WO2006090910A1

WO2006090910A1 - Novel steroid glycoside, ngf-associated active substance, process for producing the same, method of screening thereof, and cerebral dysfunction preventive agent

Info

Publication number: WO2006090910A1
Application number: PCT/JP2006/304036
Authority: WO
Inventors: Makoto Ojika; Jianhua Qi; Youji Sakagami; Takayoshi Mamiya
Original assignee: National University Corporation Nagoya University
Priority date: 2005-02-25
Filing date: 2006-02-24
Publication date: 2006-08-31
Also published as: JP5092122B2; JPWO2006090910A1

Abstract

A novel steroid glycoside having an NGF-associated activity, represented by the general formula: (1) This steroid glycoside has an NGF-associated activity.

Description

Novel steroidal glycosides, NGF-related active substances, methods for producing and searching for the same, and drugs for preventing brain dysfunction

The present invention relates to novel steroidal glycosides and NGF-related active substances isolated from honhi tode (scientific name: Acanthaster planci) etc.

The present invention relates to a method of producing and searching a coactive substance.

Field technology

One kind of starfish, honithde, is a southern starfish that inhabits coral reefs. It has 11 to 16 arms, and it has a large-sized, poisonous beak. The sea urchin starfish leaves the stomach through the mouth in the middle of the lower part of the body, secrets digestive fluid and eats living coral. In addition to eating fish corals and corals that are a tourist resource, there is a risk that dipers etc. will be stabbed with a beard and are therefore targeted for extermination. Disclosure of the invention

Problem that invention tries to solve

By the way, the effective use of the extinct vonithde is being sought. The present inventors are studying the identification and utilization of components contained in starfish through the research of marine organisms such as the starfish. For example, a novel teroid glycoside contained in the starfish has neurite outgrowth activity ( It has been reported that it has a "neural growth factor (NGF) -like activity" (J. Qi et al, Bioorganic & Medicinal Chemistry (2004), 12 (15), 4259-4265.).

The present invention has been made in view of the above-mentioned circumstances, and provides an effective utilization method which is clarified by the study of novel steroid glycosides and novel steroid glycosides which have been made clear through the study of von starfish. Means to solve the problem

A large number of saponins (steroidal glycosides) have been discovered from the sea urchin starfish and their structures have been clarified (JP-A-4-41429; JP-A-62-240697; JP 2005-520841 "^ 幸C. Pizza et al, Journal of Chemical Research, Synopses (1985), (3), 76-77 .; T. Komori et al, Liebigs Annalen der Chemie (1983), (1), 37-55. Y. Itakura et al, Liebigs Annalen der Chemie (1986), (3), 499-508.

N. Fusetani et al, Journal of Natural Products (1984), 47 (6), 997-1002.

I. Kitagawa et al, Chemical & Pharmaceutical Bulletin (1978), 26 (6), 1864-73.). The present inventors discovered novel steroid glycosides which have not been reported to them, and discovered that the novel steroid glycosides show NGF-like activity. Furthermore, it has been discovered that there are also cases where the effect on NGF is enhanced (hereinafter referred to as “NGF enhancing activity”) (herein, NGF-like activity and NGF enhancing activity are combined to Associated with NGF-related activity

Called "NGF related active substance"). The following invention was completed based on these findings. Generally, steroid is a general term for compounds having a penolehydrocyclopentaphenanthrene ring, but in the present specification, part or all of hydrogen constituting the perhydrocyclopentaphenanthrene ring is removed. Separately, those having a skeleton in which a carbon-carbon double bond is formed are also described as steroids. In particular, compounds in which the bond between the carbons at positions 4 and 5 of the perhydrodicyclic pentafucanthrene ring is a double bond are specifically described.

(1) (a) That is, the novel steroidal glycoside of the present invention is a compound having a structure shown in the following general formula (1) or formula 39A.

(In the formula (1), X is X ¹ or X ^2; any one of the Y is one of Y ¹ and Y ³ when X is X ^1, when X is _X ² Υ 1~γ 3 In addition, χ 1 to 2 and γ 1 to 3 are substituents bonded at a part of *.

(b) In addition, the other novel steroidal glycosides of the present invention can be represented by the following general formula (1 ′) or (1 ′ ′), or by the formula 64-3, 65-3, 69-11 or 101-3 It is a steroid glycoside represented.

(In the formula (1,), Y is any one of Y ⁴ to ^7. R is hydrogen if Υ is Υ ⁴ and methyl if 場合⁵ to ⁷ . ^{And 4} to ^{7 each} represents a substituent bonded to a part of *.

(In the formula (1 "), Y is Y ⁴ ^'6 ~ is any one of the ^8. R is When Y is Y ⁴ in the case of hydrogen, Y ⁵ ~ ⁷ is a methyl group. Note , Υ ⁴ , ⁶ to ⁸ combine in the part of *

(c) The compounds shown in (a) and (b) are compounds which have not been conventionally isolated from von starfish and have a structure in which one monosaccharide is bonded to one end or both ends of the steroid skeleton. . Generally, steroid glycosides contained in starfish are known to have strong toxicity, but these compounds show strong NGF-like activity and pino or NGF enhancing activity below the concentration at which toxicity appears. . Accordingly, the steroid glycosides represented by the general formulas (1), (1 ′) and (1 ′ ′) and the formulas 39A, 64-3, 65-3, 69-1 and 10-13 have NGF-related activity In the present specification, the “substance” such as “NGF-related active substance” or the like may be a pure compound or a mixture containing a plurality of compounds. In the general formula (1), it can be sufficiently predicted that compounds in which X is X ¹ and Y is Y ² also have NGF related activity (at least NGF enhancing activity). Also, the general formula (1 Among ') and (1 "), regardless of the kind of Y, it can be sufficiently predicted that compounds freely selected from hydrogen and methyl group also have NGF related activity (at least NGF enhancing activity).

In addition, as a result of comparing the structures of known steroid glycosides derived from honistarde and the steroid glycosides of the present invention exhibiting NGF-related activity, we succeeded in deducing the conditions for exhibiting NGF-related activity. Completed the invention of

That is, the NGF-related active substance of the present invention has a tetracyclic fused nucleus described in the following formula (2) as a skeleton, and one or two monosaccharides are directly or indirectly bound to each of a nucleus and a nucleus. And a steroid glycoside.

(In the formula (2), each nucleus of A to D may have a double bond, and any hydrogen atom may be substituted by one OR group (R is hydrogen, an alkyl group or a group) or a methyl group. can do. )

In addition, “a monosaccharide directly binds to the A nucleus (D nucleus)” means that one or two monosaccharides (in the case of two disaccharides including monosaccharides bound by monosaccharides) are OH in the monosaccharides. It means that it is bound to A nucleus etc. by group. And, “a monosaccharide is indirectly linked to A nucleus (D nucleus)” means that one or two monosaccharides (in the case of two, including a disaccharide linked by monosaccharides) are at least one It means that it is bound to A nucleus etc. by OH group in the monosaccharide via the group consisting of carbon atom etc. The group to be interposed may have a ketone, an ether, a double bond and the like in part or all in addition to the vanolexylene group.

Here, a compound in which one monosaccharide is bound to each of the A nucleus and the D nucleus exhibits an NGF-like activity and an NGF enhancing activity, and one or two of the A nucleus and the D nucleus are one or two. It has been found that compounds to which a monosaccharide is bound mainly exhibit NGF enhancing activity. Therefore, as a steroid glycoside having NGF enhancing activity, a compound in which one or two monosaccharides are directly or indirectly bound to either one of the A nucleus and the D nucleus. is there.

And, at least one of the following formulas 33B, 34B2, 39A2, 39A3, 74-2 and 74-4 can be exemplified as the above-mentioned steroid glycosides mainly exhibiting NGF enhancing activity. All of these compounds are known compounds isolated from starfish other than starfish, but this time they have been clarified by the present inventors to have NGF related activity.

Here, the steroid glycoside can be a compound contained in an extract from an organism belonging to the starfish class.

(2) And, it has been found that the extract from von starfish contains a parting rain that expresses NGF related activity. These fractions exhibit NGF-related activity without isolating the novel steroid glycosides described above. In addition, the above-mentioned novel steroid glycoside is isolated from a part of the extract of von starfish extract, and the NGF-related activity is also high in the fraction expected to be free of the above-mentioned novel steroid glycoside. It is known to indicate. The following inventions were completed based on these findings.

That is, as a method of obtaining an NGF-related active substance, a step of separating a hydrophobic fraction from an organic solvent extract of an organite extracted with an organic solvent comprising alcohol or acetone;

Fractionating the hydrophobic fraction by chromatography using silica gel and a dextran type carrier.

Separate the hydrophobic fraction from the organic solvent extract to obtain the fraction containing steroid glycosylate It is speculated that it is possible to obtain a fraction containing a sterol glycoside having NGF-related activity in the subsequent chromatography method.

In particular, as the chromatography method, it is a component whose R f value measured by TLC (carrier: silica gel, eluent: chloroform / methanol = 8 Z 2) is in the range of not less than 0.20 and not more than 0.64. A step of separating a fraction from the hydrophobic fraction by a K-well chromatograph method can be employed. By separating the fractions exhibiting this R f value, it is possible to obtain NGF related active substances showing high NGF related activity.

Further, as a chromatography method, a fraction having an R f value of less than 0.20 measured by TLC (carrier: silica gel, eluent: chloroformomethanol = 8 Z 2) is chromatographed from the hydrophobic fraction. An exclusion process can be employed. The highly toxic fraction can be removed by excluding the fraction showing this R f value. In addition, as the step of separating by the chromatography method, it is possible to obtain an NGF related active substance exhibiting higher NGF related activity by including the step of separating the fraction having the R f value of 0.52 or more. it can. Furthermore, the step of separating by the chromatography method can also include the step of separating a fraction having an R f value of 0.39 or less. The NGF-related active substance obtained by these production methods preferably contains at least one compound in the group consisting of the above-mentioned novel steroid glycosides.

(3) Furthermore, we invented a method for efficiently searching for NGF-related active substances.

That is, the method for searching for an NGF-related active substance of the present invention is characterized in that the tetracyclic condensation nucleus described in the above-mentioned formula (2) is a skeleton, and at least one of A nucleus and D nucleus directly or 1 monosaccharide It is important to determine the presence or absence of NGF-related activity by whether or not it contains a steroid glycoside that is indirectly linked.

The compound in which the tetracyclic fused nucleus represented by the formula (2) is bonded to a monosaccharide is a compound which is easy to determine whether it contains or not, as represented by saponin and the like. Then, the position and number of bonds of the monosaccharide are determined to determine whether or not the compound corresponds to a compound in which the tetracyclic condensation nucleus represented by the above-mentioned formula (2) is bonded to a predetermined monosaccharide. Good. Here, since the number of bonds and the position of bonds of monosaccharides can be determined relatively easily, the method is a simple method as a whole, as compared with the conventional methods for screening a wide variety of GF-related active substances. : ' Therefore, the relatively easy operation of detecting glycosides (such as steroid glycosides) in which a sugar is bonded to a 4-ring condensed nucleus is a promising possibility that it may become an NGF-related active substance and its source animal (plant). The candidates can be narrowed down roughly, and the time required for the search can be greatly reduced.

In other words, by efficiently narrowing down biological materials containing steroid glycosides such as Saboyun etc. by conventional methods etc., it becomes possible to reduce the number of compounds to be subjected to structural analysis, and search for NGF related active substances Becomes easier. After that, PC12 cell assembly is performed by determining whether or not the structure has the above-mentioned structure with respect to the substance whose structural component has been separated by separating the target component from the biological material or the like which has been narrowed down. Instead, NGF-related active substances can be searched. In addition, NGF-related active substances are searched without performing PC12-cell assay by determining whether the structure of the many steroid glycosides that have been discovered so far has the above-mentioned structure or not. can do.

In particular, it is desirable that the steroid glycoside is searched from an extract from a starfish class. Effect of the invention

The NGF-related active substance of the present invention has an unprecedented high activity. And, since it can be easily separated from ornithite which is required to be eliminated, it can also be a motivation for the elimination of ornithodete beyond its excellent availability.

Then, the method for searching for an NGF-related active substance of the present invention has an effect of being able to easily search for an NGF-related active substance other than searching for an active ingredient in a natural resource. Brief description of the drawings

Fig. 1 Analysis of the active fraction fr. A obtained from the extract of the sea urchin starfish obtained in the example by HPLC (1st round) and a diagram showing the steroid glycoside corresponding to each peak It is. Fig. 2 is a copy of a photomicrograph showing the appearance of adding Acanthasteroside 40 A and NGF to PC12 cells.

Fig. 3 The results of analysis of the active fraction fr. C obtained from the extract of honichdee obtained in the example with H PLC (1st round) and the corresponding steroid glycosides are shown. FIG.

FIG. 4 is a graph showing the evaluation results of the effect on learning memory impairment in aged male mice of the Example. BEST MODE FOR CARRYING OUT THE INVENTION

(New steroidal glycosides)

The novel steroidal glycosides of the present invention are six compounds of acanthasterosides 34 A, 34 B, 34 C, 39 A, 4 OA and 4 OB shown below, and a compound of the following formula 80-3: , 92-2, 78-3, 101-3, 64-3, 65-2, 65-3, 76-3, 62-3, 69-1 1, 42-2 (acanth terror side 39 B), 35 12 compounds of -2 (acanthasteroside 39 A4).

(NGF related active substance)

The NGF-related active substances of the present invention include the above-mentioned novel steroid glycosides. And a compound having a tetracyclic fused nucleus ({perhydro} cyclopentaphenenantrene ring and its derivative) represented by the above formula (2) as a skeleton, and each of A nucleus and D nucleus is 1 Or a steroid glycoside to which two monosaccharides are directly or indirectly linked. In the formula (2), each nucleus of A to D can independently have a double bond. In addition, any hydrogen atom can be substituted with one OR group (R is hydrogen, an alkyl group or an asyl group) or a methyl group. The alkyl group is preferably from about 3 to 3 carbon atoms, particularly preferably a methylole group, and the acyl group is preferably from about 1 to 3 carbon atoms, and more preferably an acetyl group. It is also desirable not to have a tetracyclic fused nuclei one OS 0 ₃ H group bonded directly to the (N a salts, including such K salt).

Furthermore, since the tetracyclic fused nucleus represented by the formula (2) is a nucleus moiety of a steroid :, any hydrogen can be substituted by any group. For example, many known As the steroid has, a compound in which a substituent having a skeleton as shown in the following formula (A) is bonded to the 17 position of the cyclopentaphenanthrene ring can be exemplified.

Specifically, as the steroid glycoside having a tetracyclic condensation nucleus represented by the formula (2), the above-mentioned formulas 33 B, 34 B 2, 39 A 2, 39 A 3 can be mentioned. , 7 4-2 and 7 4-4 are exemplified.

Here, when it is desired to obtain a compound exhibiting NGF-like activity, a compound in which a monosaccharide is bound to both the A nucleus and the D nucleus is used. When it is desired to obtain a compound exhibiting an NGF enhancing activity, a compound in which a monosaccharide or a disaccharide is bound to either one of A nucleus and D nucleus is used. The site to which the monosaccharide is attached is not particularly limited, and it may be attached at any site of carbon possessed by the monosaccharide. Monosaccharides are bound either directly or indirectly when binding to the A nucleus or D nucleus. In the case of indirect bonding, the group interposed between them may be a group having a ketone, an ether, a double bond and the like in part or all in addition to the alkylene group.

The type of monosaccharide is not particularly limited. It may be of any carbon number such as pentose or hexose. In addition, it may be a disaccharide in which two monosaccharides are linked, and further, an OH group is partially an OR group (R is an alkyl group (for example, having about 1 to 3 carbon atoms, preferably a methyl group)) or It may be substituted by a group (for example, about 1 to 3 carbon atoms, preferably an acetyl group is preferable), and may be substituted by Z or hydrogen. Examples of monosaccharides include xylose and the like.

Here, the steroid glycoside is preferably a compound contained in an extract from an organism belonging to the starfish class.

(Manufacturing method of NGF related active substance)

As a method for obtaining the NGF-related active substance of the present invention, there is a step of separating a hydrophobic fraction from an organic solvent extract of an ornithode extracted with an organic solvent consisting of alcohol or acetone, silica gel and Z or dextran type carrier And a step of fractionating the hydrophobic fraction by a chromatography method using The NGF-related active substance obtained by this production method is a group consisting of the above-mentioned novel steroid glycosides. It is desirable to contain at least one of these compounds.

• Extraction operation

The organic solvent extract can be obtained by mashing or pulverizing von ichthite and then immersing in an organic solvent, or by mashing or pulverizing von xfish in the presence of an organic solvent. It is desirable to carry out filtration to remove contaminants in order to facilitate the subsequent operation. You may use for the extraction operation in any state, such as frozen state and dried state, as it is. In particular, it is preferable to use lyophilization for the purpose of improving the handleability and degrading the contained components and suppressing loss. The organic solvent can be appropriately selected from methanol, ethanol, alcohols such as _n- , iso-propanol and the like (preferably methanol is selected).

• Separation of hydrophobic fractions

Any method that can separate the hydrophobic fraction may be employed. For example, it is a step of adsorbing and separating the hydrophobic fraction using a reverse phase carrier. As the carrier of the reverse phase system, an ODS system can be exemplified. Examples of chemical structures and product names include ODS (C18), reverse phase carriers other than 0DS, and synthetic adsorption resins such as DIAI 0 N and SEPABEADS.

When a method of adsorbing to a reverse phase carrier is employed, water or the like is added to the aforementioned organic solvent extract to adjust the polarity so that the hydrophobic fraction is adsorbed to the carrier. For example, in the case of employing an ODS-based carrier and employing a developing solvent of methanol and a single water system, the hydrophobic fraction is generally adsorbed by mixing in a ratio of about 1: 1 methanol: water = 1: 1 and contacting with the carrier It is separated.

After that, by increasing the concentration of methanol in the developing solvent, the hydrophobic fraction adsorbed on the carrier can be eluted and separated quickly. Under the above conditions, in particular, it is desirable to use the component contained in the fraction eluted with 70% -90% methanol as the hydrophobic component.

• Process to further fractionate hydrophobic fraction

In this step, the hydrophobic fraction obtained in the previous step is fractionated by chromatography using a silica gel and / or dextran carrier (normal phase system). By sequentially changing the polarity of the solvent, the components contained in the hydrophobic fraction can be rapidly separated. , The preferred carrier Examples of the chemical structure and product name as the body include dextran based carriers such as silica gel and S mark hadex LH20.

(a) As a method of determining the degree of separation, in principle, it can be performed by determining the strength of the NF-related activity and the strength of toxicity in the obtained fraction. The measurement of NGF-related activity is determined by whether to induce neurite outgrowth using a test system using a general cell such as PC 12 cells. When inducing neurite outgrowth alone, it is a fraction showing NGF-like activity, and when enhancing the activity of NGF, it is a fraction showing NGF enhancing activity. When PC12 cells are damaged, it can be judged as toxic.

(b) There is a method to judge the degree of separation simply by the R f value of TLC. As the conditions for TLC, the carrier is silica gel, and the eluent is black hole form Z methanol = 8/2. If the R f value of the separated fraction is within the range of 0.20 or more and 0.64 or less, it can be judged as the target fraction. By separating the fractions exhibiting this R f value, it is possible to obtain an NGF-related active substance exhibiting high NGF-related activity.

Also, similar TLC conditions can be adopted, and the fraction having an R f value of less than 0.20 can be excluded from the aforementioned hydrophobic fraction and the remaining fraction can be used as the objective fraction. Specifically, separation conditions can be controlled so that components having an R f value of less than 0.2 do not mix. Here, a fraction having a more desirable NGF-related activity can be obtained by including a fraction having a value of 0.52 or more. It is also desirable to include fractions with an R f value less than 0.39.

• Other

Other separation methods can be added as needed. For example, the above-mentioned operation can be applied repeatedly, separated by chromatography using another carrier, or separated using HPLC.

(Method for searching for NGF related active substances)

The method for searching for an NGF-related active substance according to the present invention is characterized in that the tetracyclic fused nucleus described in the above formula (2) is used as a skeleton, and at least one of A nucleus and D nucleus directly or indirectly. Determine the presence or absence of NGF-related activity by whether or not it contains a steroid glycoside to be bound Do. As for these compounds, the items described in the section on NGF-related active substances above are valid as they are, and thus further description is omitted.

In particular, it is efficient to search from organisms belonging to the starfish class. A compound (aglycone having a tetracyclic condensation nucleus) in which a tetracyclic condensation nucleus represented by the formula (2) is bonded to a monosaccharide (determination of whether or not it is contained as represented by saponin etc.) Is an easy compound. Therefore, the relatively easy operation of detecting aglycone having a four-ring condensation nucleus can roughly narrow down potential candidates for NGF-related active substances and their source animals (plants), and the time required for the search can be obtained. It can be greatly shortened.

Thereafter, the bonding position and the number of bonds of the monosaccharide may be determined to determine whether the compound corresponds to the above-mentioned formula (2). Here, since the number of linkages and the linkage position of monosaccharides can be determined relatively easily, it is a method that is simple overall as compared with the conventional methods of screening NGF related active substances widely. For example, the content ratio of monosaccharides is measured, and the value is compared with the concentration of these steroid glycosides, etc., to calculate the number of bonds of monosaccharides to the tetracyclic condensation nucleus represented by the formula (2). In addition, it is desirable to search for compounds that do not have one OSO ₃ H group (including Na salt, K salt and the like).

(Example)

[Test 1: Method for separating NGF-related active substance from starfish]

(Extraction)

Lyophilized at 1.5 kg (wet mass) collected at Oki-shi, dried with 1.2 L of methanol and 16.5 kg of the dried product was broken with a mixer and left in a plastic tank at room temperature for 1 week. The mixture is suction filtered using filter paper and the residue is washed with methanol and combined with the filtrate. The filtrate of 19 L was concentrated under reduced pressure to obtain 314 g of methanol extract.

(Separation of hydrophobic fraction)

Dissolve 314 g of the above methanol extract in 1 L of 50% aqueous methanol solution, reverse phase column (Co smo sil 140 C 18-OPN, Nacalai Tester, 1 kg, The chromatography was carried out using 105 × 20 O mm) (flow velocity is free fall velocity). The eluent and volume, fraction (fr) numbers are as follows: 50% Me OH (1.5 L, fr. 1-3), 60% Me OH (1.5 L, fr. 4-6) 70% Me OH (3 L, fr. 7-12), 80% Me OH (2.1 L, fr. 13-17), 90% Me OH (1.5 L, fr. 1 8-20) _Λ Me OH (1.5 L, f r. 21-24).

Fractions eluted with 70% -90% methanol (fr. 1 1 1 9) were combined and concentrated under reduced pressure to obtain 4.76 g of a portion containing a steroid glycoside (saponin).

(Chromatography method)

4.76 g of the above-mentioned steroid glycoside-containing portion is dissolved in 10 OmL of chloroform-form monomethanol (95: 5), and a silica gel column (Hi-F 1 ash 2 L, Yamazen, 165 g, φ 48 X 1 The chromatography was performed using 7 Omm) (flow rate 20 mL / min). The eluate was separated every 3 minutes (6 OmL) using a linear gradient (120 minutes) from chloroform in methanol (95: 5) to the same (45: 55). 24-26 (69-78 minutes) were combined and concentrated under reduced pressure to obtain a fraction f hr .A 277 mg showing neurite outgrowth activity.

This substance (5 μg / mL) depletes neurite outgrowth to 26% of PC 12 cells and does not extend the neurite in the presence of trace NGF (1.5 ng / mL). The protrusion extension activity was enhanced to 77%.

In addition, f r. B (572 mg, f r. 27-29) and f r. C (93 mg, fr. 30-34) alone did not show protrusion extension activity, but NGF (1.5 The activity of ng / mL was increased to 27% (fr. B) and 45% (fr. C). The subsequent high polar fractions f r. D (23 lmg) and f r. E (988 mg) showed cytotoxicity before exhibiting NGF-related activity.

(Method of measuring NGF related activity)

Rat adrenal medulla pheochromocytoma-derived PC 12 cells were obtained from Riken Cell Bank. Cryopreserved cells (2 xl 0 ⁴ cells) medium - and washed with (MEME- 10% fetal bovine serum 5% horse serum), with medium 1 OML plated on 9 cm Petri dish, 5% Rei_0 ₂ atmosphere under 37 Stationary culture was performed for 1 week. Harvest cells and use fresh medium 2 x 10 ⁵ After repeating the subculturing procedure of 1 week of culture dilution for 1 week after dilution into cells / dish, harvested cells were used for the test (up to 12 passages). In the following, culture is performed at 37 ° C. in a 5% CO ₂ atmosphere.

In each well of a 24-well microtiter plate, 1 mL of a medium containing PC 12 cells of 2 × 10 ⁴ cells was placed, and static culture was performed for 24 hours. The medium was changed to 1 mL (containing 1% DMSO) of serum-free MEME medium containing an appropriate concentration of samples, and the appearance of cells was observed every 24 hours for 1 week. Three fields within which approximately 100 cells can be observed were randomly selected, and the number of cells with a protrusion length longer than the cell diameter was counted, and the ratio was converted to a percentage to obtain neurite extension activity.

The neurite outgrowth activity is the value of NGF-like activity when added alone, and the neurite when further added with NGF added at a concentration that does not induce neurite outgrowth (1.5 ng / mL). The elongation activity was taken as the value of NGF enhancing activity.

[Test 2: Method for isolating novel steroidal glycoside from f r. A]

• HPLC (1st time)

The above active fraction f r. A 277 mg was purified by HPLC. The conditions are as follows: Column TSK g e l OD S- 120T (φ 20 x 25 Omm, YMC),

0% MeOH, flow rate 8 mL / min, detection wavelength 205 nm, 1/3 pouring. 25 fractions were obtained for each peak.

• HPLC (2nd time onwards)

Among the above fractions, f r. 21 (17. 6 mg) was purified by HPLC (solvent 40% MeCN, other conditions such as column are the same as above), and a novel steroid glycoside, xanthasteloside 40 B ( 15. Obtained Omg). In addition, fr. 22 (52.5 mg) was purified by HPLC (under the same conditions) to obtain acantasterosteroside 40A (10.7 mg), which is a novel steroidal glycoside. Similarly, fantaleuside 34 C (5.6 mg), which is a novel steroid glycoside derived from f r. 13 (12.6 mg), and two novel compounds from f r. 20 (29. 2 mg) The steroid glycosides Acanthasteroside 34 B (4.3 mg) and 39 A (10. 2 mg) were obtained. In addition, four known products are available from f r. 12 (1 1.4 mg), f r. 17 (7.8 mg), f r. 19 (1 4.8 mg) and f r. 22 (52.5 mg). I got the substance. that's all The results are shown in Figure 1. FIG. 1 shows the values of the steroid glycosides and NGF-related activities isolated from the peaks obtained in the first HP LC and their respective peak powers. In FIG. 1, a line is drawn from the peak where the steroid glycoside is isolated. Peaks from which these steroid glycosides were isolated are, from the left (small retention time) under the above conditions, peakl (fr. 12): 53-56 minutes; peak2 (fr. 13): 56 -60 minutes; peak 3 (fr. 1 7): 77-81 minutes; peak 4 (fr. 1 9): 87-90 minutes; peak 5 (fr. 20): 90-96 minutes; peak 6 (fr. 21): 96 -105; peak7 (fr. 22): 106-125 It can be distinguished from the fact that it is a large peak appearing in the range of about 125 minutes. The target compound can be easily isolated by separating the peaks with reference to these retention times.

Further, a photomicrograph showing the appearance of PC12 cells after inducing neurite outgrowth using Acanthasteroside 4 OA and NGF is shown in FIG. The upper right of Fig. 2 is the control, and when nothing is added, the cell shape does not change, but when Aquantasteloside 4 OA (lower left) and NGF (lower right) are added. From the cells, it can be seen that many projections are elongated.

Below, the results of optical rotation, iH-NMR ¹³ C-NMR and mass spectrometry of the obtained novel steroidal racemate are shown below. Although the details are not shown here, the existence of the compound shown in the above 34A is also confirmed.

Acanthasteroside 40A: [a] ²⁵ _D -20 (c 0.06, MeOH); ^X H NMR (600 MHz, CD ₃₀ D) δ 0.89 (6 H, d, J = 7.2 Hz), 0.93 (3 H, d, J = 6.6 Hz), 0.95 (1 m), 0.96 (1 H, m), 1.00 (1 H, m), 1.05 (1 H, d, J = 10.8 Hz), 1.10 (3 H, s), 1.16 (3 H, s) ), 1.19 (1H, m), 1.20 (2H, m), 1.22 (1H, m), 1.37 (1H, m), 1.49 (1H, ra), 1.50 (1H, ra), 1.55 (1H, dd, J = 14.4, 3.0 Hz), 1.58 (1 H, m), 1. 59 (1 H, m), 1.72 (1 H, m), 1. 74 (1 H, m), 1. 80 (1 H, ra), 1.81 (1 H, ra), 1.82 (1H, m), 1.83 (2H, ra), 1.96 (1H, m), 2.43 (1H, dd, J = 14.7, 2.4 Hz), 2.81 (1 H, dd, J = 9.0, 7.8 Hz), 2.81 (1H, dd, J = 9.0, 7.8 Hz), 3.01 (1H, t, J = 9.0 Hz), 3.18 (2H, m), 3.22 (1H, m), 3.29 (1H, m), 3.41 (1H, m) m), 3.46 (1H, ra), 3.53 (1H, m), 3.57 (3H, s), 3.61 (3H, s), 3.68 (1H, m), 3.80 (2H, ra), 3: 83 (1H) , M), 3.87 (1H, ra), 3.98 (1H, dd, J = 7.2, 2.4 Hz), 4.15 (1H, dd, J = 10.8, 2.4 Hz), 4.17 (1 H, d, J = 7.8 Hz) , 4.40 (1 H, d, J = 6.6 Hz); ¹³ C NMR (150 MHz, CD ₃₀ D) δ 15.8 (q), 16.7 (q), 18.6 (q), 19.6 (t), 19.9 (q), 20.1 (q), 26.0 (t), 29.5 (d), 30.3 (t), 31.2 (d), 33.2 (t), 34.8 (t), 36.8 (s), 41.3 (t),

43.1 (t), 45.2 (s), 45.4 (t), 46.1 (d), 49.9 (d), 57.1 (d), 60.5 (d), 60.9 (q), 61.0 (q), 63.7 (d), 66.8 (t), 66.9 (t), 70.9 (d), 71.3 (d), 72.0 (t), 74.1 (d), 74.6 (d), 76.9 (s), 77.5 (d), 80.3 (d), HR ESI-TOF-MS found m / z 797.4653 (M + Na), calcd. For 80.9 (d), 83.0 (d), 84.9 (d), 107.6 (d), 105.3 (d).

C ₄₀ H ₇₀ 0 ₁₄ Na 797.4658.

Acanthasteroside 40B: [a] ²⁵ _D- 35 (c 0.81, MeOH); ^X H NMR (600 MHz, CD ₃₀ D) 6 0.89 (6 H, d, J = 6.6 Hz), 0.93 (3 H, d, J = 6.6 Hz), 0.99 (IH, m),

1.01 (IH, d, J = 10.8 Hz), 1.02 (IH, m), 1.11 (3H, s), 1.17 (1H, m), 1.19 (IH, m), 1.20 (1H, m), 1.28 (IH) , m), 1.36 (3H, s), 1.37 (IH, m), 1.47 (1H, dd, J = 12.6, 3.0 Hz), 1.49 (2H, m), 1.62 (IH, m), 1.75 (1H, m) in), 1.79 (IH, m), 1.81 (2H, m), 1.86 (IH, dd, J = 13.2, 3.0 Hz), 1.96 (IH, ra), 1.98 (1 H, m), 2.58 (IH, dd) , J = 15.0, 2. Hz), 2.82 (IH, dd, J = 9.0, 7.8 Hz), 3.01 (IH, t, J = 9.0 Hz), 3.18 (IH, m), 3.18 (IH, m), 3.22 (1H, m), 3.29 (IH, m),

3.40 (IH, dd, J = 9.6, 6.3 Hz), 3.46 (lH, m), 3.53 (IH, m), 3.57 (3H, s), 3.61 (3H, s), 3.80 (2H, m), 3.98 (1H, dd, J = 7.8, 2.4 Hz), 4.15 (1H, dd, J = 10.8,

2.4 Hz), 3.83 (1H, m), 4.17 (1H, d, J = 7.8 Hz), 4.18 (IH, m), 4.30 (IH, s), 4.41 (IH, d, J = 7.8 Hz), 5.63 (IH, s); ¹³ C R (150 MHz, CD ₃₀ D) 516.7 (q), 18.6 (q), 19.5 (t), 19.9 (q), 20.1 (q), 22.7 (q), 26.0 ( t), 27.9 (t), 29.5 (d), 31.2 (d), 34.8 (t), 37.7 (s), 39.7 (t), 43.0 (t), 44.4 (t), 45.0 (s), 46.0 ( d), 57.8 (d), 60.5 (d), 60.9 (q), 61.1 (q), 63.6 (d), 66.8 (t, 2C), 70.9 (d), 71.2 (d), 72.0 (t), 74.6 (d), 76.2 (s), 76.4 (d), 77.5 (d), 80.9 (d), 83.0 (d), 84.9 (d), 87.6 (d), 104.6 (d), 105.3 (d), 126.9 (d ), 148.5 (s). HR ESI-TOF-MS found m / z 795. 544 (M + Na), calcd. for C 4. H ₆₈ 0 ₁₄ Na 795.4525.

Acanthasteroside 34C: [a] ²³ _D- 25 (c 0.42, MeOH); ^X H NMR (600 MHz, CD ₃₀ D) 60.94 (3 H, d, J = 6.6 Hz), 1.02 (I H, d, J = 9.6 Hz), 1.03 (IH, m),

1.05 (3H, d, J = 6.0 Hz), 1.12 (3H, s), 1.18 (1H, ra), 1.19 (IH, m), 1.22 (1H, 1) dd, J = 12.0, 5.4 Hz), 1. 28 (1 H, m), 1. 36 (3 H, s), 1. 49 (IH, dd, J = 14.4, 3.0 Hz), 1. 49 (1 H, m), 1.72 (IH, m) ), 1.75 (IH, m), 1.78 (1H, m), 1.86 (IH, m), 1.87 (1H, ra), 1.97 (IH, m), 1.98 (2H, m), 2.12 (IH, m) , 2.29 (1H, m), 2.57 (IH, dd, J = 14.7, 2.7 Hz), 2.82 (IH, dd, J = 8.7, 8.1 Hz), 3.16 (IH, m), 3.30 (1H, m), 3.35 (IH, m), 3.47 (IH, m), 3.56 (1H, m), 3.57 (3H, s), 3.81 (IH, dd, J = 11.4, 5.4 Hz), 3.98 (1 H, dd, J = 7.2, 2.4 Hz), 4.15 (IH, dd, J = 10.8, 2.4 Hz), 4.18 (IH, m), 4.30 (IH, dd, J = 4.8, 2.4 Hz), 4.41 (1 H, d, J = 9.6) Hz), 4.74 (1 H, s), 4.81 (I H, s), 5.63 (1 H, s); ¹³ C NMR (15 MHz, CD ₃₀ D) δ 16.8 (q), 17.2 (q), 18.4 (q ), 19.5 (t), 22.7 (q), 27.9 (t), 30.6 (d), 32.9 (t), 35.5 (t), 37.7 (s), 39.7 (t), 43.0 (t), 43.4 (d) ), 44.4 (t), 45.1 (s), 57.8 (d), 60.5 (d), 61.1 (q), 63.7 (d), 66.8 (t), 67.5 (t), 71.3 (d), 76.2 (s) ), 76.4 (d), 77.5 (d, 2 C), 80.9 (d), 82.8 (d), 84.9 (d), 109.2 (d), 104.6 (d), 126.9 (d), 148.6 (s), 154.0 (s). HR ESI-TOF-MS found ra / z 647.3765 (M + Na), calcd. For C ₃₄ H ₅₆ 0 ₁₀ Na 647.3706.

A Qantas terrorism side ^{_{34B: [a] 25 D -16}} (c 0.11, MeOH);: H NMR (600 MHz, CD 3 0D) 00.89 (6H, d, J = 6.6 Hz), 0.93 (3H, d, J = 6.6 Hz), 1.01 (IH, ra), 1.02 (IH, m), 1.04 (1H, m), 1.11 (3H, s), 1.16 (1H, m), 1.17 (1H, m), 1.20 (IH) , m), 1.22 (1H, m), 1.28 (IH, m), 1.36 (3H, s), 1.45 (IH, m), 1.49 (2H, ra), 1.61 (IH, m), 1.75 (IH, m) ra), 1.79 (IH, ra), 1.81 (1H, ra), 1.83 (1H, ra), 1.87 (IH, m), 1.96 (IH,), 1.98 (IH, m), 2.58 (1H, dd, J = 15.0, 3.0 Hz), 2.82 (1 H, dd, J = 9.0, 7.8 Hz), 3.18 (IH, ra), 3.29 (1 H, m), 3.46 (2 H, ra), 3.53 (IH, m), 3.57 (3H, s), 3.80 (IH, ra), 3.98 (1 H, dd, J = 7.2, 2.7 Hz), 4.14 (1 H, dd, J = 10.8, 2.4 Hz), 4.17 (1 H, br t, J = 7.8 Hz), 4.30 (1 H, br s), 4.41 (1 H, d, J = 7.8 Hz), 5.63 (1 H, s); ¹³ C NMR (150 MHz, CD ₃₀ D) 616.7 (q), 18.6 (1. q), 19.7 (t), 19.9 (q), 20.1 (q), 22.7 (q), 25.6 (t), 27.9 (t), 29.0 (d), 31.2 (d), 35.8 (t), 37.7 ( s), 39.7 (t), 43.0 (t), 44.4 (t), 45.0 (s), 48.5 (d), 57.8 (d), 60.6 (d), 61.1 (q), 63.6 (d), 63.9 (t), 66.8 (t), 71.3 (d), 76.2 (s), 76.4 (d), 77.5 (d, 2 C), 80.9 (d), 83, 0 (d), 84.9 (d), 104.6 (d), 126.9 (d), 148.5 (s). HR ESI-TOF-MS found m / z 649 '. 3939 (M + Na), calcd. 4036

for C ₃₄ H ₅₈ 0 ₁₀ Na 649.3922.

Acanthasteroside 39A: [a] ²⁵ _D- 25 (c 0.13, MeOH); NMR (600 MHz, CD ₃₀ D) δθ. 90 (9 H, m), 0.94 (1 H, m), 0.95 (3 H, s), 1.00 (2H, m), 1.12 (3H, s), 1.19 (1H, d, J = 9.6 Hz), 1.22 (1H, m), 1.26 (1H, m), 1.30 (2H, m), 1.35 (1H, ra), 1.50 (1H, m), 1.54 (1H, m), 1.56 (1H, m), 1.57 (1H, m), 1.58 (2H, m), 1.73 (2H, m), 1.75 (1 H, m), 1. 79 (1 H, ra), 1. 83 (1 H, ra), 1. 86 (1 H, m), 1. 89 (1 H, m), 2.00 (1 H, m), 2. 38 (1 H, dd, J = 14.7) , 2.7 Hz), 2.86 (1 H, dd, J = 9.0, 7.8 Hz), 3.10 (1 H, t, J = 10.8 Hz), 3.17 (1 H, ra), 3.34 (1 H, ra), 3.38 (1 H, t , J = 9.0 Hz), 3.46 (3 H, s), 3.57 (3 H, s), 3.59 (1 H, m), 3.63 (1 H, dd, J = 12.0, 5.7 Hz), 3.76 (1 H, dd, J = 12.0, 3.0 Hz), 3. 85 (1 H, d, J = 2.4 Hz), 3. 94 (1 H, m), 3. 97 (1 H, m), 3.99 (1 H, m), 4.04 (1 H, dd, J = 3.6, 0.6 Hz), 4.27 (1H, td , J = 9.6, 3.6 Hz), 4.40 (1H, d, J = 7.8 Hz), 5.07 (1H, s); 1 3 C NMR (150 MHz, CD 3 0D) δ 15.4 (q), 15.9 (q), 18.4 (q), 19.0 (q), 19.8 (t), 31.5 (d), 31.7 (t), 32.9 (t), 36.3 (t), 36.3 (d), 36.7 (s), 41.4 (t), 41.7 (d), 42.9 (t), 45.5 (t), 45.5 (s), 49.0 (d), 56.0 (d), 57.2 (d), 59, 1 (s), 61.2 (s), 62.5 (t), 64.4 (t), 66.6 (d), 70.1 (d) , 72.4 (d), 74.2 (d), 77.2 (s), 77.8 (d), 80.8 (d), 83.8 (d), 84.4 (d), 84.9 (d), 92.7 (d) HR ESI-TOF-MS found m / z 767. 558 (M + Na), calcd. For C ₃₉ H ₆₈ 0 ₁₃ Na 767.4552., 105.2 (d), 107.8 (d).

[Test 3: Method for isolating novel steroid glycoside from f r. C]

• HP LC (1st time)

The above active fraction f r. C (93 Omg) was purified by HPLC. The conditions are as follows: Column ODDSevelosil-UG-5 (φ 28 x 250), solvent 70% MeOH, flow rate 18 ml / min, detection wavelength 205 nm, injection in two divided doses. As shown in FIG. 3, five fractions (Fr. C-1 to C-5) were obtained based on the chromatographic peak.

• HP LC (second time or later)

Each of the above fractions was separated and purified as shown in Table 1. table 1

8

Specifically, it is as follows. The fr. C-1 (retention time tR = 38-45 min, 14.8 mg) is purified by HPLC (conditions K, F) to give compound 80-3 (2.9 mg), compound 92-2 (1. O mg) and compound 78-3 (1.4 mg) was purified by HPLC (conditions K, F, Ε) to give compound 101-3 (2. I mg).

fr. C-2 (tR = 51-62 min, 114. 7 mg) is purified by HPLC (conditions D, E, F) and compound 62-3 (3.5 rag) is analyzed by HPLC (conditions D, I, J) The purified compound 65-2 (4.5 mg), the compound 65-3 (2. I mg) and the compound 64-3 (5.4 mg) are purified by HPLC (conditions D, F) to a compound 76-3 (3. I got Omg).

Purification of fr. C-3 (tR = 73-82 min, 57. 9 rag) by HPLC (conditions A, E, F, G), compound 69-11 (2. Omg) by HPLC (conditions A, H) Purification gave two compounds, 74-2 and 74-4.

Purification of fr. C-4 (tR = 82-103 min, 66.7 mg) by HPLC (conditions B, C, D) gave compound 42-2 (14. 2 mg).

The fr. C- 5 (tR = 103-126 rain, 24. 8 rag) was purified by HPLC (condition A) to give compound 35-2 (19. 7 mg).

HPLC conditions

A: Developil-ODS-5 (φ 20 χ 250 mm), 40% MeCN, 8 ml / min, RI detector

B: Developil-ODS-5 (φ 20 x 250 mm), 40% MeCN, 8 ml / min, UV 205 nm

C: Developil-ODS-5 (φ 20 x 250 mm), 35% MeCN, 8 ml / min, UV 205 nm

D: Capcell pak (φ 20 x 250 mm), 35% MeCN, 8 ml / min, UV 205 nm

E: Developil-ODS-UG-5 (Φ 10 x 250 mm), 35% MeCN, 2 ml / min, RI detector

F: Developil-ODS-UG-5 (φ 10 x 250 mm), 70% MeOH, 2 ml / min, RI detector

G: Developil-ODS-UG-5 (ΐθ x 250 mm), 53% EtOH, 1.5 ml / min, RI detector

H: Developil-ODS-SR-5 (φ 20 x 250 mm), 70% MeOH, 8 ml / min, RI detector

I: Capcell pak (φ 20 x 250 mm), 70% MeOH, 8 ml / min, RI detector

J: Developil-ODS-SR-5 (Φ 20 x 250 mm), 35% MeCN, 8 ml / min, RI detector

K: Developil-ODS-SR-5 (φ 20 x 250 mm), 35% MeCN, 8 ml / min, UV 04036

'Show ¹ H-R R data (CD ₃₀ D, 600 MHz) for each compound,

Table 2

Table 3

Table 4

(About NGF related activity)

(1) Table 5 shows the results of measurement of the NGF-like activity and the NGF-strong activity of each steroid glycoside shown in FIG. Furthermore, in the compound types shown in Table 5 The terroid nucleus is a structure corresponding to the tetracyclic condensation nucleus shown in Formula (2), and is slightly different for each compound. And from the top of Table 5, one in which one monosaccharide is bonded to A nucleus shown in formula (2) (a), one in which one monosaccharide is bonded to both A nucleus and D nucleus (β), And, it can be classified into three major types of those in which two monosaccharides (disaccharides) are linked to the D nucleus (γ).

Table 5

'Forgive fflSg active (7th order)

> Wasuka (»I0K) I: 示す i® (1.5» gtal) coexistence (7 BS) As is clear from Table 5, NGF-like activity is confirmed only in type j3, type E, J3 Since both of n and y show NGF enhancing activity, it can be expected that NGF-like activity can be expected from a compound having a structure of (1) type 3; It can be inferred that a compound in which one monosaccharide or disaccharide is bound to a steroid nucleus exemplified in and the like has NGF-related activity.

In addition, (l) when a compound having NGF-like activity is required, it can be obtained by searching for a compound having a structure of type / 3, (2) when a compound having NGF enhancing activity is required It can be inferred that the compound can be obtained by searching for a compound in which one monosaccharide or disaccharide is bound to the steroid nucleus.

And, the novel steroidal glycosides (acanthasteroside 4 OA, B, 34 B, C and 39 A) of the present invention show no toxicity and their activity is recognized up to about ⁴⁰ ^ M. In addition, a sufficient effect is exhibited even at a concentration of about 20 μm. The NGF-related activity of these steroidal glycosides is higher than that of compounds for which the NGF-related activity is conventionally known. (2) The NGF-like activity and the NGF enhancing activity of Compound 35-2 and Compound 42-2 were also measured in the same manner as in (1). The results are shown in Table 6.

Table 6

As is apparent from Table 6, both Compound 35-2 and Compound 42-2 were found to exhibit extremely strong NGF-like activity and NGF enhancing activity. This is also predictable from Table 5. That is, Compound 35-2 and Compound 42-2 correspond to types having a structure in which a sugar is bonded to both ends of a steroid nucleus. In addition, the compounds 80-3, 76-3, and 65-2 corresponding to type j3 also show strong NGF-like activity and NGF enhancing activity.

In addition, compound 64-3 corresponding to type y also shows high NGF-like activity and strong NGF activity. However, as predicted from Table 5, the NGF-like activity is slightly weaker than the compound corresponding to type / 3.

[Evaluation of action on learning memory impairment in elderly male mice]

The effect of the above-mentioned f r. A (hereinafter referred to as “test sample”) on learning memory impairment when administered to mice was examined.

(Method)

Eight to ten months old ICR male mice (aged group) were used. Every evening after weight measurement, each group was administered subcutaneously. The dose of the test sample, 1 kg body weight per lmg or 10 m _g and so as to test sample group was dissolved in physiological saline (10 animals each), and the group administered neat saline (10 mice) did. The administration was for 14 days. After administration for 14 days, a Y-shaped maze test was performed according to a conventional method. As a control group, 6 week old ICR male mice (Young group: 10) were similarly subcutaneously administered with physiological saline.

(Results and Discussion)

The results are shown in FIG. Figure 4 As is evident from the left side, the Young group (Control: S al) In contrast, in the age group of saline administration (S al), impairment of short-term memory was observed. No impairment of short-term memory was observed in the aged group (OJK 10) in which the test sample was administered at 10 mg / kg. In addition, the age group (OJK 1) in which the test sample was administered at lmg / kg showed a degree of short-term memory impairment intermediate between S a 1 and OJK 10 in the aged group, and a certain improvement tendency was observed. As the concentration of the test sample, the possibility that the effect can be exerted even if it is less than 1 mg / kg of body weight is fully considered.

The total arm entries, which indicate the amount of activity in the trial during the Y-shaped maze test, showed a similar trend. That is, as is apparent from the right side of Fig. 4, it is apparent that recovery to the same level as that in the Young group is evident in the OJK 10 group and the OJKl group in which the test sample dose is 1 mg / kg. became.

As described above, it was revealed that administration of test samples to mice has an effect on brain dysfunction that occurs with growth and aging. In addition, although no details are shown, no significant side effects such as behavioral abnormalities caused by administration of this test sample are observed, so this test sample may be used for prophylactic use. It became clear. Industrial applicability

The novel steroidal glycosides of the present invention can be expected to have NGF related activity and the like. Substances having NGF-related activity are expected to exert effective actions for treating dementia and improving learning ability as pharmacological actions, and their application as drugs can be expected. Therefore, they have industrial applicability as to methods for producing and searching for these NF-related active substances.

Claims

Scope of claim A steroid glycoside represented by the following general formula (1) or the formula 39A.

(In the formula (1), X is X ¹ or X ² ; Y is ^one of Y ¹ and Y ³ when X is X ¹ and any one of _Y i to _Y 3 when X is X ² there. in addition, X ²及Pi γΐ ~ ³ is a substituent attached at portions of *.)

2. A steroid glycoside represented by the following general formula (1,) or (1 ′ ′) or the formula 64-3, 65-3, 69-11 or 101-3.

(In the formula (1,), Y is any one of Y ⁴ to ^7. R is hydrogen if Υ is Υ ⁴ and methyl if 場合⁵ to ⁷ . ^{And 4} to ^{7 each} represents a substituent bonded at a part of *.

(In the formula (1 ′ ′), Υ is one of Υ ⁴ and ⁶ to ^8. R is hydrogen when Υ is Υ ⁴ and methyl is a case of Υ ⁵ to ^7. ) , And Υ ⁴ and ⁶ to ^{8 each} is a substituent bonded to the part of *.)

3. An NGF-related active substance comprising the steroid glycoside according to claim 1 or 2.

4. A steroid glycoside having a tetracyclic fused nucleus described in the lower formula (2) as a skeleton, and one or two monosaccharides directly or indirectly bound to at least one of the nucleus and the nucleus D. An NGF-related active substance characterized by comprising.

5. The NGF-related active substance according to claim 4, wherein the steroid glycoside is at least one of the following formulas 33B, 34B2, 39A2, 39A3, 7412 and 74-4.

6. The NGF-related active substance according to claim 4 or 5, wherein in the steroid glycoside, one or two monosaccharides are directly or indirectly bound to only one of the A nucleus and the D nucleus. .

7. The NGF-related active substance according to any one of claims 3 to 6, wherein the steroid glycoside is a compound contained in an extract from an organism belonging to the family Starfish.

8. separating the hydrophobic fraction from an organic solvent extract of opharynx starfish extracted with an organic solvent consisting of alcohol or acetone;

A step of fractionating the hydrophobic fraction by chromatography using silica gel and / or dextran type carrier, and a method of producing an NGF related active substance characterized in that

9. separating the hydrophobic fraction from the organic solvent extract of opharynx star extracted with an organic solvent consisting of alcohol or acetone;

Fractions containing an R f value in the range of not less than 0.20 and not more than 0.44 as determined by TLC (carrier: silica gel, eluent: chloroform Z methanol = 8/2) Separating by chromatography, and a method of producing an NGF-related active substance.

10. separating the hydrophobic fraction from the organic solvent extract of starfish extracted with an organic solvent consisting of alcohol or acetone;

A step of removing a fraction having an R f value of less than 0.20, which is measured by TLC (carrier: silica gel, eluent: chloroform nomethanol = 8/2), from the hydrophobic fraction by chromatography A method for producing an NGF-related active substance characterized by comprising.

11. The method for producing an NGF-related active substance according to claim 9, wherein the step of separating by the chromatography method comprises the step of separating a fraction having an R f value of 0.52 or more.

12. The method for producing an NGF-related active substance according to claim 9, wherein the step of separating by the chromatography method comprises the step of separating a fraction having the R f value of 0.39 or less.

13. The NGF-related active substance comprises at least one compound of the group consisting of steroid glycosides according to any one of claims 1 to 5. The manufacturing method of the NGF related active substance as described in-.

14. A steroid glycoside having a tetracyclic fused nucleus described in the following formula (3) as a skeleton and in which one or two monosaccharides are directly or indirectly bonded to at least one of A and D nuclei. A method for searching for an NGF-related active substance, which determines the presence or absence of an NGF-related activity according to whether or not it is not necessary.

(In the formula (3), each nucleus of A to D may have a double bond, and any hydrogen atom may be substituted by one OR group (R is hydrogen, an alkyl group or a group) or a methyl group. can do. )

The method for searching for an NGF-related active substance according to claim 14, wherein the steroid glycoside is searched from an extract from an organism belonging to a starfish class.

1 6. An NF-related active substance characterized in that it can be produced by the method according to any one of claims 9 to 13.

1 7. A drug having the NGF-related active substance according to any one of claims 3 to 7 and 16 and characterized in that it is a medicine for preventing or treating brain dysfunction that occurs with aging. medicine.