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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.

Background: Determining the composition of artifact residues is a central problem in ancient residue metabolomics. This is done by comparing mass spectral features in common with an experimental artifact and an ancient artifact (standard method). While this method is simple and straightforward, we sought to increase the accuracy of predicting which plant species had been used in which artifacts., Results: Here, we introduce an algorithm (new method) based on ideas from the field of natural language processing (NLP) to solve this problem. We tested our strategy on a set of modern clay pipes. To limit biases, we were not provided information on which plant species had been smoked in which clay pipes. The results indicate that our new method performed 12.5% better than the standard method in predicting the plant species smoked in each artifact., Conclusions: Utilizing an NLP-based approach, we developed a robust algorithm for characterizing the composition of artifact residues. This work also discusses other general applications in which our algorithm could be used in the field of metabolomics, such as datasets where there are a limited number of replicates., (© 2024. The Author(s).)

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.

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.

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 of Nicotiana quadrivalvis and Rhus 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., (Copyright © 2020 Brownstein, Tushingham, Damitio, Nguyen and Gang.)

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., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Introduction: Botanicals containing iridoid and phenylethanoid/phenylpropanoid glycosides are used worldwide for the treatment of inflammatory musculoskeletal conditions that are primary causes of human years lived with disability (YLDs), such as arthritis and lower back pain., Objectives: We report the analysis of candidate anti-inflammatory metabolites of several endemic Scrophularia species and Verbascum thapsus used medicinally by peoples of North America., Methods: Leaves, stems, and roots were analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) and partial least squares-discriminant analysis (PLS-DA) was performed in MetaboAnalyst 3.0 after processing the datasets in Progenesis QI., Results: Comparison of the datasets revealed significant and differential accumulation of iridoid and phenylethanoid/phenylpropanoid glycosides in the tissues of the endemic Scrophularia species and Verbascum thapsus ., Conclusions: Our investigation identified several species of pharmacological interest as good sources for harpagoside and other important anti-inflammatory metabolites., Competing Interests: Conflict of interest: the authors declare that there are no conflicts of interest.

Sutherlandia frutescens is a medicinal plant, traditionally used to treat various types of human diseases, including cancer. Previous studies of several botanicals link suppression of prostate cancer growth with inhibition of the Gli/hedgehog (Gli/Hh) signaling pathway. Here we hypothesized the anti-cancer effect of S. frutescens was linked to its inhibition of the Gli/Hh signaling in prostate cancer. We found a dose- and time-dependent growth inhibition in human prostate cancer cells, PC3 and LNCaP, and mouse prostate cancer cell, TRAMP-C2, treated with S. frutescens methanol extract (SLE). We also observed a dose-dependent inhibition of the Gli-reporter activity in Shh Light II and TRAMP-C2QGli cells treated with SLE. In addition, SLE can inhibit Gli/Hh signaling by blocking Gli1 and Ptched1 gene expression in the presence of a Gli/Hh signaling agonist (SAG). A diet supplemented with S. frutescens suppressed the formation of poorly differentiated carcinoma in prostates of TRAMP mice. Finally, we found Sutherlandioside D was the most potent compound in the crude extract that could suppress Gli-reporter in Shh Light II cells. Together, this suggests that the S. frutescens extract may exert anti-cancer effect by targeting Gli/Hh signaling, and Sutherlandioside D is one of the active compounds., (© 2015 International Federation for Cell Biology.)

Sutherlandia frutescens is a botanical widely used in southern Africa for treatment of inflammatory and other conditions. Previously, an ethanolic extract of S. frutescens (SFE) has been shown to inhibit the production of reactive oxygen species (ROS) and nitric oxide (NO) by murine neurons and a microglia cell line (BV-2 cells). In this study we sought to confirm the anti-inflammatory activities of SFE on a widely used murine macrophage cell line (i.e., RAW 264.7 cells) and primary mouse macrophages. Furthermore, experiments were conducted to investigate the anti-inflammatory activity of the flavonol and cycloartanol glycosides found in high quantities in S. frutescens. While the SFE exhibited anti-inflammatory activities upon murine macrophages similar to that reported with the microglia cell line, this effect does not appear to be mediated by sutherlandiosides or sutherlandins. In contrast, chlorophyll in our extracts appeared to be partly responsible for some of the activity observed in our macrophage-dependent screening assay., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The introduction of spiral countercurrent chromatography in the last few years using new separation columns such as the spiral tubing support rotor has enabled the application of more polar volatile solvent systems for natural products separation. This method can be applied to water soluble compounds and their metabolites. We have used spiral countercurrent chromatography with the spiral tubing support rotor to fractionate n -butanol extracts of an African plant Sutherlandia frutescens and have determined conditions by which the predominant cycloartane glycoside (sutherlandioside B) can be purified in good yield. A solvent system of ethyl acetate, methanol, and water was modified by adding n-butanol to separate sutherlandioside B from other compounds. With the optimal amount of n-butanol in the two-phase solvent system with the lower aqueous phase mobile, the target compound was eluted well separated from the other components. The purity of sutherlandioside B was determined by high performance liquid chromatography/mass spectrometry analysis and the yield compares favorably with the content in bulk material.