Gamma rays are one of the ionizing radiations, having much more penetrating capacity, mitigating malignant tumors by cell killing mechanism during radiation therapy [24]. Their usage in radiation therapy is crucial as they offer a quality of life for cancer patients. In addition, they are commonly used for the irradiation of foods to improve their quality and extend shelf life. Also, gamma-ray irradiation is commonly applied in sterilizing medical products due to its simple sterilization without heat generation [25]. A COVID-19 rapid letter stated by Venkatraman et al. [26] proposed an idea of how gamma rays are used to breakthrough COVID-19 to manage its outbreak. Despite many applications, gamma rays induce adverse effects via the generation of ROS. Due to their instability, ROS are associated with the principle of oxidative stress that induces damage to cellular components like DNA, lipids, and proteins [27]. Biological effects of ionizing radiation vary based on nature and timing after radiation exposure through direct and indirect mechanisms. Hence, it is crucial to provide awareness to the people dealing with gamma radiation about the radiation risks and a comparison of natural sources to medical procedures. The need for radioprotection is therefore paramount and resulted in the identification of various synthetic compounds, antioxidants, phytocompounds, and vitamins, which are being administered exogenously over decades. Howbeit, to date, there is no potential and safe compound that mitigates gamma radiation-induced cellular injuries. Therefore, there is an advantage in the usage of natural compounds bearing negligible side effects. A review by Mun et al. [28] evaluated the radioprotective efficacy of several plant-derived compounds extensively based on previous scientific literature. Our research group has been studying intensively on the heartwood of Pterocarpus santalinus as the tree is growing most frequently in uranium-rich regions of the Seshachalam hill ranges of the Eastern Ghats in the state of Andhra Pradesh, situated in the southeastern coastal region of India. From the previous literature, we knew that a compound with antioxidant, immunomodulatory, and free radical scavenging activities may act as a potent radioprotector and is the right choice for the researchers to evaluate its radioprotective property [10]. Hence, the current study has designed in the same line and tried to evaluate the radioprotective efficacy of PSAE.
UHPLC-ESI-HRMS/MS studies identified eleven compounds, of which most of them belong to polyphenols. Exposure to ionizing radiation elevates ROS levels and, to combat it, cells have an inbuilt antioxidant system. A previous review stated that antioxidants are efficiently used to prevent oxygen poising and radiation injury caused by ROS [29]. Polyphenols, the main compounds present in plants with good antioxidant properties, facilitate the donation or acceptance of electrons due to the presence of aromatic rings and a hydroxyl group. The total phenol content varies from plant to plant depending on the plant species, developmental stage, environmental factors, and the plant tissue [30, 31]. The findings in our previous and present studies have demonstrated that on changing the solvent system employed in the preparation of extract, there is a variation in estimated amounts of phytoconstituents. Our previous study used chloroform as a solvent system and showed that Pterocarpus santalinus Chloroform Extract (PSCE) possesses phenols (404µg/mg Gallic acid equivalents), flavonoids (22.6 µg/mg Quercetin equivalents), anthocyanins (0.066 mg Cyn3-glu/g), and tannins (12.477 g/L) [32]. In line with this report, the present study showed that PSAE contains phenols (4.061µg/mg Gallic acid equivalents), flavonoids (6.616 µg/mg Quercetin equivalents), and anthocyanins (0.008 mg Cyn3-glu/g). Furthermore, PSAE significantly reduced molybdenum (VI) to molybdenum (V) and Fe (III) to Fe (II) in a concentration-dependent manner. In congruence with an earlier report by Gupta et al. [33], this finding indicated that the polyphenols from the extract of heartwood of Pterocarpus marsupium showed an increased total antioxidant capacity in a concentration-dependent manner. Chang et al. [34] also showed that the antioxidant property of phenolics of heartwood extract of Acacia confusa is effective.
Radioactive source, Co-60, emits gamma rays and plays a prominent role in medical, commercial, and research facilities for sterilization purposes. However, it is well known that radiation-induced oxidative stress generates ROS and disturbs redox homeostasis, which is considered as one of the primary causes of chronic diseases such as chronic inflammation atherosclerosis, myocardial infarction, diabetes, rheumatoid arthritis, stroke, cancer, and other degenerative diseases in humans [35, 36]. GSH is known to be involved in the natural detoxification of gamma radiation-induced radiolytic products. It donates hydrogen to highly reactive ROS and plays a key role in their conversion to more stable compounds [37]. PSAE supported spleen lymphocytes by reducing radiation-induced intracellular ROS levels by restoring total thiols content and GSH/ GSSG ratio. This study is in consonance with an earlier report of Chang et al. [34] on heartwood extract of A. confusa, stating the reduced levels of intracellular ROS in HL-60 cells. Gamma radiation-induced excessive ROS levels can cause an elevation in lipid peroxidation levels; thus, the function and structure of biomolecules alter, leading to tissue damage [38]. PSAE (10 µg/ml, 1h) treated membrane samples showed a downfall in TBARS levels by 70% and 43% in sub-cellular and spleen lymphocytes compared to that in the 50 Gy and 20 Gy alone groups, respectively. In line with this report, a study by Patil et al. [39] reported that TBARS levels were significantly diminished with an increase in the aqueous extract concentration than that of methanolic extract. Our studies are also in accordance with the results reported by Mansour and Hafez [40]. Several conflicting results of US-FDA- approved radioprotector, amifostine, lead to a need to identify an effective radioprotector [41]. In this view, herbal extracts hold promising effects due to their less toxicity, easy accessibility, acceptability, and economical [7]. In the present study, PSAE has shown non-toxic nature towards spleen lymphocytes and RAW264.7 macrophage cell lines. It was non-toxic until 100 µg/ml and started slight toxicity from 200 µg/ml.
Ionizing radiation directly causes DNA structure effects, mainly by inducing DNA DSBs [42], which is the most perilous state of DNA induced by radiation exposure due to erroneous DNA repair or loss of genetic information [43]. Excessive production of radiation-induced intracellular ROS can also cause tissue injury through DNA DSBs [44]. Recent studies reported an increase in DNA DSBs after exposure to gamma radiation [45, 46]. In the present study, we identified that PSAE protected plasmid DNA from strand breaks and restored the original DNA confirmation, i.e., SC form. In a previous study, it was reported that pretreatment with green tea Catechin, EGCG, reduced DNA strand breaks on exposure to gamma radiation; thus, there is an increase in the S.C. form of DNA [47]. Meanwhile, PSAE treated cells (50 µg/ml, 1h) have been shown a significant reduction in % tail DNA, tail length, tail moment, and olive tail moment by 52%, 30%, 82%, and 74%, respectively. Irradiated cells pretreated with PSAE showed 83% protection of spleen lymphocytes from gamma radiation-induced cell death and a decrease in ladder formation, as evident from DNA fragmentation assay and PI staining. A study by Mansour and Hafez [48] showed that pretreatment with Withania somnifera before gamma irradiation significantly reduced DNA fragmentation. A previous in vitro study demonstrated the protective role of extract of Mesua ferrea on gamma radiation-induced cell death in spleen lymphocytes [49]. Since exposure to radiation causes tissue injuries and induces inflammation, there is a need to study its immunomodulatory property. In response to T cell mitogen, Con A, spleen lymphocytes showed significant proliferation. However, PSAE significantly inhibited the Con A (10 µg/ml)-induced proliferation of lymphocytes by 73%, as evident from CFSE dye dilution and CD69 antibody staining. It is mainly due to the augmentation of protection of spleen lymphocytes from cell death, as observed in our current study with PI staining. A study by Checker et al. [22] demonstrated the immunomodulatory and radioprotective effects of lignans and is in line with our present findings. This finding has also shown in accordance with the report of Murthuza et al. [49] that on treatment with the extracts of Mesua ferrea, Con A-induced T-cell proliferation was inhibited significantly in a dose-dependent manner. A study by Zimmermann-Klemd et al. [50] has also demonstrated that treatment of lymphocytes with Artemisia argyi extract significantly lowered CD69 expressions.