WO2021242205A1

WO2021242205A1 - Gel formulations obtained from cistus creticus extract and the use of these as hemostatic agents

Info

Publication number: WO2021242205A1
Application number: PCT/TR2021/050492
Authority: WO
Inventors: Ayşe Esra KARADAĞ; Mehmet Evren OKUR; Neslihan ÜSTÜNDAĞ OKUR; Fatih DEMİRCİ; Fatma TOSUN; Ayşegül ÇAŞKURLU
Original assignee: İstanbul Medi̇pol Üni̇versi̇tesi̇; Anadolu Üni̇versi̇tesi̇
Priority date: 2020-05-29
Filing date: 2021-05-26
Publication date: 2021-12-02
Also published as: TR202008345A2

Abstract

The present invention relates to the gel formulations obtained from the aerial parts of a Cistus species (creticus, asper, chinamadensis, horrens, ocreatus, osbeckiifolius, palmensis, sympytifolius, heterophyllus, albidus) and to the use of said gel formulations as a hemostatic agent in wound treatment.

Description

GEL FORMULATIONS OBTAINED FROM CISTUS CRETICUS EXTRACT AND THE USE OF THESE AS HEMOSTATIC AGENTS

Technical Field of the Invention

The present invention relates to the gel formulations obtained from the aerial parts of the species (creticus, asper, chinamadensis, horrens, ocreatus, osbeckiifolius, palmensis, sympytifolius, heterophyllus, albidus) belonging to Cistus plant and to the use of said gel formulations as a hemostatic agent in wound treatment.

State of the Art

The damage in the body that is caused by physical or chemical reasons and causes deformation on skin or mucous membrane is called a wound. Depending on the injury type, generally internal or external bleedings occur.

The process in which the deformed blood vessels and protect their integrity and stop the bleeding is called hemostasis. Hemostasis is a case seen upon the interaction of thrombon and coagulation factors. The materials used to realize the hemostasis is called hemostatic agents. The requirement for a hemostatic agent is an important need in the simple incidents of the daily life to more severe bleeding conditions. The fields of use of the hemostatic agents available today are limited due to their different chemical contents, and these agents cannot focus on healing the wound/tissue deformation causing the bleeding.

It is known that Cistus species are also used by public in Anatolia for wound healing and/or stopping the bleeding.

Even though the ethnobotanical benefits of Cistus species are known, today, mostly patients living in cities cannot reach to such plants and faces difficulties in preparing and using said plant preparations. It is known that plants contain, along with useful components, many other components that can affect the health of people negatively. Therefore, it is important to correctly compartmentalize the different components nested within the plants in terms of the positive contribution of the used product to the health. Therefore, there is a need for pharmaceutical preparations containing an extract obtained from the Cistus species and dose forms that are easy to use. Summary of the Invention

The present invention relates to the gel formulations obtained from the extracts of aerial parts of a species of Cistus genus, for instance Cistus creticus, Cistus asper, Cistus chinamadensis, Cistus horrens, Cistus ocreatus, Cistus osbeckiifolius, Cistus palmensis, Cistus sympytifolius, Cistus heterophyllus, Cistus albidus species.

By this was, it is seen that the herbal preparations obtained from the aerialparts of Cistus creticus and formulated in gel forms provide positive outcomes in both stopping the bleeding occurred during an injury due to its hemostatic feature and healing the wounds. It is seen that by synergistically mixing two different effects by this way, it provides more effective wound healing compared to the products that already exists and known to be used in the prior art. Furthermore, it is seen that products prepared in this form are comfortably used by patient topically and thereby contributes to the success of the treatment.

Description of the Drawings

Figure 1 shows the graphic that indicates the effects of control group (al), empty gel (a2), formulation (a3) that contains 2.5% C. creticus extract and formulation (a4) that contains 7.5% C.. creticus extract on the amount of bleeding (A).

Figure 2 shows the graphic that indicates the effects of control group (al), empty gel (a2), formulation (a3) that contains 2.5% C. creticus extract and formulation (a4) that contains 7.5% C. creticus extract on the duration of bleeding (B) in minutes.

In figures, following markers indicate the following:

A: Amount of bleeding

B: Duration of bleeding al : control group a2: empty gel a3: formulation that contains C. creticus extract at the rate of 2.5% a4: formulation (a4) that contains C. creticus extract at the rate of 7.5%. Detailed Description of the Invention

The present invention relates to the gel formulations obtained from the surface parts of the Cistus species.

In one embodiment of the invention, the Cistus species in the gel formulation according to the invention is selected from Cistus creticus, Cistus asper, Cistus chinamadensis, Cistus horrens, Cistus ocreatus, Cistus osbeckiifolius, Cistus palmensis, Cistus sympytifolius, Cistus heterophyllus, Cistus albidus species and/or subspecies of these species or the combination thereof. In a preferred embodiment of the invention, the Cistus species in the gel formulation according to the invention is Cistus creticus.

In one embodiment of the invention, the extract of the surface parts of Cistus species used in the gel formulations according to the invention is methanol extract.

In one embodiment of the invention, the gel formulation according to the invention involves at least one pharmaceutically acceptable excipient in addition to the extracts of aerial parts of Cistus species.

Said pharmaceutically acceptable excipient can be selected from; solvents, gelling agents, emulsifiers, suspending agents, thickeners, antioxidants, pH adjusters, surfactants, preservers (e.g. antibacterial agents, antifungal agents), isotonic agents, moisture retaining agents, absorption retarding agents, volumizers, preservers, stabilizers, bonding agents, crackers, lubricants, sweetener agents, aromatic agents and the combinations thereof. Specific examples of each group can be found in Remington's Pharmaceutical Sciences, 18^th Ed. Mack Printing Company, 1990 and documents appended to it.

In a preferred embodiment of the invention, the solvent is selected from the group consisting of ethanol, methanol, propanol, isopropanol, water or a binary or ternary mixture thereof. In a particularly preferred embodiment of the invention, water and isopropanol are used as solvents.

In a preferred embodiment of the invention, the surfactant is selected from a group consisting of triethanolamine, sodium lauryl sulfate, sodium dodecyl sulfate, polysorbates; for example, polysorbate 20, polysorbate 60, poloxamer, phosphatidylcholine, benzalkonium chloride, span 20, span80, span 85, cetylpyridinium chloride, cetyltrimethylammonium bromide, dodecyl amine, polyoxyethylene nonylphenol, polyoxyethylene (POE) alkyl esters, sorbitan monopalmitate, sorbitan monolaurate, and polyoxyethylene sorbitan monoesters. In a particularly preferred embodiment of the invention, triethanolamine is used as surfactant and pH adjuster.

In a preferred embodiment of the invention, the moisture retaining agent is selected from a group consisting of glycerol, glycerin, lactic acid, propylene glycol, hexylene glycol, butylene glycol, aloe vera, honey, lithium chloride, sorbitol, xylitol, maltitol, castor oil, and polydextrose. In a particularly preferred embodiment of the invention, glycerin is used as a moisture retaining agent.

In a preferred embodiment of the invention, the gelling agent is selected from a group consisting of polyacrylic acid, such as derivatives of polyacrylic acid commercially known as Carbopol, polyacrylates, hydroxy propyl cellulose, hydroxy propyl methylcellulose, carboxy methyl cellulose, gelatin, ethyl cellulose, hydroxy ethyl cellulose, poloxamers, sodium alginate, magnesium aluminum silicate, potassium alginate, ammonium alginate, calcium alginate, pectin, and agar-agar. If needed, this group can be developed with other gelling agents available in the state of the art. In a particularly preferred embodiment of the invention, polyacrylate is used as the gelling agent.

In one embodiment of the invention, in the gel formulations according to the invention; the extract of the aerial parts of Cistus creticus are used in the range of l%-50%, preferably 2%- 30%, particularly preferably 2%-10% in proportion to the weight of the gelling agent.

In another aspect, the present invention relates to the method of preparation of the gel formulation according to the invention, wherein the said method includes the steps of; a. Mixing the gelling agent, moisture retaining agent and solvent 1, b. Preparing the solution of the surfactant in solvent 2, c. Obtaining the gel mixture by mixing the mixture prepared in step a and the mixture prepared in step b, d. Obtaining the gel formulation according to the invention by adding the extract of the aerial parts of Cistus creticus species to the mixture.

In another aspect, the present invention relates to the gel formulations containing the extract of the aerial parts of Cistus species to be used as the hemostatic agent.

In another aspect, the present invention relates to the gel formulations containing the extract of the aerial parts of Cistus species to be used in the treatment of wounds and burns. In another aspect, the present invention relates to the wound dresses, tampons, pledgets and wound care products containing the gel formulations containing the extract of the aerial parts of Cistus species.

The term “wound” used within the scope of the invention means acute wounds, chronic wounds, infected wounds, wear wounds (peeling of the outer part of the skin), laceration wounds, bruise wounds (wounds consisting of swollen bruises as a result of aggregation of dead cells and blood under the skin), concussion wounds (harm to organs and tissues under skin or skull despite no visible wounds), stab wounds, skin cuts, surgical wounds (wounds intentionally opened in the skin for surgical procedures), gunshot wounds, thermal wounds (excessive hot, excessive cold, frostbite, wounds like suntan), chemical wounds (wounds on skin upon contact with chemicals or wounds on lungs upon inhalation of chemical), bites and stings (resulting from humans and animals like bees, dogs, cats, birds, bats, rodents, snakes, scorpions, spiders and louse), and wounds resulting from electric contacts.

The term “burn” within the scope of the invention means first degree bums (red, non-blistered skin), second degree burns (blistered skin and thickening of skin), third degree burns (diffuse thickening of the skin with white, leathery look) and fourth degree bums (burns extending to tendons and/or bones).

It is clear that a person skilled in the art can implement the technique explained in the invention using similar embodiments and/or apply this embodiment to other fields with similar purposes used in the related technique. Therefore, it is clear that such embodiments will be deprive of criteria of innovation and especially of exceeding the current state of the technique.

Within the framework of these main concepts, it is possible to develop a wide variety of embodiments in relation to the topic of the invention and the invention cannot be limited to the characteristics explained herein, and in its essence, it is as explained in the claims. From this point on, the invention will be explained with the examples below without being subject to any limitations.

EXAMPLES

Example 1: Obtaining extract from the aerial parts of the Cistus creticus plant Aerial parts of C. creticus are collected from (/lnarcik, Yalova, Turkey in May 2018. The plant samples collected are being stored in Anadolu University Faculty of Pharmacy Herbarium (ESSE 15549). Said herbs are air dried, aerial parts are crashed and macerated with methanol to get the extract, then filtration and evaporation steps were employed. The obtained extract is used in biological activity studies and in preparation of gel formulations.

Example 2: Preparation of topical gel containing Cistus creticus extract

The gel containing Cistus creticus extract formulated by mixing Carbopol aqueous gel. To prepare Carbopol gel, isopropyl alcohol, glycerin and Carbopol was mixed with water at 25°C. Also, triethanolamine was added to the mixture. After that, Cistus creticus extract (2.5% and 7.5%) is added and the mixture was slowly mixed.

Example 3: In vivo animal experiments

Animals:

Wistar albino rats were procured from Istanbul Medipol University, MEDITAM, Istanbul, Turkey. Rats were hold in cages containing ad libitum feed and water, at 20±1°C. Ethical approval of the experiment was taken from Istanbul Medipol University Ethical Board (Approval No: 2019-08). The study was conducted in line with the internationally admitted principles for the use and maintenance of the laboratory animals such as in the guides of European Community (1986 AET Directive; 86/609 / EEC). Rats were treated in line with ethical regulations for laboratory animals.

Wound Model and Experiment Groups: Twenty-eight rats used were randomly assigned to four groups consistent of seven animals (n=7);

1. Control group (not-treated group)

2. Empty gel (Carrier) group

3. Group of gel involving 2.5% C. creticus extract

4. Group of gel involving 7.5% C. creticus extract

Animals were anesthetized with a mixture of ketamine (lOOmg/kg, intraperitoneal (i.p.)) and xylazine (lOmg/kg, i.p.). The aim of the study was to measure the coagulation time and bleeding amount to evaluate the local hemostatic system. In vivo bleeding test was performed by tail tip amputation method in rats. Accordingly, the distal 1 cm of the tail of the rat placed face down under anesthesia was cut transversely using a scalpel. After bleeding occurred, each wound was washed with an equal volume (1 mL) of sample. Then, with the aid of a gauze bandage, a light compress was applied to the wound for 1 minute. To determine the coagulation duration, the time that passed from the beginning of the bleeding to static hemostasis was counted with the help of a chronometer. To determine the amount of bleeding; before and after each gauze bandage, the difference was recorded as the amount of bleeding (g). To evaluate the bleeding status, each rat was monitored for 30 minutes. At the end of the study, laboratory animals were killed with carbon monoxide gas under anesthesia.

In vivo hemostatic activity

The hemostatic activity of the gel formulation prepared from C. creticus extract was evaluated statistically by calculating the in vivo experimental bleeding model with the bleeding time (min) and bleeding amount (g). The amount of bleeding is the amount of blood (g) collected from the time the rat’s tail is cut until the bleeding stops. In the control group with no treatment 1.13±0.1 g blood was measured. Additionally, in the group where empty gel formulation (the carrier) was applied, 1.05±0.04 g blood on average is measured. In the treatment groups, 0.76±0.04 g of blood was measured in samples treated with 2.5% extract- loaded gel formulation and 0.99±0.02 g in samples treated with 7.5% extract-loaded gel formulation. Compared to the control group, the 2.5% formulation (p<0.01) group showed a statistically significant difference (Figure 1).

1 Bleeding time is the time expressed in minutes, from the cut of rat’s tail till the bleeding stops. Bleeding time was measured as 27.4±3.6 minutes in the control group with no treatment. In addition, the average bleeding time in the group in which the empty gel (carrier) formulation was applied was determined as 23.6±2.9 min. Bleeding time in the treatment groups was measured as 15.8±1.8 minutes in the samples treated with the 2.5% extract-loaded gel formulation and 16.6±1.2 minutes in the samples treated with the 7.5% extract-loaded gel formulation. Compared to the control group, the 2.5% formulation (p<0.01) and 7.5% formulation (p<0.01) groups showed a statistically significant difference (Figure 2). Result

As a result of the study conducted, it is seen that in terms of both the collected blood amount and the duration of bleeding gels containing C. creticus extract present significant difference compared to control and carrier groups. Therefore, this indicates that the obtained pharmaceutical effect results completely from the plant extract. Gel formulations according to the invention has provided 25% decrease in the blood amount and has shortened the bleeding period about 50%. The obtained results indicate that gel formulations containing C. creticus extract according to invention has hemostatic feature.

Claims

1. Gel formulations containing the extract of aerial parts of Cistus species.

2. A gel formulation according to Claim 1, characterized in that the Cistus species is selected from a group consisting of Cistus creticus, Cistus asper, Cistus chinamadensis, Cistus horrens, Cistus ocreatus, Cistus osbeckiifolius, Cistus palmensis, Cistus sympytifolius, Cistus heterophyllus, Cistus albidus species and/or subspecies of these species or the combinations thereof.

3. A gel formulation according to Claims 1-2, characterized in that the Cistus species is Cistus creticus.

4. A gel formulation according to Claims 1-3, characterized in that the extract of aerial parts of Cistus species is methanol extract.

5. A gel formulation according to Claims 1-4, characterized in that it comprises at least one pharmaceutically acceptable excipient in addition to the extract of aerial parts of Cistus species.

6. A gel formulation according to Claims 1-5, characterized in that the excipient is selected from solvents, gelling agents, emulsifiers, suspending agents, thickeners, antioxidants, pH adjusters, surfactants, preservers (e.g. antibacterial agents, antifungal agents), isotonic agents, moisture retaining agents, absorption retarding agents, volumizers, preservers, stabilizers, bonding agents, crackers, lubricants, sweetener agents, aromatic agents and the combinations thereof.

7. A gel formulation according to Claim 6, characterized in that the solvent is selected from the group consisting of ethanol, methanol, propanol, isopropanol, water or a binary or ternary mixture thereof.

8. A gel formulation according to Claim 6, characterized in that the surfactant is selected from a group consisting of triethanolamine, sodium lauryl sulfate, sodium dodecyl sulfate, polysorbates; for example, polysorbate 20, polysorbate 60, poloxamer, phosphatidylcholine, benzalkonium chloride, span 20, span 80, span 85, cetylpyridinium chloride, cetyltrimethylammonium bromide, dodecyl amine, polyoxyethylene nonylphenol, polyoxyethylene (POE) alkyl esters, sorbitan monopalmitate, sorbitan monolaurate, and polyoxyethylene sorbitan monoesters.

9. A gel formulation according to Claim 8, characterized in that the surfactant is triethanol amine.

10. A gel formulation according to Claim 6, characterized in that the moisture retaining agent is selected from a group consisting of glycerol, glycerin, lactic acid, propylene glycol, hexylene glycol, butylene glycol, aloe vera, honey, lithium chloride, sorbitol, xylitol, maltitol, castor oil, and polydextrose.

11. A gel formulation according to Claim 10, characterized in that the moisture retaining agent is glycerol.

12. A gel formulation according to Claim 6, characterized in that the gelling agent is selected from a group consisting of polyacrylic acid, polyacrylic acid derivatives, polyacrylates, hydroxy propyl cellulose, hydroxy propyl methylcellulose, carboxy methyl cellulose, gelatin, ethyl cellulose, hydroxy ethyl cellulose, poloxamers, sodium alginate, magnesium aluminum silicate, potassium alginate, ammonium alginate, calcium alginate, pectin, and agar-agar.

13. A gel formulation according to Claim 12, characterized in that polyacrylate is used as gelling agent.

14. A gel formulation according to Claims 6-13, characterized in that the extract of the surface parts of Cistus species is used in the range of l%-50%, preferably 2%-30%, particularly preferably 2%-10% in proportion to the weight of the gelling agent.

15. A method to be used in the preparation of a gel formulation according to Claim 1-14, characterized in that it includes the steps of: a. Mixing the gelling agent, moisture retaining agent and solvent 1, b. Preparing the solution of the surfactant in solvent 2, c. Obtaining the gel mixture by mixing the mixture prepared in step a and the mixture prepared in step b, d. Obtaining the gel formulation according to the invention by adding the extract of the aerial parts of Cistus creticus species to the mixture.

16. Gel formulations according to Claims 1-14 to be used as hemostatic agent.

17. Gel formulations according to Claims 1-14 to be used in the treatment of wounds and burns.

18. Pledget, tampon, wound care product and wound dressing containing the gel formulations according to Claims 1-14.