Phytochemical characterization and assessments of antimicrobial, cytotoxic and anti-inflammatory properties of Lavandula coronopifolia Poir. volatile oil from Palestine

2.1 Plant collection and drying

From the lowest point on the earth, Jericho-Palestine, the LC aerial parts (leaves, flowers, and stems) were collected in July 2019. The botanical recognition of LC herb was performed in the Laboratory of Natural Products by a specialist Dr. Nidal Jaradat. The sample was deposited in the same laboratory with a voucher specimen code of pharm-PCT-1367.

The collected aerial parts were deeply washed and dried for nine days in the shade at a humidity level of 50 ± 3% RH) and stable room temperature (24 ± 4 °C). After drying, the dried herbal material was ground roughly and stored in special containers for further use.

2.2 Extraction procedure

The VO of the LC herb was obtained by the water-distillation system described by (Jaradat 2016). In brief, about 100 g of the herb was mixed well with 1 L of distilled water, and the VO was extracted using a Clevenger apparatus operating at atmospheric pressure for 240 min at 100 °C. The extracted VO was chemically dried using magnesium sulfate and then kept in a refrigerator at 4–6 °C until further use.

2.3 Characterization of VO

The volatile compounds of the LC herb were recognized and quantified using gas chromatography connected with mass spectrometry (GC–MS), as described by (Jaradat et al., 2022). Briefly, the analysis was done by an HP 5890 series II gas chromatograph linked to a mass spectrometer, Perkin Elmer Elite-5-MS. However, the components were identified based on the comparison of their relative retention index and compared with the library mass spectral database (Wiley and NIST databases). The percentage composition of compounds was measured based on the peak area.

2.4 Bactericidal and fungicidal activity

Proteus vulgaris (ATCC 8427), Klebsiella pneumonia (ATCC 13883), Staphylococcus aureus (ATCC 25923), Pseudomonas aeruginosa (ATCC 9027), Escherichia coli (ATCC 25922), and clinically diagnosed methicillin-resistant Staphylococcus aureus (MRSA) were the screened bacterial species. Moreover, Candida albicans (ATCC 90028) was the fungi screened strain in this study. The bactericidal and fungicidal activity of LC-VO were carried out by the broth microdilution method as described by (Balouiri et al., 2016). This procedure was conducted in triplicate. In addition, bactericidal and fungicidal activity was validated utilizing known antibiotics, namely ciprofloxacin, ampicillin, and fluconazole.

2.5 COX inhibitory assay

Utilizing a COX inhibitor screening test kit, the capacity of the LC-VO to prevent the conversion of arachidonic acid to PGH2 by bovine COX-1 and human recombinant COX-2 was assessed according to the manufacturer's instructions (Item No: 560131, Cayman Chemical, USA). The IC₅₀ values (50 percent inhibitory concentration) of VO COX-1/COX-2 potentials were calculated using two concentrations (50 and 350 µg/mL). To assess the inhibition of the LC-VO, a standard curve of eight concentrations of prostaglandin, a non-specific binding sample, and a maximal binding sample were utilized, as indicated in the manual of the used kit, and the produced multiple regression best-fit line was employed. The percentage inhibition of the two concentrations was utilized to derive the IC₅₀ values (Jaradat et al., 2020).

2.6 Cytotoxicity procedure

In (RPMI-1640) medium was mixed with a mixture of 10% fetal bovine serum, 1% Penicillin/Streptomycin antibiotics to avoid contamination, and 1 % l-glutamine, the HeLa (cervical adenocarcinoma) tumor cells were cultured in 96-well plates (2.6 × 10⁴ cells) and grown in a humidified atmosphere containing 5% CO₂ at 37 °C. After two days, the cells were incubated with VO at various concentrations (1, 0.5, 0.25, 0.13 and 0.063 µg/mL) for 24 h. Cell viability was estimated by Cell-Tilter 96® Aqueous One Solution Cell Proliferation (MTS) Assay following the directions of the producer (Promega Corporation, Madison, WI). At the end of the treatment, 20 μL of MTS solution per 100 μL of media was added to each well and incubated at 37 °C for two h. Finally, the absorbance was measured by spectrophotometer apparatus at 490 nm (Mosmann, 1983).

2.7 Statistical analysis

All conducted experimental works in this investigation were carried out in triplicate for the LC-VO. The results were expressed as means (±) standard deviation (SD). A p-value < 0.05 was considered statistically significant.

3.1 Phytochemical composition

The hydro-distillation of the ground air-dried aerial parts of LC afforded a colorless aromatic smelling oil in 1.03% v/w. The VO components were identified by GC–MS analysis based on comparing the obtained Kovats retention index (RI) and MS fragmentation patterns to those of standard compounds and on matching with the NIST library. The identified components, their retention indices, and their percentages from the total oil are presented in Table 1.

3.2 Antimicrobial effect

The Microdilution broth technique was employed to estimate the microbicidal effect of LC-VO. The result showed that most of the microbial growth is inhibited by LC-VO, as illustrated in Table 2. Compared with pharmaceutical antifungal and antibacterial agents, including fluconazole, ampicillin, and ciprofloxacin. This test outcome showed that the LC-VO has a strong microbicidal effect against most of the tested strains, especially against C. albicans, P. vulgaris, S. aureus, and MRSA.

3.3 COX inhibitory activity

In the current investigation, the ability of the LC-VO to inhibit the COX-1 and COX-2 enzymes was established by employing a cyclooxygenase inhibitory screening assay kit, and the outcomes were compared with a commercial NSAID ketoprofen. Fig. 1 presents the inhibition percentage of both LC-VO and positive control ketoprofen at two different concentrations toward COX-1 and COX-2. It was clear that LC-VO has a potent inhibition percentage at both used concentrations. However, it was more potent against COX-2 iso-enzyme than the positive control, with values of inhibition of 88.02 and 65.02%, respectively.

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Fig. 1 Inhibition percentages of COX-1 and COX-2 by LC-VO and positive control ketoprofen (p values < 0.005).

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3.4 Cytotoxic activity

The result of the cytotoxic effect of LC-VO on HeLa (cervical adenocarcinoma) was evaluated using the MTS assay. The treatment of HeLa cells with 1 µg/mL of LC-VO induced the best cytotoxic effect (95.14%) with an IC₅₀ value of 0.71 ± 0.01 µg/mL. In addition, the growth inhibition according to each used dose was presented in Fig. 2 compared with positive control Dox (Doxorubicin).

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Fig. 2 Cytotoxic effects of the LC-VO in comparison with the positive control (Dox) against HeLa cancer cell line.

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4.1 Phytochemical components

GC–MS analysis recognized 29 components accounting for around 99.8% of the total VO content. The most LC-VO content was contributed by oxygenated monoterpenes (93.06%), of which linalyl acetate (41.65 ± 0.91%), linalool (41.41 ± 0.77%), and α-terpineol (4.60 ± 0.31%) were the main compositions of the LC-VO. Only 1.45% of total VO was made up of monoterpene hydrocarbons, with limonene (1.26 ± 0.02%) being the most abundant. The total amount of sesquiterpenes in the oil accounted for only 1.9% of the total amount of oil. A comparison of the reported volatile components extracted from LC by hydro-distillation revealed differences in the occurrence of major components, which could be attributed to environmental conditions, soil characteristics, harvest time, and drying methods (Moghaddam and Mehdizadeh, 2017).

Our outcomes are in relative agreement with those of Aburjai et al., who documented that oxygenated terpenes (80.60–85.59%) were the major constituents of VO of LC herb grown in Jordan, where linalool, camphor, terpinen-4-ol, 1,8-cineole, and borneol were found to be the major constituents. They discovered that linalool (41.2%) was the most abundant constituent in flowering tops and leaves, while 1,8-cineole (25.4%) was the most abundant constituent in the entire aerial parts oil. Most of the VOs components showed highly significant qualitative and quantitative differences for different geographical regions (Aburjai et al., 2005).

Messaoud et al. identified 29 compounds in LC-VO grown in Tunisia, with monoterpene hydrocarbons (46.2%) being the most abundant fraction, followed by oxygenated monoterpenes (27.6%). The abundant molecules were trans-β-ocimene (26.9%), carvacrol (18.5%), β-bisabolene (13.1%), myrcene (7.5%), and α-terpinolene (5.4%) (Messaoud et al., 2012).

4.2 Antimicrobial activity

Due to the presence of VOs, medicinal herbs can impede the growth of various harmful microbes. However, in the field of infectious diseases caused by microbial resistance, no one can deny the role of VOs in discovering new potential microbicidal therapeutics. 2-Isopropyl-5-methyl phenol, linalool, eugenic acid, menthols, and caryophyllene are among the significant compounds used so far to develop potent antimicrobial agents (Akthar et al., 2014, Osuntokun and Ogunleye, 2017).

However, the development of resistance to antibiotics against MRSA limits their effectiveness, which will only worsen in the future. Therefore, there is a lot of work to be done because there is an urgent need to discover novel alternative pharmaceuticals (Guo et al., 2020).

The obtained data from the microbicidal test revealed variability in the MIC of LC-VO. In fact, the antibacterial lowest MIC value was noticed against MRSA (0.33 ± 0.03 µg/mL) followed by S. aureus (0.63 ± 0.1 µg/mL), and both of them are gram-positive bacterial strains. The LC-VO exhibited a more potent effect than the commonly used positive antibiotic controls against these two virulent species. Furthermore, LC-VO inhibited the growth of P. vulgaris (Gramm-negative bacteria) significantly more than Ampicillin and Ciprofloxacin, with MICs of 1.25 ± 0.81, 18 ± 0.12, and 15 ± 0.11 µg/mL, respectively. Compared with the potent fluconazole antifungal pharmaceutical, the LC-VO showed remarkable fungicidal effects with MICs of 0.16 ± 0.06 and 1.56 ± 0.1 µg/mL, respectively. Indeed, linalool and linalool acetate-producing species have potent antimicrobial activity against both bacterial and fungal species.

Actually, linalool and linalool acetate are by far the most abundant acyclic monoterpenoid, accounting for approximately 70% of the isoprenoid content found in the scents of flowers. They were recognized in the VOs of 200 species of plants that belong to various families (Carson and Riley, 1995, Pattnaik et al., 1997).

The results of the Khayyat study demonstrated that linalool acetate strongly inhibited the growth of Microsprum canis and Microsprum gallinna grown on solid media. Furthermore, it showed that all linalyl acetate and its derivatives effectively reduced the growth of tested fungi (Khayyat, 2020).

Besides, Nelson reported that L. angustifolia, which was rich in linalool, has potential activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium (Nelson, 1997).

In addition, our microbicidal study results agree with a study by Bogdan et al. in which they found that L. angustifolia VO rich in linalool and linalool acetate manifested remarkable antibacterial potential against S. aureus (12.5 v/v) and E. coli (25 v/v) and also has a potent fungicidal effect against C. albicans (12.5 v/v) (Bogdan et al., 2021).

4.3 Anti-inflammatory activity

Inflammation is a critical physiological reaction to activation of cellular damage, infectious diseases, or insufficient immune system. Excessive production of proinflammatory mediators can result in severe and chronic inflammation, resulting in various conditions, including rheumatoid arthritis, rheumatoid arthritis, asthma, multiple sclerosis, and atherosclerosis (Nathan, 2002).

The present investigation outcomes revealed that the LC-VO has inhibitory actions on COX-1 and COX-2. Indeed, the LC-VO COX inhibitory activity estimations demonstrated that this VO has a much more specific inhibitory effect on COX-2. Hence, it had higher COX-2 inhibitory activities at 50 µg/mL than ketoprofen.

The percentage of COX-2 inhibition increased as the concentration of VO used increased, rising from 88 to 97 percent when the VO concentration was increased from 50 to 350 µg/mL. Similarly, COX-1 inhibition increased as the VO concentration rose. For example, when the LC-VO concentration was increased from 50 to 350 µg/mL and the COX-1 inhibition rose from 95 percent to 99.55 percent.

An investigation conducted by Peana et al. indicated that linalool and linalool acetate play a vital role in the anti-inflammatory effect of the VOs containing these compounds in herbal species. Furthermore, their investigation proved that linalool and linalool acetate performing plant species are potent anti-inflammatory agents (Peana et al., 2002).

Moreover, an investigation established by Husseini et al. showed that L. officinalis hydroalcoholic extract inhibited the COX activity in a dose-dependent manner, where the inhibitory effects on COX type 1 and 2 activities were 33 and 45%, respectively (Husseini et al., 2016).

4.4 HeLa cytotoxic effect

Different kinds of cytotoxic examinations, such as colorimetric, dye exclusion, fluorometric, and luminometric assays, are utilized in toxicology and pharmacology (Gerets et al., 2009). The LC-VO has a moderate cytotoxic effect because in comparison with Doxorubicin. This potent anticancer commercial drug has cytotoxic activity against HeLa cancer cells with an IC₅₀ dose of 0.17 ± 0.02 µg/mL. On the other hand, the LC-VO displayed approximately four folds’ lower cytotoxicity against HeLa cancer cells with an IC₅₀ dose of 0.71 ± 0.01 µg/mL.

L. angustifolia VO and its main components, linalool, and linalyl acetate, were found to have strong cytotoxic activity against DU145 and PC-3 cell lines, with IC₅₀ values of (0.199 ± 0.026 percent and 0.037 ± 0.011 percent (v/v)), (7.22 ± 0.28 µM, 11.74 ± 0.62 µM), and (3.06 ± 0.22 µM and 4.98 ± 0.31 µM), respectively (Zhao et al., 2017). Furthermore, Al-Sheddi discovered that MDA-MB-231 cells' survival reduced in a concentration-dependent manner when exposed to ethanol, chloroform, ethyl acetate, and petroleum ether extracts of LC plant (Al-Sheddi 2019).

Finally, we would say that the LC screened plant was collected from the Jericho region, which is located near the north of the Dead Sea in Palestine. Jericho is considered the lowest area on earth and has a unique climatic zone (Juaidi et al., 2016). It is among the Palestinian areas that are popular for its variety of bioactive plant species used by the local population for diverse goals. As expected, the Jericho region has very different ecological and environmental conditions than other places (Qumsiyeh and Abusarhan, 2021). This is why the composition of the VOs in this study is very different from other studies. Moreover, to reach the cytotoxic dose on HeLa cancer cell lines, we need about ten folds of the effective concentration dose of bacterial strains (MIC) or COX enzymes inhibitory activities. The cytotoxic effects of plants or active agents against HeLa cancer cell lines could be used instead of normal cell lines to evaluate the cytotoxicity (Hawash et al., 2020).