Thanks to a new metabolics-based analysis method that exploits the power of LC-MS/MS, researchers now know indigenous peoples living in what is now Washington State 1,400 years ago were busy smoking Rhus glabra—a plant commonly known as smooth sumac. The discovery, made by a team of Washington State University researchers, marks the first time scientists have identified residue from a non-tobacco plant in an archeological pipe.
Previously, the identification of ancient plant residues relied on the detection of a limited number of biomarkers. However, this analysis limits results and, while the presence of a biomarker like nicotine shows tobacco was used, it doesn't distinguish which species.
For example, in the Pacific Northwest (before European contact), indigenous peoples most commonly smoked the plants Arctostaphylos uva-ursi, Cornus sericea, Nicotiana attenuata, Nicotiana quadrivalvis, Nicotiana rustica, Nicotiana tabacum, Rhus glabra, and Taxus brevifolia. However, in archeological pipes as old as 5,000 years, the four Nicotiana species cannot be distinguished from each other using the traditional biomarker approach since nicotine is a metabolite common to all of the species.
Thus, the WSU researchers developed and validated a metabolomics-based approach that can provide a much higher level of precision and accuracy to investigate the evolution of drug use and today’s similar plant-human co-evolutionary dynamics.
Using both experimental and ancient pipes, including a ~1,430-year-old pipe recently unearthed in Central Washington, the researchers extracted compounds from the surface without damaging the pipe. The compounds were then run through both GC-MS and LC-MS. The chemical standards of anabasine, arbutin, caffeine, cotinine, nicotine, theobromine, and theophylline (biomarkers typically associated with plant smoke) were readily separated and detected by both MS platforms, although LC-MS performed better overall.
As described in the study, the full LC-MS chromatograms were then processed in MZmine 2, an open-source metabolics software, to generate datasets. The datasets included the biomarker nicotine in addition to a large number of non-identified mass spectral features that are useful in non-targeted metabolomics.
“If you are only looking for a few specific biomarkers, you aren't going to be able to tell what else was consumed in the artifact," said David Gang, a professor in WSU's Institute of Biological Chemistry and a co-author of the study.
Subsequent analysis revealed Rhus glabra as the plant smoked in the 1,430-year-old pipe.
“Smoking often played a religious or ceremonial role for Native American tribes and our research shows these specific plants were important to these communities in the past," said Korey Brownstein, a former WSU Ph.D. student now at the University of Chicago and lead author of the study, published in Frontiers in Molecular Biosciences. "We think the Rhus glabra may have been mixed with tobacco for its medicinal qualities and to improve the flavor of smoke."
Overall, the method and results indicate metabolomics can help identify more plant taxa and, in some cases, discriminate plant use to the species-level.
"It wouldn't be hyperbole to say that this technology represents a new frontier in archaeo-chemistry,” Gang said.
Photo: Replica pipes used to experimentally "smoke" tobacco and other native plants in WSU laboratories for the study. The charred residue is then extracted, chemically "fingerprinted", and compared to residue of ancient archaeological pipes. Credit: WSU