The present invention refers to a process to obtain an extract from inflorescences of macela-do-campo (Achyrocline satureioides). The present invention also refers to the cosmetic, pharmaceutical and veterinary compositions containing the aforesaid extract.
The present invention also refers to the use and method of application of the aforesaid extract of macela.
Achyrocline satureioides, also known as macela-do-campo, is a plant of the Asteraceae family, which is found in Argentina, Uruguay, Brazil and Paraguay and is widely used in traditional medicine. It is an annual plant, of medium height and with yellow inflorescences, commonly found in Brazil, principally in the states of the Southern region (Marques, 1995; Marques & Barros, 2000). It can be propagated from seeds (Ikuta, 1993; Marques, 1995; Marques & Barros, 2000) or by staked plant cuttings (Pardo, 1995). There is also written data about production in greenhouses (Marques & Barros, 2001) and the evaluation of the potential of the seeds for exploitation in agricultural cultivation (Ikuta, 1993; Marques, 1995; Marques & Barros, 2000).
Macela-do-campo is an annual, monoecious, branched herb, growing up to 1.5 m in height, covered in white hairs. It has alternate, complete, sessile, linear and lanceolate leaves, of up to 12 cm in length and 1.8 cm in width. It has a capitulum with two types of flowers, meeting in a corymbose panicle; golden-yellow flowers, with one or two hermaphrodite centers, of a tubulous corolla, as well as four or five marginal flowers, which are feminine with a filiform corolla. Its fruit is achene, glabrous and brown (SIMÕES et al., 1998; MEDEIROS et al., 2005).
Pharmacological investigations of extracts and their inflorescences demonstrate sedative, analgesic, anti-inflammatory and anti-spasmodic activities (Simões, 1988; Oliveira et al., 2001), anti-microbial activity against Staphylococcus aureus (Lemos, 2000), vasorelaxing activity (Hnatyszyn et al., 2004), anti-hyperglycemic activity (Carney et al., 2002) and hepatoprotective activity (Kadarian et al., 2002).
It was also reported that it is commonly used for treating different pains, digestive problems (D'Ávila, 1910; Ritter et al., 2002) and, more recently, for losing weight (Dickel et al., 2007).
Extractive processes to obtain the extract of Achyrocline satureioides (macela-do-campo) and, more specifically, its use in compositions destined for the prevention and treatment of inflammatory reactions, are already known.
In this context, the Brazilian patent document PI0103468-5 can be highlighted, which discloses an extraction process of the active compounds of Achyrocline satureioides.
The use of the product resulting from that process has the potential to be used in the preparation of medicines for the treatment of gastrointestinal disturbances, arising from indigestion (anti-spasmodic) and for the treatment of the inflammatory conditions and topical viral infections related to herpes.
Basically, the extraction process that is the object of that Brazilian patent document includes the following stages: prepare the material that consists of dried and ground inflorescences of Achyrocline satureioides; place this material in contact with a macerator containing a polar organic solvent such as C1 to C4 alcohol or mixture with water; separate the solids present in the extraction mixture from the liquid phase; treat the solution with adjuvant agents such as colloidal silicon dioxide, for example; and dry the resultant extract.
After evaluating a document written by the same inventors (Bassani et al. 1997), and prior to that patent document, it was learned that the process consists of a maceration that uses a solvent solution in the proportion of 7.5% (p/v), or rather, 7.5 g of the plant for every 100 ml of solvent solution. Furthermore, for each phase of the extraction process, object of that state of the art document, the mixture can, preferentially, be ethanol/water in the proportion of 80% ethanol and 20% water or in increasing concentrations of ethanol up to 100% ethanol. In this context, it should be observed that the above mentioned Brazilian state of the art document describes a process that requires a long time, due to the use of maceration, whose extraction occurs at an ambient temperature and can take from 24 to 160 hours, according to the document. Furthermore, the process is of low efficiency for the extraction of the compounds of interest, namely, quercetin and 3-O-methylquercetin, because the dried extract contains those compounds in concentrations of, at the most, 0.9391% and 0.7294%, respectively. It is also known that in this prior art patent document, the 3-O-methylquercetin is dosed concomitantly with luteolin, resulting in the content of the 3-O-methylquercetin being even less than 0.7294%.
In relation to the use of extracts from Achyrocline satureioides in cosmetic and pharmaceutical products, the Japanese document JP10226619, which discloses a skin preparation for external use that presents an antioxidant action in order to prevent the oxidation of the lipidic components using the extract of a plant of the genus Achyrocline, such as macela-do-campo (Achyrocline satureioides), for example, is highlighted. That Japanese document discloses a process that involves the flowers, stem, roots, leaves and seeds of the species belonging to the genus Achyrocline. However, it is known to a person skilled in the art that the substances of interest for the benefits of our patent, namely the quercetin and the 3-O-methylquercetin, are concentrated in the inflorescences and are not found in suitable quantities in the other organs, such as the leaves, stems and roots for the production of a dried extract concentrated in these substances.
The document JP 59044313 discloses an anti-bacterial composition that is useful as an effective cosmetic in the treatment of acne, etc, using quercetin as an active component and rutin and isorhamnetin or 3-O-rutinoside as essential and auxiliary components. The anti-bacterial activity of the composition is elevated in a weakly acidic pH of approximately 5.0-5.5 and the composition is suitable for use in the treatment of acne against Propionibacterium acnes. The concentration of the quercetin is preferentially 0.2-0.6% in weight.
The North American document US 2004/0170581 also discloses cosmetic preparations that contain an effective quantity of at least one ester derived from caffeic acid. One of the activities attributed to these esters is based on its anti-inflammatory potential.
This North American document also describes the sources to obtain the aforementioned caffeic acid. The anti-inflammatory mechanism of the esters of caffeic acid described by this document is based on its inhibitory action on the release of the pro-inflammatory mediators, by the mast cells, eosinophils and basophils.
The North American document U.S. Pat. No. 7,192,611 discloses a method of treatment of osteoarthritis that comprises of the administration of a composition based on flavonoids with an unsubstituted B ring and, optionally, a physiologically acceptable vehicle. The species from the genus Achyrocline are mentioned among the sources claimed as options for the extraction of the aforesaid flavonoids.
The North American document US 2008/0070875 discloses a composition for the treatment of acne that includes an anti-microbial/molecule of anti-inflammatory polyphenol(s), including quercetin and rosmarinic acid in combination with the salicylic acid and/or salicylate salts in a cosmetic vehicle. It also refers to a method for the treatment of acne by topically administering a composition in a therapeutically effective quantity in order to reduce the redness and spots associated with acne. That document does not mention macela-do-campo as a source of active ingredients to be used in the treatment of acne.
Accordingly, that North American document is directly related to the anti-inflammatory potential (inhibition of COX-2) of extracts derived from the species belonging to the genus Achyrocline.
The North American document US 2004/0220119 describes a method of the treatment of diseases mediated by the action of COX and LOX. This method is based on the local application of a composition containing flavonoids with an unsubstituted B ring and, at least, one flavan. One of the options claimed as a source to obtain the flavonoids with an unsubstituted B ring are the plant species belonging to the genus Achyrocline.
As is known to a person skilled in the art, the enzymatic chains COX and LOX are directly related to the inflammation mechanisms. Therefore, this document makes reference to the anti-inflammatory potential of the extracts from the species belonging to the genus Achyrocline.
In addition, those North American documents are not directly related to the cosmetic environment and, therefore, the bases of the compositions of topical application capable of stabilizing the flavonoids with an unsubstituted B ring are not described in detail.
From the following description of the present invention, it can be concluded that no document of the state of the art presents, as an object, a process of multiple alcoholic and/or hydro-alcoholic extractions of the inflorescences of Achyrocline satureioides (macela-do-campo), resulting in an extract containing, concurrently, quercetin and 3-O-methylquercetin in an elevated concentration. Furthermore, the extract of macela-do-campo resulting from the process of the present invention presents anti-inflammatory, anti-oxidant and anti-microbial activities.
Also, cosmetic, pharmaceutical and veterinary compositions will be described containing the aforesaid extract of macela-do-campo, used for the cosmetic and pharmaceutical prevention and treatment of the pathologies arising from inflammatory processes, microbial infections and cellular lesions by oxidative processes.
The present invention has an objective to provide a process of extraction of inflorescences of Achyrocline satureioides (macela-do-campo) that results in an extract containing elevated concentrations of quercetin and 3-O-methylquercetin presenting a high yield and a low use of ethanol.
It is also the objective of this invention to provide cosmetic, pharmaceutical and veterinary compositions containing extract of macela-do-campo with elevated concentrations of quercetin and 3-O-methylquercetin obtained by means of the process of extraction of inflorescences of Achyrocline satureioides (macela-do-campo).
It is also the objective of this invention to provide the use and method of application of an extract of inflorescences of Achyrocline satureioides in the treatment of acne, in order to clean the skin, among others.
The present invention refers a process to obtain a dried standardized extract of quercetin and 3-O-methylquercetin from inflorescences of Achyrocline comprising of the following steps:
A) grinding the inflorescences of Achyrocline satureioides to obtain a material of ground plant;
B) submitting the ground plant to at least four sequential stages of hydro-alcoholic extraction at a temperature from 60° C. to 80° C., for 3 to 4 hours for each stage, in order to obtain 4 intermediary hydro-alcoholic extracts;
C) combining the 4 intermediary hydro-alcoholic extracts;
D) concentrating the intermediary hydro-alcoholic extracts mixture; in order to obtain up to a maximum of 20% of the initial mass of the intermediary hydro-alcoholic extracts;
E) drying the material obtained in (D).
The present invention also refers to a cosmetic composition and a pharmaceutical composition comprising, in a physiologically acceptable vehicle, an extract of inflorescence of macela-do-campo obtained from the process of extraction of inflorescences of Achyrocline satureioides described above.
The present invention refers to a process for obtaining a standardized extract of quercetin and 3-O-methylquercetin from inflorescences of Achyrocline satureioides comprising of the following steps:
Preferably the sequential steps of hydro-alcoholic extractions are carried out under reflux, for 12 hours and comprise the following stages:
i) extracting an intermediary extract with a hydro-alcoholic solution 1:30 (m/m of ground plant material/extracting solution) for a period of 3 to 4 hours, preferably for at least 3 hours, at a temperature in the range of 60 to 80° C., preferably at 80° C., in order to obtain a first extracted solution;
ii) re-extracting the hydro-alcoholic extract of (i) with a hydro-alcoholic solution in the proportion of 1:20 (m/m of plant drug/extracting solution) for a period of 3 hours at a temperature of 80° C. thus obtaining a second extracted solution;
iii) combining the first and the second extracted solutions in order to obtain a third extracted solution;
iv) extracting a fresh batch of ground plant with said third extracted solution in the proportion of 1:30 (m/m of ground plant ingredient/extracted solution of stages 1 and 2) for a period of 3 hours at a temperature of 80° C.;
v) re-extracting the hydro-alcoholic extract from stage (iv) with a hydro-alcoholic solution in the proportion of 1:16 (m/m of hydro-alcoholic extract/extracting solution) for 3 hours at 80° C. in order to obtain a final extracted solution.
The process of extraction, object of the present invention, is highly efficient (up to 64.62% of yield in relation to quercetin according to the preferential embodiment) with a reduced consumption of solvent. For example, 546,875 liters of ethanol were used for 28.13 kg of inflorescences of macela-do-campo, that is, 19.44 liters of ethanol for each kg of inflorescences of macela-do-campo.
It is a clean process (without undesirable byproducts or the use of solvents harmful to the environment), presenting an optimized result in relation to the teachings of prior art. It provides an extract with 3 times more quercetin than that described in PI0103468-5, using less ethanol and also comprising of O-3-methyl-quercetin.
It presents an ideal constitution for use as an anti-inflammatory, anti-oxidant and anti-microbial, as will detailed below.
The preferable proportions of ethanol and water used in the stages of extraction are 1:1 m/methanol:water or 1.25:1 v/v ethanol:water, and the amount of water may be reduced until only ethanol is used.
According to the process of the present invention, the chemical compounds quercetin (CAS 117-39-5) and 3-O-methylquercetin (CAS 1486-70-0) are maintained and concentrated in the final resulting extract.
Data is not found in the technical literature that describes the presence of these two components, concomitantly, and in similar proportions to those described by this process, derived from the inflorescences of macela-do-campo. On evaluation of the process object of the present invention, it can be perceived that one of the advantages of the aforesaid process is its low cost because the solvents are readily available and well known. The equipment is conventional and has a reduced environmental impact due to the lower quantity of organic residues eliminated after the process. This is because the evaporated alcohol can be re-used for a new extraction of inflorescences of macela-do-campo.
In a preferred embodiment of the present invention, the process to obtain the extract of macela-do-campo comprises of the following steps:
i) Grinding the inflorescences of macela, in order to obtain the ground plant drug in a knife grinder;
ii) Mixing the ground plant drug with an extracting hydro-alcoholic solution in the proportion of 1:1 (m/m), using ethanol 96° GL as alcohol, in the initial proportion of 1 part plant ingredient to 30 parts of hydro-alcoholic solution, based on the mass of the materials. Performing the extraction of the compounds for a period of at least 3 hours at a temperature of 80° C. in a closed reactor;
iii) Filtering the mixture obtained in ii) in order to obtain a first intermediary hydro-alcoholic extract and a first residue consisting of the components of the non-extracted plant drug;
iv) Mixing the first residue consisting of the components of the non-extracted plant drug with a fresh extracting hydro-alcoholic solution, constituted of a mixture of ethanol 96° GL and water in the proportion 1:1 (m/m). The mass of extracting hydro-alcoholic solution to be added is ⅔ of the mass of the first extracting hydro-alcoholic solution for a period of 3 hours at a temperature of 80° C. in a closed reactor;
v) Filtering the mixture obtained in iv) in order to obtain a second intermediary hydro-alcoholic extract and a second residue constituted of the components of the non-extracted plant drug;
vi) Grinding a second quantity of plant drug, obtaining a second quantity of ground plant drug;
vii) Mixing the first and second intermediary hydro-alcoholic extracts to the second quantity of ground plant drug obtained in vi) in the proportion of 1:30 (m/m of plant drug/extracting hydro-alcoholic solution) for a period of 3 hours at a temperature of 80° C. (range: 60° C. to 80° C.) in a closed reactor;
viii) Filtering the mixture obtained in vii) in order to obtain a third intermediary hydro-alcoholic extract and a third residue constituted of the components of the non-extracted plant drug;
ix) Mixing the third residue constituted of the components of the non-extracted plant drug with a new hydro-alcoholic solution, constituted of a mixture of ethanol 96° GL and water in the proportion 1:1 (m/m). The mass of the new extracting hydro-alcoholic solution to be added is 11/20 of the mass of the third intermediary hydro-alcoholic extract for a period of 3 hours at a temperature of 80° C. in a closed reactor;
x) Filtering the mixture obtained in ix) in order to obtain a fourth intermediary hydro-alcoholic extract and a fourth residue constituted of the components of the non-extracted plant drug;
xi) Evaporate the ethanol from the mixture obtained in xi) by vacuum assisted evaporation (temperature of 60° C., vacuum of 700 mm Hg) until the mass corresponds to approximately 25% of the mass of the mixture obtained in x);
xii) Mixing the concentrate of xi) with an appropriate vehicle for the carrying out of the drying by spray dryer, preferably maltodextrin in the maximum proportion in mass of 50% of the solids expected in the product when dry.
xiii) Spraying the suspension obtained in xii) in a spray dryer with an entrance temperature of 185° C. and an exit temperature of 85° C.
In one of the preferential embodiments of the present invention, the final stage of the concentration is performed in a vacuum concentrator at a temperature of 60° C. and a vacuum of 600 mmHg, wherein the aforesaid final hydro-alcoholic extract is concentrated approximately 4 times. After the concentration step, a polysaccharide is added to the solution respecting a proportion of 50% in mass regarding the estimate of dry mass present in the solution, this preferentially being genetically non-modified maltodextrin. Other materials that can also be added at this stage are silicon dioxide, amide and cyclodextrin. This mixture is submitted to drying by spray dryer (entrance temperature of 180/185° C. and exit temperature of 80/85° C. under agitation) in order to obtain a dried extract.
As an example, the equipment that can be used in the process, object of the present invention, is:
The content of evaporated ethanol in the final concentration stage will be determined by alcometers and re-used in a new extractive process of macela-do-campo.
It is necessary to operate with at least 4 stages of distinct and sequential extractions in order to obtain an extract of macela-do-campo with the concentrated compounds of interest. If one simple stage of extraction was employed, the required concentration would not be achieved. Furthermore, proportionally, the volume of solvent used can be much less than that used in a single stage because the solvent can be re-used in successive stages.
The use of other solvents or the performance of other processes to obtain the extract of macela can result in the loss of at least one of the chemical components of interest: quercetin and/or 3-O-methyl-quercetin.
During the process of the present invention, the solvents used for the extraction are basically ethanol and water, in the proportion of 15.55 kg water and 15.55 kg of ethanol for each 1 kg of plant drug to be submitted to the process of extraction. For each phase of extraction of the process according to the present invention, the mixture can, preferentially, be ethanol/water (1:1 m/m) up to entirely ethanol.
With the performance of all the abovementioned stages, an extract of macela is obtained which is standardized in quercetin and 3-O-methylquercetin, obtained by an extractive process with a yield of up to 67.4% in relation to the mass of quercetin. The concentrations of quercetin and 3-O-methylquercetin are up to 301% and 262%, respectively, in relation to the mass of quercetin (CAS 117-39-5) and 3-O-methylquercetin (CAS 1486-70-0) present in the inflorescences. The actual content obtained by the process was up to 3.01% for the quercetin and up to 2.62% for the 3-O-methylquercetin. The dried extract was obtained from a plant drug with a content of 1.00% of quercetin in a dry base.
The dried extract of inflorescences of Achyrocline satureioides (macela-do-campo) resulting from the process according to the present invention, is constituted, basically, of high concentrations of the flavonoids: quercetin and 3-O-methylquercetin. This standardized dried extract presents anti-inflammatory, antioxidant and anti-microbial activities.
The factors that should be controlled in this process, in particular, are:
The perceived advantages of employing the process according to the present invention, are highlighted as follows:
The following example is a preferential embodiment of the process of preparation of the extract of macela of the present invention and should not be interpreted as a limitation to the aforesaid invention. In this context, it should be understood that the scope of the present invention has other possible embodiments and is limited only by the content of the attached claims, including all the possible equivalents therein.
i) Grind the inflorescences of macela, in order to obtain the ground plant drug in a knife grinder;
ii) Mix the ground plant ingredient with an extracting hydro-alcoholic solution in the proportion of 1:1 (m/m), using ethanol 96° GL as alcohol, in the initial proportion of 1 part plant drug to 30 parts of hydro-alcoholic solution, by mass of the materials. Perform the extraction of the compounds for a period of 4 hours at a temperature of 80° C. in a closed reactor;
iii) Filter the mixture obtained in ii) in a filter with a porosity specification of 60 mesh, in order to obtain a first intermediary hydro-alcoholic extract and a first residue constituted of the components of the non-extracted plant drug;
iv) Mix the first residue constituted of the components of the non-extracted plant drug with a fresh extracting hydro-alcoholic solution, constituted of a mixture of ethanol 96° GL and water in the proportion 1:1 (m/m). The mass of extracting hydro-alcoholic solution to be added should be a maximum ⅔ of the mass of the first extracting hydro-alcoholic solution for a period of 4 hours at a temperature of 80° C. in a closed reactor;
v) Filter the mixture obtained in iv) in a filter with a porosity specification of 60 mesh, in order to obtain a second intermediary hydro-alcoholic extract and a second residue constituted of the components of the non-extracted plant drug;
vi) Grind a second quantity of plant drug in a knife grinder, obtaining a second quantity of ground plant drug;
vii) Mix the first and second intermediary hydro-alcoholic extracts to the second quantity of ground plant drug obtained in vi) in the proportion of 1:30 (m/m of plant drug/extracting hydro-alcoholic solution) for a period of 4 hours at a temperature of 80° C. in a closed reactor;
viii) Filter the mixture obtained in vii) in a filter with a porosity specification of 60 mesh, in order to obtain a third intermediary hydro-alcoholic extract and a third residue constituted of the components of the non-extracted plant drug;
ix) Mix the third residue constituted of the components of the non-extracted plant drug with a fresh hydro-alcoholic solution, constituted of a mixture of ethanol 96° GL and water in the proportion 1:1 (m/m). The mass to be added of the fresh extracting hydro-alcoholic solution is 11/20 of the mass of the third intermediary hydro-alcoholic extract for a period of 4 hours at a temperature of 80° C. in a closed reactor;
x) Filter the mixture obtained in ix) in a filter with a porosity specification of 60 mesh, in order to obtain a fourth intermediary hydro-alcoholic extract and a fourth residue constituted of the components of the non-extracted plant drug;
xi) Evaporate the ethanol from the mixture obtained in xi) by vacuum assisted evaporation (temperature from 40° C. to 60° C., vacuum of 600 to 700 mm Hg) until the mass corresponds to approximately 25% of the mass of the mixture obtained in x);
xii) Mix the concentrate obtained in xi) with an appropriate vehicle (carrier) for the carrying out of the drying by spray dryer, this being maltodextrin (or optionally silicon dioxide, amide or cyclodextrin) in the maximum proportion in mass of 50% of the solids expected in the product when dry.
xiii) Atomize the suspension obtained in xii) in a spray dryer with an entrance temperature of 180-185° C. and an exit temperature of 80 to 85° C.
The result of the trial of comparative phytochemical profile by HPLC among the three batches obtained from the process of extraction described in the above example is illustrated in
Table 1 below shows the content of quercetin and 3-O-methylquercetin present in the aforesaid batches of extract of inflorescences of macela-do-campo.
The average content was determined by the quantitative analytical method in High Performance Liquid Chromatography (HPLC) for samples in triplicate by two consecutive injections of each aliquot. The analysis of the content of quercetin (CAS 117-39-5) and 3-O-methylquercetin (CAS 1486-70-0) was used to verify the reproducibility of the process of manufacture of the three batches. In summary, aliquots were taken from the 3 batches reaching an average between 2.6% and 3.0% of quercetin and 2.3% to 2.6% of O-methylquercetin. The samples were diluted in methanol and subjected to analysis by HPLC. Conditions of the chromatographic system:
Column: Merck Lichrospher 100RP18e (4.6×250 mm. 5 um)
System: Isocratic
Movable Phase: phosphoric acid 0.5%: methanol (1:1/v:v)
Temperature: 25° C.
Flow: 0.6 ml/min
Wavelength: 370 nm
Volume of injection: 20 μl
Run time: 55 minutes
Standard: di-hydrated quercetin (CAS 117-39-5)
Note: the concentration of 3-O-methylquercetin (CAS 1486-70-0) was dosed as quercetin (CAS 117-39-5) because it has the same chromophoric group.
The phototoxicity and cytotoxicity were evaluated for one of the batches of the extract of macela produced in accordance with the process of the above example.
The study of phototoxicity was conducted with the objective of evaluating the phototoxic potential in fibroblasts of permanent strain 3T3 in the presence and absence of UVA light.
The study of cytotoxicity was conducted with the objective of evaluating the cytotoxic potential of a substance by means of in vitro methodology, which evaluates the potential of cytotoxicity of the test substance in fibroblasts of the permanent strain 3T3 in different concentrations.
As a result, it was concluded that the extract of macela-do-campo obtained by the process in accordance with the present invention did not present phototoxic potential in any of its tested concentrations and that the cytotoxic potential is low, presenting IC50=0.5 mg/ml. Accordingly, the aforesaid dried extract of macela-do-campo can be indicated for topical use for human beings.
IV. Inhibition Halo in a Culture of Propionibacterium acnes
This trial was performed in order to determine the bacteriostatic potential of the extract of inflorescences of macela-do-campo resulting from the process according to the present invention, or rather, the capacity that the extract has to inhibit the development of the colony of Propionibacterium acnes.
For these tests, 0.5% of the dried extract of inflorescences of macela-do-campo was applied in a basic cosmetic formulation (base A).
The measurements of the inhibition halo of the dried extract of macela-do-campo applied to a basic cosmetic formulation were compared to the pharmaceutical product available in the market, BENZAC AC® produced by Galderma, which has benzoyl peroxide 5.0% as an active ingredient.
Table 2 shows the results obtained in the test:
Based on the results listed above it can be observed that the basic cosmetic composition with 0.5% of extract of macela demonstrated the same results as the referenced product BENZAC AC®, despite presenting an active anti-microbial ingredient in a concentration 10 times less in the formula. It is also demonstrated that the cosmetic base for the application of the extract does not present any capacity of inhibition of Propionibacterium acnes.
Tests were carried out for evaluating the minimum inhibitory concentrations of 3 batches of the dried extract of inflorescences of macela-do-campo produced in accordance with the process of the present invention against Propionibacterium acnes.
The procedure used for this test is the following:
Preparation of inoculum: the microorganism to be used in testing was replicated to a glass flask containing 10 ml of sterilized culture medium broth (TSB);
The dilution was assisted by microwave for 30 minutes. After dilution the samples were filtered by gravity on quality filter paper. The dilutions were calculated so that the batches of sample BDP 09960 contained the same of concentration of quercetin among themselves and in relation to the concentration of diluted pure quercetin.
The prepared concentrations failed to determine the MIC, but the results showed that for each batch, named as PI001, P1002 and PI003 MICs fall below 0.020, 0.022 and 0.019 mg/ml, respectively, as illustrated in
The MIC value found for standard quercetin with purity degree of 100% was 0.0364 mg/ml. Since the maximum dilution of the extract of macela-do-campo tested for the three batches is of about 0.002 mg/ml and knowing that the MIC for that extract is a range of values lower than this, it is concluded that the power of that extract for the activity of inhibition of the growth of Propionibacterium acnes is at least of the same magnitude of quercetin itself in a state of maximum purity. Additionally, taking into account t the concentration of quercetin batches PI001, P1002 and P1003, namely 2.95, 2.60 and 3.01%, it is noticed that the concentration of quercetin present in the maximum dilution of the macela-do-campo extract of the three batches that still have activity in inhibition of growth of Propionibacterium acnes is about 0.0006 mg/ml, that is, a concentration approximately 60 times lower than the MIC of quercetin. Thus, although it was not reached the maximum dilution of said macela-do-campo extract to determine the MIC, it is clear that the activity of growth inhibition of Propionibacterium acnes in vitro can not be attributed only to the presence of quercetinno in that extract obtained in accordance with the process of the invention. Other chemicals, such as 3-O-methylquercetin contribute to the overall effect of the extract as far as that activity is concerned.
VI. Bioautography of Propionibacterium acnes
Bioautographic trials were conducted to determine the classes of the compounds present in the extract of inflorescence of Achyrocline satureioides that perform the anti-bacterial activity against Propionibacterium acnes. The bioautography consists of the chromatographic separation of the compounds by HPTLC and the subsequent application of the inoculation to the culture medium over the chromatographic plate.
The results obtained in the bioautography trial demonstrate that the flavonoids quercetin and 3-O-methylquercetin are the main ingredients responsible for the anti-microbial activity.
The anti-inflammatory potential of the dried extract of inflorescence of macela-do-campo was evaluated by means of the dosage of pro-inflammatory cytokines, using a culture of human skin fibroblasts. Lipopolysaccharide of a gram-negative bacteria membrane (LPS) or UVB irradiation were used to induce the stimulus for production of cytokines IL-6, IL-8 and of the prostaglandin PG-E2 in human fibroblasts in a culture with different concentrations of extract.
It is concluded that, in the stimulation model with LPS, the extract presented a significant reduction in the secretion of PG-E2 in concentrations between 0.003 mg/ml and 0.03 mg/ml. In the stimulation model with UVB, a significant reduction was noticed in the secretion of IL-6 and IL-8 in the concentration of 0.03 mg/ml, and inhibition of PG-E2 in concentrations between 0.001 mg/ml and 0.03 mg/ml. In accordance with the analyzed data, the extract of macela-do-campo indicates anti-inflammatory activity in human skin (dermis) fibroblasts in vitro.
The anti-inflammatory potential of the dried extract of inflorescences macela-do-campo was evaluated by means of the dosage of pro-inflammatory cytokines, using a culture of keratinocytes of the HaCat lineage. Inactivated Propioniumbacterium acnes was used to induce the production of IL-8 in keratinocytes of the lineage HaCat in a culture with different concentrations of the extract, such as it is commonly achieved with LPS (lipopolysaccharide of a gram-negative bacteria membrane).
It is concluded that, in the stimulation model with P. acnes, the extract of inflorescences of macela-do-campo obtained in accordance with the process of the present invention presents a reduction greater than 30% in the secretion of II-8 in the concentrations between 0.003 mg/ml and 0.03 mg/ml. In accordance with the analyzed data, the extract of macela-do-campo indicates potential anti-inflammatory activity in human keratinocytes of the lineage HaCat in vitro.
Tests were performed for the antioxidant activity by DPPH of the dried extract of macela-do-campo obtained by spray dryer. The tests indicated that the IC50 of the antioxidant activity was obtained with a concentration of dried extract of macela-do-campo of 0.02 mg/ml. Trolox (a derivative of vitamin E sold by Hoffman LaRoche) was used as a standard of the aforesaid test and has its IC50 of antioxidant activity obtained with 0.005 mg/ml. Therefore, it can be concluded that the dried extract of macela-do-campo has extremely high antioxidant effectiveness, only 4 times less than Trolox.
—Cosmetic and/or Pharmaceutical Composition Comprising of the Extract of Macela-Do-Campo:
The cosmetic and/or pharmaceutical composition, object of the present invention, contains a physiologically acceptable carrier and from 0.1% to 5.0%, in mass, preferably, from 0.1% to 0.5% in mass, based on the total mass of the composition, of extract of inflorescences of macela-do-campo obtained from the process of extraction of inflorescences of Achyrocline satureioides in accordance with the present invention.
A cosmetic, pharmaceutical or veterinary composition, object of the present invention, comprising of an extract of Achyrocline satureioides obtained by the process of the present invention is the only one that presents antioxidant, anti-inflammatory and anti-microbial activity due to the high quantities of quercetin and 3-O-methylquercetin.
Due to the presence, in the composition, of the extract of macela-do-campo obtained in accordance with the process of the present invention—containing high concentrations of quercetin and 3-O-methyl-quercetin—the cosmetic and/or pharmaceutical composition of the present invention presents anti-inflammatory, antioxidant and anti-microbial activity.
The cosmetic and/or pharmaceutical composition may be used in the prevention and treatment of acne, specifically due to its anti-inflammatory and anti-microbial activities against Propionibacterium acnes and antioxidant activity. Among the examples of damage arising from inflammatory, microbial and oxidative reactions can be cited: expression wrinkles, signs of cutaneous photoaging, cellulite, acne, skin dyschromia and topical dermatitis, skin (dermis) hypersensitivity, among others.
Also, by presenting high concentrations of these chemical compounds, this composition can also be used for erectile dysfunction and for the pharmacological treatment of bronchial asthma (reference is made to scientific articles listed below).
An ethanolic extract of inflorescences of Achyrocline satureioides shows relaxant activity in the smooth muscle of guinea pig corpus cavernosum. This activity is primarily due to the molecules of quercetin and 3-O-methylquercetin present in the extract in 0.02% (m/m) and 0.07% (m/m), respectively. These compounds, in a isolated form, show the same relaxant activity (Hnatyszyn et al., 2004). The action mechanism of this activity provided by 3-O-methylquercetin and putatively also by quercetin is the inhibition of the enzyme phosphodiesterase by the subsequent increase of the cAMP and cGMP mediators, with reduction of the concentration of intracellular Ca (Ko, et al. 2001). In another study, it was evidenced that quercetin is a potent inhibitor of PDE 5A, with IC50 of 1.9 μM, while 3-O-methylquercetin is less selective for this enzyme, with IC50 of 86.9 μM (Lines & Ono, 2006). The inhibition of the PDE 5A is the same mechanism by which sildenafil assists the vasodilatation, thus assisting the erection in humans suffering from erectile dysfunction. It can be concluded that the quercetin and the 3-O-methylquercetin contained in the extract of Achyrocline satureioides are among the molecules responsible for the relaxant activity in the smooth muscle of guinea pig corpus cavernosum and because of this there could be an indication for the use of the standardized extract of Achyrocline satureioides cited in the present invention in the form of a medication for the pharmacological treatment of erectile dysfunction in humans.
In an experimental model in vitro of relaxant muscular activity in guinea pig tracheas, it was shown that 3-O-methylquercetin is capable of provoking the relaxation of the smooth muscle of tracheas that were precontracted with histamine, carbachol, KCl and forskolin by the mechanism of the competitive enzymatic inhibition of phosphodiesterase. This inhibition results in the reduction of AMPc and GMPc with the subsequent reduction of intracellular Ca+, resulting in the muscular relaxation (Ko et al., 2002). In another study, it was demonstrated that 3-O-methylquercetin is a dual inhibitor of phosphodiesterase 3 and 4 (PDE3/4), more selective for the PDE3 than for the PDE4, with respective IC50 of 1.6 μM and 28.5 μM. (Chiu & Chang, 1998 apud Ko et al., 2007). The dual inhibitors of PDE 3/4 are more effective in the control of asthma than the simple inhibitors of PDE4. These results indicate that the standardized extract obtained in accordance with this invention, can be indicated for use in pharmaceutical and veterinary products for the pharmacological treatment of bronchial asthma and other pathologies involving bronchial hyperresponsiveness.
The more relevant examples of products that can be prepared from the extract of macela of the present invention or from the cosmetic and pharmaceutical compositions comprising of said extract are:
The extract of macela-do-campo is obtained by an extraction process of inflorescences of Achyrocline satureioides in accordance with the process of the present invention as described above.
Water is the basis of several preferred embodiments of the cosmetic composition of the present invention, acting as the carrier for the other components. The compositions of the present invention comprise water, preferentially demineralized or distilled, in an appropriate percentage (q.s.) to achieve 100% of the formula based on the total weight of the present composition. Naturally, other cosmetically acceptable carriers can be used in the present invention.
Some examples of cosmetic adjuvants compatible with the properties of the composition described herein will next be listed below—in a non-restrictive manner, only for demonstrative purposes—which, additionally, can be employed in the present cosmetic and/or pharmaceutical composition destined for the prevention and treatment of damage arising from inflammatory, microbial and oxidative/lipoperoxidative reactions:
The present cosmetic and/or pharmaceutical composition presents other physical-chemical characteristics that are appropriate and safe for the user of the end product:
1. It is stable for a period of at least two years;
2. It has an appropriate texture during application that is non-stick and non-oily;
3. It is easily spread;
4. It does not make the skin oily after application;
5. It does not present comedogenicity;
6. It does not present phototoxicity and cytotoxicity;
7. It does not present allergenicity;
8. It is not sensitizing;
9. It does not provoke any type of adverse reaction or cutaneous or ocular lesion, whether in normal conditions of use or in conditions of forced perspiration;
10. It presents an excellent level of homogeneity and stability;
11. By not provoking irritation to the skin, it becomes more comfortable and allows for daily use or for even more than once per day;
12. It presents appropriate chemical stability;
13. It comprises of a multifunctional extract of macela that presents anti-microbial, antioxidant and anti-inflammatory activities. It also presents low cost in the preparation of the composition as it dispenses with the addition of complex antioxidants and preservatives;
14. The extract of macela presents a pleasant color and smell that do not impact upon the final result of the composition;
15. The extract presents sensorial characteristics (softness, texture and shine) that are ideal for cosmetic compositions of topical use;
16. The extract combats the specific inflammation induced by the chemical components present in the membrane of the Propionibacterium acnes.
Example of a Cosmetic and/or Pharmaceutical Composition Comprising of an Extract of Macela-do-Campo:
The following examples are preferential embodiments of cosmetic and/or pharmaceutical compositions destined for the prevention and treatment of the damage arising from inflammatory, microbial and oxidative reactions and should not, therefore, be interpreted as limitations of the same. In this context, it should be understood that the scope of the present invention has other possible embodiments and is limited by the content of the attached claims, including all the possible equivalents therein.
In table 5 an example is described of one embodiment (formulation) of the cosmetic and/or pharmaceutical composition, object of the present invention:
a. homogenize the components of phase 1;
b. add to the phase obtained in (a) the components of phase 2 and homogenize;
c. add to the phase obtained in (b) the previously solubilized components of phase 3 and homogenize;
d. heat the components of phase 4 until they are completely solubilized and then cool;
e. when the temperature is less than 40° C., solubilize the components of phase 5 over phase 4 and add to the principal phase (obtained in (c)) and homogenize;
f. add the previously solubilized components of phase 6 in water to the phase obtained in (e) and homogenize.
Number | Date | Country | Kind |
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0959012 | Dec 2009 | FR | national |
Filing Document | Filing Date | Country | Kind | 371c Date |
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PCT/IB11/00015 | 1/6/2011 | WO | 00 | 9/27/2012 |