IMMUNOLOGICALLY ACTIVE PHYTO-MIXTURE AND ITS USE IN THE PREVENTION AND IN A METHOD FOR TREATMENT OF EFFLORESCENCES

Abstract

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 at least one plant extract 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 simaruba, Bursera microphylla, Bursera glabrifolia. 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.

Claims

1. Immunologically active phyto-mixture which exhibits antimicrobial efficiency against transient skin flora and anti-inflammatory efficiency, wherein the phyto-mixture comprises at least one ethanolic plant extract selected from a) Bidens alba (Bid. alba), Bidens pilosa (Bid. pilosa) from genus Bidens of the Asteraceae family, and at least one further ethanolic plant extract, selected from b) Stachytarpheta jamaicensis (Sta. jamaicensis), Stachytarpheta cayennensis (Sta. cayennensis), Stachytarpheta indica (Sta. indica) from genus Stachytarpheta of the Verbenaceae family, and/or c) Bursera simaruba (Bur. simaruba), Bursera microphylla (Bur. microphylla) and Bursera glabrifolia (Bur. glabrifolia) from genus Bursera of the Burseraceae family.

2. Phyto-mixture according to claim 1, characterised in that it further comprises: 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 of the Plantaginaceae family and/or Stemodia maritima.

3. Phyto-mixture according to claim 1, which comprises three or more ethanolic plant extracts, selected from: a) and c) and d); a) and b) and d); or a), b) and c) and d), wherein d) respectively is Stemodia maritima.

4. Phyto-mixture according to claim 1, characterised in that the phyto-mixture further comprises d) at least one further plant extract of an Aloe species of the genus Aloe of subfamily Asphodeloideae.

5. Phyto-mixture according to claim 1, characterised in that the transient skin flora comprises Staphylococcus, Streptococcus, methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonads and/or Acinetobacteria.

6. Phyto-mixture according claim 1, characterised in that the phyto-mixture comprises an antibacterial efficiency at a concentration of greater than or equal to 10 g/ml to less than or equal to 10 mg/ml measured as minimal inhibitory concentration (MHK/MIC) and/or as minimum bactericidal concentration (MBK/MBC) of the respective extract mixture.

7. Phyto-mixture according claim 1, characterised in that the phyto-mixture exhibits an anti-inflammatory efficiency at less than or equal to 20010 g/ml measured as IC.sub.50 of 5-LOX inhibition of the respective extract mixture.

8. Phyto-mixture according to claim 1, characterised in that the phyto-mixture comprises at least one of the compounds comprising flavonoids, saponins, iridoids, phenolic acids, polyphenols, polysaccharides, glycosylases, terpenes, monoterpenes, sesquiterpene lactones, proazulenes, sulfides, carotenoids, vitamins A B C D E, amino acids, and/or minerals.

9. Phyto-mixture according to claim 1, characterised in that the phyto-mixture is present in liquid form comprising solution, dispersion, suspension, emulsion, tincture, syrup, juice, and tea solid form comprising tablet, powder, powder, dragee, globules, granules and lyophilisate, capsule, or as mixture comprising capsules, aerosol, spray, emulsion, lotion, and cream.

10. Phyto-mixture according to claim 1, characterised in that the phyto-mixture in liquid form, depending on the alternative, 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.

11. Phyto-mixture according to claim 1, characterised in that the liquid phyto-mixture is present as tincture, comprising greater than or equal to 1% by weight of at least one ethanolic plant extract selected from a) Bid. alba, Bid. pilosa, and one further ethanolic plant extract, selected from b) Sta. jamaicensis, Sta. cayennensis, Sta. indica, and/or c) Bur. simaruba, Bur. microphylla, Bur. glabrifolia, based on the total content of the tincture (T=100% by weight), at least one acidifier comprising acetic acid, citric acid, ascorbic acid, adipic acid, tartaric acid, mandelic acid, and/or malic acid, greater than or equal to 1% by weight of an Aloe extract, based on the total content of the tincture (T=100% by weight), 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.

12. A method for the production of an immunologically active phyto-mixture according to claim 1 comprising the steps of providing at least one plant part of at least one plant selected from a) and one further plant selected from b) and/or c), respectively above- and/or underground plant parts; respectively at least one extraction step, wherein aqueous ethanol, ethanol, greater than or equal to 70% ethanol to less than or equal to 100% ethanol is used as solvent; and obtaining at least one ethanolic plant extract of the respective plant; optionally one further processing step comprising drying, comminution, grinding using a mill, processing in a mortar, dispersing; and mixing at least two of the ethanolic plant extracts, one being selected from a) and the at least second one of b) and/or c); and optionally adding a third plant extract selected from b), c) and/or d).

13. Method according to claim 12, wherein the at least two plant extracts are obtained in liquid form, in dry form or as mixture of solid and liquid forms.

14. Immunologically active phyto-mixture according to claim 1 and/or produced according to claim 12 for use as medicament, medical product, nutritional supplement, cosmetic, and/or as an immunologically active addition to one of the afore-mentioned products.

15. Pharmaceutic or cosmetic composition comprising the immunologically active phyto-mixture according to claim 1.

16. Immunologically active-phyto-mixture for use in the prevention or treatment of efflorescences comprising at least one ethanolic plant extract selected from: a) Bid. alba, Bid. pilosa of the Asteraceae family and at least one further ethanolic plant extract, selected from, b) Sta. jamaicensis, Sta. cayennensis, Sta. indica of the Verbenaceae family, and/or c) Bur. simaruba, Bur. microphylla and/or Bur. glabrifolia of the Burseraceae family.

17. Phyto-mixture for use according to claim 16, which comprises three or more plant extracts, selected from a) and c) and d); a) and b) and d); or a), b) and c) and d), wherein d) respectively is Stemodia maritima

18. Phyto-mixture for use according to claim 16, characterised in that 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.

19. Phyto-mixture for use according to claim 16, characterised in that the phyto-mixture is present as oral formulation comprising i) solid forms, such as tablet, powder, granules, effervescent tablet, dry syrup, dragee, globules, capsule and lyophilisate, ii) liquid forms, such as suspension, solution, dispersion, tincture, concentrate, tea, or iii) mixture, such as spray, aerosol, or topical formulation comprising i) solid forms, such as powder, bath additive, hip bath powder, ii) liquid forms, such as suspension, emulsion, solution, dispersion, tincture, tea, or iii) mixture, such as spray, aerosol, lotion, semi-solid forms comprising ointments, creams and pastes.

20. Phyto-mixture for use according to claim 16, characterised in that the phyto-mixture is present as tincture, comprising greater than or equal to 1% by weight of at least one ethanolic plant extract selected from Bid. alba, Bid. pilosa, and at least one further ethanolic plant extract, selected from Sta. jamaicensis, Sta. cayennensis, Sta. indica and/or Bur. simaruba, based on the total content (T=100% by weight) of the tincture, at least one acidifier selected from acetic acid, citric acid, ascorbic acid, adipic acid, tartaric acid, mandelic acid and/or malic acid, greater than or equal to 1% by weight of an Aloe extract, based on the total content of the tincture (T=100% by weight), and at least one excipient, 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, having an ethanol concentration greater than or equal to 70%, based on the total composition of the tincture.

21. Phyto-mixture for use according to claim 16, characterised in that the oral formulation is based on a solid form, wherein the solid form comprises at least one dried ethanolic plant extract, selected from Bid. alba, Bid. pilosa, and at least one further dried ethanolic plant extract, selected from Sta. jamaicensis, Sta. cayennensis and/or Sta. indica, Bur. simaruba and optionally Aloe, the at least one plant extract is contained in a content of greater than or equal to 1% by weight, based on the total content of the solid form (F=100% by weight), the residual moisture is less than or equal to 5% by weight, based on the total content of the dry plant extract (F=100% by weight) and at least one excipient is contained. the at least one plant extract is contained in a content of greater than or equal to 1% by weight, based on the total content of the solid form (F=100% by weight), the residual moisture is less than or equal to 5% by weight, based on the total content of the dry plant extract (F=100% by weight) and at least one excipient is contained.

Description

EXAMPLES

Example 1: Source of the Used Plant Material

[0205] 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.

[0206] A: Bidens alba (Bid. alba)/B: Stachytarpheta jamaicensis (Sta. jamaicensis)

[0207] 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.

[0208] C: Bursera simaruba (Bur. simaruba)

[0209] 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.

[0210] D1: Stemodia maritima (Stem. maritima)

[0211] 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.

[0212] D2: Aloe vera

[0213] Starting material: 500 ml of an anthraquinone-free water extract of Aloe vera (allcura Naturheilmittel GmbH, Reichencker 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).

TABLE-US-00002 TABLE 1b Plant material Plant material as Dry weight Dry weight powder after crushing water extract ethanol extract Plant [g] [g] [g] A 31.5 0.931 1.464 B 38.7 0.7275 2.485 C 33.1 1.1212 5.8 D1 38.7 0.299 1.39 D2 ./. 0.450 ./.

Example 2: Crushing of the Plant Material

[0214] 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.

Example 3: Production of an Aqueous Plant Extract

[0215] A Bidens albaAqueous Extract:

[0216] 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

[0217] B Stachytarpheta jamaicensisAqueous Extract:

[0218] 8.0 g of the powder of Stachytarpheta jamaicensis were analogously processed to Bidens alba. Yield after freeze-drying: 0.7275 g

[0219] C Bursera simarubaAqueous Extract:

[0220] 8.2 g of the powder of Bursera simaruba were analogously processed to Bidens alba.

[0221] Yield after freeze-drying: 1.1212 g

[0222] D1 Stemodia maritimaAqueous Extract:

[0223] 8.1 g of the powder of Stemodia maritima were analogously processed to Bidens alba.

[0224] Yield after freeze-drying: 0.299 g

Example 4: Production of an Ethanolic Plant Extract

[0225] A Bidens albaEthanol Extract:

[0226] The powder (8.0 g) was filled into an extraction sleeve (Macherey & Nagel; MN 645; 23100 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

[0227] 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.

[0228] B Stachytarpheta jamaicensisEthanol Extract:

[0229] 8.0 g of the powder of Stachytarpheta jamaicensis were analogously processed to Bidens alba. Yield: 2.485 g dry weight

[0230] C Bursera simarubaEthanol Extract:

[0231] 8.2 g of the powder of Bursera simaruba were analogously processed to Bidens alba.

[0232] Yield: 5.8 g dry weight

[0233] D1 Stemodia maritimaEthanol Extract:

[0234] 6.7 g of the powder of Stemodia maritima were analogously processed to Bidens alba.

[0235] Yield: 1.39 g dry weight

[0236] The residual content of ethanol of the above ethanol plant extracts was less than 0.1%.

Example 5: Determination of Cytotoxicity of the Produced Extracts

[0237] Cell Culture

[0238] 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 g/ml streptomycin and 1% NEAA.

[0239] The cells were cultivated at 37 C., 5% CO.sub.2 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 and 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.

[0240] Sample Preparation:

[0241] Aqueous extracts were dissolved in water (100 mg/ml water) and ethanol extracts in dimethylsulfoxide, DMSO, (200 mg/ml DMSO).

[0242] MTT-Assay According to Mosmann, 1983 (J. of Immunological Methods 65, 55-63)

[0243] In order to determine the concentration dependent cytotoxicity 210.sup.4 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 l 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 l 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:

[00001]$\mathrm{Vitality}\left[\%\right]=\frac{\begin{array}{c}[\left(\mathrm{OD}.Math..Math.\mathrm{sample}.Math..Math.\mathrm{of}.Math..Math.\mathrm{untreated}.Math..Math.\mathrm{HaCaT}.Math.\text{-}.Math.\mathrm{cells}\right)-\\ \left(\mathrm{OD}.Math..Math.\mathrm{sample}.Math..Math.\mathrm{medium}.Math..Math.\mathrm{control}\right)]*100\end{array}}{\left[\left(\mathrm{OD}.Math..Math.\mathrm{untreated}.Math..Math.\mathrm{HaCaT}.Math.\text{-}.Math.\mathrm{cells}\right)-\left(\mathrm{OD}.Math..Math.\mathrm{medium}.Math..Math.\mathrm{control}\right)\right]}$

[0244] Statistical Analysis

[0245] Three-fold determination (n=3) with three-fold replicate was performed and the results were stated as meansstandard deviation. IC.sub.50-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 IC.sub.50-values with standard deviations are summarized in Tables 2 and 3.

[0246] Results

[0247] 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).

[0248] 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.

[0249] Only the combinations with plant extracts of little higher IC.sub.50-values were analysed again to cytotoxicity (see Table 3).

Example 6: Determination of Anti-Inflammatory Activity

[0250] 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 l of the phosphate buffer (21.2 g K.sub.3PO.sub.4 in 1 L H.sub.2O), pH 9.0 were mixed with 10 l of 1 mg/ml concentrated 5-LOX (lyophilized powder, Fluka) and 20 l various concentrations (0.48 g/ml to 250 g/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 l 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.

[0251] 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=(IA.sub.controlIA.sub.sample)/IA.sub.control100

wherein sample corresponds to an individual plant extract or a combination of at least two plant extracts and control equals to NDGA.

[0252] 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 IC.sub.50-value as the concentration at which 50% of the enzyme activity is inhibited by the respective plant extract or the combination.

[0253] Results

[0254] 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 with IC.sub.50=13213 g/ml) exhibits provably significant anti-inflammatory activity. The same applies for the respective ethanol individual extracts of Sta. jamaicensis (B-E with IC.sub.50=8410 g/ml) and Bid. alba (A-E with IC.sub.50=1397 g/ml).

[0255] 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.

TABLE-US-00003 TABLE 2 Individual extracts Cytotoxicity IC.sub.50 5-Lox-inhibition IC.sub.50 Extract Plant [g/ml] [g/ml] A-W Bid. alba >3000 >1000 A-E Bid. alba 527 68 139 7 B-W Sta. jamaicensis 1023 153 >1000 B-E Sta. jamaicensis 546 66 84 10 C-W Bur. simaruba 4670 400 >1000 C-E Bur. simaruba 1800 670 132 13 D1-W Stem. maritime 1399 321 >1000 D1-E Stem. maritima 127 5 >1000 D2 Aloe vera 3360 860 >1000 Doxorubicin 8.6 2.03 ./. NDGA ./. 0.53 0.09 W = aqueous extract; E = ethanol extract, A, B, C, D1 and D2 corresponds to nomenclature from example 1.

TABLE-US-00004 TABLE 3 Extract combinations 1:1 mixture Extract combination cytotoxicity IC.sub.50 5-Lox-inhibition IC.sub.50 1:1 mixture [g/ml] [g/ml] A-E/B-E n.b. .sup.52 1.3 A-E/C-E n.b. 78 6 B-E/C-E n.b. 57 8 A-E/D1-E 101 15 .sup.98 7.5 B-E/D1-E 117 8.7 .sup.94 4.5 C-E/D1-E 144 16 99 14 A-E/D2 n.b. 115 15 B-E/D2 n.b. 73 16 C-E/D2 2710 290 161 23 D1-E/D2 128 13 130 18 Doxorubicin 8.6 2. 03 ./. NDGA ./. 0.53 0.09 W = aqueous extract; E = ethanol extract, A, B, C, D1 and D2 corresponds to nomenclature from example 1.

Example 7: Determination of Antimicrobial Efficiency

[0256] Antibacterial efficiency of the respective plant extract as individual extract and as combination of two extracts was analysed against the following test germs:

[0257] Gram-Positive Bacteria [0258] Methicillin resistant Staphylococcus aureus MRSA NCTC 10442 (MRSA NCTC 10442) [0259] Staphylococcus aureus ATCC 25923 (S. aureus ATCC 25923) [0260] Staphylococcus epidermidis ATCC 14990 (S. epid. ATCC 14990)

[0261] Gram-Negative Bacteria [0262] Pseudomonas aeruginosa ATCC 27853 (P. aerug. ATCC 27853) and [0263] Acinetobacter baumanii ATCC BAA747 (ATCC BAA747)

[0264] Culture Preparation

[0265] 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 110.sup.8 colony forming units per milliliter (CFU/ml). Subsequently, the suspension was diluted to 110.sup.6 CFU/ml.

[0266] Determination of the Minimal Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC)

[0267] 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 510.sup.5 CFU/ml in Mller Hinton medium (Fluka) were respectively added. The accordingly fitted 96-well plates were incubated at 37 C. for 24 hours.

[0268] The MIC was determined at 600 nm on the basis of the turbidity. In order to determine the MBC, 3 l 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.

[0269] All experiments were performed as three-fold determination (Table 4 to 5). The antibiotics Vancomycin (briefly: Van) and Streptomycin (briefly: Strep) were used parallelly 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.

[0270] Results

[0271] 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.

[0272] All the following combinations (1:1 mixture) of the aqueous extracts exhibit the same efficiency against gram-positive bacteria with MIC2/2 and MBC2/2: 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

[0273] 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.

TABLE-US-00005 TABLE 4 Antimicrobial activity of the aqueous individual plant extracts MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC Test germ C-W B-W A-W D1-W D2 Van Strep MRSA NCTC 10442 >8 >8 4 >4 >4 >4 2 4 8 >8 1 2 ./. n.b. S. aureus ATCC 25923 >8 >8 4 >4 >4 >4 2 4 4 >8 0.5 0.5 2 8 S. epid. ATCC 14990 >8 >8 4 >4 >4 >4 2 4 8 >8 1 2 1 8 P. aerug. ATCC 27853 >8 >8 >4 >4 >4 >4 >4 >4 >8 >8 ./. n.b. 4 8 ATCC BAA747 >8 >8 >4 >4 >4 >4 >4 >4 8 >8 64 128 2 4

TABLE-US-00006 TABLE 5a Antimicrobial activity of the ethanolic individual plant extracts MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC Test germ C-E B-E A-E D1-E D2 Van Strep MRSA NCTC 10442 >8 >8 2 4 4 >4 0.5 1 8 >8 1 2 ./. n.b. S. aureus ATCC 25923 >8 >8 1 4 2 4 0.5 1 4 >8 0.5 0.5 2 8 S. epid. ATCC 14990 >8 >8 1 2 1 2 0.5 1 8 >8 1 2 1 8 P. aerug. ATCC 27853 >8 >8 >4 >4 >4 >4 >4 >4 >8 >8 ./. n.b. 4 8 ATCC BAA747 8 >8 4 >4 4 >4 2 4 8 >8 64 128 2 4

TABLE-US-00007 TABLE 5b Antimicrobial activity of the ethanolic plant extract combinations (1:1 mixture) MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC Test germ A-E/B-E A-E/C-E B-E/C-E C-E/D1-E B-E/D1-E A-E/D1-E MRSA NCTC 10442 2/2 >2/2 >2/2 >2/2 2/2 >2/2 1/1 2/2 05/0.5 1/1 1/1 2/2 S. aureus ATCC 25923 1/1 >2/2 2/2 >2/2 1/1 >2/2 0.5/0.5 1/1 0.5/0.5 1/1 1/1 2/2 S. epid. ATCC 14990 1/1 2/2 1/1 >2/2 1/1 2/2 1/1 2/2 05/0.5 1/1 1/1 2/2

TABLE-US-00008 TABLE 5c Positive controls (antimikro. effect) Test germ MIC MBC MIC MBC Van g/ml Strep g/ml MRSA NCTC 10442 1 2 ./. n.b. S. aureus ATCC 25923 0.5 0.5 2 8 S. epid. ATCC 14990 1 2 1 8 P. aerug. ATCC 27853 ./. n.b. 4 8 ATCC BAA747 64 128 2 4

TABLE-US-00009 TABLE 5d Composition of the controls Growth control medium + test germ without extract/without antibiotic Negative control medium + test germ + solvent (water/DMSO) without extract/without antibiotic Sterility control medium without further additions Positive control medium + test germ + solvent (water/DMSO) + antibiotic (see Table 5c)

Example 8a: Examples Formulations

[0274]

TABLE-US-00010 Application Technical Tablets Function & Acceler- Pellets ator with for Homeo- and Semi- disaggre- pathic without Hard Soft solid gation, Excipient tritur- film gelatin gelatin Solu- Suspen- prepar- Disinte- Name ation Tea coating capsules capsules tion sion ation Powder Filler grant 1,2-Propylene 1 1 1 glycol Acetyltributylcitrate 1 1 1 Agar agar 1 Alkyl-4- 1 1 1 1 1 hydroxybenzoate Aluminium-fatty 1 1 1 1 acid compounds Aluminium oxide/ 1 1 1 1 hydroxide Gum arabic 1 1 Essential oils 1 1 1 1 1 1 1 1 1 Bentonite 1 1 Calcium carbonate 1 1 1 Calcium 1 1 1 1 1 1 hydrogenphosphate Carboxymethyl- 1 1 1 1 1 1 1 cellulose sodium Carotine 1 1 1 Cellulose 1 1 1 1 Cellulose acetate 1 1 phthalate Cetylalcohol 1 Cetylstearylalcohol 1 Citric acid 1 1 1 Dextrose 1 1 1 1 Dibutyl/ 1 1 1 Diethylphthalate Dimethylpolysiloxane 1 1 Iron oxide 1 1 1 Ethylalcohol 1 1 1 1 1 1 Ethylcellulose 1 1 Gelatine 1 1 Glycerol 1 1 1 triacetate Glycine 1 1 Glycerol 1 1 1 1 1 1 Glycerol monostearate 1 1 highly dispersed 1 1 1 1 1 1 silicon dioxide Hydroxypropyl- 1 1 1 1 1 1 methylcellulose (HPMC) Hydroxypropyl- 1 1 methylcellulose phthalate Isopropylalcohol 1 1 1 1 1 1 Isopropylmyristate 1 Lactose 1 1 1 Lecithin 1 1 Macrogol 1000-glycerol 1 monolaurate, -monostearate, -monooleate Macrogol 1500- 1 glycerol triricinoleate Macrogol glycerol 1 hydroxystearate Macrogol 1 stearate 400 Magnesium stearate 1 1 Maltodextrin 1 1 1 1 Mannitol 1 1 1 1 Methylcellulose 1 1 1 Sodium 1 1 1 1 1 cetylstearylsulfate Sodium dioctyl- 1 1 sulfosuccinate (also K salts, Ca salts) Sodium dodecylsulfate 1 1 1 1 1 Sodium 1 1 1 1 hydrogencarbonate Oleic acid-oleylester 1 Pectin 1 1 1 Poloxamer 1 1 1 1 Polyacrylic acid 1 1 Polyethylene glycol 1 1 1 1 1 1 Polyethylene oxide 1 1 1 1 1 1 Polymethacrylate 1 1 1 Polyoxyl 23 1 laurylether, -20 cetostearylether, -10 oleylether Polyoxyl 40 stearate, 1 -50 stearate Polysorbate 1 1 20, 60, 80, 40 Polyvinylacetate 1 1 1 copolymers Polyvinylalcohol 1 1 1 1 1 Polyvinylpyrrolidone 1 1 1 1 1 1 Riboflavin 1 1 1 1 1 Castor oil 1 1 1 Sucrose 1 1 Sorbitan 1 monooleate, -palmitate, -stearate, -trioleate, -tristearate, -laurate Sorbitol 1 1 Starch 1 1 1 1 Starches, rice-, 1 1 1 1 maize-potato-, wheat- Stearic acid 1 1 1 Stearylalcohol 1 Talcum 1 1 1 Titanium dioxide 1 1 1 1 Tragacanth 1 1 1 Triacetin 1 1 1 Triethanol amine 1 Triethylcitrate 1 1 1 Tromethamol 1 Vaseline 1 Tartaric acid 1 1 1 Urea (Urea) Ceramides Hyaluronic acid Technical function Emul- sifier, Gel Solubi- forming lizing agent, Sorbent agent, Thick- (Humec- Basis Lubri- Solubi- ening tant- e.g. cant, lizer, agent, or for Grease, Solvent, Wetting Salt Film Desic- powder, Release Solution agent, forming forming cant, Matrix Excipient agent, acceler- Antifoam agent, agent, respec- Sweet- Color- Plasti- forming Stabi- Name Glidant ator agent Buffer Binder tively) ener ant cizer agent lizer 1,2-Propylene 1 glycol Acetyltributylcitrate 1 Agar agar 1 Alkyl-4- 1 hydroxybenzoate Aluminium-fatty 1 acid compounds Aluminium oxide/ 1 hydroxide Gum arabic 1 Essential oils 1 Bentonite 1 Calcium carbonate 1 Calcium 1 hydrogenphosphate Carboxymethyl- 1 cellulose sodium Carotine 1 Cellulose 1 1 Cellulose acetate 1 phthalate Cetylalcohol 1 Cetylstearylalcohol 1 Citric acid 1 Dextrose 1 Dibutyl/ 1 Diethylphthalate Dimethylpolysiloxane 1 Iron oxide 1 Ethylalcohol 1 Ethylcellulose 1 1 Gelatine 1 1 Glycerol 1 triacetate Glycine 1 Glycerol 1 1 1 Glycerol monostearate 1 highly dispersed 1 1 1 1 silicon dioxide Hydroxypropyl- 1 1 methylcellulose (HPMC) Hydroxypropyl- 1 1 methylcellulose phthalate Isopropylalcohol 1 Isopropylmyristate 1 Lactose Lecithin 1 Macrogol 1000-glycerol 1 monolaurate, -monostearate, -monooleate Macrogol 1500- 1 glycerol triricinoleate Macrogol glycerol 1 hydroxystearate Macrogol 1 stearate 400 Magnesium stearate 1 Maltodextrin 1 Mannitol 1 1 Methylcellulose 1 Sodium 1 cetylstearylsulfate Sodium dioctyl- 1 sulfosuccinate (also K salts, Ca salts) Sodium dodecylsulfate 1 Sodium 1 hydrogencarbonate Oleic acidoleylester 1 Pectin 1 Poloxamer 1 Polyacrylic acid 1 1 Polyethylene glycol 1 1 1 1 1 Polyethylene oxide 1 1 1 1 Polymethacrylate 1 1 Polyoxyl 23 1 laurylether, -20 cetostearylether, -10 oleylether Polyoxyl 40 stearate, 1 -50 stearate Polysorbate 1 20, 60, 80, 40 Polyvinylacetate 1 copolymers Polyvinylalcohol 1 1 Polyvinylpyrrolidone 1 1 Riboflavin 1 Castor oil 1 Sucrose 1 1 Sorbitan 1 monooleate, -palmitate, -stearate, -trioleate, -tristearate, -laurate Sorbitol 1 1 1 Starch 1 1 Starches, rice-, maize-potato-, wheat- Stearic acid 1 1 Stearylalcohol 1 Talcum 1 1 Titanium dioxide 1 Tragacanth 1 Triacetin 1 1 Triethanol amine 1 Triethylcitrate 1 Tromethamol 1 Vaseline 1 Tartaric acid 1 Urea (Urea) 1 1 1 Ceramides 1 Hyaluronic acid 1

[0275] For example, the excipients ethyl- and methylcellulose, hydroxypropylmethylcellulose, cellulose acetate phthalate and hydroxypropylmethylcellulose phthalate exhibit retarding properties.

Example 8b Formulas

[0276] 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 [0277] 40 to 97 g of the base formula [0278] 1-10 g Bursera simaruba (C-E) [0279] 1-10 g Stachytarpheta jamaicensis (B-E) [0280] 1-40 g Aloe vera (D2) [0281] (total weight 100 g=100% by weight, each based on the dry weight)
2. Formula: Moisturizing Cream with B-E, A-E and D2 [0282] 40 to 97 g of the base formula [0283] 1-10 g Stachytarpheta jamaicensis (B-E) [0284] 1-10 g Bidens alba (A-E) [0285] 1-40 g Aloe vera (D2) [0286] (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 [0287] 30 to 96 g of the base formula [0288] 1-10 g Bursera simaruba (C-E) [0289] 1-10 g Stachytarpheta jamaicensis (B-E) [0290] 1-10 g Bidens alba (A-E) [0291] 1-40 g Aloe vera (D2) [0292] (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):

[0293] 70% by weight of an Aloe extract (D2) were homogenously mixed in 100 ml [0294] 90% ethanol

Base Tincture D2b (Total: 100% by Weight Based on the Dry Weight):

[0295] 30% by weight of an Aloe extract (D2) were homogenously mixed in 100 ml [0296] 90% ethanol
4. Immunologically Active Tinctures with A-E, B-E, C-E and D2 [0297] 100 ml base tincture D2a or D2b were respectively mixed with:

TABLE-US-00011 TABLE 6 Mixture 44 45 46 47 48 49 50 51 52 53 Plant (1:1:1) (1:1) (1:1) (1:1) (1:1:1) (1:1:1) (1:1:1) (1:1:3) (3:1) (1:3:1) extract % by weight Bid. 1 1.5 1.5 2.5 2 5 7 0.5 0 0.5/5 alba (A-E) Sta. 1 0 1.5 0 2 5 7 0.5 1.5/15 1.5/15 jamaicen. (B-E) Bur. 1 1.5 0 2.5 2 5 7 1.5 0.5/5 0.5/5 simaruba (C-E)

[0298] 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

[0299] 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.

Example 9: Application on the Skin

[0300]

TABLE-US-00012 Patient Symptom Phyto- (f/m) disease mixture Dosage Application Course 4 (f) Lymphedema B + C + 5 cups per day Tea, warm, 5 to 6 hours under (re or li), upper arm mint (flavor) fresh, strong, internal observation, partially forearm very green, after 2 to 3 hours approx. 2 cm more symptomatical in diameter improvement after two days: regression, tension reduction, no lymphedema anymore 2b (m) Dry skin, pruritus, A + B + Of approx. Tincture approx. On the same day scratch marks, C + D2 200 mg 1-4/day applied, relief. After 2 days urticarial (base tincture in 5 days 2 days .fwdarw. only still scratch eczema, left, D2a + 44) maximally still 3 day. marks (crusts) left, i.p. forearm, 50 ml used, Afterwards, but dry skin. crook of the arm. coarse partially From 5. day still gently tiny remains creamed scratch marks, but (dry skin). dry skin. 1d Oral herpes, A + B + Dropwise Tincture First, no expansion area approx. C + D2 3-4/day dropwise anymore, then fast 6 4 mm, after (base tincture 3-4/day easing of the 3-4 hours D2a + 51) applied on symptoms and of already big the oral the swelling; swelling, blister herpes desiccation within 2 days; no inflammation anymore; small crust which healed after some days through humid wound care with tincture and zinc ointment Aciclovir ointment functioned worse 1e Verrucous A + B + Dropwise Dropwise approx. Day 5: Starting papillomas at 3 C + D2 1-2/day 1-2/day, treatment because areas, 5-12 (base tincture from day 5 of inflammation mm big, D2a + 51) the affected and adhesion to dermatologically area thoroughly the plaster, removed by wetted with tincture commencing Erygenum secretion on day 5; laser; after 2 the inflammatory days all 3 pains eased inflamed; day quickly. On day 2 3: plaster of the application change and first crust spraying on of formation, little skin secretion; disinfection; sometimes light day 4: itching; day 3 of inflammation the application: no increased; secretion anymore purulent fast healing, secretion inflammation formation quickly away 18.1 4-5 A + B + Dropwise Insect bites Itching eased inflammatory C + D2 several wetted with quickly; within a insect bites, 5-8 (base tincture times/day the tincture few hours to one mm big; dark D2a + 51) several day no reddening, times a day inflammation swelling, anymore; only itching, painful; small areas without partially swelling visible; on secretion and day 2 of the scratch marks application totally healed 19 (f) 38-year old with A + B + 2-3/day as A cloth was Daily improved breast cancer, C + D2 application soaked with condition, not familially (base tincture the tincture; inflammation and predisposed; D2a + 51) cloth laid pains eased tendency to in combination onto the quickly inflamed breast with a tea with breast as After 1.5 years i.a. skin Bursera simaruba application after completion of problems, (C) with 30-60 treatment no breast min exposure recurrence of the reddened, time breast hardened, inflammations etc.; painful, breast cancer was inflammatory removed (Course not known)