EP2229165A1

EP2229165A1 - Anti-glycation properties of asicatic acid and madecassic acid

Info

Publication number: EP2229165A1
Application number: EP08856329A
Authority: EP
Inventors: Caroline Segond
Original assignee: Bayer Consumer Care AG
Current assignee: Bayer Consumer Care AG
Priority date: 2007-12-06
Filing date: 2008-11-22
Publication date: 2010-09-22
Also published as: CL2008003505A1; AR069518A1; PE20091235A1; WO2009071213A1; TW200938215A

Abstract

The present invention relates to the use of the use of compounds from Centella Asiatica selected from the group consisting of Asiatic acid and Madecassic acid, and plant extracts containing both acids in cosmetics, pharmaceuticals and food supplements for preventing the glycation phenomenon and the glycation consequences in tissues, more paticularly for preventing skin aging, enhancing wounds treating, venous insufficiency etc.

Description

Anti-glycation Properties of Asiatic Acid and Madecassic Acid

The present invention relates to the use of the use of compounds from Centella Asiatica selected from the group consisting of Asiatic acid and Madecassic acid, and plant extracts containing both acids in cosmetics, pharmaceuticals and food supplements for preventing the glycation phenomenon and the glycation consequences in tissues.

Centella asiatica, also known as Violette marronne (Reunion Island), as Gotu Kola or as Indian pennywort (India), or as Centella repanda (North America) and as Talapetraka (Madagascar), is a polymorphous herb and belongs to the family of Umbelliferae (Apiaceae), particularly to the Hydrocotyle subfamily that grows wild in pantropical moist and shady regions at an ideal altitude of 600 to 1200m. Centella asiatica includes three varieties called Typica, Abyssinica and Floridana. It is known and used for healing, sedative, analgesic, antidepressant, antiviral and antimicrobial properties. The biological activity is enabled by active molecules from the triterpene series such as Asiaticoside (I), Madecassoside (under its 2 isomeric forms : madecassoside itself and terminoloside) (JX), Asiatic acid (IDf) and Madecassic Acid (IV). They contribute to the natural defense of the plants against environmental aggression thanks to the anti-bacterial properties of the genins (HI, IV), which are obtained by hydrolysis from the heterosidic reserve form (I, II).

Extracts containing these compounds are used in the pharmaceutical and cosmetic industry regarding skin care and skin diseases, respectively for treating wounds, scars and venous insufficiency and for dermis restoration and anti-inflammatory properties as disclosed in WO2004/062678.

Glycation is a slow, spontaneous, non-en2ymatic reaction which happens in organism. It corresponds to the covalent linking between a protein and an oxidation derivative from sugar, more specifically a glucose oxidation derivative. Glycation occurs in three steps with first the reaction between protein amino groups with glucose-carbonyl group to form an unstable Schiff s base, wbich then rearranges rapidly to more stable Amadori products. The Amadori structure undergoes a series of slow reactions involving rearrangement, oxidation, and dehydration to form stable, heterogeneous adducts known as glycation products - like pyrralin, pentosidine, crosline, Advanced Glycation End Products (AGEs) — which remain tightly bound to the protein. This reaction is irreversible and leads gradually to the loss of the functional activity of the glycated proteins, their stocking and the generation of oxidative secondary reactions.

In the skin, glycation occurs in the dermis and more specifically towards collagen fibres. Collagen glycation increases gradually with years leading to the gradual formation of glycation products. Consequently physico-chemical changes of collagen changes with a decrease of its solubility and more sensitive to degradation. This phenomenon leads to tissue stiffening and a loss of the skin tonicity. The topical application of an anti-glycation agent has been proved to improve the water content and elasticity of aged skin (S.Vasan et al / Archives of Biochemistry and Biophysics VoI 419, p.89-96, 2003).

Advanced Glycations End products affect nearly every type of cell and molecule in the body and are thought to be major factors in aging and age-related chronic diseases. They are also believed to have functional consequences on various biochemical and physical phenomena, for examples:

alterations of enzymatic activity, e.g. in neuronal regeneration process

reticulation/cross-linking of proteins and aggregates formation, e.g. their accumulation increases the intermolecular spaces in crystalline lens leading to its hyperpermeability and structural modifications

problems in the nucleic acid function with subsequent chromosomal break and dysfunction of the DNA repair process.

alterations of the cytoskelett (e.g. Vimentin) and negative impact on fibroblast contractility

Glycation phenomenon and consequences of AGEs are related to age but also with chronic diseases such as type π diabetes mellitus, Alzheimers' disease (damaged endothelium and collagen), cancer (acrylamide and other side products are released, peripheral neuropathy (the myelin is attacked) and other sensory losses such as deafness and blindness (microvascular damage in the retina). The negative influence on fibroblast contractility, caused by alterations of the the cytoskelett (vimentin) (T.Kueper et al. / Journal of Biological Chemistry VoI 282(32), p.23427-36, 2007), could also contribute to delayed healing and reduced reorganisation of collagen in aged and/or diabetic skin. The present invention relates to the use of compounds from Centella Asiatica selected from the group consisting of Asiatic acid and Madecassic acid for preventing the glycation phenomenon and the glycation consequences in tissues.

Asiatic acid and Madecassic acid are well-known and commercially available compounds. Their glycolated derivatives asiaticoside and madecassoside can be isolated from e.g. Centella asiatica and the free acids can be prepared by hydrolysis as described in GB923414

Subject of the present invention is the use of Asiatic acid and/or Madecassic acid or plant extracts comprising one or both acids for the manufacture of a composition for preventing the glycation phenomenon and the glycation consequences in tissues.

Preference is given to an extract of Centella asiatica comprising Asiatic acid and /or Madecassic acid.

The solvent of the extract is preferably a mixture of water and alcohol e.g. ethanol. The ratio of the volume between water and alcohol can be from 50:50 up to 90:10, preferably 75:25.

Asiatic acid and Madecassic acid can be used in combination or as single isolated substances or in combination with one or more other compounds such as asiaticoside, madecassoside and/or terminoloside.

Preference is given to a combination or plant extract comprising at least Asiatic acid and/or Madecassic acid and optionally Asiaticoside, Madecassoside and/or Terminoloside.

Compounds, mixtures and extracts of the present invention can be administered in any form by any effective route, including, e.g., oral, parenteral, enteral, intravenous, intraperitoneal, topical, transdermal (e.g., using any standard patch), ophthalmic, nasally, local, non-oral, such as aerosol, inhalation, subcutaneous, intramuscular, buccal, sublingual, rectal, vaginal, intra-arterial, and intrathecal, etc. They can be administered alone, or in combination with any ingredient(s), active or inactive. Preference is given to a topical administration.

Compounds, mixtures and extracts of the present invention can be converted in a known manner into the usual formulations such as cosmetic or pharmaceutical compositions or compositions used as food supplement. These may be liquid or solid formulations e.g. without limitation normal and enteric coated tablets, capsules, pills, powders, granules, elixirs, tinctures, solution, suspensions, suppositories, syrups, solid and liquid aerosols, emulsions, pastes, creams, ointments, milks, gels, salves, serums, foams, shampoos, sticks or lotions. Preference is given to a cosmetic composition in a form of an aqueous solution, a white or colored cream, ointment, milk, gel, salve, serum, foam, shampoo, stick, cream, paste, or lotion.

Compounds, mixtures and extracts of the present invention can be further combined with any other suitable additive or pharmaceutically acceptable carrier. Such additives include any of the substances already mentioned, as well as any of those used conventionally, such as those described in Remington: The Science and Practice of Pharmacy (Gennaro and Gennaro, eds, 20th edition,

Lippincott Williams & Wilkins, 2000); Theory and Practice of Industrial Pharmacy (Lachman et al., eds., 3rd edition, Lippincott Williams & Wilkins, 1986); Encyclopedia of Pharmaceutical

Technology (Swarbrick and Boylan, eds., 2nd edition, Marcel Dekker, 2002). These can be referred to herein as "pharmaceutically or cosmetically acceptable carriers" to indicate they are combined with the active drug and can be administered safely to a subject for therapeutic purposes.

The dosage of the compounds, mixtures and extracts of the present invention can be selected with reference to the other and/or the type of disease and/or the disease status in order to provide the desired therapeutic activity. These amounts can be determined routinely for a particular patient, where various parameters are utilized to select the appropriate dosage (e.g., type of disease, age of patient, disease status, patient health, weight, etc.), or the amounts can be relatively standard.

The amount of the administered active ingredient can vary widely according to such considerations as the particular compound and dosage unit employed, the mode and time of administration, the period of treatment, the age, sex, and general condition of the patient treated, the nature and extent of the condition treated, the rate of drug metabolism and excretion, the potential drug combinations and drug-drug interactions, and the like.

Preference is given to a composition comprising Asiatic acid and/or Madecassic acid in an amount of from 0.005% up to 10%, preferably 0.01 up to 5%, more preferably 0.1% up to 3% by weight of the total composition.

Asiatic acid and/or Madecassic acid or extracts containing one or both acids can also be combined with other anti-aging or wound healing promoting and scar reducing agents like anti-glycation active ingredients e.g. aminoguanidin, anti-wrinkles products, anti-oxidizing agents, activators or protectants of the ECM compound synthesis, anti-inflammatory agents, chelating agents, vitamins (e.g. Vitamin E, C, B3,...), UV filters or sunscreens, growth factors, (micro-)nutrients, amino acids, physiological (ceramides, cholesterol, free fatty acids) and moisturizing agents e.g. dexpanthenol, pantothenic aicd or derivatives thereof. The pharmaceutical or cosmetic composition according to the invention is administered one or more, preferably up to three, more preferably up to two times per day. Preference is given to a topical administration.

Nevertheless, it may in some cases be advantageous to deviate from the amounts specified, depending on body weight, individual behaviour toward the active ingredient, type of preparation and time or interval over which the administration is effected. For instance, less than the aforementioned minimum amounts may be sufficient in some cases, while the upper limit specified has to be exceeded in other cases. In the case of administration of relatively large amounts, it may be advisable to divide these into several individual doses over the day.

Asiatic acid and/or Madecassic acid, or extracts, combinations or compositions comprising one or both acids, can be used for preventing the glycation phenomenon and the glycation consequences in tissues.

Asiatic acid and/or Madecassic acid, or extracts, combinations or compositions comprising one or both acids, can be used for preventing or inhibiting of the glycation of the proteins in the dermis, in particular the glycation of collagen, preventing or inhibiting of the cross-linking of collagens and/or of the extracellular matrix proteins in the dermis, preventing or inhibiting of accelerate aging, impaired wound healing, and appearance of scars.

Asiatic acid and/or Madecassic acid, or extracts, combinations or compositions comprising one or both acids, can be used for preventing or inhibiting of the glycation of intracellular proteins, in particular cytoskeletal proteins like Vimentin, preventing or inhibiting of the cross-linking of Vimentin and/or other intracellular proteins, preventing or inhibiting impaired cellular contraction in skin ageing, wound healing, and scar tissue remodelling.

Asiatic acid and/or Madecassic acid, or extracts, combinations or compositions comprising one or both acids can be used for treating aging symptoms of skin, nails and/or hair in relation with glycation.

Asiatic acid and/or Madecassic acid, or extracts, combinations or compositions comprising one or both acids can be used for the prevention or treatment of capillaritis (pigmented purpura), acanthosis nigricans, Sclerosis, sclerodermiformis syndroms, xerosis, Dupuytren disease, Rubeosis, conjonctive alterations, Xanthochromia, Chronic Venous Insuffiency (Heavy legs), general disturbances of microcirculation..

Cutaneous reactions in diabetis cases are often linked to glycation and AGEs. For example, glycation phenomenon is important in microvascular angiopathy and the associated cutaneous disorders such as acanthosis nigricans, rubeosis and purpural and pigmented capillaritis (Flagothier C. et al., Rev Med Liege 2005, 60, 5-6, 553-559). Xerosis and Dupuytren diseases can be quoted among the affections linked to diabetes, glycation and microangiopathy and the consequent reduced blood flow (Diris N et al., Ann Dermatol Venereol 130(11), 1009-1014, 2003; Rosenblomm AL et al, Endocrinol Metab Clin North Am 25(2), 473-483, 1966; Melling M et al, Arch. Pathol. Lab Med. 124 (9) 1275-1281, 2000; Melling M et al, Anat.Rec 255(4) 401-406, 1999). Xanthochromia corresponds to a skin and nail coloration that can be caused by glycation.

Examples:

Example 1: In vitro evaluation of the activity of Asiatic acid on glvcation (fibres reticulation^")

Test on fibrillar collagen I (rat tail tendon model, acellular model).

Glycation is induced by addition of glucosone and the glycation level is characterized by the visual evaluation of the collagen colour (Browning effect appears with induction of glycation) and by collagen migration in SDS-PAGE electrophoresis after controlled digestion by pepsine followed by silver nitrate dyeing. Test is performed in duplicate.

Aminoguanidine is used as a reference molecule as known to inhibit glycation reaction. Asiatic acid is tested after solubilization in DMSO (Dimethyl sulfoxide) and dilution in PBS buffer (0.05M, pH 7.4). Tested concentrations are lμg/mL, lOμg/mL and 50μg/mL.

Tendons (about 100 mg) are incubated during 15 days with glucosone (2.5 mM) in presence of the tested substance and aminoguanidine (10 mM) at 37°C. Unglycated control is obtained after incubation during 15 days without other treatment and glycation control with aminoguanidine treatment only.

Collagen is then solubilized in a solution at 3% acetic acid. After complete solubilization, pepsine is added (0.05 mg/ml) for treatment overnight at room temperature. Collagen precipitation is then obtained by adding NaCl 5M before being rinsed out to eliminate pepsine.

Collagen is solubilized again in acetic acid (3%) to enable protein quantification. 2 μg of proteins are denaturated by heating (90%) in presence of sodium lauryl sulfate and β-mercaptoethanol and are laid down on a polyacrylamide gel surface. Electrophoretic migration is performed during 3 hours before the final dying with silver nitrate.

Photos are taken of the gel and a semi-quantitative measurement of the α-1 and α-2 bands is performed thanks to image analysis (ANGIOSYS software). Density of the bands (in pixels) enables a products comparison: intense α-bands corresponds to a low glycation level as it corresponds to the presence of small unglycated proteins. Visual evaluation:

0 : No browning, + : low browning, : very intense browning

Image analysis on SDS-Page:

Asiatic acid is able to prevent the collagen glycation induction by glucosone. The activity is similar and even greater than that of aminoguanidine.

Asiatic acid can therefore be used in cosmetic or pharmaceutical preparations which aim at glycation phenomena and more widely the tissue status and appearance linked to the dermal structure preservation. Example 2: In vitro evaluation of the activity of Genins on Advanced Glycation End products (AGEsVinduced apoptosis

Tests on human fibroblasts (NHDF)

1/ Treatment over 48h with CML-CoIl (Carboxymethyl lysine-collagen) for AGEs induction and with Genins (mixture of Madecassic and Asiatic acid). Test with 3 concentrations of Genins: 0.1 μg/ml, lμg/ml and 10 μg/ml.

2/ Evaluation of the apoptosis by quantifying the DNA fragmentation ratio in the cytoplasmic fractions of control or treated cells. The quantification is performed by CDDE test (Cell Death Detection ELISA) and ise= characterized by Optical Density (at 405nm) by μg proteins. Results are based on enrichment factor to evaluate the DNA fragmentation between treated culture and control culture (FE=DNA fragmentation on cultured cells/DNA fragmentation for control without CML). The protection activity is calculated versus Controls (with CML treatment) as followed:

\_OU_{Control mth CML} L⁾V _treated J

^protection — -XlOO V^iJ Control with CML ~ ^^ Control without CML J

Each part of the test is performed in triplicate.

The results confirm that CML-CoIl is able to activate DNA fragmentation and therefore induces cells apoptosis. Genins are able to reduce DNA fragmentation in a dose-dependent way. All the results are significant. Activities of 0.1 μg/ml and lμg/ml concentrations are equivalent but smaller than the highest concentrationlOμg/ml.

Genins are therefore able to protect the cells against Advanced Glycation End products and to protect the skin against glycation consequences.

Example 3: Anti-aging cream

Cream 1 :

Ingredients (ESfCI, w/w%): 1.0% of Asiatic acid, or extract of Centella asiatica according to the invention), 1.5% of beheneth-10, 1.5% of beheneth-25, 5.0% of dycaprylyl carbonate, 5.0% of hexyl laurate, 5.0% of isohexadecane, 5.0% of cetearyl isononaoate, 1.0% of dimethicone, 2.0% of behenyl alcohol, 2.0% of hydrogenated vegetable glycerides, 0.5% of phenoxyethanol and parabens, 0.5% of tocopherol acetate, 3.0% of glycerol, 2.0% butylenes glycol, 0.1% of xanthan gum, 0.2% of carbomer and water (qs 100).

Cream 2:

Ingredients (ESfCI, w/w%): 1.0% of Asiatic acid, or extract of Centella asiatica according to the invention), 5.0% of cetearyl glucoside and cetearyl alcohol, 5.0% of caprylic/capric triglyceride, 5.0% of squalane, 3.0% of cetearyl isononaoate, 2.0% of dimethicone crosspolymer, 1.5% of stearyl alcohol, 5,0% of dycaprylyl carbonate, 0.1% parabens, 0.5% of phenoxyethanol and parabens, 2.0% of glycerol, 0.3% of carbomer, 2.0% of PEG 32, 0.2% of xanthan gum, 2.0% of aluminium starch octenyl succinate and water (qs 100).

Claims

What is claimed is:

1. Use of Asiatic acid and/or Madecassic acid or a plant extract comprising one or both acids for the manufacture of a composition for preventing or reducing the glycation phenomenon and the glycation consequences in tissues.

2. Use of claim 1 for preventing or reducing the glycation of proteins.

3. Use of any of claims 1 to 2 for preventing, reducing or inhibiting the glycation of the skin and/or the nails and/or the hair.

4. Use of any of claims 1 to 3 for preventing, reducing or inhibiting the glycation of proteins of the skin dermis and dermal cellular compartments, in particular fibroblasts.

5. Use of any of claims 1 to 4 for preventing, reducing or inhibiting the glycation of collagen.

6. Use of any of claims 1 to 4 for preventing, reducing or inhibiting the glycation of keratins.

7. Use of any of claims 1 to 4 for preventing, reducing or inhibiting the glycation of vimentin or other cytoskelletal components.

8. Use of any of claims 1 to 4 for preventing, reducing or inhibiting the glycation to enhance wound healing, to reduce scarring or to remodel scars.

9. Use of any of claims 1 to 2 for preventing, reducing or inhibiting the production of Advanced Glycation End Products (AGEs) thereby reducing the deleterious effects of Advanced Glycation End Products (AGEs)

10. Use of claim 1 for treating aging symptoms of skin, nails and/or hair.

11. Use of claim 1 for the prevention or treatment of capillaritis (pigmented purpura), acanthosis nigricans, Sclerosis, sclerodermiformis syndroms, xerosis, Dupuytren disease, Rubeosis, conjonctive alterations, Xanthochromia, Chronic Venous Insuffiency (Heavy legs), general disturbances of microcirculation.

12. Use of any of claims 1 to 11 wherein in addition the composition comprises at least one compound selected from group consisting of asiaticoside, madecassoside and terminoloside.

13. Use of any of claims 1 to 12 wherein the extract is an extract of Centella asiatica.

14. Use of any of claims 1 to 13 wherein the composition is administered topically or orally.

15. Use of any of claims 1 to 14 wherein the composition comprises Asiatic acid and/or Madecassic acid in an amount of from 0.005% up to 10%, preferably 0.01 up to 5%, more preferably 0.1% up to 3% by weight of the total composition.

16. Use of any of claims 1 to 15 wherein in addition the composition comprises at least one other anti-aging or wound healing promoting and scar reducing agent like anti-glycation active ingredients, anti-wrinkles product, anti-oxidizing agent, activator of protectants of the ECM compounds, anti-inflammatory agent, chelating agent, Vitamin, UV filter or sunscreen, growth factors, (micro-)nutrients, amino acids, physiological (ceramides, cholesterol, free fatty acids) or moisturizing agent.