Patent Application
20190183953
The present invention relates to an immunologically active phyto-mixture comprising at least one plant extract selected from the family (a) Asteraceae, (b) Verbenaceae, and/or (c) Burseraceae, preferably from the genus (a) Bidens, (b) Stachytarpheta, and/or (c) Bursera. Particularly preferred species include (a) Bidens alba, Bidens pilosa, (b) Stachytarpheta jamaicensis, Stachytarpheta cayennensis, Stachytarpheta indica, and/or (c) Bursera microphylla, Bursera glabrifolia and Bursera simaruba. The phyto-mixture optionally comprises (d) at least one further biologically active plant extract, such as Aloe vera and/or Stemodia maritima. The afore-mentioned phyto-mixture according to the invention exhibits good antimicrobial and anti-inflammatory efficiency and is particularly suitable for prevention and treatment of efflorescences. Therefore, the present invention also relates to a preparation for oral and topical administration for prevention and treatment of efflorescences of the skin and the mucous membrane.
In modern medicine, comprehensive synthetically produced pharmaceutical products for prevention or treatment of skin irritations and skin diseases are provided for the patients. These are prescribed by the doctor as authorised medicaments or are available as nonprescription medical products. However, due to synthetic active agents, the pharmaceutic compositions increasingly cause side effects, cause allergic reactions or result in steadily growing resistances in infection germs causing the skin irritations or diseases.
Therefore, there are more and more aspirations for isolating new active agents, continuously having efficiency against resistant germs, causing few or no side effects or allergic reactions in human, and potentially having an improved resorption in human. For this purpose, microorganisms itself or plants may be used as source of new acting agents.
In the case of plant sources, some compound classes, such as, for example flavones, are isolated and processed into pharmaceutic preparations. RU2412719 discloses such flavone extracts for medicaments for treatment of liver diseases. CN102743651 discloses a mixture of 30 different plants in the form of a lotion for treatment of cellulitis.
In the state of the art, miscellaneous plants, in particular from traditional medicine, are tested for their efficiency, their ingredients are identified, the toxicity is analysed, but only less or no efficiency is observed (EP114709A1).
Previously, only few analyses have been performed for plant preparations and no one has both antibacterial and anti-inflammatory efficiency. Consequently, no plant alternative to synthetic active agents, such as, for example antibiotics, or synthetic inflammation inhibitors has previously been detected.
The present invention provides the object of providing new preparations having effective ingredients from natural sources, in particular effective ingredients of plant sources. A further object is to provide a plant product and preparations having antimicrobially, preferably antibacterially, and/or anti-inflammatorily effective ingredients. In this context, various possible combinations of some plant products, such as plant extracts, shall be provided, which may individually be mixed for the user. A further object is the provision of a phyto-mixture for the production of preparations for preventive use or therapeutic use in the treatment of efflorescences. For this purpose, pharmaceutic compositions, care products, nutritional supplements, as well as medical products comprising a phyto-mixture from total extracts of plants shall be provided. Moreover, the present invention provides the object of providing a natural product as alternative to synthetically produced products. The object is to provide a natural product from plant sources, in particular for humans, in which the synthetically produced products known from the state of the art exhibit strongly reduced or no more efficiency and/or increasingly cause side effects. The afore-mentioned plant products and their preparations comprising the phyto-mixture shall be provided for prevention and treatment of efflorescences.
The present invention therefore provides a natural product from plant sources, having both antimicrobial, preferably antibacterial, and anti-inflammatory effect. Therefore, a subject matter of the present invention is an immunologically active phyto-mixture comprising at least one plant extract selected from
optionally, additionally (d) at least one further extract of a biologically active plant, as described below.
In the case where the wording “plant extracts (a), (b), and/or (c)” is used without limitation to a specific species, it shall be understood to mean an abbreviation of the afore-mentioned wording. In the following, the name of the appropriate genus is abbreviated (a) Bidens by “Bid.”, (b) Stachytarpheta by “Sta.”, and (c) Bursera by “Bur.”.
Preferably, the afore-mentioned plant extracts are aqueous or ethanolic extracts (greater than or equal to 70% ethanol, remainder water) The “immunologically active phyto-mixture” according to the invention is briefly, thus synonymously, termed as “phyto-mixture”.
Within the meaning of the invention, “immunologically active” means that the phyto-mixture according to the invention, in particular the contained ingredients and compounds, prevents, inhibits or reduces immune reactions of the body. “Immunologically active” comprises preventive effect so that immune reactions of the body are not even being triggered, and therapeutic effect so that the already triggered immune reactions in the body are stopped, downregulated or reduced. Depending on administration time, the beginning immune reaction may be inhibited prior to occurring of symptoms, accompanying with immune reaction, and phenotypic appearances. Endogenous microorganisms, pathogens influencing in/on the body, such as bacteria, viruses, fungi and parasites, or toxic and/or influencing compounds triggering allergic reactions may be trigger of the afore-mentioned immune reactions.
The immune response of the immune system to an organism, in particular microorganisms, or substance, in particular toxins, is termed as immune reactions.
What triggers the afore-mentioned immune reaction depends on constitution of the organism, human or animal. Thus, a distinction is to be made between healthy organism and immunosuppressive organism.
A healthy organism, in particular human, has no congenital disease, in particular no immune weakening disease, thus having a normally working and reacting immune system.
An immune weakened organism, in particular immune weakened human, has a weakened immune system. Weakening may occur by a temporary disease, such as influenza or common cold, a long-lasting disease, such as cancer, malnourishment/undernourishment, infections with certain pathogens, as well as intake of certain medications, e.g. chemotherapeutics, or radiation. The afore-mentioned weakening is an acquired immunodeficiency which has to be distinguished from congenital immunodeficiency. Congenital immune deficiency is based on mutations in genes impairing, for example, the production or function of antibodies or phagocytes which are related to T-cell or B-cell dominated immune response.
The present invention preferably relates to an immunologically active phyto-mixture being suitable preventively or for treatment of efflorescences in healthy humans and acquired immune weakened human.
In a further embodiment, the immunologically active phyto mixture according to the invention optionally comprises additionally (d) at least one further extract of a biologically active plant comprising Aloe species of genus Aloe of the Asphodeloideae subfamily, species of genus Stemodia (briefly “Stem.”) of the Plantaginaceae family and Stem. maritima. Preferred Aloe species include Aloe vera, Aloe barbadensis, Aloe perfoliata, Aloe vulgaris, Aloe indica and Aloe chinensis.
The Asteraceae family comprises the (a) genus Bidens (=A) comprising miscellaneous Bidens species also referred to colloquially as “Beggartick”. Genus Bidens had formerly been assigned to the Compositae family by the person skilled in the art. Genus Bidens comprises the species Bid. alba, Bid. pilosa, Bid. aurea, Bid. beckii, Bid. bipinnata, Bid. biternata, Bid. parviflora, Bid. connata and Bid. tripartita. Species Bid. alba, Bid. pilosa, Bid. bipinnata and Bid. parviflora are use according the invention. Species Bid. alba and Bid. pilosa are preferred, and Bid. alba is particularly preferred as a plant extract in the phyto-mixture.
Distinction between the mentioned species is preferably made with young and full-grown plants having differentiated growth and which already have fruit organs, such as buds and blossoms, and leaves and blossoms differentiated in colour and shape. Preferably, one- to four-years-old, particularly preferably one- to three-years-old, plants are used.
Differentiated plants of the species Bid. alba and Bid. pilosa distinguish in growth height, wherein Bid. alba has a smaller maximal growth height of up to 2 m than Bid. pilosa. The blossoms of Bid. alba are small and have a radial symmetry and have an appearance similar to the daisies having yellow pollen in the center of the blossom and five, in particular white, petals. The blossoms are always arranged at the head of a twig or stem. Further features distinguishing from Bid. pilosa are known by the person skilled in the art.
The afore-mentioned Bidens species comprise flavonoids, luteolin, terpenes, polyacetylenes, phenylheptratriyne (PHT), phenylpropanoids, in particular anethole, apiol, cinnamic aldehyde, dillapiole and estragole, lipids and benzoids as secondary plant substances, and exhibit, according to the invention, antimicrobial, preferably antibacterial and/or antimycotic, as well as anti-inflammatory efficiency.
Within the meaning of the invention, the Bidens species according the invention, preferably Bid. alba and/or Bid. pilosa, exhibit antimicrobial, in particular at least antibacterial, efficiency against transient skin flora comprising staphylococcus, streptococcus, methicillin-resistant Staphylococcus aureus (MRSA), pseudomonads and/or acinetobacteria. The Bidens species according to the invention, preferably Bid. alba and/or Bid. pilosa, particularly preferably exhibit antibacterial efficiency against Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (ATCC 14990), MRSA (NCTC 10442), Pseudomonas aeruginosa (ATCC 27853) and Acinetobacter baumanii (ATCC BAA 747) (Example 7).
In particular, antimicrobial, preferably antibacterial, efficiency is achieved by plant extracts of a Bidens species, preferably Bid. alba and/or Bid. pilosa, which comprise compounds being active against microorganisms, in particular gram-positive and/or gram-negative bacteria, comprising centaurein, centauredin, polyacetylene, phenylhepatriyne (PHT), polyyne, 1,2-dihydroxytrideca-3,5,7,9,11-pentayne.
Within the meaning of the invention, the afore-mentioned Bidens species exhibit, additionally to the antimicrobial, in particular antibacterial, efficiency, anti-inflammatory efficiency at less than or equal to 200±10 μg/ml of the respective plant extract, preferably less than or equal to 180±10 μg/ml, less than or equal to 160±10 μg/ml, less than or equal to 140±10 μg/ml, particularly preferably less than or equal to 130±10 μg/ml, and less than or equal to 110±10 μg/ml measured as IC50 der 5-LOX inhibition.
In particular, anti-inflammatory efficiency is achieved by plant extracts of a Bidens species, preferably Bid. alba and/or Bid. pilosa, which comprise compounds being active against 5-lipoxygenase, comprising triterpenes, flavonoids, aurones, chalcones, luteolin, 1-phenyl-1,3-diyne-5-en-7-ol-acetate, caffeates and ethyl caffeates.
The Verbenaceae family, also referred to as vervain family, comprises about 35 genera. The (b) genus Stachytarpheta (=B) comprises the species Sta. angustifolia, Sta. cayennensis, Sta. chamissonis, Sta. glauca, Sta. glabra, Sta. jamaicensis, Sta. indica, Sta. mutabilis, Sta. steyermarkii, Sta. svensonii und Sta. urticaefolia. Sta. cayennensis, Sta. jamaicensis and Sta. indica are species within the meaning of the invention, and Sta. jamaicensis is particularly preferably in the phyto-mixture according to the invention as a plant extract.
The afore-mentioned Stachytarpheta species comprise the ingredients 3,4-dihydroxycinnamic acid (caffeic acid), flavonoids, saponins, tannins, phenols, steroids, in particular scutellarin and hispidulin, terpenes, phenylpropanoids, in particular verbascosides (also referred to as acteosides), glycosides, in particular phenylethanoid glycosides, phenylpropanoid glycosides, iridoids, iridoid glycosides, ipolamiides, tarphetalin, and 4-methoxycarbonyl-7-methylcyclopenta[c]pyran (fulvoipolamiides).
Verbascosides are phenylethanoid glycosides being an ester of phenylethanoid hydroxytyrosol, phenylethanoid 3,4-dihydroxycinnamic acid and the sugar alpha-L-rhamnopyranosyl-(1-3)-beta-D-glycopyranose.
Stachytarpheta species comprise the compounds verbascosides, flavonoids, glycosides, phenylethanoid and phenylpropanoid glycosides and anthraquinones as antimicrobially, preferably antibacterially and/or antimycotically, effective compounds.
Stachytarpheta species comprise verbascosides, flavonoids, iridoids, ipolamiides, iridoid ipolamiides, acteosides, fulvoipolamiides, sesquiterpene lactones and proazulenes as anti-inflammatorily effective compounds. Said compounds are particularly present in the leaves of the Stachytarpheta species according to the invention. Therefore, within the meaning of the invention, plant parts comprising the afore-mentioned compounds, in particular the leaves, are preferably used for the production of a plant extract, of Sta. jamaicensis, Sta. indica and/or Sta. cayennensis. The afore-mentioned plant extract is preferably obtained as ethanolic plant extract having greater than or equal to 70% ethanol.
Surprisingly, significant anti-inflammatory efficiency has respectively been proved for Bid. alba, Sta. jamaicensis and Bur. simaruba as respective representatives of the species according to the invention of (a), (b) and (c) (Example 6, Table 2). This efficiency may be attributed to the compounds, being contained in the species according to the invention and being extracted by means of the method according the invention, comprising verbascosides, flavonoids, iridoids, ipolamiides, fulvoipolamiides, sesquiterpene lactones, polyacetylenes and/or proazulenes.
Within the meaning of the invention, the plant extracts of the Stachytarpheta species according the invention exhibit good antimicrobial, at least antibacterial, efficiency against transient skin flora comprising, in particular, staphylococcus, streptococcus, methicillin-resistant Staphylococcus aureus (MRSA), pseudomonads and/or acinetobacteria. Sta. cayennensis, Sta. jamaicensis and/or Sta. indica particularly exhibit antibacterial efficiency against Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (ATCC 14990), MRSA (NCTC 10442), Pseudomonas aeruginosa (ATCC 27853) and Acinetobacter baumanii (ATCC BAA 747) (Example 7).
Within the meaning of the invention, the Stachytarpheta species according to the invention have, preferably, additionally to the antimicrobial, preferably antibacterial and/or antimycotic, efficiency, anti-inflammatory efficiency at less than or equal to 200±10 μg/ml of the respective plant extract, preferably less than or equal to 180±10 μg/ml, less than or equal to 160±10 μg/ml, less than or equal to 140±10 μg/ml, less than or equal to 120±10 μg/ml, particularly preferably less than or equal to 100±10 μg/ml, less than or equal to 85±10 μg/ml, less than or equal to 80±μg/ml measured as IC50 der 5-LOX inhibition.
The Burseraceae family comprise subtribe Burserinae to which the (c) genus Bursera (=C) is assigned. Genus Bursera comprises about 100 species comprising Bur. bipinnata, Bur. fagaroides, Bur. glabrifolia, Bur. malacophylla, Bur. microphylla, Bur. bolivarii, Bur. trifoliolata and Bur. simaruba. Bur. simaruba, Bur. microphylla and/or Bur. glabrifolia are used within the meaning of the invention. Bur. simaruba is particularly preferred as a plant extract in the phyto-mixture according to the invention.
Within the meaning of the invention, the plant extracts of the Bursera species according the invention exhibit good antimicrobial, at least antibacterial, efficiency against transient skin flora comprising, in particular, staphylococcus, streptococcus, methicillin-resistant Staphylococcus aureus (MRSA), pseudomonads and/or acinetobacteria. Bur. simaruba, Bur. microphylla and/or Bur. glabrifolia are effective against Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (ATCC 14990), MRSA (NCTC 10442), Pseudomonas aeruginosa (ATCC 27853) and Acinetobacter baumanii (ATCC BAA 747) (Example 7).
Within the meaning of the invention, the Bursera species according to the invention have, preferably, additionally to the antimicrobial efficiency, anti-inflammatory efficiency at less than or equal to 200±10 μg/ml of the respective plant extract, preferably less than or equal to 180±10 μg/ml, less than or equal to 160±10 μg/ml, less than or equal to 140±10 μg/ml, particularly preferably less than or equal to 135±μg/ml, less than or equal to 125±10 μg/ml, less than or equal to 80±10 μg/ml measured as IC50 der 5-LOX inhibition.
Antibacterial efficiency has surprisingly been detected for Sta. jamaicensis and Bid. alba, as well as Stem. maritima (see Example 7). In particular, the afore-mentioned species exhibit significant efficiency against gram-positive bacteria and particularly against MRSA. Thus, the phyto-mixtures according to the invention, described herein, exhibit at least antibacterial and, preferably, additionally anti-inflammatory efficiency.
A phyto-mixture made of a plant extract of (a) Bis. alba and/or Bid. pilosa and of a plant extract of (b) Sta. jamaicensis, Sta. cayennensis and/or Sta. indica is a particularly preferred combination within the meaning of the invention. The afore-mentioned combinations surprisingly exhibit increased antibacterial as well as anti-inflammatory efficiency (Examples 2 to 8). Particularly surprisingly, double efficiency has been observed for all afore-mentioned preferred species. The experiments described herein exemplary summarize the described efficiencies for the respective species according to the invention of genus (a) Bidens, (b) Stachytarpheta, and (c) Bursera.
An embodiment of the phyto-mixture according to the invention comprising the respective combination of plant extracts, preferably ethanolic and dry extracts, of
(a) Bidens alba and/or Bidens pilosa and
(b) Stachytarpheta jamaicensis; or
(b) Stachytarpheta jamaicensis and
(c) Bursera simaruba
surprisingly exhibits anti-inflammatory efficiency of less than or equal to 90±10 μg/ml, preferably at less than or equal to 70±10 μg/ml and particularly preferably at 50±10 μg/ml measured as IC50 of 5-LOX inhibition (Table 3). The afore-mentioned phyto mixture exhibits, deviating from expectations, good antimicrobial, in particular antibacterial, efficiency at the same time (Table 5b). The other species according to the invention (a), (b), and (c) exhibit appropriate efficiencies as listed above.
An embodiment of the phyto-mixture according to the invention comprising the combination of plant extracts, preferably ethanolic and subsequently dried extracts, of
(a) Bidens alba and (c) Bursera simaruba
exhibits anti-inflammatory efficiency of less than or equal to 90±10 μg/ml, preferably at less than or equal to 80±10 μg/ml measured as IC50 of 5-LOX inhibition (Table 3). The afore-mentioned phyto mixture exhibits good antimicrobial, in particular antibacterial, efficiency at the same time. The other species according to the invention of genus (a) Bidens and (c) Bursera respectively exhibit appropriate efficiencies.
Particularly preferred combinations of (a), (b), and/or (c) comprise
wherein ethanol extracts, greater than or equal to 70% ethanol, and, in particular after drying, having a residual content of ethanol less than or equal to 5%, less than or equal to 1%, particularly preferably less than or equal to 0.1%, are respectively preferably used in each case. The implementations described above concerning the ingredients of the several plant species apply correspondingly herein.
Preferred formulations of the phyto-mixture described above and its combinations comprise tea, tincture, solution, dispersion and suspension, powder and semi-solid forms, such as ointments, creams and pastes. Tea according to the invention is based on at least one dry and ethanol-free plant extract from (a), (b), and/or (c) and, optionally, (d).
Species of genus Stemodia (=D1) are used as further (d) biologically active plant within the meaning of the invention. These had formerly been assigned to the Scrophulariaceae family and are nowadays assigned to the Plantaginaceae family belonging to the order Lamiales. Genus Stemodia (Stem.) comprises about 40 species comprising Stem. maritima, Stem. lantana and Stem. durantifolia, which are used in the meaning of the invention. Stem. maritima is particularly preferred.
Species of genus Aloe (=D2) assigned to the Asphodeloideae subfamily which belongs to the Xanthorrhoeaceae family are used as further (d) biologically active plant. Genus Aloe comprises about 500 species. Aloe vera, Aloe barbadensis, Aloe albiflora, Aloe perfoliata, Aloe vulgaris, Aloe indica and/or Aloe chinensis are preferably used according to the invention. Aloe vera is particularly preferred, with or without anthraquinones.
A mixture of each a plant extract from (a) Bid. alba, Bid. pilosa, (b) Sta. jamaicensis, Sta. cayennensis, Sta. indica, and/or (c) Bur. simaruba, Bur. microphylla, Bur. glabrifolia, and, optionally, (d) an Aloe species (D2), preferably Aloe vera, exhibits particularly good efficiency in topical application on the skin, mucous membrane and oral mucosa, in particular for prevention and treatment of efflorescences within the meaning of the invention. Table 1a summarizes appropriate combinations of preferred species.
Bid. alba/Sta. jamaicensis
Bid. alba/Bur. simaruba
Sta. jamaicensis/Bur.
simaruba
maritima and,
Bid. alba/Stem. maritima
Sta. jamaicensis/Stem.
maritima
Bur. simaruba/Stem.
maritima
Bid. alba/Aloe vera
Sta. jamaicensis/Aloe vera
Bur. simaruba/Aloe vera
The amount of the at least one plant extract from (a), (b), and/or (c), and, optionally, (d) is greater than or equal to 1% by weight preferably to less than or equal to 50% by weight, preferably greater than or equal to 3% by weight, greater than or equal to 5% by weight, greater than or equal to 7% by weight to each less than or equal to 50% by weight, based on the respective dry weight (ad 100% by weight), wherein the total content of the at least one plant extract or of the combination of (a), (b), and/or (c) preferably is greater than or equal to 3% by weight, based on the total content of the phyto-mixture (ad 100% by weight), greater than or equal to 5% by weight, greater than or equal to 7% by weight to less than or equal to 50% by weight, preferably less than or equal to 30% by weight. In the case of adding a (d) Aloe extract (D2), the amount of D2 is preferably greater than or equal to 5% by weight, greater than or equal to 10% by weight, greater than or equal to 15% by weight, greater than or equal to 20% by weight, to each less than or equal to 70% by weight. The amounts of the other plant extracts (a), (b), and/or (c) are correspondingly low (Example 8b). The preferred amounts described herein apply correspondingly for the compositions and formulations described in the following.
In the case of combining two plant extracts, ratios of 1:1 mixtures are preferably made, as also analysed in the following examples 2 to 8. Where two plant extracts are combined, ratios may variably adjusted between 1:10 to 10:1. In the case of three or more plant extracts, ratios of 1:1:1 etc. are used. Proportions may variably adjusted, depending on availability of the material and tolerability of the patient to the respective extract. Thus, in the case of an allergy to one of the plants described herein or intolerability to one of the ingredients described above, the plant/ingredient may be excluded from the phyto-mixture. The content of the other plant extracts may be correspondingly adjusted to achieve the desired efficiency.
A further subject matter of the present invention is the immunologically active phyto-mixture according to the invention comprising at least one plant extract which exhibit antimicrobial, preferably antibacterial and/or antimycotic efficiency against transient skin flora and/or anti-inflammatory efficiency. Preferably, the phyto-mixtures according to the invention exhibit efficiency against transient skin flora and anti-inflammatory efficiency at the same time.
Within the meaning of the invention, “antimicrobial efficiency” comprises inhibition and reduction of the reproductivity, ability to divide and reproduction of microorganisms up to the inactivation or killing of microorganisms. “Antimicrobial” comprises at least antibacterial (against gram-positive and -negative bacteria) and, preferably, additionally antimycotic (against fungi and yeasts). Antimicrobial efficiency further comprises inhibition of the biosynthesis apparatus of microorganisms, such as, for example, of the synthesis of toxins, pathogenicity factors and other physiological reacts or of compounds triggering an immune response. Toxins comprises exotoxins and endotoxins, in particular exotoxins, such as the fungal toxins aflatoxin oder exofilantin A and B of Staphylococcus aureus.
Preferably, “antimicrobial efficiency” comprises at least one efficiency analogous to antibiotics, in particular to a lesser extent and, preferably, having less or no side effects compared to synthetic antibiotics.
Within the meaning of the invention, “transient skin flora” means a microbiological flora on/in the skin and/or mucosal membrane, which, due to impact of miscellaneous influencing factors, may cause an efflorescence, results in an efflorescence or may impair further tissues by systemic spread.
The skin flora of healthy skin comprises a “resident skin flora” which is also referred to as physiological, endogenous skin flora or “localised flora”. “Resident skin flora” comprises germs, not impairing the healthy organism, comprising staphylococcus, in particular Staphylococcus epidermis, propioni-, myco- and corynebacteria. Said germs preferably multiply in Stratum corneum and constitute a protection flora against pathogenic and foreign microorganisms. The composition of resident skin flora may be variable for various skin regions, such as face, hands, feet, outer ear, as well as lips and oral mucosa, the skin of the eye, in particular the cornea, conjunctiva and mucous membrane of the eye.
Transient, thus temporary, skin flora comprising pathogenic germs is to be differentiated thereof. Pathogenic germs comprise gram-negative cocci, such as Neisseria, gonococcus, meningococcus, gram-positive cocci, such as staphylococcus, streptococcus, in particular Streptococcus aureus, micrococcus, enterobacteria, pneumococcus and Clostridia, gram-negative rods, such as Bordetella, Campylobacter, Haemophilus, Heliobacter, Legionella, Salmonella, Shigella, Vibrio, Yersinia, Escherichia coli, Klebsiella, Proteus and pseudomonads, gram-positive rods, such as Bacillus, Clostridium, Corynebacterium, Listeria und mycobacteria and/or yeasts, fungi, Candida und/oder viruses.
Healthy skin flora may be impaired by different influencing factors, such as physical, chemical, pathogenic and endogenous physiological influencing factors and merge into a transient skin flora. In particular, foreign germs or pathogenic germs may disrupt or displace healthy skin flora thus multiplying and spreading, which finally results in a skin disease. In particular diseases of the cutis, preferably of the epidermis, comprising at least one alteration of at least one tissue selected from Stratum corneum, Stratum lucidum, Stratum granulosum, Stratum spinosum, Stratum basale and/or Stratum germinativum (=Stratum spinosum+Stratum basale), dermis comprising Stratum paillare and Stratum reticulare and/or of the subcutis comprising connective tissue with fibroblasts, endothelial cells, collagen and fat cells.
Consequently, transient skin flora comprises, according to the invention, disordered skin flora comprising at least one of the afore-mentioned germs phenotypically causing efflorescences of at least one of the afore-mentioned tissues on the skin and oral mucosa.
Physical influencing factors comprise the afore-mentioned disruptions and impairments of the skin structure and comprise injuries, stings, cuts, scratches, abrasions, burns or chemical burns at least of the epidermis comprising Stratum corneum, Stratum lucidum, Stratum granulosum, Stratum spinosum, Stratum basale and/or Stratum germinativum (=Stratum spinosum+Stratum basale), optionally of the dermis comprising Stratum paillare and Stratum reticulare and/or of the subcutis comprising connective tissue with fibroblasts, endothelial cells, collagen and fat cells.
In a particular embodiment of the present invention, the immunologically active phyto-mixture according to the invention comprising at least one plant extract according to the invention (a), (b), and/or (c) and, optionally, (d) having an antimicrobial, preferably antibacterial, efficiency against transient skin flora, wherein the transient skin flora comprises staphylococcus, streptococcus, methicillin-resistant Staphylococcus aureus (MRSA), pseudomonads and/or acinetobacteria.
In a particular embodiment of the present invention, the immunologically active phyto-mixture according to the invention comprises at least one plant extract exhibiting antibacterial efficiency against at least one of the afore-mentioned germs of a transient skin flora at a concentration greater than or equal to 10 μg/ml to less than or equal to 10 mg/ml measured as minimal inhibitory concentration (MIC) and/or as minimum bactericidal concentration (MBC) of the respective plant extract or the extract mixture. Preferably, the at least one plant extract exhibit antibacterial efficiency at a concentration greater than or equal to 10 μg/ml to less than or equal to 10 mg/ml, greater than or equal to 10 μg/ml to less than or equal to 8 mg/ml, greater than or equal to 100 μg/ml to less than or equal to 8 mg/ml, greater than or equal to 250 μg/ml to less than or equal to 8 mg/ml, preferably greater than or equal to 500 μg/ml to less than or equal to 8 mg/ml, in particular greater than or equal to μg/ml to less than or equal to 6 mg/ml, greater than or equal to 100 μg/ml to less than or equal to 6 mg/ml, greater than or equal to 250 μg/ml to less than or equal to 6 mg/ml, greater than or equal to 500 μg/ml to less than or equal to 6 mg/ml, greater than or equal to 10 μg/ml to less than or equal to 4 mg/ml, greater than or equal to 100 μg/ml to less than or equal to 4 mg/ml, greater than or equal to 250 μg/ml to less than or equal to 4 mg/ml, greater than or equal to 500 μg/ml to less than or equal to 4 mg/ml.
In a particular embodiment, the phyto-mixture according to the invention with the subsequent preferred combinations of plant extracts exhibits antimicrobial, preferably antibacterial, efficiency at a concentration greater than or equal to 100 μg/ml to less than or equal to 10 mg/ml (MIC and/or MBC) comprising
The afore-mentioned phyto-mixtures exhibit surprisingly good efficiencies against MRSA as it is shown in Example 7 (Table 5a and 5b). Antibacterial efficiency has surprisingly been detected for chosen species (a), (b), and (c) of the plant extracts according to the invention. The same applies for the other species, according to the invention, of genus (a) Bidens, (b) Stachytarpheta, and (c) Bursera. The immunologically active phyto-mixture according to the invention, comprising at least one of the plant extracts according to the invention (a), (b), and/or (c) described above exhibits anti-inflammatory efficiency at less than or equal to 200±10 μg/ml, preferably at less than or equal to 180±10 μg/ml, less than or equal to 160±10 μg/ml, less than or equal to 140±10 μg/ml, less than or equal to 130±10 μg/ml, particularly preferably at less than or equal to 120±10 μg/ml measured as IC50 of 5-LOX inhibition of the respective plant extract or the extract mixture. Particularly preferred embodiments exhibit anti-inflammatory efficiency at less than or equal to 100±10 μg/ml, preferably at less than or equal to 90±10 μg/ml, less than or equal to 80±10 μg/ml, less than or equal to 70±10 μg/ml and less than or equal to 60±μg/ml or less than or equal to 50±10 μg/ml.
Particularly preferably, the at least one plant extract exhibits anti-inflammatory efficiency at less than or equal to 90±10 μg/ml of the respective plant extract of the species according to the invention (a), (b) and/or (c).
The immunologically active phyto-mixture preferably comprises at least one plant extract selected from (a) Bid. alba, Bid. pilosa, (b) Sta. jamaicensis, Sta. indica, Sta. cayennensis, (c) Bur. microphylla, Bur. glabrifolia and/or Bur. simaruba, and, optionally, (d) as extract of the ingredients of the respective plant, preferably of the aboveground parts of the at least one plant. Preferably, the extract comprises ingredients of untainted, unaltered, freshly harvested or dried plant parts comprising bark, stem, twigs, branches, leaves, blossoms, buds, seeds, pods, pollen, fruit organs, photosynthetically active parts and/or storage organs. The extract of dried plant parts is preferred. The advantage of an extract of dried plant parts is the absence of water, thus obtaining more concentrated extracts of the respective plant. As a result, extracts having comparably higher contents of active compounds per measuring unit of the respective extract are obtained.
Consequently, plant extracts obtained according to the invention from dried plants have an increased amount of the compounds described above, preferably at least one antibacterially and/or anti-inflammatorily active compound comprising flavonoids, terpenes, benzoids, phenylpropanoids, glycosides and verbascosides, iridoids, ipolamiides, fulvoipolamiides, sesquiterpene lactones and/or proazulenes.
Within the meaning of the invention, “ingredients” comprise all of the plantal biologically and/or physiologically active compounds described herein.
In a further embodiment of the present invention, the immunologically active phyto-mixture comprises at least one plant extract selected from (a), (b), and/or (c), and, optionally, (d) which comprises each an extract of at least one of the subsequent compounds, in particular of the ingredients: flavonoids, saponins, iridoids, phenolic acids, polyphenols, polysaccharides, glycosylates, terpenes, monoterpenes, sesquiterpene lactones, proazulenes, sulfides, carotinoides, vitamins A B C D E, amino acids, and/or minerals. Particularly preferably, ethanolic, dried plant extracts of (a) Bid. alba, Bid. pilosa, (b) Sta. jamaicensis, Sta. cayennensis, Sta. indica, (c) Bur. microphylla, Bur. glabrifolia, Bur. simaruba and/or (d) Stem. maritima have one of the afore-mentioned compounds.
In a particular embodiment of the immunologically active phyto-mixture according to the invention, it comprises at least one plant extract selected from (a), (b), and/or (c), and, optionally, (d) which comprises at least one of the antimicrobially, preferably antibacterially, effective compounds verbascosides, flavonoids, glycosides, phenylethanoid glycosides, phenylpropanoid glycosides and/or anthraquinones. Preferably, the afore-mentioned phyto-mixture comprises at least one plant extract from (b) St. jamaicensis, Sta. cayennensis, Sta. indica, (a) Bid. alba and/or Bid. pilosa.
In a further embodiment of the immunologically active phyto-mixture according to the invention, it comprises at least one plant extract selected from (a), (b), and/or (c), and, optionally, (d) which comprises at least one of the anti-inflammatorily effective compounds verbascosides, flavonoids, iridoids, ipolamiides, fulvoipolamiides, sesquiterpene lactoces and or proazulenes. Preferably, the afore-mentioned phyto-mixture comprises at least one plant extract from (b) St. jamaicensis, Sta. cayennensis, Sta. indica, (a) Bid. alba and/or Bid. pilosa.
In a particular embodiment of the immunologically active phyto-mixture according to the invention, it comprises at least two of the afore-mentioned plant extracts selected from (a), (b), and/or (c), and, optionally, (d) preferably comprising at least one of the species (b) St. jamaicensis, Sta. cayennensis and/or Sta. indica, and one of the species (a) Bid. alba and/or Bid. pilosa which comprise extracts, preferably ethanolic extracts, of the afore-mentioned ingredients.
The plant extracts according to the invention described above, phyto-mixtures comprising the plant extracts and embodiments according to the invention are provided for the production as medicament, medical product, nutritional supplement and cosmetic. The phyto-mixture is present in different forms for these purposes.
Therefore, a further subject matter of the invention is the immunologically active phyto-mixture according to the invention, wherein the at least one plant extract (a) Bid. alba, Bid. pilosa, (b) Sta. jamaicensis, Sta. indica, Sta. cayennensis, (c) Bur. microphylla, Bur. glabrifolia and/or Bur. simaruba, and, optionally, (d) is present in
In an embodiment of the immunologically active phyto-mixture, the at least one plant extract or the phyto-mixture according to the invention in liquid form, preferably tincture, solution, dispersion and/or suspension, each alternatively has a pH tolerated by the skin greater than or equal to 3 to less than or equal to 9, a pH tolerated by the oral mucosa greater than or equal to 6 to less than or equal to 8, a pH tolerated by the nasal mucosa greater than or equal to 5 to less than or equal to 7, or a pH tolerated by the eye greater than or equal to 7 to less than or equal to 9.
Subsequent to the production of the plant extract according to the invention, in particular of the phyto-mixture, according to the method according to the invention, the direct product is present in freeze-dried form, preferably as lyophilisate, after freeze-drying. Subsequently, it may be processed into fine powders, tablets or liquid forms.
In an embodiment, the liquid immunologically active phyto-mixture according to the invention is present as tincture, in particular as solution, suspension or dispersion, comprising
wherein the tincture has a pH value of greater than or equal to 3 to less than or equal to 9 and the tincture is an aqueous/ethanolic mixture having an ethanol concentration greater than or equal to 70%, based on the total composition of the tincture (e.g. Example 8b). The total content of the plant extract or of the combination of each a plant extract (a), (b) and/or (c), respectively, preferably is greater than or equal to 3% by weight, preferably greater than or equal to 5% by weight, greater than or equal to 7% by weight, respectively based on the phyto-mixture (ad 100% by weight).
All physiologically compatible, synthetic, biobased and natural acidifiers are suitable as acidifiers. In particular, acidifiers being available as foodstuffs, such as vinegar, cider vinegar, white vine vinegar, balsamic vinegar, may be used as acidifiers. Ultrapure, pharmaceutically and/or cosmetically authorised acidifiers are preferably used.
The appropriate pH value of the respective phyto-mixture depends on the type of indication and its administration form. The acidifier is suitable for setting the pH value, without limiting it to this function. The pH value is greater than or equal to 3 to less than or equal to 9, preferably greater than or equal to 3.1, greater than or equal to 3.2, greater than or equal to 3.5 to each less than or equal to 8.7, less than or equal to 8.5 8.3 8.1 7.9, less than or equal to 7.8 and preferably less than or equal to 7.6.
Topical formulations, preferably solution, dispersion, suspension, tincture, aerosol, spray and/or semi-solid formulations, such as ointment, cream and paste, for application on the skin within the meaning of the invention, excluding oral/nasal mucosa, eye, cornea, conjunctiva and mucous membrane of the eye, comprising the phyto-mixture according to the invention comprising at least one plant extract (a), (b), and/or (c), and, optionally, (d), preferably have a pH greater than or equal to 3 to less than or equal to 9, preferably greater than or equal to 3.5 to less than or equal to 8.7, less than or equal to 8.5 8.3 8.1 7.9. In particular, respectively pH 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9 6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6 7.8 8.0 8.1 8.3 8.5 or 8.7.
Formulations according to the invention (Example 8a-b) for application on the outer ear and ear canal, preferably solution, dispersion, suspension, tincture, spray, aerosol and semi-solid formulations, respectively have a pH greater than or equal to 3 to less than or equal to 9, preferably greater than or equal to 4 to less than or equal to 7.
Formulations according to the invention (Example 8a-b) for application on the oral mucosa, preferably solution, dispersion, suspension, tincture, spray, aerosol and semisolid formulations, respectively have a pH of preferably greater than or equal to 6 to less than or equal to 8, preferably greater than or equal to 6.2 to less than or equal to 7.8. In particular 6.2 6.3 6.4 6.5 6.6 6.5 6.6 6.7 6.8 6.9 7.0 7.1 7.2 7.3 7.4 7.5 or 7.6.
Formulations according to the invention for depositing on the nasal mucosa, preferably tincture, solution, dispersion, suspension, spray, aerosol and semisolid formulations, respectively have a pH of preferably greater than or equal to 5 to less than or equal to 7. In particular pH 5.1 5.2 5.3 5.4 5.5 5.5 5.6 5.7 5.8 5.9 6.0 6.1 6.2 6.3 6.4 6.5 6.6 6.5 6.6 6.7 6.8 6.9 or 7.0.
Formulations according to the invention, preferably solution, dispersion, suspension and tincture, for applying on/in the eye, in particular on the cornea, conjunctiva and mucous membrane of the eye, respectively have a physiological pH, preferably greater than or equal to 7 to less than or equal to 9. In particular pH 7.1 7.2 7.3 7.4 7.5 7.5 7.6 7.7 7.8 7.9 8.0 8.1 8.2 8.3 8.4 or 8.5.
The pH values described above and preferred pH ranges for the respective use correspondingly apply for all described embodiment of the phyto-mixture according to the invention and of the formulations comprising at least one plant extract (a), (b), and/or (c), and, optionally, (d), without explicitly stating it.
A further embodiment of the immunologically active phyto mixture according to the invention comprises, in solid form, at least one essentially at least one essentially dry, in particular solvent-free, plant extract, selected from (a) Bid. alba, Bid. pilosa, (b) Sta. indica, Sta. jamaicensis, Sta. cayennensis, and/or (c) Bur. simaruba, Bur. microphylla, Bur. glabrifolia, and, optionally, (d) Stemodia maritima, and/or an Aloe species.
Within the meaning of the invention, “essentially dry” means that the respective plant extract (a), (b), and/or (c), and, optionally, (d) of the at least one plant has a residual moisture of less than or equal to 10% by weight, preferably less than or equal to 8% by weight, less than or equal to 5% by weight, less than or equal to 4% by weight, less than or equal to 3% by weight, less than or equal to 2% by weight, particularly preferably less than or equal to 1% by weight, less than or equal to 0.1% by weight, equal to 0% by weight, based on the total weight of the respective plant extract.
Within the meaning of the invention, “solvent-free” means a residual content of solvent less than or equal to 10% by weight, preferably less than or equal to 8% by weight, less than or equal to 5% by weight, less than or equal to 4% by weight, less than or equal to 3% by weight, less than or equal to 2% by weight, less than or equal to 1% by weight, particularly preferably less than or equal to 0.1% by weight, based on the total weight of the respective plant extract of the at least one plant, wherein solvents comprise water, 1,2-propylene glycol, aqueous ethanol, ethanol, in particular greater than or equal to 70% ethanol to less than or equal to 100% ethanol, greater than or equal to 70% ethanol, methanol, acetone, chloroform, n-butanol, hexane, ethyl acetate, diethyl ether, or a mixture of at least two of the afore-mentioned solvents. Solvents, being completely removable, in particular without harmful or irritating residuals, are preferred.
A further subject matter of the present invention is a method for the production of an immunologically active phyto-mixture comprising at least one plant extract respectively selected from the species according to the invention of genus (a) Bid. alba, Bid. pilosa, (b) Sta. jamaicensis, Sta. cayennensis, Sta. indica, (c) Bur simaruba, Bur. microphylla and/or Bur. glabrifolia, and, optionally, (d) Stemodia and/or Aloe, respectively, comprising the steps of
In the separating step, the obtained extract is separated from the remaining plant parts using the solvent, preferably by filtration, thin-layer chromatography, distillation, shaking out or evaporation. Choice of the separating step is knowledge of the person skilled in the art.
Remaining and separated plant parts may subsequently be supplied to another extraction and be previously crushed if required. The steps extraction, separating, optionally drying of the obtained extract and crushing of separated plant parts may, in a variable manner, be combined, joined with fresh plant parts and repeated. The arrangement of the method is knowledge of the person skilled in the art, achieving maximal yield of the ingredients already described above in the plant extract according to the invention (a), (b), and/or (c), and, optionally, (d), and the phyto-mixture according to the invention.
Preferably, plant parts of the plants are used, selected from
One cycle of the method comprises at least one extraction step and at least one separating step. Optionally, at least one drying step of the already obtained extract may be performed between cycles. Drying step of the separated plant parts may be performed if required. In the case of using a toxic solvent a further step for removing the solvent is preferably performed.
In an embodiment of the method according to the invention, undesired ingredients of the respective plant extract according to the invention (a), (b), and/or (c), and, optionally, (d) are removed. Preferably, compounds with laxative effect, in particular anthraquinones, are removed for oral formulations for absorption through the gastrointestinal tract.
Preferably, the at least one further processing step for homogenization of the obtained liquid or dry plant extract is performed prior to mixing of the at least two plant extracts or prior to the formulation into one of the administration forms described herein (Example 8a-b) of the phyto-mixture according to the invention.
In the method according to the invention for the production of an immunologically active phyto-mixture comprising at least one plant extract according to the invention (a), (b), and/or (c), and, optionally, (d), extraction is performed with at least one solvent, comprising water, 1,2-propylene glycol, aqueous ethanol, ethanol, greater than or equal to 70% ethanol to less than or equal to 100% ethanol, methanol, acetone, chloroform, n-butanol, hexane, ethyl acetate, and/or diethyl ether in particular a mixture of at least two of the afore-mentioned solvents.
Aqueous ethanol greater than or equal to 70% ethanol to less than or equal to 100% ethanol is preferably used as solvent for extraction. Due to low to no toxicity of ethanol, the aqueous extracts and ethanol extracts of the species according to the invention (a), (b), and/or (c), and, optionally (d) are preferred in contrast to, for example methanol, acetone or chloroform. After drying, the residual content of ethanol preferably is less than or equal to 5%, particularly preferably less than or equal to 1%, less than or equal to 0.1% of ethanol. Aqueous ethanol, ethanol, preferably greater than or equal to 70% to less than or equal to 100% are preferred solvents. In particular greater than or equal to 75%, greater than or equal to 80%, greater than or equal to 85%, greater than or equal to 90%, greater than or equal to 95%, 96%, 97%, 98% to less than or equal to 100% ethanol. Particularly preferably, Ultrapure and biobased ethanol is used.
In a preferred embodiment of the method according to the invention for the production of an immunologically active phyto-mixture within the meaning of the invention, at least one plant extract (a), (b), and/or (c), and, optionally, (d) is obtained in liquid form, in dry form or as mixture of solid and liquid forms.
In context of the obtained product according to the method according to the invention, “mixture” comprises dry and liquid ingredients extracted from the plant parts. Liquid ingredients may comprise residual moisture of the liquids from the plants, oily ingredients and residual solvent.
The method according to the invention for the production of at least one immunologically active phyto-mixture further comprises at least one mixing step, comprising
In an arrangement of the method according to the invention, a dried form of at least one plant extract, e.g. a powder, with a liquid form of at least one plant extract can be mixed. The liquid form may be, for example, an undried ethanol extract. The advantage is that the concentration of the total plant extract per volume is not reduced by additional liquids. Moreover, purity of the extract is improved since mixtures of solvents are avoided and defined concentrations and mixtures are produced.
Preferably, dry forms of extracts are mixed into an immunologically active phyto mixture comprising at least one plant extract according to the invention (a), (b), and/or (c) and subsequently processed into a liquid form, preferably solution, solution for use as tincture, tincture, suspension and/or dispersion, using a solvent, preferably ultrapure ethanol. Preferably, a phyto-mixture according to the invention made of dry extracts according to the method according to the invention is obtained, comprising
In an alternative of the method described above, the extraction is performed with a respectively appropriate solvent and this is subsequently removed and the plant extract is dried. In order to produce a solution, dispersion, suspension or tincture, the dried and solvent-free plant extract is not homogenously mixed in ethanol, but in an aqueous solvent without ethanol. The advantage is that no irritating solvent is contained in the case of application on the skin or mucous membrane, in particular on wounds. The “aqueous alternative” correspondingly applies for all formulations according to the invention and indications. In particular for formulations for application in the eye, on nasal und oral mucosa.
“Aqueous solvents” shall be understood to mean physiologically, pharmaceutically compatible solutions on the basis of water or physiologically compatible buffer systems, in which the dried plant extracts are homogenously mixable into a dispersion or solution. The person skilled in the art knows such “aqueous solvents”. Preferably plant extracts are mixed which are obtained by the method according to the invention from plant parts of plants selected from
A further subject matter of the present invention is an immunologically active phyto-mixture obtained by the method described above, wherein the phyto-mixture comprises at least one plant extract selected from the species according to the invention of genus (a) Bid. alba, Bid. pilosa, (b) Sta. jamaicensis, Sta. cayennensis, Sta. indica, (c) Bur. simaruba, Bur microphylla and/or Bur. glabrifolia, and, optionally, (d) at least one further extract of a further biologically active plant, preferably of genus Aloe and/or Stemodia, particularly preferably Stem. maritima and/or of at least one Aloe species.
Explanations on ingredients, anti-inflammatory and antimicrobial, preferably antibacterial, efficiencies correspondingly apply for the method according to the invention and the direct products obtained by means of the method. Thus, the products obtained according to the described method exhibit the efficiencies described above, as shown by examples 2 to 9.
A further subject matter of the present invention is an immunologically active phyto-mixture in the manner described above and/or produced according to the method described above comprising at least one plant extract selected from the species respectively according to the invention of genus (a) Bidens, (b) Stachytarpheta, and/or (c) Bursera, and, optionally, (d) at least one further extract of a further biologically active plant for use as medicament, medical product, nutritional supplement, cosmetic, and/or as an immunologically active addition to one of the afore-mentioned products.
All described formulation according to the invention are suitable as or for the production of medicaments, medical products, nutritional supplements and cosmetics.
“Nutritional supplements” preferably comply, according to the invention, with EU directive 2002/46/EG and comprise products for increased supply of the human body, preferably as powder, tablet or tea, each comprising the immunologically active phyto-mixture comprising at least one plant extract according to the invention (a), (b), and/or (c), and, optionally, (d), or a preferred embodiment of the phyto-mixture.
Within the meaning of the invention, “medical products” are the formulations described herein comprising the described immunologically active phyto-mixture comprising at least one plant extract according to the invention (a), (b), and/or (c), and, optionally, (d) for use in the prevention or treatment of human for medically therapeutic purposes. The medical products according to the invention preferably comply with the directive 93/42/EWG. Examples include medicinal tea, drops, tincture, ointment, cream and paste comprising the phyto-mixture. Within the meaning of the invention, “cosmetics” are topical cosmetic products for depositing on the skin comprising the phyto-mixture according to the invention comprising at least one plant extract according to the invention (a), (b), and/or (c), and, optionally, (d). Such cosmetic products include cream, soap, peeling, poultice, cleansing solution or mil, and related skin care products.
Therefore, a further subject matter of the present invention is a pharmaceutic or cosmetic composition comprising the immunologically active phyto-mixture according to the invention that comprises at least one plant extract according to the invention selected from (a) genus Bidens of the Asteraceae family, (b) genus Stachytarpheta of the Verbenaceae family, and/or (c) genus Bursera of the Burseraceae family, and, optionally, (d) at least one further extract of a further biologically active plant comprising Aloe species of genus Aloe of the Asphodeloideae subfamily, species of genus Stemodia of the Plantaginaceae family and/or Stemodia maritima. Preferably, the afore-mentioned compositions comprise at least one cosmetically and/or pharmaceutically authorised excipient. The described preferred embodiments of the phyto-mixture and the formulations correspondingly apply for the pharmaceutic or cosmetic composition.
A further subject matter of the present invention is an immunologically active-phyto mixture, preferably a composition comprising the phyto-mixture, for use in the prevention or in a method for treatment of efflorescences comprising at least one plant extract according to the invention selected from
In general, “Efflorescences” are skin alterations and comprise cosmetic efflorescences occurring without health-damaging impairment of human, but are pertinent to psychological well-being of human, and physiologically pathologic efflorescence causing health-damaging impairment right up to serious and life-threatening diseases, such as skin cancer, in humans and animals.
Cosmetic efflorescences comprise superficial scratches in the skin, in particular epidermis, irritations or abrasions of the skin surface, tearing of the skin and roughness each of the skin, in particular epidermis.
Pathologic efflorescences comprise skin diseases accompanying with an inflammatory reaction of at least one tissue of the skin and/or comprising microbial, in particular bacterial, infection. Pathologic efflorescences are also summarized as such skin alterations that originally occur as cosmetic efflorescence and evolve into pathologic efflorescences in the course of disease, in particular through changes in the constitution of the skin cells.
According to the invention, “efflorescences” comprise irritations and inflammations of skin and mucous membrane, bacterial infections, bacterial infections accompanying with viral infections, mycoses, after-effects and secondary infection of infectious and parasitic diseases, diseases of the outer ear, Otitis extema (H60., H62.1, H62.2 ICD-10-GM), abscess of the outer ear (H60.0 ICD-10-GM), furuncle of the outer ear, local infections of skin and subcutis, Impetigo (L01.-ICD-10-GM), skin abscesses, furuncles and carbuncles (L02.-, L02.0 bis L02.9 ICD-10-GM), inflammations of the skin accompanying with pilonidal cyst (L05.-), erythrasma (L08.1 ICD-10-GM), bullous dermatoses (L10-L14), dermatitis and eczemas (L20-L30), diaper dermatitis (L22), allergic contact dermatitis (L23), toxic dermatitis (L24), pruritus (L29.-), other dermatitis (L30- to L30.09), Lichen ruber (L43.-), papulosquamous skin diseases (L45), urticaria und erythema (L50-54), symptoms affecting the skin and the subcutaneous tissue, burns, chemical burns, diseases of the skin and the subcutis through exposure to radiation (L55-L59) including sunburn, Dermatitis solaris acuta 1. to 2. degree (L55), frostbites, complications trough medical and surgical treatment and impairments accompanying therewith comprising wound healing, scars and wounds.
The abbrevation “ICD-10-GM” stands for “International Statistical Classification Of Diseases And Related Health Problems, 10th revision, German Modification” (ICD-10-GM) and is regularly updated. Herewith, explicit reference is made to this classification and to the description of skin diseases and, in particular, the content of chapter XII, L00-L59 is incorporated into the disclosure of the present invention.
Within the meaning of the invention, “skin” comprises the skin of the limbs, of the extremities, of the joints, of the upper head, of the head, of the outer ear, nose, nasal mucosa, as well as lips and oral mucosa, and the skin of the eye, in particular the cornea, conjunctiva and mucous membrane of the eye. In particular, “skin” comprises at least one tissue layer comprising Stratum corneum, Stratum lucidum, Stratum granulosum, Stratum spinosum, Stratum basale and/or Stratum germinativum (=Stratum spinosum+Stratum basale), optionally of the dermis comprising Stratum papillare and Stratum reticulare and/or of the subcutis comprising conjunctive tissue with fibroblasts, endothelial cells, collagen and fat cells.
Preferably, the immunologically active phyto-mixture, preferably the composition comprising the phyto-mixture, is used for use in the prevention or in a method for treatment of the afore-mentioned efflorescences, wherein the at least one plant extract is selected from
(a) Bid. alba, Bid. pilosa,
(b) Sta. jamaicensis, Sta. cayennensis, Sta. indica and/or
(c) Bur. simaruba, Bur. microphylla and/or Bur. glabrifolia, and, optionally,
(d) an Aloe species of genus Aloe and/or of genus Stemodia, preferably Stem. maritima, Aloe vera, Aloe barbadensis, Aloe perfoliata, Aloe vulgaris, Aloe indica and/or Aloe chinensis. A topical formulation, such as tincture, solution, suspension, dispersion, powder, ointment, paste or cream is preferred (see Example 8a-b).
Preferably, the immunologically active phyto-mixture for use in the prevention or in a method for treatment of efflorescences, as described above, comprises as (d) at least one further extract of a biologically active plant of at least one Aloe species of genus Aloe comprising Aloe vera, Aloe barbadensis, Aloe perfoliata, Aloe vulgaris, Aloe indica or Aloe chinensis, Aloe vera, a species of genus Stemodia and/or Stemodia maritima.
In a preferred arrangement of use of the immunologically active phyto-mixture according to the invention, preferably of the composition comprising the phyto-mixture, for prevention or in a method for treatment of efflorescences, efflorescences comprise cosmetic or pathologic efflorescences, skin diseases associated with the transient skin flora, bacterial and/or viral infections of the skin, furunculoses, mycoses, inflammatory reactions of the skin, impetigo, benign and malign tumor formation, dermatoses, eczemas, pruritus, psoriasis, acne, skin irritations, erythema, symptomatical efflorescences, burns, chemical burns, frostbites, efflorescences occurring by toxins, medicaments, drugs, allergens, radiation and as side effect, irritations and inflammations of the skin caused by bites and stings of insects and parasites. In particular efflorescences according to ICD-10-GM.
Efficiency of the phyto-mixture according to the invention in some of the efflorescences using the formulations according to the invention is described in Example 9 and in Table 7, without being limited to them.
The immunologically active phyto-mixture, preferably the composition comprising the phyto-mixture, for use in the prevention or in a method for treatment of efflorescences within the meaning of the invention preferably comprises at least one excipient selected from fillers, accelerators for disaggregation, lubricants, disintegrants, greases, release agents, glidants, solvents, emulsifiers, solubilizing agents, solubilizers, wetting agents, salt forming agents, buffer, gel forming agents, thickening agents, film forming agents, binders, sorbents, sweeteners, colorants, plasticizers, stabilizers, matrix forming agents, polymers, acidifiers, preservatives, scents and/or retarding agents.
Each formulation according to the invention (Examples 8a and 8b), preferably of the composition comprising the immunologically active phyto-mixture, may comprise excipients known from the state of the art. Further excipients comprise carriers, preservatives, antioxidants, stabilizers, vitamins, colorants, smell improving agents and flavors.
Flavors serve for masking a potentially unpleasant taste of the phyto-mixture, in particular of the formulation comprising the afore-mentioned phyto-mixture. For example, mint, anise, fennel, menthol, caraway, medicinal honey, honey, agave juice, sugar, sugar substitute as well as sugar replacer, propolis, and other flavors and flavourings known according to the state of the art, are well suited hereto. In cosmetic formulations, preferably of the composition comprising the phyto-mixture, comprising the immunologically active phyto-mixture from (a), (b), and/or (c), and, optionally, (d), such as ointment, paste, cream and gel, animal and vegetable fats, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicons, bentonites, silicic acid, talcum, and zinc oxide, or mixtures of at least two of the afore-mentioned substances may be used as excipients.
Excipients for powder or sprays comprising the immunologically active phyto-mixture from (a), (b), and/or (c), and, optionally, (d) comprise lactose, talcum, silicic acid, aluminium hydroxide, calcium silicate, calcium and magnesium carbonate, magnesium oxide, metal soaps, cellulose powder, pure and modified starches and polymer powder, polyamide powder, or mixtures of at least two of the afore-mentioned substances. Sprays and aerosols may additionally comprise common propellants, e.g. hydrochlorfluorocarbons, propane/butane or dimethylether, wherein preferably biologically and pharmaceutically compatible propellants are used. Powder preferably comprise lactose, silicic acid, calcium silicate and/or mineral ash, e.g. from grain.
Solutions, dispersions, suspensions and emulsions comprising the immunologically active phyto-mixture from (a), (b), and/or (c), and, optionally, (d) may comprise common excipients, such as solvents, solubilizing agents and emulsifiers, e.g. water, ethanol isopropanol, ethylcarbonate, ethylacetate, benzylalcohol, benzylbenzoate, propylene glycol, 1,3-butylglycol, oils, cottonseed oil, peanut oil, maize germ oil, olive oil, castor oil and sesame oil, glycerin fatty acid ester, polyethylene glycol and fatty acid esters of sorbitan, or mixtures of at least two of the afore-mentioned substances.
Suspensions comprising the immunologically active phyto-mixture form (a), (b), and/or (c), and, optionally, (d) comprise common excipients, such as liquid diluents, e.g. water, ethanol or propylene glycol, suspension agents, e.g. ethoxylated isosterylalcohols, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminium metahydroxide, bentonite, agar agar, and tragacanth, or mixtures of at least two of the afore-mentioned substances.
Soaps comprising the immunologically active phyto-mixture form (a), (b), and/or (c), and, optionally, (d), which comprise excipients, such as alkali salts of fatty acids, salts of fatty acid semi-esters, fatty acid protein hydrolysates, isothionates, lanolin, fatty alcohol, plant oils, plant extracts, glycerin, sugar, or mixtures of at least two of the afore-mentioned substances, are particularly well suited for daily use.
Preferred excipients for formulations of medical products in solid or liquid form for prevention or treatment of efflorescences within the meaning of the invention comprise lactose, sucrose, dextrose, mannitol, sorbitol, starch, gelatin, tragacanth, pectin, cellulose, methylcellulose, hydroxypropylmethylcellulose (HPMC), carboxymethylcellulose sodium, ethylcellulose, hydroxypropylmethylcellulose phthalate, cellulose acetate phthalate, polyvinylpyrrolidone, polyvinylalcohole, polyacrylic acid, polyethylene glycol, polyethylen oxide, sodium dodecylsulfate, sodium acetylstearylsulfate, and/or sodium dioctylsulfosuccinate (also K salts, Ca salts).
Preferred excipients for tinctures, solutions, dispersions and suspensions according to the invention comprising the immunologically active phyto-mixture from (a), (b), and/or (c) and, optionally, (d) comprise dextrose, mannitol, tragacanth, pectin, methylcellulose, hydroxypropylmethylcellulose (HPMC), carboxymethylcellulose sodium, polyvinylpyrrolidone, polyvinylalcohole, polyacrylic acid, polyethylene glycol, polyethylene oxide, sodium dodecylsulfate, sodium acetylstearylsulfate and/or Na-dioctylsulfosuccinate (also K salts, Ca salts), and, in particular for suspensions, also cellulose.
Semisolid forms or mixtures comprising the immunologically active phyto-mixture form (a), (b), and/or (c), and, optionally, (d) comprise hydroxypropylmethylcellulose (HPMC), carboxymethylcellulose sodium, polyethylene glycol, polyethylene oxide, sodium dodecylsulfate, sodium acetylstearylsulfate and/or pectin.
In a particular arrangement of the immunologically active phyto-mixture according to the invention comprising at least one plant extract from (a), (b), and/or (c), and, optionally, (d), preferably the composition comprising the phyto-mixture, for use in the prevention or in a method for treatment of efflorescences within the meaning of the invention, the phyto-mixture according to the invention is present as
Within the meaning of the invention, tablets comprise simple tablets, lozenges, sublingual and buccal tablets, oral dispersible tablets, solution tablets, microtablets, chewable tablets and effervescent tablets, spot tablet, scaffold tablet and multi-layer tablet, dragees and pellets. In the case of dissolution in mouth and oral mucosa the 15 pH value preferably is in the described pH ranges.
In context of a formulation (Example 8a and 8b), mixtures comprise semi-solid forms. Semi-solid forms of medicaments and cosmetic products include ointment-like preparations, characterised by having limited form stability. Ointments and creams include all spreadable preparations being applied on the skin or mucous membrane. Depending on character, semi-solid forms are distinguished in ointments, creams, pastes or gels.
Ointments, in narrower sense, include preparations without aqueous phase. Hydrophobic, hygroscopic and hydrophilic ointments are distinguished. Creams are ointments comprising an aqueous phase beside a lipid phase. Pastes are highly concentrated suspension ointments, representing the physical-chemical transition from suspensions to moist powders.
Preferably, the immunologically active phyto-mixture according to the invention comprising at least one plant extract according to the invention from (a), (b), and/or (c), and, optionally, (d), preferably the composition comprising the phyto-mixture, for use in the prevention or in a method for treatment of efflorescences within the meaning of the invention is present as tincture, in particular as solution, suspension, dispersion or in semi-solid form (Example 9), comprising
wherein the tincture has a pH value greater than or equal to 3 to less than or equal to 9 and the tincture is an aqueous/ethanolic mixture, in particular a solution or dispersion, having an ethanol concentration greater than or equal to 70%, based on 100 ml total volume of the tincture (ad 100%). Preferably, the tincture described above comprises greater than or equal to 3% by weight, greater than or equal to 5% by weight, preferably greater than or equal to 7% by weight, total content of at least one plant extract according to the invention or combination from (a), (b) and/or (c), and preferably greater than or equal to 15% by weight of at least one (d) Aloe extract, respectively based on the total content the phyto-mixture (ad 100% by weight).
The tincture, solution, suspension, dispersion and semi-solid forms described above has preferred pH values, depending on its respective indication, which has been described in detail in connection with the acidifiers above.
Within the meaning of the invention, “aqueous/ethanolic mixture” comprises solutions and dispersions comprising insoluble ingredients and particles. Presence of the aqueous/ethanolic mixture depends on solution behavior of the extracted ingredients, the separating step and depends on ethanol concentration.
The immunologically active phyto-mixture comprising at least one plant extract according to the invention from (a), (b), and/or (c), and, optionally, (d), preferably the composition comprising the phyto-mixture, for use in the prevention or in a method for treatment of efflorescences within the meaning of the invention, wherein the oral formulation (see Examples 8a-b and 9) is based on a solid form, is also preferred, wherein
Particularly preferred topical formulations are those for applying, depositing, massaging in, spraying on, dripping on and/or laying on the affected skin surface and thus for absorption of the immunologically active phyto-mixture through the skin or mucous membrane, in particular nasal and/or oral mucosa.
Particular preferred oral administration forms of the phyto-mixture according to the invention comprising at least one plant extract according to the invention (a), (b), and/or (c), and, optionally (d) comprise formulations for dissolving in a beverage and subsequent drinking, for swallowing (e.g. as tablet) and absorption trough the gastrointestinal tract, for oral intake and disaggregation in the oral cavity or depositing on the oral mucosa and absorption through the oral mucosa. Formulations for absorptions through the gastrointestinal tract preferably comprise no anthraquinones since they can have a laxative effect. Formulations according to the invention for indications where laxative effect is desired preferably comprise no Aloe extract or anthraquinone-free Aloe extract. The same applies for the species according to the invention of genus (a) Bidens, (b) Stachytarpheta and (c) Bursera. The following examples elucidate the invention in more detail and only represent chosen embodiments, without limiting thereto. For this purpose, some species according to the invention were used as representatives of the respective genus (a) Bidens, (b) Stachytarpheta and (c) Bursera and for the species of the respective genus, described as being preferred.
The plants Bur. simaruba, St. jamaicensis, Bid. alba and Stem. maritima disclosed and claimed within this patent application were respectively obtained from the Bahamas, Isle “Long Island”, as representatives according to the invention of genus (a) Bidens, (b) Stachytarpheta, (c) Bursera, and (d) Stemodia. The Bahamian government issued appropriate certificates for harvest of the plants used herein and their export to Germany for research and experimental purposes (“Plant Protection Service of the Bahamas” Nr. 2928 and Nr. 2929 and “Permit to conduct Scientific Research in the Bahamas”). The experiments described subsequently were performed within the scope of this research permit and export authorization.
A: Bidens alba (Bid. Alba)/B: Stachytarpheta jamaicensis (Sta. jamaicensis)
Aboveground plant parts comprising stem, leaves, shoots and blossoms each were freshly harvested from young 1- to 3-years-old plants (A and B). These were dried at 40° C. to 55° C. for 6 to 20 hours and subsequently stored in a cool dry place.
Duration of drying and temperature is variably freely selectable according to the knowledge of the person skilled in the art.
C: Bursera simaruba (Bur. Simaruba)
Leaves and branches of a 5- to 20-years-old tree. The harvested leaves were dried at 57° C. to 62° C. for 8 to 20 hours and subsequently stored in a cool dry place.
D1: Stemodia maritima (Stem. maritima)
Aboveground approx. 1-year-old herb. The harvested herb was dried at 40° C. for 7 hours and subsequently stored in a cool dry place.
D2: Aloe vera
Starting material: 500 ml of an anthraquinone-free water extract of Aloe vera (allcura Naturheilmittel GmbH, Reichenäcker 7, 97877 Wertheim). 20 ml of the water extract were freeze-dried (Christ LMC-1, Delta 1-20 KD). Yield: dry weight 450 mg powder (about 22.5 mg/ml).
The plant material described above with the described plant parts were provided, crushed and pulverized in a commercial blender for about 2 minutes. Subsequently, the respective powder was processed into, on the one hand, an aqueous and, on the other hand, an ethanolic extract.
A Bidens alba—Aqueous Extract:
The powder was transferred into a three-necked flask with distilled water (8.2 g powder to 120 ml distilled water) and stirred at 40° C. for twice 4 hours each or 8 hours in all. A clear solution was obtained after subsequent filtration of the extract. The clear solution was freeze-dried for 64 hours (Christ LMC-1, Delta 1-20 KD). Yield after freeze-drying: 0.931 g
B Stachytarpheta jamaicensis—Aqueous Extract:
8.0 g of the powder of Stachytarpheta jamaicensis were analogously processed to Bidens alba. Yield after freeze-drying: 0.7275 g
C Bursera simaruba—Aqueous Extract:
8.2 g of the powder of Bursera simaruba were analogously processed to Bidens alba.
Yield after freeze-drying: 1.1212 g
D1 Stemodia maritima—Aqueous Extract:
8.1 g of the powder of Stemodia maritima were analogously processed to Bidens alba.
Yield after freeze-drying: 0.299 g
A Bidens alba—Ethanol Extract:
The powder (8.0 g) was filled into an extraction sleeve (Macherey & Nagel; MN 645; 23×100 mm) and extraction with reflux was performed using a Soxhlet extraction with 280 ml 96% ethanol at room temperature for 8 hours. The organic phase was filtrated through glass wool (company Migge No. 1408/3). Subsequently, the ethanol extract was evaporated until dryness using a Rotavapor (Heidolph; 50° C. water bath temperature, 112 bar). Yield: 1.464 g dry weight
The dry weight was dissolved in 15 ml Ethanol in order to detach dry residues adhering to the bottom of the round-bottom flask. Subsequently, the solution was divided to sample containers for storing at −20° C. and dried by blowing through of nitrogen gas.
B Stachytarpheta jamaicensis—Ethanol Extract:
8.0 g of the powder of Stachytarpheta jamaicensis were analogously processed to Bidens alba. Yield: 2.485 g dry weight
C Bursera simaruba—Ethanol Extract:
8.2 g of the powder of Bursera simaruba were analogously processed to Bidens alba.
Yield: 5.8 g dry weight
D1 Stemodia maritima—Ethanol Extract:
6.7 g of the powder of Stemodia maritima were analogously processed to Bidens alba.
Yield: 1.39 g dry weight
The residual content of ethanol of the above ethanol plant extracts was less than 0.1%.
Cell Culture
HaCaT-Cells (human in vitro spontaneous transformed keratinocytes from histologically normal skin) of DMSZ (German Collection of Microorganisms and Cell Cultures) were kept in Dulbecco's modified Eagle Medium (DMEM) with glutamax (Invitrogen/Gibco, Karlsruhe, Germany) with 10% fetal calf serum (Sigma Aldrich, Germany), 100 U/ml penicillin and 100 μg/ml streptomycin and 1% NEAA.
The cells were cultivated at 37° C., 5% CO2 and 95% humidity. The cells were subcultivated by removing the medium, adding 0.075% EDTA-solution in 1-fold PBS (phosphate-buffered salt solution) and subsequently incubated at 37° C. for 10 min. EDTA was removed und the HaCaT-cells were detached form the culture vessel by addition of 0.25% trypsin and 0.02% EDTA at 37° C. for 5 min. Afterwards, addition of fresh medium, centrifugation, exhausting of the medium and transferring in a new flask (ratio 1:5 or 1:10) was done. All experiments were performed using cells in the logarithmic growth phase.
Sample preparation: Aqueous extracts were dissolved in water (100 mg/ml water) and ethanol extracts in dimethylsulfoxide, DMSO, (200 mg/ml DMSO).
MTT-Assay According to Mosmann, 1983 (J. Of Immunological Methods 65, 55-63)
In order to determine the concentration dependent cytotoxicity 2×104 HaCaT-cells/sample well of a 96-well plate were transferred and incubated for 24 hours. Incubation of the HaCaT-cells was performed using 100 μl of the medium containing various concentrations of the respective plant extract (see Table 2) or a 1.1 mixture of two plant extracts (see Table 3). Subsequently, 0.5 mg/ml MTT (3-(4,5-Dimethylthiazole-2-yl)-2,5-diphenyltetrazoliumbromide) were added and incubated for 4 hours. After dissolving the formazan crystals formed by the HaCaT-cells in 100 μl DMSO (10 minutes shaking), absorption at 570 nm was measured (by means of Tecan Safire II Reader, Tecan Crailsheim, Germany). Doxorubicin was measured as reference. Cell vitality was determined according to the following formula:
Statistical Analysis
Three-fold determination (n=3) with three-fold replicate was performed and the results were stated as means±standard deviation. IC50-values were calculated through the dose response curve which was created by use of a logistic regression curve with 4 parameters with the GraphPad Prism® 5.01 Software or SigmaPlot® 11.0. The individual curves are not shown herein. The determined IC50-values with standard deviations are summarized in Tables 2 and 3.
Results
None of the aqueous extracts Bid. alba (A-W), Sta. jamaicensis (B-W), Bur. simaruba (C-W), Stem. maritima and Aloe vera exhibits cytotoxicity compared to doxorubicin as cytotoxic reference compound (see Table 2).
Ethanolic plant extracts of Bid. alba and Bur. simaruba exhibit no cytotoxicity compared to doxorubicin as cytotoxic reference compound. Ethanolic extracts of Sta. jamaicensis and Stem. maritima exhibit low cytotoxicity.
Only the combinations with plant extracts of little higher IC50-values were analysed again to cytotoxicity (see Table 3).
Inhibition of 5-Lipoxygenase (5-LOX) through the individual extracts and their combinations (see Table 2 and 3) was spectroscopically determined (Baylac & Racine 2003). For this purpose 970 μl of the phosphate buffer (21.2 g K3PO4 in 1 L H2O), pH 9.0 were mixed with 10 μl of 1 mg/ml concentrated 5-LOX (lyophilized powder, Fluka) and 20 μl various concentrations (0.48 μg/ml to 250 μg/ml) of the individual plant extracts and of the combinations (1:1 mixture). The mixture was incubated at room temperature for 10 minutes. Enzymatic reaction was initiated by adding of 20 μl of 5 mM sodium linoleate (Sigma Aldrich). Reaction kinetic was determined at 234 nm every 5 seconds by means of a WPA Biowave II spectrophotometer. Nordihydroguaiaretic acid (NDGA) was used as positive control of a 5-LOX-selective inhibitor.
Initial reaction speed was respectively determined from the slope of the linear part of the curve. Inhibition of the enzyme activity was calculated from three-fold experiments. Percentage of the initial activity was determined according to the following formula:
[%] of the initial activity=(IAcontrol−IAsample)/IAcontrol×100
wherein sample corresponds to an individual plant extract or a combination of at least two plant extracts and control equals to NDGA.
Percentage [%] of the initial activity was (y-axis) graphically plotted as a function of the concentration of the respective plant extract or the combination (1:1 mixture), in order to determine the IC50-value as the concentration at which 50% of the enzyme activity is inhibited by the respective plant extract or the combination.
Results
Table 2 shows that ethanolic Bur. simaruba (C-E) extract exhibits significant inhibition of 5-Lipoxygenase. Consequently, an ethanol plant extract of Bur. simaruba (C-E withlCso=132±13 μg/ml) exhibits provably significant anti-inflammatory activity. The same applies for the respective ethanol individual extracts of Sta. jamaicensis (B-E with IC50=84±10 μg/ml) and Bid. alba (A-E with IC50=139±7 μg/ml).
Table 3 shows that the combinations of the respective extracts exhibit an increased inhibition of 5-Lipoxygenase. These experiments respectively prove significant anti-inflammatory efficiency for Bid. alba, Sta. jamaicensis and Bur. simaruba as representatives for genus (a) Bidens, (b) Stachytarpheta and (c) Bursera. This efficiency may be attributed to the compounds, being contained in the species according to the invention and being extracted by means of the method according the invention, comprising verbascosides, flavonoids, iridoids, ipolamiides, fulvoipolamiides, sesquiterpene lactones and/or proazulenes.
Bid. alba
Bid. alba
Sta. jamaicensis
Sta. jamaicensis
Bur. simaruba
Bur. simaruba
Stem. maritime
Stem. maritima
Aloe vera
Antibacterial efficiency of the respective plant extract as individual extract and as combination of two extracts was analysed against the following test germs:
Gram-Positive Bacteria
Gram-Negative Bacteria
Culture Preparation
The bacteria were precultivated on Columbia medium with 5% sheep blood at 37° C. for 24 hours. On to two colonies were suspended in saline solution (0.9% NaCl) and adjusted to a turbidity of 0.5 McFarland standard which corresponds to 1×108 colony forming units per milliliter (CFU/ml). Subsequently, the suspension was diluted to 1×106 CFU/ml.
Determination of the Minimal Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC)
The MIC was determined by means of the microbouillon dilution process according to NCCLS (2006). For this purpose, the aqueous extracts were dissolved in water (w/v) and the ethanolic extracts in 5% DMSO (w/v) with 80 mg/ml each and Bur. simaruba as well as Aloe vera were suspended with 160 mg/ml. The extracts and the combinations (1:1 mixture) of two plant extracts were respectively transferred into a sample well of a 96-well plate. The individual extracts were respectively adjusted to a concentration of 4 mg/ml to 8 mg/ml and 0.03 mg/ml and the combinations to 2:2 mg/ml to 0.3:0.3 mg/ml. Subsequently, the bacteria suspensions with about 5×105 CFU/ml in Müller Hinton medium (Fluka) were respectively added. The accordingly fitted 96-well plates were incubated at 37° C. for 24 hours.
The MIC was determined at 600 nm on the basis of the turbidity. In order to determine the MBC, 3 μl of the suspension of the respective sample well were spread on an full culture medium and incubated at 37° C. for 24 hours. The MBC was determined as lowest concentrations of the respective extract which totally kills the microorganisms.
All experiments were performed as three-fold determination (Table 4 to 5). The antibiotics Vancomycin (briefly: Van) and Streptomycin (briefly: Strep) were used parallely to each experimental approach as positive control for antimicrobial efficiency (Table 5c). Respectively one of the mentioned test germs was carried along with the respectively solvent (water, DMSO) as negative control for an inhibiting effect by the used solvent. In comparison to the growth control without any addition, no negative influence on the growth was observed for all negative controls.
A sterility control confirmed sterility of the used media (data not shown). The composition of the controls is summarized in Table 5b.
Results
Table 4a summarizes the antimicrobial activity of the aqueous individual plant extracts. Table 4a shows antibacterial efficiency for Sta. jamaicensis (B-W) and Bid. alba (A-W). Stem. maritima (D1-W) exhibits higher antibacterial efficiency compared to B-W and A-W.
All the following combinations (1:1 mixture) of the aqueous extracts exhibit the same efficiency against gram-positive bacteria with MIC≥2/2 and MBC≥2/2:
A-W/B-W, A-W/C—W, B—W/C—W, C—W/D1-W, B—W/D1-W, A-W/D1-W, A-W/D2,
B-W/D2, C-W/D2, D1-W/D2
Table 5a summarizes the antimicrobial activity of the ethanolic extracts and Table 5b the combinations of the ethanolic plant extracts. The experiments clearly prove an antibacterial efficiency of Sta. jamaicensis and Bid. alba as well as of Stem. maritima. In particular, the afore-mentioned species exhibit significant efficiency against gram-positive bacteria and particularly against MRSA.
S. aureus ATCC 25923
S. epid. ATCC 14990
P. aerug. ATCC 27853
S. aureus ATCC 25923
S. epid. ATCC 14990
P. aerug. ATCC 27853
S. aureus ATCC 25923
S. epid. ATCC 14990
S. aureus ATCC 25923
S. epid. ATCC 14990
P. aerug. ATCC 27853
For example, the excipients ethyl- and methylcellulose, hydroxypropylmethylcellulose, cellulose acetate phthalate and hydroxypropylmethylcellulose phthalate exhibit retarding properties.
Base Formula: Moisturizing Cream
100 g base cream DAC comprises:
4.0 g glycerol monostearate 60
6.0 g cetylalcohol
7.5 g medium chain triglycerides (neutral oil, Miglyol 812)
25.5 g white vaseline
7.0 g Macrogol 20-glycerol monostearate
10.0 g propylene glycol
40.0 g purified water
1. Formula: Moisturizing Cream with C-E. B-E and D2
40 to 97 g of the base formula
1-10 g Bursera simaruba (C-E)
1-10 g Stachytarpheta jamaicensis (B-E)
1-40 g Aloe vera (D2)
(total weight 100 g=100% by weight, each based on the dry weight)
2. Formula: Moisturizing Cream with B-E. A-E and D2
40 to 97 g of the base formula
1-10 g Stachytarpheta jamaicensis (B-E)
1-10 g Bidens alba (A-E)
1-40 g Aloe vera (D2)
(total weight 100 g=100% by weight, each based on the dry weight)
3. Formula: Moisturizing Cream with A-E, B-E, C-E and D2
30 to 96 g of the base formula
1-10 g Bursera simaruba (C-E)
1-10 g Stachytarpheta jamaicensis (B-E)
1-10 g Bidens alba (A-E)
1-40 g Aloe vera (D2)
(total weight 100 g=100% by weight, each based on the dry weight)
Base Tincture D2a (Total: 100% by Weight Based on the Dry Weight):
70% by weight of an Aloe extract (D2) were homogenously mixed in 100 ml 90% ethanol
Base Tincture D2b (Total: 100% by Weight Based on the Dry Weight):
30% by weight of an Aloe extract (D2) were homogenously mixed in 100 ml 90% ethanol
4. Immunologically Active Tinctures with A-E, B-E, C-E and D2
100 ml base tincture D2a or D2b were respectively mixed with:
Bid.
alba
Sta.
jamaicen.
Bur.
simaruba
The % by weight information in Table 6 is based on the dry weight of the extracts obtained according to Example 3 or 4, preferably according to Example 4.
Depending on the desired indication of the skin, the pH value of the afore-mentioned tinctures 44 to 53 was set to:
(a) tincture for the skin: pH 4.5 to 5.5
(b) tincture for the oral mucosa: pH 6.7 to 7.2
(c) tincture for the eye: pH 7.0 to 7.5
(d) tincture for the nasal mucosa: pH 5.5 to 6.5
(e) tincture for ears: pH 5.5 to 6.1
The afore-mentioned mixtures and % by weight ratios (Table 6) may vary depending on the indication. For example, in the case where strengthened antimicrobial effect is desired, e.g. in the case of bacterial infections of the (mucous membrane) skin or in the mouth (gums), it is advantageous according to the invention to combine the extracts (or dry extracts) having the strongest antimicrobial effects (Table 5a/5b), or to use only one extract, e.g. A-E, (e.g. mixture 52 or 53). In contrast, in the case of strong inflammation, e.g. eye or ear, it may advantageous according to the invention to increase the content of particularly anti-inflammatorily effective extracts, e.g. A-E and/or C-E (Table 2 and 3). In this case, e.g. mixture 45 or mixture 45 having increased % by weight of each 5-20% by weight of the respective dry extract would be conceivable. In the case of antimicrobial ant anti-inflammatory indication at the same time, the appropriate combination according to the invention is to be chosen depending on the therapeutic goal.
| Number | Date | Country | Kind |
|---|---|---|---|
| 102015102020.3 | Feb 2015 | DE | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15550600 | Aug 2017 | US |
| Child | 16191239 | US |
