Variability in the volatile constituents and biological activities of Achillea millefolium L. essential oils obtained from different plant parts and by different solvents

2.1 Chemicals and reagents

All the chemicals were of analytical grade. Anhydrous sodium sulfate, DPPH ((2,2-diphenyl-1-picrylhydrazyl), ammonium molybdate, TPTZ (2,4,6-Tris(2-pyridyl)-s-triazine, ferric chloride, ferrous chloride, ferrozine, and EDTA (ethylenediaminetetraacetic acid), were purchased from Sigma Aldrich, Germany. The V-FITC apoptosis kit was obtained from Abcam Inc. in Cambridge Science Park, Cambridge, UK.

2.2 Instruments

Clevenger apparatus was obtained from Standard Scientific Glass Industries, Mumbai, India, and was used for the volatile oil distillation; a vacuum pump (Büchi Rotavapor R-114, Büchi, Switzerland) was used for the evaporation of the solvents; a Perkin Elmer Auto System XL (Waltham, MA, USA) was used for the analysis of the volatile oil batches; and a microplate reader (BMGLABTECH®-FLUO Star Omega, Ortenberg, Germany) was used to measure the absorbances of the quantitative antioxidant assays and the protein-bound SRB stain.

2.3 Plant materials

The plant materials were collected from local farms in the northern area of the Qassim government, Saudi Arabia, during spring 2020 (March–April). A representative sample of more than 100 individual plants was gathered, which was sufficient for the current experiment. The plant was identified as Achillea millefolium L. by the taxonomists at the Department of Plant Production and Protection, College of Agriculture, Qassim University. A voucher specimen of the plant has been stored at the College of Pharmacy, Qassim University.

2.4 Distillation of the volatile oil batches

About 500 g of the fresh plant materials, i.e., leaves, stems, or aerial parts, were divided into three equal parts and separately packed into the 2 L stoppered conical flask and mixed with 700 mL of the distilled water. The flask was then connected to the Clevenger apparatus (capacity of 2000 mL, Standard Scientific Glass Industries, Mumbai, India) and fixed to the heating mantel. The distillation processes were extended to 5 h, and the distilled oil was separated by a separating funnel and collected over anhydrous sodium sulfate to remove any moisture remaining from the distillation process. The produced volatile oil batches were stored at −20 °C in a freezer for further biological and phytochemical evaluations. The distillation procedure has been conducted in triplicate. The volatile oil yield percentages were found to be 0.33, 0.65, and 0.61% from leaves, stems, and aerial portions, respectively.

2.5 Extraction of the plant materials

About 200 g of the fresh plant materials as a whole herb including the stems, aerial parts, and leaves were reduced into small pieces and divided into two portions; one portion was macerated in n-hexane and the other in diethyl ether for 24 h. The extracts were filtrated and subjected to a vacuum pump (Büchi Rotavapor R-114, Büchi, Switzerland) under reduced pressure to remove the solvents. The semi-solid, sticky extracts were kept in the −20 C freezer, awaiting further phytochemical and biological analysis. The extraction procedure has been conducted in triplicate. The volatile oil yield percentages were found to be 1.34% and 1.11% for the n-hexane and diethyl ether extracts, respectively.

2.6 Phytochemical analysis of the plant essential oils and extracts

All the volatile oil batches and extracts obtained from the plant were analyzed by the GC-FID and GC–MS spectral analysis using the Perkin Elmer Auto System XL (Waltham, MA, USA) equipped flame ionization detector in the GC-FID and the Agilent 8890 GC system attached to a PAL RTC 120 auto-sampler and equipped with a mass detector in the GC–MS. The analysis conditions in both methods exactly followed our previous literature (Mohammed, 2022; Mohammed et al., 2022c, 2021).

2.7 Antioxidant activity of the plant essential oils and extracts

The antioxidant activity of all batches (distilled oils obtained from leaves, stems, and aerial parts, and extracts obtained by n-hexane and diethyl ether) was measured using four different in vitro assays. These assays include total antioxidant activity (TAA), DPPH scavenging activity (DPPH-SA), ferric reducing antioxidant power (FRAP), and metal chelating activity (MCA) assays, which were conducted in triplicate. The TAA, DPPH-SA, and FRAP activities of all batches were calculated as a Trolox equivalent using standard calibration curves of Trolox with the molybdate reagent, DPPH, and FRAP reagent, respectively.

In the TAA, freshly prepared molybdate reagent (3.6 mL) (Aroua et al., 2021) was mixed with 400 µL of the samples (containing 200 µg of the crude oils or extracts) in glass test tubes and heated over a water bath (90 °C) for 30 min. The color of the reaction mixtures was measured at 695 nm (Aroua et al., 2021). In the DPPH-SA, a mixture of 1 mL of the samples (containing 200 µg of the crude oils or extracts) and a freshly prepared DPPH solution (12 mg of the DPPH in 100 mL of the methanol) were prepared and kept for 30 min. The DPPH color reduction was measured at 517 nm (Sulaiman et al., 2013; Mohammed et al., 2021). For the FRAP, 2 mL of the FRAP reagent (freshly prepared as mentioned in the literature (Benzie and Strain, 1996)) was mixed with 0.1 mL of the sample solution (which contained 200 µg of the crude oils or extracts). The reaction mixtures were measured at 593 nm (Aroua et al., 2023). The MCA of all batches was calculated as EDTA equivalents using the procedures of Zengin et al. (Zengin et al., 2016). The mixtures of samples (2 mL contain 200 µg of the crude oils or extracts), 2 mM FeCl₂ (25 µL), and 100 µL of ferrozine were measured at 562 nm against a blank (which contained all the mixture's ingredients except ferrozine).

2.8 Cytotoxic activity

2.9 Apoptosis assay

The most active batch, n-hexane, was used for the apoptotic test. Using a 2-fluorescent channel flow cytometer in conjunction with an Annexin V-FITC apoptosis detection kit from Abcam Inc. in Cambridge Science Park, Cambridge, UK, it was possible to determine the populations of cells that were in apoptosis and necrosis (Sulaiman, 2015). After treatment with n-hexane extract for 24/48 or 72 h, Panc-1 and MCF-7 Adr. cells (10⁵cells) were collected by trypsinization and washed twice with ice-cold PBS (pH 7.4). Then, in accordance with the manufacturer's instructions, Panc-1 and MCF-7 Adr. cells were incubated in the dark for 30 min at room temperature with 0.5 mL of Annexin V-FITC/PI solution. After staining, Panc-1 and MCF-7 Adr. cells were inoculated using an ACEA Novocyte™ flow cytometer (ACEA Biosciences Inc., San Diego, CA, USA). FITC and PI fluorescent signals were then evaluated using FL1 and FL2 signal detectors, respectively (ex/em 488/530 nm for FITC and ex/em 535/617 nm for PI). 12,000 events were collected for each sample of n-hexane extract, and the numbers of positive FITC and/or PI cells were computed using ACEA NovoExpressTM software (ACEA Biosciences Inc., San Diego, CA, USA) and quadrant analysis.

2.10 Docking method

The three main components of the active anticancer extracts, n-hexane and diethyl ethers (germacrene D, viridiflorol, and caryophyllene oxide), were docked using AutoDock Vina v1.1.2. The X-ray crystal structure of ABCB1 was downloaded from the Research Collaboratory for Structural Bioinformatics (RCSB), protein data bank (available online at https://www.rcsb.org/structure/) (Alam et al., 2019; Mohammed et al., 2022a). The AutoDock Tool (ADT), included with the MGLTools package (version 1.5.6), was used to prepare and optimize proteins and ligands for the AutoDock Vina (Morris et al., 2009). Water molecules and the co-crystallized ligands were eliminated. After making any necessary adjustments for missing residues and close interactions, polar hydrogen atoms were then added to the protein molecules. During the preparation of the protein, charges were involved. The structures of Achillea millefolium compounds were retrieved from the PubChem database (Kim et al., 2019). The grids were generated with the autogrid4 program distributed with AutoDock, v1.5.6 (Mohammed et al., 2022b). The grid centers were selected to be the proteins' active binding locations. All the atoms in the ligand set fit inside the size of the central grid box (Kalinowsky et al., 2018). Using the Discovery Studio 2017 Client, several protein–ligand interactions, including hydrogen bonds and other hydrophobic interactions, were examined. The resulting docking scores were characterized as affinity binding (Kcal/mol) (Biovia, 2016).

2.11 Statistical analysis

The mean ± standard error of the mean/standard deviation was used to express the results. Tukey's multigroup comparison in GraphPad Prism 8.0.2 was conducted as a post hoc test, with a significance value set at p < 0.05.

3.1 Phytochemical analysis

A. millefolium is a medicinal plant used in traditional medicine for several purposes, including wound healing, gastrointestinal disorders, reducing fever, epilepsy, hemorrhage, and microbial infections (Rohloff et al., 2000). The plant's volatile oil contents, which have been discovered to vary depending on the plant's growth environment, as shown in Table 1, are largely responsible for the plant's use in traditional medicine, making the economic worth of these oils an important consideration. The results revealed lower volatile oil percentages of yields that were obtained from the plant by distillation (volatile oil yield percentages were found to be 0.33, 0.65, and 0.61% from leaves, stems, and aerial portions, respectively) compared to the yields obtained by the extraction method (extract yield percentages were found to be 1.34 and 1.11% for n-hexane and diethyl ether extracts, respectively), an effect that could be related to the ability of non-polar solvents to extract other non-volatile constituents of the plant, such as sterols and fats. The current percentages of yield have been found to be high when compared to the literature. For example, the volatile oil yield of A. millefolium growing in Estonia has been reported as 0.13–0.14% from leaves and 0.02% from stems of the plant (Orav et al., 2001). However, our result for the aerial parts volatile oil yield was nearly similar to the yield obtained from the same part of the plant growing in Turkey (Candan et al., 2003). Also, the plant leaves growing in Lithuania have offered a similar volatile oil percentage (0.1–0.4%) of yield as the yield recorded for the leaves in the current study (Mockute and Judzentiene, 2003). The supercritical CO₂ extraction of A. millefolium leaf essential oils is better than the hydrodistillation process, as the supercritical CO₂ extraction of plant species growing in the Republic of Macedonia has offered a 0.65% volatile oil yield compared to the 0.33% obtained by the hydrodistillation in the current study (Bocevska and Sovová, 2007). The volatile constituents of the A. millefolium essential oils in the distillate batches from leaves, stems, and aerial parts, as well as the extracted batches by n-hexane and diethyl ether, were analyzed by gas chromatography (GC) spectroscopic techniques. The results shown in Table 2 indicated significant phytochemical variations between all the batches, which were reflected in the representation and quantification of the volatile constituents. The analysis revealed the presence of 21, 17, 15, and 24 identified compounds in the total chromatogram in the GC-FID spectra of n-hexane, diethyl ether, stem, and aerial parts, respectively. However, the highest number of the identified compounds was found in the volatile oil distillate of the leaves of the plant (33 compounds, representing 99.39% of the total peaks in the GC chromatogram). The analysis also indicated variations in the major classes of volatile oils among all batches of the plant. For example, the percentages of the monoterpene’s hydrocarbons (non-oxygenated monoterpenes) were found at the highest levels in the distillate volatile oil batches (obtained from leaves, stems, and aerial parts) compared to the batches extracted by the n-hexane and diethyl ether solvents (Table 2, Fig. 1), which might be an indication that these classes of compounds, e.g., α-pinene and myrcene, were better obtained from the plant by hydrodistillation. Furthermore, the highest class of the compounds were found as oxygenated monoterpenes; however, their representations in the plant batches were significantly varied as 68.58%, 75.34%, 60.42%, 38.91%, and 38.85% in the leaves, stem, aerial parts, n-hexane, and diethyl ether batches, respectively (Fig. 1). Variations were also found in the percentages of the oxygenated- and non-oxygenated sesquiterpenes in all batches; however, their representation in the extract batches (n-hexane and diethyl ethers) was comparatively higher than the distillated oil batches (leaves, stems, and aerial parts volatile oil batches) of the plant (Fig. 1, Table 2). The percentages of monoterpenoids and sesquiterpenoids in different batches also reflect the limited ability of non-polar solvents to extract the monoterpenes, including the oxygenated and non-oxygenated compounds. However, sesquiterpene compounds have a higher liability of being extracted by non-polar solvents, i.e., n-hexane and diethyl ether.

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Fig. 1

A cumulative histogram of the major classes of volatile oils in A. millefolium batches.

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The results in Table 2 also indicated significant variations among the major volatile constituents of the plant. The componential analysis of the major constituents, e.g., myrcene, 1,8 cineole, trans-sabinene hydrate, α-thujone, β-thujone, thymol, germacrene D, and viridiflorol, revealed distinct up down flocculation in all batched of the plant. The ketonic monoterpenes α-thujone and β-thujone were found to be the most representative compounds in the volatile oils and extract batches of the plant. They together represented 58.60%, 47.59% and 38.29% in the leaves, stems, and aerial parts volatile oil batches, respectively. They also represented 22.21% and 21.87% of the n-hexane and diethyl ether extracts of the plant, respectively. On the other hand, the sesquiterpene hydrocarbon, germacrene D and sesquiterpene alcohol, viridiflorol, were measured at significantly higher levels in the plant extracts, i.e., n-hexane and diethyl ether, compared to the plant volatile oil distillates from leaves, stems, and aerial parts.

3.2 Antioxidant activity of A. millefolium essential oil

To assess the antioxidant potency of A. millefolium essential oil, four different in vitro assays were performed as outlined in the literature (Mohammed, 2022). These assays evaluated the batches reducing ability to the metals through measuring TAA and FRAP, the scavenging ability through the DPPH assay, and the metal-chelating activity through the MCA assay. The measurements were carried out in a manner comparable to that of standard antioxidant compound, Trolox, in the TAA, DPPH, and FRAP assays, and the standard metal-chelating agent, EDTA, in the MCA assay. The results of Fig. 2 display the comprehensive findings of the study, which demonstrate the remarkable antioxidant capabilities of the volatile oils and non-polar extracts derived from A. millefolium. The results also indicated variability in the antioxidant activity of different volatile oil batches and extracts of the plant. For example, all batches of the plant were comparable in inducing MCA, FRAP, and DPPH scavenging effects; however, volatile oil batches obtained from leaves, stems, and aerial parts of the plant exhibited substantially better TAA compared to the non-polar extracts of the plant. The better reducing activity of the volatile oil batches compared to the non-polar extracts might be attributed to the presence of myrcene, 1,8 cineole, camphor, α-thujone, and β-thujone, which have been reported for their antioxidant activity (Mohammed, 2022; Mohammed et al., 2021a, 2020; Mot et al., 2022), in higher concentrations in the volatile oil batches compared to their presence in the non-polar extracts, n-hexane, and diethyl ether extracts (Fig. 2).

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Fig. 2

Histogram of antioxidant activity in the TAA, DPPH, FRAP and MCA tests of A. millefolium volatile oils in the distillate batches from leaves, stems, and aerial parts, as well as the extracted batches by n-hexane and diethyl ether, compared to reference standard antioxidant compound, Trolox, and standard metal-chelating agent, EDTA.

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The results highlighted the environmental conditions effects on the biological activities of A. millefolium essential oils. The antioxidant activity of isolated essential oils was previously determined from A. millefolium on the mountain Jahorina (Bosnia and Herzegovina) using different antioxidant methods (ABTS, DPPH, and FRAP) with the same parameters used in our experiment. By comparing the chemical profiles of the oils of Achillea species from different locations, it showed significant differences in oil composition and in their antioxidant activity. In our species, it showed a higher record in the DPPH antioxidant method than in other countries. It is assumed to be related to the different geographical origins, which have a significant impact on the composition of volatile oils and their antioxidant activity (Čulum et al., 2021).

3.3 Cytotoxic and apoptotic activity of Achillea millefolium essential oil

It is worth noting that the susceptibilities of the cell lines examined to the n-hexane and diethyl ether extracts of A. millefolium were greater compared to those of the distilled oil batches at all concentrations (Fig. 3).

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Fig. 3

Cytotoxic activity of Achillea millefolium essential oil against different cell lines: (a) A-431: human epidermoid skin carcinoma cell lines; (b) HSF: human skin fibroblast cell lines; (c) MCF-7-Adr: doxorubicin-resistant breast cell lines; (d) MCF-7: breast adenocarcinoma cell lines; (e) PANC-1: pancreatic cancer cell lines.

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The results also indicated that Panc-1 and MCF-7-Adr cell lines were more susceptible to the n-hexane and diethyl ether extracts, compared to the other cancer cell lines. This suitability of the cells was recognized by comparing the calculated IC₅₀s displayed in Table 3, which indicated lower IC₅₀ values for the Panc-1 and MCF-7-Adr (35 and 43.3 μg/mL, respectively, in the n-hexane extract and 41.3 and 57.1, respectively, in diethyl ether extract), compared to other cell lines, i.e., MCF-7, A-431, and HSF cells. In addition, the IC₅₀ values represented in Table 3 indicated the superior cytotoxic effect of n-hexane extract over diethyl ether extract and all the volatile oil distilled batches of the plant. The dose dependent cytotoxic effects of all batches are represented in Fig. 3 and indicate the remarkable higher activity of non-polar extracts, n-hexane and diethyl ether extracts, compared to the oil distilled batches obtained from leaves, stems, and aerial parts, especially at the higher doses, i.e., 10 and 100 μg/mL. The overall higher cytotoxicity activity of non-polar extracts can be attributed to the existence of higher levels of germacrene D, viridiflorol, and caryophyllene oxide, which were previously recognized as cytotoxic sesquiterpenes (Karakaya et al., 2020; Akiel et al., 2022), respectively.

The present study tested whether apoptosis induction may contribute to the growth inhibitory function of n-hexane extract in Panc-1 cells and MCF-7 Adr cells (Figs. 4 and 5). The ability of n-hexane extract to induce apoptosis in both cell lines was thus evaluated by treating cells with two concentrations of IC₅₀ for 24, 48, and 72 h. The cells were subsequently subjected to staining with FITC/PI and flow cytometry analysis, in which only annexin V-stained cells were considered apoptotic cells. The treatment with n-hexane extract at IC₅₀ 35 µg ml⁻¹ or double IC₅₀ 70 µg ml⁻¹ caused a considerable apoptotic effect in Panc-1 cells in comparison with the negative control, and double IC₅₀ 70 µg ml⁻¹ was the best in this regard. The numbers of late apoptotic and necrotic cells increased in a concentration- and time-dependent manner (Fig. 4). With respect to MCF-7 Adr. cells, treatment with IC₅₀ 43.3 µg ml⁻¹ at three time periods (24, 48, and 72 h) induced mild apoptotic changes, but they were mostly observed at the later stages (Fig. 5). The necrotic changes were still less frequent when compared with the same period recorded in Panc-1 cells, and treatment with double IC₅₀ 86.6 µg ml⁻¹ was more potent in this regard. In both cell lines, the concentrations employed in n-hexane extract were clearly more effective in inducing apoptosis or necrosis in Panc-1 cells than in MCF-7 Adr cells. According to GC analysis, the main identified chemical constituents were α-thujone, germacrene D, and heptadecane, which have potential effectiveness against cancer cell viability. Among Achillea species, the cytotoxic effects of A. alexandriregis, A. lavennae, and A. millefolium extracts have been tested on various tumor cell lines. Additionally, three human tumor cell lines were used to test the anti-tumoral effects of n-hexane, chloroform, aqueous methanol, and aqueous extracts of A. millefolium's aerial parts. Other studies in various human tumor cell lines demonstrated the anticancer potential of A. millefolium, where the chloroform extract had a mild impact on A431 cells and anticancer activity on HeLa and MCF-7 cells (Csupor-Löffler et al., 2009). Additionally, prior research has demonstrated that the methanolic fraction of A. millefolium decreased the cell proliferation of five various cancer cell lines (Abou Baker, 2020). The infusion of the plant herb has also been discovered by Dias et al. to be the most effective one in MCF-7 (Dias et al., 2013). Consequently, as shown in numerous studies, the makeup of various extracts may suggest anticancer potential (Kintzios et al., 2010; Pereira et al., 2018; Vitalini et al., 2011). Similar to this, certain sesquiterpenes from A. millefolium were isolated and demonstrated effectiveness against mouse P-388 leukemia cancer cells (TOzYO et al., 1994). HeLa cells may undergo apoptosis when exposed to A. millefolium essential oil, and pancreatic cancer cells may experience cell growth inhibition. It is found to downregulate SREBF1 and downstream molecular targets of this transcription factor, such as fatty acid synthase (FASN) and stearoyl-CoA desaturase (SCD). Previous studies on 1,8 cineole, or on its containing essential oils, reported apoptotic effects on a variety of cancer cell lines, including: human lung carcinoma (A549); human melanoma (SK-MEL-28); human Caucasian colon adenocarcinoma (Colo-205); human cervical carcinoma (SiHa); hepatocellular carcinoma (Hep-G2); breast adenocarcinoma MCF-7, T47D, MDA-MB-231; human colon carcinoma (RKO); human Caucasian colon adenocarcinoma (Caco-2); squamous cell carcinoma (A431); osteosarcoma (MG-63); and P815 (murine mastocytoma) (Abdalla et al., 2020; Akiel et al., 2022; Murata et al., 2013; Oliveira et al., 2015).

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Fig. 4

Representative cytogram of PANC-1 cells: PANC-1 cells were exposed to untreated (culture medium) and Hexane extract for (a) 24 h, (b) 48 h and (c) 72 h. Quadrant + YUI9L0\location for the representative dot plots: Q2-1: upper left—FITC^-/PI⁺ (necrotic cells); Q2-2: upper right— FITC⁺/PI⁺ (late apoptotic cells); Q2-3: lower left— FITC^-/PI^- (viable cells) and Q2-4: lower right— FITC⁺/PI^- (early apoptotic cells).

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Fig. 5

Representative cytogram of MCF-7 Adr. cells: MCF-7 Adr. cells were exposed to untreated (culture medium) and Hexane extract for (a) 24 h, (b) 48 h and (c) 72 h. Quadrant location for the representative dot plots: Q2-1: upper left—FITC^-/PI⁺ (necrotic cells); Q2-2: upper right— FITC⁺/PI⁺ (late apoptotic cells); Q2-3: lower left— FITC^-/PI^- (viable cells) and Q2-4: lower right— FITC⁺/PI^- (early apoptotic cells).

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3.4 Docking results

Several chemotherapies, including paclitaxel, doxorubicin, and vincristine, have limited efficacy due to the resistance of tumor cells through P-glycoprotein/multidrug resistance 1/adenosine triphosphate-binding cassette (ABC) B1 (P-gp/MDR1/ABCB1), which promotes drug efflux and lowers their toxic effects (Adorni et al., 2023). ABCB1 has been shown to be an overexpressed transporter in numerous drug-resistant tumors (Wang et al., 2021). Molecular docking studies were carried out using the Autodock Vina software to evaluate and understand the potential of the three main constituents (germacrene D, viridiflorol, and caryophyllene oxide) of the active cytotoxic extracts, n-hexane and diethyl ethers, to sensitize MCF-7/ADR (DOX-resistant cells). In addition to the molecular dockings of the compounds of A. millefolium, DOX and the cocrystal ligand (zosuquidar) were also docked into the substrate binding sites of ABCB1. The docking score for DOX was −8.4 kcal/mol, while the docking score for zosuquidar was −9.5 kcal/mol. Germacrene D, viridiflorol, and caryophellene oxide identified from Achillea millefolium showed good binding potentials against ABCB1, as demonstrated by the lower the docking scores, the higher the binding affinity (Sabbah et al., 2010). The docking scores for Germacrene D, viridiflorol, and caryophyllene oxide were −7.3, −6.8, and −7.6 Kcal/mol, respectively (Table 4) and were involved in hydrophobic interactions, such as, alkyl - alkyl, and π – alkyl interactions (Fig. 6). The major residues involved in hydrophobic interactions in 1FN1 with Germacrene D, viridiflorol, and caryophyllene oxide were Phe302, Ala986, Val990, Phe993, and Ala994. The oxygen atom of viridiflorol and caryophyllene oxide can generate H-bonds with Gln837. These docking results showed that germacrene D, viridiflorol, and caryophyllene oxide have high binding affinity against the ABCB1 transporter, and they can be considered to be the likely ABCB1 inhibitors. They also appeared to be involved in improving the sensitivity.

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Fig. 6

The docked poses of A) Germacrene D, B) viridiflorol, and C) caryophyllene oxide in the substrate binding sites of ABCB1 6FN1.

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