1Student Research Committee, School of Dentistry, Shiraz University of Medical Sciences, Shiraz, Iran.
2Stem Cell Technology Research Center and Pharmacology Department, Shiraz Medical School, Shiraz University of Medical Sciences, Shiraz, Iran.
3Student Research Committee, School of Dentistry, International Branch, Shiraz University of Medical Sciences, Shiraz, Iran.
4Laboratory Animal Center, Shiraz University of Medical Sciences, Shiraz, important
5Prevention of Oral and Dental Diseases Research Center, School of Dentistry, Shiraz University of Medical Sciences, Shiraz, Iran.
6Department of Oral and Maxillofacial Medicine, School of Dentistry, Shiraz University of Medical Sciences, Shiraz, Iran.
Submission: February 06, 2017; Published: March 07, 2017
*Corresponding author: Nader Tanideh, PhD, Stem Cell Technology Research Center and Pharmacology Department, Shiraz Medical School, Shiraz University of Medical Sciences, Shiraz, Iran, Tel: +9891777160017; Email: email@example.com
How to cite this article: Farrokhi F, Tanideh N, Fekri S, Koohi-Hosseinabadi O, Tadbir A A, et al. The Effect of Pistacia Atlantica and Hypericum Perforatum
as a Healing Accelerator Remedy on Induced Oral Mucositis in Male Golden Hamster. Adv Dent & Oral Health. 2017; 4(1): 555630. DOI: 10.19080/ADOH.2017.04.555630
Oral mucositis is a painful condition that occurs as a side effect of chemotherapy. P. atlantica and H. perforatum have long been known for their anti-inflammatory and anti-oxidative properties as an alternative medicine. This study evaluated the healing effects of extraction of P. atlantica and H. perforatum, on 5-FU induced oral mucositis in golden hamsters by histopathology, clinically, and anti-oxidative assessments. Oral mucositis was induced in 60 male golden hamsters by administration of 5-FU (60mg/kg), on days 0, 5, and 10 of the study. Animals were randomly divided in 5 groups, (n=12). They were treated with hydro-alcoholic extract of H. perforatum (topical gel 10%) (Group 1), P. atlantic oil extract (topical gel 10%) (Group 2), the combination of topical gel 10% of P. atlantica, and topical gel 10% of H. perforatum (Group 3), gel base group (Group 4) that were all compared with the control group (Group 5) from day 12 to day 18. On days 15 and 18 of the experiment cheek pouch were examined visually for macroscopic scoring and excisional biopsies were obtained for microscopic scoring. Tissue MDA and MPO levels were assessed. The data were statistically analyzed by analysis of variances and kruskal-wallis test. Healing of oral mucositis was significantly improved both clinically and histopathologically (P-value<0.05) in group 3 in comparison with the other four groups. It seems that treatment with H. perforatum and P. atlantica gel, specially 10% gel concentration, can facilitate the healing of oral mucositis induced by chemotherapy (5-FU).
Abbreviation: P. Atlantica: Pistacia Atlantica; H. Perforatum: Hypericum Perforatum; OM: Oral Mucositis; MDA: Malon Di Aldehyde; MPO: Myelo Per Oxidase; 5-FU: 5-Fluoro Uracil; NSAID: Non Steroid Anti-Inflammatory Drug
Oral mucositis (OM), an inflammatory response to radiotherapy/chemotherapy in patient with head and neck cancer, is very painful condition. OM has a direct effect on quality of life in patients with dysphagia, pain, and it interferes with nutrition swallowing and verbal communication, gingival bleeding, infection and weight loss. However, OM increases the hospitalization duration and treatment cost [1-3]. Until now, the available strategy therapies such as dental care, anti-oxidant agents, anti-inflammatory agents, steroids, NSAIDs, topical antiseptic, and
antimicrobial agents, cryotherapy, mucosal protectors, antibiotics, pure honey and herbal medicines are palliative, but an effective therapeutic agent for OM has long remained elusive [2,4,5].
The use of biological products such as palifermin is effective, but most patients cannot afford it because of its high costs. Therefore, herbal medicines are most commonly used for OM as they are cheaper with less toxicity than synthetic drugs. Herbal therapies with antifungal, antibacterial, anti-inflammatory, antioxidant, wound healing, and immune stimulatory characteristics such as Glycyrihiza glabra, payayor, Zizyphys jujube, calendula
officincalis carum carvi, and Curcuma longa could be used in
OM treatment. [2,3,6-8]. Pistacia atlantica (subsp. Mutica) trees
grow in the western, eastern and central parts of Iran. The fruit
of this tree has long been used as snack and also for treatment of
peptic ulcer, kidney stones, dyspepsia, wound heading, hepatica
inflammation, toothache, diabetes, expectorant antitumor, and
mycosis. P. atlantica contains saturated fatty acids like palmitic
acid, unsaturated fatty acid such as; trepenoids, plamitoleic
acid, oleic acid, lineoleic acid, and unsaponifiable matter which
is considered to be the highest tocopherols and tocoterinols,
phenolic compound such as gallic acid as antioxidant. Trepenoids
have anti-inflammatory and antinocieptive activity [9-10].
Hypericum perforatum (St. John’s Wort), as a herbal medicine,
can be identified with its golden yellow flowers and it is indigenous
to Europe, western Asia and northern Africa. Its flowers contain
red liquid which comprises of biologically active compounds. H.
perforatum plants have been used in medicine for centuries. H.
perforatum is commercially available in forms of tablet, capsule
and tea [11,12]. Wound-healing effect of H. perforatum can be
used for the treatment of burns, depression, ulcerative colitis,
analgesic, antibiotic, antiviral, and diuretic. Anti-oxidant activity
of H. perforatum is like the in vitro and in vivo activity of vitamin
E that inhibits free radical and lipid peroxidation. Several active
components of H. perforatum are hypericin, quercetin, hyperforin,
flavonoids, and xanthone derivatives . This study aims to
evaluate the efficacy of H. perforatum and P. atlantica on induced
OM in male golden hamsters.
Sixty male golden hamsters (8-10 weeks old), 105±5g, were
used after approval of the University Ethic Committee 9003. All
animals had access to normal diet, water and were placed in a
standard cage 60*40*18, at 20°±2, 55±5% humidity in a 12h light
and 12h dark cycle.
The animals were randomly divided into five groups (n=12).
All animals received intraperitoneal injections of 5-fluorouracil
(5-FU/60mg/kg) during days 0, 5, and 10. Three horizontal
superficial scratches were made on the cheek pouch with the tip
of a 19 gauge sterile needle across the everted cheek pouch once
daily on days 2 and 3 in order to simulate the clinical effect of
chronic irritation. The severity of oral mucositis was observed on
the 12th day. Subsequently, they were treated with topical gel 10%
hydro-alcoholic extract of H. perforatum (Group 1), topical gel 10%
P. atlantica fruit oil extract (Group 2), topical gel 10% P. atlantica
fruit oil extract in combination with 10% hydro-alcoholic extract
of H. perforatum (Group 3), in the 4th group the subjects received
the gel base, and finally the control group (Group 5) received no
treatment. All groups received topical gel (wound was completely
covered) except the fifth group. To ensure that topical gel was not
swallowed, animals were not given any food or drink for 90 min
after it was applied on their cheek pouches. Six hamsters were
randomly selected from each group and euthanized on the 15th
and 18th day. Both cheek pouches mucosa were harvested.
We scored lesions based on our observation. Inflammatory
aspects such as erythema, hyperemia, hemorrhagic areas,
epithelial ulcerations, and abscesses were evaluated in a singleblind
fashion for macroscopic analysis (Table 1) .
Tissues which were taken from the right cheek pouches were
fixed in 10% formalin. Sections were stained with Haematoxylin
and Eosin. The specimens were examined histopathologically and
received scores of 0–3 (Table 2) .
Tissues from the left cheek pouches were sent to the
laboratory in the faculty of Pharmacy in cryo-tubes for MDA to be
measured. MDA is an index of lipid peroxidation. Tissues weighing
between 0.15 and 0.2 g were cut and mixed and homogenized
with phosphate buffer solution in a ratio of 1/5. The homogenized
tissues (400ml) were mixed with 800ml of trichlor-oacetic acid, and centrifuged for 30 min (3000 rpm). Next, 600 ml of the samples
were mixed with 150ml of 1% thiobarbituric acid and positioned
in a water bath for 15 min. After cooling, 6ml of n-butanol was
added and they were centrifuged for 10 min (3000rpm). Finally,
butane phase absorption was read with spectrophotometer at
a wavelength of 532nm . Tetraethoxy propane was used as
control. The MDA tissue concentration was calculated as follows:
MDA (nmol/ml) = [(absorbance of the test sample - absorbance of
the negative control) + 0.0606]/0.0537.
Harvested cheek pouches were weighed and homogenized in
a solution containing 0.5% (w/v) hexadecyltrimethyl ammonium
bromide dissolved in 10 mmol/l potassium phosphate buffer (pH
7.4) and centrifuged for 30 min at 20000g at 4 °C. An aliquot of
supernatant was then removed and added to a reaction mixture
containing 1.6mmol/l tetramethylbenzidine and 0.1 mmol/l
hydrogen peroxide (H2O2). MPO activity was measured in tissues by
assays using hydrogen peroxide and o-dianisidinedihydrochloride as substrates. The absorbance rate was measured by
spectrophotometer at 650nm. MPO activity was defined as the
quantity of enzyme required to degrade 1mmol of H2O2 at 37 °C.
Enzyme activity was expressed in U/g wet tissue .
Normal distributions of data were assessed using Kolmogorov
Smirnov test. The MDA and MPO levels were compared in each
group on the 12th, 15th and 18th day, using non-parametric Kruskal-
Wallis H tests to assess the differences between groups on MDA
level and MPO activities, stereological and histopa¬thological
scores. The Mann- Whitney U-test with Bonferroni correction
(0.05:6=0.0083) was used to compare qualitative factors such
as histopathological and macroscopical scores between groups
on 12th, 15th and 18th day. The data are presented as the mean ±
standard deviation. SPSS software version 19 (SPSS Inc., Chicago,
IL, USA) was used for statistical analysis of data. Graphpad Prism
5 (GraphPad Software Inc., La Jolla, CA, USA) was used to draw the
graphs. P-value ≤0.05 was considered statistically significant.
Tissue MDA activity in study groups on 15th and 18thdays are
shown in Figure 1. On both days 15th and 18th, tissue MDA level
evaluation scores for the mixed group was significantly lower
than the gel-base group and the control group (p<0.001). MDA
level concentration for the mixed group was lower than 10% of
H. perforatum group (p=0.002 in both days). On 15th and 18th day,
MDA level concentration for the 10% P. atlantica group was lower
than gel-base group (p=0.002) and also lower than the control
group (p<0.001). MDA concentration for the 10% of H. perforatum
group was lower than the control group (p=0.002).
Tissue MPO activity in study groups on 15th and 18th day are
shown in Figure 2. On both days 15 and 18, tissue MPO level
evaluation scores for the mixed group was significantly lower than
the gel-base group and the control group (p<0.001). MPO level
concentration for the mixed group was lower than the 10% of H.
perforatum group (p=0.002 in both days). On 15th and 18thdays,
MPO level concentration for the 10% of P. atlantica group was
lower than gel-base group (p=0.002), and also lower than the
control group (p<0.001). MPO concentration for the 10% of H.
perforatum group was lower than control group (p=0.002).
Macroscopic evaluation scores in the study groups on 15th and
18thdays are shown in Figure 3. On both days 15 and 18, macroscopic
evaluation scores for the mixed group was significantly lower than
the gel-base group and the control group (p<0.001), which means
better healing in the mixed group. Macroscopic scores of 10%
P. atlantica group was lower than the control group (p<0.001 in
both days) and also lower than the gel base on days 15 and 18
(p=0.002 vs. p<0.001). On 15th and 18th days, macroscopic scores
of the control group was more than 10% of H. perforatum group
(p<0.001). On day 15th and 18th, macroscopic scores 10% of H.
perforatum group was lower than the base gel group (p=0.004 vs.
Microscopic evaluation scores in study groups on 15th and
18thdays are shown in Figure 4. On both days 15 and 18, microscopic
evaluation scores for the mixed group was significantly lower than
the gel-base group and the control group (p<0.001). Microscopic
scores of 10% P. atlantica group was lower than the control group
(p<0.001 in both days), and also lower than gel base on days 15
and 18 (p=0.001 vs. p=0.004). On day 15 and 18, microscopic
scores 10% of H. perforatum group was lower than the base gel
group (p=0.002 vs. p=0.035) and also lower than the control
group on day 15 and 18 (p<0.001 vs. p=0.013).
In this study, histopathological, macroscopical and anti
-oxidant markers of P. atlantica and H. perforatum were evaluated
in the induced oral mucositis in male golden hamsters. We showed
that the mixed gel (10% P. atlantica + 10% H. perforatum) yield
better results on healing process of OM in comparison with the
other groups both on 15th and 18thday.
NSAIDs and steroidal drugs are usually prescribed for
inflammatory diseases. Although several studies have shown the
effect of combined protein and herbal plants accelerate healing
and anti-inflammatory effects in OM in human and animal
models, but not all of them showed high efficacy for OM treatment
[12,16,17]. Tanideh et al. , showed that oral administration of
H. perforatum haydro alcoholic extract was more effective than
the topical gel on OM. Additionally, Samadi et al. , described
that using H. perforatum ointment had positive effects on cesarean
wound. H. perforatum stimulates fibroblasts to produce more
collagens, so that the wound contraction accelerates . Since normal flora of oral cavity gets the opportunity to infect the oral
mucosa lesions, hence, anti-microbial agents can be beneficial .
H. perforatum has potential antibiotic properties .
Conforti et al.  showed that the accelerated healing effect
of H. perforatum with its non-cytotoxic and anti-inflammatory
properties is due to multiple components such as flavonoids
and biapigenin. Couladis et al.  showed that the antioxidant
activities of vitamin E are as effective as those of Hypericum species
such as rutin, quercetin, and chlorogenic acid. The protective role
of H. perforatum extracts on the process of lipid peroxidation was
established by measuring the formation of MDA. Božin et al. 
showed a notable inhibitory potency of lipid peroxidation of H.
perforatum extracts. Some studies have investigated the effect of
H. perforatum on periodontitis in animal model and their findings
showed that myeloperoxidase levels can be reduced significantly
by hypericum treatment in the gingivomucosal tissues.  Dost
et al.  found that H. perforatum was effective in decreasing
MDA levels in TNBS-induced colitis in rats due to its antioxidant
In traditional Iranian medicine, different parts of Pistacia
species, such as lentiscus, atlantica, khinjuk, and terebinthus, with
their effective wound healing properties have been used in the
treatment of brain and gastrointestinal disorders and different
inflammatory diseases . Tanideh et al.  showed the efficacy
of P. atlantica on healing of acetic acid induced colitis in rats.
The fruits of P. atlantica are found to be rich in proteins, fibers,
and unsaturated fatty acids, suggesting their value as a food
source. The major components of P. atlantica are α-tocopherols
and important sterols that have antioxidant properties and health
benefits such as; being precursors of vitamin D and lowering
blood cholesterol .
There are several studies that have investigated the bioactive
agent of P. atlantica. α- pinene, tocopherol and teriterpenic acid
which are three important components of this fruit [26,27]. They
have also declared that this herbal medicine can be useful for
treating GI ulcers, strengthening gum [28,29]. Farnoosh et al. ,
expressed that 48% of composition of nonsoapebale P. atlantica
oil consist of ractopherol and tocoterion that has anti-oxidant
effect and acts similar to vitamin E. Shimizu et al. , showed
that vitamin E enriched diet could be beneficial in the treatment
of ulcerative colitis in rats. Ademoglu et al. , found that vitamin
E and selenium supplementation, significantly reduced both
the severity of colonic lesions and the level of MDA and protein
carbonyl. Ghalem et al. , showed that essential oil from the
leaves and the gum of P. atlantica has an acceptable antibacterial
and antifungal properties.
According to previous studies, it seems that therapeutics role
of P. atlantica and H. perforatum due to their components such
as anti-inflammatory properties, and synergic effects similar
to vitamin E that can accelerate healing of oral mucositis. Our
findings are in line with this theory. Based on our findings in histopathologic and clinical assessments, we observed that the
mixed gel (10% P. atlantica + 10% H. perforatum), had lower
microscopic and macroscopic scores compared to the other
groups on 15th and 18th day. This reduction in both histopathologic
and macroscopic scores indicates the healing process of these
therapeutic agents. Oxidative stress from free radical is involved
in pathophysiology of many inflammatory diseases, thus, the
protective role of H. perforatum and P. atlantica extracts on the
process of lipid peroxidation was determined by measuring
the formation of MDA and MPO. We found that MPO and MDA
concentration of mixed gel was lower than the others on both
days. This study sheds some light on this subject; however, further
studies are required to confirm the efficacy of H. perforatum and P.
atlantica in clinical trials.
This study suggests that daily use of H. perforatum and P.
atlantica gel can relieve the induced oral mucositis in hamsters.
A combination of H. perforatum and P. atlantica with their antioxidant,
anti-inflammatory and antimicrobial properties might be
a suitable choice for the treatment of oral mucositis.