CN114761031A - An antimicrobial composition from the forest variety of Olea Europaea - Google Patents

Abstract

The present application relates to a process for the preparation of compositions of trifolium elegans (trifolium oleaginum), preferably wild trifolium elegans (trifolium oleaginum forest variety), compositions of trifolium elegans or wild trifolium elegans prepared, and their use in the prevention or treatment of bacterial or fungal infections in mammals, preferably infections caused by antibiotic resistant bacteria or multidrug resistant bacteria. Other uses relate to the use of functional food additives, in particular animal feed additives, or in cosmetic products.

Description

An antibacterial composition containing eurotium commune forest variety

Technical Field

The present application relates to a process for the preparation of an olive (Olea europaea subsp. europaea) composition, preferably a wild olive (Olea europaea forest variety (Olea europaea subsp. europaea var. sylvestris)) composition, the prepared Olea europaea or wild Olea europaea composition, and the use thereof in the prevention or treatment of bacterial or fungal infections, preferably infections caused by antibiotic resistant bacteria or multi-drug resistant bacteria, in a mammal. Other uses relate to the use of functional food additives, in particular animal feed additives, or in cosmetic products.

Background

Olive tree (Olea europaea L.) is a representative plant of the family melilotaceae with a long history of planting. It includes 30 genera, 600 different species. Trifolium includes 50 species, distributed in different continents and regions, including africa, china, india, the united states and australia (Iming, 2005). Trifolium oleae (oleca oleuropaea) is divided into different subspecies, including trifolium oleae (eurolene oleuropeica), and trifolium africanum (oleca europaa subsp. Both species exist in both wild and cultivar forms (Iming, 2005). Olive trees can grow for 1000 years with very little water demand, which also explains their emergence in arid regions (Iming, 2005). Most of the world's olives have been harvested from the mediterranean sea (Lieberei, 2007).

Wild olive, the forest variety of olive, is an ancestor of all olive plants, commonly known in spain as "wild olive (acebou)". Wild trifolium oleae has irregular distribution in, for example, the spanish peninsula; they spontaneously and accidentally grow into isolated clumps, or clumps, mainly in the mediterranean region.

Trifolium oleae leaves are different from fruit tree leaves, and the leaves are sometimes used as natural medicines. Trifolium olive tree is also very resistant to fungal and bacterial attack. These antibiotic and protective properties appear to be based on a variety of active compounds produced by trifolium oleae, such as oleuropein, which is one of the most studied active compounds at present (Iming, 2005).

Trifolium oleae tree active compounds are present in leaves, fruits, buds, stems, branches, and roots at various concentrations (Effect, 2017). It is speculated that leaves of trifolium oleae may have immune enhancing, anti-inflammatory, and blood pressure lowering effects (Iming, 2005). However, little is known about the medical effects actually involved.

A large number of bioactive substances have been identified in trifolium oleae and may be classified, for example, as secoiridoid glycosides, phenolic compounds, flavonoids, monoterpenes, triterpenes, steroids, quinoline alkaloids, carotenoids, chlorophylls, phenolic acids, tannins, and vitamins (Effect, 2017).

During the harvesting of trifoliate olive, leaves of the trifoliate olive tree accumulate continuously, providing an easily available byproduct that can be used for other purposes (Cayuela, 2006). The development of new uses of byproducts in olive oil production is particularly important, especially for trifoliate olive orchards. More and more research focuses on the chemical composition of the fruit of trifoliate olive, and little is known about the chemical composition of trifoliate olive leaves, especially the leaves of wild trifoliate olive trees.

Antibiotics have been a major tool in our treatment of bacterial infections, including acquired infections in fatal hospitals, since their discovery more than 70 years ago. However, antibiotics are often improperly and routinely prescribed and administered. Antibiotics are also used in the livestock industry for therapy, disease prevention and growth promotion. Antibiotics are also found in the environment, for example in some water supply systems (WHO, 2018).

Antibiotic resistance is a natural mechanism that occurs in anticipation that antibiotics that normally prevent the growth of a particular species no longer have any effect: (

2007). Worldwide, approximately 700,000 people die annually from antibiotic resistance (AMR). In the european union, there are approximately 25,000 people. It is feared that by 2050, antibiotic resistance may resultIn more deaths than cancer (conference, 2018).

Infections caused by drug resistant bacteria are often difficult to cure, sometimes even impossible, and the number of such conditions is increasing. However, the search for new effective antibiotics is very expensive and time consuming and often results in antibiotic resistance after new antibiotics are released on the market. Currently, only very few new antibiotics are under development. If no new effective antibiotic is found and the tolerance continues to spread, the society is threatened to return to the situation before the antibiotic was found, at which time the child often dies due to a small pneumonia and the doctor is unable to do anything about meningitis. Also, without effective antibiotics for prophylaxis, a number of sophisticated medical interventions and diagnoses may not be possible.

The emergence of drug resistant bacteria is a serious problem in healthcare, including (among other problems) the induction of fatal blood and wound infections and pneumonia. Antibiotic resistance causes an increase in treatment costs due to longer hospital stays, higher antibiotic and treatment expenditures, and indirect expense costs to the home and society. In many countries in the european region, antibiotics do not require a doctor's prescription. Data on antibiotic-resistant infections are not usually collected, so there is no documentation of the severity of the problem, although there is a high degree of knowledge of this among physicians. Nosocomial infections are one of the most common infections in germany. The problem of antibiotic resistance, and the problem of its extension, closely related to these infections, is one of the most important challenges for modern medicine (Braun AG, 2018).

Ahmed et al (Ahmed, Ali & s. rabii, Nancy & Garbaj, Aboubaker & Abolghait, Said. (2014.) the Antibacterial effect of olive (Olea europae L.) the leaves extract in raw peeled undived shrimp (peaeus semisubstrate us.) the International Journal of mineral Science and medicine.2.10.1016/j. ijvsm.2014.04.002) evaluated the effect of trifolium leaf extract on the microbial load of unprocessed peeled undried shrimp and discussed the potential use of trifolium leaf extract formulations in improving microbial quality and as a natural preservative.

Paudel et al (Paudel, Shambuu & Magriti, Thakur & Lamichhane, Jay Ram. (2011) an Antimicrobial activity of wild olive extracts in vitro. International Journal of Pharma Sciences and Research) disclose screening of crude extracts of wild Olea lutea for antibacterial activity against 5 different human pathogens, with extracts obtained with methanol appearing to be most effective against all pathogens.

DE102011108948a1 discloses a clear liquid formulation of a poorly water-soluble lipophilic substance which contains only one solubilizer and can be prepared by stirring alone without further complicated working steps.

In additional studies (e.g., Korukluguloglu, Mihriban & Sahan, Yasemin & YIGIT, AYCAN & T. may Ozer, Elif & Gucer, Seref. (2010), Antibacterial activity and chemical constraints of Olea europaea L. leaf extracts. journal of Food Processing and Preservation.34.383-396.10.1111/j.1745-4549.2008.00318.x), the extract of Olea europaea leaves inhibited the growth of a large number of bacteria and molds such as E.coli (Escherichia Coli), Klebsiella pneumoniae (Klebsiella pneumoniae), Bacillus cereus (Bacillus cereus), Aspergillus flavus (Aspergillus flavus), Aspergillus parasiticus (Aspergillus parasiticus). Aqueous extracts of olive leaves have no antibacterial effect on the tested microorganisms, whereas acetone extracts show inhibitory effects on Salmonella enteritidis (Salmonella enteritidis), Bacillus cereus, Klebsiella pneumoniae, Escherichia coli, Enterococcus faecalis (Enterococcus faecalis), Streptococcus thermophilus (Streptococcus thermophilus), and Lactobacillus bulgaricus (Lactobacillus bulgaricus). In addition, some phenolic compounds were tested for antimicrobial activity against microorganisms. The most effective compound was found to be oleuropein, whereas syringic acid had no effect. Such experiments have not been performed on wild olive ".

Wang et al (Wang, Xiao-Fei & Li, Chen & Shi, Yan-Ping & Di, Duo-Long. (2009). Two new secoiridoid glycosides from the leaves of Olea europaea L. journal of Asian natural products research.11.940-4.10.1080/10286020903310979) disclose 2 secoiridoid glycosides, oleuroicine A (1) and B (2), isolated from an ethyl acetate-soluble fraction of an extract of leaves of Olea europaea, and 5 known triterpenes, β -amyrin, oleanolic acid, homoradiciol, ursol-2 β, 3 β -dihydroxy-12-en-28-oic acid, and β -maslinicic acid. The structures of these compounds were identified by a variety of spectroscopic methods, including enhanced 1D, 2D NMR, and HR-ESI-MS techniques.

Disclosure of Invention

In view of the foregoing, it is an object of the present application to provide new and effective antimicrobial compounds and compositions from trifolium or wild trifolium. Other objects and advantages of the present application will become apparent to those skilled in the art upon a reading of the following more detailed description of the present application.

In its first aspect, the present application achieves the above objects by providing a process for preparing a composition of trifoliate olive (trifoliate olive), preferably wild trifoliate olive (trifoliate olive forest variety), comprising the steps of: a) providing parts of an olive plant or a wild olive plant; and b) removing water from said parts of trifoliate or wild trifoliate olive by dehydrating or drying said parts of trifoliate olive or wild trifoliate olive; and c) crushing the part of the trifolium olive or the wild trifolium olive in the step b) by cutting, grinding, milling or crushing or other methods for crushing the trifolium olive or the wild trifolium olive to obtain powdery trifolium olive or the wild trifolium olive; d) sterilizing the powdered or wild olive suitably at 121 deg.C or 134 deg.C to obtain sterilized powdered or wild olive; and, optionally, e) mixing the sterilized powdered or wild olive with water and a thickener to obtain an orange or wild olive jelly having antibiotic or antifungal activity; or d') extracted, preferably CO₂Extracting, namely extracting an antibiotic or antifungal activity agent from the powdery olea europaea or the wild olea europaea in the step c); suspending the active agent in nontoxic solubilizer and emulsifier to obtain mixture of radix tinosporae or wild radix tinosporae with antibiotic or antifungal activityAnd (3) suspending.

Surprisingly, it was found that there are particularly significant pharmacological differences between cultured and wild olive (see below, e.g. table 2).

In its second aspect, the present application achieves the above objects by providing sterilized powdered or wild trifoliate olive obtained by steps a), b), c), or d) of the above method of the present application having antibiotic or antifungal activity.

In a third aspect thereof, the present application achieves the above objects by providing a composition of trifolium or wild trifolium prepared by the methods of the present application, optionally comprising a pharmaceutically or cosmetically acceptable carrier, diluent or excipient.

In a fourth aspect thereof, the present application achieves the above objects by providing the use of a sterilized powdered or wild olive described herein, or a composition of roots or wild olive described herein, for the prevention or treatment of a bacterial or fungal infection in a mammal, preferably for the treatment or treatment of an infection caused by an antibiotic resistant bacterium or multi-drug resistant bacterium.

In a fifth aspect thereof, the present application achieves the above objects by providing the use of a sterilized powdered or wild trifoliate olive as described herein or a composition of a powdered or wild trifoliate olive as described herein as a functional food additive, in particular an animal feed additive.

In a sixth aspect thereof, the present application achieves the above objects by providing the use of a sterilized powdered or wild trifoliate oil as described herein, or a composition of trifoliate or wild trifoliate oil as described herein, as a cosmetic product and/or in a cosmetic method.

Drawings

In the accompanying drawings, figure 1 shows the schematic process steps for the preparation of the antibiotic/antifungal jelly of the present application, or for the preparation of the antibiotic/antifungal suspension of the present application, including the respective intermediates.

FIG. 2 shows a method for removing moisture from parts of trifoliate or wild trifoliate olive by dehydration or drying; the principle of the equipment used in the method steps is that the trifoliate olive or the wild trifoliate olive part is crushed, ground, milled and crushed to obtain the powdery trifoliate olive or the wild trifoliate olive. These steps are preferably performed simultaneously by passing the trifolium elemi or wild trifolium elemi site through a venturi nozzle together with air, preferably preheated air, by subjecting the same to forces generated by a dynamic gas stream.

Figure 3 shows a comparison of the active substances (iridoids) of different trifolium cultivars.

Figure 4 shows a comparison of the active substances (flavonoids) of different trifolium cultivars.

Figure 5 shows a comparison of the active species (terpenes) of another different trifolium cultivar.

Figure 6 shows a comparison of the actives (tyrosol and hydroxytyrosol) of yet another different trifolium cultivar.

Detailed Description

As explained above, in a first aspect of the present application the above mentioned problems are solved by providing a process for the preparation of a composition of trifoliate olive (trifoliate olive), preferably wild trifoliate olive (trifoliate olive forest variety), comprising the steps of: a) providing part of an olive plant of trifolium or wild olive; and b) removing water from said parts of trifoliate olive or wild trifoliate olive by dehydrating and/or drying said parts of trifoliate olive or wild trifoliate olive; and c) crushing the trifoliate olive or the wild trifoliate olive part in the step b) by cutting, grinding, milling, crushing and/or other methods for crushing the trifoliate olive or the wild trifoliate olive part to obtain powdery trifoliate olive or the wild trifoliate olive; d) suitably sterilizing said powdered or wild sweet clover, preferably at a temperature of from about 120 ℃ to about 140 ℃, preferably at a temperature of about 121 ℃ or about 134 ℃, to obtain sterilized powdered or wild sweet clover; and, optionally, e) mixing the sterilized powdered or wild olive with water and a thickener to obtain a jelly of olive or wild olive having antibiotic or antibacterial activity; or d') extracted, preferably CO₂Extracting from the powdered or wild sweet clover olive of step c)Extracting active agent with antibiotic activity or antibacterial activity; suspending the above active agent in at least one nontoxic solubilizer and/or emulsifier to obtain suspension of trifolium oleaefolium or wild trifolium oleaefolium with antibiotic activity or antibacterial activity (see figure 1).

Thus, the present application relates to two particularly important compositions having desirable properties, i) trifoliate or wild trifoliate gum having antibiotic or antifungal activity; ii) suspensions of trifoliate or wild trifoliate with antibiotic or antifungal activity in at least one non-toxic solubilizer and/or emulsifier.

The most studied secoiridoid glycoside in the trifolium plant is oleuropein. Oleuropein is present in all parts of the trifolium plant and benefits from this compound with a variety of benefits including antibacterial, antiviral, anti-inflammatory, antirheumatic, antioxidant, or cardioprotective activity (Fleming, 1973). Leaves of trifolium oleae contain 60-90mg/g dry matter oleuropein (Khan, 2007).

In addition to oleuropein, leaves of trifolium contain other phenolic components such as dimethyl oleuropein, luteolin (ligstroside), verbascoside (verbacoside), dimethyl oleuropein, and oleuroside (Cayuela, 2006). Trifolium oliate leaf also contains flavonoids. Which comprises rutin, luteolin, and apigenin. Flavonoids reduce oxidative damage by absorbing ultraviolet light and prevent oxidation of free radicals (Cayuela, 2006).

Guinda et al describe that trifolium oleae is a suitable starting material for the production of oleanolic acid and other pentacyclic triterpenes (Guinda, 2010). It comprises ursolic acid, betulinic acid, maslinic acid, erythrodiol, and hair-blackening alcohol. Guinda et al have described that the pentacyclic triterpene content in olive leaves of trifolium is higher than that of fruit itself. The most representative are oleanolic acid and ursolic acid (Guida, 2010; Bianchi, 1992). The concentration of various triterpenes is highly dependent on the developmental stage of the fruit and plant as well as the cultivar (Guida, 2010; Stiti, 2007).

Oleanolic acid and maslinic acid are desirable raw materials that can be extracted from olea europaea leaves in the pharmaceutical and cosmetic industries (Guinda, 2010). For this reason, the leaves of the wild olive "were tested for pentacyclic triterpenes. Which may have an effect on antimicrobial efficacy.

In the context of the present application, the inventors have developed methods for optimizing the processing of trifolium olive or wild trifolium olive compositions having antimicrobial efficacy. A particular advantage of the process according to the invention is that the two decisive steps, i.e. extraction of water and comminution, are carried out in a single apparatus, which considerably reduces the investment and saves operating and energy expenditure.

Furthermore, in the context of this method, the energy consumption required is significantly lower when using a venturi nozzle.

Most importantly, and surprisingly, it has been discovered that trifoliate or wild trifoliate portions treated by a venturi apparatus can result in a powdered or wild trifoliate having significantly higher antibiotic and antifungal effects than materials treated in conventional mills (i.e., without the use of a venturi apparatus). It has also been found that powdered or wild sweet clover, produced by the venturi apparatus, has an exceptionally long shelf life.

Table 1 below shows that the CFU of the Pseudomonas aeruginosa (Pseudomonas aeruginosa) bacteria is reduced when treated with the trifolium material treated by the traditional milling process compared to the trifolium material treated by the Venturi apparatus.

The extract of leaves of trifolium oleae, as a natural antibiotic or antifungal agent with broad spectrum antibacterial activity, is a pioneering step in combating infection, especially in the case of (multiple) antibiotic resistant, and thus particularly problematic, microorganisms.

Active powdered or wild olive, or a composition of olive or wild olive, for example, may serve as a basis for an interactive wound dressing, but may also be used for antiseptic, body surface disinfection, medical cleansing of well-developed sites of microbial colonization, and in particular for reduction and/or elimination of major pathogens and their multiple tolerant variants in wound management.

The term "provided" in the context of this application means that trifolium olive and wild trifolium olive plant products are used as raw materials for the processes described herein. It may be necessary to clean the surface impurities present from the trifolium and wild trifolium plants described above, for example by water bath, washing, or spraying, or other methods known to those skilled in the art. It may also be necessary to size trifoliate and wild trifoliate plants to be suitable for manipulation of trifoliate and wild trifoliate plants. Methods are also known to those skilled in the art.

As used herein, "dehydration" or "drying" is generally and preferably associated with direct drying, for example, by simply exposing the raw material to air (also known as natural air drying), or heated air to enhance air flow drying (prepared by means such as ventilation, air blowers, or other equipment known in the art), or indirect drying, or high frequency drying, or vacuum drying, or lyophilization; wherein the indirect drying is effected by contact drying or drum drying; high frequency drying by means of, for example, microwaves; wherein these methods may be adapted to reduce drying time or to achieve a higher degree of sensitivity as may be required. The method of using elevated temperatures to shorten the water extraction time is limiting because raising the temperature too much and raising the temperature exposure time too long may cause deterioration or degradation of the antibacterial/antifungal active ingredient in the raw material. To avoid any contamination or spoilage in handling and storage of the dried material, the material is preferably dried to a degree of 95 dry matter (DS)% or more. The degree of moisture content may be optimised to lower or higher dry matter% recommended for the subsequent crushing and comminution steps.

As used herein, "crushing" is typically and preferably performed in one or more steps to achieve the desired degree of comminution. The most desirable degree of comminution is determined taking into account the cost of comminution on the one hand and the efficacy of the antibacterial or antifungal action of the resulting powdered material on the other hand. According to the invention described herein, the desired particle size of the olive or wild olive part is between 1 μm and 1000 μm, more preferably between 40 μm and 500 μm, most preferably between 125 and 250 μm. The desired degree of crushing is achieved by operating the trifoliate or wild trifoliate portion in the crushing apparatus a number of times, or by using different types of crushing apparatus which may be such that a preferred particle size is operated. Typical crushing equipment is selected from the group consisting of presses, choppers, cutting tools, mills, and mills, such as hammer mills, ball mills, impact mills, rolling ball mills, centrifugal mills, jet mills, skin mills, planetary mills, mortar mills, pulverizers, micronizers, ultrasonic fine mills, or micro mills, but is not limited thereto. Classification of the powdered material may be added to this step to classify the plant material into the desired size types, based on specification requirements. It may also be useful to regrind particles classified as too large to achieve a desired size cluster.

As used herein, the term "sterilization" refers to the removal of natural contaminants from pulegano/wild trifolium with, for example, aerobic spores using a variety of sterilization procedures. The different types of sterilization may be selected from heat sterilization, wherein the microorganisms are killed thermally, e.g. by heating in the wet state (steam sterilization), or in the dry state (hot air sterilization), or step sterilization, wherein heating is repeated continuously, or by physical sterilization techniques, or other sterilization methods known in the art. In the context of the present application, sterilization at 121 ℃ and 134 ℃ in a vacuum sterilizer is preferred, wherein the temperature is suitable for preserving the pharmaceutically active ingredient, wherein sterilization is optionally performed by steam sterilization.

As used herein, the term "mixing" refers to the mixing or homogenization of sterilized powdered or wild trifoliate with water for injection and a thickening agent, as described herein below, to obtain a gum of trifoliate or wild trifoliate having antibiotic or antifungal activity, and wherein the mixing is continued until a uniform gum is formed. There are a variety of methods known in the art that can be used to achieve the desired results.

As used herein, "extract" means to extract fromThe powdered or wild olive from the process described herein separates antibiotics or antifungal agents. According to the invention, the extraction is preferably by CO₂Extraction is carried out. The extraction can be done in batch mode, as the extractor can only be emptied and refilled at atmospheric pressure. In the extraction process, supercritical carbon dioxide (CO)₂) The raw material is infiltrated under high pressure and soluble material is extracted from powdered or wild olive (aqueous extract). The dissolved substance is separated into fractions with different composition by gradually reducing the pressure. In the first section (separator 1) there is accumulated less soluble material, while the more soluble material is collected in the subsequent separator 2. Other solid-liquid extraction types may be selected, such as, but not limited to, soaking and decanting, as long as the active agent remains fully effective.

In a preferred embodiment of the method according to the application, steps b) and c) are carried out simultaneously by subjecting said trifolium or wild trifolium locations to the forces generated by the dynamic gas flow, preferably by passing said trifolium or wild trifolium through a venturi nozzle together with air or another suitable gas, preferably preheated air. Granted patents US 7,429,008B 2, US 7,500,830B 2 and US 7,909,577B 2 disclose the comminution of materials which, during the comminution process, are subjected to continuous moisture extraction and drying by means of a gas flow generator coupled with a venturi nozzle, but generally using, for example, polymers or waste materials as starting materials. Furthermore, patent application WO2013/052583a2 generally discloses dewatering, comminution and pyrolysis of biomass such as sludge. Patent application WO2013/075003 a1 provides a process for the preparation of eggshell powder, which can be used for the preparation of biological products. The method involves pulverizing the eggshells at a high air flow rate at room temperature and separating the eggshell components from the inner membrane components. However, no method has been developed for simultaneously dehydrating and drying parts of trifoliate or wild trifoliate, and simultaneously crushing the parts, as provided herein. The active agent of trifoliate olive or wild trifoliate olive is preserved, and the powder has long shelf life.

Thus, for example, trifoliate or wild trifoliate portions are passed through a venturi nozzle with air, and the trifoliate and/or wild trifoliate portions are exposed to a dynamic airflow. In doing so, the gas stream pulls the feedstock through a venturi nozzle connected to the apparatus. The air moves through the venturi nozzle to accelerate the air and the sweet clover or wild sweet clover contained in the air flow. The airflow is generated, for example, by the rotation of blades in the turbine, or by another device adapted to drag the airflow. The gas stream and dried powdered or wild trifoliate leaves the apparatus through an outlet nozzle of the apparatus, wherein the comminuted trifoliate or wild trifoliate may be separated from the gas stream by a filter, a cyclone or similar means known to those skilled in the art. At the same time as the crushing, the air flow is heated by the energy losses (heat) of the operating turbine. The accelerated gas stream (and the comminuted or wild olive) then absorbs this heat energy, increasing its temperature. Physically, higher temperature air is able to absorb relatively more moisture than lower temperature air. Due to this natural phenomenon, the use of crushing and heat losses compensate each other in the venturi equipment specification. The material being drawn through the apparatus (including the venturi apparatus) comminutes, enlarging the surface of the material which transfers moisture to the air stream, and the increased air temperature due to the operation of the turbine causes more moisture to be transferred from the material to the air stream. The venturi device may be used to carry out steps b) and c) of the method of the invention, either completely or partially. Preferably the point of supply for trifoliate or wild trifoliate may be before the inlet pipe to which the apparatus is connected.

In other preferred embodiments of the process of the present application, the thickener used is selected from gelling agents, such as gellan gum, alginic acid, sodium alginate, potassium alginate, ammonium alginate, calcium alginate, propylene glycol alginate, agar, carrageenan, furcellaran, locust bean gum, guar gum, traganth gum, gum arabic, xanthan gum, karaya gum, tara gum, pectin, cellulose, gelatin, and modified starches.

In another preferred embodiment of the method according to the present application, the above-mentioned non-toxic solubilizer and/or emulsifier is selected from the group of surfactants, wherein the surfactant is preferably selected from the group consisting of

Most preferably

RH 40. The other surfactant may be selected from nonoxynol-9, octoxynol-9 (Triton X-

) Polysorbate 20 (Tween)

) Octyl-beta-glucoside, and n-octyl-beta-D-1-thioglucopyranoside. Other non-toxic solubilizing agents and/or emulsifiers may be selected from ethanol, acetone, hexane, chloroform, and methyl tert-butyl ether (MTBE).

The parts of trifolium oleae or wild trifolium oleae used in the methods of the present application may be selected from leaves, shoots, root bark, stem bark, fruits, or oil preparation residues, and combinations thereof. As used herein, the term "locus" refers to all appropriate components of the trifoliate and wild trifoliate plants, such as the bark of leaves, shoots, stems or roots, trifoliate or products processed from trifoliate, and residues. Parts may also refer to, for example, plants grown in soil, harvested plant parts, discarded parts of plants, processed plant parts, and inanimate plant parts.

Preferred are methods according to the present application, wherein at least one active agent is comprised in the prepared composition, which is selected from compounds having antibiotic or antifungal activity. Preferred active agents are selected from iridoids or phenols, preferably at least one organic compound selected from oleuropein, olive secoiridoid, olive oil stimulating aldehyde, tyrosol, hydroxytyrosol, hair blackening alcohol, and high root diol. In addition, in the context of the present application, the active agent may also comprise oleuropein, luteoloside, verbascoside, flavonoids, maslinic acid, apigenin, luteolin, and oleanolic acid. Most preferred are oleuropein, olive secoiridoid, olive oil stimulating aldehyde, tyrosol, hydroxytyrosol, hair blackening alcohol, and high root diol.

In its second aspect, the present application solves the above problems by providing sterilized powdered or wild trifoliate product/composition having antibiotic or antifungal activity obtained by the method of the present application.

Especially preferred is sterilized powdered or wild olive olea europaea according to the present application having antibiotic or antifungal activity, which is obtained by steps a), b), c) and d) of the method according to the present application.

The term "antibiotic activity" as used herein essentially refers to an activity acting on infections caused by microorganisms. These microorganisms are mainly bacteria, but they also include, for example, protozoa. The effect may include killing microorganisms, and/or inhibiting growth and/or reproduction. In the context of the present application, antibacterial agents are particularly active and effective against microorganisms that are resistant to commercial antibiotics, particularly multi-drug resistant microorganisms (i.e., bacteria or fungi that are resistant to at least two antibiotics, such as commercial antibiotic substances). As used herein, "antifungal activity" refers to an activity against infections caused by fungi, such as mold. The effect may include killing of fungi, or inhibition of growth or proliferation. The term "resistant" as used herein refers to the characteristics of microorganisms such that they reduce or completely neutralize the action of commercial antibiotic substances. The term "multidrug resistance" as used herein refers to a characteristic of a microorganism that reduces or completely neutralizes the effect of at least two different commercially available antibiotic substances.

Preferred is sterilized powdered wild olive according to the application having antibiotic or antifungal activity, wherein the powdered or wild olive is a powder and consists of more than 95 wt% dry biomass. The term "dry biomass" as used herein refers to a material that is free of water or other components, such as other liquids, in an actual dry amount. As used herein, "weight percent" refers to the mass ratio of a mixture, particularly powdered or wild olive. The composition of powdered or wild olive depends on the ratio of the individual components in 100 g of the mixture. In the context of the present application, preferably more than 100 grams of about 95% is made up of dry biomass.

In its third aspect, the present application solves the above problems by providing an olive or wild olive composition prepared by the process of the present application. Optionally and preferably, the above composition comprises a pharmaceutically or cosmetically acceptable carrier, diluent or excipient.

In another preferred aspect, the present application relates to an antibacterial or antifungal composition comprising at least one organic compound selected from the group consisting of olive secoiridoid, hydroxytyrosol, olive oil stimulating aldehyde, hair blackening alcohol, and high root diol. The composition is preferably a pharmaceutical or cosmetic composition as described herein.

In another preferred aspect, the present application relates to the above-mentioned composition for use in the prevention or treatment of a bacterial or fungal infection in a mammal, preferably for use in the prevention or treatment of an infection caused by an antibiotic-resistant bacterium or a multi-resistant bacterium.

As used herein, "pharmaceutically acceptable carrier, diluent or excipient" refers to an ingredient of a pharmaceutical formulation or composition other than the active ingredient, which is non-toxic to the subject. Pharmaceutically acceptable carriers, diluents, or excipients include any and all suitable physiologically compatible solvents, dispersion vehicles, coatings, other antibacterial and antifungal agents, isotonic and absorption delaying agents. Carriers include various preservatives, antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like in the compositions of the present application. In addition, sustained release of the injectable dosage form can be achieved by the use of absorption blockers such as aluminum stearate and gelatin.

Regardless of the route of administration chosen, the compositions of the present application (which may be used in the form of a suitable hydrate), and/or the pharmaceutical compositions of the present application, may be formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art. The actual dosage level of the active ingredient in the pharmaceutical composition of the present application may vary. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the specific composition of the application, the route of administration, the time of administration, the rate of excretion of the specific compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in conjunction with the specific composition being employed, the age, sex, weight, condition, general health, and prior medical history of the patient being treated, and like factors well known in the medical arts.

The composition must be sterile and fluid so that the composition can be delivered by syringe. In many cases, isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride are included in the composition.

The compositions of the present application may be administered locally or systemically. Administration is generally parenteral, e.g., intravenous. Formulations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, ringer's dextrose, dextrose and sodium chloride, lactated ringer's solution, or nonvolatile oils. Carriers for intravenous injection include liquids and nutrient replenishers, electrolyte replenishers (such as those based on ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.

As used herein, "cosmetically acceptable carrier, diluent or excipient" refers to suitable ingredients in a cosmetic formulation other than the active ingredient. Generally, such ingredients must be suitable for use in topical contact with tissue (e.g., skin) without undue toxicity, incompatibility, instability, irritation, allergic response, and the like. Cosmetically acceptable carriers, diluents, or excipients may include, for example, water, liquid or solid emollients, solvents, humectants, thickeners, and powders that facilitate distribution of the composition when applied to, for example, the skin, hair, and/or nails.

Other particularly preferred embodiments herein are trifoliate or wild trifoliate compositions according to the present application wherein the composition is a gel, preferably a homogeneous colloidal gel. In the context of this application, "colloidal gels" refer to those lipophilic powdered or wild sweet clover plants obtained by the process of this application, which are present in large amounts (up to 100%) in colloidal size particles, i.e. 1-1000 μm, resulting in solubility of the lipophilic powdered or wild sweet clover plants that are practically insoluble and ensuring uniform distribution of the powdered or wild sweet clover plants.

Other particularly preferred embodiments relate to an olive or wild olive suspension according to the application, wherein the olive or wild olive suspension is an aqueous suspension. The term as used herein specifically includes trifoliate or wild trifoliate suspensions rendered water soluble through the use of surfactants, as well as the respective aqueous solutions beneficially available through the use described above.

Further preferred is a trifoliate or wild trifoliate composition according to the application wherein the composition is a suspension wherein the amount of non-toxic solubilising agents, especially surfactants, is less than 10%, preferably less than 5%, more preferably less than 3% of the total amount of trifoliate or wild trifoliate suspension. As mentioned above, the surfactant is preferably selected from

Most preferably

RH40。

In another preferred embodiment of the present application, the composition described herein is in the form of an ointment, lotion, cream, spray, gel, liquid, drops, capsule, or suppository. The above compositions may be administered systemically, i.e., by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. For transmucosal administration, the compositions according to the application are formulated as liquid, drops, capsules or suppositories. For epidermal (transdermal) administration, the above compositions are formulated as ointments, lotions, creams, sprays, or gels, as is known in the art.

In its fourth aspect, the present application addresses the above problems by providing a sterilized powdered or wild olive described herein, or a composition of olive or wild olive described herein for use in medicine. Even more preferred is a sterilized powdered or wild olive described herein or a composition of roots as described herein for use in the prevention or treatment of infection by a microorganism, such as a bacterium or fungus, in a mammal, preferably an antibiotic-resistant bacterium or even a multi-drug resistant bacterium. Another aspect relates to the use of a sterilized powdered or wild olive described herein, or a composition of roots or wild olive described herein, in the manufacture of a medicament for the prevention or treatment of microbial, e.g. bacterial or fungal, infections (preferably infections caused by antibiotic-resistant or even multi-drug resistant bacteria) in a mammal.

As used herein, a "mammal" may be a farm animal (e.g., a horse, cow, sheep, or pig), a pet (e.g., a cat, dog, rabbit, or pig), a rodent, or, in particular, a human. Thus, the compositions according to the present application may be used to treat any of these mammals.

The term "treating" as used herein includes administering to the mammal a composition as described above, preferably in a therapeutically effective amount to slow the disease or progression of the disease. Thus, an effective amount is that amount of the composition or pharmaceutical composition described herein above that normalizes the infectious state of a mammal. This amount alleviates symptoms associated with infection and/or disorder, while being non-toxic to the subject. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, the dosage administered to any mammal will depend on many factors, including the size, body surface area, age of the mammal, the particular compound administered, sex, time and route of administration, general health, and other drugs being administered together.

As used herein, the term "preventing" includes administering to the aforementioned mammal the aforementioned composition, preferably in a prophylactically effective amount, to reduce the predisposition or risk of the subject to become infected with an antibiotic-resistant bacterium or a multi-resistant bacterium, regardless of how mild the predisposition or risk is. For prophylaxis, the mammal is preferably a mammal at risk of, or susceptible to, infection by an antibiotic-resistant bacterium or a multi-resistant bacterium, wherein the composition is preferably administered by injection. Further, enteral and transdermal administration may be included in the context of the present application and include, but are not limited to, intravenous, intramuscular, intraarterial, intradural, intraarticular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

In another preferred embodiment of the present application, sterilized powdered or wild olive according to the present application, or a composition according to the present application for use in preventing or treating a bacterial or fungal infection, preferably an infection caused by an antibiotic-resistant bacterium or a multi-drug resistant bacterium in a mammal, wherein the above-mentioned bacterium is selected from the group consisting of Escherichia coli (Escherichia coli), Staphylococcus aureus (Staphylococcus aureus), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Klebsiella pneumoniae (Klebsiella pneumoniae), Enterococcus hirae (Enterococcus hirae), Enterococcus faecalis (Enterococcus faecalis), Enterobacter (Enterobacter), Mycobacterium tuberculosis (Mycobacterium tuberculosis), Serratia (sartoria), Proteus (Proteus), Pseudomonas sp (Proteus), helicobacter sp (Mycobacterium Proteus), helicobacter sp (helicobacter pylori), helicobacter pylori (helicobacter), Mycobacterium (helicobacter), helicobacter pylori (helicobacter pylori), helicobacter pylori (helicobacter), or helicobacter pylori (helicobacter pylori), or helicobacter pylori (helicobacter), or helicobacter pylori (helicobacter pylori), or helicobacter pylori (helicobacter pylori), or helicobacter pylori (helicobacter), or helicobacter strain), or helicobacter pylori (helicobacter strain), or helicobacter strain (helicobacter pylori (helicobacter strain), or helicobacter strain, e Examples of such a compound are the use of a compound of the formula i (i) for the treatment of fungal infections in mammals, such as Candida albicans (Candida albicans) and Aspergillus brasiliensis (Aspergillus brasiliensis), staphylococcus pneumoniae (Streptococcus pneumoniae), Haemophilus influenzae (Haemophilus influenza), Shigella (Shigella), Acinetobacter baumannii (Acinetobacter baumannii), and resistant and multidrug resistant strains thereof, and for the prevention or treatment of fungal infections in mammals.

In its fifth aspect, the present application addresses the above problems by providing a functional food additive comprising a sterilized powdered or wild trifolium olive as described herein, or a composition of trifolium olive or wild trifolium olive as described herein. In addition, the above problems are solved by the use of sterilized powdered or wild olive oil as described herein, or a composition of powdered or wild olive oil as described herein, as a functional food additive, particularly an animal feed additive. As used herein, the term "animal feed additive" refers to a composition added to the feed of farm animals.

Furthermore, as used herein, the term "functional food additive" refers to the use of a composition to increase the supply of active ingredients in the composition to a mammal by supplementing the ordinary diet. The compositions may also be administered as dietary or food supplements other than basic nutrition, in the form of concentrates, or in the form of medicaments, particularly capsules, lozenges, tablets, pills, effervescent tablets, and other similar forms, powder bags, liquid ampoules, drop bottles, and other similar liquid and powder forms for administration in suitably small doses. In particular, the term "animal feed additive" refers to a composition added to the feed of farm animals, wherein feed for other animal species is not excluded.

In its sixth aspect, the present application solves the above problems by providing a cosmetic product comprising the sterilized powdered or wild olive of the present application, a jelly or suspension of olive of trifolium or wild olive according to the present application, or a pharmaceutical composition according to the present application. Another aspect relates to sterilized powdered or wild olive herein described, or a composition thereof, for use in a cosmetic product or method of beauty.

As used herein, the term "cosmetic" encompasses all areas of body care, or all areas of action to maintain, repair, or improve the appearance of a mammal. In particular, the field of application of cleaning, care and protection, and in particular of dental and mouth care, is included within the scope of the present application.

In the context of the present application, the terms "about" and "approximately" refer to an interval of accuracy that a person skilled in the art understands to still ensure the technical effect of the feature concerned. The term generally denotes a deviation of ± 20%, ± 15%, ± 10%, and for example ± 5% of the indicated value. A person skilled in the art realizes that a specific deviation of a numerical value for a given technical effect will depend on the nature of the technical features. For example, a natural or biological technical effect may have substantially greater deviation than an artificial or engineered technical effect. One of ordinary skill in the art will appreciate that the particular deviation of a numerical value for a given technical effect will depend on the nature of the technical features. For example, a natural or biological technical effect may have substantially greater deviation than an artificial or engineered technical effect. Where an indefinite or definite article is used, when referring to a singular noun e.g. "a", "an", or "the", this includes a plural of that noun unless something else is specifically stated.

The present application will be further illustrated in the following examples, but the present application is not limited thereto. For the purposes of this application, all documents, patents, and publications cited herein are incorporated by reference in their entirety.

Examples

Example 1: extracting the components of Olea europaea with supercritical carbon dioxide

The dried, pre-ground olive components are placed in an extractor. Extraction is performed in batch mode, as the extractor can only be emptied and refilled at atmospheric pressure. In the extraction process, supercritical carbon dioxide (CO)₂) The raw material is infiltrated under high pressure and soluble substances (i.e., extracts) are extracted from the raw material. The dissolved substance can be divided into fractions with different compositions by gradually reducing the pressure. In the first section (separator 1) the pressure can build up more insoluble substances, whereby in the following separator 2 more soluble substances can be collected.

For the experiments carried out, a modified extraction method with only one separation stage was chosen, whereby all dissolved material was collected in the separator 1. The amount of material depends on the extraction conditions (pressure and temperature) chosen, as well as the solubility and the amount of material contained in the feedstock.

The process flow comprises the following steps: high pressure laboratory systems (HDL4) were used in the experiments. This is a laboratory device with one extractor and two separators. The extraction pressure is up to 1000 Pa and the temperature is up to 95 ℃ for the apparatus. The extraction pressure is not higher than 1000 Pa. By pumping CO₂Liquid CO from tank₂At extraction pressure and, by heat exchange, at extraction temperature. In an extractor, supercritical CO₂Flows through the raw material and is enriched in soluble substances. After the gas pressure is reduced, the mixture separates into gaseous CO₂And an extract. The extract may be collected in a separator and removed from the apparatus. Gaseous and uncharged CO₂It can then be liquefied again in a condenser and reused in the extraction cycle.

Executing the following steps: the material was placed in an extractor of HDL4 for extraction. Next, valuable components (e.g., terpenes and terpenoids) are extracted from the natural substances. Only one separation stage is used for the next extract separation, the whole extract being collected in the separator 1. Since the extract obtained is not flowable at-60 ℃, traditional extraction of the extract through the outlet valve of the separator 1 is difficult or impossible. The extract must be taken out after each test run, after opening the separator and additionally scraped off, the solvent being partially washed out.

Example 2: treatment of trifolium material using a venturi dryer

The raw trifolium material is first thermo-mechanically processed, wherein the particle size and moisture content of the raw material is specifically altered. The result of the technical implementation is a dehydrated fine-grained product, possibly also a powdered product, for direct further use in the process chain, possibly also for intermediate process storage thereof.

Example 3: disinfection procedure

Since the starting product is naturally contaminated with aerobic spores, multiple sterilization processes are applied. Different sterilization temperatures were used to determine the optimum temperature for retention of the pharmaceutically active ingredient: 121 ℃ and 134 ℃. Sterilizing at different temperatures.

Example 4: comparison of active substances and disruption methods for cultivated and wild Olea europaea leaves

Parts of wild and cultivated trifolium oleae plants were collected manually in Casa de porros, a method in spain. The processing of each plant part is as follows: fresh leaves, shoots, bark, and root bark portions were dried in a dapogue (Taprogge) Venturi apparatus, and the other portions were crushed in a hot mixer. The sample material was stored in a sealed container protected from light at ambient temperature. For comparability, both sets of samples were screened to the same particle size range. Next, soxhlet extraction and chromatography were performed to analyze the chemical composition of the active agent in cultivated and wild trifolium olea. A positive effect was found in the Oxidative Radical Absorption Capacity (ORAC) test; active sampling after soxhlet extraction revealed that higher extraction yields were achieved for materials crushed in a darvery venturi apparatus. For materials crushed by the darvery venturi device, a favourable effect on ORAC (oxidative radical absorption capacity) was detected, which, in turn, translates into positive protective properties, and a particularly long shelf life. This can be demonstrated by measuring the antioxidant activity of leaves and shoots processed by the daphne device method compared to that of fresh samples during 30 days, the value of the samples treated by the daphne device method remaining constant. The results are shown in Table 2.

Table 2: comparison of antioxidant Activity between fresh samples and samples treated by the Dacron apparatus method over a 30 day period

By comparing the active substances in wild and cultivated trifolium oleae, it can be seen that there is a higher concentration of active substances in wild type trifolium oleae trees. Eurotium commune and forest varieties differ in many ways: the cultivated sweet clover olive has larger fruit (fixed shape), so the method is more suitable for preparing the sweet clover olive oil. However, the tendency to produce larger fruits, clearly indicates that the remainder of the trifolium plant contains less active material. Three common species based on cultivation in the adapalensis region: the published data for albugina, parsibulan and pigqer, compare two plants. Mass spectra of active species from fresh wild olive leaves were examined (Table 3).

Table 3: concentration of Individual substances (mg/g)

A comparison of active substances for different trifolium cultivars is shown in figures 3-6, where figure 3 shows a comparison of iridoids, figure 4 shows a comparison of flavonoids, figure 5 shows a comparison of terpenes, and figure 6 shows a comparison of tyrol and hydroxytyrosol.

Table 4 below compares the percentage of active substances of wild and cultivated trifolium oleae.

Table 4: the amount of active substances in wild and identified Daghestan olive

Based on the comparison of these 9 substances, it was shown that wild olive leaves have the highest concentration of phenols and monophenols, especially flavonoids. From the pharmacological point of view, it is obvious that wild olive (wild) is completely different from cultivated olive, so that the wild olive is a better raw material for potential pharmacological or cosmetic products.

Example 5: antimicrobial efficacy studies on trifoliate olive or wild trifoliate olive suspensions

The objective of the study was to test the antimicrobial efficacy of trifoliate or wild trifoliate suspensions after suspension in Kolliphor RH40 and water for injection (WFI) for expanding the spectrum of test strains (table 5).

Table 5: test bacterial Spectrum for microbiological Studies of the present application

Executing: preparation of test bacteria suspension: the test bacteria were cultured at passage numbers up to 5. The suspension of test bacteria was adjusted to approximately 1000CFU/0.1ml sterile 0.9% NaCl solution.

Sample preparation: heating 3.0g of Daghestan Sweetclover extract in 1.01g of Kolliphor RH40 in a water bath at 45 deg.C for about 5min, then in a water bath at 80 deg.C for about 15min, and shaking by hand. Trifolium oleae extract was mixed with Kolliphor RH40 in 15ml WFI to give a very viscous, dark green to dark brown suspension which could not be sucked up with a pipette. The next day, the stock solution is again heated in the water bath at 80 ℃ and another 5ml of WFI heated to 80 ℃ is added. The stock solution was heated again to 80 ℃ for about 5 minutes. Heating in water bath and shaking by hand for about 1 min. The result was a stock solution containing 125mg trifolium oleae extract/ml and 4.2% Koppliphor RH 40. After the above treatment, the trifolium oliate extract is a uniform suspension that can be absorbed by a pipette, with a dark brown to dark green color.

Evaluation of antimicrobial efficacy of trifolium oleaginum extract after preparation of stock solution: for each test strain, 1ml of stock was transformed into 1ml of double concentrated CaSo broth and sashimi broth (candida albicans). The preparation was then inoculated with 0.1ml of each test bacterial suspension adjusted to about 1000CFU/0.1 ml. The bacterial count in the test bacterial suspension was determined by means of the surface coating method on blood agar and sakefir agar. All media preparations were incubated at 30-35 ℃ for up to 72 hours. After 24h, 48h and 72h incubation, subcultures of all 0.1ml batches were performed by streaking on blood agar plates and sakheira agar, respectively. Agar plates were tested for 24h-48h (yeast) aerobic incubation at 30-35 ℃. Positive control: 1ml of WFI was added to 1ml of double concentrated Caso broth or Sasa broth (Candida albicans) before inoculation of 0.1ml of each test bacterial suspension adjusted to about 1000KBE per 0.1ml in a single determination. The positive control was used as a reference for test bacteria growth in double concentrated medium after 1: 2 dilution. The results are shown in the following table.

Table 6: results of antimicrobial efficacy of trifolium oleander suspension against the test strain candida albicans ATCC 10231; inoculum: effective inoculum bacterial count for each test batch: 1160CFU 580CFU/ml batch of culture medium

Growth of test bacteria in a nutrient medium preparation with a macroscopic degree of miscibility

Product turbidity without macroscopic test bacteria growth

Subculture (SC) by streaking 0.1ml of each nutrient medium preparation

Table 7: results of the antimicrobial efficacy of the olea europaea suspension on the test bacterium enterococcus faecium ATCC BAA 2317; inoculum: effective inoculum bacterial count for each test batch: 1810 CFU-905 CFU/ml Medium batch

Growth of test bacteria in a nutrient medium preparation with a macroscopic degree of miscibility

Product turbidity without macroscopic test bacteria growth

Subculture (SC) by streaking 0.1ml of each nutrient medium preparation

Table 8: results of the antimicrobial efficacy of the olea europaea suspension on the test bacterium enterococcus hirae ATCC 10541; inoculum: effective inoculum bacterial count for each test batch: 760CFU 380CFU/ml batch of culture medium

Growth of test bacteria in a nutrient medium preparation with a macroscopic degree of miscibility

Product turbidity without macroscopic test bacteria growth

Subculture (SC) by streaking 0.1ml of each nutrient medium preparation

Table 9: results of olea europaea suspension on antimicrobial efficacy of the test bacterium klebsiella pneumoniae subspecies CCUG 56233; inoculum: effective inoculum bacterial count for each test batch: 1200CFU 600CFU/ml batch of culture medium

Growth of test bacteria in a nutrient medium preparation with a macroscopic degree of miscibility

Product turbidity without macroscopic test bacteria growth

Subculture (SC) by streaking 0.1ml of each nutrient medium preparation

Table 10: results of the antimicrobial efficacy of the olea europaea suspension on the test bacterium klebsiella pneumoniae subspecies ATCC 10031; inoculum: effective inoculum bacterial count for each test batch: 740CFU 370CFU/ml batch of culture medium

Growth of test bacteria in a nutrient medium preparation with a macroscopic degree of mixability

Product turbidity with no visible test bacteria growth

Subculture (SC) by streaking 0.1ml of each nutrient medium preparation

Table 11: results of the antimicrobial efficacy of olea europaea suspension on the test bacterium pseudomonas aeruginosa ATCC 9027; inoculum: effective inoculum bacterial count for each test batch: culture medium batch with 640 CFU-320 CFU/ml

Growth of test bacteria in a nutrient medium preparation with a macroscopic degree of miscibility

Product turbidity with no visible test bacteria growth

Subculture (SC) by streaking 0.1ml of each nutrient medium preparation

Table 12: results of the antimicrobial efficacy of trifolium oleander suspension on the test bacterium pseudomonas aeruginosa ESBL 24600; inoculum: effective inoculum bacterial count for each test batch: 680 CFU-340 CFU/ml batch of culture medium

Growth of test bacteria in a nutrient medium preparation with a macroscopic degree of miscibility

Product turbidity without macroscopic test bacteria growth

Subculture (SC) by streaking 0.1ml of each nutrient medium preparation

Table 13: results of antimicrobial efficacy of trifolium oleander suspension on the test bacterium escherichia coli ATCC 8739; inoculum: effective inoculum bacterial count for each test batch: 860 CFU-430 CFU/ml batch of culture medium

Growth of test bacteria in a nutrient medium preparation with a macroscopic degree of miscibility

Product turbidity without macroscopic test bacteria growth

Subculture (SC) by streaking 0.1ml of each nutrient medium preparation

Table 14: results of the antimicrobial efficacy of olea europea suspension against the test strain escherichia coli DSM 22312; inoculum: effective inoculum bacterial count for each test batch: 670CFU 435CFU/ml culture medium batch

Growth of test bacteria in a nutrient medium preparation with a macroscopic degree of miscibility

Product turbidity without macroscopic test bacteria growth

Subculture (SC) by streaking 0.1ml of each nutrient medium preparation

Table 15: results of antimicrobial efficacy of trifolium oleander suspension against the test bacterium staphylococcus aureus ATCC 6538; inoculum: effective inoculum bacterial count for each test batch: 680 CFU-340 CFU/ml batch of culture medium

Growth of test bacteria in a nutrient medium preparation with a macroscopic degree of mixability

Product turbidity without macroscopic test bacteria growth

Subculture (SC) by streaking 0.1ml of each nutrient medium preparation

Table 16: results of the antimicrobial efficacy of the olea europaea suspension on the test bacterium staphylococcus aureus subspecies aurantiaca ATCC 29213; inoculum: effective inoculum bacterial count for each test batch: 2020 CFU-1010 CFU/ml culture medium batch

Growth of test bacteria in a nutrient medium preparation with a macroscopic degree of miscibility

Product turbidity without macroscopic test bacteria growth

Subculture (SC) by streaking 0.1ml of each nutrient medium preparation

For a study of two other test bacteria, acinetobacter baumannii and aspergillus brasiliensis, the Kolliphor RH40 content of the stock solution was reduced by 50%, i.e. from 4.2% to 2.1%. In addition to original stock A1 with a concentration of 127.5mg/ml of the extract of trifolium oleamen, a 1: 2 dilution was made with water for injection, so that the extract of trifolium oleamen had a concentration of 63.8mg/ml (stock A2).

Table 17: results of the antimicrobial efficacy of trifolium oleae suspension on the test bacterium acinetobacter baumannii ATCC 19606; inoculum: effective inoculum bacterial count for each test batch: 4000 CFU-2000 CFU/ml batch of culture medium

Growth of test bacteria in a nutrient medium preparation with a macroscopic degree of miscibility

Product turbidity without macroscopic test bacteria growth

Subculture (SC) by streaking 0.1ml of each nutrient medium preparation

Table 18: results of the antimicrobial efficacy of olea europaea suspension on the test bacterium acinetobacter baumannii NCTC 13420; inoculum: effective inoculum bacterial count for each test batch: 890 CFU-445 CFU/ml batch of culture medium

Growth of test bacteria in a nutrient medium preparation with a macroscopic degree of miscibility

Product turbidity without macroscopic test bacteria growth

Subculture (SC) by streaking 0.1ml of each nutrient medium preparation

Table 19: results of the antimicrobial efficacy of the olea europaea suspension against the test bacterium aspergillus brasiliensis ATCC 16404; inoculum: effective inoculum bacterial count for each test batch: culture medium batch of 310CFU/ml 620CFU

Growth of test bacteria in a nutrient medium preparation with a macroscopic degree of miscibility

Product turbidity without macroscopic test bacteria growth

Subculture (SC) by streaking 0.1ml of each nutrient medium preparation

As shown in the test, after the corresponding passages, no growth of the test bacteria was observed after 72 hours of incubation of the nutrient medium preparation, except for the test bacteria aspergillus brasiliensis ATCC 16404. Thus, it can be shown that trifolium extract has good antimicrobial efficacy at concentrations of 62.5mg/ml or 63.8mg/ml, respectively, under the conditions tested. For the test strain aspergillus brasileri, some isolated viable spores were still detectable during the test in the presence of the olea europaea suspension in stock solutions a1 and a2, but the reduction in CFU was also evident.

Example 6: study of antimicrobial efficacy of trifoliate or wild trifoliate jelly

Executing the following steps: colloidal gel containing Kelcogel CG-HA is prepared from leaf powder of Olea europaea. For this purpose, 0.1g of Kelcogel CG-HA was dissolved in 100ml of WFI by heating to 85-90 deg.C, after which 15g of Daghestan olive leaf powder was added and stirred until a visually homogeneous suspension was formed. Cooling in ice bath to obtain Daghestan sweet cloverLeaf powder colloidal gel. The adjusted test bacterium suspension (10)⁵-10⁶CFU/g) were inoculated into 10g aliquots of the gel and the number of microorganisms was tested according to the test times listed in table 20.

Table 20: CFU value according to test time

After 12 minutes, very good efficacy was shown for E.coli (T)₀h-value), staphylococcus aureus and pseudomonas aeruginosa below the detection limit of 100CFU per gram and showed overall very good antimicrobial efficacy over a short exposure period.

Example 7: study of antimicrobial efficacy of gel of olea europaea or wild olea europaea

Executing: colloidal gel containing Kelcogel CG-HA was prepared from olive leaf powder of trifolium oleae. For this purpose, 0.1g of Kelcogel CG-HA was dissolved in 100ml of WFI by heating to 85-90 deg.C, after which 15g of olea europaea leaf powder was added and stirred until a visually homogeneous suspension was formed. After cooling in an ice bath, a powdered colloidal gel of trifolium oleae leaves was obtained. The adjusted test bacterium suspension (10)⁵-10⁶CFU/g) were inoculated into 10g aliquots of the gel and tested for microbial numbers according to the test times listed in table 21.

Table 21: CFU value according to test time

Thus, very good efficacy was shown against E.coli after 6 minutes, and S.aureus was up to below the detection limit of 100CFU per gram after 12 minutes. For pseudomonas aeruginosa, after 30 minutes the bacterial load dropped below the test line and showed overall very good antimicrobial efficacy in a shorter exposure period.

Example 8: study of antimicrobial efficacy of composition dough of trifoliate olive or wild trifoliate olive

Executing the following steps: for each test, a dough piece was prepared by mixing 500mg of the sample with 1ml of WFI. After homogenization, 0.5ml of the adjusted suspension was added and mixed uniformly into the dough. Thereafter, both test strains showed very good efficacy after 6 hours reaction time at room temperature for untreated, steam sterilized powdered leaf of trifolium oleae at 121 ℃ for 15 minutes and 130 ℃ for 30 minutes.

Table 22: CFU value according to test time

Untreated leaf pieces of trifolium oleander showed 1.4X 10³CFU-2.1×10³Natural contamination on the order of CFU per 500mg of dough involving a variety of aerobic sporulating bacteria, which could not be killed during the 6 and 24 hour test periods, respectively. In the test specimen sterilized at 121 ℃ for 15 minutes, the face piece showed 1.1X 10³CFU-2.2×10²CFU per 500mg range of natural contamination. Both sterilization test specimens were sterilized at 130 ℃ for 30 minutes, and no contaminating bacteria were detected.

Example 9: study of antimicrobial efficacy of composition dough of trifoliate olive or wild trifoliate olive

Executing: for each test of each test strain, a dough piece was prepared by mixing 500mg of the olea europaea material with 1ml of WFI. After homogenization, 0.5ml of the adjusted suspension was added and mixed uniformly into the dough. Thereafter, for untreated, steam sterilized trifolium leaves at 121 ℃ for 15 minutes and 130 ℃ for 30 minutes, both test strains showed very good efficacy after 24 hours reaction time at room temperature (Table 23).

Table 23: CFU value according to test time

Untreated leaf pieces of trifolium oleander showed 2.9X 10⁴CFU's are on the order of 500mg of flour pieces that are not killed during the 24 hour test period due to natural contamination involving a variety of aerobic spore forming bacteria. No contaminating bacteria were detected in the sterilized test samples.

Example 10: study of antimicrobial efficacy of composition dough of trifoliate olive or wild trifoliate olive

Executing: for each test of each test strain, a dough piece was prepared by mixing 500mg of the olea europaea material with 1ml of WFI. After homogenization, 0.5ml of the adjusted suspension was added and mixed uniformly into the dough. Thereafter, both test bacteria showed very good efficacy already after 24 hours reaction time at room temperature for untreated stem bark/line powder preparations and steam-sterilized at 121 ℃ for 15 minutes and 130 ℃ for 30 minutes (Table 24).

Table 24: CFU value according to test time

The untreated stem bark/line in powder form showed 4.4 × 10²CFU of the order of magnitude of 500mg of flour pieceRelates to natural contamination by a number of aerobic spore forming bacteria, which cannot be killed during a 24 hour test period. In the test specimen sterilized at 121 ℃, 1.1X 10 was still detectable²CFU/piece of dough contamination. The test specimens were also sterilized at 121 ℃. In the test samples sterilized at 130 ℃, no contaminating bacteria were detected.

Example 11: study of antimicrobial efficacy of composition dough of trifoliate olive or wild trifoliate olive

Executing: for each test of each test strain, a dough piece was prepared by mixing 500mg of the olea europaea material with 1ml of WFI. After homogenization, 0.5ml of the adjusted suspension was added and mixed uniformly into the dough. Thereafter, both test bacteria showed very good efficacy already after 24 hours reaction time at room temperature for untreated stem bark/root powder preparations and steam-sterilized at 121 ℃ for 15 minutes and 130 ℃ for 30 minutes (Table 25).

Table 25: CFU value according to test time

The untreated bark/root of the stem in powder form showed 1.1X 10⁶CFU's are on the order of 500mg of flour pieces of natural contamination involving a variety of aerobic sporulating bacteria that cannot be killed during the 24 hour test period. In test specimens sterilized at 121 ℃ and 130 ℃, 1.8X 10 can still be detected³CFU/flour block and 8.8 × 10²CFU/face piece contamination.

Example 12: antimicrobial efficacy assay of hydroxytyrosol (single species) against Staphylococcus aureus ATCC 6538

Hydroxytyrosol is found in substrates and extracts from olive tree.

A stock solution was prepared by adding 0.1ml of methanol to 10.48mg of hydroxytyrosol. After dissolution of the compound, the solution was quantitatively transferred to 4.9ml of water for injection (WFI), effective concentration of hydroxytyrosol: 2.0 mg/ml.

Using the stock solution, hydroxytyrosol was detected in 5 geometric concentrations from 2mg to 0.125mg hydroxytyrosol/ml in a quantitative suspension assay.

2ml of stock and diluent (V1-V4), respectively, and 2% methanol as a positive control were inoculated with 0.1ml of Staphylococcus aureus ATCC 6538 test suspension adjusted to about 2.0X 10⁵CFU/0.1ml, about 100.000CFU/ml in each assay.

After inoculation and incubation at 20-25 ℃, samples (0.1ml) were taken at t0, 12, 30, and 60 minutes and at t24, 48, and 72 hours and diluted with 0.9ml of 0.9% NaCl solution. Samples were plated on blood agar plates and incubated at 30-35 ℃ for up to 5 days.

The results are shown in table 26 below.

Table 26: a CFU value; initial cell mass: 1.7X 10⁵CFU/ml, bacteria: staphylococcus aureus ATCC 6538, Hyd ═ hydroxytyrosol

As shown in table 26, 2% methanol had no significant antimicrobial effect at the beginning of the test. The decrease at the later time point is due to the natural loss of bacterial activity. Hydroxytyrosol showed a significant effect only after 24 hours, however, the effect was strong, i.e. below the detection limit of 100CFU/ml of the detection. Additional dilution further delays the effect.

Example 13: antimicrobial efficacy of olive oil stimulating aldehyde (singlet) against Staphylococcus aureus ATCC 6538 Analysis of

Olive oil was found to stimulate aldehydes in substrates and extracts from trifolium oleae.

Stock solutions of 2mg/ml olive oil stimulating aldehyde concentration were prepared. The compound was dissolved in ethanol (99.8%) and WFI was added.

Using this stock solution, a geometric series concentration of 1mg to 0.125mg olive oil stimulating aldehyde/ml was detected in a quantitative suspension assay.

2ml of stock and diluent (V1 to V4), respectively, and 2.85% ethanol solution as a positive control were inoculated with 0.055ml of a test suspension of Staphylococcus aureus ATCC 6538 and adjusted to about 3.6X 10⁵CFU/0.1ml, about 99.000CFU/ml in each assay.

After inoculation and incubation at 20-25 ℃, samples (0.1ml) were taken at t0, 12, 30, and 60 minutes and at t24, 48, and 72 hours and diluted with 0.9ml of 0.9% NaCl solution. Samples were plated on blood agar plates and incubated at 30-35 ℃ for up to 5 days.

The results are shown in table 27 below.

Table 27: a CFU value; initial cell mass: 9.9X 10⁴CFU/ml, bacteria: staphylococcus aureus ATCC 6538, Ole ═ olive oil stimulating aldehyde

As can be seen from table 27, 3.84% ethanol had no significant antimicrobial effect at the beginning of the test. The decrease at the later time point is due to the natural loss of bacterial activity. Olive oil stimulated aldehyde showed a fast onset of effect at higher concentrations, at the detection limit of 100CFU/ml detected. Dilution delays these effects, however, effects can still be seen.

Example 14: antimicrobial activity of olive secoiridoids (mono-species) against staphylococcus aureus ATCC 6538 Effect analysis

Olive secoiridoids are found in substrates and extracts from the olive tree, Olea europaea.

Stock solutions of 1mg/ml olive secoiridoid concentration were prepared. The compound was dissolved in ethanol (99.8%) and WFI was added.

Using this stock solution, olive secoiridoid was detected in a quantitative suspension assay at concentrations of 0.25mg olive secoiridoid/ml and 0.125mg olive secoiridoid/ml.

2ml of stock and diluent (V1 and V2), respectively, and 0.44% ethanol solution as a positive control were inoculated with 0.055ml of a test suspension of Staphylococcus aureus ATCC 6538 adjusted to about 3.5X 10⁵CFU/0.1ml, about 99.000CFU/ml in each assay.

After inoculation and incubation at 20-25 ℃, samples (0.1ml) were taken at t0, 12, 30, and 60 minutes and at t24, 48, and 72 hours and diluted with 0.9ml of 0.9% NaCl solution. Samples were plated on blood agar plates and incubated at 30-35 ℃ for up to 5 days.

The results are shown in table 28 below.

Table 28: a CFU value; initial cell mass: 9.9X 10⁴CFU/ml, bacteria: staphylococcus aureus ATCC 6538, Oci ═ Olive secoiridoid

As can be seen from table 28, 0.44% ethanol had no significant antimicrobial effect at the beginning of the test. The decrease at later time points is due to a natural loss of bacterial activity. The olive secoiridoid showed a faster onset of effect at the tested concentrations, at the detection limit of 100CFU/ml of detection.

Example 15: antimicrobial efficacy analysis of hair-blackening alcohol (single substance) against Staphylococcus aureus ATCC 6538

Hair blackening alcohols are found in substrates and extracts from olive tree.

A stock solution was prepared by adding 0.1ml of chloroform to 25mg of hair-blackening alcohol. After dissolution of the compound, the solution was quantitatively transferred to 12.4ml of water for injection (WFI), effective concentration of hair-blackening alcohol: 2.0 mg/ml.

Using the stock solution, five (5) geometric concentrations of hair-blackening alcohol ranging from 2mg hair-blackening alcohol/ml to 0.125mg hair-blackening alcohol/ml were tested in a quantitative suspension assay.

2ml of stock and diluent (V1-V4), respectively, and 0.8% chloroform solution as a positive control were inoculated with 0.1ml of a test suspension of Staphylococcus aureus ATCC 6538 and adjusted to about 2.0X 10⁵CFU/0.1ml, about 100.000CFU/ml in each assay.

After inoculation and incubation at 20-25 ℃, samples (0.1ml) were taken at t0, 12, 30, and 60 minutes and at t24, 48, and 72 hours and diluted with 0.9ml of 0.9% NaCl solution. Samples were plated on blood agar plates and incubated at 30-35 ℃ for up to 5 days.

The results are shown in table 29 below.

Table 29: a CFU value; initial cell mass: 2.0X 10⁵CFU/ml, bacteria: staphylococcus aureus ATCC 6538, Uva ═ hair-blackening alcohol

As can be seen from table 29, 0.8% chloroform had no significant antimicrobial effect during the test. After incubation at a concentration of 0.25Uva/ml or higher for longer than 24 hours, the hair-blackening alcohol showed an effect, at the detection limit of 100CFU/ml detected.

Example 16: antimicrobial efficacy analysis of high-root diols (single species) against staphylococcus aureus ATCC 6538

High root diols are found in substrates and extracts from trifolium oleae.

Stock solutions were prepared by adding 0.1ml of chloroform to 10.45mg of higher diol. After dissolution of the compound, the solution was quantitatively transferred to 4.9ml of water for injection (WFI), effective concentration of homodiol: 2.0 mg/ml.

Five (5) geometric concentrations of higher glycols ranging from 2mg higher glycols/ml to 0.125mg higher glycols/ml were tested in a quantitative suspension assay using the stock solution.

2ml of stock and diluent (V1-V4), respectively, and 2% chloroform solution as a positive control were inoculated with 0.1ml of a test suspension of Staphylococcus aureus ATCC 6538 adjusted to about 3.6X 10⁵CFU/0.1ml, about 100.000CFU/ml in each assay.

After inoculation and incubation at 20-25 ℃, samples (0.1ml) were taken at t0, 12, 30, and 60 minutes and at t24, 48, and 72 hours and diluted with 0.9ml of 0.9% NaCl solution. Samples were plated on blood agar plates and incubated at 30-35 ℃ for up to 5 days.

The results are shown in table 30 below.

Table 30: a CFU value; initial cell mass: 9.5X 10⁴CFU/ml, bacteria: staphylococcus aureus ATCC 6538, Ery ═ high root diol

As can be seen from table 30, 2% chloroform had no significant antimicrobial effect at the beginning of the test. The decrease at later time points is due to a natural loss of bacterial activity. Higher concentrations of higher diols showed stronger effects after incubation at a concentration of 2.0Ery/ml, with lower concentrations showing bacterial recurrence or no effect at later stages. It is likely that the poor effect compared to olive secoiridoid or olive oil stimulated aldehydes is caused at least in part by the lower solubility of higher diols, as encountered during the test.

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Claims (15)

1. A process for preparing a composition of trifoliate olive (Olea europaea), preferably wild Olea europaea (Olea europaea forest variety), comprising the steps of:

a) providing parts of an olive plant or a wild olive plant; and

b) removing water from parts of trifoliate olive or wild trifoliate olive by dehydrating or drying the parts of trifoliate olive or wild trifoliate olive; and

c) crushing the parts of the trifoliate olive or the wild trifoliate olive of the step b) by cutting, grinding, milling, crushing or other methods for crushing the parts of the trifoliate olive or the wild trifoliate olive to obtain powdered trifoliate olive or the wild trifoliate olive;

d) sterilizing the powdered or wild olive suitably, preferably at 121 ℃ or 134 ℃, to obtain sterilized powdered or wild olive; and, optionally,

e) mixing the sterilized powdered or wild olive with water and thickener to obtain olive or wild olive jelly with antibiotic or antifungal activity; or alternatively

d') by extraction, preferably with CO₂Extracting, namely extracting an active agent with antibiotic or antifungal activity from the powdery olea europaea or the wild olea europaea in the step c); suspending the active agent in a non-toxic solubilizer and emulsifier to obtain a suspension of trifoliate olive or wild trifoliate olive having antibiotic or antifungal activity.

2. A process as claimed in claim 1 wherein steps b) and c) are carried out simultaneously by subjecting the trifoliate or wild trifoliate to forces generated by a dynamic gas flow, preferably by passing the trifoliate or wild trifoliate through a venturi nozzle together with air, preferably preheated air.

3. The method according to any one of claims 1-2, wherein the thickening agent is selected from gelling agents, such as gellan gum, and/or wherein the non-toxic solubilizing agent and/or emulsifier is selected from surfactants.

4. A process as claimed in any one of claims 1 to 3 wherein the part of trifolium oleraceum or wild trifolium oleraceum is selected from leaves, branches, twigs, root bark, stem bark, fruits, or oil preparation residues, or combinations thereof.

5. The method of any one of claims 1-4, wherein the active agent comprises an iridoid or a phenolic, and at least one organic compound selected from oleuropein, olive secoiridoid, olive oil stimulatory aldehyde, tyrosol, hydroxytyrosol, hair blackening alcohol, and high root diol.

6. An antibacterial or antifungal composition comprises at least one organic compound selected from the group consisting of olive secoiridoid, hydroxytyrosol, olive oil stimulating aldehyde, hair blackening alcohol, and high root diol.

7. The composition according to claim 6 for use in the prevention or treatment of a bacterial or fungal infection in a mammal, preferably for use in the prevention or treatment of an infection caused by an antibiotic-resistant bacterium or a multi-resistant bacterium.

8. A sterilized powdered or wild olive of Oleaceae having antibiotic or antifungal activity obtainable by steps a), b), c) and d) of the process according to claim 1.

9. A sterilized powdered wild olive having antibiotic or antifungal activity as claimed in claim 8 wherein the powdered or wild olive is a powder and consists of more than 95% by weight dry biomass.

10. A composition comprising trifolium oleraceum or wild trifolium oleraceum prepared according to any one of claims 1 to 5, optionally together with a pharmaceutically or cosmetically acceptable carrier, diluent or excipient, wherein preferably the composition is a gel, preferably a homogeneous colloidal gel, and wherein preferably the suspension of trifolium oleraceum or wild trifolium oleraceum is an aqueous suspension.

11. An trifolium or wild trifolium composition according to claim 10 wherein the composition is a suspension and wherein the amount of non-toxic solubilising agent is less than 10%, preferably less than 5%, more preferably less than 3% of the total volume of the trifolium or wild trifolium suspension.

12. The composition according to any one of claims 10-11, in the form of an ointment, lotion, cream, spray, gel, liquid, drops, capsule, or suppository.

13. A sterilized powdered or wild olive as claimed in claim 8 or claim 9, a composition as claimed in any one of claims 10 to 12, for the prevention or treatment of a bacterial or fungal infection in a mammal, preferably an infection caused by an antibiotic-resistant bacterium or a multi-resistant bacterium, wherein preferably a) the bacteria are selected from the group consisting of escherichia coli, staphylococcus aureus, pseudomonas aeruginosa, klebsiella pneumoniae, mycobacterium tuberculosis, enterococcus hirae, enterococcus faecalis, enterobacter, serratia, proteus, paphiaceae, morganella, enterococcus faecium, helicobacter pylori, campylobacter, salmonella, neisseria gonorrhoeae, pneumococcus, haemophilus influenzae, shigella, acinetobacter baumannii, and resistant and multidrug-resistant strains thereof, and wherein b) the fungi are selected from the group consisting of candida albicans and aspergillus brasiliensis.

14. Use of sterilized powdered or wild olive as claimed in claim 8 or 9 or a composition as claimed in any one of claims 10 to 12 as a functional food additive, especially an animal feed additive.

15. Use of sterilized powdered or wild olive as claimed in claim 8 or 9 or a composition as claimed in any one of claims 10 to 12 in a cosmetic product.

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