WO2010062933A1

WO2010062933A1 - Methods for wound treatment and healing using limonene-based compositions

Info

Publication number: WO2010062933A1
Application number: PCT/US2009/065860
Authority: WO
Inventors: Sahara Adams
Original assignee: Sahara Adams
Priority date: 2008-11-26
Filing date: 2009-11-25
Publication date: 2010-06-03

Abstract

In various embodiments, the present disclosure provides compositions for healing a wound and wound treatment methods. The wound-healing compositions include a limonene-based composition and a carrier for the limonene-based composition. Methods for treating a wound include applying a limonene-based composition to a wound to inhibit growth of bacteria therein. In some embodiments, the methods include applying a bio-film to the wound, wherein the limonene-based composition is included in the bio-film.

Description

METHODS FOR WOUND TREATMENT AND HEALING USING LIMONENE-

BASED COMPOSITIONS

METHODS FOR WOUND TREATMENT AND HEALING USING LIMONENE-

BASED COMPOSITIONS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to United States provisional patent application 61/118,258, filed November 26, 2009, which is incorporated by reference herein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH [0002] Not applicable.

BACKGROUND

[0003] Limonene is a monocyclic monoterpene commonly found in nature, typically occurring in its D form in citrus and other plant species. In vitro analyses have revealed that D-limonene is effective in eradicating the following major gram-positive pathogens: Staphylococcus aureus, Staphylococcus epidermidis (both methicillin sensitive and resistant), Streptococcus pyogenes, Streptococcus mutans, and other beta hemolytic streptococci, Enter ococcus faecάlis, and Enter ococcus faecium (both vancomycin sensitive and resistant). In vitro tests have further revealed that D-limonene is effective in eradicating the following gram-negative pathogens: Escherichia coli, Enterobactor cloacae, Klebsiella pneumoniae, Serratia marcescens, Pseudomonas aeruginosa, Acinetobacter baummii/haemolyticus, Paenibacillus polymyxa, and Stenotrophomonas maltophilia. In vitro tests have also revealed that D-limonene is effective in eradicating various Bacillus species, such as Bacillus licheniformis, B. sphraericus, Bacillus cereus, and Bacillus subtilus, including the species strain of anthrax (Bacillus anthracis, both Stearns and Ames strains). Many of these microorganisms can infect wounds, potentially leading to life-threatening systemic infections in a patient.

[0004] In view of the foregoing, methods for treating wounds using limonene-based compositions could be of substantial benefit in the art. Ointments, sprays, and bio-films designed to be applied to a wound and to inhibit bacterial growth therein are contemplated within the embodiments of the present disclosure. SUMMARY

[0005] In various embodiments, methods for treating a wound are disclosed herein. The methods include applying a limonene -based composition to a wound to inhibit growth of bacteria therein.

[0006] In other various embodiments, methods for treating a wound include applying a bio- film to a wound. A limonene-based composition is included in the bio-film.

[0007] In still other various embodiments, wound-healing compositions are disclosed herein. The wound-healing compositions include a limonene-based composition and a carrier for the limonene-based composition.

[0008] The foregoing has outlined rather broadly the features of the present disclosure in order that the detailed description that follows may be better understood. Additional features and advantages of the disclosure will be described hereinafter, which form the subject of the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] For a more complete understanding of the present disclosure, and the advantages thereof, reference is now made to the following descriptions to be taken in conjunction with the accompanying drawings describing specific embodiments of the disclosure, wherein:

[0010] FIGURE 1 shows the chemical structure of D-limonene.

DETAILED DESCRIPTION

[0011] In the following description, certain details are set forth such as specific quantities, sizes, etc. so as to provide a thorough understanding of the present embodiments disclosed herein. However, it will be evident to those of ordinary skill in the art that the present disclosure may be practiced without such specific details. In many cases, details concerning such considerations and the like have been omitted inasmuch as such details are not necessary to obtain a complete understanding of the present disclosure and are within the skills of persons of ordinary skill in the relevant art. [0012] Referring to the drawings in general, it will be understood that the illustrations are for the purpose of describing particular embodiments of the disclosure and are not intended to be limiting thereto. Drawings are not necessarily to scale.

[0013] While most of the terms used herein will be recognizable to those of ordinary skill in the art, it should be understood, however, that when not explicitly defined, terms should be interpreted as adopting a meaning presently accepted by those of ordinary skill in the art. In cases where the construction of a term would render it meaningless or essentially meaningless, the definition should be taken from Webster's Dictionary, 3rd Edition, 2009. Definitions and/or interpretations should not be incorporated from other patent applications, patents, or publications, related or not, unless specifically stated in this specification or if the incorporation is necessary for maintaining validity.

[0014] Other than in the operating examples, even when not expressly stated, numeric quantities should be considered to be modified by the term 'about'.

[0015] In various embodiments, the present disclosure is directed to methods for treating a wound and wound-healing compositions. The methods for treating a wound and the wound-healing compositions each utilize limonene-based compositions. In particular, the limonene-based compositions utilize limonene as an active ingredient for killing or inhibiting the growth of a variety of bacteria known to cause a number of infectious diseases in humans and animals. In some embodiments, the limonene-based compositions comprise D -limonene. In the methods for treating a wound and the wound-healing compositions described in the present disclosure, an effective amount of limonene (e.g., D- limonene) for inhibiting bacterial growth is placed in a limonene-based composition and is then applied to the human or animal.

[0016] Without being bound by theory, Applicant envisions that wound healing afforded by using limonene-based compositions occurs at least through killing or inhibiting the growth of bacteria within a wound. Many of the bacteria inhibited by limonene are known to cause infectious diseases in humans and animals. As used herein, the term "animal" shall include humans as well as non-human animals, namely mammals and reptiles. [0017] In various embodiments, methods for treating a wound are disclosed herein. The methods include applying a limonene-based composition to a wound to inhibit growth of bacteria therein. In some embodiments, the limonene-based composition comprises D- limonene. In some embodiments, the limonene-based composition is included in a spray. In other embodiments, the limonene-based composition is included in an ointment. In still other various embodiments, the limonene-based composition is included in a bio-film such as, for example, a bandage.

[0018] In some embodiments, the wound treated by the limonene-based compositions is intra-oral (i.e., inside the oral cavity). In other embodiments, the wound treated by the limonene-based compositions is extra-oral (i.e., outside the oral cavity). As used herein, oral cavity refers to the mouth.

[0019] In other various embodiments, methods for treating a wound include applying a bio- film to a wound. A limonene-based composition is included in the bio-film. Bio-films include, for example, a bandage.

[0020] In additional embodiments, the limonene-based compositions of the present disclosure may comprise an ointment, topical spray or bio-film for intra-oral or extra-oral application to a wound.

[0021] Various embodiments of wound-healing compositions are also described by the present disclosure. The wound-healing compositions include a limonene-based composition and a carrier for the limonene-based composition. In some embodiments, the limonene-based composition includes D-limonene. In some embodiments, the carrier is a bio-film such as, for example, a bandage. In other embodiments, the carrier is a liquid. In still other embodiments, the carrier is a gel.

[0022] In some embodiments, the limonene-based compositions may be included in a mouthwash. For example, for localized infections within the mouth and throat (or for the prophylactic treatment thereof), D-limonene may be formulated in a mouthwash to be used as a rinse or a swab. In other embodiments, the limonene-based compositions may comprise a toothpaste. In some embodiments, toothpastes and mouthwashes containing limonene may be used for wound healing by killing or inhibiting the growth of common dental pathogens that are known to cause tooth decay and periodontal disease. Such dental pathogens include bacteria such as, for example, Porphyromonas gingivalis, Bacteroides species, Actinobacillus action mycetemcomitons, Prevotella intermedia, Fusobacterium nucleatum, Bacteroides forsythus and other species, Campylobacter rectus, Eikenella corrodens, Peptostreptoloccus micros, Selenomonas sp., Eubacterium sp., Streptococcus intermedins, spirochetes Treponema denticola, and Treponema pallidum and syphillis. The toothpastes and mouthwashes may also be useful in killing or inhibiting the growth of other pathogens that have been shown to colonize in the mouth, leading to various systemic diseases, such as, for example, bacterial endocarditis and arthritis.

[0023] Such toothpastes may also include base components (i.e., excipients) commonly known to those of ordinary skill in the art of toothpaste manufacture. Such toothpastes may also include calcium and/or magnesium compounds to aid in strengthening the teeth. Illustrative base components include, for example, (a) sorbitol, (b) water, (c) silica (e.g. ZEODENT, vended by Huber Corp.), (d) glycerin, (e) surfactants, (e.g., sodium lauryl sulfate or Polysorbate 20), (f) binders and viscosifying agents (e.g., CEKOL cellulose gum or xantham gum and (g) preservatives, (e.g., sodium benzoate and methyl parabens). Flavoring agents, coloring agents and/or whitening agents may be optionally employed as well. It will be appreciated by those of ordinary skill in the art that while the identified base components may indeed by employed in the embodiments of the present disclosure, other base components commonly employed in toothpaste formulations, now known or later discovered, may be used without departing from the spirit and scope of the present disclosure.

[0024] In some embodiments, the limonene-based compositions of the present disclosure may comprise at least one base component (i.e., excipient) in addition to limonene. In some embodiments, the limonene is a highly purified limonene. The limonene has a purity greater than about 98% by weight in some embodiments, greater than about 98.5% by weight in other embodiments, and greater than about 99% by weight in still other embodiments. An illustrative purification technique for limonene is described in United States Patent 6,420,435, which is incorporated by reference herein in its entirety. In various embodiments, a concentration of limonene in the limonene-based compositions ranges from about 10% to about 40% by weight.

[0025] An illustrative toothpaste formulation according to embodiments of the present disclosure includes the following components: about 10% to about 40% D-limonene, about 15% to about 35% sorbitol; about 15% to about 30% of a silica agent (e.g., ZEODENT 113 and/or ZEODENT 165), about 10% to about 20% water; about 5% to about 15% glycerin, about 2% to about 7% of surfactant (e.g., Polysorbate 20), about 1% to about 2% flavoring agent (e.g., sodium saccharin), about 0.5% to about 1.5% of titanium dioxide as a whitening agent, about 0.5% to about 1.5% binder (e.g., CEKOL 2000 gum), about 0.05% to about 0.15% of a preservative (e.g., sodium benzoate), about 0.25% to about 1.75% of calcium, and about 0.10% to about 1.75% of magnesium phosphate. In some embodiments, the toothpaste formulations comprise about 18% to about 22% D-limonene, about 1.25% to about 1.50% calcium and about 1.25% to about 1.50% magnesium phosphate.

[0026] An illustrative mouthwash formulation according to embodiments of the present disclosure includes the following components: (a) about 15% to about 25% sorbitol, (b) about 10% to about 20% of polyethylene glycol, (c) about 2.5% to about 7.5% Polysorbate 20, (d) about 2.5% to about 15% D-limonene, (e) about 45% to about 65% water, (f) about 0.2% to about 0.5% sucralose, and (g) about 1.0% to 2.0% Bel wood Wintergreen. Administration of the mouthwash is similar to conventional mouthwashes (i.e., about 30 ml is placed within the mouth and swished about therein for about 30 seconds prior to expectoration). The administrated dose and time within the mouth may be varied as desired.

[0027] In other various embodiments of the present disclosure, pure limonene may applied directly to a wound to promote healing thereof. For example, a small amount of limonene oil may be applied directly to the teeth or swabbed with a human or animal's mouth for the purpose of killing or inhibiting the growth bacteria therein. In some embodiments, limonene may be applied directly to an extra-oral wound. [0028] The present disclosure also contemplates incorporation of small amounts of pure limonene (e.g., about 0.1 mL) within a chewing gum base. Upon chewing of the gum, the limonene is released from the gum base and dispersed within the oral cavity and onto the teeth.

[0029] In further embodiments, the aforementioned limonene-based compositions may be incorporated within ointments, topical sprays, and/or bio-films for intra-oral or extra-oral application. Intra-oral applications have been described hereinabove. In an illustrative extra-oral application, such ointments, topical sprays and bio-films may be applied to a wounded area on the skin. Such extra-oral application may be further promoted by rubbing for an ointment or spraying for a topical spray. Bio -films may be used, for example, for treating wounded soldiers by applying a limonene-based composition to a wounded area. Such use of bio-films by wounded soldiers may advantageously lessen the incidence of battlefield related infections.

Experimental Examples

[0030] The following examples are provided to more fully illustrate some of the embodiments disclosed hereinabove. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represents techniques that constitute exemplary modes for practice of the disclosure. Those of ordinary skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.

[0031] Example 1: In Vitro Activity of D-limonene Against Various Bacteria. Clinical isolates (10⁵ bacteria/ml) (about 100 μl) of gram-positive pathogens (Staphylococcus aureus and epidermidis (both methicillin-sensitive and resistant) plus Enterococcus faecalis and faecium) along with a group of gram-negative pathogens (Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae and Serratia marcescens coupled with opportunistic pathogens Pseudomonas aeruginosa, Acinetobacter baummii/haemolyticus and Stenotrophomonas maltophi/a) were each inoculated into 2 ml of D-limonene, in accordance with the standard phenol-coefficient assay and other screening methodology for plant antimicrobial activity and incubated for 72 hrs. A 2 ml broth media was used as a positive control. The D-limonene used was purified to at least 98.5% via a distillation process with purity confirmed by HPLC. Aliquots were subsequently cultured at 24 hours, 48 hours, and 72 hours to determine the antimicrobial effect. Appropriate media was inoculated in accordance with NCCLS standards. Blood agar was used for the gram- positive organisms, while McConkey Agar was utilized for the gram-negative organisms. ATCC strains of S. aureus, E.faecalis, P. aeruginosa, and E. coli were used as controls organisms and compared to the clinical isolates of these pathogens.

[0032] The results of the assay are shown in Tables 1 (gram-positive organisms) and 2 (gram-negative organisms). As shown in Tables 1 and 2, all of the pathogens tested were shown to be effectively eradicated within 24 hours. Cultures were held 72 hours to ascertain if a resistant genetic code might have been facilitated. The response to subculture at 72 hours yielded no growth, thus clearly indicating that no muta-genic or plasmid transposon was noted.

Tsble 1> Antibacterial effects of d-limoaenc on gram-positive organisms

NG = no growth

Table 2. Antibacterial effects of d-limoneac on gram-negative organisms

*NG = no growth

[0033] Example 2: Preparation of a Toothpaste Comprising D-limonene. A toothpaste formulation was manufactured by combining the following components:

25.00% sorbitol

20.00% ZEODENT 1 13

20.00% D-limonene

13.39% water

10.00% Glycerin Natural Kosher

5.00% Polysorbate 20

2.70% ZEODENT 165

1.00% Flavor 484

1.00% titanium dioxide

1.00% CMC 9M31XF/Cekol 2000 (binder gum)

0.45% calcium

0.25% saccharin

0.11% magnesium phosphate

0.10% sodium benzoate

[0034] The foregoing components were combined as follows: the sodium saccharin and sodium benzoate were dissolved in the water and set aside. The Cekol and glycerin were combined, and, while mixing these two components together, the sorbitol was added. The solution of sodium saccharin and sodium benzoate was then added to the Cekol/glycerin/sorbitol mixture. Next, ZEODENT 165 was added to the combined mixture and blended in, followed by the ZEODENT 113, which in turn was also blended in until the mixture was free of lumps. Titanium dioxide, Polysorbate 20, and D-limonene were combined with the mixture and blended until the mixture was smooth. Finally, the calcium and magnesium phosphate were added, followed by the flavoring agent (Flavor 484).

[0035] Example 3: Preparation of Another Toothpaste Comprising D-limonene.

Another toothpaste formulation was manufactured by combining the following components:

25.00% sorbitol

18.00% ZEODENT 113

20.00% D-limonene

13.39% water

10.00% Glycerin Natural Kosher

5.00% Polysorbate 20

2.26% ZEODENT 165

1.00% Flavor 484

1.00% titanium dioxide

1.00% CMC 9M31XF/Cekol 2000 (binder gum)

1.50% calcium

0.25% saccharin

1.50% magnesium phosphate

0.10% sodium benzoate

[0036] The foregoing components were combined in accordance with the procedure described above in Example 2.

[0037] Example 4: Activity of Standard Topical Anti-Bacterials, Nutriceuticals and Herbal Medications on Bacillus anthracis Strains. The Steams and Ames strain of Bacillus anthracis were subjected to a battery of standard topical anti-bacterials, nutriceuticals, and herbal medications, including SILVADENE (generic silver sulfadiazine, vended by Hoescht Marion Roussel, now Par); SILVADENE with nystatin 0.025%; mafenide acetate, FURACIN (generic nitrofurazone, vended by Roberts), bacitracin with Polymyxin B (Poly B), silver nitrate, sodium hypochlorite (NaOCl), grapefruit seed extract (GSE), oleander extract with Aloe vera (Biotonics, San Antonio, Texas), and various concentrations of a new anti-infective solution called FX (Sterifx, Inc, Shreveport, Louisiana). Both B. anthracis strains were tested by Nathans Agar Well Diffusion Technique.

[0038] Results: The Steams strain of B. anthracis was susceptible to all products tested except bacitracin, Poly B and NaOCl. The most effective among the standard topicals was BACTROBAN with an average inhibition zone of 45 mm, followed by mafenide acetate at 38 mm. Furacin produced an inhibition zone of 33 mm, while that of SILVADENE was 19 mm. Both SILVADENE with Nystatin and AgNO₃ gave zones of inhibition of 18 mm. The nutraceuticals GSE and D-limonene had zones of inhibition of 25 mm and 30 mm, respectively, whereas the Oleander with Aloe vera had an inhibition zone size of 20 mm. The FX product at IX had no zone of inhibition, while the 4X and 12X zones were 25 mm and 32 mm, respectively.

[0039] The zones of inhibition for the more lethal and pathogenic Ames strain were comparable to those of the Steams strain for the standard anti-infectives, nutraceuticals (i.e. GSE and D-limonene) and herbal products. Again, mafenide acetate and BACTROBAN were at the top of the susceptibility list, with inhibition zones of 34 mm and 35 mm, respectively. The inhibitions zones of FX 4X and 12X were 35 mm and 46 mm, respectively. GSE and FX product IX zones of inhibition were both 23 mm. D-limonene's zone of inhibition was 21 mm. SILVADENE's zone of inhibition was 18 mm, while SILVADENE with Nystatin was 14 mm. The zone of inhibition for AgNO₃ was 16 mm, as was that of the Oleander Aloe vera product. Bacitracin, Polymyxin B and NaOCl were ineffective and showed no zones of inhibition.

[0040] Both strains of B. anthracis were susceptible to the standard topical antimicrobials (e.g., BACTROBAN, mafenide acetate and SILVADENE). The commercial FX product was very effective at 4X and 12X concentrations. The majority of products tested inhibited the growth of both strains of B. anthracis. [0041] Example 5: Susceptibility of Various Bacillus Species to Topical Anti- infectives. Six strains of Bacillus species were tested using the Nathans Agar Well Diffusion technique in 3 replicate assays. The strains included ATCC strains of Paenibacillus polymyxa, Bacillus licheniformis, Bacillus subtilis, Bacillus sphaericus, Bacillus cereus and a wild Bacillus strain from a burn patient. The anti-infectives tested were SILVADENE, mafenide acetate, furacin, BACTROBAN, Bacitracin plus polymyxin B, SILVADENE with Nystatin, 0.025% NaOCl, AgNO₃, Grapefruit Seed Extract (GSE), D-limonene, Oleander extract with Aloe vera and various concentrations of a new anti- infective solution FX.

[0042] All anti-infectives tested were effective against all strains of Bacillus except bacitracin with polymyxin B where none of the strains were inhibited and NaOCl, which only inhibited P. polymyxa, B. sphaericus and B. cereus with an average inhibition zone size of 16 mm. BACTROBAN's average zone of inhibition was 46 mm, followed by mafenide acetate at 36 mm. Furacin gave a zone of inhibition of 35 mm, while SILVADENE gave a zone of inhibition of 26 mm. GSE followed next with an inhibition zone of 25 mm. SILVADENE with nystatin followed next at 24 mm. Fx IX was only effective against B. subtilis, B. sphaericus and P. polymyxa, producing an inhibition zone of 22 mm. FX 5X and Fx 1OX inhibited all Bacillus strains tested with an average inhibition zone size of 32 mm and 49 mm, respectively. The Oleander extract produced an inhibition zone size of 18 mm, and D-limonene produced an inhibition zone of 21 mm. The inhibition zone Of AgNO₃ was 16 mm.

[0043] The previous results indicate that standard topicals used for treatment of wound infection can effectively treat The standard topicals used in soft tissue wound infections can effectively treat cutaneous B. anthracis. Nutriceuticals (i.e. D-limonene) and herbal medications can produce similarly effective results. Moreover, the herbals and nutraceuticals can be effectively aerosolized in a spray for treating inhalation anthrax and thus, could effectively be used as therapeutic alternatives for B. anthracis infections.

[0044] Example 6: Antibacterial Activity of Toothpaste Formulations. 0.25 to 0.50 grams of each of three different toothpaste formulations (labeled C, D, and P) were applied to clinical isolates (10⁵ bacteria/ml) of gram-positive pathogens Staphylococcus aureus and Enter ococcus faecalis and faecium as well as gram-negative pathogens Escherichia coli and Pseudomonas aeruginosa in accordance with the standard phenol-coefficient assay and other screening methodology for plant antimicrobial activity and incubated for 72 hrs. A 2 ml broth media was used as a positive control. Formulation C comprised the toothpaste formulation described herein in Example 2. Formulation D comprised at least 98% pure D- limonene (20%) and the remaining ingredients for Formulation C except for the calcium and magnesium (the remaining 0.5% being made up as water). Formulation P was a placebo formulation, comprising: (a) 31.175% sorbitol; (b) 25.0% ZEODENT 113; (c) 17.44% water; (d) 12.5% glycerin natural kosher; (e) 6.25% Polysorbate 20; (f) 3.38% ZEODENT 165; (g) 1.25% flavoring 484; (h) 1.25% titanium dioxide; (i) 1.25% CEKOL 2000; (J) 0.313% sodium saccharin; and (k) 0.125% sodium benzoate. Aliquots were subsequently cultured at 24 hours, 48 hours, and 72 hours to determine the antimicrobial effect. Appropriate media was inoculated in accordance with NCCLS standards. Blood agar was used for the gram-positive organisms, while MacConkey Agar was utilized for the gram-negative organisms.

[0045] Results of the assay at 24 hours are shown in Table 3. At 24 hours, each of the limone-containing toothpastes showed zones of inhibition for each of the pathogens tested, whereas the placebo showed no effect.

Table 3: Assay Results at 24 Hours for Limonene-Containing Toothpastes and

Placebo

[0046] Example 7: Preparation of a Mouthwash Formulation Containing D-

Limonene. A mouthwash formulation was manufactured by combining the following components: sorbitol 20.0%

PEG 61UltraPEG 300 15.0% polysorbate 20 5.0%

D-limonene 5.0% water 52.7% sucralose 0.30%

Belwood Wintergreen 2.0%

[0047] Example 8: 3-Week Clinical Study of Limone-Containing Toothpastes in Treating Periodontal Disease. A 3 week, double blind, clinical study was conducted to compare the effects of the toothpaste formulation described in Example 2 with a placebo dentifrice (i.e. without D-limonene or any other active ingredients) in treating periodontal disease. Male and female adult subjects with a baseline Quigley-Hein Plaque Index scores of 1.5 or greater were entered into the study. Each subject received a scale and root plane (S/RP) and a through periodontal screening upon entering the study. The periodontal probing pocket depth (PD), bleeding on probing (BOP), plaque accumulation (PI), and gingival status (GI) were all measured at baseline and at 3 weeks. No professional detanl hygiene was delivered during the study period.

[0048] Assays for periodontal pathogens were also collected from the buccal and lingual surfaces of the first four molars. Specifically, assays for actinobacillus actinomycetemcomtuns , bacteroides forsythus, bacteroides gingivalis, bacterdides intermedins, streptococcus mutatis, porphyromonas gingivolis and provetella intermedia were conducted. Each of these bacteria are common periodontal pathogens

[0049] All subjects received a soft-bristled toothbrush for home use and were instructed to brush their teeth twice daily (morning and evening) for 2 minutes at each tooth brushing. At the end of the 3 weeks use of their assigned dentifrice, the subjects had their teeth evaluated for plaque formulation. Mean plaque scores decreased between baseline and 3 weeks in the study group. All reductions in plaque formation were statistically significant at the 97% level of confidence or greater. Mean gingival scores decreased, and periodontal depth (PD) decreased slightly, but most significant was improvement in the bleeding on probing score (BOP). Improvements were more pronounced on teeth having the heaviest plaque formation.

[0050] It was concluded that D-limonene additive in a fluoride-containing dentifrice exhibited distinctive plaque inhibitor effects and decreased bleeding on probing in chronic periodontal patients. The results indicated that the group assigned to the D-limonene dentifrice had less plaque formulation than the group assigned to the placebo dentifrice. All reductions in plaque formation were statistically significant at the 97% level of confidence or greater.

[0051] Example 9: 6-Month Clinical Study of Limone-Containing Toothpastes in Treating Periodontal Disease. A six month, double-blind clinical study was entered by 100 male and female adult subjects previously treated for chronic periodontis in order to assess the efficacy of a dentifrice containing D-limonene on supragingival plaque formation and chronic periodontal disease, compared to a placebo dentifrice. At baseline, 8, 18, and 28 weeks, plaque accumulated (PlI) and gingival status (GI) were assessed. Probing Pocket Depth (PD) and Bleeding on Probing (BOP) were measured at baseline and week 28 using a Florida Probe. No professional dental hygiene was administered during the study period.

[0052] Table 4 summarizes the changes observed in PlI, GI, PD and BOP over the 28 week study period. Statistically insignificant changes are marked with an 'X'. There was no change to slight decreases in mean plaque score between baseline and week 28. There was also no change to slight decreases in mean gingival scores between baseline and week 28. PD and BOP did show some statistically significant reductions during the study period. Table 4: Gingival Assay Results for a Limonene-Containing Toothpaste Over a 28

Week Study Period

BASELINE 8 Weeks 18 Weeks 28 Weeks

PLAQ. ACCUM (PlI) X X X X GINGIVITIS STATUS (GI) X X X X PROBING POCKET DEPTH (PD) X X BLEEDING ON PROBING (BOP) X X

[0053] From the foregoing description, one of ordinary skill in the art can easily ascertain the essential characteristics of this disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications to adapt the disclosure to various usages and conditions. The embodiments described hereinabove are meant to be illustrative only and should not be taken as limiting of the scope of the disclosure, which is defined in the following claims.

Claims

CLAIMSWhat is claimed is the following:

1. A method for treating a wound, said method comprising: applying a limonene-based composition to the wound to inhibit growth of bacteria therein.

2. The method of claim 1, wherein the limonene-based composition comprises D- limonene.

3. The method of claim 1, wherein the limonene-based composition comprises a spray.

4. The method of claim 1, wherein the limonene-based composition comprises an ointment.

5. The method of claim 1, wherein the limonene-based composition comprises a bio- film.

6. The method of claim 5, wherein the bio-film comprises a bandage.

7. The method of claim 1 , wherein the wound is intra-oral.

8. The method of claim 1, wherein the wound is extra-oral.

9. A method for treating a wound, said method comprising: applying a bio-film to the wound; wherein a limonene-based composition comprises the bio-film.

10. The method of claim 9, wherein the bio-film comprises a bandage.

11. A wound-healing composition comprising: a limonene-based composition; and a carrier for the limonene-based composition.

12. The wound-healing composition of claim 1 1 , wherein the limonene-based composition comprises D-limonene.

13. The wound-healing composition of claim 11 , wherein the carrier is a bio-film.

14. The wound-healing composition of claim 13 , wherein the bio-film comprises a bandage.

15. The wound-healing composition of claim 11 , wherein the carrier is a liquid.

16. The wound-healing composition of claim 1 1 , wherein the carrier is a gel.