Use of a vegetal extract as an active agent in tissue re-epithelizing and cicatrizing processes

Abstract

The use of Salvia haenkei extract is described as a re-epithelizing and cicatrizing agent in the treatment of tissue lesions. Pharmaceutical compositions comprising Salvia haenkei extract and suitable pharmaceutically acceptable excipients for use in the treatment of tissue lesions are also disclosed.

Claims

1. A method of re-epithelizing and cicatrizing tissue lesions, said method comprising administering to a subject in need thereof a Salvia haenkei extract as a re-epithelizing and cicatrizing agent, and re-epithelizing and cicatrizing said tissue lesions, wherein said tissue lesions are maculae, papulae, vesicles, bullae, pustulae, cysts, erosions, abrasions, rashes, ulcers, chapping, sores, decubitus ulcers, telangiectasias, scales, erythema, crusts, lichenifications, excoriations, indurations, cuts, lacerations, diabetic lesions and ulcers, or burns.

2. The method of claim 1, wherein said extract is to be administered via external topical route.

3. The method of claim 1, wherein said extract is to be administered via external topical route in a dose of 1-1,000 mg per day.

4. The method of claim 1, wherein the Salvia haenkei extract is in the form of a pharmaceutical composition further comprising pharmaceutically acceptable vehicles.

5. The method of claim 4, said pharmaceutical composition being administered via external topical route.

6. The method of claim 5, said composition being in the form of ointment, lotion, cream, emulsion, paste, gel, aqueous solution, spray, patch, serum, soaked gauze, dressing, or a combination thereof.

7. The method of claim 4, said pharmaceutical composition comprising Salvia haenkei extract in a concentration of 0.1-500 mg/ml of said pharmaceutical composition.

8. The method of claim 4, said pharmaceutical composition further comprising collagen type II, silver and its derivatives, glycosaminoglycans, antibiotics, propolis, amino acids, growth factors, chitosan, chitin, silicates, zeolites, Triticum vulgare extract, enzymes selected from the group consisting of collagenase, protease and catalase, and mixtures thereof.

9. A pharmaceutical composition comprising Salvia haenkei extract and at least one of collagen type II, silver and its derivatives, consisting from the group consisting of silver sulfadiazine, chondroitin, chondroitin sulfate, dermatan sulfate, keratan sulphate, heparin, heparan sulfate, antibiotics, propolis, amino acids, growth factors, chitosan, chitin, silicates, zeolites, Triticum vulgare extract, enzymes such as collagenase, protease and catalase, and mixtures thereof.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) The characteristics and the advantages of the present invention will become clear from the following detailed description, the working examples provided for illustrative purposes and the accompanying figures, wherein:

(2) FIG. 1 shows the trend of reclosing the injured area of the treatment group in comparison to the positive (Iruxol®) and negative (untreated) controls, as per Example 2; and

(3) FIG. 2 shows the trend in % (calculated on the group mean) of tissue re-epithelialization of the treatment group compared to the positive (Iruxol®) and negative (untreated) controls, as per Example 2.

DETAILED DESCRIPTION OF THE INVENTION

(4) Salvia haenkei is a shrub coming from Bolivia and Peru, and although it is commonly called “prawn sage” due to the color and shape of its shrimp-like flowers, its scientific name dates from the era of Spanish exploration of the Americas at the end of the eighteenth century. Morphologically, it is characterized by lance-shaped leaves with dentate margins whose length exceeds 12 cm. Their color is light green and the surface is wrinkled. The inflorescence is very long, over 20 cm and is defined as “raceme”, i.e. the flowers are inserted at the level of the central axis through the peduncles of the same length at different heights along the same flower axis.

(5) For the preparation of the extract, the aerial parts of the plant are generally used, i.e. stem, leaves, flowers or mixtures thereof. These parts can be used fresh or after drying under controlled conditions. In both cases, the individual parts or mixtures thereof are contacted with a suitable extraction solvent, by using conventional extraction methods, such as maceration or percolation, or more complex techniques, such as for example extraction with ultrasound, microwaves, pressure or supercritical fluids.

(6) After separation of the exhausted plant, the extract can be used as such, or after substitution of the extractive solvent with one more suitable for human use (such as glycerine or glycol, if not used in the extraction phase). Preferably, the extracting solvent is removed to give a dry extract. For the removal of the extractive solvent, the preferred techniques are evaporation at reduced pressure and low temperature, and atomization. The extract can also be subjected to subsequent purification steps, to remove potential contaminants (such as lipophilic pesticides), impurities (such as chlorophyll) or to increase the concentration of secondary metabolites.

(7) The dry extract can be added with suitable excipients, for example to make it smoother, less hygroscopic or standardized in the content of secondary metabolites. Among the excipients that can be used are, for example, silica, maltodextrins, microcrystalline cellulose.

(8) Among the solvents suitable for the preparation of Salvia haenkei extract, those with a medium polarity are preferably selected, as being capable of effectively extracting the secondary metabolites of the plant. Preferably, such extraction solvents have dielectric constant of 8 to 60. The so obtained Salvia haenkei extract contains a pool of terpenoid compounds, in particular diterpenoids and triterpenoids (Almanza, G. et al., (1997) Clerodane diterpenoids and an ursane triterpenoid from Salvia haenkei Computer-assisted structural elucidation, Tetrahedron, 53 (43), pp. 14719-14728), as well as gallic acid and its derivatives, and chlorogenic acid and its derivatives. Some of these compounds are specific to this species of Salvia and differentiate it from other species of the same genus, contributing reasonably to the characteristic activity of its extracts.

(9) Examples of usable extraction solvents are alcohols having up to 4 carbon atoms, including diols and triols, aldehydes, ketones, organic esters, chlorinated compounds, and mixtures thereof. When miscible, such solvents can also be used in mixture with water. Preferred solvents include methanol, ethanol, isopropanol, butanol, ethylene glycol, propylene glycol, glycerol, acetone, ethyl acetate and mixtures thereof, as such or mixed with water.

(10) In preferred embodiments, said extraction solvent is a water-alcohol solution, even more preferably it is a 40-80% alcohol solution. Said alcohol is preferably methanol or ethanol. Embodiments in which the extraction solvent is a 60-80% ethanol solution are particularly preferred.

(11) Preferably, the preparation of said Salvia haenkei extract comprises the steps of:

(12) 1. collecting aerial parts of Salvia haenkei,

(13) 2. extracting with a solvent,

(14) 3. separating the plant exhausted from the liquid extract, and

(15) 4. removing the solvent to give the dry extract.

(16) The aerial parts of step 1. may be fresh or preliminarily dried. If the aerial parts are fresh, just harvested, the greater amount of water physiologically present in the plant shall be taken into account.

(17) The invention therefore relates to the use of Salvia haenkei extract as a re-epithelizing and cicatrizing agent in the treatment of tissue lesions.

(18) Tissue lesions are are maculae, papulae, vesicles, bullae, pustulae, cysts, erosions, abrasions, rashes, ulcers, chapping, sores, decubitus ulcers, telangiectasias, scales, erythema, crusts, lichenifications, excoriations, indurations, cuts, lacerations, diabetic lesions and ulcers, or burns, said lesions occurring to tissues both external, such as the skin, and internal, such as mucous membranes and gingival tissue.

(19) As will also be seen from the examples provided below, the Salvia haenkei extract has shown an unexpected and surprising re-epithelializing and cicatrizing effect on tissue lesions of various kinds, determined by the particular property of modulating the development of connective tissue through a targeted action on collagen and on the fundamental substance. This peculiar action results into a significant improvement in cicatrization, in an improved re-epithelization and in a normalization of the repairing processes towards an altered permeability of the blood vessels.

(20) Preferably, said extract is to be administered via topical route, more preferably via external topical, sub-cutaneous topical, mucosal topical, gingival topical, intravesical topical, vaginal topical, rectal topical, or ocular topical route.

(21) Preferably, said extract is to be administered in a dose of 0.1-1500 mg per day.

(22) In preferred embodiments, said extract is to be administered via external topical route in a dose of 1-1000 mg per day, the effective dosage being a function of the extent and severity of the tissue lesion to be treated.

(23) In another aspect, the present invention relates to a pharmaceutical composition comprising Salvia haenkei extract and pharmaceutically acceptable carriers, for use in the treatment of tissue lesions.

(24) Said pharmaceutical composition can be administered via topical route.

(25) Preferably, said pharmaceutical composition is to be administered via external topical, sub-cutaneous topical, mucosal topical, gingival topical, intravesical topical, vaginal topical, rectal topical, or ocular topical route.

(26) In preferred embodiments, said composition is to be administered via external topical route.

(27) Preferably, the pharmaceutical composition comprises Salvia haenkei extract in a concentration of 0.1-500 mg/ml of composition, more preferably 1-100 mg/ml.

(28) Said pharmaceutical composition may be in the form of ointment, lotion, cream, emulsion, paste, gel, aqueous solution, spray, patch, serum, soaked gauze, dressing, or a combination thereof.

(29) Said pharmaceutically acceptable vehicles can be rheological additives, buffering agents, antimicrobial agents, antioxidant agents, anti-isothermal agents, antistatic agents, absorbent agents, UV absorbing agents, astringent agents, chelating agents, skin conditioning agents, preservative agents, covering agents, denaturing agents, depigmenting agents, emulsifying agents, film-forming agents, gelling agents, moisturizing agents, hydrotropic agents, binders, soothing agents, smoothing agents, opacifying agents, plasticizing agents, propelling agents, skin protecting agents, reducing agents, cooling agents, sebum-restoring agents, solvents, stabilizing agents, emulsifying stabilizing agents, toning agents, wetting agents, volumizing agents or combinations thereof.

(30) In some embodiments, the pharmaceutical composition for use in the treatment of tissue lesions further comprises at least one other active ingredient, such as collagen type II, silver and its derivatives, such as silver sulfadiazine, glycosaminoglycans, antibiotics, propolis, amino acids, growth factors, chitosan, chitin, silicates, zeolites, or mixture thereof.

(31) Suitable silver derivatives are silver lactate, silver phosphate, silver citrate, silver acetate, silver benzoate, silver chloride, silver carbonate, silver iodide, silver iodate, silver nitrate, silver laurate, silver sulfadiazine, silver palmitate, silver proteinate, or combinations thereof.

(32) Suitable glycosaminoglycans are chondroitin, chondroitin sulphate, dermatan sulfate, keratan sulphate, heparin, heparan sulfate, hyaluronic acid, and mixtures thereof.

(33) In another aspect, the present invention relates to a pharmaceutical composition comprising Salvia haenkei extract and at least one of collagen type II, silver and its derivatives, such as silver sulfadiazine, chondroitin, chondroitin sulphate, dermatan sulfate, keratan sulphate, heparin, eparan sulfate, antibiotics, propolis, amino acids, growth factors, chitosan, chitin, silicates, zeolites, Triticum vulgare extract, enzymes such as collagenase, protease and catalase, and mixtures thereof.

(34) All the pharmaceutical compositions described above can be prepared by methods known in the pharmaceutical technique.

(35) It should be understood that all the aspects identified as preferred and advantageous for the Salvia haenkei extract are to be deemed as similarly preferred and advantageous also for the pharmaceutical compositions and uses thereof.

(36) It should be also understood that all the combinations of preferred aspects of the Salvia haenkei extract of the invention, as well as of the pharmaceutical compositions and uses of the same, as above reported, are to be deemed as hereby disclosed.

(37) Below are working examples of the present invention provided for illustrative purposes.

EXAMPLES

Example 1

(38) Preparation of Salvia haenkei Extracts

(39) 10 kg of aerial parts of Salvia haenkei are harvested from field crops, which are then subjected to a drying process in a ventilated dryer under controlled conditions.

(40) In this way, 1.95 kg of dried plant are obtained, which are minced into a bladed mill to give dried and ground Salvia haenkei.

(41) This is used as raw material for the subsequent solvent extraction tests carried out as described below: 1. 100 g of dried and ground Salvia haenkei are introduced into a static percolator and covered completely with 200 ml of a water and ethanol 30-70% v/v mixture. It is left to stand for 2 hours and the extraction solvent (170 ml) is recovered from the bottom of the percolator, which is set aside (extract 1); 2. the humid plant left in the percolator is covered with a new 70% aqueous ethanol (170 ml) aliquot, leaving it to rest for 2 hours. The solvent is recovered (165 ml-extract 2); 3. the extraction described in point 2 is repeated until the dry residue of the extract recovered is less than 5% of the total dry residue extracted up to that moment. At that point, the extraction is considered completed and the spent moist plant is eliminated. 6 extractions are required; 4. the extracts obtained from the individual extraction steps (from extract 1 to extract 6) are combined, filtered and concentrated in a rotary evaporator under vacuum, at a low temperature. It is proceeded until a concentrated, viscous solution (35 ml) is obtained; 5. the concentrated extract is transferred to a steel tray and inserted into a under vacuum cabinet dryer, with heating set at 30° C. After 12 hours, the solvent is completely removed (extract weight loss less than 10%, i.e. dry residue higher than 90%). 14.3 g of integral dry extract are obtained. The ratio drug:extract (DER) is 7:1 (extract 1A). 6. the dried extract obtained is added with 10 g of maltodextrin (DE 10) to improve its consistency and the mixture is milled and sieved, thus obtaining 23.7 g of ground dry extract.

(42) By applying the same procedure but different extracting solvents different native dry extracts were prepared.

(43) The table summarizes the results of the various extraction tests:

(44) TABLE-US-00001 extract extraction solvent DER 1A ethanol:water 70:30 7:1 1B ethanol:water 95:5 9.5:1.sup.  1C acetone 11:1  1D methanol 8:1 1E ethyl acetate 15:1  1F water 5:1 1G methanol:water 50:50 6:1

Example 2

(45) The aim of this study was the evaluation of the bioactivity of the hydroalcoholic extract of Salvia haenkei of Example 1A, to verify its re-epithelizing and cicatrizing power. To do this, tissue damage was induced by an excisional model in mice.

(46) In particular, the model was established in mice of the BALB/c strain of 6-7 weeks. The animals were divided into experimental groups: a positive control group (treated with a healing cream, Iruxol®, i.e. 1% collagenase+60 IU chloramphenicol), a negative (untreated) control group, and a group treated with the hydroalcoholic Salvia haenkei extract (briefly “salvia A” at 0.5%, or 5 mg/ml). The tested formulations, with the same formulation of the excipients, were topically administered every day for a period of 18 days, covering the wound with a quantity of product to cover an area equal to 19.6 mm.sup.2.

(47) Said excipients were:

(48) 10% Glycerol

(49) 7% Sorbitol 70%

(50) 1% Carbomer

(51) 0.3% Sodium hydroxide

(52) balance to 100% purified water

(53) The wound closure was monitored and documented by taking photographs on alternate days. The acquired images allowed to make measurements by using dedicated software in order to quantify the bioactivity of the tested products.

(54) The data obtained confirmed the active role of salvia A in wound repair processes.

(55) Results

(56) The cicatrizing potential of the tested formulations is expressed in terms of wound closure, i.e. the percentage ratio between the area healed and the total area of the lesion in question, normalized taking into account the absolute measurement error.

(57) With reference to FIGS. 1 and 2, the trend of the “front” of re-epithelialization on day 2 showed similarities in the two groups treated, respectively, with Salvia A 0.5% and Iruxol® (positive control), for which a greater “closure” of the epithelial borders. Treatment with the reference compound used as a positive control induced a significant reduction of the injured area compared to the untreated group, starting from the fourth day of treatment and for the entire duration of the study, leading to a statistical forecast of the closure of the 50% in only 2 days and complete re-epithelization about 2.5 days before the untreated group (13.5 vs. 15.9 days).

(58) A behavior similar to that of the positive control was observed for the treatment with Salvia A. Salvia A, in fact, induced a re-epithelization of the injured area, between day 4 and day 11, significantly greater than that observed for the untreated group. Furthermore, the trend of the re-epithelization of the injured area of the group with Salvia A was comparable to that of the group treated with Iruxol.

(59) The treatment with Salvia A, moreover, led to a re-epithelization equal to 50% of the initial injured area already after 2.7 days and to complete re-closure after 14.2 days (statistical forecast), almost 2 days before the untreated group.

(60) In conclusion, the data obtained at 3 weeks of treatment showed that the hydroalcoholic extract of Salvia haenkei at the concentration of 0.5% has a cicatrizing effect equal to that of the control drug.

(61) The data obtained in this study lead to consider positive the use of the hydroalcoholic extract of Salvia haenkei in the days following the induction of the damage. The use of the hydroalcoholic extract of Salvia haenkei 0.5% in re-epithelialization of epithelial tissues has proven to be effective since the second day of application and in determining a better and faster wound cicatrization, which is close to completion after 11 days of treatment, like positive control. In fact, the results on the re-epithelialization times are excellent and superimposable to those obtained with the treatment with the control drug Iruxol®, both in the early and in the late stages of healing. Salvia A has proven to be effective in reducing post-excisional bleeding, already on the first or second day of application, thus decreasing the discomfort present following the application of the splitting membrane and therefore showing itself useful in reducing the clinical signs of tissue inflammation. The product has therefore shown a good bioactivity in terms of anti-inflammatory and anti-hemorrhagic properties in the tissues.

(62) It can therefore be stated that the hydroalcoholic extract of Salvia haenkei can give good results as a cicatrizing and re-epithelializing agent.