Patent Application
20250034191
This disclosure relates to the deconstruction of lignin into its component monomers, and use of the monomers as cosmetic ingredients or food additives.
In general, lignin can be used as a source of phenol, both in the formulation of adhesives for wood (US 2015/0329753 A1) and in the field of carbon fiber composite materials (U.S. Pat. No. 9,133,568 B2). Indeed, for lignins whose applications depend closely on their production technique, multiple applications may be concerned with composite materials as well as with cosmetics and food ingredients. The versatility of lignin is linked in particular to a demand for aromatic compounds that are likely to replace those obtained from fossil resources.
Regarding aromatic compounds intended for cosmetics and/or food processing, these are particularly abundant when deconstruction lignin. Indeed, phenols are generally produced after the depolymerization of lignin using chemical or biochemical catalysts. The depolymerization of lignin allows access to aromatic compounds such as cresols, catechols, guaiacols, vanillin and other phenolic monomers. These compounds are of great interest in the cosmetics and/or food industry. Indeed, 75% of the global demand for vanillin, considered to be the most used flavor in the food industry, is obtained from fossil sources such as crude oil.
In view of an ever-increasing demand for more “naturalness”, alternative sources for products including vanillin and derivatives thereof, such as apocynin (acetovanillone), are sought. Apocynin has been reported as an effective inhibitor of the NADPH-oxidase complex (Zhang, Yi et al., American Journal of Hypertension, 2005, 18 (7), 910-916).
Lignin is a highly heterogeneous polymer derived from a handful of precursor lignols that crosslink in diverse ways. The lignols that crosslink are of three main types, all derived from phenylpropane: coniferyl alcohol, sinapyl alcohol, and paracoumaryl alcohol. Lignin is one of the two major components of lignocellulose after cellulose. Lignin constitutes approximately 15-30 wt. % of lignocellulosic biomass and acts as a glue that fills the space between the other components binding them together to provide structural integrity. Lignin also plays a role in protecting against biological attacks and provides for water transport in the cell walls of a plant. Most available lignins are derived from black liquors, a by-product from the kraft process when digesting pulpwood into paper pulp removing lignin, hemicelluloses and other extractives from the wood to free the cellulose fibers. Depending on the process, three kinds of technical lignins are currently available in large quantities: kraft lignin (sulfate process), lignosulfonate (bisulfite process), and organosolv lignin (processing using organic solvent(s) to solubilize lignin and hemicellulose). The chemical structure of lignin is speculative and qualitative information can only be obtained through chemical and spectroscopic analysis. Several studies have reported on the oxidative breakdown of lignin in an alkaline medium or using a catalytic system such as CuSO4 to yield simple phenolic compounds such as vanillin.
Unfortunately, the currently available methods for the deconstruction of lignin into its component monomers remain largely difficult and insufficient, often only providing access to a limited number of component monomers. As such, improved methods allowing for easier and more comprehensive access to such monomers are of commercial interest.
The present disclosure broadly relates to the deconstruction (“depolymerization”) of lignin into its component monomers. The present disclosure also relates to the use of such monomers as cosmetic ingredients and/or food additives.
In an aspect, the present disclosure relates to a process for the deconstruction or depolymerization of lignin. In an embodiment, the present disclosure relates to a process for the deconstruction or depolymerization of lignin into its component monomers. The present disclosure also relates to the use of such monomers as cosmetic ingredients and/or food ingredients.
In an aspect, the present disclosure relates to a deconstructed or depolymerized lignin, also referred to as lignin flower. In an embodiment of the present disclosure, the lignin flower comprises at least one or more of the following: vanillin (15-25 wt. %); apocynin (6-12 wt. %); acetosyringone (12-15%); syringaldehyde (13-30 wt. %); syringic acid (16-24 wt. %); and vanillic acid (5-15 wt. %).
In an aspect, the present disclosure relates to the use of lignin flower in cosmetic and/or food applications. In an embodiment of the present disclosure, the lignin flower is used in combination with a plant and/or three extract. In a further embodiment of the present disclosure, the combination may be for use as a cosmetic. In a further embodiment, the present disclosure relates to a combination comprising lignin flower and maple bark. In further embodiments, the maple bark comprises acertannin (ginnalin A).
In an aspect, the present disclosure relates to lignin flower rich in monophenols. In embodiments of the present disclosure, the monophenols comprise apocynin and vanillin. In an embodiment, lignin flower rich in apocynin may be formulated as a cosmetic composition for treating various skin conditions including effects associated with aging skin. In an embodiment, lignin flower rich in vanillin may be formulated as a food additive.
In an aspect, the present disclosure relates to a lignin monomer obtained from a deconstructed or depolymerized lignin. In embodiments, the present disclosure relates to the use of one or more lignin monomers obtained from the deconstructions or depolymerization of lignin as cosmetic ingredients and/or food additives.
It is provided a process for producing a depolymerized lignin comprising the steps of: providing a solution comprising dissolved lignin; adding an oxidation catalyst to the solution comprising dissolved lignin; and heating the solution to produce the depolymerized lignin.
In an embodiment, the solution comprising dissolved lignin further comprises an alkali.
In another embodiment, the alkali comprises sodium hydroxide.
In a further embodiment, the oxidation catalyst comprises sodium nitrite nanoparticles or carbon nanoparticles.
In an embodiment, the process described herein further comprises adding a co-catalyst to the solution comprising dissolved lignin.
In an embodiment, the co-catalyst comprises oxygen.
In a further embodiment, the pH of the solution being heated ranges from about 12 to about 14.
In an embodiment, the heating comprises temperatures ranging from about 180° C. to about 200° C.
In an embodiment, the process described herein further comprises adding an alcohol to the depolymerized lignin, filtrating said depolymerized lignin and purifying the filtrated depolymerized lignin.
In a further embodiment, the alcohol is isopropanol or ethanol.
In an embodiment, the depolymerized lignin product comprises at least one of vanillin at 15-25 wt. %; apocynin at 6-12 wt. %; acetosyringone at 12-15%; syringaldehyde at 13-30 wt. %; syringic acid at 16-24 wt. %; vanillic acid at 5-15 wt. %; and a combination thereof.
It is provided the use of a depolymerized lignin as encompassed herein in the manufacture of a cosmetic composition, a food additive, and/or an anti-inflammation composition.
It is also provided the use of a depolymerized lignin as encompassed herein as a cosmetic ingredient, a food additive, and/or an anti-inflammation ingredient.
It is further provided a cosmetic composition, a food additive, and/or an anti-inflammation composition comprising the depolymerized lignin produced by the process as described herein and a carrier.
The word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one”, but it is also consistent with the meaning of “one or more”, “at least one”, and “one or more than one” unless the content clearly dictates otherwise. Similarly, the word “another” may mean at least a second or more unless the content clearly dictates otherwise.
As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “include” and “includes”) or “containing” (and any form of containing, such as “contain” and “contains”), are inclusive or open-ended and do not exclude additional, unrecited elements or process steps.
As used in this specification and claim(s), the word “consisting” and its derivatives, are intended to be close ended terms that specify the presence of stated features, elements, components, groups, integers, and/or steps, and also exclude the presence of other unstated features, elements, components, groups, integers and/or steps.
The term “consisting essentially of”, as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and/or steps as well as those that do not materially affect the basic and novel characteristic(s) of these features, elements, components, groups, integers, and/or steps.
The terms “about”, “substantially” and “approximately” as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% of the modified term if this deviation would not negate the meaning of the word it modifies.
The deconstructed or depolymerized lignin may include a plurality of different lignin monomers. The distribution of the different lignin monomers may vary depending on the source material and process conditions used to effect deconstruction or depolymerization.
The following figures/drawings form part of the present specification and are included to further demonstrate certain aspects of the present specification.
The present disclosure broadly relates to the deconstruction (“depolymerization”) of lignin into its component monomers. The present disclosure also relates to the use of such monomers as cosmetic ingredients and/or food additives. These and other aspects of the disclosure are described in greater detail below.
In the context of cosmetics, and in accordance with various embodiments of the present disclosure, one or more plant extracts may be combined with the lignin flower of the present disclosure. In an embodiment of the present disclosure, lignin flower may act as an immunomodulator. To that effect, low molecular weight lignin fragments have been previously described as having immunomodulatory properties (Kuroki T et al., Front. Microbiol. 2018, 9, 1164). Inflammation is part of the complex biological response of body tissues to harmful stimuli, such as pathogens, damaged cells, or irritants, and is a protective response involving immune cells, blood vessels and molecular mediators. Oral administration of certain anti-inflammatory drugs such as acetylsalicylic acid can sometimes be associated with risks of internal bleeding of vascular capillaries into surrounding tissues. In an embodiment, the lignin flower of the present disclosure may be combined with acetylsalicylic acid, at least due in part to its high apocynin content, known for its anti-inflammatory properties (Liu, N. et al., Nature, 2019, 568, 344-350). In a further embodiment, the present disclosure relates to the use of a composition comprising lignin flower rich in apocynin for use as a cosmetic composition for treating various skin conditions including effects associated with aging skin.
In embodiments wherein hardwood lignin is deconstructed under alkaline conditions without the use of a catalyst, the pH of the reaction mixture decreases from about 13 to about 9, with low amounts of monophenols (e.g., 0.7 wt. % vanillin) being obtained. Phenolic acids such as vanillic acid and syringic acid are substantially consumed. In embodiments using a catalyst, the pH decrease of the reaction mixture is significantly less pronounced, decreasing from about 13 to about 12, while also yielding higher amounts of monophenols (e.g., 4.9 wt. % vanillin).
In an embodiment, the depolymerized lignin product comprises at least one of vanillin at 15-25 wt. %; apocynin at 6-12 wt. %; acetosyringone at 12-15%; syringaldehyde at 13-30 wt. %; syringic acid at 16-24 wt. %; vanillic acid at 5-15 wt. %; and a combination thereof.
In particular, the depolymerized lignin product comprises at least one of vanillin at 20-25 wt. %; apocynin at 10-12 wt. %; acetosyringone at 12-15%; syringaldehyde at 13-16 wt. %; syringic acid at 16-20 wt. %; vanillic acid at 5-7 wt. %; and a combination thereof.
In the presence of a catalyst, the oxidation of the benzylic alcohol moieties of lignin was followed by further oxidation and bond cleavage. Substantial amounts of phenolic acids such as vanillic acid and syringic acid were detected. In embodiments of the present disclosure, the catalytic oxidation may be performed in the presence of molecular oxygen acting as a co-oxidant.
Hardwood organosolv lignin (100 g; Suzano Canada Inc.) is dissolved in a sodium hydroxide solution (500 mL; 2.0 M) at a pH ranging from about 12-13. A catalyst composed of sodium nitrite or carbon nanoparticles comprising grafted nitrite groups is subsequently added to the solution (up to 5.5 wt. % relative to the lignin weight). The reaction mixture is then pressurized using oxygen (10 bars) over a period of 30 minutes and then heated at 180° C. for 1.5 h. Following the deconstruction, the reaction mixture is cooled, and the pressure slowly released. Isopropanol (250 mL) is then added to the reaction mixture and the pH is lowered to about 2 using an aqueous (15%) sulfuric acid (H2SO4) solution. The resulting solution is stirred for a period of 1 h before decantation and filtration. Following filtration over a nylon membrane (1 μm), the isopropanol is evaporated. The solid residue was subsequently treated with isopropanol 20% in water and filtration over a nylon membrane (0.22 μm) and isopropanol removal before passing over a HP20 Diaion® resin to yield lignin flower. The lignin flower is subsequently extracted from Diaion resin by dissolving in EtOH and filtered. The lignin flower after ethanol evaporation and vacuum drying corresponds to 20-30 wt. % of the initial lignin mass. Different compounds, including acetovanillone, vanillin and other monomers, can subsequently be separated by column chromatography on silica (
After grinding, bark particles (100 g) were extracted with an ethanol-water mixture (1:1, v/v; 1 L) at 85° C. over a period of 4 h. The extracted active ingredients were subsequently mixed with the lignin flower (10 wt. %). Following filtration, the mixture (maple bark extracts and lignin flower) was freeze dried and used without further treatment.
To validate the performance of lignin flower from hardwood, two types of lignin from different forest sources were tested as a model for extracting the lignin flower by oxidative depolymerization at 180° C. at basic pH and in the presence of sodium catalyst. Softwood lignin was obtained from pine while hardwood lignin was obtained from trembling aspen. The results are presented in Table 1.
The different experimental conditions allowing access to the lignin flower clearly indicate that the percentage of depolymerization is more important for an extracted hardwood (yield of 22.8%) while that of a softwood leads to 9.4%. Similarly, the polyphenol content is higher for hardwood lignin than for softwood lignin (Table 1).
To visualize the differences between extracts from hardwood and softwood lignin, an HPLC method was developed to identify the different monomers resulting from oxidative depolymerization. All HPLC analyses were performed using an Agilent 1100 system including a G1311A pump, a G1322A degasser, a G1313A autosampler and a G1315B UV-DAD detector. A SiliaChrom Plus C8 column (5 μm, 4.6×250 mm) was used to identify the molecules by comparison of retention times of commercial standards. Mobile phase A was water with 0.1% formic acid and mobile phase B was acetonitrile also containing 0.1% formic acid. The elution gradient for phase A was 85% to 60% for the first 22 min, then 60% to 45% for 3 min, 45% to 40% for 2 min, 40% to 20% for 3 min and finally 20 to 85 for 2 min. The acquisition time was 32 minutes for a fixed flow rate of 0.75 mL/min. The injection volume was 3 μL and the detection wavelengths were 254 and 280 at 40° C. (see
The comparison of HPLC profiles clearly indicates that extracts of lignin flower from hardwood are densely more abundant in terms of molecular population of monomer type than those derived of lignin flower from softwood. Indeed, in lignin flower from softwood, the guaiacyl moieties (vanillic acid, vanillin and apocynin) represent the largest proportion with 82% of the chromatogram peak area. Conversely, a proportion of 97% is measured for the lignin flower from hardwood for all monomers peak area. This represents a relatively pure proportion in terms of molecules of interest.
Cytotoxic Effect of Pure Lignin Flower and Sugar Maple Bark Extract Enriched with Lignin Flower (10 wt. %)
The cytotoxicity was determined using normal fibroblasts from human volunteers (MatTek #NHDF-CRY-AD) grown in a growth medium (MatTek #NHDF-GM). The cells were cultured at 37° C. with 5% CO2. The cells were then seeded (2.5×103 cells/well using a 96-well plate) and left to adhere overnight at 37° C., 5% CO2. The next day, different concentrations of extract were added. A positive control, camptothecin (10 UM) was used. The cells were incubated for 24, 48 and 144 hours at 37° C., 5% CO2. Four hours prior to the end of the incubation period, 20 μL/well of blue Alamar was added to the cell cultures. The latter is a non-toxic compound that contains resazurin, a non-fluorescent molecule that, in a living cell, is transformed into a fluorescent compound. The cellular activity is proportional to the number of living cells. After 4 hours of incubation with blue Alamar, the surnatant was transferred to a 96-well black plate and a fluorescence reading (EX560 nm/EM590 nm) was performed by spectrophotometry using a plate reader (Spark2, Tecan).
The cytotoxic effect of compositions comprising sugar maple bark extract enriched with lignin flower (10 wt. %) on fibroblasts was determined. The fibroblasts were obtained from a 56-year-old woman (BMI=26.8 kg/m2). Two assays of released lactate dehydrogenase (LDH) were performed on culture media (Day 1 and Day 7). The results were evaluated relative to the maximum cytotoxicity control (Triton-treated cells) and expressed as percentages: Percentage cytotoxicity=100×(Release of LDH under test conditions/LDH released under triton conditions). The cytotoxicity of the sugar maple bark extract enriched with lignin flower (10 wt. %) was evaluated on Day 3 and Day 6 on cocultures of 2D fibroblasts and 3D mature adipocytes.
The results are expressed as a percentage relative to the coculture control conditions (
Effect of Sugar Maple Bark Extract Enriched with Lignin Flower (10 wt. %) on Adiponectin Secretions.
To further characterize the effects of sugar maple bark extracts enriched with lignin flower (10 wt. %), adiponectin secretion by adipocytes was evaluated in culture media on Day 3 and Day 6 following exposure (
Effect of Sugar Maple Bark Extract Enriched with Lignin Flower (10 wt. %) on Hyaluronic Acid (HA) Secretion.
The effects of sugar maple bark extracts enriched with lignin flower (10 wt. %) on the extracellular matrix of fibroblasts was evaluated by measuring hyaluronic acid secretion by fibroblasts (
Effect of Sugar Maple Bark Extract Enriched with Lignin Flower (10 wt. %) on the Architecture of the Extracellular Matrix.
The effect of the sugar maple bark extracts enriched with lignin flower (10 wt. %) on fibronectin and collagen I was assessed. Fibroblasts were fixed at the end of the culture period, and fibronectin and collagen I proteins were labeled and measured by immunofluorescence (
Regarding the collagen quantification, it can be observed that the trends are different between the results prior and after normalization (
This study evaluated the effects of a 6-day treatment with maple bark extract enriched with 10% of lignin flower at different concentrations on a coculture model of “aged” human adipocytes and skin fibroblasts to mimic the interactions between the hypodermis and the dermis in slightly aged human skin. This extract appears to have a dose-dependent proliferative effect on dermal fibroblasts, increasing their number after 6 days of treatment. In parallel, this product tends to increase the secretion of adiponectin by adipocytes in a dose-dependent manner. At the end of treatment, it tends to increase hyaluronic acid secretions.
This active ingredient (sugar maple bark extract enriched with 10% of lignin flower) therefore has potential anti-aging effects because it stimulates the proliferation of fibroblasts while maintaining the metabolism of adipocytes and could limit skin inflammation that develops over time. In addition, it would help to improve the extracellular matrix and maintain the hydration of the skin by stimulating the secretions of hyaluronic acid by fibroblasts. Indeed, the presence of the lignin flower in the bark extract, would promote the anti-aging effect through its properties of inhibition of the secretion of the pro-inflammatory cytokine IL-6 and a potential stimulatory effect of collagen production. Maple bark extracts enriched with 10% lignin flower at tested doses (5/2.5/1 μg/mL) was not cytotoxic for coculture and promotes secretions of hyaluronic acid and collagen.
An evaluation of the anti-free radical activity of the two types of lignin was carried out with DPPH test and the results are shown in Table 2.
Compared to the softwood lignin extract which presents a polyphenol content of 28% in gallic acid equivalent, the hardwood lignin extracts leads to a higher polyphenol content with 45%. In terms of antioxidant activity, it is the lignin flower from hardwood that presents the best antioxidant profile with an IC50 of 7.7 μg/mL against 23.4 μg/mL for lignin from softwood. This antioxidant activity of the lignin flower from hardwood shows a more marked effectiveness in particular for extracts rich in syringic polyphenols.
Lignin extract decreases nitrite production from 30 μg/mL to 100 μg/mL (n=8) (see
The lignin flower extract (hardwood) was diluted in DMSO to a concentration of 10 mg/mL. The extract was subsequently diluted in the culture medium to obtain the final concentrations needed for the study. The final concentration of DMSO in the culture medium was 0.1%. According to a previous cytotoxicity study, a concentration up to 10 μg/mL did not influence cell viability. This concentration was therefore selected as a starting point. In addition to the 10 μg/mL concentration, two other concentrations of the extract (0.1 and 1 μg/mL) were used. As a positive control, recombinant human TGFβ1 (Peprotech) was used. This was diluted in the culture medium to obtain a final concentration of 10 ng/ml.
Different concentrations of lignin flower extract were used (see
To assess the effect of the test element on inflammation, we assayed extracellular concentrations of secreted IL6. The results were normalized by the number of fibroblasts and represented as a percentage of the solvent condition DMSO (
In the assays at day D3 and D6, fibroblasts alone do not secrete IL6 or hardly at all, while adipocytes alone do (14% at D3 and 52% at D6). It is when the two populations are brought into contact that secretions of the pro-inflammatory cytokine are increased. As expected at D3, with the anti-inflammatory reference (Dexamethasone), it is showed a significant decrease in secretions of the pro-inflammatory cytokine IL6 (25%). At D6, the effect of Dexamethasone is more nuanced as we only have a decreasing trend compared to the coculture control (85%). At D3, the solvent DMSO (100%) seems to increase IL6 secretions in the coculture compared to the coculture control (55%), this effect tends to disappear at D6. As the solvent induces changes in IL6 secretions, it is important to compare the conditions treated with lignin flower, compared to the DMSO solvent conditions. At D3, lignin flower induces a significant decrease in IL6 secretions, especially at doses 2 and 3 (45% and 67%). After 6 days of treatment, lignin flower tends to decrease pro-inflammatory cytokine secretions in the same proportions whatever the dose tested. This anti-inflammatory result of lignin flower in the decrease of IL6 secretions, was confirmed as illustrated in
The purpose of the test is to investigate the possible genotoxic activity exerted by the tested substance in bacterial strains of Salmonella typhimurium, with and without metabolic activation with S9.
The Ames test allows for detecting the induction of point mutations in nucleotide bases, such as deletions, insertions, transversions and frameshift mutations by using modified Salmonella typhimurium strains. These strains carry a defective gene in the histidine operon making them auxotroph for this amino acid (mutants His-which require histidine in the culture medium for growth). The method guiding principle is based on the reverse mutation phenomenon by which bacteria exposed to a mutagenic substance may change back and become again prototroph concerning histidine (His+). The bacterial cells in growth phase are exposed to different concentrations of the test agent and mutagenic activity is determined by the capability of the test substance to induce a significant increase in the number of revertant (histidine-independent mutant, His+) in comparison to the spontaneous reversions occurring in the control cultures.
Some chemical agents are not directly mutagen but become so by following transformation and metabolic activation occurring in the organism by liver enzyme activity. To study this genotoxic effect, rat liver microsomal fraction (S9) has been added. The employ of S9 allows identifying substances that act as indirect mutagen.
In this assay, 5 bacterial strains TA 1535, TA100, TA 102, TA 1537 and TA98 have been used and their characteristics are illustrated in Table 3. Each tester strains contains a different type of mutation in the histidine operon. TA 1535 and TA 100 strains are specific testers for mutagens causing base substitutions. The TA 102 strain is used to detect mutagens that require an intact excision repair system. The sensitivity of TA 100 and TA 102 is greatly enhanced by the introduction of an R factor, pKM101, which confers ampicillin resistance. Furthermore, the TA 102 strain contains the multicopy plasmid, pAQ1, which confers tetracycline resistance. The frameshift tester strains used are TA 1537 and TA 98. TA 98, like TA 100, is ampicillin resistant. All S. typhimurium strains carry, along with the defect in the histidine gene (His−), a deep rough (rfa) character, a mutation that causes partial loss of the lipopolysaccharide barrier that coats the surface of the bacteria and increases permeability to large molecules. Last, in all these strains, except TA 102, there is a deletion of a gene coding for the DNA excision repair system (uvrB), resulting in highly increased sensitivity in detecting many mutagens. For technical reason, the deletion excising the uvrB gene extends through the bio gene and, consequently, these bacteria also require biotin for growth.
S. typhimurium TA 1535
S. typhimurium TA 1537
S. typhimurium TA 100
S. typhimurium TA 98
S. typhimurium TA 102
S. typhimurium TA 1535
S. typhimurium TA 1537
S. typhimurium TA 100
S. typhimurium TA 98
S. typhimurium TA 102
The study was performed using the plate incorporation assay with and without S9 mix. S9 is composed by Aroclor 1254 induced rat liver supplemented with glucose-6-phosphate and NADP (Moltox®). The experiment was performed in duplicate Petri dishes containing basal medium and a further layer of medium containing histidine+biotin, the Salmonella typhimurium suspension, the sample to be tested at different concentrations and, if the case of metabolic activation, a 5% S9 mix. Dishes were incubated for 48 hours at 37° C. When the incubation time was over, a basal bacterial growth was achieved limited by the amount of histidine in the medium and in add the growth of revertant colonies (his+). In particular, the basic number of revertant colonies is steady, different for each strain, due to the spontaneous mutation rate of the bacterial strain. If an increase in the number of revertant colonies is observed, this is proportional to the tested sample concentration and to its mutagen capability. In each Ames test the following parameters are considered:
S. typhimurium TA 1535: sodium azide (NaN3); S. typhimurium TA 1537:9-aminoacridine (9AA); S. typhimurium TA 100: sodium azide (NaN3); S. typhimurium TA 98:4-nitroquinoline-N-oxide (NQNO); S. typhimurium TA 102:4-nitroquinoline-N-oxide (NQNO).
The following tables report the average values as number of colony forming units (UFC)/plate for revertant obtained in the assay considering the average of 3 replicates for each dilution (Tables 4-6). According to the Ames tests, the sample of maple bark extract-lignin flower did not show any evidence of mutagenicity at the tested concentrations.
The purpose of this test is to evaluate if the tested material is, or is not a skin irritant, according to the method described in OECD 439. This method uses the human artificial skin model (EPISKIN™, EpiDerm™, Episkin™ RHE) to assess the skin irritation of chemical substances, mixtures and to properly label them to this respect if applicable.
The test is based on the evaluation of cell survival after the exposure to the substance through MTT assay and by comparison with epidermis treated with phosphate buffer only (negative control). The MTT method is a colorimetric assay that allows to determine the percentage of cells alive within an in vitro cultured tissue. This assay is based on the ability of the mitochondrial succinate dehydrogenase enzyme to metabolize the nitro-blue tetrazolium salt, giving a colored compound that can be measured by spectrophotometer reading. A reconstructed artificial human skin model comprising normal human epidermal keratinocytes, growing as an integrated three-dimensional cell culture model, perfectly mimicking the human skin in vitro. The model exhibits normal barrier functions (presence of a well-differentiated stratum corneum). It was supplied by Episkin (Lion, Batch 21-RHE-098) (
The sample is tested as is, undiluted. The positive control SLS is dissolved at 5% in water. PBS alone has been used as negative control. 16 mg of the product have been applied on each epidermis unit (32 μL/cm2), in three replicates. The exposure has been carried out for 42 h at room temperature. At the end of the exposure period the product is removed with multiple washings with PBS and the tissue was further incubated at 37° C., 5% CO2 for 42 h. After 42 h of the incubation, the viability assay is performed to evaluate the cell survival in the epidermis units.
Epidermis units are treated with 1 mg/ml MTT solution (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) for 3 h at 37° C. The solution is then removed and replaced with isopropanol, with further 2 h incubation at room temperature. 2 aliquots of every sample are transferred to a 96 well plate for the reading. The absorbance is read at the wavelength of 570 nm with a colorimeter (Tecan model Infinite 200 PRO) equipped with a microplate reader.
All of the compositions and/or processes disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and processes of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or processes and in the steps or in the sequence of steps of the processes described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are chemically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.
While the present disclosure has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations including such departures from the present disclosure as come within known or customary practice within the art and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.
The present application is claiming priority from U.S. Provisional Application No. 63/264,627 filed Nov. 21, 2021, the content of which is hereby incorporated by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2022/051743 | 11/29/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63264627 | Nov 2021 | US |
