TWI587858B - Uses of butylidenephthalide - Google Patents

Description

Application of butylene benzoquinone

The present invention relates to the use of butylene benzoquinone to prevent and/or treat oral submucous fibrosis (OSF).

Oral submucous fibrosis is a pre-cancerous condition of oral cancer, but it is not oral cancer. It is mainly characterized by inflammatory reaction and fibrosis in oral submucosal tissue and deep connective tissue. It is highly likely that it will further evolve into oral cancer.

Epidemiological studies have pointed out that chewing betel nut is the most important cause of oral submucous fibrosis. The lesion is common in the buccal mucosa, followed by the Palatal portion and the posterior caries. The mucosa of the affected part will first turn white, then Repeated ulcers or blisters will eventually cause the mucous membrane to lose its elasticity, causing the patient to be unable to enlarge the mouth, seriously affecting daily oral functions such as eating, brushing, oral examination and treatment. If the lesion occurs in the soft palate between the oropharynx, it may cause difficulty in swallowing, atrophy or deformation of the uvula. The patient often has a feeling of burning, tingling, and dryness in the mouth. It is extremely sensitive to spicy, hot foods, but the taste is diminished.

There are currently no drugs that can effectively prevent and/or treat oral submucous fibrosis, usually relying on steroid injection to reduce collagen in the oral mucosa. The amount of tissue in the lower tissue, or surgical or laser to remove the diseased tissue. However, steroid injection only slows down the symptoms and does not cure oral submucous fibrosis, and often accompanied by moon face, fat, buffalo shoulder, osteoporosis, thin skin, edema, easy infection, acne, and long Side effects such as increased blood sugar, increased infection rate, oral mold infection, increased body hair, and poor wound healing. In addition, after surgically or laser surgery and laser ablation of large diseased tissue, the wound is easily contracted to form a scar when it is healed, and it is necessary to consider the limitation of mouth opening and aesthetics. Here, the dental surgeon will consider skin grafting. The way to remedy, but the site of the skin graft will have hidden concerns about early recurrence. When treating an elderly patient or a patient with a large lesion with surgery or laser, it is necessary to consider the injury of the surgery, the patient's physical resistance and quality of life.

In view of the above-mentioned problems regarding the treatment of oral submucous fibrosis, the pharmaceutical industry is still developing a drug that can prevent and/or treat oral submucous fibrosis. The inventors of the present study found that butylene benzoquinone can effectively inhibit epithelial-mesenchymal transition (EMT) in oral submucosal tissue cells, inhibit the differentiation of oral submucosal tissue cells into myofibroblasts, and inhibit The activation of myofibroblasts inhibits the accumulation of collagen in the submucosal tissues and inhibits the contraction of interstitial cells in the submucosal tissues, and is useful for the prevention and/or treatment of oral submucous fibrosis.

It is an object of the present invention to provide an use of an active ingredient for the manufacture of a medicament, wherein the active ingredient is selected from the group consisting of butylidenephthalide (BP), a pharmaceutically acceptable salt thereof, and The aforementioned group And the agent is used for the prevention and/or treatment of oral submucous fibrosis (OSF). Preferably, the medicament is an injection, a lozenge, an oral solution, or an smear.

Another object of the present invention is to provide a pharmaceutical composition for preventing and/or treating oral submucous fibrosis (OSF) comprising an effective amount of an active ingredient and a pharmaceutically acceptable substance A carrier wherein the active ingredient is selected from the group consisting of butylidenephthalide (BP), a pharmaceutically acceptable salt thereof, and combinations of the foregoing. Preferably, the pharmaceutical composition is an injection, a lozenge, an oral solution, or an smear.

A further object of the present invention is to provide a method for preventing and/or treating oral submucous fibrosis (OSF), which comprises administering an effective amount of an active ingredient to an individual in need thereof, wherein The active ingredient is selected from the group consisting of butylene benzoquinone (BP), a pharmaceutically acceptable salt thereof, or a combination of the foregoing. Wherein the active ingredient can be administered to the individual in the form of an injection, a lozenge, an oral solution, or a spread.

The detailed technical content and some of the specific embodiments of the present invention will be described in the following, and the present invention will be apparent to those of ordinary skill in the art.

Figure 1 shows buccal mucosal fibroblasts (BMFs; including "BMF-1 (ie, ◆)" and "BMF-2" in normal oral buccal mucosa treated with different concentrations of butylenephthalide (BP). (ie, ■)") and fibrotic buccal mucosal fibroblasts (fBMFs; including "fBMF-1 (ie, △)" and "fBMF-2 (ie, □)") Graph of relative survival rate (%); Figures 2A and 2B show the analysis of different concentrations of butylenephthalide (BP) by Quantitative Real-time Polymerase Chain Reaction (Q-PCR) Bar graph of the relative expression (multiple) of the Twist , Snail , and ZEB1 genes of fBMF-1 or fBMF-2, of which the second graph is the result of fBMF-1 and the second graph is the result of fBMF-2. Figure 3A is a photographic diagram showing the migration of BMFs and fBMFs treated with different concentrations of butylene benzoquinone (BP) from the upper tray to the lower tray, wherein the purple stained part is the cell; Figure 3B shows BMFs (including BMF-1 (dark gray strips), and BMF-2 (light gray strips)) and different concentrations Bar graph of the relative mobility (%) of fBMFs treated with butylene benzoquinone (BP) (including fBMF-1 (dark gray strip) and fBMF-2 (light gray strip)); 4A, 4B The relative amount of α-SMA , Col1a1 , and S100A4 genes of fBMF-1 or fBMF-2 treated with different concentrations of butylenephthalide (BP) was analyzed by real-time quantitative polymerase chain reaction (Q-PCR). a bar graph of (multiple), wherein the graph A is the result of fBMF-1, the graph 4B is the result of fBMF-2, and the graph 5A shows the BMFs and the treatment with different concentrations of butylenephthalide (BP). Photograph of a situation in which fBMFs cause colloidal contraction, where the circle enclosed by the green dashed line is the colloid; and Figure 5B shows the colloid via BMFs (including BMF-1 (dark gray strip), and BMF-2 ( Light gray strips)) and relative colloids induced by fBMFs treated with different concentrations of butylene benzoquinone (BP), including fBMF-1 (dark gray strips) and fBMF-2 (light gray strips) Bar graph of area (%).

The invention will be described in detail below with reference to the embodiments of the present invention. The present invention may be practiced in various different forms without departing from the spirit and scope of the invention. . In addition, the terms "a", "an" and "the" In addition, the words "about", "about" or "nearly" as used in this specification mean substantially the difference between the stated value and the stated value within 20%, preferably within 10%, and more preferably Within 5%.

In the present specification, the term "prevention, preventive, preventive, preventive, and prophylaxix" refers to the ability to prevent or minimize the onset or deterioration of a disease or condition before it occurs. "treatment" means the eradication, removal, reversal, mitigation, improvement, or control of a disease or condition; the so-called "effective amount" or "therapeutically effective amount" means that at least part of the treatment is effective when administered to an individual. An amount of a compound which improves the condition of a suspected individual; the term "individual" means a mammal, and the mammal can be a human or a non-human animal.

Studies have confirmed that in the process of oral submucous fibrosis, the submucosal tissue cells undergo epithelial-mesenchymal transition (EMT) and differentiate into myofibroblasts. Among them, when the cells undergo epithelial-mesenchymal transition (EMT), the genes such as Twist , Snail , and ZER1 in the cells are highly expressed, and the polarity between the cells and cells is gradually lost, and the migration ability of the cells is increased and it is easy to crawl. And invasion, participating in tissue fibrosis. Myofibroblasts are highly mobile cells that display marker genes such as α-SMA , Col1a1 , and S100A4 . When activated, they secrete excessive extracellular matrix (ECM), such as fibronectin. Collagen, and induces contraction in the interstitial tissue of the tissue, thereby promoting tissue fibrosis. It has also been confirmed that excessive accumulation of extracellular matrix is also closely related to the occurrence of oral submucous fibrosis. For example, the role of epithelial-mesenchymal transition in oral squamous cell carcinoma and oral submucous fibrosis. Clin Chim Acta. Aug; 383(1-2): 51-56 (2007), Oral submucous fibrosis: An update on etiology And pathogenesis-A review. Rama Univ J Dent Sci. Mar;2(1):24-33(2015), Oral submucous fibrosis: Review on aetiology and pathogenesis. Oral Oncol. Jul;42(6):561-568( 2006), and Molecular pathogenesis of oral submucous fibrosis-a collagen metabolic disorder. J Oral Pathol Med. Jul; 34(6): 321-328 (2005), the entire disclosure of which is hereby incorporated by reference.

Because tissue cell epithelial-mesenchymal transition (EMT) and activation of myofibroblasts are closely related to oral submucous fibrosis, Xianxin can inhibit epithelial-mesenchymal transition (EMT) and inhibit oral submucosal tissue in oral submucosal tissue cells. Differentiation of cells into myofibroblasts and/or inhibition of activation of myofibroblasts can effectively inhibit excessive accumulation of extracellular matrix, inhibit contraction of tissue interstitial cells, and prevent and/or treat oral submucous fibrosis. See, for example, Betal-drived alkaloid up-regulates keratinocyte alphavbeta6 integrin expression and promotes oral submucous fibrosis. J Pathol. Feb; 223(3): 366-377 (2011), Arecoline-induced myofibroblast transdifferentiation from human buccal mucosal fibroblasts is Mediated by ZEB1. J Cell Mol Med. Apr;18(4):698-708(2014), and Elevation of S100A4 expression in buccal mucosal fibroblasts by arecoline:involvement in the pathogenesis of oral submucous fibrosis. PLoS One .8(1 ): e55122 (2013), the entire contents of which are hereby incorporated by reference.

The inventors of the present invention have found that treatment of fibrotic oral submucosal tissue cells with p-butylphenyl hydrazine (BP) can effectively inhibit epithelial-to-mesenchymal transition (EMT) of cells in the tissue, especially inhibiting the cells. It can express the genes such as Twist , Snail , and ZEB1 , and inhibit the creep and invasion of the cells, so it can inhibit the fibrosis of the oral mucosa.

The inventors of the present invention have further found that the treatment of fibrotic oral submucosal tissue cells with butylene benzoquinone (BP) can effectively inhibit the differentiation of cells into myofibroblasts and inhibit the activation of myofibroblasts in the tissue, especially inhibiting the inhibition. The tissue cells express genes such as α-SMA, Col1a1 , and S100A4 , and inhibit the contraction of interstitial cells in the tissue, and can inhibit the fibrosis of the oral mucosa.

Accordingly, the present invention relates to medicaments, pharmaceutical compositions, and methods for providing prevention and/or treatment of oral submucous fibrosis. Wherein the pharmaceutical preparation comprises an active ingredient comprising an effective amount of the active ingredient and a pharmaceutically acceptable carrier, the method comprising administering an effective amount of the active ingredient to the need thereof. In the individual. According to the agent, pharmaceutical composition, and method of the present invention, the active ingredient is selected from the group consisting of butylenebenzaldehyde (BP), a pharmaceutically acceptable salt thereof, or a combination thereof.

In the pharmaceutical, pharmaceutical composition, and method of the present invention, examples of the pharmaceutically acceptable salt include, but are not limited to, alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts. Salts and strontium salts; transition metal salts such as zinc salts, copper salts, iron salts, cobalt salts, titanium salts, vanadium salts; aluminum salts; tin salts; alkanolamine salts such as diethanolamine salts, 2-amino groups - 2-ethyl-1,3-propanediol salt, and triethanolamine salt; heterocyclic amine salts such as morpholine salts, piperazine salts, and piperidine salts; Alkali amine salts such as ammonium salts, arginine salts, persalt salts, and histidine salts, and the like. Preferably, the active ingredient used in the medicament, pharmaceutical composition, and method of the present invention is butylene benzoquinone (BP).

When the agent or pharmaceutical composition of the present invention is used to prevent and/or treat oral submucous fibrosis, the agent or pharmaceutical composition may be in a correspondingly convenient dosage form depending on the desired form of administration. For example, but not limited thereto, the pharmaceutical or pharmaceutical composition can be administered to an individual in need via a route of administration such as intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, oral, dermal, and mucosal.

The pharmaceutical or pharmaceutical composition of the present invention can be provided in the form of an injection, but is not limited thereto, as an example of a dosage form suitable for intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, or the like. For example, examples of the injection include intravenous infusion, emulsion intravenous infusion, dry powder injection, suspension injection, and dry powder suspension injection, but are not limited thereto. In addition, the pharmaceutical or pharmaceutical composition can be prepared as a pre-injection solid, the pre-injection solid is provided in a dosage form soluble in other solutions or suspensions, or in an emulsifiable dosage form, and administered to the individual in need thereof. Previously, the pre-injection solid is dissolved in other solutions or suspensions or emulsified to provide the desired injectables.

Taking the dosage form suitable for oral administration as an example, the medicament provided by the present invention Or the pharmaceutical composition may be in the form of a solid such as a tablet, a pill, a capsule, a granule, a powder, or the like, or a liquid type such as an oral solution, a syrup, a spirit, an elixir, a tincture, or the like. Provided, but not limited to.

The dosage form of the present invention may be, for example, an emulsion, a cream, a gel (hydrogel), a paste (dispersion cream, an ointment), or the like, or a medicinal preparation, which is suitable for administration routes such as skin and mucous membranes. The form of mouth, lotion, spray, or patch (patch) is provided, but not limited to.

Preferably, the agents and pharmaceutical compositions of the present invention are provided in the form of injections, lozenges, oral solutions, or spreads.

In the present invention, depending on the route of administration and/or the form of administration, a suitable pharmaceutically acceptable carrier may be optionally provided to provide the agent of the present invention, and a pharmaceutical acceptable composition may also be included in the pharmaceutical composition of the present invention. Carrier. By way of example, examples of such pharmaceutically acceptable carriers include, but are not limited to, solvents (buffers, water, saline, dextrose, glycerol, ethanol or the like, and combinations thereof), oily solvents , diluent, stabilizer, absorption retarder, disintegrating agent, emulsifier, antioxidant, binder, binder, tackifier, solubilizer, dispersant, suspending agent, lubricant, moisture absorbent, solid carrier (eg starch, and bentonite).

Optionally, the pharmaceutical or pharmaceutical composition of the present invention may be encapsulated in a drug delivery system such as a liposome, microparticles, or microcapsules, and then administered to an individual in need thereof to increase the activity in the drug or pharmaceutical composition. The delivery efficiency of the component is such that the constituents of the drug delivery system have no adverse effect on the desired benefit of the active ingredient (i.e., butylene benzoquinone (BP), its pharmaceutically acceptable salt, or a combination of the foregoing). can.

Optionally, a suitable amount of an additive may be further added to the pharmaceutical or pharmaceutical composition provided by the present invention, for example, a flavoring agent (for example, sucrose) which improves the mouthfeel and visual sensation of the pharmaceutical or pharmaceutical composition when administered. A toner, a coloring agent, or the like, and a buffering agent, a preservative, a preservative, an antibacterial agent, an antifungal agent, and the like which can improve the stability and storage property of the pharmaceutical or pharmaceutical composition. In addition, the pharmaceutical or pharmaceutical composition may optionally contain one or more additional active ingredients (eg, steroids) or may be combined with a medicament containing the one or more other active ingredients to further enhance the efficacy or increase of the pharmaceutical or pharmaceutical composition. Flexibility and formulation of the formulation of the formulation, as long as the other active ingredient is not detrimental to the desired benefit of the active ingredient of the present invention (i.e., butylene benzoquinone (BP), its pharmaceutically acceptable salt, or a combination of the foregoing) The impact can be.

The medicament or pharmaceutical composition provided by the present invention may be administered at different frequencies, such as once a day, multiple times a day, or once every other day, depending on the age, weight, and health condition of the individual to be administered. For example, when administered orally to a body for the prevention and/or treatment of oral submucous fibrosis, it is administered in an amount of about 5 mg/kg body weight to about 500 mg per day in terms of butylene benzoquinone (BP). / kg body weight, preferably about 10 mg / kg body weight to about 120 mg / kg body weight per day, more preferably about 20 mg / kg body weight to about 90 mg / kg body weight per day, wherein the unit "mg / kg body weight" Refers to the amount of drug required per kilogram of body weight. However, for acute patients, the amount can be increased according to actual needs, for example, increased to several times or tens of times.

Further, the agent or pharmaceutical composition provided by the present invention may be used in combination with one of the following to prevent and/or treat oral submucous fibrosis: surgical treatment, and laser treatment.

According to the method of the present invention for preventing and/or treating oral submucous fibrosis, administration of the active ingredient (i.e., butylene benzoquinone (BP), a pharmaceutically acceptable salt thereof, or a combination thereof) Routes, administration forms, dosages, and related therapeutic applications are as described above.

The invention is further illustrated by the following examples. The embodiments are provided by way of illustration only and are not intended to limit the scope of the invention. The scope of the invention is shown in the appended claims.

Example

[Preparation Example]:

A. Cell culture

The two groups were taken from the normal buccal mucosa (buccal mucosal fibroblasts; hereinafter referred to as "BMF-1" and "BMF-2"; or collectively referred to as "BMFs"), and the two groups were taken from fibrosis. The fibrotic buccal mucosal fibroblasts (hereinafter referred to as "fBMF-1" and "fBMF-2"; or "fBMFs") are treated as follows: 2 x 10 ⁴ cells per well Separately seeded in 24-well plates and cultured to 8 minutes (ie, 80% confluence), then at different concentrations (0, 12.5, 25, 50, 100, or 200 μg / ml) of Aden Benzoquinone (BP) was processed for 48 hours.

B. Extraction of total RNA from cells

After suspending each group of cells provided in [Preparation Example] A with trypsin, the following steps were carried out separately: (i) centrifugation (1000 rpm, 5 minutes), removal of the supernatant; (ii) addition of 1 ml TRIzol reagent (purchased from Invitrogen Life Technologies), after mixing evenly, let stand at room temperature for 5 minutes; (iii) add Enter 100 μl of BCP (bromochloropropane) and shake it up and down to make it evenly mixed, then let it stand at room temperature for 5 minutes; (iv) Centrifugation (Eppendorf centrifuge, F45-30-11 rotor, 4 ° C, 12000 rpm, 15 After the minute, the upper liquid was transferred to a new centrifuge tube, and isopropanol was added thereto to mix well, and then allowed to stand at room temperature for 5 minutes; (v) centrifugation (Eppendorf centrifuge, F45-30-11) After removing the supernatant from the rotor, 4 ° C, 12000 rpm, 10 minutes), the RNA precipitated at the bottom of the tube was washed with 500 μl of 75% alcohol; (vi) centrifugation (room temperature, 12000 rpm, 5 minutes) to remove the alcohol layer And air-dried in a plenum; and (vii) water-resolved RNA precipitate treated with 20 microliters of diethyl pyrocarbonate (DEPC) to measure its absorbance at 260 nm. And calculate the RNA concentration.

Example 1: Evaluation of cytotoxicity of butylene benzoquinone (BP)

Take the cells of each group provided in [Preparation Example] A, and perform the following steps separately: (i) remove the supernatant in the 24-well plate, and then add 500 μl of 3-(4,5-two) to each well. 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide (MTT) buffer (final concentration) (0.5 mg/ml); (ii) Place the well plate in a 37 ° C, 5% CO ₂ incubator for 3 hours; (iii) After removing the supernatant, add 1000 μm to each well. Raise the isopropanol and place the well plate on a shaker for 10 minutes; and (iv) take 200 μl of the mixed solution from each well into a 96-well plate and measure it with a spectrophotometer. The absorbance at a wavelength of 570 nm was converted into the relative survival rate (%) of each group of cells, and the results are shown in Fig. 1.

It can be seen from Fig. 1 that a concentration of 50 μg/ml of butylene benzoquinone (BP) is added to the culture medium, and primary fibroblasts (BMFs) of the normal buccal mucosa are added. Growth will not have a significant impact. Therefore, the subsequent experiments were carried out with butylene benzoquinone (BP) at a concentration of 0 to 50 μg/ml.

Example 2: Analysis of the efficacy of butylene benzoquinone (BP) in inhibiting oral epithelial tissue cells for epithelial-mesenchymal transition (EMT)

A. Expression of marker genes in epithelial-mesenchymal transition (EMT)

It is known that during the process of epithelial-mesenchymal transition (EMT), the expression of genes such as Twist , Snail , and ZEB1 is increased, so these genes are regarded as marker genes for epithelial-mesenchymal transition (EMT). This experiment explores whether butylenebenzaphthalein (BP) affects genes such as Twist , Snail , and ZEB1 in buccal mucosa cells through quantagonistic real-time polymerase chain reaction (Q-PCR). Performance in the middle.

First, the total RNA (1 μg of each group) provided in [Preparation Example] B was taken for reverse transcription to provide complementary deoxyribonucleic acid (cDNA). Next, a real-time quantitative polymerase chain reaction (Q-PCR) was performed using a gene quantification system (PRISM ABI7700 Sequence Detecting System, available from Applied Biosystems, USA) with primers for specific genes (shown in Table 1 below) to analyze Twist , The expression of Snail , and ZEB1 genes in fBMFs (including fBMF-1, fBMF-2) treated with different concentrations (0, 25, or 50 μg/ml) of butylenephthalide (BP). Finally, the relative gene expression (multiple) of each group was calculated based on the results of cells treated without butyl benzoquinone (BP), and the results are shown in Fig. 2.

From the results of Fig. 2, it was found that the expression of genes such as fBMF-1 or fBMF-2, Twist , Snail , and ZEB1 was markedly decreased as the concentration of butylene benzoquinone (BP) was increased. The foregoing results show that butylene benzoquinone (BP) can effectively inhibit epithelial-mesenchymal transition (EMT) in oral submucosal tissue cells, and thus can be used for preventing and/or treating fibrosis of oral submucosal tissues.

B. Crawling ability and invasive ability of oral submucosal tissue cells

In this study, we conducted a penetrating cell migration test system (Transwell® system, purchased from Corning, UK) with a polycarbonate membrane with a pore size of 8 μm (purchased from Corning, UK) to investigate butylene benzoquinone. Whether (BP) can inhibit the crawling ability and invasive ability of oral submucosal tissue cells.

First, a culture solution containing 10% FBS (fetal calf serum) is added to a lower chamber, and the polycarbonate filter device is placed at the bottom of a cell culture plate, which is called an upper chamber. . On the other hand, BMF-1, BMF-2, fBMF-1, and fBMF-2 cells (2 x 10 ⁴ cells each) in [Preparation Example] A were taken, respectively, containing 0, 25, or 50 μg. /ml of butyl benzoquinone (BP) but serum-free medium (250 μl) was mixed well, and the mixed solution was injected into the upper tray device. Next, the penetrating cell migration assay system was placed in an incubator to induce cell migration for 24 hours. The penetrating cell migration test system was taken out, and the cells not moved from the upper disc membrane surface to the lower disc membrane surface were removed, and the polycarbonate filter membrane was fixed with 4% paraformaldehyde (10 minutes). And stained with 0.1% crystal violet. Finally, the polycarbonate filter was carefully cut and placed on a glass slide, and observed under a microscope at a magnification of 100 times (5 different fields of view were observed in each group), photographed, and unp-butylene benzene. Based on the results of BMFs treated with 酞 (BP), the number of cells on the membrane was converted into the relative mobility (%) of the cells. The above experiment was performed in three replicates, and the results are shown in Figures 3A and 3B, wherein the purple-stained portion of Figure 3A is the cell that migrated from the upper disk to the lower disk, and the third B-picture is the three-fold The results of the experiment averaged the relative mobility of the cells (%).

From the results of the 3A and 3B graphs, the "fBMF group" migrated from the upper tray to the lower tray significantly more than the "BMFs group". However, in the "fBMFs group", if the cell line was first treated with butylene benzoquinone (BP), the cell line that migrated from the upper disc to the lower disc significantly decreased as the concentration of butylene benzoquinone (BP) increased. The above results show that fibrotic oral submucosal tissue cells have strong crawling ability and invasive ability compared with normal oral submucosal tissue cells, and easy epithelial-mesenchymal transition (EMT), while butylenephthalide (BP) ) can effectively inhibit the crawling ability and invasive ability of oral submucosal tissue cells. That is, butylene benzoquinone (BP) can effectively inhibit epithelial-mesenchymal transition (EMT) in oral submucosal tissue cells, and thus can be used for preventing and/or treating fibrosis of oral submucosal tissues.

Example 3: Analysis of the efficacy of butylene benzoquinone (BP) in inhibiting the differentiation of oral submucosal tissue cells into myofibroblasts

It is known that in the process of fibrosis of tissues, cells in tissues are differentiated and transformed into myofibroblasts, and marker genes of myofibroblasts such as α-SMA, Col1a1 , and S100A4 . Therefore, this experiment explores whether butylenebenzaphthalein (BP) inhibits α-SMA, Col1a1 , and S100A4 in cells of fibrotic oral buccal mucosa by real-time quantitative polymerase chain reaction (Q-PCR). Performance.

The total RNA (1 μg of each group) provided in [Preparation Example] B was subjected to reverse transcription to provide complementary deoxyribonucleic acid (cDNA). Next, a real-time quantitative polymerase chain reaction (Q-PCR) was performed using a gene quantification system (PRISM ABI7700 Sequence Detecting System, available from Applied Biosystems, USA) with primers for specific genes (shown in Table 2 below) to analyze α- Genes such as SMA , Col1a1 , and S100A4 were expressed in fBMFs (including fBMF-1, fBMF-2) treated with different concentrations (0, 25, or 50 μg/ml) of butylenephthalide (BP). Finally, the relative gene expression (multiple) of each group was calculated based on the results of cells treated without butyl benzoquinone (BP), and the results are shown in Fig. 4.

From the results of Fig. 4, it was found that the expression of genes such as α-SMA, Col1a1 , and S100A4 , whether fBMF-1 or fBMF-2, significantly decreased as the concentration of butylene benzoquinone (BP) increased. The foregoing results show that butylene benzoquinone (BP) has the effect of inhibiting the differentiation of oral submucosal tissue cells into myofibroblasts, thereby inhibiting the accumulation of collagen in the oral submucosal tissue and effectively preventing and/or treating the fibers of the oral submucosal tissue. Chemical.

Example 4: Analysis of the effect of butylene benzoquinone (BP) on inhibiting interstitial contraction of oral submucosal tissue

Take BMF-1, BMF-2, fBMF-1, and fBMF-2 cells in [Preparation Example] A, and dissolve the above cells in 0.5 ml of collagen solution at a concentration of 2 mg/ml (purchased from Sigma- In Aldrich, each mixed solution was transferred to a 24-well plate and placed in an incubator at 37 ° C and 5% CO ₂ for 2 hours to coagulate the collagen colloid. The obtained gel system was called "BMFs group". And "fBMFs group". Next, the coagulated colloid was detached from the culture plate, and 0.5 ml of a cell culture medium containing different concentrations (0, 25, or 50 μg/ml) of butylene benzoquinone (BP) was added for cultivation, and after 48 hours, each was observed. The colloid shrinkage, photo recording, and image analysis software ImageJ (National Institute of Health, USA) were used to calculate the colloidal area of the "BMFs group" without butyl benzoquinone (BP). Relative colloid area (%). The results are shown in Figures 5A and 5B, in which Figure 5A shows the colloidal contraction caused by each group of cells, and Figure 5B shows the relative colloidal area (%) of each group.

As can be seen from the 5A and 5B graphs, the colloidal area of the "fBMF group" is significantly smaller than that of the "BMFs group", that is, the colloid is significantly contracted. However, in the "fBMFs group", if the cell line in the colloid is first treated with butylene benzoquinone (BP), the colloid shrinkage phenomenon is significantly improved, and the degree of improvement is accompanied by Aden. The concentration of phenylhydrazine (BP) increases as the concentration increases. The foregoing results show that butylene benzoquinone (BP) can effectively inhibit the activation of myofibroblasts and inhibit the interstitial contraction of tissue cells induced by myofibroblasts.

From the above experimental results, it can be known that butylene benzoquinone (BP) can effectively inhibit epithelial-mesenchymal transition (EMT) in oral submucosal tissue cells, inhibit the differentiation of oral submucosal tissue cells into myofibroblasts, and inhibit the activation of myofibroblasts. It can inhibit the accumulation of collagen in the submucosal tissues and inhibit the contraction of interstitial cells in the oral submucosa.

<110> Changhong Biotechnology Co., Ltd.

<120> Application of butylene benzoquinone

<130> None

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<170> PatentIn version 3.5

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Claims (8)

An use of an active ingredient for the manufacture of a medicament, wherein the active ingredient is selected from the group consisting of butylidenephthalide (BP), a pharmaceutically acceptable salt thereof, and combinations thereof, and the pharmaceutical system is For the prevention and / or treatment of oral submucous fibrosis (OSF). The use of claim 1, wherein the agent is for inhibiting epithelial-mesenchymal transition (EMT) in oral submucosal tissue cells. The use of claim 2, wherein the agent is for inhibiting the ability of the oral submucosal tissue cells to crawl and invade. The use of claim 1, wherein the agent is for inhibiting differentiation of oral submucosal tissue cells into myofibroblasts. The use of claim 1, wherein the agent is for inhibiting activation of myofibroblasts. The use according to any one of claims 1 to 5, wherein the agent is for inhibiting contraction in the interstitial cells of the oral submucosal tissue. The use of claim 6, wherein the agent is an injection, a lozenge, an oral solution, or an smear. The use of any one of claims 1 to 5, wherein the medicament is an injection, a lozenge, an oral solution, or an smear.