APTEFF, 48, 1-323 (2017) UDC: 582.477.4:543.51+543.544.3 https://doi.org/10.2298/APT1748077D BIBLID: 1450-7188 (2017) 48, 77-83 Original scientific paper

CHEMICAL PROFILE OF Taxodium distichum WINTER CONES

Nina M. Đapić1*, Mihailo S. Ristić2

1 University of Novi Sad, Technical Faculty “Mihajlo Pupin”, Đure Đakovica bb, 23000 Zrenjanin, Serbia 2 Institute for Medicinal Plant Research “Dr Josif Pančić”, Tadeuša Košćuškа 1, 11000 Belgrade, Serbia

This work is concerned with the chemical profile of Taxodium distichum winter cones. The extract obtained after maceration in absolute ethanol was subjected to qualitative analysis by / and quantification was done by gas chromatograp- hy/flame ionization detector. The chromatogram revealed the presence of 53 compounds, of which 33 compounds were identified. The extract contained oxygenated monoterpenes (12.42%), (5.18%), oxygenated sesquiterpenes (17.41%), (1.15%), and oxygenated diterpenes (30.87%), while the amount of retinoic acid was 0.32%. Mono- acylglycerols were detected in the amount of 4.32%. The most abundant compounds were: caryophyllene oxide (14.27%), 6,7-dehydro- (12.49%), bornyl acetate (10.96%), 6- deoxy-taxodione (9.50%) and trans-caryophyllene (4.20%).

KEY WORDS: Taxodium distichum, cones, GC-MS

INTRODUCTION

The Taxodium distichum tree is cultivated in urban areas (1-3). The properties and variability of ten Taxodium distichum trees grown in the Futoski Park and eighteen trees grown in the Dunavski Park are well described (2). The tree is ornamental adap- tive to grow in this region. self-propagation is done by cones. The phytoche- mical cone composition, focused on essential oil, has been determined using gas chroma- tography coupled to mass spectrometry (GC-MS) (4-7). The analysis obtained revealed monoterpenoids: α-pinene (87.3%), β-pinene (1.7%), camphene (1.0%), limonene (1.3%), myrcene (2.0%) and sesquiterpenoid thujopsene (3.7%) (4). The characteristic secondary metabolites present in essential oils of the cones from the Mediterranean Basin revealed the presence of α-pinene (71.3%) as the most abundant essential oil compound (5). The cone essential oil had a monoterpene limonene in the percentage of 18.7% (5). When milled cones were extracted with n-hexane, the analysis showed that 70% of the extract obtained were diterpenes (8). The essential oil of Taxodium distichum cone was obtained by hydrodistillation, the extraction technique which compromises elevated temperatures and can cause compound thermodegradation (4-7). The milled Taxodium distichum cones extracted with a non-

* Corresponding author: Nina M. Đapić, University of Novi Sad, Technical Faculty “Mihajlo Pupin”, Đure Đa- kovića bb, 23000 Zrenjanin, Serbia, e-mail: [email protected] 77 APTEFF, 48, 1-323 (2017) UDC: 582.477.4:543.51+543.544.3 https://doi.org/10.2298/APT1748077D BIBLID: 1450-7188 (2017) 48, 77-83 Original scientific paper polar solvent gave diterpenes as the most abundant compounds (8). In the literature, there are reports on the compounds present in Taxodium distichum cone essential oil obtained by hydrodistillaton and in n-hexane extract, while the compounds present in absolute ethanol extract have not been described up to now. This study was conducted to reveal the compounds present in winter Taxodium distichum cones, obtained by maceration in absolute ethanol.

EXPERIMENTAL

Plant material

Coppery-red bald cypress cones were collected in January 2015 in the Futoški Park, Novi Sad, Serbia. The collected cones were air dried for several weeks. After drying, the water content was found to be 7.36±0.11%. The dried cones were ground and milled in a rotating blade coffee grinder Bosch MKM 6003. The powdered plant material (20 g) was dissolved in absolute ethanol (200 ml), and allowed to stand at room temperature for 3 days, with frequent agitation. The powdered plant material was separated, the marc was pressed, and the obtained liquids were combined and filtered. After solvent evaporation, brown oil was obtained. The oil was dissolved in acetone to obtain ~1% sample solution which was subjected to further analyses.

Analysis of compounds using gas chromatography

The analysis was performed using gas chromatography followed by flame ionization detection (GC-FID) and gas chromatography-mass spectrometry (GC-MS). The GC-FID analysis was carried out on a Hewlett-Packard, HP-5890 Series II gas chromatograph (Waldbronn, Germany), equipped with a split-splitless injector, HP-5 fused silica capil- lary column (25 m x 0.32 mm id, 0.5 μm film thickness) and equipped with FID detector. The carrier gas (H2) flow rate was 1 mL/min, split ratio 1:30, injector temperature 250 °C, and detector temperature 300 °C, while column temperature was linearly program- med from 40 °-240 °C (at a rate of 4 °C). The same analytical conditions were employed for the GC-MS analysis, where an HP G 1800C GCD Series II Detector (EID) analytical system (Palo Alto, CA, USA) equipped with split-splitless injector and automatic liquid sampled (ALS) were used, and the carrier gas was helium. The injector was heated to 250 °C, and the transfer line (MSD) to 280 °C. The column temperature was linearly raised from 40 to 260 °C (at a rate of 4 °C). The mass spectra were acquired in electron ionization (EI) mode (70eV), in an m/z range of 40-400. An HP-5MS column (30 m x 0.25 mm, 0.25 μm film thickness) was used. Identification of the individual oil components was accomplished by their retention times compared to analytical standard substances of available terpenoids, by matching mass spectral data with those kept in the mass spectra library (Wiley 275.1), using computer search and the literature (9, 10). The confirmation was done using a calibrated AMDIS program for determination of experimental values for retention indices of recorded constituents and comparing them with those from the literature.

78 APTEFF, 48, 1-323 (2017) UDC: 582.477.4:543.51+543.544.3 https://doi.org/10.2298/APT1748077D BIBLID: 1450-7188 (2017) 48, 77-83 Original scientific paper

RESULTS AND DISCUSSION

The chromatogram obtained by the GC-MS analysis revealed the presence of 53 constituents, of which 33 were identified, and they are listed in Table 1. The predominant compounds in the extract analyzed were: caryophyllene oxide (14.27%), 6,7-dehydro-fer- ruginol (12.49%), bornyl acetate (10.96%), 6-deoxy-taxodione (9.50%), trans-caryophyl- lene (4.20%), humulene epoxide II (1.31%), trans-ferruginol (4.23%), sugiol (2.97%), 1- glyceryl oleate (2.88%), 2-glyceryl stearate (1.44%), while the other components were present in less than 1%. The compounds that were not identified made in total 13.70%. The oxygenated monoterpenes identified in the extract were: camphene hydrate, α-terpi- neol, α-campholenol, bornyl acetate, trans-verbenyl acetate, trans-pinocarvyl acetate, iso- dihydro carveol acetate and trans-carvyl acetate. Among these compounds, bornyl acetate was most abundant, while the other oxygenated monoterpenes were present in less than a half percent. Of sesquiterpenes trans-caryophyllene and α-humulene were identified. The amount of trans-caryophyllene in the extract was 4.20% and that of α-humulene less than one percent. The oxygenated sesquiterpenes were: caryophyllene oxide (14.27%), three humulene epoxide stereoisomers were detected where the dominant among them was hu- mulene epoxide II, then caryophylla-4(12),8(13)-dien-5β-ol (0.35%), 14-hydroxy-(Z)-ca- ryophyllene (0.81%) and iso-longifolol (0.22%). The diterpenes detected were: pimara- diene (0.39%), abietatriene (0.47%) and abietadiene (0.29%). There were six oxygenated diterpenes in the extract: manool oxide (0.75%), pimarinal (0.91%), 6,7-dehydro-ferrugi- nol (12.49%), trans-ferruginol (4.23%), 6-deoxy-taxadione (9.50%) and sugiol (2.97%). Retinoic acid was detected in the amount of 0.32%. Two monoacylglycerols were present in the extract: 1-glyceriyl oleate (2.88%) and 2-glyceryl stearate (1.44%). The previous report on the volatile oil chemical compounds in Taxodium distichum seed cones mentioned that the most abundant compound was dextro pinene (85%), along with: dextro limonene (5%), carvone (3%), tricyclic (3%) and a “pseudo alcohol” (2%) (6). Monoterpenes were the dominant compounds detected in early work on the chemical composition of Taxodium distichum cones. The essential oil compound analysis of Taxodium distichum cones, from the Mediterranean Basin, where climate is mild with rainy winters and hot and dry summers, showed that α-pinene was present in 71.3% and limonene in 18.7% (5). The North African Taxodium distichum cones essential oil contained α-pinene (87.3%), β-pinene (1.7%), camphene (1.0%), myrcene (2.0%), limonene (1.3%) and thujopsene (3.7%) (4). The Taxodium distichum cones from West Africa contained 60.5% α-pinene, 17.6% thujopsene and 29 other com- pounds (7). The compound present in all essential oils analyzed was α-pinene, while li- monene was the next compound present in the analyzed samples. The thujopsene is pre- sent in African Taxodium distichum cones essential oil in different quantities. The Taxo- dium distichum cones grown in North America, Mediterranean Basin, North and West Africa have some compounds in common, although their quantity varies. The different composition can be attributed to the different environmental effects (11). The Taxodium distichum winter cones extract obtained after maceration in absolute ethanol differ in chemical composition from previously described essential oils. The winter cones extract contained oxygenated monoterpenes, where the most abundant was bornyl acetate

79 APTEFF, 48, 1-323 (2017) UDC: 582.477.4:543.51+543.544.3 https://doi.org/10.2298/APT1748077D BIBLID: 1450-7188 (2017) 48, 77-83 Original scientific paper

(10.96%), whereas 14 components were with the sesquiterpenoid structure (~ 23%), with caryophyllene oxide being the most abundant (14.27%), while 63% comprised mainly diterpenoids with a small amount of other highly volatile substances.

Table 1. Chemical composition of Taxodium distichum winter cones macerated in absolute ethanol

% Peak Constituents KIE KIL RT/MS RRT CI m/m 1 α-Campholenal 1116.9 1122 12.78 0.37 0.338 26 2 Camphene hydrate 1138.4 1145 13.52 0.29 0.357 20 3 α-Terpineol 1183.6 1186 15.09 0.16 0.399 11 4 α-Campholenol 1197.0 1190 15.55 0.41 0.411 29 5 Bornyl acetate 1275.3 1287 18.20 10.96 0.481 768 6 trans-Verbenyl acetate 1284.5 1291 18.52 0.25 0.490 17 7 trans-Pinocarvyl acetate 1288.3 1298 18.65 0.21 0.493 15 8 iso-Dihydro carveol acetate 1323.8 1326 19.82 0.29 0.524 21 9 trans-Carvyl acetate 1327.3 1339 19.93 0.46 0.527 32 10 trans-Caryophyllene 1403.5 1417 22.39 4.20 0.592 294 11 α-Humulene 1437.4 1452 23.43 0.98 0.620 68 12 Caryophyllene oxide 1570.2 1582 27.38 14.27 0.724 1000 13 1-Hexadecene 1577.7 1588 27.60 0.18 0.730 13 14 Humulene epoxide I 1584.5 1593 27.77 0.17 0.734 12 15 Humulene epoxide II 1593.5 1608 28.07 1.31 0.742 92 16 Humulene epoxide III 1598.3 1620 28.20 0.28 0.746 20 17 Caryophylla-4(12),8(13)-dien-5β-ol 1622.5 1639 28.88 0.35 0.764 24 18 n. i. 1644.6 29.49 0.14 0.780 10 19 14-Hydroxy-(Z)-caryophyllene 1658.9 1666 29.88 0.81 0.790 57 20 iso-Longifolol 1721.4 1728 31.59 0.22 0.835 15 21 n. i. 1794.4 33.50 0.16 0.886 12 22 n. i. 1886.4 35.83 0.33 0.948 23 23 n. i. 1902.0 36.23 0.29 0.958 20 24 Pimaradiene 1939.0 1948 37.11 0.39 0.981 27 25 Manool oxide 1968.6 1987 37.82 0.75 1.000 52 26 Ethyl hexadecanoate 1978.1 1992 38.05 0.41 1.006 29 27 Abietatriene 2033.9 2055 39.35 0.47 1.041 33 28 Abietadiene 2057.8 2087 39.90 0.29 1.055 20 29 n. i. 2069.5 40.16 0.41 1.062 29 30 Pimarinal 2161.1 217X 42.20 0.91 1.116 64 31 n. i. 2209.7 43.26 0.25 1.144 17 32 λ-8(17),14-diene-6,13-diol* 2248.5 2248 44.07 0.97 1.166 68 33 M=356 2279.0 44.70 4.62 1.182 323 34 6,7-Dehydro-ferruginol 2314.5 2315 45.45 12.49 1.202 875 35 trans-Ferruginol 2317.7 2331 45.51 4.23 1.203 297 36 n. i. 2329.2 45.74 0.93 1.210 65 37 n. i. 2339.8 45.96 1.51 1.215 106 38 Retinoic acid 2353.8 46.25 0.32 1.223 22

80 APTEFF, 48, 1-323 (2017) UDC: 582.477.4:543.51+543.544.3 https://doi.org/10.2298/APT1748077D BIBLID: 1450-7188 (2017) 48, 77-83 Original scientific paper

Table 1. Continuation

% Peak Constituents KIE KIL RT/MS RRT CI m/m 39 n. i. 2361.9 46.41 0.34 1.227 24 40 n. i. 2398.4 47.15 2.68 1.247 188 41 n. i. 2407.6 47.33 0.23 1.252 16 42 n. i. 2413.9 47.45 0.25 1.255 17 43 6-Deoxy-taxodione 2450.7 2435 48.16 9.50 1.274 665 44 n. i. 2498.2 49.08 3.45 1.298 242 45 n. i. 2559.1 50.24 0.24 1.328 17 46 n. i. 2566.2 50.37 1.67 1.332 117 47 n. i. 2573.8 50.52 0.52 1.336 36 48 n. i. 2602.3 51.05 5.56 1.350 389 49 Sugiol 2620.8 2629 51.39 2.97 1.359 208 50 1-Glyceryl oleate (monoolein) 2677.8 2714 52.42 2.88 1.386 202 51 n. i. 2695.6 52.75 0.33 1.395 23 52 2-Glyceryl stearate (2-monostearin) 2702.7 n/a 52.86 1.44 1.398 101 53 n. i. 2745.0 53.61 1.93 1.418 135 Legend: KIE = Kovats (retention) index experimentally determined (AMDIS) KIL = Kovats (retention) index - literature data (9) RT/MS = Retention time of the corresponding constituent obtained by GC/MS RRT = Relative retention time to selected constituent [Caryophyllene oxide = 1.000] % m/m = Area % obtained by MSD CI = Concentration index *= Tentative identification n.i.= not identified n/a = not available

Figure 1. The GC/MS chromatogram of the extract of Taxodium distichum winter cones

81 APTEFF, 48, 1-323 (2017) UDC: 582.477.4:543.51+543.544.3 https://doi.org/10.2298/APT1748077D BIBLID: 1450-7188 (2017) 48, 77-83 Original scientific paper

CONCLUSION

The GC-MS analysis of the ethanolic extract of Taxodium distichum winter cones revealed the presence of 53 compounds, of which 33 compounds were identified. The constituents of the extract were as follows: eight oxygenated monoterpenes (12.42%), two sesquiterpenes (5.18%), oxygenated sesquiterpenes (17.41%), diterpenes (1.15%) and oxygenated diterpenes (30.87%). Oxygenated diterpenes were the most abundant class of compounds in the amount of 30.87%, then oxygenated sesquiterpenes (17.41%), followed by oxygenated monoterpenes (12.42%). Sesquiterpenes were present in 5.18%, diterpens in 1.15% and monoterpene in 0.37%. Retinoic acid was present in 0.32%. Monoacylglycerols were detected in the amount of 4.32%. The extract can be used for the isolation of most abundant compounds present: one oxygenated sesquiterpene caryo- phyllene oxide (14.27%), one oxygenated 6,7-dehydro-ferruginol (12.49%), and one oxygenated monoterpene bornyl acetate (10.96%).

REFERENCES

1. Drazic, D.; Batos, B. Mocvarni cempres Taxodium distichum (L.) Rich. u uslovima Beograda, 7th Symposium on Flora of Southeastern Serbia and Neighbouring Regions, Dimitrovgrad, 6-9 June 2002, Proceeding, p. 195 2. Ninic-Todorovic, J.; Ocokoljic, M. Varijabilnost taksodijuma (Taxodium distichum (L.) Rich.) u parkovima Novog Sada, 7th Symposium on Flora of Southeastern Serbia and Neighbouring Regions, Dimitrovgrad, 6-9 June 2002, Proceeding, p. 125 3. Sijacic-Nikolic, M.; Vilotic D.; Veselinovic, M.; Mitrovic, S.; Jokanovic, D. Bald cypress (Taxodium distichum (L.) Rich.) in the protected area “Veliko ratno ostrvo”, Bulletin of the Faculty of Forestry 2010, 103, 173-184. 4. El Tantawy, M. E.; El Sakhawy, F. S.; El Sohly, M. A.; Ross, S. A. Chemical Composition and Biological Activity of the Essential Oil of the Fruit of Taxodium distichum L. Rich. Growing in Egypt, J. Essent. Oil Res. 1999, 11 (3), 386-392. 5. Flamini, G.; Cioni, P. L.; Morelli, I. Investigation of the Essential Oil of Feminine Cones, Leaves and Branches of Taxodium distichum from Italy, J. Essent. Oil Res. 2000, 12 (3), 310-312. 6. Odell, A. F. The oil of the southern cypress, JACS, 1912, 34: 824-826. 7. Ogunwande, I. A.; Olawore, N. O.; Ogunmola, O. O.; Walker, T. M.; Schmidt, J. M.; Setzer, W. N. Cytotoxic effects of Taxodium distichum oils, Pharm. Biol. 2007, 45: 106-110. 8. Kusumoto, N.; Ashitani, T.; Murayama, T.; Ogiyama, K.; Takahashi, K. Antifungal -Type Diterpenes from the Cones of Taxodium distichum Rich, J. Chem. Ecol. 2010, 36: 1381-1386. 9. Adams, R. P. Identification of Essential Oil Components by Gas Chromato- graphy/Mass Spectrometry, 4th Ed., Allured Publishing Corp., Carol Stream, IL 60188 USA, 2007. 10. Adams, R. Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry. Allured Publishing, Illinois, USA, 1995.

82 APTEFF, 48, 1-323 (2017) UDC: 582.477.4:543.51+543.544.3 https://doi.org/10.2298/APT1748077D BIBLID: 1450-7188 (2017) 48, 77-83 Original scientific paper

11. Clark, R. J.; Menary, R. C. Environmental Effects on Peppermint (Mentha piperita L.). II. Effects of Temperature on Photosynthesis, Photorespiration and Dark Respi- ration in Peppermint with reference to Oil composition, Funct. Plant Biol. 1980, 7 (6): 693-697.

ХЕМИЈСКА ЈЕДИЊЕЊА ПРИСУТНА ЗИМИ У ШИШАРКАМА МОЧВАРНОГ ЧЕМПРЕСА

Нина М. Ђапић1, Михаило С. Ристић2

1 Универзитет у Новом Саду, Технички Факултет “Михајло Пупин”, Ђуре Ђаковића бб, 23000 Зрењанин, Србија 2 Институт за проучавање лековитог биља „Др Јосиф Панчић“, Тадеуша Кошћушка 1, 11000 Београд, Србија

Екстракт Taxodium distichum шишарки убраних током зиме добијен је након ма- церације у апсолутном етанолу. Добијени екстракт је квалитативно анализиран употребом гасне хроматографије/масене спектрометрије, док је квантитативна ана- лиза узорка урађена употребом гасне хроматографије са пламено јонизационим детектором. У добијеном хроматограму било је 53 пика и од тога идентификовано је 33 једињења. Екстракт је садржао оксидоване монотерпене (12,42%), сесквитер- пене (5,18%), оксидоване сесквитерпене (17,41%), дитерпене (1,15%) и оксидоване дитерпене (30,87%). Ретиноинска киселина је чинила 0,32% екстракта. Два моно- ацилглицерола су детектована у количини од 4,32%. Доминантна једињења екстрак- та су била: кариофилен оксид (14,27%), 6,7-дехидро-феругинол (12,49%), борнил ацетат (10,96%), 6-деокси-таксадионе (9,50%) и trans-кариофилен (4,20%).

Kључне речи: Taxodium distichum, шишарке, GC-MS

Received: 05 July 2017. Accepted: 15 October 2017

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