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A 2-year Double-Blind RCT Follow-up Study with Fermented Papaya
Preparation (FPP) Modulating Key Markers in Middle-Age Subjects with
Clustered Neurodegenerative Disease-Risk Factors
Francesco Marotta1*, Massimiliano Marcellino1, Umberto Solimene2, Biagio Cuffari3, Hariom Yadav4, Alexander N Khokhlov5, Aldo Lorenzetti1, Amelie
Mantello6, Joseph Cervi1 and Roberto Catanzaro3
1ReGenera Research Group for Aging Intervention, San Babila Clinic, Milano, Italy
2WHO-Center for Traditional Medicine and Biotechnology, University of Milano, Italy
3Department of Internal Medicine, University of Catania, Catania, Italy
4Center for Diabetes, Obesity and Metabolism, Wake Forest Medical Center, Biotech Place, Winston-Salem, USA
5Evolutionary Cytogerontology Sector, School of Biology, Moscow State University, Moscow, Russia
6Osato Research Institute and Labs, Gifu, Japan
*Corresponding author: Prof. Francesco Marotta, ReGenera Research Group for Aging Intervention, San Babila Clinic, Corso Matteotti 1/A, 20121 Milano, Italy, Tel:
+39-024077243; E-mail: fmarchimede@libero.it
Received date: Apr 28, 2017; Accepted date: May 16, 2017; Published date: May 18, 2017
Copyright: © 2017 Marotta F, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Abstract
In recent years a number of studies have reported the significant relationship between metabolic syndrome and
neurodegenerative disease. There is accumulating evidence that the interplay of combined genetic and
environmental risk factors (from diet to life style to pollutants) to intrinsic age-related oxi-inflammatory changes may
be advocated for to explain the pandemic of neurodegenerative diseases. In recent years a specific Fermented
Papaya Preparation (FPP) has been shown to significantly affect a number of redox signalling abnormalities in a
variety of chronic diseases and as well in aging mechanisms either on experimental and on clinical ground. The aim
of the present study was to evaluate FPP use in impending metabolic disease patients with potentially
neurodegenerative disease clustered risk factors. The study population consisted of 90 patients aged 45-65 years
old, with impending metabolic syndrome and previously selected as to be ApoE4 genotype negative. By applying a
RCT, double-blind method, one group received FPP 4.5 g twice a day (the most common dosage utilized in prior
clinical studies) while the other received an oral antioxidant cocktail (trans-resveratrol, selenium, vitamin E, vitamin
C). Then, after 21 month treatment period, a selected heavy metal chelator was added at the dosage of 3 g/nocte for
the final 3 months study treatment. The parameters tested were: routine tests oxidized LDL-cholesterol, anti-oxidised
LDL, Cyclophilin-A (CyPA), plasminogen activator inhibitor-1 and CyPA gene expression. From this study it would
appear that FPP, unlike the control antioxidant, significantly decreased oxidized-LDL and near normalizing the anti-
Ox-LDL/Ox-LDL ratio (p<0.001) although unaffecting the lipid profile per sè. Moreover, only FPP decreased
cyclophilin-A plasma level and plasminogen activator-inhibitor (p<0.01) together with downregulating cyclophilin-A
gene expression (p<0.01). Insulin resistance was only mildly improved. Heavy metals gut clearance proved to be
effectively enhanced by the chelator (p<0.01) and this was not affected by any of the nutraceuticals, nor it added any
further benefit to the biological action of FPP.
Keywords: Fermented Papaya Preparation (FPP); Redox balance;
Antioxidant; Neurodegenerative disease; Caviarlieri
Introduction
Starting in the early 90’ gold standard electron spin resonance
studies had shown that a functional food consisting of fermented
papaya (FPP, ORI, Oxidative Stress laboratory, Gifu, Japan) exhibited a
powerful anti-oxidative activity on
in vitro
cerebral cells [1] as well on
in vivo
epilepsy experimental model, where the epileptogenic
monoamine neural release was consistently reduced [2]. Later on, a
Japanese group proved the capacity of FPP to reduce the derangement
of oxidant/antioxidant balance at the brain level in elderly rats and in
experimental ischemia-reperfusion induced cerebral damage as well
[3,4]. In recent years our group has shown that FPP could benecially
eect on clinical ground a number of redox signaling abnormalities in
a variety of chronic diseases [5,6] as well as some key aging
mechanisms [7,8].
Recent studies using transgenic mice have shown the importance of
proinammatory Cyclophilin A (CypA)-metalloproteinase-9 in blood-
brain-barrier integrity [9] while other have proved that a specic
inhibitor of CyPA could reduce neuroinammation, improve motor
neurons activity and prolong survival in a mouse model of
amyotrophic lateral sclerosis [10]. e potential neuroprotective eects
of FPP are at the moment the target of a clinical study on Parkinson’s
disease patients by the neurology group of Nordera. is group has
reported some preliminary promising results such as a reduction of
motor scores of the Unied Parkinson Disease Rating Scale,
improvement of Activity of Daily Living performance and of redox
biochemistry (G. Nordera, personal communication at Oxidative
Stress in Health and Prevention, September 24, 2014 http://
www.centrostressossidativo.it/video-congresso-stress-ossidativo/). is
latter aspect has received further support by the ne biochemical
Clinical Pharmacology &
Biopharmaceutics Marotta et al., Clin Pharmacol Biopharm 2017, 6:2
DOI: 10.4172/2167-065X.1000170
Research Article OMICS International
Clin Pharmacol Biopharm, an open access journal
ISSN: 2167-065X
Volume 6 • Issue 2 • 1000170
analysis of same patients cohort by Bolner et al. [11]. Moreover, it has
been recently shown that FPP could dramatically decrease the
oxidative stress parameters in established Alzheimer Disease (AD)
patients [12].
In recent years a number of studies have reported the signicant
relationship between metabolic syndrome and neurodegenerative
disease [13-15]. Indeed, current views on dietary-related diseases and
also neuroimaging data have reported the association between dis-
metabolic patterns and neurodegenerative damage [16] where low-
grade inammation and insulin-resistance seem to be among main
background factors involved [17-19]. As for metabolic syndrome,
dierent groupings of the following metabolic parameters have been
listed in each classication, such as insulin resistance,
hypertriglyceridemia, low HDL-C, obesity/increased waist
circumference, altered glucose tolerance/diabetes mellitus,
hyperinsulinemia, microalbuminuria and hypertension. In the current
study we referred to the denition of MS as outlined by the American
Heart Association and the National Heart, Lung, and Blood Institute
[20].
A further open issue regarding the multifactorial pathophysiology
of neurodegenerative disease is the potentially detrimental role played
by occupational and environmental exposure to heavy metals [21-25].
Indeed, while some metals are involved in physiological enzymatic
reactions, once they accumulate in the brain, especially if overtly acting
as xenobiotic, may give raise to oxidative stress phenomena with
mitochondrial dysfunction, protein misfolding and aberrant
autophagic processes [26-28]. As a matter of fact the update literature
suggests that the aggregation of disease-related proteins during
physiological aging can be advocated for by abnormal protein
homeostasis observed. Such abnormalities may thus represent a
biomarker of aging that could modulate life span and cause
neurodegeneration. Albeit not fully unfolded as yet, it seems that the
interplay between combined genetic and environmental risk factors
(from diet to life style to pollutants) with intrinsic age-related oxi-
inammatory changes may be advocated for to explain the current
increasing surge of neurodegenerative diseases. Given all above, the
current challenging quest of medical community is mostly focused to
track down early biomarkers of likely modiable causative factors
together with possible benecial approaches within a multifunctional
therapeutic armory. e aim of the present study was to evaluate FPP
use in impending metabolic disease patients with potentially higher
risk to develop a neurodegenerative disease.
Methods
Ethics
All procedures were approved by an independent Ethical
Committee for nonpharmacological research (ReGenera Research
Group for Aging Intervention, protocol FPP-MSNEU-10/2015). Each
subject recruited for the study was fully informed and treated in
compliance with the guidelines of the Declaration of Helsinki.
FPP
e FPP used in the present study was obtained from
Carica papaya
L. cultivated in Hawaii, following yeast fermentation for 10 months
and pharmaceutical-grade batch-to-batch control at the Osato
Research Institute (Gifu, Japan). e nal composition of FPP per 100
g is as follows: 90.7 g carbohydrates, 17 μg vitamin B6, 2 μg folic acid,
2.5 mg calcium, 16.9 mg potassium, 240 μg niacin, 4.6 mg magnesium,
14 μg copper, 75 μg zinc, 16 mg arginine, 6 mg lysine, 5 mg histidine,
11 mg phenylalanine, 9 mg tyrosine, 18 mg leucine, 9 mg isoleucine, 5
mg methionine, 13 mg valine, 11 mg glycine, 8 mg proline, 37 mg
glutamic acid, 11 mg serine, 8 mg threonine, 27 mg aspartic acid, and 2
mg tryptophan. is formulation was chosen also to replicate a similar
mixture reported on the market to have antioxidant eect in humans.
Other claimed “fermented papaya” extracts available on the Italian
market were excluded since prior in-house electron spin resonance
testing had ascertained their poor to nil antioxidant eect (Mantello P.
ORI Oxidative Stress Research Lab, Gifu, Japan. In-house data).
Study design
e study population consisted 90 patients ranging from 45 to 65
years old, with impending metabolic syndrome (2 parameters out of
the above-mentioned classication) and 30 age-matched healthy
volunteers (Table 1). Patients with secondary hypertension,
cardiomyopathy, severe abnormalities of liver, thyroid and kidney
function, cerebrovascular diseases, grossly elevated total cholesterol
(>280 mg/dl or LDL >180 mg/dl), malignancies and history of
coronary bypass surgery or on insulin treatment were excluded from
this study. Exclusion criteria were also the consumption of antioxidant
or other supplements in the past 3 months.
All patients were screened for ApoE4 polymorphism to rule out the
presence of a known risk gene pattern for neurodegenerative disease.
is was a RCT, double-blind study, with FPP 4.5 g given twice a day
vs. common antioxidant cocktail (trans-resveratrol 20 mg, selenium 60
mcg, vit E 30 mg, vit C 100 mg, papaya avour) twice a day. en, aer
21 month continuous treatment period, an heavy metal chelator (talc-
size, higher potency chabasite-phillipsite zeolites naturally-occurring
mixture, Novagenics-SOL Ltd., Hong Kong) was added at the dosage of
3 g/nocte for further 3 months to complete the 24 months study
(Figure 1).
At entry 1, 3, 6, 12, 21 and 24 months, biological samples were
collected (BD Vacutainer sampling tubes) to assess biochemical
assessments as below mentioned. Blood serum was isolated by
centrifugation and stored at under −70°C until analysis. Repeated
thawing and freezing were avoided.
Parameters
Pre-recruiting gene testing: Ten millilitres of peripheral whole blood
was withdrawn from an antecubital vein of all recruited subjects.
Genomic DNA was extracted by a modied salting-out method (by
precipitating DNA with the use of 96% ethanol). is was processed to
ApoE genotyping with modications in the multiplex amplication
refractory mutation system PCR (Multi-ARMS PCR). e primer
sequences used in the PCR analysis were as follows: APOE rs7412
F:ATGCCGATGACCTGCAGA R:ACTGGCGCTGCATGTCTT
Product size (bp) 684; rs429358 F:TCGGAACTGGAGGAACAACT
R:TACACTGCCAGGCGCTTCT Product size (bp) 260. Genomic
DNA (50 ng) amplication (ABI Prism 310, Perkin Elmer, USA)
comprised 30 cycles (94ºC for 5 min, 94ºC for 30 s, 65ºC for 30 s, and
72º for 30 s), followed by a cycle of 10 min at 72°C for the nal chain
extension. Moreover, for the analysis of restriction fragment length
polymorphism, the amplication product was digested with HhaI,
followed by 5.0% agarose gel electrophoresis for 2 h and 30 min,
stained with electro-uorescent red stain (ermoFisher, USA) and
Citation: Marotta F, Marcellino M, Solimene U, Cuffari B, Yadav H, et al. (2017) A 2-year Double-Blind RCT Follow-up Study with Fermented
Papaya Preparation (FPP) Modulating Key Markers in Middle-Age Subjects with Clustered Neurodegenerative Disease-Risk Factors.
Clin Pharmacol Biopharm 6: 170. doi:10.4172/2167-065X.1000170
Page 2 of 9
Clin Pharmacol Biopharm, an open access journal
ISSN: 2167-065X
Volume 6 • Issue 2 • 1000170
UV light-visualization of the DNA fragments. As stated above, ApoE4
subjects were excluded from the study.
Parameters Healthy
Control
FPP-
treated
Antioxidant-
treated
Total no. (male/female) 30 (15/15) 45 (33/18) 45 (28/17)
Mean age 67 71 66
Mean BMI (range) 23 (21-25) 27 (23-35) 26 (24-34)
Family history of diabetes 1/30 14/45 21/45
Family history of hypertension 1/30 17/45 18/45
Overt diabetes NA 6/45 11/45
Duration of diabetes (years) NA 4 3
Dyslipidemia 0/30 28/45 31/45
Smoking 4/30 7/45 12/45
Waist (cm) 82 98 99
Waist/hip ratio 0.82 0.92 0.95
Mean Systolic Blood Pressure
(mmHg)
122 159 161
Mean Diastolic Blood Pressure
(mmHg)
65 78 81
Physical activity 15/30 6/45 12/45
Table 1: Anthropometric, clinical and biological parameters in studied
subjects.
Figure 1: Study design, see text.
Biochemical tests: routine tests (WBC, transaminases, urea,
creatinine, glucose, glycated haemoglobin, uric acid, HOMA test, LDL
and HDL cholesterol) were assayed by using the 7060 Automatic
Biochemical Analyzer (Hitachi, Ltd, Tokyo, Japan). Oxidized
cholesterol, anti-oxidised LDL, ratio of anti-ox-LDL/ox-LDL
cyclophilin-A, plasminogen activator inhibitor-1 were tested as below.
Oxidised LDL and anti-oxidised LDL antibodies assessment: ese
parameters were obtained by fractionation of LDL and preparation of
oxLDL as antigen in the antibody detection assay, according to what
reported by Ketelhuth et al. [29]. For this purpose a commercially
available ELISA kits (ChusaBiotech Co. Ltd., China), was employed.
Briey, microtiter plates were coated with 10 g/ml in PBS of either
native LDL or MDA-LDL to measure anti-oxidized LDL antibodies.
Serum samples were diluted 1:100 in 1% BSA/PBS and incubated for 2
h at 37°C and overnight at 4 C. Aerwards, the plates were washed
four times with alkaline phosphatase-conjugated anti-human IgG
(Sigma Chemical, St. Louis, MO) and le for 3 h at room temperature.
e reaction was halted aer 60 min with 1% BSA/PBS for 2 h at room
temperature. e absorbance was at 450 nm using a Multiskan RC
ELISA reader (ermo Fisher Scientic, USA). e binding of
antibodies to oxidized LDL was determined by subtracting native LDL
binding value from the one of MDA-LDL while the results were
expressed as an Optical Density (OD).
Measurement of cyclophilin A in plasma: Cyclophilin A
concentration in plasma was assayed with quantitative sandwich
enzyme immunoassay kit (Wouhan-UCSN BioScience, Wouhan,
China). Briey, a monoclonal antibody specic for CypA was pre-
coated onto a microplate. Standards and samples were put into the
wells and CypA present is bound by the immobilized antibody. An
enzyme-linked polyclonal antibody specic for CypA is added to the
wells. Plates were washed three times with PBS and a substrate solution
was added to the wells. e following dose-dependent color
appearance was then halted by an acid solution and the color intensity
measured by a reader as above. ere was no evidence of any
detectable cross-reactivity with any other tested proteins. All samples
were analyzed in triplicate and samples with a CV >10% were
discarded.
Determination of plasminogen activator inhibitor-1: PAI-1 serum
concentration was measured by sandwich enzyme ELISA (PAI-1;
DSE100; R&D Systems). Active PAI-1 levels were calculated from a
standard curve constructed by using recombinant human PAI-1. By
this method either active or latent forms of PAI-1 are detected [30].
Intra and inter-assay coecients of variation of PAI-1 antigen were
3.9% and 4.8%, respectively.
Cyclophilin-A gene expression study: Reverse
transcriptionpolymerase chain reaction (RTPCR) was used to
determine their expression at 0, 90 days and at the end of the 24 month
study period. Briey, cells were lysed by Ambion lysis buer (Ambion,
Carlsbad, CA, USA) for 20 min and the lysates were mixed with same
volume of 64% ethanol. e lysate products were put into a column
and centrifuged at 10,000 × g for 1 min. Aerwards, a buer consisting
of 700 μl buer 1 and 500 μl buer 2/3 (Genosolution, Ltd., Shenyang,
China) was used to wash the column. Aer incubation with 50 μl
buer (GenePharma Co., Ltd.), the resulting ow was collected and 50
μl elution buer was added and a further centrifugation was
performed. Next, 1 μl DNAse I was added to 20 μl of RNA solution
with suitable DNAse I buer (GenePharma Co., Ltd.) and incubated at
37˚C for 2 h. e DNAse I was removed using DNAse reagent and
puried RNA obtained by a centrifugation at 10,000 × g for 1 min. e
quantity of total RNA was measured by optical density at 260 nm and
the procedure checked through 1.5% agarose gel electrophoresis. RT
was run in a 20ml solution containing 3 μg total RNA using the Revert
cDNA Synthesis kit (Toyobo Biotech, Co., Ltd., Shanghai China). PCR
was done in a thermal cycler aer prior denaturation at 94˚C for 5
min, followed by amplication for 30 cycles of denaturation at 94˚C
for 40 s, annealing phase at 65˚C for 1 min followed by 72˚C for 1 min.
Aerwards, 5 μl PCR material was isolated by electrophoresis on a
Citation: Marotta F, Marcellino M, Solimene U, Cuffari B, Yadav H, et al. (2017) A 2-year Double-Blind RCT Follow-up Study with Fermented
Papaya Preparation (FPP) Modulating Key Markers in Middle-Age Subjects with Clustered Neurodegenerative Disease-Risk Factors.
Clin Pharmacol Biopharm 6: 170. doi:10.4172/2167-065X.1000170
Page 3 of 9
Clin Pharmacol Biopharm, an open access journal
ISSN: 2167-065X
Volume 6 • Issue 2 • 1000170
1.5% agarose gel and stained by ethidium bromide. e following
primer was used to carry out the test: CypA forward, 5'GTC AAC CCC
ACC GTG TTC TTC3', and reverse, 5'TTT CTG CTG TCT TTG GGA
CCTTG3' and glyceraldehyde3phosphate dehydrogenase (GAPDH)
forward, 5'ACC ACA GTC CAT GCC ATCAC3', and reverse, 5'TCC
ACC ACC CTG TTG CTGTA3'. e PCR products were subjected to
electrophoresis on a 1.4% agarose gel and the results were
quantitatively assessed by Gelmatrix Scan soware (BioMatrixscan
Inc., Shanghai, China).
Faecal heavy metal measurement
A 15 mg of stool sample was collected and an inductively coupled
argon plasma mass spectrometry (ICP-MS) with an Agilent 7500ce
device was used to test the following heavy metals: lead, arsenic,
mercury, cadmium, nickel and beryllium. According to the IUPAC
guideline [31] the reference value was dened within the 95%
condence interval of the 95th population percentile. e results in the
specimens were expressed as μg/L. Data were expressed on a dry
weight basis so to avoid the variability related to watery component of
the sample.
Results
Routine tests
Blood tests (WBC, creatinine, urea, glucose, glycated haemoglobin,
uric acid, total cholesterol, LDL-cholesterol, HDL-cholesterol,
triglycerides, transaminases, gamma-GT, alkaline phosphatase,
bilirubin) were not aected by any of the nutraceuticals employed,
except a not signicant trend improvement of HOMA test in the FPP-
supplemented group (data not shown).
Oxidized LDL cholesterol and anti-oxidised LDL antibodies
Whereas the group treated with antioxidant cocktail did not show
any statistically signicant dierence in the oxidised lipid asset,
patients treated with FPP showed a signicant improvement of these
parameters (Figure 2). In particular, the oxidized LDL cholesterol was
normalised by this intervention (p<0.001 vs. baseline). is variation
was signicantly detectable starting from the third month observation
period while the anti-Ox-LDL reverted to near to normal level from 12
months observation time onwards (p<0.05, Table 2). ere was no
signicant correlation between these parameter and main clinical
history data (presence or duration of diabetes, hypertension or
dyslipidemia). No signicant gender dierence was detected as for
these parameters are concerned.
Serum level of cyclophilin-A and plasminogen activator
inhibitor-1
At baseline there appeared a signicant correlation between
plasminogen activator inhibitor-1 and uric acid (r: 0.62, p<0.05, not
shown). As compared to baseline and to antioxidant cocktail control,
FPP showed to signicantly decrease serum levels of cyclophillin-A
from 1st month observation (p<0.01, Figure 3) and plasminogen
activator inhibitor-1 from 6 month observation (p<0.01, Figure 4). A
signicant correlation appeared between cyclophillin-A and oxidised-
LDL (r: 0.69, p<0.05, not shown) but not with plasminogen activator
inhibitor-1.
Cyclophilin-A gene expression study
At baseline observation, CyP-A gene expression was comparable
with what observed in healthy controls (Figure 5). However, as
compared to antioxidant cocktail-treated group, FPP enabled a
signicant upregulation at 3 and at 24 months (p<0.05, Figure 5).
Faecal heavy metal assessment
ere was no correlation between this parameter, either when
analysed as a single heavy metal or as a whole pooled value and any of
the biochemical or gene expression values measured.
Irrespective of the group, all patients showed an overall abnormal
faecal metals prole with the exception of nickel and arsenicum.
Unlike FPP or antioxidant control, the chelator-treated group
showed a signicantly greater faecal elimination of heavy metals
(namely: mercury, cadmium, lead and beryllium (Table 3, p<0.05 vs.
baseline and antioxidant combination). ere was a not signicant
trend correlation between cumulative heavy metal values (either at
baseline or aer treatment) with oxidised-LDL or its antibody (data
not shown).
Discussion
Oxidative modification of LDLs may be a prerequisite for the rapid
accumulation of LDLs within macrophages to form foam cells; indeed,
oxidized LDL has been found in extracts from atherosclerotic lesions
[3]. Oxidative modification of LDLs induces also the transformation of
immunogenic epitopes in the LDL molecule, which lead to the
formation of antibodies against oxidized LDLs in the serum [4].
Nevertheless, the clinical importance of these autoantibodies is still
under discussion. For example, in patients with diabetes, no
association has been found between anti-oxidized-LDL antibodies and
microvascular complications [11], nor has an association been found
with the levels of cholesterol in patients with heterozygous
hypercholesterolemia [32] or with the intensity of serum oxidizability
[33]. Others have reported an inverse relation between levels of
cholesterol and anti-oxidized-LDL antibodies in the general
population [34] and this seems to agree with the nding of an inverse
link between IgM autoantibodies to oxidized LDL and carotid artery
atherosclerosis [35]. e preliminary data of our study conrms that
FPP unlike the control antioxidant, specically decreased oxidized-
LDL and, on the long run, also enhanced its antibodies concentration,
although not modifying the lipid prole. Unlike what reported by
Balada et al. [36], we did not notice any signicant gender dierence as
for the levels of anti-oxidized LDL antibodies. While brain cholesterol
metabolism is separated from the systemic circulation, oxidised
cholesterol moieties may damage the functional integrity of blood-
brain barrier with potentially detrimental eects on neuronal
population [37]. e same group has also shown on an experimental
model that oxidised-LDL lipids and 27-OH-cholesterol may trigger Aβ
production by GSH depletion and β-secretase-1 activation in neuronal
cells [38]. It is noteworthy mentioning that a recent experimental study
connects a fat-based/high calorie diet with metabolic syndrome,
oxidative stress and signicant hippocampal and temporal area
degeneration together with memory dysfunction [39]. is evident
histopathological nding is also in agreement with one of the current
clinical hypothesis [40].
Citation: Marotta F, Marcellino M, Solimene U, Cuffari B, Yadav H, et al. (2017) A 2-year Double-Blind RCT Follow-up Study with Fermented
Papaya Preparation (FPP) Modulating Key Markers in Middle-Age Subjects with Clustered Neurodegenerative Disease-Risk Factors.
Clin Pharmacol Biopharm 6: 170. doi:10.4172/2167-065X.1000170
Page 4 of 9
Clin Pharmacol Biopharm, an open access journal
ISSN: 2167-065X
Volume 6 • Issue 2 • 1000170
Figure 2: Variation of plasma level of oxidised-LDL during treatment with the two nutraceuticals formulas. *p<0.001 vs. baseline values;
**p<0.01 vs. FPP-treated group.
Anti-ox-LDL EU/mL
(18.2 ± 3.4)
Entry 6 months 12 months 21 months 24 months
FPP 28.1 ± 10.2 28.2 ± 9.3 21.4 ± 14.5*21.2 ± 10.7*20.5 ± 7.9*
AntiOx mixture 26.8 ± 12.9 27.1 ± 8.8 27.4 ± 6.4 26.9 ± 13.3 25,7 ± 16.1
Table 2: Comparison of study parameters between the two treatment groups. In brackets: Values in healthy control subjects. *p<0.05 vs. baseline
and vs. Antioxidant cocktail.
Moreover, only FPP decreased cyclophilin-A plasma level and
plasminogen activator-inhibitor (PAI-1) (p<0.01). During
inammatory processes and following oxidative stress, CyPA is
released into the extracellular compartment by inamed cells [41,42]
as a tentative protection response but this factor ends up being a potent
chemoattractant for human monocytes and neutrophils. Cyclophilin A
has indeed been shown to be more strongly associated with oxidative
stress and inammation than C-reactive protein [43]. Interestingly,
prior small open pilot in-house studies testing same cohort of patients
with an omega-3-rich sturgeon-based supplement (Caviarlieri, Swiss
cap) did not benecially aect the lipid prole, oxidised lipid
parameters or CyPA. On the contrary, this latter variable showed a
worsening change either as plasma level or as gene expression, making
it a not advisable choice in metabolic syndrome or diabetes (J. Cervi,
manuscript in preparation). e brain is characterized by a low content
of antioxidant systems and the damage caused by oxidative stress is
one of the earliest pathophysiological events in the development of
Alzheimer's disease where plasma oxidized-LDL level has been
positively correlated with disease severity [44].
A further independent cardiovascular risk factor [45] as well co-
aggravating parameter in metabolic syndrome [46] is represented by
PAI-1, the major regulator of brinolysis and a stress-related factor. As
mentioned above, unlike the antioxidant cocktail and the recent
negative study testing high dose tocotrienol [47], FPP supplementation
brought about a signicant normalization of altered values of this
parameter.
Citation: Marotta F, Marcellino M, Solimene U, Cuffari B, Yadav H, et al. (2017) A 2-year Double-Blind RCT Follow-up Study with Fermented
Papaya Preparation (FPP) Modulating Key Markers in Middle-Age Subjects with Clustered Neurodegenerative Disease-Risk Factors.
Clin Pharmacol Biopharm 6: 170. doi:10.4172/2167-065X.1000170
Page 5 of 9
Clin Pharmacol Biopharm, an open access journal
ISSN: 2167-065X
Volume 6 • Issue 2 • 1000170
Figure 3: Variation of plasma level of Cyclophillin-A during
treatment with the two nutraceuticals formulas. *p<0.01 vs. baseline
values; **p<0.01 vs. FPP-treated group.
Figure 4: Variation of plasma level of plasminogen-activator
inhibitor-1 during treatment with the two nutraceuticals formulas.
*p<0.01 vs. baseline values; **p<0.01 vs. FPP-treated group.
Figure 5: Cyclophillin-A gene expression at baseline and during 24 months follow up under treatment with two dierent nutraceuticals. Only
FPP was eective in upregulating this parameter (*p<0.05 vs. baseline and vs. Antioxidant cocktail).
Citation: Marotta F, Marcellino M, Solimene U, Cuffari B, Yadav H, et al. (2017) A 2-year Double-Blind RCT Follow-up Study with Fermented
Papaya Preparation (FPP) Modulating Key Markers in Middle-Age Subjects with Clustered Neurodegenerative Disease-Risk Factors.
Clin Pharmacol Biopharm 6: 170. doi:10.4172/2167-065X.1000170
Page 6 of 9
Clin Pharmacol Biopharm, an open access journal
ISSN: 2167-065X
Volume 6 • Issue 2 • 1000170
Months Normal range FPP AntiOx mixture Chelator
Mercury
mg/kg dry wt.
21
24
<0.5 (0.3 ± 0.2)
0.4 ± 0.3
(0.4 ± 0.1)
0.4 ± 0.2
(0.3 ± 0.2)
1.1 ± 0.3*
Cadmium
mg/kg dry wt.
21
24
<0.5 (0.2 ± 0.3)
0,5 ± 0.14
(0.4 ± 0.3)
0.5 ± 0.12
(0.3 ± 0.1)
1.4 ± 0.31*
Lead
mg/kg dry wt.
21
24
<0.5 (0.2 ± 0.1)
0.2 ± 0.6
(0.2 ± 0.12)
0.3 ± 0.09
(0.3 ± 0.02)
1.1 ± 0.41*
Arsenicum
mg/kg dry wt.
21
24
<0.3 (0.1 ± 0.04)
0.09 ± 0.032
(0.1 ± 0.08)
0.1 ± 0.03
(0.3 ± 0.04)
0.2 ± 0.12
Beryllium
mg/kg dry wt.
21
24
<0.009 (0.004 ± 0.003)
0.009 ± 0.004
(0.007 ± 0.004)
0.004 ± 0.003
(0.009 ± 0.004)
0.015 ± 0.002*
Nickel
mg/kg dry wt.
21
24
<8 (5.8 ± 0.8)
5.6 ± 0.3
(6.4 ± 1.2)
6.2 ± 1.3
(6.1 ± 0.14)
6.9 ± 0.38
Table 3: Faecal heavy metal test: Eect of nutraceuticals with and without selected chelator. In brackets: baseline values corresponding to time 21
months and 24 month observation period of the trial. *p<0.05 vs. baseline and vs. antioxidant cocktail.
On the other hand, a one-year consumption of a nutraceutical rich
in resveratrol, one of the ingredient of our antioxidant cocktail, had
shown to improve PAI-1, but CyPA and oxidised lipid markers were
not tested [48]. A vascular component of neurodegenerative disease
pathophysiology is credited by several epidemiological studies [49] and
Oh et al. [50] has shown that patients with MCI and AD had
signicantly higher plasma PAI-1 levels, ese appeared to correlate
with cognitive function thus being a potentially early biomarker for
AD. e upregulation of PAI-1 has also been suggested to explain
insucient BDNF neurotrophic support and increased
neurodegeneration while suppressing PAI-1 expression/activity
increases Aβ degradation [51]. To the vascular hypothesis is likely to
cooperate also an increased CyPA which may aect endothelial cells
and vascular smooth muscle cells [52,53]. Whatever the main
mechanisms, it seems that by matching the metabolic proling and
AD, there might be three subtypes; an inammatory one, a non-
inammatory type and a zinc deciency-associated type aecting
younger population [54]. In a prior study using a sturgeon eggs-
derived compound of putative anti-inammatory eect (Caviarlieri,
Swiss cap, Switzerland) we had shown to increase circulating level of
BDNF in healthy occupationally-stressed individuals [55]. However,
internal and follow up data showed that this was a rather short-lived
eect and disappointingly unaecting PAI-1, OxLDL, Cyp-A or
proBDNF. So, its initially suggested neuroprotective eect
in vitro
(but
not at brain gene expression level in animals) had to be abandoned on
clinical ground by our group.
On the other hand, the downregulating eect played by FPP on
CyPA gene expression, but not from the antioxidant control, may be of
relevance, when considering that it has become clear that a
hyperglicemia and oxidative stress milieu may stimulate this molecule
[56] which, on its turn, drives the ox-LDL-mediated dierentiation
and activation of monocytes to foam cells [57,58].
Heavy metals gut clearance was not aected by any of the
nutraceuticals. However, the chelator, by itself remarkably increased
fecal gut discharge of heavy metals but only in those patients with
abnormal baseline values. e addition of a chelator to the control
nutraceuticals didn’t prove to yield better results while the addition to
FPP showed a trend increase of fecal discharge of cadmium (p<0.07 vs.
oral chelator alone, n.s.).
e identication and application of novel biomarkers in nutritional
and life-style interventional plans may foster healthy longevity and
benecially aect the occurrence of concomitant illnesses. Our
preliminary data suggest that FPP might play a noteworthy role within
a wider and comprehensive preventive medicine strategy plan for
impending metabolic diseases which are potential co-factors of
neurodegenerative disease. Environmental neurotoxins disrupt protein
processing as shown in experimental studies [26-28] while eective
heavy metals chelators remain a further additive avenue to possibly
pursue in selected cases.
Conclusion
As overall platform, virtuous dietary regimen such as the nutritional
model of Mediterranean diet still maintains its importance to counter
ght the cellular events involved in the atherosclerosis and
neurodegenerative process [59,60]. However, given these promising
results, longer studies addressing the issue of specic long term
evidence-based nutraceutical implementations are highly awaited.
Lastly, most recent research work points out the crucial importance of
mitochondrial dynamics in metabolic and neurodegenerative diseases
[61]. Indeed, exposure of environmental toxin, high-calorie intake,
glucolipotoxicity brings about dysfunctional mitochondria which
perpetuate ROS spilling over. Interestingly, the work of Collard et al.
[62] in diabetic rats has shown how FPP optimised mitochondrial
energetics. Such “cellular energetic” hypothesis as one of the possible
FPP mechanisms also crossing the blood-brain-barrier has been
recently envisaged by Hayashi Y. (in: Mantello A, Catanzaro C, He F,
Cuari B, Bissi L, Milazzo M, Lorenzetti A. Marotta F. Novel
Nutrigenomics Avenues in Nutraceuticals Use: e Current Status of
Fermented Papaya Preparation–Ed. E. Aguilar, Bioactive compounds:
At the frontier between nutrition and pharmacology, 2016; 94-119).
Citation: Marotta F, Marcellino M, Solimene U, Cuffari B, Yadav H, et al. (2017) A 2-year Double-Blind RCT Follow-up Study with Fermented
Papaya Preparation (FPP) Modulating Key Markers in Middle-Age Subjects with Clustered Neurodegenerative Disease-Risk Factors.
Clin Pharmacol Biopharm 6: 170. doi:10.4172/2167-065X.1000170
Page 7 of 9
Clin Pharmacol Biopharm, an open access journal
ISSN: 2167-065X
Volume 6 • Issue 2 • 1000170
Acknowledgments
is study was supported by an unbiased grant from Osato Research
Institute, Japan (91%) and with a minor grant (9%) rom San Babila
Clinic, Milano.
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Citation: Marotta F, Marcellino M, Solimene U, Cuffari B, Yadav H, et al. (2017) A 2-year Double-Blind RCT Follow-up Study with Fermented
Papaya Preparation (FPP) Modulating Key Markers in Middle-Age Subjects with Clustered Neurodegenerative Disease-Risk Factors.
Clin Pharmacol Biopharm 6: 170. doi:10.4172/2167-065X.1000170
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ISSN: 2167-065X
Volume 6 • Issue 2 • 1000170