WO2021129977A1

WO2021129977A1 - Plant extracts for treatment of increased facial vascularity

Info

Publication number: WO2021129977A1
Application number: PCT/EP2020/082154
Authority: WO
Inventors: Nahid Amini; Christina ÖSTERLUND; Michele Leonardi; Virginie LAFON-KOLB; Ia KHMALADZE; Lene VISDAL-JOHNSEN; Susanne FABRE; Nina HRAPOVIC
Original assignee: Oriflame Cosmetics Ag
Priority date: 2019-12-23
Filing date: 2020-11-13
Publication date: 2021-07-01
Also published as: SE544843C2; EP4081181A1; SE1930415A1; CN115209868A

Abstract

The present invention relates to the use of an extract of Alpinia officinarum for the treatment of increased facial vascularity. The present invention also relates to a cosmetic or pharmaceutical formulation comprising a combination of at least two different active components selected from: an extract of Alpinia officinarum, myricetin, lobaric acid, and an extract of Persicaria capitata, and the use thereof for the treatment of increased facial vascularity. The present invention also relates to a method for the production of a cosmetic or pharmaceutical formulation comprising a combination of at least two different active components selected from: an extract of Alpinia officinarum, myricetin, lobaric acid, and an extract of Persicaria capitata.

Description

PLANT EXTRACTS FOR TREATMENT OF INCREASED FACIAL VASCULARITY

The present invention provides an extract of Alpinia officinarum for the treatment of increased facial vascularity. The present invention further provides a cosmetic or pharmaceutical formulation for use in the treatment of increased facial vascularity, and a method for production of the formulation.

BACKGROUND OF INVENTION

Rosacea is a common, but poorly understood, long-term, chronic skin condition that occurs mainly on the face, typically on the nose, cheeks and forehead. The causes of rosacea are unknown and there is currently no cure.

The symptoms of rosacea typically begin with the patient suffering from episodes of flushing where the skin turns red for a short period of time. As the condition progresses, the patient may develop other symptoms including for example: burning and stinging sensations, permanent redness, spots and/or small blood vessels in the skin becoming visible. Rosacea is a relapsing condition, which means that a patient may experience periods of time where the symptoms lessen or disappear altogether, and then the symptoms return.

There are four subtypes of rosacea including: erythematotelangiectactic rosacea (ETR) which is associated with facial redness, flushing and visible blood vessels; papulopustular rosacea which is associated with acne-like breakouts; rhinophyma which is associated with thickening of the skin on the nose; and ocular rosacea which is associated with symptoms on the eye area.

Rosacea can be controlled to some degree with long term treatment using conventional medications. However, the changes in appearance caused by rosacea can have a significant psychological impact on the patient, such as for example causing a loss in confidence.

It has been found that human kallikrein-related peptidases (KLKs) are differently expressed in many tissues and exist as a subgroup of 15 serine proteases. In the epidermis, kallilkrein-5 (KLK5) and kallikrein-7 (KLK7) play a primary function. The activity of KLK5 is regulated by complex mechanisms, involving various factors such as other proteases, endogenous inhibitors and physiological environment.

In lesional rosacea skin, kallikrein-5 (KLK5) levels are increased, leading to increased levels of both LL- 37 and its proteolytic fragments. In addition to the increased abundance of KLK5 levels and LL-37 and its proteolytic fragments, these peptides also differ from those found in normal individuals. Unlike normal LL-37 peptide fragments, these abnormal peptides control functions, such as leukocyte chemotaxis, angiogenesis, and expression of extracellular matrix components. The increased levels of LL-37 and other peptides induce the clinical manifestations seen in rosacea, including inflammation and increased facial vascularity.

There is therefore a need for a cosmetic or pharmaceutical formulation for use in the treatment of rosacea with improved efficacy compared to conventional medications. In particular, there is a need for a cosmetic or pharmaceutical formulation which is capable of blocking the activity or inhibiting the production of kallikrein-5 (KLK5). By preventing the production, or blocking the activity, of kallikrein- 5, the formulation should also reduce and/or prevent the formation of LL-37 and abnormal peptide fragments, thereby inhibiting a major cascade of inflammation that has been found to be correlated with major clinical findings in rosacea.

STATEMENT OF INVENTION

According to a first aspect of the present invention, there is provided a use of an extract of Alpinia officinarum for the treatment of increased facial vascularity.

Alpinia officinarum, also known as lesser galangal, is indigenous to Southeast China (Guangdong, Guangxi, Hainan) and Indochina. Alpinia officinarum belongs to the Zingiberaceae family and is a perennial herb with thick, creeping reddish-brown rhizomes, lineolate acuminate ornamental leaves, and showy white flowers in racemes.

In one embodiment, the extract of Alpinia officinarum is used in the treatment of rosacea.

In one embodiment, the extract of Alpinia officinarum is an inhibitor of kallikrein-5 (KLK5).

The extract of Alpinia officinarum has been found to be able to treat increased vascularity, and in particular symptoms of rosacea, including inflammation and increased facial vascularity, with improved efficacy compared to conventional medications. The extract of Alpinia officinarum has been found to be capable of blocking the activity or inhibiting the production of kallikrein-5 (KLK5), which consequently reduces and/or prevents the formation of LL-37 and abnormal peptide fragments, thereby inhibiting a major cascade of inflammation that has been found to be correlated with major clinical findings in rosacea.

The extract of Alpinia officinarum preferably comprises one or more of: kaempferol, galangin, quercetin, pinocembrin, dihydrogalangin, 3-O-methylgalangin, 7-O-methylgalangin, kaempferide, 5- hydroxy-7-(4'-hydroxy-3'-methoxyphenyl)-l-phenyl-3-heptanone, 3,5-dihydroxy-l,7-bi-(3,4- dihydroxyphenyl)heptane, alpinin B, galangin 7-glucoside or any combination thereof.

The extract of Alpinia officinarum may be provided in the form of a cosmetic or pharmaceutical formulation for use in the treatment of increased facial vascularity, such as for example in the treatment of rosacea (e.g. in the treatment of symptoms associated with rosacea including for example inflammation and increased facial vascularity).

The extract is preferably an extract of the root of Alpinia officinarum.

According to a second aspect of the present invention, there is provided a cosmetic or pharmaceutical formulation comprising a combination of at least two different components selected from: an extract of Alpinia officinarum, myricetin, lobaric acid, and an extract of Persicaria capitata (PCA).

The extract is preferably an extract of the root of Alpinia officinarum and/or Persicaria capitata.

In one embodiment, the cosmetic or pharmaceutical formulation comprises an extract of Alpinia officinarum and myricetin.

In one embodiment, the cosmetic or pharmaceutical formulation comprises an extract of Alpinia officinarum and lobaric acid.

In one embodiment, the cosmetic or pharmaceutical formulation comprises an extract of Alpinia officinarum, and an extract of Persicaria capitata.

In one embodiment, the cosmetic or pharmaceutical formulation comprises myricetin and lobaric acid.

In one embodiment, the cosmetic or pharmaceutical formulation comprises lobaric acid and an extract of Persicaria capitata.

In one embodiment, the cosmetic or pharmaceutical formulation comprises myricetin and an extract of Persicaria capitata.

According to a third aspect of the present invention, there is provided the use of a cosmetic or pharmaceutical formulation as herein described in the treatment of skin inflammation, preferably in the treatment of increased facial vascularity, such as for example in the treatment of rosacea.

In one embodiment, the cosmetic or pharmaceutical formulation as herein described is used as a kallikrein-5 (KLK5) inhibitor.

According to a further aspect of the present invention, there is provided a method for the production of a cosmetic or pharmaceutical formulation as herein described, the method comprising: obtaining at least two sources of at least two different components selected from: an extract of Alpinia officinarum, myricetin, lobaric acid, and an extract of Persicaria capitata; and combining the at least two components to provide a cosmetic or pharmaceutical formulation. According to a still further aspect of the present invention, there is provided a kit for the production of a cosmetic or pharmaceutical formulation as described herein, the kit comprising: at least two sources of at least two different components selected from: an extract of Alpinia officinarum, myricetin, lobaric acid, and an extract of Persicaria capitata.

Embodiments of the present invention are described in detail with references to the accompanying Figures:

BRIEF DESCRIPTION OF FIGURES

Figures 1A-C illustrates the UFIPLC/Q-ToF-MS chromatograms of three extracts of Alpinia officinarum (ALO);

Figure 1A illustrates the UFIPLC/Q-ToF-MS chromatogram of a methanol extract of Alpinia officinarum (ALO);

Figure IB illustrates the UFIPLC/Q-ToF-MS chromatogram of a water extract of Alpinia officinarum (ALO); and

Figure 1C illustrates the UHPLC/Q-ToF-MS chromatogram of an ethanol-water extract of Alpinia officinarum (ALO);

Figure 2 illustrates the UHPLC/Q-ToF-MS chromatogram of a methanol extract of Alpinia officinarum and the identified compounds within the extract;

Figure 3 is an isobologram illustrating model lines of synergism and antagonism;

Figure 4 illustrates the percentage inhibition of the enzyme Kallikrein 5 by pure compounds and extracts;

Figure 5 is an isobologram for formulations comprising a combination myricetin/A/p/n/a officinarum (ALO);

Figure 6 is an isobologram for formualtions comprising a combination of Alpinia officinarum (ALO) and Persicaria capitata (PCA);

Figure 7 illustrates gene expression of AQP3, CASP14, and CLDN1 after 24h treatment of keratinocytes with IOmM myricetin. Treatment was compared to vehicle treated sample which was put to 1. Statistics were performed in PRISM 5 where Repeated measurement one-way ANOVA was used on the ACt values with Dunnett's post hoc test were p*<0.05, p**<0.01 and p***<0.001; Figure 8 illustrates anti-inflammatory activity in PAR2 induced proinflammatory IL-8 model using FlaCaT cells; and

Figure 9 illustrates myricetin anti-inflammatory activity in IL-lbeta induced inflammation model in dermal microvascular endothelial (DMVEC) cells.

DETAILED DESCRIPTION

Example 1 - Method of Extraction of Alpinia officinarum (ALO)

The root of the plant material ( Alpinia officinarum) was extracted using three different solvents: methanol; water; and 50:50 ethanohwater mixture. The method of extraction of the plant, as discussed below, was the same for each solvent used.

Approximately 1 g of plant material was weighed and placed in a 50ml tube. 20 ml of the appropriate solvent (as above) was added to it. The plant material/solvent mixture within the tube was then sonicated for 2 x 60 min in an ultrasonic bath at room temperature. The plant material/solvent mixture was then centrifuged for 5 min at 2000 ref. The plant material/solvent mixture was then filtered on BOchner using two filters, filter grade 1 (llpm) on the bottom and filter grade 4 (20-25 pm) on top to obtain an extract of Alpinia officinarum. The volatile solvent part of the extracts containing methanol or ethanol were first evaporated using a rotavapor (temperature = 37°C, pressure = 50 mbar) prior to freeze drying for approximately 4 days.

Table 1 shows the extraction yields of Alpinia officinarum using the three different solvents:

Table 1

The Alpinia officinarum extract prepared using a solvent comprising a 50:50 mixture of ethanol and water EtOH:H₂0 gave the highest extract yield of 22.2%

Example 2 - Characterization of extracts of Alpinia officinarum using UHPLC/Q-ToF-MS

Three solutions, one solution for each extract prepared according to the method of extraction of Alpinia officinarum Example 1, were prepared to be analysed by LC/MS. The three solutions were as follows: Solution 1 (Alpinia officinarum-001 ): 8.8 mg/1 mL 95:5 H₂O:A0N

Solution 2 (Alpinia officinarum-002 ): 7.4 mg/1 mL 95:5 H₂0:ACN

Solution 3 (Alpinia officinarum-003 ): 7.8 mg/1 mL 95:5 H₂0:ACN

Each solution was filtered using a 0.22 miti syringe filter before analysis. UHPLC analysis was carried out as follows:

UHPLC: 1290 Infinity Q-ToF LC/MS: 6520 Method: Library method

Column: Zorbax extend Ci₈2.1 x 150 mm, 1.8 miti Column temp: 35°C

Flow rate: 0.25 mL/min Injection volume: 20 mί

The UHPLC gradient results are illustrated in Table 2:

Table 2

Q-ToF ion source: -ESI Gas temp: 350°C Drying gas: 8 L/min Nebulizer: 40 psig Vcap: 3500 V

Acquisition rate: 8 spectra/s Acquisition time: 125 ms/spectrum The profiles of each of the three extracts are shown in Figures 1A-C. It can be seen that the profiles of the three extracts (using three different solvent systems as described in Example 1) are very similar.

The extract ( Alpinia officinarum-001) prepared using methanol was used to identify the compounds present within each of the extracts. These compounds are shown in Table 3 and Figure 2:

Table 3 - Compounds present within Alpinia officinarum extract using methanol as a solvent ( Alpinia officinarum-001)

Example 3 - Kallikrein-5 Enzyme Inhibition Assay

Fluman tissue Kallikrein 5 (KLK5 or hK5, also known as stratum corneum tryptic enzyme) is a serine protease expressed in the epidermis. KLK5 regulate cell shedding (desquamation) in conjunction with KLK7 and KLK14 given its ability to degrade proteins which form the extracellular component of cell junctions in the stratum corneum.

The activity of the enzyme KLK5 is measured by its ability to cleave the fluorogenic peptide substrate Boc-VPR-AMC. The assay measures the formation of AMC that is a highly fluorescent group (l exc = 380nm; l em = 460nm)

The primary screening assays are performed according to the validated standard protocol i.e. SP-SR- 201-v3 for KLK5 enzyme inhibition Assay. The combinations of active components within a formulation are performed according to the validated standard protocol i.e. SP-SR-234 for Isobologram Analysis.

Using isobolograms analysis, two methods have been used to determine if the combination of two active components within formulations of the present invention provide a synergistic effect:

Screening concentrations that gives 50% inhibition

The first step is to determine the IC₅₀s of each of the individual active components (i.e. active component A and active component B) within the formulation of the present invention. The additive isobole for 50% inhibition is then traced on Graph Pad Prism as shown in Figure 3.

The concentration of active component B that will give 50% inhibition is interpolated with a chosen concentration of active component A. The combination of the two active components A and B at these concentrations needs to then be screened.

• If the results show 50 % inhibition, it means that there is an additive effect between the two active components of the formulation at those concentration; or

• If the results show <50% inhibition, it means that the formulation needs more concentrated active components to be present in order to achieve 50 % inhibition, i.e. that the combination of the active components has an antagonist effect; or

• If the results >50% inhibition, it means that the formulation need less concentrated active components to be present within the formulation to achieve 50 % inhibition, i.e. that the combination of the active components has a synergistic effect.

Figure 4 illustrates the % inhibition of Kallikrein 5 for a number of active components and plant extracts including lobaric acid, myricetin, Alpinia officinarum (ALO) and Persicaria capitata (PCA). Lobaric acid, myricetin, Alpinia officinarum (ALO) and Persicaria capitata (PCA) were found to show a very good percentage inhibition of KLK5 and the IC₅oS of each of these active components and extracts were determined and the results are shown in Table 4.

Table 4

Example 4 - Formulation comprising two active components: myricetin and an extract of Alpinia officinarum

The isobologram of Figure 5 shows that the IC₅₀ values for the inhibition of kallikrein-5 of a number of different formulations of the present invention comprising myricetin and an extract of Alpinia officinarum. It can be seen from the isobologram that the IC₅₀ values of the formulations of the present invention are below the additive isobole, and as such it can be seen that the formulations of the present invention comprising a combination of myricetin and an extract of Alpinia officinarum provide a synergistic effect on the KLK5 inhibition activity.

Example 5 - Formulation comprising two active components: an extract of Alpinia officinarum and PCA

The isobologram of Figure 6 shows that the IC₅₀ values for the inhibition of kallikrein-5 of a number of different formulations of the present invention comprising an extract of Alpinia officinarum and PCA. It can be seen from the isobologram that the IC₅₀ values of the formulations of the present invention are below the additive isobole, and as such it can be seen that the formulations of the present invention comprising a combination of an extract of Alpinia officinarum and PCA provide a synergistic effect on the KLK5 inhibition activity.

Example 6 - Formulations comprising two active components selected from lobaric acid together with either an extract of Alpinia officinarum; an extract of Persicaria capitata; or myricetin The results for formulations of the present invention comprising a combination of lobaric acid with an extract of Alpinia officinarum and (ALO), an extract of Persicaria capitata (PCA) or myricetin were found to provide higher than 50% inhibition of kallikrein-5. As such, it was found that the formulations of the present invention comprising a combination of lobaric acid with an extract of Alpinia officinarum (ALO), an extract of Persicaria capitata or myricetin has a synergistic effect on the ability of the formulation to inhibit production of KLK5 as shown in Tables 5 and 6.

Table 5

Table 6

The extracts and formulations of Examples 3 to 6 have been found to produce improved inhibition of kallikrein-5, and to have a synergistic effect on the inhibition of kallikrein-5. The extracts and formulations of the present invention have therefore been shown to have improved efficacy for the treatment of symptoms associated with rosacea, such as for example inflammation and increased facial vascularity.

Example 7 - Effect of myricetin on Gene expression of barrier genes

Barrier genes that were analysed were OCLN, AQP3 CLDN1, IVL, CASP14, KRT1, KRT10, FLG, PNPLA and TJP1. qPCR was used to analyze the gene expression of barrier genes. Human epidermal keratinocytes (KC) were cultured in 48-well plates and treated for 24h with actives before RNA extraction followed by cDNA synthesis and then qPCR was performed for the wanted genes. GAPDH was used as the housekeeping gene.

The effect of myricetin was evaluated. The results are shown in Figure 7. Figure 7 shows that treatment with myricetin (lOpm) after 24 hours upregulated three barrier genes (AQP3, CASP14 and CLDN1) which were statistically significant when compared to the vehicle treated samples. An increase in gene expression is indicative of improvement of skin barrier in the skin.

Example 8 - Effect of Lobaric acid as an anti-inflammatory agent

TRPV1 is an important ion channel and it has been shown that it is overexpressed in sensitive skin. TRPV1 can be activated directly or indirectly via PAR2. Targeting TRPV1 via PAR2 can be done in vitro by using PAR2 specific agonistic peptide (SLIGKV-NH2) leading to inflammatory IL-8 secretion.

The aim of the study was to induce inflammatory response by targeting PAR2 in HaCaT keratinocyte cell line and test the anti-inflammatory potential of Lobaric acid (L.A) to inhibit PAR2 driven IL-8.

Plate HaCaT cells into 48 well format with respective medium from 80% confluent cells cultured in T75. (dilution factor: 1:8)

24h post or when the cells reach 50% confluence start starvation in FBS free DM EM medium 24h post starvation, pre-treat cells with Lobaric acid (3uM) in a complete medium (5% FBS in DMEM) and a control well with control antagonistic peptide (ENMD-1068, 100 mM) to block PAR 2.

30min post pre-treatment add PAR2 agonistic peptide (SLIGKV-NH2, lOOuM)

24h post treatment collects the supernatant for ELISA.

Figure 8 provides IL-8 ELISA results (of the following groups: UT (untreated), PAR2 Ago (PAR2 stimulation with agonistic peptide), PAR2 + Antago (control, to decrease PAR2 expression with antagonistic peptide pretreatment), PAR2 + Lobaric acid (L.A pretreatment prior to PAR2 stimulation)) which illustrate representative data of two identical independent experiments.

As shown in Figure 8, PAR 2 induces IL-8 upregulation in HaCaT cells and the positive control PAR2 antagonist peptide significantly down regulates it. Lobaric acid significantly inhibited PAR2 induced IL-8 and therefore is considered to be useful in the treatment of sensitive skin associated with inflammation.

Example 9 - Effect of myricetin as an anti-inflammatory agent

- Human microvascular endothelial cells (HMVEC) were grown in CADMEC medium (readymade) in 6 well format.

- When reaching 80% confluency, cells were seeded into 48 well format for the experiment (from 1 well from 6 well format plate split into 8 wells of 48 well culture plate format).

- Cells were pretreated with Myricetin (25 mM).

- 2.5h post pre-treatment, recombinant cytokine IL/lb (50 pg/ml) was added. 24h post treatment, supernatant was collected for ELISA assay.

Figure 9 illustrates IL-8 ELISA results for the following groups: UT (untreated), IL-lb (recombinant IL- lb stimulation), IL-lb + M (IL-lb + Myricetin) induced with IL-lb.

As shown in Figure 9, IL-lb induces IL-8 upregulation in HMVEC and Myricetin pretreatment, significantly inhibits cytokine induction. Thus, myricetin has been shown to be useful in the treatment of sensitive skin redness associated with proinflammatory cytokines.

Claims

1. Use of an extract of Alpinia officinarum for the treatment of increased facial vascularity.

2. Use of an extract as claimed in claim 1, for the treatment of rosacea.

3. Use of an extract as claimed in either of claims 1 and 2, in which the extract is an inhibitor of kallikrein-5 (KLK5).

4. Use of an extract as claimed in any one of claims 1 to 3, in which the extract comprises one or more of: kaempferol, galangin, quercetin, pinocembrin, dihydrogalangin, 3-0- methylgalangin, 7-O-methylgalangin, kaempferide, 5-hydroxy-7-(4'-hydroxy-3'- methoxyphenyl)-l-phenyl-3-heptanone, 3,5-dihydroxy-l,7-bi-(3,4-dihydroxyphenyl)heptane, alpinin B, galangin 7-glucoside, or any combination thereof.

5. A cosmetic or pharmaceutical formulation comprising a combination of at least two different active components selected from: an extract of Alpinia officinarum, myricetin, lobaric acid, and an extract of Persicaria capitata.

6. A cosmetic or pharmaceutical formulation as claimed in claim 5, in which the formulation comprises an extract of Alpinia officinarum and myricetin.

7. A cosmetic or pharmaceutical formulation as claimed in claim 5, in which the formulation comprises an extract of Alpinia officinarum and lobaric acid.

8. A cosmetic or pharmaceutical formulation as claimed in claim 5, in which the formulation comprises an extract of Alpinia officinarum, and an extract of Persicaria capitata.

9. A cosmetic or pharmaceutical formulation as claimed in claim 5, in which the formulation comprises myricetin and lobaric acid.

10. A cosmetic or pharmaceutical formulation as claimed in claim 5, in which the formulation comprises lobaric acid and an extract of Persicaria capitata.

11. A cosmetic or pharmaceutical formulation as claimed in claim 5, in which the formulation comprises myricetin and an extract of Persicaria capitata.

12. Use of a cosmetic or pharmaceutical formulation as claimed in any one of claims 5 to 11, in the treatment of skin inflammation.

13. Use of a cosmetic or pharmaceutical formulation as claimed in any one of claims 5 to 11, in the treatment of increased facial vascularity.

14. Use of a cosmetic or pharmaceutical formulation as claimed in any one of claims 5 to 11, in the treatment of rosacea.

15. Use of a cosmetic or pharmaceutical formulation as claimed in any one of claims 5 to 11, in which the formulation is a kallikrein-5 (KLK5) inhibitor.

16. A method for the production of a cosmetic or pharmaceutical formulation as claimed in any one of claims 5 to 11, comprising: obtaining at least two sources of at least two different components selected from: an extract of Alpinia officinarum, myricetin, lobaric acid, and an extract of Persicaria capitata; and combining the at least two components to provide a cosmetic or pharmaceutical formulation.

17. A kit for the production of a cosmetic or pharmaceutical formulation as claimed in any one of claims 5 to 11, the kit comprising: at least two sources of at least two different components selected from: an extract of Alpinia officinarum, myricetin, lobaric acid, and an extract of Persicaria capitata.