Antibacterial compounds and methods of isolating from thuja arborvitae

Abstract

The subject matter described herein is directed to antibacterial compounds and methods of isolating the compounds from extracts of Thuja arborvitae.

Description

[0001] Atty. Dkt: 015444 / 638160

[0002] ANTIBACTERIAL COMPOUNDS AND METHODS OF ISOLATING FROM THUJA ARBORVITAE

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] [1] This application claims the benefit of priority to United States Provisional Patent Application No. 63 / 721,840, filed on November 18, 2024, the content of which is incorporated by reference herein in its entirety for all purposes.

[0005] STATEMENT OF GOVERNMENT SUPPORT

[0006] [2] This invention was made with government support under Grant No. P031K190001 awarded by the U. S. Department of Education. The government has certain rights in the invention.

[0007] FIELD

[0008] [3] The subject matter described herein is directed to antibacterial compounds and methods of isolating the compounds from extracts of Thuja arborvitae.

[0009] BACKGROUND

[0010] [4] Bacterial infections are among the leading causes of health problems and have a large impact on public health (Ferri, M., et al., Antimicrobial resistance: A global emerging threat to public health systems. Crit Rev Food Sci Nutr. 2017. 57(13): p. 2857-2876). Antibacterial agents are considered the most promising chemotherapeutic agents that have been used to cure infectious diseases. By killing or reducing the metabolic activity of bacteria, their pathogenic effect in biological environments will be minimized. Today, the use of biomaterials with antibacterial effects in medical treatment is rapidly progressing.

[0011] [5] Antibacterial agents can be classified based on the types of action: bactericidal and bacteriostatic (Chen, L., S. Kumar, and H. Wu, A review of current antibiotic resistance and promising antibiotics with novel modes of action to combat antibiotic resistance. Arch Microbiol, 2023. 205(11): p. 356). Bactericidal action destroys bacteria by targeting the cell wall or cell membrane of the bacteria. Bacteriostatic action slows down or inhibits the growth of bacteria. Antibacterial agents can also be classified based on how a drug works or its mode of action. Because the major processes or functions 1

[0012] LEGAL02 / 47622964vl Atty. Dkt: 015444 / 638160

[0013] responsible for bacterial growth, for example, are cell wall synthesis, cell membrane function, protein synthesis, nucleic acid synthesis, antibacterial agents interfering or disturbing such processes in different ways can be subdivided into four groups: cell wall synthesis inhibitors (Schafer, A. B., et al., Dissecting antibiotic effects on the cell envelope using bacterial cytological profding: a phenotypic analysis starter kit. Microbiol Spectr, 2024: p. e0327523; Gupta, R., M. Singh, and R. Pathania, Chemical genetic approaches for the discovery of bacterial cell wall inhibitors. RSC Med Chem, 2023. 14(11): p. 2125-2154), inhibitors of membrane function (Schafer (2024)), inhibitors of protein synthesis (Vazquez-Laslop, N. and A. S. Mankin, Context-Specific Action of Ribosomal Antibiotics. Annu Rev Microbiol, 2018. 72: p. 185-207), and inhibitors of nucleic acid synthesis (Kirsch. S. H.. F. P. J. Haeckl, and R. Muller, Beyond the approved: target sites and inhibitors of bacterial RNA polymerase from bacteria and fungi. Nat Prod Rep, 2022. 39(6): p. 1226-1263).

[0014] [6] Although many antibacterial agents have been discovered and clinically used, diseases caused by bacterial pathogens are still challenging in public health due to the emergence of multidrug-resistant bacteria (Sofianos, G., A. Samaras, and G.

[0015] Karaoglanidis, Multiple and multidrug resistance in Botrytis cinerea: molecular mechanisms of MLR / MDR strains in Greece and effects of co-existence of different resistance mechanisms on fungicide sensitivity. Front Plant Sci. 2023. 14: p. 1273193; Wang, Z., et al., Integrated Multiomic Analysis Reveals the High-Fat Diet Induced Activation of the MAPK Signaling and Inflammation Associated Metabolic Cascades via Histone Modification in Adipose Tissues. Front Genet, 2021. 12: p. 650863; Van Boeckel, T. P., et al., Global trends in antimicrobial resistance in animals in low- and middle-income countries. Science. 2019. 365(6459); Holmes. A. H.. et al., Understanding the mechanisms and drivers of antimicrobial resistance. Lancet, 2016. 387(10014): p. 176-87; Antimicrobial Resistance, C., Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. Lancet, 2022. 399(10325): p. 629-655; Aslam, B., et al., Antibiotic resistance: a rundown of a global crisis. Infect Drug Resist, 2018. 11: p. 1645-1658). To overcome multidrug-resistant bacteria, it is important to develop novel and more effective antibacterial agents, especially with a different mechanism of action (Munita, J. M. and C. A. Arias, Mechanisms of Antibiotic Resistance. Microbiol Spectr, 2016. 4(2)). An enormous increase in the number and ty pes (e.g., structurally different and agents with a slightly different pattern of activity) of new antibacterial agents has 2

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[0017] been observed (Jacobs, L. M. C., P. Consol, and Y. Chen, Drug Discovery in the Field of beta-Lactams: An Academic Perspective. Antibiotics (Basel). 2024. 13(1)).

[0018] [7] Although researchers have primarily used synthetic compounds, natural materials could be a source of navel antibacterial agents. What is therefore needed and not addressed in the art are natural sources and methodologies that can produce new and useful antibacterial compounds. The present disclosure addresses these shortcomings in the art.

[0019] BRIEF SUMMARY

[0020] [8] In certain embodiments, the subject matter described herein is directed to an antibacterial composition comprising,

[0021] a purified extract from Thuja arborvitae leaves, wherein the extract comprises at least 20% w / w of the compound 1:

[0022]

[0023] [9] In certain embodiments, the subject matter described herein is directed to an antibacterial composition comprising from about 40 to about 50% w / w compound 1; about 15% to about 25% w / w compound 2; and about 30% to about 40% w / w compound 3, wherein the sum of the compounds is not more than 100%.

[0024]

[0010] In certain embodiments, the subject matter described herein is directed to an antibacterial composition comprising about 46% w / w compound 1.

[0025]

[0011] In certain embodiments, the subject matter described herein is directed to an antibacterial composition comprising,

[0026] 3

[0027] LEGAL02 / 47622964vl Atty. Dkt: 015444 / 638160

[0028] a purified extract from Thuja arborvitae leaves, wherein the extract comprises at least 20% w / w of the compound 3:

[0029]

[0030] HO

[0031]

[0012] In certain embodiments, the subject matter described herein is directed to an antibacterial composition comprising from about 30% to about 40% w / w compound 3.

[0032]

[0013] In certain embodiments, the subject matter described herein is directed to an antibacterial composition comprising about 33% w / w compound 3.

[0033]

[0014] In certain embodiments, the antibacterial composition further comprises a pharmaceutically acceptable excipient.

[0034]

[0015] In certain embodiments, the antibacterial composition comprising predominantly compound 1 further comprises one or more compounds selected from the group consisting of compounds 2, 3, 4 and 5; and the antibacterial composition comprising predominantly compound 3 further comprises one or more compounds selected from the group consisting of compounds 2, 1, 4 and 5:

[0035]

[0036] 4

[0037] LEGAL02 / 47622964vl Atty. Dkt: 015444 / 638160

[0038] 2

[0039]

[0040] wherein, the total amount present of compounds 2, 3, 4 and 5 combined is less than 80% w / w.

[0041]

[0016] In certain embodiments, the subject matter described herein is directed to an antibacterial composition comprising compound 1, 2 or 3, and combinations thereof, and a pharmaceutically acceptable excipient.

[0042]

[0017] In certain embodiments, the subject matter described herein is directed to a 5

[0043] LEGAL02 / 47622964vl Atty. Dkt: 015444 / 638160

[0044] method of treating a bacterial infection in a subject comprising, administering an antibacterial composition described above to the subject in need thereof.

[0045]

[0018] In certain embodiments, the subject matter described herein is directed to a method of reducing bacterial growth, comprising, contacting a bacterium with an antibacterial composition described above, wherein the growth of the bacterium is reduced.

[0046]

[0019] In certain embodiments, the subject matter described herein is directed to a method of isolating an antibacterial compound from Thuja arborvitae leaves, comprising:

[0047] contacting the leaves with a first solvent to produce a liquid extract; and, subjecting the liquid extract to column chromatography and eluting fractions to prepare a liquid eluent fraction enriched in at least one compound selected from the group consisting of:

[0048]

[0049] 6

[0050] LEGAL02 / 47622964vl Atty. Dkt: 015444 / 638160

[0051]

[0052] HO

[0053]

[0020] In certain embodiments, the subject matter described herein is directed to the above methods wherein the mobile phase is a MeOH / EtOAc mixture as described herein.

[0054]

[0021] In certain embodiments, the subject matter described herein is directed to an isolate prepared by the methods described herein.

[0055]

[0022] In certain embodiments, the subject matter described herein is directed to an isolate from Thuja arborvitae leaves comprising compound 1, having the structure:

[0056]

[0057] wherein, compound 1 is present in an amount of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% w / w of the isolate.

[0058] 7

[0059] LEGAL02 / 47622964vl Atty. Dkt: 015444 / 638160

[0060]

[0023] In certain embodiments, the subject matter described herein is directed to an isolate from Thuja arborvitae leaves comprising compound 3, having the structure:

[0061]

[0062] wherein, compound 3 is present in an amount of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% w / w of the isolate.

[0063]

[0024] In certain embodiments, the subject matter described herein is directed to an isolate from Thuja arborvitae leaves comprising compounds 1 and 3, and optionally compound 2, wherein compounds 1 and 3 combined are present in an amount of at least 40%, 50% 60%, 70%, 80%, 90% or 95% w / w of the isolate.

[0064]

[0025] Other embodiments are also described.

[0065] BRIEF DESCRIPTION OF THE FIGURES

[0066]

[0026] Figures 1 A-C depict data from a Zone of inhibition assay to detect the antibacterial activity. Zone of inhibition assay was performed with plant extracts against S. aureus (ATCC 49775) (A) and / *, aeruginosa,' (B) 1. ampicillin (5 pg); 2, chloramphenicol (1.25 pg); 3, AT grandiflora extract (5 mg); 4, T. radicans extract (5 mg); 5, G. biloba extract, (5 mg) 6, T. arborvitae extract (5 mg); and 7, F. vulgare extract (5 mg); (C) Zone of inhibition assay was performed with eicosapentaenoic acid (EP A) (0.5 mg) and Oleic acid (SA) (0.5 mg) against S. aureus (ATCC 49775).

[0067]

[0027] Figures 2A-B depict the effects of T. arborvitae (A) and T. Radicans (B) extracts on bacterial growth. Growth curves w ere determined by culturing bacteria for 18 h in the presence of various concentrations of plant extracts. The y-axis show s the turbidity' of the bacterial culture measured by spectrometry. OD600: absorbance at 600 nm. The x-axis shows the concentration of the extract. The values are plotted as mean + / - SD obtained from the experiment performed in triplicate. The following abbreviations are used in this 8

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[0069] figure and other figures: SA-1 (ATCC 49775) and SA-2 (ATCC 12600): S. aureus: PA P. aeruginosa: (ATCC 10145); AB: A. baumannii (ATCC 19606); SA: S. mutans (ATCC 25175). In each of (a) and (B), the lines can be identified at 1000 pg / ml from top to bottom: PA, SM, SA-1, AB and SA-2.

[0070]

[0028] Figure 3 depicts data showing the antibacterial effects of T. arborvitae extract on the colony-forming ability. The y-axis shows the colony numbers of the bacteria A aureus (ATCC 49775). The x-axis shows the concentrations of T. arborvitae extract. Methanol was used as the solvent to dissolve the extract. Data is represented as a mean + / - SD of three independent tests. * Significantly different (p<0.05) compared to the methanol control.

[0071]

[0029] Figure 4 depicts an exemplary scheme described herein to purify antibacterial compounds from the T. arborvitae extract.

[0072]

[0030] Figures 5A-B depict the identification of the compound in P3 as Apigenin-di-p-coumarylglucoside (Compound 3). (A) MS / MS spectra of P3; (B) Partial MS2 fragments interpretation.

[0073]

[0031] Figure 6 is the total ion chromatogram (TIC) identified P2 (Compound 1) as an isomer of P3 (Compound 3). Peaks identified from the recorded mass spectra included P3 and an isomer of P3 as indicated.

[0074]

[0032] Figure 7A-C depict P4 contained abietic acid (Compound 5) and eicosapentaenoic acid (Compound 4). (A) MS / MS spectra of P4; (B) and (C) Partial MS2 fragments interpretation.

[0075]

[0033] Figures 8A-B depict the effects of the purified P2 containing Compound 1 (A) and P4 containing Compound 4 (B) on bacterial growth. Growth curves were determined by culturing bacteria for 18 h in the presence of various concentrations of extracts. The y-axis shows the turbidity of the bacterial culture measured by spectrometry. OD600: absorbance at 600 nm. The values are plotted as mean + / - SD obtained from experiments performed in triplicate. In each of (A) and (B), the lines can be identified at 0.05 mg / ml from top to bottom: PA, SM. and overlapping SA-l / AB / SA-2.

[0076]

[0034] Figures 9A-B depict the antibacterial effects of purified P2 and P4 on bacterial viability. A: Fluorescence microscopic analysis was performed using the bacteria A aureus incubated in P3 or P4 for 1 h and stained with SYTO 9 (green: viable cells); and propidium iodide (red; dead cells). B: Fluorophotometric measurement was performed using the bacterial A. aureus treated with several concentrations of P3 or P4 for 18 h. The 9

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[0078] y-axis shows the ratio of green / red fluorescence as indicated by the intensity at the wavelength of 530 nm divided by that of 630 nm, and the horizontal axis shows the concentration of P3 or P4 compound. Data represents mean + / - SD of three independent tests. * Significantly different (p<0.05) compared to untreated control.

[0079]

[0035] Figures 10A-D depict the effects of purified P3 and P4 on intracellular nucleic acid and protein leakage in S. aureus. The y-axis shows the absorbance at 260 nm (OD260) (A) or 280 nm (OD280); (B) of the supernatant of the bacterial culture in the presence of methanol (control), P3 (10 pg / ml), and ampicillin (100 pg / ml) dissolved in methanol. The x-axis represents the incubation time. The values are plotted as mean + / -SD obtained from experiments performed in triplicate; (C) DNA analysis by agarose gel electrophoresis of the supernatant of the bacterial culture in the presence of methanol and P4 (20 pg / ml ) in the methanol with ethidium bromide staining. Ten micrograms of DNA samples were loaded on each lane. Lane 1, the 1 kb plus DNA marker (Promega). The gel was stained with ethidium bromide (0.5 pg / ml); (D) Protein analysis by DSD-polyacrylamide gel electrophoresis (SDS-PAGE) of the bacterial culture in the presence of methanol or P4 in the methanol (20 pg / ml). Ten microliters of each sample were loaded into each lane. Lane 1 shows standard protein markers (Bio-Rad). The gel was stained with Coomassie Blue R250.

[0080]

[0036] Figures 11A-B depict effects of P3 on protein and DNA synthesis. A) Photographic luciferase assays. In vitro translation assay with E. Coli S30 extracts in the presence of methanol, chloramphenicol (CAM), and P3 at the concentrations of lx MIC (10 pg / ml) and 2x MIC (20 pg / ml). Fifty microliters of the luciferase reaction were added into each well of a white 96-well plate and 50 pl of Luciferase Assay Reagent was added into each well. The image was taken by a digital camera with a 6-minute exposure. B) Immunochemical detection of BrdU in DNA by dot blot. DNA from cultured bacteria grown in the absence of BrdU and in the presence of BrdU plus methanol, chloramphenicol (CAM), or P3 at concentrations of lx MIC (10 pg / ml) and 2x MIC (20 pg / ml). The DNA sample (1 pg) was spotted onto a Zeta probe hybridization membrane and the membrane was probed with an anti -BrdU antibody and peroxidase-conjugated secondary’ antibody.

[0081]

[0037] Figures 12A-C depict cytotoxicity of P2 and P4 in human prostate cancer PC3 and primary’ keratinocytes. Methanol yvas used as the solvent to dissolve compounds and as the control. Cells were monitored with Incocyte for about three days. (A) P2 and P4 did 10

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[0083] not affect growth of PC3 cells. (B) P2 at 1 x MIC inhibited growth of keratinocytes. (C) P2 at 1 x MIC did not lead to death of keratinocytes. Human keratinocytes were treated with methanol (top) or P2 at the concentrations of 1 x MIC (middle) or 2 x MIC (bottom).

[0084]

[0038] Figure 13 depicts data for MIC of Thuja crude extract against several strains of bacteria. SA+ OLD (bacteria strain of Staphylococcus Aureus); SA+ ATTC (Staphylococcus Aureus obtained from American Type Culture Collection (ATCC)); AB-Acinetobacter baumannii; PA- Pseudomonas aeruginosa; SM- Streptococcus mutans. The lines can be identified at 0.05 from top to bottom: PA, SA+ATCC, SM, SA+OLD, and AB.

[0085]

[0039] Figure 14 depicts data for MIC of P2 extract against several strains of bacteria. SA+ OLD (bacteria strain of Staphylococcus Aureus); SA+ ATTC (Staphylococcus Aureus obtained from American Type Culture Collection (ATCC)); AB- Acinetobacter baumannii; PA- Pseudomonas aeruginosa; SM- Streptococcus mutans. The lines can be identified at 0.05 from top to bottom: PA, SM, overlapping SA-1 / SA-2 / AB.

[0086] DETAILED DESCRIPTION

[0087]

[0040] The emergence of new diseases resistant to current antibiotics has been one of the challenging problems healthcare providers encounter. Described herein are the isolation and identification of five antibacterial compounds from the Thuja arborvitae leaf extract, and their antibacterial activities. Antibacterial activity was evaluated against four common gram-positive and gram-negative bacteria. Each of the isolated compounds shows a potent inhibitory' effect on the tested bacterial strains.

[0088]

[0041] While plants are a key source for novel antibacterial agents that help the management of bacterial infectious diseases (Clardy, J. and C. Walsh, Lessons from natural molecules. Nature, 2004. 432(7019): p. 829-37; Abdallah, E. M., et al., Back to Nature: Medicinal Plants as Promising Sources for Antibacterial Drugs in the PostAntibiotic Era. Plants (Basel), 2023. 12; Ruggieri, F., et al., Antibiotics with novel mode of action as new weapons to fight antimicrobial resistance. Eur J Med Chem, 2023. 256: p. 115413) and may hold the promise of fewer side effects than synthetic antibiotics (Geng, J., et al.. Accumulation and risk assessment of antibiotics in edible plants grown in contaminated farmlands: A review. Sci Total Environ, 2022. 853: p. 158616), these agents are naturally present in an extremely complex milieu. The agents have to be identified and isolated before they can even be tested. Often, the compounds are present 11

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[0090] in complex extracts that have not been analyzed to determine the agents that provide the desired effects. Also, while the collection of plant samples from certain habitats with high species diversity can be very useful for the potential identification of novel chemical compounds (Vuorelaa, P., et al., Natural products in the process of finding new drug candidates. Curr Med Chem, 2004. 11(11): p. 1375-89), it cannot be predicted whether any such compounds will be able to be purified sufficiently or have the desired activity sufficient to be a useful therapeutic.

[0091]

[0042] As described herein, extracts of numerous plants were used to screen for antibacterial activity. Only two were found to exhibit antibacterial activity as shown in Table 1. Using these data, five chemical compounds were purified and identified from Thuja arborvitae. The spectrum and action of these antibacterial compounds are described herein.

[0092]

[0043] Thuja arborvitae are evergreen trees widely grown as ornamental trees in North America and East Asia. Thuja leave extracts have been used to treat bacterial infection. Described herein is the identification and isolation of antibiotic compounds from Thuja leave extract. The methanol extract of Thuja leaves exhibited strong antibacterial activity on gram-positive (Staphylococcus aureus and Streptococcus mutans) and gram-negative (Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa) bacteria. The five antibiotic compounds were purified and identified as isomers of apigenin-7-di-p-coumarylglucoside, abietic acid, and eicosapentanoic acid. These five compounds all showed potent antibacterial activity against the five tested microorganisms with IC50 from 0.3 to 0.6 pg / ml. More importantly, one of these compounds, compound 1 having the structure:

[0093]

[0094] showed potent inhibitory activity against the multiple-drug-resistant bacterial strain Acinetobacter baumannii. These five antibiotic compounds were described for the first

[0095] 12

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[0097] time in Thuja arborvitae and have the potential to function as antibiotics for the treatment of infections.

[0098]

[0044] The oil extract of Thuja leaves has been used to prevent and treat infectious diseases (Fu, C., et al., Research on the optimization, key chemical constituents and antibacterial activity of the essential oil extraction process of Thuja koraiensis, Nakai. J Microbiol Methods, 2022. 194: p. 106435). Antibacterial activity was detected in the crude Thuja extract (Thakur, M., et al., Comparative analysis of the antibacterial efficacy and bioactive components of Thuja occidentalis obtained from four different geographical sites. Mol Cell Biochem, 2023; Bakht, J., et al., Antibacterial activity of the crude extracts from medicinally important Thuja occidentalis. Pak J Pharm Sci, 2020. 33(2): p. 627-630; Wang, M., et al.. Antibacterial sesquiterpenes from the stems and roots of Thuja sutchuenensis. Bioorg Chem, 2020. 96: p. 103645). However, the chemical compounds that account for the observed antibacterial activity were not isolated and identified to identify which compounds provide the activity.

[0099]

[0045] Of the five chemical compounds isolated, identified and purified from Thuja leaves, two of the compounds are abietic acid and eicosapentaenoic acid, whose antibacterial activity has been reported (Baglyas, M., et al., Antimicrobial Diterpenes from Rough Goldenrod (Solidago rugosa Mill.). Molecules, 2023. 28(9); Wei, M., et al., Antimicrobial and antibiofilm effects of essential fatty acids against clinically isolated vancomycin-resistant Enterococcus faecium. Front Cell Infect Microbiol. 2023. 13: p. 1266674). The other three compounds are isomers of apigenin-di-P-coumarylglucoside, whose antibacterial activity has not been reported. These five chemical compounds show strong antibacterial activity against the three common bacterial strains tested but no effect on the bacterial strain P. aeruginosa. However, the crude Thuja leave extract showed inhibition of this bacteria at the higher concentration, indicating that additional compound(s) may be present.

[0100]

[0046] Advantageously, these compounds can inhibit the growth of the bacterial strain A. baumannii with multiple drug-resistant phenotypes. A. baumannii exhibits a multifaceted antibiotic resistance profile, often dubbed a superbug. This includes resistance to a broad spectrum of antibiotics, facilitated by enzymatic production, biofilm formation on surfaces, genetic elements enabling horizontal gene transfer, and efflux pumps actively expelling antibiotics from the bacterial cell. A. baumannii showcases a broad spectrum of resistance, extending to several classes of antibiotics. Carbapenems, once considered 13

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[0102] reliable for treating bacterial infections, are frequently ineffective against A. baumannii due to the emergence of carbapenem-resistant strains. Resistance is also encountered in aminoglycosides, fluoroquinolones, and various beta-lactam antibiotics. This extensive antibiotic resistance in A. baumannii poses a significant clinical challenge, necessitating a refined and tailored approach in the selection of antimicrobial agents for effective treatment.

[0103]

[0047] The cytoplasmic membrane serves as a selective barrier and controls the cell's internal composition. Whenever these functional roles of the cytoplasmic membrane get disturbed, macromolecules and ions will outflow, which will result in cell destruction or death. Polymyxins are active antibacterial agents, which are cyclic peptides, having a long hydrophobic tail (Nang, S. C.. et al., Rescuing the Last-Line Polymyxins:

[0104] Achievements and Challenges. Pharmacol Rev, 2021. 73(2): p. 679-728; Mohapatra, S. S., S. K. Dwibedy, and I. Padhy, Polymyxins, the last-resort antibiotics: Mode of action, resistance emergence, and potential solutions. J Biosci, 2021. 46(3)).

[0105]

[0048] Polymyxins show their specificity for polysaccharide molecules, which are present in the outer membrane of many Gram-negative bacteria. After association with the lipopolysaccharide substrate in the outer membrane of Gram-negative bacteria, polymyxins change the membrane structure so that its permeability7increases, which results in disruption of the osmotic balance. Since Gram-positive bacteria have a thick cell wall, which prohibits the access of these molecules to the Gram-positive bacterial cell membrane, polymyxins have less or even no effect on Gram-positives. We found that abietic acid and eicosapentaenoic acid could change the membrane permeability7to enhance the outflow of macromolecules (protein and DNA). These tw o molecules have bulky hydrophobic tails, which could bind the cell membrane and disrupt its structure leading to leakage of the cell membrane. Unlike polymyxins, these two antibiotics inhibited Gram-positives as well as Gram-negatives.

[0106]

[0049] Apigenin is a flavone that is abundantly present in plants and has various beneficial health effects such as antioxidant (Muhammed. T. M., et al., The Effects of Apigenin in the Treatment of Diabetic Nephropathy: A Systematic Review of Non-clinical Studies. Mini Rev Med Chem, 2024. 24(3): p. 341-354), anti-inflammatory7(Wang, F., et al., Systemic meta-analysis: apigenin's effects on lung inflammation and oxidative stress. J Asthma, 2023: p. 1-11), and chemoprevention (Fossatelli, L., et al., Resources for Human Health from the Plant Kingdom: The Potential Role of the

[0107] 14

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[0109] Flavonoid Apigenin in Cancer Counteraction. Int J Mol Sci, 2023. 25(1)). Apigenin-di-p-coumarylglucoside is one of the apigenin derivatives. As described herein, apigenin and some derivatives had no detectable antibacterial activity against the four tested bacterial strains. In contrast, apigenin-di-p-coumarylglucoside showed strong antibacterial activity against the three bacterial strains tested. Apigenin-di-p-coumarylglucoside has some structural similarities to aminoglycoside which contains two amino sugars joined by a glycosidic bond to an aminocyclitol (Tevyashova, A. N. and K. S. Shapovalova, Potential for the Development of a New Generation of Aminoglycoside Antibiotics. Pharm Chem J, 2021. 55(9): p. 860-875). Commonly used aminoglycosides are streptomycin, gentamicin, sisomicin, netilmicin, kanamycin, amikacin, neomycin, tobramycin, spectinomycin. and paromomycin, which function as protein synthesis inhibitors.

[0110] Apigenin-di-p-coumarylglucoside at a high concentration (4X MIC) also inhibited protein synthesis but it promoted protein synthesis at a low concentration (2X MIC). Under the same conditions, ampicillin also enhanced protein synthesis. The reason for the observed enhancement of protein synthesis by these two antibacterial compounds is unclear.

[0111]

[0050] One of the most important targets for antibiotics to cure infectious diseases is nucleic acid synthesis. A large difference in the enzymes that cany out DNA and RNA synthesis between eukary otic and prokaryotic cells helps to achieve selective toxicity7. Well-known example is the rifamycin family, which binds to DNA-dependent RNA polymerase, thereby inhibiting the elongation of RNA (Gugnani, J. S., et al., Effectiveness of Rifabutin-Based Regimens in Treating Helicobacter pylori Infections. Cureus, 2023.

[0112] 15(12): p. e50541). Quinolones bind to DNA gyrase, inhibiting their function, which results in inhibition of the DNA replication that ultimately results in cell death (Chen, J. P., et al., Membrane active 7-thiazoxime quinolones as novel DNA binding agents to decrease the genes expression and exert potent anti-methicillin-resistant Staphylococcus aureus activity. Eur J Med Chem, 2021. 217: p. 113340). As described herein, the BrdU incorporation assay indicated that Apigenin-di-p-coumarylglucoside strongly inhibited the DNA synthesis of the tested bacteria, suggesting that it targets DNA synthesis.

[0113]

[0051] The presently disclosed subject matter will now be described more fully hereinafter. However, many modifications and other embodiments of the presently disclosed subject matter set forth herein yy ill come to mind to one skilled in the art to which the presently disclosed subject matter pertains having the benefit of the teachings presented in the foregoing descriptions. Therefore, it is to be understood that the

[0114] 15

[0115] LEGAL02 / 47622964vl Atty. Dkt: 015444 / 638160

[0116] presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. In other words, the subject matter described herein covers all alternatives, modifications, and equivalents. In the event that one or more of the incorporated literature, patents, and similar materials differs from or contradicts this application, including but not limited to defined terms, term usage, described techniques, or the like, this application controls. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in this field. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the drawings, the relative sizes of regions or features may be exaggerated for clarity. This subject matter may, however, be embodied in many different forms and should not be construed as limited to the aspects set forth herein; rather, these aspects are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the subject matter to those skilled in the art.

[0117] I. Definitions

[0118]

[0052] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary’ skill in the art to which this subject matter belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the present application and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein. The terminology' used in the description of the subject matter herein is for the purpose of describing particular aspects only and is not intended to be limiting of the subject matter. In case of a conflict in terminology, the present specification is controlling.

[0119]

[0053] As used herein, ‘‘antibacterial” refers to any substance, compound, a combination of substances, or a combination of compounds capable of: (i) inhibiting, reducing or preventing growth of bacteria; (ii) inhibiting or reducing ability of a bacteria to produce infection in a subject; or (iii) inhibiting or reducing ability of bacteria to multiply or remain infective in the environment.

[0120]

[0054] As used herein the term “extract” refers to a process of dissolving a compound of interest from plant material where it is contained or refers to the product of an extraction

[0121] 16

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[0123] process that dissolves a product from plant material where it is contained.

[0124]

[0055] As used herein the term "‘isolate’7refers to a process of separating a compound of interest from its milieu or refers to the product of an isolation process that separates a product from its milieu.

[0125]

[0056] As used herein, the term “purified” means separated from other materials, such as plant material, e.g., protein, chitin, cellulose, or water. In certain embodiments, the term “purified” refers to a compound substantially free of other materials. In certain embodiments, the term “purified” refers to a compound(s) that is substantially free from non-antibacterial compounds found in Thuja arborvitae. In certain embodiments, the term “purified” refers to a compound that is substantially free from other antibacterial compounds found in Thuja arborvitae. In certain embodiments, the term “purified” refers to a compound that has been separated from other compounds that are typically coextracted when the purified compound is extracted from a naturally occurring source. In certain embodiments, a “purified” composition contains components in substantially different amounts and ratios than found in Thuja arborvitae. Such purified extracts and compositions can have unique properties. In certain embodiments, the term “purified” refers to a composition that has been subjected to column chromatography. In certain embodiments, the term “purified” refers to a composition that has been subjected to gas chromatography, liquid chromatography (e.g., LC, HPLC, etc.).

[0126]

[0057] As used herein, the term “enriched” refers to the quality in a preparation, formulation or composition where one or more ingredients’ proportions are significantly and artificially increased compared to their respective, natural levels. Enrichment can be achieved through purification or isolation techniques as described herein that reduces or removes unwanted components and compounds after separation from the desired compounds.

[0127]

[0058] As used herein, the term “solvent” refers to a liquid capable of dissolving antibacterial compounds, such as C1-5 alcohols, ethyl acetate, DMSO, hexane, water, and methanol.

[0128]

[0059] The term “infection” or “bacterial infection” as used herein includes the presence of bacteria, in or on a subject, which, if its growth w ere inhibited, would result in a benefit to the subject. As such, the term “infection” in addition to referring to the presence of bacteria also refers to presence of other floras, which are not desirable. The

[0129] 17

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[0131] term ‘‘infection” includes infection caused by bacteria.

[0132]

[0060] The term “treat”, “treating” or “treatment” as used herein refers to administration of a medicament, including a pharmaceutical composition, or one or more pharmaceutically active ingredients, for prophylactic and / or therapeutic purposes. The term “prophylactic treatment” refers to treating a subject who is not yet infected, but who is susceptible to, or otherwise at a risk of infection (preventing the bacterial infection). The term “therapeutic treatment” refers to administering treatment to a subject already suffering from infection. The terms “treat”, “treating” or “treatment” as used herein also refer to administering compositions, or one or more of pharmaceutically active ingredients discussed herein, with or without additional pharmaceutically active or inert ingredients, in order to: (i) reduce or eliminate either a bacterial infection, or one or more symptoms of a bacterial infection, or (ii) retard progression of a bacterial infection, or one or more symptoms of a bacterial infection, or (iii) reduce severity of a bacterial infection, or one or more symptoms of a bacterial infection, or (iv) suppress clinical manifestation of a bacterial infection, or (v) suppress manifestation of adverse symptoms of a bacterial infection.

[0133]

[0061] The terms “pharmaceutically effective amount” or “therapeutically effective amount” or “effective amount” as used herein refer to an amount, which has a therapeutic effect or is the amount required to produce a therapeutic effect in a subject. For example, a “therapeutically effective amount” or “pharmaceutically effective amount” or “effective amount” of an antibacterial agent or a pharmaceutical composition is the amount of the antibacterial agent, or the pharmaceutical composition required to produce a desired therapeutic effect as may be judged by clinical trial results, model animal infection studies, and / or in vitro studies (e.g. in agar or broth media). Such effective amount depends on several factors, including but not limited to, the microorganism (e.g. bacteria) involved, characteristics of the subject (for example height, weight, sex, age, and medical history), the severity of the infection, and the antibacterial agent used. For prophylactic treatments, a prophylactically effective amount is that amount which would be effective in preventing the bacterial infection.

[0134]

[0062] The term “administration” or “administering” refers to and includes the delivery of a composition, or one or more pharmaceutically active ingredients to a subject, including for example, by any appropriate method, that serves to deliver the composition or its active ingredients or other pharmaceutically active ingredients to the site of

[0135] 18

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[0137] infection. The method of administration may vary depending on various factors, such as the components of the pharmaceutical composition or ty pe / nature of the pharmaceutically active or inert ingredients, the site of the potential or actual infection, the microorganism involved, the severity of the infection, age and physical condition of the subject and alike. Some non-limiting examples of ways to administer a composition or a pharmaceutically active ingredient to a subject according to this invention include oral, intravenous, topical, intra-respiratory, intraperitoneal, intramuscular, parenteral, sublingual, transdermal, intranasal, aerosol, intraocular, intratracheal, intrarectal, vaginal, gene gun, dermal patch, eye drop and mouthwash. In case of a pharmaceutical composition comprising more than one ingredient (active or inert), one of the ways of administering such composition is by admixing the ingredients (e.g. in the form of a suitable dosage form such as a tablet, capsule, solution, powder or alike) and then administering the dosage form. Alternatively, the ingredients may also be administered separately (simultaneously or one after the other) as long as these ingredients reach beneficial therapeutic levels such that the composition as a whole provides a synergistic and / or desired effect.

[0138]

[0063] The term “growth” as used herein refers to a growth of one or more microorganisms and includes reproduction or population expansion of the microorganism (e.g. bacteria). The term "growth" also includes maintenance of on-going metabolic processes of the microorganism, including the processes that keep the microorganism alive. In certain embodiments of the methods described herein refer to the reduction of growth, such as a reduction by 1-10%, 5-15%, 10-20%, 15-25%, 20-30%, 25-35%, 30-40%, 35-45%, 40-50%, 45-55%, 50-60%, 55-65%, 60-70%, 65-75%, 70-80%, 75-85%, 80-90%, 85-95%, or greater than 90%.

[0139]

[0064] The term, “effectiveness” as used herein refers to the ability of a treatment, a composition, or one or more pharmaceutically active ingredients to produce a desired biological effect in a subject. For example, the term “antibacterial effectiveness” of a composition or an antibacterial agent refers to the ability' of the composition or the antibacterial agent to prevent or treat bacterial infection in a subject.

[0140]

[0065] The term “synergistic” or “synergy” as used herein refers to the interaction of two or more agents so that their combined effect is greater than their individual effects.

[0141]

[0066] The term “pharmaceutically inert ingredient” “carrier” or “excipient” refers to and includes compounds or materials used to facilitate the administration of a compound, for example, to increase the solubility of the compound. Typical, non-limiting examples of 19

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[0143] solid carriers include starch, lactose, dicalcium phosphate, sucrose, and kaolin. Typical, non-limiting examples of liquid carriers include sterile water, saline, buffers, non-ionic surfactants, and edible oils. In addition, various adjuvants commonly used in the art may also be included. These and other such compounds are described in the literature, e.g., in the Merck Index (Merck & Company, Rahway, N. J.). Considerations for the inclusion of various components in pharmaceutical compositions are described, e.g., in Gilman et al. (Goodman and Gilman's: The Pharmacological Basis of Therapeutics, 8th Ed., Pergamon Press., 1990), which is incorporated herein by reference in its entirety.

[0144]

[0067] The term “subj ect” as used herein refers to vertebrates or invertebrates, including a mammal. The term “subject’' includes a human, an animal, a bird, a fish, or an amphibian. Typical, non-limiting examples of a “subject” include humans, cats, dogs, horses, sheep, bovine cows, pigs, lambs, rats, mice, and guinea pigs.

[0145]

[0068] The term “pharmaceutically acceptable derivative” as used herein refers to and includes any pharmaceutically acceptable salt, pro-drug, metabolite, ester, ether, hydrate, polymorph, solvate, complex, and adduct of a compound described herein which, upon administration to a subject, is capable of providing (directly or indirectly) the parent compound. For example, the term “antibacterial agent or a pharmaceutically acceptable derivative thereof’ includes all derivatives of the antibacterial agent (such as salts, prodrugs, metabolites, esters, ethers, hydrates, polymorphs, solvates, complexes, and adducts) which, upon administration to a subject, are capable of providing (directly or indirectly) the antibacterial agent.

[0146]

[0069] The term “pharmaceutically acceptable salt” as used herein refers to one or more salts of a given compound that possesses the desired pharmacological activity' of the free compound, and which is neither biologically nor otherwise undesirable. In general, the term “pharmaceutically acceptable salts” refers to salts that are suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response, and the like, and are commensurate with a reasonable benefit / risk ratio.

[0147] Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. (J. Pharmaceutical Sciences, 66; 1-19, 1977), incorporated herein by reference in its entirety, describes various pharmaceutically acceptable salts in detail.

[0148]

[0070] The term “stereoisomer” as used herein refers to and includes isomeric molecules that have the same molecular formula but differ in the positioning of atoms and / or functional groups in the space. Stereoisomers may further be classified as enantiomers 20

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[0150] (where different isomers are mirror images of each other) and diastereomers (where different isomers are not mirror images of each other). Diastereomers include isomers such as conformers, meso compounds, cis-trans (E-Z) isomers, and non-enantiomeric optical isomers.

[0151]

[0071] As used herein, the term “contacting” refers to allowing the composition to come into physical contact with the subject or allowing two or more reagents to come in contact with each other.

[0152]

[0072] As used herein, the term “substantially” refers to the complete or nearly complete extent or degree of a component, or an action, characteristic, property, state, structure, item, or result. The exact allowable degree of deviation from the absolute presence of such a component, or an action, characteristic, property, state, structure, item, or result may in some cases depend on the specific context. However, generally speaking, “substantially” will be so near as to have the same overall result as if absolute and total extent or degree were obtained. The use of “substantially” is equally applicable when used in a negative connotation to refer to the complete or near complete lack of a component, or an action, characteristic, property, state, structure, item, or result. For example, a composition that is “substantially free of’ leaching would either completely lack leaching or so nearly completely lacking that the effect would be the same as if it completely lacked leaching. In other words, a composition that is “substantially free of’ leaching may still actually leach as long as there is no measurable effect thereof, for example, trace amounts. As used herein, “essentially free” means a component, or an action, characteristic, property, state, structure, item, or result is not present or is not detectable.

[0153]

[0073] As used herein, the term “predominantly” and “predominant” refer to the component that is produced in the relative highest quantity.

[0154]

[0074] Additional definitions may also be provided below.

[0155] II. Compositions and Methods

[0156]

[0075] In certain embodiments, the subject matter described herein is directed to an antibacterial composition comprising,

[0157] a purified extract from Thuja arborvitae leaves, wherein the purified extract comprises at least 20% w / w of the compound 1:

[0158] 21

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[0160]

[0161]

[0076] In certain embodiments, the subject matter described herein is directed to an antibacterial composition comprising from about 40 to about 50% w / w compound 1; about 15% to about 25% w / w compound 2; and about 30% to about 40% w / w compound 3, wherein the sum of the compounds is not more than 100%.

[0162]

[0077] In certain embodiments, the subject matter described herein is directed to an antibacterial composition comprising about 46% w / w compound 1.

[0163]

[0078] In certain embodiments, the relative amounts of compounds 1-3 are modified to increase antibacterial activity. In certain embodiments, the relative amounts of compounds 1 and 2 are modified to increase antibacterial activity to result in a synergistic effect. In certain embodiments, the relative amounts of compounds 1 and 3 are modified. In certain embodiments, the relative amounts of compounds 1 and 4 are modified. In certain embodiments, the relative amounts of compounds 1 and 5 are modified.

[0164]

[0079] In certain embodiments, the subject matter described herein is directed to an antibacterial composition comprising from about 30% to about 40% w / w compound 3.

[0165]

[0080] In certain embodiments, the subject matter described herein is directed to an antibacterial composition comprising about 33% w / w compound 3.

[0166]

[0081] In certain embodiments, the antibacterial composition comprising compound 1 further comprises one or more compounds selected from the group consisting of:

[0167] 22

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[0169] 2

[0170]

[0171] HO

[0172] wherein, the total amount present of compounds 2, 3, 4 and 5 combined is less than 80% w / w.

[0173]

[0082] In certain embodiments, the subject matter described herein is directed to an antibacterial composition comprising,

[0174] a purified extract from Thuja arborvitae leaves, wherein the purified extract 23

[0175] LEGAL02 / 47622964vl Atty. Dkt: 015444 / 638160

[0176] comprises at least 20% w / w of the compound 3:

[0177]

[0178] HO

[0179]

[0083] In certain embodiments, the antibacterial composition comprising compound 3 further comprises one or more compounds selected from the group consisting of:

[0180]

[0181] 24

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[0183]

[0184] HO

[0185] wherein, the total amount present of compounds 2, 1, 4 and 5 combined is less than 80% w / w.

[0186]

[0084] In certain embodiments, the subject matter described herein is directed to an antibacterial composition as described above further comprising a pharmaceutically acceptable excipient.

[0187]

[0085] In certain embodiments, the subject matter described herein is directed to a purified extract comprising at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% w / w of compound 1 or 3.

[0188]

[0086] In certain embodiments, the subject matter described herein is directed to an antibacterial composition as described above, wherein the purified extract is prepared by extracting the leaves with an alcohol to form a liquid extract and subjecting the liquid extract to column chromatography to prepare the purified extract.

[0189]

[0087] In certain embodiments, the alcohol is a C1-5 alcohol, such as methanol or ethanol.

[0190]

[0088] In certain embodiments, the antibacterial compositions described above have a MIC value of about 10 to about 60 μg / ml, about 10 to about 55 μg / ml, about 10 to about 50 μg / ml against S. aureus. In certain embodiments, the antibacterial compositions described above have a MIC value of about 50 μg / ml against gram-positive (Staphylococcus aureus and Streptococcus mutans) and gram-negative (Acinetobacter baumannii, and Escherichia coli) bacteria.

[0191]

[0089] In certain embodiments, the subject matter described herein is directed to methods of treating a bacterial infection in a subject comprising, administering an antibacterial composition as described above.

[0192]

[0090] In certain embodiments, the subject matter described herein is directed to methods of reducing bacterial growth, comprising, and contacting a bacterium with an antibacterial composition as described above, wherein the growth of the bacterium is reduced.

[0193]

[0091] In certain embodiments, the bacterial infection involves, or the bacteria is a Gramnegative bacterium. In certain embodiments, the Gram-negative bacteria belongs to:

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[0196] (i) a phylum selected from the group consisting of Acidobacteria, Aquificae, Chlamydiae, Bacteroidetes, Chlorobi, Cyanobacteria, Fibrobacteres, Verrucomicrobia, Planctomycetes, and Spirochetes:

[0197] (ii) a class selected from the group consisting of Alphaproteobacteria, Bacilli, Epsilonproteobacteria, Deltaproteobacteria, and Gammaproteobacteria; or

[0198] (iii) an order selected from the group consisting of Hydrogenophilales, Methylophilales, Neisseriales, Nitrosomonadales, Procabacteriales, and Rhodocyclales. In certain embodiments, the Gram-negative bacterium is Acinetobacter baumannii or Steptococcus mutans.

[0199]

[0092] In certain embodiments, the bacterial infection involves, or the bacteria is a Grampositive bacteria. In certain embodiments, the Gram-positive is selected from the group consisting of: Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus sp. (Coagulase-negative), Streptococcus pneumoniae (Viridans group), Streptococcus agalactiae (group B), Streptococcus pyogenes, Enterococcus sp., Bacillus anthracis, Bacillus oereus, Bifidobacteriu bifldum, Lactobacillus sp., Listeria monocytogenes, Nocardia sp., Rhodococcus equi (coccobacillus), Erysipelothrix rhusiopathiae, Corynebacterium diptheriae, Propionibacterium acnes, Actinomyces sp., Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Mobiluncus sp., and Peptostreplococcus sp. In certain embodiments, the Gram-positive negative bacterium is Staphylococcus aureus.

[0200]

[0093] For the prevention or treatment of bacterial infections, the appropriate dosage (when the extract, isolate or composition comprising them is used alone or in combination with one or more other additional therapeutic agents) will depend on the type of bacteria to be treated, the severity and course of the disease, whether the extract, isolate or composition is administered for preventive or therapeutic purposes, previous therapy, the subject's clinical history and response to the extract, isolate or composition, and the discretion of the attending physician. The extract, isolate or composition is suitably- administered to the subject at one time or over a series of treatments. Depending on the type and severity of the bacterial infection, about 0.01 pg / kg to 20 mg / kg (e.g. 0.1 mg / kg- 15 mg / kg) of the extract, isolate or composition can be an initial candidate dosage for administration to the subject, whether, for example, by one or more separate administrations. An initial higher loading dose, followed by one or more lower doses, may be administered. One typical daily dosage might range from about 0.1 pg / kg to 100

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[0203] mg / kg or more, depending on the factors mentioned above. For repeated administration over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs. The progress of this therapy is easily monitored by conventional techniques and assays. Such doses may be administered intermittently, e.g. every week, every' two weeks, or every three weeks (e.g. such that the subject receives from about two to about twenty, or e.g. about six doses of the polypeptide).

[0204]

[0094] In certain embodiments, the subject matter described herein is directed to a method of isolating an antibacterial compound from Thuja arborvitae leaves, comprising: contacting the leaves with a first solvent to produce a liquid extract; and,

[0205] subjecting the liquid to column chromatography and eluting fractions to prepare a liquid eluent fraction enriched in at least one compound selected from the group consisting of:

[0206]

[0207] 27

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[0209]

[0210] HO

[0211]

[0095] In certain embodiments, the first solvent is an organic solvent. In certain embodiments, the organic solvent is a C1-5 alcohol, such as methanol or ethanol, or ethyl acetate.

[0212]

[0096] In certain embodiments, the method further comprises allowing the liquid eluent fraction to undergo evaporation to prepare a concentrate.

[0213]

[0097] In certain embodiments, the method further comprises dissolving the concentrate in a mobile phase to prepare a liquid concentrate and subjecting the liquid concentrate to

[0214] 28

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[0216] high performance liquid chromatography (HPLC) to prepare an isolate comprising at least one of compounds 1. 2, 3 4 or 5.

[0217]

[0098] In certain embodiments, the HPLC is reverse phase with a linear gradient of a C1-5 alcohol. In certain embodiments, the HPLC is reverse phase with a linear gradient of about 50% methanol in water-ethyl acetate from about 10% to about 100%.

[0218]

[0099] In certain embodiments, the method further comprises after the contacting and prior to the subjecting, lyophilizing the extract to prepare a lyophilized extract; and dissolving the lyophilized extract in a second solvent to prepare a liquid solution.

[0219]

[0100] In certain embodiments, an isolate prepared by the methods described herein comprises compound 1, having the structure:

[0220]

[0221] wherein, compound 1 is present in an amount of at least 20%. 30%. 40%. 50%. 60%. 70%, 80%, 90% or 95% w / w of the isolate.

[0222]

[0101] In certain embodiments, an isolate prepared by the methods described herein comprises compound 3, having the structure:

[0223]

[0224]

[0102] wherein, compound 3 is present in an amount of at least 20%. 30%. 40%. 50%. 60%, 70%, 80%, 90% or 95% w / w of the isolate.

[0225]

[0103] In certain embodiments, the isolate is an oil or a solid.

[0226]

[0104] In certain embodiments, an isolate prepared by the methods described herein 29

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[0228] comprises predominantly compound 1 and further comprises one or more compounds selected from the group consisting of compounds 2. 3, 4 and 5; and an isolate prepared by the methods described herein comprises predominantly compound 3 and further comprises one or more compounds selected from the group consisting of compounds 2, 1, 4 and 5:

[0229]

[0230] 30

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[0232] 4

[0233] and

[0234] 5

[0235]

[0236] HO

[0237]

[0105] In certain, embodiments, the isolate comprises compound 1, having the structure:

[0238]

[0239] wherein, compound 1 is present in an amount of at least 20%. 30%. 40%. 50%. 60%. 70%, 80%, 90% or 95% w / w of the isolate.

[0240]

[0106] In certain embodiments, the isolate comprises compound 3, having the structure:

[0241]

[0242] wherein, compound 1 is present in an amount of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% w / w of the isolate.

[0243]

[0107] In certain embodiments, an isolate from Thuja arborvitae leaves comprises compounds 1 and 3, and optionally compound 2, wherein compound 1 and 3 combined are present in an amount of at least 40%, 50% 60%, 70%, 80%, 90% or 95% w / w of the 31

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[0245] isolate.

[0246]

[0108] In all embodiments, the compositions and methods described above can consist essentially of the desired compounds, such as any of compounds 1, 2, 3, 4 and 5, and combinations thereof, and any excipients, but are substantially free of other plant extract materials.

[0247]

[0109] The General Procedures and Examples provide exemplary methods for preparing compounds and compositions. Those skilled in the art will appreciate that other routes may be used to isolate the compounds. Although specific starting materials and reagents are depicted and discussed in the Schemes, General Procedures, and Examples, other starting materials and reagents can be easily substituted to provide a variety of derivatives and / or reaction conditions. In addition, many of the exemplary compounds prepared by the described methods can be further modified in light of this disclosure using conventional chemi stry well known to those skilled in the art.

[0248]

[0110] The following examples are offered by w ay of illustration and not by way of limitation.

[0249] EXAMPLES

[0250] Materials and Methods

[0251] Collection of Samples and Preparation of Plant Extracts

[0252]

[0111] Seeds of Foeniculum vulgare, Ginkgo biloba, Pimpinella anisum, and Zanthoxylum americanum were purchased from local grocery stores. Leaves of Magnolia grandiflora, Toxicodendron radicans, and Thuja arborvitae, and berries of Lycium barbarum were collected from the Northwestern region of Atlanta, GA. The plant samples w ere dried at room temperature and grinded with a blender. Ten grams of each powdered sample were mixed with 20 ml of the solvent (water, methanol, or ethyl acetate) in a 50 ml plastic tube. The extraction was performed at room temperature for 24 hours on a rotator. The suspension was filtered and dried with a lyophilizer. The lyophilized powder was stored at -20 °C until further analysis.

[0253] Bacterial Strains and Maintenance of Bacteria

[0254]

[0112] Four bacterial strains: Staphylococcus aureus (ATCC 49775; ATCC 12600), Acinetobacter baumannii (ATCC 19606), Pseudomonas aeruginosa (ATCC 10145), Streptococcus mutans (ATCC 25175), and Escherichia coli (ATCC 33694) were purchased from ATCC (American Type Culture Collection). Bacterial cultures were stored in 20% glycerol at -80 °C. Bacterial species were activated by streaking the stored 32

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[0256] culture onto LB (Lysogeny Broth) agar plates and then incubating overnight at 37 °C. The individual colony was selected from each plate and transferred to the LB broth, after which it was incubated overnight at 37 °C before use.

[0257] Antibiotics and Chemicals

[0258]

[0113] Ampicillin (A9518), chloramphenicol (C0378), eicosapentaenoic acid (44864), oleic acid (01008), abietic acid (00010), apigenin (10798), apigenin-7-glucoside (44692), and apigenin 7-O-neohesperidoside (A8906) were purchased from Sigma- Aldrich.

[0259] Apigenin 7-(2",6"-di-p-coumarylglucoside) (BCN9608) was sourced from BioCrick. These compounds were dissolved in solvents to achieve a stock concentration of 10 mg / ml, aliquoted, and stored at -20 °C until use.

[0260] Zone of Inhibition Assay

[0261]

[0114] The zone of inhibition assay was used to investigate the antibacterial activity of the crude plant extracts as well as purified compounds. Overnight LB broth cultures of the tested bacteria were seeded onto LB plates (1 x 105CFU / per plate) after which wells approximately 70 mm in diameter and 30 mm deep were made on the surface of the solid medium using a sterile disposable glass pipette. The plates were then turned upside down and the wells were labeled with a marker. The well was then filled with 50 pl of the tested sample. The solvent used to dissolve the sample was used as a negative control. Ampicillin (100 ug / ml) and chloramphenicol (25 ug / ml) were used as positive controls. The plates were incubated at 37 °C for 18 hours and then zones of inhibition were measured with a ruler. Samples with zones of inhibition greater than or equal to 100 mm diameter were considered positive.

[0262] Minimum Inhibition Concentration

[0263]

[0115] The Minimum Inhibition Concentration (MIC) was determined as described by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (www. eucast.org). Bacteria was cultured in the LB medium for 16 h and centrifuged for 5 min at 4,000 rpm (850 g) at room temperature to collect the bacterial pellet, which was re-suspended in the fresh LB medium at 1 x 105CFU / ml and 0.1 ml of the diluted bacterial culture was distributed to each well of a 96-well plate. A series of 2x dilutions of the test samples were prepared in sterile tubes or 96-well microplates, starting with the highest concentration and diluting further to achieve a range of concentrations needed. Control wells containing only the solvent used to dissolve the sample (negative) and ampicillin or chloramphenicol (positive) were included. The microplates were incubated 33

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[0265] at 37°C for 18 hours and the bacteria were re-suspended and the absorbance values at 600nm were detected with an automatic microplate reader (Synergy- Hl, BioTek. USA). The minimum concentration (pg / ml) of the test sample that completely inhibited the growth of a given strain of bacteria was deduced.

[0266] Purification of Antibacterial Compounds from Thuja arborvitae

[0267]

[0116] The dried leaves of Thuja arborvitae (800 g) were extracted with methanol (3,000 ml) at room temperature for 24 h. The mixture was then filtered by a porcelain funnel with filter paper. The filtered extract was concentrated by a rotavapor and dried via lyophilizer to obtain the crude extract (140 g). The crude extract was dissolved in methanol (14 ml) and loaded onto an alumina column (2.4 x 40 cm). The column was eluted with ethyl acetate and methanol (1:1) and fractions (10 ml each) were collected with a fraction collector (LKB 2111 MultiRac). Fifty microliters of each fraction were dried via SpeedVac (Thermo Savant) and submitted for the zone inhibition assay to detect the antibacterial activity-. The fractions with antibacterial activity were pooled and dried under the vacuum. The dried material was then dissolved in methanol (10 ml) and loaded onto a silica column (1.25 x 27.5 cm). The column was washed with ethyl acetate (800 ml) and eluted with methanol. The fractions were dried and submitted to the zone inhibition assay. The fractions with the antibacterial activity- were pooled and dried. The dried material was dissolved in methanol and loaded onto a HPLC C18 column (19 x 100 mm, Water). The column was eluted with a linear gradient of 10 to 100% acetonitrile in water. The peaks were detected with the absorbance at 280 nm, collected, dried, and submitted to the zone inhibition assay. Four peaks named Pl, P2, P3, and P4 showed antibacterial activity with retention time at 45, 46, 47, and 71 min, respectively. From 800 g dry leaves. 12.7 mg Pl, 16.8 mg P2. 7.7 mg P3. and 41.4 mg P4 were obtained. Compound No. Fraction ID

[0268] 1 P2

[0269] OH

[0270] o

[0271] \ \

[0272] o o

[0273] JL o— s-X 1 -o |[

[0274] fl T

[0275] HO'^HO OHXJC J

[0276] OH O

[0277] apigenin-7-(2‘\6’ -di-p-coumarylglucoside)

[0278]

[0279] 34

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[0281] 2 Pl

[0282] OH

[0283] o

[0284] If

[0285] 0

[0286] HO— ^L 1 o |[. OH

[0287] h° o T T T

[0288] J (i| ° O l H Y 0

[0289] o

[0290] HO

[0291] apigenin-7-(4‘’,6”-di-p-coumarylglucoside)

[0292] 3 P3

[0293] _ ZOH

[0294] 0

[0295] o

[0296] HO— v—L 1 -0 |[. OH

[0297] / T^os^\.oxY%;Y

[0298] °OHULJ

[0299] <1 ° OH o

[0300] HO

[0301] apigenin-7-(3”,6”-di-p-coumarylglucoside)

[0302] 4 P4

[0303] O1”

[0304] Eicosapentanoic acid

[0305] 5 P5

[0306] V H HO

[0307] Abietic acid

[0308]

[0309] 35

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[0311] Bacterial Colony-Forming Assay

[0312]

[0117] The bacterial culture (5. aureus. 1 x 106CFU) was inoculated in LB solution for 1 h at 37 °C. The culture was serially diluted with the medium and the 50 JJ.1 of the tested compound was added in aliquots and then plated on LB agar plates (35 mm). The plates were incubated for 16 h at 37 °C, and the number of colonies was counted manually. Viability Analysis of Bacteria

[0313]

[0118] The LIVE / DE AD" BacLight™ Bacterial Viability Kit (Life Technologies, Carlsbad, CA, USA) was used to detect both viable and dead counts of bacteria. The 100 pl aliquots of S'. aureus (IxlO6CFU / ml) in LB broth in the absence or presence of the tested compound was dispensed into each well of 96-well plates and incubated at 37 °C for 18 hrs. The subsequent reactions with dyes was performed following the manufacturer’s protocol. The fluorescence intensity of the stained samples at 530 nm and 630 nm was measured using a fluorophotometer (Gemini XPS: Molecular Devices, Sunnyvale, CA, USA) at an excitation wavelength of 485 nm. The ratio of fluorescence observed at 530 nm to 630 nm was measured to determine the ratio of the number of viable cells to the number of dead cells. For microscopic analysis, S', aureus (1 x 109CFU / ml) in LB medium were incubated at 37 °C for 1 h in the absence and presence of the tested compound. The stained samples were observed under a fluorescence microscope (AX10, ZEISS) with a 100X lens.

[0314] Immunochemical detection of BrdU in genomic DNA by dot blot

[0315]

[0119] A single colony of S. aureus was inoculated in 3 ml of LB medium and grown at 37 °C for 16 h in a shaker. The overnight culture (0.3 ml) w as diluted into fresh LB medium (3 ml) with and without BrdU (20 pM) in the presence or absence of the test compound. The culture was incubated for one doubling of optical density at 600 nm (ODGOO) (about 1 h). The bacterial genomic DNA was isolated according to the guanidium isothiocyanate protocol (McOrist, A. L., M. Jackson, and A. R. Bird, A comparison of five methods for extraction of bacterial DNA from human faecal samples. J Microbiol Methods, 2002. 50(2): p. 131-9). The DNA preparation was treated with RNase A and DNA concentration was determined by ethidium bromide-stained agarose gel and absorbance at 260 nm (OD260) using a NANODROP 2000C (Thermo Scientific). Twenty microliters (1.0 pg) of genomic DNA from each culture was denatured at 95 °C with the addition of 2.2 pl of 4N NaOH and renatured by the addition of 2.4 pl of 4N HC1

[0316] 36

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[0318] and then spotted onto a Zeta probe hybridization membrane. The DNA was cross-linked to the membrane by the GS Gene Linker (Bio-Rad) and baked for 2 h at 80 °C. The membrane was washed three times with TBS-0.5% Tween 20 and blocked by 3% non-fat milk in TBS-0.5% Tween 20 for 30 min. The membrane was incubated with the anti-BrdU antibody (BD; 1:1,000) for 2 h and washed with TBS-0.5% Tween 20 three times. The membrane was then incubated with the second antibody (peroxidase-conjugated goat anti-mouse IgGl, BD, 1:5,000) for 90 min. The membrane was washed three times with TBS-0.5% Tween and antibody binding was visualized by ChemiDoc Imaging System (Bio-Rad) using a Western blot chemiluminescence detection system (Cytiva, Amersham).

[0319] In vitro translation assay

[0320]

[0120] The E. coli S30 Extract System (Promega) was used for in vitro translation analysis. The pBESTluc vector (Promega) (1 pg) was linearized by Xhol digestion and the linearized template was transcribed by T7 RNA polymerase to generate mRNA of the luciferase gene. The luciferase mRNA was then translated to the luciferase protein using 25 pl of E. Coli S30 extract by following the manufacturers' instructions. A 10 pl aliquot of each reaction was placed in a well of a 96-well white plate and 50 pl of the luciferase assay reagent was added. The plate was immediately placed in the ChemiDoc Imaging System to take the image. The luciferase activity was also measured by the luminometer (BioTek Hl Hybrid Reader).

[0321] Mass spectrometry analysis

[0322]

[0121] Four purified compounds (Pl, P2, P3, and P4) were submited for the high-resolution mass spectrometry' (HRMS) analysis. The Agilent 1290 HPLC system was connected to a diode array detector followed by an Agilent 6545 quadruple time of flight mass spectrometry (Agilent Technologies, Santa Clara, CA) equipped with dual electrospray ionization interface. The LC / QTOF-MS analysis was performed on a reversed-phase Hypersil Gold-C 18 analytical column of 2.1 x 50 mm and 1.9 pm particle size (Thermo Fisher). An aliquot (1 pL) of sample solution in acetonitrile was injected in each run. The mobile phase consisted of 0.1% formic acid in both water (A) and acetonitrile (B). The chromatography was run at isocratic 60% B at 0.4 ml / min. For HRMS, the following operation parameters were used in a positive mode: capillary voltage, 3500 V; nozzle voltage 1000 V; gas temperature 300 °C; drying gas 10 L / min; nebulizer pressure, 35 psig; sheath gas temperature, 350 °C; sheath gas flow 12 L / min.

[0323] 37

[0324] LEGAL02 / 47622964vl Atty. Dkt: 015444 / 638160

[0325] For untargeted MS / MS analysis, a formula of 8 x mz / 100 + 4 was used to determine collision energy for fragmentation. The molecular formula was predicted using the Agilent MassHunter Qualitative Analysis software based on the accurate mass of adduct complex, isotope space, and relative intensity. The samples were also sent to the Mass Spectrometry Facility' at Georgia State University for the routine mass spectrometry analysis to determine the purity of HPLC peaks.

[0326] Cell cytotoxicity assay

[0327]

[0122] Normal human primary epidermal keratinocytes (PCS-200-010) and prostate cancer PC3 cells (CRL-1435) were purchased from ATCC and cultured in dermal cell basal medium and RPMI 1640 medium containing 10% fetal bovine serum, respectively. Cells were plated in 24-well plates (lx 104cells / well) and treated with purified compounds for 96 h. The treated cells were monitored with Incucyte (Sartorius).

[0328] Example 1

[0329] Screening for Antibacterial Activity

[0330]

[0123] Seeds of Foeniculum vulgare, Ginkgo biloba, Pimpinella anisum, and Zanthoxylum americanum, leaves of Magnolia grandiflora, Toxicodendron radicans, and Thuja arborvitae, and berries from Lycium barbarum were collected. Four species of bacteria (2 Gram-positive and 2 Gram-negative), mostly involved in common infections were used to screen for the antibacterial activity. Methanol was used to prepare the crude extracts of different plant species for screening.

[0331]

[0124] The methanol extracts from Al. grandiflora, T. arborvitae, and T. radicans were active against the bacteria strains S. aureus. (Fig. 1 A; Table 1).

[0332] Table 1. Zone of Inhibition of Different Plant Extracts

[0333] Plot £ aureas P. aeruginosa F. vutgare NA NA

[0334] G. biloba NA NA

[0335] L. barbaaan NA NA

[0336] M. grandiflara 2.0 + / - 0.2 NA

[0337] P. amsitm NA NA

[0338] T, arborvitae 1.9 ^ / - 0.1 + / - 0A

[0339] Z radicals 1.7 N- 0.1 1.74 / - O.2

[0340]

[0341] Z, amcricaminn NA

[0342]

[0125] P. aeruginosa exhibits inherent resistance to certain antibiotics, including penicillin and chloramphenicol, owing to its unique structural and biochemical characteristics (13). We found that the T. arborvitae and T. radicans extract slightly 38

[0343] LEGAL02 / 47622964vl Atty. Dkt: 015444 / 638160

[0344] inhibited the bacteria strain P. aeruginosa which is resistant to ampicillin and chloramphenicol (Fig. IB). The extract of T. arborvitae (Fig. 2A) was more potent than that of T. radicans (Fig. 2B) to inhibit the growth of the four bacteria strains tested. Both extracts strongly inhibited the growth of bacterial strains of A aureus, A. baumannii, and S. mutans but had less effect on the growth of the P. aeruginosa bacteria. Under a high concentration (4,000 pg / ml). the extract of T. arbovitae could completely suppress the growth of the bacteria P. aeruginosa. The leaf extract of T. arbovitae inhibited the colony formation ability of bacteria S', aureus in a dosage-dependent manner (Fig. 3). At the concentration of 125 pg / ml, the colony formation was completely abolished.

[0345] Consistent with our observations, the antibacterial activity’ yvas reported in Toxicodendron and Thuja (Puskarova, A., et al., The antibacterial and antifungal activity of six essential oils and their cyto / genotoxicity to human HEL 12469 cells. Sci Rep, 2017. 7(1): p. 8211; Saravanakumar, K., et al., Antioxidant, Anti-Lung Cancer, and Anti-Bacterial Activities of Toxicodendron vemicifluum. Biomolecules, 2019. 9(4)) previously but the antibacterial compounds that are responsible for the observed antibacterial activity have not been purified and identified from these plants. Several antibacterial compounds were identified from M. grandiflora (Zeutsop, J. F., et al., Phytochemical, antibacterial, antioxidant and cytoxicity investigation of Tarenna grandiflora. Z Naturforsch C J Biosci, 2021. 76(7-8): p. 285-290). Since leaves of T. arbovitae are largely available and have the most significant antibacterial activity, we decided to use them as the material to purify and identify chemical compounds that contribute to the observed antibacterial activity.

[0346] Example 2

[0347] Purification of antibacterial chemical compounds from T. arborvitae

[0126] Dried leaves of T. arborvitae were extracted wdth water, methanol, or ethyl acetate at room temperature overnight. Ethyl acetate and methanol but not water extract showed antibacterial activity against S. aureus (Table 2), suggesting that the antibacterial constituents are not water-soluble.

[0348] Table 2. The antibacterial activity of different solvent extracts of Thuja arborvitae testing for antibiotic compounds against Gram-positive bacteria S. aureus +.

[0349] Solvents MIC tomb

[0350] Ethyl acetate 1028 + 41

[0351] _ Methanol _ 500 ± 2.0 -.

[0352]

[0353] _ Water _

[0354]

[0127] The purification scheme is illustrated in Figure 4. The ethanol extract w as 39

[0355] LEGAL02 / 47622964vl Atty. Dkt: 015444 / 638160

[0356] separated on an alumina column with the mobile phase of ethyl acetate and methanol (1:1). The zone inhibition assay was used to detect the antibacterial activity of column fractions. Fractions containing the antibacterial activity were pooled, condensed, and freeze-dried under a vacuum. The dried materials were dissolved in ethyl acetate and loaded onto a Silica column. The unbound materials were eluted from the column with ethyl acetate and the bound materials were eluted with methanol. The antibacterial activity was detected only in the methanol fraction. The materials in the methanol fraction were further separated by an HPLC C18 reverse phase chromatography with a linear gradient of water-acetonitrile from 10-100%. Twenty-three major peaks were collected and dried under a vacuum. The zone inhibition assay indicated that four peaks designed as Pl, P2, P3, and P4 with retention times of 45, 46. 47. and 71 minutes contained the antibacterial activity, suggesting that T. arborvitae contains multiple antibacterial constituents. The P4 was further purified by a HPLC C18 reverse phase chromatography with a linear gradient of 50% methanol in water-ethyl acetate from 10-100%.

[0357]

[0128] MeOH / EtOAc Mixture: performed best in terms of efficiency and separation quality. MeOH / EtOAc + 1 / 150 Hexane: a small proportion of hexane added to the MeOH / EtOAc mix to determine effects, refine the separation. Pure Solvents (MeOH and EtOAc): tested individually but found to be insufficient in achieving optimal separation due to either poor elution or resolution of the compounds. EtOAc + Cyclohexane: tested combination at different concentrations to adjust polarity and study impact, but did not reach performance of MeOH / EtOAc mixture.

[0358]

[0129] From 800 grams of dry leaves of T. arborvitae, \NQ obtained about 12.7 mg of Pl, 16.8 mg of P2, 7.7 mg of P3. and 41.4 mg of P4. The purified compounds showed MIC of about 10 to about 50 pg / ml against the bacterial strain S. aureus (Table 3).

[0359] Table 3. The minimum inhibition concentration assay from purified compounds and purchased compounds Gram-positive bacteria against S. aureusv.

[0360] 40

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[0362] Purified Compounds j MiC fpg / ml)

[0363] _ Crude _ T _ 500 + 20

[0364] Pi j 20 t. G8 ~~

[0365] 50 + 2

[0366] 18 + 0.4

[0367] Purchased Compounds _ | _ MiC ft-ig / ml) _

[0368] Apig&nkvp^G) >5)00 ^ 80

[0369] APG-7-Giucosrde I >2000 + 80

[0370] . t. 50 + 2.

[0371] APG-7-O neospendossd^ i >2000 ± 80

[0372] Abiebc acid I 20 + G3

[0373]

[0374] Eicosapenianoic acid j. Saias...

[0375] Example 3

[0376] Identification of purified antibacterial compounds

[0377]

[0130] The four purified samples (Pl, P2, P3, and P4) were subjected to HPLC-QTOF and high-resolution mass spectrometry (HRMS) analysis. MS / MS analysis revealed that P3 contained the major peak showing a molecular mass of 724.1721 g / mole and gave the predicted molecular formula C39H22O14 (Fig. 5B). The P3 sample matched with apigenin-di-p-coumaryglucoside (the molecular mass: 724.1792 g / mole) from searching against the Dictionary of Natural Products. Fragmentation analysis revealed that ten fragments were matched with that of apigenin-di-p-coumaryglucoside (Fig. 5B). The P2 sample contained the compound found in the P3 sample and an isomer of the P3 compound (Fig. 6). These analyses showed that Pl was not pure and contained multiple compounds. Based on these analyses, the candid compounds for P2 and P3 are isomers of apigenin7- di-p-coumaryglucoside.

[0378]

[0131] The major peak in the P4 sample showed a molecular mass of 302.2320 g / mole and gave the predicted molecular formula C20H30O2. From searching against the Dictionary of Natural Products, P4 matched well with abietic acid and eicosapentaenoic acid, which are isomers with the same molecular formula (Fig. 7A). Fragmentation analysis revealed that fragments were matched with that of eicosapentaenoic acid (Fig.

[0379] 7B) and abietic acid (Fig. 7C). Based on these analyses, the P4 sample contains both abietic acid and eicosapentaenoic acid.

[0380]

[0132] It was reported that both abietic acid (Baglyas, M., et al., Antimicrobial Diterpenes from Rough Goldenrod (Solidago rugosa Mill.) Molecules, 2023. 28(9)) and eicosapentaenoic acid (Wei, M., et al.. Antimicrobial and antibiofilm effects of essential fatty acids against clinically isolated vancomycin-resistant Enterococcus faecium. Front 41

[0381] LEGAL02 / 47622964vl Atty. Dkt: 015444 / 638160

[0382] Cell Infect Microbiol, 2023. 13: p. 1266674) have antibacterial activity but the antibacterial activity of apigenin-di-p-coumaryglucoside has not been documented. The commercial apigenin 7-(3”, 6”-di-p-coumarylglucoside), abietic acid, and eicosapentaenoic acid showed antibacterial activity to a similar extent as the purified P3 and P4 preparations (See, Table 3). We conclude that purified P2 and P3 are isomers of apigenin 7-di-p-coumarylglucoside and P4 is the mixture of abietic acid, and eicosapentanoic acid. The apigenin 7-di-p-coumarylglucoside has three isomers existing in nature, i.e. apigenin-7-(2”, 6” - di- p-coumarylglucoside), apigenin-7-(3”, 6” - di -p-coumarylglucoside), and apigenin-7-(4”, 6” - di -p-coumarylglucoside). It is more likely that Pl is one of these isomers.

[0383] Example 4

[0384] Effects of the purified compounds on the bacterial growth and viability

[0133] Apigenin and its derivative compounds were purchased and tested for the antibacterial activity against the bacteria S. aureus. Apigenin, apigenin-7-glucoside, and apigenin-7-O-neosperidoside showed no detectable antibacterial activity (See, Table 3), suggesting that apigenin-di-p-coumarylglucoside might be the unique compound that has antibacterial activity. Even though P4 is a mixture of two compounds (eicosapentaenoic acid and Abietic acid) no synergism was observed between these two compounds in the inhibition of bacterial growth. Although eicosapentaenoic acid and Abietic acid belong to the fat acid family, the other fat acid Oleic acid (OA) did not show antibacterial activity in the same assay (Fig.1C).

[0385]

[0134] The purified P3 and P4 compositions strongly inhibited the growth of bacterial strains of S. aureus, A. baumannii, and S. mutans at concentrations below' 0.05 mg / ml. P3 had less effect on the growth of the bacterial strain P. aeruginosa but P4 had no effect on the growth of this bacterial at the concentration up to 0.4 mg / ml (400 pg / ml. Fig. 8). Similar effects of the purchased compounds [apigenin 7-(3”, 6’'-di-p-coumarylglucoside, abietic acid, and eicosapentaenoic acid] on the growth of these bacterial strains w ere observed.

[0386]

[0135] The LIVE / DEAD viability kit, which employs two nucleic acid-binding dyes (SYTO 9™, and propidium iodide), was used to determine the effect of purified compounds on bacterial cells' viability and membrane integrity. SYTO 9™ is a green, fluorescent dye that can enter both live and dead bacterial cells. It binds to the DNA of these cells, resulting in bright green fluorescence. On the other hand, propidium iodide is 42

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[0388] a red fluorescent dye that can only enter cells with damaged membranes, such as dead or dying cells. It binds to the DNA of these cells, resulting in red fluorescence. When both dyes are used together, live cells fluorescence green, while dead or dying cells fluorescence red. The purified compounds P3 and P4 significantly induced cell death of the bacterial strain S. aureus at concentrations at or above 5 pg / ml (Fig. 9A). By quantifying the ratio of green to red fluorescence, the effectiveness of the purified compounds was shown in Figure 10B. In the presence of 2.5 pg / ml or more of P3 and P4, the intensity of the red fluorescence was significantly high, indicating that the bacterial membrane was disrupted. In contrast, little red fluorescence was observed in the presence of 2.5 pg / mL or lower P3 or P4 (Fig. 9B). The results showed the purified antibacterial compounds effectively induced cell death or damage to the bacterial membrane of S. aureus.

[0389] Example 5

[0390] Effects of the purified compounds on the permeability of bacterial membrane

[0136] The leakage of nucleic acid and protein in the supernatant of bacterial culture in the presence of the purified compounds was determined to evaluate the effects on the cell permeability of the bacterial membrane. The leakage of nucleic acid (Fig. 10 A) and protein (Fig. 10B) was significantly higher than that in the control group at 1-, 2-, and 3-hours post-treatment with P4. The nucleic acid and protein in the supernatant of the cultured bacterial cells were also analyzed by agarose electrophoresis (Fig. 10C) and SDS-PAGE (Fig. 10D). The treatment with P4 significantly increased the levels of nucleic acid and protein in the bacterial culture. The results indicated that the P4 compounds led to the leakage of intracellular nucleic acid and protein in the bacteria. Under the same conditions, P3 and chloramphenicol did not affect the permeability of the bacteria (Fig. 10A and 10B).

[0391] Example 6

[0392] The effects of P2 on translation and transcription of the bacterial

[0137] The in vitro cell-free translation system was used to determine the effect of the purified compounds on bacterial protein synthesis. Chloramphenicol, a well-known inhibitor of bacterial translation, was used as a positive control. As expected, chloramphenicol strongly inhibited the bacterial translation at the concentration of lx MIC (0.6 pg / ml) and 2x MIC (1.6 pg / ml) (Fig. 11 A). P3 enhanced the bacterial translation at the concentration of 1 x MIC. However, it enhanced the bacterial translation 43

[0393] LEGAL02 / 47622964vl Atty. Dkt: 015444 / 638160

[0394] at the concentration of 2x MIC. Therefore, P3 might inhibit bacterial translation at a higher concentration.

[0395]

[0138] The effect of P3 on bacterial DNA synthesis was determined. The bacterial DNA was isolated from the bacterial cells cultured in the presence of BrdU as well as in the absence or presence of P3. The DNA synthesis (BrdU-incorporation) was monitored by immunoblot with the anti-BrdU antibody. P3 strongly inhibited the bacterial DNA synthesis at the concentrations of lx MIC and 2x MIC (Fig. 1 IB). Under the same conditions, chloramphenicol did not affect the bacterial DNA synthesis (Fig. 11B).

[0396] Example 7

[0397] Cell toxicity of purified compounds on human cells

[0398]

[0139] Cytotoxicity of purified P2 and P4 compounds on prostate cancer PC3 and normal epidermal keratinocytes were examined and the concentrations of lx MIC and 2x MIC were used. No cell toxicity and proliferative effects on PC3 cells were observed (Fig. 12A). How ever, P2 at the concentration of lx MIC inhibited growth of epidermal keratinocytes (Fig. 12B) and did not lead to cell death (Fig. 13C, middle panel). At the higher concentration (2x MIC), P2 led to cell death after 24 h of the treatment (Fig. 12C, bottom panel). P4 treatment led to more dramatic death of epidermal keratinocytes at the concentrations of both lx and 2x MIC after 24 h treatment (Fig. 12C).

[0399]

[0140] Five antibacterial compounds were purified from the T. arborvitae plant and showed strong antibacterial activity against three bacterial strains. Two antibacterial compounds induced damage to the cell membrane, leading to leakage of DNA and protein. The other identified compounds inhibited DNA synthesis. The identified antibacterial compounds could provide a useful tool for controlling bacterial pathogens and infectious diseases by inhibiting bacterial DNA synthesis or inducing cell membrane permeability.

[0400]

[0141] Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for.

[0401]

[0142] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in practicing the subject matter

[0402] 44

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[0404] described herein. The present disclosure is in no way limited to just the methods and materials described.

[0405]

[0143] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this subject matter belongs, and are generally consistent with the Compendium of Chemical Terminology, IUPAC Recommendations, 2ndEd. 2019, available at

[0406] https: / / goldbook. iupac.org.

[0407]

[0144] Throughout this specification and the claims, the words '‘comprise,” “comprises,” and “comprising” are used in a non-exclusive sense, except where the context requires otherwise. It is understood that embodiments described herein include “consisting of’ and / or “consisting essentially of’ embodiments. Consisting essentially of refers to any composition as described herein that contains the compounds in proportions described herein and does not contain any substantial amount of other plant extract materials. In other words, the compositions and methods utilize the compounds and any excipients but not other plant materials.

[0408]

[0145] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0409]

[0146] The term “about” as used herein when referring to a measurable value, such as, for example, an amount or concentration and the like, is meant to encompass variations of 20%, ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of the specified amount. A range provided herein for a measurable value may include any other range and / or individual value therein.

[0410]

[0147] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limit of the range and any other stated or intervening value in that stated range, is encompassed. The upper and lower limits of these small ranges which may independently be included in the smaller ranges is also encompassed, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included.

[0411]

[0148] Many modifications and other embodiments set forth herein will come to mind to one skilled in the art to which this subject matter pertains having the benefit of the 45

[0412] LEGAL02 / 47622964vl Atty. Dkt: 015444 / 638160

[0413] teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0414] 46

[0415] LEGAL02 / 47622964vl

Claims

Atty. Dkt: 015444 / 638160THAT WHICH IS CLAIMED:

1. An antibacterial composition comprising,a purified extract from Thuja arborvitae leaves, wherein the extract comprises at least 20% w / w of the compound 1:

2. The antibacterial composition of claim 1, further comprising a pharmaceutically acceptable excipient.

3. The antibacterial composition of claim 1, wherein the extract comprises at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% w / w of compound 1.

4. The antibacterial composition of claim 1, wherein the extract is prepared by extracting the leaves with alcohol to form a liquid extract and subjecting the liquid extract to column chromatography to prepare the purified extract.

5. The antibacterial composition of claim 4, wherein the alcohol is a C1-5 alcohol.

6. The antibacterial composition of claim 1, further comprising one or more compounds selected from the group consisting of:47LEGAL02 / 47622964vlAtty. Dkt: 015444 / 638160HOwherein, the total amount present of compounds 2, 3, 4 and 5 combined is less than 80% w / w.

7. An antibacterial composition comprising,48LEGAL02 / 47622964vlAtty. Dkt: 015444 / 638160a purified extract from Thuja arborvitae leaves, wherein the extract comprises at least 20% w / w of the compound 3:

8. The antibacterial composition of claim 7, further comprising a pharmaceutically acceptable excipient.

9. The antibacterial composition of claim 7, wherein the extract comprises at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% w / w of compound 1.

10. The antibacterial composition of claim 7, wherein the extract is prepared by extracting the leaves with an alcohol to form a liquid extract and subjecting the liquid extract to column chromatography to prepare the purified extract.

11. The antibacterial composition of claim 10, wherein the alcohol is a C1-5 alcohol.

12. The antibacterial composition of claim 7, further comprising one or more compounds selected from the group consisting of:49LEGAL02 / 47622964vlAtty. Dkt: 015444 / 6381602HOwherein, the total amount present of compounds 2, 1, 4 and 5 combined is less than 80% w / w.

13. The antibacterial composition of any one of claims 1-12, wherein the composition has a MIC value of about 10 to about 60 pg / ml against gram-positive (Staphylococcus aureus and Streptococcus mutans) and gram-negative (Acinetobacter baumannii. and 50LEGAL02 / 47622964vlAtty. Dkt: 015444 / 638160Escherichia coli) bacteria.

14. The antibacterial composition of any one of claims 1-12, wherein the composition has a MIC value of about 50 pg / ml against gram-positive (Staphylococcus aureus and Streptococcus mutans) and gram-negative (Acinetobacter baumannii, and Escherichia coli) bacteria.

15. A method of treating a bacterial infection in a subject comprising,administering the antibacterial composition of any one of claims 1-14 to the subject in need thereof.

16. The method of claim 15, wherein the bacterial infection involves Gram-negative bacteria.

17. The method of claim 16, wherein the gram-negative bacteria belongs to:(i) a phylum selected from the group consisting of Acidobacteria, Aquificae, Chlamydiae, Bacteroidetes, Chlorobi, Cyanobacteria, Fibrobacteres, Verrucomicrobia, Planctomycetes, and Spirochetes;(ii) a class selected from the group consisting of Alphaproteobacteria, Bacilli, Epsilonproteobacteria, Deltaproteobacteria, and Gammaproteobacteria; or(iii) an order selected from the group consisting of Hydrogenophilales, Methylophilales, Neisseriales, Nitrosomonadales, Procabacteriales, and Rhodocyclales.

18. The method of claim 17, wherein the Gram-negative bacterium is Acinetobacter baumannii or Steptococcus mutans.

19. The method of claim 15, wherein the bacterial infection involves Gram-positive bacteria.

20. The method of claim 19, wherein the Gram-positive bacteria is selected from the group consisting of: Staphylococcus aureus. Staphylococcus epidermidis.51LEGAL02 / 47622964vlAtty. Dkt: 015444 / 638160Staphylococcus sp. (Coagulase-negative), Streptococcus pneumoniae (Viridans group). Streptococcus agalactiae (group B). Streptococcus pyogenes, Enterococcus sp.. Bacillus anthracis. Bacillus oereus, Bifidobacteriu bifldum.Lactobacillus sp., Listeria monocytogenes, Nocardia sp., Rhodococcusequi (coccobacillus), Erysipelothrix rhusiopathiae, Corynebacterium diptheriae, Propionibacterium acnes, Actinomyces sp., Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Mobiluncus sp., and Peptostreptococcus sp.

21. The method of claim 20, wherein the Gram-positive bacterium is Staphylococcus aureus.

22. A method of reducing bacterial growth, comprising,contacting a bacterium with the antibacterial composition of any one of claims 1-14, wherein the growth of the bacterium is reduced.

23. The method of claim 22, wherein the bacterial infection involves Gram-negative bacteria.

24. The method of claim 23, wherein the Gram-negative bacteria belongs to:(i) a phylum selected from the group consisting of Acidobacteria, Aquificae, Chlamydiae, Bacteroidetes, Chlorobi, Cyanobacteria, Fibrobacteres, Verrucomicrobia, Planctomycetes, and Spirochetes;(ii) a class selected from the group consisting of Alphaproteobacteria, Bacilli. Epsilonproteobacteria, Deltaproteobacteria, and Gammaproteobacteria; or(iii) an order selected from the group consisting of Hydrogenophilales, Methylophilales, Neisseriales, Nitrosomonadales, Procabacteriales, and Rhodocyclales.

25. The method of claim 24, wherein the Gram-negative bacterium is Acinetobacter baumannii or Steptococcus mutans.

26. The method of claim 22, wherein the bacterial infection involves Gram-positive bacteria.52LEGAL02 / 47622964vlAtty. Dkt: 015444 / 63816027. The method of claim 26, wherein the Gram-positive bacteria is selected from the group consisting of Staphylococcus aureus, Staphylococcus epidermidis.Staphylococcus sp. (Coagulase-negative), Streptococcus pneumoniae (Viridans group), Streptococcus agalactiae (group B), Streptococcus pyogenes, Enterococcus sp., Bacillus anthracis, Bacillus oereus, Bifidobacteria bifidum, Lactobacillus sp., Listeria monocytogenes. Nocardia sp., Rhodococcusequi (coccobacillus), Erysipelothrix rhusiopathiae, Corynebacterium diptheriae, Propionibacterium acnes, Actinomyces sp., Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Mobiluncus sp., and Peptostreptococcus sp.

28. The method of claim 27, wherein the Gram-positive bacterium is Staphylococcus aureus.

29. A method of isolating an antibacterial compound from Thuja arborvitae leaves, comprising:contacting the leaves with a first solvent to produce a liquid extract; and, subjecting the liquid to column chromatography and eluting fractions to prepare a liquid eluent fraction enriched in at least one compound selected from53LEGAL02 / 47622964vlAtty. Dkt: 015444 / 638160HO30. The method of claim 29, wherein the first solvent is an organic solvent.

31. The method of claim 30, wherein the organic solvent is a C1-5 alcohol or ethyl acetate, or a mixture thereof.54LEGAL02 / 47622964vlAtty. Dkt: 015444 / 63816032. The method of claim 29, further comprising:allowing the liquid eluent fraction to undergo evaporation to prepare a concentrate.

33. The method of claim 32, further comprising:dissolving the concentrate in a mobile phase to prepare a liquid concentrate and subjecting the liquid concentrate to high performance liquid chromatography (HPLC) to prepare an isolate comprising at least one of compounds 1, 2, 3 4 or 5.

34. The method of claim 33, wherein the HPLC is reverse phase with a linear gradient of a C1-5 alcohol.

35. The method of claim 34, wherein the HPLC is reverse phase with a linear gradient of about 50% methanol in water-ethyl acetate from about 10% to about 100%.

36. The method of claim 33, wherein the isolate comprises compound 1, having the1wherein, compound 1 is present in an amount of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% w / w of the isolate.

37. The method of claim 33, wherein the isolate is an oil or a solid.

38. The method of claim 29, further comprising:after the contacting and prior to the subjecting.55LEGAL02 / 47622964vlAtty. Dkt: 015444 / 638160lyophilizing the extract to prepare a lyophilized extract; and, dissolving the lyophilized extract in a second solvent to prepare a liquid solution.

39. An isolate prepared by the method of claim 29, comprising one or more compounds selected from the group consisting of:56LEGAL02 / 47622964vlAtty. Dkt: 015444 / 638160andHO40. The isolate of claim 39, wherein the isolate comprises compound 1, having thewherein, compound 1 is present in an amount of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% w / w of the isolate.

41. The isolate of claim 39, wherein the isolate comprises compound 3, having the structure:57LEGAL02 / 47622964vlAtty. Dkt: 015444 / 638160wherein, compound 1 is present in an amount of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% w / w of the isolate.

42. An isolate from Thuja arborvitae leaves comprising compound 1, having the1wherein, compound 1 is present in an amount of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% w / w of the isolate.

43. An isolate from Thuja arborvitae leaves comprising compound 3, having the structure:58LEGAL02 / 47622964vlAtty. Dkt: 015444 / 638160wherein, compound 3 is present in an amount of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% w / w of the isolate.

44. An isolate from Thuja arborvitae leaves comprising compounds 1 and 3, and optionally compound 2, wherein compounds 1 and 3 combined are present in an amount of at least 40%, 50% 60%, 70%, 80%, 90% or 95% w / w of the isolate.59LEGAL02 / 47622964vl

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