SE1930415A1 - Plant extracts for treatment of increased facial vascularity - Google Patents

Abstract

PLANT EXTRACTS FOR TREATMENT OF INCREASED FACIAL VASCULARITYThe 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 facialvascularity. 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 onthe 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 wherethe skin turns red for a short period of time. As the condition progresses, the patient may developother symptoms including for example: burning and stinging sensations, permanent redness, spotsand/or small blood vessels in the skin becoming visible. Rosacea is a relapsing condition, which meansthat 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 isassociated with facial redness, flushing and visible blood vessels; papulopustular rosacea which isassociated 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. lt has been found that human kallikrein-related peptidases (KLKs) are differently expressed in manytissues and exist as a subgroup of 15 serine proteases. ln the epidermis, kallilkrein-5 (KLK5) andkallikrein-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. ln lesional rosacea skin, kallikrein-5 (KLK5) levels are increased, leading to increased levels of both LL-37 and its proteolytic fragments. ln addition to the increased abundance of KLK5 levels and LL-37 andits proteolytic fragments, these peptides also differ from those found in normal individuals. Unlikenormal 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 ofrosacea with improved efficacy compared to conventional medications. ln particular, there is a needfor a cosmetic or pharmaceutical formulation which is capable of blocking the activity or inhibiting theproduction 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 peptidefragments, 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 lndochina. Alpinia officinarum belongs to the Zingiberaceae family and is aperennial herb with thick, creeping reddish-brown rhizomes, lineolate acuminate ornamental leaves, and showy white flowers in racemes.ln one embodiment, the extract of Alpinia officinarum is used in the treatment of rosacea.ln 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 inparticular symptoms of rosacea, including inflammation and increased facial vascularity, withimproved efficacy compared to conventional medications. The extract of Alpinia officinarum has beenfound to be capable of blocking the activity or inhibiting the production of kallikrein-5 (KLK5), whichconsequently 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)-1-phenyl-3-heptanone, 3,5-dihydroxy-1,7-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 pharmaceuticalformulation 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 prefera bly an extract of the root of Alpinia officinarum and/or Persicaria capitata. ln one embodiment, the cosmetic or pharmaceutical formulation comprises an extract of Alpinia officinarum and myricetin. ln one embodiment, the cosmetic or pharmaceutical formulation comprises an extract of Alpinia officinarum and lobaric acid. ln one embodiment, the cosmetic or pharmaceutical formulation comprises an extract of Alpinia officinarum, and an extract of Persicaria capitata. ln one embodiment, the cosmetic or pharmaceutical formulation comprises myricetin and lobaricacid.ln one embodiment, the cosmetic or pharmaceutical formulation comprises lobaric acid and an extract of Persicaria capitata. ln 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 orpharmaceutical 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. ln 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 UHPLC/Q-ToF-MS chromatograms of three extracts of Alpinia officinarum(ALO); Figure 1A illustrates the UHPLC/Q-ToF-MS chromatogram of a methanol extract ofAlpinia officinarum(ALO); Figure 1B illustrates the UHPLC/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 Alpiniaofficinarum (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/Alpinia 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 keratinocyteswith 10uM 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*s0.05, p**s0.0l and p***s0.00l; Figure 8 illustrates anti-inflammatory activity in PAR2 induced proinflammatory IL-8 model usingHaCaT cells; andFigure 9 illustrates myricetin anti-inflammatory activity in |L-1beta induced inflammation model in dermal microvascular endothelial (DMVEC) cells.

DETAILED DESCRIPTIONExample 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 ethanol:water 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 appropriatesolvent (as above) was added to it. The plant material/solvent mixture within the tube was thensonicated for 2 x 60 min in an ultrasonic bath at room temperature. The plant material/solventmixture was then centrifuged for 5 min at 2000 rcf. The plant material/solvent mixture was thenfiltered on Büchner using two filters, filter grade 1(11um)on the bottom and filter grade 4 (20-25 um)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: Extract Extraction solvent Extraction yield %Alpinia officinarum -001 MeOH 10.7Alpinia officinarum -002 H20 16.3Alpinia officinarum -003 50:50 EtOH:H2O 22.2 Table 1 The Alpinia officinarum extract prepared using a solvent comprising a 50:50 mixture of ethanol and water EtOH:H2O 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 ofAlpinia 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:ACNSolution 2 (Alpinia officinarum-002): 7.4 mg/1 mL 95:5 H2O:ACN Solution 3 (Alpinia officinarum-003): 7.8 mg/1 mL 95:5 H2O:ACN Each solution was filtered using a 0.22 um syringe filter before analysis.

UHPLC analysis was carried out as follows: UHPLC:1290|nfinity Q-ToF LC/MS: 6520 Method: Library method Column: Zorbax extend C18 2.1 x 150 mm, 1.8 umColumn temp: 35°C Flow rate: 0.25 mL/min Injection volume: 20 uL The UHPLC gradient results are illustrated in Table 2: Table 2 Q-ToF ion source: -ESIGas temp: 350°CDrying gas: 8 L/minNebulizer: 40 psigVcap: 3500 V Acquisition rate: 8 spectra/s Acquisition time: 125 ms/spectrum Time (min) A% (H20 + 91% FA) 8% (AcN + o.1% FA)o 98 z 98 z 70 40 eo 90 z 98 95 z 98 96 98 z The profiles of each of the three extracts are shown in Figures 1A-C. lt 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: Compound Retentiontime (min) Formula Volume % Pinocembrin 39.8 C15H1204 2.14Galangin 40.3 C15H1005 7.00Dihydrogalangin 29.8 C15H1205 3.50Dihydrogalangin 29.3 C15H1205 0.04Dihydrogalangin 29.6 C15H1205 0.283-O-methylgalangin or 7-O-methylgalangin 42.1 C16H1205 2.22keampferol 30.3 C15H1006 0.52kaempferide 40.8 C16H1206 1.99quercetin 26.1 C15H1007 0.075-hydroxy-7-(4'-hydroxy-3'-methoxyphenyl)-1-phenyl-3- heptanone 38.3 C20H2404 1.883,5-dihydroxy-1,7-bis(3,4-dihydroxyphenyl)heptane 19.4 C19H2406 1.12Alpinin B 19.5 C20H2607 2.96Galangin 7-glucoside 23.9 C21H20010 0.12 Table 3 - Compounds present within Alpinia officinarum extract using methanol as a solvent (Alpinia officinarum-001) Example 3 - Kallikrein-5 Enzyme |nhibition Assay Human 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 substrateBoc-VPR-AMC. The assay measures the formation of AMC that is a highly fluorescent group (Ä exc =380nm; Ä 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 aformulation are performed according to the validated standard protocol i.e. SP-SR-234 for |sobologram 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 |C50s of each of the individual active components (i.e. activecomponent 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 chosenconcentration of active component A. The combination of the two active components A and B at these concentrations needs to then be screened. °|f 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 °|f the results show <50% inhibition, it means that the formulation needs more concentrated activecomponents to be present in order to achieve 50 % inhibition, i.e. that the combination of the active components has an antagonist effect; or °|f the results >50% inhibition, it means that the formulation need less concentrated activecomponents 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 avery good percentage inhibition of KLK5 and the |C50s of each ofthese 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 ICSO values for the inhibition of kallikrein-5 of a number ofdifferent formulations of the present invention comprising myricetin and an extract of Alpiniaofficinarum. lt can be seen from the isobologram that the ICSO values of the formulations of thepresent invention are below the additive isobole, and as such it can be seen that the formulations ofthe 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 ICSO values for the inhibition of kallikrein-5 of a number ofdifferent formulations of the present invention comprising an extract of Alpinia officinarum and PCA.lt can be seen from the isobologram that the ICSO values of the formulations of the present inventionare below the additive isobole, and as such it can be seen that the formulations of the presentinvention 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 anextract of Alpinia officinarum and (ALO), an extract of Persicaria capitata (PCA) or myricetin werefound to provide higher than 50% inhibition of kallikrein-5. As such, it was found that the formulationsof the present invention comprising a combination of lobaric acid with an extract ofAlpinia 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. zawv f Atol -Miftssuzvi' :sura f :Hm negativaz Table 5 ": åífl Myršcetin bzxšïarâc acšaš PCR MÅ) ' Nïyršeetín íaâæarâc acid _ ma: " Table 6 The extracts and formulations of Examples 3 to 6 have been found to produce improved inhibition ofkallikrein-5, and to have a synergistic effect on the inhibition of kallikrein-5. The extracts andformulations of the present invention have therefore been shown to have improved efficacy for thetreatment 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 CLDN 1, IVL, CASP14, KRT1, KRT10, FLG, PNPLA andTJP1. 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 bycDNA 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 (10um) 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 culturedin T75. (dilution factor: 1:8) - 24h post or when the cells reach 50% confluence start starvation in FBS free DMEM medium - 24h post starvation, pre-treat cells with Lobaric acid (3uM) in a complete medium (5% FBS inDMEM) and a control well with control antagonistic peptide (ENMD-1068, 100 uM) to block PAR2. - 30min post pre-treatment add PAR2 agonistic peptide (SLIGKV-NH2, 100uM) - 24h post treatment collects the supernatant for ELISA.

Figure 8 provides IL-8 ELISA results (of the following groups: UT (untreated), PAR2 Ago (PAR2stimulation with agonistic peptide), PAR2 + Antago (control, to decrease PAR2 expression withantagonistic 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 PAR2antagonist peptide significantly down regulates it. Lobaric acid significantly inhibited PAR2 inducedIL-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 uM). - 2.5h post pre-treatment, recombinant cytokine |L/1b (50 pg/ml) was added. 11 - 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. 12

Claims (7)

1. 1. 0. 11. 1

2. 1

3. 1

4. 1

5. Use of an extract of Alpinia officinarum for the treatment of increased facial vascularity.Use of an extract as claimed in claim 1, for the treatment of rosacea. Use of an extract as claimed in either of claims 1 and 2, in which the extract is an inhibitor ofkallikrein-5 (KLK5). Use of an extract as claimed in any one of claims 1 to 3, in which the extract comprises oneor more of: kaempferol, galangin, quercetin, pinocembrin, dihydrogalangin, 3-0-methylgalangin, 7-O-methylgalangin, kaempferide, 5-hydroxy-7-(4'-hydroxy-3'-methoxyphenyl)-1-phenyl-3-heptanone, 3,5-dihydroxy-1,7-bi-(3,4-dihydroxyphenyl)heptane,alpinin B, galangin 7-glucoside, or any combination thereof. A cosmetic or pharmaceutical formulation comprising a combination of at least two differentactive components selected from: an extract of Alpinia officinarum, myricetin, lobaric acid,and an extract of Persicaria capitata. A cosmetic or pharmaceutical formulation as claimed in claim 5, in which the formulationcomprises an extract of Alpinia officinarum and myricetin. A cosmetic or pharmaceutical formulation as claimed in claim 5, in which the formulationcomprises an extract of Alpinia officinarum and lobaric acid. A cosmetic or pharmaceutical formulation as claimed in claim 5, in which the formulationcomprises an extract of Alpinia officinarum, and an extract of Persicaria capitata. A cosmetic or pharmaceutical formulation as claimed in claim 5, in which the formulationcomprises myricetin and lobaric acid. A cosmetic or pharmaceutical formulation as claimed in claim 5, in which the formulationcomprises lobaric acid and an extract of Persicaria capitata. A cosmetic or pharmaceutical formulation as claimed in claim 5, in which the formulationcomprises myricetin and an extract of Persicaria capitata. Use of a cosmetic or pharmaceutical formulation as claimed in any one of claims 5 to 11, inthe treatment of skin inflammation. Use of a cosmetic or pharmaceutical formulation as claimed in any one of claims 5 to 11, inthe treatment of increased facial vascularity. Use of a cosmetic or pharmaceutical formulation as claimed in any one of claims 5 to 11, inthe treatment of rosacea. 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. 13 1

6. 1

7. 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. A kit for the production of a cosmetic or pharmaceutical formulation as claimed in any oneof 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. 14