Ancient Chinese Marijuana (Gushi Shaman)
Phytochemical and genetic analyses of ancient cannabis from Central Asia
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.
Key words: Archaeology, botany, cannabis, cannabinoids, archaeobotany, ethnopharmacology, genetics, medical history, phytochemistry
Uighur farmers cultivating the land at the base of the Huoyan Shan (‘Flaming Mountains’) in the Gobi Desert near Turpan, Xinjiang-Uighur Autonomous Region, China some 20 years ago uncovered a vast ancient cemetery (54 000 m2) that seemingly corresponds to the nearby Aidinghu, Alagou, and Subeixi excavations (Ma and Wang, 1994; Chen and Hiebert, 1995; Davis-Kimball, 1998; Kamberi, 1998; An, 2008) (see Supplementary Fig. S1 at JXB online) attributed to the Gsh culture (later rendered Jüshi, or Cheshi) (Academia Turfanica, 2006). The first written reports concerning this clan, drafted about 2000 years BP (before present) in the Chinese historical record, Hou Hanshu, described nomadic light-haired blue-eyed Caucasians speaking an Indo-European language (probably a form of Tocharian, an extinct Indo-European tongue related to Celtic, Italic, and Anatolic (Ma and Sun, 1994). The Gsh tended horses and grazing animals, farmed the land and were accomplished archers (Mallory and Mair, 2000). The site is centrally located in the Eurasian landmass (Fig. 1A, B), 2500 km from any ocean and located in the Ayding Lake basin, the second lowest spot on Earth after the Dead Sea (Fig. 1A, B). Formal excavations completed in 2003 revealed some 2500 tombs dating from 3200-2000 years BP (Xinjiang Institute of Cultural Relics and Archaeology, 2004). Other evidence from chipped stone tools and other items indicate a possible human presence in the area for some 10 000-40 000 years (Kamberi, 1998; Academia Turfanica, 2006). Due to a combination of deep graves (2 m or more), an extremely arid climate (16 mm annual rainfall), and alkaline soil conditions (pH 8.6-9.1 (Pan, 1996), the remarkable preservation of the human remains resulted in the mummification of many bodies without a need for chemical methods. Numerous artefacts from the tombs included equestrian equipment and numerous Western Asian crops such as Capparis spinosa L. (capers) (Jiang et al., 2007), Triticum spp. (wheat), Hordeum spp. (naked barley), and Vitis vinifera L. (grapevines) (Jiang, 2008), often centuries before their first descriptions in Eastern China (Puett, 1998).
Microscopic botanical analysis
Gross examination of the 11 g sample of cannabis provided by the Chinese Academy of Sciences revealed loose dry vegetative material (Fig. 2A). The impression that the vegetative material had been lightly pounded was supported by examination of the wooden bowl, whose internal surface was worn smooth, apparently from use as a mortar (see Supplementary Fig. S5B at JXB online). The cannabis retained a surprisingly green colour in its leafy parts and displayed visible glandular trichomes (Fig. 2B), the phytochemical factory of the plant and site of manufacture of cannabinoids and terpenoids (Potter, 2004; McPartland and Russo, 2001; Kim and Mahlberg, 2003). However, the ancient sample lacked the typical cannabis odour. Microscopic examination confirmed the presence of intact sessile trichomes with an amber tint (Fig. 2B), while higher resolution documented the retention of visible secretory cells within the trichomes (Fig. 2C). Achenes (‘seeds’) averaged 2.2-3.6 mm in length (Jiang et al., 2006), were light in colour with some striations, but demonstrated rough, non-concave fruit attachment (Fig. 2D), all traits of domestication (Schlumbaum et al., 2008) associated with cultivated cannabis strains (Vavilov, 1926). In contrast, achenes of wild strains are typically smaller and darker with concave attachment zones that favour shattering and easy spread (Vavilov, 1926). Germination was attempted with 100 achenes in compost, but no emergence was observed after 21 d.
The results presented collectively point to the most probable conclusion which is that the Gsh culture cultivated cannabis for pharmaceutical, psychoactive or divinatory purposes. In examining the botanical evidence from this ‘old and cold’ site with its unique degree of preservation, the cannabis consisted of a processed (pounded) sample whose seed size, colour, and morphology, at least according to principles of Vavilov (Vavilov, 1926), suggest that it was cultivated rather than merely gathered from wild plants. The considerable amount of cannabis present (789 g) without any large stalks or branches would logically imply a pooled collection rather than one from a single plant. Importantly, no obvious male cannabis plant parts (e.g. staminate flowers, not infrequently observed in Indian herbal cannabis, or bhang (Russo, 2007) were evident, implying their exclusion or possible removal by human intervention, as these are pharmacologically less psychoactive.
The HPLC, GC, and MS analyses confirm the identity of the supplied plant sample as Cannabis sativa L. The predominance of CBN indicates that the original plants contained 9-tetrahydrocannabinol (THC) as the major phytocannabinoid constituent. The presence of CBO and numerous CBN-related substance peaks further supports this view. CBD and CBC, together with their known thermo-oxidative degradation products CBE and CBL (Brenneisen, 2007), are present, but the GC analysis would appear to indicate that, in both cases, CBC and CBL are represented in greater quantities, as expected in a high-THC cannabis strain wherein CBD is only a minor component. In addition, there is a peak for CBNV which confirms that the plant also contained 9-tetrahydrocannabivarin (THCV), a propyl phytocannabinoid. All of these observations are consistent with strains of cannabis with a high THC content and in an alternative taxonomy suggests it should be assigned to Cannabis indica Lamarck (Hillig and Mahlberg, 2004).
While chromatography elution times may vary with temperature, column type, and other factors, confirmation was evident with corroboratory mass spectra values that were identical to those seen daily in assays performed on fresh cannabis extracts in this laboratory.
The presence of so many recognized cannabinoid degradants is consistent with very old cannabis samples. The very low concentration levels measured in the HPLC analysis may indicate that the sample provided contained significantly more leaf and twig material than flower material, rather than being evidence in itself that the sample was of low potency originally. This plant material is therefore conclusively cannabis derived from a population of plants within which THC was the dominant cannabinoid. By contrast, a sample taken from a mix of wild-type Cannabis sativa would customarily harbour a more equal mixture of THC and CBD (de Meijer et al., 2003). It would appear, therefore, that humans selected the material from plants on the basis of their higher than average THC content. To elaborate, a chemotaxonomy of cannabis previously outlined indicates three types (Small and Beckstead, 1973): chemotype I (drug) strains with high-THC:CBD ratios, chemotype II low-THC, higher-CBD (fibre) strains, and chemotype II with more equal ratios. THC and CBD production are mediated by co-dominant alleles BT and BD, respectively (de Meijer et al., 2003). By comparison, pooled samples from cannabis fields in Morocco and Afghanistan will normally produce 25% high-THC plants, 25% high-CBD plants, and 50% with lower, mixed titres, combining to yield roughly equivalent amounts of the two phytocannabinoids (Russo, 2007), a pattern not observed in our specimen.
Isotopic analysis of cellulose from this cannabis sample might conceivably be used in comparison with other samples in an attempt to establish its geographic origin.
While multi-purpose cannabis plants used simultaneously for food (seed), fibre (stalks), and pharmaceutical uses (flowering tops) have been recently reported from Darchula in far western Nepal (Clarke, 2007), more customarily, a given plant is best suited toward a single purpose. Of additional key importance is the absence of hemp artefacts from the Yanghai Tombs. The Gsh fabricated clothing from wool (see Supplementary Fig. S6B at JXB online) and ropes from Phragmites (reed) spp. fibres (see Supplementary Fig. S6C at JXB online). Whereas hemp textiles have been collected from the Northern China Yangshao Culture from 6000-7000 years BP, their appearance in the west was not documented before 2000 years BP, for example, 1500 years BP in Kucha, 600 km west of Turpan (Mallory and Mair, 2000).
Previous phytochemical analyses of antique cannabis preparations have demonstrated THC remnant fingerprints from 19th century cannabis preparations (Harvey, 1990) including a 140-year-old sample of Squire’s Extract (Harvey, 1985). A study in 1992 reported the presence of 8-THC (previously termed 6-THC) from burned cannabis that was reportedly inhaled as an aide to childbirth in a Judean cave 1700 years BP (Zias et al., 1993), supported by the finding of cannabinoid residues in an adjacent glass vessel (Zias, 1995). In the Mustang region of Nepal, mummified human remains of probable Mongolian ancestry have been dated 2200-2500 years BP in association with cannabis, probably transported from elsewhere (Knörzer, 2000; Alt et al., 2003), but with insufficient detail to ascertain its use. Rudenko recovered cannabis seeds, censers, and hempen clothing in Pazyryk, Siberia from Scythian kurgans (burial mounds) from 2400-2500 years BP (Rudenko, 1970; Brooks, 1998), closely matching Herodotus’ descriptions of funeral rites for that culture (Herodotus, 1998). Sarianidi also claimed cannabis use in the Bactria-Margiana Archaeological Complex (BMAC) (present day Turkmenistan) (Sarianidi, 1994, 1998), but this interpretation has been debated (see discussion in Russo, 2007).
Another independent genetic analysis of this material published subsequent to our analysis (Mukherjee et al., 2008) confirmed the presence of THCA synthase, but not the single nucleotide polymorphisms. The authors posited a European-Siberian origin for the material.
Current genetic data also confirm that the plant material examined is Cannabis sativa L. according to ITS and cpDNA analysis. The results also support the hypothesis of the existence of at least two THCA-synthase nucleotide sequences in the ancient plant material examined. One of these sequences perfectly matches the corresponding sequence of already-known THCA-synthases deposited in GenBank, both as gene and protein sequences; the second sequence is a novel one, with two single nucleotide polymorphisms (SNPs) encoding for a protein with presumably very similar characteristics. Whether these two sequences coexisted in a single cannabis plant or a strain heterozygous at the B locus, or belong to different plants, could not be concluded.
THC represents one of the possible phytocannabinoid end-products manufactured by cannabis plants; THC (or, in its native form, THCA) is synthesized by a well-characterized enzyme (THCA- or THC-synthase) from a precursor (CBG or CBGA) common to most chemotypes that represents the metabolic ‘switching point’, downstream of which the variability of the different chemotypes is concentrated. The agents of such variability found in cannabis germplasm are exclusively the different synthases, among which THC(A)-synthase is the only one responsible for making that specific cannabinoid, THC. Therefore, the presence of the allelic variant responsible for coding the THC(A)-synthase may well be considered to be diagnostic, or at least strongly suggestive of a THC-producing plant. The fossil cannabis plants found were therefore genetically equipped to produce THC. How much THC they actually produced, cannot of course be specified because they depend on a number of anatomical, environmental, and nutritional factors that remain unknown.
Numerous questions remain. Current data do not permit it to be ascertained how the cannabis from the tomb was administered. If used orally, perhaps it was combined in some fashion with Capparis spinosa L., as these plants were found together in a nearby but later tomb at Yanghai (Jiang et al., 2007). That date for that tomb was initially reported as 2700 years BP via radiocarbon methods, and since corrected to 2200-2400 years BP with additional calibration employing tree ring data. If this cannabis were smoked or inhaled, no mechanism for so doing has been excavated in the area. The Gsh could have sifted the cannabis through fabric after pounding, then fumigated it, much as described for the alleged cannabis candidate, the Sumerian A.ZAL.LA, administered medicinally for ‘hand of ghost'(Thompson, 1923, 1949), since posited as nocturnal epilepsy (Russo, 2007; Wilson and Reynolds, 1990). While this culture could have arrived from the earlier BMAC region as ‘oasis hoppers’ (Barber, 1999), and certain cultural relationships are apparent to the Scythian culture with respect to cannabis use and equestrian prowess, those peoples were Iranian speakers (Mallory and Mair, 2000). In addition, Gsh cultural affinities and burial practices much more closely resemble those of the presumed proto-Tocharian speaking, incense-burning (Kuzmina, 1998) Afanasievo peoples in the Yenisei Valley to the north (Anthony, 1998, 2007; Mallory, 1998; Renfrew, 1998; Mallory and Mair, 2000), whose putative southward migration some authorities have attributed to ‘global cooling’ c. 4000 years BP (Hsü, 1998), and to their proto-Indo-European-speaking Yamnaya forebears further west, dating to 6000 years BP (Mallory, 1989; Anthony, 1998; Winter, 1998). Abundant mysteries remain as to the origins and customs of the Gsh. Additional answers may accrue from future archaeological excavations or human genetic analyses that elucidate relationships with other ancient cultures and modern peoples of the region. The unique SNPs discovered in this ancient sample may yet be of critical importance in tracing the phylogeny and geographic spread of cannabis and the humans who used it.
The excellent preservation of the cannabis from this tomb allowed an unprecedented level of modern botanical investigation through biochemistry and genetics to conclude that the plant was cultivated for psychoactive purposes. While cultivation of hemp for fibre has been documented in Eastern China from a much earlier date (vide supra Mallory and Mair, 2000), the current findings represent the most compelling physical evidence to date for the use of cannabis for its medicinal or mystical attributes.