KR100999227B1 - A composition of complex herb medicine for treatment and control of alzheimer's disease - Google Patents

Abstract

PURPOSE: A composition of complex herb medicinal formulation for treating and suppressing dementia is provided to suppress gene expression relating Alzheimer disease and dementia without side effects. CONSTITUTION: A composition of complex herb medicinal formulation for treating and suppressing dementia contains mixture extracts of 4.0 weight parts of Acori graminei Rhizoma, 2.0 weight parts of Polygalae Radix, 4.0 weight parts of Hoelen cum Radix, 2.0 weight parts of Crataegi Fructus and 2.0 weight parts of Morindae Radix. An agent for suppressing and treating dementia is manufactured from the composition.

Description

  A composition of complex herbal medicine for treatment and control of Alzheimer's disease

 The present invention relates to a composite pharmaceutical composition obtained by extracting from a mixture of 4.0 parts by weight of Seokchangpo, 2.0 or 4.0 parts by weight of paper, 4.0 or 2.0 parts by weight of baekxin, 2.0 parts by weight of livestock and 2.0 parts by weight of rupture cloth, and a pharmaceutical containing the composition. will be.

  Recently, the social cost burden for senile disease has increased significantly due to the rapid increase of the elderly population and the advancement of society, and the development of new drugs in this field is considered as an essential field in order to improve the quality of life. In particular, the elderly population is increasing due to the extension of life expectancy, and a significant proportion of them are estimated to be more than 8%, and the potential market for the treatment of dementia is gradually increasing. In addition, dementia is not yet developed, so it is expected that the market will expand rapidly when a new drug is developed.

    However, there is no foundation for the development of a unique treatment for dementia, and research on the pathogenesis of dementia and the role of genes and proteins related to dementia are underdeveloped. Therefore, if a bioinformatics system for dementia-related genes and proteins is established at the national level to identify new target substances and identify the pathogenesis, it will be possible to develop novel new dementia treatments.

    Alzheimer's dementia (senile dementia) is a disease that causes abnormalities in the central nervous system, which causes memory loss and language disorders as early symptoms, and as the disease progresses, nothing can be done without surrounding help, leading to death. The dementia treatments for senile dementia developed so far are acetyl cholin esterase (AChE) inhibitors, choline-producing precursors, receptor inhibitors, and antioxidants. Among them, the only commercially available dementia treatment agent, tacrine, tacrine, which is an AChE inhibitor, is commercialized. Aricept consisting of Cognex and donepezil, which are based on a series of compounds, is known, but none of the drugs have yet been fully effective. In the case of AChE inhibitors, many substances are difficult to cross the blood brain barrier, and the inhibitory constant (Ki) falls to the nM range. On the other hand, in the case of β-amyloid accumulation, which is being studied in recent years, α and γ secretase inhibitors and metalloendopeptidase inhibitors are important, but these mechanisms are used clinically and there are no drugs.

    On the other hand, Chong Myeong-tang (聰明 湯) has been used for the symptoms of dementia and dementia caused by cardiac and biliary diseases since it was described as "chida mang, bok bok seong cheon eon" as a control made up of Wonji, Seokchangpo, and Baekboksin. Neuropsychiatric Medicine, College of Oriental Neuropsychiatry, Seoul Munmundang, 2005: 311-6,432-5,451.), Kim et al. (Kim, Young-Wook.Chong Myeong-tang's effect on the class and memory of the induction white paper, Daejeon University Graduate School 1999.) It has been reported that there is a significant effect on learning and memory of judo white paper. Ha et al. (Ha Soo-young et al. 2, Effects of Chongmyung-tang and Sansa Chongmyung-tang on Alzheimer's Disease Condition Model, Journal of the Korean Society for Neuropsychology. 2006; 17 (1): 59-78) , IL-6, TNF-α expression, PC-12 cell line APP and AChE gene expression was inhibited and AChE activity and APP expression in the PC-12 cell line cellular protein. In addition, the effects of hot water extracts of Chongmyung-tang and Pageukcheon Chongmyeong-tang and ultra-fine powder formulations on microglia and memory decay pathologic models, and the Korean Journal of Oriental Physiology and Pathology. 2006. 20 (4): 997-1008) Inhibited the expression of IL-1β, IL-6, TNF-α, NOS-II and COX-2 genes in BV2 microglial cell lines and culture supernatants, reduced NO and ROS production, and reduced IL-10 and TGF-β1 MRNA expression was increased and Morris water maze showed significant memory improvement. Therefore, it can be used as a therapeutic agent for memory loss caused by Alzheimer's dementia and choline neuron degeneration caused by overproduction of β-amyloid and overexpression of proinflammatory cytokynes in microglial cells. do.

Ha, et al. (Ha Soo-young et al., 2, Chong Myung-tang and Sansa Chong-myeong-tang, on the effect of Alzheimer's Disease condition model. Journal of the Korean Society for Neuropsychology. 2006; 17 (1): 59-78) It inhibited the expression of IL-1β, IL-6, and TNF-a, inhibited the expression of APP and AChE genes in PC-12 cell lines, and inhibited AChE activity and APP expression in PC-12 cell line cellular proteins. -Alzheimer's dementia, which may be caused by overproduction of amyloid, overexpression of proinflammatory cytokynes in microglial cells, and memory loss caused by degeneration of choline neurons I've done it.

Considering the research suggesting the possibility that Chongmyung-tang can be applied to the treatment and prevention of Alzheimer's dementia and the study of the effect of Pagecheoncheon on the dementia condition model, Pageokcheon Chongmyeongtang made by applying Paekcheoncheon to Chongmyeongtang It is also thought to be effective in the treatment and prevention of dementia. Im, et al. (Effect of Im, Jung-Hwa et al. 2, Chongmyung-tang and Pageukcheon Chongmyung-tang hot water extracts and ultra-fine powder formulations on microglia and memory loss condition model, Journal of Oriental Physiology and Pathology. 2006. 20 (4): 997-1008) Inhibited the mRNA expression of IL-1β, IL-6, TNF-a, NOS-II and COX-2 in the BV2 microglial cell line, and increased the mRNA expression of IL-10 and TGF-β1 in BV2 microglial cell line. The production of IL-1β, IL-6, TNF-a, NO and ROS was decreased in the culture supernatant of the cell line. In addition, serum glucose in scopolamine-induced memory depleted mice significantly reduced AChE activity and significantly improved memory in the Morris water maze experiment. Pageuk Chun-myung-tang was used to prevent and treat Alzheimer's dementia. It seems to be possible.

  The present invention differs in the blending ratio of raw paper and Seokchangpo in Chongmyeongtang (2g base, 2g Seokchangpo, 2g Baeksin), which is described in "東 醫 寶 鑑 · 內景" as "治 多 忘 久服 能 日 誦 千 言". It is a prescription that adds 2g, 2g of livestock breeding, and can be used in various ways in the treatment of forgetfulness and dementia in the clinic. In addition, the experimental results of dementia in Pagecheoncheon and Sansaengdeok confirmed the efficacy of both drugs, and further studies on Chongmyeongtang, Pagecheoncheon or Sansa Chong, and Sansa Chongmyeongtang further inhibited memory loss, anti-inflammatory effect, and brain cell protection. The effects and behavioral effects were confirmed.

    Currently, the treatment of dementia currently sold in Korea has been required to develop drugs for fundamental treatment since it delays disease progression or alleviates symptoms. Therefore, the present inventors have described a conventional combination pharmaceutical composition. By combining various composition ratios of Seokchangpo, Wonji, Paeksin, Pagekcheon, and mountain cultivation in the same amount, complex pharmacology such as inhibiting β-amyloid production, protecting brain nerve cells, and anti-inflammatory action, compared to the same composition The invention was completed by finding a combination of the best combination drugs and their composition ratio.

 The present invention is to provide a composition exhibiting an excellent dementia suppression effect by presenting the optimal combination and the optimal composition ratio as a composite herbal preparation that is more effective than the conventional composite herbal preparation composition.

The present invention provides a composite pharmaceutical composition obtained by extracting from a mixture of 4.0 parts by weight of Seokchangpo, 2.0 parts by weight of base paper, 4.0 parts by weight of baekboksin, 2.0 parts by weight of mountain breeding and 2.0 parts by weight of rupture cloth.

 In another aspect, the present invention provides a composite pharmaceutical composition obtained by extracting from a mixture of 4.0 parts by weight of Seokchangpo, 4.0 parts by weight of base paper, 2.0 parts by weight of baekboksin, 2.0 parts by weight of mountain breeding and 2.0 parts by weight of rupture cloth.

 In another aspect, the present invention provides a medicament prepared from the combined biopharmaceutical composition.

  The combination pharmaceutical composition according to the present invention inhibits the expression of genes related to Alzheimer's prevention and dementia treatment in cells, and has a superior effect in the case of the combination herbal composition according to the present invention than the composition of the conventional proportion among the combination herbal composition. Indicates.

  In other words, when the composite herbal composition according to the present invention is used as an agent for inhibiting dementia, it is possible to eliminate the user's rejection because the formulation component of the herbal drug is applied, and has no negative effect on other body parts and organs according to the dementia suppression. Product becomes possible.

  1 is a table showing the combination of various combination herbal compositions according to the present invention and the highest percentage of inhibition of the β-amyloid production activity of the cells in the composition combination.

  Hereinafter, the present invention will be described in more detail with reference to Examples and Application Examples. The following examples are merely illustrative of the present invention in detail, and thus, the nature of the technical idea of the present invention is not changed or reduced in scope. It will be apparent to those skilled in the art that various embodiments and applications not shown in the following examples are possible.

    Hereinafter will be described the drug as a component of the present invention.

1) Seokchangpo

   Seokchangpo is a root stem of a perennial herb that is a perennial herb of Chonnam Province. Beta-asarone, asarone, caryophyllene, a-humulene, sekison ), Contains amino acids, organic acids and sugars, and is effective in activating bile scab, succinct, hydration. Yang, tinnitus, jeongan, plaque, abundant rain, skin swelling.

2) original paper

      Dried roots of perennial grasses of the dicotyledonous rats, the perennial herb, are contained saponin, tenuigenin A, B, polygalytol, and tenuidine. Efficacy is Ahn Shin Gyu, Ning Shim An Sin, True, Conscience, Geodam Li Gyu, Hwadam Jihae, Sosan Ongjong, Sojong Detox, Traffic Psychology, Kang Ji Ikjeong, Ripe, and Lee Myung-mok. It is described in the book of the first book as a delusion network, border boycott, Xinjihwang Hall, Haedam Buddha statue, Changyang poisoning, breast pain, etc.

3) Bai Fuxin

   Baekboksin is a fungus belonging to the family of fungi, which covers the roots of pine trees.The quality is solid and white, the sex is flat, the beauty is daring, and the heart and the parentage are returned. , Risu's efficacy to treat the cardiac border, forgetfulness, cotton, span, urine and so on.

4) Mountain Breeding

   Mountain cultivation is also called hawthorn, hawthorn, etc. It is a mature fruit of Sanlihong, Sansa or Yasansa, a deciduous tree belonging to the Rosaceae family. many.

The cultivation of mountain breeding is lukewarm, sensational, and rain, stomach and liver. In addition, it is used for diseases such as flesh, obesity, carbonic acid, and saree because it has the efficacy of dry airplane, hwadam firefish, and news, and it is judged that it can be applied to dementia caused by gaseous blood, stagnant stagnation, etc. do.

5) Pagokcheon

   Pageukcheon is a boil medicine that is dried on the roots of Paekcheoncheon, a perennial plant that belongs to Cheoncheon family.

Paekukcheon's temper is sensational and lukewarm, and it is considered to be applicable to non-new demented dementia because of its efficacy in Boshinyang.In modern times, Paekukcheon is classified as a remedy among Boikyang. Efficacy in the treatment of stomach oil well, cold quenching, small stomach cold pain, urinary incontinence, customs pains, pains such as uric acid is being used for symptoms caused by lack of Xinyang.

    Hereinafter, the preparation examples and experimental data of the present invention will be described in detail.

<Preparation Example 1> Preparation of the composite herbal formulation composition

 The composite pharmaceutical composition according to the present invention can be used in powder form by extracting a mixture of each component by hot water extraction according to a conventional method, then filtering and freeze drying. For example, it can be prepared by the following method, first, mixed and trimmed to a suitable size of dried gchangchangpo 4.0g, raw paper 2.0g, baekboksin 4.0g, mountain breeding 2.0g and Pagukcheon 2.0g. 1.3 L of pure water was added to the mixture, followed by hot water extraction at 100 ° C. for 3 hours, followed by rapid freezing at minus 40 ° C. for 15 hours, vacuum drying at a pressure of 0.23 Torr or lower for 35 hours, and then pulverized to obtain a composite pharmaceutical composition according to the present invention. 1.99 g of powder was obtained.

<Preparation Example 2> Preparation of the composite herbal formulation composition

  In comparison with Formulation Example 1, except that 4.0g instead of 2.0g of base paper and 2.0g instead of 4.0g of Vaccine, respectively, 1.82g of the composite pharmaceutical composition powder was obtained under the same conditions as in Formulation Example 1.

<Comparative Formulation Examples 1 to 9>

 Formulation example for comparison with the present invention was prepared in the same manner as the formulation example by varying the mixing amount of the drug in various ways as shown in Table 1, in particular Comparative Example 1 is Seokchangpo, original paper, baeksinsin, mountain breeding and rupture cloth It is a formulation by the conventional ratio of 2 g.

Formulation ratio and dry extract for each comparative formulation (g) Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Example 7 Example 8 Example 9 Seokchangpo 2.0 2.0 4.0 2.0 2.0 2.0 2.0 4.0 2.0 Paper 2.0 4.0 2.0 2.0 2.0 2.0 4.0 4.0 2.0 Avenger 2.0 2.0 2.0 4.0 2.0 2.0 4.0 4.0 2.0 Mountain breeding 2.0 2.0 2.0 2.0 4.0 2.0 2.0 2.0 4.0 Pagokcheon 2.0 2.0 2.0 2.0 2.0 4.0 2.0 2.0 4.0 Sum 10.0 12.0 12.0 12.0 12.0 12.0 14.0 16.0 14.0 Dry extract 2.26 2.06 2.04 1.99 2.47 2.58 2.05 2.03 3.32

Test Example 1 Analysis of Amyloid precusor protein mRNA gene expression inhibitory effect of the combination pharmaceutical composition through in vitro experiments.

The BV-2 cell line, previously used in the experiments by conventional methods, is a cell line produced by infecting a primary microglial cell with v-raf / v-myc oncogene. The cell line is cultured to a concentration of about 2 × 10 ⁶ cells / ml. Then, 100 μg of the composite pharmaceutical composition according to the present invention per 1 ml of the cell culture solution was added and after 1 hour, LPS (10 μg / ml) and 25 μM synthetic βAP fragment, residues 25-35 (BAP; Bachem California, Torrance, CA). After 24 hours, the cells were washed with DMEM, added with a new culture medium and a combination biopharmaceutical composition (100 ㎍ / ml), incubated in a CO ₂ tissue incubator for 40 hours, and then extracted with mRNA to reverse transcription polymerase chain reaction (reverse transcription). Gene expression level of APP mRNA was examined using -polymerase chain reaction (Table 2).

APP mRNA gene expression inhibition rate (%) according to the original invention and comparative formulation Headquarters 1 2 Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Example 7 Example 8 Example 9 54.1 42.7 21.8 41.8 6.4 12.0 24.7 14.4 28.6 24.7 40.9

* Relative inhibition rate (%) of the mixed herbal preparation composition (100㎍ / ml) for the experimental control group (Ratio APP mRNA / GAPDH) stimulated with LPS and βAP only in BV-2 cell line

[Table 2] shows the results of analyzing the APP mRNA gene expression by adding a combination pharmaceutical composition with different composition ratios to BV-2 cell lines and stimulating with LPS and βAP, as shown in the table above, the combination pharmaceutical composition Among the conventional ratio of the composition (Example 1), the APP mRNA gene expression of the multi-pharmaceutical composition of the present invention showed an APP mRNA expression inhibitory effect almost twice or more.

Therefore, it was confirmed that the composite herbal composition according to the present invention exhibited an excellent inhibitory effect on APP mRNA gene expression compared to the conventional proportion of the composition (Example 1).

<Test 2> Analysis of β-Amyloid protein production inhibitory effect of the combination drug composition in vitro experiment.

The BV-2 cell line, previously used in the experiments by conventional methods, is a cell line made by infecting a primary microglial cell with v-raf / v-myc oncogene. The cell line is cultured to a concentration of about 2 × 10 ⁶ cells / ml. Then, 100 후 of the combined biopharmaceutical composition according to the present invention per 1 ml of the cell culture solution was added and LPS (10 ㎍ / ml) and 25 μM synthetic βAP fragment, residues 25-35 (βAP; Bachem California, Torrance, CA) ) After 24 hours, the cells were washed with DMEM culture medium, and the new culture medium and the co-product composition (100 µg / ml) were added and incubated in a CO ₂ tissue incubator for 40 hours. After the incubation period, the whole culture solution was centrifuged at 2000 rpm for 5 minutes to recover the supernatant, and the β-amyloid protein production was determined by ELISA (Code No. 27720, Mouse / Rat Amyloidβ (1-40) High Specific Assay Kit-IBL, USA). Measured.

β-myloid protein production inhibition rate (%) Headquarters 1 2 Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Example 7 Example 8 Example 9 80.1 77.9 42.0 41.7 40.2 46.7 56.4 60.5 47.3 46.9 60.4

* Relative inhibition rate (%) of dry extract of herbal composition (100㎍ / ml) for experimental control group stimulated with LPS and βAP alone in BV-2 cell line

Table 3 shows the results of analyzing the production of β-amyloid protein in the culture supernatant by adding the combined pharmaceutical composition with different composition ratios to BV-2 cell lines and stimulating with LPS and βAP. Β-amyloid protein production of the present invention compared to the composition (Example 1) of β-amyloid protein production showed an excellent inhibitory effect of β-amyloid protein production more than 85.5%.

Therefore, it was confirmed that the composite pharmaceutical composition according to the present invention exhibited an excellent β-amyloid protein production inhibitory effect compared to the composition of the conventional ratio.

<Test Example 3> Analysis of β-Amyloid precursor protein activity inhibitory effect of the combination pharmaceutical composition through in vitro experiments.

The BV-2 cell line, previously used in the experiments by conventional methods, is a cell line made by infecting a primary microglial cell with v-raf / v-myc oncogene. The cell line is cultured to a concentration of about 2 × 10 ⁶ cells / ml. Next, 100 μg of the composite pharmaceutical composition according to the present invention per 1 ml of the cell culture solution was added and after 1 hour, LPS (10 μg / ml) and 25 μM synthetic βAP fragment, residues 25-35 (BAP; Bachem California, Torrance, CA) and calpain protease inhibitor MDL 28170 (35 μM, MDL 28170) as a positive control. After 24 hours, the cells were washed with DMEM culture medium, a new culture medium and a combination biopharmaceutical composition (100 µg / ml) were added thereto, and cultured in a CO ₂ tissue incubator for 40 hours. Cellular lysate was obtained to measure β-APP activity. 50 μl of lysis buffer (Tris-HCl (200 mM; pH 8.0), 150 mM NaCl, 0.5% (v / v) Nonidet P-40, 0.1 mM EGTA, 1 mM PMSF, 0.1 mM DTT, 10 µg) / Ml leupetin}, incubated for 30 minutes on ice and centrifuged for 5 minutes to obtain cellular lysate. Bradford staining (Bradford, M. "A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein -Dye Binding "Anal. Biochem. 1976; 72: 248-254), the protein was quantified and adjusted to 50 μg. Cell lysate (50 μg) was transferred to PDVF membrane on 12% SDS-PAGE gel. After blocking for 2 hours with 2% BSA, the mice were treated overnight with anti-mouse β-APP N-terminal antibody. After the reaction with anti-mouse Ig HRP-conjugated sceondary Ab (1: 4000, Amersham, Arlington Heights, IL), the analysis was performed by immunoblotting with an ECL-Hybond film.

In FIG. 1, the stomach was analyzed by β-APP immunoblotting in BV-2 cell line. The control group was stimulated with LPS (10 μg / ml), 25 μM synthetic βAP fragment, residues 25-35, and anti-β-APP. Shows a marked increase in the amount of expression, and the anti-β-APP of the composite pharmaceutical composition (No. 8, 9) according to the present invention than the composition (No. 1) of the conventional proportion of the composite pharmaceutical composition according to the present invention It was found that the expression level was significantly suppressed. In addition, as shown in Figure 1, by analyzing the expression of β-APP by Western blot, the composite herbal composition according to the present invention (No. 1) of the conventional composition (No. 1) of the composite herbal formulation according to the present invention ( Nos. 8 and 9) showed significantly more inhibitory effect on the expression level of β-APP.

Therefore, it was confirmed that the composite pharmaceutical composition according to the present invention exhibited an excellent inhibitory effect on β-APP expression compared to the composition of the conventional ratio, and accordingly, the combined pharmaceutical composition according to the present invention could be usefully applied to the treatment and suppression of dementia. It could be confirmed.

Claims (4)

A composite pharmaceutical composition for treating and inhibiting dementia, which is obtained by extracting from a mixture of 4.0 parts by weight of Seokchangpo, 2.0 parts by weight of paper, 4.0 parts by weight of baekxin, 2.0 parts by weight of livestock, and 2.0 parts by weight of rupture cloth.
   Dementia treatment and suppression agent prepared from the composite herbal formulation according to claim 1.  A composite herbal composition for treating and inhibiting dementia, which is obtained by extracting from a mixture of 4.0 parts by weight of Seokchangpo, 4.0 parts by weight of raw paper, 2.0 parts by weight of baekxin, 2.0 parts by weight of livestock, and 2.0 parts by weight of rupture cloth. An agent for treating and inhibiting dementia prepared from the combination herbal preparation composition according to claim 3.

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