Essential oil profilingThe GC-MS analysis of the essential oil of C. maritimum was performed in the current study to profile the volatile constituents for the plant species growing in the coastal region of east Libya and demonstrate the qualitative and quantitative variations in these constituents compared to the constituents of the plant species growing in different regions and under different climatic conditions. The plants’ responses to environmental conditions and biotic stresses, including their liability to biosynthesis-specific constituents, have been documented in several reports33,34,35. There is also a literature proving that volatile constituents of plants have great sensitivity, compared to other plants secondary metabolites, to the abiotic and biotic stresses that affect the plants during their growth25,36,37,38. In the current findings, twenty-five compounds were identified in the volatile oil sample of the plant (Table 1). Among the identified compounds, the most abundant compound in C. maritimum as profiled by the GC-MS was the thymyl methyl ether with a relative percentage of 56.86%, indicating its dominance in the plant volatile composition (Table 1). This compound has also been reported as one of the major volatile constituents of C. maritimum species growing in different regions14,22,39,40. However, the Libyan species of the plant has the highest amount of the compound. For instance, the relative concentration of thymyl methyl ether in the C. maritimum Tunisian species was found at 20.13–40.40% 14,22; in the Turkish growing plant, the percentages of the compound were found at 7.7–29.8% 40,41; in the species from Portugal, the percentages of the compound were 12.90–15% 42; and in Palagonia, the percentages of the compound were found at 25.48% 39. The variations in the thymyl methyl ether and other volatile concentrations of the plant, C. maritimum, growing in different locations, indicated the plant’s sensitivity to changes in the environmental conditions related to its growing areas which is expecting a variation in its biological activity comparing with the same species in other geographical locations.Furthermore, γ-terpinene (16.17%) was also found at a high relative percentage in the current Libyan species. The compound has also been identified as a major constituent in several C. maritimum species growing around the world; however, its percentages in those species were flocculated up and down15,22,39. Compared to the relative percentages of γ-terpinene in plant species around the world, current findings indicate a relatively lower concentration in the Libyan species of C. maritimum. For instance, γ-terpinene has been found at percentages of 33.60%, 19.3–30.62%, and 8.8–32.4% in the species growing in Portugal, Tunisia, and Turkey, respectively15,22,40.Elucidating the GC-MS analysis also revealed the presence of ledene oxide, γ-guaiene and terpinen-4-ol at the concentration levels of 4.32, 3.32 and 2.89, respectively, and at relatively higher concentrations compared to other identified constituents, except for the existence of γ-terpinene and thymyl methyl ether. The analysis also revealed the presence of several other biologically active volatile constituents in considerable percentages. For example, carvacrol and thymol, the antioxidant and antimicrobial phenolic monoterpene volatile oils25,43, were found at the relative percentages levels of 0.91, and 1.15%, respectively. All the previously mentioned compounds and the other identified volatile constituents of the plant in Table 1 such as germacrene D (2.17%) and cuparene (0.69%) are contributing to the overall complexity of the plant aroma.Grouping of the identified compounds indicated the presence of the oxygenated monoterpenes in significant combined higher concentration (63.85%) compared to other groups of constituents including the non-oxygenated monoterpenes, the oxygenated sesquiterpenes, and the non-oxygenated sesquiterpenes, which were calculated with the relative percentages of 17.37, 9.26, and 6.18%, respectively (Fig. 2).The presence of these bioactive volatile constituents in the plant implicated its importance in both the food and medicine application. These bioactive volatile constituents could be participated in the beneficial application of the plant in the cosmetic preparation and in traditional medicine including its application as carminative and anti-inflammatory agent14,15,20. GC-MS Chromatogram of the essential oil of C. maritimum in supplementary file.Fig. 2Relative percentages of the collective classes of volatile oils in Crithimum maritimum.Table 1 Volatile oil constituents of Crithimum maritimum growing in the coastal area of Libya.Antioxidant activity of C. Maritimum
As part of the C. maritimum quality evaluation, the antioxidant activity of the volatile oils of the plant has been measured using three different in vitro assays, i.e., DPPH, FRAP, and ORAC. These methods were selected to evaluate the transition metals reducing power and free radical capturing effect of the plant volatile oils23. The results demonstrated in Fig. 3 indicated that the plant volatile oils have exerted substantial DPPH and peroxyl radical scavenging effect in the DPPH and ORAC assays at the values of 34.30 ± 0.10 and 27.89 ± 0.93 µM TE/ mg of the plant volatile oils, respectively. C. maritimum volatile oils also exhibited remarkable reducing power effect to the ferric ions in the FRAP test at the level of 38.90 ± 0.51 µM TE/ mg of the plant volatile oils.The results also indicated the higher antioxidant activity of the C. maritimum Libyan species compared to the plant species growing in different locations. For instance, the extract of the plant species growing in Croatia has demonstrated lower reducing power (FRAP) and peroxyl radical scavenging effects compared to the Libyan species18. This antioxidant potential may related to variable contents of the oil and its enrichment of thymyl methyl ether and other volatile concentrations as γ-terpinene comparing with the same species in other geographical locations. The plant species growing in Tunisia have also exerted antioxidant effects against DPPH free radicals and reduced power for ferric ions at the values of IC50 0.44–3.3 and EC50 2.44–3.08, respectively22, which seem to have lower effects compared to the present antioxidant results recorded for the Libyan species of the plant. However, an exact comparison to the reported antioxidant results is difficult due to the variations in the assay analysis, as the reported methods calculated the IC50/EC5022, and in the current methods, the antioxidant results were expressed as Trolox equivalents (Fig. 3).Fig. 3Antioxidant activities of essential oils from C. maritimum. The values indicated are three equivalent measurements’ means ± SD. TE: Trolox equivalent; FRAP: Ferric reducing antioxidant power; ORAC: Cupric reducing antioxidant capacity; DPPH: 2,2-diphenyl-1-picrylhydrazyl.Tyrosinase and AChE inhibitory effect of C. Maritimum essential oilEnzyme inhibition is an important therapeutic approach for the treatment of a number of issues. For example, AChE has been linked to neurological disorders that are implicated in the etiology of Alzheimer’s disease (AD) and is involved in the hydrolysis of neurotransmitters, specifically acetylcholine, which terminates neurotransmission. On the other hand, melasma, age spots, freckles, and other skin hyperpigmentation problems are thought to be best treated by regulating the manufacture of melanin. Using common in vitro assays, the anti-tyrosinase and anti-AChE properties of C. maritimum essential oil were assessed; the results of Enzyme inhibitory activity of essential oils from C. maritimum are shown in Table 2.Table 2 Enzyme inhibitory activity of essential oils from C. Maritimum.Values expressed are means ± S.D. of three equivalent measurements. AChE: Acetylcholinesterase; Ar: Arbutin equivalent.Acetylcholinesterase (AChE) inhibitors find extensive biological use in the treatment of neurological conditions, particularly Alzheimer’s disease. By impeding AChE activity, these inhibitors elevate acetylcholine levels in the brain, temporarily improving cognitive function in affected individuals. This approach aims to address the neurotransmitter imbalance characteristic of Alzheimer’s, offering symptomatic relief44,45.The mechanism of action of acetylcholinesterase (AChE) inhibitors in Alzheimer’s disease treatment revolves around the enhancement of cholinergic neurotransmission. In Alzheimer’s, there is a deficiency of the neurotransmitter acetylcholine due to increased breakdown by AChE. AChE inhibitors, such as donepezil, rivastigmine, and galantamine, work by blocking the activity of AChE46. By inhibiting AChE, these drugs allow acetylcholine to accumulate in the synaptic cleft, facilitating increased stimulation of cholinergic receptors. This elevated acetylcholine level helps improve neurotransmission and temporarily alleviates cognitive symptoms associated with Alzheimer’s disease, such as memory loss and cognitive decline. While AChE inhibitors do not halt the progression of Alzheimer’s, they provide symptomatic relief and can enhance cognitive function, thereby improving the quality of life for individuals affected by the disease.Tyrosinase enzyme inhibitors, on the other hand, are commonly explored in skincare and cosmetics due to their role in melanin synthesis. By impeding tyrosinase activity, these inhibitors can mitigate hyperpigmentation and even out skin tone. The efficacy of such inhibitors depends on factors like formulation, concentration, and individual skin characteristics47.The mechanism of action of tyrosinase inhibitors in skin disease treatment lies in their ability to regulate melanin production. Tyrosinase is a key enzyme involved in the melanin synthesis pathway. Melanin is the pigment responsible for skin, hair, and eye color. Overactivity of tyrosinase can lead to hyperpigmentation disorders, such as melasma, age spots, and certain types of hyperpigmentation48. Tyrosinase inhibitors, commonly used in skincare products, work by interfering with the enzymatic activity of tyrosinase. By inhibiting tyrosinase, these compounds reduce the production of melanin, leading to a decrease in pigmentation and a more even skin tone. This is particularly beneficial in treating conditions where excessive melanin production results in uneven skin pigmentation49. The application of tyrosinase inhibitors in skincare underscores their significance in addressing cosmetic concerns related to hyperpigmentation and promoting a more uniform complexion.Both AChE and tyrosinase enzyme inhibitors underscore the importance of enzyme modulation in diverse fields, ranging from neuroscience to dermatology, showcasing their potential therapeutic applications.It was previously reported that EOs from different Croatian sea fennel were highly efficient against cholinesterase enzymes, the flower extract from sea fennel proved to have strong vasodilatory properties. Anti-acetylcholinesterase activities of essential oils of aerial parts of Tunisian Crithmum maritimum L. showed nearly the same activity 31.16 ± 0.012 mg/ml compared with the species under investigation; 34.43 ± 0.25 mg/ml19. A previous study evaluated the Anti-Tyrosinase activity of essential oils of Crithmum maritimum L. from France and Croatia, it reported the inactivity of the French one while (IC50 = 649 µg/mL) of Croatian sample. In the other side Libyan species activity is 12.449 ± 0.68, which indicates that geographical variability played an essential role in oil composition and its activity50. In vitro and intracellular antioxidant capacity of thymyl methyl ether was evaluated in Druce leaves and it exhibited a significant intracellular antioxidant capacity and considered as hepatoprotective agent51. These characteristics may indicate the possibility of using sea fennel in the culinary, medicinal, and other industries. Particularly, the flowers and stems have not always been used.One of the most prominent Artemisia campestris essential oil constituents; γ-terpinene was reported both tyrosinase (38.36 ± 3.86%) and AChE inhibition (53.95 ± 5.55%) compared to Kojic acid and Galantamine, respectively52.Molecular docking study on apiole from essential oil of Petroselinum crispum (Mill, ) Fuss in study of some Moroccan Apiaceae species, it showed an AChE inhibition with binding energy (-5.9 kcal/mol) for the interactions with the acetylcholinesterase53.The biological features under investigation were found to be affected differently by the essential oils isolated from aerial parts of Libyan sea fennel, which contain wide variety of phytochemicals of different classes where thymyl methyl ether, γ-terpinene and apiole were the most abundant ones.Docking resultsThis study investigated the potential of Crithmum maritimum essential oils as inhibitors for two important therapeutic targets: the tyrosinase and acetylcholinesterase. To validate the docking procedure, the two co-crystal structures of Donepezil and tropolone were redocked, yielding an RMSD value of 0.69 and 1.96 Å, respectively, as represented in Fig. 4. It is proposed that an RMSD values, falling beneath the 2 Å limit, validates the reliability of the docking protocol for subsequent examinations54. The results suggest that several compounds exhibit promising binding affinities towards these targets, with binding energies ranging from − 5.81 to -13.14 Kcal/mol (Supplementary file).Fig. 4Stick representations of co-crystal structures in grey color and the docked configurations in blue color against (a) Acetylcholinesterase and (b) Tyrosinase enzymes. Generated by BIOVIA Discovery Studio visualizer.Column chart summarizes the molecular docking results of the compounds against both tyrosinase and acetylcholinesterase (AChE) provide significant insights into their potential inhibitory activities. The binding energies for tyrosinase range from − 6.24 to -13.14 kcal/mol. Compounds such as γ-santonin, stigmastene, and Apiol exhibit remarkably low binding energies, suggesting strong binding to tyrosinase. On the other hand, the binding energies for AChE range from − 5.81 to -9.6 kcal/mol. Interestingly, again both stigmastene and γ-santonin demonstrate the strongest binding to AChE among the compounds tested (Fig. 5).Fig. 5Column chart showing the lowest binding energy for the Crithmum maritimum essential oils against tyrosinase and acetylcholinesterase (AChE).According to the initial docking results, γ-santonin and stigmastene emerged as frontrunners due to their exceptional binding affinities towards both enzymes. A more detailed investigation into the intermolecular interactions within the binding pocket of these compounds could elucidate the specific molecular forces governing their remarkable binding behaviors. Table 3; Figs. 6 and 7 showed the detailed intermolecular interactions for both compounds in the respected binding sites.Table 3 Docking scores (LBE, Kcal/mol) and putative interacting amino acids in the binding pocket of compounds exhibiting highest affinity for acetylcholinesterase and tyrosinase enzymes.Fig. 6Stick representation of (a) stigmastene, (b) γ- santonin, and (c) donepezil, docked within acetylcholinesterase (PDB ID: 4EY7) binding site. Generated by BIOVIA Discovery Studio visualizer.Fig. 7Stick representation of (a) tropolone, (b) γ- santonin, and (c) stigmastene, docked within tyrosinase (PDB ID: 2Y9X) binding site. Generated by BIOVIA Discovery Studio visualizer.Stigmastene displayed the highest binding affinity among the three (-13.14 kcal/mol) against acetylcholinesterase enzyme, followed by donepezil (-11.04 kcal/mol) and γ-santonin (-10.31 kcal/mol). Interestingly, stigmastene lacks predicted hydrogen bonds with AChE, while both donepezil and γ-santonin form hydrogen bonds with key amino acid residues. This suggests that stigmastene’s exceptional binding might be driven by other factors like hydrophobic interactions. Additionally, both stigmastene and γ-santonin interact with a similar set of hydrophobic amino acids (Trp86, Tyr124, Trp286, Val294, Phe297, Tyr337, Tyr341, His447). Donepezil also interacts with some of these residues but lacks interactions with Val294 and His447. The extensive hydrophobic interactions observed for stigmastene could be a significant contributor to its strong binding affinity. Even though none of the compounds were predicted to form π-π interactions with AChE residues. Donepezil, however, performs aromatic interactions with Trp87, Trp286, and Tyr341, which might contribute to its binding. These docking results highlight the importance of both hydrophobic interactions and hydrogen bonding in AChE inhibition.On the other hand, stigmastene and γ-santonin demonstrate stronger binding affinity towards tyrosinase compared to the co-crystalized control, tropolone. While γ-santonin forms a hydrogen bond with His259, similar to tropolone, its overall stronger binding could be attributed to additional hydrophobic interactions with other amino acid residues. Again, stigmastene, despite lacking hydrogen bonds, exhibits the strongest binding due to its extensive hydrophobic interactions across the active site. Both stigmastene and γ-Santonin engage in extensive hydrophobic interactions with tyrosinase residues, indicating favorable binding.These findings suggest the potential of stigmastene and γ-santonin as tyrosinase and acetylcholinesterase inhibitors,
Chemical composition, antioxidant, and enzyme inhibition activities of Crithmum maritimum essential oils: the first chemo-biological study for species grown in North Africa
