Journal of Essential Oil Bearing Plants ISSN Print: X Online:

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1 Journal of Essential Oil Bearing Plants ISSN Print: X Online: Chemical Composition of the Hydrosol and the Essential Oil of Three Different Species of the Pinaceae Family : Picea glauca (Moench) Voss., Picea mariana (Mill.) B.S.P., and Abies balsamea (L.) Mill. François-Xavier Garneau *, Guy Collin, Hélène Gagnon and André Pichette Laboratoire LASEVE, Université du Québec à Chicoutimi, 555, boul. Université, Saguenay (Québec) Canada, G7H 2B1 Abstract: The chemical composition of the oils and hydrosols collected from wild plantations of three species of the Pinaceae family of conifers in the Grondines region of the province of Quebec, Canada, were determined by GC-FID and GC-MS analyses. The components obtained from the hydrosol of Picea glauca (Moench) Voss., (White Spruce) were mainly monoterpene alcohols, aldehydes and ketones. The major compound by far was camphor (65 %) with borneol (10 %) as an important minor constituent. In contrast, the essential oil was composed mainly of β-pinene (20 %) and camphor (20 %) followed by α- pinene (12 %) and bornyl acetate (12 %). Bornyl acetate (34.2 %) was the main compound identified in the Picea mariana (Mill.) B.S.P., (Black Spruce) oil and monoterpenes, mainly α-pinene (12.9 %) and camphene (16.4 %), constituted at least 45 % of the oil. On the contrary, due to their very low water solubility, monoterpene hydrocarbons were barely present in the hydrosol in which alcohols were the main components. They were α-terpineol and borneol (each ~14 %), terpinen-4-ol and camphene hydrate (each ~6 %) as well as several aliphatic alcohols (cis-3-hexen-1-ol %). Bornyl acetate was also a significant component (7.8 %). Monoterpenes were the main components of the essential oil of Abies balsamea (L.) Mill. (Canadian Fir oil). They were β-pinene (33.5 %), Δ 3 -carene (14 %), α-pinene (11.3 %) as well as limonene and β- phellandrene (each ~7 %). Bornyl acetate was also a significant component (10 %). α-terpineol (~41 %) was by far the main component observed in the hydrosol followed by borneol (6.4 %). Of particular interest was the presence of maltol (~15 %). To our knowledge, this is the first report on the hydrosol composition obtained from a coniferous tree. Key words: Picea glauca, Picea mariana, Abies balsamea, Pinaceae, hydrosol, essential oil, composition, camphor, maltol, β-pinene. Introduction Picea glauca (White Spruce), P. mariana (Black Spruce), and Abies balsamea (Balsam fir oil) are very common to the northern part of North America and is found mainly from Alaska to Newfoundland and to the south in Montana, Michigan and Maine 1. The first settlers benefited from a recipe given to them by the First Nations whereby a decoction of the twigs and needles of white spruce was used to treat scurvy. Nowadays, White and Black Spruces are very important economically in Canada for their wood and are exploited by the Pulp and Paper industries. The leaves and branches, or the essential oils, can also be used to brew spruce beer. The chemical composition of the essential oils of White and Black Spruce were *Corresponding author (François-Xavier Garneau) < fxgarnea@uqac.ca > 2012, Har Krishan Bhalla & Sons

2 reported in the early sixties by E. Von Rudloff 2. The chemical composition of the essential oil from the same plant material was also determined for comparison purposes. Experimental Plant material Branches of the three species of the Pinaceae family were collected from a plantation belonging to Aliksir located at Grondines in the province of Quebec. The plant material hydro-distilled was composed of branches of ¾ in. in diameter and three to four feet long from 50 year-old trees. Samples were identified and are available at l Herbier Louis-Marie, Laval University, Quebec (Que) Canada. Oil and Hydrosol Extraction The branches were steam-distilled the day after the collection of fresh material cut into sections of two to four inches. Distillation of 312 kg of branches yielded 840 ml of essential oil and 50 kg of hydrosol. 100 ml hydrosol are submitted to extraction using chloroform as solvent. An internal standard, 400 μl (tetradecane/chloroform solution - 0.4:100) was added to the extract. Quantification of the total organic carbon A sample of the Picea glauca hydrosol was submitted to a Total Organic Carbon Analysis (TOC) using the Combustion-Infrared Non-Purgeable Organic (NPOC) method. The instrument used was a Shimadzu TOC-VCPH combustion-infrared TOC analyzer 3. Oil Analysis The refractive index of the oil was measured according to standard methods NF ISO 279 and 280. Essential oils were analyzed by gas chromatography on a HP 5890, equipped with a flame ionisation detector(gc-fid), and two capillary columns: a Supelcowax 10 and a DB-5 column (30 m 0.25 mm 0.25 μm). The oils were also analyzed by gas chromatography, HP 5890, coupled with an HP 5972 mass spectrometer at 70 ev (GC-MS), and equipped either with a DB- 5 or Supelcowax column (same as above). The temperature program for both GC-FID and GC- MS was 40 C for 2 min, then 2 C/min until 210 C and held constant for 33 min. Identification of the components was done by comparison of their retention indices with standards and by comparison of their mass spectra with literature data 4,5, 6 and with our own data bases. Quantitative data were obtained electronically from GC-FID area percentages. The FID response factors for compounds relative to tetradecane were taken as one. Hydrosol Analysis the hydrosol was submitted to GC and GC-MS analyses using the same procedure as that used for the essential oils. Results and discussion The essential oils described in this paper are well known. For example, they are described in Guenther s books 7 and are available in barrel quantities on the market. Dr. Von Rudloff carried out very extensive studies on the oils of Picea glauca (Moench) Voss (White Spruce) 8 and P. mariana (Mill.) B.S.P. (Black Spruce) 2 as well as Abies balsamea (L.) Mill. (Canadian Balsam Fir) 9. Our laboratory studied the influence of sample preparation on the composition of the foliage of Black Spruce 10 as well as the composition of oils from 29 individual Balsam Fir trees 11. On the other hand, papers describing the hydrosol phase are rather rare. Table 1 gives the main characteristics and yields of the hydrosols obtained concurrently with the essential oils from three major trees growing in the Canadian forest. One should point out the acidity of the hydrosols: 3.5 < ph < 4.0. This is in line with data available in the literature 12, 13. The measured quantities of the volatile organic compounds of the hydrosols depend strongly on the species. The concentrations are around 1 gram per litre. Of course these figures must be strongly dependent on the duration of the extraction process: the longer the extraction process, the lower is the concentration of the organic compounds. Again, these figures are in line with those reported in the literature 13, 14. Of course, the hydrosol concentration of the organic compounds is limited by the solubility of each individual compound (see below). In order to verify the quantitative aspect of the analyses of the organic matter in the hydrosols,

3 Table 1. Main characteristics of the hydrosols produced Picea glauca Picea mariana Abies balsamea Foliage quantity (kg) Production of essential oil (ml) Refraction index of the oil Production of hydrosol (kg) ph of hydrosol Organic products in hydrosol (mg/l ±10%) 930* * See text: the response factor is supposed to be unity relatively to the tetradecane reference we compared the Total Organic Carbon (TOC) of a sample of the Picea glauca hydrosol with the measured weight of the residue obtained by extraction and evaporation of another sample of the hydrosol. The main components of the Picea glauca hydrosol are monoterpenes (94 %), mainly ketones and alcohols. Using the normal method involving combustion, it is possible to determine the TOC. The TOC of the hydrosol is 1000 mg/l. This result is equivalent to1266 mg of camphor. All the results appearing in the Tables are obtained by comparison with the tetradecane reference without the introduction of any correction factor. In 2008, an Italian team showed that monooxygenated compounds such as alcohol, ketones, ethers gives a signal that must be corrected by a response factor of 1.3 in comparison with unity for CH monoterpenes and sesquiterpenes molecules 15. Molecules with two oxygen atoms (esters), the response factor if 1.6. These very simple rules can be applied to the results appearing in the Table 1. In this hydrosol, all the compounds except hexanoic acid and exo-2-hydroxycineole acetate (see details later in the Table 3) belong to the monooxygenated molecules. Taking into account the 1.3 response factor identified above, the quantity of volatile compounds is 1209 mg/l rather than 930 mg/l. This calculated value is more in line with those obtained by the TOC measurement. In order to test the validity of the extraction method using a volatile solvent (chloroform) and its evaporation, we weighed the residue obtained with a rotating evaporator under vacuum. The concentration found was 1230 mg/l instead of 1266 mg/l, calculated as camphor equivalents. The concordance of the results confirms the validity of the extraction - evaporation method. The difference between the TOC measured by the usual method involving combustion and the extraction method described above can be explained by several factors such as: 1. Even though more than 65 % of the constituents are monoterpene ketones (10 carbons), the molecular mass of the other constituents including the number of carbons can be different. 2. Part of the organic matter, the more volatile constituents, can be lost during a prolonged evaporation under vacuum. Further evaporation for an hour reduced the weight of the residue to 1000 mg/l. In the case of a hydrosol rich in hexane - type molecules with a higher vapor pressure than the monoterpenes, the loss by evaporation can be facilitated. 3. It should also be recalled that in spite of all the precautions taken, it is possible that traces of organic compounds subsist in the form of an emulsion in the hydrosol. This possibility would artificially increase the quantity of analyzed organic matter even though we have no reason to believe that this possibility would significantly affect the results presented herein. The White Spruce hydrosol The composition of the essential oil and the hydrosol are shown in Table 2 and Table 3. The first observation that can be made from the results shown in these tables is the large difference in the composition of the essential oil and the organic content of the hydrosol. Depending on the relative concentration of one organic compound in the essential oil and its water solubility measured at 25 C

4 16, several cases are possible. For example, more than 99 % of a very hydro soluble product appearing in small quantities in the distillation process will be observed only in the hydrosol. This is the case for furfural and many other components. On the other hand, an almost water insoluble product will be found only in the oil. This is the case for monoterpenes such as limonene and camphene whose water solubility is lower than 20 mg/l. Their concentration will be so low in the hydrosol than they will remain either below or close to the detection limits of the used analytical procedure. In between these two opposite cases, the products are observed in both oil and hydrosol. Examples of this latter case are 1,8-cineole, bornyl acetate, camphor and several alcohols such as linalool, borneol and citronellol. Camphor is particularly interesting since it is a major compound both in the hydrosol (~ 65 %) and in the essential oil (~20 Table 2. Composition of the essential %). oil of white spruce, Picea glauca DB-5 Supelcowax Water Identification KI % area % area KI solubility b Santene Tricyclene α-thujene α-pinene Camphene Sabinene β-pinene Myrcene α-phellandrene δ-3-carene α-terpinene para-cymene Limonene a β-phellandrene a ,8-Cineole x 10 3 γ-terpinene Terpinolene Linalool x 10 3 Camphor x 10 3 Camphene hydrate Borneol a Terpin-1-en-4-ol α-terpineol a Citronellol Piperitone Bornyl acetate Longifolene β-caryophyllene α-humulene γ-cadinene a δ-cadinene a Total a : at least two products have the same retention time. b : in mg/l at 25 C, US National Library of Medicine. 16

5 Table 3. Comparative composition of the hydrosol and essential oil of Picea glauca Hydrosol Essential oil Solubility in Identification a mg/l b % area % area Water b Furfural x 10 4 cis-3-hexen-1-ol Hexanol x 10 3 Unidentified Camphene Hexanoïc acid x 10 4 Limonene ,8-Cineole x 10 3 Benzyl alcohol x 10 3 Benzeneacetaldehyde x trans-linalool oxide (fur.) Camphenilone cis-linalool oxide (fur.) Fenchone Linalool x 10 3 α-campholenal Fenchol-exo Nopinone Camphor x 10 3 Camphene hydrate Isoborneol x 10 3 Borneol Unknown Terpinen-4-ol p-cymen-8-ol α-terpineol Piperitenol Borneol isomer Verbenone trans-carveol Citronellol Piperitone Isopiperitone + unidentified A* Bornyl acetate Unknown Puleganolide (isomer 2) Exo-2-hydroxycineole acetate Methyl eugenol Oplopanone Total a : Traces of hydroxycitronellol and vanillin are also observed in the hydrosol. b : These figures are given using a response factor of unity relatively to the tetradecane reference. See text. c: in mg/l at 25 C, US National Library of Medicine 16. *: m/z: 71(100). 41(20), 122(16), 55(16), 83(12), 182(3),...

6 On a theoretical point of view, we must consider a two phase system: one phase is acidic water and the second phase (the upper one) is a liquid rich in camphor. For any other organic compound its distribution between the two phases is governed by its activity in each phase. Thus, the ratio of the concentrations of one organic compound is given by the ratio of the activities of the same compound in both phases. The solubility of a compound in water at a neutral ph may be different than at a ph between 3.5 and 4. Although very interesting, the water solubility approach is a rather limited one. The Black Spruce and Canadian Balsam Fir hydrosols Similar observations and comparisons can be made between the hydrosols and the essential oils obtained from the foliage of Black Spruce and Canadian Balsam Fir (Table 4). For example, the Canadian Balsam Fir oil (A. balsamea) mainly contains monoterpenes: pinenes (α- and β-) as well as Δ 3 -carene and β-phellandrene. It also contains between 8 and 16 % of esters calculated as bornyl acetate 17. Also, Black Spruce oil (P. mariana) contains 37 to 45 % of esters calculated as bornyl acetate 18 as well as camphene (16 %) and α-pinene (13 %) and several other monoterpenes. All components that have solubility in water higher than 10 g/l are not found in the essential oil. This is the case of aliphatic alcohols such as isoamylic alcohol and pentanol or even hexanol which has a solubility of 5.9 g/l. On the contrary, the C 10 H 16 monoterpenes such as α-pinene, camphene, β-pinene are only present in low concentrations in the hydrosol: their known solubility is less than 10 mg/l. The higher the percentage in the oil, the higher is the concentration in the hydrosol. This is the case for camphene and α-pinene in the Black Spruce hydrosol. Between these two extremes, several compounds have water solubility around 1 g/l. Monoterpenols such as linalool, isoborneol, borneol, and α-terpineol belong to this group of compounds. Bornyl acetate, an ester, has low water solubility, around 23 mg/l. However, due to its high content in the black spruce oil, its concentration in the hydrosol is important (7.8 %). Finally, due to its high water solubility maltol is not observed in the Canadian Fir needle oil. It is an important product of the hydrosol: ~11 %. Industrial aqueous and solvent extracts of the wood of this tree are an important source of this compound 19. Finally, it is valuable to recall a study made on the glycosidically bound volatile compounds of P. mariana. Several of the alcohols present in the hydrosol and not observed in the oil were also observed after hydrolysis of glucosidic compounds through the use of the enzymes β-glucosidase or cellulase 20. This group of alcohols includes cis- 3-hexen-1-ol, hexanol, linalool, benzyl alcohol, perillic alcohol, vanillin, and some aldehydes such as benzaldehyde, α-campholenal, and a ketone: acetovanillone. Table 4. Comparative compositions of the hydrosols and essential oils obtained from two foliages of Pinaceae Picea marianaa Abies balsamea Hydrosol Oil Hydrosol Oil Solubility in Identification mg/l % % mg/l % % water a 3-Hydroxy-2-butanone b x10 6 Isoamylic alcohol b x Methyl-1-butanol b x10 3 Pentanol b x10 3 Dihydro-2-methyl-3(2H)-furanone b tr Penten-3-ol/2-penten-1-ol b x Methyl-2-buten-1-ol b Hexanal x 10 3 Furfural x 10 4

7 table 4. (continued). Picea marianaa Abies balsamea Hydrosol Oil Hydrosol Oil Solubility in Identification mg/l % % mg/l % % water a trans-3-hexen-1-ol cis-3-hexen-1-ol d trans-2-hexen-1-ol Hexanol d x Heptanone x10 3 α-pinene Camphene Benzaldehyde d x10 3 β-pinene Myrcene Hexanoic acid b x10 3 Δ 3 -Carene ,4-Cineole tr Unidentified A e Limonene β-phellandrene tr ,8-Cineole tr tr - tr 3.5 x10 3 Benzyl alcohol d x10 3 Lavender ketone Benzeneacetaldehyde tr x10 3 Acetophenone x10 3 Unidentified B cis-linalool oxide (fur.) trans-linalool oxide (fur.) Unidentified C Unidentified D Terpinolene tr Fenchone tr Linalool d x10 3 Unidentified E Ethyl-2-methyl-2,5-furandione Maltol exo-fenchol Dehydrosabinacetone cis-para-menth-2-en-1-ol α-campholenal d Nopinone trans-pinocarveol trans-para-menth-2-en-1-ol Camphor x 10 3 Isopulegol Camphene hydrate Neo-isopulegol ~ p-mentha-1,5-dien-8-ol isomer Isoborneol x10 3 trans-pinocamphone

8 table 4. (continued). Picea marianaa Abies balsamea Hydrosol Oil Hydrosol Oil Solubility in Identification mg/l % % mg/l % % water a Borneol d p-mentha-1,5-dien-8-ol isomer ~ δ-terpineol p-mentha-1,5-dien-8-ol Isopinocamphone Terpinen-4-ol d m-cymen-8-ol Cryptone p-cymen-8-ol d α-terpineol Myrtenal Myrtenol d Unidentified F Verbenone trans-piperitol trans-carveol cis-carveol Citronellol d Piperitone Geraniol d x10 3 Bornyl acetate trans-linalool oxide acetate (pyr.) Unidentified G Hydroxy-neo-menthol Exo-2-hydroxycineole acetate b Methyl eugenol Vanillin tr - - tr x Hydroxy-4-oplopanone Perillic alcohol Thymol x 10 3 Puleganolide (isomer 1) c Total a : in mg/l at 20/25 C, experimental or estimated values, US National Library of Medicine 16. b : NBS data bank identification 4 ; c : MassFinder3 identification 6. Other products were determined by comparison with at least two MS data banks: one of the two previously mentioned banks and Adams data bank 5. These include I.K. values on DB-5 column indicated either in MassFinder3 or/and in Adams data bank. d : products also observed in the form of glycosidically bound product; see text 19. e : Unidentified compounds - m/z (int.): A: 95(100), 43(61), 123(49), 79(44), 109(40), 138(43), 67(38), 105(34), (77(27), B: 79(100), 93(69), 137(44), 94(42), 43(41), 77(28), 152(22), 91(20), 41(16), C: 71(100), 43(98), 72(98), 67(60), 97(54), 55(50), 79(48),, 150(2), D: 108(100), 93(90), 43(75), 79(55), 67(50), 152(2), E: 79(100), 94(98), 77(23), 91(20, 138(16), 93(16), F: 95(100), 93(55), 121(50), 79(37), 91(35), 77(26), 154(4), G: 94(100), 95(66), 138(30), 70(25), 149(2), 164(1).

9 Use or applications Except for the industrial preparation of maltol, see above, to our knowledge there is no industrial use or application of the hydrosols of coniferous trees although a hot water extract of a soft powder of Pinus halepensis L. was proposed as a hair growth enhancer product 21. This extract may contain VOC and also higher molecular compounds. In one extraction plant in this Province, the hydrosol obtained from Abies balsamea is used to prepare craft made soap. Of course, the use of such hydrosols may be limited by their stability. Current work on their shelf life is under way and will be published in due time. Acknowledgements We would like to express our gratitude to the Aliksir inc., Grondines (Quebec) for providing us with the samples of essential oils and hydrosols. References 1. Frère Marie-Victorin. (1964). Flore Laurentienne, 2 nd Edition, Les Presses de l Université de Montréal, Montreal (Quebec), Canada. pp Von Rudlov, E. (1962). Gas Liquid Chromatography. Part V. The Volatile Oils of the Leaves of Black, White and Colorado Spruce. Tappi.. 45(3): Shimadzu Corporation (2001), PC Controlled Total Organic Carbon Analyzer TOC-VCPH\CPN & TOC Control V Software User Manual. 4. NBS 75 k Data bank. 5. Adams, R.P. (2005). Identification of Essential Oil Components by Gas Chromatography/ Mass Spectrometry, 4 th Edition, Allured Publ. Co., Carol Stream, IL. 6. König, W.A., Hochmuth, D.H., Joulain, D. (2004). Terpenoids and Related Constituents of Essential Oils, Version 3, Hamburg, Germany ( 7. Guenther, E. (1976) The essential Oils, Krieger Publ. Co., Malabar, Florida. Reprint Edition, Vol. 6, pp and Von Rudloff, E. (1967). Chemosystematic Studies in the Genus Picea (Pinaceae). II. The Leaf Oil of Picea glauca and P. mariana. Can. J. Bot. 45: Von Rudloff, E. and Granat, M. (1982). Seasonal Variation of the Terpenes of the Leaves, Buds, and Twigs of Balsam Fir (Abies balsamea). Can. J. Bot. 60: Hachey, J.-M., Collin, G.J. and Simard, S. (1989). Influence of sample preparation on the composition of the essential oil of the needles and twigs of Picea mariana. J. Wood Chem. & Tech. 9: Simard, S., Hachey, J.-M. and Collin, G.J. (1988). The variations of essential oil composition during the extraction process. The case of Thuja occidentalis L. and Abies balsamea (L.) Mill. J. Wood Chem. & Tech. 8: Régimbal, J.M. and Collin, G.J. (1994). Essential oil of Balsam fir, Abies balsamea (L.) Mill. J. Essent. Oil Res. 6: Catty, S. (2001). Hydrosols. The Next Aromatherapy. Healing Arts Press, Rochester, Vermont. 14. Price, L. and Price, S. (2004) Understanding Hydrolats: The Specific Hydrosols for Aromatherapy. Churchill Livinsgtone, London, U.K. 15. Edris, Amr E. (2009) Identification and absolute quantification of the major water-soluble aroma components isolated from the hydrosols of some aromatic plants. J. Essent. Oil Bearing Plants. 12(2): Costa, R.., Zellner, B. d A., Crupi, M.L., De Fina, M.R., Valentino, M.R., Dugo, P., Dugo, G., and Mondello. L. (2008). GC-MS, GC-O and enantio-gc investigation of the essential oil of Tarchonanthus camphoratus L. Flavour Fragr, J. 23: Bicchi, C., Liberto, E., Matteodo, M., Sgorbini, B., Mondello, L., Zellner, B. d A., Costa, R. and Rubiolo, P., (2008). Quantitative analysis of essential oils: a complex task. Flavour Fragr, J.

10 23: US National Library of Medicine, verified on December 14, chemical.html 18. Essential Oil of America, N Essential Oil of America, N For examples: Heintz, D.N., Roos, C.W. and Knowles, W. S. Extracting maltol from bark. US patent199532, Arsenault, R., Trottier, M., Chornet, E. and Jollez, P. Process of aqueous extraction of maltol. US Patent , ; Guzek, D.B., Dickey, K.D and Hausman, R.J. Extraction of maltol from aqueous solutions containing it. US Patent , Garneau, F.-X., Bouhajib, M., Collin, G.J. and Gagnon, M. (1994). The glycosydically bound volatile compounds of Picea mariana (Mill.) B.S.P. J. Essent. Oil Res. 6: El Amri and Alla Eddine, (2007). Hair growth enhancer product comprising coniferous tree extract, patent, accession Number: 148: CA.