This article examines the cytotoxic effects of perezone and its isomer isoperezone on human leukemia cells. The key findings are: 1. Both perezone and isoperezone induce cytotoxicity in a concentration-dependent manner through both caspase-dependent and caspase-independent mechanisms. 2. The compounds cause changes in cell size, granularity, phosphatidylserine translocation, mitochondrial membrane potential disruption, and do not induce apoptosis inducing factor. 3. Perezone showed greater cytotoxic effects than isoperezone, but the mechanisms of cell death induction differed between the two compounds depending on concentration.

1. This article appeared in a journal published by Elsevier. The attached
copy is furnished to the author for internal non-commercial research
and education use, including for instruction at the authors institution
and sharing with colleagues.
Other uses, including reproduction and distribution, or selling or
licensing copies, or posting to personal, institutional or third party
websites are prohibited.
In most cases authors are permitted to post their version of the
article (e.g. in Word or Tex form) to their personal website or
institutional repository. Authors requiring further information
regarding Elsevier’s archiving and manuscript policies are
encouraged to visit:
http://www.elsevier.com/copyright

2. Author's personal copy
ARTICLE IN PRESS
Phytomedicine 17 (2010) 614–620
Contents lists available at ScienceDirect
Phytomedicine
journal homepage: www.elsevier.de/phymed
Perezone and its isomer isoperezone induce caspase-dependent and
caspase-independent cell death
L.E. Sanchez-Torres a,n, J.A. Torres-Martınez a, M. Godınez-Victoria a,b, J.-M. Omar c, B. Velasco-Bejarano c
´ ´ ´
a
´ ´ ´ ´ ´
Departamento de Inmunologıa, Escuela Nacional de Ciencias Biologicas-IPN, Prol. Carpio y Plan de Ayala s/n, Colonia Santo Tomas, Mexico, D.F., C.P. 11340, Mexico
b
´ ´ ´ ´az ´ ´
Departamento de Bioquımica y Seccion de Estudios de Posgrado e Investigacion, Escuela Superior de Medicina-IPN, Salvador Dı Miron y Plan de San Luis, Colonia Santo Tomas,
´ ´
Mexico, D.F., C.P. 11340, Mexico
c
´ ´ ´ ´ ´ ´ ´
Departamento de Ciencias Quımicias, Seccion Quımica Organica Facultad de Estudios Superiores Cuautitlan, Universidad Nacional Autonoma de Mexico, Av. 1 de mayo S/N,
´ ´ ´
Cuautitlan Izcalli, Estado de Mexico, C.P. 54740, Mexico
a r t i c l e in f o a b s t r a c t
In this study, we investigated the cytotoxic effect of perezone, a constituent isolated from the roots of
Keywords: Perezia spp. and of its synthetic isomer isoperezone on the K562 human leukemia cell line. Perezone
Perezone showed greater cytotoxic effect than isoperezone but both compounds were found to induce
Isoperezone cytotoxicity trough a caspase-dependent and a caspase-independent mechanisms; important changes
Cytotoxic in their light scattering properties, phosphatidylserine translocation and mitochondrial membrane
Sesquiterpenes potential disruption were detected by cytometry. The mechanism of death induction of each compound
Cell death showed interesting concentration-dependent differences. Neither compound induced the apoptosis
inducing factor.
& 2009 Elsevier GmbH. All rights reserved.
Introduction with more accurate descriptions of the biochemical and cellular
parameters that are actually measured. This is because, there are
The therapeutic goal of cancer treatment has been to trigger events that are not exclusive to one type of cell death (Kroemer
tumor selective cell death. Until now, agents inducing apoptosis et al., 2009). In the present report, the recommendations of the
remain the main chemotherapeutic drugs in medical oncology NCCD were taken into account.
(Lefranc et al., 2007; Ghobrial et al., 2005; Howley and Fearnhead, There is growing interest in the ability of phytochemicals to
2008). Apoptosis plays an essential role in regulating develop- prevent and/or cure chronic diseases such as cancer (Howells
ment, growth, immune response and eliminating redundant or et al., 2007). The widespread occurrence and function of quinones
abnormal cells in organisms (Reviewed in Lockshin and Zakeri, in diverse biological systems with particular reference to their
2007 and Lamkanfi et al., 2007). The induction and execution of role in oxygen activation have been used to know the mechanisms
apoptosis require the cooperation of a series of molecules and significance of oxidation–reduction reactions and the energy
including signal molecules, receptors, enzyme and gene regulat- conserving systems in living cells (Nohl et al., 1986). Perezone
ing proteins (Howells et al., 2007; Fan et al., 2005). In addition to (2-(1,5-dimethyl-4-hexenyl)-3-hydroxymethyl-p-benzoquinone),
apoptosis, other types of death have been described. Cell death a sesquiterpene quinone, is recognized as the first secondary
has been classified according to morphological appearance, metabolite isolated in crystalline form in the New World, by Rıo ´
enzymological criteria, functional aspects or immunological ´
de la Loza in 1852 from roots of plants of the genus Perezia (Rıo de
characteristics. Due to the complexity of the subject, the la Loza, 1852). Several pharmacological effects of perezone have
Nomenclature Committee on Cell Death (NCCD) has proposed ´
been reported (Enrıquez et al., 1980; Alarcon-Aguilar et al., 1997;
unified criteria for the definition of cell death and recommends to ˜ ˜
De La Pena et al., 2001; Burgueno-Tapia et al., 2008). Interesting,
avoid expressions like ‘‘percentage apoptosis’’ and to replace them perezone promotes the release of intramitochondrial Ca2 + , thus,
maintaining ATP production during experimental reperfusion
(Tellez et al., 1999), and induce Ca2 + efflux by inducing collapse
´
Abbreviations: AIF, apoptosis inducing factor; DMSO, dimethylsulfoxide; FBS, of the membrane potential and oxidation of pyridine nucleotides,
fetal bovine serum; FITC, fluorescein isothiocyanate; FSC, forward scatter; SSC, producing a cytotoxic effect through an impairment in Ca2 +
side scatter; GSH, glutathione; PE, phycoerythrin; PI, propidium iodide; Rho 123,
rhodamine 123; TP, transition pore
homeostasis (Cuellar et al., 1987). It is clear that perezone acts as
n
Corresponding author. Tel./fax: +52 55 57296300x62489. an electron sink which results in inhibition of electron transport
´
E-mail address: luviasanchez@hotmail.com (L.E. Sanchez-Torres). (Carabez and Sandoval, 1988), but its cytotoxic activity has not
0944-7113/$ - see front matter & 2009 Elsevier GmbH. All rights reserved.
doi:10.1016/j.phymed.2009.12.011

3. Author's personal copy
ARTICLE IN PRESS
´
L.E. Sanchez-Torres et al. / Phytomedicine 17 (2010) 614–620 615
been evaluated yet in the view of its possible use as anti-neoplasic ´
perezone according to protocol previously described (Martınez
agent. In order to clarify the mechanism of the sesquiterpenes et al., 2008). The complete 1H and 13C NMR data assignment was
perezone- and isoperezone-induced cell death, modification in made by comparison with previous data reported (Burgueno- ˜
cell size and granularity, translocation of phosphatidylserine, ´
Tapia and Joseph-Nathan, 1997; Martınez et al., 2008).
activation of caspase-3, mitochondrial potential and the presence
of the inducing apoptosis factor (AIF) in the cytoplasm were
evaluated. Cultures
Perezone and isoperezone were dissolved in a minimum
Materials and methods concentration of dimethylsulfoxide (DMSO, Sigma) and added to
the culture medium at final concentrations of 6.25, 12.5, 25, 50
Cell culture and 100 mM for 24 h. The final concentration of DMSO in the
culture medium was 0.1%. As a positive control of apoptosis K562
K562 cells were cultured in RPMI 1640 medium with 10% heat- were culture with cisplatin (CISP, 30 mg/ml). Treated and
inactivated fetal bovine serum (FBS), 100 units/ml penicillin, untreated (controls) cells were grown in 24-well plates with
100 mg/ml streptomycin and 2 mM L-glutamine. Cells were 5 Â 105 cells/well under the same conditions.
routinely grown at 37 1C in a humidified atmosphere of 5% CO2.
Perezone and isoperezone Viability assay
Perezone (Fig. 1a) was isolated from the dry roots of Perezia In order to investigate if the tested compounds were cytotoxic,
´
spp. as described previously (Enrıquez et al., 1980). The non- cells treated for 24 h were evaluated in their viability by Trypan
natural derivative isoperezone (Fig. 1b) was obtained from blue exclusion analysis.
Fig. 1. Chemical structure of perezone (a) and isoperezone (b).
120
Perezone
100 Isoperezone
*
*
80
*
Viability (%)
60 *
40
*
20
*
*
0
RPMI DMSO CISP 12,5 25 50 100
Controls Concentration (μM)
Fig. 2. Perezone and isoperezone induce cell death. Cytotoxicity induced by different concentrations of perezone and isoperezone was evaluated in K562 cells treated for
24 h by the Trypan blue exclusion assay. As a positive control K562 were culture with cisplatin (CISP, 30 mg/ml). Data represent the mean 7 SD of three different
experiments.

4. Author's personal copy
ARTICLE IN PRESS
616 ´
L.E. Sanchez-Torres et al. / Phytomedicine 17 (2010) 614–620
Phosphatidylserine translocation V-fluorescein isothiocyanate (FITC) and propidium iodide (PI)
according to the manufacturer’s instructions (Caltag Laboratories).
To investigate whether the compound-induced decrease in cell Samples were acquired on a FACScalibur flow cytometer (Becton
viability is accompanied with phosphatidylserine translocation, ´,
Dickinson, San Jose California, USA) and analyzed using Cellquest
cells treated as described above were dual stained with Annexin software (Becton Dickinson). A total of 10,000 cells were recorded.
RPMI DMSO
Controls
CISP
30 μg/ml
Perezone Isoperezone
6.25 μM
12.5 μM
25 μM
50 μM
100 μM
Fig. 3. Perezone and isoperezone induce changes in FSC and SSC parameters. Cells untreated (RPMI and DMSO) and treated for 24 h with cisplatin (CISP, 30 mg/ml) or with
perezone or isoperezone at different concentrations (6.25, 12.5, 25, 50 and 100 mM) were analyzed in their FSC and SSC parameters by flow cytometry. Representative dot
plots are shown of at least three performed (arrow, lower FSC parameter).

5. Author's personal copy
ARTICLE IN PRESS
´
L.E. Sanchez-Torres et al. / Phytomedicine 17 (2010) 614–620 617
Active caspase-3 washed with PBS and incubated with 20 mg/mL Rhodamine
123 (Rho123, Sigma) at room temperature for 20 min.
To evaluate if the cells death induced was caspase-dependent, After washing cells twice, Rho123-related fluorescence was
cells treated as described previously and untreated cells were recorded and analyzed using a FACSCalibur cytometer (Becton
stained with a FITC-conjugated anti-active caspase-3 antibody Dickinson). Viable cells retain the Rho123 and apoptotic cells
according to the manufacturers specifications (Pharmingen). The release the dye and are detected as cells with reduced fluores-
fluorescent signal intensity was recorded and analyzed in a cence. Results are shown as percentage of cells without Rho123
FACSCalibur flow cytometer (Becton Dickinson). For each sample retention.
10,000 events were collected.
AIF determination
Mitochondrial membrane potential
In order to evaluate if the AIF factor was involved in the cell
To evaluate if the induced cell death on K562 cells altered the death induced by perezone or isoperezone, a PE-labeled anti-AIF
mitochondrial membrane potential, cells were treated with antibody was used. Cells were processed according the manu-
perezone or isoperezone for 24 h. After culture, K562 cells were facturer’s specifications (Pharmingen). Cells were acquired and
120
Perezone
100 * Isoperezone
*
Annexin V positive cells (%)
* *
*
80 *
60
*
*
40
*
20
0
RPMI DMSO CISP 6,25 12,5 25 50 100
Controls Concentration (μM)
Fig. 4. Perezone and isoperezone induce phosphatidylserine translocation. Cells untreated and treated with cisplatin (CISP, 30 mg/ml) or with perezone or isoperezone at
different concentrations for 24 h were stained with Annexin V and PI and analyzed by flow cytometry. Data represent the mean 7 SD of three different experiments
(nPr 0.05).
Perezone
120
Isoperezone
*
100 * *
Cells with low ΔΨm (%)
*
80
*
60
40
*
20 * *
0
RPMI DMSO CISP 6,25 12,5 25 50 100
Controls Concentration (μM)
Fig. 5. Perezone and isoperezone modify the mitochondrial membrane potential. Cells untreated and treated with cisplatin (CISP, 30 mg/ml) or with perezone or
isoperezone at different concentrations for 24 h were stained with Rho 123 and analyzed by flow cytometry. Data represent the mean 7SD of three different experiments,
(nPr 0.05).

6. Author's personal copy
ARTICLE IN PRESS
618 ´
L.E. Sanchez-Torres et al. / Phytomedicine 17 (2010) 614–620
analyzed in a FACSCalibur flow cytometer (Becton Dickinson). Results and discussion
Results are shown as the percentage of cell with cytoplasmic AIF.
Cytotoxic activity
The results of cell viability after 24 h incubation of the K562
Reproducibility of the results and statistical analysis cell line with the compounds tested at different concentrations
are displayed in Fig. 2. It can be seen that both compounds were
All values were presented as the mean7SD of at least three cytotoxic when added at concentrations of 25, 50 and 100 mM.
independent assays. Statistical analyses were performed by using Perezone had a higher cytotoxic activity than its isomer as it can
SPSS 11.0 software. A P-value equal or less than 0.05 was see clearly at 50 mM. As showed, the vehicle (DMSO) does not
considered statistically significant. affect cell viability at the concentration used.
Fig. 6. Perezone and isoperezone induce cell death through mechanisms dependent and independent of caspases. Cells untreated and treated for 24 h with cisplatin (CISP,
30 mg/ml) or with perezone or isoperezone at different concentrations (6.25, 12.5, 25, 50 and 100 mM) were stained with anti-active caspase-3-FITC antibody and analyzed
by flow cytometry. The data represent the mean 7SD of three different experiments, (nP r0.05), (a). Representative histograms are shown (b).

7. Author's personal copy
ARTICLE IN PRESS
´
L.E. Sanchez-Torres et al. / Phytomedicine 17 (2010) 614–620 619
Modifications in forward and side scatter parameters mitochondrial electron transport inducing the intrinsic pathway
of apoptosis
Cell death induces changes in size and granularity in the cell
which can be detected by analyzing variations in their light scattering
properties by flow cytometry. When K562 cells treated with both
Active caspase-3
compounds were analyzed, important changes in Forward Scatter
(FSC) and Side Scatter (SSC) parameters were detected. Fig. 3 clearly Mitochondrial damage may result in the release of apopto-
shows the modification induced by both perezone and isoperezone in genic molecules to cytosol and further activation of caspases. In
their light-scattering properties. In the particular case of the cells order to evaluate if the cell death process induced by perezone
treated with perezone it can be observed that at 6.25 and 12.5 mM or isoperezone involved a caspase-dependent pathway, we
appeared a new population with lower size (arrow, lower FSC determined by cytometry the percentage of cell with cytoplasmic
parameter) whereas K562 cells showed an increased in granularity active caspase-3, since this enzyme is a key executioner of
(higher SSC parameter) mainly at the highest concentrations (50 and apoptosis and it is activated both in the intrinsic and in the
100 mM). At 25 mM both changes can be detected indicating that, at extrinsic pathway of apoptosis. Fig. 6 shows that the presence of
this concentration, perezone induces two different events. The same the active caspase-3 did not show dose-dependent changes. The
changes were observed in cells treated with isoperezone but the presence of the active enzyme was evident in the lowest
decreased in FSC parameter was also observed at 50 mM. At this concentrations of both compounds but at higher concentrations,
concentration, the increased in granularity was also evident, but the the percentage of positive cells decreased. The results showed
majority of the cells showed this effect when 100 mM was employed. that at lower doses both compounds induced cell death through a
It is important to note that when isoperezone was added at 25 mM, caspase-dependent mechanism. In the particular case of
almost all the cells show a reduced size. The aforementioned changes isoperezone, the caspase-3 participation to induce cell death
are consistent with those mentioned for the apoptotic (lower FSC was evident even at 50 mM. At higher doses, the cell death
with or without changes in SSC parameter) and necrotic (higher FSC induced by both compounds was mediated through a caspase-
and SSC parameters) cell death. As expected, and in correlation with independent pathway mainly in perezone-treated cells. It is
the results obtained with the Trypan blue exclusion assay (Fig. 2), a evident that the position of the hydroxyl substituent in the
proportion of the cisplatin-treated cells showed a clear reduction in isoperezone molecule diminishes the cytotoxic activity of the
the FSC parameter. perezone but favors the caspase-dependent apoptotic cell
death.
Phosphatidylserine translocation
To investigate whether the compound-induced decrease in cell Apoptosis inducing factor (AIF)
viability was accompanied with phosphatidylserine exposure, we
measured Annexin V-FITC/PI positive cells after incubation with Due to at higher concentrations of both compounds, the active
or without perezone or isoperezone at varying concentrations form of the caspase-3 was not detected, we determined the possible
(6.25, 12.5, 25, 50 and 100 mM). Similar to cisplatin treated-K562 participation of AIF in the cell death induced by both compounds at
cells, both compounds had the capacity to induce the shift of higher concentrations. AIF is one of the apoptogenic molecules that
phosphatidylserine from the inner to the outer layer of the can be release from the mitochondria and it is translocated into the
cytoplasmic membrane. The translocation was more profound nucleus during the induction of apoptosis. Within the nucleus, in
with increasing concentrations. As seen in cell viability, perezone cooperation with Endo G, it induces apoptosis without the
showed the more potent activity. Fig. 4 shows that the participation of caspases necessarily. As shown in Fig. 7 the cell
significative increase began at 12.5 mM for both compounds. For death induced by both compounds is not related to the release of AIF
perezone-treated cells at 12.5 mM it was possible to detect at any of the concentration tested. These results suggest that the
Annexin V+ /PI- referred as ‘‘early apoptosis’’, but at higher perezone- or isoperezone-induced cell death through a caspase-
concentrations only Annexin V+ /PI+ (late apoptosis or independent pathway was related to necrosis.
secondary necrosis) was present (data not shown). These
observations showed that the phosphatidylserine translocation
is involved in the cell death induced. This event is related to a
‘‘silent’’ clearance of dying cells in vivo (Ghobrial et al., 2005;
50
Lockshin and Zakeri, 2007).
Perezone
Isoperezone
AIF positive cells (%)
Mitochondrial membrane potential 30
Mitochondria play a critical role in cell death caused by drugs
such as chemotherapeutics. Because mitochondria manifest signs
of outer and/or inner membrane permeabilization when exposed 10
to a variety of cytotoxic drugs as cisplatin, the drug used as a
positive control, we determined the mitochondrial membrane
potential in K562 cells using the fluorescent dye Rho123 and RPMI DMSO CISP 6,25 12,5 25 50 100
-10
analyzed it by flow cytometry. Fig. 5 shows that perezone and Controls Concentration (μM)
isoperezone produced a reduction in the mitochondria membrane
potential in a dose-dependent manner that correlate with the Fig. 7. Cell death induced by perezone and isoperezone is an AIF-independent
event. Cells untreated and treated for 24 h with cisplatin (CISP, 30 mg/ml) or with
cytotoxic activity. Once again perezone had the highest activity in perezone or isoperezone at different concentrations were stained with anti-AIF-PE
comparison with its isomer as it can be seen clearly at 25 mM. Our antibody. Cells were analyzed by flow cytometry. The data represent the
results suggest that both sesquiterpene quinones modulate the mean 7SD of three different experiments (nP r0.05).

8. Author's personal copy
ARTICLE IN PRESS
620 ´
L.E. Sanchez-Torres et al. / Phytomedicine 17 (2010) 614–620
Conclusion ´
Enrıquez, R., Ortega, J., Lozoya, X., 1980. Active components in Perezia roots. J.
Ethnopharmacol. 2, 389–393.
Fan, T.J., Han, L.H., Cong, R.S., Liang, J., 2005. Caspase family proteases and
Perezone showed greater cytotoxic effect than isoperezone but apoptosis. Acta Biochim. Biophys. Sin. (Shanghai) 37, 719–727.
both compounds induce the cell death trough caspase-dependent Ghobrial, I.M., Witzig, T.E., Adjei, A.A., 2005. Targeting apoptosis pathways in
and caspase-independent mechanisms. cancer therapy. CA Cancer J. Clin. 55, 178–194.
Howells, L.M., Moiseeva, E.P., Neal, C.P., Foreman, B.E., Andreadi, C.K., Sun, Y.Y.,
Hudson, E.A., Manson, M.M., 2007. Predicting the physiological relevance of
in vitro cancer preventive activities of phytochemicals. Acta Pharmacol. Sin.
Acknowledgements 28, 1274–1304.
Howley, B., Fearnhead, H.O., 2008. Caspases as therapeutic targets. J. Cell Mol. Med.
´
This research was financially supported by SIP-IPN. Godınez- 12, 1502–1516.
Kroemer, G., Galluzzi, L., Vandenabeele, P., Abrams, J., Alnemri, E.S., Baehrecke,
´
Victoria thanks CONACyT for the scholarship. Luvia Enid Sanchez-
E.H., Blagosklonny, M.V., El-Deiry, W.S., Golstein, P., Green, D.R., Hengartner,
´
Torres, is a fellow of COFAA-IPN and EDI-IPN. Benjamın Velasco ˜
M., Knight, R.A., Kumar, S., Lipton, S.A., Malorni, W., Nunez, G., Peter, M.E.,
Bejarano is a fellow of CONACYT-SNI-08/89717. We thank Alan Tschopp, J., Yuan, J., Piacentini, M., Zhivotovsky, B., Melino, G., 2009.
Larsen for reviewing the use of English in this manuscript. Classification of cell death: recommendations of the Nomenclature Committee
on Cell Death 2009. Cell Death Differ. 16, 3–11.
Lamkanfi, M., Festjens, N., Declercq, W., Vanden Berghe, T., Vandenabeele, P., 2007.
References Caspases in cell survival, proliferation and differentiation. Cell Death Differ. 14,
44–55.
Lefranc, F., Facchini, V., Kiss, R., 2007. Proautophagic drugs: a novel means to
Alarcon-Aguilar, F.J., Roman-Ramos, R., Jimenez-Estrada, M., Reyes-Chilpa, R.,
combat apoptosis-resistant cancers, with a special emphasis on glioblastomas.
Gonzalez-Paredes, B., Flores-Saenz, J.L., 1997. Effects of three mexican
Oncologist 12, 1395–1403.
medicinal plants (Asteraceae) on blood glucose levels in healthy mice and
Lockshin, R.A., Zakeri, Z., 2007. Cell death in health and disease. J. Cell Mol. Med.
rabbits. J. Ethnopharmacol. 55, 171–177.
11, 1214–1224.
˜
Burgueno-Tapia, E., Joseph-Nathan, P., 1997. Detailed studies of perezone
´ ´
Martınez, J., Velasco-Bejarano, B., Delgado, F., Torres-Domınguez, H.M., Ferrara-
rearrangements. Monatsh. Chem. 128, 651–658.
˜ ´
Burgueno-Tapia, E., Castillo, L., Gonzalez-Coloma, A., Joseph-Nathan, P., 2008. Trujillo, J.G., Arroyo, G.A., Miranda, R., 2008. Eco-contribution to the chemistry
Antifeedant and phytotoxic activity of the sesquiterpene p-benzoquinone of perezone, a comparative study using different modes of activation and
perezone and some of its derivatives. J. Chem. Ecol. 34, 766–771. solventless conditions. Nat. Prod. Commun. 3, 1465–1468.
Carabez, A., Sandoval, F., 1988. The action of the sesquiterpenic benzoquinone, Nohl, H., Jordan, W., Youngman, R.J., 1986. Quinones in biology: functions
perezone, on electron transport in biological membranes. Arch. Biochem. in electron transfer and oxygen activation. Adv. Free Radic. Biol. Med. 2,
Biophys. 260, 293–300. 211–279.
Cuellar, A., Carabez, A., Chavez, E., 1987. Ca2 + releasing effect of perezone on
´ ´ ´ ´
Rıo de la Loza, L., 1852. Speach to the Medical Academy, Mexico, Escritos de
adrenal cortex mitochondria. Life Sci. 41, 2047–2054. Leopoldo Rio de la Loza compiled by J.M. Noriega. Imprenta Ignacio Escalante,
˜ ˜
De La Pena, A., Izaguirre, R., Banos, G., Viveros, M., Enriquez, R.G., Fernandez, J.M., ´
Mexico 1911, 94–100.
2001. Effect of perezone, aminoperezone and their corresponding isomers ´ ´ ´
Tellez, J.F., Carvajal, K., Cruz, D., Carabez, A., Chavez, E., 1999. Effect of perezone on
isoperezone and isoaminoperezone upon in vitro platelet aggregation. arrhythmias and markers of cell injury during reperfusion in the anesthetized
Phytomedicine 8, 465–468. rat. Life Sci. 65, 1615–1623.