For the first time, archaeologists have used advanced scientific techniques on 4,000-year-old dental plaque to confirm traces of betel nut chewing in ancient Thai communities.
Betel nuts are usually chewed as “quids,” a mix of slaked lime and ground betel nuts—which contain psychoactive compounds that boost energy, alertness, euphoria, and relaxation—wrapped in a betel leaf.
The stimulant, which can leave a red, brown or black stain on the teeth, is thought to be the world’s fourth most commonly used psychoactive substance, after caffeine, alcohol, and nicotine, with hundreds of millions of users globally.
In the past, betel nuts have been identified at archaeological sites via plant fragments or stained teeth, offering circumstantial evidence that its use goes back at least 8,000 years.
But using advanced scientific techniques, an international team of researchers has identified betel nut chewing in an individual with no dental discoloration.
The study, published Thursday in Frontiers in Environmental Archaeology, provides the earliest direct biochemical proof of betel nut consumption in Southeast Asia, predating previous evidence by at least 1,000 years, said author Piyawit Moonkham, an archaeologist at Chiang Mai University in Thailand.
The discovery of “invisible” traces of betel nut chewing in the molars demonstrates that for some prehistoric practices, “the visible evidence that we have might not tell us the whole story,” Moonkham said.
Highly sensitive and minimally invasive, the method requires only tiny samples of plaque and offers a “fascinating” way of finding more clues about the past, said Thanik Lertcharnrit, an associate professor at Silpakorn University in Bangkok, Thailand, and an expert in Southeast Asian archaeology, who was not involved in the study.
“In terms of methodology, we have very few, if any, archaeologists using that kind of scientific technique, the residue analysis, to infer the life, the tradition, the culture of the (prehistoric) people,” said Lertcharnrit.
“This paper represents a pioneer; it’s state of the art in terms of archaeological research in mainland South Asia, particularly in Thailand.”
Researchers began collecting ancient dental plaque, known as calculus, from Nong Ratchawat, a Neolithic burial site in central Thailand, in 2021. The team removed tiny, five-milligram scrapes of plaque from 36 dental samples, taken from six individuals.
The method, called liquid chromatography-mass spectrometry (LC-MS), extracts, separates, and identifies chemical compounds by measuring how heavy the molecule is compared with its electrical charge.
But before testing the ancient samples, the team needed a control sample — something they could compare the results with and demonstrate what traces of betel nut liquid might look like.
“We tried to mimic the culture of chewing,” said Moonkham, adding that in addition to the core ingredients of dried betel nut, red limestone paste, and piper betel leaves, they included catechu bark and tobacco in some of their control samples, and ground the ingredients together with human saliva.
The modern control samples were tested first to validate the method before the dental samples were analyzed.
They detected trace plant alkaloids – including betel nut’s main psychoactive compounds, arecoline and arecaidine – in three samples from one individual known as “Burial 11,” likely a woman aged around 25.
Researchers say the benefit of the technique is that it doesn’t destroy the original samples, leaving the remains intact for future study.
‘Nobody has done this before’
LC-MS is currently used in a variety of fields, including pharmaceuticals, food safety, and environmental testing.
But its use in archaeology so far has been limited, said Dr. Melandri Vlok, bioarchaeologist and a lecturer in anatomy and physiology at Charles Sturt University in Australia.
“A lot of the work that’s been done using this (method) is looking for proteins in dental calculus for dietary reasons. So, using it to pick up these compounds that get trapped in the dental plaque, that’s what’s really innovative here. Nobody has done this before,” said Vlok.
There’s a reason it isn’t common: the method requires expensive machinery—such as an Orbitrap, one of the most advanced mass spectrometers on the market, which identifies molecules by measuring the mass-to-charge ratio—that many researchers don’t have access to, she added.
“It’s starting to be used more routinely by some of the bigger labs, like Harvard and Max Planck — which makes this research even more amazing, because this is a paper with a Thai first-author, which is great,” she said. “Seeing this research come from within the region is actually the thing that excites me the most.”
The team on this paper included researchers from eight institutions across three continents, and the chemical residue analysis was conducted at Washington State University, where Moonkham studied for his PhD.
The study’s control samples, which created a “standard” to test against, are another novelty, and future studies could refine this even further by considering how the compounds degrade over thousands of years, said Vlok.
“This is a method that I can definitely see being used quite frequently from now on in the region,” she added.
While betel nuts have long been linked to hospitality and religious rituals, much of the research in recent years has focused on its classification as a carcinogen and the correlation between betel quid use and oral cancers.
“Betel nut chewing has significant implications for people’s health,” said Vlok. “It’s something that affects millions of people in tropical Asia-Pacific today, but we don’t really know how long people have been doing this for.”
Better understanding where the tradition comes from, and how and why people are using it, could help address some of these concerns, she added.
In Thailand, Moonkham says the practice has been strongly discouraged by the government since the 1940s, and while it’s still popular in rural areas, it’s now uncommon in cities and with younger generations.
Although he recognizes the potential health hazards, Moonkham believes the practice has been overly “demonized” and hopes research like this can show the long history of betel nuts in Thailand, and their importance in society. He has a personal attachment to the practice, too: he has childhood memories of his grandparents often chewing betel quids, usually while gossiping with friends or relaxing after a family meal.
“I asked my grandmother once, ‘Why do you chew it?’ And she responded, because it cleans the teeth and it helps me relax,” Moonkham recalled. “When she chewed it, she tended to share with a friend, family, or colleagues. I think it’s significant in the way it creates a social bond.”
Researchers are still exploring possible reasons for the absence of tooth stains in the individual they examined, which they speculate could be due to different chewing methods, cleaning habits, or decay over the thousands of years since.
Further research could help narrow down the possibilities. The team plans to analyze more individuals from the Nong Ratchawat site, where a further 150 individuals could be tested for signs of betel nut use, and Moonkham intends to dig deeper into the social, religious and medicinal roles of betel nut in ancient societies in future projects.
The technique could also be applied to a wide range of plant and food residues, opening new avenues for understanding ancient practices.
“I think people tend to neglect the social and cultural aspect of plants,” said Moonkham. “It’s important to understand the whole perspective.”