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Chemical composition and anticancer activity of Elsholtzia ciliata essential oils and extracts prepared by different methods
Industrial Crops & Products 107 (2017) 90–96
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Chemical composition and anticancer activity of Elsholtzia ciliata essential oils and extracts prepared by different methods
MARK
Lauryna Pudziuvelytea, Mantas Stankeviciusb, Audrius Maruskab, Vilma Petrikaited,e, ⁎ Ona Ragazinskienec, Gailute Draksienea, Jurga Bernatonienea, a
Department of Drug Technology and Social Pharmacy, Lithuanian University of Health Sciences, Eivenių str. 4, LT-50161, Kaunas, Lithuania Department of Biology, Vytautas Magnus University, Vileikos str. 8, LT-44404, Kaunas, Lithuania c Kaunas Botanical Garden of Vytautas Magnus University, Kaunas, Lithuania d Department of Drug Chemistry, Faculty of Pharmacy, Lithuanian University of Health Sciences, LT-44307, Kaunas, Lithuania e Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Vilnius University, LT-10222, Vilnius, Lithuania b
A R T I C L E I N F O
A B S T R A C T
Keywords: Elsholtzia ciliata Anticancer activity Essential oil Ethanolic extracts Hydrodistillation GC–MS
E. ciliata (Lamiaceae) is very interesting and promising herb mainly for chemical composition and pharmacological activities. The aim of this study was to determine chemical composition of the essential oils of fresh, frozen and dried herbal materials of E. ciliata and compare different extraction methods This is the first study on composition of E. ciliata volatile compounds from fresh, frozen and dried herbal samples. The samples were prepared by hydrodistillation (HD), extraction with polar solvent-ethanol (ESE) and dynamic headspace solidphase micro extraction (SPME) and analyzed by gas chromatography-mass spectrometry (GC–MS) method. A total of 48 compounds were identified by GC–MS. Dehydroelsholtzia ketone, elsholtzia ketone, sesquiterpenes βbourbonene, caryophyllene, α-caryophyllene, germacrene D and α-farnesene were identified and found to be predominant compounds in SPME composition of the fresh, frozen and dried herbal samples. The major amounts of ketones (dehydroelsholtzia and elsholtzia) were determined in dried herbal samples where they made up 24.94% (p < 0.05) and 71.34% (p < 0.05) of the SPME composition. Artemisia ketone was determined only in fresh herb. No previous report exists regarding this ketone in E. ciliata fresh, frozen and dried herbal materials or essential oil. There were 26 components identified in the essential oil obtained by HD. The main compounds of this essential oil were dehydroelsholtzia ketone (78.28%) and elsholtzia ketone (14.58%). Essential oil showed antiproliferative activity on three tested cancer cell lines (human glioblastoma (U87), pancreatic cancer (Panc-1) and triple negative breast cancer (MDA-MB231)) in vitro. EC50 (half maximal effective concentration) values of essential oil against those cells were in the range of 0.017–0.021%. The viability of human normal fibroblasts exposed to the same concentrations of the essential oil was statistically significantly higher compared to the viability of cancer cells (p < 0.05). The extracts did not show any effect on U87 cells, and only slightly decreased MDA-MB231 cell viability (up to 76.4%) at the highest concentration of 10 mg/mL. The impact that different extraction methods have on the yield of the essential oil from the tested herbal materials requires a further research. It would enable production of pharmaceutical forms enriched by these essential oils, which are notable for their anticancer activity.
1. Introduction Essential oils are considered to be one of the most important substances in plants and to possess antimicrobial, antiviral, antifungal, antioxidant and anti-inflammatory activities (Bey-Ould Si Said et al., 2016; Raut and Karuppayil, 2014; Teixeira et al., 2013; Buchbauer, 2010). Essential oils are complex mixtures of various terpenes and their oxygenated derivatives such as alcohols, phenols, ketones. Essential oils ⁎
are usually obtained by HD, steam distillation or solvent extraction (Bakkali et al., 2008; Longaray Delamare et al., 2007; Raut and Karuppayil, 2014). For the experiments herbal materials of E.ciliata grown in Lithuania (Europe) were chosen. A flowering plant Elsholtzia ciliata (Thunb.) Hylander of the family Lamiaceae is native to Asia and is also found in Europe, Africa, North America, India (Guo et al., 2012; Korolyuk et al., 2002). Lamiaceae family plants are well known for their antiviral, antimutagenic, chemotherapeutic, antimicrobial, antioxidant
Corresponding author. E-mail addresses: [email protected], [email protected] (J. Bernatoniene).
http://dx.doi.org/10.1016/j.indcrop.2017.05.040 Received 11 January 2017; Received in revised form 19 May 2017; Accepted 21 May 2017 0926-6690/ © 2017 Elsevier B.V. All rights reserved.
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2.5. GC–MS analysis
and anti-inflammatory activities (Raut and Karuppayil, 2014). E. ciliata is widely used in folk medicine for antibacterial, anticancer and antiinflammatory properties (Tian, 2013). In traditional Chinese medicine the herb has been used to treat the common cold, headaches, pharyngitis, fever, edema, diarrhea, rheumatic arthritis, digestion disorders, nephritises (Guo et al., 2012; Sung et al., 2011). The pharmacological activities, such as antibacterial, antiviral, antioxidant, anti-inflammatory, diuretic and anti-obese, of extracts and pure compounds from E. ciliata are under investigation (Guo et al., 2012; Sung et al., 2011). The major chemical constituents in Elsholtzia are flavonoids, phenylpropanoids, phytosterols, cyanogenic glycosides and triterpenes (Guo et al., 2012; Liu et al., 2012). Essential oils accumulating herbal materials are used to produce various pharmaceutical forms such as tablets, capsules, microcapsules, ointments, creams or gels. Usually materials used in production are air dried; however, using fresh or frozen materials could also be suitable. There has not been a research done yet comparing chemical composition of essential oils from freshly collected and from frozen herbal materials, and it is not known what effect the material preparation has on the volatile composition. The aim of this study was to investigate the effects of different methods of material preparation and extraction on chemical composition of volatile compounds from E. ciliata. Also, one of the objectives was to evaluate the toxicity and anticancer activity in vitro of the plant extracts as well as the essential oil.
The analysis was carried out using a GCMS-QP2010 system (Shimadzu, Tokyo, Japan). For separation of volatiles a low polarity RTX-5MS (Restec, USA) (30 m × 0.25 mm i.d. × 0.25 μm film thickness) GC column was used. The oven temperature gradient started at 60 °C and was raised to 150 °C at 5 °C/min, and then raised to 280 °C at 20 °C/min and was held at it for 3 min. The carrier gas helium (99.999%, AGA Lithuania) was used at a flow rate of 1.2 mL/min. The injector temperature was kept at 230 °C in a split mode (1:20). The mass detector electron ionization was 70 eV. The ion source and interface temperatures were set at 220 °C and 260 °C correspondingly. The compounds preliminary were identified by mass spectra library search (NIST, v1.7) and by comparing with the mass spectral data from literature (Adams, 1995). The repeatability of solid-phase micro extraction – gas chromatography mass spectrometry method was evaluated by injecting the same sample three times. The relative standard deviation for the peak area was 5.15%.
2.6. Cell lines Anticancer activity was tested on three selected cancer cell lines: human glioblastoma (U87), pancreatic ductal adenocarcinoma (Panc-1) and triple-negative mammary gland adenocarcinoma (MDA-MB231). Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) high glucose (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% antibiotics (10,000 units/mL penicillin and 10 mg/mL streptomycin) (Gibco) at 37 °C in a humidified atmosphere containing 5% CO2. Human fibroblasts were grown in a Medium 106 with Low Serum Growth Supplement (LSGS) (Gibco) supplemented with 1% antibiotics (10,000 units/mL penicillin and 10 mg/mL streptomycin) (Gibco) at 37 °C in a humidified atmosphere containing 5% CO2. All cell lines were grown to 70% confluence and trypsinized with 0.125% TrypLE™ Express solution (Gibco) before passage. They were used until passage 20.
2. Materials and methods 2.1. Plant material E. ciliata aerial parts were collected in Vilnius, Lithuania, in July 2016 and were purchased as fresh and dried herbs from “Žolynų namai” (Vilnius, Lithuania). The herbs were identified by Dr. Prof. Nijole Savickiene, Medical Academy, Lithuania University of Health Sciences, Kaunas, Lithuania. A voucher specimen (L 17710) was placed for storage at the Herbarium of the Department of Drug Technology and Social Pharmacy, Lithuanian University of Health Sciences, Lithuania. Fresh and dried materials were mechanically ground in a laboratory mill to a homogenous powder or paste. A sample of fresh herb was frozen in a freezer (−18 °C) until preparation of extracts and SPME by GC–MS method.
2.7. Determination of cell viability Cell viability was studied using the method of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma). 100 μL of cells were seeded in 96-well plates in triplicates (5000 cells/well for U87, MDA-MB231 and human fibroblasts cells, and 3000 cells/well for Panc-1 cells) and incubated at 37 °C for 24 h. Then serial dilutions of different extracts and the essential oil were made in microplates. The essential oil before the experiment was mixed with 10% of Tween 80 to enhance its solubilization in culture medium. In order to avoid the essential oil evaporation, 0.05% of methylcellulose (Sigma) was added into the medium. Cells treated only with medium containing the same concentration of ethanol (in the case of extracts) or Tween 80 (in the case of essential oil) served as a negative control. Only medium without cells was used as a positive control. After 72 h incubating at 37 °C, 10 μL of MTT was added in each well. After 3 h the liquid was aspirated from the wells and discarded. Formazan crystals were dissolved in 100 μL of DMSO, and absorbance was measured at a test wavelength of 490 nm and a reference wavelength of 630 nm using a multidetection microplate reader. The experiments were repeated three times independently.
2.2. Isolation of the essential oil 30 g dried E. ciliata sample was mixed with 500 mL bi-distilled water and submitted to HD for 4 h using a Clevenger-type apparatus (European pharmacopoeia). A yellow colored oil with specific aroma was obtained. The essential oil was collected with water and stored in a refrigerator at +4 °C until needed. 2.3. Preparation of ethanolic extracts Fresh, frozen and dried powdered materials (Fig. 1) of plant’s areal parts (0.5 g each) were extracted for 24 h with 20 mL 96% ethanol in TiterTek shaker (Germany). The extracts were filtered through a paper filter and 0.22 μm pore size PVDF membrane filter and stored at +4 °C in a refrigerator until GC–MS analysis. 2.4. Dynamic headspace SPME Samples for gas chromatography analysis were prepared using SPME. Extraction of E. ciliata volatiles was performed on 65 μm PDMS/DVB (polydimethylsiloxane/divinylbenzene) Stable Flex fibre (Supelco, Bellefonte, USA). 10 mg of fresh, frozen and dried samples were added into 10 mL vials and placed in the AOC-5000 autosampler. The samples were thermostated for 10 min at 40 °C and the fiber was exposed in the headspace.
2.8. Statistical analysis Data are presented as mean ± SEM. Statistical analysis was performed by using Student’s t-test. A value of p < 0.05 was taken as the level of significance. 91
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Fig. 1. Fresh (A.), frozen (B.) and dried (C.) herbal materials of E. ciliata areal parts.
3. Results
found in dried herbal samples and made up 21.94% and 71.34% of SPME composition accordingly (p < 0.05 vs fresh and frozen material) (Table 1). Artemisia ketone was identified only in fresh herbal sample and made up 1.83% of SPME composition. Sesquiterpenes were obtained from dried (3.3%), frozen (1.73%) and fresh (1.95%) E. ciliata samples (Table 1). Predominant sesquiterpenes were α-caryophyllene and β-bourbonene in fresh (1.04% and 0.53%), frozen (0.84% and 0.49%) and dried (1.6% and 0.97%) herbal materials. Three alcohols were identified in frozen samples only and one alcohol ((S)-3,4-dimethylpentanol (0.08%)) in dried samples (Table 1). The predominant compound in fresh herb was eucalyptol (0.38%). The tests showed that 2-propenoic acid and 5-methyl-furan-2carboxylic acid (1H-[1,2,4]-triazol-3-yl)-amide were not present in airdried herbal material SPME composition. However, they were identified in fresh and frozen material SPME composition. 2,3-Dimethyl-5(2,6,10-trimethylundecyl)-furan was present only in dried material. The
In the first experiments the chemical compositions of essential oils in the fresh, frozen and dried E. ciliata herbal materials were determined. To our knowledge, this is the first study of E. ciliata volatile compounds extracted from fresh, frozen and dried herbal samples. The volatile compounds found in tested samples are listed in Table 1, their relative percent contents were calculated from the peak areas. E. ciliata grown in Lithuania is not rich in quantity and variety of the volatile compounds. From all SPME samples there were 16 different compounds identified (Table 1). Dehydroelsholtzia ketone, elsholtzia ketone, sesquiterpenes β-bourbonene, caryophyllene, α-caryophyllene, germacrene D and α-farnesene were identified and found to be predominant compounds in fresh, frozen and dried herbal samples. The major amounts of ketones (dehydroelsholtzia and elsholtzia) were 92
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Table 1 SPME composition of fresh, frozen and dried E. ciliata herbal materials. Compounds
3-Methyl-3oxetanemethanol 3-Octanol Eucalyptol 2-Propenoic acid, 2-methyl-, ethenyl ester Elsholtzia ketone 2,3-Dimethyl-5-(2,6,10trimethylundecyl) furan 5-Methyl-furan-2-carboxylic acid (1H-[1,2,4]triazol3-yl)-amide Dehydroelsholtzia ketone Artemizia ketone Beta-Bourbonene Caryophyllene Alpha-Caryophyllene Germacrene D Alpha-Farnesene 1,3,6,10-Dodecatetraene, 3,7,11-trimethyl-, (Z,E)(S)-3,4-Dimethylpentanol Sesquiterpenes Oxygenated monoterpenes Ketones Other Total
Fresh (%)
Frozen (%)
Dried (%)
Table 2 Chemical composition of essential oil obtained by hydrodistillation from E. ciliata dried herb. Retention index
–
0.22
–
935
– – 0.12
0.11 0.38 0.16
– – –
944 963 1052
23.09 –
33.64 –
24.94 0.26
1067 1082
0.28
0.23
–
1083
72.64 1.83 0.53 0.23 1.04 0.07 0.08 0.09
63.31 – 0.49 0.23 0.84 0.09 0.08 –
71.34 – 0.97 0.42 1.60 0.14 0.17 –
1118 1123 1152 1167 1181 1193 1199 1205
– 1.95 – 97.01 0.49 99.45
– 1.73 0.38 96.95 0.72 99.78
0.08 3.3 – 96.28 0.34 99.92
1256
SPME – solid-phase micro extraction.
results presented in Table 1 show that the methods of herb preparation affected the chemical composition. Further tests were aimed to determine the effect of HD method on the chemical composition of the essential oil. In our study, 26 components of the essential oil obtained by HD were identified (Table 2). The main compounds in the essential oil were dehydroelsholtzia ketone (78.28%) and elsholtzia ketone (14.58%). These ketones were also predominant in SPME composition of fresh, frozen and dried herbal samples. The second major identified group of volatile compounds was sesquiterpenes (5.02%), which included α-caryophyllene (1.87%), α-farnesene (0.66%), β-bourbonene (0.57%), isocaryophyllene (0.57%), trans-α-bergamotene (0.55%), δ-cadinene (0.28%), germacrene D (0.24%), γ-cadinene (0.15%), β-cubebene (0.06%), ledene (0.05%), and α-cubebene (0.02%). Some chemical compounds, not present in the herbal material SPME composition, were identified in the essential oil: isocaryophyllene, β-cubebene, ledene, α-cubebene, γcadinene and δ-cadinene. Eucalyptol, caryophyllene oxide, palmitic acid were determined only in the essential oil as well. A total of 23 components were identified in E. ciliata ethanolic extracts (Fig. 2). The main compounds in fresh herb extracts were dehydroelsholtzia ketone (45.74%), cis,cis,cis-7,10,13-hexadecatrienal (16.94%), elsholtzia ketone (7.34%), palmitic acid (4.69%), α-caryophyllene (0.87%). Dehydroelsholtzia ketone (58.47%), elsholtzia ketone (11.49%), linolenic acid (8.5%), palmitic acid (3.95%), α-caryophyllene (1.3%) were obtained as the main components from frozen herb extracts. Predominant compounds from dried herb extracts were dehydroelsholtzia ketone (57.94%), linolenic acid (11.79%), elsholtzia ketone (11.40%), palmitic acid (3.19%), α-caryophyllene (1.31%). This extraction method was better for extraction of organic acids, esters and ketones from herbal samples. The essential oil showed antiproliferative activity (Fig. 3) on all tested cancer cell lines (human glioblastoma (U87), pancreatic cancer (Panc-1) and triple negative breast cancer (MDA-MB231)) in vitro. EC50 values of the essential oil were in the range of 0.017–0.021%. The viability of human normal fibroblasts exposed to the same concentra-
Compounds
Retention index
Composition (%)
Eucalyptol Cyclohexene, 2-ethenyl-1,3,3-trimethyl 2-propenoic acid, 2-methyl-, ethenyl ester Elsholtzia ketone Furane-2-carboxaldehyde, 5(nitrophenoxymethyl)(−)-1R-8-Hydroxy-p-menth-4-en-3-one Dehydroelsholtzia ketone Beta-Bourbonene Isocaryophyllene Beta-Cubebene Ledene Alpha-Caryophyllene Alpha-Cubebene Naphthalene Germacrene D Trans-alpha-Bergamotene Alpha-Farnesene Gamma-Cadinene Delta-Cadinene Caryophyllene oxide Nonane 3-Tetradecen-5-yne, (Z)Palmitic acid Phytol Methyl (Z)-5,11,14,17-eicosatetraenoate 2,6-octadiene, 2,7-dimethylSesquiterpenes Oxygenated monoterpenes Oxygenated sesquiterpenes Ketones Others Total
963 1011 1053 1066 1079
0.05 0.15 0.06 14.58 0.43
1110 1117 1152 1166 1170 1174 1180 1186 1190 1192 1197 1202 1205 1208 1224 1243 1268 1275 1286 1289 1294
0.08 78.28 0.57 0.57 0.06 0.05 1.84 0.02 0.13 0.24 0.55 0.66 0.15 0.28 0.21 0.05 0.05 0.16 0.06 0.61 0.08 4.99 0.05 0.21 92.86 1.86 99.97
tions of essential oil was statistically significantly higher compared to the viability of cancer cells (p < 0.05). The extracts were not very active against tested cell lines (Fig. 4). They did not show any effect on U87 cells, and only slightly decreased the viability of MDA-MB231 cells (up to 76.4%) at the highest concentration of 10 mg/mL. However, they also did not decrease the viability of human fibroblasts at the same dilutions. 4. Discussion In this study the fresh, frozen and dried herbal samples and different extraction methods were analyzed. Our results show that E. ciliata main volatile components are dehydroelsholtzia, elsholtzia ketones and sesquiterpenes α-caryophyllene, β-bourbonene. We determined the composition of extracts obtained from dried herbal samples using HD method in percentages of these major components. Other authors reported that α-caryophyllene demonstrated anti-inflammatory, antiviral activities (Djilani and Dicko, 2012; Fernandes et al., 2007). Caryophyllene oxide (12.45%) rich essential oil of Helichrysum fulgidum showed antibacterial and antifungal activities (Bougatsos et al., 2004). Surprisingly, sesquiterpene trans-α-bergamotene in E. ciliata essential oil has not been reported earlier, but was determined for the first time in our study (0.55%). This volatile compound is attractive to insects (Schnee et al., 2006) and was identified as the main component in the oil of Cichorium intybus (Rustaiyan et al., 2011). Artemisia ketone was determined only in fresh herb SPME composition. No previous report exists regarding this ketone in E. ciliata fresh, frozen and dried herbal materials or essential oil. Artemisia ketone is a major constituent of essential oil from some cultivars of A. annua and may have antimalarial, antibacterial and antifungal activities (Bilia et al., 2014; Weathers et al., 2014). 93
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Fig. 2. A. Identified sesquiterpenes in ethanolic extracts from E. ciliata. B. Identified ketones in ethanolic extracts from E. ciliata. C. Identified organic acids in ethanolic extracts from E. ciliata. D. Identified other compounds in ethanolic extracts from E. ciliata. *Hexadecahydrocyclopenta[a]phenanthrene-3, 17-diol, 16-(1,3-dimethyl-1H-pyrazol-4-ylmethylene)-10, 13-dimethyl-
lene (11.13%), β-linalool (10.86%), eugenol (9.32%), and caryophyllene oxide (8.83%) (Tian, 2013). Ketones have not been identified by these researchers. GC–MS analysis showed the influence of different techniques of extraction on the quantity and quality of volatile components composition. More volatile compounds were obtained using HD (26 components). HD has been earlier reported as a better extraction method for C. aurantium (58 components) and E. fruticosa (35 components) herbal samples. HD was more suitable for monoterpene and sesquiterpene extraction (Jiang et al., 2011; Saini et al., 2010). ESE extracted more organic acids, esters and other compounds. Similar results were obtained from the research where herbal material was extracted by ethyl acetate (Jiang et al., 2011). Volatile compounds composition differences are related to the extraction methods and other factors, such as growing habitats,
There is a considerable similarity reported by different authors about the main components of E. ciliata essential oil. Dehydroelsholtzia ketone and elsholtzia ketone have been reported to be the main ingredients (Kharina et al., 1995; Thappa et al., 1999). Researchers also identified dehydroelsholtzia (66.1–72.4%) and elsholtzia (3.3–19.3%) ketones as the main volatile constituents of essential oil prepared by HD of fresh E. ciliata material from suburb area of Novosibirsk (Korolyuk et al., 2002). There is some difference between our data and other researcher’s results about the main identified compounds. Dung et al. (1996) reported geranial (19.5–26.5%), neral (15.2–20.5%), and limonene (10.9–14.2%) as the main ingredients of E. ciliata essential oils from the leaves were β-linalool (12.06%), caryophyllene (11.02%), eugenol (9.67%), and caryophyllene oxide (9.61%), from the flowers β-linalool (11.52%), β-lonone (11.08%), eugenol (10.56%), and caryophyllene (9.57%), and from the seeds caryophyl94
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(Loizzo et al., 2007; Kim et al., 2014). We could not find any data in the official literature sources about the anticancer effect of dehydroelsholtzia and elsholtzia ketones. However, there are published data that the essential oil from E. ciliata did not possess significant toxicity on rat pheochromocytoma (PC12) cell line at doses of 10, 25, and 50 μg/mL (Choi et al., 2015). It was found that the water extract of E. ciliata could inhibit cytokine expression through NF-κB and p38 MAPK pathway (Kim et al., 2011). p38 MAPK participates in the regulation of cell survival/death, is important for cell invasion and migration, and its levels are usually associated with short survival in cancer patients (Koul et al., 2013). Thus, it is very important to study the mechanism of action of E. ciliata active components. Anticancer activity may be possessed not only by volatile compounds but maybe by polyphenols as well, which can be extracted by different solvents. At this moment it is not easy to answer the question which constituent is the most important for the anticancer activity and at the same time is not very toxic. It could be that not one substance only but the whole complex is responsible for this effect, as different substances may have different mechanisms of action and they may act synergistically (Bakkali et al., 2008).
Fig. 3. The activity of E. ciliata essential oil on human normal fibroblasts and cancer cells. *p < 0.05. Human fibroblasts (HF), human glioblastoma (U87), pancreatic cancer (Panc1) and triple negative breast cancer (MB231).
5. Conclusion Volatile compounds from E. ciliata herbal samples were extracted by different extraction methods and then analyzed by GC–MS. The data shows that E. ciliata grown in Lithuania is rich in volatile compounds ketones. Dehydroelsholtzia ketone and elsholtzia ketone were predominant compounds in all samples, and were obtained by using HD and SPME. Sesquiterpenes are the second major group of compounds identified in the essential oil obtained by using HD. The impact that different extraction methods have on the yield of the essential oil from the tested herbal materials requires a further research. The essential oil from E. ciliata could be further investigated for application of its constituents as anticancer substances. Fig. 4. The activity of E. ciliata extracts on human glioblastoma (U87) and triple-negative breast cancer (MB231) cell lines. 1 – extract from frozen fresh herbal material, 2 – extract from fresh herbal material, 3 – extract from dried herbal material.
Conflicts of interest The authors declare that there are no competing interests regarding the publication of this paper.
seasonal variation, parts of the plant, storage conditions and others. Our experimental data showed that the ethanolic extracts from the fresh, dried and frozen E. ciliata herbal samples were not very active against different tested cancerous and non-cancerous cell lines. We have shown for the first time that the essential oil from E. ciliata shows an anticancer activity against human glioblastoma, breast and pancreatic cancer. However, the oil was significantly less toxic against human normal fibroblasts. Similar effects have been obtained with the extract and essential oil of the plant from the same Lamiaceae family by Fekrazad et al. (2017). They showed that the essential oil from Thymus caramanicus Jalas possesses antiproliferative properties. The activity of this essential oil (IC50 = 0.44 μL/mL) against human oral epidermoid carcinoma KB cells was also much lower compared to that of the extract. However, it was about 2–3 times more active compared to the essential oil from E. ciliata against human breast, pancreatic cancer or glioblastoma cells. Interestingly, cytotoxic effect of essential oil from Thymus caramanicus Jalas was greater than the effect on normal gingival cells, what was seen also in our experiments. In another experiment Russo et al. (2015) showed that the essential oil from Salvia verbenaca was also active against human melanoma cell line M14 and possessed lower activity against normal human nonimmortalised buccal fibroblast cells. The activity of that essential oil mainly has been explained by the presence of sesquiterpenes and carvacrol. α-Caryophyllene and caryophyllene, which are found in the essential oil of E. ciliata as the major constituents, also could contribute to the anticancer activity against tested cell lines as there is some data published about its in vitro and in vivo anticancer effect
Acknowledgments The authors would like to thank Open Access Centre for the Advanced Pharmaceutical and Health Technologies (Lithuanian university of Health Sciences) for providing the opportunity to use infrastructure and perform this research and Instrumental Analysis Open access research center of Vytautas Magnus University of Natural Sciences. Also, the authors want to thank Dr. Manel Esteller (Bellvitge Biomedical Research Institute (IDIBELL), Spain) for kindly providing the MDA-MB231, Panc-1 and U87 cell lines; and Dr. Ramunas Valiokas (Center for Physical Sciences and Technology, Lithuania) for providing human fibroblasts cell line. References Adams, R.P., 1995. Identification of Essential Oil Components by Gas Chromatography/ Mass Spectrometry. Allured Publishing Corporation, Carol Stream, IL. Bakkali, F., Averbeck, S., Averbeck, D., Idaomar, M., 2008. Biological effects of essential oils – a review. Food Chem. Toxicol. 46, 446–475. http://dx.doi.org/10.1016/j.fct. 2007.09.106. Bey-Ould Si Said, Z., Haddadi-Guemghar, H., Boulekbache-Makhlouf, L., Rigou, P., Remini, H., Adjaoud, A., Khoudja, K.N., Madani, K., 2016. Essential oils composition, antibacterial and antioxidant activities of hydrodistillated extract of Eucalyptus globulus fruits. Ind. Crops Prod. 89, 167–175. http://dx.doi.org/10.1016/j.indcrop. 2016.05.018. Bilia, A.R., Santomauro, F., Sacco, C., Bergonzi, M.C., Donato, R., 2014. Essential oil of artemisia annua L.: an extraordinary component with numerous antimicrobial
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Koul, H.K., Pal, M., Koul, S., 2013. Role of p38 MAP kinase signal transduction in solid tumors. Genes Cancer 4, 342–359. http://dx.doi.org/10.1177/1947601913507951. Liu, X., Jia, J., Yang, L., Yang, F., Ge, H., Zhao, C., Zhang, L., Zu, Y., 2012. Evaluation of antioxidant activities of aqueous extracts and fractionation of different parts of Elsholtzia ciliate. Molecules 17, 5430–5441. http://dx.doi.org/10.3390/ molecules17055430. Loizzo, M.R., Tundis, R., Menichini Saab, A.M., Statti, G.A., Menichini, F., 2007. Cytotoxic activity of essential oils from labiatae and lauraceae families against in vitro human tumor models. Anticancer Res. 27, 3293–3300. Longaray Delamare, A.P., Moschen-Pistorello, I.T., Artico, L., Atti-Serafini, L., Echeverrigaray, S., 2007. Antibacterial activity of the essential oils of Salvia officinalis L. and Salvia triloba L. cultivated in South Brazil. Food Chem. 100, 603–608. http://dx.doi.org/10.1016/j.foodchem.2005.09.078. Raut, J.S., Karuppayil, S.M., 2014. A status review on the medicinal properties of essential oils. Ind. Crops Prod. 62, 250–264. http://dx.doi.org/10.1016/j.indcrop.2014.05. 055. Russo, A., Cardile, V., Graziano, A.C.E., Formisano, C., Rigano, D., Canzoneri, M., Bruno, M., Senatore, F., 2015. Comparison of essential oil components and in vitro anticancer activity in wild and cultivated Salvia verbenaca. Nat. Prod. Res. 29, 1630–1640. http://dx.doi.org/10.1080/14786419.2014.994212. Rustaiyan, A., Masoudi, S., Ezatpour, L., Danaii, E., Taherkhani, M., Aghajani, Z., 2011. Composition of the essential oils of Anthemis Hyalina DC., Achillea Nobilis L. and Cichorium intybus L. Three asteraceae herbs growing wild in Iran. J. Essent. Oil Bear. Plants 14, 472–480. http://dx.doi.org/10.1080/0972060X.2011.10643603. Saini, R., Guleria, S., Kaul, V.K., Lal, B., Garikapati, G.D.K., Singh, B., 2010. Comparison of the volatile constituents of Elsholtzia fruiticosa extracted by hydrodistillation, supercritical fluid extraction and head space analysis. Nat. Prod. Commun. 5, 641–644. Schnee, C., Köllner, T.G., Held, M., Turlings, T.C.J., Gershenzon, J., Degenhardt, J., 2006. The products of a single maize sesquiterpene synthase form a volatile defense signal that attracts natural enemies of maize herbivores. Proc. Natl. Acad. Sci. U. S. A. 103, 1129–1134. Sung, Y.Y., Yoon, T., Yang, W., Kim, kyung, Kim, S.J., 2011. Inhibitory effects of Elsholtzia ciliata extract on fat accumulation in high-fat diet-induced obese mice. J. Appl. Biol. Chem. 54, 388–394. http://dx.doi.org/10.3839/jksabc.2011.061. Teixeira, B., Marques, A., Ramos, C., Neng, N.R., Nogueira, J.M.F., Saraiva, J.A., Nunes, M.L., 2013. Chemical composition and antibacterial and antioxidant properties of commercial essential oils. Ind. Crops Prod. 43, 587–595. http://dx.doi.org/10.1016/ j.indcrop.2012.07.069. Thappa, R.K., Agarwal, S.G., Kapahi, B.K., Srivastava, T.N., 1999. Chemosystematics of the Himalayan Elsholtzia. J. Essent. Oil Res. 11, 97–103. Tian, G., 2013. Chemical constituents in essential oils from Elsholtzia ciliata and their antimicrobial activities. Chin. Herb. Med. 5, 104–108. http://dx.doi.org/10.3969/j. issn.1674-6348.2013.02.004. Weathers, P.J., Towler, M., Hassanali, A., Lutgen, P., Engeu, P.O., 2014. Dried-leaf Artemisia annua: a practical malaria therapeutic for developing countries? World. J. Pharmacol. 3, 39–55.
properties. Evid. Based Complement. Altern. Med. 2014, 1–7. http://dx.doi.org/10. 1155/2014/159819. Bougatsos, C., Ngassapa, O., Runyoro, D.K.B., Chinou, I.B., 2004. Chemical composition and in vitro antimicrobial activity of the essential oils of two Helichrysum species from Tanzania. Zeitschrift Fur Naturforschung Sect. C J. Biosci. 59, 368–372. Buchbauer, G., 2010. Biological activities of essential oils. In: Bas er, K.H.C., Buchbauer, G. (Eds.), Handbook of Essential Oils: Science, Technology, and Appli- Cations. CRC Press/Taylor & Francis Group, Boca Raton, pp. 235–280. Choi, M.S., Choi, B., Kim, S.H., Pak, S.C., Jang, C.H., Chin, Y., Kim, Y., Kim, D., Jeon, S., Koo, B., 2015. Essential oils from the medicinal herbs upregulate dopamine transporter in rat pheochromocytoma cells. J. Med. Food 18, 1–9. http://dx.doi.org/ 10.1089/jmf.2015.3475. Djilani, A., Dicko, A., 2012. The therapeutic benefits of essential oils. In: Bouayed, J., Bohn, T. (Eds.), Nutrition, Well-Being and Health. InTech, Croatia, pp. 155–178. Dung, N.X., Van, H.L., Le, H.H., Leclercq, P.A., 1996. Composition of the essential oils from the aerial parts of Elsholtzia ciliata (Thunb.) Hyland. from Vietnam. J. Essent. Oil Res. 8, 107–109. Fekrazad, R., Afzali, M., Pasban-Aliabadi, H., Esmaeili-Mahani, S., Aminizadeh, M., Mostafavi, A., 2017. Cytotoxic effect of Thymus caramanicus Jalas on human oral epidermoid carcinoma KB cells. Braz. Dent. J. 28, 72–77. http://dx.doi.org/10.1590/ 0103-6440201700737. Fernandes, E.S., Passos, G.F., Medeiros, R., Cunha da, F.M., Ferreira, J., Campos, M.M., Pianowski, L.F., Calixto, J.B., 2007. Anti-inflammatory effects of compounds alphahumulene and (−)-trans-caryophyllene isolated from the essential oil of Cordia verbenacea. Eur. J. Pharmacol. 3, 228–236. http://dx.doi.org/10.1016/j.ejphar. 2007.04.059. Guo, Z., Liu, Z., Wang, X., Liu, W., Jiang, R., Cheng, R., She, G., 2012. Elsholtzia: phytochemistry and biological activities. Chem. Cent. J. 6, 147. http://dx.doi.org/10. 1186/1752-153X-6-147. Jiang, M., Yang, L., Zhu, L., Piao, J., Jiang, J., 2011. Comparative GC/MS analysis of essential oils extracted by 3 methods from the bud of Citrus aurantium L. var. amara. Engl. J. Food Sci. 76, 1219–1225. http://dx.doi.org/10.1111/j.1750-3841.2011. 02421.x. Kharina, T.G., Kalinkina, G.I., Dembitsky, A.D., Maksimenk, N.B., 1995. Essential oil composition and morphological and biological characteristics of Elsholtzia ciliata (Thumb.). Hyl. Rastit. Resur. 31, 58–64. Kim, H.H., Yoo, J.S., Lee, H.S., Kwon, T.K., Shin, T.Y., Kim, S.H., 2011. Elsholtzia ciliata inhibits mast cell-mediated allergic inflammation: role of calcium, p38 mitogenactivated protein kinase and nuclear factor-{kappa}B. Exp. Biol. Med. (Maywood) 236, 1070–1077. http://dx.doi.org/10.1258/ebm.2011.011017. Kim, C., Cho, S.K., Kapoor, S., Kumar, A., Vali, S., Abbasi, T., Kim, S.H., Sethi, G., Ahn, K.S., 2014. β-Caryophyllene oxide inhibits constitutive and inducible STAT3 signaling pathway through induction of the SHP-1 protein tyrosine phosphatase. Mol. Carcinog. 53, 793–806. http://dx.doi.org/10.1002/mc.22035. Korolyuk, E.A., König, W., Tkachevc, A.V., 2002. Composition of essential oil of Elsholtzia ciliata (Thunb.) Hyl. From the Novosibirsk region, Russia. Химия растительного сырья 1, 31–36.
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Chemical composition and anticancer activity of Elsholtzia ciliata essential oils and extracts prepared by different methods
MARK
Lauryna Pudziuvelytea, Mantas Stankeviciusb, Audrius Maruskab, Vilma Petrikaited,e, ⁎ Ona Ragazinskienec, Gailute Draksienea, Jurga Bernatonienea, a
Department of Drug Technology and Social Pharmacy, Lithuanian University of Health Sciences, Eivenių str. 4, LT-50161, Kaunas, Lithuania Department of Biology, Vytautas Magnus University, Vileikos str. 8, LT-44404, Kaunas, Lithuania c Kaunas Botanical Garden of Vytautas Magnus University, Kaunas, Lithuania d Department of Drug Chemistry, Faculty of Pharmacy, Lithuanian University of Health Sciences, LT-44307, Kaunas, Lithuania e Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Vilnius University, LT-10222, Vilnius, Lithuania b
A R T I C L E I N F O
A B S T R A C T
Keywords: Elsholtzia ciliata Anticancer activity Essential oil Ethanolic extracts Hydrodistillation GC–MS
E. ciliata (Lamiaceae) is very interesting and promising herb mainly for chemical composition and pharmacological activities. The aim of this study was to determine chemical composition of the essential oils of fresh, frozen and dried herbal materials of E. ciliata and compare different extraction methods This is the first study on composition of E. ciliata volatile compounds from fresh, frozen and dried herbal samples. The samples were prepared by hydrodistillation (HD), extraction with polar solvent-ethanol (ESE) and dynamic headspace solidphase micro extraction (SPME) and analyzed by gas chromatography-mass spectrometry (GC–MS) method. A total of 48 compounds were identified by GC–MS. Dehydroelsholtzia ketone, elsholtzia ketone, sesquiterpenes βbourbonene, caryophyllene, α-caryophyllene, germacrene D and α-farnesene were identified and found to be predominant compounds in SPME composition of the fresh, frozen and dried herbal samples. The major amounts of ketones (dehydroelsholtzia and elsholtzia) were determined in dried herbal samples where they made up 24.94% (p < 0.05) and 71.34% (p < 0.05) of the SPME composition. Artemisia ketone was determined only in fresh herb. No previous report exists regarding this ketone in E. ciliata fresh, frozen and dried herbal materials or essential oil. There were 26 components identified in the essential oil obtained by HD. The main compounds of this essential oil were dehydroelsholtzia ketone (78.28%) and elsholtzia ketone (14.58%). Essential oil showed antiproliferative activity on three tested cancer cell lines (human glioblastoma (U87), pancreatic cancer (Panc-1) and triple negative breast cancer (MDA-MB231)) in vitro. EC50 (half maximal effective concentration) values of essential oil against those cells were in the range of 0.017–0.021%. The viability of human normal fibroblasts exposed to the same concentrations of the essential oil was statistically significantly higher compared to the viability of cancer cells (p < 0.05). The extracts did not show any effect on U87 cells, and only slightly decreased MDA-MB231 cell viability (up to 76.4%) at the highest concentration of 10 mg/mL. The impact that different extraction methods have on the yield of the essential oil from the tested herbal materials requires a further research. It would enable production of pharmaceutical forms enriched by these essential oils, which are notable for their anticancer activity.
1. Introduction Essential oils are considered to be one of the most important substances in plants and to possess antimicrobial, antiviral, antifungal, antioxidant and anti-inflammatory activities (Bey-Ould Si Said et al., 2016; Raut and Karuppayil, 2014; Teixeira et al., 2013; Buchbauer, 2010). Essential oils are complex mixtures of various terpenes and their oxygenated derivatives such as alcohols, phenols, ketones. Essential oils ⁎
are usually obtained by HD, steam distillation or solvent extraction (Bakkali et al., 2008; Longaray Delamare et al., 2007; Raut and Karuppayil, 2014). For the experiments herbal materials of E.ciliata grown in Lithuania (Europe) were chosen. A flowering plant Elsholtzia ciliata (Thunb.) Hylander of the family Lamiaceae is native to Asia and is also found in Europe, Africa, North America, India (Guo et al., 2012; Korolyuk et al., 2002). Lamiaceae family plants are well known for their antiviral, antimutagenic, chemotherapeutic, antimicrobial, antioxidant
Corresponding author. E-mail addresses: [email protected], [email protected] (J. Bernatoniene).
http://dx.doi.org/10.1016/j.indcrop.2017.05.040 Received 11 January 2017; Received in revised form 19 May 2017; Accepted 21 May 2017 0926-6690/ © 2017 Elsevier B.V. All rights reserved.
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2.5. GC–MS analysis
and anti-inflammatory activities (Raut and Karuppayil, 2014). E. ciliata is widely used in folk medicine for antibacterial, anticancer and antiinflammatory properties (Tian, 2013). In traditional Chinese medicine the herb has been used to treat the common cold, headaches, pharyngitis, fever, edema, diarrhea, rheumatic arthritis, digestion disorders, nephritises (Guo et al., 2012; Sung et al., 2011). The pharmacological activities, such as antibacterial, antiviral, antioxidant, anti-inflammatory, diuretic and anti-obese, of extracts and pure compounds from E. ciliata are under investigation (Guo et al., 2012; Sung et al., 2011). The major chemical constituents in Elsholtzia are flavonoids, phenylpropanoids, phytosterols, cyanogenic glycosides and triterpenes (Guo et al., 2012; Liu et al., 2012). Essential oils accumulating herbal materials are used to produce various pharmaceutical forms such as tablets, capsules, microcapsules, ointments, creams or gels. Usually materials used in production are air dried; however, using fresh or frozen materials could also be suitable. There has not been a research done yet comparing chemical composition of essential oils from freshly collected and from frozen herbal materials, and it is not known what effect the material preparation has on the volatile composition. The aim of this study was to investigate the effects of different methods of material preparation and extraction on chemical composition of volatile compounds from E. ciliata. Also, one of the objectives was to evaluate the toxicity and anticancer activity in vitro of the plant extracts as well as the essential oil.
The analysis was carried out using a GCMS-QP2010 system (Shimadzu, Tokyo, Japan). For separation of volatiles a low polarity RTX-5MS (Restec, USA) (30 m × 0.25 mm i.d. × 0.25 μm film thickness) GC column was used. The oven temperature gradient started at 60 °C and was raised to 150 °C at 5 °C/min, and then raised to 280 °C at 20 °C/min and was held at it for 3 min. The carrier gas helium (99.999%, AGA Lithuania) was used at a flow rate of 1.2 mL/min. The injector temperature was kept at 230 °C in a split mode (1:20). The mass detector electron ionization was 70 eV. The ion source and interface temperatures were set at 220 °C and 260 °C correspondingly. The compounds preliminary were identified by mass spectra library search (NIST, v1.7) and by comparing with the mass spectral data from literature (Adams, 1995). The repeatability of solid-phase micro extraction – gas chromatography mass spectrometry method was evaluated by injecting the same sample three times. The relative standard deviation for the peak area was 5.15%.
2.6. Cell lines Anticancer activity was tested on three selected cancer cell lines: human glioblastoma (U87), pancreatic ductal adenocarcinoma (Panc-1) and triple-negative mammary gland adenocarcinoma (MDA-MB231). Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) high glucose (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% antibiotics (10,000 units/mL penicillin and 10 mg/mL streptomycin) (Gibco) at 37 °C in a humidified atmosphere containing 5% CO2. Human fibroblasts were grown in a Medium 106 with Low Serum Growth Supplement (LSGS) (Gibco) supplemented with 1% antibiotics (10,000 units/mL penicillin and 10 mg/mL streptomycin) (Gibco) at 37 °C in a humidified atmosphere containing 5% CO2. All cell lines were grown to 70% confluence and trypsinized with 0.125% TrypLE™ Express solution (Gibco) before passage. They were used until passage 20.
2. Materials and methods 2.1. Plant material E. ciliata aerial parts were collected in Vilnius, Lithuania, in July 2016 and were purchased as fresh and dried herbs from “Žolynų namai” (Vilnius, Lithuania). The herbs were identified by Dr. Prof. Nijole Savickiene, Medical Academy, Lithuania University of Health Sciences, Kaunas, Lithuania. A voucher specimen (L 17710) was placed for storage at the Herbarium of the Department of Drug Technology and Social Pharmacy, Lithuanian University of Health Sciences, Lithuania. Fresh and dried materials were mechanically ground in a laboratory mill to a homogenous powder or paste. A sample of fresh herb was frozen in a freezer (−18 °C) until preparation of extracts and SPME by GC–MS method.
2.7. Determination of cell viability Cell viability was studied using the method of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma). 100 μL of cells were seeded in 96-well plates in triplicates (5000 cells/well for U87, MDA-MB231 and human fibroblasts cells, and 3000 cells/well for Panc-1 cells) and incubated at 37 °C for 24 h. Then serial dilutions of different extracts and the essential oil were made in microplates. The essential oil before the experiment was mixed with 10% of Tween 80 to enhance its solubilization in culture medium. In order to avoid the essential oil evaporation, 0.05% of methylcellulose (Sigma) was added into the medium. Cells treated only with medium containing the same concentration of ethanol (in the case of extracts) or Tween 80 (in the case of essential oil) served as a negative control. Only medium without cells was used as a positive control. After 72 h incubating at 37 °C, 10 μL of MTT was added in each well. After 3 h the liquid was aspirated from the wells and discarded. Formazan crystals were dissolved in 100 μL of DMSO, and absorbance was measured at a test wavelength of 490 nm and a reference wavelength of 630 nm using a multidetection microplate reader. The experiments were repeated three times independently.
2.2. Isolation of the essential oil 30 g dried E. ciliata sample was mixed with 500 mL bi-distilled water and submitted to HD for 4 h using a Clevenger-type apparatus (European pharmacopoeia). A yellow colored oil with specific aroma was obtained. The essential oil was collected with water and stored in a refrigerator at +4 °C until needed. 2.3. Preparation of ethanolic extracts Fresh, frozen and dried powdered materials (Fig. 1) of plant’s areal parts (0.5 g each) were extracted for 24 h with 20 mL 96% ethanol in TiterTek shaker (Germany). The extracts were filtered through a paper filter and 0.22 μm pore size PVDF membrane filter and stored at +4 °C in a refrigerator until GC–MS analysis. 2.4. Dynamic headspace SPME Samples for gas chromatography analysis were prepared using SPME. Extraction of E. ciliata volatiles was performed on 65 μm PDMS/DVB (polydimethylsiloxane/divinylbenzene) Stable Flex fibre (Supelco, Bellefonte, USA). 10 mg of fresh, frozen and dried samples were added into 10 mL vials and placed in the AOC-5000 autosampler. The samples were thermostated for 10 min at 40 °C and the fiber was exposed in the headspace.
2.8. Statistical analysis Data are presented as mean ± SEM. Statistical analysis was performed by using Student’s t-test. A value of p < 0.05 was taken as the level of significance. 91
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Fig. 1. Fresh (A.), frozen (B.) and dried (C.) herbal materials of E. ciliata areal parts.
3. Results
found in dried herbal samples and made up 21.94% and 71.34% of SPME composition accordingly (p < 0.05 vs fresh and frozen material) (Table 1). Artemisia ketone was identified only in fresh herbal sample and made up 1.83% of SPME composition. Sesquiterpenes were obtained from dried (3.3%), frozen (1.73%) and fresh (1.95%) E. ciliata samples (Table 1). Predominant sesquiterpenes were α-caryophyllene and β-bourbonene in fresh (1.04% and 0.53%), frozen (0.84% and 0.49%) and dried (1.6% and 0.97%) herbal materials. Three alcohols were identified in frozen samples only and one alcohol ((S)-3,4-dimethylpentanol (0.08%)) in dried samples (Table 1). The predominant compound in fresh herb was eucalyptol (0.38%). The tests showed that 2-propenoic acid and 5-methyl-furan-2carboxylic acid (1H-[1,2,4]-triazol-3-yl)-amide were not present in airdried herbal material SPME composition. However, they were identified in fresh and frozen material SPME composition. 2,3-Dimethyl-5(2,6,10-trimethylundecyl)-furan was present only in dried material. The
In the first experiments the chemical compositions of essential oils in the fresh, frozen and dried E. ciliata herbal materials were determined. To our knowledge, this is the first study of E. ciliata volatile compounds extracted from fresh, frozen and dried herbal samples. The volatile compounds found in tested samples are listed in Table 1, their relative percent contents were calculated from the peak areas. E. ciliata grown in Lithuania is not rich in quantity and variety of the volatile compounds. From all SPME samples there were 16 different compounds identified (Table 1). Dehydroelsholtzia ketone, elsholtzia ketone, sesquiterpenes β-bourbonene, caryophyllene, α-caryophyllene, germacrene D and α-farnesene were identified and found to be predominant compounds in fresh, frozen and dried herbal samples. The major amounts of ketones (dehydroelsholtzia and elsholtzia) were 92
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Table 1 SPME composition of fresh, frozen and dried E. ciliata herbal materials. Compounds
3-Methyl-3oxetanemethanol 3-Octanol Eucalyptol 2-Propenoic acid, 2-methyl-, ethenyl ester Elsholtzia ketone 2,3-Dimethyl-5-(2,6,10trimethylundecyl) furan 5-Methyl-furan-2-carboxylic acid (1H-[1,2,4]triazol3-yl)-amide Dehydroelsholtzia ketone Artemizia ketone Beta-Bourbonene Caryophyllene Alpha-Caryophyllene Germacrene D Alpha-Farnesene 1,3,6,10-Dodecatetraene, 3,7,11-trimethyl-, (Z,E)(S)-3,4-Dimethylpentanol Sesquiterpenes Oxygenated monoterpenes Ketones Other Total
Fresh (%)
Frozen (%)
Dried (%)
Table 2 Chemical composition of essential oil obtained by hydrodistillation from E. ciliata dried herb. Retention index
–
0.22
–
935
– – 0.12
0.11 0.38 0.16
– – –
944 963 1052
23.09 –
33.64 –
24.94 0.26
1067 1082
0.28
0.23
–
1083
72.64 1.83 0.53 0.23 1.04 0.07 0.08 0.09
63.31 – 0.49 0.23 0.84 0.09 0.08 –
71.34 – 0.97 0.42 1.60 0.14 0.17 –
1118 1123 1152 1167 1181 1193 1199 1205
– 1.95 – 97.01 0.49 99.45
– 1.73 0.38 96.95 0.72 99.78
0.08 3.3 – 96.28 0.34 99.92
1256
SPME – solid-phase micro extraction.
results presented in Table 1 show that the methods of herb preparation affected the chemical composition. Further tests were aimed to determine the effect of HD method on the chemical composition of the essential oil. In our study, 26 components of the essential oil obtained by HD were identified (Table 2). The main compounds in the essential oil were dehydroelsholtzia ketone (78.28%) and elsholtzia ketone (14.58%). These ketones were also predominant in SPME composition of fresh, frozen and dried herbal samples. The second major identified group of volatile compounds was sesquiterpenes (5.02%), which included α-caryophyllene (1.87%), α-farnesene (0.66%), β-bourbonene (0.57%), isocaryophyllene (0.57%), trans-α-bergamotene (0.55%), δ-cadinene (0.28%), germacrene D (0.24%), γ-cadinene (0.15%), β-cubebene (0.06%), ledene (0.05%), and α-cubebene (0.02%). Some chemical compounds, not present in the herbal material SPME composition, were identified in the essential oil: isocaryophyllene, β-cubebene, ledene, α-cubebene, γcadinene and δ-cadinene. Eucalyptol, caryophyllene oxide, palmitic acid were determined only in the essential oil as well. A total of 23 components were identified in E. ciliata ethanolic extracts (Fig. 2). The main compounds in fresh herb extracts were dehydroelsholtzia ketone (45.74%), cis,cis,cis-7,10,13-hexadecatrienal (16.94%), elsholtzia ketone (7.34%), palmitic acid (4.69%), α-caryophyllene (0.87%). Dehydroelsholtzia ketone (58.47%), elsholtzia ketone (11.49%), linolenic acid (8.5%), palmitic acid (3.95%), α-caryophyllene (1.3%) were obtained as the main components from frozen herb extracts. Predominant compounds from dried herb extracts were dehydroelsholtzia ketone (57.94%), linolenic acid (11.79%), elsholtzia ketone (11.40%), palmitic acid (3.19%), α-caryophyllene (1.31%). This extraction method was better for extraction of organic acids, esters and ketones from herbal samples. The essential oil showed antiproliferative activity (Fig. 3) on all tested cancer cell lines (human glioblastoma (U87), pancreatic cancer (Panc-1) and triple negative breast cancer (MDA-MB231)) in vitro. EC50 values of the essential oil were in the range of 0.017–0.021%. The viability of human normal fibroblasts exposed to the same concentra-
Compounds
Retention index
Composition (%)
Eucalyptol Cyclohexene, 2-ethenyl-1,3,3-trimethyl 2-propenoic acid, 2-methyl-, ethenyl ester Elsholtzia ketone Furane-2-carboxaldehyde, 5(nitrophenoxymethyl)(−)-1R-8-Hydroxy-p-menth-4-en-3-one Dehydroelsholtzia ketone Beta-Bourbonene Isocaryophyllene Beta-Cubebene Ledene Alpha-Caryophyllene Alpha-Cubebene Naphthalene Germacrene D Trans-alpha-Bergamotene Alpha-Farnesene Gamma-Cadinene Delta-Cadinene Caryophyllene oxide Nonane 3-Tetradecen-5-yne, (Z)Palmitic acid Phytol Methyl (Z)-5,11,14,17-eicosatetraenoate 2,6-octadiene, 2,7-dimethylSesquiterpenes Oxygenated monoterpenes Oxygenated sesquiterpenes Ketones Others Total
963 1011 1053 1066 1079
0.05 0.15 0.06 14.58 0.43
1110 1117 1152 1166 1170 1174 1180 1186 1190 1192 1197 1202 1205 1208 1224 1243 1268 1275 1286 1289 1294
0.08 78.28 0.57 0.57 0.06 0.05 1.84 0.02 0.13 0.24 0.55 0.66 0.15 0.28 0.21 0.05 0.05 0.16 0.06 0.61 0.08 4.99 0.05 0.21 92.86 1.86 99.97
tions of essential oil was statistically significantly higher compared to the viability of cancer cells (p < 0.05). The extracts were not very active against tested cell lines (Fig. 4). They did not show any effect on U87 cells, and only slightly decreased the viability of MDA-MB231 cells (up to 76.4%) at the highest concentration of 10 mg/mL. However, they also did not decrease the viability of human fibroblasts at the same dilutions. 4. Discussion In this study the fresh, frozen and dried herbal samples and different extraction methods were analyzed. Our results show that E. ciliata main volatile components are dehydroelsholtzia, elsholtzia ketones and sesquiterpenes α-caryophyllene, β-bourbonene. We determined the composition of extracts obtained from dried herbal samples using HD method in percentages of these major components. Other authors reported that α-caryophyllene demonstrated anti-inflammatory, antiviral activities (Djilani and Dicko, 2012; Fernandes et al., 2007). Caryophyllene oxide (12.45%) rich essential oil of Helichrysum fulgidum showed antibacterial and antifungal activities (Bougatsos et al., 2004). Surprisingly, sesquiterpene trans-α-bergamotene in E. ciliata essential oil has not been reported earlier, but was determined for the first time in our study (0.55%). This volatile compound is attractive to insects (Schnee et al., 2006) and was identified as the main component in the oil of Cichorium intybus (Rustaiyan et al., 2011). Artemisia ketone was determined only in fresh herb SPME composition. No previous report exists regarding this ketone in E. ciliata fresh, frozen and dried herbal materials or essential oil. Artemisia ketone is a major constituent of essential oil from some cultivars of A. annua and may have antimalarial, antibacterial and antifungal activities (Bilia et al., 2014; Weathers et al., 2014). 93
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Fig. 2. A. Identified sesquiterpenes in ethanolic extracts from E. ciliata. B. Identified ketones in ethanolic extracts from E. ciliata. C. Identified organic acids in ethanolic extracts from E. ciliata. D. Identified other compounds in ethanolic extracts from E. ciliata. *Hexadecahydrocyclopenta[a]phenanthrene-3, 17-diol, 16-(1,3-dimethyl-1H-pyrazol-4-ylmethylene)-10, 13-dimethyl-
lene (11.13%), β-linalool (10.86%), eugenol (9.32%), and caryophyllene oxide (8.83%) (Tian, 2013). Ketones have not been identified by these researchers. GC–MS analysis showed the influence of different techniques of extraction on the quantity and quality of volatile components composition. More volatile compounds were obtained using HD (26 components). HD has been earlier reported as a better extraction method for C. aurantium (58 components) and E. fruticosa (35 components) herbal samples. HD was more suitable for monoterpene and sesquiterpene extraction (Jiang et al., 2011; Saini et al., 2010). ESE extracted more organic acids, esters and other compounds. Similar results were obtained from the research where herbal material was extracted by ethyl acetate (Jiang et al., 2011). Volatile compounds composition differences are related to the extraction methods and other factors, such as growing habitats,
There is a considerable similarity reported by different authors about the main components of E. ciliata essential oil. Dehydroelsholtzia ketone and elsholtzia ketone have been reported to be the main ingredients (Kharina et al., 1995; Thappa et al., 1999). Researchers also identified dehydroelsholtzia (66.1–72.4%) and elsholtzia (3.3–19.3%) ketones as the main volatile constituents of essential oil prepared by HD of fresh E. ciliata material from suburb area of Novosibirsk (Korolyuk et al., 2002). There is some difference between our data and other researcher’s results about the main identified compounds. Dung et al. (1996) reported geranial (19.5–26.5%), neral (15.2–20.5%), and limonene (10.9–14.2%) as the main ingredients of E. ciliata essential oils from the leaves were β-linalool (12.06%), caryophyllene (11.02%), eugenol (9.67%), and caryophyllene oxide (9.61%), from the flowers β-linalool (11.52%), β-lonone (11.08%), eugenol (10.56%), and caryophyllene (9.57%), and from the seeds caryophyl94
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(Loizzo et al., 2007; Kim et al., 2014). We could not find any data in the official literature sources about the anticancer effect of dehydroelsholtzia and elsholtzia ketones. However, there are published data that the essential oil from E. ciliata did not possess significant toxicity on rat pheochromocytoma (PC12) cell line at doses of 10, 25, and 50 μg/mL (Choi et al., 2015). It was found that the water extract of E. ciliata could inhibit cytokine expression through NF-κB and p38 MAPK pathway (Kim et al., 2011). p38 MAPK participates in the regulation of cell survival/death, is important for cell invasion and migration, and its levels are usually associated with short survival in cancer patients (Koul et al., 2013). Thus, it is very important to study the mechanism of action of E. ciliata active components. Anticancer activity may be possessed not only by volatile compounds but maybe by polyphenols as well, which can be extracted by different solvents. At this moment it is not easy to answer the question which constituent is the most important for the anticancer activity and at the same time is not very toxic. It could be that not one substance only but the whole complex is responsible for this effect, as different substances may have different mechanisms of action and they may act synergistically (Bakkali et al., 2008).
Fig. 3. The activity of E. ciliata essential oil on human normal fibroblasts and cancer cells. *p < 0.05. Human fibroblasts (HF), human glioblastoma (U87), pancreatic cancer (Panc1) and triple negative breast cancer (MB231).
5. Conclusion Volatile compounds from E. ciliata herbal samples were extracted by different extraction methods and then analyzed by GC–MS. The data shows that E. ciliata grown in Lithuania is rich in volatile compounds ketones. Dehydroelsholtzia ketone and elsholtzia ketone were predominant compounds in all samples, and were obtained by using HD and SPME. Sesquiterpenes are the second major group of compounds identified in the essential oil obtained by using HD. The impact that different extraction methods have on the yield of the essential oil from the tested herbal materials requires a further research. The essential oil from E. ciliata could be further investigated for application of its constituents as anticancer substances. Fig. 4. The activity of E. ciliata extracts on human glioblastoma (U87) and triple-negative breast cancer (MB231) cell lines. 1 – extract from frozen fresh herbal material, 2 – extract from fresh herbal material, 3 – extract from dried herbal material.
Conflicts of interest The authors declare that there are no competing interests regarding the publication of this paper.
seasonal variation, parts of the plant, storage conditions and others. Our experimental data showed that the ethanolic extracts from the fresh, dried and frozen E. ciliata herbal samples were not very active against different tested cancerous and non-cancerous cell lines. We have shown for the first time that the essential oil from E. ciliata shows an anticancer activity against human glioblastoma, breast and pancreatic cancer. However, the oil was significantly less toxic against human normal fibroblasts. Similar effects have been obtained with the extract and essential oil of the plant from the same Lamiaceae family by Fekrazad et al. (2017). They showed that the essential oil from Thymus caramanicus Jalas possesses antiproliferative properties. The activity of this essential oil (IC50 = 0.44 μL/mL) against human oral epidermoid carcinoma KB cells was also much lower compared to that of the extract. However, it was about 2–3 times more active compared to the essential oil from E. ciliata against human breast, pancreatic cancer or glioblastoma cells. Interestingly, cytotoxic effect of essential oil from Thymus caramanicus Jalas was greater than the effect on normal gingival cells, what was seen also in our experiments. In another experiment Russo et al. (2015) showed that the essential oil from Salvia verbenaca was also active against human melanoma cell line M14 and possessed lower activity against normal human nonimmortalised buccal fibroblast cells. The activity of that essential oil mainly has been explained by the presence of sesquiterpenes and carvacrol. α-Caryophyllene and caryophyllene, which are found in the essential oil of E. ciliata as the major constituents, also could contribute to the anticancer activity against tested cell lines as there is some data published about its in vitro and in vivo anticancer effect
Acknowledgments The authors would like to thank Open Access Centre for the Advanced Pharmaceutical and Health Technologies (Lithuanian university of Health Sciences) for providing the opportunity to use infrastructure and perform this research and Instrumental Analysis Open access research center of Vytautas Magnus University of Natural Sciences. Also, the authors want to thank Dr. Manel Esteller (Bellvitge Biomedical Research Institute (IDIBELL), Spain) for kindly providing the MDA-MB231, Panc-1 and U87 cell lines; and Dr. Ramunas Valiokas (Center for Physical Sciences and Technology, Lithuania) for providing human fibroblasts cell line. References Adams, R.P., 1995. Identification of Essential Oil Components by Gas Chromatography/ Mass Spectrometry. Allured Publishing Corporation, Carol Stream, IL. Bakkali, F., Averbeck, S., Averbeck, D., Idaomar, M., 2008. Biological effects of essential oils – a review. Food Chem. Toxicol. 46, 446–475. http://dx.doi.org/10.1016/j.fct. 2007.09.106. Bey-Ould Si Said, Z., Haddadi-Guemghar, H., Boulekbache-Makhlouf, L., Rigou, P., Remini, H., Adjaoud, A., Khoudja, K.N., Madani, K., 2016. Essential oils composition, antibacterial and antioxidant activities of hydrodistillated extract of Eucalyptus globulus fruits. Ind. Crops Prod. 89, 167–175. http://dx.doi.org/10.1016/j.indcrop. 2016.05.018. Bilia, A.R., Santomauro, F., Sacco, C., Bergonzi, M.C., Donato, R., 2014. Essential oil of artemisia annua L.: an extraordinary component with numerous antimicrobial
95
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L. Pudziuvelyte et al.
Koul, H.K., Pal, M., Koul, S., 2013. Role of p38 MAP kinase signal transduction in solid tumors. Genes Cancer 4, 342–359. http://dx.doi.org/10.1177/1947601913507951. Liu, X., Jia, J., Yang, L., Yang, F., Ge, H., Zhao, C., Zhang, L., Zu, Y., 2012. Evaluation of antioxidant activities of aqueous extracts and fractionation of different parts of Elsholtzia ciliate. Molecules 17, 5430–5441. http://dx.doi.org/10.3390/ molecules17055430. Loizzo, M.R., Tundis, R., Menichini Saab, A.M., Statti, G.A., Menichini, F., 2007. Cytotoxic activity of essential oils from labiatae and lauraceae families against in vitro human tumor models. Anticancer Res. 27, 3293–3300. Longaray Delamare, A.P., Moschen-Pistorello, I.T., Artico, L., Atti-Serafini, L., Echeverrigaray, S., 2007. Antibacterial activity of the essential oils of Salvia officinalis L. and Salvia triloba L. cultivated in South Brazil. Food Chem. 100, 603–608. http://dx.doi.org/10.1016/j.foodchem.2005.09.078. Raut, J.S., Karuppayil, S.M., 2014. A status review on the medicinal properties of essential oils. Ind. Crops Prod. 62, 250–264. http://dx.doi.org/10.1016/j.indcrop.2014.05. 055. Russo, A., Cardile, V., Graziano, A.C.E., Formisano, C., Rigano, D., Canzoneri, M., Bruno, M., Senatore, F., 2015. Comparison of essential oil components and in vitro anticancer activity in wild and cultivated Salvia verbenaca. Nat. Prod. Res. 29, 1630–1640. http://dx.doi.org/10.1080/14786419.2014.994212. Rustaiyan, A., Masoudi, S., Ezatpour, L., Danaii, E., Taherkhani, M., Aghajani, Z., 2011. Composition of the essential oils of Anthemis Hyalina DC., Achillea Nobilis L. and Cichorium intybus L. Three asteraceae herbs growing wild in Iran. J. Essent. Oil Bear. Plants 14, 472–480. http://dx.doi.org/10.1080/0972060X.2011.10643603. Saini, R., Guleria, S., Kaul, V.K., Lal, B., Garikapati, G.D.K., Singh, B., 2010. Comparison of the volatile constituents of Elsholtzia fruiticosa extracted by hydrodistillation, supercritical fluid extraction and head space analysis. Nat. Prod. Commun. 5, 641–644. Schnee, C., Köllner, T.G., Held, M., Turlings, T.C.J., Gershenzon, J., Degenhardt, J., 2006. The products of a single maize sesquiterpene synthase form a volatile defense signal that attracts natural enemies of maize herbivores. Proc. Natl. Acad. Sci. U. S. A. 103, 1129–1134. Sung, Y.Y., Yoon, T., Yang, W., Kim, kyung, Kim, S.J., 2011. Inhibitory effects of Elsholtzia ciliata extract on fat accumulation in high-fat diet-induced obese mice. J. Appl. Biol. Chem. 54, 388–394. http://dx.doi.org/10.3839/jksabc.2011.061. Teixeira, B., Marques, A., Ramos, C., Neng, N.R., Nogueira, J.M.F., Saraiva, J.A., Nunes, M.L., 2013. Chemical composition and antibacterial and antioxidant properties of commercial essential oils. Ind. Crops Prod. 43, 587–595. http://dx.doi.org/10.1016/ j.indcrop.2012.07.069. Thappa, R.K., Agarwal, S.G., Kapahi, B.K., Srivastava, T.N., 1999. Chemosystematics of the Himalayan Elsholtzia. J. Essent. Oil Res. 11, 97–103. Tian, G., 2013. Chemical constituents in essential oils from Elsholtzia ciliata and their antimicrobial activities. Chin. Herb. Med. 5, 104–108. http://dx.doi.org/10.3969/j. issn.1674-6348.2013.02.004. Weathers, P.J., Towler, M., Hassanali, A., Lutgen, P., Engeu, P.O., 2014. Dried-leaf Artemisia annua: a practical malaria therapeutic for developing countries? World. J. Pharmacol. 3, 39–55.
properties. Evid. Based Complement. Altern. Med. 2014, 1–7. http://dx.doi.org/10. 1155/2014/159819. Bougatsos, C., Ngassapa, O., Runyoro, D.K.B., Chinou, I.B., 2004. Chemical composition and in vitro antimicrobial activity of the essential oils of two Helichrysum species from Tanzania. Zeitschrift Fur Naturforschung Sect. C J. Biosci. 59, 368–372. Buchbauer, G., 2010. Biological activities of essential oils. In: Bas er, K.H.C., Buchbauer, G. (Eds.), Handbook of Essential Oils: Science, Technology, and Appli- Cations. CRC Press/Taylor & Francis Group, Boca Raton, pp. 235–280. Choi, M.S., Choi, B., Kim, S.H., Pak, S.C., Jang, C.H., Chin, Y., Kim, Y., Kim, D., Jeon, S., Koo, B., 2015. Essential oils from the medicinal herbs upregulate dopamine transporter in rat pheochromocytoma cells. J. Med. Food 18, 1–9. http://dx.doi.org/ 10.1089/jmf.2015.3475. Djilani, A., Dicko, A., 2012. The therapeutic benefits of essential oils. In: Bouayed, J., Bohn, T. (Eds.), Nutrition, Well-Being and Health. InTech, Croatia, pp. 155–178. Dung, N.X., Van, H.L., Le, H.H., Leclercq, P.A., 1996. Composition of the essential oils from the aerial parts of Elsholtzia ciliata (Thunb.) Hyland. from Vietnam. J. Essent. Oil Res. 8, 107–109. Fekrazad, R., Afzali, M., Pasban-Aliabadi, H., Esmaeili-Mahani, S., Aminizadeh, M., Mostafavi, A., 2017. Cytotoxic effect of Thymus caramanicus Jalas on human oral epidermoid carcinoma KB cells. Braz. Dent. J. 28, 72–77. http://dx.doi.org/10.1590/ 0103-6440201700737. Fernandes, E.S., Passos, G.F., Medeiros, R., Cunha da, F.M., Ferreira, J., Campos, M.M., Pianowski, L.F., Calixto, J.B., 2007. Anti-inflammatory effects of compounds alphahumulene and (−)-trans-caryophyllene isolated from the essential oil of Cordia verbenacea. Eur. J. Pharmacol. 3, 228–236. http://dx.doi.org/10.1016/j.ejphar. 2007.04.059. Guo, Z., Liu, Z., Wang, X., Liu, W., Jiang, R., Cheng, R., She, G., 2012. Elsholtzia: phytochemistry and biological activities. Chem. Cent. J. 6, 147. http://dx.doi.org/10. 1186/1752-153X-6-147. Jiang, M., Yang, L., Zhu, L., Piao, J., Jiang, J., 2011. Comparative GC/MS analysis of essential oils extracted by 3 methods from the bud of Citrus aurantium L. var. amara. Engl. J. Food Sci. 76, 1219–1225. http://dx.doi.org/10.1111/j.1750-3841.2011. 02421.x. Kharina, T.G., Kalinkina, G.I., Dembitsky, A.D., Maksimenk, N.B., 1995. Essential oil composition and morphological and biological characteristics of Elsholtzia ciliata (Thumb.). Hyl. Rastit. Resur. 31, 58–64. Kim, H.H., Yoo, J.S., Lee, H.S., Kwon, T.K., Shin, T.Y., Kim, S.H., 2011. Elsholtzia ciliata inhibits mast cell-mediated allergic inflammation: role of calcium, p38 mitogenactivated protein kinase and nuclear factor-{kappa}B. Exp. Biol. Med. (Maywood) 236, 1070–1077. http://dx.doi.org/10.1258/ebm.2011.011017. Kim, C., Cho, S.K., Kapoor, S., Kumar, A., Vali, S., Abbasi, T., Kim, S.H., Sethi, G., Ahn, K.S., 2014. β-Caryophyllene oxide inhibits constitutive and inducible STAT3 signaling pathway through induction of the SHP-1 protein tyrosine phosphatase. Mol. Carcinog. 53, 793–806. http://dx.doi.org/10.1002/mc.22035. Korolyuk, E.A., König, W., Tkachevc, A.V., 2002. Composition of essential oil of Elsholtzia ciliata (Thunb.) Hyl. From the Novosibirsk region, Russia. Химия растительного сырья 1, 31–36.
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