Earliest Direct Evidence of Bronze Age Betel Nut Use: Biomolecular Analysis of Dental Calculus from Nong Ratchawat, Thailand

Piyawit Moonkham^1*Shannon Tushingham^2,3*Mario Zimmermann⁴Anna Berim⁵Korey J Brownstein⁶Charmsirin Devanwaropakorn⁷Suphamas Duangsakul⁸David R Gang⁵

• ¹Department of History, Faculty of Humanities, Chiang Mai University, Chaing Mai, Thailand
• ²California Academy of Sciences, San Francisco, United States
• ³Archaeological Research Facility, University of California, Berkeley, Berkeley, United States
• ⁴Department of Anthropology, Boise State University, Boise, United States
• ⁵Institute of Biological Chemistry, Washington State University, Pullman, United States
• ⁶Oak Ridge Institute, USDA, Peoria, United States
• ⁷Unearthed Heritage Australia, Melbourne, Australia
• ⁸The Second Regional Office of Fine Arts Department, Suphanburi, Thailand

The temporal and social dimensions of psychoactive plant use remain poorly understood in prehistoric Southeast Asia. This study employs novel biomolecular methods to detect compounds associated with betel nut chewing in ancient dental calculus. We implemented a two-phase approach: (1) Method validation through LC-MS analysis of modern betel nut chew plant control samples and (2) Archaeological testing of 36 dental calculus samples from human burials (4,000 ) at Nong Ratchawat, Central Thailand. Method validation confirmed reliable detection of arecoline derivatives using LC-MS in control samples.Archaeological analysis identified diagnostic compounds in three dental calculus samples, all associated with a single female burial dating to approximately 4,000 BP (4080-3850 cal BP; 4410-3070 cal BP) (Lertwinitnan, 2022:26; Tanompolkrang, 2017:59), representing the earliest direct biochemical evidence for betel nut use in Southeast Asia. While traditional archaeological markers of betel nut use (stained teeth, betel nutshell, and associated artifacts) have not been identified at Nong Ratchawat, our biomolecular analysis revealed this practice through dental calculus residues. This study presents the first successful detection of betel nut compounds in dental calculus, validating this approach for identifying prehistoric psychoactive substance use at the individual level. The results demonstrate the antiquity of betel nut consumption in mid-Holocene Thailand and establish new methodological protocols for investigating prehistoric psychoactive plant use in Southeast Asia.