Dental calculus – the mineralized plaque that builds up on teeth – is emerging as a valuable archive of ancient dietary and cultural practices. In a new study published in Frontiers in Environmental Archaeology, researchers applied liquid chromatography–mass spectrometry (LC-MS) to dental calculus samples from six Bronze Age burials at Nong Ratchawat, central Thailand. The results uncovered the molecular signature of arecoline, a key alkaloid in betel nut, providing the earliest direct biomolecular evidence of betel chewing in Southeast Asia – around 4,000 years ago.
The study establishes betel chewing in Southeast Asia more than a millennium earlier than earlier records and shows that such practices can be identified even in the absence of visible archaeological markers. To learn more about the project’s origins, the analytical hurdles, and the wider implications for understanding human–plant relationships, we spoke with Piyawit Moonkham of Chiang Mai University, Thailand, co-lead author of the study.
What initially motivated you to investigate ancient dental calculus for evidence of plant use?
It started with an NSF granted project back in 2018, where our team at Washington State University began looking at how various psychoactive plants were used, seen, and understood by indigenous communities. I’ve also watched my grandparents use and chew these plants ever since I was young, which has only added to my curiosity. As betel nut chewing cultures are still present in many parts of rural Thailand, I was inspired to find out more about the historical and social background and transformations of betel nut plants and to learn more about a specific and complex human-plant interaction.
How did you approach the challenge of detecting trace plant compounds in plaque more than 4,000 years old?
We conducted an initial control experiment with modern samples of betel nut quid by creating and mimicking betel nut quid chewing with human saliva. From this, we observed the generation of chemical compounds – arecoline, arecaidine, and tetrahydropyridine – which provided a reference, before analyzing the samples retrieved from the archaeological site.
Was there a key breakthrough during your research?
The breakthrough came when we detected arecoline – the key compound in betel nut – in dental calculus, matching our standardized residue samples. This confirmed to us that this individual had indeed chewed betel nut.
What was the biggest analytical challenge you faced during your research – and how did you overcome it?
The human burials at Nong Ratchawat, despite resulting in a high number of discoveries, have seen significant degradation due to the site’s hot, humid climate. Thus, during our data collection we retrieved dental calculus from the healthiest teeth of six individuals. The chemical compound detected in the archaeological samples matched arecoline's molecular weight and formula. However, its MSn fragmentation pattern differed from both reference samples and the arecoline standard. We conducted targeted analysis at the USDA ARS laboratory, which confirmed the arecoline derivative peaks with the same molecular weight and formula with greater accuracy – though the divergent fragmentation pattern still persisted. Given the age of the Nong Ratchawat site (4,080–3,850 cal BP*; 4,410–3,070 cal BP), we suggest this difference likely reflects the structural transformation of the original compound.
What are some of the broader implications this research might have for understanding ancient lifeways, ritual practices, or plant–human relationships?
Our work represents the first dental calculus analysis study on betel nut residues and provides the earliest biomolecular evidence of betel nut use in Southeast Asia. The absence of traditional markers, such as black-stained teeth and betel nut fragments, in our research demonstrates the value of biomolecular studies in revealing invisible behaviors. We hope the protocol we applied will provide a platform for analyzing human remains across more sites in Southeast Asia.
Overall, our study broadens understanding of psychoactive plant use patterns and communal practices, enhances our comprehension of sociocultural worldviews, and offers a fresh approach to studying gender-based consumption patterns and disparities – as well as human-environment interactions.
Where do you see this kind of biomolecular archaeology going next?
There are more psychoactive plants used in local communities here in rural Thailand and Southeast Asia – such as cannabis, tobacco, kra-tom leaves, or opium, as well as other indigenous herbal plants – that we’re hoping to look at in further research. Our focus will be on local knowledge, plant utilization, social and cultural aspects of these plants and their transformation from archaeological sites in Thailand and Southeast Asia.
In our next project, we’re considering exploring the complex relationships between tobacco and betel nut use – opposed to focusing on gender disparities, religious spaces or psychoactive plants – in archaeological contexts likely dating to the same period as the Nong Ratchawat site.
*cal BP = calibrated radiocarbon years before present (with ‘present’ defined as 1950 AD)
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