Soxhlet Extraction of Cannabinoids (CBD and THC) From Four Different Strains of Cannabis Grown in Four Different Regions

The objective of this work was to investigate the cannabinoid content of extracts which was conducted by soxhlet extraction. In the extraction system hexane, ethyl acetate and ethanol were used sequentially to extract cannabis plant. When all species were evaluated in terms of CBD, all results in Meram region were the highest. As it can be understood, if an ecient extract in terms of CBD is desired, sowing can be done in Meram. Narlı strain provided the highest (65.88%) yield in terms of CBD in accordance with its cultivation purpose. When evaluated THC active ingredient, it was seen that each strain gave high yields in different regions. Elnur strain yielded the highest yields in Altınekin (66.21%), Papatya in Beyşehir (52.05%), Narlı (14.49%) and Gökçeağaç in Çumra (56.13%). It is the rst study to show the different climatic conditions effect on the yield of CBD and THC in different strains.

The main cannabinoids are Δ 9 -Tetrahydrocannabinol (THC) and its structurally analogue, although not psychoactive, cannabidiol (CBD). They are present in the plant as inactive acids (THC-A and CBD-A) and they can be converted in the corresponding non-acidic forms via thermal decarboxylation [8].
As the analysis of cannabis has gained new global importance, mainly for quality control within the legalized recreational and medical cannabis industry, and also for forensic differentiation between drugtype cannabis and legal products such as bre hemp and CBD-rich and THC-poor cannabis the importance of the correct choice of an extraction method for the extraction of cannabinoids from various matrices e.g. cannabis plant extracts, hemp food products, biomass, cannabis oils, whole blood, plasma, oral uids, hair and so on, has become paramount [9,10]. There are a wide variety of cannabinoids in cannabis. The occurrence of these cannabinoids in different parts of the cannabis is strongly dependent on the genetic expression and growing environmental conditions. Such as, female plants gives higher quantity of cannabinoids at the stage of maturity. While, male plants are preferable for higher yields of the non-cannabinoids and avonoids i.e. hemp form [11].
The choice of an extraction method relies on the nature of the source material, e.g. dried plant powder, biological materials, soil or water, as well as the target compounds, e.g. cannabinoids [12]. Prior to deciding on a particular extraction method for naturally occurring cannabinoids, one must consider the following: purpose of extraction, quantity of extraction, puri cation steps to be carried out, purity level of cannabinoids, possible artefact formation, stability of target cannabinoids, physicochemical properties of target cannabinoids, and obviously the cost and environmental impacts. In addition to traditional methods like maceration, distillation or boiling, several other modern extraction methods and techniques can be applied for the extraction of naturally occurring cannabinoids, and those methods include, soxhlet, accelerated solvent extraction, pressurized liquid extraction (PLE), microwave-assisted extraction (MAE), ultrasound assisted extraction (UAE), supercritical uid extraction (SFE), solid phase extraction (SPE) and micro solid-phase extraction (MSPE) [12,13]. The choice of solvent for extraction is equally important to have maximum extraction yield. For the extraction of naturally occurring cannabinoids, most often, organic solvents are preferred, because of the lipophilic nature of most of the naturally occurring cannabinoids.
Traditional methods, like maceration, percolation, and soxhlet, are known to have some limits such as time and solvent consumption, and decomposition of heat sensitivity bioactive compounds [14].
However, soxhlet technique is still common in laboratories and industries being involved in a wide variety of o cial methods [15].
According to the scanning results of the literature, generally hemp seed, seed oil yield, essential oil composition or mostly non-psychoactive ingredient CBD and sometimes only THC have been researched.
And also there are a few researches by ultrasonic and microwave extractions. Apart from extraction methods, various analytical methods (such as GC-MS and HPLC) have been reported for the analysis of cannabinoids in cannabis plant. There was no publication in the literature that cultivated several strains at the same time, developed a soxhlet extraction procedure, and analyzed CBD and THC at the same time.
Searching "cannabidiol" in literature scanning web-sites and ltering results just for the last 20 years, it is amazing to realize that most of the work, about 60%, are interested on pharmacological activity of the compound, 30% are still researching and progressive analytical methods able to separate the presence of other cannabinoids. Finally, only 6% of the published researches is interested in the extraction protocols. Therefore in this context, the aim of the present study was to develop and a soxhlet extraction procedure to get CBD and THC extracts from the different four strains which planted from different four regions of Konya, Turkey. It is the rst study to show the different climatic conditions effect on the yield of CBD and THC.

Chemicals
Soxhlet extraction solvents which are hexane, ethyl acetate and ethanol were purchased from Sigma, Germany. Cannabinoid standards (CBD and THC) were supplied at 1 mg/mL stock solutions in methanol by Sigma, Germany, Acetonitrile (Sigma, Germany), water (Ultrapure for chromatographic analysis), Acetic acid (100% anhydrous, glacial, Merck) and methanol (Sigma, Germany).

Seeds
Four different cannabis seeds were purchased from local markets in Turkey. Two of them from Konya (Elnur and Papatya) and one of them from Ankara (Gökçeağaç) and the last one is "Narlı" from Narlısaray district from Samsun. Only the Narlı is genus, the others are the population. Narlı, the rst domestic and national seed variety, was o cially registered at the end of 2019 with the work carried out within the scope of the "Development of Genotypes with Low THC in Cannabis Populations Project" initiated by Samsun Ondokuz Mayıs University Faculty of Agriculture in Turkey. The purpose of developing this variety is to produce hemp with low THC and to transform it into products such as ber.

Cultivation Regions
The seeds were planted at four different regions which have different climates in Konya, Turkey. You can see the characteristics of the planted regions in supplementary material (Table S1). In the table (S1), The harvested plants were kept in the greenhouse without direct sunlight, according to the regions, so that the male and female did not interfere. The plants were then hand-picked without using a mechanical tool so that the leaves were separated from their stems. The drying process will continue in the oven at 30°C for 48 hours (low temperature and for a long time) until the samples reach the grinding conditions.

Sample preparation
The leaves were splintered at 3 degrees speed for 3 minutes using Waring Commercial Laboratory Blender (Sigma, Germany). Every strain was weighed 100.0 g portions and stored at -18°C until to use.

Decarboxylation process
The plants were exposed to the decarboxylation process at 140°C for 30 min before extraction according to the Grijo et al. [5].

Soxhlet extraction procedure
The CBD and THC present in the raw material was extracted using the Soxhlet method. The extractions were performed under reduced pressure to avoid the thermal degradation of the target compound. The extractions were carried out in a six-hour period, using a solvent polarity gradient -rst hexane, then ethyl acetate, and nally ethanol. Brie y, 10.0 g of cannabis plant material was extracted with hexane, obtaining the non-polarity fraction. Subsequently, the residual cake was extracted with ethyl acetate, producing the middle-polarity fraction. Finally, the next residual cake was extracted with ethanol, thus getting the polar fraction. All fractions were concentrated under vacuum and analyzed by HPLC.
The extraction yields were calculated according to the equation: Yield (% w/w) = mass dried extract (g) / mass dried matrix (g) x 100 Eq-1 The results were expressed as the average of two replicates of the extraction.

Determination of CBD and THC with HPLC
Cannabinoid composition was analyzed with HPLC (Waters e2695) equipped with a ACE-5 C18 250×4,6 mm id 5µm column at 30°C oven temperature. Mobile phase solvents were acetonitrile with 0.1% (v/v) acetic acid and water were run at isocratic system. Analysis were run at 210 nm. Injection volume was 20 µL. Flow rate was 1 mL/min. Run time was 65 min. HPLC system has a 2489 UV/vis detector. This conditions were selected based on the literature with some modi cations. Concentrations of CBD and THC were quanti ed by their peak areas against those of standards. All the samples concentrations were 1 mg/1 mL in methanol then diluted 50 times. Concentration of the standards were 12.5 ppm CBD and 10 ppm THC.

Extract yields
Choosing the right cannabis soxhlet extraction solvent can be complicated. Prices can vary widely, different processes require different levels of expertise to operate, and not all ground facilities will be suitable to handle some of the more hazardous solvents that are seen in select extraction systems. The cannabinoids are non-polar compounds, and so can be removed effectively from the cannabis plant using other non-polar substances. But some terpenes, a class of compound responsible for much of cannabis' avor and aroma, are polar compounds.
The extract yields obtained for the four strains grown in four different regions are reported in Table 1. As you can see from the table, all yields of different strains (Elnur, Gökçeağaç, Narlı, Papatya) and their genders comparisons and soxhlet solvent comparisons according to the different cultivation regions are reported. Table 1 Extract yields * (%) of the all strains which grown in different regions. Looking at the table in terms of differences between species, it is seen that the highest extract yields were obtained from Gökçeağaç (13.83% and 8.42%). Although the second highest yield (11.27%) is obtained when the female of the Elnur species grows in the Beyşehir region, the second highest yields are all regions of the female of the Narlı species (9.90%, 8.84%, 6.74%, and 5.48%) considering the high yields obtained from each region.
When looking at the table in general terms, it is seen that the highest yields were obtained with hexane solvent (13.83% Gökçeağaç strain in Altınekin region) and the lowest yields were obtained with ethanol solvent. This idea is disrupted by the higher yields of ethanol than hexane yields of all species in Çumra region. Although higher amounts of cannabinoids are obtained with ethanol, which is considered here, we will explain in the section that we examine the content that this idea is not correct. It is understood that other components other than cannabinoids dissolved in the plant with ethanol solvent also come at this stage.
According to the extract yields, soxhlet seems to be the suitable technique for the extraction of plants aerial parts. However, it should be pointed out that this extraction technique carried out at high temperature allows the co-extraction of the bers. These contribute to the dry extract weight. While the species with high yields cannot be considered to have high content of cannabinoids, the cannabinoid content of Papatya and Elnur species with low yields should not be considered to be low. Because in soxhlet extraction, hexane, ethyl acetate, and ethanol solvents chosen to make a transition from nonpolar to polar in terms of polarity will not only extract the cannabinoid species in the cannabis plant. If the cannabinoid content of the highest yield species is also high, then we can say that we have made a selective extraction.
Palmieri et al., (2020) [16] were found that the yields of hemp which conducted soxhlet extraction system for 6 hours, used ethanol for solvent was 10.00%. This result is tting with our ethanol extraction result part.
The difference in extract yields can also be evaluated in terms of climatic differences. Differences in latitude, longitude, temperature, rainfall and humidity of the cultivated regions given in Table S1 cause differences in the development and content of the species. When evaluated from this point of view, it is seen that the highest yields are obtained from the regions with the highest rainfall and the lowest temperature in Altınekin and Beyşehir regions.

Composition determination of extracts with HPLC
According to the method described in section 2.7, the calibration charts were drawn by running the CBD and THC standards at different concentrations. According to the standard chromatograms, all extracts were quanti ed. Analyses were done by duplicate and values are expressed as mean ± SD.

The same strains which grown in different regions
Four different cannabis strains cultivated in four different regions were used throughout the study.

Elnur strain
In Table 2, we can see how the Elnur strain, the rst of these plants, differs in content when planted in different regions. According to the data in the table; when interpreted in general, we can say that female strain contain higher amounts of CBD and THC than males. When evaluated in terms of the solvent used in the soxhlet system, hexane was the solvent with the highest CBD and THC contents as expected. It can be said that there is a linear decrease in ethyl acetate and ethanol results according to polarity ranking.
As we said when evaluating the extract yields, it cannot be said that high CBD and THC are obtained when the contents of the species with high extract yield are considered. To be more precise, high extract yields do not necessarily mean a high selective extraction. When the differentiation of content of Elnur strain according to different regions is examined from Figure  1A-B-C-D and Figure S3-A, the highest CBD yield in Meram region (52.11%); we see that the highest THC yield is obtained in the Altınekin region (66.21%). These results were examined in terms of female species. Because the general trend is that the results of the female species are higher than the male species. When Figure 1-A-B-C-D is examined, it is seen that hexane, ethyl acetate and ethanol results are obtained by decreasing and female species generally contain higher CBD and THC results than males.
Only in the extracts obtained with hexane in the Beyşehir region, the CBD and THC contents of the female and male strains are close to each other. More THC was obtained in the Elnur species than expected and allowed by legal limits.
The climatic differences of the Meram and Altınekin regions where the highest yields were obtained are given in Table S1. It is Meram, which receives the least rainfall and is one of the regions with the highest altitude, and Altınekin, one of the regions with the highest rainfall and the lowest altitude.

Papatya strain
The results of the contents of Papatya, another of the species, are given in Table 3. It is seen that the highest CBD yield was obtained in the Meram region (47.39%) and the highest THC yield was obtained from the Beyşehir region (52.05%) (Table S3-

Narlı strain
Narlı, the rst domestic and national cannabis strain as we said in section 2.2, is the most e cient strain in terms of CBD among all cultivated strains for the purpose of production and cultivation. Looking at Table 3, it is seen that the hexane results of female species are 65.88% in Beyşehir region, 61.53% in Çumra region, 54.67% in Altınekin region and 54.51% in Meram region. Even the idea that female species results were higher than male species did not t for this species, and even male species yielded CBD yields as much as females. (CBD yield obtained with hexane solvent in Meram region is 64.79%. CBD yield obtained with hexane solvent in Altınekin region is 59.38%.) The rst noticeable content is CBD when looking at Figure 3A-B-C-D. In fact, in line with the cultivation logic, THC content is considerably less than all other species. When Figure S3-C is examined, it is seen that there will be no problem in terms of CBD when it is desired to be grown in all regions, but it will be obtained with a yield of over 60 percent in Beyşehir and Çumra regions. When it is considered as an extract with a lot of CBD but low in terms of THC, it is seen that the Beyşehir region is the most suitable region for growing Narlı strain. It can be seen from Table S1 that the Beyşehir region receives the most rainfall and is the coolest region. The Narlı strain is the rst for Turkey, as it is among the species that are allowed to be produced in terms of legal limits. In order to increase production and avoid legal obligations, incentives should be provided for the production of species that do not exceed legal limits in terms of THC rich in CBD.

Gökçeağaç strain
When we look at Table 5 to examine the CBD and THC results of Gökçeağaç, it can be said that the highest CBD yield is in the Meram region (56.18%), followed by the female species obtained with hexane solvent in the Altınekin region (50.49%). In general, low contents were obtained from Çumra region ( Figure   4A-B-C-D). It seems that this region is not suitable for cultivating this strain. In the Beyşehir and Altınekin regions, it is seen that the CBD results are almost close to the THC results (obtained with hexane in female strains). In the Beyşehir and Meram regions, it is seen that quite a lot of CBD and THC are produced in male species. If a production rich in CBD is desired, if a THC-rich production is desired in the Meram region, Çumra and Altınekin regions should be preferred ( Figure S3-D). Although the production of THC-rich species is prohibited in most of the countries, it can be ensured that the production of THC can be produced in a controlled manner, since it is used as a drug active substance in the eld of medicine and especially as an analgesic.
The yield results of CBD and THC contents of all strains in the different regions in dry cannabis sample are shown in Table 6. In the Çumra region, the highest amount of CBD in the dry plant was obtained from Narlı strain with 4.15%. In Altınekin region, the highest amount of CBD in dry plant is again Narlı strain with 4.83%. In the Meram region, the highest amount of CBD in the dry plant is the Elnur strain with 3.67%, but also the Narlı type is still at a considerable amount with 2.97%. In the Beyşehir region, the highest amount of CBD in dry plant was obtained from Narlı strain with 6.52%. In line with the production logic of the Narlı strain, it is an expected result that the highest yields of CBD amounts in dry plants were obtained from the Narlı strain. After the Narlı strain, higher yields are Gökçeağaç and Elnur strains, respectively, grown in Altınekin and Beyşehir regions, which are obtained with the hexane solvent extraction of the females. Considering the percentages in dry plants in terms of THC, according to the data in Table 6, at least percentages of THC were obtained in Narlı strain. Even though it is still above the legal limits, if a poor production of THC rich in CBD is desired, the necessity of choosing the Narlı strain is proven in all circumstances.

Different strains which grown in same regions
See Figure S2 to show which strains grow best in which region. Considering the polarity of the CBD and THC active ingredients, it was determined that the highest results were obtained with hexane and at the same time it was determined that the female species were more e cient than the male species, so we made the interpretation of which species should be cultivated in which region by considering these parameters. Figure S2 A-B-C-D shows the differences of different species in the same region.
Considering the Meram region, it is seen that the most productive strains in terms of CBD are Gökçeağaç and Narlı strains. At the same time, it is seen that THC is the lowest in Narlı strain ( Figure S2-A).
It is seen that the most productive type in terms of CBD in Çumra region is Narlı, followed by Elnur strains with the highest percentage of CBD. In terms of THC, Gökçeağaç yielded the highest yield in Çumra region, while the lowest yield was obtained in Narlı strain ( Figure S2-B).
It is seen that the most e cient strain in terms of CBD in the Beyşehir region is Narlı and the most e cient strain in terms of THC is Papatya ( Figure S2-C).
It is seen in Figure S2-D that the most e cient strain in terms of CBD growing in Altınekin region is also Narlı strain, as in other regions, followed by Gökçeağaç strain. In terms of THC, it is seen that Elnur strain give very high result.

Conclusions
In accordance with the purpose of the study, the CBD and THC active ingredients extracted by the soxhlet extraction method from the leaves of four different cannabis strains harvested after planting seeds in four different regions in Konya region were analyzed by HPLC. Extract amounts and CBD / THC contents of dry plant both between regions and strains were examined. Based on the apolar character of the CBD and THC active ingredients, the extract yields obtained with the hexane solvent, which is the apolar solvent, are the highest, as expected. The lowest yields were obtained with ethanol solvent in all regions.
In general, the highest extract yields were obtained from female species in accordance with the literature.
Although there are very low results in the literature in male strains, the yields obtained in our study are quite high. We can relate this to the close planting of male and female strains in the same environment.
Because male and female species can pollinate by wind in the same environment and form an average strain. Under normal circumstances, male and female species would have to be planted in areas too far away to cause pollination. In the yields obtained by soxhlet extraction, the results of Gökçeağaç male strains using hexane solvent in Altınekin region (13.83%) and Gökçeağaç female strains in Beyşehir region (8.42%) were the highest results. The results of the Elnur female strains in Beyşehir region are in the second place with 11.27%. However, if evaluated in terms of the species with high results in all regions, the extract yields of the female of the Narlı strains obtained with hexane solvent are the highest (9.90%, 8.84%, 6.74% and 5.48%).
When all species are evaluated in terms of CBD content from HPLC analysis results after soxhlet extraction, all results in the Meram region are the highest. As it can be understood from here, if it is desired to obtain a CBD-enriched extract, it can be planted in Meram region. Narlı strain provided the highest yield (65.88%) in terms of CBD in accordance with the purpose of cultivation. When evaluated in terms of THC active ingredient, it was seen that each strain gave high yields in different regions after soxhlet extraction. Elnur strains gave the highest yields in Altınekin region (66.21%), Papatya strains in Beyşehir region (52.05%), Narlı strains (14.49%) and Gökçeağaç strains in Çumra (56.13%). It has been observed that the regions with the highest temperature and the lowest annual average humidity are more suitable for cannabis cultivation (Meram).

Declarations
Author contributions The design of experiments, all the analysis, evaluations of results, and also drafting and writing manuscript were done by Umut Karğılı and Dr. Ezgi Aytaç.

Con icts of Interest
The authors declare that they have no known competing nancial interests or personal relationships that could have appeared to in uence the work reported in this paper.