KR20210033609A - Especially extraction and use it for application of antithrombotic functional low molecular collagen from Fig - Google Patents

Abstract

The present invention relates to a pharmaceutical composition and health functional food for preventing or treating thrombosis containing a fig extract as an active ingredient. The fig raw ingredient extract is prepared by extracting unripe and semi-dried figs (ripe and unripe dried fruits), discarded fig tree trunks, fig tree roots and fig leaves which are fig raw ingredients in a water-soluble organic acid and sequentially fractionating the extract using nucleic acids, ethyl acetate and butanol. Among the fractions prepared thereby, a dilute organic acid fraction exhibiting excellent antithrombotic activity by direct inhibition of thrombin and inhibition effects of blood clotting factors has effects of: effectively inhibiting thrombus formation; and being used for prevention and treatment of thrombosis such as ischemic stroke and hemorrhagic stroke through improvement of blood circulation. Specifically, the fig raw ingredient extract according to the present invention does not exhibit acute oral toxicity, exhibits thermal stability, and does not exhibit a loss of antithrombotic effects even under an acid condition of pH 2 and even in blood, thereby exhibiting an excellent platelet aggregation inhibiting rate, and thus can be usefully applicable for preventing and treating a disease which involves thrombus formation.

Description

[Especially extraction and use it for application of antithrombotic functional low molecular collagen from Fig}

The present invention relates to a method for extracting a food component with antithrombotic function from a fig and an antithrombotic extract thereof, and more particularly, to an unripe fruit and semi-dried fig (ripe fruit and unripe fruit) as a raw material of a fig, and a fig tree to be discarded. Fig gel can be obtained in high yield by pulverizing the stem fig tree root and fig leaf, and then adding an organic acid solution to extract it easily at room temperature. In other words, the yield is improved by more than 3 times compared to the existing method of extraction with water or alcohol, and the process is fast because it is easily filtered well by using unripe fruits and semi-dried figs, and a large amount of organic solvent is used to extract and precipitate figs with hot water. However, in this process, the optimum extraction method is promoted even with a small amount of organic acid, organic solvent and acid alkali treatment, so that high purity and high yield can be ascertained, so it can be said to be highly feasible for industrial use. Therefore, it relates to a method for extracting figs having antithrombotic function that exhibits anticoagulant activity against platelets that are activated by stimulation of aggregation inducers such as collagen and causes agglutination reaction from figs, and an antithrombotic extract extracted according to the method. Use of cosmetic materials or pharmaceutical compositions for preventing or treating thrombosis, as well as cleaning agents for skin disease treatment and mouthwash

Normal blood circulation facilitates blood circulation as the blood coagulation reaction system and the thrombolysis reaction system in the body are complementarily regulated. Among them, the mechanism of the blood coagulation reaction system is after platelets adhere and aggregate to the blood vessel wall to form platelet thrombi. , It has been reported that the blood coagulation system is activated and fibrin thrombi are formed around the platelet agglutinator.

On the other hand, the formation of fibrin thrombi causes thrombin, which is involved in fibrin coagulation, through several step reactions of numerous blood coagulation factors, and finally produces fibrin monomers from fibrinogen, and fibrin monomers are polymerized by calcium and platelets. It binds to endothelial cells and forms a fibrin polymer cross-linked by factor XIII, creating a permanent blood clot. In addition, thrombin plays a central role in the formation of blood clots by activating platelets, factors V, and VII to promote blood coagulation. Therefore, thrombin activity inhibitors can be used as very useful prophylactic and therapeutic agents for various thrombotic diseases caused by excessive blood coagulation. Meanwhile, in the endogenous thrombogenic pathway, the sequential activation of factor XII, factor XI, factor IX, and factor X, followed by the activation of prothrombin, is known to ultimately activate thrombin. Has become.

To date, various anticoagulants such as heparin, coumarin, aspirin, and urokinase have been used for the prevention and treatment of thrombotic diseases, but these are not only very expensive, but also bleeding side effects, gastrointestinal disorders and irritability, etc. As a result, its use is limited.

However, studies on the useful physiological activity of figs, a natural product, have been very actively conducted in recent years, and as a patent document related to figs,

Patent Registration No. 10-1080994 relates to a composition comprising a hot water extract of Goji berry, fig and pear chorion, and may have antioxidant activity, immunity enhancement, whitening, wrinkle improvement, and collagen decomposing enzyme inhibitory effects, but It can be seen that the utilization is low due to the problem that the activity decreases due to hot water extraction and side effects are accompanied by a complex substance. Application No. 10-2017-0029586: According to the method of removing blood clots using electromagnetic field generation and control, magnetic nanoparticles are attached to blood clots. Thus, the magnetic blood clots are pulled through the generation of an electromagnetic field, and the blood flow can be restored by moving through the propagation of the electromagnetic field through the vascular path. This is an electronic device, and there is an external application problem, and there may be a problem that it cannot be used as an injection agent or an improvement material directly inside the blood vessel.

Registration No. 10-0512285: Patent for a medicinal soap composition containing a natural fig separating component is a state in which the present inventors directly conduct an experiment on the overall separation of fig tree leaves, and obtain a component spectrum. It was intended to be applied to the present application by improving the extraction method. Korea/10-2017-0133212: A cosmetic composition for anti-allergic and atopic dermatitis improvement comprising a Hoeriwind flower extract as an active ingredient. 10-2016-0085387: A cosmetic composition for improving atopic itching comprising a bioconversion extract and a method for producing the same. 10-2013-0152314: Atopic or skin trouble prevention and improvement composition. A study published in the patent how to contain the food component of the anti-thrombotic functions from fig 10-2012-0019968 and antithrombotic standing extract, but subjected to the aqueous solution of the low-temperature extraction is extraction without application of results attempted by applying physical methods, such as column In addition, it is supposed to extract fractions with ethyl acetate during the purification process, but it cannot be extracted due to the formation of a layer with an aqueous solution. In particular, it was not possible with an azotropic mixture (azeotropic mixture phenomenon), and it was considered impossible to extract with ethyl acetate without water. So, it can be said that the pretreatment was performed with the organic acid aqueous solution mentioned in the present manufacturing process, and it was completed in the optimum conditions according to the invention in high yield.

Domestic patent application (application number 10-2010-0093901): A composition for the prevention or treatment of atopic dermatitis containing a herbal extract or a fermented product thereof, and its manufacturing method is different from the present invention, and registration number 10-1181402: Collagen powder 3 To 7 parts by weight; And the edible plant mixture consisting of Ogapi, Nogeun, Chinese quince, cherry, grape seed, and edible plant extract concentrated under reduced pressure after extracting the edible plant mixture with a solvent. It is different from the present invention and relates to an antithrombotic composition containing a phenolic compound as an active ingredient is known to have a solubility problem due to its high molecular weight. Laid-Open Patent 10-2004-0027283: Diversification of raw materials for producing silver collagen and a method for producing vegetable or marine collagen peptides through a more simplified process than conventional techniques. It can be said that the entanglement phenomenon is remarkable depending on the ability of the bonding and the Van der Waals force dipole, and there is a disadvantage that it cannot be easily used because it is oxidized by air contact and becomes rice cake. Therefore, the inventors also studied fig enzymes in the form of peptides formed by about 35 amino acid bonds, and as a result of intensive research, they were able to extract more efficiently because they had the advantage of being partially hydrolyzed by an aqueous organic acid solution treatment.

In this regard, researchers have reported antithrombotic activity from various natural products, and this activity has also been reported from ethanol extracts isolated from figs (Anwarul HG et.al., Journal of Ethnopharmacology119 (2008) 1-5). In the active ingredients obtained from natural products, the types of the extracted ingredients differ depending on the polarity according to the solvent selection, and the functional effects may also vary.

Even if the fig is cut and extracted, it is immediately softened and killed to the extent that the shape is unknown. Even if the target compound is attempted to be extracted, there is a problem that it is not filtered even if a diatomaceous earth or a bed filter is used. Even if centrifugation is carried out, the fig aqueous solution is a unit that is obtained from a large amount to a small amount. Even if the porridge is centrifuged, the separation is not clean due to hydrogen bonding between the water and the gel. Is being caused. This process is difficult as a problem that can be confirmed in the actual separation process by breaking away from the theoretical method, and even if it is obtained, the yield is remarkably low, and it can be said that the value of industrial use is low.

Therefore, as a result of intensive research, the present inventors crushed fig raw materials, unripe and semi-dried figs (finished and unripe), and discarded fig tree trunks, fig tree roots, and fig leaves, and recovered even after extraction at room temperature with an aqueous organic acid solution. After discovering a method, extracting it with sufficient stirring, filtering insoluble figs, concentrating the recovered filtrate under reduced pressure, neutralizing it by adding a small amount of basic aqueous solution, filtering the gel compound produced by adding alcohol, and washing it several times with alcohol. A pure fig gel was obtained with a higher yield of 70% or more than the hot water extraction method. Therefore, the present inventors aim to develop a functional food material that exhibits antithrombosis and blood flow improvement activity, after preparing a fig raw material extract, using human plasma and human thrombin to evaluate the thrombiogenesis inhibitory activity according to direct thrombin inhibition, and human concentration. The present invention was completed by measuring the platelet aggregation inhibitory activity of figs using platelets, and confirming very excellent antithrombotic activity in the fig raw material extract and sequential fractions thereof.

And on the other hand, vesicular stomatitis virus (VSV), a member of the family Rhabdoviridae, has an unsegmented, negative-sense, single-stranded RNA genome. Its 11 kb genome has 5 genes encoding 5 structural proteins of the virus; Nucleocap seed protein (N), which is required in stoichiometric amounts for encapsulation of the replicated RNA; Phosphoprotein (P), which is a cofactor of RNA-dependent RNA polymerase (L); Matrix protein (M) and binding glycoprotein (G) (see, for example, Gallione et al., 1981, Rose and Gallione, 1981; Rose and Schubert,.1987 and Schubert et al., 1985; US Patent 6,033,886; US Patent 6,168,943 ).

VSV is an arthropod-derived virus that can be transferred to various mammalian hosts, most commonly cattle, horses, pigs and rodents. VSV infection in humans is not common and is usually characterized by mild cold-like symptoms that accompany no asymptomatic or complications for 3 to 8 days. Since VSV is not considered a human lesion, existing immunity to VSV is rare in the human population, and the development of VSV-derived vectors focuses on areas such as immunogenic compositions and gene therapy. And stomatitis is an inflammation that occurs in the oral mucosa, which is also called stomatitis, and is often caused by infections such as bacteria, viruses and fungi, and also occurs when resistance is weakened due to malnutrition, respiratory diseases, gastrointestinal diseases, and pregnancy. Symptoms include increased salivation, pain, bad breath and blisters, and severe symptoms include ulcers in the mouth and tongue. For mild symptoms, it is treated by applying fiogutanine solution, mercurochrome or borax, glycerin and 2% silver nitrate to the treatment area. However, in severe symptoms, treatment is possible by dropping triamcinolone acetonide.

Currently, as a formulation containing triamcinolone acetonide as a treatment for stomatitis, ointments (Olamedi OintmentTM, Dongguk Pharm) and oral tablets (Aptachi TabletTM, Donghwa Pharm) are commercially available. Triamcinolone acetonide ointment is a product formulated with active ingredients such as cetyl alcohol or soft meat. This is a formulation in which the ointment is applied to the treatment area with a strabismus swab or gauze. It is inconvenient to use and does not stick well to the treatment area. have. In addition, oral tablets are tableted with polymers such as ethyl cellulose and hydroxypropyl cellulose, and the surface of the tablet is slightly swollen with water or saliva, and then attached to the administration site to express the medicinal effect. However, although this product is well attached, it has problems such as feeling foreign bodies and feelings of rejection in the oral cavity in order for the drug to develop for a long time. Therefore, in order to solve the discomfort of the existing ointment or the feeling of foreign body and rejection of the oral tablet, it is urgently required to develop a new treatment for stomatitis, and ethanol is used as a solvent. Ethanol has the effect of disinfecting the wound after spraying, and it volatilizes quickly, helping to deposit the drug quickly.

The composition of the present invention can be sprayed by being prepared by putting it in a propellant equipped with a large bowl. Therefore, if this composition is sprayed on the treatment area, it is sprayed only on the site of stomatitis, not the entire oral cavity by the daerong, and has superior convenience and bioadhesiveness compared to the existing triamcinolone acetonide formulation. It is also a natural product, so it can be eaten as it is. It can be said that there are advantages to this. In addition, the ethanol solvent not only instantly sterilizes at the site where stomatitis occurs, but also immediately volatilizes to form a molecular film, so that the fig alcoholic ethyl acetate fraction extract is superior to the existing drugs at the site where the stomatitis occurs, although it has an excellent medicinal effect. Represents.

The formulation method of the present invention is a method well known to those in the relevant industry, and can be prepared in a pharmaceutical form including conventional excipients and diluents such as fragrances.

Hereinafter, the composition of the present invention will be described in more detail by the following examples, but the composition of the present invention is not limited to the following examples.

Patent Document 1 (Korean Patent Registration No. 10-1080994) Patent Document 2 (Korean Application No. 110-2017-0029586) Patent Document 3 (Korean Patent Registration No. 10-0512285) Patent Document 4 (Korean Application No. 10-2016-0085387) Patent Document 5 (Korean Application No. 10-2013-0152314) Patent Document 6 (Korean Application No. 10-2010-0093901) Patent Document 7 (Korean Patent Registration No. 10-1181402) Patent Document 8 (Korean Application No. 10-2016-0085387) Patent Document 9 (Korean Application No. 10-2016-0085387) Patent Document 8 (Korean Application No. 10-2015-0014410) Patent Document 8 (Korean Application No. 10-2004-0027283)

The present invention was derived to solve the problems of the prior art as described above, the present invention is the fig raw material immature and semi-dried figs (ripe and unripe), and discarded fig tree stem fig tree root, and fig leaves By using (hereinafter referred to as a fig raw material) to provide a pharmaceutical composition for preventing or treating thrombosis containing as an active ingredient. Selecting the fig raw material selectively, cutting it finely, extracting useful water-soluble components at room temperature using an organic acid aqueous solution as an extraction solvent, filtering the extraction solution to remove insoluble components, and concentrating the recovered solution under reduced pressure to obtain a saturated solution. Then, a small amount of basic dilute solution is added to neutralize it, then put in ethanol, washed, filtered and washed several times with an alcohol solution to extract antithrombotic components from figs, and to antithrombotic extracts of figs with excellent antithrombotic effect. . The antithrombotic extract of the fig showed very excellent antithrombotic effect as a result of measuring the antiplatelet aggregation ability using the impedance method in an in vitro experiment.

The problem to be solved in the present invention is a pharmaceutical composition for the prevention or treatment of thrombotic diseases caused by direct inhibition of human thrombin, inhibition of prothrombin, and inhibition of endogenous coagulation factors containing fig raw material extracts used for edible and medicinal purposes And a method for extracting an active ingredient that has excellent antithrombotic effect and does not cause side effects by containing the above extract, and to provide an antithrombotic extract extracted according to the extraction method, but also applied to stomatitis to examine the efficacy As a result, it was confirmed that the effect was better than that of the chemical synthesis agent, and it was intended to show the method of application.

According to the features of the present invention for achieving the above object,

The present invention relates to a method for efficient extraction of low-molecular collagen with antithrombotic function from a fig raw material and its use,

i. Figs (unripe fruits, semi-dried figs) selective pulverization step (100), ii. organic acid hot water extraction and filtration step (200), III. extraction solution recovery, neutralization and concentration under reduced pressure and then production step step (300) as a saturated solution, iv. After alcohol washing, to prepare a dried fig through a manufacturing method consisting of a gelation step 400, v. filtration and several times of alcohol washing step 500, vi. Fig Gel-1 recovery step 600.

The fig raw material extract as an active ingredient in the pharmaceutical composition for preventing or treating thrombosis and the health functional food of the present invention prevents the activation of platelets that cause an agglutination reaction by stimulation of an aggregation inducer such as collagen, thereby causing blood coagulation. It can prevent the formation of blood clots in blood vessels. In addition, since it uses leaves that are discarded after harvesting figs, it has the advantage of contributing to the reuse of unused resources and preservation of the environment.

The fig raw material extract as an active ingredient of the pharmaceutical composition for preventing or treating thrombosis and the health functional food of the present invention, the ethyl acetate fraction that exhibits antithrombotic activity due to an excellent blood coagulation factor inhibitory effect can effectively inhibit thrombus formation. It has an excellent effect that can be used for the prevention and treatment of thrombosis such as ischemic stroke and hemorrhagic stroke through improvement of blood circulation. In particular, the fig raw material extract of the present invention does not exhibit acute oral toxicity, has excellent thermal stability, and does not directly inhibit human thrombin, inhibit prothrombin, and inhibit blood coagulation factor even in acidic conditions of pH 2 and plasma. Since it has an excellent effect that it can be prepared in a form that can be taken at all times by being processed into various forms such as powder, pill, and tablet, it is a very useful invention in the pharmaceutical industry and food industry, and it is possible to stably promote the consumption of figs. It is expected that there will be benefits that can increase the income of growers.

1 shows the extraction experiment process process: the extraction process of extracting fig leaves (other fig raw materials are obtained using the same process in parallel, respectively, depending on the selection),
2 shows the extraction process of unripe figs and semi-dried figs,
3 shows an extraction process for extracting fig stems and fig roots,
4 shows a fig leaf crushing extraction process

Hereinafter, the present invention will be described in detail.

The present invention relates to a pharmaceutical composition for preventing or treating thrombosis having direct inhibition of human thrombin, prothrombin inhibition, and endogenous coagulation factor inhibitory activity, and a health functional food containing a fig raw material extract. More specifically, the inventors of the present invention recovered the antithrombotic active ingredient from the fig raw material extract obtained by a certain method, and these ingredients did not show oral acute toxicity, and confirmed that it has excellent characteristics of thermal stability and acid stability. By doing so, it was intended to use the extract as a pharmaceutical composition and health functional food for the prevention or treatment of thrombosis.

Specifically, the present inventors prepared ethanol extracts of figs to develop pharmaceutical compositions and health functional foods for the prevention or treatment of thrombosis using figs, and sequentially fractionated with organic solvents to obtain nucleic acid fractions, ethyl acetate fractions, butanol fractions. And water residues, and the like, respectively, to human plasma and human thrombin (Thrombin Time), prothrombin inhibition (Prothrombin Time) and active partial thromboplastin time (activated Partial Thromboplastin Time: aPTT), platelets The ability to inhibit aggregation was evaluated. As a result, the thrombin time, prothrombin time, and active portion of the thrombin time prolonging activity in the fig ethanol extract were hardly recognized, but in the organic solvent fraction, especially the ethyl acetate fraction, the thrombin time more powerful than aspirin (commercial antithrombotic agent). The present invention was completed by confirming the prolonging effect and prothrombin time similar to that of aspirin, the active part thromboplastin time prolonging effect, and platelet aggregation inhibitory effect.

In order to confirm the effect of such figs, the inventors of the present invention prepared a fig raw material extract and an organic solvent fraction, and the direct inhibitory effect of human thrombin, prothrombin inhibitory effect, blood coagulation factor (factor VIII, factor IX, By evaluating the effect of prolonging the active part thromboplastin time by inhibition of factor XI and factor XII), the extract of the present invention has superior antithrombotic activity than a commercially used antithrombotic agent aspirin (brand name: Protect). Was confirmed, human red blood cell hemolytic activity did not appear, and the heat and acid stability of the substance were investigated, and then the substance was orally administered to rats to conduct an acute toxicity test, thereby confirming that the extract or fraction had little toxicity to the human body. . Accordingly, the present invention is characterized by providing a pharmaceutical composition for preventing or treating thrombosis and a health functional food containing the fig raw material extract as an active ingredient.

The pharmaceutical composition containing the fig raw material extract of the present invention is formulated according to a conventional method according to the purpose of each use, oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc. It may be formulated and used in various forms such as injections, and may be administered orally or administered through various routes including intravenous, intraperitoneal, subcutaneous, rectal, and topical administration. In addition, the pharmaceutical composition of the present invention may further include a filler, an anti-aggregating agent, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent, a preservative, and the like.

As a preferred embodiment, solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and as a preferred embodiment, the effective amount of the fig raw material extract in the pharmaceutical composition of the present invention is the age and sex of the patient. , It may vary depending on the body weight, and in general, 1 to 5,000 mg per kg of body weight, preferably 100 to 3,000 mg may be administered daily or every other day, or divided into 1 to 3 times a day. However, since it may increase or decrease depending on the route of administration, the severity of the disease, sex, weight, age, etc., the dosage amount is not limited by any method. The pharmaceutical composition of the present invention can be administered to a subject through various routes. All modes of administration can be expected and can be administered, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dura mater or intracerebroventricular injection. The specific method of the present invention will be described in more detail through examples. The following examples are only one preferred specific example of the present invention, and the scope of the present invention is not limited to the scope of the following examples.

Hereinafter, the present invention will be described in more detail through examples.

[Example]

Example 1: Unripe fruit and semi-dried figs (ripe fruit and unripe fruit) are pulverized in fig raw materials, and using a solution having a volume ratio of water and citric acid as an organic acid solution as an extraction solvent (0.01 to 0.5: 1) at room temperature Extracting water-soluble components from the extract, filtering the extraction solution, concentrating under reduced pressure to make a saturated solution, cooling, and using a small amount of basic compound. Add dilute 2N-sodium hydrogen carbonate solution, which is a weak base, to filter the precipitate, and then an alcohol solution or ethyl acetate. It was washed quickly with and then separated and freeze-dried, or immersed in a separate alcohol solution and stored to prepare a pharmaceutical composition for preventing or treating thrombosis.

Example 2: A step of pulverizing the whole fig roots and stems from fig raw materials and extracting water-soluble components at room temperature using a solution having a volume ratio of water and citric acid (0.01 to 0.5: 1) as an organic acid solution as an extraction solvent, Filter the extract solution, concentrate under reduced pressure to make a saturated solution, cool, and use a small amount of basic compound. Add dilute 2N-sodium hydroxide solution, which is a weak base, to filter the precipitate, wash quickly with alcohol solution or ethyl acetate, and then separate and separate each A pharmaceutical composition for preventing or treating thrombosis was prepared by lyophilizing or immersing it in a separate alcohol solution and storing it.

Example 3: A step of extracting water-soluble components at room temperature by pulverizing fig leaves from fig raw materials and using a solution having a volume ratio of water and citric acid (0.01 to 0.5: 1) as an organic acid solution as an extraction solvent. Filter, concentrate under reduced pressure to make a saturated solution, cool, and use a small amount of basic compound. Add diluted potassium hydroxide solution, which is a weak base, to filter the precipitate, quickly wash it with alcohol solution or ethyl acetate, and separate and freeze-dry them, respectively. A pharmaceutical composition for preventing or treating thrombosis was prepared while immersed in a separate alcohol solution and stored.

Example 4: Preparation of Fig Raw Material Extracts and Organic Solvent Fractions Figs cultivated in Gochang, Jeollabuk-do in 2019 were purchased, about 10 times the volume of water-soluble organic acid was added to the figs, and extracted twice at room temperature for 5 hours. Thereafter, the ethanol extract was suspended in distilled water, followed by sequential fractionation with nucleic acid, ethyl acetate, and butanol, and finally a water residue was obtained. The yields of the nucleic acid fraction, ethyl acetate fraction, butanol fraction and water residue of the fig ethanol extract were 1.62%, 2.42%, 18.98% and 78.33%, respectively. In particular, the amount of the ethyl acetate fraction was very small, which means that about 46 mg of the ethyl acetate fraction was obtained for 100 g of figs, and the ethyl acetate fraction was judged to be considerably purified.

Next, the antithrombotic activity of the fig raw material extract was evaluated. Antithrombotic activity was evaluated according to previously reported methods (Sohn et al., 2004. Kor. J. Pharmacogn 35. 52-61; Kwon et al., 2004. J. Life Science, 14. 509-513; Ryu et al. 2010. J. Life Science, 20. 922-928), thrombin time, prothrombin time and APT time were measured. Plasma was prepared from the volunteer's whole blood, and immediately after blood collection, it was centrifuged at 4°C for 5 minutes to separate the plasma and stored in a frozen state (fresh frozen plasma), and if necessary, thawed at room temperature and used. Thrombin time, prothrombin time, and AP measurement were performed as follows.

Thrombin Time [0059] 50 μl of 0.5 U thrombin (Sigma Co., USA) and 50 μl of 20 mM CaCl2, and 10 μl of sample extracts of various concentrations at 37°C were placed in a tube of Amelung coagulometer KC-1A (Japan). After mixing and reacting for 2 minutes, 100 μl of plasma was added and the time until the plasma coagulated was measured. Aspirin (Sigma Co., USA) was used as a control, and DMSO was used instead of the sample as a solvent control. In the case of DMSO, the coagulation time was 32.1 seconds. In the case of thermal stability measurement, sample solutions of various concentrations were heat-treated at 100° C. for 30 minutes, allowed to cool at room temperature for 1 hour, and then the residual activity was measured. The thrombin inhibitory effect was expressed as the average value of the experiment repeated three or more times, and the coagulation time at the time of sample addition was divided by the coagulation time of the solvent control and multiplied by 100 and expressed as %. Add 70 μl of prothrombin time standard plasma (MD Pacific Co., China) and 10 μl of sample solution of various concentrations to the tube of Amelung coagulometer KC-1A(Japan), warm at 37°C for 3 minutes, and then 130 μl The time until the plasma coagulation after addition of the PT reagent of was expressed as the average value of the experiment in which 3 times were repeated. Aspirin (Sigma Co., USA) was used as a control, and DMSO was used instead of the sample as a solvent control. In the case of DMSO, the coagulation time was 18.1 seconds. 100 μl of aPTT (activated Partial Thromboplastin Time) plasma and 10 μl of sample extracts of various concentrations were added to the tube of Amelung coagulometer KC-1A(Japan), heated at 37°C for 3 minutes, and then 50 μl of aPTT reagent (Sigma, ALEXINTM). ) Was added and incubated again at 37° C. for 3 minutes.

Thereafter, 50 μl CaCl2 (35 mM) was added and the time until plasma coagulated was measured. DMSO was used instead of the sample as a solvent control, and in this case, the coagulation time was 55.1 seconds. The result of aPTT was expressed as the average value of the experiment repeated three times, and the coagulation time at the time of sample addition was divided by the coagulation time of the solvent control and multiplied by 100 to express a %. The antithrombosis measurement results according to the above-described thrombin time, prothrombin time, and AP time measurement methods are shown in Table 2 below. Aspirin used as a control showed excellent antithrombotic effect at a concentration of 5 mg/ml, and ethanol extracts from the whole root, skin, and edible parts of figs were used as thrombin time, prothrombin time, and AP up to a concentration of 5 mg/ml. It didn't change the time significantly. Therefore, it is judged that it is difficult to detect the antithrombotic activity when the fig raw material extract is prepared and the antithrombotic activity is evaluated.

Example 5: Component analysis and antithrombotic activity evaluation of sequential organic solvent fractions of fig raw material extract

After solvent fractionation of the fig ethanol extract of Example 1, the fraction

Table 2. Antithrombotic activity of whole stem roots, unripe and semi-dried figs, edible skins, and aqueous solution residues among fig materials

Components and antithrombotic activity were investigated. Table 3 shows the results of measuring the total flavonoid, total polyphenol, total sugar and reducing sugar content of the fraction.

Table 3: Fractionation efficiency and analysis of fig material organic solvent extract

In the organic solvent fractionation of the fig ethanol extract, water residues accounted for most, and some of them were transferred to the butanol fraction, and the nucleic acid fraction and ethyl acetate fraction showed very low content of 1.62% and 0.42%, respectively. Most of the water and butanol fractions were composed of sugars, and showed low polyphenol content of 2.27-3.51 mg/g and flavonoid content of 0.57-8.16 mg/g. The nucleic acid fraction also contained about 16% sugar, and showed a polyphenol content of 2.48 mg/g and a relatively low flavonoid content of 3.25 mg/g. However, in the ethyl acetate fraction, the sugar content was at the level of 8.5%, but it was confirmed that the polyphenol content of 4.59 mg/g and the flavonoid content of 147.36 mg/g were very high. From these results, it is judged that the ethyl acetate fraction of figs has most of the flavonoids of figs, and is expected to show strong antioxidant power and useful physiological activity, and actually shows strong antioxidant power.

As a result of evaluating the antithrombotic activity of the fractions, as shown in Table 4 below, the nucleic acid fraction exhibited a 15-fold increase in thrombin time and a 2.5-fold increase in prothrombin time at a concentration of 5 mg/ml, indicating excellent thrombin inhibitory activity. However, compared with aspirin used in clinical practice at the same concentration, it showed relatively weak inhibitory activity. This was similar in the butanol fraction and the water residue, and a weak antithrombosis was confirmed compared to aspirin. However, in the case of the ethyl acetate fraction, thrombin time, prothrombin time, and AP time all showed a 15-fold or more prolonging effect at a concentration of 5 mg/ml, confirming a very strong antithrombotic activity. In particular, even at a concentration of 1.25 mg/ml lower than 1.5 mg/ml of aspirin used in the experiment, the thrombin time prolongation effect increased by more than 15 times and prothrombin time and AP time increased by 1.47 and 1.5 times, and the ethyl acetate fraction of figs It was confirmed that it can be developed as a composition that inhibits thrombogenesis better than aspirin. In addition, this result suggests that the ethyl acetate sequential solvent fraction has a very low fractional yield, but exhibits strong activity, and is therefore very efficient as a method of purifying the antithrombotic active substance of figs.

Table 4 Antithrombotic activity of organic solvent extracts of fig materials and water residues

Example 6: Human Platelet Aggregation Inhibitory Activity of Fig Raw Material Extracts and Sequential Organic Solvent Fractions As part of the antithrombotic activity evaluation of fig raw material extracts and fractions, the aggregation inhibitory activity against human platelets was evaluated, and the results are shown in FIGS. 1 and 5 Shown in. First, aspirin showed excellent human platelet aggregation inhibitory activity, and concentration-dependent aggregation inhibitory activity was confirmed. The fig ethanol extract and the organic solvent fraction had excellent overall platelet aggregation inhibitory activity at 0.25 mg/ml concentration, which was comparable to the aggregation inhibitory activity exhibited by aspirin 0.25 mg/ml concentration. In particular, the nucleic acid fraction and ethyl acetate fraction were platelet aggregation. It showed more potent platelet aggregation inhibitory activity than the effect of aspirin 0.5 mg/ml concentration in the extended time and descending area. Therefore, it was confirmed that the fig raw material extract and fraction could replace aspirin as an antithrombotic agent. Platelet aggregation inhibitory activity was evaluated according to the following method. Platelet aggregation inhibition activity [0075] Platelets were mixed with 3.2% sodium citrate solution from healthy human whole blood in a ratio of 1:9, and then centrifuged at 1,100 rpm for 10 minutes to obtain platelet rich (PRP) in the upper layer. plasma), which was washed once with a washing buffer (138 mM NaCl, 2.7 mMKCl, 12 mM NaHCO3, 0.36 mM NaH2PO4, 5.5 mM Glucose, 1 mM EDTA, pH 6.5). Thereafter, re-suspended in suspending buffer (138 mM NaCl, 2.7 mM KCl, 12 mM NaHCO3, 0.36 mM NaH2PO4, 5.5 mM Glucose, 0.49 mMMgCl2, 025% gelatin, pH 7.4), and centrifuged at 3,000 rpm for 10 minutes. It was resuspended in the suspending buffer again, and the platelet count was adjusted to be 4x109/ml. Then, 2.5 ul collagen was added to the 1 ml suspension and reacted for 5 minutes, and platelet aggregation was measured at 37°C using a whole-blood aggregometer (Chrono-log, USA).

Table 5. Inhibitory Activity of Human Platelet Aggregation of Ethanol Extracts and Manures of Fig Materials

Example 7: Chemical properties and stability of the active substance of the fig ethylacetate fraction After the ethyl acetate fraction of the fig obtained in Example 1 was pressure-dried to prepare a powder, the recovered active substance was subjected to human erythrocyte hemolytic activity, plasma stability, Thermal stability and acid stability were confirmed. Adjusted active substance is treated at 100℃ for 2 hours, pH 2 (0.01 M HCl) for 2 hours, and plasma for 2 hours, the ethylacetate fraction does not inhibit thrombin and platelet aggregation inhibitory activity, resulting in high stability. Shown. In addition, hemolytic activity against human red blood cells was not shown up to the concentration of 5 mg/ml (Table 6).

Table 6. Human Erythrocyte Hemolytic Activity of Ethanol Extracts and Manures of Fig Materials

Example 8: Test for measuring antiplatelet aggregation ability using the first purified mixture after extraction of fig organic acid aqueous solution

In order to compare the anticoagulant activity of the extract according to the extraction temperature of the solvent during extraction, the fig material is selected and pulverized, and extracted with an organic acid aqueous solution at a temperature of 10℃, 30℃, 50℃, 70℃, and 100℃, respectively. After filtration and concentration under reduced pressure to obtain a saturated solution, ethanol was added to filter the crystalline compound, washed several times, and the antiplatelet aggregation ability was measured using an impedance method. The impedance method is a method of measuring the change in electrical resistance due to platelet aggregation by passing an electric current into the blood, and a method capable of measuring in whole blood was used. For the experiment, a 4-week-old rat (Slc, ICR Mouse) was supplied from the Central Experimental Animal Co., Ltd., at a temperature of 23±3 ℃, a relative humidity of 50±10%, and an illuminance of 150~300 Lux for 12 hours (9 am ~ 9 p.m.) after acclimatization for 2 weeks in an animal laboratory controlled by 4 weeks old SD (Sprague-Dewley) rat, and 0.38% sodium citrate is 1:9 (v/v) to the blood. Whole blood was used so that the ratio of And after diluting the extract by temperature of figs according to the above extraction method to a concentration of 5 mg/mL, treating each of the whole blood, inducing aggregation by collagen, measuring each resistance value, and then calculating the antiplatelet activity below in the mathematical formula. Each was calculated by applying. The calculated antiplatelet activity is shown in Table 7.

Equation Antiplatelet activity (%) = 1- Resistance value of sample / Resistance value of control X 100

Table 7. Antiplatelet activity by extraction temperature.

From the results of Table 1, it was found that the antiplatelet activity decreased as the extraction temperature increased by 30-40 °C or more.

Example 9: Toxicity test by single intake of ethyl acetate fraction using the first purified mixture after extraction of fig organic acid aqueous solution

One-time oral toxicity of the ethyl acetate fraction obtained from Example 1 was evaluated. First, the samples were dissolved in DMSO and water, and then orally administered at a concentration of 400, 800, 1500, and 2000 mg/kg, respectively, 3 animals, and then survival and symptoms of pathological abnormalities were observed for a week. As a control group, only DMSO was administered orally. As a result, the survival rate was 100%, and there were no signs of pathological abnormalities. Therefore, the ethyl acetate fraction of figs, which have been used for medicinal purposes, is classified as either low toxicity or non-toxic, and it was judged that the use of a low concentration thereof would not cause additional problems.

Example 10: According to the extraction method of the present invention (Fig.-1), using the fig raw material immature fruits and semi-dried figs (dried ripe fruits and unripe fruits) and discarded fig tree trunk fig tree roots, and fig leaves according to the process method As a result, fig leaves based on the concentrated solution of 100g (not dried before thinning) were obtained primarily from 5.6 g of the butanol layer and 12 g from the organic layer according to the extraction and separation process with partial solvent residual content, respectively, and unripe and semi-dried figs (ripe fruit and dried unripe fruit). In the same method, 62.1 g or more was obtained from 15.6 g of a butanol layer and an organic layer. And fig tree trunk and fig tree roots were mixed in the same way to obtain 41.8 g of butanol layer in 34.1 g organic layer.

Example 11: The superiority of the water-soluble organic acid fig raw material extract according to the present invention was confirmed by comparing the anti-platelet activity of the fig raw material extract prepared according to the present invention with the previously reported fig ethanol extract. . The preparation of fig ethanol extract was faithfully prepared according to the method described in the previous report. The antiplatelet activity was measured according to the experimental method described in Example 1.

Table 8. Comparison of antiplatelet activity activity

From the results of Table 8, it was confirmed that the yield was improved and the antiplatelet activity was more excellent depending on the selection of the fig raw material and the selection of the solvent. However, in the case of the previously reported fig raw material extract, extraction with water or alcohol is a common theoretical concept. Due to the physical properties of the fig enzyme picin, a useful thrombolytic substance is not extracted, and even if it is extracted, the yield is too high. I don't think it has any commercial value. In particular, the material to be obtained by the present inventors was difficult to obtain in a high yield due to physical properties due to the change of the material into an insoluble material due to oxygen bonding due to the fusion in the form of a sticky wax. However, as a result of studying figs for many years, the present inventors have found a method of easily extracting even at low temperatures by applying an aqueous organic acid solution, and as a result of conducting an antiplatelet activity experiment using this, the conventional alcohol extraction method or the method of extracting using water has a remarkable yield. It was too low to obtain a useful compound, and the antiplatelet activity test was compared and identified as a control, but it showed little activity. This is judged by the difference in the pretreatment and recovery method for compound extraction used in this example. In particular, in the previous known report, PRP (Platelet Rich Plasma) was used to confirm antiplatelet activity, and ADP and Adrenaline were used as platelet aggregation inducers, but in this example, only whole blood platelets were used, and platelet aggregation inducers were compared using collagen. It was identified and confirmed by previously known methods.

Example 12: Stomatitis drug efficacy test

On the day of infection, anesthetize the rat, remove the hair on the back with an electric razor, and make a 2 cm wound with a knife on the back and stretch the skin epidermal layer to the skin muscle layer. After infecting the wound with Staphylococcus aureus ATCC 6538P (104~106 cfu (inoculum size; 1.12x106 cfu)) directly, the wound area injected with the bacteria was stitched once with a thread and temporarily closed. From the first day after infection, the original ointment (Oramedi OintmentTM) and the composition of Example 9 were each administered three times a day (0.1 g/kg) from the day 1 after infection, confirming that the anesthesia had broken and the same area did not touch other areas (0.1 g/kg /time). On days 2 and 3 of infection, the tissue including the wound site (2x2 cm) was carefully removed. After homogenizing the skin tissue plate in 10 ml of sterile saline for a long time, the supernatant is serially diluted and smeared on an agar plate, and the bacteria grown after 18 hours are counted to determine the therapeutic effect. In addition, rats (7 weeks, 200-250 g) were freely supplied with water and food, and were subjected to the experiment by dividing into a drug-free control group, an existing ointment treatment group, and the composition treatment group of Example 1 by random extraction. As a result, as shown in Table 5 below, the number of bacteria on the 2nd day after infection was the highest in the drug-free rats, but the number of bacteria started to decrease on the 3rd day after infection, and spontaneously healed on the 7th day after infection. Indicated. The composition of Example 1 and the existing ointment had the highest number of bacteria on the first day of infection, and the number of bacteria started to decrease from the second day of infection, and showed a healing phenomenon on the third day. In addition, as shown in Table 5, there was no significant difference, but the fig extract composition of Example 9 was judged to have an excellent infection treatment effect compared to the conventional ointment when compared with the existing ointment. Therefore, the composition for treating stomatitis was judged to have a better treatment effect for stomatitis more quickly when compared to conventional ointments.

Table 9. Comparison of stomatitis treatment efficacy

Unripe and semi-dried figs, which are raw materials for figs, and fig tree stems, fig tree roots, and fig leaves that are discarded were pulverized and extracted with an organic acid solution at room temperature. Next, the insoluble figs were filtered, the recovered filtrate was concentrated under reduced pressure, neutralized by adding a small amount of basic aqueous solution, and then the gel compound produced by adding alcohol was filtered and washed several times with alcohol. A pure fig gel was obtained in yield. Therefore, the present inventors aim to develop a functional food material that exhibits antithrombosis and blood flow improvement activity, after preparing a fig raw material extract, using human plasma and human thrombin to evaluate the thrombiogenesis inhibitory activity according to direct thrombin inhibition, and human concentration. The present invention was completed by measuring the platelet aggregation inhibitory activity of figs using platelets, and confirming very excellent antithrombotic activity in the fig raw material extract and sequential fractions thereof. Therefore, it is possible to use the resources discarded in large quantities by fig cultivation farmers as raw materials for prototypes, thereby contributing to the future industrial development by increasing the income of fig cultivation farmers as well as promoting national health.

(100): Fig (unripe fruit, semi-dried fig) selective crushing step
(200): Organic acid hot water extraction and filtration step
(300): Extraction solution recovery, neutralization and concentration under reduced pressure, and then preparation step as a saturated solution
(400): gelation step after alcohol washing
(500): filtration and alcohol washing step
(600): Fig Gel-1 recovery step
(700) Insoluble solid removal step

Claims (5)

Unripe and semi-dried figs, which are raw materials for figs, and fig tree trunks, fig tree roots, and fig leaves that are discarded are pulverized, and an organic acid solution (acetic acid, citric acid, vitamin C, alpha-beta-dichloroacetate) is used as an extraction solvent. Tec acid (α, β-dichloromaleic acid), alpha-chloro acetec acid (α-chloromaleic acid), acetic anhydride, maleic anhydride, maleic acid, oxalic acid), that is, the volume ratio of water and organic acids is (0.5~5) : 1) Extracting water-soluble components at room temperature using phosphorus solution. Filter the extraction solution, concentrate under reduced pressure to make a saturated solution, cool, filter the precipitate, wash with alcohol solution or ethyl acetate, separate and freeze-dry each A pharmaceutical composition for preventing or treating thrombosis containing fig extract used while immersing it in a separate alcohol solution and used as an active ingredient. The method of claim 1,
The fig raw material extract is a pharmaceutical composition for preventing or treating thrombosis, characterized in that the fig raw material is selected and an organic acid aqueous solution extract. The method of claim 1, wherein a small amount of a basic compound is used as a neutralizing agent after removing insoluble substances from the aqueous organic acid solution, concentration under reduced pressure, and a weak base such as dilute aqueous ammonia, sodium carbonate, sodium hydrogen carbonate, dilute sodium hydroxide, dilute potassium hydroxide, dilute calcium hydroxide, etc. A method for extracting an antithrombotic component from a fig, comprising the step of obtaining a neutralized and purified gel compound by using. It is characterized in that it is obtained by the extraction method of any one of the above claims 1 to 3 by obtaining the organic acid aqueous solution extract, followed by alcohol fractionation, followed by fractionation with ethyl acetate and ethanol mixed solvent (1:1)) solvent. Cosmetic material or pharmaceutical composition for preventing or treating thrombosis, material for treating skin diseases, and use of mouthwash A propellant composition for treating stomatitis, comprising 0.1 to 1% by weight of the organic acid aqueous solution fraction obtained in claim 4 and ethanol. And natural drug stomatitis treatment use

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