Journal of Dietary Supplements, 12(1):1–10, 2015 C 2015 by Informa Healthcare USA, Inc. Available online at www.informahealthcare.com/jds DOI: 10.3109/19390211.2014.887601
ARTICLE
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Potential Oil Yield, Fatty Acid Composition, and Oxidation Stability of the Hempseed Oil from Four Cannabis sativa L. Cultivars Carla Da Porto, Deborah Decorti, & Andrea Natolino Department of Food Science, University of Udine, Udine, Italy
ABSTRACT. The cultivation of four industrial hemp cultivars (Felina 32, Chamaeleon, Uso31, and Finola) was investigated for oil production in the north-east of Italy along two years. The oils of all cultivars resulted in rich amount of linoleic acid (ω-6) and α-linolenic acid (ω-3). Felina 32 and Chamaeleon oils exhibited the highest amount of linoleic acid (59%) and α-linolenic acid (18%). Finola and Uso31 oils resulted in the richest of γ -linolenic acid (5–6%). All hempseed oils presented high oxidation stability and an acceptable initial quality. It is suggested that these oils can be used to produce EFA dietary supplements high in ω-6 and ω-3 of vegetal origin. KEYWORDS. Cannabis sativa L., cultivar, essential fatty acids, hempseed oil, oxidation stability
INTRODUCTION The cultivation of industrial hemp has nothing to do with the cultivation of cannabis plants for the production of cannabis as an intoxicant. The former is legally regulated, and offers many advantages in the agricultural, economic, and ecological field (Acosta Casas & Rieradevall i Pons, 2005; Alexakhin, 1993; Callaway, 2004; Campbell, Paquin, Jonathan, Awaya, & Li, 2002; Kos & Letan, 2004; Lser, Zehnsdorf, Fussy, & Strk, 2002; Mohanty, Misza, & Drzol, 2002; Pate, 1999; Vandenhove & Van Hees, 2005; Van der Werf, 2004). Only varieties of industrial hemp named in the EU Approved Common Catalogue of Cultivars (Regulation EC No 1251/99 and subsequent amendments), can be planted in Europe. These cultivars grown for fiber and seed have less than 0.2% of δ-9-tetrahydrocannabinol (THC) (Regulation EC No. 1124/2008), the principal psychoactive constituent of the cannabis plant. In Italy, hemp cultivation reintroduction took place in 1998 (Circular of the Ministry of Agriculture, 2nd December 1997) on approximately 350 hectares, a very small area considering that 30 years previously Italy was second in the world after Address correspondence to: Department of Food Science, University of Udine, via Sondrio 2/A, 33100 Udine, Italy (E-mail: [email protected])
1
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Da Porto et al.
Russia for the areas under hemp cultivation, and the World’s best for the quality of the products obtained. Traditionally, hemp is grown for its long bast fibers, which are used for textile production (clothing, linen, ropes, and sails). Nowadays, the potential applications of hemp are many including, paper, biodegradable plastics, construction sector, and biofuel. One new opportunity for hemp presently under development is targeted at the hempseed oil market for nutritional or personal care use. Hempseed oil is a rich source of polyunsaturated fatty acids (PUFAs), which are present mainly as linoleic acid and α-linolenic acid. These two fatty acids are known as essential fatty acids (EFAs) because they are essential for humans and other animals for good health but they cannot synthesize them. Essential fatty acids play a part in many metabolic processes and there is evidence to suggest that low levels of essential fatty acids, or the wrong balance of types among the essential fatty acids, may be a factor in a number of illnesses (Albert et al., 2005; Mokuno et al., 1990; Nalini et al., 2008; Riemersma, 2001; Simopoulos & Artemis, 2002; Willett, 2012). Besides, hempseed oil contains tocopherols, which may reduce the risk of cardiovascular diseases, cancer, and age-related macular degeneration (Leger, 2000). Tocopherols act as antioxidants and prevent the oxidation of unsaturated fatty acids. The information on the variability of oil content and fatty acid patterns in hemp is limited (Kriese et al., 2004; Oomah, Busson, Godfey, & Drover, 2002). Based on these considerations, in 2011 and 2012, four cultivar trials of hemp (Cannabis sativa L.), Felina 32, Chamaeleon, Uso31, and Finola, were carried out in two locations of Friuli Venezia-Giulia region (Italy) to evaluate potential oil yields and to investigate the fatty acids composition and oxidation stability of the oils obtained.
MATERIALS AND METHODS Cultivar Trials Certified seeds of hemp variety Felina 32 (French origin), Uso 31 (Ukrainian origin), Chamaeleon (The Netherlands origin), and Finola (Finland origin) were used. The field trials were set up by the University of Udine—Department of Food Science—in Friuli Venezia-Giulia region (Italy), at Prato Carnico (PC) (80 km NO of Udine, latitude 46 N, longitude 12 E, and altitude 856 m above sea level) and Forni di Sotto (FS) (90 km NO of Udine, latitude 46 N, longitude 12 E, and altitude 568 m above sea level) during 2011 and 2012. The morphology of the region is mountainous and is characterized by wet climate, with abundant rainfall mainly concentrated in the autumn and spring (1500–1700 mm per year). Mean air temperatures generally increase from April to July with mean minimum temperature of 5◦ C, and mean maximum temperature of 20◦ C. During summer (July–half August), a dry period generally occurs with low rainfall and high air temperatures often above 25◦ C. The soil has a gravel composition at Prato Carnico, and a loamyclay composition at Forni di Sotto. In 2011 and 2012, fertilization was carried out by organic fertilizers allowed in organic farming. A total of nitrogen (N) 100, phosphorus (P) 30, and potassium (K) 40 kg per hectare were used. The cultivar trials were carried out using a randomized block plot design with four varieties and three
Hemp Seed Oil a Great Health Resource
3
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replicates. Each plot had a size of 4 × 2 m. Seeding rate was 20 kg per hectare with plant density of 40 plants per m2 and row distance was 40 cm. The sowing was generally performed in the last of May. Usually, 5–7 days after sowing, a rapid and uniform emergence of seedlings was obtained. No pesticides were supplied. The variety Finola was the earliest to flower (end of June), while blooming for the other varieties occurred after 40–50 days from sowing. The seeds of Felina 32, Uso31, and Chamaeleon were collected during the second half of September, while those of the variety Finola were collected scalarly. Afterward, the seeds were properly cleaned and dried naturally at constant moisture in a cool, dry, and ventilated room. The final content of moisture of the seeds was about 8% (w/w). After the drying step, hemp seeds were stored at 4◦ C before the extraction step. Analytical Methods Soxhlet Extraction Thirty grams of hemp seeds were ground in a stainless steel blender, transferred into a filter paper extraction thimble, and extracted with 240 ml n-hexane for 8 h at a maximum temperature of 70◦ C in a Soxhlet apparatus. After extraction was completed, n-hexane was removed at 50◦ C under reduced pressure using a rotary evaporator (Rotavapor R210, Buchi, Flawil, Switzerland). Subsequently, the flask was placed into a desiccator chamber for 1 h. The oil obtained was weighed and the yield was calculated. Determination was done in triplicate. GC Analysis of Fatty Acids The fatty acid methyl esters (FAME) were prepared by trans-esterification of oil with 2N KOH in methanol and n-hexane. Gas chromatographic (GC) analysis of FAME were performed in a Varian 3400 gas chromatograph equipped with a SP2380 fused-silica column (Supelco, Bellafonte, PA) (30 m × 0.32 mm i.d., film thickness 0.20 μm), a split injector at 250 ◦ C; flame ionization detector at 260◦ C. Helium was used as carrier gas and the split ratio used was 1:50. The programmed temperature was: 2 min at 50◦ C, 50◦ C–250◦ C at 4◦ C min−1 . The identification of FAME was based on external standards using commercial reference compounds (Sigma Aldrich, Milan, Italy). Each result presents the mean and the standard deviation for a minimum of three analyses. Spectroscopic Indices in the UV Region Spectroscopic indices, K232 and K270 in the UV region, were determined as outlined in the Standard methods for the Analysis of Oils, Fats, and Derivatives (International Union of Pure and Applied Chemistry, 1985). K232 corresponds to a measure of the formation of hydroperoxides due to the linoleic acid oxidation, as long as K270 is indicative of the content in α-diaketones and ketones αβ- unsaturated. Each result presents the mean and the standard deviation for a minimum of three experiments Oxidation Stability Assay The oxidation stability of the hempseed oil samples was evaluated by the total free radical scavenger capacity (RSC) following the methodology described by Esp´ın
4
Da Porto et al.
et al. (Espn, Soler-Rivas, & Wichers, 2000) with slight modification. In brief, 10 μl of ethyl acetate sample solution at different concentrations was added with 1,990 μl of fresh ethyl acetate DPPH solution (93 μM). Then, the mixture was shaken vigorously and left in darkness for 60 min. Finally, the absorbance of the mixture was measured against pure ethyl acetate (blank) at 515 nm using a UV-Visible spectrophotometer (Shimadzu UV 1650, Italia srl). The RSC is the variation of the concentration of DPPH· free radical (CDPPH·,i) previously dissolved in ethyl acetate, after 60 min of reaction with the samples (CDPPH·f ):
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RSC = CDPPH• ,i − CDPPH• ,f .
(1)
The oxidation stability of the hemp seed oil samples was expressed both as RSC (μM) and as α-tocopherol equivalents, the concentration of α-tocopherol solution which gives rise to the same RSC. A calibration curve was built using a series of tocopherols standard solutions in ethyl acetate. All determinations were done in triplicate. Statistical Analysis Statistica 7.0 software (StatSoft Italia srl) was used for statistical analysis for Analysis of variance (ANOVA with Tukey’s HSD multiple comparison) and Correspondence Analysis. RESULTS AND DISCUSSION Cultivar Trials Results reported in Table 1 show significant differences between varieties in terms of seed yield expressed as kg per hectare (kg ha−1 ) and oil yield expressed as% by weight of seeds (%w/w) for both locations (Prato Carnico and Forni di Sotto) and crop years (2011 and 2012). Table 1. Seed yield (kg per hectare) (kg ha−1 ) and oil yield (% by weight of seeds) (% w/w) of the cultivar trials at Prato Carnico and Forni di Sotto in 2011 and 2012. Location Year
Variety
Prato Carnico
2011
Felina 32 Chamaeleon Uso31 Finola
740ba 520b 410b 320a
2012
Felina 32 Chamaeleon Uso31 Finola
630b 230a 370b 220a
Forni di Sotto
Prato Carnico
Forni di Sotto
Seed Yield (kg ha -1) ± 10.3 1120a ± ± 8.5 630a ± ± 11.3 530a ± ± 4.3 150b ±
9.3 8.1 5.8 6.3
20.7b 25.9b 25.7b 28.4b
± ± ± ±
Oil Yield (% w/w) 0.22 30.7a 0.38 29.9a 0.27 29.7a 0.29 30.1a
± ± ± ±
0.32 0.21 0.31 0.41
± ± ± ±
9.8 2.5 5.9 3.1
20.8b 21.5b 20.6a 24.5b
± ± ± ±
0.25 0.23 0.19 0.27
29.5a 27.8a 19.5b 26.3a
± ± ± ±
0.31 0.35 0.24 0.25
8.8 3.2 6.1 3.1
1040a 200b 398a 100b
± ± ± ±
Each data represents the mean of three replicates ± standard deviation. a Values with different letter within rows indicate significant differences (p β> α-tocopherol. Gamma-tocopherol was reported to be the predominant tocopherol in hempseed oil (Jones, 1995; Ramadan & Mo-
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Da Porto et al.
Figure 2. Results of correspondence analysis between cultivars and hempseed 1 oils fatty acid composition.
ersel, 2006; Yu, Zhou, & Parry, 2005). This could explain the high oxidation stability detected for all hempseed oils. Table 3 shows the growing season effect on fatty acid composition, spectroscopic indices, and oxidation stability of hempseed oils. In 2012, the unfavorable climatic conditions that occurred after sowing did not affect the fatty acid composition of the oils. In fact, each cultivar was found to give oil characterized by its own fatty ˘ Bruehl, Aitzetmuller, & Altan, 2003) acid profile, genetically determined (Bagci, as shown in Figure 2. However, the high values of the oxidation stability and K232 and K270 highlighted that hempseed oils underwent no oxidative damage.
CONCLUSIONS Cultivar trials carried out for hempseed oil production showed that Felina 32 was the most suitable cultivar for the pedoclimatic conditions of Carnia region. Hempseed oil from Felina 32 and Chamaeleon varieties exhibited the highest amount of EFAs, while Uso31 and Finola resulted as the richest in γ -linolenic acid. Felina 32 and Chamaeleon cultivar could be used to obtain oil helpful as dietary source of EFAs for health promotion and disease prevention. Instead, Uso31 and Finola cultivar could be used to obtain oil rich of γ -linolenic acid and so particularly suitable as ingredient for light body oils and lipid-enriched creams. The oils of all cultivars could be used to produce EFA dietary supplements of vegetal origin, alternative to fish oil supplements, potentially dangerous for problems of contamination toxins such as mercury, dioxins, and polychlorinated biphenyls (PCBs). Declaration of interest: The authors report no conflict of interest. The authors alone are responsible for the content and writing of this paper.
Hemp Seed Oil a Great Health Resource
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ABOUT THE AUTHORS Carla Da Porto, Dr., is Assistant Professor at the Department of Food Science, University of Udine, Italy. Deborha Decorti Postdoctoral Researcher at Department of Food Science, University of Udine, Italy. Andrea Natolino, is a PhD student at Department of Food Science, University of Udine, Italy.
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REFERENCES Acosta Casas X, Rieradevall i Pons J. Environmental analysis of the energy use of hemp—analysis of the comparative life cycle: diesel oil vs. hemp–diesel. Int J Agric Resour Gov Ecol. 2005;4:133–139. Albert CM, Oh K, Whang W, Manson JAE. Dietary α-linolenic acid intake and risk of sudden cardiac death and coronary heart disease. Circulation. 2005;112:3232–3238 doi: 10.1161 Alexakhin RM. Countermeasures in agricultural production as an effective means of mitigating the radiological consequences of the Chernobyl accident. Sci Total Environ. 1993;137:9–20 ˘ E, Bruehl L, Aitzetmuller K, Altan YA. Chemotaxonomic approach to the fatty acid and Bagci tocochromanol content of Cannabis sativa L. (Cannabaceae). Turk J Bot. 2003;27:141–147. Callaway JC. Hempseed as a nutritional resource: An overview. Euphytica. 2004;140:65–72 ¨ ¨ ¨ Callaway J, Schwab U, Harvima I, Halonen P, Mykkanen O, Hyvonen P, Jarvinen T. Efficacy of dietary hempseed oil in patients with atopic dermatitis. J Dermatol Treat. 2005;16:87–94. Callaway JC, Tennila T, Pate DW. Occurrence of “omega-3” stearidonic acid (cis-6,9,12,15- octadecatetraenoic acid) in hemp (Cannabis sativa L.) seed. J Int Hemp Assoc. 1996;3:61–63. Campbell S, Paquin D, Jonathan D, Awaya JD, Li QX. Remediation of Benzoa.pyrene and chrysene-contaminated soil with industrial hemp (Cannabis sativa). Int J Phytoremediation. 2002;4:157–168 Esp´ın JC, Soler-Rivas C, Wichers HJ. Characterization of the total free radical scavenger capacity of vegetable oils and oil fractions using 2, 2-diphenyl-1-picrylhydrazyl radical. J Agric Food Chem. 2000;48:648–656 Huang Y-S, Ziboh A. Gamma-linolenic acid: recent advances in biotechnology and clinical applications. Champaign, IL: AOCS Press, 2001. International Union of Pure and Applied Chemistry. Standard methods for the analysis of oils, fats and derivatives (7th ed.). Oxford, UK: Author, 1985 Jones K. Nutritional and medicinal guide to hemp seed. Gibsons, BC, Canada: Rainforest Botanical Laboratory, 1995. Kos B, Leˇstan D. Soil washing of Pb, Zn and Cd using biodegradable chelator and permeable barriers and induced phytoextraction by Cannabis sativa. Plant and Soil. 2004;263:3–51 ¨ L, Matthaus ¨ B. Oil content, tocopherol comKriese U, Schuman E, Weber WE, Beyer M, Bruhl position and fatty acid patterns of the seeds of 51 Cannabis sativa L. genotypes. Euphytica. 2004;137:339–351. Leger CL. La vitamine E: etat actuel des connaissances, role dans la prevention cardiovasculaire, biodisponibilite. Oleagineux Corps Gras Lipide 2000;7:235–265. ¨ ¨ HJ. Conditioning of heavy metal polluted river sediment Loser C, Zehnsdorf A, Fussy M, Stark by Cannabis sativa L. Int J Phytoremediation. 2002;4:27–45. Mediavilla V, Bassetti P, Leupin M. Agronomic characteristics of some hemp genotypes. J Int Hemp Assoc. 1999;6:45–53 Meijer WJM, Vanderwerf HMG, Mathijssen E, Vandenbrink PWM. Constraints to dry-matter production in fiber hemp (Cannabis sativa L). Eur J Agron. 1995;4:109–117. Mohanty AK, Misza M, Drzol LT. Sustainable bio-composites from renewable resources: opportunities and challenges in the green materials world. J Polym Environ. 2002;10, 19–26 Mokuno H, Yamada N, Sugimoto T, Kobayashi T, Ishibashi S, Shimano H, et al. Cholesterolfree diet with a high ratio of polyunsaturated to saturated fatty acids in heterozygous familial hypercholesterolemia: significant lowering effect on plasma cholesterol. Horm Metab Res. 1990;4:246–251
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Nalini K, Kreml RJ, Austria A, Richard MN, Edel AL, Dibrov E, Hirono S, Zettler ME. Supplementation on selected parameters of cardiovascular health in healthy volunteers. J Am Coll Nutr. 2008;27:51–58. Oomah BD, Busson M, Godfey DV, Drover JCG. Characteristics of hemp (Cannabis sativa L.) seed oil. Food Chem. 2002;76:33–43 Pate DW. Hemp seed: a valuable food source. In P Ranalli (Ed), Advances in hemp research (pp. 243–255). Binghamton, NY: The Haworth Press, 1999. ISBN 978-1-56022-872-9, Ramadan MF, Moersel JT. Screening of the antiradical action of vegetable oils. J Food Comp Anal. 2006;19:838–842 Riemersma RA. The demise of the n-6 to n-3 fatty acid ratio? A dossier. Eur J Lipid Sci Technol. 2001;103:372–373. Ruggeri S, Cappelloni M, Gambelli L, Nicoli S, Carnovale E. Chemical composition and nutritive value of nuts grown in Italy. Ital J Food Sci. 1998;10:243–252. ˇ Pokorny J. Potential application of oilseeds as sources of antioxidants for food Schmidt S, lipids—a review. Czech J Food Sci. 2005;23, 93–102. ¨ Schuster WH. Olpflanzen in Europa. DLG-Verlag, Frankfurt/Main, Germany, 1992. Simopoulos C, Artemis P. The importance of the ratio of omega-6/omega-3 essential fatty acids. Biomed Pharmacother. 2002;56:365–379 Vandenhove H, Van Hees M. Fibre crops as alternative land use for radioactively contaminated arable land. J Environ Radioact. 2005;81:131–141. Van der Werf HMG. Life cycle analysis of field production of fibre hemp, the effect of production practices on environmental impacts. Euphytica. 2004;1–2:13–23. Vogl R, Lissek-Wolf G, Surbock A. Comparing hemp seed yields (Cannabis sativa L.) Of an onfarm scientific field experiment to an on-farm agronomic evaluation under organic growing conditions in lower Austria. J Ind Hemp. 2004;9:37–49. Willett WC. Dietary fats and coronary heart disease. J Int Med. 2012;272:13–14 Yu L, Zhou KK, Parry J. Antioxidant properties of cold-pressed black caraway, carrot, cranberry, and hemp seed oils. Food Chem. 2005;91:723–729
ARTICLE
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Potential Oil Yield, Fatty Acid Composition, and Oxidation Stability of the Hempseed Oil from Four Cannabis sativa L. Cultivars Carla Da Porto, Deborah Decorti, & Andrea Natolino Department of Food Science, University of Udine, Udine, Italy
ABSTRACT. The cultivation of four industrial hemp cultivars (Felina 32, Chamaeleon, Uso31, and Finola) was investigated for oil production in the north-east of Italy along two years. The oils of all cultivars resulted in rich amount of linoleic acid (ω-6) and α-linolenic acid (ω-3). Felina 32 and Chamaeleon oils exhibited the highest amount of linoleic acid (59%) and α-linolenic acid (18%). Finola and Uso31 oils resulted in the richest of γ -linolenic acid (5–6%). All hempseed oils presented high oxidation stability and an acceptable initial quality. It is suggested that these oils can be used to produce EFA dietary supplements high in ω-6 and ω-3 of vegetal origin. KEYWORDS. Cannabis sativa L., cultivar, essential fatty acids, hempseed oil, oxidation stability
INTRODUCTION The cultivation of industrial hemp has nothing to do with the cultivation of cannabis plants for the production of cannabis as an intoxicant. The former is legally regulated, and offers many advantages in the agricultural, economic, and ecological field (Acosta Casas & Rieradevall i Pons, 2005; Alexakhin, 1993; Callaway, 2004; Campbell, Paquin, Jonathan, Awaya, & Li, 2002; Kos & Letan, 2004; Lser, Zehnsdorf, Fussy, & Strk, 2002; Mohanty, Misza, & Drzol, 2002; Pate, 1999; Vandenhove & Van Hees, 2005; Van der Werf, 2004). Only varieties of industrial hemp named in the EU Approved Common Catalogue of Cultivars (Regulation EC No 1251/99 and subsequent amendments), can be planted in Europe. These cultivars grown for fiber and seed have less than 0.2% of δ-9-tetrahydrocannabinol (THC) (Regulation EC No. 1124/2008), the principal psychoactive constituent of the cannabis plant. In Italy, hemp cultivation reintroduction took place in 1998 (Circular of the Ministry of Agriculture, 2nd December 1997) on approximately 350 hectares, a very small area considering that 30 years previously Italy was second in the world after Address correspondence to: Department of Food Science, University of Udine, via Sondrio 2/A, 33100 Udine, Italy (E-mail: [email protected])
1
J Dietary Suppl Downloaded from informahealthcare.com by McMaster University on 03/13/15 For personal use only.
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Da Porto et al.
Russia for the areas under hemp cultivation, and the World’s best for the quality of the products obtained. Traditionally, hemp is grown for its long bast fibers, which are used for textile production (clothing, linen, ropes, and sails). Nowadays, the potential applications of hemp are many including, paper, biodegradable plastics, construction sector, and biofuel. One new opportunity for hemp presently under development is targeted at the hempseed oil market for nutritional or personal care use. Hempseed oil is a rich source of polyunsaturated fatty acids (PUFAs), which are present mainly as linoleic acid and α-linolenic acid. These two fatty acids are known as essential fatty acids (EFAs) because they are essential for humans and other animals for good health but they cannot synthesize them. Essential fatty acids play a part in many metabolic processes and there is evidence to suggest that low levels of essential fatty acids, or the wrong balance of types among the essential fatty acids, may be a factor in a number of illnesses (Albert et al., 2005; Mokuno et al., 1990; Nalini et al., 2008; Riemersma, 2001; Simopoulos & Artemis, 2002; Willett, 2012). Besides, hempseed oil contains tocopherols, which may reduce the risk of cardiovascular diseases, cancer, and age-related macular degeneration (Leger, 2000). Tocopherols act as antioxidants and prevent the oxidation of unsaturated fatty acids. The information on the variability of oil content and fatty acid patterns in hemp is limited (Kriese et al., 2004; Oomah, Busson, Godfey, & Drover, 2002). Based on these considerations, in 2011 and 2012, four cultivar trials of hemp (Cannabis sativa L.), Felina 32, Chamaeleon, Uso31, and Finola, were carried out in two locations of Friuli Venezia-Giulia region (Italy) to evaluate potential oil yields and to investigate the fatty acids composition and oxidation stability of the oils obtained.
MATERIALS AND METHODS Cultivar Trials Certified seeds of hemp variety Felina 32 (French origin), Uso 31 (Ukrainian origin), Chamaeleon (The Netherlands origin), and Finola (Finland origin) were used. The field trials were set up by the University of Udine—Department of Food Science—in Friuli Venezia-Giulia region (Italy), at Prato Carnico (PC) (80 km NO of Udine, latitude 46 N, longitude 12 E, and altitude 856 m above sea level) and Forni di Sotto (FS) (90 km NO of Udine, latitude 46 N, longitude 12 E, and altitude 568 m above sea level) during 2011 and 2012. The morphology of the region is mountainous and is characterized by wet climate, with abundant rainfall mainly concentrated in the autumn and spring (1500–1700 mm per year). Mean air temperatures generally increase from April to July with mean minimum temperature of 5◦ C, and mean maximum temperature of 20◦ C. During summer (July–half August), a dry period generally occurs with low rainfall and high air temperatures often above 25◦ C. The soil has a gravel composition at Prato Carnico, and a loamyclay composition at Forni di Sotto. In 2011 and 2012, fertilization was carried out by organic fertilizers allowed in organic farming. A total of nitrogen (N) 100, phosphorus (P) 30, and potassium (K) 40 kg per hectare were used. The cultivar trials were carried out using a randomized block plot design with four varieties and three
Hemp Seed Oil a Great Health Resource
3
J Dietary Suppl Downloaded from informahealthcare.com by McMaster University on 03/13/15 For personal use only.
replicates. Each plot had a size of 4 × 2 m. Seeding rate was 20 kg per hectare with plant density of 40 plants per m2 and row distance was 40 cm. The sowing was generally performed in the last of May. Usually, 5–7 days after sowing, a rapid and uniform emergence of seedlings was obtained. No pesticides were supplied. The variety Finola was the earliest to flower (end of June), while blooming for the other varieties occurred after 40–50 days from sowing. The seeds of Felina 32, Uso31, and Chamaeleon were collected during the second half of September, while those of the variety Finola were collected scalarly. Afterward, the seeds were properly cleaned and dried naturally at constant moisture in a cool, dry, and ventilated room. The final content of moisture of the seeds was about 8% (w/w). After the drying step, hemp seeds were stored at 4◦ C before the extraction step. Analytical Methods Soxhlet Extraction Thirty grams of hemp seeds were ground in a stainless steel blender, transferred into a filter paper extraction thimble, and extracted with 240 ml n-hexane for 8 h at a maximum temperature of 70◦ C in a Soxhlet apparatus. After extraction was completed, n-hexane was removed at 50◦ C under reduced pressure using a rotary evaporator (Rotavapor R210, Buchi, Flawil, Switzerland). Subsequently, the flask was placed into a desiccator chamber for 1 h. The oil obtained was weighed and the yield was calculated. Determination was done in triplicate. GC Analysis of Fatty Acids The fatty acid methyl esters (FAME) were prepared by trans-esterification of oil with 2N KOH in methanol and n-hexane. Gas chromatographic (GC) analysis of FAME were performed in a Varian 3400 gas chromatograph equipped with a SP2380 fused-silica column (Supelco, Bellafonte, PA) (30 m × 0.32 mm i.d., film thickness 0.20 μm), a split injector at 250 ◦ C; flame ionization detector at 260◦ C. Helium was used as carrier gas and the split ratio used was 1:50. The programmed temperature was: 2 min at 50◦ C, 50◦ C–250◦ C at 4◦ C min−1 . The identification of FAME was based on external standards using commercial reference compounds (Sigma Aldrich, Milan, Italy). Each result presents the mean and the standard deviation for a minimum of three analyses. Spectroscopic Indices in the UV Region Spectroscopic indices, K232 and K270 in the UV region, were determined as outlined in the Standard methods for the Analysis of Oils, Fats, and Derivatives (International Union of Pure and Applied Chemistry, 1985). K232 corresponds to a measure of the formation of hydroperoxides due to the linoleic acid oxidation, as long as K270 is indicative of the content in α-diaketones and ketones αβ- unsaturated. Each result presents the mean and the standard deviation for a minimum of three experiments Oxidation Stability Assay The oxidation stability of the hempseed oil samples was evaluated by the total free radical scavenger capacity (RSC) following the methodology described by Esp´ın
4
Da Porto et al.
et al. (Espn, Soler-Rivas, & Wichers, 2000) with slight modification. In brief, 10 μl of ethyl acetate sample solution at different concentrations was added with 1,990 μl of fresh ethyl acetate DPPH solution (93 μM). Then, the mixture was shaken vigorously and left in darkness for 60 min. Finally, the absorbance of the mixture was measured against pure ethyl acetate (blank) at 515 nm using a UV-Visible spectrophotometer (Shimadzu UV 1650, Italia srl). The RSC is the variation of the concentration of DPPH· free radical (CDPPH·,i) previously dissolved in ethyl acetate, after 60 min of reaction with the samples (CDPPH·f ):
J Dietary Suppl Downloaded from informahealthcare.com by McMaster University on 03/13/15 For personal use only.
RSC = CDPPH• ,i − CDPPH• ,f .
(1)
The oxidation stability of the hemp seed oil samples was expressed both as RSC (μM) and as α-tocopherol equivalents, the concentration of α-tocopherol solution which gives rise to the same RSC. A calibration curve was built using a series of tocopherols standard solutions in ethyl acetate. All determinations were done in triplicate. Statistical Analysis Statistica 7.0 software (StatSoft Italia srl) was used for statistical analysis for Analysis of variance (ANOVA with Tukey’s HSD multiple comparison) and Correspondence Analysis. RESULTS AND DISCUSSION Cultivar Trials Results reported in Table 1 show significant differences between varieties in terms of seed yield expressed as kg per hectare (kg ha−1 ) and oil yield expressed as% by weight of seeds (%w/w) for both locations (Prato Carnico and Forni di Sotto) and crop years (2011 and 2012). Table 1. Seed yield (kg per hectare) (kg ha−1 ) and oil yield (% by weight of seeds) (% w/w) of the cultivar trials at Prato Carnico and Forni di Sotto in 2011 and 2012. Location Year
Variety
Prato Carnico
2011
Felina 32 Chamaeleon Uso31 Finola
740ba 520b 410b 320a
2012
Felina 32 Chamaeleon Uso31 Finola
630b 230a 370b 220a
Forni di Sotto
Prato Carnico
Forni di Sotto
Seed Yield (kg ha -1) ± 10.3 1120a ± ± 8.5 630a ± ± 11.3 530a ± ± 4.3 150b ±
9.3 8.1 5.8 6.3
20.7b 25.9b 25.7b 28.4b
± ± ± ±
Oil Yield (% w/w) 0.22 30.7a 0.38 29.9a 0.27 29.7a 0.29 30.1a
± ± ± ±
0.32 0.21 0.31 0.41
± ± ± ±
9.8 2.5 5.9 3.1
20.8b 21.5b 20.6a 24.5b
± ± ± ±
0.25 0.23 0.19 0.27
29.5a 27.8a 19.5b 26.3a
± ± ± ±
0.31 0.35 0.24 0.25
8.8 3.2 6.1 3.1
1040a 200b 398a 100b
± ± ± ±
Each data represents the mean of three replicates ± standard deviation. a Values with different letter within rows indicate significant differences (p β> α-tocopherol. Gamma-tocopherol was reported to be the predominant tocopherol in hempseed oil (Jones, 1995; Ramadan & Mo-
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Da Porto et al.
Figure 2. Results of correspondence analysis between cultivars and hempseed 1 oils fatty acid composition.
ersel, 2006; Yu, Zhou, & Parry, 2005). This could explain the high oxidation stability detected for all hempseed oils. Table 3 shows the growing season effect on fatty acid composition, spectroscopic indices, and oxidation stability of hempseed oils. In 2012, the unfavorable climatic conditions that occurred after sowing did not affect the fatty acid composition of the oils. In fact, each cultivar was found to give oil characterized by its own fatty ˘ Bruehl, Aitzetmuller, & Altan, 2003) acid profile, genetically determined (Bagci, as shown in Figure 2. However, the high values of the oxidation stability and K232 and K270 highlighted that hempseed oils underwent no oxidative damage.
CONCLUSIONS Cultivar trials carried out for hempseed oil production showed that Felina 32 was the most suitable cultivar for the pedoclimatic conditions of Carnia region. Hempseed oil from Felina 32 and Chamaeleon varieties exhibited the highest amount of EFAs, while Uso31 and Finola resulted as the richest in γ -linolenic acid. Felina 32 and Chamaeleon cultivar could be used to obtain oil helpful as dietary source of EFAs for health promotion and disease prevention. Instead, Uso31 and Finola cultivar could be used to obtain oil rich of γ -linolenic acid and so particularly suitable as ingredient for light body oils and lipid-enriched creams. The oils of all cultivars could be used to produce EFA dietary supplements of vegetal origin, alternative to fish oil supplements, potentially dangerous for problems of contamination toxins such as mercury, dioxins, and polychlorinated biphenyls (PCBs). Declaration of interest: The authors report no conflict of interest. The authors alone are responsible for the content and writing of this paper.
Hemp Seed Oil a Great Health Resource
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ABOUT THE AUTHORS Carla Da Porto, Dr., is Assistant Professor at the Department of Food Science, University of Udine, Italy. Deborha Decorti Postdoctoral Researcher at Department of Food Science, University of Udine, Italy. Andrea Natolino, is a PhD student at Department of Food Science, University of Udine, Italy.
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