WO2006115307A1

WO2006115307A1 - Method of extracting ginsengnoside rg2m, pharmaceutical composition including ginsengnoside rg2, and uses thereof

Info

Publication number: WO2006115307A1
Application number: PCT/KR2005/001204
Authority: WO
Inventors: Long Yun Li
Original assignee: Ko, Boong-Kyung; Academy Of Traditional Chinese Medical Science And Medicine Of Jilin Province
Priority date: 2005-04-26
Filing date: 2005-04-26
Publication date: 2006-11-02
Also published as: US7884195B2; JP4773512B2; JP2008539225A; EP1895966A4; EP1895966A1; US20080227760A1

Abstract

The present invention provides a method of extracting ginsenoside Rg2, which is applied to mass production. The method of the present invention employs gradient salting-out instead of conventional silica gel chromatography, and therefore enables convenient performance and mass production of ginsenoside Rg2. Provided are also a pharmaceutical composition comprising ginsenoside Rg2 as an active ingredient, and prophylactic and therapeutic agents for preventing and treating dementia, depression, peripheral circulation disorder and related diseases, utilizing the pharmaceutical composition.

Description

METHOD OF EXTRACTING GINSENGNOSIDE Rg2, PHARMACEUTICAL COMPOSITION INCLUDING GINSENGNOSIDE Rg2, AND USES THEREOF

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to a method of extracting ginsenoside Rg₂, a pharmaceutical composition comprising ginsenoside Rg₂ as an active ingredient, and prophylactic and therapeutic agents for preventing and treating dementia, depression, peripheral circulation disorder, and related diseases, utilizing the same.

Description of the Related Art Ginseng contains various ginsenosides (ginseng saponins) and there are around 30 kinds of ginsenosides which have been known hitherto. Among these, ginsenoside Rg₂ is a substance belonging to protopanaxatriol glycoside which was isolated and named a long time ago.

It is known through previous research and study that, in the course of red ginseng processing, a glycosidic bond at the C-20 position of ginsenoside Rg₂ is readily susceptible to hydrolysis and at the same time, hydroxyl radicals lead to anti-equilibrium, i.e., breakdown of an equilibrium state, thereby forming a ginsenoside isomer C-20(S) or C-20(R). In addition, it is generally published that red ginseng contains mixed-type ginsenoside Rg₂ C-20(RS) in a substantially higher content than fresh ginseng or white ginseng. Meanwhile, since medical use of such ginsenoside Rg₂ is as yet unknown, extracting and separating Rg₂ has been conventionally carried out, on a laboratory-scale, by extracting ginseng total saponin (GTS) with an organic solvent, and separating ginsenoside Rg₂ via silica gel column chromatography or high performance liquid chromatography (HPLC).

Firstly, a silica gel column chromatography process refers to a method involving adsorbing the ginseng total saponin extracts on a silica gel chromatography column, and separating ginsenoside Rg₂ therefrom using the organic solvent, in which chloroform, methyl alcohol, ethyl acetate and water are mixed in a predetermined ratio, as a washing agent.

In this connection, the separation method utilizing the above-mentioned silica gel column chromatography mainly suffers from disadvantages such as trace amounts of substance of interest separated from the column, very slow separation speed, a need for an additional separation process due to use of a large quantity of harmful organic solvents, and being unsuitable for large scale production due to high production costs, low yield and unsafety.

Whereas, high performance liquid chromatography (HPLC) enables separation of high-purity ginsenoside Rg₂, but presents problems such as high equipment and operation costs and increased production costs associated with use of a large quantity of expensive organic solvents such as methyl alcohol or acetonitrile. Consequently, conventional methods of separating ginsenoside Rg₂ are not suitable for mass production of Rg₂, thus making it expensive and difficult to enter practical commercial products.

That is, a process suitable for industrial-scale production is preferred to satisfy requirements such as simple and convenient manipulation and processes, easy practical application, high yield and purity, and harmlessness and safety to humans. In spite of such circumstances, research into such a desired process is insignificant.

Meanwhile, there are few published studies and investigations into medicinal usage of ginsenoside Rg₂ and application thereof. Even though the present inventors have proposed medical use of ginsenoside Rg₂, in Chinese

Patent No. ZLOl 102117.9, entitled "Application of ginsenoside Rg₂ in preparation of a therapeutic drug for cardio- and cerebrovascular diseases

(CCVD), use of this therapeutic drug is restricted only to treatment of cardio- and cerebrovascular diseases such as myocardial ischemia, cerebral ischemia and shock diseases. As such, there is a need in the art for more advanced and improved research into new medical uses of ginsenoside Rg₂.

SUMMARY OF THE INVENTION

Therefore, the present invention has been made in view of the above problems, and it is an object of the present invention to provide a novel method of extracting ginsenoside Rg₂, suitable for industrial-scale production.

It is another object of the present invention to provide a pharmaceutical composition comprising ginsenoside Rg₂.

It is a further object of the present invention to provide a therapeutic agent comprising ginsenoside Rg₂, for preventing or treating dementia, depression, peripheral circulation disorder and related diseases. In accordance with an aspect of the present invention, the above and other objects can be accomplished by the provision of a method of extracting ginsenoside Rg₂ from a raw material of Araliaceae panax, comprising: subjecting the raw material to hot water extraction and concentration to obtain concentrates, adding a lower alcohol to the concentrates to precipitate and remove foreign materials; decolorizing the remaining supernant and recovering the lower alcohol; adding a salting-out agent to a saturation concentration so as to obtain precipitates and re-dissolving the precipitates in water, followed by gradient salting-out to obtain differential precipitates corresponding to different concentrations of the salting-out agent; analyzing to divide the precipitates into ginsenoside Rg₂-enriched fractions and ginsenoside Rg₂ non-enriched fractions; destaining and decolorizing the ginsenoside Rg₂-enriched fractions with a destaining agent, followed by concentration to obtain concentrates; and adding the lower alcohol to re-crystallize the resulting concentrates, thereby obtaining ginsenoside Rg₂.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

Fig. 1 is a graph showing HPLC analysis results of a crude product of ginsenoside Rg₂, isolated from ginseng leaves (Panax ginseng); Fig. 2 is a graph showing HPLC analysis results of mixed-type ginsenoside Rg₂, isolated from ginseng leaves {Panax ginseng);

Fig. 3 is a graph showing HPLC analysis results of S-type ginsenoside Rg₂, isolated from ginseng leaves {Panax ginseng); Fig. 4 is a graph showing HPLC analysis results of R-type ginsenoside

Rg₂, isolated from ginseng leaves {Panax ginseng);

Fig. 5 is a graph showing HPLC analysis results of mixed-type ginsenoside Rg₂, isolated from leaves of American ginseng {Panax quinquefolius Linn.); and Fig. 6 is a graph showing HPLC analysis results of mixed-type ginsenoside Rg₂, isolated from ginseng berries {Panax ginseng).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Hereinafter, the present invention will be described in more detail.

As already known, ginsenoside Rg₂ has a molecular formula of C₄₂H₇₂Oi₃ and a molecular weight of 784. Structural formula 1 of ginsenoside Rg₂ is as shown below:

The present invention relating to extraction of ginsenoside Rg₂ represented by structural formula 1 employs gradient salting-out, instead of conventional silica gel column chromatography, and thereby can be simply and conveniently performed and is suited for mass production of ginsenoside Rg₂. As the salting-out agent which can be utilized in the present invention, mention may be made of sodium chloride, calcium chloride, sodium sulfate, sodium sulfite, ammonium sulfate and any other non-toxic inorganic salts or any combination thereof. The salting-out agent exhibits low-toxicity, is inexpensive, is easily available, and is recoverable thereby being easily re- utilizable.

In addition, the method of the present invention enables acquisition of high-purity ginsenoside Rg₂, even with use of relatively inexpensive lower alcohols instead of conventional expensive organic solvents, and also enables obtaining of other ginseng total saponin (GTS) other than Rg₂, in the course of gradient salting-out.

Ginsenoside Rg₂, which is sought to be extracted in the present invention, can be extracted from raw materials of Araliaceae panax plants as described above, and the method preferentially adopted for the raw materials includes the following steps: a. Raw materials from Araliaceae panax plants are added to a 16-fold excess of water and are heat-extracted for 3 hours, repeatedly 3 times. Herein, the raw materials of Araliaceae panax include leaves, roots and berries of Panax ginseng and American ginseng {Panax quinquefolius Linn.); b. The extracts obtained in step a are concentrated and mixed with a 3 to 4-fold excess of a lower alcohol. Herein, the lower alcohol is selected from the group consisting of methanol, ethanol, isopropyl alcohol, amyl alcohol and any combination thereof; c. The resulting mixture is stirred to precipitate foreign materials and filtered to obtain a supernant. The supernant is decolorized and alcohol used as the solvent is recovered; d. A salting-out agent is added to the solution from which the solvent was removed, to a saturation concentration, followed by precipitation for 8 hours, thereby obtaining precipitates. The thus-obtained precipitates are re-dissolved in a 20-fold excess of water and are subjected to gradient salting-out while adding the salting-out agent again, thereby separating and harvesting differential precipitates formed corresponding to different concentrations of the salting-out agent. Herein, the salting-out agent is selected from the group consisting 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 of the salting-out agent by gradient salting-out, are subjected to thin layer chromatography analysis to separately collect ginsenoside Rg₂-enriched fractions and ginsenoside Rg₂ non-enriched fractions, respectively; f. The ginsenoside Rg₂-enriched fractions are isolated and 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 Rg₂-enriched fractions are dissolved in a 10-fold 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 Rg₂-enriched fractions are dissolved in a 20-fold excess of water and passed through the adsorbent resin to remove inorganic materials. This is followed by destaining through 50% ethanol wash; g. An alcohol such as ethanol or methanol is added to the destained and concentrated ginsenoside Rg₂ solution, thereby obtaining crude products of ginsenoside Rg₂, precipitating from a point of higher than 50% concentration; h. The crude products of ginsenoside Rg₂ are recrystallized under different alcohol concentrations on the basis of solubility difference, thereby obtaining respective isomers of high-purity ginsenoside Rg₂. For example, the crude products of ginsenoside Rg₂ are first dissolved in 70% alcohol and allowed to stand for 12 hours at room temperature, thereby precipitating isomeric C-20(R) ginsenoside Rg₂. The alcohol concentration of the resulting supernatant is adjusted to 80%. This is followed by recrystallization at 4 ^"C for 24 hours to precipitate crystals. The crystals are added to 90% alcohol and heated to 80 ^°C so as to dissolve crystals, followed by standing at 10 ^°C for 24 hours to precipitate mixed-type C-20(SR) ginsenoside Rg₂. The resulting supernant is allowed to stand for 12 hours at -20 ^°C , thereby precipitating isomeric C-20(S) ginsenoside Rg₂. As such, the extraction method in accordance with the present invention requires no special equipment, is easily performed, provides high purity and yield, is harmless and safe to humans, and therefore is suitable for industrial- scale production. In addition, the method of the present invention does not affect recovery of other ginseng total saponin (GTS). Further, the present invention provides a pharmaceutical composition comprising ginsenoside Rg₂ extracted by the above-mentioned extraction method. The pharmaceutical composition comprises 1 to 99% by weight of

R- or S-isomer ginsenoside Rg₂, or a mixture thereof, and 99 to 1% by weight of a pharmaceutically acceptable carrier and adjuvant. More preferably, the pharmaceutical composition comprises more than 50% by weight of ginsenoside Rg₂ as an active ingredient.

The pharmaceutically acceptable carrier and adjuvant may be selected from the group consisting of starch, talcum powder, sugar, flavoring substance, dextrin, medical-grade ethanol, polyethylene glycol, propylene glycol, vegetable oil, gelatin and Tween 80 (Polyoxyethylene Sorbitan Monooleate) and other surfactants.

More specifically, the pharmaceutical composition may further comprise a diluting agent and excipient such as water; fillers such as starch and sucrose; binding agents such as cellulose derivatives, alginate, gelatin and polyvinyl pyrrolidone; a wetting agent such as glycerin; disintegrating agents such as agar, calcium carbonate, and sodium hydrogen carbonate; an absorption promoter such as a quaternary ammonium salt; a surfactant such as hexadecanol; adsorbent carriers such as kaolin and crude clay; lubricating agents such as talcum powder, calcium stearate, magnesium and polyethylene. Besides these, other adjuvants such as flavoring agents and sweetening agents may be added as constituent substances.

Further, in accordance with the present invention, the pharmaceutical composition containing ginsenoside Rg₂ can be formulated into injectable preparations, capsules, pills, tablets, nebula, oral solutions, patches, suppositories, microcapsules and micro balls, all of which are conventionally used in Chinese and Western medicines.

More specifically, the pharmaceutical composition may be applied to injectable preparations such as powder for injection and injectable solution; tablets such as dragee, chewing tablets and film-coated tablets; capsules such as normal and micro capsules; nebula; suppositories such as anal and vaginal suppositories; buccal mucous membrane patches; chewable pills; syrups and other oral solutions.

Meanwhile, it was confirmed through the following experimental examples that the pharmaceutical composition prepared in accordance with the method of the present invention had therapeutic action on dementia, including presenile dementia, typically Alzheimer and vascular dementia, typically multiple infarct dementia (MID) and also had anti-depressant activity and improved effects of excitation center and peripheral circulation. By medical definition, dementia belongs to a category of disorders stemming from deterioration of mental and nervous functions. In other words, dementia is also called intellectual (perception, memory, speculation, inference and judgment, emotion, etc.) inability due to brain organic mental disorders, and is known to be caused by degeneration or death of nerve cells. However, use of anti-dementia drugs known hitherto provided slight improvement in symptoms via enhanced action of a neurotransmitter, i.e., acetylcholine, but failed to prevent underlying pathogenic causes, i.e., degeneration and death of nerve cells. Consequently, as acetylcholinergic neurons are damaged and glutaminergic neurons are also damaged, at the early stage of dementia, enhancement of acetylcholine activity has at most transient effects on patients in the early stage of dementia development.

On the other hand, glutamic acid is the most abundant neurotransmitter in the brain, and thus it is expected that if a glutamic acid activator is developed, therapeutic effects upon dementia thereof will be superior to those of an acetylcholine activator. However, similar to the acetylcholine activator, the glutamic acid activator also plays a role in enhancing interaction between remaining nerve cells after occurrence of nerve cell damage, rather than preventing damage of nerve cells. Therefore, if the glutamic acid activator is administered in an excess amount, this may result in damage of nerve cells and thus cannot serve as a substantial treatment for dementia, a primary cause of which is cell death.

Recent study further demonstrates that damage of nerve cells induced by insoluble beta-amyloid is a primary cause of intellectual disability. Therefore, the key point for treatment of dementia is to enhance action of neurotransmitters and at the same time to prevent degeneration or death of nerve cells.

Meanwhile, depression is a clinically frequent psychotic disease, and also manifests mental disorder-causing general emotional symptoms. At present, etiology of depression is not exactly known, but decreased synthesis of the neurotransmitters belonging to intracerebral monoamines is known to be involved in development of depression.

The known anti-depressant imipramine increases concentrations of noradrenalin (NA) and 5-hydroxytryptamine (5-HT) in synaptic clefts. Some species of monoamine oxidase inhibitors also increase concentrations of intracerebral monoamine neurotransmitters.

Currently, an animal model of depression is established on the basis of a monoamine neurotransmitter theory. Therefore, control of intracerebral monoamine neurotransmitters has become a major focus in treatment of psychotic diseases or mental disorders.

In compliance with such circumstances, when ginsenoside Rg₂, obtained by the extraction method of the present invention, was applied to the following animal experiments, ginsenoside Rg₂ exerted excellent therapeutic action on dementia, exhibited significantly improved action on memory disorders caused by both insoluble beta-amyloid proteins and multiple cerebral infarction, controlled apoptosis of cerebral nerve cells, inhibited expression of apoptosis- related genes/proteins, and prevented loss and death of nerve cell bodies while simultaneously inhibiting damage of correlated nerve cells caused by glutamic acid and excitatory amino acids.

Further, it was also found through various other animal experiments that ginsenoside Rg₂ in accordance with the present invention remarkably increased the concentration of intracerebral monoamine neurotransmitters, thereby exerting pronounced anti-depressant effects, and remarkably reduced sleeping time exerted by pentobarbital sodium, thus possessing cerebral cortex-stimulating action.

EXAMPLES Now, the present invention will be described in more detail with reference to the following examples. These examples are provided only for illustrating the present invention and should not be construed as limiting the scope and spirit of the present invention.

Example 1

- Extraction of ginsenoside Rg₂ from ginseng leaves {Panax ginseng)

First, 100 kg of ginseng leaves were added to a 16-fold amount of water and boiled for 3 hours. This procedure was repeated 3 times. The resulting liquids were combined and filtered through a filter. The filtrates were concentrated to approximately 100 kg in a concentrator. The resulting concentrates were added to 3-fold excess of 95% ethanol, stirred to precipitate foreign materials and re-filtered to remove precipitated foreign materials. The thus-separated supernant was decolorized using activated charcoal and the ethanol solvent used was then recovered.

The supernant, from which ethanol was removed, was dissolved in a 10- fold excess of water, and the resulting solution was saturated with high-purity ammonium sulfate, followed by precipitation for 8 hours. The thus-obtained precipitates were re-dissolved in a 20-fold excess of water and subjected to gradient salting-out with continuous addition of high-purity ammonium sulfate. At 5%, 10%, 15%, 20% and 30% concentrations of ammonium sulfate in the solution, the resulting respective precipitates were collected. The precipitates collected according to different concentrations of the salting-out agent were aliquoted and subjected to thin layer chromatography analysis, thereby being divided into ginsenoside Rg₂-enriched fractions (precipitates at 15% and 20% concentrations of ammonium sulfate) and other ginsenoside Rg₂ non-enriched fractions (precipitates at 5%, 10% and 30% concentrations of ammonium sulfate). Precipitates of ginsenoside Rg₂- enriched fractions were isolated and dissolved in a 10-fold excess of 95% ethanol and filtered to obtain filtrates. The filtrates thus obtained were diluted 1-fold with distilled water, followed by destaining and decolorizing via an ion exchange resin. The thus-treated filtrates were concentrated to 1,000 ml.

The concentrates were added again to 95% ethanol to obtain crude products of ginsenoside Rg₂ - beginning to precipitate from the point at which the ethanol concentration in the solution rose above 50%. Purity of precipitated crude products of ginsenoside Rg₂ was measured at 203 nm, via high performance liquid chromatography (HPLC). The results are shown in Table 1 and Fig. 1 , respectively.

Table 1

As can be seen from the data of Table 1 and Fig. 1, purity of ginsenoside Rg₂ crude products, as measured in terms of the sum of Area % for peaks 20 and 21, with a retention time ranging from 18 to 22 min, was 75.81%. In addition, the thus-obtained ginsenoside Rg₂ crude products were dissolved in 70% ethanol and allowed to stand at room temperature for 12 hours, thereby precipitating isomeric C-20(R) ginsenoside Rg₂ in the form of crystals. Then, the ethanol concentration of the resulting supernatant was adjusted to 80%, followed by recrystallization at 4^°C for 24 hours to precipitate crystals. The crystals were added to 90% ethanol, heated to 80⁰C , and left at 10 ^°C for 24 hours, thereby precipitating mixed-type C-20(SR) ginsenoside Rg₂ in the form of crystals. Then, the resulting supernant was allowed to stand at - 20 ^°C for 12 hours, thereby precipitating isomeric C-20(S) ginsenoside Rg₂ as crystals.

The respective different isomers of ginsenoside Rg₂ crystals were finally purified, filtered under reduced pressure and spontaneously dried in the air at room temperature for 24 hours, thereby sequentially obtaining 20 g of isomeric C-20(S) ginsenoside Rg₂, 238 g of mixed-type C-20(SR) ginsenoside Rg₂ and 30 g of isomeric C-20(R) ginsenoside Rg₂. Additionally, 2100 g of ginseng saponin was obtained in Rg₂ non-enriched fractions.

When the respective isomers of ginsenoside Rg₂ thus obtained were determined by a gravimetric method (expressed as % mass of extracted and separated ginsenoside Rg₂ for 100 kg of ginseng leaves used as raw materials), yield of C-20(SR) ginsenoside Rg₂ was 0.24%, yield of C-20(S) ginsenoside Rg₂ was 0.02%, and yield of C-20(R) ginsenoside Rg₂ was 0.03%.

In addition, purity of the respective different isomers of ginsenoside Rg₂ was measured at 203 nm, via high performance liquid chromatography (HPLC). The results are shown in Tables 2 through 4 and Figs. 2 through 4, respectively. Table 2

As can be seen from the data of Table 2 and Fig. 2, purity of mixed- type C-20(SR) ginsenoside Rg₂, as measured in terms of the sum of Area % for peaks 25 and 26, with a retention time ranging from 18 to 22 min, was 96.80%.

Table 3

As can be seen from the data of Table 3 and Fig. 3, purity of isomeric C-20(S) ginsenoside Rg₂, as measured in terms of the sum of Area % for peaks 20 and 21, with a retention time ranging from 18 to 22 min, was 75.25%. Table 4

As can be seen from the data of Table 4 and Fig. 4, purity of isomeric C-20(R) ginsenoside Rg₂, as measured in terms of the sum of Area % for peaks 20 and 21, with a retention time ranging from 18 to 22 min, was 98.05%.

Example 2 - Extraction of ginsenoside Rg₂ from American ginseng leaves {Panax quinquefolius Linn.)

75 g of mixed-type C-20(SR) ginsenoside Rg₂ was obtained using 50 kg of American ginseng leaves {Panax quinquefolius Linn.), according to the same procedure as in Example 1. In the same manner as in Example 1, yield and purity of C-20(SR) ginsenoside Rg₂ were determined. Yield was 0.15%. Purity was 97.41%, as can be seen from the data of Table 5 and Fig. 5

Table 5

Example 3

- Extraction of ginsenoside Rg₂ from ginseng berry {Panax ginseng)

50 kg of ginseng berry was added to small quantities of water. Fruit flesh was crushed to remove seeds and concentrated to prepare a syrup. In the same manner as in Example 1, 125 g of mixed-type C-20(SR) ginsenoside Rg₂ was obtained from the syrup. In the same manner as in Example 1, yield and purity of C-20(SR) ginsenoside Rg₂ were determined. Yield was 0.25%. Purity was 98.55%, as can be seen from the data of Table 6 and Fig. 6.

Table 6

Hereinafter, experiments on therapeutic effects of dementia, antidepressant action and improving effects of excitation center and peripheral circulation were conducted, using ginsenoside Rg₂ extracted in Examples 1 through 3.

Experimental Example 1

- Therapeutic effects of ginsenoside Rg₂ on beta-amyloid (Aβi._4O) dementia

This experiment was carried out employing an Alzheimer Dementia (AD) model. 70 rats were divided into 7 groups consisting of 10 rats each and were anesthetized with chloral hydrate. Except for a placebo group, the remaining 6 groups were rendered dementia models by injecting 4 μg of beta- amyloid (Aβi._4O) and 1 μg of Ibotenic acid (IBA) into the hippocampus CAl region of each rat. The placebo group was infused with an equal amount of physiological saline, instead of beta-amyloid and Ibotenic acid (IBA). The above-mentioned dementia model groups were subdivided into a control group to which physiological saline was administered, four experimental groups to which ginsenoside Rg₂ was administered and a comparative group to which a medical dementia therapeutic agent, nimodipine was administered. Each group received the corresponding respective reagents via intravenous injection for 7 days, once a day, according to the dosages set forth in Table 7 below. Herein, reagents, which are administered to experimental groups, are isomeric C-20(S) ginsenoside Rg₂ and C-20(R) ginsenoside Rg₂, and mixed- type C-20(SR) ginsenoside Rg₂ (in the following Tables, referred to as S, R and SR, respectively).

On day 6 of respective reagent administration, animals were subjected to a Y-shaped electronic maze test. Learning-memory ability of rats was determined according to the following method. Firstly, the order of learning is taken as the number of stimulus times necessary for 9 to 10-time successive and exact response of rats. After 24 hours, the stimulus times necessary for 9 to 10- time successive and exact response of rats were recorded again as the order of memory. Based on the recordings thus obtained, a t-test was made between respective groups. The results are shown in Table 7 below.

Table 7

Comparative Nimodipine 0.05 8.60+4.58 4.10±4.80

(Compared with the placebo group; ^AAP<0.01; compared with the dementia model groups: ^**P<0.01)

As can be seen from Table 7, upon examining learning-memory ability of rats to which beta-amyloid (Aβ_1-40) and Ibotenic acid (IBA) were intracerebrally administered, experimental groups to which respective different structures of ginsenoside Rg₂ were administered exhibited significantly improved learning-memory ability, as compared to the control group exhibiting symptoms of typical beta-amyloid dementia.

In addition, 24 hours after examination of learning-memory ability, the front and rear of the injected site of the hippocampus CAl region were successively sliced and subjected to Nissl staining, HE staining, and chemical examination of immune tissues and in situ cell death, thereby observing morphological changes of hippocampus neuron cell zones, necrosis, degree of cell loss, rate of cell death, and degree of expression of Bax/Bcl-2 and C-fos genes. The results are shown in Tables 8 through 11.

Table 8

(Note: Compared with the control group: P<0.01)

As can be seen from Table 8, Nissl staining revealed that intracerebral injection of Aβj.₄o and IBA caused a wide range loss of nerve cell bodies, as demonstrated by the control group, but obvious inhibitory effects against loss of the nerve cell bodies were observed in experimental groups to which the respective isomers of ginsenoside Rg₂ were administered.

Table 9

(Note: Compared with the control: ^*P<0.05) As can be seen from Table 9, examination of in situ cell death

(apoptosis) by TUNEL (Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling) staining revealed that injection of Ap_1-40 and IBA into the hippocampus induced death of nerve cells, as demonstrated by the control group, but death of nerve cells was remarkably reduced in experimental groups to which the respective isomers of ginsenoside Rg₂ were administered.

Table 10

(Note: Compared with the placebo group: ^{A Λ}P<0.01; compared with the control: ^*P<0.05, ^**P<0.01)

Table 11

(Note: Compared with the placebo group: ^ΛΛP<0.01; compared with the control: ^*P<0.05, ^**P<0.01)

As can be seen from Tables 10 and 11, upon examining expression of apoptosis-promoting regulator (Bax), apoptosis-inhibiting regulator (Bcl-2) and transcriptional regulator (C-fos), the control group with beta-amyloid (Aβ_1-40) dementia symptoms exhibited increased expression of Bax and decreased expression of Bcl-2. Whereas, experimental groups to which the respective isomers of ginsenoside Rg₂ were administered exhibited decreased expression of Bax and enhanced expression of Bcl-2 and C-fos. These results demonstrate that the respective isomers of ginsenoside Rg₂ have regulatory action on expression of abnormal Bax and Bcl-2 genes.

In addition, results of HE staining showed that the placebo group exhibited less damage to hippocampal neurons, slight damage around sites to which an injection needle penetrated, small quantities of gliocyte infiltration, but intactness and no loss of pyramidal cell zones.

On the other hand, the control group manifesting beta-amyloid dementia symptoms exhibited large quantities of gliocyte infiltration, significant loss of hippocampal neurons. The region of cell death extended a great distance from CAl near an injection point, and thus a very long pyramidal-granular cell zone was observed to be lost. Upon examination under a high magnification microscope, rupture of the neuronal cell membrane, cellular atrophy, reduction of cell nuclei, and apoptotic bodies were apparently observed.

In contrast, experimental groups, to which the respective isomers of ginsenoside Rg₂ were administered, did not exhibit local infiltration of gliocytes, damage to pyramidal cell zones and granular cell zones, diminishing of cells and loss of cell zones. Upon examination under a high magnification microscope, cell membranes were intact, apoptotic bodies were very rare or nearly non-existent in a region more distant than CAl and pathological changes were also observed to be very slight.

In conclusion, for dementia models showing AD behavior and pathological changes caused by injection of beta-amyloid (Aβi_-40) and Ibotenic acid (IBA) mixtures into the hippocampus region of the rat brain, the respective isomers of ginsenoside Rg₂ can significantly improve learning and memory ability in AD rats, as with an anti-dementia drug, nimodipine, prevent loss of nerve cell bodies, increase expression of C-fos and bcl-2 in the brain, but inhibit expression of bax. Therefore, nerve cell bodies are protected and death thereof is prevented.

Experimental Example 2

- Therapeutic effects of ginsenoside Rg₂ on multiple infarct dementia This experiment was carried out employing a multiple infarct dementia (MID) model. 56 rats were divided into 7 groups consisting of 8 rats each and were anesthetized with 10% chloral hydrate. Except for a placebo group, the remaining 6 groups were established as dementia models by injecting 0.13 ml/100g of a composite thrombosis-inducing agent into the carotid artery of each rat,^" restoring anatomical structures thereof and suturing skins along layers. The placebo group was injected with an equal amount of physiological saline, instead of the composite thrombosis-inducing agent.

The above dementia model groups were subdivided into a control group to which physiological saline was administered, four experimental groups to which ginsenoside Rg₂ was administered and a comparative group to which a medical dementia therapeutic agent, nimodipine was administered. Each group received the corresponding respective reagents via intravenous injection for 7 days, once a day, according to dosages set forth in Table 12 below. Herein, reagents, which are administered to experimental groups, are isomeric C-20(S) ginsenoside Rg₂ and C-20(R) ginsenoside Rg₂, and mixed-type C-20(SR) ginsenoside Rg₂ (in the following experiments, referred to as S, R and SR, respectively).

On day 6 of respective reagent administration, learning-memory ability of animals was determined according to the same manner as in Experimental Example 1. The results are shown in Table 12 below.

Table 12

(Compared with the placebo group; Δ, P<0.01; compared with the control group: ^*P<0.05)

As can be seen from Table 12, upon examining and determining learning-memory ability of rats, the control group with typical multiple infarct dementia (MID) symptoms exhibited increased order of learning and memory, but experimental groups to which respective structures of ginsenoside Rg₂ were administered exhibited significantly decreased learning and memory orders. These results demonstrate that the respective isomers of ginsenoside Rg₂ have significantly improved therapeutic effects on multiple infarct dementia.

In addition, 24 hours after determination of learning-memory ability, the heads of animals were dissected, and a 2 x 5 mm incision was made in the bregma, at front and rear sides, respectively, and fixed with 4% paraformaldehyde, covered with paraffin and sectioned into sections having a thickness of 7 μm using a paraffin microtome. The sections were subjected to Nissl staining, and chemical tests were carried out on glutamate (GIu), apoptosis promoting regulator (Bax), an apoptosis inducer Caspase-3 and a protease Calpain II-positive neurons in a CPU region in rats of immunized groups. The results are shown in Tables 13 and 14.

Table 13

(Compared with the placebo group; ^{Λ A}P<0.01; compared with the control group: ^*P<0.05, ^**P<0.01)

Table 14

(Compared with the placebo group; ^{A A}P<0.01; compared with the control group: ^**P<0.01)

From chemical examinations of immunized groups, as shown in Tables 13 and 14, it was confirmed that rats of experimental groups to which respective different structures of ginsenoside Rg₂ were administered exhibited significant reduction in expression of GIu, Bax, Caspase-3, Calpain II-positive neurons in the CPU region, as compared with the control group having typical MID dementia symptoms, demonstrating that respective structures of ginsenoside Rg₂ have regulatory action on expression of the above abnormal genes.

In conclusion, for dementia models with MID behavior and pathological changes caused by injection of the composite thrombosis-inducing agent to rats, it was confirmed that the respective isomers of ginsenoside Rg₂ can significantly improve learning and memory ability in MID rats, similar to another anti- dementia drugs, nimodipine, and such action was correlated with declined expression of GIu, Calpain II, Caspase-3 and Bax involved in protein death.

Experimental Example 3 - Effects of ginsenoside Rg₂ on MTT metabolism after damage of PC12 cells

100 μβ/well of PC 12 culture having a cell density of 10⁵ cells/mL was inoculated into a 96-well plate and PC 12 cells were allowed to adhere to walls of the plate. Wells were divided into 6 groups to which 6 wells were assigned each.

A serum-free medium was added to a non-treatment group, 1 mmol/L of a serum-free medium containing glutamic acid and an equal volume (1 mmol/L) of 20% 1,2-polyethylene glycol were added to a control group, 1 mmol/L of a serum-free medium containing glutamic acid and 0.1 mmol/L of respective structures of ginsenoside Rg₂ were added to experimental groups, and 5 pmol of nimodipine, instead of ginsenoside Rg_2, was added to a comparative group. Then, PC12 cells of respective groups were cultured at 37^°C for 20 hours.

20 /^/well of MTT (5 g/L) was added to respective groups and cells were continuously cultured at 37 ^°C for 4 hours. Then, the original medium was aspirated, and 0.1 ml/well of dimethyl sulfoxide (DMSO) was added to dissolve red granules. Next, optical density (OD) at 490 nm was measured using an enzyme-linked immuno-assay and then a Mest was made between groups. Results are shown in Table 15.

Table 15

(Compared with the non-treatment group; Δi P<0.05; compared with the control group: ^*P<0.05)

As can be seen from Table 15, the control group exhibited significantly reduced MTT metabolism, confirming that glutamic acid partially damaged PC 12 cells. Whereas, in comparison with the control group, it was confirmed that both experimental and comparative groups, to which respective structures of ginsenoside Rg₂ and nimodipine were administered, respectively, exhibited significantly increased MTT metabolism of PC 12 cells, thus demonstrating that all of the respective isomeric ginsenoside Rg₂ can inhibit damage of PC 12 cells affected by glutamic acid.

Experimental Example 4

- Therapeutic effects of ginsenoside Rg₂ on depression A general model of depression adopts drug- or stress stimulation, but this experiment adopts ischemia/reperfusion to lower contents of norepinephrine (NE), dopamine (DA) and 5-hydroxytryptamine (5-HT) in brain tissues, thereby establishing a depression model.

48 rats were divided into 6 groups consisting of 8 rats each and were anesthetized with 10% chloral hydrate. Except for a placebo group, the remaining 5 groups were established as depression models as follows. A nylon line was inserted from a left external carotid artery into an internal carotid artery of each rat and a middle cerebral artery (MCA) was occluded. 30 min after blockage of blood flow, the nylon line was removed and reperfusion was allowed, thereby establishing depression models of rats.

Among the above depression models, 10 mg-kg^"1 of physiological saline was administered to a control group, and respective structures of ginsenoside Rg₂ were intravenously injected into experimental groups for 5 consecutive days, once a day, according to dosages set forth in Table 16 below.

After 48 hours of respective reagent administration, learning-memory ability of rats was determined according to the same manner as in Experimental Example 1. The results are shown in Table 16 below.

Table 16

(Compared with the placebo group; Δ Δi P<0.01; compared with the control group: ^*P<0.01)

In addition, 6 days after examination of learning-memory ability, heads of rats were dissected to remove the brain which was then homogenized. Contents of cerebral neurotransmitters, such as norepinephrine (NE), dopamine (DA) and 5-hydroxytryptamine (5-HT) were determined, and thereby a Mest was made between respective groups. The results are shown in Table 17 below.

Table 17

(Compared with the placebo group; ^{Λ Λ}P<0.01; compared with the control group: ^*P<0.05, ^**P<0.01) As can be seen from Tables 16 and 17, the control group exhibited remarkable decreases in learning-memory ability and cerebral NE and DA contents, while all of the experimental groups, to which respective isomers of ginsenoside Rg₂ were administered, exhibited remarkable increases in learning/memory ability and cerebral NE, DA and 5-HT contents.

According to the monoamine theory, it can be confirmed that respective isomers of ginsenoside Rg₂ have pronounced ameliorating action on depression or diseases resulting from disturbance in stimulation /suppression of central nervous system.

Experimental Example 5

- Effects of ginsenoside Rg₂ on pentobarbital sodium-induced sleeping time

38 male mice were divided into three groups as set forth in Table 18 below. Mixed-type ginsenoside Rg₂ was intravenously administered in an amount of 10 and 20 mg-kg^"1 to experimental groups, respectively. At the same time, 50 mg-kg^"1 of pentobarbital sodium was intravenously administered again. Whereas, for the control group, 10 mg-kg^'1 of 20% 1,2-polyethylene glycol was intravenously administered, instead of ginsenoside Rg₂. Using loss of lighting reflex as an indicator, a sleeping time of the mice belonging to the respective groups was recorded and a t-test was made between the respective groups. The results are shown in Table 18. Table 18

(Compared with the control group: P<0.05)

In comparison with the control group, as shown in Table 18, all of the experimental groups, to which mixed-type ginsenoside Rg₂ was administered, exhibited great reduction in the sleeping time of mice induced by pentobarbital sodium, demonstrating that ginsenoside Rg₂ stimulates the central nervous system (CNS).

Experimental Example 6

- Therapeutic effects of ginsenoside Rg₂ on peripheral circulation disorders of auricle

68 male mice were divided into 6 groups as set forth in Table 19 below and were anesthetized with urethane. Under a microscope, each strand of the venule (V) and arteriole (A) in the auricle was located. A diameter of each blood vessel was measured and state of blood flow was observed and recorded.

For the above groups, a control group was injected with physiological saline, and a comparative group was injected with an injectable preparation containing 0.2 g/niL of red ginseng and Big Blue Lily turf (Liriope platyphylla Wang et Tang) mixed extract. For experimental groups, respective structures of ginsenoside Rg₂ were intravenously injected according to dosages set forth in Table 19 below. 5 min later, 0.01 mg-kg^"1 of adrenalin was re-administered. Diameters of blood vessels, state of blow flow and numbers of vascular network intersection points were measured every hour.

Calculating percentage changes in adrenaline based on results thus measured, a Mest between respective groups was carried out. The results are shown in Tables 19, 20 and 21 below.

Table 19

♦ ♦-_{^ Λ Λ} - ***

(Compared with the control group: P<0.05, PO.01, P<0.001)

Table 20

**-~ „ „ . ***_τ

(Compared with the control group: P<0.05, PO.01, PO.001)

Table 21

(Compared with the control group: PO.05, P<0.01, P<0.001)

As can be seen from Tables 19 through 21, experimental groups, to which respective isomers of ginsenoside Rg₂ were administered, exhibited significant inhibition against reduction of blood vessel diameter of the arterioles and venules of mouse auricles caused by adrenalin and reduction of the number of vascular network intersection points, as compared with the control group.

Additionally, as compared to the control group to which the preparation containing a mixture of red ginseng and Big Blue Lily turf was injected, the ginsenoside Rg₂-administered experimental groups displayed a much less decrease in terms of a degree of reduction in blood vessel diameters and a degree of reduction in the number of vascular network intersection points, with respect to the passage of time. In particular, inhibitory action against reduction in the blood vessel diameter of venules was much stronger than in arterioles.

Experimental Example 7

- Effects of ginsenoside Rg₂ on ear perfusion of rabbit

33 white rabbits were divided into 4 groups as set forth in Table 22 below and were anesthetized with pentobarbital sodium. Arteries of the ear roots were stripped and tubes were inserted therein. The remaining blood was washed with a Locke solution and the rabbit ears were dissected, followed by perfusion.

For the above groups, a control group was injected with physiological saline, and a comparative group was injected with an injectable preparation containing 0.2 g/mL of red ginseng and Big Blue Lily turf {Liriope platyphylla Wang et Tang) mixed extract. For experimental groups, mixed-type ginsenoside Rg₂ was administered via the tube according to dosages set forth in Table 22 below. Before administration of reagents and at intervals of 3 min after administration, changes in the number of perfusion drops were expressed as a degree of peripheral vasodilation and then t-test between groups was carried out. The results are shown in Table 22 below.

Table 22

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

As can be seen from Table 22, experimental groups, to which mixed- type ginsenoside Rg₂ was administered, displayed significant increase in the number of perfusion drops after administration of Rg₂, as compared to the control group, demonstrating that mixed-type ginsenoside Rg₂ has vasodilating action on peripheral vessels.

Industrial Applicability

As disclosed from the foregoing, a method of extracting ginsenoside Rg₂ in accordance with the present invention employs gradient salting-out substituting conventional silica gel column chromatography separation, and therefore is suitable for mass production of Rg₂ due to easy and convenient operation and manipulation, enables extraction of high-purity finished products even with use of relatively cheap lower alkane alcohol instead of expensive mixed organic solvents, and thereby provides effects such as low production costs, harmlessness, safety and no influence on recovery of other ginseng saponins from the remaining materials.

In addition, the present invention reveals that respective isomers of ginsenoside Rg₂ have significantly improved action on memory disorders of animals with dementia caused by insoluble beta-amyloid proteins and multiple cerebral infarction, can control expression of genes or proteins involved in apoptosis/anti-apoptosis of cerebral neurons, prevent loss and death of neurons and inhibit damage of PC 12 cells caused by glutamic acid. In addition, the present invention has confirmed that respective isomers of ginsenoside Rg₂ significantly increase concentrations of monoamine neurotransmitters such as intracerebral NE, 5-HT and DA in cerebral blood deficit-reperfusion rats, and ginsenoside Rg₂ has distinctive anti-depressant action according to a monoamine theory. Additionally, ginsenoside Rg₂ significantly reduces pentobarbital sodium-induced sleeping time, demonstrating that ginsenoside Rg₂ exerts stimulatory effects upon the cerebral cortex.

Further, the present invention demonstrates that respective isomers of ginsenoside Rg₂ have significantly improved effects on peripheral circulation disorders of the mouse auricle and increasing effects of perfusion rate in rabbit ears, representing that ginsenoside Rg₂ has vasodilating action on peripheral vessels.

Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

Claims

CLAIMS:

1. A method of extracting ginsenoside Rg₂ from a raw material of a Araliaceae panax plant, comprising: subjecting the raw material to hot- water extraction and concentration to obtain concentrates, adding a lower alcohol to the concentrates to precipitate and remove foreign materials; decolorizing the remaining supernant and recovering the lower alcohol; adding a salting-out agent to a saturation concentration so as to obtain precipitates and re-dissolving the precipitates in water, followed by gradient salting-out to obtain differential precipitates corresponding to different concentrations of the salting-out agent; analyzing to divide the precipitates into ginsenoside Rg₂-enriched fractions and ginsenoside Rg₂ non-enriched fractions; destaining and decolorizing the ginsenoside Rg₂-enriched fractions with a destaining agent, followed by concentration to obtain concentrates; and adding the lower alcohol to re-crystallize the resulting concentrates, thereby obtaining ginsenoside Rg₂.

2. The method according to claim 1, wherein the raw material is selected from leaves and berries of Araliaceae panax and American ginseng {Panax quinquefolius Linn.)_plants.

3. The method according to claim 2, wherein hot- water extraction includes adding the raw material from Araliaceae panax to a 16-fold excess of water and heat-extracting for 3 hours, repeatedly, 3 times.

4. The method according to claim 3, wherein the lower alcohol is selected from the group consisting of methyl alcohol, ethyl alcohol, isopropyl alcohol, amyl alcohol and any combination thereof.

5. The method according to claim 4, wherein the salting-out agent is selected from the group consisting of sodium chloride, sodium sulfate, sodium sulfite, ammonium sulfate, other non-toxic inorganic salts and any combination thereof.

6. The method according to claim 5, wherein separating ginsenoside Rg₂-enriched fractions and ginsenoside Rg₂ non-enriched fractions, from the precipitates, is carried out by thin layer chromatography analysis.

7. The method according to claim 6, wherein the salting-out agent is selected from an anion exchange resin, a cation exchange resin, an adsorbent resin and a filler.

8. The method according to claim 7, wherein the recrystallizing process includes a first precipitation step to obtain crystals precipitating from more than 50% concentration of an alcohol in the solution while adding 95% alcohol to the ginsenoside Rg₂-concentrates, and a second precipitation step to obtain respective different crystals by recrystallizing the crystals under different alcohol concentrations on the basis of solubility difference.

9. The method according to claim 8, wherein the second precipitating step is carried out by dissolving the first precipitated crystals in 70% alcohol and allowing to stand for 12 hours at room temperature, so as to precipitate isomeric C-20(R) ginsenoside Rg₂, adjusting the alcohol concentration of the resulting supernant to 80%, followed by recrystallization at 4 ^"C for 24 hours to precipitate crystals, adding the resulting crystals to 90% alcohol and heating the mixture to 80 °C so as to dissolve crystals, followed by standing at 10^°C for 24 hours to precipitate mixed-type C-20(SR) ginsenoside Rg₂, and allowing the resulting supernant to stand for 12 hours at -20 ^"C, thereby precipitating isomeric

C-20(S) ginsenoside Rg₂.

10. A pharmaceutical composition, comprising 1 to 99% by weight of isomeric or mixed-type ginsenoside Rg₂ extracted by the method according to any one of claims 1 through 9; and 99 to 1% by weight of a pharmaceutically acceptable carrier and adjuvant.

11. The composition according to claim 10, wherein the content of ginsenoside Rg₂ is more than 50% by weight.

12. A pharmaceutical composition formulated into an injectable preparation, a capsule, a pill, nebula, a tablet, an oral solution, a patch, a suppository, a microcapsule or a micro ball, using the pharmaceutical composition of claim 10.

13. A therapeutic agent for treatment of dementia, comprising the pharmaceutical composition of claim 11 or 12.

14. A therapeutic agent according to claim 13, wherein the dementia is presenile dementia or vascular dementia.

15. A therapeutic agent for treatment of depression, comprising the pharmaceutical composition of claim 11 or 12.

16. A therapeutic agent for treatment of a peripheral circulation disorder, comprising the pharmaceutical composition of claim 11 or 12.