DERIVED FROM PLANTS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of and priority to U.S. Patent Application No. 16/818,488 filed on March 13, 2020, which is a continuation-in-part application of U.S. Patent Application No. 14/333,279 filed on July 16, 2014, which claims benefit of and priority to U.S. Provisional Patent Application No. 61/846,784 filed on July 16, 2013, all of which are incorporated by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under AI24738 awarded by National Institutes of Health. The government has certain rights in the invention.

TECHNICAL FIELD OF THE INVENTION

This invention is generally related to compositions and methods of treating and preventing infections from alcohol-resistant human pathogens such as nonenveloped viruses and bacterial spores.

BACKGROUND OF THE INVENTION

Noroviruses are a group of single-stranded, positive sense RNA non-enveloped viruses constituting the Norovirus genus in the family Caliciviridae . Noroviruses have been recognized as the most important cause of viral epidemic acute gastroenteritis affecting people of all ages.

In the United States noroviruses cause 19 to 21 million cases of acute gastroenteritis each year, lead to 1.7 to 1.9 outpatient visits and 400,000 emergency department visits each year, and contribute to about 56,000 to 71,000 hospitalizations and 570 to 800 deaths, mostly among young children and the elderly. Norovirus is the leading cause of foodborne illness in the United States. On a worldwide basis noroviruses lead to 218,000 deaths in developing countries and 1.1 million episode of pediatric gastroenteritis in developed countries annually. Thus, norovirus associated diseases have been a heavy burden to public healthcare. Noroviruses are difficult to control owing to their widespread nature and the lack of effective vaccines and antivirals.

In 2011, the CDC updated the Guidelines for the Prevention and Control of Norovirus Gastroenteritis Outbreaks in Healthcare Settings. The current guideline for hand hygiene is “during outbreaks, use soap and water for hand hygiene after providing care or having contact with patients suspected or confirmed with norovirus gastroenteritis”. There is very low-quality evidence available to suggest that hand hygiene using alcohol-based hand sanitizers may reduce the likelihood of symptomatic norovirus infection. Since 2011, rather than a decline in the number of norovirus outbreaks reported to the CDC, there was a spike during 2013-2014, possibly due to the lack of alternatives other than alcohol-based hand sanitizer (CDC NoroSTAT, 2009-2015). In fact, the use of alcohol -based hand sanitizer was found to be a risk factor for norovirus spread vs. soap and water wash in long-term care facilities (Blaney, et ah, Am J Infect Control , 39(4):296-301 (2011); Stebbins, et ah, Pediatr Infect Dis J, 30(11):921-926 (2011)). Alcohol -based hand sanitizers are not effective against norovirus. In a human study, 30 second incubation with alcohol (3% - 95%) was ineffective in reducing the infectivity of norovirus (genomic copy reduction <0.5 logio), less effective than liquid soap or water rinse (Liu, et ah, Appl Environ Microbiol, 76(2):394-399 (2010)). Therefore, there is a need for improved hand hygiene approaches that are more effective than hand washing with water and soap, in order to reduce outbreaks and infections from norovirus and other nonenveloped or enveloped viruses.

It is an object of the invention to provide compositions and methods for of treating and preventing infections from nonenveloped or enveloped viruses.

It is another object of the invention to provide methods of preventing the transmission of nonenveloped or enveloped viruses.

It is yet another object of the invention to provide a germicidal composition that can rapidly kill or reduce the transmission of disease resulting from alcohol-resistant human pathogens such as non-enveloped viruses and bacterial spores.

SUMMARY OF THE INVENTION

Compositions and methods for rapidly killing or preventing the replication of alcohol- resistant human pathogens such as viruses and bacterial spores are disclosed. An exemplary composition includes alcohol, a modified green tea polyphenol, and other plant-derived ingredients. One embodiment provides a germicidal composition including 78% alcohol, 0.2% modified green tea polyphenol, 8% glycerin, and 14% water containing 0.4% Ultrez 20 and 0.04% triethanolamine, wherein the composition achieves at least a 4-log reduction in viral particles or bacterial spores within sixty seconds of contacting the viral particles or endospores.

In one embodiment, the modified green tea polyphenol is (-)-epigallocatechin-3-gallate (EGCG) modified at the 4’ position with palmitic acid in combination with alcohol. The compositions can also include one or more additional components selected from the group consisting of bioactive agents, therapeutic agents, excipients, carriers, fillers, additives, binders, disintegration agents, lubricants, flavoring agents, and combinations thereof.

Also disclosed are compositions and methods of killing or preventing the replication of viruses or bacterial spores in as few as 60 seconds, or as few as 30 seconds from contact. An exemplary method of rapidly killing, inactivating, or preventing the replication of viruses or bacterial spores includes steps of contacting viruses or spores with a composition having at least one modified green tea polyphenol, one additional plant-derived compound, and ethanol, in an amount effective to prevent or reduce replication and transmission of the viruses or endospores.

Compositions and methods of sterilizing objects or surfaces suspected of being contaminated with viruses and endospores are also disclosed herein. The methods include steps of contacting the object or surface with a composition having at least one modified green tea polyphenol, one additional plant-derived compound, and alcohol in an amount effective to sterilize the object or surface, wherein at least a 4-log reduction in viral particles or bacterial spores is achieved within sixty seconds of contacting the object or surface with the composition. One embodiment provides hand hygiene products such as hand sanitizers and surgical hand rubs that include the disclosed compositions.

The compositions and methods can be used in a variety of applications, for example, to decontaminate a device or surface contaminated with viruses or spores. Exemplary objects or surfaces that can be decontaminated or sterilized include but are not limited to a food item, a food preparation surface, a food contact surface, a surgical surface, a surgical tool, a medical device, a hospital or veterinary facility surface, hospital and surgical linens and garments, or a skin surface.

BRIEF DESCRIPTION OF THE DRAWING

Figure l is a bar graph showing Logio reduction of spore re-germination by media (control), different concentrations of EtOH (70%, 78%, and 85%), or 0.2% EGCG-P with different concentrations of EtOH after 60 seconds treatment. Means are shown with standard deviation (n=3). This result shows that alcohol plus EGCG-P (a modified EGCG with palmitate ester) at various concentrations failed to reduce spore germination by >4 logio.

Figure 2 is a bar graph showing Logio reduction of spore re-germination by five EGCG-P formulations and positive control (80% EtOH) after 60 seconds treatment. Means are shown with standard deviation (n=3). This result shows that with additional plant-derived compound in FI (glycerol added) and F2 (citric acid added), the spore germination was reduced by 4 logio.

Figures 3 A-3F are scanning electron microscope images showing B. cereus and C. sporogenes endospores untreated (Figs. 3 A and 3D, respectively), FI -treated (3B and 3E, respectively), or F-2 treated (Figs. 3C and 3F, respectively). This result demonstrates that the bacterial spores were rpidly damaged by FI and F2 formulations.

Figure 4 is a bar graph showing Logio reduction of spore re-germination by improved formulations #1 (cFl) & #2 (cF2) after 30 seconds. cFl-N and cF2-N refer to the formulations neutralized with PBS (1:9 v/v). Mean are shown with standard deviation (n=3). This result show that with further improvement with the combinations of plant-derived compounds, these alcohol- based formulations reached >5 i _ogi o reduction of spore germination.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

It should be appreciated that this disclosure is not limited to the compositions and methods described herein as well as the experimental conditions described, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing certain embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any compositions, methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All publications mentioned are incorporated herein by reference in their entirety.

The use of the terms "a," "an," "the," and similar referents in the context of describing the presently claimed invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.

Use of the term "about" is intended to describe values either above or below the stated value in a range of approx. +/- 10%; in other embodiments the values may range in value either above or below the stated value in a range of approx. +/- 5%; in other embodiments the values may range in value either above or below the stated value in a range of approx. +/- 2%; in other embodiments the values may range in value either above or below the stated value in a range of approx. +/- 1%. The preceding ranges are intended to be made clear by context, and no further limitation is implied. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non- claimed element as essential to the practice of the invention.

As used herein, “virus” refers to a particle composed of nucleic acids surrounded by proteins.

Enveloped viruses, which include human immunodeficiency virus (HIV), hepatitis B virus (HB V), influenza viruses, coronavirus, and herpes simplex virus (HSV), are considered more sensitive to germicides. Their characteristics depend on structure including the external lipid bilayer envelope, which contains proteins (usually glycoproteins, or proteins with linked carbohydrate groups). The infectious unit of a virus is the virion, envelope + nucleocapsid. Enveloped viruses are easily destroyed by agents affecting lipids such as alcohols, ether, 2- phenlphenol, cationic surfactants, and chlorhexidine.

Non-enveloped viruses, which include the norovirus, poliovirus, human papilloma virus (HPV), enterovirus (EV), adenovirus, and human hepatitis A virus (HAV), are composed of a nucleocapsid without an envelope. In these viruses, the virion is the nucleocapsid itself. The small, non-enveloped viruses are extremely resistant to most disinfectants. Despite the lack of a lipid envelope, these organisms have a very resistant viral capsid which is made out of protein. The protein capsid is resistant to both lipophilic disinfectants (i.e. quaternary ammonium compounds) as well as solvents (i.e., alcohol). As used herein, “Norovirus” refers to a group of single-stranded, positive sense RNA non-enveloped viruses constituting the Norovirus genus in the family Caliciviridae.

Transmission of Norovirus occurs primarily through contaminated food or water, but also through person-to-person contact and exposure to objects that have been contacted with the virus. Symptoms of norovirus include fever, cramps, head and body aches, along with profound gastroenteritis, diarrhea and vomiting. Symptoms can arise gradually or abruptly and usually resolve within 48 to 72 hours. There are currently no treatments for norovirus. During an active norovirus infection it is important for the infected person to intake a sufficient amount of fluids to avoid dehydration. Intravenous fluid delivery is necessary if the infected person cannot drink enough fluids. Loss of fluid due to vomiting and diarrhea can lead to severe dehydration, and if untreated, more severe complications and even death.

As used herein, “viral gastroenteritis” refers to a viral infection of the intestines that causes inflammation, swelling, and irritation to the lining of the intestines. Symptoms of viral gastroenteritis include but are not limited to fever, body aches and cramps, nausea, vomiting, diarrhea, and stomach pain. Common viruses that cause viral gastroenteritis include but are not limited to norovirus, sapovirus, rotavirus, and adenovirus.

As used herein “spore” refers to a reproductive cell capable of developing into a new individual without fusion with another reproductive cell. Spores are produced by bacteria, fungi, algae, and plants. Some bacteria, such as various Bacilli and Clostridia species, are able to undergo a sporulation process under prolonged and unfavorable conditions to form dormant spores. Spores have features which enable them to become resistant to various harsh environmental conditions, including heat, chemical solvents, and UV radiation, potentially surviving for hundreds of years or even longer.

As used herein, “virucide” refers to any physical or chemical agent that deactivates or destroys viruses. “Virucidal” refers to the ability to deactivate or destroy viruses.

As used herein, “germicide” refers to a substance or process that kills bacteria, viruses, bacterial spores and other microorganisms that can cause infection and disease.

As used herein, the term “sporicidal” refers to the ability to kill spores.

As used herein, the term “sterilization” refers to a process that destroys or eliminates all forms of microbial life, including bacterial spores. “Disinfection” refers to a process that eliminates many or all pathogenic microorganisms, except bacterial spores, on inanimate objects. Unlike sterilization, disinfection is not sporicidal. A few disinfectants will kill spores with prolonged exposure times (3-12 hours); these are called chemical sterilants. Sterilization and disinfection are important techniques used in the medical field to prevent the transmission of microbes to patients.

As used herein, the terms “treat”, “treating”, “treatment” and “therapeutic use” refer to the elimination, reduction, or amelioration of one or more symptoms of a disease or disorder.

As used herein, the term “prophylactic agent” refers to an agent that can be used in the treatment of a disorder or disease prior to the detection of any symptom of such disorder or disease. A “prophylactically effective” amount is the amount of prophylactic agent sufficient to mediate such protection. A prophylactically effective amount may also refer to the amount of the prophylactic agent that provides a prophylactic benefit in the prevention of disease.

As used herein, the term “effective amount” and “therapeutically effective amount” refer to the amount of a therapeutic agent sufficient to mediate a clinically relevant elimination, reduction or amelioration of such symptoms. An effect is clinically relevant if its magnitude is sufficient to impact the health or prognosis of a recipient subject. A therapeutically effective amount may refer to the amount of therapeutic agent sufficient to delay or minimize the onset of disease, e.g., delay or minimize the spread of cancer. A therapeutically effective amount may also refer to the amount of the therapeutic agent that provides a therapeutic benefit in the treatment or management of a disease.

As used herein, the terms “individual,” “subject,” and “patient” are used interchangeably, and refer to a mammal, including, but not limited to, humans, rodents, such as mice and rats, and other laboratory animals.

The term “Green Tea Polyphenols” and “GTP” refers to polyphenolic compounds present in the leaves of Camellia sinensis. Green tea polyphenols include, but are not limited to (-)- epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin-3-gallate (ECG), (-)- epigallocatechin-3-gallate (EGCG), proanthocyanidins, enantiomers thereof, epimers thereof, isomers thereof, combinations thereof, and prodrugs thereof. Modified green tea polyphenols refers to a green tea polyphenol having one or more hydrocarbon chains, for example Ci to C3 ₀ and the compounds according to Formula I and II disclosed herein.

“Lipid soluble” as used herein refers to substances that have a solubility of greater than or equal to 5g / 100ml in a hydrophobic liquid such as castor oil. The term “lipid-soluble green tea polyphenol” and “LTP” refers to a green tea polyphenol having one or more hydrocarbon chains having for example Ci to C30 groups linked to the polyphenol. Ci to C30 groups include for example cholesterol. Representative lipid-soluble green tea polyphenols include those according to Formula I and Formula II disclosed herein.

The term is used interchangeably with “modified green tea polyphenol”.

As used herein, “hand hygiene” is one of four standard precautions that are used in addition to disinfection and sterilization to prevent against disease transmission in a hospital or clinical setting. Hand hygiene means cleaning ones hands by using either handwashing (washing hands with soap and water), antiseptic hand wash, antiseptic hand rub (i.e. alcohol-based hand sanitizer including foam or gel), or surgical hand antisepsis. The Centers for Disease Control and Prevention state that alcohol-based hand sanitizers are the most effective products for reducing the number of germs on the hands of healthcare providers and are the preferred method for cleaning ones hands in most clinical situations.

“Log reduction” refers to the measurement of how thoroughly a decontamination process reduces the concentration of a contaminant. It is defined as the common logarithm of the ratio of levels of contamination before and after the process. An increment of 1 corresponds to a reduction in concentration by a factor of 10. So for example, a 0-log reduction is no reduction at all, while a 1-log reduction corresponds to a reduction of 90 percent from the original concentration, and a 2-log reduction corresponds to a reduction of 99 percent from the original concentration, etc.

II. Compositions and Methods of Improving the Germicidal Activity of Alcohols

Alcohols, such as ethanol, possess antimicrobial activities. Alcohols are able to inactivate many bacteria and viruses. Therefore, alcohol-based hand hygiene products/surface disinfectants are able to effectively eliminate pathogens sensitive to alcohols. However, some species of pathogenic microorganisms such as alcohol-resistant viruses and bacterial spores are not sensitive to alcohols. These pathogens pose higher risks to human population due to their resistant to alcohols and other conventional disinfectants. Among these alcohol-resistant human pathogens, non-enveloped viruses and bacterial spores are associated with high transmission rate and mortality.

Disclosed herein are alcohol-based compositions including plant-derived, non-toxic compounds that are useful in rapidly killing or inactivating alcohol -resistant human pathogens such as but not limited to enveloped and nonenveloped viruses and bacterial spores. In one embodiment, the disclosed alcohol-based compositions rapidly kill, inactivate, or otherwise reduce viruses in as few as 60 seconds. In another embodiment, the disclosed alcohol-based compositions and methods achieve at least a 4-log reduction in viruses within sixty seconds of contacting the object or surface with the composition. In another embodiment, the disclosed alcohol-based compositions and methods achieve at least a 5-log reduction in bacterial spore germination within thirty seconds of contacting the object or surface with the composition.

A. Alcohol-Based Germicidal Compositions

Methods of rapidly killing, inactivating or otherwise reducing the number of viruses, or inhibiting, reducing, or preventing viral replication typically include contacting viruses, or a surface thought to be contaminated by viruses, with one or more of the alcohol-based compositions described herein. The disclosed germicidal compositions can also can be used to rapidly kill, inactivate, or otherwise reduce bacterial spores. The alcohol-based compositions include two or more plant-based components or ingredients. The compositions can also include additional active agents, carriers, devices, fillers, etc., as discussed in more detail below.

The disclosed compositions can be suitable for use as a hand-hygiene product, a surface sanitizer, or an antiseptic depending on the additional components or ingredients added to the composition, and one of skill in the art can select the additional components based on the intended use. It will also be appreciated that if the composition is to be used for coating surgical or medical devices any additional active or inert components of the composition should be compatible with the intended use of the surgical or medical device, for example, introduction or implantation into or onto the body of the subject.

1. Green Tea Polyphenols and Modified Green Tea Polyphenols

In one embodiment, the disclosed alcohol-based compositions include at least one green tea polyphenols, preferably one or more green tea polyphenols modified with one or more hydrocarbon chains having Ci to C30 groups. Representative green tea polyphenols include, but are not limited to (-)-epigallocatechin-3-gallate, (-)-epicatechin, (-)-epigallocatechin, and (-)- epicatechin-3-gallate. Preferred modified GTPs include modified (-)-epigallocatechin-3-gallate, a pharmaceutically acceptable salt, prodrug, or derivative thereof.

A modified green tea polyphenol, a derivative or a variant of a green tea polyphenol includes green tea polyphenols having chemical modifications to increase solubility or bioavailability in a host. In certain embodiments, these chemical modifications include the addition of chemical groups having a charge under physiological conditions. In other embodiments the modifications include the conjugation of the green tea polyphenol to other biological moieties such as polypeptides, carbohydrates, lipids, or a combination thereof. Preferred modifications include modifications with one or more hydrocarbon chains having Ci to C30 groups.

Another embodiment provides an alcohol-based germicidal composition including one or more green tea polyphenols, modified green tea polyphenols, optionally in combination with one or more of a pharmaceutically acceptable carrier, diluent, excipient, filler, or other inert or active agents. In some embodiments, the active ingredient in the composition consists essentially of (- )-epigallocatechin-3-gallate, (-)-epigallocatechin-3-gallate modified with one or more hydrocarbon chains having Ci to C30 groups, or a combination thereof, a pharmaceutically acceptable salt or prodrug thereof. The active ingredient can be in the form a single optical isomer. Typically, one optical isomer will be present in greater than 85%, 90%, 95%, or 99% by weight compared to the other optical isomer. It will be appreciated that the composition can also include at least one additional active ingredient, for example a second therapeutic. Additional description of the disclosed pharmaceutical compositions is provided below.

Green tea polyphenols have poor solubility in lipid medium. Therefore, lipophilic tea polyphenols are also disclosed for use in lipid-soluble medium. Lipophilic tea polyphenols (LTP or Modified green tea polyphenols) can be prepared by catalytic esterification of a green tea polyphenols (GTP).

Compositions containing green tea polyphenols modified to increase the permeability of the green tea polyphenols to skin and cell membranes or increase their solubility in hydrophobic media relative to unmodified green tea polyphenols are therefore provided. Green tea polyphenols that can be modified include, but are not limited to (-)-epicatechin (EC), (-)- epigallocatechin (EGC), (-)-epicatechin-3-gallate (ECG), (-)-epigallocatechin-3-gallate (EGCG), proanthocyanidins, enantiomers thereof, epimers thereof, isomers thereof, combinations thereof, and prodrugs thereof. One embodiment provides a green tea polyphenol having an ester-linked Ci to C30 hydrocarbon chain, for example a fatty acid, at one or more positions. Another embodiment provides a green tea polyphenol having one or more cholesterol groups linked to the polyphenol. The cholesterol group can be linked for example by an ether linkage directly to the polyphenol or a Ci to Cio linker can connect the cholesterol group to the polyphenol.

Another embodiment provides a green tea polyphenol compound having one or more acyloxy groups, wherein the acyl group is Ci to C3 _0. It is believed that the addition of alkyl, alkenyl, or alkynyl chains, for example via fatty acid esterification, to green tea polyphenols increases the stability of the green tea polyphenols and increases the solubility of the green tea polyphenols in hydrophobic media including lipids, fats, soaps, detergents, surfactants or oils compared to unmodified green tea polyphenols. Green tea polyphenols having one or more hydrocarbon chains, for example ester-linked Ci to C3 ₀ groups or Ci to C ₃₀ acyloxy groups are believed to more permeable to skin or cell membranes and thereby enable the ester-linked hydrocarbon chain containing or acyloxy containing green tea polyphenol to readily enter a cell and have a biological effect on the cell, for example modulating gene expression, compared to unmodified green tea polyphenols.

It will be appreciated that one or more hydrocarbon chains can be linked to the green tea polyphenol using linkages other than ester linkages, for example thio-linkages. Esterified green tea polyphenols can be combined with oils, detergents, surfactants, or combinations thereof to produce compositions which clean the skin and deliver green tea polyphenols to the skin. The oils, detergents, or surfactants advantageously increase the stability of green tea polyphenols by reducing contact of the green tea polyphenols with aqueous media. Certain embodiments provide single optical isomers, enantiomers, or epimers of the disclosed modified green tea polyphenols. Other embodiments provide compositions containing single optical isomers, enantiomers, or epimers or the disclosed modified green tea polyphenols.

One embodiment provides a compound according to Formula I: wherein Ri, R2, R ₃ , R4, Rs, and R7 are each independently H, OH, wherein Rs is a linear, branched or cyclic, saturated or unsaturated, substituted or unsubstituted C1-C30 group, wherein if Rx is cyclic, Rx is a C3-C30 group; and

R6 is O, -NR9R10, or S, wherein R9 and Rio are independently hydrogen, or a linear, branched, or cyclic, saturated or unsaturated, substituted or unsubstituted C1-C30 group, wherein if R9 and/or Rio are cyclic, R9 and/or Rio are C3-C30 groups; wherein at least one of Ri, R2, R3, R4, Rs, R7, R9, or Rio is or a pharmaceutically acceptable salt or prodrug thereof, optionally in combination with an excipient.

In preferred embodiments of Formula I, Rx is a linear or branched alkyl chain. In more preferred embodiments of Formula I, Rx is a linear or branched C16-C25 alkyl group. In particularly preferred embodiments of Formula I, Rx is a C17H35 group.

One embodiment provides a compound according to Formula I as described above, provided R.4 is not when Ri, R2, R3, Rs, and R7 are OH; or a pharmaceutically acceptable salt or prodrug thereof, optionally in combination with an excipient.

One embodiment provides a compound according to Formula I as described above

O O wherein at least two of Ri, R2, R3, R4, Rs, or R7 are independently , _? · or a pharmaceutically acceptable salt or prodrug thereof, optionally in combination with an excipient. Another embodiment provides a compound according to Formula I as described above

O O wherein at least three of Ri, R2, R3, R4, Rs, or R7 are independently or a pharmaceutically acceptable salt or prodrug thereof, optionally in combination with an excipient.

Still another embodiment provides a compound according to Formula I as described

O above wherein at least four of Ri, R2, R3, R4, Rs, or R7 are independently ® ^R s , or O

0-R ₈ · or a pharmaceutically acceptable salt or prodrug thereof, optionally in combination with an excipient.

Another embodiment provides a compound according to Formula II:

Formula II wherein Ri, R2, R3, R4, R7, Rx, R9, and Rio are each independently H, OH,

R11 is a linear, branched, or cyclic, saturated or unsaturated, substituted or unsubstituted C1-C30 group, wherein if R11 is cyclic, R11 is a C3-C30 group;

R5 and R6 are independently O, -NR12R13 or S, wherein R12 and R13 are independently hydrogen, or a linear, branched, or cyclic, saturated or unsaturated, substituted or unsubstituted C1-C30 group, wherein if R12 and/or R13 are cyclic, R12 and/or R13 are C3-C30 groups; and wherein at least one of Ri, R2, R3, R4, R7, Rx, R9, and Rio are independently or a pharmaceutically acceptable salt or prodrug thereof, optionally in combination with an excipient.

In preferred embodiments of Formula II, Rn is a linear or branched alkyl chain. In more preferred embodiments of Formula II, Rn is a linear or branched C16-C25 alkyl group. In particularly preferred embodiments of Formula II, R11 is a C17H35 group.

Another embodiment provides a compound according to Formula II wherein at least two

O O of Ri, R2, R3, R4, R7, R8, R9, and Rio are independently — O— ¹ R

Ί1 or O R ^-1111 · or a pharmaceutically acceptable salt or prodrug thereof, optionally in combination with an excipient.

Another embodiment provides a compound according to Formula II as described above

O wherein at least three of Ri, R2, R3, R4, R7, Rx, R9, and Rio are independently 1 , or

O

^{0-Rl 1} ;optionally in combination with an excipient.

Another embodiment provides a compound according to Formula II as described above

O wherein at least four of Ri, R2, R3, R4, R7, Rx, R9, and Rio are independently or

O

^L O-R ₁₁ optionally in combination with an excipient.

Another embodiment provides a compound according to Formula II wherein Ri, R2, R3, R4, R7, R8, R9, and Rio are each independently H, OH,

R11 is a linear, branched, or cyclic, saturated or unsaturated, substituted or unsubstituted C1-C30 group, wherein if R11 is cyclic, R11 is a C3-C30 group;

R5 and R6 are independently O, -NR12R13 or S, wherein R12 and R13 are independently hydrogen, or a linear, branched, or cyclic, saturated or unsaturated, substituted or unsubstituted C1-C30 group, wherein if R12 and/or R13 are cyclic, R12 and/or R13 are C3-C30 groups; and wherein at least one of Ri, R2, R3, R4, R7, Rx, R9, and Rio are independently o and wherein R.4 is not when Ri, R2, R3, R7, Rs, R9, and Rio are OH; or a pharmaceutically acceptable salt or prodrug thereof, optionally in combination with an excipient.

Another embodiment provides a composition including a compound according to Formula II wherein at least two of Ri, R2, R3, R4, R7, Rx, R9, and Rio are independently or a pharmaceutically acceptable salt or prodrug thereof, optionally in combination with an excipient.

Another embodiment provides a composition including a compound according to Formula II as described above wherein at least three of Ri, R2, R3, R4, R7, Rx, R9, and Rio are

O o independently , optionally in combination with an excipient.

Another embodiment provides a composition including a compound according to Formula II as described above wherein at least four of Ri, R2, R3, R4, R7, Rx, R9, and Rio are independently , optionally in combination with an excipient.

Another embodiment provides a composition including a compound according to Formula II wherein Ri, R2, R3, R4, R7, Rx, R9, and Rio are each independently H, OH,

R11 is a linear, branched, or cyclic, saturated or unsaturated, substituted or unsubstituted C1-C30 group, wherein if R11 is cyclic, R11 is a C3-C30 group;

R5 and R6 are independently O, -NR12R13 or S, wherein R12 and R13 are independently hydrogen, or a linear, branched, or cyclic, saturated or unsaturated, substituted or unsubstituted C1-C30 group, wherein if R12 and/or R13 are cyclic, R12 and/or R13 are C3-C30 groups; and wherein at least one of Ri, R2, R3, R4, R7, Rx, R9, and Rio are independently o and wherein R.4 is not when Ri, R2, R3, R7, Rs, R9, and Rio are OH; or a pharmaceutically acceptable salt or prodrug thereof, optionally in combination with an excipient.

One embodiment provides a compound according to Formula III:

Formula III wherein Ri, R2, R3, R4, Rs, and R7 are each independently H, OH, wherein Rs is a linear or branched C1 ₆ -C25 alkyl group.

R6 is O, -NR9R10, or S, wherein R9 and Rio are independently hydrogen, or a linear, branched, or cyclic, saturated or unsaturated, substituted or unsubstituted C1-C30 group, wherein if R9 and/or Rio are cyclic, R9 and/or Rio are C3-C30 groups; wherein at least one of Ri, R2, R3, R4, Rs, R7, R9, or Rio is or a pharmaceutically acceptable salt or prodrug thereof, optionally in combination with an excipient.

In particularly preferred embodiments of Formula III, Rs is a C17H35 group.

One embodiment provides a compound according to Formula III as described above, wherein one or more of Ri, R2, R3, R4, Rs, or R7 is

— 0-U-C ₁₇ H _{35 or} — LO-C _{1 7} H ₃₅ . or a pharmaceutically acceptable salt or prodrug thereof, optionally in combination with an excipient. One embodiment provides a compound according to Formula III as described above,

O wherein at least two of Ri, R2, R3, R4, Rs, or R7 are independently ₀ r or a pharmaceutically acceptable salt or prodrug thereof, optionally in combination with an excipient.

Another embodiment provides a compound according to Formula III as described above

O wherein at least three of Ri, R2, R3, R4, Rs, or R7 are independently ₀ r o

— ^{1 L} O-C ₁₇ H ₃₅ . or a pharmaceutically acceptable salt or prodrug thereof, optionally in combination with an excipient.

Still another embodiment provides a compound according to Formula III as described

O above wherein at least four of Ri, R2, R3, R4, Rs, or R7 are independently ^{0 C} 17 ^H 35 ₀ r or a pharmaceutically acceptable salt or prodrug thereof, optionally in combination with an excipient.

Another embodiment provides a compound according to Formula IV: wherein Ri, R2, R3, R4, R7, Rx, R9, and Rio are each independently H, OH, R11 is a linear or branched C16-C25 alkyl group;

R5 and R6 are independently O, -NR12R13 or S, wherein R12 and R13 are independently hydrogen, or a linear, branched, or cyclic, saturated or unsaturated, substituted or unsubstituted C1-C30 group, wherein if R12 and/or R13 are cyclic, R12 and/or R13 are C3-C30 groups; and wherein at least one of Ri, R2, R3, R4, R7, Rx, R9, and Rio are independently or a pharmaceutically acceptable salt or prodrug thereof, optionally in combination with an excipient.

In particularly preferred embodiments of Formula IV, R11 is a C17H35 group.

One embodiment provides a compound according to Formula IV as described above, wherein one or more of Ri, R2, R3, R4, R7, Rs, R9, and Rio is or a pharmaceutically acceptable salt or prodrug thereof, optionally in combination with an excipient.

Another embodiment provides a compound according to Formula IV wherein at least two of Ri, If _! , R- ₃ , R- ₄ , If _? , R-8, If _? , and Rio are independently or a pharmaceutically acceptable salt or prodrug thereof, optionally in combination with an excipient.

Another embodiment provides a compound according to Formula IV as described above

O wherein at least three of Ri, R2, R3, R4, R7, Rs, R9, and Rio are independently 7 ^H 35 , ₀ r optionally in combination with an excipient.

Another embodiment provides a compound according to Formula IV as described above

O wherein at least four of Ri, R2, R3, R4, R7, Rx, R9, and Rio are independently — O C ₁₇ H 35, or

O _· optionally in combination with an excipient _. Another embodiment provides a composition including a compound according to

Formula IV wherein at least one of Ri, R2, R3, R4, R7, Rx, R9, and Rio are independently or a pharmaceutically acceptable salt or prodrug thereof, optionally in combination with an excipient.

Another embodiment provides a composition including a compound according to

Formula IV wherein at least two of Ri, R2, R3, R4, R7, Rx, R9, and Rio are independently or a pharmaceutically acceptable salt or prodrug thereof, optionally in combination with an excipient.

Another embodiment provides a composition including a compound according to Formula IV as described above wherein at least three of Ri, R2, R3, R4, R7, Rx, R9, and Rio are O O independently ; optionally in combination with an excipient.

Another embodiment provides a composition including a compound according to Formula IV as described above wherein at least four of Ri, R2, R3, R4, R7, Rx, R9, and Rio are independently ^. optionally in combination with an excipient _.

In one embodiment, a green tea polyphenol esterified with one fatty acid is provided. Another embodiment provides a green tea polyphenol esterified with at least two fatty acids. Certain embodiments provide a green tea polyphenol esterified with one or more fatty acids having a hydrocarbon chain greater than 16 carbons. Some embodiments provide a green tea polyphenol esterified with one or more fatty acids having a hydrocarbon chain of between 17 and 25 carbons in length. Some embodiments provide a green tea polyphenol esterified with one or more stearic acid or palmitic acid chains.

Representative green tea polyphenols include, but are not limited to (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin-3-gallate (ECG), (-)-epigallocatechin-3-gallate (EGCG). Representative fatty acids include, but are not limited to butanoic acid, hexanoic acid, octanoic acid, decanoic acid, dodecanoic acid, tetradecanoic acid, hexadecanoic acid (palmitic acid), 9-hexadecenoic acid, octadecanoic acid (stearic acid), 9-octadecenoic acid, 11- octadecenoic acid, 9,12-octadecadienoic acid, 9,12,15-octadecatrienoic acid, 6,9,12- octadecatrienoic acid, eicosanoic acid, 9-eicosenoic acid, 5,8,11,14-eicosatetraenoic acid,

5,8,11, 14, 17-eicosapentaenoic acid, docosanoic acid, 13-docosenoic acid, 4,7, 10,13,16,19- docosahexaenoic acid, and tetracosanoic acid. a. Methods of Esterifying Green Tea Polyphenols Lipid esters of EGCG can be formed either enzymatically or chemically (Chen, et al., Journal of Zhejiang University Science. 2003; 6:714-718).

EGCG-ester was purified previously by Chen et al in China. This was accomplished from a catalytic esterification between green tea polyphenols and Cl 6-fatty acid. The esterification was obtained by mixing 4 grams of green tea polyphenols and 6.5 grams of hexadecanoyl chloride. Next, 50 mLs of ethyl acetate and a catalyst at 40°C were added to the mixture. After 3 hours of stirring, the solution was washed three times with 30 mLs of deionized water. The organic layer was then allowed to evaporate and further dried by using a vacuum at 40°C. This resulted in 8.7g of powder product. A schematic of the synthesis of a likely esterification between GTP and Hexadecanoyl Chloride is shown below. (Chen, et al., Journal of Zhejiang University Science, 2003; 6:714-718.) galloyl

Next, high current chromatography separation was used to purify the EGCG-ester product. A two-phase solvent composed of (1:1) n-hexane-ethyl acetate-methanol-water was used in the separation column. Five grams of EGCG-ester was dissolved in 50 mL of the upper phase solution. After purification and HPLC analysis, it was seen that EGCG ester was successfully purified. The structure of an EGCG acyl -derivative is shown below. (Chen, et ah, Journal of Zhejiang University Science, 2003; 6:714-718.)

In a preferred embodiment, EGCG is esterified at the 4' position according to the structure above with stearic acid (as shown) or palmitic acid.

2. Bioactive Ingredients

Alcohol-based germicidal compositions containing the disclosed modified green tea polyphenols optionally include one more bioactive agents or additional therapeutic agents. In certain embodiments, one or more bioactive agents can be conjugated to the green tea polyphenol. Bioactive agents include therapeutic, prophylactic and diagnostic agents. These may be organic or inorganic molecules, proteins, peptides, sugars, polysaccharides, tea saponin, vitamins, cholesterol, or nucleic acid molecules. Representative vitamins include, but are not limited to lipid soluble vitamins such as vitamin D, vitamin E, or combinations thereof.

Examples of therapeutic agents include proteins, such as hormones, antigens, and growth effector molecules; nucleic acids, such as antisense molecules; and small organic or inorganic molecules such as antimicrobials, antihistamines, immunomodulators, decongestants, neuroactive agents, anesthetics, amino acids, and sedatives.

Various active agents that can be used in combination with the disclosed modified green tea polyphenol compositions are disclosed in U.S. Published Application Nos. 2012/0172423 and 2012/0076872 each of which are specifically incorporated by reference herein in their entities. The active agents can be, for example, anti-fungal agents, anti -bacterial agents, antiseptic agents, skin protectants, anti -psoriasis agents, local anesthetics, antihistamines, and antioxidants.

In one embodiment, the composition includes one or more additional antibacterial agents. A variety of known antibacterial agents can be used to prepare the described compositions. A list of potential antibacterial agents can be found in “Martindale - The Complete Drug Reference”, 32nd Ed., Kathleen Parfitt, (1999) on pages 112-270. Classes of useful antibacterials include aminoglycosides, antimycobacterials, cephalosporins and beta-lactams, chloramphenicols, glycopeptides, lincosamides, macrolides, penicillins, quinolones, sulphonamides and diaminopyridines, tetracyclines, and miscellaneous. In a preferred embodiment, the antibacterial agent is selected from the group consisting of metronidazole, timidazole, secnidazole, erythromycin, bactoban, mupirocin, neomycin, bacitracin, cicloprox, fluoriquinolones, ofloxacin, cephalexin, dicloxacillin, minocycline, rifampin, famciclovir, clindamycin, tetracycline and gentamycin.

Suitable aminoglycosides include antibiotics derived from Streptomyces and other actinomycetales, including streptomycin, framycetin, kanamycin, neomycin, paramomycin, and tobramycin, as well as gentamycin, sissomycin, netilmycin, isepamicin, and micronomycin.

Suitable antimycobacterials include rifamycin, rifaximin, rifampicin, rifabutinisoniazid, pyrazinamide, ethambutol, streptomycin, thiacetazone, aminosalicylic acid, capreomycin, cycloserine, dapsone, clofazimine, ethionamide, prothionamide, ofloxacin, and minocycline.

Cephalosporins and beta-lactams generally have activity against gram-positive bacteria and newer generations of compounds have activity against gram -negative bacteria as well. Suitable cephalosporins and beta-lactams include: First generation; cephalothin, cephazolin, cephradine, cephaloridine, cefroxadine, cephadroxil, cefatrizine, cephalexin, pivcephalexin, cefaclor, and cefprozil; second generation; cephamandole, cefuroxime axetil, cefonicid, ceforanide, cefotiam, and cephamycin; third generation; cefotaxime, cefmenoxime, cefodizime, ceftizoxime, ceftriaxone, cefixime, cefdinir, cefetamet, cefpodoxime, ceftibuten, latamoxef, ceftazidime, cefoperazone, cefpiramide, and cefsulodin; and fourth generation: cefepime and cefpirome.

Other cephalosporins include cefoxitim, cefmetazole, cefotetan, cefbuperazone, cefminox, imipenem, meropenem, aztreonam, carumonam, and loracarbef.

Chloramphenicols inhibit gram positive and gram negative bacteria. Suitable cloramphenicols include chloramphenicol, its sodium succinate derivative, thiamphenicol, and azidamfenicol.

Suitable glycopeptides include vancomycin, teicoplanin, and ramoplanin. Suitable lincosamides include lincomycin and clindamycin, which are used to treat primarily aerobic infections. Macrolides have a lactam ring to which sugars are attached. Suitable macrolides include erytjhromycin, as well as spiromycin, oleandomycinjosamycin, kitamycin, midecamycin, rokitamycin, azithromycin, clarithromycin, dirithromycin, roxithromycin, flurithromycin, tylosin; and streptgramins (or synergistins) including pristinamycin, and virginiamycin; and combinations thereof.

Suitable penicillins include natural penicillin and the semisynthetic penicillins F, G, X, K, and V. Newer penicillins include phenethicillin, propicillin, methicilin, cloxacillin, dicloxacillin, flucloxacillin, oxacillin, nafcillin, ampicillin, amoxicillin, bacampicillin, hetacillin, metampicillin, pivampicillin, carbenecillin, carfecillin, carindacillin, sulbenecillin, ticarcillin, azlocillin, mezlocillin, piperacillin, temocillin, mecillinam, and pivemecillinam. Lactamase inhibitors such as clavulanic acid, sulbactam, and tazobacytam are often co-administered.

Suitable quinolones include nalidixic acid, oxolinic acid, cinoxacin, acrosoxacin, pipemedic acid, and the fluoroquinolones flumequine, ciprofloxacin, enoxacin, fleroxacin, grepafloxacin, levofloxacin, lomefloxacin, nadifloxacin, norfloxacin, ofloxacin, pefloxacin, rufloxacin, sparfloxacin, trovafloxacin, danofloxacin, enrofloxacin, and marbofloxacin.

Sulphonamides and diaminopyridines include the original of the “sulfa” drugs, sulphanilamide, and a large number of derivatives, including sulfapyridine, sulfadiazine, sulfafurazole, sulfamethoxazole, sulfadimethoxine, sulfadimethoxydiazine, sulfadoxine, sulfametopyrazine, silver sulfadiazine, mafenide acetate, and sulfasalizine, as well as related compounds including trimethoprim, baquiloprim, brodimoprim, ormetoprim, tetroxoprim, and in combinations with other drugs such as co-trimoxazole.

Tetracyclines are typically broad-spectrum and include the natural products chlortetracycline, oxytetracycline, tetracycline, demeclocycline, and semisynthetic methacycline, doxycycline, and minocycline.

Suitable antibacterial agents that do not fit into one of the categories above include spectinomycin, mupirocin, newmycin, fosfomycin, fusidic acid, polymixins, colistin, bacitracin, gramicidin, tyrothricin, clioquinol, chloroquinaldol, haloquinal, nitrofurantonin, nitroimidazoles (including metronizole, timidazole and secnidazole), and hexamine.

The antibiotic and antifungal agents may be present as the free acid or free base, a pharmaceutically acceptable salt, or as a labile conjugate with an ester or other readily hydrolysable group, which are suitable for complexing with the ion-exchange resin to produce the resinate.

3. Additional Components

In some embodiments, the composition include one or more excipients, carriers, fillers, additives, binders, disintegration agents, lubricants, flavoring agents, and combinations thereof.

For example, in certain embodiments, a composition can include one or more of the following: a binder, such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; an excipient, such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate or combinations thereof; a disintegrating agent, such as, for example, com starch, potato starch, alginic acid or combinations thereof; a lubricant, such as, for example, magnesium stearate; a sweetening agent, such as, for example, sucrose, lactose, saccharin or combinations thereof; a flavoring agent, such as, for example peppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc.; or combinations thereof.

Typical formulae for compositions are well known in the art. In addition to proteinaceous and farinaceous materials, the compositions of the invention generally may include vitamins, minerals, and other additives such as flavorings, preservatives, emulsifiers and humectants.

Other exemplary ingredients include animal protein, plant protein, farinaceous matter, vegetables, fruit, egg-based materials, undenatured proteins, food grade polymeric adhesives, gels, polyols, starches, gums, flavorants, seasonings, salts, colorants, time-release compounds, prebiotics, probiotics, aroma modifiers, textured wheat protein, textured soy protein, textured lupin protein, textured vegetable protein, breading, comminuted meat, flour, comminuted pasta, water, and combinations thereof.

If the composition is intended to be ingested by a subject, the nutritional balance, including the relative proportions of vitamins, minerals, protein, fat and carbohydrate, and other components can be determined according to dietary standards known in the veterinary and nutritional art.

In embodiments where the composition is in a liquid form, a carrier can be a solvent or dispersion medium. Exemplary carriers include, but are not limited to, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), lipids (e.g., triglycerides, vegetable oils, liposomes) and combinations thereof. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin; by the maintenance of the required particle size by dispersion in carriers such as, for example liquid polyol or lipids; by the use of surfactants such as, for example hydroxypropyl cellulose; or combinations thereof such methods. In many cases, it will be preferable to include isotonic agents, such as, for example, sugars, sodium chloride or combinations thereof.

Also useful herein, as an optional ingredient, is a filler. The filler can be a solid, a liquid or packed air. The filler can be reversible (for example thermo-reversible including gelatin) and/or irreversible (for example thermo-irreversible including egg white). Non limiting examples of the filler include gravy, gel, jelly, aspic, sauce, water, air (for example including nitrogen, carbon dioxide, and atmospheric air), broth, and combinations thereof.

Additional suitable compounds, agents, and ingredients that can be used in combination with the disclosed modified green tea polyphenol compositions are found in U.S. Published Application Nos. 2013/0035361, 2012/0251700, 2011/0207818, 2009/0297672, and 2009/0196939.

4. Exemplary Compositions

Exemplary alcohol-based germicidal compositions are described herein. In one embodiment, the compositions contains 0.01% to 20% (v/v) epigallocatechin-3-gallate (EGCG) esterified at the 4’ position. The composition can include 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0,7%, 0.8%, 0.9%, 1.0%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% esterified EGCG. In another embodiment, the alcohol -based germicidal composition includes 0.1% to 1% (-)-epigallocatechin-3-gallate (EGCG) esterified at the 4’ position with palmitic acid (referred to as EC 16).

In one embodiment, the alcohol-based germicidal composition includes 62% to 90% (v/v) alcohol. The composition can include 62%, 63%, 64%, 65%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% (v/V) alcohol. In some embodiments, the alcohol is ethanol or isopropanol and is present in 62% to 90% (v/v).

In another embodiment, the alcohol-based germicidal composition additionally includes 0% to 20% (v/v) glycerin. The composition can include 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% glycerin. The alcohol-based germicidal compositions can also include citrate. In one embodiment, the composition includes 0% to 1% (v/v) citrate. The composition can include 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% (v/v) citrate.

In yet another embodiment, the alcohol-based germicidal composition additionally includes benzalkonium chloride. The composition can include 0% to 1% benzalkonium chloride. In one embodiment, the composition includes 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% (v/v) benzalkonium chloride.

The composition can also optionally include 0% to 40% water. In one embodiment, the composition includes 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40% water. In some embodiments, the compositions contains 10% to 38%.(v/v) water.

In one embodiment, the composition includes 78% alcohol, 0.2% EGCG-palmitate, 8% glycerin, and 14% water containing 0.4% Ultrez 20 and 0.04% triethanolamine. Table 1 describes additional exemplary formulations.

Table 1. Alcohol-based germicidal compositions.

B. Contacting Pathogens with Alcohol-Based Compositions

The disclosed alcohol-based germicidal compositions rapidly kill, inactivate, or otherwise reduce viruses and bacterial spores in as few as thirty seconds. In some embodiments, the disclosed alcohol-based germicidal compositions are formulated as a sterilization agent for use on objects and surfaces. The compositions can also be formulated into germicidal hand and skin hygiene products.

Currently, there are no effective alcohol-based hand hygiene products on the market that can effectively eliminate alcohol-resistant human pathogens such as nonenveloped viruses like Norovirus, and bacterial spores. Hand wash with soap and water is the only recommended method to prevent the spread of Norovirus or bacterial spores either in healthcare settings or at home. This is because commonly used hand sanitizers, hand rubs or scrubs, either containing alcohol or bactericidal agents, are not able to eradicate nonenveloped viruses or bacterial spores, which are resistant to alcohol and other bactericidal agents. The use of alcohol-based hand sanitizer was found to be a risk factor for norovirus spread vs. soap and water wash in long-term care facilities (Blaney, et ah, Am J Infect Control , 39(4):296-301 (2011); Stebbins, et ah, Pediatr Infect Dis J , 30(11):921-926 (2011)). In a human study, 30 second incubation with alcohol (3%

- 95%) was ineffective in reducing the infectivity of norovirus (genomic copy reduction <0.5 logio), less effective than liquid soap or water rinse (Liu, et ah, Appl Environ Microbiol, 76(2):394-399 (2010)).

Therefore, the disclosed methods of rapidly killing, inactivating, or otherwise reducing alcohol-resistant human pathogens typically includes contacting pathogens with an effective amount of one or more of the disclosed alcohol-based germicidal compositions to kill pathogens, thereby reducing or preventing pathogen replication and transmission. The alcohol-based germicidal compositions can include one or more modified green tea polyphenol compositions and one or more additional inert or active ingredients. Therefore, in some embodiments, a method of preventing pathogen replication and transmission includes contacting pathogens with an effective amount of a composition including alcohol and one or more green tea polyphenols, one or more modified green polyphenols, one or more plant-derived inert, or combinations thereof to reduce or prevent pathogenic replication and transmission. The one or more green tea polyphenols, one or more modified green polyphenols, one or more plant-derived inert, or combinations thereof, can be effective to reduce or prevent pathogen replication or to reduce or prevent pathogen transmission. The contacting can be for minutes, hours, days, weeks or longer. For example, in some embodiments, the contacting is for 30 seconds, 35 seconds, 40 seconds, 45 seconds, 50 seconds, 55 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 12 minutes, 18 minutes, 24 minutes, 36 minutes, 48 minutes, or more minutes, hours, days, weeks, or months. In another embodiment, the disclosed alcohol-based germicidal compositions inhibits pathogen replication in as few as 60 seconds.

In one embodiment, the alcohol-based germicidal compositions and methods achieve at least a 4-log reduction in pathogens within at least thirty seconds of contacting the object or surface with the composition. The compositions and methods can achieve a 4-log, 5-log, 6-log, 7-log, 8-log, 9-log, 10-log or more than 10-log reduction in pathogen activities.

In some embodiments, the compositions kill, inactivate, or otherwise reduce the number of total pathogen or the number of active pathogen. In some embodiments, the compositions reduce or prevent one or more steps of the viral life cycle, including, but not limited, blocking or preventing attachment, penetration, viral uncoating, viral RNA entry and replication within the host nucleus, assembly of new virus, and release of viral particles from the host cell. In another embodiment, the compositions reduce or prevent one or more hallmarks of germination, outgrowth, or a combination thereof, including, but not limited to, an increase in metabolic activity of the spore/bacterium, rupture or absorption of the spore coat, swelling of the spore, loss of resistance to environmental stress, the core of the spore manufacturing new chemical components, exiting the old spore coat, formation of a fully functional vegetative bacterial cell, and vegetative bacterial cell division.

The effect of the one or more alcohol-based germicidal compositions can be compared to a control. Controls are known and understood by one of skill in the art and can include, for example, untreated pathogen or pathogen treated with an alternative germicidal composition. An exemplary in vitro test that can used to measure pathogen infectivity in the presence or absence of alcohol-based germicidal compositions is described in the Examples below.

As discussed above, the methods disclosed herein typically include contacting a pathogen, or a surface thought to be infected or contaminated with pathogens, with an effective amount of one or more of the disclosed alcohol-based germicidal compositions containing modified green tea polyphenols and other plant-based compounds. The viruses can be contacted with a composition that is less than 1%, 1%, 2%, 5%, 10%, 25%, or more than 25% modified green tea polyphenol. As illustrated in the Example below, the amount of modified green tea polyphenol that is need to effectively reduce or prevent viral infectivity can depend on factors including the composition(s) of the modified green tea polyphenol, the composition of other plant-derived compounds, the species of virus to be contacted, and the environmental conditions (i.e., temperature, availability of nutrients, etc.).

C. Pathogens to be Treated

The disclosed compositions and methods are used to reduce or prevent viral replication and transmission, for example to reduce or prevent replication and transmission of enveloped viruses or nonenveloped viruses. The viruses can be DNA viruses or RNA viruses.

The disclosed compositions and methods are also useful for rapidly killing, inactivating, or otherwise reducing bacterial spores. The spores can be endospores, fungal spores, or exospores.

1. Viruses

Viruses cannot replicate outside of a host because they rely on enzymes within the host for replication. For the virus to reproduce and thereby establish infection, it must enter cells of the host organism and use those cells' materials. To enter the cells, proteins on the surface of the virus interact with proteins of the cell. Attachment, or adsorption, occurs between the viral particle and the host cell membrane. A hole forms in the cell membrane, then the virus particle or its genetic contents are released into the host cell, where replication of the viral genome may commence. Next, a virus must take control of the host cell's replication mechanisms. It is at this stage a distinction between susceptibility and permissibility of a host cell is made. Permissibility determines the outcome of the infection. After control is established and the environment is set for the virus to begin making copies of itself, replication occurs quickly by the millions. After a virus has made many copies of itself, it has usually exhausted the cell of its resources. The host cell is now no longer useful to the virus, therefore the cell often dies and the newly produced viruses must find a new host. The process by which virus progeny are released to find new hosts, is called shedding. This is the final stage in the viral life cycle.

Some viruses can "hide" within a cell, either to evade the host cell defenses or immune system, or simply because it is not in the best interest of the virus to continually replicate. This hiding is deemed latency. During this time, the virus does not produce any progeny, it remains inactive until external stimuli — such as light or stress — prompts it to activate. In one embodiment, the disclosed alcohol-based compositions are used to inactivate or kill latent viruses on a surface or in a cell. a. Nonenveloped Virus

In one embodiment, the disclosed compositions and methods are used to reduce or prevent replication and transmission of nonenveloped viruses. Exemplary nonenveloped viruses include but are not limited to adenoviruses, human papillomavirus, parvovirus B19, human astrovirus, Norwalk virus (norovirus), hepatitis E virus, rotavirus, orbivirus, coltivirus, poliovirus, Coxsackie viruses, echoviruses, hepatitis A virus, and Banna virus.

In one embodiment, the disclosed compositions and methods are used to reduce or prevent replication and transmission of Picomaviruses. Picomaviruses are small (20-30 nm) nonenveloped viruses composed of an icosahedral nucleocapsid and a single-stranded RNA genome. Picomaviruses replicate in the cytoplasm of cells. They are not inactivated by lipid solvents, such as ether, because they do not have an envelope. The picornavirus family includes two groups of medical importance: the enteroviruses and the rhinoviruses. Among the major enteroviruses are poliovirus, Coxsackie viruses, echoviruses, and hepatitis A vims.

Poliovirus has tropism for epithelial cells of the alimentary tract and cells of the central nervous system. Infection is asymptomatic or causes a mild, undifferentiated febrile illness. Spinal and bulbar poliomyelitis occasionally occurs. Paralytic poliomyelitis is not always preceded by minor illness. Paralysis is usually irreversible, and there is residual paralysis for life. All three poliovirus serotypes (1 to 3) can give rise to paralytic poliomyelitis.

Most coxsackievirus infections are inapparent or mild. Rashes and vesicular lesions are most commonly caused by group A coxsackieviruses and pleurodynia and viral pericarditis/myocarditis by group B coxsackieviruses. The coxsackievirus A24 variant causes epidemic and pandemic outbreaks of acute hemorrhagic conjunctivitis. Occasionally, coxsackieviruses are associated with paralytic and encephalitic diseases. Coxsackieviruses are characterized by their pathogenicity for suckling mice. They are classified by antibody neutralization tests as coxsackievirus group A (A1 to A24) and coxsackievirus group B (B1 to B6).

Echoviruses have been associated with febrile and respiratory illnesses, aseptic meningitis, rash, occasional conjunctivitis, and paralytic diseases.

Enterovirus types 68 and 69 cause respiratory illnesses; type 70 causes acute hemorrhagic conjunctivitis and occasionally polio-like radiculomyelitis; type 71 can cause meningitis, encephalitis and outbreaks of hand-foot-mouth disease with or without encephalitis. There is only one serotype of Hepatitis A virus. This virus causes gastroenteritis infections and hepatitis A, a disease of the liver. Many cases have few or no symptoms, especially in the young. The time between infection and symptoms, in those who develop them, is between two and six weeks. When symptoms occur, they typically last eight weeks and may include nausea, vomiting, diarrhea, jaundice, fever, and abdominal pain.. Hepatitis A is transmitted via fecal-oral route.

Rhinoviruses cause mainly respiratory infections including the common cold. There are to date 115 serotypes. Immunity is type specific. Rhinovirus infections are among the most prevalent of acute respiratory illnesses in humans. More than 90 percent of susceptible individuals infected with rhinoviruses succumb to the infection. Although most rhinovirus infections manifest as mild common colds with rhinorrhea, nasal obstruction, fever, sore throat, coughs, and hoarseness lasting for a few days, serious lower respiratory tract illnesses in infants are common. The incubation period is a few days. Viral shedding begins several days after infection peaks shortly after the onset of symptoms, and may persist for a few weeks.

In another embodiment, the disclosed compositions and methods are used to reduce or prevent replication and transmission of adenoviruses. Adenoviruses are medium-sized (90-100 nm), nonenveloped viruses with an icosahedral nucleocapsid containing a double stranded DNA genome. In humans, there are 57 accepted human adenovirus types (HAdV-1 to 57) in seven species (Human adenovirus A to G). Different types and serotypes of adenoviruses are associated with different conditions and diseases. For example, species HAdv-B and C cause respiratory disease, species HAdv-B and HAdv-D cause conjunctivitis, species HAdv-F types 40 and 41 and HAdv-Gtype 52 cause gastroenteritis. Adenoviruses are unusually stable to chemical or physical agents and adverse pH conditions, allowing for prolonged survival outside of the body and water. Adenoviruses are spread primarily via respiratory droplets, however they can also be spread by fecal routes. In one embodiment, the disclosed compositions and methods can kill or reduce the number of adenoviruses on a contaminated surface or object.

In some embodiments, the disclosed compositions and methods are used to reduce or prevent replication and transmission of parvoviruses. In one embodiment, the parvovirus is parvovirus B 19. The virus is primarily spread by infected respiratory droplets, but has been shown to be transmittable through blood-borne transmission. Symptoms begin about six days after exposure (between 4 and 28 days, with the average being 16 to 17 days) and last about a week. Infected subjects with normal immune systems are contagious before becoming symptomatic, but generally are not contagious after showing symptoms. Fifth disease or erythema infectiosum is one of several expressions of parvovirus B19.

In another embodiment, disclosed compositions and methods are used to reduce or prevent replication and transmission of norovirus. Noroviruses are a genetically diverse group of single-stranded positive-sense RNA, non-enveloped viruses belonging to the family Caliciviridae. Noroviruses are transmitted directly from person to person, and indirectly via contaminated water and food. They are extremely contagious, and fewer than twenty virus particles can cause an infection. In humans, the virus is replicated within the small intestine.

One to two days after infection with norovirus, symptoms can appear. The principal symptom is acute gastroenteritis that develops between 12 and 48 hours after exposure, and lasts for 24-72 hours. The disease is usually self-limiting, and characterized by nausea, forceful vomiting, watery diarrhea, and abdominal pain, and in some cases, loss of taste. General lethargy, weakness, muscle aches, headache, cough, and low-grade fever may occur. Hand washing with soap and water is the only widely-accepted method for reducing the transmission of norovirus pathogens. In one embodiment, the disclosed compositions and methods can kill or reduce the number of noroviruses on a contaminated surface or object. b. Enveloped Virus

In another embodiment, the disclosed compositions and methods are used to reduce or prevent replication and transmission of enveloped viruses. Exemplary enveloped viruses include but are not limited to herpes simplex type 1, herpes simplex type 2, varicella-zoster virus, Epstein-Barr virus, human cytomegalovirus, human herpesvirus type 8, smallpox, hepatitis B virus, Severe acute respiratory syndrome-related coronaviruses including Severe acute respiratory syndrome virus and Severe acute respiratory syndrome coronavirus 2, hepatitis C virus, yellow fever virus, dengue virus, West Nile virus, TBE virus, rubella virus, human immunodeficiency virus, influenza virus, Lassa virus, Crimean-Congo hemorrhagic fever virus, Hantaan virus, Ebola virus, and Marburg virus.

2. Spores

Certain microorganisms are able to form spores which help them survive under harsh environmental conditions. For example, in spore-forming bacteria, when a bacterium detects environmental conditions are becoming unfavorable, it may initiate endosporulation. First, DNA is replicated and a membrane wall called a spore septum forms between it and the rest of the cell. The plasma membrane of the cell surrounds this wall and pinches off to leave a double membrane around the DNA. The developing structure is referred to as a forespore. Calcium dipicolinate is incorporated into the forespore and a peptidoglycan cortex forms between the two layers. The bacterium adds a spore coat to the outside of the forespore. The now mature endospore is released when the surrounding vegetative cell degrades.

Bacterial spores are highly resistant to environmental challenges such as temperature differences, absence of air, water and nutrients, chemicals insults, heat, mechanical disruption, UV irradiation, and enzymes. Most agents that would normally kill the vegetative cells they formed from are ineffective against spores. For example, nearly all household cleaning products, alcohols, quaternary ammonium compounds and detergents have little effect endospores. Through sporulation, bacteria can adapt to unfavorable conditions surviving for years before reactivation via spore germination and outgrowth.

In one embodiment, the disclosed compositions and methods are used to reduce or prevent reactivation and germination of spores, including but not limited to endospores. In some embodiments the spores are fungal spores. In some embodiments, the compositions and methods disclosed herein are used to reduce or prevent reactivation of exospores, such as those formed by Methylosinus. The difference between endospores and exospores is mainly in how they form. Endospores form inside the original bacterial cell, as described above. Exospores form outside by growing or budding out from one end of the cell. Exospores also do not typically have all the same components as endospores, but are similarly resistant to environment insults.

In some embodiments, the spores are cysts. Members of the Azotobacter, Bdellovibrio, Myxococcus and Cyanobacteria genera can form protective structures called cysts. Cysts are thick-walled structures that, like spores, protect bacteria from harm. Cysts can be less durable than endospores and exospores. a. Spore-Forming Microorganisms

The spores treated with the disclosed compositions and methods are typically protective spores. In a preferred embodiment, the spores are formed by spore-forming bacteria. Examples of spore-forming bacteria include the genera: Acetonema, Alkalibacillus, Ammoniphilus, Amphibacillus, Anaerobacter, Anaerospora, Aneurinibacillus, Anoxybacillus, Bacillus, Brevibacillus, Caldanaerobacter, Caloramator, Caminicella, Cerasibacillus, Clostridium, Clostridiisalibacter, Cohnella, Dendrosporobacter, Desulfotomaculum, Desulfosporomusa, Desulfosporosinus, Desulfovirgula, Desulfunispora, Desulfurispora, Filifactor, Filobacillus, Gelria, Geobacillus, Geosporobacter, Gracilibacillus, Halonatronum, Heliobacterium, Heliophilum, Laceyella, Lentibacillus, Lysinibacillus, Mahella, Metabacterium, Moorella, Natroniella, Oceanobacillus, Orenia, Ornithinibacillus, Oxalophagus, Oxobacter, Paenibacillus, Paraliobacillus, Pelospora, Pelotomaculum, Piscibacillus, Planifilum, Pontibacillus, Propionispora, Salinibacillus, Salsuginibacillus, Seinonella, Shimazuella, Sporacetigenium, Sporoanaerobacter, Sporobacter, Sporobacterium, Sporohalobacter, Sporolactobacillus, Sporomusa, Sporosarcina, Sporotalea, Sporotomaculum, Syntrophomonas, Syntrophospora, Tenuibacillus, Tepidibacter, Terribacillus, Thalassobacillus, Thermoacetogenium, Thermoactinomyces, Thermoalkalibacillus, Thermoanaerobacter, Thermoanaeromonas, Thermobacillus, Thermoflavimicrobium, Thermovenabulum, Tuberibacillus, Virgibacillus, and Vulcanobacillus.

In a preferred embodiment, the bacteria is Baceillus, Clostridium, Sporolactobacillus, or Sporosarcina.

In another embodiment, the spore forming microorganism is not bacteria. For example, the microorganism can be Microsporidia. Microsporidia constitute a phylum (Microspora) of spore-forming unicellular parasites with over 1,500 species. Microsporidia can cause chronic, debilitating diseases and in some cases lethal infections in humans.

III. Methods of Preventing Pathogenic Contamination

A. Collection, Preparation, and Distribution of Food

The disclosed alcohol-based germicidal compositions can be used at manufacturing or processing sites handling food or foodstuffs. For example, the alcohol -based germicidal compositions can be used on food transport lines (e.g., as belt sprays); boot and hand-wash dip- pans; food storage facilities; anti-spoilage air circulation systems; refrigeration and cooler equipment; beverage chillers and warmers, blanchers, cutting boards, third sink areas, and meat chillers or scalding devices. The alcohol-based germicidal compositions can be used to treat produce transport waters such as those found in flumes, pipe transports, cutters, slicers, blanchers, retort systems, washers, and the like.

The alcohol-based germicidal compositions can also be used on food packaging materials and equipment. The alcohol-based germicidal compositions can also be used on or in ware wash machines, dishware, bottle washers, bottle chillers, warmers, third sink washers, cutting areas (e.g., water knives, slicers, cutters and saws) and egg washers. Particular treatable surfaces include packaging such as cartons, bottles, films and resins; dish ware such as glasses, plates, utensils, pots and pans; ware wash machines; exposed food preparation area surfaces such as sinks, counters, tables, floors and walls; processing equipment such as tanks, vats, lines, pumps and hoses (e.g., dairy processing equipment for processing milk, cheese, ice cream and other dairy products); and transportation vehicles.

The alcohol-based germicidal compositions can also be used on or in other industrial equipment and in other industrial process streams such as heaters, cooling towers, boilers, retort waters, rinse waters, aseptic packaging wash waters, and the like. The alcohol-based germicidal compositions can be used to treat microbes and odors in recreational waters such as in pools, spas, recreational flumes and water slides, fountains, and the like.

B. Medical Devices

In some embodiments the disclosed alcohol-based germicidal compositions can be coated onto, or incorporated into, a medical device to reduce or prevent viral contamination of the device. The device can be a device that is inserted into the subject transiently, or a device that is implanted permanently.

Examples of medical devices include, but are not limited to, needles, cannulas, catheters, shunts, balloons, and implants such as stents and valves. In some embodiments, the medical device is a vascular implant such as a stent. Stents are utilized in medicine to prevent or eliminate vascular restrictions. The implants may be inserted into a restricted vessel whereby the restricted vessel is widened.

In some embodiments, the device is a surgical device. Surgical devices include, but are not limited to articulator, bone chisel, cottle cartilage crusher, bone cutter, bone distractor, ilizarov apparatus, bone drill, bone extender, bone file, bone lever, bone mallet, bone rasp, bone saw, bone skid, bone splint, bone button, caliper, cannula, catheter, cautery, clamps, curette, depressor, dilator, dissecting knife, distractor, dermatome, forceps, acanthulus or acanthabolos, hemostat, hook, lancet (scalpel), luxator, lythotome, lythotript, mallet, mouth prop, mouth gag, mammotome, needle holder, occlude, osteotome, elevator, probe, retractor, rake, rib spreader, rongeur, scissors, spatula, speculum, sponge bowl, sterilization tray, tubes, knife, mesh, needle, snare, sponge, spoon, stapler, suture, syringe, tongue depressor, tonsillotome, tooth extractor, towel clamp, towel forceps, tracheotome, tissue expander, subcutaneous inflatable balloon expander, trephine, trocar, and tweezers.

The alcohol-based germicidal compositions can be formulated to permit its incorporation onto the device. The composition can be included within a coating on the device. There are various coatings that can be utilized such as, for example, polymer coatings that can release the composition over a prescribed time period. The composition can be embedded directly within the medical device. In some embodiments the composition is coated onto or within the device in a delivery vehicle such as a microparticle or liposome that facilitates its release and delivery.

C. Hand Hygiene

The disclosed alcohol-based germicidal compositions can be formulated into hand hygiene products for use in a hospital or clinical setting, schools, airports, cruise ships, nursing homes, or any other setting in which the transmission of microbes from one person to another, or a surface to a subject is possible. In one embodiment, the hand hygiene product is a gel, spray, foam, soap, wipe, lotion, sanitizer, or other product that can be applied to the skin.

Current hand hygiene guidelines by the CDC recommend alcohol-based hand sanitizers for medical professionals to use in most clinical situations. In some embodiments, the disclosed alcohol-based germicidal compositions are more effective than products currently available to healthcare professionals.

EXAMPLES

Example 1. Formulations for surface disinfectant against norovirus

Materials and Methods

Cell culture: Fcwf cells (from feline fetus) were maintained in Eagle’s Minimum Essential Medium (EMEM) medium containing 10% FBS and 1% antibiotics (Penicillin Streptomycin Solution, 100 X, MediaTech, Inc. VA) at 37°C with 5% CO2. Prior to confluency, the cells were harvested using 0.25% (w/v) trypsin - 0.53 mM EDTA (Life Technologies, CA), sub-cultured in 96-well tissue culture plates, and allowed to become 80-90% confluent before infection.

FCV propagation: Fcwf cells (10 ⁵ /cm ² ) were seeded in a 75 cm ² tissue culture plate and grown for 24-48 hrs until the monolayer became 90% confluent. FCV at an MOI of 0.1 for infection was prepared by diluting a titered virus suspension in HBSS. The monolayer of Fcwf cells was washed briefly with HBSS prior to adding diluted virus in 3 ml HBSS. The flask was incubated in a cell culture incubator for 1 hr with gentle rocking every 10 min to spread the virus evenly. EMEM (9 ml) was then added to the flask, and incubation continued for 24 hrs before observation for cytopathic effect (CPE). When >80% cells showed CPE, the flask was frozen at - 80°C and thawed for two cycles, followed by centrifugation at 400 x g for 20 min. The supernatant was filtered using a 2 pm tube top filter (50 ml, Corning Inc., Corning, NY) and the virus was dispensed into cryogenic vials in 1 ml aliquots and stored at -80° C. Viral titer was determined by TCID50 assay.

TCID50 assay: To obtain a viral titer, 50 pi of virus suspension was added to 450 pi HBSS (i.e., 10-1 viral dilution). A series of 10-fold dilutions of this dilution up to 10 ¹¹ was made with HBSS. From each dilution, 100 pi was loaded in quadruplicate into a 96-well plate of Fcwf cells, followed by 1 hr incubation for viral absorption. The overlying liquid containing virus was then removed and replaced with EMEM. Incubation was continued for 24 hrs prior to observation for CPE. It took 4-7 days to complete the viral infection cycle when there was no new CPE emerging in the wells. The number of wells associated with CPE were entered into Reed & Muench calculation calculator software (Lindenbach, BD., MethodMol Biol , 510:329- 336 (2009)).

Suspension assay: Suspension tests for virucidal activity were performed according to the protocol described previously by Zhang et al. ( Antivirals , Antiretroviral Research and Therapy , 1 :002 (2016)). For each test formulation, 450 pi of the test sample was placed in a plastic centrifuge tube, 50 pi of FCV was added and mixed by shaking the tube for 60 sec. The test sample was then immediately neutralized by dilution with HBSS (i.e., 10 ² viral dilution), and a series of dilutions was made from this mix up to 10 ⁸ , A TCID50 assay was performed on quadruplicate 100 pi aliquots from each dilution. A viral titer was simultaneously performed on the same plate with tested samples as untreated viral infectivity control.

Results

A synergistic effect of plant-derived compounds increased the activity of alcohol in formulation that contain 70% alcohol, 0.1% EGCG-palmitate, and 0.3% citric acid, pH 3-4. The following result shows that only two formulations increased the virucidal activity to > 4 log (99.99% reduction of feline calicivirus infectivity in TCID50 suspension test). Table 2. Comparison of different formulations with 75% alcohol against feline calicivirus (surrogate for human norovirus).

The results demonstrate that among various combinations of plant-derived compounds, the combination of EGCG-p and citric acid significantly enhanced the virucidal activity of alcohol against norovirus surrogate. Alcohol alone, alcohol with EGCG-p alone, or alcohol with EGCG-p plus glycerin, did not effectively inactivate feline calicivirus. Only at certain combination and concentration levels did the formulation become potent.

After further testing, the formulation with 70% alcohol, 0.1% EGCG-p and 0.3% citric acid was selected for a third party test in Good Laboratory Practice (GLP) certified laboratory. The test is based on CIS Environmental Protection Agency (EPA) guidelines for surface disinfectant spray and wipe products. Table 3 shows the result from carrier test (not suspension test) required by EPA. The result demonstrates that the composition inactivated feline calicivirus by >4 log reduction, exceeded EPA requirement for norovirus virucidal surface disinfectant.

Table 3. GLP certified laboratory results.

+ CPE (cytopathic/cytotoxic effect) present

0 CPE (cytopathic/cytotoxic effect) not detected NT Not tested N/A Not applicable

IP Initial Population CC Cell Control

Test Formulation: PST70 Spray, Batch #1 Lot # 1, Batch #2 Lot # 2, Vims: Feline Calicivirus (ATCC #VR-782) Host Cell Line: CRFK Host Cell Line ATCC #CCL-94, Volume Plated per Well: 1.0 mL. Exposure Time: 1 minute The Test Formulation spray, Batch #1 Lot # 1 and Batch #2 Lot #2 demonstrated a >3 logio reduction on each test surface following a 1-minute exposure in the presence or absence of cytotoxicity. Feline Calicivirus infectivity was reduced by 4.75 logio and 4.50 logio by the Test Formulation spray, Batch #1 Lot # 1, and was reduced by 4.00 logio and 4.25 logio by Batch #2 Lot # 2, following a 1 -minute exposure. Example 2. Formulations for hand sanitizer against norovirus

Materials and Methods

See Example 1 for details regarding materials and methods.

Results A synergistic effect of plant-derived compounds increased the activity of alcohol in a formulation that contains 78% alcohol, 0.2% EGCG-palmitate, and 8% glycerin, and 14% water containing 0.4% Ultrez 20 and 0.04% triethanolamine.

The following result shows that only two formulations increased the sporicidal activity to > 4 log (99.99% reduction of feline calicivirus infectivity in TCID50 suspension test). Below formulations all contain 0.4% Ultrez 20 and 0.04% trimethylamine and water.

Table 4. Comparison of different formulations with 78% alcohol against feline calicivirus

(surrogate for human norovirus).

The results demonstrate that among various combinations of plant-derived compounds, the combination of EGCG-p and 8% glycerin significantly enhanced the virucidal activity of alcohol against norovirus surrogate. Alcohol alone, alcohol with glycerin, alcohol with EGCG-p, or alcohol with EGCG-p plus <8% glycerin, did not effectively inactivate feline calicivirus. Only at certain combination and concentration levels did the formulation become potent.

The formula with 78% alcohol, 0.2% EGCG-palmitate, and 8% glycerin, and 14% water containing 0.4% Ultrez 20 and 0.04% triethanolamine was selected for additional testing.

In addition, it was discovered that the addition of Aloe Vera and vitamin E at 0.01% and blue or yellow artificial color did not alter the virucidal activity. Example 3. Effect of EGCG-P, different concentrations of ethanol and EGCG-P/alcohol combinations on the spore germination.

Materials and Methods

Spore Enrichment and Purification : B. cereus was incubated on modified nutrient agar plates (supplemented with 0.06 g of MgSCri and 0.25 g of KEhPCri per liter) at 37°C for 10 days to enhance endospore formation. After 10 days, Schaeffer Fulton differential staining was conducted to observe the endospore and vegetative cells. The endospores were then purified by centrifugation at room temperature for 10 min at 10,000 rpm twice. The supernatant was discarded and the endospores were suspended in sterile deionized water and vortexed to create a homogenous suspension. The suspension was heated for 20 min at 75°C to eliminate any remaining vegetative cells and obtain pure endospores. Purified endospores from B. cereus were mixed for 1 min (60 sec) with the formulations #1, 2, 3, 4 or 5 (Table 5). After treatment, serial 10 X dilutions were made immediately, plated out and incubated for 24 hours. The CFU was counted, the % of inhibition and logio reduction were calculated, plated onto nutrient agar plates, and subsequently incubated at 37°C for 24 h. After incubation, the colony forming unit (CFU) value was obtained. Non-treated endospore samples (i.e., suspended in media for 60 sec) were used as a negative (treatment) control. The 80% EtOH was used as positive control. Three independent experiments were carried out and the mean and standard deviation of the results were calculated. The logio (fold) reduction was calculated with the following equation:

Log reduction = Logio (CFU control/ CFU treated).

Table 5. Modified green tea polyphenol compositions.

Results:

The spore suspensions were treated with media (control), or different concentrations of EtOH (70%, 78% and 85 %) respectively for 60 seconds. The combination of 0.2% EGCG-P with different concentrations of EtOH was also used to treat the spore suspension for 60 seconds. Results are shown in Figure 1. The different concentrations of EtOH alone inhibited spore germination of B. cereus, the percentage inhibition in for all three concentrations was above 99%. The average logio reduction for 70%. 78% and 85% EtOH was 2.43, 2.58 and 2.45, respectively. The average logio reduction of EGCG-P+70% EtOH, EGCG-P+78% EtOH and EGCG-P+85% EtOH was 2.45, 2.94 and 2.77, respectively. Although the combination of 0.2% EGCG-P with different concentrations of EtOH gave higher mean values than alcohol alone at that concentration, the differences were not statistically significance (p>0.05). The best combination with the highest log reduction was EGCG-P with 78% EtOH. These results suggested that this combination was not ideal as a sporicidal formulation, which aims for more than log 4 reduction. However, this was used as a base for the novel formulations with addition of other agents .

Example 4. Effect of different sporicidal formulations on spore germination

Materials and Methods:

Spore Enrichment and Purification : B. cereus was incubated on modified nutrient agar plates (supplemented with 0.06 g of MgSCri and 0.25 g of KH2PO4 per liter) at 37°C for 10 days to enhance spore formation. The spores were then purified by centrifugation at room temperature for 10 min at 10,000 rpm twice. The supernatant was discarded and the spores were suspended in sterile deionized water and vortexed to create a homogenous suspension. The suspension was heated for 20 min at 75°C to eliminate any remaining vegetative cells and obtain pure spores. Purified spores from B. cereus were mixed for 30 sec with the formulations #1 (FI) and #2 (F2), respectively. After treatment, serial 10 X dilutions were made immediately, plated onto nutrient agar plates, and subsequently incubated at 37°C for 24 h. After incubation, the colony forming unit (CFU) value was counted, the % of inhibition and logio reduction were calculated. Non- treated spore samples were used as a negative (treatment) control. Three independent experiments were carried out and the mean and standard deviation of the results were calculated. The logio (fold) reduction was calculated with the following equation:

Log reduction = Logio (CFU control/ CFU treated)

Scanning Electron Microscopy.

Endospores were treated with FI and F2 for 60 seconds. Once the treatment time was up, 100 pL of the samples were dispensed and vacuum filtered using 0.2 pm polycarbonate membrane (EMD Millipore Isopore #GTTP01300). Samples were rinsed with PBS (pH 7.2) or 0.1 mol G ¹ sodium cacodylate buffer [Na(CH3)2As02-3H20] three times for 5 min each; fixed with 2.5% glutaraldehyde in 0.1 mol-G ¹ cacodylate buffer for 30 min at room temperature. The samples were further fixed, went through a series of dehydration and were immersed in ethanol, followed by drying with liquid CO2 at 1072 psi and 31°C in Denton Critical Point Dryer.

Samples were mounted on a stub and coated with a thin layer of copper metal film using Denton IV Sputter Coater. Images were captured with a Hitachi S-3400N Scanning Electron Microscope

Results:

Five different EGCG-P formulations were used in this study to determine and evaluate the sporicidal activities of these formulations. As shown in Figure 2, the formulations were able to reduce spore germination by >4 logio, with two formulations (F2 and F3) reducing spore germination by >5 logio (99.999%), after a 60-sec incubation with endospores of B. cereus. All formulations showed significantly (p= 0.0093; 0.0008; 0.0001; 0.0128; and 0.0052 for F1-F5) higher log reduction when compared with the positive control (80% EtOH). As described above, F2 and F3 only contain food grade plant-derived ingredients that are commonly found in popular beverages. These formulations exhibited extremely powerful sporicidal activity against endospores of B. cereus , and demonstrated that our new formulations improved the sporicidal activity by about 1000-fold compared to previously published data, even without considering the shortened time from 5 min to 60 sec.

Two different EGCG-P formulations were used in this study to determine and evaluate the sporicidal activities of these formulations. In comparison to the untreated control, FI -treated spores of B. cereus exhibit altered morphology with smaller size and porous spore shell along with film-like appearance among the spores, while F2-treated spores show a complete destruction of spore structures (Figs. 3A-3C). Similarly, morphology changes are apparent after formula treatments in spores of C. sporogenes. While FI -treated spores become clustered with disfigured spores within the clusters, F2-treated spores exhibit disfigurement with collapsed spore shells, completely different from the untreated control spores, which have a ping-pong ball-like morphology (Figs. 3D-3F). These structural alterations suggest both FI and F2 are able to rapidly alter the spore morphology, leading to inactivation of these bacterial spores by collapsing the spore shells and allow ethanol to penetrate the outer layer of the spores, therefore denature the inner biological molecules.

A pilot study was conducted to improve the germicidal activity of FI (hand sanitizer formulation) and F2 (hand rub formulation). It was found that when glycerol was added to FI within a certain concentration range, the germicidal activity was significantly improved. This result was surprising, because glycerol was added as an emollient to reduce the irritant effect of alcohol to the skin, rather than as an antimicrobial agent. Accordingly, glycerol was incorporated at an appropriate concentration in FI to make “cFl”. For F2 improvement, it was found the germicidal activity increased significantly with the addition of citric acid within a certain concentration range. This was not due to the lowered pH because other pH reducing acids were not able to increase the antimicrobial activity of F2. In fact, alcohol-based formulations containing different acids, with pH <1, failed to reduce bacterial spore germination by >2.5 log after 5 min exposure. In addition, this low concentration of citric acid did not lead to a pH drop to <3.3. Thus, it is possible the combination of citric acid and EGCG-P results in a synergistic effect in the alcohol-based formulation now referred to as ‘cF2’.

As shown in Figure 4, the EGCG-P formulations were able to reduce spore germination by an average of >5 logio (99.999%), after a 30-sec incubation with spores of B. cereus. Both formulations showed significantly (cFl: p=0.01 and cF2: p=0.007) higher logio reduction when compared with the control. Neutralization tests were carried out as well. Similar suspension tests were conducted with cFl and cF2 neutralized with PBS (1:9 v/v). Both neutralized formulations were ineffective for the sporicidal activity.

While in the foregoing specification this invention has been described in relation to certain embodiments thereof, and many details have been put forth for the purpose of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein can be varied considerably without departing from the basic principles of the invention.

All references cited herein are incorporated by reference in their entirety. The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof and, accordingly, reference should be made to the appended claims, rather than to the foregoing specification, as indicating the scope of the invention.