Metabolomics-Based Analysis of Miniature Flask Contents Identifies Tobacco Mixture Use among the Ancient Maya

A particular type of miniature ceramic vessel locally known as “veneneras” is occasionally found during archaeological excavations in the Maya Area. To date, only one study of a collection of such containers successfully identified organic residues through coupled chromatography–mass spectrometry methods. That study identified traces of nicotine likely associated with tobacco. Here we present a more complete picture by analyzing a suite of possible complementary ingredients in tobacco mixtures across a collection of 14 miniature vessels. The collection includes four different vessel forms and allows for the comparison of specimens which had previously formed part of museum exhibitions with recently excavated, untreated containers. Archaeological samples were compared with fresh as well as cured reference materials from two different species of tobacco (Nicotiana tabacum and N. rustica). In addition, we sampled six more plants which are linked to mind-altering practices through Mesoamerican ethnohistoric or ethnographic records. Analyses were conducted using UPLC-MS metabolomics-based analytical techniques, which significantly expand the possible detection of chemical compounds compared to previous biomarker-focused studies. Results include the detection of more than 9000 residual chemical features. We trace, for the first time, the presence of Mexican marigold (Tagetes lucida) in presumptive polydrug mixtures

Changes in the Harpagide, Harpagoside, and Verbascoside Content of Field Grown Scrophularia lanceolata and Scrophularia marilandica in Response to Season and Shade

Scrophularia lanceolata Pursh and Scrophularia marilandica L. are two common species within the Scrophulariaceae family that are endemic to North America. Historically, these species were used by indigenous peoples and colonialists to treat sunburn, sunstroke, frostbite, edema, as well as for blood purification, and in women’s health. Several iridoid and phenylethanoid/phenylpropanoid glycosides detected in these species, such as harpagoside and verbascoside, possess anti-inflammatory and anti-nociceptive properties. Due to the presence of anti-inflammatory metabolites and the historical uses of these species, we performed a two-year field study to determine the optimal production of these important compounds. We subjected the plants to shade treatment and analyzed differences in the metabolite composition between the two species and each of their leaves, stems, and roots at various times throughout the growing seasons. We determined that S. lanceolata plants grown in full sun produced 0.63% harpagoside per dried weight in their leaves compared to shade-grown plants (0.43%). Furthermore, S. lanceolata accumulated more harpagoside than S. marilandica (0.24%). We also found that verbascoside accumulated in the leaves of S. lanceolata and S. marilandica as the growing season progressed, while the production of this metabolite remained mostly seasonally unchanged in the roots of both species

An Ancient Residue Metabolomics-Based Method to Distinguish Use of Closely Related Plant Species in Ancient Pipes

Residues from ancient artifacts can help identify which plant species were used for their psychoactive properties, providing important information regarding the deep-time co-evolutionary relationship between plants and humans. However, relying on the presence or absence of one or several biomarkers has limited the ability to confidently connect residues to particular plants. We describe a comprehensive metabolomics-based approach that can distinguish closely related species and provide greater confidence in species use determinations. An ~1430-year-old pipe from central Washington State not only contained nicotine, but also had strong evidence for the smoking ofNicotiana quadrivalvisandRhus glabra, as opposed to several other species in this pre-contact pipe. Analysis of a post-contact pipe suggested use of different plants, including the introduced trade tobacco,Nicotiana rustica. Ancient residue metabolomics provides a new frontier in archaeo-chemistry, with greater precision to investigate the evolution of drug use and similar plant-human co-evolutionary dynamics

Biomolecular archaeology reveals ancient origins of indigenous tobacco smoking in North American Plateau

Chemical analysis of residues contained in the matrix of stone smoking pipes reveal a substantial direct biomolecular record of ancient tobacco (Nicotiana) smoking practices in the North American interior northwest (Plateau), in an area where tobacco was often portrayed as a Euro-American–introduced postcontact trade commodity. Nicotine, a stimulant alkaloid and biomarker for tobacco, was identified via ultra-performance liquid chromatography-mass spectrometry in 8 of 12 analyzed pipes and pipe fragments from five sites in the Columbia River Basin, southeastern Washington State. The specimens date from 1200 cal BP to historic times, confirming the deep time continuity of intoxicant use and indigenous smoking practices in northwestern North America. The results indicate that hunting and gathering communities in the region, including ancestral Nez Perce peoples, established a tobacco smoking complex of wild (indigenous) tobacco well before the main domesticated tobacco (Nicotiana tabacum) was introduced by contact-era fur traders and settlers after the 1790s. This is the longest continuous biomolecular record of ancient tobacco smoking from a single region anywhere in the world—initially during an era of pithouse development, through the late precontact equestrian era, and into the historic period. This contradicts some ethnohistorical data indicating that kinnikinnick, or bearberry (Arctostaphylos uva-ursi) was the primary precontact smoke plant in the study area. Early use likely involved the management and cultivation of indigenous tobaccos (Nicotiana quadrivalvis or Nicotiana attenuata), species that are today exceedingly rare in the region and seem to have been abandoned as smoke plants after the entry of trade tobacco

Extraction, Purification and Characterization of an Arabinogalactan from Frost (Riverbank) Grape (Vitis riparia Michx.) Stems

This study investigated the extraction and properties of an arabinogalactan polysaccharide from frost grape (FGP) as a potential alternative to gum arabic (GA). Collection date, solvent:feed ratio (S:F), chip size, C-18 filtration, ultrafiltration, freeze drying versus spray drying, methanol pre-extraction, and water absorption were examined. Sugar composition, elemental analysis, dietary fiber content, emulsification activity index (EAI), emulsification stability index (ESI), and viscosity were used to evaluate the extracts. Exudates collected in March from live stems were viscous with high percentage solids and FGP, while May collections were watery with low percentage solids and FGP. Frost grape stems were collected, chipped, and classified by size. The extraction system utilized pressure or vacuum to increase contact between the chips and extraction water. A S:F ratio of ca. 24% gave an excellent yield of FGP. Chips between 1.8 and 3.8 mm gave the highest mass yields. Pre-extracting the chips with methanol and C-18 filtration of the water extract both yielded a lighter product. The EAI for the FGP was higher than that for GA; however, its ESI was lower. Ultrafiltration of the crude extract separated glucose, fructose, and sucrose from the FGP. FGP with glucose, fructose, and sucrose adsorbed water and became darker

Dental calculus as a source of ancient alkaloids: Detection of nicotine by LC-MS in calculus samples from the Americas

Dental calculus has been shown to be a repository of a variety of exogenous organic materials, including bacterial DNA, proteins, phytoliths, and starch grains. Here we show that certain alkaloids, nicotine in this case, can also be trapped and preserved in ancient dental calculus. We present Ultra-Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS) results of analyses of ten archaeological calculus samples from eight individuals from Central California. Two samples tested positive for the presence of nicotine, including one from an individual buried with a pipe. UPLC-MS analyses of dental calculus could provide an alternative means to trace the spread and consumption of tobacco and other plants with distinctive alkaloid products in ancient societies. As shown here for the first time in ancient samples, the ability to detect nicotine alkaloids in calculus has enormous potential to help us better understand the consumption of intoxicant plants by ancient humans on the individual level, for example by giving us the ability to test assumptions about the age, sex, and status of individual tobacco users in the past