Phytochemistry Letters 18 (2016) 162–167

Contents lists available at ScienceDirect

Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol

Short communication

Biosynthesis of ( )-ent-kaurenoic acid in Smallanthus sonchifolius and

its effect against microbial biofilms
Adriana A. Lopesa,* , Edieidia S. Pinaa , Talita T. Nadera , Fernando B. Da Costab ,
Ana Maria S. Pereiraa , Mônica T. Pupob,*
a
Unidade de Biotecnologia, Universidade de Ribeirão Preto, Av. Costábile Romano, 2201, 14096-900, Brazil
b
Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Avenida do Café s/n, 14040-903, Brazil

A R T I C L E I N F O A B S T R A C T

Article history:
Received 29 April 2016 The biosynthetic pathway of (–)-ent-kaurenoic acid (1) was investigated by incorporation of 1-D-13C-
Received in revised form 20 September 2016 glucose in Smallanthus sonchifolius (Asteraceae) plantlets. The 13C-enrichment pattern indicated that
Accepted 11 October 2016 methylerythritol-4-phosphate (MEP) pathway is the biosynthetic pathway involved in diterpenoid
Available online 17 October 2016 biosynthesis. Our studies in S. sonchifolius reinforce that the biosynthesis of different classes of terpenes
should not be compartmentalized into cytosol and plastids. Additionally, (–)-ent-kaurenoic acid showed
Keywords: antimicrobial activity against Staphylococcus aureus biofilm.
Diterpene ã 2016 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
Ent-kaurenoic acid
Biosynthesis
Methylerythritol-4-phosphate (MEP)
pathway
Smallanthus sonchifolius

1. Introduction (Asteraceae) is biosynthesized by MEP pathway (Steliopoulos et al.,

Smallanthus sonchifolius (Poepp & Endl.) H. Robinson (Aster-
aceae), popularly known as yacón, biosynthesizes ent-kaurenoic
acid (1) (Valentová and Ulrichová, 2003), a biologically active
diterpenoid that is a key intermediate in the biosynthesis of some
plant secondary metabolites, including gibberellins (Kasahara
et al., 2002; Bömke and Tudzynski, 2009). Plants can synthesize all
terpenoids by the mevalonate (MVA) or methylerythritol-4
phosphate (MEP) pathways operating in the cytoplasm and
plastids, respectively. Thereby, two independent pathways estab-
lished in separate intracellular compartments are involved in the
biosynthesis of isoprene units (DMAPP/IPP) (Vranová et al., 2013).
For years, triterpenoids and sesquiterpenoids were considered
biosynthesized by MVA pathway, while monoterpenoids, diterpe-
noids, tetraterpenoids were designed exclusively MEP pathway as
proposed by some authors (Vranová et al., 2012; Kirby and
Keasling, 2009), but this statement can no longer hold true
(Hemmerlin et al., 2012). Precursors of germacrene D in Tanacetum
vulgare L. (Asteraceae) are synthesized by MVA pathway (Umlauf
et al., 2004), while the germacrene D in Solidago canadensis
2002). We recently showed that S. sonchifolius biosynthesizes
sesquiterpene lactones by MEP pathway (Lopes et al., 2013).
Therefore, MVA and MEP metabolic pathways are compartmen-
talized into cytosol and plastids, but not the classes of terpenoids.
So far, there is only one report in the literature about
gibberellins and ent-kaurene precursor’s biosynthesis by incorpo-
ration of 14C-mevalonic acid in Phaseolus coccineus (Faboideae)
cell-free system (Turnbull et al., 1986). Years later, studies of
steviol, an ent-kaurene derivative from Stevia rebaudiana Bertoni
(Asteraceae), showed that the MEP pathway is involved in the
diterpenoid biosynthesis (Totté et al., 2000). Gibberellin diter-
penes are biosynthesized by contribution of the MEP pathway by
which the intermediate ent-kaurene is produced from GGPP
(trans-geranylgeranyl diphosphate) and its regulation occurs in
proplastids (Hedden and Thomas, 2012).
Herein, we present the (–)-ent-kaurenoic acid biosynthesis by
incorporation of 1-13C-D-glucose precursor. To the best of our
knowledge, (–)-ent-kaurenoic acid biosynthesis by 13C-precursor is
being described for the first time. Additionally, the effect of (–)-ent-
kaurenoic acid on biofilm formation against Staphylococcus aureus
has been evaluated.

* Corresponding authors.
E-mail addresses: alopes@unaerp.br (A.A. Lopes), mtpupo@fcfrp.usp.br
(M.T. Pupo).

http://dx.doi.org/10.1016/j.phytol.2016.10.011
1874-3900/ã 2016 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
 A.A. Lopes et al. / Phytochemistry Letters 18 (2016) 162–167 163

2. Results and discussion kaurenoic acid also displayed antimicrobial activity against oral
pathogens (Ambrosio et al., 2008; Porto et al., 2009; Carvalho et al.,
With the aim to elucidate the biosynthetic origin of the 2011; Andrade et al., 2011; Moreira et al., 2016). Recently, ent-
isoprene units in the ( )-ent-kaurenoic acid, we used in vitro kaurenoic acid isolated from Aralia continentalis (Araliaceae)
cultures of Smallanthus sonchifolius (Lopes et al., 2013). The pattern inhibited biofilm formation of Streptococcus mutans (Jeong et al.,
of isotopic incorporation was determined by quantitative 13C NMR 2013). Considering this previous antibacterial potential, we
(Fig. 2). The 13C-enrichment pattern of 1 showed that the positions decided to investigate the activity of 1 against Gram-positive
C-2, C-6, C-11, C-14, C-15, C-17, C-19 and C-20 (Table 1) were highly Staphylococcus aureus biofilm, once it is necessary to understand
labeled after 13C-glucose metabolism (3.1% up to 6.3%). These data the relation between bacterial biofilms and human diseases
confirm that ent-kaurene was biosynthesized exclusively by (Archer et al., 2011).
isoprenoid units from MEP pathway once C-positions labeled The minimum biofilm inhibitory concentration (MBIC) assay
were correspondent to C-1 and C-5 from IPP (Fig. 1). The metabolic showed that the minimal concentration of 0.206 mM seems to be
pathway of glycolysis converts 1-13C-D-glucose to 1-deoxy-D- sufficient to inhibit 100% of S. aureus biofilm, whereas vancomycin
xylulose-5-phosphate (DXP), the first intermediate from MEP at 2.760  10 3 mM inhibited 100% and high concentration of
pathway, from pyruvate and glyceraldehyde 3-phosphate by a gentamicin at 62.814 mM inhibited 85% of S. aureus biofilm
thiamine diphosphate-dependent synthase (Dewick, 2009; Totté (Table 2). The minimum biofilm eradication concentration (MBEC)
et al., 2000). The condensation of dimethylallyl diphosphate assays showed that the concentrations 0.412 mM and 0.206 mM
(DMAPP) with three IPP units generates the geranylgeranyl inhibited 56% and 52% of strip biomass, respectively. Interestingly,
pyrophosphate (GGPP). The cyclization of GGPP to ent-kaurene (–)-ent-kaurenoic acid showed the best biomass eradication when
scaffold occurs by the Wagner-Meerwein rearrangement in the compared to gentamicin that showed 13% biofilm eradication at
chloroplasts and one specific oxidation at position C-18 occurs by 62.814 mM (Table 3), and this antibiotic is used to treat bovine
microsomal mono-oxygenases at endoplasmic reticulum surface subclinical mastitis (Nader et al., 2014). Regarding the eradication
(Brandle and Telmer, 2007). Interestingly, diterpenes and sesqui- of bacterial population, the concentrations 0.412 mM and
terpene lactones biosynthesized by S. sonchifolius share the same 0.206 mM decreased about five logarithm cycles of standard
MEP biosynthetic pathway. strain. These data were similar when compared to vancomycin at
Ent-kaurenoic acid (1) has previously shown biological poten- the concentration of 22.080  10 3 mM, and gentamicin at
tial in different assays: it displayed cytotoxic and embryotoxic concentration higher of 62.814 mM after approximately three
effects (Costa-Lotufo et al., 2002; Cavalcanti et al., 2009; Dutra logarithmic cycles of the of S. aureus population (Table 3). In fact,
et al., 2014), induced genotoxicity (Cavalcanti et al., 2006), our data showed that 1 exhibited a better antimicrobial action
inhibited vascular smooth muscle contractility (Ambrosio et al., when compared to gentamicin. Biofilm represents the complex
2006), demonstrated promising antinociceptive activity in inflam- association of bacteria attached to several surfaces. The biofilm
matory pain models (Mizokami et al., 2012), and leishmanicidal formation can increase the pathogenicity of the bacteria and
activity (Miranda et al., 2015). In addition, there are evidences that protects the bacteria from external treatment (Gupta et al., 2016).
1 exhibits antibacterial activities against Staphylococcus aureus Thus, searching for molecules with anti-biofilm properties can be
(Velikova et al., 2000; Padla et al., 2012), Bacillus cereus (Wilkens useful to inhibit biofilm development and bacterial infectivity.
et al., 2002), Staphylococcus epidermidis (Padla et al., 2012) and Here we report, for the first time, the biosynthesis of (–)-ent-
Staphylococcus aureus ATCC 6538 (Pereira et al., 2012). Ent- kaurenoic acid by MEP pathway as well as we had already observed
for sesquiterpene lactones in S. sochifolius (Lopes et al., 2013).
Table 1 Therefore, diterpenes e sesquiterpene lactones share the same
13
C NMR data of (–)-ent-kaurenoic acid (1) isolated from S. sonchifolius after terpenoid biosynthetic route in this plant. Additionally, (–)-ent-
incorporation of 1-13C-D-glucose into cultures (CDCl3, 25  C). kaurenoic acid exhibited a better antibacterial action when
C d 1-13C-D-glucose compared to gentamicin.
Relative intensity of signal DC
3. Experimental
U L
1 40.7 0.9 1.0 1.2 3.1. Chemicals
2 19.1 0.8 3.1 4.3
3 37.8 0.8 0.9 1.2
4 43.7 2.2 2.0 1
The labeled precursor 1-13C-D-glucose, vancomycin and genta-
5 57.1 1.2 1.1 1 micin were purchased from Sigma-Aldrich1.
6 21.8 0.9 2.8 3.4
7 33.1 0.8 0.9 1.2 3.2. Culture condition
8 44.2 2.1 1.4 0.7
9 55.1 1.1 1.0 1
10 39.7b 2.1 2.6 1.4 The source of S. sonchifolius was described previously (Lopes
11 18.4 0.8 2.6 3.6 et al., 2013). The plantlets were grown in glass tubes (8.5 cm  2.5
12 41.3 1.0 1.1 1.2 cm) containing 5 mL of Murashige & Skoog culture medium,
13 43.8 1.6 1.4 1 supplemented with 3% of D-glucose (w/v) and solidified with 0.2%
14 39.7b 2.1 2.6 1.4
15 48.9 0.9 2.7 3.3
Phytagel1 (pH 6.0). Cultures were stored at 25  2  C (55–60%
16a 155.9 1.4 1.4 1.1 relative humidity with a 16-h photoperiod of 40 mmol m 2 s 1
17 103.0 1.0 3.3 3.6 intensity, provided by 85 W cool-white GE fluorescent lamps) and
18 28.9 1.0 1.1 1.2 subcultured at intervals of 8 weeks until feeding experiments.
19 184.3 0.4 2.0 5.5
20 15.6 1.1 3.2 3.2
3.3. Labeled kaurenoic acid from plantlets fed with 1-13C-D-glucose
L: labeling experiment with 13C precursor; U: control experiments with unlabeled
precursor; DC = 1.1%  L/U: increase in the relative intensity (significant increases in
bold for enriched carbons).
Nodal segments of S. sonchifolius plantlets were inoculated in
a
Used as reference. liquid medium supplemented with 1-13C-D-glucose (3% m/v) and
b
Overlapping resonance signals. transferred into glass tubes (8.5 cm  2.5 cm) containing 2.5 mL of
164 A.A. Lopes et al. / Phytochemistry Letters 18 (2016) 162–167

Fig. 1. Biosynthesis of 1 after feeding with 1-13C-glucose via MEP pathway.

Murashige & Skoog medium. After inoculation, the culture flasks
were stored at 25  2  C and incubated for 9 weeks. After this
period, the fresh shoots (n = 30) from S. sonchifolius were extracted
with acetone for 12 h to obtain the crude extract. The crude extract
(212 mg) was fractionated by column chromatography over silica
gel (70–230 mesh; Merck, column size 8.0  1.0 cm) to yield eight
fractions, using n-hexane and increasing amounts of EtOAc (up to
30%) as eluent. Fraction 2 (7.0 mg) was further purified via column
chromatography over silica gel (70–230 mesh; Merck, column size
 A.A. Lopes et al. / Phytochemistry Letters 18 (2016) 162–167 165

Fig. 2. 13C NMR spectrum (100 MHz, CDCl3) of 1 with natural isotopic abundance (a) and 13C NMR spectrum (100 MHz, CDCl3) for 13C-enriched 1 after feeding experiments
with 1-13C D-glucose. Enriched carbons have their intensities increased and are numbered in the spectrum (b).

13
3.0  1.2 cm) using n-hexane-EtOAc (9:1) yielding C-labeled 1
Table 2
Minimum biofilm inhibitory concentration (MBIC) assays of 1 and antibiotics (2.0 mg), white powder, [a]D 115 (c 1.0, MeOH).
against S. aureus using crystal violet.
3.4. NMR analysis
Compounds (mM) % minimal biofilm inhibition concentrations (MBIC)
1 (0.825) 100a
NMR spectra were obtained on a Bruker 400 MHz spectrometer
1 (0.412) 100a
1 (0.206) 100a using CDCl3 as solvent and internal standard. The relative 13C
1 (0.103) 45c enrichments were calculated by the comparison of the peak
gentamicin (62.814) 85b intensities of labeled versus non-labeled 13C NMR spectra.
vancomycin 100a Unequivocal 1H and 13C NMR chemical shifts assignments for
(2.760  10 3)
(–)-ent-kaurenoic acid were based on the previous data and also on
1 = (–)-ent-kaurenoic acid; mean values followed by dissimilar letters are HSQC and HMBC data (Enriquez et al., 1997).
significantly different according to the Scott–Knott test (P\0.05).
166 A.A. Lopes et al. / Phytochemistry Letters 18 (2016) 162–167
Table 3
Minimum biofilm eradication concentration (MBEC) assays of 1 and antibiotics against S. aureus using crystal violet and counting of bacterial colony forming units (CFUs).
Compounds (mM) Eradication of biomass by
crystal violet (%)
1 (0.412) 56b
1 (0.206) 52b
gentamicin (62.814) 13c
3
vancomycin (22.080  10 ) 100a
1 = ent-kaurenoic acid; mean values followed by dissimilar letters are significantly different according to the Scott–Knott test (P\0.05).
3.5. MBIC and MBEC assays
The Staphylococcus aureus strain ATCC 25923 was cultured in
Brain Heart Infusion (BHI-Himedia1) agar plate at 37  C for 24 h.
After that, the bacteria were diluted with BHI-Himedia medium
supplemented with glucose 2% (to promote biofilm formation) to
1 105 CFU/ml (optical density at 546 nm), transferred to 96-well
microplate and grown at 37  C for 24 h under 130 rpm rotation. Ent-
kaurenoic acid was evaluated in 1.6; 0.8; 0.4; 0.2 and 0.1 mM, as
well as, 2.8  10 3 mM of vancomycin and 62.8 mM of gentamicin
against biofilm development (triplicate), and assays were per-
formed in 96-well plates for 24 h. Natural compound (1) and
antimicrobial agents (vancomycin and gentamicin) were prepared
in 1.25% dimethyl sulfoxide (DMSO) followed by serial dilutions
and the final concentration of DMSO in well was 0.156%. Then,
planktonic cells were removed carefully, and the biofilm was
washed twice with 200 mL of ultrapure water. After exposure, the
biofilms were washed with 2600 mL of 0.9% NaCl, then 200 mL of
methanol was added and fixed for 15 min. After that, methanol was
removed and added 200 mL and crystal violet (Dinamica1) for
5 min. The wells were washed with ultrapure water, acetic acid 33%
(v/v) was added, and the optical density was measured at 570 nm
(Gomes et al., 2009). The MBIC assay was performed with either
standard inoculum with ent-kaurenoic acid, or antibiotic at the
same time. The MBEC assay was performed after biofilm formation.
3.6. Statistical analysis
The data from all assays were compared using one-way analysis
of variance (ANOVA) by applying SISVAR V.5.1 software (Ferreira,
2005). Triplicate measurements and multiple comparisons of
mean values were performed using the Scott Knott test at a 5%
confidence level.
Acknowledgments
Authors are grateful to the São Paulo Research Foundation
(FAPESP, grant #2013/07600-3, CEPID-CIBFar), to the Conselho
Nacional de Desenvolvimento Científico e Tecnológico (CNPq), to
the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
(CAPES) and to the Biotechnology Unit (UNAERP) for their financial
support. A.A.L. thanks FAPESP for providing postdoctoral fellow-
ship (grant #2008/01220-6).
References
Ambrosio, S.R., Trapelli, C.R., Da Costa, F.B., 2006. Kaurane and pimarane-type
diterpenes from the Viguiera species inhibit vascular smooth muscle
contractility. Life Sci. 79, 925–933.
Ambrosio, S.R., Furtado, N.A.J.C., De Oliveira, D.C.R., Da Costa, F.B., Martins, C.H.G.,
Carvalho, T.C., et al., 2008. Antimicrobial activity of kaurane diterpenes against
oral pathogens. Z. Naturforsch. 63, 326–330.
Andrade, B.B., Moreira, M.R., Ambrosio, S.R., Furtado, N.A.J.C., Cunha, W.R., Heleno,
V.C.G., Silva, A.N., Simão, M.R., Da Rocha, E.M.P., Martins, C.H.G., Veneziani, R.C.
S., 2011. Evaluation of ent-kaurenoic acid derivatives for their anticariogenic
activity. Nat. Prod. Commun. 6 (6), 777–780.
Reduction in bacterial population by CFUs
(logarithm scale)
5.1a
4.7a
3.1b
4.6a
Archer, N.K., Mazaitis, M.J., Costerton, W., Leid, J.G., Powers, M.E., Shirtliff, M.E., 2011.
Staphylococcus aureus biofilms: properties, regulation and roles in human
disease. Virulence 2 (5), 445–459.
Bömke, C., Tudzynski, B., 2009. Diversity, regulation, and evolution of the gibberellin
biosynthetic pathway in fungi compared to plants and bacteria. Phytochemistry
70, 1876–1893.
Brandle, J.E., Telmer, P.G., 2007. Steviol glycoside biosynthesis. Phytochemistry 68,
1855–1863.
Carvalho, T.C., Simão, M.R., Ambrósio, S.R., Furtado, N.A.J.C., Veneziani, R.C.S.,
Heleno, V.C.G., Da Costa, F.B., Gomes, B.P.F.A., Souza, M.G.M., Reis, E.B., Martins,
C.H.G., 2011. Antimicrobial activity of diterpenes from Viguiera arenaria against
endodontic bacteria. Molecules 16, 543–551.
Cavalcanti, B.C., Costa-Lotufo, L.V., Moraes, M.O., Burbano, R.R., Silveira, E.R., Cunha,
K.M.A., Rao, V.S.N., Moura, D.J., Rosa, R.M., Henriques, J.A.P., Pessoa, C., 2006.
Genotoxicity evaluation of kaurenoic acid, a bioactive diterpenoid present in
copaiba oil. Food Chem. Toxicol. 44, 388–392.
Cavalcanti, B.C., Bezerra, D.P., Magalhães, H.I.F., Moraes, M.O., Lima, M.A.S., Silveira,
E.R., Câmara, C.A.G., Rao, V.S., Pessoa, C., Costa-Lotufo, L.V., 2009. Kauren-19-oic
acid induces DNA damage followed by apoptosis in human leukemia cells. J.
Appl. Toxicol. 29, 560–568.
Costa-Lotufo, L.V., Cunha, G.M.A., Farias, P.A.M., Viana, G.S.B., Cunha, K.M.A., Pessoa,
C., Moraes, M.O., Silveira, E.R., Gramosa, N.V., Rao, V.S.N., 2002. The cytotoxic
and embryotoxic effects of kaurenoic acid: a diterpene isolated from Copaifera
langsdorffii oleo-resin. Toxicon 40, 1231–1234.
Dewick, P.M., 2009. In: Dewick, P.M. (Ed.), Medicinal Natural Products: A
Biosynthetic Approach. 3rd ed. John Wiley & Sons Ltd, pp. 235–240 (Ch. 5).
Dutra, L.M., Bomfim, L.M., Rocha, S.L.A., Nepel, A., Soares, M.B.P., Barison, A., Costa, E.
V., Bezerra, D.P., 2014. Ent-kaurane diterpenes from the stem bark of Annona
vepretorum (Annonaceae) and cytotoxic evaluation. Bioorg. Med. Chem. Lett. 24,
3315–3320.
Enriquez, R.G., Barajas, J., Ortiz, B., Lough, A.J., Reynolds, W.F., Yu, M., Leon, I., Gnecco,
D., 1997. Comparison of crystal and solution structures and 1H and 13C chemical
shifts for grandiflorenic acid kaurenoic acid, and monoginoic acid. Can. J. Chem.
75, 342–347.
Ferreira, D.F., Statistical analysis by Sisvar for Windows. Sisvar 5.1. 2005.
Gomes, F.I.A., Teixeira, P., Azeredo, J., Oliveira, R., 2009. Effect of farnesol on
planktonic and biofilm cells of Staphylococcus epidermidis. Curr. Microbiol. 59,
118–122.
Gupta, P., Sarkari, S., Das, B., Bhattacharjee, S., Tribedi, P., 2016. Biofilm: pathogenesis
and prevention—a journey to break the wall: a review. Arch. Microbiol. 198, 1–
15.
Hedden, P., Thomas, S.G., 2012. Gibberellin biosynthesis and its regulation. Biochem.
J. 44, 11–25.
Hemmerlin, A., Harwood, J., Bach, T.J., 2012. A raison d’être for two distinct
pathways in the early steps of plant isoprenoid biosynthesis? Prog. Lipid Res. 51,
95–148.
Jeong, S., Kim, B., Keum, K., Lee, K., Kang, S., Park, B., Lee, Y., You, Y., 2013. Kaurenoic
acid from Aralia continentalis inhibits biofilm formation of Streptococcus mutans.
Evid.—Based Complement. Altern. Med. 2013, 1–9.
Kasahara, H., Hanada, A., Kuzuyama, T., Takagi, M., Kamiya, Y., Yamaguchi, S., 2002.
Contribution of the mevalonate and methylerythritol phosphate pathways to
the biosynthesis of gibberellins in Arabidopsis. J. Biol. Chem. 277, 45188–45194.
Kirby, J., Keasling, J.D., 2009. Biosynthesis of plant isoprenoids: perspectives for
microbial engineering. Annu. Rev. Plant Biol. 60, 335–355.
Lopes, A.A., Pina, E.S., Silva, D.B., Pereira, A.S., Silva, M.F.G.F., Da Costa, F.B., Lopes, N.
P., Pupo, M., 2013. A biosynthetic pathway of sesquiterpene lactones in
Smallanthus sonchifolius and their localization in leaf tissues by MALDI imaging.
Chem. Commun. 49, 9989–9991.
Miranda, M.M., Panis, C., Silva, S.S., Macri, J.A., Kawakami, N.Y., Hayashida, T.H.,
Madeira, T.B., Junior, V.R.A., Nixdorf, S.L., Pizzatti, L., Ambrosio, S.R., Cecchini, R.,
Arakawa, N.S., Junior, W.A.V., Costa, I.C., Pavanelli, W.R., 2015. Kaurenoic acid
possesses leishmanicidal activity by triggering a NLRP12/IL-1b/cNOS/NO
pathway. Mediators Inflamm. 2015, 1–10 (Hindawi Publishing Corporation).
Mizokami, S.S., Arakawa, N.S., Ambrosio, S.R., Zarpelon, A.C., Casagrande, R., Cunha,
T.M., Ferreira, S.H., Cunha, F.Q., Junior, W.A.V., 2012. Kaurenoic acid from
Sphagneticola trilobata inhibits inflammatory pain: effect on cytokine
production and activation of the NO-cyclic GMP-protein kinase G-ATP-sensitive
potassium channel signaling pathway. J. Nat. Prod. 75, 896–904.
Moreira, M.R., Souza, A.B., Soares, S., Bianchi, T.C., Eugênio, D.S., Lemes, D.C., Martins,
C.H.G., Moraes, T.S., Tavares, D.C., Ferreira, N.H., Ambrosio, S.R., Veneziani, R.C.S.,
 A.A. Lopes et al. / Phytochemistry Letters 18 (2016) 162–167
2016. Ent-kaurenoic acid-rich extract from Mikania glomerata: in vitro activity
against bacteria responsible for dental caries. Fitoterapia 112, 211–216.
Nader, T.T., Coppede, J.S., Amaral, L.A., Pereira, A.M.S., 2014. Atividade antibiofilme
de diterpeno isolado de Croton antisyphiliticus frente Staphylococcus aureus. Ars
Vet. 30 (1), 32–37.
Padla, E.P., Solis, L.T., Ragasa, C.Y., 2012. Antibacterial and antifungal properties of
ent-kaurenoic acid from Smallanthus sonchifolius. Chin. J. Nat. Med. 10, 408–
4014.
Pereira, S., Taleb-Contini, S., Coppede, J., Pereira, P., Bertoni, B., França, S., Pereira, A.
M., 2012. An ent-kaurane-type diterpene in Croton antisyphiliticus mart.
Molecules 17, 8851–8858.
Porto, T.S., Rangel, R., Furtado, N.A.J.C., De Carvalho, T., Martins, C.H.G., Veneziani, R.
C.S., Da Costa, F.B., Vinholis, A.H.C., Cunha, W.R., Heleno, V.C.G., Ambrosio, S.R.,
2009. Pimarane-type diterpenes: antimicrobial activity against oral pathogens.
Molecules 14, 191–199.
Steliopoulos, P., Wüst, M., Adam, K.-P., Mosandl, A., 2002. Biosynthesis of the
sesquiterpene germacrene D in solidago canadensis: 13C and 2H labeling
studies. Phytochemistry 60, 13–20.
Totté, N., Charon, L., Rohmer, M., Campernolle, F., Baboeuf, I., Geuns, J.M.C., 2000.
Biosynthesis of the diterpenoid steviol, an ent-kaurene derivative from Stevia
167
rebaudiana Bertoni, via the methylerythritol phosphate pathway. Tetrahedron
Lett. 41, 6407–6410.
Turnbull, C.G.N., Crozier, A., Schwenen, L., Graebe, J.E., 1986. Biosynthesis of
gibberellin A12-aldehyde, gibberellin A12 and their kaurnoid precursors from
[14C] mevalonic acid in a cell-free system from immature seed of Phaseolus
coccineus. Phytochemistry 25, 97–101.
Umlauf, D., Zapp, J., Becker, H., Adam, K.P., 2004. Biosynthesis of the irregular
monoterpene artemisia ketone: the sesquiterpene germacrene D and other
isoprenoids in Tanacetum vulgare L. (Asteraceae). Phytochemistry 65, 2463–
2470.
Velikova, M., Bankova, V., Tsvetkova, I., Kujumgiev, A., Marcucci, M.C., 2000.
Antibacterial ent-kaurene from Brazilian propolis of native stingless bees.
Fitoterapia 71, 693–696.
Vranová, E., Coman, D., Gruissem, W., 2012. Structure and dynamics of the
isoprenoid pathway network. Mol. Plant 5, 318–333.
Vranová, E., Coman, D., Gruissem, W., 2013. Network analysis of the MVA and MEP
pathways for isoprenoid synthesis. Annu. Rev. Plant Biol. 64, 665–700.
Wilkens, M., Alarcón, C., Urzúa, A., Mendoza, L., 2002. Characterization of the
bactericidal activity of the natural diterpene kaurenoic acid. Planta Med. 68 (5),
452–454.