The genus
Although there are some studies using the essential oil of
Yarrow was collected from its habitats near Kayseri Ali mountain (a registered example is CV4851, Erciyes University Herbarium). Collected plants were dried at room temperature. After weighing of the dried plant, about 10 times more water was added (i.e. 1 liter per 100 grams). Distillation method was applied as described in the procedure of Raal et al (2006) to obtain essential oils (9). The essential oils extracted were stored at 4 oC for cell culture analysis. 500 μL of the essential oil of
500 μL of the essential oil was prepared for GC-MS analysis (Shimadzu-QP2010 ULTRA). The analysis were performed at Bozok University Science and Technology Application and Research Center. GC analysis was performed on SLB-5m colum (Spelco, Milan, Italy). Oven temperature was from 50 to 280 oC with an increments of 4 oC per min, as the carrier gas Helium being used in flowrate of 2 mL per min. C5-C24 n-alkane library library was used to identify the spectras. In order to establish a link between cancer cells and the molecules, the function of the GC-MS identified compounds was investigated by Ingenuity Pathway Analysis (IPA, QIAGEN) software.
In this study, we used ATCC HeLa (CCL-2) cell line to investigate the anticancerogenic effect of essential oil. HeLa cells were cultured in RPMI-1640 (Biological Industries) medium containing 10% FBS (Biological Industries), 1% L-glutamine (Biological Industries) and 1% penicillin-streptomycin (Biological Industries).
For the apoptosis assay, MUSE Annexin V / Dead Cell kit was used. After the essential oil application, HeLa cells were liberalized by trypsinization and dissolved in 100 μL of DPBS with 2% FBS. 100 μL of Annexin V and Dead Cell Reagent were added on suspension and incubated for 20 minutes. The analyses were performed by using MUSE TM Cell Analyzer (Merck, Germany).
HeLa cells were dissolved in 100 μL of 2% FBS containing DPBS and test groups were fixed with 70% ethanol in and incubated at -20 oC overnight. In the following step, ethanol was removed by centrifugation and 100 μL of PI (Propodeum iodide)-containing dye was added. After 30 minutes incubation, cells were analyzed by using MUSETM Cell Analyzer.
In order to complete Ki-67 proliferation assay, HeLa cells were dissolved in 100 μL DPBS containing 2% FBS and washed by centrifugation. Cells were fixed with 4% paraformaldehyde at room temperature for 15 minutes. In order to get rid of the fixative solution, cells were washed with assay buffer. Fixed cells were incubated with permeabilization buffer and washed with assay buffer once. Phycoerythrin (PE)-conjugated Ki-67 antibody was added to cells and incubated for 30 mins. PE-conjugated Hu-IgG were used as isotype control during threshold determination. Analyses was completed by using the MUSE TM Cell Analyzer.
In statistical calculations, the changes of the experimental groups according to the control groups were compared by using One-Way Anova Test. Post-Hoc Tukey test was applied to determine the general differences, multiple comparisons were made. The results were determined according to mean ± standard deviation (X ± SD) and showed a statistical significance of p <0.05 SPSS (16.0-2010) package program was used in all calculations.
In GC-MS analysis, 10 different major molecules were identified from yarrow essential oil. Retention time and peak area percentage were provided at Peak report total ion chromatography table (
GC-MS analysis of essential oil components from
Peak Report Total Ion Chromatogram | ||||
---|---|---|---|---|
Peak # | Molecule | R. Time | Area | Area % ↓ |
3 | 1,8-Cineole | 8.127 | 83328771 | 27.34 |
4 | Camphor (CAS) | 9.585 | 74017016 | 24.28 |
10 | .beta.-Eudesmol (CAS) | 14.158 | 57129364 | 18.74 |
8 | Caryophyllene oxide | 13.595 | 25349728 | 8.32 |
5 | Bicyclo[2.2.1]heptan-2-ol. 1.7.7-trimethyl-, (1S-endo)- | 9.832 | 16313677 | 5.35 |
1 | Camphene | 7.053 | 10954991 | 3.59 |
9 | Viridiflorol | 13.681 | 10656458 | 3.50 |
2 | Bicyclo[3.1.1]heptan. 6.6-dimethyl-2-methylene-. (1S)- | 7.424 | 9919490 | 3.25 |
7 | .alpha.-Terpineol | 10.005 | 8602416 | 2.82 |
6 | 3-Cyclohexen-1-ol. 4-methyl-1-(1-methylethyl)-. (R)- | 9.880 | 8528233 | 2.80 |
304800144 | 100.00 |
The three compounds were more dominant than rest of other compounds in essential oil. These dominant yarrows essential oil content was determined as 1.8 cineole (27.4%), Camphor (24.28%) and β-Eudesmol (18.74%). Additionally with the chemically similar structure carrying compound, Camphene (3.9%), medically noticeable, were also been detected. Although we analyzed all identified compounds of essential oil, only four of the all compunds were recognized by IPA databases. Therefore the biological effects of these four molecules (1,8-Cineole, Camphor, β-Eudesmol and Camphene) were analyzed by using Ingenuity Pathway Analysis software for the network analysis. Data obtained from these four biomolecules were evaluated in terms of biological significance and related canonical pathways were determined.
Our results indicated that the major 4 molecules in essential oil have potential to influence various pathway and network which were crucial for cellular metabolism and viability. Most important of these pathways were cAMP, Protein Kinase A (PKA), AMPK, MAPK, ATM and Apoptosis Signaling Pathways. IPA results also showed that the components of essential oil can influence Type II Diabetes signaling, Superoxide radical degradation process and estrogen biosynthesis (
Following IPA analysis, it was determined that cineole molecule blocks the Cng channel and increases the expression of Superoxide Dismutase 1. The Cng channel is associated with cAMP signaling pathway and Protein Kinase A pathway. (B.) it was found that the camphor molecule increased the expression of some hepatic enzymes associated with the Pyruvate fermentation and increased expression of the CYP2B6 molecule related to estrogen biosynthesis. (C.) Camphene molecule increased the expression of the ADIPOQ molecule associated with PPARα/RXRα Activation and Glutathione Redox Reactions. Adiponectin also is closely associated with AMPK signaling pathway and Type II Diabetes signaling pathway. (D.) β-Eudesmol molecule influences the cAMP, ATM, Apoptosis and GPCR signaling pathways through MAPK, inositol phosphate, Calcium and CREB1.
It was observed that
This ratio of apoptotic cells was found statistically significant (p <0.05) between the application of 0.5% essential oil and DMSO control. It was observed that 0.5% essential oil application was successful in inducing apoptosis in HeLa cells compared to other doses.
When applied, the
According to the results obtained from the cell cycle test, the high amount of DMSO blocks the cells in the G0/G1 phase. Difference between ACH and DMSO control groups were found to be significant (P<0.05). The current results indicated that 0.5% (v/v)
The results of Ki-67 test showed that the application of essential oil decreased the proliferation of HeLa cells when compared to the control groups (
Our study indicated that the components present in yarrow essential oil were associated with various molecular mechanisms. The mixture of these compounds induced apoptosis and inhibited cell proliferation by reducing proliferation in HeLa cells. It has been shown that one or more of the components in yarrow essential oil might have anticancer properties (10).
Various plants have been used medical purpose and applied for different treatments in traditional medicine. The components of the essential oils of plants are shown to be effective in complementary and alternative therapy (1). Some of the well-known components in the essential oils of medicinal plants are monoterpenes and derivatives having a property of anticancer agent that are used in chemotherapy. There are examples of the medical uses of yarrow for complementary and alternative therapies that are related to cancer, diabetes, atherosclerosis and metabolism (11).
In this study, GC-MS analysis detected the presence of monoterpene, sesquiterpene and terpenes in the essential oil of
In our GC-MS analysis, camphor molecule was found to be the second most abundant ones. According to IPA analysis, it was detected that camphor was related to the expression of CYP2B6 molecule (
Considering IPA analysis, β-Eudesmol was determined to be affecting G-protein mediated receptor pathways, calcium and inositol 3-phosphate secondary messenger pathways and MAPK pathway (
Another molecule identified by GC-MS analysis was the camphene. A recent study showed the antidiabetic effect of
As a second part of study, we investigated the effects of essential oil on cervix cancer cell line HeLa. When apoptosis test performed on HeLa cells, ACH treatment at different concentrations increased the number of total apoptotic cells. The treatment of 0.5% (v/v) ACH showed an approximately 15% increase in the percentage of apoptotic cells when compared to the control group, which containing the same amount of DMSO. Similarly, our results, the study by Bali et al. showed that the phenolic compounds containing ACH extracts increased the activation of apoptosis genes such as Bax and Cas3 in PC-3 cells (19). The viability of The number of HeLa cells was reduced about 10-15% with the treatment of 0.5% (v/v) ACH essential oil. The aforementioned study done by Bali et al. (19) also tested membrane integrity that shares common grounds with the dead cell assay we conducted. In order to deeply investigate the essential oil effects on apoptosis pathway, it needs to be investigated the expression of certain genes such as Bax, Bad, Bcl2, and Cas3/7 at transcriptomic level in an extensive study.
We also observed that the application of essential oil blocked cell cycle of HeLa cells on G0/G1 phase of cycle. In accordance with cell cycle data, essential oil application reduced the expression of Ki-67, a marker for cell proliferation. Anti-proliferative properties of
Furthermore, individual analysis of identified molecules by GC-MS could lead valuable data that could be used for anticancerogenic drug discovery studies.
As a consequence, essential oil of
Therapeutic effects of the yarrow essential oils were investigated on cervical cancer cell line, which is important for women’s health (21). We reported that essential oil components may interact with molecular pathways that are closely related to cancers. The yarrow’s essential oil has potential to induce apoptosis in HeLa cells and inhibit cell proliferation. It was found that at least one of the components of the essential oil have anticancer properties. Concurrently, components of the yarrow’s essential oils should be subject to more detailed research. Examination of 1,8-Cineole, Camphor, Beta-eudesmol and Camphene molecules may provide valuable data to lead anti-cancer drug discoveries. Explaining the relationship of these components to molecules, which involved in disease-related mechanisms, may help complementary and alternative therapies.