Anti-inflammatory and anti-apoptotic effects of (RS)- glucoraphanin bioactivated with myrosinase in murine sub-acute and acute MPTP-induced Parkinson's disease

Article abstract of DOI:10.1016/j.bmc.2013.05.065

This study was focused on the possible neuroprotective role of (R _S)-glucoraphanin, bioactivated with myrosinase enzyme (bioactive R_S-GRA), in an experimental mouse model of Parkinson's disease (PD). R_S-GRA is one of the most important glucosinolates, a thiosaccharidic compound found in Brassicaceae, notably in Tuscan black kale seeds. R _S-GRA was extracted by one-step anion exchange chromatography, further purified by gel-filtration and analyzed by HPLC. Following, pure R _S-GRA was characterized by ¹H and ¹³C NMR spectrometry and the purity was assayed by HPLC analysis of the desulfo-derivative according to the ISO 9167-1 method. The obtained purity has been of 99%. To evaluate the possible pharmacological efficacy of bioactive R_S-GRA (administrated at the dose of 10 mg/kg, ip +5 μl/mouse myrosinase enzyme), C57BL/6 mice were used in two different sets of experiment (in order to evaluate the neuroprotective effects in different phases of the disease), according to an acute (2 injections·40 mg/kg MPTP) and a sub-acute (5 injections·20 mg/kg MPTP) model of PD. Behavioural test, body weight changes measures and immunohistochemical localization of the main PD markers were performed and post-hoc analysis has shown as bioactive R _S-GRA is able to reduce dopamine transporter degradation, tyrosine hydroxylase expression, IL-1β release, as well as the triggering of neuronal apoptotic death pathway (data about Bax/Bcl-2 balance and dendrite spines loss) and the generation of radicalic species by oxidative stress (results focused on nitrotyrosine, Nrf2 and GFAP immunolocalization). These effects have been correlated with the release of neurotrophic factors, such as GAP-43, NGF and BDNF, that, probably, play a supporting role in the neuroprotective action of bioactive R_S-GRA. Moreover, after PD-induction mice treated with bioactive R_S-GRA are appeared more in health than animals that did not received the treatment both for phenotypic behaviour and for general condition (movement coordination, presence of tremors, nutrition). Overall, our results suggest that bioactive R_S-GRA can protect neurons against the neurotoxicity involved in PD via an anti-apoptotic/anti-inflammatory action.

Full text of DOI:10.1016/j.bmc.2013.05.065

Bioorganic & Medicinal Chemistry 21 (2013) 5532–5547
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Anti-inflammatory and anti-apoptotic effects of (RS)-glucoraphanin
bioactivated with myrosinase in murine sub-acute and acute
MPTP-induced Parkinson’s disease
Maria Galuppo a, Renato Iori b, Gina Rosalinda De Nicola b, Placido Bramanti a, Emanuela Mazzon a,
⇑
a IRCCS Centro Neurolesi ‘Bonino-Pulejo’, Via Provinciale Palermo, S.S. 113, Contrada Casazza, 98124 Messina, Italy
b Consiglio per la Ricerca e la sperimentazione in Agricoltura, Centro di Ricerca per le Colture Industriali (CRA-CIN), Via di Corticella 133, 40128 Bologna, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
This study was focused on the possible neuroprotective role of (RS)-glucoraphanin, bioactivated with
myrosinase enzyme (bioactive RS-GRA), in an experimental mouse model of Parkinson’s disease (PD).
RS-GRA is one of the most important glucosinolates, a thiosaccharidic compound found in Brassicaceae,
notably in Tuscan black kale seeds.
RS-GRA was extracted by one-step anion exchange chromatography, further purified by gel-filtration
and analyzed by HPLC. Following, pure RS-GRA was characterized by 1H and 13C NMR spectrometry
and the purity was assayed by HPLC analysis of the desulfo-derivative according to the ISO 9167-1
method. The obtained purity has been of 99%.
Received 11 March 2013
Revised 23 May 2013
Accepted 28 May 2013
Available online 11 June 2013
Keywords:
Parkinson’s disease
RS-Glucoraphanin
Glucosinolates
Myrosinase
To evaluate the possible pharmacological efficacy of bioactive RS-GRA (administrated at the dose of
10 mg/kg, ip +5 ll/mouse myrosinase enzyme), C57BL/6 mice were used in two different sets of experi-
ment (in order to evaluate the neuroprotective effects in different phases of the disease), according to an
acute (2 injections Á 40 mg/kg MPTP) and a sub-acute (5 injections Á 20 mg/kg MPTP) model of PD.
Behavioural test, body weight changes measures and immunohistochemical localization of the main PD
markers were performed and post-hoc analysis has shown as bioactive RS-GRA is able to reduce dopa-
mine transporter degradation, tyrosine hydroxylase expression, IL-1b release, as well as the triggering
of neuronal apoptotic death pathway (data about Bax/Bcl-2 balance and dendrite spines loss) and the
generation of radicalic species by oxidative stress (results focused on nitrotyrosine, Nrf2 and GFAP
immunolocalization). These effects have been correlated with the release of neurotrophic factors, such
as GAP-43, NGF and BDNF, that, probably, play a supporting role in the neuroprotective action of bioac-
tive RS-GRA. Moreover, after PD-induction mice treated with bioactive RS-GRA are appeared more in
health than animals that did not received the treatment both for phenotypic behaviour and for general
condition (movement coordination, presence of tremors, nutrition).
Inflammation
Nitrosative stress
Overall, our results suggest that bioactive RS-GRA can protect neurons against the neurotoxicity
involved in PD via an anti-apoptotic/anti-inflammatory action.
Ó 2013 Published by Elsevier Ltd.
1. Introduction
As neurodegenerative disorder, PD is characterized by progres-
sive and selective loss of dopaminergic (DA) neurons in the Sub-
Parkinson’s disease (PD) is a motor system disorder that affects
between 100 and 200 per 100,000 people over 40,1 more com-
monly men than women.2 The incidence of the disease increases
rapidly over 60 years, with a mean age at diagnosis of 70.5 years.1
stantia Nigra pars compacta (SNpc) and striatum,3 accompanied
by the appearance of Lewy bodies,4 abnormal aggregates mainly
composed by alpha-synuclein, a mutated protein found in this5
as well as many other neurodegenerative diseases.6
Clinically, resting tremor, rigidity, bradykinesia, and postural
instability (difficulty with walking and gait) later followed by
dementia, cognitive and behavioural problems are all symptoms
that occur in the course of the PD.7
The mechanisms regulating neuronal cell death in PD have been
extensively investigated. Oxidative stress, protein misfolding and
aggregation are involved in the pathogenesis of the disease.8
Abbreviations: GL, glucosinolate; PD, Parkinson’s disease; DAT, dopamine
transporter; TH, tyrosine hydroxilase; DA, dopaminergic; IL-1b, interleukin-1beta;
SNpc, Substantia Nigra pars compacta; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahy-
dropyridine; Myr, myrosinase; ITC, isothiocyanates; IHC, immunohistochemical.
⇑
Corresponding author. Tel.: +39 09060128708; fax: +39 09060128850.
E-mail address: emazzon.irccs@gmail.com (E. Mazzon).
0968-0896/$ - see front matter Ó 2013 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmc.2013.05.065

M. Galuppo et al. / Bioorg. Med. Chem. 21 (2013) 5532–5547
5533
a procedure developed at CRA-CIN of Bologna, Italy.17 In the context
of our continuous program concerning biochemical investigation on
GLs, this work was aimed to study the possible anti-neurodegener-
ative role of the bioactive RS-GRA in an experimental mouse model
of sub-acute/acute MPTP-induced PD, through a mechanism that in-
volves a modulation of the inflammatory pathway and the subse-
quent physiologic responses.
Figure 1. Chemical structure and enzymatic reaction for bioactivation of RS-GRA by
Myr. (RS)-glucoraphanin purified from Tuscan black kale seed, bio-activated by
myrosinase enzyme: in situ release of (RS)-sulforaphane.
2. Methods
2.1. Animals
In particular, a deficit in dopamine seems correlated with phys-
iopathology of the disease.
Male C57BL/6 mice (n = 50, 4–5 weeks old, 20–25 g weight)
were purchased from Harlan (Italy). Animals were housed in indi-
vidually ventilated cages with food and water ad libitum. The room
was maintained at a constant temperature and humidity on a 12 h/
12 h light/dark cycle. Procedures involving animals and their care
were conducted in conformity with the international and national
(D.L. 116/92) law and policies (European Communities Council
Directive of 24 November 1986, 86/609/EEC).
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced
neurotoxicity is the most common experimental model used
through which mice show typical neuropathological defects as ob-
served in human PD. MPTP is converted to 1-methyl-4-phenylpy-
ridinium (MPP+) by monoamine oxidase B and it is carried by the
dopamine transporter (DAT) into DA cells, causing the block of
mitochondrial complex I activity.9
Neurodegenerative potential of MPTP-administration depends
by the dosage.10 In particular, differently regimes of administration
have showed that the dose of 40 mg/kg, repeated twice, causes tox-
icity and severe behavioural degeneration, while sub-acute doses
of five injections of 20 mg/kg MPTP generate different Parkinson-
like symptoms.11
All efforts were made, during experimental procedures, to min-
imize animal suffering and to reduce the number of animal used.
2.2. MPTP-induced acute and sub-acute PD
PD was induced by MPTP hydrochloride (Sigma–Aldrich, Italy)
injection, a neurotoxin that causes a selective destruction of DA
neurons. The selective toxicity of MPTP for the nigrostriatal tract
was recognized in the mid-1980s by Langston et al.18
In our experimental design, we performed two different sets of
experiment: the first one to induce an acute (two injections for
MPTP 40 mg/kg) Parkinsonian syndrome, while the second one
(five injections for MPTP 20 mg/kg) to produce sub-acute
symptoms.
Among the phytochemicals present in cruciferous vegetables,
glucosinolates (GLs) are the major constituents and by 2012 the esti-
mated number of the identified compounds is around 132.12 Their
structures share a common core of a b-
via a sulfur atom to a (Z)-N-hydroximinosulfate ester and a variable
aglycon side chain derived from the -amino acid biosynthetic pre-
D-glucopyrano moiety linked
a
cursor. Several methionine-derived GLs, which constitute the larg-
est group of GLs, bear in their side chain an additional sulfur atom
at different oxidation states (sulfide, sulfoxide, or sulfone functions).
Among them, one of the most studied is RS-(À)-glucoraphanin
(RS-GRA; 4(RS)-methylsulfinylbutyl glucosinolate) a thiosaccharidic
compound found in Brassicaceae, notably in Tuscan black kale.
The hydrolysis of GLs by myrosinase (Myr; EC 3.2.1.147), an
enzyme found both in the same plants and in human bowel micro-
flora, leads to production of several biologically active compounds
such as isothiocyanates (ITCs), thiocyanates and nitriles, depending
on the hydrolytic conditions.
At neutral pH condition Myr catalyses the GLs hydrolysis pro-
ducing the corresponding ITCs.13 The enzymatic action on natural
RS-GRA by Myr releases RS-sulforaphane (RS-SFN; (À)1-isothiocy-
anato-(RS)-(methylsulfinyl)-butane)14 which is considered the
most active phase II enzyme inducer among analogues. The bio-
logical activity of RS-SFN, as an electrophilic compound, activates
the nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant
response element (ARE) pathway and consequently exerts anti-
oxidant functions, regulates glutathione synthesis and regenera-
tion, enhances xenobiotic metabolism and elimination as well
as it inhibits cytokine mediated inflammation (reviewed in
14,15).
To date, most studies on the biological activities of SFN have been
conducted using the racemic form RS,SS-SFN. Conversely, marked
differences in the modulation of cytochrome P450 and of the phase
II enzymes by the two enantiomers of SFN have been recently re-
ported. In particular, in cut tissue slices of both liver and lung of rats,
RS-SFN, generated in situ from natural RS-GRA by Myr-catalysed
hydrolysis, enhanced quinone reductase and glutathione S-transfer-
ase activities, whereas the SS-enantiomer was either ineffective or
provoked a much weaker response.16 Gram-scale production of nat-
ural RS-GRA has been recently set up starting from Tuscan black kale
Cavolo nero (Brassica oleracea L. var. acephala sabellica) according to
2.3. GLs and myrosinase purification
RS-GRA was isolated according to a procedure developed at
CRA-CIN of Bologna, Italy.17 Briefly, seeds of B. oleracea L. var. acep-
hala sabellica (Tuscan black kale) supplied by Subaseeds (Longiano,
Italy) were grinded to a fine powder and subsequently defatted
with hexane. The solvent was removed and the defatted seed meal
was treated with boiling 70% ethanol in order to quickly deactivate
the endogenous enzyme Myr. RS-GRA was extracted using an Ultra-
turrax homogeniser at medium speed for 15 min. The resulting
homogenate was centrifuged at 17,700g for 30 min. The isolation
of the RS-GRA from the extract was carried out by one-step anion
exchange chromatography, and the purity was further improved
by gel-filtration performed using a XK 26/100 column packed with
Sephadex G10 chromatography media (GE Healthcare, Italy), con-
nected to an AKTA-FPLC System (GE Healthcare, Italy). Individual
fractions were analyzed by HPLC and those containing pure
RS-GRA were pooled and freeze-dried.19 RS-GRA was characterized
by 1H and 13C NMR spectrometry and the purity was assayed by
HPLC analysis of the desulfo-derivative according to the ISO
9167-1 method.20 The purity was 99% (peak purity HPLC) and
>95% weight basis (hydrated potassium salt).
The enzyme Myr was isolated from seeds of Sinapis alba L. as
described by Pessina et al.21 with some modification. The specific
activity of the stock solution used in the present study was 60 U/
mg of soluble protein. The enzymatic activity was 32 U/ml and
the solution was stored at 4 °C in sterile saline solution at neu-
tral pH until use. One Myr unit was defined as the amount of
enzyme able to hydrolyze at pH 6.5 and 37 °C 1 lmol/min of
sinigrin.22

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M. Galuppo et al. / Bioorg. Med. Chem. 21 (2013) 5532–5547
Figure 2. Bioactive RS-GRA-treatment prevents body weight loss and preserves DA neurons by MPTP-induced damage. Body weight (A) and behavioural test is performed to
assess bioactive RS-GRA-neuroprotection in sub-acute and acute MPTP-administrated mice. 40 mg/kg MPTP-injected mice show a significant body weight loss when delta
body weight is compared with 40 mg/kg MPTP mice treated with bioactive RS-GRA (A). The same occurs in 20 mg/kg MPTP-administrated mice that, caused by the
progression of the disease, clearly lose weight too. Also in this case, bioactive RS-GRA-control after sub-acute injection of MPTP (20 mg/kg) prevents MPTP-induced weight
loss (A). No sign of any decrease in body weight is detected in bioactive RS-GRA-treated control group (A). Open field is performed separately and in a different way for 40 mg/
kg (without latency-time) (B) and 20 mg/kg (with latency-time) (C, C1) MPTP set groups. In acute MPTP-administrated animals, a reduced degree of mobility and a little
number of changes is noted (B), while bioactive RS-GRA-treatment after MPTP-administration preserve the mice by the neurological changes found in the behaviour (B), also
comparing the results with naïve group (B). Moreover, please, note that also mice no subjected to disease but injected with bioactive RS-GRA as control show a reduced
number of field changed compared with naïve (B): this result is due probably due to a stress condition caused by the daily handling of the animals. This could lead to a
reduced mobility of the mice when put on the table just for fear and not for side effects of the molecule, that is, safe and well tolerated. In any cases, the difference with
40 mg/kg MPTP-administrated mice is significant. During latency-time, 20 mg/kg MPTP-administrated mice causes an immobility of around 40 s (C), while the latency-time
of bioactive RS-GRA-treated is around equal to zero (C). Conversely, confirming this data, during open field 20 mg/kg MPTP-administrated mice totalize a total number of
changed fields lower compared to the bioactive RS-GRA-treated group (C1). The same experimental group examined in a pole test gives significant results. 20 mg/kg MPTP-
administrated mice treated with bioactive RS-GRA show a reduced bewilderment when placed head-up on top of the vertical wooden pole and compared with the untreated
groups (D). Moreover, mice pharmacologically injected with bioactive RS-GRA show a faster drop time, if we compare the results with sub-acute MPTP-administrated group
that don’t receive this treatment (Fig. 1D). Delta body weight (A) ⁄P <0.05 versus MPTP 40 mg/kg; Open field 40 mg/kg (B) #P <0.05 versus MPTP 40 mg/kg; ⁄⁄⁄P <0.0006 versus
NAÏVE; Latency (C) ⁄P <0.05 versus MPTP 20 mg/kg; Open field 20 mg/kg (C1); ⁄⁄P <0.005 versus MPTP 20 mg/kg; Pole test MPTP 20 mg/kg (D) ⁄P <0.05 versus bioactive RS-
GRA-control group.

M. Galuppo et al. / Bioorg. Med. Chem. 21 (2013) 5532–5547
5535
Figure 3. Neurotrophic factors level expression in sub-acute and acute MPTP-induced PD. GAP-43 expression by immuhistochemical localization shows that subacute (C) and
acute (D) MPTP injection causes a decreased GAP-43 expression, while following bioactive RS-GRA administration, GAP-43 staining results strongly and significantly positive
both in subacute (E) and acute (F) MPTP-injured mice. Naive (A) and bioactive RS-GRA control (B) groups show a basal expression for GAP-43. NGF immunolocalization
indicates that subacute (I) and acute (L) MPTP administration decrease NGF expression, opposed to bioactive RS-GRA treatment (M and N, respectively). Naive (G) and
bioactive RS-GRA control (H) groups show a basal expression for NGF.
2.4. Enzyme bio-activation of RS-GRA and animal treatment
Schematically, a plan of the experiment is shown below:
RS-GRA (10 mg/kg) was dissolved in PBS solution pH 7.2 and
mouse treatment required the enzyme bioactivation of the phyto-
chemical. The in situ action of the Myr enzyme (5 ll/mouse) for
15 min allowed us to have a bioactive RS-GRA quickly (Italian Pat-
Days
0–7
Sub-acute PD
Days
Acute PD
Bioactive RS-GRA 0–7
pretreatment
(10 mg/kg)
Bioactive RS-GRA
pretreatment
(10 mg/kg)
MPTP (40 mg/
kg) + bioactive RS-GRA
(10 mg/kg)
ent pending MI2012A001774), before the ip treatment (Fig. 1).
8–12 MPTP (20 mg/
kg) + bioactive
RS-GRA (10 mg/
kg)
13–14 Bioactive
RS-GRA (10 mg/
kg)
8–9
2.5. Timetable and experimental animal groups
Mice were randomly divided according to the following
scheme:
10–14 Bioactive
RS-GRA (10mg/kg)
(1) Acute PD group (n = 10): Mice received two injections of
MPTP (40 mg/kg, ip) 16 h apart.
15
Sacrifice
15
Sacrifice
(2) Acute PD + bioactive RS-GRA group (n = 10): Mice were daily
pre-treated for 1 week with bioactive RS-GRA (10 mg/kg, ip),
before the two injections of MPTP (40 mg/kg, ip) 16 h apart.
Bioactive RS-GRA treatment was protracted until the
sacrifice.
2.6. Immunohistochemical (IHC) localization of IL-1b, DAT,
nitrotyrosine, Bax, Bcl-2, TH
(3) Sub-acute PD group (n = 10): Mice received five injections of
MPTP (20 mg/kg, ip) 24 h apart.
After the sacrifice, brain tissues were collected and fixed in
10% (w/v) PBS-buffered formaldehyde. Sections (6 lm thickness)
(4) Sub-acute PD + bioactive RS-GRA group (n = 10): Mice were
daily pre-treated for 1 week with bioactive RS-GRA (10 mg/
kg, ip), before the five injections of MPTP (20 mg/kg, ip)
24 h apart. Bioactive RS-GRA treatment was protracted until
the sacrifice.
(5) Naïve group (n = 5): Mice no subjected to MPTP damage or
to any injection, sacrificed as control.
(6) Bioactive RS-GRA-CONTROL group (n = 5): Mice no subjected
to MPTP damage but injected with bioactive RS-GRA (10 mg/
kg), sacrificed as control of drug safety and tolerance as well
as to evince eventually behavioural changes on the animals.
were prepared from paraffin embedded tissues. After deparaffini-
zation, endogenous peroxidase was quenched with 0.3% (v/v)
hydrogen peroxide in 60% (v/v) methanol for 30 min. The sections
were permeabilized with 0.1% (w/v) Triton X-100 in PBS for
20 min. Nonspecific adsorption was minimized by incubating
the section in 2% (v/v) normal goat serum in PBS for 20 min.
Endogenous biotin or avidin binding sites were blocked by
sequential incubation for 15 min with avidin–biotin peroxidase
complex. Sections were incubated overnight with different
antibody:

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M. Galuppo et al. / Bioorg. Med. Chem. 21 (2013) 5532–5547
Figure 4. Bioactive RS-GRA prevents DAT loss in sub-acute and acute MPTP-induced PD. IHC localization of DAT shows a positive staining in Naïve (A) and bioactive RS-GRA-
control (B) brain tissue sections. Likewise, a recovery in DAT expression after MPTP-injection is found in animals treated with bioactive RS-GRA in sub-acute administration of
MPTP (E), as well as in acute regime (F). On the contrary, a low or totally absent DAT tissue expression is detected in pharmacologically untreated sub-acute (C) and acute (D)
MPTP-administrated mice.
(1) mouse monoclonal anti-GAP-43(1:50 in PBS, wt/vol) (Santa
Cruz Biotechnology.INC);
(2) rabbit monoclonal anti-NGF (1:100 in PBS, wt/vol)
(Epitomics.INC);
(3) rabbit polyclonal anti-BDNF (1:50 in PBS, wt/vol) (Santa
Cruz Biotechnology.INC);
(4) rabbit polyclonal anti-DAT (1:250 in PBS, wt/vol) (Santa Cruz
Biotechnology.INC);
(5) rabbit polyclonal anti-TH (1:100 in PBS, wt/vol) (Santa Cruz
Biotechnology.INC);
(6) rabbit polyclonal anti-IL-1b (1:100 in PBS, wt/vol) (Santa
Cruz Biotechnology.INC);
(7) rabbit polyclonal anti-nitrotyrosine (1:500 in PBS, wt/vol)
(Millipore);
(8) rabbit polyclonal anti-NrF2 (1:100 in PBS, wt/vol) (Santa
Cruz Biotechnology.INC);
goat anti-rabbit IgG and avidin-biotin peroxidase. The counter
stain was developed with avidin-biotin peroxidase (brown stain-
ing) and Mayer’s hematoxylin (blue background). A positive stain-
ing (brown colour) was found in the sections, indicating that the
immunoreactions were positive. To verify the binding specificity
for IL-1b, DAT, nitrotyrosine, Bax, Bcl-2, NrF2 and GFAP some sec-
tions were also incubated with only the primary antibody (no sec-
ondary) or with only the secondary antibody (no primary). In these
situations no positive staining was found in the sections indicating
that the immunoreactions were positive in all the experiments car-
ried out.
All sections were obtained using light microscopy (LEICA DM
2000 combined with LEICA ICC50 HD camera). Leica Application
Suite V4.2.0 software was used as image computer program to ac-
quire pictures and to perform densitometric analysis.
(9) goat polyclonal anti-GFAP (1:50 in PBS, wt/vol) (Santa Cruz
Biotechnology.INC);
2.7. Golgi stain
(10) rabbit polyclonal anti-Bcl-2 (1:100 in PBS, wt/vol) (Santa
Cruz Biotechnology.INC);
FD Neurotech kit (FD NeuroTechnologies, Ellicott City, MD, USA)
was used for Golgi impregnation of tissue.
(11) rabbit polyclonal anti-Bax (1:100 in PBS, wt/vol) (Santa Cruz
Biotechnology.INC).
Brain samples were placed directly into solution (A + B) con-
taining mercuric chloride, potassium dichromate and potassium
chromate, without rinsing, and remained there for 2 weeks in the
dark at room temperature. Forty-eight hours after placing in
solution C (4 °C), brains were frozen on dry ice and stored at
Sections were washed with PBS, and incubated with secondary
antibody. Specific labelling was detected with a biotin-conjugated

M. Galuppo et al. / Bioorg. Med. Chem. 21 (2013) 5532–5547
5537
Figure 5. TH protein expression in the striatum after MPTP lesioning. At 8 days after MPTP-injection both in sub-acute and acute regime, immunohistochemistry analysis
using an antibody against TH protein demonstrates an increased expression in striate brain section of animals treated with bioactive RS-GRA (E and F). Brain sections, taken
from bioactive RS-GRA-untreated sub-acute (C) and acute (D) MPTP-administrated mice sample slides, stained for TH protein, show a negative staining. Conversely, a positive
expression has been found in naive mice group sections (A) as well as in bioactive RS-GRA control group (B).
À70 °C until sectioning. Cryostat sections (100
l
m) were cut at
Moreover, each induction of MPTP was followed by the behav-
ioural tests listed below:
À25 °C and mounted onto gelatinized slides. Slides were allowed
to dry in the dark, and the rest of the staining process done as pre-
viously described.23
Open field: Motor function of the animal is measured by quanti-
fied spontaneous activity in the open field. Locomotor activity was
monitored for 3 min in an open field, a white Plexiglas box
50 Â 50 cm with its floor divided into 16 squares. Four squares
were defined as the center and 12 squares along the walls as the
periphery. Each mouse was gently placed in the center of the box
and activity was scored as a line crossing when a mouse removed
all four paws from one square and entered another. Immediately
after each test, the apparatus was thoroughly cleaned with cotton
pad wetted with 70% ethanol. The line crossings and the time spent
in the center were counted and scored. Just for 20 mg/kg MPTP-
administrated group and the relative bioactive RS-GRA-treated
group, the test was performed after a latency period of 5 min, dur-
ing which mice were placed on the table and the time elapsing first
change of square was noted.
Neurons were chosen for the analysis if completely impreg-
nated with Golgi stain and unobscured by other impregnated neu-
rons or precipitant. To perform spine count, the longest dendrite
trees visible within the plane of focus, with continuous unbroken
dendrites, well impregnated cells and clearly distinguishable from
adjacent, were chosen. Spines were counted under oil (X100),
using light microscopy (LEICA DM2000, equipped with a LEICA
ICC50 HD camera), and the entire visible dendrite length mea-
sured by Imaging computer program (Leica Application Suite
V4.1). Spine density was calculated referring to the length of the
dendrite.
2.8. Body weight loss and behavioural tests
Pole test: The pole test is used to assess basal ganglia related
movement disorders in mice. In particular, it is a useful method
in mice PD model to measure bradykinesia.24
In addition, the measure of the body weight was daily assessed,
and any loss was evaluated as marker of pathology. The graph rep-
resents
D
value for each group obtained calculating the difference
Briefly, after 20 mg/kg MPTP-injection and the open field test,
animals are placed head-up on top of a vertical wooden pole
(diameter 5 mm, height 55 cm, with rough surface). The base of
in body weight between the measure taken the day of sacrifice and
that one taken the day of the disease induction.

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M. Galuppo et al. / Bioorg. Med. Chem. 21 (2013) 5532–5547
Figure 6. Bioactive RS-GRA-treatment modulates IL-1b tissue localization after MPTP-induced lesioning. Cytokine tissue localization has been performed in brain tissue
sections at 8 days after MPTP-injection. Inflammatory signs of MPTP neurotoxicity were been counteracted by bioactive RS-GRA-treatment both in sub-acute (E) and acute (F)
MPTP-regime. On the contrary, IL-1b tissue localization show a significantly intense staining in section of tissue taken from bioactive RS-GRA-untreated mice, both in sub-
acute (C) and acute (D) MPTP-regime. Please, note that naive (A) and bioactive RS-GRA-control mice (B) don’t present any positive staining for IL-1b IHC localization.
the pole is placed in a cage filled with bedding material. When
placed on the pole, animals orient themselves downward and des-
cend the length of the pole back into the cage. During the behav-
ioural test, animals were videotaped and timed. The time that
mice needed to turn down completely and climbed down and four
feet reached the floor was recorded.
acute experimental set (40 mg/kg MPTP) when animal delta body
weight has been compared with that one of mice subjected to
40 mg/kg MPTP + treatment with bioactive RS-GRA.
Moreover also comparing sub-acute 20 mg/kg MPTP group with
bioactive RS-GRA-control group, and MPTP-administrated animals
treated with bioactive RS-GRA there are difference in delta body
weight. In fact, caused by the progression of the disease, 20 mg/
kg MPTP-administrated mice clearly lose weight.
2.9. Statistical analysis
On the contrary, after PD induction, bioactive RS-GRA-treated
mice increased their body weight.
No sign of any decrease in body weight was detected in bioac-
tive RS-GRA-treated control group.
All statistical comparisons of values between two animal
groups were carried out by unpaired Student’s t-test and multiple
group comparisons were conducted by one-way analysis of vari-
ance (ANOVA) with Bonferroni test with the Prism software
(GraphPad Software, Inc., La Jolla, CA, USA). Data are represented
as mean S.E.M. In all cases, a P value of <0.05 was considered to
be significantly different.
3.2. Motor behaviour evidences about bioactive RS-GRA-
treatment protective role in sub-acute and acute PD
development
A square table divided in sub-squares by a grid was used for
open-field test.
3. Results
For 40 mg/kg MPTP-administrated mice, the test was performed
without any latency time, since the animals showed a constant
behavioural immobility. The results show that reduced degree of
mobility and a little number of changes was noted in acute
MPTP-administrated animals, while bioactive RS-GRA-treatment
3.1. Bioactive RS-GRA-treatment avoids body weight changes
resulting in sub-acute and acute MPTP-induced PD
Figure 2A summarizes the results of body weight changes at the
end of the experiment. Significant body weight loss was found in

M. Galuppo et al. / Bioorg. Med. Chem. 21 (2013) 5532–5547
5539
Figure 7. Bioactive RS-GRA-treatment prevents nitrosative stress damage in DA neurons. Nitrotyrosine immunolocalization results negative in naive (A) and bioactive RS-
GRA-control (B) brain tissue sections. Likewise, a low or totally absent nitrotyrosine tissue expression was detected in pharmacologically bioactive RS-GRA-treated sub-acute
(E) and acute (F) MPTP-administrated mice. On the contrary, a significant nitrotyrosine expression was found in animals untreated with bioactive RS-GRA in sub-acute (C), as
well as in acute (D) after administration of MPTP.
after MPTP-administration preserve the mice by the neurological
behavioural changes (Fig. 2B), also compared to the results of naïve
and bioactive RS-GRA-control group. Moreover, please, consider
that bioactive RS-GRA-control group consists of animals that are
daily handled for bioactive RS-GRA injection so that, the behav-
ioural differences with naïve animals could be explained in this
way. In any case, the difference with 40 mg/kg MPTP-adminis-
trated mice remain significant.
For 20 mg/kg MPTP-administrated mice, the test was started by
placing the mice at the center of the table to be familiar with the
situation for a time of 5 min (latency-time, Fig. 2C), then the total
horizontal ambulatory route and the spatial analysis activity with-
in the period of the following 3 min was measured by observers.
Looking at the latency-time (Fig. 2C), sub-acute MPTP-administra-
tion causes an immobility time of around 40 s, while bioactive
RS-GRA-treated mice have showed a latency-time around equal
to zero.
To confirm these results, immediately after the open field, the
same animals were examined for evaluating the degree of PD-re-
lated bradykinesia, performing a pole test (Fig. 2C1).
20 mg/kg MPTP-administrated mice treated with bioactive
RS-GRA don’t show bewilderment when placed head-up on top of
the vertical wooden pole and compared with the untreated groups.
Moreover, pharmacological injection of bioactive RS-GRA causes
faster drop time, if we compare the results with sub-acute MPTP-
group that didn’t receive this treatment (Fig. 2C1).
3.3. Bioactive RS-GRA-treatment enhances neurotrophic factor
release
By neurotrophic factors analysis, authors have hypothesized
that, following both subacute and acute MPTP regime, a possible
mechanism of action of bioactive RS-GRA treatment can be related
to the release of these mediators supporting the neuroprotective
action.
In particular, we have detected GAP-43 expression in SN by
immuhistochemical localization. Results clearly show that suba-
cute (Fig. 3C, see densitometric analysis Fig. 13A) and acute
(Fig. 3D, see densitometric analysis Fig. 13A) MPTP injection causes
a decreased GAP-43 expression, while under the same damaging
conditions, bioactive RS-GRA administration increased GAP-43
(Fig. 3E and F, respectively, see densitometric analysis Fig. 13A).
Please, note that naive (Fig. 3A, see densitometric analysis
Fig. 13A) and bioactive RS-GRA control (Fig. 3B, see densitometric
analysis Fig. 13A) groups show a basal expression for GAP-43.
We also detected NGF expression. Our results indicate that sub-
acute (Fig. 3I, see densitometric analysis Fig. 13B) and acute
(Fig. 3L, see densitometric analysis Fig. 13B) MPTP administration

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M. Galuppo et al. / Bioorg. Med. Chem. 21 (2013) 5532–5547
Figure 8. Bioactive RS-GRA-treatment stimulates Nrf2 overexpression after sub-acute and acute MPTP damage. Histological slides of subacute (C) and acute (D) MPTP-
administrated mice untreated with bioactive RS-GRA were negative for Nrf2 antibody. On the contrary, when after PD-induction bioactive RS-GRA was given (E and F), it
causes Nrf2 expression at high levels, preserving tissues by MPTP-damage. Note that brain sections taken from in naive (A) and bioactive RS-GRA (B) animals no subjected to
MPTP-administration and changes didn’t stain after Nrf2 incubation. This was considered normal considering that animals weren’t subjected to any stress or damage (quiet
state).
decrease NGF, which was reversed by bioactive RS-GRA treatment
(Fig. 3M and N, respectively, see densitometric analysis Fig. 13B).
Please, note that naive (Fig. 3G, see densitometric analysis
Fig. 13B) and bioactive RS-GRA control (Fig. 3H, see densitometric
analysis Fig. 13B) groups, not undergoing to MPTP-damage, show
just a basal expression for NGF.
Moreover, bioactive RS-GRA administration following MPTP-
damage enhanced BDNF level showing the same trend of the afore-
mentioned neurotrophic factors (data not shown).
sis Fig. 13C) and acute (Fig. 4F, see densitometric analysis
Fig. 13C) MPTP-regime prevents the loss of DA transporter
expression in brain section. Sections from naïve group (Fig. 4A,
see densitometric analysis Fig. 13C) or drug control group
(Fig. 4B, see densitometric analysis Fig. 13C), incubated with
anti-DAT antibody, show no reduction in the level of expression
of this marker.
The pathophysiology of PD is largely correlated with a decrease
in tyrosine TH activity, TH synthesis and TH mRNA in the striatum.
Thus, due to its importance as rate limiting enzyme in the conver-
3.4. Bioactive RS-GRA-treatment prevents DAT loss and TH
sion of tyrosine into L
-DOPA during dopamine synthesis,27 we
decrease in nigrostriatal neuronal cells
decided to assess the changes in the expression levels of TH. Both
20 mg/kg and 40 mg/kg MPTP-injections followed by bioactive
RS-GRA-treatment (Fig. 5E and F, respectively, see densitometric
analysis Fig. 13D) evoke an increased TH expression in striate brain
section, that correlated with the return of striatal dopamine. Con-
versely, brain sections taken from bioactive RS-GRA-untreated sub-
acute (Fig. 5C, see densitometric analysis Fig. 13D) and acute
(Fig. 5D, see densitometric analysis Fig. 13D) MPTP-administrated
mice and incubated with anti-TH antibody, show negative staining
for this marker. An intense positive staining was found in naive
(Fig. 5A, see densitometric analysis Fig. 13D) as well as in bioactive
RS-GRA-control (Fig. 5B, see densitometric analysis Fig. 13D)
samples.
Scientific literature about PD feature and pathway largely
shows that the plasma membrane DAT is a reliable marker of pre-
synaptic DA terminal loss.25 So, each cellular role currently associ-
ated with the DAT depends on plasma membrane expression.26
As showed in the Figure 4, one of the first signs of MPTP-induced
neurotoxicity is the neuronal damage in the substantia nigra, that
causes a decreasing of DAT expression, both in sub-acute (Fig. 4C,
see densitometric analysis Fig. 13C) and acute (Fig. 4D, see densito-
metric analysis Fig. 13C) PD brain tissue sections.
On the contrary, the neuroprotective action of bioactive
RS-GRA-treatment in sub-acute (Fig. 4E, see densitometric analy-

M. Galuppo et al. / Bioorg. Med. Chem. 21 (2013) 5532–5547
5541
Figure 9. Bioactive RS-GRA-treatment prevents GFAP expression in neuronal cells during PD GFAP works as pro-oxidative marker during neurodegenerative diseases. As
expected, sub-acute (C) and acute (D) MPTP-administrated mice exhibited positive immunolocalization for GFAP, whereas bioactive RS-GRA-treatment reduced the degree of
positive staining for GFAP in mice subjected to MPTP-induced sub-acute (E and acute F) injury. Naive (A) and bioactive RS-GRA-control (B) brain samples section did not stain
for GFAP too.
3.5. Bioactive RS-GRA-treatment inhibits expression of IL-1b,
modulating inflammatory pathway
3.6. Bioactive RS-GRA-treatment preserves neuronal cells by
oxidative stress after sub-acute and acute MPTP-induced PD:
evidences by nitrotyrosine, Nrf2 and GFAP expression
A key role in PD development was attributed to cytokine release
that was associated with a progressive condition of disease in ani-
mals MPTP-administrated.
One consequence of upstream inhibition of the inflammatory
pathway is the possible blockage of the oxidative stress triggering.
Starting from the supposed anti-inflammatory power of bioactive
RS-GRA and since an increased nitration of proteins leading to
nitrosative stress has been identified in motor neurons of patients
with neurodegenerative disease,29 we choose as indirectly marker
for peroxynitrite formation in vivo the IHC detection of nitrotyro-
sine expression.
As showed in the Fig. 7, bioactive RS-GRA inhibits MPTP-in-
duced oxidant production and oxidative DNA damage in the SNpc.
20 mg/kg (Fig. 7C, see densitometric analysis Fig. 13F) and
40 mg/kg (Fig. 7D, see densitometric analysis Fig. 13F) MPTP-
administrated mice showed significant positive staining for
nitrotyrosine. The corresponding bioactive RS-GRA-treated animal
groups present a reduced tissue expression for this marker in both
sub-acute (Fig. 7E, see densitometric analysis Fig. 13F) and acute
(Fig. 7F, see densitometric analysis Fig. 13F) conditions.
In particular, mice expressing a dominant-negative inhibitor of
IL-1b-converting enzyme or those deficient in TNF-a or iNOS seem
resistant to MPTP-induced neurotoxicity.28
Visibly, our results show that brain sections cut from sub-acute
(Fig. 6E, see densitometric analysis Fig. 13E) and acute (Fig. 6F, see
densitometric analysis Fig. 13E) MPTP-administrated mice treated
with bioactive RS-GRA show a reduced IL-1b IHC localization.
Conversely, sub-acute (Fig. 6C, see densitometric analysis
Fig. 13E) and acute (Fig. 6D, see densitometric analysis Fig. 13E)
MPTP-administrated mice that didn’t received bioactive RS-GRA-
treatment have high levels of IL-1b expression, supporting the
idea according to which one of the possible mechanism of action
of bioactive RS-GRA should be an anti-inflammatory role of the
molecule.
Please, note that IL-1b staining detection was negative in naive
(Fig. 6A, see densitometric analysis Fig. 13E) as well as in bioactive
RS-GRA-treated control mice sections (Fig. 6B, see densitometric
analysis Fig. 13E).
Naive (Fig. 7A, see densitometric analysis Fig. 13D) and bioac-
tive RS-GRA control (Fig. 7B, see densitometric analysis Fig. 13F)
groups show no positive staining for nitrotyrosine.

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M. Galuppo et al. / Bioorg. Med. Chem. 21 (2013) 5532–5547
Figure 10. Bioactive RS-GRA-treatment modulates apoptotic pathway, decreasing Bax tissue expression. IHC localization for Bax demonstrates that sub-acute (C) and acute
(D) MPTP-administration activates pro-apoptotic proteins at the level of nigrostriatal brain sections. On the contrary, no staining is present in sections of animals subjected to
subacute and acute MPTP-induced injury and treated with bioactive RS-GRA (E and F, respectively). A negative staining for naive (A) and bioactive RS-GRA-control mice
sections (B) is detected too.
Moreover, to confirm the reduced oxidative stress after bioac-
tive RS-GRA-treatment, Nrf2 IHC localization was performed.
A negative expression was found in histological slides of suba-
cute (Fig. 8C, see densitometric analysis Fig. 13G) and acute
(Fig. 8D, see densitometric analysis Fig. 13G) MPTP-administrated
mice untreated with bioactive RS-GRA, that, when given (Fig. 8E
and F, respectively see densitometric analysis Fig. 13G), on the con-
trary, keeps Nrf2 nuclear expression at high levels, preserving tis-
sues by MPTP-damage.
Note that brain sections taken from naive (Fig. 8A, see densito-
metric analysis Fig. 13G) and bioactive RS-GRA (Fig. 8B, see
densitometric analysis Fig. 13G) animals no subjected to MPTP-
administration and changes didn’t stain after Nrf2 incubation. This
was believed normal considering that animals weren’t subjected to
any stress or damage (quiet state).
To confirm this data, we stained brain section with an anti-
GFAP antibody. As expected in agreement with previous results,
sub-acute (Fig. 9C, see densitometric analysis Fig. 13H) and acute
(Fig. 9D, see densitometric analysis Fig. 13H) MPTP-administrated
mice exhibited positive immunolocalization for cytosolic GFAP,
whereas bioactive RS-GRA-treatment reduced the degree of posi-
tive staining for GFAP in mice subjected to MPTP-induced sub-
acute (Fig. 9E, see densitometric analysis Fig. 13H) and acute
(Fig. 9F, see densitometric analysis Fig. 13H) injury. Naive
(Fig. 9A, see densitometric analysis Fig. 13H) and bioactive RS-
GRA-control brain samples section (Fig. 9B, see densitometric anal-
ysis Fig. 13H) did not stain for GFAP too.
3.7. Bioactive RS-GRA-treatment protects DA neurons against
apoptotic cell death
Brain tissue samples were also collected 8 days after MPTP-
administration for IHC detection of Bax and Bcl-2.
Sections cut from naive (Fig. 10A, see densitometric analysis
Fig. 13I) as well as from bioactive RS-GRA-control mice (Fig. 10B,
see densitometric analysis Fig. 13I) samples did not stain for Bax,
whereas tissue MPTP-administrated sub-acute (Fig. 10C, see densi-
tometric analysis Fig. 13I) and acute (Fig. 10D, see densitometric
analysis Fig. 13I) mice exhibited positive immunolocalization. Bio-
active RS-GRA-treatment reduced the degree of positive staining
for Bax in mice subjected to MPTP-induced sub-acute (Fig. 10E,
see densitometric analysis Fig. 13I) and acute (Fig. 10F, see densi-
tometric analysis Fig. 13I) injury.
On the contrary, brain tissue sections cut from naive (Fig. 11A,
see densitometric analysis Fig. 13L) and bioactive RS-GRA-treated
control mice demonstrate positive staining for Bcl-2 (Fig. 11B,

M. Galuppo et al. / Bioorg. Med. Chem. 21 (2013) 5532–5547
5543
Figure 11. Bioactive RS-GRA-treatment modulates apoptotic pathway, avoiding Bcl-2 tissue downregulation. Brain tissue sections cut from naive (A) and bioactive RS-GRA-
treated control (B) mice demonstrated positive staining for Bcl-2, while in sub-acute (C) and acute (D) MPTP-administrated mice staining for Bcl-2 was absent. Bioactive RS-
GRA-treatment significantly attenuates the loss of positive staining for Bcl-2 in samples cut from mice subjected to sub-acute and acute MPTP-induced injury (E and F).
see densitometric analysis Fig. 13L), while in MPTP-administrated
sub-acute (Fig. 11C, see densitometric analysis Fig. 13L) and acute
(Fig. 11D, see densitometric analysis Fig. 13L) mice staining for Bcl-
2 was absent. Bioactive RS-GRA-treatment significantly attenuated
the loss of positive staining for Bcl-2 in the samples cut from mice
subjected to MPTP-induced sub-acute and acute injury (Fig. 11E
and F, respectively see densitometric analysis Fig. 13L).
In the dentate gyrus of MPTP-injured animals in both sub-acute
(Fig. 12C, see mean of dendritic spines/10 fields, Fig. 12G) and
acute (Fig. 12D, see mean of dendritic spines/10 fields, Fig. 12G)
trend, the number of tree branches results significantly reduced.
Conversely, bioactive RS-GRA-treatment protects the neuronal
functional disruption (Fig. 12E and F, respectively see mean of den-
dritic spines/10 fields, Fig. 12G), with a dendritic spine count for
field that resembling the results obtained for naïve and bioactive
RS-GRA-control groups (Fig. 12A and B, respectively see mean of
dendritic spines/10 fields, Fig. 12G).
3.8. Bioactive RS-GRA-treatment protects DA neurons against
impaired synaptic functionality and plasticity
4. Discussion
An abnormal number of dendrite spines is often associated with
brain disorders and correlated to abnormal neuronal cell function
in terms of biochemical signals, in particular Ca2+-mediated synap-
tic plasticity.30 Also Bezard, in a short commentary published in
2010, highlights that in advanced PD there is a marked atrophy
of dendrites and spines in striatofugal neurons—indicative of dys-
functional signal integration—and underlies the importance of pre-
vention of spine loss for effective therapy based on dopamine cell
replacement.31
Quantitative analysis of dendrite spine density was performed
through Golgi impregnation in hypothalamic dental nucleus sec-
tions. Microscopic images were acquired and following, the num-
ber of spines for a total number of 10 fields was obtained.
Established evidences, combined with the most recent litera-
ture, show the beneficial power of Brassicaceae consumption,32
very likely attributed to the dominant presence of GLs-derived
ITCs, where the RS-SFN was the most evoked protective
phytochemical.
Our approach is to develop an exciting way to deliver a highly
unstable compound such as RS-SFN. This natural substance has to
be kept at À20 °C and it is poor soluble in water. Moreover, SFN
is usually tested in vitro and in vivo dissolved in corn oil or in
water solution containing DMSO. Bioactivation of RS-GRA with
Myr enzyme allows overcoming the above mentioned drawbacks.

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M. Galuppo et al. / Bioorg. Med. Chem. 21 (2013) 5532–5547
Figure 12. Dendritic spine and apoptosis. Golgi stain for dendrite spine detection shows a marked recovery in bioactive RS-GRA-treated mice in PD mice. Brain samples taken
from naive (A) and bioactive RS-GRA-control (B) animals show a normal number of dendrite spines. Degree of MPTP lesions in the SNpc is scored on the basis of neuronal
dendrite spine disruption. Nigral DA neurons are preserved by bioactive RS-GRA-treatment in sub-acute (E) and acute (F) MPTP-injured mice sections. A severe loss of
termination at level of the dendrite tree is marked in sub-acute (C) MPTP-damaged section and more significantly evident in acute (D) MPTP-administration when bioactive
RS-GRA wasn’t administrated. Bioactive RS-GRA-treatment (B) preserves dendrite spines by neuronal degeneration following sub-acute and acute MPTP-administration.
DENDRITIC SPINES (G) ⁄P <0.05 versus NAÏVE; ⁄⁄P <0.05 versus NAÏVE; ⁄⁄⁄⁄P <0.0001 versus NAÏVE; ####P <0.0001 versus 20 mg/kg MPTP; °°°°P <0.0001 versus 40 mg/kg MPTP.
In Tuscany, cabbage cultivations represent a thriving resource, and
the production in this area of the famous black kale (B. oleracea var.
acephala subvar. sabellica) is of particular interest due to the high
level of RS-GRA concentration in this vegetable which is correlated
to antioxidant33 and anti-cancerogenic properties.34 Starting from
the idea that RS-GRA is easily available at CRA-CIN of Bologna
(Italy) together with homogeneous Myr, our goal was to demon-
strate a possible neuroprotective power of bioactive RS-GRA, as a
novel important field of action potentially applicable in the pre-
vention of PD, widely associated both to inflammatory response

M. Galuppo et al. / Bioorg. Med. Chem. 21 (2013) 5532–5547
5545
Figure 13. Densitometric analysis summary. For IHC images, densitometric analysis was carried out to quantify and highlight significant differences among experimental
groups. Summarizing: (A) GAP-43; (B) NGF; (C) dopamine transporter (DAT); (D) tyrosine hydroxylase; (E) interleukin-1beta; (F) nitrotyrosine; (G) nuclear factor-erythroid 2
p45-related factor 2 (Nrf2); (H) glial fibrillary acidic protein (GFAP); (I) Bax; and (L) Bcl-2. A P value <0.05 was considered significant.
and oxidative mechanism, as well as to apoptotic pathway and
neurodegenerative feature.
model, but after the initial response to the first injection, cells seem
to become resistant to MPTP and remain in a silent state.11
At any rate, wanting us keep here on a classic line of research
focused just on DA neurons, we have obtained evidences to believe
that, despite subacute conditions, a milder but, not for this reason,
less worrying state of illness occurs.
Thus, this study was designed to provide new knowledge about
bioactive RS-GRA action and, in particular, to understand whether
its capability to inhibit the progression of PD could cover a wide
range of advancement of the pathology. Current literature shows
that MPTP can be injected according to a variety of regimes.35
Our results show that acute induction of PD is characterized by
an higher toxic power (as confirmed in other studies36) with more
marked symptoms of pathology, both in terms of weight loss and
in behavioural attitudes as well as in damage by dendrite spine
loss. Moreover, the severity of the pathology induced under acute
regime is translated also in a more serious panel of inflammatory
and apoptotic mediator expression.
He et al. going over the notion that the neurotoxicity of MPTP is
restricted to DA neurons in the substantia nigra, not only reported
evidences of rapid cell death in the subventricular zone (SVZ) and
rostral migratory stream (RMS) in the adult C57BL/6 mouse brain
in response to acute or subacute treatment with MPTP but they
have also speculated that continuous cell death should be detect-
able following consecutive injections of MPTP in the subacute
Acute model seems to trigger a necrotic mechanism of neuronal
cell death unlike what happens in the subacute model, in which
apoptotic events occur. So, differences in these two MPTP neuro-
toxicity paradigms lead to different responses in terms of oxidative
damage extension as well as of different microglia activation in ini-
tiating and/or perpetuating DA cell death. Also, in acute than in
subacute MPTP regime there is a greater degree of oxidative stress
and a more robust microglia activation,37–39 possibly due to a
markedly increased in the mitochondrial complex I inhibition. This
causes the secretion of a complex array of cytokines, chemokines,
and reactive oxygen/nitrogen species that create a profound oxida-
tive stress leading to a necrotic cell death in SNpc.36 In both cases,
it is noteworthy that the bioactive RS-GRA administration has
shown a trend of neuroprotection depending of MPTP dose-
dependent.

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M. Galuppo et al. / Bioorg. Med. Chem. 21 (2013) 5532–5547
A possible mechanism, that we here suggest to understand the
MPTP-injected mice, we found a neuroprotection due to prophy-
lactic pretreatment by bioactive RS-GRA, commensurate to the
damage power of the toxic molecule.
Interestingly, Bax/Bcl-2 balance isn’t altered after bioactive
RS-GRA-treatment both in sub-acute and acute MPTP-regime. Like-
wise, further demonstration of neuroprotection and maintenance
of the neuronal functionality was the unaffected number of den-
drite spines, preserved following the pharmacological treatment
but severely decreased, according to a MPTP-dosage dependence
manner in untreated mice.
bioactive RS-GRA role, is based on the idea that these effects could
be due to the stimulation of neurotrophic factors release.
BDNF as well as GAP-43 and NGF, a survival-promoting factor,
have been yet hypothesized to bear neurotrophic support against
neurodegenerative effects of MPTP.40–42 In particular, high levels
of NGF seem counteract the death mechanisms of cell damage by
ROS in MPTP-induced parkinsonism, leading to nigrostriatal DA
neuron degeneration.41 The loss of DA neurons in the SNpc43 is
unanimously recognized as a pathological hallmark of idiopathic
PD. DAT deficiency is an important illness factor in PD, being re-
duced by 50–70%44 and playing a key role in the occurrence of mo-
tor complications.45 The treatment with bioactive RS-GRA
represents a beneficial protective factor, into avoiding the molecu-
lar deficit of DAT and this data is supported by the evidence that
TH, involved into metabolism of dopamine, is preserved by bioac-
tive RS-GRA administration too.
Visibly, this has resulted in a good motor capability, absence of
bewilderment, bradykinesia and tremor as well as of all behav-
ioural symptom of disease.
Despite we don’t know exactly the mechanism through which
PD signs are reduced by bioactive RS-GRA, we hypothesized that
it could modulate the triggering of the inflammatory cascade and
the responses to it correlated.
5. Conclusions
Taken together, our results show a protective role of bioactive
RS-GRA against MPTP neurotoxicity in mice, suggesting that it
could represent a potential treatment in early, not advanced or
chronic phases of PD.
The DA neurodegeneration in PD caused by a cascade of events
involving different inflammatory mediators and downstream cas-
cade both due to radicalic species and apoptotic intermediates is
affected by bioactive RS-GRA-treatment, the benefits of which can
be obtained either in the form of readily available dietary supple-
ment or as a therapeutic agent safe and well tolerated.
Thus, this assumption was confirmed by the investigation of
key regulators of (1) inflammatory response (2) oxidative damage
and (3) apoptotic pathway.
Acknowledgements
Inflammation has been implicated in the pathogenesis of PD.43
In particular, microglia activation, following NFkB translocation
during PD, lead to the related release of Cox-2, IL-1b, and toll-like
receptor activation, resulting in increased degeneration of DA neu-
rons of the SNpc.46
The authors would like to thank the secretary office of IRCCS
Centro Neurolesi ‘‘Bonino-Pulejo’’, Messina for their excellent tech-
nical assistance and all staff of Institute of Experimental Zoopro-
phylaxy of Sicily ‘‘A. Mirri’’, Palermo, Italy for the experimental
assistance during this study.
By our analysis of one among these hallmarks, it was resulted
that, effectively, there is a dowregulation of IL-1b in animals trea-
ted with bioactive RS-GRA, showing that, one of the possible action
ways of this molecule is the control of the cytokine production.
This data was open to counterproof. Other evidences were given
by the analysis of stress oxidative degree after bioactive RS-GRA
treatment.
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