Chemical composition of Z. coccineum EO
Hydrodistillation of
Z.
coccineum aerial portions yielded a pale yellowish EO with a percentage of 0.049% (v/w), comparable to the 0.044% yield reported for
Z.
album (Kchaou et al., 2016). The extracted EO was investigated utilizing GC-MS to establish its chemical composition (Fig 1). GC-MS analysis identified 22 components, representing 99.97% of the total oil composition (Table 1), a number similar to those identified in the EO of
Z.
album (Kchaou et al., 2016).
The EO constituents of
Z.
coccineum were analyzed, revealing a diverse range of chemical compounds classified into different groups (Table 1). Monoterpenes were present in moderate amounts, with limonene (7.31%) being the most abundant (Fig 2), followed by β-pinene (2.31%), thymol (2.14%) and safranal (1.13%). The phenolic compounds included 4-vinylphenol (1.06%) and eugenol (2.27%). Caryophyllene oxide, a sesquiterpene, accounted for 4.23% of the total composition. The alkyl benzene group was represented by (E)-2-(buta-1,3-dien-1-yl)-1,3,4-trimethylbenzene (3.04%) and 1-(2,3,6-trimethylphenyl)-2-butanone (15.62%), the latter being the second most abundant compound overall (Figure 2). Aldehydes were dominated by (E)-3-(2,6,6-trimethylcyclohex-1-en-1-yl) acrylaldehyde (11.47%), with nonanal contributing 1.68%.
Fatty acids and their derivatives constituted a significant portion, with palmitic acid (20.36%) being the maximum abundant substance in the oil (Fig 2), followed by oleic acid (7.31%), docosanoic acid (3.28%), 2-mono-linolein (3.42%) and 2-mono-olein (2.8%). Steroids were detected in lower amounts, including β-sitosterol-3-O-β-D-glucopyranoside (0.91%), β-sitosterol (0.76%) and stigmasterol (1.57%). Additionally, the hydrocarbon cyclic alcohol 3,3a,7,7-tetramethyl-3a,5,6,7-tetrahydro-4H-inden-4-ol was present at 3.87% (Table 1).
Palmitic acid was found in a superior concentration (20.36%) in the EO of
Z.
eichwaldii (Mazoochi et al., 2021). Additionally, several identified compounds, including thymol and oleic acid, were also reported as constituents of
Z.
eichwaldii EO.
Kchaou et al., (2016) reported that nonanal, safranal, thymol and eugenol are commonly dispersed compounds in EOs of
Zygophyllum album. In this study, oleic acid was found in moderate amounts (7.31%) in the plant extracts, consistent with its presence in the EOs of
Z.
oxianum and
Z.
eichwaldii as reported by previous studies
(Sasmakov et al., 2012; Mazoochi et al., 2021). Additionally,
Mazoochi et al., (2021) documented caryophyllene oxide in
Z.
eichwaldii. Furthermore, limonene, β-pinene, docosanoic acid, 2-mono-linolein and 1-(2,3,6-trimethylphenyl)-2-butanone are commonly found compounds in EOs of various plants, including
Moringa oleifera (
Kuete, 2017),
Pistacia atlantica (Tahir et al., 2019) and
Cannabis sativa (
Judzentienë et al., 2023).
With the exception of the presence of palmitic and oleic acids, the chemical makeup of the EO in this investigation was completely different from that reported for
Z.
eichwaldii EO
(Mazoochi et al., 2021). The genetic distinctions between the two species might be the cause of this variance (
Abd-ElGawad et al., 2021). Furthermore, it has been established that climatic and ecological variables have exert a substantial influence on the composition of EO (
Abd-El Gawad et al., 2020). The EO of
Z.
coccineum contains various chemical classes, with fatty acids and derivatives making up 40.91% of the composition. Alkyl benzenes account for 18.66%, followed by aldehydes and monoterpenes at 13.15% and 13.14%, respectively. Monoterpenes, known for their fragrance, may aid in plant defense and therapeutic uses. Sesquiterpenes (4.53%), hydrocarbons (3.87%), phenols (3.33%) and steroids (3.24%) are also present, contributing to the oil’s stability and pharmacological activities, including antioxidant and anti-inflammatory properties
(Salama et al., 2022).
Antioxidant activity of Z. coccineum EO
The EO of
Z.
coccineum exhibited strong antioxidant capacity in both DPPH and ABTS assays (Table 2). A one-way ANOVA indicated significant differences in scavenging activities across concentrations (p£0.05) and Duncan’s post hoc test revealed that values at 20 and 25 µg/mL were significantly higher than at lower concentrations (p<0.05), confirming a dose-dependent trend. At a concentration of 25 µL/L,
Z.
coccineum EO reduced the DPPH and ABTS colors by 64.05% and 73.59%, correspondingly. Based on IC50 values, the EO exhibited IC50 values of 19.96 µL/L and 16.70 µL/L for DPPH and ABTS, correspondingly, while vitamin C displayed IC50 values of 0.73 µL/L and 0.41 µL/L for the same assays.
The pronounced antioxidant potency of
Z.
coccineum EO in this work may be ascribed to its major constituents, including palmitic acid, oleic acid, 1-(2,3,6-trimethylphenyl)-2-butanone, caryophyllene oxide, limonene and docosanoic acid, which may act either individually or synergistically
(Abd-ElGawad et al., 2020; Salama et al., 2022; El Hachlafi et al., 2024). Notably, palmitic acid, the predominant compound, has been reported at a high concentration (14.85%) in
Reichardia tingitana, which exhibited strong antioxidant activity
(Salama et al., 2022).
Antibacterial activity of Z. coccineum EO
The EO demonstrated differential antibacterial effects against the tested strains (Table 3). With an inhibitory zone of 20.88 mm,
S.
typhimurium showed the greatest sensitivity to the EO among the tested bacterial strains, demonstrating its strong antibacterial action. Similarly,
E.
coli and
B.
cereus exhibited considerable sensitivity, with clear zones of 16.84 mm and 17.91 mm, correspondingly. These findings suggest that the EO possesses potent antibacterial properties, particularly against certain enteric and foodborne pathogens (Table 3).
On the other hand,
P.
aeruginosa exhibited the lowest sensitivity to the EO, with an inhibition zone of only 8.47 mm, suggesting a higher level of resistance. This is consistent with previous reports indicating that
P.
aeruginosa is often more resistant to plant-derived antimicrobials due to its robust efflux pump system and biofilm formation capabilities. Among the Gram-positive bacteria tested,
S.
aureus demonstrated moderate sensitivity, with an inhibition zone of 10.97 mm, whereas
B.
cereus showed a stronger inhibitory response (Table 3). ANOVA confirmed significant differences in bacterial inhibition zones (p≤0.05). Duncan’s test identified
S.
typhimurium as significantly more susceptible than
P.
aeruginosa and
S.
aureus (p<0.05).
The selected antibiotics demonstrated varying degrees of activity versus the tested bacterial strains, with Gram -ve bacteria generally exhibiting higher resistance than Gram-positive strains. This trend aligns with findings from previous research
(Breijyeh et al., 2020; Salama et al., 2022; Abduljabbara et al., 2024), which attribute the observed resistance to the structural differences in bacterial cell walls
(Breijyeh et al., 2020). Gram -ve bacteria’s outer membrane serves as an extra barrier, preventing antibiotics from penetrating and enhancing their resistance. The reference antibiotic tetracycline exhibited the strongest inhibition across all tested bacteria, with MIC values extending from 0.035 to 0.058 mg/ml. Azithromycin and ampicillin, however, showed inconsistent effects, with
S.
typhimurium and
P.
aeruginosa being resistant to at least one of these antibiotics. Interestingly, the EO demonstrated higher efficacy against
S.
typhimurium compared to both azithromycin and ampicillin, indicating its potential as an alternative antimicrobial agent.
The antibacterial activity of the EO may be attributed to its major bioactive components, including limonene, 1-(2,3,6-trimethylphenyl)-2-butanone. (E)-3-(2,6,6-trimethylcyclohex-1-en-1-yl) acrylaldehyde, palmitic acid and oleic acid. These compounds may exert their antimicrobial effects either individually or through synergistic interactions. A similar study by
Han et al., (2021) demonstrated potent inhibitory activity against
S.
aureus. Additionally, according to
Kuete (2017), 1-[2,3,6-trimethylphenyl]-2-butanone is a significant component of
Moringa oleifera EO and has strong microbicidal, anti-inflammatory and antidiabetic effects. Moreover, palmitic and oleic acids have also shown antibacterial activity against various Gram-positive and Gram-negative oral pathogens
(Huang et al., 2010). Furthermore,
Sharma et al., (2021) identified palmitic acid as a major compound (15.57%) in
Capsicum annuum oleoresin, where it exhibited antimicrobial potential towards
B.
subtilis,
E.
coli,
P.
aeruginosa and S.
aureus.
EOs with high concentrations of caryophyllene oxide have also exhibited significant antimicrobial properties
(Bhaisare et al., 2016; Abd-ElGawad et al., 2022). Moreover, EOs intense in fatty acids and monoterpenes have been reported to possess substantial antimicrobial activity
(Marchese et al., 2017).
Cytotoxic activity of Z. coccineum EO
The EO of
Z.
coccineum exhibited dose-dependent cytotoxicity against MCF-7 breast cancer and normal WI-38 cells at different concentrations, with doxorubicin as a standard (Fig 3). The highest inhibition rate was recorded at 70.96% at 800 µg/mL, while the IC50 value for MCF-7 cells was calculated to be 48.26 µg/mL. In contrast, its effect on normal WI-38 cells was minimal, with greatest inhibition of 9.32% at 1000 µg/mL and an IC50 value exceeding 100 µg/mL, indicating selective toxicity toward cancer cells. Doxorubicin exhibited stronger cytotoxicity, achieving complete inhibition (100%) at 800 µg/mL, with an IC50 of 5.24 µg/mL for MCF-7 cells. However, it also affected normal cells, showing 11.61% cytotoxicity at 1000 µg/mL.
These findings indicate that EOs exhibits promising anticancer potential with selective cytotoxicity, suggesting its potential as a natural medicinal agent to treat breast cancer. According to published guidelines, cytotoxicity is classified based on IC
50 values as follows: IC
50 less than 10 µg/mL is considered very toxic, IC
50 between 10 and 100 µg/mL is classified as potentially toxic, IC
50 ranging from 100 to 1000 µg/mL is deemed hypothetically harmful and IC
50 greater than 1000 µg/mL is regarded as potentially non-toxic (
Gad-Shayne, 2009). This classification provides a valuable reference for evaluating the cytotoxic potential of phytoconstituents of extracts and essential oils in biomedical applications.
The principal chemical substance in the EOs analyzed are responsible for the cytotoxicity of the oils employed in this investigation. Several factors influence the cytotoxic mechanism, such as the sample intensity, nature of the isolated components, phytochemical content, nanoparticle model, size and collection and the nature of the tumor and non-cancerous cell line. Cytotoxicity may also be related to protein loss (
El-Amier et al., 2023).
The main component of EOs, palmitic acid, has cytotoxic properties, especially against cancer cells, via a number of pathways, including oxidative stress and apoptosis induction
(Biswas et al., 2022). According to
Kuete (2017), 1-[2,3,6-trimethylphenyl]-2-butanone, derived from
Moringa oleifera essential oil, exhibited anticancer activity. In addition, limonene induced cytotoxic effects on multiple cancer cell lines, including those of the colon, prostate, lung and breast
(Chaudhary et al., 2012). Oleic acid and limonene have also been said to enhance the action of conventional chemotherapy drugs such as doxorubicin and paclitaxel
(Menendez et al., 2005).