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. 2021 Mar 8;37(2):192–198. doi: 10.1007/s12291-021-00964-3

Antioxidant Properties of Trianthema Portulacastrum and Protection Against Ionizing Radiation-Induced Liver Damage Ex vivo

Uttam Das 1, Tanmay Saha 1, Subir Kumar Das 1,✉
PMCID: PMC8993979  PMID: 35463107

Abstract

Antioxidants in fruits and vegetables protect cells against radiation induced damage. Trianthema portulacastrum is used as vegetables from ancient time. The effects of T. Portulacastrum ethanolic extracts against γ-radiation induced liver tissue damage ex vivo were evaluated in this study. Antioxidant phytochemicals present in T. Portulacastrum includes flavonoids [3.3 ± 0.15 to 10 ± 0.16 mg catethin equivalent (CE)/g fresh weight (fw)], ascorbic acid (0.15 ± 0.03 to 0.21 ± 0.03 mg/g fw), glutathione s-transferase (GST) (1.57 ± 0.06 to 3.59 ± 0.05 nmole/mg fw/min), superoxide dismutase (SOD) (1.6 ± 0.03 to 1.79 ± 0.04 U/min), peroxidase (3.26 ± 0.18 to 6.38 ± 0.03 U/g fw) and catalase (0.51 ± 0.03 to 2.84 ± 0.15 mg H2O2 decomposed/g fw/min). Total phenolic content varied from 122.9 ± 8.7 to 302.8 ± 15.7 mg gallic acid equivalent/g extract, and flavonoid content varied from 316.7 ± 33.3 to 800.7 ± 28.9 CE mg/g extract. The IC50 value of Nitric oxide (NO•) scavenging activity of extracts varies from 208.7 to 387.4 µg/ ml. Pre-treatment with the T. portulacastrum extracts mitigated the 4-Gy gamma(γ) radiation-induced oxidative stress related parameters in hepatic tissue such as TBARS, catalase, nitrite, Glutathione reductase (GR), SOD and GST in dose dependent manner. The ethanolic extract of the stem from T. Portulacastrum demonstrated highest protection in comparison to leaf and whole plant extracts. This study demonstrated the hepatoprotective efficacy of T. portulacastrum extracts against γ-radiation in ex-vivo condition was possibly due to its potential antioxidant properties of phenolic and flavonoids present in extracts.

Keywords: Antioxidant, Flavonoids, Hepato-protective, Liver, Phenolics, Scavenging activity, Trianthema portulacastrum, Γ-radiation

Introduction

Ionizing radiation (IR) has been described as a double-edged sword and is used for both diagnostic and therapeutic purposes [1]. Exposure to IR is also responsible for undesired effects towards normal tissues. It is not only responsible for generation of mutagens and carcinogens but also cause significant damage to the normal tissues due to bystander effects [2]. This triggers cells signalling that leads to either mutation or cell death [3]. IR is responsible for the formation of free radicals mainly by water hydrolysis and resultant hydroxyl radicals (•OH) act as strong oxidizers [4]. Hydrated electrons react with oxygen and subsequently form superoxide anion radicals which further form singlet oxygen and hydrogen peroxide. ROS-induced oxidative stress leads to damage of lipid membrane, protein and DNA [5]. Radiation-induced liver injury is a well-known phenomenon for conventionally fractionated radiotherapy [6].

Several natural, semi-synthetic and synthetic chemicals have been investigated for the protection against radiation-induced damage in biological systems [4]. However, only a few have demonstrated appreciably good radio-protectors but with limited practical application at their optimum protective dose due to toxicity [4]. This fact has remained the mainstay for the search of non-toxic or less toxic compounds from biological sources. Studies revealed that fruits and vegetables are rich in antioxidants and minimally toxic [1]. Naturally occurring phytochemicals like curcumin, genistein, parthenolide, ellagic acid, withaferin, plumbagin, resveratrol etc. showed considerable antioxidant activity [1].

Trianthema portulacastrum (also called Trianthema monogyna L., family of Aizoaceae) is a well-known medicinal plant used from ancient time to treat several diseases. It is also known as horse purslane, Bishkhapra, carpetweed, Punarnava, Gadabani and Labuni [4]. The herb is found worldwide e.g. Southeast Asia (India, Bangladesh, Sri Lanka, Pakistan, etc.), Africa (like Ghana and Tanzania) and America. It contains approximately 9% crude protein, 3% carbohydrate, and supplies nearly 76 kcal energy/100 g [7].

Different parts of T. portulacastrum are rich in pharmacological components which are used to treat enumerable pathological conditions. The T. portulacastrum leaves have been used as diuretics in edema and ascites. The ethanolic extract of whole plant of T. portulacastrum showed anti-inflammatory response against formaldehyde-induced arthritis in rats. Chloroform, aqueous and alcoholic extract of T. portulacastrum leaves, stem and roots individually acts as a potential pregnancy interceptive [7].

T. portulacastrum ethanolic extract also had demonstrated distinct protection against CCl4- induced DNA damage and chromosomal aberration [8]. Due to its antioxidant potential ethanolic leaf extract of T. portulacastrum had been found hepatoprotective against thioacetamide, paracetamol [9] and aflatoxin B1-induced toxicity in animal model [10]. Methanolic extract of T. portulacastrum from whole plant had exhibited protective effect against atherosclerosis and renal hepatic disorders in rats [11].

The hepato-protective efficacy of T. portulacastrum extracts against gamma radiation in ex-vivo condition was evaluated in this study and examined its antioxidant and scavenging properties.

Materials and Methods

Plant

T. portulacastrum was collected from the fields in Kalyani and authenticated from Department of Botany, University of Kalyani, Kalyani, Nadia (Voucher No. UD-101). The leaves were collected young and fresh with petiole from this perennial weed. The young thin green stem having no bark, and the upper portion of the root without petiole and leaf were collected. The fresh young whole plant includes leaf, stem and root.

Activities of catalase (CAT; EC 1.11.1.6) [12], peroxidase (POD; EC 1.11.1.7) [12], superoxide dismutase (SOD, EC 1.15.1.1) [13] and glutathione S-transferase (GST; EC 2.5.1.18) [14] were measured from the fresh weight of T. portulacastrum.

Total Flavonoids Content

500 mg of sample was ground well in 10-time of 80% ethanol. The homogenate was centrifuged at 5000 rpm for 30 min, and the supernatant was collected. The residue was re-extracted with 80% ethanol, centrifuged and the supernatant was pooled. The supernatant was evaporated to dryness, and the residue was dissolved in distilled water (5 ml). 100 μl extracts were added to 0.4 ml distilled water followed by NaNO2 (0.03 ml, 5%). After incubation for 5 min at 25 °C, AlCl3.6H2O (0.03 ml, 10%) was added. The reaction mixtures were treated with NaOH (0.2 ml, 1 mM) after 6 min, diluted to 1 ml with water, mixed well and the absorbance was read at 510 nm using catechin as standard [15].

Ascorbic Acid

5 g fresh plant material was extracted with 5% metaphosphoric acid, filtered and volume was made to 50 ml. 20 ml standard ascorbic acid (50 mg ascorbic acid in 250 ml 5% metaphosphoric acid) solution was titrated with 2,6-dichlorophenol indophenol (DCPIP) solution (75 mg of DCPIP and 100 mg of sodium bicarbonate in 500 ml) till a faint pink colour appeared. Similarly, 20 ml of diluted plant extracts was also titrated using the dye solution. The volume of dye used in each case was noted. Calculation: If v ml of dye is required for the oxidation of ascorbic acid in 20 ml of the standard solution and y ml of dye is required for the oxidation of T. portulacastrum juice, the ascorbic acid content in 100 ml of diluted juice was calculated as y/v mg; and in 100 ml of undiluted juice was 10 × y/v mg [16].

T. portulacastrum Extract Preparation

Dry powder leaves, stem and full plant (100 g) was extracted with 500 ml petroleum ether for 24 h with constant shaking and then filtered. The same procedure was repeated again. Ethyl acetate, acetone and ethanol solvents were used sequentially followed by petroleum ether. Solvents were evaporated and dried. Approximate yield were 1.3 g, 0.8 g and 1.1 g from 100 g dried weight of leaves, stems and whole plants respectively. Total phenolic content [17], flavonoids content [15] and nitric oxide radical scavenging activity [1] were determined from the extracts.

Ex vivo Studies

Goat liver was collected in chilled phosphate buffered saline (PBS) from the slaughter house immediately after the sacrifice. Liver sample was sliced at approximately 1 mm thick and 1 g of weight and kept at 4 ml of sterile PBS in each of ten flasks [18]. The ethanolic T. portulacastrum extracts (at 50, 100, 200 μg/g liver tissue) were added and incubated for 2 h at 37 °C with mild shaking. Appropriate controls were also set up. The samples were irradiated at UGC-DAE Consortium for Scientific Research, Kolkata Centre, Salt Lake City, Kolkata (dose rate 3.05 kGy/h). The radiation exposure time was adjusted to final exposure of 4-Gy. The samples were incubated for another 2 h at room temperature (25 °C) after irradiation. The goat liver slices were then homogenized in 1X PBS (137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 2 mM KH2PO4, pH 7.4) and stored at − 80 °C for further experiments. The study was approved by the Animal ethics committee (27/3/2019) of the University of Kalyani following CPCSEA guidelines. The homogenate was used for assaying thiobarbituric acid reactive substances (TBARS) [19], and nitrite [20] content, as well as activities of CAT [21], glutathione reductase (GR; EC 1.6.4.2) [22], SOD [23] and GST [14].

Statistical Analysis

All analyses related to T. portulacastrum polyphenols extracts were performed five times and the results are expressed as the mean ± standard error (SE). Significant differences were assessed through the one-way analysis of variance (ANOVA), followed by the Tukey’s test for individual differences with SPSS version 20. A value of P < 0.05 was used to evaluate statistical significance.

Results

Higher concentrations of ascorbic acid and flavonoids contents were found in stem compared to whole plant and leaf (Table 1). Total flavonoid contents varied from 3.33 to 9.98 mg catechin equivalent (CE) in different extracts (Table 1). SOD and CAT activities differed significantly (P < 0.01) in different parts of extracts (Table 1), and stem exhibited highest activity compared to other parts. Peroxidase and GST showed maximum activity in whole plant followed by stem and leaf (Table 1). The phenolic content varied from 122.9 ± 8.7 to 302.8 ± 15.7 mg gallic acid equivalent (GAE)/g of extract and flavonoids content varied from 316.7 ± 33.3 to 800.7 ± 28.9 mg CE/g of extract (Table 2).

Table 1.

Flavonoids, ascorbic acid content and Catalase, peroxidase, superoxide dismutase (SOD) and glutathione s-transferase (GST) activity of different parts of T. portulacastrum

Parameters Stem Leaf Whole Plant
Flavonoids (CE mg/g fresh wt.) 9.98 ± 0.16dg 3.33 ± 0.15ah 6.53 ± 1.29ae
Ascorbic Acid (mg/ g fresh wt.) 0.21 ± 0.04e 0.15 ± 0.03bh 0.21 ± 0.03e
Catalase (mg H2O2 decomposed/g fresh weight/ min) 2.84 ± 0.15dg 0.51 ± 0.03ag 2.38 ± 0.02ad
Peroxidase (Ua/ min) 4.92 ± 0.10dg 3.26 ± 0.18ag 6.38 ± 0.03ad
SOD (Ub/g fresh wt) 1.79 ± 0.04e 1.6 ± 0.03bi 1.67 ± 0.04f
GST (nmole CDNB conjugate formed/min/mg Fresh wt) 3.11 ± 0.12dg 1.57 ± 0.06ag 3.59 ± 0.05ad
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Values are mean ± SE (standard deviation). P values: a ≤ 0.001, b ≤ 0.01 and c ≤ 0.05 compare to Stem, d ≤ 0.001, e ≤ 0.01 and f ≤ 0.05 compare to Leaf, g ≤ 0.001, h ≤ 0.01 and i ≤ 0.05 compare to whole plant of T. portulacastrum

CE catechin equivalent

aOne unit of peroxidase is defined as the change in absorbance/min at 430 nm

bOne Unit of SOD enzyme is defined as the enzyme concentration required to inhibit the OD at 560 nm of chromogen produced by 50% / min under assay condition

Table 2.

Flavonoids and total phenolic content of different parts of T. portulacastrum extracts

Parameters Stem Leaf Whole plant
Flavonoids (CE mg/g extract) 800.7 ± 28.9d 316.7 ± 33.3ag 667.3 ± 43.6d
Total phenolic content (mg GAE/g extract) 302.8 ± 15.7d 122.9 ± 8.7 299.3 ± 10.2d
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Values are mean ± SE (standard deviation). P values: a ≤ 0.001, b ≤ 0.01 and c ≤ 0.05 compare to Stem, d ≤ 0.001, e ≤ 0.01 and f ≤ 0.05 compare to Leaf, g ≤ 0.001, h ≤ 0.01 and i ≤ 0.05 compare to whole plant of T. portulacastrum

CE catechin equivalent, GAE gallic acid equivalent

Different parts of the plant exhibited concentration-dependent NO• scavenging abilities of T. portulacastrum extracts (Table 3). 4-Gy of γ-irradiation showed varying degrees of oxidative stress in goat liver tissues ex-vivo (Table 4). Gamma irradiation at 4-Gy significantly (P < 0.001) decreased activities of catalase by 44.4%, GR by 47.8%, SOD by 38.6%, GST by 22.4%, while TBARS level was increased by 1.5-fold and nitrite level by 42.8% against control (Table 4).

Table 3.

IC50 values of NO scavenging properties of T. portulacastrum

IC 50 (μg/ml) LE SE PE
NO 387.38 347.66 208.69
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Table 4.

Effect of Trianthema portulacastrum extracts on TBARS and nitrite content and activities of catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR) and glutathione s-transferase (GST) in 4 Gy γ-radiation treated liver tissue ex vivo

Concentration of extract (µg/g liver) TBARS (Mg/dl protein) Catalase (Ua/mg protein) Nitrite (μg/mg Protein) GR (μmole NADPH breakdown/min/100 mg protein) SOD (Ub/mg protein) GST (μmole CDNB conjugate formed/ min/mg protein)
Control 0.62 ± 0.03 23.96 ± 0.78 0.67 ± 0.02 0.76 ± 0.07 33.89 ± 0.86 0.49 ± 0.019
4 Gy Control 1.74 ± 0.029* 10.32 ± 0.25* 2.48 ± 0.12* 0.12 ± 0.007* 15.06 ± 0.42* 0.14 ± 0.01*
Stem 50 1.09 ± 0.04*# 15.6 ± 0.41*# 1.14 ± 0.09*# 0.16 ± 0.007* 19.46 ± 0.53*# 0.18 ± 0.005*
100 0.94 ± 0.04*# 19.66 ± 0.53*#& 1.0 ± 0.07# 0.35 ± 0.019*#& 24.36 ± 0.55*#& 0.23 ± 0.007*#
200 0.81 ± 0.03*#& 23.2 ± 0.36#&@ 0.8 ± 0.02# 0.51 ± 0.017*#&@ 28.62 ± 0.44*#&@ 0.33 ± 0.007*#&@
Leaf 50 1.3 ± 0.04*# 13.3 ± 0.29*# 1.76 ± 0.81*# 0.13 ± 0.003* 17.0 ± 0.41* 0.15 ± 0.007*
100 1.15 ± 0.04*# 14.56 ± 0.42*# 1.48 ± 0.09*# 0.24 ± 0.015*# 18.78 ± 0.25*# 0.17 ± 0.007*
200 0.94 ± 0.26*#&$ 16.46 ± 0.26*#& 1.2 ± 0.04*#& 0.32 ± 0.12*#& 21.28 ± 0.35*#&@ 0.21 ± 0.011*#&
Plant 50 1.13 ± 0.27*# 14.58 ± 0.3*# 1.5 ± 0.08*# 0.14 ± 0.005* 19.0 ± 0.35*# 0.18 ± 0.004*
100 1.02 ± 0.033*# 16.42 ± 0.27*#& 1.24 ± 0.05*# 0.18 ± 0.01* 22.63 ± 0.67*#& 0.21 ± 0.009*#
200 0.89 ± 0.03*#& 18.14 ± 0.54*#& 1.1 ± 0.07*#& 0.31 ± 0.014*#&@ 25.28 ± 0.21*#&@ 0.26 ± 0.012*#&@
F value 72.02 94.95 47.48 72.94 122.9 112.08
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Values are mean ± SE (standard Error) of five samples

P values: * < 0.05 compared to control group; # < 0.05compared to experimental (radiation exposed) control group; & < 0.05 against 50 µg extract/g liver tissue; @ < 0.05 against 100 µg extract/g liver tissue of that corresponding extracts

aOne Unit of Catalase enzyme is defined as µmole H2O2 decomposed/min

bOne Unit of SOD enzyme is defined as the enzyme concentration required to inhibit the OD at 560 nm of chromogen produced by 50%/min under assay condition

Extracts of leaf, stem and full-plant at a concentration of 200 µg/g decreased TBARS level by 28.1%, 42.2%, and 37.5%, respectively; while, nitrite content decreased by 25%, 18.7% and 21.2%, respectively. Enzyme activities also restored with increased concentrations of extracts. Leaf, whole plant and stem extracts at a concentration of 200 µg/g increased catalase activity by 33.3%, 41.7% and 66.7%, respectively; while SOD activity by 46%, 35% and 25% respectively, GR activity by 1.77-fold, 0.92-fold and 1.2-fold respectively and GST activity by 93.2%, 53% and 81.8% respectively compared to radiation exposed liver slices (Table 4).

Discussions

T. portulacastrum contains phenolic compounds, flavonoids, antioxidant enzymes and demonstrated scavenging properties. T. portulacastrum extracts at doses of 50 μg/g liver tissues, 100 μg/g liver tissue and 200 μg/g liver tissues were used to evaluate its response against radiation. Due to limited availability of root extracts, other parts including whole plant was included in the study to evaluate its efficacy.

Free radical scavengers and the antioxidant enzymes may provide a defensive mechanism against the deleterious actions of ROS. Some of the antioxidant enzymes that are found to protect against the ROS are CAT and total peroxidase [24]. Catalases are responsible for the four-electron reduction of molecular oxygen to water with concomitant one-electron oxidation of the substrate [25]. Peroxidase (POD) is one of the most thermo stable enzymes responsible for performing the single electron oxidation on a wide variety of compounds, in the presence of H2O2. POD reduces H2O2 to water while oxidizing a variety of substrates in a multistep reaction [26].

H2O2 acts an important signal molecule in plant development and environmental responses. Plants contain several types of H2O2 neutralizing enzymes such as CAT, which is highly active and does not require cellular reductants as it primarily catalyses a dismutase reaction [27]. The presence of significant amount of SOD protects cells against oxygen toxicity [28] and limits hydroxyl radical formation [29]. Plant GSTs are a family of multi-functional enzymes involved in the intracellular detoxification of xenobiotics and toxic compounds produced endogenously [30]. The higher level of GST activity in different parts of plant is capable of detoxifying higher concentrations of toxins in the presence of sufficient GSH.

The presence of phenolic compounds, flavonoids and ascorbic acid in T. portulacastrum is responsible for antioxidant properties and are related to their chelating ability with metal, capturing free radicals, inhibition of lipoxygenase [31]. These compounds prevent or delaying the oxidation or auto-oxidation and through free radicals scavenging [31]. These phytochemicals specifically bind or react through polyphenol ring structure with methoxyl and hydroxyl groups and sometimes with the base or others groups in DNA backbone and mitigate the damaging effects of IR at the cellular, molecular and tissue level [32].

The antioxidant potential of phytochemicals is generally linked to their ability to scavenge free radicals [33]. In the present study, the antioxidant properties of TP extracts were evaluated by nitric oxide (NO•) scavenging activity. Nitrogen monoxide (NO•) is a diffusible free radical that acts as an effecter molecule in diverse biological systems including vasodilation, inhibition of platelet aggregation and regulation of cell medicated toxicity [34]. Over production of NO• beyond threshold level results in vascular collapse due to cytotoxicity. It has been established that chronic exposure to increased level of NO• provokes various inflammatory responses by tissues [35]. IR increases NO• level which is due to increased level of inducible nitric oxide (iNOS) [36] even at low dose of radiation [37]. This in vitro ex vivo study has demonstrated that the T. portulacastrum extract can scavenge NO radicals.

Liver irradiation heads towards the assemblage of pathologies collectively known as radiation-induced liver disease (RILD), which includes the alteration of liver enzyme activities, fibrosis, cirrhosis, and cancer [1, 38]. 4-Gy γ-radiation dose has been considered as standard for experimental purpose in earlier studies [1]. Decreased activities of antioxidant enzymes (CAT, GR and GST) and the increased levels of TBARS and NO in the liver tissues ex-vivo due to IR could be attributed to enhanced production of ROS [12, 39].

γ-radiation induced free radicals alter the structure and the function of lipids and proteins by lipid peroxidation (LPO), formation of lipid-lipid crosslinks, hydrolysis of membrane phospholipids, disulfide bridge formation and damage of the amino acid residues in the membrane proteins which eventually results in loss of the membrane integrity, increased permeability and rigidity [40, 41]. LPO inhibit the membrane transport and enzyme activity which may arise due to structural alteration such as oxidation of thiol groups, modification of spectrin/actin complex responsible for maintenance of cytoskeleton structure of membrane [7, 41, 42]. T. portulacastrum extracts decrease radiation-induced malondialdehyde (MDA) formation.

GST eliminates the toxic compounds from the liver by conjugating with GSH. Decreased GST and GR activities play an important role in sustaining the pathogenic activity for oxidative stress [43]. CAT protects cells from oxidative stress through the decomposition of H2O2 and produces water and oxygen [24]. T. portulacastrum extracts partially restored concentration dependent enzyme activities. Among all the three extracts the stem showed best protection followed by whole plant and leaf.

Conclusion

Plants have developed an antioxidant system for protection against potentially toxic effects of xenobiotics and ROS. The extracts of T. portulacastrum have demonstrated concentration-dependent protection against γ-radiation induced alteration in TBARS level and antioxidant abilities of enzymes. The antioxidant potential of T. portulacastrum extract varies in different parts of plant and its efficacy is directly related to the concentration and part of the extract.

Acknowledgements

Financial assistance received from the Department of Atomic Energy-Board of Research in Nuclear Studies (35/14/39/2016-BRNS/35174) and radiation facilities received from UGC-DAE-CSR, Salt Lake City, Kolkata are gratefully acknowledged.

Footnotes

Publisher's Note

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