EXTRACTS OF SANTOLINA CHAMAECYPARISSUS

Abstract

The invention relates to an extract of Santolina chamaecyparissus, to a process for preparing said extract, to a composition comprising it, and to its use a preservative, particularly as antioxidant, bactericidal and/or fungicidal.

Claims

1. Extract of Santolina chamaecyparissus L. obtained by extraction of the aerial parts with a solvent chosen from methanol, dichloromethane, ethyl acetate, acetone, tetrahydrofuran, supercritical carbon dioxide and a mixture ethanol:water in a ratio of from 70:30% v/v to 99:1% v/v.

2. Extract of Santolina chamaecyparissus L. according to claim 1 further decolorized and/or deodorized.

3. Process for preparing an extract of Santolina chamaecyparissus L. according to claim 1, comprising the following steps: a) mixing aerial parts of Santolina chamaecyparissus L. with a solvent chosen from methanol, dichloromethane, ethyl acetate, acetone, tetrahydrofuran, supercritical carbon dioxide and a mixture ethanol:water in a ratio of from 70:30% v/v to 99:1% v/v, b) macerating the mixture obtained in a) during at least 1 h, c) filtering the mixture obtained in b), so as to obtain the filtrate and the retentate, the filtrate being the crude extract.

4. Process according to claim 3 which comprises a further step c1) comprising decoloration and/or deodorization of the crude extract using activated carbon

5. Process according to claim 3, wherein the mixture ethanol:water of step a) has a ratio of 96:4% v/v.

6. Process according to claim 3 wherein the maceration of the mixture in step b) is at ambient temperature or at around 50 C.

7. Process according to claim 3, which comprises a further step d) comprising: d1) mixing the retentate of step c) with the same solvent as the one used in step a), d2) macerating the mixture obtained in d1) during at least 1 h, d3) filtering the mixture obtained in d2), so as to obtain the filtrate and the retentate, and d4) mixing the filtrate of step c) with the filtrate of step d3), said final mixture being the extract.

8. Process according to claim 3, which comprises a further step e) of evaporation of the solvent of the extract, such as vacuum-concentration.

9. Process according to claim 8, wherein step e) comprises: e1) the addition of a cosmetic compound chosen from polyols, alkylglucosides and alkyl polyglucosides and monoglycerides to the extract, and e2) the evaporation of the solvent of the extract obtained in e1), such as vacuum-concentration.

10. Process according to claim 9, wherein the cosmetic compound of step e1) is chosen from ethylhexylglycerin, caprylyl glycol and their mixtures.

11. Extract of Santolina chamaecyparissus L. obtainable by the process according to claim 3.

12. Extract according to claim 1, comprising the compound of formula (I): ##STR00006##

13. Extract according to claim 1, wherein it is not an essential oil.

14. Composition comprising, in a physiologically acceptable medium, an extract of Santolina chamaecyparissus L. according to claim 1.

15. Use of an extract of Santolina chamaecyparissus L. according to claim 1, as preservative, antioxidant, bactericide and/or fungicide.

16. Use of the compound of formula (I): ##STR00007## as preservative, antioxidant, bactericide and/or fungicide.

17. Use of a composition comprising the compound of formula (I): ##STR00008##

18. Use of a composition according to claim 14 to prevent and/or treat the cutaneous disorders related to Propionibacterium acnes.

19. Use of a composition according to claim 18 to prevent and/or treat wherein the cutaneous disorders related to Propionibacterium acmes is the acne.

Description

EXAMPLE 1

Plant Extraction of Santolina chamaecyparissus L.

[0112] Aerial parts of Santolina chamaecyparissus L. (also called santolina in the examples, unless indicated otherwise) were harvested, dried, ground into powder and extracted under mechanical stirring using a mixture of water and ethanol (75:25 v/v) (steps a) and b)). The extraction was repeated once (step d)), and both extractions were performed at 25 C. during 2 hours (step c)). The obtained extracts were filtered, stirred together and vacuum-concentrated (step e)).

[0113] Crude extracts were analyzed by HPLC using an Agilent 1200 system equipped with a DAD and an ELSD detectors. Separation was performed using a Luna C18 column (Phenomenex, 2504.6 mm; 5 m). HPLC grade acetonitrile, water and 2-propanol (Sigma-Aldrich), acidified with formic acid 0.1%, were used according to the following gradient (Table 1):

TABLE-US-00001 TABLE 1 Solvent gradient used for HPLC-ELSD analysis Time (min) Water (%) Acetonitrile (%) 2-propanol (%) 0-5 95 5 5-35 0 100 35-45 0 100 45-50 40 60 50-60 40 60 60-65 5 95 65-70 5 95

[0114] The characteristic analytical profile (HPLC-ELSD) of S. chamaecyparissus was obtained (data not shown).

EXAMPLE 2

Evaluation of the Antimicrobial and Antioxidant Activities of the Crude Extract Obtained in Example 1

[0115] The crude extracts mentioned in this example were obtained in example 1.

[0116] 1) Antimicrobial Activity Assay

[0117] Background and Samples Preparation:

[0118] Antimicrobial activity of the crude extracts was determined using a 96-well microtiter plate assay based on growth inhibition. The assay was performed on four different microbial strains, chosen according to the European Pharmacopoeia (ATCC references): Staphylococcus aureus (gram-negative bacteria), Escherichia coli (gram-negative bacteria), Pseudomonas aeruginosa (gram-positive bacteria), Propionibacterium acnes (anaerobic gram-positive bacteria), Aspergillus niger (fungus) and Candida albicans (yeast).

[0119] Samples for antimicrobial assay were prepared as follows: crude extracts were diluted at 200 mg/mL in a mixture of appropriate solvents, e.g. ethanol and water (60:40 v/v) or pure dimethylsulfoxide. After solubilization, the solutions were filtrated on 0.45 m syringe filters.

[0120] Assay Procedure:

[0121] The assay was performed for each strain according as follows: samples (at 200 mg/mL) were first diluted to 4% in water, then to 2% and 0.2% in wells. Final concentrations were also 0.4 and 0.04% of crude extracts in wells. Each concentration was assessed in replicate with one supplementary control well containing no microbial strain. For the assay at 2%, samples were mixed up in the wells with growth medium (95 L) and microbial suspension (representing an absorbance of 0.6 for C. albicans, S. aureus and P. aeruginosa and 40 spores/L for A. niger). For the assay at 0.2%, samples were first diluted to 0.2% with water in the wells. Negative controls constituted of the solvent mixture used to prepare the sample solutions, and positive controls with a synthetic preservative (methyl-paraben and/or phenoxyethanol, positive standard) were also prepared. Results were also compared with those obtained using a commercial natural preservative (commercial preservative). The 96-well plates were incubated at 25 C.

[0122] The absorbance was read for each plate at 620 nm 24, 48 and 72 hours after the beginning of the incubation to evaluate the growth of each microorganism in presence of crude extracts.

[0123] The results were expressed as a percentage of growth inhibition by the samples for each microorganism.

[0124] In a second step for the optimized extract of S. chamaecyparissus, the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) were also measured including E. coli and P. acnes. The MIC is defined as the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after incubation from 24 h to 72 h according to the microbial strains. The MBC is the lowest concentration of antibacterial agent required to kill a particular bacterium, that is to say to reduce the viability of the initial bacterial inoculum by >99.9%. The dilutions were run in triplicate for the MIC and MBC test. At the end of the incubation, the tubes were read for the growth inhibition and then the MBC was determined by sampling all the macroscopically clear tubes and the first turbid tube in the series.

[0125] The results were expressed in mg/mL of extract.

[0126] 2) Results for S. chamaecyparissus Crude Extracts

[0127] S. chamaecyparissus crude extracts exhibited antimicrobial activity as shown in Table 2 (the results presented correspond to one reference crude extract, which is representative of most of extracts that have been evaluated):

TABLE-US-00002 TABLE 2 Antimicrobial activity of S. chamaecyparissus crude extracts and commercial preservatives Growth inhibition (%) 24 h, 48 h, 72 h, Sample Microbial strain 2% 2% 2% Review Crude Aspergillus niger 102 101 +++ extract Candida albicans 100 100 +++ Pseudomonas aeruginosa 1 55 Staphylococcus aureus 97 98 +++ Commercial Aspergillus niger 65 99 preservative Candida albicans 0 10 Pseudomonas aeruginosa 48 19 Staphylococcus aureus 3 54 Positive Aspergillus niger 100 100 +++ standard Candida albicans 99 100 +++ (0.4%) Pseudomonas aeruginosa 75 81 ++ Staphylococcus aureus 80 93 ++ Numbers in bold & underlined: medium inhibition greater or equal to 60% +++: medium inhibition greater or equal to 90% ++: medium inhibition comprised between 70% and 90% +: medium inhibition comprised between 60% and 70% ~: medium inhibition comprised between 20% and 60% : medium inhibition less or equal to 20%

[0128] Positive Standard: Methylparaben

[0129] => As antimicrobial activity was considered to be interesting above 60% of growth inhibition, Santolina chamaecyparissus crude extracts showed a very interesting activity on 3 of the 4 microbial strains, e.g. S. aureus, A. niger and C. albicans. It was interesting to notice that a commercial extract sold for its preservative properties did not show any activity, in contrast to the positive standard (methylparaben).

[0130] 3) Description of the Antioxidant Assays

[0131] Sample solutions for antioxidant assays were prepared in methanol, sonicated and filtered on 0.45 m syringe filters.

[0132] Total Phenolic Content (Folin-Ciocalteu Assay)

[0133] Total phenolic content of santolina crude extracts was determined using the Folin-Ciocalteu method based on oxidation of a phenolate ion from an antioxidant sample and reduction of the phosphotungstic-phosphomolybdate reagent (Folin-Ciocalteu reagent) under alkaline conditions. As a result of this reduction, the chromophore moiety turns into from a yellow complex to a blue phosphotungstic-phosphomolybdic, representative of the presence of phenols (antioxidant biological species) in the sample. The concentration of these biological species is proportional to the light absorption at a wavelength of 750 nm, where other biological species do not absorb. The absorbance values of the samples were compared to a standard, which is commonly gallic acid. Total phenolic content is thus expressed as gallic acid equivalent (GAE) in milligrams per gram of plant extract.

[0134] Antiradical Power: DPPH Assay

[0135] The antiradical power of the sample was measured by the DPPH (2,2-diphenyl-1-picryl-hydrazyl) electron-transfer assay. DPPH is a free radical, which is purple in solution and reduced and getting yellow to colorless in presence of an antioxidant sample. The antiradical power of a sample is thus measured by the capacity to neutralize free radicals responsible for oxidation. DPPH reduction is observed by a spectrophotometric measurement of absorbance value at 520 nm (the lower the absorbance, the stronger the antioxidant activity). The IC.sub.50 (inhibiting concentration 50%) is the parameter determined with this assay. It corresponds to the concentration of antioxidant sample that reduces 50% of the initial DPPH concentration, expressed in micrograms of sample per milliliters of methanol.

[0136] 4) Antioxidant Activity of Crude Extracts

[0137] Different S. chamaecyparissus crude extracts were evaluated (different origins and harvesting periods) and showed a total phenolic content (GAE) varying between 33 and 70 mg/g. As standards, natural rosemary extracts commercialized for their high antioxidant properties were evaluated and expressed a total phenolic content of about 85 mg/g. Another commercial antioxidant extract made of wasabi was evaluated and expressed only about 27 mg/g GAE.

[0138] Concerning the DPPH assay, the lowest IC.sub.50 of S. chamaecyparissus extracts was 118 g/mL. As a comparison, an extract of rosemary, known for its antioxidant potential, presented a very similar IC.sub.50, namely 103 g/mL.

[0139] => As a conclusion, the S. chamaecyparissus crude extracts according to the invention seem to present an interesting antioxidant potential.

EXAMPLE 3

Evaluation of the Antimicrobial and Antioxidant Activity of the Fractions Isolated from the Crude Extract of Example 1

[0140] 1) Fractionation of the Crude Extract

[0141] As the reference S. chamaecyparissus extract (ethanol:water 75:25 v/v) exhibited a strong antimicrobial activity, bioguided fractionation was performed to identify the fraction(s) containing active metabolites. Open column chromatographic fractionation of the crude extract was performed.

[0142] Fractionation was performed on a silica gel (normal phase) open column and yielded five fractions: cyclohexane fraction (F1), cyclohexane:ethyl acetate fraction (F2), ethyl acetate fraction (F3), ethyl acetate:methanol fraction (F4) and methanol fraction (F5).

[0143] All obtained fractions were analyzed by HPLC-ELSD in the same conditions as the crude extracts to have a qualitative idea of the different metabolites constituting each fraction.

[0144] 2) Antimicrobial Activity of santolina Fractions

[0145] Fractions obtained from S. chamaecyparissus crude extract were submitted to antimicrobial activity assays (as explained in example 2) if sufficient quantity was present. Sample solutions were prepared at 100 mg/mL in DMSO and assessed the same way as crude extracts.

[0146] The results were as following in Table 3 (only fractions with an activity, even weak, are shown):

TABLE-US-00003 TABLE 3 Antimicrobial activity of S. chamaecyparissus fractions of interest Growth inhibition (%) 24 h, 48 h, 72 h, Sample Microbial strain 2% 2% 2% Review F2 Aspergillus niger 100 100 +++ Candida albicans 99 98 +++ Pseudomonas aeruginosa 94 90 +++ Staphylococcus aureus 105 113 +++ F3 Aspergillus niger 90 103 +++ Candida albicans 96 44 ~ Pseudomonas aeruginosa 6 12 Staphylococcus aureus 24 15 F4 Aspergillus niger 68 7 ~ Candida albicans 17 13 Pseudomonas aeruginosa 13 8 Staphylococcus aureus 36 56 Bold & underlined: medium inhibition greater or equal to 60% +++: medium inhibition greater or equal to 90% ++: medium inhibition comprised between 70% and 90% +: medium inhibition comprised between 60% and 70% ~: medium inhibition comprised between 20% and 60% : medium inhibition less or equal to 20%

[0147] => F2 cyclohexane:ethyl acetate fraction showed an increased activity. The presence of one major metabolite in this fraction that is not present in the other normal-phase fractions, is noticed, and this metabolite could be responsible for the antimicrobial activity (or part of) of santolina crude extracts.

EXAMPLE 4

Isolation of the Active Metabolite and Properties Thereof

[0148] Semi-preparative HPLC was performed on the active cyclohexane:ethyl acetate santolina fraction in order to purify the major metabolite supposed to be responsible for the antimicrobial activity. It was carried out on a Luna C18 column (Phenomenex, 25010 mm; 5 m). Elution profile was optimized and the single component of interest was successfully isolated using a gradient of non-acidified HPLC grade water and acetonitrile. The single component was vacuum-concentrated and submitted to structure elucidation techniques.

[0149] 1) Structure Elucidation

[0150] 1D- and 2D NMR

[0151] NMR experiments were performed using a BRUKER Avance spectrometer (.sup.1H 500, 13 MHz). All 1D (.sup.1H, .sup.13C) and 2D (COSY, HSQC, HMBC, NOESY) spectra were acquired in chloroform (CDCl.sub.3). The following structure of formula (I) was identified:

##STR00005##

[0152] This molecule has following molecular formula: C.sub.15H.sub.12O.sub.4 and following molecular weight: 256,2534 g/mol. It is already known as 1,6-dioxaspiro[4,4]nona-2,8-dien-4-ol,7-(2,4-hexadiyn-1-ylidene)-,4-acetate and belongs to the secondary metabolites polyacetylenes family.

[0153] UPLC-HRMS

[0154] The isolated metabolite was analyzed by UPLC-ESI-HRMS using a Waters Acquity system. The inventors used UPLC-HRMS to identify the active metabolite in santolina crude extract. With the developed method, the mass-to-charge ratio (m/z) of its ion in positive mode was [M+H].sup.+=257.0816 g/mol (data not shown).

[0155] This information confirmed the hypothesis of the proposed molecule, since the molecular weight of the known compound was established as M=256.2534 g/mol.

[0156] 2) Validation of the Activity of the Pure Compound (Formula (I))

[0157] The pure compound appeared to be unstable once isolated, and particularly in aqueous and acid conditions. Since antimicrobial assays were performed in water, its antimicrobial activity could not be evaluated as for crude extracts or plant fractions. Thus, the inventors tried to establish a relationship between antimicrobial activity and active substance content in crude extracts.

[0158] Relationship between Antimicrobial Activity and Active Substance Content

[0159] A quantitation of the active substance was performed by HPLC-UV. The same solvent gradient as for HPLC-ELSD analysis was applied but stopped at 43 min. A calibration curve was determined using a trans-cinnamic acid standard (that has a retention time relatively close to the active substance retention time) prepared at different concentrations (from 0.0078 to 1 mg/mL) in methanol. Different santolina crude extracts (E1 to E2) were diluted to 20 mg/mL in methanol and analyzed by HPLC-UV at 310 nm, which is the absorption maximum of the active substance (and which allows comparison between the different extracts). The linear regression equation generated by the calibration curve permitted to calculate the value of active substance by corresponding integration and area peak measurement.

[0160] Antimicrobial activity assays (as explained in example 2) were also performed on said crude extracts, and the following results were obtained (table 4):

TABLE-US-00004 TABLE 4 Active substance concentration and antimicrobial activity of santolina crude extracts Relative active Antimicrobial activity Crude substance A. C. P. S. extracts concentration (mg/g) niger albicans aeruginosa aureus E1 17.60 + + + + E2 0.00 + +: strong activity, ~: weak activity, : no activity

[0161] => The results show that a relationship between active substance concentration in crude extracts and their antimicrobial activity can be observed, especially on A. niger and C. albicans.

[0162] However, the activity on S. aureus seems to be independent of the presence of the active substance (formula (I)).

[0163] To confirm this hypothesis, the inventors wondered if other plants of the genus Santolina also containing the active substance would present the same results.

[0164] Antimicrobial Activity of other Santolina Species Containing the Active Substance

[0165] Two other Santolina species (Santolina rosmarinifolia L. and Santolina pinnata L.) were investigated. Crude extracts were prepared and analyzed by HPLC-ELSD to confirm the presence of the active substance. They were then submitted to the same HPLC-UV quantitation as described in the previous paragraph, and to antimicrobial activity assays, and the following results were obtained (table 5):

TABLE-US-00005 TABLE 5 Active substance concentration (mg of active/g of extract) and antimicrobial activity of Santolina species. Relative active substance Antimicrobial activity Crude concentration A. C. P. S. extracts (mg/g) niger albicans aeruginosa aureus S. 3.97 + + + rosmarinifolia S. pinnata 4.56 + +: strong activity, : no activity

[0166] => The results show differences of activity between these extracts and S. chamaecyparissus extracts. It can be explained by differences of chemical composition of the species.

[0167] => The presence of the active substance (formula (I)) and the antimicrobial activity can although be correlated.

EXAMPLE 5

Formulation of the Extract as a Natural Preservative

[0168] I) Optimization of the Extraction Process

[0169] Optimization of Solvent Extraction

[0170] As the inventors demonstrated that antimicrobial activity is in relation with active metabolite (compound of formula (I)) concentration, optimization of the extraction process was performed with modification of solvents.

[0171] Ethanol/Water Gradient

[0172] The initial solvent of extraction was a mix of ethanol/water 75:25 v:v. First step was also to change this gradient from 100% of ethanol to 100% of water. Seven new solvents were evaluated as described in Table 6:

TABLE-US-00006 TABLE 6 EtOH/Water solvent extraction gradient Active substance (formula (I)) Ethanol (%) Water (%) concentration (mg/g) 100 (absolute EtOH) 0 8.5 100 (96 EtOH) 0 15.0 80 20 19.9 60 40 2.9 40 60 0.3 20 80 0 100

[0173] These crude extracts were then analyzed using UV-HPLC at 310 nm and the active metabolite was quantified following the method described in example 4. Higher concentration was found for EtOH/Water 80:20 v:v gradient. No active was observed in water extract, neither for weak percentage of water in extraction solvent. Higher active substance concentration was found for 80% and 96% of ethanol and decreased then for higher purity of ethanol.

[0174] => Optimal gradient was thus between 60 and 96% v/v of ethanol.

[0175] Other Solvents

[0176] The second step was to compare other solvents to extract the plant and then quantify the metabolite concentration. Tested solvents were tetrahydrofuran, acetone, dichloromethane, methanol, absolute ethanol, ethanol 96, ethyl acetate and methyltetrahydrofuran.

TABLE-US-00007 TABLE 7 Active substance concentration in function of extraction solvents Active substance (formula (I)) Ethanol (%) concentration (mg/g) Absolute ethanol 8.5 1.2 Ethanol 96 15.0 1.0 Methanol 44.3 2.6 Ethyl acetate 20.3 0.8 Tetrahydrofuran 12.0 0.9 Acetone 34.0 0.9 Dichloromethane 40.8 0.8 Methyltetrahydrofuran 16.4 0.7

[0177] As shown in Table 7, methanol was the extraction solvent giving the higher concentration of metabolite before dichloromethane, acetone and ethyl acetate which gave very good data too.

[0178] All these 4 solvents gave higher active concentrations than ethanol/water 80:20 v/v. Methanol was also selected for the rest of the optimization of the extraction process to produce a cosmetic ingredient.

[0179] 2) Optimization of a Cosmetic Preservative

[0180] Solvent extract of Santolina chamaecyparissus was not an easy to use ingredient for cosmetic as it was thick and doughy. To remove this drawback, a liquid and specific cosmetic raw material was added. In order to do this, extraction process was modified as follows:

[0181] After the two methanolic extractions processing of aerial parts of Santolina chamaecyparissus L. (see example 1), the obtained extracts were filtered and stirred together. Then, one selected liquid cosmetic raw material was added to the extract under stirring. Finally, methanol was eliminated under vacuum.

[0182] Several cosmetic raw materials were selected. They were polyols (like glycols, such as glycerol) and monoglyceride esters.

[0183] After concentration of the extract, the inventors obtained a product easier to use in cosmetic formulations.

[0184] Extract Solubility in Liquid Cosmetic Raw Material

[0185] The list of cosmetic raw materials added during the extraction process is described in Table 8.

[0186] Santolina chamaecyparissus was extracted (methanol extraction) according to the above protocol, and each of the cosmetic raw materials was added. Then, a classification was done in function of crude extract solubility therein as follows:

TABLE-US-00008 TABLE 8 List of cosmetic raw materials tested for the cosmetic ingredient optimization and Santolina chamaecyparissus solubility in these products. Santolina chamaecyparissus Raw material solubility Ethylhexylglycerin Very soluble Ethylhexylglycerin/Caprylyl Very soluble glycol Glyceryl caprate Very soluble Caprylyl glycol Very soluble Capryloyl glycine Soluble Glyceryl caprylate Soluble Heptyl glucoside Soluble Glycerin Moderately soluble Propylene glycol Moderately soluble Butylene glycol Moderately soluble Pentylene glycol Moderately soluble Propanediol Moderately soluble

[0187] Ethylhexylglycerin was the raw material in which the crude extract of Santolina chamaecyparissus presented the best solubility. Three of the best raw materials for the plant extract were also selected for antimicrobial activity evaluation: [0188] Ethylhexylglycerin, [0189] Ethylhexylglycerinlcaprylyl glycol (Sensiva SC10 from Schlke & Mayr) and [0190] Caprylyl glycol.

[0191] These 3 ingredients were liquid and easy to use for cosmetic formulations.

[0192] Metabolite Concentration in Optimized Extract

[0193] Before biological test, HPTLC analysis were done on these three optimized extracts and compared with the crude extract without any cosmetic raw material.

[0194] HPTLC Analysis

[0195] Metabolite relative quantification was done using a Camag HPTLC system (Muttenz, Switzerland) equipped with an automatic TLC sampler (ATS4), an automatic developing chamber (ADC 2), a visualizer and a TLC scanner 4 controlled with WinCATS software. Sample solutions were applied on silica gel 60 F.sub.254 plates (2010 cm0.20 mm) purchased from Merck. All plates were developed until 70 mm from the lower edge, with humidity control from 33 to 38% and 20 min of saturation. Mobile phase for plates development was toluene/ethyl acetate/formic acid 96:4:1 v/v. Plates were scanned at 315 nm in reflectance mode, with D2 and W lamp, slit dimension of 8.00 mm0.40 mm, scanning speed of 20 mm/s and data resolution of 100 m/step.

[0196] Calibration curve was performed with stock solutions of Santolina chamaecyparissus (crude methanolic extract) at 1 mg/mL in methanol. Several amount of the stock solution (1, 2, 6, 8 and 12 L) were applied on plates which were then developed and scanned. The calibration plots of peak areas versus concentration were polynomial equation (second degree). R.sup.2 coefficient was acceptable above 0.99. The metabolites eluted with a Rf of 0.42.

[0197] Identification of the metabolite through TLC development was performed using isolated compounds.

[0198] HPTLC Dosage

[0199] Active compound concentration was compared in each sample as described in Table after pilot extraction. A very high metabolite concentration in all products (between 31.3 and 38.9 mg of active/g of extract) was observed. Also, the addition of cosmetic raw material during the extraction process did not affect the active compound.

TABLE-US-00009 TABLE 9 active substance concentration in Santolina chamaecyparissus extract without and with addition of cosmetic raw materials during extraction process Active substance concentration Ingredient in the extract (mg/g) Crude extract of Santolina 38.9 1.5 chamaecyparissus Santolina chamaecyparissus + RM1 31.3 1.2 Santolina chamaecyparissus + RM2 36.2 1.8 Santolina chamaecyparissus + RM3 33.6 0.9 RM1: raw material 1 (ethylhexylglycerin), RM2: raw material 2 (ethylhexylglycerin/caprylyl glycol), RM3: raw material 3 (caprylyl glycol).

[0200] For the rest of the study, the three evaluated products are named as follows: [0201] Ingredient 1: Santolina chamaecyparissus+Ethylhexylglycerin, [0202] <<Ingredient 2>>: Santolina chamaecyparissus+Ethylhexylglycerin/caprylyl glycol and [0203] <<Ingredient 3>>: Santolina chamaecyparissus+caprylyl glycol.

[0204] Antimicrobial Activity of Santolina chamaecyparissus Extract with Selected Cosmetic Raw Materials

[0205] Antimicrobial activity of the methanolic crude extract and the three ingredients containing the crude extract and selected cosmetic raw materials was evaluated against five microorganisms at 0.4 and 0.04% during 48 to 72 h: [0206] Aspergillus niger, [0207] Escherichia coli, [0208] Candida albicans, [0209] Pseudomonas aeruginosa, and [0210] Staphylococcus aureus.

[0211] Details of results are described in Tables 10 to 13 below.

TABLE-US-00010 TABLE 10 Inhibition percentage of microorganisms with crude methanolic extract of Santolina chamaecyparissus at 0.4 and 0.04% at 24, 48 and 72 h. Inhibition >90% was noted +++, inhibition >80% and at less 70% was noted ++, inhibition >60% was noted +, inhibition >60% but lots was notes ~, Inhibition <60% was noted . Inhibition percentage (%) 24 h 48 h 72 h Analyze Sample Strains 0.4% 0.04% 0.4% 0.04% 0.4% 0.04% 0.4% 0.04% Santolina chamaecyparissus Aspergillus niger 93 50 91 12 +++ Crude extract Escherichia coli 76 35 29 24 12 24 Extraction solvent: MeOH Candida albicans 103 2 41 17 ++ Pseudomonas aeruginosa 26 14 18 0 Staphylococcus aureus 102 62 100 38 +++

TABLE-US-00011 TABLE 11 Inhibition percentage of microorganisms with ingredient 1 at 0.4 and 0.04% at 24, 48 and 72 h. Inhibition >90% was noted +++, inhibition >80% and at less 70% was noted ++, inhibition >60% was noted +, inhibition >60% but lots was notes ~, Inhibition <60% was noted . Inhibition percentage (%) 24 h 48 h 72 h Analyze Sample Strains 0.4% 0.04% 0.4% 0.04% 0.4% 0.04% 0.4% 0.04% Santolina chamaecyparissus Aspergillus niger 96 75 97 60 +++ + Ingredient 1 Escherichia coli 100 39 101 54 98 61 +++ ~ Extraction solvent: MeOH Candida albicans 107 3 101 10 +++ Pseudomonas aeruginosa 63 10 53 19 ~ Staphylococcus aureus 95 55 98 9 +++

TABLE-US-00012 TABLE 12 Inhibition percentage of microorganisms with ingredient 2 at 0.4 and 0.04% at 24, 48 and 72 h. Inhibition >90% was noted +++, inhibition >80% and at less 70% was noted ++, inhibition >60% was noted +, inhibition >60% but lots was notes ~, Inhibition <60% was noted . Inhibition percentage (%) 24 h 48 h 72 h Analyze Sample Strains 0.4% 0.04% 0.4% 0.04% 0.4% 0.04% 0.4% 0.04% Santolina chamaecyparissus Aspergillus niger 96 71 99 22 +++ ~ Ingredient 2 Escherichia coli 50 39 88 38 103 45 ++ Extraction solvent: MeOH Candida albicans 104 14 85 13 ++ Pseudomonas aeruginosa 85 11 84 13 ++ Staphylococcus aureus 66 99 93 3 +

TABLE-US-00013 TABLE 13 Inhibition percentage of microorganisms with ingredient 3 at 0.4 and 0.04% at 24, 48 and 72 h. Inhibition >90% was noted +++, inhibition >80% and at less 70% was noted ++, inhibition >60% was noted +, inhibition >60% but lots was notes ~, Inhibition <60% was noted . Inhibition percentage (%) 24 h 48 h 72 h Analyze Sample Strains 0.4% 0.04% 0.4% 0.04% 0.4% 0.04% 0.4% 0.04% Santolina chamaecyparissus Aspergillus niger 93 51 97 16 +++ Ingredient 3 Escherichia coli 114 32 98 28 97 36 +++ Extraction solvent: MeOH Candida albicans 97 6 97 5 +++ Pseudomonas aeruginosa 103 26 101 8 +++ Staphylococcus aureus 102 105 101 4 +++

[0212] Crude extract was active against A. niger, C. albicans and S. aureus at 0.4%.

[0213] Ingredient 1 was active against A. niger and E. coli at 0.4 and 0.04% and against C. albicans and S. aureus at 0.4% of Santolina chamaecyparissus extract. Finally, it was moderately active against P. aeruginosa.

[0214] Ingredient 2 was active against the five microorganisms at 0.4% of Santolina chamaecyparissus extract and additionally it was active against A. niger at 0.04% during 48 h.

[0215] Ingredient 3 was active against the five microorganisms at 0.4% of Santolina chamaecyparissus extract.

[0216] => Finally, a better inhibition of all ingredients was observed in comparison to the crude extract alone. These ingredients were also optimized for antimicrobial activity and cosmetic use.

[0217] 3) Evaluation of the Antimicrobial Activity for Cosmetic Preparations

[0218] Challenge Test (Description of the Test)

[0219] The challenge test is designed to provide the level of biological activity possessed by the preservative system of cosmetic products. Briefly, a controlled amount of specific microorganisms is inoculated in products. Mixtures are stocked in controlled conditions and microorganisms are counted at known times for 28 days.

[0220] Antimicrobial preservative effectiveness of the formulations was performed following the method recommended by the European Pharmacopeia (7.sup.th edition2011).

[0221] Microorganisms

[0222] Tested Microorganisms were:

[0223] Bacteria [0224] Pseudomonas aeruginosa ATCC 9027 [0225] Staphylococcus aureus ATCC 6538 [0226] Escherichia coli ATCC 8739

[0227] Fungi & Mold [0228] Candida albicans ATCC 10231 [0229] Aspergillus brasiliensis ATCC 16404

[0230] These one are potential pathogenic germs and could contaminate products as they are issued from biotypes such as water, skin, intestinal or environmental.

[0231] Culture Media

[0232] Tested strains were prepared as described in Table 14:

TABLE-US-00014 TABLE 14 Strain preparation Preservation Storage Densities Incubation Strains temperature Culture media (CFU/mL) temperature P. aeruginosa 80 C. 5 C. Tryptic Soy Agar 10.sup.7-10.sup.8 32.5 C. 2.5 C. (TSA) S. aureus 80 C. 5 C. Tryptic Soy Agar 10.sup.7-10.sup.8 32.5 C. 2.5 C. (TSA) E. coli 80 C. 5 C. Tryptic Soy Agar 10.sup.7-10.sup.8 32.5 C. 2.5 C. (TSA) C. albicans 80 C. 5 C. Sabouraud 10.sup.7-10.sup.8 22.5 C. 2.5 C. Dextrose A. brasiliensis 80 C. 5 C. Sabouraud 10.sup.6-10.sup.7 22.5 C. 2.5 C. Dextrose

[0233] Procedure

[0234] The test organisms specified were to be tested separately in cosmetic products.

[0235] The product to be tested is distributed in single-use sterile flasks (20 g of product/flask) and every flask is inoculated with the suspension of one strain to be tested. Final concentration is about 10.sup.5 to 10.sup.6 microorganisms/g.

[0236] Inoculated products were to be held at 22.5 C.2.5 C. in darkness during the test.

[0237] Neutralization

[0238] Neutralization of preservative system was validated and performed on the 5 strains with LT 100 Broth at 1/10.sup.th and 1/100.sup.th dilution.

[0239] Interpretation

[0240] Sampling and analysis are done after 2-day, 7-day, 14-day and 28-day period. Each sample was neutralized and enumeration method was applied (results in CFU/g). The microbial densities obtained during the follow-up for each of the tested strains are counted and compared with the thresholds of the logarithmic reduction imposed by the reference table (table 15).

TABLE-US-00015 TABLE 15 Table of logarithmic reduction imposed by the European Pharmacopeia European Pharmacopeia Logarithmic reduction Strains Criteria Day-2 Day-7 Day-14 Day-28 Bacteria A 2 3 / NI B / / 23 NI Fungi/mold A / / 2 NI B / / 1 NI NI: No Increase; /: No minimal reduction required

[0241] Cosmetic Formulations

[0242] Challenge tests were realized with oil-in-water (O/W) emulsions comprising or not Santolina chamaecyparissus extract. The O/W emulsion formulations are described in Table 16.

TABLE-US-00016 TABLE 16 O/W emulsions with Santolina chamaecyparissus extract Raw material (INCI) % (w/w) Procedure A Aqua Qsp Heat A at 55 C. Glycerin 4.00 (formula 1) or 10.00 (formula 2) B Acrylates/C10-30 alkyl acrylate crosspolymer 0.15 Add B C Polyoxyethylene (2), stearyl ether 2.00 Heat C at 75 C., heat Steareth-21, polyethoxylated alcohol 2.00 A + B at 75 and Stearyl alcohol 1.50 emulsified C in A + B Glyceryl stearate 3.00 Octyldodecanol 3.00 Squalane 2.50 Isohexadecane 3.00 Simmondsia chinensis (Jojoba) seed oil 1.50 Triticum vulgare (weat) germ oil 1.50 Butylhydroxytoluene 0.05 Butyrospermum parkii butter 2.00 Dimethicone 1.50 Butyl methoxydibenzoyl-methane 1.00 Ethylhexyl methoxycinnamate 1.50 Dimethicone 2.00 D Triethanolamine 0.11 Add D and E at 60 C. E Titane dioxyde, paraffin oil 1.25 F Panthenol, propylene glycol 0.20 Add F, G and H at Tocopheryl 0.50 30 C. G Sodium hyaluronate 5.85 H Perfume 0.10 I Santolina chamaecyparissus Extract 0.5-2% Add the preservative system at 30 C.

[0243] In the experiments, Formula 1 comprised 4% of glycerin and Formula 2 comprised 10% of glycerin.

[0244] Challenge tests were systematically performed: [0245] on formulations 1 and 2 comprising either Ingredients 1, 2 or 3. As Ingredients 1, 2 and 3 comprised 50% by weight of Santolina chamaecyparissus extract, these formulations comprised in fact 1% (for 0.5% Santolina chamaecyparissus extract) or 4% (for 2% Santolina chamaecyparissus extract) of Ingredient 1, 2 or 3, and [0246] on formulations 1 and 2 comprising 0.5% or 2% of ethylhexylglycerin alone (S1), of the mixture ethylhexylglycerinlcaprylyl glycol alone (S2) or of caprylyl glycol alone (S3).

[0247] Antimicrobial Activity

[0248] Antimicrobial Activity in Formulation I

[0249] Santolina chamaecyparissus extract was first tested at 0.5% and 2% in formulation 1 with Ingredient 2 or 3. As ingredients 1 to 3 contained 50% of plant extract, they were introduced at 1 and 4% respectively.

[0250] The results are in Table 17.

TABLE-US-00017 TABLE 17 Challenge test results with Santolina chamaecyparissus extract at 0.5 and 2% with different cosmetic supports. Comparison with formula 1 without any plant extract. Interpretation following the European Pharmacopeia (Logarithmic reduction and interpretation) Strains P. aeruginosa S. aureus E. coli Days D2 D7 D14 D28 D2 D7 D14 D28 D2 Criteria A 3 3 NI* 2 3 NI 2 Criteria B 3 NI 3 NI Formula 1 1.3 >3 1.1 NCo 0.2 0.0 0.2 NCo 0.5 Interpretation B B NC NCo B B NC NCo B Formula 1 + S2 (2%) >3 >3 / >3 >3 >3 / >3 >3 Interpretation A A A A A A A A A Formula 1 + ingredient 2 >3 >3 / >3 >3 >3 / >3 >3 (4%) Interpretation A A A A A A A A A Formula 1 + S3 (2%) >3 >3 / >3 >3 >3 / >3 >3 Interpretation A A A A A A A A A Formula 1 + ingredient 3 >3 >3 / >3 >3 >3 / >3 >3 (4%) Interpretation A A A A A A A A A Formula 1 + S2 (0.5%) >3 >3 / NCo 0.3 0.7 2.6 NCo >3 Interpretation A A A NCo B B NC NCo A Formula1 + ingredient2 >3 >3 / NCo 0.5 >3 >3 NCo >3 (1%) Interpretation A A A NCo B A A NCo A Formula 1 + S3 (0.5%) >3 >3 / NCo 0.5 >3 >3 NCo >3 Interpretation A A A NCo B A A NCo A Formula1 + ingredient3 >3 >3 / NCo 1.1 >3 >3 NCo >3 (1%) Interpretation A A A NCo B A A NCo A Formula 2 0.9 >3 >3 NCo 1.1 2.0 >3 NCo 0.9 Interpretation B A A NCo B B A NCo B Formula 2 + ingredient 1 >3 >3 / >3 >3 >3 >3 >3 >3 (2%) Interpretation A A A A A A A A A Strains E. coli C albicans A. brasiliensis Days D7 D14 D28 D2 D7 D14 D28 D2 D7 D14 D28 Criteria A 3 NI / / 2 NI / / 2 NI Criteria B 3 NI / / 1 NI / / 1 NI Formula 1 0.7 0.0 NCo / / 0.0 NCo / / 0.1 NCo Interpretation B NC NCo NC NCo NC NCo Formula 1 + S2 (2%) >3 / >3 / / >3 >3 / / 0.1 0.1 Interpretation A A A A A NC NC Formula 1 + ingredient 2 >3 / >3 / / >3 >3 / / 0.9 2.0 (4%) Interpretation A A A A A B A Formula 1 + S3 (2%) >3 / >3 / / >3 >3 / / 0.4 0.9 Interpretation A A A A A NC NC Formula 1 + ingredient 3 >3 / >3 / / > >3 / / 2.5 >3 (4%) Interpretation A A A A A A A Formula 1 + S2 (0.5%) >3 / NCo / / 1.1 NCo / / 0.1 NCo Interpretation A A NCo B NCo NC NCo Formula1 + ingredient2 >3 / NCo / / >3 NCo / / 0.1 NCo (1%) Interpretation A A NCo A NCo NC NCo Formula 1 + S3 (0.5%) >3 / NCo / / >3 NCo / / 0.0 NCo Interpretation A A NCo A NCo NC NCo Formula1 + ingredient3 >3 / NCo / / >3 NCo / / 0.1 NCo (1%) Interpretation A A NCo A NCo NC NCo Formula 2 0.9 1.1 NCo / / 0.2 NCo / / 0.1 NCo Interpretation B NC NCo NC NCo NC NCo Formula 2 + ingredient 1 >3 / >3 / / >3 >3 / / 0.8 0.9 (2%) Interpretation A A A A A B B /: No minimal reduction required, NI: No Increase, NC: not compliant; NCo: Test not continued

[0251] The results show that 0.5% of Santolina chamaecyparissus extract was sufficient to be in accordance with criteria A of European pharmacopeia against P. aeruginosa, E. coil and C. albicans and in accordance with criteria B against S. aureus.

[0252] At 2% of crude extract in Ingredient 2 (=4% of ingredient 2), challenge test on formula 1 was in accordance with criteria A against all bacteria and yeast, and with criteria B against mold. It was noted that after 28 days of incubation, formula 1 was in keeping with criteria A against all microorganisms. The antifungal activity against A. brasiliensis was only slower than expected in the method.

[0253] With 2% of Santolina chamaecyparissus in ingredient 3 (=4% of ingredient 3), challenge test on formula 1 was in accordance with criteria A against all microorganisms.

[0254] Antimicrobial Activity in Formulation 2

[0255] Ingredient 1 was tested at 2% in formulation 2 (=1% of crude extract). As shown in Table 17, 1% of Santolina chamaecyparissus extract was sufficient to be in accordance with criteria A of European pharmacopeia against P. aeruginosa, S. aureus, E. coli and C. albicans and with criteria B against A. brasiliensis.

EXAMPLE 6

Validation of the Optimized Extract for a High Source of Compound (I) into Santolina chamaecyparissus L.

[0256] Optimization of Solvent Extraction

[0257] As the inventors demonstrated that antimicrobial activity is in relation with active metabolite (compound of formula (I)) concentration, optimization of the extraction process was performed with modification of solvents, temperature of extraction and the time of extraction.

[0258] Ethanol/Water Gradient

[0259] The initial solvent of extraction was a mix of ethanol/water 75:25 v:v. A different plant batch, more abundant incompound (I) was used, to which the extraction gradient ranging from 60% to 100% of ethanol was assessed, as described in Table 18. Two successive extraction steps were performed prior to extract concentration and analysis.

TABLE-US-00018 TABLE 18 EtOH/Water solvent extraction gradient Active substance (formula (I)) concentration Ethanol (%) Water (%) (mg/g) 100 (absolute EtOH) 0 74.5 100 (96 EtOH) 0 86.8 80 20 51.5 60 40 24.0

[0260] These crude extracts were then analyzed using UV-HPLC at 310 nm and the active metabolite was quantified following the method described in example 4. Higher concentration was found for EtOH/Water 99:1 v:v gradient. Higher active substance concentration was found for 80% and 98% of ethanol and decreased then for higher purity of ethanol.

[0261] => Optimal gradient was thus confirmed between 75 and 98% v/v of ethanol.

[0262] Temperature of Extraction

[0263] The second step involved an evaluation of the impact of different temperatures of extraction on extraction yield and compound (1) concentration. Only one extraction step was performed during the optimization of the temperature of extraction.

TABLE-US-00019 TABLE 19 Active substance concentration vs the temperature of extraction Active substance (formula (I)) Temperature ( C.) concentration (mg/g) Ambient temperature 80.4 50 C. 93.8 Under Reflux 77.4

[0264] As shown in Table 19, a higher concentration in the active substance (I) was reached at 50 C. rather than when lower temperatures were used.

[0265] Extraction Time

[0266] The plant extraction was performed at 50 C., as previously optimized.

TABLE-US-00020 TABLE 20 Active substance concentration in function of the extraction time Active substance Extraction (formula (I)) time concentration (mg/g) 30 min 14.5 1 h 30 53.4 3 h 93.8 4 h 73.7

[0267] As mentioned in Table 20, only 3 hours and no more are required to reach the highest concentration in active substance (I) contained within the said Santolina chamaecyparissus L. extract.

EXAMPLE 7

Evaluation of the Antimicrobial Activity of the Crude Extract of Example 6

[0268] The optimized extract was then assessed for its antimicrobial activity and more particularly in order to determine the minimum bactericidal concentration for the following microbial strains: Staphylococcus aureus (gram-negative bacteria), Escherichia coli (gram-negative bacteria), Pseudomonas aeruginosa (gram-positive bacteria), Aspergillus niger (fungus) and Candida albicans (yeast).

[0269] Due to a higher active substance (I) content, it convinces to evaluate the impact on its antimicrobial activity.

TABLE-US-00021 TABLE 21 Minimum bactericidal concentration of microorganisms MBC (mg/mL) Crude optimized A. C. P. S. extract niger albicans aeruginosa aureus E. coli S. chamaecyparissus nd 10.1 5 1.2 20.3 nd: not determined

EXAMPLE 8

Evaluation of the Antimicrobial Activity of the Crude Extract (of Example 6) on P. acnes

[0270] The optimized extract was then assessed for its antimicrobial activity and more particularly in order to determine the minimum inhibitory concentration for Propionibacterium acnes (gram-positive bacteria). Methyl paraben and phenoxyethanol, as universal antimicrobial agents, were used as positive control. Due to the a higher active substance (I) content, the impact on the antimicrobial activity and more particularly on an anaerobic bacteria was evaluated.

TABLE-US-00022 TABLE 22 Minimum inhibitory concentration of P. acnes Crude optimized MIC (mg/mL) extract P. acnes S. chamaecyparissus 1.27 Methyl paraben 2.53 Phenoxyethanol 10.13

[0271] Surprisingly, the said extract is able to strongly inhibit the growth of this anaerobic bacteria acting only at 1.27 mg/mL.

EXAMPLE 9

Evaluation of the Antimicrobial Activity of the Decolored and Deodorised Extract of Example 6

[0272] In order to decrease its color and odor impact in a cosmetic formulation, the Santolina extract was (crude extract) deodorized and decolored using activated carbon. This step was performed at ambient temperature or at 50 C. and the treatment time lasted at least 30 min using from 1% w/w extract to 20% w/w extract of activated carbon. Preferably, 5-15% w/w extract of activated carbon was used. After this step, a decrease on the active substance (I) content was observed. The optimized extract was then assessed for its antimicrobial activity and more particularly in order to determine the minimum bactericidal concentration for the following microbial strains: Staphylococcus aureus (gram-negative bacteria), Escherichia coli (gram-negative bacteria), Pseudomonas aeruginosa (gram-positive bacteria), Aspergillus niger (fungus) and Candida albicans (yeast). The following conditions of decoloration/deodorization were assessed with activated carbon:

TABLE-US-00023 TABLE 23 Various conditions for an efficient decoloration/deodorization step Charcoal w/w Temperature Dry extract % extract 50 C. 2% 5% 50 C. 2% 10% 50 C. 2% 15% 50 C. 5% 5% 50 C. 5% 10% 50 C. 5% 15% Reflux 2% 5% Reflux 2% 10% Reflux 2% 15% Reflux 5% 5% Reflux 5% 10% Reflux 5% 15%

[0273] As mentioned below in the Table 24, the decolorized/deodorized extract used is representative to the above conditions of decoloration/deodorization. Despite the decrease of the active substance (I) content, the level of active substance (I) remained sufficient to maintain a protection against large antimicrobial spectrum.

TABLE-US-00024 TABLE 24 Minimum bactericidal concentration of microorganisms Relative active substance concen- MBC (mg/mL) tration A. C. P. S. E. (mg/g) niger albicans aeruginosa aureus coli Crude 81.0 nd 10.1 5 1.2 20.3 extract Decolored/ 67.0 nd 20.4 5.1 2.5 10.2 deodorized extract nd: not determined

EXAMPLE 10

Evaluation of the Antimicrobial Activity on P. acnes of the Decolored and Deodorised Extract of Example 9

[0274] The optimized extract was then assessed for its antimicrobial activity and more particularly in order to determine the minimum inhibitory concentration for Propionibacterium acnes (gram-positive bacteria). Methyl paraben and phenoxyethanol, as universal antimicrobial agents, were used as positive control. Due to the a higher active substance (I) content, it convinces to evaluate the impact on its antimicrobial activity and more particularly on an anaerobic bacteria.

[0275] As mentioned below in the Table 25, despite the decrease of the active substance (I) content, the level of active substance (I) remained sufficient to inhibit the microbial growth of P. acnes.

TABLE-US-00025 TABLE 25 Minimum inhibitory concentration of P. acnes Crude optimized MIC (mg/mL) extract P. acnes Decolored/deodorized 1.27 0.03 extract of S. chamaecyparissus Methyl paraben 2.53 Phenoxyethanol 10.13

REFERENCES

[0276] Cunliffe, W. J., Shuster, S., 1969. Pathogenesis of acne. The Lancet 293, 685-687.

[0277] Feldman, S., Careccia, R. E., Barham, K. L., Hancox, J., 2004. Diagnosis and treatment of acne. American Family Physician 69, 2123-2138.

[0278] Gollnick, H., Cunliffe, W., Berson, D., Dreno, B., Finlay, A., Leyden, J. J., Shalita, A. R., Thiboutot, D., 2003. Management of acne: a report from a Global Alliance to Improve Outcomes in Acne. Journal of the American Academy of Dermatology 49, S1-S37.