KR20080103321A - Pharaceutical composition containing extracts from polygonatum falcatum for the preventing or treating dementia and method of production thereof - Google Patents

Abstract

A pharmaceutical composition for preventing or treating dementia is provided to suppress an activity of the acetylcholine esterase using extracts derived from Polygonatum falcatum as an active ingredient and to prevent or treat the dementia. A pharmaceutical composition for preventing or treating dementia includes Polygonatum falcatum extracts by an organic solvent as an active ingredient. The Polygonatum falcatum extract is manufactured by being extracted by a chloroform after the split Polygonatum falcatum is extracted in a methanol one or more times. The methanol is diluted to a concentration of 80% by being mixed with water.

Description

Pharmaceutical composition for the prevention or treatment of dementia comprising extracts from citrine, and a method for producing the same

Figure 1-Separation flow chart for separating and extracting the Jinhwang Jung extract with several organic solvents.

Figure 2-Flow chart of primary purification of citrine extract.

Figure 3-Chromatogram graph of chloroform fractions of citrine extract extracted with silica-gel column.

FIG. 4-Flowchart of Secondary Purification Separation of Citrine Extract.

Figure 5-Chromatogram graph through Sephadex LH-20 column of citrine extract.

Figure 6-Chromatogram graph through Bio-gel-P2 column of Citrine extract.

Figure 7-Chromatogram profile of HPLC of C-3.

8-UV spectral graph of compound I.

Figure 9-IR spectral graph of compound I.

10-Spectral graph of ¹ H-NMR of Compound I isolated from Qin yellow extract.

FIG. 11-Spectral graph of ¹³ C-NMR of Compound I isolated from Qin yellow extract.

12-GC / Mass spectrum graph of Compound I isolated from Ginseng Extract.

Figure 13-Bar graph of the passive avoidance experiment of the Jinhwangjeong extract.

Figure 14-Bar graph of the Y-maze test of the extract of Qinhuang Jing.

The present invention relates to a pharmaceutical composition for the prevention or treatment of dementia comprising an extract derived from cinnabar goose extract from an organic solvent in a citrate, and a method for preparing the same. More specifically, a cactus extract composed of benzokinone The present invention relates to a pharmaceutical composition for preventing or treating dementia and a method for producing the same, including the active ingredient, and inhibiting the activity of acetylcholinesterase (hereinafter referred to as AChE).

As human ages, the function of cerebral brain cells gradually decreases and the size of the brain decreases, and the probability of developing senile disease increases. As the elderly population increases, the population suffering from senile disease increases rapidly. Difficult to do, the situation is difficult to prevent or treat.

In particular, dementia among senile diseases has a significant mental and economic loss due to mental disorders such as memory impairment and loss of judgment, leading to serious social problems called 'domestic destruction'. have. In Korea, it is reported that the population aged 65 years or older becomes 7.2% of the total population in 2000, but 740,000 in 2015. According to the aging population, the incidence of infringement increases from 60 years to 5 years. It has been reported that there is an exponential increase of 2 times. In other words, assuming a prevalence of 1% between 60 and 64 years old is 2% for 65-69 years old, 4% for 70-74 years old, and 8% for 75-79 years old. Therefore, since senile diseases such as dementia are expected to increase rapidly with the increase of the elderly population, the need for research on the prevention or treatment of dementia has emerged.

Dementia is a brain disease caused by impaired cognitive function of the whole brain. As a result of memory and thinking deterioration, symptoms such as cerebral dysfunction such as understanding, calculation, learning, and judgment appear. There are three main types: Alzheimer's disease caused by Alzheimer's disease, vascular dementia caused by cerebrovascular disorders, and metabolic diseases such as infections, trauma, tumors, thyroid diseases, infectious diseases, and hydrocephalus. It is classified as secondary dementia. In foreign countries, Alzheimer's dementia is 70-80%, and the remainder is mainly vascular dementia, while in Korea, vascular and Alzheimer's dementia account for 50%.

When looking at the structural abnormalities of the brain of many patients with dementia, including Alzheimer's disease, cerebral atrophy is commonly observed, and substances composed of abnormal proteins firmly scavenged in brain tissues are observed. These substances are caused by the destruction of chemical transporters in the brain, called the senile plaque and the neurorefibrillary tangle. The protein that forms the senile plaque is a toxic protein called β-amyloid, which is known to be an important cause of brain neurotoxins, such as inflammatory reactions that accumulate in the brain and destroy normal signaling systems.

In addition, biochemical changes in patients with dementia have been reported to be caused by a significant decrease in acetylcholine levels in the temporal lobe of the brain. Acetylcholine is biosynthesized from acetylcoa and choline by cholineacetylsperase, but is degraded and destroyed by AChE. Therefore, AChE deactivation is closely related to the degree of dementia.

Therefore, the hypothesis that it is possible to prevent dementia or to treat dementia by inhibiting the production of beta amyloid and blocking the toxicity, or inhibiting the activity of AChE to maintain the acetylcholine content. Recently, various drugs that increase the concentration of a substance capable of inhibiting AChE activity have been developed. Related to this, drugs developed so far include lecithin as an acetylcholine precursor. RS-86 is a receptor agonist, and nicotine is an acetylcholinesterase inhibitor. In addition, there are tacrine (Tacrine) and Aricept (Aricept) that is commercially available in Korea, but the price is expensive, there is a problem that can not be used for a long time. In addition, Donepezil, Rivastigmine, Galantamine, etc. have been developed as AChE inhibitors, but there are limitations to practical use due to toxicity and side effects.

On the other hand, polygonatum fakatum a gray - odoratum ) is a perennial perennial, with rhizomes extending laterally, bloated fleshy, slightly flat cylindrical, yellowish white. Ginseng tablets are a source of essential ingredients for the human body, which is a raw material of round tea, and it contains sugars, especially fructose, alkaloids, and essential amino acids. Since ancient times, Jinhwangjeong has been known to be effective in hyperlipidemia, arteriosclerosis, cerebrovascular disease, etc., but little research has been done on the AChE inhibitory effect and the pharmaceutical composition for the prevention and treatment of dementia using the same.

The present inventors have completed the present invention by finding a component that is effective in dementia while studying the extract extracted from the jinhwangjeong various organic solvents, and using such a component as a composition for the prevention or treatment of dementia.

The present invention has been made to solve the above problems, and an object of the present invention is to provide a pharmaceutical composition for preventing or treating dementia, and a method for producing the same, as an active ingredient of a citrate extract that can inhibit AChE activity.

It is another object of the present invention to provide a pharmaceutical composition which has no side effects and is effective in preventing or treating dementia.

In order to achieve the above object, the present invention includes the extract of the true yellow jeongjeong by the organic solvent as an active ingredient.

Here, the cinnabar cinnabar extract is characterized in that the fine cinnabar cinnabar extract is extracted with methanol as a solvent one or more times, and then extracted with chloroform as a solvent.

The methanol is characterized in that it is mixed with water to form a concentration of 80%.

The citrine extract, the chemical structural effect of preventing or treating dementia extracted from the citrine:

It is characterized by comprising a compound having a.

In addition, the first step of separating the methanol extract by extracting the finely divided Jinhwangjung; A second step of separating the chloroform extract by extracting the methanol extract with chloroform; A third step of separating the first fraction by extracting the chloroform extract with a solvent mixed with methanol, chloroform and hexane; A fourth step of separating the second fraction by extracting the first fraction with methanol; A fifth step of separating the third fraction by extracting the second fraction with a solvent mixed with acetonitrile and water; And a sixth step of extracting the third fraction with a solvent mixed with methanol, acetonitrile and water.

In the first step, the methanol extract is separated by repeating the methanol three more times with a solvent.

And, after the step 2, characterized in that it further comprises the step of fractionating the chloroform extract with a solvent of 4: 6 methanol and chloroform.

The third step, methanol, chloroform and hexane is characterized in that 2: 1: 1.

The fourth step is characterized in that the methanol is mixed with water to 30% concentration.

In the fifth step, methanol, acetonitrile, and water are 30:15:35.

Example  One: AChE  Identify organic solvents with high activity inhibitory activity

Example 1-1: Methanol Extract Separation of Ginseng Extract

1 is a separation flow chart for separating and extracting a true yellow vera extract with various organic solvents.

Referring to Figure 1, the extract of the citrine of the present invention is 100g dried dried citrine ( Polygonatum odoratum ), cooled by 80 ° C at 80 ℃ extracted three times with 10% of 80% methanol sample, the filtrate was combined and filtered After concentration under reduced pressure, the mixture was vacuum frozen and separated into 22.4 g of 80% methanol extract and the residue.

Example 1-2 Separation of Hexane Layer from Ginseng Extract

After dispersing 22.4 g of the methanol extract obtained in Example 1-1 in an appropriate amount of water, an equal amount of n-hexane was added to the Separation Funnel, and then the same process was repeated three times. Each fraction was filtered and concentrated under reduced pressure to obtain a 2.53 g hexane layer (Fraction. H: hereinafter Fr.) as an n-hexane soluble fraction and a first water layer as a fraction soluble in water.

Example 1-3 Separation of Ethyl Acetate Layer of Ginseng Extract

After adding and adding ethyl acetate to the first water layer obtained in Example 1-2, the mixture was separated into fractions dissolved in ethyl acetate and fractions dissolved in water. The process was repeated three times, and each fraction was filtered and concentrated under reduced pressure to separate 5.64 g of ethyl acetate layer (Fr. E) and the second water layer.

Example 1-4 Separation of Chloroform Layer of Ginseng Extract

Chloroform was added to the second water layer obtained in Example 1-3, mixed, and then separated into fractions soluble in chloroform and fractions soluble in water. The process was repeated three times, and each fraction was filtered and concentrated under reduced pressure to separate the chloroform layer (Fr. C) and the third water layer.

Example 1-5: Butanol Layer Separation of Ginseng Extract

Butanol (n-butanol) was added to the third water layer obtained in Example 1-4, followed by mixing. The process was repeated three times, and each fraction was filtered and concentrated under reduced pressure to obtain 3.41 g of butanol layer (Fr. B) and 6.19 g of fourth material layer (Fr. A).

Example 1-6 Determination of Talc Inhibitory Effect of Acetylcholinesterase (AChE)

The brain tissues of the rats to which the respective fractions obtained in Examples 1-1 to 1-6 were added are used as the enzyme source. The brain tissue was separated by cutting the skull using a sharp surgical scissors, washed with physiological saline, and then gently pressed to remove foreign substances. Brain tissue was added with 4 times homogenization buffer (containing 125 mM sodium phosphate butter pH 7.0, 0.4 M NaCl) per gram and ground with a glass teflon homogenizer at 0 ° C. Centrifuge the ground homogenate at 600 × g for 10 minutes to remove the unbroken part, and then centrifuge it again at 10,000 × g for 20 minutes, and then use the supernatant postmitochondria fraction as an enzyme for measuring AChE inhibitory activity. Used as a circle. All the above operations were performed at 0-4 degreeC.

AChE activity in brain cells was measured by adding 300 µl of 0.1M Tris butter, pH 8.0 (Trizma HCl + Trizma base) and 10 µl of an enzyme suspension to each microplate well, and then again 0.1M acetylthiocholine 10 µl of Acetylthiocholine chloride was added. A microplate reader (ELISA reader) was used for 30 minutes after the change in absorbance at 405 nm, and the activity of AChE (unit / min / mg protein) was measured. A summary of the AChE activity of the citrate extracts according to the various organic solvents is as follows.

menstruum Extraction rate (%, w / w) Acetylcholinesterase (mg, protein / min) 80% methanol 100 198.1 ± 6.4 Hexane (Fr. H) 1.30 142.6 ± 4.3 Ethyl Acetate (Fr. E) 25.19 150.4 ± 2.5 Chloroform (Fr. C) 20.64 216.4 ± 4.7 Butanol (Fr. C) 15.21 183.7 ± 6.2 Water (Fr.A) 27.66 140.3 ± 5.1

As shown in Table 1, the inhibitory activity of AChE, which separated 80% methanol as a solvent, was 198.1 ± 6.4 mg, protein / min, and 80% methanol extract was extracted with sequential organic solvent method, chloroform (Fr). C) was the highest with 216.4 ± 4.7mg, protein / min, butanol (Fr. B) was the highest with 183.7 ± 6.2mg, protein / min, and the rest of the extract was ethylacetate (Fr.E). > Hexane (Fr. H)> water (Fr. A). Therefore, among the extracts sequentially fractionated by various organic solvents, the most excellent inhibitory activity of AChE was the chloroform layer (Fr. C).

Example  2 From True Yellow Extract AChE Purification as an active inhibitory substance

Example 2-1 Methanol Fraction Separation

Figure 2 is a flow chart of the primary purification of the extract of ginwangjeong. As shown in FIG. 2, 10 kg of Ginseng (Polygonatum odoratum) was chopped to obtain a sample required for the experiment, and then extracted with 80% methanol twice more with 80% methanol, followed by evaporation of methanol. 224 g of fractions were isolated.

Example 2-2 Separation of Chloroform Fraction

The chloroform fraction obtained by extracting chloroform three times to inhibit the activity of AChE by the methanol fraction about 10 times the weight of the methanol fraction was separated again.

Example 2-3 Fractionation Through Silica Gel Column

Next, the chloroform fraction is injected into a silica gel column (Silica gel column: Kiselgel, 60 GF 354, Merck Co. (100 cm x 4.5 cm)) equilibrated with distilled water, and the components not adsorbed are washed with distilled water, followed by elution. To increase the non-polarity of the solvent to methanol: chloroform eluted with 9: 1, 8: 2, 4: 6, 2: 8, respectively, the active portion was concentrated. At this time, the flow rate was 10ml / h, these fractions were collected in the fraction (Fraction) measured by UV only after collecting the active part, concentrated in vacuo and freeze-dried to Fr. I (tube No. 10-70) 3.6 g, Fr. II (tube No. 75 to 115) 12.6 g, Fr. III (tube No. 116-165) 24 g and Fr. 6 g of IV (No. 202-270) was obtained.

Figure 3 is a graph of silica gel column chromatograms of the extracts of the jinhwangjeong extract with chloroform and methanol as a solvent.

As shown in FIG. 3, the absorbance per tube (hereinafter referred to as tube) was measured. The absorbance of 126 (hereinafter referred to as O.D) was the highest at 1.93, followed by tube no. 86 had an O.D of 1.82 and tube No. O.D of 228 is shown in the order of 1.26. Fr. collecting only the active part. I, Fr. II, Fr. III, Fr. AChE inhibitory activity of Ⅳ was determined to be 52.9%, 64.3%, 67.2% and 56.4%, respectively. III was the highest.

Example 2-4 Fractionation Through Sephadex LH-20 Columns

Figure 4 is a flow chart of the primary purification of citrate extracts that inhibit the activity of AChE in citrine. As shown in Figure 4, the 24g Fr. III was injected into a Sephadex LH-20 column equilibrated with 10% methanol (95 cm x 2 cm) and eluted at a rate of 12 ml / hr to fraction tube No. Aliquots were from 1 to 120. In this case, using a solvent in which methanol: chloroform: hexane is 10: 1: 1, Fr. 1 (tube No. 30 to 70) were isolated, and eluted with a solvent of 4: 1: 1 methanol: chloroform: hexane to give Fr. 2 (tube No. 80-110) were separated.

The eluted fractions were measured by UV and tube no. 38 had the highest O.D of 1.63, followed by tube No. The O.D of 86 was in the order of 1.45. Among these fractions, only 16.8 g of Fr. 1 and 7.2 g of Fr. 2 was obtained respectively.

5 shows a chromatogram graph of Sephadex LH-20 column. As shown in Figure 5, the inhibitory activity of AChE of each fraction is Fr. 1 is 58.3%, and Fr. Divalent to 60.5% of Fr. The inhibitory activity of divalent AChE was shown to be higher.

Example 2-5 Fractionation through Biogel P-2

Referring to Figure 4, the fraction of Fr. AChE inhibitory activity higher. 2 was added to the Bio gel P-2 column (30cm × 1.8cm) equilibrated with 20% methanol, and methanol: chloroform: hexane was dissolved in 10: 1: 1 solvent at 5ml / hr at a flow rate of 5ml / tube. Eluted to concentration under reduced pressure, and vacuum-dried Fr. 3 (tube No. 10-35) and Methanol: Chloroform: Hexane were separated by a solvent of 2: 1: 1 Fr. 4 (tube No. 36-70) eluting with vacuum concentration only 3.2g Fr. 3 and 4.0 g of Fr. Got 4.

6 is a graph of Bio gel P-2 chromatogram. As shown in Figure 6, the Fr, 3 and Fr. 4 was concentrated in vacuo to test the inhibitory activity of AChE. Tetravalent 62.5% with 58.2% of Fr. Higher than 3.

For reference, the method of eluting by injecting the Bio gel P-2 column can remove the fraction of the lower portion of the lower activity and has a size exclusion effect, but Sephadex G-25 (fraction range molecular weight 1,000 ~) The fractionation of the active moiety is better than that of Sephadex LH-20, which is 5,000).

4, the Fr. 4 was dissolved in an ODS column equilibrated with 30% methanol (23 cm x 1.5 cm), and injected.Fr. 5 separated by a solvent of methanol: chloroform: hexane and 10: 1: 1, methanol: chloroform: hexane 2 The solvent was eluted with an insoluble fraction separated by a solvent of 1: 1, and the active fraction was concentrated in vacuo, followed by vacuum drying to obtain 3.7 g of Fr. Got 5.

Example  3 extracted from Qinhuang Fraction Of acetylcholinesterase  Activity Inhibition Measurement

Example 3-1 Purification of Fractions by FPLC

Finally, to confirm the purification, the active ingredient obtained from the ODS column was passed through a Sep-Pak (C18) cartridge and then fractionated again using FPLC (Fast Protein Liquid Chromatography). Fractionation conditions were the Pharmacia FPLC system using a Pharmacia, Monitor UV-M (254nm) detector and PepRPCTM AR 5/5 (C ₂ / C ₁₈ , reverse phase) column. Nitrile (acetonitrile): 300 mL of 1: 9, 300 mL of 2: 8, 200 mL of 3: 7, 200 mL of 4: 6 and 200 mL of 7: 3 with H ₂ O, respectively, at a flow rate of 7.5 mL / h Elution and lyophilization respectively gave 1.23 mg of fractions C-1, 850 mg, C-2, 640 mg, C-3, 800 mg, C-4, 180 mg and C-5.

Example 3-2 Inhibitory Effect of Acetylcholinesterase

AChE Inhibitory Effect of 10μl of Citrine Extract AChE Inhibitory Effect of Ginseng Extract of 20μl Control 159.1 ± 2.4 163.4 ± 2.9 C-1 176.4 ± 3.6 197.6 ± 1.4 C-2 180.2 ± .9 213.0 ± 0.6 C-3 191.16 ± 4.7 217.3 ± 0.4 C-4 174.6 ± 2.5 213.4 ± 1.7

Average value of three experiments

Table 2 shows the untreated control group fed with water, and the Citrine extracts C-1, C-2, C-3, and C-4 into the brain of rats, and inhibited AChE activity (unit / mg, protein / min) is measured.

As shown in Table 2, these fractions were diluted under the same conditions, and 10 μl each of C-1, C-2, C-3, and C-4 was added to measure AChE inhibition activity. C-1, C-2, C-3, and C-4 were 176.4 ± 3.6, 180.2 ± .9, 191.16 ± 4.7 and 174.6 ± 2.5 unit / mg, and protein / min, 10.9 compared to / mg and protein / min, respectively. %, 13.3%, 20.2% and 9.7% increased, indicating that the C-3 group had the highest AChE inhibitory activity.

In addition, the enzyme inhibition activity was measured by adding 20 μl to each sample, and the enzyme inhibitory activity of C-1, C-2, C-3, and C-4 was 163.4 ± 2.9 unit / mg and protein / min of the control group. Was 181.2 ± 1.4, 184.6 ± 0.6, 201 ± 0.4 and 182.4 ± 1.7 unit / mg and protein / min respectively, increasing by 20.9%, 30.4%, 33.0% and 30.6%, respectively. Appeared.

Example  4 Analysis of Compound I Extracted from Jinhwang Jung

Example 4-1 Fractionation by Pre-HPLC

C-3, a fraction of high AChE inhibitory activity, was dissolved in 30% methanol and filtered through 0.45㎛ membrane filter, followed by Prep-HPLC (Model 510, solvent delivery system, Agillent, LC 1100, USA).

The column used was Develosil ODS (8 × 250mm, Nomura co., Japan). The mobile phase used 30% methanol and the flow rate was 1 ml / min. Detection was performed using a UV detector. It was carried out at 254nm and 280nm.

Fraction C-3 was fractionated by Pre-HPLC with methanol: acetonitrile: water (30:15:35) to obtain compound I, 340 mg, and the structure thereof was identified. 7 is a profile graph of the chromatogram of HPLC of C-3. As shown in FIG. 7, the C-3 was purified by HPLC and showed a peak at 6.2 minutes.

Example 4-2 Measurement of UV-VIS and IR Absorption Spectrum

UV-VIS absorption spectra of the separated fractions were measured using a UN-VIS Spectrophotometer (Hitachi, u-3000 Japan). Measurement conditions were 20 mV, scanning rate was 60 nm / min, and slit 120 nm / min. The IR absorption spectra was measured using an IR spectrophotometer (Hitachi, 260-30, Japan), and 2 mg of the freeze-dried sample was mixed with 200 mg of KBr to make a tablet.

8 is a UV spectral graph of Compound I. Referring to FIG. 8, Compound I shows absorbance at 225 and 280 nm in the UV spectrum, which shows a similar tendency to UV absorbance of benzozuinone as a standard.

9 is an IR spectral graph of Compound I. As shown in FIG. 9, the IR spectrum showed a peak corresponding to a hydroxy group at 3400 cm ⁻¹ as measured using KBr Pellet, and C = conjugated carbonyl group at 1680 cm ⁻¹ . O peak appeared, and CO stretching (1210 cm ⁻¹ ).

Example 4-3 GC-MS Fraction

The Joel JMS-AX 505 WA mass spectrometer equipped with a direct injection port (DIP) was used for direct probe analysis under ionized EI (70er) and ion source temperature 200. The results of the GC-MS spectra show that the molecular ion peaks are obtained at m / z 152 and high intensity peaks at m / z, 69 and 124.

Example 4-4 Protein Quantification of Compound I

Compound I was light brown with a molecular formula of C ₈ H ₁₃ O ₃ and a molecular weight of Compound I was 156.

To measure the molecular weight, Bovine serum albumin was used as a standard according to the Lowry protein quantification method. First, 0.1 ml of 2N NaOH is added to a 1.0 ml sample or standard solution, and heated at 100 ° C. for 10 minutes for hydrolysis. After the hydrolyzate is allowed to cool, 1.0 ml of Lowry reagent is added, mixed, and left to stand for 10 minutes. 0.1 ml of Folin reagent was added and left to mix for 20 to 60 minutes, and then absorbance was measured at 660 nm.

Example 4-5 NMR Measurement of Compound I

Next, compound I was analyzed using a Varian Unity Plus-300 (300 ¹ H, ¹³ C) -NMR spectrometer, DMSO was used as a solvent, and tetramethylsilane (TMS) was used as an internal standard.

Carbon number H NMR C NMR One - 187.39 2 - 158.81 3 6.03 (d, 1 H) 147.27 4 - 181.28 5 - 107.04 6 6.52 (q, 1 H) 107.04 7 3.30 (S, 3H) 56.36 8 3.75 (S, 3 H) 56.89

Table 3 is the ¹ H-NMR and ¹³ C-NMR spectral data of Compound I in solvent DMSO-D ₆ . Where s is a singlelet, d is a double, t is triple and q is a curetet. And, Figure 10 is a spectral graph of ¹ H-NMR of compound I separated and separated in the Jinhwang Jung extract.

As shown in Table 3 and FIG. 10, in the ¹ H-NMR spectrum, sigma (hereinafter referred to as δ) measured using DMSO-d ₆ solvent was 6.03 ppm (1H, d) and 6.52 ppm ( In 1H, q), two protons and four protons were connected, resulting in an aromatic ring proton signal, and δ was 3.30 ppm (S, 3H) and 3.75 ppm (S). , 3H) showed proton signals of two CH ₃ groups

From these results, the structure of the substance is that two methyl groups are connected to the benzoquinone ring, and through the difference in chemical shift, one methyl group provides electrons like oxygen. It turns out that it is a substituted structure.

In addition, as shown in Table 3 and FIG. 11, ¹³ C-NMR spectra were measured for carbon signals belonging to carbonyl at 187.39 ppm and 181.28 ppm. Four enolic signals appeared at 158.81 ppm (C-1, C-2) and 147.2 ppm (C-4, C-5).

 In conclusion, in addition to the results of molecular ions, hydrogen and carbon NMR data and IR spectra, Compound I has a methyl group and a methoxy group linked to the benzoquinone nucleus. The benzoquinone nucleus is identified as 2-methoxy, 5-methyl or 4-hydroxy, and has a structure similar to a substance having a benzoquinone nucleus.

Example  5 Dementia Treatment or Prevention Effect of Ginseng Extract

Example 5-1 Experimental Animal Breeding Method

Sprague-Dawley female rats weighing 180-220 g were divided into four groups of seven in each group, and then placed in the bregma of mice with a normal control group fed with water, and a 26-gauge syringe with a Hamilton syringe. Β-amyloid, which was induced by injecting β-amyloid in the ventricle and induced dementia alone, and β-amyloid group that induced dementia, 1.80 µg / μl amyloid and 2 mg / kg Four groups were administered for four weeks after the administration of the true yellow extract and four groups of 1.80 μg / μl amyloid and 5 mg / kg of the true yellow extract, followed by manual avoidance test and maze test. It was. Four weeks later, one day of training, the second day of manual hedging, and the Y-maze, three days of training and four days of testing.

Example 5-2 Passive avoidance test of experimental animals

The manual evasion test evaluated learning and memory. It consists of a brightly lit room and a dark room, and a passive evacuation box is used on the floor to provide electric shock. First, the mice were placed in a bright room, and as soon as the mice entered the dark room, they were subjected to electric shock for 1 second at 0.25 mA. After 24 hours of training, the mice were placed in a bright room and the time to enter the dark room was measured as a passive avoidance response time. The maximum time limit was 300 seconds, and the passive avoidance response time was determined to be 300 seconds unless the user entered the dark room for more than 300 seconds.

As shown in FIG. 13, the passive avoidance response time of the normal control group was 230 seconds, but the dementia-induced β-amyloid group was slowed to 145 seconds, and the passive avoidance of the group to which 2 mg of β-amyloid and citrate extract were administered. The response time was 170 seconds, which was 25 seconds longer than the β-amyloid group. In addition, the passive avoidance response time of the group administered with β-amyloid and 5 mg / kg was 180 seconds, indicating that memory cognition was improved compared to the group administered with β-amyloid and 2 mg.

That is, compared with the normal control group, the group treated with β-amyloid induced by dementia showed a decrease in passive avoidance response time, resulting in a decrease in cognitive learning and memory, resulting in dementia. In addition, when β-amyloid and citrine extract were administered together, passive evasion reaction time increased and brain metabolism was smoother than β amyloid-administered group. .

Example 5-3 Y-maze test

Normal control group, β-amyloid injection group, β-amyloid injection group and Qin yellow extract 2mg / kg, β-amyloid injection group and 5mg / kg-treated mice were bred for 4 weeks, and then Y-maze test was performed. Voluntary alternative behavior was assessed.

The Y-maze is composed of three channels shaped like an English Y. At the end of the passage, the mouse head was faced and allowed to travel freely in the three passages for eight minutes. In this case, the number of crossings can be measured once the mouse has crossed the three passages in a row and checks whether the mouse remembers the way the mouse had gone once. Spontaneous cross activity was calculated as a percentage of the number of crossings and the number of arm entries through the passage. Statistical analysis of all data used here was performed using analysis of variance and tested.

As shown in Figure 14, the dementia-induced β amyloid injection group showed a decrease in cross activity compared to the control group in the normal state, while dementia was observed, whereas in the group administered with β amyloid injection and 2 mg of the true yellow extract, Although less than that, the amount of cross-activation increased compared to the β-amyloid group. In addition, the group administered with β amyloid injection and 5 mg of citrate extract showed a higher cross-acting activity than the group administered with the β amyloid injection group and 2 mg of citrate extract, and the extract of citrate was effective in preventing or treating dementia. Appeared to be.

Example  6: Preparation of Hard Capsules and Tablets Containing Ginseng Extract

Example 6-1 Preparation of Tablets

Ginseng extract 100.0 mg, starch 90.0 mg, lactose 175.0 mg, L-hydroxypropyl cellulose 15.0 mg, polyvinyl chirrolidone 5.0 mg and a suitable amount of ethanol, granulated by wet granulation method and magnesium stearate 1.8 After adding mg and mixing, one tablet was compressed to 400 mg.

Example 6-2 Preparation of Capsules

A raw material of 100 mg of true yellow extract, 83.2 mg of starch, 175.0 mg of lactose, and 1.8 mg of magnesium stearate was homogeneously mixed and filled to contain 360 mg in one capsule.

As described above, the extracts and compounds derived therefrom in Qinhuangzheng have the effect of inhibiting the activity of acetylcholinesterase, thereby preventing or treating diseases that may be caused by the decrease of acetylcholine, in particular, dementia. .

Claims (9)

A pharmaceutical composition for preventing or treating dementia comprising an extract of citrate from an organic solvent as an active ingredient. According to claim 1, The citrine extract, The finely divided citrate tablets are extracted with methanol at least once, and then extracted with chloroform as a solvent. The method of claim 2, wherein the methanol, A pharmaceutical composition for preventing or treating dementia, wherein the composition is mixed with water to form a concentration of 80%. According to claim 1, The citrine extract, Chemical formulas for the prevention or treatment of dementia extracted from Qinhuangjung:

Dementia prevention or treatment pharmaceutical composition comprising a compound having a. A first step of separating the methanol extract by extracting the shredded Jinhwang Jung with methanol; A second step of separating the chloroform extract by extracting the methanol extract with chloroform; A third step of separating the first fraction by extracting the chloroform extract with a solvent mixed with methanol, chloroform and hexane; A fourth step of separating the second fraction by extracting the first fraction with methanol; A fifth step of separating the third fraction by extracting the second fraction with a solvent mixed with acetonitrile and water; And extracting the third fraction with a solvent mixed with methanol, acetonitrile, and water; and a method for preparing a pharmaceutical composition for preventing or treating dementia. The method of claim 5, wherein the first step is Method for producing a pharmaceutical composition for preventing or treating dementia, characterized in that the methanol extract is repeated by repeating the methanol three more times as a solvent. The method of claim 5, After the second step, the chloroform extract is fractionated with a solvent of methanol and chloroform 4: 6; a method for producing a pharmaceutical composition for preventing or treating dementia, further comprising. The method of claim 5 or 7, wherein the third step, Method for producing a pharmaceutical composition for preventing or treating dementia, wherein the methanol, chloroform and hexane are 2: 1: 1. 8. The method of claim 5, wherein the fifth step comprises: Method for preparing a pharmaceutical composition for preventing or treating dementia, wherein methanol, acetonitrile and water are 30:15:35

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