Immunopharmacology and Immunotoxicology

ISSN: 0892-3973 (Print) 1532-2513 (Online) Journal homepage: https://www.tandfonline.com/loi/iipi20

Protective effect of a topical sunscreen

formulation fortified with melatonin against UV-
induced photodermatitis: an immunomodulatory
effect via NF-κB suppression

Nilutpal Sharma Bora, Bhaskar Mazumder, Santa Mandal, Yangchen D.

Bhutia, Sanghita Das, Sanjeev Karmakar, Pronobesh Chattopadhyay & Sanjai
K. Dwivedi

To cite this article: Nilutpal Sharma Bora, Bhaskar Mazumder, Santa Mandal, Yangchen D.
Bhutia, Sanghita Das, Sanjeev Karmakar, Pronobesh Chattopadhyay & Sanjai K. Dwivedi (2019):
Protective effect of a topical sunscreen formulation fortified with melatonin against UV-induced
photodermatitis: an immunomodulatory effect via NF-κB suppression, Immunopharmacology and
Immunotoxicology, DOI: 10.1080/08923973.2019.1566358

To link to this article: https://doi.org/10.1080/08923973.2019.1566358

Published online: 11 Feb 2019.

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IMMUNOPHARMACOLOGY AND IMMUNOTOXICOLOGY
https://doi.org/10.1080/08923973.2019.1566358

ORIGINAL ARTICLE

Protective effect of a topical sunscreen formulation fortified with melatonin

against UV-induced photodermatitis: an immunomodulatory effect via NF-jB
suppression
Nilutpal Sharma Boraa,b , Bhaskar Mazumderb , Santa Mandala,c , Yangchen D. Bhutiaa, Sanghita Dasa,
Sanjeev Karmakara, Pronobesh Chattopadhyaya and Sanjai K. Dwivedia
a
Division of Pharmaceutical Technology, Defence Research Laboratory, Tezpur, India; bDepartment of Pharmaceutical Sciences, Dibrugarh
University, Dibrugarh, India; cSchool of Pharmaceutical Sciences, IFTM University, Moradabad, India

ABSTRACT ARTICLE HISTORY

Objective: Melatonin and pumpkin seed oil, along with US FDA approved UV filters were incorporated Received 26 July 2018
into a formulation for enhancement of UV protection by exerting an antioxidant effect. The objective Revised 10 December 2018
of this study was to assess the protective effect of this formulation against ultraviolet (UV) radiation- Accepted 3 January 2019
induced photo dermatitis in rats, which is an established model to study the aetiopathogenic mecha-
KEYWORDS
nisms in psoriasis vulgaris, as the former exhibits the same features to those of clinical psoriasis vulga- Melatonin; photodermatitis;
ris in humans. pumpkin seed oil;
Materials and methods: The animals were segregated into five groups (6/group) and all received sunscreen; US FDA
their respective formulations dermally prior to chronic UV irradiation for 28 days. The test, placebo, approved UV filters
and standard groups; received the test, placebo, and standard formulations respectively; whereas the
positive control group received only UV radiation. A normal control group was also maintained.
Disease and treatment status were analyzed using various techniques by euthanizing the rats
after 28 days.
Results: The test formulation was able to ameliorate the UV-induced increase in skin fold, epidermal
thickness, and skin edema; inhibit the reduction of hydroxyproline content and incidence of LPO
within the skin tissues of exposed animals. The formulation was also able to inhibit the release of
proinflammatory cytokines; IFN-c, IL-1b, IL-6, and TNF-a; and upregulation of NF-jB and COX-2 genes
caused by chronic UV exposure.
Conclusion: It can be stated that melatonin included in the newly formulated sunscreen was able to
inhibit the induction of photodermatitis via immunoregulation of inflammatory cytokines along with
NF-jB and COX-2 genes.

Introduction NFjB, a transcription factor and COX-2, its downstream tar-

Skin disease is a frequently occurring health problem afflict-
ing all ages from the neonates to the elderly. Psoriasis is
a chronic skin disease, which is immune-mediated and
is caused by activation of cellular immune system like
T-lymphocytes [1]. A characteristic feature of psoriasis is
keratinocyte hyperproliferation marked by thickening of the
epidermis [2]. Psoriasis not only cause physical discomfort
like itching, burning, and bleeding but also causes psycho-
logical stress due to chronic unpleasant visible psoriatic
inflammation on the scalp, face, hairline, and nails, both
lower and upper limbs and can manifest long-term effects
on self-esteem, self-image, and emotional stability [3].
Existing treatments of psoriasis include emollients, dithranol,
coal tar etc., which are both cosmetically unesthetic and pos-
ses low efficacy. Alongside, systemic therapies such as cyclo-
sporine, acitretin methotrexate, were found to possess
significant side effects [1]. Increased ROS productions along
with diminished activity of antioxidant mechanism have
been found to be related with psoriasis pathogensis [4].
get has been found to be upregulated in cases of psoriasis.
Moreover, many immune-derived cytokines, including interlu-
kins (IL-1, IL-6, IL-17, IL-19, IL-20, IL-22), tumor necrosis factor
(TNF), and interferons (IFNs) have been found to play an
important role in psoriasis via regulation of keratinocyte pro-
liferation [5,6].
Exposure to ultraviolet (UV) radiation generate reactive
oxygen species (ROS) which leads to edema, erythema, skin
aging, skin tanning, hyperpigmentation, etc., which have
become more frequent lately as a result of diminution of
ozone layer and climate change. In view of this, an effective
treatment must be instituted against the perilous effects of
UV rays like redness, rashes, swelling, etc., which is possible
by using some physical and/or chemical UV filters [7]. The
various inflammatory responses caused due to UV radiation
exposure results in well-differentiated brownish-red scaly
lesions combined with various histological features which
closely resemble the aspects of clinical psoriasis vulgaris in
humans. This attribute can be exploited for various

CONTACT Pronobesh Chattopadhyay chattopadhyay.drl@gmail.com Division of Pharmaceutical Technology, Defence Research Laboratory, Tezpur Assam
784001, India.
ß 2019 Informa UK Limited, trading as Taylor & Francis Group
2 N. S. BORA ET AL.

Table 1. Composition of the dermal formulations for evaluation of protective activity against UV radiation induced photodermatitis.
% w/w
Sl. No. Ingredients Use Test formulation Placebo formulation Challenge formulation
Phase A
Titanium Dioxide Active ingredient 3.0–6.0 – 3.0–6.0
Ethylhexyl Methoxycinnamate 1.5–2.25 – 1.5–2.25
Butyl Methoxydibenzoylmethane 0.9–1.35 – 0.9–1.35
Benzophenone-3 0.6–0.9 – 0.6–0.9
Pumpkin Seed Oil 5.6–10.0 – –
Phase B
Melatonin Active ingredient 0.9–2.5 – –
Aquagel 35VR Primary emulsifying agent 0.5–2.0 0.5–2.0 0.5–2.0
Demineralised water Solvent q.s. q.s. q.s.
Cream base
Butylated Hydroxy Toluene Preservative, antioxidant 0.8–1.5 0.8–1.5 0.8–1.5
Disodium EDTA Chelating agent 1.0–2.5 1.0–2.5 1.0–2.5
Phenoxyethanol Preservative, stabilizer 1.25–2.0 1.25–2.0 1.25–2.0
Xanthan Gum Binder, stabilizer, surfactant 7.0–10.0 7.0–10.0 7.0–10.0
Parabens Preservatives 2.5–5.0 2.5–5.0 2.5–5.0
Glyceryl monostearate Emollient, emulsifier 4.5–7.0 4.5–7.0 4.5–7.0
Dimethicone crosspolymer Dispersing agent, emulsion stabilizer 5.0–9.0 5.0–9.0 5.0–9.0
Light liquid paraffin Emollient, stabilizer 7.0–12.0 7.0–12.0 7.0–12.0
Glycerin Antifreeze, solublilizer 9.5–14.0 9.5–14.0 9.5–14.0
Glycols Antifreeze, humectant 10.5–13.0 10.5–13.0 10.5–13.0
Polysorbate 20 Surfactant 3.0–5.0 3.0–5.0 3.0–5.0
Isopropyl myristate Emollient, thickening agent 7.5–11.0 7.5–11.0 7.5–11.0

investigations related to the mechanisms and prophylaxis of
psoriasis vulgaris [7]. Apart from using only UV filters to curb
the adverse effects of UV radiation reaching the skin, it is
also necessary to avert the ROS from reacting with the bio-
molecules [8]. Owing to the protective effects of antioxidants
our study envisaged on using antioxidant-rich sunscreen
with United States Food and Drug Administration (US FDA)
approved UV filters. Newly formulated sunscreen was incor-
porated with pumpkin seed oil and melatonin for better UV
protection along with their antioxidant property. Pumpkin
belongs to Cucurbitaceae family and contains active substan-
ces of active non triacylglycerol origin. Pumpkin seed is rich
in vitamin E and contains high levels of a- and c-tocoferol. In
addition, it also contains a high level of ß-carotene. The
sodium, calcium, phosphor, magnesium, and potassium con-
tent of pumpkin seed oil are greater than that of pumpkin
seeds [9]. In view of these aforementioned constituents,
pumpkin seed oil exhibit antioxidant as well as anti-inflam-
matory activity [10]; wherein melatonin a pineal hormone,
lipid-soluble compound scavenges hydroxyl radical, and lipid
peroxidyl radicle [8] found to be twice more active than vita-
min E and believed to possess lipophilic antioxidant prop-
erty [11].
Therefore, the present study is aimed at evaluating a com-
binational dermal sunscreen formulation as a prophylactic
measure against broad spectrum UV radiation-induced psor-
iasis with special reference to its molecular aspects of kera-
tinocyte proliferation and inflammatory responses.
Materials and methods
Chemicals
Titanium dioxide (SKU 14027), benzophenone-3 (SKU
PHR1074), pumpkin seed oil (SKU W530274), butyl methoxy-
dibenzoylmethane (SKU PHR1073), and ethylhexyl
methoxycinnamate (SKU 78848) were purchased from Sigma
Aldrich Co., St. Louis, MO, US. Melatonin (CAS 73–31-4) was
purchased from Santa Cruz Biotechnology, CA, US. All other
reagents, pharmaceutical excipients and kits used in this
study were purchased from reputed companies and were of
highest grade purity.
Test formulation design protocol
The sunscreen formulation evaluated in this study is a novel
sunscreen formulation containing an amalgamation of four
US FDA sunscreen ingredients along with two synergistic
natural ingredients, namely melatonin and Cucurbita pepo
(pumpkin) seed oil (PSO). The formulation was prepared with
minor modifications; as per the method previously described
[12] and stored in borosilicate glass containers, protected
from light. A placebo formulation (using only excipients) was
also prepared to serve as a suitable negative control and
standard test formulation (only UV filters and excipients;
without melatonin and pumpkin seed oil) was designed to
evaluate their synergistic action in combination with the UV
filters. The formulae for all the compositions are listed in
Table 1. The complete pre-clinical toxicity assessment of the
test formulation was carried out previously [12] and no
adverse reactions have been reported.
Animals
Healthy adult Wistar rats (weighing 210–250 g, 5–8 weeks of
age, male) were obtained from the institutional animal hous-
ing facility and allowed to acclimatize for 7 days prior to the
study. During the entire experimental period, the animals
were housed in accordance with the Guide for the Care and
Use of Laboratory Animals, National Institutes of Health
(NIH). Standard food (Pranav Agro Industries Limited, Sangli,
 IMMUNOPHARMACOLOGY AND IMMUNOTOXICOLOGY 3

Table 2. Experimental groups and dosage.

Group No. Group Name Description Dose
I Normal group Normal animals –
II Positive control group UV irradiated only 3.80 J cm2
III Test group UV irradiated þ test formulation (US 3.80 J cm2 þ 200 mg kg1 b.w. day1
FDA approved UV filters in combination
with melatonin and pumpkin seed oil)
IV Placebo group UV irradiated þ placebo formulation 3.80 J cm2 þ 200 mg kg1 b.w. day1
(without any actives)
V Standard group UV irradiated þ standard formulation 3.80 J cm2 þ 200 mg kg1 b.w. day1
(containing only US FDA approved
UV filters)

Maharastra, India) and purified water ad libitum were pro-
vided to the animals which were housed in clean and
hygienic polypropylene cages in air-conditioned rooms at
22–25
C, 40–70% humidity with 12 h light-dark cycles and
ventilation of 15–21 air changes/h.
Compliance with ethical standards
All experiments carried out in this study had the approval of
the Institutional Animal Ethical Committee (IAEC, Registration
no. 1227/bc/07/CPCSEA) vide approval no. IAEC/02/2015.
Experimental groups and dosage
Animals were divided into the following five groups (contain-
ing 6 animals each) as shown in Table 2. All animals except
the normal group were irradiated with the same UV source.
The dose of 200 mg/kg/day was selected as the 1/10th of the
LD50 value found in studies previously reported on the same
formulation [12].
UV radiation-induced photo dermatitis model for
psoriasis in vivo
Controlled irradiation of depilated backs of experimental ani-
mals have been known to cause a biphasic erythematous
reaction, which includes an initial faint erythema usually dis-
appearing after 30 min of exposure, and a second phase of
erythema starts 6 h post irradiation and gradually increases,
peaking between 24 and 48 h. Sharply demarcated reaction
response area is observed which is brownish-red in color.
From 48 to 72 h, thick silvery white scales appear on the ery-
thematous lesions which bear a close pathological resem-
blance to psoriasis vulgaris, with only one differentiating
factor. UV induced photodermatits is a self-healing, non-
recurring disorder as opposed to psoriasis vulgaris, which
can relapse even after treatment [2]. Although the inflamma-
tory responses produced in this model are triggered artifi-
cially, their close resemblance to the symptoms exhibited in
psoriasis provides us with an excellent model to examine
drugs that have a prospective in the reduction of the inflam-
matory reactions associated with psoriasis.
The fur on the back of the acclimatized animals was
clipped carefully with scissors and shaved without injuring
the skin. The animals were restrained in suitable restrainers
so as to avoid movement and facilitate the exposure of the
depilated area to UV radiation. As described previously by
Vijayalakshmi and Geetha [2]. The animals prior to irradiation
were covered with a UV resistant film with an opening in the
center which only allowed an area of 1.5  2.5 cm of the
depilated backs to be exposed to UV radiation, using a
300 W Ultra-Vitalux UV lamp (Osram GmbH, Augsburg,
Germany) at a distance of 60 cm. The UV radiation was meas-
ured using a UV light meter (Model: UV-340A, Lutron
Electronic Enterprise Co. Ltd., Taipei, Taiwan) so that a fixed
radiation rate of 0.45 mW cm2 and a dosage of 3.80 J cm2
was maintained. The UV source was allowed to warm up for
10 min before measurement of light intensity or exposure
of subjects.
The entire experiment was carried out daily over a period
of 14 days, where each of the animals from group III to V
was administered the respective formulations onto an area
of 1.5  2.5 cm on the depilated backs. After allowing the for-
mulation to dry for 15–20 min, the rats were irradiated. Post
every experiment, the formulation was removed from the
application site using a cotton pad soaked in 50% isopropyl
alcohol. 24 h the final irradiation; i.e., on the 15th day, the
animals were euthanized and UV exposed skin samples were
excised from the animal. A part of the tissue was processed
for fixation in 10% buffered formalin for histopathological
examination and the remaining part was washed in phos-
phate buffer saline (PBS) and stored at 80
C for further
analysis. A schematic representation of the experimental
design is shown in Figure 1(a).
Macroscopic examination and skin thickness evaluation
The experimental animals were evaluated by a severity index
of inflammatory psoriatic inflammatory reactions every third
day during the entire course of experimentation using a vis-
ual scoring system based on Severity Index (SI) as described
by Nagar et al. [1]. SI was scored on a scale from 0 to 3: 0—
none (clear); 1—mild (redness); 2—moderate (redness and
erythema); 3—severe (redness, erythema, and scaling). The
thickness of the excised skin post euthanasia of the animals
was measured using electronic calipers (Tanotis, Bangalore,
India). The values were expressed as the percentage of skin
thickness increase compared to controls: ((skin thickness 24 h
after irradiation  skin thickness before irradiation)/skin thick-
ness before irradiation)  100. Epidermal thickness of the
skin was analyzed within hematoxylin-eosin stained photomi-
crographs using ImageJ (Fiji) software [13].
4 N. S. BORA ET AL.

Figure 1. (a) Schematic representation of the experimental design; (b) Effect of the formulations in prevention of skin thickness increment occurring due to chronic
UV radiation exposure in Wistar albino rats. n ¼ 6; Results are expressed as mean ± SD Statistical analysis were carried out using ordinary one-way ANOVA followed
by Dunnett’s multiple comparison tests. (p ˂ .001), (p ˂ .05); when compared with the control group; (c) Effect of the formulations on lipid peroxidation levels
of the skin of Wistar albino rats exposed to chronic UV radiation exposure. n ¼ 6; Results are expressed as mean ± SD Statistical analysis were carried out using
ordinary one-way ANOVA followed by Dunnett’s multiple comparison tests. (p ˂ .01), (p ˂ .05); when compared with the control group; (d) Effect of the formula-
tions on hydroxyproline levels of the skin of Wistar albino rats exposed to chronic UV radiation exposure. n ¼ 6; Results are expressed as mean ± SD Statistical ana-
lysis were carried out using ordinary one-way ANOVA followed by Dunnett’s multiple comparison tests. (p ˂ .001); when compared with the control group.

Histopathological examination 10% (w/v) trichloroacetic acid to induce precipitation of the

After euthanasia of the animals at the end of the experimen-
tal period, the skin samples from the back of the animals
were excised, collected, and stored in 10% formalin solution.
5 mm thick longitudinal sections were prepared for micro-
tomy and stained using hematoxylin-eosin dye for examin-
ation of histological architecture. The presence of the
cardinal histological features like Munro’s microabscesss,
elongation of rete ridges, and capillary loop dilation by direct
microscopy as described previously [14]. The vertical epider-
mal thickness (between the dermoepidermal junction and
the lowest part of the stratum corneum); mean thickness of
stratum corneum and stratum granulosum were also meas-
ured (n ¼ 3 measurements per scale, n ¼ 3 scales per animal,
n ¼ 6). The relative epidermal thickness in the percentage of
all the groups was calculated in comparison with the positive
control group considered as 100% (n ¼ 54 measurements per
treatment) [2].
Lipid peroxidation (LPO) assay of skin tissue
The extent of lipid peroxidation (LPO) as an indirect marker
of oxygen metabolite production, was estimated using the
thiobarbituric acid (TBAR) reactivity assay [15]. For the meas-
urement of the levels of LPO on the skin tissues, methods
described previously was used with minor modifications. The
excised skin tissues were homogenized in ice-cold Tris–HCl
buffer (50 mM, pH 7.4) for 2 min at 5000 rpm in a tissue hom-
ogenizer (MM 400, Retsch, GmbH, Germany). The supernatant
was collected and mixed with two volumes of cold
proteins. This reaction mixture was centrifuged for removal
of the precipitate following which an aliquot of the super-
natant was reacted with an equal volume of 0.67% TBA in a
boiling water bath for 10 min. After cooling, the extent
of LPO was determined by measuring the amounts of
malondialdehyde (MDA); a secondary product resulting
due to the oxidation of polyunsaturated fatty acids, which
reacts with two molecules of TBA, yielding a pinkish
red chromogen with an absorbance maximum at 532 nm,
against an appropriate blank. The malondialdehyde concen-
tration of the sample can be calculated using the below-
mentioned formula with an extinction coefficient of
1.56  105 M1 cm1 [16,17].
MDA levels ¼ Absorbance at 532nm=1:56  105 (1)
Hydroxyproline assay
The hydroxyproline content of the excised skin samples was
determined using the colorimetric method recommended by
ISO 3496(E) [1,18].
Estimation of pro-inflammatory markers
The excised skin samples were analyzed for the content of
inflammatory cytokines, namely IL-1b, IL-6, TNF-a, and IFN-c
using rat-specific enzyme-linked immunosorbent assay
(ELISA) kits as per manufacturer’s instructions (BD
Biosciences, NJ, US).
 IMMUNOPHARMACOLOGY AND IMMUNOTOXICOLOGY 5

Table 3. Effect of the topical formulations on macroscopic features in UV radiation induced psoriasis in rats
scored on the basis of Severity index (SI).
Groups Day 0 Day 3 Day 6 Day 9 Day 12 Day 15
Normal 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
Control 0.00 ± 0.00 0.33 ± 0.52 0.83 ± 0.75 1.33 ± 1.21 1.50 ± 1.05 2.17 ± 0.75
Test 0.00 ± 0.00 0.17 ± 0.41 0.33 ± 0.52 0.33 ± 0.52 0.50 ± 0.55 0.50 ± 0.55
Placebo 0.00 ± 0.00 0.33 ± 0.52 0.50 ± 0.55 0.83 ± 0.98 1.50 ± 0.84 1.83 ± 0.41
Challenge 0.00 ± 0.00 0.16 ± 0.41 0.67 ± 0.82 1.17 ± 0.98 1.33 ± 0.82 1.67 ± 0.52
n ¼ 6; Results are expressed as mean ± SD Statistical analysis were carried out using ordinary two-way
ANOVA followed by Dunnett’s multiple comparison tests. (p ˂ .001), when compared with the con-
trol group.

Estimation of NF-jB and COX-2 genes by Western 3. On every 3rd day, starting from Day 0 to Day 15, all the
immunoblot technique animals were observed for visible psoriatic lesions by naked
eyes. It was observed that the test formulation was able to
The skin samples were also analyzed for the expression of
induce a statistically significant decrease (p ˂ .001) in all the
NF-jB and COX-2 genes using Western protein immunoblot
observation days. The test formulation was able to induce a
technique. The samples were homogenized in radioimmuno-
decrease of 48.48%, 60.24%, 75.18%, 66.66%, and 76.96% on
precipitation assay (RIPA) buffer (Sigma Aldrich Co., St. Louis,
the 3rd, 6th, 9th, 12th, and 15th day of the experimentation,
MO, US) in tissue homogenizer (MM 400, Retsch, Newtown,
respectively, when compared with the control group.
USA). Equal amounts of protein were loaded on the sample
However, the placebo and standard formulations were not
wells of a 10% acrylamide resolving gel (TGX Stain-FreeTM
able to induce any significant reduction on the psori-
FastCastTM Acrylamide Kit, Bio-Rad, California, United States)
atic lesions.
and electrophoresed using a Mini-PROTEANV Tetra System
R

Figure 1(b) depicts the effect of the formulations on the

and PowerPacTM HC electrophoresis power supply system
reduction of skin thickness which correlates with the inflam-
(Bio-Rad, CA, US). The separated proteins were transferred
mation caused within the skin due to UV radiation. It was
onto an Immun-BlotV PVDF Membrane (Bio-Rad, CA, US)
R

observed that both the test and standard formulation were

using Trans-BlotV SemiDry Cell coupled with PowerPacTM HC
R

able to reduce the increase in skin thickness of the experi-

electrophoresis power supply system (Bio-Rad, CA, US). The
nonspecific antibodies were blocked using a 3% bovine
serum albumin (BSA) in tris-buffered saline and Tween 20
(TBST) solution for 1 h at room temperature on rocking
shaker (Rockymax, Tarsons, Kolkata, India). The membranes
were probed separately with NF-jB, COX-2 and b-actin pri-
mary antibodies (Sigma Aldrich Co., St. Louis, MO, US) (1:500
dilution in 3% BSA in TBST) overnight at 4
C; followed by
1 h incubation with horse radish peroxidase (HRP)-linked sec-
ondary antibodies (1:1000 dilution in 3% BSA in TBST) on
rocking shaker at 4
C. The membranes were then treated
with an enhanced chemilumescent (ECL) substrate (ClarityTM
and Clarity MaxTM, Bio-Rad, CA, US) and visualized and
imaged in G:Box Chemi-XRQ gel doc system (Syngene,
Cambridge, UK).
Statistical analysis
All statistical analysis was performed using GraphPad Prism
version 5 (GraphPad Software, La Jolla, CA, US). One way
ANOVA followed by various post-hoc tests was used to ana-
lyze the difference among multiple dosage groups. All values
are expressed as Mean ± SD A p < .05 value, evaluated at
95% level of confidence was considered as statistically signifi-
cant; unless otherwise indicated in the results.
Results
Macroscopic examination and skin thickness evaluation
The effect of the topical sunscreen formulation on severity
index during the course of experiment is tabulated in Table
mental animals. However, it was observed that the test for-
mulation was able to induce a 69.49% (p ˂ .001) decrease,
whereas the standard formulation showed a 24.50% (p ˂ .01)
decrease in skin thickness when compared with the control
group. The placebo group did not show any significant effect
in decreasing the skin thickness.
Histopathological examination
Figure 2 demonstrates the histopathological features of the
excised skin samples of the back of the experimental rats.
Figure 2(a,b) reveals the presence of Munro’s microabscess,
rete ridges elongation, and capillary loop dilation along with
the presence of thick and confluent parakeratosis, neutrophil,
and lymphocyte infiltration; with the UV exposed group.
These manifestations were not present in the normal and
test formulation treated groups (Figure 2(c,d), respectively).
The epidermal thickness of the UV exposed group was also
observed to be increased up to 169.81% when compared
with the normal group, whereas the test formulation showed
a significant decrease (p ˂ .01) in epidermal thickness when
compared with the control (UV) group. The placebo-treated
group (Figure 2(e)) also showed the presence of elongated
rete ridges and dilated capillary along with a severe increase
(163.45%) in epidermal thickness. The standard formulation
(Figure 2(f)) showed a decrease in epidermal thickness when
compared with the control (UV) group; however, these differ-
ences were not found to be statistically significant. Moreover,
the presence of dilated capillary was evident in the stand-
ard group.
6 N. S. BORA ET AL.

Figure 2. Histological evaluation of the dorsal skin biopsies of experimental animals to estimate the protective effect of the formulations against UV induced psor-
iasis (H&E stain). (a,b) Control group; (c) Normal group; (d) Test formulation treated group; (e) Placebo formulation treated group; (f) Standard formulation treated
group. Magnification: 100 for main images; 400 for inset images. ERR: Elongation of rete ridges; MMA: munro microabcess; NI: neutrophil infiltration; DC: dilated
capillary; TPLI: thick parakeratosis with lymphocyte infiltration; CP: confluent keratosis; NC: normal capillary. Representative measurements are shown in all images
for measurement of epidermal thickness using ImageJ software.

Lipid peroxidation (LPO) assay of skin tissue
Figure 1(c) displays the results for the LPO assay in the
excised skin of the experimental animals in terms of MDA
levels (ng/ml). It was observed that the LPO levels were sig-
nificantly higher in the UV only (control) group when com-
pared with the normal group (p ˂ .01). The test formulation
was able successfully inhibit the LPO significantly (p ˂ .05)
when compared with the control group, whereas the pla-
cebo and standard formulation were not able to induce
such effects.
Hydroxyproline assay
The levels of hydroxyproline in terms of mg per gram of skin
tissue is displayed in Figure 1(d). It was observed that the
hydroxyproline content was significantly reduced in the con-
trol (UV only) group. The test formulation was able to inhibit
this reduction in hydroxyproline levels significantly (p ˂ .01)
when compared with the control group. The placebo and
standard group, however, expressed results, which were simi-
lar to that of the control group, therefore, were ineffective in
preventing the reduction of hydroxyproline content due to
UV radiation exposure.
Estimation of pro-inflammatory markers
The increase in the levels of IL-1b, IL-6, TNF-a, and IFN-c
induced by chronic UV radiation exposure is significantly
reduced in the test formulation treated group (p ˂ .001), as
shown in Figure 3. The placebo formulation did not show
any such effects. The standard formulation was not able to
reduce the increase of IFN-c but was able to inhibit the
increase of IL-1b, IL-6, and TNF-a in a statistically significant
manner (p ˂ .01). It was thus observed that the test formula-
tion was significantly more effective in protecting the skin
from UV radiation-induced inflammation that is mediated via
production of IL-1b, IL-6, TNF-a, and IFN-c.
Western blot analysis
Quantitative estimation of the inflammatory mediator genes,
namely COX-2 and NF-jB, showed that the test formulation
was able to induce a protective effect against UV radiation-
induced inflammation. As seen in Figure 4, chronic UV
 IMMUNOPHARMACOLOGY AND IMMUNOTOXICOLOGY 7

Figure 3. Effect of the formulations in modulation of UV-induced production of proinflammatory markers within dorsal skin tissue homogenate of Wistar albino
rats, namely (a) TNF-a; (b) IFN-c; (c) IL-6, and (d) IL-1b. n ¼ 6; Results are expressed as mean ± SD Statistical analysis were carried out using ordinary one-way
ANOVA followed by Dunnett’s multiple comparison tests. (p ˂ .001), (p ˂ .01); when compared with the control group.

Figure 4. Effect of the formulations on (a) NF-jB and (b) COX-2 expression within dorsal skin tissue of Wistar albino rats. n ¼ 6; Results are expressed as
mean ± SD Statistical analysis were carried out using ordinary one-way ANOVA followed by Dunnett’s multiple comparison tests. (p ˂ .001); when compared
with the control group.

irradiation of the experimental animals results in the upregula-
tion of the transcription factor NF-jB and the inflammatory
gene COX-2 within the skin. This is found to be reduced in the
test formulation treated group animals which showed a statis-
tically significant decrease (p ˂ 0.001) in both COX-2 and NF-jB.
The placebo and standard formulation were however ineffective
in this regard, as both the groups exhibited very high levels of
both genes which were comparable with the control (UV only)
group. This indicated that the test formulation was able to
affect the functions of COX-2 and NF-jB, thereby providing
optimum protection from UV radiation-induced inflammation.
Discussion
The aim of the present study was to determine the protect-
ive effects of a cosmeceutical sunscreen formulation against
UV induced psoriasis-like symptoms. The formulation tested
herein is a first of its kind and has successfully passed the
preclinical toxicity profiling which has been reported earlier
[12]. The inclusion of pumpkin seed oil and melatonin is
expected to provide a boost to the antioxidant potential of
the formulation and exert a synergistic sun protective effect.
Table 1, shows the composition of the formulations, namely
test, placebo nad standard. As seen, the test formulation is
comprised of the test formulation contains one US FDA
approved inorganic UV filter, titanium dioxide; three organic
US FDA approved UV filters, namely benzophenone-3, ethyl-
hexyl methoxycinnamate, and butyl methoxydibenzoylme-
thane and as active ingredients. The antioxidant cocktail of
melatonin (N-acetyl-5-methoxytryptamine) and pumpkin
seed oil along with these UV filters is aimed at countering
the generation of ROS and its unwanted effects. The pineal
8 N. S. BORA ET AL.
Table 4. Effect of the topical formulations on histopathological features of UV-induced psoriasis in rats.
Groups Epidermal thickness (lm) Munro’s microabscess
Normal 19.29 ± 3.40
Control 32.74 ± 3.74
Test 21.41 ± 5.29
Placebo 31.53 ± 4.95
Challenge 27.33 ± 3.28
n ¼ 6; values are expressed as mean ± SD; þ: mild or slight grade lesion; þþ: moderate grade lesion; þþþ: severe
grade lesion; : no lesion. Data were analyzed using ordinary two-way ANOVA followed by Dunnett’s multiple compari-
son tests; (p ˂ .01), when compared with the control group.
hormone melatonin has been found to be potent antioxi-
dant, immunomodulator, and dose-dependent sunscreen
whereas pumpkin seed oil has been found to contain
43–53% of linoleic acid and two classes of antioxidant com-
pounds, namely tocopherols and phenolics, which when
used even in small proportions in topical formulations can
have significant beneficial properties [19–21].
The effect of the formulation on the appearance of the
dorsal skin of the experimental animals was scored on the
basis of the severity index visually by naked eyes, on every
3rd day during the course of the experiment (Table 3). It was
observed that the animals in the control group showed
severe signs of inflammation characterized by redness, ery-
thema, and scaling. While redness and erythema were evi-
dent from the 3rd day onward, the incidences of scaling
were observed to suffice from the 12th day onward. The test
formulation was able to completely inhibit these undesirable
effects during the entire duration and the results were com-
parable to the normal group. The results of the test formula-
tion treated group were significantly different from the
control (UV only) group at p < .001. The placebo formulation
did not show any inhibitory effects as it was predicted due
to the absence of any active constituents. The standard for-
mulation was, however, able to elicit some protective effects
on the skin of the experimental rats from chronic UV radi-
ation exposure. While the incidences of scaling were not
observed in this group, mild redness and erythema were vis-
ible. Therefore, the results though lower than the UV only
group were not significantly different. This difference in
results of the test and the standard formulation treated
groups may be attributed to the presence of melatonin and
pumpkin seed oil, which is predicted to exert an antioxidant
and emollient activity to the skin.
The test formulation was also able to reduce the increase
in the thickness of the skin due to chronic UV exposure. As
evident from Figure 1(a), the control group exhibited an
increase of 40% of the normal skin thickness due to UV radi-
ation. The test formulation treated groups, however, exhib-
ited an increase of only 15% which was significantly different
from the control group (p < .001). The placebo formulation
treated group exhibited no significant difference when com-
pared with the control group, whereas the standard formula-
tion treated group exhibited an increase of 29% which was
significantly different from the control group (p < .05). This
superiority of the test formulation in reducing the thickness
of the skin may also be attributed to the presence of mela-
tonin and pumpkin seed oil as both these ingredients have
been known to possess anti-inflammatory activities [22,23].
Elongation of rete ridges Capillary loop dilation
  
þþ þþþ þ
  
 þ þþ
  þ
The effect of the formulations on the histological features
of the skin of the animals exposed to UV radiation was also
studied and depicted in Figure 2, and the observations tabu-
lated in Table 4. The control (UV only) animals showed the
presence of Munro’s microabscess, elongated rete ridges and
dilated capillary loop with a significant increase in the epi-
dermal thickness. The test formulation treated group did not
show the presence of any of the above adverse effects and
the epidermal thickness of the skin of the experimental ani-
mals were significantly lower (p < .01) when compared with
the control group. The placebo formulation treated group
showed increased epidermal thickness and presence of elon-
gated rete ridges and dilated capillary loops. The standard
formulation exhibited a somewhat prominent protective
activity and the symptoms of Munro’s microabscess and
elongated rete ridges were not present. The epidermal thick-
ness of the standard formulation treated group was not
found to statistically different from the control group. It was
thus observed that when the histological factors were con-
sidered, chronic UV radiation resulted in the induction of
Munro’s microabscesses, elongated rete ridges, and dilated
capillary loop which are the classical features of psoriasis
[22]. The test formulation was found to be significantly bet-
ter than the standard formulation in mitigating all these
adverse effects and at the same time inhibited the increase
in the skin thickness of the experimental animals. This effi-
ciency of the test formulation, therefore, may also be attrib-
uted to the presence of the cocktail of melatonin and
pumpkin seed oil.
As depicted in Figures 1(c) and 1(d), the test formulation
was able to inhibit the reduction of hydroxyproline content
and incidence of LPO within the skin tissues of UV exposed
animals. The test formulation was able to significantly reduce
the skin MDA levels (p < .05) when compared with the con-
trol group. In this regard, the placebo and the standard for-
mulations were not able to produce any protective effect.
The increase in MDA levels have been observed and
reported in patients with psoriasis [4] and therefore the
effects of the test formulation in this regard is highly desir-
able since the presence of melatonin and pumpkin seed oil
has delivered a significant antioxidant activity to the skin of
the experimental animals. The test formulation was also able
to directly inhibit the UV radiation-induced collagen degen-
eration in the skin of the experimental animals, which was
assessed by estimating the hydroxyproline content of the
skin samples. Hydroxyproline content is the hydroxylated
form of the collagen-specific amino acid proline [24], which
is observed to be significantly reduced in the control group
(p < .001) in Figure 1(d). Reduced collagen content of the

skin has been considered as an important pathological sign
of psoriasis [25]. The test formulation was able to signifi-
cantly (p < .001) mitigate this UV dependent decrease in the
collagen content. These effects were not observed with the
placebo or standard formulation. These results suggest that
the presence of melatonin and pumpkin seed oil may be a
crucial factor for the maintenance of the collagen levels in
the skin of the experimental animals.
Psoriasis, which is an autoimmune hyperproliferative dis-
ease, is characterized by epidermal hyperproliferation, abnor-
mal differentiation, and inflammatory infiltration of the
epidermis and dermis. The transcription factor NF-jB and its
downstream target COX-2 is reported to play a major role in
psoriasis pathogenesis [6]. This activation of NF-jB is sup-
ported by the increase in the expression of endogenous
inducers like cytokines (IL-6, IL-1 b), interferons (IFN-c), and
TNF-a [26–29]. Therefore, the effect of the test formulation in
modulating the keratinocyte proliferation via activation of
NF-jB pathway was studied at the molecular level. Figure 3
displays the effects of the formulation on the expression of
TNF-a, IFN-c, IL-6, and IL-1b. It was observed that there was
an increase in the expression of all these factors in the skin
samples of the experimental animals that were exposed to
chronic UV radiation (p < .001). This increase of TNF-a, IFN-c,
IL-6, and IL-1b were significantly reduced in the test formula-
tion treated group (p < .001). This activity was not observed
in the placebo formulation treated groups. The standard for-
mulation was able to inhibit the overexpression of TNF-a, IL-
6, and IL-1b (p < .001). The increase in the levels of IFN-c
was, however, not affected in the standard formulation
treated group. The cytokines TNF-a and IFN-c are involved in
the Th1/Th17 pro-inflammatory axis causing inflammation
and keratinocye hyperproliferation, respectively. IL-6 and IL-
1b are produced by the keratinocytes, which, in turn, causes
the activation of myeloid dendritic cells to T helper
cells [30,31].
The levels of NF-jB and COX-2 were tested using Western
immunoblot techniques in all the experimental group and
results are portrayed in Figure 4. As reported earlier, the lev-
els of NF-jB and COX-2 were found to be increased in
human skin samples with psoriatic like lesions. Similarly, in
this study, both these genes were found to be upregulated
in the skin samples of the experimental rats. The test formu-
lation treated group exhibited a significant decrease in the
intensity of both NF-jB and COX-2 (p < .001). These benefi-
cial effects were not visible in the placebo group. The stand-
ard formulation was able to inhibit the increase of COX-2
levels up to an extent but the difference was not significant
in comparison to the control group. However, the NF-jB lev-
els remained unchanged in the standard formulation treated
groups when compared with the control group. These data
may be indicative of the fact that the inclusion of melatonin
and pumpkin seed oil within the test formulation has
increased the protective effect of the formulation against
psoriasis-like symptoms induced by chronic UV radi-
ation exposure.
Psoriasis is an inflammatory process driven by the Th1/
Th17 pathway which leads to keratinocyte hyperproliferation
IMMUNOPHARMACOLOGY AND IMMUNOTOXICOLOGY 9
and inflammation. The three main T helper cell effector sub-
strates, namely Th1, Th2, and Th17 are all involved in the
development of this condition, wherein Th1/Th17 are
involved in a positive regulatory manner, and Th2 is involved
in a counter regulatory manner. This, in turn, draws in the
cytokines, namely IL-1b, IL-6, IL-12, IL-17, IL-22, IL-23, TNF- a,
IFN-c, IL-10, and IL-4 [31]. The data obtained in this study
only predict the activity of the fortified sunscreen against UV
induced dermatitis. Further molecular studies in relation to
this research work are anticipated which can substantiate the
amelioration of psoriasis-like symptoms via the modulation
of the Th1/Th17 pro-inflammatory axis.
Conclusion
The above study demonstrated that the combination of the
US FDA approved UV filters along with melatonin and pump-
kin seed oil in a dermal formulation can induce a protective
effect against the induction of psoriasis-like symptoms
caused due to exposure of the skin to chronic UV radiation
in adult Wistar rats. The groups treated with the test formu-
lation exhibited an increased level of in vivo antioxidant cap-
acity within the skin along with intact collagen levels and
demonstrated skin conditions which were comparable to the
normal skin, both macroscopically and microscopically, even
when exposed to UV radiation. Reduced expression of
inflammatory markers along with the absence of erythema
and scaling further proved the protective activity of the for-
mulation. Modulation of the transcription factor NF-jB and
its downstream target, COX-2, an enzyme responsible for
inflammation, was also observed in the test formulation
treated group which may have led to non-psoriatic skin.
Furthermore, the absence of key pathogenic symptoms
within the histopathological architecture of the skin of the
experimental animals gave a stronger perception of the abil-
ity of the formulation to be used as a palliative treatment
against UV-radiation induced dermatitis. Therefore, it is evi-
dent from the above study that, that melatonin and pumpkin
seed oil, when used topically along with sunscreens, can
shield UV rays; visibly reduce UV induced dermatitis and can
improve psoriasis-like symptoms that arise due to UV radi-
ation exposure. Further studies on the ability to modulate
the Th1/Th17 axis, the key pathway to the development of
psoriasis, are necessary in the future.
Acknowledgments
All the authors are grateful to the Defence Research and Development
Organization (DRDO), Ministry of Defence, Government of India for pro-
viding the necessary laboratory facilities, financial aid and support to
carry out this research work. Nilutpal Sharma Bora specifically acknowl-
edges Defence Research and Development Organization, Ministry of
Defence, Govt. of India for providing assistance in the form of research
fellowship (Letter no. DRL/1206/TC/03) and Dibrugarh University,
Dibrugarh, Assam, India for necessary support to carry out doctoral
research work. The authors also extend their heartfelt gratitude to; Dr.
Danswrang Goyary, Scientist 'D', Division of Pharmaceutical Technology,
Defence Research Laboratory, Tezpur, Assam, India for scientific editing
and revision of the article.
10 N. S. BORA ET AL.
Ethical approval
All procedures performed in studies involving animals were in accord-
ance with the ethical standards of the institution at which the studies
were conducted. All experiments carried out in this study had the
approval of the Institutional Animal Ethical Committee (IAEC,
Registration no. 1227/bc/07/CPCSEA) vide approval no. IAEC/02/2015.
Disclosure statement
No potential conflict of interest was reported by the authors.
ORCID
Nilutpal Sharma Bora http://orcid.org/0000-0001-8648-0823
Bhaskar Mazumder http://orchid.org/0000-0002-5731-0435
Santa Mandal http://orcid.org/0000-0002-3386-8370
Pronobesh Chattopadhyay http://orcid.org/0000-0001-7770-6341
References
[1] Nagar HK, Srivastava AK, Srivastava R, et al. Evaluation of potent
phytomedicine for treatment of psoriasis using UV radiation
induced psoriasis in rats. Biomed Pharmacother. 2016;84:
1156–1162.
[2] Vijayalakshmi A, Geetha M. Anti-psoriatic activity of flavonoids
from cassia tora leaves using the rat ultraviolet B ray photoder-
matitis model. Brazilian J Pharmacogn. 2014;24:322–329.
[3] Aldeen T, Basra M. Management of psoriasis and its comorbidities
in primary care. Br J Nurs. 2011;20:1186–1192.
[4] Baz K, Cimen MYB, Kokturk A, et al. Oxidant/antioxidant status in
patientes with psoriasis. Yonsei Med J. 2003; 44:987–990.
[5] Lowes MA, Bowcock AM, Krueger JG. Pathogenesis and therapy
of psoriasis. Nature. 2007;445:866–873.
[6] Bakry OA, Samaka RM, Shoeib MAM, et al. Nuclear factor kappa B
and cyclo-oxygenase-2: two concordant players in psoriasis
pathogenesis. Ultrastruct Pathol. 2015;39:49–61.
[7] Nakaguma H, Kambara T, Yamamoto T. Rat ultraviolet ray B pho-
todermatitis: an experimental model of psoriasis vulgaris. Int J
Exp Pathol.
[8] Dreher F, Gabard B, Schwindt DA, et al. Topical melatonin in
combination with vitamins E and C protects skin from ultraviolet-
induced erythema: a human study in vivo. Br J Dermatol. 1998;
139:332–339.
[9] Eraslan G, Kanbur M, Aslan O, € et al. The antioxidant effects of
pumpkin seed oil on subacute aflatoxin poisoning in mice.
Environ Toxicol. 2013;28:681–688.
[10] Xanthopoulou MN, Nomikos T, Fragopoulou E, et al. Antioxidant
and lipoxygenase inhibitory activities of pumpkin seed extracts.
Food Res Int. 2009;42:641–646
[11] Pieri C, Marra M, Moroni F, et al. Melatonin: A peroxyl radical
scavenger more effective than vitamin E. Life Sci. 1994;55:
271–276.
[12] Bora NS, Pathak MP, Mandal S, et al. Safety assessment and toxi-
cological profiling of a novel combinational sunprotective dermal
formulation containing melatonin and pumpkin seed oil. Regul
Toxicol Pharmacol. 2017;89:1–12.
[13] Sirerol JA, Feddi F, Mena S, et al. Topical treatment with pterostil-
bene, a natural phytoalexin, effectively protects hairless mice
against UVB radiation-induced skin damage and carcinogenesis.
Free Radic Biol Med. 2015;85:1–11.
[14] Vijayalakshmi A, Geetha M. Anti-psoriatic activity of Givotia rottler-
iformis in rats. Indian J Pharmacol. 2014;46:386.
[15] Rocha-Pereira P, Santos-Silva A, Rebelo I, et al. The inflammatory
response in mild and in severe psoriasis. Br J Dermatol. 2004;150:
917–928.
[16] Rahnama S, Rabiei Z, Alibabaei Z, et al. Anti-amnesic activity of
Citrus aurantium flowers extract against scopolamine-induced
memory impairments in rats. Neurol Sci. 2015;36:553–560.
[17] Janero DR. Malondialdehyde and thiobarbituric acid-reactivity as
diagnostic indices of lipid peroxidation and peroxidative tissue
injury. Free Radic Biol Med. 1990;9:515–540.
[18] Hou H, Li B, Zhao X, et al. The effect of pacific cod (Gadus macro-
cephalus) skin gelatin polypeptides on UV radiation-induced skin
photoaging in ICR mice. Food Chem. 2009;115:945–950.
[19] Narendhirakannan RT, Hannah MAC. Oxidative stress and skin
cancer: an overview. Indian J Clin Biochem. 2013;28:110–115.
[20] Goswami S, Haldar C. Melatonin as a possible antidote to UV
radiation induced cutaneous damages and immune-suppression:
an overview. J Photochem Photobiol B Biol. 2015;153:281–288.
[21] Thiele J, Elsner P, editors. Oxidants and antioxidants in cutaneous
biology. 29th ed. Basel (Switzerland): Karger Medical and
Scientific Publishers; 2001.
[22] Fahim A. Effect of pumpkin-seed oil on the level of free radical
scavengers induced during adjuvant-arthritis in rats. Pharmacol
Res. 1995;31:73–79.
[23] EL-Shenawy SM, Abdel-Salam OME, Baiuomy AR, et al. Studies on
the anti-inflammatory and anti-nociceptive effects of melatonin
in the rat. Pharmacol Res. 2002;46:235–243.
[24] Hou Y, Guo Z, Li J, et al. Seleno compounds and glutathione per-
oxidase catalyzed decomposition of s-nitrosothiols. Biochem
Biophys Res Commun. 1996;228:88–93.
[25] Kruglikov I, Wollina U. Local effects of adipose tissue in psoriasis
and psoriatic arthritis. Psoriasis Targets Ther. 2017;7:17–25
[26] Wei L, Debets R, Hegmans JJ, et al. IL-1b and IFN-c induce the
regenerative epidermal phenotype of psoriasis in the transwell
skin organ culture system. IFN-Ç up-regulates the expression of
keratin 17 and keratinocyte transglutaminase via endogenous IL-
1 production. J Pathol. 1999;187:358–364.
[27] Terajima S, Higaki M, Igarashi Y, et al. An important role of tumor
necrosis factor-alpha in the induction of adhesion molecules in
psoriasis. Arch Dermatol Res. 1998;290:246–252.
[28] Hong K, Chu A, Ludviksson BR, et al. IL-12, independently of IFN-
gamma, plays a crucial role in the pathogenesis of a murine psor-
iasis-like skin disorder. J Immunol. 1999;162:7480–7491.
[29] Lin Z-Q, Kondo T, Ishida Y, et al. Essential involvement of IL-6 in
the skin wound-healing process as evidenced by delayed wound
healing in IL-6-deficient mice. J Leukoc Biol. 2003;73:713–721.
[30] Kofoed K, Skov L, Zachariae C. New drugs and treatment targets
in psoriasis. Acta Derm Venerol. 2015;95:133–139.
[31] Wong T, Hsu L, Liao W. Phototherapy in psoriasis: a review of
mechanisms of action. J Cutan Med Surg. 2013;17:6–12.