Radioprotective Effect of Hesperidin: A Systematic Review

Background and objectives: Ionizing radiation (IR) has been of immense benefit to man, especially for medical purposes (diagnostic imaging and radiotherapy). However, the risks of toxicity in healthy normal cells, leading to cellular damage as well as early and late side effects, have been major drawbacks. The aim of this study was to evaluate the radioprotective effect of hesperidin against IR-induced damage. Materials and Methods: The preferred reporting items for systematic reviews and meta-analyses (PRISMA) were applied in reporting this study. A search was conducted using the electronic databases PubMed, Scopus, Embase, Google Scholar, and www.ClinicalTrials.gov for information about completed or ongoing clinical trials. Results: From our search results, 24 studies involving rats, mice, and cultured human and animal cells were included. An experimental case—control design was used in all studies. The studies showed that the administration of hesperidin reduced oxidative stress and inflammation in all investigated tissues. Furthermore, it increased 30-day and 60-day survival rates and protected against DNA damage. The best radioprotection was obtained when hesperidin was administered before irradiation. Conclusions: The results of the included studies support the antioxidant, anti-inflammatory, and antiapoptotic abilities of hesperidin as a potential radioprotective agent against IR-induced damage. We recommend future clinical trials for more insights.


Introduction
Since the discovery of ionizing radiation (IR) in the early 1900s, its use has been on the increase. Nowadays, the use of IR can be found in industrial and agricultural sectors. However, it is mostly utilized in medicine for diagnostic as well as treatment aims. The use of IR for diagnostic purposes is responsible for the majority of radiation doses received by man [1,2]. Moreover, radiotherapy (cancer treatment using IR) is more utilized compared to other cancer therapeutic modalities [3].
Despite the numerous benefits of IR to man, the risks of toxicity to healthy normal cells leading to cellular damage as well as early and late side effects have been major drawbacks. IR causes damage to cells via direct and indirect effects. Direct effects occur when IR interacts directly with DNA. Indirect effects take place as a result of the interaction between free radicals, including reactive oxygen species (ROS) and reactive nitrogen species (RNS), and neighboring DNA molecules [4]. The most important biological molecule in the cell for sustaining life, DNA, is the primary target for radiation-induced cell death.
In addition to radiation exposure for medical purposes, several side effects due to radiation exposure from nuclear accidents as well as radiation disasters have been reported. The Hiroshima and Nagasaki events during World War II led to the deaths of over 150,000 people who were exposed to sublethal radiation doses [5]. The Chernobyl nuclear disaster was also another event that led to chronic biological changes to the immune system as well as subsequent cancer development in exposed persons [6].
Most radiation-induced side effects are due to the free radicals produced by IR in cells [7]. Radioprotectors (or radioprotective agents) have been proposed for preventing or reducing these side effects. A radioprotector is most suitable for use if it has minimal toxicity while protecting healthy cells and not cancer cells [8]. It is also important that they are easily accessible and not expensive [9]. The use of amifostine, the first Food and Drug Administration (FDA)-approved radioprotector, has been mostly restricted as a result of possible toxicity [10]. Therefore, recent studies on radioprotectors have been majorly centered on natural substances such as flavonoids, due to their minimal side effects.
Flavonoids of varying phenolic structures are present in natural substances with varying phenolic structures such as fruits, vegetables, grains, bark, roots, stems, flowers, tea, and wine [11]. It has been shown that the potent antioxidant effects of flavonoids are a result of their high redox abilities, making them efficient hydrogen donors and reducing agents, in addition to their metal-chelating capabilities and singlet oxygen quenchers [12]. Some flavonoids that have been explored for radioprotection include curcumin, sesamol, hesperidin, rutin, ocimum sanctum, quercetin, and resveratrol [13].
Hesperidin (hesperetin-7-rhamnoglucoside, Figure 1) is a bioflavonoid found in citrus fruits such as tangerine, orange, and lemon as well as in plant extracts such as tea and olive oil. Citrus fruit peels have the highest concentrations of hesperidin. It has shown promising results in the treatment of inflammatory as well as allergy diseases [14]. Its potential in the treatment of cardiovascular and neurological disorders has also been investigated [15,16]. Studies have shown that hesperidin possesses antimicrobial, anticarcinogenic, antioxidant, and anti-inflammatory effects and decreased capillary fragility [17]. The purpose of this systematic review was to evaluate the radioprotective effect of hesperidin against radiation-induced damage to cells and organs.

Search Strategy
The reporting of this systematic review was done according to the statement of preferred reporting items for systematic reviews and meta-analyses (PRISMA) [18]. A computer-based literature search was conducted in January 2019 using PubMed, Scopus, Embase, and Google Scholar for articles published in English. No limit in publication year was applied. The following keywords were used for our literature search: "hesperidin", "radiation", "radiation protection", and "radioprotector". We also searched www.ClinicalTrials.gov for completed or ongoing clinical trials. In addition, references of retrieved studies were manually screened to obtain relevant studies.

Inclusion Criteria
The articles retrieved were based on the following inclusion criteria: • Studies that were conducted to determine the radioprotective effect of hesperidin and were published in the English language; • Studies in which ionizing radiation was used; and • Experimental and clinical studies with full texts.

Exclusion Criteria
We excluded studies based on the following criteria: • Studies in which hesperidin was not used; • Studies in which hesperidin was used in combination with other agents; • Studies that made use of other forms of radiation such as ultraviolet (UV), fluorescence, cosmic, etc.; • Studies that evaluated the effect of hesperidin with chemotherapy instead of radiation therapy; and • Conference abstracts, simulation studies, review articles, case reports, letters, editorials, unpublished data, articles without full texts, and non-English articles.

Study Selection
All retrieved articles from electronic as well as manual searches were entered into endnote software (EndNote version X6, Thomson Reuters, New York, NY, USA). Thereafter, duplicates were removed. Afterwards, two authors (A.E.M. and G.O.) independently reviewed the titles and abstracts of the retrieved studies for eligibility. Studies were then selected based on the predetermined inclusion and exclusion criteria. For any disagreements concerning the inclusion of studies, all authors agreed on a consensus based on factual evidence.

Data Extraction
Data from each eligible study were extracted by A.E.M. and G.O. and checked by F.E. and D.S. The following information was obtained: Author name, year of publication, subject, organ (or tissue) of interest, radiation type and dose, hesperidin dose, as well as time for outcome assessment. Furthermore, the main outcomes were summarized and included.

Literature Search
The PRISMA flow diagram showing our search results is presented in Figure 2. Our initial search gave a total of 229 records, with the breakdown as follows: 225 records from electronic databases and 4 records obtained through a manual search. Our search of the online database of www.ClinicalTrials.gov showed that there were no completed or ongoing clinical trials evaluating the radioprotective effect of hesperidin. From these figures, 143 records were retained after removing duplicates. Following careful examination and screening of their titles and abstracts as well as the application of the inclusion and exclusion criteria, a further 114 records were excluded. The full texts of the remaining 29 records were assessed. We excluded two articles for non-English language publication, while three more records were removed for not having full texts. Finally, a total of 24 studies were included in this systematic review.

Study Characteristics
The summary of data showing the characteristics of included studies is presented in Table 1. These articles, published between 2006 and 2018, employed an experimental case-control design. Furthermore, they include 4 in vitro, 17 in vivo, 1 in vitro/in vivo, and 2 in vivo/in vitro studies using rats, mice, and cultured human and animal cells. Gamma (γ)-radiation was utilized in 21 studies (with 1 study making use of a γ-ray from Technetium sestamibi ( 99m Tc-MIBI) radiopharmaceuticals), and X-ray radiation was used in 3 studies. The doses of the radiation were between 1 and 18 Gy. Hesperidin was administered orally in 18 studies and intraperitoneally in 2 studies. Countered radiation-induced free radicals post-irradiation, decreased prolonged oxidative stress, and protected against radiation-induced DNA damage.
Hosseinimehr et al. [20] Cultured human blood lymphocytes Lymphocytes γ-ray, 1.5 250 mg/kg body weight 0-3 h Significant decrease in the incidence of micronuclei of blood lymphocytes collected 1 h after oral administration of hesperidin compared to those collected at 0 h. Maximum protection and decrease in frequency of micronuclei (33%) was observed at 1 h after ingestion of hesperidin.
Kalpana et al. [21] Cultured human lymphocytes Lymphocytes γ-ray, 1-4 3.27-19.65 µM 30 min Here, 16.38 µM hesperidin pretreatment prior to irradiation had the maximum radioprotective effect, which included a significant decrease in the levels of MN and DC counts, as well as TBARS. Reduction in tail length, tail moment, olive tail moment, and % DNA in the tail. Increased levels of enzymatic (SOD, CAT, and GPx) and non-enzymatic (glutathione (GSH)) antioxidants and restored DNA damage to near-normal levels.
Hosseinimehr et al. [22] Cultured human lymphocyte cells Lymphocytes  Hesperidin administration led to significant decrease in radiation-induced inflammation and inflammatory cells at 24 h post-irradiation. Furthermore, there was a reduction in radiation pneumonitis and radiation fibrosis in the lung tissue at 8 weeks post-irradiation.  Said et al. [41] Albino rats Brain γ-ray, 5 50 mg/kg body weight 14 days Significant reduction in oxidative stress, monoamine alterations, and mitochondrial damage, and hence a reduction in the severity of radiation-induced biochemical brain disorders.
Park et al. [42] Sprague-Dawley rats Heart and kidney γ-ray, 5 50 and 100 mg/kg body weight 7 days Treatment with hesperidin post-irradiation led to significant reduction in levels of lipid peroxidation, improvements in activities of endogenous antioxidants (SOD, CAT, GPx, and GSH), and minimal damage to the heart and kidney tissues.

Hesperidin Dosage
A hesperidin dose of 100 mg/kg body weight was mostly used in the included studies to assess its radioprotective effect. In addition, this dose has been shown to be the most effective in reducing the healing time of radiation-induced wounds by two days [24]. Results from another study by Haddadi et al. also showed that this same oral dose of hesperidin was effective in accelerating wound healing from radiation-induced skin damage [31].
Different effective hesperidin doses have also been reported in several studies. In a study by Hosseinimehr and Nemati, a hesperidin dose of 80 mg/kg showed a maximum reduction in the frequencies of micronucleated polychromatic erythrocytes (MnPCEs) [30]. In a later study by Hosseinimehr et al., they observed that maximum radioprotection was obtained 1 h after oral ingestion of 250 mg/kg hesperidin [20].
Kalpana et al. detected maximum protection against radiation-induced reproductive death for a hesperidin concentration in a cell medium of 16.38 µM (9.99 mg/L) [21]. In another study, 25 mg/kg hesperidin was the most effective dose, restoring antioxidant status to normal levels as well as decreasing lipid peroxidation and preventing DNA damage [28]. Furthermore, Shaban et al. showed that the best radioprotection by hesperidin was obtained when administered before irradiation [33].

Toxicity and Survival Analysis
In all included studies, there was no reported case of toxicity or side effects following the administration of hesperidin. In an in vivo/in vitro study by Hosseinimehr et al., five human subjects each received a hesperidin dose of 250 mg/kg orally before collection of their blood samples. They showed no adverse signs 0 (before), 1, 2, and 3 h post-hesperidin ingestion [20].
In a study by Rezaeyan et al., rats treated with 100 mg/kg hesperidin before exposure to an 18-Gy single-dose X-ray showed significant improvement in survival rates compared to those in the radiation group (with a median survival period of 55 days). A 60-day follow-up indicated that five rats survived in the radiation group, while eight survived in the hesperidin-pretreated group [38].
Hesperidin also improved survival in a study investigating its protective effect against radiation-induced lung damage. Rats were administered 100 mg/kg hesperidin orally for seven consecutive days before exposure to 18-Gy γ-rays. After a 60-day period of observation, 4 out of 10 rats in the radiation group survived, while 7 out of 10 rats survived in the hesperidin + radiation group [32].
In evaluating the radioprotective effect of hesperidin against radiation-induced hepatic damage, Kalpana et al. showed that mice pretreatment with 25 mg/kg hesperidin before exposure to 10-Gy γ-rays increased the median survival period to 15 days compared to the nontreated groups (6 days) exposed to the same radiation dose. Daily monitoring of these rats was done for 30 days [28].

Discussion
Exposure to IR can affect cellular components of living tissues, leading to early and late side effects. The severity of IR-induced complications on normal tissues varies with radiation dose as well as cell or organ type [43]. Early effects such as apoptosis, lymphocyte adhesion and infiltration, vascular permeability, increased endothelial cell swelling, and edema occur within hours after radiation exposure [44]. Late effects including necrosis, organ dysfunction, death, cancer, etc., occur months to years following exposure [45]. These radiation effects pose serious concerns to humans, especially to children, who are more radiosensitive [46]. If adequate protective measures are not put in place, they may also negatively impact the quality of life of patients exposed to either diagnostic or therapeutic doses of IR.
One of the strategies for countering radiation-induced damage is the use of radioprotectors. The efficacy of natural radioprotective agents such as hesperidin has been explored in recent times. Thus, in the present study, using a systematic review design, we searched for studies that made use of hesperidin as a radioprotective agent against IR-induced damage.
Results from the included studies showed that hesperidin demonstrated a protective effect against both gamma and X-rays in the healthy tissue of mice, rats, and cultured human and animal cells (Table 1). These effects were observed in hesperidin doses as low as 10 mg/kg administered orally. Furthermore, hesperidin treatment reduced biochemical markers of oxidative stress and improved histopathological outcomes of exposed tissues [27]. Moreover, its antiapoptotic activities were demonstrated in mouse testes exposed to IR [33].
Skin, the body's largest organ, is inevitably exposed to IR during radiotherapy. It has been observed that bone marrow and skin epithelia have high susceptibility to IR-induced side effects [47]. It has been estimated that 90-95% of patients who receive radiotherapy show varying grades of radiation-induced skin reactions [48,49]. Skin complications arising after radiotherapy are referred to as radiodermatitis. Radiodermatitis is a common side effect after radiotherapy for breast cancer. Furthermore, acute radiodermatitis, with indications including scaling, edema erythema, erosion, and ulcers, could arise 90 days after radiotherapy. Chronic radiodermatitis can be observed a few months after radiotherapy, with symptoms such as changes in skin texture, hypopigmentation or hyperpigmentation, teleangiectasis, and poikiloderma. Hesperidin has been shown to prevent radiation-induced skin burns via initiating the formation of new vessels and a microvascular network through vascular endothelial growth factor (VEGF) gene induction [31].
Cardiovascular diseases are the leading causes of mortality worldwide [50]. It has been projected that by the year 2030, cardiovascular diseases will account for an annual mortality of more than 23.3 million people [51]. The risk of radiation-induced heart diseases increases with IR doses to heart tissues after radiotherapy for lung, breast, or esophageal cancers (due to the close proximities of these organs to the heart), as well as in nuclear disasters. The functionalities of coronary vessels, valves, the pericardium, and the myocardium are adversely affected after irradiation [52]. Our investigation of the included studies showed potent radioprotective and anti-inflammatory effects of hesperidin on heart tissues. The administration of hesperidin before irradiating the thorax with a high dose of gamma radiation showed an improvement in survival as well as a reduction in oxidative damage, vascular leakage, inflammation, the fibrosis and infiltration of macrophages, lymphocytes, and mast cells [38]. It was also effective in ameliorating serum heart disease markers [42] and preventing cellular damage to the heart [40]. Similar outcomes were obtained for lung tissues [32,35,36].
Promising radioprotective effects have been observed for some natural antioxidants, such as melatonin, selenium, Coenzyme Q10, α-tocopherol, caffeic acid, and ascorbic acid. [13]. Various clinical trials have also confirmed the efficacy of some of these natural products [53,54]. Since experimental findings have shown encouraging radioprotective results for hesperidin, it would be interesting to see how it competes with other radioprotectors.
The present review has some limitations. First, we reviewed studies that used animals as well as cultured human and animal cells due to the nonavailability of clinical trials. Furthermore, in the studies included, the radioprotective effect of hesperidin was only investigated in healthy tissues. It would be desirable to observe these effects in tumor cells.

Conclusions
In the results of the included studies in this review, it was shown that hesperidin has the potential to be an effective radioprotector against IR-induced damage to cellular components of healthy tissues. It showed promising antioxidant, anti-inflammatory, and as antiapoptotic abilities. There were no reported side effects due to its administration. We suggest future clinical trials to further assess its efficacy as well as its optimal dose. This is necessary in order to assess the clinical effects of experimental melatonin doses as well as for more insight into possible variations between experimental outcomes using cells or animals and those in humans. Comparative studies with other radioprotectors will be required in order to investigate its effectiveness. The effects of hesperidin treatment on cancer cells exposed to ionizing radiation should also be investigated. Lastly, more studies of the molecular mechanisms of radioprotection by hesperidin would be desirable.

Conflicts of Interest:
The authors declare no conflicts of interest.