Extracts of L. tulipifera, A. spinosa, and Q. alba displayed inhibitory activity against bacteria that cause skin and soft tissue infections, substantiating their use as antiseptics during the American Civil War. These medicinal plants may be useful in modern medicine as treatments for antibiotic-resistant bacteria. Of particular interest are 618B and 620 as S. aureus biofilm inhibitors and 619, 619-F2, and 620 as growth inhibitors of carbapenem-resistant Klebsiella pneumoniae.

While a 1947 survey of antibacterial properties of plants found no activity in A. spinosa and L. tulipifera15, the positive results in this experiment may be explained by differences in a number of factors. The previous study used H 2 O extracts whereas this experiment used MeOH extracts15; L. tulipifera bark was historically prepared for treatment by dissolving in EtOH5, which produces an extraction profile similar to MeOH16. Additionally, given the role of endophytic microorganisms in the synthesis of secondary metabolites, the chemical composition of plant extracts can vary based on differences in the plant microbiome17. Other possible sources of variation include collection date and location, assay method, and extract concentration tested. Finally, given the variability in how different laboratories may perform one type of extraction, results can vary between related studies. For example, of two studies that evaluated Aralia nudicaulis root (a traditional Native American remedy ingredient) for growth inhibition of mycobacteria, only one reported moderate antibacterial effects while the other reported little activity18,19.

In his report, Porcher recommended the entire genus Quercus as a source of antiseptics5. This activity is confirmed not only by the results of the experiments reported herein, but also by multiple other studies showing antibiotic effects by Quercus spp. extracts20,21,22,23,24. A European herbal remedy referred to as Quercus cortex (originating from Q. robur, Q. petrea, and Q. pubescens bark) has shown weak antibacterial and quorum sensing inhibition effects25. Acorn extract from a variety of oaks has shown inhibition of both Gram-positive and Gram-negative bacteria26.

However, the activity of various Quercus spp. extracts is far from uniform. For example, the Q. alba gall extract (620) in this study inhibited growth of drug-resistant K. pneumoniae whereas a study of Q. infectoria galls found no significant inhibition of drug-resistant K. pneumoniae24.

Antibacterial activity in oak extracts is frequently attributed to tannins27, compounds that typically interfere with biological processes by binding to proteins28. In Quercus, tannin content is typically highest in galls, with a reported 70% tannin content in Q. infectoria galls27. In this experiment, higher activity in 620 (gall crude extract) over 619 (bark crude extract) and 619-F2 (bark tannin fraction) over 619-F1 (bark non-tannin fraction) suggests that Q. alba’s growth inhibitory activity is due to tannins. However, quorum sensing inhibition by 619-F1 suggests that additional compounds could contribute to the antibacterial activity of crude oak extract, the medicine used during the Civil War.

LC-FTMS analysis of 619-F2 and 620 confirmed the existence of a variety of tannins in both extracts (Supplementary Tables S6 and S7). Of particular interest are ellagitannin isomers, 2, found in 620; as well as related ellagitannins 12a and 12b. Ellagitannins have been reported to have antibiotic activity against antibiotic-resistant S. aureus9. While only three MS peaks were found in common between 619-F2 and 620, both extracts are rich in tannins. 619-F2 is enriched in procyanidin condensed tannins and 620 contains many ellagitannins and triterpenes.

Tannins have been shown to inhibit growth in a wide range of bacteria, fungi, and viruses. Suggested mechanisms of action include inactivation of microbial enzymes, inhibition of membrane transport, and sequestering essential metal ions in complexes28. Tannins may also act as biofilm inhibitors by binding to matrix proteins29. However, tannins have also been found to bind with digestive enzymes and nutrients such as proteins and starches, and as such are generally considered as anti-nutritive; a variety of animals have shown gastrointestinal distress and decreased growth when fed on high-tannin diets28. Because of this nondiscriminatory binding, external applications of Q. alba extracts would be preferable to internal or systemic applications; Porcher recommended that powdered oak bark be applied in a wash for gangrene and a poultice for wounds5.

Leaves of several Quercus species (Q. cerris, Q. ilex, Q. virginiana, Q. incana) have also shown antibacterial properties, including biofilm and quorum sensing inhibition20,22,30. One future research direction could be to compare the antibacterial properties of Q. alba leaves with the activity identified in bark and gall extracts.

While A. spinosa has several reported uses in traditional medicine31, it has not frequently been studied for medicinal properties. The most notable results of this experiment for A. spinosa are significant biofilm inhibition by 618B (leaf hexane partition) and quorum sensing inhibition by 618 C (leaf ethyl acetate partition). The presence of these adjuvant properties rather than simple growth inhibitory activity in A. spinosa leaves may explain the 1947 report of no significant antibiotic activity in A. spinosa15.

Other Aralia species have exhibited antibacterial activity in roots18 and aerial parts (flowers, leaves, and stems)32, including biofilm inhibition by A. cachemirica32. In his list, Porcher also ascribed antiseptic activity to A. racemosa5.

L. tulipifera has been widely studied and its various parts have exhibited a variety of medicinal effects including antibacterial33, anti-malarial34, and anti-cancer35,36 activity. The other species of Liriodendron, L. chinense, is used in Chinese traditional medicine and has been shown to have antibacterial effects37. Additionally, an extract from a hybrid of L. tulipifera and L. chinense has been shown to exhibit inhibition of biofilm production and quorum sensing38.

In the present study, L. tulipifera extracts demonstrated activity in the inhibition of growth, biofilm production, and quorum sensing. However, the root bark extract (617), which is generally more bioactive than the leaf extract (616) and branch bark extract (621) in our models, displayed significant mammalian cytotoxicity (IC 50 : 16 µg/mL). It may therefore be ill-suited for medicinal use, or at least dose-limited. A study of L. tulipifera for antiplasmodial activity also found high cytotoxicity in active fractions but it has been suggested that, given the use of L. tulipifera in traditional medicine, toxicity may not be problematic in vivo at therapeutic doses34. Porcher recommended root bark as the medicinal part of L. tulipifera to be harvested5; perhaps preparation techniques or dosage made the potency/toxicity trade-off worthwhile in a wartime context. Interestingly, Porcher also suggested L. tulipifera bark as a substitute for Cinchona bark in malaria treatment, an application supported by recent research34.

Perhaps the most notable L. tulipifera extract with low toxicity is 616-F1 (leaf non-tannin fraction), which displayed little growth inhibition but significant biofilm and quorum sensing inhibition—an adjuvant effect similar to the A. spinosa extracts tested.

Further study should focus on bioassay-guided fractionation, a recursive process of fractionation and bioassay to identify individual active compounds and synergistic relationships. Of the extracts tested, 616-F1, 618B, 618 C, 619-F2, and 620 exhibit the most promise for antibiotic NCEs and are good candidates for this process. Specifically, the HPLC methods developed for 619-F2 and 620 could be used to produce further fractions with adaptation to preparative liquid chromatography.

In vivo testing of the antibacterial properties of extracts active in vitro is also a logical next step in this research. Given the potential of some of these extracts as adjuvants rather than direct antibiotics, they may be tested as adjuvants with existing, FDA-approved antibiotics for the potentiation of antibacterial activity in wound infections.

Finally, given the activity seen in the extracts tested in this study, it may be worthwhile to investigate the antibacterial properties of other plants recorded as antiseptics in Porcher’s book. In total, 37 plant species were described as having antiseptic applications5. As the global spread of antibiotic-resistant strains of bacteria continues, it is increasingly important to consider all possible sources of new, and perhaps old, treatments.