Elucidating the Structure and Identity of Pinckneyin, a Historical Natural Product from Pinckneya bracteata

Abstract

Pinckneya bracteata (Georgia bark) was historically used in the southeastern United States to treat intermittent fevers and has long been believed to contain quinine. In 1885, a bitter crystalline substance termed pinckneyin was isolated, but its structure remained unknown. Reproduction of the original extraction procedure combined with modern spectroscopic and single-crystal X-ray diffraction analysis definitively identifies pinckneyin as 7-hydroxycoumarin (umbelliferone). This work resolves a long-standing ambiguity in the phytochemical literature and clarifies the chemical basis for reassessing the historical medicinal use of P. bracteata.

Introduction

During the 19th century, physicians in the southeastern United States sought indigenous plant-based substitutes for quinine to treat intermittent fevers and malaria. Among the most widely discussed candidates was Georgia bark (Pinckneya bracteata Raf.; syn. Pinckneya pubens Michx.), a subtropical flowering tree indigenous to the American South. Its geographic range extends from southern South Carolina through Georgia and into Florida. Historically, P. bracteata was widely used as a medicinal plant, particularly as an antimalarial agent for the treatment of intermittent fevers. − Owing to its presumed close relationship to Peruvian bark (Cinchona spp.), Georgia bark was believed to contain quinine and was prescribed in the United States Pharmacopoeia and recommended to surgeons as a treatment for malaria during the first half of the 19th century. However, repeated attempts to isolate quinine were unsuccessful, and preparations of the bark were instead administered as tinctures or powders. − Clinical observations reported in 1864 noted that although Georgia bark exhibited antiperiodic activity, its effects were considerably slower than those of quinine sulfate. Interest in the plant increased during the American Civil War, when shortages of imported cinchona bark and quinine prompted physicians in the Confederate states to investigate indigenous plant-based substitutes for antimalarial therapy. , Contemporary medical reports describe P. bracteata among several native plant species examined as potential antiperiodic remedies during this period. Although such substitutes were generally considered less effective than quinine, their use illustrates the historical importance of Georgia bark in attempts to identify locally available treatments for malaria and related febrile illnesses.

Further investigation of Georgia bark was carried out by Naudain in 1885, who demonstrated the absence of quinine and other alkaloid constituents. In that study, a light brown crystalline substance was isolated and named pinckneyin, which was tentatively proposed to be a glucoside based on rudimentary chemical tests and organoleptic properties. Pinckneyin was speculated to be the bioactive component of the bark, although its chemical structure could not be determined due to the limited material. A subsequent study in 1943 confirmed the absence of quinine and other alkaloids using standardized analytical techniques described in the US Pharmacopeia XI. These methods included classical alkaloid precipitation assays and fluorescence tests; however, such techniques lacked the sensitivity and structural resolution afforded by modern spectroscopic methods. Despite these efforts, the identity of the bioactive constituents of P. bracteata remained unclear and the structure of pinckneyin was left unresolved.

Malaria remains a major global public health challenge, with an estimated 282 million cases and 610,000 deaths worldwide in 2024. Plasmodium falciparum, the species responsible for the majority of malaria-related mortality, has developed resistance to many current antimalarial therapies. , Resistance to quinine itself was first reported as early as 1910, underscoring the long-standing need for new therapeutic agents. In this context, natural product-based drug discovery has experienced renewed interest as a source of structurally diverse and biologically relevant chemical scaffolds. ,

Motivated by the unresolved structural identity of pinckneyin, we sought to reproduce Naudain’s original extraction procedure and to perform a definitive structural elucidation using modern spectroscopic and crystallographic techniques. Although rapid dereplication could be achieved by comparison of ¹H and ¹³C NMR data with literature values and spectral databases, definitive confirmation via single-crystal X-ray diffraction was pursued, given the historical ambiguity surrounding pinckneyin. The results clarify the chemical nature of pinckneyin and provide a foundation for reassessing the phytochemical basis of the medicinal use of P. bracteata.

Results and Discussion

Isolation of Pinckneyin

Extraction of dried inner bark of P. bracteata following the procedure originally described by Naudain afforded an off-white precipitate after sequential treatment of the ethanolic extract with lead acetate and lead subacetate. The appearance and behavior of this material were consistent with historical descriptions of the substance referred to as pinckneyin. Recrystallization from water yielded a crystalline material suitable for spectroscopic and single-crystal X-ray diffraction analysis.

Structural Elucidation of Compound 1

Initial spectroscopic screening (LC–MS, NMR, and IR) suggested a low-molecular-weight aromatic compound rather than a glycosidic species, contrary to early speculation. Single-crystal X-ray diffraction analysis of compound 1 unambiguously established its identity as 7-hydroxycoumarin (umbelliferone) (Figure ). The observed molecular geometry and crystallographic parameters are consistent with previously reported X-ray diffraction data for this compound. The structure consists of a planar benzopyran-2-one (coumarin) core with a hydroxyl substituent at the C-7 position. Bond lengths and angles fall within expected ranges for substituted coumarins, and the molecular formula (C₉H₆O₃) determined crystallographically is consistent with high-resolution mass spectrometric data. Although the crystal structure of 7-hydroxycoumarin has been reported previously, its identification as the compound historically referred to as pinckneyin has not previously been reported. While NMR and HRMS data strongly supported the structural assignment, single-crystal X-ray diffraction was employed to provide historically definitive confirmation, eliminating any potential ambiguity arising from spectral overlap or substitution pattern interpretation.

1.

ORTEP representation of compound 1 (pinckneyin, 7-hydroxycoumarin) with displacement ellipsoids drawn at the 50% probability level; hydrogen atoms are shown as spheres of arbitrary radius.

The crystal packing reveals intermolecular hydrogen bonding involving the phenolic hydroxyl group, contributing to the observed lattice arrangement (Supporting Information). NMR and IR spectra further corroborate the presence of a coumarin scaffold with characteristic resonances and absorptions corresponding to the conjugated lactone and phenolic functional groups. Taken together, these data conclusively identify pinckneyin as 7-hydroxycoumarin, resolving a structural ambiguity that has persisted since its initial isolation in the late 19th century.

7-Hydroxycoumarin is a well-known natural product that has been reported to exhibit a broad range of biological activities, including anti-inflammatory, antioxidant, antimicrobial, neuroprotective, and antiproliferative effects. A summary of representative pharmacological activities reported for this compound is provided in the Supporting Information (Table S2). Notably, despite extensive study, no prior reports to our knowledge have demonstrated direct antiplasmodial activity for 7-hydroxycoumarin. Historical accounts indicate that preparations of Georgia bark were administered orally as tinctures or powdered infusions. Given the limited oral bioavailability reported for simple coumarins such as 7-hydroxycoumarin, this may partially explain the comparatively slow or inconsistent therapeutic effects described in 19th century clinical observations.

Although direct antiplasmodial activity has not been reported, coumarins are known to exhibit anti-inflammatory, antioxidant, and antimicrobial effects, which could plausibly have contributed to symptomatic relief in febrile illnesses historically attributed to malaria.

It is also conceivable that simple coumarins may have contributed to historical assumptions of similarity to cinchona alkaloids. Coumarins can exhibit bitterness and characteristic fluorescence under certain conditions, properties that may have superficially resembled the sensory attributes of quinine-containing preparations. In the absence of modern analytical techniques, such organoleptic similarities could reasonably have reinforced the early suppositions of chemical equivalence.

Effect of Extraction Conditions on Pinckneyin Isolation

Modification of the historical extraction protocol revealed a pronounced sensitivity of the isolated material to the processing conditions. Specifically, replacement of the original solvent-removal steps involving simple distillation with rotary evaporation consistently failed to produce a crystalline material. Instead, a brown amorphous solid was obtained, which LC-CAD analysis showed to be a complex mixture of at least five components (Supporting Information). These additional components were not individually characterized in the present study as the primary objective was structural clarification of pinckneyin under historically faithful extraction conditions. Comprehensive identification of all minor constituents is beyond the scope of the present study. This observation suggests that the conditions employed during solvent removal and concentration may influence the chemical composition of the isolated material. Speculatively, the differing outcomes may reflect the combined influence of the temperature, solvent removal conditions, and exposure to air on the stability of extract components. Higher temperatures (80–100 °C) during simple distillation may promote thermally induced transformations consistent with processes such as hydrolysis, whereas rotary evaporation (40 °C) under reduced pressure and an ambient atmosphere may permit oxidative or light-sensitive processes, potentially contributing to the observed differences in composition. While further study is required to distinguish between these effects, these results underscore the importance of historical methodology in reproducing legacy phytochemical isolations.

Isolation and Identification of Compound 2

Analysis of the remaining ethanolic filtrate after removal of pinckneyin revealed the presence of additional constituents. Fractionation by column chromatography afforded a second compound (compound 2), which was characterized by NMR and IR spectroscopy as 6-methoxy-7-hydroxycoumarin (scopoletin). The structures of both isolated coumarins are shown in Figure . This compound is another previously identified, well-documented plant-derived coumarin with known biological activity. ,

2.

Chemical structure of compound 1, 7-hydroxycoumarin and compound 2, 6-methoxy-7-hydroxycoumarin.

The co-occurrence of at least two substituted coumarins in P. bracteata suggests that the historical medicinal effects attributed to Georgia bark are unlikely to arise from a single compound alone. Instead, they may reflect the combined or synergistic activity of several phenolic constituents along with other unidentified metabolites present in the bark. The present study was designed to resolve the structural identity of pinckneyin rather than to evaluate its pharmacological efficacy. While in vitro antiplasmodial assays would be of interest, structural identification is independent of biological evaluation. Future work may explore the comparative bioactivity of crude extracts and isolated constituents.

Conclusion

In this study, the compound historically termed pinckneyin was definitively identified as 7-hydroxycoumarin through reproduction of the original extraction procedure from 1885 and comprehensive modern spectroscopic and crystallographic analysis. This result resolves a long-standing ambiguity in the phytochemical literature surrounding P. bracteata and clarifies that the medicinal reputation of Georgia bark was not attributable to quinine or related alkaloids. Although 7-hydroxycoumarin is a well-studied natural product, no prior evidence directly supports the antimalarial activity of this compound. The identification of pinckneyin as 7-hydroxycoumarin, coupled with the absence of demonstrated antiplasmodial activity, suggests that historical therapeutic effects may have arisen from alternative mechanisms, additional constituents, or combined effects within complex extracts. The sensitivity of the isolated material to extraction and processing conditions further highlights the challenges inherent in reproducing legacy phytochemical studies. Together, these findings underscore the value of revisiting historically significant natural products using modern analytical tools and provide a foundation for future investigations into the broader phytochemical composition and bioactivity of P. bracteata.

Materials and Methods

General Experimental Procedures

All chemicals were obtained from commercial sources and used without further purification unless specified. The melting point was measured with a Barnstead Electrothermal Mel-Temp. ¹H NMR and ¹³C NMR were recorded on a Bruker Avance III-HD 400 MHz instrument equipped with a Broadband Prodigy Cryoprobe (400 MHz for ¹H, 100.6 MHz for ¹³C). Chemical shifts were reported in ppm with the solvent resonance and/or TMS as internal standard (¹H NMR DMSO-d ₆ δ = 2.51 ppm, TMS δ = 0.00 ppm; ¹³C NMR DMSO-d ₆ δ = 39.99 ppm). NMR spectra were analyzed with NMRium. IR spectra were acquired using a ThermoFisher Nicolet iS5 infrared spectrometer on neat samples (8 scans from 400 to 4000 cm^–1, automatic averaging, and baseline correction). High-resolution electrospray ionization mass spectrometry (HR-ESI-MS) analyses were performed on a Thermo Scientific DelosProbe mass spectrometer operated in either negative or positive ion mode. Samples were introduced by direct infusion. Accurate mass measurements were obtained under high-resolution conditions, with a measured mass accuracy within 4.7 ppm. LC-CAD analyses were performed using a Thermo Scientific Dionex UltiMate 3000 RSLC system equipped with a Dionex Corona Veo RS charged aerosol detector. Thin-layer chromatography was run on Macherey-Nagel precoated TLC-sheets ALUGRAM Xtra SIL G/UV₂₅₄. Column chromatography was performed using silica gel from Himedia (60–120 mesh).

Plant Material

Branches of P. bracteata were collected from “Big Bertha” at the South Carolina Botanical Gardens in spring 2023. A major limb of the specimen was also collected in Chatham County, GA, in summer 2024. It was identified by Misty Shealy, the nursery manager at the South Carolina Botanical Gardens, based on local knowledge and expertise. Identification was confirmed by comparison with Clemson University Herbarium voucher specimen 2903585 (P. bracteata Raf.; syn. P. pubens Michx.). The bark was whittled from the branches and limbs and dried in an oven set to 50 °C for 7 days.

Extraction and Isolation

116 g of the dried bark was blended in a coffee grinder and crushed with a mortar and pestle, resulting in a fine powder. This fine powder was refluxed with 300 mL of petroleum ether for 4 h, allowed to cool, and vacuum filtered. The resulting residue was spread out on a glass dish and dried in an oven at 100 °C for 4 days, until all petroleum ether was removed. The residue was transferred into a 500 mL round-bottom flask and refluxed with 250 mL of 80% aqueous ethanol for 3 h, vacuum filtered, and returned to the 500 mL flask for further refluxing with 200 mL of 80% aqueous ethanol for 3 h. The mixture was once again vacuum filtered and rinsed with 50 mL of 80% aqueous ethanol. The filtrates were combined and concentrated with simple distillation down to 50 mL of tincture. 40 mL of this deep red tincture was combined with 120 mL of 1 N Lead acetate in 70% ethanol, causing a white precipitate to form. The precipitate was filtered out, and the filtrate was freed from lead with a stream of hydrogen sulfide gas until no further red/black precipitate formed. The precipitate was filtered out, and the filtrate was treated with 50 mL of 1 N Lead Subacetate in 70% ethanol, again yielding a white precipitate. The precipitate was filtered out, and the filtrate was once more freed from lead with a stream of hydrogen sulfide, forming a red/black precipitate. Upon filtration, the filtrate was concentrated with simple distillation to a volume of 100 mL. Upon cooling, a tan precipitate gradually formed, consistent with the substance known as Pinckneyin. This precipitate was filtered out and recrystallized from water to yield 0.243 g of Pinckneyin (compound 1). TLC of the filtrate (50:50 EtOAc/Hexane) showed several compounds still present, most notably a spot at a Rf of 0.62 corresponding to compound 1, and another substance with an Rf of 0.45. The filtrate was further concentrated and dry loaded onto silica gel, and upon column chromatography (50:50 EtOAc/Hexane), concentration of fractions with a rotavap, a further 0.137 g of compound 1 and 0. 101 g of compound 2 were isolated. Unlike in the original paper, our laboratory did not taste any residue to determine bitterness. NOTE: the extraction and isolation procedure was repeated on three independent samples from both collected specimens. Although isolated yields varied (no optimization was performed), the overall chemical profile of the extracts and the formation of pinckneyin were consistent across experiments, indicating reproducible isolation.

X-ray Crystallographic Analysis

Off-white crystals of compound 1 were obtained by recrystallization in water. X-ray intensity data from a platelike single crystal were collected at 304(2) K using a Bruker D8 QUEST diffractometer equipped with a PHOTON-II area detector and an Incoatec microfocus source (Mo Kα radiation, λ = 0.71073 Å). , The structure was solved with SHELXT. Subsequent difference Fourier calculations and full-matrix least-squares refinement against F² were performed with SHELXL-2025/1 by using OLEX2. , The compound crystallizes in the monoclinic system. The pattern of systematic absences in the intensity data was uniquely consistent with space group P2₁/c, which was confirmed by structure solution. The asymmetric unit consists of one molecule. All non-hydrogen atoms were refined with anisotropic displacement parameters. Hydrogen atoms bonded to carbon were located in difference Fourier maps before being placed in geometrically idealized positions and included as riding atoms with d­(C–H) = 0.93 Å and Uiso­(H) = 1.2Ueq­(C). The hydroxy hydrogen atom H3 was located and refined freely. The largest residual electron density peak in the final difference map is 0.16 e^–/Å3, located 0.61 Å from C4. See Supporting Information for further details. Crystallographic data were deposited with the Cambridge Crystallographic Data Centre. The deposition number 2525967 contains the supplementary crystallographic data for 1. This data can be obtained free of charge via http://www.ccdc.cam.ac.uk/data_request/cif Crystallographic data for 1: C₉H₆O₃, M = 162.14 g·mol^–1, monoclinic, space group P2₁/c (No. 14), a = 3.9174(2) Å, b = 11.0106(7) Å, c = 16.6658(10) Å, β = 90.397(2)°, V = 718.83(7) ų, Z = 4, T = 304(2) K, μ = 0.114 mm^–1, D _calc = 1.498 g·cm^–3. A total of 10,330 reflections were measured (θ = 2.217–26.379°). Final refinement gave R ₁ = 0.0468 [I > 2σ­(I)], wR ₂ = 0.1315 (all data), and goodness-of-fit on F ² = 1.034.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.6c01169.

• Characterization of compound 1 and 2; SC-XRD data for pinckneyin; bioactivity summary of compound 1; FTIR Spectra; ¹H and ¹³C NMR spectra; spectral comparison to literature; HRMS spectra; LC-CAD spectrum for amorphous extract (PDF)

• Crystallographic data for 1 (CIF)

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

This project was generously funded by the Office of the Vice President for Research at the University of South Carolina through a Research Initiative for Summer Engagement (RISE) Grant (#961000–23–63469) and a Palmetto Undergraduate Research Experience (PURE) Grant. It was also partially funded by the University of South Carolina Sumter.

The authors declare no competing financial interest.