Phytochemical and genetic analyses of ancient cannabis from Central Asia

Abstract

The Yanghai Tombs near Turpan, Xinjiang-Uighur Autonomous Region, China have recently been excavated to reveal the 2700-year-old grave of a Caucasoid shaman whose accoutrements included a large cache of cannabis, superbly preserved by climatic and burial conditions. A multidisciplinary international team demonstrated through botanical examination, phytochemical investigation, and genetic deoxyribonucleic acid analysis by polymerase chain reaction that this material contained tetrahydrocannabinol, the psychoactive component of cannabis, its oxidative degradation product, cannabinol, other metabolites, and its synthetic enzyme, tetrahydrocannabinolic acid synthase, as well as a novel genetic variant with two single nucleotide polymorphisms. The cannabis was presumably employed by this culture as a medicinal or psychoactive agent, or an aid to divination. To our knowledge, these investigations provide the oldest documentation of cannabis as a pharmacologically active agent, and contribute to the medical and archaeological record of this pre-Silk Road culture.

Fig. 1.

Area maps. (A) Map of Turpan, Xinjiang, China and its location in Central Asia. (B) Map of Yanghai Tombs site and surrounding area (adapted from Xinjiang Institute of Cultural Relics and Archaeology, 2004).

Fig. 2.

Photomicrographs of ancient cannabis. (A) Photograph of the whole cannabis sample being transferred in laminar flow hood. (B) Photomicrograph of leaf fragment at low power displaying non-glandular and amber sessile glandular trichomes. Note retention of chlorophyll and green colour, scale bar=100 μm. (C) Higher power photomicrograph of a single sessile glandular trichome. At least 4 of its 8 secretory cells are clearly visible on the right, and the scar of attachment to the stype cells in the centre, scale bar=25 μm. (D) Low power photomicrograph of a cannabis achene (‘seed’) including the base with a non-concave scar of attachment visible, scale bar=1 mm.

Fig. 3.

Complete high performance liquid chromatography (HPLC) of ancient cannabis.

Fig. 4.

Complete gas chromatography-flame ionization detection (GC-FID) of ancient cannabis.

Fig. 5.

Gas chromatography of ancient cannabis subsections. (A) GC of the 30–34 min region demonstrates several phytocannabinoids: cannabidiol (CBD), cannabichromene (CBC), cannabicyclol (CBL), and cannabinavarin (CBNV). (B) GC of the 34–36.3 min region displays the highest peak, cannabinol (CBN), the direct non-enzymatic oxidative metabolite of THC, with possible cannabielsoin (CBE) at 34.2 min. (C) GC of the 36.3–40.5 min region displays cannabitriol (CBO) a THC degradant, and CBN variants (see text).

Fig. 6.

Mass spectra of ancient cannabis. Subsections demonstrate the phytocannabinoids cannabinol (CBN), cannabidiol (CBD), cannabicyclol (CBL), cannabinolivarin (CBNV), cannabichromene (CBC), cannabielsoin (CBE), 1′-oxcannabinol, and 1′-hydroxycannabinol.

Fig. 7.

DNA analysis of ancient cannabis. (A) Nucleotide sequences of the wild-type tetrahydrocannabinolic acid synthase, China F, and the mutant sequence, China F(h), with two single nucleotide polymorphisms highlighted in lower case yellow. (B) Amino acid translation of China F and China F(h), demonstrating divergence in a change from serine (wild-type) to threonine (mutant), highlighted in blue.