Journal of Archaeological Science (2002) 29, 000–000 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 February 2001, revised manuscript accepted 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 reasons 2002 Elsevier Science Ltd Keywords: BETEL NUT, VIETNAM, TEETH, GC/MS, SEM Stowers (1994) suggest that the higher frequency of foregut cancers in habitual betel nut chewers indicates that the habit can cause diabetes Inokuchi et al (1986) have highlighted the antihypertensive properties of some constituents of betel nut Perry (1980: 302) lists a host of uses for Areca catechu, both leaves and nut in various preparations, including: treatment of diarrhoea, dropsy, sunstroke, beri-beri, throat inflammations, oedema, lumbago, bronchial catarrh and urinary disorders Moreover, tape worm (Taenia saginata) infestations have been treated with betel nut preparations at least as far back as 1400 years (Perry, 1980) The chewing of betel nut itself is not known to etch or otherwise damage the surface of teeth Howden (1984) has demonstrated that a betel nut stain on teeth, caused through the chewing of betel nut, is instrumental in protecting the enamel from acid dissolution However, Flynn (1977) has described the process of purposely staining the dentition in modern Vietnamese as including a form of enamel etching Anecdotal evidence is cited (Flynn, 1977) that the traditional procedure of tooth etching and blackening serves as a Introduction he 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 suggests 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 periodontal disease (Jeng et al., 1996; Chang et al., 1998), the pre-malignant condition oral squamous fibrosis and other subsequent malignant cancerous conditions (Maher et al., 1994; Murti et al., 1995; Fang et al., 1997; Jeng et al., 1999) Boucher, Ewen & T *E-mail: moxenham@coloradocollege.edu 000 0305–4403/02/000000+00 $35.00/0 2002 Elsevier Science Ltd M F Oxenham et al 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 not display etching are representative of incidental staining 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– 900 years (Hocart & Fankhauser, 1996) The earliest Southeast Asian evidence for betel nut in an archaeological context comes from the upper levels of Spirit Cave in Thailand and is dated to at least 7600 years (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 nut during Han dynasty times (206 – 220) For instance, in his translation of early Chinese historical material Maspero (1918 cited in Hickey, 1982: 60) says 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 and quantification of very small sample amounts (10 up to 10 12 g) In the gas chromatograph (GC) a compound mixture is separated under suitable conditions (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 ionization process further aid the structural analysis GC/MS has been successfully used in a number of recent studies to identify bio-molecules in archaeological material (Balabanova et al., 1996; Wischmann, 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 tetrahydropyridine alkaloids arecoline, arecaidine, guvacine and guvacoline as its active principles (Dar & Khatoon, 1997), which can be used as marker compounds in GC/MS analysis (Hocart & Fankhauser, 1996) In addition to identifying the presence of betel 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 beginning in 1977 (Institute of Archaeology HS 251; 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 available, is dated to between 3000 and 2500 years The next group, for which again only two individuals are preserved, is dated to between 2400 and 2000 years The last group, including the remaining 30 individuals, has a radio carbon date of 1670 85 years (ZK378) 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 is tooth faces affected, was recorded as was the 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 stained, with presumed preparatory etching, or 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 examined in an attempt to establish a link with characteristic Residues on the Dentitions of Bronze Age Individuals 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 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 following the same protocol, using 15 ml solvent mix, as well as ml CHCl3 and H2O each To increase the volatility and therefore detectability of the extracted compounds, both samples were derivatized by heating for 20 at 90 C in a mixture of pyridine and bis(trimethylsilyl)trifluoroacetamide (BSTFA) containing 1% trimethylchlorosilane (TCMS); for the betel nut sample, ml pyridine and ml silylation mix were used, for the tooth extract 0·1 ml of each 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 240 C over 13 min) Figure 77NNM10ka: 20–29 year old Nui Nap male Note darkly stained anterior teeth and unstained enamel bands near the cemento-enamel junctions The general pattern of staining manifests as a concentration 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 illustrates 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 the teeth in this photograph Figure shows the greater frequency of anterior tooth staining and the gradual anterior-posterior decline in staining frequency 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 individuals with assessable teeth displayed the effects 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%) individuals Of those individuals displaying carious lesions, seven also had betel nut stained teeth Only 8/31 Percent stained teeth Results 90 75 60 45 30 15 I1 I2 C P3 P4 Tooth class M1 M2 M3 Figure 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) (25·8%) individuals did not display betel nut staining and none of these individuals displayed carious lesions Figure 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 cervical margin) of the same tooth (Figure 4) Hanson & Butler (1997) have examined crosssections of betel nut stained teeth from archaeological 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 M F Oxenham et al and non-silylated parent compounds, 133 as an indication for a chroman skeleton) and its fragmentation 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 Figure 77NNM13aka: roughened surface morphology of the stained enamel area of an isolated mandibular first incisor from Nui Nap (SEM, 1000) Figure 77NNM13aka: relatively smooth surface morphology of an unstained section of the same tooth shown in Figure (SEM, 1000) Note, markings in the top left of the image are surface scratches 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 major component in the betel nut extract (Figure 6a & b) This finding is important in providing the common chemical link between the two samples Based on some characteristic mass fragments (387 and 314 as silylated What appears to be a tannin derivative was detected in a residue staining a human incisor dated to between 2400 and 2000 years and was also detected as a major compound in a fresh Areca catechu extract Despite being less specific than the characteristic alkaloids, 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 successful 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 concentration in the stain consequently dropped under the instrument’s detection limit Only more robust compounds, 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 The caries prophylactic effects of betel nut use are 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 added during mastication (Hanson & Butler, 1997) Furthermore, various constituents of betel nut have been linked to both plaque prevention (Iwamoto et al., 1991) and bacterial suppression (De Miranda et al., 1996) Deliberately applying betel nut juices to the 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 Residues on the Dentitions of Bronze Age Individuals (a) 50% Relative signal intensity T 1.66 3.33 4.99 6.66 8.33 Retention time (in minutes) Abundance (b) 10.00 11.66 299 – 73 314 133 59 50 100 150 211 387 283 200 250 300 350 Molecular weight of fragments 447 400 450 495 500 Figure (a) Gas chromatogram of silylated tooth extract (T=presumed tannin derivative) (b) Mass spectrum of compound with a retention time of in silylated tooth extract 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 M F Oxenham et al (a) Relative signal intensity 100% SGC T AL SAD 1.66 3.33 4.99 6.66 8.33 Retention time (in minutes) 9.99 11.66 299 (b) Abundance SGL – 73 314 133 158 59 50 100 150 211 387 283 200 250 300 350 Molecular weight of fragments 447 400 450 495 500 Figure (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 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 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