Factors Affecting Cannabinoid Contents for Classification of Hemp (Cannabis sativa L.) in Northern Thailand

Abstract

The Thai government has recognized the possibility for legitimate cultivation of hemp. The study of certain cannabinoid characteristics is necessary in establishing criteria for regulation of cannabis cultivation in Thailand. For this purpose, factors affecting characteristics of cannabinoid composition of Thai-grown cannabis were investigated. Plants were cultivated from seeds derived from the previous studies under the same conditions. Three hundreds and seventy two cannabis samples from landraces, three different trial fields and the seized marijuana were collected and analyzed for THC, CBD and CBN contents by GC-FID. The results showed that the cannabis grown during March-June, 2008 which had longer vegetative stages and longer photoperiod exposure, had higher cannabinoid contents than those grown in August. The male plants grown in the trial fields had the range of THC contents from 0.722-0.848 %d.w and average THC/CBD ratio of 1.9. Cannabis in landraces at traditional harvest time of 75 days had a range of THC contents from 0.874-1.480 %d.w and an average THC/CBD ratio of 2.6. The THC contents and THC/CBD ratios of cannabis in the second generation crops grown in the same growing season were found to be lower than those grown in the first generation, unless fairly high temperatures and a lesser amount of rainfall were present. The average THC content in the seized fresh marijuana was 2.068% d.w while THC/CBD ratios were between 12.6 and 84.09, which is 10-45 times greater than those of similar studied cannabis samples from the previous study. Thus, the use of the THC/CBD ratio or log10 values of THC/CBD may give an advantage for distinguishing hemp from marijuana. Furthermore, at the harvest time of 70-75 days which is appropriate time for collecting good quality fiber, the cannabis plants grown in landraces and the trial fields appeared to have the mean actual THC content less than 1%. Therefore, the permitted THC content of 0.5-1% in cannabis for fiber usage would be acceptable. Moreover, both chemical and physical characters of Thai-grown cannabis were studied to provide information for breeders to discriminate their phenotypes and accessions in order to select the low intoxicant with high fiber producing cultivars. The cannabinoids on the basis of THC, CBD and CBN content of 750 plants from eight accessions derived from five local cannabis variants were analyzed individually and their morphological features were also determined. According to the individual plants belonging to the same accessions showing distinct THC/CBD ratios were classified into different phenotypes, it is impossible to classify only single plant for defining the phenotype or determine cannabinoid contents on the single analysis. The minimum number of 50 plants is considerable as representative to be sampled. The THC content was found to correlate negatively to their physical characters such as plant height, stem diameter and fiber weight. The principal component analysis showed that the fiber weight, core weight and stem diameter of the plant as well as the chemical features such a THC content, CBD content, THC/CBD ratio and log10(THC/CBD ratio) explained most of the total variation which could distinguish accession and phenotype of the cannabis plants. The stepwise discriminant analysis confirmed that the cannabinoids and some physical properties could be used to classify the phenotype of cannabis plants into drug, intermediate and fiber types as well, whereas the accessions of the cannabis could not be discriminated clearly by using only their physico-chemical parameters. Pre-defined phenotype of cannabis plants based on the log10 (THC/CBD ratio) were also evaluated with the developed multiplex PCR method. Although the total relative accuracy of identification obtained was only 94%, the pre-defined as fiber and drug type exhibited the 100% of identify. Therefore, the use of the log10 (THC/CBD ratio) to classify the phenotype of cannabis plant is preferable. From the chemical fingerprints study, the results not only revealed the difference between the authorized cannabis grown for fiber usage and marijuana but also indicated that some terpenoids especially sesquiterpenoids and some cannabinoids could be used to distinguish the marijuana from the cannabis grown for fiber usage as well as among the variants Nonetheless, more samples should be further investigated to confirm this conclusion as well as the genetic relationships among the cannabis accessions grown in northern Thailand should be performed in further study. However, the expanded information provided by the current study will assist the development of criteria for regulation of hemp cultivation in Thailand.