The results presented collectively point to the most probable conclusion which is that the Gūshī
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
) 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 Δ9
-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 Δ9
-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
, 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 Gūshī
fabricated clothing from wool (see Supplementary Fig. S6B
online) and ropes from Phragmites
(reed) spp. fibres (see Supplementary Fig. S6C
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 Δ8
-THC (previously termed Δ6
-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
; 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
), 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 Gūshī
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
), 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, Gūshī
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
; Mallory, 1998
; Renfrew, 1998
; Mallory and Mair, 2000
), whose putative southward migration some authorities have attributed to ‘global cooling’ c
. 4000 years BP
), and to their proto-Indo-European-speaking Yamnaya forebears further west, dating to 6000 years BP
; Anthony, 1998
; Winter, 1998
). Abundant mysteries remain as to the origins and customs of the Gūshī
. 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.