R Maharaj, G Singh, C Thomas, D John
cannabis, gland, thc, δ9 tetrahydrocannabinol
R Maharaj, G Singh, C Thomas, D John. Physical And Chemical Characteristics Of Cannabis Found In Trinidad And Tobago. The Internet Journal of Forensic Science. 2006 Volume 2 Number 2.
The analysis in 2002 of representative samples taken from flowering and fruiting tops of cannabis material seized by law enforcement officials in Trinidad and Tobago showed that the mean content of Δ9 tetrahydrocannabinol (THC) was 5.26%, range 0.53 – 11.58% with a standard deviation of 2.98%. The mean THC content of the Cannabis samples seized in 2002 was significantly higher than the content found Cannabis representative samples seized and analyzed in Trinidad and Tobago in 1993 (mean content of Δ9 tetrahydrocannabinol (THC) was 0.079%, range 0.01 – 0.18% with a standard deviation of 0.04%) .The specialized glands present on the aerial structures of the plant responsible for the biosynthesis of the cannabinoids were observed in all the samples.
Much confusion exists over the various terms used to describe the Cannabis plant. Terms such as
Secondary metabolic compounds are produced by Cannabis. A variety of alkanes have been identified (Adams Jr., Jones, 1973), as well as nitrogenous compounds (El Sohly et al, 1982), flavonoids (Gellbert, 1974)) and other miscellaneous compounds (Hanus, 1976). Terpenes appear in abundance and contribute to the characteristic odor of the plant (Hendricks et al, 1975) and some of its crude preparations, such as hashish. The compounds which comprise the active drug ingredients are apparently unique to this genus and are termed cannabinoids. There are over 60 of these type compounds present in the plant (Turner et al, 1980).
The major sites of cannabinoid production appear to be epidermal glands (Fairbain, 1972). These epidermal glands seem to fall into two broad categories: stalked and sessile. The glandular cells are covered with a “sheath” under which the resins are secreted via vesicles (Mahlberg and Kim, 1992). A spherical structure forms as the resins accumulate until the sheath bulges away from the secretory cells. The resin is then released by rupture of the membrane or through pores in its surface (De Pasquale et al, 1974). Bracts subtending the female flowers contain a greater density of glands than the leaves. The bracteole enclosing the pistil has the highest cannabinoid content of any single plant part (Kimura and Okamoto, 1970).
The widespread use of the sinsemilla growing technique (Marnell, 1997)) has increased the average concentration of THC from 1 – 3% in the 1960s to 6-14% in the 90s (DEA, 1997). There are several reports in the literature on the THC content of cannabis and some of these are summarized in table I. In the present context the origin refers to the country in which the plant was ground and not the country of origin of the seeds. The quoted THC levels are expressed as a percentage of dry weight of cannabis.
Most of the data in table are derived from cannabis legitimately grown and processed as part of scientific studies of cannabinoids in the 1970s.
The present work is concerned with the identification of the epidermal glands (both stalked and sessile) as well as non-glandular trichomes in plant material seized by law enforcement officials in Trinidad and Tobago in the years 1993 and 2002. The level of THC content in the representative samples will be quantitated and the mean concentration of each year statistically compared to detect any changes in THC concentration over the period.
Materials and Method
Fifty (50) and Sixteen (16) samples of plant material were collected in 1993 and 2002 respectively. These samples were seized from different parts of Trinidad and Tobago by Police Officers and submitted to the Trinidad and Tobago Forensic Science Centre for analysis.
Segments of leaves, stem, bracts and roots were immersed in 5% chloral hydrate solution and gently heated for 30 mins. The sections were examined with a Zeis Microscope (magnification 100). Samples of Cannabis were removed from the flowering and fruiting tops of the plant and were chopped and mixed to give a homogeneous mixture prior to analysis by gas chromatography.
The extraction process was carried out for each sample as follows:
Plant material (3g) was placed in a conical flask and approximately 50mls toluene added. The conical flask was stoppered and mounted on a Gallenkamp orbital shaker, at 140 revolutions per minute for two hours. The solution was filtered undergravity. The filtrate was transferred to a round bottom flask and evaporated to dryness, under vacuum, using a Buchi Rotary Evaporator at 70°C.
Each extract was made up to 50ml using an internal standard solution of 0.50mg/ml tetracosane in chloroform: methanol (1:1) and 2ml of this solution analysed by gas chromatography (Fairbairn and Liebmann, 1973). A Hewlett Packard model 6890 instrument was fitted with a HP-5 capillary column cross link 5% PHME siloxane (30m x 3.2mm x 0.25um film thick). The carrier gas flow rate was 2ml/min and the column temperature was 250ºC. Detection was by flame conisation and the detector was calibrated for THC response using a primary standard of 0.50 mg/ml. THC in an internal standard solution of 0.50mg/ml tetracosane in chloroform:methanol (1:1).
Results and Discussion
The microscopic analysis of the segments of plant material revealed the presence of glandular and non-glandular trichomes on most of the aerial parts of the plant. None of the characteristic features were observed on the root surfaces. The non-glandular trichomes observed included the cystolithic and non-cystolithic types. The stalked glands appeared as a single cell or a small group of cells on a single or multicellular pedestal whereas the sessile glands possess no stalk. Cystolithic and non-cystolithic Most of the structures were found at the apical tip and on the underside of the leaves. These observations are consistent with previous work done (De Pasquale et al, 1974).
The results generated from the gas chromatography analysis revealed that the mean THC (%) content for the sixteen (16) samples collected in 2002 was 5.26%, range 0.53 – 11.58% with a standard deviation of 2.98% and that the mean THC (%) content for the fifty (50) samples collected in 1993 was 0.079%, range 0.01-0.18% with a standard deviation of 0.04%
Statistical analysis of the data was done suing the Statistical Package for Social Scientist (SPSS) version 13.0. An independent two-sample t-test also referred to as the Aspin-Welch Unequal-Variance Test was conducted to evaluate the hypothesis that the purity of drugs produced in 2002 was higher to that produced in 1993. The test was significant at the conventional levels of significance of 0.0001, 0.01, 0.05 and 0.10. The value of the t-test statistic computed was 6.964 (degrees of freedom = 15) and the p-value obtained was 0.000002. As a result, one can strongly conclude that the concentration of the psychoactive drug THC in the samples seized in 2002 was significantly higher than those produced in 1993 (Mean = 0.0790, SD = 0.04032, n = 50).
The widespread use of the sensimella technique (Marnell, 1997) as well as other heredity factors (Small and Beckstead, 1973) can be attributed to the increase in THC content of Cannabis found in Trinidad and Tobago. The Sinsemilla
Ecological factors have long been thought to have an important influence in THC concentration by stressing the
The epidermal glands responsible for the biosynthesis were observed in the Cannabis samples examined in the study. The level of THC in the samples analyzed in 2002 was higher than that observed in 1993. Although the chemistry of