Introduction
Radiation dermatitis (RD) refers to a series of skin alterations
caused by radiation exposure and is observed
in around 95% of patients receiving radiotherapy (RT).[] RD is characterized by acute and chronic skin
changes. Its findings are erythema, dry desquamation,
itching, wet desquamation, necrosis, and infection. The
severity of RD is measured by various scoring systems
used in the clinic, and treatment recommendations are made accordingly.[] General skin care recommendations,
moisturizing with hydrophilic moisturizers, topical
corticosteroids, topical/systemic antibiotics, silicone
foam bandages, and laser therapy are recommended for
treatment.[] Although there is no gold standard for its
treatment, patients often use traditional methods.[]
Given that traditional techniques are typically derived
from herbal sources, individuals may choose not to
disclose their use to healthcare professionals on the assumption
that they are devoid of any adverse effects.[]
Hypericum perforatum (HP), also referred to as
St. John's Wort, is a perennial plant that has its origins
in Europe, West Asia, and North Africa. It has
gained significant popularity for its extensive utilization
in traditional ointments.[] Especially oil-based
HP preparations are frequently used in various skin
inflammations, burns, wounds, and scars.[] Also, in
Turkish folk medicine, the utilization of olive oil macerate
derived from flowering herbs is a commonly used
home remedy for the treatment of gastrointestinal ailments
as well as other dermatological conditions such
as skin inflammations, wounds, and burns.[] It is also
an alternative method for the treatment of depression
and mood disorders.[] Hyperforin is the compound
accountable for its anti-inflammatory, antibacterial,
and keratinocyte differentiation properties.[] It also
possesses anti-cancer properties and may inhibit cancer
invasion and metastasis.[] The many properties
of HP could render it an optimal therapeutic agent for
the management of RD, a condition commonly observed
in individuals with cancer. Hence, Franco et
al.[] assessed HP wort in a prospective trial on head
and neck cancer patients and showed it was safe and
had therapeutic benefits for RD.[]
In addition to the therapeutic effects of HP, it
also has photosensitivity-enhancing effects due to
hypericin.[] Hypericin has been found to exhibit
absorption of UVA radiation at a wavelength of 300
nm, as well as absorption of visible light within the
range of 550 to 590 nm. This absorption behavior is
responsible for inducing photosensitizing effects.
[] This phenomenon is called "hypericism." There
are studies reporting the increased therapeutic effects
of HP with photodynamic therapy in bladder cancer
and anaplastic thyroid cancer.[,] Again, studies
on human melanoma cells and human skin have reported
increased phototoxicity with ultraviolet light
and solar-simulated radiation.[-] Although the
photosensitizing effects of HP have been widely studied,
there are merely two case reports in the literature
that address the possibility of enhanced radiation sensitivity.[,] As of yet, there are no pre-clinic or
prospective trials on this issue. In this study, we investigated
the possible radiation sensitization-enhancing
effect of prophylactic and simultaneous use of HP oil
in a radiation-induced rat radiodermatitis model.
Methods
Animals
All animal experiments were performed in accordance
with Turkish laws on animal welfare and approved
by the local ethics board (approval number:
428). To establish the rat RD model, 16 male Sprague-
Dawley rats (12-week-old, 200-250 g) were used, and
these rats were obtained from Kobay Laboratory, Ankara-
Turkey. The experimental animals were housed
in artificially lit rooms with a 12-hour light/dark cycle
and temperatures ranging from 20°C to 24°C until
euthanasia. The standard rat diet and water were
supplied ad libitum. Rats were anesthetized with an
intraperitoneal ketamine/xylazine cocktail before RT
administration (90/10 mg/kg).
Hypericum Perforatum Oil Application
A quantity of 50 grams of HP was placed in a clear glass
jar together with 500 ml of olive oil. The container was
then exposed to sunlight for a duration of 12 hours
each day for a period of 4 weeks throughout the summer
season. Following the acquisition of HP oil, rats
were randomized to control (n=8) and HP oil-treated
(n=8) arms at the start of the experiment, and a 3×3
cm patch of their backs was shaved (Figs. 1, 2). The HP
oil arm was treated with 100 ?l of HP oil three times a
day, starting on the 3rd day before RT. Treatment was
continued up to the 14th day following RT.
Fig. 1. Experimental design. The study involved a total of 16 rats, which were randomized into 8 control groups and 8
groups treated with hypericum perforatum (HP). The application of HP oil began three days before to the RT in the
group treated with HP oil. During radiotherapy (RT), two rats from the group treated with HP oil succumbed to
problems related to anesthesia. The sacrification was performed on the 14th day following the RT procedure.
Radiation-induced Dermatitis Model and Sacrification
RT was administered in a single fraction of 30 Gy at a
dose rate of 600 MU with 4 MeV electrons under anesthesia
(Fig. 2). Electrons were used only to irradiate
the skin and protect the organs at risk underneath. The
RT application was performed using the Varian Clinac
DHX (Varian Medical System, Palo Alto, California,
USA) linear accelerator at Hacettepe University Department
of Radiation Oncology. Two animals allocated to
the HP oil-treated group succumbed to problems related
to anesthesia. Skin changes during the two weeks after
RT were assessed using a semi-quantitative skin damage
score in 14 animals (Table 1).[] The rats were euthanized
after two weeks, and skin samples were harvested.
Fig. 2. (a) A patch measuring 3×3 cm after shaving on the dorsal area of each rat's back. After shaving, 100 µl of HP oil was
applied three times daily, commencing on the third day prior to radiation therapy started in HP oil-treated arm. (b)
Treatment position of the rat.
HP: Hypericum perforatum.
Table 1 Semi-quantative skin damage scores
Histopathology
Irradiated rat skin samples were examined histomorphologically.
The tissues were fixed with 4% paraformaldehyde
overnight; paraffin blocks were prepared
and stained with hematoxylin & eosin, and sections of
4 µm were cut. The stained preparations were evaluated
by the pathologist using a light microscope by scoring
the presence of scarring, and the severity and extent of
inflammation. The presence of scarring was categorized as present, absent, and focal. The severity of inflammation
was scored as 1, 2, and 3 as no/mild, moderate, and
severe, respectively. The extent of inflammation was
scored as 1, 2, 3, 4, and 5 as <10%, 11-25%, 26-50%, 51-
75%, and >75% inflammation prevalence, respectively.
Statistical Analysis
Statistical analyses were performed with SPSS software
version 23 (SPSS Inc., Chicago, IL, USA). Descriptive statistics were presented as median and
minimum?maximum. Differences between groups
were analyzed with the Mann-Whitney U and the
chi-square test. An overall 5% type-I error level was
used to infer statistical significance.
Results
Post-radiation Skin Damage Score
The HP oil-treated group (n=6) and the control group
(n=8) were observed for two weeks to assess acute
skin changes after RT. In both groups, skin reactions
increased gradually over the first 14 days. On the 14th
day, the maximum dermatitis score recorded in the
HP oil group was 4 (patchy moist desquamation),
while the highest score recorded in the control group was 2.5 (dry desquamation). Figure 3 presents the
skin damage scores for each group's minimum, maximum,
and median values throughout a 14-day period.
Accordingly, the difference in skin reactions between
the two arms was evident (p=0.001). More skin reactions
were observed in the HP oil-treated arm.
Fig. 3. Median (minimum-maximum) skin damage scores over a 14-day period for each group (p<0.05). (a) Hypericum
perforatum oil-treated group (b) control group.
Histopathological Evaluation
All samples treated with HP oil (n=6) showed signs of
diffuse scarring, severe inflammation (score 3), and inflammation
with a prevalence of at least 50%. Looking at
the extent of inflammation scores in more detail, 4 (66%)
had >75% inflammation, and 2 (33%) had 51-75% inflammation.
In the control arm, on the other hand, scarring
was absent in 5 (62%), and in 3 (37%) cases only
focal scarring was observed. Inflammation was absent
or mild in 50% of cases (n=4), with an extent of <10%.
In the other half of the cases (n=4), inflammation was
moderate, with an extent ranging from 11% to 25% (Fig.
4). Harvested skins treated with HP oil had statistically
higher scores than the control arm in terms of the presence
of scarring, and the severity and extent of inflammation
(p=0.001, p=0.001, p=0.003, respectively).
Fig. 4. (a-c) Representative rat skin from Hypericum Perforatum oil-treated group, scarring and diffuse-severe inflammation,
(a, c) 40x field (b) 100x field. (d) Representative rat skin from the control group, no inflammation, 100x field.
(e, f) Another representative image form control group with focal-moderate inflammation, 100x field.
Discussion
To the best of our knowledge, this is the first preclinical
study showing the radiation-enhancing effect of HP oil
in the RD model. In our study, whether HP oil could
prevent RD and diminish RD-related symptoms in prophylactic
and post-RT therapeutic use was investigated.
However, we observed increased RD and increased inflammation in the histopathological examination
with HP oil. HP oil is an over-the-counter treatment
method that is frequently used by patients. It remains
unaddressed during the standard medical history assessment,
with individuals opting not to provide this information
to their attending healthcare provider. Therefore,
the use of HP oil should be questioned before RT
and should not routinely be recommended to patients.
In a single-arm prospective observational study in
which HP oil was used to treat RD, an ointment containing
HP oil and neem oil was started when bright
erythema, moderate edema, or patchy moist desquamation
was observed in patients who underwent chemoradiotherapy (CRT) in the head and neck (H&N) region,
and this treatment was found to be safe and effective.
[,] The contradictory outcomes obtained in our
study and the H&N studies investigating the same
question might be attributed to two main factors. First,
in Narayanan et al.[],'s study, HP oil was used as an
ointment together with neem oil. Consequently, the
concentration of HP oil in the ointment is diminished,
resulting in a corresponding decrease in the concentration
of hypericin. Neem oil is an agent whose antibacterial
properties have been well demonstrated. Neem oil
might have compensated for the increased RD effect of
HP oil. The second main difference is the prophylactic initiation of HP oil in our study. Franco et al.[] started
to apply the remedy containing HP oil after grade 2
dermatitis was observed. The presence of inconsistent
findings could possibly be attributed to variations in the
methodologies employed across different research projects.
In order to obtain a more definitive understanding
of the matter at hand, it is imperative to conduct future
prospective clinical investigations specifically focusing
on the application of an ointment including only HP oil.
The possibility that HP wort may cause increased radiosensitivity
first emerged in the literature with a case
report published in 2006. Putnik et al.[] reported a
case of recall dermatitis in a patient who had undergone
adjuvant RT for laryngeal cancer. The dermatitis
occurred one year after RT and was associated with
the administration of hypericin. Eichkorn et al.[] reported
that HP oil produced significant photon radiosensitivity
and that severe folliculitis capitis was found
on the scalp of the patient who underwent whole-brain
RT. The existing literature consists solely of two case
reports that demonstrate radiosensitivity with HP oil.
Nevertheless, research was conducted to analyze the
levels of plasma hyperforin and hypericin during hospitalization,
revealing positive results in 11.3% of patients.[] The fact that this herbal remedy, which is
frequently self-prescribed by patients, has an enhanced
photosensitizing impact that is not addressed in the literature
might be for a number of various reasons. Due
to the frequent utilization of this wort as a therapeutic
agent for skin lesions, it is plausible that the augmented
radiosensitivity effects may go unnoticed by patients or
remain undisclosed to their healthcare providers. Additionally,
it is conceivable that a threshold exists for the
radiation-sensitizing effect of products containing HP.
Unlike the photon radiosensitizing effect of HP, its
photosensitizing effects have been frequently studied
in the literature. The first publication showing increased
photosensitivity with the topical application of
HP wort belongs to Schempp et al.[] In this study,
the effects of hypericum oil (hypericin 110 mg/mL)
and hypericum ointment (hypericin 30 mg/mL) on
skin sensitivity to solar radiation were examined, and
hypericum oil has been shown to cause an increased
erythema index. Increased photosensitivity with HP
oil strengthens the idea that hypericin has an effect
on a certain threshold value, as was previously stated.
The photosensitivity-enhancing effects of HP have
also been demonstrated in preclinical studies of transitional
cell bladder cancer.[,] Although HP's collaboration
with photodynamic therapy has also been
demonstrated in anaplastic thyroid and melanoma cells, in one particular clinical study, no photosensitizing
effect was demonstrated when HP extract was
applied to skin lesions.[,,] The contradictory results
in the studies and the heterogeneity of the literature
cause confusion. Therefore, in studies where HP
wort will be used, especially in topical applications, the
poor penetration rate due to the high molecular weight
and high melting point of hypericin and the possible
photo/radiosensitizing cut-off value should be kept in
mind, and studies should be designed accordingly.[]
Limitations of the Study
Our study has several limitations. The most significant is
the small sample size and the loss of two animals in the
HP oil-treated group during the experiments. Additionally,
our study focused exclusively on HP oil, so different
outcomes might be observed with other HP extracts.
Conclusion
To conclude, our findings indicate that the use of HP
oil simultaneously with RT resulted in an elevation of
the post-radiation skin damage score in rats over the
follow-up period. Histopathological analyses revealed
that this combination led to an augmentation in both
the occurrence and intensity of scar formation and inflammation.
Our data suggest that HP oil worsens RD
when administered concurrently with RT. In routine
clinical practice, it is advisable to inquire about patients"
use of HP supplements, and caution should be
exercised while recommending it.
Ethics Committee Approval: The study was approved
by the Kobay Local Ethics Committee (no: 428, date:
11/11/2019).
Authorship contributions: Concept - M.T.Y., F.Y.Y.,
S.Y.S., O.K., M.C., F.Z., G.Y.; Design - M.T.Y., F.Y.Y., S.Y.S.,
O.K., M.C., F.Z., G.Y.; Supervision - S.Y.S., O.K., M.C., F.Z.,
G.Y.; Funding - S.Y.S., M.C., F.Z., G.Y.; Materials - M.T.Y.,
F.Y.Y., S.Y.S., O.K., M.C., F.Z., G.Y.; Data collection and/or
processing - M.T.Y., F.Y.Y., S.Y.S., O.K., M.C., F.Z., G.Y.;
Data analysis and/or interpretation - M.T.Y., F.Y.Y., S.Y.S.,
O.K., M.C., F.Z., G.Y.; Literature search - M.T.Y., F.Y.Y.,
G.Y.; Writing - M.T.Y., F.Y.Y., G.Y.; Critical review - M.T.Y.,
F.Y.Y., S.Y.S., O.K., M.C., F.Z., G.Y.
Conflict of Interest: All authors declared no conflict of interest.
Use of AI for Writing Assistance: No AI technologies utilized.
Financial Support: None declared.
Peer-review: Externally peer-reviewed.
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