KR101763670B1 - Pharmaceutical composition for treatment of contusion comprising Rehmanniae Radix preparata faction - Google Patents
Pharmaceutical composition for treatment of contusion comprising Rehmanniae Radix preparata faction Download PDFInfo
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- KR101763670B1 KR101763670B1 KR1020150139554A KR20150139554A KR101763670B1 KR 101763670 B1 KR101763670 B1 KR 101763670B1 KR 1020150139554 A KR1020150139554 A KR 1020150139554A KR 20150139554 A KR20150139554 A KR 20150139554A KR 101763670 B1 KR101763670 B1 KR 101763670B1
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- A—HUMAN NECESSITIES
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- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
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- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
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Abstract
The present invention relates to a composition for the treatment of contusions comprising a safflower fraction as an active ingredient, wherein the safflower fraction exhibits an effect of increasing the activity of HO-1 and HO-2 in the hepatocyte and kidney glomerular endothelial cells, And can be usefully used as a pharmaceutical composition.
Description
The present invention relates to a composition for the treatment of contusion, which comprises a safflower fraction as an active ingredient, and more particularly, to a composition for treating an contusion, which comprises an effective ingredient for promoting the expression of hemoxysginase-1 and hemoxyoenase- The present invention relates to a pharmaceutical composition for treating bruises which contain fractions as an active ingredient.
Contusion is a disease in which subcutaneous bleeding (bruising) and edema occur due to damage to soft tissues and muscles due to external impact or blunt force (beating or falling), and is sometimes referred to as ejaculation. In the case of a bruise, internal bleeding is always present and inflammation and swelling of the ligaments or muscles occur in sequence. Once over time, inflammation and edema are reduced and gradually recovered, but hemorrhagic leukocytes often remain in the muscle . These eosinophils, when old, interfere with the blood circulation and turn into feces. When the eosinophils and feces are left in the muscles, secondary inflammatory diseases due to necrosis of the blood together with the pain occur.
In the treatment of such bruises, physiotherapy is important, for example, by releasing hemorrhoids by using a medicament therapy or a circulation therapy, preventing inflammation, and promoting blood circulation by vasodilation. However, it is not easy to continue practicing physical therapy in a busy modern society. Even if you are treated to relieve eosinophilia, it is only about 80% relieving pain and inflammation caused by eosinophilia. Fundamental treatment should be done to smooth blood circulation so that inflammation does not occur at all.
On the other hand, heme oxygenase (HO) is an enzyme that catalyzes an oxidation reaction involving oxygen molecules in a heme composed of a porphyrin ring containing an iron (Fe) atom. (Noguchi et al. , 1983, J. Biochem., 93 (4)), as well as hemolysin (HO) decomposes hemoglobin as biliverdin, iron (Fe) and carbon monoxide : 1027-1036 .; Yoshida et al. , 1980, J. Biol. Chem., 255 (10): 4418-4420.).
[Reaction Scheme 1]
Heme b + 3O 2 + 3½NADPH + 3½H + → biliberdin +
Such a reaction is that the red heme is converted to green bilirubin through the green bilirubin, slowly disappearing, resulting in bruising. In the steady state, HO is the most active spleen, where old red blood cells are captured and destroyed.
Heme oxygenase (HO) exists in two forms: heme oxygenase-1 (HO-1) and heme oxygenase-2 (HO-2). The heme oxygenase-1 (HO-1) was induced by a reaction with oxidative stress, hypoxia, heavy metals and the like, and thus the hemoglobin level was decreased due to bleeding or bruising. (Maines et al. , 1977, J. Exp. Med., 145 (4): 1054-1059 .; Agarwal et < RTI ID = 0.0 > al. , 2013, Trans. Am. Clin. Climatol. Assoc., 124: 111-122.). The hemoxic oxygenase-2 (HO-2) is an enzyme that is always present to maintain normal homeostasis and affects the hematopoietic function.
In this regard, studies on HO-1 and HO-2 have shown that Paletum has an anti-inflammatory effect by inducing HO-1 expression in macrophages stimulated by LPS. HO-1 is known to exhibit oxidative tissue And deficiency of HO-1 is known to weaken the resistance to oxidative erythropoiesis by lowering the level of CD49d of erythroblast in the spleen (Oh et al. , 2014, Int. J Gen. et al. , 2013, World J. Hepatol., 5 (10): 541-549 .; Agarwal et al. , 2013, Trans Am. Clin. Clinatol Assoc., 124: 111-122; Cao et al. , 2011, PLoS One., 6 (5): e20634.). It is also known that arsenite deactivates HO-1 while vitamin C (Vit-C) is known to induce HO-1. Induction of HO-1 also exerts an antioxidative protective effect in the heart, and capsaicin capsaicin protects against cisplatin-induced renal toxicity through the induction of HO-1 and protects brain cells from brain damage such as cerebral hemorrhage (Reichard et al. , 2008, J. Bio. Chem., 283 (33):.. 22363-22370 .; Zhao et al, 2014, PLoS One, 9 (6):.. e99134 .; Issan et al, 2014, PLoS One, 9 (3): e92246 .; Jung et al. , 2014, Mol. Cells., 37 (3): 234-240 .; Wang et al., 2007, Brain., 130 (Pt 6): 1643-1652).
In addition, HO-2 has been reported to be selectively activated by the antidepressant menadion. Carbon monoxide (CO) generated during the metabolism of heme, like HO-1, induces NO synthetase In the case of HO-2 deficiency, iron accumulates and toxicity is evident. When HO-2 is released by inflammation in the nephron, its metabolites biliverdine and carbon monoxide (CO) Inhibition of nutrient re-uptake by inhibiting tubuloglomerular feedback has been shown to result in nutritional deficiencies and polyuria and to increase survival of vascular endothelial cells in hypoxia (Wang et al. , 2007, Brain., 130 (Pt6):... 1643-1652 .; Vukomanovic et al, 2014, Mod Gas Res, 4 (1):...... 4 .; Zakhary et al, 1996, Prox Natl Acad Sci, 93: 795-798 .; Leffler et al, 2011: .... Am J. Physiol Heart Cric Physiol, 301 (1):.. H1-H11 .; Dennery et al, 1 998, J. Clin. Invest., 101 (5): 1001-1011 .; Hong et al. , 2011, Am. J. Physiol. Heart Cric Physiol., 300 (4): H1320-H1326 .; He et al . , 2010, J. Biol. Chem., 285 (13): 9452-9461.). Therefore, induction of HO-1 expression can prevent the deterioration of all inflammation associated with hemorrhage through antioxidant protective effect, and induction of H0-2 expression can release eosinophilia caused by bruising through homeostasis and vasodilation effect .
However, most of the substances inducing the expression of HO-1 or HO-2 are mostly chemical substances, which cause problems such as osteoporosis, atherosclerosis, the possibility of carcinogenesis, and the deterioration of symptoms due to resistance. Therefore, it is urgently required to develop a composition capable of treating the bruise by promoting the expression of HO-1 and HO-2 while safely and safely reducing the side effects derived from natural products and thereby improving or preventing inflammation caused by hemorrhage and hemorrhage.
Therefore, the inventors of the present invention have found that, while trying to develop a composition for treating bruises from natural materials, the safflower fraction can be used for treating bruise by promoting the expression of HO-1 and HO-2, .
It is an object of the present invention to provide a pharmaceutical composition for the treatment of bruise which comprises a safflower fraction as an active ingredient.
The present invention provides a pharmaceutical composition for the treatment of bruises, which comprises a safflower fraction as an active ingredient.
As used herein, the term "contusion treatment" is intended to solve the bleeding caused by damage to the soft tissues and muscles caused by an external impact or blunt force (beating, falling), and to prevent and improve inflammation caused by subcutaneous bleeding it means.
The Rehmanniae Radix preparata used in the present invention is a dried form of the root of Rehmannia glutonosa Liboschitz, which is dried in the state of yellow, white or sine, and is dried to improve the blood flow, thereby eliminating ischemia. And at the same time supplement the aura of the relatively difficult blood flow, and it is effective in the Shinyang tribe, the yoshgawa, the oil well, and the bone marrow of the bone is seen, and the various an immune cell and the blood cell proliferation and the bone marrow cell are activated, And it is known that it has an effect of strengthening and strengthening cellular immunity. However, as in the present invention, there is almost no research on the efficacy of treatment of bruise caused by promoting the expression of HO-1 and HO-2 in the safflower fraction.
In the present invention, the safflower fraction is obtained by filtering an extract obtained from a safflower using a hot water extraction method and then fractionating the same into a C 1 to C 4 lower alcohol solution. In the hepatocyte and kidney glomerular endothelial cells, HO-1 and Refers to a substance exhibiting an effect of increasing the activity of HO-2.
The C 1 to C 4 lower alcohol solution may be methanol, ethanol, propanol, butanol, n-propanol, iso-propanol, n-butanol, 1-pentanol, 2-butoxyethanol or ethylene glycol Is preferably ethanol, more preferably 50% to 70% ethanol, even more preferably 60% ethanol.
In one specific embodiment, the safflower fraction of the present invention is obtained by wetting the safflower sourdough with 10 times the total weight of steamed sulfur, heating it at 95 to 100 ° C for 2 to 4 hours, filtering the safflower fraction, To 99.5% ethanol and then fractionated.
The safflower fraction obtained by eluting with 60% ethanol in the obtained safflower fractions increased the expression of HO-1 in the hepatocyte and kidney glomerular endothelial cells by about 1.6 to 3 times and increased the expression of HO-2 by about 1.4 to 3.2 times , Respectively.
Accordingly, the safflower fraction of the present invention can be usefully used as a pharmaceutical composition for treating contusion.
The composition of the present invention may contain 0.001 to 30% by weight, preferably 0.0001 to 10% by weight, of the safflower fraction based on the total weight of the composition. When the weight of the safflower fraction is less than 0.00001 wt%, the effect of promoting the expression of HO-1 and HO-2 is weak. When the weight of the safflower fraction is more than 30 wt%, the expression of HO-1 and HO-2 There is a problem in that the increase in the effect of promoting the growth of the microorganism is not sufficient and the safety of the microorganism is not secured.
The pharmaceutical composition according to one embodiment of the present invention may contain a pharmaceutically acceptable carrier other than the safflower fraction as an active ingredient. The pharmaceutically acceptable carriers are those conventionally used in pharmaceutical preparations and include carbohydrate compounds such as lactose, amylose, dextrose, sucrose, sorbitol, mannitol, starch, cellulose, acacia rubber, calcium phosphate, (Such as corn oil, cottonseed oil, soybean oil, olive oil, coconut oil) such as, for example, water, syrup, salt solution, alcohol, gum arabic, , Polyethylene glycol, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like.
The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc., in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).
The pharmaceutical composition of the present invention may be administered by various routes, such as oral or parenteral, and all manner of administration may be expected, for example, by oral, rectal or intravenous, muscular, subcutaneous injection have. At this time, transdermal administration is preferable for the parenteral route, and topical application by application is most preferable.
The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on such factors as formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, route of administration, excretion rate, . Oral doses of the pharmaceutical compositions of the invention are in the range of 0.001-100 mg / kg (body weight) on an adult basis. When the composition is an external preparation, it is preferable to apply the composition in an amount of 1.0 to 3.0 ml on an adult basis once to five times a day for one month or longer. However, the dose is not intended to limit the scope of the present invention.
The pharmaceutical composition of the present invention may be formulated into a unit dosage form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container.
The pharmaceutical composition of the present invention can be used as an oral preparation or an external preparation in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, according to conventional methods. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like. Examples of liquid formulations for oral use include suspensions, solutions, emulsions and syrups. Common diluents such as water, In addition to liquid paraffin, various excipients such as wetting agents, sweetening agents, fragrances, preservatives and the like may be included. Examples of the external preparation for parenteral administration include non-aqueous solutions, and propylene glycol, polyethylene glycol and vegetable oil such as olive oil may be used as the suspending agent.
On the other hand, the safflower fraction of the present invention can be safely applied to the above-mentioned pharmaceutical composition since it is fractionated from natural safflower and is harmless to the human body and has little toxicity and side effects.
Since the safflower fraction used as an active ingredient in the present invention has an effect of increasing the activity of HO-1 and HO-2 in hepatocytes and kidney glomerular endothelial cells, it can be usefully used as a pharmaceutical composition for treating contusion.
Figure 1 is a graph showing the results of the determination of the total amount of (A) standard product (5-HMF), (B) Sukjiro extract, (C) Fr-1 (water fraction), (D) Fr- 60% EtOH fractions), (F) Fr-4 (99.5% EtOH fractions).
FIG. 2 is a graph showing the effect of 5-HMF and Fr-1 (water fraction), Fr-2 (30% EtOH fractions), Fr-3 (60% EtOH fractions), Fr-4 (99.5% EtOH fractions) 1 activity in human hepatocyte (HH) and human kidney glomerular endothelial cells (HRGEC) treated.
3 is a graph showing the activity of HO-1 in human hepatocyte (HH) and human kidney glomerular endothelial cells (HRGEC) treated with Fr-3 (60% EtOH fraction) and / or hemin .
FIG. 4 shows the results obtained by measuring the concentration of 5-HMF and Fr-1 (water fraction), Fr-2 (30% EtOH fraction), Fr-3 (60% EtOH fraction), Fr- 2 activity in treated human hepatocyte (HH) and human kidney glomerular endothelial cells (HRGEC).
5 is a graph showing the activity of HO-2 in human hepatocyte (HH) and human kidney glomerular endothelial cells (HRGEC) treated with Fr-3 (60% EtOH fraction) and / or hemin .
Hereinafter, embodiments of the present invention will be described in detail to facilitate understanding of the present invention. However, the embodiments according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the invention are provided to more fully describe the present invention to those skilled in the art.
Example 1. Preparation of samples and analysis of components
1-1. Preparation of Sukjipu Extract or Succipyl Fractions
The extracts of Sukjipphi extract were prepared by adding 1 kg of Sukjipu and 10 L of water into an extractor, extracting it at 95 ~ 100 ℃ for 3 hours, filtering and drying.
1 kg of Sukjihwang and fractions of 10 L of water were added to an extractor, and the mixture was extracted at 95 to 100 ° C for 3 hours and filtered. Then, 100 ml of the filtered extract was placed in a column filled with 500 g of
1-2. Analysis of components of Sukjiophorus fractions
Fr-1 (water fraction), Fr-2 (30% ethanol fraction), Fr-3 (60% ethanol fraction) or Fr-4 (99.5% ethanol fraction) Were concentrated under reduced pressure. The fractions were dissolved in 10 mL of methanol and used as a test solution. In addition, 5-HMF (5-Hydroxymethyl-2-furaldehyde) was dissolved in 10 mL of methanol and used as a standard solution.
Next, 10 μL of each of the test solution and standard solution was packed into octadecylsilica gel for a liquid chromatograph having a particle size of 5 μm, and the 5-HMF component was analyzed by HPLC (high performance liquid chromatography). The results are shown in Fig.
As shown in Fig. 1, the content of 5-HMF contained in each fraction was found to be 0.2 mg / g in the water fraction Fr-1, 1.1 mg / g in the 30% ethanol fraction Fr-2, It was confirmed that 5-HMF was contained in 56.5 mg / g of Fr-3, which is a 99.5% ethanol fraction, and 5.0 mg / g, of Fr-4.
Example 2. Cell experiment
2-1. Preparation of experimental material
Ethanol fraction (Fr-1), 30% ethanol fraction (Fr-2), 60% ethanol fraction (Fr-3) or 99.5% ethanol fraction (Fr-4) prepared in Example 1-1 were dissolved in ethanol The concentration of each solution was adjusted to 1 mg / ml and diluted with media to make 100, 10, 1, 0.1 ug / ml. Then, it was added to the cell solution so as to be diluted by 10 times, and prepared so as to have final concentrations of 0.01, 0.1, 1.0, and 10.0 ug / ml, respectively.
2-2. Cell culture
(HGS, Cat. No. 5252) and 1% penicillin / streptomycin solution (Penicillin / Streptomycin) were added to a test tube containing 5% fetal bovine serum, hepatocyte growth supplement was prepared by culturing in 500 ml of hepatocyte medium (ScienCell Research Laboratories, USA) containing 5% CO 2 at 37 ° C and humidified condition of 5% CO 2 / streptomycin solution (P / S, Cat. No. 0503).
HRGEC human renal glomerular endothelial cells (ScienCell Research Laboratories, USA) were incubated with 5% fetal bovine serum and endothelial growth supplement (ECGS, Cat. No. 1052) and 1% penicillin / streptomycin was prepared by culturing in 500 ml of endothelial Cell Medium (ScienCell Research Laboratories, USA) containing a solution (P / S, Cat. No. 0503) at 37 ° C and humidified 5% CO 2 .
2-3. Measure HO-1 and HO-2 activity
The HH human hepatocytes and HRGEC human renal glomerular endothelial cells prepared in 2-2 above were inoculated (1 × 10 6 cells / well) on a 12-well plate and cultured at 37 ° C. for 16 hours at 5% CO 2 .
The first experiment was carried out on the hepatocytes and endothelial cells cultured in the above-mentioned 2 ~ 1 scallopsulfate fraction (Fr-1), 30% ethanol fraction (Fr-2), 60% ethanol fraction (Fr- The fraction (Fr-4) or 5-HMF was treated to 0.01, 0.1, 1, 10 ug / ml concentration and then cultured for 24 hours.
In the second experiment, the hepatocytes and endothelial cells were divided into two groups. One group was treated with 1 μM of hemin and the other group was incubated for 2 hours without any treatment. 1 and 10 ug / ml of 60% ethanol fraction (Fr-3), respectively, and cultured for 24 hours. As a control, HH human hepatocytes and HRGEC human kidney glomerular endothelial cells prepared in 2-2 above were used.
First, the HO-1 activity was measured by measuring the activity of HO-1 using the HO-1 assay kit (Heme oxygenase-1 ELISA kit, MyBioSource, USA) Respectively.
The activity of HO-2 was collected from the wells of the first and second experiments and the activity of HO-2 was measured using HO-2 assay kit (Heme oxygenase-2 ELISA kit, MyBioSource, USA). The results are shown in Table 1 and Figs. 2 to 5.
(ug / ml)
Hepatocyte (HH) and kidney glomerular endothelial cells (HRGEC) treated with 60% ethanol fraction (Fr-3) in the Sulfuric acid fractions of hepatocyte (HH) treated with different fractions, as shown in Tables 1 and 2 to 5, 1 and HO-2 were significantly higher than those of kidney glomerular endothelial cells (HRGEC), and the concentrations of HO-1 and HO-2 increased with increasing concentrations of 60% ethanol fraction (Fr-3) -2 expression was increased in a concentration-dependent manner. Especially, the expression levels of HO-1 and HO-2 were further increased in hepatocyte (HH) and kidney glomerular endothelial cells (HRGEC) treated with hemin.
On the other hand, hepatocyte (HH) and kidney glomerular endothelial cells (HRGEC) treated with SukjungHwang water fraction (Fr-1), 30% fraction of sour orange juice (Fr-2) or 99.5% And HO-2, which are similar to the control group, and that the expression levels of H0-1 and H0-2 did not increase even when the concentration was increased.
Claims (3)
Wherein the C 1 to C 4 lower alcohol solution is a 50% (v / v) to 70% (v / v) ethanol solution.
Characterized in that the safflower fraction promotes protein expression of hamoxygenase-1 (HO-1) and heme oxygenase-2 (HO-2) Composition.
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| KR20220050470A (en) | 2020-10-16 | 2022-04-25 | 최영아 | Composition for removing bruises and skin external materials comprising the same |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1085791A (en) * | 1993-07-30 | 1994-04-27 | 高峰 | Medicine of a kind of strengthening bone, pain killing and removing blood stasis and preparation method thereof |
| CN102363022A (en) * | 2011-11-03 | 2012-02-29 | 李光彩 | Chinese medicine for treating postpartum anemic fainting |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1085791A (en) * | 1993-07-30 | 1994-04-27 | 高峰 | Medicine of a kind of strengthening bone, pain killing and removing blood stasis and preparation method thereof |
| CN102363022A (en) * | 2011-11-03 | 2012-02-29 | 李光彩 | Chinese medicine for treating postpartum anemic fainting |
| CN102363022B (en) * | 2011-11-03 | 2013-03-27 | 李光彩 | Chinese medicine for treating postpartum anemic fainting |
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| KR20220050470A (en) | 2020-10-16 | 2022-04-25 | 최영아 | Composition for removing bruises and skin external materials comprising the same |
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