WO2007072233A2

WO2007072233A2 - Improved mimosa tenuiflora extracts, methods for its obtaining and use for the treatment of ulcers in mammals

Info

Publication number: WO2007072233A2
Application number: PCT/IB2006/050915
Authority: WO
Inventors: Xavier Lozoya Legorreta; Marco Antonio Chavez Soto; Erika Rivera Arce
Original assignee: Xavier Lozoya Legorreta; Marco Antonio Chavez Soto; Erika Rivera Arce
Priority date: 2005-12-19
Filing date: 2006-03-24
Publication date: 2007-06-28
Also published as: MXPA05013894A; WO2007072233A3

Abstract

The invention is related to the obtaining of phytomedicaments containing standardized extracts derived from the tepescohuite plant (Mimosa tenuiflora), particularly from its bark, useful for the treatment of several skin afflictions such as lesions of difficult treatment associated to blood circulation dysfunctions, as for example, ulcers in the inferior extremities (UIE). Cicatrizant, antimicrobial, anti-inflammatory and immunomodulatory properties of the clinically evaluated phytomedicaments of the invention, as well as their null toxicity and high tolerance allow their clinical use for a safe, controlled and efficient treatment of difficult eradication ulcers.

Description

Description Improved Mimosa tcnuiflora extracts, methods for its obtaining and use for the treatment of ulcers in mammals Field of the invention.

[1] The present invention is related to the obtaining of plant extracts for medical use, specifically to the obtaining of standardized extracts of Mimosa tenutflora and more particularly to the obtaining of standardized extracts from the cortex of the Mimosa tenutflora tree with the purpose of obtaining useful phytomedicaments for the treatment of ulcers of difficult management, particularly for the treatment of ulcerations in the inferior extremities.

Background of the invention.

[2] The skin is the most exposed tissue of the human body given its direct interaction with the environment. In consequence skin lesions are the most common events experienced by humans; in fact, it is assumed that at least once in its lifetime a person has experienced skin injuries or wounds as well as its healing process. Even though the appearance of wounds and their healing are everyday events, there is an infinity of factors that allows its cure or aggravation.

[3] Healing of wounds implies a complex and tidy sequence of cellular events that end in the restoration of the structural integrity of the damaged tissue. The presence of cells that stimulate the inflammatory process and the production of cellular growth factors, the in situ development of the blood vessels and the production of an extra-cellular matrix are all important factors for a fast and efficient healing of the wound.

[4] Even though these events of cell repair allow the normal restoration of the damaged tissue as well as its functioning, there exist multiple factors that hinder an adequate restoration of the wounds, causing for example, cutaneous ulcers that may turn chronic. Cutaneous ulcers are breakings of the continuity of the external epidermal layer with a loss of tissue and a consequent exposition of the internal epidermal tissue (open wound) which may turn chronic when some resistance to complete the healing process or regeneration of the epidermal tissue appears.

[5] Cutaneous ulcers are a common and chronic problem that manifest themselves through skin pain that is caused by pressure and are generally associated to the presence of some pathological event or physiological disorder in the patient that impedes or delays the healing process and normal epidermal tissue regeneration, as for example, in those patients with diabetes or those exposed to repeated steroid therapy to treat autoimmune diseases or topic dermatitis, or as result of cancer treatment by chemotherapy, to mention a few. [6] Independently of the causes that generate the appearance of ulcers, these regularly provoke a deficiency in the formation of granulation tissue and re-epithelization of the affected area.

[7] Given that ulcers are open wounds exposing the most inner parts of the epithelial tissue, considerable discomfort to the patient appears as well as increments in the frequency of infections or more serious complications.

[8] When the cause of the ulcer appearance suspend or importantly reduces the blood flow to the affected area, necrosis of tissue appears as a result of progressive deterioration of cells by lack of nutrients; this pathological effect is commonly the cause of the appearance of ulcers in the inferior extremities (UIE).

[9] UIE is a commonly disabling condition that regularly emerges as a consequence of chronic vascular insufficiency. This disease affects nearly 1% of adults during their lifetime; early symptoms (deterioration of the vascular wall, valve modification of the veins and formation of varicose veins) are related to venous hypertension that end in a cascade of local metabolic events generating the formation of skin ulcers in distant

1 9 regions of the legs ' .

[10] Ulcers in the inferior extremities cause an important loss of leg or foot skin. They occur in association with a range of diseases most commonly related to the circulatory system. Ulcers in low extremities can be acute or chronic, acute are those that follow the normal stages of the curing process and are expected to show signs of heal in less than 4 weeks, including traumatic and postoperatory wounds. Chronic ulcers are those that persist for more than 4 weeks and show a complex and poorly understood origin.

[11] Chronic ulceration of the inferior extremities commonly occurs after a minor skin lesion associated with chronic venous insufficiency (45-80% of cases), chronic arterial insufficiency (5-20% of cases) and diabetes (15-25% of cases).

[12] Venous insufficiency is caused by incorrect functioning of the unidirectional valves of the veins that drain the blood of feet and legs up to the heart. Two mechanisms cause this ascendant flux of the blood: a muscular pump that pushes the blood to the heart during exercise and unidirectional valves that prevent the flux of blood downwards. Faults in valves and in the action of the muscular pumping have as result the deterioration of blood influx to the lowest part of the leg, which is below the ankle. The increase in the venous pressure causes the deposition of fibrin around the capillary tubes acting as a barrier to oxygen and nutrient flux to the muscular and epithelial tissue causing tissue necrosis which in the long term leads to ulceration.

[13] Arterial insufficiency refers to the poor circulation of blood to the feet and legs and is most frequently caused by arteriosclerosis. Arteriosclerosis provokes the narrowing of the arteries due to deposition of fatty substances on arterial walls owed to high levels of circulating cholesterol which aggravates because of high arterial pressure (hypertension) and smoking. For this reason, the arteries discontinue the delivering of oxygen and nutrients to the feet and legs resulting in necrosis and rupture of the tissue.

[14] Diabetic ulcers are caused by a combination of arterial blockage and damage to the nerve. Although diabetic ulcers can occur in other parts of the body they commonly appear on the feet. The damage to nerves or sensorial neuropathy that appears with diabetes decreases the sensibility to pressure, heat or some other lesion. The constant friction and pressure on the foot passes therefore unnoticed, causing damage to the skin and the subsequent appearance of neuropathic ulceration.

[15] A significant number of UIE treatments that cover the basics for the healing of wounds have been reported: in the first place, the importance of daily washes with sterile water and neutral soap, in second place, the roll of oxygen in the healing process of wounds, the effect of pH, the use of the antimicrobial medicine and the use of simple first-aid bandage compression on the wounds .

[16] Even though it is recognized that the standard treatment for ulcers in the inferior extremities is graduated compression bandage, patients experiment some complications because of the use of too bulky bandages, being necessary the use of graduated compression socks below the knees. Despite the improvement observed in patients treated with the former methods, high rates of recurrence are also observed, especially when no compression is used .

[17] Medical treatments available to date are therefore focussed on reducing the risk of appearance of ulceration for which an appropriate diagnostic of the physiopathology of ulceration is needed, especially to be able to provide an adequate treatment for the disease.

[18] Ulcers in the inferior extremities not related with arterial insufficiency are generally treated with exercise, leg elevation during rest and compression. In those cases with significant arterial disease, compression is not recommended because it can aggravate the already inadequate provisioning of blood. In some cases surgery is used to relieve the narrowing of the vascular lumen.

[19] Despite the existence of multiple treatments for ulcers in the inferior extremities, several studies suggest that none of these treatments are completely effective. Unfortunately, the treatments used in many of these cases result inefficient and may even hinder the adequate management of the affliction, promoting the permanence of ulcers that stay open for months or years producing chronic pain and impairment .

[20] Costs of treatments for UIE are available for several countries. In Sweden, for example, an average of 52 weeks of treatment varies in cost between $1,300 and $2,500 dollars. According to epidemiological data the estimated annual costs for the treatment is of 100 to 120 million pounds⁶. Given the high costs of treatments new procedures are necessary to reduce the healing period, and therefore, especially welcomed are new products that assist in the process of wound curing and skin cicatrization.

[21] A variety of treatments and its modalities, that include the use of antibiotics, occlusive layers, bandages, mechanic devices that reduce water evaporation and several others, are available in the market to treat lesions and ulcers. Nevertheless, all these treatments base their therapeutic effect in the stimulation of the proper body mechanisms to restore the epithelial tissue, which represents an important disadvantage in the case of UIE given that the physiological state of the patient does not work in an optimal manner. For those cases several therapeutic products focused on provoking the active regeneration of the epithelial tissue including its several internal layers have been developed.

[22] It has been observed that a number of extracts or products derived of plants stimulate the epithelial restoration in diverse skin afflictions such as wounds or ulcers, therefore multiple treatment options derived from plants have been developed. For example, the use of the biologically active polysaccharides of Aloe , pulverized herb materials for the treatment of eczema or psoriasis , Melia azadirachta and Cen- tratherum anthelminthicum extracts for the treatment of psoriasis , Chinese rhubarb and Phellodendron amurense bark compositions¹⁰, Acacia gum or Colophonium and curcuma extracts , Huangqin sesame oil extract and pharmaceutical preparations containing alcoholic extracts of 5 different plants {Scutellaria baicalensis Georgi, Phellodendron amurense Rupr, Coptis chinensis Franch, Dϊlong and Opuntia)¹³. Nevertheless, none of these products are completely safe for its topic application given the presence of substances that may cause diverse allergic or unwanted side effects or else are not well tolerated by the patient.

[23] In this sense medications prepared with plants of the Mimosa genera have major advantages in the treatment of skin afflictions in comparison with the above mentioned products, given that Mimosa tenuiflora extracts were those that proportioned the best results.

[24] Mimosa tenuiflora Poiret (Leguminosae) is a tree known in Mexico as te- pescohuite. According to Mexican ethnobotanic medicine, the dried and pulverized bark of tepescohuite applied directly over the wound is an efficient remedy for the treatment of wounds and lesions ' ' . In Mexico in 1984 this material was empirically used for relieving the pain of hundreds of victims left by an explosion of a natural gas deposit. In that occasion the use of the pulverized Mimosa bark on the lesions of many of the burned patients by the medical emergency staff, resulted in the improvement of skin regeneration and the avoidance of scars .

[25] Pharmacological and phytochemical studies performed in Mexico and France gave support to the existence of natural compounds with cicatrization properties in the bark of the plant. Basic pre-clinical studies report that the water and alcoholic extracts of the dried bark are particularly rich in tannins and show in vitro antimicrobial properties against an ample group of gram-positive and gram-negative microorganisms, yeasts and dermatophytes¹⁷; this same extracts induced the growth of several human cells

1 S under culture conditions .

[26] Further studies identified a group of triterpenoid saponins designated as mi- monosides A, B and C ' , that according to in vitro observations induce proliferation of cultured human cells, possess immunomodulation capacity, and are therefore attributed with at least part of the potential cicatrization properties of the plant bark ' .

[27] Based on published biomedical literature that gives support to the cicatrizing properties of tepezcohuite, some herbal products have been developed and commercialized for its topic administration in the treatment of skin ulcers and bums.

[28] Leon ' ' describes the obtaining of a pulverized bark of one Mimosa genera plant.

The mentioned powder is useful in the treatment of bums, wounds, as skin medication or used for cosmetic purposes. The powder obtained from the plant bark exhibits strong analgesic and soothing effects, aiding thereby in the process of skin cicatrization.

[29] Tellez²⁶ describes the obtaining of an extract from the Mimosa tenuiflora bark with skin regeneration properties. The dried extract demonstrated to be useful in the treatment of bums, abrasions and skin lesions, both in humans and mice.

[30] Izundegui²⁷ describes the obtaining of a Mimosa tenuiflora watery extract that can be used directly, as a cosmetic mixture, pomade or well as a concentrate solution to promote hair growth. It is also mentioned that the powder obtained from the tree cortex subjected to high temperature can be directly applied on the skin affected by burns or ulcers.

[31] Izundegui describes the obtaining of a Mimosa tenuiflora bark extract. It is mentioned that the extract is useful as a healing agent when applied to wounds or cuts in animal skin, without leaving marked or keloid scars.

[32] Yoshitani describes a Mimosa tenuiflora extract that shows antioxidant properties when incorporated to the material that is pretended to be protected against free radicals.

[33] Heyda describes a compound containing a Mimosa tenuiflora glycolic extract blended with soy derived isoflavones for vaginal hygiene. It is mentioned that the compound can be directly applied in the vaginal tract given its anti-inflammatory, antibacterial and re-epithelization properties.

[34] Anderson describes the obtaining of Mimosa pudica extracts that possess phenolic compounds, useful for cosmetic applications, which exhibit biological activity, maintaining and enhancing skin collagen levels. [35] Nevertheless, despite of the mentioned efforts to obtain Mimosa derived phy- tomedicaments, and particularly those from Mimosa tenuiflora, the mentioned products have not been completely efficient in its controlled clinical use , are not considered official drugs because they haven't been clinically evaluated and do not contain standardized quantities of its active ingredients.

[36] It is therefore important to develop drugs for the treatment of UIE that can be used in combination with conventional wound healing therapies, which do not present toxic or unwanted side effects for the patient, which can be administered in a safe and controlled manner and undertake clinical evaluation.

Objectives of the invention.

[37] One of the objectives of the present invention is to provide standardized extracts from the Mimosa tenuiflora plant for the management of difficult treatment skin ulcers.

[38] Another objective of the present invention is to provide standardized extracts from the Mimosa tenuiflora plant for the treatment of ulcers in the inferior extremities, particularly those of venous ulceration in the inferior extremities.

[39] Another objective of the present invention is to provide standardized extracts from the Mimosa tenuiflora plant without toxicity for the patient which permits the relive the diverse symptoms accompanying ulcerous skin lesions.

[40] Another objective of the present invention is to provide standardized extracts from the Mimosa tenuiflora plant by means of standardization of its tannin concentration for the treatment of ulcers in the inferior extremities.

[41] Another objective of the present invention is to provide standardized extracts from the Mimosa tenuiflora plant for the obtaining of phytomedicaments that can be administered in a controlled manner in diverse therapeutic regimes of wound treatments.

[42] Another objective of the present invention is to provide phytomedicaments for the treatment of ulcers in the inferior extremities containing standardized extracts from the Mimosa tenuiflora plant as a therapeutic agent.

[43] Another objective of the present invention is to provide phytomedicaments containing standardized extracts from the Mimosa tenuiflora plant in diverse pharmaceutical presentations for its controlled administration in patients.

[44] Another objective of the present invention is to provide efficient and low cost methods for the obtaining of standardized extracts from the Mimosa tenuiflora plant which conserves its therapeutic activity.

[45] Another objective of the present invention is to provide efficient methods that permit the standardization of the active ingredient concentration contained in the Mimosa tenuiflora extract by means of normalizing its tannin concentration.

[46] Another objective of the present invention is to provide an efficient treatment method for ulcers in the inferior extremities using the standardized extracts from the Mimosa tenuiflora plant as the therapeutic agent.

Brief description of the figures.

[47] Figure 1. Shows a microscopic view of the Mimosa tenuiflora bark. The inner and external cortex can be recognized (A), the details of that indicated in A (B), details of the longitudinal-tangential section of the inner cortex (C), details of the longitudinal section of the inner cortex (D), dissociated (E and F), sclereids and macrosclereids with crystalifer parenchymatic pod (E) and fibres and fibre with crystalifer parenchymatic pod (F).

[48] Figure 2. Shows a graphic depicting the results obtained from the clinical study indicating the effects of the auxiliary treatment with the phytomedicament of the invention in patients with ulcers in the inferior extremities (UIE). Shows results obtained by treatment with placebo (A) and by treatment with the phytomedicament of the invention (B).

[49] Figure 3. Shows the ulcers in the inferior extremities in some patients mentioned in example number 5, before (1) and after (2) the treatment with the phytomedicament of the invention, at time of the 15 (A) and 19 (B) weeks of treatment.

[50] Figure 4. Shows the ulcers in the inferior extremities in some patients mentioned in example number 5, before (1) and after (2) the treatment with the phytomedicament of the invention, at time of the 12 (C) and 15 (D) weeks of treatment.

[51] Figure 5. Shows the ulcers in the inferior extremities in some patients mentioned in example number 5, before (1) and after (2) the treatment with the phytomedicament of the invention, at time of the 15 (E) and 26 (F) weeks of treatment.

[52] Figure 6. Shows a graphic where the following can be observed: the antiinflammatory effect produced by different concentrations of the extract of the invention (Extract) and different concentrations of the A (saponins), B and C (tannins) fractions, against ear oedema of mice induced by

12-O-tetradecanoylphorbol-13-acetate (TPA), using dexametasone (Dex) as positive control. Observed results represent the mean of 6 observations (±S.E., *=p<0.05 and **=p<0.001) according to one way ANOVA test followed by a Bonferrioni test.

Detailed description of the invention.

[53] Cicatrizant, antimicrobial, anti-inflammatory and immunomodulatory properties of extracts derived from the Mimosa tenuiflora bark justify the use of this vegetal drug for the development of phytomedicaments for the treatment of cutaneous ulcers, as for example ulcers in the inferior extremities and particularly those venous ulcers in the inferior extremities that present a high rate of healing resistance of the injured epithelial area. Before the present invention no efficient and safe medications existed that lacked of collateral effects and which could be applied to patients with cutaneous ulcers of difficult treatment allowing controlled dosage regimes at the same time. [54] According the established international regulation of the World Health Organization for the development of phytomedicaments , among the several requisites that has to fulfil a medicine of this type, the next stand out:

• The scientific grounding of the traditional use of the plant sustained by pharmacological and chemical studies supporting its medicinal properties,

• The control and standardization of vegetal drug derived from the medicinal plant for commercialization purposes, and

• The chemical standardization of the active ingredients present in the proposed medicine that allow its dosage for its clinical test.

[55] In the present application a phytomedicament derived from the standardized extracts obtained from theMimosa tenutflora bark is presented, that reunite the toxi- cological and standardization studies established for the production of phytomedicaments, based on clinical studies in patients. The phytomedicament of the present invention is a safe medicine given its absence of toxicity, and can therefore be administered to patients with cutaneous ulcers of difficult treatment, as for example, ulcers in the inferior extremities.

[56] The phytomedicament of the invention is very useful for the treatment of cutaneous ulcers including those of difficult treatment.

[57] The combined effects that produce the Mimosa tenuiflora extracts, is an advantageous characteristic that stands out in comparison with other medications, thus with the sole application of the phytomedicament several exposed skin lesions with different characteristics can be treated, including ulcers of difficult treatment as the UIE, without the necessity to apply several medicines or treatments for each one of the clinical symptoms presented by the patients. The phytomedicament of the invention can also be used as a coadjuvant of traditional treatments for this kind of lesions, as for example, with the application of common hygiene methods and, compressions on the affected area by graduated compression bandages.

[58] On the other hand the phytomedicament described in this document, represent a natural origin product designed as a coadjuvant for the treatment of skin ulcers which offers important benefits given its complete therapeutic effect and the absence of adverse effects for the patients. Moreover, its controlled dosage and administration in addition to its efficient therapeutic effect in patients with skin ulcers, allows its usage in conventional medical treatments.

[59] Before the present invention no Mimosa tenuiflora extract existed, particularly not from its bark, which contained determined and standardized quantities of the active ingredients responsible for the characteristic therapeutic effect of the extract. This distinctive characteristic of the invented extract allows the frequent and controlled obtaining of pharmaceutical compositions containing it, making it possible to control the quantities of active ingredients found in such compositions. In consequence it is also possible to obtain phytomedicaments with predetermined and known dosages able to be administered in adequate presentations. In addition, with the administering of this phytomedicaments there is certainty that the patient receives constant and controlled quantities of the phytomedicament according to different treatment regimes and in accordance with the type of skin ulcer treated. This phytomedicament allows the physician to design controlled treatments for patients with chronic skin lesions and to make an adequate following of them.

[60] Furthermore, the standardized extract of the invention can be easily combined with multiple compounds for the manufacturing of diverse conventional pharmaceutical forms, as could be powder, cream or liquid depending of the administering and regime requirements. The extract conserves its pharmacological properties independently of the process to which it is submitted for the obtaining of convenient pharmaceutical forms, allowing a great versatility in its management.

[61] The extract contains pre-established quantities of its active ingredients (tannins) standardized as equivalents of tannic acid, using the quantitative determination of the tannic acid molecule in the extract for this purpose, as for example by means of a UV- Vis spectrophotometry.

[62] For purposes of the invention the extract described here possesses a 12 to 36% w/w

(weight/weight) tannin concentration as active ingredient, measured as total tannic acid concentration.

[63] Studies performed so far regarding the diverse compounds present in the Mimosa bark have shown that the existence of a direct relationship between all the therapeutic effects of the plant extracts with a single molecule or group of molecules existent in the particular extract , can not be assured with precision. Nevertheless, independently of the method used for its obtaining, the majority of extracts obtained so far exhibit antimicrobial and cicatrizant effects, being reported that the group of molecules present in a major concentration are tannins¹⁷'¹⁸.

[64] Despite that the multiple compositions using or containing Mimosa tenuiflora extracts showed favourable effects in the treatment of wounds ' ' ' , these are usually obtained in the form of powder directly applicable over the damaged skin preventing the administration of the powder in a hygienic way and under a controlled dosage regime. In this sense the Mimosa tenuiflora bark is still used in a rudimentary way by means of spreading over the wound the powder derived from the grinded bark, which does not permit the adequate use of its active ingredients properties nor its adequate dosage; which is the reason why physicians show a reluctance to use this healing method. [65] On the other hand, the mentioned extracts do not contain determined quantities of the active ingredients responsible for the observed therapeutic effect, hindering the administration of therapeutically effective and controlled quantities of active ingredients to the affected area of the patients. This situation complicates the adequate design of controlled therapeutic regimes for the patients using such extracts. Besides, the treatments that use this type of non-standardized extracts are not always efficient and show varying results depending on the kind of affection treated, on the patients' characteristics and basically on the characteristics of the used extract. It has been reported that diverse Mimosa tenuiflora preparations produced with the sterile powder of the tree bark showed hepatotoxic effects and a very low therapeutic efficiency in rabbits with chemically induced burns . In this same sense the mentioned extracts are not applied as pharmaceutical compositions containing standardized Mimosa extracts, particularly Mimosa tenuiflora extracts, and have not been proved in clinical studies among human patients. Given the impressive therapeutic effects observed with the use of the Mimosa bark powder since the 80's , the preparations made out of this plant have undergone multiple adulterations which turn them therapeutically inefficient in the best of cases.

[66] On the other hand diverse compositions containing a mixture of several compounds including tannins, have been reported, such as those containing bee pollen , zinc , chatecol and galic acid³⁷, and also those containing tannin compounds from different plants, as for example the Origanum vulgaris and Salvia officinalis ³⁸. Nevertheless none of the mentioned compositions present the whole range of observed effects of Mimosa tenuiflora extracts in the treatment of wounds and ulcers of difficult treatment such as UIE; they also doesn't contain complete standardized plant extracts which limit their therapeutic application and efficiency over this kind of wounds.

[67] In contrast with that known to date, the present invention describes the obtaining of the Mimosa tenuiflora bark extracts with the purpose of standardizing the concentration of its active ingredients which permits the obtaining of adequate plant extracts from which phytomedicaments can be obtained. The standardization of the extract permits to control its active ingredients concentration to obtain adequate pharmaceutical forms containing them. This also permits to perform precise clinical and pre-clinical studies to explore the therapeutic effect of the extract as well as its mechanisms of pharmacological action.

[68] The pharmacological composition using the extract of the invention as a therapeutic agent can be prepared in accordance with whichever conventional methods and procedures. Any convenient formulation for the local administration of therapeutic can be used in accordance with the invention. The pharmaceutical compositions of the invention can be formulated to provide a rapid, sustained or prolonged liberation of the active ingredients after its administration to a mammal, employing any of the methods and/or procedures well known in the art.

[69] Particularly, the pharmaceutical composition of the invention can be topically applied on the skin proportioning a localized effect. The active ingredients contained in the standardized extract of the invention can also be administered in any appropriated manner for its topic use. Such pharmaceutical forms include solutions, colloidal dispersions, emulsions (oil/water or water/oil), suspensions, powders, creams, lotions, gels, foams, mousses, aerosols and similar presentations.

[70] Compositions for local use include solutions, liposome suspensions, and water/oil or oil/water emulsions. Compositions for topic use include solutions, lotions, suspensions, liposome suspensions, water/oil or oil/water emulsions, gels, ointments, creams, pomades and others.

[71] In the case of the emulsions and suspensions the composition can contain adequate surfactants of the non-ionic, zwitterionic, anionic o cationic type commonly used in the formulation of medicines. Lipophylic water/oil emulsions are preferable for topic use.

[72] On the other hand, compositions of the invention can contain optional additives as isotonic agent such as sugars or polyalcohols, buffers, quelant agents, antioxidants and preservatives.

[73] Topic use compositions include liquid or semisolid forms. Liquid forms include solutions or lotions which should be preferably watery and obtained by solubilization of the used extract.

[74] Alternatively the solutions containing the extract of the invention can also be formulated in a gel form by the addition of the known gelling agents, for example, pharmaceutical gelling agents.

[75] In one of the preferred embodiments of the invention, the pharmaceutical compositions of the invention can be formulated as hydrogels using cross-linking polymers of high molecular weight of the acrylic acid, as for example carbopol resins, NOVEON AA-I USP and emulsifying polymers as PEMULEN, among which carbopol polymers are preferred because of their good bio-adhesive capacity. Carbopol polymers are essentially synthetic hydrophilic colloids, dispersible in water and available in the form of a white fluid acid powder, with a low oral toxicity, not irritant or allergenic, and with a molecular weight of 450,000 to 5,000,000. For the purposes of the present invention, carbopol polymers that can be used for the obtaining of hydrogels are those that provide an adequate viscosity compatible with the one of the extract described in this document, as for example carbopol 934, 940, 941 and Ultrez which posses a viscosity of 1,000 to 50,000 cP, among which carbopol 940 is preferred. The gelling agent is aggregated to the hydrogel composition of the invention previously dispersed in water in a 0.2 to 1% w/w concentration, preferably in a 0.5 to 1% w/w and most preferably in a 0.75% w/w concentration.

[76] For a convenient dissolving of the extract of the invention and its further incorporation to the hydrogel, dissolvent polymers are used such as polyethylene glycols of different molecular weights, preferably polyethylene glycol 200, blending the extract and the dissolving polymer in a weight proportion that allows the complete dilution of the extract, preferably in a 1 : 1 weight proportion.

[77] Then, an alkaline neutralizing agent of the group that comprise trietanolamine, iso- propilamine, diisopropilamine and morfolin, preferably trietanolamine is added to the hydrogel composition in a quantity that permits the adjusting of the hydrogel pH to a physiological pH as for example at pH 7.

[78] In the particular case of the hydrogel containing the phytomedicament of the invention, it can be said that this pharmaceutical form has major advantages in its use as a coadjuvant in the treatment of UIE because it provides a humid environment adequate for the wound. In this case, the humid environment that form the phytomedicament of the invention over the ulcer, isolates the wound from the outside, limits the entrance of pathogenic microorganisms and allows the contact with the bactericidal exudates rich in cellular growth factors, creating an ideal environment for the survivorship and restoration of the epidemic cells. In addition to this, the pharmacological properties exhibited by the active ingredients of the phytomedicament of the invention (standardized extract of M. tenutflora) provide to the patient with an effective local treatment of the wound and a greater bio-availability of the active ingredient, which simplifies the treatment rendering it more acceptable. The pharmacological effect exhibited by the phytomedicament is very complete and highly efficient in comparison with conventional treatments for the healing of wounds. In that sense the phytomedicament permits the physiological removal of the necrotized tissue from the wound (autolytic debryding and cleaning), the control and removal of potential infections, stimulation of the cicatrization process and cellular regeneration of the wound (occlusion), and elimination of inflammation, to cite some of the observed effects.

[79] Hydrogels commonly used in the conventional treatment of wounds are, in contrast with the phytomedicament described here, commonly applied to dry wounds given that its application is contradicted over open wounds with exudates. Generally, they require the use of a bandage or secondary dressing because of its lack of adhesiveness³⁹ and some part of its constitutive elements result irritating to the exposed wounds (alcohols).

[80] The phytomedicament of the invention works as a biocompatible non-allergenic apposite that provides protection to the wound from physical, chemical and bacteriological external aggressions without adhering itself on the epidermal surface, it maintains the wound under an adequate temperature and a humid environment, eliminates the exudates and necrotic tissue by means of absorption, it is easily applied and removed, and its change is painless and spaced apart.

[81] The mentioned compositions permit the active ingredients of the standardized extract to arrive to its site of action in a safe and controlled manner allowing the skin regeneration process to function in an adequate manner.

[82] Other kind of compositions to topic use are emulsions or suspensions in form of pomades, pastes or creams. Water/oil emulsions are preferable, providing a more rapid absorption. Examples of lipophilic excipients are liquid paraffin, anhydride lanolin, white vaseline, cethylic alcohol, estearic alcohol, vegetable oils or mineral oils. Physiologically acceptable agents that can be favourably used to increase cutaneous permeability and facilitate absorption are polyvinilic alcohol, polyethylene glycol or dimethylsulfoxide (DMSO).

[83] Other additives used in topic compositions are isotonic agents such as sugars or polyalcohols, buffers, quelant agents, antioxidants, preserving, thickening and dispersant agents.

[84] Controlled liberation compositions for topic or systemic use can also be useful, and include polymers like polylactate, polymethacrylate, polyvinylpyrrolidone, car- boxymethylcellulose, methylcellulose and other substances well known in the art. Controlled liberation compositions in the form of subcutaneous implants, based on polylactate or other biodegradable polymers can be useful too.

[85] The pharmaceutical compositions of the present invention containing the M. tenutflora extract described here, contain a 1 to 3% w/w of tannins measured as total concentration of tannic acid, preferably a 1.5 to 2% w/w of the mentioned tannins. With respect to the diverse pharmaceutical presentations that can be obtained using the extract, the concentration of active ingredients can vary within the mentioned ranges depending upon the pharmaceutical presentation required.

[86] Pharmaceutical compositions containing the extract of the invention are stable under storage at room temperature for a minimum period of 18 months, conserving its therapeutic efficiency and active ingredient concentration. This permits the removal of high contamination risks present in the Mimosa tenuiflora extracts obtained in the traditional way.

[87] The composition of the invention can be directly administered on the wound, ulcer or affected skin. A daily dose of the extract equivalent of 5 to 10 mg of tannins applied over the affected zone, is adequate to be administered in human patients, preferably a daily dose of the extract equivalent to 7 to 9 mg of tannins over the affected zone; such doses can be administered only once or daily in spaced doses until the complete disappearance of the clinical symptoms or the complete healing of the wound. The exact quantity of the composition administered to the patient can vary according to age, sex, weight, severity of illness, size of the affected area and type of formulation administered.

[88] The extract of the invention is very useful in curing skin wounds, especially cutaneous ulcers of difficult treatment as for example leg ulcers associated to dysfunctions in blood circulation.

[89] Given that the extract of the invention lacks of toxicity and is well tolerated during the treatment, it can be administered in a safe manner among patients that suffer skin ulcers. It can also be used in conjunction with other traditional lesion treatments, increasing the efficiency of the treatment by reducing healing period, especially in the case of chronic wounds. The extract of the invention as well as the phytomedicaments containing it can be used for the efficient and successful management of ulcers of difficult treatment such as UIE which show healing resistance and high treatment costs.

[90] Given the cicatrizant, antimicrobial and immunomodulatory properties of the phy- tomedicament of the invention, cutaneous ulcers can be conveniently treated eliminating the need to resort to the use of antibiotics, anti-inflammatories and analgesics. The therapeutic effects exhibited by the phytomedicament of the invention allow the efficient and complete treatment of the mentioned diseases.

[91] Before the present invention, no similar phytomedicament existed that provided the mentioned therapeutic effects in a single product. For example, chronic lesions were generally treated with broad spectrum antibiotics intended to fight potential opportunists infections, requiring also the application of local analgesics and antiinflammatory to control the pain.

[92] The extract of the invention has in common with the products normally used in the treatment of skin lesions their antimicrobial, analgesic and cicatrizant effects, but differs from them by its lack of toxicity while keeping the mentioned effects, which in conjunct permits its efficiency and therapeutic security. As a result, the mentioned treatment becomes more tolerated and accepted by the patient with UIE in contrast with the conventional management techniques consisting in the application of multiple drugs.

[93] Despite the use of several plant extracts, such as those obtained from Matricaria recutitia ⁴⁰ , Calendulae officinalis ⁴¹ , Limnanthes alba ⁴² , Prunus armeniaca in combination with Morus albae, Artemisia argyi and others , and Mimosa pudica , for the treatment of diverse skin disorders like ulcers, burns and even against skin aging, they do not present the same combined effects provided by the Mimosa tenuiflora extracts of the invention.

[94] The method employed to obtain the extract of the invention allows the obtaining of Mimosa tenuiflora alcoholic extracts useful for the treatment of cutaneous ulcers given its confirmed antimicrobial, cicatrizant and immunomodulatory properties and supported by results provided by clinical tests done in patients using the extract of the invention.

[95] The extract of the present invention is prepared from the dried pulverized Mimosa tenuiflora bark with a minimum tannin concentration (estimated as total tannic acid) no less than 12% w/w in its vegetal matter. The obtained product is put into contact with ethanol for a 24 to 72 hr at room temperature; the obtained extract is then filtered and concentrated by dissolvent distillation until its elimination, conserving it in closed containers until its further use.

[96] This extract is standardized in its tannin content, quantified as equivalents of a 12 to

40% w/w tannic acid concentration through addition of convenient quantities of pharmaceutically acceptable excipients that allow the obtaining of the most convenient pharmaceutical presentation containing determined quantities of active ingredients.

[97] In one of the preferred embodiments of the invention, the extract of the invention is blended with a pharmaceutical excipient in such a way that the tannin composition found in the extract is of 1 to 3% w/w, preferably of 1.5 to 2% w/w of the mentioned tannins measured as total tannic acid concentration. Components of the mixture are blended by conventional methods to obtain compositions for topic application, preferably hydrogels containing carbopol which are directly applied over the lesion or skin ulcer for its treatment.

[98] The process of the invention allows the obtaining of Mimosa tenuiflora extracts with such adequate concentrations of its components that it is afterward possible to standardize the tannin concentration contained in the extract with the aim of administering it in effective doses leading to adequate therapeutic effects for the patient under treatment.

[99] Just as for the present invention, several general procedures for the obtaining of

Mimosa tenuiflora extracts have been reported.

[100] Leon ' ' describes the obtaining of a powder from the bark of a plant of the

Mimosa genus. The cortex obtained from the plant is treated with an oxidant substance such as hydrogen peroxide to eliminate irritant and pyrogenic substances and then dried at room temperature. In the next step the cortex is submitted at a temperature of 80 to 140⁰C and grinded to reach a particle size of 100 mesh.

[101] Tellez describes the obtaining of an extract with skin regeneration activity obtained from the Mimosa tenuiflora bark. The extract is obtained by subjecting the cortex to chloroform, ethanol and water at a temperature of 50 to 100°C. After solvent evaporation, the powder obtained from the extract showed a 54 to 57% carbon, 4.5 to 5.4% hydrogen and a 36.5 to 37.5% oxygen percentage and was characterized by UV and C-NMR analysis.

[102] Izundegui²⁸ describes the obtaining of a Mimosa tenutflora bark extract with dimethylsulfoxide.

[103] Yoshitani²⁹ describes the obtaining of a Mimosa tenuiflora extract using different dried parts of the plant, extracted by means of low molecular weight alcohols. The resulting mixture of the extraction is then concentrated by means of solvent evaporation obtaining a concentrated extract that presents antioxidant activity when incorporated to the material that is tried to protect against free radicals.

[104] Anderson describes the obtaining of extracts from Mimosa pudica containing phenolic compounds. Extracts are obtained from several parts of the plant using a mixture made from a diversity of solvents such as heptane, ethanol, ethyl acetate and water.

[105] Despite that Mimosa tenuiflora extracts with proved pharmacological activity can be obtained with the above mentioned methods, all procedures involve the use of solvents that can have toxic effects as well as low concentrations of their active ingredients, therefore, it is not possible to obtain standardized extracts that allow the manufacture of phytomedicaments directly applicable in the clinic. On the other hand, none of the described methodologies provide data from clinical studies performed in patient to asses the therapeutic efficiency and security of the obtained extract.

[106] With the method of the invention the concentration of components in the extract are so adequate, that its concentration can be adjusted manipulating volume and weight proportions both in the concentrated extract and in the obtained pharmaceutical compositions.

[107] Like a way to illustrate the present invention, the following examples are presented without them limiting the reach of the same.

Example 1. Obtaining and selection of the vegetal matter.

[108] Mimosa tenuiflora (Willd) Poir (Leguminosae) bark was collected from a controlled harvest performed in the state of Chiapas in Mexico. A backup sample was prepared and deposited for reference in the ethnobotanic herbarium of the Instituto Mexicano del Seguro Social (Mexican Institute of Social Security) under the code 14841.

[109] Portions of 50-60 cm long from tree bark were collected detaching them from the trunk of the tree and then submitted to drying at room temperature. Once dehydrated, portions were cut in 10-15 cm long and 5-7 cm wide fragments which were grinded in an electrical mill until the obtaining of a homogenous reddish-maroon powder that passes through a mesh #8 when sieved. The obtained powder was then packed for its processing protecting it from direct light. The powder presented the typical morphological characters that permit to botanically identify the Mimosa tenuiflora bark as well as their typical anatomical characters which are mentioned next:

[110] a) Organoleptic properties.

[Ill] The external surface of the cortex had a dark maroon to reddish-maroon tint, a characteristic smell and a stringent and bitter taste. Internal surface was fine, smooth to touch and had a shiny aspect. The pulverized drug presented a fine texture, reddish- maroon tint, opaque aspect and a characteristic smell.

[112] b) Macroscopic description.

[113] It was observed that the cortex was integrated by curve fragments of variable longitude and a thickness of 6-7 mm wide of sharp fracture and shiny aspect, the external surface presented longitudinal striate that delimited small plaques with more or less deep cracks.

[114] c) Microscopic description.

[115] External cortex. The presence of 3 to 5 periderms was observed, arranged in a imprecated form and constituted by 2 to 5 suber cell layers with thin walls and inactive phloem (see figure IA), the layers were alternated with an interrupted row of rectangular brachysclereids with a wide lumen and 700 μm long sclerenchymatic fibres disposed in small groups in a parallel and tangential form. Tannin deposits were found on the walls. Fibres were accompanied by a parenchymatous pod and cells presented rhombohedric calcium oxalate crystals in its enlarged tangential intern surface (see figure IE and IF). In its tangential section numerous tanniniferous parenchymatous radiuses were identified conformed by four cells in its wide part and eleven to fourteen cells in its height. They were characterized by being conformed by axial parenchymatic elements with wide punctuation fields disposed in a scalariform manner (see figure IB and ID).

[116] Internal cortex. The tissue situated between the periderm and the internal cortex corresponded to active secondary phloem. Biseriated and triseriated radius of a sinuous path that expanded and disposed themselves tangentially in the most superficial region were observed (see figure IA). Small packages of fibbers disposed in parallel stratums finding narrow tubes between them were also observed. Fibbers were surrounded by crystalifer parenchyma containing rhombohedric calcium oxalate crystals (see figure ID). The micrographic aspect of the identified cortex can be observed in figure 1.

[117] The former procedure allows the morphological and anatomical identification of the

Mimosae tenutflorae cortex ftepescohuite bark) which permits the authentication of the botanic matter and discarding of adulterated or contaminated botanical material.

Example 2. Obtaining of the extract.

[118] The cortex obtained and characterised by the example 1 (500 g) was then extracted through percolation with 70% ethanol at room temperature for 72 hours, the extract was then filtered through Whatman #1 paper and concentrated under low pressure at 60°C temperature in a rotavapor until the complete elimination of the alcohol, obtaining a brown, odourless viscous extract of colloidal aspect. The previously obtained extract was weighted and the concentration of its active ingredients analysed by the method mentioned in the example 4. Afterwards it was stored for its future use to obtain the desired pharmaceutical presentation.

[119] The concentrated extract containing a concentration of active ingredients no less than 12% w/w was afterwards used for the obtaining of the pharmaceutical compositions of the invention.

Example 3. Obtaining of a hydrogelfor topic application containing the extract of the invention.

[120] A portion of the concentrated extract (50 g) of example 2 was blended with 50 g of polyethylene glycol 200 (PEG 200) (J.T. Baker), then 0.75% w/w Carbopol 940 (Noveon®) previously dispersed in sterile distilled water (900 g) by means of mechanic agitation, was added to the mixture. The final mix was adjusted at pH 7 with the use of trietanolamine (Sigma), the stirring was continued until the obtaining of a gel which M. tenutflorα concentrated extract concentration was 5% w/w. Using the same methodology, but excluding the addition of the M. tenuiflorα extract, and including the addition of red 3 and green 3 to provide the same maroon aspect of the phytomedicament, a control gel (placebo) was obtained to perform the clinical study described in example 5.

[121] Another hydrogel was obtained in the same way as described before but instead of using polyethylene glycol, 5% ethanol was used to dissolve the extract. Nevertheless, the gel obtained this way resulted irritant to some patients who thereby rejected the application of the gel over the ulcer.

Example 4. Quantification of the active ingredients.

[122] The concentration of active ingredients in the cortex, concentrated extract and gel was quantified as tannin concentration using the following methodology:

[123] A convenient quantity of the material to determine was weighted and introduced into an Erlenmeyer flask with 100 ml of distilled water, the material was then totally dissolved through magnetic stirring. The obtained solution was filtered and rinsed with distilled water until completing a volume of 250 ml. Next, in a 25 ml flask, 250 μL of the previous solution, 20 ml of water and 1.25 ml of Folin-Denis reactive were mixed, then after 5 min waiting, 2.5 ml of 35% w/w Na CO was added. The previous mixture was gauged with water, blended and left to rest, protected from the light for 1 hr. Once finished this period, a sample from the previous solution was taken to read the absorbance at 700 nm employing as control a solution prepared according the described procedure but that did not contain the matter to determine. The tannin content (% w/w) was calculated according to the next equation:

[124] ((Ct x 250 ml x 100 μg) x 100) / Pm μg [125] Where: [126] Ct = Tannin concentration in μg/ml, interpolated in the Standard curve. [127] Pm = Weight of the sample in μg. [128] For the previous determination about 200 mg of the concentrated ethanolic extract described in example 2 and 4 g of the gel described in example 3 were used.

[129] The fractions of tannins were quantified by means of UV- Vis spectrophotometry at 700 nm using the Folin-Denis method, employing tannic acid as reference standard. Calibration curve showed a lineal relation between the tannic acid concentration and 700 nm absorbance in the range of 1.02 to 8.12 μg/mL. The correlation coefficient was 0.9940 (R= 0.9940). As showed in Table 1, the tannin content in the M. tenutflora bark was 15.8% w/w, which complies with the limits established in the Mexican Pharmacopeia⁴⁷ that maintains that the tannin content in the bark of Mimosa tenutflora Wild Poiret will have to be equal or greater than 12% w/w. The tannin content in the concentrated ethanolic extract of the invention obtained from the bark was 36% w/w.

[130] The tannin concentration in the phytomedicament of the invention mentioned in example 3 was 1.73% w/w, being observed a correspondence between the theoretically estimated tannin content (1.8% w/w) and the real value obtained (1.73% w/w) according to the procedure described here.

[131] The phytomedicament containing the standardized extract with a tannin concentration of 1.8% w/w and the placebo described in example 3, were packed in plastic packages similar as those containing 100 g of the gel for topical used afterwards for the clinical essay of example 5. Table 1. Tannin content.

Example 5. Clinical evaluation of the phytomedicament.

[132] A double-blind, randomized controlled clinical study was undertaken with a group of patients with venous ulcers in the inferior extremities. Patients were informed about the possible risks and advantages of the study and also about their right to leave the protocol at any time or in response to the occurrence of adverse reactions to the treatment. A written consent was obtained from each of the participants and the study obtained the approval of the ethical committee of the medical institution where the study was performed.

[133] The study was performed with ambulatory patients of the Regional Hospital No. 1 of the Instituto Mexicano del Seguro Social (IMSS) in the city of Cuernavaca, Morelos, Mexico. With the project approval by the Institution, a total of 40 patients with UIE diagnosis were included. Control and experimental treatments were randomly assigned. Adult patients of both sexes between 30 and 70 years old that complied with the following criteria were included: a) resident of the city of Cuernavaca, b) diagnosed with UIE by the medical equipment of the project, c) no treatment for at least 1 month before the beginning of the study, and d) to provide signed consent for the participation in this clinical test. Those patients with UIE that were pregnant or breastfeeding were not included in the study.

[134] The experimental study was conducted over a period of 12 weeks. Each patient received the phytomedicament of the invention or the placebo described in example 3, and was also provided with instructions about the daily use of the treatment that consisted in daily washing of the ulcerated area with boiled water and neutral soap followed by the application of the phytomedicament or placebo (about 500 mg of the gel under essay); and the subsequent covering of the lesion with a simple compression bandage.

[135] Patients were cited under a weekly scheme for continual medical following. During the first and last medical consult, blood samples of the patients were obtained to monitor their hepatic-renal function (TGO and TGP, creatinin and urea) to determine the therapeutic safety of the administered treatments.

[136] The clinical evolution was determined for all the patients measuring the size of the wounded area once a week during the period of the clinical essay with the aid of a digital photographic camera and processing of the data in an image analyzer (LSM5 browser, Carl Zeiss). Quantification of the healing effect was established comparing the size of the wounded area in cm at the beginning of the study with the size of the lesion at each weekly consult; the obtained values were expressed as cicatrization percentage.

[137] Results obtained with the measuring instruments were analyzed with STATA software. Deductive analysis consisted in comparing the variables evaluated in the experimental group with those obtained for the control group. X test was used to determine proportion differences, ANOVA test was employed for the establishment of significant differences and p<0.05 or p=0.05 values were considered to establish the significance.

[138] Table 2 shows the general characteristics of the population under study, statistic analysis of selected parameters did not exhibit important differences between the groups under treatment; 40 patients diagnosed with UIE were included in the essay: randomly 20 patients were assigned to the experimental group and 20 to the control group.

[139] Mean age of the population under study was 61 years, showing that the affliction is more common during the second half of life in humans. Body mass index (BMI) showed a mean value of 30.6 for the total sample, indicating that great proportions (82.5%) of patients were overweight or obese. Only 22.5% of patients studied, declared antecedents of UIE among their relatives.

[140] All patients presented varicose veins in the same leg afflicted by ulceration, but the time of evolution of this symptom varied with a mean value of 16.5 years, on the other hand the mean value of time of evolution for the UIE was of 8.5 years. In 72.5% of cases, patients reported the previous use of another drug without success: 51% of the patients were parenterally administered with diverse products while 20% of patients reported the local application of diverse remedies (see table 2).

[141] A great proportion of patients of both sexes showed oedema, ochre dermatitis and local pain in the ulcerated area, free of infections in most cases.

[142] Table 3 and figures 2 to 5 show that the size of the ulcer clearly reduced its size by the end of the observation period among patients treated with the phytomedicament of the invention containing M. tenutflora extract. Within this group we found, by comparing the size of the lesion at the beginning and end of the study, that the wounded area was reduced in 73% by the end of the study. The curative effect of the coadjuvant phytomedicament was observed since the first weeks of treatment and the process of complete cicatrization varied in time between different patients of this group depending on the size of the wounded area. Table 2. Basic clinical characteristics of the population under study.

Nevertheless, at the end of the eight week the majority of the cases completely cicatrized (see figure 2). Within the control group that received the placebo hydrogel, no reduction in the size of the lesion was observed during the first 6 weeks in none of the cases. In contrast, after the seventh week of treatment an increase in the lesion size was detected with greater mean values of ulcerated surface in comparison with the conditions at the beginning of the study. Table 3. Effect of the coadjuvant treatments in patients with UIE.

[144] *Expressed as percentage with respect to the size of the original wounded area.

[145] Results demonstrated that the extract derived from the M. tenuiflora bark facilitate the cicatrization of skin ulcers when used as a coadjuvant in the treatment of UIE in ambulatory patients, in most cases depending on the size of the wounded area the complete cicatrization of the ulcer was obtained at the end of 8 to 12 weeks of treatment with the phytomedicament of the invention containing the M. tenuiflora bark extract. The efficiency of this phytomedicament was demonstrated among patients with ulcerations who suffered for many years (mean value, 8-9 years) from this affliction and who tried to heal them without success with several remedies and topic procedures, with both traditional and allopathic medicine. In the course of the clinical study no secondary or adverse reactions were detected during the 3 months of daily use of the phytomedicament containing the extract, which shows the safety and good tolerance presented by the phytomedicament of the invention.

Example 6. Analyses of the pharmacological activity of the Mimosa tenuiflora extract of the invention and some of its derived fractions.

[146] With the aim to elucidate the therapeutic effect presented by the extract of the invention in patients with UIE (see example 5), basic pharmacological studies were done, with both the complete extract and with some of the fractions derived from it. These studies permitted the evaluation of its anti-inflammatory and antimicrobial effects, as well as its effects upon cellular growth.

[147] I. Obtaining of the fractions of the extract of the invention.

[148] a) Obtaining of the saponin fraction (fraction A).

[149] The dry pulverized bark of Mimosa tenuiflora (500 g) was extracted twice with hexane to eliminate the fats, to be subsequently extracted twice with methanol at room temperature. Methanol was eliminated by vacuum and the resultant residue was dissolved in 1% NaOH water solution to eliminate the tannins. The resultant alkaline suspension was portioned with water saturated n-butanol. After the azeotropic concentration of the butanolic extract, a portion that was again suspended in a minimal volume of methanol added with diethyl ether was used; the overflow of the obtained methanolic suspension was concentrated under vacuum to obtain the fraction rich in saponins (fraction A). [150] b) Obtaining of the tannin fractions (fraction B and fraction C).

[151] The extract obtained in example 2 was separated in a Sephadex LH-20 column⁴⁸'⁴⁹ previously prepared in 80% ethanol. The small phenolic compounds were eluted with 95% ethanol, while tannins were eluted with watery acetone (1:1, v/v). Later the remanent acetone was eliminated by means of reduced pressure rotatory evaporation and the resulting portion was extracted with ethyl acetate in a separation funnel. The organic superior stage was concentrated until dryness and after the evaporation of the ethyl acetate traces at a temperature below 30°C, the water soluble fraction was lyophilized obtaining a light maroon powder (fraction B) containing tannins of high molecular weight (tetramers, oligomers and polymers). The portion of ethyl acetate essentially contained small proantocianidoles and galotannins (fraction C).

[152] II. Biological studies of the extract of the invention and its fractions.

[153] a) Anti-inflammatory activity.

[154] ICR female and male mice (28-30 g) were arranged in groups of six members each one. The right ear of each mouse was treated with 2.5 g of

12-O-tetradecanoylphorbol-13-acetate (TPA) dissolved in 20 μl acetone, being applied 10 μl in the anterior and posterior surface. The left ear (used as control) received only the vehicle in the same form and volume. The extract of the invention and the A, B and C fractions (dissolved in 70% ethanol) as well as the drug of reference (dissolved in acetone), were administrated topically 15 minutes after the treatment with TPA. Ninety minutes later the animals were sacrificed and their ears were cut. The central sections of the ear were obtained using punches with a diameter of 6 mm and then weighted. The weight increase of the right ear samples in relation to the weight of the left ear samples indicated the magnitude of the oedema ' ' .

[155] The obtained results are showed in figure 6, where the effect produced by the different concentrations (10, 20, 40, 80 and 160 mg/ml) of the extract of the invention can be observed. The results show that the extract of the invention was able to reduce the oedema induced by TPA, being the effect directly dependent upon the concentration of the extract, and that the highest concentrations (80 and 160 mg/ml) were significantly (p<0.001) different with respect to the control group.

[156] All the fractions derived from the extract of the invention exhibited an antiinflammatory effect in the concentrations used for the test, but the observed effect was dependent upon the concentration only in the C fraction. In the case of A and C fractions an important anti-inflammatory effect was observed but the dependency of the effect with the concentration of these fractions was not clear. It is important to mention that all the fractions of the extract of the invention showed significant differences with respect to the control group (p<0.001) and did not show significant differences among them (p>0.05). Nevertheless, the more consistent and clear antiinflammatory activity was observed only with the complete extract of the invention.

[157] The previous results showed that the extract of the invention exhibit capacity to reduce the TPA induced ear oedema, indicating the presence of active ingredients with anti-inflammatory properties, especially among the proantocianidoles and galotannins group. Given the characteristics of the used experimental model, it is possible to conclude that the extract of the invention may be interfering with the metabolism and liberation of the arachidonic acid, which is the underlying TPA effect action mechanism through the protein kinase C receptor site in this model ³.

[158] b) Antimicrobial activity.

[159] The following bacterial strains were used: Staphylococcus aureus ATCC 6358

(Rockville, MD); Streptococcus faecalis (ATCC 10231); Escherichia coli (ATCC 8937); Klebsiella pneumoniae (ATCC 13883) Salmonela typhi (ATCC 06539) and Candida albicans (ATCC 10231). Bacteria were maintained and tested in trypticase soy agar (TSA, Merck) and the yeast in Sabourad-dextrose agar (SDA, Merck).

[160] The antimicrobial activity of the extract of the invention and A, B and C fractions, was measured determining the minimum inhibitory concentration (MIC) of each one of them by means of the agar dilution method. The extract and its fractions were dissolved in dimethylsulfoxide (Merck) until respectively obtaining a 70 mg/ml and 10 mg/ml concentration. According to the internationally accepted techniques for this kind of studies, the dilution of these products were prepared to obtain final concentrations of 0.5 to 8.0 mg/ml for the extract and 0.250 to 2.0 mg/ml for its fractions. The inoculum for each organism was prepared of cultures that contained 108 colony forming units (CFU)/ml. The diluted inoculum (1:20) was applied on a culture plate as a dot by means of a handle calibrated to apply 0.002 ml, given by result an inoculum dot that covered a 5-8 mm diameter circle containing 10⁴ CFU. Plates containing bacteria were incubated for 24 hrs and that containing yeasts for 48 h, both at 37°C. Gentamicin and nystatin (1.0-128.0 μg/ml) (Sigma) were used as reference standards. Observations were performed twice and results expressed as lowest plant extract concentration capable of completely suppressing the growth of microbial colony. Minimal inhibitory concentration (MIC) was defined as the lowest extract concentration that inhibits visible growth in agar.

[161] Table 4 shows the obtained results. The extract of the invention inhibited all microorganisms under essay, showing its broad spectrum antimicrobial action. Its strongest inhibitory antimicrobial action was against gram positive bacteria under a low MIC (less than 1.0 mg/ml), while gram negative bacteria and yeast were inhibited under a MIC value of 8 mg/ml. In the case of the fractions it was observed that the fraction rich in saponins (fraction A) lacked of antimicrobial activity within the acceptable limits in this test, while the fraction rich in tannins (B and C) had a clear effect over gram positive bacteria given that inhibition was observed under a MIC of 250 μg/ml. However the best antimicrobial activity spectrum was obtained only with the complete extract of the invention.

Table 4. Minimal inhibitory concentrations (MIC, mg/ml) of the extract of the invention and its fractions.

[162] Sa = Staphylococcus aureus, Sf = Streptococcus faecalis, Ec _^Escherichia coli, Kp

= Klebsiella pneumoniae, St = Salmonella typhi, Ca = Candida albicans, NT = Not tested.

[163] c) Effect upon cellular growth.

[164] We used MRC-5 cells (normal fibroblasts from embrionary lung) obtained from

ATCC that were maintained in an RPMI culture environment with foetal bovine serum at 10% to be cultured at 37°C with an air atmosphere of 5% of CO 2 with a 100% ₂ humidity. Cells in logarithmic growth phase were placed in culture plates of 2.25 cm (multi 24) at 2.5 X lO cells/plaque in 1 ml of complete culture media. On day 0, the extract of the invention and fractions A, B and C, were dissolved in DMSO and aggregated to each one of the culture plates in a 2 mg/ml concentration. No extract or fractions were aggregated to the control groups. All cultures were incubated for four days at 37°C, with an air atmosphere of 5% of CO and 100% humidity. The experiment was conducted three times and cultured cells were collected on days 2, 3 and 4 to determine its growth status. Obtained results are showed in table 5. Table 5. Effect produced by the extract of the invention and its fractions upon growth of the MRC-5 cellular line.

[165] Results show that the non-fractioned extract of the invention can induce a clear increase in cell proliferation of in vitro cultivated fibroblasts. On the other hand, fractions A and B also exhibited the mentioned effect in the same direction but more attenuated that the complete extract, whereas fraction C does not have the effect of cell proliferation, being observed on the contrary inhibition of the cellular growth.

[166] As can be observed from the previous results, the pharmacological effects produced by the extract of the invention are owed to the multiplicity of components present in the extract which all contribute to the anti-inflammatory, antimicrobial and cellular growth inducing properties of it. Despite that some fractions derived from the extract exhibited a greater or lesser effect on some of the proved pharmacological properties, none of them possess all the pharmacological effects observed in the complete extract of the invention.

[167] Despite that Tellez described the obtaining of an topic application hydrogel containing Mimosa tenuiflora extracts, it does not contain the complete extracts from the tree bark because chloroform and heat treatment (50 to 70°C) was used to eliminate alkaloids, fats and other chemical compounds from the cortex. With such treatment great part of the tannins and the other substances responsible for the observed pharmacological effects of Mimosa tenuilfora extracts are eliminated. Moreover, the described hydrogel does not contain standardized quantities of none of its components including its active ingredients.

[168] In contrast with the hydrogel described by Tellez²⁶, which contains an incomplete

M. tenuiflora extract because its lack of alkaloids, the phytomedicament of the invention owes its pharmacological effects to the combined components present in the standardized M. tenuiflora extract described here and not only to a fraction of them (see example 6). In addition, the hydrogel described by Tellez²⁶ uses alcoholic substances in its composition (isopropanol), generating irritation to the patients with exposed wounds such as in the case of ulcers in the inferior extremities (UIE). Moreover, the use of methyl p-hydroxybenzoate affects the stability and viscosity of the composition so that it does not conserve its hydrogel form affecting in the long time the release of the active ingredients on the wound under treatment.

[169] Due to the previous, the application of the hydrogel described by Tellez²⁶ does not allow the establishment of prolonged clinical treatments in patients with chronic skin diseases and neither the establishment of adequate dosage regimes for each of the pathologies under treatment. It thereby results particularly difficult to give an adequate clinical following to patients. Especially in the case of skin pathologies that affect the tissue for several years as in the case of ulcers in the inferior extremities (UIE), is important the administration of controlled quantities of the active ingredients responsible for the therapeutic effect in the affected zone. Moreover, the mentioned document does not show effective clinical results in patients affected with chronic skin alterations (as in the case of ulcers) limiting itself solely to show the effect of the extract in solution on burned patients with several degrees of damage.

[170] Efficient alternative treatments for ulcers in the inferior extremities have been presented that employ in vitro cultivated allogenic epidermal laminae ⁴, nevertheless before the application of this laminae to the ulcer, it is necessary to administer antibiotics to eradicate infections in the affected zone and so that the treatment can have the desired effect. In contrast, the phytomedicament of the invention can be directly applied to the affected zone allowing the effective treatment of the ulcer with no need to administer antibiotics to prevent potential infections (see example 5).

[171] With respect to the possible curative mechanisms of the used M. tenuiflora bark extract, these are actually very difficult to determine. The healing effect could be related to the presence of saponins and/or tannins in the extract. In the case of saponins, although showing very low concentrations in the bark, it has been described that mimonosides possess in vitro cicatrizant properties and also induce certain im- munostimulant effects²². On the other hand, the high tannin concentration in the same extract is considered as the predominant composition implicated in the curative effect observed, as demonstrated by the results described in this document.

[172] It is well known that natural products containing condensed tannins are used in the medical practice to assist the healing of wounds and burns⁵⁵; moreover according to the Modern Herbal Vademedum, plant extracts from Centella asiatica, Tabebuia im- petiginosa or Quercus robur (among several others) contain as much as 2.0% of condensed tannins and are comercially used for the treatment of skin ulcers . However the precise rol that plays this group of compounds in cicatrization processes are not entirely clear.

[173] It is reported that tannins possess ample antimicrobial characteristics through diverse mechanisms that include enzymatic inhibition, oxidative phosphorylation reduction and iron privation among others ⁷. Also, astringency as a well known property of tannins has not been explored pharmacologically but it is clear that the effects produced by them are radically different of those observed during the leather tanning and dying process. [174] Microcirculation protection by activation of endothelial inhibition is reported as a mechanism of action of diverse tannin rich extracts with therapeutic efficiency in the management of chronic venous insufficiency . The medical use of the bark extract from Hamamelis virginiana in the treatment of vascular and anal mucus inflammation is based on the fact that its procyanidins (ETl inhibitors) restore endothelial function . Is reported that the anti-inflamatory and cicatrizant activity of the Echinacea pallida root extracts is related with the antihyaluronidase activity of its echinosides, a group of compounds derived from caffeic acid ⁹.

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Claims

[I] A Mimosa tenutflora standardized extract, characterized because containing a 12 to 36% w/w tannin concentration, measured as tannic acid concentration.

[2] The extract of claim 1 characterized because it is in colloidal form.

[3] A pharmaceutical composition for the treatment of skin lesions in mammals, characterized because comprise a therapeutically effective quantity of the extract of the claim 1 to 2 in a pharmaceutically acceptable vehicle.

[4] The pharmaceutical composition of claim 3, characterized because is in a pharmaceutical presentation selected from the group comprise gels and creams.

[5] The pharmaceutical composition of claim 4, characterized because is in the form of gel.

[6] The pharmaceutical composition of claims 3 to 5, characterized because containing a tannin concentration of 5 to 10 mg measured as tannic acid concentration, in every 500 mg of the composition.

[7] A pharmaceutical composition for the treatment of skin lesions in mammals, characterized because comprise: a) A therapeutically effective quantity of the extract of claims 1 to 2, b) A cross-linking polymer of acrylic acid of high molecular weight as gelling agent, c) A dissolvent polymer of the extract, d) A neutralizing alkaline agent, and e) Water.

[8] The pharmaceutical composition of claim 7, characterized because the acrylic acid polymer is selected from the group comprise carbopol with a 1,000 to 50,000 cP viscosity, carbopol 934, carbopol 940, carbopol 941, Ultrez, NOVEON AA-I and PEMULEN.

[9] The pharmaceutical composition of claim 8, characterized because the acrylic acid polymer is carbopol 940.

[10] The pharmaceutical composition of claim 8, characterized because the acrylic acid polymer is in 0.2 to 1% weight concentration with respect of the total weight of the composition.

[II] The pharmaceutical composition of claim 7, characterized because the dissolvent polymer of the extract is polyethylene glycol 200.

[12] The pharmaceutical composition of claim 11, characterized because the dissolvent polymer of the extracts is in a weight proportion of 1 : 1 with respect to the extract.

[13] The pharmaceutical composition of claim 7 characterized because the alkaline neutralizing agent is selected from the group comprise trietanolamine, iso- propilamine, diisopropilamine and morfolin.

[14] The pharmaceutical composition of claim 13, characterized because the alkaline neutralizing agent is trietanolamine.

[15] The pharmaceutical composition of claim 13, characterized because the alkaline neutralizing agent is found in a sufficient quantity to adjust the pH of the composition to a physiological pH.

[16] A method for the obtaining of the extract of claims 1 to 2, characterized because comprising the following steps: a) Selection of the Mimosa tenutflora bark, botanically identified and previously dried, with a minimum tannin content of 12% w/w, measured as tannic acid concentration, b) Grinding of the cortex selected in step a) until obtaining a particle size, of at least mesh 8, c) Extracting of the powder of step b) with ethanol at room temperature during a period of 24 to 72 hrs, d) Filtering of the obtained mixture and concentrate the filtrate through evaporation.

[17] The method of claim 16, characterized because in step c), ethanol is found in a

70% w/w proportion.

[18] The use of the extract of claim 1 to 2, for the manufacturing of a phy- tomedicament for the treatment of cutaneous ulcers in mammals.

[19] The use of claim 18, characterized because a dose of the extract equivalent to

5-10 mg of tannins is directly administered to the ulcer to be treated.

[20] The use of claim 19, characterized because a dose of the extract equivalent to 6-9 mg of tannins is directly administered to the ulcer to be treated.

[21] The use of claims 18 to 20, characterized because the extract is topically administered.

[22] The use of claim 21, characterized because the extract is administered in a single dose.

[23] The use of claim 22, characterized because the extract is administered in several doses, at least once a day and during 8 to 12 weeks.

[24] The use of claims 18 to 23, characterized because the extract is administered until the complete disappearance of the clinical symptoms.

[25] The use of a Mimosa tenutflora extract for the manufacturing of a medicament for the treatment of cutaneous ulcers in mammals which are associated to dysfunctions in the circulatory system.

[26] The use of claim 25, characterized because the circulatory system dysfunctions are selected from the group comprise chronic venous insufficiency, chronic arterial insufficiency and diabetes.

[27] The use of claim 26, characterized because the circulatory system dysfunction is chronic venous insufficiency.

[28] The use of claim 26, characterized because the circulatory system dysfunction is chronic arterial insufficiency.

[29] The use of claim 26, characterized because the circulatory system dysfunction is diabetes.

[30] The use of claim 25, characterized because the cutaneous ulcers are ulcers in the inferior extremities.

[31] The use of claims 25 to 30, characterized because a dose of the extract equivalent to 5-10 mg of tannins is directly administered to the ulcer to be treated.

[32] The use of claim 31 , characterized because a dose of the extract equivalent to 6-9 mg of tannins is directly administered on the ulcer to be treated.

[33] The use of claim 31 to 32, characterized because the extract is topically administered.

[34] The use of claim 33, characterized because the extract is administered in a single dose.

[35] The use of claim 33, characterized because the extract is administered in several doses, at least once a day and during 8 to 12 weeks.

[36] The use of claims 25 to 35, characterized because the extract is administered until the complete disappearance of the clinical symptoms.

[37] A method for standardizing the concentration of active ingredients in the Mimosa tenuiflora bark extracts, characterized because it comprise the determination of the tannin concentration in the extract, measured as tannic acid concentration.

[38] The method of claim 37, characterized because comprising the following steps: a) Determination of the tannin concentration, measured as tannic acid concentration, present in the Mimosa tenuiflora bark to be used for the obtaining of the extract, and b) Determination of the tannin concentration, measured as tannic acid concentration, present in the extract.

[39] The method of claim 38, characterized because the minimum tannin concentration, measured as tannic acid concentration present in the bark is of 12% w/w.

[40] The method of claim 38 characterized because the minimum tannin concentration, measured as tannic acid concentration, present in the obtained extract is of 12% w/w.