TWI377949B - A chinese herb extract for treating dementia and preparation method thereof - Google Patents

Description

1377949 IX. DESCRIPTION OF THE INVENTION: TECHNICAL FIELD OF THE INVENTION The present invention relates to a traditional Chinese medicine extract and a preparation method thereof. The invention also has the use of the extract and the active moiety for the treatment or prevention of dementia such as Alzheimer's disease. [Prior Art] Dementia is caused by brain damage or disease that is expected beyond normal aging. (4) Progressive decline in knowledge. The most well-known type of dementia is Alzheimer's disease. 'Alzheimer's disease (AD) is an increasingly common form of neurodegenerative disease, in more than (4): humans account for about 50%-60% of the total cases of dementia. Due to the relative increase in population and age among the elderly, it is estimated that about 240 million people around the world are affected. Its prevalence may increase over the next 20 to 30 years. Gossip is a progressive condition with an average duration of approximately 85 years between the onset of clinical symptoms and death. In the brain associated with higher intellectual function, the death of pyramidal Φ-shaped neurons and the loss of neuronal synapses lead to typical symptoms, which are characterized by general and progressive impairment of cognitive function (see Francis, P. Ding. Et al., J. Neuro1. Neur〇surg. pSyChiatry (1999) 66: 137-147). The clinical signs of Alzheimer's disease are characterized by progressive cognitive decline, as well as decreased daily activity and neuropsychiatric symptoms or behavioral changes. In neuropathology, the main hallmark of AD is the presence of two characteristic diseases. .¥. Starch-like age spots and neurofibrillary tangles (NFT). The plaque is deposited outside the nerves of the sac, and the tangles are observed in the neurons in the brain after death. Class U5989.doc 1377949 A major component of the amyloid plaque core is the pathologically deposited small starchy beta-peptide (Αβ), which is cleaved from the starch-like precursor protein (Αρρ) by secreting enzymes (see Selkoe, DJ, Hv. (2001) 81: 741-766; Hardy, J. and Selkoe, DJ., Sciewce (2002) 297: 353-356). Αβ, a self-aggregating peptide of 39_43 residues (MW about 4 kDa), synthesized as part of the larger app " (11(M2〇 kDa). ApP is a type I integral membrane glycoprotein with

• Large N-terminal extracellular domain, single transmembrane region and short cytoplasmic tail region. The Αβ region is located outside the extracellular and trans-sacral regions of the sputum. The most common hypothesis that ΑΡΡ participates in neuronal cell death in AD is the amyloid-like hypothesis. This hypothesis assumes that plaque-like starch deposition or partially agglutinated soluble Αβ causes a cascade of neurotoxicity, thereby leading to neurodegeneration similar to AD pathology (see Se(1)〇e, D J.' (2001) 81: 741-766; Hardy , J_ and Selkoe, DJ, Sczewe (2002) 297: 353-356). Currently available drugs can help alleviate the symptoms of Alzheimer's disease, but they cannot change the underlying pathological process. Alzheimer's disease usually cannot be cured. There is still a need to provide a method of treating and/or preventing age-related dementia, particularly Alzheimer's disease. He Shouwu (4) Qing is also commonly known as Shouwu (10) (10) wu > he shou wu . f〇-ti , f〇.ti.teng , Ch.nese kn〇tweed ^ climbing knotweed, fl〇wery kn〇tweed, (5) called and similar Name n - The name of the Chinese man of Shouwu is the first food to be recorded for this herb, and it lives to 132 years old. Polygonum multiflorum is traditionally used as a nourishing and anti-aging drug in Chinese music, especially for hair loss and hair early. Shouwuweiji, Ganzi, and sputum, and has a slight warming effect when ingested I15989.doc 1377949

Should:, can be used as a main drug or nourishing drug < main components. The main factors of Polygonum multiflorum are / can be Nanzhou, Sichuan, Hunan, Shandong and other places. According to the earliest record of the use of Polygonum multiflorum, the Chinese version can be traced back to the Han Dynasty (206 B.C.-220 A.D.). The extract of Polygonum multiflorum has been shown to prolong the life cycle of diploid cells/month to prolong the lifespan of Drosophila and old ticks, reduce the amount and activity of superoxide dismutase (SOD) and enhance the repair in aging organisms. The ability of power (Vag ,, w〇Hd Science and

Modernization of Traditional Chinese Medicine and Materia Medica (2005) 7(5): 63-67^86) 〇, US 2002/146467 A1 and US 7,083,81 1 B2 disclose a herbal composition for preventing and treating dementia, Contains Polygonum and other herbs. CN 1 582975 discloses a Chinese medicine for treating cerebrovascular diseases and Alzheimer's disease, which is prepared by extracting and drying five kinds of Chinese herbal medicines including Polygonum multiflorum, lotus leaf and earthworm. CN 1621〇78 relates to a traditional Chinese medicine composition which is composed of red ginseng, ginsenoside, fieece fi〇wer root, polygonum, and yellow seal. Haw and rhubarb are prepared to treat mental retraction and treatment. ' CN 1742968 relates to a drug for treating vascular spasm disease' which is composed of Chinese herbal medicines Heshouwu, Astragalus root, Salvia root, Lungusticum root, Acorus root, and puzzle. Alphinia fruit, ginkgo leaf total flavone, earthworm, leech and gastrodia root were prepared. 115989.doc 1377949 A major component of Polygonum multiflorum is 2,3,5,4'-tetramethylene stilbene-2-0-β-indole-glycoside (TSG) as shown. TSG can use the microwave extraction method (see Wang, J. 荨人, (2〇〇3) z/iongcaoyao 34 (4): 314· 317) to reverse the technique (see Zhang, T. et al. / /owrwa/ 〇/ • - Chromatography & Related Technologies (2QQ3) 26[9 & \Qy 1565-1577), and methods using liquid phase extraction and chromatography (see Yan, χ, (1981) 8(2): 123 6&amp ;cn _ 1215728) Extraction and separation. CN 1 159015 C discloses the use of reverse phase chromatography to extract stilbene glycoside compound extracted from Polygonum multiflorum with chloroform followed by ethanol. It has been proved in the physical experiments that the stilbene glycoside compound can prevent brain cell damage caused by β-amyloid protein and hydrogen peroxide and enhance the memory ability of mice suffering from dementia and Αβ dementia. Zhang, L. et al. (Clin. Pharm. J., 2005, 40 (10): 749-752) found 2,3,5,4'-tetrahydroxystilbene_2_〇_p_d_ in Polygonum multiflorum Glycoside (TSG) has the effect of treating choline-induced injury in rats. At doses as low as 3〇mg/kg, TSG Φ can enhance choline acetyltransferase (C h AT) activity and improve Μ-choline Receptor - binding ability. CN 1 15901 5C^ teaches that 3,4,5-trihydroxy-2-pyrphenyl-P-D-glycoside can be used as an agent for the treatment of Alzheimer's disease, vascular dementia and - cognitive dysfunction. • The literature also indicates that green tea can prevent strokes (see Kakuda, Τ., Foods & Food Ingredients Journal of Japan (2001) 191: 51-5 5). The catechins in green tea have been found to have a free radical scavenging effect and can prevent dementia indirectly. Jg 2005-082495 reveals that catechins and butylamine (GABA) contained in green tea have the effect of improving memory and treating dementia and psychosis. The complexity of grass exists not only in the composition, but also in the possible interactions between the early music. Therefore, it is different from the role of a new western medicine that is different from the ancient ones, and it is difficult to use the method to identify all the components of the single-medicine medicine. The synergistic and/or replenishing effect of each active ingredient in the dry side 3-8 is the effect of researching Φ座+ a μ 丄a # 研九中乐 and preparing therapeutic agents from Zhongle, it is necessary to study effective methods to separate 'Identify and purify the active ingredients in herbal medicines. Polygonum multiflorum raw materials must be processed before use to obtain emodin and emodin from the material/causing side effects. In the method of transmission, the Polygonum multiflorum will be dried (~ (10) w ww / h / / or_) dip, in ... 'large juice, and heated with steam, and then dried to obtain a product called "Radix P〇lyg〇ni Multifl〇ri". However, the therapeutically active ingredient degrades during processing. Therefore, there is still a need to develop effective methods for processing Polygonum multiflorum. In addition, in order to improve the efficacy of Polygonum multiflorum extract, it is necessary to develop an economical and effective method to obtain a product containing the maximum amount of catechin and the lowest amount of emodin. SUMMARY OF THE INVENTION One object of the present invention is to provide a method for preparing an extract of Polygonum multiflorum. Another object of the present invention is to provide an extract of Polygonum multiflorum. Another object of the present invention is to provide a composition comprising the Shouwu extract of the present invention. A further object of the present invention is to provide a dementia for preventing or treating an individual D5989.doc 1377949 (such as aging-related dementia, or light η "know a to Hermès, vascular dementia, its inclusion to the need The subject of the present invention is a composition of the present invention. Another object of the present invention is to provide a σ top prevention or treatment dementia (such as aging-related dementia, Az). A drug of the present invention is described in detail in the following section. "Re-track 1 is not invented. Other features, objects and advantages of the present invention can be readily seen in the present invention.实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 实施 이

And the rules should be clear; however, in any potential ambiguity, the definitions provided herein are superior to any dictionary or extrinsic definition. ::: External reference [Otherwise the following terms as used in this disclosure should be understood to have the following meanings. Shows progressive decline beyond the expected performance of natural aging, such as old, per-tube dementia, light essence as used herein, the term "dementia," the brain damage or disease caused by cognitive work, related Dementia, Alzheimer's disease and intellectual disorders and the like. The term "prevention" as used herein refers to the symptoms of an individual who has been delayed from suffering from a disease. Symptoms of a diseased individual As used herein, the term "treatment" indicates a relief or improvement of feeling 115989.doc 1377949. * The term "individual" as used herein denotes an animal, in a preferred embodiment, the term •• individual", especially a mammal. In the expression of human ". The term "therape effective amount" The combination of drugs used in households is $ _ _, 9 early alone or with other treatments/quantity. ', the terminology of active ingredients, carrier, or " pharmaceutically acceptable to those skilled in the art For the preparation of a medical (4) means that the agent, the recipient or the like is generally used. ...D thinner of the substance, the shape of the plant, the root of the plant, and the whole plant, preferably refers to the root of the plant. *,,, Roots, leaves, stems and / or anhydrous alcohol or by Hungarian and water. According to the present invention, the alcohol has a cold carbon content of from 至 to 6 = 从 s from ', preferably from 1 to 4. The anti-atomic and more preferably 〗 〖to 3 carbon, the alcohol is methanol, ethanol Li Shi Xuanji. For example or ethanol. Compound. The alcohol is preferably methanol. Unless otherwise required herein, the singular terms shall include the singular. The method for preparing the extract of Polygonum multiflorum of the present invention comprises the following steps:) extracting the extract of Polygonum multiflorum with alcohol solution to obtain a crude extract; and diluting the crude extract obtained by step a) with water Obtaining the diluted extract; step c) loading the diluted crude extract of step b) into the resin column; I15989.doc d) extracting the extract of Polygonum multiflorum from the column t; and e) collecting the extract Obtained purified Polygonum multiflorum extract. For this reason, the concentration of the alcohol in the alcohol solution used in the step is 1 〇〇 ν / νβ / In the preferred embodiment, the alcohol solution is about 10 to V V . The alcohol solution, preferably about 2 to 9 is preferably about 2 to 5" / v% of the methanol solution ... 4 of the solution A, the solution of the alcohol is about 10 to 95 v / v% of the Ethylene" X In the examples, the ethanol solution of ',7. /V/〇, preferably about 10 to 30 v/v% ethanol solution, preferably about 2 〇v/v% ethanol solution. "According to the method of the present invention, the step-like crude extract can be obtained by any conventional stroke method, such as by boiling, dipping, ultrasonic treatment, searching, searching, or a combination thereof. The extraction time is about hours, Preferably, it is about 12 to 24 hours, preferably about 17 hours or more. The extraction temperature is about 5 to 95. 〇, preferably about, preferably at room temperature. The extraction pH is about 25 to 91, compared with: About 2.5 to 5.3, preferably about 5.3 β In a preferred embodiment, the Polygonum multiflorum herb is extracted by cold extraction (ultrasonic treatment at room temperature for 丨 hours and stirring for Μ hours). According to the method of the present invention, In step b), the crude extract is diluted with water such that the alcohol in the diluted extract has an indifference of about wv%, preferably about 2 to 5 v/v%. In the preferred embodiment, the mixture is diluted. The alcohol concentration of the extract is "2 v/v/〇». In another preferred embodiment, the alcohol of the diluted crude extract has an elongation of about 5 v/v%. According to the method of the present invention, step c) The resin used in the selection is selected from, but not limited to, macroporous resins and gel resins. The macroporous resin is preferably selected from the diii〇n series and the AmberHte series. Dowex sorbent, better mAi 〇 N series., II59S9.doc 13 1377949 The gum tree is preferably selected from the group consisting of Sephadex series, Toyopearl and Sephacryl, and more preferably in series. In a preferred embodiment, the resin DIAION HP-20. In another preferred embodiment, the resin is Sephadex-LH-20. * According to the method of the present invention, the diluted crude extract is added to the step c) Rinse with a rinse solution having the same alcohol concentration as the diluted crude extract obtained in step b). Pain according to the method of the invention, prior to the stripping step d), the column is obtained with v (b) Rinse the rinse solution with the same alcohol concentration as the diluted crude extract. According to the method of the present invention, in step d), in order to obtain the extract of Polygonum multiflorum, the column is about 1 〇 to 丨〇〇v/v%, Preferably, the alcohol solution is extracted from an alcohol bath of about 3 〇 to 7 〇 % and more preferably about A and then eluted with an acetone solution of about 7 〇 to 1 〇〇 v/v%. A series of alcohol solutions. In a preferred embodiment, the column is treated with a 50 v/v% methanol solution followed by • ι〇00/° And finally the acetone is extracted. In another preferred embodiment, the column is in a 50 v/v% ethanol solution, followed by a 95 v/v% ethanol solution and finally 100%. Acetone flushing. In a further preferred embodiment, the column is used. A 35 v/v% ethanol solution followed by a 50 v/v% ethanol solution followed by a 75-v/v% ethanol solution followed by 95 Wv. /. ethanol solution and finally 7 〇 v / v% of acetone. The present invention provides a useful method for preparing extract of Polygonum multiflorum. The extract of Polygonum multiflorum obtained by the method of the present invention contains more 2,3,5,4,-tetrahydroxystilbene-2-indole-β_〇_糖普115989 than the traditionally processed Polygonum multiflorum extract. Doc 14 1377949 (TSG) and a small amount of emodin, and less emodin (emd) and emodin 8-〇_b D-sugar (EMDG). The extract of Polygonum multiflorum prepared by the method of the present invention preferably contains TSG and catechin. Preferably, the Polygonum multiflorum extract, which is carried out by the method of the present invention, contains KG greater than about 65 and greater than about $w/w. /. Child's life, less than about 〇 5 w/w% of emd and less than about 〇 2

w/w% of EMDG. The extract of Hessian extract by the method of the present invention preferably contains about 65 to 85 w/w% of TSG and about 5 to 1 () w/w% of catechin. The recovery of tetrahydroxystilbene in the extract of Polygonum multiflorum according to the method of the present invention is greater than about qing, preferably greater than about 9%, more preferably greater than about 95%, and most preferably compared to crude extract of Polygonum multiflorum. More than about 97%. The recovery rate of catechin in the extract of Polygonum multiflorum is greater than about 鸠, preferably greater than about 75% and more preferably greater than about 8%, compared to the crude extract of Polygonum multiflorum. The extracted extract obtained in accordance with the method of the present invention, step e), can be condensed by any conventional solution concentration method, such as concentration using a reduced pressure rotary vaporization. Composition The present invention provides a composition of a Polygonum multiflorum extract. The composition of the present invention may be administered by any suitable route of administration (such as oral administration) to an individual. Suitable formulations include, but are not limited to, money, oral preparations. , hard or soft capsules, aqueous suspensions or oily suspensions, emulsions, sexual (four) or granules. If necessary, they can be sterilized, such as stabilizers, humectants and their analogues. .doc 1377949 Carrier Mixing. The la compounds of the present invention can be prepared by conventional procedures using known pharmaceutical excipients well known in the art. Thus, compositions for oral use may contain, for example, one or A variety of coloring agents, sweeteners, flavoring agents and/or preservatives.

In the treatment of AD, the compositions of the present invention may be compatible with other existing anti-dementia agents, such as cholinesterase inhibitors (e.g., Reminyl, Exelon, Aricept & Combination of c〇gnex and memantine in combination with the effect of the present invention for preventing or treating dementia, such as aging-related dementia, P-Kehmer's disease, vascular Dementia, mild mental and intellectual disorders and the like.

Those skilled in the art should be able to choose the appropriate route and dosage for treatment. According to the present invention, the preferred route is oral administration. The dosage will depend on the nature of the condition and the condition, the age and condition of the patient, the route of administration, and any prior treatment. Those skilled in the art will recognize that the dosage will vary depending on the age, size, health and other relevant factors of the individual. In a preferred embodiment, the optimal dose of the extract of Polygonum multiflorum of the present invention is from about 1 to about 25 mg per kilogram of body weight. The following examples are not provided to assist those skilled in the art to practice the invention, as those skilled in the art are not to be construed as limiting the scope of the invention. In this case, modifications and variations are made to the embodiments described herein. Example 115989.doc Preparation of Polygonum multiflorum extract #When the dried Polygonum multiflorum plant is ground, it is mixed with a highly polar solvent (such as water, methanolic ethanol, isopropanol or a mixture thereof) and soaked for 24 hours. The sieve 2 or I paper was used to remove the plant tissue residue' to obtain a clear & brown solution (Heshouwu crude extract). Month Fill a large pore resin or gel resin into the separation column. The tubular string can be made of glass, non-mineral steel or high molecular weight polymers such as pp. If a macroporous resin is used, the resin is filled into the column by dry filling. If gel resin is used, the resin is immersed in water and the expanded resin is filled into the column. The weight of the tree is more than 1〇 of the original dry plant weight. The filled ruthenium column was rinsed with 100 〇/〇 methanol or 95 v/v% ethanol solution to remove impurities, and then equilibrated with 2 to 5 Wv% aqueous alcohol solution. The crude Shouwu extract of S. sinensis is diluted with water to a concentration of about 2 to 5 v/v%. The diluted solution is injected into the separation column. After loading all of the diluted solution into the column, the column is rinsed with a 2 to 5 v/v% alcohol solution at 5 column volumes and then with a different ratio of solvent water mixture. Official column. The extracts were separately concentrated in a reduced pressure rotary evaporator and lyophilized to obtain an extract powder. Composite analysis of Polygonum multiflorum extracts and equipment: HPLC system (Shimadzu HPLC system, including SCL-lOAvp system controller, LC-i〇ATvp pump, FCV-1〇ALvp four-way valve, SIL-lOADvp autosampler, SPD_M1〇Avp diode array detector, Alltech 2000 ELSD Evaporetive light scattering detector and ERC-3415a degasser) 115989.doc 17 1377949 Analytical column: Cosmosil C1 8-5MS (4.6x250mm) Condition: Flow rate :1 .〇ml/min Analysis time: 60 min Sample size: 1 〇μ 1 PDA conditions: sampling period: 〇·64 sec; wavelength range: 190-370 nm; channel z 254 nm

ELSD conditions: temperature i〇(TC; nitrogen flow rate: 3 〇ml/min Control sample: TSG, catechin, EMDG and emodin mobile phase: B=l〇〇% acetonitrile; C=methanol; D=l 〇mM NH4OAc, pH 3.05 time (min); δ B(CAN) C(MeOH) D(10 mM NH4OAc, pH 3.05) 0 10 90 5 ~~ 0 10 90 _ 35 100 0 0 50 100 0 0 _ 53 &quot ; 0 10 90 60 0 10 90 one

Method for testing the activity of extracts of Polygonum multiflorum The activity of extracts of Polygonum multiflorum was tested using an animal model of Nabeshima, T. and Nitta, A. (Tohoku J. Exp. Med.' 1994, 174(3): 241-249). Alzheimer's disease rats induced by intraventricular injection of amyloid (Αβ40) were used to evaluate the therapeutic effect of Polygonum multiflorum extract on intelligence and learning disability induced by amyloid peptides. Further, the biological activity of the purified substance in the extract of Polygonum multiflorum L. was analyzed using an active screening technique using an in vitro neuronal cell antagonistic amyloid 神经β neurotoxin. Example 1 Effect of extraction temperature and solvent 115989.doc • 18· 1377949 5 g of Polygonum (PM) powder was added to about 50 ml of water and extracted in a 100 ° C water bath for 1 hour. The mixture was centrifuged at 8,000 rpm for 1 5 minutes at room temperature, followed by suction filtration through a No. 1 filter paper. 34 ml of filtrate (PMWH) was collected.

2.5 g of Polygonum powder was extracted in four different solvents, namely, double distilled water, 20 v/v% ethanol solution, 50 v/v% ethanol solution and 95 v/v% ethanol solution (in the room). Under the temperature, the ultrasonic treatment was performed for 1 hour and then rotated at 45 rpm for 16 hours). Subsequently, the mixture was centrifuged at 3,000 rpm for 15 minutes at room temperature, and then filtered with a No. 1 filter paper to collect the first extract (PMWL-1, PM20EL-1, PM50EL-1, and PM95EL-1). . The remaining dregs were re-extracted by the cold extraction step described above and the second extracts (PMWL-2, PM20EL-2, PM50EL-2 and PM95EL-2) were collected. The extraction yield of each extract was analyzed and the emodin (EMD), emodin-8-0-β-ϋ-glycoside (EMDG) and 2,3,5,4'- in the extract were analyzed by HPLC. The amount of tetrahydroxystilbene-2-0-β-indole-glycoside (TSG). The extraction yields for each extraction method and batch are summarized in Table 1. Table 1 Extraction rate using different extraction methods and multiple extractions Samples First extraction Second extraction method Extraction amount (mg/ml) Extraction rate (%) The amount of extract (mg/ml) Extraction Rate (%) PMWH (hot water extraction) 34 23.1 - - PMWL (room temperature water extraction) 24.9 19.1 3.7 pm PM20EL (ethanol extraction at room temperature 20 ° /.) 30.3 23.1 5.5 5.1 PM50EL (room temperature 50 ° /. Ethanol extraction) 33.4 26.2 5.1 4.5 PMEL (95% ethanol extraction at room temperature) 20.7 19.7 2.5 2.8 As shown in Table 1, the extraction rate of hot water extraction is about 20% higher than the extraction rate of room temperature water extraction. If an ethanol solution is used as the extraction solvent, the method using 50% of the 115989.doc -19-1377949 ethanol solution has the highest extraction rate. In summary, the extraction rate for the first extraction is about 19 to 26%, while the extraction rate for the second extraction is only 3 to 5%. The analysis results of the amounts of EMD, EMDG and TSG are summarized in Table 2. Table 2 EMD, EMDG and TSG in different extracts Quantity of ingredients (mg) PMWH PMWL PM20EL PM50EL PM95EL EMD 0.00 0.13 0.00 1.88 3.88 EMDG 1.34 0.00 0.00 0.00 8.72 TSG 20.40 28.78 32.61 56.28 40.08

The amount of TSG in the extract extracted with room temperature water was about 40% higher than the amount of TSG in the extract extracted with hot water. The extract extracted with room temperature water contains EMD, and the extract extracted with hot water contains EMDG. When extracted with an ethanol solution, the extract extracted with a 50% ethanol solution contains the maximum amount of TSG. However, the amount of EMD and EMDG increases as the concentration of ethanol increases. Although the TSG content of the extract extracted with a 20% ethanol solution is not the highest, it does not contain EMD or EMDG. Therefore, the extraction method using a 20% ethanol solution at room temperature has the greatest benefit as assessed by the TSG extraction rate and the quality of the extract.

Example 2 Effect of extraction solvent ratio and pH Three different solvents (10 v/v%, 20 v/v%, and 30 v/v% ethanol solution) were prepared. 2 g of Polygonum multiflorum powder was added to 20 ml of each solvent, and a three-tube mixture was prepared for each solvent. The three tube mixtures of the respective solvents were adjusted to pH values of 2.5, 5.3 and 9.1 with NaOH and HCl, respectively. Subsequently, each tube was ultrasonicated for 1 hour and rotated and mixed at a speed of 45 rpm for 16 hours. Each sample was centrifuged at 3,000 rpm for 15 minutes and the supernatant was collected. The composition analysis of the supernatant is summarized in Table 3. 115989.doc -20- 1377949 Table 3 EMD, EMDG and TSG in extracts of Polygonum multiflorum (PM) extracted with different solvents and pH pH 2.5 5.3 9.1 Solvent (ethanol concentration) 10% 20% 30% 10% 乂20% 30% 10% 20% 30% Amount of ingredients < :mg/g PM powder) EMD 0.14 0.18 0.36 0.19 0.26 0.38 1.22 1.61 2.03 EMDG 0.98 1.05 1.20 ;0.00 0.00 0.00 0.60 1.04 1.60 TSG 22.12 22.01 25.20 1 25.75 25.35 27.24 0.00 0.00 2.43

As shown in Table 3, the amount of EMD at three pH values increased with increasing ethanol concentration (10% < 20% < 30%). The amount of EMD extracted with a 30% ethanol solution was about twice the amount of EMD extracted with a 20% ethanol solution. At a pH of 9.1, extraction of Polygonum multiflorum with three solvents will hardly extract TSG. At pH 5.3 and 2.5, the amount of D8 extracted as the ethanol concentration increased was similar and slightly increased (10%$20% < 30%). Under the condition of 5.31^ value 5.3, EMDG could not be extracted by the three solvents; however, under the condition of μΗ2.5 and 9.1, some EMDG could be extracted, and the amount increased with the increase of ethanol concentration (10% <;20%<30°/〇).

The results of Example 2 show that the optimum conditions for extracting Polygonum multiflorum are 10 to 20 °/ at pH 5.3. The ethanol solution is extracted. Under these conditions, the TSG can be effectively extracted and the minimum amount of EMD and EMDG extracted. When the extraction pH is lowered to 2.5, the amount of TSG is reduced by about 10%, the amount of EMD is reduced by about 30%, and the amount of EMDG is increased. Example 3 Extraction of active ingredients of Polygonum multiflorum using macroporous resin 1000 g of Polygonum multiflorum powder was cold extracted with 1 liter of methanol (ultrasonic treatment at room temperature for 1 hour and then stirred for 16 hours). The extracted solution was suction-filtered on a No. 1 filter paper and 900 ml of a filtrate (PM901) was collected. I15989.doc 1377949

100 g of DIAION HP-20 resin (Mitsubishi Chemicals) was packed into a chromatography column (ID = 40 mm; L = 200 mm), and the column was recirculated with 1 liter of decyl alcohol for 1 hour. The column was then rinsed with 2 liters of 5 v/v% methanol solution. 400 ml of PM901 was diluted with pure water to a final volume of 8 liters to a final concentration of 5% methanol. The diluted solution was added to the DIAION column at a flow rate of 15 ml/min and the unadsorbed liquid was collected. The column was rinsed with 1 liter of 5 v/v% sterol solution and the rinse was combined with the non-adsorbed liquid, and then the combined liquid was concentrated to 50 ml (PM902) with a reduced pressure rotary evaporator.

The column was then flushed with 1.5 liters of 50 v/v% sterol solution. The extracted liquid was collected and concentrated to 200 ml (PM903) in a reduced pressure rotary evaporator. The column was further stripped with 1.5 liters of 100% sterol. The extracted liquid was collected and concentrated to 200 ml (PM904) in a reduced pressure rotary evaporator. Finally, the column was flushed with 1 liter of 100% acetone. The extracted liquid was collected and concentrated to 50 ml (PM905) in a reduced pressure rotary evaporator. The amount and recovery of TSG and catechin in the sample (before the column separation or after the column separation) are shown in Table 4. Table 4 TSG and catechin amount and recovery rate in PM901 to PM905 Total amount of sample catechin (mg) Recovery rate of catechin (%) Percentage of catechin in sample (%) Total amount of TSG ( g) Recovery of T^G (%) Percentage of TSG in the sample (%) PM901 10.8 100 0.13 1.7 100 21.2 PM902 1.6 14.8 0 0 0 0 PM903 8.6 79.6 0.31 1.5 88.2 54.3 PM904 0 0 0 0.1 5.8 14.6 PM905 0 0 0 0 0 0 As shown in Table 4, the percentages of TSG and catechin in the methanol-extracted sample (PM901) were 21.2% and 0.13%, respectively. The extracted sample PM901 can be separated from the column by 50°/ by 115989.doc -22- 1377949. The methanol solution was further purified to give 2.8 g of PM903. The percentage of TSG in PM903 was 54.3%, which was 2.6 times the initial percentage and the percentage of catechin in PM903 was 0.31%, which was 3 times the original percentage. The recovery rates of TSG and catechin were 88.2% and 79.6%, respectively. Example 4 Extraction of active ingredients of Polygonum multiflorum using macroporous resin

180.10 g of Polygonum multiflorum powder was cold extracted with 1.8 liters of 20 v/v% ethanol solution (ultrasonic treatment at room temperature for 1 hour and then stirred for 16 hours). The extracted solution was centrifuged at 8,000 rpm for 15 minutes at 25 ° C, and then suction filtered with a No. 1 filter paper. 1,840 ml of filtrate (PM20EL) was collected and the amount of solid matter was measured.

300.08 g of DIAION HP-20 resin was packed into a chromatography column (ID = 80 mm; L = 295 mm), and the column was recirculated for 1.5 hours with a 2.0 liter 95 v/v 0/〇 ethanol solution. The column was then rinsed with 4.38 liters of 2 v/v% ethanol solution. Dilute 1,533 ml of PM20EL to 15,330 m in pure water so that the final concentration of ethanol is 2%. The diluted solution was added to the DIAION column at a flow rate of 15 ml/min and the unadsorbed liquid was collected. The column was rinsed with a 3.84 liter 2 v/v% sterol solution and the rinse was combined with the non-adsorbed solution to a total volume of 16,840 ml (PM801). The column was then flushed with 6.5 liters of 50 v/v% ethanol solution. The initial 200 ml yellow-orange extract liquid was collected and the collected liquid was combined with the remaining extraction liquid into PM802. The amount and recovery of TSG and catechin in the sample (collected before or after separation of the column) are shown in Table 5. Table 5 The amount of TSG and catechins in PM20EL, PM801 and PM802 and back 115989.doc -23- 1377949 Yield The total amount of sample catechins (mg) The recovery rate of catechins (%) The catechins in the sample Percentage (%) Total amount of TSG (g) Recovery rate of TSG (%) Percentage of TSG in the sample (%) PM20EL 41.4 100.0 0.11 5.2 100.0 14.3 PM801 0.6 1.5 0.02 0.0 0.0 0.0 PM802 28.9 69.9 0.32 4.8 91.2 52.3

As shown in Table 5, the percentages of TSG and catechin in the 50% ethanol solution sample (PM20EL) were 14.3% and 0.11%, respectively. The extracted sample PM20EL was further purified by column separation using a 50% decyl alcohol extracting solution to obtain 9.1 g of PM802. The percentage of TSG in PM802 is 52.30/. , which is 3.6 times the initial percentage' and the percentage of catechins in PM802 is 0.32%, which is three times the initial percentage. The recovery rates of TSG and catechin were 91.2% and 69.9%, respectively. According to the results of Example 4, the preparation method of the present invention can effectively separate TSG and catechin, and control the loss rate as low as 8.8 ° / 〇 (TSG) and 3 0.1 ° /. (catechin). Example 5 extraction of active ingredients of Polygonum multiflorum using gel resin

30 g of Sephadex LH-20 resin (Pharmacia) was packed into a chromatography column and recycled in a 500 ml 95 v/v% ethanol solution at a flow rate of 20 ml/min for 2 hours. The column was then rinsed with 500 ml 2 v/v% ethanol solution. 132 ml of PM20EL-1 obtained in Example 4 was diluted with M.Q. water to a final volume of 1000 ml. The diluted solution was placed in an LH-20 column and the unadsorbed light brown turbid liquid was collected. The column was then rinsed again with 500 ml 2 v/v% ethanol solution. The first 150 m 1 rinse was combined with the non-adsorbed liquid, and the combined liquid was concentrated to 25 ml (PM211) with a reduced pressure rotary evaporator. The remaining 345 ml of light yellow clarified rinse was collected and concentrated to 10 ml (PM212) using a reduced pressure rotary evaporator. Combine PM211 and 115989.doc •24- 1377949 PM212 into PMLH201. The column was first flushed with 250 ml of 35 v/v% ethanol solution, and 244 ml of the extracted liquid was collected (the first 80 ml was light yellow and the remaining 164 ml was light yellowish brown) and was compressed in a reduced pressure vaporizer. It is concentrated to 25 ml (PMLH202c) °

The column was then flushed with 250 ml of 50 v/v% ethanol solution and 244 ml of the extracted liquid was collected (the first 60 ml was light yellowish brown and the remaining 184 ml was yellowish brown) and was compressed in a reduced pressure vaporizer. It was concentrated to 25 ml (PMLH203C) ° and the column was then 25 0 ml 75 v/v. /. The ethanol solution was extracted and 242 ml of the extracted liquid was collected (the first 70 ml was light yellowish brown, followed by 56 ml of yellowish brown, and the remaining 11 6 ml was light orange) and concentrated to a reduced pressure rotary evaporator. 25 ml (PMLH204c).

The column was then stripped with 250 ml of 95 v/v% ethanol solution and 246 ml of the rinse liquid was collected (the first 70 ml was light orange and the remaining 176 ml was colorless) and was compressed with a reduced pressure rotary evaporator. Concentrate to 25 ml (PMLH205c) ° Finally, pipette was flushed with 250 ml 70 v/v% acetone solution, 246 extracts were collected and compressed to 25 ml (PMLH206c) with a reduced pressure rotary evaporator. The ratio of the amount of solid matter to the active ingredient in each concentrated sample (flush) was analyzed and the results are shown in Table 6. Table 6 The amount and recovery of TSG, EMD, EMDG and catechin in each of the extracts 115989.doc 25- 1377949 The amount of the TSG mass of the solvent solid TSG (mg/ml) (mg /ml) Yield (%) TSG EMD EMD Equivalent to the solid matter (μβ/πιΐ) mass ratio (%) Ratio (%) EMDG amount (Mg/ml) EMDG and solid matter Ratio (°/〇) The ratio of the amount of catechin catechin to the solid (mg/ml) substance (%) PM20EL-1 20% EtOH 26.4 2.8 100 10.6 8.0 0.03 0 0.00 0 0.00 PMLH201 20% EtOH (unbound 84.0 0.6 4.7 0.7 <3.5 <0.004 <5 <0.01 0.7 0.83 PMLH202C 35% EtOH 8.5 6.9 53.6 81.2 <3.5 <0.041 7 0.08 0.7 8.24 PMLH203C 50% EtOH 7.1 5.6 43.5 78.9 21.9 0.31 <5 <0.07 <0.5 <7.04 PMLH204C 75% EtOH 3.2 0 0 0 4.0 0.13 0 0.00 0 0.00 PMLH205C 95% EtOH 1.2 0 0 0 0 0.00 0 0.00 0 0.00 PMLH206C 70% Acetone 10.8 0 0 0 0 0.00 0 0.00 0 0.00

As shown in Table 6, most TSGs can be extracted with 35 to 50% ethanol solution, and the total recovery is over 97%. The catechins and EMDG can be extracted from the same portion, but most of the EMD is eluted with a 50% ethanol solution. The results of Example 5 indicate that when LH-resin was used as the separation material, the optimum elution solution was a 35 to 40 v/v% ethanol solution. Example 6 Induction of neurodegeneration in rats by the cognitive behavioral assessment of Alzheimer's rat model ΡΜ903 induced by Αβ injection Αβ 1-40 was injected into the rat model as a rapid screening for the treatment of Alzheimer's Animal model of the disease drug. The neurodegeneration of the rat model was induced by slowly injecting Αβ 1-40 into the third lateral ventricle of the rat to cause cognitive behavioral disorder by injuring the hippocampus region by the cerebrospinal fluid (CSF) system. To prepare Alzheimer's disease rats, a Αβ 1-40 solution was injected into an Alzet osmotic pump (Alzet 1002; DURECT Co., USA) and the PE tube of the cerebral perfusion set was connected to a pump. The rats were anesthetized with sodium pentobarbital (45 mg/kg) and placed on a stereotaxic instrument -26- 115989.doc 1377949

(31'0£1/1' with 0,1;3-8;1^〇(161 51650) to locate the lateral ventricle. Insert the cerebral perfusion set into the third lateral ventricle of the rat, and the Alzet osmotic pump The subcutaneous area of the rat's neck was implanted. The surgical area was sutured and the rats were placed in a cage. In the control group (Sham), the Αβ 1-40 solution in the Alzet osmotic pump was 35% acetonitrile / 0.1%. Trifluoroacetic acid replacement. Rats were subjected to passive avoidance testing 8 to 9 days after implantation. The cognitive behavior of the rats was assessed using a Morris water maze 1 to 15 days after implantation. From the experimental group, rats were given placebo, different doses of PM903 (1 to 25 mg/kg) and 1 〇〇mg/kg of PMWH. Passive avoidance test constructed passive avoidance test a-type system according to the dark test method (step-through method) (see Jarvik Μ. Ε· and Kopp R. and ep. (1967) August; 21(1): 221-224). The device consisting of a clear box is separated by a gate. The wall is connected to the black box. During the training period, the acquisition trial is performed in the following manner: the rat is allowed to acclimate in the clear box for 2 min, and then the gate is opened. Thereafter, when the rat passed through to the dark box, an unavoidable electric shock (〇8 mA, 3 seconds) was transmitted. This test was repeated until it was determined to be obtained. When the rat finally stayed in the clear box for more than 300 seconds to avoid entering the dark box Can be determined to be determined. The rats were removed and returned to the cage. The data was analyzed by an〇va.

Construction of the Morris Water Maze In neuroscience, the Morris water maze is designed to test the behavior of spatial memory. It was developed by neuroscientist Richard GM〇rrissi984 (see Morris R. J Neurosci Methods (1984) 1 1 (l)· 47- 115989.doc • 27· 1377949 6〇), which is now commonly used to detect hippocampus The role of spatial memory. Add a cylindrical water tank (160 (10) in diameter and 60 cm deep) to a water depth of 35 (10) and maintain the water temperature at 23 ± rc. The pool is divided into four quadrants (north, southwest, northeast, and southeast). Fix the circular stainless steel life-saving platform in the " southwest quadrant and hide it on the water surface with TI(10). Place visual cues (such as colored shapes) around the sink in the animal's line of sight. The camera is placed above the pool to capture and record the image of the test track. Test of animal behavior Practice = = only rats. Rats were trained for 3 days, 1 training per day = 2: 4 cycles. In each cycle, each of the _ large _ private I was in the southwest " quadrant platform, and the rats were guided to the flat rats and no lifesaving seconds were found. Let the rest of the 15 15 hours by the computer before the cycle. *, first. It has recorded the entire nm path including the swimming path and the time (latency) used for the platform, and the ancient ten is a large process. Day 4 'The time taken to move the life-saving platform. ι Γ,. Within 5 minutes to reach the "Southwestern" quadrant and to reach other data such as the latency time and the ice travel distance obtained by the '卩 most (four) behavior test to indicate the large △ and ANOVA analysis data. By memory. (4) The results of the reference space test of the rat are shown in Figure 2. The results of learning and memory ability are not shown in Figure 3, Figure 4 of the monthly b-knife. From Figure 2 to L-L The results of the reference memory of type 6 were shown to be seen in the 'sham' group. The rats in the control group (Sham) did not show recognition of 115989.doc -28·1377949, but with placebo (the Αβ ι_4〇 group shown in the figure) The treatment of Αβ 1 -40 induced dementia rats showed severe cognitive behavior disorder. It was found that ΡΜ903 (1 '5 and 25 mg/kg body weight) can effectively improve the passive avoidance and spatial memory of Αβ 1_40 induced dementia rats.ΡΜ903 The effective dose is 1 to 25 mg per kg of body weight. Compared with the positive control (the PMWH group shown in the figure), the effective dose of ΡΜ903 is reduced by more than 20 times. [Simplified explanation]

囷1 shows (Α): 2,3,5,4'-tetrahydroxystilbene-;2-〇-pD-purine glycoside (TSG); (B): catechin; (C): emodin (EMD); and (D): chemical structure of emodin-8-〇_p_D_glycoside (Em〇g). Figure 2 shows the results of the passive avoidance test.圊3 shows the results of the space maneuverability of the water maze test. Figure 4 shows the results of the working memory of the water maze test. 115989.doc 29-

Claims (1)

Patent application No. 097122258, tb 4_ Syrian gold 1 々Ar na From · <_ · ίρυ, /κζζ:>δ is looking for; f'J A please tired J>«, apply for special paste: wna The preparation of the Polygonum multiflorum (p0!yg0num muitifl〇rum) extract ~^~^- method comprises the following steps: a) extracting Polygonum multiflorum with about 1〇 to 30 Wv% ethanol solution or methanol to obtain Polygonum multiflorum Extracting; b) diluting the crude extract obtained in step a) with water to obtain a diluted crude extract having a final alcohol concentration of about 2 to 5 v/v%; C) diluting the crude extract of step b) Loading the resin column;) extracting the extract of Polygonum multiflorum from the column with one or more alcohol solutions of about 1 Torr to 1 〇〇 ν / ν 〇 / β 2; and 2. 4. e) collecting the extracted The extracts were combined and combined to obtain purified Polygonum multiflorum extract. For example, the method of claim 1 wherein the Polygonum multiflorum herb is a dried or processed Polygonum multiflorum herb. A method of claim 1, wherein the alcohol solution is an ethanol, .anol or isopropanol solution. The method of claim 1, wherein the resin is a macroporous resin or a gel tree. 5. The method of claim 4, wherein the macroporous resin is DiAION HP. The method of item 4, wherein the gel resin is Sephadex LH-20 tree 7. The extraction system of the step 1) of claim 1 is carried out at room temperature. 0 115989-1010627.doc 丄:wmy 8. If the claim i is ♦, the step a) is carried out at a pH of about 5.3. 9. The alcohol solution of claim 1, wherein the alcohol solution of step d) has a concentration of from about 30 to 70 v/v%. The method of A U item 1 wherein the alcohol solution of step d) has a concentration of about 35 to 50 v/v%. 11. For example, in the case of the item 1 of the formula, the alcohol solution of the step d) is a 50 v/v% methanol solution. 12. The method of claim 1, wherein the step of the alcohol solution is an ethanol solution of about h v/v%. 13. The method of claim 1, wherein the alcohol solution in the step is an ethanol solution of about % ν / ν %. 14. The method of claim 1, wherein the extract of Polygonum multiflorum obtained in step e) comprises about 2, 3, 5, 4, _ tetrahydroxy diphenyl bromide of about 65 to 85 w/w 〇 /0 2·〇-ρ_ D-sugar tone (TS.G) and about 5 to l〇w/w% of catechin. 15. An extract of Polygonum multiflorum prepared by the method of claim 丨. 16. The extract of Polygonum multiflorum according to claim 15 which comprises about "to a TSG of ~% and a catechin of about 5 to 10 w/w%." 17. The extract of Polygonum multiflorum according to claim 15 which comprises less than大 之 之 emodin and less than about 0.2 w / w 〇 / emodin · 8_ 〇 _p_D sugar buds. 18. A pharmaceutical composition comprising a therapeutically effective dose of the claimant Extracts and, where appropriate, pharmaceutically acceptable carriers, diluents or excipients 19. The pharmaceutical composition of claim 18, which additionally comprises one or more additional anti-I15989-1010627.doc 1377949 dementia agents. 20. Use of a pharmaceutical composition according to claim 18 for the manufacture of a medicament for the prevention or treatment of dementia. 21. The use of claim 20, wherein the dementia is selected from the group consisting of aging-related dementia, Alzheimer's disease, such as schizophrenia, and mild mental and intellectual conditions. . twenty two. The use of claim 2 wherein the pharmaceutical product is about 1 to 25 mg of Polygonum multiflorum extract. For every kilogram of body I15989-1010627.doc