur-11-en-19-
oic acid (6) were isolated using a bioassay-guided fractionation method. The known com-
pounds (1–6) were identified by comparison of their spectroscopic data with reported values
in the literature. In an attempt to increase the resultant antimicrobial activity of 1 and 4, a se-
ries of reactions was performed on ent-trachyloban-19-oic acid (1) and xanthorrhizol (4), to
obtain derivatives 1a, 1b, and 4a–4d. All the isolated compounds (1–6) and the derivatives
1a, 1b, and 4a–4d were evaluated for their antimicrobial activity against two oral pathogens,
Streptococcus mutans and Porphyromonas gingivalis associated with caries and periodontal
disease, respectively. Compounds 1, 1b, 2+3, 4 and 4d inhibited the growth of S. mutans
with concentrations ranging from 4.1 μg/mL to 70.5 μg/mL. No significant activity was found
on P. gingivalis except for 4 with an MIC of 6.8 μg/mL. The ability of 1, 1b, 2+3, 4 and 4d to in-
hibit biofilm formation by S. mutans was evaluated. It was found that 1, 1b, 4 and 4d interfered
with the establishment of S. mutans biofilms, inhibiting their development at 32.5, 125.0, 14.1
and 24.4 μg/mL, respectively.
Keywords:
Streptococcus mutans
Biofilm
Iostephane heterophylla
Diterpenes
Xanthorrhizol
Ent-trachyloban-19-oic acid
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
on the joints, and to make tea for diabetes, gastrointestinal
complaints and liver ailments [2]. Previous phytochemical in-
Iostephane heterophylla (Cav.) Benth Hemsl is a traditional
medicinal plant that grows in the pine–oak–juniper forested
mountains of western, central and southern Mexico where
is commonly known as “tecpahtli”, “bauji”, “corsoner”, “liga”,
“tlalpopolote” and “raíz del manso” [1, 2]. The roots of I. het-
erophylla are widely commercialized species in Mexico for
the treatment of bacterial infections, in fresh wounds and
sores in the form of a poultice, to alleviate rheumatism and
arthritis by rubbing an alcoholic tincture or aqueous extract
vestigation on I. heterophylla roots resulted in the isolation
and identification of several diterpenoids, bisabolenes and
coumarins [3–5]. The traditional uses of I. heterophylla sug-
gested the presence of antibacterial agents. Furthermore, a
preliminary test revealed that the CHCl3 extract prepared
from the roots of the plant showed MIC values of 77 and
105 μg/mL for Streptococcus mutans and Porphyromonas gin-
givalis, respectively. Accordingly, in our continuous efforts
to investigate Mexican plants widely used to treat oral dis-
eases [6], the aim of the present investigation was to isolate
and characterize the antibacterial principles against oral
pathogens from I. heterophylla. Following a bioassay-guided
isolation procedure, ent-trachyloban-19-oic acid (1), the
mixture of ent-kaur-16-en-19-oic acid (2) and ent-beyer-
⁎
Corresponding author. Tel.: +52 55 5622 5281; fax: +52 55 5622 5329.
E-mail addresses: joserc@unam.mx, laurents@unam.mx
(J.F. Rivero-Cruz).
1
Taken in part from her BS Thesis.
0367-326X/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.fitote.2011.12.022

528
D.M. Hernández et al. / Fitoterapia 83 (2012) 527–531
Table 1
15-en-19-oic acid (3), xanthorrhizol (4), 16α-hydroxy-ent-
kaurane (5) and 16α-hydroxy-ent-kaur-11-en-19-oic acid
(6) were isolated. The structures of the known compounds
(1–6) were determined by comparison of their spectroscopic
data with reported values in the literature [3, 7, 8].
MIC and MBC values of compounds isolated from Iostephane heterophylla
and its derivatives against Streptococcus mutans and Porphyromonas
gingivalis.
Compound
Minimum inhibitory
Minimum bactericidal
concentration (μg/mL)a
concentration (μg/mL)a
The antibacterial activity of compounds 1–6 and deriva-
tives 1a, 1b, and 4a–4d was evaluated against S. mutans and
P. gingivalis quantitatively. The compounds ent-trachyloban-
19-oic acid (1), ent-trachyloban-19-ol (1b), xanthorrhizol (4)
and 12,13-dihydroxanthorrhizol (4d) also interfered with the
establishment of S. mutans biofilms, inhibiting their develop-
ment at concentrations ranging from 14.1 to 125.0 μg/mL.
The structures of the known compounds (1–6) and the
derivatives (1a, 1b, and 4a–4d) were determined by compar-
ison of their spectroscopic data with reported values in the
literature [3, 7–11].
S. mutans
P. gingivalis
S. mutans
P. gingivalis
CHCl3 extract
77.0
37.6
64.1
105.0
154.1
187.2
57.6
>1000
125
308.0
150.4
512.8
35.6
>1000
250
720.0
432.8
697.6
70.8
>1000
1000
F-02
F-05
1
1a
1b
2+3
4
4a
4b+4c
4d
5
8.9
>1000
70.5
34.4
4.1
95.3
6.8
340.4
8.2
362.4
18.2
>1000
>1000
13.6
>1000
125
>1000
>1000
138.9
>1000
>1000
3.2
>1000
>1000
270.3
>1000
>1000
10.8
>1000
>1000
466.8
>1000
>1000
12.8
2. Experimental
6
CHXb
1.2
a
2.1. General procedures
Values represent the average obtained from
a
minimum of three
experiments.
b
CHX: chlorhexidine gluconate.
NMR were recorded on a Varian VNMRS spectrometer op-
erating at 400 MHz. EIMS was obtained in a positive mode in
a Thermo Electron DFS spectrometer. CC was performed
using silica gel (300–400 mesh). TLC was carried out by
using silica gel plates prepared with GF254 (Merck); spots
were visualized by UV light or 1% sulfuric vanillin.
NMR, 13C NMR and EIMS) data comparable to published values
[3].
2.4.2. Preparation of ent-trachyloban-19-ol (1b)
To a stirring solution of LiAlH4 (7.9 mmol) in anhydrous
THF (5 mL) at 4 °C was added the ent-trachyloban-19-oic
methyl ester (1a) (1, 1.654 mmol) in 15 mL of THF drop
wise over a period of 1 h. The solution was stirred at refluxing
temperature under N2 for 12 h. The solution was cooled to
4 °C by ice bath, and 1 mL of 1 N NaOH followed by H2O
(2 mL) was slowly added to the solution to quench the reac-
tion. The solution was stirred at 23 °C for 1 h and then filtered
to remove the solid material. The solution was concentrated.
Purification by TLC (hexanes/acetone 95:5) gave the ent-
trachyloban-19-ol (1b). This compound exhibited spectro-
scopic (1H NMR, 13C NMR and EIMS) data comparable to
published values [9–11].
2.2. Plant material
Fresh roots of I. heterophylla were purchased in Mercado
de Sonora of Mexico City. One of the authors (R. Bye), au-
thenticated the plant material (R. Bye and E. Linares 17986).
A voucher specimen is deposited in the Ethnobotanical Col-
lection in the Institute of Biology, National Autonomous Uni-
versity of Mexico, Mexico City.
2.3. Extraction and isolation
The air dried and powdered roots (2.8 kg) were extracted
with CHCl3 by maceration, for up to 1 week. The resultant ex-
tract was concentrated in vacuo at 40 °C. A portion of CHCl3-
soluble extract (313 g) was subjected to silica gel vacuum
column chromatography (VLC) and eluted with a gradient
mixture of n-hexane-EtOAc (10:0→0:10) to give twenty
pooled fractions. Fractions F-02 and F-05 were active when
tested against S. mutans and P. gingivalis (Table 1). The most
active fraction F-02 was chromatographed over a silica gel
column and eluted with n-hexane-EtOAc to yield 1 (4.6 g).
Fraction F-05, eluted with n-hexane-EtOAc (9:1), was chro-
matographed over a silica gel column, using a mixture of n-
hexane-EtOAc (9:1) as solvent system, to give a mixture of
2 and 3 (100 mg) and 4 (120.2 mg). From inactive fractions
F-08 and F-20 compounds 5 and 6 were respectively isolated.
2.4.3. Preparation of 1-O-acetylxanthorrizhol (4a)
To a stirred solution of 4 (15 mg) in pyridine (0.1 mL),
0.15 mL of acetic anhydride was added. When TLC indicated
complete consumption of starting material, the reaction solu-
tion was diluted with EtOAc (3 mL), washed with 1 N HCl
(3×3 mL), saturated NaHCO3 solution (3×4 mL), and water
(3×3 mL), then dried over anhydrous Na2SO4, filtered, and
evaporated to dryness to yield an acetyl derivative (4a,
10 mg). Compound 4a exhibited spectroscopic data (1H
NMR, 13C NMR and EIMS) comparable to published values
[7, 8].
2.4.4. Preparation of (12R/12S)-12,13-epoxy-xanthorrhizols
(4b+4c)
2.4. Preparation of derivatives
To a suspension of m-chloroperbenzoic acid (23.32 mg),
NaHCO3 (5 mg), and CHC13 (2.5 mL), a solution of xanthor-
rhizol (15.0 mg) in CHC13 (1 mL) was dropwise added and
stirred, keeping the reaction at 5 °C. After 1 h, 50% sodium
sulfite in water (2.5 mL) was added and 30 min later, water
(3×1 mL) was added. The organic layer was separated and
2.4.1. Preparation of ent-trachyloban-19-oic methyl ester (1a)
Ent-trachyloban-19-oic acid (20.0 mg) was treated with
ethereal diazomethane to yield ent-trachyloban-19-oic methyl
ester (1a, 19.5 mg). Compound 1a exhibited spectroscopic (1H

D.M. Hernández et al. / Fitoterapia 83 (2012) 527–531
529
washed with 5% aqueous sodium carbonate (3×2.5 mL). The
aqueous layers were extracted with CH2Cl2 (3×2.5 mL) and
the organic solution dried with Na2SO4. The organic residue
was chromatographed (Si Gel, using n-hexane-ethyl acetate
gradient elution system) to obtain a 1:l mixture of 4b+4c
(50% yield). Compounds 4b and 4c exhibited spectroscopic
(1H NMR, 13C NMR and EIMS) data comparable to published
values [3, 8].
5, 10, 50 and 200 μg/mL of the test compounds, the content
of each well was removed.
Biofilm formation was quantified by a crystal violet assay
[14]. Briefly, the biofilm-coated wells of microtiter plates as
described above for biofilm formation were vigorously shak-
en in order to remove all non-adherent bacteria. The remain-
ing attached bacteria were washed twice with 200 μL of
50 mM PBS (pH=7.0) and air dried for 45 min, and 200 μL
of absolute ethyl alcohol was transferred to each well, in
order to fix the adherent cells, and allowed to contact during
15 min. Then, each well washed was stained with 100 μL of
0.4% aqueous crystal violet solution for 45 min. Afterwards,
each well was washed twice with 200 μL of sterile distilled
water and immediately de-stained with 200 μL of 90% etha-
nol. After 45 min of de-staining, 100 μL of de-staining solu-
tion was transferred to a new well and the amount of the
crystal violet stain in the de-staining solution was measured
with a tunable microplate reader at 595 nm. The activity of
the tested compounds was expressed as the percentage of
the absorbance of biofilm treated compared with the control
(untreated).
2.4.5. Preparation of the 12,13-dihydroxanthorrhizol (4d)
A solution of xanthorrhizol (4) (19 mg) dissolved in ethyl
acetate (2 mL) was hydrogenated using 10% Pd/C (5 mg), as
catalyst. The reaction was monitored by TLC (petroleum
ether/ethyl acetate 85:15), and the reaction product was pu-
rified by preparative chromatography as a yellowish oil.
Compound 4d exhibited spectroscopic (1H NMR, 13C NMR
and EIMS) data comparable to published values [7].
2.5. Determination of MIC and MBC
The in vitro antibacterial activity of compounds 1–6 was
determined against S. mutans and P. gingivalis according to
the National Committee of Clinical Laboratory Standards
(NCCLS) recommended minimum inhibitory concentration
(MIC) protocol with modifications [6, 9, 10]. Briefly, 2-fold di-
lution series were made from all tested antibacterial agents
starting from 2000 μg/mL in a 96-well plate. S. mutans strain
ATCC 10499 was grown at 37 °C under aerobic conditions in
brain heart infusion (BHI) broth media (Becton Dickinson,
Sparks, MD). An aliquot of 20 μL of bacterial suspension at a
concentration of 106 colony-forming units/mL was added to
180 μL of antibacterial dilution. Chlorhexidine (0.12%) was
used as positive control and the untreated suspension as neg-
ative control. The MIC was defined as the lowest concentra-
tion of the test agent that had restricted growth to a
levelb0.05 at 660 nm after incubation at 37 °C for 16–24 h.
For the determination of MBC, an aliquot of 50 μL of all the in-
cubated test samples was subcultured on BHI agar supple-
mented with 5% of defibrinated sheep blood. MBC was
defined as the lowest concentration that allows no growth
on the agar.
3. Results and discussion
In this study, the in vitro antimicrobial activity of the
CHCl3 extract and isolated compounds from I. heterophylla
against S. mutans and P. gingivalis was investigated. S. mutans,
a Gram-positive facultative anaerobic coccus is commonly ac-
knowledged as the main bacteria responsible for the forma-
tion of the dental plaque and dental caries and P. gingivalis,
the Gram-negative anaerobic oral bacterium most commonly
associated with gum diseases [6]. In traditional Mexican
medicine the hot water extract of I. heterophylla has been
used for the treatment of oral cavity infections. In our data,
77 and 105 μg/mL of the CHCl3 extract showed antibacterial
activity against S. mutans and P. gingivalis. This result sup-
ports the scientific rationale that native inhabitants used
the extract for the treatment of dental diseases. Purification
of the active fractions F-02 and F-05 (Table 1) led to the iso-
lation of ent-trachyloban-19-oic acid (1), the mixture of ent-
kaur-16-en-19-oic acid (2) and ent-beyer-15-en-19-oic acid
(3), and xanthorrhizol (4). From the inactive fractions F-08
and F-20, 16α-hydroxy-ent-kaurane (5) and 16α-hydroxy-
ent-kaur-11-en-19-oic acid (6) were isolated (Fig. 1). The
structures of the known compounds were identified by
their spectroscopic data (1H NMR, 13C NMR, DEPT, COSY,
HMQC, HMBC, UV and IR) and by comparison with published
values [3, 7, 8].
The selected microorganisms were predictive of potential
applications against human diseases caused by bacteria. The
results in Table 1 indicate that the extract from I. heterophylla
inhibited the growth of S. mutans and P. gingivalis with MIC
values of 77 and 105 μg/mL, respectively. Among the evaluat-
ed metabolites, compounds 1, the mixture of 2+3 and 4 dis-
played the highest antibacterial activity against S. mutans and
P. gingivalis with MICs ranging from 4.1 to 95.3 μg/mL. The
MBC values of the tested compounds shown in Table 1 were
higher (four to ten times) than the MIC values, ranging
from 8.2 to 362.4 μg/mL. Concerning S. mutans, compounds
1, and 4 also interfered with the establishment of its biofilms,
2.6. In vitro biofilm formation, treatment and quantification
The formation of S. mutans biofilms was studied in com-
mercially available presterilized, flat-bottom 96-well microti-
ter plates by the method described previously [12, 13] with
modifications. The plates were conditioned with 200 μL of ar-
tificial saliva solution. The plates were then incubated at
room temperature for 2 h with gentle shaking and air-dried
after removal of the excess artificial saliva solution. The
growth of biofilm formation was initiated by addition of
175 μL of BHI broth supplemented with 3% (w/v) of sucrose
to each well of a 96-well microtiter plate. A 96-well microti-
ter plate containing 175 μL of BHI broth supplemented with
sucrose per well was inoculated with 25 μL of a previously
prepared inoculum cell suspension at a density of 2×105
cells/mL. The final density of inoculum in each well of a 96-
well microtiter plate was 2.5×104 cells/mL. The microtiter
plates were then incubated at 37 °C during 24 h for biofilm
development. After the biofilm growth in the presence of 0,

530
D.M. Hernández et al. / Fitoterapia 83 (2012) 527–531
structure. In fact, the presence of two HBDs in the decalin
ring system of compound 6 really decreases the antimicrobial
activity displayed by these diterpenes.
To find out if activity could be increased in the two most
active terpenoids (1 and 4), derivatives 1a, 1b, and 4a–4d
were obtained. As Table 1 shows, the acidic moiety in trachy-
lobanoic acid (1) is essential to keep antimicrobial activity,
meanwhile blocking of phenol group and double bond C12-
13 in xanthorrhizol, also leads to loss of activity but hydroge-
nation of this bond keeps activity but at a lesser extent. Com-
pounds 1b and 4d also interfered with the establishment of
S. mutans biofilms, inhibiting their development at 125.0
and 24.4 μg/mL, respectively.
Earlier in vitro studies have shown that ent-trachyloban-
19-oic acid (1) has antimicrobial activity against methicillin-
resistant Staphylococcus aureus and Mycobacterium smegmatis
[16]. This diterpene inhibits also larval development of Home-
osoma electullum (sunflower moth) and the three Lepidoptera
species Heliotis virscens, Heliotis zea, and Pectinophera gossy-
piella (pink bollworm) [17]. Xanthorrhizol (4) has been
isolated from Zingiberaceae [18, 19] and Asteraceae families
of higher plants [7]. Several pharmacological properties of
xanthorrhizol (4) have been demonstrated, including anti-
bacterial, anti-biofilm, antifungal, cytotoxic and as inhibitor
of the tonic contraction of rat uterus [20–23].
In conclusion, the compounds isolated from I. heterophylla
are effective antibacterial agents and represent an alternative
to commonly used compounds to treat oral infections. The
results in this study confirmed that the roots of I. heterophylla
have a significant antimicrobial activity against oral patho-
gens and justifies its ethnomedical use as such. The roots
are also safe for consumption as traditional remedy, as toxic-
ity tests have demonstrated [24].
Fig. 1. Isolated compounds from I. heterophylla and ent-trachyloban-19-oic
acid (1) and xanthorrhizol (4) derivatives.
inhibiting their development at concentrations of 32.5 and
14.1 μg/mL, respectively.
Regarding with the degree of activity of the tested com-
pounds, it can be noted that the MIC values varied from 77
to 105 μg/mL, 4.1 to the concentration limit of 1000 μg/mL,
and 1.2 to 3.2 μg/mL for the crude extract, pure compounds
and reference antibiotic, respectively. The MIC and MBC
values obtained with compounds 1, 1b, the mixture of 2+3
and 4 were less potent than that for chlorhexidine on the cor-
responding microorganisms, being the most active xanthor-
rhizol (4) (Table 1). The diterpenes 5 (16α-hydroxy-ent-
kaurane) and 6 (16α-hydroxy-ent-kaur-11-en-19-oic acid)
were inactive whereas the mixture of 2+3 showed signifi-
cant activity (34.4 μg/mL) against S. mutans. Even though
the diterpene trachylobanoic acid (1) was less active than
sesquiterpene xanthorrhizol (4), it showed MIC of 8.9 μg/mL
and was more active than the mixture of kaurenoic acids
2+3. Recently, Urzúa et al. [15] have established that a lipo-
philic decalin ring system, with a strategically positioned
hydrogen-bond-donor group (HBD; hydrophilic group), is
very important for the antimicrobial activity displayed by
diterpenes. Moreover, the authors pointed out that a second
HBD introduced in the decalin ring system led to a reduction
in or suppression of the activity. Two reasons may explain
these observations. The first, the presence of two HBDs de-
creases the lipophilicity of the hydrophobic moiety, hindering
its interaction with the bacterial membrane. A second expla-
nation, the intramolecular HBD group interactions compete
with intermolecular hydrogen-bonds between each HBD
and the cell membrane. A careful observation of the results
in Table 1 reveals that compounds 1, 1b, 2 and 3, which con-
tain HBD at C-19, display MIC and MBC values lower than
that of compound 5, which has no HBD in its chemical
Acknowledgments
This investigation was supported by PAPIIT 205709 and
223411 (National Autonomous University of Mexico). The
authors thank the USAI-UNAM staff for recording the NMR
and mass spectrometry spectra used in this investigation.
References
[1] Argueta V, Cano A, Rodarte ME. Atlas de las Plantas Medicinales de la
Medicina Tradicional Mexicana. 772: Instituto Nacional Indigenista;
1994. Tomo II.
[2] Martínez M. Las Plantas Medicinales de México. México: Editorial
Botas; 1969.
[3] Aguilar MI, Delgado G, Bye R, Linares E. Bisabolenes, polycyclic diterpe-
noids and other constituents from the roots of Iostephane heterophylla.
Phytochemistry 1993;33:1161–3.
[4] Aguilar MI, Delgado G. Novel bisabolene glycoside and other constitu-
ents from the roots of the medicinal plant Iostephane heterophylla
(Asteraceae). Nat Prod Lett 1995;7:155–62.
[5] Aguilar MI, Osorio N, Bernal I, Navarrete A, Bye R. Development and val-
idation of a LC analytical method to quantify xanthorrhizol in roots of
Iostephane heterophylla (Cav.) Benth ex Hemsl. J AOAC Int 2007;90:
892–6.
[6] Rivero-Cruz JF. Antimicrobial compounds isolated from Haematoxylon
brasiletto. J Ethnopharmacol 2008;119:99–103.
[7] Aguilar MI, Delgado G, Hernández ML, Villarreal ML. Bioactive com-
pounds from Iostephane heterophylla (Asteraceae). Nat Prod Lett
2001;15:134–6.
[8] Aguilar MI, Delgado G, Villarreal ML. New bioactive derivatives of
xanthorrhizol. Rev Soc Quím de Méx 2001;45:56–9.

D.M. Hernández et al. / Fitoterapia 83 (2012) 527–531
531
[9] Arnone A, Mondelli R. 13C NMR spectroscopy of natural substances.
IV-13C NMR studies of trachylobane diterpenes: complete carbon as-
signment. Org Magn Reson 1979;12:429–31.
[18] John TK, Krisna-Rao GS. Studies in terpenoids. Part LXIII. Absolute con-
figuration of naturally occurring (−)-xanthorrhizol. Indian J Chem
1985;24B:35–7.
[10] da Costa FB, Alburquerque S, Vichnewski W. Diterpenes and synthetic
derivatives from Viguiera aspillioides with trypanomicidal activity.
Planta Med 1996;62:557–9.
[11] Takahashi JA, Pereira CR, Pimenta LPS, Boaventura MAD, Silva LGFE.
Antibacterial activity of eight Brazilian Annonaceae plants. Nat Prod
Res 2006;20:21–6.
[19] Rimpler H, Hansel R, Kochendoerfer LZ. Xanthorrhizol, a new sesquiter-
pene from Curcuma xanthorrhiza. Z Naturforsch B 1970;25:995–8.
[20] Ponce-Monter H, Campos M, Aguilar MI, Delgado G. Effect of xanthor-
rhizol, xanthorrhizol glycoside and trachylobanoic acid isolated from
cachani complex plants upon the contractile activity of uterine smooth
muscle. Phytother Res 1999;13:202–5.
[12] Rivero-Cruz JF, Zhu M, Kinghorn AD, Wu CD. Antimicrobial constituents
of Thompson seedless raisins (Vitis vinifera) against selected oral path-
ogens. Phytochem Lett 2008;1:151–4.
[21] Campos MG, Oropeza MV, Villanueva T, Aguilar MI, Delgado G, Ponce
HA. Xanthorrhizol induces endothelium-independent relaxation of rat
thoracic aorta. Life Sci 2000;67:327–33.
[13] Methods for dilution antimicrobial susceptibility tests for bacteria that
grow aerobically. Approved Standard. 8th ed. NCCLS; 2009.
[14] Rukayadi Y, Hwang JK. In vitro activity of xanthorrhizol against Strepto-
coccus mutans biofilms. Lett Appl Microbiol 2006;42:401–4.
[15] Urzúa A, Rezende MC, Mascayano C, Velásquez L. A structure–activity
study of antibacterial diterpenoids. Molecules 2008;14:191–9.
[16] Zgoda-Pols JR, Freyer AJ, Killmer LB, Porter JR. Antimicrobial diterpenes
from the stem bark of Mitrephora celebica. Fitoterapia 2002;73:434–8.
[17] Alliger CA, Zinkel DF, Chang BG, Waiss AC. Diterpene acids as larvae
growth inhibitors. Experientia 1976;32:1364–6.
[22] Rukayadi Y, Hwang JK. Effect of coating the wells of a polystyrene mi-
crotiter plate with xanthorrhizol on the biofilm formation of Strepto-
coccus mutans. J Basic Microbiol 2006;46:410–5.
[23] Hwang JK, Shim JS, Pyun YR. Antibacterial activity of xanthorrhizol
from Curcuma xanthorrhiza against oral pathogens. Fitoterapia
2000;71:321–3.
[24] Déciga-Campos M, Rivero-Cruz I, Arriaga-Alba M, Castañeda-Corral G,
Angeles-López GE, Navarrete A, et al. Acute toxicity and mutagenic ac-
tivity of mexican plants used in traditional medicine. J Ethnopharmacol
2007;110:334–43.