Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
  • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
  • A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
  • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
  • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Preparation or treatment thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
  • A61K36/18—Magnoliophyta (angiosperms)
  • A61K36/185—Magnoliopsida (dicotyledons)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
  • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
  • A61K8/9783—Angiosperms [Magnoliophyta]
  • A61K8/9789—Magnoliopsida [dicotyledons]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q19/00—Preparations for care of the skin
  • A61Q19/08—Anti-ageing preparations
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/04—Plant cells or tissues

Definitions

• the present invention relates to Harpagophytum procumbens plant cell cultures, cell lines and extracts, as well as methods for their preparation and uses thereof.
• Harpagophytum 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 et al., 2012; Journal of Ethnopharmacology 143 p775- 771). Harpagophytum 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 pl71-178) 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).
• H. procumbens In Sun, 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 Sun. 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.
• procumbens plant material has reportedly been produced by cell culture techniques
• 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 phenylethanoid glycosides include 2-O-AcetylAcetoside.
• 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 (WO2004069218) and for its skin pigmentation activity (JP2005082522 and WO2001026670). 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:
• step c Culturing the callus resulting from step c on solid medium, said medium and the culture conditions being suitable for callus growth;
• step d 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;
• 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:
• step a A cell line of H. procumbens produced as above, wherein the cells used in step a are provided as a sample of material from a callus produced by
• step c Culturing the callus resulting from step c on solid medium, said medium and the culture conditions being suitable for callus growth;
• BAP 6-benylaminopurine
• NAA alpha-naphtalene acetic acid
• a method for the production of secondary metabolites in cells of H. procumbens 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 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 extraction of one or more phenylethanoid glycoside from cells of H. procumbens 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, 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-O-AcetyleAcetoside is at least 1.5% w/w of the extract.
• elicitation of secondary metabolite production may be achieved by adding a concentration of 100 ⁇ Methyl Jasmonate towards the end of the growth cycle and at least 4 days prior to harvesting.
• step b or step c 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 the invention as described herein.
• 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;
• the yield of verbascoside is at least 1%, at least 2%, or preferably at least 3% w/w of the extract;
• 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.
• 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 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 or composition of the invention for use in method of treatment of a disease or condition.
• a plant extract or composition of the invention for treating, reducing or preventing in an individual at least one sign of skin ageing or skin damage associated with one or more of: mal pigmentation, collagen expression, inflammation, glycation, disrupted barrier function or any combination thereof.
• use of the plant extract or composition of the invention is a cosmetic use.
• a method of treating, reducing or preventing in an individual at least one sign of skin ageing or at least one sign of skin damage associated with skin ageing comprising administering to said individual an effective amount of a plant extract or composition of the invention.
• a method of treating, reducing or preventing in an individual at least one sign of skin ageing or at least one sign of skin damage associated with skin ageing comprising administering to said individual an effective amount of a plant extract or composition of the invention, 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.
• a cosmetic method of treating, reducing or preventing in an individual at least one sign of skin ageing or at least one sign of skin damage associated with skin ageing comprising administering to said individual an effective amount of a plant extract or composition of the invention.
• a cosmetic method of treating, reducing or preventing in an individual at least one sign of skin ageing or at least one sign of skin damage associated with skin ageing comprising administering to said individual an effective amount of a plant extract or composition of the invention, 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.
• 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 wavelengths: 330nm. Separation was carried out on a Zorbax Eclipse CI 8 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.
• FIG. 4 Gene expression of HMOX1 in fibroblasts stimulated with H.procumbens (ALD97) or Verbascoside (VB). TGFb is an assay positive control.
• Figure 5 Normalized % inhibition of UV induced (50mJ/cm 2 UVB) H2O2 release in primary fibroblasts that were pre-treated with actives lhr prior to UV radiation.
• Figure 6 Data showing % inhibition of the Collagenase enzyme after treatment with H. procumbens extract (0,2mg/ml) or the positive control (ImM)
• UVB no pre-treatment with active agent, followed by addition of UV stimulated KC media.
• UVB+ H procumbens pre- treatment for lh with H procumbens extract, followed by addition of UV stimulated KC media.
• 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 UV- conditioned media.
• 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 (CML) by
• 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.
• 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.
• 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.
• a callus is an amorphous mass of unorganised thin-walled parenchyma cells.
• callus formation occurs at the cut surfaces and is thought to be a protective response to seal off damaged tissues.
• 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.
• 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 et al, 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 Litvay'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.
• supply 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:
• cytokinin typically selected fromZeatin (Z), zeatinriboside (ZR), isopentenyladenine (IP), 6-benzylaminopurine (BAP), 6-furfurylaminopurine (kinetin), N 6 - (meto-hydroxybenzyl)adenine (topolin), thidiazuron (TDZ), forchlorfenuron (CPPU or 4PU- 30);
• 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 (IBA), 1- 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.
• 2,4-D is not to be used.
• 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.
• 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.
• the cytokinin is preferably 6-benylaminopurine (BAP)
• the auxin is preferably alpha-naphtalene acetic acid (NAA)
• the sugar is preferably sucrose.
• BAP 6-benylaminopurine
• NAA alpha-naphtalene acetic acid
• sucrose is preferably about 20g/l to about 50g/l, preferably about 30g/l.
• 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.
• 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.
• 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 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:
• the method of suspension culture of the invention comprises inoculating H.
• 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.
• 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.
• supplied 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:
• cytokinin typically selected fromZeatin (Z), zeatinriboside (ZR), isopentenyladenine (IP), 6-benzylaminopurine (BAP), 6-furfurylaminopurine (kinetin), N 6 - (meto-hydroxybenzyl)adenine (topolin), thidiazuron (TDZ), forchlorfenuron (CPPU or 4PU- 30);
• 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 (IBA), 1- naphthaleneacetic acid (NAA), phenylacetic acid (PAA), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), picloram, dicamba, p-chlorophenoxyacetic acid (CPA); and
• the cytokinin is preferably 6-benylaminopurine (BAP)
• the auxin is preferably alpha-naphtalene acetic acid (NAA)
• the sugar is preferably sucrose.
• BAP 6-benylaminopurine
• NAA alpha-naphtalene acetic acid
• 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.
• 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 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
• 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:
• a biotic elicitor e.g. oligosaccharides, chitosan, glucans, glycoproteins, autoclaved mycelium of pathogenic fungi, inactivated enzymes, plant signalling compounds
• Suitable immobilizers include calcium alginate,
• polyphenyleneoxide fibrous polypropylene, alginate + nylon, reticulate polyurethane, agarose, agar, carrageenan, gelatin, polyacrylamide, alginate + gelatine.
• altering comprises addition of a substance to the medium
• altering comprises adjustment of the conditions
• suitable adjustment the skilled person is also aware of suitable methods for adsorption of secondary metabolites and immobilisation of a suspension culture.
• 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 ⁇ to about 500 ⁇ , most preferably about ⁇ .
• 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.
• 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 NCEVIB 42467.
• 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.
• 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.
• 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 ⁇ (e.g MiraclothTM).
• 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.
• 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
• 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 ⁇ . An exemplary method is described in the Examples.
• 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
• Time range 1-72 hours, preferably about 48hours
• the plant extract may be incorporated in a product as part of a freeze-drying or spray- drying process, by freeze-drying or spray-drying with a carrier suitable for cosmetic, therapeutic, nutraceutical, foodstuff and/or animal feed use.
• a carrier suitable for cosmetic, therapeutic, nutraceutical, foodstuff and/or animal feed use include maltodextrin, starches, cellulosics, and cyclodextrin.
• 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.
• 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.
• free radicals 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.
• antioxidants in skin tissue serves as an efficient protection system against the destructive action of these reactive free radicals.
• 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.
• 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.
• 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 non- therapeutic.
• the method may be described as a cosmetic method or, in other words, the method is not a method of treatment by therapy.
• 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.
• 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.
• 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.
• 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.
• Preferred macro and micro element and vitamin composition for MS medium used in the methods of the invention.
• MS Murashige & Skoog
• BAP 6-benzylaminopurine
• NAA alpha- naphtalene acetic acid
• G5 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 alpha- naphtalene acetic acid (NAA), 30 g/1 sucrose.
• BAP 6-benzylaminopurine
• NAA alpha- naphtalene acetic acid
• the Petri dishes were sealed and incubated at 24°C in the dark.
• the plant tissue used to initiate each callus was a stem internode 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 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.
• 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 alpha- naphtalene acetic acid (NAA), 30 g/1 sucrose.
• MS Murashige & Skoog
• BAP 6-benzylaminopurine
• NAA alpha- naphtalene acetic acid
• G5 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 alpha- naphtalene acetic acid (NAA), 30 g/1 sucrose.
• BAP 6-benzylaminopurine
• NAA alpha- naphtalene acetic acid
• 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.
• 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:
• MS Murashige & Skoog
• BAP 6-benzylaminopurine
• NAA alpha-naphtalene acetic acid
• G5 medium 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.
• BAP 6-benzylaminopurine
• NAA alpha-naphtalene acetic acid
• 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 5 Induction of secondary metabolite production
• Example 5 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 ⁇ ), 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/H 2 0; 10 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.
• hydro-alcoholic solution EtOH/H 2 0; 10 to 95% v/v
• 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
• the secondary metabolite profile of a plant extract obtained in accordance with Example 6 was elucidated using reverse phase HPLC-ESTQ-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, 5 ⁇ X 4.6mm X 250mm) was employed.
• 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, ⁇ (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.
• 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.
• 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 et al Lebenson Wiss Technol 1995;28:25- 30).
• 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.
• DPPH solution Sigma
• ALD53 and ALD64 achieved similar, good levels of anti-oxidant activity at all tested concentrations, reaching 50- 60% 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 ⁇ -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.
• ⁇ 1 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
• cDNA samples were diluted 1:5 with water prior to qPCR.
• qPCR reactions were prepared with 5 ⁇ 1 cDNA, ⁇ SsoAdvanced SYBR green + ⁇ specific primer + 4 ⁇ 1 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.
• Example 10 Capacity of H. procumbens extract to modulate UV-induced production of reactive oxygen species in human cells
• 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/cm 2 ) 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.
• MMP matrix metalloproteinase
• Clostridium Histolyticum in a cell free system Clostridium Histolyticum in a cell free system.
• collagenase (0,3U/ml) was incubated with active (ImM) for 15 minutes at
• 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.
• 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.
• IL-6 and GM-CSF are standard markers for inflammation.
• MMPl and MMP3 are also markers for
• MMP matrix metalloprotease
• 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 (2 ⁇ g/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.
• 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.
• IL-lbeta 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.
• MMP1, MMP3, IL-6 and GM-SCF inflammatory markers
• 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).
• 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.
• DEJ dermal-epidermal junction
• 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.
• 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.
• 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.
• both H. procumbens extract and verbascoside significantly increase Collagen I protein production in the presence of UV stress.
• DAPI lOng/ml
• 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.
• 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).
• DMEM + 10% FBS normal fibroblast media
• 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.
• Example 16 Capacity of H. procumbens extract to modulate protein production in aged human cells
• 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.
• H. procumbens extract was 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.
• AGEs Advanced glycation end-products
• proteins e.g collagen
• 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 Bovine Serum Albumin
• BSA Bovine Serum Albumin
• Rutin positive control
• left untreated negative control
• 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 cultured for a further 72hrs.
• cells were treated with Metformin (ImM) for 72h.
• CML Carboxymetyl lysin
• 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.
• 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
• H. procumbens or verbascoside on Laminin V subunit gamma gene code LAMC2
• 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).
• 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.
• H. 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.
• Example 19 Capacity of the H. procumbens extract to modulate epidermal gene expression in human cells without UV stimulation
• Example 20 Skin lightening properties of the H. procumbens extract
• 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).
• 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.
• ⁇ is under the control of major melanogenic pathways, including the a- melanocyte 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)
• ⁇ -catenin In the Wnt/p-catenin pathway, a key control is the level of intracellular ⁇ -catenin. In the absence of a Wnt signal, ⁇ -catenin is sequentially phosphorylated by glycogen synthase kinase-3P (GSK-3P), and the phosphorylated ⁇ -catenin is recognized by an ubiquitin ligase complex, resulting in degradation via an ubiquitin- dependent mechanism.
• GSK-3P glycogen synthase kinase-3P
• cytoplasmic ⁇ -catenin which translocates to the nucleus and forms a complex with both T- cell factor (TCF) and lymphocyte enhancer factor- 1 (LEF) to up-regulate expression of ⁇ .
• TCF T- cell factor
• LEF lymphocyte enhancer factor- 1
• activation of the Wnt/ ⁇ -catenin pathway stimulates melanogenesis via the up- regulation of MITF activity (Bellei, Pitisci, Catricala, Larue, & Picardo, 2011; Cadigan, 2008; Yaar & Park, 2012).
• procumbens extract might suggest a possible whitening effect through a slow-down of the Wnt/p-catenin melanogeneis pathway.
• 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 C02 5%.
• 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/cm 2 of UVA and 200mJ/cm 2 UVB (+ 15 minutes), with an illuminator Biosun (Vilbert Lourmat, FR). After the irradiation, the epidermis were re-incubated with the extract during 6 hours.
• NHEKs Keratinocytes
• 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.
• 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-to- cDNA kit (Applied Biosystems) from 2 ⁇ g 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 (MITF- Hs01117294_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).
• 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 were obtained for each gene.
• Results files were exported from the Real-Time qPCR device using the SDS RQ Manager Software (v 1.2.1 , Applied
• Example 21 Further skin lightening properties of the H. procumbens extract
• procumbens extract and verbascoside have properties that can lead directly to skin lightening effects.
• 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.
• MTS assay 3-(4,5-dimethythiazol-2-yl)-5-(3- carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium).
• kojic acid 250 ⁇ 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.
• 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).

Landscapes

• Life Sciences & Earth Sciences (AREA)
• Health & Medical Sciences (AREA)
• Engineering & Computer Science (AREA)
• Biotechnology (AREA)
• Developmental Biology & Embryology (AREA)
• Botany (AREA)
• General Health & Medical Sciences (AREA)
• Cell Biology (AREA)
• Veterinary Medicine (AREA)
• Public Health (AREA)
• Animal Behavior & Ethology (AREA)
• Environmental Sciences (AREA)
• Natural Medicines & Medicinal Plants (AREA)
• Microbiology (AREA)
• Chemical & Material Sciences (AREA)
• Epidemiology (AREA)
• Mycology (AREA)
• Alternative & Traditional Medicine (AREA)
• Biomedical Technology (AREA)
• Dermatology (AREA)
• Gerontology & Geriatric Medicine (AREA)
• Medical Informatics (AREA)
• Medicinal Chemistry (AREA)
• Zoology (AREA)
• Pharmacology & Pharmacy (AREA)
• Wood Science & Technology (AREA)
• Genetics & Genomics (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Organic Chemistry (AREA)
• Birds (AREA)
• Nutrition Science (AREA)
• Food Science & Technology (AREA)
• Polymers & Plastics (AREA)
• Physiology (AREA)
• Biochemistry (AREA)
• General Engineering & Computer Science (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Cosmetics (AREA)
• Medicines Containing Plant Substances (AREA)

Applications Claiming Priority (2)

Publications (1)

Family

ID=56891605

Family Applications (1)

Country Status (6)

Cited By (1)

Families Citing this family (5)

Family Cites Families (6)

• 2016
  • 2016-06-29 GB GB1611285.6A patent/GB2552926A/en not_active Withdrawn
• 2017
  • 2017-06-27 MX MX2019000137A patent/MX2019000137A/es unknown
  • 2017-06-27 WO PCT/EP2017/065814 patent/WO2018002029A1/en unknown
  • 2017-06-27 EP EP17732918.2A patent/EP3478051A1/en not_active Withdrawn
  • 2017-06-27 CN CN201780039538.5A patent/CN109922657A/zh active Pending
  • 2017-06-27 EA EA201990128A patent/EA201990128A1/ru unknown

Also Published As

Similar Documents

Legal Events