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Cannabinoid content in industrial hemp (Cannabis sativa L.) varieties grown in Slovenia
Industrial Crops & Products 145 (2020) 112082
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Industrial Crops & Products journal homepage: www.elsevier.com/locate/indcrop
Cannabinoid content in industrial hemp (Cannabis sativa L.) varieties grown in Slovenia
T
Taja Glivara,*, Jan Erženb, Samo Kreftc, Marjeta Zagožend, Andreja Čerenakd, Barbara Čehd, Eva Tavčar Benkovićb,c a
Biotechnical Faculty, University of Ljubljana, Jamnikarjeva ulica 101, 1000, Ljubljana, Slovenia Freyherr d.o.o., Kersnikova 10, 1000, Ljubljana, Slovenia c Faculty of Pharmacy, University of Ljubljana, Aškerčeva cesta 7, 1000, Ljubljana, Slovenia d Slovenian Institute of Hop Research and Brewing, Cesta Žalskega tabora 2, 3310, Žalec, Slovenia b
A R T I C LE I N FO
A B S T R A C T
Keywords: Cannabis sativa L. Hemp Cannabinoids THC CBD Varieties
Cannabinoid content in different hemp varieties from the Common catalogue of varieties of agricultural plant species is not well known. Hemp (Cannabis sativa L., subsp. sativa) contains a wide range of cannabinoids, where cannabidiol (CBD) and (−)-trans-Δ9-tetrahydrocannabinol (Δ9-THC) are the constituents with known therapeutic activity. Also, Δ9-THC is recognized as an illicit drug; therefore, cultivation of hemp is restricted to a 0.2 % limit of THC content in many European countries. In this study, the cannabinoid profiles of 15 hemp varieties, accessible to our research group, were analysed. The content of 13 cannabinoids was determined with HLPC (highperformance liquid chromatography) analysis. Large variations in cannabinoid content among varieties that grew in uniform conditions (ANOVA p < 0.05) and also within a single variety were found, which shows on ununiform genetic profiles of the seed material. The varieties Fedora 17, USO 31, Tisza, Tiborszallasi, and Antal all displayed good response to growth conditions, related to cannabinoid content, in Slovenia.
1. Introduction Hemp (Cannabis sativa L. subsp. sativa) is a subspecies of the genus Cannabis (Cannabaceae), which also includes medicinal cannabis (Cannabis sativa L. subsp. indica). It is an annual plant that occurs in versatile forms, depending on the variety and availability of space for growth. It is mostly dioecious, meaning that the male and female flowers are located on separate plants. Seeds can only be found on female plants. Cannabis plants contains two main cannabinoids; one is a psychoactive cannabinoid substance (−)-trans-Δ9-tetrahydrocannabinol, better known as THC, which earns medicinal cannabis its classification as an illicit drug. The second is cannabidiol (CBD), represented primarily in hemp. Cannabidiol is known as a pharmacologically active substance and is becoming more and more important for use in medicinal applications. A broad chemical profile of cannabinoids, that can be found in cannabis, exhibits the typical C21 terpenophenolic skeleton, including their derivatives and transformation products (Pertwee, 2014). Cannabinoids are initially formed as carboxylic acids (eg, Δ9-
tetrahydrocannabinolic acid (Δ9-THC-A), cannabidiolic acid (CBD-A), cannabichromenic acid (CBC-A), and tetrahydrocannabivarin carboxylic acid (Δ9-THCV-A)) that, when heated, are decarboxylated to their corresponding neutral forms. Another process caused by heating, light exposure or ageing, is oxidative degradation, wherein the conversion of Δ9-THC to cannabinol (CBN) and isomerization of Δ9-THC to Δ8-tetrahydrocannabinol (Δ8-THC) occurs. The concentrations of cannabinolic acid (CBN-A) and CBN, primary chemical degradants of Δ9THC-A and Δ9-THC, increase during storage, if exposed to light at 22 °C as compared with storage in darkness at 4 °C. The concentration of CBDA, when exposed to light and 22 °C, decreases with cyclization from CBD-A to Δ9-THC-A, followed by the degradation of Δ9-THC-A to CBN-A (Thomas and Elsohly, 2016). Cannabinoid production in hemp can be affected by numerous biotic and abiotic factors such as the sex and maturity of the plant, daily light integral, ambient temperature, nutrient availability, and intensity of ultraviolet light (Hillig and Mahlberg, 2004). The content of cannabinoids was found to be differently distributed in plant parts with the highest percentage in secretory cells inside glandular trichomes (up to 60 %), highly concentrated in unpollinated female flowers prior to
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Corresponding author. E-mail addresses: [email protected] (T. Glivar), [email protected] (J. Eržen), samo.kreft@ffa.uni-lj.si (S. Kreft), [email protected] (M. Zagožen), [email protected] (A. Čerenak), [email protected] (B. Čeh), [email protected] (E. Tavčar Benković). https://doi.org/10.1016/j.indcrop.2019.112082 Received 22 August 2019; Received in revised form 22 December 2019; Accepted 29 December 2019 0926-6690/ © 2019 Elsevier B.V. All rights reserved.
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senescence (up to 30 %), pollinated flowers (up to 13 %), leaves (0.05 %), and least in the stem (0.02 %). No cannabinoids have been found in the roots or seeds (Russo and Marcu, 2017) (Russo, 2011). The highest cannabinoid concentration (in % of dry weight plant material) can be found in the bracts of the flowers that contain abundant glandular trichomes (Turner et al., 1981). Better knowledge of the cannabinoid production in glandular trichomes would help the breeder to identify the optimum time for harvest (Potter and D., 2009). Mature female plants are preferred for cannabis extract production, since they produce higher amounts of cannabinoids. The highest yield of cannabinoids are achieved if grown without male plants to prevent pollination and the formation of seeds (Thomas and Elsohly, 2016). Hemp is becoming well-known for its versatile use and great economic importance. Recently, there has been a dramatic increase in CBD-rich supplementation in the food supplement and cosmetic industries. Even more potential is reported on the pharmaceutical use of cannabinoids. Since the pharmaceutical industry requires the highest quality of materials, it is of the upmost importance to ensure consistency in the chemical profiles of plants intended for such use (Thomas and Elsohly, 2016). Chemotypes are highly dependent on the genetics, and in addition the plants are exposed to many natural factors, which can be very variable among seeds within a single variety. As a result, a constant profile of cannabinoids is not easy to achieve with sowing seed. The best way for the propagation to produce uniform plants is with cuttings (clones). Current legislation in Slovenia allows cultivation of hemp for the purpose of seeds production and further propagation, production of food and beverages, the production of substances for cosmetic purposes, production of fibres, animal feed, and for other industrial purposes. This applies to all varieties listed in the Common catalogue of varieties of agricultural plant species, whose THC content does not exceed 0.2 % (“Rules on conditions for obtaining a permit for hemp and poppy cultivation,” 2018) in the upper third part of the dried plant or in a 30 cm dried plant part, containing at least one female inflorescence (“Regulation (EU) No 809/2014,” 2014Regulation (EU) No 809/, 2014Regulation (EU) No 809/2014,” 2014). The aim of this study was to determine the chemical profile of 13 cannabinoids in 15 different hemp varieties, grown in Slovenia.
In 2017, three inflorescences, each from different female plants of each variety were studied. Table 2 illustrates the experimental procedure of the sample preparation. The preparation of the samples began with careful removal of the seeds from each inflorescence. The bracts were separated from the stems, leaflets, and supporting leaves. Stems, leaflets, and supporting leaves of the inflorescence were ground with a laboratory grinder (30 s, 20.000 rpm) to a size of ≤ 0.5 mm. Samples from the first and second plants were prepared identically, while the third inflorescence was additionally separated into upper and lower parts. The third flower of the varieties Finola and Ferimon was not separated into an upper and lower part; it was analysed as a whole flower since not enough material was available. Developing seeds were present in most plants, so if the seeds would not be removed the mass ratio of seeds to other parts of the inflorescences would differ from plant to plant, which would influence the representability of the results of cannabinoid content. By removal of the seeds representative results were achieved. For additional comparison, bracts were used to obtain the most accurate and comparable results on cannabinoid profiles among the plants. Also, glandular trichomes contained the highest content of cannabinoids and were mostly located on the bracts (Turner et al., 1981). In 2018, one inflorescence of each variety was prepared according to the procedure described above, but only bracts were analysed. For the second sample, inflorescences from another plant of each variety were used with prior removal of seeds and ground with a laboratory grinder (30 s, 20.000 rpm) to a size of ≤ 0.5 mm as shown in Table 2.
2. Materials and methods
2.3. Cannabinoid content evaluation
2.1. Plant material
The following cannabinoids were analysed: Cannabidivarinic acid (CBDV-A), Cannabidivarin (CBDV), Tetrahydrocannabivarin (THCV), Cannabidiolic acid (CBD-A), Cannabidiol (CBD), Cannabigerolic acid (CBG-A), Cannabigerol (CBG), Cannabinol (CBN), (−)trans-Δ9-tetrahydrocannabinol (Δ9-THC), Δ8-tetrahydrocannabinol (Δ8-THC), Cannabicyclol (CBL), Δ9-tetrahydrocannabinolic acid (Δ9-THC-A), and Cannabichromene (CBC).
were not irrigated during the growth phase. Compared with the year 2017, 2018 was cooler with more precipitation (Fig. 1B). Sampling of the female flowers was carried out on the 30 August 2018. The vegetation period lasted for 93 days. Plants were dried, transferred, and stored with the same procedure as in 2017. In this study, only few plants were sampled (see Section 2.2), while 50 plants/ha should be sampled according to the legislation directive and the average content should not exceed 0.2 % (“Rules concerning the requirements for obtaining permission to cultivate hemp,” 2004). 2.2. Preparation of plant material for chemical characterization
A total of 15 industrial hemp varieties (Cannabis sativa L., subsp. sativa) from the Common catalogue of varieties of agricultural plant species were grown on the fields of the Slovenian Institute of Hop Research and Brewing (IHPS) in Žalec in the year 2017 and in Gornja Radgona (approximately 100 km from Žalec) in 2018. The plants were collected at the phase of maturity at which most Slovenian hemp farmers usually harvest. The seeds of the same producer were used for each variety in both years. Properties of investigated varieties, obtained from producers, web pages (“Zadruga konopko, 2019),(“Ihempfarms, 2019). and data source (Pacifico et al., 2008), are summarized in Table 1. On the 3 May 2017, 13 varieties of hemp (Table 1), were sown on 9 m2 large parcels, with each variety in a separate plot, but with no physical obstacles between them. The plants were not irrigated during the growth phase. During the growth period, the weather was warmer than usual; this was one of the warmest summers in Žalec since 1961 (Fig. 1A). The vegetation phase lasted for 135 days. Sampling of the female plants was carried out on the 15 September 2017. Plants were dried for 24 h at 35 °C and transferred to the analytical laboratory at the Faculty of Pharmacy, University of Ljubljana, to be stored in a cold room in paper bags, protected from light until preparation for analytical testing. The second study began on the 29 May 2018, when 14 varieties of hemp (Table 1) were sown. The parcels were 2 m2 large. The plants
2.4. Cannabinoid extraction The samples were extracted by adding 10 mL of ethanol (96 %, Kefo Slovenia) to the 100−200 mg of the accurately weighted ground or non-ground material in closed plastic tubes. Samples were incubated for 30 min in an ultrasonic bath at 50 °C. The supernatant was removed and filtered through a 25 μm filter in the HPLC (high-performance liquid chromatography) vial. 2.5. Chromatographic analysis Samples were analysed using HPLC as described in one of our other articles (submitted for publication). The amount of each cannabinoid (%w/w [%mg of cannabinoid/100 mg herbal drug]) in the original sample was calculated by using calibration curves of standard compounds. Additionally, to show the total concentration of Δ9-THC, CBD, and 2
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Table 1 Basic characteristics of hemp varieties. Variety
Grown in research study in years
Country origin
Genotypic expression
Climate adaptation
Vegetative cycle
CBD content
Δ9-THC content
Fedora 17 KC Dora Monoica USO 31 Helenaa
2017 2017 2017 2017 2017
and and and and and
2018 2018 2018 2018 2018
France Hungary Hungary Germany Serbia
Monoecious Dioecious Dioecious Monoecious Dioecious
Atlantic Continental Continental Atlantic No data available
Early < 125 days Late < 145 days Medium < 135 days Early < 125 days Early < 125 days
< 0.06 % < 0.12 % < 0.12 % < 0.06 % No data available
Santhica 27 Tisza Tiborszallasi Antalb Carmagnolac
2017 2017 2017 2017 2017
and and and and and
2018 2018 2018 2018 2018
France Hungary Hungary Hungary Italy
Monoecious Dioecious Dioecious Dioecious Dioecious
Atlantic Continental Continental Continental Mediterranean
Medium < 135 days Late < 145 days Late < 145 days Late < 145 days Very late < 160 days
Finola
2017 and 2018
Finland
Dioecious
Continental
Kompolti hibrid TC Ferimon Novosadskaa
2017 and 2018 2017 2018
Hungary France Serbia
Dioecious Monoecious No data available
Continental Atlantic No data available
Very early < 110 days Medium < 135 days Early < 125 days Very late < 160 days
Marinaa
2018
Serbia
Dioecious
No data available
Late < 145 days
1.5 – 2.0 % 1.5 – 2.0 % 1.5 – 2.0 % 0.5 – 1.0 % No data available 1.0–1.5 % 2.0–3.0 % 2.0–3.0 % 2.0–3.0 % No data available No data available 2.0–3.0 % 1.0–1.5 % No data available No data available
< < < < <
0.02 0.12 0.20 0.12 0.30
% % % % %
No data available < 0.12 % < 0.06 % No data available No data available
Cannabidiol (CBD), (−)-trans-Δ9-tetrahydrocannabinol (Δ9-THC). a Varieties Novosadska, Marina, and Helena are new and have never been included into the Common catalogue of varieties of agricultural plant species. Data regarding those varieties was gained at producers. b Antal was removed from the Common catalogue of varieties of agricultural plant species after experiments were completed. c Carmagnola was deleted from Common catalogue of varieties of agricultural plant species in 2019, with market extension until 30 June 2021.
3. Results and discussion
CBG in %w/w [%mg of cannabinoid/100 mg herbal drug], the following equations were used:
The cannabinoid content of varieties grown in 2017 and 2018 are summarized in Tables 3,4, and 5. Additionally, total CBD, total Δ9-THC, total CBG, and the ratio of total Δ9-THC/total CBD content is presented. The differences in the content of CBD-A, CBG-A, CBN, Δ9-THC-A, CBC, total CBD, total CBG, and total Δ9-THC between the varieties were significant (ANOVA p < 0.05). The content of remaining cannabinoids were not significantly different between the varieties (ANOVA p > 0.05).
% of Total Δ9-THC = % Δ9-THC + (% Δ9-THC-A x 0.877) % of Total CBD = % CBD + (% CBD-A x 0.877) % of Total CBG = % CBG + (% CBG-A x 0.878) The conversion factors 0.877 and 0.878 represent a loss of molecular mass during the decarboxylation reaction (transformation of carboxylic acid derivatives [Δ9-THC-A, CBD-A, CBG-A] to non-carboxylic acid forms [Δ9-THC, CBD, CBG]).
3.1. Total CBD and total Δ9-THC content 2.6. Statistical analysis It is well known that Cannabis sativa L. is a highly variable species in terms of botany, genetics, and its chemical profile (Thomas and Elsohly, 2016). Considering that higher parts of the inflorescences are more exposed to sunlight and exhibit differential hormonal distribution than lower parts, it is expected that cannabinoid concentration in upper parts would be higher compared with lower parts. Variability between the upper part and the lower part of the inflorescence was examined in the year 2017, as summarized in Table 4. Evaluation was carried out
All statistical analyses were performed by using Excel software Office Home and GraphPad Prism 6. Concentrations, mean values, and standard deviations were calculated with Excel. In addition, for results from study in 2017 (number of repetitions: N = 3), statistical analysis ANOVA and t-tests, were performed with IBM SPSS. For study from 2018, results have not been statistically proceeded since no repetitions were done (N = 1).
Fig. 1. Weather conditions of precipitation and temperature (average of the Tmin and Tmax of the daily temperature) during the growth period presented in decades (data source: ARSO - Slovenian Environment Agency): A.) from May to September in 2017 in Žalec, compared to the average of 30 years, B.) from May to August in 2018 in Gornja Radgona, compared to the average of 49 years. 3
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Table 2 Experimental design in 2017 and 2018 for inflorescences presenting year of sowing, sequential flower, part of the flower, and sample condition. Year
Inflorescence
Part of flower
Grinded/non-grinded sample
2017
first first second second third third third third first second
Bracts from whole inflorescence Rest of the inflorescence Bracts from whole inflorescence Rest of the inflorescence Bracts from upper inflorescence part Bracts from lower inflorescence part Rest of the upper inflorescence part Rest of the lower inflorescence part Bracts from whole inflorescence Whole inflorescence
non-grinded grinded non-grinded grinded non-grinded non-grinded grinded grinded non-grinded grinded
2018
with comparison of the total CBD and total Δ9-THC content, as their ratios are the most interesting of all the analyzed cannabinoids. The distribution of total CBD in bracts from the upper and lower inflorescence parts was found to be equal. A considerable difference was observed only with the variety Antal (upper part 5.970 %, lower part 13.460 %, mean 9.715 % ± 3.745). If compared with the results from bracts of whole inflorescence (of first and second flower) analysis (mean value was found to be 9.513 % ± 1.572), results are similar. The difference could be explained by ununiformed quality of the inflorescences. It could be theorized that the upper parts stopped maturing during growth because of excessive sunlight exposure. Concentration of total Δ9-THC had shown no important differences between parts of the inflorescences for any variety. With two varieties, Tiborszallasi (mean value 3.304 % ± 0.097) and Carmagnola (mean value 2.457 % ± 0.089), extreme values of high total Δ9-THC content were detected. High values are presumably due to the non-uniform genetic material, possibly due to cross-pollination in a previous generation between seed-producing female plants and male plants from neighboring fields with contrasting genetics, causing higher Δ9-THC content. According to the obtained results, it can be concluded that no significant differences in the total Δ9-THC/total CBD content between the upper and lower bracts of the uppermost inflorescences was observed (paired Student t-test: p = 0.44). Additionally, in a research study by Namdar et al. where cannabinoids were analysed in the uppermost, middle, and lowermost inflorescences of hemp, it has been found that the uppermost inflorescences contained a higher content of cannabinoids compared with the lowermost inflorescences (Namdar et al., 2018). In this study, the uppermost inflorescences were used. The highest content of total CBD (> 5 % reported as %w/w) produced in both years (Table 4 and Table 5) was found to be in the bracts of the varieties Antal (9.580 % ± 1.287 in 2017 and 6.159 % in 2018), Carmagnola (7.069 % ± 3.339 in 2017 and 5.726 % in 2018), Helena (5.873 % ± 1.816 in 2017 and 8.108 % in 2018), and Tiborszallasi (4.666 % ± 1.931 and 4.876 % in 2018). The lowest total CBD content was detected with the varieties USO 31 (1.207 % ± 0.356 in 2017 and 0.735 % in 2018) and Santhica 27 (0.038 % ± 0.042 in 2017 and 0.010 % in 2018) (Fig. 2A). Important information about a variety is its uniformity in terms of cannabinoid content (chemotype), which can be evaluated with the standard deviation in total CBD, in our case from inflorescences from three different plants in experiment from 2017. High uniformity within the population (standard deviation < 0.5) was found in Fedora 17, USO 31, and Santhica 27 (Table 4). The varieties Carmagnola, Monoica, Helena, Tisza, and Tiborszallasi displayed low uniformity of the seeds with high standard deviation (sd > 2) within the population (Table 4). In hemp cultivation, special focus must also be given to the allowed concentration of Δ9-THC as different countries have different regulations. The limit of 0.2 % Δ9-THC is prescribed by Slovenian legislation (“Rules on conditions for obtaining a permit for hemp and poppy cultivation,” 2018). The highest content of total Δ9-THC (> 0.2 %) was
detected in bracts of the following varieties in 2017: Tiborszallasi (2.509 % ± 1.627), Carmagnola (1.015 % ± 1.020), Tisza (0.895 % ± 1.004), KC Dora (0.812 % ± 0.991), and Antal (0.317 % ± 0.042). Other varieties showed high uniformity (sd < 0.07) and lower total THC content (< 0.2 %) (Fig. 2B) (Table 4). Results from 2018 confirm that levels of total Δ9-THC in bracts for all listed varieties except for Tiborszallasi (0.172 %) exceeded suitable quantities, with the addition of the variety Helena (0.280 %) with a value slightly above the limit (Fig. 2B) (Table 5). It was shown that total Δ9-THC and total CBD reached higher concentrations in 2017 for the majority of the varieties. In 2017, environmental conditions were warmer and dryer, compared with year 2018. From this study it can be concluded that these environmental conditions are more suitable for CBD production, but it could not be summarized, which of the above mentioned weather parameters contributed most to the higher content of total CBD. The data of total Δ9THC content in the bracts are higher than they would be with mechanical harvesting since with manual harvesting only inflorescences were removed and agitation of the inflorescences is minimized. With mechanical harvesting of bigger stems, their contribution to the final weight of the sample is added. In order to confirm that total Δ9-THC values do not exceed the allowed levels, we verified the content of total CBD and total Δ9-THC in the remaining parts of the inflorescences (stems, leaflets, and supporting leaves) compared with bracts from inflorescences in the study from 2017, as shown in Figs. 3A and 3B. Information gathered from publications indicate that it is expected that cannabinoid ratios between isolated glandular trichomes and whole pollinated flowers is approximately 1:4 (Russo and Marcu, 2017) in favor of glandular trichomes. From our results, as shown by evidence in Figs. 3A and 3B, it can be proven that the content of total CBD and total Δ9-THC in the bracts is between two to eight times (on average four times) higher than in other parts of the inflorescence (without bracts). It can be stated that the remaining parts of the inflorescence present an additional mass with lower cannabinoid concentration. In the case of analysing the whole inflorescence, leaflets and stems present a contributing factor to detection of lower cannabinoid concentration than the ones detected in bracts. To further confirm this finding, in 2018, cannabinoid comparisons between bracts and whole inflorescence (with bracts, stems, leaflets, and supporting leaves) had been performed (Table 5). Results showed up to four times lower values detected in whole inflorescences (with the exception of USO 31). Also, Turner et al. reported about the highest levels of cannabinoids being found in bracts (Turner et al., 1981). The content of total Δ9-THC in case of whole inflorescences would exceed the limit of < 0.2 % only with KC Dora (0.878 %). According to previous findings regarding the KC Dora uniformity, this phenomenon is probably caused due to a different chemotype of the variety. Relating to the above facts it can be assumed that, according to current Slovenian legislation, varieties wouldn’t presumably exceed the required limit, especially when plants are harvested mechanically, since
4
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Table 3 Cannabinoid content of bracts from the first, second, and third flower from a study in the year 2017, with results of statistical analysis performed by ANOVA. Cannabinoids CBG (Cannabigerol), CBDV (Cannabidivarin), THCV (Tetrahydrocannabivarin), CBL (Cannabicyclol), and Δ8-THC (Δ8-tetrahydrocannabinol) were not detected in any of the varieties. The values of the third flower was previously averaged (up and low) before the total averaging and the calculation of the standard deviation was performed. Variety
Flower
first second thirdup thirdlow Mean ± KC Dora first second thirdup thirdlow Mean ± Monoica first second thirdup thirdlow Mean ± USO 31 first second thirdup thirdlow Mean ± Helena first second thirdup thirdlow Mean ± Santhica 27 first second thirdup thirdlow Mean ± Tisza first second thirdup thirdlow Mean ± Tiborszallasi first second thirdup thirdlow Mean ± Antal first second thirdup thirdlow Mean ± Carmagnola first second thirdup thirdlow Mean ± Kompolti hibrid first TC second thirdup thirdlow Mean ± Finola first second thirda Mean ± Ferimon first second thirda Mean ± p (ANOVA of the differences between varieties)
Fedora 17
sd
sd
sd
sd
sd
sd
sd
sd
sd
sd
sd
sd
sd
CBDV-A (%w/w)
CBD-A (%w/w)
CBD (%w/w)
CBG-A (%w/w)
CBN (%w/w)
Δ9-THC (%w/w)
Δ9-THC-A (%w/w)
CBC (%w/w)
0.112 0.016 0.016 0.020 0.049 0.016 0.007 0.051 0.040 0.023 0.050 0.020 0.018 0.015 0.029 0.026 0.009 0.021 0.018 0.018 0.032 0.032 0.045 0.034 0.034 0.002 ND 0.003 0.003 0.002 0.024 0.017 0.020 0.020 0.020 0.026 0.024 0.023 0.017 0.023 0.193 0.058 0.110 0.098 0.118 0.052 0.050 0.528 0.498 0.205 0.017 0.015 0.036 0.042 0.024 0.052 0.115 0.050 0.072 0.024 0.017 0.018 0.020 0.192
1.587 2.281 2.040 3.520 2.216 ± 0.489 2.232 2.699 5.618 4.351 3.305 ± 1.203 5.113 9.819 6.993 6.685 7.257 ± 1.944 0.785 1.224 1.452 1.563 1.172 ± 0.297 8.618 6.863 4.190 3.392 6.424 ± 1.995 0.009 0.110 0.006 0.014 0.043 ± 0.047 3.466 8.398 3.480 3.421 5.105 ± 2.329 4.241 7.667 3.182 2.925 4.987 ± 1.956 8.747 12.119 6.420 14.743 10.483 ± 1.378 8.459 10.918 2.840 2.739 7.389 ± 3.404 3.882 4.251 5.884 6.433 4.764 ± 0.998 1.126 1.727 1.659 1.504 ± 0.269 4.455 6.096 4.719 5.090 ± 0.719 0.001
0.071 0.416 0.071 0.132 0.196 0.407 0.440 0.136 0.088 0.320 0.168 0.566 0.340 0.279 0.348 0.027 0.472 0.008 0.071 0.180 0.259 0.335 0.147 0.100 0.239 ND ND ND ND ND 0.081 0.472 0.112 0.109 0.221 0.156 0.601 0.132 0.105 0.292 0.270 0.456 0.340 0.530 0.387 0.789 0.893 0.085 0.082 0.589 0.094 0.242 0.189 0.209 0.178 0.670 1.413 0.187 0.757 0.250 0.521 0.275 0.349 0.164
0.021 0.040 0.029 0.044 0.032 0.039 0.095 0.217 0.114 0.100 0.173 0.343 0.145 0.107 0.214 0.012 0.029 0.033 0.038 0.025 0.184 0.244 0.212 0.124 0.199 0.090 2.585 2.371 3.424 1.857 0.185 0.188 0.189 0.183 0.186 0.417 0.210 0.208 0.190 0.275 0.598 0.512 0.263 0.503 0.497 0.154 0.211 0.054 0.076 0.143 0.072 0.438 0.473 0.334 0.305 0.009 0.011 0.007 0.009 0.045 0.023 0.064 0.044 0.002
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 0.003 0.005 ND 0.003 ± 0.002 ND ND ND ND /
ND ND ND ND ND 0.027 0.528 ND ND 0.185 0.025 0.043 0.026 0.030 0.032 ND ND ND ND ND 0.024 0.028 0.010 0.022 0.022 ND ND ND ND ND 0.083 0.028 0.013 0.016 0.042 0.172 0.036 0.182 0.145 0.124 0.024 0.034 0.022 0.042 0.030 0.058 0.068 0.138 0.105 0.082 ND ND ND ND ND 0.034 0.059 0.017 0.037 0.009 0.033 0.014 0.019 0.315
ND 0.070 0.060 0.092 0.049 0.068 1.922 0.176 0.133 0.715 0.137 0.320 0.224 0.210 0.225 0.021 0.058 0.047 0.048 0.042 0.263 0.197 0.126 0.097 0.190 ND 0.006 ND ND 0.002 2.543 0.254 0.128 0.118 0.973 4.345 0.235 3.671 3.491 2.720 0.274 0.380 0.199 0.458 0.327 0.237 0.292 2.745 2.581 1.064 0.123 0.147 0.179 0.195 0.152 0.031 0.039 0.045 0.038 0.116 0.181 0.118 0.138 0.037
0.020 0.022 0.018 0.016 0.020 0.021 0.017 ND ND 0.013 0.020 0.026 0.018 0.019 0.022 0.040 0.054 0.051 0.052 0.048 0.010 0.013 0.011 0.012 0.012 ND 0.005 ND ND 0.002 ND ND ND ND ND ND 0.033 0.013 0.012 0.015 ND ND ND ND ND 0.038 0.049 0.008 0.011 0.032 0.009 0.013 0.020 0.021 0.014 0.022 0.067 0.055 0.048 0.003 0.020 0.004 0.009 0.001
± 0.045
± 0.017
± 0.015
± 0.007
± 0.004
± 0.001
± 0.003
± 0.003
± 0.056
± 0.218
± 0.011
± 0.030
± 0.003
± 0.156
± 0.147
± 0.165
± 0.207
± 0.088
± 0.178
± 0.219
± 0.083
± 0.360
± 0.062
± 0.504
± 0.122
± 0.008
± 0.052
± 0.093
± 0.010
± 0.033
± 1.256
± 0.001
± 0.100
± 0.088
± 0.060
± 0.165
± 0.002
± 0.017
± 0.243
± 0.008
± 0.005
± 0.030
± 0.062
± 0.004
± 0.028
± 0.017
± 0.010
± 0.034
± 0.854
± 0.075
± 0.015
± 0.062
± 0.003
± 1.111
± 1.785
± 0.043
± 1.131
± 0.026
± 0.006
± 0.030
± 0.002
± 0.009
± 0.003
± 0.006
± 0.001
± 0.002
± 0.013
± 0.016
± 0.005
± 0.019
± 0.008
Cannabidivarinic acid (CBDV-A), Cannabidiolic acid (CBD-A), Cannabidiol (CBD), Cannabigerolic acid (CBG-A), Cannabinol (CBN), (−) trans-Δ9-tetrahydrocannabinol (Δ9-THC), Δ9-tetrahydrocannabinolic acid (Δ9-THC-A), Cannabichromene (CBC), ND (not detected), up (upper), low (lower). a in the Finola and Ferimon varieties the third flower was not separated into upper and lower parts and was analysed as a whole flower (see Section 2.2).
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Table 4 Total CBG, total CBD, total Δ9-THC content, and the ratio of total Δ9-THC/total CBD in the bracts of inflorescence from the first, second, and third flowers from a study in the year 2017, with results of statistical analysis performed by ANOVA. The values of the third flower was previously averaged (up and low) before the total averaging and the calculation of the standard deviation was performed. Variety
Flower
Total CBG (%w/w)
Total CBD (%w/w)
Total Δ9-THC (%w/w)
Ratio of total Δ9-THC/ total CBD
Fedora 17
first second thirdup thirdlow Mean ± sd first second thirdup thirdlow Mean ± sd first second thirdup thirdlow Mean ± sd first second thirdup thirdlow Mean ± sd first second thirdup thirdlow Mean ± sd first second thirda Mean ± sd first second thirdup thirdlow Mean ± sd
0.018 0.035 0.026 0.038 0.028 ± 0.007 0.152 0.301 0.128 0.094 0.188 ± 0.082 0.162 0.214 0.186 0.109 0.175 ± 0.029 0.163 0.165 0.166 0.160 0.164 ± 0.001 0.525 0.449 0.231 0.442 0.437 ± 0.078 0.008 0.009 0.006 0.008 ± 0.002 0.064 0.385 0.415 0.293 0.268 ± 0.145 (%w/w) 0.034 0.083 0.191 0.100 0.088 ± 0.045 0.010 0.026 0.029 0.033 0.022 ± 0.009 0.079 2.269 2.082 3.006 1.631 ± 1.103 0.366 0.184 0.182 0.167 0.241 ± 0.088 0.135 0.185 0.047 0.067 0.126 ± 0.053 0.040 0.020 0.056 0.039 ± 0.015 0.002
1.463 2.416 1.861 3.220 2.140 ± 0.481 4.652 9.177 6.473 6.141 6.712 ± 1.870 7.817 6.354 3.821 3.075 5.873 ± 1.816 3.121 7.837 3.165 3.109 4.698 ± 2.219 7.941 11.085 5.970 13.460 9.580 ± 1.287 1.658 2.928 1.643 2.076 ± 0.602 3.498 3.970 5.350 5.850 4.356 ± 0.900 (%w/w) 2.365 2.807 5.063 3.904 3.219 ± 0.913 0.716 1.545 1.282 1.442 1.207 ± 0.356 0.008 0.096 0.005 0.012 0.038 ± 0.042 3.876 7.325 2.923 2.671 4.666 ± 1.931 8.208 10.468 2.576 2.484 7.069 ± 3.339 4.158 5.867 4.413 4.812 ± 0.753 0.001
ND 0.061 0.052 0.081 0.043 ± 0.030 0.145 0.324 0.222 0.214 0.229 ± 0.073 0.254 0.201 0.120 0.107 0.189 ± 0.058 2.313 0.251 0.125 0.119 0.895 ± 1.004 0.264 0.367 0.196 0.444 0.317 ± 0.042 0.061 0.093 0.057 0.071 ± 0.016 0.108 0.129 0.157 0.171 0.133 ± 0.023 (%w/w) 0.087 2.213 0.155 0.117 0.812 ± 0.991 0.019 0.051 0.041 0.042 0.037 ± 0.014 ND 0.005 ND ND 0.002 ± 0.002 3.983 0.241 3.401 3.207 2.509 ± 1.627 0.265 0.324 2.545 2.368 1.015 ± 1.020 0.111 0.191 0.117 0.140 ± 0.037 0.042
N/A 1:39 1:35 1:40 1:50 1:32 1:28 1:29 1:29 1:29 1:31 1:32 1:32 1:29 1:31 1:1 1:31 1:25 1:26 1:5 1:30 1:30 1:30 1:30 1:30 1:27 1:31 1:29 1:29 1:32 1:31 1:34 1:34 1:33
Monoica
Helena
Tisza
Antal
Finola
Kompolti hibrid TC
KC Dora
USO 31
Santhica 27
Tiborszallasi
Carmagnola
Ferimon
p (ANOVA of
first second thirdup thirdlow Mean ± sd first second thirdup thirdlow Mean ± sd first second thirdup thirdlow Mean ± sd first second thirdup thirdlow Mean ± sd first second thirdup thirdlow Mean ± sd first second thirda Mean ± sd the differences between varieties)
1:27 1:1 1:33 1:33 1:4 1:38 1:30 1:31 1:34 1:33 N/A 1:20 N/A N/A 1:23 1:1 1:30 1:1 1:1 1:2 1:31 1:32 1:1 1:1 1:7 1:38 1:31 1:38 1:34 /
Cannabidiol (CBD), (−)-trans-Δ9-tetrahydrocannabinol (Δ9-THC), Cannabigerol (CBG), ND (not detected), up (upper), low (lower). a in the Finola and Ferimon varieties the third flower was not separated into upper and lower part and it was analysed as a whole flower (see Section 2.2).
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Table 5 Cannabinoid content and the ratio of total Δ9-THC/total CBD in bracts from inflorescences and whole inflorescences from a study in the year 2018. Cannabinoids CBG (Cannabigerol), Δ8-THC (Δ8-tetrahydrocannabinol), and CBL (Cannabicyclol) were not detected in any of the varieties. Variety
Fedora 17 KC Dora Monoica USO 31 Helena Santhica 27 Tisza Tiborszallasi Antal Carmagnola Kompolti hibrid TC Finola Novosadska Marina
B/WI
B WI B WI B WI B WI B WI B WI B WI B WI B WI B WI B WI B WI B WI B WI
CBDV-A
CBDV
THCV
CBD-A
CBD
CBG-A
CBN
Δ9-THC
Δ9-THC-A
CBC
Total CBG
Total CBD
(%w/w)
(%w/w)
(%w/w)
(%w/w)
(%w/w)
(%w/w)
(%w/w)
(%w/w)
(%w/w)
(%w/w)
(%w/w)
(%w/w)
0.230 0.065 ND 0.004 0.030 0.005 0.008 0.007 0.045 0.009 ND ND 0.017 0.007 0.033 0.006 0.030 0.007 0.073 0.137 0.024 0.004 0.047 0.021 0.076 0.019 0.008 ND
0.120 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 0.061 ND ND ND 0.010 ND ND ND ND
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 0.152 ND ND ND ND ND ND ND ND
3.158 1.301 3.343 0.439 1.490 0.497 0.635 0.813 8.825 2.504 0.011 ND 3.152 1.489 3.625 1.073 5.153 1.234 4.120 0.161 6.012 1.047 1.409 0.573 6.485 1.434 1.460 0.332
1.754 0.492 2.582 0.311 1.156 0.530 0.178 0.176 0.368 0.178 ND ND 1.235 0.606 1.697 0.681 1.639 0.744 2.113 0.112 0.858 0.459 1.545 0.474 0.292 0.122 0.890 0.389
0.104 0.041 0.219 0.030 0.049 0.018 0.012 0.029 0.283 0.057 3.566 1.423 0.109 0.066 0.362 0.095 0.491 0.078 0.184 0.007 0.201 0.076 0.017 0.005 0.146 0.022 0.081 0.019
0.005 0.001 0.016 0.012 ND 0.001 0.001 ND ND ND ND ND 0.005 0.001 0.003 ND 0.006 ND 0.003 0.003 ND 0.002 0.004 0.001 ND ND ND 0.002
0.090 0.028 0.723 0.545 0.057 0.024 0.009 0.010 0.035 0.016 ND ND 0.432 0.040 0.112 0.040 0.769 0.037 0.150 0.105 0.071 0.027 0.067 0.020 0.026 0.011 0.052 0.016
0.056 0.025 0.532 0.379 0.021 0.006 0.015 0.020 0.280 0.075 ND ND 0.413 0.029 0.068 0.020 1.314 0.032 0.120 0.063 0.140 0.018 0.022 0.011 0.191 0.038 0.027 0.004
0.099 0.031 0.173 0.038 0.085 0.042 0.009 0.014 0.026 0.015 0.020 0.021 0.094 0.044 0.102 0.038 0.159 0.042 0.140 0.026 0.064 0.031 0.068 0.024 0.020 0.012 0.057 0.026
0.091 0.036 0.193 0.026 0.043 0.016 0.010 0.025 0.249 0.050 3.131 1.249 0.095 0.058 0.318 0.083 0.431 0.068 0.162 0.006 0.177 0.067 0.015 0.004 0.128 0.019 0.071 0.017
4.523 1.633 5.513 0.696 2.462 0.966 0.735 0.889 8.108 2.374 0.010 ND 4.000 1.912 4.876 1.622 6.159 1.827 5.726 0.254 6.131 1.377 2.781 0.977 5.979 1.380 2.170 0.680
Total Δ9 THC (%w/ w)
Ratio total Δ9−THC/ total CBD
0.139 0.050 1.190 0.878 0.075 0.029 0.022 0.027 0.280 0.082 ND ND 0.794 0.066 0.172 0.058 1.922 0.065 0.255 0.161 0.193 0.043 0.086 0.030 0.193 0.044 0.075 0.020
1:33 1:33 1:5 1:1 1:33 1:33 1:34 1:32 1:29 1:29 N/A N/A 1:5 1:29 1:28 1:28 1:3 1:28 1:22 1:2 1:32 1:32 1:32 1:33 1:31 1:31 1:29 1:34
Cannabidivarinic acid (CBDV-A), Cannabidivarin (CBDV), Tetrahydrocannabivarin (THCV), Cannabidiolic acid (CBD-A), Cannabidiol (CBD), Cannabigerolic acid (CBG-A), Cannabinol (CBN), (−)-trans-Δ9-tetrahydrocannabinol (Δ9-THC), Δ9-tetrahydrocannabinolic acid (Δ9-THC-A), Cannabichromene (CBC), B (bracts), WI (whole inflorescence), ND (not detected).
expected. Based on the obtained total CBD and total Δ9-THC values in whole inflorescences, the variety Santhica 27 is a less appropriate variety for CBD production since both cannabinoids were present in minimal amounts.
due to the diverse nature of cannabis varieties, more plants within the sample population represent reliable results. Influence of Slovenian soil and climate on selected varieties was not studied up to now, which makes comparisons of the content of CBD and Δ9-THC from whole inflorescences in year 2018 with declared values published in publications (summarized in Table 1) interesting. For the varieties Fedora 17, USO 31, Tisza, Tiborszallasi, and Antal comparable values of cannabinoid concentrations were found. From this it can be concluded that varieties reacted to the cultivation environment as
3.2. Ratio of total Δ9-THC/total CBD The ratio of total Δ9-THC/total CBD can primarily be used for the identification of fiber or the indica type of the hemp. A ratio of total Δ9-
Fig. 2. Cannabinoid content from bracts: average value of three inflorescences from 2017 (mean value %w/w with standard deviation) and value in one inflorescence from 2018 (single value %w/w): A.) total CBD (Cannabidiol), B.) total Δ9-THC ((−)-trans-Δ9-tetrahydrocannabinol). 7
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Fig. 3. Content of cannabinoids in bracts (average value of inflorescences from three plants) and in parts from the rest of inflorescences, study from 2017 (mean value %w/w with standard deviation): A.) total CBD (Cannabidiol), B.) total Δ9-THC ((−)-trans-Δ9-tetrahydrocannabinol).
THC/total CBD lower than 1.0 is attributed to “fiber-type’’ plants (Hillig and Mahlberg, 2004), which applies to all varieties considered in our study. This ratio can also be used to measure seed uniformity. This is important information for breeders, who must know if genetic sowing material is consistent. When compared, the ratio of total Δ9-THC /total CBD in bracts from the first, second, third upper, and third lower flower as shown in Table 4 had high uniformity for the varieties Antal (with standard deviation ± 0), Helena ( ± 1), Monoica ( ± 1), Kompolti hibrid TC ( ± 1), Finola ( ± 2), Ferimon ( ± 3), and USO 31 ( ± 3) has been recognized. Varieties with low uniformity were found to be Carmagnola ( ± 15), KC Dora ( ± 13), Tiborszallasi ( ± 13), and Tisza ( ± 12). In order to confirm these findings, the ratio of total Δ9-THC/total CBD from bracts and from whole inflorescences from 2018 (Table 5) was added for comparison. When averaging all values, it was found that the varieties Helena ( ± 1), Kompolti hibrid TC ( ± 1), Finola ( ± 2), Monoica ( ± 2), and USO 31 ( ± 3) are highly uniform between both seasons. For the mentioned varieties we could assume that homogenous plant genetics are advantageous for farmers that are cultivating hemp for production of CBD, since consistent content was shown between both years. Low uniformity was detected with the same varieties as listed above when compared inside year 2017. For the varieties Novosadska, Marina, and Ferimon ratios could not be compared since these varieties were sown in only one season. The varieties Novosadska and Marina showed high uniformity between the ratio of total Δ9-THC/ total CBD in bracts and whole inflorescence within year 2018.
The concentration of CBDV-A in 2017 was represented in all varieties. In Carmagnola (0.205 % ± 0.218) and Antal (0.118 % ± 0.056) levels were higher since in other varieties concentrations were found to be lower than 0.07 %. In 2018 the highest levels of CBDV-A was found in Fedora 17 (0.230 %). In other varieties, concentrations were found to be under < 0.07 % or even nondetectable (Santhica 27 and KC Dora). Only in Fedora 17, CBDV was detected (0.120 % in 2018); in others varieties it was undetectable during both years. In trace amounts CBC was detected (< 0.05 %) or even not detected in 2017. Interestingly, in 2018 CBC was detected in higher quantities in the varieties KC Dora (0.173 %), Antal (0.159 %), Carmagnola (0.140 %) and Tiborszallasi (0.102 %), while in other varieties the levels were less than < 0.1 %. From the results it can be concluded that CBC content is higher in less mature flowers since flowers in 2018 were harvested earlier than those in 2017. Trace amounts of CBN were detected in both years (< 0.02 %) or were undetectable. Since CBN is an oxidative product of Δ9-THC, which is more prominent in aged cannabis samples (Russo, 2011), (Andre et al., 2016), results suggest that plants were collected at their maturity. The content of the cannabinoid CBL rises by exposure of CBC to UVradiation (Hazekamp, 2007). Results from both years indicate that storage of flowers after harvesting was appropriate (with no exposure to light), since the concentration of CBL was found to be undetectable in 2017 and 2018. Cannabinoids Δ8-THC and THCV were not detected at any of the varieties in either year. 4. Conclusion
3.3. Other cannabinoids in bracts The response of hemp varieties used in research regarding growth conditions in Slovenia are not well known since they are produced in different European countries or breeding programs in geographical areas. The purpose of this study was to compare and to show the chemical profile of cannabinoids in varieties that were accessible to our research group. The major focus of the study was on the total concentration of CBD and Δ9-THC in the bracts of fertilized female flowers. Some of them were already over the phase of their vegetative cycle and were therefore treated as the cannabinoid production reached the plateau phase. From the results presented in this article it can be concluded that:
The following section describes the content of cannabinoids in 2017 and 2018 found in bracts (Tables 3–5). Besides the total CBD and total Δ9-THC in 2017, the most represented cannabinoid was found to be CBG-A with the highest content in Santhica 27 (1.857 % ± 1.256), which did not contain significant amounts of CBD, CBD-A, THC, or Δ9-THC-A. It is known the THCA- and CBDA-synthases that metabolize cannabinoids from CBG-A are not well expressed. This could be described and confirmed with results from whole inflorescence and bracts in 2018, where the concentration of CBG-A in Santhica 27 was found to be 3.566 % and levels of CBD-A and Δ9-THC-A were minimal. Cannabinoid CBG was not detected in any varieties in either year. Relatively high CBG-A content in Antal (0.497 % ± 0.088 in 2017, 0.491 % in 2018) and in Tiborszallasi (0.362 % in 2018) could show immaturity of the flowers. Concentration levels of CBG-A lower than 0.3 % correspond to others varieties for both years.
• The response of hemp varieties to growth conditions in Slovenia was
good in the sense of CBD production in the varieties Fedora 17, USO 31, Tisza, Tiborszallasi, and Antal since the values of CBD and Δ9THC in whole inflorescences were found to be comparable to
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declared values.
Declaration of Competing Interest
production since only trace amounts of CBD and Δ9-THC were detected. High uniformity of the seeds, according to preserved ratio of total Δ9-THC/total CBD between years, was found with the varieties Kompolti hibrid TC, USO 31, Finola, Helena, and Monoica. Low uniformity was found in the varieties Carmagnola, Tiborszallasi, Tisza, and KC Dora. For these varieties, it is possible that infinite phenotypic change occurred. All tested varieties belong to the ‘fiber-type’’ plants according to the ratio of total Δ9-THC/total CBD. No significant differences in cannabinoid concentration were found between bracts from upper and lower inflorescence parts of hemp. It can be assumed that for all varieties the < 0.2 % limit of total Δ9THC will not be exceeded with appropriate sampling. The concentration of total CBD and total Δ9-THC proved to be highest in bracts from inflorescence. It was found that the concentration was on average four times higher compared with the rest parts of the inflorescence (without bracts) and up to four times higher compared with the concentration of the whole inflorescence. High ratios (> 1:27) of total Δ9-THC/total CBD in bracts from both years were found in the varieties USO 31, Monoica, Helena, Finola, and Kompolti hibrid TC.
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
• The variety Santhica 27 was found to not be appropriate for CBD •
• • • • •
Acknowledgements Research was supported by the Slovenian Research Agency and Slovenian Ministry of Agriculture, Forestry and Food (research project reference: V4-1611). We would like to offer special thanks to Matevž Štefančič, for his laboratory work. References Andre, C.M., Hausman, J.-F., Guerriero, G., 2016. Cannabis sativa: the plant of the thousand and one molecules. Front. Plant Sci. 7, 1–17. https://doi.org/10.3389/fpls. 2016.00019. Fischedick, J.T., Hazekamp, A., Erkelens, T., Choi, Y.H., Verpoorte, R., 2010. Metabolic fingerprinting of Cannabis sativa L., cannabinoids and terpenoids for chemotaxonomic and drug standardization purposes. Phytochemistry 71, 2058–2073. https://doi.org/ 10.1016/j.phytochem.2010.10.001. Hazekamp, A., 2007. Cannabis; Extracting the Medicine. Dr Thesis. Department of Pharmacognosy (IBL), Faculty of Science, Leiden Universityhttps://doi.org/10.1081/ JLC-200028170. Hillig, K.W., Mahlberg, P.G., 2004. A chemotaxonomic analysis of cannabinoid variation in Cannabis (Cannabaceae). Am. J. Bot. 91, 966–975. https://doi.org/10.1002/cncr. 25812. Ihempfarms [WWW Document], (2019) n.d. URL www.ihempfarms.com/ (accessed 6. 4.19). Namdar, D., Mazuz, M., Ion, A., Koltai, H., 2018. Variation in the compositions of cannabinoid and terpenoids in Cannabis sativa derived from inflorescence position along the stem and extraction methods. Ind. Crop. Prod. 113, 376–382. https://doi.org/10. 1016/j.indcrop.2018.01.060. Pacifico, D., Miselli, F., Carboni, A., Moschella, A., Mandolino, G., 2008. Time course of cannabinoid accumulation and chemotype development during the growth of Cannabis sativa L. Euphytica 160, 231–240. https://doi.org/10.1007/s10681-0079543-y. Pertwee, R.G., 2014. Handbook of Cannabis, 1st ed. Oxford University Presshttps://doi. org/10.1093/acprof:oso/9780199662685.001.0001. Potter, J.P., D, 2009. The Propagation, Characterisation and Optimisation of Cannabis sativa L as Phytopharmaceutical. Department of Pharmaceutical Science Research. King’s College London. Regulation (EU) No 809/2014, 2014. Off J Eur Union. pp. 69–124. Rules concerning the requirements for obtaining permission to cultivate hemp, 2004. Off Gaz Repub Slov 44/04. Rules on conditions for obtaining a permit for hemp and poppy cultivation, 2018. Off Gaz Repub Slov 40/11. Russo, E.B., 2011. Taming THC: potential cannabis synergy and phytocannabinoid-terpenoid entourage effects. Brit. J. Pharmacol. 163, 1344–1364. https://doi.org/10. 1111/j.1476-5381.2011.01238.x. Russo, E.B., Marcu, J., 2017. Cannabis Pharmacology: The Usual Suspects and a Few Promising Leads, 1st ed. Adv Pharmacol. Elsevier Inc.https://doi.org/10.1016/bs. apha.2017.03.004. Thomas, B.F., Elsohly, M.A., 2016. The Analytical Chemistry of Cannabis, 1st ed. Elsevier Inchttps://doi.org/10.1016/C2014-0-03861-0. Turner, J.C., Hemphill, J.K., Mahlberg, P.G., 1981. Interrelationships of glandular trichomes and cannabinoid content. I. Developing pistillate bracts of Cannabis sativa L. (Cannabaceae). B Narcotics 33, 59–69. Zadruga konopko [WWW Document], n.d. URL http://www.konopko.si/ (accessed 6. 4.19).
The use of genetically identical plant material from representative clones under strictly controlled environmental conditions could ensure reproducible cannabis material by reaching the same growth stages and by controlling glandular trichomes profiles. Chemical differences can be seen within the growth cycle, harvesting, and storage due to environmental conditions. The major focus should be genotype consistency of a specific chemical profile of cannabis to produce a reproducible and stable chemical composition (Fischedick et al., 2010). With this information, the farmers and breeders will know more precisely what the risks and benefits of cultivating a particular variety are in terms of compliance with Slovenian legislation, depending on the production purpose. This could enable a better understanding and an in-depth knowledge of cannabis cultivation in Slovenia for increased yields of dioecious and monoecious varieties. CRediT authorship contribution statement Taja Glivar: Conceptualization, Formal analysis, Data curation, Writing - original draft, Visualization. Jan Eržen: Methodology, Investigation. Samo Kreft: Formal analysis, Resources, Writing - review & editing. Marjeta Zagožen: Investigation. Andreja Čerenak: Writing - review & editing. Barbara Čeh: Resources, Writing - review & editing, Project administration, Funding acquisition. Eva Tavčar Benković: Conceptualization, Methodology, Validation, Writing - review & editing, Supervision.
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Cannabinoid content in industrial hemp (Cannabis sativa L.) varieties grown in Slovenia
T
Taja Glivara,*, Jan Erženb, Samo Kreftc, Marjeta Zagožend, Andreja Čerenakd, Barbara Čehd, Eva Tavčar Benkovićb,c a
Biotechnical Faculty, University of Ljubljana, Jamnikarjeva ulica 101, 1000, Ljubljana, Slovenia Freyherr d.o.o., Kersnikova 10, 1000, Ljubljana, Slovenia c Faculty of Pharmacy, University of Ljubljana, Aškerčeva cesta 7, 1000, Ljubljana, Slovenia d Slovenian Institute of Hop Research and Brewing, Cesta Žalskega tabora 2, 3310, Žalec, Slovenia b
A R T I C LE I N FO
A B S T R A C T
Keywords: Cannabis sativa L. Hemp Cannabinoids THC CBD Varieties
Cannabinoid content in different hemp varieties from the Common catalogue of varieties of agricultural plant species is not well known. Hemp (Cannabis sativa L., subsp. sativa) contains a wide range of cannabinoids, where cannabidiol (CBD) and (−)-trans-Δ9-tetrahydrocannabinol (Δ9-THC) are the constituents with known therapeutic activity. Also, Δ9-THC is recognized as an illicit drug; therefore, cultivation of hemp is restricted to a 0.2 % limit of THC content in many European countries. In this study, the cannabinoid profiles of 15 hemp varieties, accessible to our research group, were analysed. The content of 13 cannabinoids was determined with HLPC (highperformance liquid chromatography) analysis. Large variations in cannabinoid content among varieties that grew in uniform conditions (ANOVA p < 0.05) and also within a single variety were found, which shows on ununiform genetic profiles of the seed material. The varieties Fedora 17, USO 31, Tisza, Tiborszallasi, and Antal all displayed good response to growth conditions, related to cannabinoid content, in Slovenia.
1. Introduction Hemp (Cannabis sativa L. subsp. sativa) is a subspecies of the genus Cannabis (Cannabaceae), which also includes medicinal cannabis (Cannabis sativa L. subsp. indica). It is an annual plant that occurs in versatile forms, depending on the variety and availability of space for growth. It is mostly dioecious, meaning that the male and female flowers are located on separate plants. Seeds can only be found on female plants. Cannabis plants contains two main cannabinoids; one is a psychoactive cannabinoid substance (−)-trans-Δ9-tetrahydrocannabinol, better known as THC, which earns medicinal cannabis its classification as an illicit drug. The second is cannabidiol (CBD), represented primarily in hemp. Cannabidiol is known as a pharmacologically active substance and is becoming more and more important for use in medicinal applications. A broad chemical profile of cannabinoids, that can be found in cannabis, exhibits the typical C21 terpenophenolic skeleton, including their derivatives and transformation products (Pertwee, 2014). Cannabinoids are initially formed as carboxylic acids (eg, Δ9-
tetrahydrocannabinolic acid (Δ9-THC-A), cannabidiolic acid (CBD-A), cannabichromenic acid (CBC-A), and tetrahydrocannabivarin carboxylic acid (Δ9-THCV-A)) that, when heated, are decarboxylated to their corresponding neutral forms. Another process caused by heating, light exposure or ageing, is oxidative degradation, wherein the conversion of Δ9-THC to cannabinol (CBN) and isomerization of Δ9-THC to Δ8-tetrahydrocannabinol (Δ8-THC) occurs. The concentrations of cannabinolic acid (CBN-A) and CBN, primary chemical degradants of Δ9THC-A and Δ9-THC, increase during storage, if exposed to light at 22 °C as compared with storage in darkness at 4 °C. The concentration of CBDA, when exposed to light and 22 °C, decreases with cyclization from CBD-A to Δ9-THC-A, followed by the degradation of Δ9-THC-A to CBN-A (Thomas and Elsohly, 2016). Cannabinoid production in hemp can be affected by numerous biotic and abiotic factors such as the sex and maturity of the plant, daily light integral, ambient temperature, nutrient availability, and intensity of ultraviolet light (Hillig and Mahlberg, 2004). The content of cannabinoids was found to be differently distributed in plant parts with the highest percentage in secretory cells inside glandular trichomes (up to 60 %), highly concentrated in unpollinated female flowers prior to
⁎
Corresponding author. E-mail addresses: [email protected] (T. Glivar), [email protected] (J. Eržen), samo.kreft@ffa.uni-lj.si (S. Kreft), [email protected] (M. Zagožen), [email protected] (A. Čerenak), [email protected] (B. Čeh), [email protected] (E. Tavčar Benković). https://doi.org/10.1016/j.indcrop.2019.112082 Received 22 August 2019; Received in revised form 22 December 2019; Accepted 29 December 2019 0926-6690/ © 2019 Elsevier B.V. All rights reserved.
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senescence (up to 30 %), pollinated flowers (up to 13 %), leaves (0.05 %), and least in the stem (0.02 %). No cannabinoids have been found in the roots or seeds (Russo and Marcu, 2017) (Russo, 2011). The highest cannabinoid concentration (in % of dry weight plant material) can be found in the bracts of the flowers that contain abundant glandular trichomes (Turner et al., 1981). Better knowledge of the cannabinoid production in glandular trichomes would help the breeder to identify the optimum time for harvest (Potter and D., 2009). Mature female plants are preferred for cannabis extract production, since they produce higher amounts of cannabinoids. The highest yield of cannabinoids are achieved if grown without male plants to prevent pollination and the formation of seeds (Thomas and Elsohly, 2016). Hemp is becoming well-known for its versatile use and great economic importance. Recently, there has been a dramatic increase in CBD-rich supplementation in the food supplement and cosmetic industries. Even more potential is reported on the pharmaceutical use of cannabinoids. Since the pharmaceutical industry requires the highest quality of materials, it is of the upmost importance to ensure consistency in the chemical profiles of plants intended for such use (Thomas and Elsohly, 2016). Chemotypes are highly dependent on the genetics, and in addition the plants are exposed to many natural factors, which can be very variable among seeds within a single variety. As a result, a constant profile of cannabinoids is not easy to achieve with sowing seed. The best way for the propagation to produce uniform plants is with cuttings (clones). Current legislation in Slovenia allows cultivation of hemp for the purpose of seeds production and further propagation, production of food and beverages, the production of substances for cosmetic purposes, production of fibres, animal feed, and for other industrial purposes. This applies to all varieties listed in the Common catalogue of varieties of agricultural plant species, whose THC content does not exceed 0.2 % (“Rules on conditions for obtaining a permit for hemp and poppy cultivation,” 2018) in the upper third part of the dried plant or in a 30 cm dried plant part, containing at least one female inflorescence (“Regulation (EU) No 809/2014,” 2014Regulation (EU) No 809/, 2014Regulation (EU) No 809/2014,” 2014). The aim of this study was to determine the chemical profile of 13 cannabinoids in 15 different hemp varieties, grown in Slovenia.
In 2017, three inflorescences, each from different female plants of each variety were studied. Table 2 illustrates the experimental procedure of the sample preparation. The preparation of the samples began with careful removal of the seeds from each inflorescence. The bracts were separated from the stems, leaflets, and supporting leaves. Stems, leaflets, and supporting leaves of the inflorescence were ground with a laboratory grinder (30 s, 20.000 rpm) to a size of ≤ 0.5 mm. Samples from the first and second plants were prepared identically, while the third inflorescence was additionally separated into upper and lower parts. The third flower of the varieties Finola and Ferimon was not separated into an upper and lower part; it was analysed as a whole flower since not enough material was available. Developing seeds were present in most plants, so if the seeds would not be removed the mass ratio of seeds to other parts of the inflorescences would differ from plant to plant, which would influence the representability of the results of cannabinoid content. By removal of the seeds representative results were achieved. For additional comparison, bracts were used to obtain the most accurate and comparable results on cannabinoid profiles among the plants. Also, glandular trichomes contained the highest content of cannabinoids and were mostly located on the bracts (Turner et al., 1981). In 2018, one inflorescence of each variety was prepared according to the procedure described above, but only bracts were analysed. For the second sample, inflorescences from another plant of each variety were used with prior removal of seeds and ground with a laboratory grinder (30 s, 20.000 rpm) to a size of ≤ 0.5 mm as shown in Table 2.
2. Materials and methods
2.3. Cannabinoid content evaluation
2.1. Plant material
The following cannabinoids were analysed: Cannabidivarinic acid (CBDV-A), Cannabidivarin (CBDV), Tetrahydrocannabivarin (THCV), Cannabidiolic acid (CBD-A), Cannabidiol (CBD), Cannabigerolic acid (CBG-A), Cannabigerol (CBG), Cannabinol (CBN), (−)trans-Δ9-tetrahydrocannabinol (Δ9-THC), Δ8-tetrahydrocannabinol (Δ8-THC), Cannabicyclol (CBL), Δ9-tetrahydrocannabinolic acid (Δ9-THC-A), and Cannabichromene (CBC).
were not irrigated during the growth phase. Compared with the year 2017, 2018 was cooler with more precipitation (Fig. 1B). Sampling of the female flowers was carried out on the 30 August 2018. The vegetation period lasted for 93 days. Plants were dried, transferred, and stored with the same procedure as in 2017. In this study, only few plants were sampled (see Section 2.2), while 50 plants/ha should be sampled according to the legislation directive and the average content should not exceed 0.2 % (“Rules concerning the requirements for obtaining permission to cultivate hemp,” 2004). 2.2. Preparation of plant material for chemical characterization
A total of 15 industrial hemp varieties (Cannabis sativa L., subsp. sativa) from the Common catalogue of varieties of agricultural plant species were grown on the fields of the Slovenian Institute of Hop Research and Brewing (IHPS) in Žalec in the year 2017 and in Gornja Radgona (approximately 100 km from Žalec) in 2018. The plants were collected at the phase of maturity at which most Slovenian hemp farmers usually harvest. The seeds of the same producer were used for each variety in both years. Properties of investigated varieties, obtained from producers, web pages (“Zadruga konopko, 2019),(“Ihempfarms, 2019). and data source (Pacifico et al., 2008), are summarized in Table 1. On the 3 May 2017, 13 varieties of hemp (Table 1), were sown on 9 m2 large parcels, with each variety in a separate plot, but with no physical obstacles between them. The plants were not irrigated during the growth phase. During the growth period, the weather was warmer than usual; this was one of the warmest summers in Žalec since 1961 (Fig. 1A). The vegetation phase lasted for 135 days. Sampling of the female plants was carried out on the 15 September 2017. Plants were dried for 24 h at 35 °C and transferred to the analytical laboratory at the Faculty of Pharmacy, University of Ljubljana, to be stored in a cold room in paper bags, protected from light until preparation for analytical testing. The second study began on the 29 May 2018, when 14 varieties of hemp (Table 1) were sown. The parcels were 2 m2 large. The plants
2.4. Cannabinoid extraction The samples were extracted by adding 10 mL of ethanol (96 %, Kefo Slovenia) to the 100−200 mg of the accurately weighted ground or non-ground material in closed plastic tubes. Samples were incubated for 30 min in an ultrasonic bath at 50 °C. The supernatant was removed and filtered through a 25 μm filter in the HPLC (high-performance liquid chromatography) vial. 2.5. Chromatographic analysis Samples were analysed using HPLC as described in one of our other articles (submitted for publication). The amount of each cannabinoid (%w/w [%mg of cannabinoid/100 mg herbal drug]) in the original sample was calculated by using calibration curves of standard compounds. Additionally, to show the total concentration of Δ9-THC, CBD, and 2
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Table 1 Basic characteristics of hemp varieties. Variety
Grown in research study in years
Country origin
Genotypic expression
Climate adaptation
Vegetative cycle
CBD content
Δ9-THC content
Fedora 17 KC Dora Monoica USO 31 Helenaa
2017 2017 2017 2017 2017
and and and and and
2018 2018 2018 2018 2018
France Hungary Hungary Germany Serbia
Monoecious Dioecious Dioecious Monoecious Dioecious
Atlantic Continental Continental Atlantic No data available
Early < 125 days Late < 145 days Medium < 135 days Early < 125 days Early < 125 days
< 0.06 % < 0.12 % < 0.12 % < 0.06 % No data available
Santhica 27 Tisza Tiborszallasi Antalb Carmagnolac
2017 2017 2017 2017 2017
and and and and and
2018 2018 2018 2018 2018
France Hungary Hungary Hungary Italy
Monoecious Dioecious Dioecious Dioecious Dioecious
Atlantic Continental Continental Continental Mediterranean
Medium < 135 days Late < 145 days Late < 145 days Late < 145 days Very late < 160 days
Finola
2017 and 2018
Finland
Dioecious
Continental
Kompolti hibrid TC Ferimon Novosadskaa
2017 and 2018 2017 2018
Hungary France Serbia
Dioecious Monoecious No data available
Continental Atlantic No data available
Very early < 110 days Medium < 135 days Early < 125 days Very late < 160 days
Marinaa
2018
Serbia
Dioecious
No data available
Late < 145 days
1.5 – 2.0 % 1.5 – 2.0 % 1.5 – 2.0 % 0.5 – 1.0 % No data available 1.0–1.5 % 2.0–3.0 % 2.0–3.0 % 2.0–3.0 % No data available No data available 2.0–3.0 % 1.0–1.5 % No data available No data available
< < < < <
0.02 0.12 0.20 0.12 0.30
% % % % %
No data available < 0.12 % < 0.06 % No data available No data available
Cannabidiol (CBD), (−)-trans-Δ9-tetrahydrocannabinol (Δ9-THC). a Varieties Novosadska, Marina, and Helena are new and have never been included into the Common catalogue of varieties of agricultural plant species. Data regarding those varieties was gained at producers. b Antal was removed from the Common catalogue of varieties of agricultural plant species after experiments were completed. c Carmagnola was deleted from Common catalogue of varieties of agricultural plant species in 2019, with market extension until 30 June 2021.
3. Results and discussion
CBG in %w/w [%mg of cannabinoid/100 mg herbal drug], the following equations were used:
The cannabinoid content of varieties grown in 2017 and 2018 are summarized in Tables 3,4, and 5. Additionally, total CBD, total Δ9-THC, total CBG, and the ratio of total Δ9-THC/total CBD content is presented. The differences in the content of CBD-A, CBG-A, CBN, Δ9-THC-A, CBC, total CBD, total CBG, and total Δ9-THC between the varieties were significant (ANOVA p < 0.05). The content of remaining cannabinoids were not significantly different between the varieties (ANOVA p > 0.05).
% of Total Δ9-THC = % Δ9-THC + (% Δ9-THC-A x 0.877) % of Total CBD = % CBD + (% CBD-A x 0.877) % of Total CBG = % CBG + (% CBG-A x 0.878) The conversion factors 0.877 and 0.878 represent a loss of molecular mass during the decarboxylation reaction (transformation of carboxylic acid derivatives [Δ9-THC-A, CBD-A, CBG-A] to non-carboxylic acid forms [Δ9-THC, CBD, CBG]).
3.1. Total CBD and total Δ9-THC content 2.6. Statistical analysis It is well known that Cannabis sativa L. is a highly variable species in terms of botany, genetics, and its chemical profile (Thomas and Elsohly, 2016). Considering that higher parts of the inflorescences are more exposed to sunlight and exhibit differential hormonal distribution than lower parts, it is expected that cannabinoid concentration in upper parts would be higher compared with lower parts. Variability between the upper part and the lower part of the inflorescence was examined in the year 2017, as summarized in Table 4. Evaluation was carried out
All statistical analyses were performed by using Excel software Office Home and GraphPad Prism 6. Concentrations, mean values, and standard deviations were calculated with Excel. In addition, for results from study in 2017 (number of repetitions: N = 3), statistical analysis ANOVA and t-tests, were performed with IBM SPSS. For study from 2018, results have not been statistically proceeded since no repetitions were done (N = 1).
Fig. 1. Weather conditions of precipitation and temperature (average of the Tmin and Tmax of the daily temperature) during the growth period presented in decades (data source: ARSO - Slovenian Environment Agency): A.) from May to September in 2017 in Žalec, compared to the average of 30 years, B.) from May to August in 2018 in Gornja Radgona, compared to the average of 49 years. 3
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Table 2 Experimental design in 2017 and 2018 for inflorescences presenting year of sowing, sequential flower, part of the flower, and sample condition. Year
Inflorescence
Part of flower
Grinded/non-grinded sample
2017
first first second second third third third third first second
Bracts from whole inflorescence Rest of the inflorescence Bracts from whole inflorescence Rest of the inflorescence Bracts from upper inflorescence part Bracts from lower inflorescence part Rest of the upper inflorescence part Rest of the lower inflorescence part Bracts from whole inflorescence Whole inflorescence
non-grinded grinded non-grinded grinded non-grinded non-grinded grinded grinded non-grinded grinded
2018
with comparison of the total CBD and total Δ9-THC content, as their ratios are the most interesting of all the analyzed cannabinoids. The distribution of total CBD in bracts from the upper and lower inflorescence parts was found to be equal. A considerable difference was observed only with the variety Antal (upper part 5.970 %, lower part 13.460 %, mean 9.715 % ± 3.745). If compared with the results from bracts of whole inflorescence (of first and second flower) analysis (mean value was found to be 9.513 % ± 1.572), results are similar. The difference could be explained by ununiformed quality of the inflorescences. It could be theorized that the upper parts stopped maturing during growth because of excessive sunlight exposure. Concentration of total Δ9-THC had shown no important differences between parts of the inflorescences for any variety. With two varieties, Tiborszallasi (mean value 3.304 % ± 0.097) and Carmagnola (mean value 2.457 % ± 0.089), extreme values of high total Δ9-THC content were detected. High values are presumably due to the non-uniform genetic material, possibly due to cross-pollination in a previous generation between seed-producing female plants and male plants from neighboring fields with contrasting genetics, causing higher Δ9-THC content. According to the obtained results, it can be concluded that no significant differences in the total Δ9-THC/total CBD content between the upper and lower bracts of the uppermost inflorescences was observed (paired Student t-test: p = 0.44). Additionally, in a research study by Namdar et al. where cannabinoids were analysed in the uppermost, middle, and lowermost inflorescences of hemp, it has been found that the uppermost inflorescences contained a higher content of cannabinoids compared with the lowermost inflorescences (Namdar et al., 2018). In this study, the uppermost inflorescences were used. The highest content of total CBD (> 5 % reported as %w/w) produced in both years (Table 4 and Table 5) was found to be in the bracts of the varieties Antal (9.580 % ± 1.287 in 2017 and 6.159 % in 2018), Carmagnola (7.069 % ± 3.339 in 2017 and 5.726 % in 2018), Helena (5.873 % ± 1.816 in 2017 and 8.108 % in 2018), and Tiborszallasi (4.666 % ± 1.931 and 4.876 % in 2018). The lowest total CBD content was detected with the varieties USO 31 (1.207 % ± 0.356 in 2017 and 0.735 % in 2018) and Santhica 27 (0.038 % ± 0.042 in 2017 and 0.010 % in 2018) (Fig. 2A). Important information about a variety is its uniformity in terms of cannabinoid content (chemotype), which can be evaluated with the standard deviation in total CBD, in our case from inflorescences from three different plants in experiment from 2017. High uniformity within the population (standard deviation < 0.5) was found in Fedora 17, USO 31, and Santhica 27 (Table 4). The varieties Carmagnola, Monoica, Helena, Tisza, and Tiborszallasi displayed low uniformity of the seeds with high standard deviation (sd > 2) within the population (Table 4). In hemp cultivation, special focus must also be given to the allowed concentration of Δ9-THC as different countries have different regulations. The limit of 0.2 % Δ9-THC is prescribed by Slovenian legislation (“Rules on conditions for obtaining a permit for hemp and poppy cultivation,” 2018). The highest content of total Δ9-THC (> 0.2 %) was
detected in bracts of the following varieties in 2017: Tiborszallasi (2.509 % ± 1.627), Carmagnola (1.015 % ± 1.020), Tisza (0.895 % ± 1.004), KC Dora (0.812 % ± 0.991), and Antal (0.317 % ± 0.042). Other varieties showed high uniformity (sd < 0.07) and lower total THC content (< 0.2 %) (Fig. 2B) (Table 4). Results from 2018 confirm that levels of total Δ9-THC in bracts for all listed varieties except for Tiborszallasi (0.172 %) exceeded suitable quantities, with the addition of the variety Helena (0.280 %) with a value slightly above the limit (Fig. 2B) (Table 5). It was shown that total Δ9-THC and total CBD reached higher concentrations in 2017 for the majority of the varieties. In 2017, environmental conditions were warmer and dryer, compared with year 2018. From this study it can be concluded that these environmental conditions are more suitable for CBD production, but it could not be summarized, which of the above mentioned weather parameters contributed most to the higher content of total CBD. The data of total Δ9THC content in the bracts are higher than they would be with mechanical harvesting since with manual harvesting only inflorescences were removed and agitation of the inflorescences is minimized. With mechanical harvesting of bigger stems, their contribution to the final weight of the sample is added. In order to confirm that total Δ9-THC values do not exceed the allowed levels, we verified the content of total CBD and total Δ9-THC in the remaining parts of the inflorescences (stems, leaflets, and supporting leaves) compared with bracts from inflorescences in the study from 2017, as shown in Figs. 3A and 3B. Information gathered from publications indicate that it is expected that cannabinoid ratios between isolated glandular trichomes and whole pollinated flowers is approximately 1:4 (Russo and Marcu, 2017) in favor of glandular trichomes. From our results, as shown by evidence in Figs. 3A and 3B, it can be proven that the content of total CBD and total Δ9-THC in the bracts is between two to eight times (on average four times) higher than in other parts of the inflorescence (without bracts). It can be stated that the remaining parts of the inflorescence present an additional mass with lower cannabinoid concentration. In the case of analysing the whole inflorescence, leaflets and stems present a contributing factor to detection of lower cannabinoid concentration than the ones detected in bracts. To further confirm this finding, in 2018, cannabinoid comparisons between bracts and whole inflorescence (with bracts, stems, leaflets, and supporting leaves) had been performed (Table 5). Results showed up to four times lower values detected in whole inflorescences (with the exception of USO 31). Also, Turner et al. reported about the highest levels of cannabinoids being found in bracts (Turner et al., 1981). The content of total Δ9-THC in case of whole inflorescences would exceed the limit of < 0.2 % only with KC Dora (0.878 %). According to previous findings regarding the KC Dora uniformity, this phenomenon is probably caused due to a different chemotype of the variety. Relating to the above facts it can be assumed that, according to current Slovenian legislation, varieties wouldn’t presumably exceed the required limit, especially when plants are harvested mechanically, since
4
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Table 3 Cannabinoid content of bracts from the first, second, and third flower from a study in the year 2017, with results of statistical analysis performed by ANOVA. Cannabinoids CBG (Cannabigerol), CBDV (Cannabidivarin), THCV (Tetrahydrocannabivarin), CBL (Cannabicyclol), and Δ8-THC (Δ8-tetrahydrocannabinol) were not detected in any of the varieties. The values of the third flower was previously averaged (up and low) before the total averaging and the calculation of the standard deviation was performed. Variety
Flower
first second thirdup thirdlow Mean ± KC Dora first second thirdup thirdlow Mean ± Monoica first second thirdup thirdlow Mean ± USO 31 first second thirdup thirdlow Mean ± Helena first second thirdup thirdlow Mean ± Santhica 27 first second thirdup thirdlow Mean ± Tisza first second thirdup thirdlow Mean ± Tiborszallasi first second thirdup thirdlow Mean ± Antal first second thirdup thirdlow Mean ± Carmagnola first second thirdup thirdlow Mean ± Kompolti hibrid first TC second thirdup thirdlow Mean ± Finola first second thirda Mean ± Ferimon first second thirda Mean ± p (ANOVA of the differences between varieties)
Fedora 17
sd
sd
sd
sd
sd
sd
sd
sd
sd
sd
sd
sd
sd
CBDV-A (%w/w)
CBD-A (%w/w)
CBD (%w/w)
CBG-A (%w/w)
CBN (%w/w)
Δ9-THC (%w/w)
Δ9-THC-A (%w/w)
CBC (%w/w)
0.112 0.016 0.016 0.020 0.049 0.016 0.007 0.051 0.040 0.023 0.050 0.020 0.018 0.015 0.029 0.026 0.009 0.021 0.018 0.018 0.032 0.032 0.045 0.034 0.034 0.002 ND 0.003 0.003 0.002 0.024 0.017 0.020 0.020 0.020 0.026 0.024 0.023 0.017 0.023 0.193 0.058 0.110 0.098 0.118 0.052 0.050 0.528 0.498 0.205 0.017 0.015 0.036 0.042 0.024 0.052 0.115 0.050 0.072 0.024 0.017 0.018 0.020 0.192
1.587 2.281 2.040 3.520 2.216 ± 0.489 2.232 2.699 5.618 4.351 3.305 ± 1.203 5.113 9.819 6.993 6.685 7.257 ± 1.944 0.785 1.224 1.452 1.563 1.172 ± 0.297 8.618 6.863 4.190 3.392 6.424 ± 1.995 0.009 0.110 0.006 0.014 0.043 ± 0.047 3.466 8.398 3.480 3.421 5.105 ± 2.329 4.241 7.667 3.182 2.925 4.987 ± 1.956 8.747 12.119 6.420 14.743 10.483 ± 1.378 8.459 10.918 2.840 2.739 7.389 ± 3.404 3.882 4.251 5.884 6.433 4.764 ± 0.998 1.126 1.727 1.659 1.504 ± 0.269 4.455 6.096 4.719 5.090 ± 0.719 0.001
0.071 0.416 0.071 0.132 0.196 0.407 0.440 0.136 0.088 0.320 0.168 0.566 0.340 0.279 0.348 0.027 0.472 0.008 0.071 0.180 0.259 0.335 0.147 0.100 0.239 ND ND ND ND ND 0.081 0.472 0.112 0.109 0.221 0.156 0.601 0.132 0.105 0.292 0.270 0.456 0.340 0.530 0.387 0.789 0.893 0.085 0.082 0.589 0.094 0.242 0.189 0.209 0.178 0.670 1.413 0.187 0.757 0.250 0.521 0.275 0.349 0.164
0.021 0.040 0.029 0.044 0.032 0.039 0.095 0.217 0.114 0.100 0.173 0.343 0.145 0.107 0.214 0.012 0.029 0.033 0.038 0.025 0.184 0.244 0.212 0.124 0.199 0.090 2.585 2.371 3.424 1.857 0.185 0.188 0.189 0.183 0.186 0.417 0.210 0.208 0.190 0.275 0.598 0.512 0.263 0.503 0.497 0.154 0.211 0.054 0.076 0.143 0.072 0.438 0.473 0.334 0.305 0.009 0.011 0.007 0.009 0.045 0.023 0.064 0.044 0.002
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 0.003 0.005 ND 0.003 ± 0.002 ND ND ND ND /
ND ND ND ND ND 0.027 0.528 ND ND 0.185 0.025 0.043 0.026 0.030 0.032 ND ND ND ND ND 0.024 0.028 0.010 0.022 0.022 ND ND ND ND ND 0.083 0.028 0.013 0.016 0.042 0.172 0.036 0.182 0.145 0.124 0.024 0.034 0.022 0.042 0.030 0.058 0.068 0.138 0.105 0.082 ND ND ND ND ND 0.034 0.059 0.017 0.037 0.009 0.033 0.014 0.019 0.315
ND 0.070 0.060 0.092 0.049 0.068 1.922 0.176 0.133 0.715 0.137 0.320 0.224 0.210 0.225 0.021 0.058 0.047 0.048 0.042 0.263 0.197 0.126 0.097 0.190 ND 0.006 ND ND 0.002 2.543 0.254 0.128 0.118 0.973 4.345 0.235 3.671 3.491 2.720 0.274 0.380 0.199 0.458 0.327 0.237 0.292 2.745 2.581 1.064 0.123 0.147 0.179 0.195 0.152 0.031 0.039 0.045 0.038 0.116 0.181 0.118 0.138 0.037
0.020 0.022 0.018 0.016 0.020 0.021 0.017 ND ND 0.013 0.020 0.026 0.018 0.019 0.022 0.040 0.054 0.051 0.052 0.048 0.010 0.013 0.011 0.012 0.012 ND 0.005 ND ND 0.002 ND ND ND ND ND ND 0.033 0.013 0.012 0.015 ND ND ND ND ND 0.038 0.049 0.008 0.011 0.032 0.009 0.013 0.020 0.021 0.014 0.022 0.067 0.055 0.048 0.003 0.020 0.004 0.009 0.001
± 0.045
± 0.017
± 0.015
± 0.007
± 0.004
± 0.001
± 0.003
± 0.003
± 0.056
± 0.218
± 0.011
± 0.030
± 0.003
± 0.156
± 0.147
± 0.165
± 0.207
± 0.088
± 0.178
± 0.219
± 0.083
± 0.360
± 0.062
± 0.504
± 0.122
± 0.008
± 0.052
± 0.093
± 0.010
± 0.033
± 1.256
± 0.001
± 0.100
± 0.088
± 0.060
± 0.165
± 0.002
± 0.017
± 0.243
± 0.008
± 0.005
± 0.030
± 0.062
± 0.004
± 0.028
± 0.017
± 0.010
± 0.034
± 0.854
± 0.075
± 0.015
± 0.062
± 0.003
± 1.111
± 1.785
± 0.043
± 1.131
± 0.026
± 0.006
± 0.030
± 0.002
± 0.009
± 0.003
± 0.006
± 0.001
± 0.002
± 0.013
± 0.016
± 0.005
± 0.019
± 0.008
Cannabidivarinic acid (CBDV-A), Cannabidiolic acid (CBD-A), Cannabidiol (CBD), Cannabigerolic acid (CBG-A), Cannabinol (CBN), (−) trans-Δ9-tetrahydrocannabinol (Δ9-THC), Δ9-tetrahydrocannabinolic acid (Δ9-THC-A), Cannabichromene (CBC), ND (not detected), up (upper), low (lower). a in the Finola and Ferimon varieties the third flower was not separated into upper and lower parts and was analysed as a whole flower (see Section 2.2).
5
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Table 4 Total CBG, total CBD, total Δ9-THC content, and the ratio of total Δ9-THC/total CBD in the bracts of inflorescence from the first, second, and third flowers from a study in the year 2017, with results of statistical analysis performed by ANOVA. The values of the third flower was previously averaged (up and low) before the total averaging and the calculation of the standard deviation was performed. Variety
Flower
Total CBG (%w/w)
Total CBD (%w/w)
Total Δ9-THC (%w/w)
Ratio of total Δ9-THC/ total CBD
Fedora 17
first second thirdup thirdlow Mean ± sd first second thirdup thirdlow Mean ± sd first second thirdup thirdlow Mean ± sd first second thirdup thirdlow Mean ± sd first second thirdup thirdlow Mean ± sd first second thirda Mean ± sd first second thirdup thirdlow Mean ± sd
0.018 0.035 0.026 0.038 0.028 ± 0.007 0.152 0.301 0.128 0.094 0.188 ± 0.082 0.162 0.214 0.186 0.109 0.175 ± 0.029 0.163 0.165 0.166 0.160 0.164 ± 0.001 0.525 0.449 0.231 0.442 0.437 ± 0.078 0.008 0.009 0.006 0.008 ± 0.002 0.064 0.385 0.415 0.293 0.268 ± 0.145 (%w/w) 0.034 0.083 0.191 0.100 0.088 ± 0.045 0.010 0.026 0.029 0.033 0.022 ± 0.009 0.079 2.269 2.082 3.006 1.631 ± 1.103 0.366 0.184 0.182 0.167 0.241 ± 0.088 0.135 0.185 0.047 0.067 0.126 ± 0.053 0.040 0.020 0.056 0.039 ± 0.015 0.002
1.463 2.416 1.861 3.220 2.140 ± 0.481 4.652 9.177 6.473 6.141 6.712 ± 1.870 7.817 6.354 3.821 3.075 5.873 ± 1.816 3.121 7.837 3.165 3.109 4.698 ± 2.219 7.941 11.085 5.970 13.460 9.580 ± 1.287 1.658 2.928 1.643 2.076 ± 0.602 3.498 3.970 5.350 5.850 4.356 ± 0.900 (%w/w) 2.365 2.807 5.063 3.904 3.219 ± 0.913 0.716 1.545 1.282 1.442 1.207 ± 0.356 0.008 0.096 0.005 0.012 0.038 ± 0.042 3.876 7.325 2.923 2.671 4.666 ± 1.931 8.208 10.468 2.576 2.484 7.069 ± 3.339 4.158 5.867 4.413 4.812 ± 0.753 0.001
ND 0.061 0.052 0.081 0.043 ± 0.030 0.145 0.324 0.222 0.214 0.229 ± 0.073 0.254 0.201 0.120 0.107 0.189 ± 0.058 2.313 0.251 0.125 0.119 0.895 ± 1.004 0.264 0.367 0.196 0.444 0.317 ± 0.042 0.061 0.093 0.057 0.071 ± 0.016 0.108 0.129 0.157 0.171 0.133 ± 0.023 (%w/w) 0.087 2.213 0.155 0.117 0.812 ± 0.991 0.019 0.051 0.041 0.042 0.037 ± 0.014 ND 0.005 ND ND 0.002 ± 0.002 3.983 0.241 3.401 3.207 2.509 ± 1.627 0.265 0.324 2.545 2.368 1.015 ± 1.020 0.111 0.191 0.117 0.140 ± 0.037 0.042
N/A 1:39 1:35 1:40 1:50 1:32 1:28 1:29 1:29 1:29 1:31 1:32 1:32 1:29 1:31 1:1 1:31 1:25 1:26 1:5 1:30 1:30 1:30 1:30 1:30 1:27 1:31 1:29 1:29 1:32 1:31 1:34 1:34 1:33
Monoica
Helena
Tisza
Antal
Finola
Kompolti hibrid TC
KC Dora
USO 31
Santhica 27
Tiborszallasi
Carmagnola
Ferimon
p (ANOVA of
first second thirdup thirdlow Mean ± sd first second thirdup thirdlow Mean ± sd first second thirdup thirdlow Mean ± sd first second thirdup thirdlow Mean ± sd first second thirdup thirdlow Mean ± sd first second thirda Mean ± sd the differences between varieties)
1:27 1:1 1:33 1:33 1:4 1:38 1:30 1:31 1:34 1:33 N/A 1:20 N/A N/A 1:23 1:1 1:30 1:1 1:1 1:2 1:31 1:32 1:1 1:1 1:7 1:38 1:31 1:38 1:34 /
Cannabidiol (CBD), (−)-trans-Δ9-tetrahydrocannabinol (Δ9-THC), Cannabigerol (CBG), ND (not detected), up (upper), low (lower). a in the Finola and Ferimon varieties the third flower was not separated into upper and lower part and it was analysed as a whole flower (see Section 2.2).
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Table 5 Cannabinoid content and the ratio of total Δ9-THC/total CBD in bracts from inflorescences and whole inflorescences from a study in the year 2018. Cannabinoids CBG (Cannabigerol), Δ8-THC (Δ8-tetrahydrocannabinol), and CBL (Cannabicyclol) were not detected in any of the varieties. Variety
Fedora 17 KC Dora Monoica USO 31 Helena Santhica 27 Tisza Tiborszallasi Antal Carmagnola Kompolti hibrid TC Finola Novosadska Marina
B/WI
B WI B WI B WI B WI B WI B WI B WI B WI B WI B WI B WI B WI B WI B WI
CBDV-A
CBDV
THCV
CBD-A
CBD
CBG-A
CBN
Δ9-THC
Δ9-THC-A
CBC
Total CBG
Total CBD
(%w/w)
(%w/w)
(%w/w)
(%w/w)
(%w/w)
(%w/w)
(%w/w)
(%w/w)
(%w/w)
(%w/w)
(%w/w)
(%w/w)
0.230 0.065 ND 0.004 0.030 0.005 0.008 0.007 0.045 0.009 ND ND 0.017 0.007 0.033 0.006 0.030 0.007 0.073 0.137 0.024 0.004 0.047 0.021 0.076 0.019 0.008 ND
0.120 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 0.061 ND ND ND 0.010 ND ND ND ND
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 0.152 ND ND ND ND ND ND ND ND
3.158 1.301 3.343 0.439 1.490 0.497 0.635 0.813 8.825 2.504 0.011 ND 3.152 1.489 3.625 1.073 5.153 1.234 4.120 0.161 6.012 1.047 1.409 0.573 6.485 1.434 1.460 0.332
1.754 0.492 2.582 0.311 1.156 0.530 0.178 0.176 0.368 0.178 ND ND 1.235 0.606 1.697 0.681 1.639 0.744 2.113 0.112 0.858 0.459 1.545 0.474 0.292 0.122 0.890 0.389
0.104 0.041 0.219 0.030 0.049 0.018 0.012 0.029 0.283 0.057 3.566 1.423 0.109 0.066 0.362 0.095 0.491 0.078 0.184 0.007 0.201 0.076 0.017 0.005 0.146 0.022 0.081 0.019
0.005 0.001 0.016 0.012 ND 0.001 0.001 ND ND ND ND ND 0.005 0.001 0.003 ND 0.006 ND 0.003 0.003 ND 0.002 0.004 0.001 ND ND ND 0.002
0.090 0.028 0.723 0.545 0.057 0.024 0.009 0.010 0.035 0.016 ND ND 0.432 0.040 0.112 0.040 0.769 0.037 0.150 0.105 0.071 0.027 0.067 0.020 0.026 0.011 0.052 0.016
0.056 0.025 0.532 0.379 0.021 0.006 0.015 0.020 0.280 0.075 ND ND 0.413 0.029 0.068 0.020 1.314 0.032 0.120 0.063 0.140 0.018 0.022 0.011 0.191 0.038 0.027 0.004
0.099 0.031 0.173 0.038 0.085 0.042 0.009 0.014 0.026 0.015 0.020 0.021 0.094 0.044 0.102 0.038 0.159 0.042 0.140 0.026 0.064 0.031 0.068 0.024 0.020 0.012 0.057 0.026
0.091 0.036 0.193 0.026 0.043 0.016 0.010 0.025 0.249 0.050 3.131 1.249 0.095 0.058 0.318 0.083 0.431 0.068 0.162 0.006 0.177 0.067 0.015 0.004 0.128 0.019 0.071 0.017
4.523 1.633 5.513 0.696 2.462 0.966 0.735 0.889 8.108 2.374 0.010 ND 4.000 1.912 4.876 1.622 6.159 1.827 5.726 0.254 6.131 1.377 2.781 0.977 5.979 1.380 2.170 0.680
Total Δ9 THC (%w/ w)
Ratio total Δ9−THC/ total CBD
0.139 0.050 1.190 0.878 0.075 0.029 0.022 0.027 0.280 0.082 ND ND 0.794 0.066 0.172 0.058 1.922 0.065 0.255 0.161 0.193 0.043 0.086 0.030 0.193 0.044 0.075 0.020
1:33 1:33 1:5 1:1 1:33 1:33 1:34 1:32 1:29 1:29 N/A N/A 1:5 1:29 1:28 1:28 1:3 1:28 1:22 1:2 1:32 1:32 1:32 1:33 1:31 1:31 1:29 1:34
Cannabidivarinic acid (CBDV-A), Cannabidivarin (CBDV), Tetrahydrocannabivarin (THCV), Cannabidiolic acid (CBD-A), Cannabidiol (CBD), Cannabigerolic acid (CBG-A), Cannabinol (CBN), (−)-trans-Δ9-tetrahydrocannabinol (Δ9-THC), Δ9-tetrahydrocannabinolic acid (Δ9-THC-A), Cannabichromene (CBC), B (bracts), WI (whole inflorescence), ND (not detected).
expected. Based on the obtained total CBD and total Δ9-THC values in whole inflorescences, the variety Santhica 27 is a less appropriate variety for CBD production since both cannabinoids were present in minimal amounts.
due to the diverse nature of cannabis varieties, more plants within the sample population represent reliable results. Influence of Slovenian soil and climate on selected varieties was not studied up to now, which makes comparisons of the content of CBD and Δ9-THC from whole inflorescences in year 2018 with declared values published in publications (summarized in Table 1) interesting. For the varieties Fedora 17, USO 31, Tisza, Tiborszallasi, and Antal comparable values of cannabinoid concentrations were found. From this it can be concluded that varieties reacted to the cultivation environment as
3.2. Ratio of total Δ9-THC/total CBD The ratio of total Δ9-THC/total CBD can primarily be used for the identification of fiber or the indica type of the hemp. A ratio of total Δ9-
Fig. 2. Cannabinoid content from bracts: average value of three inflorescences from 2017 (mean value %w/w with standard deviation) and value in one inflorescence from 2018 (single value %w/w): A.) total CBD (Cannabidiol), B.) total Δ9-THC ((−)-trans-Δ9-tetrahydrocannabinol). 7
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Fig. 3. Content of cannabinoids in bracts (average value of inflorescences from three plants) and in parts from the rest of inflorescences, study from 2017 (mean value %w/w with standard deviation): A.) total CBD (Cannabidiol), B.) total Δ9-THC ((−)-trans-Δ9-tetrahydrocannabinol).
THC/total CBD lower than 1.0 is attributed to “fiber-type’’ plants (Hillig and Mahlberg, 2004), which applies to all varieties considered in our study. This ratio can also be used to measure seed uniformity. This is important information for breeders, who must know if genetic sowing material is consistent. When compared, the ratio of total Δ9-THC /total CBD in bracts from the first, second, third upper, and third lower flower as shown in Table 4 had high uniformity for the varieties Antal (with standard deviation ± 0), Helena ( ± 1), Monoica ( ± 1), Kompolti hibrid TC ( ± 1), Finola ( ± 2), Ferimon ( ± 3), and USO 31 ( ± 3) has been recognized. Varieties with low uniformity were found to be Carmagnola ( ± 15), KC Dora ( ± 13), Tiborszallasi ( ± 13), and Tisza ( ± 12). In order to confirm these findings, the ratio of total Δ9-THC/total CBD from bracts and from whole inflorescences from 2018 (Table 5) was added for comparison. When averaging all values, it was found that the varieties Helena ( ± 1), Kompolti hibrid TC ( ± 1), Finola ( ± 2), Monoica ( ± 2), and USO 31 ( ± 3) are highly uniform between both seasons. For the mentioned varieties we could assume that homogenous plant genetics are advantageous for farmers that are cultivating hemp for production of CBD, since consistent content was shown between both years. Low uniformity was detected with the same varieties as listed above when compared inside year 2017. For the varieties Novosadska, Marina, and Ferimon ratios could not be compared since these varieties were sown in only one season. The varieties Novosadska and Marina showed high uniformity between the ratio of total Δ9-THC/ total CBD in bracts and whole inflorescence within year 2018.
The concentration of CBDV-A in 2017 was represented in all varieties. In Carmagnola (0.205 % ± 0.218) and Antal (0.118 % ± 0.056) levels were higher since in other varieties concentrations were found to be lower than 0.07 %. In 2018 the highest levels of CBDV-A was found in Fedora 17 (0.230 %). In other varieties, concentrations were found to be under < 0.07 % or even nondetectable (Santhica 27 and KC Dora). Only in Fedora 17, CBDV was detected (0.120 % in 2018); in others varieties it was undetectable during both years. In trace amounts CBC was detected (< 0.05 %) or even not detected in 2017. Interestingly, in 2018 CBC was detected in higher quantities in the varieties KC Dora (0.173 %), Antal (0.159 %), Carmagnola (0.140 %) and Tiborszallasi (0.102 %), while in other varieties the levels were less than < 0.1 %. From the results it can be concluded that CBC content is higher in less mature flowers since flowers in 2018 were harvested earlier than those in 2017. Trace amounts of CBN were detected in both years (< 0.02 %) or were undetectable. Since CBN is an oxidative product of Δ9-THC, which is more prominent in aged cannabis samples (Russo, 2011), (Andre et al., 2016), results suggest that plants were collected at their maturity. The content of the cannabinoid CBL rises by exposure of CBC to UVradiation (Hazekamp, 2007). Results from both years indicate that storage of flowers after harvesting was appropriate (with no exposure to light), since the concentration of CBL was found to be undetectable in 2017 and 2018. Cannabinoids Δ8-THC and THCV were not detected at any of the varieties in either year. 4. Conclusion
3.3. Other cannabinoids in bracts The response of hemp varieties used in research regarding growth conditions in Slovenia are not well known since they are produced in different European countries or breeding programs in geographical areas. The purpose of this study was to compare and to show the chemical profile of cannabinoids in varieties that were accessible to our research group. The major focus of the study was on the total concentration of CBD and Δ9-THC in the bracts of fertilized female flowers. Some of them were already over the phase of their vegetative cycle and were therefore treated as the cannabinoid production reached the plateau phase. From the results presented in this article it can be concluded that:
The following section describes the content of cannabinoids in 2017 and 2018 found in bracts (Tables 3–5). Besides the total CBD and total Δ9-THC in 2017, the most represented cannabinoid was found to be CBG-A with the highest content in Santhica 27 (1.857 % ± 1.256), which did not contain significant amounts of CBD, CBD-A, THC, or Δ9-THC-A. It is known the THCA- and CBDA-synthases that metabolize cannabinoids from CBG-A are not well expressed. This could be described and confirmed with results from whole inflorescence and bracts in 2018, where the concentration of CBG-A in Santhica 27 was found to be 3.566 % and levels of CBD-A and Δ9-THC-A were minimal. Cannabinoid CBG was not detected in any varieties in either year. Relatively high CBG-A content in Antal (0.497 % ± 0.088 in 2017, 0.491 % in 2018) and in Tiborszallasi (0.362 % in 2018) could show immaturity of the flowers. Concentration levels of CBG-A lower than 0.3 % correspond to others varieties for both years.
• The response of hemp varieties to growth conditions in Slovenia was
good in the sense of CBD production in the varieties Fedora 17, USO 31, Tisza, Tiborszallasi, and Antal since the values of CBD and Δ9THC in whole inflorescences were found to be comparable to
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declared values.
Declaration of Competing Interest
production since only trace amounts of CBD and Δ9-THC were detected. High uniformity of the seeds, according to preserved ratio of total Δ9-THC/total CBD between years, was found with the varieties Kompolti hibrid TC, USO 31, Finola, Helena, and Monoica. Low uniformity was found in the varieties Carmagnola, Tiborszallasi, Tisza, and KC Dora. For these varieties, it is possible that infinite phenotypic change occurred. All tested varieties belong to the ‘fiber-type’’ plants according to the ratio of total Δ9-THC/total CBD. No significant differences in cannabinoid concentration were found between bracts from upper and lower inflorescence parts of hemp. It can be assumed that for all varieties the < 0.2 % limit of total Δ9THC will not be exceeded with appropriate sampling. The concentration of total CBD and total Δ9-THC proved to be highest in bracts from inflorescence. It was found that the concentration was on average four times higher compared with the rest parts of the inflorescence (without bracts) and up to four times higher compared with the concentration of the whole inflorescence. High ratios (> 1:27) of total Δ9-THC/total CBD in bracts from both years were found in the varieties USO 31, Monoica, Helena, Finola, and Kompolti hibrid TC.
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
• The variety Santhica 27 was found to not be appropriate for CBD •
• • • • •
Acknowledgements Research was supported by the Slovenian Research Agency and Slovenian Ministry of Agriculture, Forestry and Food (research project reference: V4-1611). We would like to offer special thanks to Matevž Štefančič, for his laboratory work. References Andre, C.M., Hausman, J.-F., Guerriero, G., 2016. Cannabis sativa: the plant of the thousand and one molecules. Front. Plant Sci. 7, 1–17. https://doi.org/10.3389/fpls. 2016.00019. Fischedick, J.T., Hazekamp, A., Erkelens, T., Choi, Y.H., Verpoorte, R., 2010. Metabolic fingerprinting of Cannabis sativa L., cannabinoids and terpenoids for chemotaxonomic and drug standardization purposes. Phytochemistry 71, 2058–2073. https://doi.org/ 10.1016/j.phytochem.2010.10.001. Hazekamp, A., 2007. Cannabis; Extracting the Medicine. Dr Thesis. Department of Pharmacognosy (IBL), Faculty of Science, Leiden Universityhttps://doi.org/10.1081/ JLC-200028170. Hillig, K.W., Mahlberg, P.G., 2004. A chemotaxonomic analysis of cannabinoid variation in Cannabis (Cannabaceae). Am. J. Bot. 91, 966–975. https://doi.org/10.1002/cncr. 25812. Ihempfarms [WWW Document], (2019) n.d. URL www.ihempfarms.com/ (accessed 6. 4.19). Namdar, D., Mazuz, M., Ion, A., Koltai, H., 2018. Variation in the compositions of cannabinoid and terpenoids in Cannabis sativa derived from inflorescence position along the stem and extraction methods. Ind. Crop. Prod. 113, 376–382. https://doi.org/10. 1016/j.indcrop.2018.01.060. Pacifico, D., Miselli, F., Carboni, A., Moschella, A., Mandolino, G., 2008. Time course of cannabinoid accumulation and chemotype development during the growth of Cannabis sativa L. Euphytica 160, 231–240. https://doi.org/10.1007/s10681-0079543-y. Pertwee, R.G., 2014. Handbook of Cannabis, 1st ed. Oxford University Presshttps://doi. org/10.1093/acprof:oso/9780199662685.001.0001. Potter, J.P., D, 2009. The Propagation, Characterisation and Optimisation of Cannabis sativa L as Phytopharmaceutical. Department of Pharmaceutical Science Research. King’s College London. Regulation (EU) No 809/2014, 2014. Off J Eur Union. pp. 69–124. Rules concerning the requirements for obtaining permission to cultivate hemp, 2004. Off Gaz Repub Slov 44/04. Rules on conditions for obtaining a permit for hemp and poppy cultivation, 2018. Off Gaz Repub Slov 40/11. Russo, E.B., 2011. Taming THC: potential cannabis synergy and phytocannabinoid-terpenoid entourage effects. Brit. J. Pharmacol. 163, 1344–1364. https://doi.org/10. 1111/j.1476-5381.2011.01238.x. Russo, E.B., Marcu, J., 2017. Cannabis Pharmacology: The Usual Suspects and a Few Promising Leads, 1st ed. Adv Pharmacol. Elsevier Inc.https://doi.org/10.1016/bs. apha.2017.03.004. Thomas, B.F., Elsohly, M.A., 2016. The Analytical Chemistry of Cannabis, 1st ed. Elsevier Inchttps://doi.org/10.1016/C2014-0-03861-0. Turner, J.C., Hemphill, J.K., Mahlberg, P.G., 1981. Interrelationships of glandular trichomes and cannabinoid content. I. Developing pistillate bracts of Cannabis sativa L. (Cannabaceae). B Narcotics 33, 59–69. Zadruga konopko [WWW Document], n.d. URL http://www.konopko.si/ (accessed 6. 4.19).
The use of genetically identical plant material from representative clones under strictly controlled environmental conditions could ensure reproducible cannabis material by reaching the same growth stages and by controlling glandular trichomes profiles. Chemical differences can be seen within the growth cycle, harvesting, and storage due to environmental conditions. The major focus should be genotype consistency of a specific chemical profile of cannabis to produce a reproducible and stable chemical composition (Fischedick et al., 2010). With this information, the farmers and breeders will know more precisely what the risks and benefits of cultivating a particular variety are in terms of compliance with Slovenian legislation, depending on the production purpose. This could enable a better understanding and an in-depth knowledge of cannabis cultivation in Slovenia for increased yields of dioecious and monoecious varieties. CRediT authorship contribution statement Taja Glivar: Conceptualization, Formal analysis, Data curation, Writing - original draft, Visualization. Jan Eržen: Methodology, Investigation. Samo Kreft: Formal analysis, Resources, Writing - review & editing. Marjeta Zagožen: Investigation. Andreja Čerenak: Writing - review & editing. Barbara Čeh: Resources, Writing - review & editing, Project administration, Funding acquisition. Eva Tavčar Benković: Conceptualization, Methodology, Validation, Writing - review & editing, Supervision.
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