Identification of areca catechu betel nu

doi:10.1006/jasc.2001.0767, available online at http://www.idealibrary.com onIdentification of Areca catechu Betel Nut Residues on the Dentitions of Bronze Age Inhabitants of Nui Nap, No

doi:10.1006/jasc.2001.0767, available online at http://www.idealibrary.com on

Identification of Areca catechu (Betel Nut) Residues on the

Dentitions of Bronze Age Inhabitants of Nui Nap,

Northern Vietnam

Marc F Oxenham*

Department of Anthropology, Colorado College, U.S.A.

Cornelia Locher

Faculty of Science, Information Technology and Education, Northern Territory University, Australia

Nguyen Lan Cuong and Nguyen Kim Thuy

Department of Palaeoanthropology, Institute of Archaeology, Hanoi, Vietnam

(Received 2 February 2001, revised manuscript accepted 9 October 2001)

The dentitions of 31 individuals excavated from the Bronze Age site Nui Nap, Thanh Hoa province, Vietnam were

examined for the presence of Areca catechu (betel nut) residues The chief investigatory method involved GC/MS

analysis of a dark reddish-brown stain commonly observed on the labial aspect of the anterior teeth of individuals from this site The results of this work indicate that these teeth were stained by way of betel nut Enamel surface morphology and the distribution of staining suggests purposeful application of betel nut residues to the teeth, perhaps for aesthetic

Keywords: BETEL NUT, VIETNAM, TEETH, GC/MS, SEM.

Introduction

The chewing of betel nut (Areca catechu) on its own or in combination with pepper leaf (Piper

betel) and/or a lime paste (calcium hydroxide) is

a ubiquitous habit in Southeast Asia and the Pacific

today The stimulant properties of betel nut are likely

its main attraction, in addition to low cost and ready

availability

There is a growing literature on both health benefits

and costs to betel nut users Mounting evidence

sug-gests that the regular mastication of betel nut has a

cariostatic effect (Moller, Pindborg & Effendi, 1977;

Schamschula et al., 1977; Nigam & Srivastava, 1990),

although others have reported contrary evidence

(Reichart & Gehring, 1984; Williams et al., 1996)

Numerous studies have linked betel nut chewing with

fibrosis and other subsequent malignant cancerous

et al., 1997; Jeng et al., 1999) Boucher, Ewen &

Stowers (1994) suggest that the higher frequency of foregut cancers in habitual betel nut chewers indicates

have highlighted the antihypertensive properties of

lists a host of uses for Areca catechu, both leaves and

nut in various preparations, including: treatment of diarrhoea, dropsy, sunstroke, beri-beri, throat inflam-mations, oedema, lumbago, bronchial catarrh and

urinary disorders Moreover, tape worm (Taenia

saginata) infestations have been treated with betel nut

1980)

The chewing of betel nut itself is not known to etch

(1984)has demonstrated that a betel nut stain on teeth, caused through the chewing of betel nut, is instrumen-tal in protecting the enamel from acid dissolution

purposely staining the dentition in modern Vietnamese

as including a form of enamel etching Anecdotal

procedure of tooth etching and blackening serves as a

*E-mail: moxenham@coloradocollege.edu

000

0305–4403/02/000000+00 $35.00/0  2002 Elsevier Science Ltd

caries prophylaxis Tooth blackening is seen in many

Asian countries, Africa and South America (Phan Hai

Linh, 1998) and the process can involve the use of betel

nut at various stages It might then be expected that

stained archaeological specimens that do not

dis-play etching are representative of incidental

stain-ing through betel nut mastication On the other hand,

stained specimens that also display etching are

likely the result of deliberate etching and subsequent

intentional application of betel nut residues

While the beginning of the human use of betel nut in

Southeast Asia is unknown, a betel nut and lime slurry

has been reported to have been used to deliberately

stain human teeth in the Mariana Islands some 300–

Southeast Asian evidence for betel nut in an

archaeo-logical context comes from the upper levels of Spirit

(Yen, 1977) It is unclear if the presence of betel nut

remains in this context is fortuitous or indicates human

use of this plant The antiquity of the use of betel nut in

what is now Vietnam is also unclear However, Chinese

historical sources suggest the inhabitants of what was

then Annam (modern northern Vietnam) did use betel

instance, in his translation of early Chinese historical

of the northern Vietnamese of the time:

They tattooed their bodies so as to ‘‘avoid crocodiles’’,

they chewed betel and areca and they blackened their teeth

Betel and areca were prescribed offerings for the bride

at marriage

The reference to betel here, in distinction to areca, is to

the Piper betel leaf.

The chief technique used in this study to identify the

human use of betel nut in northern Vietnam 2300–1700

years ago is gas-chromatography/mass spectrometry

(GC/MS) GC/MS can be used for the identification

compound mixture is separated under suitable

condi-tions (column type, temperature), and the various

retention times obtained from this procedure can be

used for identification Coupled to a mass spectrometer

(MS), the characteristic fragmentation patterns of the

separated compounds undergoing the subsequent

ion-ization process further aid the structural analysis

GC/MS has been successfully used in a number of

recent studies to identify bio-molecules in

Hummel & Herrmann, 2000; Agozzino et al., 2001;

Copley et al., 2001) Next to amino acids, tannins and

gallic acid, betel nut is known to contain the

tetra-hydropyridine alkaloids arecoline, arecaidine, guvacine

Khatoon, 1997), which can be used as marker

nut in association with human biological remains, this study sought to determine if the nut was masticated, presumably for its narcotic effect, or utilized in some other manner, perhaps as a cosmetic

Materials

The archaeological dental sample derives from the northern Vietnamese Bronze period site Nui Nap Nui Nap is a high limestone mountain in Dong Son Commune, Dong Hieu District, Thanh Hoa Province

It is situated some 10 km to the west of the eponymous Dong Son site It was first discovered and surveyed in

1962, with a follow up survey in 1976 and excavation

Ha Van Phung & Pham Ninh Huyen, 1978;Vo Hung

& Nguyen Lan Cuong, 1979; Ha Van Phung, 1984) Most of the burials were rich in artefacts Bronze objects included spear-heads, arrow-heads, daggers, axes, harpoons, vessels, earrings, drums, and beads

A variety of pottery and ceramic vessels were also found Other artefacts included a spindle whorl, stone earrings, glass beads and minted Han coins

There are three separate groups of dates for this site The earliest, for which only two individuals are

next group, for which again only two individuals are

The last group, including the remaining 30 individuals,

(ZK-378) The dental assemblage includes all preserved

teeth (n=558 teeth, 31 individuals) from this site An

isolated maxillary central incisor from this sample (individual identification number 77NNM13aka) was used for scanning electron microscopy (SEM) and gas-chromatography coupled with mass spectrometry

Method

The entire preserved dentition of all individuals was assessed for staining The location of the stain, that

distribution of the stain on the most severely affected face

The surface morphology of an incisor was examined

in order to assess whether the tooth was deliberately

incidentally stained through betel nut mastication An epoxy cast was made of the labial aspect of an isolated maxillary incisor dated to between 2400 and 2000 years

 After coating in gold the stained and unstained areas of this cast were examined using SEM The surface morphologies of the stained and unstained areas were compared

The aforementioned tooth was also used for the GC/MS analysis whereby organic compounds extracted from the tooth’s stained surface were exam-ined in an attempt to establish a link with characteristic

betel nut compounds The stained surface was carefully

ground off using a dental drill and the pale orange

powder collected (4 mg) To extract the organic matter,

that sample was suspended in a mixture of CHCl3/

MeOH/H2O (1:2:0·8) and sonicated for 2 min CHCl3

(0·5 ml) as well as H2O (0·5 ml) were subsequently

added, followed in each case by further sonication The

mixture was centrifuged and the supernatant (CHCl3

and MeOH/H2O) evaporated under N2 To obtain a

reference sample a whole betel nut was cut into very

small pieces and the organic material extracted

follow-ing the same protocol, usfollow-ing 15 ml solvent mix, as well

as 4 ml CHCl3and H2O each To increase the volatility

and therefore detectability of the extracted

com-pounds, both samples were derivatized by heating for

20 min at 90C in a mixture of pyridine and

bis(tri-methylsilyl)trifluoroacetamide (BSTFA) containing 1%

trimethylchlorosilane (TCMS); for the betel nut

sample, 1 ml pyridine and 1 ml silylation mix were

used, for the tooth extract 0·1 ml of each 1
l of each

sample was analysed by GC/MS (on a Varian Star

3400 CX Spectrometer with a SGE 30QC2.5/BPX1.0

column and a temperature ramp from 150 to 240C

over 13 min)

Results

The general pattern of staining manifests as a

concen-tration on the buccal aspect of the anterior teeth, with

a general marked lessening in density on the premolars

and a general absence or only very slight staining of the

molars.Figure 1illustrates the concentration of a dark

reddish-brown stain on the anterior teeth of an adult

male excavated at Nui Nap In life, the gum has

protected the enamel at the cervical margin of the

tooth from being stained This has resulted in the

narrow band of white unstained enamel evident on

greater frequency of anterior tooth staining and the

gradual anterior-posterior decline in staining

fre-quency Of the 558 teeth, 281 (50·4%) displayed some

form of staining Of the total sample, 50·9% (140/275)

of male teeth, 55·0% (120/218) of female teeth and

32·3% (21/65) unsexed teeth were stained Examining

individuals, rather than separate teeth, 74·2% (23/31)

of betel nut staining Slightly more females (81·8%,

9/11) than males (69·2%, 9/13) displayed discoloured

teeth

Given the evidence for a relationship between betel

nut use and reduced caries risk, the co-occurrence of

each condition was explored for the Nui Nap sample

Of the 558 teeth, 15 (2·7%) displayed carious lesions,

with all but one of these lesions affecting the occlusal

and interstitial aspects of premolars and molars These

lesions were distributed between 8/31 (25·8%)

individ-uals Of those individuals displaying carious lesions,

seven also had betel nut stained teeth Only 8/31

(25·8%) individuals did not display betel nut staining and none of these individuals displayed carious lesions Figure 3 illustrates the surface morphology of the stained labial area of the archaeological maxillary incisor later used in GC/MS analysis The surface of the stained region of the tooth is extremely rough and uneven, in contrast to the unstained region (close to the

Hanson & Butler (1997) have examined cross-sections of betel nut stained teeth from archaeologi-cal contexts in the Mariana Islands They illustrate (Hanson & Butler, 1997: 282, Figure 6) that seepage of the stain into the enamel is coincident with changes to the enamel prism morphology It is unclear if the stain itself is responsible for these changes It is not improbable that deliberate etching of the enamel

Figure 1 77NNM10ka: 20–29 year old Nui Nap male Note darkly stained anterior teeth and unstained enamel bands near the cemento-enamel junctions.

0 90

Tooth class

I1

75 60 45 30 15

M3 M2 M1 P4 P3 C I2

Figure 2 Frequency of stained teeth by tooth class for the total assessable Nui Nap sample of 558 teeth representing 31 individuals (I=incisor, C=canine, P=premolar, M=molar).

surface preparatory to staining is the aetiological agent

implicated here

As anticipated, GC/MS analysis of the betel nut

extract showed, next to a number of other peaks

that were not investigated further, the aforementioned

alkaloids, which were identified by their typical MS

fragmentation pattern The archaeological tooth

sample also presented a large number of peaks,

although no evidence for tetrahydropyridine alkaloids

could be found However, the main compound of the

archaeological sample (Figure 5a & b) also occurs as a

chemical link between the two samples Based on some

characteristic mass fragments (387 and 314 as silylated

and non-silylated parent compounds, 133 as an indica-tion for a chroman skeleton) and its fragmentaindica-tion pattern (314, 299, 283, 211, 73), the compound might

be a tannin, a common constituent of betel nut However, in the absence of any further attempts at structural analysis this tentative identification remains speculative

Discussion and Conclusions

What appears to be a tannin derivative was detected in

a residue staining a human incisor dated to between

major compound in a fresh Areca catechu extract.

Despite being less specific than the characteristic alka-loids, the detection of this tannin in both samples is significant It can be seen as an indication that the stain was caused by betel nut residues coming into contact with the surface of the tooth It can be argued that the 20-year hiatus between the excavation and analysis of the tooth material may have prevented the success-ful detection of the alkaloids Organic archaeological material is known to change quickly when exposed to air and light It is therefore probable that the alkaloids have decomposed to a large degree, and their concen-tration in the stain consequently dropped under the instrument’s detection limit Only more robust com-pounds, such as tannin derivatives, would remain intact under those conditions and can still be identified and used as a link between the two samples The implications are that the distinctive reddish-brown stain commonly observed on the labial aspect of the anterior teeth of the inhabitants of Nui Nap can be traced to the incidental or deliberate application of betel nut compounds

not apparent in the Nui Nap sample A possible reason for this is that betel nut was not chewed, but rather betel nut residues were deliberately applied to the teeth The cariostatic properties of actually chewing betel nut are believed to be associated with the cleansing action of the fibrous wad, increased saliva flow and also neutralization of acidogenic bacteria, if lime is

Furthermore, various constituents of betel nut have

visible aspects of the anterior teeth as a single event, or

on an infrequent basis, would perhaps not assist in caries protection

The concentration of staining on the labial aspect of the anterior teeth, the reduced intensity and frequency

of posterior tooth staining, and the complete absence

of occlusal and buccal staining are not consistent with incidental staining due to habitual betel nut chewing This pattern would be expected if betel nut residues were deliberately applied to the outer surfaces of the

Figure 3 77NNM13aka: roughened surface morphology of the

stained enamel area of an isolated mandibular first incisor from Nui

Nap (SEM, 1000).

Figure 4 77NNM13aka: relatively smooth surface morphology of

an unstained section of the same tooth shown in Figure 3 (SEM,

1000) Note, markings in the top left of the image are surface

scratches.

anterior teeth, perhaps for aesthetic purposes The

eroded appearance of the stained portion of these teeth

also suggests that they were likely purposefully etched

preparatory to the application of the betel nut stain

The human use of betel nut has been demonstrated

in Northern Vietnam at least 2000 years  This is

the earliest substantiated use of Areca catechu in

Southeast Asia to date An examination of the

500

50

–

450 400

350 300

250 200

150 100

Molecular weight of fragments

(b)

50%

11.66 10.00

8.33 6.66

4.99 3.33

1.66

Retention time (in minutes) (a)

59

73

495 447

299 T

Figure 5 (a) Gas chromatogram of silylated tooth extract (T=presumed tannin derivative) (b) Mass spectrum of compound with a retention time of 5 min in silylated tooth extract.

dentition (personal observation by M.O.) of the

remains of individuals excavated in an archaeological

site in the far south of Vietnam, Giong Co Vo dated

to some 2500 years , indicates the widespread

human use of this plant in the early Bronze period of

Southeast Asia

Acknowledgements

The authors wish to thank a number of individuals and departments for assistance in this project: Professor Ha Van Tan, Director of the Institute of Archaeology, Hanoi, Vietnam for access to the dental material used

500

50

–

450 400

350 300

250 200

150 100

Molecular weight of fragments

(b)

100%

11.66 9.99

8.33 6.66

4.99 3.33

1.66

Retention time (in minutes) (a)

59

73

314

387

495 447

299

T AL

SGL SAD

SGC

158

Figure 6 (a) Gas chromatogram of silylated betel nut extract (T=presumed tannin; AL=non-silylated arecoline; SAD=silylated arecaidine; SGL=silylated guvacoline; SGC=silylated guvacine) (b) Mass spectrum of compound with a retention time of 5·00 minutes in silylated betel nut extract.

in this study; Elie Haywood with S.I.T.E at the

Northern Territory University (Australia) for handling

the SEM work; Dr Dirk Megirian, of the Museums

and Art Galleries of the Northern Territory, for access

to equipment used to obtain samples for GC/MS

analysis; and Dr Barry Fankhauser for comments on

technique

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