GB2552926A - Method - Google Patents

Abstract

Methods for preparing callus and/or cell suspension cultures of Harpagocytum procumbens (devils claw, grapple plant, cats claw or wood spider) are claimed, wherein the callus culture is established from stem internodes originated from plantlets grown in vitro. Alternatively claimed are method for secondary metabolite production in H procumbens cells or methods of extracting one or more phenylethanoid glycosides from H procumbens cells and methods of treatment and medical use claims of the extract for skin damage or aging, preferably caused by exposure to UV radiation or pollution.

Description

(71) Applicant(s):

Oriflame Cosmetics AG

Global Management AG, Bleicheplatz, Schaffhausen, CH-8200, Switzerland (72) Inventor(s):

Lene Visdal-Johnsen Michele Leonardi Christina Osterlund Virginie Lafon-Kolb Johanna Gillbro Susanne Fabre Alain Mavon Tamara Al-Bader (74) Agent and/or Address for Service:

Sirius IP

Office 2, Eight Bells House, 14 Church Street, TETBURY, Gloucestershire, GL8 8JG,

United Kingdom (51) INT CL:

A01H 4/00 (2006.01) A01H 5/04 (2018.01)

C12N 5/04 (2006.01) (56) Documents Cited:

Plant Cell Reports, Vol 11 ,1992, KS McLean et al, Callus induction and adventitious organogenesis of kenaf (Hibiscus cannabinus L.), 532-534 Plant Sciences, Vol 60,1989, C Mollers & S Sarkar, Regeneration of healthy plants from Cathranthus roseus infected with mycoplasma-like organisms through callus culture, 83-89

South African Journal of Botany, Vol 103, 2016, R Grabkowska et al, Callus cultures of Harpagophytum procumbens(Burch.) DC. ex Meisn.; production of secondary metabolites and antioxidant activity,

41-48

South African Journal of Botany, Vol 77, 2011, MW Bairu et al, Comparative phytochemical analysis of wild and-derived greenhouse-grown tubers,shoots and callus-like basal tissues of Harpagophytum procumbens, 479-484

Applied Microbiology and Biotechnology, Vol 89,

2011, M Georgiev et al, Bioactive metabolite production and stress-related hormones in Devil's claw cell suspension cultures grown in bioreactors, 1683-1691

Plant Cell Tiss Organ Cult, Vol 105, 2011, N Stancheva et al, Phytochemical and flow cytometric analyses of Devil's claw cell cultures, 79-84

South African Journal of Botany, Vol 104, 2016, D Liskova et al, Alternatives to improve long-term cultures of Harpagophytum procumbens in vitro, 55-60 (58) Field of Search:

INT CLA01H, C12N

Other: ONLINE: EPODOC, WPI, BIOSIS, MEDLINE (54) Title ofthe Invention: Method

Abstract Title: Harpagocytum procumbens plant cell cultures, cell lines and extracts, as well as methods for their preparation and uses thereof.

(57) Methods for preparing callus and/or cell suspension cultures of Harpagocytum procumbens (devil’s claw, grapple plant, cat’s claw or wood spider) are claimed, wherein the callus culture is established from stem internodes originated from plantlets grown in vitro. Alternatively claimed are method for secondary metabolite production in H procumbens cells or methods of extracting one or more phenylethanoid glycosides from H procumbens cells and methods of treatment and medical use claims of the extract for skin damage or aging, preferably caused by exposure to UV radiation or pollution.

At least one drawing originally filed was informal and the print reproduced here is taken from a later filed formal copy.

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CELL LINE CULTURES FROM PLANTS BELONGING TO THE

HARPAGOCYTUM GENUS

Field of the Invention

The present invention relates to Harpagocytum procumbens plant cell cultures, cell lines and extracts, as well as methods for their preparation and uses thereof.

Background to the Invention

Harpagocytum is a genus of plants within the Pedaliaceae, or sesame, family. Most commonly called Devils Claw or Cat’s Claw, it is a weedy, perennial tuberous plant with visually striking fruits (Mncwangi etal., 2012; Journal of Ethnopharmacology 143 p775771). Harpagocytum procumbens (H. procumbens) (Burch.) DC. Ex Mesin is native to the Kalahari Desert region of South Africa and is considered to be of high medicinal value (Gyurkovska et al., 2011 Food Chemistry 125 pi71 -178) with the secondary tuber from the plant being harvested for its medicinal properties. Up to 36 chemical compounds have been isolated from H. procumbens (Mncwangi, 2012); however, it was not reported if the chemicals were isolated from the entire plant or from the secondary tuber. H. procumbens tubers have been used as herbal medicines for thousands of years to treat more than 30 medical disorders and have been demonstrated to have strong anti-inflammatory properties (Georgiev et al., 2010; Food Chemistry 121 p967-972).

In Namibia, wild-harvesting of H. procumbens is the livelihood of many rural communities. The increased demand for this medicinal plant brings greater opportunities for primary producers but also strains the natural resource. H. procumbens was listed in 1977 as a protected species under the Nature Conservation Ordinance of 1975 in Namibia. In terms of this ordinance, permits are required to harvest and export H. procumbens. It is also protected through similar legislation in both Botswana and South Africa. Wild-harvesting by knowledgeable harvesters tended to protect the species but with increased demand, and therefore financial motivation, has caused over-harvesting by more (and less knowledgeable) harvesters with significant effects on the H. procumbens resource base. In Namibia, it is said that there is only one plant left per hectare, where previously there were 1000-2000 plants in a natural population. Consequently stricter regulations and conservation was applied and H. procumbens (known locally as gamagu) became listed as a protected species in 1977 by the former Ministry of Environment and Tourism under the Nature Conservation Ordinance of 1975.

It has proven difficult to cultivate H. procumbens to replace the need for wildharvesting and in any case the yield of bioactive compounds from both wild and cultivated plants cannot be guaranteed. Yield is wholly dependent on the plant type and growth conditions to which they have been subjected, and variation cannot be eliminated even when environmental factors are optimised as for cultivated plants. In addition, although H. procumbens plant material has reportedly been produced by cell culture techniques (Georgiev, 2010), the methods used are unsuitable for industrial production of bioactive compounds due to problems with reproducibility and incidence of infection. Finally, irrespective of the source of the plant material, extraction of bioactive compounds from H. procumbens in commercially viable quantities and in a form which is suitable for use presents further difficulties.

Thus there is a need for alternative methods of production of H. procumbens plant material and for alternative methods of extraction of bioactive compounds from said material.

Summary of the Invention

The present inventors have found that it is possible to produce H. procumbens by cell culture techniques, thus avoiding the need to harvest wild or cultivated plants. The methods of the invention provide for callus and suspension culture, and are particularly suitable for scale-up to industrial levels of production. The present inventors have also produced a stable cell line of H. procumbens which is particularly suitable for use in these methods.

The present inventors have further developed a method for secondary metabolite production in cultured cells of H. procumbens, and a method for obtaining a plant extract from said cells, which extract is also provided by the invention. The said extract may be a stem cell extract. The said extract comprises commercially viable yields of a particular class of bioactive compound, the phenylethanoid glycosides (including in particular verbascoside). The method may optionally comprise isolating one or more phenylethanoid glycoside from the extract. The extract, or a composition comprising said extract, may be used for therapeutic and cosmetic (non-therapeutic) purposes.

Phenylethanoid glycosides are a type of bioactive compounds, naturally occurring in plants and structurally characterized as derivatives of the benzoic acid, containing a phenylethyl ring, to which is added beta-glycopyranose (apiose, galactose, rhamnose or xylose) by ester or glycosidic bond. Phenylethanoid glycosides can be subdivided into five main compounds: verbascoside, isoverbascoside, forsythoside B, jionoside D and leucosceptoside B. Other phenyl ethanoid glycosides include 2-0-Acetyl Acetoside.

Pharmacological studies in vitro and in vivo have shown that these compounds possess a broad array of biological activities including antibacterial, antitumor, antiviral, antiinflammatory, neuro-protective, antioxidant, hepatoprotective, immunomodulatory, and tyrosinase inhibitory actions. Verbascoside, also known as Kusaginin, is a caffeic acid derivative that was originally elucidated in 1963 under the name acteoside. Verbascoside has been implicated in many therapeutic and cosmetic applications. It has been described as an anti-aging ingredient (W02004069218) and for its skin pigmentation activity (JP2005082522 and W02001026670). The plant extract of the invention (and compositions comprising it) is suitable for use in any of these applications.

The present invention provides:

A method for the culture of a callus of H. procumbens comprising:

a. Originating a plantlet in vitro from a sterilised seed of H. procumbens',

b. Extracting at least one stem internode from said plantlet;

c. Inducing callus formation by culture of said intemode on solid medium, said medium and the culture conditions being suitable for callus initiation;

d. Culturing the callus resulting from step c on solid medium, said medium and the culture conditions being suitable for callus growth;

and optionally:

e. Maintaining the callus resulting from step d in culture by periodically transferring healthy parts of the callus to fresh solid medium and maintaining conditions suitable for callus growth; or

f. Storing the resulting callus by freezing a sample of a healthy part of said callus.

- A callus of H. procumbens produced according to a method of the invention.

A method for the suspension culture of cells of H. procumbens comprising:

a. Inoculating H. procumbens cells into liquid medium suitable for the growth of cells in suspension;

b. Culturing the resulting cells under conditions suitable for the growth of cells in suspension;

and optionally:

c. Maintaining the resulting cells in suspension culture by periodically dividing any compact clumps, resuspending said cells in fresh medium or adding fresh medium, and maintaining conditions suitable for the continued growth of cells in suspension; or

d. Storing the resulting cells as a cell line, optionally by freezing a sample of said cells.

- A cell line of H. procumbens produced according to a method of the invention.

- A cell line of H. procumbens which is deposited with the NCIMB depositary institution with Accession number NCIMB 42467.

- A method for the production of secondary metabolites in cells of H. procumbens, the method comprising first culturing cells of H. procumbens in suspension in medium and under conditions suitable for growth of said cells, and subsequently altering the culture conditions and/or the medium such that they are suitable for the production of secondary metabolites by said cells.

- A method for the extraction of one or more phenylethanoid glycoside from cells of H. procumbens, the method comprising obtaining a sample of said cells in medium, producing a plant extract from said cells which comprises one or more phenylethanoid glycoside, and optionally isolating one or more phenylethanoid glycoside from said plant extract, preferably verbascoside.

- A plant extract produced according to a method of the invention.

- A composition comprising the plant extract of the invention and a carrier suitable for cosmetic, therapeutic, nutraceutical, foodstuff and/or animal feed use.

- A method of treating, reducing or preventing in an individual at least one sign of skin aging or at least one sign of skin damage associated with aging, the method comprising administering to said individual an effective amount of a plant extract of the invention or of a composition comprising said extract.

- A plant extract of or composition of the invention for use in method of treatment of a disease or condition.

- Use of a plant extract or composition of the invention in the manufacture of a medicament.

Brief Description of the Figures

Figure 1: illustrates the structure of verbascoside, an example of a phenylethanoid glycoside.

Figure 2: is a representative RP-HPLC Chromatogram of Harpagophytum procumbes cell line extract (Water/Ethanol 25%w/w). UV wavelenghts: 330nm. Separation was carried out on a Zorbax Eclipse C18 column (4.6X250mmX5mm; Agilent Technology) at 35°C with a gradient of Water and Acetonitrile.

Figure 3: DPPH assay screening on several extracts. Two batches of H. procumbens were tested in this assay, ALD53 and ALD64, which showed similar free scavenging results in all concentrations tested.

Figure 4: Gene expression of HM0X1 in fibroblasts stimulated with H.procumbens (ALD97) or Verbascoside (VB). TGFb is an assay positive control.

Figure 5: Normalized %inhibition of UV induced (50mJ/cm² EiVB) H2O2 release in primary fibroblasts that were pre-treated with actives lhr prior to EiV radiation.

Figure 6: Data showing % inhibition of the Collagenase enzyme after treatment with H. procumbens extract (0,2mg/ml) or the positive control (ImM)

Figure 7A: Data showing % Inhibition of UV-induced inflammation markers after pre-treatment with H. procumbens extract (0,2mg/ml). MMP 1, MMP 3, IF-6 and GM-CSF protein levels were measured by Multiplex analysis. Summary data of n=4 experiments on different donors.

Figure 7B: Data showing % Inhibition of UV-induced MMP1 production after pretreatment with H. procumbens extract (0,2mg/ml) or Verbascoside (30uM). MMP 1 protein levels were measured by ELISA. Summary data of n=5 experiments on different donors.

Figure 7C: Data showing % Inhibition of pollution-induced (DPM 2pg/ml) MMP1 production after pre-treatment with H. procumbens extract (0,2mg/ml) or Verbascoside (50uM). MMP 1 protein levels were measured by EFISA. Summary data of n=3 different donors.

Figure 8: Gene expression analysis of Collagen I, Collagen III, Collagen IV and Fibrillin in UV-paracrine stimulated fibroblasts that were pretreated with H. procumbens extract (0,2mg/ml). Data is a summary of n=3, on different donors. Significance compared to UVB treatment: * p<0.05, **p<0.01, p***<001. Untreated = no pre-treatment with active agent, followed by addition of non-stimulated KC media. UVB = no pre-treatment with active agent, followed by addition of UV stimulated KC media. UVB+ H procumbens = pretreatment for lh with H procumbens extract, followed by addition of UV stimulated KC media.

Figure 9A: Intracellular production of Collagen I protein by UV-paracrine stimulated fibroblasts pre-treated with H. procumbens extract (0,2mg/ml) or Verbascoside (16μΜ) for 48hrs.Analysis performed by immunofluorescence, data shown is a summary of n=2 on different donors. Significance compared to UVB only: p<0.01 * *, p<0.001 * * *.

Figure 9B and C: Fluorescence images showing Collagen I staining, representative of one donor only treated with or without H. procumbens, and in the presence of UVconditioned media.

Figure 10: Gene expression analysis of Collagen I, Collagen III, Collagen IV and Fibrillin in fibroblasts after 24 hr stimulation with H. procumbens extract (0,2mg/ml) or Verbascoside (16μΜ) in non-UV conditioned media. Data is a summary of n=3, on different donors (Ages represented 54, 56, 74 years old). Significance compared to untreated p<0.05*, p<0.01**.

Figure 11 A: Intracellular production of Collagen I protein by fibroblasts treated with H. procumbens extract (0,2mg/ml) or Verbascoside (16μΜ) for 48hrs in non-UV conditioned media. Analysis performed by immunofluorescence, data shown is a summary of n=2 on different donors. Significance compared to untreated p<0.05*.

Figure 11B and C: Fluorescence images showing Collagen I staining, representative of one donor only treated with or without H. procumbens, and in the presence of non-UV conditioned media.

Figure 12A: Data shows % inhibition of glycation in a cell free system. Protein BSA was mixed with the sugar D-ribose (0,5M) and the assay control Rutin (ImM) or extracts of H. procumbens (0,2mg/ml) and the production of AGE (advanced glycation end products) were quantified spectrofotometrically. Summary data of n=4. Significance compared to untreated samples (only BSA and sugar). p<0.01**, p<0.001**.

Figure 12B: Data shows % inhibition of glycation on human fibroblasts. Cells were treated with sugar 0,5mM Glyoxal to induce 100% glycation. Glycated cells were then treated with extracts of H. procumbens (0,2mg/ml) or assay control (ImM Metformin) and then stained for the presence of glycated protein carboxymetyl lysin (CMF) by immunofluorescence. Summary data of n=4, from different donors. Significance compared to Glyoxal treatement: p<0.01**, p<0.001**.

Figure 13: Gene expression analysis of UV-stimulated Keratinocytes treated with H. procumbens extract (0,2mg/ml) or Verbascoside (16μΜ). Data is summary of n=2, on different donors. Significance compared to UVB: p<0.05*, p<0.01**, p<0.001**.

Figure 14: Gene expression analysis of Keratinocytes treated with H. procumbens extract (0,2mg/ml) or Verbascoside (16μΜ). Data is summary of n=2, on different donors. Significance compared to untreated *p<0.05

Figure 15 A: Quantification of melanin following extraction with the solvable reagent, from human pigmented epidermis treated from day 4 to day 10 with H. procumbens, verbascoside and kojic acid, in the absence of UV challenge. The histograms represent values obtained from 3 tissues (n=3) for each condition. Significance to Solvent control *p<0.01.

Figure 15B: Quantification of melanin following extraction with the solvable reagent, from human pigmented epidermis treated from day 4 to day 10 with H. procumbens, verbascoside and kojic acid, in the presence of UV challenge. The histograms represent values obtained from 3 tissues (n=3) for each condition. Significance to Solvent control *p<0.01.

Detailed Description of the invention

All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

It is to be understood that different applications of the disclosed products and methods may be tailored to the specific needs in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.

In addition as used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a cell” includes “cells”, and the like. A “plant extract” as referred to herein means a preparation obtained from plant material (typically dried plant material) including cultured plant cells or callus, which preparation may be in liquid, semisolid or solid form.

Provided herein is a method for the culture of a callus of H. procumbens. A callus is an amorphous mass of unorganised thin-walled parenchyma cells. When a plant is wounded, callus formation occurs at the cut surfaces and is thought to be a protective response to seal off damaged tissues. In vitro, callus is initiated by placing a fragment of plant tissue (an explant) on solid culture media under aseptic conditions. Callus is induced and formed from proliferating cells at the cut surface of the explant tissue. The present invention also provides a callus produced and maintained by the method of the invention. Said callus is stable, meaning that the plant cells remain undifferentiated.

In the method of the invention, the plant tissue to be used is a stem internode from a plantlet originated in vitro from a sterilised seed of H. procumbens. This ensures the absence of virus or bacterial infections from the originating plant material, with seeds being substantially easier to sterilize by comparison to leaves or other plant cuttings as used in many cell culture techniques including that described in Georgiev etal, 2010.

A stem internode is the tissue between the nodes of a stem, the nodes typically holding leaves, buds or inflorescences. Sterilisation of a seed and origination of a plantlet from a sterilised seed may be achieved by any suitable method known in the art. Suitable methods are disclosed in Plant Cell Culture; Evans et al; published by Taylor & Francis 2003 ISBN 185996320X (see in particular Protocol 6.1 parts A and B).

The method of the invention comprises extracting at least one stem internode from the plantlet and plating onto solid medium. Any suitable solid medium may be used. Suitable media include but are not limited to Murashige and Skoog’s medium (MS), Gamborg B5 medium (GB5), McCowan’s Woody Plant Medium (McC), Andersons Rhododendrum Medium (AR), Shenk and Hildebrandt Medium (SH), and Titvay’s medium, each of which may be solidified with any appropriate solidifying agent. Preferred media for use in the method of the invention include Murashige and Skoog’s (MS) or Gamborg B5 (GB5) medium solidified with plant agar. The media used in the methods of the invention preferably include the macro and micro element and vitamin compositions as set out in Tables A and B, respectively.

Callus formation is induced by maintaining the medium at conditions which are suitable for callus induction, and/or by supplementing the medium with particular components which are suitable for callus induction. By “supplementing” it is meant that the specified component may be included in the medium prior to initiation of the method, or may be added to the medium at an appropriate stage in the method. Appropriate conditions for callus induction and growth/maintenance may readily be determined by the skilled person. Typical conditions are a temperature of about 22 to about 28°C under low intensity fluorescent light with a dark/light cycle of about 8hrs: about 16hrs. A preferred method of the invention uses a temperature of about 23 °C to about 25 °C, preferably at about 24°C, in the dark.

The supplementary components for the medium typically include:

- at least one cytokinin, typically selected from Zeatin (Z), zeatinriboside (ZR), isopentenyladenine (IP), 6-benzylaminopurine (BAP), 6-furfurylaminopurine (kinetin), N⁶(meto-hydroxybenzyl)adenine (topolin), thidiazuron (TDZ), forchlorfenuron (CPPU or 4PU30);

- at least one auxin which is not 2,4-dichlorophenoxyacetic acid (2,4-D) and which is typically selected from Indole-3-acetic acid (IAA), indole-3-butyric acid (IB A), 1naphthaleneacetic acid (NAA), phenylacetic acid (PAA), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), picloram, dicamba, p-chlorophenoxyacetic acid (CPA); and

- a sugar which is typically selected from sucrose, glucose, fructose and maltose.

In all methods of the invention which require inclusion of an auxin, 2,4-D is not to be used. Although this auxin is commonly used in plant cell culture, the present inventors have determined that it is an unsuitable component for use in the methods of the present invention, which are intended to produce a stable callus or suspension cell culture of plant cells of H. procumbens. 2,4-D is unsuitable because it may cause mitotic activity, as well as changes in chromosome and chromatin structure, and also changes during the cell cycle leading to abnormalities in plant cells.

The appropriate concentrations of each component may be readily determined by the skilled person. However, the cytokinin : auxin ratio in the medium at initiation of the culture is preferably between about 2:1 and about 20:1. The ratio may, for example, be about 2:1, about4:l, about5:l, about6:l, about 10:1 or about 20:1, but preferably the ratio is about 2:1 or about 5:1. In the method of the invention the cytokinin is preferably 6-benylaminopurine (BAP), the auxin is preferably alpha-naphtalene acetic acid (NAA) and the sugar is preferably sucrose. The quantity of BAP is preferably about 5mg/l. The quantity of NAA is preferably about lmg/1. The quantity of sucrose is preferably about 20g/l to about 50g/l, preferably about 30g/l.

Once induced, callus is grown/maintained by continuing to culture callus tissue on the solid medium. This is achieved by maintaining the medium at conditions which are suitable for callus growth and/or by altering the medium composition such that is suitable for callus growth. Appropriate conditions for callus growth may readily be determined by the skilled person and may be the same as the conditions used for callus induction. In a preferred method of the invention, callus growth conditions typically include culture in the dark at about 22°C to about 28°C, preferably about 23°C to about 25°C, most preferably at about 24°C. Altering the medium composition may include adding different levels of the supplementary components to the existing medium, or transferring healthy callus material to fresh medium in which the levels of the various components are already suitable for callus growth.

Appropriate changes to the medium composition to render it suitable for growth may include changing the cytokinin : auxin ratio relative to that used for initiation of the culture. The ratio is typically maintained between about 2:1 and about 20:1, but is preferably increased relative to the ratio used for callus induction. The ratio may be, for example, about 2:1, about 4:1, about 5:1, about 6:1, about 10:1 or about 20:1, provided it is the same or preferably higher than the ratio used for callus induction. The most preferred cytokinin :

auxin ratio is about 20:1. The amount of sugar is typically maintained at a similar level to that required for callus induction. In the methods of the invention, the quantity of BAP is preferably reduced to about 2mg/l. The quantity of NAA is preferably reduced to about 0.1mg/l. The quantity of sucrose is preferably about 20g/l to about 50g/l, preferably about 30g/l.

The callus may optionally be maintained in culture by periodically transferring healthy callus material to fresh solid medium which is suitable for callus growth and maintaining the conditions suitable for callus growth. The skilled person is readily able to determine the appropriate interval for periodic transfer. However, in the method of the invention said transfer typically takes place approximately every 2-3 weeks.

The callus may optionally be stored by freezing a sample of a healthy part of said callus, such as cells from said callus suspended in culture medium. Freezing of callus or other plant material may be conducted by any suitable method known in the art.

Also provided herein is a method for the suspension culture of cells of H. procumbens. By suspension culture it is meant that the cells are grown in liquid medium.

Any suitable liquid medium may be used. Suitable media include but are not limited to Murashige and Skoog’s medium (MS), Gamborg B5 medium (GB5), McCowan’s Woody Plant Medium (McC), Andersons Rhododendrum Medium (AR), Shenk and Hildebrandt Medium (SH), and Litvay’s medium. Preferred media for use in the method of the invention include Murashige and Skoog’s (MS) or Gamborg B5 (GB5). The media used in the methods of the invention preferably include the macro and micro element and vitamin compositions as set out in Tables A and B, respectively.

The present invention also provides cells produced and/or maintained by the suspension culture method of the invention. Said cells are stable, meaning that they remain undifferentiated, and may be deposited as a cell line. A “stable cell line” is defined as a cell culture line having a high and constant proliferation rate over time, preservation of the same phenotypic characteristics throughout various subcultures (cell colour, aggregate friability, size etc.) and having a reproducible secondary metabolite levels over the course of various subculture steps. An exemplary cell line, which is provided by the invention, has been deposited with the following depositary institution:

NCIMB Ltd

Ferguson Building

Craib stone Estate

Bucksburn

Aberdeen

AB21 9YA

Scotland.

Date of deposit: 18 September 2015; Accession number of deposit: NCIMB 42467.

Depositor: Oriflame Cosmetics AG, c/o Global Management AG, Bleicheplatz 3, CH-8200 Schaffhausen, Switzerland.

The method of suspension culture of the invention comprises inoculating H. procumbens cells into liquid medium suitable for the growth of cells in suspension and culturing the resulting cells under conditions suitable for the growth of cells in suspension. The inoculated cells may preferably comprise a sample of callus material produced by a method of callus culture in accordance with the invention, in which case the callus material to medium ratio is preferably between about 1:2 and about 1:10. Alternatively, the inoculated cells may be from any suitable source, such as a stable H. procumbens cell line which may previously have been cultured in accordance with the methods of the invention. An example of such a cell line is the deposited cell line NCIMB 42467.

Appropriate conditions for cell growth in suspension may readily be determined by the skilled person and may be the same as the conditions used for callus growth.

Typical conditions are a temperature of about 22°C to about 28°C under low intensity fluorescent light with a dark/light cycle of about 8hrs: about 16hrs. A preferred method of the invention uses a temperature of about 23°C to about 25 °C, preferably about 24°C, in the dark. A suspension cell culture is typically gently agitated throughout, for example with orbital shaking or on a wave reactor.

A liquid medium is suitable for growth of cells in suspension if it is supplemented with components which are suitable for said growth. By “supplemented” it is meant that the specified component may be included in the medium prior to initiation of the method, or may be added to the medium at an appropriate stage in the method. The supplementary components for the medium typically include:

- at least one cytokinin, typically selected from Zeatin (Z), zeatinriboside (ZR), isopentenyladenine (IP), 6-benzylaminopurine (BAP), 6-furfurylaminopurine (kinetin), N⁶(meto-hydroxybenzyl)adenine (topolin), thidiazuron (TDZ), forchlorfenuron (CPPU or 4PU30);

- at least one auxin which is not 2,4-dichlorophenoxyacetic acid (2,4-D) and which is typically selected from Indole-3-acetic acid (IAA), indole-3-butyric acid (IB A), 112 naphthaleneacetic acid (NAA), phenylacetic acid (PAA), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), picloram, dicamba, p-chlorophenoxyacetic acid (CPA); and

- a sugar which is typically selected from sucrose, glucose, fructose and maltose.

The appropriate concentrations of each component may be readily determined by the skilled person. However, the cytokinin : auxin ratio in the medium at initiation of the culture is preferably between about 2:1 and about 20:1. The ratio may, for example, be about 2:1, about 4:1, about 5:1, about 6:1, about 10:1 or about 20:1, but preferably the ratio is about 2:1 or about 5:1. In the method of the invention the cytokinin is preferably 6-benylaminopurine (BAP), the auxin is preferably alpha-naphtalene acetic acid (NAA) and the sugar is preferably sucrose. The quantity of BAP is preferably about 5mg/l. The quantity of NAA is preferably about lmg/1. The quantity of sucrose is preferably about 20g/l to about 50g/l, preferably about 30g/l.

The cells may optionally be maintained in suspension culture by periodically dividing any compact clumps, resuspending said cells in fresh medium or adding fresh medium, and maintaining the conditions suitable for the continued growth of cells in suspension. This process may be referred to as “sub-culturing”. The skilled person is readily able to determine the appropriate interval for these periodic activities based on the growth rate of the cells. However, in the method of the invention the interval is typically approximately 1-3 weeks.

Maintenance of the cells may also include altering the medium composition. Altering the medium composition may include adding different levels of the supplementary components to the existing medium, or transferring cells to fresh medium in which the levels of the various components are already altered.

Appropriate changes to the medium composition to render it suitable for maintenance of cells in suspension culture may include changing the cytokinin : auxin ratio relative to that used for initiation of the culture. The ratio is typically maintained between about 2:1 and about 20:1, but is preferably increased relative to the ratio used for initiation of the culture. The ratio may be, for example, about 2:1, about 4:1, about 5:1, about 6:1, about 10:1 or about 20:1, provided it is the same or preferably higher than the ratio used for initiation. The most preferred cytokinin : auxin ratio is about 20:1. The amount of sugar is typically maintained at a similar level to that required for callus induction. In the methods of the invention, the quantity of BAP is preferably reduced to about 2mg/l. The quantity of NAA is preferably reduced to about 0. lmg/1. The quantity of sucrose is preferably about 20g/l to about 50g/l, preferably about 30g/l.

The cells may optionally be stored by freezing a sample of healthy cells according to any suitable method known in the art, or by deposit as a cell line at a recognised depositary institution.

The invention also provides a method for the production of secondary metabolites in cells οΐΗ. procumbens, the method comprising first culturing cells of //. procumbens in suspension in medium and under conditions suitable for growth of said cells, and subsequently altering the culture conditions and/or the medium such that they are suitable for the production of secondary metabolites by said cells. Said first culturing in suspension may be conducted in accordance with the methods of the invention as set out above. The cells used may be from a cell line produced in accordance with the methods of the invention as set out above, such as cells of the deposited cell line NCIMB 42467. Said altering will typically occur towards the end of a growth phase of said culturing in suspension, which may be readily determined by the skilled person. In the methods of the invention, said altering typically occurs after 1-3 weeks of growth in suspension, but may occur sooner or later depending on the rate of growth of the suspended cells.

Said altering may comprise any one or more of the following:

- Increase in the concentration of precursor compounds in the medium, e.g. adding sucrose;

- Reduction in the concentration of secondary metabolites in the medium, e.g. by in situ adsorption of the metabolites;

- Addition of a biotic elicitor to the medium, e.g. oligosaccharides, chitosan, glucans, glycoproteins, autoclaved mycelium of pathogenic fungi, inactivated enzymes, plant signalling compounds

- Addition of an abiotic elicitor to the medium, e.g. heavy metal salts, pH adjuster

- Adjustment of the temperature and/or light conditions

- Immobilisation of the suspension culture by any suitable method, e.g. entrapment within an inert matrix. Suitable immobilizers include calcium alginate, polyphenyleneoxide, fibrous polypropylene, alginate + nylon, reticulate polyurethane, agarose, agar, carrageenan, gelatin, polyacrylamide, alginate + gelatine.

Where said altering comprises addition of a substance to the medium, the skilled person is readily able to determine a suitable concentration. Where said altering comprises adjustment of the conditions, the skilled person is readily able to determine a suitable adjustment. The skilled person is also aware of suitable methods for adsorption of secondary metabolites and immobilisation of a suspension culture.

In a preferred method of the invention, said altering comprises the addition of a biotic elicitor to the medium. Said biotic elicitor is preferably a plant signalling compound, most preferably methyl jasmonate. The biotic elicitor is preferably added at a concentration of about 0.1 μΜ to about 500μΜ, most preferably about ΙΟΟμΜ.

Said altering is typically followed by culture in the dark for at least 4 days at about 22°C to about 28°C, preferably about 23°C to about 25°C, most preferably at about 24°C, with orbital shaking or on a wave reactor.

The method may be a semi-continuous or continuous process, optionally comprising periodic medium exchange and/or periodic addition of fresh callus material or cells and/or periodic resuspension of a portion of the cells in fresh medium and/or periodic addition of biotic elicitor.

The invention also provides a method for the extraction of one or more phenylethanoid glycoside from cells of H. procumbens or other plant cells comprising said compounds, the method comprising obtaining a sample of said cells in medium, producing a plant extract from said cells which comprises one or more phenylethanoid glycoside, wherein said phenylethanoid glycoside is preferably verbascoside. The extract most preferably comprises verbascoside and 2-O-Acetylacteoside. The method may alternatively be described as the production of a plant extract from cells of H. procumbens, said plant extract comprising one or more phenylethanoid glycoside, which is preferably verbascoside and most preferably wherein the extract comprises verbascoside and 2-O-Acetylacteoside. The method may optionally comprise isolating one or more phenylethanoid glycoside from said plant extract.

The sample of cells used in the method may be obtained from any suitable source, such as a stable H. procumbens cell line which may previously have been cultured in accordance with the methods of the invention. An example of such a cell line is the deposited cell line NCIMB 42467. Alternatively the cells may be obtained by conducting a suspension cell culture method of the invention and taking a sample of the resulting cells.

The extraction method of the invention is an optimised cold extraction method which uses gentle extraction conditions and a sustainable solvent. By “sustainable” it is meant that the solvent is advantageous in terms of low toxicity, low price, widespread availability, and good renewability (e.g. easily produced from biomass feedstock). The choice of gentle conditions and sustainable solvent ensures high yields and an optimal secondary metabolite profile, with resulting products free from potentially harmful contaminants.

The method comprises first harvesting the cells. Harvesting may be conducted by any suitable method. An exemplary method is described in the Examples.

A preferred method of harvesting cells comprises filtration. For example, cells may be harvested by suction filtration through a Buchner funnel flask, with a suitable filter material having a pore size of about 22 - 25 pm (e.g Miracloth™). The filtered cellular material is then typically rinsed with water (preferably distilled water) prior to drying under mild temperature conditions, typically between about 0°C and about 38°C, preferably between about 20°C and about 30°C.

The resulting dried material is then suspended in a suitable hydro-alcoholic solution as solvent, e.g. Water/Ethanol, Water/Propanol, Water/Isopropanol, Water/n-Buthanol, preferably Water/Ethanol. The hydro-alcoholic solution is typically at 10 - 95%v/v, preferably about 25% v/v. The resulting suspension is ultra-sonicated (exposed to ultrasound) at about 45-65Hz, preferably about 45Hz, at room temperature for between about 1 hour and about 24 hours, preferably about 1 to 2 hours. Following ultra-sonication, the suspension is left to macerate for between about 0.5 hours and about 48 hours, preferably about 0.5 to 1 hours, before being filtered again to remove solids. Filtering may be conducted using any suitable method known in the art. Such methods include use of a quantitative paper filter with a pore size of about 11 pm. An exemplary method is described in the Examples.

After filtration, the plant extract may then optionally be concentrated by evaporating remaining solvent at low temperatures, typically using rotary evaporation between about 0°C and about 30°C, until the azeotropic point is reached. This typically requires between about 5 and about 20 minutes. The presence of a light brown residue may be taken as an indicator that the desired plant extract has been obtained.

The concentrated extract may then be freeze-dried or spray-dried by any suitable method. A preferred freeze-drying protocol involves freezing the concentrated plant extract at about -20 °C to about -150 °C, preferably about -80 °C, for about lhr to about 3 hours, preferably for about 1 hour. The frozen solution is then placed in a freeze drier in order to obtain a dried crude extract. Typical parameters for this process are as follows:

Condenser Temperature Range: -50 to -120 °C, preferably about -110 °C

Pressure Range: lOmillibar to lmicrobar, preferably about 7microBar

Time range: 1-72 hours, preferably about 48hours

The plant extract may be incorporated in a product as part of a freeze-drying or spraydrying process, by freeze-drying or spray-drying with a carrier suitable for cosmetic, therapeutic, nutraceutical, foodstuff and/or animal feed use. Suitable carriers in this context include maltodextrin, starches, cellulosics, and cyclodextrin. Alternatively, the plant extract may be incorporated in a product by suspending in a carrier suitable for cosmetic, therapeutic, nutraceutical, foodstuff and/or animal feed use.

Suitable carriers include glycerol, glycerine, butylene glycol, propylene glycol, sorbitol either alone or in combination with any of the above, denatured alcohol, PEG-40, hydrogenated castor oil, and isopropyl myristate.

In a plant extract produced by the method of the invention, typically:

- the yield of phenylethanoid glycosides is at least 1.5%, at least 2%, at least 3%, at least 4%, or preferably at least 5% w/w of the biomass of the extract; and/or

- the yield of verbascoside is at least 1%, at least 2%, or preferably at least 3% w/w of the biomass of the extract; and/or

- the yield of 2-O-AcetyleAcetoside is at least 0.5%, at least 1%, or preferably at least 1.5% w/w of the biomass of the extract.

The invention also provides a said plant extract and a composition comprising it. Said extract and said composition may be suitable for any cosmetic, therapeutic, nutraceutical, foodstuff and/or animal feed use. Said product may optionally incorporate an excipient or carrier suitable for a said use.

The invention also provides a method of treating, reducing or preventing in an individual at least one sign of skin aging or at least one sign of skin damage associated with aging, the method comprising administering to said individual an effective amount of a plant extract of the invention or of a composition comprising said extract. The individual is typically human. The skin may be on any part of the body, but is preferably the face or scalp. Administration of the extract or composition will typically be direct to the skin, i.e. topical, and the extract or composition will be formulated accordingly.

Human skin is made up of two major layers that play an important role maintaining overall health of the skin. The top outer layer, the epidermis, functions to provide a protective barrier and is the first line of defense against mechanical insults, foreign microorgansims and other external aggressors such as UV light and pollution. The dermis lies underneath the epidermis and makes up approx. 90% of the skins thickness. The dermis contains a dense network of proteins that give the skin its strength and elasticity. Fibroblasts are the principal cell type found in the dermis and are responsible for producing collagens that are one of the most abundant proteins found in the dermis.

Skin aging is biological process that results from the influence of a combination of internal (natural biological aging) and external factors that lead to structural changes of the skin, usually by degrading various protein biomarkers in both the epidermal and dermal layers of the skin. External factors include exposure to UV light (photo-induced or actinic aging), exposure to pollution or other irritants, and stress. Each of these factors results in the exposure of skin to abiotic oxidative stress, which in turn leads to skin aging and skin damage associated with aging. The method of the invention may thus alternatively be described as a method of treating, reducing or preventing damage to skin caused by abiotic oxidative stress.

Said damage is believed to arise in part due to the formation of free radicals in skin. For example, repeated exposure to UV results in the formation of peroxyl free radicals which break down to form malondialdehyde, this subsequently cross-links and polymerizes collagen. Free radicals are also reported to activate metalloproteases, such as collagenases, that are responsible for breaking down skin collagen and elastin. Free-radical damage can also cause a reduction in dermal layer thickness, which in turn would cause the skin to slacken. The combined actions of free-radicals on skin lead to one of the first and most visible signs of signs of aging - the detrimental loss of skin elasticity followed by pre-mature wrinkle formation. The presence of antioxidants in skin tissue serves as an efficient protection system against the destructive action of these reactive free radicals. However, with age the antioxidant defence capacity in skin diminishes thereby enhancing the damaging effects of the free radicals. Other mechanisms also contribute to skin aging and skin damage associated with aging, and these may include an impaired ability to synthesise and/or repair proteins in the dermis and epidermis.

As is demonstrated in the Examples, the plant extract of the invention functions as an anti-oxidant (reducing free radical effects), as an enhancer of endogenous anti-oxidant activity, and as a stimulator of dermal and epidermal protein production, and thus the method of the invention directly acts upon multiple factors associated with skin aging and skin damage associated with aging, thereby treating, reducing or preventing signs of skin aging or of skin damage associated with aging.

The method of the invention may treat, reduce or prevent at least one of the following signs of skin aging or of skin damage associated with aging: wrinkles, skin with fine lines, wizened skin, lack of skin elasticity, lack of skin tone, thinned skin, skin suffering from degradation of collagen fibres, flaccid skin, sagging skin, skin suffering from internal degradation, inflammation, redness, blotchiness, puffy eyes or dark circles, telangiectases, solar elastosis, leathery appearance, and/or or skin pigmentation disorders.

The method may also be used for at least one of: improving wound healing in skin; promoting evenness of skin tone by reducing skin redness, blotchiness or inflammation; lightening (whitening) the colour of the skin and/or reducing the appearance of skin pigmentation; promoting skin regeneration to produce more homogenous, firmer, more toned and more elastic skin; promoting cell longevity in skin; promoting skin brightness; promoting skin texture and tone uniformity; the treatment or prevention of ulcerated areas or areas of cutaneous stress or damage brought about by exposure to UV or exposure to an irritant or pollution; and treating acne or other skin blemishes.

It will be appreciated that the signs of skin aging and skin damage associated with aging described above may result from no underlying disease pathology, and so methods of treating, reducing or preventing these signs may be regarded as solely cosmetic, that is nontherapeutic. Thus the method may be described as a cosmetic method or, in other words, the method is not a method of treatment by therapy. Similarly, the invention may be described as the cosmetic use of a plant extract or composition of the invention to treat, reduce or prevent in an individual at least one sign of skin aging or at least one sign of skin damage associated with aging.

However, it will also be appreciated that certain diseases and conditions may give rise to the same or similar effects in skin as aging. As such, in certain embodiments, methods of treating, reducing or preventing these signs may be regarded as methods for treating, reducing or preventing the symptoms of such underlying diseases or conditions. Therefore, the invention also provides a plant extract or composition of the invention for use in a method for treating, reducing, or preventing a symptom of such a disease or condition. The invention also provides use of a plant extract or composition of the invention in the manufacture of a medicament for treating, reducing or preventing a symptom of such a disease or condition. The disease or condition may be any disease or condition which gives rise to one or more of the signs of skin aging, the signs of skin damage associated with aging, or of skin damage caused by abiotic oxidative stress described above. Such diseases and conditions include solar lentigo, melasma, vitiligo and seborrheic keratosis.

The plant extract and composition of the invention are also suitable for the treatment or prevention of inflammation, and associated diseases and conditions such as rheumatoid arthritis. Thus, the invention provides a method of treating inflammation, the method comprising administering to said individual an effective amount of a plant extract of the invention or of a composition comprising said extract. The invention provides a plant extract of the invention or of a composition comprising said extract for use in a method of treating inflammation. The invention provides use of a plant extract or composition of the invention in the manufacture of a medicament for treating inflammation. Said inflammation may be due to a disease or condition including rosacea, acne, atopic dermatitis and psoriasis.

Table A

Preferred macro and micro element and vitamin composition for MS medium used in the methods of the invention.

Micro Elements	mg/l	μΜ
COCI2.6H2O	0.025	0.11
CuSO4.5H2O	0.025	0.10
FeNaEDTA	36.70	100.00
H3BO3	6.20	100.27
KI	0.83	5.00
MnSO4.H2O	16.90	100.00
Na2MoOd. 2H2O	0.25	1.03
ZnSO4.7H2O	8.60	29.91
Macro Elements	mg/l	μΜ
CaCl2	332.02	2.99
KH2PO4	170.00	1.25
KNO3	1900.00	18.79
MgSO4	180.54	1.50
NH4NO3	1650.00	20.61
Vitamins	mg/l	μΜ
Glycine	2.00	26.64
myo-Inositol	100.00	554.94
Nicotinic acid	0.50	4.06
Pyridoxine HCI	0.50	2.43
Thiamine HCI	0.10	0.30

Table B

Preferred macro and micro element and vitamin composition for GB5 medium used in the methods of the invention.

Mier» Elements	mg/l	μΜ
CoCl2.6H2O	0.025	0.11
CuSO4.5H2O	0.025	0.10

FeNaEDTA	36.70	100.00
H3BO3	3.00	48.52
KI	0.75	4.52
M11SO4.H2O	10.00	59.16
Na2MoOd. 2H2O	0.25	1.03
ZnSO4.7H2O	2.00	6.96
Macro Elements	mg/1	μΜ
CaCl2	113.23	1.02
KNO3	2500.00	24.73
MgSO4	121.56	1.01
NaH2PO4	130.44	1.09
NH4NO3	134.00	1.01
Vitamins	mg/1	μΜ
myo-Inositol	100.00	554.94
Nicotinic acid	1.00	8.12
Pyridoxine HC1	1.00	4.86
Thiamine HC1	10.00	29.65

The present invention is further illustrated by the following examples which should not be construed as further limiting.

Examples

All cell culture described herein was conducted in a laminar air flow cabinet under sterile conditions.

Example 1: Initiation of static cultures (callus)

Callus was initiated from plant tissue by plating the tissue on to Petri dishes containing:

A) 4.4g/l Murashige & Skoog (MS) medium as defined in Table A, solidified with 6.5g/l plant agar, supplemented with 5 mg/1 6-benzylaminopurine (BAP) and 1 mg/1 alphanaphtalene acetic acid (NAA), 30 g/1 sucrose.

or

B) 4.4g/l Gamborg B5 medium (GB5) medium as defined in Table B, solidified with 6.5g/l plant agar, supplemented with 5 mg/1 6-benzylaminopurine (BAP) and 1 mg/1 alphanaphtalene acetic acid (NAA), 30 g/1 sucrose.

The Petri dishes were sealed and incubated at 24°C in the dark.

The plant tissue used to initiate each callus was a stem intemode cut from sterile H. procumbens plantlets originated in vitro from sterilised seed in accordance with standard protocols. See, for example, Plant Cell Culture; Evans et al; published by Taylor & Francis 2003 ISBN 185996320X (in particular Protocol 6.1 parts A and B).

Example 2: Continued growth of static cultures (callus)

The same media as in Example 1 were used for the initial growth cycles of the callus.

The callus was thereafter grown on Petri dishes containing media with reduced levels of cytokinin and auxin. Specifically:

A) 4.4g/l Murashige & Skoog (MS) medium as defined in Table A, solidified with 6.5g/l plant agar, supplemented with 2 mg/1 6-benzylaminopurine (BAP) and 0.1 mg/1 alphanaphtalene acetic acid (NAA), 30 g/1 sucrose.

or

B) 4.4g/l Gamborg B5 medium (GB5) medium as defined in Table B, solidified with 6.5g/l plant agar, supplemented with 2 mg/1 6-benzylaminopurine (BAP) and 0.1 mg/1 alphanaphtalene acetic acid (NAA), 30 g/1 sucrose.

The Petri dishes were sealed and incubated at 24°C in the dark.

The callus was typically sub-cultured every third week (for faster growing cultures a shorter cultivation time may be required). Sub-culture involved transfer of callus material to fresh medium and removal of any aberrant cells or cells that had started to differentiate. If the callus was friable a sample of material was simply spread out on a fresh Petri dish containing new medium. If the callus was compact, it was first divided into clumps of approximately 5mm diameter and transferred to a fresh Petri dish with new medium. This process was repeated until a friable callus was obtained. Friable callus is desirable when seeking to initiate suspension culture, since the callus fragments easily during agitation in liquid medium.

Example 3: Initiation of suspension cultures from callus on static medium

Liquid medium was inoculated with callus material obtained in accordance with Examples 1 and 2, at a callus (g) to medium (ml) ratio between 1:2 and 1:10. The liquid medium used consisted of:

A) 4.4g/l Murashige & Skoog (MS) medium as defined in Table A, supplemented with 5 mg/1 6-benzylaminopurine (BAP) and 1 mg/1 alpha-naphtalene acetic acid (NAA), 30 g/1 sucrose.

or

B) 4.4g/l Gamborg B5 medium (GB5) medium as defined in Table B, supplemented with 5 mg/1 6-benzylaminopurine (BAP) and 1 mg/1 alpha-naphtalene acetic acid (NAA), 30 g/1 sucrose.

Callus material was obtained by scraping from the Petri dish and then weighing, prior to transfer to a sterile 250-1000ml Erlenmeyer flask. Medium was then added to achieve the appropriate ratio. The flask was then sealed with a gas permeable seal, and incubated at 24°C in the dark with orbital shaking rotating at 70-90 rpm (lab scale) or on a wave reactor (large scale).

Example 4: Continued growth of suspension cultures

Growth of suspension cultures prepared in accordance with Example 3 was continued by periodic division of compact clumps and adding fresh MS or GB5 medium as appropriate, with a modified supplement profile of 30g/l sucrose, 2 mg/1 BAP and 0.1 mg/1 NAA. This was done typically every 2-3 weeks (when growth entered a lag phase), whilst maintaining the cells in sterile 250-1000ml Erlenmeyer flasks in the dark at 24°C with orbital shaking rotating at 70-90 rpm (lab scale) or on a wave reactor (large scale).

Example 5: Induction of secondary metabolite production

Secondary metabolite production was induced in suspension cultures prepared in accordance with Examples 3 and 4 by adding 100 μΜ Methyl Jasmonate towards the end of a growth cycle (that is in a lag phase) and at least 4 days prior to harvesting. The cells were maintained in 250-100ml flasks in the dark at 24°C with orbital shaking rotating at 70-90 rpm (lab scale) or on a wave reactor (large scale).

Example 6: Extraction of products

Cellular material resulting from Example 5 was filtered by suction filtration through a Buchner funnel flask with a Miracloth® filter (Typical pore size: 22-25 pm), rinsed in distilled water and dried at about 30°C, until the weight of was stable.

The dried cell material was then suspended in hydro-alcoholic solution (EtOH/TEO;

to 95% v/v) and the mixture ultra-sonicated at 45Hz at room temperature for between 1 hr and 24 hrs. Post sonication, the suspension was left to macerate for between 1 and 48 hrs at room temperature before performing a further filtration step to remove solids.

The resulting plant extract was concentrated by rotary evaporation of solvent at low temperature (0 to 30°C), until the minimum azeotropic point was reached, typically after between 5 and 20 minutes. The presence of the resultant plant extract was indicated by a light brown residue. The concentrated extract was then typically frozen at about -80 °C for about lhr. The frozen solution was then placed in a freeze drier in order to obtain a dried crude extract. Typical parameters for this process are as follows:

Condenser Temperature Range: -50 to -120 °C, preferably about -110 °C

Pressure Range: lOmillibarto lmicrobar, preferably about 7microBar

Time range: 1-72 hours, preferably about 48hours

The secondary metabolite profile of the dried extract was subsequently analysed and elucidated

Example 7: Secondary Metabolite Profile Analysis

The secondary metabolite profile of a plant extract obtained in accordance with Example 6 was elucidated using reverse phase HPLC-ESI-Q-TOF on an Agilent 1200 series HPLC system equipped with an Agilent 6520 Q-TOF Mass Detector. A reverse phase column (Agilent, Zorbax C-18, 5pm X 4.6mm X 250mm) was employed.

The HPLC condition were as follows: flow rate, 0.218 ml/min; oven temperature, 35°C; Solvent A, 0.1% formic acid in water; Solvent B, Acetonitrile; Gradient: O.OOmin, 5% (B), 60.00min, 40% (B), 70.00min, 5% (B), 75.00min, 5% (B); Injection volume, ΙΟμΙ (lOmg/ml). Mass spectral data were acquired in the range m/z 100-1000, with an acquisition rate of 1.35 spectra/sec, averaging 10.000 transients. The source parameters were adjusted as follows: drying gas temperature, 250°C; drying flow rate 5L/min, nebulizer pressure 45psi, and fragmentator voltage 150V. Data acquisition and processing were done by Agilent Mass Hunter.

Exemplary yields are shown in the following table:

Compound	Yield of the Extraction	Ratio Verbscoside/2-O-Acetylacteoside
(%w/w)	(g/Kg)
Verbascoside	0.94	9.43	15.70
2-O-Acelylacteoside	0.06	0.60
Total Phenylethanoids	1.00	10.03

“%w/w” = weight of the indicated compound(s) as a percentage of the plant material extracted.

“g/Kg” is the weight in grams of the indicated compound(s) per Kg of plant material extracted.

A representative RP-HPLC Chromatogram for an extract is shown in Figure 2.

Example 8: Antioxidant capacity of extract - biochemical in vitro assay

The anti-oxidant activity of a sample of H. procumbens extract taken from two separate batches (ALD53 and ALD64) prepared as described above was determined with a standard DPPH assay (see, for example, Brand-Williams W etalLebenson Wiss Technol 1995;28:2530). This assay determines level of anti-oxidant effect of a test substance by measuring the change in colour from the purple radical state of DPPH (absorbing at 516nm) to the reduced yellow product DPPHH after addition of the test substance. Vitamin C and a green tea extract were used as positive controls. Verbascoside alone was also tested for the purposes of comparison and a green tea extract of the purposes of comparison. The assay involved mixing DPPH solution (Sigma) with each of the test substances at a range of different concentrations, then incubating for 30 minutes before measurement of absorbance at 516nm. The results are shown in Figure 3. Both//, procumbens extracts (ALD53 and ALD64) achieved similar, good levels of anti-oxidant activity at all tested concentrations, reaching 5060% at 0.2nM. Verbascoside alone was found to achieve results comparable to the positive controls.

Example 9: Capacity of H. procumbens extract to alter gene expression in human cells

The aim of these experiments was to determine the gene expression of cells treated with test substance and controls by quantitative polymerase chain reaction (qPCR), which measures the amount of specific RNA in the samples. The method includes procedure for preparation of RNA and cDNA from all samples. Human primary cell cultures, including fibroblasts or keratinocytes, were cultured to approximately 80% confluence in 48 well cell culture plates. The cells were subsequently treated for 12-48hrs with test substance (H. procumbens extract or verbascoside) or with controls (vehicles or TGFb), prior to RNA extraction and cDNA synthesis followed by qPCR. Details of primers and other materials are provided in Annex 1.

The experiments specifically measured the expression of HM0X1, encoding for the protein HO-1 or heme oxygenase, belongs to the family of Vitagenes which further include heat shock proteins and thioredoxin systems. These genes are up-regulated in response to stress, and play a crucial role in the protection and maintenance of cellular homeostasis. The HO-1 protein is involved in the degradation of heme (with pro-oxidant properties) to carbon monoxide, ferrous iron and biliverdin. These last two components are precursors of antioxidant compounds named bilirubin and ferritin. HM0X1 is well-known for its protective role against oxidative stress (Vile, Basu-Modak, Waltner, & Tyrrell, 1994).

RNA extraction

Following cell treatments with H. procumbens extract or verbascoside, cells where subsequently lysed directly on the cell culture plate by adding RTL Lysis buffer (RNeasy Mini kit, Qiagen). The cell lysate was then processed for RNA extraction according to manufacturer’s protocol. The final concentration and purity of the RNA content was measured by reading the absorbance ratio 260nm/280nm with Nanodrop.

cDNA synthesis cDNA was synthesized from RNA using iScript Advanced (BioRad) according to manufacturer’s protocol. RNA was incubated at 42°C for 30 min followed by inactivation of the enzyme at 85°C for 5 min in the Thermal cycler (BioRad). 5-15ml of RNA sample was used in each 20 μΐ reaction, depending on the abundance of target transcript. The 20ml cDNA samples was then diluted 1:5 directly in the PCR plate by adding 80ml of RNase free water, and stored at -80°C prior to qPCR analysis.

qPCR cDNA samples were diluted 1:5 with water prior to qPCR. qPCR reactions were prepared with 5μΐ cDNA, 1 Ομί SsoAdvanced SYBR green + 1 μΐ specific primer + 4μ1 RNase free water. GAPDH is used as internal reference gene. PCR cycling parameters; 95 °C hot start for 3min, 40 cycles of 95°C for 10 sec and 60°C for 30 sec. Results are analyzed according to AACt method normalized to the reference gene.

Results - HM0X1

The H. procumbens extract and pure verbascoside significantly increase the expression of ΗΜΌΧ1 in human fibroblasts. The results from two separate experiments are summarised in the following table and in Figure 4A.

Gene name	H. procumbens	Verbascoside
HMOX1: Heme oxygenase (decycling) 1	2.37 (p=0.012)	2.89 (p=0.007)

Upregulation of HM0X1 by the H. procumbens extract and by verbascoside suggests that both have protective properties against free radicals by upregulating the endogenous antioxidant response.

Example 10 - Capacity of H. procumbens extract to modulate UV-induced production of reactive oxygen species in human cells

These experiments tested the ability of H. procumbens extract or verbascoside to inhibit UV induced production of hydrogen peroxide in human dermal fibroblasts.

Fibroblasts were cultured in DMEM/10%FBS media according to standard cell culture procedures. The experiment was set up in 48 well plates with a cell confluency of 80-90%. The cells were pretreated with H. procumbens extracts, verbascoside or controls (Trolox (vit E) and resveratrol) for lh before the UVB treatment (50mJ/cm²) followed by 2h incubation at 37C. H2O2 levels were then analysed using the ROS-Glo kit from Promega according to manufacturer’s instructions. The results are shown in Figure 5. Both the H. procumbens extract and verbascoside alone were found to have higher capacity to inhibit UV-induced hydrogen peroxide production than the other well-known antioxidants used as positive controls (Trolox (vit E) and resveratrol).

Example 11 - Capacity of H. procumbens extract to modulate collagenase activity

The amount of collagen in the skin is maintained by a balance between collagen synthesis and collagen breakdown that is mediated by collagenases, enzymes in the matrix metalloproteinase (MMP) family. The aim of this study was to determine the ability of H. procumbens extract or Verbascoside to inhibit a representative collagenase, NB4 from Clostridium Histolyticum in a cell free system.

Briefly, collagenase (0,3U/ml) was incubated with active (ImM) for 15 minutes at 25°C. Synthetic peptide of sequence FALGPA (mimics the structure of collagen) was added as substrate at 3mM, and the resulting quantity measured by spectrophotometry at 345nm.

The results are shown in Figure 6. The H. procumbens extract inhibited the collagenase enzyme by 20%.

Materials

Collagenase NB 4 from Clostridium histolyticum SERVA17454.02 500mg 2-furanacryloyl-Leu-Gly-Pro-Ala (FALGPA) Sigma, Cat. F5135-25mg Calcium chloride Sigma

Sodium chloride Sigma

70% ethanol Histolab

Tricine buffer Sigma

Example 12 - Capacity of extract to modulate inflammatory markers in human cells

The aim of these assays was to determine the effect of H. procumbens extract or verbascoside on the inhibition of UV- or pollution-mediated production of inflammatory markers in human dermal fibroblasts.

The assay measures levels of IL-6, GM-CSF, MMP1 and MMP3. IL-6 and GM-CSF are standard markers for inflammation. MMP1 and MMP3 are also markers for inflammation, but are particularly relevant for the present invention in that they are also directly involved in modulating damage to skin. Each is a matrix metalloprotease (MMP), that is an enzyme which is responsible for the breakdown of collagen in skin. MMP-1 (Collagenase) and MMP-3 (Stromelysin) play a major role in the degradation of dermal architecture, and are known to be stimulated by environmental stresses such as UV and pollution. The pollution used in these studies consisted of Diesel Particulate matter (DPM).

Keratinocyte-conditioned (KC) media

Keratinocytes were cultured in EpiLife/HKGS media according to standard cell culture procedures. When reaching 80-90% confluency the cells were stimulated with either UVB (50mJ/cm2) or DPM (2pg/ml), or left unstimulated, and cultured for a further 24h. The cell culture media was then collected and used for subsequent stimulation of fibroblasts. The UV or pollution treated keratinocytes are believed to trigger responses in fibroblasts via paracrine signaling, providing a model of external stress damage to skin.

Fibroblast stimulation and treatment with actives

Fibroblasts were cultured in DMEM/10%FBS media according to standard cell culture procedures. The experiment was set up in 48 well plates with a cell confluency of 80-90%. The cells were pretreated with H. procumbens extract or verbascoside for lh before the addition of KC-conditioned media from the UV or pollution treated keratinocytes.

As a positive control the cells were treated with IL-lbeta (lOOng/ml). After 24hr treatment of fibroblasts, levels of inflammatory markers (MMP1, MMP3, IL-6 and GM-SCF) in the fibroblast media was then determined by ELISA or Luminex, according to manufacturer’s instructions.

Results

H. procumbens extract inhibits the production of each of the UV-stimulated inflammatory markers MMP-1, MMP-3, IL-6 and GM-CSF in fibroblasts (see Figure 7A). H. procumbens extract completely inhibits the production of MMP-1 under UV stress (see Figure 7B), and inhibits the pollution induced production of MMP-1 by 75% (see Figure 7C).

Materials

EpiLife keratinocyte media HKGS supplement DMEM fibroblast media FBS

Cell culture flasks 75cm 48 well cell culture plates 6 well cell culture plates UV light source DPM

IL1 beta

IL 1 receptor antagonist MMP1 El A

Invitrogen Cat.no S0015 Gibco Cat.no 31966047 Gib co

Gibco

Coming

Coming

Opsytec Dr. Grobel GmbH

National Institute of Standards and technology Cat.nol650b Sigma Cat.no SRP3083-10pg Sigma Cat.no SRP3327-100pg Sigma RAB0361-1KT

Example 13 - Capacity of H. procumbens extract to modulate gene expression in human cells after UV stimulation

The aim of these assays was to determine the effect of H. procumbens extract or verbascoside on gene expression in a model of skin exposed to UV-stress. Fibroblasts were pre-treated with H. procumbens extract, before exposure to UV-stimulated (or unstimulated) cell culture media as in Example 11. RNA extraction and cDNA synthesis followed by qPCR to determine gene expression levels were conducted as in Example 9. Levels of expression for Collagen I, Collagen III, Collagen IV and Fibrillin were determined.

Collagen is a constituent protein of the extracellular matrix, and is made up of 29 different types, with Collagen I being the most predominate protein present in the dermis, at approx. 85-90%. Collagen III approximately makes up 10-15% of the extracellular matrix. Both these proteins play an important role in giving the skin its structural integrity, in photoaged/UV damaged skin both collagen I and III levels are significantly reduced (Talwar HS 1995). Collagen IV is present in the dermal-epidermal junction (DEJ) where it helps form the cohesion between the dermis and epidermis. Reduction in this protein is described to lead to deeper wrinkles and contributes to the loss of elasticity of the skin. Photoaged skin is characterized by the disorganization of tropoelastin and its associated microfibrillar component fibrillin. Moreover, depletion of fibrillin in the dermal-epidermal junction has been observed in photoaged vs non photoaged skin. Changes resulting in the breakdown of Collagen I, Collagen III, Collagen IV and Fibrillin are mainly responsible for the eventual signs of aged skin. Thus, new production of these proteins is important to help skin repair damage due to continual external stresses.

As shown in Figure 8, fibroblasts pre-treated with H. procumbens extract that are subsequently exposed to UV-media conditions, are able to significantly increase the gene expression of Collagen I, III, IV and Fibrillin relative to controls. Upregulation of these genes by the H. procumbens extract suggests that it will help to repair skin from damage which may occur in the presence of continued environmental stresses.

Example 14 - Capacity of H. procumbens extract to modulate protein production in human cells after UV stimulation

The finding that H. procumbens extract stimulates the gene expression of Collagen I after UV stress (see Figure 8) was further explored to determine whether collagen I gene expression translates into biologically relevant protein production. Human fibroblasts were pre-treated with actives and stimulated with UV as in Example 13. The presence of collagen 1 protein in the cells was then visualized with immunofluorescence (see details below) using an anti-Collagen antibody. As shown in Figure 9, both H. procumbens extract and verbascoside significantly increase Collagen I protein production in the presence of UV stress.

Immunofluorescence staining

After treatment of cells, these were then fixed for 5 minutes with 100% methanol prior to immunostaining. Unspecific staining was inhibited by blocking with 10% goat serum in PBS for lh. Collagen I was then measured by incubating with primary antibody; rat anti human pro Collagen I (1:100 dilution) in PBS and 1% goat serum, for lh followed by secondary antibody, goat anti rat Alex 488 (1:100 dilution) in PBS and 1% goat serum for lh.

DAPI, lOng/ml, was used for counterstaining to visualize the cell nuclei. Image analysis was performed by using the Cellular Imaging/Image statistic tool in the Cytation 3 software (Biotek) resulting in the ratio of labelling intensity/cell.

Materials

DAPI

Rat anti human pro Collagen I Goat anti rat alexa 488 TGF beta

DMEM glutamax 500ml ml FBS (heat inactivated) 10%

TryplEx

PBS -I- Mg2+ and Ca2+ CellTiter-Glo kit Cell culture flasks 75cm 96 well cell culture plates Cytation 3

Santa Cruz Cat. Sc-3598

Millipore Cat. MAB1912

Invitrogen Cat. Al 1006 Dilution 1:100 Sigma

Gibco Cat. 10566-16,

Gibco Cat 26140

Gibco Cat. 12604-013,

Gibco Cat. 14190-094,

Promega Cat. G7570

NUNC

Coming

Biotek

Example 15 - Capacity of H. procumbens extract to modulate gene expression in aged human cells

The aim of these assays was to determine the effect of H. procumbens extract or verbascoside on gene expression in fibroblasts from aged donors (three donors tested: 54, 56 and 74 years old). Fibroblasts were treated with H. procumbens extract (or left untreated) and cultured in normal fibroblast media (DMEM + 10% FBS). Thus this is a model of the capacity of the H. procumbens extract to affect changes in cells due to chronological aging rather than external stresses such as UV. RNA extraction and cDNA synthesis followed by qPCR to determine gene expression levels were conducted as in Example 9. Levels of expression for Collagen I, Collagen III, Collagen IV and Fibrillin were determined.

As shown in Figure 10, cells pre-treated with H. procumbens extract expressed higher levels of all four genes than verbascoside-treated cells. H. procumbens treated cells also expressed higher levels of Collagen I, Collagen III, Collagen IV than untreated controls, although the increase was only significant for Collagen I and Collagen IV. As these biomarkers are reduced in the chronological aging process, these findings on cells obtained from older individuals suggests that H. procumbens extract can play a role in helping to re31 build / strengthen the dermal skin layer, which would in turn help alleviate the visible signs of aging.

Example 16 - Capacity of H. procumbens extract to modulate protein production in aged human cells

The finding that H. procumbens extract stimulates the gene expression of Collagen I in aged cells (see Figure 10) was further explored to determine whether collagen I gene expression translates into biologically relevant protein production in such cells. Fibroblasts from two aged donors (49 and 54 years old) were pre-treated with actives and exposed to non-stimulated KC media (see Example 11) as in Example 15. Collagen I levels were determined by immunofluorescence as in Example 14. The results are shown in Figure 11. It is clearly demonstrated that only H. procumbens extract significantly increased the production of collagen I protein. This confirms the result of Example 15, namely that H. procumbens extract is able to stimulate skin fibroblasts to produce biologically relevant Collagen I protein in aged skin cells.

Example 17 - Capacity of the H. procumbens extract to prevent glycation

Glycation is the damage to proteins caused by sugar molecules. It is a process that plays a significant role in skin aging, whereby sugar molecules bind to cells that are bonding to fats and proteins. In turn the proteins become damaged and change the shape of cells, eventually leading a disruption of cellular metabolism. Collagen is a protein that is particularly vulnerable to glycation, which if damaged manifests as wrinkles and facial lines. Experiments were conducted to test the ability of H. procumbens extract to inhibit glycation both in a cell free system and in human fibroblasts. The results show that H. procumbens extract is able to inhibit glycation in a cell-free system by 56% (Figure 12A) and in cells by 35% (Figure 12B). This indicates that the H. procumbens extract may be useful to prevent or reverse skin damage associated with aging.

Cell free assay

Advanced glycation end-products (AGEs) are formed during non-enzymatic reactions involving proteins (e.g collagen) and sugars, i.e. Maillard reaction. Anti-glycation activity can be monitored by measuring the fluorescence of AGEs formed from the glycation of model proteins and sugars. This assay measured glycation of Bovine Serum Albumin (BSA) by the sugar D-ribose. BSA (10 mg/mF) and D-ribose (0.5 M) in Sodium Phosphate Buffer pH 7.4 was treated with H. procumbens extract, or Rutin (positive control) or left untreated (negative control) prior to incubation at 37 °C for 24 h. Fluorescent AGEs were then measured by kexc 370 nm; kem 440 nm with a spectrophotometer micro plate reader.

Materials

BSA

D-ribose

Sodium phosphate dibasic anhydrous

Rutin hydrate

DMSO

Sigma Cat. A2153-50G Sigma Cat. R1757-25G-A Fluka Cat. 71636 Sigma Cat. R5143-50G Fisher Cat. D/4121/PB15

Cellular assay (immunofluorescence)

Fibroblasts were cultured in DMEM/10%FBS media according to standard cell culture procedures. Cultured fibroblasts at 50-70% confluency were treated for 48hrs with 0.5mM sugar Glyoxal to induce glycation. Control cells were not treated with Glyoxal.

After exposure to Glyoxal, the media was replaced with fresh media containing H. procumbens extract or Verbascoside and cultured for a further 72hrs. As a positive control for this assay, cells were treated with Metformin (ImM) for 72h.

After treatment of cells, these were then fixed for 5 minutes with 100% methanol prior to immunostaining. Unspecific staining was inhibited by blocking with 10% goat serum in PBS for lh. Carboxymetyl lysin (CML), an adduct formed on proteins as a result of glycation, was measured by incubating with primary antibody; mouse anti human CML (3pg/ml) in PBS and 1% goat serum for lh followed by secondary antibody, goat anti mouse Alex 488 (1:100 dilution) in PBS and 1% goat serum for lh. DAPI, lOng/ml, was used for counterstaining to visualize the cell nuclei. Image analysis was performed by using the Cellular Imaging/Image statistic tool in the Cytation 3 software (Biotek) resulting in the ratio of labelling intensity/cell.

Materials

DAPI

Rabbit anti human vimentin Mouse anti CML 0,lmg/ml Goat anti mouse alexa 488 Glyoxal 40% in H2O DMEM glutamax 500ml 50 ml FBS (heat inactivated) 10%

Santa Cruz Cat. Sc-3598 AbCam Cat. ab92547 AbCam Cat. ab 125145 Invitrogen Al 1001 Dilution 1:100 Sigma Cat. 128465-100g Gibco Cat. 10566-16,

Gibco Cat 26140

TryplEx

PBS -/- Mg2+ and Ca2+ CellTiter-Glo kit Metformin

Cell culture flasks 75cm 96 well cell culture plates Cytation 3

Gibco Cat. 12604-013, GibcoCat. 14190-094, Promega Cat. G7570 Sigma Cat. PHR1084-500MG NUNC

Coming

Biotek

Example 18 - Capacity of the H. procumbens extract to modulate epidermal gene expression in human cells after UV stimulation

The aim of these assays was to determine the effect of H. procumbens extract or verbascoside on gene expression in keratinocytes subsequently exposed to UV.

Keratinocytes were cultured in EpiLife/HKGS media according to standard cell culture procedures, and treated with H. procumbens extract or verbasocoside (or left untreated). When reaching 80-90% confluency the cells were stimulated with UVB (50mJ/cm2) and cultured for a further 24h. RNA extraction and cDNA synthesis followed by qPCR to determine gene expression levels were conducted as in Example 9. Levels of expression for Laminin V, Aquaporin 3, Involucrin and Hornerin were determined.

Aged human skin exhibits morphological differences in the epidermis as well as the dermis. Some of this changes that occur in the epidermis include: epidermal thickness reduction, flattening of the dermal-epidermal junction and decreased keratinocyte proliferation. Laminins are a family of glycoproteins that play an integral role in the maintenance of the architecture of the skin. They are found in abundance in the basal lamina of the skin, and are very important for maintaining the dermal-epidermal junction. The biological acitivity of laminins help influence various cell functions such as cell differentiation, migration, adhesion and skin regeneration - mechanisms necessary for the maintenance and healing of the skin. In this study we sought to measure the effect of H. procumbens or verbascoside on Laminin V subunit gamma (gene code LAMC2). In the presence of UV, H. procumbens significantly stimulated the production of Laminin which suggests that this my may exert a potentially strengthening effect on the dermo-epidermal junction, enhancing firmness and smoothness of the skin.

Aquaporins are protiens that facilitate the transport of water across cell membranes. Aquaporin 3 (AQP3) water channels are strongly affected by chronological aging and chronic UV exposure. Reduction in AQP3 levels have been observed in older people which could account for the skin dryness that occurs as we age. Therefore AQP is a key protein target to improve UV-induced dryness. In our studies, we observed that H. procumbens stimulates the production of AQP3 in UV-stimulated epidermal keratinocytes.

Hornerin is a protein that makes up the epidermal cornified envelopes. It possesses a similar structural organization with that of pro-fillagrin - an essential component of the stratum corneum. Reduced expression of Hornerin in the epidermis of patients with atopic dermatitis is suggested to contribute to the epidermal barrier defects seen in these individuals (Wu Z et al. 2009). A recent study also demonstrated that in patients with chronic hand eczema, hornerin was significantly downregulated, coorborating the assumption that hornerin plays an important role in skin barrier function (Molin S et al. 2015). In our study, we observed that H. procumbens extract treated keratinocytes significantly stimulate hornerin gene expression. This finding suggests that H. procumbens extract may help maintain skin barrier function under UV stress conditions.

Involucrin is a marker of keratinocyte differentiation and maturation. There are conflicting reports on the expression of involucrin after UV exposure, with some authors reporting that involucrin expression increases after UV radiation (Bertrand-Vallery 2010) and others suggesting that involucrin expression decreases (Mammone et al. 2000). The differences in results appear to depend on the UVB source and biological model.

Nonetheless, in our studies we observed that//, procumbens extract stimulates the production of Involucrin in UV-exposed keratinocytes compared to cells stimulated with UV alone. This finding together with the effects seen on the other epidermal biomarkers described above further supports that H. procumbens extract possesses a potent ability to regulate important epidermal markers that may help improve the condition, hydration and function of the skin barrier under UV stress.

As shown in Figure 13, treatment with H. procumbens extract resulted in significantly higher gene expression for each of Laminin V, Aquaporin 3, Involucrin and Hornerin relative to untreated cells. This indicates that the extract may be useful to prevent or reverse skin damage associated with aging, such as damage caused by external stresses.

Example 19 - Capacity of the H. procumbens extract to modulate epidermal gene expression in human cells without UV stimulation

The aim of these assays was to determine the effect of H. procumbens extract or verbascoside on gene expression in keratinocytes which are not exposed to UV or another external stress. Thus this is a model of the capacity of the extract to affect changes in cells due to chronological aging rather than external stresses such as UV. Keratinocytes were cultured in EpiLife/HKGS media according to standard cell culture procedures, and treated with H. procumbens extract or verbasocoside (or left untreated). RNA extraction and cDNA synthesis followed by qPCR to determine gene expression levels were conducted as in Example 9. Levels of expression for Aquaporin 3, were determined. As shown in Figure 14, both H. procumbens extract and verbascoside treatment resulted in a significant increase in AQP3 expression relative to untreated cells.

This indicates that the extract may be useful to prevent or reverse skin damage associated with aging.

Example 20 - Skin lightening properties of the H. procumbens extract

Reconstituted epidermis containing NHEMs-dark pigmented melanocytes were used in studies to determine whether H. procumbens extract or verbascoside are able to inhibit melanogenesis. Gene analysis showed that H. procumbens extract and Verbascoside decrease the expression of WNT16 (Wingless-type MMTV integration site family, member 16) in the absence of UV irradiation (see results in following table).

TGene name	H. procumbens	Verbascoside
WNT16	-6.49 (p=0.002)	-3.72 (p=0.019)

MITF, a key transcription factor involved in the differentiation, growth and survival of the melanocytes, regulates more than 25 pigmentation genes, including keys enzymes for melanin synthesis (i.e. tyrosinase, tyrosine-related protein-1, dopachrome tautomerase), structural components of the melanosomes and proteins involved in their maturation and transport along the melanocyte dendrites.

MITF is under the control of major melanogenic pathways, including the amelanocyte stimulating hormone/melanocortin-1 receptor, the stem cell factor/c-Kit, the endothelin/protein kinase C and Wnt/p-catenin pathways. It is itself regulated at the transcription level and by specific phosphorylation (Levy, Khaled, & Fisher, 2006; Shibahara et al., 2001)

In the Wnt/p-catenin pathway, a key control is the level of intracellular β-catenin. In the absence of a Wnt signal, β-catenin is sequentially phosphorylated by glycogen synthase kinase-3β (ϋ8Κ-3β), and the phosphorylated β-catenin is recognized by an ubiquitin ligase complex, resulting in degradation via an ubiquitin-dependent mechanism. In contrast, activation of the Wnt pathway negatively regulates GSK-33, leading to the accumulation of cytoplasmic β-catenin, which translocates to the nucleus and forms a complex with both Tcell factor (TCF) and lymphocyte enhancer factor-1 (LEF) to up-regulate expression of MITF. Hence, activation of the Wnt/ β-catenin pathway stimulates melanogenesis via the upregulation of MITF activity (Bellei, Pitisci, Catricala, Larue, & Picardo, 2011; Cadigan, 2008; Yaar & Park, 2012).

The marked decrease of the expression of WNT ligand in cells treated with H. procumbens extract might suggest a possible whitening effect through a slow-down of the WntT-catenin melanogeneis pathway.

Experimental method was as follows:

Reconstituted human epidermis containing normal human epidermal melanocytes darkly pigmented (NHEMs-DP; phototype V-VI) were cultured at air-liquid interface for 14 days (fully differentiated) in a serum free culture medium in a humid atmosphere at 37°C with CO2 5%. The epidermis were transferred to a 12-wells plate before the treatment with H. procumbens extract, in triplicate cultures (n=3).

For the experiment with UVs irradiation, H. procumbens extract was applied during 18 hours in the culture medium of differentiated epidermis. Then tissues were placed in PBS and irradiated with 4J/cm² of UVA and 200mJ/cm² UVB (±15 minutes), with an illuminator Biosun (Vilbert Lourmat, FR). After the irradiation, the epidermis were re-incubated with the extract during 6 hours.

Materials		Suppliers	References
Keratinocytes (NHEKs)	Cells	Lonza	00192906
Melanocytes (NHEMs)	Cells	Gibco/ Life Technologies	C-202-5C
DMEM		Culture medium	31885-049
Foetal bovine serum (FBS)		Supplement	10270-106
Penicillin/Streptomycin		Antibiotics	157140-122
Epilife		Culture medium	M-Epi-500-A
HKGS		Supplement	S-001-5
M254		Culture medium	M-254-500
HMGS		Supplement	S-002-5
Gentamycin		Antibiotic	15710-049
CaC12	Supplement	Merck	1.02378.0500
KGF/FGF-7	Supplement	R&D systems	251-KG

Ascorbic acid (Vitamin C) Supplement VWR BDH Prolabo 83568-180

At the end of treatments, total RNA were extracted and sample integrity was analyzed by spectrophotometry and capillary electrophoresis. cDNA were then synthesized from mRNA by reverse transcription. Gene expression changes were addressed by qPCR.

Total RNA extraction and cDNA synthesis

Total RNAs were extracted using the Qiagen RNeasy kit. After treatments, cells were rinsed with PBS and lysed in buffer while epidermis were immersed directly in the lysis buffer (triplicate culture were performed for each condition). Extraction of RNA was performed from cells and tissues according to the supplier’s recommendations. The RNAs collected were stored at -80°C. Reverse transcription was performed with the high capacity RNA-tocDNA kit (AppliedBiosystems) from 2pg of total RNA according to the manufacturer's instructions. The cDNA were then stored at -20°C.

qPCR

The different probes were manufactured by Applied Biosystems (MITFHsOl 117294 ml, WNT16-Hs00365138_ml). Real-time qPCR was used to quantify the expression of specific targets using the 7900HT Fast Real-Time System (Applied Biosystems). In brief, 4pl ADNc (4 ng) were mixed with 1 Opl of TaqMan Fast Universal Master Mix (Applied Biosystems), 1 μΐ of TaqMan Gene Expression Assay and 5pl of water RNAse free. In order to normalize the results, gene expression was normalized to housekeeping genes β2-ηύ(η^1ο1ηι1ΐηβ, B2M. The thermal cycles were programmed with one incubation step at 50°C during 2 min followed by a first denaturation step at 95°C during 10 min. The amplification protocol was followed with 40 cycles (15sec at 95°C and lmin at 60 °C).

Threshold cycles (Ct) were obtained for each gene. Results files were exported from the Real-Time qPCR device using the SDSRQ Manager Software (vl.2A, Applied Biosystems) and data were analyzed by using the DataAssist Software (v3.0, Applied Biosyslems) designed to perform relative quantification of gene expression using the comparative Ct (Ct) method (Pfaffl, 2001 & Livak and Schmittgen, 2001), through a combination of statistical analysis. For the epidermis, data were compared to the reference condition (EtOH 1%) with β2-ηΰ(η^ΙοΙ)ηΙΐη (B2M) as the housekeeping gene. Maximum allowable Ct value used as a detection threshold or Ct cut-off value was fixed at 36 cycles.

Example 21 - Further skin lightening properties of the H. procumbens extract

A study was carried out to test the whitening properties of H. procumbens extract and verbascoside using reconstituted human epidermis with phototype ΠΙ-IV melanocytes, in the presence and absence of UV challenge. Kojic acid as a positive control decreased the melanin content of epidermis in both the presence and absence of UV, validating the test and analysis method. A significant decrease in melanin deposition was observed for the epidermis treated with H. procumbens extract or verbascoside in comparison to the EtOH negative control, in both the absence (Figure 15A) and presence (Figure 15B) of UV challenge. This finding supports the conclusion of Example 19, and indicates that H. procumbens extract and verbascoside have properties that can lead directly to skin lightening effects.

Experimental method was as follows:

Human epidermis were reconstituted at the air-liquid interface, from day 0 to day 4 in the growth culture medium containing BPE 100% (Bovine Pituitary Extract). The culture process was then continued from day 4 to day 7 with a growth culture medium containing BPE 50% and from day 7 to day 14 without BPE.

The H. procumbens extract was applied in the culture medium for a total of 10 days, from day 4 to day 14 (n=3), with medium refreshes at day 7 to day 11 and in presence of aMSH (ΙμΜ) and UVA/B challenge (UVA lJ/cm2, UVB 50mJ/cm2) as illustrated after.

The absence of cytotoxicity was checked by a MTS assay (3-(4,5-dimethythiazol-2-yl)-5-(3carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium).

The effect of kojic acid (250μΜ) was analyzed in parallel as whitening reference compound to validate the analysis. Ethanol, at the corresponding concentration of 0.1%, used as solvent for the preparation of H. procumbens extract in the culture medium, was tested in parallel. At day 14, tissues were removed from their inserts and immerged in a solvable extraction solution (Perkin Elmer) and heated at 80°C during lh. The optical density of the supernatants was been measured at 490 nm and the melanin content was determined by comparison with a standard curve of synthetic melanin (Sigma Aldrich M8631).

Annex 1

Details of primers and other materials for qPCR (Examples 9, 13, 15, 18, 19)

Item	Source	Item	Source
RNeasy Mini kit	Qiagen Cat. 74106	Human IVL primer	BioRad Cat. qHsaCED0046054
RNase-free DNase Set	Qiagen Cat. 79254	Human BIRC5 primer	BioRad Cat. qHsaCED0001615
Ethanol 96 %	Histolab	Human CLDN4 primer	BioRad Cat. qHsaCED0038265
14,3 Μ β- Mercaptoethanol	Sigma Cat. M3148	Human SIRT1 primer	BioRad Cat. qHsaCID0006484
iScript Advanced	Biorad Cat. 170-8843	Human SIRT4 primer	BioRad Cat. qHsaCIDOO 14472
TGFb	Sigma Cat. T7039	Human LAMC2 primer	Qiagen Cat. PPH09460A
SsoAdvanced SYBR green	Biorad Cat. 172-5274	Human HMOX1 primer	Qiagen cat no PPH00161F
Human GAPDH primer	Qiagen Cat. PPH00150F	Cell culture plates, 48 well	Coming
Human C0L1A1 primer	Qiagen Cat. PPH01299F	DMEM Glutamax medium	Gibco Cat.no 31966-021
Human COL3A1 primer	BioRad Cat. qHsaCIDOO 14986	Foetal bovine serum	Gibco Cat.no 26140-079
Human Col4A3 primer	BioRad Cat. qHsaCID0013308	Nanodrop
Human FBN1 primer	BioRad Cat. qHsaCID0021556	Biorad Thermal cycler CFX96™
Human CASP14 primer	BioRad Cat. qHsaCIDOO 17034	Verbascoside 10-50μΜ	Extrasynthese
Human AQP3 primer	BioRad Cat. qHsaCED0046291	H. procumbens extract 0.2mg/ml
Human HRNR primer	BioRad Cat. qHsaCIDOO 11602

Claims (33)

1. A method for the culture of a callus of H. procumbens comprising:

a. Originating a plantlet in vitro from a sterilised seed of H. procumbens',

b. Extracting at least one stem internode from said plantlet;

c. Inducing callus formation by culture of said intemode on solid medium, said medium and the culture conditions being suitable for callus initiation;

d. Culturing the callus resulting from step c on solid medium, said medium and the culture conditions being suitable for callus growth;

and optionally:

e. Maintaining the callus resulting from step d in culture by periodically transferring healthy parts of the callus to fresh solid medium and maintaining conditions suitable for callus growth; or

f. Storing the resulting callus by freezing a sample of a healthy part of said callus.

2. The method of claim 1, wherein the solid medium of steps c, d and e is Murashige and Skoog (MS) or Gamborg B5 (GB6) medium solidified with plant agar and supplemented with at least one cytokinin, at least one auxin which is not 2,4dichlorophenoxyacetic acid (2,4-D), and sugar, wherein the cytokinin is preferably 6-benylaminopurine (BAP), the auxin is preferably alpha-naphtalene acetic acid (NAA) and the sugar is preferably sucrose.

3. The method according to claim 2, wherein the ratio of cytokinin : auxin in the medium of steps c and/or d and/or e is each independently between 2:1 and 20:1.

4. The method according to claim 2 or 3, wherein the medium in step c is supplemented with 5mg/ml BAP, lmg/ml NAA and 3Og/1 sucrose and/or the medium in steps d and e is supplemented with 2mg/ml BAP, 0. lmg/ml NAA and 3 Og/1 sucrose.

5. The method of any one of the preceding claims, wherein the conditions in steps c, d and e comprise culture in the dark at approximately 22 to 28°C and/or wherein the periodic transfer in step e takes place approximately every 2-3 weeks.

6. The method of any one of the preceding claims, wherein the medium has a macro and micro element and vitamin composition as set out in Table A or B.

7. A callus of H. procumbens produced according to the method of any one of the preceding claims.

8. A method for the suspension culture of cells of H. procumbens comprising:

a. Inoculating H. procumbens cells into liquid medium suitable for the growth of cells in suspension;

b. Culturing the resulting cells under conditions suitable for the growth of cells in suspension;

and optionally:

c. Maintaining the resulting cells in suspension culture by periodically dividing any compact clumps, resuspending said cells in fresh medium or adding fresh medium, and maintaining conditions suitable for the continued growth of cells in suspension; or

d. Storing the resulting cells as a cell line, optionally by freezing a sample of said cells.

9. A method according to claim 8, wherein the cells used in step a are provided as a sample of material from the callus of claim 7, preferably wherein the callus material to medium ratio is between 1:2 and 1:10 w/w.

10. A method according to claim 8 or 9, wherein the medium of step a and/or c is MS or GB5 medium supplemented with at least one cytokinin, at least one auxin which is not 2,4-dichlorophenoxyacetic acid (2,4-D), and sugar, wherein the cytokinin is preferably 6-benylaminopurine (BAP), the auxin is preferably alpha-naphtalene acetic acid (NAA) and the sugar is preferably sucrose.

11. The method according to claim 10, wherein the ratio of cytokinin : auxin in the medium of step a and/or c is each independently between 2:1 and 20:1.

12. The method according to claim 10 or 11, wherein the medium in step a is supplemented with 5mg/ml BAP, lmg/ml NAA and 3Og/1 sucrose and/or the medium in step c is supplemented with 2mg/ml BAP, 0. lmg/ml NAA and 3Og/1 sucrose.

13. The method of any one of claims 7 to 12, wherein the conditions in steps b and c comprise culture in the dark at approximately 22 to 28°C, with orbital shaking or on a wave reactor, and/or wherein the periodic resuspension or addition of medium in step c takes place approximately every 2-3 weeks.

14. The method of any one of claims 7 to 13, wherein the medium has a macro and micro element and vitamin composition as set out in Table A or B.

15. A cell line of H. procumbens produced according to the method of any one of claims 7 to 14.

16. A cell line of H. procumbens which is deposited with the NCIMB depositary institution with Accession number NCIMB 42467.

17. A method for the production of secondary metabolites in cells of H. procumbens, the method comprising first culturing cells of H. procumbens in suspension in medium and under conditions suitable for growth of said cells, and subsequently altering the culture conditions and/or the medium such that they are suitable for the production of secondary metabolites by said cells.

18. A method according to claim 17 wherein said first culturing in suspension is conducted in accordance with the method any one of claims 7 to 14 and said altering takes place after step b or step c, and/or wherein said cells for said first culturing are a sample from the cell line of claim 15 or 16.

19. A method according to claim 17 or 18 wherein said altering comprises the addition of a biotic elicitor to the medium

20. A method according to claim 19, wherein following said addition of elicitor the cells are culture in the dark for 2 to 6 days at approximately 24°C, with orbital shaking or on a wave reactor.

21. The method of claim 19 or 20 wherein the elicitor is methyl jasmonate, preferably at a concentration of 0.1 μΜ to 500μΜ, most preferably ΙΟΟμΜ.

22. A method according to any one of claims 17 to 21, which is a semi-continuous or continuous process, optionally comprising periodic medium exchange and/or periodic addition of fresh callus material or cells and/or periodic resuspension of a portion of the cells in fresh medium and/or periodic addition of elicitor.

23. A method for the extraction of one or more phenylethanoid glycoside from cells of H. procumbens, the method comprising obtaining a sample of said cells in medium, producing a plant extract from said cells which comprises one or more phenylethanoid glycoside, and optionally isolating one or more phenylethanoid glycoside from said plant extract, preferably verbascoside.

24. A method according to claim 23, wherein said sample of cells is obtained from the medium resulting from the method of any one of claims 17 to 22.

25. A method according to claim 23 or 24, wherein said plant extract is produced by:

a. Filtering cellular material from the said sample and rinsing with water prior to drying at between about 0°C and about 30°C;

b. Suspending the resulting dried material in hydro-alcoholic solution;

c. Exposing the resulting suspension to ultrasound for between about 1 hour and about 24 hours at room temperature prior to leaving said suspension to macerate for between about 1 hr and about 48 hours at room temperature; and

d. Filtering the resulting suspension to remove solids; and optionally

e. Concentrating the resulting extract by evaporating the remaining solvent at between about 0°C and about 30°C.

26. A method according to claim 25, further comprising freeze-drying or spray-drying the plant extract resulting from step d or e with a carrier suitable for cosmetic, therapeutic, nutraceutical, foodstuff and/or animal feed use; or suspending the plant extract resulting from step d or e in a carrier suitable for cosmetic, therapeutic, nutraceutical, foodstuff and/or animal feed use.

27. A method according to any one of claims 23 to 26 wherein in the plant extract resulting from step d or e:

- the yield of phenylethanoid glycosides is at least 1.5%, at least 2%, at least 3%, at least 4%, or preferably at least 5% w/w of the extract; and/or

- the yield of verbascoside is at least 1%, at least 2%, or preferably at least 3% w/w of the extract; and/or

- the yield of 2-O-AcetyleAcetoside is at least 0.5%, at least 1%, or preferably at least 1.5% w/w of the extract.

28. A plant extract produced according to a method of any one of claims 23 to 27, optionally wherein the yield of phenylethanoid glycosides in the extract is at least 5% w/w, the yield of verbascoside is at least 3% w/w, and the yield of 2-0AcetyleAcetoside is at least 1.5% w/w of the extract.

29. A composition comprising the plant extract of claim 28 and optionally a carrier suitable for cosmetic, pharmaceutical, nutraceutical, foodstuff and/or animal feed use.

30. A method of treating, reducing or preventing in an individual at least one sign of skin aging or at least one sign of skin damage associated with aging, the method comprising administering to said individual an effective amount of a plant extract of claim 28 or composition of claim 29.

31. A method according to claim 30, wherein said skin damage is due to exposure of the skin to abiotic oxidative stress, which optionally is caused by exposure to UV radiation or pollution.

32. A plant extract of claim 28 or composition of claim 29 for use in method of treatment of a disease or condition.

33.

Use of a plant extract of claim 28 or composition of claim 29 in the manufacture of a medicament.

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Application No: GB1611285.6 Examiner: Dr Patrick Purcell