Oral Human Administration of Red Grape Polyphenol in Nickel-mediated Allergic Contact Dermatitis: an in Vitro Study
Phase: NA
Conditions: Allergic Contact Dermatitis
Sponsor: University of Bari

                                                                                                                      October, 31  2018
Oral human administration of red grape polyphenol in nickel-mediated 
allergic contact dermatitis: an in vitro study
Thea Magrone* 
Departement of Basic Medical Sciences, Neuroscience and Sensory Organs, School of Medicine, 
University of Bari, Bari, Italy 
*Correspondence: 
Thea Magrone
thea.magrone@uniba.it
 Abstract
Nickel (Ni)-mediated allergic contact dermatitis (ACD) is a very common disease worldwide. Our 
previous findings demonstrated that in vitro supplementation of polyphenols, extracted from seeds of 
red grape (Nero di Troia cultivar), to peripheral lymphomonocytes from Ni-mediated ACD patients 
could reduce release of T helper (h)1 [interferon (IFN)-] and Th2 [interleukin (IL)-4] cytokines, on 
the one hand. On the other hand, IL-10 (an anti-inflammatory cytokine) levels increased with a 
reduction of IL-17 (an inflammatory cytokine). Also levels of nitric oxide (NO) decreased in 
response to polyphenol pretreatment (1). On these grounds, 25 Ni-mediated ACD patients were orally 
administered with 300 mg polyphenols prodie extracted from seeds of red grape (Nero di Troia 
cultivar) (NATUR-OX®) for 3 months. Other 25 matched Ni-mediated ACD patients received 
placebo only for the same period of time. Serum biomarkers analyzed before (T0) and after (T1) 
treatment were represented by IFN-, IL-4, IL-10, IL-17, pentraxin (PTX)3 and NO. Patch test scores 
to evaluate sensitization to Ni were recorded at T0 and T1, respectively. In both groups seven drop 
outs were recorded. At T1 in comparison to T0, in treated patients, values of IFN-, IL-4 and IL-17 
decreased, while IL-10 levels increased. Also PTX3 and NO concentrations decreased at T1 when 
compared with T0 values. Conversely, in placebo-treated patients elevated amounts of IFN-, IL-4, 
IL-17, PTX3 and NO at T0 did not change at T1. Also decreased levels of IL-10 at T0 remained 
unmodified when compared to those determined at T1. Clinically, in 16 out of 18 treated patients 
patch test positivity decreased at T1, while in placebo patients positivity score remained unchanged. 
Conclusively, present laboratory and clinical data are justified by the anti-oxidant, anti-inflammatory 
and anti-allergic properties of polyphenols, as also demonstrated in previous experimental and 
clinical settings. 
Patients and Methods
A total of 50 females patients with Ni-mediated ACD were enrolled in the Section of Dermatology 

                                                                                                                      October, 31  2018
Department of Biomedical Sciences and Human Oncology, University of Bari, School of Medicine, 
Bari (Italy). At the time of enrollment (T0), data about, age, onset of the related eczematous events, 
date of diagnosis, magnitude of the positivity of the reaction to the nickel patch test were collected.

Patients were orally administered for three months (T1) with one capsule daily of NATUR-OX® 
[Farmalabor, s.r.l., Canosa (Italy)] which is a dietary supplement containing grape seed extracts 
from Nero di Troia (Vitis vinifera). Each capsule contains 280 mg of proanthocyanidins where Ni 
contamination of capsule is below 0.24 ppm. The other group of patients was administered with 
placebo  one capsule/daily for three months.  The placebo capsules had the same appearance and 
composition of the supplement except for the active ingredient (polyphenols). At T1, all patients 
were examined by dermatologists for patch test evaluation.
In both groups peripheral venous blood sampling (5 ml test tubes without anticoagulant) was carried 
out before T0 and after T1 treatment.
Serum samples were collected and stored at -30°C until use.
Patch testing
Patch tests were performed with the S.I.D.A.P.A. (Italian Society of Allergological, Occupational 
and Environmental Dermatology) baseline series (Euromedical, Calolziocorte, Italy) by Al Test® 
(Euromedical) on Scanpor® Tape (Norgesplaster, Vennesla, Norway).  They were applied on the 
back and left in occlusion for 2 days. Readings were performed at days 2, 4 and 7 following 
European Society of Contact Dermatitis guidelines (2). All patients were positive and degree of 
sensitization to Ni was indicated with (+).
Clinical evaluation
In particular, positive patients were graded as following: i. asymptomatic patients at the moment of 
the enrollment but with a past history of Ni-mediated ACD; ii. patients with weak erythema; iii. 
patients with even erythema, edema and possibly papules or hints of vesicles; iv. patients with 
erythema, edema, papules and vesicles which can flow to form bubbles (3).
In both groups of patients, 7 dropouts were recorded not due to side effects but spontaneous 
discontinuation of the treatment.
Determination of serum cytokines
Serum samples collected by both groups and at different times, T0 and T1 were used to detect by 
means of an ELISA kit (TEMA Ricerca, Bologna, Italy) according to manufacturer’s instructions 
the following cytokines: IFN-, IL-4, IL-10, IL-17 and PTX3. Cytokine concentrations were read at 
550  nm  by  means  of  an  ELISA reader  (iMarkTM  Microplate  Absorbance  Reader,  BioRad, 
Hercules, California, U.S.A.).
Determination of serum nitric oxide 
Determination of serum nitric oxide (NO) was performed by a colorimetric assay.
Evaluation of serum cytokines
Polyphenols significantly diminish levels of IFN- and IL-4, thus, suggesting that Ni-mediated 
ACD is a disease characterized by the involvement of both Th1 and Th2 cell subsets. Moreover, in 
the same patients, investigation on the IL-10/IL-17 ratio demonstrates in a clear cut way that the 
polyphenol-mediated enhancement of IL-10 production with the parallel dampening of IL-17 
amounts may represent one of the key mechanisms through which these substances switch off the 
inflammatory/allergic reactions in Ni-mediated ACD. 
Evaluation of serum PTX3

In our experiments, PTX3 is significantly reduced by polyphenol administration. Conversely, in the 
placebo group, no modifications of the above cited immune parameters are detectable at the end of 
the trial.
Evaluation of serum NO
Serum NO concentration decreased at T1 in the group of patients who assumed polyphenols. 
Conversely, NO serum levels in the placebo group were unchanged at both times.
Conclusion
The present study clearly demonstrates that polyphenol administration to Ni-induced ACD patients 
is able to reduce the inflammatory allergic pathway, while enhancing the tolerogenic response 
mediated by IL-10. Of note, NO is also able to inhibit cutaneous DCs, thus, down modulating the 
effect  related  to  their  interaction  with  Ni.  Laboratory  findings  are  corroborated  by  clinical 
improvement of symptomatic patients.
References

(1) Magrone T, Romita P, Verni P, Salvatore R, Spagnoletta A, Magrone M, et al. In vitro Effects of 
Polyphenols on the Peripheral Immune Responses in Nickel-sensitized Patients. Endocr Metab 
Immune Disord Drug Targets (2017) 17:324-31. doi: 10.2174/1871530317666171003161314.
(2) Johansen JD, Aalto-Korte K, Agner T, Andersen KE, Bircher A, Bruze M, et al. European 
Society of Contact Dermatitis guideline for diagnostic patch testing - recommendations on best 
practice. Contact Dermatitis (2015) 73:195-221. doi: 10.1111/cod.12432
(3) Summer B, Ständer S, Thomas P. Cytokine patterns in vitro, in particular IL-5/IL-8 ratio, to 
detect patients with nickel contact allergy. J Eur Acad Dermatol Venereol (2018) 32:1542-8. 
doi: 10.1111/jdv.14931