Yielding Morphological Characteristics and Biochemical Analysis of “Karma Lemon” Cannabis Producing Cannabinoids in Thessaloniki-Greece

Cannabis has been widely used by humans over many centuries as a source of fiber, oil and for medicinal purposes. Its use was illicit in numerous countries, including Greece and Lebanon. “Karma Lemon”, one of the newest cannabis strain originated from Italy, is selected in this study to analyze its components using various techniques starting from the extraction, isolation and identification of cannabinoids using separatory compounds and NMR techniques as well as the main important morphological traits of this strain to be harvested at an appropriate time for medicinal uses in Greece and later on, in Lebanon. Thirty different samples were selected from the field of respected “Hemp Way Company” in Thessaloniki and studied for morphological traits. These were related to the length of the plant at harvest time (1.809 m) needed for the use of combines and the weight of inflorescence (213.5 g fresh and 40.8 g dry) for oil or seed production. Three samples of Karma Lemon cannabis strain inflorescence were analyzed at the Laboratory of Pharmacognosy, School of Pharmacy, in the Aristotle University of Thessaloniki in Greece, after proper extraction and isolation using ethanol and other separation compounds. TLC and NMR techniques were used to visualize and identify cannabinoids present after isolation. Cannabinoid acids, CBG, CBN, THC, CBD and other cannabinoids were identified and isolated.


Introduction
The genus "Cannabis" contains some of the oldest plants used for food, fiber, and medicine. Cannabis indica belongs to the family Cannabaceae. Its earliest cultivation originated in central Asia, wherefrom the crop spread to the Middle East, China, Europe, and the Americas during the early 16 th Century (Radwan et al., 2017).
The appearance of thirty plants of Karma Lemon cannabis strain were selected randomly, in a field of one dunum from thirty three hectares owned by "The Hemp Way" company in Thessaloniki (Kalamariá) in Greece (37°48′35.6″ North, 23°51′35.2″ East and has an average elevation of 50 m above sea level).
This field is divided into several sections with 13 rows exist. The main cause of the adoption of this system is to harvest Cannabis in a continuous way because these sections contain different growth stages of one week time difference from seeding to maturity. These offered samples are from the last year cultivation and they were harvested, dried and put in sealed packages.
Some traits were determined on each of these plants. The length of the plant at maturity was measured by taking the length (in meter) of Karma Lemon plant at harvesting time using a measuring tape (in cm); the height was taken from the surface of the soil to the top of the plant. This trait is important for setting the cut at an appropriate height at the harvesting time, using the combines, approximately 2 meters for Karma Lemon. It is very important that the plants have almost the same length when cultivated to reduce the loss of productivity in the field.
The weight of inflorescence (gram fresh and dry weight) for the 30 different samples was also measured on fresh and was labeled to be taken again after drying (two weeks were necessary for complete drying at room temperature). This indicates the productivity of the Cannabis. These two traits were averaged and a standard deviation was calculated. Standard error and coefficient of variation were also calculated in case the standard deviation is high at p = 0.1.

Chemical Analysis
Ten grams of dried Karma Lemon plant materials were kept at -20 o C along with the ethanol bottles for one overnight, grinded and subsequently soaked, sonicated and filtered for two more times. The filtrate was poured and diluted (1:2). Half of the filtrate volume was directly condensed and to the other half; a binding compound was added to remove the chlorophyll before the condensation. The condensed extract materials were and kept at -20 o C for further analysis.
Isolation of cannabinoids was done using a separation compound to seize all chemicals excluding cannabinoids of interest and was confirmed by TLC and NMR analysis.
Cannabinoids were separated on thin-layer chromatography (Si 60F 254 plate stationary phase TLC). Samples were applied on TLC plates as six spots of minimum size with a homogeneous distribution of material on one cm line of its starting zone using a glass capillary. Four developing solvents were eluted. The plates were put in a chamber containing 50 ml of the following eluents: dichloro-methane/methanol (98:2 v/v), DM (100%), hexane-ethyl acetate (85:15 v/v) and acetone/dichloro-methane/tert-butyl methyl ether/hexane (4:4:12:80 v/v). Plates were put under U.V light, poured with vanillin and separated compounds of interested were marked and subjected to NMR analysis for identification. The 1H 1 NMR spectra (500 MHz) and 13C NMR spectra (125.0 MHz) were recorded in CDCl 3 using AGILENT DD2 500 spectrometer. Chemical shifts are reported in  (ppm) values relative to TMS and 1 H NMR spectrum with signals was observed for the identification of aromatic and olefinic hydrogen atoms of cannabinoids (Brenneisen, 2007).

Morphological Characteristics at Harvest Time
The samples of Karma Lemon in the field were between 1.50 and 1.90 meters length with an erect stem that is sometimes hollow. The basal leaves were opposite but the highest one was alternate, palmate, lanceolate, sharp spikes up to 10 cm long and their color was deep green with hints of yellow and brown. Petiolules were 0.5-1.5 jas.ccsenet.org Journal of Agricultural Science Vol. 12, No. 7;2020 cm long. The leaves were palmately 3-9-lobed, showing actinodromous venation and the youngest leaves were sometimes unlobed. The lobes were narrowly oblong-lanceolate, 3-20 cm long, up to 1.8 cm wide, dark green above, paler beneath, attenuate at base, caudate-acuminate at apex, and serrate along the margins. The serrations along the margins were prominent, curved and pointed towards the tips of the leaf blades. Each lobe had a primary midrib and several secondary veins at either side. Each of the secondary veins run out obliquely from the midrib and entered into a serration of the margin. The veins were prominently raised forming ridges on the abaxial side whereas they were impressed on the adaxial side forming grooves. The lowest pair of lobes was usually much smaller than the others and pointing backwards. In seedlings, the first pair of leaves was 1-foliolate and the second and third pairs were three and five-foliolates, respectively.
Male flowers were pale green, borne on axillary laxly branched cymose panicles. Flowers in the panicles occured solitarily, in clusters, or in 3-flowered cymules. Each flower consisted of five tepals, five stamens and a slender pedicel. The tepals were ovate-oblong, 2-4 cm long, yellowish-or whitish-green, spreading, and minutely hairy. The stamens were drooping and consist of slender filaments and oblong, greenish anthers. Pollen grains were liberated through terminal pores in the anthers.
Female flowers were dark green, subsessile and were borne in pairs. The flowers were closely aggregated at the apex of short spike inflorescences, which were densely formed in the upper axils of branches. Each flower consisted of ovary with a style that ends in a pair of long slender feathery stigmas at apex, a membranous perianth surrounding the ovary, and a bract. The style-stigma portion of the pistil in wild-growing plants generally measured about 3 mm long and the styles were usually 2-branched.
Male and female flowers occur in separate plants; they generally bloom during July-August. Male plants are usually taller and the female plants are usually more robust than male plants. Several cultivars with varying features occur in cultivation.
The achene fruit was ovoid, ellipsoid or subglobose, about 4-6 mm long and 3-4 mm in diameter, smooth, somewhat compressed, brownish grey and mottled, containing a single seed with a hard shell. Sometimes, the cannabis "seed" of commerce was actually the enclosed fruit in its hooded floral bract.
Morphological characteristics of Karma Lemon studied plants confirmed the data recuperated in the year 2018 with very small differences that may be influenced by the strain as well as by environmental factors such as soil type, light, water, nutrients and space . Among the apparent modifications we cite in the table below (Table 1), the average length of thirty Karma Lemon plant samples selected randomly at harvesting time (in meter), their average weight of inflorescence (gram fresh and dry weight) and related measures of variability (minimum, maximum, median, standard deviation, standard error and coefficient of variation). The average length of studied Karma Lemon strains at harvest is 1.809 ±0.080 m. The use of combine harvesters was adequate to cut hectares of crops using a sharp at 1.80 m ( Table 1). The optimal length of Karma was reported to be between 1.25 m and 2.00 meters .
The average fresh weight inflorescences ± standard deviation of the studied plants is about 213.5±4.783 g and (Table 1). For the weight of fresh inflorescence, the very low standard variation means the stability of yield at plants level. The optimal Karma weight average published ranged between 190 g and 235 g .
Regarding the dry weight of inflorescence (40.8±3.377 g), the standard variation was a bit high and this is due to the trimming of some organs (leaves and not flowers or seeds) that may affect its weight and making the difference almost double to that of fresh weight. The optimal weight according to Chandra et al. (2017) was 30 g to 50 g.   According to NMR technique, the presence of the most important cannabinoids (CBG, CBD, CBN and THC) in Karma Lemon was clear. As well, it was referred that the percentage of CBDA, CBGA, CBG, CBD, CBN, 9-THC and 9-THCA in Karma Lemon cannabis strain was approved by the Food Chemistry Laboratory for The Extraction and Quantitative Analysis of the University of Naples Federico II-Department of Pharmacy, to be 92.625 mg/g, 0.184 mg/g, 0.044 mg/g, 2.077 mg/g, 0.001 mg/g, 0.545 mg/g and 0.746 mg/g respectively (Appendices). The cannabinoid profile of this strain of interest Karma Lemon was accurately determined at 0.262 indicating that Karma Lemon is an "oil and seed plant" .

Conclusions and Recommendations
This work explained the methods of Finola strain (Cannabis sativa L.) growth and morphological traits used for hemp harvest at the appropriate time in order to extract, isolate and identify cannabinoids from panicle samples.
Extraction and isolation were done using ethanol and oil was obtained from the samples. Oil was examined by TLC and NMR spectroscopy for analysis using TLC plates and NMR spectroscopy graphs. With TLC, it was possible to detect the presence of cannabinoid acids, Cannabidiol (CBD), Tetrahydrocannabinol (THC) and Cannabinol (CBN) when compared with other TLCs but this method did not offer their purification. The best TLC plate obtained was the one containing the mixture (Acetone:Dichloromethane:Tert-Butyl-methylether: Hexane) = (4%:4%:12%:80%), when compared to others which gave fair good results. NMR spectroscopy confirmed the presence of CBD, THC and CBN in the analyzed samples through peaks present in the graph and position of hydrogen molecules in the cannabinoid structures. Cannabis is accepted as a medicinal plant due to the impressive amount of therapeutic and pharmacological properties of cannabinoids.
In Lebanon, Legalization of cannabis has recently taken place and its regulation has to be settled the sooner the better especially when having on hand previous results of specific trials of cultivating Finola in addition to other strains. Specific facilities, protocols and analytical methods of identification and quantification of cannabinoids should be executed so that all the cultivation and production of drugs could be perfectly controlled from the field to the manufacturing companies.