Journal of Psychoactive Drugs

ISSN: 0279-1072 (Print) 2159-9777 (Online) Journal homepage: http://www.tandfonline.com/loi/ujpd20

Betel Nut Chewing in Iron Age Vietnam? Detection

of Areca catechu Alkaloids in Dental Enamel

Simone Krais M.A., Miriam Klima M.Sc., Laura M. Huppertz B.Sc., Volker
Auwärter Ph.D., Markus J. Altenburger M.D. & Merja A. Neukamm Ph.D.

To cite this article: Simone Krais M.A., Miriam Klima M.Sc., Laura M. Huppertz B.Sc., Volker
Auwärter Ph.D., Markus J. Altenburger M.D. & Merja A. Neukamm Ph.D. (2016): Betel Nut
Chewing in Iron Age Vietnam? Detection of Areca catechu Alkaloids in Dental Enamel, Journal
of Psychoactive Drugs, DOI: 10.1080/02791072.2016.1264647

To link to this article: http://dx.doi.org/10.1080/02791072.2016.1264647

Published online: 20 Dec 2016.

Submit your article to this journal

Article views: 2

View related articles

View Crossmark data

Full Terms & Conditions of access and use can be found at

http://www.tandfonline.com/action/journalInformation?journalCode=ujpd20

Download by: [Gazi Universitesi] Date: 22 December 2016, At: 20:51

JOURNAL OF PSYCHOACTIVE DRUGS
http://dx.doi.org/10.1080/02791072.2016.1264647

Betel Nut Chewing in Iron Age Vietnam? Detection of Areca catechu Alkaloids in
Dental Enamel
Simone Krais, M.A.a, Miriam Klima, M.Sc.b, Laura M. Huppertz, B.Sc.c, Volker Auwärter, Ph.D.d,
Markus J. Altenburger, M.D.e, and Merja A. Neukamm, Ph.D.f
a
Doctoral Candidate, Physical Anthropology, Faculty of Medicine, University of Freiburg, Freiburg, Germany; bDoctoral Candidate, Institute
for Forensic Medicine, Medical Center–University of Freiburg, Faculty of Medicine, Hermann Staudinger Graduate School, University of
Freiburg, Freiburg, Germany; cResearch Associate, Institute for Forensic Medicine, Medical Center–University of Freiburg, Faculty of Medicine,
University of Freiburg, Freiburg, Germany; dProfessor, Institute for Forensic Medicine, Medical Center–University of Freiburg, Faculty of
Medicine, University of Freiburg, Freiburg, Germany; eProfessor, Department of Operative Dentistry and Periodontology, University Medical
Center, Freiburg, Germany; fAssistant Professor, Institute for Forensic Medicine, Medical Center–University of Freiburg, Faculty of Medicine,
University of Freiburg, Freiburg, Germany

ABSTRACT ARTICLE HISTORY

The betel quid is one of the most commonly consumed psychoactive substances in the world. By Received 7 July 2016
archaeological evidence like the occurrence of areca nuts in archaeological sites, the typical overall Revised 11 October 2016
reddish-brown staining on prehistoric human teeth or specific artifacts linked with the habit, it is Accepted 8 November 2016
assumed that this tradition reaches back to prehistoric times. Since this kind of evidence is indirect, KEYWORDS
it is frequently doubted. The present study provides the earliest direct analytical indication of betel Analytical toxicology;
nut chewing in human history. A typical stained tooth from an Iron Age skeleton (site Gò Ô Chùa in arecoline; dental analysis;
Southern Vietnam, 400–100 BC) was analyzed by liquid chromatography-tandem mass spectro- Gò Ô Chùa; LC-MS/MS;
metry (LC-MS/MS) and liquid chromatography-high-resolution mass spectrometry (LC-HR-ToF-MS) prehistoric drug
and the alkaloid arecoline which is specific for Areca catechu L. (Arecaceae) was detected. consumption

Currently, approximately 600 million people practice the
habit of betel nut chewing (Gupta and Warnakulasuriya
2002). Little known in the Western world, along with
alcohol, tobacco, and coffee, it is globally one of the most
commonly used drugs. Its geographical distribution is
centered in South and Southeast Asia and extends from
Madagascar to the Solomon Islands. The areca nut is the
seed of the areca palm (Areca catechu L., Arecaceae) and
is commonly referred to as betel nut. Usually, betel is
consumed as so-called betel quid; a small package con-
taining pieces of betel nut and slaked lime (calcium
hydroxide) wrapped in a betel leaf (Piper betle L.,
Piperaceae). Sometimes, spices are added. Typical visible
indication of regular betel quid chewing is the overall
reddish staining of the teeth surface. Due to its mild
stimulating effects, it is most often used as a recreational
drug, but it is also part of rituals and traditional medi-
cine (Zumbroich 2008). In many traditional societies,
drug use plays an important role in religious ceremonies
and for healing purposes.
The origin of betel nut chewing in Southeast Asia
can be traced back for more than 2000 years by
historical, linguistic, and ethnographic sources. In
archaeology, artifacts of material culture, like specific
jars, remains of the areca plant, and reddish-brown
stained teeth, are associated with the habit, and claims
to the origin of the habit reach back even some millen-
nia ago (see Zumbroich 2008). Unfortunately, these
lines of evidence are indirect and depend on current
archaeological interpretations. The issue can also be
approached by biochemical analysis, which is more
direct and independent.
On a biochemical level, upon chewing a betel quid,
the active tetrahydropyridine alkaloids of the betel nut
(arecoline, arecaidine, guvacoline, and guvacine) are
released and can be detected in high concentrations in
saliva (Franke et al. 2015). During mastication with
slaked lime, the ester bonds of arecoline and guvacoline
are cleaved, yielding arecaidine and guvacine, respec-
tively (see Figure 1). Additionally, areca red, a tannin
formed during mastication, produces reddish-brown
saliva covering all surfaces of the teeth, resulting in
red staining in the oral cavity. Habitual use can lead
to permanent staining of all enamel surfaces. These

CONTACT Merja A. Neukamm merja.neukamm@uniklinik-freiburg.de Institute for Forensic Medicine, Medical Center–University of Freiburg, Faculty
of Medicine, Albertstrasse 9, 79104 Freiburg, Germany.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/ujpd.
© 2016 Taylor & Francis Group, LLC
2 S. KRAIS ET AL.
Figure 1. Chemical structures of the characteristic areca catechu alkaloids arecoline, arecaidine, guvacoline, and guvacine and their
reactions with lime.
discolorations range from red to black, depending on
the frequency and duration of chewing and dental
hygiene (Reichart et al. 1985; Rooney 1993).
This unintentional staining has to be distinguished
from the staining resulting from tooth blackening,
which is the custom of intentional dental staining. It is
a common type of body decoration in parts of Asia,
Micronesia, and Melanesia. For social and aesthetic rea-
sons, mainly the labial surface of the front teeth is
stained. Ethnobotanic studies show that, among many
other plants, and organic or inorganic preparations,
areca nut can serve as a dying agent for deliberate
tooth blackening. However, due to the “Western ideal
of white teeth,” the importance of this custom is declin-
ing (Zumbroich 2009). While intentional tooth black-
ening results in reddish-brown staining of mainly the
labial surface of the front teeth, betel nut chewing results
in unintentional reddish-brown irregular staining of all
enamel surfaces. These staining patterns have to be dis-
tinguished thoughtfully in the archaeological record,
since they imply different customs (Zumbroich 2008).
The biochemical analysis of prehistoric tooth black-
ening is reported from the Bronze Age site Nui Nap in
northern Vietnam (Oxenham et al. 2002) and the Latte
Phase (1100–1700 AD) of the Alaguan site on Rota,
Mariana Islands (Hocart and Fankhauser 1996).
Oxenham et al. (2002) detected a tannin in the labial
staining of a skeleton dating to between 2400 to
2000 years before the present and in a betel nut sample
(Areca catechu L.) by analysis with gas chromatography-
mass spectrometry (GC-MS), suggesting that betel nut
was used for prehistoric tooth blackening. However, the
detected tannin may not be specific for betel nut, since it
may also occur in other plants. By GC-MS analysis,
Hocart and Fankhauser detected arecaidine and guvacine
in stained enamel (Hocart and Fankhauser 1996). These
alkaloids are specific for Areca catechu L. To exclude
possible overlapping of tooth blackening and betel nut
chewing (Zumbroich 2008), Hocart and Fankhauser addi-
tionally tested an unstained buccal surface of a tooth from
the same skeleton, but the alkaloids were only found on
the stained labial surface. However, Hocart and
Fankhauser demonstrated that betel nut was used as a
staining agent for prehistoric tooth blackening. Since
arecaidine and guvacine are cleavage products of areco-
line or guvacoline, respectively, it can also be concluded
that the staining paste used for the intentional blackening
might have been produced including mastication with
slaked lime, possibly in a similar way as a betel quid.
The objective of this study was to analyze dental
tissue from an Iron Age skeleton with irregular red-
dish-brown dental staining of all enamel surfaces com-
monly associated with betel nut chewing. In comparison
to more indirect approaches, the chemical proof of betel-
nut-specific substances in this kind of dental staining
would be more straightforward. Moreover, it would sup-
port the common association of the stains with the habit
of betel nut chewing. In addition to the analysis of the
Iron Age tooth, chewing of a betel quid was simulated in
an in-vitro experiment to shed more light on the ques-
tion of which betel alkaloids are actually incorporated in
different types of dental tissue.
Materials
The site of Gò Ô Chùa is situated in the Mekong Delta
in Long An province in Southern Vietnam, close to the
 JOURNAL OF PSYCHOACTIVE DRUGS 3

present Vietnamese-Cambodian border. Five excava-

tion campaigns from 1997 to 2008 unburied the
remains of 68 inhumations and seven jar burials in
three contiguous mounds, providing one of the largest
prehistoric collections on the Vietnamese side of the
Mekong Delta. The seven jar burials contained remains
of infants and, according to their stratigraphic position,
the lack of iron offerings, and radiocarbon dating, it is
very likely that they belong in the end period of the
Bronze Age or the beginning of the Iron Age (sixth to
fourth centuries BC). The majority of the 68 inhuma-
tions seemed undisturbed and were in a good condi-
tion. The graves were equipped with pottery and often
tools or weapons of iron or antler. Some graves con-
tained ornaments of precious stones or glass, exotic raw
materials like turtle shell, predator teeth, or ivory and
food deposition in the form of small pots with remains
Figure 2. Overall reddish-brown staining of the teeth of skele-
of animal bones. Bronze objects like bracelets were ton M6 from Gò Ô Chùa.
present to a minor degree. Calibrated radiocarbon
dates and the typical Iron Age ceramics suggest that
these inhumations belong to the Early Iron Age
caries, premature suture closure, and periosteal reac-
between 400 and 100 BC. By burial custom and cera-
tions as indicators of unspecific physiological stress.
mics, the cemetery of Gò Ô Chùa belongs to the Pre-
Since no archaeobotanical remains of areca nut or
Funan culture, which was distributed from Southern
artifacts that might be related with the habit of betel
Vietnam at least to Northwestern Cambodia. Next to
nut chewing were found in the site, the extraordinary
the cemetery, archaeological finds prove an earlier per-
dental staining of this skeleton is the only indication
iod of occupation of Gò Ô Chùa as a salt-boiling center.
that the habit of betel nut chewing was practiced in
Radiocarbon dating of the related ceramic pedestals
Iron Age Gò Ô Chùa.
suggests a continuous occupation between 1000–500
BC (Reinecke 2004, 2009).
Until now, 61 of the 68 Iron Age inhumations have
been anthropologically examined. Seven of them are Methods
unsexed infants or juveniles (0–19 years), 23 were
In-vitro experiment
sexed as female and 24 as male. Seven adults remained
unsexed due to insufficient preservation for morpholo- Bovine enamel and dentin pellets obtained from freshly
gical sex determination (Francken, Wahl, and Reinecke extracted bovine incisors were used. The pellets were
2010). The majority of the adult skeletons display red- produced as previously described (see Spinner et al.
dish-brown staining of the labial surface of the front 2014). Round specimens, 4.5 mm in diameter, were
teeth, resembling typical tooth blackening. Since infant drilled out of each tooth’s root after removal of the
skeletons do not display this type of dental staining, it cementum to obtain dentin or the crown region to
has obviously been socially restricted to adult persons. obtain enamel using a custom-made trephine bur.
However, one of the skeletons (08GOC HI M6) dis- After removing tissue residues, the samples were
plays the dental staining commonly associated with polished to create uniform pellets. The pellets were
betel nut chewing. All enamel surfaces of the 21 treated with 0.1% citric acid for 30 seconds to erode
erupted teeth display the typical overall irregular red- the surface, washed and covered with betel nut paste.
dish-brown staining (Figure 2). By morphological char- For the betel nut paste, dried betel nut was ground and
acteristics and tooth cementum annulation, the age at mixed with calcium hydroxide powder in equal
death of this individual was estimated to be about amounts. Aqueous buffer was added to produce a
15 years. While the grave position and burial goods of spreadable paste. The samples were incubated at 37°C
this individual did not show any notable features, the in a humidity chamber. After about 24 hours, the pel-
skeleton itself exhibits pathological characteristics that lets were rinsed with water and again treated with 0.1%
are unusual in comparison to the rest of the skeletal citric acid for 30 seconds. In a period of two weeks, the
collection (Francken, Wahl, and Reinecke 2010): severe pellets were repeatedly treated with betel nut paste for
4 S. KRAIS ET AL.
five consecutive days, followed by untreated incubation
at 37°C in a humidity chamber for two days.
Preparation and analysis of specimens
For analysis of the Iron Age tooth, the stained dental
enamel and preserved dentin material of one molar
(35) of individual 08GOC HI M6 were used. All of
the stained enamel was removed from the whole sur-
face of the tooth with a fine dental drill to gain enamel
powder, and the inside of the tooth was hollowed out to
gain pulp-oriented dentin powder.
All specimens were powdered using a ball mill. The
extraction procedure of the prehistorical material, the
pellets, and the betel nut was according to Spinner et al.
(2014). Briefly, 50 mg of powder was ultrasonicated
with 500 µl of methanol three times for 60 minutes.
The combined extracts were evaporated to dryness,
resuspended in liquid chromatography mobile phase,
and analyzed by liquid chromatography-tandem mass
spectrometry (LC-MS/MS).
All samples were semi-quantitatively analyzed by a
liquid chromatography-tandem mass spectrometry (LC-
MS/MS) method (Spinner et al. 2014). The LC-MS/MS
system comprised a Dionex Ultimate 3000 HPLC
(Thermo Scientific, Dreieich, Germany) coupled to a
SCIEX API 5000 (SCIEX, Darmstadt, Germany). Separa-
tion was performed at 40°C on a Luna PFP column
(150 × 2 mm, 5 μm) with a guard column with the same
stationary phase (4 × 2 mm) (Phenomenex, Aschaffenburg,
Germany), and with mobile phases consisting of aqueous
formate buffer (mobile phase A, 0.1% formic acid and
1.0 mmol/l ammonium formate, pH 2.7) and methanol
with 0.1% formic acid (mobile phase B). The following
gradient was used: initially, 5% of solvent B at a flow rate
of 0.4 ml/min was kept for 1 min. During 14 min, solvent B
increased up to 98% at a flow rate of 0.6 ml/min, which was
kept for 2 min. Initial conditions were restored and held for
3 min to re-equilibrate the system. Injection volume was
20 µl. The mass spectrometer was operated in positive ESI-
mode. Ion-spray voltage was set to +1,500 V. The gas
settings were as follows: curtain gas 20 psi, collision gas 9
psi, ion source gas (1) 60 psi, ion gas (2) 70 psi, source
temperature 500°C. The following transitions in multiple
reaction monitoring mode (MRM) were recorded: areco-
line: 156 > 81, 156 > 53, 156 > 156 (pseudo-transition);
arecaidine: 142 > 81, 142 > 53; guvacoline: 142 > 113,
142 > 59; guvacine: 128 > 81, 128 > 53. Deuterated methy-
lenedioxymethamphetamine (d5-MDMA) was used as
internal standard (MRM: 199 > 165). For all MRMs, declus-
tering potential (DP) was 70 V, entrance potential was 10 V,
collision energy was 20 V, and cell exit potential was 13 V.
Extracts of dentin powder previously spiked with 0.1 ng/mg
arecoline, arecaidine, guvacoline, and guvacine, respec-
tively, were used for single-point calibration.
Additionally, the enamel and dentin samples of the
prehistoric tooth were analyzed by liquid chromatogra-
phy-high-resolution time-of-flight mass spectrometry (LC-
HR-ToF-MS). For LC-HR-ToF-MS, a Dionex UltiMate
3000 RSLC HPLC system (HPG–3400RS binary pump
with solvent selection valve, WPS–3000TRS autosampler,
TCC–3000RS column compartment, and SRD–3600 sol-
vent rack degasser; Thermo Fisher Scientific, Dreireich,
Germany) coupled to a maXis impact Q–ToF instrument
(Bruker Daltonik, Bremen, Germany) was used.
Data acquisition and evaluation was performed with
HyStar and Data Analysis software (Bruker Daltonik).
LC conditions were as follows: mobile phase A deio-
nized water/MeOH 90:10 (v/v) with 5 mM ammonium
formate and 0.01% HCOOH, mobile phase B MeOH
with 5 mM ammonium formate and 0.01% HCOOH.
For chromatographic separation, a Dionex Acclaim
RSLC 120 C18 column (100 × 2.1 mm, 2.2 μm) was
used. Gradient elution was applied starting at 1%
mobile phase B for 1 min with a flow rate of 0.2 ml/
min, increased to 39% mobile phase B in 2 min, and
increased to 99.9% mobile phase B at a flow rate of
0.4 ml/min in 9 min followed by a 2 min hold at a flow
rate of 0.48 ml/min. Starting conditions were restored
within 0.1 min with a flow rate reduction to 0.2 ml/min
after 3 min; these conditions were held for 0.9 min
prior to the injection of the next sample. The autosam-
pler and column oven temperatures were set to 5 and
30°C, respectively. The ToF was operated in positive
ESI mode acquiring full-scan and broadband collision-
induced dissociation (bbCID) specta. For bbCID colli-
sion energy stepping from 24 to 36 eV was applied.
Results
In the reddish-brown stained enamel of the prehistoric
tooth, arecoline, one of the characteristic alkaloids of
Areca catechu L., was identified by LC-HR-ToF-MS
(acquired m/z 156.1018, calculated m/z 156.1019, deviation
0.06 ppm) and LC-MS/MS. Arecolidine, another character-
istic alkaloid ingredient of Areca catechu L., could be tenta-
tively identified by LC-HR-ToF-MS (acquired m/z
156.1021, calculated m/z 156.1019, deviation −1.2 ppm;
no reference standard to compare retention time or possible
fragmentation was available). However, no alkaloids at all
were detected in the dentin of the prehistoric tooth. In the
bovine enamel and dentin samples treated with betel nut
paste in-vitro, arecoline and guvacoline were detected in the
enamel pellets. In the dentin pellets, in contrast, arecaidine
was the main finding along with smaller amounts of guva-
cine (see Table 1 and Figure 3).
 JOURNAL OF PSYCHOACTIVE DRUGS 5

Table 1. Findings of the betel alkaloids in different tooth structures of the characteristic dental staining with betel nut
with LC-MS/MS. chewing and the assumption that this prehistoric per-
Arecoline Arecaidine Guvacoline Guvacine son chewed betel nut, and strengthens the theory that
Prehistoric enamel + – – –
Prehistoric dentin – – – –
areca nut was already used as a psychoactive stimulant
Enamel in-vitro + – + – in prehistory.
Dentin in-vitro – ++ – +
However, this interpretation is disturbed by one impor-
tant fact. The in-vitro experiment showed that next to the
incorporation of arecoline and guvacoline into dental
Discussion
enamel, the incorporation of betel alkaloids arecaidine
The detection of arecoline in the dental enamel of and guvacine into dentin is possible. But in the prehistoric
individual 08GOC HI M6 from Gò Ô Chùa proves dentin, no alkaloids were found. This absence of arecaidine
that this individual used betel nut orally. This bio- and guvacine in the prehistoric dentin could be explained
chemical evidence supports the common association by different reasons.

Figure 3. MRM-chromatograms of arecoline, arecaidine, guvacoline, and guvacine in the different materials.
6 S. KRAIS ET AL.
Firstly, we cannot exclude that the alkaloids were
absent in dentin because they never came in touch
with it. This might be the case if the staining was
applied by a procedure similar to tooth blackening
where betel nut is not chewed and alkaloids do not
enter the blood stream in larger amounts. In the in-
vitro experiment, dentin was directly exposed to the
betel preparation and so to the alkaloids in very high
concentrations. But, in contrast, while chewing the
betel quid, the betel alkaloids are absorbed by the
mucosa of the oral cavity or swallowed and subse-
quently distributed in the blood stream. Successively,
via the blood vessels in the dental pulp, the alkaloids
could theoretically diffuse into dentin—but, secondly, it
is not known if they are incorporated in a relevant and
measurable concentration via the pulp. As alkaloid
concentrations in saliva (Franke et al. 2015) are
approximately three orders of magnitude higher than
in blood plasma (Wang et al. 2014), diffusion into
dentin via the dental pulp might be much less pro-
nounced than the incorporation into enamel from the
oral cavity. Thirdly, the absence of alkaloids in dentin
could also be the result of structural changes by
destructive taphonomic processes and the subsequent
loss of alkaloids. Due to its higher porosity, dentin is
more exposed to such processes compared to enamel.
Thus, even though the presence of betel nut sub-
stances in dentin would be evidence for betel nut chew-
ing, their absence cannot be valued as disproof.
Therefore, these results are very likely to be the earliest
biochemical evidence of betel nut chewing in human
history.
In Iron Age Gò Ô Chùa, out of 68 skeletons, only
individual 08GOC HI M6 shows the overall reddish-
brown staining characteristic for betel nut chewing. The
available archaeological and anthropological data does
not permit any conclusion on a social restriction of this
pattern, for example to a specific age groups or gender,
nor show the burial goods of individual 08GOC HI M6
any unusual features. Since the only outstanding fea-
tures of individual 08GOC HI M6 are the extraordinary
pathological lesions that have probably been painful
(premature suture closure can lead to strong headache
and severe caries to tooth pain), it could be hypothe-
sized that this person chewed betel nut to achieve a
feeling of relief or well-being in context of healing or
ritual purposes.
The findings underline the value of stained teeth as
biological artifacts capable of providing deep cultural
insights. Archaeological human teeth reflect past cul-
tural practices in an extraordinary way on preserved
biological tissue. Additionally, the methodological
approach of detection of specific betel nut alkaloids in
dental enamel and dentin contributes to the assessment
of betel nut use by biomarkers in modern contexts.
Acknowledgments
The authors wish to thank the following persons and institu-
tions for their support: Andreas Reinecke for archaeological
background information; Bùi Phát Diệm, director of the
Long An province museum, vice-director Vương Thu
Hồng, and staff member Đỗ Thị Lan for access to the mate-
rial and the friendly cooperation.
Funding
The authors wish to thank the Deutsche Forschungsgemeinschaft
for funding the project “Determination of Drugs in Dental
Material” (NE 1879/2-1, AL 1665/3-1).
References
Francken, M., J. Wahl, and A. Reinecke. 2010. Reflections of
a hard life: Burials from Go O Chua (Vietnam). In
Proceedings of the Fourth Meeting of Junior Scientists in
Anthropology, ed. C. Buhl, F. Engel, L. Hartung, M.
Kästner, A. Rüdell, and C. Weisshaar, 16–23. Freiburg,
Germany: University of Freiburg.
Franke, A. A., A. J. Mendez, J. F. Lai, C. Arat-Cabading, X. Li,
and L. J. Custer. 2015. Composition of betel specific che-
micals in saliva during betel chewing for the identification
of biomarkers. Food and Chemical Toxicology 80:241–46.
doi:10.1016/j.fct.2015.03.012.
Gupta, P. C., and S. Warnakulasuriya. 2002. Global epide-
miology of areca nut usage. Addiction Biology 7:77–83.
doi:10.1080/13556210020091437.
Hocart, C. H., and B. Fankhauser. 1996. Betel nut residues in
archaeological samples of human teeth from the Mariana
Islands. Experientia 52:281–85. doi:10.1007/BF01920724.
Oxenham, M. F., C. Locher, L. C. Nguyen, and K. T. Nguyen.
2002. Identification of Areca catechu (betel nut) residues
on the dentitions of Bronze Age inhabitants of Nui Nap,
northern Vietnam. Journal of Archaeological Science
29:909–15. doi:10.1006/jasc.2001.0767.
Reichart, P. A., H. Lenz, H. König, J. Becker, and U. Mohr.
1985. The black layer on the teeth of betel chewers: A light
microscopic, microradiographic and electronmicroscopic
study. Journal of Oral Pathology 14:466–75. doi:10.1111/
j.1600-0714.1985.tb00518.x.
Reinecke, A. 2004. Reiche gräber: Frühes salz: 600 Tage
feldforschungen auf dünen und reisfeldern (Vietnam)
[Rich graves: Early Salt: 600 days of field studies on
dunes and rice fields (Vietnam)]. In Expeditionen in ver-
gessene Welten: 25 Jahre archäologische forschungen in
Amerika, Afrika und Asien. Vol. 10, ed. Commission for
Archaeology of Non-European Cultures, 209–241. Aachen,
Germany: Linden Soft Verlag e.K.
Reinecke, A. 2009. Early cultures in Vietnam (first millen-
nium B.C. to second century A.D.). In Arts of ancient Viet
Nam: From river plain to open sea, ed. N. Tingley, 23–25.
Houston, TX: Museum of Fine Arts, Houston.

Rooney, D. F. 1993. Betel chewing traditions in Southeast
Asia. Oxford, UK: Oxford University Press.
Spinner, J., M. Klima, J. Kempf, L. M. Huppertz, V. Auwärter,
M. J. Altenburger, and M. A. Neukamm. 2014.
Determination of drugs of abuse in bovine dentin using
liquid chromatography–electrospray ionization tandem
mass spectrometry. Journal of Mass Spectrometry 49:1306–
13. doi:10.1002/jms.3464.
Wang, T. N., M. S. Huang, M. C. Lin, T. H. Duh, C. H. Lee, C. C.
Wang, P. H. Chen, S. L. Chiang, C. C. Sheu, V. C. Chen, C. C.
JOURNAL OF PSYCHOACTIVE DRUGS 7
Wu, C. P. Ferri, R. Stewart, and Y. C. Ko. 2014. Betel chewing
and arecoline affects Eotaxin-1, asthma and lung function.
PLOS One 9:e91889. doi:10.1371/journal.pone.0091889.
Zumbroich, T. J. 2008. The origin and diffusion of betel chew-
ing: A synthesis of evidence from South Asia, Southeast Asia
and beyond. Ejournal of Indian Medicine 1:87–140.
Zumbroich, T. J. 2009. “Teeth as black as a bumble bee’s
wings”: The ethnobotany of teeth blackening in Southeast
Asia. Ethnobotany Research and Applications 7:381–98.
doi:10.17348/era.7.0.381-398.