JPWO2020046477A5 - - Google Patents

Description

TECHNICAL FIELD OF THE INVENTION The present invention relates generally to plant extracts for use in skin care applications containing inhibitors of tyrosinase, and more specifically to plant extracts from cashew seed coats and to the use of such plant-based The invention relates to the use of inhibitors as skin protectants and/or for skin tightening.

The cashew tree (Anacardium occidentale Linn) is originally from the Amazon and has since been transplanted to India, East Africa and other countries for cultivation. The tree produces a very distinctive apple or fruit in the shape of a swollen peduncle. Externally, at the end of this peduncle, the cashew nut (i.e. the seed) grows inside its own gray kidney-shaped hard shell. This shell has a soft, leathery outer skin and a thin, hard inner skin called the foreskin or seed coat that surrounds the seed. Between these two skins there is a honeycomb structure containing the liquid of the cashew nut shell. This liquid contains anacardic acid, cardanol and cardol, among other ingredients. Anacardic acid is a salicylic acid, while cardanol and cardol are substituted phenols.

Various parts of the fruit have been studied for their uses. In addition to being an edible food, the juice from cashew apples is used in beverages, while the fruit extract has shown benefits in weight management. Cashew nut shell liquids can be used in a variety of applications including friction linings, paints, laminating resins, rubber compound resins, cashew cement, polyurethane-based polymers, surfactants, epoxy resins, foundry chemicals, chemical intermediates, pesticides and fungicides. It has been extracted for many industrial and agricultural uses. Cashew seed coats have been used as a tanning material.

Disclosed herein is a plant extract containing catechins, wherein the extract is standardized to a catechin content of about 15.0% or more by weight based on the total weight of the extract, and the plant extract has a tyrosinase inhibitory content. activity and the plant extracts include extracts from at least the genus Anacardium. In particular, the plant extract is an extract from at least Anacardium occidentale L., more specifically from at least Anacardium occidentale L. (Anacardium occidentale L.) is an extract from the seed coat of seeds.

In one embodiment, at least Anacardium occidental L. Plant extracts from the pericarp of seeds exhibit antioxidant activity due to increased expression of the MT1A gene.

In further embodiments, at least Anacardim occidental L. Plant extracts from seed coats of seeds exhibit antioxidant activity due to increased expression of the NQO1 gene.

In another embodiment, at least Anacardim occidental L. Plant extracts from seed coats of seeds exhibit antioxidant activity due to increased expression of the TXNRD1 gene.

In yet another embodiment, at least Anacardim occidental L. A plant extract from the seed coat of seeds exhibits wound healing activity by suppressing the expression of the CTGF gene.

In further embodiments, at least Anacardim occidental L. Plant extracts from the pericarp of seeds exhibit anti-inflammatory activity due to increased expression of IL1α and IL1β genes.

The present invention is based on Anacardim Occidental L. There is further provided a composition comprising a plant extract of the pericarp of seeds of the invention, wherein the plant extract exhibits tyrosinase inhibitory activity. Preferably, the plant extract is present in an amount of about 1.0 μg/mL or greater. More preferably, the plant extract is present in an amount of about 1.0 μg/mL to about 2000.0 μg/mL. In one embodiment, the composition is a cosmetic composition.

Also provided herein is a dietary supplement having tyrosinase inhibitory properties and comprising a therapeutically effective amount of cashew husk extract. Preferably, the cashew seed coat extract is present in an amount of about 1.0 μg/mL or greater.

In addition, Anacardim Occidental L. Provided herein is a method of inhibiting melanin production in a subject by administering a composition comprising a botanical extract of the pericarp of a seed at a concentration of about 1.0 μg/mL to about 2000.0 μg/mL.

FIG. 1 is an HPLC chromatogram of cashew husk extract at a wavelength of 275 nm over a retention time of 0 minutes (start) to 20 minutes.

FIG. 2 is an LC/MS and LC/PDA (wavelength 280 and 350 nm) chromatogram of cashew seed coat extract.

FIG. 3 is a graph showing mushroom tyrosinase inhibition using cashew seed coat extract at various (10 different) concentrations.

The present invention is based on the surprising discovery that the seed coat of cashew (Anacardium occidentalis Linn) is substantially high in certain flavonoids. In particular, it has been discovered that extracts of cashew seed coats contain catechin and epicatechin as major components, as well as procyanidins. The data described herein indicate that cashew seed coat extract may have tyrosinase inhibition utility.

As used herein, the term "extract" or "plant extract" refers to at least the Anacardium plant (e.g., Anacardium humile, Anacardium othonianum, Anacardium giganteum, Anacardium nanum, Anacardium negren). se or Anacardium occidental), preferably Anacardium Occidental L. A solid, viscous or liquid substance or preparation containing one or more active ingredients of a substance. Preferably, the active ingredient is derived from an extract of cashew seed coat . Extracts can be extracted from water, lower alcohols of 1 to 4 carbon atoms (e.g. methanol, ethanol, butanol, etc.), ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, dichloromethane, N,N-dimethylformamide. (“DMF”), dimethyl sulfoxide (“DMSO”), 1,3-butylene glycol, propylene glycol and combinations thereof; It's also a part of it. Any extraction method may be used as long as it ensures extraction and preservation of the active ingredient.

The present invention provides plant-based inhibitors capable of inhibiting tyrosinase. More specifically, the present invention is directed to a botanical extract of cashew seed coats from the genus Anacardium. It has been found that such plant extracts are able to inhibit tyrosinase.

Useful plant extracts capable of inhibiting tyrosinase of the present invention include plant extracts from the genus Anacardium. More specifically, the plant-based inhibitor is one of Anacardium humile, Anacardium othonianum, Anacardium giganteum, Anacardium nanum, Anacardium negrense and/or Anacardium occidental. It is a plant extract selected from the above species. Preferably, the plant extract is from the species Anacardium occidentalis. In one embodiment, the plant extract is from the seed coat of the species Anacardium occidentalis.

Generally, one or more parts of a plant can be used to produce a plant extract, including, but not limited to, roots, stems, leaves, flowers, fruits, seeds, and seed coats of seeds. . In the present invention, at least the pericarp of seeds is used alone or together with other plant parts to produce plant extracts. Seed coats from Anacardim plants are commercially available from a variety of sources. Extracts of cashew seed coats can be obtained using any suitable extraction technique.

In this regard, one or more parts of the plant, in particular the seed coat of the plant, can be collected and ground. The ground material can then be extracted using a suitable solvent. The solvent can be removed in a concentration step. For example, the extracted material can be screened or filtered to produce a supernatant and a filter cake. The sieve can be expressed to remove a substantial portion of the liquid, which can be added to the supernatant. The sieve can then be dewatered and used as a fiber source. The supernatant can be distilled to remove the solvent or a portion thereof to form a plant extract concentrate. The removed solvent can be recycled. The concentrate can be dried (eg, by spray drying) to provide a dry plant extract. This dried plant extract can be assayed or standardized as described herein. Preferably, the dried plant extract is derived from the seed coat of Anacardium occidentalis, especially Anacardium occidental plants.

In one embodiment, the plant extract can be obtained using organic solvent extraction techniques. In another embodiment, continuous solvent fractionation can be used to obtain plant extracts. Total hydroethanol extraction techniques can also be used to obtain plant extracts. This is commonly referred to as bulk extraction.

In one embodiment, the plant extract is about 4 μg/mL or more, preferably about 4.0 μg/mL to about 2000.0 μg/mL, more preferably about 15.0 μg/mL to about 1000.0 μg/mL, More preferably, it is present in the composition in a therapeutically effective amount, such as an amount of about 30.0 μg/mL to about 500.0 μg/mL. The composition can be administered as a single dose or in multiple doses. In one example, the compound is administered in up to three doses per day. For example, the compound may be administered before, during, or after a meal. In one embodiment, the composition is a dietary supplement with tyrosinase inhibitory properties comprising a therapeutically effective amount of cashew husk extract.

Example 1 Preparation of cashew husk extract using 70% ethanol solvent Dried cashew husk powder (Anacardium occidentalis) (60 g) was placed in three 100 mL stainless steel tubes and placed in a Thermo Scientific™ Dionex™ tube. ) Extracted twice with a solvent containing 70% ethanol in deionized water at a temperature of 80° C. and a pressure of 1500 psi in an ASE350 accelerated solvent extractor. The extract was filtered and collected. The combined ethanol extracts were evaporated on a rotary evaporator under vacuum to obtain a crude cashew seed coat extract.
The extraction results are shown in Table 1 below.

Example 2 Catechin quantification in cashew seed coat extract Free catechins present in cashew seed coat extract were quantified using a C18 Measurements were made using a reverse phase column (Luna® 5 μm C18(2) 100 angstrom LC column 250 x 4.6 mm, available from Phenomenex® (Torrance, CA, USA). Mobile phase A. The solvent for is 0.10% phosphoric acid (“H ₃ PO ₄ ”) aqueous solution, and the solvent B for mobile phase B is acetonitrile (“ACN”), which has a flow rate of 1.0 mL/min, UV absorbance at 275 nm and column temperature of 35° C. were used for elution. The catechin standards used were from Sigma-Aldrich. The standards were prepared in methanol (“MeOH”): 0.1% of H ₃ PO ₄ (ratio 1:1), the catechin (C1251) concentration was 0.5 mg/mL and the epicatechin (E1753) concentration was 0.1 mg/mL. Prepare to 2 mg/ _mL in 50% MeOH in 0.1% _HPO , sonicate until dissolved (approximately 10 min), then cool to room temperature, mix well, and filtered with a 0.45 μm nylon syringe filter. HPLC analysis was performed by injecting 20 μL of sample into the HPLC. Table 2 below shows the gradient table for the HPLC analysis method.

HPLC catechin quantification results of cashew pericarp extract showed catechin content of 9.40% by weight and epicatechin content of 6.12% by weight, total catechin content of 15.52% by weight, based on the total weight of the extract. provided the content. Accordingly, the cashew husk extract can be standardized to a total catechin content of about 15.00% or more by weight based on the total weight of the extract. The HPLC chromatogram of the cashew seed coat extract at a wavelength of 275 nm is shown in FIG.

Example 3 Chemical Profiling of Cashew Peel Extract The flavonoid compounds present in cashew seed coat extract were determined by ultra-high pressure liquid chromatography (“HPLC”) and mass spectrometry (ACQUITY® UPLC I class and XEVO® ) GS-XT-QTof system, both available from Water Corporation (Milford, Mass., USA). The column used was ACQUITY (registered trademark) UPLC HSS T3 2.1 x 100 mm, 1.8 μm, the column temperature was 40°C, and the sample temperature was 15°C. For mobile phase, solvent A was 10% acetonitrile ("ACN") in water (0.1% formic acid) and solvent B was ACN. The capture range was 100-1500 Daltons ("Da") and the capture mode was electrospray ionization ("ESI") (-). Table 3 below shows the HPLC conditions.

Peak identification was based on exact mass only. Digallocatechin, catechin and epicatechin were identified as the main components of cashew seed coat extract. Procyanidins, including type A and B procyanidins, procyanidin tetramers and procyanidin trimers, were also detected in the extract, with type B procyanidins being the main component of procyanidins. In addition to the above-mentioned compounds, the identified compounds included digallocatechin, bacsihain A, 6″-p-coumaroylpurunin and zunarianoside B, among others. Of the cashew pericarp extract obtained from the analysis The LC/MS and LC/PDA chromatograms are shown in Figure 2.

Examples - Bioassay Extracts of cashew seed coats were prepared in food grade ethanol, then filtered and dried as described above. Research grade reagents were used for the remaining assay preparations. Extracts were dissolved in dimethyl sulfoxide ("DMSO") to a final concentration of 50 mg/mL and then diluted to working concentrations with the appropriate buffer for each bioassay.

Cashew seed coat extract was analyzed for its inhibitory effect on mushroom tyrosinase enzyme. Cashew seed coat extract was diluted in assay buffer (100 mM sodium phosphate, pH 6.8) with 10% dimethyl sulfoxide (“DMSO”) to test the concentration. L-DOPA amino acid substrate solution was prepared in fresh assay buffer. Samples and L-DOPA (1 mM final concentration) were mixed in a microplate before adding 10 units of mushroom tyrosinase enzyme. Absorbance at 492 nm was read at 5 and 10 minutes and calculations were made using the 5 minute data. Percent inhibition was calculated relative to untreated controls.

Mushroom tyrosinase activity was measured by mixing mushroom tyrosinase with substrate (L-DOPA) in the presence of potential inhibitors and measuring the presence of the reaction product at 492 nm. FIG. 3 shows the mushroom tyrosinase inhibition rate of the cashew seed coat extract. From FIG. 3, it can be seen that cashew seed coat extract has the effect of inhibiting mushroom tyrosinase enzyme. As shown, mushroom tyrosinase inhibition of at least 50% occurs at cashew seed coat extract concentrations of about 15.0 μg/mL or higher. More specifically, mushroom tyrosinase inhibition of at least 50% is about 15.0 μg/mL or more, preferably about 15.0 μg/mL to about 2000.0 μg/mL, more preferably about 125.0 μg/mL to about Occurs at a concentration of 1000.0 μg/mL. In particular, cashew seed coat extract exhibits an IC ₅₀ of approximately 391 μg/mL.

Cashew seed coat extract was investigated to determine how it affects gene expression in the skin when added to the culture medium. The study was conducted using a full-thickness in vitro skin culture model containing epidermal and dermal cell layers (EpiDermFT™ (full-thickness) tissue model manufactured by MatTek Corporation (Ashland, MA, USA)). . Gene expression was assessed in full-thickness tissue after 24 hours of exposure.

A control ("Vehicle Control") was prepared as a blend of dimethyl sulfoxide ("DMSO"), ethanol and water in a 70:20:10 ratio. To measure cell viability, three different concentrations (500 μg/mL, 1000 μg/mL and 2000 μg/mL) of cashew seed coat extract were applied to the cell culture medium. Equivalent amounts of vehicle controls were also applied to each at 0.5%, 1.0% and 2.0%, respectively. One culture was treated with 1% Triton X-100 for 24 hours and was used as a negative (-) MTT control. Untreated cultures served as positive (+) controls. After 24 hours of incubation, MTT assay was performed.

Cell viability was less than 80% (74%) at a concentration of 2000 μg/mL of cashew seed coat extract, but 100% at a concentration of 500 μg/mL (126%) and 1000 μg/mL (105%). It was more expensive. A concentration of 500 μg/mL was chosen for gene expression analysis.

Treatment and Maintenance of Cultures EFT-400 tissues (Mattech) were equilibrated overnight at 37° C., 5% CO ₂ and approximately 95% relative humidity. The next day, the equilibration medium was removed and replaced with cashew seed coat extract medium. A volume of 2.5 mL of medium was added to each EFT-400 culture well.

MTT portion Each group contained three homogeneous cultures. For MTT(+) tissues, three tissues were cultured in untreated EFT-400-ASY medium. For MTT(-) tissues, a volume of 100 μL of 1% Triton X-100 was applied to the surface of each EFT culture and incubated with untreated EFT-400-ASY medium. After adding the cashew seed coat extract medium, the culture was returned to the incubator with 5% _CO2 and approximately 95% relative humidity for 24 hours.

Gene expression section Each group contained four replicates. After adding the cashew seed coat extract medium, the culture was returned to the incubator at 37 °C, 5% _CO2 , and approximately 95% relative humidity for 24 hours. After 24 hours, extract media from each well was collected for LDH assay (described below). Each EFT-400 culture was then cut into quarters and incubated in tubes containing RNAlater storage solution for 2 hours at room temperature before being transferred to 4°C. After 4 days of incubation at 4°C in RNAlater storage solution, RNA was isolated from each tissue as described below.

LDH Cytotoxicity Assay The collected EFT medium (cashew seed coat extract test medium) was diluted 1:10 with sterile phosphate buffered saline (“PBS”). LDH reaction mixture (TaKaRa Bio Inc.) was prepared and added to an aliquot of diluted medium in an optically clear flat bottom 96 well plate (1:1). background control (diluted EFT medium not used for cell culture), "low control" (diluted EFT medium collected from untreated EFT culture wells), and "high control" (treated with 1% Triton X-100). Diluted EFT media collected from culture wells) were included in the analysis. Each sample was analyzed in two reaction wells.

Statistical data analysis using TIBCO Spotfire software. Tibco Spotfire software was used with and without applying the optional Benjamini-Hochberg false discovery rate (FDR) p-value adjustment. An unpaired t-test was performed. Statistically significant (unpaired t-test, FDR p≦0.05, N=4) changes in gene expression are reported in Table 4 below.
"n.s" = not statistically significant. Linear changes greater than 1.5 times are shown in bold. Linear changes less than 1.5 times are shown in parentheses. Shaded values were passed through the Benjamini-Hochberg false discovery rate (FDR) to adjust the p-value (both the unadjusted p-value and the FDR p-value are p≦0.05).

Table 4 above shows statistically significant (unpaired t-test, FDR p≦0.05, N=4) changes in gene expression of cashew husk extract compared to vehicle control group. Gene expression was analyzed in a full-thickness in vitro skin culture tissue model as described above. Additional analysis of separate layers (epidermis and/or dermis) may identify additional changes in gene expression or changes in already regulated ones that may have been masked by analyzing both cell types together. May increase the fold change value.

Metallothionein 1A (“MT1A”) and metallothionein 2A (“MT2A”) are involved in antioxidant detoxification. Increased metallothionein leads to reduced free radical activity, reduced oxidative damage and reduced inflammation. Table 4 above shows a significant increase in MT1A expression in the presence of cashew seed coat extract, indicating both antioxidant and anti-inflammatory activity in the skin.

NAD(P)H:quinone oxidoreductase-1 (“NQO1”) is an antioxidant stress-responsive gene that encodes an enzyme that protects cells from oxidative damage induced by chemicals and radiation. AHR and NRF2 transcription factors control the expression of NQO1. The level of NQO1 correlates with the expression of p63 in epidermal keratinocytes and dermal fibroblasts, and plays a role in regulating cell proliferation and differentiation. NQO1 is involved in antioxidant detoxification and can protect cells from further damage by slowing down tyrosinase degradation. Table 4 above shows that cashew seed coat extract significantly increases the expression of NQO1 and therefore functions as an antioxidant.

Thioredoxin (“TXN”) and thioredoxin reductase 1 (“ _TXNRD1 ”) are involved in the antioxidant response of fibroblasts, as seen upon exposure to _H2O2 , to repair and protect against further injury. , opening redox-sensitive signaling pathways. This allows cells to adapt to oxidative stress. TXNRD1 expression is downstream of NFE2L2 (nrf2) activation. TXNRD1 is also induced by exposure to resveratrol. Table 4 above shows that cashew seed coat extract significantly increases the expression of TXNRD1 and therefore functions as an antioxidant.

Connective tissue growth factor (“CTFG”) is a gene that encodes a protein. Diseases associated with CTGF include systemic scleroderma and renal fibrosis. Associated pathways include ERK signaling and gene expression. Gene Ontology (GO) annotations associated with this gene include growth factor activity and integrin binding. Overexpression of CTGF in fibroblasts promotes fibrosis in the dermis, kidney and lung, and deletion of CTGF in fibroblasts and smooth muscle cells significantly reduces bleomycin-induced skin fibrosis. Table 4 shows a significant decrease in the expression of CTFG in the presence of cashew seed coat extract, indicating that the extract reduces fibrosis, possibly preventing the more significant increase in collagen thickening in dermal fibrosis. This indicates that it may have the activity of

Interleukin 1, beta (“IL1β”) is a cytokine that is upregulated during inflammatory responses and upon barrier disruption. In skin diseases such as psoriasis, IL1β may disrupt epidermal homeostasis by blocking differentiation while proliferation persists. This cytokine is produced by activated macrophages as a proprotein and is proteolytically processed into its active form by caspase 1 (CASP1/ICE). This cytokine is an important mediator of the inflammatory response and is involved in a variety of cellular activities including cell proliferation, differentiation, and apoptosis. Cashew seed coat extract significantly increases the expression of IL-1β, indicating its potential use in mediating inflammatory activities in the skin.

BIRC5 (Survivin) is an anti-apoptotic protein expressed in basal keratinocytes, possibly a subpopulation of keratinocyte stem cells. B1RC5 has an anti-apoptotic effect on cells exposed to UV, and BIRC5 can inhibit apoptosis by inhibiting the activation of caspases. Overexpression of BIRC5 can rescue cells from p53-induced apoptosis. BIRC5 is upregulated in psoriasis and overexpressed in aging dermal fibroblasts. As shown in Table 4, the decreased expression of BIRC5 suggests an anti-aging effect of cashew seed coat extract.

The above data is based on Anacardim Occidental L. It has been demonstrated that plant extracts of the seed coats of the present invention have one or more compounds that exhibit inhibition of tyrosinase. Additionally, in vitro studies of cashew seed coat extracts have shown that cashew seed coat extracts lead to changes in genes related to antioxidant protection, cellular renewal and regeneration, wound healing, anti-aging, and immune/inflammatory responses. It has been suggested that it increases the expression of antioxidants and anti-inflammatory related genes present in the skin while suppressing genes involved in scarring.

Claims (10)

Anacardim Occidental L. (Anacardium occidentale L.) is a composition containing a plant extract of the outer seed coat of seeds, the plant extract is standardized to a catechin content of 15.0% by weight or more based on the total weight of the extract, and is a plant. A composition in which the extract is present in an amount of 125.0 μg / mL to 1000.0 μg / mL and the composition exhibits tyrosinase inhibitory activity. The composition according to claim 1, further comprising a carrier . The composition according to claim 1, wherein the composition is a cosmetic composition. The composition according to claim 1, wherein the composition is a dietary supplement. The composition according to claim 1 , wherein the composition exhibits antioxidant activity due to increased expression of the MT1A gene. The composition according to claim 1 , wherein the composition exhibits antioxidant activity due to increased expression of the NQO1 gene. The composition according to claim 1 , wherein the composition exhibits antioxidant activity due to increased expression of the TXNRD1 gene. The composition according to claim 1 , wherein the composition exhibits wound healing activity by suppressing the expression of the CTGF gene. The composition according to claim 1 , wherein the composition exhibits anti-inflammatory activity due to increased expression of the IL1α gene and the IL1β gene. Anacardim Occidental L. A method of inhibiting melanin production in a subject, comprising administering a composition comprising a plant extract of the outer seed coat of (Anacardium occidentale L.), wherein the plant extract is 15 relative to the total weight of the extract. A method in which a plant extract is present in an amount of 125.0 μg / mL-1000.0 μg / mL, standardized to a catechin content of 0.0% by weight or more .