Functional Foods Science 2024; 4(1): 29-41
Research article

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Open-Access

Neutrophil-lymphocyte ratio and update conclusions from a 2year double-blind rct testing fermented papaya preparation in
healthy middle-aged/elderly subjects
Aldo Lorenzetti1, Antonio Ayala2, Fang He3, Cristiana Aperio1, Leila Haghshenas1, Maki Osato4, Umberto Solimene5┼,
Saida Rasulova1, Francesco Marotta1*
1

ReGenera R&D International for Aging Intervention, Milano, Italy; 2Department of Biochemistry and Molecular Biology,
Faculty of Pharmacy, University of Seville, Spain; 3West China School of Public Health, Dept of Nutrition, Sichuan University,
Chengdu, P.R. China. 4Redox Molecular Biology Laboratory, ORI Research Institute, Gifu, Japan; 5WHO-collaborating Center
for Traditional Medicine & Biotechnology, University of Milano, Milano, Italy.
*Corresponding Authors: Francesco Marotta, ReGenera R&D International for Aging Intervention, Milano, Italy
Submission Date: December 11th, 2023; Acceptance Date: December 27th, 2023; Publication Date: January 9th, 2024
Please cite this article as: Lorenzetti A., Ayala A., He F., Aperio C., Haghshenas L., Osato M., Solimene U., Rasulova S.,
Marotta F. Neutrophil-lymphocyte ratio and update conclusions from a 2-year double-blind rct testing fermented papaya
preparation in healthy middle-age/elderly subjects. Functional Food Science 2024; 4(1): 29-41. DOI:
https://www.doi.org/10.31989/ffs.v4i1.1270
ABSTRACT
Background: This study represents the second part of a 2-year interventional study in middle-aged /elderly healthy
subjects treated either with an antioxidant supplementation (200mg trans-resveratrol, 100mg Centella asiatica extract,
80mg ubiquinol, 50mg epigallo-cathechin-gallate, 20 mg anthocyanidins, 5mg zinc, and 200 IU vitamin E) or with aproprietary fermented papaya preparation (FPP). The choice of the fermented papaya preparation was based on over
12 years of experimental and clinical data proving its effectiveness on redox and immune regulation. The aim of thisstudy was to complete a 24-month investigation of the potential beneficial effect of this functional food on key agerelated immune system parameters.
Methods: Study population: From a total of 106 subjects, we analyzed data collected from ninety-eight clinically stable,
healthy community dwelling males and females aged 58 to 76 years. The investigation followed a double-blind
methodology involving two groups, A and B. Group A received FPP® 4.5 grams, one sachet twice a day, coupled with aplacebo capsule in the morning. Meanwhile, Group B was administered a papaya-flavored sachet twice a day, with anadditional antioxidant mixture capsule in the morning, referred to as the AA group. Morning blood samples werecollected and tested for: Asymmetric dimethylarginine (ADMA), apoptosis of PBMCs and blood including Neutrophil-Functional Foods Science 2024; 4(1): 29-41

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Lymphocyte Ratio (NLR) after 12 months, i.e. corresponding to 24 months of the 2-year study (Visit VI of the initial
protocol).
Results: As compared to the first preliminary published year of the study, this further 12-month study enabled a morecomprehensive analysis with a significant increase of analyzed overweight (≥25 and ≤29.9 kg/m2) subjects (58 vs 31,
P<0.05). On the 24-month mark, AA-group showed a significant increase of NLR (P<0.05 vs entry level and FPP group),
whereas FPP-group maintained a stable value. Urinary 8-OHdG values maintained, as in the preliminary study, a wide
dispersion of data but both treatments equally reduced its value in over 27BMI/r over 70y old subjects, which as taken
separately, had significantly elevated values (p<0.05 vs younger subjects). ADMA and frequency of apoptotic cells values
were beneficially reduced only by FPP supplementation throughout all 24 months of the study (P<0.05 vs baseline andvs AA supplementation). A significant correlation between NLR and Apoptosis of PBMCs appeared in FPP-treated subjects
and this was more pronounced in subjects with >27BMI or over 70 years of age.
Conclusions: The novelty of the present work was the finding that FPP represents a substantial pharma-grade, non-GMO
technological advancement over uncontrolled traditional plant extracts. This functional food affects oxi-inflammatory
aging phenotype and NLR, a marker worth great attention for its preventive interventional potential in middleage/elderly communities health care.
Key words: Fermented Papaya Preparation, NLR, ADMA, apoptosis, antioxidants, SARS-Cov19©FFC 2023. This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0
License (http://creativecommons.org/licenses/by/4.0)

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INTRODUCTION

comprehensive data compilation on NLR could be

For the last 13 years, a proprietary functional food known

undertaken, given that elevated values are indicative ofas fermented papaya preparation (FPP®, Osato Research

unfavorable outcomes in various contexts, including

Institute, Gifu, Japan, Patent Number: 6401792) has

chronic inflammatory conditions, acute infectious

undergone rigorous validation through numerous

diseases, monitoring severity in SARS-COVID-19, andexperimental and clinical trials, showcasing its enduringassessing injury induced by environmentally retrievableimmune and redox modulator nutrigenomics effects [1-toxins [13-19].Given the promising first-year beneficial

6].The Functional Food Center's (FFC) recent consensus

effect of FPP supplementation in a number of crucial

has provided a distinct and enhanced definition forredox parameters and apoptosis regulation, the present

evidence-based functional foods, elucidating theirstudy intended to confirm the stability of that data along

impactful bioactivity and the associated biomarkers

a further year of observation to study the profile of NLR[7].This year a brief but stringent position paper further

and its possible relationships with the other variables.

focused on the fundamental regulatory pillars in the field[8]. In the previously published 1-year preliminary report

MATERIALS AND METHODS[9] of the present 2-year investigation, we demonstrated

The study design, conducted in compliance with thethat,

Declaration of Helsinki guidelines, was approved by aascompared

supplementation,

to

FPP®

the

antioxidant-mix

intervention

significantly

decreased the asymmetric dimethylarginine (ADMA), an

joint ethical committee of the ReGenera Research
Association in January. 19, 2020.

endogenous NOS inhibitor and the percentage ofapoptotic PBMC, together with a more satisfactory

Study population: As stated in the initial section of ourquality of life profile in middle-age/elderly subjects. On

inclusion criteria, participants were required to be

the other hand, the dynamics of other variables such as

clinically stable, healthy, community-dwelling individuals

neutrophil lymphocyte ratio (NLR) values, no matter theof both genders, testing negative for Covid-SARS2, andtreatment group, did not reach substantial differences. A

with a BMI within the range of 23.9 to 32.5 kg/m². Mild

recent study by the renowned inChianti group has founddietary-controllable

that NLR emerges as a superior predictor of healthy aging

hyperglycemia were not grounds for exclusion, provided

compared to the isolated assessment of neutrophil orthat participants did not require pharmacologicallymphocyte counts. This observation adds valuable

treatment for these conditions. Exclusion criteria were

insights to the understanding of aging biomarkers [10].

BMI ≥ 36, past tumors, major hepato-gastrointestinal,

Moreover, it appeared that NLR variations were

neuropsychiatric, renal or endocrinological disorders,associatedsurrounding

and relevant lung or cardiovascular events in the last 6

environmental abnormalities, undergoing diseases andmonths. Antibiotic use as well as fish oils, vitamins,

the status of immune system [11]. This work

probiotics, nutraceuticals, nicotine/caffeine dependence,corroborated and expanded the NLR-survival rate in non-or high drinking habit (4+ alcohol-containing beverages

cancer subjects already shown in 2016 [12]. In the secondon any day or 5+ alcohol-containing beverages per week

year of the study, besides monitoring the stability of thefor women and 7 or more alcohol-containing beverages

results previously achieved, we managed to pool all 98

per week for men), strenuous physical activity in the pastpatients’ data thus enabling a thorough and updated

4 weeks or ongoing were regarded as exclusion factors.

analysis of whole study protocol parameters. Specifically,[9]. From the original start of the study, one year earlier,withlifestyle

and

its

dyslipidemia

andborderline

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both supplementations did not stop. This second year oflower detection limit of 0.01 mg/L, serum ADMA level by

the study, analyzed 98 subjects, (8 of 106 lost at follow

a commercially available enzyme-linked immunosorbent

up or dropped out for personal reasons or trauma), allassay (ELISA) kit for ADMA (DLD Diagnostika GmbH,clinically stable, healthy community dwelling males andHamburg, Germany), yielding a sensitivity 0.01 μmol/L

females (m/f: 42/56) aged 58 to 77 years, Due to theand a the inter-assay coefficients of variation of less thanextensive

participants'

3% [21], urinary 8-oxo-7,8-dihydro-2’-deoxyguanosine

engagement in regular daily activities was monitored(8OHdG) concentration adjusted for urinary creatinine

using a comparable watch-pedometer application,

values [21] by a competitive assay (Oxis Health Products,

aiming to maintain an average of 6000 steps/day withoutInc.). In particular, spot urine samples were collected by

specific performance targets.

subjects in sterile boxes upon awakening (7.00-7.30),

durationofthestudy,

before consuming any water or beverages and thenDesign of the study: During the second year of the study,

taken to the laboratory by 9.00am where they were

the original double-blind protocol was consistently

stored at -70 until the date of assay. Sample aliquots

applied, assessing two distinct treatments. The FPP®

were tested as above for the quantification of 8-OHdG

group (group A) received FPP® 4.5g, 1 sachet twice a day,(ng/mL). The urinary creatinine concentration (mg/mL)with an additional placebo capsule (vegan cellulose)

was quantified by using an ELISA assay kit (Afinion-kit ACR,

before breakfast. Meanwhile, the AA group (group B) was

Italy). The creatinine-adjusted 8-OHdG concentrations

administered a fruit-flavored 4.5g, 1 sachet twice a day,(ng/mg) were used for the analysis. To normalize the 8-with the morning dose associated with a capsule

OHdG

containing a comprehensive antioxidant mixture (200mg

transformation was used. Univariate associationstrans-resveratrol, 100mg Centella asiatica extract, 80mg

between subjects’ characteristics (age, gender, BMI, fruit,ubiquinol, 50mg epigallo-cathechin-gallate, 20 mg

and vegetable consumption) and 8-OHdG concentration

anthocyanidins, 5mg zinc, and 200 IU vitamin E). Thislevels wase evaluated with a linear mixed-effects

formulation was done by the researchers in aregression model for repeated measurements.

concentration

distribution,

the

Box-Coxcompounding pharmacy by putting together ingredients

Annexin V staining was carried out using anti-

and dosages much popular in the market. Planned blood

annexin V antibody (eBioscience) in annexin V binding

tests were set as follows: Visit V at 18 months (onlybuffer kept for 15 min. DAPI staining served to exclude

clinical assessments) and Visit VI (final clinical andthe presence of dead cells and the apoptosis frequency

biochemical assessment) at 24 months.

was calculated by using BD LSRFortessa (BD Biosciences,
San Jose, CA, USA). Apoptosis of peripheral Blood

Methods: Morning blood samples were collected fromMononuclear Cells (PBMCs, isolated from the whole

patients after overnight fasting. In addition to routine

blood by Ficoll-Paque gradient centrifugation. Profiles ofclinical laboratory tests, performed blindly, the following

each population were analyzed by Cytomics FC 500

parameters were tested: Neutrophil-lymphocyte ratio(Beckman Coulter).

(NLR), an indicator of systemic inflammation [20] wasmeasured by dividing the number of neutrophils by theStatistical analysis: All tests were performed by SPSS

one of lymphocytes. As in the previous preliminary study,

version 22 (IBM Inc., Armonik, NY, USA). The normality of

the following tests were also carried out: Ultra sensitivity

the data was checked and confirmed by the Shapiro-Wilk

C-reactive Protein (by a highly sensitive ELISA with amethod. Results are presented with means ± standard

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FFS

deviations (SD) for continuous variables. We also usedthebaseline

value

of

FPP®

or

Page 33 of 41

and ≤29.9 kg/m2). As compared to the first year of theantioxidant

study, these data showed a significant increase of

supplementation, as assessed at the start of the study 24

overweight (≥25 and ≤29.9 kg/m2) subjects (58 vs 31,

months earlier, to analyze the time-course changes.

P<0.05). No significant weight change was noted at the

Differences between the two study groups wereend of the study.

2

calculated by t-test and Chi -test while a clear-cut alphalevel of 0.05 was set for all analyses.

NLR values were comparable between the twotreatment groups till the 11th month observation. During
this observation period, a notable increasing trend was

RESULTSobserved in the AA group, culminating in a statistically

Forty subjects had normal body mass index (18.5–24.9

significant difference at the 24-month control markkg/m2), and fifty-eight were classified as overweight (≥25

(Figure 1, P<0.05 vs entry level and FPP group).

Figure 1. Time-course profile of NLR in both treatments’ groups, AA group (red stripe) and FPP-group (green stripe).
* P<0.05.
Urinary 8-OHdG values exhibited the previously observed

significantly elevated compared to the overall entry level

extensive variability between the two groups at baseline(P<0.05). Both supplementations’ regimes equally(data presented in the previous publication). However,improved this variable (Fig 2, P<0.05) with FPP® effectwhen analyzing these values in subjects aged over 70 or

started being significant already at 6months till the end

with a BMI exceeding 27, urinary 8-OHdG values wereof the study (24 weeks).

Figure 2. Time-course effect of AA supplemented group (red bars) and FPP-supplemented group (green bars) on the profile
of urinary 8OHdG level in in the cohort of participants older than 70 years or with BMI >27. Both supplementations enabled
a significant and comparable decrease of this parameter as compared to entry level (P<0.05).

Functional Foods Science 2024; 4(1): 29-41

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In contrast, ADMA values remained unaffected by AAfrom the 6-month checkpoint onward (P<0.05 compared

supplementation (Fig. 3), while FPP administration

to baseline and AA supplementation).

exhibited a notable and significant decrease, observed

Figure 3. Time-course changes of plasma ADMA level in AA supplemented group (red bars) and in FPP-treated group (greenbars). Differently from AA treatment, FPP® supplementation yielded a significant decrement till to 24 months control(*P<0.05 vs baseline and AA group).

At the study's onset, there was no notable difference insignificant increase (P<0.05 compared to baseline). In

the frequency of apoptotic cells in PBMCs between thecontrast, the FPP®-administered group showed only a

two treatment groups (Fig. 4). Following the 6th monthslight, non-significant trend increase while maintainingof the trial, the AA group exhibited a gradual andan overall stable lower percentage (P<0.05 compared to
AA group values observed at 6, 11, and 24 months).

Figure 4. Frequency of apoptotic cells (Annexin V+ DAPI+) at baseline, 6, 11 and 24 months. While this parameter remained
fairly stable throughout the 24 month follow up in the FPP® group (green bars) (#P<0.05 vs AA group), ), In the AA group(red bars), a notably steep increase was observed at 6 months, and more prominently at 11 and 24 months, reachingstatistical significance (P< 0.01 compared to baseline).

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Table 1 shows the level of statistical significance whenmonths and 24 months, the two parameters were highly

correlating the values of NLR and Apoptosis of PBMCs

correlated, and this was more pronounced in thosethroughout the study period. It appeared that, at 11

participants older than 70 years or with BMI >27.

Table 1. Correlation between NLR and Apoptosis of PBMCs in FPP-supplemented group
Elements of correlation
NLR

Apoptosis (PBMCs)

2 weeks

6 weeks

11 weeks

24 weeksr = 0.545

r = 0.524

r = 0.759*

r= 0.772*

ns

ns*P<0.05*P< 0.05

vs baseline

vs baseline

participants older than 70 years or with BMI >27

NLR

Apoptosis (PBMCs)r = 0.443

r = 0.546

r = 0.791*

r = 0.813**

ns

ns*P<0.005**P<0.005

vs baseline

vs baseline

No correlation appeared between NLR and hCRP, Ur-

content analysis [28-31].

8OHdG or ADMA at any of the time-check, irrespective of

duration of the study confirmed the stability of thethe gender (data not shown). No correlation appeared

results observed during the initial year, persisting until

when analyzing AA group separately, irrespective of age

the 24-month observation. . This is significant whenand BMI, ns: not statistically significant.

considering

theincreased

However, the extended

numberof

recruited

overweight patients (58 vs 31, P<0.05) and aged subjects.
DISCUSSION

This data is in agreement with the observation of drastic

The initial broad range of data observed during theimprovement of this plasma and urinary parameter afterurinary 8-OHdG level assessments in the first year of theweight loss surgery [32]. NLR can be regarded as astudy did not allow for definitive conclusions regarding

correlated marker of oxi-inflammatory imbalance,

the

This

associated with cardiovascular disease risk, and serves as

observation persisted until the conclusion of the second-a prognostic factor for atherosclerosis, aging, overall

year investigation (data not presented). This finding is not

mortality, cancer progression, and rheumatoid arthritis

surprising, when considering the disappointing or modest[33-39]. This has also been reported in advanced chronic

results reported in the past by using vitamin C or E [24-gastritis by Helicobacter Pylori [40]. Nevertheless, even25] and where also the role of diet [26] was notthough none of our subjects reported gastric symptoms,

specifically ruled out. For instance, dietary regimens

considering the susceptibility of the aged populationprescribed for chronic benign diseases have indirectly

group to oxidative damage [41], we cannot exclude theshown to act as cancer protectors [27]. Moreover, suchpotential presence of gastric atrophy with various

studies, to optimize the homogeneity of the diet, would

immune-inflammatory mucosal rearrangements [42].

need a detailed food bioactive components and probiotic

The anticipated positive effects of FPP [43] at this specificimpactofnutraceuticalinterventions.

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level are evident, as demonstrated in a clinical study [44].

influencing brain physiology [58-64], this signifies a step

Interestingly, NLR and ADMA, although not yielding abeyond the raised caution associated with intrinsic

statistical correlation between each other, showed amethodological variables in in vitro analysis [65]. In thesimilar higher level and, unlike the AA group, bothquest for a rational science-validated functional food [66]parameters significantly responded to FPP® at 11 and 24

and more controlled selection of bioactive compounds

monthss. Exercising due caution due to the severity of[67], FPP represents a substantial demonstration of

the disease, a recent study conducted on hospitalized

pharma-grade, non-GMO technological advancement

SARS-CoV-2 patients revealed an association between

over traditional carica papaya use [68]. The ongoingmortality and concurrent abnormalities in NLR andpursuit of healthy living inevitably involves impacting a

ADMA [45]. This supports the prognostic value of ADMA

fundamental oxi-inflammatory phenotype to varying

assessments

duringdegrees [69-71]. In this context, NLR monitoring [72]post-COVID

may be a marker worth great attention [73-75] andinhospitalization

these

and

patients,

throughout

both
the

syndrome period, which is still not fully understood [46-preventive

interventional

potentialinmiddle-50]. Although none of our patients had diabetes, we did

aged/elderly communities health care [76-77]. The

not conduct HOMA testing. In this expanded group of

novelty of the present study resides in a long-term

overweight individuals, HOMA testing might havemonitoring of key age-related parameters while using a

revealed insulin resistance in some cases. Existing

functional food treatment.

literature clearly highlights that NLR abnormality is
associated with this condition [51] and is responsive to

CONCLUSION

antioxidant treatment [52]. Although our study did notThis groundbreaking study represents the first two-year

address cerebrovascular pathophysiology, a recent

monitoring of the effects of antioxidants and a novel

review from Framingham Heart Study has reported thatfunctional food (FPP) on key redox parameters, PBMCs

subjects with higher NLR are at a greater risk of later

apoptosis, and the NLR, with a particular emphasis on thedementia during a 5.9-year follow-up period [53]. This

long-term impact. While the antioxidants mixture

area is understandably of extremely complex metabolic

exerted a significant effect on redox parameters, on top

interrelations, either environmentally (exotoxins) [54, 55]of this, FPP was shown to bring about age/health- andor internally (endotoxins) [56].

longevity-associated immune system markers.

In

this

nutritional

interventionalsetting,

Belpomme’s group [57], by using FPP®, has demonstrated

Thisfinding may help substantiate health maintenancestrategies.

a statistically significant clinical and redox benefit
together with an increase in intracerebral tissue pulse
metric index in the temporal lobes through ultrasonicbrain

tomosphygmography andwhose

long-term

Abbreviations: NLR: neutrophil-lymphocyte ratio, i.e. thenumber of neutrophils divided by the number oflymphocytes; ADMA: Asymmetric dimethylarginine;
PBMCs: Perypheral Blood Mononuclear Cells; FPP®:

relevance warrants follow up studies. Amid theFermented Papaya Preparation; BMI: Body Mass Index;

accumulating evidence of effective functional foods

8OHdG: 8-oxo-7,8-dihydro-2’-deoxyguanosine

Functional Foods Science 2024; 4(1): 29-41

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modification by nutraceutical intervention. J Biol Regul

Contribution: OM, SU discussed the research plan, LA

Homeost Agents. 2011 Apr-Jun;25(2):221-9.

and RS followed the clinical aspects FH and AC discussed
and evaluated nutritional aspects, AA and US discussed

Page 37 of 41

7.

Adany, A. Kanya, H. Martirosyan, D. Japan’s health foodindustry: An analysis of the efficacy of the FOSHU system.

and evaluated the biochemistry.

Bioactive Compounds in Health and Disease 2021; 4(4):6378. DOI: https://www.doi.org/10.31989/bchd.v4i4.795

Conflict of interest: Authors declare no personal or8.

institutional conflict of interest.

Martirosyan, D., Alvarado, A. Functional Foods Regulation
System: Proposed Regulatory Paradigm by Functional Food
Center.

addressee.

Food

Science(inpress).

DOI:

https://www.doi.org/10.31989/ffs.v2i2.890

Acknowledgment: We gladly acknowledge the scientificsupport of prof. Marotta who acts as a correspondence

Functional

9.

Lorenzetti, A., Maki Osato, M., He, F., Aperio, C., Ayala, A.,
Rasulova, S., Barbagallo, M. Interim report from a 2-year
double-blind rct testing fermented papaya preparation on
immune enhancement, endothelial health and qol in elderly
adults. Functional Food in Health & Disease, 2023; 12(2):68-

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