Oral mucositis is a common and irritating complication of chemotherapy and radiotherapy for malignancies. Current treatments have failed to achieve complete remission of this complication. The St. John’s wort plant ( Hypericum perforatum ) has long been known for its anti-inflammatory and antibacterial effects. The current study was designed to investigate the therapeutic efficacy of the topical and systemic administration of H. perforatum extract on oral mucositis. Oral mucositis was induced in 72 male golden hamsters by administration of 5-fluorouracil (60 mg/kg), on days 0, 5, and 10 of the study. The cheek pouch was scratched with a sterile needle on days 1 and 2. On days 12–17, H. perforatum extract topical gel 10%, oral H. perforatum extract (300 mg/kg), and gel base groups were treated and then compared with a control group. Weights and blood samples were evaluated, biopsies from buccal lesions were examined histopathologically, and tissue malondialdehyde (MDA) was measured. Both of the H. perforatum extract treatment groups saw a significant relief in oral mucositis compared to the control and base gel groups; the systemic form was superior to the topical form. H. perforatum extract, administered orally or topically, expedited the healing of chemotherapy-induced oral mucositis in hamsters.
Oral mucositis is a common, painful, and dose-limiting complication of chemoradiotherapy for head and neck cancers, bone marrow transplantation, and certain chemotherapeutic agents for a variety of malignancies. It typically manifests as erythema or breakdown of the oral mucosa, with the development of ulcerative lesions. This condition can adversely affect the patient’s treatment regimen, nutrition, and quality of life. In addition, the loss of integrity of the oral mucosa often provides a route for microbial contamination and sepsis. Furthermore, it can increase the use of health care resources. Unfortunately, despite the clinical significance of oral mucositis, there is no effective strategy for its management, and treatment focuses on a palliative management approach.
Hypericum perforatum , known botanically as St. John’s wort, is a medicinal plant with yellow flowers. It is used for the treatment of anxiety, depression, cuts, burns, cancer, and bacterial and viral diseases, and as an antioxidant, analgesic, and neuroprotective agent. H. perforatum also possesses remarkable wound healing and anti-inflammatory properties. The anti-inflammatory effects of H. perforatum can be related in part to its inhibition of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2). Pseudohypericin and hyperforin are the primary anti-inflammatory constituents of H. perforatum ; along with the flavonoids, they can exert their effect by inhibiting prostaglandin E 2 production. Hyperforin also acts as a dual inhibitor of 5-lipooxygenase and COX-1 in intact cells. It has significant antibiotic activity against some Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus , and it becomes bioavailable by oral ingestion. Hyperoside, another compound found in H. perforatum , has been shown to have an anti-inflammatory action by suppressing the production of tumour necrosis factor, interleukin 6, and nitric oxide.
Considering the debilitating effects that oral mucositis induced by chemoradiotherapy can have on cancer patients and the lack of a definite treatment strategy, and based on the data implicating the anti-inflammatory, analgesic, and wound healing activities of H. perforatum , we decided to study the effects of H. perforatum extract on oral mucositis induced in golden hamsters.
Materials and methods
The methodology used in this study was approved by the research and ethics committee of the study university; all relevant animal rights considerations were addressed. This randomized animal trial was performed in the university laboratory animal centre.
Seventy-two male golden hamsters (6–8 weeks old, weighing 95–130 g; Laboratory Animal Centre of Shiraz University of Medical Sciences) were used. All animals had free access to a standard laboratory diet and water.
Chemotherapy-induced oral mucositis
The hamsters were divided randomly into four groups (18 hamsters in each group). All animals received three intraperitoneal injections of 5-fluorouracil (5-FU) on days 0, 5, and 10 at a dose of 60 mg/kg with insulin needles, following the protocol proposed by Sonis et al. To mimic the clinical effect of chronic irritation, the cheek pouch mucosa was scratched superficially with the tip of an 18-gauge sterile needle after anesthetizing the area. Two horizontal linear superficial scratches were made across the everted cheek pouch once daily on days 1 and 2. The maximum severity of oral mucositis was seen on day 12 ( Fig. 1 A ). Thus, the treatment was started on day 12.
The first group of hamsters served as controls and received no treatment. Group 2 were treated with topical H. perforatum extract gel 10% and group 3 received the gel base used in the preparation of the topical gel (as a placebo match for the second group), on the cheek pouch mucosa of both sides once a day (morning), and the affected area was completely covered. To make sure that it was not swallowed, animals were not allowed to eat or drink for 30 min after the gel applications. We also examined the hamsters’ mouths to check for the presence of the thick gel layer over the lesions after 30 min. The thick gel base does not dissolve easily in saliva and even if the topical gel was swallowed, the dose would be much lower than the therapeutic dose. Group 4 were fed H. perforatum extract 300 mg/kg once a day (morning). Animals were weighed daily. Six hamsters from each group were selected randomly and killed on days 13, 15, and 17 ( Fig. 1 B).
At the time of sacrifice, the hamsters were anesthetized with an overdose of ether. A heart puncture was performed for the collection of blood samples and a complete blood count (CBC) test was done on the samples. Biopsies of the cheek pouch mucosa of both sides were obtained. The whole cheek pouch and the underlying mucosa were excised.
Tissue obtained from the right side was placed in 10% formalin. Tissues were then prepared for histopathological examination in the standard fashion; sections were stained with haematoxylin and eosin. The specimens were examined histopathologically and received scores of 0–3, as described by Lima et al. : (1) score 0 = normal epithelium and connective tissue without vasodilatation; absent or mild inflammatory infiltrate; absence of bleeding, ulcers and abscesses; (2) score 1 = mild vascular hyperaemia; areas of re-epithelialization; mild inflammatory infiltrate with a prevalence of mononuclear cells; no haemorrhagic areas, ulcerations or abscesses; (3) score 2 = moderate vascular redness; areas of epithelial degeneration; inflammatory infiltration with prevalence of neutrophils; haemorrhagic areas, oedema and occasional ulcerations; absence of abscesses; and (4) score 3 = severe hyperaemia and vascular vasodilatation; inflammatory infiltration with prevalence of neutrophils; haemorrhagic areas, oedema, extensive ulcerations, and abscesses.
Measurement of tissue malondialdehyde (MDA)
Tissue from the left side was sent to the toxicology laboratory of the faculty of pharmacy of the study university in cryotubes for subsequent measurement of MDA. MDA is an index of lipid peroxidation. To measure tissue MDA, tissue weighing between 0.15 and 0.2 g was cut and mixed and homogenized with phosphate buffer solution (PBS) in a ratio of 1/5. The homogenized tissue (400 μl) was then mixed with 800 μl of trichloroacetic acid (TCA) and centrifuged for 30 min (3000 rpm). Next 600 μl of the sample was mixed with 150 μl of 1% thiobarbituric acid (TBA) and was placed in a water bath for 15 min. After cooling down, 6 ml of n -butanol was added and it was centrifuged for 10 min (3000 rpm). Subsequently butane phase absorption was read at a wavelength of 532 nm. Tetraethoxypropane 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.
After assessing the normal distribution of the data with the Kolmogorov–Smirnov test, comparisons of the tissue MDA levels, blood factors, and weight changes (between day 12 and days 13, 15, and 17) in the different groups (control, gel base, topical gel, and systemic treatment) on the different days, were made using two-way analysis of variance (ANOVA) and the post hoc test of least significant difference (LSD). The paired t -test was used to assess weight changes in each group between the different days. A P -value of ≤0.05 was considered to be statistically significant. To compare qualitative factors such as histopathological scores between the different groups on different days, the Mann–Whitney U -test was used, with Bonferroni correction (0.05:6 = 0.0083). A P -value of ≤0.0083 was considered to be statistically significant. The data are presented as the mean ± standard error. SPSS 11.5 software (SPSS Inc., Chicago, IL, USA) was used to analyze the data and GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA) was used to draw the graphs.
The mean change in weight in each group on different days, and among the different groups on distinct days, is shown in Fig. 2 . On day 13, only the weight gain of the topical gel group was greater than that of the systemic treatment group ( P = 0.04). On day 15, the weight gain in the systemic treatment group was greater than that of the control group ( P = 0.01). On day 17, the weight gain of the topical gel ( P = 0.02), systemic treatment ( P < 0.001), and gel base ( P < 0.001) groups was greater than that of the control group, and the weight gain of the systemic treatment group was greater than that of the topical gel group ( P = 0.01).
During the treatment period, significant weight gain was observed in the systemic treatment group. Weight gain was greater on days 15 ( P = 0.02) and 17 ( P = 0.002) than on day 13. On the other hand, comparing days 13–17, significant weight loss occurred in the control group ( P = 0.04). There was no significant weight gain in the topical gel group, despite weight gain in the gel base group between days 13 and 17 ( P = 0.04).
Histopathological changes according to the determined scores are shown in Fig. 3 . There was a significant histopathological difference between the treatment groups and the control groups. No histopathological difference was seen between the systemic treatment group and the topical gel group. Also, no difference was observed between the control group and the gel base group on the different days. Over the whole treatment period, histopathological scores were lower in the topical gel and systemic treatment groups than in the gel base and control groups, indicating less severe inflammation in the topical gel and systemic treatment groups ( Fig. 4 A–C ). Epithelialization and wound healing were detected in the topical gel and systemic treatment groups, whereas extensive infiltration of inflammatory cells, haemorrhage, and ulcers were observed in the control and gel base groups. On days 13, 15, and 17, histopathological scores for the topical gel ( P = 0.004, P = 0.002, and P = 0.001, respectively) and systemic treatment ( P = 0.004, P = 0.002, and P = 0.005, respectively) groups were lower than those of the gel base group. Also on days 13, 15, and 17, histopathological scores for the topical gel ( P = 0.008, P = 0.008, and P = 0.006, respectively) and systemic treatment ( P = 0.008, P = 0.008, and P = 0.006, respectively) groups were lower than those of the control group.
Tissue MDA level
Differences in tissue MDA concentrations in the different groups on different days are shown in Fig. 5 . There were no differences in MDA concentrations between the gel base and control groups on the different days. The MDA concentration was higher in the control and gel base groups than in the topical gel and systemic treatment groups on all days ( P < 0.001), indicating more oxidative stress in the gel base and control groups; the MDA concentration in the systemic treatment group was higher than that in the topical gel group on day 13 ( P = 0.027), day 15 ( P < 0.001), and day 17 ( P = 0.005).
Differences in white blood cell (WBC) counts are shown in Fig. 6 A . On day 15, the WBC count of the gel base group was higher than that of the control group ( P = 0.03) and topical gel group ( P = 0.04). On day 17, the WBC count of the control group was higher than that of all of the other groups ( P < 0.001). No WBC count changes were detected in the groups treated with H. perforatum extract during the treatment period.