REFERENCE BACTERICIDAL ENDPOINT (RBE) LIMONENE AND A PROCESS FOR THE PREPARATION THEREOF

Abstract: A process for producing a stable, bactericidal and fungicidal composition which consists essentially of bubbling air or oxygen into limonene, to produce an oxidized limonene having a rapid bactericidal activity as evidenced by the capability of killing substantially all of the bacteria in a suitable culture medium inoculated with 10<6> colony forming units of Staphylococcus aureus ATCC 25923 per ml and the product produced thereby.

Description: REFERENCE BACTERICIDAL ENDPOINT (RBE) LIMONENE AND A PROCESS ' FOR THE PREPARATION THEREOF TECHNICAL FIELD This invention relates to an oxidized li onene having a potent, rapidly acting antimicrobial activity that persists when stored; and an economical process for the preparation of that product. BACKGROUND OF THE INVENTION FIELD OF THE INVENTION During the study of fresh limonene as a hand cleaner, the applicants found that fresh limonene is an excellent sol¬ vent, but it is not bactericidal. Conversely, limonene which has undergone auto-oxidation, unexpectedly, was found to have a minimal, inconsistent antibacterial activity which varied in potency from batch to batch. Therefore, it was decided to purposely oxidize limonene to try to enhance and stabilize the antimicrobial properties of the limonene, The applicants found that by bubbling air (or oxygen) into limonene to purposely oxidize (or oxygenate limonene which is the same) the oil, that it became bactericidal at progressively decreasing concentrations until it reached a point beyond which further oxidation caused a decrease in its bactericidal activity. As the purposeful oxidation of limo¬ nene progressed, it was noted that the physical properties of the limonene changed as its antimicrobial activity increased. Limonene, which was purposely oxidized was found to be effec¬ tive in killing bacteria, yeasts, and fungi and its anti¬ microbial activity persisted unchanged when the purposely- oxidized limonene was stored at 2-4°C in the dark, for periods exceeding one year. SUBSTITUTE SHEET The ability to produce limonene with a stable antimicrobial activity was unexpected and is not appreciated nor delineated in the prior art. In fact it is recognized in the prior art that the addition of air (or oxygen) to terpenes produces volatile mixtures of oxides and peroxides which antibacterial effect that is not rapidly bactericidal. It evaporates to a thick, non- usable gel before it becomes an effective antimicrobial when auto-oxidation is allowed to occur over a large surface area of the oil. On the other hand, limonene that is purposely oxidized becomes an excellent, stable bactericide and fungicide that is bactericidal and fungicidal in bactericidal and fungicidal concentrations and its activity can be demonstrated with even shortT interval application. Thus, the object of this invention- is to economically produce oxidized limonene: (1) with a potent, rapidly acting antimicrobial activity, (2) that will retain its antimicrobial activity when stored, and (3) that is superior in its antimicrobial activity to the antimicrobial activity of auto-oxidized limonene. Limonene is a terpene with the following formula: SUBSTITUTESHEET d-limonene 1-limonene Limonene is an oil with a terpenic aroma. It is soluble in alcohol and miscible in oil, but it is insoluble in glycerine. Limonene is usually derived from either a pine or citrus origin. The 1-isomer is derived from pine and has a characteristic pine aroma while the d-isomer is derived from citrus and has a characteristic citrus aroma. The 1-isomer is also made synthetically but the synthetic 1-limonene has no characteristic aroma. SUBSTITUTE SHEET Commercial limonene is usually obtained from the peel of citrus fruit. Pressed liquor is obtained from the peel of any citrus fruit, but most commonly from oranges and grapefruit, after which, it is passed through a flash evaporation at 240° F at atmospheric conditions. Limonene is collected from the condensation of the flash vaporization. Limonene is used commercially as a flavoring agent, is used in large quantities to produce carvone which has a spearmint flavor, is used as a solvent, can be used as a base for soap or perfume, is used in the manufacture of rubber, and is useful as a penetrating oil. Limonene undergoes auto-oxidation when it is exposed to air or oxygen. After one to two weeks exposure to air at room temperature, limonene becomes rancid with a foul smelling odor. The temperature has to be above about 40° F for auto- oxidation to occur while temperatures above 90° F increase the rate of evaporation. Because auto- oxidation limits the commercial value of limonene by causing it to become rancid with a foul smelling odor, limonene is usually stored in 55 gallon drums which, have their interior painted with phenolic resin base enamel especially formulated for terpenes. The drums are completely filled. and capped in order to limit exposure to SUBSTITUTE SHEET air (and oxygen) which causes auto-oxidation. Auto-oxidized limonene develops antibacterial activity during auto-oxidation but does not become a rapidly acting bactericide. Auto-oxidation does not produce the potent, rapidly bactericidal limonene described in this patent. (2) Description of the prior art The oxidation of limonene produces several chemical compounds which have been enumerated by several different investigators. For instance Bain (in US Patent 2,SS3,882 and 3,014.047) showed a method of producing and recovering terpenic -oxidation products in which he delineated the oxidation products of limonene as: limonene-1,2- oxide, limonene-8,9-oxide, l-menthene-9-ol, -2,8- p-menthadiene-1-ol, -2,8-p-menthadiene-l-ol, dihydrocarvone, -cymenol, carvone, cis-carveol, and trans-carveol. He never studied the antimicrobial activity of oxidized limonene nor oxidized limonene to the point that it became stable with a potent antimicrobial activity. In fact, because limonene is chemically unstable during its oxidation, Bain thought that in order to obtain the maximum number of oxidation products from limonene, that he should add alkali to SUBSTITUTE SHEET limonene when the peroxide value was between 1000 and 2000 and should cease adding air,or oxygen to limonene. He thought the peroxide value was the sole indicator of the formation of limonene oxidation products (oxides and peroxides) and that these products could be obtained only when the oxidation of limonene was stopped as the peroxide value rose to its maximum. He never realized that by continuing to oxidize limonene that the maximal bactericidal activity achievable through the oxidation of limonene is obtained before maximal peroxide formation occurs. Blumann listed the compounds formed by the auto-oxidation of limonene in Chemical Abstracts, Volume 63, 1965, on page 1819, and found minute amounts of cis and trans-carveol, trans-p-menth-8- ene-l,2-diol, limonene 1,2-epoxide, limonene 8,9- epoxide, cis and trans-p-mentha-2,8-dien-l-ol, and perillyl alcohol, but he never tried to purposely oxidize limonene and never tried to develop limonene with a stable, rapidly bactericidal activity. Bardyshev studied the chemical compounds in photorinitiated, auto-oxidized, 1-limonene and found tiny amounts of the following compounds: carvone, carveol, trans-p-menth-8-ene-l,2,-diol, hydroperoxide, p-menthane, p-cymene, terpinolene, SUBSTITUTE SHEET p-mentha-2,4(8)-diene, cyclohexene, cuminic aldehyde, dihydrocarvone, piperitone, p-mentha- 2,8-dien-l-ol, pulegone, carvomenthone, PhCOMe, 4- MeC g H 4 C0Me, cyclohexanone, perillyl alcohol, and p-CH 2 :C eC 6 H 4 CH 2 OH as was outlined in Chemical Abstracts, Volume 80, 1974, page 359. Like Blumann, Bardyshev never purposely oxidized limonene, and never studied the antimicrobial activity of fresh nor auto-oxidized limonene. Zuckerman studied the effect of d-limonene on bacteria and found that fresh limonene had no effect on bacteria. He found that when limonene became auto-oxidized, it developed minimal, inconsistent, weakly inhibitory (not bactericidal) properties. He noted that, at best, auto-oxidized limonene was only weakly inhibitory in that it only "shifts the time of the lag phase of bacterial growth without any influence on the logarithmic phase of bacterial growth." He noted that auto-oxidized limonene is unstable, and loses its' bacteriostatic effect on keeping as was discussed in Nature 168: 517 (1951). He also recognized that the addition of alkali, as advocated by Bain in his method of oxidizing limonene, destroys the bacteriostatic properties of auto-oxidized limonene. He never recognized that limonene can be purposely oxidized to a point SUBSTITUTE SHEET that it becomes a potent, stable antimicrobial which, retains its antimicrobial activity when stored. In short, he could not make oxidized limonene with a stable rapidly bactericidal activity. In US Patent 3,595,975 Gauvreau showed a means of producing disinfecting compositions by combining cetyl pyridinium with, terpenes to form antiseptics, but he never learned that limonene which had been purposely oxidized, becomes a potent, stable bactericide and fungicide. The active ingredient in his disinfecting compositions was cetyl pyridinium chloride (and not the terpenes) . Disclosure of the Invention This invention relates to an economical and safe process of producing oxidized limonene that has a potent, stable antimicrobial activity which is retained when it is stored. Its antimicrobial activity is superior to, and distinct from, the antimicrobial activity of auto-oxidized limonene. In one preferred use the oxidized limonene of this invention is a icrobicide. Wherever used herein, the term "microbe" as used alone or in derivations such as microbicide and antimicrobial, means a SUBSTITUTE SHEET microscopic organism, such as bacteria, fungi, yeast, germs, viruses, and rickettsia. During the purposeful oxidation of limonene it was found that oxidized limonene became bactericidal as oxidation progressed. When the bactericidal activity of oxidized limonene: (a) became greater than that which could be accomplished by the auto-oxidation of limonene and (b) could be demonstrated at a concentration of 0.06 or less against Staphylococcus aureus ATCC 25923 when incubated for sixty minutes, the oxidized limonene was defined as reference bactericidal endpoint limonene (i.e. RBE limonene) . RBE limonene is a potent, stable, rapidly bactericidal compound. The production of oxidized limonene that is bactericidal at a concentration of 0.06 or less against Staphylococcus aureus ATCC 25923 when incubated for sixty minutes can be produced by continuously bubbling air (method one) or bubbling oxygen (method two) through limonene at room temperature for three to eight weeks. The applicants prefer to bubble air into limonene because oxygen is more expensive to use and oxygen has a great potential to explode, making it much more hazardous to handle. After limonene is oxidized and becomes a potent, stable bactericide, SUBSTITUTE SHEET regardless of whether it is produced by the addition of air or oxygen, it retains its antimicrobial activity when stored, maintains its pleasant aroma (as opposed to the rancid odor of auto-oxidized limonene) , and changes physical characteristics with an increased viscosity and an increased specific gravity. While the majority of limonene specimens which are purposely oxidized develop a potent, rapidly bactericidal activity, occasional limonene specimens do not become bactericidal with oxidation regardless of the length of, or the method of, oxidation. Thus, every batch of limonene which is purposely oxidized (to produce RBE limonene) must be individually tested for bactericidal activity. In short, the applicants's preferred method of producing oxidized limonene with a potent, stable bactericidal activity is to bubble air into limonene, because bubbling air through limonene is less expensive that using oxygen, and it is less hazardous to use than oxygen. Oxidized limonene is slightly viscous and readily adheres to glass, metal, wood, cloth, rope, book covers, cement, canvas, ceramic tile, plant surfaces, skin, mucous membranes and teeth leaving a film of oil. In practice, any surface on which it is desirable to kill or prevent SUBSTITUTE SHEET bacterial or fungal growth, is contacted directly with bactericidal or fungicidal concentrations of limonene which has been oxidized until it has developed a minimum bactericidal concentration of at least 0.06 or less for Staphylococcus aureus ATCC 25923 when incubated for sixty minutes. It can be applied by swabbing, wiping, painting, washing, brushing, spraying, or any other direct application technique. Alternatively, it can be incorporated in creams, ointments, tinctures, gels, suppositories, paints, sprays, aerosols, tooth-pastes, solutions, emulsions, surgical soaps, mouthwashes, or antiseptics and can be applied anywhere it is desirable to kill or prevent the growth of bacteria, fungi, or yeast. Best Mode for Carrying Out the Invention The following examples are illustrative of the best mode for carrying out the invention. They are, obviously, not to be construed as limitative of the invention since various other embodiments can readily be evolved in view of the teachings provided herein. SUBSTITUTE SHEET Example 1 In Example 1, the several methods of producing purposely oxidized limonene are demonstrated. In the first example, the process of bubbling air into limonene to produce oxidized limonene that is bactericidal at a concentration of 0.06 or less for Staphylococcus aureus ATCC 25923 when incubated for 60 minutes is shown in Table A below. The method of producing auto- oxidized limonene is shown in Table B. The process of producing oxidized limonene and auto-oxidized limonene was as follows: Four gallons of fresh d-limonene (Florida Chemical Company, Inc., Lake Alfred, Florida) were divided - into one gallon portions in small-mouthed metal containers. Two portions were treated at room temperature (~25°C) while two were treated at 50°C (in a water bath) . For each temperature, one portion was left open to room air (auto-oxidized) while the second portion was actively oxidized by continuously bubbling air through it with a Second Nature Challenger I Pump aerator (purposely oxidized) . Aliquots were removed from each of the four portions at 0, 2, 4, 6, 8, 10, 12, 14, and 16 weeks. For each aliquot, the viscosity, specific, gravity, peroxide value, and the antibacterial SUBSTITUTE SHEET activity against Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922 were determined as outlined in Tables A, B, C, D, and E below. For those portions treated at room temperature (Tables A and B) there was a clear difference in the type and potency of the antibacterial activity produced through the purposeful oxidation and auto-oxidation of d- limonene. The purposely oxidized limonene (Table A) showed maximal bactericidal activity by 6 to 8 weeks of treatment. It was rapidly bactericidal (10 minutes exposure) at dilutions up to 1:6.25 for §____ aureus ATCC 25923 and 1:800 for _______ coli ATCC 25922. It was bactericidal at dilutions of 1:1600 for both organisms with more prolonged exposure time (24 hours) . SUBSTITUTE SHEET Table A. Changes in Physical Parameters, Peroxide Value, and Bactericidal Activity of d-Limonene During The Purposeful Oxidation of the Oil at Room Temperature. In contrast, limonene that had been allowed to auto-oxidize at room temperature (Table B) showed maximal bactericidal activity at 14 to 16 weeks of treatment, but never reached the potency of the purposely oxidized oil. Rapid bactericidal activity (at 10 minutes) against _______ aureus ATCC 25923 was never seen with any dilution of the auto-oxidized oil and only weakly diluted oil (1:2.5 - 1:10) was rapidly bactericidal for _______ coli ATCC 25922. Even with prolonged incubation (24 hours), the auto-oxidized oil's bactericidal potency was 17 times less than that of the purposely oxidized oil against S___ aureus ATCC 25923 and four times less against _______ coli ATCC 25922. Increasing the treatment time of the oil beyond 16 weeks resulted in an overall decrease in potency. SUBST I TUTE SHEET Table B. Changes in Physical Parameters, Peroxide Value, and Bactericidal Activity of d-Limonene During Auto-Oxidation of the Oil at Room Temperature. Bactericidal Concentration For Auto-Oxidation at 5, aureus ATCC 25923 After £\ coli ATCC 25922 After Room Temperature Incubation for It jn Weeks l Min. 60 Min. 24 Hr. 10 Min. 0 (fresh) 1 2 1 c 4 0.5 6 1 CO 8 05 21 10 1 H 12 0.10 σ, I 14 0.40 £. 16 0.10 rπ 21 0.16 For the purposely oxidized limonene (Table A) , neither the highest peroxide value nor kinematic viscosity corresponded to the greatest antibacterial potency. This suggested that the antibacterial activity was not due to mere concentration of antibacterial substances resultant from evaporation that occurred during treatment. For the auto-oxidized limonene, antibacterial activity followed the peroxide value and kinematic viscosity up to 16 weeks. This suggested that the antibacterial may have been due, in part, to peroxides and concentration of antibacterial substances during treatment. A comparison of the antibacterial effects of the two oils when each had a similar peroxide value clearly reveals the distinct nature of the activity of the purposely oxidized limonene (Table C) . The auto-oxidized oil was not bactericidal at any dilution for either test organism after short exposure (10 minutes) while the purposely oxidized oil was bactericidal at a dilution 1:4.2 for S. aureus ATCC 25923 and 1:800 for _______ coli ATCC 25922. Even with prolonged exposure (24 hours), the bactericidal potency of the auto-oxidized oil was four to eight times less than that of the purposely oxidized oil. SUBSTITUTE SHEET Table C. Comparison of the Antibacterial Effects of Purposely Oxidized d-Limonene and Auto-Oxidized d-Limonene. , Bactericidal Concentration For S, aureus ATCC 25923 After Incubation For E. coli ATCC 29522 After Incubation For d-Limonene Preparation Peroxide Value lO Min. 6 Min. 24 Hr. 10 Min. 60 Min. 24 Hr. Purposely Oxidized, 8 Weeks 334.2 0.24 0.06 0.0006 0.0012 0,0006 0.0003 Auto-Oxidized, 10 Weeks 336.9 > 1 1 0,005 1 0.06 0,0012 cø c 130 i rn Compiled from Tables A and B I i rπ x m Increasing the temperature of oxidation from room temperature (~25°C) to 50°C did not uniformly decrease the time necessary to produce potent, antibacterial activity in the oils (Tables D and E) . For the purposely oxidized limonene, maximum bacterial activity was reached after only 3 weeks oxidation (Table D) . This was in contrast to the 6 to 8 weeks required for the purposeful oxidation at room temperature (Table A) . However, the rapid bacterial potency (10 minutes exposure) was 6 to 83 times less for oil purposely oxidized at 50°C in comparison to that purposely oxidized at room temperature. Thus, the two processes appeared to be generating oils with unique antibacterial effects. The oil purposely oxidized at 50°c became so viscous at 6 weeks, it could not be tested further for antimicrobial activity since it would not mix with the broth diluent in the assay. SUBSTITUTE SHEET Table D. Changes in Physical Parameters, Peroj ide Value, and Bactericidal Activity of d-Limonene During The Purposeful Oxidation of the Oil at 50°C. 1 Bactericidal Concentration For 5. aureus ATCC 25923 After E. coli ATCC 25922 After Purposeful Oxidation Incubation for Incubation for Perox iiddee S --.ppeecciifiπcc K Kiinneemmaattiic at 50°C in Weeks 10 Min. 60 Min. 24 Hr. lO Min. 6 Min. 24 Hr. Value 4 Oravitv 5 Viscosity 6 Oil too viscous to permit testing for antibacterial act vity after 6 weeks and for other parameters after 10 weeks. Auto-oxidation of limonene at 50°C produced an oil with even more distinct antibacterial activity (Table E) . Fourteen weeks of treatment are required to maximize its rapid bacterial effects (10 minutes exposure) while two to four weeks of treatment were required to maximize its longer interval (24 hours) killing. This was similar to the time interval required to maximize the rapid and longer interval bactericidal effects of limonene auto-oxidized at room temperature (Table B) . However, for the limonene auto- oxidized at 50°C, the bactericidal activity determined after long interval exposure (24 Hours) decreased after 4 weeks treatment when the test organism was S^ aureus ATCC 25923 but not when it was Ei coli ATCC 25922. These results indicated that auto-oxidation of limonene at 50°C could not produce the same bacterial activity as purposeful oxidation of limonene at room temperature. t--3. SSTITUTE SHE! Table E. Changes in Physical Parameters, Peroxide Value, and Bactericidal Activity of d-Limonene During Auto-Oxidation of the Oil at 50°C. Bactericidal Concentration For I t t I lowest concentration) killing at least 99.99% of the original inoculum. 'Undiluted oil was not bactericidal for 99.99% of the original inoculum. A summary of the maximal bactericidal activity obtained following treatment of limonene by the four processes described in the previous paragraphs is given in Table F. This activity has been separated into rapid bactericidal effects (10 minutes exposure) and longer interval bactericidal effects (24 hours exposure) to emphasize the unique nature of the activity produced by the purposeful oxidation of limonene at room temperature. From this summary it is clear that the rapid bactericidal activity can be produced only with purposeful oxidation of limonene at room temperature. The substances in the oil responsible for the rapid bactericidal activity appear to be heat labile (since they were not generated at 50°C) and are clearly not peroxides. Longer interval bactericidal effects can be produced in limonene by a variety of treatments, and these too appear unrelated to peroxide content of the oils. However, only purposeful oxidation of limonene at room temperature results in an oil with both rapid and longer interval bactericidal activity at low concentrations. SUBSTITUTE SHEET Table F. Maximal Bactericidal Activity and Peroxide Value of Various Limonene Preparations. Rapid Bactericidal Activity Against 3 • Long-Interval Bactericidal Activity Against Limonene Preparation S. a reus ATCC 25923 E. coli ATCC 25922 S. aureus ATCC 25923 E. coli ATCC 25922 A. Maximal Bactericidal Activit Purposely Oxidized, Room Temperature 0,16 (6) ' 0.0025 (6) 0,0012 (6) Purposely Oxidized, 50°C 1 (2) 0.1 (2) 0.0012 (2) Auto-Oxidized, Room Temperature 1 (16) 0.1 (16) 0,01 (2) Auto-Oxidized, 50°C 1 (14) 0.01 (14) 0,0025 (2) B. Peroxide Value of Oils with Maximal Bactericidal Activity CO Purposely Oxidized, Room Temperature 353.5 353.5 353,5 I H Purposely Oxidized, 50°C 286,1 286,1 286.1 t H Auto-Oxidized, Room Temperature 393,8 393.8 163.5 Auto-Oxidized, 50°C 2U.0 211.0 308.8 rπ ω 1 " Data from Tables A, B, D, and E IT! Measured after 10 min. exposure of the test organism to the oil rn ^Measured after 24 hr. exposure of the test organism to the oil Shown as highest dilution of oil killing 99.99% of bacterial inoculum ^Milliequivalents peroxide/kg sample Number in parentheses indicates weeks of treatment of the oil. The appearances of the oils following six weeks of the four treatment regimens are quite different. For purposely oxidized limonene, the oil treated at 50°C was darker yellow and more viscous than that treated at room temperature. For auto-oxidized limonene, the oil treated at room temperature was essentially unchanged in its appearance from un¬ treated (fresh) limonene. Limonene auto-oxidized at 50°C was similar in appearance to that purposely oxi¬ dized at room temperature. From the data presented in Tables A, B, D, and E, a single point was selected as a reference point to indicate that any future oxidized limonene was "fully oxidized". The reference bactericidal endpoint (RBE) selected was a bactericidal activity at a dilution of at least 1:16.7 (0.06) when tested against J--. aureus ATCC 25923 for 60 minutes exposure. This endpoint was selected because it (a) was found to be highly reproducible and required a greater dilution of oil than any 10 minute exposure endpoint, and (b) was an indicator for the presence of a rapidly bactericidal effect against S___ aureus and E^ coli and (c) was not obtained with any of the treatments of d-limonene except purposeful oxidation at room temperature. Thus, hereafter, SUBSTITUTE SHEET all oxidized limonene achieving this reference bactericidal endpoint is referred to as RBE limonene. The standard assay used to test the bactericidal activity against Staphylococcus aureus ATCC 25923 in Tables A, B, C, D, E, F, G, H, I, & J was as follows: various volumes of the desired limonene were added to Mueller-Hinton broth, mixed vigorously (Vortex Genie Mixer, Scientific Products, Evanston, 111.), inoculated with 10 6 colony forming units (CFU)/ml of Staphylococcus aureus ATCC 25923 or Escherichia coli ATCC 25922, remixed and incubated at 37°C in air. In varying the volume of the desired limonene to broth, concentrations of 1 (undiluted) to 0.01 (1:100 dilution) could be tested in a final volume of one to five ml (Table G) . Higher dilutions of limonene (up to 1:51,200) were tested by preparing serial two-fold dilutions from the 0.01 dilution (i.e. equal volumes of limonene- containing broth and broth were mixed serially) . At various time intervals, each tube was mixed vigorously and a 0.01 aliquot removed and subcul- tured on 5% sheep blood agar plates to determine the approximate number of viable CFU/ml in each tube. Results of these assays were expressed as the highest dilution of limonene with no detec- SUBSTITUTE SHEET table viable colonies on subculture i.e. the bactericidal concentration. This represented the lowest concentration killing 99.99 per cent of the original inoculum. TABLE G Dilutions of limonene used in fixed volume assays Dilutions Tested Limonene concentration Undiluted 1 1:2 0.5 1:2.5 0.4 1:3.3 0.3 1:4.2 0.24 1:5 0.2 1:6.25 0.16 1:10 0.1 1:16.25 0.06 1:50 0.02 1:100 0.01 To ascertain if a more potent bactericide could be produced through auto-oxidation of d- limonene when the process occurred over a large surface area of the oil, the following experiment was performed: Five hundred milliliters.of fresh d-limonene (Intercit Inc., Safety Harbor, Florida) was equally divided into 250 ml portions. One portion was placed into a 500 ml Erlenmeyer flask while the second portion was placed in a 500 ml SUBSTITUTE SHEΞ " beaker. The beaker was left open, exposed to air at room temperature (auto-oxidized) , while the sample in the flask was purposely oxidized at room temperature by continuously bubbling air through it with a Second Nature Challenger I Pump aerator (purposely oxidized limonene) . An aliquot of 5 ml was removed from each (beaker or flask) at one, two, and three weeks. The viscosity, specific gravity, peroxide value, and the bactericidal activity against S^. aureus ATCC 25923 of each aliquot was determined as previously outlined. In this experiment, auto-oxidation could take place over a larger surface area of the oil than in the prior experiment (performed in the small-mouthed container) . Purposeful oxidation was still performed in a small-mouthed container in this experiment to prevent any ancillary auto- oxidation. As shown in Table H, auto-oxidation of d- limonene at room temperature over a large surface area of the oil, did not produce a highly bactericidal oil. The characteristics of the oil found at three weeks were most similar to those of limonene auto-oxidized over a small surface area after six weeks (Table B) . However, oxidation over a large surface area could not be continued for prolonged periods because evaporation of the SUBSTITUTE SHEET oil led to a product too viscous to examine for bactericidal activity. Thus, auto-oxidation of limonene over a large surface area did not produce a rapidly bactericidal oil and evaporation precluded long-term oxidation under these conditions. SUBSTITUTE SHEET Table H Changes in the physical parameters, peroxide value, and bacterici d al activity^of d-limonene 1 during auto-oxidation of the oil over a lar g e surface area. Auto-oxidation 1 Bactericidal Concentration 2 At Room Temper- After Incubation for: Peroxide Specific Viscosity 5 ature in Weeks 10 Min 60 Min 24 Hrs Value 3 Gravity 4 c ro 3 H . >1 >1 0.01 231.3 I -j 1 250 ml of fresh limonene (interσit Inc., Safety Harbor, Florida, lot rπ ω 509801 in an open beaker at room temperature T rπ 2 Bactericidal concentration shown as the highest dilution (i.e. lowest concentration) killing at least 99.99% of the original inoculum of S__. aureus ATCC 25923 3 Milliequivalents peroxide/kg sample 4 at 25°C 5 Cst at 25°C ° Undiluted oil was not bactericidal The bactericidal activity of purposely oxidized limonene in this experiment is shown in Table I. As was seen before, a rapidly bactericidal oil was produced by this process. The source of the oil was completely different than that shown in Table A, and the reference endpoint (i.e. bactericidal at a concentration of 0.06 or less for S___ aureus ATCC 25923) was reached sooner than the previous experiment. These results suggested that different lots of limonene may require different treatment times for purposeful oxidation to reach the defined reference endpoint. SUBSTITUTE SHEET TABLE I Changes In Physical Parameters, Peroxide Value, and B Activity of d-Limonene 1 During The Purposeful Oxidati Room Temperature Purposeful Bactericidal Concentration 3 Peroxide Sp Oxidation 2 For S^. aureus ATCC 25923 Value 4 Gr at Room After Incubation For: Tem erature lOMin 60M 24Hrs 68.7 CO 2 Air bubbled continuously into 250 ml of limonene in -X Erlenmeyer flask with aerator (Second Nature Challe IT! room temperature. m "» 3 Bactericidal concentration shown as the highest dil lowest concentration) killing at least 99.99% of th inoculum of S. aureus ATCC 25923 ^ Milliequivalents peroxide/kg sample 5 at 25°C - 6 -- Cst at 25°---C ' ' 7 Undiluted oil was not bactericidal The ability of purposeful oxidation of limonene to produce a rapidly bactericidal oil was determined using limonene from various sources. As shown in Table J, not all samples of limonene became bactericidal with purposeful oxidation at room temperature. Two samples from Aldrich did not acquire bactericidal activity even after purposeful oxidation for five weeks while one sample obtained form this source became rapidly bactericidal in as short as three weeks. Also, as shown in Table J, both d-and 1- isomers of limonene can be purposely oxidized to become a rapid bactericide. These data emphasize the need to test every batch of purposely oxidized limonene -after three to eight weeks treatment to ensure that the reference bactericidal endpoint has been reached. SUBSTITUTE SHEE " TABLE J Antibacterial Activity Of Various Limonene Samples As Determined Against Staphylococcus aureus ATCC 25923 During Oxidation at Room Temperature. Purposeful MBC for S. Aureus ATCC Sample & Oxidation 25923 Measured At Source in weeks 10 Mins 60 Mins 24 Hrs d-limonene Aldrich 1 5 weeks >1 : >1 >1 1-limonene Aldrich 1 5 weeks >1 >1 >1 1-limonene SCM 2 3 weeks >1 >1 >1 d-limonene Intercit 3 3 weeks 0.1 0.005 0.0003 1-limonene SCM 2 3 weeks 0.1 0.005 0.0006 d-limonene Aldrich 1 3 weeks 0.005 0.0024 0.0003 d-limonene FCC 4 6 weeks 0.16 0.06 0.0012 1 Aldrich, Milwaukee, Wisconsin 2 SCM Glidden Organics, Jacksonville, Florida 3 Intercit, Inc. , Safety Harbor, Florida 4 Florida Chemical Company, Lake Alfred, Florida 5 Undiluted oil was not bactericidal The second method of producing RBE limonene is by continuously bubbling oxygen into limonene as outlined in Table below. This process, like bubbling air through limonene, produces a rapidly bactericidal oil and requires 6 to 7 weeks treatment for the reference endpoint to be achieved. SUBSTITUTE SHEET TABLE K Bactericidal Activity d-limonene 1 During Oxygenation As Determined Against Staphylococcus aureus ATCC 25923 at 25°C. 1 Fresh d-limonene (Intercit Inc. , Safety Harbor, Florida 509801) 2 Results expressed as the highest dilution of limonene killing at least 99.99% of the original inoculum. 3 Oxygen bubbled continuously into d-limonene at a rate of 3 liters per minute at 25°C. 4 Undiluted oil was not bactericidal. SUBSTITUTE SHEET Example 2 In Example 2 the bactericidal and fungicidal activity of RBE d-limonene and RBE 1-limonene was determined against bacteria, yeast, and fungi as outlined in Tables L & M below. TABLE L BACTERICIDAL ACTIVITY OF RBE d-LIMONENE ORGANISM BACTERICIDAL CONCENTRATION AFTER INCUBATION FOR: A. Bacteria 10 Min 60 Min 24 Hrs 1. Staphylococcus aureus, ATCC 25923 0.30 0.06 0.0012 2. Staphylococcus aureus 3 1.00 0.10 0.001 3. Staphylococcus aureus 27 ** ^ 1.00 0.10 0.001 4. Staphylococcus aureus 37 0.30 0.10 0.0025 5. Staphylococσus aureus 39 " *^ 0.30 0.06 " 0.0012 6. Staphylococcus aureus 52T 0.30 0.06 0.0012 7. Staphylococcus aureus 53T 0.30 0.06 0.0025 8. Staphylococcus aureus 54T 0.30 0.06 0.0025 9. Staphylococcus epidermitis 10 1.00 0.01 0.001 10. Sarcinia lutea 1.00 0.01 0.001 11. Streptococcus mitus 15 (alpha) 0.01 0.01 0.001 12. Streptococcus pyoqenes 1 (group A) 0.01 0.01 0.01 SUBSTITUTE SHE! 13. Streptococcus salivarius (gamma) 14. Streptococcus mutans 15. Streptococcus faecalis 15 16. Lactobacillus casi M7 17. Listeria monocvto enes M9 0.01 0.01 0.01 18. Bacillus subtilis ATCC 6633 19. Escherichera coli 7 20. Klebsiella SP 12 21. Salmonella 14 (para B) 22. Shiqella sonnei 8 23. Proteus mirabilis 2 24. Proteus vulcraris 127 25. Proteus rettqeri 106 26. Proteus morαanii 99 27. Providencia stuartii 97 28. Enterobacter cloacae 20 29. Citrobacter SP 8 30. Pseudomonas aeriqinosa 115 0.10 0.10 0.01 31. Pseudomonas mendocinaa 129 32. Neisseria gonorrhea 5 33. Neisseria gonorrhea (Pen R)* 34. Hemophilus influenza (Amp R)** SUBSTITUTE SHEET 35. Hemophilus influenza (Amp S)*** 0..001 0.001 0.001 36. Neisseria meninqitidis 0.001 0.001 0.001 37. Xanthomonas campestris pv vesicatoria 1 0.0012 0.0003 0.0003 38. Xanthomonas campestris pv vesicatoria 2 0.0012 0.0003 0.0003 39. Xanthomonas (SP)* + isolated from a Nebraska farmer 0.0025 0.0003 0.0003 40. Xanthomonas campestris pv citri 0.005 0.0003 0.0003 41. Mycobacterium tuberculosis var hominis H37 RV 0.001 0.001 0.0001 42. Mycobacterium kansasii Tarn 30 0.1 0.01 0.0001 43. Mycobacterium scrofulaceum 0.1 0.01 0.0001 44. Mycobacterium intracellulare 0.1 0.10 0.0001 45. Mycobacterium fortuitum ATCC 6841 1.0 0.10 0.0001 B. YEAST 1. Candida albicans 0.01 0.001 0.001 2. Sacchromyces SP 0.01 0. 01 0.001 C. FUNGI FUNGICIDAL CONCENTRATION AFTER INCUBATION FOR: 10 Min 60 Min 24Hrs 7-45 Days 1. Epidermophyton floccosum 0.001 2. Microsporum audouini 0.001 3. Microsporum canis 0.01 0.0025 0.0006 0.001 SUBSTITUTE SHEET 4. Trichophvton mentaqrophvtes 0.001 5. Aureoblasidium pullulans OM 279C 0.01 0.005 0.0012 6. Cladosporium cladosporiodes OM 489 0.01 0.005 0.0012 7. Philopohora lingnicola OM 5922 0.10 0.01 0.0025 * ** Penicillin resistant *** Ampicillin resistant Ampicillin sensitive *+ Species unidentified TABLE M BACTERICIDAL AND FUNGICIDAL ACTIVITY OF RBE 1- LIMONENE ORGANISM BACTERICIDAL CONCENTRATION AFTER INCUBATION FOR: A. Bacteria 10 Min 60 Min 24 Hrs 1. Staphylococcus aureus, ATCC 25923 0.5 0.06 0.0025 2. Staphylococcus aureus 37 (Pen R) 0.5 0.40 0.005 3. Staphylococcus aureus 39 (Meth R) 0.3 0.06 0.0025 4. Staphylococcus aureus 52T 0.5 0.10 0.01 5. Staphylococcus aureus 53T 0.50 0.10 0.0025 6. Staphylococcus aureus 54T 0.5 0.06 0.01 7. Staphylococcus epidermitis 10 0.1 0.1 0.0025 8. Streptococcus mutans M42 0.3 0.16 0.005 9. Streptococcus SUBSTITUTE SHEET faecalis 15 0.1 0.01 0.0025 10. Bacillus subtilis ATCC 6633 0.1 0.1 0.1 11. Escherichera coli 7 0.005 0.0025 0.0012 12. Salmonella 14 (para B) 0.0025 0.0025 0.0006 13. Shiqella sonnei 8 0.01 0.0025 0.0012 14. Pseudomonas aeri inosa 115 0.1 0.1 0.005 15. Neisseria meninqitidis 40 0.0012 0.006 0.00004 16. Xanthomonas campestris pv vesicatoria 1 0.0025 0.00012 0.0012 17. Xanthomonas campestris pv vesicatoria 2 0.0025 0.0012 0.0003 18. Xanthomonas (SP)* + isolated from a Nebraska farmer 0.0006 0.0006 0.0003 19. Xanthomonas campestris pv citri 0.005 0.0025 0.0006 20. Mycobacterium tuberculosis var hominis H37 RV 0.0005 0.000125 0.000125 21. Mycobacterium fortuitum ATCC 6841 0.1 0.1 0.0006 B. YEAST 1. Candida albicans 0.0012 0.0012 0.0006 C. FUNGI FUNGICIDAL CONCENTRATION AFTER INCUBATION FOR: 10 Min 60 Min 24Hrs 1. Microsporum canis 0.01 0.0025 0.0006 SUBSTITUTE SHEET 2. Aureoblasidium pullulans OM 279C 0.1 0.005 0.0012 3. Cladosporium cladosporiodes OM 489 0.01 0.005 0.0012 4. Philopohora lin nicola OM 5922 0.1 0.01 0.005 * Penicillin resistant ** Methcillin resistant * + Species unidentified The standard assay used to test the bactericidal activity of the d and 1 isomers of limonene which had been oxidized until they reached the reference bactericidal endpoint was as follows: various dilutions of the desired limonene were separately prepared in an appropriate broth medium for the test strain. Ah inoculum of 10 6 colony-forming units (CFU)/ml was used. Each test was incubated at the proper temperature for each organism and subcultured (0.01 ml) at 10 minutes, 60 minutes, and 24 hours onto agar media free of limonene. Results were expressed as the bactericidal concentration, i.e., the lowest concentration of RBE limonene (ml limonene/total ml of test) killing at least 99.99% of the inoculum. The activity of RBE limonene was evaluated against Mvcobacteria using undiluted oil, and oil diluted up to 1:8000 in Proskauer Beck liquid medium. Each test was inoculated with 10 6 CFU/ml SUBSTITUTE SHEET of Mycobaσteria and incubated at 37 degrees C in 7% C0 2 in air. At various time intervals, each test was shaken vigorously and a 0.01 ml aliquot removed. This was subcultured onto Dubos oleic acid-albumin agar plates to determine the number of viable Mycobacteria remaining. Each test was sampled in this fashion after incubation for 10 minutes, 60 minutes, 24 hours, 1, 2, 3, and 4 weeks. The bacterial concentration was defined as the lowest concentration of oil killing 99.99% of the original inoculum. The standard assay used to test certain fungi (dermatophytes) was as follows: RBE d-limonene and RBE 1-limonene were tested in air on sabouraud dextrose agar slants supplemented with 0.05% yeast extract. Each of the two compounds was individually added to different samples of molten agar in dilutions of 1:10, 1:15, 1:100, 1:1000 and then mixed vigorously after which the agar was allowed to solidify. The inoculum was prepared as follows: 10 ml of saline (0.85%) and four drops of tween 80 were added to sabouraud dextrose agar slants containing the fungus grown for 14 days at room temperature. After vigorous shaking, the culture was filtered through sterile gauze and adjusted to the density of a MacFarland Standard No. 1 with 0.85% saline. The limonene containing SUBSTITUTESHEET -43- slants were inoculated with 0.1 ml of this adjusted suspension, incubated at room temperature and checked for growth daily for 43 days. After 7 days, confluent growth of each fungus was observed on limonene-free control slants. No growth was observed on any limonene containing slants during the 43 days. The fungicidal concentration of RBE limonene was defined as the lowest concentration preventing growth on the slants for 43 days. The test conditions used to assay the antimicrobial activity of RBE limonene are listed in Table N which follows. SUBSTITUTE SHEET TABLE N SUBCULTURE INCUBATION ORGANISM BROTH MEDIUM AGAR MEDIUM CONDITIONS 1. Staphylococcus, Mueller- 5% sheep Air @ Sarcinia, Hinton blood 37°C Bacillus Enterobac- teriaceae. and Pseudomonas Streptococcus r Lactobacillus- Listeria Neisseria meninqitidis 4. Neisseria gonorrhea Hemophilis influenza 6. Yeast 7. Xanthomonas campestris vesicatoria & citri 8. Xanthomonas Mueller- blood agar air at species iso¬ Hinton 37°C lated from a Nebraska farmer (species not identified) 9. Mvcobacteria Proskauer Dubos oleic 7% CO-- 10. Fungi SUBSTITUTE SHEl El -45- Exa ple 3 In example 3, formulations, containing RBE limonene are listed. RBE d-limonene and RBE 1- limonene are prepared in the following formulations including liquids, gels, soaps, paints, pastes, creams, ointments, suppositories, tampons, aerosols, and emulsions. When bacteria, fungi or yeasts are treated with formulations that contain RBE d-limonene or RBE 1-limonene, the formulations kill or prevent the growth of bacteria, yeasts, and fungi. SUBSTITUTE SHEET -46- 2. GEL CHEMICAL OF TOTAL RANGE ACTION 100.05 SUBSTITUTE SHEE" 3. PASTE CH I A % OF TOTAL RANGE AC I 100.0% SUBSTITUTE SKEE C. OINTMENTS S SUPPOSITORIES WITH AND WITHOUT HYDROCORTISONE 1. OINTMENT WITH HYDROCORTISONE CHEMICAL % OF TOTAL RANGE ACTION RBE d-limonene 1.0% 0.1-15.0% bacteri- cide Polyethylene glycol 3350 59.5% 48.5-59.7% bodying agent & emulsi- fier Polyethylene glycol 400 39.5% 31.5-39.7% bodying agent & emulsi- fier Hydrocortisone 1.0% 0.5-5.0% anti- 100.0% inflam¬ matory 2. OINTMENT WITHOUT HYDROCORTISONE CHEMICAL % OF TOTAL RANGE ACTION RBE 1-limonene 1.0% 0.1-15.0% fungi¬ cide Polyethylene glycol 3350 59.5% 51.0-59.95% bodying _____ agent & emulsi- fier Polyethylene glycol 400 39.5% 34.0-39.95% bodying 100.0% agent & emuls- ifier SUBSTITUTE SHEET -50- 3. SUPPOSITORY WITHOUT HYDROCORTISONE CHEMICAL % OF TOTAL RANGE ACTION RBE 1-limonene 1.0% 0.1-15% bacteri- cide Polyethylene glycol 1000 9.5% 51.0-59.95% bodying agent & emulsi- fier Polyethylene glycol 3350 39.5% 34.0-39.95% bodying 100.0% agent & emulsi- fier 4. SUPPOSITORY WITH HYDROCORTISONE CHEMICAL % OF TOTAL RANGE ACTION RBE d-limonene 1.0% 0.1-15% anti- yeast Polyethylene glycol 1000 74.0% 60.0-75.2% bodying agent & eroulsi- fier Polyethylene glycol 3350 24.0% 20.0-24.2% bodying agent & emulsi- fier Hydrocortisone 1.0' 0.5-5.0? anti- 100.0? inflam- matory D. CREAMS WITHOUT HYDROCORTISONE CHEMICAL % OF TOTAL RANGE ACTION RBE 1-limonene 1.0% 0.1-15.0% bacteri- cide Cetyl alcohol 15. 0% 12. 0-18. 0? thickener Arlacel 165 ** 5. 0% 3 .5-7 .5% emulsi- fier Sorbitol 70% solution 5. 0? 3 .5-8 . 0% humectant Water 74. 5? 51.5-80. 9? diluent 100. 0? SUBSTITUTE SHEE " .-51- CHEMICAL % OF TOTAL RANGE ACTION RBE 1-limonene 1.0% 0.1-15.0% fungi¬ cide Spermaceti wax 12.5% 10.0-15.0% thickener Sorbitan monostearate 100.0% E. CREAMS WITH HYDROCORTISONE Water 73.0% 46.5-80.4% diluent 100.0% * Croda, Inc., 51 Madison Ave. , New York, New York 10010 ** Glycerol monostearate and polyoxyethylene stearate ICI of America (Formerly Atlas Chemical Industries) , Wilmington, Delaware 19899 STITUTE SHEET -52- G. AEROSOLS WITHOUT HYDROCORTISONE CHEMICAL % OF TOTAL RANGE ACTION RBE 1-limonene 6.0% 0.5-50% bacteri- cide Ethyl alcohol 94.0? 50-99.5% diluent 100.0% Pressurized nitrogen propellant at 100-125 psig CHEMICAL % OF TOTAL RANGE ACTION RBE d-limonene 10.0% 0.5-50.0% fungi¬ cide Soybean Oil 90.0% 50.0-99.5% diluent 100.0% Pressurized nitrogen propellant at 100-125 psig H. AEROSOL WITH HYDROCORTISONE Pressurized nitrogen propellant at 100-125 psig SUBSTITUTE SHEET I. OIL IN WATER EMULSION CHEMICAL (1) RBE d-limonene (2) Corn oil (2) Arlacel 40** (2) Tween 40 (3) Water Heat (2) to 70°C. Heat (3) to 72°C. Add (3) to (2) with continuous agitation. When (3) and (2) cool to 40°C, add (1) with continous agitation until room temperature is reached. J. OIL IN WATER EMULSION WITH SOAP (FUNGICIDAL SOAP) CHEMICAL (1) RBE d-limonene (2) Corn oil (2) Arlacel 40** (2) Tween 40 (2) Liquid soap concentrate (3) Water Heat (2) to 70°C. Heat (3) to 72°C. Add (3) to (2) with continuous agitation. When (3) and (2) cool to 40°C, add (1) with continous agitation until room temperature is reached. SUBSTITUTE SHEET Heat (2) to 70°C. Heat (3) to 72°C. Add (3) to (2) with continuous agitation. When (3) and (2) cool to 40°C, add (1) with continous agitation until room temperature is reached. SUBSTITUTE SHEET 2 . LATEX While only certain preferred embodiments of this invention have been shown and described by " way of illustration, many modifications will occur to those skilled in the art and it is, therefore,- desired that it be understood that it is intended herein, to cover all such modifications that fall within the true spirit and scope of this invention. SUBSTITUTE SHEET

Claims: CLAIMS: WE CLAIM: 1. Reference bactericidal endpoint (RBE) limonene as a compound of matter comprising oxidized limonene that is bactericidal at a concentration of 0.06 ml. per ml. of a broth culture medium inoculated with 10 6 colony forming units of Staphylococcus aureus ATCC 25923 after being incubated at 37°C in air for 60 minutes. 2. A process for producing a stable, bactericidal and fungicidal composition that is bactericidal and fungicidal in bactericidal and fungicidal concentrations, which process consists essentially of bubbling air into limonene, or bubbling oxygen into limonene, to produce an oxidized limonene having a rapid bactericidal activity as evidenced by the capability of killing substantially all of the bacteria in a suitable culture medium inoculated with 10 6 colony forming units of Staphylococcus aureus ATCC 25923 per ml. of said broth, containing not more than 0.06 ml of said oxidized limonene per ml. of said broth and incubated at 37°C in air for 60 minutes. 3. The process of Claim 1 wherein said rapid bactericidal activity remains substantially unchanged upon storage at 4°C in a dark covered container for at least 6 months. S UB STITUTESHEET 4. The process of claim 1 wherein said process consists essentially of bubbling air into limonene until said rapid bactericidal activity is reached. 5. The process of claim 1 wherein said process consists essentially of bubbling oxygen into limonene until rapid bactericidal activity is reached. 6. The process of Claim 3 wherein said air is bubbled into limonene for 3 - 8 weeks at room temperature. 7. The process of Claim 4 wherein said oxygen is bubbled into limonene for 3 - 8 weeks at room temperature. 8. A process for producing a stable microbicide which process consists essentially of bubbling air or oxygen into limonene to produce an oxidized limonene having a rapid bactericidal activity as evidenced by the capability of killing substantially all- of the bacteria in a suitable culture medium inoculated with 10 6 colony forming units of Staphylococcus aureus ATCC 25923 per ml. of said broth, containing not more than 0.06 ml of said oxidized limonene per ml. of said broth and incubated at 37°C in air for 60 minutes. 9. The process of Claim 8 wherein said rapid bactericidal activity remains substantially unchanged upon storage at 4°C in a dark covered _, SUBSTITUTE SHEET container for at least 6 months. 10. The process of Claim 8 wherein said process consists essentially of bubbling air into limonene until said rapid bactericidal activity is reached. 11. The process of Claim 8 wherein said process consists essentially of bubbling oxygen into limonene until said rapid bactericidal activity is reached. 12. The process of Claim 10 wherein the said air is bubbled into limonene for about 3-8 weeks at room temperature. 13. The process of Claim 10 wherein the said oxygen is bubbled into limonene for about 3-8 weeks at room temperature. SUBSTITUTE SHEET

Inventors: CHASTAIN DOYLE E, SANDERS CHRISTINE C, SANDERS W EUGENE JR, CHASTAIN, DOYLE, E., SANDERS, CHRISTINE, C., SANDERS, W., EUGENE, JR.
Applicants: CHASTAIN DOYLE E, CHASTAIN, DOYLE, E.
Application year: 1992
Main classification: A01N27
IPC codes: A01N27, A01N61