Molecular fingerprint and anti-carcinogenic properties of Turkish immortelle (Helichrysum italicum) essential oil

Abstract Immortelle (Helichrysum italicum) essential oil is one of the most valuable essential oils with its commercial importance and favorable characteristic properties in various cosmetic products and food products. Current research performed a comprehensive characterization of Immortelle essential oil using powerful and robust analytical techniques. The relative volatile composition of the Immortelle essential oil from Turkey was determined by GC-MS (gas chromatography-mass spectrometry) technique. Thirty-eight volatile compounds were identified. Average total phenolic content and DPPH radical scavenging activities (IC50) were determined as 54.24 mg GAE/mL essential oil and 0.27 mg/ml, respectively. FTIR and Raman spectroscopy fingerprints were determined for chemical characterization. The thermal profile was obtained by DSC (differential scanning calorimetry) analysis. The Immortelle essential oil (12.5 μg/mL) inhibited the growth of Caco 2 adenocarcinoma cells at the ratio of 48% without any deterioration in healthy L929 fibroblast cells according to the cytotoxicity analyses.


Introduction
The genus Helichrysum has consisted of more than 400 species across the world.The Immortelle (H.italicum) has been known as a typical Mediterranean plant belonging to the Asteraceae plant 1 .The name Helichrysum originated from the Greek "helios", meaning the sun and "chrysos" which means gold.Helichrysum is also known as Immortelle or Everlasting because the flowering tops keep their yellow color even as they dry out.This sun gold plant has been valued and concerned by herbalists and researchers for centuries 2 .
Extracts of Helichrysum species have antiinflammatory, antimicrobial, antioxidant, antiallergic, cytotoxic and wound healing properties 3 .Especially the essential oil (EO) obtained by hydro-distillation of Immortelle is highly demanded by food, cosmetic and pharmaceutical industries due to its skin regenerative, anti-aging and wound-healing properties 4 .Growing market value of Immortelle is related to its collagen regeneration properties.Thus Immortelle essential oil finds a place as an ingredient in many anti-aging cosmetic products such as hand creams and skincare products 1 .Additionally, the Immortelle essential oil has high commercial importance in the aromatherapy and perfumery industries.Furthermore, food quality and food safety have been known as major concerns in the food industry and essential oils are promising natural extracts that can be used as food preservatives because of their antifungal, antiviral and antibacterial properties 5 .
In recent years, Immortelle essential oil gained importance due to its desirable functional properties.The composition and chemical properties of essential oil change related to the geographical origin, climatic conditions and soil properties in which the essential oil plant is cultivated.The chemical properties of essential oils affect their application areas and their industrial importance.The first goal of this research was to obtain Immortelle essential oil from Karacabey (Bursa, Turkey) area by using the industrial process and the second goal was to determine the quality properties based on important chemical characteristics such as FTIR (Fourier transform infrared spectroscopy) spectrum, Raman spectrum, GC-MS (gas chromatography-mass spectrometry) chromatogram, DSC (differential scanning calorimetry) plot, cytotoxic properties, antioxidant and anti-radical properties.

Steam distillation of Immortelle essential oil
The Immortelle essential oils were obtained from Karacabey (Bursa, Turkey).Flowers were harvested from an exclusive Immortelle field which was planted by a natural product producer company in 2020.The Helichrysum italicum chemotype was especially rich in α-pinene, limonene, nerylacetate, γ-curcumene and β-selinen.The extraction of Immortelle essential oil was performed in a distillation plant which was located near the Immortelle field in Karacabey (Bursa, Turkey) by using a stainless steel steam distillation machine with 50 L of herb capacity.The temperature of the steam was between 100 and 110°C and steam distillation took approximately 3.5 hours.Distillation was performed with the full capacity of the steam-distillation machine and the yield of essential oil obtained by the steam distillation was 0.1%.Flower parts of the herb were used in the distillation process.After distillation, the pure essential oils were stored in amber vials at 4°C before further analysis.100% pure Immortelle essential oils were used in this study.The geographical origin and appearance of Immortelle (Helichrysum italicum) are presented in Fig. 1.

Determination of total phenolic contents of essential oils
The analysis for determination of the total phenolic content was performed by using the Folin-Ciocalteu method according to the previous study 12 .Folin-Ciocalteu's phenol reagent (1:10) and Na 2 CO 3 (7.5 g/100 g) were added to diluted methanolic essential oils (1:1000, 0.5 mL) in amounts of 2.5 mL and 2 mL, respectively.The liquid was then combined in a tube and left in a dark environment for 30 min.The absorbance values of samples were determined at 765 nm on the basis of the gallic acid calibration curve.

Determination of the antioxidant (antiradical) activity of essential oils
Antioxidant activities were measured according to the study of Ozkan et al. 12 .Briefly, the diluted essential oils (100 µL) were mixed with 4.9 mL of 1,1-diphenyl-2-picrylhydrazyl radical (DPPH, Sigma) standard.The absorbance of each essential oil was measured at 517 nm using a Shimadzu 1800 spectrometer.The IC 50 values were determined and all measurements were Acquisition of FTIR spectra of essential oils FTIR spectra were obtained by using FTIR-ATR (Fourier transform infrared spectroscopyattenuated total reflectance) equipment.Pure EO samples (50 uL) were directly placed on the diamond crystal of the ATR accessory by using an automatic pipette.The spectra were acquired at 4 cm -1 resolution with 16 scans.Each scan was performed at room temperature against a background air spectrum.

Acquisition of raman spectra of essential oils
The collection of Raman spectra was performed by using a portable Raman spectrometer equipped with a 1064 nm YAG laser (Rigaku, USA).EO samples were placed in glass Raman vials then these samples were located in the sample compartment of the Raman spectrometer.0.25 W laser was applied to each sample for 0.851 s.Each sample was scanned six times and an average Raman spectrum was obtained.

DSC (Differential Scanning Colorimetry) analysis
The DSC analysis was performed with slight modifications in the study of Monteiro et al. 15 .The DSC (TA Q20, DE, USA) equipped with a thermal analysis automatic program was used to obtain thermal curves.Samples of approximately 10 mg were weighed in the aluminum hermetic pan and sealed with its lid.An empty pan was used as a reference.The triple DSC measurements of Immortelle essential oil were carried out by heating the sample at a rate of 10°C/min 25 to 500°C under a nitrogen stream with a flow rate of 20 mL/min.

GC-MS (gas chromatography-mass spectrometry) measurements
The samples were analyzed according to the previous study by Cebi et al. 18 .In the sample preparation, diethyl ether was used as a diluent (1:50).Rtx-5MS capillary column (30 m × 0.25 mm × 0.25 μm) was used in measurements.The oven temperature increased gradually, the temperature was 40°C for 3 min at the beginning of the temperature program, increased to 100°C at 8°C/min rate, raised to 200°C at the rate of 5°C/min, and finally to 250°C at the rate of 10°C/ min.The temperature of the injection block and the flow rate of carrier gas were 250°C and 1 mL/ min, respectively.The samples were scanned at the mass range of 35 and 650 (m/z).
The composition of essential oil was determined by comparing the obtained GC-MS total ion chromatogram with those included in the NIST (National Institute of Standard and Technology) and Wiley Library of GCMS-QP2010 equipment (Shimadzu, Milan, Italy).The relative abundance of each compound was calculated on the basis of the percentage area of each identified compound chromatogram compared to the areas of total peaks (100%).Samples were scanned three times and all peaks detected in at least two of the total ion chromatograms (TIC) were used for the calculation of relative abundance.The retention indices are presented in Table 1 according to the literature data 13 .The total ion chromatogram of the GC-MS analysis is presented in Fig. S1.

Cytotoxicity analysis using XTT assay and trypan blue assay
Cytotoxicity analyses of the Immortelle essential oil samples were performed using XTT assay with some modifications as in the study of Cakir-Koc et al. 14 .Caco-2 adenocarcinoma cell line and L929 fibroblast cell line were used for the evaluation of the cytotoxic effects of samples.In the first step, cell lines were incubated at 37°C for 3-4 days in a 5% CO 2 incubator.After incubation, the cells were removed by a trypsinization process and counted with a Thoma cell counting chamber.Then, 10 4 cells/well are plated in the 96-well flat-bottom microplates and incubated at 37°C for 24 h to adhere to the bottom of the well.After the incubation process, the different concentrations of the samples (200, 100, 50, 25, 12.5, 6.75, and 3.375 µg/mL) were added to each well and incubated again for 24 h.The part of the medium in the well was removed and 100 μL of XTT solution was put in the well at 0.5 mg/ mL.Following the incubation of the microplates at 37°C for 3 hours, optical density (OD) was measured at 450 nm using a microplate reader (Elisa reader Elx800, Biotek, Vermont, USA) and the number of viable cells was calculated according to the following equation (Eq.1): In addition to XTT assay, Trypan blue test was performed to determine the number of viable cells.For this purpose, cell lines were incubated at 37°C for 3-4 days in a 5% CO 2 incubator.After incubation, the cells were removed by a trypsinization process and counted with a Thoma cell counting chamber.Then, 10 4 cells/well are plated in the 96-well flat-bottom microplates and incubated at 37°C for 24 h to adhere to the bottom of the well.After the incubation process, the different concentrations of the samples (200, 100, 50, 25, 12.5, 6.75, and 3.375 µg/mL) were added to each well and incubated again for 24 h.The part of the medium in the well was removed and after that the cells were also removed by a trypsinization process and resuspended in PBS solution.Cell suspension and 0.4% trypan blue solution were mixed at a ratio of 1:1 (v/v).Then, viable cells were counted with a Thoma cell counting chamber and the results were given as cell viability%.
The analyses were carried out in triplicate.Statistical analysis (analysis of variance, ANOVA) was performed using a Windows based statistical analysis software (SAS 8.2, SAS Institute, Cary, North Carolina, USA).The significance of the differences between the mean data was assessed using Duncan's multiple comparison test at a significance level of 95%.

Volatile compound composition of Immortelle (Helichrysum italicum) essential oil
The volatile composition of the Immortelle essential oil was determined on the basis of the GC-MS total ion chromatograms of EO.The relative abundance of the detected compounds is presented in Table 1.Determination of each compound was accomplished by comparison of the total ion chromatogram with the commercial GC-MS libraries of NIST 27 and WILEY 7. The relative abundance of the compounds was calculated based on the related peak areas.1.

Evaluation of the immortelle (Helichrysum italicum) essential oil FTIR spectrum
Fig. 2 shows the FTIR spectrum of immortelle essential oil from Bursa (Turkey).The most significant bands were observed at 2918, 1741, 1644, 1447, 1376, 1231, 1016, 954, 886, 825 and 787 cm -1 .According to the previous contributions, the band at 2918 cm -1 corresponds to the C-H asymmetric stretching vibrations of C-H and CH 2 groups 16 .The band with a peak point at 1741 cm -1 arises from the C=O ester group stretching vibrations 17 .The low-intensity sharp peak at 1644 cm -1 is due to the C=C stretching vibrations of alkanes 18 .
The significant bands at 1447, 1376 and 1231 cm -1 correspond to the CH 2 deformation and asymmetrical CH 3 deformation bands, C-H asymmetric and symmetric bending vibrations and CH 2 stretching vibrations, respectively 16,18 .
The peak around 1016 cm -1 may be due to the CH 2 group stretching vibrations 19 .Three significant bands around 886, 825 and 787 cm -1 wavelengths are may arise from the C-H stretching vibrations, C-H out-of-plane bending vibrations and skeletal C-C vibrations, respectively 16,19,20 .

Evaluation of the Immortelle (Helichrysum italicum) essential oil raman spectrum
Fig. 3 shows the Raman spectrum of the immortelle essential oil.Major bands were observed at 1667, 1610, 1450, 1381, 1311, 1084, 1043, 763 and 663 cm -1 wavelengths at the infrared range of 2000-200 cm -1 .The two bands at 1667 and 1610 cm -1 correspond to the C=C stretching vibrations and ring quadrant stretching vibrations, respectively 21 .The most significant band with a peak point at 1450 cm -1 arises from the CH 3 /CH 2 bending vibration 18 .
The peaks at 1381, 1311, 1084 and 1043 cm -1 may be due to the CH 3 bending vibrations, =CH rocking vibrations, C-H bending vibrations and C-H twisting vibrations of the CH 2 group, respectively 18,22 .The sharp peak at 663 cm -1 is due to the ring deformation vibrations 22 .

Dsc analysis results of immortelle essential oil
The DSC profile of Immortelle essential oil is presented in Fig. 4. The endothermic peak was observed as a result of the evaporation process at 248.22°C.The major phase change was observed between 249 and 270°C with an enthalpy change of 206.7 J/g, and the second one was observed between 271 and 276°C with an enthalpy change of 1.610 J/g in the two-dimensional DSC diagram.
The major transition may be attributed to the main volatilization and the second transition between 271 and 276°C may be related to the minor volatile compounds.

Total phenolic content and anti-radical activity of essential oils
The average total phenolic content of the Immortelle essential oil samples was determined as 54.24 mg GAE/mL essential oil.Additionally, the average anti-radical activity of the Immortelle essential oil samples was determined.The IC 50

Discussion
Immortelle (H.italicum) essential oil is one of the most valuable and vital essential oil in the cosmetic industry.Also, it takes place as a highvalue natural ingredient in the composition of various functional foods 24 .Previous studies reported that the geographical origin of the plant is one of the most important properties affecting the quality and composition of essential oils 25 .
In the current research, chemical fingerprint and quality assessment of the Immortelle essential oil was performed on the basis of FTIR and Raman fingerprinting, total phenolic content, radical scavenging activity (DPPH analysis), volatile compound composition, cytotoxicity analysis, antidiabetic activity and differential scanning calorimetry (DSC) analyses.To the best of our knowledge, this is the first attempt that characterizes the Immortelle essential oil from Bursa (Turkey) on the basis of mentioned multiple quality and functionality properties.FTIR and Raman spectra of the Immortelle essential oil are presented in Fig. 2 and Fig. 3. To the best of our knowledge, this study is the first attempt for characterization of Immortelle essential oil on the basis of the FTIR and Raman spectra.Vibrational spectroscopy (FTIR and Raman) has been known as accurate, fast, specific and economic technique that provides unique fingerprint information on the basis of chemical functional groups of the investigated compounds 18 .The observed FTIR and Raman bands are in accordance with those observed by Taylan et al. 16 , Cebi et al. 18 , Jentzsch et al. 21and Berechet et al. 19 .Essential oil-related FTIR and Raman bands were observed in the FTIR and Raman spectra of Immortelle essential oil.Average total phenolic content and DPPH radical scavenging activities (IC 50 ) were determined as 54.24 mg GAE/g essential oil and 0.27 mg/ml, respectively.These findings are coherent with that of Dzamic et al. 3 , who reported the total phenolic content and anti-radical activity (IC 50 ) of Immortelle essential oil as 72.33 mg GAE/g of extract and 0.35 mg/mL.In the current research, higher total phenolic content and lower anti-radical activity values were observed when compared to those reportedin a previous study as 22.7 mg GAE/g and 0.438 mg/ml 11 .
In this study, the most abundant volatile compounds were detected as γ-curcumene (16.02%), α-pinene (9.01%), β-selinen(6.70%),α-selinen (4.73%), caryophyllene (4.12%), γ-selinen (3.82%), dl-limonene (3.37%).Detected compounds explained 81.25% of the total volatile compound in the composition of Immortelle essential oil.These results are consistent with those of Andjić et al. 26 who reported the major volatile compounds as α-pinene, trans-caryophyllene, neryl acetate, limonene, γ-curcumene, β-selinen and α-selinene.Especially the γ-curcumene compound was the most abundant one among all volatiles in most of the previous studies 24,25,27 .Mollova et al. 11 presented the volatile compound composition of Immortelle essential oil from Bosnia and France; results showed that the most abundant compounds were α-pinene (20.84%) and neryl acetate (33.87%) for Bosnia and France, respectively.Several factors could explain the differences in quantities and species of the detected compounds by GC-MS analysis.Firstly, geographical origin and climate conditions directly affect the volatile compounds of Immortelle essential oil that is cultivated in different locations around the world.Secondly, variety in the analysis conditions such as method parameters, column properties, and sample preparation may affect the quantity and variety of detected volatile compounds.Up to now, none of the studies have reported the evaporation enthalpy (ΔH vap ) of the Immortelle essential oil.However, Martins et al. 23 reported the ΔH vap values of the orange, lemongrass and basil essential oils as 277.39 J/g, 336.02 J/g and 305.89J/g, respectively.The DSC profile is affected by the volatile compound composition of the investigated essential oil; that DSC technique can be utilized for the characterization and discrimination of pure essential oils from non-authentic or fraudulent ones.
Cytotoxicity results showed that Immortelle essential oil had cytotoxic activity against Caco 2 human colorectal adenocarcinoma cells at the dose range of 3.375 and 12.5 µg/mL without any deterioration in healthy L929 fibroblast cells.To the best of our knowledge, this is the first attempt that examines the cytotoxic effect of Immortelle essential oil cytotoxic activity against Caco 2 cells.However, these results support previous research into cytotoxic properties, which showed the cytotoxic activity of Immortelle essential oil against B16F10 murine melanoma cells, MCF-7 and HeLa cells 27 .In the present research, even the lowest concentration (3.375 µg/mL) showed cytotoxic activity (9.82%) on the Caco 2 human colorectal adenocarcinoma cells without any deterioration in the healthy L929 fibroblast cells.

Conclusion
This study presents new data on the characterization of Immortelle essential oil (Bursa, Turkey) on the basis of FTIR, Raman, GC-MS and DSC analysis.FTIR and Raman's results presented the unique fingerprint of the natural product.GC-MS results revealed the major and minor volatile compounds in the composition of Immortelle essential oil.Additionally, total phenolic content, anti-radical activity and cytotoxicity were evaluated to define additional quality properties of the Immortelle essential oil.The high anti-radical activity of 55.03±0.20%was observed by DPPH analysis.Significantly, at the concentration of 12.5 µg/mL of Immortelle essential oil, the growth of Caco 2 cells was inhibited at the ratio of 48% also, this dose didn't inhibit the growth of healthy L929 fibroblast cells.Overall, results showed that the Immortelle essential oil could be utilized in various sectors such as food and beverage, pharmaceutical, aromatherapy and cosmetics due to its flavoring, antioxidant, anticarcinogenic and multi-functional properties.
Enlightening the chemical fingerprint, chemical composition and characteristics of the Immortelle essential oil will help researchers for further investigations and explorations.Additionally, DSC technique can be used as an effective tool for authentication and certification of economically high-valued essential oils due to the sensitivity of DSC technique to the volatile composition of essential oils.
Industrial applicators may utilize these results for the development of new commercial products such as functional foods and drinks, dietary supplements, and beauty or age-rewind cosmetic products.Cytotoxicity findings may shed light on further research for the prevention or curing of cancer disease.Additionally, essential oils have high potential to be utilized for a wide variety of valuable purposes such as control of airborne microorganisms, preservation of food, feed, pharmaceutical and cosmetic industries 28,29 .From the point of view of economic development, these findings may motivate farmers or local producers to tend to the cultivation of the Immortelle herb.

Figure 2 .
Figure 2. FTIR spectrum of the Immortelle essential oil at the spectral range of 4000-400 cm -1

Figure 3 . 4 Figure 4 .
Figure 3. Raman spectrum of the Immortale essential oil at the spectral range of 200-2000 cm -1