Phytochemical and genetic analyses of ancient cannabis from Central Asia

12 Pages·0.851 MB·English

by  Russo E.B. et al.

JournalofExperimentalBotany,Vol.59,No.15,pp.4171–4182,2008
doi:10.1093/jxb/ern260
Thispaperisavailableonlinefreeofallaccesscharges(seehttp://jxb.oxfordjournals.org/open_access.htmlforfurtherdetails)
RESEARCH PAPER
Phytochemical and genetic analyses of ancient cannabis
from Central Asia
Ethan B. Russo1,2,3,*, Hong-En Jiang4,5,Xiao Li5, Alan Sutton2, Andrea Carboni6, Francesca del Bianco6,
GiuseppeMandolino6,DavidJ. Potter2, You-XingZhao7, Subir Bera8,Yong-Bing Zhang5,En-GuoLu¨9, David
K.Ferguson10,FrancisHueber11,Liang-ChengZhao12,Chang-JiangLiu4,Yu-FeiWang4andCheng-SenLi5,13,*
1 Visiting Professor, Institute ofBotany, Chinese Academy ofSciences,[email protected]
2 GW Pharmaceuticals, Porton Down Science Park,Salisbury, Wiltshire SP4 OJQ, UK
3 Faculty Affiliate, Department of PharmaceuticalSciences, Universityof Montana, Missoula, MT, USA
4 Laboratory of Systematicand Evolutionary Botany,Institute ofBotany,Chinese Academy ofSciences,Beijing
100093, China
5 Bureau of Cultural Relics of Turpan Prefecture,Turpan 838000, Xinjiang, China
6 CRA-Centro di Recerca per leColture Industriali, viadi Corticella 133, 40128, Bologna, Italy
7StateKeyLaboratoryofPhytochemistryandPlantResourcesinWestChina,KunmingInstituteofBotany,Chinese
Academy ofSciences,Kunming 650204, China
8 Departmentof Botany, Universityof Calcutta,Kolkata 700019, India
9 Xinjiang Institute ofArchaeology, 4-5 South Beijing Road, U¨ru¨mqi, Xinjiang 830011, China
10Institute of Palaeontology, Universityof Vienna, Althanstrasse 14, A-1090 Vienna, Austria
11Department ofPaleobiology,Smithsonian Institutions, Washington, DC 20560-0121, USA
12College ofBiologicalScience and Biotechnology, Beijing Forestry University, Beijing 100083, China
13Beijing Museum ofNatural History, Beijing 100050, China
Received7August2008;Revised24September2008;Accepted25September2008
Abstract psychoactive agent, or an aid to divination. To our
knowledge, these investigations provide the oldest
The Yanghai Tombs near Turpan, Xinjiang-Uighur
documentation of cannabis as a pharmacologically
Autonomous Region, China have recently been exca-
active agent, and contribute to the medical and
vated to reveal the 2700-year-oldgrave of aCaucasoid
archaeologicalrecordofthispre-SilkRoadculture.
shaman whose accoutrements included a large cache
of cannabis, superbly preserved by climatic and burial
Key words: Archaeology, botany, cannabis, cannabinoids,
conditions.Amultidisciplinaryinternationalteamdem-
archaeobotany, ethnopharmacology, genetics, medical
onstrated through botanical examination, phytochemi-
history,phytochemistry.
cal investigation, and genetic deoxyribonucleic acid
analysis by polymerase chain reaction that this mate-
rial contained tetrahydrocannabinol, the psychoactive
Introduction
component of cannabis, its oxidative degradation
product, cannabinol, other metabolites, and its syn- UighurfarmerscultivatingthelandatthebaseoftheHuoyan
thetic enzyme, tetrahydrocannabinolic acid synthase, Shan(‘FlamingMountains’)intheGobiDesertnearTurpan,
as well as a novel genetic variant with two single Xinjiang-UighurAutonomousRegion,Chinasome20years
nucleotidepolymorphisms.Thecannabiswaspresum- ago uncovered a vast ancient cemetery (54 000 m2) that
ably employed by this culture as a medicinal or seeminglycorrespondstothenearbyAidinghu,Alagou,and
*Towhomcorrespondenceshouldbeaddressed:E-mail:[email protected];[email protected]
ª2008TheAuthor(s).
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionNon-CommercialLicense(http://creativecommons.org/licenses/by-nc/2.0/uk/)which
permitsunrestrictednon-commercialuse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
4172 Russoetal.
Subeixi excavations (Ma and Wang, 1994; Chen and ormore),anextremelyaridclimate(16mmannualrainfall),
Hiebert, 1995; Davis-Kimball, 1998; Kamberi, 1998; An, and alkaline soil conditions (pH 8.6–9.1 (Pan, 1996), the
2008) (see Supplementary Fig. S1 at JXB online) attributed remarkable preservation of the human remains resulted in
to the G(cid:1)ushı(cid:1) culture (later rendered Ju¨shi, or Cheshi) the mummification of many bodies without a need for
(Academia Turfanica, 2006). The first written reports chemical methods. Numerous artefacts from the tombs
concerning this clan, drafted about 2000 years BP (before included equestrian equipment and numerous Western
present) in the Chinese historical record, Hou Hanshu, Asian crops such as Capparis spinosa L. (capers) (Jiang
describednomadiclight-hairedblue-eyedCaucasiansspeak- et al., 2007), Triticum spp. (wheat), Hordeum spp. (naked
ing an Indo-European language (probably a form of barley), and Vitis vinifera L. (grapevines) (Jiang, 2008),
Tocharian, an extinct Indo-European tongue related to often centuries before their first descriptions in Eastern
Celtic, Italic, and Anatolic (Ma and Sun, 1994). The G(cid:1)ushı(cid:1) China(Puett,1998).
tended horses and grazing animals, farmed the land One tomb, M90 (GPS coordinates: 42(cid:1) 48.395# N, 89(cid:1)
and were accomplished archers (Mallory and Mair, 2000). 38.958#E;elevation,58m)(seeSupplementaryFig.S2A,B
The site is centrally located in the Eurasian landmass at JXB online), contained the skeletal remains of a male
(Fig. 1A, B), 2500 km from any ocean and located in the ofhighsocialstatusofanestimatedageof45years,whose
Ayding Lake basin, the second lowest spot on Earth after accoutrements included bridles, archery equipment, a kon-
the Dead Sea (Fig. 1A, B). Formal excavations completed gou harp, and other materials supporting his identity as
in 2003 revealed some 2500 tombs dating from 3200–2000 a shaman (see Supplementary Figs S3A, B, 4A–C at JXB
years BP (Xinjiang Institute of Cultural Relics and Archae- online). His burial as a disarticulated skeleton, as opposed
ology, 2004). Other evidence from chipped stone tools and to a mummified body as more frequently was found,
other items indicate a possible human presence in the area suggestedthatheprobablydiedinthehighlandsoftheTi(cid:1)an
for some 10 000–40 000 years (Kamberi, 1998; Academia Sh(cid:1)an (‘Heavenly Mountains,’ or Ta¨ngri Tagh in Uighur)
Turfanica,2006).Duetoacombinationofdeepgraves(2m (Fig. 1), and his bones were later interred at Yanghai, as
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
(adaptedfromXinjiangInstituteofCulturalRelicsandArchaeology,2004).
Ancientcannabis 4173
nearby tombs contained large timbers of Picea (spruce) laboratoryglovesinalaminar-flowhood,andtransferredwithaclean
spp. that grow at 3000 m elevation. Modern Uighur metalspatula(Fig.2A).Twolevelsoflightmicroscopywereusedin
this study. For the observations on the achenes (Fig. 2D), a low
pastoralists follow a similar annual migratory path to
power Brunel MX3 microscope (Chippenham, Wiltshire, UK) was
summer grazing lands some 60–80 km distant from the
used and a 33 objective utilized in conjunction with an Olympus
tombs. Near the head and foot of the shaman’s bier lay SP350 8 megapixel camera, stereo insert 30 mm lens tube, and
alargeleatherbasketandwoodenbowl(seeSupplementary PhotonicPL2000–doublearmcoldlightsource.Greatermagnifica-
Fig. S5A, B at JXB online) filled with 789 g of vegetative tion was required for more detailed observations of trichomes
(Fig.2B,C):ahighpowerstereolightmicroscopewithaTrinocular
matter, initially thought to be Coriandrum sativum L.
Head for camera attachment (STE UK, Sittingbourne, Kent, UK)
(coriander), but which, after meticulous botanical examina-
with an eye piece graticule for specimen size measurement fitted
tion, proved to be Cannabis sativa L. (Jiang et al., 2006). with 34, 310, and 340 objectives. The camera’s 33 optical zoom
An initial radiocarbon date of 2500 years BP has sub- capabilityprovidedadditionalmagnification.
sequently been corrected to a calibrated figure of 2700 The observations on the seed were made on unmounted speci-
mens.Forthese,smallpiecesofplanttissuewereplaceddirectlyonto
yearsBPbasedonadditionalanalysesofequestriangearand
the low-power microscope plate. When using the high power
correlation to tree ring data (dendrochronology) in China.
microscope,samples were dry mounted ona glass slide.Toachieve
Whileanearlierpublication(Jiangetal.,2006)emphasized viewswherelargeproportionsofthematerialweresimultaneouslyin
morphological features in identifying the cannabis, the focus,flatsamplesspecimens(asshown)gavethegreatestsuccess.
current study used additional botanical, phytochemical, and On the low power microscope the seed sample was illuminated
with incident light, using a Photonic PL2000 – double arm ‘cold
genetic investigations to demonstratethat this cannabis was
light source’ (Fig. 2D). Some samples, when placed on the high
psychoactive and probably cultivated for medicinal or
power microscope, were also illuminated using the cold light
divinatory purposes. Great care was taken to prevent source. Others were illuminated from below. When viewing
contamination ofthesamplethroughout the analyses. samples mounted beneath a cover slip, it is common to set up
a microscope using the Ko¨hler illumination method (Delly, 1988).
This ensured that light from the condenser lens was focused
correctly on the microscope slide. For uncovered specimens, the
Materials and methods condenser height and aperture were adjusted while viewing
the subjectuntiloptimumresolution wasachieved. Inall cases,the
Photomicrographymethods specimensweremeasuredusingagraticulewithintheeyepiece.
Upon courier delivery from China, a polythene bag containing 11 g To enable photographs to be taken through the low power
of ancient cannabis was sterilized with ethanol, handled with microscope, one eyepiece was replaced with a compatible 30 mm
Fig. 2. Photomicrographs of ancient cannabis. (A) Photograph of the whole cannabis sample being transferred in laminar flow hood. (B)
Photomicrographofleaffragmentatlowpowerdisplayingnon-glandularandambersessileglandular trichomes.Noteretentionofchlorophylland
green colour, scale bar¼100 lm. (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 bar¼25 lm. (D) Low power photomicrograph of
acannabisachene(‘seed’)includingthebasewithanon-concavescarofattachmentvisible,scalebar¼1mm.
4174 Russoetal.
lens tube to which single lens reflex or digital cameras would be (Taberletetal.,1991).Thereactionmixturesweresubjectedfirstlyto
attached. As in ordinary photography, the depth of field is an initial heat denaturation at 94 (cid:1)C for 3 min; then, they were
considered to be the distance from the nearest object plain to the subjectedto35cyclesofheatdenaturationat94(cid:1)Cfor30s,1minof
farthest object plain that is in focus. When objects are a long primerannealingat55(cid:1)CfortheITSregion,and50(cid:1)CforcpDNA,
distance from the camera lens the depth of field is large. However, and DNA extension at 72 (cid:1)C for 40 s. Finally, the samples were
depthdecreasesastheimagecomesclosertothelens.Whentaking maintainedat72(cid:1)Cfor5minforthefinalextension.PCRreactions
photomicrographs, depth of field is measured in microns (Delly, were performed in an MJ Research PTC-100 thermal cycler (MJ
1988).Tomaximizethechanceoffindingsubstantialareasoftissue Research, USA). The amplification products were separated by
simultaneously in focus within this narrow depth of field, multiple electrophoresis in a 1.5% agarose gel. The bands were excised and
samples were laid as flat as possible onto glass slides. In all cases, purifiedwiththeMinEluteGelExtractionKit(Qiagen).PCR-purified
photomicrographs were taken on a solid bench and the shutter products were quantified and directly forward- and reverse-
activatedremotelytoreducemanually-inducedcamera-shake. sequenced,usingtheGenomeLab(cid:2)DyeTerminatorCycleSequenc-
In no instance was any image modification technique used in ingwithaQuickStartKitonaCEQ8000Geneticanalyser(Beckman
thesephotographs. Coulter). Primer sequences were identified and removed manually,
and database searches were performed with the BLASTN algorithm
Phytochemistrymethods (Altschul et al., 1990). The sequences results proved that the
pulverized dried tissue was from Cannabis sativa L., despite our
Approximately 2 g of the dried plant material was extracted with
observation in the mixed sample of some small seeds of different
200 ml methanol:chloroform (9:1 v/v) by sonication at room
species, removed before the DNA extraction; no differences were
temperature (21 (cid:1)C), the standard extractive technique for this
observed between the sequencesobtained and those deposited atthe
laboratory (GW Pharmaceuticals), a method that recruits >95% of
NCBI gene-bank (for THCA-and CBDA-synthases, GeneBank
phytocannabinoid content. The solvent layer was then transferred
accessionnumbersE55108/GI18529739andE33091/GI18623981).
through a paper filter into a rotary evaporator flask. The flask was
Bycontrast,noamplificationwasobtainedfromDNAextractedfrom
evaporated to dryness at 40 (cid:1)C, under reduced pressure, prior to
seedsofbothcannabisandtheother,unidentifiedspecies.Theallelic
resuspension in 4 ml of methanol:dichloromethane (3:1 v/v). This
status at a single locus, B, known to be the major gene determining
sample was transferred to two autosampler vials to be analysed by
the CBD/THC ratio in cannabis (de Meijer et al., 2003), was
GC-FID-MSandHPLC-UV.Atallstages,thecleanglasswarewas
investigated in the ancient material. The primer pairs described (de
extractedwiththesamesolventstoensurethatnoneoftheobserved
Meijeretal.,2003)arenotsufficientlyassociatedwiththechemotype
peaks would be a result of contamination. GC-FID-MS analyses
(Pacifico et al., 2006), and the sequence-based primers described
wereperformedonaHP6890gaschromatograph,coupledtoa5975
therein (Pacifico et al., 2006) failed to yield any amplification,
inert mass spectrometer. The system was controlled with Agilent
probably due to the limited integrity of DNA from ancient cannabis
MSD chemstation D.03.00.611. The GC was fitted with a Zebron
tissues, which did not sustain the amplification of a 1100 Da DNA
fused silica capillary column (30 m30.32 mm inner diameter)
fragment. Therefore, three different primer pairs (Fw1503Rev328,
coatedwithZB-5atafilmthicknessof0.25lm(Phenomenex).The
Fw1663Rev318, and Fw1543Rev318) were used. These primers
oventemperaturewasprogrammedfrom70(cid:1)Cto305(cid:1)Catarateof
were designed on two conserved small regions of a zone varying
5(cid:1)Cmin(cid:1)1.Theinjectorportandthetransferlineweremaintained
betweentheknownsequencesofTHCandCBDalleles.Whentested
at275(cid:1)Cand300(cid:1)C,respectively.Heliumwasusedasthecarrier
onfreshcannabistissues,theseprimersweredemonstratedtobeable
gasatapressureof55kPa.Theinjectionsplitratiowas5:1.HPLC
to amplify both alleles (PCR and sequences data not shown). Using
profiles were obtained using an Agilent 1100 series HPLC system
different primer pair combinations, the risk of a no-match or
controlled by Chemstation version A09.03 software. Cannabinoid a mismatch because of possible mutations in the 3# end of primer
profilesweregeneratedusingaC (15034.6mm,5lm)analytical
18 regionwasovercome.TheprimersequencesarelistedinSupplemen-
column fitted with a C (1034.6 mm, 5 lm) guard column. The
18 taryFig.S8atJXBonline.Allreactionmixturesweresubjectedfirst
mobile phase consisted of acetonitrile, 0.25% w/v acetic acid and
to heat denaturation at 94 (cid:1)C for 3 min and then to 35 cycles
methanolataflowrateof1.0mlmin(cid:1)1andthecolumnwaskeptat
consistingofheatdenaturationat94(cid:1)Cfor15s,primerannealingat
35(cid:1)C.TheUVprofileswererecordedat220nm.
54 (cid:1)Cfor30s,andDNAextension at 72 (cid:1)Cfor1 min.Finally,the
samplesweremaintainedat72(cid:1)Cfor5minforthefinalextensionof
Geneticmethods DNA. PCR products were separated by electrophoresis in a 1.5%
DNA was extracted from pulverized dried leaves, from two seeds agarosegel.ThebandswereexcisedandpurifiedwithMinEluteGel
probablybelongingtoCannabisspp.,andfromthreeseedsprobably Extraction Kit (Qiagen). PCR-purified products were quantified and
fromotherunidentifiedspecies.TheDNeasyPlantMiniKit(Qiagen) directly sequenced in forward and reverse, using the GenomeLab(cid:2)
was used, according to the Qiagen protocol, but with some Dye Terminator Cycle Sequencing with Quick Start Kit on
modificationtoincreasethefinalDNAamountandtoavoidexternal aCEQ8000Geneticanalyser(BeckmanCoulter).
and artificial contamination. For this reason, pre-PCR and post-PCR
operations were physically separated and carried out in different
environments. Ancient DNA extraction and other pre-PCR works Results
wereperformedunderaUV-filteredventilationsystemandapositive
pressure airflow. Filtered pipette tips and sterile tubes and plastics Microscopic botanical analysis
were always used; gloves, masks, and laboratory coats were always
Grossexaminationofthe11gsampleofcannabisprovided
worn. The quality of DNA obtained was estimated by A /A
260 280
absorbance ratio. In order to obtain the highest possible fidelity by the Chinese Academy of Sciences revealed loose dry
duringPCRsynthesis,PCRreactionswereperformedusingthePwo vegetative material (Fig. 2A). The impression that the
Master ready-to-use proofreading master mix (Roche Applied vegetative material had been lightly pounded was sup-
Science) according to their protocol. The primers designed to test
ported by examination of the wooden bowl, whose internal
DNA integrity and suitability for PCR analysis and species
surface was worn smooth, apparently from use as a mortar
identification were from the ITS region of nuclear ribosomal DNA
(Blattner, 1999), and from a non-coding region of chloroplast DNA (see Supplementary Fig. S5B at JXB online). The cannabis
Ancientcannabis 4175
retained a surprisingly green colour in its leafy parts and material produced 67.9 mg of solids after the removal of
displayed visible glandular trichomes (Fig. 2B), the solvents. Using high performance liquid chromatography
phytochemical factory of the plant and site of manufacture (HPLC), the largest cannabinoid peak was cannabinol
of cannabinoids and terpenoids (Potter, 2004; McPartland (CBN) at 7.4 min, but concentration levels were very low,
and Russo, 2001; Kim and Mahlberg, 2003). However, the averaging 0.007% w/w. CBN is an oxidative breakdown
ancient sample lacked the typical cannabis odour. Micro- product THC, generated non-enzymatically, with increas-
scopic examination confirmed the presence of intact sessile ing age (Brenneisen, 2007). There were also peaks
trichomes with an amber tint (Fig. 2B), while higher corresponding to expected retention times for cannabidiol
resolution documented the retention of visible secretory (CBD) at 4.9 min and cannabichromene (CBC) at 12 min
cells within the trichomes (Fig. 2C). Achenes (‘seeds’) (Fig. 3). Both are phytocannabinoids resulting from
averaged 2.2–3.6 mm in length (Jiang et al., 2006), were alternative enzymatic pathways than that yielding THC
light in colour with some striations, but demonstrated (de Meijer et al., 2003). There were very few peaks in the
rough, non-concave fruit attachment (Fig. 2D), all traits of first 20 min of the gas chromatogram where mono- and
domestication (Schlumbaum et al., 2008) associated with sesquiterpenes elute (Fig. 4). This lack of terpenoid
cultivated cannabis strains (Vavilov, 1926). In contrast, volatiles supports the physical observation that the plant
achenes of wild strains are typically smaller and darker material lacked the herbal smell traditionally associated
with concave attachment zones that favour shattering and with cannabis (McPartland and Russo, 2001). Shown in
easy spread (Vavilov, 1926). Germination was attempted Fig. 5A–C, (and in Supplementary Fig. S7A, B at JXB
with 100 achenes in compost, but no emergence was online) are breakdowns of sub-regions of the gas
observed after 21d. chromatogram. The major peaks in the 13–30.5 min
region are free fatty acids (see Supplementary Fig.S7A at
Phytochemicalanalysis JXB online). The largest peak identified as palmitic acid
Phytochemical and genetic teams were initially blinded to was the most abundant in the sample. Methyl and propyl
one another’s results. The extraction of 2 g of plant cannabinoids eluted in the 27–30 min region and the
Fig.3. Completehighperformanceliquidchromatography(HPLC)ofancientcannabis.
Fig.4. Completegaschromatography-flameionizationdetection(GC-FID)ofancientcannabis.
4176 Russoetal.
Fig.5. Gaschromatographyofancientcannabissubsections.(A)GCofthe30–34minregiondemonstratesseveralphytocannabinoids:cannabidiol
(CBD),cannabichromene(CBC),cannabicyclol(CBL),andcannabinavarin(CBNV).(B)GCofthe34–36.3minregiondisplaysthehighestpeak,
cannabinol(CBN),thedirectnon-enzymaticoxidativemetaboliteofTHC,withpossiblecannabielsoin(CBE)at34.2min.(C)GCofthe36.3–40.5
minregiondisplayscannabitriol(CBO)aTHCdegradant,andCBNvariants(seetext).
peaks marked as 286 Da and 302 Da all had MS spectra samples were supplied). A number of phytocannabinoids
consistent with propyl cannabinoids. There were two were identified in the 30–34 min region (Fig. 5A)
phthalate peaks at approximately 23.5 min (believed to including cannabidiol (CBD), cannabichromene (CBC),
have originated from the polythene bags in which the cannabicyclol (CBL, a heat-generated artefact of CBC
Ancientcannabis 4177
(Brenneisen, 2007), and cannabinavarin (CBNV, a propyl in yellow in Fig. 7A), which can be considered trans-
analogue of CBN). In the 34–36.3 min region (Fig. 5B), versions: from guanine to cytosine, and from cytosine to
apart from cannabinol (CBN), the largest individual adenine. The first of these two nucleotide substitutions is
phytocannabinoid component, there were at least four synonymous, i.e. it does not change the amino acid
peaks of 330 Da with cannabielsoin (CBE, an artefact sequence, while the second one is a non-synonymous
derived from CBD (Brenneisen, 2007) a likely identifica- substitution, leading to a serine-threonine exchange
tion of the peak at 34.2 min. In the 36.3–40.5 min region (highlighted in light blue in Fig. 7B) in the encoded
(Fig. 5C), the known THC degradant cannabitriol (CBO) amino acid sequence; these two amino acids, however,
(Brenneisen, 2007) was seen, as well as a series of peaks have similar physico-chemical properties. No CBDA
with spectral similarities to CBN, three of which are synthase, the biosynthetic enzyme for CBD (de Meijer
tentatively identified by the NIST database as either et al., 2003), was identified in the sample.
hydroxyl- or oxo-CBN. The last region (42–50 min; see
Supplementary Fig. S7B at JXB online), contained
phytosterols and triterpene alcohols with beta-sitosterol
Discussion
the most abundant compound.
Mass spectra (MS) of selected phytocannabinoids The results presented collectively point to the most
corresponding to the above are displayed (Fig. 6). Values probable conclusion which is that the G(cid:1)ushı(cid:1) culture
are all in agreement with those in NIST and GW cultivated cannabis for pharmaceutical, psychoactive or
Pharmaceutical databases. divinatory purposes. In examining the botanical evidence
There was a very small peak detected at the correct from this ‘old and cold’ site with its unique degree of
retention time in the sample for THC, but the spectra preservation, the cannabis consisted of a processed
could not confirm its identity. (pounded) sample whose seed size, colour, and morphol-
ogy, at least according to principles of Vavilov (Vavilov,
Geneticanalysis 1926), suggest that it was cultivated rather than merely
Because of the unique degree of preservation of the gathered from wild plants. The considerable amount of
cannabis, a genetic analysis was undertaken. The two cannabis present (789 g) without any large stalks or
ancient DNA sequences determined were labelled China branches would logically imply a pooled collection rather
F and China F(h). Alignment of these paleo-sequences than one from a single plant. Importantly, no obvious
(excluding the primers’ region, in red in Fig. 7A) with the male cannabis plant parts (e.g. staminate flowers, not
presently available databases demonstrated: infrequently observed in Indian herbal cannabis, or bhang
(Russo, 2007) were evident, implying their exclusion or
possible removal by human intervention, as these are
(i) China F (Fig. 7A) is identical 134/134 nucleotide pharmacologically less psychoactive.
agreement to other deposited sequences: AB212841, The HPLC, GC, and MS analyses confirm the identity
AB212839, AB212836, AB212833, AB212830, all be- of the supplied plant sample as Cannabis sativa L. The
longing to tetrahydrocannabinolic acid synthases (THCA- predominance of CBN indicates that the original plants
synthases), a species-specific genetic region (Schlumbaum contained D9-tetrahydrocannabinol (THC) as the major
et al., 2008) from Cannabis sativa L. phytocannabinoid constituent. The presence of CBO and
numerous CBN-related substance peaks further supports
(ii) ChinaF(h)(Fig.7A)isanewvariant,notpreviously
this view. CBD and CBC, together with their known
present in the genetic databases (submitted to NCBI,
thermo-oxidative degradation products CBE and CBL
GenBank accession number EU839988), showing a maxi-
(Brenneisen, 2007), are present, but the GC analysis
mum identity of 132/134 nucleotides with the above-
would appear to indicate that, in both cases, CBC and
mentioned sequences and with China F.
CBL are represented in greater quantities, as expected in
Utilizing BLASTX, i.e. performing searches through a high-THC cannabis strain wherein CBD is only a minor
amino acid translation, it was again shown that the China component. In addition, there is a peak for CBNV which
F(h)aminoacidsequenceisnotregisteredinthedatabase, confirms that the plant also contained D9-tetrahydrocanna-
and this is an obvious but necessary confirmation of the bivarin (THCV), a propyl phytocannabinoid. All of these
originality of this variant of the THCA synthase allele. observations are consistent with strains of cannabis with
These results also prove that both sequences encode for a high THC content and in an alternative taxonomy
THCA synthase, the biosynthetic enzyme for THCA that suggests it should be assigned to Cannabis indica
decarboxylates via heat or ageing to yield psychoactive Lamarck (Hillig and Mahlberg, 2004).
THC (Russo, 2007). Direct comparison of the two ancient While chromatography elution times may vary with
sequences, identified the nature of the small differences temperature, column type, and other factors, confirmation
observed: the samples have two ‘mutations’ (highlighted was evident with corroboratory mass spectra values that
4178 Russoetal.
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,and1#-hydroxycannabinol.
were identical to those seen daily in assays performed on ofplantswithinwhichTHCwasthedominantcannabinoid.
fresh cannabis extracts in this laboratory. By contrast, a sample taken from a mix of wild-type
The presence of so many recognized cannabinoid Cannabis sativa would customarily harbour a more equal
degradants is consistent with very old cannabis samples. mixtureofTHCandCBD(deMeijeretal.,2003).Itwould
The very low concentration levels measured in the HPLC appear, therefore, that humans selected the material from
analysis may indicate that the sample provided contained plants on the basis of their higher than average THC
significantly more leaf and twig material than flower content. To elaborate, a chemotaxonomy of cannabis
material,ratherthanbeingevidenceinitselfthatthesample previously outlined indicates three types (Small and
was of low potency originally. This plant material is Beckstead, 1973): chemotype I (drug) strains with high-
therefore conclusively cannabis derived from a population THC:CBD ratios, chemotype II low-THC, higher-CBD
Ancientcannabis 4179
Fig.7. DNAanalysisofancientcannabis.(A)Nucleotidesequencesofthewild-typetetrahydrocannabinolicacidsynthase,ChinaF,andthemutant
sequence,ChinaF(h),withtwosinglenucleotidepolymorphismshighlightedinlowercaseyellow.(B)AminoacidtranslationofChinaFandChina
F(h),demonstratingdivergenceinachangefromserine(wild-type)tothreonine(mutant),highlightedinblue.
(fibre) strains, and chemotype II with more equal ratios. from 19th century cannabis preparations (Harvey, 1990)
THC and CBD production are mediated by co-dominant including a 140-year-old sample of Squire’s Extract
alleles B and B , respectively (de Meijer etal., 2003). By (Harvey, 1985). A study in 1992 reported the presence of
T D
comparison, pooled samples from cannabis fields in D8-THC(previouslytermedD6-THC)fromburnedcannabis
Morocco and Afghanistan will normally produce 25% that was reportedly inhaled as an aide to childbirth in
high-THC plants, 25% high-CBD plants, and 50% with a Judean cave 1700 years BP (Zias et al., 1993), supported
lower, mixed titres, combining to yield roughly equivalent by the finding of cannabinoid residues in an adjacent glass
amounts of the two phytocannabinoids (Russo, 2007), vessel (Zias, 1995). In the Mustang region of Nepal,
a patternnot observedinour specimen. mummifiedhumanremainsofprobableMongolianancestry
Isotopic analysis of cellulose from this cannabis sample have been dated 2200–2500 years BP in association with
might conceivably be used in comparison with other cannabis, probably transported from elsewhere (Kno¨rzer,
samples in an attempt to establish its geographic origin. 2000; Alt et al., 2003), but with insufficient detail to
While multi-purpose cannabis plants used simulta- ascertain its use. Rudenko recovered cannabis seeds,
neously for food (seed), fibre (stalks), and pharmaceutical censers, and hempen clothing in Pazyryk, Siberia from
uses (flowering tops) have been recently reported from Scythiankurgans(burialmounds)from2400–2500yearsBP
Darchula in far western Nepal (Clarke, 2007), more (Rudenko, 1970; Brooks, 1998), closely matching Herodo-
customarily, a given plant is best suited toward a single tus’ descriptions of funeral rites for that culture (Herodotus,
purpose. Of additional key importance is the absence of 1998). Sarianidi also claimed cannabis use in the Bactria–
hemp artefacts from the Yanghai Tombs. The G(cid:1)ushı(cid:1) Margiana Archaeological Complex (BMAC) (present day
fabricated clothing from wool (see Supplementary Fig. Turkmenistan) (Sarianidi, 1994, 1998), but this interpreta-
S6BatJXBonline)andropesfromPhragmites(reed)spp. tionhas been debated(see discussioninRusso,2007).
fibres (see Supplementary Fig. S6C at JXB online). Another independent genetic analysis of this material
Whereas hemp textiles have been collected from the published subsequent to our analysis (Mukherjee et al.,
Northern China Yangshao Culture from 6000–7000 years 2008) confirmed the presence of THCA synthase, but not
BP, their appearance in the west was not documented the single nucleotide polymorphisms. The authors posited
before 2000 years BP, for example, 1500 years BP in a European–Siberian origin for the material.
Kucha, 600 km west of Turpan (Mallory and Mair, 2000). Current genetic data also confirm that the plant material
Previous phytochemical analyses of antique cannabis examined is Cannabis sativa L. according to ITS and
preparations have demonstrated THC remnant fingerprints cpDNA analysis. The results also support the hypothesis
4180 Russoetal.
of the existence of at least two THCA-synthase nucleotide Yenisei Valley to the north (Anthony, 1998, 2007;
sequences in the ancient plant material examined. One of Mallory, 1998; Renfrew, 1998; Mallory and Mair, 2000),
these sequences perfectly matches the corresponding whose putative southward migration some authorities
sequence of already-known THCA-synthases deposited in have attributed to ‘global cooling’ c. 4000 years BP (Hsu¨,
GenBank, both as gene and protein sequences; the second 1998), and to their proto-Indo-European-speaking Yam-
sequence is a novel one, with two single nucleotide naya forebears further west, dating to 6000 years BP
polymorphisms (SNPs) encoding for a protein with (Mallory, 1989; Anthony, 1998; Winter, 1998). Abundant
presumably very similar characteristics. Whether these mysteries remain as to the origins and customs of the
two sequences coexisted in a single cannabis plant or G(cid:1)ushı(cid:1). Additional answers may accrue from future
a strain heterozygous at the B locus, or belong to different archaeological excavations or human genetic analyses that
plants, could not be concluded. elucidate relationships with other ancient cultures and
THC represents one of the possible phytocannabinoid modern peoples of the region. The unique SNPs discov-
end-productsmanufacturedbycannabisplants;THC(or,in ered in this ancient sample may yet be of critical
its native form, THCA) is synthesized by a well-character- importance in tracing the phylogeny and geographic
ized enzyme (THCA- or THC-synthase) from a precursor spread of cannabis and the humans who used it.
(CBG or CBGA) common to most chemotypes that Theexcellentpreservationofthecannabisfromthistomb
represents the metabolic ‘switching point’, downstream of allowed an unprecedented level of modern botanical
which the variability of the different chemotypes is investigation through biochemistry and genetics to con-
concentrated. The agents of such variability found in clude that the plant was cultivated for psychoactive
cannabisgermplasmareexclusivelythedifferentsynthases, purposes. While cultivation of hemp for fibre has been
among which THC(A)-synthase isthe only one responsible documented in Eastern China from a much earlier date
for making that specific cannabinoid, THC. Therefore, the (vide supra Mallory and Mair, 2000), the current findings
presence of the allelic variant responsible for coding the representthe most compellingphysicalevidencetodatefor
THC(A)-synthase may well be considered to be diagnostic, the use of cannabisfor its medicinalor mystical attributes.
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
Supplementary data
produced, cannot of course be specified because they
depend on a number of anatomical, environmental, and Photographs and diagrams of the Yanghai Tombs site,
nutritionalfactors thatremain unknown. Tomb M90 contents including fabric and ropes, and
Numerous questions remain. Current data do not permit additional chromatographic and genetic analysis primer
it to be ascertained how the cannabis from the tomb was sequenceinformationarepresentedinSupplementaryFigs
administered. If used orally, perhaps it was combined in S1–S8, available online.
some fashion with Capparis spinosa L., as these plants Fig. S1. Study site at the Yanghai tombs with Huoyan
were found together in a nearby but later tomb at Yanghai Shan mountain range in background (photo EBR).
(Jiang et al., 2007). That date for that tomb was initially Fig. S2. Diagrams of the Yanghai Tombs (adapted from
reported as 2700 years BP via radiocarbon methods, and Xinjiang Institute of Cultural Relics and Archaeology,
since corrected to 2200–2400 years BP with additional 2004, with permission).
calibration employing tree ring data. If this cannabis were Fig. S3. The shaman’s tomb, M90 [previously pub-
smoked or inhaled, no mechanism for so doing has been lished in Mandarin (Xinjiang Instgitute of Cultural Relics
excavated in the area. The G(cid:1)ushı(cid:1) could have sifted the and Archaeology, 2004), used with permission].
cannabis through fabric after pounding, then fumigated it, Fig. S4. The shaman’s skull (photos EBR).
much as described for the alleged cannabis candidate, the Fig. S5. Containers in which cannabis was stored in
Sumerian A.ZAL.LA, administered medicinally for ‘hand tomb [previously published in Mandarin (Xinjiang In-
of ghost’(Thompson, 1923, 1949), since posited as stitute of Cultural Relics and Archaeology, 2004] used
nocturnal epilepsy (Russo, 2007; Wilson and Reynolds, with permission.
1990). While this culture could have arrived from the Fig. S6. Re-excavation of Tomb M90. This was
earlier BMAC region as ‘oasis hoppers’ (Barber, 1999), undertaken to re-examine artefacts, measure GPS co-
and certain cultural relationships are apparent to the ordinates, and assess environment conditions (photos
Scythian culture with respect to cannabis use and EBR).
equestrian prowess, those peoples were Iranian speakers Fig. S7. Chromatography subsections from phytochem-
(Mallory and Mair, 2000). In addition, G(cid:1)ushı(cid:1) cultural ical analysis.
affinities and burial practices much more closely resemble Fig. S8. Primer sequences employed in the genetic
those of the presumed proto-Tocharian speaking, incense- analysis to amplify THC- and CBD-allele specific frag-
burning (Kuzmina, 1998) Afanasievo peoples in the ments and their sequences (5#/3#).