Cadmium biosorption: Lake waters in Bengaluru-mitigation of cadmium-induced oxidative stress by Selaginella bryopteris

ABSTRACT

Heavy metals cause alarming levels of environmental and health problems and among them Cadmium has become a threat to organisms and natural resources like soil and water alike. It accumulates in living systems thereby causing oxidative stress. Efforts are made for bioremediation of heavy metals by employing biosorption, which is a well-known economic method for removal and in the current study Selaginella bryopteris was used as biosorbent. The biosorption capacity was optimized by its physicochemical parameters such as pH, dosage, contact time, and temperature. Cadmium-induced Reactive Oxygen Species levels and the antioxidant potential of S. bryopteris in ameliorating them were studied in Drosophila melanogaster. Water-quality analysis was performed using Chemical Oxygen Demand(COD) and Biological Oxygen Demand (BOD) and effect of S.bryopteris on these parameters were also analyzed. Further the concentration of Cadmium via colorimetric assay and Atomic Absorption Spectroscopy(AAS) was employed to quantify the Cadmium in lake water samples before and after treatment with biosorbent. Fourier Transform Infrared Spectroscopy (FTIR) and Scanning Electron Microscope (SEM) were performed to characterize the surface properties for adsorptive removal of metal ions, and antioxidant studies were conducted to assess the role of S. bryopteris in suppressing oxidative stress.

KEYWORDS:

• Biosorption
• Cadmium
• heavy metal toxicity
• oxidative stress
• Selaginella bryopteris

Introduction

Among heavy metals of environmental and occupational concern, Cadmium has significant importance. It has a low average concentration in the earth’s crust, at around 0.1 mg/kg, and the highest concentration of cadmium compounds is found in sedimentary rocks, while marine phosphates have 15 mg L⁻¹ Cadmium content. Numerous industrial processes routinely employ Cadmium thus enabling its leaching into the water and soil through effluents. Cadmium is primarily used in the production of alloys, pigments, and batteries in industry. The implementation of rigorous effluent limits from plating businesses and, more recently, the introduction of general restrictions on cadmium consumption in some countries have both been connected. Administration of Cadmium acetate to rats was found to inhibit the antioxidant enzyme Super Oxide Dismutase (SOD) in liver and kidney thereby leading to enhanced lipid peroxidation in these organ systems. Through cigarette smoke, Cadmium was also found to enter the lungs and cause toxicity. Exposure to Cadmium through water and rice during gestation leads to issues in pregnancy and lactation. Cadmium is also found to have implications in decrease in male fertility, atherosclerosis, and osteotoxicity due to decreased Calcium absorption (Sharma, Rawal, & Mathew, 2015).

Heavy metals such as Cadmium are known to enhance oxidative stress by producing free radicals. Cadmium is known to produce reactive oxygen species (ROS) and reactive nitrogen species (RNS) by which they induce oxidative damage such as lipid peroxidation, protein carbonylation, DNA damage, and also depletion of cellular reductants such as glutathione (Briffa, Sinagra, & Blundell, 2020; Malik, Singh, Thakur, Kaur, & Nijhawan, 2016). Cellular accumulation of Cd(II) leads to upregulation of enzymes involved in the production of free radicals and inhibits the action of antioxidant enzymes such as Superoxide Dismutase (SOD), catalase, and glutathione reductase (Flora, Mittal, & Mehta, 2008).

Many artificial and natural chelators have antioxidant potential and were thus proposed to be used for mitigation of metal-induced oxidative stress (Malik, Singh, Thakur, Kaur, & Nijhawan, 2016). Many edible herbal chelators such as Moringa oleifera and Bacopa monnieri have been used in the removal of heavy metal from polluted water (Reddy, Seshaiah, Reddy, & Lee, 2012; Sinha & Chandra, 1990). Permissible level of Cadmium in water is 0.003 mg L⁻¹ and its Acceptable Daily Intake (ADI) in food is 0.001 mg L⁻¹. It was also found that many lake waters contain Cd(II) in them which may lead to biomagnifications of the metal toxicity. The levels of Cadmium in water can be monitored and mitigated through low cost and ecofriendly bioremediation methods such as adsorption. In silico studies can also be performed with ligands from remedial plants with antioxidant potential to screen for in vitro and in vivo antioxidant capability (Costa et al., 2018). S.bryopteris is a pteridophyte known to possess antioxidant and anti-inflammatory properties apart from its wound-healing properties and also had a potential for phytoremediation of polluted water and soil. It is also known as Sanjeevani or Laxman Booti and is well sought after for its Ayurvedic importance to combat arthritis and other inflammatory diseases, in wound healing and even as an antiviral preparation (Paswan, Srivastava, & Rao, 2020; Setyawan, 2011). In the current study, S. bryopteris was assessed for its abilities for biosorption of Cd(II) from solution and its antioxidant potential to combat metal-induced oxidative stress.

Major lakes in and around Bangalore city in Karnataka state of India were found to contain Cadmium and other heavy metals beyond permissible limits, and these metals were also found to contaminate soils and biotic population growing in the polluted ecosystem (Hamsa & Prakash, 2020; Ramesh & Krishnaiah, 2014). Cadmium is a known neurotoxin and is found to induce multiple organ toxicity in exposed organisms (Sharma, Rawal, & Mathew, 2015). Biosorptive uptake of heavy metals from water was found to be a promising measure for reduction of levels of heavy metal pollutants in water (Cheraghi, Ameri, & Moheb, 2015; Imran et al., 2019; Reddy, Seshaiah, Reddy, & Lee, 2012; Shamim, 2018). Various model organisms including Drosophila melanogaster have been extensively studied for the manifestation of heavy metal–induced oxidative stress and possible amelioration mechanisms (Fasae & Abolaji, 2022; Yang et al., 2022). Leaves from herbs have been used for biosorptive studies (Gyamfi, Yonamine, & Aniya, 1999; Reddy, Seshaiah, Reddy, & Lee, 2012; Sinha & Chandra, 1990) and in other cases for the amelioration of neurotoxicity in organisms like D. melanogaster (Flora, Mittal, & Mehta, 2008; Hosamani & Muralidhara, 2009). The current study combines the evaluation of biosorptive potential of Selaginella bryopteris along with its ameliorative effect on neurotoxicity induced by Cadmium exposure in D. melanogaster.

Materials and methods

Preparation of biosorbent

S. bryopteris was procured from local vendors at Vishakhapatnam, Andhra Pradesh, and was authenticated by Dr. V. Rama Rao, Regional Ayurveda Research Institute for Metabolic Disorders, Bengaluru. Powdered S. bryopteris was stored in air-tight containers for further biosorption studies. Biosorbent surface modification was carried out according to the procedure followed by Reddy, Seshaiah, Reddy, and Lee (2012) to enhance the cadmium biosorption. Biosorbent was pre-treated with Calcium hydroxide in a slurry of 1 mol L⁻¹ calcium hydroxide solution. The slurry was left undisturbed for 24 h at room temperature. Further, pre-treated biosorbent was washed thoroughly with deionized water to remove calcium hydroxide and dried overnight at 70°C. Biosorbent was prepared into four types of grinds by varying the size and texture. Biosorption studies were carried out on each grind to check and verify biosorption capacity. The surface characterization for functional groups and adsorption cavities was done using Fourier Transform Infrared (FTIR) Spectroscopy and Scanning Electron Microscopy (SEM- Hitachi SU 3500), respectively. The chemical characteristics of biosorbent before exposure to the metal ions were analyzed. The spectra were obtained by FT-IR (Shimadzu Corporation, Japan) within the range of 500–4500 cm⁻¹ by KBr pellet method (Imran et al., 2019).

Biosorption studies

Cadmium sulfate solution was prepared in the range of 0.2–1 mg L⁻¹ for S.bryopteris. 1000 mg L⁻¹ Cadmium solution was prepared in 1 L of distilled water and serial diluted to 0.2–1 mg L⁻¹ Cadmium salt solution of 100 ml volume each. 10 mg L⁻¹ of Cadmium was taken in a conical flask and treated with biosorbent and later the biosorbent with adsorbed metal was removed by filtration using Whatman Filter paper No. 41.

The method for measuring cadmium in trace amounts using dithizone in chloroform solution has been updated and modified to produce colors that are more stable and tolerable to interference from other metals (Saltzman, 1953). The separation of interfering substances was improved using tartaric acid as a stripping medium and trace amounts of cyanide as a suppressant. A strong alkaline solution yielded two phases in extraction. By employing hydroxylamine in the extraction and cutting down on the amount of time, the chloroform spends in contact with the alkali, stable colors were obtained and losses caused by decomposition was avoided. The reagents used were Sodium Potassium Tartrate, Sodium Hydroxide, Potassium cyanide, Hydroxylamine hydrochloride, Chloroform, Dithizone, and Tartaric acid. Chloroform layer was filtered into a cuvette. Intensity of the color developed was measured at 518 nm Optical Density (OD) against blank.

Optimization of physicochemical parameters

The physicochemical parameters required for biosorption that influence the extent of Cd uptake from the solution were varied to assess the optimum variables required for biosorption. The parameters such as pH, temperature of biosorption, contact time, and dosage were varied, and the value for which highest percentage of biosorption was recorded was used as the optimum parameter. pH influences the availability of adsorption sites for binding Cadmium and the attainment of equilibrium are influenced by temperature and availability of more active sites (dosage of biosorbent) and minimum time of contact between metal ions and biosorbent.

Percentage biosorption was calculated using the formula mentioned in equation (1)(1) $\%\mathit{biosorption}=\frac{\mathit{Co}-\mathit{Ce}}{\mathit{Co}}$(1) below:

(1) $\%\mathit{biosorption}=\frac{\mathit{Co}-\mathit{Ce}}{\mathit{Co}}$(1)

where C_o is the initial concentration and C_e is the concentration of metal ions after biosorption.

Measurement of chemical and biological oxygen demand

The Chemical Oxygen Demand, or COD, is a measurement of the amount of material that can be oxidized (combined with oxygen) in the presence of a strong chemical oxidizing agent. The method for estimation of COD which uses dichromate as oxidant was carried out by taking a wastewater sample of known volume in an excess of Potassium dichromate in the presence of sulphuric acid in presence of Silver sulfate as catalyst (Fulgheci, Rudaru, Stavarache, & Lucaciu, 2023). The Biological Oxygen Demand (BOD) is an empirical test which measures the oxygen required by the microorganisms for the biochemical degradation of organic matter to carbon dioxide and water at 20°C temperature. The test consists of determination of dissolved Oxygen (DO) prior to and the following period of 5 days. The difference between DO of the first day and fifth day is the amount of BOD (Kadakolmath, Saravanakumar, Parthiban, & Anju, 2021).

Assessment of biosorption in lake waters

Lakewater was collected from four different points of Puttenahalli lake, Madiwala lake, and Hulimavu lake in sterile bottles from 6 inches below the surface and was acidified with nitric acid as per the American Public Health Association (APHA) standards for examination of water samples (Rice & Bridgewater, 2012). The lake water was treated with S. bryopteris with optimized parameters: 1 g of biosorbent at pH 7 at 40℃ for 60 min. The treated water sample was estimated for Cd²⁺ by Atomic Absorption Spectroscopy after filtration with Whatman filter paper No. 41. The testing was done at Environmental Health and Safety Consultants Pvt. Ltd., Bangalore, according to protocol followed by Reddy, Seshaiah, Reddy, and Lee (2012).

In vitro antioxidant assays

Ethanolic extract of S. bryopteris was obtained using 70% ethanol by soxhlet extraction at 60°C for 52 h (Alara, Abdurahman, Ukaegbu, & Kabbashi, 2019). Phytochemical analysis revealed the presence of flavonoids and steroids based on which antioxidant assays were performed. Nitric oxide Radical Scavenging assay was performed using Griess Ilosvay reagent and was modified using naphthyl ethylene diamine hydrochloride (0.1% w/v). The reaction mixture containing sodium nitroprusside, phosphate buffer saline, and methanolic extract alongside standard rutin incubated at 25°C. After incubation, the reaction mixture was mixed with sulfanilic acid reagent followed by naphthyl ethylene diamine dihydrochloride and allowed to stand. The absorbance of these solutions was measured at 540 nm against the corresponding blank solution (Garat, 1964).

Hydroxyl Radical Scavenging Assay was assayed using the reaction mixture containing 2-deoxy-2 ribose, 500 μl was added to varying concentrations of the extract along with 200 μM FeCl₃ and 1.04 mM EDTA, H₂O₂ (1.0 mM) and 1 mM ascorbic acid. After incubation period, the extent of deoxyribose degradation was measured by the TBA reaction. The absorbance was measured at 532 nm against blank. Vitamin E was used as a positive control (Halliwell, Gutteridge, & Aruoma, 1987). The percentage of scavenging activity was calculated by following Equation (2)(2) $\%\mathit{scavenging}\mathit{activity}=\left[\frac{\mathit{Ao}-\left(\mathit{A}1-\mathit{A}2\right)}{\mathit{Ao}}\right]\times 100\mathrm{\%}$(2)

(2) $\%\mathit{scavenging}\mathit{activity}=\left[\frac{\mathit{Ao}-\left(\mathit{A}1-\mathit{A}2\right)}{\mathit{Ao}}\right]\times 100\mathrm{\%}$(2)

where A₀ is the absorbance of the control without a sample. A₁ is the absorbance after adding the sample and 2-deoxy-D-ribose. A₂ is the absorbance of the sample devoid of 2-deoxy-D-ribose. Then, the percentage of inhibition was plotted against concentration.

The antioxidant activity was also assayed by the DPPH (2,2-diphenyl-1-picrylhydrazyl) assay by procedure given by (Gyamfi, Yonamine, & Aniya, 1999). 50 μl of ethanolic extract yielding 100 μg/ml in each reaction mixture was mixed with 0.1 mM DPPH in methanol solution and 50 mM Tris-HCl buffer (pH 7.4). 50 μl of methanol was used as the experimental control. After 30 min of incubation at room temperature, the reduction in the number of DPPH free radicals was measured reading absorbance at 517 nm. α-tocopherol was used as control against which the antioxidant potential was compared and evaluated. The percent inhibition was calculated from following Equation (3)(3) $\%\mathit{Inhibition}=\text{break}\frac{\left[\mathit{Absorbance}\mathit{of}\mathit{control}-\mathit{Absorbance}\mathit{of}\mathit{test}\mathit{sample}\right]}{\mathit{Absorbance}\mathit{of}\mathit{control}}\ast 100\mathrm{\%}$(3) :

(3) $\%\mathit{Inhibition}=\text{break}\frac{\left[\mathit{Absorbance}\mathit{of}\mathit{control}-\mathit{Absorbance}\mathit{of}\mathit{test}\mathit{sample}\right]}{\mathit{Absorbance}\mathit{of}\mathit{control}}\ast 100\mathrm{\%}$(3)

In vivo antioxidant assays

Fruit flies were obtained from Manasagangothri, Mysore University, and the medium was prepared by dissolving corn flour, D-Glucose, Sugar, Agar, and yeast powder in distilled water and after autoclaving and cooling to 55°C Triethylene glycol (TEGO) solution and ortho-phosphoric acid and propionic acid were added to the medium (Sundararajan et al., 2019). Cadmium sulfate was dissolved in the water used for medium to expose the flies for intake of metal ions. S. bryopteris dried extract powder was used for pretreatment of water used for the medium to adsorb Cadmium. The flies raised in the metal containing medium were exposed to various concentrations of metal ions ranging from 0 to 100 mg L⁻¹ in various tubes to determine LD₅₀ value of Cd(II) for Drosophila. Each culture tube was inoculated with 10 flies, and negative geotaxis assay was performed at concentration below LD₅₀ to assess the ability of flies to cross 15 cm of height in the culture tube (Yang et al., 2022). Further, the exposed flies were anesthetized and the heads were excised from the rest of the body using scalpel. The brain samples were homogenized by adding phosphate buffer to assess the levels of oxidative stress induced by Cadmium exposure. The mitigation of Cadmium uptake after administration of S. bryopteris along with metal ions in the water used for preparation of the medium also may indicate reduction in oxidative stress in the flies. The assays were performed in triplicates along with negative controls (Hosamani & Muralidhara, 2009) to assess the antioxidant potential of S. bryopteris.

Catalase test

The presence of the enzyme catalase is evident when a small concentration of hydrogen peroxide is introduced into homogenate, and the rapid elaboration of oxygen bubbles occurs. Catalase activity was determined by the method of Aebi (1984). The enzyme activity was expressed as µmol H₂O₂ consumed. Briefly, the reaction mixture consisting of homogenized and thawed brain sample and phosphate buffer (0.1 M, pH 7.0) and 8.8 mM H₂O₂ were incubated, and decrease in absorbance at 240 nm was measured using a UV-Visible Spectrophotometry.

Protein carbonyl test

Protein carbonyl content was determined according to the method of Levine et al. (1990). Briefly, a homogenate in 20 mM Tris-HCl- 0.14 M NaCl (pH 7.4) was made and centrifuged at 10,000 g for 10 min at 4ºC. 100 µl of the supernatant 100 µl of 20% TriChloro Acetic acid (TCA) was added and centrifuged at 10,000 g for 10 min at 4ºC. The supernatant was discarded and pellet was re-suspended in 10 mM DNPH (Dinitro Phenyl Hydrazine) and kept at dark for 1 h with occasional mixing. 20%TCA was added to precipitate protein, and pellet was washed in acetone and dissolved in 2% Sodium Dodecyl Sulphate (SDS) prepared in 20 mM Tris-HCl. The absorbance was read at 570 nm and the results expressed as ƞ moles carbonyls/mg protein using Molar Extinction Coefficient (MEC)-22.0 mM⁻¹cm⁻¹.

Results and discussion

The initial biosorption studies reveal that S.bryopteris was successful in removing Cd(II) with an average biosorption percentage of 82%. Different grinds of S. bryopteris were tested to study which had the best biosorption capacity 1 g of fine-grinded, semi-grinded, large-grinded, and Ca(OH)₂ treated fine-grind biosorbent were assessed for metal uptake. It was observed that the fine-ground biosorbent had the most biosorption potential, with an average removal percentage of 94.44% (Figure 1). There have been studies on spent coffee grounds indicating the difference in adsorption of cadmium based on adsorbant particle size (Gora et al., 2022).

Figure 1. Biosorption of Cadmium by various grades of ground Selaginella bryopteris.

Characterization of biosorbent

FTIR spectra showed prominent bands of absorbance at around 606, 688, 1046, 1249, 1452, 1610, 1640, 1734, 2199, 2870, 2927, 3423, 3701, and 3783 cm⁻¹(Figure 2). The observed peaks denote -CH₃ rocking vibration; C-H, C=N, N-H, and O-H stretching vibrations which correspond to methyl benzenes, ketones, oximes/imines, amides, ether, and aromatic rings on the surface of S. bryopteris.

Figure 2. FTIR spectrum of Selaginella bryopteris fine ground powder.

SEM was used to characterize surface morphology and topography. The biosorbent was observed to have a rough surface structure with flaky micro-particles of which can be inferred to mean the possible presence of cavities for biosorption. Cheraghi, Ameri, and Moheb (2015) observed that the sesame leaves used as biosorbent displayed rough and irregular texture which could increase the area for interaction between the pollutants ions and the adsorbent surface, and was later smoothened out after cadmium adsorption. Many other studies also have noticed similar changes in texture after metal adsorption (Ighalo & Adeniyi, 2020). In the current study, the SEM was performed for magnification of 5, 10, 20, 50, and 100 μm (Figure 3).

Figure 3. Scanning electron microscopy images of Selaginella bryopteris.

Biosorption studies

Investigation of biosorption parameters

One of the crucial factors in biosorption is pH because it changes the ionization state of the binding groups of both the adsorbent and the adsorbate. Metal ions being positively charged bind to the adsorbent effectively between pH 7 and 8 due to optimal presence of protons in the solution. The effect of pH on biosorption of cadmium onto S. bryopteris was evaluated and results represented graphically (Figure 4). From pH 2 to 6, there was an increase in the biosorption and 94.62% removal was observed at pH 6–8 for 10 mg L⁻¹ concentration of cadmium. The biosorption decreased from pH 8 to 12.

Figure 4. Optimization of physicochemical factors for biosorption of Cadmium.

The biosorption capability changes as a result of changes in temperature. From 10 to 40°C, it was seen that the percentage of biosorption increased. For a concentration of 10 mg L⁻¹, 99.46% removal was seen at 40°C. Adsorbent dosage is one of the important factors which affect the adsorption process significantly. The presence of many sites or cavities for adsorption enhances biosorption but also reduces the extent of biosorption due to crowding effect. It was observed that for a biosorbent dosage of 1 g at optimum pH and temperature, the average biosorption percentage after three trials was 95.16%. Various studies have reported the importance of optimum temperature and pH for heavy metal removal using non-living organic materials (Shamim, 2018).

The duration of the contact time between metal ions and biosorbent ranged from 15 to 90 min. When S. bryopteris biosorbent and metal ions were in contact for 60 min, the average percentage of Cadmium biosorption was observed to be 97.31%. Increase in contact time has a saturating effect on the binding of metal ions. With increasing time, more ions bind with greater affinity which does not change after prolonged incubation. These results are consistent with the observations of Reddy, Seshaiah, Reddy, and Lee (2012) in case of Cd(II) biosorption by Moringa oleifera. Maximum biosorption was, however, observed at pH 6–8 and the effect of contact time also at an early time of 20 min and other parameters were also in line with the observations made by Reddy, Seshaiah, Reddy, and Lee (2012) and Ahalya, Kanamadi, and Ramachandra (2005). This result illustrates the fact that for optimum biosorption, additional sites are to be available. Thus in the current study, by increasing the biomass levels, number of sites accessible for biosorption site were augmented.

Biosorption was enhanced sharply with contact time in the first 20 min, and equilibrium was attained within 60 min. It was observed that as contact time increases, metal uptakes elevate initially and become approximately stable, depicting equilibrium. This trend of metal uptake may be because, initially, all adsorption sites were unoccupied and the metal ion concentration was high. The optimized parameters based on highest percentage biosorption were recorded and further utilized for removal of Cadmium for lake waters. The recorded parameters are shown in the table below in Table 1.

Table 1. Optimum parameters for biosorption of Cd²⁺ by Selaginella bryopteris.

Parameters	pH	Temperature	Dosage	Contact time
Optimum value	6–8	40°C	1 g	60 minutes

Chemical oxygen demand and biological oxygen demand

COD and BOD are indications of the organic pollutants that are oxidizable and are often indicative of organic pollutants that may be biodegradable or chemically oxidizable. High levels of COD may be indicative of high content of decaying plant biomass, human waste, or even industrial effluents in water bodies. A COD value of 60–2000 mg O₂ L⁻¹ is usually found in industrial effluents. The results of treatment with biosorbent were evidence for the presence of these contaminants in sewage treatment plant found in the campus and also in partially treated water used for watering plants in the campus. The decrease in COD of lake water (Table 2) could indicate the adsorption of organic contaminants by the biosorbent (Ramesh & Krishnaiah, 2014).

Table 2. COD levels of treated and untreated samples.

	Sample 1 DW blank	Sample 2 Sewage water raw	Sample 3 Sewage water filtered	Sample 4 Lake water untreated	Sample 5 Lake water Selaginella treated
COD (mg/L)	-	60	40	32	0

Although the lake water did not show any indication of BOD levels, treated sewage water from the plant showed decrease in BOD levels taken in a 5 day study indicating the presence of microbial contaminants. High BOD indicates requirement of higher dissolved oxygen for the breakdown of contaminants. A level of 20–100 mg L⁻¹ indicates slight level of pollution and clearly the reduction of BOD 5 days after treatment with biosorbent (Table 3) indicates removal of microbial pollutants according to APHA standard testing protocols (Rice & Bridgewater, 2012).

Table 3. BOD levels of treated and untreated samples.

	Control		Samples
	Sample 1 Sewage water raw	Sample 2 Sewage water filtered	Sample 3 Lake water before treatment	Sample 4 Lake water treated with Selaginella
Day 1	7.9	8	7.3	5.7
Day 5	5.9	6.9	7.1	-
BOD (mg/L)	40	2	-	-

Assessment of Cadmium in lake waters

Lake waters collected from six different locations of every lake were acidified and analyzed for the presence of Cd(II). However, Madiwala lake and Hulimavu lake did not show the presence of Cadmium in their waters. However, the presence of Cd(II) was slightly above the permissible limits of 0.003 mg L⁻¹ (Table 4) which makes it unsuitable for consumption.

Table 4. AAS results of treated and untreated lake water sample: BDL indicates below detection levels (<0.044ng/L or < 0.044 × 10⁻³ ppm).

	Madiwala lake (mg/L)	Puttenahalli lake (mg/L)	Hulimavu lake (mg/L)
Untreated Lake water	BDL	0.006	0.007
Treated Lake water	BDL	BDL	BDL

The results of COD, BOD, and AAS analysis emphasize the role of Cadmium uptake and uptake of other organic pollutants and microbes from polluted water by S. bryopteris. This may be explained by the antioxidant activity assays performed further and due to the presence of hydroxyl, ketone and ester groups on the surface which may anchor to the pollutants and other positively charged metal ions.

In vitro antioxidant assays

Phytochemical analysis revealed that ethanolic extract of S.bryopteris contained proteins and carbohydrates. Secondary metabolites such as alkaloids, flavonoids, steroids, saponins, glycosides, and tannins were also found to be present. This substantiates the presence of antioxidants well known as classes of compounds belonging to the class flavonoids, glycosides, and steroids required to reduced cadmium-induced oxidative stress and thereby protect the organism from oxidative damage caused by cadmium (Paswan, Verma, Azmi, Srivastava, & Venkateswara Rao, 2021). Dietary intake of antioxidants in regular form is known to reduce the bioavailability of toxic compounds and also mitigate the toxicity caused by the free radicals released due to oxidative stress (Asejeje, Ogunro, Asejeje, Adewumi, & Abolaji, 2023; Briffa, Sinagra, & Blundell, 2020).

Nitric oxide radical scavenging assay

Nitric oxide is classified as a free radical because of its unpaired electron and displays important reactivity with certain types of proteins and other free radicals such as superoxide (Balakrishnan et al., 2013). The toxicity of NO increases greatly when it reacts with the superoxide radical, forming the highly reactive peroxy nitrite anion (ONOO−). Nitric oxide has been presented to be directly scavenged by flavonoids (Buxton, Greenstock, Helman, & Ross, 1988).

It can be seen from the nitric oxide scavenging assay results (Figure 5) that the extract possessed an antioxidant potential similar to rutin which was already reported to mitigate Cr (VI)-induced oxidative stress (Calabrese, Boyd-Kimball, Scapagnini, & Butterfield, 2004). The results depict an increase in nitric oxide scavenging with increasing concentration of the extract. 100 µg/ml concentration of the extract was found to depict upto 98% inhibition of nitric oxides which leaves a very reduced amount of free radicals (induced by nitric oxide) and thereby lesser extent of oxidative damage (Oyeleke & Owoyele, 2022).

Figure 5. Inhibition of Nitric Oxide by methanolic extract of S. bryopteris and standard (Rutin).

Hydroxyl radical scavenging assay

Hydroxyl radicals are generated by Fenton-like reaction. Many flavonoids, glycosides, and steroids were earlier reported to scavenge hydroxyl radical formation (.OH) generated by ultraviolet (UV) photolysis of hydrogen peroxide. Further, the results of Nitric oxide scavenging and Hydroxyl radical scavenging assays have supported the antioxidant potency of the extract; as the extract has demonstrated a free radical scavenging ability on par with antioxidants such as rutin and α- tocopherol. The increasing inhibition of hydroxyl radicals with increasing concentrations of plant extract as well as α-tocopherol may be attributed to the presence of double bonds, hydrogens and free electrons as in the latter (Zhou et al., 2020). 100 µg/ml of plant extract exhibited 90% inhibition compared to α-tocopherol showing 88% inhibition of hydroxyl radicals (Figure 6). The ethanolic extract of S. bryopteris depicts a dose-dependent scavenging of reactive oxygen species as is evident from the experimental data. While 20 µg/ml of the extract elicits 20.65% inhibition, it was also observed that 100 µg/ml of the extract demonstrated 90.65% inhibition of free radicals which amounts to approximately 4.5-fold increase in hydroxyl radical inhibitory activity for a fivefold increase in concentration.

Figure 6. Hydroxyl radical scavenging activity by S.Bryopteris extract and Vitamin E as standard.

DPPH assay

DPPH(1,1-Diphenyl-2-picrylhydrazyl) is a stable free radical with red color (absorbed at 517 nm). If free radicals have been scavenged, DPPH will generate its color to yellow. This assay uses this character to show biosorbent free radical scavenging activity. DPPH is a stable nitrogen-centered free radical commonly used for testing radical scavenging activity of the compound or plant extracts. When the stable DPPH radical accepts an electron from the antioxidant compound, the violet color of the DPPH radical was reduced to yellow-colored diphenylpicryl hydrazine radical which was measured colorimetrically. Substances which are able to perform this reaction can be considered as antioxidants and therefore radical scavengers. A potential antioxidant that depicts promising DPPH radical scavenging effect may also inhibit some of the various mechanisms of lipid peroxidation. It is, therefore, essential that plant extracts show dose-dependent inhibition of DPPH activity proportional to the activity of standard antioxidant molecules (Zhou et al., 2020).

These results indicate the potency of S.bryopteris on par with α-tocopherol which is a form of vitamin E highly known for its antioxidant activity which may further aid the reduction of oxidative free radicals. With increasing concentration of the plant extract, it is found that 100 µg/mL of the extract was found to elicit highest percentage of 94% inhibition of free radicals similar to that of rutin (Figure 7).

Figure 7. DPPH assay using ethanolic extract of S. bryopteris and α-tocopherol.

Oxidative stress parameters

Oxidative stress may pose a hallmark of various neurodegenerative diseases such as Alzheimer’s disease (AD), Huntington’s disease (HD), and Parkinson’s disease (PD). Collectively, ROS can lead to oxidation of proteins and DNA, peroxidation of lipids, and, ultimately, cell death. The assay of carbonyl groups in proteins, MDA levels for lipid peroxidation and reactive oxygen species by colorimetric methods provides a convenient technique for detecting and quantifying oxidative modification of proteins. Drosophila melanogaster were cultured in culture vials containing growth medium and further 10 flies were transferred into each vial to study the toxicity of Cadmium and the reversal of toxicity by S.bryopteris.

Vials in which 50 mg L⁻¹ of Cd(II) concentration was found in medium showed that 5 flies had survived out of 10 initially inoculated, whereas in the positive control vial with 0 mg L⁻¹ concentration of Cd(II), all 10 flies survived until the medium was exhausted after 96 h. From the observation of the above tests, it was inferred that the LD₅₀ value of Cd(II) in Drosophila was 50 mg L⁻¹. The LD₅₀ value determines the dose–response relationship required to study oxidative stress in the flies in which Cadmium mediates toxicity (Figure 8). This was further validated by the negative geotaxis assay (Figure 9) which highlights the decline in locomotor ability of the flies in the figure shown below. The time required by the flies to move a distance of 25 cm was recorded against an exposure of Cadmium, and it was found that increasing concentration deprived the locomotor abilities of the flies in an increasing extent thereby indicating metal-induced neurotoxicity. The impaired ability of the metal exposed flies is an indication of oxidative stress built up in the nervous system and the loss of functional macromolecules leading to impaired neuromuscular function (Asejeje, Ogunro, Asejeje, Adewumi, & Abolaji, 2023; Hosamani & Muralidhara, 2009).

Figure 8. Fly survival curve of Cadmium exposure in Drosophila melanogaster.

Figure 9. Negative Geotaxis assay for behavioral disorder in Drosophila melanogaster.

This was followed by assessment on the level of Cadmium uptake from water in the medium thereby mitigating its intake and effect on the flies. The water used in medium preparation was subjected to biosorption at optimal parameters obtained earlier and 1 g of biosorbent was used to treat 100 ml of water containing 30 mg L⁻¹ of Cadmium. The results of negative geotaxis indicate that biosorbent exposed water usage has improved the fly performance. Oxidative stress parameters (protein carbonyls) and assays of antioxidant enzymes such as catalase further validated our findings from in vitro antioxidant potency.

Catalase activity

The flies were assessed for scavenging potential of the enzyme catalase in restoring the oxidative balance in brain of D.melanogaster. The biosorbent-induced catalase activity did not show significant increase in the brain of the fruit flies but the increase was consistently observed as much as 3.5–5% in various concentrations of Cadmium-treated water (Figure 10). This was in contrast with the results obtained when hesperidin was used to ameliorate Cd(II)-induced oxidative stress in D. melanogaster where up to 33% enhancement of catalase was observed due to the administration of the flavonoid (Asejeje, Ogunro, Asejeje, Adewumi, & Abolaji, 2023). In control flies that were not exposed to Cadmium concentration nor to the biosorbent, the catalase activity was found to be 0.022 ± 0.00042 µmol H₂O₂ where as in the flies exposed to 20 mg L⁻¹ of Cd(II) along with the biosorbent showed an activity of 0.026 ± 0.00346 µmol H₂O₂ which is not significantly high compared to the control group without oxidative stress. The in vivo antioxidant activity of S. bryopteris was still not found to be as significant as expected.

Figure 10. Catalase assay in brain samples of Drosophila before and after exposure to biosorbent in water containing various concentrations of Cadmium (II).

Protein carbonyl assay

Carbonylation is frequently used as an oxidative stress biomarker, which is a non-enzymatic post-translational modification. Heavy metals are indirect causes of oxidative stress in plants, which is a direct cause of increased carbonylation of proteins. Protein carbonyls activity showed that a concentration-dependent measure in activity. Protein carbonyls are produced due to the oxidative stress induced by Cd(II) which is concentration-dependent. The amount of protein carbonyls produced due to modification of side chain increased with increase in Cd(II) concentration. However, it was observed that there was a significant reduction in protein damage up to 14% at 0.1, 0.15, and 0.2 µg/ml of Cadmium water which was used in Drosophila medium. The control showed no improvement in protein carbonyls as there was an absence of metal ion before (0.35 ± 0.015 nmol/mg of protein) and also after treatment of water with S. bryopteris biosorbent (0.256 ± 0.015 nmol/mg of protein) (Figure 11). In the flies exposed to the highest metal concentration of 20 mg L^−1,it was found that there was 14.28% reduction in the protein carbonyl content after treatment of water with S. bryopteris. The levels of protein carbonyls reduced from 1.54 ± 0.0115 nmol/mg protein before treatment to 1.32 0.0115 nmol/mg protein after treatment. These results are similar to the reduction in protein carbonyls as observed by Asejeje, Ogunro, Asejeje, Adewumi, and Abolaji (2023) who reported up to 20% reduction in protein carbonyls with the administration of the flavonoid hesperidin in the food of D. melanogaster.

Figure 11. Protein Carbonyls in brain samples of Drosophila before and after exposure to biosorbent in water containing various concentrations of Cadmium (II).

Conclusions

Cadmium is a known toxin in the environment resulting from many human activities. Its deposition in lake waters poses serious health hazards and immediate measures are required for mitigation from polluted water. Plant-based biosorbents are ecofriendly, economic, ubiquitous, and known for their reusability and therapeutic effects. In our study, we aim to study the biosorption and antioxidant potency which has been successfully validated by in vitro antioxidant assays and in vivo oxidative stress markers. S. bryopteris also reduces pollutants in water as seen with BOD, COD, and lakewater analysis. Many such antioxidants can be screened further for amelioration of metal-induced oxidative stress in various model organisms such as zebra fish and rats. The potential of plant materials in uptake of metal ions and pollutants from water bodies is a field that requires vast exploration and the biomagnification of metal ions across various species in the ecosystem also needs to be checked for reduced toxicity. The plant may also contain the ability to rejuvenate free radical-injured tissues back to normalcy, thereby facilitating exit of Cadmium from accumulated tissues and repair of damage induced by oxidative stress, thus rejuvenating the tissues.

Acknowledgments

The authors are thankful to the Management and staff of Dayananda Sagar College of Engineering for supporting the work. We are also grateful to the Management of Kristu Jayanti College, Autonomous for their constant support.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by the no funders [NA].