FOMENTARIUS EXTRACT

Technical Field

The invention is related to a production method of a gel formulation that has haemostatic effect obtained from Femes Fomentarius ( tinder fungus ) extract that contains positively charged polysaccharides and a polysaccharide-protein complex.

Known State of the Art (Prior Art)

All of the events that occur at the starting of bleeding in any place of the organism due to any reason, and the stopping of bleeding are called hemostasis. In other words, hemostasis is the physiological mechanism that ensures that the blood stays in the liquid form during circulation. The haemostatic (anti-hemorrhagic) mechanism comprises four different mechanisms including the vascular system, thrombocytes, coagulation proteins and the fibrinolytic system. The blood circulating without coagulation in the vessels, covered with endothelial cells immediately coagulates when it encounters a foreign surface other than the endothelium. Post hemorrhagic coagulation is connected to the correct functioning of the biochemical balance between various parts of the haemostatic system.

Even though the hemostatic process is a single process, it can be studied under different sub stages as primary and secondary hemostasis. The thrombocytes that form a thrombus where there is vascular damage is called a primary hemostasis and the formation of a fibrin clot after the activation of the coagulation system is called a secondary hemostasis. Thrombocytes are blood cells which initiate the first stage of primary hemostasis, which is coagulation, by means of their adhesion, secretion and aggregation functions. Their purpose is to occlude the hemorrhaging area by coagulation.

With the studies made in the last decade, it has been reported that some haemostatic agents achieved a dramatic decrease in posttraumatic mortality (posttraumatic death) rates by means of topical application (applied on a particular skin or mucosal area; superficial) in external (outer, exterior) hemorrhagic (bleeding) injuries. Although clinical studies on the products providing the haemostatic control with the least amount of side effects still continue, in the last few years, studies on the effects of products having zeolite, kaolin and chitosan as active agents have become prominent.

Topical hemostats recommended for use in posttraumatic hemorrhagic cases can be divided into three groups based on their mechanism of action as factor concentrators, mucoadhesive agents and coagulation promoters.

1. Factor concentrators

Plasma constitutes 55% of the total blood volume. The liquid part of the blood besides the blood cells is called the plasma. 90-92% of plasma is water and the rest is formed of elements such as organic and inorganic materials of plasma proteins, amino acids, carbohydrates, fats, hormones, urea, uric acid, lactic acid, enzymes, antibodies, sodium, potassium, iodine, iron, bicarbonate etc. These agents are delivered to the related tissues via the plasma. Plasma proteins constitute the majority of the organic agents of the plasma. These proteins are albumin, globulin and fibrinogen. 7 - 8 g of plasma protein is found in 100 grams (g) of blood, most of which is albumin. The formed elements of the blood form approximately 45% of the blood volume. Blood cells are named erythrocytes, leukocytes and thrombocytes.

Factor concentrators support the formation of coagulation by quickly absorbing the blood plasma and concentrating the plasma proteins and formed elements of the blood in the area of the injury. With this purpose, hemostatic agents having zeolite, which is a volcanic mineral, as the active agent have been prepared. Zeolites are natural minerals that are formed by three- dimensional infinite bonding of the [S1O4] ^"4 and [AIO4] ^"5 tetrahedrons, which are the smallest units of crystal structures, having high ion exchange and adsorption capacity, molecular sieve function by means of the three-dimensional lattice structure, containing micropores comprising alkaline and/or alkaline-earth cations and water molecules.

The first granular zeolite form used for topical hemostatic purposes was launched under the name QuikClot™. Due to the side effects resulting from temperature increases occurring with the exothermic reaction the QC-ACS® form having developed haemostatic product attributions that could be easily and effectively applied and that can be debrided was developed. QC-ACS® was used in a total of 103 cases of hemorrhagic injury in Iraq in 2006 and it was reported that it was 92% effective; however, side effects such as thermal injuries as a result of exothermic reaction and pain was reported. 2. Mucoadhesive agents

The fundamental attribution of products with chitosan effective agent, is the fact that they firmly seal the site of hemorrhage physically with their high adhesion capacity with the tissue. Chitosan is a polymer which is obtained by the deacetylation of the polysaccharide-structure chitin (Poly-N-acetyl glucosamine), which is especially found in shellfish.

The product called HemCon™ that is prepared by the deacetylation of chitosan acetate salt has been started to be used in the American and British army in 2003. It is the first topical hemostat that is deemed appropriate for field use by The Committee on Tactical Combat Casualty Care (CoTCCC). In a study that has been conducted, 74% success was reported with 3 minutes of application in 34 patients with external hemorrhaging, where bleeding control could not be achieved with direct pressure and gauze in a civilian emergency room. However, studies that show low effectiveness compared to other products in the femoral artery injury model are present.

WoundStat® is a smectite mineral in the form of a sodium, calcium and aluminosilicate and it concentrates coagulation factors with a powerful water absorption effect. It swells upon coming in contact with blood, and turns into a clay-like consistency and it covers the hemorrhaging wound surface, due to its high tissue adhesion attribute. It speeds coagulation by activating the coagulation cascade intrinsically (internal) as the smectite granules are negatively charged. However it has been reported that, in the porcine caratoid artery and jugular vein injury model, WoundStat™ created severe endothelium and transmural damage even though it did not cause temperature increase, that it was difficult in terms of debridement and that it caused thromboembolic complications.

3. Coagulation promoters

Dry Fibrin Sealant Dressing™ (DFSD) comprises highly purified human fibrinogen, thrombin, calcium and factor XIII. It was not granted clinical use approval due to viral transmission risks, even though there are high technological capabilities. It was reported to be effective in kidney injuries, liver injuries, aorta injuries, and lethal extremity artery injuries; however, it is very expensive.

The product produced by means of soaking kaolin nanoparticles of smectite minerals having an aluminosilicate structure in a cloth containing 50% polyester and 50% artificial silk, that was released to the market under the name QuikClot™-Combat Gauze by the Company Z- Medica in 2008, was accepted to be the standard topical hemostat recommended for use in the field, the same year by CoTCCC. In the study published regarding this product used by the Israeli and American army in the field, it was reported that said product was used in 14 cases in Gaza in 2009, and that creating hemostasis was successfully achieved in 11 cases; however hemorrhage control could not have been achieved in three cases with neck, hip and femur injuries. No complications and side effects have been detected; however, since the product exhibits its effect by activating the intrinsic coagulation cascade of the patient, which differs from mucoadhesive agents, it was emphasized that it has latent effect, and therefore that it could have low effects on patients suffering from coagulopathy.

The technical report prepared at the Medical Research Unit of the American Naval Forces in 2012 indicates that the effectiveness of the QuikClot™-Combat Gauze accepted as the standard topical hemostat by CoTCCC is low with 30% hemostasis and 60% survival rate. In the porcine method wherein femoral artery injury was created, the subjects were followed in terms of survival, hemostasis and blood loss for 150 minutes. Blood loss was more common in the group where QuikClot™-Combat Gauze and Celox™ Trauma Gauze was applied, and the survival rates were detected to be less. As a result of this study, it was indicated that the list of topical hemostats every soldier participating in the operations should carry, recommended by CoTCCC, should be reconsidered.

In addition, in Turkey, a hemostatic agent prepared by using five botanical ingredients in various amounts used in the Turkish medicine conventionally under the name Ankaferd® Blood Stopper (ABS) is produced. This botanical mixture also has effects on the vessel endothelium, blood cells, angiogenesis and cell proliferation. It comprises, Glycrrhiza Glabra (Licorice), Vitis Vinifera (Unripe grape), Alphina Officinarum’s (Galangal) dried leaf extracts, Urtica Dioica’s (Nettle) dried root extract, and Thymus Vulgaris’ s (Thyme) dried weed extract. Another local production used in Turkey, Mecsina, has similar structure and contents. Both products are in the form of a solution and bandage. However, the problems of easy oxidation and tendency for microbiological reproduction can be experienced in addition to transportation and packaging problems of the solutions. They hydrolyze and oxidate rapidly compared to other pharmaceutical forms. In addition, dosing is difficult.

Today, using extracts obtained from various plants and shellfish, in addition to conventional coagulation methods has become popular. As a result of the studies carried out, the characteristic attributions of ideal topical hemostat were determined as follows:

• It should be able stop arterial and/or venous hemorrhaging in 2 minutes,

• It should not require special conditions for storage,

• It should be durable and light, · It should have a long shelf life (>2 years),

• It should not require special training to use,

• It should be easy to use during war or combat,

• It should be applicable by the injured person themselves,

• It should be applicable during harsh weather conditions (rain, wind, at night, etc.), · It should be effective on wounds that are complex and where tourniquets cannot be applied,

• It should be effective for patients using antiaggregant or anticoagulants,

• It should be effective on hypothermic injured people,

• It should not cause pain or damage on the applied tissue, · It should not delay wound healing,

• It should be easily cleaned from the application area and should be bioabsorbant,

• It should not have a local or systemic side effect,

• It should create an antimicrobial, antibacterial environment,

• It should be inexpensive and cost- effective. In the last 10 years, although important studies have been conducted, and great developments were achieved, a product that includes all of the characteristics of an ideal topical hemostat and which is agreed upon not only in clinical studies but also in major animal studies has not been developed yet.

Forties fomentarius (tinder fungus) Femes fomentarius is a parasite fungus that is found on live and drying trees and it is inedible.

The fungus that is widespread in Turkey and in Europe, West Siberia and India as a household remedy, is indicated to be used as a coagulant in small injuries and as an agent that relieves external inflammation. The cities where said fungus grows in Turkey are Adiyaman, Agri, Alanya, Antalya, Artvin, Aydin, Balikesir, West Anatolia, West Black Sea, Batman, Bayburt, Bitlis, Bursa, Canakkale, Denizli, Elazig, Erzurum, Gumushane, Isparta, Izmir, Kahramanmaras, Karaman, Kastamonu, Kirklareli, Konya, Malatya, Mersin, Mugla, Nigde, Ordu, Samsun, Sinop, Tokat, Van, and Zonguldak.

Tinder fungus is a fungus used as a caogulant by surgeons, barbers, and dentists since many years and therefore it is called ‘surgeon’s fungus’. In addition, it applied on external wounds and burns by a dressing with a certain amount of iodine in Europe, West Siberia and Indian people’s medicine. In the Alpine region where the German language is spoken, tinder fungus is called ‘Wundschwamm’ or ‘Chirurgenschwamm’ and this preparation is sold in pharmacies in the form of a clotting bandage has been used by Austrian farmers up until the 19 ^th century. Apart from these external applications, it is indicated that the active agent of the tinder fungus, fomitininin dismenore, is used as a remedy for hemorrhoid and bladder diseases. In addition, information regarding the use of tinder fungus in the treatment of esophagus, stomach and uterine cancer is available. In traditional Chinese medicine, it has been used for the treatment of oral ulcers, gastroenteric diseases, inflammation and treatment of various cancers. In some studies, it has been reported that F. fomentarius carried out hypoglycemic, antinociceptive, anti-inflammatory, anti-infective and anti-tumor activity.

Even though its coagulation effects are known, tinder fungus has only been used by directly cutting the fungus and applying it on the wound or by placing the previously cut fungus on the wound and applying a bandage over it, up to this day. Innovation is required in order to provide ease of use and to increase coagulation effect.

Tinder fungus was defined by classifying its metabolites: The primary metabolites are formed of proteins, polysaccharides, polysaccharide -protein complexes. The secondary metabolites are formed of triterpene glycosides, esters, lactones, alcohols (7-ergostenol, -sitosterol), aldehyde and ketones, organic acids, benzofuranes, coumarins, and volatile compounds. Their clinically beneficial composition are glucans. In vitro studies suggest that macromolecule heavy glucans have direct phagocytic, cytotoxic antioxidant, and antimicrobial activity.

In 2009, polysaccharides of Fomes fomentarius were inspected in a study. In this study, crude polysaccharides were prepared with ethanol precipitation from fungus liquid method and then, the polysaccharides were purified with ion exchange chromatography and gel chromatography. The structure and composition of the polysaccharide structure was analyzed. As a result, it was determined that is has high homogeneity and it was formed with polymerization of galactose. The structure was analyzed with infrared (IR) and it has been reported that polysaccharide had pyranose form.

Pyranose is a general term used for carbohydrates which have the structure of six bound rings having one oxygen atom for five carbons. The pyranose ring if formed with the hemiacetal combination of the C-5 alcohol group of sugar with the C-l aldehyde group.

The pyranose ring is also found in chitosan. Crystallographic dimension of the pyranose ring allows us to see chitosan as a weak polyelectrolyte element. As a difference from the products where the chitosan polymer obtained by the deacetylation of chitin polysaccharide is used as an active agent, products where the polysaccharides formed by galactose polymerization of Fomes fomentarius are used as the active agent are not available.

The chitosan ionization constant (Pka) is 12,93 as a strong acid character. The Pka of the saccharide in the structure of Fomes fomentarius is 11.8 as a strong acid character. Due to their electrolyte structures, they are forced to perform cationic charging in biological environments.

Similar to chitosan, the presence of the polymerized galactose that is found in Fomes fomentarius and that has pyranose ring, supports the presence of the positively charged polysaccharide, its behavior in the blood and its coagulation effect as a mucoadhesive agent.

It can be said that the hemostatic effect results completely from polysaccharides and even though the gel prepared from Fomes fomentarius exhibits a haemostatic effect due to cationic charged polysaccharides similar to chitosan, it is faster in terms of haemostatic action time, it is less complex in terms of the production process and less expensive in terms of cost- effectiveness compared to chitosan formulations of the prior art.

Description and the Invention and its Aims

The invention is related to a gel formulation production method which exhibits a coagulation effect by means of the positively charged polysaccharide and polysaccharide-protein complex contents contained therein; by using the extract obtained from Fomes Fomentarius fungus (tinder fungus), which is local and natural element and which is not used in the production of pharmaceuticals in Turkey. The aim is to obtain a product with high added value from the fungus which is widespread but and completely unused in Turkey.

It exhibits a coagulation effect by means of the positively charged polysaccharide and polysaccharide-protein complex structure that bonds the negatively charged erythrocytes with chemical and mechanical effect and activates the thrombocytes, therefore creating an adhesive structure on the tissue and forming a physical barrier around the wounded vessel.

The gel formulation created exhibits a plastic flow. Therefore, it is easy to apply. In contrast with the hemostatic agents in the solution form of the prior art, it does not drain off from its container immediately, and it does not oxidate and hydrolyze easily. In addition, the dosing is easier.

In contrast with the hemostatic agents of the prior art which are expensive since a lot of chemicals are used, or the molecules having natural content are hard to find, the haemostatic gel of the invention obtained from the Fomes Fomentarius fungus is inexpensive and easy to produce. It does not require special conditions for storage and therefore has a long shelf life. It is easily used and transported. It is durable and light.

Thanks to the gel structure, it attaches to and remains on the skin easily; therefore it does not drain off the skin easily in rainy situations. Since the gel is produced from a biocompatible material, it does not cause damage or pain in the application area. Thanks to the content of the tinder fungus, it creates an antimicrobial environment in the application area. Since the formulation is composed of one active agent and three auxiliary agents, it is easily prepared and practical in terms of evaluating the dynamics.

Detailed Description of the Invention

The production method of the gel formulation of the invention having a coagulation effect obtained from fungus extract comprises the following procedure steps: i. Mixing the fungus dust with distilled water, ii. Extracting the fungus dust - distilled water mixture in a boiling water bath, iii. Preparing the fungus extract by filtering the fungus residuals from the extracted fungus dust - distilled water mixture, iv. Adding the gel forming agent to the fungus extract which is being stirred at medium speed and mixing the same until its appearance is homogenized, v. Adding a pH-changing agent on the fungus extract-gel forming agent mixture and creating the haemostatic gel.

The haemostatic gel of the invention comprises 2-5% Fomes Fomentarius (tinder fungus) extract by weight, 1-2% gel forming agent by weight, and 93-97% water by weight.

First of all a tinder fungus extract that is 5-10% by weight is prepared by adding water. 2,5g dust obtained from the shell part of the tinder fungus which is in the powdered state is weighed on the analytical scale for preferably a tinder fungus extract (50 mL water + 2,5g tinder fungus) of 5% . The tinder fungus dust can be obtained by pulverizing the shell part of the fungus by hand, in a mortar or in a blender.

Following the weighing process, the tinder fungus dust is placed into an Erlenmeyer flask containing 50 mL distilled water. The fungus dust-distilled water mixture is extracted in a boiling water bath at 100-120°C for an hour by stirring at intervals. At the end of the extraction, the fungus dust-distilled water mixture is removed from the water bath and filtered through a muslin cloth, first to remove the coarse particles and then is filtered through a filter paper, thereby, tinder fungus residual is removed from the extract. The missing volume is completed with the addition of 50 mL distilled water. At the end of this procedure, a tinder fungus extract purified from residual tinder fungus is obtained.

The extract is placed into a beaker to prepare a gel formulation from the 5% extract obtained. Powdered carbomer (carbopol) 940 is used as 2% by weight of the gel formulation as the gel forming agent. The amount of carbopol (carbomer) required for 50 mL extract is 1-2% (preferably lg for 50 mL) by weight and it is weighed on an analytical scale. Among the carbopol derivatives as gel forming agents, 71G NF, 97 IP NF, 974P NF, 980 NF, 981 NF, 5984 EP, ETD 2020 NF, Ultrez 10 NF, 934 NF, 934P NF, 940 NF; and methyl cellulose, hydroxyl methyl cellulose, hydroxyl propyl cellulose, hydroxyl propyl methyl cellulose, ethyl cellulose, hydroxy ethyl cellulose, propyl cellulose, carregeene, agar, chitosan, hyaluronic acid, pectin, gelatin, starch alginic acid, sodium alginate, potassium alginate, ammonium alginate, calcium alginate, guar gum, xanthan gum, collagen, or egg white can be used apart from Carbomer 940.

The beaker containing the 5% extract is placed on a magnetic stirrer and a Teflon coated stirrer is placed therein, the stirring speed is set at medium. As the extract is stirred at medium speed, lg of carbomer weighed is added into the beaker very slowly. When the carbomer dissolves in the extract and its appearance is homogenized, the stirring is stopped and the beaker is taken off the magnetic stirrer.

In order to achieve gelling, 149.19 g/mol trietanolamine is used as the pH changing agent. To obtain a product at a pH applicable to the skin, 5 to 5,5 pH is chosen. The pH changing agent (preferably trietanolamine) is added by single drops with a plastic dropper on the fungus extract - gel forming agent mixture in the beaker and stirred rapidly with a glass rod. By adding a sufficient amount of a pH changing agent until gelling is observed, the desired gel consistency is obtained and the addition of the pH changing agent is stopped. The gel formulation is placed into a plastic container and labelled for storage. The physical properties of the gel formulation are as follows:

. pH Measurement Results (3 repetitions): 5,52

. Viscosity Measurement Results (5 repetitions): 34, IP was reached by taking the average of 5 different measurements at 29,4°C using a Vibrational Viscometer.

0,1 N sodium hydroxide solution, pyridine, calcium carbonate magnesium carbonate, calcium hydroxide, calcium bicarbonate, magnesium bicarbonate or sodium tetra borate can be used as alkalizing pH changing agents in addition to trietanolamine. The gel forming agent carbopol can be used in a range between 1-2%. A sufficient amount of pH changing agent is added until gel formation is observed. Water is used as a carrier in the preparation of the extract.

The haemostatic gel obtained from Fomes Fomentarius (tinder fungus) contains Fomes Fomentarius (tinder fungus) extract in a range of 2-5% , and gel forming agent between 1-2% by weight and water at a range of 93-97%.

2 mice were procured from Trakya University Faculty of Medicine Experimental Animals Unit in order to conduct hemorrhage and coagulation time tests. Approval from Trakya University Experimental Animals Local Ethics Committee was taken for the studies that were to be conducted.

The efficacy of the haemostatic gel formulation obtained from Fomes Fomentarius fungus of the invention is given in the following tables. One of the 2 mice obtained for hemorrhage and coagulation time tests is used as control for the detection of regular hemorrhage and coagulation time and 5% gel formulation is applied to the other in order to examine the effects of the formulation on the hemorrhage and coagulation time. Animal experiments are conducted in two different ways: i. Detecting the hemorrhage and coagulation time by creating a wound on the back of the neck with a 5mm biopsy punch

The mice were anesthetized with an I.M. ketamine and xylazine mixture. Then, the hair on the neck and back of the neck region was trimmed and the area was cleaned with 70% alcohol. For the detection of hemorrhage and coagulation time, a wound was created on the back of the necks of the mice with a 5mm biopsy punch. Before starting the procedures for the experiment conducted by creating a wound on the back of the neck, the amount of 5% and 10% gel formulations to be applied on the wound was determined. The surface of the wound where the 5mm biopsy punch is to be made was found (from the 7tr2 formula). 0,lg formulation was applied on the wound of this size. In terms of ease of application during application, 0,lg weight of formulation were taken and packed beforehand.

The hemorrhage time indicates the period that lasts until the end of bleeding on the wound after the wound was made, and the coagulation time is the time that lasts until a drop of blood taken from the wound onto a lamella is frozen (clotted). After the control animal were wounded, no other application was made. The other animal was wounded and 0,lg of 5% formulation is applied. The results of the experiment conducted by creating a wound on the back of the neck of the mice are given in Table 1.

Table 1.

According to the results obtained in this experiment, significant healing was observed regarding both the hemorrhage and the coagulation time of the mouse on the tail of which, the formulation was applied. According to the data in Table 1, the hemorrhage time of the control subject was calculated to be 2.5 times longer than the hemorrhage time of the mouse to which 5% Formulation was applied. Similarly, the coagulation time of the control subject was 3.5 times longer than the coagulation time of the mouse to which the 5% Formulation was applied. ii. Detecting the hemorrhage and coagulation time by cutting 5mm off the tail of the mice A 5mm thickness point is detected on the tail of one of the mice anesthetized with an FM. ketamine and xylazine mixture and the tail was cut from this point with a 5mm biopsy punch. 5% formulation was applied on the point which was cut and the hemorrhage and coagulation time were determined. After the application was completed, the tail which was cut was ligatured and then cleaned with iodized antiseptic. The results obtained in the experiment conducted by cutting the tails of the mice are shown in Table 2.

Table 2.

According to the results obtained in this experiment, significant healing was observed in both the hemorrhage and coagulation time of the mouse on the tail of which the formulation was applied. According to the data in Table 2, the hemorrhage time of the control subject was 2.25 times longer than the hemorrhage time of the mouse to which 5% Formulation was applied. Similarly the coagulation time of the control subject was 3.32 times longer than the coagulation time of the mouse to which 5% Formulation is applied.

The results shown in Table 1 and Table 2 show that the haemostatic gel obtained, significantly decreases hemorrhage and coagulation time.