Antioxidant and cytoprotective effects of sequentially extracted Terminalia prunioides pods

ABSTRACT Oxidative stress has been linked to a wide array of health-debilitating diseases. To alleviate oxidative stress, antioxidants especially of plant origin are desired due to their potency and low toxicity. The current study, therefore, evaluated the antioxidant properties and cytoprotective effects of Terminalia prunioides pods. Hexane, chloroform, ethyl acetate and methanol extracts from Terminalia prunioides pods were evaluated for flavonoid and total phenol contents. Their effects on 2, 2-diphenyl-1-picryl hydrazyl (DPPH), 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS), Superoxide dismutase (SOD), Catalase (CAT), reduced glutathione (GSH) and Ferric reducing antioxidant power (FRAP) were evaluated. The extracts were also tested for their cytoprotective effects on HeLa cells against H2O2, using 4-[−3(4-Iodophenyl)-2-(4-nitro-phenyl)-2 H-5-tetrazolio]-1,3-benzene sulfonate (WST-1) assay. The methanol extract possessed 67.8 ± 10.4 mg QE/g and 113.2 ± 7.6 mg GAE/g of total flavonoid (TFC) and total phenolic contents (TPC) respectively. Methanol extracts from Terminalia prunioides pods recorded IC50 values of 0.06 mg/mL, 0.07 mg/mL, and 0.24 mg/mL for DPPH, FRAP and ABTS assays respectively. Catalase (CAT), reduced glutathione (GSH) and Superoxide Dismutase (SOD) activities were significantly increased (p < 0.05) in HeLa cells treated with Terminalia prunioides pods extracts. The current study’'s findings indicate the high antioxidant activity of Terminalia prunioides pods extracts and cytoprotection of HeLa cells from H2O2 induced cell death.


Introduction
Reactive oxygen species (ROS) dysregulation has repeatedly been associated with the emergence of a variety of illnesses, including heart disease, liver disease, pancreatic disease and cancers [1,2].Normal physiological processes like oxidative phosphorylation and other cell signaling pathways can generate reactive oxygen species, a family of highly reactive and very unstable oxygen-based radicals [3].Oxidative stress, which is caused by excessive ROS, is a destructive cellular environment [4].Lipids, proteins, and nucleic acids are subject to oxidative damage by ROS due to interaction and electron/hydrogen reduction [5].Given that it has been shown to impair macromolecules, oxidative stress is harmful to the structure and function of cells [6].Oxidative stress has been demonstrated to limit the activity of endogenous antioxidants, reducing the cell's antioxidant defenses and increasing the cell's susceptibility to oxidative damage [7].
The body has antioxidants as an adaptation response to the prevention, and to lessen the adverse consequences of oxidative stress.When present in small amounts, antioxidants, which are chemicals and proteins that significantly slow down or stop oxidative damage, can protect cells from oxidants [8,9].Endogenous antioxidants, such as non-enzymatic reduced glutathione (GSH), enzymatic superoxide dismutase (SOD), and enzymatic catalase (CAT), serve as the body's first line of defense [10,11].In many circumstances, the physiologically necessary quantities of endogenous antioxidants alone are insufficient to properly protect cells [12].Considering this, exogenous antioxidant supplements are necessary to augment the endogenous antioxidant system.Superoxide anion is reduced by SOD into diatomic oxygen and hydrogen peroxide [13].Hydrogen peroxide is converted by catalase (CAT) into water and, occasionally, hydroxyl anion through the Fenton reaction [14].Hydroxyl radical is reduced by GSH to water [15].GSH is reduced to GSSG (oxidized GSH) as a result of the hydroxyl radical reaction [16].In order to convert back GSSH into GSH, it needs either electron and/or hydrogen atom donors.Exogenous antioxidants from medications and plants have been demonstrated in studies to convert GSSH to GSH by electron and hydrogen reduction [17].It has been discovered that bioactive compounds extracted from plants are efficient free radical reducing agents through several mechanisms such as hydrogen atom transfer (HAT) and single electron transfer (SET) [18].
Terminalia prunioides is one of the least investigated members of the genus Terminalia.Terminalia prunioides is native to eastern and some parts of Southern Africa [19].This plant can grow up to heights of 2.5 meters to 15meters, with a bark that is pale gray to gray black, spur shoots at the end of lateral shoots, and has creamy or white flowers.Leaves are present from November to June.The pods are vissible from late February to late August.Terminalia prunioides bears purple pods.In some parts of Southern Africa, Terminalia prunioides is used as traditional medicine to relief abdominal pains, and the pods are used as tea [20].Analysis of other species of the genus Terminalia such as Terminalia neotaliala and Terminalia Arjuna revealed that they have biological activities such as radical scavenging properties and modulatory effects on enzymes [21,22].Though the pods of Terminalia prunioides are used as tea, there is limted scientific information about this plant's pods.Therefore, this study aimed at evaluating the antioxidant properties and cytoprotective effects of Terminalia prunoides pods on HeLa cells.

Sample preparation
The pods of Terminalia prunioides shown in Figure 2 were washed with distilled water and dried in a Lasec MMMERT UF 110 laboratory oven at 40°C for a period of 48 hours.The dried samples were ground to powder using a Salton SB400E blender.The powdered samples were then extracted sequentially in hexane, chloroform, ethyl acetate, and methanol following protocol by [23], with some modifications.3 kg of dried powder was soaked in 7 L of N-hexane for 76 hours in round bottom flasks and then filtered using a Whatman filter paper, Grade 1 (Merck, South Africa).The plant residue from N-hexane was air-dried for about 6 hours and then soaked again in chloroform (7 L) for 76 hours and the procedure was repeated for ethyl acetate and methanol.The filtrates were evaporated to dryness using Heidolph Hei-VAP Platinum 2 rotary evaporator, and samples were then stored at −20°C awaiting analysis.

Determination of total flavonoid content (TFC)
The TFC of Terminalia prunioides pods was determined using a published method [24]. 2 mL of methanol, 200 µL of 10% aluminum chloride, 200 µL of 1 M potassium acetate, 600 µL of distilled water, and 1 mL of quercetin at concentrations of 0.0312, 0.0625, 0.125, 0.25, 0.5 and 1 mg/mL were mixed in test tubes.After mixing, the solution was incubated in the dark for 30 minutes at room temperature, to allow the ingredients to react completely.Absorbance was measured at 420 nm using a Thermo Scientific™ SPECTRONIC™ 200 Spectrophotometer.The results of quercetin were used to plot a standard curve which was used for calculating TFC of extracts.2 mL of methanol, 200 µL of 10% aluminum chloride, 200 µL of 1 M potassium acetate, 600 µL of distilled water, and 1 mL of extracts at a concentration of 1 mg/mL, were mixed in test tubes and incubated for 30 mins in the dark at room temperature.Absorbance was measured at 420 nm using a Spec200E UV-V (Thermofisher Scientific, United States of America).Quercetin standard curve (R 2 = 0.91) was used in determining TFC.TFC was calculated as, Where c= concentration of extract equivalence to that of quercetin (quercetin standard curve), V= volume of extracts in mL and m= mass of extracts in grams.

Determination of total phenolic content (TPC)
The TPC of Terminalia prunioides pods were determined using a method by [25].200 µL of gallic acid at concentrations 0.0312, 0.0625, 0.125, 0.25, 0.5 and 1 mg/mL, 2 mL distilled water and 200 µL of 10% Folin-ciocalteu reagent were mixed and incubated for 8 minutes in the dark.600 µL of 20% sodium carbonate was added and left to incubate in the dark for 2 hours at room temperature.Absorbance was read at 760 nm using a Spec200E UV-V, and the results for gallic acid were used to plot a standard curve that was used in calculating TPC of extracts.200 µL of 1 mg/mL extracts, 2 mL distilled water and 200 µL of 10% Folin-ciocalteu reagent, were mixed and incubated for 8 minutes in the dark.600 µL of 20% sodium carbonate was added and left to incubate in the dark for 2 hours at room at temperature.Absorbance was read at 760 nm using a Spec200E UV-V (Thermofisher Scientific, United States of America).Gallic acid standard curve (R 2 = 0.99) was used in determining TPC.The TPC was calculated as,

TPC ¼ cV m
Where c= concentration of extract equivalence to that of gallic acid (from gallic acid standard curve), V= volume of extracts in mL and m = mass of extracts in grams.
For wells designated as treatment, DPPH was treated with 50 µL of plant extracts.For wells designated as positive control, DPPH was treated with 50 µL of gallic acid.For wells designated as negative control, DPPH was not treated.The reactions were incubated for 30 mins in the dark at room temperature.Optical density (OD) was measured after 30 minutes at 518 nm using a MULTISKAN FC microplate reader (Thermo Scientific, USA).The percentage inhibition (decolorization) was determined as follows, DPPH inhibitionð%Þ ¼ ODsample À OD control OD control �100

Determination of antioxidant activity using ferric reducing antioxidant powder (FRAP)
This assay was used as described by [27] with some modifications, by running the experiment in serial dilutions instead of 1 concentration, and evaluating FRAP reduction in percentages.1.0 N hydrochloric acid (HCl) was prepared by adding 8.3 mL of HCl into 100 mL of distilled water.Plant extracts and gallic acid were serial diluted into concentrations of 0.0312, 0.0625, 0.125, 0.25, 0.5 and 1 mg/mL.40 µL of each concentration of plant extracts and gallic acid, were added to the wells of a 96 well plate.100µL of 1.0 N HCl was added to the 96 well plates.Followed by adding 20 µL of 1% sodium dodecylsulphate and 30 µL 1% of potassium ferrocyanide.The reaction was left to incubate for 20 minutes at 50°C.Absorbance was read at 750 nm using a MULTISKAN FC microplate reader (Thermo Scientific, USA).

Determination of antioxidant activity using the 2,2'-azino-bis (3-ethylbenzothiazoline6sulfonic acid (ABTS) method
ABTS method was executed as described by [28].This method is based on ABTS radical cation decolorization.100 mL of 7 mM ABTS and 100 mL of 2.4 mM of potassium persulfate (1:1) were mixed to make ABTS working solution, and stored in the dark for 16 hours at room temperature.1 mL of ABTS solution was diluted gradually with methanol until an absorbance of 0.706 ± 0.01 was obtained at 734 nm.Then 1 mL of the ABTS solution was added to 0.0312, 0.0625, 0.125, 0.25, 0.5 and 1 mg/mL concentrations of extracts and gallic acid.After mixing, the reaction was left to incubate for 7 minutes in the dark at room temperature.Absorbance was measured at 734 nm using a MULTISKAN FC microplate reader (Thermo Scientific, USA).Gallic acid was used as standard.Percentage inhibition was determined as follows:

Total antioxidants capacity (TAC) using phosphomolybdenum
As described by [29], TAC is an assay that is based on the formation of a stable green phosphate-Mo(V) complex, as a result of electron reduction of Mo (VI) to Mo(V).0.1 mL of extracts and gallic acid was mixed with 1 mL of phosphomolybdenum (prepared by mixing 31.4 mL of 0.6 M H 2 SO 4 , 397.49 mg of 28 mM sodium phosphate and 494.36 mg of 4 mM of ammonium molybdate).The mixture was incubated at 95°C in a IncoCool LABOTEC (LAMWORLD TECHNOLOGIES PTY LTD) for 90 minutes, then cooled to room temperature.Absorbance was measured at 950 nm using a Spec200E UV-V (Thermofisher Scientific, United States of America).Gallic acid was used to make a standard (R 2 = 0.97), from which TAC of extracts was interpolated from.

Preparation of plant extracts concentrations
100 mg of plant extracts was dissolved in 10 mL of 10% dimethyl-sulfoxide (DMSO) to make a stock solution of 10 mg/mL.10 mL of stock solution was added to 25 mL Dulbecco's minimal essential medium (DMEM) to make a concentration of 400 µg/mL.The 400 µg/mL was adopted from [30].The 400 µg/mL was serial diluted to make concentrations of 12.5, 25, 50, 100, 200, and 400 µg/mL.

Cell culture
HeLa cells were cultured in preparation for performing several experiments on the cytoprotective effects of Terminalia prunioides pods extracts.HeLa cells were grown in high glucose Dulbecco's minimal essential medium (DMEM), supplemented with penicillin-streptomycin (10 000 U/mL: 10 000 µg/mL) and 10% of fetal bovine serum (FBS).Cells were incubated in a Thermo Scientific Forma SERIES 11 Water Jacket CO 2 Incubator at 37°C and 5% CO 2 .A Countess ™ 3 Automated Cell Counter was used to count cells before seeding them into flat bottom 96-well plates, and the concentrations of cells were prepared as per the output of the cell counter to attain a cell concentration of 1 × 10 5 /mL.

Effects of extracts on oxidative stress biomarkers
About 80-90% confluent HeLa cells (day 6 of passage of number 5) at a concentration of 1 × 10 5 /mL were treated with 100 µL of extracts (400 µg/mL) for 2 hours, and untreated cells were used as a control for the experiment.Cells were detached using trypsin and centrifuged at 1000 g for 10 minutes.The pellet was resuspended in cold PBS and centrifuged at 1000 g for 10 minutes.The pellet was suspended in cold PBS, then frozen at − 20°C for about 3 hours.After freezing, the pellet was thawed, frozen again, and thawed again, this was repeated three times to break the cells.The cell homogenate was centrifuged at 1500 g for 10 minutes and preserved in ice for further studies.The effects of extracts on the activity of superoxide dismutase (SOD), catalase (CAT), and reduced glutathione (GSH) were evaluated using ELISA kits (Elabscience, USA) following the manufacturer's instructions.

Determination of the cytoprotective effect of extracts against hydrogen peroxide-induced cell death using WST-1
About 90-98% confluent HeLa cells at day 6 of passage number 5 were trypsinized, seeded in flat bottom 96-well plates at a concentration of 1 × 10 5 /mL, and left to attach to the bottom of the plate for 72 hours.After the incubation, media was aspirated and replaced with 100 µL Terminalia prunioides pods extracts (400 µg/mL), for wells designated as treatment.For positive control wells, cells were treated with PBS only.
After treatment cells were incubated for 2 hours.After 2 hours, cells were exposed to hydrogen peroxide induced oxidative stress, by adding 10 µL of 1 mM hydrogen peroxide (H 2 O 2 ) to the wells containing extracts and PBS (positive control) and incubated for 4 hours.The 1 mM H 2 O 2 was adopted from literature [31].For negative control wells, cells were not treated.After incubation, 10 µL of WST-1 was added to the wells and incubated for 3 hours.Absorbance was taken at 480 nm using a MULTISKAN FC microplate reader (Thermo Scientific, USA).Absorbance of wells containing media and 10 µL of WST-1 was regarded as background and was subtracted from that of treatment and control.The protective effect of extracts was determined as percentage of viable cells which was calculated as absorbance comparison with that of control.

¼
OD negative control À OD treated cells OD negative control � 100

Data analysis
All results were analyzed using Origin Pro 2018 software.Data was presented as mean ± standard error (n = 3).Means were compared using One way-analysis of variance (ANOVA) with Tukey-Kramer's range test.Means were considered significant at 95% confidence level (p < 0.05).

Total flavonoid content (TFC) and Total phenolic content (TPC) of extracts from Terminalia prunioides pods
Hexane extracts had the lowest TFC values of 4.3 ± 2.9 mg QE/g.Methanol extracts had the highest TFC value of 67.8 ± 10.4 mg QE/g, followed by ethyl acetate extracts (59.4 ± 3.8 mg QE/g) and chloroform extracts (19.4 ± 5.2 mg QE/g).The TFC value of hexane and chloroform extracts were significantly different from each other (p < 0.05) as shown in Figure 3a. Figure 3b shows that TPC values increased with increasing extraction solvent polarity.Hexane extracts recorded the lowest TPC value of 3.6 ± 5.0 mg GAE/g.Methanol extracts showed the highest TPC value of 113.2 ± 7.6 mg GAE/g, followed by ethyl acetate extracts (89.6 ± 2.4 mg GAE/g), and chloroform extracts (36.9 ± 14.5 mg GAE/g).The TPC values of all extracts were significantly (p < 0.05) different from each other.
After DPPH, FRAP and ABTS assay, TAC of extracts was then determined.Figure 4d shows that there was a positive relationship between extraction solvent polarity and TAC.Methanol extracts had the highest TAC value of 43.9 ± 0.7 mg GAE/g, followed by ethyl acetate (41.73 ± 0.36 mg GAE/g), however TAC value of methanol extracts was not significantly different (p < 0.05) from the TAC value of ethyl acetate extracts.They were followed by chloroform (15.63 ± 0.64 mg GAE/g) and hexane extracts (4.03 ± 0.31 mg GAE/g).

Effects of extracts on oxidative stress markers
The activities of SOD, CAT and GSH in cells treated with extracts were also evaluated.Figure 5a shows that all extracts of Terminalia prunioides pods significantly increased (p < 0.05) the activity of SOD.There was no significant difference (p < 0.05) between the activities of SOD in cells treated with chloroform (8.44 ± 0.54 U/mL), ethyl acetate (9.06 ± 0.7 U/mL) and Figure 5b shows that all extracts significantly (p < 0.05) elevated the activity of CAT.Extracts did not exhibit extraction solvent polarity dependent activity; chloroform recorded the least effect on the activity of CAT (47.98 ± 2.68 U/ mL).Hexane extracts showed the third highest increase in CAT activity behind ethyl acetate and methanol extracts (54.14 ± 4.11 U/mL).Methanol extracts had the most effect on CAT activity.There was no significant difference (p < 0.05) between the effects of hexane, chloroform, ethyl acetate and methanol extracts on CAT activity.Figure 5c shows that all extracts significantly increased GSH activity.However, the effects of all four extracts on GSH activity were not significantly different (p < 0.05) from each other.Methanol extracts showed the most effect on GSH activity with 47.97 ± 2.30 mmol/L, followed by ethyl acetate extracts (46.27 ± 5.03 mmol/L), chloroform extracts (45 ± 4.46 mmol/ L) and hexane extracts (44.66 ± 1.15 mmol/L).

Determination of the cytoprotective effects of Terminalia prunioides pods extracts against H 2 O 2 induced cell death
Cell protective effects against ROS was evaluated using WST-1 assay.Figure 6 shows that HeLa cells that were exposed to 1 mM of H 2 O 2 without any protective agent (positive control) had only 25.3 ± 0.01% of HeLa cells being viable.HeLa cells that were only treated with PBS only (negative control) had 96.3 ± 0.6% of HeLa cells being viable.At a concentration of 400 µg/mL (the highest concentration), methanol extracts had the highest protective activity, resulting in 95.9 ± 2.3% cells being viable, which was not significantly different (p < 0.05) from negative control.Hexane extracts had the least protective activity with 83 ± 3.1% viable cells.The protective activity of the extracts decreased with their decreasing concentrations.At a concentration of 12.5 µg/mL (the lowest concentration), methanol extracts had the highest protective activity with 54 ± 2.76% viable cells, which was not significantly different (p < 0.05) from the protective activity of ethyl acetate extracts.Hexane extracts had the least cytoprotective activity which was numerically (29 ± 3.1%) higher, but not significantly different (p < 0.05) from that of positive control.

Discussion
The role of plants in human health has been linked to their capacity to detoxify toxic substance and replenish other bodily proteins such as endogenous antioxidants.This property may be the reason why different cultures all over the world have been using plants to alleviate oxidative stressed based diseases.However, many plants have not been scientifically studied.This study evaluated the antioxidant properties of Terminalia prunioides pods, and their cell protective effects against H 2 O 2 .The results (Figures 3a,b) showed that the methanol and ethyl acetate extracts recorded higher total flavonoid contents (TFC) and total phenolic contents (TPC) than the hexane and chloroform extracts.This is consistent with published findings were high TPC was recorded in ethanolic extract of Terminalia prunioides herbal tea [32].This is also supported by other studies [33,34] which found outthat nonpolar solvents extract lower TFC and TPC compared to more polar solvents.While the phytochemistry of Terminalia prunioides is not yet fully characterized, the observed TFC and TPC may be due bioactive compounds such as tannins, flavonoids, phenols, terpenoids, and steroids which have been reported in other species of the genus Terminalia [21,22].
The extracts of Terminalia prunioides pods showed potent antioxidant activity against DPPH with the methanol extract recording the lowest IC 50 value of 0.06 mg/mL.These findings are supported by previous studies [32] where Terminalia prunioides pods herbal tea, recorded an IC 50 of 0.16 µg/mL.For the FRAP assay, Terminalia prunioides pods methanol extracts recorded an IC 50 value of 0.07 mg/mL, which concurs with the IC 50 value of 0.49 mg/mL for Terminalia catappa leaf extracts [35], Terminalia prunioides pods also scavenged the ABTS radical with the methanol extract recording an IC 50 value of 0.24 mg/mL which agrees with published findings where Terminalia avicennioides leaf extracts recorded IC 50 of 3.50 µg/mL [36].The effectiveness of the methanol extract is also demonstrated in the TAC assay, where they recorded the highest TAC with a value of 43.9 ± 0.7 mg GAE/g.This is consistent with published works of [37,38], as they reported TAC of Terminalia arjuna and Terminalia citrina extracts to be 62.74 mg GAE/g and 10.12 AAE/ gm respectively.The high radical scavenging activity of methanol extracts may be due to high total flavonoid and phenolic contents of Terminalia prunioides pods.
Results also reveal that HeLa cells treated with extracts of Terminalia prunioides pods have upregulated the activities of both the endogenous enzymatic antioxidants (Superoxide dismutase and Catalase) and nonenzymatic antioxidant (Glutathione) as shown in Figure 5.While methanol extracts showed the highest TFC, TPC and antioxidant activities compared to other extracts, the recorded effects on SOD, CAT and GSH activities were not significantly different from other extracts except that of hexane on SOD activity.Results suggest that all extracts except those of hexane may possibly possess antioxidant compounds with the ability to quench free radicals, hence a rise in the activities of SOD, CAT, and GSH.The increase in SOD, CAT and GSH activities after treatment by plant extracts, confers with results published by [39][40][41], where Terminalia arjuna, Terminalia catappa, and Terminalia bellirica elevated activities of SOD, CAT and GSH.In the absence of hydrogen/electron donors, peroxide radicals can act as both oxidizing or reducing agents, as they have been reported to inhibit SOD, CAT, and GSH activities.The inhibition happens when peroxide radicals oxidize and bind to amino acids residues in the spatial proximity to the active sites, in reactions termed 'peroxide-peroxide inhibition [7,42,43].The antioxidant activities of the extracts may have upregulated the activities of SOD, CAT and GSH by reducing hydrogen peroxide bound to the endogenous antioxidants, through binding to the free electrons of hydrogen peroxide, by so doing, setting the SOD, CAT and GSH free from hydrogen peroxide.Additionally, extracts were investigated for their capacity to prevent cell death induced by H 2 O 2 .Methanol extracts were the most effective in protecting HeLa cells from H 2 O 2 as 95.9 ± 2.3% of HeLa cells remained viable after exposure, compared to other extracts.This outcome was also found in previous investigations by [44,45], in which Bucida buceras and Fructus chebulae (COMBRETACEAE family) extracts protected RPE and PC12 rat cell lines from H 2 O 2 induced cell death.Other than upregulated SOD, CAT and GSH activities, cytoprotective effects of extracts may be due to high antioxidant activity.Extracts may have directly reduced H 2 O 2 before cell membrane damage.

Conclusion
Extracts from Terminalia prunioides pods have radical scavenging properties, positive modulatory effects on SOD, CAT and GSH activities.Terminalia prunioides pods extracts also have cell protective activity against H 2 O 2 .

Figure 3 .
Figure 3.Total flavonoid content (TFC) (a) and Total phenolic content (TPC) (b) of one gram of Terminalia prunioides pods extracts, expressed in mg of quercetin equivalence per one gram of dry mass of an extract (mg QE/g) and mg of gallic acid equivalence per 1 gram of dry mass of an extract (mg GAE/g), respectively.Data presented as mean ± standard error (n = 3).The letters above bars indicate significant differences between the means (p< 0.05), where different letters depict significant differences, and the same letters indicate that means are not significantly different from each other.

Figure 4 .
Figure 4. DPPH inhibition (a) FRAP reduction (b) ABTS inhibition (c) by Terminalia prunioides pods extracts at different concentrations, expressed as % inhibition.Total antioxidant capacity, TAC (d) of one gram of Terminalia prunioides pods extracts, expressed in mg of gallic acid equivalence per one gram of dry mass of an extract (mg GAE/g).Data presented as mean ± standard error (n = 3).Significant differences (p< 0.05) are indicated by letters above the bars, where different letters depict significant differences while the same letters indicate that means are not significantly different.