Patent Publication Number: US-2015064227-A1

Title: Sporicidal Formulation Including Botanical Extracts/Botanical-Derived Ingredients

Description:
BACKGROUND 
     Spores are metabolically dormant microbes that remain viable under a wide range of environmental conditions. Spores are typically heat-, acid-, and desiccation-resistant and can persist in the environment for years. Because of their stability, contamination by spores is very common in hospital, clinical, long-term care or nursing home environments. Often, it can be cultured from almost any surface in a hospital. Patient-to-patient transmission of spores occurs by sharing the medical equipment or facilities in hospitals, nursing homes, and other extended-care facilities. Transmission in community settings also occurs. 
     Given the pathogenesis of various spore-forming microorganisms, judicious use of antibiotics, strict infection control and environmental measures are keys to the prevention and outbreak of disease. The implementation of antibiotic stewardship programs has been associated with decreased incidence of spore related diseases. To prevent spread of spores, environmental cleaning and patient isolation are needed. Several disinfectants commonly used in hospitals may be ineffective against spores, and may actually promote spore formation. 
     For example,  Clostridium difficile,  also known as “CDF/cdf”, or “ C. diff. ”, a species of gram-positive, spore-forming anaerobic bacillus, can lead to severe complications ranging from antibiotic-associated diarrhea to severe life-threatening pseudomembranous colitis, a severe infection of the colon. In fact,  C. diff.  is the cause of approximately 25 percent of all cases of antibiotic-associated diarrhea. Most cases of  C. diff.  associated disease occur in hospitals or long-term care facilities causing more than 300,000 cases per year in the United States alone. The total US hospital costs for  C. diff.  associated disease management have been estimated to be $3.2 billion per year. 
     Health care workers should avoid using only alcohol hand sanitizers, especially in outbreak settings, because alcohol is not effective at killing spores. Due to their resistant nature, spores are very difficult to eliminate with standard measures. Consumer and health care applications are taking measures with large amounts of harsh chemicals including ethylene oxide, aldehydes and highly reactive oxidizing agents such as peracetic acid, chlorine dioxide and ozone which are either carcinogenic or corrosive. It would be virtually impossible to use the current technologies/tactics on skin and delicate devices or surfaces. There is a need to develop a sporicide disinfectant that is nonharmful to human skin and the environment but still provides the sporicidal efficacy required by the products necessary to reduce the spore-forming bacteria. 
     SUMMARY 
     Formulations and wipes for imparting a sporicide to a surface are disclosed herein. Unexpectedly, a set of naturally derived ingredients have been found to combat and treat spore-based bacteria without the use of harsh chemicals. To achieve the sporicidal efficacy of the product, naturally derived botanical extracts and/or botanical-derived ingredients have been incorporated into a sporicidal formulation. Example botanical extract that demonstrated sporicidal activity include  Garcinia morella, Setaria italica, Eucalypti globulus, Salvia miltiorrhiza, Coptis teeta,  and  Psoralea corylifolia.  Example botanical-derived ingredients that demonstrated sporicidal activity include gambogic acid, neogambogic acid, and cryptotanshinone. Incorporating these botanicals and/or botanical-derived ingredients into products that were then tested and found to provide a sporicidal benefit is an unexpected observation. Other botanicals and/or botanical extracts were not found to have sporicidal efficacy. 
     Typically, the sporicidal formulation contains botanical extracts or botanical-derived ingredients to provide sporicidal efficacy in an amount from about 0.1 to about 300 mg/ml (by volume of the sporicidal formulation), more typically from about 0.1 to about 250 mg/ml (by volume of the sporicidal formulation), and more typically from about 5 to about 50 mg/ml (by volume of the sporicidal formulation). Desirably, the formulation contains from about 0.1 to about 50 mg/ml (by volume of the sporicidal formulation) of the botanical-derived ingredient. Desirably, the formulation contains from about 50 to about 300 mg/ml (by volume of the sporicidal formulation) of the botanical extract. 
     In some embodiments, it is beneficial for the sporicidal formulation to also include an antimicrobial agent. The antimicrobial agent may be selected from alcohols, quaternary ammonium compounds, biguanides, phenols, oxidants, alkylating agents, silver, copper, isothiazalones, short-chain acids, or a combination thereof 
    
    
     DETAILED DESCRIPTION 
     Generally stated, formulations and wipes for imparting a sporicide to a surface is disclosed herein. Unexpectedly, a set of naturally derived ingredients have been found to combat and treat spore-based bacteria without the use of harsh chemicals. To achieve the sporicidal efficacy of the product, Botanicals and/or botanical-derived ingredients have been incorporated into a sporicidal formulation. Example botanical extracts that demonstrated sporicidal activity include  Garcinia morella, Setaria italica, Eucalypti globulus, Salvia miltiorrhiza, Coptis teeta,  and  Psoralea corylifolia.  Example botanical-derived ingredients that demonstrated sporicidal activity include gambogic acid, neogambogic acid, and cryptotanshinone. Incorporating these botanicals and/or botanical-derived ingredients into products that were then tested and found to provide a sporicidal benefit is an unexpected observation. Other botanicals and/or botanical extracts were found to not have sporicidal efficacy. 
     The sporicidal formulation described herein may be used in combination with a product. More particularly, the sporicidal formulation may be incorporated into or onto a substrate, such as a wipe substrate, an absorbent substrate, a fabric or cloth substrate, or a tissue substrate, among others. For example, the sporicidal formulation may be incorporated into cleansing products, such as wipes, absorbent articles, cloths, and the like. More particularly, the sporicidal formulation may be incorporated into wipes such as wet wipes, dry wipes, hand wipes, face wipes, cosmetic wipes, and the like. In one preferred embodiment, the sporicidal formulation is a liquid composition that may be used in combination with a wipe substrate to form a wet wipe, or may be a wetting composition for use in combination with a dispersible wet wipe. 
     Reference will now be made in detail to the presently preferred embodiments of the invention. Each example is provided by way of explanation and is not meant as a limitation. For example, features illustrated or described as part of one embodiment can be used on another embodiment or figure to yield yet another embodiment. It is intended that the present disclosure include such modifications and variations. 
     As described above, the sporicidal formulation requires certain botanical extracts or botanical-derived ingredients to provide sporicidal efficacy. Example botanical extracts that demonstrated sporicidal activity included:  Garcinia morella, Setaria italica, Eucalypti globulus, Salvia miltiorrhiza, Coptis teeta,  and  Psoralea corylifolia.  Example botanical-derived ingredients that demonstrated sporicidal activity include gambogic acid, neogambogic acid, and cryptotanshinone. The botanical extracts and botanical-derived ingredients listed above may also be used in combination to provide the sporicidal efficacy. 
     Typically, the sporicidal formulation contains botanical extracts or botanical-derived ingredients to provide a sporicidal efficacy insoluble oxidant in an amount from about 0.1 to about 300 mg/ml (by volume of the sporicidal formulation), more typically from about 0.1 to about 250 mg/ml (by volume of the sporicidal formulation), and more typically from about 5 to about 50 mg/ml (by volume of the sporicidal formulation). Desirably, the formulation contains from about 0.1 to about 50 mg/ml (by volume of the sporicidal formulation) of the botanical-derived ingredient. Desirably, the formulation contains from about 50 to about 300 mg/ml (by volume of the sporicidal formulation) of the botanical extract. 
     In some embodiments, it is beneficial for the sporicidal formulation to also include an antimicrobial agent. The antimicrobial agent may be selected from alcohols, quaternary ammonium compounds, biguanides, phenols, oxidants, alkylating agents, silver, copper, isothiazalones, short-chain acids, or a combination thereof. 
     Typically, the sporicidal formulation may contain an antimicrobial agent in an amount from about 0.01 to about 85 percent (by weight of the sporicidal formulation), more typically from about 0.01 to about 70 percent (by weight of the sporicidal formulation), and more typically from about 0.5 to about 65 percent (by weight of the sporicidal formulation). 
     The sporicidal formulation exhibits at least a 90 percent reduction of viable spores within about 5 minutes of application of said cleaning medium using the sporicidal efficacy test described herein. Other efficacy tests, including those on skin and hard surfaces, may also be employed to demonstrate at least a 90 percent reduction in viable spores within about 5 minutes. 
     As noted above, the sporicidal formulation may be incorporated into personal care compositions and wipes to improve the antibacterial benefit of these products. Generally, the wipes including the sporicidal formulation can be wet wipes or dry wipes. As used herein, the term “wet wipe” means a wipe that includes greater than about 70 percent (by weight substrate) moisture content. As used herein, the term “dry wipe” means a wipe that includes less than about 10 percent (by weight substrate) moisture content. Specifically, suitable wipes for use with the sporicidal composition described herein can include wet wipes, dry wipes, hand wipes, face wipes, cosmetic wipes, household wipes, industrial wipes, and the like. Particularly preferred wipes are wet wipes, and other wipe types that include a solution. 
     Materials suitable for the substrate of the wipes are well known to those skilled in the art, and are typically made from a fibrous sheet material which may be either woven or nonwoven. For example, suitable materials for use in the wipes may include nonwoven fibrous sheet materials which include meltblown, coform, air-laid, bonded-carded web materials, hydroentangled materials, and combinations thereof Such materials can contain synthetic or natural fibers, or a combination thereof Typically, the wipes define a basis weight of from about 25 to about 120 grams per square meter and desirably from about 40 to about 90 grams per square meter. 
     In one particular embodiment, the wipes may be a coform basesheet of polymer fibers and absorbent fibers having a basis weight of from about 45 to about 80 grams per square meter and desirably about 60 grams per square meter. Such coform basesheets are manufactured generally as described in U.S. Pat. No. 4,100,324, issued to Anderson, et al.; U.S. Pat. No. 5,284,703, issued to Everhart, et al.; and U.S. Pat. No. 5,350,624, issued to Georger, et al., which are incorporated by reference to the extent to which they are consistent herewith. Typically, such coform basesheets contain a gas-formed matrix of thermoplastic polymeric meltblown fibers and cellulosic fibers. Various suitable materials may be used to provide the polymeric meltblown fibers, such as, for example, polypropylene microfibers. Alternatively, the polymeric meltblown fibers may be elastomeric polymer fibers, such as those provided by a polymer resin. For instance, Vistamaxx® elastic olefin copolymer resin designated PLTD-1810, available from ExxonMobil Corporation (Houston, Tex.) or KRATON G-2755, available from Kraton Polymers (Houston, Tex.) may be used to provide stretchable polymeric meltblown fibers for the coform basesheets. Other suitable polymeric materials, or combinations thereof, may alternatively be utilized as known in the art. 
     The coform basesheet additionally may contain various absorbent cellulosic fibers, such as, for example, wood pulp fibers. Suitable commercially available cellulosic fibers for use in the coform basesheets can include, for example, NF 405, which is a chemically treated bleached southern softwood Kraft pulp, available from Weyerhaeuser Co. (Federal Way, Wash.); NB 416, which is a bleached southern softwood Kraft pulp, available from Weyerhaeuser Co.; CR-0056, which is a fully debonded softwood pulp, available from Bowater, Inc. (Greenville, S.C.); Golden Isles 4822 debonded softwood pulp, available from Koch Cellulose (Brunswick, Ga.); and SULPHATATE HJ, which is a chemically modified hardwood pulp, available from Rayonier, Inc. (Jesup, Ga.). 
     The relative percentages of the polymeric meltblown fibers and cellulosic fibers in the coform basesheet can vary over a wide range depending upon the desired characteristics of the wipes. For example, the coform basesheet may contain from about 10 to about 90 percent (by weight substrate), desirably from about 20 to about 60 percent (by weight substrate), and more desirably from about 25 to about 35 percent (by weight substrate) of the polymeric meltblown fibers based on the dry weight of the coform basesheet being used to provide the wipes. 
     In another embodiment, the wipe substrate may be an airlaid nonwoven fabric. The basis weights for airlaid nonwoven fabrics may range from about 20 to about 200 grams per square meter with staple fibers having a denier of about 0.5-10 and a length of about 6 to about 15 millimeters. Wet wipes may generally have a fiber density of about 0.025 to about 0.2 g/cc. Wet wipes may generally have a basis weight of about 20 to about 150 grams per square meter. More desirably the basis weight may be from about 30 to about 90 grams per square meter. Even more desirably the basis weight may be from about 50 to about 75 grams per square meter. 
     Processes for producing airlaid nonwoven basesheets are described in, for example, published U.S. Pat. App. No. 2006/0008621, herein incorporated by reference. 
     In an alternative embodiment, the wipes may be a composite which includes multiple layers of materials. For example, the wipes may include a three layer composite which includes an elastomeric film or meltblown layer between two coform layers as described above. In such a configuration, the coform layers may define a basis weight of from about 15 to about 30 grams per square meter and the elastomeric layer may include a film material such as a polyethylene metallocene film. Such composites are manufactured generally as described in U.S. Pat. No. 6,946,413, issued to Lange, et al. (Sep. 20, 2005), which is hereby incorporated by reference to the extent it is consistent herewith. 
     As mentioned above, one type of wipe suitable for use in combination with the sporicidal formulation is a wet wipe. In addition to the wipe substrate, wet wipes also contain a liquid composition. The liquid composition can be any liquid, which can be absorbed into the wet wipe basesheet and may include any suitable components, which provide the desired wiping properties. For example, the components may include water, emollients, surfactants, fragrances, preservatives, organic or inorganic acids, chelating agents, pH buffers, or combinations thereof, as are well known to those skilled in the art. Further, the liquid may also contain lotions, medicaments, and/or antimicrobials. 
     The wet wipe composition may desirably be incorporated into the wipe in an add-on amount of from about 10 to about 600 percent (by weight of the treated substrate), more desirably from about 50 to about 500 percent (by weight of the treated substrate), even more desirably from about 100 to about 400 percent (by weight of the treated substrate), and especially more desirably from about 200 to about 300 percent (by weight of the treated substrate). 
     The desired liquid composition add-on amounts may vary depending on the composition of the wipe substrate. Typically, however, for coform basesheets, the composition add-on amount will be from about 250 to about 350 percent (by weight of the treated substrate), and more typically about 330 percent (by weight of the treated substrate). For air-laid basesheets, the composition add-on amount will typically be from about 200 to about 300 percent (by weight of the treated substrate), and more typically will be about 235 percent (by weight of the treated substrate). 
     These add-on amounts will preferably result in a wet wipe comprising sporicidal formulation in an add-on amount of from about 1 to about 5 percent (by weight of the treated substrate), and more preferably from about 1.65 to about 4.95 percent (by weight of the treated substrate). 
     In another embodiment, the wipe is a dry wipe. In this embodiment, the wipe can be wetted with an aqueous solution just prior to, or at the point of, use of the wipe. The aqueous solution can be any aqueous solution known in the art to be suitable for use in wipe products. Generally, the aqueous solution includes mainly water, and can further include additional components, such as cleansers, lotions, preservatives, fragrances, surfactants, emulsifiers, dyes, humectants, emollients, oils, sunscreens, and combinations thereof. The sporicidal formulation may be present in the aqueous solution used to wet the dry wipe prior to use. 
     Alternately, the dry wipe may be prepared by applying by any suitable means (e.g., spraying, impregnating, etc.) a composition comprising a sporicidal formulation described herein onto a wipe substrate. The composition may contain 100 percent of the sporicidal formulation, or alternately, the sporicidal formulation may be present in the composition in combination with a carrier and/or other skin benefit agent, as described herein. In embodiments where the sporicidal formulation used to prepare the dry wipe contains water or moisture, the resulting treated substrate is then dried so that the wipe contains less than about 10 percent (by weight substrate) moisture content, and a dry wipe is produced. The treated substrate can be dried by any means known to those skilled in the art including, for example by use of convection ovens, radiant heat sources, microwave ovens, forced air ovens, and heated rollers or cans, or combinations thereof. 
     The dry wipe may contain the sporicidal formulation in an add-on amount composition of from about 40 to about 250 percent (by weight of the treated substrate), more desirably about 100 percent (by weight of the treated substrate). One may use a wipe sheet to clean various different kinds of surfaces either in a clinical or other type of setting. These may include, for instance, various desk, table or countertops or other parts of furniture surfaces, bath and lavatory surfaces, floor and wall surfaces, medical instruments or devices, bedding and linens or even human skin. In a liquid form, the sporicidal formulation may be employed in bath or rinse to wash medical instruments, linens, bedclothes, or human skin. One may even incorporate use the formulation in a disinfecting or sanitary solution to wash hands or medical instruments. 
     Test Method 
     Sporicidal Efficacy Test 
     Objective: 
     To determine the kill rate of solutions of interest against bacterial spores. 
     Materials:
         1. Test solutions of interest   2.  Clostridium difficile  ATCC 43593   3.  Geobacillus stearothermophilus  CICC 10142   4. Filter sterilized MilliQ water   5. Neutralization broth (prepared according to directions below)   6. Brain Heart Infusion (BHI) agar plates with 0.15% Sodium Taurocholate   7. Tryptic Soy Agar (TSA) plates   8. Taurocholic acid sodium salt hydrate (Sigma-Aldrich T-4009)   9. Phosphate buffered saline (PBS) pH 7.2   10. Fetal Bovine Serum (FBS)   11. Sterile plating beads   12. Sterile Eppendorf vials (1.5 mL)   13. Sterile tubes (15 mL)   14. Pipettes and sterile pipette tips (100 μL and 1000 μL)   15. Incubator capable of 37±3° C.   16. Anaerobic rectangular jar (7 Liter AnaeroPack System commercially available from Mitsubishi Gas Chemical Co.) with 3 GasPak satchels (BD GasPak EZ, #260678)       

     Procedure:
         1. Prepare culture stock of organism of interest to 10 7  CFU/ml in PBS.   2. Thoroughly vortex the stock culture for ten minutes at medium-high speed.   3. Dilute a small portion of the stock culture in MilliQ filter sterilized water and add FBS soil load to the inoculum to achieve a concentration of 5% v/v FBS.   4. Place the inoculum in a sonicating water bath for five cycles of one minute on, one minute off   5. Add 100 μL of spore culture to sterile vial containing 900 μL of test solution and vortex.   6. After the test exposure time, vortex the vials.   7. Transfer 100 μL of test solution and spore mixture to sterile tubes containing 900 μL neutralization broth to neutralize.   8. Place the neutralized samples in a sonicating water bath for five cycles of one minute on, one minute off   9. Vortex each tube. For  C. difficile,  pipette 100 μL of each neutralized sample on BHI+0.15% sodium taurocholate media plates (prepared in lab according to dehydrated powder instructions). For  G. stearothermophilus,  pipette 100 μL of each neutralized sample onto TSA plates. Spread using sterile beads.   10. Prepare a control sample by adding the spore culture to 900 μL of filter sterilized MQ water and repeat steps 3-6 above. Dilute control code to achieve 10 2  CFU/ml.   11. For  C. difficile,  place plates in anaerobic jars or boxes (with appropriate number of catalase pouches) or in the anaerobic chamber and incubate for 48±8 hours at 37±3° C. For  G. stearothermophilus,  incubate plates aerobically for 48±8 hours at 37±3° C.   12. After incubation, enumerate colonies and record results. Calculate Log 10  reduction by comparing the number of colonies recovered from the test solution versus those recovered with the control.       

     Neutralization Broth Preparation:
         1. Mix the following ingredients:
           1 L Letheen broth   0.3% ppm Lecithin   3% ppm Tween 80   0.1% ppm Histidine   
           2. Solution is sterilized by autoclave.   3. Catalase (0.1-0.2%) is added when completely cooled and then filter sterilized.       

     EXAMPLES 
     Example 1 
     In this example, sporicidal formulations were prepared by placing botanical extracts in a solution of 75 percent by volume of ethanol and 25 percent by volume of water. The following botanical extracts were shown to provide sporicidal efficacy. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Sporicidal activity of botanical extracts against C.  diff . spores. 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Log Reduction 
               
               
                   
                 Botanical Extract 
                 Concentration 
                 (5 min contact time) 
               
               
                   
                   
               
               
                   
                 
                   Setaria italica 
                 
                 100 mg/ml 
                 2.08 
               
               
                   
                 
                   Garcinia morella 
                 
                 200 mg/ml 
                 4.14 
               
               
                   
                 
                   Eucalypti globulus 
                 
                 200 mg/ml 
                 3.25 
               
               
                   
                 
                   Salvia miltiorrhiza 
                 
                 100 mg/ml 
                 1.63 
               
               
                   
                 
                   Psoralea corylifolia 
                 
                 100 mg/ml 
                 1.03 
               
               
                   
                 
                   Coptis teeta 
                 
                 100 mg/ml 
                 1.85 
               
               
                   
                   
               
            
           
         
       
     
     Example 2 
     In this example, sporicidal formulations were prepared by placing botanical-derived ingredients in a solution of 75 percent by volume of ethanol and 25 percent by volume of water. The following botanical-derived ingredients were shown to provide sporicidal efficacy. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Sporicidal activity of botanical-derived 
               
               
                 ingredients against C.  diff . spores 
               
            
           
           
               
               
               
               
            
               
                   
                 Botanical 
                   
                 Log Reduction 
               
               
                   
                 Extract 
                 Concentration 
                 (5 min contact time) 
               
               
                   
                   
               
               
                   
                 Neogambogic 
                 10 mg/ml 
                 3.96 
               
               
                   
                 acid 
               
               
                   
                 Gambogic acid 
                 10 mg/ml 
                 3.96 
               
               
                   
                 Cryptotanshinone 
                 5.9 mg/ml  
                 3.43 
               
               
                   
                   
               
            
           
         
       
     
     Example 3 
     Comparative 
     The following botanical extracts were tested to determine the sporicidal activity: osthole, usnic acid, kaempferol, genistein, luteolin, protopine, noroxylin, salidroside, quercetin, puerarin, p-hydroxy-cinnamic acid,  Artemisia argyi  levl. et vant.,  Smilax china  l.,  Ginkgo biloba  l.,  Gletilla striata  (thunb.) reichb.f,  Ajuga decumbens  thunb.,  Chelidonium majus  l.,  Paeonia lactiflora  pall.,  Atractylodes macrocephala  koidz.,  Litsea cubeba  (lour.) pers,  Alpinia officinarum  hance,  Schizonepeta tenuifolia  brig.,  Portulaca oleracea  l.,  Elsholtzia splendens  nakai ex f.maekawa,  Scrophularia ningpoensis  hemsl.,  Anemarrhena asphodeloides  bunge,  Cirsium japonicum  dc.,  Stephania cepharantha, Angelica dahurica  (fisch. ex hoffin.) benth. et hook. f,  Stemona japonica  (bl).miq.,  Stemona sessilifolia  (miq.) miq.,  Lobelia chnensis  lour.,  Macleaya cordata  (willd.) r.br.,  Amomum tsao - ko  crevost et lemaire,  Camellia sinensis  kuntze,  Plantago asiatica  l.,  Paeonia veitchii  lynch,  Andrographis paniculata  (burm.f.) nees.,  Acanthopanax senticosus  (rupr.et maxim.) harms,  Rheum palmatum  l.,  Codonopsis tubulosa  kom,  Juncus effusus  l.,  Kochia scoparia  (l.) schrad.,  Sanguisorba officinalis  l.,  Curcuma phaeocaulis  val,  Curcuma kwangsiensis  S. G. Lee et C. F. Liang,  Pueraria iobata  (willd.) ohwi,  Pogostemon cablin  (blanco) benth.,  Cinnamomum cassia  presl.,  Polygonum flaccidum  meissn.,  Polygonum cuspidatum  sieb. et zucc.,  Scutellaria baicalensis  georgi, aloe vera l.,  Ephedra sinica  stapf,  Ephedra intermedia  schrenk et C. A. Mey.,  Ephedra equisetina  bge.,  Lasiosphaera fenzlii  reich.,  Vitex trifolia  l.,  Belamcanda chinensis  (l.),  Evodia rutaecarpa  (juss.) benth.,  Cyperus rotundas  l.,  Cynanchum paniculatum  (bunge) kitag,  Inula japonica  thunb.,  Inula Britannica  l.,  Daemonorops draco  bl.,  Thalictrum baicalense  turcz.,  Baphicacanthus cusia  (nees) brem.  Litsea cubeba  (lour.) pers,  Berberis vernae  schneid. None of these botanical extracts illustrated sporicidal activity under the Sporicidal Efficacy Test described herein. 
     The following ingredients derived from botanical extracts were also tested: tanshinone IIA, dihydrotanshinone, L-alanine, β-alanine, L-proline, isoquercetrin, isoquercitrin, ferulic acid, caffeic acid, hyperoside, cineole, protocatechuic acid, p-hydroxybenzoic acid, stigmasterol, neobavaisoflavone, oleanolic acid, isoimperatorin, imperatonin, geniposide, bavachin, bavachinin, icaritin. None of these ingredients derived from botanical extracts illustrated sporicidal activity under the Sporicidal Efficacy Test described herein. 
     Unexpectedly, as illustrated by the large number of botanicals and botanical-derived ingredients that did not show sporicidal efficacy, the botanical extracts and botanical-derived ingredients described herein illustrate sporicidal efficacy. Furthermore, botanical-derived ingredients very similar in structure did not perform similarly. For example, cryptotanshinone was found to have sporicidal activity against  C. diff.  while tanshinone IIA and dihydrotanshinone were found to have no detectable effect. These compounds are very similar in structure; for example cryptotanshinone differs in structure from tanshinone IIA only by having two less hydrogen atoms on its pentyl ring. It would be expected that such similar structured chemicals would have a similar effect, but only certain botanical extracts and botanically derived ingredients functioned against spores. 
     Other modifications and variations to the appended claims may be practiced by those of ordinary skill in the art, without departing from the spirit and scope as set forth in the appended claims. It is understood that features of the various examples may be interchanged in whole or in part. The preceding description, given by way of example in order to enable one of ordinary skill in the art to practice the claimed invention, is not to be construed as limiting the scope of the invention, which is defined by the claims and all equivalents thereto.