Nutritive quality of romanian hemp varieties (Cannabis sativa L.) with special focus on oil and metal contents of seeds
© Mihoc et al.; licensee Chemistry Central Ltd. 2012
Received: 22 July 2012
Accepted: 12 October 2012
Published: 23 October 2012
The study aims to determine the nutritional value of hemp seed expressed by the oil content and by the concentration of metals (Ca, Mg, K, Fe, Mn, Zn and Cd), for five varieties of monoecious and dioecious hemp seeds approved in Romania, comparative with the concentration of these metals in the soil.
The content of oil in hempseed registers a slight decrease in the production records of 2011, losses due to drought and low levels of precipitation during the growth period. The greatest loss is found in Diana monoecious variety (26.54-20.82%) followed by Zenit varieties (27.37-22.97%), Armanca (29.27-25.32%), Silvana (28.89-25.04%) and Denise (26.96-25.30%). Siccative hemp oil has a yellowish green color and an iodine index of 140–156 g I2/100 g oil. Hemp seed are rich in mineral based Ca (144–955 mg/100 g seed), Mg (237–694 mg/100 g seed), K (463–2821 mg/100 g seed), Fe (1133-2400 mg.kg-1), Mn (63–110 mg.kg-1) and Zn (42-94 mg.kg-1). For the soil the following macroelements concentrations were determined: Ca (2100–2520 mg.kg-1), Mg (320–376 mg.kg-1) and K (232–257 mg.kg-1). Mn (156–197 mg.kg-1) and Zn (54–67 mg.kg-1) remain within normal limits for Romania. The soils in the experience area contain large amounts of Fe (19000–20430 mg.kg-1). The presence of K in large quantities determines the accumulation of large quantities of Fe in the soil.
Hempseed belonging to the five Romanian varieties are rich source of nutrients (Ca, Mg, K) and unsaturated oil easily digestible by the body, but the presence of Cd concentrations above the upper limit puts a question mark over the use of seeds in various food products. Hemp extracts easily certain metals from the soil. Significant amounts of Fe (1133–2400 mg.kg-1), Mn (63–110 mg.kg-1), Zn (42–94 mg.kg-1) and Cd (1.3-4.0 mg.kg-1) are found in hemp seeds. Hemp (Cannabis sativa L.) is included among plants suitable for phytoremediation of soil contaminated with cadmium, zinc and iron.
In recent years, the desire to adopt a healthy diet, draws attention to hempseed and food products derived from hempseed (oil, flour, milk, bakery products, chocolate, beer, etc.). Hemp (Cannabis sativa L.) is a highly variable crop with varieties both for fiber and oil. Hemp seeds and hemp oil have a high nutritional value and both are used for human consumption and animal feeding [1–3]. The nutritional value of hemp seeds is based on the content of protein and oil. Hemp oil is particularly valuable for the health because of its content in fatty acid, mostly being unsaturated acids, completed by high contents of phytosterols, but the properties of hemp oil are still little known in Romania [4–6]. The high price and reduced duration of keeping of the hemp oil translate in a low interest by the people for this alimentary supplement. Reduced supplies and the desire to get rich fast can bring a forged hemp oil on the market with an oil like the rapeseed oil. The authentication of the fatty acid in vegetable oils requires chromatography technique or FT-IR spectroscopy [7, 8]. The 3:1 perfect ratio of omega 6 and omega 3 brings results as treatment for various diseases such as cardiovascular problems, reduction of fibrinogen (in atherosclerosis), in the production of prostaglandins series 3 (PG3) [9, 10]. The reduction of the cholesterol is a major benefit of β-sitosterols present in the unsaponifiable of the vegetable oils like rapeseed, soybean and the hemp oil . This acid γ-linolenic acid (GLA; 18:3ω6) in unsaturated oils such as hemp, flax or evening primrose oil refreshes and moisturizes the skin even with anti aging effects . Fat calories in your diet should not exceed 2500 calories/day. The daily 9–18 g/day LA (18:2 ω6) and 6–7 g/day LNA (18:3 ω3) provides the optimum fatty acid ration the body requires [9, 13].
Hemp is an excellent soil phytoremediation agent because it extracts heavy metals [14, 15]. The use of hemp seeds in various derived foods and the consumption of supplements of hemp seeds bring attention to the potential negative effects of heavy metals possibly contained. Heavy metals are present naturally in soil in low concentrations. The presence of heavy metals in soil, as the natural background from pedogenetic processes or from human activities brings the current need to analyze their content in soil, water, air, crops and food. The capacity to absorb heavy metals depends on the geochemical characteristics of the soil and the nature of crops, some of them have a high potential to accumulate higher concentrations of heavy metals . However, most crop plants can not survive on soils heavily contaminated with heavy metals .
Plants have different storage centers in vegetative and reproductive organs. Leaves can contain large quantities of metal, while the seeds have low metal amounts . Thus, cereal grains accumulate low amounts of metals, while vegetables accumulate higher amounts especially in leaves [18, 19]. Heavy metals are related to certain components of cell membranes, enzyme systems in the body or have the role of catalyst. Nutritional deficiency caused by lack of metals in food or the amount of metals exceeding a certain threshold, produces toxic effects. The implementation of procedures and control mechanism imposed by national and international regulations reduce considerably the maximum levels of toxic metals in food.
Hemp seeds are major sources of magnesium necessary for the body. Also, they bring a substantial contribution of phosphorus, iron and manganese . Hemp (Cannabis sativa L.) along with rape or spinach is among the plants appropriate for decontamination of soil contaminated with Zn and Cd . Hemp mainly extracts Ni concentrating it in leaves, not so much in the seeds . Hemp is great consumer of nutrients (N, P, K), because during the growing season it develops a large vegetative mass and accumulates 70-75% of the dry matter in the first period of vegetation. The lignification process of the stem and the increase in resistance of the plant requires significant calcium intake. Potassium is also a key nutrient to ensure resistance to cold, drought and pests, but primary for the initial stage of seed formation. Magnesium is a component of oil and it contributes to the formation of hemp seeds [22–24]. Magnesium deficiency induces leaf chlorosis and the plants are small.
Manganese is an essential element in the activation of plant enzymes, it increases hemp plants resistance to drought and cold and helps to assimilate nitrogen from the soil. Increasing the concentration of manganese in the soil over 1500–3000 mg.kg-1 induce over 300–350 mg.kg-1 in the plant as a result of fertilization or due to pollution sources such as sewage sludges [24–26]. Excess manganese is found in acid soils (pH less than 5.5), with low organic matter and excess water. Iron is another plant nutrient involved in nitrogen metabolism and photosynthesis. The concentration of iron minerals in different layers of soil is ranged between 100–100000 μg∙g-1. Fe and Mn intake by hemp seed must be within the upper tolerable daily intakes (UL) level of 45–11 mg/day/person . Zinc is the cofactor of many enzymes, proteins and influences the electronic transfer in Krebss cycle reactions affecting the plant’s energy production. In accordance with the zinc transfer coefficient from soil to plants of 1–10 , the daily intake required by the plant is 70 mg/g Zn, and the critic one is 170 mg/g for soil containing 50–100 mg/g . The recommended daily intake for adults is 8–11 mg Zn/day and an excess reduces the levels of high density lipoproteins and reduces the immune function . Cadmium in soil poses a risk both for human receptors, as well as for the ecological ones, because of its relatively high toxicity and uptake in plants. Cd binds rapidly to the intra and extra cellular proteins, leading to the rupture of cell membranes and to the stoppage of the cells functions. As the most important natural cadmium sources we can mention integral cereal, potatoes and seafood. As a result maximum admitted cadmium content in cereal is 100 μg.kg-1 and in medicinal plants 300 μg.kg-1. The medium Cd content in soil is of 0.5-0.8 mg.kg-1, and the maximum limit admitted for the total content of Cd tolerated by the plants is between 3–5 mg.kg-1, while in Romania the maximum concentration of Cd allowed in soil is 1 mg.kg-1[8, 29, 31, 32].
Results and discussion
Oil content and fatty acids from whole hemp seeds
Oil content * (%) in hemp seed varieties and iodine value
Oil content % (w/w)
27.37 ± 0.11
26.54 ± 0.22
26.96 ± 0.06
29.27 ± 0.37
28.89 ± 0.08
22.97 ± 0.96
20.82 ± 0.85
25.30 ± 0.14
25.32 ± 1,21
25.04 ± 0.34
140.5 ± 1.50
141.3 ± 1.24
144.4 ± 1.41
155.5 ± 1.62
150.6 ± 1.36
(g of I 2 / 100 g of oil)
Iodine value from the literature (g of I 2 / 100 g of oil)
Metal content in soil and hemp seeds
Content of Ca, Mg, K and heavy metals (Fe, Mn, Zn and Cd) of gleyed chernozem moderately low on hemp cultivation area dioecious and monoecious hemp
Gleyed chernozem moderately weak dioecious hemp cultivation area
Gleyed chernozem moderately weak monoecious hemp cultivation area
Normal metal contents, for Romania (NC)*mgKg-1
Using the F test (F(4,24)=26.5, p<0.05, F5%=2.78) it can be observed that the variety and the fertilization interaction with the variety (F(8,24)=4.1, p<0.05, F5%=2.34) determines significant differences in the Ca content from the studied hemp seeds.
Metal content for five varieties hemp seed under influence fertilization and seeding distance (mg.kg -1 )
Average –factor B
Average factor A
maximum allowable limits MAL- Ca unregulated
Average factor A
maximum allowable limits MAL – Mg unregulated
Average factor A
maximum allowable limits MAL- K unregulated
Average factor A
maximum allowable limits MAL- Fe unregulated
Average factor A
maximum allowable limits MAL-Mn 300–350 mg.kg-1
Average factor A
maximum allowable limits MAL- Zn 170 mg.kg-1
Average factor A
maximum allowable limits MAL –Cd 0.300 mg.kg-1
Magnesium is a component of several primary and secondary minerals in the soil, which are essentially insoluble, for agricultural considerations. These materials are the original sources of the soluble or available forms of Mg. Magnesium is also present in relatively soluble forms, and is found in ionic form (Mg++) retained to the soil colloidal complex. The ionic form is considered to be available to crops. Being a major cation, Mg availability is related to the soil CEC, and it is in competition with other major cations such as calcium (Ca++), potassium (K+), sodium (Na+), ammonium (NH4+), iron (Fe++), and aluminum (Al+++). It appears that potassium is a stronger competitor with Mg than it is sometimes considered to be. Cambic chernozem on which experiments were made has good magnesium supply, ranged between 320–376 mg.kg-1. Ideal soil Ca:Mg ratio range between 5:1 and 8:1. Like in calcium case, Zenit variety, Armanca variety at 50 cm and Silvana variety at 20 cm had a higher magnesium content after fertilization with Fertileader Viti B:P:K=1:6:12. Diana variety, Denise variety at 50 cm and Silvana variety at 50 cm have the highest magnesium content in unfertilized variant. Although soil has a good magnesium supply, its uptake could be disturbed by calcium and potassium presence in soil. Application of Corona K (N:P:K=8:11:39) fertilizer lead to highest magnesium content in hempseed Denise and Armanca variety at 20 cm (Figure 1).
The statistical processing by the ANOVA method of the Mg results generated in the trifactorial experience with three repetitions shows the effects of the variety on the Mg content in the seeds. The average Mg content in the Silvana seeds is significantly higher thant the concentration in the Diana, Denise and Armanca varieties. Changing the treatment and the seeding spacing doesn’t produce significant differences of the Mg concentration in hemp seeds.
The experimental values F calculated for the separate action of the factors and their interaction in this experiment proves the fact that the variety (F(4,24)=17.8, p<0.05; F1% =4.22) and the variety’s interaction with the seeding distance (F(4,30)=4.4, p<0.05; F1% =4.02) have a significant influence on the K content from the analyzed seed samples.
After the use of the Tukey test the differences of the K concentration are considered significant for the Armanca –Denise(*), high significant for the Armanca-Diana (**) and extremely significant for the Silvana –Diana and Silvana –Denise (***) using as control the Zenit variety.
After the analysis of variance for the separate action and interaction of the factors it can be observed that neither the variety nor the seeding distance or the applied treatments have no influence on the Fe concentration in studied the hemp seeds.
Zinc content of soil is medium (Table 2), cambic chernozem having a good supply in organic matter which can chelate inorganic sources of Zn and increase their availability to fiber hemp. Zinc availability to hemp can be decreased by phosphorus fertilization. Plant roots appear to absorb Zn and Cu by the same mechanism. This causes interference in the uptake of one when the other is in excess in the root zone. Application of Fertileader Viti B:P:K=1:6:12 fertilizer lead to a decrease of zinc content in all hemp varieties, excepting Denise variety. In Zenit and Silvana variety and also in Diana variety at 20 cm, highest zinc content of hempseed was calculated in unfertilized variant. In this varieties application of fertilizers containing phosphorus inhibited zinc absorption by plant and its translocation to seeds. In only one variant, Armanca variety at 20 cm, application of Corona K (N:P:K=8:11:39) fertilizer containing zinc, lead to increase of zinc content.
By examining the factors and their interaction in the ANOVA factorial test of the Zn concentrations, it can be observed that the F values calculated are smaller that the theoretical ones F5%. So the differences between the averages are insignificant.
Comparing the Zn content in two varieties by the limit differences bring to attention the Zenit variety which has lower Zn content than Armanca and Silvana (*).
As a conclusion for the statistical data determined through the ANOVA 5x3x2 technique, the different components of the independent variable (variety, fertilization and seeding distance) and their interaction doesn’t influence significantly the Mn content in the analyzed hemp seeds.
The F ratio of the independent variable and their interaction on the Mn content isn’t significant statistically, so as a result there aren’t differences between the averages of these variables.
Cadmium is a heavy metal known to have harmful effects on soil microorganisms and plants . Experimental soil has higher content of cadmium, than admitted limit in soil (Table 2). Cadmium is often included in the fertilizer as a component of the phosphate component of the fertilizer. Removal of cadmium from the phosphate is considered expensive and rarely undertaken, so one of the cadmium source in soil could be fertilizers. The average content of Cd in the hemp seeds is ranged between 1.9 mg∙kg-1 (Zenit) and 3.4 mg∙kg-1 for the Armanca and Silvana varieties.
After the analysis of variance (ANOVA one-way) with dependent variable of Cd showed that the variety, as an influence factor and the interaction of the variety with the seeding distance determines statistically highly significant differences (**) and extremely significant differences (***) of the Cd content in the seeds.
The researches made on typical chernozem soil moderately gleyed from Timisoara, at the same nitrogen doses, but in the presence of phosphorus and potassium fertilizers, at different levels, we observed a decrease of available content of Zn, Mn, Cu and Ni because of pH increase (that limit metal availability) and by the other hand, because of metal-phosphate combination which are forming in P excess condition. The most affected by mineral fertilization seems to be the available zinc content which decrease with almost 50% with increasing phosphorus and potassium doses. Available iron content increases with increasing the nitrogen, phosphorus and potassium. With increasing phosphorus and potassium doses, the available iron content decrease mainly because the formation of iron phosphates take place. The concentrations of monitored metals from hemp seeds studied under the influence of foliar fertilization and planting distance are found in Table 3.
The Romanian hemp varieties can be the base of a diet containing lots of oils an a large intake of fatty acids. The utilization of hemp seeds and hemp seed by-products requires careful monitoring of metal content from soil and from the seeds. Hemp is one of the plants that extract easily metals such as Cadmium. P and K intake with foliar fertilizers like Fertileader Viti lightly enhances the average calcium concentration in seeds. Although over a certain value of K in soil the hemp will extract easier K cations instead of Ca. The increase of the K content in seeds determines a growth in the Mg content so that the K:Mg ratio in the monoic and dioic unfertilized and fertilized seeds are to be maintained in certain limits. Neither fertilizing nor distance don’t modify substantially the metal content such as Fe, Mn, Zn and Cd content.
Samples collection and preparation
Soil samples were taken from the two sites with dioecious respectively monoecious hemp. Soil samples are air dried, ground and sieved through a 2 mm sieve. Hemp seed samples were taken from experimentally obtained seed production on the unfertilized parcel associated with each variety at a distance of 20 cm.
Hempseed oil content
Hemp seed oil extracted by Soxhlet method was with petroleum ether . The fully automated Foss Soxtec 2055 system is used for fast and safe determinations of oil content of 6 samples of seed. Seed samples subjected to extraction are from yields of the five varieties of hemp produced in 2011, on unfertilized plots and sown at 20 cm, but also from the biological material used in seeding to achieve the experiment. Grind about 20 g of whole hemp seeds, mix and take three samples of 5.0 g powder which is subject to extraction with petrolium ether. Iodine value was determined in according by standard AOAC 920.158 methods .
Macro and microelements content
Soil samples, in three replicates for each variant, were air dried at room temperature, ground and sieved through a 2 mm sieve. From each soil sample 0.1 g was weight and subject it to mineralization with a mixture of HNO3 : HCl = 1 : 3 in the microwave with a method adapted after the 3050B method of the United States Environmental Protection Agency . After mineralization the sample is diluted to 50 mL volume with 0.5N HNO3.
Samples of 3 g of ground seed were burned 8 h at 550°C in furnace (Nabertherm B150, Lilienthal, Germany). The ash was dissolved in HCl 20% and are brought to 20 ml in a volumetric flask.
The macroelements (K, Ca, Mg) and microelements (Fe, Cd, Zn and Mn) were determined by AAS (Varian 220 FAA equipment). The standard solution for calibration curve were prepared by diluting the standards (1000 mg/L). Mix standard solutions (ICP Multielement Standard solution IV CertiPUR) were purchased from Merck. Double distilled water was used for the preparation of reagents and standards. Concentrate nitric acid (HNO3 65%), and concentrate HCl (37%), were obtained from Merck Germany. All chemicals were trace metal grade (Suprapur).
Method detection limits (MDL mg/L) for analyzed elements were: 0.02 mg/L for Mg and K; 0.06 mg/L for Fe; 0.03 mg/L for Ca; 0.04 mg/L for Zn; 0.01 mg/L for Cd and Mn. The average recoveries ± SD (%) for each element were: Fe (92±3.421%), Mn (95±0.897%), Zn (102±1.083%), Cd (105±0.764%), K (99±0.543%), Ca (92±2.121%), Mg (89±3.211%).
Metal concentration was obtained as the arithmetic average of three readings.
The results of the present study were processed by ANOVA one-way and the least significant difference test, in order to compare the mean values of the investigated parameters. The statistical processing and interpretation of the metal results of the trifactorial experience had as variation source hemp varieties, three different agrofonds, two seeding distances used and the three repetitions obtained after the parallel analysis of the samples. Computations Tukey post-hoc means comparisons and Levene’s test for equal variance was also included. Statistically significant differences are marked (*) and indicate a p value <0.05. Statistically highly significant differences are marked (**) and indicate a p value <0.01. Statistically extremely significant differences are marked (***) and indicate a p value <0.001. Statistical processing of data was performed using the Statistical Analysis System - SAS (Software Version 8.1. SAS Institute, Inc., Cary, NC).
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