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 2014
NPC Natural Product Communications Vol. 9
No. 3
Distribution of (-)-Hamanasic Acid A in South American Species of 341 - 345
Flourensia and Phytotoxic Effects of Leaf Aqueous Extracts
Daniela Lópeza, Leonardo A. Piazzaa*, Mariana P. Silvaa, Marisa J. López Rivillia, Juan J. Canterob,
Graciela M. Tourna,c and Ana L. Scopela,d*
a
Estación de Biología Sierras, Facultad de Agronomía-Sede Punilla, Universidad de Buenos Aires, Casilda S/N,
Huerta Grande, 5174, Córdoba, Argentina
b
Departamento Biología Agrícola, Facultad de Agronomía y Veterinaria, Universidad de Río Cuarto, Río Cuarto,
5800, Córdoba, Argentina; Instituto Multidisciplinario de Biología Vegetal (CONICET-UNC), 5000. Córdoba,
Argentina
c
Cátedra de Botánica Agrícola, Agronomía, Universidad de Buenos Aires, Buenos Aires, Argentina
d
Instituto de Investigaciones en Biociencias Agrícolas y Ambientales (INBA), Consejo Nacional de Investigaciones
Científicas y Técnicas (CONICET) y Facultad de Agronomía, Universidad de Buenos Aires, Buenos Aires,
Argentina

leonardoalbertopiazza@yahoo.com.ar; scopel.ana@gmail.com

Received: August 31st, 2013; Accepted: September 30th, 2013

The presence of the phytotoxic sesquiterpene (-)-hamanasic acid A {(-)HAA; 7-carboxy-8-hydroxy-1(2), 12(13)-dien-bisabolene} isolated from Flourensia
campestris (FC), was investigated in the South American species of the genus, together with the evaluation of the phytotoxic activity of their leaf aqueous
extracts. (-)HAA was identified and isolated from F. fiebrigii (FF) and F. oolepis (FO), being chemically (GC-MS, NMR, [α]D) and biologically (bioassayed
on lettuce) indistinguishable from that of FC, while no (-)HAA was found in F. hirta (FH), F. riparia (FR) and F. niederleinii (FN). Its leaf content in FF was
similar to that found in FC (ca. 15 mg g-1 WT) and significantly higher than in FO (0.8 mg g-1 WT). The screening for the presence of (-)HAA in other species
of Flourensia communities showed that its natural occurrence is restricted only to Flourensia species. No (-)HAA could be detected in any of the 37 -most
representative- species of these communities (26 natives, 11 exotics), despite many of them belong to the same family and tribe as Flourensia spp. Leaf
aqueous extracts of all Flourensia species exhibited strong inhibitory effects on lettuce germination and on root and shoot growth, regardless of the presence
and content of (-)HAA. These results strongly suggest the existence of other powerful phytotoxic compounds in those Flourensia spp lacking (-)HAA. Our
results clearly show that (-)HAA only pertains to some species of the genus Flourensia. Relative to previous exomorphologic groupings of the genus, our
chemotaxonomic data would give support to the close link described between FC and FF, but not with FR. In addition, the fact that (-)HAA was also found in
FO, which belongs to a second different line, also points out that species position in this lineage would deserve to be revisited. The restricted production of
(-)HAA by Flourensia in their communities suggests its special link with the genus, and sustains its putative allelochemical role.

Keywords: Flourensia, (-)-Hamanasic acid A, Phytotoxic activity, Sesquiterpenes, Chemotaxonomy.

The genus Flourensia (Family Asteraceae; Subfamily Asteroideae;
Tribe Heliantheae; Subtribe Enceliinae) comprises 25 species
distributed throughout America, from which 18 are found in South
America, and 12 in Argentina [1-3]. As part of our ongoing studies
on the adaptive ecophysiological traits of native species of
Argentina [4-6], we found that F. campestris Griseb. exhibited
strong phytotoxic effects [7]. This species, endemic to the inland
central region of the country, produces elevated amounts of a
phytotoxic compound identified as (-)-hamanasic acid A (7-
carboxy-8-hydroxy-1(2), 12(13)-dien-bisabolene {(-)HAA}), a new
levorotatory form of the (+)-sesquiterpene previously described by
Hashidoko et al. [8] in Rosa rugosa leaves (Rosaceae). At present,
(-)HAA is only available by extraction as its chemical synthesis is
still lacking. In bioassays using Lactuca sativa as a test system,
(-)HAA drastically inhibited seed germination (by 90%), and
completely arrested root and shoot growth at the higher
concentrations tested (ca. 4 mM). This bisabolanoid is accumulated
on plant surfaces in exceptionally high concentrations (1.6% of total
leaf biomass), and is easily leached out by water from fresh and dry
tissues suggesting a putative allelochemical role. Other Flourensia
species have also been found to synthesize mixtures of phytotoxic
compounds, where sesquiterpenes are always present, as was
revealed in F. cernua [9], in FC -namely (-)HAA [7]-, and more
recently in F. oolepis [10]. These findings pose a number of
questions regarding the distribution and specificity of (-)HAA in
South American species of the genus and the potential phytotoxic
activity of their leaf aqueous extracts.
Prenylflavonoids and benzofuran derivatives [11,12,13a,b], together
with similar surface deposited and volatile mono and sesquiterpenes
[7,14-16] have been characterized in Flourensia species. Even
though some of these molecules and structural types are repeatedly
found among species, a clear terpene pattern implying
chemosystematic correlations could not be detected. In this sense,
the study of the sesquiterpene (-)HAA as an (-)-α-bisabolol
derivative in Flourensia spp., and in coexisting species, may bring
additional chemotaxonomical insights, as well as valuable
information about its ecophysiological significance for the genus.
In the present work we addressed the following questions: a) is
(-) HAA widely distributed among South American species of
Flourensia? b) is (-)HAA production genus specific? c) are the
phytotoxic effects of leaf aqueous extracts a common trait in
Flourensia species? d) is phytotoxicity related to the amount of
(-)HAA present?
342 Natural Product Communications Vol. 9 (3) 2014 López et al.

HO 10
CH3 possessing 5-8 ray florets. This lineage tends to occupy more arid
8
H
4
3
2
environments at lower elevations. FR seems basal within the line.
9
11 5
OH
The other lineage, composed of Flourensia suffrutescens, F.
6 7
12 1
tortuosa, F. macroligulata, F. heterolepis and FO is characterized
13
O by large capitula with broad phyllaries and 13-21 ray florets. The
15 14

1
(-)HAA with numbering and absolute configuration.
The occurrence of (-)HAA was screened in F. oolepis (FO), which
grows in the same biome as FC, but in different communities, and
four other South American Flourensia species distantly distributed:
F. hirta (FH), F. riparia (FR), F. niederleinii (FN) and F. fiebrigii
(FF), which together with FC comprises 50% of the total taxa
described in Argentina [17]. The detection and quantification of
(-)HAA were accomplished through a screening 2D-TLC technique
and GC-FID, as described previously [7]. The identity of the
compounds was confirmed through their isolation (LC), and further
chemical (1H NMR, 13C NMR, DEPT, COSY, HMBC, HSQC and
[α]D), and biological (bioassay on lettuce seeds) characterizations.
Bioassays of leaf aqueous extracts from Flourensia species were
performed on lettuce seeds [7]. The role of (-)HAA is discussed in
relation to its value as a taxonomical character, as key to Flourensia
phytotoxicity and the ecophysiological significance for the genus.
In Flourensia species, the (-)HAA moiety was first revealed by 2D-
TLC in FO and FF, and was absent in FH, FN and FR.
Taking into account the existence of the enantiomer (+)HAA
already described in Rosaceae [8], and the possibility of other
similar sesquiterpenes with similar chromatographic behavior, the
identified compounds were isolated and chemically and biologically
characterized, and results were compared with those from FC
(-)HAA. FF was primarily selected owing to its higher
concentration. The compound from FF was isolated in a similar
yield to that from FC (ca. 3% vs. 4% DW basis, respectively).
Obtained as a colorless syrup, the 1H, 13C, and 1H-1H COSY NMR
spectroscopic data were the same as those previously described for
FC (-)HAA (7-carboxy-8-hydroxy-1(2),12(13)-dien-bisabolene;
[7]), and consistent with those for its enantiomer (+)-hamanasic acid
A [8]. The rotational value, [α]D - 64.9, was technically the same as
that of the bioactive sesquiterpene found in FC ([α]D - 63.0; [7]), in
contrast to the [α]D - 94.4 value that was documented previously as
preliminary data [18]. The isolated (-)HAA from FF was also
biologically characterized through bioassays on lettuce, giving
ECg50=4.8±1.0 mM, ECr50=2.8±0.6 mM and ECs50=3.6±0.9 mM.
These results were not significantly different from those exerted by
FC (-)HAA in the same experimental test system [7]. Overall
results showed that FF (-)HAA was chemically and biologically
indistinguishable from FC (-)HAA, discarding possible
chromatographic misidentifications of optical isomers and of other
related sesquiterpenes. Very similar results were obtained for FO
(data not shown).
occurrence of prenylflavonoids was observed in at least four
species: F. heterolepis [11], FC, FR [13a], and FF [13b], suggesting
a chemotaxonomical relationship. FC was found to resemble FR in
chemical constituents like benzofuranes and flavonoids. However,
sesquiterpene lactones of the eudesmanolide type {(E,E)-farnesyl
cation} identified in FR were not found in FC [13a]. The fact that
(-)HAA, as a bisabolene derivative {(E,Z)-farnesyl cation}, is
present in FC, but not in FR indicates the activation of different
biosynthetic pathways of sesquiterpenes in both species.
Our data would give support to the close link between FC and FF,
but this was not the case with other members of this line, namely
FR, FH and FN. In addition, (-)HAA was also found in FO, which
integrates a different lineage, posing the question as to whether FO
should be included in this line. Future phytochemical studies
comprising the screening of (-)HAA in the vast majority of species
within the genus will certainly provide new insights for a revision
of species composition within lineages.
Previous work demonstrated that in FC, the specific phytotoxic
activity detected in the bioassays performed with the aqueous
extracts was 3-fold higher than that observed with the isolated
(-)HAA [7]. The difference in bioactivity observed could not be
explained on the basis of (-)HAA concentration (alone). Taking into
account that recovery of (-)HAA through EtOAc extraction from
the aqueous phase was very efficient (more than 95%), the higher
phytotoxicity of the aqueous extracts could be explained by the
presence of other phytotoxins which have not been extracted with
EtOAc (the phase used for bioassay-guided fractionation) and/or the
action of natural detergents that would raise the solubility of
(-)HAA, and thus its bioactivity. In order to ascertain the
relationship between the presence of (-)HAA and the possible
phytotoxic effects of the different Flourensia species, bioassays of
leaf aqueous extracts from (-)HAA containing (FO and FF) and
lacking (FR) Flourensia species were investigated, and compared
with the corresponding content of (-)HAA detected in each species.

As regards to the genus, the bioactive sesquiterpene failed to serve

as a chemotaxonomic marker, as it was only detected in three of the
six Flourensia species investigated; 50 % of those described in our
country. Dillon´s work [17] is the only comprehensive revision of
the genus Flourensia and has not been revisited until now. He based
the grouping of Flourensia species primarily on the comparison of
exomorphic features and partial phytochemical data (Figure 2). In
Figure 2: Dillon [17] phylogram of Flourensia species.
his phylogram, South American taxa are divided into three weakly
differentiated lines; whilst our studied species are present in two of An enlarged version of the South American lines of Flourensia species relationships is
them. One of these lines is integrated by eight species shown. Key to abbreviations: FA= F. angustifolia; FB= F. blakeana; FC= F.
campestris; FF= F. fiebrigii; FH= F. hirta; FHe= F. heterolepis; FHt= F. hirtissima;
geographically distributed from southern Bolivia to southern FL= F. leptopoda; FM= F. macroligulata; FMp= F. macrophylla; FN= F. niederleinii;
Argentina: FR, FC, F. leptopoda, FN, FH, FF, and F. blakeana; all FO= F. oolepis; FP= F. peruviana; FPo= F. polycephala; FR= F. riparia; FS= F.
species exhibit a trend toward fewer ray florets, with the majority suffrutescens; FT= F. tortuosa; FTh= F. thurifera.
Phytotoxicity of South American Flourensia sp. Natural Product Communications Vol. 9 (3) 2014 343

Table 1: (-)HAA leaf content and biological activity of aqueous extracts from Table 2: FC and FO community coexisting species.
Flourensia species.
Species Family
Flourensia Leaf (-)HAA Phytotoxicity of leaf aqueous extracts Natives
species  content  (% w/v)  Lithraea molleoides (Vell.) Engl. Anacardiaceae
-1
(mg g DW)  ECg50 ECr50 ECs50 Acanthostyles buniifolium (Hook and Arn.) King and Rob Asteraceae
FC    16.0 ± 0.7 ** a*
3.1 ± 0.4   1.4 ± 0.1 b * **  2.2 ± 0.1 a ** Achyrocline satureioides (Lam.) DC. Asteraceae
a ** b *** Ambrosia tenuifolia Spreng. Asteraceae, H
FO    0.80 ± 0.01  *
4.9 ± 0.1   4.0 ± 0.1   4.3 ± 0.2 b ***
Baccharis articulata (Lam.) Pers. Asteraceae
FF  15.2 ± 0.7 **  4.6 ± 0.1 a **  2.2 ± 0.4 b **  1.9 ± 0.3 b * ** Bidens pilosa L. Asteraceae
FR  Not detected  2.5 ± 0.1 a *  1.0 ± 0.02b *  1.3 ± 0.3 b * Eupatorium viscidum Hook.et Arn. Asteraceae
Data are expressed as mean ± se of triplicates {(-)HAA content}, and from dose- Heterothalamus alienus (Spreng.) Kuntze Asteraceae
response curves (EC50) of two different bioassays with three replicates for each Parthenium hysterophorus L. Asteraceae, H
concentration (aqueous extracts). Different letters (a, b, c) and symbols (*, **, ***) Viguiera tucumanensis (Hook. Et Arn.) Griseb. Asteraceae, H
indicate significant differences between phytotoxic effects and species, respectively Vernonanthura nudiflora (Less.) H.Rob. f. nudiflora Asteraceae
(p<0.05). Thelesperma megapotamicum (Spreng.) Kuntze Asteraceae
Xanthium spinosum L. var. spinosum Asteraceae, H
Zinnia peruviana (L.) L. Asteraceae, H
As far as we reviewed, the herbicidal bioactivity of leaf aqueous Croton lachnostachyus Baill Euphorbiaceae
Acacia caven (Mol.) Molina Fabaceae
extracts of FO, FF and FR on lettuce had never been assessed Apurimacia dolichocarpa (Gris.) Burkart Fabaceae
before. The concentration of (-)HAA (mg g-1 DW) in the leaves of Collaea argentina Griseb. Fabaceae
FF was similar to that in FC, and significantly higher than that Sophora linearifolia Griseb. Fabaceae
Colletia spinosissima J. F. Gmel. Rhamnaceae
found in FO (Table 1). As shown in Table 1, all the leaf aqueous Condalia microphylla Cav. Rhamnaceae
extracts from the Flourensia species tested showed bioactivity at Kageneckia lanceolata Ruiz and Pav. Rosaceae
CE50 below 5% (g of leaves per 100 mL of water). These values fall Cestrum parqui L´ Hér. Solanaceae
Celtis ehrenbergiana (Klotzch) Liebm. Ulmaceae
within the range found in species with potential allelopathic activity Aloysia gratissima (Gillies and Hook. Ex Hook.) Tronc. Verbenaceae
[19, 20], as compared with other species whose leaf aqueous Larrea divaricata Cav. Zygophyllaceae
Exotics
extracts would not exhibit bioactivity on lettuce even at Carduus acanthoides L. Asteraceae
concentrations as high as 10 or 20% [21, 22]. FC and FR showed Xanthium cavanillesii Schouw. Asteraceae, H
similar inhibitory effects on germination, and were significantly Gleditsia triacanthos L. Leguminosae
Robinia pseudoacacia L. Leguminosae
higher compared with both FO and FF (p<0.05, Table 1). The Melia azedarach L. Meliaceae
phytotoxic effects of leaf extracts on germination among (-)HAA Ligustrum lucidum W.T. Aiton Oleaceae
Ligustrum sinense Lour. Oleaceae
containing species (FC, FO and FF) was not consistent with their Pinus taeda L. Pinaceae
respective (-)HAA contents, confirming the presence of other Cotoneaster franchetii Bois Rosaceae
phytotoxic compounds in the case of FO, as was already described Pyracantha atalantioides (Hance) Stapf Rosaceae
Ulmus pumila L. Ulmaceae
[10]. Instead, the response of root and shoot growth were shown to
List of 37 coexisting species studied from local Flourensia communities H, Heliantheae
be correlated with the (-)HAA concentration in these species; ECr50 tribe.
and ECs50 were ca. 2x lower in FC and FF compared with FO
(Table 1). Leaf extracts of FR also exerted a drastic inhibition of investigated is partially supported by previous results highlighting
root and shoot growth, similar to that of FC and FF. Root growth the high amount of (-)HAA accumulated on leaves surface and the
was the most sensitive parameter affected by the leaf extracts of feasibility of being extracted with water [7], and reinforced by the
most of the tested species (FC, FF and FR), a result that is present results showing that the compound is solely produced by
commonly reported in many studies dealing with allelochemicals Flourensia species. However, in order to confirm this hypothesis
[23, 24]. The unexpected strong phytotoxic effects of leaf aqueous further investigations should be carried out in order to test species´
extracts found in FR, a non (-)HAA containing species, clearly response to (-)HAA at the community level.
indicate the presence of powerful bioactive compounds which
deserves further elucidation. As concluding remarks, present work showed that (-)HAA
originally isolated from FC was also identified in FF and FO, and
The natural occurrence of the bioactive compound {(-)HAA was absent in other South American species of the genus, like FR,
moiety} was restricted to Flourensia species in their communities, FH and FN. The isolated compounds were chemically and
being absent in all the 37 FC and FO coexisting species tested biologically characterized as (-)HAA, discarding the possibility of
(26 natives and 11 exotics, Table 2). From the 15 Asteraceae its isomer (+)HAA or other related sesquiterpenes with similar
species studied, 6 belonged to Heliantheae, which correspond chromatographic behavior. In local communities, the sesquiterpene
to the same tribe as Flourensia (Table 2). Despite similar C-7, was not widely distributed, but restricted to the species of the genus
C-8-dioxygenated bisabolanes having been isolated from other Flourensia. According to previous genus exomorphologic
Asteraceae [24, 25], our results show that Flourensia was the only groupings, our chemotaxonomic data support the close link between
(-)HAA producing Asteraceae in these communities. The compound FC and FF, but not with FR. The phytotoxic effects of leaf aqueous
was not found in the three coexisting Rosaceae studied either, a extracts observed seem to be a common trait in this genus, and were
family where its enantiomer (-)HAA had been firstly described by not restricted to (-)HAA containing Flourensia species. This was
Hashidoko et al. [8]. Moreover, a screening performed in other the case of FR where the presence of other phytotoxic compounds is
domestic Rosaceae (Spiraea cantoniensis Lour., Malus pumila under investigation. Taken together with previous findings, the
Mill., Prunus persica (L.) Stokes, and Cydonia oblonga Mill.) also restricted production of (-)HAA by Flourensia in their communities
failed to detect (-)HAA in any of these species (data not shown). It confirms a specific link with the genus, and sustains its putative
is puzzling how the two different HAA enantiomeric forms were allelochemical role in the communities studied.
found in phylogenetically distant plants such as those belonging to
Asteraceae and Rosaceae. This aspect highlights the complexity of Experimental
the sesquiterpene biochemical pathways characteristic of plants.
Plant material: Air dried leaves of adult plants from natural areas
The possibility that (-)HAA may play a role as an alellopathic were used. Endemics Flourensia campestris Griseb. and F. oolepis
agent involved in plant-plant interactions within the communities S.F. Blake were harvested in Punilla Valley (700-800 MASL),
344 Natural Product Communications Vol. 9 (3) 2014
Córdoba province, Central Argentina. Another four Flourensia
species were collected within the geographic range of distribution
of each species. F. hirta S.F. Blake (endemic), was collected in La
Rioja province (1640 MASL), where it grows as a companion
species in different bush-dominated communities (ca. 400 km
northwest from Punilla Valley). F. niederleinii S.F. Blake
(endemic) was also collected in La Rioja, on the slopes of Sierras de
Velazco (1543 MASL), from almost pure stands. F. riparia Griseb
(endemic) and F. fiebrigii S.F. Blake were both collected in the
province of Salta (ca. 900 km north from Punilla Valley, 1000
MASL and 3000 MASL; respectively).
In order to evaluate the specificity of (-)HAA in the genus, the
compound was screened in local Flourensia communities. The most
conspicuous native species found growing together with FC and FO
in their natural shrub dominated environments [26, 27], as well as
exotic species that invade the same sites were collected (Table 2).
All the species were determined by Drs J.J. Cantero and G.M.
Tourn.
Identification and quantification of (-)hamanasic acid A by 2D-
TLC: Briefly [7], powdered air dried leaves (50 mg) were extracted
twice using 1.5 mL and 0.5 mL EtOAc, vortexed for 2 min and
centrifuged (10 min, 300 g). EtOAc extracts were pooled and 3 µL
was loaded on chromatograms (Silica gel 60 (Merck), aluminum
sheets, F254). Once developed in two dimensions using
corresponding solvents, (-)HAA was elucidated at a final Rf=0.35 as
a single blue spot through UV254 light, which stained strongly with
I2 vapors. Semi-quantitative results were obtained using different
concentrations of pure (-)HAA as standard, which were in
agreement with the accurate concentration of (-)HAA obtained by
the GC-FID standard curve in plant extracts [7].
GC-FID: EtOAc extracts were re-suspended in toluene plus N,O-
Bis(trimethylsilyl)trifluoroacetamide (950 + 50 µL, respectively)
prior to analysis. The (-)HAA concentration was assessed by
GC-FID (GC-hydrogen flame ionization detector, Agilent GC 6890)
using a calibration standard curve (0.005–1.0 mg mL-1), as already
described [7]. The column was ZB-5HT (15 m x 0.32 mm i.d.). The
injector temperature was set to 200°C with an injection volume of 4
µL, split ratio of 10 and N2 flow of 2 mL min-1. The oven
temperature program began at 60°C with a ramp rate of 3°C min-1.
The final temperature was 290°C (200-245 ºC, 40ºC min-1;
245-255ºC, 1ºC min-1; 255-290ºC, 50ºC min-1), which was held for
5 min making a total run time of 18 min per sample.
(-)HAA isolation: In order to increase the yield of (-)HAA
originally isolated from FC through bio-guided fractionation [7], a
more efficient methodology was devised, and then applied
successfully to other plant materials where (-)HAA had been
previously detected by 2D-TLC.
Powdered air dried FC leaves (25 g) were shaken vigorously with
200 mL EtOAc for 2 min and filtered. The obtained extract was
partitioned with a polar solvent (200 mL H20 + 10 mL MeOH + 4
mL of H4NOH), mixed vigorously for 2 min and let stand for 24 h
at 4ºC. The lower polar phase (ca. 250 mL) was partitioned
with 255 mL of a solvent described by Hashidoko et al. [8] (125 mL
n-hexane + 125 mL EtOAc + 5 mL formic acid), mixed vigorously
for 2 min and let stand for 20 min at room temperature. The upper
organic phase was evaporated in vacuo. The resulting extract (1.3 g)
was fractionated by silica gel CC (1.5 cm i.d., 47 cm; 50 g silica gel
60 (Merck), 70-230 mesh) eluted at a flow rate of 8 mL min-1 using
the described Hashidoko´s solvent. After eluting with 60 mL of
solvent the collected 1 min fractions were subjected to TLC using
López et al.
CHCl3–EtOAc–MeOH (5:1:0.25; Solvent 1), and pure (-)HAA as
standard, detected under UV254 and with I2 vapors. Selected
fractions (Fr. 3-6) were pooled, dried at 30ºC with N2 flow (ca. 200
mg) and further fractionated by an equivalent CC using Solvent 1.
After eluting with 150 mL of solvent, the collected and dried 2 min
fractions (Fr. 2-18; revealed by TLC) afforded ca. 100 mg of pure
(-)HAA (ca. 4 %, DW basis). The described methodology improved
200 times the extraction yield in FC as compared with that
originally performed [7], and, once applied to other species, yielded
sufficient amounts of the compound for further chemical and
biological characterizations.
(-)HAA chemical characterization: The putative (-)HAA identified
and isolated from other species was chemically characterized from
1
H NMR, 13C NMR, DEPT, COSY, HMBC and HSQC spectra in
CDCl3, by GC–MS and polarimetry, and compared with previously
reported data, as already described [7]. Solvents were used from
commercial sources without purification. 1H NMR (400 MHz), 13C
NMR (100 MHz), DEPT, COSY, HMBC and HSQC spectra were
recorded at room temperature with Bruker AC 400 spectrometers.
The spectra were recorded in CDCl3 and the solvent signals (7.26
ppm for 1H NMR and 77.16 ppm for 13C-NMR) were used as
reference [28]. Coupling constants, J, are reported in Hertz (Hz).
Optical rotation was measured on a Jasco P-1010 polarimeter for
solutions in acetone (ca. 0.5%) at 25°C. GC-MS identification was
obtained on GC-17A Shimadzu and QP-5000 MS Shimadzu
machines. The GC column was HP 5% phenylmethylsilicone
(Alltech) (30 m x 0.32 mm i.d.). The linear temperature program
was from 150 to 300ºC, at the rate of 10ºC min-1, and the carrier gas
was He (1 mL min-1). UV spectra were recorded on a Metrolab
2500.
The compound isolated from FF was obtained as a colorless syrup;
[α]D25 - 64.9° (c 0.70 , acetone). Results from 1H NMR, 13C NMR,
DEPT, COSY, HMBC and HSQC spectra in CDCl3, and GC–MS
analysis, were the same as those already reported for (-) 7-carboxy-
8-hydroxy-1(2),12(13)-dien-bisabolene [7].
(-)HAA biological characterization: Pure and dried (-)HAA
isolated from other species was dissolved in EtOAc and bioassayed
at 0.5, 1.0 and 1.5 mg mL-1 (1.0, 2.0 and to 3.0 mM, respectively).
Bioactivity was evaluated on seeds of lettuce (Lactuca sativa,
Grand Rapids) [7]. Briefly, 50 seeds of lettuce were placed in a 9.5
cm Petri dish lined with one sheet of filter paper previously
moistened with each test solution (3 mL), and allowed to germinate
in a growth chamber in darkness at 22 ± 1ºC. Solvents were
evaporated at 40ºC before adding H2O (3 mL) to both samples and
controls. Two different bioassays with 3 replicates for each
concentration were performed. Alternatively, bioassays using 25
seeds of lettuce placed in a 5.0 cm Petri dish lined with one sheet of
filter paper previously moistened with each test solution (1.5 mL)
were used. For comparison purposes, and as experimental controls,
bioassays using pure FC (-)HAA were also performed.
Seed germination and the lengths of roots and shoots were assessed
at 3 days, as previously described [7]. Controls showed (mean ±
s.e.) 93.7 ± 0.5 % of germination and 2.1 ± 0.1 cm of root and 1.1 ±
0.1 cm of shoot growth. Effective concentrations capable of
inhibiting 50% germination, root growth or shoot growth were
calculated as ECg50, ECr50 and ECs50, respectively. The results were
analyzed by ANOVA and Tukey´s test (p < 0.05).
Biological activity of aqueous extracts from Flourensia species:
Air dried leaves were extracted in distilled H2O (6 g of plant
material in 100 mL) for 24 h at 22ºC. Aqueous extracts were
Phytotoxicity of South American Flourensia sp. Natural Product Communications Vol. 9 (3) 2014 345

bioassayed at 6%, w/v, as well as serial dilutions with H2O at 3.0 Tecnológica (ANPCyT) Grant PICT 0411, the Universidad de
and 1.5%, as described above. Buenos Aires, UBACyT 0566, and the Ministerio de Ciencia y
Tecnología – Córdoba, Grant PID 2010. We are most grateful to Dr
For comparison purposes, and as experimental controls, bioassays Pablo Ortega-Baes for his help in the collection of plant material,
using aqueous extracts from FC leaves were also performed. Dr M. Laura Uriburu for providing initial plant samples, and
Augusto Maillet for his dedication and technical assistance in the
Acknowledgments - This research was supported financially by laboratory.
grants from the Agencia Nacional de Promoción Científica y
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 Natural Product Communications Vol. 9 (3) 2014
Published online (www.naturalproduct.us)

New Schiartane-type Triterpene from Kadsura heteroclita and their Cytotoxic Activities
Pham Thi Hong Minh, Do Tien Lam, Nguyen Quyet Tien, Nguyen Ngoc Tuan, Vu Phuong Nhung, Nong Van Hai,
Phan Van Kiem, Nguyen Xuan Nhiem, Chau Van Minh, Park Seon Ju and Kim Seung Hyun 373
C-Lactam Derivatives of Oleanolic Acid. Hydrolysis and Further Acylation of Methyl Acetyloleanolate C-Lactam and
C-Thiolactam
Barbara Bednarczyk – Cwynar and Lucjusz Zaprutko 375
New Acylated Triterpene Glycosides from the Roots of Polygala tenuifolia
Minpei Kuroda, Takaaki Shizume and Yoshihiro Mimaki 379
Cucurbitane-type Triterpene Glycosides from the Fruits of Momordica charantia
Pham Hai Yen, Duong Thi Dung, Nguyen Xuan Nhiem, Hoang Le Tuan Anh, Dan Thi Thuy Hang, Duong Thi Hai Yen,
Nguyen Thi Cuc, Ninh Khac Ban, Chau Van Minh and Phan Van Kiem 383
Cytotoxic Effects of Four Aescin Types on Human Colon Adenocarcinoma Cell Lines
Ewa Seweryn, Michał Gleńsk, Kamila Środa-Pomianek, Ireneusz Ceremuga, Maciej Włodarczyk and Andrzej Gamian 387
Structures of Violaceusosides C, D, E and G, Sulfated Triterpene Glycosides from the Sea Cucumber
Pseudocolochirus violaceus (Cucumariidae, Dendrochirotida)
Alexandra S. Silchenko, Anatoly I. Kalinovsky, Sergey A. Avilov, Pelageya V. Andryjaschenko, Pavel S. Dmitrenok,
Vladimir I. Kalinin, Ekaterina A. Yurchenko and Salim S. Dautov 391

Review/Account

Phytotoxic Terpenes Produced by Phytopathogenic Fungi and Allelopathic Plants

Alessio Cimmino, Anna Andolfi and Antonio Evidente 401
Pungent and Bitter, Cytotoxic and Antiviral Terpenoids from Some Bryophytes and Inedible Fungi
Yoshinori Asakawa, Fumihiro Nagashima, Toshihiro Hashimoto, Masao Toyota, Agnieszka Ludwiczuk, Ismiarni Komala,
Takuya Ito and Yasuyuki Yagi 409
Terpenoids and Sterols from Some Japanese Mushrooms
Yasunori Yaoita, Masao Kikuchi and Koichi Machida 419
Phytochemicals and Biological Activities of Poisonous Genera of Ericaceae in China
Xiaohong Wang, Rui Jiang, Zizhen Liu, Weirui Liu, Meng Xie, Shengli Wei and Gaimei She 427
 Natural Product Communications
2014
Volume 9, Number 3
Contents

Bioactive Terpenes – From Isolation to Chemical Synthesis

Original Paper Page

An Expedient Synthesis of Linden Ether

Stefano Serra and Alessandra A. Cominetti 293
Simple and Short Synthesis of Trans-(R)-Nerolidol, a Pheromone Component of Fruit Spotting Bug
Thanh C. Le and Kamlesh R. Chauhan 297
Monoterpene Citral Derivatives as Potential Antimalarials
Soni Singh, Reena P. Khandare, Manish Sharma, Virendra K. Bhasin and Sujata V. Bhat 299
A General Synthetic Approach to Hydroquinone Meroterpenoids: Stereoselective Synthesis of (+)-(S)-Metachromin V
and Alliodorol
Stefano Serra, Alessandra A. Cominetti and Veronica Lissoni 303
Synthetic Approach to the Psoracorylifols
George A. Kraus and Pengfei Dong, 309
Chemotaxonomic Value of Magastigmane Glucosides of Cichorium calvum
Klaudia Michalska, Alex Beharav and Wanda Kisiel 311
Cyclonerol Derivatives from Trichoderma longibrachiatum YM311505
Qi-Cun Xuan, Rong Huang, You-Wei Chen, Cui-Ping Miao, Kai-Xia Ma, Tang Wang and Shao-Hua Wu 313
Anti-influenza Sesquiterpene from the Roots of Reynoutria japonica
Nguyen Xuan Nhiem, Phan Van Kiem, Chau Van Minh, Nguyen Thi Hoai, Ho Viet Duc, Bui Huu Tai, Tran Hong Quang,
Hoang Le Tuan Anh, Sang-Gu Yeo, Jae-Hyoung Song, Doo-Sung Cheon, Moon Ho Park, Hyun-Jeong Ko and Seung Hyun Kim 315
Lactarane Sesquiterpenes from the European Mushrooms Lactarius aurantiacus, L. subdulcis, and Russula sanguinaria
Gianluca Gilardoni, Omar Malagòn, Solveig Tosi, Marco Clericuzio and Giovanni Vidari 319
Laurane-, Cyclolaurane-, and Cuparane-type Sesquiterpenes from the Marine Red Alga Laurencia okamurai
Yi Liang, Xiao-Ming Li, Chun-Shun Li, Hong Sun and Bin-Gui Wang 323
The First Isolation of Furanoeremophilane from Ligularia nelumbifolia
Hiroshi Hirota, Yurie Horiguchi, Satoru Kawaii, Ryo Hanai, Xun Gong and Chiaki Kuroda 325
Use of (S)-trans-γ-Monocyclofarnesol as a Useful Chiral Building Block for the Stereoselective Synthesis of Diterpenic
Natural Products
Stefano Serra, Alessandra A. Cominetti and Veronica Lissoni 329
Bioactivity-guided Isolation of Antiproliferative Compounds from the Roots of Onopordum acanthium
Boglárka Csupor-Löffler, István Zupkó, Judit Molnár, Peter Forgo and Judit Hohmann 337
Distribution of (-)-Hamanasic Acid A in South American Species of Flourensia and Phytotoxic Effects of Leaf Aqueous Extracts
Daniela López, Leonardo A. Piazza, Mariana P. Silva, Marisa J. López Rivilli, Juan J. Cantero, Graciela M. Tourn and Ana L. Scopel 341
neo-Clerodane Diterpenoids from Scutellaria galericulata
Petko I. Bozov, Plamen N. Penchev and Josep Coll 347
Seed Dormancy Breaking Diterpenoids, Including Novel Brassicicenes J and K, from Fungus Alternaria brassicicola, and
their Necrotic/Apoptotic Activities in HL-60 Cells
Hiromichi Kenmoku, Sayaka Takeue, Megumi Oogushi, Yasuyuki Yagi, Takeshi Sassa, Masao Toyota and Yoshinori Asakawa 351
Facile Access to Optically Active Ring C Aromatic Diterpene Derivatives from (+)-Manool. First Synthesis of
13,14-Dihydroxy-8,11,13-podocarpatrien-7-one
María L. Nóvoa, Franklin J. Salazar, Carlos Gámez, Ana Y. Angarita, Eleonora Tropper, Nieves Canudas and José E. Villamizar 355
Antiproliferative Effects of 12-Oxoheteronemin vs Heteronemin
Siriporn Kittiwisut, Cristina C. Rohena, Supreeya Yuenyongsawad, Susan L. Mooberry and Anuchit Plubrukarn 359
New Cembranoids from the Soft Coral Sinularia arborea
Li-Hsueh Wang, Kuan-Hua Chen, Chang-Feng Dai, Tsong-Long Hwang, Wei-Hsien Wang, Zhi-Hong Wen,
Yang-Chang Wu and Ping-Jyun Sung 361
T-DNA Insertion Alters the Terpenoid Content Composition and Bioactivity of Transgenic Artemisia annua
Netiya Karaket, Suthep Wiyakrutta, Marie-Aleth Lacaille-Dubois and Kanyaratt Supaibulwatana 363
A New Cytotoxic Tirucallane from the Twigs of Walsura trichostemon
Jirapast Sichaem, Pongpun Siripong, Santi Tip-pyang and Jatuporn Phaopongthai 367
A New Ergostane Triterpenoid from Cultures of the Basidiomycete Inocybe lilacina
Dong-Ze Liu, Qi Liu, Ping Yang, and Wen-Xia Jiang 369
New Ursane Triterpene from the Fruits of Terminalia arjuna
Rashadul Hossain, Rajia Sultana, Aychout Adhikari, Muhammad Iqbal Choudhary, Yusuff Ali and Shahed Zaman 371

Continued inside backcover