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Effect of nitrogen on tetrahydrocannabinol (THC) content in hemp (Cannabis sativa L.) leaves at different positions

I. Bócsa, P. Máthé, and L. Hangyel

GATE "Fleischmann R." Research Institute, Kompolt 3356, Hungary

        Bócsa, I., P. Máthé, and L. Hangyel 1997. Effect of nitrogen on tetrahydrocannabinol (THC) content in hemp (Cannabis sativa L.) leaves at different positions. Journal of the International Hemp Association 4(2): 78 -79. The effect of different levels of nitrogen fertilizer, of physiological age of leaves and of the interaction between these factors on the Δ⁹-tetrahydrocannabinol (THC) content of leaves from different positions on the hemp (Cannabis sativa L.) plant were analyzed by gas liquid chromatography. High nitrogen levels reduced the THC content of leaves, and older leaves contained less THC than younger ones. There was no significant interaction between these two factors.

Introduction
        Hemp (Cannabis sativa L.) is a traditional and important raw material for the textile industry and is currently of interest as a wood fiber supplement in the paper industry (de Meijer and van der Werf 1994). A significant increase in cultivation of hemp in Europe is anticipated for future fiber production. Furthermore, drug-type Cannabis may play an important role in future therapeutics (Clarke and Pate 1994.). However, since one of its cannabinoid compounds is Δ⁹-tetrahydrocannabinol (THC), the psychoactive agent of the plant, its cultivation is presently limited. Breeders have developed low-THC and high-fiber content varieties, but some of these still contain a THC concentration verging on the EU limit for cultivation subsidy in Europe (de Meijer et al. 1992).
        Since hemp has a high nitrogen (N) requirement, it is important to determine the relationship between N fertilization and THC content, and (for the purpose of analytical sampling) the interaction between N fertilizer and leaf position, in relation of leaf THC content.
        Cannabinoid content of the leaves is known to decrease gradually from the top to the bottom of the plant (Hemphill et al. 1980). Nitrogen content in vegetative parts of the plant has been thought to correlate positively with its THC content (Coffman and Gentner 1975, Haney and Kutscheid 1973).

Table 1. Fertilizer treatments in mg/kg soil.

Table 2. The effect of nitrogen treatment on the fresh weight and plant height of hemp and on the N content of the phytomass.

Materials and methods
        The experiment was performed using 5.5 liter pots, with two plants of the variety ‘Kompolti Hibrid TC’ per pot. This high-fiber variety has a THC content (0.5-0.7%) exceeding the EU 0.3% subsidy limit. Pots were placed in a glasshouse under ambient environmental conditions. A chernozem brown forest soil from Kompolt was used (Krisztian and Hollo 1992). Treatments are shown in Table 1.
        Chemicals used for the nutrient supply : anal. grade NH₄NO₃, KH₂PO₄, KCl. Doses were supplied as solutions. Date of sowing: 22 April. Date of harvesting: 13 August. The lowest N level treatment was considered as the control.
        The number of replications was six. Plants were grown until the end of flowering for staminate plants. Leaf samples were collected on 13 August in the following way: every leaf of both the staminate and pistillate plants was collected, dried at 40°C for 24 hr, ground, weighed, homogenized, and stored in a refrigerator at 3°C for 90 days. Leaves were placed into three groups: (a) older leaves that occur along the middle part of stem, (b) leaves from the side branches, and (c) younger leaves that occur near the top of the stem.
        THC was extracted from the dried leaves with petroleum ether for 3 hr at RT. (Hanus et al. 1981). Analyses were performed with a Hewlett-Packard 5890 series II gas liquid chromatograph. Parameters of analyses were: HP-1 capillary column, 0.3 ID x 25 m length; injector temperature 260°C ; split ratio 1:70; detector temperature 300oC. Analyses were programmed from 190 to 265°C at 15C/min with an 11 min and a 5 min internal plateau at 235°C and 260°C, respectively.
        Initially, an analytically pure THC standard was not available, so areas of THC peaks were used for characterization of THC content. The peak identification of cannabidiol and THC was carried out according to Hanus et al. (1981). Subsequently, CBD and THC standards were obtained and the original data were verified with these standards. The back-calculation of the data to an absolute value is analytically incorrect in our opinion, so this determination was not made.
        The statistical significance of factors presumably affecting the THC content of leaves was determined by analysis of variance. The first factor was (a.) the leaf position on the plant, and the second factor was (b.) the nitrogen treatment.

Table 3. Analysis of variance of N fertilizer experiment (in a randomly arranged bifactorial split-plot experiment with six replications).

Results
        There was a significant increase in fresh weight of shoot (80-130%) and plant height (28-39%) due to N supplimentation (Tab. 2). THC was highest in leaves near the shoot tip and on side branches, and lowest in oldest leaves (Fig. 1a). THC content of leaves of each plant part decreased in response to N fertilizer (Fig. 1b).
        The decrease was significant in the case of the highest N dose (Fig. 1b). THC contents of leaves from various plant regions were significantly different, independent of the N level (Tab. 3, see: F-value of Factor "a") The other factor, N fertilizer treatment, also had a statistically significant effect on THC content of leaves (Tab. 3, F-value of Factor "b"). There was no interaction between the two factors (Tab. 3, F-value of Factor "a X b") in relation to leaf THC content.

Figure 1. Effect of examined factors on THC content of hemp leaves :
a.) THC content of leaves from different positions on the hemp plant (means of three levels of N fertilizer).
b.) Effect of N fertilization on the THC content of hemp leaves (means of three positions on the plant).

Discussion
        These experiments show that the THC content of leaves decreases with increasing N doses. This phenomenon is favorable for agricultural production, because nitrogen fertilization will increase stem yield and simultaneously decrease THC content of the plant significantly. Additional studies are necessary to determine optimal N dose/ha, time of application, fertilizer type and the lowest THC content achievable under field conditions.

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Acknowledgments
        The authors thank the National Research Fund of Hungary (OTKA), for financial support of this research, Prof. Paul G. Mahlberg (Indiana University, Bloomington, U.S.A) and Mr. László Pummer ("Fleischmann R." Institute, Hungary) for their help.