Beruflich Dokumente
Kultur Dokumente
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
a r t i c l e i n f o a b s t r a c t
Article history: Ferula communis (L.), a plant belonging to Apiaceae, is widely present in Sardinia, Italy. Currently, interest
Received 3 August 2012 in F. communis focuses on the presence of two chemotypes in the wild. One chemotype is poisonous to
Received in revised form 28 December 2012 animals, whereas the other chemotype is non-poisonous. Polyphenol oxidase (PPO) has been extracted
Available online 21 March 2013
and partially puried from the two chemotypes of F. communis. The biochemical characterization of
the enzymes showed signicant differences. In particular, while the two PPOs were not able to use
Keywords: 6- and 7-hydroxycoumarin as substrates, they showed distinct specicity for 6,7- and 7,8-dihydroxy-
Ferula communis
coumarin. Signicant differences in the enzyme behavior towards common PPO inhibitors were also
Apiaceae
Giant fennel
observed. In addition, activation energy and activation energy for denaturation were determined, show-
Poisonous chemotype ing signicant differences between FP-PPO and FNP-PPO, particularly for denaturation kinetics. The pos-
Polyphenol oxidase sible roles of the two PPOs in determining differences in composition and toxicity of the two F. communis
Enzyme characterization chemotypes are also discussed.
Hydroxycoumarins 2013 Elsevier Ltd. All rights reserved.
0031-9422/$ - see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.phytochem.2013.02.019
P. Zucca et al. / Phytochemistry 90 (2013) 1624 17
and physiology of plants is not yet well dened (Mayer, 2006), this
enzyme is certainly involved in the polymerization of oxidized
phenolic molecules and most likely also the biosynthesis of sec-
ondary metabolites. Worthy of note, Sollai and colleagues have
shown that a preparation of F. communis was able to oxidize 6,7-
dihydroxycoumarin (Sollai et al., 2008). The presence of hydroxy-
coumarin derivatives and PPO in F. communis poses the problem
of the interactions, if any, between the enzyme and such a chemi-
cal family. In fact, several hydroxycoumarin derivatives have been
found in F. communis (Aragno et al., 1988; Valle et al., 1987), whose
biochemical behaviors should be quite different. In particular,
4-hydroxycoumarins usually show anti-vitamin K activity not
found in 6- and/or 7-hydroxyl derivatives. Consequently, signi-
cant differences among these substances with respect to substrate
or inhibitor behavior for PPO could be reasonably anticipated. The
phenolic hydroxyls of these substances are often masked by meth-
ylation and glycoside formation, but this masking does not exclude
their possible inhibitory activity towards PPO. In addition, the pos-
sible presence of glycosidases in the plant tissues could cause the
release of the aglycone moiety, restoring a free hydroxyl group
on the coumarin derivative. Most likely, coumarins and PPO have
different intracellular localizations within plant cells. However, di-
rect interaction becomes quite likely in ruminants, where the long
rumination phase allows cell rupture under conditions (pH approx-
imately 6.5, temperature ranging between 38 and 40 C) favoring
enzyme action. Differences in PPO activity could possibly lead to
the differing chemical compositions of the two chemotypes of Fer-
ula spp., which are related to their distinct toxicities. In this paper,
using the criteria distinguishing the two chemotypes as above
(Marchi et al., 2003; Sacchetti et al., 2003; Valle et al., 1987), we
Fig. 2. Native PAGE of homogenate from FP (lane a) and FNP (lane b). The activity
selected a few specimens of both the poisonous and non-poisonous
staining was performed using a method based on the formation of the adduct
chemotype of F. communis, extracted PPO from the aerial parts of between 4-tert-butyl-1,2-benzoquinone (TBBQ) and 4-amino-N,N-diethylaniline
the plant and partially puried it. Biochemical properties such as (ADA) (Rescigno et al., 1997).
18 P. Zucca et al. / Phytochemistry 90 (2013) 1624
Table 1
Summary of purication of PPO from poisonous (P) and non-poisonous (NP) chemotypes of F. communis.
Purication step Chemotype Vtot (mL) Total activity (nkat) Total protein (mg) Specic activity (nkat/mg prot) Purication (fold) Yield (%)
Homogenate P 120 12,617 240.2 52.5 1 100
NP 110 8305 525.5 15.8 1 100
(NH4)2SO4 precipitation P 36 14,540 178.0 81.7 1.5 115
NP 24 9672 140.7 68.7 2.1 116
Phenyl sepharose (HIC) P 29 6703 13.9 482 9.3 46
NP 5 2721 2.2 1237 39.0 32
DEAEcellulose (IEC) P 25 2,280 2.46 927 17.9 18
NP = = = = = =
HiLoad Superdex (SEC) P 1 127 0.073 1736 33.5 1
NP 1 117 0.037 3154 99.5 1.4
used as the rst purication step, leading, in both cases, to a slight that increased hydrophobicity of the substrates results in greater
increase in the specic activity. During this procedure, the number activity for both enzymes, while more hydrophilic substrates
of total units increased. This increase could have occurred because (chlorogenic acid, pyrogallol or DOPA, for instance) are slowly oxi-
the fractional precipitation and subsequent dialysis removed the dized by both PPOs, in strict accordance with the hydrophobic nat-
reduced glutathione (GSH) used to limit the browning of the solu- ure of the enzyme active site (Rescigno et al., 2011a). PPOs showed
tion during the homogenization step (GSH apparently decreases MichaelisMenten-like kinetics for the best diphenolic substrates,
PPO activity, reducing quinone products back to diphenols). After and the calculated parameters are reported in Table 3. The afnity
(NH4)2SO4 precipitation, chromatographic steps were performed. of the enzymes for catechols was similar to that observed for other
Hydrophobic interaction (HIC) and size exclusion (SEC) chromatog- PPO isoforms in Ferula spp. (Erat et al., 2006), whereas our puried
raphy were common steps for both PPOs, while for the FP-PPO, an enzymes showed a greater afnity for diphenolic phenylpropa-
additional step by means of anion exchange chromatography was noids (the KM was almost one order of magnitude lower for caffeic
required. HIC was performed using a phenyl-sepharose column. and chlorogenic acids, (Shimizu et al., 2011). All values are signif-
During this step, PPOs were eluted at a very low ammonium sulfate icantly higher compared with non-plant PPOs (Whitaker, 1995).
concentration (approximately 10%), due to the high hydrophobic- The MichaelisMenten constants of FP-PPO and FNP-PPO were
ity and possible membrane localization of these enzymes (Mayer, quite similar for the same substrate.
2006). Ion exchange chromatography onto DEAEsepharose gave Both PPOs showed strong pH-dependent activity, as reported in
quite poor results with FNP-PPO and was thus removed from the Fig. 3. An optimum was observed at neutrality, and only slight dif-
standard purication protocol for this chemotype. Conversely, this ferences between FP and FNP-PPO were observed at the same pH
procedure nearly doubled the specic activity of FP-PPO. However, value. Other PPOs from Ferula sp. and other plant sources have
both enzymes were able to reversibly bind DEAE at pH 7.0, show- shown similar features (Erat et al., 2006; Palma-Orozco et al.,
ing an acidic pI, according to similar isoforms from other plants. 2011), although acid-preferring (Dincer et al., 2002; Nagai and
Native IEF on polyacrylamide gel conrmed this nding, as the Suzuki, 2001) and even slightly basic-preferring PPOs have been
comparison with commercial standards showed a pI of approxi- described (Park and Luh, 1985).
mately 5.0. This purication procedure completely eliminated con- FP-PPO and FNP-PPO showed temperature-dependent behavior,
taminant pigments and signicantly reduced the amount of other as activity increased up to 55 C in both cases prior to thermal inac-
protein (as demonstrated by the increase in specic activity in tivation. FP-PPO also showed quite good thermostability when
Table 1). At this stage of purication, the enzymes could be stored stored for 1 h at increasing temperatures (slightly higher compared
for months at 4 C without an appreciable loss of activity; storage to other Ferula sp. enzymes (Erat et al., 2006)). No loss of catalytic
at 20 or 80 C led to a quick denaturation. activity was observed up to 35 C. On the contrary, slight thermal
activation was apparent. This behavior was previously reported
2.2. PPO characterization for other copper-containing oxidases (Zucca et al., 2011), whereas
over 60 C, no remaining activity was present after 1 h incubation.
The partially puried PPOs were then used to characterize The loss of activity was more signicant for FNP-PPO, showing par-
enzyme activity. Neither FP-PPO and FNP-PPO showed any detect- ticularly poor performances from 25-40 C, in strict accordance
able activity with monophenols such as tyrosine, 4-tert-butylphe- with a higher Ea of denaturation and a lower z value (Table 4). In
nol, 6-hydroxycoumarin or 7-hydroxycoumarin, even in the the same table, all kinetic data about activation energy and activa-
presence of detergents and activating concentrations of catechols. tion energy for denaturation are reported; these data show signif-
Conversely, the enzymes were able to oxidize a wide variety of icant differences between FP-PPO and FNP-PPO, particularly with
o-diphenols, including catechol and substituted catechols (Table respect to the denaturation kinetics. More similar values of Ea were
2). The substrate specicity was quite different between the two calculated, as expected, using 4-tert-butylcatechol (TBC) as the
isolated PPOs. 4-Methylcatechol was the best substrate for FP- substrate. All of these data are in the range of those reported for
PPO; a similar nding has been described for other plant PPOs other plant PPOs (Chaves et al., 2011; Zheng et al., 2012).
(Ni Eidhin et al., 2006; Siddiq et al., 1992). Conversely, FNP-PPO The effects of selected detergents on PPO activity were also
presented a lower KM for catechol (Table 3), as have other plant studied (Fig. 4). Despite the activation by means of low concentra-
PPOs, including those from Ferula sp. (Erat et al., 2006; Todaro tions of sodium dodecyl sulfate (SDS) described elsewhere
et al., 2010). Both enzymes, however, showed a poor ability to (Jimenez and Garcia-Carmona, 1996; Shimizu et al., 2011), our par-
transform chlorogenic acid and pyrogallol, compounds elsewhere tially puried isoforms were not activated by SDS, whereas a
reported as the best substrates for other PPOs (Palma-Orozco degree of activation by 3-[(3-cholamidopropyl)dimethylammo-
et al., 2011; Shimizu et al., 2011). These ndings could reasonably nio]-1-propanesulfonate (CHAPS) was observed, up to a 0.2%
contribute to different patterns of secondary metabolites in FP and CHAPS concentration for FP-PPO and a 0.7% CHAPS concentration
FNP (Appendino et al., 2001; Valle et al., 1987). It is worth noting for FNP-PPO, which was signicantly more stable and active in
P. Zucca et al. / Phytochemistry 90 (2013) 1624 19
Table 2
Substrate specicity for puried PPOs.a,b
HO
Chlorogenic acid O 420 945 1140
HO HO COOH
O
HO
HO OH
Catechol 420 4100 3170
HO
OH
4-tert-Butylcatechol CH3 420 1040 1040
HO CH3
CH3
HO
(+)-Catechin OH 420 90 25
HO
O OH
HO
OH
Caffeic acid O 420 130 70
OH
HO
OH
Dopamine HO 475 80 3
NH2
HO
L-Dopa O 475 70 170
HO
OH
NH2
HO
Gallic acid O 420 60 4
HO OH
HO
OH
Pyrogallol HO 420 140 130
HO
OH
Tyrosine NH2 305 n.d n.d.
HO HO O
c
4-tert-Butylphenol CH3 n.d. n.d.
HO CH3
CH3
20 P. Zucca et al. / Phytochemistry 90 (2013) 1624
Table 4
Table 3 Kinetic parameters of thermal effect on FP-PPO and FNP-PPO oxidation of 4-tert-
Calculated kinetic parameters for selected diphenols.a butylcatechol.a
Fig. 4. Effects of CHAPS (above) and SDS (below) on PPO activity. The reaction mixture contained 50 mM sodium phosphate buffer pH 7, 5 mM 4-tert-butylcatechol (TBC),
0.75 mM ADA, the indicated amount of detergent and 2 nkat PPO in a nal volume of 1 mL. n = 4.
Table 6
Name, structure and related kinetic parameters of tested dihydroxycoumarin derivatives.a.
HO
4-Methyl-6,7-dihydroxycoumarin P nd nd
HO O O NP nd nd
HO
HO O O
HO O O
3-Carbetoxypropyl-4-methyl-7,8-dihydroxycoumarin P nd nd
OH NP nd nd
HO O O
OEt
O
a
P = poisonous chemotype; NP = non-poisonous chemotype. n = 4.
whereas its afnity for FNP-PPO was not signicantly altered. On physiological substrates of the two PPOs are still awaiting
the whole, the data for o-dihydroxycoumarins conrmed slightly determination.
different catalytic activity for FP-PPO and FNP-PPO, which poten-
tially contributes to different patterns of secondary metabolites 4. Experimental
in FP and FNP. This observation is corroborated by the fact that
both FP and FNP showed b-glucosidase activity ranging from 0.33 4.1. Chemicals and plant material
and 0.93 nkat per gram of acetone powder. This enzymatic activity
could release the aglycone moiety, restoring a free hydroxyl group Umbelliferone (7-hydroxycoumarin) and esculetin (7,8-dihydr-
on the coumarin glycosides. Unmasking the hydroxy substituent in oxycoumarin) were purchased from SigmaAldrich (Milan, Italy).
o-dihydroxycoumarins produces new catechol-type substrates, The other coumarin derivatives were synthesized according to pre-
further increasing the pattern diversity between the two vious reports (Le-Thi-Thu et al., 2011). All other reagents used
chemotypes. were of the highest grade available, purchased from SigmaAldrich
(Milan, Italy) and used without further purication. Aerial parts of
3. Conclusions F. communis were harvested before owering in April 2011 and
transferred to our laboratory within 2 h of harvest. Then, plant
Partial purication of PPOs from poisonous and non-poisonous material (leaves and stems) were gently washed with deionized
chemotypes of F. communis, allowing biochemical characterization water, wiped with blotting paper, frozen in liquid N2 and stored
of the enzymatic activity, was accomplished. The two enzymes at 80 C until use. FP specimens were collected close to Lago Corsi
showed signicant differences, particularly with respect to their (Iglesias, southern Sardinia), exsiccated and stored at Orto Botanico
specicity towards common diphenolic substrates of catecholases. of Cagliari (Herbarium n. 612), whereas FNP specimens were col-
In addition, PPOs showed varied behavior towards coumarin deriv- lected at Seneghe (Oristano, central Sardinia), exsiccated and
atives. 6- and 7-hydroxycoumarin are neither substrates nor inhib- stored at Orto Botanico of Cagliari (Herbarium No. 612/b).
itors for the PPOs contained in the two chemotypes, but 6,7- and
7,8-dihydroxycoumarins are substrates. However because both 4.2. Enzyme extraction and purication
FNP-PPO and FP-PPO behaved differently with the studied hydrox-
ycoumarins, a relationship between the behavior of these enzymes A typical extraction procedure requires the leaves (100 g) to be
and the quite different pattern of the known secondary metabolites homogenized in a blender in the presence of 250 mL of cold ace-
can be hypothesized. This relationship is certainly complex, as the tone ( 50 C). The homogenate was ltered through a Buchner
P. Zucca et al. / Phytochemistry 90 (2013) 1624 23
funnel equipped with fast-ow lter paper and washed with 2 L of cmixture contained 50 mM sodium phosphate buffer pH 7.0,
cold acetone until the ltrate was colorless. The solid residue was 5 mM TBC, 0.75 mM ADA and a suitable enzyme concentration in
dried by an air ow and ground with a mortar and pestle. The ob- a nal volume of 1 mL. b-Glucosidase activity was also determined
tained powder was divided into 5 g aliquots and stored at 20 C. using a previously described spectrophotometric procedure,
Each aliquot was later suspended in 100 mL of 100 mM sodium involving p-nitrophenyl-b-D-glucopyranoside (p-NPG) as the sub-
phosphate buffer, pH 7.0, containing 4 mM reduced glutathione strate (Rescigno et al., 2007). Six samples of acetone powder from
(GSH) and 25 lL Antifoam A (Fluka, Cat. No. 10794), under gentle FP and FNP were inspected for this purpose. Protein content was
stirring for 30 min at 4 C. The suspension thus obtained was determined using Coomassie Brilliant Blue G250 (Bradford meth-
passed through lter paper and the ltrate centrifuged (10,000g od), with bovine serum albumin as a standard. No interfering con-
for 20 min). This solution was dialtrated and concentrated in a taminant activity capable of altering the analytical response of the
Vivaow 200 apparatus (Vivascience AG, Hannover, Germany) PPO assays was detected in the puried PPOs (Rescigno et al.,
equipped with a Hydrosart membrane module (nominal MW 2007).
cut-off 10,000 Da) and a Masterex L/S system pump (Cole-Parmer,
USA). During this step, 2 L of 15 mM sodium phosphate buffer pH
4.3.2. Kinetic studies on PPO enzyme activity
7.0 containing 1 mM GSH was added to the enzyme solution. The
Monophenolase activity was studied using tyrosine, 4-tert-
nal enzyme solution thus obtained had a nal volume of approx-
butylphenol, 6-hydroxycoumarin or 7-hydroxycoumarin as the
imately 100120 mL. The sample was then treated with ammo-
substrate, according to previously described methods (Rescigno
nium sulfate (35% of the saturating concentration) for 30 min at
et al., 1998, 2007). Each substrate was used at 2 mM nal
4 C under gentle stirring. After centrifugation for 20 min at
concentration.
8,000g, the supernatant was collected, and the salt was removed
Diphenolase activity was studied by adding each substrate at
by dialtration using the same apparatus described above. The en-
2 mM nal concentration to the reaction mixture and observing
zyme solution was then subjected to hydrophobic interaction chro-
the increase in absorbance at the kmax of the specic product
matography. The sample was treated with 1 M ammonium sulfate
(Murugesan et al., 2006). For comparison, one PPO arbitrary unit
and loaded onto a Hiprep 16/10 Phenyl FF (GE Healthcare, Milan,
(E.U.) was dened as the amount of enzyme that caused an in-
Italy) column using FPLC Akta Prime (Amersham Bioscience, Milan,
crease in the absorbance of 0.001/min (Erat et al., 2006). Arbitrary
Italy) equipped with a UV detector. Column equilibration was per-
units were necessary because, for many substrates, a complex mix-
formed with 50 mM sodium phosphate buffer (pH 7.0) containing
ture of products, often unknown, arises, with wide-ranging e val-
1 M ammonium sulfate at a constant ow of 1 mL/min. Elution was
ues that cannot be compared (Erat et al., 2006).
then obtained with a linear gradient of 50 mM sodium phosphate
MichaelisMenten kinetic parameters were calculated by vary-
buffer (0 ? 100% B in 20 min). Ammonium sulfate was removed
ing the substrate concentration and using recursive regression
from the enzymatic solution through dialtration, using the appa-
using R 2.5.1 software (R Foundation for Statistical Computing,
ratus described above.
Vienna). The activity at different pH values was determined by
In the case of FP, PPO-containing fractions were loaded onto a
repeating the catalytic assay in the presence of various 25 mM
Hiprep 16/10 DEAE FF for anion exchange chromatography. Col-
BrittonRobinson buffers ranging from pH 4.0 to 8.0. PPO activity
umn equilibration was performed with 10 mM sodium phosphate
was determined over a suitable range of temperatures, using 4-
buffer pH 7.0 at a constant ow of 3 mL/min. Elution was per-
tert-butylcatechol (TBC) and ADA as the substrates. The activation
formed with a linear gradient of 10 mM sodium phosphate buffer
energies (Ea) were calculated from the Arrhenius equation, using
containing 1 M NaCl (0 ? 100% B in 60 min). Sodium chloride
ascending activities from 25 C up to the optimum reaction tem-
was removed from the enzymatic solution by dialtration. The last
perature. Inactivation kinetics were assayed by incubating the en-
purication step was carried out by means of size exclusion chro-
zyme for 1 h at the indicated temperature in a water bath (1 C).
matography using a HiLoad 16/60 Superdex 75 column (GE Health-
Enzyme samples were quickly brought to 25 C and the remaining
care, Milan, Italy). FPLC Akta Prime was used with a 0.4 mL/min
catalytic activity was assayed as previously described, using TBC
constant ow equipped with a UV detector. Column equilibration
and ADA as the substrates. The rst-order rate constant (k) for
and sample elution were achieved with 50 mM sodium phosphate
inactivation was calculated from the slope of the time course of
buffer at pH 7.0 with 0.15 M NaCl. Collected PPO-containing frac-
denaturation, using the equation: ln(A/Ao) = kt, where Ao is the
tions were concentrated prior to use with an ultralter membrane
initial enzyme activity, and A is the activity measured at time t.
Centriprep 30 (Amicon, Stonehouse, UK), MW cut-off 10,000 Da,
The half-life of the enzyme was calculated as t1/2 = 0.693/k. The
and nally stored at 4 C until use.
decimal reduction time (D value) was estimated as D = 2.303/k.
Purity was veried by native PAGE and SDSPAGE using 12%
The z value, which is the temperature increase required for a
polyacrylamide gels. Isoelectrofocusing (IEF) was performed using
log10 reduction (90% decrease) in the D value, was determined
a 5% polyacrylamide gel and a pH range of 310 (Ampholine, Sig-
from a plot of log10 D against temperature. The slope of the graph
ma, Milan, Italy). Comparison with pI standards allowed the deter-
is equal to 1/z, and the apparent activation energy of denaturation
mination of the pI of PPO.
(Ea) was calculated by multiplying the slope of the Arrhenius plot
(ln k vs 1/T) by the universal gas constant R.
4.3. Characterization of Ferula PPO
4.3.4. Oxidation of coumarins Murugesan, K., Arulmani, M., Nam, I.H., Kim, Y.M., Chang, Y.S., Kalaichelvan, P.T.,
2006. Purication and characterization of laccase produced by a white rot
The oxidation of mono- and dihydroxycoumarin was also stud-
fungus Pleurotus sajor-caju under submerged culture condition and its potential
ied by following enzyme action spectrophotometrically from 300 in decolorization of azo dyes. Appl. Microbiol. Biotechnol. 72, 939946.
to 700 nm. The reaction mixture contained 100 mM sodium phos- Nagai, T., Suzuki, N., 2001. Partial purication of polyphenol oxidase from Chinese
phate buffer pH 7.0, 0.1 mM coumarin derivative and suitable en- Brassica rapa L. J. Agric. Food Chem. 49, 39223926.
Neeley, E., Fritch, G., Fuller, A., Wolfe, J., Wright, J., Flurkey, W., 2009. Variations in
zyme concentrations in a nal volume of 1 mL. In a series of IC50 values with purity of mushroom tyrosinase. Int. J. Mol. Sci. 10, 38113823.
experiments, 0.5 mM 3-methyl-2-benzothiazolinone hydrazone Ni Eidhin, D.M., Murphy, E., OBeirne, D., 2006. Polyphenol oxidase from apple
(MBTH) was added to increase sensitivity (Sollai et al., 2008). (Malus domestica Borkh. cv Bramleys seedling): purication strategies and
characterization. J. Food Sci. 71, C51C58.
Nurchi, V.M., Crisponi, G., Lachowicz, J.I., Murgia, S., Pivetta, T., Remelli, M.,
Rescigno, A., Niclos-Gutierrez, J., Gonzalez-Perez, J.M., Dominguez-Martin, A.,
Appendix A. Supplementary data Castineiras, A., Szewczuk, Z., 2010. Iron(III) and aluminum(III) complexes with
hydroxypyrone ligands aimed to design kojic acid derivatives with new
Supplementary data associated with this article can be found, in perspectives. J. Inorg. Biochem. 104, 560569.
Palma-Orozco, G., Ortiz-Moreno, A., Dorantes-lvarez, L., Sampedro, J.G., Njera, H.,
the online version, at http://dx.doi.org/10.1016/j.phytochem.2013. 2011. Purication and partial biochemical characterization of polyphenol
02.019. These data include MOL les and InChiKeys of the most oxidase from mamey (Pouteria sapota). Phytochemistry 72, 8288.
important compounds described in this article. Park, E.Y., Luh, B.S., 1985. Polyphenol oxidase of kiwifruit. J. Food Sci. 50, 678684.
Rescigno, A., Bruyneel, F., Padiglia, A., Sollai, F., Salis, A., Marchand-Brynaert, J.,
Sanjust, E., 2011a. Structureactivity relationships of various amino-hydroxy-
References benzenesulfonic acids and sulfonamides as tyrosinase substrates. Biochim.
Biophys. Acta Gen. Sub. 1810, 799807.
Rescigno, A., Casaola-Martin, G.M., Sanjust, E., Zucca, P., Marrero-Ponce, Y., 2011b.
Appendino, G., 1997. The toxins of Ferula communis L. In: Verotta, L. (Ed.), Virtual Vanilloid derivatives as tyrosinase inhibitors driven by virtual screening-based
Activity, Real Pharmacology: Different Approaches to the Search for Bioactive QSAR models. Drug Test. Anal. 3, 176181.
Natural Compounds, vol. 1. Research Signpost, pp. 115. Rescigno, A., Sanjust, E., Montanari, L., Sollai, F., Soddu, G., Rinaldi, A.C., Oliva, S.,
Appendino, G., Cravotto, G., Sterner, O., Ballero, M., 2001. Oxygenated Rinaldi, A., 1997. Detection of laccase, peroxidase, and polyphenol oxidase on a
sesquiterpenoids from a nonpoisonous Sardinian chemotype of giant fennel single polyacrylamide gel electrophoresis. Anal. Lett. 30, 22112220.
(Ferula communis). J. Nat. Prod. 64, 393395. Rescigno, A., Sanjust, E., Pedulli, G.F., Valgimigli, L., 1999. Spectrophotometric
Aragno, M., Tagliapietra, S., Nano, G.M., Ugazio, G., 1988. Experimental studies on method for the determination of polyphenol oxidase activity by coupling of 4-
the toxicity of Ferula communis in the rat. Res. Commun. Chem. Pathol. tert-butyl-o-benzoquinone and 4-amino-N,N-diethylaniline. Anal. Lett. 32,
Pharmacol. 59, 399402. 20072017.
Bruneton, J., 1999. Toxic Plants Dangerous to Human and Animals. Lavoiser Rescigno, A., Sanjust, E., Soddu, G., Rinaldi, A.C., Sollai, F., Curreli, N., Rinaldi, A.,
Publishing, Inc., Paris. 1998. Effect of 3-hydroxyanthranilic acid on mushroom tyrosinase activity.
Casaola-Martin, G.M., Marrero-Ponce, Y., Khan, M.T.H., Khan, S.B., Torrens, F., Biochim. Biophys. Acta Gen. Sub. 1384, 268276.
Prez-Jimnez, F., Rescigno, A., Abad, C., 2010. Bond-based 2D quadratic Rescigno, A., Zucca, P., Flurkey, A., Inlow, J., Flurkey, W.H., 2007. Identication and
ngerprints in QSAR studies: virtual and in vitro tyrosinase inhibitory activity discrimination between some contaminant enzyme activities in commercial
elucidation. Chem. Biol. Drug Des. 76, 538545. preparations of mushroom tyrosinase. Enzyme Microb. Technol. 41, 620627.
Casaola-Martin, G.M., Marrero-Ponce, Y., Tareq-Hassan-Khan, M., Torrens, F., Rompel, A., Bldt-Karentzopoulos, K., Molitor, C., Krebs, B., 2012. Purication and
Perez-Gimenez, F., Rescigno, A., 2008. Atom- and bond-based 2D TOMOCOMD- spectroscopic studies on catechol oxidase from lemon balm (Melissa ofcinalis).
CARDD approach and ligand-based virtual screening for the drug discovery of Phytochemistry 81, 1923.
new tyrosinase inhibitors. J. Biomol. Screen. 13, 10141024. Sacchetti, G., Appendino, G., Ballero, M., Loy, C., Poli, F., 2003. Vittae uorescence as
Chaves, I.R., De Souza Ferreira, E., Da Silva, M.A., Neves, V.A., 2011. a tool to differentiate between poisonous and non-poisonous populations of
Polyphenoloxidase from atemoya fruit (Annona cherimola mill. Annona giant fennel (Ferula communis) of the island Sardinia (Italy). Biochem. Syst. Ecol.
squamosa L.). J. Food Biochem. 35, 15831592. 31, 527534.
Dincer, B., Colak, A., Aydin, N., Kadioglu, A., Gner, S., 2002. Characterization of Shetty, S.M., Chandrashekar, A., Venkatesh, Y.P., 2011. Eggplant polyphenol oxidase
polyphenoloxidase from medlar fruits (Mespilus germanica L., Rosaceae). Food multigene family: cloning, phylogeny, expression analyses and
Chem. 77, 17. immunolocalization in response to wounding. Phytochemistry 72, 22752287.
Egber, A., Perevolotsky, A., Yonatan, R., Shlosberg, A., Belaich, M., Landau, S., 1998. Shimizu, M.M., Melo, G.A., Brombini dos Santos, A., Bottcher, A., Cesarino, I., Arajo,
Creating aversion to giant fennel (Ferula communis) in weaned orphaned lambs. P., Magalhes Silva Moura, J.C., Mazzafera, P., 2011. Enzyme characterisation,
Appl. Anim. Behav. Sci. 61, 5162. isolation and cDNA cloning of polyphenol oxidase in the hearts of palm of three
Erat, M., Sakiroglu, H., Kufrevioglu, O.I., 2006. Purication and characterization of commercially important species. Plant Physiol. Biochem. 49, 970977.
polyphenol oxidase from Ferula sp. Food Chem. 95, 503508. Siddiq, M., Sinha, N.K., Cash, J.N., 1992. Characterization of polyphenoloxidase from
Halaouli, S., Asther, M., Sigoillot, J.C., Hamdi, M., Lomascolo, A., 2006. Fungal stanley plums. J. Food Sci. 57, 11771179.
tyrosinases: new prospects in molecular characteristics, bioengineering and Sollai, F., Zucca, P., Sanjust, E., Steri, D., Rescigno, A., 2008. Umbelliferone and
biotechnological applications. J. Appl. Microbiol. 100, 219232. esculetin: inhibitors or substrates for polyphenol oxidases? Biol. Pharm. Bull.
Jimenez, M., Garcia-Carmona, F., 1996. The effect of sodium dodecyl sulphate on 31, 21872193.
polyphenol oxidase. Phytochemistry 42, 15031509. Solomon, E.I., Sundaram, U.M., Machonkin, T.E., 1996. Multicopper oxidases and
Lannehoa, Y., Harry, P., Michau, C., Fareta, A., Merad, R., 1998. Anal bleeding due to oxygenases. Chem. Rev. 96, 25632605.
hypovitamin K caused by ingesting fassoukh. Presse Med. 27, 1579. Todaro, A., Peluso, O., Catalano, A.E., Mauromicale, G., Spagna, G., 2010. Polyphenol
Le-Thi-Thu, H., Cardoso, G.C., Casaola-Martin, G.M., Marrero-Ponce, Y., Puris, A., oxidase activity from three sicilian artichoke [Cynara cardunculus L Var.
Torrens, F., Rescigno, A., Abad, C., 2010. QSAR models for tyrosinase inhibitory scolymus L (Fiori)] cultivars: studies and technological application on
activity description applying modern statistical classication techniques: a minimally processed production. J. Agric. Food Chem. 58, 17141718.
comparative study. Chemometr. Intell. Lab. Syst. 104, 249259. Valgimigli, L., Pedulli, G.F., Cabiddu, S., Sanjust, E., Rescigno, A., 2000. Formation of a
Le-Thi-Thu, H., Casaola-Martn, G.M., Marrero-Ponce, Y., Rescigno, A., Saso, L., blue adduct between 4-tert-butyl-1,2-benzoquinone and 4-amino-N,N-
Parmar, V.S., Torrens, F., Abad, C., 2011. Novel coumarin-based tyrosinase diethylaniline. Tetrahedron 56, 659662.
inhibitors discovered by OECD principles-validated QSAR approach from an Valle, M.G., Appendino, G., Nano, G.M., Picci, V., 1987. Prenylated coumarins and
enlarged, balanced database. Mol. Divers. 15, 507520. sesquiterpenoids from Ferula communis. Phytochemistry 26, 253256.
Marchi, A., Appendino, G., Pirisi, I., Ballero, M., Loi, M.C., 2003. Genetic Whitaker, J.R., Polyphenol oxidase. In: Wong, D.W.S. (Ed.), Food Enzymes
differentiation of two distinct chemotypes of Ferula communis (Apiaceae) in Structure and Mechanism, New York, 1995, pp. 271307.
Sardinia (Italy). Biochem. Syst. Ecol. 31, 13971408. Zheng, Y., Shi, J., Pan, Z., 2012. Biochemical characteristics and thermal inhibition
Mayer, A.M., 2006. Polyphenol oxidases in plants and fungi: going places? A review. kinetics of polyphenol oxidase extracted from Thompson seedless grape. Eur.
Phytochemistry 67, 23182331. Food Res. Technol. 234, 607616.
Mazzafera, P., Robinson, S.P., 2000. Characterization of polyphenol oxidase in coffee. Zucca, P., Rescigno, A., Olianas, A., Maccioni, S., Sollai, F.A., Sanjust, E., 2011.
Phytochemistry 55, 285296. Induction, purication, and characterization of a laccase isozyme from Pleurotus
Monti, M., Pinotti, M., Appendino, G., Dallocchio, F., Bellini, T., Antognoni, F., Poli, F., sajor-caju and the potential in decolorization of textile dyes. J. Mol. Catal. B
Bernardi, F., 2007. Characterization of anti-coagulant properties of prenylated Enzym. 68, 216222.
coumarin ferulenol. Biochim. Biophys. Acta Gen. Sub. 1770, 14371440.