WO2008125231A1

WO2008125231A1 - Ginsenosides and extracts containing them combined with dexpanthenol

Info

Publication number: WO2008125231A1
Application number: PCT/EP2008/002679
Authority: WO
Inventors: Caroline Segond; Raymond De Bony; Armelle Magnet
Original assignee: Bayer Consumer Care Ag
Priority date: 2007-04-17
Filing date: 2008-04-04
Publication date: 2008-10-23
Also published as: PE20090073A1; CL2008001065A1

Abstract

The present invention relates to the combination comprising at least one ginsenoside and pantothenic acid and/or a derivative of pantothenic acid for cosmetically or pharmaceutical use, a cosmetically or pharmaceutical composition containing such a combination and its manufacturing process.

Description

Ginsenosides and extracts containing them combined with dexpanthenol

The Panax plant family comprises numerous species such as Panax ginseng (C.A.Meyer), Panax quinquefolium, and Panax notoginseng which are cultivated and used industrially in food supplements, pharmaceuticals and cosmetics. They contain saponins as active substances which are called ginsenosides. Ginsenosides of each species are basically the same, but are contained in different proportions in each species.

Panax notoginseng (also called San Qi) is a straight herbaceous perennial plant which grows e.g. in the southwest of China. Traditionally people use the roots of Panax notoginseng not only as a tonic but also for the treatment of many symptoms and diseases such as trauma, inflammation, hepatitis, heart and vascular diseases as well as aging. Up to now more than 30 ginsenosides could be isolated from the roots of Panax notogingseng. The major ginsenosides are ginsenoside RgI and ginsenoside RbI followed by ginsenosides Rd, Re, Rg2 and notoginsenoside Rl . Many scientific works have confirmed the pharmacological properties and biological activities of Panax notoginseng such as ditropism regulating effects to organic function (Y.M. Luo et al., Acta Pharmacologica Sinica, 1993, 14(5), 401-404), effects on the central nervous system (Y. Ying et al., Acta Pharmaceutica Sinica, 1994, 29(4), 241-245), cancer prevention (T. Konoshima et al., Chemical and Pharmaceutical Bulletin, 1992, 40, 531-533; L. Xu et al., Journal of WCUMS, 1991, 22(2), 124-127) and antiviral activity (J. Li et al., Journal of Norman Bethune University of Medical Sciences, 1992, 18(1), 24-26).

In the cosmetic industry, Panax notoginseng and/or ginsenosides are used for various applications. Panax notoginseng extract is claimed for skin elasticity activation to improve wrinkles and prevent chronological and UV-induced aging (JP 2006-028150).

Ginsenoside RbI or Rb-I like substances are described for stimulation of elastin synthesis (WO 99/07338), for treating hair (FR 9300899, US 5,663,160), for stimulating the regeneration of tissues after the wound (JP 2002-255826), for the reconstruction of tissues suffering from skin aging (WO 2002/072599), for treating wounds in the case of burns (JP 2004-077456). Ginsenoside RbI is also used in combination with Ginsenosides Rc and Rd (JP 2003-070496) to activate endonuclease for the repair of UV damaged DNA. Ginsensosides Rh2 and Rg3 are blended in a UV -blocking cosmetic composition due to their UV absorbing properties (KR 2004-0098177). Langerhans cells are responsible for the immune protection of skin. When skin is exposed to UV light, Langerhans cells disappear from the epidermis and as a result it becomes less protected against infectious diseases and cancer (T. Schwarz, Photodermatol Photoimmunol Photomed 2002, 18, 141-145).

Heme oxygenase HO is an enzyme which catalyzes the ring opening of heme (a molecule found in cells) with the formation of carbon monoxide CO, biliverdin (which is rapidly transformed into the antioxidant bilirubin) and free iron (which leads to the induction of the iron-binding protein ferritin). Heme oxygenase has two forms: HO-2 which is the constitutive form mainly in neural tissues and HO-I which is the inducible form. They are considered to be cytoprotective enzymes due to the antioxidant, anti-inflammatory, anti-apoptotic and anti-proliferative effects (L. E. Otterbein et al., Trends Immunol. 2003, 24(8), 449-55). HO-I induction is considered to improve burn injury healing (Gan HT et al., Surgery., 2006, 141(3):385-93) and its induction by 20(S)- Protopanaxadiol is proved to decrease NO production for anti-inflammatory activity (Lee SH et al., Planta Med., 2005, 71(12), 1 167-70). It has also been found that carbon monoxide generated by HO-I, which itself is generated by UV-A exposure, can protect Langerhans cells from photoimmunosuppression caused by UV-B irradiation (M. Allanson et al., J. Invest. Dermatol., 2005, 124(3), 644-650) .

Pantothenic acid, which is also known as D(+) N-(2,4-dihvdroxy-3,3-dimethylbutyryl)p-alanine, is a member of the B complex vitamins and is sometimes referred to as vitamin B5. Pantothenic acid plays a key role in cellular metabolism, as after incorporation into coenzyme A (CoA), it participates in the synthesis of fatty acids, cholesterol and sterols. Through its participation in the Krebs cycle, coenzyme CoA is also instrumental in the generation of energy by the cells. Pantothenic acid is hence essential for epithelial regeneration and development in the event of damage to the skin when a high rate of lipids and cellular renewal is needed.

Dexpanthenol, the alcohol of the pantothenic acid, is well absorbed by the skin. After penetration in the skin it is rapidly transformed into pantothenic acid. Dexpanthenol has therefore been used for many years in topical products such as ointments and creams. The clinical effectiveness of the topical application of dexpanthenol in promoting wound healing has been confirmed by several studies in cases of wounds, burns, cracked nipples, ulcers, and bedsores (e.g. P. Girard, A. Beraud, C. Goujon, A. Sirvent, J-L Foyatier, B. Alleaume, R. de Bony, Les Nouvelles Dermatologiques, 17,1998, pp. 559-570).

Pantothenic acid and its derivatives are also known for treating dermatological disorders which involve the degranulation process of mastocytes as mentioned in WO 2001/19330. It has been now surprisingly found that the combination comprising at least one ginsenoside and pantothenic acid and/or a derivative of pantothenic acid shows a synergistic effect.

The present invention relates to a combination comprising at least one ginsenoside and pantothenic acid and/or a derivative of pantothenic acid.

According to the invention ginsenosides include but are mot limited to the ginsenosides G-RaI , G- Ra2, G-Ra3, G-RbI , G-Rb2, G-Rb3, G-Rc, G-Rd, G-Re, G-Rf, G-RgI, G-Rg2, G-Rg3, G-RhI , G- Rh2, G-RsI , G-Rs2, G-Ro, MG-RbI, MG-Rb2, MG-Rc, MG-Rd, Q-Rl, N-Rl, N-R4 and 20 GIc- Rf. Preference is given to ginsenoside G-RgI, G-RbI, G-Rd, G-Re, G-Rg2 and N-Rl . Most preferably the ginsenoside is selected from the group consisting of ginsenoside G-RgI and G-RbI .

All mentioned ginsenosides are known and can be isolated from the Panax plants by standard methods e.g. by extraction and chromatography.

In particular the combination according to the invention comprises a mixture of different ginsenosides. Preference is given to a mixture of ginsenosides comprising ginsenoside G-RgI and G-RbI, more preferably a mixture of ginsenoside G-RgI, G-RbI, G-Rd, G-Re, G-Rg2 and N-Rl.

Furthermore the combination according to the invention comprises a plant extract containing the ginsenosides according to the invention.

Plant extracts according to the invention are extracts of plants of the Panax family which include but are not limited to Panax ginseng (C.A.Meyer), Panax quinquefolium, and Panax notoginseng (San Qi). Preference is given to Panax notoginseng (San Qi).

The extraction can be performed on all parts of the plant. Preferably the roots or rhizomes are extracted.

The extraction can be done by standard extraction methods. Preferably roots or rhizomes are extracted with a polar solvent applicable for extraction optionally by several times. The crude extract can be purified by chromatography. Optionally the fractions can be dried by spray-drying.

An extract according to the invention is normally a dry extract. Nevertheless the extract can also be used as solution, i.e. that the final drying step of the described extraction process is omitted and the product can optionally be encapsulated, e.g. in a gelatine capsule.

The polar solvent used for extraction is preferably alcohol or a mixture of water and alcohol wherein the alcohol is preferably ethanol. - A -

Preference is given to a dry plant extract containing ginsenosides in an amount between 0.1 and 100%, preferably between 10 and 100%, preferably above 80%.

The extract according to the invention preferably contains G-RbI in an amount of from 10 to 60%, G-RgI in an amount of from 10 to 60%, G-Rd in an amount of from 0 to 15%, N-Rl in an amount of from 0 to 15 % and/or G-Re in an amount of from 0 to 10% by weight of the total extract.

Ginsenosides can be isolated and/or purified from the extract containing it by standard isolation methods. Standard isolation methods include but are not limited to chromatographic methods.

In the combination of the present invention any pharmaceutically and/or cosmetically acceptable derivative of pantothenic acid can be used. Examples of derivatives of pantothenic acid include alcohols, aldehydes, alcohol esters, acid esters, salts and the like. The preferred derivative of pantothenic acid is pantothenyl alcohol (panthenol), particularly the D(+) of pantothenyl alcohol which is more commonly known as dexpanthenol. Calcium pantothenate is a preferred salt and as preferred alcohol ester, pantothenyl triacetate can be chosen.

The combination according to the invention can also be combined with at least one further active substance or plant extract e.g. substances or plant extracts usually employed for dermatological use.

Further active substances include but are not limited to desquamating and/or moisturizing agents, UV filtering or blocking agents, depigmenting or propigmenting agents, antiglycation agents, antiinflammatory agents, anti-microbial agents, agents stimulating the synthesis of dermal, epidermal, hair or nail macromolecules and/or preventing the degradation thereof, agents stimulating the differentiation of keratinocytes, muscle relaxants, antipollution and/or anti-free radical agents, slimming agents, agents acting on the microcirculation, agents acting on the energy metabolism of the cells, tightening agents, agents preventing the loss or stimulating the growth of hair, agents preventing grey or white hair, or a mixture thereof. Preferably that combination is contained in a topically dermatological or cosmetically composition.

The combination according to the invention can also be combined with an alpha-hydroxy acid, a salicylic acid or derivatives thereof such as acetylsalicylic acid, a cystein derivative, a ceramide, a steroid, tocopherol, tocotrienol, arbutin or derivatives thereof, ascorbic acid or a derivative thereof, retinol or derivatives thereof, retinoid or derivatives thereof, a carotenoid, glycyrrhetinic acid, glycyrrhizic acid or its salts, a centella extract or isolated ingredients thereof, a plectranthus extract, a boswellia extract, a ginger extract, an aloes extract, an angelica extract, an eleutherococcus extract, a rhodiola extract, an hippophae extract, a cyanotis extract, a vegetable oil, an oligopeptide, coenzyme QlO, ubiquinone, caffeine, theophylline, a tea extract, a cacao extract, a yeast extract, a soybean extract, a resveratrol and/or a procyanidin oligomer. Preferably that combination is contained in a topically dermatological or cosmetic composition.

The combination according to the invention can also be combined with an agent for supporting the cosmetic qualities of skin and/or hair, supporting to stay slim, retaining muscular strength, improving memory, reducing cholesterol, reducing menopause side effects, preventing harmful effects of sunlight and/or preventing cardiovascular problems. Preferably that combination is contained in an orally applicable composition.

The combination according to the invention can also be combined with polyunsaturated fatty acids or derivatives thereof, vitamins, oligoelements, a calcium salt, a carotenoid, a phytohormone, a polyphenol, a medicinal plant extract, a carnosine and/or caffeine. Preferably that combination is contained in an orally applicable composition.

The combination according to the invention can be used in the field of food supplement or in the dermatological field, which include cosmetically and pharmaceutically use, for the protection of the skin against deleterious effects of stress or irradiation e.g. sunlight or UV irradiation, preferably UV-A or UV-B irradiation.

Preference is given to a combination comprising at least one ginsenoside, and pantothenic acid and/or a derivative of pantothenic acid, and glycine.

Glycine is an amino-acid naturally occurring in most animal species. It inhibits the histamine release by mastocytes. The effect of the glycine on the mastocyte degranulation is described, for instance, in M. Paubert-Braquet, G. Lefranois, S. Picquot, D. Rod, Therapetitique, 95, 1992, 2. An inhibition effect on the degranulation process of the mastocytes has been observed, wherein a decrease in the amount of mediators released into the extra-cellular environment (e.g. histamine) is observed.

The use of the glycine for treating dermatological disorders which involve the degranulation process of the mastocytes is furthermore described in A. Siboulet, JM Bohbot, Gyn. Obs., 1987, 166 by means of a study on pruritis in the genital area.

According to a preferred embodiment of the present invention, the ratio between the pantothenic acid and/or its derivatives and the glycine varies between 0. 13 and 13.3 wt/wt.

Combinations according to the present invention are not limited to application forms which comprise all active substances (so called fixed combinations), and combination packages keeping the components in separated compositions, but include also the administration of the active ingredients or components at the same time or at different times, provided it is used for the treatment or prophylaxis of the same disorder or disease. For example, the compounds can be administered simultaneously, e.g., as a single composition or dosage unit (e.g., a pill or liquid containing both compositions), or they can be administered as separate compositions, but at the same time (e.g., where one drug is administered intravenously and the other is administered orally or intramuscularly). The drugs can also be administered sequentially at different times. Agents can be formulated conventionally to achieve the desired rates of release over extended period of times, e.g., 12-hours, 24-hours. This can be achieved by using agents and/or their derivatives which have suitable metabolic half-lives, and/or by using controlled release formulations.

The dosage of each agent of the combination 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. For example, the active agents in the combination can be present and administered in a fixed combination. "Fixed combination" is intended here to mean pharmaceutical forms in which the components are present in a fixed ratio that provides the desired efficacy. 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. Preference is given to a fixed combination.

Compositions:

The combination according to the invention can be converted in a known manner into the usual formulations such as cosmetically, dermatological, pharmaceutical or food supplement compositions. These may be liquid or solid formulations e.g. without limitation normal and tablets, (e.g. enteric coated tablets), capsules, pills, powders, granules, hard and soft gelatine capsules, 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 dermatological or 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.

Salves and creams contain oily, absorbent, water-soluble and/or emulsifying carrier materials such as vaseline, paraffin oil, propylene glycol, cetylalcohol, glycerine monostearate, alkyl-branched fatty acids and the like. Lotions are liquid preparations and can vary from simple solutions to aqueous or aqueous/alcoholic preparations which contain the substances in finely divided form. The preparations contain suspended or dispersing substances such as, for example, sodium carboxymethyl cellulose which suspend or disperse the active substance in a carrier prepared from water, alcohol, glycerine and the like.

Gels are semi-solid preparations which are obtained by gelling a Solution or suspension of the active substance in a carrier material. The carrier materials, which can be hydrophilic or hydrophobic, are gelled using a gelling agent in form of polymers of biological, natural or synthetically origin.

Aerosols are solutions or suspensions of the active substance in a carrier material which are applied using spray generators. Usually used carriers are, for example, trichloromonofluoromethane, trichlorodifluoromethane, volatile silicones, nitrogen, etc..

Spays are solutions, suspensions or powders of the active substance in a carrier material which are applied using mechanical pumps.

Suitable excipients for soft gelatine capsules are e.g. vegetable oils, waxes, fats, semi-solid and liquid Io polyols etc. Suitable excipients for the manufacture of solutions and syrups are e.g. water, polyols, saccharose, invert sugar, glucose etc. Moreover, the pharmaceutical and/or cosmetical compositions can contain preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavorants, salts for varying the osmotic pressure, buffers, masking agents or antioxidants. They can also contain still other therapeutically valuable substances, such as further vitamins, minerals, sun filters, phytotherapeutic extracts, etc. For topical administration, the above active compounds are conveniently used in the form of pharmaceutical and/or cosmetical preparations or compositions which further contain an acceptable carrier material.

The composition according to the invention can be further combined with any other suitable additive or pharmaceutically acceptable carrier, preferably dermatological and/or cosmetically 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 acceptable carriers" to indicate they are combined with the active drug and can be administered safely to a subject for therapeutic purposes. It is also an object of the present invention to provide a process for preparing the above pharmaceutical and/or cosmetical compositions, comprising mixing pantothenic acid and/or its derivatives with ginsenoside or an extract containing ginsenoside and further excipients and optionally further active substances.

Administration:

The combination or the composition comprising the combination according to the 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 aerosal, 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 or orally administration.

The term "topical" as used in the present specification relates to the use of the combination or composition, which are processed with a suitable carrier material and which is applied to the skin or mucous membrane, so that it can display local activity. Accordingly, the topical forms embrace pharmaceutical and/or cosmetically dosage forms which are suitable for external use, so that a direct contact with the skin results. The topical dosage forms embrace gels, creams, lotions, salves, powders, aerosols and other conventional forms which are suitable for the direct application of products on the skin or mucous membrane.

These dosage forms can be manufactured by mixing the above compounds with known carrier materials which are suitable for topical use.

The dosage of the combination of the present invention can be selected with reference to the effects to be treated 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 a ginsenoside extract according to the invention in an amount of more than 0.01% up to 10% by weight of the total composition, more preferably between 0.1% and 1%.

Furthermore the composition according to the invention preferably comprises G-RbI in an amount of from 0.001 to 6%, G-RgI in an amount of from 0.001 to 6%, G-Rd in an amount of from 0 to 1.5%, N-Rl in an amount of from 0 to 1.5 % and/or G-Re in an amount of from 0 to 1 % by weight of the total composition.

The composition according to the invention comprises the pantothenic acid and/or its derivatives in an amount varying between 0,1 and 10% of its total weight and, preferably, in an amount varying between 2 and 5% of the total weight of the composition.

Topical dosage forms provided by the invention generally contain 0.1 to 10 weight percent of active compound, based on the total weight of the dosage form. However, higher or lower concentrations can also be present depending on the dosage form which is used. The topical preparations of the present invention can be applied in an amount and under a time schedule varying with the needs of the patient.

The compositions according to the present invention are used by applying an amount of the active compounds sufficient to provide a therapeutic and/or cosmetic effect to the skin to be treated. This application can be effected in the usual manner by rubbing, spraying or by a plaster.

The topical compositions are usually applied in an amount to provide from 0.1 to 5, preferably from 1 to 3 mg, most preferably around 2 mg of active ingredient per cm² skin per application.

The 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 or orally 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.

Use:

The combination or composition according the invention can be used for wound healing, dry skin, regeneration or reconstruction of the skin, anti-aging of the skin and epithelial regeneration and development in the event of damage to the skin when a high rate of lipids and cellular renewal is needed. In particular the combination can be used f or wound healing in the case of burns, cracked nipples, ulcers and bedsores.

Furthermore the combination or composition according to the invention can be used for treating prurigineous conditions. It has been observed that the combination or composition inhibit the degranulation process of the mastocytes, thus diminishing the amount of mediators released into the extra-cellular environment.

The combination or composition according to the invention can be therefore used for all dermatological disorders which involve the degranulation process of the mastocytes, such as atopic dermatitis, psoriasis, contact eczema, skin allergies, skin inflammation due to insect bites, skin allergies, senile pruritus, etc..

The combination or composition according to the invention can be used in the dermatological field, which include cosmetically and pharmaceutically use, for the protection of the skin against deleterious effects of stress or irradiation e.g. sunlight or UV irradiation, preferably UV-A or UV- B irradiation.

Protection of the skin according to the invention include but is not limited to protection of the immune system of the skin, protection of the skin against oxidative or other stress, protection against inflammatory skin diseases, protection against apoptosis, protection against senescence, protection against hyperproliferation of skin cells and protection against imperfectly controlled respiration in mitochondriae.

Furthermore the combination or composition according to the invention can be used for the protection of Langerhans cells, inducing an increase of HO-I m-RNA expression in human skin fibroblasts, increasing the endogenous synthesis of heme oxygenase, increasing the endogenous production of carbon monoxide and bilirubin, preventing and/or limiting endogenous reactive oxygen species and/or eliminating reactive oxygen species from cells, preventing and/or limiting the photoimmunodepression in the skin e.g. caused by UV light exposure, preventing and/or limiting cellular apoptosis, senescence or loss of functionality of the cells, and/or for the treatment or prevention of diseases or conditions affected thereby.

It has been now surprisingly found that the pharmaceutically or cosmetically effect is strongly increased by the combination. A synergic effect by the combination in comparison with the single compounds is therefore observed. The synergic effect obtained by mixing the above compounds together lead to several important advantages. For the same effect, it is possible to reduce the concentrations of the active compounds, thus decreasing the risk of intolerance for the patients. On the other hand when using standard amounts of active ingredients the achieved synergistic effect of the combination is higher then the additional effect of the single active ingredients.

A lower concentration of the active compounds lead also to lower manufacture costs and, therefore, to lower selling prices of the compositions. With the same therapeutic effect, the compositions according to the present invention are therefore more tolerable and more competitive than the conventional ones.

Examples

Example 1 :

Roots or rhizomes of Panax notoginseng are extracted with ethanol. The fraction containing the ginsenosides is isolated by column-chromatography before spray-drying.

The corresponding product is in powder form and contains more than 90% of ginsenosides. The purity can be controlled by HPLC and shows extracts which can contain 10 - 60% of G-RbI , 10 - 60% of G-RgI , 0 - 15% of G-Rd, 0 - 15 % of N-Rl and 0 - 10% G-Re by weight of the total extract.

Example 2:

The activity of the Panax notoginseng extract (according to Example 1) is evaluated in an ex vivo human skin model against UV-induced Langerhanscell toxicity.

Skin punches are taken from abdominal skin biopsy and cultured in a medium composed by DMEM (Dulbecco's Modified Eagle's Medium), Glutamine, penicillin-streptomycin and fetal calf serum. The Panax notoginseng extract diluted in DMSO is added in the culture medium twice, 24h and one hour before UV-irradiation. Two other biopsies series without any treatment and with or without irradiation are prepared as controls with or without UV.

Langerhans cells are then specifically labelled by AntiCD Ia-FITC antibodies. Skin biopsies are frozen and sliced off. Then the sections are observed in epifluorescence in order to count the fluorescent Langerhanscells.

The percentage of protection is calculated compared to control without UV according to the following formula:

₌ (Control _+υv - Treated ^y ) x 100 Control__υv

In the control + UV the number of Langerhans cells located in the epidermis decreases by 95% and are labelled. A 24 h prior incubation with 0.04, 0.2 and 1 mg/ml of a Panax notoginseng root ginsenoside fraction prevents 32%, 55% and 63 % of the decrease of Langerhans cells.

As the Panax notoginseng extract (according to Example 1) does not absorb UV A or UVB, the tested activity on Langerhanscells is indeed linked to biological activity. Example 3 : Heme-Oxygenase activation

Activation of Heme-Oxygenase 1 by Panax notoginseng extract (according to Example 1), dexpanthenol and the combination thereof is determined by PCR (Polymerase Chains Reaction) amplification. Fibroblasts are cultured in an assay medium containing or not the test compound/combination for e.g. 4 h, 8 h or 24 h. At the end of each incubation time, the cells are washed in PBS buffer, placed in Tri-reagent and frozen with G3PDH as reference.

The PCR is performed in triplicate using the LightCycler® system. The incorporation of fluorescence in amplified DNA is measured continuously during the PCR cycles. The 'fluorescence intensity' versus 'PCR-cycle' plot allows the evaluation of a relative expression value for each marker.

Results:

The expression of HO-I m-RNA in the cells after 4 h of contact with the P.Notoginseng extract is significantly increased to the base level.

Furthermore the results show a synergistic effect of the combination.

Example 4: Inhibition of the metal loproteinases

The purpose of this study is to assess the modulating effects of Panax notoginseng extract, dexpanthenol and the combination thereof on the production of matrix metalloproteinases (MMPs) in an in vitro model (equivalent to dermis). Study is performed on human fibroblasts cultured in a three dimensional collagen lattices (dermal equivalent).

The normal human dermal fibroblasts monolayers (line F04.3H5/8) established using the explant method from foreskins is obtained from plastic surgery. A cell suspension is diluted to the desired concentration. 3D collagen gels are prepared by mixing concentrated DMEM (Dulbecco's Modified Eagle's Medium) medium (1.76x), type I collagen solution (extracted from rat tail tendon in acetic acid) and cell suspension. This solution is poured into 24-well plates (ImI/ well). Polymerization of the gels occurred at 37°C within 30 minutes and gels contracted over 24 to 72h to form discs grossly less than 50% the original size. Incubation is continued at 37°C under humidified 5% CO2 atmosphere. After the 3D gel formation, the cells are first treated with the test compound/combination for 24 hours (preventive treatment). In a second step, MMPs production is induced by adding TNFα at 1 Ong/ml in culture medium. After 24hrs incubation, the cell viability and MMPs concentrations in supernatants of control and treated fibroblast cell cultures are measured. Concentration of MMP are measured by ELISA method.

Furthermore the results show a synergistic effect of the combination.

Example 5: Activation of the extra-cellular matrix compounds

Biopsies from abdominal plastic surgery are used in this ex vivo experiment. They are cultured in a specific survival explants medium: BEM (BIO-ECs Explants Medium).

2 mg of a formulation containing Panax notonginseng (according to Example 1), dexpanthenol or combination thereof is applied versus a control formulation (only excipient without active compounds) at the following days: DO, D2, D4, D6 and D8.

Histological studies are performed at the following after 0 h, 6 days and 10 days.

After dehydration and paraffin impregnation, explants are fixed with Bouin's solution. They are then cut and stained by Masson's trichome stain.

Specific immunomarking are performed on frozen cryostat cut tissues for collagen, hyaluronic acid and elastin and enables to visualize the stimulation of this compounds in comparison of the excipient.

Furthermore the results show a synergistic effect of the combination.

Example 6:

The evaluation of the protective effect of the Panax notoginseng extract (according to Example 1 ) against stress-induced premature senescence is tested in vitro on cultured human diploid fibroblasts. Stress-induced premature senescence can be defined as the long term effects of subtoxic stress on proliferative cell types and can be represented in vitro by H₂O₂- induction. The senescence-associated β-galactosidase is regarded as the biomarker of senescent, non-proliferative cells.

In the present study the fibroblasts treatment happens in 3 phases. In phase 1 fibroblasts are treated for 24 h with the culture medium containing the Panax notoginseng extract at three different concentrations of 50, 150 or 300 μg/ml (pre-treatment phase). Thereafter in phase 2 the medium is changed by a medium containing H₂O₂ (200 μM) for the stress induction. After 2 h the medium is changed again by the initial culture medium having again the three different concentrations of 50, 150 or 300 μg/ml (phase 3, recovery period). After 72 h recovery time the senescence-associated β-galactosidase is determined at pH 6 by colorimetric and fluorimetric tests.

According to the results exposure of fibroblasts to sub-toxic H₂O₂ dose leads to an increase of the amount of β-galactosidase positive cells from 17.6% to 43.5%. A concentration of 150 or 300 μg/ml in the culture medium 24 h before induction and after the period of recovery after the stress significantly decreases the percentage of stress-induced β-galactosidase positive cells, respectively to 37.5% and 33.2%.

Example 7: Cream

Claims

What is claimed is:

1. Combination comprising at least one ginsenoside and pantothenic acid and/or a derivative of pantothenic acid.

2. Combination of claim 1 wherein the ginsenoside is selected from the group consisting of ginsenoside G-RaI , G-Ra2, G-Ra3, G-RbI, G-Rb2, G-Rb3, G-Rc, G-Rd, G-Re, G-Rf, G-

RgI , G-Rg2, G-Rg3, G-RhI , G-Rh2, G-RsI, G-Rs2, G-Ro, MG-RbI , MG-Rb2, MG-Rc, MG-Rd, Q-Rl , N-Rl, N-R4, 20 GIc-Rf or a mixture thereof.

3. Combination of any of claims 1 to 2 comprising an extract of Panax notoginseng (San Qi), Panax ginseng (C.A.Meyer) and/or Panax quinquefolium.

4. Combination of claim 3 wherein the extract contains ginsenosides in an amount between 0.1% and 100% by weight of the total extract

5. Combination of any of claims 1 to 4 comprising G-RbI in an amount of from 10 to 60%, G-RgI in an amount of from 10 to 60%, G-Rd in an amount of from 0 to 15%, N-Rl in an amount of from 0 to 15 % and/or G-Re in an amount of from 0 to 10% by weight of the total extract.

6. Combination of any of claims 1 to 5 wherein the derivative of pantothenic acid is selected from alcohols, aldehydes, alcohol esters, salts and acid esters.

7. Combination of any of claims 1 to 6 wherein the derivative of pantothenic acid is dexpanthenol.

8. Combination of any of claims 1 to 7 comprising a further active substance.

9. Combination of claim 8 comprising glycine.

10. Combination of any of claims 1 to 9 which is a fixed combination.

1 1. Composition comprising a combination of any of claims 1 to 10 and one or more suitable additive or pharmaceutically, dermatological and/or cosmetically acceptable carrier.

12. Composition of claim 1 1 which is a dermatological or 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.

13. Composition of any of claims 11 to 12 comprising G-RbI in an amount of from 0.001 to 6%, G-RgI in an amount of from 0.001 to 6%, G-Rd in an amount of from 0 to 1.5%, N- Rl in an amount of from 0 to 1.5 % and/or G-Re in an amount of from 0 to 1 % by weight of the total composition.

14. Composition of any of claims 1 1 to 13 comprising the pantothenic acid and/or its derivatives in an amount varying between 0,1 and 10% of its total weight of the composition.

15. Composition of any of claims 11 to 14 which is a topical or oral composition.

16. Use of a combination of any of claims 1 to 10 for the manufacture of a composition for wound healing, dry skin, regeneration or reconstruction of the skin, anti-aging of the skin and epithelial regeneration and development in the event of damage to the skin when a high rate of lipids and cellular renewal is needed.

17. Use of a combination of any of claims 1 to 10 for the manufacture of a composition for dermatological disorders which involve the degranulation process of the mastocytes.

18. Use of claim 17 which is atopic dermatitis, psoriasis, contact eczema, skin allergies, skin inflammation due to insect bites, skin allergies or senile pruritus.

19. Use of a combination of any of claims 1 to 10 for the manufacture of a composition for the protection of the skin against deleterious effects of stress or irradiation.

20. Use of claim 19 wherein the irradiation is sunlight, UV-A or UV-B radiation.

21. Use of a combination of any of claims 1 to 10 for the manufacture of a composition for the protection of Langerhans cells, inducing an increase of HO-I m-RNA expression in human skin fibroblasts, increasing the endogenous synthesis of heme oxygenase, increasing the endogenous production of carbon monoxide and bilirubin, preventing and/or limiting endogenous reactive oxygen species and/or eliminating reactive oxygen species from cells, preventing and/or limiting the photoimmunodepression in the skin caused by UV light exposure, preventing and/or limiting cellular apoptosis, senescence or loss of functionality of the cells, and/or for the treatment or prevention of diseases or conditions affected thereby.

22. Use of a combination of any of claims 1 to 10 for the manufacture of a composition for protection of the immune system of the skin, protection of the skin against oxidative stress, protection against inflammatory skin diseases, protection against apoptosis, protection against senescence, protection against hyperproliferation of skin cells and/or protection against imperfectly controlled respiration in mitochondriae.

23. A method of treating wound healing, dry skin, regeneration or reconstruction of the skin, anti-aging of the skin and epithelial regeneration and development in the event of damage to the skin when a high rate of lipids and cellular renewal is needed comprising administering effective amounts of a combination of claims 1 to 10.