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Evaluation of the in vitro and in vivo antioxidant activity of Albizia adianthifolia Schumach (Fabaceae) stem bark
, Florence Nwakaego Mbaoji1, Collins Azubuike Onyeto1
*Corresponding author: Chinonyelum Emmanuel Agbo, B.Pharm, Department of Pharmacology and Toxicology, University of Nigeria, Nsukka 410001 Enugu State, Nigeria chinonyelumagbo1010@gmail.com
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Received: ,
Accepted: ,
How to cite this article: Peter IM, Emenalo MC, Agbo CE, et al. Evaluation of the in vitro and in vivo antioxidant activity of Albizia adianthifolia Schumach (Fabaceae) stem bark. Am J Pharmacother Pharm Sci. 2026:013
Abstract
Objectives:
This study assessed the in vitro and in vivo antioxidant activities of the methanol extract of A. adianthifolia stem bark (MEAA).
Materials and Methods:
The in vitro antioxidant parameters assayed included diphenylpicrylhydrazine (DPPH), ferric reducing antioxidant power (FRAP), total antioxidant capacity (TAC), metal ion chelating activity, thiobarbituric acid reactive species (TBARS) and nitric oxide (NO) while superoxide dismutase (SOD), reduced glutathione (GSH), malondialdehyde (MDA) and catalase (CAT) were evaluated in vivo in adult Wistar albino rats after oral administration of the extract (100, 200 and 400 mg/kg) and distilled water (5 ml/kg) for 14 days. Acute toxicity test and phytochemical analysis were also conducted.
Results:
The acute toxicity (LD50) was estimated to be 4246 mg/kg. Phytochemical analysis of the extract revealed the presence of flavonoids, phenolic compounds, tannins, alkaloids, saponin, terpenoids and steroids. The IC50 values of the in vitro antioxidant studies were 4.1 ± 0.23, 8.70 ± 2.48, 232.10 ± 69.11, 32.29 ± 16.70, 27.99 ± 5.96, and 73.32 ± 49.39 µg/ml for DPPH, FRAP, TAC, metal ion, TBARS, and NO, respectively. The extract significantly (p <0.05) elevated SOD, GSH and CAT levels, whereas MDA was significantly (p <0.05) reduced when compared to the negative control. The MEAA showed considerable in vitro and robust in vivo antioxidant properties.
Conclusion:
Our findings justify the use of A. adianthifolia for the management of oxidative stress-related conditions in traditional medicine. Studies are ongoing to isolate the active molecule(s).
Keywords
Albizia adianthifolia
Antioxidant
In vitro
In vivo
Oxidative stress
INTRODUCTION
The generation of oxidants is a common occurrence during aerobic metabolism. Reactive oxygen species (ROS) or free radicals, such as superoxide anions, hydroxyl radicals, and hydrogen peroxide, are produced when there is an imbalance between the generation of ROS and the antioxidant defense system.[1] The accumulation of free radicals in body organs or tissues can cause oxidative damage to cell biomolecules and membranes, which can lead to inflammation, chronic diseases, and other disorders.[2] Radiation, bacterial and viral toxins, smoking, alcohol, and psychological or emotional stress have all been linked to the production of ROS in cells.[3] An imbalance between the production and breakdown of ROS promotes inflammation-induced vascular damage and endothelial dysfunction, which leads to nitric oxide (NO) deficiency and facilitates vasoconstriction and elevated blood pressure.[4,5] Oxidative stress results from a chemical reaction between superoxide anion and NO. This leads to the formation of peroxynitrite, which contributes to oxidative stress by enhancing lipid peroxidation and mitochondrial dysfunction.[6]
Antioxidants play an important role in suppressing ROS activity and preventing oxidative damage from free radicals.[7] Through this, there is a maintenance of cellular health and redox homeostasis, which is essential in reducing the risk of chronic diseases.[7] Various synthetic agents that serve as antioxidants have been developed and have shown efficacy in reducing oxidative stress and scavenging ROS. However, their use is limited by some shortcomings, such as high cost, poor availability, and intolerable adverse effects.[8] Therefore, research has focused on developing safe novel agents to solve these challenges. Given the recent interest in medicinal plants as alternatives in the treatment of various ailments, researchers are in constant search for safe, efficacious, cost-effective, and readily available materials with antioxidant properties.
Albizia adianthifolia Schumach is a tree in the family of Fabaceae, commonly known as flat-crown.[9] It is characterized by fluffy flowers and heavy-scented green leaves with a spreading, flat-crown shape, reaching 35 m in height, with axillary inflorescences of reddish to greenish-white flowers, and flat, dehiscent pods upon maturity.[9] A. adianthifolia has been used in tropical Africa as a herbal medicine for a range of conditions, including diabetes, gastrointestinal complaints, headaches, respiratory problems, and skin diseases.[10] A. adianthifolia has been studied experimentally for its pharmacological effects. Studies have reported its antiplasmodial,[11] antimicrobial,[12] antiepileptic,[13] apoptotic,[14] memory-enhancing,[15] anxiolytic, and antidepressant[16] properties.
Despite the extensive studies on this plant, there is a lack of a comprehensive evaluation of its in vivo and in vitro antioxidant properties. Given the traditional use of the extract of this plant for antioxidant purposes, and the need to develop novel agents from natural sources, we performed an experimental evaluation of the antioxidant properties of the methanol extract of A. adianthifolia stem bark.
MATERIALS AND METHODS
Plant collection and extraction
The stem bark of A. adianthifolia was collected in November 2021 from the Orba Local Government Area of Nsukka, Enugu State, Nigeria. The plant was identified and authenticated by Mr. Alfred Ozioko, a botanist at the International Centre for Drug Discovery and Development, Nsukka, Enugu State. A voucher specimen (voucher number CEDD/16046) was deposited in the department of pharmacology for future reference.
The stem bark of A. adianthifolia was chopped into small pieces using a table knife and air-dried under ambient laboratory conditions. Afterward, the dried materials were crushed and powdered using a grinding mill. The powdered plant material (2 kg) was weighed out and macerated with 10 L of methanol for 72 h at room temperature with intermittent shaking. At the end of 72 h, the filtrate was first separated from the marc using a filter cloth and further filtered by using Whatman No. 1 filter paper to obtain the methanol extract of the stem bark of A. adianthifolia (MEAA). The filtrate was thereafter concentrated using a rotary evaporator under reduced pressure.
Experimental animals
One hundred and twenty adult Wistar albino rats weighing 120–150 g of both sexes were used in this study. All the animals were obtained from the animal house of the Department of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, University of Nigeria, Nsukka. Throughout the experiment, the animals were kept in sterile gauze cages under regular laboratory conditions. They were provided with standardized pellets and clean portable water ad libitum. The institutional ethical rules for animal research were followed in all animal trials. Furthermore, ethical approval was obtained from the Faculty of Pharmaceutical Sciences Research Ethics Committee with reference number: FPSRA/UNN/22/0042.
Phytochemical analysis
The qualitative and quantitative phytochemical analyses were conducted using the methods described by Harborne[17] and Madhu et al.[18]
Acute toxicity test
An acute toxicity test was conducted using Lorke’s method.[19] For the first phase, three animals each were randomized into three groups. Group A, Group B, and Group C received 10, 100, and 1000 mg/kg of MEAA, respectively. The animals were constantly observed for the first 2 h, intermittently for the next 4 h, and then overnight. Then, at the end of 24 h, the number of mortalities was noted for each group. The second phase was conducted using the results obtained from the first phase, where four groups made up of one animal each received doses of 1600, 2900, 3600, and 5000 mg/kg, respectively. Appropriate calculations were done to obtain the lethal (LD)50.
In vitro antioxidant studies
2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay
Using methods adapted from Gyamfi et al.,[20] the scavenging activity on DPPH free radicals by MEAA was determined. Various concentrations of 1 mL MEAA diluted 2-fold in 80% methanol were mixed with 0.5 mL of 0.076 mM DPPH in methanol. After a thorough shake, the mixture was allowed to stand for 25 min in the dark at room temperature. The 1 mL of 0.076 mM DPPH in methanol served as the negative control, while L-ascorbic acid was used as the positive control. Thereafter, the absorbance of the assay mixture was measured at 517 nm using a ultraviolet-visible spectrophotometer, and methanol was used as the reference blank. Appropriate calculations of DPPH radical scavenging activity were made using the equation.[20]
Total antioxidant capacity (TAC) assay
The TAC assay of MEAA was conducted using methods described by Prieto et al.,[21] A 0.1 mL aliquot of MEAA at different concentrations and ascorbic acid was mixed with 1 mL of reagent solution containing 600 mM sulphuric acid, 28 mM sodium phosphate, and 1% ammonium molybdenum. To ensure completion of the reaction, the test tubes were incubated in a water bath at 95°C for 10 min. Cooling of the extracts to room temperature preceded the measurement of the absorbance of the mixture at 765 nm against a blank containing 1 mL of the reagent solution. Ascorbic acid was used as a standard. Appropriate calculations were made, and the TAC was estimated using the formula:
Ferric reducing antioxidant power (FRAP) assay
The FRAP assay was performed following the procedure of Benzie and Strain,[22] with minor modifications. Briefly, 2 mL of MEAA was mixed with 2 mL of 0.2 M phosphate buffer (pH 6.6) and 2 mL of 0.1% (w/v) potassium ferricyanide. The mixture was then incubated in a water bath at 50°C for 20 min. After incubation, 2 mL of 10% (w/v) trichloroacetic acid (TCA) was added to stop the reaction. Finally, 2 mL of the resulting mixture was diluted with 2 mL of distilled water for subsequent analysis.
Thiobarbituric acid reactive species (TBARS) assay
The method described by Banerjee et al.[23] was used to conduct the TBARS assay. 100 µL of MEAA (10–250 µL/mL) and egg homogenate were added to a test tube, which was then made up to 1.0 mL with distilled water. To initiate peroxidation, 50 µL of 0.075 M FeSO4 and 20 µL of 0.1 M L-ascorbic acid were added, and the mixture was incubated at 37°C for 1 h. After incubation, 0.2 mL of 0.1 M ethylenediaminetetraacetic acid (EDTA) and 1.5 mL of thiobarbituric acid reagent were added, and then heated at 100°C for 15 min. The samples were then cooled and centrifuged at 3000 rpm for 10 min using a centrifuge. Butylated hydroxytoluene (BHT) was used as the reference standard antioxidant. The absorbance of the resulting supernatant was measured at 532 nm, and the percentage inhibition of lipid peroxidation was determined using the appropriate formula.
NO assay
To 100 µL of MEAA, 3 mL of 10 mmoL/L sodium nitroprusside in phosphate buffer saline 0.2 mmoL/L was added. After 150 min of incubation at 25°C, in accordance with established NO scavenging assay protocols, 500 µL of Griess reagent was added. A standard curve was used to extrapolate the concentration and measure the absorbance at 546 nm.
Metal ion chelating activity
The metal chelating capacity of the extract was determined following the method of Santos et al.[24] with slight modifications. Briefly, 200 µL of MEAA at different concentrations (10–250 µg/mL) was mixed with 2 mL of methanol in triplicate test tubes. To each mixture, 50 µL of 2 mM Fe2+ solution was added, followed by 50 µL of 50 mM perchloric acid to prevent hydrolysis. The reaction was initiated by adding 0.2 mL of 5 mM ferrozine or thiocyanate reagent, after which the mixture was shaken thoroughly and left to stand at room temperature for 10 min. The absorbance of the resulting pink-violet ferrozine complex was read at 562 nm, while that of the thiocyanate complex was measured at 490 nm using a spectrophotometer.
In vivo antioxidant studies
The in vivo antioxidant studies conducted include the catalase (CAT) assay, reduced glutathione (GSH), malondialdehyde (MDA), and superoxide dismutase (SOD) assay.
Adult Wistar rats of both sexes weighing 120–150 g were randomized into five groups of five animals per group as follows: Group 1 received 5 mL/kg distilled water and served as the negative control, while Group 2 received 100 mg/kg ascorbic acid, used as the positive control. Groups 3, 4, and 5 received 100, 200, and 400 mg/kg of MEAA, respectively. All treatments were administered orally once daily for 14 days. On the 14th day, animals were anesthetized using ketamine (50 mg/kg), and blood samples were collected by cardiac puncture into plain (non-heparinized) sample tubes and were left for about 2–3 h for the blood to coagulate. At 3000 rpm and a temperature of 4°C, the coagulated blood samples were centrifuged for 10 min, and the serum was carefully separated and used for biochemical analysis.
CAT assay
CAT activity was determined according to the method described by Góth.[25] A 0.2 mL serum sample was incubated for 1 min at 37°C in 1.0 mL of substrate (65 µmoL/mL hydrogen peroxide in 60 mmoL/L sodium-potassium phosphate buffer, pH 7.4). Using 1.0 mL of 32.4 mmoL/L ammonium molybdate [(NH4)6 Mo7O24•4H2O], the enzymatic reaction was halted. Using a spectrophotometer, the yellow complex of hydrogen peroxide and molybdate was detected at 405 nm in comparison to reagent blanks.
GSH assay
This was determined according to the method described by Moron et al.[26] Serum (0.5 mL) was mixed with 0.1 mL of 25% TCA, kept on ice, and centrifuged at 3000 × g for 10 min. The supernatant (0.3 mL) was combined with 0.7 mL of 0.2 M phosphate buffer (pH 8.0) and 2 mL of 0.6 mM (5,5’-dithiobis-(2-nitrobenzoic acid). After 10 min, the absorbance of the yellow complex was read at 412 nm. GSH concentration was obtained from a standard curve (0–100 nmoL) and expressed as nmoL/mL.
MDA assay
Serum MDA level was measured based on the method of Stocks and Dormandy.[27] TBA reacts with MDA to produce a stable chromogen that is quantified by spectrophotometry. The chromogen’s color intensity, which is directly correlated with the MDA content, is measured at 532 nm. By assessing the production of TBARS, the amount of lipid peroxidation in serum was measured. An equal volume of serum (0.5 mL) was mixed with 20% TCA (1:1) and incubated at room temperature. Samples were centrifuged at 2500 × g for 10 min. Then, 1.0 mL of 1% TBA was added to the supernatant, and the mixture was heated in a boiling water bath (100°C) for 15 min. After heating, the samples were cooled on ice and centrifuged at 2500 × g for 15 min. The absorbance of the resulting supernatant was measured at 532 nm against a reagent blank using a spectrophotometer. A standard calibration curve was constructed using varying concentrations (0–20 nmoL) of MDA.
SOD assay
SOD activity in the serum was determined according to the method described by Misra and Fridovich.[28] Using 0.5 mL of distilled water, 0.5 mL of serum was diluted, after which 0.25 mL of ice-cold ethanol and 0.15 mL of ice-cold chloroform were introduced. A cyclo-mixer was used to thoroughly mix this, and it was centrifuged for 10 min at 2500 rpm. Then, 1.5 mL of carbonate buffer (0.05M, pH 10.2) and 0.5 mL of a 0.5 mM EDTA solution were combined with the supernatant. Through the addition of 0.4 mL of 3 mM epinephrine, the reaction was initiated, and the change in absorbance per minute was measured at 480 nm against a reagent blank. When SOD inhibits epinephrine to adrenochrome by 50%, the change in absorbance per minute is the enzyme unit. A calibration curve for the enzyme was created using SOD units ranging from 0 to 195.
Statistical analysis
GraphPad Prism was used to analyze the data, and Dunnett’s multiple comparisons were applied post hoc. Mean ± standard error of the mean was used to express values. Significance was set at p < 0.05.
RESULTS
Phytochemical analysis
Phytochemical analysis MEAA revealed the presence of secondary metabolites such as alkaloids, flavonoids, tannins, saponins, steroids, and terpenoids, in varying amounts, with alkaloids having the greatest abundance. However, reducing sugars and glycosides are absent in Table 1.
| Phytochemicals | mg/100 g |
|---|---|
| Alkaloids | 3631.00±7.35 |
| Steroids | 4.54±0.01 |
| Terpenoids | 133.90±13.23 |
| Flavonoids | 53.13±1.88 |
| Tannins | 15.83±0.36 |
| Phenolics | 2381.00±68.45 |
| Saponin | 0.69±0.00 |
| Reducing sugar | - |
| Glycosides | - |
Values are expressed as mean±SEM
Oral acute toxicity of methanol extract of the stem bark of A. adianthifolia.
No death was recorded in the first phase of the acute toxicity test. However, mortality was recorded at 5000 mg/kg in the second phase of the acute toxicity test after 24 h of observation. The acute toxicity (LD50) was therefore estimated to be 4246 mg/kg.
Effect of MEAA on in vitro antioxidant parameters
In the DPPH radical scavenging assay, the half maximal inhibitory concentration (IC50) was estimated to be 4.16 ± 0.23 µg/mL, and this represented the concentration of the extract required to inhibit 50% of the free radicals. Although this value was higher than that of the standard antioxidant, ascorbic acid (0.38 ± 0.22 µg/mL), it nonetheless indicates strong free radical scavenging activity [Figure 1]. In the FRAP assay, the extract produced a concentration-dependent increase in inhibition. The IC50 was estimated to be 8.70 ± 2.48 µg/mL, and this is the concentration of the extract required to reduce Fe3+ -TPTZ (tripyridyltriazine) to Fe2+-TPTZ at a low pH [Figure 2].
In the TBARS assay, tMEAA caused a concentration-dependent inhibition of lipid peroxidation. Statistically significant inhibition of lipid peroxidation was observed at all tested concentrations of MEAA when compared with the control, with exact p = 0.0261 (15.63 µg/mL), 0.0035 (31.26 µg/mL), 0.0040 (62.26 µg/mL), 0.0001 (125 µg/mL), 0.0016 (250 µg/mL), and 0.0001 (500 µg/mL). The IC50 of the extract was estimated to be 27.99 ± 5.96 µg/mL compared to that of BHT with an estimated IC50 of 93.39 ± 6.54 µg/mL [Figure 3].
The extract also produced a concentration-related inhibition of the NO radical, as shown in Figure 4. The extract had an IC50 of 73.32 ± 49.39 µg/mL compared to the ascorbic acid (6.28 ± 1.42 µg/mL).
In the metal ion chelating activity assay, the extract, as well as the positive control (ascorbic acid), produced a concentration-dependent increase in chelating activity. The IC50 of the extract was obtained as 32.29 ± 16.70 µg/mL, while that of the positive control was 3.06 ± 0.53 µg/mL [Figure 5].
For the TAC, the extract caused a concentration-dependent increase in inhibition, with an IC50 estimated to be 232.10 ± 69.11 µg/mL [Figure 6].
Effect of MEAA on in vivo antioxidant biomarkers
The extract increased the CAT level dose-dependently with a significant elevation at 400 mg/kg (p < 0.01). The CAT level at 400 mg/kg MEAA (431.3 ± 59.78 kU/L) was higher than that of ascorbic acid (321.6 ± 60.52 kU/L) [Figure 7].
Similarly, the extract significantly elevated GSH level at 200 mg/kg (P = 0.009) and 400 mg/kg (p = 0.020). The GSH level obtained for MEAA at 200 mg/kg was higher than that of ascorbic acid [Figure 8].
In addition, the extract significantly elevated SOD levels at 200 mg/kg (p = 0.007) and 400 mg/kg (p = 0.034). The SOD level obtained at 200 mg/kg of MEAA was higher than that of ascorbic acid [Figure 9].
For the MDA assay, the extract significantly reduced MDA levels at 200 mg/kg (p = 0.005) and 400 mg/kg (p = 0.006). The reduction in MDA level at the 200 mg/kg MEAA was comparable to that of ascorbic acid [Figure 10].
DISCUSSION
Oxidative stress has been implicated as a process in the pathogenesis of several diseases and can lead to disruption of the normal function of the body.[29] Due to some limitations of the synthetic antioxidants, researchers have sought alternative agents with safe and efficacious antioxidant effects, especially using natural product sources. Oxidative stress occurs through several mechanisms in the body, including the formation of ROS, impairment of antioxidant defenses, mitochondrial leakage, and transition metal reactions.[30,31] Therefore, it is essential to evaluate antioxidant properties using several appropriate models to determine the spectrum of activity. This study utilized the in vitro evaluation comprising the DPPH, TAC, FRAP, TBARS, NO assays, metal chelating ion activity, and in vivo evaluation comprising the CAT, SOD, GSH, and MDA assays. The results show that the methanol extract of the stem bark of A. adianthifolia possesses a strong antioxidant effect.
The DPPH assay is used to evaluate the capacity of antioxidants or test substances to scavenge free radicals. This works on the principle of neutralization of the DPPH radical through electron or hydrogen donation of the test substance.[32] In this study, the IC50 obtained for the extract showed a strong antioxidant power and its hydrogen-donating capacity. The concentration required to inhibit 50% of the DPPH free radicals is low; however, the standard drug, ascorbic acid, was lower, showing a higher potency of ascorbic acid compared to MEAA. These findings are consistent with earlier reports on A. adianthifolia, where significant antioxidant activities were demonstrated using similar in vitro models. Tamokou et al. reported strong DPPH radical scavenging and TAC of the stem bark extract of A. adianthifolia, attributing these effects to its phenolic constituents.[33]
For the FRAP assay, the MEAA’s ability to reduce ferric ions to ferrous ions was evaluated. In general, reducing agents or antioxidants act by donating hydrogen ions or electrons, leading to a reduction in oxidation number.[34] The IC50 obtained for this assay was 8.70 ± 2.48 µg/mL, which indicates strong redox power. Comparable antioxidant potentials have been reported in other plant extracts using the FRAP assay. For instance, Pistacia lentiscus methanolic extract exhibited an FRAP EC50 of 6.63 ± 1.41 µg/mL, indicating strong ferric-reducing activity similar to the value obtained in our study.[35]
In the TBARS assay, MEAA’s ability to inhibit lipid peroxidation was evaluated. Lipid peroxidation occurs due to oxidative damage to the lipid membrane, which may lead to the alteration of cell integrity, tissue injury, mitochondrial dysfunction, organ damage, and several chronic diseases.[36] This assay works based on the reaction between TBA and MDA, which is obtained from lipid peroxidation. This reaction leads to the formation of a complex between TBA and MDA that absorbs light strongly and can be measured by spectrophotometric determination.[37] Our results show a strong antioxidant power in this model, evidenced by the IC50. MEAA’s IC50 was over 3-fold smaller than the one obtained for the standard agent, BHT. This shows MEAA’s high potency and efficacy in inhibiting lipid peroxidation and preventing cellular damage.
On the other hand, the inhibitory effect of MEAA on NO is moderate compared to the models and assays described above. NO, when released at normal and physiological levels, is an important signaling molecule that plays an essential role in vasodilation, platelet aggregation, and immune regulation.[38] However, when produced at excess levels, it causes oxidative stress and initiates reactions that are detrimental to the cells. The NO assay evaluates the capacity of MEAA to scavenge NO, and it works by preventing the formation of nitrite in the cells. Our study shows a considerably higher IC50 for MEAA compared to ascorbic acid, showing that this extract is less efficient in neutralizing reactive nitrogen species. Moderate NO scavenging activity has also been observed in several plant extracts, where antioxidant efficacy was more pronounced against ROS than reactive nitrogen species.[39]
Another in vitro assay conducted was the metal ion chelating activity, which evaluated the ability of MEAA to chelate metals. These metals, especially the ferrous and copper (II) ions, can cause oxidative stress through the Fenton reaction.[40] Despite showing a considerable chelating activity, its IC50 was remarkably lower than that shown by ascorbic acid, denoting a reduced efficacy in sequestering metals. An IC50 of 232.10 ± 69.11 µg/mL was obtained for the TAC assay, indicating that the overall antioxidant capacity of MEAA is moderate. The TAC assay depicts both the enzymatic and non-enzymatic antioxidants present in the sample. Despite MEAA showing strong antioxidant capacity for the DPPH, FRAP, and the TBARS assay, the moderate TAC capacity suggests that the overall concentration of antioxidant constituents in the extract is relatively lower compared to the pure standard antioxidants. This indicates that MEAA contains highly potent antioxidant compounds in limited quantities, which may be capable of effectively scavenging specific radicals and inhibiting lipid peroxidation, but has a smaller total pool of reducing agents that contribute to overall antioxidant potential.
To evaluate the antioxidant effect of MEAA under real biological conditions, in vivo assays such as CAT, SOD, GSH, and MDA were conducted. CAT is an enzyme that catalyzes the breakdown of hydrogen peroxide into water and oxygen. This reaction is important because the accumulation of hydrogen peroxide can initiate oxidative stress through the generation of ROS.[41] Hence, a high CAT activity or levels after the administration of a substance indicate an antioxidant property. GSH is another compound that donates a hydrogen atom from its sulfhydryl group to scavenge ROS and prevent oxidative stress. The increase of GSH conveys strong antioxidant protection, while a decrease indicates oxidative stress and depletion due to ROS generation. Similarly, SOD is a metalloenzyme that catalyzes the formation of oxygen and hydrogen peroxide from a very reactive free radical known as the superoxide radical. Across these models, MEAA led to the elevation of the enzymatic component of the antioxidant defense system. In the CAT assay, there was a dose-dependent elevation of the CAT level. At the 400 mg/kg dose of MEAA, the CAT levels were elevated to levels above those of ascorbic acid. This reflects the efficacy of the extract in mopping up oxidative stress initiated by excess hydrogen peroxide production. However, for the GSH and SOD assays, the 200 mg/kg MEAA resulted in the highest elevation, even more than the standard. In certain scenarios and models using plant extracts as the test substance, a biphasic response is obtained. This could result from antagonizing combinations of phytochemicals at higher doses.[42] For the MDA assay, MEAA treatments reduced the MDA levels, especially at higher doses. MDA is a biomarker of lipid peroxidation, where elevated levels denote oxidative stress. Therefore, the ability of MEAA to reduce MDA levels, comparable to pure standard drugs, shows its antioxidant property. These in vivo findings are in agreement with earlier reports by Beppe et al. demonstrating enhancement of endogenous antioxidant enzymes following treatment with antioxidant-rich plant extracts.[16]
The antioxidant property exhibited by MEAA may be explained by the presence of flavonoids, terpenoids, and the abundance of phenolics. Several studies have reported the antioxidant effect of these phytochemicals and their potential to maintain cellular homeostasis. Flavonoids and terpenoids have been reported to inhibit oxidative stress by preventing the generation of ROS and enhancing endogenous antioxidant systems.[43] Furthermore, alkaloids have been postulated to exhibit antioxidant properties through the activation of the Nrf2 transcription factor, which protects the cells from free radicals.[44]
Given the results obtained from the acute toxicity test, MEAA showed a favorable safety profile and can be applied in the management of some oxidative stress-related conditions through the enhancement of endogenous antioxidant systems, inhibition of lipid peroxidation, and scavenging of ROS. However, to obtain a more reliable result on its safety profile, a chronic toxicity test is needed. Future studies should focus on the isolation and characterization of the active moiety so as to enhance standardization.
CONCLUSION
The MEAA showed considerable in vitro and robust in vivo antioxidant properties. This justifies the use of A. adianthifolia in the management of oxidative stress-related conditions in traditional medicine.
Authors’ contributions:
IEP: Conceptualization; IEP, MCE, CEA: Methodology; IEP, MCE, CEA, AMO, HNA: Formal analysis; IEP, MCE, CEA, AMO, HNA, MCE, EMA, ONI, MNO, FNM, CAO: Investigation; IEP, MCE, CEA, AMO, HNA, MCE, EMA, ONI, MNO, FNM, CAO: Writing – original draft preparation; IEP, MCE, CEA, AMO, HNA, MCE, EMA, ONI, MNO, FNM, CAO: Writing – review & editing, supervision; IEP, MCE, CEA, AMO, HNA, MCE, EMA, ONI, MNO, FNM, CAO: Project administration.
Ethical approval:
The research/study was approved by the institutional review board at faculty of Pharmaceutical Sciences Research Ethics Committee with reference number FPSRA/UNN/22/0042, dated March 12, 2025.
Declaration of patient consent:
Patient’s consent not required as there are no patients in this study.
Conflicts of interest:
There are no conflicts of interest.
Use of artificial intelligence (AI)-assisted technology for manuscript preparation:
The authors confirm that there was no use of artificial intelligence (AI)-assisted technology for assisting in the writing or editing of the manuscript and no images were manipulated using AI.
Financial support and sponsorship: None.
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