Mitragyna KORTH. is a small genus in family Rubiaceae consisting of ten species in the world. Six species, M. speciosa (KORTH.) HAVIL., M. tubulosa (ARN.) HAVIL., M. parvifolia (ROXB.) KORTH., M. hirsuta HAVIL., M. diversifolia (WALL. ex G. DON) HAVIL., M. rotundifolia (ROXB.) O. KUNTZE, widely grow in Thailand, Vietnam, Philippines, Malay Peninsula, Sumatra, Borneo, and New Guinea islands. 1,2) In Thailand, M. hirsuta, M. diversifolia and M. rotundifolia are seen commonly. M. speciosa distributed from central to southern part, is a narcotic plant, so called "Kratom," but with specific medicinal importance.1,3) The leaves of M. speciosa have been chewed habitually by Thai natives for the opium-like effect and coca-like stimulant to overcome the burdens of their hard work. In Thai traditional medicine it has also been used to treat diarrhea, stop coughing and relieve injury pain, and in modern it has been used as a substitute for opium and for treatment of addiction to morphine.4) Phytochemical studies revealed that mitragynine, an indole alkaloid, was the major constituent of this plant, accounting for about half of the total alkaloid contents. Its analogues, speciogynine, speciociliatine, and paynanthine etc. were also found. 5,6) Recent pharmacological studies reported the antinociceptive effect of this species due to containing of mitragynine and its analogues. Especially, 7-hydroxymitragynine, a minor constituent, revealed significantly potent antinociceptive activity through opioid receptors.5-7) Its antinociceptive activity was many times more potent than morphine when administrated orally and subcutaneously. 8,9) The use of M. speciosa has been forbidden in Thailand due to its narcotic effects. The Thai government passed the law to make planting of M. speciosa illegal. However, since the species is indigenous to Thailand and widely distributed, the law is hard to raise its efficiency. On the other hand, the leaves of M. diversifolia or others are frequently used as substitutes but are not considered as effective. Therefore, authentication of M. speciosa is essential for both forensic and medicinal purpose.In recent years, molecular identification of herbal drugs by nucleotide sequence of various DNA regions has been demonstrated to be a powerful way.10-16) Especially, DNA regions with high evolutionary rate have been widely used to discriminate species and investigate phylogenetic relationship among closely-related taxa, such as chloroplast trnK/matK, 11) rpl14-16, 12) trnT-F, 13) and nuclear internal transcribed spacers (ITS), [13][14][15] 5S spacer region, 16) etc. In a preliminary study, we found that there was no nucleotide difference in chloroplast matK gene and nuclear 18S rRNA gene regions between M. speciosa and M. hirsuta (unpublished data). In the present study, we investigated the sequence of ITS region for characterizing four Mitragyna species in Thailand and further to discriminate M. speciosa from the other species.
MATERIALS AND METHODS
Plant MaterialsTotal of seventeen specimens of four Mi...
