Extract of undifferentiated cells of <i>Mimosa pudica </i>and uses thereof in dermo-cosmetics

Abstract

The present invention relates to a preparation obtained from an in vitro culture of undifferentiated cells of Mimosa pudica, as well as to the preparation method thereof; a cosmetic or dermatological composition comprising said preparation; and the uses thereof for the treatment of inflammatory skin conditions, as an antioxidant agent in the treatment of oxidative stress caused by environmental pollution, and as an anti-aging agent.

Claims

1. A process for in vitro preparation of a Mimosa pudica cell extract having a mimosine content of less than 5 ng/g dry weight, comprising the following steps: a. providing sterile plant material of Mimosa pudica, b. dedifferentiating cells from the plant material, c. forming a suspension culture of the undifferentiated cells in a liquid medium for maintaining said cells in the undifferentiated state, d. propagating an undifferentiated cell biomass in the culture medium, e. stopping the propagation and obtaining a cell extract having a mimosine content of less than 5 ng/g dry weight; wherein the culture medium of step c) and/or step d) contains a phenyl-ammonia-lyase substrate.

2. The process according to claim 1, wherein the Mimosa pudica plant material is selected from the group consisting of leaf, stem, petiole, root, seed, flower and bud.

3. The process according to claim 1, wherein step b) is carried out on a solid medium containing one or more growth factors.

4. The process according to claim 1, wherein step b) is repeated so as to obtain calluses of dedifferentiated cells.

5. The process according to claim 1, wherein step c) is carried out in a liquid medium containing one or more growth factors.

6. The process according to claim 5, wherein the one or more growth factors are the same as that or those of the dedifferentiation medium.

7. The process according to claim 1, further comprising the following additional steps: f. liquid/solid separation, g. recovering a cell extract consisting of biomass separated from the culture medium having a mimosine content of less than 5 ng/g dry weight.

8. The process according to claim 1, further comprising an additional step of crushing the extract and recovering a crushed cell material having a mimosine content of less than 5 ng/g dry weight.

9. The process according to claim 1, wherein the resulting extract contains at least one N-phenylpropenoyl amino acid.

10. The process according to claim 9, wherein the N-phenylpropenoyl amino acid is selected from the group consisting of: P1: 1-O-(4-coumaroyl)--D-glucose C.sub.15H.sub.18O.sub.8 ##STR00016## P2: N-p-Coumaroylaspartic acid or Aspartic acid; (S)-form, N-(4-Hydroxycinnamoyl C.sub.13H.sub.13NO.sub.6 ##STR00017## P3: N-cis-(p-Coumaroyl)glutamic acid or Glutamic acid; (S)-form, N-(4-Hydroxy-Z-cinnamoyl) C.sub.14H.sub.16NO.sub.6 ##STR00018## P5: 4-hydroxycinnamide C.sub.9H.sub.9NO.sub.2 ##STR00019## P6: Glutamic acid; (S)-form, N-cinnamoyl C.sub.14H.sub.14NO.sub.5 ##STR00020## mixtures thereof.

Description

EXAMPLE: PLANT CELL CULTURE IN A WAVE BIOREACTOR (5 L)

(1) Materials and methods: The WAVE reactor (Sartorius) with a useful volume of 5 L containing 4 L of MS SENS medium (see table above for composition) was inoculated with 1 L of M. pudica cell culture suspension (cell density between 300 and 400 g/L in fresh weight (FW))

(2) Parameters:

(3) Temperature: 27 C. Air volume: 0.5 L/min (lpm) Pressure: 5 mPa to 10 mPa Rocking angle: 7. pO.sub.2 maintained at 75% by increasing rpm between D0 (19 rpm) and D9 (27 rpm) pO.sub.2 maintained at 75% by enriching the air with O.sub.2 between D9 (5% O.sub.2) and D15 (20% O.sub.2) at 25 rpm

(4) By following the growth kinetics, an increase in biomass evaluated by fresh weight (FW) or dry weight (DW), correlated with sugar consumption (decrease in the curve), is observed until the 13.sup.th day of culture, D13, when the bioconversion medium is added.

(5) Bioconversion and Harvesting

(6) After 13 days (D13) of batch culture of a 4 L culture, the cell concentration is between 300 and 350 g/L (FW), equivalent to 12 to 14 g/L (DW). When this cell concentration is reached, bioconversion is carried out by injecting the precursors by sterilizing filtration: we have selected the various amino acids (aspartic acid, cinnamic acid, glutamic acid or L-phenylalanine), and under our culture conditions, L-phenylalanine gave the best results in terms of NPA bioconversion yield. Below is an example of the following precursor concentrations:

(7) TABLE-US-00002 Extemporaneous Concentration Concentration Final volume of preparation (DW (Suspension) DW (ml) NH.sub.4H.sub.2PO.sub.4 50 g/L 0.1 g/L 10 L-Phenylalanine 300 mM 6 mM 100 Sucrose 500 g/L 10 g/L 100
Bioconversion Medium Favouring Hydroxycinnamic Acids Including their Derivatives, N-phenylpropenoyl Amino Acids (NPAs)

(8) Dilute the compounds in H.sub.2O and add the mix to the bioreactor by sterilizing filtration. After 48 h, i.e. between D15 and D16, harvest the biomass by direct filtration using a 20 m nylon bag for example. The biomass is washed once with a volume of sterile demineralized water equivalent to the volume of biomass harvested. The cell concentration evaluated at D13 must be equivalent (+/10%) to D15. Throughout the culture, the decrease in suspension volume is mainly due to the samples taken.

(9) A volume of the culture is taken at D13, D14, D15 and D16, then extracted with a solvent and the various extracts are dissolved in before being analysed by HPLC coupled with mass spectrometry. The figure shows, relative to D13 (the day the precursor L-phenylalanine was added), the gradual appearance of the new peaks D14, D15 until the maximum intensity is reached at D16.

(10) Peaks P1, P2, P3, P4, P5 and P6 were isolated at D16, analysed by mass spec. and NMR to determine the structure of the molecules. Peak P4 being in a very small amount, we were unable to determine its structure. For the first time, and surprisingly, we discover that the precursor L-phenylalanine was the best amino acid for bioconversion.

(11) NPA products P1, P2, P3, P5 and P6 were identified by mass spectroscopy and NMR.

(12) List of NPA Names:

(13) P1: 1-O-(4-coumaroyl)--D-glucose C.sub.15H.sub.18O.sub.8 P2: N-p-Coumaroylaspartic acid or Aspartic acid; (S)-form, N-(4-Hydroxycinnamoyl) C.sub.13H.sub.13NO.sub.6 P3: N-cis-(p-Coumaroyl)glutamic acid or Glutamic acid; (S)-form, N-(4-Hydroxy-Z-cinnamoyl) C.sub.14H.sub.15NO.sub.6 P5: 4-hydroxycinnamide C.sub.9H.sub.9NO.sub.2 P6: Glutamic acid; (S)-form, N-cinnamoyl C14H14NO5

EXAMPLE 2: ASSAYS OF MIMOSINE IN MIMOSA PUDICA PLANT CELLS Versus Leaves

(14) The objective is to develop the analytical conditions for identifying and quantifying mimosine in the extracts. Biomass derived from suspension culture of M. pudica cells (with bioconversion) was extracted with ETOH80 or ETOH60. Dried leaves of M. pudica were dried, crushed and extracted with the same solvents. L-Mimosine from Sigma is used as reference for identification and quantification. The samples were analysed by HPLC/mass spectrometry.

(15) Materials and Methods: Analytical Conditions

(16) ABSciex TripleTOF 4600 mass spectrometer Method (ES+): TOF (100 ms)/MRM (50 ms) Column: Acquity HSS C18, 1.8 m, 2.1100 mm, sintered 0.5 m Eluents: A (H.sub.2O-0.1% HCO.sub.2H)/B (CH.sub.3CN-0.1% HCO.sub.2H) Flow rate: 500 l/min Injections: 10 l Gradient:

(17) TABLE-US-00003 Time (min) % A % B 0.0 100 0 1.0 100 0 4.0 10 90 4.01 0 100 5.0 0 100

(18) The quantitative assays established that the lower limit of quantification (LLOQ) reached in the control solution is 5 ng/mL. This LLOQ and the preparation method of our hydro-ethanolic samples confirm the absence of mimosine in our cell culture samples at the limit of detection (1 ng of mimosine per g of biomass): EtOH80 and EtOH60 extracts of cells (20% w/w)<1 ng/g of fresh cells EtOH60 extract of freeze-dried cells (5% w/w)<1 ng/g of freeze-dried cells EtOH60 extract of plants=2160 ng/g of dry plant. EtOH80 extract of plants=780 ng/g of dry plant

(19) The analyses were carried out in the same way on the PCCs without bioconversion (i.e. without addition of NPA precursors) and showed that the mimosine content is also <1 ng/g of fresh or freeze-dried cells.

(20) In conclusion, in our M. pudica plant cell cultures, surprisingly, we did not detect any undesirable element such as mimosine.

EXAMPLE 2ANTI-INFLAMMATORY ACTIVITIES: COMPARISON OF PLANT EXTRACT VS. PCC Extract

(21) In this example, we compare the anti-inflammatory activity of 2 Mimosa pudica extracts (E11 and E13). Extract E11 comes from ethyl acetate extraction of the dried and crushed Mimosa pudica leaves and extract E13 comes from extraction with the same solvent of the crushed materials derived from Mimosa pudica plant cell culture without bioconversion in an Erlenmeyer flask. The 2 extracts were weighed after evaporation of the solvent to dryness. They were taken up in DMSO. To evaluate and compare the anti-inflammatory activity of the 2 extracts in the same concentrations, we have an in vitro pharmacological test which consists in stimulating murine macrophages, RAW264.7 cells, that express on their surface the TLR4 receptor by the bacterial LPS according to (Kang et al. 2002. J Pharmacol Exp Ther. 302:138-144). Several parameters were studied and compared to a reference anti-inflammatory, dexamethasone: nitrite production by the Griess technique. The nitrite level reflects the level of NO synthesis induced by inducible nitrite oxide synthase (iNOS). cytokine secretion: interleukin-6 by ELISA TNF-alpha production by multiplex assay
Cell Culture

(22) RAW264.7 cells are mouse macrophages in lineage. These cells are adherent and cultured in a 12-well plate at 100000 cells/cm.sup.2 (Millipore Scepter count) in DMEM medium supplemented with 10% FCS, 2 mM L. glutamine and 50 g/mL gentamicin.

(23) At about 70% confluence, the cells are treated with Mimosa pudica extracts E11 (plant) or E13 (PCC) in the same concentrations (dry weight) or with the reference anti-inflammatory 1 M dexamethasone (Biovison 1042-1) 1 h before being activated by 0.2 g/mL [E. coli 055:B5] LPS. The cells are incubated at 37 C. under 5% CO2. After 24 h, the cell supernatants are recovered in ice, centrifuged for 5 min at 3000 rpm at 4 C., aliquoted then stored at 80 C. The cell tests were repeated 3 times, including twice in duplicate. The average values of the results are presented in the graphs.

(24) Cell viability is monitored by a metabolic test with MTT (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide) using the SIGMA CGD1 Kit (kit based on the activity of an enzyme, mitochondrial succinate dehydrogenase). This test was done beforehand to determine the doses of the extracts to be tested in this model.

(25) Nitrite Assay

(26) The level of NO synthesis is evaluated in fresh or frozen (80 C.) cell supernatants, without affecting the assay. The principle of the assay is based on the Griess (diazotization) reaction which produces a pink azo compound with absorbance at 540 nm.

(27) IL6 Assay

(28) IL-6 is assayed in the cell supernatants diluted to 1/200.sup.th by colorimetric ELISA, according to the supplier's protocol (R&D Systems, kit M6000B).

(29) TNF Alpha Assay

(30) Simultaneous assay of TNF-alpha in the cell supernatants was performed using the Luminex xMAP (multi-analyte profiling) technology which is based on the principles of flow cytometry and ELISA in a 96-well microplate. The microbeads used as substrate incorporate two fluorochromes in a precise ratio, which gives them an identifying colour code (different fluorescences). The optical system of the cytometer (Bio-Plex 200) consists of two lasers: a red laser (=635 nm) excites in each microbead its defining dye mixture, and thus identifies the cytokine to be assayed. The second laser, green, (=532 nm) excites the reporter fluorochrome attached to the specific detection antibody in order to quantify the cytokine. The system is controlled by a computer equipped with data acquisition and analysis software (Bio-Plex Manager version 4.1).

(31) After thawing, the supernatants were tested diluted to 1/10.sup.th and 1/40.sup.th in culture medium, using a Milliplex Kit (MILLIPORE, item number MCYTOMAG-70K-04). The latter includes specific beads, detection antibodies and standards for assaying cytokines.

(32) Results

(33) Concentrations are expressed as averages. The inhibition percentages, when cited for the extracts, are relative to the 0.2% DMSO control. DMSO is used to dissolve the dry sample beforehand, before dilution in aqueous buffer for the cell tests.

(34) Figure Legends:

(35) FIG. 1: NITRITE ASSAY

(36) FIG. 2: IL6 ASSAY

(37) FIG. 3: TNF-alpha ASSAY

(38) The various samples are listed on the X-axis (from left to right): O: negative controlculture of RAW246.7 cells activated by 0.2 g/mL LPS. DEXA: positive controlincubation of cells with 1 M dexamethasone (Biovison 1042-1) 1 h before being activated by 0.2 g/mL LPS. DMSO: incubation with the solvent control used to solubilize (E11 and E12) 1 h before being activated by 0.2 g/mL LPS. E11: incubation with extract derived from M. pudica leaves (50 g/mL) 1 h before being activated by 0.2 g/mL LPS. E13: with extract derived from plant cells (50 g/mL) 1 h before being activated by 0.2 g/mL LPS.
Nitrite Assay

(39) NO production is exclusively inducible and significantly inhibitable by dexamethasone (% i=35). 0.2% DMSO has no impact on the assay. Plant extract E11 weakly inhibits (% i=16%) nitrite production while the extract E13 inhibits nitrite production in an equivalent way and close to the inhibition obtained with the reference anti-inflammatory (% i=32%).

(40) IL-6 ELISA

(41) Cells activated (O) with LPS secrete IL6. Dexamethasone (DEXA) inhibits IL-6 secretion (% i=38%). The extracts inhibit IL-6 secretion for E11 (% i=24%) and for E13 (% i=31%)

(42) Luminex Assay of TNF-Alpha

(43) RAW264.7 cells secrete TNF-alpha in the basal state (0.23 ng/mL on average). This production is activated by 0.2 g/mL LPS up to 10 ng/ml TNF-alpha. This activation is weakly inhibitable by dexamethasone (% i=16%). PCC extract E13 reduces TNF-alpha production (% i=40%). Conversely, plant extract E11 increases TNF-alpha production and seems to potentiate the activity of LPS (+112%).

(44) In conclusion, the results show that the 2 extracts E11 and E13 at the same concentration strongly inhibit inflammation, reflected in this model by: inhibition of nitrites NO, inhibition of pro-inflammatory cytokine IL6 like the control, DEXA. Surprisingly, under these experimental conditions, PCC extract E13, better than DEXA, inhibits TNF-alpha while plant extract E11 potentiates it.

EXAMPLE 3: ANTIOXIDANT ACTIVITYORAC TEST

(45) Antioxidant activity was evaluated using an oxygen radical absorbance capacity (ORAC) test (Dvalos A. et al.; Polish journal of food and nutrition sciences; 2003; 12/53:133-136). The ORAC value is used to evaluate the antioxidant capacity of an extract. AAPH (2,2-azobis-2-methyl-propanimidamide, dihydrochloride) is the source of peroxyl free radicals. In this test it is used to mimic the kinetics of fluorescein degradation by free radicals. This results in a decrease in fluorescence over time and the concentration of the extract or reference tested (here Trolox). Trolox (an analogue of vitamin E) is known as a strong antioxidant. The addition of Trolox (standard solutions) or extract protects fluorescein from degradation. This makes it possible to evaluate an anti-oxidant activity measured against Trolox standard solutions. Extracts of Mimosa pudica PCC with a bioconversion step (NPAs). Batch WO2. Samples taken on D13, bioconversion day, on D14 at 10 am, on D14 at 4 pm, D15, 16 and D17. Dilution of sampled extracts in water.

(46) ORAC Test

(47) Solutions used: all solutions are prepared in phosphate buffer 75 mM, pH 7.6. 1.17 M fluorescein (use of a 117 mM stock solution, storage 1 week at 4 C.). 125 mM AAPH to be prepared extemporaneously. 1 mM Trolox (storage 20 C.)

(48) Preparation of Trolox Standard Solutions:

(49) TABLE-US-00004 1 mM Trolox Phosphate Trolox [ ] pmoles of Tubes (l) buffer (l) in M Trolox 1 40 960 40 800 2 30 970 30 600 3 20 980 20 400 4 10 990 10 200 5 5 995 5 100 6 2.5 997.5 2.5 50 7 0 1000 0 0
Reaction volume: 200 l

(50) Place the following in a black 96-well plate: 20 l of antioxidant (Trolox standard solutions or extract to be tested (crushed cell culture medium))+160 l of 1.17 M fluorescein. Incubate the film-covered plate 15 min at 37 C. Add 20 l of 125 mM AAPH/well. Immediately incubate at 37 C. in a spectrofluorometer (SpectraMax) and take a reading every minute for 90 min at an excitation wavelength of 485 nm and an emission wavelength of 520 nm. Each test is carried out in triplicate. Calculate the areas under the curve (AUCs) for each of the tests (Trolox standard solutions or samples). Determine net AUCs=AUC standard solution point or sampleblank AUC. Plot the calibration curve: Trolox concentration (M) as a function of net AUC.

(51) The linear equation is used to determine a Trolox equivalent (M) for each of the samples assayed.
Eq Trolox in M=a(Auc net)2+b(AUC net)

(52) (a and b of which are determined by the linear equation).

(53) The ORAC value corresponds to pmoles Trolox/100 g extract
ORAC value (TEAC)=Trolox eq in M20(100 000/[of the extract tested]20)

(54) FIG. 4 shows the antioxidant activity of the extract containing 75 mg dry weight per ml, diluted to 1/200th; before and after the bioconversion step.

(55) Results:

(56) On the x-axis, a PCC extract WO2 taken after X days of culture (time) D13, D14 at 10 am, D14 at 4 pm, and every 24 h after D15, D16, D17 and the activity of the last sample, the 40% ETOH blank control alone (no antioxidant activity). It is seen that at D14, after 13 days of culture and 1 day after bioconversion (addition of AA), the TEAC (M eq of TROLOX) quintupled to stabilize until D16-D17. In conclusion, bioconversion made it possible to potentiate the antioxidant activity by virtue of the presence of NPAs.

EXAMPLE 4: PHARMACOLOGYACTIVITY: IN VITRO MODEL OF INDUCED ATOPIC Dermatitis (AD)

(57) Multiparametric evaluation of the anti-inflammatory activity of Mimosa pudica in an in vitro model exhibiting an atopic dermatitis phenotype. The pharmacological model was described by Castex-Rizzi et al. (Br J Dermatol. 2014. 170 Suppl 1:12-8)

(58) 4.1. Extracts and Compounds

(59) PCC extracts collected from crushed, dried, standardized Mimosa pudica culture medium W01D15 (with bioconversion) were dissolved/diluted in solution in 40% EtOH at initial concentrations of 75 mg/ml. The compounds were solubilized extemporaneously for viability tests as well as at the appropriate dose in order to measure pharmacological action on the model of induced atopic dermatitis.

(60) 4.2 Cell Type

(61) Normal human epidermal keratinocytes (NHEKs) from Lonza. The cells are amplified under standard culture conditions.

(62) 4.3 Induction of an Atopic Dermatitis Phenotype

(63) NHEK cells are seeded and cultured in Keratinocyte-SFM culture medium. The culture medium is then replaced with medium containing the compounds and extracts to be tested or solvent used as control (EtOH40% in concentrations equivalent to those used during treatment with the compounds). After a pre-incubation of 1 h the inflammation inducer mixture (Poly (I:C), IL4, IL13) is added and the cells are cultured for 24 h.

(64) A control without inducer and without compound is also carried out in parallel, allowing us to validate the induced model (NHEK vs NHEK+inducers).

(65) NHEK cells are also treated with a reference product, 0.29 mM dexamethasone, and used as efficacy control.

(66) RNA is extracted from the cells after 24 hours of incubation with the inducer mixture.

(67) 4.5 Analysis of Differential Expression by RT-qPCR

(68) 4.5.1. Extraction of Total RNAs and cDNA Synthesis

(69) Extraction was carried out using RNABle (Eurobio) and the RNeasy Mini Kit from QIAGEN. The total RNAs extracted are assayed on a spectrophotometer (NanoDrop, ND1000, Thermo Scientific) and their qualities analysed on agarose gel.

(70) 4.5.2. The Quantitative PCR Technique

(71) Principle

(72) Real-time PCR is a precise, sensitive and rapid method that allows the relative quantification of the rate of expression of a target gene relative to that of a ubiquitously expressed reference gene. This technique makes it possible to quantify messenger RNA. This operation is carried out after reverse transcription of the RNAs into complementary DNA by extension of two primers located on either side of the target to be amplified using a DNA polymerase. Incorporation of a fluorophore (SYBR Green) during the hybridization step of exponential amplification allows quantification and real-time monitoring of the amount of neo-formed amplification product. Quantitative values are obtained from the number of threshold cycles (Ct: for Cycle threshold) at which the signal increase, associated with exponential growth of the PCR product, begins to be detected using a Biosystems PE analysis program according to the manufacturer's manual. Thus, the greater the amount of cDNA of the target gene at time zero, the lower the Ct number (cycle number to reach the threshold).

(73) In order to standardise the quantitative RT-PCR analyses, it is necessary to quantify in the same experiment at least one endogenous control called the reference gene. This reference gene must have a constitutive expression independent of the treated or untreated condition of the cells. The relative expression of the selected target genes is calculated by the Ct method using the RQ (Relative Quantification) software provided by the manufacturer (Applied Biosystems). The expression values of each gene induced by a given compound are also normalized so that the value of the control NHEK cells (untreated cells and cells treated with DMSO in % identical to that used as carrier for the compound) is equal to 1. Therefore, the RQ value obtained for each compound for a given gene represents the relative expression of this gene after treatment in relation to the control cells whose expression is 1.

(74) Choice of Primers

(75) The primers were chosen with the assistance of computer programs including Oligo 4 (National Biosciences, Plymouth, Minn.). The selection criteria concern the size of the fragment to be amplified (between 80 and 120 nucleotides), the size of the primers (between 21 and 25 nucleotides), the hybridization temperature of the primers (about 65 C.) and the position of the primers; indeed, the primers are designed such that one of the 2 primers straddles an intron and an exon or that the 2 primers are in two different exons if the intron separating them is greater than 2 kbp. The choice of the quite specific position of the primers avoids the amplification of genomic DNA in case of sample contamination even if no contamination has been observed on the dissociation curves (ABI, 7900HT). All these precautions stem from the fact that the least contamination by genomic DNA can have a very significant impact on the results obtained by a technology as sensitive as quantitative RT-PCR. Another important selection criterion is to ensure that the selected primer pair does not form a duplex that would interfere non-specifically with the specific PCR product obtained and thus distort the result (inherent in the SYBR Green technique used). Finally, the primers are chosen so that they do not contain consensus regions and/or polymorphisms. The total specificity of the nucleotide sequences chosen as primers of the target gene is tested by preparing a collage (nucleotide-nucleotide blast) over the entire human genome (Altschul et al., J. Mol. Biol., 215:403-410, 1990).

(76) Standard Amplification Curve

(77) The efficiency of the primers was tested by 5-fold serial dilutions (4 points) prepared in duplicate on RNA extracted from the NHEK cells. Only primer pairs with an efficiency close to 100% (slope equal to 3.32) are retained. A no-template control (NTC) amplification is also performed to ensure that no duplexes are formed that could distort the quantitative PCR results obtained by the SYBR Green method. The dissociation curve obtained on the 7900HT device is used to ensure that the amplification product obtained is unique. A regular check of the standard amplification curve is carried out in order to prevent any decrease in primer efficiency.

(78) Amplification

(79) The amplifications are performed on an ABI Prism 7900 Sequence Detection System (Applied Biosystems) using the SYBR Green method (SYBR Green PCR Core Reagents Kit, Applied Biosystems). Amplification consists of a denaturation step (10 min at 95 C.) then by the repetition of 40 cycles of hybridization (15 sec at 95 C.) and extension (1 min at 65 C.) which ensure an exponential duplication of each strand.

(80) Genes Quantified

(81) The genes characteristic of an AD phenotype that were quantified are listed in the left column in Table 2. The response values are normalized. From left to right in the table, the column labelled AD indicates the level of induction of each gene (in the absence of active agent). The column labelled ETOH40 corresponds to the values generated by the solvent alone without active agent in order to detect possible response interference due to the solvent. The next 2 columns, labelled W01, are the test sample at two concentrations, 1.5 mg/ml and 0.75 mg/ml, respectively, after induction. The last column shows the values generated by the positive control, 0.28 mM dexamethasone.

(82) Results

(83) TABLE-US-00005 TABLE 2 W01 W01 0.28 mM AD EtOH40 (1.5 mg/ml) (0.75 mg/ml) DEXA Anti-microbial peptide, innate immunity, receptor DEFB103A Defensin, beta 103B 27.19 11.05 498.69 32.02 107.56 RNASE7 Ribonuclease, RNase A family, 7 31.69 27.32 117.21 13.69 43.23 S100A7 S100 calcium binding protein A7 75.22 79.10 102.11 98.50 79.12 TLR3 Toll-like receptor 3 15.17 15.03 0.30 0.75 2.79 Interleukins IFNB1 Interferon, beta 1, fibroblast 30.55 14.88 0.24 0.13 1.90 IL1A Interleukin 1, alpha 56.38 41.86 19.39 7.55 8.54 IL1B Interleukin 1, beta 67.18 33.58 4.77 3.52 2.63 IL4R Interleukin 4 receptor 9.63 9.33 4.35 3.14 7.03 TSLP Thymic stromal lymphopoietin 50.67 31.31 0.04 0.04 1.80 Chemokines CCL13 Chemokine (C-C motif) ligand 13 3.31 2.01 0.90 0.41 1.98 CCL11 Chemokine (C-C motif) ligand 11 0.97 0.93 0.56 0.12 1.14 CCL20 Chemokine (C-C motif) ligand 20 85.57 55.47 0.60 0.19 28.94 CCL27 Chemokine (C-C motif) ligand 27 16.82 12.08 0.77 2.34 0.71 CCL5 Chemokine (C-C motif) ligand 5 78.90 99.37 2.15 2.58 49.90 IL15 Interleukin 15 31.54 34.53 1.44 1.50 29.16 IL8 Interleukin 8 133.07 78.39 0.23 0.11 7.89 CX3CL1 Fractalkine 62.38 119.24 5.90 1.83 36.63

(84) The results presented in the tables show the inhibitory power of the pro-inflammatory and inflammatory genes (interleukins and chemokines) and also the induction of antimicrobial peptides (which are deficient in atopic dermatitis). The antimicrobial peptides that are strongly induced are: DEFB103, RNASE7, and notably psoriasin (S100A7) by W01 in a dose-dependent manner. The inhibited interleukins are: IFNB1, IL1A and IL1B. IL4R and TLR3 receptors are suppressed, the latter correlates well with the near-complete inhibition of the chemokine TSLP. These 2 factors are overexpressed in AD patients and more particularly in the case when pruritis appears (Miyagaki et al. 2015. J Dermatol Science 78:89-94). This is the first time that we have demonstrated such a strong anti-TSLP inhibition by the Mimosa pudica PCC extract in this in vitro model of AD. The chemokines CCL11, CCL13, CCL20, CCL27, CCLS, CX3CL1, IL15 and notably IL8 were strongly inhibited by extract WO1 regardless of the dose. In summary, this experiment shows that the Mimosa pudica PCC extract inhibits the majority of pro-inflammatory and inflammatory factors as well as, if not better than, the dexamethasone control, in particular the factor TSLP which causes pruritus in patients with AD.