(12) United States Patent (10) Patent No.: US 7,884, 195 B2 Li (45) Date of Patent: Feb

US007884195 B2

(12) United States Patent (10) Patent No.: US 7,884, 195 B2 Li (45) Date of Patent: Feb. 8, 2011

(54) METHOD OF EXTRACTING KR 2002 00843.11 11, 2002 GINSENGNOSIDE RGM, KR 2003 0049984 6, 2003 PHARMACEUTICAL COMPOSITION KR 2005 OO18226 2, 2005 INCLUDING GINSENGNOSIDE RG AND WO OO O4912 2, 2000 USES THEREOF WO OO 37481 6, 2000 WO O1 15717 3, 2001 (75) Inventor: Long Yun Li, Dong 2 Hong tong, Hong OTHER PUBLICATIONS Qi Street, Chao Yang suburb, Changchun MIT “7.6. Two-Solvent Recrystallization Guide', published Jan. (CN) 130-021 2004, also available at http://ocw.mit.edu/NR/rdonlyres/Chemistry 5-301 January--IAP-2004/DDE9861 E-3411-4EBD-8210 (73) Assignees: Long Yun Li, Changchun (CN); 512B41F785C6/0/8 6 recrystallization.pdf, last viewed Feb. 4, Boong-Kung Ko, Seoul (KR) 2010.* Ma, T. C. et al., “Effect of 200S)-Ginsenoside-Rg and (*) Notice: Subject to any disclaimer, the term of this Cyproheptadine on Two-way Active Avoidance Learning and patent is extended or adjusted under 35 Memory in Rats' In; Arzneimittel Forschung Drug Research, vol. 43 U.S.C. 154(b) by 92 days. (II), No. 10, pp. 1049-1052, (1993). Kaku, T. et al., “Isolation and Characterization of Ginsenoside-Rg, (21) Appl. No.: 11/912,804 20R-Prosapogenin, 20S-Prosapogenin and Delta 20-Prosapogenin. Chemical studies on Saponins of Panax ginseng C. A. Meyer, Third (22) PCT Filed: Apr. 26, 2005 report” In; Arzneimittelforschung, vol. 30, No. 6, pp. 936-943, (1980). (86). PCT No.: PCT/KR2OOS/OO1204 Kudo, Kenzo et al., “Properties of ginseng Saponin inhibition of catecholamine Secretion in bovine adrenal chromaffin cells'. Euro pean Journal of Pharmacology, Elsevier, vol. 341, No. 2-3, pp. 139 S371 (c)(1), 144, (1998). (2), (4) Date: Oct. 26, 2007 Tachikawa, Eiichi et al., “Effects of ginseng Saponins on responses induced by various receptor stimuli”, European Journal of Pharma (87) PCT Pub. No.: WO2006/115307 cology, Elsevier, vol. 369, No. 1, pp. 23-32, (1999). Qun Fang et al. "Micelle-mediated extraction and preconcentration PCT Pub. Date: Nov. 2, 2006 of ginsenosides from Chinese herbal medicine.” Journal of Chroma tography A. vol. 904, XP004221275, Dec. 2000, pp. 47-55. (65) Prior Publication Data * cited by examiner US 2008/O22776O A1 Sep. 18, 2008 Primary Examiner Shaojia Anna Jiang (51) Int. Cl. Assistant Examiner Bahar Schmidtmann C07J 75/00 (2006.01) (74) Attorney, Agent, or Firm Oblon, Spivak. McClelland, (52) U.S. Cl...... 536/5 Maier & Neustadt, L.L.P. (58) Field of Classification Search ...... None See application file for complete search history. (57) ABSTRACT (56) References Cited The present invention provides a method of extracting ginse noside Rg, which is applied to mass production. The method U.S. PATENT DOCUMENTS of the present invention employs gradient salting-out instead 3,418,311 A 12, 1968 Sakai et al...... 536,123 of conventional silica gel chromatography, and therefore 6,083,932 A 7/2000 Pang et al. enables convenient performance and mass production of gin 7,438,935 B2 * 10/2008 Wei et al...... 424,728 senoside Rg. Provided are also a pharmaceutical composi 2004/O247703 A1 12, 2004 Rhim et al. tion comprising ginsenoside Rg as an active ingredient, and 2005/0031711 A1 2/2005 Park ...... 424,728 prophylactic and therapeutic agents for preventing and treat ing dementia, depression, peripheral circulation disorder and FOREIGN PATENT DOCUMENTS related diseases, utilizing the pharmaceutical composition. CN 1309.969 8, 2001 DE 28 28.851 1, 1980 8 Claims, 6 Drawing Sheets U.S. Patent Feb. 8, 2011 Sheet 1 of 6 US 7,884,195 B2 F. G.

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U.S. Patent Feb. 8, 2011 Sheet 6 of 6 US 7,884,195 B2 F.G. 6

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£170 US 7,884, 195 B2 1. 2 METHOD OF EXTRACTING nient manipulation and processes, easy practical application, GINSENGNOSIDE RGM, highyield and purity, and harmlessness and safety to humans. PHARMACEUTICAL COMPOSITION In spite of Such circumstances, research into Such a desired INCLUDING GINSENGNOSIDE RG AND process is insignificant. USES THEREOF Meanwhile, there are few published studies and investiga tions into medicinal usage of ginsenoside Rg and application CROSS-REFERENCE TO RELATED thereof. Even though the present inventors have proposed APPLICATIONS medical use of ginsenoside Rg, in Chinese Patent No. ZL01102117.9, entitled "Application of ginsenoside Rg in This application is a 371 of PCT/KR05/01204 filed Apr. 10 preparation of a therapeutic drug for cardio- and cerebrovas 26, 2005. cular diseases (CCVD), use of this therapeutic drug is restricted only to treatment of cardio- and cerebrovascular BACKGROUND OF THE INVENTION diseases such as myocardial ischemia, cerebral ischemia and shock diseases. As such, there is a need in the art for more 1. Field of the Invention 15 advanced and improved research into new medical uses of The present invention relates to a method of extracting ginsenoside Rg2. ginsenoside Rg, a pharmaceutical composition comprising ginsenoside Rg as an active ingredient, and prophylactic and SUMMARY OF THE INVENTION therapeutic agents for preventing and treating dementia, depression, peripheral circulation disorder, and related dis Therefore, the present invention has been made in view of eases, utilizing the same. the above problems, and it is an object of the present invention 2. Description of the Related Art to provide a novel method of extracting ginsenoside Rg. Ginseng contains various ginsenosides (ginseng Saponins) suitable for industrial-scale production. and there are around 30 kinds of ginsenosides which have It is another object of the present invention to provide a been known hitherto. Among these, ginsenoside Rg is a 25 pharmaceutical composition comprising ginsenoside Rg. Substance belonging to protopanaxatriol glycoside which was It is a further object of the present invention to provide a isolated and named a long time ago. therapeutic agent comprisingginsenoside Rg, for preventing It is known through previous research and study that, in the or treating dementia, depression, peripheral circulation dis course of red ginseng processing, a glycosidic bond at the order and related diseases. C-20 position of ginsenoside Rg is readily susceptible to 30 hydrolysis and at the same time, hydroxyl radicals lead to In accordance with an aspect of the present invention, the anti-equilibrium, i.e., breakdown of an equilibrium state, above and other objects can be accomplished by the provision thereby forming a ginsenoside isomer C-20(S) or C-20(R). In ofa method of extractingginsenoside Rg from a raw material addition, it is generally published that red ginseng contains of Araliaceae panax, comprising: mixed-type ginsenoside Rg C-20(RS) in a substantially 35 Subjecting the raw material to hot water extraction and higher content than fresh ginseng or white ginseng. concentration to obtain concentrates, adding a lower alcohol Meanwhile, since medical use of Such ginsenoside Rg is to the concentrates to precipitate and remove foreign materi as yet unknown, extracting and separating Rg has been con als; ventionally carried out, on a laboratory-scale, by extracting decolorizing the remaining Supernant and recovering the ginseng total Saponin (GTS) with an organic solvent, and 40 lower alcohol; separating ginsenoside Rg via silica gel column chromatog adding a salting-out agent to a saturation concentration So raphy or high performance liquid chromatography (HPLC). as to obtain precipitates and re-dissolving the precipitates in Firstly, a silica gel column chromatography process refers water, followed by gradient salting-out to obtain differential to a method involving adsorbing the ginseng total Saponin precipitates corresponding to different concentrations of the extracts on a silica gel chromatography column, and separat 45 salting-out agent; ing ginsenoside Rg therefrom using the organic solvent, in analyzing to divide the precipitates into ginsenoside Rg which chloroform, methyl alcohol, ethyl acetate and water are enriched fractions and ginsenoside Rg non-enriched frac mixed in a predetermined ratio, as a washing agent. tions; In this connection, the separation method utilizing the destaining and decolorizing the ginsenoside Rg-enriched above-mentioned silica gel column chromatography mainly 50 Suffers from disadvantages such as trace amounts of Sub fractions with a destaining agent, followed by concentration stance of interest separated from the column, Very slow sepa to obtain concentrates; and ration speed, a need for an additional separation process due adding the lower alcohol to re-crystallize the resulting to use of a large quantity of harmful organic solvents, and concentrates, thereby obtaining ginsenoside Rg. being unsuitable for large scale production due to high pro 55 duction costs, low yield and unsafety. BRIEF DESCRIPTION OF THE DRAWINGS Whereas, high performance liquid chromatography (HPLC) enables separation of high-purity ginsenoside Rg, The above and other objects, features and other advantages but presents problems such as high equipment and operation of the present invention will be more clearly understood from costs and increased production costs associated with use of a 60 the following detailed description taken in conjunction with large quantity of expensive organic solvents such as methyl the accompanying drawings, in which: alcohol or acetonitrile. Consequently, conventional methods FIG. 1 is a graph showing HPLC analysis results of a crude of separating ginsenoside Rg are not suitable for mass pro product of ginsenoside Rg, isolated from ginseng leaves duction of Rg, thus making it expensive and difficult to enter (Panax ginseng); practical commercial products. 65 FIG. 2 is a graph showing HPLCanalysis results of mixed That is, a process Suitable for industrial-scale production is type ginsenoside Rg, isolated from ginseng leaves (Panax preferred to satisfy requirements such as simple and conve ginseng); US 7,884, 195 B2 3 4 FIG. 3 is a graph showing HPLC analysis results of S-type Araliaceae panax plants as described above, and the method ginsenoside Rg, isolated from ginseng leaves (Panax gin preferentially adopted for the raw materials includes the fol Seng); lowing steps: FIG. 4 is a graph showing HPLC analysis results of R-type a. Raw materials from Araliaceae panax plants are added ginsenoside Rg, isolated from ginseng leaves (Panax gin 5 to a 16-fold excess of water and are heat-extracted for 3 hours, Seng); repeatedly 3 times. Herein, the raw materials of Araliaceae FIG. 5 is a graph showing HPLC analysis results of mixed panax include leaves, roots and berries of Panax ginseng and type ginsenoside Rg, isolated from leaves of American gin American ginseng (Panax quinquefolius Linn.); Seng (Panax quinquefolius Linn.); and b. The extracts obtained in step a are concentrated and FIG. 6 is a graph showing HPLC analysis results of mixed 10 mixed with a 3 to 4-fold excess of a lower alcohol. Herein, the type ginsenoside Rg, isolated from ginseng berries (Panax lower alcohol is selected from the group consisting of metha ginseng). nol, ethanol, isopropyl alcohol, amyl alcohol and any combi nation thereof; DESCRIPTION OF THE PREFERRED c. The resulting mixture is stirred to precipitate foreign EMBODIMENTS 15 materials and filtered to obtain a Supernant. The Supernant is decolorized and alcohol used as the solvent is recovered; Hereinafter, the present invention will be described in more d. A salting-out agent is added to the Solution from which detail. the solvent was removed, to a saturation concentration, fol As already known, ginsenoside Rg has a molecular for lowed by precipitation for 8 hours, thereby obtaining precipi mula of CH7O and a molecular weight of 784. Structural tates. The thus-obtained precipitates are re-dissolved in a formula I of ginsenoside Rg is as shown below: 20-fold excess of water and are subjected to gradient salting out while adding the salting-out agent again, thereby separat

ing and harvesting differential precipitates formed corre sponding to different concentrations of the salting-out agent. 25 Herein, the salting-out agent is selected from the group con sisting of sodium chloride, calcium chloride, Sodium sulfate, Sodium sulfite, ammonium sulfate, other non-toxic inorganic salts and any combination thereof; e. The precipitates, obtained from different concentrations 30 of the salting-out agent by gradient salting-out, are Subjected to thin layer chromatography analysis to separately collect ginsenoside Rg-enriched fractions and ginsenoside Rg non enriched fractions, respectively; f. The ginsenoside Rg-enriched fractions are isolated and 35 destained/decolorized using a destaining agent, followed by re-concentration. As the destaining agent, anion or cation exchange resins, adsorbent resins, fillers and the like may be employed. Upon using the ion exchange resin, precipitates of ginsenoside Rg-enriched fractions are dissolved in a 10-fold 40 excess of 95% ethanol and filtered. The resulting filtrates are diluted with an equal volume of distilled water and then destained. Where the adsorbent resin is used as the salting-out agent, precipitates of ginsenoside Rg-enriched fractions are dissolved in a 20-fold excess of water and passed through the OH 45 adsorbent resin to remove inorganic materials. This is fol OH lowed by destaining through 50% ethanol wash: g. An alcohol Such as ethanol or methanol is added to the The present invention relating to extraction of ginsenoside destained and concentrated ginsenoside Rg solution, thereby Rg represented by structural formula 1 employs gradient obtaining crude products of ginsenoside Rg, precipitating salting-out, instead of conventional silica gel column chro 50 from a point of higher than 50% concentration; matography, and thereby can be simply and conveniently h. The crude products of ginsenoside Rg are recrystallized performed and is Suited for mass production of ginsenoside under different alcohol concentrations on the basis of solu Rg. As the salting-out agent which can be utilized in the bility difference, thereby obtaining respective isomers of present invention, mention may be made of sodium chloride, high-purity ginsenoside Rg. For example, the crude products calcium chloride, Sodium sulfate, sodium sulfite, ammonium 55 of ginsenoside Rg are first dissolved in 70% alcohol and Sulfate and any other non-toxic inorganic salts or any combi allowed to stand for 12 hours at room temperature, thereby nation thereof. The salting-out agent exhibits low-toxicity, is precipitating isomeric C-20(R) ginsenoside Rg. The alcohol inexpensive, is easily available, and is recoverable thereby concentration of the resulting Supernatant is adjusted to 80%. being easily re-utilizable. This is followed by recrystallization at 4°C. for 24 hours to In addition, the method of the present invention enables 60 precipitate crystals. The crystals are added to 90% alcohol acquisition of high-purity ginsenoside Rg, even with use of and heated to 80° C. so as to dissolve crystals, followed by relatively inexpensive lower alcohols instead of conventional standing at 10° C. for 24 hours to precipitate mixed-type expensive organic solvents, and also enables obtaining of C-20(SR) ginsenoside Rg. The resulting Supernant is other ginseng total saponin (GTS) other than Rg in the allowed to stand for 12 hours at -20°C., thereby precipitating course of gradient salting-out. 65 isomeric C-20(S) ginsenoside Rg. Ginsenoside Rg, which is sought to be extracted in the As such, the extraction method in accordance with the present invention, can be extracted from raw materials of present invention requires no special equipment, is easily US 7,884, 195 B2 5 6 performed, provides high purity and yield, is harmless and early stage of dementia, enhancement of acetylcholine activ safe to humans, and therefore is suitable for industrial-scale ity has at most transient effects on patients in the early stage production. In addition, the method of the present invention of dementia development. does not affect recovery of otherginseng total Saponin (GTS). Further, the present invention provides a pharmaceutical On the other hand, glutamic acid is the most abundant composition comprising ginsenoside Rg extracted by the neurotransmitter in the brain, and thus it is expected that if a above-mentioned extraction method. The pharmaceutical glutamic acid activator is developed, therapeutic effects upon composition comprises 1 to 99% by weight of R- or S-isomer dementia thereofwill be superior to those of an acetylcholine ginsenoside Rg, or a mixture thereof, and 99 to 1% by weight activator. However, similar to the acetylcholine activator, the of a pharmaceutically acceptable carrier and adjuvant. More 10 glutamic acid activator also plays a role in enhancing inter preferably, the pharmaceutical composition comprises more action between remaining nerve cells after occurrence of than 50% by weight of ginsenoside Rg as an active ingredi nerve cell damage, rather than preventing damage of nerve ent. cells. Therefore, if the glutamic acid activator is administered The pharmaceutically acceptable carrier and adjuvant may in an excess amount, this may result in damage of nerve cells be selected from the group consisting of starch, talcum pow 15 and thus cannot serve as a Substantial treatment for dementia, der, Sugar, flavoring Substance, dextrin, medical-grade etha a primary cause of which is cell death. nol, polyethylene glycol, propylene glycol, vegetable oil, Recent study further demonstrates that damage of nerve gelatin and Tween 80 (Polyoxyethylene Sorbitan cells induced by insoluble beta-amyloid is a primary cause of Monooleate) and other surfactants. intellectual disability. Therefore, the key point for treatment More specifically, the pharmaceutical composition may of dementia is to enhance action of neurotransmitters and at further comprise a diluting agent and excipient Such as water, the same time to prevent degeneration or death of nerve cells. fillers such as starch and Sucrose; binding agents such as cellulose derivatives, alginate, gelatin and polyvinyl pyrroli Meanwhile, depression is a clinically frequent psychotic done; a Wetting agent Such as glycerin; disintegrating agents disease, and also manifests mental disorder-causing general Such as agar, calcium carbonate, and sodium hydrogen car 25 emotional symptoms. At present, etiology of depression is not bonate; an absorption promoter Such as a quaternary ammo exactly known, but decreased synthesis of the neurotransmit nium salt; a surfactant such as hexadecanol; adsorbent carri ters belonging to intracerebral monoamines is known to be ers such as kaolin and crude clay; lubricating agents such as involved in development of depression. talcum powder, calcium Stearate, magnesium and polyethyl The known anti-depressant imipramine increases concen ene. Besides these, other adjuvants such as flavoring agents 30 trations of noradrenalin (NA) and 5-hydroxytryptamine and Sweetening agents may be added as constituent Sub (5-HT) in synaptic clefts. Some species of monoamine oxi Stances. dase inhibitors also increase concentrations of intracerebral Further, in accordance with the present invention, the phar monoamine neurotransmitters. maceutical composition containing ginsenoside Rg can be formulated into injectable preparations, capsules, pills, tab 35 Currently, an animal model of depression is established on lets, nebula, oral Solutions, patches, Suppositories, microcap the basis of a monoamine neurotransmitter theory. Therefore, sules and micro balls, all of which are conventionally used in control of intracerebral monoamine neurotransmitters has Chinese and Western medicines. become a major focus in treatment of psychotic diseases or More specifically, the pharmaceutical composition may be mental disorders. applied to injectable preparations such as powder for injec 40 In compliance with Such circumstances, when ginsenoside tion and injectable solution; tablets such as dragee, chewing Rg, obtained by the extraction method of the present inven tablets and film-coated tablets; capsules such as normal and tion, was applied to the following animal experiments, gin micro capsules; nebula; Suppositories Such as anal and vagi senoside Rg exerted excellent therapeutic action on demen nal Suppositories; buccal mucous membrane patches; chew tia, exhibited significantly improved action on memory able pills; Syrups and other oral Solutions. 45 disorders caused by both insoluble beta-amyloid proteins and Meanwhile, it was confirmed through the following experi multiple cerebral infarction, controlled apoptosis of cerebral mental examples that the pharmaceutical composition pre nerve cells, inhibited expression of apoptosis-related genes/ pared in accordance with the method of the present invention proteins, and prevented loss and death of nerve cell bodies had therapeutic action on dementia, including presenile 50 while simultaneously inhibiting damage of correlated nerve dementia, typically Alzheimer and vascular dementia, typi cells caused by glutamic acid and excitatory amino acids. cally multiple infarct dementia (MID) and also had anti Further, it was also found through various other animal depressant activity and improved effects of excitation center experiments that ginsenoside Rg in accordance with the and peripheral circulation. present invention remarkably increased the concentration of By medical definition, dementia belongs to a category of 55 intracerebral monoamine neurotransmitters, thereby exerting disorders Stemming from deterioration of mental and nervous pronounced anti-depressant effects, and remarkably reduced functions. In other words, dementia is also called intellectual (perception, memory, speculation, inference and judgment, sleeping time exerted by pentobarbital Sodium, thus possess emotion, etc.) inability due to brain organic mental disorders, ing cerebral cortex-stimulating action. and is known to be caused by degeneration or death of nerve 60 cells. EXAMPLES However, use of anti-dementia drugs known hitherto pro vided slight improvement in Symptoms via enhanced action Now, the present invention will be described in more detail of a neurotransmitter, i.e., acetylcholine, but failed to prevent with reference to the following examples. These examples are underlying pathogenic causes, i.e., degeneration and death of 65 provided only for illustrating the present invention and should nerve cells. Consequently, as acetylcholinergic neurons are not be construed as limiting the scope and spirit of the present damaged and glutaminergic neurons are also damaged, at the invention. US 7,884, 195 B2 7 Example 1 TABLE 1-continued Extraction of Ginsenoside Rg from Ginseng Leaves Retention time Area (peak value x peak Theoretical (Panax ginseng) Pkhi (min) time duration) Area 90 plate First, 100 kg of ginseng leaves were added to a 16-fold 22 26.232 316288 3.713 O.OO amount of water and boiled for 3 hours. This procedure was 23 27.902 111465 1.309 O.OO repeated 3 times. The resulting liquids were combined and filtered through a filter. The filtrates were concentrated to Total 8518505 1OOOOO approximately 100 kg in a concentrator. The resulting con 10 centrates were added to 3-fold excess of 95% ethanol, stirred As can be seen from the data of Table 1 and FIG. 1, purity to precipitate foreign materials and re-filtered to remove pre of ginsenoside Rg crude products, as measured in terms of cipitated foreign materials. The thus-separated Supernant was the sum of Area % for peaks 20 and 21, with a retention time decolorized using activated charcoal and the ethanol solvent ranging from 18 to 22 min, was 75.81%. used was then recovered. 15 In addition, the thus-obtained ginsenoside Rg crude prod The Supernant, from which ethanol was removed, was dis ucts were dissolved in 70% ethanol and allowed to stand at solved in a 10-fold excess of water, and the resulting solution room temperature for 12 hours, thereby precipitating iso was Saturated with high-purity ammonium Sulfate, followed meric C-20(R) ginsenoside Rg in the form of crystals. Then, by precipitation for 8 hours. The thus-obtained precipitates the ethanol concentration of the resulting Supernatant was were re-dissolved in a 20-fold excess of water and subjected adjusted to 80%, followed by recrystallization at 4°C. for 24 to gradient salting-out with continuous addition of high-pu hours to precipitate crystals. The crystals were added to 90% rity ammonium sulfate. At 5%, 10%, 15%, 20% and 30% ethanol, heated to 80° C., and left at 10° C. for 24 hours, concentrations of ammonium sulfate in the solution, the thereby precipitating mixed-type C-20(SR) ginsenoside Rg resulting respective precipitates were collected. in the form of crystals. Then, the resulting Supernant was The precipitates collected according to different concen 25 allowed to stand at -20°C. for 12 hours, thereby precipitating trations of the salting-out agent were aliquoted and Subjected isomeric C-20(S) ginsenoside Rg as crystals. to thin layer chromatography analysis, thereby being divided The respective different isomers of ginsenoside Rg crys into ginsenoside Rg-enriched fractions (precipitates at 15% tals were finally purified, filtered under reduced pressure and and 20% concentrations of ammonium sulfate) and other spontaneously dried in the air at room temperature for 24 ginsenoside Rg non-enriched fractions (precipitates at 5%, 30 hours, thereby sequentially obtaining 20 g of isomeric C-20 10% and 30% concentrations of ammonium sulfate). Precipi (S) ginsenoside Rg, 238g of mixed-type C-20(SR) ginseno tates of ginsenoside Rg-enriched fractions were isolated and side Rg and 30 g of isomeric C-20(R) ginsenoside Rg. dissolved in a 10-fold excess of 95% ethanol and filtered to Additionally, 2100 g of ginseng Saponin was obtained in Rg obtain filtrates. The filtrates thus obtained were diluted 1-fold non-enriched fractions. with distilled water, followed by destaining and decolorizing 35 When the respective isomers of ginsenoside Rg thus via an ion exchange resin. The thus-treated filtrates were obtained were determined by a gravimetric method (ex concentrated to 1,000 ml. pressed as % mass of extracted and separated ginsenoside Rg The concentrates were added again to 95% ethanol to for 100 kg of ginseng leaves used as raw materials), yield of obtain crude products of ginsenoside Rg beginning to pre C-20(SR) ginsenoside Rg was 0.24%, yield of C-20(S) gin cipitate from the point at which the ethanol concentration in 40 senoside Rg was 0.02%, and yield of C-20(R) ginsenoside the solution rose above 50%. Purity of precipitated crude Rg was 0.03%. products of ginsenoside Rg was measured at 203 nm, via In addition, purity of the respective different isomers of high performance liquid chromatography (HPLC). The ginsenoside Rg was measured at 203 nm, via high perfor results are shown in Table 1 and FIG. 1, respectively. mance liquid chromatography (HPLC). The results are shown 45 in Tables 2 through 4 and FIGS. 2 through 4, respectively. TABLE 1. TABLE 2 Retention time Area (peak value x peak Theoretical Pkhi (min) time duration) Area 90 plate Retention time Area (peak value x peak Theoretical Pkhi (min) time duration) Area 90 plate 1 3.034 2955 O.O3S O.OO 50 2 3.377 32346 O.380 1583.78 1 3.361 2492 O.O14 2492.06 3 3.673 99726 1.171 1431.46 2 3.663 53477 O.306 2161.39 4 4.211 191479 2.248 1458.2O 3 4.177 40047 O.230 O.OO 5 4.482 125818 1477 3877.20 4 4.486 2446 O.O14 800S.15 6 4.860 8O246 O.942 1459.10 5 4.98O 1684 O.O1O 2585.79 7 5.355 301.88 O3S4 1848.53 55 6 6.693 4707 O.O27 O.OO 8 6.085 4651 0.055 O.OO 7 7.298 8494 O.049 O.OO 9 6.666 4788 O.OS6 3306. S1 8 7.682 4710 O.O27 1918.52 10 7.293 23324 O.274 1438.15 9 7.893 1724 O.O1O O.OO 11 7.739 41530 O488 2408.69 10 8.800 4037 O.O23 285.71 12 10.216 668782 7.851 2S42.60 11 8.869 2534 O.O15 O.OO 13 11.133 7692 O.O90 10971.31 12 9.08O 2565 O.O15 1393.54 14 12.139 17798 O.209 SO89.94 60 13 9.2O7 2181 O.O12 221. SO 15 13.260 193484 2.271 3968. SS 14 9.318 1710 O.O1O 1199.20 16 14.08O 11252 O.132 O.OO 15 9.703 10485 O.O60 394.82 17 14.595 61696 O.724 3420.54 16 10.164 65134 0.373 2495.55 18 16.030 21353 O.251 4112.83 17 10.967 23 O.OOO 735389.56 19 16.701 12897 O.151 2347.50 18 11.033 615 O.OO)4 O.OO 2O 18.561 3437.480 40.353 5085.97 65 19 11.091 2.472 O.O14 4875.31 21 21.269 3021268 35.467 S180.98 2O 12.096 10381 O.O59 864.65 US 7,884, 195 B2 9 10

TABLE 2-continued TABLE 4-continued

Retention time Area (peak value x peak Theoretical Retention time Area (peak value x peak Theoretical Pkhi (min) time duration) Area 90 plate Pkhi (min) time duration) Area 90 plate 21 13.2SO 115126 O660 3274.46 22 14.SS6 9969S 0.571 321861 12 25.710 30713 O.827 O.OO 23 16.O16 42O54 O.241 O.OO 13 26.805 3746 O.101 O.OO 24 16.622 42576 O.244 115.97 25 18.446 8261910 47.349 4799.02 26 21116 8629243 49.454 4757.97 10 Total 3715968. 1OOOOO 27 23.079 72 O.OOO O.OO 28 23.233 2O O.OOO 3136816.OO 29 24.3SS 9418 O.OS4 O.OO 30 25.953 9923 0.057 O.OO As can be seen from the data of Table 4 and FIG.4, purity 31 28.515 17110 O.098 O.OO of isomeric C-20(R) ginsenoside Rg, as measured interms of 15 the sum of Area % for peaks 20 and 21, with a retention time Total 17449064 1OOOOO ranging from 18 to 22 min, was 98.05%. As can be seen from the data of Table 2 and FIG. 2, purity Example 2 of mixed-type C-20(SR) ginsenoside Rg, as measured in terms of the sum of Area % for peaks 25 and 26, with a Extraction of Ginsenoside Rg from American retention time ranging from 18 to 22 min, was 96.80%. Ginseng Leaves (Panax quinquefolius Linn.) TABLE 3 75 g of mixed-type C-20(SR) ginsenoside Rg was Retention time Area (peak value x peak Theoretical 25 obtained using 50 kg of American ginseng leaves (Panax Pkhi (min) time duration) Area 90 plate quinquefolius Linn.), according to the same procedure as in 1 3.037 121 O.OO1 O.OO Example 1. In the same manner as in Example 1, yield and 2 3.220 92 O.OO1 95.38.11 purity of C-20(SR) ginsenoside Rg were determined. Yield 3 3.433 454 O.OO3 1972.74 4 3.670 323O2 O.224 2289.08 was 0.15%. Purity was 97.41%, as can be seen from the data 30 5 4.154 7107 O.049 1141.86 of Table 5 and FIG. 5 6 4.497 2088 O.O14 SSO9.86 7 4.903 7983 0.055 1492.92 8 6.230 7394 O.OS1 200341 TABLE 5 9 6.664 98.46 O.O68 1263.64 10 7.673 27398 O.190 S4.15 Retention time Area (peak value x peak Theoretical 11 7.940 15722 O.109 O.OO 35 Pkhi (min) time duration) Area 90 plate 12 10.129 166788 1157 12233 1 3.049 66979 1.007 O.OO 13 10.483 59464 O412 186.53 2 3.422 477 O.007 102O7.29 14 11.063 87894 O610 383.29 3 3.677 22110 O.332 2462.35 15 12.OOS 265257 1840 1998.89 4 4.173 904 O.O14 8237.63 16 13.094 14O3S30 9.73S 3804.62 5 4.494 14288 O.215 7087.08 17 14.433 663.230 4.600 204764 40 6 4.912 894 O.O13 O.OO 18 15.781 342779 2.378 1902.34 7 6.687 953 O.O14 4984.06 19 16.483 231848 1608 1212.25 8 7.672 1661 O.O2S 71.9144 2O 18.230 1 O305761 71.484 SOS/40 9 9.888 1653 O.O2S 10264. SO 21 20.988 S43020 3.767 3841.79 10 13.156 3551 O.OS3 O.OO 22 24.228 190829 1.324 34.66.42 11 13.903 1023 OO15 10016.99 23 25.656 45929 O.319 O.OO 45 12 18.398 219 1765 32.948 5575..19 13 21.035 4288045 64.462 SSS344 Total 14416834 1OOOOO 14 24.184 S421 O.O82 O.OO 15 25.672 29789 O448 O.OO As can be seen from the data of Table 3 and FIG. 3, purity 16 26.906 22585 O.340 O.OO of isomeric C-20(S)ginsenoside Rg, as measured interms of 50 Total 6652099 1OOOOO the sum of Area % for peaks 20 and 21, with a retention time ranging from 18 to 22 min, was 75.25%.

TABLE 4 Example 3 55 Retention time Area (peak value x peak Theoretical Extraction of Ginsenoside Rg from Ginseng Berry Pkhi (min) time duration) Area 90 plate (Panax ginseng) 1 3.222 99 O.OO3 8425.45 2 3.413 124 O.OO3 98.11.11 3 3.671 2S369 O.683 2331.91 50 kg of ginseng berry was added to Small quantities of 4 4.145 3S4 O.O10 7352.22 60 water. Fruit flesh was crushed to remove seeds and concen 5 4.479 5032 O.13S 7809.87 6 6.664 1740 O.047 5713.31 trated to prepare a syrup. In the same manner as in Example 1, 7 7.633 1231 O.O33 7793.26 125 g of mixed-type C-20(SR) ginsenoside Rg was obtained 8 14.429 2982 O.08O O.OO from the syrup. In the same manner as in Example 1, yield and 9 15.294 822 O.O22 O.OO 10 18.361 382O28 10.281 S601.45 65 purity of C-20(SR) ginsenoside Rg were determined. Yield 11 20.987 3261726 87.776 SSS4.10 was 0.25%. Purity was 98.55%, as can be seen from the data of Table 6 and FIG. 6. US 7,884, 195 B2 11 12 sive and exact response of rats. After 24 hours, the stimulus TABLE 6 times necessary for 9 to 10-time Successive and exact response of rats were recorded again as the order of memory. Retention time Area (peak value x peak Theoretical Based on the recordings thus obtained, a t-test was made Pkhi (min) time duration) Area 90 plate between respective groups. The results are shown in Table 7 1 3.2O1 122 O.OO1 12340.68 below. 2 3.666 28434 O.204 2308.68 3 4.212 2874 O.O21 2468.14 4 4.481 43S4 O.O31 6820.24 TABLE 7 5 5.017 2284 O.O16 O.OO 6 6.689 1429 O.O10 O.OO 10 Amount 7 7.233 921 O.OO7 O.OO administered Order of Order of 8 7.668 6803 O.049 5732.65 Groups Reagents (mg kg) Learning Memory 9 O.232 749 O.OOS O.OO Placebo 10.8 473 3.1O-312 10 O.313 509 O.OO)4 8705.97 Control Saline 10 27.9 +3.35AA 26.0 +4.69AA 11 O.472 592 O.OO4 13533.43 Experi- Rg(S) 7.50 15.8 6.08* 13.66.59** 12 O.860 98 O.OO1 123277.41 15 mental Rg2(R) 7.50 14.8 S.88* 12.67.89** 13 1.109 1636 O.O12 20884.91 Rg(SR) 7.50 16.8 7.45** 14.69.58** 14 1.300 157 O.OO1 O.OO Rg(SR) 1S.O 12.46.87** 3.30 2.79** 15 1431 103 O.OO1 O.OO Compara- Nimodipine O.OS 8.60 - 4.58* 4.104.80** 16 1.780 286 O.OO2 27707.96 tive 17 1873 283 O.OO2 O.OO 18 2.OOO 622 O.OO)4 O.OO (Compared with the placebo group; AAP-00 1; compared with the dementia model groups: 19 2.131 1791 O.O13 O.OO **P<0.01) 2O 2.467 77 O.OO1 238945.98 21 2.62O 77 O.OO1 287686.34 As can be seen from Table 7, upon examining learning 22 3.271 47850 O.343 4493.61 23 4.570 47079 0.337 O.OO memory ability of rats to which beta-amyloid (AB) and 24 6.009 15535 O-111 O.OO Ibotenic acid (IBA) were intracerebrally administered, 25 6.678 16021 O.115 O.OO 25 experimental groups to which respective different structures 26 8.488 6388832 45.758 S169.40 of ginsenoside Rg were administered exhibited significantly 27 21.1SO 7385339 52.895 S126.25 improved learning-memory ability, as compared to the con 28 26.08.1 7433 O.OS3 O.OO trol group exhibiting symptoms of typical beta-amyloid Total 13962290 1OOOOO dementia. 30 In addition, 24 hours after examination of learning Hereinafter, experiments on therapeutic effects of demen memory ability, the front and rear of the injected site of the tia, anti-depressant action and improving effects of excitation hippocampus CA1 region were successively sliced and Sub center and peripheral circulation were conducted, using gin jected to Nissl staining, HE staining, and chemical examina senoside Rg extracted in Examples 1 through 3. tion of immune tissues and in situ cell death, thereby observ 35 ing morphological changes of hippocampus neuron cell Experimental Example 1 Zones, necrosis, degree of cell loss, rate of cell death, and degree of expression of Bax/Bcl-2 and C-fos genes. The Therapeutic Effects of Ginsenoside Rg on Beta results are shown in Tables 8 through 11. Amyloid (AB) Dementia 40 TABLE 8

This experiment was carried out employing an Alzheimer Amount Dementia (AD) model. 70 rats were divided into 7 groups administered Length of nerve cell consisting of 10 rats each and were anesthetized with chloral Groups Reagents (mg kg) body loss (Lm) hydrate. Except for a placebo group, the remaining 6 groups 45 Control Saline 10 648.16 61.52 were rendered dementia models by injecting 4 ug of beta Experimental Rg(S) 7.50 303.21 59.00** amyloid (A3) and 1 ug of Ibotenic acid (IBA) into the Rg2(R) 7.50 283.23 69.60** hippocampus CA1 region of each rat. The placebo group was Rg(SR) 7.50 343.23 - 59.60** Rg(SR) 1S.O 275.3356.59** infused with an equal amount of physiological saline, instead Comparative Nimodipine O.OS 259.14 - 60.36* of beta-amyloid and Ibotenic acid (IBA). 50 The above-mentioned dementia model groups were Subdi (Note: Compared with the control group: **P<0.01) vided into a control group to which physiological saline was administered, four experimental groups to which ginsenoside As can be seen from Table 8, Nissl staining revealed that Rg was administered and a comparative group to which a intracerebral injection of AB and IBA caused a wide range medical dementia therapeutic agent, nimodipine was admin 55 loss of nerve cell bodies, as demonstrated by the control istered. Each group received the corresponding respective group, but obvious inhibitory effects against loss of the nerve reagents via intravenous injection for 7 days, once a day, cell bodies were observed in experimental groups to which according to the dosages set forth in Table 7 below. Herein, the respective isomers of ginsenoside Rg were administered. reagents, which are administered to experimental groups, are isomeric C-20(S) ginsenoside Rg and C-20(R) ginsenoside 60 TABLE 9 Rg, and mixed-type C-20(SR) ginsenoside Rg (in the fol Amount lowing Tables, referred to as S. R and SR, respectively). administered On day 6 of respective reagent administration, animals Groups Reagents (mg kg) Rate of nerve cell death were subjected to a Y-shaped electronic maze test. Learning Control Saline 10 SO.67 6.15 memory ability of rats was determined according to the fol 65 Experimental Rg(S) 7.50 24.805.73* lowing method. Firstly, the order of learning is taken as the Rg2(R) 7.50 23.834.62 number of stimulus times necessary for 9 to 10-time succes US 7,884, 195 B2 14 damage around sites to which an injection needle penetrated, TABLE 9-continued Small quantities of gliocyte infiltration, but intactness and no loss of pyramidal cell Zones. Amount administered On the other hand, the control group manifesting beta Groups Reagents (mg kg) Rate of nerve cell death 5 amyloid dementia symptoms exhibited large quantities of Rg(SR) 7.50 25.005.73* gliocyte infiltration, significant loss of hippocampal neurons. Rg(SR) 1S.O 23.333.988 The region of cell death extended a great distance from CA1 Comparative Nimodipine O.OS 1983 2.93* near an injection point, and thus a very long pyramidal-granu lar cell Zone was observed to be lost. Upon examination under (Note:: CCompared d with ththe control:1: *P<0.05*P<0.05) 10 a high magnificationY microscope, rupture of the neuronal cell As can be seen from Table 9, examination of in situ cell membrane, cellular atrophy, reduction of cell nuclei, and death (apoptosis) by TUNEL (Terminal deoxynucleotidyl apoptotic bodies were apparently observed. Transferase Biotin-dUTP Nick End Labeling) staining In contrast, experimental groups, to which the respective revealed that injection of Afa and IBA into the hippocam- isomers of ginsenoside Rg were administered, did not pus induced death of nerve cells, as demonstrated by the 15 exhibit- local infiltration of gliocytes, damage to pyramidal control group, but death of nerve cells was remarkably cell Zones and granular cell Zones, diminishing of cells and reduced in experimental groups to which the respective iso- loss of cell Zones. Upon examination under a high magnifi mers of ginsenoside Rg were administered. cation microscope, cell membranes were intact, apoptotic

TABLE 10 Expression Amount Expression Intensity of administered Intensity of bax bcl-2 Groups Reagents (mg kg) - + +++++ ------Placebo 1 8 1 O O 1 9 O Control Saline 10 O 1 3 6AA O & 2 OAA Experimental Rg(S) 7.50 O 7 3 O* * O 3 6 * * Rg(R) 7.50 O 6 3 18: O 2 8 O** Rg(SR) 7.50 O 7 1* * O 2 7 1* * Rg(SR) 1S.O O 8 2 O** O 1 9 O** Comparative Nimodipine O.OS O 7 3 O* * O 5 4 1* * (Note: Compared with the placebo group: AAP < 0.01; compared with the control: *P < 0.05, **P<0.01) 35 bodies were very rare or nearly non-existent in a region more TABLE 11 distant than CA1 and pathological changes were also observed to be very slight. Amount Expression Intensity In conclusion, for dementia models showing AD behavior administered of C-fos 40 and pathological changes caused by injection of beta-amyloid Groups Reagents (mg kg) ------(AB) and Ibotenic acid (IBA) mixtures into the hippoc ampus region of the rat brain, the respective isomers of gin Placebo 6 4 O O senoside Rg can significantly improve learning and memory Control Saline 10 1 4 5 OAA Experimental Rg(S) 7.50 O O 6 4** 45 ability in AD rats, as with an anti-dementia drug, nimodipine, Rg(R) 7.50 O 1 6 3* * prevent loss of nerve cell bodies, increase expression of C-fos Rg(SR) 7.50 O O 6 4** and bcl-2 in the brain, but inhibit expression of bax. There Rg(SR) 1S.O O 1 4. 5* * fore, nerve cell bodies are protected and death thereof is Comparative Nimodipine O.OS O 3 4 38 prevented. (Note: Compared with the placebo group: AAP <0.01; compared with the control: *P<0.05, 50 **P<0.01) Experimental Example 2 As can be seen from Tables 10 and 11, upon examining Therapeutic Effects of Ginsenoside Rg on Multiple expression of apoptosis-promoting regulator (Bax), apopto Infarct Dementia sis-inhibiting regulator (Bcl-2) and transcriptional regulator 55 (C-fos), the control group with beta-amyloid (AB) demen This experiment was carried out employing a multiple tia symptoms exhibited increased expression of Bax and infarct dementia (MID) model. 56 rats were divided into 7 decreased expression of Bcl-2. Whereas, experimental groups consisting of 8 rats each and were anesthetized with groups to which the respective isomers of ginsenoside Rg 10% chloral hydrate. Except for a placebo group, the remain 60 ing 6 groups were established as dementia models by inject were administered exhibited decreased expression of Bax and ing 0.13 ml/100g of a composite thrombosis-inducing agent enhanced expression of Bcl-2 and C-fos. These results dem into the carotid artery of each rat, restoring anatomical struc onstrate that the respective isomers of ginsenoside Rg have tures thereof and Suturing skins along layers. The placebo regulatory action on expression of abnormal Bax and Bcl-2 group was injected with an equal amount of physiological genes. 65 saline, instead of the composite thrombosis-inducing agent. In addition, results of HE staining showed that the placebo The above dementia model groups were subdivided into a group exhibited less damage to hippocampal neurons, slight control group to which physiological Saline was adminis US 7,884, 195 B2 15 16 tered, four experimental groups to which ginsenoside Rg was administered and a comparative group to which a medi TABLE 1.4 cal dementia therapeutic agent, nimodipine was adminis Amount tered. Each group received the corresponding respective adminis reagents via intravenous injection for 7 days, once a day, tered Number of Number of according to dosages set forth in Table 12 below. Herein, (mg Caspase-3 Calpain II reagents, which are administered to experimental groups, are Groups Reagents kg) positive cells positive cells isomeric C-20(S) ginsenoside Rg and C-20(R) ginsenoside Placebo 1.5 - 2.58 1.O. O.91 Rg, and mixed-type C-20(SR) ginsenoside Rg (in the fol Control Saline 10 33.0 + 13.01AA 13.9 +3.14AA lowing experiments, referred to as S. Rand SR, respectively). 10 Experi- Rg(S) S.O O41.12** 4.01.07** mental Rg2(R) S.O 0.22.12** 3.02.07** On day 6 of respective reagent administration, learning Rg(SR) S.O O.20.34** 3.5 + 1.09:* memory ability of animals was determined according to the Rg(SR) 1O.O 0.7.1.16** 3.01.73** same manner as in Experimental Example 1. The results are Com- Nimodipine O.OS 2.3 2.72** 3.9 0.67** shown in Table 12 below. para 15 tive TABLE 12 (Compared with the placebo group; AAP < 0.01; compared with the control group: **P< 0.01) Amount administered Order of Order of Groups Reagents (mg kg) Learning Memory From chemical examinations of immunized groups, as shown in Tables 13 and 14, it was confirmed that rats of Placebo 2.13.18 2.13.18 Control Saline 10 14+ 11.634A 7.39.52AA experimental groups to which respective different structures Experi- Rg(S) S.O 1.3 : S.S8* O.7 141* of ginsenoside Rg were administered exhibited significant mental Rg2(R) S.O 3.34.58* O.6 3.24 reduction in expression of Glu, Bax, Caspase-3, Calpain II Rg(SR) S.O 3.75.10* O.7 141* positive neurons in the CPU region, as compared with the Rg(SR) 1O.O 4.6 - 4.16* O.1 0.32 25 Compara- Nimodipine O.OS 2.53.70: O.O.O.O* control group having typical MID dementia symptoms, dem tive onstrating that respective structures of ginsenoside Rg have (Compared with the placebo group; AP < 0.01; compared with the control group: *P-0.05) regulatory action on expression of the above abnormal genes. In conclusion, for dementia models with MID behaviorand As can be seen from Table 12, upon examining and deter 30 pathological changes caused by injection of the composite mining learning-memory ability of rats, the control group thrombosis-inducing agent to rats, it was confirmed that the with typical multiple infarct dementia (MID) symptoms respective isomers of ginsenoside Rg can significantly exhibited increased order of learning and memory, but experi improve learning and memory ability in MID rats, similar to mental groups to which respective structures of ginsenoside another anti-dementia drugs, nimodipine, and Such action Rg were administered exhibited significantly decreased 35 learning and memory orders. These results demonstrate that was correlated with declined expression of Glu, Calpain II, the respective isomers of ginsenoside Rg have significantly Caspase-3 and Bax involved in protein death. improved therapeutic effects on multiple infarct dementia. In addition, 24 hours after determination of learning Experimental Example 3 memory ability, the heads of animals were dissected, and a 40 2x5 mm incision was made in the bregma, at front and rear Effects of Ginsenoside Rg on MTT Metabolism sides, respectively, and fixed with 4% paraformaldehyde, after Damage of PC12 Cells covered with paraffin and sectioned into sections having a thickness of 7 Lum using a paraffin microtome. The sections 100 ul/well of PC12 culture having a cell density of 10 were subjected to Nissl staining, and chemical tests were 45 carried out on glutamate (Glu), apoptosis promoting regulator cells/mL was inoculated into a 96-well plate and PC12 cells (Bax), an apoptosis inducer Caspase-3 and a protease Calpain were allowed to adhere to walls of the plate. Wells were II-positive neurons in a CPU region in rats of immunized divided into 6 groups to which 6 wells were assigned each. groups. The results are shown in Tables 13 and 14. A serum-free medium was added to a non-treatment group, 50 1 mmol/L of a serum-free medium containing glutamic acid TABLE 13 and an equal volume (1 mmol/L) of 20%. 12-polyethylene Amount glycol were added to a control group, 1 mmol/L of a serum adminis free medium containing glutamic acid and 0.1 mmol/L of tered Number Number of (mg of Glu- Bax-positive 55 respective structures of ginsenoside Rg were added to Groups Reagents kg) positive cells cells experimental groups, and 5 pmol of nimodipine, instead of ginsenoside Rg, was added to a comparative group. Then, Placebo 5.5 - 1.4 O.O2S O.OS Control Saline 10 16.2 + 3.43AA 10.2 + 1.48AA PC12 cells of respective groups were cultured at 37°C. for 20 Experi- Rg(S) S.O 6.53.65** 4.0 1.87** hours. mental Rg2(R) S.O 5.7 2.63** 3.41.62* 60 Rg(SR) S.O 6.1 + 2.65** 4.0 1.87** 20 ul/well of MTT (5 g/L) was added to respective groups Rg(SR) 1O.O 6.33.46* 1.7 0.978.* and cells were continuously cultured at 37° C. for 4 hours. Com- Nimodipine O.OS 11.83.89* 3.22.47:8: Then, the original medium was aspirated, and 0.1 ml/well of para tive dimethyl sulfoxide (DMSO) was added to dissolve red gran 65 ules. Next, optical density (OD) at 490 nm was measured (Compared with the placebo group; AAP <0.01; compared with the control group: P ×0.05, **P<0.01) using an enzyme-linked immuno-assay and then a t-test was made between groups. Results are shown in Table 15. US 7,884, 195 B2 18

TABLE 1.5 TABLE 16

Amount Amount administered administered Order of Order of Groups Reagents (mmol/L) MTT (OD) Groups Reagents (mg kg) Learning Memory

Non-treatment O.O94 O.O16 Placebo 5.0 - 141 3.41.19 Control Polyethylene glycol 1 0.060 + 0.010A Control Saline 10 28.3 + 2.194A 26.0 + 2.67AA Experimental Rg(S) O.1 O.O87 O.O16* Experimental Rg(S) 7.50 17.53.28** 13.8 2.98** Rg2(R) O.1 O.O83 O.O16* Rg2(R) 7.50 19.03.89** 15.83.69** Rg(SR) O.1 O.O83 OO15* 10 Rg(SR) 7.50 12.O 2.97** 8.9 2.42** Comparative Nimodipine 5 pmol 0.086 + 0.017* Rg(SR) 1S.O 10.9 2.42** 7.93.04** (Compared with the non-treatment group; AP < 0.05; compared with the control group: *P < 0.05) (Compared with the placebo group; AAP <0.01; compared with the control group: P ×0.01) As can be seen from Table 15, the control group exhibited significantly reduced MTT metabolism, confirming that In addition, 6 days after examination of learning-memory glutamic acid partially damaged PC12 cells. Whereas, in ability, heads of rats were dissected to remove the brain which comparison with the control group, it was confirmed that both was then homogenized. Contents of cerebral neurotransmit experimental and comparative groups, to which respective ters, such as norepinephrine (NE), dopamine (DA) and 5-hy structures of ginsenoside Rg and nimodipine were adminis- 20 droxytryptamine (5-HT) were determined, and thereby a tered, respectively, exhibited significantly increased MTT t-test was made between respective groups. The results are metabolism of PC12 cells, thus demonstrating that all of the shown in Table 17 below.

TABLE 17

Amount administered Groups Reagents (mg kg) NE (ng/g) DA (ng/g) 5-HT (ng/g) Placebo 1212.8 49.50 321.833.0 1911 - 33.24 Control Saline 10 783.8 + 74.25AA 1650+ 24.75AA 0.12 + 33.24AA Experimental Rg(S) 7.50 958.066. O** 298.033.O. 106.3 48.17** Rg2(R) 7.50 858.O 66.O* 198.033.O. 108.041.SS** Rg(SR) 7.50 973.549.5** 239.3 + 41.25** 116.3 58.17** Rg(SR) 1S.O 1014.8 132.08: 272.3 24.75** 157.924.93** (Compared with the placebo group; AAP- 0.01; compared with the control group: *P < 0.05, **P<0.01) respective isomeric ginsenoside Rg can inhibit damage of As can be seen from Tables 16 and 17, the control group PC12 cells affected by glutamic acid. exhibited remarkable decreases in learning-memory ability and cerebral NE and DA contents, while all of the experimen Experimental Example 4 40 tal groups, to which respective isomers of ginsenoside Rg Therapeutic Effects of Ginsenoside Rg on were administered, exhibited remarkable increases in learn Depression ing/memory ability and cerebral NE, DA and 5-HT contents. According to the monoamine theory, it can be confirmed A general model of depression adopts drug- or stress 45 that respective isomers of ginsenoside Rg have pronounced stimulation, but this experiment adopts ischemia/reperfusion ameliorating action on depression or diseases resulting from to lower contents of norepinephrine (NE), dopamine (DA) disturbance in Stimulation/suppression of central nervous and 5-hydroxytryptamine (5-HT) in brain tissues, thereby system. establishing a depression model. 48 rats were divided into 6 groups consisting of 8 rats each 50 Experimental Example 5 and were anesthetized with 10% chloral hydrate. Except for a placebo group, the remaining 5 groups were established as Effects of Ginsenoside Rg on Pentobarbital depression models as follows. A nylon line was inserted from Sodium-Induced Sleeping Time a left external carotid artery into an internal carotid artery of each rat and a middle cerebral artery (MCA) was occluded. 30 55 min after blockage of blood flow, the nylon line was removed 38 male mice were divided into three groups as set forth in and reperfusion was allowed, thereby establishing depression Table 18 below. Mixed-type ginsenoside Rg was intrave models of rats. nously administered in an amount of 10 and 20 mgkg' to Among the above depression models, 10 mg kg of physi experimental groups, respectively. At the same time, 50 ological Saline was administered to a control group, and 60 mgkg' of pentobarbital sodium was intravenously adminis respective structures of ginsenoside Rg were intravenously tered again. Whereas, for the control group, 10 mgkg' of injected into experimental groups for 5 consecutive days, 20% 1.2-polyethylene glycol was intravenously adminis once a day, according to dosages set forth in Table 16 below. tered, instead of ginsenoside Rg. Using loss of lighting reflex After 48 hours of respective reagent administration, learn as an indicator, a sleeping time of the mice belonging to the ing-memory ability of rats was determined according to the 65 respective groups was recorded and a t-test was made same manner as in Experimental Example 1. The results are between the respective groups. The results are shown in Table shown in Table 16 below. 18. US 7,884, 195 B2

microscope, each strand of the venule (V) and arteriole (A) in TABLE 18 the auricle was located. A diameter of each blood vessel was measured and state of blood flow was observed and recorded. Amount Sleeping administered Number of time Groups Reagents (mg kg) animals (n) (min) 5 Control Polyethylene 1O.O 12 17.09 - 1041 For the above groups, a control group was injected with glycol Experimental Rg(SR) 1O.O 13 9.41 S.91* physiological saline, and a comparative group was injected Rg(SR) 2O.O 13 8.39 6.55* with an injectable preparation containing 0.2 g/mL of red 10 ginseng and Big Blue Lily turf (Liriope platyphylla Wang et (Compared with the control group: *P<0.05) Tang) mixed extract. For experimental groups, respective In comparison with the control group, as shown in Table 18, all of the experimental groups, to which mixed-type gin structures of ginsenoside Rg were intravenously injected senoside Rg, was administered, exhibited great rediction in according to dosages set forth in Table 19 below. s min later, the sleeping time of mice induced by pentobarbital sodium, is 0.01 mgkg' of adrenalin was re-administered. Diameters of demonstrating that ginsenoside Rg stimulates the central blood vessels, state of blow flow and numbers of vascular nervous system (CNS). network intersection points were measured every hour. Experimental Example 6 E.EE E. I 2O Calculating percentage changes in adrenaline based on eripheral Circulation D1sorders of Auricle results thus measured, a t-test between respective groups was 68 male mice were divided into 6 groups as set forth in carried out. The results are shown in Tables 19, 20 and 21 Table 19 below and were anesthetized with urethane. Under a below.

TABLE 19

Number Amount of Changes in diameter of venous capillary administered animals vessel with respect to the time (%)

Groups Reagents (mg kg) (n) 1 min 5 min 10 min 15 min 20 min

Control Saline 1O.O 12 27.O. 14.9 27.O. 11.52 16.7 841 1S.S 7.53 11989.89 Comparative Ginseng- 10.0 x 10 11 10.02 12.41** 6.24 1543** -3.93 + 12.03*** -506 12.54*** -1.66:11.72** Big Blue Lily Turf Experimental Rg(S) 1O.O 11 14.91 - 1826 9.1510.52** S.O2 10.30° 2.25 10.39* -1.01 - 10.94 Rg2(R) 1O.O 11 1S.S1 17.26 10.151O.S2** 41.42 11.12* 3.258.39* -0.81 - 0.94* Rg(SR) 1O.O 11 13.68 - 1951 9.42 1.8.00* 4.1513.OO* 2.29 - 11.72** -0.77 - 0.72*** Rg(SR) 2O.O 12 5.249.96*** 6.30 - 12.78*** 0.42 12.13*** -0.899.63*** -1.31 + 1142*.*

(Compared with the control group: *P<0.05, **P<0.01, ***P<0.001)

TABLE 20 Amount Number of Changes in diameter of arterial capillary administered animals vessel with respect to the time (% Groups Reagents (mg kg) (n) 1 min 5 min 10 min 15 min 20 min Control Saline 1O.O 12 27.6 22.8 1917 - 12.5 15.02 12.61 15.91 - 14.14 9.86 - 15.28 Comparative Ginseng-Big 10.0 x 10 11 23.75 20.91 12.21 - 12.66 8.09 - 12.15 6.82 - 12.25 5.86 - 11.03 Blue Lily Turf Experimental Rg(S) 1O.O 11 25.6S 19.93 8.60 - 13.48** 6.75 10.24 4.57 7.18 -2.04 7.OS* Rg2(R) 1O.O 11 27.6S 18.30 9.60 - 12.40** 7.75 11.24 3.57 8.30 -1.04 6.15* Rg(SR) 1O.O 11 33.74 19.62 9.42 1.8.00 7.39 - 12.04 3.57 8.30 O.O.O.O** Rg(SR) 2O.O 12 26.65 20.23 16.88 - 14.48 6.14 - 10.73* 3.177.25* 0.753.20 * * (Compared with the control group: *P<0.05, **P<0.01, ***P<0.001)

TABLE 21 Amount Number of Changes in intersection points of peripheral administered animals circulation with respect to the time (% Group Reagents (mg kg) (n) 1 min 5 min 10 min 15 min 20 min Control Saline 1O.O 12 78.29 22:36 47.78 - 12.58 30.12 17.13 24.26 - 17.07 22.26 - 21.06 Comparative Ginseng-Big 10.0 x 10 11 33.64. 19.87*** 11.11 - 16.41*** 6.25 + 11.85*** 2.08 + 7.22*** 1.043.61** US 7,884, 195 B2 22

TABLE 21-continued Amount Number of Changes in intersection points of peripheral administered animals circulation with respect to the time (% Group Reagents (mg kg) (n) 1 min 5 min 10 min 15 min 20 min Blue Lily Turf Experimental Rg(S) 1O.O 11 33.05 - 12.02*** 4.76 7.74*** 0.13 - 0.51*** 0.561.24*** O.O.O.O** Rg2(R) 1O.O 11 31.55 11.32*** 5.769.04*** 1.13 - 150*** 1.56 2.24*** O.O.O.O** Rg(SR) 1O.O 11 27.79 20.67*** 436 8.32*** O.O.O.O*** O.O.O.O*** O.O.O.O** Rg(SR) 2O.O 12 1451 - 16.25*** 2.83 5.93*** 1.103.69*** 1929.93*** O.O.O.O** (Compared with the control group: *P < 0.05, **P<0.01, ***P<0.001)

15 As can be seen from Tables 19 through 21, experimental a degree of peripheral vasodilation and then t-test between groups, to which respective isomers of ginsenoside Rg were groups was carried out. The results are shown in Table 22 administered, exhibited significant inhibition against reduc- below.

TABLE 22

Amount administered Number of drops after administration Group Reagents (mg kg) 0-3 min 4-6 min 7-9 min Control Saline 0.675 8 69.7 14.76 2.O. 1.51 -29 4.25 -3.3 4.03 Comparative Ginseng-Big 250 8 63.O. 11.9 44.O. 12.24*** 24.O 7.80*** 1748.07*** Blue Lily Turf Experimental Rg(S) O.63 8 67.4 18.01. 16.1 8.84*** 8.54.07*** 5.05.21** Rg(R) 1.25 9 68.6 16.8S 14.17.04*** 5.8 6.70** 1.84.92* (Compared with the control group: *P < 0.05, **P<0.01, ***P<0.001) tion of blood vessel diameter of the arterioles and venules of As can be seen from Table 22, experimental groups, to mouse auricles caused by adrenalin and reduction of the which mixed-type ginsenoside Rg was administered, dis number of vascular network intersection points, as compared 35 played significant increase in the number of perfusion drops with the control group. after administration of Rg, as compared to the control group, Additionally, as compared to the control group to which the demonstrating that mixed-type ginsenoside Rg has vasodi preparation containing a mixture of redginseng and Big Blue lating action on peripheral vessels. Lily turf was injected, the ginsenoside Rg-administered experimental groups displayed a much less decrease in terms 40 INDUSTRIAL APPLICABILITY of a degree of reduction in blood vessel diameters and a degree of reduction in the number of vascular network inter As disclosed from the foregoing, a method of extracting section points, with respect to the passage of time. In particu ginsenoside Rg in accordance with the present invention lar, inhibitory action against reduction in the blood vessel employs gradient salting-out Substituting conventional silica diameter of Venules was much stronger than in arterioles. gel column chromatography separation, and therefore is Suit able for mass production of Rg due to easy and convenient Experimental Example 7 operation and manipulation, enables extraction of high-purity finished products even with use of relatively cheap lower Effects of Ginsenoside Rg on Ear Perfusion of 50 alkane alcohol instead of expensive mixed organic Solvents, Rabbit and thereby provides effects such as low production costs, harmlessness, safety and no influence on recovery of other 33 white rabbits were divided into 4 groups as set forth in ginseng Saponins from the remaining materials. Table 22 below and were anesthetized with pentobarbital In addition, the present invention reveals that respective sodium. Arteries of the ear roots were stripped and tubes were 55 isomers of ginsenoside Rg have significantly improved inserted therein. The remaining blood was washed with a action on memory disorders of animals with dementia caused Locke solution and the rabbit ears were dissected, followed by insoluble beta-amyloid proteins and multiple cerebral inf by perfusion. arction, can control expression of genes or proteins involved For the above groups, a control group was injected with in apoptosis/anti-apoptosis of cerebral neurons, prevent loss physiological saline, and a comparative group was injected 60 and death of neurons and inhibit damage of PC12 cells caused with an injectable preparation containing 0.2 g/mL of red by glutamic acid. ginseng and Big Blue Lily turf (Liriope platyphylla Wang et In addition, the present invention has confirmed that Tang) mixed extract. For experimental groups, mixed-type respective isomers of ginsenoside Rg significantly increase ginsenoside Rg was administered via the tube according to concentrations of monoamine neurotransmitters such as dosages set forth in Table 22 below. Before administration of 65 intracerebral NE, 5-HT and DA in cerebral blood deficit reagents and at intervals of 3 min after administration, reperfusion rats, and ginsenoside Rg has distinctive anti changes in the number of perfusion drops were expressed as depressant action according to a monoamine theory. Addi US 7,884, 195 B2 23 24 tionally, ginsenoside Rg significantly reduces pentobarbital 3. The method according to claim 2, wherein the lower Sodium-induced sleeping time, demonstrating that ginsenos alcohol is selected from the group consisting of methyl alco ide Rg exerts stimulatory effects upon the cerebral cortex. hol, ethyl alcohol, isopropyl alcohol, amyl alcohol and any Further, the present invention demonstrates that respective combination thereof. isomers of ginsenoside Rg have significantly improved 4. The method according to claim 3, wherein the salting effects on peripheral circulation disorders of the mouse auricle and increasing effects of perfusion rate in rabbit ears, out agent is selected from the group consisting of sodium representing that ginsenoside Rg has vasodilating action on chloride, sodium sulfate, sodium sulfite, ammonium sulfate, peripheral vessels. other non-toxic inorganic salts and any combination thereof. Although the preferred embodiments of the present inven 10 5. The method according to claim 4, wherein separating tion have been disclosed for illustrative purposes, those ginsenoside Rg-enriched fractions and ginsenoside Rg non skilled in the art will appreciate that various modifications, enriched fractions, from the precipitates, is carried out by thin additions and Substitutions are possible, without departing layer chromatography analysis. from the scope and spirit of the invention as disclosed in the 6. The method according to claim 5, wherein the destaining accompanying claims. 15 agent is selected from the group consisting of an anion The invention claimed is: exchange resin, a cation exchange resin, an adsorbent resin 1. A method of extractingginsenoside Rg from leaves and and a filler. berries of Araliaceae panax and American ginseng (Panax quinquefolius Linn.) plants, comprising: 7. The method according to claim 6, wherein the recrystal Subjecting the raw material to hot-water extraction and lizing comprises a first precipitation and a second precipita concentration to obtain concentrates, adding a lower tion, wherein the first precipitation comprise adding 95% alcohol to the concentrates to precipitate and remove lower alcohol to the ginsenoside Rg-concentrates, and foreign materials; wherein the second precipitation comprises adding a different decolorizing the remaining Supernatant and recovering the concentration of a lower alcohol compared to the first pre lower alcohol; 25 cipitation to obtain respective different crystals compared to adding a salting-out agent to a saturation concentration so the first precipitation. as to obtain precipitates and re-dissolving the precipi 8. The method according to claim 7, wherein the second tates in water, followed by gradient salting-out to obtain precipitating step is carried out by dissolving the first precipi differential precipitates corresponding to different con tated crystals in 70% alcohol and allowing to stand for 12 centrations of the salting-out agent; 30 hours at room temperature, so as to precipitate isomeric C-20 analyzing to divide the precipitates into ginsenoside Rg (R) ginsenoside Rg, adjusting the alcohol concentration of enriched fractions and ginsenoside Rg non-enriched the resulting supernant to 80%, followed by recrystallization fractions; destaining and decolorizing the ginsenoside Rg-enriched at 4°C. for 24 hours to precipitate crystals, adding the result fractions with a destaining agent, followed by concen 35 ing crystals to 90% alcohol and heating the mixture to 80°C. tration to obtain concentrates; and so as to dissolve crystals, followed by standing at 10°C. for 24 adding the lower alcohol to re-crystallize the resulting hours to precipitate mixed-type C-20(SR) ginsenoside Rg, concentrates, thereby obtaining ginsenoside Rg. and allowing the resulting Supernant to stand for 12 hours at 2. The method according to claim 1, wherein hot-water -20°C., thereby precipitating isomeric C-20(S) ginsenoside extraction includes adding the raw material from Araliaceae 40 Rg2. panax to a 16-fold excess of water and heat-extracting for 3 hours, repeatedly, 3 times. UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION PATENT NO. : 7,884, 195 B2 Page 1 of 1 APPLICATIONNO. : 1 1/912804 DATED : February 8, 2011 INVENTOR(S) : Long Yun Li It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:

On the title page, Item (73), the second Assignee's name is incorrect. Item (73) should read:

-- (73) Assignee: Long Yun Li, Changchun (CN); Boong-Kyung Ko, Seoul (KR) --

Signed and Sealed this Twelfth Day of April, 2011

David J. Kappos Director of the United States Patent and Trademark Office