Comparative in vitro Evaluation of the Antioxidant Potential of Apomorphine and Pyrazolones for Hepatocellular Carcinoma

INTRODUCTION

Hepatocellular Carcinoma (HCC) represents a major global health concern and contributes substantially to the burden of liver cancer (Kudo et al., 2023; Llovet et al., 2021). Its pathogenesis involves complex interactions among cell-cycle dysregulation, immune escape mechanisms and oxidative stress-induced damage. The emergence of CDK4/6 inhibitors and immune checkpoint modulators has revolutionized targeted therapy for HCC by providing dual-action tumour suppression through cell-cycle arrest and immune system reactivation. However growing evidence suggests that Oxidative stress contributes significantly to the progression of tumours, thereby necessitating antioxidant strategies to mitigate Reactive Oxygen Species (ROS)-driven carcinogenesis. Ayurvedic and Unani medical systems have historically recognized the therapeutic benefits of herbal compounds in preventing and managing cancer. The Charaka Samhita (1000 B.C.) is one of the earliest texts documenting over 2,000 medicinal plants, many of which are known to exhibit antioxidant, anti-inflammatory and antineoplastic properties (Kudo et al., 2023; Llovet et al., 2021; Charaka, 1000 B.C.). Among these, Oxalis corniculata Linn. (Family: Oxalidaceae) has gained attention for its broad-spectrum biological activities, including hepatoprotective, immunomodulatory and free radical-scavenging properties (Unani Medical Council, 2020; Singh and Patel, 2019; Halliwell, 2022). Plant-derived antioxidants play a pivotal role in reducing oxidative damage, which is a known contributor to cancer, atherosclerosis, cardiovascular diseases and inflammation (Rojo de la Vega et al., 2018; George et al., 2022; Finn et al., 2015). The accumulation of ROS can lead to DNA mutations, lipid peroxidation and protein degradation, thereby exacerbating tumour development and progression (Parker and Finn, 2023; Cai et al., 2024). Therefore, integrating antioxidant-rich compounds with modern therapeutic strategies may enhance the overall efficacy of cancer treatment. The regulation of free radicals in hepatic cells involves a delicate balance between enzymatic and non-enzymatic antioxidant systems, including Superoxide Dismutase (SOD), Catalase and glutathione (Qiu et al., 2024; Zamio, 2024; Beaufrère et al., 2024). The nuclear transcription factor Nrf2 plays a crucial role in the regulation of antioxidant genes and the maintenance of cellular redox homeostasis. To assessed the antioxidant potential of the investigated compounds, the present study employed five key antioxidant assays, namely the DPPH assay for determining free radical scavenging efficiency, the FRAP assay for measuring ferric reducing antioxidant power, the TBARS assay for assessing lipid peroxidation levels, the nitric oxide assay for evaluating nitric oxide radical scavenging capacity and the hydrogen peroxide assay for assessing ROS scavenging activity and oxidative damage control. CDK4/6 inhibitors such as Palbociclib suppress uncontrolled cellular proliferation by inducing G1-phase cell-cycle arrest, thereby restricting tumour progression (He et al., 2021; Zhang et al., 2023; Wu et al., 2023; He et al., 2021). Simultaneously, immune checkpoint restores T-cell-mediated immune responses and counteract immune evasion strategies employed by HCC cells modulators focusing on the PD-1/PD-L1 and CTLA-4 pathways (Toyokuni and Akatsuka, 2020; Brand-Williams et al., 1995; Benzie and Strain, 1996; Ohkawa et al., 1979). Given the intricate relationship among redox homeostasis, immune activation, and cell-cycle regulation, evaluating antioxidant parameters alongside targeted therapeutic approaches may provide valuable insights into improving treatment outcomes in HCC. The primary objective of the present study was to evaluate the antioxidant profile of Apomorphine and Pyrazolones in HepG2 cells and to analyse the regulation of oxidative stress using DPPH, FRAP, TBARS, nitric oxide and hydrogen peroxide scavenging assays. The ongoing study may contribute to a better understanding of the antioxidant properties of these compounds and their potential relevance to oxidative stress modulation in hepatocellular carcinoma.

MATERIALS AND METHODS

Materials

Cell Culture

The human hepatocellular carcinoma cell line was procured from the National Center for Cell Science (NCCS), Pune, India. The cells were set in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% Foetal Bovine Serum (FBS) and 1% Penicillin-Streptomycin solution. The cultures were in-cubated at 37ºC in a humidified atmosphere comprising 5% CO₂. HepG2 cells are extensively used as an in vitro model for Hepatocellular carcinoma research due to their stable hepatic characteristics and reproducible growth pattern (Llovet et al., 2021).

Drug Treatment

Apomorphine and Pyrazolones were procured from a commercial source and evaluated for their antioxidant potential in the present study. Unless otherwise specified, Pyrazolones were dissolved in sterile distilled water, whereas Apomorphine was dissolved in Dimethyl Sulfoxide (DMSO). Equal volumes of solvent were maintained in the control groups and the final concentration of DMSO in the culture medium did not surpass 0.1% (v/v). All experimental solutions were freshly prepared and sterilized using 0.22 µm syringe filters before use.

Treatment Protocol

Depending on the experimental assay, cells were seeded in either 6-well or 96-well plates and allowed to attach for 24 hr before being treated with various concentrations of Apomorphine or Pyrazolones dissolved in DMSO or distilled water. Control cells received the same volume of the vehicle solution and the highest concentration of DMSO remained below 0.1% (v/v).

Methods

DPPH Assay

The test substances antioxidant activity was evaluated using the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging test. For the test, 100 μL of a methanol-prepared 0.1 mM DPPH solution was added to a 96-well microplate along with 5 μL of the test compounds at the respective concentrations. Methanol used for the assay was procured from SD Fine Chemicals. Ascorbic acid served as the reference antioxidant. All concentrations were evaluated in quadruplicate (n=4), and the results were specified as mean±Standard Deviation (SD). Blank wells were prepared by adding 200 μL methanol and 10 μL of test compound or standard without DPPH. Wells containing only DPPH solution served as the control, whereas wells without DPPH were considered blank. A combination of reactions containing 20 μL deionized water was use as the negative control (Abdulrasheed-Adeleke and Musa, 2020). The microplate was kept at room temperature in the dark for 30 min before recording the absorbance at 517 nm with a microplate reader. DPPH radical inhibition was expressed as a percentage relative to the control. The IC₅₀ values were obtained using GraphPad Prism 6 software by plotting the test concentration along the X-axis against the corresponding percentage inhibition on the Y-axis (Imam et al., 2011).

Ferric Reducing Antioxidant Power (FRAP) Assay

The FRAP assay was carried out to determine the declining ability of the test compounds. Briefly, 40 μL of 0.2 M sodium phosphate buffer, 50 μL of 1% potassium ferricyanide [K₃Fe(CN)₆] and 10 μL of various concentrations of the test compounds or standard ascorbic acid were added into a 96-well plate. After mixing, the reaction mixture was in-cubated at 50ºC for 20 min. Untreated wells containing the reaction mixture without test compounds were used as controls. Subsequently, 50 μL of 10% trichloroacetic acid was added to each well. To develop the colour response, 50 μL deionized water and 50 μL of 0.1% ferric chloride solution were mixed according to the standard FRAP protocol (Rao et al., 2013). Absorbance was calculated at 700nm thorugh a microplate reader. Anti-oxidant activity was expressed as IC₅₀ values obtained using GraphPad Prism 6 software. A dose-response curve was plotted between concentration and absorbance (Xavier and Reddy, 2013). Each measurement was carried out in quadruplicate (n=4) and the results are indicated as mean±SD.

Thiobarbituric Acid Reactive Substances (TBARS) Assay

Lipid peroxidation was evaluated using the TBARS assay. Approximately 50 mg of HepG2 cell pellet obtained after treatment was homogenized in 1 mL Phosphate-Buffered Saline (PBS) to prepare the test solution. From the homogenate, 100 μL was mixed with 500 μL of Solution A and vortexed thoroughly. The mixture was centrifuged at 2500 rpm for 5 min and the supernatant was obtained. Subsequently, 1800 μL of Thiobarbituric Acid (TBA) reagent was added and mixed thoroughly. The reaction combination was heated in a water bath for 15 min to facilitate colour development. Absorbance was recorded at 532 nm against a reagent blank using a spectrophotometer or microplate reader. The concentration of Malondialdehyde (MDA), an indicator of lipid peroxidation, was assessed using the molar extinction coefficient of 1.56 × 10⁵ M⁻¹cm⁻¹ (Olszewska-Slonina et al., 2011). All measurements were performed in quadruplicate (n=4) and the results were expressed as mean±SD.

Nitric Oxide (NO) Scavenging Assay

Nitric oxide scavenging activity was assessed by following the sodium nitroprusside method. For the reaction, 50 μL of 10 mM sodium nitroprusside was combined with 40 μL of distilled water and 10 μL of the test sample, standard, or blank solution. Untreated wells served as controls. The combinations were kept at ambient temperature and exposed to light for 15 min to facilitate nitric oxide generation. Thereafter, 100 μL of Griess reagent was included to each well and incubated for an additional 5-10 min for chromophore development. Absorbance was measured at 540 and 660 nm using a microplate reader. The percentage inhibition of nitric oxide radicals was computed relative to the management. IC₅₀ values were calculated via GraphPad Prism 6 software (Rao et al., 2013). Each experiment was carried out in quadruplicate (n=4), and the data are presented as mean±SD.

Hydrogen Peroxide (H₂O₂) Scavenging Assay

The hydrogen peroxide scavenging efficacy of the test compounds was evaluated using a colorimetric method. In a 96- well plate, 10 μL of test sample or standard was added into designated wells. Wells in SET 1 and SET 3 received 90 μL of 6% hydrogen peroxide solution prepared in distilled water, whereas SET 2 and SET 4 received 90 μL buffer solution. The plate was incubated at 37ºC for 5 min according to previously reported methods (Rahman et al., 2015). Following incubation, 100 μL of ammonium molybdate reagent was included to each well. The plates were then incubated at 37ºC for 2 min to allow development of the chromogenic response. Absorbance was recorded at 405-415 nm using a microplate reader. Hydrogen peroxide scavenging was expressed as percentage inhibition relevant to the control. The IC₅₀ values were estimated using GraphPad Prism 6 software by plotting the concentration against the corresponding percentage inhibition (Hazra et al., 2008). Measurements were carried out in quadruplicate (n=4), and the data are presented as mean±SD.

Statistical Analysis

Each experiment was directed using four replicates (n=4), and the findings are reported as mean±Standard Deviation (SD). GraphPad Prism 6.0 (GraphPad Software Inc., San Diego, CA, USA) was used for statistical analysis. An unpaired Student's t-test was used to compare the two groups. One-way variance analysis (ANOVA) and Tukey's post hoc test were employed when comparing more than two groups. When the p-value was less than 0.05, differences were deemed statistically significant.

RESULTS

DPPH Radical Scavenging Activity

The antioxidant activity of Apomorphine and Pyrazolones was assessed utilizing the DPPH radical scavenging test and contrasted with ascorbic acid, a common antioxidant. The IC₅₀ values of ascorbic acid, Pyrazolones, and Apomorphine were found to be 19.25±0.023 µg/mL, 97.26±0.031 µg/mL and 66.90±0.028 µg/mL, respectively. Among the tested compounds, Apomorphine exhibited a lower IC₅₀ value than Pyrazolones, indicating comparatively greater DPPH radical’s detoxification activity Figure 1.

Click to enlarge

Ferric Reducing Antioxidant Power (FRAP) Assay

The ferric reduction antioxidant activity of Apomorphine and Pyrazolones was assessed using the FRAP assay. The IC₅₀ values of ascorbic acid, Pyrazolones and Apomorphine were 2.258±0.023 µg/mL, 4.127±0.832 µg/mL and 4.822±0.247 µg/mL. Both compounds demonstrated ferric reducing activity under the experimental conditions employed. The results are shown in Figure 2.

Click to enlarge

Thiobarbituric Acid Reactive Substances (TBARS) Assay

Lipid-peroxidation was evaluated via measuring Malondialdehyde (MDA) levels by using TBARS assay. The MDA concentrations observed in the Pyrazolones- and Apomorphine-treated groups were 6.795 nmoles/mL and 4.615 nmoles/mL, respectively. A lower MDA level was observed in the Apomorphine-treated group compared with the Pyrazolones-treated group. The results are presented in Figure 3.

Click to enlarge

Nitric Oxide (NO) Scavenging Activity

The nitric oxide scavenging activity of Apomorphine and Pyrazolones was determined and assessed with gallic acid as the reference standard. The IC₅₀ values of gallic acid, Pyrazolones and Apomorphine seemed 18.96±0.026 µg/mL, 417.9±0.047 µg/mL and 1844±0.110 ng/mL, respectively. Both compounds demonstrated nitric oxide scavenging activity within the tested concentration range. The results are presented in Figure 4.

Click to enlarge

Hydrogen Peroxide (H₂O₂) Scavenging Activity

The H₂O₂ activity of Apomorphine and Pyrazolones was evaluated utilizing the hydrogen peroxide radical scavenging test and contrasted with gallic acid as the reference typical. Gallic acid exhibited an IC₅₀ value of 84.16±0.020 µg/mL. However, the IC₅₀ values of both Apomorphine and Pyrazolones could not be determined within the tested concentration range and were therefore recorded as above the maximum tested dose. These results suggest limited hydrogen peroxide scavenging activity of both compounds under the experimental conditions employed. The results are presented in Figure 5.

Click to enlarge

DISCUSSION

Oxidative stress is recognized as an important contributor to the development and progression of Hepatocellular Carcinoma (HCC). Excessive production of Reactive Oxygen Species (ROS) can induce DNA damage, lipid peroxidation, protein oxidation and alterations in cellular signalling pathways, thereby promoting tumour progression. Consequently, the identification of compounds with antioxidant properties may provide valuable support in strategies aimed at reducing oxidative stress-associated cellular damage.

In the present study, the antioxidant potential of Apomorphine and Pyrazolones was evaluated using multiple in vitro antioxidant assays, including DPPH, FRAP, TBARS, nitric oxide and hydrogen peroxide scavenging assays. The use of multiple assays was important because antioxidant activity is a complex phenomenon and no single method can comprehensively evaluate all antioxidant mechanisms. The DPPH assay demonstrated that both compounds possessed free radical scavenging activity with Apomorphine exhibiting greater activity than Pyrazolones. The DPPH method primarily measures the capacity of a compound to donate electrons or hydrogen atoms to neutralize free radicals. Similar observations have been reported in previous studies, where compounds with effective electron-donating properties showed enhanced DPPH radical scavenging capacity (Brand-Williams et al., 1995; Halliwell, 2022).

The FRAP assay further confirmed the antioxidant potential of both compounds by demonstrating their ferric ion reducing capacity. Since the FRAP assay is based on electron transfer reactions rather than direct radical scavenging, differences between DPPH and FRAP results are expected. Previous studies have shown that antioxidant compounds may perform differently across assays because each method evaluates distinct mechanisms of antioxidant action (Benzie and Strain, 1996). The results obtained in the present study support this observation and highlight the importance of employing multiple antioxidant assays for comprehensive evaluation.

Lipid peroxidation is a major consequence of oxidative stress and contributes to membrane damage and cellular dysfunction. The TBARS assay revealed lower Malondialdehyde (MDA) levels in the Apomorphine-treated group compared with the Pyrazolones-treated group, indicating reduced lipid peroxidation. Since MDA is a widely accepted biomarker of oxidative damage, the observed findings suggest that Apomorphine may provide greater protection against oxidative membrane injury under the experimental conditions employed (Ohkawa et al., 1979; Rojo de la Vega et al., 2018).

Nitric oxide scavenging analysis demonstrated that both compounds were capable of interacting with reactive nitrogen species. Nitric oxide plays an important role in inflammation and oxidative stress and excessive production has been associated with cancer progression and tissue injury. The observed scavenging activity therefore indicates an additional antioxidant property of the tested compounds. Similar findings have been reported for several natural and synthetic antioxidants capable of reducing nitric oxide-mediated oxidative damage (Rao et al., 2013).

In contrast, both Apomorphine and Pyrazolones exhibited limited activity in the hydrogen peroxide scavenging assay, as their IC₅₀ values could not be determined within the tested concentration range. This finding highlights the assay-dependent nature of antioxidant activity, where a compound may demonstrate effectiveness against certain reactive species while exhibiting limited activity against others. Such differences are commonly observed because individual antioxidants interact differently with specific oxidants and free radical systems (Hazra et al., 2008; Rahman et al., 2015).

Overall, the variations observed among DPPH, FRAP, TBARS, nitric oxide and hydrogen peroxide assays emphasize that antioxidant activity involves multiple mechanisms, consisting of minimizing power, neutralizing radicals and preventing lipid peroxidation. Collectively, the results demonstrate that both Apomorphine and Pyrazolones possess measurable antioxidant properties with Apomorphine demonstrating comparatively stronger antioxidant activity in several of the evaluated parameters. Further studies are necessary to investigate the underlying molecular mechanisms and to determine the potential biological significance of these findings in hepatocellular carcinoma models.

CONCLUSION

In conclusion, Apomorphine and Pyrazolones exhibited measurable antioxidant activity in multiple in vitro assays, including DPPH, FRAP, TBARS and nitric oxide scavenging assays. Among the tested compounds, Apomorphine demonstrated comparatively stronger antioxidant activity in several evaluated parameters. These findings suggest that both compounds possess antioxidant properties that may contribute to the modulation of oxidative stress. Future investigations involving mechanistic studies, ROS analysis and in vivo validation are required to establish their potential role in hepatocellular carcinoma and related oxidative stress-associated conditions.

ACKNOWLEDGEMENT

We sincerely thank Teerthankar Mahaveer College of Pharmacy, Teerthankar Mahaveer University, Moradabad for their support. Special appreciation to Krishana Kumar Sharma for his invaluable guidance throughout the research.

ABBREVIATIONS

CDK4/6

Cyclin-Dependent Kinase 4 and 6

HCC

Hepatocellular Carcinoma

Rb

Retinoblastoma Protein

DMSO

Dimethyl Sulfoxide

FBS

Fetal Bovine Serum

MTT

3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide

MMP

Mitochondrial Membrane Potential

PBS

Phosphate-Buffered Saline

TBARS

Thiobarbituric Acid Reactive Substances

MDA

Malondialdehyde

DPPH

2,2-Diphenyl-1-picrylhydrazyl

FRAP

Ferric Reducing Antioxidant Power

NO

Nitric Oxide

H₂O₂

Hydrogen Peroxide

ROS

Reactive Oxygen Species

IC₅₀

Half-Maximal Inhibitory Concentration

Nrf2

Nuclear Factor Erythroid 2-Related Factor 2

PD-1/PD-L1

Programmed Cell Death Protein 1/Programmed Cell Death Ligand 1

CTLA-4

Cytotoxic T-Lymphocyte-Associated Protein 4.

CONFLICT OF INTEREST

The authors declare that there is no conflict of interest.

FUNDING SOURCE

No outside funds were used to carry out this investigation.

ETHICAL STATEMENT

HepG2 human hepatocellular carcinoma cell lines used in the present investigation were obtained from the National Center for Cell Science (NCCS), Pune, India. The study was carried out exclusively under in vitro conditions and did not involve any human subjects or experimental animals; therefore, ethical committee approval was not applicable for this work.