PLANT EXTRACTING METHOD

Abstract

The present disclosure provides a method of preparing a cosmetic active ingredient from patchouli, as well as the cosmetic active ingredient thus obtained and cosmetic compositions comprising the same.

Claims

1. A method of preparing a cosmetic active ingredient, said method comprising the steps of: a) providing exhausted patchouli aerial parts; and b) performing a sub-critical water extraction on the exhausted patchouli aerial parts.

2. The method of claim 1, wherein sub-critical water extraction is performed at a temperature of 100 to 175 C., and a pressure of 1 to 50 bar.

3. The method of claim 1, further comprising the step of: c) solid-liquid separation.

4. The method of claim 1, further comprising the step of: d) adding a preservative, preferably phenethyl alcohol.

5. The method of claim 1, further comprising the step of: e) sterilization.

6. A cosmetic active ingredient obtained by the method of claim 1.

7. The cosmetic active ingredient of claim 6, wherein the cosmetic active ingredient comprises a total sugar content of about 3-15 wt % on a dry basis.

8. A cosmetic composition comprising the cosmetic active ingredient of claim 6 and a carrier.

9. The cosmetic composition of claim 8, wherein the cosmetic composition is a scalp care composition.

10. The cosmetic composition of claim 9, wherein the cosmetic composition is a shampoo, an anti-dry dandruff product or a dry scalp lotion.

11. A method of stimulating the sebum production and/or of reducing the formation of dry flakes by applying the cosmetic active ingredient according to claim 6 to human scalp.

12. The method of claim 2, wherein the sub-critical water extraction is performed at a temperature of 110 to 150 C.

13. The method of claim 12, wherein the sub-critical water extraction is performed at a temperature of about 125 C.

14. The method of claim 2, wherein the sub-critical water extraction is performed at a pressure of 10 to 30 bar.

15. The method of claim 14, wherein the sub-critical water extraction is performed at a pressure of about 20 bar.

16. The method of claim 3, further comprising the step of: d) adding a preservative.

17. The method of claim 4, wherein the preservative is phenethyl alcohol.

18. The method of claim 4, further comprising the step of: e) sterilization.

19. The method of claim 5, comprising the step of: e) sterile filtration.

20. The cosmetic active ingredient of claim 7, wherein the cosmetic active ingredient comprises a total sugar content of at least 10 wt %.

21. A method of stimulating the sebum production and/or of reducing the formation of dry flakes by applying the cosmetic composition according to claim 8 to human scalp.

Description

EXAMPLE 1: ETHANOL/WATER EXTRACTION OF EXHAUSTED PATCHOULI AERIAL PARTS (COMPARATIVE Example)

[0085] Dried steam distilled patchouli aerial parts were crushed to obtain a powder. 75 g of powder were extracted at 20 C. in 550 g of ethanol (70% in water) for 60 min under stirring. The extract was filtered through a 0.7 m cellulosic filter, concentrated five times to remove ethanol and finally filtered through a 0.35 m cellulosic filter. The extract (1.078 g) was then freeze dried.

[0086] Fractionation of the Crude Extract by Centrifugal Partition Chromatography (CPC)

[0087] The crude extract (1.078 g) was dissolved in 30 ml a biphasic solvent system consisting of methyl-tert-butylether (MTBE), acetonitrile and water in a 3:3:4 ratio (v/v). Centrifugal Partition Chromatography (CPC) was performed using an FCPE300 instrument (Rousselet Robatel Kromaton) with a column of 303 ml, a column rotation speed of 1200 rpm and a flow rate of 20 ml/min.

[0088] An isocratic elution of the mobile phase (lower phase of the two-phase solvent system) was performed in the ascending mode for 75 min (with an initial flow rate ramp from 0 to 20 ml/min during 5 min). The column was finally extruded by switching the mode selection valve for 20 min. The CPC chromatogram was monitored at 220 nm. Fractions of 20 ml were collected over the whole experiment, and combined according to their thin layer chromatography (TLC) profiles. TLC was performed on pre-coated silica gel 60 F254 Merck plates with the migration solvent system EtOAc/toluene/formic acid/acetic acid (70/30/11/11; v/v), visualized under UV light at 254 nm and 360 nm and revealed by spraying the dried plates with 50% H2504 and vanillin followed by heating. As a result, 16 sub-fractions were collected.

[0089] NMR Analyses and Identification of the Major Metabolites

[0090] An aliquot of each fraction from F1 to F16 (up to J 20 mg) was dissolved in 700 l DMSO-d6 and analyzed by .sup.13C NMR at 298 K on a Bruker Avance AVIII-600 spectrometer (Karlsruhe, Germany) equipped with a TXI cryoprobe. Spectra were manually phased and baseline corrected using the TOPSPIN 3.2 software (Bruker) and calibrated on the central resonance of DMSO-d6 ( 39.80 ppm). The absolute intensities of all .sup.13C NMR signals were automatically collected and binned across the spectra of the fraction series by using a locally developed computer script. The resulting table was imported into the PermutMatrix version 1.9.3 software (LIRMM, Montpellier, France) for Hierarchical Clustering Analysis (HCA). The resulting .sup.13C chemical shift clusters were visualized as dendrograms on a two-dimensional map. For metabolite identification, each .sup.13C chemical shift cluster obtained from HCA was manually submitted to the structure search engine of the database management software ACD/NMR Workbook Suite 2012 software, ACD/Labs, Ontario, Canada) comprising the structures and predicted chemical shifts of low molecular weight natural products (nJ 2950 in March 2018). In parallel, a literature survey was performed to obtain the names and chemical structures of a maximum of metabolites already reported in the species Pogostemon cablin (n70). Additional 2D NMR experiments (HSQC, HMBC, and COSY) were performed on fractions containing putatively identified compounds in order to confirm the molecular structures proposed by the database at the end of the dereplication process.

[0091] The following major metabolites were identified:

TABLE-US-00001 Verbascoside Pachypodol Apigenin-7-O-glucuronide Cytosporone V Succinic acid p-hydroxybenzoic acid Glucosyl-cytosporone V (2 isomers) Protocatechuic acid Lactic acid Syringaresinol Choline Vanillin -D-fructose Rhamnocitrin -D-fructose Luteolin-7-O-glucuronide -D-glucose Hydroxymethylglutaric acid -D-glucose 12-hydroxyjasmonic acid -D-fructopyranose a caffeoyl derivate 7,3-dimethyleriodictyol apigenin-7-sugars

[0092] The composition of the CPC fractions was as follows (Maj=major; Med=medium; Min=minor):

TABLE-US-00002 CPC- Mass % crude Fractions (mg) extract Composition 1 7.9 0.7 7,3-dimethyleriodictyol (Maj); Pachypodol (Maj); Elution Rhamnocitrin (Min) 2 16.7 1.5 7,3-dimethyleriodictyol (Maj); Pachypodol (Maj); Elution Cytosporone V (Med); Rhamnocitrin (Min) 3 7.7 0.7 p-hydroxybenzoic acid (Maj); Syringaresinol (Min); Vanillin Elution (Med) 4 10.2 0.9 Protocatechuic acid (Maj); Syringaresinol (Min); Vanillin Elution (Med) 5 14.0 1.3 12-hydroxyjasmonic acid (Maj) + caffeoyl derivatives and Elution Apigenin-7-sugars 6 11.8 1.1 Apigenin-7-O-glucuronide (Min); Succinic acid (Min); caffeoyl Elution derivatives and Apigenin-7-sugars 7 10.6 1.0 Verbascoside (Min); Apigenin-7-O-glucuronide (Maj); Elution Succinic acid (Maj); Glucosyl-cytosporone V (Min) 8 16.0 1.5 Verbascoside (Med); Apigenin-7-O-glucuronide (Med); Elution Succinic acid (Maj); Glucosyl-cytosporone V (Min) 9 26.7 2.5 Verbascoside (Maj); Apigenin-7-O-glucuronide (Min); Elution Succinic acid (Med); Glucosyl-cytosporone V (Med); Luteolin-7-O-glucuronide (Min) 10 26.9 2.5 Verbascoside (Maj); Apigenin-7-O-glucuronide (Min); Elution Succinic acid (Min); Glucosyl-cytosporone V (Med); Luteolin- 7-O-glucuronide (Min); Hydroxymethylglutaric acid (Med) 11 56.0 5.2 Apigenin-7-O-glucuronide (Min); Succinic acid (Min); Lactic Elution acid (Maj); Luteolin-7-O-glucuronide (Min); Hydroxymethylglutaric acid (Maj) 12 97.5 9.0 Choline (Min); -D-fructose (Min); -D-fructose (Med); -D- Extrusion glucose (Maj); -D-glucose (Maj); -D-fructopyranose (Min) 13 329.4 30.3 Choline (Min); -D-fructose (Med); -D-fructose (Maj); -D- Extrusion glucose (Maj); -D-glucose (Maj); -D-fructopyranose (Med) 14 306.0 28.2 Choline (Min); -D-fructose (Med); -D-fructose (Maj); -D- Extrusion glucose (Maj); -D-glucose (Maj); -D-fructopyranose (Med) 15 68.0 6.3 Choline (Min); -D-fructose (Med); -D-fructose (Maj); -D- Extrusion glucose (Maj); -D-glucose (Maj); -D-fructopyranose (Med) 16 81.1 7.5 Choline (Min); -D-fructose (Min); -D-fructose (Med); -D- Extrusion glucose (Med); -D-glucose (Med); -D-fructopyranose (Min)

EXAMPLE 2: SUB-CRITICAL WATER EXTRACTION OF EXHAUSTED PATCHOULI AERIAL PARTS

[0093] Patchouli aerial parts Pogostemon cablin (Blanco) Benth were supplied by PT Indowangi Nusajaya (Indonesia).

[0094] The plants were cultivated in Sulawesi (Indonesia). Harvest takes place all year long. Patchouli aerial parts were dried during 3 days then gathered into bundles and kept a few days before distillation. This storage allows for a light fermentation process to release some key odour components of the plant. The patchouli aerial parts were then subjected to a first steam distillation locally by the farmers and to a subsequent redistillation in stainless steel vessels to obtain patchouli oil, leaving behind the exhausted patchouli aerial parts that can be used in the method of the present invention.

[0095] The exhausted aerial part of patchouli were dried under direct sunlight for at least 3 days (8 hours per day: 07.00-15.00). Additional drying time may be needed if it rains during the drying process. The ratio between leaves and stems is about 70%/30% (w/w).

[0096] Sub-Critical Water Extraction

[0097] For each production lot, between 0.75 kg and 1.1 kg of dried exhausted patchouli aerial parts were placed into a 5 l extraction chamber. Demineralized water is pumped through the extraction chamber by means of a high pressure pump (HPLC) at 150 ml/min until a pressure of 20 bar is reached. The water circulating through the extraction chamber is previously heated to a temperature of 125 C. by means of a heating resistor. The temperature is measured by means of a thermocouple (K-type) connected to a temperature control. At the exit of the extraction chamber, the extract is cooled using an ice bath and then collected in a vessel at atmospheric pressure.

[0098] In general, the extraction was performed at a ratio of plant to solvent of 1:5 (wt/wt), such that 12.75 kg of aqueous extract were obtained from 2.55 kg of plant, for instance.

[0099] 2 kg of the thus obtained crude aqueous extract was diluted by the addition of 2 kg of demineralized water and filtered through a 1 m filter cloth, followed by sterile filtration over a 0.2 m filter plate to obtain 4 kg of an extract according to the present invention.

[0100] Fractionation

[0101] The sub-critical water extract was subjected to Centrifugal Partition Chromatography (CPC). Twelve fractions were obtained, each of which was analyzed by .sup.13C NMR. It was found that fractions 10 to 12 contained 84.5% of the dry matter present in the crude extract.

[0102] The composition of the CPC fractions was as follows (Maj=major; Med=medium; Min=minor; recovery=97.2%):

TABLE-US-00003 CPC- Mass % crude Fractions (mg) extract Composition 1 1.8 0.95 Linear free fatty acids (Maj); 7,3-dimethyleriodictyol (Med); Elution Pachypodol (Med) 2 1.8 0.95 Linear free fatty acids (Maj); 7,3-dimethyleriodictyol (Med); Elution Pachypodol (Med) 3 11.7 0.6 Syringaresinol (Min); Cytosporone V (Maj); Linear free fatty Elution acids (Med); p-hydroxybenzoic acid (Min) 4 15.8 0.8 Protocatechualdehyde (Maj); Linear free fatty acids (Med); Elution Caffeic acid (Med); p-hydroxybenzoic acid (Maj); Protocatechuic acid (Med) 5 22.2 1.2 Vomifoliol (Med); Protocatechuic acid (Med); 12- Elution Hydroxyjasmonic acid (Med) 6 30.4 1.6 Succinic acid (Med); Apigenin-7-O-glucuronide (Maj) Elution 7 82.5 4.3 Succinic acid (Maj); 3-Hydroxy-3-methylglutaric acid (Min); Elution Apigenin-7-O-glucuronide (Med); Glucosyl-cytosporone V (Med); Luteolin-7-O-glucuronide (Min); Verbascoside (Min) 8 32.1 1.7 Succinic acid (Min); 3-Hydroxy-3-methylglutaric acid (Maj); Elution Apigenin-7-O-glucuronide (Min); Glucosyl-cytosporone V (Med); Lactic acid (Med); Pentonic acid 2-desoxy-y-lactone (Min); Luteolin-7-O-glucuronide (Med); Verbascoside (Min) 9 30.5 1.6 3-Hydroxy-3-methylglutaric acid (Maj); Lactic acid (Maj); Elution Pentonic acid 2-desoxy--lactone (Maj); Luteolin-7-O- glucuronide (Med) 10 732.3 38.7 Mixture of oliogosacchardies (not well soluble in DMSA-d6, Extrusion not fully identified); Glycerol 11 758.6 40.0 Mixture of oliogosacchardies (not well soluble in DMSA-d6, Extrusion not fully identified); Glycerol 12 110.6 5.8 Mixture of oliogosacchardies (not well soluble in DMSA-d6, Extrusion not fully identified); Glycerol (Maj)

EXAMPLE 3: DETERMINATION OF SUGAR CONTENT

[0103] The sugar content of the extract of the present invention, which was obtained by sub-critical water extraction, was compared to those of extracts prepared by classic extraction using water at high temperature and an ethanol/water mixture, respectively.

[0104] The extract of the present invention, Sample A, was prepared as described in Example 2 above.

[0105] The first comparative Sample B was prepared by extracting 40 g of exhausted patchouli aerial parts with 2400 ml of water at reflux temperature for 22 h, followed by filtration over an AF6 Filtrox filter plate (15-35 m) and over an AF31 H Filtrox filter plate (5-12 m), using perlite as a filtration aid.

[0106] The second comparative Sample C was prepared as described in comparative Example 1 above.

[0107] The sugar content of the three samples was determined by high performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD) using calibration curves of rhamnose, arabinose, galactose, glucose, and mannose. To this end, 100 mg of each sample was placed into a 20 ml tube, and 2.5 ml of water and 0.5 ml of hydrochloric acid (37%) were added. The resulting mixture was mixed well and heated to 100 C. for 6 h. After cooling to reoom temperature, the mixture was transferred to a 10 ml volumetric flasked, which was filled up with water. 1 ml of the resulting diluted mixture was further diluted with 19 ml of water, passed through a 0.45 m RC filter and an OnGuard Ag/H cartridge, before injection into the IC system.

TABLE-US-00004 Extract Total sugar content on a dry basis Sample A 45.6 wt % Sample B 14.9 wt % Sample C 8.42 wt %

[0108] It was found that the extract of the present invention (Sample A) had a significantly higher total sugar content than the two conventional extracts, namely about triple for Sample B and more than five-fold for Sample C.

EXAMPLE 4: COSMETIC ACTIVE INGREDIENT

[0109] 495 g of the liquid sub-critical water extract obtained in Example 2 was diluted with 495 g of water, and 10 g of phenethyl alcohol was added. The thus obtained cosmetic active ingredient contained 1.2% of patchouli extract (based on dry matter), 0.94% of phenethyl alcohol, and 98.04% of water.

[0110] The thus obtained cosmetic active ingredient of the present invention is a water-soluble, brown to dark brown, liquid with a characteristic smell. It has a pH of 4-6, a Gardner Index of 16-18, a dry matter content of 1-5%, a phenethyl alcohol content of 0.9-1.2%, and a total sugar content of 0.07-0.4%. Microbiological specifications with regard to total plate count and yeast and mould count were both below 100 U.F.C./g.

EXAMPLE 5: WOUND HEALING

[0111] Normal Human Keratinocytes (NHEKs) were seeded at 200000 cells per well pre-coated with Type I collagen in 12-wells culture plates in the presence of keratinocyte growth medium (KGM, Lonza) supplemented with growth factors such as hydrocortisone, transferrin, epinephrine, bovine pituitary extract (BPE), recombinant human epidermal growth factor (rhEGF) and insulin. At confluence, cells were pre-incubated with the patchouli extracts and some of the fractions thereof described in examples 1 and 2 above, each at 0.5% (v/v), overnight in keratinocyte basal medium without supplement. After this pre-conditioning phase, the cell monolayer sticking to the bottom of the well was scratched with a P200 sterile cone, followed by two washes with phosphate buffered saline (PBS). The NHEK were then again stimulated with the same samples for 24 h in basal medium.

[0112] The wound healing process was analyzed by pictures recording at T.sub.0 and T.sub.24 h using inverting optical microscope (Zeiss). After image analysis, the percentage of scratch closing was determined relative to untreated condition.

[0113] Results are shown in the following table:

TABLE-US-00005 Scratch Closing [%] Average SEM Untreated 100 7 Whole patchouli extract from example 1 2 1 (comparative example) Whole patchouli extract from example 2 1 1 Fraction 2 from example 2 93 5 Fraction 3 from example 2 100 18 Fraction 4 from example 2 83 8 Fraction 5 from example 2 86 26 Fraction 6 from example 2 128 25 Fractions 7-8 from example 2 75 4 Fraction 9 from example 2 94 3 Fractions 10-12 from example 2 17 7

[0114] As can be seen from the above, both whole patchouli extracts led to a complete inhibition of the keratinocytes migration. They are therefore both suitable for wound healing control. An inhibition of wound healing is particularly important in conditions involving a hyperproliferation of keratinocytes, e.g. hyperkeratosis or dry dandruff.

[0115] Within the patchouli extract according to the present invention, fractions 10-12 were found to be responsible for the inhibition of keratinocytes migration.

EXAMPLE 6: ANTI-OXIDANT ACTIVITY

[0116] NHEKs were seeded in a black plate with a glass bottom at 20000 cells per well pre-coated with type I collagen. Cells were incubated at 37 C. with 5% CO.sub.2 for 24 h. On the next day, the cells were incubated for 24 h with the patchouli extracts and some of the fractions thereof described in examples 1 and 2 above, each at 0.5% (v/v), or Resveratrol (positive control) at 200 M. After this pre-incubation, the cells were incubated with dichloro-dihydro-fluorescein (DCFH) probe at 50 M for 30-45 min. The cells were then rinsed two times with PBS and incubated with PBS alone or with PBS containing 5 mM tert-butyl peroxide (TBP) to induce an oxidative stress. Fluorescence reading was performed every 10 min for 1 h, exciting at 488 nm and emitting at 525 nm.

[0117] The results are shown in the following table:

TABLE-US-00006 ROS Production [% of TBP, untreated] Average SEM No TBP Untreated 32 2.7 TBP Untreated 100 4.8 Resveratrol 18 1.1 Patchouli Extract of Example 1 69 3.4 Patchouli Extract of Example 2 51 4.0 Fractions 10-12 of Example 2 77 3.3

[0118] As can be seen from the above, the whole patchouli extract of example 2 led to a reduction in reactive oxygen species (ROS) production by 49% while the comparative whole patchouli extract of example 1 showed a reduction of ROS by only 31%. Thus, the extract of the present invention is significantly more efficient than the comparative sample.

[0119] Interestingly, it was found that the fractions 10-12 were also involved in the antioxidant property of the patchouli extract.

EXAMPLE 7: CLINICAL STUDY 1: REACTIVATION OF SEBUM PRODUCTION ON DRY SCALP BY APPLICATION OF a Shampoo (Rinse-Off)

[0120] This first clinical study was performed to evaluate the efficacy of the cosmetic active ingredient of the present invention in a rinse-off application on volunteers having dry scalp, itching, scalp discomfort and white flakes. The patchouli extract was tested in a shampoo at a concentration of (v/v) versus a placebo. The volunteers applied the shampoo every other day for 28 days.

[0121] INCI Formula and Composition of Shampoos [0122] Placebo: AQUA/WATER, SODIUM LAURETH SULFATE, COCAMIDOPROPYLBETAINE, SODIUM CHLORIDE, PHENOXYETHANOL, FRAGRANCE [0123] Active: AQUA/WATER, SODIUM LAURETH SULFATE, COCAMIDOPROPYLBETAINE, PATCHOULI EXTRACT, SODIUM CHLORIDE, PHENOXYETHANOL, FRAGRANCE

TABLE-US-00007 Placebo (v/v) Active (v/v) AQUA/WATER 57.5% 57.0% SODIUM LAURETH SULFATE 28.9% 28.9 COCAMIDOPROPYLBETAINE 12.0% 12.0% SODIUM CHLORIDE 0.5% 0.5% PHENOXYETHANOL 0.6% 0.6% FRAGRANCE 0.5% 0.5% PATCHOULI EXTRACT 0.5%

[0124] Panel Description

[0125] A single-center study with 40 volunteers exhibiting dry and itchy scalp was carried out. Volunteers were divided in two groups of 20 volunteers as following: [0126] Group 1:20 volunteers in total, including 10 women and 10 men with an average age of years [0127] Group 2: 20 volunteers in total, including 14 women and 6 men with an average age of 5111 years

[0128] The study was conducted according to the standard operating procedure of Centro de Tecnologia Capilar S.L. and in compliance with the regulations established in Gula para investigaciones con seres humanos (Guidelines for Research on Human Beings) and the guidelines of the Scientific Committee on Consumer Safety (SCCS).

[0129] The volunteers applied the active (containing 0.5% of the extract of example 2 above) or placebo shampoo every other day for 28 days.

[0130] Statistical Analysis

[0131] For all clinical studies described herein, the following statistical analysis was performed: [0132] Comparison before/after: [0133] a Shapiro-Wilk test was performed to determine the normality of differences (=0.05) [0134] according to the result of the normality tested, either a paired Student t test or a Wilkoxon signed rank test was performed [0135] results with p0.05 were rejected as not statistically significant [0136] results with p0.05 were considered statistically significant [0137] Comparison product/placebo: [0138] a Shapiro-Wilk test was performed to determine the normality of data within each group (=0.05) [0139] according to the result of the normality tested, either an unpaired Student t test or a Mann-Whitney test was performed [0140] results with p0.05 were rejected as not statistically significant [0141] results with p0.05 were considered statistically significant [0142] For the analysis of self-assessment results, a Chi-square test was done (dichotomous analysis which consists in comparing the number of associated answers)

[0143] Sebum Analysis by Sebufix Scoring

[0144] Scalp sebum is measured at different times to assess whether the tested products have sebum regulating properties. In this case, the SEBUFIX (Sebufix, CKelectronic, Cologne, Germany) is applied to the upper part of the scalp to achieve the absorption of the sebum present on the scalp. The microcamera is a professional diagnostic piece of equipment consisting of a probe for exploring the scalp and the software for capturing and displaying images. The microcamera's function is to allow a direct, magnified view of a scalp area to observe its state: whether flakiness, greasiness or redness are present, etc.

[0145] The attributes under studydandruff, redness, greasiness, etc.were evaluated on a scale of 0-5: The lowest value (0) corresponds to the level absence of the attribute and the highest value (5) corresponds to the level very high. The intermediate values 1, 2, 3 and 4 correspond to the levels low, average, considerable and high, respectively.

[0146] Results

[0147] After 28 days of shampoo application, it was observed that the placebo shampoo led to a decrease in sebum by 25% compared to D.sub.0, while the shampoo containing 0.5% of the patchouli extract of example 2 led to a slight increase by +9%.

[0148] In addition, a significant improvement of the sebum production was observed with the active by +34% in comparison to the placebo.

[0149] These effects were also confirmed by illustrative pictures showing lipid droplets caused by sebum on Sebufix: Indeed, an increase of lipid droplets was observed for the active, while the placebo led to a decrease, as observed by the distribution of dark spots on illustrative pictures.

[0150] These results demonstrate that the patchouli extract of the present invention is able to reactivate sebum production on dry scalp.

EXAMPLE 8: CLINICAL STUDY 2: IMPACT ON SCALP MICROBIOTA COMPOSITION ON DRY SCALP BY Application of a Shampoo (Rinse-Off)

[0151] Microbiota Sampling and Storage

[0152] Two groups of 20 volunteers each (same panel as in example 7), exhibiting dry and itching scalp presenting white flakes, had to apply on their scalp and hair a shampoo formula either containing the active (vehicle+patchouli extract of example 2 at 0.5%) or a placebo (vehicle only), every other day for 28 days.

[0153] Scalp samples of microflora were collected from the occiput by a non-invasive swabbing method using sterile swabs moistened with a sterile solution of 0.15 M NaCl. Swabs were transferred at 20 C. and kept frozen until DNA extraction. Samples were taken before treatment (D.sub.0), and after 28 days of treatment (D.sub.28), using a standardized procedure.

[0154] DNA Extraction

[0155] DNA extraction was performed, for 92 samples (220 test samples plus 12 negative control samples), using the DNeasy PowerLyzer PowerSoil DNA Isolation Kit with Qiacube device (Qiagen, Hilden, Germany), with the following modifications: The tip of each swab was detached with a sterile surgical blade and transferred into a 1.5 ml tube containing 750 l of Bead Solution. The sampled biomass was suspended by stirring and pipetting, and then transferred to a bead beating tube. The remaining steps were performed according to the manufacturer instructions. DNA concentration was determined using the QuBit dsDNA HS fluorometric quantitation kit (Invitrogen, ThermoFisher Scientific, Courtaboeuf, France) according to the manufacturer's instructions.

[0156] Sequencing and Data Analysis

[0157] 16S rRNA Gene Sequencing

[0158] Sequencing was performed using a MiSeq device (Illumina, Inc., San Diego, CA, USA) through a 500 cycles paired-end run, targeting the V3V4 16S variable regions using the following primers: 16S-Mi341F forward primer 5-CCTACGGGNGGCWGCAG-3 and 16S-Mi805R reverse primer producing about 460 bp amplicons.

[0159] PCR1s were performed as follows: 8 l of template DNA (0.2 ng) were mixed with 5 l of each reverse and forward primers (1 M), 5 l of KAPA HiFi Fidelity Buffer (5), 0.8 l of KAPA dNTP Mix (10 mM each), 0.7 l of RT-PCR grade water (Ambion), and 0.6 l of KAPA HiFi hotstart Taq (1 U/l), for a total volume of 25 l. Each amplification was duplicated, and duplicates were pooled after amplification. PCR1 cycles consisted of 95 C. for 3 min and then 32 cycles of 95 C. for 30 s, 59 C. for 30 s, and 72 C. for 30 s, followed by a final extension at 72 C. for 3 min, with a BioRad CFX1000 thermocycler. Negative and positive controls were included in all steps to check for contamination. All duplicate pools were controlled by gel electrophoresis, and amplicons were quantified using fluorometry.

[0160] Libraries ready for analysis were then produced following the Illumina guidelines for 16S metagenomics libraries preparation. Briefly, the PCR1 amplicons were purified and controlled using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, USA). To enable the simultaneous analysis of multiple samples (multiplexing), Nextera XT indexes (Illumina) were added during PCR2 using between 15 to 30 ng of PCR1 amplicons. PCR2 cycles consisted of 94 C. for 1 min and then 12 cycles of 94 C. for 60 s, 65 C. for 60 s, and 72 C. for 60 s, followed by a final extension at 72 C. for 10 min. Indexed libraries were purified, quantified and controlled using an Agilent 2100 Bioanalyzer. Validated indexed libraries were pooled in order to obtain an equimolar mixture.

[0161] The run (500 cycles) was achieved on MiSeq sequencer (Illumina) using the MiSeq Reagent Kit v3 600 cycles (Illumina). The sequencing run produced an output of 12.7 million of paired-end reads of 250 bases, i.e. up to 3.2 Gigabases.

[0162] After the MiSeq run, raw data sequences were demultiplexed and quality-checked to remove all the reads with ambiguous bases. Indexes and primers sequences were removed with cutadapt (v1.9; http://cutadapt.readthedocs.io/en/stable/index.html) and reads with fastq score lower than 28 were trimmed. The forward and reverse sequences were paired using bbmerge (https://jgi.doe.gov/data-and-tools/bbtools/). Samples with less than 5000 paired-sequences were discarded. The remaining paired sequences were then treated using an in-house pipeline that uses vsearch (Rognes et al., 2016) to remove chimeras and amplicons with PCR errors. Sequences were then split into Operational Taxonomic Units (OTUs, a cluster of similar sequence variants of the 16S rRNA marker gene sequence) at a 1% dissimilarity level using swarm (v2.6Mah et al., 2015). Unique amplicons were mapped to the SILVA SSU Ref NR 99 (non-redundant) database (release 132; https://www.arb-silva.de/) for taxonomic assignation using the RDP classifier (Wang et al., 2007). Data normalization and analyses were done using R statistical computing environment (v3.2.0; https://www.r-project.org/R core team (2014) using Bioconductor package (mainly Phyloseq, DESeq2 and Vegan libraries; http://www.bioconductor.org/).

[0163] Data were then compared using Wilcoxon's test for paired samples (Wilcoxon, 1945). Due to multiple testing, p-value were adjusted using false discovery rate (FDR) correction (Binyamini and Hochberg, 1995)

[0164] Results

[0165] The impact of sebum level on scalp microbiota composition was studied using a 16S sequencing approach. A significant variation on scalp microbiota composition was observed, which is related to the amount of sebum. More particularly, an important increase of the Cutibacterium abundance was observed on oily scalp with +54%. The high dominance of Cutibacterium on oily scalp would explain the decrease of alpha diversity through the selective pressure mediated by the increased quantity of sebum. Other significant modifications were also observed, such as a strong decrease of Corynebacterium, Streptococcus, Micrococcus and Kocuria by 71%, 21%, 33% and 74%, respectively.

[0166] This shows that a high abundance of Cutibacterium can be used as marker to evaluate the impact of sebum on the scalp microbiota composition. Indeed, a dry scalp presents a low level of sebum linked to an important alpha diversity and a low abundance of Cutibacterium.

[0167] During the clinical evaluation of the patchouli extract, scalp microbiota samples were collected by swabbing scalps in order to assess the improvement of dry scalp.

[0168] After treatment with the active, a decrease in alpha diversity (Shannon Index) by 19.5% was observed, while the placebo treatment led to a decrease by only 4.4%. Thus, the patchouli extract of the present invention induces the sebum production on dry scalp and triggers a decrease of microbiota diversity.

[0169] Furthermore, after placebo treatment, an important decrease of Cutibacterium proportion by 18.5% was observed after 28 days of application, while the application of the active led to a stabilising of the Cutibacterium proportion (slight increase by +3.2%). Thus, the patchouli extract of the present invention increased the abundance of Cutibacterium on dry scalp, which is an indicator for a better scalp condition.

[0170] Overall, the above results demonstrate that the patchouli extract of the present invention is able to increase the sebum production on dry scalp, to decrease the alpha diversity, and to increase the Cutibacterium abundance. These observations clearly indicate that the extract is suitable for treating dry scalp condition.

EXAMPLE 9: CLINICAL STUDY 3: REDUCTION OF WHITE FLAKES ON DRY SCALP BY APPLICATION OF A Shampoo (Rinse-Off)

[0171] INCI Formula and Composition of Shampoos [0172] Placebo: AQUA/WATER, SODIUM LAURETH SULFATE, COCAMIDOPROPYLBETAINE, SODIUM CHLORIDE, PHENOXYETHANOL, FRAGRANCE [0173] Active: AQUA/WATER, SODIUM LAURETH SULFATE, COCAMIDOPROPYLBETAINE, PATCHOULI EXTRACT, SODIUM CHLORIDE, PHENOXYETHANOL, FRAGRANCE

TABLE-US-00008 Placebo (v/v) Active (v/v) AQUA/WATER 57.5% 57.0% SODIUM LAURETH SULFATE 28.9% 28.9 COCAMIDOPROPYLBETAINE 12.0% 12.0% SODIUM CHLORIDE 0.5% 0.5% PHENOXYETHANOL 0.6% 0.6% FRAGRANCE 0.5% 0.5% PATCHOULI EXTRACT 0.5%

[0174] Panel Description

[0175] This third clinical study was carried out on 41 volunteers aged over 18 years, exhibiting dry and itchy scalp with the presence of white flakes. The volunteers were divided in two groups: [0176] Group 1: 21 volunteers including 5 men and 16 women with an average age of 332 years [0177] Group 1: 20 volunteers including 4 men and 16 women with an average age of 331 years

[0178] They willingly signed the consent form which was written in compliance with the Declaration of Helsinki and the Dec. 20, 1988 act of the Code de la Sant Publique.

[0179] During this study, volunteers applied a shampoo containing 0.5% of active or placebo every two days for 28 days.

[0180] White Flakes Reduction Analysis by Scoring

[0181] For the clinical evaluation, four regions of the head were assessed separately: front left, front right, back left, and back right. Scores were then calculated as the mean of the four regions for each volunteer.

[0182] Two days after application of the shampoo, non-adherent dandruff was evaluated on a scale of 0-5: The lowest value (0) corresponds to no dandruff, and the highest value (5) corresponds to a very large quantity of dandruff. The intermediate values 1, 2, 3 and 4 correspond to a few dispersed dandruff flakes, a small quantity of dandruff, a moderate quantity of dandruff and a large quantity of dandruff, respectively.

[0183] It was found that the shampoo containing the patchouli extract of example 2 at 0.5% significantly reduced the number of white flakes by 23% and 33% after 14 and 28 days of application, respectively.

[0184] By comparison, the application of the placebo shampoo only slightly reduced the number of white flakes by 9% after 14 days, and the reduction was only significant after 28 days of application, showing a reduction by 20% compared to Do.

[0185] The difference between the active of the present invention and the placebo was found to be significant.

[0186] These results demonstrate that the patchouli extract of the present invention is able to significantly reduce white flakes on dry scalp in a rinse-off application (shampoo).

[0187] Illustrative Pictures Taken by C-Cube

[0188] C-Cube 2 (Pixience) dermoscope allows skin image acquisition in Ultra High Definition (4K UHD video streaming; 18 million pixels image resolution). It incorporates True Color patented technology, which optimally renders the full spectrum of the skin's natural colours. The size of image acquisition is 1216 mm and the magnification is 5.

[0189] It was found that the application of placebo shampoo for 28 days had only a very slight effect on white flakes, while the shampoo containing the patchouli extract at 0.5% was efficient drastically reduced white flakes.

EXAMPLE 10: CLINICAL STUDY 4: IMPACT ON DRY SCALP AND WHITE FLAKES USING A LEAVE-ON PRODUCT (Hair Lotion)

[0190] INCI Formula and Composition of Hair Lotions [0191] Placebo: AQUA/WATER, SODIUM BENZOATE, PPG-26 BUTETH-26, PEG-40 HYDROGENATED CASTOR OIL, CITRIC ACID, FRAGRANCE, CITRONELLOL, BUTYLPHENYL, METHYLPROPIONAL, D-LIMONENE, ALPHA-ISOMETHYL IONONE [0192] Active: AQUA/WATER, PATCHOULI EXTRACT, SODIUM BENZOATE, PPG-26 BUTETH-26, PEG-40 HYDROGENATED CASTOR OIL, CITRIC ACID, FRAGRANCE, CITRONELLOL, BUTYLPHENYL, METHYLPROPIONAL, D-LIMONENE, ALPHA-ISOMETHYL IONONE

TABLE-US-00009 Placebo (v/v) Active (v/v) AQUA/WATER 99.1% 98.6% SODIUM BENZOATE 0.5% 0.5% PPG-26 BUTETH-26, PEG-40 0.2% 0.2% HYDROGENATED CASTOR OIL, WATER (Solubilizing LRI solution) CITRIC ACID 0.1% 0.1% FRAGRANCE, CITRONELLOL, 0.08% 0.08% BUTYLPHENYL, METHYLPROPIONAL, D-LIMONENE, ALPHA-ISOMETHYL IONONE PATCHOULI EXTRACT 0.5%

[0193] Panel Description

[0194] This fourth clinical study was done in a double-blind, randomized and placebo controlled setting. The assessment was based on an intra-subject comparison.

[0195] All subjects received verbal and written information concerning the study. This information emphasized that participation in the study is voluntary and that the subject may withdraw from the study at any time and for any reason. All subjects were given the opportunity to ask questions about the study and were given sufficient time to consider their participation before consenting. The subject's written informed consent to participate in the study was obtained prior to any study-related procedure being carried out.

[0196] 30 volunteers, aged between 18 and 75, with a mean age of 43.413.3 years, where split into two groups of 15 each. Volunteers were exhibiting white flakes, itching on the scalp and with a dry scalp (Corneometer<50 a.u on the forehead/scalp).

[0197] Volunteers applied the respective products twice a day for 28 days to their scalp according to a 12-point pattern (3 lines of 4 points each on left, middle and right, going from front to back). After application of the products, the application points were massaged until full product penetration.

[0198] The reduction of white flakes was evaluated by scoring and self-assessment.

[0199] White Flakes Reduction Analysis by Scoring

[0200] Evaluation of the amount of white flakes was done by scoring using the following scale from 1 to

TABLE-US-00010 Grade 1 Grade 2 Grade 3 Grade 4 Grade 4 No presence No visible dry Some visible dry Important visible Important dry of dry dandruffs but dandruffs and dry dandruffs and dandruffs on scalp dandruffs visible after visible after visible after and clothes without scratching scratching scratching and with scratching

[0201] After 28 days of application of the active, a significant reduction of white flakes by 21% was observed compared to Do.

[0202] The application of the placebo formula, on the other hand, led to a slight but not significant increase of white flakes by 10%.

[0203] Thus, a significant reduction of white flakes by 31% with the active lotion compared to the placebo lotion was observed after 28 days of application.

[0204] These results demonstrate the strong efficacy of the extract of the present invention on the reduction of white flakes from dry and itching scalp.

[0205] Self-Assessment

[0206] After 28 days of application, only 13% of volunteers applying the active lotion had intense and frequent itching, compared to 47% of those applying the placebo lotion. These differences were significant, demonstrating that the lotion containing the patchouli extract of the present invention significantly reduces the frequency and intensity of itching in comparison with placebo.

[0207] It was further observed that only 7% of volunteers who applied the active lotion found white flakes on their clothes, compared to 33% of those applying the placebo lotion. This observation demonstrates that the cosmetic active ingredient of the present invention is able to significantly reduce the white flakes found on clothes in comparison to the placebo effect.

EXAMPLE 11: CLINICAL STUDY 5: EMOTIONAL EVALUATION BY NEUROSCIENCE METHODS (LEAVE-ON Product)

[0208] This fifth clinical study was performed in the same setting as the fourth clinical study described in example 12 above.

[0209] Prosody Analysis

[0210] The participants were asked to verbalize how they feel about their scalp at the beginning and end of the 28-day study. From the audio recording of their free verbal productions, a prosody analysis was applied. The prosody analysis consists in capturing the vocal signal of the verbal expression of subjects and analyzing the physical parameters of the voice in terms of emotional expression.

[0211] Using computer-based solutions, two main variables were extracted: [0212] Loudness (i.e. vocal intensity), represented by the mean amplitude measured in dB [0213] Pitch (i.e. tone), represented by the coefficient of variation of the fundamental frequency (cvFO) measured in Hz)

[0214] The combination of these two variables allows for assessing the degree of emotional valence and arousal expressed vocally. An increase of frequency and amplitude in this context suggest that volunteers felt more positive emotions.

[0215] It was found that, after 28 days of application, the active lotion led to a stronger increase in the frequency and amplitude than the placebo lotion. Furthermore, the active showed a significant increase of frequency and amplitude in comparison with placebo by +15.4% and +12.4% respectively.

[0216] Wherefore, the extract of the present invention was able to significantly improve the emotional response after 28 days of application, as observed by prosody evaluation.

[0217] In order to complete this analysis, an emotional valence analysis was performed, permitting to study the emotional distribution considering stimulating/unstimulating and pleasant/unpleasant emotions scale.

[0218] It was found that the active lotion triggered stimulating and pleasant emotions, while the placebo lotion triggered neutral emotions.

[0219] Finally, the percentage of volunteers who have triggered negative and positive emotions was analysed for each group. It was found that volunteers applying the active lotion have triggered 80% of positive emotions and 20% negative ones. In contrast, volunteers who applied the placebo lotion, have triggered only 33% of positive emotions and 66% negative ones.

[0220] Mood Portraits

[0221] The Mood Portraits method was used to measure the emotional responses of consumers at the beginning and end of the 28-day study. Participants were asked to think about the condition of their current scalp and the emotions that this brought to their minds. They were then asked to select pictures representing these emotions.

[0222] Day 0 results showed that participants had a very negative emotional response when thinking about their scalp. Day 28 results showed that participants had a significantly more positive emotional response when thinking about their scalp, with the active group evoking more significant positive mood responses than the placebo.

[0223] On D.sub.0, participants felt very negative, and significantly not happy, when thinking about their scalp.

[0224] On D.sub.28, participants in the active group felt significantly less negative after using the hair lotion for 28 days and significantly more refreshed.

[0225] Participants in the placebo group did not feel as negatively about their scalp on D.sub.28; however, there was no significant difference. No positive mood was significantly evoked, but participants tended to feel more refreshed.

[0226] There is a significant difference between the active group, the placebo group and the D.sub.0 results for refreshed and negative moods.

[0227] This effect was also visible through the images selected by volunteers at D.sub.0 and D.sub.28: At Do, volunteers illustrated their feelings about their scalp condition by images having negative and non-happy connotations.

[0228] After 28 days of active lotion application, a strong decrease of negative images associated was observed, as well as new images with a connotation of refreshing and self-confidence.

[0229] The group who applied placebo did not show the same efficacy, as shown by the selected images which are still negative.

[0230] This results show that the patchouli extract of the present invention delivers a positive emotional response after 28 days of application on volunteers who have dry itching scalp with white flakes. The improvement of the volunteers' mood is correlated to a significant reduction of white flakes and itching and, thus, to an improvement of their scalp condition.

[0231] Non-Verbal Communication Using Non-Verbal Decoding System

[0232] Measurement of the unconscious emotional power of the lotion on the consumers was carried out by Marina Cavassilas of Semiopolis, an expert in non-verbal communication. To determine the emotional impact of the tested lotions, the body language of 14 users who used the active lotion, compared to that of 15 users who used the placebo.

[0233] The participants were interviewed on their use of the lotion and filmed face to face for about 5 minutes. A study of their expressiveness had been organized before the study to check their normal abilities to express their emotions by the face. This methods uses a unique grid to analyse over 200 non-verbal reactions (facial reactions, postures, gestures, voice), and remove all verbalisation barriers.

[0234] After 28 days of active lotion application, it was observed that 19 positive emotions and only 4 negative emotions were triggered in the volunteers. In contrast, the volunteers applying placebo lotion presented an inverse emotional response, with 14 negative emotions and only 2 positive emotions after 28 days.

[0235] These results demonstrate that the patchouli extract of the present invention was able to deliver a positive emotional response compared to the placebo lotion. Interestingly, it was found that this difference is statistically significant.

[0236] This method permits also to identify the type emotions after product application.

[0237] It was observed that volunteers who applied the active lotion showed positive emotions including comfort, care and happiness, while no any specific emotions were attributed to the placebo lotion. In addition, some emotions were found to be common to both the active and the placebo, including vigor, energy, power, and annoyance.

[0238] Thus, using non-verbal communication, it was found that the patchouli extract delivered a positive emotional response after 28 days of application on volunteers who have dry itching scalp with white flakes.