The present invention relates to the treatment of microbial infections, especially the prevention and treatment of digestive tract infections in humans and animals, by orally administering a single terpene, a terpene mixture or a liposome-terpene (s) composition before or after the onset of the infection.

Digestive tract infections are mainly caused by pathogenic and opportunistic microorganisms and toxins produced by them. These illnesses are present in all types of animals and humans.

Diseases caused by organisms pathogenic to humans and animals are very common and encompass a range from the trivial to the lethal. With the arrival of the so-called'antibiotic age'following World War II, it was hoped that the scourge of infection

would be I largely controlled on a permanent basis.

However, this has not proved to be the case and in recent years many formerly useful prior art anti-bacterials have become ineffective as resistance has emerged. In the case of fungal infections the armamentarium has always been limited and the need remains for additional and more effective treatments.

In recent years, a number of particularly difficult problems have emerged and these have engaged considerable public concern. For instance, the rapidly rising prevalence of multiply resistant Staphylococcus aureus (MRSA) in hospitals in Western countries which has led to many deaths and, to all intents and purposes, only Vancomycin now stands as a fall-back treatment. Another example is outbreaks of severe E. coli infection, such as that in Scotland in the late nineteen-nineties which killed over 150 people. In the case of E. coli, there are particular problems in respect of treatment in that, even if the organism is killed quickly, the patient may die as the result of endotoxins being released from the organism if it is lysed as a result of anti-microbial attack.

Not all the mechanisms governing the emergence of resistance to anti-bacterials are understood but sufficient is known to suggest strongly that whilst a fairly simple game of molecular roulette will produce new anti-bacterials, any such product will

not remain free of resistance for long. Thus, it would appear that any solution to this apparently intractable problem of reduced effectiveness in anti-bacterials would need to be radically different to those employed in the prior art.

Recently with the scare of bio-terrorism there has been an increased concern with pathogens that can produce deadly outbreaks. This is the case with anthrax. Anthrax is considered a potential agent for use in biological warfare. Anthrax is an acute infectious disease caused by the spore-forming bacteria Bacillus anthracis. Anthrax is primarily a disease of domesticated and wild animals, particularly herbivorous animals. Humans become infected with anthrax by handling products from infected animals or by inhaling anthrax spores from contaminated animal products. Anthrax can also be spread by eating undercooked meat from infected animals. Anthrax infection can occur in three forms: cutaneous, inhalation, and gastrointestinal.

The most common form is the cutaneous anthrax infection, which occurs when bacteria enter a cut or abrasion on the skin. This infection begins as a raised itchy bump that develops into a vesicle and then a painless ulcer, usually 1-3 cm in diameter, with a characteristic black necrotic area in the center. About 20% of untreated cases of cutaneous anthrax result in death. Deaths may be prevented with prompt antimicrobial treatment. The inhalation form has early symptom similar to a

common cold which progressively results in severe breathing problems. This type of anthrax is usually fatal. The intestinal form is characterized by an acute inflammation of the intestinal tract. The initial signs are nausea, loss of appetite, vomiting, and fever followed by abdominal pain, vomiting of blood and severe diarrhea. Intestinal anthrax results in death in 25% to 60% of cases. Anthrax is treated with antimicrobials and can be prevented with vaccination. The Department of Defense in the USA has a mandatory anthrax vaccination of all active military personnel.

Another digestive infection in humans is traveller's diarrhea, which affects over seven million visitors to high-risk tropical and semitropical areas every year. Others suggest that the incidence of traveller's diarrhea is 15-56% among international travelers. Approximately 1% of the sufferers are hospitalized, at least 20% are confined to bed for a day and nearly 40% have to change plans in their travel itinerary.

Traveler's diarrhea, defined as the passage of more than 3 unformed stools in a 24-hour period, is a self-limiting illness lasting 3-5 days. The illness may be presented either as (1) acute watery diarrhea (2) diarrhea with blood (dysentery) or (3) chronic diarrhea, often with clinical nutrient malabsorption.

Several factors contribute to the development of diarrhea in travelers, including personal (age, socioeconomic status, body weight, preexisting gastrointestinal illnesses), behavioral (mode of travel, standard of accommodation, eating in public places, dietary errors) and travel related (destination, duration of stay, country of origin, season). Approximately 85% of lithe diarrheas among international travelers are produced by bacterial enteropathogens. These pathogens are usually acquired through ingestion of fecally contaminated food or water. Sometimes dirty hands or insects are the vectors of fecal contamination.

Cooked food is safe to consume as long as the temperature at the interior of the food reaches 160°F or more. An undercooked hamburger is risky food, because ground meat can become contaminated at the processing plant and during preparation.

The common pathogens that produce traveler's diarrhea include Clostridium difficile, Yersenia enterolitica, Shigella sp., Campylobacter sp., Salmonella sp., ETEC (enterotoxigenic) and EAEC (enteroaggregative) Escherichia coli. Traveler's diarrhea produced by Shigella sp. or Salmonella sp. tend to cause a more severe and longer lasting disease than that caused by the most common cause, enterotoxigenic E. coli (ETEC). Campylobacter jejuni is a relativelly common cause of traveler's diarrhea especially in the winter. Viruses such as

rotavirus, cytomegalovirus and Norwalk agent are less common causes.

There are several groups of pathogenic E. coli.

They include Enterotoxigenic (ETEC), which produce a range of toxins, heat-stable or heat-labile in nature. ETEC is the most common cause of diarrhoea disease in children in the developing world; it also causes many travelers'diarrhoea cases.

Verocytotoxic E. coli (VTEC) strains produce toxins that destroy the gut mucosa and can cause kidney damage; E. coli 0157 H: 7 is the most publicised example of this type.

Enteropathogenic E. coli (EPEC) do not appear to produce toxins but may attach the microvilli, this group often causes infection in babies and young children.

Enteroinvasive E. coli (EIEC) attaches to the mucosal lining of the large intestine and invade the cells, causing tissue destruction and inflammation. EIEC are usually food borne pathogens and are an important cause of disease in areas of poor hygiene.

The severity of the disease symptoms are dependent on the strain encountered and the underlying health of the individual. EIEC and VTEC strains can cause very serious disease (haemorrhagic colitis and

renal failure) and require hospitalisation. Milder cases are usually treated by fluid and electrolyte replacement and rest.

The use of antibiotics limits the course of diarrhea to a little over a day compared with an average of over 3-5 days when diarrhea remains untreated. The widespread resistance of the traditional antimicrobial agent, Trimethoprim plus sulfamethoxazole (TMP/SMX), and fluoroquinolones are the main reasons of concern about the continuous use of antimicrobials for the treatment of traveler's diarrhea (Dupont et al, 1998). The extensive use of antibiotics can also lead to overgrowth syndromes, Candida vaginitis can occur, the overgrowth of Clostridium difficile due to less competitive environment in the gastrointestinal tract can also result in diarrhea.

Short-term travelers that have experience diarrhea do not develop protection, since it requires continued exposure to enteropathogens to develop immunological protection against traveler's diarrhea. Vaccination is a promising option, but vaccines against all enteropathogens that cause traveler's diarrhea have not been developed. Other protection methods to treat traveler's diarrhea are: the use of nonabsorbed antimicrobials, which have fewer side effects and should be safer to use in children and pregnant women in whom quinolones are contraindicated; antisecretory and antimotility

agent (loperamide); the use of attapulgite, a hydrated aluminum silicate clay preparation; and probiotics i. e. lactobacillus, which appear to be useful in the prevention or treatment of travelers diarrhea. In all cases the restoration of water and electrolyte balance is necessary. The following table shows the current treatments for Traveler's Diarrhea: Agent Efficacy Comments Activated charcoal Not May absorb important efficacious medications Lactobacillus Not proven Safe Bismuth 65% Rinse mouth to avoid subsalicylate protective black tongue preparations Trimethropim-70-80% Resistance rising Sulfamethoxazole protective worldwide Fluoroquinolones 90% Currently most (norfloxacin, protective effective antimicrobial ciprofloxacin, or but resistance rising Ofloxacin) better worldwide.

Ericsson, Charles (1998) In humans and animals, peptic ulcers are open sores produced by a bacteria. These open sores can be present on the entire gastro-intestinal tract, mainly esophagus, stomach and proximal part of the small intestine. There is evidence that support the role of H. pylori as the etiologic agent of chronic gastritis and peptic ulcer. H. pylori, a

gram-negative, microaerophilic spiral bacteria is the major cause of gastro-duodenal disease, including chronic gastritis, gastric and duodenal ulcers and gastric neoplasia. Greater than 50% of North American adults over 50 years of age are infected with H. pylori. In contrast, in some developing and newly industrialized countries virtually all adults are infected. In developing countries almost all children are infected by age 10, whereas in developed countries only the children of lower socioeconomic levels are infected. H. pylori is characterized by very high urease activity that may be associated with virulence, in the absence of urea H. pylori is sensitive to acidic pH. Urease activity may be an important colonization and survival factor by generating ammonia in the immediate bacterial microenvironment. H. pylori has been classified as a type 1 carcinogen by the World Health Organization because of the danger of persistent infection with the bacterium causing gastric cancer. H. pylori infection is of extreme importance in the causation of peptic ulcer disease. By initiating a gastritis or dyspeptic symptoms, it can predispose to subsequent episode of either gastric lymphoma or stomach cancer.

The eradication of H. pylori has been obtained with combination therapy, triple therapy using bismuth plus two antibiotics (metronidazole and either amoxicillin or tetracycline has been effective).

Problems due to development of antimicrobial resistant and side effects (diarrhea, nausea, abdominal pain and others) may explain why the use of antibiotics has not become a preferred treatment for gastritis and peptic ulcers due to H. pylori.

Antibacterial treatment of H. pylori is difficult because of the habitat occupied by the organism below the layer of the mucus adherent to the gastric mucosa. Access of antibacterial agents to this site is limited from the lumen of the stomach and also from the gastric blood supply.

The use of medium chain fatty acids and medium chain triglycerides has been shown to inhibit the growth of H. pylori in vitro. The mechanism by which they exert antibacterial effect is thought to involve: 1) damage to the bacterial outer membrane leading the increase membrane fluidity and permeability, 2) Incorporation of these fatty acids, making the bacterial membrane unstable, 3) Production of peroxides due to oxidation of fatty acids.

The mode of transmission of H. pylori in humans is still poorly understood. There are reports of detection of this microorganism in the oral cavity and in the feces. If H. pylori is harvested in the oral cavity or bowel, these might represent important reservoir for the reinfection and transmission with consequences from treatment. One

vector for the transmission of H. pylori are flies, they can carry viable H. pylori in their external surfaces and alimentary tracts.

In animals, the presence of scours in calves is of economic importance. It is estimated that the death lost of calves less than 6 months of age is approximately 2.5% or over 100,000 a year. Most of the mortality and morbidity of the calves are due to infectious diseases, mainly scours. More than 90% of scours in calves is produced by E. coli and Salmonella. Clostridia has proved to be fatal in the majority of cases. There are preventive methods like (I) vaccination of the mothers in order to passively transfer antibodies in colostrum; (2) the use of immunological supplements for milk replacers; (3) the use of probiotics to create a gastro-intestinal healthy environment (4) changes in calf management. None of these protective measures are 100% effective.

Another animal of economic importance is swine.

The incidence of diarrhea in neonates and weaned piglets is very high. Again, E. coli and Salmonella are the main microorganisms involved in diarrhea in swine. There are losses in the nursery while piglets are still lactating and after weaning. There are similar preventive methods as in calves. One of the preferred methods is segregated early weaning (SEW). The basis of early weaning is that the earlier piglets are weaned from

the sow the less are the chances of crossover diseases between sow and piglets. This method requires the use of antibiotics.

In both cases, calf and piglet scours, the preferred method of treatment is antibiotics. The European Community has banned the use of 5 antibiotics and in the Unites States the FDA is banning the use of fluoroquinolone in animals due to the development of Campylobacter resistant to this antibiotic. Bacteria resistance has encouraged the development of antibiotic- alternative products.

Terpenes are widespread in nature, mainly in plants as constituents of essential oils. Their building block is the hydrocarbon isoprene (C5H8) n. Terpenes have been found to be effective and nontoxic dietary antitumor agents which act through a variety of mechanisms of action (Crowell and Gould, 1994 and Crowell et al, 1996). Terpenes, i. e. geraniol, tocotrienol, perillyl alcohol, b-ionone and d-limonene, suppress hepatic HMG-COA reductase activity, a rate limiting step in cholesterol synthesis, and modestly lower cholesterol levels in animals (Elson arid Yu, 1994). D-limonene and geraniol reduced mammary tumors (Elegbede et al, 1984 and 1986 and Karlson et al, 1996) and suppressed the growth of transplanted tumors (Yu et al, 1995).

Terpenes have also been found to inhibit the in-vitro growth of bacteria and fungi (Chaumont and Leger, 1992, Moleyar and Narasimham, 1992 and Pattnaik, et al, 1997) and some internal and external parasites (Hooser, et al, 1986). Geraniol was found to inhibit growth of Candida albicans and Saccharomyces cerevisiae strains by enhancing the rate of potassium leakage and disrupting membrane fluidity (Bard, et al, 1988). B-ionone has antifungal activity which was determined by inhibition of spore germination, and growth inhibition in agar (Mikhlin et al, 1983 and Salt et al, 1986). Teprenone (geranylgeranylacetone) has an antibacterial effect on H. pylori (Ishii, 1993).

Solutions of 11 different terpenes were effective in inhibiting the growth of pathogenic bacteria in in-vitro tests; levels ranging between 100 ppm and 1000 ppm were effective. The terpenes were diluted in water with 1% polysorbate 20 (Kim et al, 1995).

Diterpenes, i. e. trichorabdal A (from R.

Trichocarpa) has shown a very strong antibacterial effect against H. pylori (Kadota, et al, 1997).

Rosanol a commercial product with 1% rose oil has been shown to inhibit the growth of several bacteria (Pseudomona, Staphylococus, E. coli and Hpylori). Geraniol is the active component (75%) of rose oil. Rose oil and geraniol at a concentration of 2 mg/litre inhibited the growth of H pylori in vitro. Some extracts from herbal medicines have been shown to have an inhibitory

effect on H. pylori, the most effective being decursinol angelate, decursin, magnolol, berberine, cinnamic acid, decursinol and gallic acid (Bae, et al 1998). Extracts from cashew apple, anacardic acid and (E)-2-hexenal, have shown bactericidal effect against H. pylori.

There may be different modes of action of terpenes against H. pylori. They could (1) interfere with the phospholipid bilayer of the cell membrane (2) impair a variety of enzyme systems (HMG-reductase) and (3) destroy or inactivate genetic material.

SUMMARY OF THE INVENTION Prevention and treatment of digestive tract infections by orally administering a biocidal terpene, a biocidal terpene mixture or a liposome-terpene (s) composition before of after the onset of the infection.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Digestive tract infections not only are an uncomfortable illness for humans but also are of economic importance for the animal industry. In some cases the illness can cause death in children, elderly and immune-compromised people. The preferred treatment of the disease is antibiotics.

The extensive use of antibiotics in humans and the animal industry has created the development of antibiotic-resistant bacteria. The increased

antibiotic resistance has beer the main reason to seek new antimicrobial alternatives. The European Community has banned the use of 5 antibiotics in animals and in the Unites States the FDA is banning the use of fluoroquinolone in animals due to the development of Campylobacter resistant to this antibiotic.

Terpenes, which are GRAS (Generally Recognized As Safe) have been found to inhibit the growth of cancerous cells, decrease tumor size, decrease cholesterol levels and have a biocidal effect on microorganisms in vitro. Onawunmi (1989) showed that growth media with more than 0.01 % citral reduced the concentration of E. coli and at 0.08% there was a bactericidal effect. Barranx, et al (1998) teach us a terpene formulation, based on pine oil, used as a disinfectant or antiseptic cleaner. Koga, et al (1998) teach that a terpene found in rice has antifungal activity. Iyer, et al (1999) teach us an oral hygiene antimicrobial product with a combination of 2 or 3 terpenes that showed a synergistic effect. Neither of them suggested the use of a terpene, terpene mixture or liposome-terpene (s) combination for the prevention or treatment of gastro-intestinal infections i. e. traveler's diarrhea.

Several US Patents (US#5, 547,677, US#5, 549,901, US#5, 618,840, US#5, 629,021, US#5, 662,957, US#5, 700,679, US&num 5, 730, 989) teach us that certain

types of oil-in-water emulsions have antimicrobial, adjuvant and delivery properties.

Thus, the present invention provides a composition for preventing or treating gastro-intestinal infections, wherein said composition comprises a terpene or a mixture of terpenes. We have found that certain mixture of terpenes are synergistically effective, relative to the effects of the component terpenes administered separately.

Thus terpenes having biocidal activity which in combination with two or more other terpenes synergistically increase the biocidal effectiveness are of especial interest.

One composition of interest comprises a mixture of carvone and geraniol, optionally together with other terpenes. The content of carvone and geraniol may each be from 10 to 90% (by weight), but is preferably 10 to 60% by weight. Other terpenes which may be present include citral, b- ionone, eugenol, terpeniol, carvacrol, anethole or the like. These optional additional terpenes may be present at 5 to 50% by weight, for example 10 to 40% by weight.

Optionally, the terpenes may be presented in the form of liposomes.

Liposomes are microscopic structures consisting of concentric lipid bilayer enclosing an aqueous

space. Liposomes are classically prepared from phospholipids which occur naturally in animal cell membranes, but several synthetic formulations are now commonly used. The lipid composition of the liposome can be varied to give liposomes different physical characteristics i. e. size and stability.

Liposomes can be prepared by the reverse-phase evaporation or dehydration-rehydration vesicle methods using a mixture of dipalmitoyl phosphatidyl choline, cholesterol, dipalmitoyl phosphatidyl glycerol, dipalmitoyl phosphatidyl ethanolamine and other synthetic fatty acids and emulsifiers. When making liposomes first multilamellar vesicles are formed spontaneously when amphipathic lipids are hydrated in an aqueous medium. Unilamellar vesicles are often produced from multilamellar vesicles by the application of ultrasonic waves.

Multilamellar vesicles can be prepared by the procedure known as dehydration-rehydration.

Briefly, egg phosphatidylcholine and cholesterol are mixed in chloroform, dried in a rotary evaporator, dilute with water and sonificated to form unilamellar vesicles. The solution is freeze dried and rehydrated with the terpene solution in order to embed the terpene inside the liposome.

Another method to produce liposomes is by mixing together lipids, an emulsifier and the terpenes.

The emulsion is obtained by using a Polytron homogenizer with special flat rotor that creates an emulsion. The lipids could consist of soybean oil,

any commercial or pharmaceutical oil; the emulsifier consist of egg yolk lecithin, plant sterols or synthetic including polysorbate-80, polysorbate-20, polysorbate-40, polysorbate-60, polyglyceryl esters, polyglyceryl monooleate, decaglyceryl monocaprylate, propylene glycol dicaprilate and triglycerol monostearate. The lipid concentration in the oil phase is 75-95% and the emulsifier concentration from 5-25%. When preparing the emulsion a ratio oil to water could vary from 10-15 parts lipid to 35-40 parts terpenes diluted in water at a concentration of 0.5% to 50%.

Once the emulsion is formed this is combined with a carrier in order to be use as a humectant, cream or other suitable carrier for topical applications.

The emulsion concentration use for topical application varies from 0.0055 through 1.0% of the final product. Several modifications to the emulsion can be achieved by simply varying the concentration and type of terpenes used. This modification can give us different products with different antimicrobial specificity.

By encapsulating terpenes within these emulsions the antimicrobial effect will be increased: (1) the liposome will disrupt the bacterial membrane and (2) the terpenes will be more effective in disrupting cytoplasmatic enzymes.

It will be apparent for those skilled in the art that the aforementioned objects and other

advantages may be further achieved by the practice of the present invention.

EXAMPLE 1: Preparation of the terpene mixture The terpene, terpene mixture or liposome-terpene (s) combination consists of a blend of generally recognized as safe (GRAS) terpenes with a GRAS surfactant. The ratio of terpenes is from 1-99% and the surfactant ratio from 1-99% of the mixture.

The terpenes, comprised of natural or synthetic terpenes, are citral, b-ionone, geraniol, eugenol, carvone, terpeniol, carvacrol, anethole or other terpenes with similar properties. The surfactant is preferably polysorbate-80 or other suitable GRAS surfactants.

EXAMPLE 2: Preparation of liposomes containing terpenes Any standard method for the preparation of liposomes can be followed with the knowledge that the lipids used are all food-grade or pharmaceutical-grade. A set amount of lipids, an emulsifier and the terpenes was used to prepare an emulsion. The emulsion was obtained by using a Polytron homogenizer with special flat rotor that created an emulsion. The lipids consisted of soybean oil, any commercial or pharmaceutical oil; the emulsifier consist of egg yolk lecithin, plant sterols or synthetic emulsifiers including

polysorbate-80, polysorbate-20, polysorbate-40, polysorbate-60, polyglyceryl esters, polyglyceryl monooleate, decaglyceryl monocaprylate, propylene glycol dicaprilate and triglycerol monostearate. A solution containing 75-95% lipids (oil) and 5-25% emulsifier consisted of the oil phase. The aqueous phase consisted of the terpene diluted in water at a rate of 0.5% to 50%. To form the emulsion a ratio of oil to water varying from 10-15 parts lipid (oil phase) to 35-40 parts terpenes (aqueous phase) was mixed. Any standard method for the preparation of liposomes can be followed with the knowledge that the lipids used are all food-grade or pharmaceutical-grade. The suspension containing a lipid, an emulsifier and the terpenes is emulsified with a Posytron homogenizer until a complete milky solution is obtained.

EXAMPLE 3: Preparation of liposomes This step consists of the preparation of the terpene (s)-liposome combination by mixing 99% of liposome and 1% of terpene mixture. Several combinations of this formulation can be obtained by varying the amount of terpene and liposome from 1% to 99%. The liposomes are prepared as in Example 2 without the addition of terpenes in the formulation.

EXAMPLE 4: In-vitro effectiveness of terpenes against E. coli

This example demonstrates the effect of terpenes on the cell membrane fragility of E. coli, which is considered indicative of other pathogenic bacteria such as Salmonella and Listeria. Lysis of the cell membrane was monitored by the determination of galactosidase activity. 8-galactosidase is a well-characterized cytosolic enzyme in bacteria.

This enzyme is inducible in the presence of isopropyl-1-thiogalactosidase (IPTG) and assayed colorimetricaly with substrate o-nitro-phenyl-p-D-galactoside (ONPG). ONPG is cleaved to release o-nitrophenol with peak absorbance at 420 nm. Since intact E. coli is impermeable to both ONPG and the enzyme, the cells have to be lysed prior to enzymatic assay.

Therefore the ability of terpenes to lyse E. coli can be measured with this enzymatic assay and compared to known lysing agents.

The procedure used was as follows: E. coli strains AW574 or AW405 were cultured overnight in 10 ml tryptone broth with 1 nM IPTG at 35°C. Cells were allowed to grow until an absorbance equal to 0.9 was reached. Cells were harvested, washed with phosphate buffer and resuspended to an absorbance equal to 0.5.0.1 ml of the bacteria culture was added to 0.9 ml of buffer, warmed to 30°C and then 80 go-ouf terpenes (85% terpenes and 15% polysorbate-80), 80 Ill water (background) or 40 Rl chloroform plus 40 Wl 1% SDS in water (positive

control) were added. After the addition of the lysing agents the tubes were mixed for 10 seconds and 0.2 ml of ONPG (4 mg/ml water) was added, then incubated for 5 minutes. The enzyme activity was stopped with 0.5 ml of 1 M sodium carbonate. After being centrifuged for 3 minutes at 1,500 x g, supernatant was transferred to cuvettes and read at 420 nm. The relative degree of lysis caused by terpenes was calculated as follows: 100 x (OD terpenes-OD water)/ (OD chloroform-OD water) This shows that dosages can be manipulated to either lyse the cell outright, or in the case of lower dosages, stop bacterial growth without lysis of the cell membrane. The advantage of this controllable result is the ability to prevent lysis and the resultant release of endotoxins where contraindicated.

Table 1: Lysis of E. coli by Terpenes Terpenes (µM) Relative lysis % Carvone 404, 000 NM* 40,400 54 4,040 22 404 3. 2 Geraniol 363, 000 NM 36,300 96 3,630 98 363 34 36.3 4 3.63 2.4 b-Ionone 308, 000 NM 30,800 NM 3,080 NM 308 52 30.8 44 3.08 23 0.308 4.78 0.0308 1.3 80 gl Polysorbate-80 3.2 80 Rl Polysorbate-80 + 100 SDS + Chloroform SDS + Chloroform 100* *Lysis due to chloroform and SDS combination was considered to be 100%.

*NM, not measurable due to formation of turbid colloidal solution.

EXAMPLE 5: In in-vitro effectiveness of terpenes against several microorganisms This example demonstrate the effectiveness of terpenes against Escherichia coli, Salmonella typhimurium, Pasteurella mirabilis, Staphylococcus aureus, Candida albicans and Aspergillius fumigates. Each organism, except A. fumigatus, was grown overnight at 35-37°C in tryptose broth. A. fumigates was grown for 48 hours. Each organism was adjusted to approximately 105 organisms/ml with sterile saline. For the broth dilution test, terpenes were diluted in sterile tryptose broth to give the following dilutions: 1: 500,1: 1000,1: 2000,1: 4000,1: 8000, 1: 16,000,1: 32,000,1: 64, 000 and 1: 128,000.

Each dilution was added to sterile tubes in 5 ml amounts. Three replicates of each series of dilutions were used for each test organism. 0.5 ml of the test organism was added to each series and incubated at 35-37°C for 18-24 hours. After incubation the tubes were observed for growth and plated onto blood agar. The tubes were incubated an additional 24 hours and observed again. The A. fumigates test series was incubated for 72 hours.

The minimum inhibitory concentration for each test organism was determined as the highest dilution that completely inhibits the organism.

Table 2: Results of the inhibitory activity of different dilutions Visual assessment Growth after of subculture to Mean growth * agar plates * inhibitory Organism dilution 1 2 3 1 2 3 S. typhimurium 500 500 500 500 500 500 500 E.coli 1000 1000 1000 1000 1000 1000 1000 P. mirabilis 1000 1000 1000 1000 1000 1000 1000 S.aureus 1000 1000 1000 1000 1000 1000 1000 Calbicans 1000 1000 1000 1000 1000 1000 1000 A.fumigatus 8000 16000 16000 8000 16000 16000 13300 * The results of the triplicate tests with each organism as the reciprocal of the dilution that showed inhibition/killing ** NI = not inhibited

EXAMPLE 6: In in-vitro effectiveness of terpenes against Escherichia coli over time.

This example demonstrates the effectiveness of terpenes at several concentrations against Escherichia coli and cultured over time. Terpene dilutions (1: 500,1: 1000,1: 2000,1: 4000,1: 8000, and 1: 16,000) were prepared in BHI broth and in saline. These were prepared in 25 ml amounts. E. coli was grown overnight in BHI broth and diluted to a MacFarland 0.5 concentration in saline. This solution was diluted 1: 100 to be used to inoculate

(0.5 ml) each terpene dilution tube. The series that contained the terpene dilution in BHI was tested at 30 min, 90 min, 150 min and 450 min.

Each tube was mixed and serially diluted in saline.

0.5 ml of each dilution was spread plated onto MacConkey (MAC) agar plates. Also, 3 drops of the undiluted and the 1: 100 dilution was added into respective tubes of BHI broth. The tubes and plates were incubated overnight at 35°C. The series that contained the terpene's dilution in saline were tested at 60 min, 120 min, 180 min and 480 min. Each tube was mixed and serially diluted in saline. 0.5 ml of each dilution was spread plated onto MacConkey (MAC) agar plates. Also, 3 drops of the undiluted and the 1: 100 dilution were added into respective tubes of BHI broth. The tubes and plates were incubated overnight at 35°C.

Table 3: Subculture from the tubes containing various dilutions of terpenes in broth Time Dilution 1:500 1:1000 1:2000 1:4000 1:8000 1:16,000 Undiluted NG + + + + + 30 min 1: 100 NG + + + + + Undiluted NG NG + + + + 90 min 1 : 100 NG NG NG + + + 150 Undiluted NG NG + + + + min 1: 100 NG NG NG + + + 450 Undiluted NG NG + + + + min 1: 100 NG NG + + + +

NG: no growth, +: growth

Table 4: Subculture from the tubes containing various dilutions of terpenes in saline Time Dilution 1: 500 1: 1000 1: 2000 1 : 40001 : 8000 Control 60 min Undiluted NG + + + + + 1:100 NG NG NG + 120 min Undiluted NG NG NG + + + 1: 100 NG NG NG NG + + 180 min Undiluted NG NG NG + + + 1:100 NG NG NG NG + + 480 min Undiluted NG NG NG NG + + 1:100 NG NG NG NG NG +

NG: no growth, +: growth Table 5: The quantitative results of the activity of various terpene dilutions against E. coli (cfu) Media Time 1: 500 1: 1000 1: 2000 1: 4000 1: 8000 Control 30 min 0 0 660 3600 3600 4600 90 min 0 0 12 4600 5400 7600 Broth 150 min 0 0 10 8000 12,000 14,000 450 min 0 0 15, 000 28 x 103 23 x 107 16 x 108 60 min 0 4 140 4000 2000 1300 120 min 0 0 0 90 3800 2600 Saline 180 min 0 0 0 2 2000 5000 480 min 0 0 0 0 104 8000

NG: no growth, +: growth

EXAMPLE 7: In vitro effectiveness of selected terpenes on Helicobacter pylori.

This example shows the bactericidal effect of selected terpenes on the viability of H. pylori.

Five terpenes (anethole, carvone, citral, geraniol and b-ionone) were used for this study. Terpenes were mixed to a ratio of 90% terpene plus 10% polysorbate-80. The H. pylori, used was strain &num 26695 of porcine origin, this bacteria is a motile, cag A, vac A cytotoxin-positive gram negative bacteria which colonizes gnotobiotic piglets and indefinitely persists within the gastric microenvironment as a superficial infection of the gastric mucosa and mucus layer.

The study was as follows: 1) Stock solutions of each terpene with polysorbate-80 were prepared (1.8 ml terpene plus 0.2 ml polysorbate-80).

2) Stock solutions were diluted in Brucella broth 10% (v/v) fetal calf serum to a final concentration of stock at 1: 10,1: 50,1: 100,1: 500,1: 1000, 1: 5000 and 1: 10000. Controls consisted of 10% (v/v) polysorbate-80 in Brucella broth, Brucella broth alone and bacteria in Brucella broth.

3) A total of 1.0 x 106 bacteria (30 jll) was added to 970 jll terpene dilutions (final volume of 1.0

ml) in loosely capped tubes and incubated for 24 hours at 37 °C with continuous mixing.

4) Duplicate samples (0.1 ml) from each test dilution was titrated onto blood agar plates and incubated for 48 hours at 37°C on 10% C02 environment. Bacterial colony forming units (cfu) were determined by visual (counting) inspection.

Recovered bacteria were confirmed to be H. pylori by catalase and urease enzyme activities.

The results are summarized in the following table: Table 6: Effect of different terpenes on H. pylori growth Final dilution tested for antimicrobial effects against 106 cfu Terpene 1 : 10 1: 50 1: 100 1: 500 1: 1000 1: 5000 1: 10000 1 : 50000 Polysorbate NG* NG NG 103 104 TNTC** TNTC TNTC -80 Anethole NG NG NG NG 103 103 TNTC TNTC Carvone NG NG NG NG NG 104 TNTC TNTC Geraniol NG NG NG NG NG NG 102 TNTC Citral NG NG NG NG NG NG NG TNTC b-ionone NG NG NG NG NG NG NG TNTC

* NG = no growth ** TNTC = Too Numerous To Count EXAMPLE 8: In vitro effectiveness of single or combination of terpenes against E. coli.

The objective of this example was to determine an optimum terpene mixture which could have a greater

biocidal effect. E. coli strain AW574 was grown in tryptone broth to an exponential growth phase (O. D. between 0.4 and 1.0 at 590 nm). One tenth of this growth was inoculated to 10 ml of tryptone broth followed by the addition of individual terpenes as indicated in Example 5; then incubated for 24 hours at 35-37°C and the O. D. determined in each tube.

The concentration of terpenes was 1 or 2 pool.

Each treatment was repeated in triplicate. The results are expressed as percentage bacterial growth as compared to the control treatment. It is observed that the combination of terpenes give better biocidal effect than single terpenes, with geraniol and carvone better than b-ionone.

Table 7: Effect of single terpene or their combination against on E. coli growth pmol terpenes % Growth B-ionone Carvone Geraniol 0 0 0 100. 00 2 0 0 84. 00 0 2 0 63. 00 0 0 2 54. 00 1 1 1 41. 00 1 2 1 31. 10 1 1 2 14. 80 1 2 2 15. 90 2 1 1 48. 60 2 2 1 44. 30 2 1 2 30. 20 2 2 2 1. 50 EXAMPLE 9: In vitro effectiveness of a combination of terpenes against different E. Coli strains Both well-test and broth test methods were used to assess the effect of terpene formulations against a variety of strains of E. coli. The broth test method was judged to be a more applicable simulation of gastrointestinal tract conditions than the well plate (zone of inhibition) method. A series of broth tests was conducted on a selected test formulation to determine its activity in an aqueous environment.

Test micro-organisms Bacteria were sub-cultured from original American Type Culture Collection (ATCC) freeze-dried material. They included E. coli strains 8739, 25922 and 700728 (Serotype group O : 157 H: 7), which are BioSafety class 1 organisms and E. coli 12795 (Serotype group O : 26) which is a BioSafety class 2 organism. All the bacteria were cultured on Tryptone Soya Agar (TSA), supplied by Oxoid Ltd, Hampshire and Mueller Hinton Agar (MHA), supplied by Merck Ltd. The incubation temperature was 35 °C.

Broth Test Procedure E. coli cultures were prepared in nutrient broth and allowed to grow until exponential growth phase was achieved (16 hours at 35 °C). 1 ml of this culture was transferred to each of a series of pre-sterilised Duran bottles containing 100 ml nutrient broth, 0.5 % w/v Polysorbate 80 and this gave an initial inoculum of approximately 108 microbial cells per ml of broth.

The Duran bottles were agitated on a vortex shaker to produce good mixing and the Optical Density (OD) at 590 nm read on a calibrated Unicam W 300 spectrophotometer controlled by Vision 32 software.

The OD of a sample of a placebo broth was also recorded.

The bottles were then placed in an incubator at 35°C. The bottles were removed at 30 minute intervals and placed on a vortex shaker at level three for 30 seconds. The bottles were then returned to the incubator. The OD was recorded at hourly intervals, for up to 24 hours.

After completion of the tests, the broths were autoclaved on programme 4 of an AVX240 autoclave (132 °C for 30 minutes) to sterilise them.

The terpenoids tested in this series of exemplary experiments included I-carvone, citral and geraniol in varying proportions. One exemplary formulation, constituting the test formulation, is given in Table 8, below Table 8 % w/w I-Carvone 40 Citral 40 Geraniol 15 Polysorbate 80 5 Total 100 This exemplary test formulation was highly active and clear inhibition of E. coli growth was observed in broth tests conducted at 50 jll and 100 ul doses in 100 ml broth.

Table 9: E. coli 8739 Broth Test of Formulation (Optical Density at 590 nm) Time Post Volume of Test Formulation (l) Inoculation 500 100 50 10 5 Control 0 0. 958 0. 063 0.019 0.007 0.007 0.003 1 0. 708 0. 025 0.028 0. 024 0.021 0.022 2 0. 762 0. 023 0.053 0.081 0.087 0.102 3 0. 547 0 0. 094 0.158 0.179 0.191 4 0. 486 0 0. 140 0. 270 0.286 0.323 5 0. 594 0 0. 181 0.316 0.311 0.345 6 0. 522 0. 011 0.238 0.361 0.367 0.401 6. 5 0. 579 0. 014 0. 262 0.376 0.372 0.411 23 0. 617 0. 031 0.285 0.619 0.747 0.654 24 0. 553 0. 058 0.286 0.606 0.740 0.683 The 16 hour old E. coli culture used as the inoculum had an OD at 590 nm of 0.697 units.

Further broth tests were conducted against two pathogenic strains of E. coli (700728,12795) and an antimicrobial agent test strain at 50 jll and 100 fil.

Table 10: ODs at 16 hours of E. coli Cultures used as the Inoculum for the Multi-Strain Broth Test Optical density at E.coli test strain 590 nm (absorbance units) 25922 0. 852 700728 0. 423 12795 0. 395

Table 11: Results of a multi-strain Trial of Test formulation Time Post E. coli strain/Volume of Test formulation (gel) Inocul- ation (hours) 25922 700728 12795 0 50 100 0 50 100 0 50 100 0 0. 006 0.040 0.088 0. 006 0.032 0.077 0.005 0.040 0.074 1 0. 034 0.021 0.031 0.020 0.030 0.028 0.017 0.024 0.035 2 0. 129 0.039 0.017 0.075 0.055 0.023 0.055 0.046 0.026 30. 263 0.074 0. 018 0.197 0.106 0.013 0.133 0.101 0.024 4 0. 365 0.01380.011 0.335 0.188 0.019 0.311 0.198 0.044 5 0. 366 0.174 0.004 0.312 0.240 0.050 0.364 0.205 0.049 6 0. 395 0.234 0.009 0.371 0. 264 0.044 0.399 0.264 0. 038 7 0. 427 0.256 0. 009 0.406 0. 287 0.040 0.436 0.282 0.042 23.5 0.688 0.351 0.049 0.545 0. 323 0.096 0.564 0.296 0.079 24.5 0.683 0.349 0.054 0.561 0.323 0.106 0.582 0.279 0.083

Table 9 summarises the results of the 50 and 100 fol/100 ml broth test. These results indicated good activity against E. coli 8739. Table 11 indicates that the test formulation showed good activity when challenged with other strains of E. coli including two pathogenic strains 700728,12795.

The 100 Fl dose of the test formulation had the lowest OD readings, therefore indicating greater inhibition of cell proliferation. 50 jell/100 ml broth of the test formulation appeared to have both slowed cell proliferation and reduced the final number of cells present in the broth. Where no test formulation was present, growth was rapid for all strains tested, especially in the first 4 hours after inoculation.

The test formulation is only one of a range of terpene formulations investigated so far and it is clearly very active. Clear inhibition of E. coli growth has been observed in broth tests conducted at 50 gl and 100 {il/100 ml broth, both against anti microbial assay strains and against pathogenic strains.

Formulations have been developed now which show very great activity against potentially lethal strain 0157: H7 of E. coli, both at very high innocula which are not sustainable in life and at levels which, though likely to be fatal, are found.

Three 100 ml bottles were each filled with McConkey

broth to which was added one of either 20 Fg/ml oxacillin, or 10 llg/ml of amoxicillin, or 1 ug/ml of the exemplary test terpene formulation. Each bottle was then inoculated with 104 E. coli 0157: H: 7 and incubated for 24 hours at 35°C.

Following incubation, the McConkey broth containing the oxacillin had lost its magenta colour and become yellowish and turbid, indicating that the antibiotic had been overwhelmed by the E. coli.. The McConkey broth in the bottle containing the amoxicillin had only slightly reduced magenta colour, indicating that the antibiotic had contained the E. coli, whereas the McConkey broth in the bottle containing the terpene sample had an undiminished magenta colour.

This experiment was then repeated under the same conditions, except that the inoculum of E. coli 0157: H : 7 was 108. In this case, both the oxacillin and amoxicillin samples were overwhelmed but the McConkey broth in the bottle containing the terpene sample had an undiminished magenta colour, indicating that, even with this extremely high inoculum, no growth had occurred.

Experiments have been carried out on xanthomonads including assay strains such as Xylefa maltifolia and plant pathogens such as X. fastidiosa. The latter causes Pierce's disease which has devastated grape culture in Southern California and threatens the wine growing areas of Napa Valley and Sonoma

Valley. The organisms are highly susceptible to terpene formulations according to the present invention.

It will be apparent for those skilled in the art that a number of modifications and variations may be made without departing from the scope of the present invention as set forth in the appending claims.

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