Extract of herbal composition as antimicrobial and / or anti-biofilm agent

Abstract

Described herein is an extract of a herbal composition comprising at least two different dry plants, the compounds are useful as antimicrobial and / or antibiotic film agents in the treatment or prevention of microbial infections caused by bacteria (e.g., Escherichia, Klebsiella, Listeria, Pseudomonas, Salmonella, Streptococcus, or Staphylococcus) or by fungi, as well as in the treatment or prevention of microbial infections caused by bacteria (e.g., Escherichia, Klebsiella, Listeria, Pseudomonas, Salmonella, Streptococcus, or Staphylococcus). It has been found that in such an extract, the active ingredients exert their biological effects in a synergistic manner. The extracts may form active ingredients of food supplements, nutraceuticals, pharmaceutical or cosmetic compositions or functional foods or food additives. Also described herein are methods for preparing the extracts.

Description

[0001] This application is a divisional application of the invention patent 201780067636.X of the same name filed on November 1, 2017.

Technical field

[0002] The present invention relates to an extract of a herbal composition comprising at least two different dried plants, which can be used as an antimicrobial and / or anti-biofilm agent. It has also been found that in such extracts, the active ingredients exert their biological effects in a synergistic manner. [Background Technology]

[0003] Like any living organism, plants contain many chemical entities, including low molecular weight chemicals, peptides, polysaccharides, and enzymes. Therefore, plant extracts are simply examples of mixtures of naturally occurring chemical entities.

[0004] The biological and therapeutic activities of the many mixtures of chemical entities obtained from plants are secular knowledge and are compiled in many manuscripts (see, for example, Andrew Chevallier, "Encyclopedia of Herbal Medicine: The Definitive Home Reference Guide to 550 Key Herbs with all their Uses as Remedies for Common Ailments Hardcover" - November 15, 2000).

[0005] Modern science allows observing the biological and therapeutic effects of combinations of chemical entities obtained from plants on a large number of organisms.

[0006] For example, Scheggi (Scheggi S. et al., Pharm. Biol. 2016 Jan 5: 1-11. [Epub ahead of print]) describes how a rat model allows testing the effects of combinations of chemical entities obtained from plants against depression.

[0007] Zhipeng QU (Oncotarget, September 1, 2016, electronic version) demonstrates how it is possible to use functional genomics to identify candidate anticancer molecular mechanisms of compound sophora flavescens injection. Compound sophora flavescens injection (CKI) has been used clinically in China for over 15 years for the treatment of various types of solid tumors. However, because this type of traditional Chinese medicine (TCM) preparation is a complex mixture of plant secondary metabolites, its underlying molecular mechanisms must be explored in a systematic manner. This study demonstrates that CKI inhibits MCF-7 cell proliferation and induces apoptosis in a dose-dependent manner. Multiple pathways were found to be perturbed, and the cell cycle was identified as a potential major target pathway of CKI in MCF-7 cells. CKI can also induce apoptosis in MCF-7 cells through a p53-independent mechanism. In addition, they identified novel lncRNAs and showed that many of them are expressed in response to CKI treatment.

[0008] The effect of mixtures of chemical entities obtained, for example, from plants, fungi or bacteria, on the growth of microorganisms such as bacteria and fungi has been described several times, for example in Venkatadri (Venkatadri B. et al., Indian J. Pharm. Sci. 2015 Nov-Dec; 77(6):788-91).

[0009] The influence of combinations of chemical entities obtained, for example, from plants, fungi or bacteria, on biofilm formation (microbial communities embedded in a polymer matrix attached to a surface) has been described many times, for example in Süntar (Süntar I. et al., Pharmaceutical Biol. 2016 Jun;54(6):1065-70. Epub 2015 Oct 29).

[0010] Shimizu M. et al. (Antimicrob Agents Chemother. 2001 Nov;45(11):3198-201) found that extracts of the bearberry tree (Arctostaphylos uva-ursi) significantly reduced the MIC (minimum inhibitory concentration) of β-lactam antibiotics (such as oxacillin and cefmetazole) against methicillin-resistant Staphylococcus aureus. The same group also isolated an effective compound and identified it as corilagin.

[0011] Another group (Denev P. et al., Acta Biochim. Pol. 2014;61(2):359-67. Epub 2014 Jun 18) investigated the antioxidant, antimicrobial, and neutrophil modulating activities of extracts from six medicinal plants—blackberry (Rubus fruticosus) leaves, aronia (Aronia melanocarpa) leaves, hawthorn (Crataegus monogyna) leaves, lady's mantle (Alchemilla glabra) aerial parts, meadowsweet (Filipendula ulmaria) aerial parts, and raspberry (Rubus idaeus) leaves. The antimicrobial activity of the investigated extracts against 11 human pathogens was studied using three different methods, and the results of this study allowed to conclude that meadowsweet and blackberry leaf extracts had the highest antimicrobial effect and the lowest minimum inhibitory concentrations (MICs) against the tested microorganisms.

[0012] Yam T. et al. (FEMS Microbiol Lett. 1997 Jul 1;152(1):169-74) focused on the microbial activity of whole and fractionated crude extracts and tea components of tea (Camellia sinensis). Aqueous extracts of different types and origins of tea (Camellia sinensis) were tested and found to inhibit a variety of pathogenic bacteria, including methicillin-resistant Staphylococcus aureus. The tea extracts were bactericidal against Staphylococci and Yersinia enterocolitica at concentrations well below 'a cup of tea'. Activity was restricted to one of four fractions obtained from green tea extract by partition chromatography. Tests of pure tea compounds and closely related chemicals showed that the antimicrobial activity of green tea extract was explained by the content of epigallocatechin, epigallocatechin gallate, and epicatechin gallate. In black tea extract, theaflavins and their gallates are additional antimicrobial active components.

[0013] WO 2009 / 031041 reports a composition comprising (a) an antimicrobial compound having a given chemical formula, (b) an antimicrobial material selected from the group consisting of lanthionine bacteriocins, tea [Camellia sinensis] extract, hops [Humulus lupulus] extract, grape skin extract, grape seed extract, bearberry [Uva arctium] extract, and combinations thereof.

[0014] WO 99 / 20289 reports a clear herbal extract solution comprising a concentrated herbal extract and a filler solution. The concentrated herbal extract is reportedly selected from a long list of plant extracts, including Filipendula ulmoides, Camellia sinensis, and Uva ursi. However, WO 99 / 20289 does not mention any specific therapeutic or medical uses for the claimed herbal solution.

[0015] US 2015 / 0056255 reports a product for pharmaceutical, cosmetic, coloring or dermatological use, comprising a layer of a fibrous plant product and a plant extract coated on the fibrous plant product layer. The plant extract is said to comprise a substance from one or more specific parts of one or more plants, wherein the plants are selected from at least one of a long list of plants.

[0016] Azanida NN (Med. Aromat. Plants, 4:3, pp. 1–6, 2015) published a review describing and comparing the most commonly used methods for preparing extracts of medicinal plants. The authors concluded their article by stating that "no universal extraction method is ideal, and each extraction procedure is unique to the plant" (see "Conclusion," lines 5–7).

[0017] Selman A. Wachsman et al. (Bacteriology, 31, pp. 157-164, 1945) reported that different strains of the same bacterial species can have widely varying resistance to streptomycin. In this paper, different strains of Staphylococcus aureus were used to test the activity of streptomycin. However, plant extracts were not tested on the same strains, so no clear guidance can be drawn from this paper.

[0018] Biological effects such as those considered in the previous examples can be induced by a single chemical entity (as in a standard drug) or by a synergistic effect of different chemical entities (as in an extract). In some cases, the biological effect is almost entirely attributable to a single chemical entity contained in the extract. In such cases, the extract can be replaced with a purified active chemical entity.

[0019] The biological effect of a chemical entity can be due to a chemical reaction (transformation) with a chemical entity constituting an organism, or due to a transient or permanent binding to a chemical entity constituting an organism, in particular a macromolecule. Such binding modulates the activity of a chemical entity, in particular a macromolecule, in the organism, for example increasing or decreasing the activity of an enzyme.

[0020] Unfortunately, the effects of chemical entities on organisms are never singular. In particular, interactions with macromolecular components are numerous. Indeed, it is well known that any chemical entity, whether natural or chemically synthesized, can produce deleterious effects on organisms (humans, animals, plants, microbial flora, etc.) in a dose-related manner. Finally, dose-related positive or therapeutic effects can also be observed. Clearly, for chemical entities of interest to the industry, the dose required to observe a positive or therapeutic effect needs to be lower than the dose at which deleterious effects become unacceptable.

[0021] Chemical entities that differ in chemical composition or structure will have different affinities (or binding constants) for binding to the different macromolecules that make up an organism, and therefore different modes of action, leading to observed deleterious, beneficial, and therapeutic effects.

[0022] The macromolecules involved in the deleterious effects and the macromolecules involved in the positive or therapeutic effects are in many cases different. Chemical entities that differ in chemical composition will in most cases have very different affinities (or binding constants) for binding to the many different macromolecules that make up an organism.

[0023] In most cases, different chemical entities will have different affinities (or binding constants) for binding to a given macromolecule. In rare cases, different chemical entities will have similar affinities (or binding constants) for binding to the same macromolecule or group of macromolecules involved in a positive or therapeutic effect. In these cases, it will be rare that these different chemical entities will have comparable affinities (or binding constants) for binding to a macromolecule involved in a given adverse effect.

[0024] Thus, a mixture of different chemical entities can have a synergistic positive or therapeutic effect without a cumulative effect of a given adverse effect. Thus, for a given positive or therapeutic effect, the dose required for each individual different chemical entity in the mixture will be lower than the dose required for each chemical entity considered individually to achieve the given positive or therapeutic effect. In cases where the adverse effect induced by each different chemical compound is different or has a different macromolecular etiology, each adverse effect of the mixture will be lower than the adverse effect of the chemical entity considered individually because it is used at a lower dose.

[0025] Note that the synergistic situation of simultaneously administering different chemical entities with reduced doses is different from the co-administration of different drugs (chemical entities) with different therapeutic effects administered at standard doses. In the latter case, it is well known that the side effects caused by drug interactions increase. One of the causes of side effects is the possible chemical interactions of different drugs (chemical entities). In the synergistic situation, each chemical entity is present in a reduced concentration and therefore has a much lower chemical interaction rate. For illustrative purposes, each chemical entity in a synergistic mixture of 10 different chemical entities (drugs) is administered at 1 / 10 the concentration (dose) required for the chemical entity (drug) administered alone. As suggested by the universal law of mass action, the rate of chemical reaction between drugs will be reduced by 100 times.

[0026] Therefore, it is of interest to use synergistic mixtures of different chemical entities to achieve a given positive or therapeutic effect with reduced deleterious effects, or to achieve an increased positive or therapeutic effect at a given level of different deleterious effects. Indeed, several extracts are known to have synergistic activity relative to the isolated chemical entities (see, e.g., Deharo E. et al., Malaria J. 2011 Mar 15;10 Suppl 1:S5).

[0027] Natural extracts consisting of a mixture of synergistic chemical entities can be found, for example, by systematically testing many natural extracts of a single plant powder or of an herbal composition containing at least two or more different plant powders. [Specific implementation method]

[0028] It has now been discovered that extracts of herbal compositions comprising at least two dried plants selected from the group consisting of Filipendula ulmaria, Camellia sinensis, and Uva ursi are useful as antimicrobial and / or anti-biofilm agents in the treatment or prevention of microbial infections caused by bacteria (e.g., Escherichia, Klebsiella, Listeria, Pseudomonas, Salmonella, Streptococcus, or Staphylococcus) or fungi (e.g., Candida). It has also been discovered that in such extracts, the active ingredients exert their biological effects in a synergistic manner.

[0029] Therefore, an object of the present invention is a method for treating or preventing the above-mentioned microbial infections, said method comprising administering to a subject in need thereof an effective amount of an extract of a herbal composition comprising at least two dried plants selected from the group consisting of Filipendula ulmaria, Camellia sinensis and Uva ursi., a lyophilizate of said extract or a pharmaceutical or veterinary composition comprising said extract or said lyophilizate.

[0030] Another object of the present invention is an extract of a herbal composition comprising at least two dried plants selected from the group consisting of Filipendula ulmoides, Camellia sinensis and Uva ursi and at least one plant selected from the group consisting of Vitis vinifera var. tinctoria, Eugenia caryophyllus and Desmodium adscendens.

[0031] The herbal composition may further comprise one or more additional dried plants selected from the group consisting of:

[0032] Yarrow (Achillea millefolium), Sweet calamus (Acorus calamus), European agrimonia (Agrimonia eupatoria), Creeping ice grass (Agropyrum repens), Creeping ice grass, Alchemilla vulgaris, Alkanna tinctoria, Marshmallow (Althaea officinalis), Dill (Anethum graveolens), Angelica archangelica, Strawberry tree (Arbutus unedo), Arnica montana, Artemisia pontica, Artemisia vulgaris, Asparagus (Asparagus officinalis), Asparagus, Woodruff (Asperula odorata), Weeping birch (Betulapendula), Borage (Borrago officinalis), Buxus sempervirens, Calamintha officinalis, Calendula officinalis, Calluna avulgaris, Caraway (Carum carvi), Cassia angustifolia, Centaurea cyanus, Centaurium erythraea, Centella asiatica, Cetraria islandica, Roman chamomile (Chamaemelum nobile), German chamomile (Chamomilla recutita), Chrysanthellum americanum, Cichorium endivia, Chicory (Cichorium intybus), Ceylon cinnamon (Cinnamomum zeylanicum), Citrus aurantium, Combretum micranthum, Crataegus oxyacantha, Cuminum cyminum, Cupressus sempervirens), Curcumazedoaria, Cynara scolymus, Cytisusscoparius), cardamom (Elettaria cardamomum), Eleutherococcus senticosus, Epilobium parviflorum, Erysimum officinale, Eucalyptus globulus, Eupatorium cannabinum, Foeniculum vulgare, Fraxinus excelsior, Fucus vesiculosus, Fumaria officinalis, woodruff (Galium odoratum), Gentiana lutea, Geranium robertianum, Ginkgo biloba, Glechoma hederacea, Glycyrrhiza glabra, Handroanthus impetiginosus), Harpagophytum procumbens, Hieracium pilosella, Humulus lupulus, Hypericum perforatum, Hyssopus officinalis, Illicium verum, Inula helenium, Juglans regia, Juniperus communis, Lamium album, Lavandula angustifolia, Levisticum officinale, Lippia citriodora, Lotus corniculatus, Lythrum salicaria, Malvasylvestris, Marrubium vulgare, Medicago sativa, Melissa officinalis), Mentha × piperita, Morus nigra, Myrtle (Myrtus communis), Olive (Olea europaea), Marjoram (Origanummajorana), Panax ginseng, Papaver rhoeas, Parietaria officinalis, Passiflora incarnata, Petroselinum crispum, Peumusboldus, Phaseolus vulgaris, Pimpinella anisum, Plantago lanceolata, Plantago ovata, Potentilla anserina, Quercus robur, Rhamnus frangula, Rheum palmatum, Rosa centifolia, Rosmarinus officinalis, Rubia tinctorum, Raspberry, Salix alba, Salvia officinalis, Sambucus sambucus nigra), mountain savory (Satureja montana), milk thistle (Silybummarianum), white sage (Solanum dulcamara), variegated bellflower (Tabebuia impetiginosa), chrysanthemum wormwood (Tanacetum vulgare), dandelion (Taraxacum officinalis), red thyme (Thymus serpyllum), silver thyme (Thymus vulgaris), silver-haired linden (Tilia tomentosa), small-leaved linden (Tilia cordata), fenugreek (Trigonella foenum-graecum), coltsfoot (Tussilago farfara), bilberry (Vaccinium myrtillus), valerian (Valeriana officinalis), mullein (Verbascum thapsus), verbena (Verbena officinalis), white mistletoe (Viscum album), corn (Zea mays), and ginger (Zingiber officinale).

[0033] Another object of the present invention is an extract of a herbal composition comprising three to seven dried plants selected from the group consisting of Filipendula ulmaria, Camellia sinensis, Uva ursi, Rheum palmatum, Rosemary, Grape variegata, Desmodium variegata, Clove and Eucalyptus globulus, wherein at least two of such plants are selected from the group consisting of Filipendula ulmaria, Camellia sinensis and Uva ursi, and at least one of such plants is selected from the group consisting of Grape variegata, Desmodium variegata and Clove.

[0034] Another object of the present invention is an extract of a herbal composition comprising the dried plants of: Rosemary, Filipendula ulmoides, Camellia sinensis, Grapefruit and Uva ursi.

[0035] Another object of the present invention is an extract of a herbal composition comprising the dried plants of: Rheum palmatum, Filipendula ulmoides, Camellia sinensis, Grape hue and Uva ursi.

[0036] Another object of the present invention is an extract of a herbal composition comprising the following dried plants: Rheum palmatum, Rosemary, Filipendula ulmoides, Camellia sinensis, Grapefruit, Clove and Bearberry.

[0037] Another object of the present invention is an extract of a herbal composition comprising the following dried plants: Rheum palmatum, Rosemary, Filipendula ulmaria, Camellia sinensis, Grapefruit, Eucalyptus globulus, Bearberry, Spearmint and Rubia tinctorium.

[0038] Another object of the present invention is an extract of a herbal composition comprising at least fourteen or fifteen dried plants selected from the following sixteen: Rheum palmatum, Rosemary, Filipendula ulmoides, Wild savory, Valerian, Tea tree, Grape, Fucus vesiculosus, Fennel, Bearberry, Strawberry tree, Clove, Juniperus chinensis, Pinus parviflora, Lemongrass, and Artemisia selengensis.

[0039] Another object of the present invention is an extract of a herbal composition comprising the following dried plants: Rheum palmatum, Rosemary, Filipendula ulmoides, Wild savory, Valerian, Tea tree, Grape, Fucus vesiculosus, Fennel, Bearberry, Strawberry tree, Clove, Juniperus chinensis, Pinus parviflora and Lemongrass.

[0040] Another object of the present invention is an extract of a herbal composition comprising the following dried plants: Rheum palmatum, Rosemary, Filipendula ulmaria, Wild savory, Valerian, Tea tree, Grape, Fucus vesiculosus, Fennel, Bearberry, Strawberry tree, Clove, Juniperus chinensis, Pinus parviflora, Lemongrass, and Artemisia selengensis.

[0041] All the above-mentioned herbal compositions are also an object of the present invention as intermediates for the preparation of the corresponding extracts.

[0042] The extract may constitute the active ingredient of a food supplement, nutraceutical, pharmaceutical or cosmetic composition or a functional food or food additive.

[0043] The extract of the present invention can be used as such or in a dried or lyophilized form.

[0044] A food supplement, nutraceutical, pharmaceutical, veterinary or cosmetic composition or a functional food or food additive containing the extract defined above and suitable excipients is a further object of the present invention.

[0045] Such food supplements, nutraceuticals, pharmaceuticals, veterinary or cosmetic compositions or functional foods or food additives may be in any suitable form, e.g., a form suitable for oral administration, generally including solid forms such as powders, granules, capsules, pills, tablets (normal or controlled release), chewing gum; or liquid forms such as syrups, drops, elixirs, solutions and suspensions.

[0046] Furthermore, the extracts according to the present invention may be incorporated into other formulations such as creams, ointments, gels, emulsions, pastes, water-based creams, lotions, serums, masks, balms, fluids, sprays, suppositories, vaginal suppositories, skin patches and toothpastes, periodontal gels, mouthwashes.

[0047] According to the present invention, the expression "herbal composition" refers to a powder containing one or more different ground dried plants. The powder of a single dried plant can be obtained from the whole plant or in the form of a specific part thereof, such as flowers, buds, fruits, stems, leaves, seeds, roots or others.

[0048] According to the present invention, the extract can be obtained by simply incubating the herbal composition with the extraction solvent (with or without added sugar) at room temperature.

[0049] Suitable extraction solvents according to the present invention are water and all non-aqueous solvents authorized for use in the production of food and food ingredients. For example, according to Directive 2009 / 32 / EC, the list of such non-aqueous solvents includes propane, butane, ethyl acetate, ethanol, carbon dioxide, acetone, nitrous oxide. According to a particular embodiment of the present invention, the extraction solvent is selected from water, ethanol, ethyl acetate and any mixture thereof.

[0050] According to another alternative embodiment, the extract may be obtained by incubating the herbal composition with an extraction solvent (with or without added sugar) and subsequently heating (with or without centrifugation and filtration).

[0051] According to an alternative embodiment, the aqueous extract may be obtained by incubating the herbal composition with water (with or without added sugar) and subsequently heating (e.g. by heating the resulting liquid preparation at a temperature of 60°C to 134°C, preferably 119°C to 121°C, for a period of 5 to 60 minutes, preferably 20 to 30 minutes).

[0052] By the term "sugar" we mean herein pharmaceutical grade or nutritional grade sugars, such as dextrose, lactose, anhydrous lactose, mannitol, sorbitol, maltose, galactose or sucrose.

[0053] According to a particular embodiment of the invention, the sugar is sucrose.

[0054] The sugar may be used in a concentration of 1 to 100 g / l, optionally 50 to 75 g / l.

[0055] The term "food supplement" as used herein refers to a food product in unit dosage form intended to supplement the usual diet, which is a concentrated source of nutrients such as vitamins and minerals or other substances having a nutritional or physiological effect, (cf. Directive 2002 / 46 / EC of 10 June 2002.

[0056] The expression "functional food" is intended to include foods characterized by having additional effects due to the presence of naturally occurring or added components (usually non-nutrients), which more or less selectively interact with one or more physiological functions of the organism (bioregulation), thereby having a positive effect on maintaining health and / or preventing disease. If it is well documented that a food has a beneficial effect on one or more functions of the body in addition to sufficient nutritional effects, such as related to health and wellness or a reduced risk of disease, the food can be considered "functional". Beneficial effects can include maintaining a healthy state or health and / or reducing the risk of a disease process or disease (see AT Diplock et al.: Scientific concepts of functional foods in Europe: a consensus document, British Journal of Nutrition 1999, 81 (Supplement 1), S1-S27).

[0057] According to the present invention, the expression "nutraceutical composition" is intended to mean a product that not only supplements the diet but also contributes to the prevention and / or treatment of diseases and / or conditions. This expression was coined in 1989 by Stephen L. DeFelice, MD, founder and chairman of the Foundation for Innovation in Medicine (FIM), combining the concepts of "nutrition" and "pharmaceutical".

[0058] The term "excipient" as used herein refers to conventional excipients, ie compounds that are inert toward the active ingredients of the composition.

[0059] Excipients can be divided into various functional categories, depending on their role in the resulting formulation, for example, in solid dosage forms, they can act as diluents (e.g., lactose, microcrystalline cellulose), disintegrants (e.g., sodium starch glycolate, croscarmellose sodium), binders (e.g., PVP, HPMC), lubricants (e.g., magnesium stearate), glidants (e.g., colloidal SiO ), or flavorings (e.g., peppermint, lemon oil). In liquid dosage forms, they can act as solvents, cosolvents, or solubilizers (e.g., propylene glycol, glycerol, ethanol, sorbitol, dextrose), buffers (e.g., sodium phosphate), antimicrobials (e.g., potassium sorbate), wetting agents (e.g., polysorbates or Tweens), defoamers (e.g., polydimethylsiloxane-silicon dioxide), thickeners (e.g., methylcellulose or hydroxyethylcellulose), or sweeteners (e.g., sorbitol, acesulfame potassium).

[0060] As mentioned above, the extract of the present invention is an antimicrobial and / or antibiofilm agent and it is advantageously used in the treatment, prevention and / or diagnosis of microbial infections in humans and animals.

[0061] Therefore, another object of the present invention is the use of the extract of the present invention in the preparation of a medicament for the treatment, prevention or diagnosis of microbial infections. Such medicament can be used in animals or humans.

[0062] According to the present invention, the expression "treatment" intends activities designed to cure the disease or pathological condition, alleviate its symptoms or delay its progression.

[0063] The expression "prevention" is intended to mean activities designed to minimize the incidence or effects of a disease or pathological condition. For example, in the context of a microbial infection, prevention herein specifically refers to preventing pathogenic microorganisms from adhering to cells, tissues and organs and colonizing.

[0064] The expression "diagnostic" is intended to mean activities designed to identify a series of physiological parameters in order to assess the presence and extent of a defined disease or pathological condition.

[0065] The expression "antimicrobial agent" is used herein to mean an agent capable of inhibiting the growth of microbial cells. If the microbial cells are pathogenic microbial cells, the antimicrobial agent can be used as an active ingredient of a pharmaceutical or veterinary composition for treating microbial infections in humans or animals.

[0066] The expression "anti-biofilm agent" is used herein to refer to an agent capable of inhibiting the formation of biofilms by microbial cells and / or promoting the dispersion of pre-formed biofilms. Compared to conventional antimicrobial agents, anti-biofilm agents do not directly affect microbial survival and are therefore less likely to develop resistance to these agents.

[0067] According to the present invention, the term "synergistic" is intended to mean that the biological effect or activity (herein antimicrobial or antibiofilm) exhibited by the extracts of the herbal composition of the present invention (i.e. the herbal composition comprising at least two dried plants selected from Filipendula ulmaria, Camellia sinensis and Uva ursi) is greater than the sum of the effects or activities of the extracts of the corresponding single dried plants.

[0068] This greater biological effect or activity can be demonstrated, for example, in terms of inhibition of planktonic growth (antimicrobial activity), inhibition of biofilm formation (antibiofilm activity), or strain numbers (where one or both of these activities can be identified).

[0069] According to a specific embodiment of the present invention, the microbial infection is caused by bacteria, such as Escherichia coli, Klebsiella pneumoniae, Listeria innocua, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella enterica subsp. enterica serovar Enteritidis, Salmonella enterica subsp. enterica serovar Typhimurium, Streptococcus pneumoniae, Streptococcus equisubsp. zooepidemicus, Streptococcus pyogenes; or fungi, such as Candida albicans.

[0070] According to another embodiment of the present invention, the microbial infection is caused by Staphylococcus aureus, Staphylococcus epidermidis or Staphylococcus pseudintermedius.

[0071] Staphylococcus aureus, a commensal Gram-positive bacterium, is a major pathogen that can cause a wide range of human illnesses, from skin and soft tissue infections to severe sepsis, toxic shock, or pneumonia. S. aureus is a significant cause of hospital-associated infections, and many nosocomial strains of S. aureus have acquired resistance to antibiotics, particularly methicillin. While no vaccine is currently available, significant work has been conducted to develop active and passive immunotherapies against various targets in S. aureus.

[0072] In order to evaluate such biological activities of the extracts of the present invention and comparative samples, a number of different strains have been used and will be mentioned in the Examples section.

[0073] In particular, the following bacterial strains have been used to test the antimicrobial and antibiofilm activities of the extracts of the present invention.

[0074] ATCC 25923, also known as Seatle 1945, is a Staphylococcus aureus strain widely used in academic research (cited in 627 publications as of October 2016 (see NIH-NCBI-Pubmed)). Its antibiotic resistance behavior has been well characterized (see, for example, Reimer LG et al., Antimicrob Agents Chemother 1981 Jun;19(6):1050-5).

[0075] ATCC 29213 is also a widely used S. aureus strain in academic research (cited in 327 publications as of October 2016 (see NIH-NCBI-Pubmed)). It has been used in biofilm research (see, for example, Ceri H. et al., J. Clin. Microbiol. 1999 Jun; 37(6): 1771-6).

[0076] ATCC 1228 is a strain of Staphylococcus epidermidis (cited in 101 publications as of October 2016 (see NIH-NCBI-Pubmed)). The Federal Regulations (FDA) list this strain as a preferred analytical organism for neomycin (see Robertson JH et al., Appl. Microbiol. 1971 Dec;22(6):1164-5).

[0077] ATCC 43300 is a S. aureus strain isolated from clinical studies (cited in 81 publications as of October 2016 (see NIH-NCBI-Pubmed), including Lubenko et al., J. Antimicrob. Chemother. 2008 Nov;62(5):1065-9).

[0078] The Newman strain of Staphylococcus aureus was isolated from a human infection in 1952 and has been widely used in animal models of staphylococcal infection due to its potent virulence phenotype (see Baba T. et al., Journal of bacteriology, 190:300-310 (2008)). Compared with hospital-acquired MRSA (methicillin-resistant Staphylococcus aureus), Newman strain carries only a small number of insertion sequences (IS) and lacks known antibiotic resistance determinants.

[0079] The other strains used in the examples are also commonly used in academic research or industry for quality control (QC). Most of them are also methicillin-resistant.

[0080] For reference to ATCC 33591, see Reyes N. et al., J. Antimicrob. Chemother. 2006 Aug;58(2):462-5, epub 2006 May 30. For reference to ATCC 33592, see Teka A. et al., BMC Complement Altern Med. 2015 Aug 18;15:286. For reference to NCTC 12493, see Carey BE et al., J. Antimicrob. Chemother. 2006 Aug;58(2):455-7, epub 2006 Jun 20. For reference to ATCC 700698, see Kirker KR et al., Int. J. Antimicrob Agents, 2015 Oct;46(4):451-5, epub 2015 Jul 9. For reference to ATCC 700699, see Sy CL et al., J. Clin. Microbiol. 2016 Mar;54(3):565-8, epub 2015 Dec 16. For reference to ATCC 9144, see Carson CF et al., Antimicrob. Agents Chemother. 2002 Jun;46(6):1914-20. For references to ATCC BAA-44, see Monecke S. et al., PLoS One. 2011 Apr 6;6(4):e17936. For references to ATCC 14990, see Bernardo TH et al., Scientific World Journal. 2015;751791, electronic version 2015 Jun 3). For references to ATCC 49444, see Ramsey KJ et al., J Food Prot. (2010). For references to ATCC 60193, see Petrikaite V et al., Medicina (Kaunas). 2007;43(8):657-63. For references to ATCC 11775, see Usman H et al., Afr J Tradit Complement Altern Med. 2007 Jun 10;4(4):488-94.For references to ATCC 13883, see Supardy NA et al., J Microbiol Biotechnol. 2012 Jun;22(6):872-81. For references to ATCC 33090, see Le Marc Y et al., Int J Food Microbiol. 2002 Mar;73(2-3):219-37. For references to ATCC 19115, see Vodnar DC., Chem Cent J. 2012 Jul 28;6(1):74. doi:10.1186 / 1752-153X-6-74. For reference to ATCC 27853, see Reimer LG et al., Antimicrob Agents Chemother. 1981 Jun;19(6):1050-5. For reference to ATCC 13076, see Fioretto F et al., Braz J Med Biol Res. 2005 Aug;38(8):1259-65. Electronic version 2005 Jul 30. For reference to ATCC 13311, see Leguérinel I et al., Int J Food Microbiol. 2007 May 1;116(1):88-95. Electronic version 2007 Jan 13. For reference to ATCC 27336, see Chen S et al., Appl Environ Microbiol. 2013 Jul;79(13):4015-23. doi:10.1128 / AEM.00704-13. Epub 2013 Apr 19. For reference to ATCC 43079, see Farrow JA and Collins MD., Syst. Appl. Microbiol. 5:483-493, 1984. For reference to ATCC BAA-1323, see Svensson Mikael D et al., Microbiology 148:3933-3945, 2002. The food supplement, nutraceutical, pharmaceutical or cosmetic composition of the present invention can be appropriately formulated for different routes of administration. These routes of administration include: auricular, oral, skin, dental, nasal, ocular, buccal, oropharyngeal, rectal, respiratory (inhalation), sublingual, topical, or vaginal.

[0081] Typically, compositions comprising the extracts of the present invention are administered in a "therapeutically effective amount."

[0082] For the purposes of this specification and the appended claims, the term "comprising" also includes the terms "consisting essentially of" or "consisting of.

[0083] The actual amount of composition administered is generally determined by a physician based on the relevant circumstances, including the condition to be treated, the route of administration chosen, the actual composition administered, the age, weight, and response of the individual subject, the severity of the subject's symptoms, etc. For any composition, the therapeutically effective dose can be estimated initially in cell culture assays or in animal models (usually mice, rats, guinea pigs, rabbits, dogs, or pigs).

[0084] Animal models can also be used to determine suitable concentration ranges and routes of administration. This type of information can then be used to determine useful doses and routes for human administration. When calculating the human equivalent dose (HED), it is recommended to use the conversion table provided in the Guidance for Industry and Reviewers document (2005, U.S. Food and Drug Administration, Rockville, Maryland, USA).

[0085] According to the present invention, the dosage of the extract administered to humans can be adjusted with reference to the herbal composition used to prepare it, and therefore the amount of ground individual dried plants present in the herbal composition. Typically, 0.001 to 500 g of each ground dried plant is used to prepare the final extract, optionally 0.01 to 100 g. This extract can be used as is or in a lyophilized, dried, or freeze-dried form in a final composition for human or animal use, or can be further diluted.

[0086] The precise effective dose for human subjects will depend on the severity of the disease or condition, the general health of the subject, the age, weight and sex of the subject, diet, time and frequency of administration, other concurrent treatments, reaction sensitivity and tolerance / response to treatment. Such amounts can be determined by routine experimentation and are within the judgment of the clinician.

[0087] Dosage treatment may be a single dose regimen or a multiple dose regimen.

[0088] Another object of the present invention is a method for preparing the extract of the present invention. Such a process schematically comprises the following steps:

[0089] a) chopping or grinding separately or in a mixture of at least two single dried plants to obtain the corresponding plant powder;

[0090] b) mixing at least two different plant powders to obtain a herbal composition;

[0091] c) adding an extraction solvent to the herbal composition to obtain a corresponding liquid preparation (or extract);

[0092] d) incubating the liquid formulation at room temperature for 5 to 15 minutes; optionally,

[0093] e) heating the liquid formulation at a temperature of 60° C. to 134° C. for 5 to 60 minutes; optionally,

[0094] f) centrifuging, collecting the supernatant and filtering the collected supernatant; and, optionally,

[0095] g) concentrating and / or drying or freeze-drying the liquid preparation obtained from step e) or f).

[0096] According to the present application, the term "lyophilisate" refers to a freeze-dried (dehydrated) and dried extract according to the present invention.

[0097] Single dried plants are commercial products that can be purchased as part of a dried plant collection at a pharmacy or herbal shop. A list of dried plant collections is available at each pharmacy of your choice of herbal shop, and the dried plants available in these collections may vary from country to country.

[0098] For example, Belgium, France and Italy decided in 2012 to join forces to create a common list of plants that manufacturers can use as long as they meet the quality requirements of European law (EC Regulation 852 / 2004). Thanks to the hard work of three recognized experts (Robert Anton, Luke Delmulle and Mauro Serafini), the project created a list of 1,029 plants and 11 mushrooms (see

[0099] http: / / www.economie.gouv.fr / files / files / directions_services / dgccrf / imgs / breve / 2014 / documents / harmonized_list_Section_A.pdf)

[0100] The invention will now be described with reference to the following non-limiting examples.

[0101]

Example

[0102]

List of plants used in the example

[0103] The plants and plant parts in the following list have been used in different examples. For the sake of simplicity, each plant is identified by the code used in the example. The weight corresponding to 200 μl (the volume of the calibration spoon) of plant powder is also shown (Table 0-1). The dried plants of pharmaceutical grade PA00 to PB12 are obtained from "Pharmacie Fontgiève" (Clermont-Ferrand, France), and the dried plants of pharmaceutical grade PB13 to PD08 are obtained from "Pharmacie St Herem" (Clermont-Ferrand, France). When indicated in the examples, plants can also be obtained from "Herboristerie Cailleau" (Chemillé, France) or Siccarapam (Aubiat, France).

[0104] Table 0-1: List of plants with related codes and weight of one calibrated scoop of dry powder

[0105]

[0106]

[0107]

[0108]

[0109] [Method for preparing herbal composition (Method A)]

[0110] Each dried plant was ground using a coffee bean grinder (Model 8100, SEB, France). The resulting powder (plant powder) was sieved using a 2 mm sieve and stored in a 60 ml container (Labbox, France).

[0111] Mixtures of different ground dry plants (or plant powders) were prepared by collecting a fixed volume of plant powder using a calibrated spoon that allowed the collection of 200 μL of powder. The weight of the collected volume depended on the specific plant powder (see Table 0-1).

[0112] Such mixes or mixtures of different ground dry plants (or plant powders) as well as single dry plants are also referred to herein as "herbal compositions".

[0113] Method for preparing an aqueous extract (also referred to as a "processed sample," "extract," or "mixture extract" in this Examples section) (Method B)

[0114] These herbal compositions were placed in 50 ml falcon tubes (Falcon, USA) and added to 20 ml or 40 ml (as indicated in the examples) of a solution of 100 g / l digestive grade sucrose in digestive grade spring water (Volvic, France) (Daddy, France). The choice of digestive grade sucrose and water over their scientific grade counterparts was based on the aim of obtaining an edible composition that falls into the category of "condiments" and is intended for direct consumption according to Regulation (EC) No. 1334 / 2008 of 16 December 2008 on condiments and certain food ingredients having flavouring properties for use in and on foodstuffs.

[0115] Each falcon tube containing these liquid formulations was agitated for 10 seconds using a vortex mixer (ThermoFisher, USA), incubated at room temperature for 10 minutes, and placed in an autoclave (MED12, JPSelecta, Spain) and sterilized for 20 minutes at 119°C or 121°C, as indicated. After cooling, the sterilized formulations were kept at -20°C until use.

[0116] On the same day of operation, the preparation was thawed at 37°C. The preparation was centrifuged at 4000rpm for 10 minutes (Multifuge35-R, Heraeus, Germany) or incubated at room temperature to decant (as shown in the examples). 2mL supernatant was collected with a 10mL syringe and filtered using a 0.2μM filter (Minisart Syringe Filters, Sartorius, Germany) and collected on a 2mL Eppendorf tube. The remaining sterilized preparation was maintained at -20°C for further use.

[0117] The samples so obtained are also referred to herein as "processed samples" and will be identified by the same name or code given to the corresponding herbal composition (e.g., the name "Mixture\[2P]-1" will be used to identify the herbal composition and the corresponding extract (or processed sample) in which the biological activity is determined).

[0118] Method for determining antimicrobial and antibiofilm activity (Method C)

[0119] The biological activity of each processed sample was determined by using the strain indicated in each experiment (or example). For each measurement, each bacterium stored on cryobeads (Technical Service, UK) at -80°C was inoculated on TS agar plates (Tryptic Soy Broth, Sigma, USA; Agar, Fisherbrand, USA) and incubated overnight at 37°C. Several colonies were collected using an inoculating loop and suspended in saline solution. The optical density was measured and diluted in TS broth to obtain 2×10 5.5 bacteria / mL (for Staphylococcus aureus and Staphylococcus pseudintermedius strains), 2×10 5 The concentration of bacteria / mL (for Staphylococcus epidermidis) was determined.

[0120] The bioactivity of each processed sample was measured in 96-well plates using the strains indicated in each experiment (or example) and a control containing no bacteria. Each well contained 190 μL of TS broth, inoculated with 10 5.5 bacteria / mL, for Staphylococcus epidermidis, 10 5 Bacteria / mL (or the concentrations indicated in the Examples for all other species) or no bacteria for controls, and processed samples or negative controls (100 g / l sucrose solution without any plant matter) were diluted at 1:10, 1:20, 1:40 and 1:80 final concentrations or at 1:20, 1:63 and 1:200 (as indicated in the Examples).

[0121] Each plate was incubated at 37°C in an incubator (HettCube400, Hettich, Germany) for 17 hours.

[0122] The measurements were performed based on analysis of images of the flat plates obtained using a commercial document scanner (V220, Epson, Japan) using ImageJ software (NIH, USA) (any other equivalent software may also be used). Images of the flat plates located on the scanned area were acquired at 600 dpi using the software provided with the scanner. For each flat plate, three images were obtained: the first before incubation (T0), the second after 17 hours of incubation (Tf), and the third after crystal violet (CV) staining as described in the optimization of the microtiter plate assay, which is used to test biofilm formation ability in different Salmonella serotypes (Agarwal, RK et al., International Food Research Journal 18(4):1493-1498, 2011).

[0123] For each well of interest, an ImageJ macro was used to characterize a circular area (14, 14, and 76 pixels, respectively) relative to the diameter of the well set, based on the first, second, and third images, to calculate the mean values ​​M1 and M2 of 1% of the weak-intensity pixels in that area, and the mean value M3 of all pixels in that area, respectively. The turbidity of the bacterial solution in the well was deduced from M1 and M2 using a reference curve obtained in an independent experiment, in which the mean value of 1% of the weak-intensity pixels in a 14-pixel diameter area was measured from a well containing a bacterial solution of known turbidity. The optical density of the crystal violet solution in the well was deduced from M3 using a reference curve obtained in an independent experiment, in which the mean value of pixels in a 76-pixel diameter area was measured from a well containing a crystal violet solution of known optical density.

[0124] For each process sample and a given bacterium, the MIC was determined from the highest inhibitory dilution (HID) among the four tested (1:10, 1:20, 1:40, 1:80) process samples that prevented visible growth within 17 hours. When no dilution tested prevented bacterial growth, a "0" was assigned.

[0125] For each well containing processed sample and a given bacterium, the percent inhibition of bacterial planktonic growth was determined according to the following formula:

[0126]

[0127] Where Tf(processed sample) is the turbidity of the second image from the sample well, T0(processed sample) is the turbidity of the first image of the same well, Tf(negative control) is the turbidity of the second image from the well containing the same bacteria but without the processed sample, and T0(negative control) is the turbidity of the first image of the same well.

[0128] For each well containing a processed sample and a given bacterium, the percent inhibition of biofilm formation (IBF) was determined according to the following formula:

[0129]

[0130] Where CV(processed sample) is the OD from the third image of the sample well, CV(negative control) is the OD from the third image of the well containing the same bacteria but without the processed sample, and CV(control sample) is the OD from the third image of the well containing the same dilution of the same sample or negative control without any bacteria. A negative value of IBF means that the tested mixture promotes biofilm formation.

[0131]

Example 1

[0132] In this example, we demonstrate the simultaneous antimicrobial and antibiofilm activity of the disclosed combination of two of the three plants, Filipendula ulmoides, Camellia sinensis, and Uva ursi against Staphylococcus aureus. Pharmaceutical grade dried plants were obtained from "Herboristerie Cailleau" (Chemillae, France).

[0133] Starting from these three different plant powders, six herbal compositions were prepared according to the previously reported method A, and their ingredients are recorded in Table 1-1.

[0134] [Table 1-1: Herbal composition of processed samples]

[0135] Herbal composition PA13 (nb spoon) PA21(nb spoon) PB12 (nb spoon) PA13 1 PA21 1 PB12 1 Mixture [2P]-1 1 1 Mixture [2P]-2 1 1 Mixture [2P]-3 1 1

[0136] As shown in Table 1-1, the first three herbal compositions contained only a single plant powder, while the last three compositions contained two of the three different plant powders.

[0137] Starting from each of the herbal compositions reported in Table 1-1, the corresponding processed samples were prepared by water extraction using 20 mL of water containing 100 g / l sucrose according to previously reported method B, including a centrifugation step, followed by filtration.

[0138] The bacterial planktonic growth and anti-biofilm formation inhibition percentages of the processed samples of the two plant mixtures and the related isolated plants of this Example 1 at a 1:10 dilution were determined using the following Staphylococcus aureus strains: ATCC 25923 and ATCC 29213 according to the previously reported Method C and are given in Tables 1-2.

[0139] Table 1-2: Inhibition of planktonic growth and biofilm formation (IBF) of samples processed from a two-plant mixture and a single plant at a dilution of 1:10

[0140]

[0141]

[0142] PA13, PA21, and PB12 have known antibiotic effects. Limited inhibition of planktonic growth (<27%) and biofilm formation (<51%) was measured at a 1:10 dilution, while three aromatic formulations of the two plant mixture extracts showed antimicrobial effects (>66%) and antibiofilm effects (>78%) on the two test strains ATCC 25923 and ATCC 29213.

[0143] For all mixtures (Mixture [2P]-1, Mixture [2P]-2, Mixture [2P]-3), a synergistic effect of the two plants was observed with respect to the antimicrobial action of the two strains tested.

[0144] [Comparative Example 1]

[0145] As comparative examples, we illustrate the activity of some processed samples that have been found to have no or poor antimicrobial and antibiofilm activity against Staphylococcus aureus and that were obtained from corresponding herbal compositions containing powders of 2 different plants.

[0146] Five processed samples were prepared using two other plant powders, PA74 (blackcurrant fruit powder) and PC72 (Coleoptera: Camellia sinensis flower pollen).

[0147] Single dried plant powders were processed as described in Example 1 and five herbal compositions were prepared following the protocol reported in the previously described Method A. These five herbal compositions had the ingredients reported in Tables 1-3.

[0148] [Table 1-3: Herbal composition of processed samples]

[0149] Herbal composition PA74 (nb spoon) PC72 (nb spoon) PA13 (nb spoon) PA21(nb spoon) PA74 1 PC72 1 Mixture 1 1 1 Mixture 2 1 1 Mixture 3 1 1

[0150] Each herbal composition was processed according to the protocol reported in Method B for water extraction using 20 mL of water containing 100 g / l sucrose and including a centrifugation step followed by filtration to obtain the corresponding processed samples.

[0151] The bioactivity of each processed sample was determined by using the following Staphylococcus aureus strains ATCC 25923 and ATCC 29213 according to Method C described previously.

[0152] The percentage inhibition of bacterial planktonic growth and anti-biofilm formation of processed samples of the 2-plant mixture and the relevant isolated plants at a dilution of 1:10 are given in Tables 1-4.

[0153] Table 1-4: Inhibition of planktonic growth and biofilm formation (IBF) of samples processed from a two-plant mixture and a single plant at a 1:10 dilution

[0154]

[0155]

[0156] PC72 and PA74, as well as the related mixture 1, showed no antimicrobial activity and limited antibiofilm activity against both strains tested at a 1:10 dilution. In each case, no synergistic effect was observed.

[0157] For all mixtures containing PA13 and PA21 (mixtures 2 and 3), no synergistic effect was observed with respect to the antimicrobial action against the two strains tested at a 1:10 dilution when these mixtures also contained PC72 or PA74.

[0158] Notably, both Ribes nigra and Camellia sinensis are known to have antimicrobial properties.

[0159] This comparative example demonstrates that any random combination of single dried plants with known antimicrobial and / or antibiofilm activity did not result in an aqueous extract in which synergistic activity was observed.

[0160]

Example 2

[0161] In this example, we demonstrate the simultaneous antimicrobial and antibiofilm activities of different aqueous extracts obtained from a herbal composition containing two of the three plants reported in Example 1 against Staphylococcus aureus when these aqueous extracts were prepared according to different methods.

[0162] Pharmaceutical grade dried plants were obtained from "Herboristerie Cailleau" (Chemilles, France).

[0163] Twenty herbal compositions were prepared according to the previously reported method A, and their ingredients are recorded in Table 2-1.

[0164] [Table 2-1: Herbal composition of processed samples]

[0165] Herbal composition PA13 (nb spoon) PA21(nb spoon) PB12 (nb spoon) Mixture 1 1 1 Mixture 2 1 1

[0166] For each herbal composition, 20 liquid (water) formulations were prepared as follows:

[0167] The collected powder was placed in 20 50 ml falcon tubes (Falcon, USA). Under each condition, 20 ml of digestive grade spring water (Volvic, France) solution containing 0, 25 g / l, 50 g / l, 75 g / l or 100 g / l digestive grade sucrose (Daddy, France) was added to 4 flacon tubes. Each falcon tube containing this liquid preparation was stirred for 10 seconds using a vortex mixer (ThermoFisher, USA), incubated at room temperature for 10 minutes, and then 4 different heat treatments were applied to each condition:

[0168] -Cold infusion (do not warm before centrifugation and filtration):

[0169] After incubation at room temperature, the preparation was centrifuged at 4,000 rpm for 10 minutes (Multifuge 35-R, Heraeus, Germany), and 2 mL of the supernatant was collected with a 10 mL syringe and filtered using a 0.2 μM filter (Minisart Syringe Filters, Sartorius, Germany) into a 2 mL Eppendorf tube and kept at −20° C. until use. On the day of operation, the preparation was thawed at 37° C.

[0170] -Heat at 60°C for 30 minutes:

[0171] After incubation at room temperature, the preparation was placed in an autoclave (VWR, USA) and circulated at 60°C for 20 minutes. After cooling, the treated preparation was centrifuged at 4000 rpm for 10 minutes (Multifuge35-R, Heraeus, Germany), 2 mL of the supernatant was collected with a 10 mL syringe and filtered using a 0.2 μM filter (Minisart Syringe Filters, Sartorius, Germany), and collected on a 2 mL Eppendorf tube and kept at -20°C until use. On the day of operation, the preparation was thawed at 37°C.

[0172] -Heat at 119°C for 20 minutes:

[0173] After incubation at room temperature, the preparation was placed in an autoclave (VWR, USA) and sterilized at 119°C for 20 minutes. After cooling, the treated preparation was kept at -20°C until use. On the day of operation, the preparation was thawed at 37°C, centrifuged at 4000 rpm for 10 minutes (Multifuge35-R, Heraeus, Germany), and 1 mL of supernatant was collected with a 10 mL syringe and filtered using a 0.2 μM filter (Minisart Syringe Filters, Sartorius, Germany) and collected on a 2 mL Eppendorf tube.

[0174] These samples are referred to herein as "processed samples."

[0175] The bioactivity of each processed sample was determined by using Staphylococcus aureus strain ATCC 25923 according to Method C described above.

[0176] Table 2-2 shows the percentage of bacterial planktonic growth at a 1:10 dilution for 20 processed samples prepared starting from the herbal composition after each of the different heat treatments presented previously.

[0177] [Table 2-2: Planktonic growth of ATCC 25923 of 20 processed samples at a 1:10 dilution prepared from herbal compositions (Mixture 1 and Mixture 2 of Table 2-1)]

[0178]

[0179] All processed samples prepared from Mix 1 showed an antimicrobial effect against ATCC 25923 only after temperature treatment at 119°C, while Mix 2 showed limited antimicrobial effect after cold infusion of all sucrose concentrations (except the condition without sucrose) (31% to 52% inhibition) and after all tested temperature treatments (except the condition without added sucrose treated at 60°C) (>82% inhibition), with the processed samples showing only 16% inhibition of planktonic growth. Temperature treatment enhanced the antimicrobial effect of Mix 2. The best temperature treatment tested for both Mix 1 and Mix 2 was 119°C for 20 minutes.

[0180] The effect of sucrose concentration on the antimicrobial effect depended on the temperature treatment applied. For Mixture 1 treated at 119°C for 20 minutes, the same antimicrobial effect was achieved with 100, 75, 25, and 0 g / l of sucrose, with lower activity observed with 50 g / l. For Mixture 2, the same antimicrobial effect was achieved regardless of the sucrose concentration used for the 119°C temperature treatment.

[0181] In Tables 2-3 are given the percentage inhibition of biofilm formation (IBF) at 1:10 dilution for 20 processed samples prepared starting from the herbal composition after each of the different heat treatments presented previously.

[0182] [Table 2-3: Inhibition of biofilm formation (IBF) of ATCC 25923 in 20 processed samples prepared from herbal compositions (Mixture 1 and Mixture 2 of Table 2-1) at a dilution of 1:10]

[0183]

[0184]

[0185] Under all conditions tested, the processed samples showed some anti-biofilm effect.

[0186] For mixture 1, limited anti-biofilm activity was observed at cold infusion and 60°C temperature treatment (22% to 55% inhibition) for all concentrations of sucrose. High anti-biofilm activity (>90% inhibition) was observed at 119°C temperature treatment for all concentrations of sucrose, except for 50 g / l, which showed 75% inhibition.

[0187] For mixture 2, equivalent anti-biofilm activity (>77% inhibition) was obtained regardless of the sucrose concentration used for the 60°C and 119°C temperature treatments. After cold infusion, mixture 2 showed moderate to good anti-biofilm effects (50% to 74% inhibition), depending on the sucrose concentration: the higher the sucrose concentration, the more active the formulation.

[0188]

Example 3

[0189] In this example, we demonstrate the simultaneous antimicrobial and antibiofilm activity of 12 mixtures containing 2 to 7 species against Staphylococcus aureus and Staphylococcus epidermidis.

[0190] Pharmaceutical grade dried plants were obtained from "Herboristerie Cailleau" (Chemilles, France).

[0191] Herbal compositions containing powders of 2 to 7 plants were prepared by collecting a fixed volume of plant powder using a calibrated spoon that allowed the collection of 200 μL of powder.

[0192] According to the aforementioned method A, twelve herbal compositions were prepared as follows (Table 3-1).

[0193] [Table 3-1: Herbal composition of processed samples]

[0194]

[0195] According to the aforementioned method B, starting from each herbal composition, 12 corresponding processed samples were prepared by water extraction using 20 mL of water containing 100 g / l sucrose, including a centrifugation step and then filtration.

[0196] The biological activity of the processed samples was determined according to the aforementioned method C by using the following Staphylococcus aureus strains: ATCC 25923, ATCC 29213, NCTC 12493, ATCC 33591, ATCC 33592, ATCC 43300, ATCC 700698, ATCC 700699, ATCC 9144 and ATCC BAA-44 and the following Staphylococcus epidermidis strains: ATCC 12228, ATCC 700296, ATCC 49461 and ATCC14990.

[0197] The minimum inhibitory concentrations (MICs) observed for the 12 processed samples are given in Table 3-2 as HIDs, along with the average efficacy against all strains of S. aureus and S. epidermidis.

[0198]

Table 3-2:HID of 12 processed samples against 10 strains of Staphylococcus aureus and 4 strains of Staphylococcus epidermidis

[0199]

[0200] Since the HID observed in this example was between 1:10 and 1:20 in most cases, a 1:20 dilution was selected as the reference dilution for calculating the percent inhibition of bacterial planktonic growth and percent inhibition of biofilm formation. Table 3-3 shows the percent inhibition of planktonic growth at a 1:20 dilution for 12 processed samples, as well as the average efficacy against all strains of Staphylococcus aureus and Staphylococcus epidermidis.

[0201] Table 3-3: Planktonic growth inhibition of 10 Staphylococcus aureus and 4 Staphylococcus epidermidis by 12 processed samples at a dilution of 1:20

[0202]

[0203]

[0204] The percent inhibition of biofilm formation (IBF) for the 12 processed samples at a 1:20 dilution, as well as the average efficacy for all strains of S. aureus and S. epidermidis, are given in Tables 3-4. The average efficacy of the percent inhibition of bacterial planktonic growth and biofilm formation for all tested strains is a good indicator of antimicrobial and antibiofilm efficacy against different bacterial strains.

[0205] Table 3-4: Inhibition of biofilm formation (IBF) of 12 processed samples at a dilution of 1:20 against 10 strains of Staphylococcus aureus and 4 strains of Staphylococcus epidermidis

[0206]

[0207]

[0208] The 2 plant mixture extracts showed complete antimicrobial activity against 10 Staphylococcus aureus strains at a 1:10 dilution, except for mixture [2P]-3, which had no antimicrobial effect against 2 of the 10 Staphylococcus aureus strains tested (ATCC 29213 and ATCC 43300) at the tested dilution (see Table 3-2).

[0209] The three 2-plant mixture extracts showed different antimicrobial activities against the four strains of Staphylococcus epidermidis tested. Mixture [2P]-1 had a HID of 1:10 on three strains (ATCC 12228, ATCC 49461, and ATCC 14990) and a HID of 1:20 on the ATCC 700296 strain. Mixture [2P]-2 had a HID of 1:10 on two strains (ATCC 12228 and ATCC 49461) and a HID of 1:20 on the other two strains (ATCC 700296 and ATCC 14990). The mixture [2P]-3 had a HID of 1:10 on two strains (ATCC 12228 and ATCC 14990) and a HID of 1:20 on the other two strains (ATCC 700296 and ATCC 49461) (see Table 3-2).

[0210] Mixture [3P]-1, obtained from the three plants included in mixture [2P]-1, mixture [2P]-2, and mixture [2P]-3, showed a broader antimicrobial activity than the three two-plant mixtures (HID of 1:10 on 9 strains of Staphylococcus aureus, HID of 1:20 on ATCC 700699, and HID of 1:20 on 4 tested Staphylococcus epidermidis (see Table 3-2). The increase in antimicrobial activity was confirmed by calculating the percentage of planktonic growth inhibition for each plant mixture (see Table 3-3).

[0211] Three different plants, PA11, PA12, and PA22, were added to the 3-plant mixture [3P]-1 to produce three 4-plant mixture extracts, namely mixture [4P]-1, mixture [4P]-2, and mixture [4P]-3. These 4-plant mixture extracts showed the same or better HID as the 3-plant mixture extract mixture [3P]-1 (see Table 3-2), and a higher percentage of planktonic growth inhibition: compared to 23% for the 3-plant mixture extract, the 4-plant mixture extract achieved an average of 28% to 45% on 10 strains of Staphylococcus aureus (see Table 3-3). They also showed a higher percentage of biofilm formation inhibition: compared to 25% for the 3-plant mixture extract, the 4-plant mixture extract achieved an average of 34% to 42% on 10 strains of Staphylococcus aureus (see Table 3-4).

[0212] A 5-plant mixture was prepared by adding 2 plants to the 3 selected additional plants. All three 5-plant mixture extracts showed higher antimicrobial and anti-biofilm activities than the 4-plant mixture extract (see Tables 3-2, 3-3, and 3-4).

[0213] Mixture [6P]-1 consists of the six plants used to prepare the previously tested processed samples. Interestingly, the 6-plant mixture extract showed lower antimicrobial and antibiofilm activity than the 5-plant mixture extract, but showed higher antimicrobial efficacy and equivalent antibiofilm efficacy than the 4-plant mixture extract. The addition of a new plant (PA79) restored and enhanced the antimicrobial and antibiofilm activities (see Tables 3-2, 3-3, and 3-4).

[0214] Increasing the number of plants in the mixture allowed the preparation of corresponding aqueous extracts with enhanced antimicrobial and antibiofilm activity and an increased number of strains in which one or both of these activities could be identified.

[0215]

Example 4

[0216] In this example, we demonstrate the simultaneous antimicrobial and antibiofilm activity against Staphylococcus aureus of 196 aqueous extracts obtained from a herbal composition comprising at least 2 of the 3 plants described in Example 1 and additional plants forming a mixture of 3 to 10 plants.

[0217] Pharmaceutical grade dried plants were obtained from "Pharmacie Fontgiève" (Clermont-Ferrand, France, plants PA00 to PB12) and "Pharmacie St Herem" (Clermont-Ferrand, France, plants PB13 to PD08).

[0218] Four reference herbal compositions, Mixture [2P]-1, Mixture [2P]-2, Mixture [2P]-3, Mixture [3P]-1 and 196 new herbal compositions were prepared according to the aforementioned method A, and they had the ingredients reported in List 4-1.

[0219] [List 4-1: Herbal composition of processed samples]

[0220] Mixture [2P]-1: PA13; PA21;

[0221] Mixture [2P]-2: PA21; PB12;

[0222] Mixture [2P]-3: PA13; PB12;

[0223] Mixture [3P]-1: PA13; PA21; PB12;

[0224] Mixture [3P]-2: PA11; PA13; PA21;

[0225] Mixture [3P]-3: PA21; PB12; PC63;

[0226] Mixture [3P]-4: PA13; PB12; PC63;

[0227] Mixture [3P]-5: PA13; PA21; PC63;

[0228] Mixture [3P]-6: PA13; PA21; PB12;

[0229] Mixture [3P]-7: PA21; PB12; PC29;

[0230] Mixture [3P]-8: PA13; PB12; PC29;

[0231] Mixture [3P]-9: PA13; PA21; PB12;

[0232] Mixture [4P]-1: PA11; PA13; PA21; PB12;

[0233] Mixture [4P]-2: PA12; PA13; PA21; PB12;

[0234] Mixture [4P]-3: PA13; PA21; PA22; PB12;

[0235] Mixture [4P]-4: PA11; PA13; PA21; PA22;

[0236] Mixture [4P]-5: PA13; PA21; PB12; PC63;

[0237] Mixture [4P]-6: PA11; PA12; PA21; PB12;

[0238] Mixture [4P]-7: PA11; PA12; PA13; PB12;

[0239] Mixture [4P]-8: PA11; PA13; PA21; PB12;

[0240] Mixture [4P]-9: PA12; PA13; PA21; PB12;

[0241] Mixture [4P]-10: PA13; PA21; PB12; PC29;

[0242] Mixture [5P]-1: PA12; PA13; PA21; PA22; PB12;

[0243] Mixture [5P]-2: PA11; PA13; PA21; PA22; PB12;

[0244] Mixture [5P]-3: PA11; PA12; PA13; PA21; PB12;

[0245] Mixture [5P]-4: PA11; PA12; PA21; PA22; PB12;

[0246] Mixture [5P]-5: PA11; PA12; PA13; PA22; PB12;

[0247] Mixture [5P]-6: PA11; PA12; PA13; PA21; PA22;

[0248] Mixture [5P]-7: PA13; PA21; PA22; PB12; PC63;

[0249] Mixture [5P]-8: PA13; PA21; PB12; PC29; PC63;

[0250] Mixture [6P]-1: PA11; PA12; PA13; PA21; PA22; PB12;

[0251] Mixture [6P]-2: PA12; PA13; PA21; PA22; PB12; PC63;

[0252] Mixture [6P]-3: PA11; PA13; PA21; PA22; PB12; PC63;

[0253] Mixture [6P]-4: PA11; PA12; PA13; PA21; PB12; PC63;

[0254] Mixture [6P]-5: PA12; PA13; PA21; PA22; PB12; PC63;

[0255] Mixture [6P]-6: PA11; PA13; PA21; PA22; PB12; PC63;

[0256] Mixture [6P]-7: PA11; PA12; PA21; PA22; PB12; PC63;

[0257] Mixture [6P]-8: PA11; PA12; PA13; PA22; PB12; PC63;

[0258] Mixture [6P]-9: PA11; PA12; PA13; PA21; PB12; PC63;

[0259] Mixture [6P]-10: PA11; PA12; PA13; PA21; PA22; PC63;

[0260] Mixture [6P]-11: PA11; PA12; PA13; PA21; PA22; PB12;

[0261] Mixture [7P]-1: PA11; PA12; PA13; PA21; PA22; PB12; PA79;

[0262] M[7P]-2: PA11; PA12; PA13; PA21; PA22; PB12; PB49;

[0263] M[7P]-3: PA11; PA12; PA13; PA21; PA22; PB12; PC29;

[0264] M[7P]-4: PA11; PA12; PA13; PA21; PA22; PB12; PC63;

[0265] M[9P]-11: PA12; PA13; PA21; PA22; PA79; PB12; PB49; PC29; PC63; M[9P]-12: PA11; PA13; PA21; PA22; PA79; PB12; PB49; PC29; PC63; M[9P]-13: PA11; PA12; PA2​​​​​​​​​​​​​​​​

[0271] M[9P]-20:PA11;PA12;PA13;PA21;PA22;PA79;PB12;PB49;PC29;

[0272] M[10P]-40:PA11;PA13;PA14;PA20;PA21;PA23;PA67;PB12;PC27;PC60;

[0273] M[10P]-44:PA13;PA40;PA41;PA62;PA75;PB18;PC22;PC63;PC64;PD05;

[0274] M[10P]-71:PA21;PA85;PB14;PB18;PB85;PC19;PC61;PC63;PD05;PD08;

[0275] M[10P]-75:PA02;PA10;PA12;PA13;PA18;PA21;PA27;PA79;PB86;PC26;

[0276] M[10P]-84:PA10;PA21;PA27;PB12;PB15;PC26;PC68;PD05;PD06;PD08;

[0277] M[10P]-98:PA11;PA12;PA13;PA14;PA20;PA21;PA27;PB16;PB18;PD06;

[0278] M[10P]-102:PA12;PA13;PA19;PA21;PA41;PA79;PB10;PC26;PC61;PC64;

[0279] M[10P]-103:PA13;PA17;PA21;PA24;PB08;PB49;PB85;PC15;PC29;PD05;

[0280] M[10P]-105:PA10;PA11;PA12;PA18;PA19;PA21;PA22;PB12;PC63;PD08;

[0281] M[10P]-109:PA10;PA12;PA13;PA21;PA27;PA75;PB14;PB16;PC45;PC63;

[0282] M[10P]-112:PA08;PA13;PA15;PA17;PA18;PA21;PA27;PB18;PB41;PB81;

[0283] M[10P]-115:PA13;PA20;PA21;PA22;PA62;PA79;PB08;PB19;PC15;PC61;

[0284] M[10P]-119:PA11;PA13;PA14;PA24;PB12;PB41;PB49;PC51;PD06;PD08;

[0285] M[10P]-123:PA10;PA13;PA15;PA20;PA21;PA75;PB12;PB18;PB49;PC63;

[0286] M[10P]-124:PA08;PA13;PA21;PA24;PA27;PB08;PB41;PB49;PC64;PD05;

[0287] M[10P]-125:PA13;PA14;PA19;PA21;PA75;PB12;PC15;PC26;PC61;PC63;

[0288] M[10P]-129:PA08;PA12;PA18;PA21;PA41;PB08;PB85;PC63;PC64;PD08;

[0289] M[10P]-130:PA12;PA13;PA21;PA24;PA27;PB10;PB41;PB49;PC15;PC61;

[0290] M[10P]-132:PA12;PA20;PA21;PA24;PC15;PC26;PC61;PC63;PC64;PD08;

[0291] M[10P]-134:PA21;PA22;PA24;PA27;PB10;PB12;PB49;PC15;PC29;PD05;

[0292] M[10P]-136:PA10;PA11;PA12;PA13;PA18;PA21;PB16;PB18;PC26;PC29;

[0293] M[10P]-140:PA13;PA15;PA21;PA24;PA62;PA79;PB10;PB12;PC63;PC64;

[0294] M[10P]-142:PA11;PA12;PA13;PA19;PA21;PB12;PB85;PC15;PC26;PC64;

[0295] M[10P]-144:PA13;PA15;PA21;PA24;PA75;PB49;PC51;PC61;PC64;PD05;

[0296] M[10P]-145:PA12;PA13;PA17;PA20;PA21;PA79;PB08;PB16;PB85;PC63;

[0297] M[10P]-148:PA10;PA13;PA20;PA24;PB08;PB12;PB49;PC26;PC51;PC64;

[0298] M[10P]-149:PA13;PA14;PA15;PA19;PA21;PB08;PB10;PB41;PC15;PC61;

[0299] M[10P]-150:PA10;PA12;PA13;PA14;PA21;PA62;PB10;PC26;PC64;PD06;

[0300] M[10P]-151:PA10;PA13;PA21;PA22;PA24;PA41;PB49;PC15;PC61;PC63;

[0301] M[10P]-152:PA11;PA12;PA13;PA15;PA21;PA27;PA75;PB12;PB49;PB85;

[0302] M[10P]-154:PA11;PA12;PA13;PA21;PA22;PA79;PB12;PB49;PC29;PC63;

[0303] M[10P]-156:PA15;PA21;PA22;PB10;PB85;PC26;PC29;PC61;PC63;PC64;

[0304] M[10P]-157:PA11;PA13;PA15;PA19;PA21;PB18;PB41;PC15;PC51;PD08;

[0305] M[10P]-158:PA10;PA17;PA21;PA24;PA27;PA41;PB49;PC26;PC61;PC63;

[0306] M[10P]-160:PA10;PA12;PA13;PA15;PA18;PA20;PA21;PA24;PB12;PC26;

[0307] M[10P]-162:PA12;PA13;PA21;PA27;PA79;PB12;PB16;PB18;PC63;PC64;

[0308] M[10P]-164:PA12;PA13;PA15;PA19;PA21;PA27;PB08;PB12;PB49;PD05;

[0309] M[10P]-165:PA13;PA15;PA22;PA75;PA79;PB12;PB18;PC15;PC26;PC29;

[0310] M[10P]-167:PA12;PA13;PA75;PB08;PB12;PB85;PC15;PC51;PC61;PC63;

[0311] M[10P]-168:PA11;PA13;PA17;PA21;PA27;PA62;PA79;PB16;PB49;PC64;

[0312] M[10P]-169:PA12;PA17;PA21;PA24;PA75;PA79;PB10;PB12;PB85;PC15;

[0313] M[10P]-170:PA11;PA13;PA15;PA20;PA21;PA22;PC26;PC29;PC63;PC64;

[0314] M[10P]-172:PA13;PA20;PA21;PA75;PB10;PB12;PB18;PB49;PC29;PC61;

[0315] M[10P]-173:PA10;PA12;PA13;PA14;PA19;PA20;PA21;PA79;PC15;PC63;

[0316] M[10P]-174:PA11;PA12;PA13;PA27;PA62;PB12;PB16;PB49;PB85;PC26;

[0317] M[10P]-175:PA11;PA12;PA13;PA21;PA22;PA75;PB10;PB49;PC15;PC63;

[0318] M[10P]-176:PA11;PA13;PA21;PA24;PA62;PB12;PC15;PC26;PC61;PC64;

[0319] M[10P]-178:PA11;PA14;PA15;PA18;PA19;PA21;PA22;PB16;PC15;PC63;

[0320] M[10P]-180:PA08;PA11;PA12;PA13;PA15;PA17;PA21;PA62;PB12;PC63;

[0321] M[10P]-181:PA13;PA19;PA21;PA22;PA79;PB08;PB10;PB18;PC15;PC29;

[0322] M[10P]-182:PA11;PA15;PA20;PA21;PA22;PA75;PB12;PC29;PC63;PC64;

[0323] M[10P]-183:PA11;PA12;PA13;PA27;PA79;PB16;PB18;PB49;PC15;PC63;

[0324] M[10P]-184:PA11;PA13;PA15;PA20;PA21;PA22;PA27;PA75;PB12;PC29;

[0325] M[10P]-186:PA11;PA15;PA19;PA20;PA21;PA22;PA27;PB10;PB12;PB85;

[0326] M[10P]-188:PA11;PA12;PA13;PA19;PA21;PA75;PA79;PB49;PC15;PC63;

[0327] M[10P]-190:PA11;PA13;PA21;PA22;PA27;PA75;PB10;PB12;PB49;PC63;

[0328] M[10P]-191:PA13;PA19;PA20;PA21;PA79;PB10;PB18;PB85;PC29;PC61;

[0329] M[10P]-192:PA12;PA13;PA19;PA21;PA27;PA75;PB12;PB49;PC63;PC64;

[0330] M[10P]-193:PA13;PA15;PA20;PA21;PA79;PB16;PC15;PC26;PC29;PC61;

[0331] M[10P]-194:PA11;PA17;PA21;PA22;PA27;PB12;PB49;PC15;PC63;PC64;

[0332] M[10P]-196:PA11;PA12;PA13;PA15;PA21;PA75;PB10;PB18;PC51;PC63;

[0333] M[10P]-197:PA11;PA12;PA13;PA15;PA21;PA24;PA79;PB49;PC61;PC64;

[0334] M[10P]-198:PA12;PA15;PA21;PA22;PB12;PB16;PB85;PC15;PC26;PC29;

[0335] M[10P]-199:PA11;PA12;PA13;PA15;PA21;PA75;PB12;PC15;PC63;PC64;

[0336] M[10P]-201:PA11;PA13;PA20;PA21;PA22;PA27;PA75;PB12;PB49;PC64;

[0337] M[10P]-202:PA11;PA21;PA75;PB10;PB12;PB61;PC26;PC29;PC42;PC63;

[0338] M[10P]-205:PA11;PA13;PA19;PA21;PA22;PA27;PA75;PB12;PB41;PC63;

[0339] M[10P]-207:PA11;PA13;PA21;PA75;PB10;PB12;PB41;PB51;PC45;PC63;

[0340] M[10P]-209:PA12;PA13;PA21;PA24;PA75;PB12;PB16;PB49;PB60;PC15;

[0341] M[10P]-210:PA13;PA15;PA20;PA22;PA27;PB08;PB10;PB12;PB18;PC63;

[0342] M[10P]-212:PA11;PA13;PA21;PA22;PA27;PA75;PB12;PB16;PC15;PC63;

[0343] M[10P]-214:PA11;PA12;PA15;PA21;PA22;PA75;PB12;PB49;PC15;PC61;

[0344] M[10P]-215:PA11;PA12;PA13;PA21;PA25;PB12;PC02;PC29;PC51;PC64;

[0345] M[10P]-216:PA11;PA12;PA13;PA21;PA27;PA75;PA85;PB18;PB49;PC49;

[0346] M[10P]-217:PA18;PA20;PA21;PA22;PA75;PB10;PB12;PB16;PB85;PC64;

[0347] M[10P]-218:PA10;PA13;PA15;PA20;PA22;PA27;PB18;PB49;PC51;PC63;

[0348] M[10P]-219:PA12;PA13;PA15;PA19;PA21;PA22;PA75;PB10;PB12;PC26;

[0349] M[10P]-220:PA11;PA12;PA13;PA19;PA21;PA22;PA27;PB12;PB49;PC63;

[0350] M[10P]-221:PA13;PA15;PA19;PA20;PA21;PA41;PA75;PB12;PC15;PC60;

[0351] M[10P]-222:PA05;PA13;PA21;PA22;PA75;PA79;PB49;PC26;PC29;PC63;

[0352] M[10P]-223:PA11;PA12;PA13;PA22;PA25;PA75;PB12;PB49;PC61;PC64;

[0353] M[10P]-225:PA12;PA13;PA21;PA27;PB10;PB12;PC29;PC63;PC64;PD08;

[0354] M[10P]-226:PA11;PA12;PA21;PA39;PA79;PB18;PB49;PB85;PC61;PC63;

[0355] M[10P]-227:PA11;PA12;PA15;PA20;PA21;PA22;PB12;PB42;PC15;PC26;

[0356] M[10P]-229:PA13;PA20;PA62;PA79;PB12;PB46;PB85;PC29;PC64;PD08;

[0357] M[10P]-230:PA11;PA12;PA13;PA21;PA22;PA75;PB10;PB12;PB58;PC61;

[0358] M[10P]-232:PA12;PA13;PA15;PA22;PA27;PA83;PB12;PB49;PC26;PC63;

[0359] M[10P]-235:PA12;PA13;PA15;PA20;PA21;PA22;PC29;PC51;PC63;PC65;

[0360] M[10P]-236:PA12;PA13;PA20;PA21;PA27;PA75;PB50;PB85;PC29;PC64;

[0361] M[10P]-237:PA11;PA13;PA15;PA21;PA27;PA75;PB10;PB12;PC63;PC70;

[0362] M[10P]-239:PA10;PA11;PA12;PA20;PA21;PB10;PB41;PB49;PC63;PD08;

[0363] M[10P]-240:PA11;PA12;PA15;PA21;PA27;PA79;PB46;PC29;PC51;PC63;

[0364] M[10P]-241:PA11;PA14;PA20;PA21;PA22;PA75;PB12;PB18;PC15;PC29;

[0365] M[10P]-242:PA11;PA12;PA13;PA21;PA22;PA79;PB18;PB49;PC51;PC63;

[0366] M[10P]-244:PA11;PA12;PA13;PA20;PA22;PA27;PB10;PB12;PC63;PC64;

[0367] M[10P]-246:PA11;PA12;PA13;PA21;PA61;PA79;PB10;PB12;PB18;PB85;

[0368] M[10P]-247:PA11;PA12;PA21;PA27;PB16;PB49;PC21;PC51;PC61;PC63;

[0369] M[10P]-248:PA11;PA13;PA14;PA19;PA21;PA75;PA79;PB14;PB49;PC15;

[0370] M[10P]-250:PA11;PA12;PA15;PA20;PA21;PA41;PA75;PB49;PC26;PC63;

[0371] M[10P]-251:PA12;PA13;PA21;PA22;PA27;PA79;PB10;PB12;PB13;PB16;

[0372] M[10P]-252:PA11;PA13;PA20;PA21;PA44;PB08;PB12;PB18;PB88;PD06;

[0373] M[10P]-253:PA10;PA11;PA12;PA13;PA15;PA21;PA22;PA27;PA79;PC63;

[0374] M[10P]-255:PA11;PA13;PA21;PA75;PB12;PB18;PB49;PC15;PC26;PC63;

[0375] M[10P]-256:PA11;PA12;PA13;PA21;PA22;PA23;PA75;PA79;PC29;PC64;

[0376] M[10P]-257:PA13;PA20;PA21;PA27;PB12;PB49;PB83;PC29;PC63;PD00;

[0377] M[10P]-258:PA12;PA13;PA20;PA21;PA22;PA75;PB10;PB12;PC15;PC63;

[0378] M[10P]-262:PA11;PA12;PA21;PA31;PA75;PB49;PB85;PC61;PC63;PC64;

[0379] M[10P]-264:PA21;PA24;PA27;PA31;PA79;PB10;PB18;PB49;PC15;PC63;

[0380] M[10P]-267:PA11;PA12;PA21;PA22;PA75;PC29;PC51;PC52;PC61;PC63;

[0381] M[10P]-268:PA11;PA12;PA13;PA15;PA27;PA75;PB10;PB12;PB45;PC64;

[0382] M[10P]-269:PA13;PA20;PA21;PA22;PA79;PB10;PB11;PB49;PC61;PC63;

[0383] M[10P]-270:PA11;PA12;PA13;PA15;PA21;PA75;PB12;PB14;PB18;PC49;

[0384] M[10P]-271:PA11;PA13;PA20;PA22;PA27;PA45;PB08;PB12;PB49;PC64;

[0385] M[10P]-272:PA13;PA21;PA24;PA79;PB10;PB12;PB41;PC15;PC29;PC63;

[0386] M[10P]-273:PA11;PA15;PA21;PA22;PA27;PA75;PB12;PB14;PC64;PD00;

[0387] M[10P]-274:PA12;PA13;PA21;PA22;PB12;PB49;PC26;PC51;PC63;PC64;

[0388] M[10P]-278:PA13;PA15;PA21;PA22;PA75;PA79;PB10;PB12;PB49;PC63;

[0389] M[10P]-280:PA11;PA12;PA15;PA21;PB08;PB12;PB18;PB49;PB58;PC51;

[0390] M[10P]-281:PA11;PA12;PA20;PA21;PA22;PA27;PA29;PA75;PB16;PC63;

[0391] M[10P]-282:PA10;PA13;PA21;PA22;PA27;PA79;PB12;PC15;PC29;PC61;

[0392] M[10P]-284:PA11;PA13;PA20;PA21;PA22;PA27;PA95;PC15;PC29;PC63;

[0393] M[10P]-285:PA11;PA13;PA14;PA21;PA79;PB10;PB12;PB18;PB49;PB82;

[0394] M[10P]-286:PA11;PA12;PA13;PA14;PA20;PA21;PA79;PC51;PC64;PC82;

[0395] M[10P]-287:PA12;PA13;PA19;PA20;PA75;PB10;PB12;PC26;PC63;PD08;

[0396] M[10P]-289:PA12;PA13;PA15;PA19;PA20;PA21;PB10;PB12;PC29;PC51;

[0397] M[10P]-290:PA11;PA12;PA13;PA22;PA27;PA75;PB85;PC29;PC63;PD07;

[0398] M[10P]-292:PA12;PA13;PA15;PA75;PA79;PB10;PB12;PC29;PC45;PC63;

[0399] M[10P]-293:PA00;PA11;PA12;PA13;PA21;PA27;PA75;PB18;PB85;PC26;

[0400] M[10P]-294:PA12;PA13;PA15;PA21;PA22;PA79;PB12;PB49;PC51;PC63;

[0401] M[10P]-296:PA11;PA13;PA15;PA17;PA21;PA62;PA79;PB10;PB12;PC63;

[0402] M[10P]-300:PA12;PA13;PA17;PA48;PB10;PB12;PB16;PC26;PC29;PC63;

[0403] M[10P]-301:PA11;PA12;PA13;PA20;PA21;PA75;PB08;PC15;PC35;PC51;

[0404] M[10P]-302:PA11;PA12;PA13;PA21;PA70;PB12;PB16;PB18;PC26;PC63;

[0405] M[10P]-303:PA11;PA12;PA15;PA21;PA75;PA79;PB10;PB12;PC29;PC64;

[0406] M[10P]-307:PA12;PA13;PA21;PB10;PB12;PB18;PB78;PC51;PC63;PD08;

[0407] M[10P]-308:PA17;PA21;PA22;PA27;PB12;PB49;PB85;PC15;PC26;PC29;

[0408] M[10P]-309:PA13;PA22;PA27;PA53;PA62;PA75;PB10;PC51;PC63;PD06;

[0409] M[10P]-311:PA12;PA15;PA19;PA21;PA79;PB12;PB49;PC26;PC63;PC83;

[0410] M[10P]-312:PA00;PA11;PA13;PA21;PA22;PA29;PA75;PB10;PB85;PC29;

[0411] M[10P]-313:PA13;PA15;PA21;PA22;PA27;PA75;PB49;PC51;PC61;PC63;

[0412] M[10P]-314:PA08;PA12;PA13;PA21;PB10;PB12;PB49;PC15;PC32;PC64;

[0413] M[10P]-315:PA11;PA13;PA21;PA47;PA75;PB16;PB83;PC15;PC51;PC63;

[0414] M[10P]-317:PA11;PA12;PA19;PA21;PA22;PA79;PB10;PB16;PC29;PC63;

[0415] M[10P]-319:PA12;PA13;PA22;PA75;PA79;PA98;PB08;PB10;PB85;PC63;

[0416] M[10P]-321:PA11; PA12; PA13; PA20; PA21; PA22; PB49; PB85; PC55; PC63.

[0417] The corresponding aqueous extracts (or processed samples) of these herbal compositions were prepared by aqueous extraction using 20 mL of water containing 100 g / l sucrose according to the aforementioned method B, including a centrifugation step, followed by filtration.

[0418] The biological activity of the processed samples was determined according to the aforementioned method C by using the following Staphylococcus aureus strains: ATCC 25923, ATCC 49476, ATCC 6538, ATCC 51740, ATCC 29213 and ATCC 14775, and the final dilutions of the processed samples were as follows: for processed samples containing less than 10 spoons of plant powder (M[2P] to M[9P]), the final dilutions were 1:10, 1:20, 1:40 and 1:80; or for processed samples containing 10 spoons of plant powder (M[10P]), the final dilutions were 1:20, 1:63 and 1:200.

[0419] The mean values ​​of the percent inhibition of bacterial planktonic growth and the percent inhibition of biofilm formation for processed samples obtained from two plants of the mixture [2P]-1 at a dilution of 1:20 for all tested strains are given in Table 4-1.

[0420] Table 4-1: Average values ​​of percent inhibition of planktonic growth and percent inhibition of biofilm formation of 6 tested Staphylococcus aureus strains by processed samples based on mixture \[2P]-1 at a dilution of 1:20

[0421]

[0422]

[0423] Twenty processed samples obtained from herbal compositions containing 5 or 10 plants were found to have higher anti-biofilm and antimicrobial activities than the reference mixture extract mixture [2P]-1 (Table 4-1A). Four 10-plant mixture extracts were found to have higher anti-biofilm activity and comparable or lower antimicrobial activity (Table 4-1B). Five processed samples containing 3, 4, or 10 plants were found to have higher antimicrobial activity and comparable or lower anti-biofilm activity (Table 4-1C).

[0424] The mean bacterial planktonic growth inhibition percentage and the mean biofilm formation inhibition percentage of processed samples obtained from the herbal composition of two plants containing the mixture [2P]-2 at a dilution of 1:20 for all tested strains are given in Table 4-2.

[0425] Table 4-2: Average efficacy of processed samples based on mixture [2P]-2 at a 1:20 dilution against six tested Staphylococcus aureus strains for percent inhibition of planktonic growth and percent inhibition of biofilm formation. (A) Mixture extracts exhibiting higher antimicrobial and antibiofilm activity. (B) Mixture extracts exhibiting antibiofilm activity and comparable or lower antimicrobial activity. (C) Mixture extracts exhibiting antimicrobial activity and comparable or lower antibiofilm activity.

[0426]

[0427]

[0428] Thirty-three processed samples obtained from herbal compositions containing 3, 5, 9, or 10 plants were found to have higher anti-biofilm and antimicrobial activities than the reference mixture extract, mixture [2P]-2 (Table 4-2A). Six 10-plant mixture extracts were found to have higher anti-biofilm activity and comparable or lower antimicrobial activity (Table 4-2B). One 4-plant mixture extract was found to have higher antimicrobial activity and comparable anti-biofilm activity (Table 4-2C).

[0429] The mean bacterial planktonic growth inhibition percentage and the mean biofilm formation inhibition percentage of processed samples obtained from the herbal composition of two plants containing the mixture [2P]-3 at a dilution of 1:20 for all tested strains are given in Table 4-3.

[0430] Table 4-3: Average efficacy of processed samples based on mixture [2P]-3 at a 1:20 dilution against six tested Staphylococcus aureus strains for percent inhibition of planktonic growth and percent inhibition of biofilm formation. (A) Mixture extracts exhibiting higher antimicrobial and antibiofilm activity. (B) Mixture extracts exhibiting antimicrobial activity and comparable or lower antibiofilm activity.

[0431]

[0432] Twenty-three processed samples obtained from herbal compositions containing 3, 5, 9, or 10 plants were found to have higher anti-biofilm and antimicrobial activities than the reference mixture extract mixture [2P]-3 (Table 4-3A). Four processed samples obtained from herbal compositions containing 3, 4, or 10 plants were found to have higher anti-biofilm activity and comparable or lower antimicrobial activity (Table 4-3B).

[0433] The mean bacterial planktonic growth inhibition percentage and the mean biofilm formation inhibition percentage of processed samples obtained from the herbal composition of three plants containing mixture [3P]-1 at a dilution of 1:20 for all tested strains are given in Table 4-4.

[0434] Table 4-4: Average efficacy of processed samples based on the mixed mixture [3P]-1 at a 1:20 dilution against six tested Staphylococcus aureus strains for percent inhibition of planktonic growth and percent inhibition of biofilm formation. (A) Mixture extracts exhibiting higher antimicrobial and antibiofilm activity. (B) Mixture extracts exhibiting both antibiofilm activity and comparable or lower antimicrobial activity. (C) Mixture extracts exhibiting both antimicrobial activity and comparable or lower antibiofilm activity.

[0435]

[0436]

[0437]

[0438] 80 processed samples obtained from herbal compositions containing 4, 5, 6, 7, 9 or 10 plants were found to have higher anti-biofilm and antimicrobial activities than the reference mixture extract mixture [3P]-1 (Table 4-4A). 18 processed samples obtained from herbal compositions containing 3, 4 or 10 plants were found to have higher anti-biofilm activity and comparable or lower antimicrobial activity (Table 4-4B). Two processed samples obtained from herbal compositions containing 4 and 10 plants were found to have higher antimicrobial activity and comparable anti-biofilm activity (Table 4-4C).

[0439] [Comparative Example 2]

[0440] As comparative examples, we describe several extracts of mixtures of three plants or ten plants obtained from a herbal composition containing only one of the three plants of Example 1 (PA13, PA21, PB12), which were found to have no or poor antimicrobial and antibiofilm activity against Staphylococcus aureus.

[0441] Ten herbal compositions were prepared according to the aforementioned method A, and their ingredients are listed in Tables 4-8.

[0442] [List 4-8: Herbal composition of processed samples]

[0443] M[3P]-31: PA21; PA05; PB06;

[0444] M[3P]-35: PA21; PA76; PB50;

[0445] M[3P]-37: PA21; PB06; PB50;

[0446] M[3P]-43: PB12; PA05; PC34;

[0447] M[3P]-45: PB12; PA76; PB50;

[0448] M[10P]-65: PA01; PA08; PA25; PA29; PA37; PB12; PB50; PC15; PC37; PD05;

[0449] M[10P]-70: PA10; PA16; PA21; PA25; PA37; PA75; PB18; PB45; PC04; PC57;

[0450] M[10P]-72: PA08; PA21; PA62; PA70; PA85; PB19; PB31; PB45; PB49; PC26;

[0451] M[10P]-89: PA10; PA13; PA18; PA19; PA37; PA41; PA75; PB49; PC21; PC67;

[0452] M[10P]-306:PA15;PA21;PA22;PA62;PA75;PB18;PB49;PB85;PC49;PC61;

[0453] Ten aqueous extracts (or processed samples) were prepared starting from the corresponding herbal compositions.

[0454] The mean percent inhibition of bacterial planktonic growth and the mean percent inhibition of biofilm formation for these processed samples at a 1:20 dilution for all tested strains were determined according to previously reported Method C and are presented in Tables 4-5.

[0455] Table 4-5: Average values ​​of percent inhibition of planktonic growth and percent inhibition of biofilm formation of processed samples at 1:20 dilution against 6 tested Staphylococcus aureus strains

[0456]

[0457] All tested processed samples obtained from herbal compositions consisting of 3 or 10 plants, which included one of the 3 plants of Example 1 (PA13, PA21, PB12), showed lower antimicrobial activity than the three 2-plant mixture extracts of the reference mixture [2P]-1 (obtained from PA13 and PA21), mixture [2P]-2 (obtained from PA21 and PB12), and mixture [2P]-3 (obtained from PA13 and PB12), and lower anti-biofilm activity than mixture [2P]-1 and mixture [2P]-3.

[0458]

Example 5

[0459] In this example, we demonstrate the simultaneous antimicrobial and antibiofilm activity against Staphylococcus aureus of 390 mixture extracts obtained from an herbal composition comprising at least 2 of the 3 plants described in Example 1 (see Table 1-1) and additional plants used to assemble a mixture of 9 to 20 plants.

[0460] Pharmaceutical grade dried plants were obtained from "Pharmacie Fontgiève" (Clermont-Ferrand, France, plants PA00 to PB12) and "Pharmacie St Herem" (Clermont-Ferrand, France, plants PB13 to PD02).

[0461] According to the aforementioned method A, a herbal composition containing 9 to 20 kinds of plant powders was prepared.

[0462] Four reference herbal compositions, mixture [2P]-1, mixture [2P]-2, mixture [2P]-3, mixture [3P]-1 and 390 new herbal compositions were prepared, and their ingredients are shown below (Table 5-3).

[0463] [List 5-3: Herbal composition of processed samples]

[0464] Mixture [2P]-1: PA13; PA21;

[0465] Mixture [2P]-2: PA21; PB12;

[0466] Mixture [2P]-3: PA13; PB12;

[0467] Mixture [3P]-1: PA13; PA21; PB12;

[0468] M[09P]-1: PA13; PA14; PA20; PA21; PA23; PA67; PB12; PC27; PC60;

[0469] M[09P]-2: PA11; PA14; PA20; PA21; PA23; PA67; PB12; PC27; PC60;

[0470] M[09P]-3: PA11; PA13; PA20; PA21; PA23; PA67; PB12; PC27; PC60;

[0471] M[09P]-4: PA11; PA13; PA14; PA21; PA23; PA67; PB12; PC27; PC60;

[0472] M[09P]-6: PA11; PA13; PA14; PA20; PA23; PA67; PB12; PC27; PC60;

[0473] M[09P]-6: PA11; PA13; PA14; PA20; PA21; PA67; PB12; PC27; PC60;<0001​​​​​​​​​​​​​M[09P]-21:PC49;PC37;PA13;PA13;PA13;PA11;PA29;PA21;PB12;

[0479] M[10P]-41:PA13;PA20;PA21;PB02;PB12;PB27;PB33;PB60;PB88;PC20;

[0480] M[10P]-303:PC49;PC37;PA13;PA13;PA13;PA11;PA29;PA21;PB12;PC33;

[0481] M[11P]-1:PA13;PA20;PA21;PB02;PB12;PB27;PB33;PB60;PB88;PC20;PA11;

[0482] M[11P]-2:PA13;PA20;PA21;PB02;PB12;PB27;PB33;PB60;PB88;PC20;PA14;

[0483] M[11P]-3:PA13;PA20;PA21;PB02;PB12;PB27;PB33;PB60;PB88;PC20;PA25;

[0484] M[11P]-4:PA13;PA20;PA21;PB02;PB12;PB27;PB33;PB60;PB88;PC20;PA26;

[0485] M[11P]-5:PA13;PA20;PA21;PB02;PB12;PB27;PB33;PB60;PB88;PC20;PA28;

[0486] M[11P]-6:PA13;PA20;PA21;PB02;PB12;PB27;PB33;PB60;PB88;PC20;PA29;

[0487] M[11P]-7:PA13;PA20;PA21;PB02;PB12;PB27;PB33;PB60;PB88;PC20;PA67;

[0488] M[11P]-8:PA13;PA20;PA21;PB02;PB12;PB27;PB33;PB60;PB88;PC20;PA75;

[0489] M[11P]-9:PA13;PA20;PA21;PB02;PB12;PB27;PB33;PB60;PB88;PC20;PC49;

[0490] M[11P]-10:PA13;PA20;PA21;PB02;PB12;PB27;PB33;PB60;PB88;PC20;PC60;

[0491] M[11P]-11:PC49;PC37;PA13;PA13;PA13;PA11;PA29;PA21;PB12;PC33;PA39;

[0492] M[11P]-12:PA11;PC37;PC33;PA39;PB60;PC26;PB12;PA13;PA13;PA13;PA21;

[0493] M[12P]-1:PC49;PC37;PA13;PA13;PA13;PA11;PA29;PA21;PB12;PC33;PA39;PA22;

[0494] M[12P]-2:PA11;PC37;PC33;PA39;PB60;PC26;PB12;PA13;PA13;PA13;PA21;PA29;

[0495] M[13P]-1:PC49;PC37;PA13;PA13;PA13;PA11;PA29;PA21;PB12;PC33;PA39;PA22;PA20;

[0496] M[13P]-2:PA11;PC37;PC33;PA39;PB60;PC26;PB12;PA13;PA13;PA13;PA21;PA29;PC20;

[0497] M[14P]-1:PC49;PC37;PA13;PA13;PA13;PA11;PA29;PA21;PB12;PC33;PA39;PA22;PA20;PC26;

[0498] M[14P]-2:PA11;PC37;PC33;PA39;PB60;PC26;PB12;PA13;PA13;PA13;PA21;PA29;PC20;PC49;

[0499] M[15P]-1:PA10;PA11;PA13;PA15;PA20;PA21;PA23;PA26;PA33;PB08;PB12;PB60;PB88;PC12;PC20;

[0500] M[15P]-5:PC49;PC37;PA13;PA13;PA13;PA11;PA29;PA21;PB12;PC33;PA39;PA22;PA20;PC26;PC20;

[0501] M[16P]-1:PA00;PA10;PA20;PA21;PA23;PA24;PA29;PA57;PB07;PB12;PC11;PC12;PC20;PC51;PC55;PC67;

[0502] M[16P]-2:PA11;PA12;PA13;PA14;PA20;PA21;PA22;PA29;PA39;PB12;PB60;PC20;PC26;PC33;PC37;PC49;

[0503] M[16P]-3:PA11;PA12;PA14;PA14;PA20;PA21;PA22;PA29;PA39;PB12;PB60;PC20;PC26;PC33;PC37;PC49;

[0504] M[16P]-5:PC49;PC37;PA13;PA13;PA13;PA11;PA29;PA21;PB12;PC33;PA39;PA22;PA20;PC26;PC20;PA12;

[0505] M[16P]-6:PA11;PC37;PC33;PA39;PB60;PC26;PB12;PA13;PA13;PA13;PA21;PA29;PC20;PC49;PA14;PA14;

[0506] M[17P]-1:PA00;PA10;PA20;PA21;PA23;PA24;PA29;PA57;PB07;PB12;PC11;PC11;PC12;PC20;PC51;PC55;PC67;

[0507] M[17P]-2:PA11;PA12;PA13;PA14;PA14;PA20;PA21;PA22;PA29;PA39;PB12;PB60;PC20;PC26;PC33;PC37;PC49;

[0508] M[17P]-3:PA11;PA12;PA13;PA13;PA13;PA20;PA21;PA22;PA29;PA39;PB12;PB60;PC20;PC26;PC33;PC37;PC49;

[0509] M[17P]-5:PA00;PA10;PA20;PA21;PA23;PA24;PA29;PA57;PB07;PB12;PC11;PC11;PC12;PC20;PC51;PC55;PC67;

[0510] M[17P]-4:PA10;PA10;PA11;PA13;PA15;PA20;PA20;PA21;PA23;PA33;PB08;PB12;PB12;PB60;PB88;PC12;PC20;

[0511] M[17P]-6:PA10;PA11;PA14;PA18;PA21;PA23;PA29;PA67;PB00;PB07;PB12;PB33;PB77;PC11;PC52;PC60;PC71;

[0512] M[17P]-8:PA11;PC37;PC33;PA39;PB60;PC26;PB12;PA13;PA13;PA13;PA21;PA29;PC20;PC49;PA14;PA14;PA22;

[0513] M[17P]-9:PA13;PA13;PA13;PC49;PC37;PA11;PA20;PA22;PA12;PC26;PB60;PA39;PC33;PC20;PA14;PA14;PA21;

[0514] M[18P]-1:PA10;PA11;PA13;PA14;PA18;PA21;PA23;PA29;PA67;PB00;PB07;PB12;PB33;PB77;PC11;PC52;PC60;PC71;

[0515] M[18P]-2:PA11;PA11;PA12;PA13;PA14;PA14;PA20;PA21;PA22;PA29;PA39;PB12;PB60;PC20;PC26;PC33;PC37;PC49;

[0516] M[18P]-3:PA12;PA13;PA13;PA13;PA14;PA14;PA20;PA21;PA22;PA29;PA39;PB12;PB60;PC20;PC26;PC33;PC37;PC49;

[0517] M[18P]-4:PA11;PA13;PA13;PA13;PA14;PA14;PA20;PA21;PA22;PA29;PA39;PB12;PB60;PC20;PC26;PC33;PC37;PC49;

[0518] M[18P]-5:PA11;PA12;PA13;PA13;PA14;PA14;PA20;PA21;PA22;PA29;PA39;PB12;PB60;PC20;PC26;PC33;PC37;PC49;

[0519] M[18P]-6:PA11;PA12;PA13;PA13;PA13;PA14;PA20;PA21;PA22;PA29;PA39;PB12;PB60;PC20;PC26;PC33;PC37;PC49;

[0520] M[18P]-7:PA11;PA12;PA13;PA13;PA13;PA14;PA14;PA21;PA22;PA29;PA39;PB12;PB60;PC20;PC26;PC33;PC37;PC49;

[0521] M[18P]-9:PA11;PA12;PA13;PA13;PA13;PA14;PA14;PA20;PA22;PA29;PA39;PB12;PB60;PC20;PC26;PC33;PC37;PC49;

[0522] M[18P]-9:PA11;PA12;PA13;PA13;PA13;PA14;PA14;PA20;PA21;PA29;PA39;PB12;PB60;PC20;PC26;PC33;PC37;PC49;

[0523] M[18P]-10:PA11;PA12;PA13;PA13;PA13;PA14;PA14;PA20;PA21;PA22;PA39;PB12;PB60;PC20;PC26;PC33;PC37;PC49;

[0524] M[18P]-11:PA11;PA12;PA13;PA13;PA13;PA14;PA14;PA20;PA21;PA22;PA29;PB12;PB60;PC20;PC26;PC33;PC37;PC49;

[0525] M[18P]-12:PA11;PA12;PA13;PA13;PA13;PA14;PA14;PA20;PA21;PA22;PA29;PA39;PB60;PC20;PC26;PC33;PC37;PC49;

[0526] M[18P]-13:PA11;PA12;PA13;PA13;PA13;PA14;PA14;PA20;PA21;PA22;PA29;PA39;PB12;PC20;PC26;PC33;PC37;PC49;

[0527] M[18P]-14:PA11;PA12;PA13;PA13;PA13;PA14;PA14;PA20;PA21;PA22;PA29;PA39;PB12;PB60;PC26;PC33;PC37;PC49;

[0528] M[18P]-15:PA11;PA12;PA13;PA13;PA13;PA14;PA14;PA20;PA21;PA22;PA29;PA39;PB12;PB60;PC20;PC33;PC37;PC49;

[0529] M[18P]-16:PA11;PA12;PA13;PA13;PA13;PA14;PA14;PA20;PA21;PA22;PA29;PA39;PB12;PB60;PC20;PC26;PC37;PC49;

[0530] M[18P]-17:PA11;PA12;PA13;PA13;PA13;PA14;PA14;PA20;PA21;PA22;PA29;PA39;PB12;PB60;PC20;PC26;PC33;PC49;

[0531] M[18P]-18:PA11;PA12;PA13;PA13;PA13;PA14;PA14;PA20;PA21;PA22;PA29;PA39;PB12;PB60;PC20;PC26;PC33;PC37;

[0532] M[18P]-23:PA00;PA10;PA20;PA21;PA23;PA24;PA29;PA57;PB07;PB12;PB12;PC11;PC11;PC12;PC20;PC51;PC55;PC67;

[0533] M[18P]-19:PA11;PA13;PA15;PA20;PA20;PA21;PA23;PA26;PA26;PA26;PA33;PB08;PB12;PB12;PB60;PB88;PC12;PC20;

[0534] M[18P]-20:PA10;PA10;PA11;PA13;PA15;PA21;PA23;PA26;PA26;PA26;PA33;PB08;PB12;PB12;PB60;PB88;PC12;PC20;

[0535] M[18P]-21:PA10;PA10;PA11;PA13;PA15;PA20;PA20;PA21;PA23;PA26;PA33;PB08;PB12;PB12;PB60;PB88;PC12;PC20;

[0536] M[18P]-22:PA10;PA10;PA11;PA13;PA15;PA20;PA20;PA21;PA23;PA26;PA26;PA26;PA33;PB08;PB60;PB88;PC12;PC20;

[0537] M[18P]-24:PA00;PA10;PA20;PA21;PA23;PA24;PA29;PA57;PB07;PB12;PB12;PB12;PB12;PC12;PC20;PC51;PC55;PC67;

[0538] M[18P]-25:PA10;PA11;PA13;PA14;PA18;PA21;PA23;PA29;PA67;PB00;PB07;PB12;PB33;PB77;PC11;PC52;PC60;PC71;

[0539] M[18P]-31:PA13;PA13;PA13;PA14;PA14;PA26;PA26;PA28;PA52;PA67;PB00;PB12;PB27;PB28;PB38;PB60;PC37;PC52;

[0540] M[18P]-33:PA11;PA11;PA13;PA14;PA14;PA26;PA26;PA28;PA52;PA67;PB00;PB12;PB27;PB28;PB38;PB60;PC37;PC52;

[0541] M[18P]-34:PA11;PA11;PA13;PA13;PA13;PA26;PA26;PA28;PA52;PA67;PB00;PB12;PB27;PB28;PB38;PB60;PC37;PC52;

[0542] M[18P]-34:PA11;PA11;PA13;PA13;PA13;PA14;PA14;PA28;PA52;PA67;PB00;PB12;PB27;PB28;PB38;PB60;PC37;PC52;

[0543] M[18P]-35:PC49;PC37;PA13;PA13;PA13;PA11;PA29;PA21;PB12;PC33;PA39;PA22;PA20;PC26;PC20;PA12;PA14;PA14;

[0544] M[18P]-36:PA11;PC37;PC33;PA39;PB60;PC26;PB12;PA13;PA13;PA13;PA21;PA29;PC20;PC49;PA14;PA14;PA22;PA20;

[0545] M[18P]-37:PA13;PA13;PA13;PC49;PC37;PA11;PA20;PA22;PA12;PC26;PB60;PA39;PC33;PC20;PA14;PA14;PA21;PA29;

[0546] M[19P]-1:PA13;PA14;PA20;PA21;PA25;PA26;PA28;PA29;PA67;PA75;PB02;PB12;PB27;PB33;PB60;PB88;PC20;PC49;PC60;

[0547] M[19P]-2:PA11;PA14;PA20;PA21;PA25;PA26;PA28;PA29;PA67;PA75;PB02;PB12;PB27;PB33;PB60;PB88;PC20;PC49;PC60;

[0548] M[19P]-3:PA11;PA13;PA20;PA21;PA25;PA26;PA28;PA29;PA67;PA75;PB02;PB12;PB27;PB33;PB60;PB88;PC20;PC49;PC60;

[0549] M[19P]-4:PA11;PA13;PA14;PA21;PA25;PA26;PA28;PA29;PA67;PA75;PB02;PB12;PB27;PB33;PB60;PB88;PC20;PC49;PC60;

[0550] M[19P]-5:PA11;PA13;PA14;PA20;PA25;PA26;PA28;PA29;PA67;PA75;PB02;PB12;PB27;PB33;PB60;PB88;PC20;PC49;PC60;

[0551] M[19P]-6:PA11;PA13;PA14;PA20;PA21;PA26;PA28;PA29;PA67;PA75;PB02;PB12;PB27;PB33;PB60;PB88;PC20;PC49;PC60;

[0552] M[19P]-7:PA11;PA13;PA14;PA20;PA21;PA25;PA28;PA29;PA67;PA75;PB02;PB12;PB27;PB33;PB60;PB88;PC20;PC49;PC60;

[0553] M[19P]-8:PA11;PA13;PA14;PA20;PA21;PA25;PA26;PA29;PA67;PA75;PB02;PB12;PB27;PB33;PB60;PB88;PC20;PC49;PC60;

[0554] M[19P]-9:PA11;PA13;PA14;PA20;PA21;PA25;PA26;PA28;PA67;PA75;PB02;PB12;PB27;PB33;PB60;PB88;PC20;PC49;PC60;

[0555] M[19P]-10:PA11;PA13;PA14;PA20;PA21;PA25;PA26;PA28;PA29;PA75;PB02;PB12;PB27;PB33;PB60;PB88;PC20;PC49;PC60;

[0556] M[19P]-11:PA11;PA13;PA14;PA20;PA21;PA25;PA26;PA28;PA29;PA67;PB02;PB12;PB27;PB33;PB60;PB88;PC20;PC49;PC60;

[0557] M[19P]-12:PA11;PA13;PA14;PA20;PA21;PA25;PA26;PA28;PA29;PA67;PA75;PB12;PB27;PB33;PB60;PB88;PC20;PC49;PC60;

[0558] M[19P]-13:PA11;PA13;PA14;PA20;PA21;PA25;PA26;PA28;PA29;PA67;PA75;PB02;PB27;PB33;PB60;PB88;PC20;PC49;PC60;

[0559] M[19P]-14:PA11;PA13;PA14;PA20;PA21;PA25;PA26;PA28;PA29;PA67;PA75;PB02;PB12;PB33;PB60;PB88;PC20;PC49;PC60;

[0560] M[19P]-15:PA11;PA13;PA14;PA20;PA21;PA25;PA26;PA28;PA29;PA67;PA75;PB02;PB12;PB27;PB60;PB88;PC20;PC49;PC60;

[0561] M[19P]-16:PA11;PA13;PA14;PA20;PA21;PA25;PA26;PA28;PA29;PA67;PA75;PB02;PB12;PB27;PB33;PB88;PC20;PC49;PC60;

[0562] M[19P]-17:PA11;PA13;PA14;PA20;PA21;PA25;PA26;PA28;PA29;PA67;PA75;PB02;PB12;PB27;PB33;PB60;PC20;PC49;PC60;

[0563] M[19P]-18:PA11;PA13;PA14;PA20;PA21;PA25;PA26;PA28;PA29;PA67;PA75;PB02;PB12;PB27;PB33;PB60;PB88;PC49;PC60;

[0564] M[19P]-19:PA11;PA13;PA14;PA20;PA21;PA25;PA26;PA28;PA29;PA67;PA75;PB02;PB12;PB27;PB33;PB60;PB88;PC20;PC60;

[0565] M[19P]-20:PA11;PA13;PA14;PA20;PA21;PA25;PA26;PA28;PA29;PA67;PA75;PB02;PB12;PB27;PB33;PB60;PB88;PC20;PC49;

[0566] M[19P]-21:PA00;PA10;PA20;PA21;PA23;PA24;PA29;PA57;PB07;PB12;PB12;PB12;PB12;PC11;PC12;PC20;PC51;PC55;PC67;

[0567] M[19P]-22:PA10;PA11;PA13;PA13;PA14;PA18;PA21;PA23;PA29;PA67;PB00;PB07;PB12;PB33;PB77;PC11;PC52;PC60;PC71;

[0568] M[19P]-23:PA11;PA12;PA13;PA13;PA13;PA14;PA14;PA20;PA21;PA22;PA29;PA39;PB12;PB60;PC20;PC26;PC33;PC37;PC49;

[0569] M[19P]-24:PA11;PA11;PA12;PA13;PA13;PA13;PA14;PA20;PA21;PA22;PA29;PA39;PB12;PB60;PC20;PC26;PC33;PC37;PC49;

[0570] M[19P]-25:PA10;PA11;PA13;PA15;PA20;PA20;PA21;PA23;PA26;PA26;PA26;PA33;PB08;PB12;PB12;PB60;PB88;PC12;PC20;

[0571] M[19P]-26:PA10;PA10;PA13;PA15;PA20;PA20;PA21;PA23;PA26;PA26;PA26;PA33;PB08;PB12;PB12;PB60;PB88;PC12;PC20;

[0572] M[19P]-27:PA10;PA10;PA11;PA15;PA20;PA20;PA21;PA23;PA26;PA26;PA26;PA33;PB08;PB12;PB12;PB60;PB88;PC12;PC20;

[0573] M[19P]-28:PA10;PA10;PA11;PA13;PA20;PA20;PA21;PA23;PA26;PA26;PA26;PA33;PB08;PB12;PB12;PB60;PB88;PC12;PC20;

[0574] M[19P]-29:PA10;PA10;PA11;PA13;PA15;PA20;PA21;PA23;PA26;PA26;PA26;PA33;PB08;PB12;PB12;PB60;PB88;PC12;PC20;

[0575] M[19P]-30:PA10;PA10;PA11;PA13;PA15;PA20;PA20;PA23;PA26;PA26;PA26;PA33;PB08;PB12;PB12;PB60;PB88;PC12;PC20;

[0576] M[19P]-31:PA10;PA10;PA11;PA13;PA15;PA20;PA20;PA21;PA26;PA26;PA26;PA33;PB08;PB12;PB12;PB60;PB88;PC12;PC20;

[0577] M[19P]-32:PA10;PA10;PA11;PA13;PA15;PA20;PA20;PA21;PA23;PA26;PA26;PA33;PB08;PB12;PB12;PB60;PB88;PC12;PC20;

[0578] M[19P]-33:PA10;PA10;PA11;PA13;PA15;PA20;PA20;PA21;PA23;PA26;PA26;PA26;PB08;PB12;PB12;PB60;PB88;PC12;PC20;

[0579] M[19P]-34:PA10;PA10;PA11;PA13;PA15;PA20;PA20;PA21;PA23;PA26;PA26;PA26;PA33;PB12;PB12;PB60;PB88;PC12;PC20;

[0580] M[19P]-35:PA10;PA10;PA11;PA13;PA15;PA20;PA20;PA21;PA23;PA26;PA26;PA26;PA33;PB08;PB12;PB60;PB88;PC12;PC20;

[0581] M[19P]-36:PA10;PA10;PA11;PA13;PA15;PA20;PA20;PA21;PA23;PA26;PA26;PA26;PA33;PB08;PB12;PB12;PB88;PC12;PC20;

[0582] M[19P]-37:PA10;PA10;PA11;PA13;PA15;PA20;PA20;PA21;PA23;PA26;PA26;PA26;PA33;PB08;PB12;PB12;PB60;PC12;PC20;

[0583] M[19P]-38:PA10;PA10;PA11;PA13;PA15;PA20;PA20;PA21;PA23;PA26;PA26;PA26;PA33;PB08;PB12;PB12;PB60;PB88;PC20;

[0584] M[19P]-39:PA10;PA10;PA11;PA13;PA15;PA20;PA20;PA21;PA23;PA26;PA26;PA26;PA33;PB08;PB12;PB12;PB60;PB88;PC12;

[0585] M[19P]-40:PA10;PA20;PA21;PA23;PA24;PA29;PA57;PB07;PB12;PB12;PB12;PB12;PC11;PC11;PC12;PC20;PC51;PC55;PC67;

[0586] M[19P]-41:PA00;PA20;PA21;PA23;PA24;PA29;PA57;PB07;PB12;PB12;PB12;PB12;PC11;PC11;PC12;PC20;PC51;PC55;PC67;

[0587] M[19P]-42:PA00;PA10;PA21;PA23;PA24;PA29;PA57;PB07;PB12;PB12;PB12;PB12;PC11;PC11;PC12;PC20;PC51;PC55;PC67;

[0588] M[19P]-44:PA00;PA10;PA20;PA21;PA24;PA29;PA57;PB07;PB12;PB12;PB12;PB12;PC11;PC11;PC12;PC20;PC51;PC55;PC67;

[0589] M[19P]-45:PA00;PA10;PA20;PA21;PA23;PA29;PA57;PB07;PB12;PB12;PB12;PB12;PC11;PC11;PC12;PC20;PC51;PC55;PC67;

[0590] M[19P]-46:PA00;PA10;PA20;PA21;PA23;PA24;PA57;PB07;PB12;PB12;PB12;PB12;PC11;PC11;PC12;PC20;PC51;PC55;PC67;

[0591] M[19P]-47:PA00;PA10;PA20;PA21;PA23;PA24;PA29;PB07;PB12;PB12;PB12;PB12;PC11;PC11;PC12;PC20;PC51;PC55;PC67;

[0592] M[19P]-48:PA00;PA10;PA20;PA21;PA23;PA24;PA29;PA57;PB12;PB12;PB12;PB12;PC11;PC11;PC12;PC20;PC51;PC55;PC67;

[0593] M[19P]-49:PA00;PA10;PA20;PA21;PA23;PA24;PA29;PA57;PB07;PB12;PB12;PB12;PC11;PC11;PC12;PC20;PC51;PC55;PC67;

[0594] M[19P]-50:PA00;PA10;PA20;PA21;PA23;PA24;PA29;PA57;PB07;PB12;PB12;PB12;PB12;PC11;PC12;PC20;PC51;PC55;PC67;

[0595] M[19P]-51:PA00;PA10;PA20;PA21;PA23;PA24;PA29;PA57;PB07;PB12;PB12;PB12;PB12;PC11;PC11;PC20;PC51;PC55;PC67;

[0596] M[19P]-52:PA00;PA10;PA20;PA21;PA23;PA24;PA29;PA57;PB07;PB12;PB12;PB12;PB12;PC11;PC11;PC12;PC51;PC55;PC67;

[0597] M[19P]-53:PA00;PA10;PA20;PA21;PA23;PA24;PA29;PA57;PB07;PB12;PB12;PB12;PB12;PC11;PC11;PC12;PC20;PC55;PC67;

[0598] M[19P]-54:PA00;PA10;PA20;PA21;PA23;PA24;PA29;PA57;PB07;PB12;PB12;PB12;PB12;PC11;PC11;PC12;PC20;PC51;PC67;

[0599] M[19P]-55:PA00;PA10;PA20;PA21;PA23;PA24;PA29;PA57;PB07;PB12;PB12;PB12;PB12;PC11;PC11;PC12;PC20;PC51;PC55;

[0600] M[19P]-56:PA11;PA13;PA13;PA13;PA14;PA18;PA21;PA23;PA29;PA67;PB00;PB07;PB12;PB33;PB77;PC11;PC52;PC60;PC71;

[0601] M[19P]-57:PA10;PA13;PA13;PA13;PA14;PA18;PA21;PA23;PA29;PA67;PB00;PB07;PB12;PB33;PB77;PC11;PC52;PC60;PC71;

[0602] M[19P]-58:PA10;PA11;PA13;PA13;PA14;PA18;PA21;PA23;PA29;PA67;PB00;PB07;PB12;PB33;PB77;PC11;PC52;PC60;PC71;

[0603] M[19P]-59:PA10;PA11;PA13;PA13;PA13;PA18;PA21;PA23;PA29;PA67;PB00;PB07;PB12;PB33;PB77;PC11;PC52;PC60;PC71;

[0604] M[19P]-60:PA10;PA11;PA13;PA13;PA13;PA14;PA21;PA23;PA29;PA67;PB00;PB07;PB12;PB33;PB77;PC11;PC52;PC60;PC71;

[0605] M[19P]-62:PA10;PA11;PA13;PA13;PA13;PA14;PA18;PA23;PA29;PA67;PB00;PB07;PB12;PB33;PB77;PC11;PC52;PC60;PC71;

[0606] M[19P]-62:PA10;PA11;PA13;PA13;PA13;PA14;PA18;PA21;PA29;PA67;PB00;PB07;PB12;PB33;PB77;PC11;PC52;PC60;PC71;

[0607] M[19P]-63:PA10;PA11;PA13;PA13;PA13;PA14;PA18;PA21;PA23;PA67;PB00;PB07;PB12;PB33;PB77;PC11;PC52;PC60;PC71;

[0608] M[19P]-64:PA10;PA11;PA13;PA13;PA13;PA14;PA18;PA21;PA23;PA29;PB00;PB07;PB12;PB33;PB77;PC11;PC52;PC60;PC71;

[0609] M[19P]-65:PA10;PA11;PA13;PA13;PA13;PA14;PA18;PA21;PA23;PA29;PA67;PB07;PB12;PB33;PB77;PC11;PC52;PC60;PC71;

[0610] M[19P]-66:PA10;PA11;PA13;PA13;PA13;PA14;PA18;PA21;PA23;PA29;PA67;PB00;PB12;PB33;PB77;PC11;PC52;PC60;PC71;

[0611] M[19P]-67:PA10;PA11;PA13;PA13;PA13;PA14;PA18;PA21;PA23;PA29;PA67;PB00;PB07;PB33;PB77;PC11;PC52;PC60;PC71;

[0612] M[19P]-68:PA10;PA11;PA13;PA13;PA13;PA14;PA18;PA21;PA23;PA29;PA67;PB00;PB07;PB12;PB77;PC11;PC52;PC60;PC71;

[0613] M[19P]-69:PA10;PA11;PA13;PA13;PA13;PA14;PA18;PA21;PA23;PA29;PA67;PB00;PB07;PB12;PB33;PC11;PC52;PC60;PC71;

[0614] M[19P]-70:PA10;PA11;PA13;PA13;PA13;PA14;PA18;PA21;PA23;PA29;PA67;PB00;PB07;PB12;PB33;PB77;PC52;PC60;PC71;

[0615] M[19P]-71:PA10;PA11;PA13;PA13;PA13;PA14;PA18;PA21;PA23;PA29;PA67;PB00;PB07;PB12;PB33;PB77;PC11;PC60;PC71;

[0616] M[19P]-72:PA10;PA11;PA13;PA13;PA13;PA14;PA18;PA21;PA23;PA29;PA67;PB00;PB07;PB12;PB33;PB77;PC11;PC52;PC71;

[0617] M[19P]-73:PA10;PA11;PA13;PA13;PA13;PA14;PA18;PA21;PA23;PA29;PA67;PB00;PB07;PB12;PB33;PB77;PC11;PC52;PC60;

[0618] M[19P]-89:PA11;PA13;PA13;PA13;PA14;PA14;PA26;PA26;PA28;PA52;PA67;PB00;PB12;PB27;PB28;PB38;PB60;PC37;PC52;

[0619] M[19P]-91:PA11;PA11;PA13;PA13;PA14;PA14;PA26;PA26;PA28;PA52;PA67;PB00;PB12;PB27;PB28;PB38;PB60;PC37;PC52;

[0620] M[19P]-92:PA11;PA11;PA13;PA13;PA13;PA14;PA26;PA26;PA28;PA52;PA67;PB00;PB12;PB27;PB28;PB38;PB60;PC37;PC52;

[0621] M[19P]-93:PA11;PA11;PA13;PA13;PA13;PA14;PA14;PA26;PA28;PA52;PA67;PB00;PB12;PB27;PB28;PB38;PB60;PC37;PC52;

[0622] M[19P]-93:PA11;PA11;PA13;PA13;PA13;PA14;PA14;PA26;PA26;PA52;PA67;PB00;PB12;PB27;PB28;PB38;PB60;PC37;PC52;

[0623] M[19P]-95:PA11;PA11;PA13;PA13;PA13;PA14;PA14;PA26;PA26;PA28;PA67;PB00;PB12;PB27;PB28;PB38;PB60;PC37;PC52;

[0624] M[19P]-96:PA11;PA11;PA13;PA13;PA13;PA14;PA14;PA26;PA26;PA28;PA52;PB00;PB12;PB27;PB28;PB38;PB60;PC37;PC52;

[0625] M[19P]-96:PA11;PA11;PA13;PA13;PA13;PA14;PA14;PA26;PA26;PA28;PA52;PA67;PB12;PB27;PB28;PB38;PB60;PC37;PC52;

[0626] M[19P]-98:PA11;PA11;PA13;PA13;PA13;PA14;PA14;PA26;PA26;PA28;PA52;PA67;PB00;PB12;PB28;PB38;PB60;PC37;PC52;

[0627] M[19P]-100:PA11;PA11;PA13;PA13;PA13;PA14;PA14;PA26;PA26;PA28;PA52;PA67;PB00;PB12;PB27;PB38;PB60;PC37;PC52;

[0628] M[19P]-100:PA11;PA11;PA13;PA13;PA13;PA14;PA14;PA26;PA26;PA28;PA52;PA67;PB00;PB12;PB27;PB28;PB60;PC37;PC52;

[0629] M[19P]-101:PA11;PA11;PA13;PA13;PA13;PA14;PA14;PA26;PA26;PA28;PA52;PA67;PB00;PB12;PB27;PB28;PB38;PC37;PC52;

[0630] M[19P]-102:PA11;PA11;PA13;PA13;PA13;PA14;PA14;PA26;PA26;PA28;PA52;PA67;PB00;PB12;PB27;PB28;PB38;PB60;PC52;

[0631] M[19P]-103:PA11;PA11;PA13;PA13;PA13;PA14;PA14;PA26;PA26;PA28;PA52;PA67;PB00;PB12;PB27;PB28;PB38;PB60;PC37;

[0632] M[19P]-104:PC49;PC37;PA13;PA13;PA13;PA11;PA29;PA21;PB12;PC33;PA39;PA22;PA20;PC26;PC20;PA12;PA14;PA14;PB60;

[0633] M[19P]-105:PA11;PC37;PC33;PA39;PB60;PC26;PB12;PA13;PA13;PA13;PA21;PA29;PC20;PC49;PA14;PA14;PA22;PA20;PA12;

[0634] M[19P]-106:PA13;PA13;PA13;PC49;PC37;PA11;PA20;PA22;PA12;PC26;PB60;PA39;PC33;PC20;PA14;PA14;PA21;PA29;PB12;

[0635] M[20P]-29:PA10;PA11;PA12;PA13;PA14;PA15;PA16;PA17;PA18;PA19;PA20;PA21;PA22;PA23;PA24;PA25;PA26;PA27;PA28;PA29;

[0636] M[20P]-240:PA21;PA24;PA26;PA33;PA37;PA39;PA67;PB00;PB10;PB12;PB26;PB28;PB33;PB82;PB88;PC20;PC21;PC52;PC60;PC97;

[0637] M[20P]-296:PA00;PA11;PA13;PA15;PA21;PA22;PA24;PA26;PA28;PA33;PA39;PA94;PB08;PB12;PB20;PB28;PB33;PB99;PC26;PC52;

[0638] M[20P]-297:PA10;PA11;PA13;PA14;PA20;PA21;PA25;PA26;PA29;PA33;PA57;PA67;PA69;PB07;PB12;PB60;PC20;PC37;PC49;PC98;

[0639] M[20P]-335:PA00;PA10;PA11;PA21;PA26;PA27;PA31;PA34;PA67;PA69;PA81;PA89;PB01;PB10;PB12;PB25;PB58;PC11;PC86;PC97;

[0640] M[20P]-340:PA11;PA13;PA15;PA18;PA21;PA26;PA34;PA36;PA69;PA79;PA95;PB00;PB05;PB07;PB25;PB48;PB60;PC35;PC74;PC84;

[0641] M[20P]-349:PA10;PA11;PA13;PA16;PA20;PA33;PA69;PA94;PB07;PB08;PB12;PC01;PC27;PC28;PC49;PC50;PC53;PC55;PC60;PC67;

[0642] M[20P]-371:PA05;PA14;PA15;PA21;PA22;PA23;PA25;PA27;PA32;PA34;PA35;PA57;PA72;PA89;PB12;PB39;PB88;PC20;PC55;PC74;

[0643] M[20P]-374:PA06;PA13;PA21;PA24;PA25;PA26;PA27;PA57;PB12;PB24;PB28;PB29;PB35;PB38;PB61;PC11;PC37;PC54;PC74;PC86;

[0644] M[20P]-375:PA11;PA15;PA19;PA20;PA21;PA22;PA28;PA29;PA67;PA81;PB10;PB12;PB26;PB28;PC23;PC52;PC58;PC60;PC73;PC98;

[0645] M[20P]-377:PA07;PA10;PA11;PA13;PA14;PA21;PA22;PA24;PA29;PA32;PA33;PA35;PA57;PB10;PB28;PB36;PB82;PC49;PC52;PC94;

[0646] M[20P]-380:PA13;PA15;PA16;PA21;PA24;PA28;PA33;PA38;PA40;PA72;PB12;PB26;PB27;PB82;PC14;PC33;PC55;PC57;PC58;PC98;

[0647] M[20P]-382:PA00;PA12;PA13;PA16;PA21;PA24;PA25;PA26;PA28;PA29;PA35;PA57;PA75;PB33;PB88;PC14;PC24;PC36;PC58;PC66;

[0648] M[20P]-383:PA10;PA21;PA22;PA23;PA25;PA27;PA81;PA85;PB12;PB26;PB28;PB39;PB77;PB82;PC01;PC17;PC37;PC50;PC52;PC67;

[0649] M[20P]-386:PA13;PA14;PA28;PA29;PA33;PA35;PA40;PA57;PA67;PA79;PA88;PB00;PB01;PB07;PB12;PB33;PB77;PB85;PC50;PC66;

[0650] M[20P]-387:PA00;PA05;PA10;PA21;PA23;PA24;PA29;PA40;PA94;PA99;PB12;PB82;PB88;PB99;PC11;PC20;PC24;PC34;PC37;PC61;

[0651] M[20P]-388:PA12;PA13;PA14;PA15;PA20;PA21;PA26;PA29;PA35;PA40;PA67;PA89;PA98;PB12;PB33;PC01;PC14;PC58;PC68;PC88;

[0652] M[20P]-392:PA11;PA14;PA21;PA23;PA26;PA29;PA30;PA57;PA81;PA85;PA94;PB03;PB07;PB12;PB29;PC33;PC51;PC53;PC71;PC73;

[0653] M[20P]-396:PA05;PA14;PA15;PA17;PA21;PA28;PA29;PA35;PA37;PA39;PA40;PA69;PA72;PA89;PB10;PB12;PC34;PC67;PC71;PC97;

[0654] M[20P]-401:PA13;PA15;PA16;PA23;PA26;PA29;PA33;PA34;PA85;PB12;PB26;PB33;PC21;PC50;PC52;PC53;PC55;PC58;PC67;PC71;

[0655] M[20P]-403:PA13;PA14;PA20;PA22;PA24;PA26;PA33;PA35;PB08;PB12;PB26;PB33;PB50;PB61;PC21;PC27;PC37;PC52;PC54;PC58;

[0656] M[20P]-407:PA20;PA21;PA22;PA25;PA29;PA48;PA51;PA57;PA67;PA75;PA94;PB07;PB12;PC21;PC34;PC41;PC51;PC53;PC55;PC97;

[0657] M[20P]-408:PA11;PA12;PA13;PA15;PA16;PA20;PA21;PA22;PA25;PA26;PA27;PA28;PA29;PB10;PB26;PC20;PC21;PC24;PC25;PC58;

[0658] M[20P]-409:PA10;PA15;PA21;PA28;PA35;PA69;PA89;PA94;PA98;PB07;PB12;PB28;PB35;PB36;PC11;PC23;PC49;PC52;PC73;PC98;

[0659] M[20P]-416:PA11;PA13;PA14;PA20;PA21;PA25;PA26;PA28;PA29;PA67;PA75;PB02;PB12;PB27;PB33;PB60;PB88;PC20;PC49;PC60;

[0660] M[20P]-418:PA10;PA11;PA12;PA13;PA21;PA22;PA23;PA24;PA26;PA39;PB07;PB12;PB38;PC11;PC12;PC20;PC23;PC24;PC49;PC55;

[0661] M[20P]-420:PA10;PA14;PA21;PA22;PA23;PA29;PA30;PA33;PA37;PB01;PB12;PB26;PB33;PB99;PC20;PC21;PC36;PC37;PC73;PC98;

[0662] M[20P]-421:PA13;PA15;PA21;PA25;PA26;PA29;PA39;PA40;PA75;PA94;PB20;PB55;PB82;PC06;PC20;PC24;PC49;PC71;PC74;PC99;

[0663] M[20P]-422:PA10;PA11;PA13;PA18;PA21;PA23;PA25;PA29;PA33;PA57;PA70;PB01;PB07;PB12;PB88;PC11;PC27;PC33;PC55;PC97;

[0664] M[20P]-427:PA06;PA13;PA15;PA21;PA24;PA25;PA27;PA29;PA33;PA75;PA98;PB00;PB28;PB60;PC11;PC20;PC26;PC50;PC55;PC74;

[0665] M[20P]-429:PA11;PA19;PA21;PA23;PA26;PA33;PA40;PA75;PB00;PB03;PB12;PB60;PC11;PC21;PC24;PC26;PC36;PC37;PC68;PC98;

[0666] M[20P]-430:PA00;PA07;PA13;PA23;PA26;PA27;PA34;PA40;PA75;PB07;PB12;PB18;PB28;PB88;PC20;PC41;PC66;PC67;PC71;PC97;

[0667] M[20P]-431:PA00;PA06;PA11;PA12;PA13;PA15;PA18;PA21;PA22;PA25;PA28;PA33;PA81;PB09;PB32;PB33;PB38;PC11;PC33;PC52;

[0668] M[20P]-432:PA12;PA14;PA19;PA21;PA24;PA26;PA33;PA67;PB05;PB09;PB12;PB28;PB32;PB46;PB60;PC20;PC49;PC58;PC71;PC73;

[0669] M[20P]-435:PA10;PA13;PA14;PA15;PA21;PA28;PA33;PA40;PA55;PA57;PB07;PB12;PB58;PB88;PB99;PC12;PC21;PC29;PC66;PC67;

[0670] M[20P]-439:PA10;PA13;PA16;PA21;PA24;PA27;PA29;PA34;PA40;PA60;PA75;PB12;PB27;PB35;PB38;PC25;PC26;PC36;PC51;PC55;

[0671] M[20P]-443:PA00;PA10;PA13;PA15;PA17;PA20;PA29;PA33;PA35;PA40;PB07;PB12;PB26;PB27;PB82;PC12;PC24;PC50;PC60;PD02;

[0672] M[20P]-444:PA11;PA13;PA14;PA18;PA21;PA24;PA28;PA39;PA40;PA57;PA60;PA67;PA88;PB20;PB85;PB88;PC06;PC52;PC57;PC58;

[0673] M[20P]-445:PA11;PA13;PA17;PA20;PA21;PA29;PA30;PA33;PA35;PA40;PA57;PB00;PB12;PB28;PB60;PC11;PC20;PC35;PC50;PC51;

[0674] M[20P]-446:PA13;PA15;PA16;PA21;PA22;PA23;PA25;PA26;PA27;PA29;PA39;PA67;PA75;PB12;PB26;PB27;PC36;PC49;PC60;PC98;

[0675] M[20P]-447:PA11;PA13;PA17;PA19;PA24;PA28;PA30;PA33;PA39;PA40;PA57;PA92;PB07;PB08;PB12;PB33;PB61;PB85;PC58;PC94;

[0676] M[20P]-449:PA10;PA11;PA13;PA14;PA15;PA20;PA21;PA24;PA28;PA40;PA67;PA75;PA95;PB12;PB20;PB35;PC14;PC20;PC52;PC60;

[0677] M[20P]-453:PA10;PA13;PA15;PA18;PA21;PA25;PA29;PA34;PA35;PA67;PA93;PB06;PB07;PB09;PB12;PB20;PB26;PB35;PB99;PC55;

[0678] M[20P]-455:PA00;PA00;PA11;PA11;PA13;PA14;PA18;PA20;PA25;PA28;PA33;PA70;PA75;PB00;PB12;PC20;PC23;PC26;PC67;PC67;

[0679] M[20P]-456:PA14;PA21;PA33;PA34;PA39;PB00;PB07;PB07;PB12;PB27;PB28;PB38;PB88;PC20;PC23;PC26;PC49;PC58;PC67;PC71;

[0680] M[20P]-457:PA00;PA10;PA11;PA12;PA13;PA13;PA14;PA29;PA29;PA57;PA67;PB02;PB07;PB09;PB12;PB38;PC11;PC60;PC97;PC98;

[0681] M[20P]-458:PA10;PA11;PA11;PA13;PA13;PA18;PA21;PA26;PA33;PA57;PA75;PA98;PB12;PB33;PB33;PB38;PB82;PC20;PC55;PC73;

[0682] M[20P]-459:PA00;PA06;PA21;PA26;PA26;PA29;PA33;PA75;PB10;PB10;PB12;PB18;PB32;PB85;PC20;PC24;PC49;PC55;PC66;PC98;

[0683] M[20P]-461:PA06;PA21;PA29;PA29;PA33;PA33;PA39;PA57;PA67;PB07;PB07;PB12;PB26;PB32;PB32;PB85;PC20;PC27;PC52;PC58;

[0684] M[20P]-465:PA11;PA11;PA13;PA16;PA21;PA22;PA27;PA29;PA33;PA69;PB07;PB26;PB28;PB32;PB77;PB88;PB88;PC23;PC60;PC73;

[0685] M[20P]-467:PA00;PA06;PA21;PA21;PA23;PA26;PA33;PA33;PA40;PA67;PB12;PB38;PB88;PC11;PC23;PC23;PC27;PC55;PC71;PC97;

[0686] M[20P]-469:PA00;PA12;PA13;PA13;PA13;PA21;PA29;PA33;PA33;PA34;PA57;PB07;PC11;PC11;PC20;PC23;PC23;PC55;PC74;PC97;

[0687] M[20P]-470:PA13;PA14;PA21;PA21;PA22;PA23;PA24;PA24;PA27;PA29;PA33;PA67;PA75;PB07;PB07;PB33;PB33;PB77;PC37;PC52;

[0688] M[20P]-472:PA10;PA10;PA11;PA13;PA21;PA25;PA25;PA28;PA33;PA39;PB07;PB12;PB88;PC12;PC12;PC20;PC20;PC24;PC55;PC71;

[0689] M[20P]-474:PA00;PA13;PA20;PA21;PA26;PA28;PA29;PA33;PA57;PA67;PA81;PA81;PA98;PB02;PB12;PB38;PB60;PB88;PC11;PC20;

[0690] M[20P]-475:PA11;PA12;PA12;PA13;PA28;PA28;PA29;PA29;PA32;PA52;PA52;PA75;PA81;PB12;PB20;PB33;PB60;PC21;PC49;PC51;

[0691] M[20P]-478:PA15;PA16;PA21;PA21;PA21;PA23;PA25;PA32;PA34;PA39;PB12;PB77;PB85;PB88;PB88;PB88;PB88;PC11;PC33;PC37;

[0692] M[20P]-479:PA10;PA11;PA13;PA13;PA18;PA20;PA21;PA28;PA32;PA40;PA67;PA69;PB07;PB07;PB10;PB12;PB20;PC55;PC60;PC98;

[0693] M[20P]-480:PA00;PA06;PA13;PA22;PA23;PA26;PA26;PA27;PA29;PA40;PA81;PB07;PB10;PB10;PB12;PB12;PB18;PB32;PC49;PC71;

[0694] M[20P]-481:PA11;PA13;PA26;PA29;PA29;PA35;PA69;PA75;PA75;PB02;PB12;PB33;PC20;PC23;PC23;PC37;PC41;PC50;PC52;PC98;

[0695] M[20P]-482:PA06;PA10;PA11;PA11;PA11;PA13;PA13;PA13;PA14;PA14;PA20;PA25;PA26;PA28;PA39;PA67;PB12;PC11;PC24;PC74;

[0696] M[20P]-484:PA11;PA13;PA23;PA28;PA29;PA29;PA35;PA39;PA40;PA60;PA75;PA98;PB02;PB12;PB38;PB38;PB82;PC20;PC35;PC50;

[0697] M[20P]-485:PA13;PA14;PA14;PA21;PA21;PA21;PA26;PA26;PA26;PA26;PA29;PA29;PA52;PA57;PB00;PC11;PC11;PC21;PC51;PC67;

[0698] M[20P]-486:PA11;PA13;PA23;PA24;PA35;PA35;PA35;PA69;PA69;PA75;PB07;PB12;PB27;PC20;PC20;PC21;PC23;PC50;PC55;PC73;

[0699] M[20P]-490:PA10;PA11;PA11;PA14;PA21;PA24;PA26;PA26;PA39;PA40;PA69;PA75;PB00;PB01;PB12;PB12;PB18;PB28;PB60;PC20;

[0700] M[20P]-493:PA06;PA14;PA20;PA21;PA21;PA28;PA29;PA33;PA69;PA81;PA98;PB12;PB38;PB60;PC11;PC12;PC49;PC55;PC71;PD02;

[0701] M[20P]-494:PA06;PA10;PA11;PA11;PA13;PA13;PA15;PA23;PA26;PA35;PB01;PB12;PB60;PC12;PC20;PC37;PC41;PC49;PC49;PC52;

[0702] M[20P]-495:PA10;PA10;PA13;PA14;PA18;PA20;PA21;PA26;PA29;PA29;PA57;PB02;PC11;PC11;PC20;PC20;PC24;PC33;PC49;PC49;

[0703] M[20P]-496:PA00;PA18;PA21;PA24;PA26;PA27;PA29;PA39;PA39;PA52;PA69;PA70;PA98;PB00;PB02;PB12;PC12;PC24;PC55;PC67;

[0704] M[20P]-497:PA11;PA11;PA13;PA13;PA15;PA21;PA23;PA24;PA25;PA26;PA28;PA39;PA52;PA67;PB00;PB12;PB27;PB28;PC12;PC37;

[0705] M[20P]-499:PA13;PA21;PA21;PA21;PA29;PA30;PA69;PA75;PB07;PB07;PB07;PB60;PC20;PC20;PC21;PC37;PC37;PC49;PC55;PD02;

[0706] M[20P]-503:PA06;PA06;PA10;PA10;PA13;PA13;PA20;PA21;PA25;PA26;PA29;PA33;PA39;PA70;PB28;PC11;PC20;PC23;PC49;PC97;

[0707] M[20P]-506:PA11;PA16;PA21;PA23;PA29;PA39;PA57;PB12;PB27;PB32;PB88;PB88;PB88;PC12;PC20;PC21;PC37;PC71;PD02;PD02;

[0708] M[20P]-510:PA13;PA21;PA25;PA26;PA27;PA33;PA39;PA40;PA69;PA81;PB01;PB28;PB60;PB85;PB88;PC27;PC41;PC41;PC50;PC55;

[0709] M[20P]-514:PA11;PA12;PA13;PA13;PA21;PA21;PA28;PA29;PA33;PA35;PA39;PA40;PA40;PA67;PA67;PB12;PC14;PC20;PC41;PC55;

[0710] M[20P]-516:PA07;PA10;PA13;PA14;PA17;PA21;PA28;PA29;PA39;PA40;PA40;PA69;PB07;PB12;PC21;PC21;PC27;PC37;PD02;PD02;

[0711] M[20P]-520:PA11;PA11;PA12;PA13;PA14;PA15;PA29;PA33;PA33;PA57;PA67;PA70;PA98;PB12;PC20;PC23;PC33;PC52;PC55;PD02;

[0712] M[20P]-523:PA10;PA11;PA18;PA20;PA21;PA33;PA39;PA67;PA67;PA69;PB07;PB12;PB28;PB88;PB88;PC21;PC33;PC35;PC49;PC50;

[0713] M[20P]-524:PA00;PA11;PA11;PA12;PA13;PA20;PA29;PA40;PB07;PB12;PB28;PB38;PB60;PB60;PB77;PC20;PC20;PC49;PC49;PC98;

[0714] M[20P]-525:PA11;PA11;PA13;PA15;PA21;PA26;PA26;PA27;PA28;PA29;PA29;PA30;PA40;PA52;PA75;PB07;PB07;PC11;PC33;PC37;

[0715] M[20P]-527:PA10;PA10;PA11;PA13;PA15;PA20;PA20;PA21;PA23;PA26;PA26;PA26;PA33;PB08;PB12;PB12;PB60;PB88;PC12;PC20;

[0716] M[20P]-532:PA14;PA20;PA20;PA21;PA22;PA25;PA26;PA27;PA28;PA29;PA33;PA40;PA75;PB00;PB12;PB12;PB60;PB85;PC12;PC51;

[0717] M[20P]-533:PA13;PA21;PA26;PA27;PA27;PA28;PA32;PA60;PA70;PA75;PB18;PB32;PB38;PC23;PC41;PC50;PC50;PC51;PC52;PC97;

[0718] M[20P]-535:PA11;PA21;PA26;PA28;PA29;PA29;PA29;PA34;PA57;PB07;PB12;PB12;PB12;PB33;PB60;PC20;PC20;PC51;PC55;PC67;

[0719] M[20P]-536:PA00;PA10;PA11;PA12;PA12;PA13;PA14;PA23;PA29;PA29;PA35;PA40;PA81;PB07;PB07;PB12;PB27;PB33;PB60;PC20;

[0720] M[20P]-538:PA21;PA23;PA25;PA26;PA27;PA28;PA75;PB07;PB12;PB27;PB32;PB88;PC11;PC11;PC12;PC20;PC33;PC49;PC55;PC97;

[0721] M[20P]-541:PA10;PA11;PA13;PA20;PA24;PA28;PA29;PA67;PA70;PB10;PB12;PB77;PC20;PC20;PC23;PC41;PC51;PC52;PC52;PC97;

[0722] M[20P]-548:PA10;PA13;PA14;PA14;PA22;PA27;PA27;PA28;PA29;PA29;PA33;PA39;PB10;PB12;PB60;PB88;PC20;PC37;PC50;PC55;

[0723] M[20P]-551:PA12;PA13;PA13;PA20;PA22;PA23;PA24;PA24;PA26;PA33;PA33;PA39;PA67;PB07;PB07;PB12;PB33;PC20;PC55;PC98;

[0724] M[20P]-552:PA06;PA11;PA11;PA13;PA14;PA14;PA20;PA21;PA21;PA21;PA24;PA35;PB00;PB02;PB12;PB60;PB60;PC12;PC21;PC98;

[0725] M[20P]-555:PA00;PA10;PA11;PA18;PA20;PA20;PA21;PA25;PA26;PA26;PA39;PA39;PA39;PA89;PB12;PB12;PC20;PC20;PC49;PC74;

[0726] M[20P]-557:PA00;PA10;PA20;PA21;PA23;PA24;PA29;PA57;PB07;PB12;PB12;PB12;PB12;PC11;PC11;PC12;PC20;PC51;PC55;PC67;

[0727] M[20P]-559:PA00;PA11;PA12;PA13;PA20;PA22;PA24;PA25;PA25;PA26;PA28;PA57;PA70;PA70;PB07;PB12;PB88;PB88;PC11;PC60;

[0728] M[20P]-560:PA10;PA10;PA11;PA12;PA13;PA14;PA20;PA25;PA28;PA28;PA29;PA67;PB12;PB38;PB60;PC24;PC35;PC41;PC51;PC97;

[0729] M[20P]-563:PA12;PA13;PA20;PA20;PA21;PA24;PA25;PA25;PA29;PA29;PA33;PA81;PB33;PB60;PB60;PB88;PB88;PB88;PC11;PC41;

[0730] M[20P]-564:PA07;PA13;PA13;PA21;PA26;PA37;PA39;PA67;PA67;PB02;PB07;PB07;PB12;PB12;PB28;PC12;PC37;PC97;PC97;PC97;

[0731] M[20P]-565:PA07;PA11;PA13;PA20;PA29;PA29;PA32;PA75;PA95;PA98;PB02;PB12;PB32;PB33;PB60;PB85;PC20;PC24;PC35;PC41;

[0732] M[20P]-567:PA10;PA13;PA14;PA16;PA21;PA26;PA28;PA75;PA81;PB07;PB07;PB12;PB88;PC23;PC24;PC24;PC33;PC52;PC55;PC67;

[0733] M[20P]-569:PA13;PA14;PA21;PA26;PA27;PA39;PA57;PA67;PB02;PB07;PB12;PB28;PC12;PC21;PC49;PC55;PC55;PC67;PC67;PC98;

[0734] M[20P]-572:PA13;PA14;PA21;PA27;PA29;PA33;PA67;PA69;PA79;PB00;PB01;PB12;PB33;PB38;PC37;PC41;PC49;PC51;PC55;PD02;

[0735] M[20P]-573:PA10;PA11;PA13;PA13;PA13;PA14;PA18;PA21;PA23;PA29;PA67;PB00;PB07;PB12;PB33;PB77;PC11;PC52;PC60;PC71;

[0736] M[20P]-574:PA13;PA21;PA22;PA23;PA25;PA29;PA35;PA67;PB01;PB12;PB12;PB35;PB88;PC24;PC37;PC37;PC51;PC52;PC52;PC66;

[0737] M[20P]-575:PA11;PA11;PA13;PA13;PA13;PA14;PA14;PA26;PA26;PA28;PA52;PA67;PB00;PB12;PB27;PB28;PB38;PB60;PC37;PC52;

[0738] M[20P]-576:PA11;PA11;PA12;PA13;PA13;PA13;PA14;PA14;PA20;PA21;PA22;PA29;PA39;PB12;PB60;PC20;PC26;PC33;PC37;PC49;

[0739] M[20P]-577:PA06;PA13;PA13;PA14;PA22;PA22;PA23;PA26;PA28;PA69;PA70;PB07;PB12;PB12;PB32;PC12;PC20;PC24;PC24;PC98;

[0740] M[20P]-579:PA12;PA13;PA13;PA13;PA14;PA26;PA26;PA28;PA57;PA81;PB02;PB07;PB12;PC11;PC12;PC20;PC51;PC55;PD02;PD02;

[0741] M[20P]-580:PA00;PA11;PA12;PA13;PA20;PA21;PA21;PA25;PA26;PA26;PA33;PA75;PA75;PA75;PB28;PB88;PC20;PC76;PC98;PC98;

[0742] M[20P]-581:PA10;PA11;PA13;PA21;PA22;PA25;PA29;PA67;PB07;PB60;PB88;PB88;PC20;PC21;PC24;PC37;PC41;PC55;PC66;PC98;

[0743] M[20P]-582:PA10;PA11;PA12;PA13;PA20;PA21;PA23;PA25;PA39;PA75;PB07;PB07;PB38;PB38;PB60;PB60;PC20;PC24;PC41;PC66;

[0744] M[20P]-583:PA11;PA13;PA14;PA23;PA25;PA25;PA26;PA26;PA28;PA67;PB12;PB18;PB88;PC24;PC24;PC37;PC41;PC49;PC55;PC60;

[0745] M[20P]-584:PA00;PA14;PA21;PA26;PA26;PA28;PA39;PA39;PA67;PA75;PB12;PB12;PB27;PB33;PB60;PC11;PC41;PC49;PC55;PC97;

[0746] M[20P]-588:PA07;PA13;PA14;PA21;PA26;PA26;PA26;PA26;PA32;PA67;PA67;PA75;PB12;PB27;PB33;PB60;PC20;PC20;PC23;PC24;

[0747] M[20P]-589:PA13;PA13;PA22;PA25;PA25;PA27;PA29;PA29;PA35;PA39;PA52;PA75;PB12;PB32;PB38;PB85;PB88;PC11;PC23;PC98;

[0748] M[20P]-590:PA11;PA13;PA13;PA14;PA25;PA29;PA33;PA35;PA35;PA35;PA39;PB02;PB12;PC11;PC20;PC20;PC21;PC27;PC55;PC55;

[0749] M[20P]-592:PA10;PA10;PA11;PA11;PA13;PA14;PA21;PA21;PA26;PA26;PA27;PA29;PA30;PA75;PB07;PB07;PB12;PB38;PC20;PC24;

[0750] M[20P]-594:PA13;PA21;PA21;PA21;PA23;PA27;PA28;PA39;PA60;PA69;PA70;PB02;PB09;PB12;PB12;PB88;PC24;PC49;PC97;PC98;

[0751] M[20P]-595:PA11;PA13;PA22;PA23;PA25;PA28;PA28;PA28;PA29;PA29;PA39;PA67;PA69;PB12;PB60;PC12;PC20;PC24;PC49;PC55;

[0752] M[20P]-596:PA11;PA11;PA13;PA13;PA14;PA21;PA22;PA23;PA26;PA26;PA67;PB12;PB60;PC11;PC20;PC20;PC37;PC50;PC55;PC60;

[0753] M[20P]-599:PA13;PA13;PA14;PA16;PA26;PA29;PA75;PA75;PB07;PB12;PB60;PB60;PB60;PB60;PC12;PC20;PC20;PC37;PC37;PC52;

[0754] M[20P]-601:PA11;PA11;PA13;PA13;PA13;PA23;PA26;PA29;PA29;PA35;PB02;PB10;PB12;PB33;PC20;PC21;PC41;PC49;PC60;PC67;

[0755] M[20P]-602:PA00;PA10;PA10;PA14;PA14;PA14;PA21;PA25;PA26;PA29;PA29;PB07;PB07;PB07;PB12;PB12;PC20;PC52;PC55;PD02;

[0756] M[20P]-603:PA11;PA13;PA16;PA20;PA21;PA23;PA23;PA26;PA26;PA28;PA33;PA39;PA39;PA67;PB60;PB60;PC20;PC23;PC49;PC55;

[0757] M[20P]-604:PA10;PA13;PA14;PA14;PA18;PA21;PA22;PA23;PA26;PA28;PA28;PA29;PB12;PB33;PC11;PC11;PC51;PC51;PC60;PC71;

[0758] M[20P]-605:PA11;PA11;PA11;PA11;PA13;PA13;PA13;PA20;PA25;PA29;PA39;PA39;PA69;PA81;PB07;PB12;PB88;PC20;PC41;PC55;

[0759] M[20P]-607:PA10;PA11;PA11;PA13;PA13;PA13;PA22;PA22;PA26;PA33;PA35;PA75;PA75;PA81;PB12;PB88;PC20;PC49;PC55;PC97;

[0760] M[20P]-608:PA00;PA13;PA13;PA13;PA18;PA26;PA27;PA29;PA35;PA57;PA69;PA75;PA98;PB12;PB60;PC11;PC20;PC20;PC55;PC60;

[0761] M[20P]-609:PA11;PA14;PA21;PA22;PA23;PA26;PA34;PA52;PA67;PA69;PA69;PB07;PB12;PB12;PB27;PB77;PC12;PC20;PC55;PC97;

[0762] M[20P]-612:PA11;PA13;PA14;PA14;PA14;PA21;PA22;PA23;PA52;PA81;PB00;PB07;PB12;PB33;PB38;PB60;PC12;PC24;PC33;PC37;

[0763] M[20P]-613:PA10;PA13;PA13;PA14;PA21;PA21;PA22;PA23;PA26;PA28;PA28;PA29;PB12;PB12;PB27;PB60;PC20;PC21;PC21;PC60;

[0764] M[20P]-616:PA13;PA26;PA28;PA29;PA39;PA57;PA67;PB07;PB12;PB38;PB60;PB77;PB77;PB88;PC33;PC49;PC52;PC52;PC98;PC98;

[0765] M[20P]-618:PA11;PA12;PA13;PA13;PA14;PA20;PA21;PA22;PA23;PA26;PA29;PA67;PA79;PB88;PC11;PC20;PC20;PC37;PC41;PC55;

[0766] M[20P]-620:PA12;PA13;PA20;PA22;PA22;PA23;PA26;PA26;PA28;PA39;PA69;PA95;PB12;PB60;PB77;PC20;PC21;PC52;PC60;PC98;

[0767] M[20P]-621:PA10;PA13;PA14;PA24;PA24;PA26;PA27;PA29;PA32;PA35;PA67;PB12;PB12;PB12;PB27;PB33;PB33;PB38;PB38;PB38;

[0768] M[20P]-622:PA10;PA12;PA14;PA14;PA15;PA20;PA21;PA26;PA27;PA32;PA40;PB10;PB12;PB12;PB88;PC20;PC20;PC21;PC52;PC60;

[0769] M[20P]-623:PA10;PA11;PA13;PA14;PA20;PA24;PA25;PA26;PA26;PA28;PA39;PA81;PB09;PB12;PB28;PB88;PC24;PC41;PC52;PC98;

[0770] M[20P]-626:PA11;PA11;PA13;PA13;PA20;PA21;PA21;PA25;PA27;PA27;PA28;PA39;PA39;PA40;PA57;PB12;PB60;PC20;PC37;PC41;

[0771] M[20P]-631:PA10;PA13;PA20;PA21;PA21;PA23;PA23;PA26;PA29;PA39;PA39;PA67;PB10;PB38;PB60;PC11;PC20;PC37;PC49;PC66;

[0772] M[20P]-632:PA10;PA11;PA18;PA21;PA22;PA23;PA28;PA29;PA33;PA34;PA69;PB07;PB12;PB60;PC20;PC20;PC20;PC37;PC41;PC55;

[0773] M[20P]-633:PA10;PA11;PA11;PA13;PA20;PA34;PA39;PB00;PB07;PB12;PB12;PB60;PB88;PB88;PC11;PC12;PC23;PC35;PC49;PC52;

[0774] M[20P]-634:PA13;PA13;PA13;PA21;PA22;PA22;PA25;PA25;PA28;PA28;PA29;PA67;PA81;PB00;PB12;PB12;PB27;PB33;PC11;PC98;

[0775] M[20P]-635:PA00;PA14;PA17;PA20;PA21;PA23;PA26;PA27;PA75;PA81;PB00;PB12;PB82;PC11;PC20;PC26;PC51;PC52;PC55;PC71;

[0776] M[20P]-638:PA00;PA14;PA17;PA21;PA22;PA24;PA28;PA33;PA52;PA57;PA75;PB00;PB07;PB12;PB28;PB88;PC20;PC37;PC41;PC97;

[0777] M[20P]-649:PA06;PA10;PA11;PA12;PA13;PA15;PA16;PA20;PA24;PA30;PA33;PA98;PB10;PB12;PB28;PB32;PB60;PC11;PC20;PC66;

[0778] M[20P]-654:PA00;PA11;PA15;PA20;PA21;PA26;PA32;PA34;PA35;PA39;PA57;PB10;PB12;PB18;PC11;PC20;PC21;PC50;PC55;PC66;

[0779] M[20P]-655:PA11;PA13;PA14;PA20;PA22;PA26;PA28;PA30;PA40;PA52;PA57;PA81;PB12;PB32;PB85;PC41;PC50;PC55;PC66;PC71;

[0780] M[20P]-658:PA10;PA11;PA12;PA13;PA14;PA15;PA17;PA20;PA21;PA28;PA29;PA52;PA67;PB10;PB12;PB18;PB60;PC20;PC41;PC66;

[0781] M[20P]-659:PA00;PA11;PA21;PA22;PA24;PA26;PA27;PA28;PA29;PA33;PA35;PA81;PB07;PB12;PB18;PB28;PB85;PC37;PC49;PC74;

[0782] M[20P]-663:PA06;PA13;PA20;PA25;PA26;PA27;PA28;PA40;PA67;PA81;PB00;PB12;PB18;PB33;PB61;PC20;PC24;PC37;PC41;PC52;

[0783] M[20P]-666:PA06;PA13;PA21;PA23;PA27;PA28;PA30;PA40;PA75;PB00;PB07;PB10;PB12;PB18;PB28;PB33;PC20;PC49;PC52;PC71;

[0784] M[20P]-668:PA10;PA16;PA17;PA21;PA24;PA27;PA28;PA33;PA40;PA57;PA75;PA79;PB07;PB08;PB12;PB33;PB88;PC20;PC35;PC97;

[0785] M[20P]-670:PA11;PA12;PA13;PA14;PA20;PA23;PA27;PA28;PA35;PA39;PA67;PA79;PA81;PA89;PB10;PB12;PB18;PC20;PC21;PC51;

[0786] M[20P]-678:PA06;PA11;PA12;PA13;PA21;PA23;PA29;PA35;PA39;PA40;PB00;PB07;PB10;PB12;PB33;PB60;PB77;PC11;PC67;PC76;

[0787] M[20P]-681:PA07;PA10;PA11;PA21;PA24;PA26;PA30;PA35;PA39;PA57;PA75;PB07;PB12;PB28;PB88;PC11;PC49;PC55;PC58;PC66;

[0788] M[20P]-686:PA00;PA10;PA13;PA22;PA24;PA27;PA28;PA33;PA39;PA40;PA69;PB12;PB32;PB38;PB85;PB88;PC20;PC55;PC60;PC66;

[0789] M[20P]-692:PA07;PA10;PA13;PA21;PA22;PA26;PA34;PA39;PA40;PA81;PB12;PB18;PB32;PB33;PB88;PC35;PC37;PC52;PC55;PC76;

[0790] M[20P]-694:PA07;PA11;PA13;PA14;PA15;PA16;PA21;PA27;PA28;PA29;PA35;PA39;PA40;PA75;PB12;PB33;PB88;PC50;PC51;PC52;

[0791] M[20P]-697:PA00;PA13;PA16;PA19;PA21;PA22;PA25;PA27;PA28;PA39;PA79;PA81;PB00;PB12;PB32;PB60;PB99;PC51;PC58;PC76;

[0792] M[20P]-703:PA00;PA13;PA14;PA15;PA24;PA26;PA33;PA35;PA52;PA75;PA81;PB07;PB12;PB28;PB85;PC20;PC21;PC35;PC41;PD02;

[0793] M[20P]-705:PA11;PA13;PA30;PA33;PA40;PA67;PA75;PB07;PB12;PB28;PB35;PB88;PC20;PC21;PC24;PC35;PC37;PC41;PC50;PC55;

[0794] M[20P]-708:PA07;PA13;PA23;PA25;PA27;PA29;PA30;PA33;PA52;PA57;PA75;PB00;PB08;PB12;PB38;PB60;PB77;PC50;PC76;PD02;

[0795] M[20P]-709:PA11;PA13;PA17;PA20;PA21;PA23;PA26;PA27;PA28;PA40;PA69;PA81;PA92;PB00;PB07;PB12;PC20;PC41;PC52;PC66;

[0796] M[20P]-712:PA12;PA13;PA15;PA20;PA22;PA26;PA27;PA29;PA35;PA52;PA81;PB07;PB10;PB12;PB33;PB88;PC11;PC20;PC41;PC52;

[0797] M[20P]-714:PA07;PA10;PA11;PA16;PA21;PA26;PA33;PA40;PA67;PA81;PA98;PB00;PB07;PB12;PB28;PC11;PC41;PC49;PC50;PC98;

[0798] M[20P]-716:PA11;PA13;PA15;PA19;PA22;PA26;PA33;PA37;PA39;PA57;PB07;PB12;PB28;PB33;PB88;PB99;PC14;PC23;PC35;PC67;

[0799] M[20P]-718:PA12;PA13;PA15;PA16;PA20;PA22;PA25;PA26;PA39;PA40;PA92;PB07;PB12;PB27;PB35;PB38;PB99;PC11;PC27;PC41;

[0800] M[20P]-721:PA19;PA20;PA21;PA24;PA25;PA27;PA29;PA30;PA55;PA57;PB12;PB32;PB58;PB60;PB88;PB99;PC41;PC50;PC60;PC67;

[0801] M[20P]-723:PA12;PA13;PA16;PA17;PA21;PA28;PA29;PA33;PA34;PA40;PA60;PA89;PB28;PB60;PB61;PC14;PC24;PC41;PC51;PC74;

[0802] M[20P]-724:PA13;PA23;PA25;PA35;PA57;PA67;PB07;PB12;PB32;PB33;PB88;PB99;PC12;PC20;PC23;PC49;PC50;PC66;PC67;PC94;

[0803] M[20P]-725:PA00;PA13;PA14;PA20;PA22;PA25;PA26;PA28;PA33;PA40;PA57;PA81;PB12;PB16;PB33;PB88;PC11;PC24;PC51;PC74;

[0804] M[20P]-733:PA11;PA13;PA15;PA21;PA28;PA30;PA33;PA40;PB12;PB32;PB33;PB99;PC11;PC12;PC20;PC60;PC73;PC74;PC94;PD02;

[0805] M[20P]-737:PA00;PA11;PA13;PA15;PA17;PA21;PA24;PA28;PA29;PA30;PA67;PB07;PB27;PB28;PB77;PC20;PC21;PC41;PC51;PC94;

[0806] M[20P]-740:PA11;PA13;PA14;PA15;PA22;PA23;PA28;PA33;PA39;PA57;PA75;PA89;PB02;PB12;PB28;PB33;PC12;PC21;PC24;PC55;

[0807] M[20P]-745:PA11;PA21;PA22;PA24;PA26;PA27;PA28;PA29;PA57;PA89;PB07;PB12;PB28;PB33;PC08;PC11;PC41;PC66;PC67;PC98;

[0808] M[20P]-746:PA20;PA21;PA25;PA26;PA28;PA33;PA39;PA40;PA81;PB06;PB07;PB12;PB32;PB60;PC20;PC21;PC24;PC37;PC50;PC55;

[0809] M[20P]-748:PA11;PA13;PA14;PA17;PA23;PA26;PA32;PA57;PA60;PA81;PB07;PB12;PB28;PB32;PB33;PB85;PB99;PC20;PC21;PC35;

[0810] M[20P]-752:PA11;PA13;PA18;PA26;PA28;PA29;PA33;PA35;PA39;PA67;PA75;PB07;PB12;PB32;PB88;PB99;PC12;PC50;PC54;PC62;

[0811] M[20P]-753:PA11;PA12;PA13;PA14;PA15;PA29;PA32;PA40;PA57;PA81;PB00;PB12;PB33;PB60;PC20;PC21;PC49;PC52;PC66;PD02;

[0812] M[20P]-757:PA11;PA13;PA21;PA22;PA23;PA27;PA34;PA35;PA40;PA57;PA67;PB10;PB12;PB22;PB58;PC11;PC14;PC23;PC55;PC98;

[0813] M[20P]-758:PA10;PA11;PA13;PA20;PA21;PA23;PA28;PA29;PA30;PA40;PA69;PA75;PA81;PB12;PC06;PC20;PC41;PC52;PC57;PC74;

[0814] M[20P]-764:PA11;PA13;PA22;PA29;PA35;PA40;PA57;PA73;PA75;PA81;PB08;PB12;PB33;PB82;PC11;PC20;PC21;PC29;PC41;PC52;

[0815] M[20P]-765:PA10;PA11;PA13;PA15;PA26;PA27;PA30;PA35;PA40;PA89;PB12;PB22;PB58;PB60;PC34;PC37;PC55;PC58;PC67;PD02;

[0816] M[20P]-766:PA11;PA13;PA17;PA22;PA24;PA25;PA29;PA30;PA33;PA34;PA35;PA81;PA92;PB00;PB12;PB82;PC24;PC50;PC74;PC97;

[0817] M[20P]-767:PA11;PA21;PA22;PA28;PA29;PA33;PA37;PB12;PB60;PB85;PB88;PB99;PC14;PC20;PC21;PC41;PC51;PC55;PC57;PC98;

[0818] M[20P]-771:PA10;PA11;PA13;PA14;PA22;PA26;PA27;PA28;PA29;PA35;PA86;PA89;PB07;PB12;PB28;PB88;PB99;PC12;PC21;PC50;

[0819] M[20P]-777:PA11;PA14;PA15;PA20;PA21;PA24;PA25;PA28;PA29;PA33;PA75;PB12;PB85;PB99;PC21;PC51;PC55;PC62;PC67;PD02;

[0820] M[20P]-779:PA15;PA19;PA20;PA21;PA22;PA23;PA24;PA25;PA29;PA30;PA57;PA67;PA75;PA89;PB07;PB12;PB32;PC20;PC51;PC74;

[0821] M[20P]-781:PA10;PA15;PA21;PA22;PA39;PA57;PA75;PB08;PB09;PB12;PB28;PB35;PB58;PB60;PC12;PC33;PC37;PC66;PC67;PC74;

[0822] M[20P]-782:PA10;PA11;PA12;PA13;PA14;PA20;PA26;PA30;PA75;PA89;PA92;PB05;PB07;PB12;PB32;PB82;PB99;PC51;PC58;PC73;

[0823] M[20P]-785:PA16;PA17;PA21;PA28;PA29;PA30;PA32;PA33;PA55;PB07;PB12;PB77;PB85;PB88;PB99;PC12;PC20;PC35;PC67;PC98;

[0824] M[20P]-786:PA11;PA13;PA14;PA18;PA19;PA21;PA22;PA27;PA28;PA29;PA33;PA40;PA52;PA57;PA81;PB28;PC11;PC20;PC21;PC50;

[0825] M[20P]-787:PA10;PA11;PA13;PA22;PA23;PA35;PB07;PB12;PB22;PB85;PB99;PC11;PC20;PC41;PC49;PC52;PC53;PC55;PC73;PD02;

[0826] M[20P]-789:PA10;PA11;PA13;PA22;PA23;PA27;PA40;PA57;PA75;PA81;PB07;PB12;PB27;PB60;PB88;PC11;PC20;PC23;PC35;PC41;

[0827] M[20P]-791:PA10;PA11;PA13;PA15;PA21;PA33;PA35;PA40;PA57;PA69;PA85;PA98;PA99;PB07;PB12;PB20;PB99;PC12;PC20;PC58;

[0828] M[20P]-793:PA11;PA13;PA14;PA15;PA19;PA26;PA29;PA55;PA67;PA89;PB12;PB33;PB88;PC14;PC20;PC21;PC34;PC37;PC67;PC97;

[0829] M[20P]-794:PA21;PA23;PA28;PA29;PA34;PA35;PA57;PA75;PA81;PB07;PB12;PB18;PB32;PB58;PC11;PC14;PC41;PC55;PC62;PC74;

[0830] M[20P]-795:PA10;PA13;PA20;PA23;PA27;PA29;PA57;PA60;PA69;PB00;PB07;PB12;PB18;PB32;PB85;PB88;PC20;PC66;PC74;PD02;

[0831] M[20P]-803:PA11;PA13;PA15;PA22;PA25;PA26;PA29;PA30;PA33;PA40;PA75;PB07;PB12;PB28;PC21;PC23;PC34;PC55;PC71;PC73;

[0832] M[20P]-804:PA11;PA12;PA13;PA20;PA22;PA23;PA30;PA33;PA34;PA79;PB12;PB18;PB58;PB60;PB85;PB88;PC20;PC50;PC67;PC68;

[0833] M[20P]-806:PA06;PA10;PA11;PA13;PA19;PA20;PA21;PA23;PA25;PA28;PA52;PA73;PA75;PA98;PB12;PB33;PB82;PB85;PB88;PC11;

[0834] M[20P]-816:PA11;PA13;PA19;PA25;PA28;PA29;PA33;PA57;PA81;PA89;PB00;PB10;PB12;PB26;PB28;PB99;PC11;PC12;PC76;PC97;

[0835] M[20P]-819:PA10;PA11;PA13;PA15;PA26;PA34;PA60;PA81;PA92;PB07;PB12;PB38;PB85;PC01;PC14;PC20;PC52;PC58;PC66;PC67;

[0836] M[20P]-823:PA00;PA11;PA12;PA13;PA14;PA17;PA27;PA29;PA40;PA60;PA75;PA79;PA89;PB07;PB12;PB99;PC20;PC52;PC66;PC71;

[0837] M[20P]-830:PA13;PA20;PA25;PA26;PA28;PA40;PA75;PA89;PB00;PB07;PB12;PB32;PB88;PC06;PC12;PC20;PC41;PC52;PC76;PC98;

[0838] M[20P]-831:PA11;PA15;PA21;PA25;PA33;PB12;PB28;PB32;PB33;PB38;PB60;PB77;PB88;PC20;PC23;PC35;PC37;PC49;PC58;PC66;

[0839] M[20P]-841:PA10;PA11;PA13;PA18;PA22;PA28;PA29;PA30;PA35;PA40;PA55;PA75;PB07;PB12;PB28;PB32;PB33;PC41;PC49;PC60;

[0840] M[20P]-844:PA11;PA15;PA21;PA24;PA26;PA29;PA33;PA40;PA69;PA81;PA98;PB12;PB28;PB61;PC49;PC67;PC71;PC73;PC74;PD02;

[0841] M[20P]-845:PA11;PA13;PA14;PA15;PA21;PA30;PA34;PA39;PA60;PA79;PA81;PA89;PB07;PB18;PC14;PC20;PC21;PC34;PC68;PC97;

[0842] M[20P]-846:PA11;PA13;PA25;PA27;PA29;PA32;PA33;PA35;PA37;PA39;PA40;PB08;PB12;PB33;PB88;PC24;PC41;PC52;PC58;PC62;

[0843] M[20P]-847:PA10;PA19;PA20;PA21;PA28;PA29;PA30;PA67;PA75;PA89;PB00;PB12;PB28;PB33;PB60;PC12;PC55;PC73;PC74;PD02;

[0844] M[20P]-848:PA07;PA11;PA13;PA15;PA20;PA22;PA26;PA27;PA30;PA40;PA69;PA75;PB12;PB32;PB85;PB88;PC34;PC37;PC66;PC68;

[0845] M[20P]-849:PA00;PA13;PA25;PA27;PA28;PA29;PA34;PA40;PA57;PA81;PB00;PB12;PB18;PB77;PC20;PC24;PC35;PC41;PC50;PC51;

[0846] M[20P]-852:PA00;PA11;PA13;PA15;PA19;PA22;PA26;PA35;PA89;PB00;PB12;PB58;PB77;PC12;PC20;PC29;PC62;PC71;PC94;PC98;

[0847] M[20P]-853:PA00;PA11;PA21;PA24;PA25;PA33;PA57;PA60;PB07;PB12;PB28;PB32;PB60;PB82;PB88;PC24;PC41;PC50;PC51;PC58;

[0848] M[20P]-857:PA00;PA11;PA13;PA26;PA28;PA75;PA76;PA79;PB12;PB28;PB60;PB88;PC11;PC12;PC20;PC37;PC50;PC52;PC67;PD02;

[0849] M[20P]-873:PA10;PA13;PA26;PA27;PA30;PA34;PA85;PA98;PB05;PB07;PB12;PB28;PB29;PB32;PB60;PC14;PC20;PC26;PC41;PC76;

[0850] M[20P]-880:PA11;PA13;PA17;PA20;PA22;PA24;PA26;PA27;PA29;PA38;PA57;PB07;PB12;PB20;PB29;PB97;PC24;PC37;PC51;PC71;

[0851] M[20P]-888:PA10;PA12;PA15;PA16;PA21;PA27;PA28;PA33;PA44;PB00;PB12;PB13;PB27;PC11;PC20;PC51;PC58;PC73;PC75;PD02;

[0852] M[20P]-890:PA00;PA11;PA13;PA15;PA16;PA25;PA34;PA75;PA82;PA86;PB00;PB12;PB16;PB27;PB33;PC20;PC49;PC50;PC57;PC98;

[0853] M[20P]-894:PA12;PA18;PA21;PA24;PA26;PA27;PA34;PA57;PA67;PA82;PB01;PB12;PB33;PB76;PB98;PB99;PC11;PC24;PC33;PC83;

[0854] M[20P]-902: PA12; PA13; PA21; PA26; PA29; PA39; PA40; PA57; PA96; PB00; PB07; PB12; PB27; PC20; PC50; PC53; PC61; PC62; PC75; PC94;

[0855] M[20P]-904: PA06; PA13; PA20; PA23; PA25; PA27; PA35; PA46; PA52; PA60; PA81; PA90; PA97; PB03; PB12; PB57; PB99; PC20; PC52; PC73;

[0856] M[20P]-907: PA00; PA07; PA14; PA21; PA24; PA25; PA45; PA75; PA83; PA97; PB12; PB26; PB32; PB60; PB92; PC06; PC20; PC35; PC37; PD02;

[0857] M[20P]-909: PA12; PA14; PA20; PA21; PA29; PA39; PA72; PA93; PB12; PB22; PB28; PB32; PC21; PC23; PC24; PC41; PC52; PC73; PC79; PD02.

[0858] NOTE: If, in similar lists above and below, a plant code appears more than once in the same herbal composition, this means that more than one calibrated scoop of the corresponding ground dry plant was used.

[0859] Starting from the herbal composition listed in Table 6-3, the corresponding aqueous extract (or processed sample) was prepared according to the aforementioned method B, using 40 mL of water containing 100 g / l sucrose and including a decantation step before filtration.

[0860] The bioactivity of each processed sample was determined according to the aforementioned method C using the following Staphylococcus aureus strains: ATCC 25923, ATCC 49476, ATCC 6538, ATCC 51740, ATCC 29213 and ATCC 14775, wherein the processed samples had the following final dilutions: 1:20, 1:63 and 1:200.

[0861] The mean bacterial planktonic growth inhibition percentage and the mean biofilm formation inhibition percentage of processed samples obtained from the herbal composition of two plants including the mixture [2P]-1 at a dilution of 1:20 for all tested strains are given in Table 5-1.

[0862] Table 5-1: Average efficacy of the processed samples based on the mixture [2P]-1 at a 1:20 dilution against six tested Staphylococcus aureus strains for percent inhibition of planktonic growth and percent inhibition of biofilm formation. (A) Mixture extracts exhibiting higher antimicrobial and antibiofilm activity. (B) Mixture extracts exhibiting antibiofilm activity and comparable or lower antimicrobial activity. (C) Mixture extracts exhibiting antimicrobial activity and comparable or lower antibiofilm activity.

[0863]

[0864] Twenty processed samples obtained from herbal compositions containing 9, 18, 19, or 20 plants were found to have higher anti-biofilm and antimicrobial activities than the reference mixture extract mixture [2P]-1 (Table 5-1A). Fourteen extracts from the 20-plant mixture were found to have higher anti-biofilm activity and comparable or lower antimicrobial activity (Table 5-1B). Two processed samples obtained from herbal compositions containing 17 and 18 plants were found to have higher antimicrobial activity and comparable or lower anti-biofilm activity (Table 5-1C).

[0865] Table 5-2 shows the mean bacterial planktonic growth inhibition percentage and the mean biofilm formation inhibition percentage of processed samples obtained from the herbal composition of two plants including the mixture [2P]-2 at a dilution of 1:20 for all tested strains.

[0866] Table 5-2: Average percent inhibition of planktonic growth and biofilm formation for six tested Staphylococcus aureus strains by processed samples based on the mixture [2P]-2 at a 1:20 dilution. (A) Mixture extracts exhibiting higher antimicrobial and antibiofilm activity. (B) Mixture extracts exhibiting antimicrobial activity and comparable or lower antibiofilm activity.

[0867]

[0868]

[0869] 58 processed samples obtained from herbal compositions containing 9, 16, 17, 18, 19 or 20 plants were found to have higher anti-biofilm and antimicrobial activities than the reference mixture extract mixture [2P]-2 (Table 5-2A). 25 processed samples obtained from herbal compositions containing 16, 17, 19 or 20 plants were found to have higher anti-biofilm activity and comparable or lower antimicrobial activity (Table 5-2B).

[0870] Table 5-3 shows the mean bacterial planktonic growth inhibition percentage and the mean biofilm formation inhibition percentage of processed samples obtained from the herbal composition of two plants including the mixture [2P]-3 at a dilution of 1:20 for all tested strains.

[0871] Table 5-3: Average efficacy of processed samples based on mixture [2P]-3 at a 1:20 dilution against six tested Staphylococcus aureus strains for percent inhibition of planktonic growth and percent inhibition of biofilm formation. (A) Mixture extracts exhibiting higher antimicrobial and antibiofilm activity. (B) Mixture extracts exhibiting antimicrobial activity and comparable or lower antibiofilm activity.

[0872]

[0873]

[0874]

[0875] 70 processed samples obtained from herbal compositions containing 9, 19 or 20 plants were found to have higher anti-biofilm and antimicrobial activities than the reference mixture extract mixture [2P]-3 (Table 5-3A). 37 processed samples containing 18 to 20 plants were found to have higher anti-biofilm activity and comparable or lower antimicrobial activity (Table 5-3B).

[0876] Table 5-4 shows the mean bacterial planktonic growth inhibition percentage and the mean biofilm formation inhibition percentage of processed samples obtained from the herbal composition of 3 plants including the mixture [3P]-1 at a dilution of 1:20 for all tested strains.

[0877] Table 5-4: Average efficacy of processed samples based on mixture [3P]-1 at a 1:20 dilution against six tested Staphylococcus aureus strains for percent inhibition of planktonic growth and percent inhibition of biofilm formation. (A) Mixture extracts exhibiting higher antimicrobial and antibiofilm activity. (B) Mixture extracts exhibiting antimicrobial activity and comparable or lower antibiofilm activity.

[0878]

[0879]

[0880]

[0881] 51 processed samples obtained from herbal compositions containing 9, 16, 17, 18, 19 or 20 plants were found to have higher anti-biofilm and antimicrobial activities than the reference mixture extract mixture [3P]-1 (Table 5-4A). 113 processed samples containing 9 to 20 plants were found to have higher anti-biofilm activity and comparable or lower antimicrobial activity (Table 5-4B).

[0882] [Comparative Example 3]

[0883] As comparative examples, we report that several 20-plant mixture extracts containing only one of the three plants of Example 1 (PA13, PA21, PB12) have been found to have no or poor antimicrobial and antibiofilm activity against Staphylococcus aureus.

[0884] Ten herbal compositions having the ingredients reported in Lists 5-8 were prepared according to Method A described above.

[0885] [List 5-8: Herbal composition of processed samples]

[0886] M[20P]-39: PB00; PB01; PB02; PB03; PB04; PB05; PB06; PB07; PB08; PB09; PB10; PB11; PB12; PB13; PB14; PB15; PB16; PB17; PB18; PB19;

[0887] M[20P]-239: PA00; PA05; PA21; PA23; PA26; PA33; PA39; PA60; PA85; PB00; PB06; PB32; PB79; PC02; PC26; PC34; PC38; PC62; PC78; PC84;

[0888] M[20P]-337: PA00; PA10; PA11; PA15; PA18; PA21; PA22; PA24; PA26; PA28; PA52; PA94; PA98; PB10; PB20; PB26; PB29; PB82; PC67; PC97;

[0889] M[20P]-362: PA21; PA23; PA25; PA33; PA37; PA57; PA69; PA81; PA92; PB16; PB25; PB26; PB28; PB29; PB78; PC20; PC21; PC71; PC83; PD02;

[0890] M[20P]-364: PA15; PA21; PA25; PA28; PA39; PA75; PA80; PA85; PA89; PA98; PB01; PB06; PB24; PB28; PB35; PC24; PC25; PC57; PC75; PC82;

[0891] M[20P]-540: PA06; PA10; PA12; PA14; PA20; PA21; PA26; PA27; PA27; PA57; PA57; PA57; PA69; PB07; PB82; PC11; PC20; PC23; PC55; PC98;

[0892] M[20P]-647: PA10; PA11; PA24; PA26; PA28; PA33; PA35; PA40; PA57; PA69; PB00; PB07; PB12; PB28; PB33; PB38; PB60; PC55; PC67; PC97;

[0893] M[20P]-652: PA00; PA07; PA13; PA15; PA16; PA23; PA26; PA28; PA33; PA39; PA57;

[0894] M[20P]-862: PA11; PA15; PA18; PA21; PA25; PA27; PA28; PA29; PA35; PA38; PA52; PA86; PA89; PA92; PB07; PB28; PB88; PC11; PC55; PC98;

[0895] M[20P]-872: PA13; PA14; PA16; PA28; PA29; PA30; PA33; PA35; PA39; PA81; PA90; PA92; PB00; PB32; PC12; PC34; PC37; PC67; PC97; PD02.

[0896] According to the aforementioned method B, water extraction was performed using 40 mL of water containing 100 g / l sucrose and including a decantation step before filtration, thereby preparing 10 water extracts (or processed samples) from the corresponding herbal compositions.

[0897] For each processed sample, the biological activity was determined according to Method C described previously, with final dilutions of the processed sample being 1:20, 1:63, and 1:200.

[0898] Table 5-5 shows the mean percentage inhibition of bacterial planktonic growth and biofilm formation for all tested strains for processed samples at a 1:20 dilution.

[0899] Table 5-5: Average values ​​of percent inhibition of planktonic growth and percent inhibition of biofilm formation of processed samples at 1:20 dilution against 6 tested Staphylococcus aureus strains

[0900]

[0901]

[0902] All tested processed samples obtained from the herbal composition containing 20 plants but including only one of the three plants of Example 1 (PA13, PA21, PB12) showed lower antimicrobial activity than the three 2-plant mixture extracts of the reference mixture [2P]-1 (obtained from PA13 and PA21), mixture [2P]-2 (obtained from PA21 and PB12) and mixture [2P]-3 (obtained from PA13 and PB12) and lower anti-biofilm activity than mixture [2P]-1 and mixture [2P]-3.

[0903]

Example 6

[0904] In this example, we report the simultaneous antimicrobial and antibiofilm activities of two processed samples selected from the optimal mixtures described in Examples 4 and 5 against S. aureus, S. epidermidis and S. pseudintermedius.

[0905] Pharmaceutical grade dried plants were obtained from "Pharmacie Fontgiève" (Clermont-Ferrand, France) and "Pharmacie St Herem" (Clermont-Ferrand, France).

[0906] Two herbal preparations containing the dry plant powders reported in Table 6-1 below were prepared according to Method A described above.

[0907]

Table 6-1

[0908] Herbal composition M[20P]-576 M[10P]-154 Plant 1 2 scoops PA11 1 scoop of PA11 Plant 2 1 scoop of PA12 1 scoop of PA12 Plant 3 3 scoops of PA13 1 scoop of PA13 Plant 4 2 scoops of PA14 1 scoop of PA21 Plant 5 1 scoop of PA20 1 scoop of PA22 Plant 6 1 scoop of PA21 1 scoop of PA79 Plant 7 1 scoop of PA22 1 scoop of PB12 Plant 8 1 scoop of PA29 1 scoop of PB49 Plant 9 1 scoop of PA39 1 scoop PC29 Plant 10 1 scoop of PB12 1 scoop of PC63 Plant 11 1 scoop of PB60 Plant 12 1 scoop of PC20 Plant 13 1 scoop PC26 Plant 14 1 scoop of PC33 Plant 15 1 scoop PC37 Plant 16 1 scoop of PC49

[0909] Starting from these herbal compositions, two corresponding aqueous extracts (or processed samples) were prepared according to the aforementioned method B, using 40 mL for M[20P]-576 and 20 mL for M[10P]-154 of water containing 100 g / l sucrose for aqueous extraction, including a centrifugation step and then filtration.

[0910] The biological activity of the processed samples was determined according to the aforementioned method C by using the following strains:

[0911] - Staphylococcus aureus strains: ATCC 25923, ATCC 49476, ATCC 6538, ATCC 51740, ATCC 29213, ATCC 14775, NCTC 12493, ATCC 33591, ATCC 33592, ATCC 43300, ATCC 700698, ATCC 700699, ATCC 9144, and ATCC BAA-44

[0912] - Staphylococcus epidermidis strains: ATCC 14990, ATCC 12228, ATCC 700296, ATCC 49134, and ATCC 49461

[0913] - Staphylococcus pseudintermedius strain: ATCC 49444

[0914] The minimum inhibitory concentrations (MICs) observed with the two processed samples are given in Table 6-2 as HIDs, along with the average efficacy against all strains of S. aureus and S. epidermidis.

[0915] [Table 6-2: HID of 2 treated samples against 14 strains of Staphylococcus aureus, 5 strains of Staphylococcus epidermidis, and 1 strain of Staphylococcus pseudintermedius]

[0916]

[0917] M[10P]-154 and M[20P]-576 showed complete antimicrobial activity against all tested S. aureus and S. epidermidis. M[10P]-154 showed complete antimicrobial activity against one S. epidermidis strain at 1:80, four S. aureus strains, four test strains of S. epidermidis and S. pseudintermedius at 1:40, nine S. aureus strains at 1:20, and one S. aureus strain at 1:10.

[0918] M[20P]-576 showed full antimicrobial activity against 1 S. epidermidis strain at 1:80, against 6 S. aureus strains and a test strain of S. pseudintermedius at 1:40, against 7 S. aureus strains and 4 S. epidermidis at 1:20, and against 1 S. aureus strain at 1:10.

[0919] Table 6-3 shows the percentage inhibition of bacterial planktonic growth and percentage inhibition of biofilm formation (IBF) for all strains of S. aureus and S. epidermidis at a dilution of 1:20 for the two processed samples, as well as the average efficacy.

[0920] Table 6-3: Planktonic growth inhibition and biofilm formation inhibition (IBF) of two processed samples at a dilution of 1:20 against 14 strains of Staphylococcus aureus, 5 strains of Staphylococcus epidermidis, and 1 strain of Staphylococcus pseudintermedius

[0921]

[0922]

[0923] At a 1:20 dilution, M[10P]-154 showed an average of 88% inhibition of biofilm formation against all tested S. aureus strains, while M[20P]-576 showed an average of 90%. Both mixtures showed lower antimicrobial activity against one strain: ATCC 6538 (45% for M[10P]-154 and 60% for M[20P]-576). M[10P]-154 showed an average of 91% inhibition of planktonic growth against all tested S. epidermidis strains, while M[20P]-576 showed an average of 88%. (See Table 6-3). Both mixtures showed percentage inhibition of 95% (M[10P]-154) and 93% (M[20P]-576), respectively, against the tested S. pseudintermedius. Thus, at a 1:20 dilution, M[10P]-154 and M[20P]-576 have demonstrated antibiofilm activity against S. aureus, S. epidermidis, and S. pseudintermedius.

[0924]

Example 7

[0925] Solutions of the mixture [20C]-576 (described in Example 6) for the following formulations were prepared according to Methods A and B above, except that in this example the filtration step was performed by suction filtration through a Buchner funnel filled with a 5 cm thick layer of hydrophilic cotton. For clarity, this cotton-filtered solution is referred to as SK576 in the following examples.

[0926] An ointment and two creams for topical application were prepared using the following emulsification method:

[0927] - Prepare Phase A and Phase B by mixing the following ingredients in two separate beakers using orbital shaking:

[0928]

[0929]

[0930] - Heat both beakers to 70°C using a warm water bath and keep at 70°C for 10 minutes to avoid crystallization of the beeswax.

[0931] Phase B is then slowly poured into phase A while keeping the beaker in a warm water bath and stirring continuously by means of a mechanical immersion mixer (Moulinex "Infinyforce_handblender", France).

[0932] The beaker containing the mixture of phases A and B is then removed from the warm bath and formed into an ointment using a dip-type "mayonnaise blender" (Moulinex "Infinyforce_handblender", France).

[0933] Filter through a hydrophilic syringe filter (0.22 μm)

[0934] - 20.6 g of SK576 was used to prepare a gel for topical application.

[0935] - 0.75g xanthan gum powder (Hyteck, "AromaZone TM ”, France) and 0.25 g ascorbic acid (Hyteck, “AromaZone TM ", France) was added to the filtered solution and mixed with a spatula for 5-10 minutes to form.

[0936] Prepare the spray solution for topical application by the following method:

[0937] - 5.5g of SK576 were mixed with 11.9g of anhydrous ethanol (Sigma-Aldrich, France) in a 20 mL beaker and stored in a 5°C refrigerator for 3 hours.

[0938] - Place the beaker with the cold mixture in an ice water bath and gently add 0.7 g of P-407 (Sigma-Aldrich, France).

[0939] - After stirring for 30 minutes in an ice-water bath, the solution was filled into a reagent sprayer (CAMAG, Switzerland) for application.

[0940] The protocol for each of these five formulations can be easily scaled to different formulation volumes by maintaining the same relative amounts of each ingredient.

[0941]

Example 8

[0942] [Bioactivity of the aqueous extract of the present invention in a mouse model of lethal Staphylococcus aureus infection]

[0943]

introduce

[0944] S. aureus strain Newman was isolated from a human infection in 1952 and has been widely used in animal models of staphylococcal infection due to its potent virulence phenotype.

[0945] Female OF1 mice have been widely used to study the pathogenesis of S. aureus-induced infection. Early studies on the pathogenesis of S. aureus infection used an intraperitoneal challenge route to infect and treat mice because it is technically easy.

[0946] The objective of this example was to evaluate the antimicrobial activity of the aqueous extract of the present invention in female OF1 mice infected with a lethal dose of Staphylococcus aureus. Treatment was administered orally from 2 days before infection to 7 days after infection, according to a schedule of 10 days. Mortality was monitored from D0 to D15.

[0947] Vehicle was used as a negative control.

[0948] Materials and Methods

[0949] Test substances and controls

[0950] Test substance: aqueous extract M[20C]576 (obtained starting from 120 g / l of herbal composition, prepared as described in Example 6). It is provided in 11 vials, each containing 1,200 μl of aqueous extract. It is stored at -20°C.

[0951] Vehicle: sucrose 10% aqueous solution. It will be stored at -20°C.

[0952] Bacterial strain: Staphylococcus newmannii strain (provided by Dr. T. Foster, Trinity College Dublin, Ireland, batch number 14156, ST4L).

[0953] Animal Purchase and Cage

[0954] Twenty (20) 6-week-old Crl:OF1 (outbred) female mice were purchased from Charles River (L'Arbresle, France).

[0955] Animal care was authorized by the French Ministry of Agriculture and Research (protocol number B35-288-1). Animal experiments were performed in accordance with the ethical guidelines for animal experimentation (Principes d'éthique de l'expérimentation animale, Directive n°86 / 609 CEE of 24 November 1986, Décrêt n°87 / 848 of 19 October 1987, Arrêt d'Application du 19 April 1988).

[0956] The animals were kept in room "A2" under controlled conditions of temperature (22 ± 3°C), humidity (50 ± 20%), photoperiod (12 h light / 12 h dark), air exchange, and hypobaric pressure. The air handling system could be programmed to 14 air changes per hour without recirculation.

[0957] Food and water were provided ad libitum and placed in a metal lid on top of the cage.

[0958] Treatment and experimental design

[0959] Preparation of bacteria - amplification of bacteria for stimulation

[0960] Thaw a vial of bacteria and culture 100 μl of the inoculum in 50 ml of Tryptic Soy Broth (TSB, Ref. 43592 or 22902, Sigma). Incubate the bacteria at 37°C on a horizontal shaker with agitation at 110 rpm for 20 hours. Dilute the bacterial suspension to 1 / 20 (100 μl + 1.9 ml TSB) and read the optical density at 680 nm (OD680 nm).

[0961] Then, 10 ml of bacteria were centrifuged at 5,200 rpm (3400 g) at 4°C for 10 minutes. The supernatant was discarded and the bacteria were dissolved in 10 ml of DPBS (Ref. BE17-512F, Lonza, France). The bacterial suspension (C0) was diluted 1 / 20 (100 μl + 1.9 ml DPBS) and the OD680 nm was measured.

[0962] The OD680nm measured before and after centrifugation must be similar.

[0963] The CFU / ml concentration of the bacterial solution is calculated as follows:

[0964] CFU / ml = (OD680nm x 20 (dilution factor) x 6.5.108 (CFU / UDO)

[0965]

Mucin preparation

[0966] Mucin (Sigma, France) was prepared at 20% 14-16 days before treatment as follows:

[0967] Preheat DPBS (Dulbecco's Phosphate-Buffered Saline) at 56°C and transfer ¾ of the final volume to a blender, then add porcine mucin and mix. Rinse the blender with the remaining volume of DPBS. Then, transfer the 20% mucin solution to an Erlenmeyer flask and incubate at 37°C with stirring for 1 hour and 30 minutes. Transfer ¾ of the 20% mucin solution to a sterile bottle. Store the remaining ¼ of the solution separately. Then sterilize both ¾ and ¼ of the solution by autoclaving. Store the sterile 20% porcine mucin solution at +4°C.

[0968] One day before infection, adjust the pH of ¾ of the 20% mucin solution to 7.4 by adding filtered 30% NaOH. If necessary, use a portion of the remaining ¼ of the solution to adjust the pH.

[0969]

Bacteria preparation

[0970] Based on the OD680nm results, the bacteria (C0) were diluted in DPBS to reach 6.8 x 10 7 These suspensions were then diluted 1 / 2 in 20% porcine mucin to obtain 3.4 × 10 CFU / ml in PBS-10% porcine mucin. 7 The LD80 concentration of CFU / ml was used. The suspension to be treated was placed in a tube containing sterile glass beads (4 mm, Ref. 068502, Dutscher, France). The suspension was not vortexed, but mixed by inverting the tube. The bacteria were placed on ice before treatment and kept on ice during treatment.

[0971]

CFU determination

[0972] To determine the exact concentration of the bacterial suspension before and after inoculation into the animal, the bacterial suspension was diluted in a deep well in DPBS-0.5% Tween 20 (Sigma, France) in a 10-fold serial dilution (50 μl bacteria + 450 μl DPBS-0.5% Tween 20) as described in Table 8-1. Then, 6×50 μl of bacterial dilutions 3, 4, 5, and 6 were inoculated on tryptic soy agar plates (Tryptic soy agar, Sigma) and placed overnight at 37°C. Colonies were counted to determine the true CFU (colony forming units) of the inoculum.

[0973] Counting was performed on drops (50 μl) containing 20 to 100 colonies. The shaded boxes correspond to the dilutions inoculated on the plates.

[0974] Table 8-1: Dilutions of bacterial suspensions used to determine the true CFU (viability) of the inoculum. The shaded boxes correspond to the dilutions inoculated on the plates.

[0975]

[0976] Randomization

[0977] Before the experiment (D-2), 20 healthy OFI female mice were randomly divided into two groups according to the weight standard, with 10 mice in each group, so that there was no statistical difference in the average body weight of the groups.

[0978]

deal with

[0979] Treatment was performed by oral (PO) administration from D-2 to D7 (D0: bacterial inoculation day). Bacterial inoculation was performed by intraperitoneal injection (IP).

[0980]

Vaccination

[0981] The mice were inoculated with bacterial suspension at D0. Each inoculation consisted of 3.4×10 7 CFU / ml(1.7×10 7 CFU / mouse) were injected intraperitoneally with 500 μl of the suspension.

[0982] Experimental Design

[0983] Animals were treated by oral administration of the test substances from D-2 to D7. At D0, treatment was performed 1 hour after bacterial inoculation.

[0984] The definitions of the experimental groups are described below and summarized in Table 8-2.

[0985] • Group 1 of 10 mice will be treated from D-2 to D7 by oral administration of vehicle according to the treatment schedule Q1DX10.

[0986] • Group 2 of 10 mice will be treated from D-2 to D7 by oral administration of the test substance according to the treatment schedule Q1DX10.

[0987]

Table 8-2: Experimental design

[0988]

[0989] Animal Monitoring

[0990] From D0 to D1, the mortality of mice was observed twice a day.

[0991] From D2 to D15, the mortality of mice was observed once a day.

[0992] The body weights of the mice were recorded twice a week until the end of the experiment.

[0993] Detailed clinical observations (skin, fur, eyes, mucous membrane changes, secretions and discharges, respiratory function, gait changes, posture) were monitored twice weekly as described in Table 8-3 below. This list is not exhaustive and will be supplemented by other observations as necessary.

[0994]

Table 8-3: Clinical observations / parameters

[0995]

[0996] Killing of mice

[0997] On D15, surviving mice were sacrificed by exsanguination followed by cervical dislocation when indicated. No necropsy was performed.

[0998]

result

[0999] Mouse survival analysis showed the results reported in Tables 8-4 and 8-5 below.

[1000]

Table 8-4: Number of mice surviving each day

[1001]

[1002]

Table 8-5: Percentage of mice surviving each day

[1003]

[1004] NA = Not Available

[1005] This treatment reduced the mortality rate of mice from 100% to 70%.Thus, this animal study shows that the claimed antimicrobial and antibiofilm activities observed in vitro are translated into a therapeutic effect when the aqueous extract of the invention is administered to animals by the oral route.

[1006]

Example 9

[1007] In this example, we demonstrate the simultaneous antimicrobial and antibiofilm activity of the disclosed composition against Staphylococcus aureus, comprising two of three plant species: Filipendula ulmoides PA13, Camellia sinensis PA21, and Uva arctica PB12, and at least one of three plant species: Syringa syringae PC20, Vitis vinifera var. tinctoria PA22, and Dexiella corylifolia PB07. Pharmaceutical-grade dried plants were obtained from "Herboristerie Cailleau" (Chemilles, France).

[1008] Starting from these three different plant powders, six herbal compositions were prepared according to Method A reported previously.

[1009] [List 9-1: Herbal composition of processed samples]

[1010] M[02P]-064:PA13;PA21;

[1011] M[03P]-050: PA13; PA21; PC20;

[1012] M[03P]-053: PA13; PA21; PB07;

[1013] M[03P]-056: PA13; PA21; PA22;

[1014] M[02P]-065:PA13;PB12;

[1015] M[03P]-051: PA13; PB12; PC20;

[1016] M[03P]-054: PA13; PB12; PB07;

[1017] M[03P]-057: PA13; PB12; PA22;

[1018] M[02P]-066:PA21;PB12;

[1019] M[03P]-052: PA21; PB12; PC20;

[1020] M[03P]-055: PA21; PB12; PB07;

[1021] M[03P]-058: PA21; PB12; PA22;

[1022] M[03P]-059: PA13; PB12; PA21;

[1023] M[04P]-025: PA13; PB12; PA21; PC20;

[1024] M[04P]-026: PA13; PB12; PA21; PB07;

[1025] M[04P]-027: PA13; PB12; PA21; PA22.

[1026] Starting from the herbal composition listed in Table 6-3, the corresponding aqueous extract (or processed sample) was prepared according to the aforementioned method B, using 40 mL of water containing 100 g / l sucrose and including a decantation step before filtration.

[1027] The bioactivity of each processed sample was determined using the following Staphylococcus aureus strain (ATCC 6538) at the following final dilutions of processed samples (1:5 for biofilm formation inhibition and 1:10 for growth inhibition) according to the aforementioned Method C. Note that for each mixture at a 1:5 dilution, growth inhibition approached 100%, and for all mixtures at a 1:10 dilution, biofilm formation inhibition approached 0%.

[1028] The percentage inhibition of bacterial planktonic growth and biofilm formation of processed samples obtained from different herbal compositions against all tested strains are reported in Table 9-1 below.

[1029] [Table 9-1: Based on the composition given in Table 9-1, the processed samples were effective against Staphylococcus aureus strains 6538 TM The average of the percent planktonic growth inhibition and percent biofilm formation inhibition efficacy was calculated. Processed samples were diluted 1:10 for planktonic growth inhibition and 1:5 for biofilm formation inhibition.

[1030]

[1031] (A) List of extracts obtained from a herbal composition containing PA13 and PA21, wherein one of PC20, PB07 and PA22 was added to improve the activity; (B) List of extracts obtained from a herbal composition containing PA13 and PB12, wherein one of PC20, PB07 and PA22 was added to improve the activity; (C) List of extracts obtained from a herbal composition containing PA13, PA21 and PB12, wherein one of PC20, PB07 and PA22 was added to improve the activity; (D) List of extracts obtained from a herbal composition containing PA13 and PA21, wherein one of PC20, PB07 and PA22 was added to improve the activity.

[1032]

Example 10

[1033] In this example, we demonstrate the simultaneous antimicrobial and antibiofilm activities of 324 mixture extracts obtained from the disclosed herbal composition comprising two of three plants, Filipendula ulmoides (PA13), Camellia sinensis (PA21), Uva arctica (PB12), and at least one of three plants, Syringa syringae (PC20), Vitis vinifera var. tinctoria (PA22), Dexiella variegata (PB07), against Staphylococcus aureus.

[1034] For the mixtures M[20C]-2083 to M[20C]-20798, pharmaceutical-grade dried botanicals were obtained from "Herboristerie Cailleau" (Chemilles, France). For the mixtures M[20C]-2805 to M[20C]-2957, pharmaceutical-grade dried botanicals were obtained from "Pharmacie Fontgiève" (Clermont-Ferrand, France, plants PA00 to PB12) and "Pharmacie St Herem" (Clermont-Ferrand, France, plants PB13 to PD02).

[1035] According to the aforementioned method A, a herbal composition containing 3 to 20 kinds of plant powders is prepared.

[1036] Starting from the herbal composition, the corresponding aqueous extract (or processed sample) was prepared by aqueous extraction according to the aforementioned method B using 40 mL of water containing 100 g / l sucrose and including a decantation step before filtration.

[1037] The bioactivity of each processed sample was determined according to Method C described above by using the following S. aureus strains (ATCC 25923, ATCC 49476, ATCC 6538, ATCC 51740, ATCC 29213, and ATCC 14775) at a final dilution of the processed sample of 1:20.

[1038] Tables 10-1, 10-2, 10-3, and 10-4 show the average percent inhibition of bacterial planktonic growth and the average percent inhibition of biofilm formation for all tested strains obtained from the processed samples obtained from the following herbal compositions, respectively, listed in List 10-1, List 10-2, List 10-3, and List 10-4. In each group, extracts obtained from herbal compositions containing more than three or four plants were found to exhibit greater inhibition of planktonic growth or inhibition of biofilm formation (or both) than any mixture containing only three or four plants.

[1039] [List 10-1: Extracts from the following herbal composition, the herbal composition containing two of the three plants of Filipendula ulmaria (PA13) and Camellia sinensis (PA21) and at least one of the three plants of Syringa syringae (PC20), Vitis vinifera (PA22), and Desmodium variegatum (PB07)]

[1040] M[03P]-050: PA13; PA21; PC20;

[1041] M[03P]-053: PA13; PA21; PB07;

[1042] M[03P]-056: PA13; PA21; PA22;

[1043] M[20C]-2808: PA11; PA12; PA13; PA13; PA18; PA21; PA23; PA24; PA28; PA69; PB07; PB88; PC23; PC24; PC49; PC52; PC55(2 spoons); PC60; PC97;

[1044] M[20C]-2820: PA10; PA11(2 spoons); PA12; PA13; PA14; PA18; PA20; PA21; PA25; PA29; PA33; PA35; PA67; PA75; PB07; PB60; PC33; PC35; PC55;

[1045] M[20C]-2821: PA11; PA13(2 spoons); PA19; PA20; PA21; PA22; PA25; PA29; PA33; PA35; PA75; PB07; PB36; PC20; PC33(2 spoons); PC49; PC55; PC97;

[1046] M[20C]-2826: PA11; PA13(2 spoons); PA20; PA21; PA24; PA25; PA29; PA33; PA34; PA35; PA52; PA70; PA75; PB07; PC11; PC21; PC33; PC37; PC74;

[1047] M[20C]-2827: PA10; PA11; PA13(2 spoons); PA20; PA21; PA25; PA26; PA29; PA33; PA35; PA57; PA75; PB02; PB07; PB28; PC11; PC23; PC33; PC49;

[1048] M[20C]-2830: PA10; PA11; PA12; PA13; PA14; PA18; PA21; PA22; PA25; PA29; PA32; PA34; PA67; PB07; PB60; PB88; PC33; PC35; PC55; PC98;

[1049] M[20C]-2831: PA13; PA13; PA19; PA21; PA22; PA22; PA25; PA29; PA32; PA34; PB07; PB36; PB88; PC20; PC33; PC33; PC49; PC55; PC97; PC98;

[1050] M[20C]-2833: PA10; PA11; PA13; PA14; PA21; PA22; PA24; PA25; PA26; PA29; PA32; PA34; PB33; PB88; PB88; PC12; PC23; PC27; PC33; PC98;

[1051] M[20C]-2836: PA13; PA13; PA21; PA22; PA24; PA25; PA29; PA32; PA34; PA34; PA52; PA70; PB07; PB88; PC11; PC21; PC33; PC37; PC74; PC98;

[1052] M[20C]-2837: PA10; PA13 (2 spoons); PA21; PA22; PA25; PA26; PA29; PA32; PA34; PA57; PB02; PB07; PB28; PB88; PC11; PC23; PC33; PC49; PC98;

[1053] M[20C]-2838: PA12; PA13 (2 spoons); PA)18; PA21; PA21; PA22; PA24; PA25; PA29; PA32; PA34; PB88; PC23; PC33; PC49; PC52; PC55; PC97; PC98;

[1054] M[20C]-2839: PA13 (2 spoons); PA14; PA21; PA22; PA25; PA25; PA26; PA29; PA32; PA34; PA39; PA52; PA57; PB33; PB88; PC33; PC71; PC97; PC98;

[1055] M[20C]-2858: PA12; PA13; PA18; PA20; PA21; PA23; PA24; PA24; PA67; PB07; PB28; PB33; PC12; PC20; PC21; PC23; PC49; PC52; PC55; PC97;

[1056] M[20C]-2861: PA13; PA19; PA21; PA22 (2 spoons); PB07; PB28; PB36; PB)60; PCll; PC20; PC20; PC23; PC33; PC49; PC55 (2 spoons); PC60; PC74; PC97;

[1057] M[20C]-2866: PA13; PA21; PA22; PA24; PA34; PA52; PA70; PB07; PB28; PB60;

[1058] M[20C]-2867: PA10; PA13; PA21; PA22; PA26; PA57; PB02; PB07; PB28; PB28; PB60; PC11 (2 scoops); PC20; PC23; PC23; PC49; PC55; PC60; PC74;

[1059] M[20C]-2868: PA12; PA13; PA18; PA21; PA21; PA22; PA24; PB28; PB60; PC11; PC20; PC23 (2 scoops); PC49; PC52; PC55 (2 scoops); PC60; PC74; PC97;

[1060] M[20C]-2869: PA13; PA14; PA21; PA22; PA25; PA26; PA39; PA52; PA57; PB28; PB33; PB60; PC11; PC20; PC23;

[1061] M[20C]-2871: PA10; PA11; PA13; PA19; PA21; PA22; PA35; PA57; PB07; PB07; PB36; PB77; PC20 (2 scoops); PC23; PC33; PC49; PC55; PC71; PC97;

[1062] M[20C]-2876: PA10; PA11; PA13; PA21; PA24; PA34; PA35; PA52; PA57; PA70; PB07 (2 scoops); PB77; PC11; PC20; PC21; PC23; PC37; PC71; PC74;

[1063] M[20C]-2877: PA10; PA10; PA11; PA13; PA21; PA26; PA35; PA57; PA57; PB02; PB07; PB28; PB77; PC11; PC20; PC23 (2 scoops); PC49; PC71;

[1064] M[20C]-2878: PA10;

[1065] M[20C]-2879: PA10; PA11; PA13; PA14; PA21; PA25; PA26; PA35; PA39; PA52; PA57 (2 scoops); PB07; PB33; PB77; PC20; PC23; PC71 (2 scoops); PC97;

[1066] M[20C]-2889: PA10; PA12; PA13; PA14; PA18; PA21; PA26; PA26; PA28; PA29; PA33; PA39; PA75; PB36; PC20; PC52; PC55 (2 scoops); PC60;

[1067] M[20C]-2894: PA11 (2 scoops); PA13; PA21; PA28; PA39; PA39; PA70; PB02; PB02; PB07; PB99 (2 scoops); PC14; PC20; PC49; PC71; PC97; PC98;

[1068] M[20C]-2944: PA10; PA11 (2 scoops); PA13; PA14; PA21; PA28; PA39; PA70; PB02; PB07; PB27; PB99; PC11; PC20; PC23; PC67; PC71; PC97; PC98;

[1069] M[20C]-2954: PA11;

[1070] Table 10-1: Average of the percent inhibition of planktonic growth and percent inhibition of biofilm formation of the processed samples against the six tested Staphylococcus aureus strains based on the extracts of List 10-1 diluted 1:20

[1071]

[1072]

[1073] [List 10-2: Extracts from the following herbal composition, the herbal composition containing Filipendula ulmaria (PA13), Urtica bearberry (PB12), and at least one of three plants: Syringa syringae (PC20), Vitis vinifera (PA22), and Desmodium variegatum (PB07)]

[1074] M[03C]-051: PA13; PB12; PC20;

[1075] M[03C]-054: PA13; PB12; PB07;

[1076] M[03C]-057: PA13; PB12; PA22;

[1077] M[20C]-2083: PA02; PA13; PA28 (2 scoops); PA31; PA36; PA40; PA79; PA93; PB01; PB12; PB17; PB18; PB31; PB51; PB77; PC20; PC58; PC64; PC66;

[1078] M[20C]-2084: PA13;

[1079] M[20C]-2085: PA12; PA13; PA14; PA28; PA30; PA31; PA36; PA79; PA94; PB00; PB01; PB08; PB12; PB17; PB31; PC02; PC14; PC20; PC64; PC66;

[1080] M[20C]-2090: PA08; PA13; PA20; PA23; PA26; PA32; PA46; PA52; PA57; PA79; PA95; PB10; PB12; PB33; PB76; PB82; PC08; PC20; PC21; PD00;

[1081] M[20C]-2091: PA08; PA13; PA26; PA52; PA57; PA72; PA79; PA88; PA92; PA95; PB12; PB60; PB76; PB83; PC08; PC11; PC20; PC26; PC68; PD00;

[1082] M[20C]-2092:PA08;PA13;PA17;PA26;PA52;PA53;PA57;PA79;PA95;PB06;PB12;PB50;PB76;PC08;PC20;PC20;PC27;PC97;PD00;PD02;

[1083] M[20C]-2100:PA13;PA20;PA23;PA32;PA41;PA46;PA53;PA79;PA80;PA90;PB08;PB10;PB12;PB33;PB82;PB85;PC20;PC21;PC28;PC75;

[1084] M[20C]-2102:PA13;PA17;PA41;PA53;PA53;PA79;PA80;PA90;PB06;PB08;PB12;PB50;PB85;PC20;PC20;PC27;PC28;PC75;PC97;PD02;

[1085] M[20C]-2104:PA13;PA41;PA53;PA61;PA76;PA76;PA79;PA80;PA82;PA90;PB08;PB10;PB12;PB57;PB85;PC20;PC28;PC36;PC41;PC75;

[1086] M[20C]-2105:PA12;PA13;PA14;PA30;PA41;PA53;PA79;PA80;PA90;PA94;PB00;PB08;PB08;PB12;PB85;PC02;PC14;PC20;PC28;PC75;

[1087] M[20C]-2110:PA00;PA10;PA13;PA20;PA23;PA25;PA30;PA32;PA46;PA52;PA79;PB05;PB10;PB12;PB33;PB82;PC01;PC20;PC21;PD07;

[1088] M[20C]-2111:PA00;PA10;PA13;PA25;PA30;PA52;PA72;PA79;PA88;PA92;PB05;PB12;PB60;PB83;PC01;PC11;PC20;PC26;PC68;PD07;

[1089] M[20C]-2120:PA11;PA13;PA16;PA20;PA23;PA32;PA44;PA46;PA60;PA67;PA73;PA79;PB10;PB12;PB33;PB48;PB82;PC17;PC20;PC21;

[1090] M[20C]-2122:PA11;PA13;PA16;PA17;PA44;PA53;PA60;PA67;PA73;PA79;PB06;PB12;PB48;PB50;PC17;PC20;PC20;PC27;PC97;PD02;

[1091] M[20C]-2131:PA05;PA13;PA18;PA37;PA55;PA72;PA72;PA79;PA88;PA92;PA97;PB04;PB12;PB60;PB83;PC11;PC20;PC26;PC35;PC68;

[1092] M[20C]-2135:PA05;PA12;PA13;PA14;PA18;PA30;PA37;PA55;PA72;PA79;PA94;PA97;PB00;PB04;PB08;PB12;PC02;PC14;PC20;PC35;

[1093] M[20C]-2137:PA05;PA13;PA18;PA27;PA37;PA46;PA55;PA72;PA79;PA82;PA97;PB00;PB04;PB11;PB12;PC20;PC35;PC45;PC52;PC67;

[1094] M[20C]-2140:PA13;PA14;PA20;PA23;PA32;PA37;PA46;PA79;PA85;PB10;PB12;PB33;PB49;PB82;PB98;PC20;PC21;PC55;PC62;PC98;

[1095] M[20C]-2141:PA13;PA14;PA37;PA72;PA79;PA85;PA88;PA92;PB12;PB49;PB60;PB83;PB98;PC11;PC20;PC26;PC55;PC62;PC68;PC98;

[1096] M[20C]-2142:PA13;PA14;PA17;PA37;PA53;PA79;PA85;PB06;PB12;PB49;PB50;PB98;PC20;PC20;PC27;PC55;PC62;PC97;PC98;PD02;

[1097] M[20C]-2143:PA02;PA13;PA14;PA28;PA37;PA40;PA79;PA85;PA93;PB12;PB18;PB49;PB51;PB77;PB98;PC20;PC55;PC58;PC62;PC98;

[1098] M[20C]-2144:PA13;PA14;PA37;PA61;PA76;PA76;PA79;PA82;PA85;PB10;PB12;PB49;PB57;PB98;PC20;PC36;PC41;PC55;PC62;PC98;

[1099] M[20C]-2150:PA13;PA20;PA23;PA31;PA32;PA46;PA60;PA74;PA79;PB03;PB10;PB12;PB33;PB82;PC20;PC21;PC29;PC37;PC60;PC86;

[1100] M[20C]-2151:PA13;PA31;PA60;PA72;PA74;PA79;PA88;PA92;PB03;PB12;PB60;PB83;PC11;PC20;PC26;PC29;PC37;PC60;PC68;PC86;

[1101] M[20C]-2152:PA13;PA17;PA31;PA53;PA60;PA74;PA79;PB03;PB06;PB12;PB50;PC20;PC20;PC27;PC29;PC37;PC60;PC86;PC97;PD02;

[1102] M[20C]-2153:PA02;PA13;PA28;PA31;PA40;PA60;PA74;PA79;PA93;PB03;PB12;PB18;PB51;PB77;PC20;PC29;PC37;PC58;PC60;PC86;

[1103] M[20C]-2154:PA13;PA31;PA60;PA61;PA74;PA76;PA76;PA79;PA82;PB03;PB10;PB12;PB57;PC20;PC29;PC36;PC37;PC41;PC60;PC86;

[1104] M[20C]-2155:PA12;PA13;PA14;PA30;PA31;PA60;PA74;PA79;PA94;PB00;PB03;PB08;PB12;PC02;PC14;PC20;PC29;PC37;PC60;PC86;

[1105] M[20C]-2163:PA06;PA13;PA17;PA20;PA23;PA72;PA79;PA80;PA92;PB01;PB03;PB10;PB12;PB33;PB57;PB82;PC20;PC62;PC86;PC97;

[1106] M[20C]-2164:PA13;PA17;PA20;PA41;PA79;PA80;PA86;PA92;PB03;PB10;PB12;PC20;PC26;PC27;PC29;PC55;PC60;PC62;PC86;PC97;

[1107] M[20C]-2214:PA13;PA37;PA41;PA79;PA86;PB00;PB10;PB12;PB49;PB82;PC20;PC26;PC27;PC29;PC55;PC60;PC62;PC66;PC86;

[1108] M[20C]-2234:PA13;PA41;PA79;PA85;PA86;PB12;PB31;PB50;PB77;PC14;PC20;PC26;PC27;PC29;PC55;PC55;PC60;PC62;PC98;

[1109] M[20C]-2241:PA10;PA13;PA14;PA23;PA36;PA37;PA46;PA79;PB00;PB10;PB11;PB12;PB17;PB57;PC20;PC27;PC29;PC37;PC64;PC68;

[1110] M[20C]-2242: PA13; PA14; PA17; PA23; PA36; PA37; PA60; PA61; PA76; PA79; PA82; PB00; PB06; PB12; PB49; PC20; PC27; PC29; PC68; PC98;

[1111] M[20C]-2243: PA13; PA14; PA23; PA36; PA37; PA52; PA53; PA73; PA79; PA92; PB00; PB01; PB12; PB18; PB60; PC20; PC20; PC27; PC29; PC68;

[1112] M[20C]-2248: PA13; PA14(2 spoons); PA23; PA25; PA36; PA36; PA37; PA46; PA79; PA85; PB00; PB12; PC02; PC20; PC27; PC29; PC29; PC55; PC68;

[1113] M[20C]-2249: PA10; PA13; PA14; PA23; PA36; PA37; PA53; PA74; PA79; PA92; PB00; PB12; PC20; PC27; PC29; PC62; PC67; PC68; PC86; PC97;

[1114] M[20C]-2250: PA13; PA23; PA37; PA76; PA79; PB10; PB12; PB33; PB50; PB82; PB82; PB85; PB98; PC01; PC11; PC20; PC45; PC55; PC60; PD02;

[1115] M[20C]-2253: PA13; PA37; PA52; PA53; PA73; PA79; PA92; PB01; PB10; PB12; PB18; PB33; PB50; PB60; PB82; PB85; PC01; PC20; PC20; PC45;

[1116] M[20C]-2254: PA11; PA13; PA17; PA27; PA37; PA79; PB00; PB10; PB12; PB33; PB50; PB82; PB83; PB85; PB92; PB98; PC01; PC20; PC27; PC45;

[1117] M[20C]-2255:PA13;PA36;PA37;PA53;PA72;PA79;PA80;PB03;PB10;PB12;PB33;PB49;PB50;PB82;PB82;PB85;PC01;PC20;PC45;PC68;

[1118] M[20C]-2256:PA13;PA23;PA37;PA53;PA61;PA79;PA85;PB10;PB12;PB33;PB50;PB60;PB82;PB85;PC01;PC11;PC20;PC29;PC45;PD02;

[1119] M[20C]-2257:PA13;PA37;PA37;PA60;PA72;PA79;PB10;PB12;PB33;PB49;PB50;PB82;PB85;PC01;PC08;PC20;PC45;PC68;PC98;PD02;

[1120] M[20C]-2258:PA13;PA14;PA25;PA36;PA37;PA46;PA79;PA85;PB10;PB12;PB33;PB50;PB82;PB85;PC01;PC02;PC20;PC29;PC45;PC55;

[1121] M[20C]-2259:PA10;PA13;PA37;PA53;PA74;PA79;PA92;PB10;PB12;PB33;PB50;PB82;PB85;PC01;PC20;PC45;PC62;PC67;PC86;PC97;

[1122] M[20C]-2261:PA10;PA13;PA23;PA36;PA37;PA46;PA53;PA73;PA79;PA97;PB10;PB11;PB12;PB17;PB57;PC20;PC26;PC29;PC37;PC64;

[1123] M[20C]-2278:PA10;PA13;PA14;PA25;PA28;PA30;PA36;PA37;PA41;PA46;PA79;PA85;PB12;PB50;PB60;PC02;PC20;PC29;PC55;PC66;

[1124] M[20C]-2281:PA10;PA13;PA17;PA46;PA52;PA79;PA82;PA92;PA96;PB10;PB11;PB12;PB17;PB57;PB98;PC20;PC36;PC37;PC64;PC86;

[1125] M[20C]-2321:PA13;PA46;PA53;PA79;PA82;PA92;PB03;PB10;PB11;PB12;PB83;PB85;PC20;PC20;PC21;PC55;PC60;PC86;PC97;PD02;

[1126] M[20C]-2406:PA13;PA37;PA46;PA53;PA79;PA85;PB01;PB05;PB10;PB11;PB12;PB17;PB49;PB98;PC20;PC36;PC37;PC45;PC58;PC67;

[1127] M[20C]-2420:PA10;PA13;PA14;PA36;PA46;PA79;PB10;PB10;PB11;PB11;PB12;PB17;PB17;PB33;PB82;PC20;PC54;PC55;PC68;PC98;

[1128] M[20C]-2441:PA00;PA13;PA14;PA17;PA17;PA37;PA37;PA46;PA74;PA79;PB00;PB03;PB10;PB12;PB49;PC08;PC20;PC35;PC62;PC86;

[1129] M[20C]-2443:PA00;PA13;PA14;PA14;PA17;PA36;PA37;PA79;PB00;PB01;PB10;PB12;PC02;PC08;PC11;PC20;PC29;PC62;PC67;PC86;

[1130] M[20C]-2449:PA00;PA13;PA14;PA17;PA36;PA37;PA60;PA79;PA85;PB00;PB10;PB12;PB82;PB98;PC08;PC20;PC29;PC62;PC86;PC98;

[1131] M[20C]-2451:PA10;PA13;PA17;PA23;PA37;PA46;PA74;PA76;PA79;PB00;PB03;PB08;PB10;PB12;PB49;PB49;PC20;PC35;PC97;PC98;

[1132] M[20C]-2460:PA08;PA13;PA14;PA36;PA36;PA46;PA79;PB10;PB11;PB12;PB17;PB17;PB98;PC20;PC36;PC54;PC62;PC68;PC68;PC86;

[1133] M[20C]-2488:PA10;PA13;PA37;PA46;PA53;PA74;PA79;PA82;PB00;PB00;PB12;PB49;PC20;PC29;PC37;PC45;PC54;PC74;PC86;PC98;

[1134] M[20C]-2545:PA00;PA08;PA10;PA13;PA46;PA73;PA76;PA79;PA90;PB01;PB08;PB10;PB12;PB17;PB33;PB33;PC20;PC55;PC68;PD02;

[1135] M[20C]-2551:PA13;PA14;PA16;PA27;PA36;PA79;PA82;PA92;PA95;PB10;PB12;PB57;PB85;PC11;PC20;PC29;PC29;PC54;PC64;PC98;

[1136] M[20C]-2553:PA13;PA16;PA23;PA60;PA79;PA82;PA95;PB00;PB10;PB10;PB12;PB33;PB49;PB50;PB85;PC02;PC11;PC20;PC29;PC98;

[1137] M[20C]-2555:PA00;PA10;PA13;PA16;PA79;PA82;PA90;PA95;PB08;PB10;PB10;PB12;PB33;PB85;PC11;PC20;PC29;PC68;PC98;PD02;

[1138] M[20C]-2580:PA10;PA13;PA25;PA37;PA40;PA46;PA79;PB11;PB12;PB33;PB49;PB82;PB85;PB98;PC20;PC29;PC29;PC36;PC45;PC97;

[1139] M[20C]-2589:PA13;PA14;PA17;PA40;PA46;PA79;PA80;PB11;PB12;PB33;PB49;PB85;PB98;PC20;PC26;PC29;PC37;PC86;PC97;PD02;

[1140] M[20C]-2622:PA13;PA25;PA36;PA36;PA37;PA46;PA46;PA79;PA82;PB12;PB49;PB82;PC20;PC20;PC54;PC55;PC64;PC68;PC86;PC98;

[1141] M[20C]-2627:PA13;PA14;PA36;PA46;PA76;PA79;PA80;PA96;PB12;PB49;PB49;PC20;PC20;PC45;PC54;PC55;PC64;PC97;PC98;PC98;

[1142] M[20C]-2641:PA05;PA13;PA16;PA27;PA36;PA37;PA41;PA79;PA96;PB05;PB10;PB12;PB82;PB85;PB98;PC11;PC20;PC54;PC68;PD02;

[1143] M[20C]-2651:PA13;PA36;PA41;PA46;PA79;PA92;PB00;PB01;PB05;PB12;PB33;PB82;PB82;PB85;PB98;PC20;PC54;PC55;PC86;PD02;

[1144] M[20C]-2654:PA13;PA36;PA46;PA79;PA92;PB00;PB00;PB01;PB08;PB11;PB12;PB33;PB82;PC01;PC20;PC55;PC55;PC64;PC86;PC86;

[1145] M[20C]-2717:PA13;PA14;PA46;PA79;PB00;PB10;PB12;PB49;PB50;PB77;PB85;PB85;PC02;PC20;PC29;PC64;PC64;PC68;PC98;PD02;

[1146] M[20C]-2728:PA05;PA13;PA16;PA30;PA46;PA60;PA61;PA79;PA85;PB04;PB10;PB12;PB49;PB82;PB82;PB85;PB98;PC11;PC20;PC37;

[1147] M[20C]-2730:PA13;PA36;PA37;PA46;PA61;PA79;PA82;PA85;PB00;PB12;PB17;PB33;PB82;PB85;PC01;PC02;PC20;PC29;PC64;PC98;

[1148] M[20C]-2738:PA05;PA13;PA37;PA46;PA61;PA61;PA79;PA85;PB10;PB12;PB82;PB82;PB85;PB85;PB98;PC02;PC20;PC29;PC37;PC64;

[1149] M[20C]-2788:PA00;PA05;PA13;PA27;PA46;PA61;PA79;PA82;PB00;PB10;PB12;PB82;PB85;PB85;PB98;PC20;PC37;PC37;PC58;PC97;

[1150] M[20C]-2805:PA11;PA13;PA13;PA14;PA22;PA23;PA28;PA33;PA35;PA69;PB07;PB12;PB38;PB88;PB88;PC20;PC24;PC55;PC55;PC60;

[1151] M[20C]-2812:PA11;PA11;PA13;PA20;PA23;PA29;PA30;PA83;PA98;PB02;PB12;PB27;PB38;PC11;PC11;PC20;PC23;PC37;PC51;PC60;

[1152] M[20C]-2815: PA11; PA13; PA14; PA22; PA23; PA29; PA33; PA35; PA98; PB02; PB12; PB27; PB38; PB38; PB88; PC11; PC20; PC23; PC37; PC55;

[1153] M[20C]-2840: PA10; PA11; PA12 (2 scoops); PA13; PA14; PA18; PA28; PA33; PA67; PA67; PB07 (2 scoops); PB12; PB60; PC23; PC35; PC37; PC55; PC67;

[1154] M[20C]-2841: PA12; PA13 (2 scoops); PA19; PA22; PA28; PA33; PA67; PB07 (2 scoops); PB12; PB36; PC20; PC23; PC33; PC37; PC49; PC55; PC67; PC97;

[1155] M[20C]-2842: PA11; PA12; PA13 (2 scoops); PA20; PA28; PA30; PA33; PA67; PA83; PB07; PB12 (2 scoops); PC11; PC20; PC23; PC37; PC51; PC60; PC67;

[1156] M[20C]-2843: PA10;

[1157] M[20C]-2844: PA12; PA13; PA13; PA15; PA18; PA20; PA28; PA32; PA33; PA67 (2 scoops); PB07; PB12; PB12; PC20; PC23; PC37; PC49; PC55; PC67;

[1158] M[20C]-2845: PA12; PA13 (2 scoops); PA14; PA22; PA28; PA33; PA33; PA35; PA67; PB07; PB12 (2 scoops); PB38; PB88; PC20; PC23; PC37; PC55; PC67;

[1159] M[20C]-2847: PA10; PA12; PA13 (2 scoops); PA26; PA28; PA33; PA57; PA67; PB02; PB07; PB07; PB12; PB28; PC11; PC23; PC23; PC37; PC49; PC67;

[1160] M[20C]-2849: PA12; PA13 (2 scoops); PA14; PA25; PA26; PA28; PA33; PA39; PA52; PA57; PA67; PB07; PB12; PB33; PC23; PC37; PC67; PC71; PC97;

[1161] M[20C]-2852: PA11; PA13; PA20 (2 scoops); PA23; PA24; PA30; PA67; PA83; PB07; PB12; PB28; PB33; PC11; PC12; PC20; PC20; PC21; PC51; PC60;

[1162] M[20C]-2854: PA13; PA15; PA18; PA20 (2 scoops); PA23; PA24; PA32; PA67 (2 scoops); PB07; PB12; PB28; PB33; PC12; PC20 (2 scoops); PC21; PC49; PC55;

[1163] M[20C]-2855: PA13; PA14; PA20; PA22; PA23; PA24; PA33; PA35; PA67; PB07; PB12; PB28; PB33; PB38; PB88; PC12; PC20; PC21; PC55;

[1164] M[20C]-2893: PA00; PA06; PA11; PA12; PA13; PA23; PA26; PA28; PA33; PA39; PB02; PB07 (2 scoops); PB12; PB99; PC14; PC20; PC49; PC98; PD02;

[1165] M[20C]-2897: PA11 (2 scoops); PA13; PA14; PA24; PA29; PA39; PA83; PB02; PB07; PB12; PB60; PB99; PC11; PC14; PC20; PC20; PC49; PC51; PC98;

[1166] M[20C]-2930: PA11; PA13; PA14; PA23; PA26; PA28; PA33; PA35; PA83; PB12; PB28; PB33; PB38; PB77; PB88; PC20; PC23; PC37; PC60; PC60;

[1167] M[20C]-2933: PA00; PA06; PA11; PA12; PA13; PA14; PA23; PA26; PA28 (2 scoops); PA33; PA33; PB07; PB12; PB12; PB28; PB38; PB77; PC60; PD02;

[1168] M[20C]-2935: PA10; PA11; PA13; PA14; PA28; PA29; PA33; PA57; PA67; PB07; PB12; PB28 (2 scoops); PB38; PB77; PB77; PC11; PC20; PC52; PC60;

[1169] M[20C]-2936: PA11 (2 scoops); PA13; PA14; PA18; PA28; PA33; PA57; PA67; PB02; PB12; PB28; PB38 (2 scoops); PC11; PC20; PC27; PC52; PC60;

[1170] M[20C]-2937: PA11; PA11; PA13; PA14 (3 scoops); PA28; PA29; PA33; PA83; PB12 (2 scoops); PB28; PB38; PB60; PB77; PC11; PC20; PC51; PC60;

[1171] M[20C]-2939: PA10; PA11; PA12; PA13 (2 scoops); PA14; PA18; PA26; PA28; PA33 (2 scoops); PA39; PB12; PB28; PB38; PB77; PC20; PC52; PC55; PC60;

[1172] M[20C]-2943: PA00; PA06; PA10; PA11; PA12; PA13; PA14; PA23; PA26; PA28; PA33; PB07 (2 scoops); PB12; PB27; PC11; PC20; PC23; PC67; PD02;

[1173] M[20C]-2947: PA10; PA11 (2 scoops); PA13; PA14 (2 scoops); PA24; PA29; PA83; PB07; PB12; PB27; PB60; PC11 (2 scoops); PC20 (2 scoops); PC23; PC51; PC67;

[1174] M[20C]-2953: PA00; PA06; PA12 (2 scoops); PA13; PA14; PA23 (2 scoops); PA26; PA28; PA33 (2 scoops); PB01; PB07 (2 scoops); PB12; PC20; PC27; PC52; PD02;

[1175] M[20C]-2957: PA11; PA12; PA13; PA14 (2 spoons); PA23; PA24; PA29; PA33; PA83; PB01; PB07; PB12; PB60; PC11; PC20 (2 spoons); PC27; PC51; PC52.

[1176] Table 10-2: Average values ​​of percent inhibition of planktonic growth and percent inhibition of biofilm formation of processed samples against six tested Staphylococcus aureus strains based on a 1:20 dilution of the mixture of Table 10-2

[1177]

[1178]

[1179] [List 10-3: Herbal compositions of the following processed samples, comprising: Camellia sinensis (PA21), Uva ursi (PB12), and at least one of three plants: Syringa syringae (PC20), Vitis vinifera (PA22), and Desmodium variegatum (PB07)]

[1180] M[03C]-052: PA21; PB12; PC20;

[1181] M[03C]-055: PA21; PB12; PB07;

[1182] M[03C]-058: PA21; PB12; PA22;

[1183] M[20C]-2888: PA14; PA15; PA18; PA21; PA21; PA23; PA26; PA28; PA28; PA29; PA39; PA57; PA75; PB07; PB12; PB36; PC11; PC55; PC55; PC60;

[1184] M[20C]-2913: PA00; PA06; PA12; PA21; PA23; PA26; PA26; PA27; PA28; PA33; PB07; PB07; PB12 (2 scoops); PB33; PC20; PC24; PC41; PC71; PD02;

[1185] M[20C]-2918: PA15; PA18; PA21 (2 scoops); PA23; PA26; PA27; PA28; PA57; PB07 (2 scoops); PB12 (2 scoops); PB33; PC11; PC20; PC24; PC41; PC55; PC71;

[1186] M[20C]-2920: PA20; PA21; PA23; PA26; PA28; PA35; PA35; PA67; PA83; PB12; PB33; PB88; PC12; PC20; PC23; PC37; PC37; PC60 (2 scoops); PC74;

[1187] M[20C]-2923: PA00; PA06; PA12; PA20; PA21; PA23; PA26; PA28 (2 scoops); PA33; PA35; PA67; PB07; PB12 (2 scoops); PC12; PC37; PC60; PC74; PD02;

[1188] M[20C]-2928: PA15; PA18; PA20; PA21 (2 scoops); PA23; PA28 (2 scoops); PA35; PA57; PA67; PB07; PB12 (2 scoops); PC11; PC12; PC37; PC55; PC60; PC74;

[1189] M[20C]-2887: PA11;

[1190] M[20C]-2914: PA11; PA21 (2 scoops); PA26; PA27; PA28; PA39; PA70; PB02; PB07; PB12; PB33; PB99; PC20; PC24; PC41; PC71 (2 scoops); PC97; PC98;

[1191] M[20C]-2915: PA10; PA21; PA26; PA27; PA29; PA57; PA67; PB07 (2 scoops); PB12; PB28; PB33; PB77; PC11; PC20 (2 scoops); PC24; PC41; PC52; PC71;

[1192] M[20C]-2916: PA11;

[1193] M[20C]-2917: PA11; PA14; PA21; PA24; PA26; PA27; PA29; PA83; PB07; PB12 (2 scoops); PB33; PB60; PC11; PC20 (2 scoops); PC24; PC41; PC51; PC71;

[1194] M[20C]-2925: PA10; PA20; PA21; PA28; PA29; PA35; PA57; PA67 (2 scoops); PB07; PB12; PB28; PB77; PC11; PC12; PC20; PC37; PC52; PC60; PC74;

[1195] M[20C]-2926: PA11; PA18; PA20; PA21; PA28; PA35; PA57; PA67 (2 scoops); PB02; PB12; PB38; PC11; PC12; PC20; PC27; PC37; PC52; PC60; PC74;

[1196] M[20C]-2927: PA11; PA14; PA20; PA21; PA24; PA28; PA29; PA35; PA67; PA83; PB12; PB12; PB60; PC11; PC12; PC20; PC37; PC51; PC60; PC74;

[1197] M[20C]-2910: PA21; PA23; PA26 (2 scoops); PA27; PA35; PA83; PB07; PB12; PB33 (2 scoops); PB88; PC20 (2 scoops); PC23; PC24; PC37; PC41; PC60; PC71.

[1198] Table 10-3: Average values ​​of percent inhibition of planktonic growth and percent inhibition of biofilm formation of processed samples against six tested Staphylococcus aureus strains based on a 1:20 dilution of the mixture of Table 10-2

[1199]

[1200] [List 10-4: Extracts from the following herbal composition, the herbal composition containing: Filipendula ulmaria (PA13), Camellia sinensis (PA21), Uva ursi (PB12), and at least one of three plants: Syringa syringae (PC20), Vitis vinifera (PA22), and Desmodium variegatum (PB07)]

[1201] M[04C]-025: PA13; PA21; PB12; PC20;

[1202] M[04C]-026: PA13; PA21; PB12; PB07;

[1203] M[04C]-027: PA13; PA21; PB12; PA22;

[1204] M[20C]-2898: PA11; PA13; PA15; PA18; PA21; PA23; PA28; PA39; PA57; PB02; PB07 (2 scoops); PB07; PB12; PB99; PC11; PC14; PC20; PC49; PC55; PC98;

[1205] M[20C]-2900: PA10; PA11; PA13; PA21; PA23; PA25; PA26; PA35; PA69; PA83; PB12; PB32; PB33; PB88; PC20 (2 scoops); PC23; PC37; PC55; PC60;

[1206] M[20C]-2903: PA00; PA06; PA10; PA11; PA12; PA13; PA21; PA23; PA25; PA26; PA28; PA33; PA69; PB07; PB12 (2 scoops); PB32; PC20; PC55; PD02;

[1207] M[20C]-2908: PA10; PA11; PA13; PA15; PA18; PA21 (2 scoops); PA23; PA25; PA28; PA57; PA69; PB07; PB12 (2 scoops); PB32; PC11; PC20; PC55 (2 scoops);

[1208] M[20C]-2938: PA11; PA13; PA14; PA15; PA18; PA21; PA23; PA28 (2 scoops); PA33; PA57; PB07; PB12 (2 scoops); PB28; PB38; PB77; PC11; PC55; PC60;

[1209] M[20C]-2948: PA10; PA11; PA13; PA14; PA15; PA18; PA21; PA23; PA28; PA57; PB07 (2 scoops); PB12; PB27; PC11 (2 scoops); PC20; PC23; PC55; PC67;

[1210] M[20C]-2951: PA12; PA13 (2 scoops); PA14; PA21; PA23; PA24; PA33; PA39; PA67; PB01; PB07; PB12; PB77; PC20; PC27; PC33; PC49; PC52; PC60;

[1211] M[20C]-2952: PA12; PA13 (2 scoops); PA14; PA21; PA23; PA25; PA26; PA33; PA37; PA67; PB01; PB07 (2 scoops); PB12; PC20; PC27; PC49; PC52; PC71;

[1212] M[20C]-2958: PA12; PA13; PA14; PA15; PA18; PA21; PA23 (2 scoops); PA28; PA33; PA57; PB01; PB07 (2 scoops); PB12; PC11; PC20; PC27; PC52; PC55;

[1213] M[20C]-2822: PA11 (2 scoops); PA13 (2 scoops); PA20 (2 scoops); PA21; PA25; PA29; PA30; PA33; PA35; PA75; PA83; PB12; PC11; PC20; PC33; PC51; PC60;

[1214] M[20C]-2824: PA11; PA13 (2 scoops); PA15; PA18; PA20 (2 scoops); PA21; PA25; PA29; PA32; PA33; PA35; PA67; PA75; PB12; PC20; PC33; PC49; PC55;

[1215] M[20C]-2825: PA11; PA13 (2 scoops); PA14; PA20; PA21; PA22; PA25; PA29; PA33 (2 scoops); PA35 (2 scoops); PA75; PB12; PB38; PB88; PC20; PC33; PC55;

[1216] M[20C]-2834: PA13 (2 scoops); PA15; PA18; PA20; PA21; PA22; PA25; PA29; PA32 (2 scoops); PA34; PA67; PB12; PB88; PC20; PC33; PC49; PC55; PC98;

[1217] M[20C]-2835: PA13 (2 scoops); PA14; PA21; PA22 (2 scoops); PA25; PA29; PA32; PA33; PA34; PA35; PB12; PB38; PB88 (2 scoops); PC20; PC33; PC55; PC98;

[1218] M[20C]-2848: PA12 (2 scoops); PA13 (2 scoops); PA18; PA21; PA24; PA28; PA33; PA67; PB07; PB12; PC23 (2 scoops); PC37; PC49; PC52; PC55; PC67; PC97;

[1219] M[20C]-2862: PA11; PA13; PA20; PA21; PA22; PA30; PA83; PB12; PB28; PB60; PC11 (2 scoops); PC20 (2 scoops); PC23; PC51; PC55; PC60 (2 scoops); PC74;

[1220] M[20C]-2864: PA13; PA15; PA18; PA20; PA21; PA22; PA32; PA67; PB12; PB28; PB60; PC11; PC20 (2 scoops); PC23; PC49; PC55 (2 scoops); PC60; PC74;

[1221] M[20C]-2865: PA13; PA14; PA21; PA22 (2 scoops); PA33; PA35; PB12; PB28; PB38; PB60; PB88; PC11; PC20 (2 scoops); PC23; PC55 (2 scoops); PC60; PC74;

[1222] M[20C]-2872: PA10; PA11 (2 scoops); PA13; PA20; PA21; PA30; PA35; PA57; PA83; PB07; PB12; PB77; PC11; PC20 (2 scoops); PC23; PC51; PC60; PC71;

[1223] M[20C]-2874: PA10; PA11; PA13; PA15; PA18; PA20; PA21; PA32; PA35; PA57; PA67; PB07; PB12; PB77; PC20 (2 scoops); PC23; PC49; PC55; PC71;

[1224] M[20C]-2875: A10; A11; A13; A14; A21; A22; A33; A35; A35; A57; B07; B12; B38; B77; B88; C20 (2 scoops); C23; C55; C71;

[1225] M[20C]-2882: PA13; PA14; PA21 (2 scoops); PA25; PA26 (2 scoops); PA28; PA29; PA37; PA39; PA67; PA75; PB07; PB12; PB36; PC49; PC55; PC60; PC71;

[1226] M[20C]-2891: PA11; PA13 (2 scoops); PA21; PA24; PA39; PA39; PA67; PB02; PB07; PB12; PB77; PB99; PC14; PC20; PC33; PC49 (2 scoops); PC60; PC98;

[1227] M[20C]-2892: PA11; PA13 (2 scoops); PA21; PA25; PA26; PA37; PA39; PA67; PB02; PB07 (2 scoops); PB12; PB99; PC14; PC20; PC49 (2 scoops); PC71; PC98;

[1228] M[20C]-2901: PA10; PA11; PA13 (2 scoops); PA21 (2 scoops); PA24; PA25; PA39; PA67; PA69; PB12 (2 scoops); PB32; PB77; PC20; PC33; PC49; PC55; PC60;

[1229] M[20C]-2902: PA10; PA11; PA13 (2 scoops); PA21 (2 scoops); PA25 (2 scoops); PA26; PA37; PA67; PA69; PB07; PB12 (2 scoops); PB32; PC20; PC49; PC55; PC71;

[1230] M[20C]-2904: PA10; PA11 (2 scoops); PA13; PA21 (2 scoops); PA25; PA28; PA39; PA69; PA70; PB02; PB12; PB32; PB99; PC20; PC55; PC71; PC97; PC98;

[1231] M[20C]-2905: PA10 (2 scoops); PA11; PA13; PA21; PA25; PA29; PA57; PA67; PA69; PB07; PB12; PB28; PB32; PB77; PC11; PC20 (2 scoops); PC52; PC55;

[1232] M[20C]-2906: PA10; PA11 (2 scoops); PA13; PA18; PA21; PA25; PA57; PA67; PA69; PB02; PB12; PB32; PB38; PC11; PC20 (2 scoops); PC27; PC52; PC55;

[1233] M[20C]-2907: PA10; PA11 (2 scoops); PA13; PA14; PA21; PA24; PA25; PA29; PA69; PA83; PB12 (2 scoops); PB32; PB60; PC11; PC20 (2 scoops); PC51; PC55;

[1234] M[20C]-2909: A10 (2 scoops); A11; A12; A13 (2 scoops); A18; A21; A25; A26; A33; A39; A69; B12; B32; C20 (2 scoops); C52; C55 (2 scoops);

[1235] M[20C]-2911: PA13; PA21 (2 scoops); PA24; PA26; PA27; PA39; PA67; PB07; PB12 (2 scoops); PB33; PB77; PC20; PC24; PC33; PC41; PC49; PC60; PC71;

[1236] M[20C]-2912: PA13; A21 (2 scoops); PA25; PA26 (2 scoops); PA27; PA37; PA67; PB07 (2 scoops); PB12 (2 scoops); PB33; PC20; PC24; PC41; PC49; PC71 (2 scoops);

[1237] M[20C]-2919: PA10; PA12; PA13; PA18; PA21; PA26 (2 scoops); PA27; PA33; PA39; PB07; PB12; PB33; PC20 (2 scoops); PC24; PC41; PC52; PC55; PC71;

[1238] M[20C]-2922: PA13; PA20; PA21 (2 scoops); PA25; PA26; PA28; PA35; PA37; PA67 (2 scoops); PB07; PB12 (2 scoops); PC12; PC37; PC49; PC60; PC71; PC74;

[1239] M[20C]-2929: PA10; PA12; PA13; PA18; PA20; PA21; PA26; PA28; PA33; PA35; PA39; PA67; PB12; PC12; PC20; PC37; PC52; PC55; PC60; PC74;

[1240] M[20C]-2932: PA11; PA13 (2 scoops); PA14; PA21; PA25; PA26; PA28; PA33; PA37; PA67; PB07; PB12 (2 scoops); PB28; PB38; PB77; PC49; PC60; PC71;

[1241] M[20C]-2941: PA10; PA11; PA13 (2 scoops); PA14; PA21; PA24; PA39; PA67; PB07; PB12; PB27; PB77; PC11; PC20; PC23; PC33; PC49; PC60; PC67;

[1242] M[20C]-2942: PA10; PA11; PA13 (2 scoops); PA14; PA21; PA25; PA26; PA37; PA67; PB07 (2 scoops); PB12; PB27; PC11; PC20; PC23; PC49; PC67; PC71;

[1243] Table 10-4: Average of the percent inhibition of planktonic growth and percent inhibition of biofilm formation of the processed samples against the six tested Staphylococcus aureus strains based on the mixture of Table 10-4 diluted 1:20

[1244]

[1245]

[1246] [Comparative Example 4]

[1247] As comparative examples, we report extracts of several herbal compositions containing only one of three plants: Filipendula ulmaria (PA13), Camellia sinensis (PA21), and Uva ursi (PB12), and only one of three plants: Syringa syringae (PC20), Vitis vinifera (PA22), and Dexiella variegata (PB07).

[1248] Five herbal compositions having the contents reported in List 10-5 were prepared according to the aforementioned Method 1.

[1249] [List 10-5 Extracts from herbal compositions containing the following plants]

[1250] M[20P]-318: PA15; PA21; PA34; PA35; PA82; PA83; PA85; PA92; PA96; PB03; PB07; PB26; PB30; PB36; PB79; PC11; PC49; PC91; PC92; PD10;

[1251] M[20P]-329: PA10; PA28; PA34; PA36; PA55; PA72; PA83; PA85; PA91; PB03; PB12; PB16; PB28; PB35; PB49; PB92; PC20; PC23; PC64; PC85;

[1252] M[20P]-333: PA04; PA05; PA13; PA16; PA22; PA39; PA51; PA55; PA57; PA72; PA82; PA92; PA98; PB05; PB20; PB36; PB43; PB74; PB77; PC37;

[1253] M[20P]-362: PA21; PA23; PA25; PA33; PA37; PA57; PA69; PA81; PA92; PB16; PB25; PB26; PB28; PB29; PB78; PC20; PC21; PC71; PC83; PD02;

[1254] M[20P]-513: PA12;

[1255] An herbal composition containing 20 kinds of plant powders was prepared according to the aforementioned method A.

[1256] Starting from the herbal composition, the corresponding aqueous extract (or processed sample) was prepared by aqueous extraction according to the aforementioned method B using 40 mL of water containing 100 g / l sucrose and including a decantation step before filtration.

[1257] The bioactivity of each processed sample was determined according to Method C described above by using the following S. aureus strains (ATCC 25923, ATCC 49476, ATCC 6538, ATCC 51740, ATCC 29213, and ATCC 14775) at a final dilution of the processed sample of 1:20.

[1258] Table 10-5 shows the average percentage inhibition of bacterial planktonic growth and biofilm formation for all tested strains for processed samples obtained from the herbal compositions of Table 10-4. Each percentage is the average of two measurements under identical conditions in two different wells. Negative percentages reflect growth promotion and biofilm enhancement, rather than the desired inhibition. It should be understood that this extract exhibits very poor, or even no, inhibitory effect compared to the extract of Example 10.

[1259] [Table 10-5: Average percentage inhibition of bacterial planktonic growth and biofilm formation for all tested strains of the processed samples obtained from the herbal composition of Table 10-4]

[1260]

[1261]

Example 11

[1262] In this example, we demonstrate the antimicrobial activity of 10 mixed extracts obtained from the herbal composition disclosed in Table 11-1, which contains at least two of the three plants Filipendula ulmoides (PA13), Camellia sinensis (PA21), and Uva ursi (PB12) and at least one of the three plants Syringa syringae (PC20), Vitis vinifera (PA22), and Desmodium variegatum (PB07), against 14 different microbial species described in Table 11-1.

[1263] [Table 11-1: Microbial species and strains used in Example 11]

[1264]

[1265] [List 11-1: In Example 11, extracts from the following herbal composition were used, the herbal composition containing: Filipendula ulmaria (PA13), Camellia sinensis (PA21), and at least one of three plants: Syringa syringae (PC20), Vitis vinifera (PA22), and Desmodium variegatum (PB07)]

[1266] M[20P]-297: PA10; PA11; PA13; PA14; PA20; PA21; PA25; PA26; PA29; PA33; PA57; PA67; PA69; PB07; PB12; PB60; PC20; PC37; PC49; PC98;

[1267] M[20P]-416: PA11; PA13; PA14; PA20; PA21; PA25; PA26; PA28; PA29; PA67; PA75; PB02; PB12; PB27; PB33; PB60; PB88; PC20; PC49; PC60;

[1268] M[20P]-418: PA10; PA11; PA12; PA13; PA21; PA22; PA23; PA24; PA26; PA39; PB07; PB12; PB38; PC11; PC12; PC20; PC23; PC24; PC49; PC55;

[1269] M[20P]-527: PA10; PA10; PA11; PA13; PA15; PA20; PA20; PA21; PA23; PA26 (3 scoops); PA33; PB08; PB12 (2 scoops); PB60; PB88; PC12; PC20;

[1270] M[20P]-557: PA00; PA10; PA20; PA21; PA23; PA24; PA29; PA57; PB07; PB12 (4 scoops); PC11 (2 scoops); PC12; PC20; PC51; PC55; PC67;

[1271] M[20P]-573: PA10; PA11; PA13 (3 scoops); PA14; PA18; PA21; PA23; PA29; PA67; PB00; PB07; PB12; PB33; PB77; PC11; PC52; PC60; PC71;

[1272] M[20P]-576: PA11 (2 scoops); PA12; PA13 (3 scoops); PA14 (2 scoops); PA20; PA21; PA22; PA29; PA39; PB12; PB60; PC20; PC26; PC33; PC37; PC49;

[1273] M[19P]-576: PA11 (2 scoops); PA12; PA13 (3 scoops); PA14 (2 scoops); PA20; PA21; PA22; PA29; PA39; PB12; PB60; PC20; PC26; PC33; PC37;

[1274] M[5P]-1: PA12; PA13; PA21; PA22; PB12;

[1275] M[5P]-2: PA11; PA13; PA21; PA22; PB12;

[1276] For mixtures M[5P]-1, M[5P]-2, and M[19P]-576, pharmaceutical grade dried plants were obtained from "Herboristerie Cailleau" (Chemilles, France). For the other mixtures, pharmaceutical grade dried plants were obtained from "Pharmacie Fontgiève" (Clermont-Ferrand, France, plants PA00 to PB12) and "Pharmacie St Herem" (Clermont-Ferrand, France, plants PB13 to PD02).

[1277] All herbal compositions of 20 plants were prepared according to Method A described above.

[1278] Starting from the herbal composition, the corresponding aqueous extract (or processed sample) was prepared according to the aforementioned method B, using 40 mL of water containing 100 g / l sucrose and including a decantation step before filtration.

[1279] As shown in Table 11-2, herbal compositions M[19P]-576, M[5P]-1 and M[5P]-2 were prepared by weighing each dried herb.

[1280] Table 11-2: Weight (mg) of dried plants used in the herbal compositions of M[19P]-576, M[5P]-1, and M[5P]-2 used in Example 11

[1281]

[1282] Starting from the herbal compositions, aqueous extracts (or processed samples) were prepared according to the aforementioned method B, using 30 mL, 40 mL and 40 mL of sucrose-free water in the case of M[19P]-576, M[5P]-1 and M[5P]2, respectively, and including a decantation step before filtration.

[1283] Table 11-3 shows the biological activity of each processed sample determined according to the aforementioned method C using the following strains at the specified final dilution of the processed sample.

[1284] The average values ​​of the percentage inhibition of microbial planktonic growth are given in Table 11-3.

[1285] [Table 11-3: Percentage of inhibition of planktonic growth of 14 test microbial strains by processed samples of the composition disclosed in Table 11-1]

[1286]

[1287]

[1288] Note: "ND" stands for "Not Determined"

[1289]

Example 12

[1290] In this example, we demonstrate the antimicrobial activity of the following plant mixtures, where the extraction step was performed with the non-aqueous solvents acetone, ethyl acetate, and ethanol, used alone or in binary mixtures as shown in Table 12-1. A comparison with water is provided.

[1291] [Table 12-1: Composition of the binary mixture used for non-aqueous extraction in Example 12]

[1292]

[1293] The M[20P]-576 dry plant mixture was composed of the following Table 12-2 and placed in a 500 mL vial and mixed thoroughly by manual inversion and rotation. The dried herbs were obtained from "Herboristerie Cailleau" (Chemilles, France), except PC49 which was obtained from Siccarapam (Aubia, France).

[1294] Table 12-2: List of dried plants constituting M[20P]-576 used in Example 12

[1295]

[1296] For each solvent tested, 5g of dry plant mixture is put into the 100ml colored vial (Schott (Schott), Germany) with a magnetic bar, and 50ml of solvent is added. At room temperature, the liquid suspension is stirred at maximum speed for 48 hours with a magnetic stirrer (RO 15PIKA, Germany). Use the 250mm folded filter paper (J025106, Prats Dumas, France) placed on a glass funnel (Schott, Germany) to filter each kind of suspension / solution by gravity, and the glass funnel itself is placed in an evaporating flask. The flask containing the liquid through the filter paper is placed in a rotary vacuum evaporator ( The extract was placed on a R205+V800 vacuum controller, Büchi, Switzerland) and evaporated at 35°C with the vacuum adjusted below the boiling point of the solvent until all the solvent was evaporated. The remaining material was weighed and dissolved in DMSO (Acros organic, USA) qsp at 10 g / L to form an extraction mixture.

[1297] Two aqueous extracts were prepared according to the aforementioned method B, wherein one spoonful of dry plant was replaced with 2 g of the M[20P]-576 dry plant mixture of Example 12, and the aqueous extraction was performed using 40 mL of water and a decantation step was included before filtration. The dry residue content of one of the plant mixture extracts was determined by lyophilizing the extract using a FreeZone TM The determination was performed by weighing the lyophilized residues of the frozen extracts obtained from RZ6 Vacuubrand, Germany).

[1298] These dry residue contents were used to infer the specific activities of the different extracts.

[1299] The biological activity of each extracted mixture was determined using the strains shown in Table 12-3 according to Method C above, wherein the dilutions of the extracted mixture were 1:20, 1:40, 1:80, and 1:160. The wells were visually observed, and the lowest concentration at which no growth was observed was noted as the minimum inhibitory concentration (MIC).

[1300] [Table 12-3: List of bacterial strains used in Example 12]

[1301]

[1302] The results are compiled in Table 12-4 for different solvents.

[1303] Table 12-4: CMI (in mg / L) of mixtures extracted using A) acetone, ethyl acetate, ethanol, and water as solvents, B) different ratios of acetone and ethanol as binary solvents, as shown in Table 12-2-A, C) different ratios of ethyl acetate and ethanol as binary solvents, as shown in Table 12-2-B, and D) different ratios of ethyl acetate and acetone as binary solvents, as shown in Table 12-2-C.

[1304]

[1305]

[1306]

Example 13

[1307] In this example, we demonstrate the antimicrobial activity of a botanical mixture prepared according to the aforementioned method B, wherein the added carbohydrates are selected from sucrose, dextrose, maltose and galactose.

[1308] The dried plant mixture of M[19P]-576, M[5P]-1 and M[5P]-2 was composed according to Table 13-1 and placed in a 500 mL vial and mixed thoroughly by manual inversion and rotation. The dried herbs were obtained from "Herboristerie Cailleau" (Chemilles, France), except PC49, which was obtained from Siccarapam (Aubia, France).

[1309] Table 13-1: List of dried plants constituting A) M[20P]-576 mixture, B) M[5P]-1 mixture, and C) M[5P]-2 mixture used in Example 13

[1310]

[1311] For each dry plant mixture, an extract was prepared according to the aforementioned method B, wherein one spoonful of dry plants was replaced with 2 g of the plant mixture of Example 13, using 40 mL of water and the amount of carbohydrates shown in Table 13-3 for aqueous extraction, and including a decantation step before filtration.

[1312] The dry residue content of the carbohydrate-free plant mixture was determined separately by lyophilizing the plant mixture using a freeze-drying device (FreeZone TMThe specific activity of the extracts was determined by weighing 40 mL of the freeze-dried residue obtained from a frozen extract obtained from a plant extract company (RZ6 Vacuubrand, Germany) according to the aforementioned method B, wherein one spoonful of dry plant matter was replaced by 2 g of the plant mixture of Example 13, 40 mL of water was used for the aqueous extraction, and a decanting step was included before filtration. These dry residue contents were used to infer the specific activity of the different extracts, where carbohydrates were considered as adjuvants and were not considered to be part of the active ingredient.

[1313] The biological activity of each extracted mixture was determined using the strains shown in Table 13-2 according to Method C above, wherein the dilutions of the extracted mixture were 1:20, 1:40, 1:80, and 1:160. The wells were visually observed, and the lowest concentration at which no growth was observed was marked as the minimum inhibitory concentration (MIC).

[1314] [Table 13-2: List of bacterial strains used in Example 13]

[1315]

[1316] The results are compiled in Table 13-4.

[1317] Table 13-3: MICs (in mg / L) of the following mixtures extracted with sucrose, lactose, dextrose, maltose, or galactose: A) M[20P]-576 mixture, B) M[5P]-1 mixture, and C) M[5P]-2 mixture

[1318]

[1319]

Example 14

[1320] In this example, we demonstrate that the M[20P]-576 herbal mixture extract has enhanced antimicrobial activity against commonly used antibiotics.

[1321] [Table 14-1: Bacterial species and strains used in Example 14]

[1322]

[1323] [List 14-1: Herbal composition used for processing samples in Example 14:]

[1324] M[19P]-576: PA11; PA12; PA13; PA14; PA20; PA21; PA22; PA29; PA39; PB12; PB60; PC20; PC26; PC33; PC37.

[1325] Pharmaceutical grade dried plants were obtained from "Herboristerie Cailleau" (Chemilles, France).

[1326] The herbal composition M[19P]-576 was prepared by weighing each dried herb as shown in Table 14-2.

[1327] Table 14-2: Weight of dried plants used in the herbal composition of M[19P]-576 (mg)

[1328] PA11 PA12 PA13 PA14 PA20 PA21 PA22 PA29 PA39 PB12 PB60 PC20 PC26 PC33 PC37 M[19P]-576 409 145 573 316 277 271 207 380 207 247 206 236 198 187 154

[1329] Starting from the herbal composition, a water extract (or processed sample) was prepared using 2.06 g of the herbal composition and 40 mL of water containing 100 g / L sucrose according to the aforementioned method B, including a decantation step before filtration. In a separate water extract preparation without sucrose, 2.06 g / 40 mL of the herbal composition was measured, which is equivalent to 14.5 g / L derived from the weight of the freeze-dried herbal extract.

[1330] The biological activity of each processed sample was determined using the following strains at the specified final dilution of the processed sample according to the aforementioned method C and is given in Table 14-3. In Example 14, we calculated the growth of bacteria in the presence of antibiotics and / or herbal mixture relative to the growth in the absence of antibiotics and herbal mixture extracts.

[1331] The mean values ​​of percent inhibition of bacterial planktonic growth are given in Table 14-3.

[1332] It is important to note that the presence of the herbal extract M[19P]-576 had three effects at the concentrations used: 1) it reduced bacterial growth at low concentrations and in the absence of antibiotics, 2) it reduced the increase in bacterial growth induced by low antibiotic concentrations relative to growth in the absence of antibiotics, and 3) it reduced the minimum inhibitory concentrations (MICs) of vancomycin against B-A17 and ampicillin and penicillin against B-A21 at concentrations of M[20C]-576 starting at 90.6 μg / mL, 181.3 μg / mL, and 90.6 μg / mL, respectively.

[1333] Table 14-3: Planktonic growth in the presence of different extract concentrations and antibiotic concentrations relative to bacterial growth in the absence of extract and antibiotic on two different strains

[1334]

[1335]

Example 15

[1336] In this example, we illustrate how to adjust the temperature in Method B to address industrial constraints.

[1337] Organic grade dried plants were obtained from "Herboristerie Cailleau" (Chemilles, France).

[1338] As shown in Table 15-1, the herbal composition M[5P]-1 was prepared by weighing each dried herb.

[1339] [Table 15-1: Weight (mg) of dried plants used in the herbal composition of M[5P]-1]

[1340] PA12 PA13 PA21 PA22 PB12 M[5P]-1 395.2 688.8 978.9 552.5 884.6

[1341] The first extract, M[5P]-1-A-, was prepared by incorporating 120 g of the herbal composition into 600 mL of water contained in a 2 L beaker (Schott, Germany). The second extract, M[5P]-1-B, was prepared by placing 120 g of the herbal composition into a 25 x 30 cm organic grade cotton bag (Ecobags, USA), and placing the filled bag into 600 mL of water contained in a 2 L beaker (Schott, Germany). The third extract, M[5P]-1-C, was prepared by placing 60 g of the herbal composition twice into a 25 x 30 cm organic grade cotton bag (Ecobags, USA), and placing both filled bags into 600 mL of water contained in a single 2 L beaker (Schott, Germany). Each formulation (M[5P]-1-A, M[5P]-1-B, and M[5P]-1-C, respectively) was allowed to stand at room temperature for 3 hours to allow for maceration, and then placed in its beaker in an autoclave (VWR Vapour Line Eco 25, USA) and maintained at 121°C, 121°C, and 134°C for 30 minutes, respectively. For each of M[5P]-1-B and M[5P]-1-C, a manually operated 5 L fruit squeezer (Brouwland, Belgium) was used to squeeze the cotton bag and the recovered liquid was returned to the 2 L beaker. Each beaker was decanted, and the necessary amount of supernatant was used to measure its biological activity.

[1342] [Table 15-2: Bacterial species and strains used in Example 15]

[1343]

[1344] The biological activity of each processed sample was determined at final dilutions of 1:40, 1:80, 1:160, and 1:320 using the strains listed in Table 15-2 according to the aforementioned Method C. The dilution corresponding to the minimum inhibitory concentration (MIC) was recorded as the lowest concentration at which no growth was observed by visual observation of the wells and is reported in Table 15-3.

[1345] Table 15-3: Dilutions corresponding to the minimum inhibitory concentration (MIC) of the extracts achieved with different industrial details and temperatures

[1346] M[5P]-1-A at 121°C M[5P]-1-B at 121°C At 134℃, M[5P]-1-C B-A17 1 / 80 1 / 40 1 / 80 B-A21 1 / 80 1 / 40 1 / 80 B-A49 1 / 80 1 / 40 1 / 160

[1347] It is noteworthy that the introduction of the cotton bag made the industrial process easier, but slightly reduced the activity of the extract, compared to the unsatisfactory method without the bag. Raising the temperature of the autoclave during the hot extraction phase allowed the full activity to be restored.

Claims

1. An extract of a herbal composition for use as an antimicrobial and / or anti-biofilm agent for treating and / or preventing microbial infections, the herbal composition comprising as active ingredients: Dried plant parts of the flowers of Filipendula ulmaria, the leaves of Camellia sinensis, and the leaves of Arctostaphylos uva-ursi, and at least one plant part selected from the group consisting of leaves of Vitis vinifera var. tinctoria and roots and rhizomes of Rheumpalmatum, The extract is prepared by the following method: (a) chopping or grinding at least two single dried plants individually or in a mixture to obtain the corresponding plant powders; (b) mixing at least three different plant powders to obtain a herbal composition; (c) adding water to the herbal composition to obtain the corresponding liquid preparation (or extract); (d) incubating the liquid formulation at room temperature for a period of 5 to 15 minutes; Optionally, (e) heating the liquid formulation at a temperature of 60° C. to 134° C. for a period of 5 to 60 minutes; Optionally, (f) centrifuging, collecting the supernatant, and filtering the collected supernatant; Optionally, (g) concentrating and / or drying or freeze-drying the liquid preparation obtained from step (e) or (f), and The infection is caused by Staphylococcus.

2. The extract according to claim 1, wherein the herbal composition further comprises one or more additional dried plants selected from the group consisting of: Agropyrum repens, Arbutus unedo, Buxus sempervirens, Calendula officinalis, Cassia angustifolia, Centaurea cyanus, Centella asiatica, Chamomilla recutita, Cinnamomum zeylanicum, Combretum micranthum, Crataegus oxyacantha, Cupressus sempervirens, Desmodium adscendens, Epilobium parviflorum, Eucalyptus globulus, Eugenia caryophyllus), fennel (Foeniculum vulgare), European ash (Fraxinus excelsior), fucus vesiculosus, ginkgo (Ginkgo biloba), mint (Glechoma hederacea), handroanthus impetiginosus, Hypericum perforatum, walnut (Juglansregia), juniper (Juniperus communis), sesame (Lamium album), lemon vine (Lippia citriodora), honeysuckle (Melissa officinalis), olive (Olea europaea), ginseng (Panax ginseng), wall grass (Parietaria officinalis), plantain (Plantago lanceolata), buckthorn (Rhamnus frangula), rhubarb (Rheum palmatum), rosemary (Rosmarinus officinalis), rubia officinalis tinctorum), raspberry (Rubus idaeus), European elderberry (Sambucus nigra), mountain savory (Satureja montana), chrysanthemum (Tanacetumvulgare), dandelion (Taraxacum officinalis), red thyme (Thymus serpyllum), silver thyme (Thymus vulgaris), small-leaved lime (Tiliacordata), bilberry (Vaccinium myrtillus), and valerian (Valeriana officinalis).

3. The extract according to any preceding claim, wherein the herbal composition comprises: Filipendula ulmaria, Camellia sinensis, and Arctostaphylos uva-ursi, At least one plant selected from the group consisting of: Vitis vinifera and Rheumpalmatum, and One to three dried plants are selected from the following: rosemary (Rosmarinus officinalis), variegated leech (Desmodium adscendens), clove (Eugenia caryophyllus) and blue gum (Eucalyptus globulus).

4. The extract according to any preceding claim, wherein the herbal composition comprises dried plants selected from the group consisting of Rosemary (Rosmarinus officinalis), Filipendula ulmaria, Camellia sinensis, Vitis vinifera and Arctostaphylos uva-ursi.

5. The extract according to any preceding claim, wherein the herbal composition comprises dried plants of: Rheum palmatum, Filipendula ulmaria, Camellia sinensis, Vitis vinifera and Arctostaphylos uva-ursi.

6. The extract according to any preceding claim, wherein the herbal composition comprises the following dried plants: Rheum palmatum, Rosmarinus officinalis, Filipendula ulmaria, Camellia sinensis, Vitis vinifera, Eugenia caryophyllus and Arctostaphylos uva-ursi.

7. The extract according to any preceding claim, wherein the herbal composition comprises the following dried plants: Rheum palmatum, Rosmarinus officinalis, Filipendula ulmaria, Camellia sinensis, Vitis vinifera, Eucalyptus globulus, Arctostaphylos uva-ursi, Mentha spicata and Rubia tinctorum.

8. The extract according to any preceding claim, wherein the herbal composition comprises at least fourteen or fifteen dried plants selected from the group consisting of: Rheum palmatum, Rosmarinus officinalis, Filipendula ulmaria, Satureja montana, Valeriana officinalis, Camellia sinensis, Vitis vinifera, Fucus vesiculosus, Foeniculum vulgare, Arctostaphylos uva-ursi, Arbutus unedo, Eugenia caryophyllus, Juniperus communis, Combretum micranthum, Lippia citriodora, and Tanacetum vulgare.

9. The extract according to any preceding claim, wherein the herbal composition comprises the following dried plants: Rheum palmatum, Rosmarinus officinalis, Filipendula ulmaria, Satureja montana, Valerian (Valeriana officinalis), Camellia sinensis, Vitis vinifera, Fucus vesiculosus, Foeniculum vulgare, Arctostaphylos uva-ursi, Arbutus unedo, Eugenia caryophyllus, Juniperus communis, Combretum micranthum, and Lippia citriodora.

10. The extract according to any preceding claim, wherein the herbal composition comprises the following dried plants: Rheum palmatum, Rosmarinus officinalis, Filipendula ulmaria, Satureja montana, Valerian (Valeriana officinalis), Camellia sinensis, Vitis vinifera, Fucus vesiculosus, Foeniculum vulgare, Arctostaphylos uva-ursi, Arbutus unedo, Eugenia caryophyllus, Juniperus communis, Combretum micranthum, Lippia citriodora, and Tanacetum vulgare.

11. The extract according to any one of claims 1 to 10, wherein the infection is caused by a microorganism selected from the group consisting of Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus pseudintermedius.

12. A lyophilisate obtained by freeze-drying the extract according to any one of claims 1 to 11.

13. A pharmaceutical, veterinary or cosmetic composition for use as an antimicrobial and / or antibiofilm agent for treating and / or preventing microbial infections, comprising as an active ingredient an extract according to any one of claims 1 to 11, or The lyophilized product according to claim 12, and At least one excipient.

14. The pharmaceutical, veterinary or cosmetic composition according to claim 13, which is formulated for oral or topical administration.

15. Use of an extract according to any one of claims 1 to 11 in the preparation of an antimicrobial and / or antibiofilm agent for treating and / or preventing microbial infection, wherein the infection is caused by a microorganism selected from the group consisting of Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus pseudintermedius.

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