Patent Application
20240041054
The present invention relates to the realization of a natural extract with high antimicrobial activity. This extract, which can be diluted in water at different concentrations, constitutes an antimicrobial solution that can be used in various fields: in environmental disinfection, both in humans and animals, as a base for antibacterial/antifungal preparations for topical, cutaneous or mucosal use.
The extract, object of the present invention can be used in the preparation of disinfectant solutions that can be used for: handpieces and surfaces in the outpatient-hospital and domestic field, air conditioner filters, environments used for preparing food, as an ingredient in mouthwashes, toothpastes and skin creams. In general in all those areas where it is necessary to adopt antimicrobial prophylaxis.
Antibiotic resistance is currently a global problem involving virtually the entire world population. In addition to the causes related to abuse and incorrect methods of intake/administration and the appearance of multi-resistant microbial strains (Multidrug Resistant MDR strains), there is also the lower investment by the pharmaceutical industries in the study of new active molecules [1-4]. In this regard, the World Health Organization (WHO) published in 2017 an alarm on the presence, in the health sector, of super-bacteria now refractory to current treatments, among them high priority, some nosocomial pathogens are mentioned, among which: Pseudomonas aeruginosa and several species belonging to the Enterobacteriaceae family. There is therefore a need for the development of new antimicrobials [5].
A similar problem is detected in the use of disinfectants in which the use of synthetic substances, often highly toxic, also raises the problem of environmental contamination.
The alarm on antimicrobial controls involves, in parallel, apparently different but cohesive fields in the same problem; we think, for example, of the veterinary sector as well as the agricultural sector where some newly acquired bacterial infections are bringing entire production chains to their knees, such as the spread of Xylella fastidiosa [6] in the olive growing sector or infections in sheep due to mastitis from P. aeruginosa [7].
There are estimated to be 250,000 to 500,000 plant species on Earth. A relatively small percentage, between (1 and 10%) of these, are used as food by humans and/or other animal species. It is possible that this percentage will increase further due to increased use for their medicinal properties.
Although natural remedies for microbes and infections have been in use for thousands of years, the use of plant extracts, in whole or in parts of them, has never involved the development of antimicrobials. The reasons are different and often depend on the difficulty of using a single species, on the variability of the extract and consequently on the biological activity of the vegetable preparation obtained from it and-an important aspect-the processing system used to obtain the formulation [8].
Currently, plant extracts of very different origins are used both for the phylogenetic belonging of the plant and for the different type of plant organs subjected to extraction eg. leaves, fruits, seeds etc. In recent years, however, the use and research of drugs derived from plants has been progressively increasing; in particular to obtain derivatives that could be used against infectious diseases. Currently (25-30)% of pharmaceutical products in use derive from plants, but few have entered the market against infections, as disinfectants or as drugs for human use, despite the fact that plants are rich in a great variety of secondary metabolites (substances belonging to the classes of tannins, terpenoids, alkaloids and flavonoids, which show in vitro discrete antimicrobial properties) [9].
Various techniques are used to extract active compounds, starting from the plant product, which play a critical role in determining the results of biological assays. In fact, the literature reports a vast range of different technologies in terms of extract composition, yield, environmental impact, costs, feasibility of scale up to the development of an industrial process [10, 11].
The main extraction methods known to date can be listed as follows: hydrodistillation, steam distillation, maceration, infusion, percolation, decoction, extraction with organic solvents, microwave assisted extraction (MAE), accelerated extraction with solvents (Accelerated solvent extraction, ASE), ultrasound-assisted extraction (UAE), extraction using supercritical fluids (Supercritical Fluid Extraction, SFE) [12].
In general, the shortcomings in the isolation of antimicrobial biocompounds from plant matrices can be highlighted in the pre-extraction, extraction and post-extraction (analytical) phases as follows:
The technical problem posed by the known art is the shortage of antibiotics effective against microorganisms that have developed antibiotic-resistance, in particular with regard to bacterial and fungal strains resistant and multi-resistant (MDR) to different antibiotics/antifungals/disinfectants, such as for example: S. aureus methicillin-resistant, P. aeruginosa ATCC 15442, Klebsiella pneumoniae, fungal strains of Candida ssp multidrug-resistant.
With reference to Rosa canina, extraction and analytical methods are known as follows.
In Szentmihályi K. et al. 2002 [16]-work whose purpose was to obtain the isolation of the oily fraction by means of different extraction techniques-the results relating to the analysis of the fatty acids and dyes contained in the oil obtained by CO2-SFE at 250 bar, 35° C. for the duration of 80 minare reported. The authors do not specify whether the desired oily extract was obtained together with an aqueous solution. The obtained extracts have not been tested as antimicrobials, nor are any uses of extracts as antimicrobials indicated.
In the work of Taneva S. et al. 2017 [17]-which set out to ascertain the optimal conditions for extracting seed oil R. Canina-the quantities of tocopherol and carotenoids and the percentages of fatty acids determined on extracts obtained by extraction with hexane or with CO2 Supercriticalat (350 and 450) bar. By means of CO2-SFE the authors obtain an oily fraction and an aqueous fraction which, however, they choose to throw away without subjecting it to chemical and biological investigation. Also in this case, no uses of the extracts obtained are indicated, nor are uses of extracts as antimicrobials indicated.
The choice not to consider the aqueous fraction is well explained in a paper by Rebolleda et al. [18] where its removal is recommended and its use is not recommended as the presence of water in a lipid extract favors the rancidity process and the oxidation of fatty acids. It follows that whenever extraction with CO2 in the supercritical phase is used to obtain a lipid fraction from oil seeds, the authors focus on optimizing the operating parameters in order to maximize yields and quality of the recovered oils.
These considerations discouraged the use of aqueous phases deriving from supercritical extractions with CO2.
The technical problem posed by the known art therefore remained unsolved, i.e. the shortage of antibiotics effective against microorganisms that have developed antibiotic-resistance, in particular with regard to bacterial and fungal strains resistant and multi-resistant (MDR) to different antibiotics/antifungals/disinfectants, such as: S. aureus methicillin-resistant, P. aeruginosa ATCC 15442 used as a standard of resistance to disinfectants, a nosocomial isolate of Klebsiella pneumoniae, a clinical isolate of Candida albicans multidrug-resi stantto azoles.
The inventors, investigating the aqueous fractions, have now unexpectedly and experimentally found a marked antimicrobial activity of the aqueous phase of the extracts from supercritical fluids which has led them to enhance a by-product of the extract that was considered undesirable by the scientific community, for which they were chosen. operating conditions such as to minimize its production and which in any case was discarded as unwanted.
In the works of Szentmihályi K. et al. 2002 and Taneva S. et al. 2017 [17], previously cited, the same technique used by the inventors on the same type of matrix (seeds Rosa canina) is used, but adopting in the extraction and separation section of the plant operating conditions different from those of the present invention.
The inventors have now unexpectedly and experimentally found that, by suitably modulating the temperature but above all the pressure-which has a much wider range of variability-it is possible to regulate the solvent power of the extracting fluid, both in the extraction section and in the release section of the extracts, allowing to obtain an aqueous extract particularly active for antimicrobial activity and thus solving the problems of the known art.
The aim of the present invention is therefore to develop a R. Canina seed extract obtained by extraction with CO2 in the supercritical state.
The basic stages of the extraction, performed on a starting plant matrix consisting of seeds of Rosa canina, are the following:
Then store in the refrigerator (typically at +4° C.) waiting to be analyzed. At an industrial level, on a pilot scale and in artisanal production, separation can be achieved by decantation associated with cooling or, more efficiently and quickly, by centrifugation.
The extract consists of an aqueous solution. The water content, determined by TGA and gravimetric analysis, is between 90% and 99.9%. The dry fraction, which is in complement to 100, comprises organic compounds. The main organic components of the extract, based on GC-MS analysis with HP5-MS apolar column, are the following: methyl linoleate (31.4%), methyl oleate (25.1%), methyl palmitate (7.5%), ethyl linoleate (4.7%) and ethyl linolenate (3.3%) (Table 1). The content of these compounds is therefore: methyl linoleate: 0.031-3.1%, methyl oleate: 0.025-2.5%, methyl palmitate: 0.0075-0.75%, ethyl linoleate: 0.0033-0.33%.
Other compounds, identified by ESI-MS analysis, are:
The extract and the process are identified in the attached claims and their use is in both the cosmetic and pharmaceutical fields.
Another object of the invention are the compositions comprising effective quantities of the R. Canina extract as a cosmetic and pharmaceutical active ingredient.
Further objects will become apparent from the detailed description of the invention.
The present invention relates to obtaining an extract from seeds of Rosa canina means of carbon dioxide in the supercritical state and its use as an antimicrobial.
An antimicrobial is a natural or synthetic substance that can kill or inhibit the growth of bacteria, fungi, viruses and parasites. Therefore, the extract according to the present invention can be defined as antibacterial, antifungal, antiviral and disinfectant. The latter terms mean, respectively: natural or synthetic substance having the ability to kill bacteria or to inhibit their growth; natural or synthetic substance capable of killing fungi or capable of inhibiting their growth; natural or synthetic substance having the ability to kill viruses or inhibit their growth; natural or synthetic substance capable of drastically reducing the presence of bacteria, fungi, viruses and protozoa from surfaces and environments.
More specifically, the invention relates to the use of the extract from seeds of Rosa canina for antibacterial/antifungal preparations as defined above for cutaneous or mucosal use eg. in the preparation of creams or mouthwashes, as well as environmental disinfectant, both in the human, animal and vegetable fields.
In the experimental works reported below, standardized international methodologies were used as basic analytical methods for the assay of antibiotics, such as the EUCAST protocols (The European Committee on Antimicrobial Susceptibility Testing-EUCAST) [19, 20].
The results of the in vitro experimentation showed a remarkable antimicrobial activity of the extract against Gram positive bacteria (Staphylococcus aureus MRSA), pathogenic streptococci and Gram negative bacteria (Escherichia coli), as well as some clinical isolates of nosocomial origin such as Pseudomonas aeruginosa and Klebsiella pneumoniae. A high antifungal activity against Candida albicans azole-resistant was also observed. The preparation was found to be bactericidal and antibiofilm (antimicrobial or antimicrobial substance capable of inhibiting biofilm structuring) at low concentrations, in the order of μg/mL, towards all tested strains.
The invention consists in obtaining an extract from seeds of rosehip by means of carbon dioxide in the supercritical state, and in the subsequent use as an antimicrobial. In fact, the preparation obtained has a very high microbicidal activity, using the standard method of the European committee for antimicrobial susceptibility tests (EUCAST-ISO 20776-2) [19-20], against the following multi-resistant pathogens to antibiotics (MDR): Staphylococcus aureus Methicillin-resistant(MSRA); Streptococcus pyogenes; Streptococcus mutans, Escherichia coli; Pseudomonas aeruginosa (mucoid strain, nosocomial clinical isolate); Klebsiella pneumonia (nosocomial isolate); Candida albicans, Candida krusei and Candida glabrata (azole-resistant clinical isolates). Furthermore, considering the presence of bioactive lipids in the extract, in particular the esters of saturated or polyunsaturated fatty acids, an antiviral activity of the preparation against various viruses, including Sars-CoV-2 is desirable [21-23].
The extraction technique that uses high pressure carbon dioxide (SFE) is still little used in industrial applications due to the greater complexity of the instrumentation and greater difficulty of management than the existing one, despite it offers numerous advantages, such as better product quality (free of contaminating solvent residues, thermal degradation products and compounds that are generated as a result of the chemical-physical treatment, called artifacts) and guarantees a high degree of eco-sustainability. Carbon dioxide is an easily available, low-cost, non-toxic substance that spontaneously separates-moving away in the form of gas-from the extract and exhausted matrix in the final phase of the extraction process. Although considered one of the responsible for the greenhouse effect, in the extraction processes it is not produced but that coming from natural reserves or recovered as a by-product of other production cycles is used; in industrial plants, moreover, it is usually recycled.
Other extraction techniques, tested by us on the same matrix, have provided formulations having antiproliferative activity far lower than those of the extract in question.
The starting plant matrix consists of seeds of Rosa canina, a plant of the Rosaceae family that is easily available and cultivable in the case of massive productions. The seeds are present in the rose hips of the plant and undergo the extraction procedure which includes the following basic stages:
The extract according to the invention consists only of the aqueous fraction, obtained together with the oily fraction from the seeds of the rose hips of the specie Rosa canina. The extraction is carried out using pure CO2, without the use of organic solvents acting as co-solvents or entrainer.
To the best of the inventors' knowledge, no information is reported in the literature regarding the composition and antimicrobial activity of similar aqueous extracts obtained from the seeds of Rosa canina.
The efficacy of the extract has been tested against bacterial and fungal strains resistant and multidrug-resistant (MDR) to different antibiotics/antifungals/disinfectants, such as: S. aureus ATCC BAA-811 methicillin-resistant, P. aeruginosa ATCC 15442 used as disinfectant resistance standard, a nosocomial isolate of Klebsiella pneumoniae NC5, a clinical isolate of Candida albicans BF1 multidrug-resistant to azoles.
In fact, it should be noted that the extract showed a relevant antimicrobial activity, according to the EUCAST methods and with respect to different pathogenic microorganisms, including some prone to develop antibiotic resistance: Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus mutans, Staphylococcus aureus and Candida albicans.
The resistance profiles of the microbial strains mentioned above, evaluated by the inventors and available in the literature, are reported in Tables 4 and 5 [24-26].
The extract according to the invention can be formulated in liquid form, as a cream or lotion or as a gel or spray for topical applications on animals and humans, including children and the elderly, as well as on vegetables. Topical applications include applications on the skin and mucous membranes. The carriers can be all those used in the pharmaceutical and cosmetic fields. The application can also be done on plants with the carriers typically used for applications on plants. The extract can also be formulated in liquid, semi-liquid or gel form to be applied on the surfaces to be treated. The application can also be a spray. The extract can also be formulated in semi-liquid, creamy, semi-solid or solid formulations such as creams, suspensions, milks or soaps.
Adjuvants and carriers are cosmetically and pharmaceutically acceptable, as well as adjuvants and carriers used in the phytopharmaceutical field. The carriers include single and multi-lamellar liposomes.
The use of the SFE technique-a technique considered green-for the extraction of seeds of rosehip allows the obtaining of bioactive preparations free from contaminants.
The extract thus obtained can be used as a functional antimicrobial base in preparations for human, animal and vegetable use, as indicated below:
The extract can be used as a functional antimicrobial base in the veterinary field, in gelled and liquid preparations (to be used as such or nebulized) for:
The extract can be used as a functional antimicrobial base in the agricultural field:
The following examples are provided for the sole purpose of illustrating the invention and are not to be considered in any way limitative of its scope.
The whole and dried fruits—named rose hips of Rosa canina L., belonging to the Rosaceae family, were purchased by the Minardi company (Bagnacavallo-RA-Italy) Lot N°MP0480, from Albania, collected from the wild in 2018.
From receipt, the matrix was stored in a cool and dry place at a temperature not exceeding 25° C.
To prepare the feed to be extracted (
The carbon dioxide (purity, v/v>99.7%) in 30 kg cylinders equipped with a dip tube, was supplied by Air Liquide-Italy.
The extracts object of this patent proposal were obtained using a Supercritical Phase Extraction laboratory, SFE, using carbon dioxide, CO2, as a solvent. The scheme is shown in
The extract in question was obtained, starting only from seeds of rosehip finely ground, using the plant just described with the single separator setup. The operating conditions are indicated below: pressure 90 bar and temperature 40° C., in the extraction section; 20 bar and 40° C. in the separation section; extraction time 4 h; CO2, flow rate 1.2 kg h−1. At 90 bar and 40° C. the CO2 has a high density and solvent power while at 20 bar and 40° C. the CO2 returns to the state of sub-critical gas, loses its solubilizing power and releases the extract to the inside the separator. By opening the lower valve of the separator it is possible to collect the final extract from which the gaseous CO2 at ambient pressure and temperature moves away spontaneously. In each of the tests carried out, an average of 300 g of matrix was loaded into the extractor.
Three repeated extractions were performed, using the same matrix lot, in order to verify the reproducibility of the obtained results.
The extract obtained consisted of two clearly distinguishable phases (
The aqueous extracts thus obtained from the seeds of R. canina were subjected to instrumental investigation in order to determine their chemical composition.
From the analysis of the traces that make up the thermograms, it was possible to obtain the fraction, by mass, of dry matter present in the aqueous extracts between (0.1 and 10)% (w/w).
At the operating conditions adopted, chromatograms were obtained which show sufficiently resolved peaks.
The chromatographic data allows to calculate the percentage composition of each organic component of the sample under study. They are reported in Table 1 as a function of retention time, tr and retention indices defined in accordance with Kovats, IK.
Among the components identified by GC-MS there is the presence of apiol and methyl or ethyl esters of palmitic, oleic, stearic and linoleic fatty acids, having 16, or 18 carbon atoms and none, one or two unsaturations. The peaks of the esters appear in the chromatogram at tr between (40 and 50) min and at IKteo between 1900 and 2200. At higher tr and IK some long-chain hydrocarbon compounds saturated or containing an unsaturation have been identified, typical constituents of plant cuticular waxes.
In negative ionization mode, performed in ESI-MS it was possible to observe in the mass spectrum of the mixture, peaks with an m/z ratio 255, 277, 279, 281 and 283 corresponding respectively to palmitic acid, linolenic acid, acid linoleic, oleic acid and stearic acid also identified with the exact mass determined using an OrbitrapElite high resolution mass spectrometer (Table 2).
Again with reference to the spectra performed in negative ionization mode, the peaks showing an m/z ratio of 121, 151, 171 and 187 are respectively attributable to the following compounds, typical degradation products of tannins: benzoic acid, methoxybenzoic acid, acid 1-hydroxy-i, j-dimethylcyclohexanecarboxylic acid and 3,4-dimethoxycyclohexanecarboxylic acid. The identification was confirmed by the exact mass measurements obtained by OrbitrapElite (Table 3)
The most abundant peaks detected with positive ionization show an m/z ratio of 127 attributable to a trihydroxybenzene while the peaks with an m/z ratio 141 and 163 correspond to a methoxyhydroxybenzene and the peaks with an m/z ratio 181 and 203 are attributable to an ethoxy phenyl-3-propanol all derived from tannins and identified by the exact mass provided by the OrbitrapElite.
The 1H spectra of the samples are consistent, confirming the good reproducibility of the extraction technique adopted. The spectra appear quite complex; numerous signals can be identified in the aliphatic zone between (1 and 3) ppm, signals attributable to aromatic groups, between (7 and 8) ppm; typical signals of aldehyde groups, between (9 and 10) ppm. The characteristic signals of esters, between (4 and 5) ppm, are masked by the large water peak at 4.7 ppm.
These indications confirm the identifications obtained through the previously mentioned techniques. In fact, apiol (a phenylpropanoid) and most of the tannin derivatives contain an aromatic ring; while the alkyl groups constitute the skeleton of acids and esters; as well as the substituents of some aromatic compounds.
The extract consists of an aqueous solution. The water content, determined by TGA and gravimetric analysis, is between 90% and 99.9% mass/mass. The main organic components of the extract, based on GC-MS analysis with HP5-MS apolar column (5% phenyl-95% methylpolysiloxane), are the following: methyl linoleate (31.4%), methyl oleate (25.1%), methyl palmitate (7.5%), ethyl linoleate (4.7%) and ethyl linolenate (3.3%) with respect to the dry fraction of the extract, between 0.1% and 10% (mass/mass) (Table 1).
Other compounds, identified by ESI-MS analysis, are:
The activity on bacterial or fungal (yeast) strains isolated from biological samples from hospitalized or collection patients was evaluated, the nosocomial derivation of the majority of these strains characterized their resistance to different antimicrobials as reported in Tables 4 and 5.
In particular:
All strains, belonging to the collection of the Molecular Biology laboratory, were stored at −80° C. in 2.5 mL Eppendorf microtubes, in the respective liquid culture media enriched in 15% glycerol to protect them from low temperatures. Each bacterium was contained in the tubes in the exponential growth phase, at a titer equal to 108 CFU/mL (where CFU: Colony Forming Units). The inocula for the growth assays were made from these tubes, 106 CFU/mL for bacteria and 105 CFU/mL for yeasts.
In particular,were evaluated in vitro the following parameters, measured by standardized methods:
PI %=(Bi×100/Ac)−100
Where Ac represents the absorbance value at λ=600 nm of the positive control (untreated biofilm) and Bi that relating to the sample, after exposure to the formulation.
In preliminary form, an antimicrobial sensitivity test was performed on solid culture medium (agar) by means of the diffusion method in agar or the Kirby-Bauer method. In practice, for each formulation, 100 μL of solution were deposited in a well positioned in the center of a Petri dish, previously seeded with the pathogen under examination. After 24 h of incubation, the possible growth inhibition zone on the surface of the medium was evaluated (
The Kirby-Bauer method, as structured, provided a non-definitive result of the antibacterial efficacy, that is an end-point and not a quantitative value.
In this context, it was subsequently necessary to evaluate a more sensitive dose-response system, that is, able to relate the time and concentration of the compound with the biological response. In the cells suspended in a special liquid medium, scalar concentrations of each preparation were tested. After a 24 h incubation, the presence/absence of microbial growth was evaluated based on the turbidity/clarity of the growth medium. Subcultures in agar medium were prepared from the suspensions in which complete inhibition of growth was observed, in order to evaluate the possible residual viability of the microorganism under examination.
For this purpose, scalar concentrations of each preparation were tested on a bacterial suspension equal to 106 CFU/mL. After incubation in 96-well multi-well plates (Corning) for 48 h at 37° C., in the liquid media previously described for the various strains. The presence/absence of microbial growth was then evaluated on the basis of the turbidity/clarity of the growth medium, by reading with a spectrophotometer at a wavelength, 1, of 550 nm (SLT-Spectra II™, SLT Instruments, Germany). Subcultures in agar medium were prepared from the suspensions in which complete inhibition of growth was observed to evaluate the possible residual viability of the microorganism (MCB). As already described, the formulations under examination were tested at serial concentrations calculated according to the scheme [D=0.5 X], where D stands for dilution and X represents an integer between 1 and 10. The minimum concentration was evaluated for each formulation inhibitor (MIC) and minimal bactericidal concentration (MBC).
In this assay, together with the parameters that evaluate the activity of an antimicrobial on microorganisms in suspended form (planktonic), we have introduced an evaluation which tends to measure the minimum biofilm inhibitory concentration (MBIC). It represents the lowest concentration of formulation (highest dilution) capable of inhibiting the structuring of the biofilm of a microorganism. The inhibition of the biofilm, for all strains, by the preparation was evaluated after 3 days of incubation in 96-well multi-well plates, using the same growth procedure for the suspension tests for the evaluation of the MIC. The residual biofilm was calculated using the method described by the “Center for Biofilm Engineering” (CBE) of the University of Montana (USA), http://www.biofilm.montana.edu/ [28].
The method involved: I) elimination of the supernatant, II) 2 washes in PBS buffer and III) subsequent staining of the wells with 0.3% Crystal Violet, IV) followed by 2 washes in PBS and V) subsequent addition of 200 mL of a aqueous solution 30% acetic acid. The amount of biofilm present in each well was evaluated by measuring absorbance at=620 nm in a multiplate reader (SLT-Spectra II™, SLT Instruments, Germany).
The evaluation of experimental errors and uncertainty in the measurements of the microbial biofilm was performed following the procedure reported in our previous work [29]. In particular, both standard deviations and variances were taken into consideration in the spectrophotometric measurements, calculating for the same concentration of extract, the average of the absorbance values over at least 3 experimental replicates. In particular, thewas used F test to ascertain, for each microbe, at which concentration significant differences occur in the biological activity under examination [30]
As previously described, using the agar diffusion method (Kirby-Bauer) , the preparation obtained by supercritical phase extraction compared with other extraction methods, was more active towards all the tested strains, as reported in Table 6 and
The extract obtained by SFE has a broad spectrum of action for both Gram positive and Gram negative bacteria as well as blastomycetes (Candida spp.). The result suggests that the formulation under examination acts according to a mechanism of action independent of the structure of the cell wall or of the pathogenic species under examination; result shown also in
The results obtained with the activity test in solid medium (Kirby-Bauer) were then confirmed with the assays in liquid medium designed to evaluate the MIC and MBC. In these trials additional pathogenic strains were also evaluated such as: Streptococcus mutans, Candida krusei and C. glabrata, Tables 7 and 8.
From the tables it is clear that the MIC and MBC values are rather low. In fact, these values (relative to the concentrations of extract in aqueous suspensions) are between (6.25 and 1.5)% (w/v) for the MICs, and in a range between (12.5 and 3.125)% (w/v) for the MBC.
Like the MIC and MBC values, the extract under examination has shown a strong ability to inhibit bacterial and fungal biofilms, with MBIC values ranging between (12.5 and 6.25)% (w/v) of extract. These results suggest a broad-spectrum use of the formulation, for products aimed at an industrial environment, eg. to eliminate/prevent contamination by P. aeruginosa in food industry plants, or as anticaries, in consideration of the remarkable activity shown against S. mutans.
The graphs in
As Table 4 shows, the extracts obtained with procedures other than that of the invention proved to be less active.
S. aureus
E. coli
S. pyogenes
P. aeruginosa*
K. pneumoniae
C. albicans
C. krusei
C. glabrata
Candida albicans BF1
Escherichia coli ATCC 7075
Streptococcus pyogenes NC4
Staphylococcus aureus ATCC BAA-811
Pseudomonas aeruginosa ATCC 15442
Klebsiella pneumoniae NC5
Pseudomonas aeruginosa
Klebsiella pneumoniae NC5
Staphylococcus aureus TCC
Escherichia coli ATCC 7075
Streptococcus pyogenes NC4
Streptococcus mutans
Candida albicans BF1
Candida krusei BF2
Candida glabrata BF3
Pseudomonas aeruginosa ATCC
Klebsiella pneumoniae NC5
Staphylococcus aureus ATCC
Escherichia coli ATCC 7075
Streptococcus pyogenes NC4
Streptococcus mutans CIP103220
Candida albicans BF1
Candida krusei BF2
Candida glabrata BF3
G, Mameli A, Tramontano E, Frau A. (2017). Broad-range potential of Asphodelus microcarpus leaves extract for drug development. BMC Microbiol, 17(1):159.
This application is the national phase entry of International Application No. PCT/IB2020/062317, filed on Dec. 22, 2020, the entire contents of which are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2020/062317 | 12/22/2020 | WO |
