Standardization and DNA barcoding of cannabis in Thailand and pharmacological properties of cannabis-based Thai traditional medicine formula extract (Kealomkeasan)

Abstract

Cannabis sativa L. subsp. Indica (Cannabaceae) has been used as a medicinal plant in various aspects. C. sativa has been part of Thai medicine for more than three centuries and its Ingredients in herbal medicine recipe. Kealomkeasan (KLKS) remedy is one of the Thai traditional medicines, consists of seven plants with an unequal part by weight (1:2:3:4:5:20:40) as follows: Cuminum cyminum L. fruits, Nigella sativa L. seeds, Foeniculum vulgare Miller fruits, Zingiber officinale Roscoe rhizomes, Plumbago indica L. roots, Cannabis sativa L. leaves and Piper nigrum L. seeds for relief of muscle pain and anti-inflammation. Recently, medical cannabis was officially legalized for research and medical purpose in Thailand. Therefore, scientific evidence to support the use of medical cannabis is still limited. This study was divided the aim of the study into 3 parts; 1. To evaluate and develop a pharmacognostic standardization of C. sativa leaves collected from 12 various locations of Thailand according to WHO guidance including pharmacognostic specification, macroscopic-microscopic examination, TLC fingerprinting, physicochemical parameter, and preliminary phytochemical screening using standard methods. 2. To establish DNA barcoding of C. sativa from 20 samples and suggested the most suitable one for C. sativa identification. 3. To evaluate in vitro biological activities of ethanolic extract of KLKS remedy including antioxidant, anti-inflammatory, anti-diabetic and acute toxicity activities. The results indicated that 1. pharmacognostic standardization evaluation of the loss on drying, water content, total ash and acid insoluble ash were 4.07 ± 0.09, 7.95 ± 0.12, 14.36± 0.13, 2.73 ± 0.09% , respectively. Water and ethanol extractive vales were 11.10 ± 0.15 and 23.04 ± 0.16%, respectively. Phytochemical screening showed the present of alkaloids, phenolics, flavonoids, triterpenes, steroids and diterpenes. 2. Establishing DNA barcoding of 20 samples of C. sativa from three candidate DNA regions (ITS, matK, and rbcL), the phylogenetic tree was constructed and genetic distance was investigated using a maximum likelihood and the result found that the ITS region was suitable marker for C. sativa species identification. 3. In vitro biological activities of ethanolic extract of KLKS remedy, the results indicated that KLKS remedy and its ingredients showed antioxidant, anti-inflammatory, and anti-diabetic properties. The ethanolic extract of KLKS remedy against brine shrimp lethality tasting showed week toxicity (LC50 = 503.13 µg/ml) whereas each ingredient of KLKS remedy showed median toxicity to non-toxic (LC50 = 150.07 - > 1,000 µg/ml). The finding results provide highly useful information for establishing standardization and identification of C. sativa leaves, and also contribute to the quality control, safety and effectiveness of medical cannabis for health benefits.