Antioxidant and Anti-inammatory Activities Mediate the Radioprotective Effect of Trianthema Portulacastrum L. Extracts

Ionizing radiation (IR) generates reactive oxygen species (ROS) which leads to oxidative stress and often leads to inammatory responses in organisms. Trianthema portulacastrum L., a plant commonly growing in India, is rich in antioxidant phytochemicals which is responsible for scavenging free radicals, and may provide radio-protective and anti-inammatory effects in response to ionizing radiation. The effect of T. portulacastrum extracts was studied in hepatic cells, which are susceptible to radiation-induced damage, and in macrophages which are the primary inammatory cells of the body. T. portulacastrum stem extracts showed ecient free radical scavenging activity in hepatocytes and reduction of radiation-induced lipid peroxidation in cell and mitochondrial membranes. Treatment of irradiated cells with T. portulacastrum stem extracts enhanced cell viability, although at higher concentrations there was reduction in cell viability. Treatment with low concentration of T. portulacastrum stem extract also reduced cellular ROS generation and increased cellular concentration of the anti-oxidant glutathione. T. portulacastrum extracts also showed a marked anti-inammatory effect in macrophages activated by the inammatory agonist bacterial lipopolysaccharide (LPS) by reducing inammatory gene expression and nitric oxide (NO) production, and increasing glutathione content. LPS treatment lowered expression of Nrf2, a transcription factor involved in regulation of multiple anti-oxidant genes, while treatment with low concentration of T. portulacastrum stem extract signicantly restored it. Together, these observations demonstrated a potential radioprotective role of T. portulacastrum extract mediated by both its antioxidant activity on hepatic epithelial cells and its anti-inammatory activity on immune cells


Introduction
Exposure to ionizing radiation (IR) causes cellular damage either by direct impairment of biomolecules or indirectly by generation of free radicals. Water radiolysis leads to generation of reactive oxygen species (ROS) which causes lipid peroxidation in membrane, DNA strand breaks and oxidation of cellular protein (Wang et al. 2018). Radiation not only affects the irradiated cells but also non-irradiated cells and tissues undesirably due to bystander effect through the activation of in ammatory responses (Shemetun and Pilins'ka 2007). IR sensitizes lymphocytes, macrophages, monocytes and other immune cell (Carvalho and Villar 2018). Radiation exposure shows immune-modulatory properties through the production of reactive oxygen and nitrogen species (RONS), and release of in ammatory cytokines such as tumor necrosis factor-alpha (TNF-α), tumor growth factor-beta (TGF-β), interleukins, nucleoside, high mobility group box-1 molecule (HMGB1) and heat shock proteins (HSPs) (Carvalho and Villar 2018). As ionizing radiation is a mainstay of therapy in many cancers, methods of protection against radiation-induced damage are an important requisite for cancer radiotherapy.
Radioprotectors may be naturally occurring antioxidants that can protect normal cells and tissues from radiation-induced damage. Sulfhydryl agents such as glutathione, cysteine, cystamine and other antioxidants have shown protective activity against the lethal effect of radiation and also increased the survival rate of irradiated mice (Obrador et al. 2020). Sulfhydryl group containing erdosteine protect rats against gamma radiation through antioxidants and anti-in ammatory properties (Elkady and Ibrahim 2016). Sulfhydryl compounds protect cellular DNA through a combination of free radical scavenging, modulation of repair process, and hydrogen donation ability (Kumar et al. 2002). and Cu 2+ to Fe 2+ and Cu + respectively and inducing Fenton reaction (Maurya and Devasagayam 2010). Therefore, further exploration of naturally occurring compounds in plants with antioxidant properties is warranted.
Trianthema porulacastrum L., a well-known medicinal plant from the family of Aizoaceae, is a natural source of antioxidant and phytochemicals and has been used for treatment of numerous disease conditions in Indian and African traditional medicine (Shivhare et al. 2012;Das et al. 2020). T. portulacastrum is also well-known for its hepatoprotective activity against chemical-induced toxicity such as carbon tetrachloride (CCl 4 ) (Sarkar et al. 1999), paracetamol and thioacetamide (Kumar et al. 2004).
The hepatoprotective activity of T. portulacastrum was marked by enhancement of antioxidant enzymes, suggesting that the protection of liver cells from oxidative damage may be a mode of hepatoprotection by T. portulacastrum extract. However, no studies have been performed on the anti-in ammatory role of T. portulacastrum, although in ammatory responses are known to be a major contributor to hepatic damage. Therefore, the objective of this study was to evaluate the radioprotective activity of T. portulacastrum in hepatocytes and its anti-in ammatory effects using murine macrophages. Preparation of TP extracts: Dried powder of different parts of T. portulacastrum such as leaves, stem and whole plant (100 g) was extracted with 500 ml petroleum ether for 24 h with constant shaking and ltered. This process was repeated twice. Ethyl acetate, acetone and ethanol solvent were used twice sequentially followed by petroleum ether. All the solvents were evaporated and dried. Further studies were carried out with ethanolic fractions.

Antioxidant capacity study
Antioxidant capacity of the different extracts of the TP was measured using 2,2'-azino-bis ( Evaluation of radioprotective property of T. portulacastrum extract Radioprotective property of T. portulacastrum extract was evaluated using sub-cellular and cellular model systems. For sub-cellular assay, we have used murine mitochondrial membrane whereas for cellular assay we have employed human hepatic cells (WRL 68) as a model system.

Evaluation IR-induced lipid peroxidation
For evaluation of lipid peroxidation mouse mitochondrial membrane and human hepatic cells were used.
Mitochondrial membrane fractions were isolated from the liver of male Swiss mice as described (Checker et al. 2010). Damage to the mitochondrial membrane fraction after radiation exposure was assessed in terms of lipid peroxidation (Maurya and Devasagayam 2010). Mitochondrial membrane fraction (a protein equivalent of 300 µg) was suspended in 300 µl of 10 mM potassium phosphate buffer, pH 7.4, and exposed to 50 Gy radiations in the absence and presence of different concentrations of TP extracts (pre-treated for 30 min at 37°C). After treatment, 900 µl TBA reagent (0.375% TBA, 0.25 M HCl, 15% trichloroacetic acid (TCA) and 6 mM Na 2 -EDTA) was added. The reaction mixture was incubated at 95 0 C for 20 min, cooled to ambient temperature and centrifuged at 12,000 g for 5 min at 25 0 C.
Malondialdehyde (MDA) equivalents in the supernatant were estimated by measuring the uorescence (as uorescence provide more sensitivity) with excitation at 530 nm and emission at 590 nm using a microplate reader.
WRL 68 (2x10 6 ) cells were collected and treated with different T. portulacastrum extracts for 1 h at 37 0 C for IR-induced cellular lipid peroxidation study inhuman hepatic cells. Subsequently cells were exposed to 8 Gy of IR. IR-induced cell membrane damage was assessed in terms of lipid peroxidation as described above [(Maurya and Devasagayam 2010).

MTT assay
To study the cytotoxicity and proliferation of cells after IR exposure, MTT assay (3-[4,5-dimethylthiazol-2yl]-2,5-difenyl-tetrazolium bromide) was used. In brief, 15×10 3 WRL 68 cells were seeded in 96-well plate one day prior to addition of extract. Next day cells were treated with different concentration of the extracts and incubated for 1 h. These extract treated cells were exposed to 4 Gy of IR. After completion of incubation period, MTT assay was performed by adding 10 µl MTT solution (10 mg/ml) to each well. Formazan crystals formed inside the cells were dissolved by adding 100 µl of solubilizing buffer (0.01 N HCl in 10% SDS) and incubated for overnight at 37°C. The absorbance was measured at 570 nm using Synergy Bio-Tek (USA) microplate reader (Maurya et al. 2011).

Clonogenic assay
The clonogenic assay was used to evaluate the radioprotective e ciency of TP extract using WRL 68 cells. In brief, 2x10 3 exponentially growing cells were plated in a 6-well plate for overnight. Next day, cells were treated with different concentration of T. portulacastrum extracts 1h before 4 Gy IR-exposure. After irradiation, cells were cultured for 12 days at 37°C in CO 2 incubator for the development of macroscopic colonies. The colonies were xed with methanol, stained with 0.5% crystal violet and counted using a colony counter (Oxford Optronix, UK) (Jayakumar et al. 2015).

Evaluation Of The Cellular Redox Status
To study the mechanism of the T. portulacastrum extracts for radioprotection, cellular redox study was carried out. For this cellular ROS and intracellular thiol (GSH) levels were monitored using H 2 DCFDA and monochlorobimane (MCB) uorescence dye respectively.

Evaluation Of Anti-in ammatory Property Of Tp Extract
The anti-in ammatory effects of T. portulacastrum extract in LPS-stimulated RAW 264.7 macrophages were evaluated by nitric oxide (NO) assays and quantitative real-time reverse transcription-polymerase chain reaction analysis of expression of in ammatory genes. For studying the cytotoxicity in RAW 264.7 activated with LPS and treated with the T. portulacastrum extract, MTT assay was performed as described previously. GSH concentration was measured through Ellman's reagent and calculated from standard curve using pure GSH (Moron et al. 1979).
Nitric oxide (NO) assay RAW 264.7 cells (0.5×10 5 cells/well) were seeded into 96 well plates for 24 h. Next day, cells were preincubated with different concentrations of T. portulacastrum extracts (0, 31.25, 62.5, 125 µg/ml) for 1 h and further stimulated with 500 ng/ml of LPS. The culture supernatants were collected 24 h after the LPS stimulation, and the concentrations of NO were measured using Griess reagent. 100 µl of culture supernatant was mixed with 100 µl of Griess reagent (sulfanilamide 1%, 2% phosphoric acid and 0.1% NEDD in water) and the mixture was incubated at room temperature for 10 min before measuring the absorbance at 550 nm. In all experiments, fresh culture medium was used as the blank and sodium nitrite was used as the standard (Kacem et al. 2015).
Semi-quantitative PCR: For semi-quantitative PCR, RAW 264.7 cells were pretreated with T. portulacastrum extract for 1 h followed by LPS treatment. Total cellular RNA was extracted using Trizol following manufacturer's protocol. cDNA was synthesized from RNA using oligo-(dT) primer by M-MLV reverse transcriptase (Thermo Fisher, 28025-013). Speci c primers for TNF-α, Nrf-2 and iNOS were used for PCR reactions and then run on 1.5% Agarose gel. GAPDH primers were used for normalization of mRNA quantity respectively (Ahuja et al. 2016). The following primers were used for semi-quantitative

Results
T. portulacastrum extracts show antioxidant activities by free radical scavenging and transient metal reduction The major form of oxidants in cells are oxidative free radicals such as hydroxyl and peroxide and transition metal ions such as Fe 2+ and Cu + , both of which oxidize a variety of biomolecules and cellular components and cause oxidative damage. Therefore, we investigated the free radical scavenging and transition metal ion reducing activities of T. portulacastrum extracts. Extracts of leaf, stem and whole plant of T. portulacastrum exhibited concentration-dependent scavenging activity in model free radicals such as DPPH and ABTS radicals (ABTS •+ ), in free radical scavenging assays (Fig. 1). The half inhibition concentration [IC 50 ] of stem extract (SE) was found to be the lowest (245.04 and 290.79 in the ABTS and DPPH assays respectively) in free radical scavenging activity (Table 1). We also determined the transition metal ion reducing activity of these extracts by FRPA and MRA assays based on reduction of iron and molybdenum ions respectively. All the T. portulacastrum extracts showed reducing capacity in a dosedependent manner, with the highest activity being shown by leaf extracts (LE) (Fig. 1). Together, these observations showed signi cant antioxidant capacity of T. portulacastrum, with different parts of the plant contributing to the same. Further assays were therefore carried out with stem extracts (SE). T. portulacastrum stem extracts protect against lipid peroxidation and cell death consequent to radiation exposure Radiation exposure is known to damage the structure of cell membranes through degradation of lipids, mainly mediated via lipid peroxidation (LPO) of WRL68 hepatocyte cell and mouse mitochondrial membranes were observed after exposure to 8 Gy and 50 Gy radiation respectively which increased the formation of malondialdehyde (MDA). In both cases, addition of T. portulacastrum stem extracts (SE) mitigated the radiation-induced lipid peroxidation in a dose-dependent manner, showing the protective activity against radiation-induced membrane lipid peroxidation (Fig. 2).
For further evaluation of radioprotective activity of the extract, cell viability of irradiated cells, untreated or treated with T. portulacastrum stem extracts, was estimated using MTT and clonogenic assays. Exposure to 12 Gy radiation signi cantly reduced cell viability after 72 h and cells treated with T. portulacastrum extract showed enhanced cell viability compared to irradiated cells in the MTT assay (Fig. 3a). However, only 31.25 µg/ml concentration of the extract demonstrated signi cant enhancement of cell viability and increased concentrations of the extracts did not show signi cant difference in cell viability in comparison to the irradiated cells.
In the clonogenic assay, exposure to 4 Gy radiation resulted in a 66% reduction in colony-forming ability of the cells (Fig. 3b). Treatment with high concentrations (to125 µg/ml) of T. portulacastrum extracts alone did not show any reduction in colony formation (Fig. 3c). However, on addition of T. portulacastrum stem extracts to irradiated cells, signi cant rescue in colony formation was only observed in cells treated with 31.25 µg/ml extract (Fig. 3d). Together these observations suggest a radioprotective activity of T. portulacastrum stem extract, although at a higher concentration the level of protection may decrease due to the presence of compounds with non-speci c toxic effect on radiation-induce damaged cells.

T. portulacastrum stem extracts reduces cellular ROS and enhances GSH
To explore the mechanism of radioprotection by T. portulacastrum stem extract, we evaluated the cellular redox status by measuring reactive oxygen species (ROS) and reduced Glutathione (GSH) which are important markers of the redox status of cells. Determination of cellular ROS level by DCFDA uorescence intensity showed that 4 Gy of radiation increased the cellular ROS level by nearly 2-fold, whereas, pretreatment with the T. portulacastrum stem extract reduced the ROS level in a dose-dependent manner (Fig. 4a). Similarly, exposure to 4 Gy of radiation signi cantly reduced the concentration of GSH, one of the most important cellular antioxidant molecules, whereas treatment with the lowest concentration of T. portulacastrum stem extract signi cantly upregulated the GSH content (Fig. 4b).
Higher concentrations of T. portulacastrum extract failed to signi cantly enhance the GSH content, most likely by inhibition of GSH biosynthesis by high concentration of some non-speci c molecules. Thus, these observations demonstrated that the observed radioprotection by T. portulacastrum extract is due to modi cation of cellular redox status.

T. portulacastrum stem extract exerts anti-in ammatory effects on LPS-activated macrophages
Another arm of the radiation induced tissue damage is mediated by secondary in ammatory responses, caused by the activation of in ammatory cells such as neutrophils and macrophages. Therefore, we also investigated the effect of T. portulacastrum stem extract in mitigating the in ammatory response by evaluating its effect on mouse macrophage cells (RAW264.7) activated by the in ammatory agonist LPS. RAW264.7 cells were stimulated with 500 ng/ml LPS with and without 1 h pretreatment with T. portulacastrum extracts. T. portulacastrum stem extract did not show any autonomous cytotoxicity in RAW264.7 cells at the highest concentration (125 µg/ml). However, MTT assays showed that treatment with TP stem extract reduced the LPS-stimulated proliferation of RAW 264.7 cells in a dose-dependent manner (Fig. 5a).
One of the major in ammatory mediators released by macrophages after LPS stimulation is nitric oxide (NO) generated by upregulation of inducible nitric oxide synthase (iNOS). We therefore determined NO generation by LPS-stimulated RAW264.7 cells in presence and absence of treatment with T. portulacastrum stem extract. LPS stimulation increased the secreted NO level by 4.6 fold while treatment with T. portulacastrum stem extract dose-dependently reduced the secreted NO level (Fig. 5b). LPS induced activation of macrophages not only elevates NO level but also downregulated GSH concentration. Treatment with T. portulacastrum stem extract also enhanced GSH level in LPS-treated RAW264.7 cells, but the highest enhancement was observed at the lowest concentration of T. portulacastrum stem extract as in the case of hepatocytes (Fig. 5c).
We then investigated the expression of some of the genes responsible for the synthesis of the major in ammatory mediators in LPS stimulated macrophages in presence and absence of treatment with T. portulacastrum extract. LPS stimulation increased the mRNA level of iNOS, the enzyme responsible for NO synthesis, by 1.5 fold while treatment with T. portulacastrum stem extract brought down the iNOS mRNA to basal level of expression (Fig. 6a). iNOS expression is mediated through activation of TNF-α, a pro-in ammatory cytokine. TNF-α mRNA level was increased signi cantly after 24 h of LPS treatment and while treatment with T. portulacastrum stem extract also signi cantly decreased the TNF-α level in a dose-dependent manner (Fig. 6b).
We also checked the level of Nrf2 mRNA as Nrf2 is a well-known transcription factor which regulates a number of antioxidant genes in cells, including GSH, and is involved in protection against oxidative stress and in ammation. Treatment of the cells with LPS lowers the Nrf2 mRNA level while treatment with T. portulacastrum stem extract signi cantly restored it although the mRNA level reduced with increasing concentration of the T. portulacastrum extract (Fig. 6c), re ecting the effect on GSH level as observed before.
Together, these observations demonstrate a potential radioprotective role of T. portulacastrum extract mediated by both its antioxidant activity on epithelial cells and its anti-in ammatory activity on immune cells. to exhibit hepatoprotective activity against chemical-induced toxicity (Yamaki et al. 2016). Therefore, in this study we have demonstrated the radioprotective role of TP extract on hepatocytes, mediated via its activity of reducing oxidative damage to cells. Moreover, we have demonstrated an anti-in ammatory role of T. portulacastrum extract in the case of activated macrophages, which is likely to enhance its radioprotective function by mitigating the in ammatory response induced by radiation damage.

Discussion
A major mode of antioxidant function is via scavenging of oxidative free radicals. Radical scavenging capacity of an antioxidant lies on its proton donating or accepting ability (Singha et al. 2020). T.
portulacastrum extracts showed e cient free radical scavenging activity in both DPPH and ABTS •+ assays which are based on electron transfer ability used to measure antioxidant capacity. Moreover, T. portulacastrum extracts showed e cient reducing ability to reduce Mo (VI) to Mo (V) and Fe (III) to Fe (II) which is a marker of its antioxidant activity.
Exposure to radiation causes damage to different biomolecules such as lipids, proteins and nucleic acids. Cellular membranes are one of the major targets of the oxidative free radicals, generated due to radiation exposure. Damage of the lipids present in the cell membranes changes its uidity status and also activates several critical signaling pathways (Nicolson and Ash 2014). ROS is responsible for thiol oxidation which initiates lipid peroxidation. Radiation induced free radicals react with macromolecules and damage membranes of intracellular organelles (Singha and Das 2015). It has been reported that 50 Gy of gamma radiation impairs mitochondrial membrane function by damaging complex I (NADH dehydrogenase) and III (cytochrome c reductase) (Pearce et al. 2001). In our study T. portulacastrum stem extract showed e cient protection against lipid peroxidation by ionizing radiation in a MDA formation assay, further substantiating its role in radioprotection.
The nal effect to check in case of radioprotection is the reduction of cell death in response to radiation exposure. Treatment with T. portulacastrum extract showed a survival advantage of irradiated cells in both short term (MTT) and long term (colony formation) cell viability assays. As it has been reported that lower radiation dose gives false-positive result in MTT as high formazan is deposited in irradiated cells compared to control (Rai et al. 2018), the clonogenic assay is a good measure to study the ability of T. portulacastrum extract to protect against radiation-induced cell damage. However, interestingly, higher concentration of T. portulacastrum extract failed to show this survival advantage and whether this is due to a pro-oxidant activity demonstrated at a higher concentration as reported for other natural products  (Mun et al. 2018). Changes in the cellular oxidative stress level play a pivotal role in in ammation (Han et al. 2019). However, the effect of natural products on radiation-induced in ammation has not been explored su ciently. Therefore, we investigated the effect of TP extract on the in ammatory response in LPS-treated macrophages, one of the major immune cells involved in radiation-induced in ammation. Treatment with T. portulacastrum extract was able to reduce the secretion of NO from these cells, as well as reduce the expression, of inducible nitric oxide synthase (iNOS) gene, which are important mediators of the in ammatory response (Cao et al. 2019). Excess NO induces in ammation and nitrosative stress (Calabrese et al. 2004). These results showed that T. portulacastrum extract may protect from radiation damage by modulating the in ammatory response in the body. ). LPS stimulation of macrophages decreased the Nrf2 mRNA level as well as the concentration of GSH but treatment with T. portulacastrum extract markedly upregulated the mRNA expression of Nrf2 and GSH concentration, suggesting a molecular mechanism for upregulation of GSH in cells treated with TP extract. Interestingly, the same effect of higher dose of T. portulacastrum extract failing to enhance Nrf2 expression and GSH concentration was noted, re ecting the similarity with radiation induced GSH concentration, and cell viability in hepatocytes. This warrants further investigation into the concentration dependent effect of T. portulacastrum extract, and its bioactive molecules, on regulation of expression of anti-oxidant genes in cells.

Conclusion
This study for the rst time shows a radioprotective activity of extracts from the plant Trianthema portulacastrum, mediated via its dual effect in modifying the redox status of irradiated cells and the in ammatory response of immune cells activated by the in ammatory agonist LPS. Both these effects together may strongly support the role of T. portulacastrum extract as a natural product with signi cant radioprotective ability.