Evaluation of the Anti- Yersinia Activity of Botanicals Used During the Black Plague

Evaluation of the Anti- Yersinia Activity of Botanicals Used During the Abstract Background: During the fourteenth century, the Black Plague ravaged through Europe. At this time, records described using specific botanical formulations, the “Four Thieves” recipe and Nostradamus’ “Rose Petal Lozenges”, as successful therapies for preventing plague-associated bacterial infections. Purpose: This study describes the characterization of the antibacterial activity associated with botanical extractions prepared from these medieval formulations against Yersinia pseudotuberculosis , a related species of Y. pestis , the causative agent of the Plague. Methods: Ethanol and acetic acid extracts were prepared from16 botanicals that may have been used in masks or formulated into tinctures to protect medieval populations from contracting the Plague. These extracts were tested in a dose-dependent manner related to inhibition of the growth of Y. pseudotuberculosis . Results and Conclusions: The results suggest which botanicals may have been useful in reducing infection during the historically detrimental pandemic of Y. pestis . With a renewed and emerging threat of Yersinia -related infections, the results provide insight into the history of botanical medicine and indicate which botanicals may act as another defensive measure against future Yersinia -related infections.


Background
Across Europe in the early 1300's, the Black Death infection ended the lives of millions of humans during a plethora of epidemics [1,2]. In medieval Europe, fleas were the primary disease vector and the reservoir of the fleas were rats, especially flea-bitten rat [3]. During the 19 th century, Swiss-French physician Alexandre Yersin was studying the disease, hence the name Yersinia pestis [3]. Slightly behind him, in 1894 Japanese physician-bacteriologist Kitasato Shibasaburo was also working with the bacteria [1,3]. Ultimately, it was determined the routes of Y. pestis infection included fleabite, or the presence of Yersinia on a cut, wound, skin lesion, mucosa of the mouth, nose or conjunctival mucus membrane of eye [1,3]. Recent research suggests Y. pestis may be carried by more than 200 species of rodents all over the world (except Australia and Antarctica) and at least 80 species of flea [1]. The disease has two primary forms, bubonic and pneumonic plague. Bubonic occurs typically after the bacteria invades the body following contact with inflected fleas leading to the onset of painful swelling of lymph nodes and lymph glands days to weeks after exposure [3,4]. Pneumonic is the deadliest and most contagious. It affects the lungs with victim developing severe pneumonia-like symptoms including cough, chest pain, difficulty breathing [1,3]. Pneumonic symptoms will present quickly with death occurring as rapidly as 24 hours post onset [1,3].
Metanalyses on the Plague during the 1300's revealed two acclaimed recipes, both very similar [5,6], and another attributed to Nostradamus, that may have been used in a mask or formulated into tinctures to protect the population from contracting the deadly illness [7]. During the epidemic of Y. pestis, tales are told of a facial mask containing herbs being used to house herbs near the Volume  nose to protect people from getting the infection. Some peddlers drafted cure-alls to make money during these epidemics, and others reported actual cures. In the Tale of the Four Thieves, it was said that 4 men repeatedly robbed the homes of the dead, but never contracted the illness themselves. Historical investigation indicates the tale originated in either Marseilles or Toulouse [7] which is corroborated by a sizeable plague outbreak during 1720 in Marseilles [3]. According to their tale, a mother of the thieves was an herbalist that concocted an herbal vinegar recipe. She instructed them to soak a handkerchief in the herbal extract and wear it over their face so they could safely continue robbing houses [7]. The herbal vinegar recipe was used to rub on the face and hands as an insect repellant, to consume orally, and to carry in a small jar to inhale as vapors [3,7]. The recipe became known as the "Four Thieves" recipe. French aromatherapy doctor, Jean Valnet, has two "Four Thieves" recipes in his book [5][6][7]. He claims his original recipe was revealed by corpse robbers who were caught redhanded in the area around Toulouse between 1628 and 1631 [7].
Another treatment purportedly used for the plague, was developed by the prominent French physician, Nostradamus, also known as Michel de Nostredame [8]. He survived the plague outbreak (living from 1503 to 1566) and treated many others, supposedly with his "Rose Petal Lozenges" formulation [7].
The goal of this project was to investigate how the individual botanicals present in these two formulations may inhibit the replication of Yersinia bacteria when extracted in ethanol and vinegar-based tinctures. These extraction solutions were selected in an attempt to make tinctures in a similar fashion as those possibly used historically by medieval apothecaries. The genus Yersinia includes 11 species, three of which are human pathogens: Y. pestis, Y. enterocolitica, and Y. pseudotuberculosis [4]. Y. pestis evolved from parent strain Y. pseudotuberculosis [3], both of which are gram-negative bacteria [1]. Since Y. pestis is classified as a Biosafety Level 3 agent, preliminary experiments performed this study utilized Y. pseudotuberculosis as a model organism to investigate the antibacterial properties of the botanicals described in the "Four Thieves" and "Rose Petal Lozenge" formulations. Our results support the antibacterial properties of several of these botanicals against Y. pseudotuberculosis and supports the need for future studies to investigate the antibacterial activity against Y. pestis to support the efficacy of these botanical blends in inhibiting Y. pestis infections during the 1300s.

Bacteria source
The bacterial culture of Y. pseudotuberculosis (ATCC 6904), strain NCTC 2476, was obtained from American Type Culture Collection (Manassas, VA).

Botanical extract preparation
Plant material obtained from Starwest Botanicals and AmeriHerb was authenticated by HPLC profiling by the supplier with documentation of authenticity. All plant material was subsequently verified by qualified botanical specialists using herbal pharmacopoeia monographs and reference keys. A voucher specimen of all plant material was deposited in a repository/ herbarium at the SCNM Botanical Institute. The botanical formulations, including the scientific and common names, of the botanicals used in the "Four Thieves" and Nostradamus's "Rose Petal Lozenge" formulations are shown in Tables  For extract preparation, the dried botanicals were ground to a fine powder, resuspended in extraction solution (1:6 wt:vol) and incubated at room temperature with constant mixing for 48 hours. The botanicals were extracted in 2.5% acetic acid (to simulate a historical vinegar solution) or 50% ethanol. The extract was filtered through 3 layers of netting cloth over a beaker, the collected liquid centrifuged for 3,000xg for 10 minutes and then 20,000xg for 20 minutes to remove plant debris. The supernatant was sterilized by filtration through a 0.2 µM filter. To standardize the botanical treatment doses, a sample of each extract was dried by rotary evaporation to determine the concentration (mg/ml) of the non-volatile constituents present/ml.

Bacterial growth inhibitory assay:
To calculate the minimum bactericidal concentration (MBC) with >99.9% killing, 18-hour cultures grown at 37 o C in Tryptic Soy Broth (TSB) (ranging from 1 to 5 x 10⁸ colony-forming units/ ml (CFU/mL)) were diluted into TSB (1:1000 dilution) followed by the addition of the indicated concentrations of each botanical extract or control. Controls included TSB with only the botanical extract or vehicle (equivalent doses of 2.5% acetic acid or 50% ethanol), TSB with only the bacterial inoculum, or TSB with the bacterial inoculum and the vehicle (equivalent doses of 2.5% acetic acid or 50% ethanol). The cultures were incubated at 37 o C with aeration by continuous rotation for 24 hours. After 24 hours, each sample was then titrated by serial dilution. MBC endpoints were calculated as the lowest dilution of botanical extract with >99.9% killing (> 3-logs) after overnight incubation at 37 °C

Results
Based on historical literature, two botanical recipes are most commonly discussed as being used during the Black Plague. Although the exact recipes of these botanical recipes are not known, the most consistent formulations are shown in Table 1A for the "Four Thieves" blends and the Nostradamus "Rose Petal Lozenges" shown in Table 1B. Much of the literature suggests these herbs may have been used in a solid form or extracted with a solvent [3,7]. For our assays, the herbs were extracted in acetic acid (to represent the historical use of vinegar) or ethanol (to represent historical alcohol extractions).
A:  As a control and to confirm that the ethanol and acetic acid vehicles in the extracts did not have any anti-Yersinia effects at the concentrations tested, a control experiment was done at the equivalent volumes for the mg/ml concentrations of the botanical extracts, but solely containing starting solutions of the 50% ethanol or 2.5% acetic acid. As compared to the untreated sample (bacteria only), both the ethanol and acetic acid vehicle solutions did not lead to any significant decrease in bacterial titers ( Figure 1). Upon testing the anti-Yersinia activity of the various botanicals described in these historical blends, several were found to be efficacious. However, the method of extraction, whether prepared with ethanol or acetic acid, did seem to influence the antibacterial activity. As shown in Figure 2 in either acetic acid or ethanol where over a 7-log reduction was observed with the acetic acid extraction and a 3-log reduction with the ethanol extract ( Figure 2). Comparatively, F. ulmaria was more effective when extracted with ethanol showing a MBC at 90 mg/ ml and a 5-log reduction at 110 mg/mL compared to a 2.5-log reduction at 110 mg/mL of the acetic acid extract. The conserved activity of these botanical extracts when prepared in acetic acid or ethanol may suggest the presence of different active constituents or active constituents which are soluble in both extraction solutions. Alternatively, two botanical extractions were found to have anti-Yersinia activity only when extracted in acetic acid. These botanicals included Allium sativum and Angelica pubescens ( Figure 3). As shown in Figure 3, A. sativum extracted in acetic acid demonstrated a dose dependent inhibition in Yersinia growth with a MBC at 70 mg/mL and over a 7-log reduction at the highest dose tested (110 mg/mL). When A. sativum was extracted in ethanol, no reduction in bacterial growth was observed (Figure 3). Similarly, A. pubescens extracted in acetic acid gave a MBC of <70 mg/mL with a reduction in Yersinia growth by over 4-logs, while the ethanol extract had no antimicrobial activity at all doses tested ( Figure 3). These results suggest that an active anti-Yersinia constituent was present in both A. sativum and A. pubescens, but this compound was soluble only in acetic acid, and not ethanol.  Of the sixteen botanicals evaluated, I. helenium, J. communis, C. rapunculoides, A. calamus, R. canina, and A. agallocha were found to have no substantial anti-Yersinia activity when extracted in either ethanol or acetic acid ( Figure 5). Historically, these botanicals may have been present in the formulations described not to provide antibacterial activity, but potentially for other roles.

Discussion
Y. pestis outbreaks have occurred within the human population for hundreds of years. Recently, in 1994, the Indian cities of Surat (Gujarat) and Beed (Maharashtra) had 150 pneumonic cases and 600 bubonic cases from which 50-60 people died [3]. Between November to December of 2014, a plague outbreak occurred in Madagascar leading to 60 deaths [3]. In July of 2014, the City of Yemen in the province of Gansu in China reported one human death from Y. pestis following contact with an infected marmot [3] and there are typically human and animal epidemics each year in China [2,4]. According to the CDC, there are still up to 17 confirmed cases of bubonic plague in the US each year, and as recently as 2015, as many as 4 deaths [9]. Worldwide as recently as 2015 there were 783 cases of plague causing 126 deaths [3]. The current standard of treatment for this disease is a broad antibiotic regime which can decrease chances of death from 50-90% to 10-20% [1,3]. Some antibiotics used in this regimen include streptomycin, gentamicin, tetracycline, doxycycline and oxytetracycline, however Y. pestis is becoming increasingly antibiotic resistant [1,3].
Historical claims report that grave robbers, Nostradamus and several medieval physicians survived the Black Death utilizing "Rose Petal Lozenges" and the "Four Thieves" blends [3,8].
Identifying the potential anti-Yersinia activity of the botanicals present in these formulations may validate the historical efficacy of these blends and may provide an alternative treatment or prophylaxis to those currently living in high risk regions. Due to Y. pestis being classified as a Biosafety Level 3 agent, these initial studies had to be performed using the model organism Y. pseudotuberculosis. From this study, of the 16 botanicals examined, 10 were found to have potential antibacterial activity against Y. pseudotuberculosis, with several having very strong activity. These results support the need for future studies to investigate the antibacterial activity against Y. pestis to validate the efficacy of these botanical blends in inhibiting Y. pestis infections during the 1300s. To date, there has been limited studies on the effectiveness of these botanicals on Yersinia and how these botanicals may have influenced and saved the lives of people during the Black Plague. As described, two extraction solutions were tested to simulate what would be most commonly available in medieval apothecary formulations. For the inefficacious botanicals, these herbs may have had other roles in the historical formulation, since it has been proposed that these herbs also may have acted as insect repellants. Additionally, the mode of action of the inefficacious botanicals could have been to act in a supportive role on the immune system rather than direct inhibition of the pathogen.
Based on historical use, botanicals can be categorized by their route of administration including oral, inhaled, topical or a combination of the three routes. This is vital to the use of our botanical extractions because of disease transmission of Yersinia pestis via fleabite to the skin or contact with the respiratory system via aerosol inhalation [1]. Historical accounts mention the use of plants from these botanical blends applied topically, orally and via Volume  inhalation [3,10,11].
In our current study, the botanicals that demonstrated activity against Yersinia were separated by the extraction solution in which they performed best. These anti-Y. pseudotuberculosis assays were performed using crude botanical extracts, so the quantitative values are based on the concentration of non-volatile components in the extracts (mg/mL) and not the actual concentration of the active constituent(s). This should be noted due to the likelihood that the active anti-Y. pseudotuberculosis constituents are likely different compounds and/or at varying concentrations between the different extracts. Since many of the botanicals described in these historical formulations had different activity based on the extraction method, the results suggest that several of these extracts likely have structurally different and unique active anti-Yersinia constituents. When combined into the historical formulations described, these different constituents likely would produce positive synergistic effects. For the results shown, the "Four Thieves" blend of botanicals appeared to have the most potent botanicals and the broadest activity based on extraction methods. Based on this, it may be predicted that the "Four Thieves" blend was more potent related to antibacterial activity and sheds some light on how these thieves may have survived plague ridden houses of the dead.

Conclusions
The goal of this study was to investigate the potential antibacterial activity of ancient botanical formulations, the "Four Thieves" and "Rose Petal Lozenge" formulations, reportedly used during the 1300's for the prevention of the Black Plague. Our results, using a related and model bacterium, Yersinia pseudotuberculosis, suggested that the "Four Thieves" blend especially had very potent antibacterial activity and may shed some historical validation on how thieves using this formulation may have survived being exposed to the bacteria in plague ridden houses. Life threatening infections with Yersinia pestis are still prevalent worldwide, especially in China [1,12]. Some of the antibiotics used against Y. pestis are becoming ineffective due to resistance [1]. In this study, various botanical extracts were found to inhibit the growth of Y. pseudotuberculosis, a related species to Y. pestis [1,3,4]. Based on our results, future studies are warranted to test the antibacterial activity against Y. pestis where these results could support a potential new source of antibacterial therapeutics for the treatment of Y. pestis infections. These therapies could be beneficial as crude botanical extracts or offer a source for the isolation of novel pharmaceuticals. In a recent issue of Naturopathic Doctor News & Review, the World Health Organization's definition of Anti-Microbial Resistance was discussed, calling to action for naturopathic medicine to utilize tools against the next pandemic where resources are scarce and the threat to the population is legitimate [13]. Utilizing botanical medicine against Yersinia may offer a legitimate way to move forward toward reaching the goals of the Global Action Plan on Antimicrobial Resistance that the World Health Organization proposes [13].