The protective and antioxidant effects of Hygrophila schulli seeds on oxidative damage of DNA and RBC cellular membrane.

Medicinal plants are sources of antioxidant which may protect the body against oxidative stress related diseases and can be used as human food supplements. In this investigation, seeds of Hygrophila schulli (M. R. Almeida & S. M. Almeida) (Fam.-Acanthaceae), a herbaceous plant well known for its medicinal properties, has been examined for antioxidant activity of crude methanolic extract (CME) and its fraction using in vitro and in vivo assay as well as their protective activity against oxidative damage of DNA and RBC. Total phenolic and flavonoid content have also been estimated using the aluminum chloride colorimetric and Folin-Ciocalteu method. Among the different fractions of CME, Ethyl acetate fraction (EAF) had higher antioxidant activity in vitro assay and was selected for in vivo antioxidant activity in cadmium intoxicated mice. The EAF showed a significant (p < 0.05) increase in serum catalase and SOD activity compared to the control group. TBARS levels were restored to 17.42 and 19.19 nmol/mg protein, respectively, after treatment with EAF and standard ascorbic acid (AA); compared to the normal group (14.96 nmol/mg protein). Similarly, levels of albumin, bilirubin, uric acid, and alkaline phosphatase were also brought back to normal levels. EAF's protective role against oxidative damage of DNA has shown a significant reduction in destroying of nicked DNA. RBC as a target of oxidation by H2O2 and HOCl, EAF showed inhibition of oxidation in a concentration-dependent manner, compared to standard gallic acid. In this study, we confirmed that EAF could scavenge reactive oxygen species (ROS) thus preventing DNA strand scission and the extract can be used as a functional food or nutraceutical product for health benefits.

Introduction

Medicinal plants are a source of a wide variety of bioactive compounds which act as antioxidants and protect the human body against different oxidative stress related diseases, and are being used as food supplements. Among various medicinal plants, Hygrophila schulli (M. R. Almeida & S. M. Almeida) (English- Marsh Barbel; Local Bangla- Shulamardan; family-Acanthaceae), has gained much importance in the recent past [1, 2]. The species, a thorny sub-herb among which many grow as aquatic plants [3], is distributed throughout the Indian subcontinent, Myanmar and tropical Africa. In traditional medicine of Ayurveda, the extracts of the plant are used for treatment of various ailments like diabetes and dysentery [4]. Moreover, it has been reported that since ancient times, along with the whole plant, roots, seeds and ashes are commonly used in traditional medicine as antibacterial, diuretic, demulcent, stomachic and for the treatment of jaundice, rheumatism, tonic, hypnotic, diarrhea, dysentery, urinary calculi, urinary discharge, anti-inflammatory, joint pain, biliousness, eye problems, abdominal troubles, anemia, cough, arthritis and gastric disorders [5]. The species has been placed under 'least concern' category (IUCN 2011).

Various types of chemical compounds are confirmed and isolated by scientific reports from this plant like saponins, alkaloids, steroids, tannins, flavonoids, phenols, and triterpenoids [6]. Similarly, the literature review revealed some notable pharmacological effects like anti-nociceptive, anti-tumor, antioxidant, hepatoprotective, hypoglycemic, haematinic, diuretic, free radical scavenging, anthelmintic, anti-inflammatory, antipyretic, anabolic, and androgenic activities [7]. It has also been reported that the free radical scavenging activity of H. schulli seed extract is comparable to that of reference standard antioxidants. In addition, it could prevent oxidative damage of various biomolecules, and provide notable protection against oxidative damage [8].

Plants are the vital sources of various bioactive substances [9] and medicinal plants still now to be the main resource against many diseases [10]. In the literature review, there are some scientific reports about seeds but no reports on protection against oxidative damage to DNA and RBC. Regular cellular functions, like oxidative phosphorylation and lipid peroxidation, generate ROS that can induce oxidative damage to DNA. It is known that about ten thousand oxidation reactions every day affect the DNA in each of our cells [11]. In the literature review, there are some scientific report on H. schulli seeds but no report on the protection of oxidative damage of DNA and Erythrocytes. H. schulli seeds are believed to be effective and have been utilized in many systems of traditional medicine's formula by various ethnic communities [12]. Therefore, the goal of this project was to explore the protective activity against oxidative damage on DNA and erythrocytes with antioxidant activities for future investigation toward the finding a new, potent and safe antioxidant compound as well as the possibility of using extracts of this species as potential food supplements.

Materials and methods

Plant material

The seeds of H. schulli from matured fallen were collected from the ground of Chapainawabganj, Bangladesh in December 2016. It was taxonomically identified and preserved by the Dept. of Botany, University of Rajshahi, Bangladesh with specification number 102019-37.

Preparation of extract

Collected air-dried seeds without removing the seeds coat were ground into a coarse powder using a grinder machine and the coarse powder (400 ​g) was soaked in chilled methanol (1:4; w/v) and kept in a cold room (4 °C) for 10 days with occasional stirring. The mixture was then filtered through cotton and Whatman No. 1 filter paper and concentrated in a rotary evaporator (model C-303; Sibata, Japan) at 150 mbar pressure and 40 °C temperature. The process was repeated two times. The concentrated crude methanolic extract (CME) was fractionated with the organic solvent of different potentials into petroleum ether fraction (PEF), chloroform fraction (CHF), ethyl acetate fraction (EAF), and the rest of the residue left was designated as aqueous fraction (AQF).

Experimental animal model

Healthy albino male mice (24–31 ​g) were procured from the branch of animal research, Dept. of Pharmacy, Jahangirnagar University, Dhaka, Bangladesh. The animals were kept under 12 ​h light/dark cycles at 25 °C in conventional plastic cages and nourished with regular pellet diet and tap water. All the mice were allowed 1 week to acclimate to the housing conditions before starting the experiment. The experiment on mice was conducted under the rules of the Institute of Biological Sciences (IBS), University of Rajshahi. The experimental procedure was authorized by the Institutional Animal, Medical Ethics, Biosafety, and Biosecurity Committee (IAMEBBC) at the IBS, University of Rajshahi. (Approval memo no-82/320/IAMEBBC/IBSc).

Phytochemical analysis

Total phenolic content (TPC)

Folin-Ciocalteu colorimetric method described by Meda et al. [13] was used to determine the phenolic compounds in CME and fractions. Briefly, an aliquot of 1 ml of CME/fractions solution (50 μg/ml) was mixed with 2.5 ml of Folin-Ciocalteu reagent. Then, 14% of sodium carbonate solution (0.3 ml) was added to the above mixture and incubated at room temperature for 20 min. Afterwards, the absorbance was taken at 760 nm in a spectrophotometer (Shimadzu UV Mini-1240, Japan). A calibration curve for gallic acid in the range of 10–200 μg/ml was prepared in the same manner and results were expressed as milligrams of gallic acid equivalent per gram (mg of GAE/g) of extract.

Total flavonoid content (TFC)

The total flavonoid was determined according to the aluminum chloride colorimetric method [14]. Briefly, 0.5 ​ml CME/fractions solution (200 μg/ml) was mixed with 5% NaNO2 (0.15 ​ml) solution in a test tube. After 5 min of incubation, 10% AlCl3 (0.3 ​ml), 1 ml NaOH (1 mM), and 550 μl distilled water were added. The same procedure was followed for the standard solution of quercetin and calibration curve was constructed. The absorbance was read at 510 nm and the results expressed as milligramg of quercetin equivalent per gram (mg of QE/g) of extract.

In vitro antioxidant activity

DPPH radicals scavenging activity

The radical scavenging activity of the CME/fractions was determined as described by N. Jahan et al [15]. CME and various fractions at 5–100 μg/ml were mixed separately with 1 ml of methanol solution of DPPH (0.008% w/v) The mixure was homogenized and incubated in dark for 20 min, and absorbance was measured at 517 nm. Ascorbic acid was used as the antioxidant standards. The percentage of free radical scavenging was estimated using the following equation-

Concentration providing 50% inhibition (IC50) is calculated from the graph plotted for scavenging percentage against the sample concentration.

Hydrogen peroxide scavenging activity

The ability of the CME/fractions to scavenge hydrogen peroxide was assessed by the replacement titration method [16]. Reaction medium containing 0.1 ​mM H2O2, 3% ammonium molybdate, 2M H2SO4, 1.8M KI and CME/fractions (200 μg/ml) were mixed and the solution was titrated with Na2S2O3 (5.09 ​mM) until the yellow color vanished. Scavenging activity was calculated by the following equation.

Reducing power activity

The assay was based on the reduction of colorless potassium ferricyanide [K3Fe (CN) 6] to its blue ferrous-colored form due to electrons donation by antioxidants. The ferric reducing power was expressed in terms of absorbance of the sample [17].

Total antioxidant capacity

The total antioxidant capacity of CME/fractions was determined by the spectrophotometric phosphomolybdenum method [18]. Precisely, solutions of the extracts (2 ml, 5–100 ug/ml) in methanol were incubated with 3 ml of the reaction composition (containing 0.6M sulfuric acid, 28 ​mM sodium phosphate, and 1% w/v ammonium molybdate) and incubated at 95 °C for 90 min. Then the absorbance of the cooled aqueous solution of each was measured at 695 nm against a blank.

In vivo antioxidant activity

Animal grouping

In vivo antioxidant activity was determined by using the cadmium (Cd)-induced hepatotoxicity of the mice model. After seven days of acclimatization, mice were randomly divided into seven groups of five mice each. Group-I served as normal control and group-II received 8 mg/kg body weight (b.w.) of Cd, Group–III–V were simultaneously treated with 8 mg/kg body weight (b.w.) of each and 100, 200, and 400 mg/kg of b.w of the EAF, respectively. Mice in group-VI treated with CME (400 mg/kg) and groups-VII treated with ascorbic acid (100 mg/kg) of b.w along with Cd 8 mg/kg b.w.

Preparation of serum and tissue homogenates

At the end of 7 days of treatment, all the animals were sacrificed and blood serum was collected by centrifugation (Biobase BKC-TL4IV, China) at 3000 rpm for 10 min at 4 °C temperature. The serum was used immediately for the biochemical analysis. The animals were quickly dissected and the liver and brain removed. Then these organs were homogenized and supernatants obtained after centrifugation were stored frozen overnight and used for analysis of antioxidant enzyme activities (SOD and CAT), and concentration of thiobarbituric acid reactive substances (TBARS).

Estimation of biochemical parameters

The concentration of albumin, total bilirubin, uric acid and alkaline phosphatase were determined in accordance with the principles described by Doumas et al., [19], Weigl et al., [20] and Fossati et al., [21], Akpinar [22], respectively.

Antioxidant enzymatic activities

Superoxide dismutase (SOD) activity was determined at 560 nm as described by Marklund et al., [23] and the enzyme activity was expressed as unit (U)/mg Tissue.

Another antioxidant enzyme, catalase (CAT) activity was measured at 240 nm by the Aebi method [24], and the values were expressed as μmol H2O2 decomposed/min/mg of tissue.

Thiobarbituric acid reactive substances (TBARS) assay

The mixture of tissue homogenate (10%, w/v) with SDS (8.1%, w/v) was vortexed. After 5 min, acetic acid (20%) and TBA (0.8, w/v) was added to the mixture and heated at 95 °C for 60 min. Then the mixture was centrifuged at 4000 rpm for 15 min at room temperature. Finally, the colored layer was measured at 532 nm and the concentration of MDA was calculated using the following equation [25].

Protection of human erythrocytes against oxidative damage

Protection of erythrocytes

Heparinized blood samples were obtained from healthy volunteers via venapuncture and erythrocytes were isolated by centrifugation at 2500 rpm for 10 min, washed two times with PBS and finally re-suspended using the same buffer to a hematocrit level of 5%. The erythrocytes suspension was then incubated at 37 °C in the presence or absence of CME/fractions (CHF, EAF) at different concentration with 100 μM H2O2 or 125 μM HOCl (final concentrations). CME/fractions were added to the erythrocytes suspension 10 min prior to addition of the oxidizing agents. The erythrocyte suspension incubated with PBS served as control. After 15 min of incubation the absorbance of the supernatant was taken at 540 nm and hemolysis percentage was calculated using the formula [26] –

Lipid peroxidation

Erythrocyte suspension (25%) was incubated 30 min at 37 °C with CME/fractions and gallic acid as standard, separately. Then, H2O2 (20 mM), PBS, SDS, trichloroacetic acid (0.75 ​ml 0f 28%), and TBA (0.5 ml, 1%) was added to the reaction mixtures were kept at 90 °C for 30 min. The absorbance of the resulting pink-colored layer was taken at 532 nm with centrifugation [25]. The percent inhibition was calculated using the following formula:

Protection of DNA against oxidative damage

Protection of plasmid DNA from harmful effects of Fenton's reagent was evaluated by DNA nicking assay [27] with minor modifications. Different concentrations of the sample (250 μg/ml to 50 μg/ml) and plasmid DNA (5 μg) were mixed with Fenton's reagent (0.5 μl) and incubated for 30 min at 37 °C. After incubation the reaction mixture (10 μl) was taken and electrophoresed using 1 % agarose in TBE buffer at 90 V for 1 h. The gel was stained with EtBr, viewed utilizing trans-illuminating UV light, and finally photographed were taken by gel documentation system, (Acinterlab, USA).

Statistical analysis

All the experiments are expressed as means ± SD of the responses of triplet per sample and the results of in vivo tests are expressed as means ± SD of the number of experimental animals of each group. One-way ANOVA coupled with Tukey's post hoc test at p < 0.05 was used to determine the statistical significance between groups. Microsoft Excel 2013 and Graph Pad Prism 8 software were used for the statistical analysis.

Results

The antioxidant constituents like total phenolic content were 270.63 ± 3.64 mg of GAE/gm. extract for EAF followed by CME, AQF, CHF, PEF, and 234.70 ± 5.46 mg of QE/gm. of dry extract for the total flavonoid content of EAF followed by AQF, CME, PEF, and CHF (Table 1).

Table 1

Total phenolic and flavonoid content of CME and fractions of H. schulli seeds.

Fractions	TPC (mg of GAE/g)	TFC (mg of QE/g)
CME	226.06 ± 4.35	154.45 ± 2.79
PEF	27.67 ± 4.04	104.53 ± 3.02
CHF	87.03 ± 1.80	55.75 ± 2.53
EAF	270.63 ± 3.64	479.70 ± 5.46
AQF	173.20 ± 5.82	223.61 ± 5.77

All values are expressed as mean ± SD (n = 3), SD = Standard deviation, CME = Crude methanol extract, CHF = Chloroform fraction, EAF = Ethyl acetate fraction, AQF = Aqueous fraction, GA = Gallic acid, TPC = Total phenolic content. TFC = Total flavonoid content.

The DPPH scavenging activity of the sample measured by the spectrophotometric method in terms of IC50 values were 3.7, 14, 19, 40.5, and 58.3 μg/ml for EAF, CME, AQF, CHF, and PEF respectively; compared with standard BHT (3.1 μg/ml). Further, at a concentration of 200 μg/ml, the % of scavenging exhibited by EAF (57.82 %) was most comparable with a reference standard (BHT) exhibited 86.04% in the hydrogen peroxide scavenging assay model (Table 2).

Table 2

In vitro antioxidant activity of CME, fractions, and standard.

Name of Experiment/Fractions	DPPH radical scavenging assay (IC50)	Hydrogen peroxide scavenging assay (% SCV for 200 μg/ml)	Reducing power capacity (Absorbance for 50 μg/ml)	Total Antioxidant Assay (Absorbance for 200 μg/ml)
CME	14	35.88	2.171 ± 0.002	0.811 ± 0.013
PEF	58	24.96	0.722 ± 0.005	0.322 ± 0.007
CHF	40	28.45	0.716 ± 0.003	0.410 ± 0.013
EAF	3.1	57.82	2.861 ± 0.040	1.096 ± 0.059
AQF	19	43.35	2.383 ± 0.002	0.504 ± 0.022
Standard	2 (BHT)	86.04 (BHT)	3.565 ± 0.050 (GA)	1.608 ± 0.059 (GA)

All values are expressed as mean ± SD, (n = 3). Here, SD = Standard deviation, CME = Crude methanol extract, CHF = Chloroform fraction, EAF = Ethyl acetate fraction, AQF = Aqueous fraction,.GA = Gallic acid, BHT = Butylated hydroxyl toluene.

For reducing power, the highest absorbance activity was found in EAF (3.26) with moderate AQF (2.58) compared with standard GA (4.66) at the concentration of 50 (μg/ml). Similarly for the total antioxidant assay, at the concentration of 200 (μg/ml) the highest absorbance activity were found in EAF (1.296 ± 0.059) and CME (0.811 ± 0.013) showed moderate activity as compared to reference standard GA (1.608 ± 0.059). Therefore, the EAF demonstrated greater scavenging compared with others (Table 2; Figure 1).

Figure 1

DPPH radical scavenging activity of CME and its fractions of H. schulli seeds. a: % of Scavenging activity on DPPH radical; b: IC50 value. Each value is expressed as mean ± SD (n = 3), SD = Standard deviation.

Antioxidant activity (In vivo) was performed in cadmium (Cd) intoxicated mice, and administration of Cd significantly decreased the activity of SOD (0.408 U/mg of tissue) as compared with normal mice (0.792 U/mg of tissue). Treatment with CME and EAF at 400 mg/kg in Cd intoxicated mice improved the level of SOD (0.621 and 0.774 U/mg) of tissue, respectively. Also, a significant (p < 0.05) reduction in catalase activity was noticed in liver homogenate of cadmium-induced mice (2.411) compared to the control grouped mice (4.312) as well. TBARS level showed a significant increase in Cd intoxicated mice (35.48 ± 2.214 nmol/mg tissue) comparing to the normal value (17.192 ± 0.689 nmol/mg protein). After treatment with EAF, a significant (p < 0.05) decline in TBARS levels was observed as standard ascorbic acid-treated mice (Table 3).

Table 3

In vivo antioxidant assay of CME, EAF, and standard AA.

Name of Experiment	SOD (Enzyme unit (U)/mg Tissue)	Catalase (H2O2 decomposed/min./mg of tissue)	LPO (TBARS level nmol/mg wet tissue)	Albumin (gm/dl)	Bilirubin (mg/dl)	Uric acid (mg/dl)	Alkaline phosphatase (U/l)
Positive control/Normal group	0.792	4.312 ± 0.644	17.192 ± 0.689	17.52 ± 0.526	2.36 ± 0.343	104.1 ± 3.978	20.6 ± 2.408
Negative Control(Cd 8 mg/kg)	0.408*	2.411 ± 0.557*	35.48 ± 2.215*	10.37 ± 0.873*	1.57 ± 0.306*	59.97 ± 4.725*	04.0 ± 2.646*
Cd with EAF(100 mg/kg)	0.516**	2.860 ± 0.771	29.30 ± 1.976**	11.88 ± 1.271**	1.675 ± 0.340	66.6 ± 6.195	11.75 ± 2.217**
Cd with EAF(200 mg/kg)	0.594**	3.245 ± 0.595	23.63 ± 1.186**	13.64 ± 2.230	1.84 ± 0.230**	73.40 ± 3.140**	14.60 ± 2.074**
Cd with EAF(400 mg/kg)	0.676**	3.937 ± 0.628	21.17 ± 1.018**	16.05 ± 0.354**	2.05 ± 0.212	90.8 ± 3.394**	17.00 ± 2.828**
Cd with CME(400 mg/kg)	0.568**	3.042 ± 0.543	24.65 ± 1.270**	13.03 ± 2.346	1.76 ± 0.251	65.4 ± 7.077	16.67 ± 1.528**
Standard AA(100 mg/kg)	0.686**	4.056 ± 0.339**	19.19 ± 0.901**	15.70 ± 1.098**	2.02 ± 0.330	87.42.42 ± 3.129**	18.50 ± 2.646**

All values are expressed as mean ± SD (n = 2–5); analysis of variance followed by unpaired t-test. SD = Standard deviation, CME = Crude methanol extract, CHF = Chloroform fraction, EAF = Ethyl acetate fraction, AQF = Aqueous fraction, Cd= Cadmium, AA = Ascorbic acid, SOD = Superoxide dismutase, LPO = Lipid peroxide.

*p < 0.05, significant compared with the normal group.

**p < 0.05, significant compared with Cd (negative control) group.

Similarly, in EAF400 mg/kg b.w. and CME400 mg/kg b.w. treated mice albumin (18.05 and 13.03 gm/dl) and total bilirubin (2.15 and 1.8 m mg/dl) levels were also restored towards normal value (17.19 gm/dl) and (2.26 mg/dl), respectively. Likewise, uric acid and ALP levels were decreased in the Cd-treated animals and EAF (400 mg/kg b. w.), as well as CME (400 mg/kg b.w.) of the extract, produced uric acid and ALP levels significantly (P < 0.05) Table 3.

RBC as the target of oxidation by H2O2, EAF showed 60.81% inhibition of oxidation followed by CME (48.26%), and CHF (44.77%) in the decreasing order, comparing with the standard gallic acid (63.14%). EAF various fractions also suppressed HOCl-induced oxidation of erythrocytes moderately. Similarly, a significant decrease in lipid peroxidation in erythrocytes was observed treated with EAF, CME, and CHF (Table 4 & Figure 2).

Table 4

Effect of CME, CHF, and EAF against oxidative damage of human erythrocytes.

Sample with dose	H2O2 induced assay (% of inhibition)	HOCl induced assay (% of inhibition)	Lipid peroxidation (% of inhibition)
CME (500 μg/ml)	48.26	41.19	49.61
CHF (500 μg/ml)	41.77	30.11	35.05
EAF (500 μg/ml)	62.81	57.13	70.50
GA (500 μg/ml)	86.14	84.37	91.68

All values are expressed as mean ± SD (n = 3). Here, SD = Standard deviation, CME = Crude methanol extract, CHF = Chloroform fraction, EAF = Ethyl acetate fraction, AQF = Aqueous fraction,.GA = Gallic acid.

Figure 2

Protection of Human Erythrocyte against H2O2 (a) and HOCl (b) induced oxidative damage and lipid peroxidation (c). All values are expressed as mean ± SD (n = 3) SD = Standard deviation.

The protective effect of samples by Fenton's reagent-induced oxidative DNA damage was determined in the plasmid DNA of E. coli cells based on the DNA Nicking assay. The qualitative visualization of DNA by gel electrophoresis showed that EAF at the various concentration (L3 to L7, 300 to 100 μg/ml, respectively) and GA as standard (L8, 100 μg/ml) protect DNA from destruction as compared to Fenton's reagent treated DNA (L2). Here, supercoiled DNA was broken into an open circular form compared to the untreated DNA (Figure 3).

Figure 3

DNA nicking assay. Lane-1: Untreated DNA; Lane-2: DNA + Fenton's reagent; Lane-3: DNA + Fenton's reagent + EAF (300 mg/ml); Lane-4: DNA + Fenton's reagent + EAF (250 mg/ml); Lane-5: DNA + Fenton's reagent + EAF (200 mg/ml); Lane-6: DNA + Fenton's reagent + EAF (150 mg/ml); Lane-7: DNA + Fenton's reagent + EAF (100 mg/ml); Lane-8: DNA + Fenton's reagent + Gallic Acid (100 mg/ml).

Discussion

As far as our concern, this study is the first report on the oxidative induced DNA, RBC cellular membrane protective effect, and in vivo antioxidant activities of H. schulli seeds along with repeated in vitro antioxidant activity.

Phenolic compounds or polyphenols are thought to be strong chain-breaking antioxidants [28]. Phytochemical analysis in the EAF of seeds has shown that the amount of total flavonoids and flavonols is low but the amount of total phenolic component is relatively high.

For in vitro antioxidant activity, four methods were used including the most widely used DPPH method [29] as well as hydrogen peroxide scavenging for direct measurement of radical scavenging activity. H. schulli has shown good radical quenching activity against both DPPH and hydrogen peroxide radicals. In the present study, CME and its fraction showed moderate to potent DPPH free radicals scavenging activity. As the concentration of extracts was increased the DPPH scavenging activity also increased, as indicated by percentage inhibition. EAF had the lowest IC50 value when compared with other fractions or CME and very near to standard BHT (Table 2). In H2O2 scavenging assay, all the extracts showed different levels of scavenging activity but EAF again exhibited strongest H2O2 scavenging activity compared to other fractions or CME. Although H2O2 is not very reactive, high penetrability of the cellular membrane leads to OH˚ formation when it reacts with Fe2+ or the superoxide anion radical in the cell which can initiate lipid peroxidation and cause DNA damage in the body [30].

So, the extract of H. schulli seeds specially EAF may serve as an important source of free radical scavengers Moreover, our findings observed for radical scavenging support the findings of Vijaya [31], which stated that the ethanolic extract of H. schulli seeds display potent DPPH antioxidant activities and high total phenolic and flavonoid contents.

On the other hand, reducing power and total antioxidant capacity assay methods indirectly evaluates antioxidant activity. The reducing properties of antioxidants are generally related to the presence of reductants and based on the reduction of Fe3+ to Fe2+, which are shown to break down radical chains by donating an atom [32] to act as antioxidants. It was observed that CME/fractions were able to donate electrons to ferric ions which converted to ferrous ions to form a blue complex and measured at 532 nm. The total antioxidant capacity was indicated by the reduction of molybdenum (VI) to molybdenum (V), with the formation of the green complex by the CME and its fractions. In both cases the activity was expressed in terms of absorbance. Reducing power and total antioxidant capacity were increased with increase in concentration of CME/fractions. Table 2 shows the absorbance value of 50μgm/ml for reducing power and 200μgm/ml for total antioxidant capacity assay. Among the fractions and CME, EAF showed the highest activity in both assay as it was observed for the phenolic and flavonoid contents and DPPH activity. So the results revealed that the EAF is rich in phenolic compounds, well-known reducing agents responsible for the antioxidant capacity of this fraction.

For the function of the enzymatic antioxidants, some exogenous antioxidants like vitamin A, C, E, and some metals are essential which are of dietary origin [33]. Also, dietary sources enhanced the level of non-enzymatic antioxidants such as ALP, albumin, bilirubin, uric acid at the extracellular fluid of the body that binds with extracellular free radical generating metals (iron and copper) and ensures the limited survival of reactive oxygen species such as superoxide and H2O2 in the extracellular fluid. Enzymatic antioxidants also provide natural protection against oxidative stress of the cell [34]. Therefore, simultaneous use of Cd and extractives on the animal model showed an intention of balancing the amounts of these enzymes in animal cells. In Cd-treated mice the level of SOD and catalase was decreased whereas administration of EAF at different doses significantly improves to the level of normal mice in a dose dependent manner.

Another marker of in vivo antioxidant assay is lipid peroxidation (LPO) which is thought of as the primary mechanism for Cd toxicity despite its inability to directly generate free radicals [35]. Cd raises the LPO and changes the antioxidant defense mechanism, creating oxidative damage, and treatment with EAF along with Cd showed protective effects against LPO [36]. In this study, EAF treated groups showed a dose-dependent rise of TBARS level which indicates the lipid peroxidation inhibition ability of the extractives. This evidence is in agreement with scientific reports describing the inhibitory potential of different plant extracts against lipid peroxidation [37, 38]. Thus, the potential of anti-lipid peroxidation also signs of their polyphenol content including flavonoids.

Due to the presence of a high concentration of polyunsaturated fatty acids and oxygen transport in cell membranes, erythrocytes are the main targets for ROS attack [39]. Thus, erythrocytes are very convenient for the assessment of the intracellular antioxidant activities of various compounds. Many reagents can be used to induce oxidative stress in erythrocyte test models like hydrogen peroxide, hypochlorous acid which can easily penetrate through the cell membrane. Erythrocytes were affected both from inside and outside [40, 41]. The plant extracts are capable of protecting erythrocytes against oxidative damage, thereby maintaining the basal levels of GSH and MDA concentration [42]. EAF of H. schulli showed concentration-dependent protection against evaluated hydrogen peroxide, hypochlorous acid as well as lipid peroxidation. So far no reports are available on protecting erythrocytes cell membrane damage by H. schulli seeds.

Another noble experiment of this study was the evaluation of antioxidant activity using DNA models like DNA Nicking assay in which plasmid DNA was exposed to the Fenton's reaction. Fenton's reaction produced hydroxyl radicals that induced oxidative DNA strands breaking and yield fragmented forms of DNA [43]. The addition of EAF resulted in a moderate decrease in the formation of nicked DNA and it increased the supercoiled form of DNA as well as protected from its oxidative destruction by Fenton's reagent (Figure 3). The protection of DNA indicated the presence of flavonoids and phenolic compounds like trycin and quercetin in H. shulli seeds which can prevent the production of ROS by complexing cations [44]. Extracts prepared from black raspberry [45] seed and various natural resources [46] could either stop or slow down the process of DNA damage. So these results indicate that H. schulli seeds is a potential source of antioxidants that prevent DNA damage. To the simplest of our knowledge, no previous literature has reported the DNA protecting activity by H. schulli seeds. Therefore, the results reported here would appear to be the first report.

Conclusion

Taken together our findings indicate that the methanolic extract and its fraction EAF of H. schulli (Buch.-Ham.) could have strong antioxidant activity both in vitro and in vivo. Our results also proveded that the EAF was able to protect erythrocytes and DNA against oxidative damage. Therefore these results showed that the EAF has a potential antioxidant activity that would be used for the human being. Besides, it would be a potential food supplement as it is used in various systems of traditional medicine for centuries. However, further studies are still required to isolate the novel compounds and to elucidate the molecular mechanisms underlying the effects.

Declarations

Author contribution statement

Md. Shoriful Islam: Performed the experiments; Wrote the paper.

Mst. Shahnaj Parvin: Analyzed and interpreted the data.

Md. Ekramul Islam: Conceived and designed the experiments.

Funding statement

This work was supported by National Science and Information & Communication Technology (NSICT) of Bangladesh.

Data availability statement

Data included in article/supplementary material/referenced in article.

Declaration of interests statement

The authors declare no conflict of interest.

Additional information

No additional information is available for this paper.