EP4240375A1 - Antiseptische zusammensetzung - Google Patents
Antiseptische zusammensetzungInfo
- Publication number
- EP4240375A1 EP4240375A1 EP21814686.8A EP21814686A EP4240375A1 EP 4240375 A1 EP4240375 A1 EP 4240375A1 EP 21814686 A EP21814686 A EP 21814686A EP 4240375 A1 EP4240375 A1 EP 4240375A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- composition
- extract
- silver
- weight
- gram
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
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- PLXBWHJQWKZRKG-UHFFFAOYSA-N Resazurin Chemical compound C1=CC(=O)C=C2OC3=CC(O)=CC=C3[N+]([O-])=C21 PLXBWHJQWKZRKG-UHFFFAOYSA-N 0.000 description 1
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- CAYJBRBGZBCZKO-BHGBQCOSSA-N ethyl (e,4s)-4-[[(2r,5s)-2-[(4-fluorophenyl)methyl]-6-methyl-5-[(5-methyl-1,2-oxazole-3-carbonyl)amino]-4-oxoheptanoyl]amino]-5-[(3s)-2-oxopyrrolidin-3-yl]pent-2-enoate Chemical compound C([C@@H](/C=C/C(=O)OCC)NC(=O)[C@@H](CC(=O)[C@@H](NC(=O)C1=NOC(C)=C1)C(C)C)CC=1C=CC(F)=CC=1)[C@@H]1CCNC1=O CAYJBRBGZBCZKO-BHGBQCOSSA-N 0.000 description 1
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- IDSXLJLXYMLSJM-UHFFFAOYSA-N morpholine;propane-1-sulfonic acid Chemical compound C1COCCN1.CCCS(O)(=O)=O IDSXLJLXYMLSJM-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/24—Heavy metals; Compounds thereof
- A61K33/38—Silver; Compounds thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/02—Medicinal preparations containing materials or reaction products thereof with undetermined constitution from inanimate materials
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/56—Materials from animals other than mammals
- A61K35/63—Arthropods
- A61K35/64—Insects, e.g. bees, wasps or fleas
- A61K35/644—Beeswax; Propolis; Royal jelly; Honey
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
- A61K36/282—Artemisia, e.g. wormwood or sagebrush
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/35—Caprifoliaceae (Honeysuckle family)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/38—Clusiaceae, Hypericaceae or Guttiferae (Hypericum or Mangosteen family), e.g. common St. Johnswort
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/88—Liliopsida (monocotyledons)
- A61K36/896—Liliaceae (Lily family), e.g. daylily, plantain lily, Hyacinth or narcissus
- A61K36/8962—Allium, e.g. garden onion, leek, garlic or chives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/10—Antimycotics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
Definitions
- the invention relates to a composition for use as a medicament comprising at least silver in colloidal form, an extract of Sambucus Nigra, an extract of Primulae flos cum calycibus and/or an extract of Hypericum perforatum, a pelargonium sidoides extract.
- said composition is used in antiviral, antibacterial and antifungal prophylaxis and/or treatment. Even more preferably, said composition is in the form of an antifungal and antibacterial cream.
- the invention relates to the field of antifungal, antiviral and antibacterial treatments.
- Colloidal silver makes it possible to improve the functioning of the immune system, because it acts as a catalyst, by deactivating the specific enzymes necessary for the metabolism of numerous bacterial, fungal and viral species.
- Colloidal silver is mainly used as an antiseptic in dermatology for the treatment of certain types of lesions. It is also known for its application as a potabilizing agent for water and as a preservative in the cosmetics industry.
- colloidal silver is not effective against all fungi, viruses and bacteria.
- the invention relates to a composition for use as a medicament comprising:
- colloidal ionic silver is understood according to the invention a suspension in water of extremely fine silver particles.
- said silver in colloidal form has a particle size of between 0.1 and 30 nanometers.
- composition for use according to the invention comprises:
- Img of silver is equivalent to Ippm in a 1 liter solution, i.e. 20mg to 250mg per liter of solution.
- Sambucus Nigra is a shrub-like plant from the Caprifoliaceae family.
- the extracts from this plant can come from its flowers or its fruits, and be dry, aqueous and/or alcoholic extracts.
- composition for use according to the invention comprises:
- composition for use according to the invention comprises:
- Hypericum perforatum or perforated St. John's wort is a perennial herbaceous plant of the Clusiaceae family. Said extracts come from the plant or the flowering tops which can be ground, optionally in water and/or alcohol.
- said composition for use according to the invention comprises: - Between 0.5 and 11% by weight of the composition of an extract of Hypericum perforatum.
- Pelargonium sidoides or Umckaloabo, is a plant native to South Africa. Extracts from this plant can take the form of dry, aqueous and/or alcoholic extracts of the roots or other parts of the plant.
- composition for use according to the invention comprises:
- composition for use according to the invention comprises:
- composition for use according to the invention further comprises:
- ionic silver By ionic silver is understood according to the invention silver whose particles have been dissolved in water, on the contrary colloidal silver where said particles are suspended in water.
- composition for use according to this latter embodiment comprises:
- composition Between 20 mg and 250 mg of total silver per liter of composition, divided into i. Between 70 and 99.99% by total weight of silver, silver in colloidal form; ii. Between 0.01 and 30% by total weight of silver, silver in ionic form.
- total silver is understood according to the invention all of the silver included in the composition according to the invention, whatever its form.
- said composition for use comprises:
- said composition for use according to the invention further comprises: - An extract of Echinacea, and/or
- Echinacea designate a genus of plants of the Asteraceae family.
- Said extracts take the form of dry, aqueous and/or alcoholic extracts, produced from the root, the whole plant or the aerial parts.
- composition for use according to the invention comprises:
- Allium sativum a plant more commonly known as “garlic”, is a species of perennial plant. Extracts can take the form of a dry, aqueous and/or alcoholic extract, or an aged garlic extract.
- composition for use according to the invention comprises:
- Propolis is a product derived from plant resins and exudates, which can be secreted by plants or produced by bees from plant resin and wax. As propolis depends on the species of plants from which it is produced, standardized propolis is produced, often defined by their color. Preferably, yellow, green or red propolis will be used here. Said propolis can be presented in the form of a dry, purified, aqueous and/or alcoholic extract.
- composition for use according to the invention comprises:
- Artemisia or Mugwort is a genus of plant species comprising herbaceous plants, shrubs and shrubs, with pinnate leaves.
- the species used according to the invention is Artemisia annua.
- the latter is an aromatic branched herbaceous plant. Extracts from this plant can come from the whole plant or from the aerial parts, and be dry, aqueous and/or alcoholic extracts.
- composition for use according to the invention comprises:
- composition for use according to the invention comprises:
- said composition for use according to the invention comprises at least one other dermatologically and/or pharmaceutically acceptable agent.
- said composition for use according to the invention is intended to be used in prophylaxis and/or antiviral, antibacterial and antifungal treatment.
- said composition for use according to the invention is intended to be used in prophylaxis and/or antiviral treatment.
- said composition for use according to the invention is intended to be used in prophylaxis and/or antibacterial treatment.
- said composition for use according to the invention is intended to be used in prophylaxis and/or antifungal treatment.
- said composition for use according to the invention is intended to be used in prophylaxis and/or antibacterial and antifungal treatment.
- said composition for use according to the invention can be administered orally, intravenously, subcutaneously, nasally, ophthalmically, auricularly, rectally, vaginally, via the respiratory route, or by application to the skin.
- the oral route can take the form of a tablet, a syrup, a drinkable solution or suspension, a capsule,
- the intravenous route can take the form of a solution.
- the subcutaneous route can take the form of a solution.
- the route of application to the skin can take the form of an ointment, a cream, a patch or a gel.
- the administration is carried out by the respiratory or nasal route.
- said composition for use according to the invention is administered by the respiratory or nasal route, in the form of nasal drops, nasal spray, nasal powder, aerosol, for example aerosols and/or nasal spray compressed gas, or a nebulizer.
- the pharmaceutical composition when suitable for nasal or respiratory administration, it can advantageously be for broncho-pulmonary or sinus purposes.
- said composition for use according to the invention is administered by aerosol in the nasal passages.
- composition according to the present invention can be administered to a patient daily, once a day, twice a day, three times a day or even more times a day.
- the composition of the present invention is administered to a patient twice a day and every 12 hours.
- said composition comprises, by weight of the composition:
- said composition comprises, by weight of the composition:
- composition for use according to the invention takes the form of a cream for topical application.
- said topical application cream is an antifungal cream.
- said topical application cream is an antibacterial cream.
- said topical application cream is an antifungal and antibacterial cream.
- composition for use according to the present invention in the form of an antifungal and/or antibacterial cream for topical application, can be applied topically to a patient daily, once a day, twice a day, three times a day or even more times a day.
- the composition of the present invention is administered to a patient twice a day and every 12 hours.
- said composition is used as an antibiotic.
- said composition is used as an antibiotic to treat antibiotic-resistant patients.
- Antibiotic resistance is a phenomenon which consists, for a pathogenic organism, in becoming resistant to antibiotics. These organisms evolve and develop defense mechanisms that allow them to prevent antibiotic treatments from working. These treatments become ineffective.
- said composition for use as an antibiotic can be administered orally, intravenously, subcutaneously, nasally, ophthalmically, auricularly, rectally, vaginally. , through the respiratory tract, or by application to the skin.
- the oral route can take the form of a tablet, a syrup, a drinkable solution or suspension, a capsule,
- the intravenous route can take the form of a solution.
- the subcutaneous route can take the form of a solution.
- the route of application to the skin can take the form of an ointment, a cream, a patch or a gel.
- the invention relates to a composition
- a composition comprising:
- composition according to the invention comprises:
- composition according to the invention comprises:
- composition according to the invention comprises:
- composition according to the invention comprises:
- composition according to the invention comprises:
- said composition comprises:
- composition according to the invention further comprises:
- composition according to this latter embodiment comprises:
- said composition comprises:
- composition according to the invention further comprises:
- composition according to the invention comprises:
- composition according to the invention comprises:
- composition according to the invention comprises:
- composition according to the invention comprises:
- composition according to the invention comprises:
- said composition comprises, by weight of the composition:
- said composition comprises, by weight of the composition:
- the invention relates to the use of the composition according to the second aspect of the invention for disinfecting a surface.
- disinfecting a surface means the application to inert surfaces of the composition according to the invention to obtain an anti-fungal and/or antiviral and/or antibacterial effect on the surface on which it is used.
- the surfaces on which this composition is used can generally be found in all places where it may be necessary to clean and disinfect a surface or objects or devices, for example in the food industry, stores, restaurants, a medical environment or even in private homes.
- said surface is a surface present in a medical environment, for example in a doctor's office, a hospital, an analysis laboratory, a veterinary office.
- surface present in a medical environment is meant in particular the floors, the walls, the benches and the medical instruments.
- Example 1 Example of composition:
- composition comprises, for 100 mL of composition:
- Example 2 Antibacterial activity of the composition of Example 1.
- the antibacterial activity is studied by monitoring in vitro the densities of cultivable Staphylococcus aureus after seeding the strains tested and bringing them into contact with the composition of example 1.
- the densities expressed in CFU/ml are measured for periods of 1, 3 and 24 hours, and make it possible to characterize a bactericidal effect.
- composition exhibits a bactericidal effect.
- Example 3 Anti-viral effect of the composition of example 1.
- the composition of example 1 is tested according to the test for evaluating potential activity against HRV-A16 (a rhinovirus responsible for colds) on a culture of fully differentiated human airway epithelial cells.
- the compositions are first applied before infection for one hour and replication is carried out for 4 hours. 3 rinsing steps with the formulations are carried out. The rest is collected and the RNA is assayed after cell lysis. The same collection step is performed 24 hours later and the same assay step is performed. The percentage expresses the changes in viral RNA, resulting in percentage inhibition of HRV-A16 replication, leading to anti-viral efficacy.
- the composition according to example 1 is tested against rupintrivir (a known antiviral compound, providing an almost complete response). The results are superior for the composition according to Example 1.
- Example 4 Antifungal activity of the composition according to Example 1:
- a strain of Scytalidium dimidiatum is obtained by separation and identification of the stratum corneum of patients suffering from superficial mycosis.
- Fungi pre-cultivated on SDA medium are suspended in a sterile saline solution containing 0.1% (w/v) Tween 80 and the suspension is filtered through cheesecloth to collect the arthrospores.
- This is suspended in a sterile saline solution containing 0.1% (w/v) Tween 80, then added to a medium containing 20% Alamar blue at 2 x 10 4 cells/ml to give fungal inoculum solution.
- Example 1 The composition according to Example 1 was used, as well as luliconazole.
- the minimum inhibitory concentration is measured by a broth microdilution method. That is, RPMI1640 medium (Sigma-Aldrich) was buffered with 0.165 M morpholine propanesulfonic acid (MOPS, Wako Pure Chemical Industries, Ltd.) at pH 7.0, and a given amount of A solution of the composition according to Example 1 or luliconazole is added to prepare two-fold dilution series in the range of 0.00098-4 pg/mL. 100 ⁇ l are added to a 96-well microplate, then a solution of fungal inoculum (100 ⁇ l, final concentration of fungi to be added: 1.0 ⁇ 10 4 cells/ml) and the mixture is cultured at 35°C.
- An Alamar blue reagent is previously added to the culture medium (final concentration of the addition: 10%) and, at the moment when the Alamar blue reagent in the growth control group without drug changes from blue to red , the culture is interrupted and the absorbance (differential optical density at 570 nm on the basis of 590 nm served as a reference) measured.
- the minimum concentration of the composition according to Example 1 or of luliconazole which inhibited the growth of the fungus in the growth control group by 80% or more is taken as the MIC.
- the concentration to be the MIC (MIC90) in 9 strains out of the 10 strains measured (90%) is determined.
- the MIC of the composition according to example 1 is higher than luliconazole, demonstrating a specifically strong antifungal activity.
- Example 5 In vivo antiviral, antibacterial, antifungal efficacy test
- the treatment consisted of spraying the composition of example 1 and PLACEBO twice a day. The treatment lasted two weeks. Efficacy of treatment was assessed using a total symptom score, linked to swab samples. The scores, evaluated at the beginning and at the end of the treatment with the Student's t-test, were used to assign an efficacy score with the same statistical method.
- Example 6 Protocol for studies of the in vitro antimicrobial activity for the combination of extracts of pelargonium sidoides, Sambucus nigra and Hypericum perforatum with silver in colloidal form:
- Plant extracts The antimicrobial effect of extracts of African geranium (Pelargonium sidoides DC.), St. John's wort (Hypericum perforatum L.) and black elderberry (Sambucus nigra L.) in colloidal silver (AgNPs) at a concentration of 30 ppm was tested.
- Control The broad-spectrum antibiotic thiamphenicol (Nikovet - Sofia) was used as a positive control, to which the microorganisms tested did not show resistance.
- Microorganisms Pure cultures of 7 pathogenic strains were tested: Esherichia coli ATCC - 8739 (NBIMCC 3397), Salmonella enterica subsp. enterica ATCC 1304 (NBIMCC 8691), Staphylococcus aureus subsp. aureus ATCC - 6538 (NBIMCC 3359), Clostridium perfringens ATCC 13124 (NBIMCC 8615) and Candida albicans ATCC 10231 (NBIMCC 74).
- the other two (Pseudominas aeruginosa and Streptococcus pyogenes) were isolated from inflammatory skin secretions of dogs in the microbiology laboratory of the University Clinic of the Faculty of Veterinary Medicine of the University of Forestry in Sofia.
- the culture of the microorganisms was carried out at 35-37° C. for 18-24 and 72 hours in an anaerobic medium for C. perfringens and in aerobic conditions for the other microbial species.
- the Anaerob Pack system with palladium catalyst - H2 + CO2 (BUL BIO NCIPD - Sofia) in a jar was used to create anaerobic conditions.
- the Indic Strip indicator (BUL BIO NCIPD - Sofia) was used to prove the achievement of anaerobiosis.
- the combination of plant extracts contains 2 mg of P. sidoides, 2 mg of S. nigra, 0.4 mg of H. perforatum and 24 ppm of AgNPs in 0.1 ml, and thiamphenicol - 30 pg in 0.1ml (as needed). After a 3-4 hour incubation at room temperature for diffusion, the cultures were incubated at 35-37°C for 18-24 and 72 hours. Results were read by measuring the diameters of the inhibitory zones in millimeters, including the diameter of the well to the nearest 1 mm, with a transparent ruler outside the bottom of the plates.
- the minimum inhibitory concentrations were determined by the method of double serial dilutions in Zeissler agar for C. perfringens and Mueller-Hinton agar for the other microorganisms, described by Ericsson and Sherris (1971 ) and NCCLS (1999). Bacterial suspensions were applied at a dose of 10 6 cells/ml. The combination of plant extracts tested, composed of P. sidoides, S. nigra and H. perforatum, as well as the control antibiotic were administered at different doubling increasing final concentrations per ml of agar. After incubation at 35-37°C for 18-24 hours, the number of colonies developed was determined.
- the MIC50s were calculated mathematically based on the number of colonies inhibited on the agar with the respective dilution of the test compound compared to the colonies on the media with controls without plant extracts or antibiotics.
- the growth inhibition range (D) was defined as the concentration with no visible growth.
- the Gram-negative bacteria studied showed greater sensitivity compared to Gram-positive microorganisms (P>0.05, Student's t criterion). The lowest sensitivity by this method was reported in C. perfringens and S. aureus, and the highest - in E. coli and P. aeruginosa. All microorganisms tested showed high sensitivity to thiamphenicol used as a positive control, even the strain of C. albicans tested. However, the differences in the diameter of the inhibitory zones of the microorganisms studied between the antibiotic and the combination tested with colloidal silver were not statistically significant (P > 0.05, Student's t criterion).
- the microorganisms tested were suppressed by very low concentrations of the plant extracts applied in combination.
- the MIC50s of P. sidoides, S. nigra and H. perforatum were 5.0 + 0.0, 5.0 + 0.0 and 50.0 + 0.0 respectively for Gram-negative bacteria.
- the MIC50 of P. sidoides, S. nigra and H. perforatum were 10.0 + 0.0, 10.0 + 0.0 and 100.0 + 0 respectively, 0.
- Example 7 Additional study protocol of the in vitro antimicrobial activity for the combination of extracts of pelargonium sidoides, Sambucus nigra and Hypericum perforatum with silver in colloidal form:
- John's wort in colloidal nano-silver and aqueous extracts against Gram-negative and Gram-positive microorganisms, one of the most common causes common hard-to-treat infections in humans and animals.
- Control The broad-spectrum antibiotic thiamphenicol (Nikovet - Sofia) was used as a positive control, to which the microorganisms tested did not show resistance.
- NBIMCC Bulgarian National Bank of Industrial Microorganisms and Cell Cultures
- the other two (Pseudominas aeruginosa and Streptococcus pyogenes) were isolated from inflammatory skin secretions of dogs in the microbiology laboratory of the university clinic of the University of Forestry, Faculty of Veterinary Medicine in Sofia.
- the culture of the microorganisms was carried out at 35-37° C. for 18-24 and 72 hours in an anaerobic environment for C. perfringens and under aerobic conditions for the other microbial species.
- the Anaerob Pack system with palladium catalyst - H2 + CO2 (BUL BIO NCIPD - Sofia) in pot was used to create anaerobic conditions.
- the Strip indicator (BUL BIO NCIPD - Sofia) was used to prove the achievement of anaerobiosis.
- Plant extracts in colloidal nanosilver and in water, as well as the control antibiotic were applied by 0.1 ml in 9 mm wells in the agar.
- Plant extracts alone and in combination contained 2 mg of P. sidoides, 2 mg of S. nigra, 0.4 mg of H. perforatum and 24 ppm of AgNPs or water in 0.1 ml, respectively, and thiamphenicol - 30 ⁇ g in 0.1 ml (as needed). After a 3-4 hour incubation at room temperature for diffusion, the cultures were incubated at 35-37°C for 18-24 and 72 hours.
- Results were read by measuring the diameters of the inhibitory zones in millimeters, including the diameter of the well to the nearest 1 mm, with a transparent ruler on the outside of the bottom of the plates.
- an inhibitory effect of plant extracts with or without AgNPs was observed in areas > 12 mm, and of thiamphenicol - at > 17 mm.
- the susceptibility of the microorganisms tested was determined as for non-antibiotic preparations such as sulfonamides, namely: resistant (R) - in areas with a diameter of ⁇ 12 mm, moderately sensitive - intermediate (I) - in areas between 13 and 16 mm and sensitive (S) to > 17 mm.
- the corresponding limits are as follows: R ⁇ 12 mm, I - 13 - 17 mm and S - > 18 mm (NCCLS, 1997, 1999).
- the minimum inhibitory concentrations were determined by the method of double serial dilutions in Zeissler agar for C. perfringens and Mueller-Hinton agar for the other microorganisms, described by Ericsson and Sherris ( 1971) and NCCLS (1999).
- the bacterial suspensions were applied at a dose of 10 6 cells/ml.
- the tested plant extracts of P. sidoides, S. nigra and H. perforatum, alone or in combination, in water or with AgNPs, as well as the control antibiotic were administered in different doubling increasing final concentrations per ml of agar. After incubation at 35-37°C for 18-24 hours, the number of colonies developed was determined.
- the MIC50s were calculated mathematically based on the number of colonies inhibited on the agar with the respective dilution of the test compound compared to the colonies on the media with the controls without plant extracts or antibiotic.
- the growth inhibition range (D) was defined as the concentration with no visible growth.
- the results obtained during the determination of the minimum inhibitory concentrations are presented in table 7. They correspond to those of the agar-gel diffusion method.
- the studied extracts of the three plants showed significant antimicrobial activity.
- the effect of P. sidoides and S. nigra was similar.
- Their MIC50 for Gram-negative bacteria was low - 10 mg/ml, and for Gram-positive microorganisms - 20 mg/ml.
- the growth of the microbial strains studied was inhibited by higher concentrations of H. perforatum - 50 mg/ml for Gram-negative strains and 100 mg/ml for Gram-positive strains.
- the combination of plant extracts also inactivated the Gram-positive microorganisms studied in a suspension with a density of 106 cells/ml, but for a longer period - C. perfringens for 30 to 60 min, S. pyogenes for at least 1 hour, C. albicans - for at least two hours, and S. aureus - for more than two hours.
- C. perfringens for 30 to 60 min
- S. pyogenes for at least 1 hour
- C. albicans - for at least two hours
- S. aureus - for more than two hours.
- the reduction of the tested microorganisms of all groups occurred more slowly than when the plant combination contained colloidal nanosilver.
- Table 11 shows the results of the suspension method tests of the extract of black elderberry (S. nigra) & H. perforatum and the extract of H. perforatum & P. sidoides. All Gram-negative bacteria tested in a suspension with a density of 106 cells/ml died within 15-30 minutes, but much faster when the extract contained nanosilver. These extracts also inactivated the Gram-positive microorganisms studied, but for a longer period - more than two hours. In the case of the silver-containing extracts, the reduction of the tested microorganisms of all groups occurred faster compared to the aqueous extract.
- the combinations studied had a synergistic effect against Gram-negative and Gram-positive microorganisms.
- the presence of colloidal nano-silver in the extracts and in the combination between them increased their antimicrobial activity.
- Example 8 Protocol for additional studies of the in vitro antimicrobial activity of extracts of African geranium, black elderberry and St. John's wort in nano colloidal silver: [0190]
- the objective of the present work was to carry out studies to evaluate the in vitro antimicrobial activity of extracts from the berries of Pelargonium sidoides, Hypericum perforatum and Sambucus nigra, alone and in combination with each other, in the form of colloidal nano-silver and in the form of aqueous extracts, against Gram-negative and Gram-positive microorganisms, which are among the most common causes of difficult-to-treat infections in humans and animals.
- Control The broad-spectrum antibiotic thiamphenicol (Nikovet - Sofia) was used as a positive control, to which the microorganisms tested did not show resistance.
- Microorganisms Pure cultures of 7 pathogenic strains were tested. Five of them are references from the Bulgarian National Bank of Industrial Microorganisms and Cell Cultures (NBIMCC): Esherichia coli ATCC - 8739 (NBIMCC 3397), Salmonella enterica subsp. enterica ATCC 1304 (NBIMCC 8691), Staphylococcus aureus subsp.
- NBIMCC 3359 Clostridium perfringens ATCC 13124 (NBIMCC 8615) and Candida albicans ATCC 10231 (NBIMCC 74).
- the other two Pseudominas aeruginosa and Streptococcus pyogenes were isolated from inflammatory skin secretions of dogs in the microbiology laboratory of the university clinic of the University of Forestry, Faculty of Veterinary Medicine in Sofia.
- the culture of the microorganisms was carried out at 35-37° C. for 18-24 and 72 hours in an anaerobic environment for C. perfringens and under aerobic conditions for the other microbial species.
- the Anaerob Pack system with palladium catalyst - H2 + CO2 (BUL BIO NCIPD - Sofia) in pot was used to create anaerobic conditions.
- the Strip indicator (BUL BIO NCIPD - Sofia) was used to prove the achievement of anaerobiosis.
- Plant extracts in colloidal nanosilver and in water, as well as the control antibiotic were applied by 0.1 ml in 9 mm wells in the agar.
- Plant extracts alone and in combination contained 2 mg of P. sidoides, 2 mg of S. nigra, 0.4 mg of H. perforatum and 24 ppm of AgNPs or water in 0.1 ml, respectively, and thiamphenicol - 30 ⁇ g in 0.1 ml (as needed). After a 3-4 hour incubation at room temperature for diffusion, the cultures were incubated at 35-37°C for 18-24 and 72 hours.
- Results were read by measuring the diameters of the inhibitory zones in millimeters, including the diameter of the well to the nearest 1 mm, with a transparent ruler on the outside of the bottom of the plates.
- an inhibitory effect of plant extracts with or without AgNPs was observed in areas > 12 mm, and of thiamphenicol - at > 17 mm.
- the susceptibility of the microorganisms tested was determined as for non-antibiotic preparations such as sulfonamides, i.e.: resistance aunt (R) - in areas with a diameter of ⁇ 12 mm, moderately susceptible - intermediate (I) - in areas between 13 and 16 mm and susceptible (S) at > 17 mm.
- R resistance aunt
- I moderately susceptible - intermediate
- S susceptible
- the minimum inhibitory concentrations were determined by the method of double serial dilutions in Zeissler agar for C. perfringens and Mueller-Hinton agar for the other microorganisms, described by Ericsson and Sherris ( 1971) and NCCLS (1999).
- the bacterial suspensions were applied at a dose of 10 6 cells/ml.
- the tested plant extracts of P. sidoides, S. nigra and H. perforatum, alone or in combination, in water or with AgNPs, as well as the control antibiotic were administered in different doubling increasing final concentrations per ml of agar. After incubation at 35-37°C for 18-24 hours, the number of colonies developed was determined.
- the MIC50s were calculated mathematically based on the number of colonies inhibited on the agar with the respective dilution of the test compound compared to the colonies on the media with the controls without plant extracts or antibiotic.
- the growth inhibition range (D) was defined as the concentration with no visible growth.
- the results obtained during the determination of the minimum inhibitory concentrations (MIC) are presented in table 15. They correspond to those of the agar-gel diffusion method.
- the studied extracts of the three herbs showed significant antimicrobial activity.
- the effect of P. sidoides and S. nigra was similar.
- Their MIC5o for Gram-negative bacteria was low - 10 mg/ml, and for Gram-positive microorganisms - 20mg/ml.
- the growth of the microbial strains studied was inhibited by higher concentrations of H. perforatum - 50 mg/ml for Gram-negative strains and 100 mg/ml for Gram-positive strains.
- Tables 20 and 21 show the results of the tests in the suspension method of the extract of black elderberries (S. nigra) All the Gram-negative bacteria tested in a suspension with a density of 10 6 cells/ ml are dead in 30 min, but much faster when the extract contained nanosilver. S. nigra berry extract also inactivated the Gram-positive microorganisms studied in a suspension with a density of 10 6 cells/ml, but for longer - C. perfringens for 2 hours, and the other bacterial species and the oval fungus C. albicans for more than two hours (Table 20). In the case of S. nigra berry extract with silver content, the reduction of the tested microorganisms of all groups occurred faster compared to the aqueous extract.
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