Therapeutic potential of Hyoscyamus niger-derived compounds: Targeting ovarian cancer through antioxidant activity and EGFR tyrosine kinase inhibition

2.1 Plant material

The aerial components of H. niger were harvested from the willaya of biskra in the southeastern of Algeria. The meticulous botanical identification of the plant was performed by Dr Azzedine Zeraib associate professor at department of agronomy in Abbes Laghrour University of Khenchela, Algeria. In adherence to rigorous scientific protocols, the Biotechnology, Water, Environment and Health Laboratory attributed a distinct voucher code (No. 454./ 40) to the plant specimen.Subsequently, the collected material underwent air-drying and was then finely ground into a powder. The extraction process closely followed the procedures outlined by (Bendjedid et al., 2021; Benslama et al., 2023; Lekmine et al., 2022, 2021, 2020). To obtain hydro-alcoholic extracts (70 % ethanol), we employed 10 g of powdered plant material. The powdered plant material was mixed with ethanol and left to dissolve for 24 h at ambient room temperature. Subsequently, the mixture underwent filtration, and the resulting filtrate was concentrated utilizing a rotary evaporator (Hahnvapor) at 40 °C. The resulting residue from the hydro-alcoholic extract was gathered and stored at 4 °C for subsequent utilization (Lekmine et al., 2023).

2.2 Equipment and Chromatographic parameters

The extracts were introduced into a Shimadzu® 8045 UPLC system (Kyoto, Japan). connected to a triple quadrupole mass analyzer from Shimadzu® Corporation. These extracts were solubilized in methanol of HPLC-grade quality and subsequently filtered through a 0.2 µm PTFE filter. Chromatographic separation was accomplished utilizing a Shimpack C18 reversed-phase column (2.7 µm, 2 x 150 mm). A gradient elution was administered at a flow rate of 0.2 mL/min, utilizing solvent A (water) and solvent B (acetonitrile). The elution initiated at 10 % B for 5 min, incrementally ascending to 30 % B until 15 min, followed by a progression to 70 % B until 22 min, then elevated to 80 % B until 30 min, and eventually restored to 10 % B by 35 min. The mass detection was conducted under the negative Electrospray Ionization (ESI) mode, wherein the interface temperature was set at 300 °C, desolvation temperature at 526 °C, cone gas flow was adjusted to 50 L/h, and the nebulizing gas flow was fine-tuned to 3 L/min. For the purpose of collision-induced dissociation (CID) MS/MS measurements, collision energy was modulated individually for each peak within the range of 20–50 eV. The mass detection spanned the range of m/z 100–1200. Data processing was undertaken using the Lab Solutions software (Lekmine et al., 2023).

2.3 Antioxydant activities

The details of all biological activity studies are provided as Supplementary Material. CUPRAC, Reducing Power, Beta Carotene, DMSO Alcalin, SNP (IC50), Phenonthroline, and Hydroxyl Radical method were used to estimate the antioxidant potential.

2.4 Statistical evaluation

The tests were conducted in triplicate on the samples. The data are expressed as means ± standard deviation. Variance analysis was carried out using SPSS version 13.0. A P value of less than 0.05 was deemed statistically significant, and pairwise comparisons were conducted using Tukey's test.

2.5 Computational analysis

3.1 LC-ESI-MS/MS analysis of the H. Niger

The LC-ESI-MS/MS analysis of the H. niger extract yielded notable findings, as illustrated in Fig. 1 and Table 1. The investigation unveiled the presence of 21 phenolic compounds, with the most substantial concentrations observed in Luteolin (22,241.0 ± 1.4 µg/g extract) and p-Coumaric acid (22,228.3 ± 3.1 µg/g extract). Notably, no significant difference was observed between these two compounds (p < 0.05). Furthermore, the analysis identified several other compounds with high concentrations, including Chlorogenic acid (16,627.3 ± 3.7 µg/g extract), Gallic acid (15,123 ± 3.1 µg/g extract), Rutin (14,548.2 ± 1.8 µg/g extract), and Apigenin (10,229.9 ± 1.7 µg/g extract). Moderately concentrated compounds, such as Rosmarinic acid, Hyperoside, Kaempferol, and Naringenin, were also detected, with concentrations ranging between 6598 ± 3.2 and 1223 ± 2.2 µg/g extract. Additionally, Tannic acid, Hesperetin, Quercetin, Quinic Acid, Fisetin, Vanillin, and Protocatechuic Acid were registered with values between 971 ± 1.3 and 125.3 ± 1.7 µg/g extract. In contrast, Salicylic Acid, tr-Caffeic acid, Malic acid, and Coumarin were detected in trace amounts, all measuring less than 100 µg/g extract.

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Fig. 1

LC-MS/MS chromatogram of H. niger ethanolic extract.

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According to the literature, these findings align with the study conducted by Amir Reza Jassbi, (Jassbi et al., 2014) who also identified similar compounds, albeit with variations in their concentrations. Notably, among these compounds, chlorogenic acid, rutin, and Quercetin-3O-Glucoside-Rhamnoside-Rhamnoside (QGRR) were detected using HPLC-Diode Array Detector-Electrospray Ionization Mass Spectroscopy (LC-DAD ESIMS) in both studies.

The phenolic compounds found in H. niger (commonly known as black henbane) can vary depending on various factors such as the plant's growth conditions and location.

Rutin had been previously detected in the seeds of H. niger (Sengupta et al., 2011). However, in our study, the sample we analyzed consisted only of the aerial parts of the plant, excluding the seeds. This variation could potentially be attributed to differences in the source of the plant material or variations in the extraction procedure (Ma et al., 2002).

3.2 Antioxidant potential

The antioxidant activity of plant extracts plays a pivotal role in numerous processes, encompassing their ability to diminish and neutralize free radicals, along with their capacity to absorb oxygen radicals. As a result, we assessed the antioxidant potential of the ethanolic extract derived from H. niger through seven antioxidant tests (as detailed in Table 2).

To the best of our knowledge, there is no existing research in the literature regarding the use of antioxidant techniques on H. niger. As illustrated in Table 2, the terms “IC50″ and ”A0.50 levels“ pertain to the concentration at which absorbance reaches 0.50 and the percentage of inhibitory activity at 50 %, respectively. These values were determined through a linear regression approach and are presented as mean ± standard deviation (n = 3).

The results indicate that the H. niger extract exhibited potent activity in the Reducing Power assay, with a notably high half-maximal inhibitory concentration of 4.5 ± 1.3 µg/mL. This concentration surpassed that of α-Tocopherol, Ascorbic Acid, Tannic Acid, and Trolox, which served as standards for the Reducing Power method, with values of 23.53 ± 2.18, 6.37 ± 1.5, 5.57 ± 1.4, and 5.33 ± 1.8 µg/mL, respectively.

Additionally, the H. niger extract demonstrated a strong presence in the Beta Carotene assay, with a concentration of 7.17 ± 2.8 µg/mL. It also displayed notable activity in other assays, including SNP, Phenonthroline, CUPRAC, Hydroxyl Radical, and DMSO Alkaline assays, with values of 10.5 ± 2.5 µg/mL, 13 ± 2.8 µg/mL, 15.57 ± 1.2 µg/mL, 19.95 ± 2.1 µg/mL, and 22 ± 2.2 µg/mL, respectively. As shown in Table of LC-MS-MS, p-Coumaric acid and Luteolin are both present in very high concentrations in the H. niger extract.

These compounds have been widely demonstrated by numerous studies as effective bioactive antioxidants, According to Ismail Kiliç and Yeşim Yeşiloğlu (Kiliç and Yeşiloğlu, 2013), at a concentration of 45 μg/mL, p-Coumaric acid exhibited a remarkable inhibitory effect, reducing lipid peroxidation by 71.2 % in a linoleic acid emulsion.

Furthermore, p-Coumaric acid demonstrated significant scavenging activity against DPPH radicals, ABTS radicals, superoxide anions, hydrogen peroxide, and exhibited strong reducing power for ferric ions (Fe3+). It also displayed chelating activity for ferrous ions (Fe2+).

Collectively, these findings indicate that p-Coumaric acid possesses substantial antioxidant properties, making it a promising candidate for applications in the pharmaceutical and food industries.

On the other hand, Luteolin is a flavonoid, a type of polyphenolic compound, that is naturally found in various plants, including fruits, vegetables, and herbs. It is known for its antioxidant properties, which contribute to its potential health benefits.(Wang et al., 2011). Luteolin has the ability to neutralize and scavenge free radicals in the body. Free radicals are highly reactive molecules that can cause oxidative stress and damage to cells, proteins, and DNA. By scavenging these radicals, luteolin helps protect cells from oxidative damage.(Xiong et al., 2017).

Luteolin has anti-inflammatory properties, which can be linked to its antioxidant effects. Chronic inflammation is often associated with oxidative stress, and luteolin's ability to reduce inflammation contributes to its overall antioxidant activity (Azeem et al., 2023). Therefore, it may be suggested that the observed activity is caused by p-Coumaric acid and Luteolin and their synergic effects with other phenolic compounds.

Natural compounds found in medicinal plants, especially those with antioxidant activities, can play a significant role in cancer prevention and treatment. Antioxidant compounds like flavonoids, polyphenols, and carotenoids found in many medicinal plants help neutralize free radicals. Free radicals can damage cells and DNA, potentially contributing to cancer development. By eliminating these radicals, antioxidants can reduce the risk of cancer.(Sadiq, 2023).

3.3 Molecular docking study

Ovarian cancer stands as the most prevalent and lethal gynecological malignancy. Globally, roughly 230,000 women receive an ovarian cancer diagnosis annually, leading to the unfortunate loss of 150,000 lives (Funingana et al., 2023).

Given the detrimental side effects and the resistance observed with existing chemotherapeutic agents, the quest for the creation of more effective and less harmful anticancer drugs has become a crucial focus in drug development (Zhao et al., 2022). The results of this research constitute a crucial component of the preliminary evaluation of our compounds, which could potentially be advanced for further development as anticancer agents targeting ovarian tumors.

In the present study, we provide structural insights into the protein interactions of H. niger phenolic compounds exhibiting binding energies lower than that of the co-crystallized inhibitor, 4-anilinoquinazoline. Consequently, they are considered potentially active against EGFR. The docking results for this dataset of phycompounds are presented in Table 3.The docking of these phytocompounds was conducted within the receptor's auto-phosphorylation site, a significant binding site for many kinase potent inhibitors. This analysis unveiled a protein–ligand interaction map, highlighting all potential hydrophobic and hydrophilic interaction.

In our molecular docking analysis, several phenolic compounds derived from H. niger were evaluated for their binding affinity with the EGFR, a pivotal target in cancer therapy. Among the tested compounds, hyperoside exhibited the highest binding energy, surpassing the reference compound 4-anilinoquinazoline. It formed multiple hydrogen interactions with key residues in the EGFR binding site, demonstrating strong potential as an EGFR inhibitor. Luteolin, apigenin, quercetin, and fisetin also displayed notable binding affinities and engaged in diverse hydrogen and hydrophobic interactions within the receptor's active site. Furthermore, hesperetin, chlorogenic acid, naringenin, kaempferol, rutin, and rosmarinic acid exhibited considerable binding energies, indicating their potential as EGFR inhibitors (Table 3).

In addition, the outcomes of our docking analysis indicate the significance of residues Thr830 and Met769 in forming crucial hydrogen and hydrophobic bonds with EGFR. This observation aligns with prior docking studies on various compound (Nasab et al., 2018) affirming the consistency of our findings. Interestingly, when compared to reference inhibitor 4-anilinoquinazoline, certain compounds demonstrated a favorable fit within the ATP binding gorge of the EGFR (Fig. 2). These results suggest that phenolic compounds from H. niger may hold promise as candidates for further investigation as anticancer agents targeting EGFR.

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Fig. 2

3D interaction model of phenolic compounds from H. niger binding to EGFR active sit.

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