WO2010045969A1 - New synergic association for the treatment of deep skin or mucosa injuries - Google Patents

Abstract

The invention concerns a synergistic composition comprising at least two compounds chosen in the group comprising: - an extract of Alchemilla vulgaris; - an extract of Mimosa tenuiflora; and - an extract of Echinacea purpurea. It also concerns a medicament comprising said synergistic composition for the treatment of skin or mucosa injuries, particularly deep skin or mucosa injuries, and a method for promoting the growth of fibroblast and epithelial cells, comprising the step of administering to a subject said synergistic composition.

Description

NEW SYNERGISTIC ASSOCIATION FOR THE TREATMENT OF DEEP

SKIN OR MUCOSA INJURIES

The present invention concerns a synergistic composition comprising at least two particular plants extracts, said association of plant extracts having synergistic effect on the growth of fibroblast and epithelial cells. It also concerns a medicament comprising said synergistic composition for the treatment of skin or mucosa injuries, particularly deep skin or mucosa injuries, notably of traumatic or infectious origin. The skin is the outermost part of our body and is therefore exposed to all types of physical

(injuries for example) and physiological (excessive pressure, continuous heal, for example) aggressions. Due to these reasons, the occurrence of skin injuries is the most common event in the life of a human being.

The skin is made up of three parts. The epidermis containing dead and dying cells (keratinocytes) and acts as the outermost; the dermis located below the epidermis containing nourishing matrix rich in water and glycoproteins, the fundamental substance. The dermis hydrates and supply nutrients to the deeper cell layer which generates the skin and called as the basal cell layer. The basal cell layer contains epithelial type of cells. These cells grow very fast and continue to migrate upwards to constitute the epidermis. During migration, these cell loose slowly and slowly their substance and finally transform into keratinocytes. The keratinocytes loose the nuclear substance and constitute the outermost corneal dead cell layer.

The hypoderm is found below the dermis and is rich in fat containing cells, and in fibroblasts which synthesise fundamental substance but also the elastin and collagen fibres which offer resistance and elasticity to the skin. Below the hypoderm, depending upon the type of organ, we often find fibroblast cells as muscles and occasionally the bone, ligaments or tendons.

Skin injuries can be considered superficial when injury reaches only up to the level of hypodermis and deep when the structures such as the muscles, situated below the skin are also injured. The superficial skin lesions heal very rapidly through the growth of the cells located in the basal cell layer. In the case of superficial injury, the liberation and the drying of cellular and intercellular liquid help to cover the lesion and protect it from external aggressions. Broken blood vessels liberate platelets which adhere to the injured surface to prepare the blood clot which reduced blood loss. These platelets under inflammatory conditions liberate a cytokine, the platelet derived growth factor (PDGF) which promotes the growth of superficial epithelial cells and eventually the deeply situated fibroblast cells to close the injury.

In addition to the PDGF, platelets also release several other growth factors such as platelet derived epidermal growth factor (PDEGF), transforming growth factor alpha and beta (TGF), epidermal growth factor (EGF) and Insulin-like growth factor - 1 (IGF-I). These growth factors attract macrophages and neutrophils to clean the injury thereby preparing a favourable ground for cell growth and subsequent healing.

Fibroblasts are the major cells for the production of tropocollagen, collagen, elastin, and proteoglycans that makes up the intercellular matrix. In this way fibroblast cells prepare a ground for their own future growth. During healing, the fibroblast cells located in the deeper part start growing, and reach up to the level of dermis. At this stage, the fibroblasts secrete EGF and keratinocyte growth factor (KGF) which sparks the growth of skin epithelial cells to close the injury. The primary reduction in the depth of the injury is due to the growth of deeper fibroblast cells followed by the growth of superficial epithelial cells to complete the healing process.

But this ideal healing process is not followed very often, particularly in cases of deep injuries because such injuries are often infected. Certain pathological conditions also delay the healing process such as some hereditary diseases, diseases involving disturbances of microcirculation, old age, prolonged immobility, diabetes, corticosteron or cancer treatment, presence of antiseptics or antibiotics in the injury, viral infections such as Herpes type I and II, drying of the injured surface, and the pressure applied during cleaning the injuries.

Deep injuries such as bedsore, pressure ulcers or diabetic ulcers are known to occur in physically dull or old persons and are extremely difficult to heal. The death rate in bedsore patients is estimated to be above 40%. Deep injuries are painful and require regular analgesic treatment. Due to microbial growth and the presence of dead and dying cells, deep the injuries require regular dressing which is expensive, time consuming and painful for the patient. This is the reason why daily dressings and patient care constitute above 90% cost of deep injury treatment and this is one of the most time consuming factor in the hospitals. The average period of hospitalisation for a bedsore or for a diabetic ulcer patient in the

European hospitals is estimated to be 10-12 months and each patient costs on an average 60000 Euro per year (1). This is comprehensible as according to the inventors the healing of deep injuries depends upon the growth of fibroblast and epithelial cells and up to today there is no single product in the market, which is directed to stimulate the growth of these two cell types.

According to the inventors, the healing of deep mucosa or skin injuries require initially the growth of deeply situated fibroblast cells to reduce the depth of the injury followed by the growth of epithelial cells located in the upper part of the injury. According to the inventors, this process is essential to heal the injury rapidly without excessive formation of the scar tissue.

Deep injuries are often infected with micro-organisms which delay healing. To reduce infection and pain, often chemical agents such as analgesics and antiseptics or antibiotics are used which create a toxic environment into the injury for cell growth and repair. Often the cells start growing locally only in those areas where the concentration of these cytotoxic substances is less which leads to the formation of localised cell growth, formation of buds, pain due to the drying of buds and their subsequent debridement which again retards healing. This process continues over a very long period of time and requires extensive human efforts to obtain healing with above 40% death during the period of treatment. According to the inventors, the only way to accelerate deep injury healing is to promote the growth of fibroblast cells situated in the deeper part of the injury followed by the epithelial cells located at the level of skin. The contaminating agents, such as bacteria or viruses present in the injury should also be removed. Unfortunately, currently there is no treatment available which takes into consideration these two parameters simultaneously.

A variety of treatments are available in the market such as

- the antiseptics and antibiotics to check infection but due to cytotoxicity they interfere with the cell growth and promote development of buds in areas where the concentration of these chemicals is less requiring regular debridement; - the alginate preparations to absorb exudates but they have no effect on cell growth and are difficult to clean;

- hydrocolloid and polymer membranes to protect the injury and to keep the injury humid but they have no effect on the cell growth;

- collagen membranes to facilitate autolytic debridement of localised excessive cell growth common in deep injuries but they retard healing;

- enzymatic debriders to remove tissue outgrowth but they simultaneously inhibit fibroblast and epithelial cell growth, - exudates absorbers to rehydrate and to fill the dead space without any effect on the healing process,

- foams and gauzes to clean the injuries, and many other products,

- antiviral treatments to reduce viral load such as Acycloguanosine and other virus neutralising agents with adverse effects on the cell growth.

But the therapeutic base of all the above mentioned treatments is to clean the injury or to keep it hydrated thereby reducing pain but none of these treatments are directed to stimulate the growth of epithelial and fibroblast cells which is the basic requirement for a rapid injury healing

(2). Many trials have also been conducted with tissue engineered skin substitutes such as cultured epidermal autografts, processed human cadaver skin, autologous fibroblast cells grown in tissue cultures, direct treatment with EGF, KGF, PDGF, VEGF (vascular endothelial growth factor), TGF (transforming growth factor) and topical or systemic antiviral agents but none of these products were found to be significantly effective, they require sophisticated technology, they are expensive and are not suitable for daily treatment. Recent scientific publications consider these products more as a complement to the basic treatment and not as a replacement product (3).

In the absence of any effective treatment, many plant extracts such as the extracts of aloe, A. vulgaris, Centella asiatica, Calundella officinalis, Vitis vinifera, Mimosa, tenuiflora, Echinacea sp, polyphenols, polyphenols, acacia gum, curcuma, honey, saesame oil, and many other plant products have been suggested to treat injuries, either alone or in associations, but the absence of widespread use of these plants suggest that none of these plant preparations have yet shown any real effect on deep injury healing.

The absence of real efficacy of plant extracts is probably due to the fact that most of the plants have an antiseptic or antimicrobial activity and thereby they create a favourable ground for healing but none of these plant extracts which are employed for injury treatment possess dual properties of promoting the growth of fibroblast as well as of epithelial cells. It is also important to recollect that there are nearly no comparative scientific studies on the cell growth properties of different plants, using different parts of the same plant and employing different type of extraction methods.

M. tenuiflora (tepezcohuite) bark is traditionally used to treat various skin infections, injuries, as an analgesic, antiseptic or antimicrobial agent. The bark contains polyphenols, particularly the Oligomeric Proanthocyanidins (OPCs), saponins, an alkaloide fraction, lipids, phytosterols, glucosides, xylose, rhamnose, arabinose, lupeol, methoxychalcones, and kukulkanins (5).

The plant extract of A. vulgaris also contains many types of polyphenols and numerous other phyto-chemical moleciles and therefore it is extremely difficult to identify the ingredients which may affect cell growth.

Echinaceae sp. whole plant extracts are traditionally used as immunostimulant (6), antiinflammatory, anti- viral, or as anti-tumour agent (7) but the cell growth promoting properties of Echinaceae were never described. Meckes-Lozova et al. (8) studied pharmacological properties in vitro of various extracts of

M. tenuiflora and concluded that the plant extract possesses antibacterial activity probably due to the presence of polyphenols, while alkaloids fraction, abundant in the butanol extract strongly inhibited the intestinal peristalsis and produced contraction of uterine and gastric strips of rat and guinea pig. Saponins were detected in butanol and methanol extracts producing hemolysis (red blood cell death). The screenings performed showed the diversity of bioactive compounds present in these plant extracts.

No clinical trials are cited in the literature to evaluate the skin injury healing properties of the associations of M. tenuiflora, A vulgaris and E. purpurea or two of these plants.

Rivera-Arce E et al (9) conducted a randomized, double-blind, placebo-controlled clinical trial to evaluate therapeutic effectiveness of a M. tenuiflora cortex extract alone in venous leg ulceration treatment with ambulatory patients distributed into two groups (10 patients). One group received a hydrogel containing 5% of a crude extract standardized in its polyphenol concentration (1.8%), while the control group, was administered the same hydrogel but without addition of the extract. In both aseptic washings were performed initially followed by topical application of the corresponding hydrogel and dressing. Follow-up lasted 13 weeks and ulcer healing was determined through measurement of the lesion area by digital-photographic parameters. The results of this study show that the application of the extract of M. tenuiflora probably helps to reduce the healing period to 8 instead of 10-12 weeks, but the injury healing duration still remained too long. This is probably due to the fact that the local application of M. tenuiflora extract helps only the growth of fibroblast cells without affecting the growth of epithelial cells. Anton R et al. (10) evaluated different pharmacological properties of bark extracts of M. tenuiflora in vitro and found a clear inhibition growth effect in all the gram positive and gram negative organisms, yeasts and dermatophytes.

In vitro cell proliferation studies published in the literature argue that the effect of M. tenuiflora on cell growth depends upon the type of cells used. In some studies, a cell growth inhibition effect was observed while in some studies a cell proliferation effect was noticed. Villarreal et al. (11) found that the ethylacetate and butanol extracts produced growth inhibition on WB 8 normal embryonic fibroblasts and KB cells while the petroleum ether extract markedly increased proliferation of the same cells in vitro. Yulin Jiang et al. (12) studied the saponosides: mimonosides A, B and C, isolated from the bark of M. tenuiflora for their influence on cell growth on a panel of lymphoma cells from human and murine origins and on murine lymphocytes in vitro. The three saponins did not exhibit any growth influence on two tumour cell lines (Molt 4 and RDM 4). However, they exhibit an important increase of incorporation of tritiated thymidine into DNA of cultured lymphocytes in vitro. Synergic effects of the saponins with concanavaline A (Con A) and lipopolysaccharide (LPS) on the lymphocytes were observed.

These results indicate that the M. tenuiflora extract can have a cell growth or cell inhibition properties depending upon the presence of other ingredients in the composition used for the studies. The dried bark powder or the bark extract of M. tenuiflora is traditionally used in Mexican medicine as a treatment of injuries and burns. Standardised extract of M. tenuiflora bark was also suggested as a standardised phytomedication for the treatment of ulceration in the inferior extremities (Patent N° WO/2007/072233; Ref .13).

With respect to the extract of A. vulgaris, the inventors were the first to observe that the extract of this plant helps to accelerate epithelial cell growth and the healing of superficial injuries in rats (14). Initially the inventors observed that a standard extract of A. vulgaris in glycerine, exposed to Chang human liver, MDBK and vascular smooth muscle cell (VSMC) cultures in vitro, increase cell number in a concentration dependent fashion between 0.1 to 1 % concentrations with 21.3 and 10.6 % increases in MDBK and Chang liver cells, respectively compared to corresponding control cultures. These cell proliferating and putative healing properties of A. vulgaris (once a day topical application for 7 days) were then confirmed on superficial dorsal skin lesions in adult male rats. A. vulgaris (3 %) treated lesions were significantly reduced in size by the second day of treatment and complete healing occurred a day earlier than in controls. These initial results were confirmed in a pilot clinical trial (15) by applying a product containing 3% A. vulgaris in glycerine on 48 healthy volunteers suffering from superficial common minor oral ulcers. It was observed that the topical application of this gel to minor mouth ulcers twice a day relieved discomfort and produced complete healing in the majority of patients (60.5 %) within 2 days and in 75 % within 3 days, compared to 10.4 and 33.3 % respectively without treatment and 15 and 40 % with commonly available treatments.

Experiments conducted in our laboratory showed that although the extract of A. vulgaris was effective in healing of superficial skin or mouth ulcers, it was not very active for the treatment of deep ulcers or scars (unpublished data). This is because this product contained only the extract of A. vulgaris which promotes the growth of superficial epithelial cells but not the deeper fibroblast cells.

An international application filed on the basis of initial research results by the inventors (Shrivastava et al. US 6964783; PCT WO 00/74668 Al (16)) concerns a composition, in particular a non-solid pharmaceutical composition for local application comprising, as active principle, at least glycerol or a concentrated solution of glycerol, saccharose, sorbitol or mannitol, the active principle concentration of said composition being osmotically active towards plasma. The osmotic activity of glycerol is considered responsible for the healing effects. It was possible to associate different plant extracts in the said composition such as the extracts of Quercus, Asculus hippocastanum, Geranium robertianum, Cupressus sempervirens, Vitis vinifera, Ribus nigrum, M. tenuiflora, A. vulgaris, Matricaria chamomilla or Centilla asiatica, or any other antiseptic or antibiotic like substance, so as to increase product efficacy. In this patent the inventors have not mentioned about the synergistic activity of plant extracts to obtain specifically the growth of fibroblasts and epithelial cells. Therefore, such preparations may be good for the treatment of superficial injuries contaminated with micro-organisms and localised superficially at the level of the skin but are not suitable for the treatment of deep injuries with wide skin opening and involving muscle cell injury.

A Mexican patent by Lozoya Legorreta et al. (17) concerning a process for preparing a powder with a high injury- healing capacity from the bark of M. tenuiflora explains a method of preparing active ingredient rich bark extract where the bark and wood is chemically and manually washed, then roasted to separate the curative active ingredients from the cells containing the active ingredients, and then ground into the powder of the invention. The ground powder is then sterilized. The powder is used for application on burns, injuries, lesions and other skin injuries. This patent explains a method of preparation of a standardised extract of Mimosa but do not mention the synergistic fibroblast and epithelial cell growth promoting properties of M tenuiflora with A. vulgaris extracts.

An another patent by Anderson Jon et al. (18) concerns enriching Mimosa pudica extract in flavonoid or flavone aglycone for topical cosmetic and pharmaceutical use to enhance collagen levels in skin cells due to the presence of phenolic compound. This discovery is related to a method of preparing a plant extract rich in phenolic compounds and not to any injury healing effects of synergistic plant extracts.

Among all the existing plant extracts or plant ingredients, traditionally used to treat skin injuries or skin microbial or viral infections, none of the plant extracts have shown the three basic properties which are essential according to the inventors for rapid wound healing i.e. to promote the growth of fibroblast cells and simultaneously the growth of epithelial cells and to exert an anti- viral or anti-bacterial effect, essential for rapid healing of deep injuries.

According to the inventors, an ideal treatment for deep injury healing should have the following properties: - It should promote the growth of fibroblast cells. Fibroblast cells are located in the deeper parts of the injury and promoting the growth of these cells is essential to reduce the depth of the injury.

- It should simultaneously promote the growth of superficial epithelial cells to reduce the width of the injury. If the width of the injury is not reduced and only deeper fibroblast cell growth is promoted, a shallow but open injury will persist with a depressed skin surface. On the contrary, if only the epithelial cell growth is enhanced, the injury will close at the top and a fistula, often containing pus, necrotic tissue and infection will remain below the skin. Therefore, an ideal treatment must promote the growth of fibroblast as well as of epithelial cells simultaneously. Unfortunately, up till today, there is no treatment having these dual properties. - In addition, an ideal product should be easy to apply, should be colourless so as to avoid daily injury cleaning, should be capable to adhere to the injury surface, should neutralise infection whether of bacterial or viral origin, should be of natural origin if possible so as to avoid cytotoxicity, should not be irritant and should not contain alcohol or chemicals which interfere with the cell growth. Its is thus interesting to develop a product having dual properties to promote the growth of fibroblast as well as the growth of epithelial cells, for the treatment of skin or mucosa injuries, and particularly of deep skin or mucosa injuries, which is non cytotoxic, non irritant, not expensive, suitable for daily treatment, which enables avoiding infection whether of bacterial or viral origin of the injury without being toxic and which does not contain compounds which interfere with the cell growth.

One of the objectives of the present invention is to provide a synergistic composition capable to promote the growth of fibroblast cells situated at the bottom of the injury and the epithelial cells located in the upper part of the injury, for the treatment of deep skin injuries.

Another objective of the present invention is to provide a synergistic composition for the treatment of bedsore, pressure ulcers and diabetic ulcers as well as the skin injuries involving improper functioning of cutaneous or the subcutaneous tissues and injuries of viral origin.

Another objective of the present invention is to provide a safe treatment for deep skin or mucosa injuries,

Another objective of the present invention is to provide a topical treatment which is transparent and do not require repeated injury cleaning.

Another objective of the present invention is to provide a synergistic composition, particularly a synergistic association of two plant extracts in a defined excipient as a medicament for deep skin or mucosa injuries.

Another objective of the present invention is to provide an efficient and low cost composition, for the treatment of injuries on human and animal bodies requiring repair of damaged skin fibroblast and epithelial cells.

The inventors have solved in whole or part the problems mentioned above. During in vitro research the inventors observed that only a very few plant extracts (3 over

198 tested plant extracts) promote the growth of epithelial and/or fibroblast cells. An extract of A. vulgaris promotes preferably the growth of epithelial cells, an extract of M. tenuiflora preferentially promotes the growth of fibroblast cells, and that an extract of E. purpurea promotes both the growth of epithelial cells and of fibroblast cells. Surprisingly, the inventors have shown that the association of at least two of said plant extracts (Alchemilla vulgaris extract, Mimosa tenuiflora extract and Echinacea purpurea extract), and particularly 50% of the most active concentrations of said extracts, do not produce an additive but an extraordinary synergistic effect on the growth of both the fibroblast as well as the epithelial cells, without any cytotoxicity. The association of at least two of said plant extracts {Alchemilla vulgaris extract, Mimosa tenuiflora extract and Echinacea purpurea extract) to obtain a synergistic effect on the growth of epithelial and fibroblast cells is not know or obvious in view of the above-mentioned documents. This synergistic effect is extremely useful to treat skin or mucosa injuries, particularly deep skin or mucosa injuries where healing rate is proportional to the growth of fibroblast and epithelial cells.

The present invention relates to a synergistic composition comprising at least two compounds chosen in the group comprising: - an extract of Alchemilla vulgaris;

- an extract of Mimosa tenuiflora; and

- an extract of Echinacea purpurea.

The synergistic composition according to the present invention has the advantage to be safe given its absence of toxicity and natural origin and can therefore be applied on the injuries without the risk of inducing local cytotoxicity. Due to synergistic properties of the at least two plant extracts, the quantity of each product applied can be reduced by 50%. Moreover, being a natural and safe synergistic association, such a product can also be used as a coadjuvant of traditional treatments such as with the use of compression bandage, hydrogels, polymer films, antiseptics and antibiotics, if required. Said extracts of Alchemilla vulgaris (A. vulgaris) , of Mimosa tenuiflora (M. tenuiflora) and of Echinacea purpurea (E. purpurea) can be prepared using traditional extraction techniques, well known by one skilled in the art.

Solvents used in the extraction step according to the invention, can be, water, alcohol, glycerol, ethylene glycol, preferably glycerol. Preferably, said extracts are hydro glycerinated extracts or glycerinated extracts.

In one embodiment of the synergistic composition according to the invention, at least one of said extract is a glycerinated or a hydroglycerinated extract.

The synergistic composition according to the invention can also comprise glycerol. The synergistic composition can comprise between 50 % and 99 %, preferably between 60 % and 95 % by weight of glycerol relative to the total weight of the synergistic composition.

The term "glycerol" is intended to mean glycerin, which corresponds to the formula C₃H₈O₃ or propane- 1,2,3-triol or 1,2,3-propanetriol (CAS Number 8013-25-0)

The glycerol (glycerin) extraction has the advantage of optimizing the epithelial and fibroblast cell growth-promoting properties of the synergistic composition according to the invention as glycerol molecules have a complex structure and are bigger in size, therefore, they can not cross the cell membrane and can not be toxic to the cells. Use of glycerol has another advantage of being colorless which do not color the injury facilitating examination of deep injuries and glycerol is known to be a safe and non toxic product used in food preparations.

Pharmacological studies conducted in the inventor's laboratory show that glycerol alone accelerates slightly the healing of superficial injuries but has very little or no effect to accelerate the healing of deep and large injuries.

Before extraction, the plant can be crushed, or cut into pieces and can be fresh, frozen, dried or lyophylized.

It is possible to carry out several successive extractions until complete exhaustion of the active ingredient by the solvant used. The time of extraction varies according to solvent, the temperature and possibly the pressure used for extraction. In practice, this time can be limited to less than two hours for a profitable industrial exploitation.

Any part of the plant can be used to prepare the plant extracts according to the invention.

Preferably, the extract of Alchemilla vulgaris is an extract of at least one aerial part, and more preferably a whole plant extract of Alchemilla vulgaris, the extract of Mimosa tenuiflora is a bark extract of Mimosa tenuiflora and the extract of Echinacea purpurea is a whole plant extract of Echinacea purpurea.

A whole plant extract of Alchemilla vulgaris, a bark extract of Mimosa tenuiflora and a whole plant extract of Echinacea purpurea have the advantage to optimize the fibroblast and epithelial cells growth-promoting properties. The term "aerial part of said plant", is intended to mean any part of the plant which is not underground. Aerial part of a plant, comprise leaves, fruits, flowers, stems, seeds, preferably the leaves and branches.

The term "whole plant extract" is intended to mean an extract of the whole aerial parts of a plant. Particularly, said extracts of Alchemilla vulgaris, Mimosa tenuiflora and Echinacea purpurea can be obtained by at least one extraction step chosen in the group comprising extraction with solvent using techniques such as solid-phase, supercritical CO2 or fluid, pressurised-liquid extraction, microwave-assisted extraction, surfactant-mediated extraction and a combination of those, preferably extraction with solvents and all preferentially glycerol (glycerin) extraction.

These extracts can be prepared using different methods as described in the review article written by Carmen W Huie (19). Some of said plant extracts are sold, for example, by Quimdis company in France (Z.I. rue Anatole France, 92532 Lavallois Perret cedex) under the reference Echinacea PE E080715, NTL 4% polyphenol.

Particularly, the extract of Alchemilla vulgaris according to the invention can comprise between 1 to 20 %, preferably between 2 to 12 % and more preferably between 3 to 8 % by weight of polyphenols relative to the total weight of the extract, expressed in dry extract.

Particularly, the extract of Mimosa tenuiflora according to the invention can comprise between 1 to 25 %, preferably between 2 to 14 % and more preferably between 3 to 12 % by weight of polyphenols relative to the total weight of the extract, expressed in dry extract. The polyphenols, also called as tannins, are a group of chemical substances found in some plants, characterized by the presence of more than one phenol unit. They are either hydrosoluble polyphenol (gallic acid for example) or condensed polyphenol such as proanthocyanidins, which are rich in plants.

Particularly, the extract of Echinacea purpurea according to the invention can comprise between 0,5 to 12 %, preferably between 1 to 8 % and more preferably between 2 to 7 % by weight of echinacosides relative to the total weight of the extract, expressed in dry extract.

The term "echinacosides" is intended to mean the water soluble polysaccharide fraction in the extract.

As plants and plant extracts contain hundreds of ingredients, it is extremely difficult to isolate each ingredient and to elucidate the synergistic mode of action of these plants. It is postulated that probably these plant extracts do not have any specific pharmacological effects on cellular parameters but probably they act by inhibiting the activity of certain proteolytic enzymes which are known to degrade intercellular matrix essential for a rapid cell growth. The plant extracts contain different types of polyphenols and polysaccharides which are known to precipitate big molecule. Therefore, the inventors assume that the plant polyphenols or polysaccharides could bind to proteolytic enzymes and neutralise these proteolytic enzymes. The proteolytic enzymes destroy cellular matrix on which cells attach and grow. Therefore the absence of proteolytic enzymes may enhance cell attachment and cell growth. As there are different types of proteolytic enzymes (such as collagenase, elastase, matrix metaloproteins) they require different types of specific polyphenols to be neutralised. It is therefore possible that associating these at least two specific plant extracts bring two or three different type of polyphenols or polysaccharides which neutralise different types of proteolytic enzymes, thereby stopping the degradation of intercellular matrix and producing a synergistic effect on cell growth. As the polyphenols and polysaccharides have a strong affinity for proteins, it is also likely that some polyphenols or polysaccharides bind to the bacterial or the viral proteins and thereby help to reduce infection, liberation of toxic substances of microbial origin and thereby promote the cell growth. The synergistic composition according to the invention preferably comprises between 0.1 to 20%, particularly between 0,5% to 6%, and more particularly between 2% and 4% by weight of each plant extract relative to the total weight of the synergistic composition, particularly depending upon the concentration of polyphenols or echinacosides in the extract used.

A percentage of each plant extract by weight of each plant extract relative to the total weight of the synergistic composition comprises between 2% and 4% has the advantage that it optimize the fibroblast and epithelial cell growth-promoting properties of the synergistic composition. Particularly, the final product is particularly active to promote healing while there is sufficient quantity of the base (for example an ointment, gel, oil, or glycerol) to prepare a formulation for topical application. Taking into consideration the results of pharmacological in vitro and in vivo studies and the results of the clinical observation, it is important to associate at least two plant extracts among A vulgaris, M. tenuiflora and E. purpurae.

Taking into consideration the concentration of polyphenols in A. vulgaris extract (for example 9% by weight of polyphenols relative to the total weight of the extract) and M tenuiflora (for example 11% by weight of polyphenols relative to the total weight of the extract) and the concentration of echinacosoides in E purpurae (for example 4,5% by weight of echinacosoides relative to the total weight of the extract) the active association of plant extracts should be close to a ratio of 1 :1 for a synergistic composition according to the invention containing A vulgaris and M tenuiflora; or in a ratio of 1 :3 {A vulgaris extract : E purpura extract) for a synergistic composition according to the invention containing A vulgaris and E purpura, or in a ratio of 1 :3 (M. tenuiflora extract and E. purpurea extract,) for a synergistic composition according to the invention containing M. tenuiflora and E. purpurea. When these ratios are respected, it has the advantage to optimize the fibroblast and epithelia cells growth- promoting properties of the synergistic composition according to the invention. Particularly, the amount of A vulgaris polyphenols per 100 ml of a synergistic composition according to the invention can be comprised between 50 to 200 mg, preferably between 110 to 170 mg and more preferably between 125 to 145 mg. Particularly, the amount of M. tenuillora polyphenols per 100 ml of a synergistic composition according to the invention can be comprised between 100 to 300 mg, preferably between 170 to 250 mg and more preferably between 210 to 230 mg.

Particularly, the amount of E. purpura echinacosides per 100 ml of a synergistic composition according to the invention can be comprised between 2 to 50 mg, preferably between 15 to 35 mg and more preferably between 20 to 30 mg.

The synergistic composition according to the invention can also comprise another injury healing agent, like synthetic or natural from a plant, as a combination product for a simultaneous or separate use, or for use spread out over time. Said injury healing agents can be chosen in the group comprising injury healing plants such as Piper betel, Emblica officinalis; analgesic drugs such as aspirin or paracetamol; antiinflammatory plant extracts such as Cassica angustifolia; anti- inflammatory drugs such as Ibuprofen; injury healing antiseptics and antibiotics such as Povidone Iodine, chlorhexidine; growth factors such as FGF-2 and FGF-7, EGF (fibroblast and epithelial growth factors); nucleoside inhibitors such as Acyclovir, or topical heparin sulphate receptor binding inhibitors either given orally or applied topically.

The synergistic compositions according to the invention can comprise said plant extract(s) and said injury healing agent(s) in a molar ratio between 10/1 and 1/10.

The synergistic composition can also comprise at least one anti-infectious agent as a combination product for a simultaneous or separate use, or for use spread out over time.

Said anti-infectious agent can be chosen in the group comprising antibiotics, antiseptics such as bacitracin, neomycin, and polymyxin.

The synergistic compositions according to the invention can comprise said plant extract(s) and said anti- infectious agent(s) in a molar ratio between 10/1 and 1/10. The synergistic composition according to the invention can be chosen from the group comprising food, pharmaceutical, cosmetic compositions, preferably pharmaceutical and cosmetic compositions.

These synergistic compositions can be formulated in different ways. Such formulations and their method of preparation are well known to one skill in the art. The present invention also relates to a synergistic composition according to the invention, for use as a medicament for the treatment of skin or mucosa injuries, said synergistic composition comprising at least two compounds chosen in the group comprising:

- an extract of Alchemilla vulgaris; - an extract of Mimosa tenuiflora; and

- an extract of Echinacea purpurea.

The invention also relates to the use of the synergistic composition according to the invention, for the manufacture of a medicament for the treatment of skin or mucosa injuries, said synergistic composition comprising at least two compounds chosen in the group comprising: - an extract of Alchemilla vulgaris;

- an extract of Mimosa tenuiflora; and

- an extract of Echinacea purpurea.

The term "skin" is intended to mean the outermost tissue layer of human and animal bodies comprising epidermis, dermis and hypodermis. The term "mucosa" is intended to mean the tissue layer that covers transition zone between the skin that covers our body and the soft (and moist) thin tissue that covers inner side of mouth, nose, ear, and ventral opening.

The term "injury" is intended to mean any damage or harm done to the skin or the mucus membrane in which the skin or mucus membrane is broken, cut, pierced, torn or damaged. Injury can be traumatic, non-traumatic, of infectious origin, of non infectious origin.

Particularly, skin or mucosa injuries can be deep skin or mucosa injuries.

The term "deep skin or mucosa injury" is intended to mean any skin or mucosa injury, in which the lesion extends not only to the superficial tissue structures such as the epidermis and dermis but also to at least one deeper structure such as hypodermis and the muscles or other tissues present below the skin or the mucus membrane, and which can be improved and/or treated by the growth of epithelial and/or fibroblast cells.

Deep skin or mucosa injury can include bedsore, pressure ulcers, diabetic ulcers, injuries of viral origin containing as cold sore and vaginal herpes, injuries caused due to deep burns, atopic dermatitis, psoriasis, eczyma, chronic uenous insufficiency ulcers, canker sore, vaginal herpes lesions on the mucosa, and traumatic deep injuries of accidental origin. The synergistic composition and the medicament according to the invention can be administered via different administration mode, which is adequate for the required effect, including oral, topical administration either directly on the injury or as a gel on the polymer hydrogel films, as a patch with slow diffusion, as a spray, as a film, or as a cotton gauze soaked with the synergistic composition according to the invention. Preferably, the administration can be made orally or topically and more preferably topically and particularly to an injured site to be improved and/or treated.

The plant extracts and subsequent pharmaceutical synergistic composition or medicament according to the invention for topical application can be prepared by employing any of the methods or procedures well known in the art. Their form depends upon the type of the injury to be treated and may be presented as solution, emulsion, suspension, cream, lotion, gel, aerosols, spray, viscous liquid, ointment, pomades or others. The synergistic composition and medicament according to the invention can also be used as a powder to be applied on the injuries directly by spreading or the powder can also be mixed with a suitable carrier such as the glycerol, clay, an ointment, gel or a lotion. They can also be incorporated in a polymer film, in a hydrogel, in a bandage like cotton badage, or in an antiseptic or antibiotic preparation formulated according to usual methods.

To further improve its efficiency, the preparation for local application can be associated with any other topical or oral treatment such as associated with wound healing preparation, with an antiseptic, anti-inflammatory or with an antibiotic cream or gel.

The invention also relates to a medical device comprising the synergistic composition according to the invention, said synergistic composition comprising at least two compounds chosen in the group comprising:

- an extract of Alchemilla vulgaris; - an extract of Mimosa tenuiflora; and

- an extract of Echinacea purpurea.

The term "medical device is intended" to mean any product (the association of plant extracts for example) or article that exerts its action mainly through its physical properties (for example, binding with the proteolytic enzymes or other proteins in the cavity of the injury without affecting the functions of the body cells). Medical device can include compression bandage, hydrogels, polymer films, solution, suspension, cream, gel, ointment, lotion, slow release devise, or a spray.

The present invention also relates to the non-therapeutic use of a synergistic composition according to the invention, for improving the aesthetic appearance of injured skin or mucosa. The term "aesthetic appearance" is intended to mean the cosmetic appearance.

The present invention also relates to the non-therapeutic use of a synergistic composition according to the invention for promoting the growth of fibroblast and epithelial cells.

Particularly, it relates to the non-therapeutic use of a synergistic composition according to the invention for promoting the growth of fibroblast and epithelial cells in dermis, hypodermis and below the tissues situated below the hypodermis, particularly of injured skin or mucosa.

The invention also relates to a method of treatment of skin or mucosa injuries, comprising the step of administering to a subject a synergistic composition according to the invention, said synergistic composition comprising at least two compounds chosen in the group comprising:

- an extract of Alchemilla vulgaris; - an extract of Mimosa tenuiflora; and

- an extract of Echinacea purpurea.

The terme "subject" includes human and animals.

In one embodiment of the method according to the invention, the synergistic composition according to the invention can be administered to an injured site to be treated. In one embodiment of the method according to the invention, the step of administering to a subject a synergistic composition according to the invention can be a step of topically applying to skin or mucosa, a synergistic composition according to the invention. The synergistic composition according to the invention can be topically applied to skin or mucosa, preferably to an injured site. The invention also relates to a method for promoting skin or mucosa injuries healing comprising the step of administering to an injured site to be treated a synergistic composition according to the invention.

In one embodiment of the methods according to the invention, skin or mucosa injuries are deep skin or mucosa injuries. The invention also relates to a method for promoting the growth of fibroblast and epithelial cells, comprising the step of administering to a subject a synergistic composition according to the invention, said synergistic composition comprising at least two compounds chosen in the group comprising:

- an extract of Alchemilla vulgaris;

- an extract of Mimosa tenuiflora; and - an extract of Echinacea purpurea.

Particularly, said method according to the invention is a method for promoting the growth of fibroblast and epithelial cells in dermis, hypodermis and below the tissues situated below the hypodermis, particularly of injured skin or mucosa.

In one embodiment of the method according to the invention, the synergistic composition according to the invention can be administered to an injured site to be treated.

In one embodiment of the method according to the invention, the step of administering to a subject a synergistic composition according to the invention can be a step of topically applying to skin or mucosa, a synergistic composition according to the invention. The synergistic composition according to the invention can be topically applied to skin or mucosa, preferably to an injured site.

The synergistic composition according to the invention can be applied for example at least twice a day on the injury, according to the size of the injury, for example 1 to 5 ml of the synergistic composition per day as presented in the example number 4.

The present invention also covers the method of preparation of synergistic compositions described previously characterised by mixing the plant extracts with excipients or acceptable solvents, notably pharmaceutically or cosmetically acceptable solvents, according to the established methods.

The synergistic effect of the association of at least two of said plant extracts {Alchemilla vulgaris extract, Mimosa tenuiflora extract and Echinacea purpurea extract) on the growth of epithelial and fibroblast cells is apparent from the examples. The examples illustrate the invention, but they should not be considered to limit the scope of the invention in any way. These compositions are elaborated on the basis of pharmacological studies showing that they help to promote and create a favourable ground for promoting the growth of epithelial and fibroblast cells. All pant extracts used had standardised quantities of polyphenols or echinacosides. All preparations were mixed by gentle stirring for a period of 24h before filling in tubes (10 or 30-ml), in bottles (100-ml) or in plastic ampoules (10-ml).

EXAMPLES

I. Examples of formulation of synergistic composition according to the invention

In the absence of evidence to the contrary, the quantity of compounds comprised in the different synergistic compositions is expressed in % by volume of compound relative to the total volume of the composition.

EXAMPLE 1

Water 45,0%

Xanthan gum 0,5%

Methyl parahydrobenzoate 0,15%

Glycerinated extract of A. vulgaris 2,5%

Glycerinated extract of M. tenuiflora 3,5%

Honey 15,0%

Glycerol 33,35%

EXAMPLE 2

Glycerol 94,0%

Hydroalcoholic extract of A. vulgaris 3,0%

Glycerinated extract of M. tenuiflora 3,0% EXAMPLE 3

Glycerol 64,0%

Honey 30,0%

Glycerinated extract of A. vulgaris 3,0% Glycerinated extract of M. tenuiflora 3,0%

EXAMPLE 4

Glycerol 90,5%

Xanthan gum 0,5% Glycerinated extract of A. vulgaris 4,5%

Glycerinated extract of M. tenuiflora 4,5%

EXAMPLE 5

Dry powder of A. vulgaris 3,0 g Glycerinated extract of M. tenuiflora 3,0 ml

Glycerol qsp 100,0 ml

EXAMPLE 6

Dry powder of A. vulgaris 12,0 g Dry powder of M. tenuiflora 3,0 g

Glycerol qsp 100,0 ml

EXAMPLE 7

Glycerinated extract of A. vulgaris 16,0 % Glycerinated extract of M. tenuiflora 12,0%

Honey 14,0%

Hydroalcoholic extract of C. asiatica 4,5% Cammiphora mukul gum 2,0%

Glycerol qsp 100,0 ml

EXAMPLE 8 Glycerol 96,0%

Glycerinated extract of A. vulgaris 3,0%

Glycerinated extract of E purpurea 1 ,0%

EXAMPLE 9 Glycerol 64,0%

Honey 29,0%

Glycerinated extract of M. tenuiflora 5,0%

Glycerinated extract of M. tenuiflora 2,0%

EXAMPLE 10

Glycerol 96,5%

Xanthan gum 0,5%

Glycerinated extract of A. vulgaris 1,5%

Dried powdered extract of E. purpura 1 ,5g The preparation was mixed by stirring for 2h before filling 10 ml tubes;

EXAMPLE 11

Dry powder M. Tenuiflora bark extract 1,5 g

Glycerinated extract of E. purpura 3,0 ml Glycerol qsp 100,0 ml

EXAMPLE 12 Dry powder of A. vulgaris 5,5 g

Dry powder of E. purpura 1,5 g

Glycerol qsp 100,0 ml

EXAMPLE 13

Glycerinated extract of M. tenuiflora 9,0 %

Glycerinated extract of E. purpura 4,0%

Honey 14,0%

Hydroalcoholic extract of C. asiatica 2,5% Cammiphora mukul gum 1,0%

Glycerol qsp 100,0 ml

EXAMPLE 14

Glycerinated extract of A. vulgaris 2,0 % Glycerinated extract of M. tenuiflora 1,5%

Dried powder extract of E. Purpura l,2g

Glycerol qsp 100,0 ml

EXAMPLE 15 Glycerinated extract of A. vulgaris 2,0 %

Glycerinated extract of M. tenuiflora 1,5%

Dried powder extract of E. Purpura 1 ,2g

Carbopol gel qsp 100,0 ml

EXAMPLE 16

Water extract of A. vulgaris 3,0 %

Water extract of M. tenuiflora 3,0% Honey 24,0%

Leaf extract of V. vinifera 4,0%

Glycerol qsp 100,0 ml

The preparation was mixed by gentle stirring for 24h and 10-ml of the preparation was applied on a cotton gauze. The gauze was used for application on the injuries.

EXAMPLE 17

Dry powder of the extract of A. vulgaris

(containing 24% polyphenols) 2,0 %

Dry powder of the extract of M. tenuiflora

(containing 32% polyphenols) 3,0 %

Glycerol qsp 100,0 ml

EXAMPLE 18

Glycerinated extract of A. vulgaris 3,0 %

Glycerinated extract of M. tenuiflora 2,0%

Xanthan gum 0,2%

Honey 29,0%

Glycerol qsp 100,0 ml

EXAMPLE 19

Gel for topical application

Aqua 68,91%

Glycerin 17,0%

Bark extract of Mimosa tenuiflora 3,0% Glycerinated extract of E. purpurea 2,5%

Propylene glycol 2,74%

Glyceryl Polymethacrylate 2,07% PEG-40 1,0%

Xanthan gum 0,80%

Triethanolamine 0,80% Carbomer 0,50%

Phenoxyethanol 0,36%

Fragrance 0,20%

Methylparaben 0,08%

Butylparaben 0,01% Butyl paraben 0,01%

Propylparaben 0,01%

Isobutylparaben 0,01%

EXAMPLE 20 Cream for topical application

Sorbitan sterate 5,40% Glycerinated extract of A. Vulgaris 3,0% Glycerinated extract of E. purpura 3,0%

Octyl octanoate 2,0% Saccharide isomerate 2,0%

Caprylic Capric triglyceride 1,5%

Octyldodecyl myristate 1,5%

Triethhanolamine 0,66%

Sucrose cocoate 0,60% Carbomer 0,55%

Cetryl alcohol 0,50%

Cetyl palmitate 0,50% Glyceryl sterate 0,50%

Phenoxyethanol 0,36%

Propylene glycol 0,20%

Disodium EDTA 0, 10% Methylparaben 0,08%

Ethylparaben 0,02%

Butylparaben 0,01%

Isobutyl apraben 0,01%

Propylparaben 0,01% Aqua QSP 100 ml

II. Pharmacological, toxicological and stability studies

ILl. In vitro toxicity and cell growth properties of A vulgaris or M. tenuiflora extracts alone or in association on fibroblast and epithelial cells.

The aim of this study was to determine the toxicity and the mitotic effect of the Alchemilla vulgaris (A. vulgaris) and Mimosa tenuiflora (M. tenuiflora) glycerinated extracts, either alone or in combinations of different concentrations, on vascular smooth muscle cell fibroblasts and MDBK (Madin Durby Bovine Kidney) epithelial cell line in vitro.

Initially, cells were grown in 75 cm² tissue culture flasks (Corning, USA), dispersed with trypsin (0.25%, Difco, USA) and a 100 μl of cell suspension containing 3 x 10⁵ cells per ml in

2% serum containing growth medium was put in each well of the 96 well microplate. After 48 h incubation at 37°c in a 5% CO2 - 95% air atmosphere, cells were exposed to new growth medium containing appropriate concentration of the test product.

For each test product, after initial cytotoxicity measurements, test concentrations between 0,3 to 5% were used for cell growth studies. Culture plates were then incubated at 37°c - 5% CO2 humidified environment for a period of 48h . Untreated controls correspond to cell controls were not exposed to ant test product. I After 48h culture, cell morphology was examined by phase microscopy and live cell count was determined using MTT (4, 5 - dimethylthiazol - 2 yl) - 2, 5 diphenyl tretrazolium bromide) vital stain which quantifies the mitochondrial dehydrogenase activity of the live cells. The optical density (OD) of each microplate well was measured at 560 nm using an automatic micro-well reading apparatus (Dynatech MR 400).

The average values of optical density for each experiment for the same dilution in two lines (16 microwells repeated at least 3 times) were taken into consideration to calculate mean values.

Results were compared with corresponding controls. The term "n % extract of a plant" is intended to mean the volume of the plant extract which was present in the tissue culture medium during experimentation. 1% indicate that 1 ml of extract was added to 99 ml of culture medium.

The inventors observed that a 1% glycerinated extract of the whole plant of A. vulgaris alone, specifically promoted the growth of epithelial cells (+21,30%; p<0,05) with little effect on the growth of fibroblast cells (+ 3,39%) while a 3% glycerinated bark extract of M. tenuiflora alone, specifically promoted the growth of fibroblast cells (+ 26,67%; p<0,05) with very little effects on the growth of epithelial cells (+ 7,96%).

A 1 % dried whole plant extract of E. purpurea increased the growth of epithelial cells by 14.2 % and of fibroblast cells by 12.6 % compared to controls. Other plant extracts tested had no significant effect on any cell type.

Associating half the most active concentrations of A. vulgaris extract (0,5%) and M. tenuiflora extract (1,5%) in the culture medium, do not produce a simple additive effect but a strong synergistic effect on both cell types, increasing the number of fibroblast cells by 42,89%

(p<0,005) and that of the epithelial cells by 57,70% (p<0,005) within 48h compared to corresponding control cultures.

E. purpurea extract (0.5 %) with M. tenuiflora extract (1.5 %) increased epithelial cell growth by 26.6 % and fibroblast cell growth by 34.2 %. E. purpurea extract (0.5 %) with A. vulgaris extract (0.5 %) enhanced the growth of fibroblast cells by 36.3 % but had no significant effect on epithelial cell growth. These results show that these three plant extracts possess a promitotic activity where

A. vulgaris preferentially promotes the growth of epithelial cells, while M. tenuiflora bark extract and E. purpurea to some extent, preferentially exerts a pro-mitotic activity on fibroblast cells at non cytotoxic concentrations. Therefore, this synergistic association of at least two of these plant extracts can be highly useful to treat injuries requiring the growth promoting of both fibroblasts as well as of epithelial cells.

II.2. Effect of the addition of the extracts of A. Vulgaris, M. teniflora and E. purpura on the growth of epithelial cells (MDBK cell line) and of fibroblast cells (VSMC), compared to other plant extracts

The inventors have evaluated the epithelial and fibroblast cell growth promoting properties of different plant extracts and have compared them to the properties of the synergistic composition according to the invention comprising at least two plant extract chosen in the group comprising an extract of A. Vulgaris, an extract of M. teniflora and an extract of E. purpura.

The fibroblast and epithelial cell culture models were employed to test growth-promoting properties of different plant extracts prepared using different extraction techniques. Initially, the cytotoxic concentration of each plant extract was evaluated on each cell type and afterwards various non-cyto toxic concentrations of different plant extracts were tested on MDBK (Madin Derby Bovine Kidney) cell line as a model of epithelial cells and primary cultures of vascular smooth muscle cells (VSMC) as a model of fibroblast cells as described by Shrivastava et al. (4).

3xlO⁵ cells were initially seeded in lOOμl culture medium (MEM) with 10% foetal calf serum (FCS) in 96 well tissue culture plates (Corning, USA) for 24h in a 5% CO2 - 37°c environment. After 24h, initial medium was replaced by a new medium containing lOOμl fresh growth medium (2% FCS) containing appropriate concentration of the test product (plant extract) was added to each culture well. Cells were incubated for further 48h and cell number was measured using MTT vital stain. 24 wells were used for each experiment with corresponding untreated controls and experiments were repeated at least 3 times. In total 128 plant extracts were selected on the basis of their known traditional use in ancient medicine and the parts of the plant which are commonly employed and are known to be non-toxic to human beings and to the skin tissue were used to prepare the extracts. All extracts tested were glycerinated or hydroglycerinated as other solvants (such as alcohol) may affect the cellular parameters measured and may have undesired effect on the body cells. Some examples of the plant extracts tested are the extract of Allium sativum, Medicago sativa, Angelica archangelica, Artemisia vulgaris, Cynara scolymus, Bambousa arundinacea, Arctium lappa, Peumus boldus, Betula pendula, Vitis vinifera, Thymus vulgaris, Camella sinensis, Salix alba, Filipendula ulmaria, Rosmarinus officinalis, Raphanus sativus, Vinca minor, Pinus sylvestris, Carica papaya, Tenacetum parthenium, Utrica dioica, Hypercum perforatum, Asculus hippocastanum, Cola nitida, Hemamelis virginiana, Humulus lupulus, Harpagophytum procumbens, Paullinia cupana, Commiphora mukul, Panax ginseng, Garcinia combogia, Fumaria officinalis. All plant extracts used were standardized by quantifying a specific marker for each plant for example gingerol for Zingiber officinalis, caffeine for Paulina cupana, proteasic enzymes for Cynara scolymus, polyphenols for Alchemilla vulgaris (A. vulgaris) and for Mimosa tenuiflora (M. tenuiflora), echinacosides for Echinacea, and catachins for Cameilla sinensis. Glycerinated whole plant extract of Alchemilla vulgaris (A. vulgaris) and glycerinated bark extract of Mimosa tenuiflora (M. tenuiflora), standardised for 10% polyphenols, were in the list of plant extracts tested.

The extracts used were standardised by identifying the concentration of a specific marker present in the plant. For example, the extracts of A. vulgaris and M. tenuiflora were standardised by measuring the concentration of tannic acid (a polyphenol) with the use of UV- Vis spectrophotometry, the extract of Echinacea purpura (E purpura) by measuring the concentration of echinacoside by UV method.

The glycerinated extract of A. vulgaris described contained between 4 to 20% by weight of polyphenols relative to the total weight of the extract, the concentration of which was adjusted to obtain a glycerinated extract containing 9 % by weight of polyphenols relative to the total weight of the extract. The bark extract of M. tenuiflora contained between 5 to 25% by weight of polyphenols relative to the total weight of the extract, the concentration of which was adjusted to 11% for the experiments. The tops of the Echinacea purpura contains between 0,5 to 6% by weight of echinacosides relative to the total weight of the extract, the concentration of which was adjusted to obtain 4,8% by weight of echinacosides relative to the total weight of the extract. These concentrations were selected on the basis of the cell growth promoting properties and cytotoxicity results obtained in vitro on both cell types. All the plant extracts of A. Vulgaris, E purpura and M. tenuiflora currently used in traditional preparations are rarely standardised and were never associated to study the synergistic effect on fibroblast and epithelial cell growth simultaneously. The inventors observed that nearly 98% of plant extracts studied had no activity on cell growth. Only a very few plant extracts (3 out of 128 tested) promoted specifically either the growth of fibroblast cell or that of epithelial cells but none of the plant extracts tested had significant properties of stimulating the growth of fibroblast and epithelial cells simultaneously. This probably explains that why there are no plant drugs available in the market capable to heal injuries within a shortest period of time.

Surprisingly the inventors observed that a glycerinated extract of the whole plant of A. vulgaris between 1 to 3% specifically promoted the growth of epithelial cells (+ 21,30%, p<0,05) with little effect on the growth of fibroblast cells (+ 3,39%) while a 3% glycerinated bark extract of M. tenuiflora specifically promoted the growth of fibroblast cells (+ 26,67%; p<0,05) with very little growth promoting effects on epithelial cells (+ 7,96%). The dried whole plant extract of Echinacea purpurea (1.0 %) containing 4,8 % echinacoside, promoted the growth of epithelial cells by 14.2 % and that of fibroblast cells by 12.6 % compared to the corresponding untreated cultures.

As plant extracts contain hundreds of ingredients, it was scientifically interesting to associate half the most active concentration of the fibroblast cell growth promoting plant with the epithelial cell growth promoting plant extract so as to find a composition capable to promote simultaneous growth of both cell types required for injury healing. The inventors were astonished to observe that associating half the most active concentrations of A. vulgaris extract (1,5% instead of 3,0%) with M. tenuiflora extract (1,5% instead of 3,0%) in the culture medium, do not produce a simple additive effect but a strong synergistic effect on both cell types, increasing the number of fibroblast cells by 42,89% (p<0,005) and that of the epithelial cells by 57,70% (p<0,005) within 48h compared to corresponding control cultures. An association of 0.5 % Echinacea purpurea extract (E. purpurea) with 1.5 % M. tenuiflora extract, promoted the growth of epithelial cells by 26.6 % (p<0.05) and that the fibroblast cells by 34.2 % (p<0.05) while associating E. purpurea extract (0.5 %) with A. vulgaris extract (1.5 %) had no additional significant effect on the growth of epithelial cells (+26.6 %, NS) while the growth of fibroblast cells was enhanced (36.3 %; p<0.05) compared to corresponding cultures.

This synergistic effect of the association of at least two of these plant extracts to enhance the growth of both fibroblast and epithelial cells were never discovered and is a major breakthrough, particularly, for the treatment of deep skin or mucosa injuries.

II.3. Effect of the addition of the extracts of A. Vulgaris, M. teniflora and E. purpura on the growth of vero cells in the presence of 50% cytopathic concentrations of herpes simplex 1 virus in vitro. The aim of this experiment was to evaluate the cell growth promoting effects of the extracts of the 3 selected plant extracts, alone or in association, in the presence of an infective agent (HSV-I virus) which retards the cell growth.

In short, a vera cell culture suspension (purchased from ATCC, USA) were prepared in EMEM medium supplemented with earls salt and 5% foetal bovine calf serum containing 10 000 cells per ml.

8 suspensions each containing 35 ml preparation were prepared. One suspension (plate 1) culture was used as a negative cell control culture. In all other cultures, a pre-titrated HSV having 50% cytopathic effects within 72h was added. The 1^st virus infected culture was used as a positive virus control suspension (plate 2), the 2^nd, 3^rd and 4^th virus cell suspensions (plates 3,4) received either extract of A vulgaris, M.tenuiflora, or extract of E. purpura, (plate 5). For each plant, the concentrations of 0,1; 0,3; 1.0; and 3,0 μg/ml were used for testing. (16 wells per dilution were used, all experiments were repeated at least 3 times). Other cultures were added either with half the concentration of a mixture of A vulgaris + M. tenuiflora (plate 6), A. Vulgaris + E. purpura (plate 7) , or M. tenuiflora + E. purpurea (plate 8). lOOμl of each suspension was then placed in 96 well tissue culture micro-plates and plates were incubated at 37°C, 5% CO2 environment for a period of 72h. After 72h culture, the plates were stained with MTT live cell coloration method and the increase or decrease in the number cells in each plate was measured using the optical density values. The results of the study showed that none of the plant extract alone with the CT50 concentration of the virus, produced any significant effect on cell growth or reduced viral cytopathic effects. On the contrary, the association of A. vulgaris and E. purpurea increased the number of cells by 33,3; 69,5; 85,6; and 72, 4% respectively at concentrations of 0,1; 0,3, 1,0 and 3,0 μg/ml of each plant respectively compared to virus control cell numbers. Similarly, the association of M.tenuiflora with E; purpurea increased the cell number by 17,5; 37,1; 69,1; and 56,3% at concentrations of 0,1; 0,3; 1,0; and 3,0 μg/ml for each plant respectively compared to the CT50 virus controls. The results were less pronounced with the association of A. Vulgaris with M. tenuiflora although a slight increase of cell growth (10-21%) was observed.

These results show that the it is not the individual plant extract but it is the association of two plant extracts among the three plant extracts used in this study helps to protect the cells against virus induced cytotoxicity.

The mode of action of this protective effect is not clear but it may be related to the fact that the test products inhibit the proteolytic enzymes in the culture medium preparing a better surface for the cell growth or the association of the plant extracts binds to the viral proteins thereby inhibiting viral attachment with the cells. Further studies are required to better elucidate the mechanism of this action.

II.4. Healing of experimental injuries in rats 30 male Sprague Dawley rats (IFFA-CREDO, France) were separated at random in 3 batches. On the day of the application of products, the back skin of the rats was shaved with precaution using a shaver adjusted to 0,05 mm of height to obtain a sufficient surface. After general anaesthesia, 6 circular identical deep lesions of 0,9 mm diameter were created on the back of the skin, 3 on each side (right and left of the vertebral column), with the help of a special apparatus (Dermatom) which helps to incise the dermis and epidermis part of the skin. The skin inside this lesion was than carefully removed with the help of a scissors. 1 hour after the creation of the lesions, 0,1 ml of the glycerinated extracts of the test products containing either 3% extract of A. vulgaris alone or 3% extract of M. tenuiflora alone or 1,5% A vulgaris and 1,5% M. tenuiflora extract in glycerine, were applied on the lesions of the respective groups with the help of a ImI sterile syringe. Products were applied only on 3 right side lesions of each animal while the left side lesions were kept as internal controls. The lesions on the left side were treated with 3% water in glycerol solution as control.

The injured area of the back was then covered with a 8-layered sterile bandage which was loosely secured by adhesive tape. The same procedure was repeated every day for 12 consecutive days.

The diameter of each lesion was measured with the help of a transparent ruler, added by a magnifying glass and values were meaned for the three ipsilateral lesions per rat during 14 consecutive days.

The mean diameter of lesions (mm) was as follows:

(± (± (± (± (± (± (±

0 ,2) 0 3) 0 ,5) 0 ,6) 0 ,6) 0 ,4) 0 ,2)

A v (50%) 9,1 7,8 5,6 3,4 1,2 0,0 0,0

+ M t (50%)

(± (± (± (± (± (± (±

0 ,3) 0 ,3) 0 ,5) 0 A) 0 ,2) 0 ,0) 0 ,0)

(Av= Alchemilla vulgaris, Mt= Mimosa tenuiflora)

These results indicate that although A vulgaris 3% alone or M tenuiflora 3% extracts help to accelerate deep injury healing, half the concentration of these two plant extracts mixed together produces a synergistic effect and accelerate injury healing.

The following safety and stability tests were conducted with the formula AS21 containing 3% glycerinated extract of the whole plant of A. vulgaris + 3% glycerinated bark extract of M. tenuiflora in glycerol (97%) or with AS22 containing 3 % glycerinated bark extract of M. tenuiflora with 0.5 % dried hydroglycerinated extract of the whole plant of E. purpurea or with the formula AS 23 containing 3% glycerinated extract of the whole plant of A. Vulgaris with 1,0% dried hyrdoglycerinated extract of the whole plant of E. purpurea.

II.5. Skin irritation tests The skin irritation tests were conducted on rabbit skin as per the OCDE standards related to

Good Laboratory Practices concerning mutual acceptance of data for the chemicals dated 12^th May 1981 (C(81) 30 Final).

0,5 ml of test products were applied onto the intact and scarified skin of 6 rabbits and the product was held in contact with the skin using a patch for 24 consecutive hours. The results showed a Primary Cutaneous Irritation Indices (P.C.I.) of 0 for the 3 compositions tested qualifying the products as non- irritant to the skin.

II.6. Ocular irritation test

The BCOP (Bovine Corneal Ocular Irritation) in vitro test classify all the products as slightly irritant. II.7. Stability tests

The products AS21, AS 22 and AS23 were subjected to stability studies at 20-25°c and 40⁰C (+/- 2°c) and the measurements of physical parameters such as the volume, taste, colour, pH, density, viscosity, and the chemical parameters such as the polyphenol content indicated that the products are stable for a minimum period of 3 years.

π.8. Pilot clinical trial

A test product as described in the example number 3 was used to study the effects on diabetic injury healing. The study was conducted at two hospitals and clinical research centres at

Ranchi in India. A total of 68 diabetic patients having deep injuries on foot and limbs were included in the study after initial written consent. The width of each injury was measured with the help of a scale and the depth was evaluated by an experienced doctor using a 1 (very superficial lesion) to 10 (very deep lesion of approximate average depth of 5 cm) rating system. The first group of 34 patients received different treatments including hydrocolloid bandages (27) or topical antiseptics (7). The second group of remaining 34 patients were treated with 2 to 10 ml of the preparation as given in the example number 3 according to the size of the injury, twice a day, up to complete healing.

In the first group, the average width of the injury was 7,7 cm and the mean lesion depth score was 8,2 on the day 0 (first treatment). After 14 weeks of treatment the average width was

5,2 cm (- 32,46%) and mean depth score was 6,3 (- 23,17%) in the first group. In the second group, the average width of injuries on the first day was 7,9 cm and the mean lesion depth score was 8,4. After 14 weeks of treatment, the average injury width was 3,1 cm and the mean depth score was 2,6 indicating an average reduction of 60,76% in the width and of 69,05% in the depth of the injury over the same period of time. These results clearly show that promoting the growth of fibroblast and epithelial cells is essential for deep injury healing and that the test product markedly reduced the healing period of deep injuries.

Double blind clinical trial is under progress.

References

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Claims

Claims:What is claimed is:

1. A synergistic composition comprising at least two compounds chosen in the group comprising:

- an extract of Alchemilla vulgaris;

- an extract of Mimosa tenuiflora; and

- an extract of Echinacea purpurea, for use as a medicament for the treatment of skin or mucosa injuries.

2. The synergistic composition according to Claim 1, wherein skin or mucosa injuries are deep skin or mucosa injuries.

3. The synergistic composition according to Claim 1 or 2, wherein skin or mucosa injuries are chosen in the group comprising bedsore, pressure ulcers, diabetic ulcers, injuries of viral origin containing cold sore and vaginal herpes, injuries caused due to deep burns, atopic dermatitis, psoriasis, eczyma, chronic uenous insufficiency ulcers, canker sore, vaginal herpes lesions on the mucosa, and traumatic deep injuries of accidental origin.

4. Use of the synergistic composition as defined in Claim 1, for the manufacture of a medicament or a medical device for the treatment of skin or mucosa injuries.

5. Non-therapeutic use of a synergistic composition comprising at least two compounds chosen in the group comprising:

- an extract of Alchemilla vulgaris;

- an extract of Mimosa tenuiflora; and

- an extract of Echinacea purpurea, for improving the aesthetic appearance of injured skin or mucosa.

6. Non-therapeutic use of a synergistic composition as defined in Claim 5, for promoting the growth of fibroblast and epithelial cells.

7. A method of treatment of skin or mucosa injuries, comprising the step of administering to a subject a synergistic composition as defined in Claim 1.

8. A method for promoting skin or mucosa injuries healing comprising the step of administering to an injured site to be treated a synergistic composition as defined in Claim 1.

9. A method for promoting the growth of fibroblast and epithelial cells, comprising the step of administering to a subject a synergistic composition as defined in Claim 1.