Process of producing reference bactericidal endpoint (RBE) limonene

TECHNICAL FIELD
The object of this invention is to show an economical process of producing oxidized limonene which has a potent, rapidly acting antimicrobial activity that persists when stored, and which is superior in antimicrobial activity to that of auto-oxidized limonene.
BACKGROUND OF THE INVENTION
(1) Field of the Invention
During the study of fresh limonene as a hand cleaner, the applicants found that fresh limonene is an excellent solvent, but it is not bactericidal. Conversely, limonene which has undergone auto-oxidation, unexpectedy, 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) to 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 limonene 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 effective in killing bacteria, yeasts, and fungi and its antimicrobial activity persisted unchanged when the purposely oxidized limonene was stored at 2.degree.-4.degree. C. in the dark, for periods exceeding one year.
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 that are unstable. Limonene which undergoes auto-oxidation has an inconsistent 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 fungicidal that is bactericidal and fungicidal in bactericidal and fungicidal concentrations and its activity can be demonstrated with even short 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: ##STR1##
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 l-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 l-isomer is also made synthetically, but the synthetic l-limonene has no characteristic aroma.
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.degree. 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 exposure to air at room temperature, limonene becomes rancid with a foul smelling odor. The temperature has to be above about 40.degree. F. for auto-oxidation to occur while temperatures above 90.degree. 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 that 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 air (and oxygen) that 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 that have been enumerated by several different investigators. For instance Bain (in U.S. Pat. Nos. 2,863,882 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, 1-menthene-9-ol, .alpha.-2,8-p-menthadiene-1-ol, .beta.-2,8-p-menthadiene-1-ol, dihydrocarvone, .beta.-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 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 the maximal activity achievable through the oxidation of limonene is obtained before maximum 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-1,2-diol, limonene 1,2-epoxide, limonene 8,9-epoxide, cis and trans-p-mentha-2,8-dien-1-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 photo-initiated, auto-oxidized, 1-limonene and found tiny amounts of the following compounds: carvone, carveol, trans-p-menth-8-ene-1,2,-diol, hydroperoxide, p-menthane, p-cymene, terpinolene, p-mentha-2,4(8)-diene, cyclohexene, cuminic aldehyde, dihydrocarvone, piperitone, p-mentha-2,8-dien-1-ol, pulegone, carvomenthone, PhCOMe, 4-MeC.sub.6 H.sub.4 COMe, cyclohexanone, perillyl alcohol, and p-CH.sub.2 :CMeC.sub.6 H.sub.4 CH.sub.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 inhibitory 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 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 U.S. Pat. No. 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 microbicide. Wherever used herein, the term "microbe" as used alone or in derivatives such as microbicide and antimicrobial, means a microscopic organism, such as bacteria, fungi, yeast, germs, viruses, and rickettsia.
During the purposeful oxidation of limonene it was found that oxidized limonene became rapidly 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 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, regardless of whether it is produced by the addition of air or oxygen, it retains its antimicrobial activity when stored, maintains its pleasant terpenic 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 applicant'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 than using oxygen, and 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 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, toothpastes, 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 yeasts.

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.
EXAMPLE 1
In Example 1, the several methods of producing purposely oxidized limonene that is bactericidal 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 purposely oxidized limonene and auto-oxidized limonene was as follows: Four gallons of fresh d-limonene (Florida Chemical Company, Inc., Lake Alfred, Fla.) were divided into one gallon portion in small-mouthed metal container. Two portions were treated at room temperature (.about.25.degree. C.) while two were treated at 50.degree. 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 activity against Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922 were determined as outlined in Tables A, B, D, and E below.
For those portions treated at room temperatures (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 maximum bactericidal activity by 6 to 8 weeks of treatment. It was rapidly bactericidal (10 minutes exposure) at dilutions up to 1:6.25 for S. aureus ATCC 25923 and 1:800 for E. coli ATCC 25922. It was bactericidal at dilutions of 1:1600 for both organisms with more prolonged exposure time (24 hours).
TABLE A__________________________________________________________________________Changes in Physical Parameters, Peroxide Value, and Bactericidal Activityof d-Limonene.sup.1During the Purposeful Oxidation of the Oil at Room Temperature.Purposeful Bactericidal Concentration.sup.3 ForOxidation.sup.2 at S. aureus ATCC 25923 After E. coli ATCC 25922 AfterRoom Temperature Incubation for Incubation for Peroxide Specific Kinematicin Weeks 10 Min. 60 Min. 24 Hr. 10 Min. 60 Min. 24 Hr. Value.sup.4 Gravity.sup.5 Viscosity.sup.6__________________________________________________________________________ 0 (fresh) >1.sup.7 >1 0.2 1 1 0.10 50.2 0.8439 1.035 2 1 0.5 0.0025 0.24 0.02 0.0012 344.3 0.8535 1.176 4 1 0.10 0.0006 0.06 0.0012 0.0006 327.2 0.9118 2.847 6 0.16 0.06 0.0012 0.0025 0.0012 0.0006 353.5 0.9679 10.910 8 0.24 0.06 0.0006 0.0012 0.0006 0.0003 334.2 1.0122 51.8510 1 0.10 0.0012 0.10 0.0025 0.0012 405.8 1.0420 199.712 0.4 0.10 0.0025 0.005 0.0025 0.0006 389.8 1.0633 500.414 0.4 0.10 0.0025 0.06 0.010 0.0012 848.3 1.0665 1,268.0016 0.4 0.16 0.0025 0.10 0.005 0.0006 929.9 1.0784 2,562.00__________________________________________________________________________ .sup.1 Florida Chemical Co., Inc., Lake Alfred, Florida .sup.2 Air was bubbled continuously into 1 gallon of oil in a smallmouthe metal container with an aerator (Second Nature Challenger I Pump). .sup.3 Bactericidal concentration shown as the highest dilution of limonene (i.e. lowest concentration) killing at least 99.99% of the original inoculum. .sup.4 Milliequivalents peroxide/kg sample .sup.5 at 25.degree. C. .sup.6 at 25.degree. C. cSt .sup.7 Undiluted oil was not bactericidal for 99.99% of the original inoculum. In contrast, limonene that had been allowed to auto-oxidize a room temperature (Table B) showed maximum 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 S. 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 E. 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 E. coli ATCC 25922. Increasing the treatment time of the oil beyond 16 weeks resulted in an overall decrease in potency.
TABLE B__________________________________________________________________________Changes in Physical Parameters, Peroxide Value, and Bactericidal Activityof d-Limonene.sup.1During Auto-Oxidation of the Oil at Room Temperature. Bactericidal Concentration.sup.3 ForAuto-Oxidation.sup.2 at S. aureus ATCC 25923 After E. coli ATCC 25922 AfterRoom Temperature Incubation for Incubation for Peroxide Specific Kinematicin Weeks 10 Min. 60 Min. 24 Hr. 10 Min. 60 Min. 24 Hr. Value.sup.4 Gravity.sup.5 Viscosity.sup.6__________________________________________________________________________ 0 (fresh) >1.sup.7 >1 0.2 1 1 0.10 50.2 0.8439 1.035 2 >1 >1 0.01 1 0.24 0.005 163.5 0.8440 1.066 4 >1 >1 0.01 0.5 0.16 0.0025 199.5 0.8477 1.082 6 >1 >1 0.01 1 0.06 0.0025 229.5 0.8486 1.090 8 1 1 0.01 0.5 0.06 0.0012 288.2 0.8510 1.12410 >1 1 0.005 1 0.06 0.0012 336.9 0.8548 1.16712 1 0.5 0.01 0.10 0.01 0.0025 325.7 0.8570 1.19814 1 0.4 0.01 0.40 0.0025 0.0012 366.1 0.8587 1.24716 1 0.4 0.01 0.10 0.01 0.0025 393.8 0.8626 1.29921 >1 1 0.005 0.16 0.005 0.0025__________________________________________________________________________ .sup.1 Florida Chemical Co., Inc., Lake Alfred, Florida .sup.2 1 gallon of oil in smallmouthed metal container left open to room air. .sup.3 Bactericidal concentration shown as the highest dilution of limonene (i.e. lowest concentration) killing at least 99.99% of the original inoculum. .sup.4 Milliequivalents peroxide/kg sample .sup.5 at 25.degree. C. .sup.6 at 25.degree. C. cSt .sup.7 Undiluted oil was not bactericidal for 99.99% of the original inoculum.
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 solely to peroxides formed during oxidation nor was it due to mere concentration of antibacterial substances resultant from the 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 large 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 of 1:4.2 for S. aureus ATCC 25923 and 1:800 for E. 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.
TABLE C__________________________________________________________________________Comparison of the Antibacterial Effects of Purposely Oxidized d-Limoneneand Auto-Oxidized d-Limonene..sup.1 Bactericidal Concentration For Peroxide S. aureus ATCC 25923 After Incubation E. coli ATCC 29522 After Incubation Ford-Limonene Preparation Value 10 Min. 60 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 0.0003Auto-Oxidized, 10 Weeks 336.9 >1 1 0.005 1 0.06 0.0012__________________________________________________________________________ .sup.1 Compiled from Tables A and B
Increasing the temperature of oxidation from room temperature (.about.25.degree. C.) to 50.degree. 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 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.degree. 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.degree. C. became so viscous at 6 weeks, it could not be tested further for antibacterial activity since it would not mix with the broth diluent in the assay.
TABLE D__________________________________________________________________________Changes in Physical Parameters, Peroxide Value, and Bactericidal Activityof d-Limonene.sup.1During the Purposeful Oxidation of the Oil at 50.degree. C. Bactericidal Concentration.sup.3 For S. aureus ATCC 25923 After E. coli ATCC 25922 AfterPurposeful Oxidation.sup.2 Incubation for Incubation for Peroxide Specific Kinematicat 50.degree. C. in Weeks 10 Min. 60 Min. 24 Hr. 10 Min. 60 Min. 24 Hr. Value.sup.4 Gravity.sup.5 Viscosity.sup.6__________________________________________________________________________ 0 (fresh) >1.sup.7 >1 0.2 1 1 0.1 50.2 0.8439 1.035 2 1 0.16 0.0012 0.1 0.0006 0.0006 286.1 0.9549 8.203 4 1 0.5 0.0012 0.1 0.0025 0.0006 273.2 1.0300 141.9 6.sup.8 1 0.4 0.01 0.01 0.005 0.0025 307.9 1.0670 1293 8 226.2 1.0784 314910 241.9 1.0863 10,526__________________________________________________________________________ .sup.1 Florida Chemical Co., Inc., Lake Alfred, Florida .sup.2 Air was bubbled continuously into 1 gallon of oil in a smallmouthe metal container with an aerator (Second Nature Challenger I Pump). .sup.3 Bactericidal concentration shown as the highest dilution of limonene (i.e. lowest concentration) killing at least 99.99% of the original inoculum. .sup.4 Milliequivalents peroxide/kg sample .sup.5 at 25.degree. C. .sup.6 at 25.degree. C. cSt .sup.7 Undiluted oil was not bactericidal for 99.99% of the original inoculum. .sup.8 Oil too viscous to permit testing for antibacterial activity after 6 weeks and for other parameters after 10 weeks.
Auto-oxidation of limonene at 50.degree. C. produced an oil with even more distinct antibacterial activity (Table E). Fourteen weeks of treatment were 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.degree. 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 E. coli ATCC 25922. These results indicated that auto-oxidation of limonene at 50.degree. C. could not produce the same bacterial activity as purposeful oxidation of limonene at room temperature.
TABLE E__________________________________________________________________________Changes in Physical Parameters, Peroxide Value, and Bactericidal Activityof d-Limonene.sup.1 During Auto-Oxidation of the Oil at 50.degree. C. Bactericidal Concentration.sup.3 For S. aureus ATCC 25923 After E. coli ATCC 25922 AfterAuto-Oxidation.sup.2 Incubation for Incubation for Peroxide Specific Kinematicat 50.degree. C. in Weeks 10 Min. 60 Min. 24 Hr. 10 Min. 60 Min. 24 Hr. Value.sup.4 Gravity.sup.5 Viscosity.sup.6__________________________________________________________________________ 0 (fresh) >1.sup.7 >1 0.2 1 1 0.1 50.2 0.8439 1.035 2 >1 1 0.0025 0.5 0.02 0.0012 308.8 0.8743 1.559 4 >1 1 0.0025 0.4 0.02 0.0012 268.0 0.8906 2.010 6 >1 1 0.02 0.16 0.0025 0.0025 217.5 0.9005 2.406 8 1 1 0.01 0.2 0.0025 0.0012 189.6 0.9056 2.70010 1 1 0.01 0.1 0.005 0.0012 182.5 0.9135 3.16812 > 1 1 0.02 0.1 0.0025 0.0025 192.6 0.9201 3.74814 1 0.5 0.02 0.01 0.0025 0.0025 211.0 0.9282 4.54416 1 0.5 0.06 0.1 0.0025 0.0025 219.6 0.9363 5.51621 >1 1 0.02 0.06 0.005 0.005__________________________________________________________________________ .sup.1 Florida Chemical Co., Inc., Lake Alfred, Florida .sup.2 1 gallon of oil in a smallmouthed metal container left open to roo air. .sup.3 Bactericidal concentration shown as the highest dilution of limonene (i.e. lowest concentration) killing at least 99.99% of the original inoculum. .sup.4 Milliequivalents peroxide/kg sample .sup.5 at 25.degree. C. .sup.6 at 25.degree. C. cSt .sup.7 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 bacterial 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.degree. 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.
TABLE F__________________________________________________________________________Maximal Bactericidal Activity and Peroxide Value of Various LimonenePreparations..sup.1 Rapid.sup.2 Bactericidal Activity Against Long-Interval.sup.3 Bactericidal Activity AgainstLimonene Preparation S. aureus ATCC 25923 E. coli ATCC 25922 S. aureus ATCC E. coli ATCC__________________________________________________________________________ 25922A. Maximal Bactericidal Activity.sup.4 Purposely Oxidized, Room Temperature 0.16 (6).sup.6 0.0025 (6) 0.0012 (6) 0.0006 (6) Purposely Oxidized, 50.degree. C. 1 (2) 0.1 (2) 0.0012 (2) 0.0006 (2) Auto-Oxidized, Room Temperature 1 (16) 0.1 (16) 0.01 (2) 0.0025 (4) Auto-Oxidized, 50.degree. C. 1 (14) 0.01 (14) 0.0025 (2) 0.0012 (2)B Peroxide Value.sup.5 of Oils with Maximal Bactericidal Activity Purposely Oxidized, Room 353.5 353.5 353.5 353.5 Temperature Purposely Oxidized, 50.degree. C. 286.1 286.1 286.1 286.1 Auto-Oxidized, Room Temperature 393.8 393.8 163.5 199.5 Auto-Oxidized, 50.degree. C. 211.0 211.0 308.8 308.8__________________________________________________________________________ .sup.1 Data from Tables A, B, D, and E .sup.2 Measured after 10 min. exposure of the test organism to the oil .sup.3 Measured after 24 hr. exposure of the test organism to the oil .sup.4 Shown as highest dilution of oil killing 99.99% of bacterial inoculum .sup.5 Milliequivalents peroxide/kg sample .sup.6 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.degree. C. was darker yellow and more viscous than that treated at room temperature. For auto-oxidized, the oil treated at room temperature was essentially unchanged in its appearance from untreated (fresh) limonene. Limonene auto-oxidized at 50.degree. C. was similar in appearance to that purposely oxidized 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 S. aureus ATCC 25923 for 60 minutes exposure. This end point 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 rapid 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, all oxidized limonene achieving this reference bacterial endpoint is referred to as RBE limonene.
The standard assay used to test the bactericidal activity against Staphylococcus aureus ATCC 25923 or Escherichia coli ATCC 25922 in Tables A, B, C, D, E, F, 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, Ill.), inoculated with 10.sup.6 colony forming units (CFU)/ml of Staphylococcus aureus ATCC 25923 or Escherichia coli ATCC 25922, remixed and incubated at 37.degree. 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 subcultured 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 detectable viable colonies on subculture i.e. the bactericidal concentration. This represented the lowest concentration killing 99.99 percent of the original inoculum.
TABLE G______________________________________Dilutions of limonene used in fixed volume assaysDilutions Tested Limonene Concentration______________________________________Undiluted 11:2 0.51:2.5 0.41:3.3 0.31:4.2 0.241:5 0.21:6.25 0.161:10 0.11:6.25 0.061:50 0.021:100 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, Fla.) 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 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 antibacterial 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 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.
TABLE H__________________________________________________________________________Changes in the physical parameters, peroxide value, andbactericidal activity of d-limonene.sup.1 during auto-oxidation ofthe oil over a large surface area. Bactericidal Concentration.sup.2Auto-oxidation at After Incubation for Peroxide.sup.3 Specific.sup.4Room Temperature in Weeks 10 Min 60 Min 24 Hrs Value Gravity Viscosity.sup.5__________________________________________________________________________0 (Fresh) .sup. >1.sup.6 >1 >1 68.7 0.8432 1.0241 >1 >1 >1 131.2 0.8438 1.0362 >1 >1 >1 170.5 0.8434 1.0523 >1 >1 0.01 231.3 0.8454 1.071__________________________________________________________________________ .sup.1 250 ml of fresh limonene (Intercit Inc., Safety Harbor, Fla. lot 509801) in an open beaker at room temperature .sup.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 .sup.3 Millequiv. peroxide/kg sample .sup.4 at 25.degree. C. .sup.5 cSt at 25.degree. C. .sup.6 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.
TABLE I__________________________________________________________________________Changes in the physical parameters, peroxide value, and bactericidalactivity of d-limonene.sup.1 during purposeful oxidation of the oil. Bactericidal Concentration.sup.3Purposeful oxidation at After Incubation for Peroxide.sup.4 Specific.sup.5Room Temperature in Weeks.sup.2 10 Min 60 Min 24 Hrs Value Gravity Viscosity.sup.6__________________________________________________________________________0 (Fresh) .sup. >1.sup.7 >1 >1 68.7 0.8432 1.0241 0.1 0.1 0.1 131.2 0.8438 1.0362 0.1 0.1 0.0025 170.5 0.8434 1.0523 0.1 0.1 0.00125 231.3 0.8454 1.071__________________________________________________________________________ .sup.1 Intercit, Inc., Safety Harbor, Florida, lot 509801 .sup.2 Air bubbled continuously into 250 ml of limonene in a 500 ml Erlenmeyer flask with an aerator (Second Nature Challenger I Pump) at roo temperature. .sup.3 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. .sup.4 millequiv. peroxide/kg sample .sup.5 at 25.degree. C. .sup.6 cSt at 25.degree. C. .sup.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 from this same source became rapidly bactericidal in as short as three weeks. Also, as shown in Table J, both d- and l- 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.
TABLE J______________________________________Antibacterial Activity Of Various Limonene Samples AsDetermined Against Staphylococcus aureus ATCC 25923During Purposeful Oxidation at Room Temperature. Duration of Pur- Bactericidal Concentration poseful After Incubation ForSample Source Oxidation 10 Mins 60 Min 24 Hours______________________________________d-limonene Aldrich.sup.1 5 weeks >1.sup.5 >1 >1l-limonene Aldrich.sup.1 5 weeks >1 >1 >1l-limonene SCM.sup.2 3 weeks >1 >1 >1d-limonene Intercit.sup.3 3 Weeks 0.1 0.005 0.0003l-limonene SCM.sup.2 3 weeks 0.1 0.005 0.0006d-limonene Aldrich.sup.1 3 weeks 0.005 0.0024 0.0003d-limonene FCC.sup.4 6 weeks 0.16 0.06 0.0012______________________________________ .sup.1 Aldrich, Milwaukee, Wisconsin .sup.2 SCM Glidden Organics, Jacksonville, Florida .sup.3 Intercit, Inc., Safety Harbor, Florida .sup.4 Florida Chemical Company, Inc., Lake Alfred, Florida
The second method of producing RBE limonene is by continuously bubbling oxygen into limonene as outlined in Table K 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.
TABLE K______________________________________Bactericidal Activity of d-limonene.sup.1 During Oxygenation AsDetermined Against Staphylococcus aureus ATCC 25923Days d-Limonene Bactericidal Concentration.sup.2Oxidized.sup.3 at Following Incubation For:25.degree. C. 15 Min 60 Min 24 Hours______________________________________ 0 days >1.sup.4 >1 >1 1 day >1 >1 0.01 2 days >1 >1 0.01 3 days >1 >1 0.01 4 days >1 >1 0.01 7 days >1 >1 0.005 9 days >1 >1 0.00515 days 0.5 0.5 0.00517 days 0.5 0.5 0.00124 days >1 0.16 0.00536 days 0.5 0.10 0.000645 days 0.16 0.06 0.00149 days 0.16 0.06 0.000353 days 0.3 0.06 0.000359 days 0.10 0.02 0.000366 days 0.16 0.01 0.000373 days 0.01 0.01 0.0003______________________________________ .sup.1 Fresh dlimonene (Intercit Inc., Safety Harbor, Florida lot 509801) .sup.2 Results expressed as the highest dilution of limonene killing at least 99.99% of the original inoculum. .sup.3 Oxygen bubbled continuously into dlimonene at a rate of 3 liter pe min at 25.degree. C. .sup.4 Undiluted oil was not bactericidal.
EXAMPLE 2
In Example 2 the bactericidal and fungicidal activity of RBE d-limonene and RBE l-limonene was determined against bacteria, yeasts, and fungi as outlined in Tables L and M below.
TABLE L______________________________________BACTERICIDAL AND FUNGICIDAL ACTIVITYOF RBE d-LIMONENE______________________________________ BACTERICIDAL CONCENTRATION AFTER INCUBATION FORORGANISM 10 Min 60 Min 24 Hours______________________________________A. BACTERIA 1. Staphylococcus aureus, 0.3 0.06 0.0012 ATCC 25923 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 0.06 0.0025 5. Staphylococcus aureus 39** 0.30 0.06 0.0015 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 epidermidis 10 1.00 0.10 0.00110. Sarcinia lutea 1.00 0.01 0.00111. Streptococcus mitis 15 (alpha) 0.01 0.01 0.00112. Streptococcus pyogenes 1 (group A) 0.01 0.01 0.0113. Streptococcus salivarius (gamma) 0.01 0.01 0.0114. Streptococcus mutans 0.16 0.06 0.001215. Streptococcus faecalis 15 1.00 0.10 0.0116. Lactobacillus casei M7 0.01 0.01 0.0117. Listeria monocytogenes M9 0.01 0.01 0.0118. Bacillus subtilis ATCC 6633 0.01 0.001 0.00119. Escherichia coli 7 0.01 0.01 0.0120. Klebsiella SP12 0.01 0.01 0.00121. Salmonella 14 (paraB) 0.01 0.01 0.0122. Shigella sonnei 8 0.01 0.001 0.00123. Proteus mirabilis 2 0.01 0.01 0.0124. Proteus vulgaris 127 0.10 0.01 0.0125. Proteus rettgeri 106 0.10 0.01 0.1026. Proteus Morganii 99 0.01 0.01 0.0127. Providencia stuartii 97 0.01 0.01 0.0128. Enterobacter cloacae 20 0.01 0.01 0.1029. Citrobacter SP8 0.01 0.01 0.1030. Pseudomonas aeruginosa 115 0.10 0.10 0.0131. Pseudomonas mendocina 129 1.0 0.10 0.0132. Neisseria gonorrhea 5 0.001 0.001 0.00133. Neisseria gonorrhea (Pen R)* 0.001 0.001 0.00134. Hemophilus influenzae (Amp R)** 0.01 0.001 0.00135. Hemophilus influenzae (Amp S)*** 0.001 0.001 0.00136. Neisseria meningitidis 0.001 0.001 0.00137. Xanthomonas campestris PV 0.0012 0.0003 0.0003 vesicatoria 138. Xanthomonas campestris PV 0.0012 0.0003 0.0003 vesicatoria 239. Xanthomonas (SP) *+ isolated 0.0025 0.0003 0.0003 from a Nebraska farmer40. Xanthomonas campestris PV citri 0.005 0.0025 0.000641. Mycobacterium tuberculosis 0.001 0.001 0.0001 var hominis H37 RV42. Mycobacterium kansasii Tam 30 0.1 0.01 0.000143. Mycobacterium scrofulaceum 0.1 0.01 0.000144. Mycobacterium intracellulare 0.1 0.10 0.000145. Mycobacterium fortuitum 1.0 0.10 0.0001 ATCC 6841B. YEASTS 1. Candida albicans 0.01 0.001 0.001 2. Sacchromyces SP 0.01 0.01 0.001______________________________________ FUNGICIDAL CONCENTRATION AFTER INCUBATION FOR 10 Min 60 Min 24 Hours 7-45 Days______________________________________C. FUNGI 1. Epidermophyton 0.001 floccosum 2. Microsporum audouini 0.001 3. Microsporum canis 0.01 0.0025 0.0006 4. Trichophyton 0.001 mentagrophytes 5. Aureoblasidium 0.01 0.005 0.0012 pullulans OM 279C 6. Cladosporium 0.01 0.005 0.0012 cladosporiodes OM 489 7. Philopohora lignicola 0.10 0.01 0.0025 OM 5922______________________________________ *Penicillin resistant **Ampicillin resistant **Ampicillin sensitive *+ Species unidentified
TABLE M______________________________________BACTERICIDAL AND FUNGICIDAL ACTIVITYOF RBE 1-LIMONENE______________________________________ BACTERICIDAL CONCENTRATION AFTER INCUBATION FORORGANISM 10 Min 60 Min 24 Hours______________________________________A. BACTERIA 1. Staphylococcus aureus 0.5 0.06 0.0025 ATCC 25923 2. Staphylococcus aureus 37 0.5 0.40 0.005 (Pen R)* 3. Staphylococcus aureus 39 0.3 0.06 0.0025 (Meth R)** 4. Staphylococcus aureus 52T 0.5 0.10 0.01 5. Staphylococcus aureus 53T 0.5 0.10 0.0025 6. Staphylococcus aureus 54T 0.5 0.06 0.01 7. Staphylococcus epidermidis 10 0.1 0.1 0.0025 8. Streptococcus mutans M42 0.3 0.16 0.005 9. Streptococcus faecalis 15 0.1 0.01 0.002510. Bacillus subtilis ATCC 6633 0.1 0.1 0.111. Escherichia coli 7 0.005 0.0025 0.001212. Salmonella 14 (Para B) 0.0025 0.0025 0.000613. Shigella sonnei 8 0.01 0.0025 0.001214. Pseudomonas aeruginosa 115 0.1 0.1 0.00515. Neisseria meningitidis 40 0.0012 0.0006 0.0000416. Xanthomonas campestris 0.0025 0.0012 0.0012 vesicatoria 117. Xanthomonas campestris 0.0025 0.0012 0.0003 vesicatoria 218. Xanthomonas SP isolated 0.0006 0.0006 0.0003 from a Nebraska farmer *+19. Xanthomonas campestris 0.005 0.0025 0.0006 PV citri20. Mycobacterium tuberculosis 0.0005 0.000125 0.000125 var. hominis H37 RV21. Mycobacterium fortuitum 0.1 0.1 0.0006 ATCC 6841B. YEAST 1. Candida albicans 0.0012 0.0012 0.0006______________________________________ FUNGICIDAL CONCENTRATION AFTER INCUBATION FOR 10 Min 60 Min 24 Hours______________________________________C. FUNGI 1. Microsporum canis 0.01 0.0025 0.0006 2. Aureobasidium pullulans OM 279C 0.1 0.005 0.0012 3. Cladosporium cladosporiodes 0.01 0.005 0.0012 OM 489 4. Phialophora lignicola OM 5922 0.1 0.01 0.005______________________________________ *Penicillin resistant *Methicillin resistant *+ Unidentified species
The standard assay used to test the bactericidal activity of the d and l isomers 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. An inoculum of 10.sup.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 bacterial inoculum.
The activity of RBE limonene was evaluated against Mycobacteria using undiluted oil, and oil diluted up to 1:8000 in Proskauer Beck liquid medium. Each test was inoculated with 10.sup.6 CFU/ml of Mycobacteria and incubated at 37 degrees C in 7% CO.sub.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 at least 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 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. 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.
TABLE N______________________________________ SUB- INCU- CULTURE BATION BROTH AGAR CONDI-ORGANISM MEDIUM MEDIUM TIONS______________________________________ 1. Staphylococcus, Mueller- 5% sheep air @ 37.degree. C. Sarcina, Bacillus, Hinton blood Enterobacteriacea, and Pseudomonas 2. Streptococcus, Lac- Todd- 5% sheep 10% CO.sub.2 in tobacillus, Listeria Hewitt blood air @ 37.degree. C. 3. Neisseria Mueller- 5% sheep 10% CO.sub.2 in meningitidis Hinton blood air @ 37.degree. C. 4. Neisseria gonorrhea Eugon chocolate 10% CO.sub.2 in air @ 37.degree. C. 5. Hemophilus Levinthal chocolate 10% CO.sub.2 in influenza air @ 37.degree. C. 6. Yeast Sabouraud 5% sheep air @ 37.degree. C. dextrose blood 7. Xanthomonas Nutrient blood agar air @ 25.degree. C. campestris vesicatoria & citri 8. Xanthomonas Mueller- blood agar air @ 37.degree. C. species isolated Hinton from Nebraska farmer (species not identified) 9. Mycobacteria Proskauer Dubos-oleic 7% CO.sub.2 @ Beck acid-albumin 37.degree. C. agar10. Fungi Sabourauds dextrose with 0.05% yeast extract______________________________________
EXAMPLE 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.
______________________________________ % OFCHEMICAL TOTAL RANGE ACTION______________________________________A. LIQUIDS1. SOLUTIONS OR SPRAYSa. RBE d-Limonene 6.0% 0.1-50% bactericide Corn Oil 94.0% 50-99.9% diluent 100.0%b. RBE 1-Limonene 1.0% 0.1-50% fungicide Ethyl Alcohol 99.0% 50-99.9% diluent 100.0%2. MOUTHWASHa. RBE d-limonene 50.0% 0.1-50% antiyeast Flavor 2.0% 1-5% flavor Ethyl Alcohol 48.0% 45-98.9% diluent 100.0%B. DENTIFRICE1. LIQUIDLiquid soap concentrate 5.0% 2-10% surfactantSaccharin 0.2% 0.1-1.0% flavorClove Oil 1.0% 0.5-3.0% flavorCinnamon Oil 0.5% 0.5-3.0% flavorPeppermint Oil 0.5% 0.5-3.0% flavorEthyl Alcohol 42.6% 29.5-95.3% diluentColor 0.2% 0.1-0.5% colorRBE 1-limonene 50.0% 1-50% fungicide 100.0%2. GELSodium monofluoro- 0.8% 0.5-1.5% antiplaquephosphateRBE d-limonene 50.0% 1-50% antibacterialHydrated silica xerogel 10.0% 8-15% abrasiveHydrated thickening 8.5% 5-10% bindersilicaSorbitol 70% solution 18.8% 5-73.3% humectantPolyethylene glycol 32 5.0% 3-7% bodying agentSodium lauryl sulfate 1.5% 1-2% surfactantCarboxymethyl cellulose 1.0% 0.5-2% bindergumS D alcohol 1.0% 0.5-2% stabilizerFlavor 3.0% 2-4% flavorSaccharin 0.2% 0.1-0.5% flavorF D & C Green #3 0.1% 0.1-0.5% colorF D & C Yellow #10 0.1% 0.1-0.5% color 100.0%3. PASTESodium monofluoro- 0.8% 0.5-1.5% antiplaquephosphateRBE 1-limonene 50.0% 1-50% fungicideDicalcium phosphate 22.0% 20.4-30% abrasivedihydrateWater 16.0% 11.1-69.5% diluentGlycerine 5.1% 4.5-12.5% bodying agentFlavor 2.0% 2-3% flavorSodium lauryl sulfate 1.5 1-2% surfactantCarboxymethyl cellulose 1.4% 0.5-2.0% bindergumTetrasodium pyrophos- 1.0% 0.5-2.0% binderphateSodium saccharin 0.2% 0.1-0.5% flavor 100.0%C. OINTMENTS & SUPPOSITORIES WITH AND WITHOUTHYDROCORTISONE1. OINTMENT WITH HYDROCORTISONERBE d-limonene 1.0% 0.1-15.0% bactericidePolyethylene glycol 59.0% 48.5-59.7% bodying agent3350 & emulsifierPolyethylene glycol 39.0% 31.5-39.7% bodying agent400 & emulsifierHydrocortisone 1.0% 0.5- 5.0% anti- 100.0% inflammatory2. OINTMENT WITHOUT HYDROCORTISONERBE 1-limonene 1.0% 0.1-15.0% fungicidePolyethylene glycol 59.5% 51.0-59.95% bodying agent3350 & emulsifierPolyethylene glycol 39.5% 34.0-39.95% bodying agent400 100.0% & emulsifier3. SUPPOSITORY WITHOUT HYDROCORTISONERBE 1-limonene 6.0% 0.1-15% bactericidePolyethylene glycol 56.5% 51.0-59.95% bodying agent1000 & emulsifierPolyethylene glycol 37.5% 34.0-39.95% bodying agent3350 100.0% & emulsifier4. SUPPOSITORY WITH HYDROCORTISONERBE d-limonene 1.0% 0.1-15% antiyeastPolyethylene glycol 74.0% 60.0-75.2% bodying agent1000 & emulsifierPolyethylene glycol 24.0% 20.0-24.2% bodying agent3350 & emulsifierHydrocortisone 1.0% 0.5-5.0% anti- 100.0% inflammatoryD. CREAMS WITHOUT HYDROCORTISONE1. RBE 1-limonene 6.0% 0.1-15.0% bactericide Cetyl alcohol 15.0% 12.0-18.0% thickener Arlacel 165** 5.0% 3.5-7.5% emulsifier Sorbitol 70% solution 5.0% 3.5-8.0% humectant Water 69.0% 51.5-80.9% diluent 100.0%2. RBE 1-limonene 1.0% 0.1-15.0% fungicide Spermaceti wax 12.5% 10.0-15.0% thickener Sorbitan monostea- 10.0% 7.5-12.5% emulsifier rate* Polyethylene 20 Sorbitan monostea- 6.0% 4.0-8.0% emulsifier rate* Water 70.5% 49.5-78.4% diluent 100.0%E. CREAMS WITH HYDROCORTISONE1. RBE d-limonene 1.0% 0.1-15.0% fungicide Cetyl alcohol 15.0 12.0-18.0% thickener Arlacel 165** 5.0 3.5-7.5% emulsifier Sorbitol 70% solution 5.0% 3.5-8.0% humectant Hydrocortisone 1.0% 0.5-5.0% anti- inflammatory Water 73.0% 46.5-80.4% diluent 100.0%F. TAMPONS1. RBE d-limonene 8% 1-15% bactericide2 cc 2 Gm Tampon 23 Gm 92% 85-99% reservoir for 100% fungicideG. AEROSOLS WITHOUT HYDROCORTISONE1. RBE 1-limonene 6.0% 0.5-50% bactericide Ethyl alcohol 94.0% 50-99.5% diluent 100.0% Pressurized nitrogen propellant at 100-125 psig2. RBE d-limonene 10.0% 0.5-50.0% fungicide Soybean Oil 90.0% 50.0-99.5% diluent 100.0% Pressurized nitrogen propellant at 100-125 psigH. AEROSOL WITH HYDROCORTISONE1. RBE 1-limonene 6.0% 0.5-50% bactericide Soybean oil 93.0% 45-99.0% diluent Hydrocortisone 1.0% 0.5-5.0% anti- 100.0% inflammatory Pressurized nitrogen propellant at 100-125 psigI. OIL IN WATER EMULSION1. RBE d-limonene 0.1% 0.1-50% fungicide2. Corn oil 10.0% 10-15% oil Arlacel 40** 2.0% 1-3% emulsifier Tween 40 3.0% 2-4% emulsifier3. Water 84.9% 28-86.9% diluent 100.0%Heat 2 to 70.degree. C. Heat 3 to 72.degree. C. Add 3 to 2 withcontinuousagitation. When 3 and 2 cool to 40.degree. C., add 1 with continuousagitation until room temperature is reached.J. OIL IN WATER EMULSION WITH SOAP(FUNGICIDAL SOAP)1. RBE d-limonene 10.0% 0.1-25% bactericide2. Corn oil 21.0% 20.0-40.0% oil Arlacel 40** 2.0% 1.0-3.0% emulsifier Tween 40 3.0% 2.0-4.0% emulsifier Liquid soap concen- 3.5% 2.5- 5.0% surfactant trate3. Water 60.5% 23-74.4% diluent 100.0%Heat 2 to 70.degree. C. Heat 3 to 72.degree. C. Add 3 to 2 withcontinuousagitation. When 3 and 2 cool to 40.degree. C., add 1 with continuousagitation until room temperature is reached.K. WATER IN OIL EMULSION1. RBE 1-limonene 1.0 0.1-25% fungicide2. Arlacel 186** 3.0 2.0-4.0% emulsifier Soybean oil 15.0% 10.0-25.0% oil Ceresin wax 0.5% 0.3-0.6% thickener Beeswax 0.5% 0.3-0.6% thickener Tween 80 0.5% 0.3-0.6 emulsifier3. Water 79.5% 44.2-87.0% diluent 100.0%Heat 2 to 70.degree. C. Heat 3 to 72.degree. C. Add 3 to 2 withcontinuousagitation. When 3 and 2 cool to 40.degree. C., add 1 with continuousagitation until room temperature is reached.L. PAINT1. ENAMELRBE d-limonene 1.00% 1-10% fungicideTitanium dioxide 14.91% 12-16% pigmentCalcium carbonate 29.83% 25-35% pigmentSilicate 4.81 3-6% pigmentSoya alkyd resin 25.72% 22-28% pigment (binder)Mineral spirits 23.73% 5-37% solvent 100.00% (thinner)2. LATEXRBE 1-limonene 1.00% 1-10% fungicideTitanium dioxide 10.76% 8-12% pigmentSilicate 12.91% 10-16% pigmentCalcium carbonate 20.91% 15-25% pigmentVinyl acrylic resin 12.22% 10-16% vehiclesolids (binder)Glycol 8.30% 6-10% solvent (thinner)Water 34.00% 12-50% solvent 100.00% (thinner)______________________________________ *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
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.

Number	Name	Date	Kind
RE28263	Gtauvreau	Dec 1974
2863882	Bain et al.	Dec 1958

Process of producing reference bactericidal endpoint (RBE) limonene

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