JP2009234962A - Depressant for absorbing neutral lipids obtained from daisy, and saponin compound and its application - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a depressant for absorbing neutral lipids obtainable from an inflorescence of daisy which is an oxeye, a medicine or a food for a human or an animal having depressing effect for absorbing neutral lipids, and further a novel saponin compound obtainable by separating and purifying an extract from an inflorescence of daisy. <P>SOLUTION: The depressant for absorbing neutral lipids includes an inflorescence of daisy, an extracted liquid obtained by extracting the inflorescence of daisy with water, an aqueous lower aliphatic alcohol or the like, an extracted essential obtained by concentrating the extracted liquid, or a saponin compound obtained by separating and purifying the extracted liquid. The medicine or the food for a human or an animal includes the depressant for absorbing neutral lipids. Further, the novel saponin compound can be obtained by separating and purifying the extracted liquid. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a neutral lipid absorption inhibitor containing a flower part of a daisy which is an Asteraceae plant, an extract or extract thereof, or a saponin compound obtained therefrom, and a neutral lipid absorption inhibitor thereof. It relates to pharmaceuticals and health foods. The present invention further relates to a novel saponin compound obtained from a daisy flower, its extract or extract.

Daisy (scientific name: Bellis perennis L.) is a perennial plant whose Japanese name is also called daisies (chicken chrysanthemum), enmaykeiku (long life chrysanthemum), butterfly daisy (long life chrysanthemum) and the like. In Western countries, young daisy leaves, buds and flower parts have been used for food in addition to salads, and their roots have also been used for the treatment of rheumatism, wounds, and expectorants. Non-patent document 1). In Non-Patent Document 1 and the like, several saponin components and the like have been reported so far for the roots as daisy-containing components. However, few scientific studies have been carried out such as searching for detailed components of flower parts and evaluating their biological activity.
Li W., Asada Y., Koike K., Nikaido T., Furuya T., Yoshikawa T., Tetrahedron, 61, 2921-2929 (2005)

  The present invention was made based on the search for detailed ingredients relating to daisy flower parts and research on their action as a drug, and is obtained from daisy flower parts and has a neutral lipid absorption inhibitory action. An object is to provide a (neutral lipid absorption inhibitor). Another object of the present invention is to provide a human or veterinary drug containing these and a food containing this neutral lipid absorption inhibitor. It is another object of the present invention to provide a novel saponin compound that can be obtained from a daisy flower.

  The inventor of the present invention relates to a daisy flower part, an extract obtained by extracting the daisy flower part with water, a lower aliphatic alcohol or the like, an extract obtained by concentrating the extract, and the extraction. For compounds obtained by separating and purifying from liquids and extracts, we investigated the neutral oil absorption-inhibiting effect on olive oil-induced blood neutralization and lipase-inhibiting activity using mice as an index for evaluating the activity of inhibiting neutral lipid absorption. did. As a result, it has been found that an extract or extract of daisy flowers and daisy flowers with water or a lower aliphatic alcohol exhibits this action. Furthermore, it discovered that the compound which shows neutral lipid absorption inhibitory activity was contained in the compound obtained by isolate | separating and refine | purifying from an extract and an extract. Furthermore, it discovered that there existed a novel saponin compound in the compound obtained by isolate | separating and refine | purifying from an extract and an extract. The invention of the aspect shown below is completed based on these results.

  The present invention provides, as a first aspect thereof, an extract obtained by extracting a daisy flower, a daisy flower with water, a lower aliphatic alcohol or a hydrated product of a lower aliphatic alcohol, or the extract A neutral lipid absorption inhibitor comprising an extract extracted by concentration as an active ingredient is provided (claim 1).

  The present inventor further separated and purified the extract or extract, measured various physical properties of the separated and purified compound, and as a result of structural analysis, the extract or extract was , A novel saponin compound represented by any one of the following structural formulas (1) to (39), and a known represented by any one of the following structural formulas (40) to (58) It was found that the saponin compound was contained.

  That is, the novel saponin compound obtained by extracting the daisy flower part is a compound represented by any one of structural formulas (1) to (39) shown below.

  In addition, the novel saponin compounds represented by any one of structural formulas (1) to (39) were named as follows.

Compound of structural formula (1): Perennisoside I
Compound of structural formula (2): perenisoside II
Compound of structural formula (3): perenisoside III
Compound of Structural Formula (4): Perenisoside IV
Compound of structural formula (5): perenisoside V
Compound of Structural Formula (6): Perenisoside VI
Compound of Structural Formula (7): Perenisoside VII
Compound of structural formula (8): Perenisoside VIII
Compound of structural formula (9): Perenisoside IX
Compound of structural formula (10): Perenisoside X
Compound of structural formula (11): Perenisoside XI
Compound of Structural Formula (12): Perenisoside XII
Compound of structural formula (13): perenisoside XIII
Compound of Structural Formula (14): Perenisoside XIV
Compound of structural formula (15): Perenisoside XV
Compound of Structural Formula (16): Perenisoside XVI
Compound of structural formula (17): perenisoside XVII
Compound of structural formula (18): perenisoside XVIII
Compound of Structural Formula (19): Perenisoside XIX
Compound of structural formula (20): Perennisaponin A
Compound of Structural Formula (21): Perenisaponin B
Compound of structural formula (22): Perennisaponin C
Compound of Structural Formula (23): Perenisaponin D
Compound of structural formula (24): Perennisaponin E
Compound of Structural Formula (25): Perenisaponin F
Compound of structural formula (26): perenisaponin G
Compound of Structural Formula (27): Perenisaponin H
Compound of Structural Formula (28): Perennisaponin I
Compound of structural formula (29): Perennisaponin J
Compound of structural formula (30): Perennisaponin K
Compound of Structural Formula (31): Perenisaponin L
Compound of Structural Formula (32): Perennisaponin M
Compound of Structural Formula (33): Perenisaponin N
Compound of Structural Formula (34): Perenisaponin O
Compound of Structural Formula (35): Perenisaponin P
Compound of Structural Formula (36): Perennisaponin Q
Compound of Structural Formula (37): Perenisaponin R
Compound of structural formula (38): Perennisaponin S
Compound of structural formula (39): Perennisaponin T

  Moreover, the known saponin compound obtained by extracting the flower part of a daisy is a compound represented by any one of structural formulas (40) to (58) shown below.

Structural formula (40): In the above structural formula (A), R ¹ and R ² are both hydrogen.
Structural formula (41): In the structural formula (A), R ¹ is a substituent represented by the following formula I, and R ² is hydrogen.
Structural formula (42): In the structural formula (A), R ¹ is a substituent represented by the following formula II, and R ² is hydrogen.
Structural formula (43): In the structural formula (A), R ¹ is a substituent represented by the following formula II, and R ² is an acetyl group (CH ₃ CO—).

Structural formula (44): In the above structural formula (B), R ¹ and R ⁴ are both β-D-glucopyranosyl, R ² and R ⁵ are both acetyl groups, and R ³ is α-L-rhamnopyranosyl. If there is.
Structural formula (45): In the above structural formula (B), R ² , R ³ and R ⁴ are all hydrogen, and R ¹ and R ⁵ are both β-D-glucopyranosyl.
Structural formula (46): In the above structural formula (B), R ¹ , R ² and R ⁴ are all hydrogen, R ³ is α-L-rhamnopyranosyl, and R ⁵ is β-D-glucopyranosyl. .
Structural formula (47): In the structural formula (B), R ¹ and R ⁵ are both β-D-glucopyranosyl, R ² and R ⁴ are both hydrogen, and R ³ is α-L-rhamnopyranosyl. If.

Structural formula (48): In the above structural formula (C), R is hydrogen.
Structural formula (49): In the above structural formula (C), R is β-D-glucopyranosyl.
Structural formula (50): In the above structural formula (C), R is β-D-glucuronopyranosyl.
Structural formula (51): In the above structural formula (C), R is 6-methyl β-D-glucuronopyranosyl.

Structural formula (52): In the above structural formula (D), R ¹ is α-L-rhamnopyranosyl (1 → 6) β-D-glucopyranosyl and R ² is hydrogen.
Structural formula (53): In the above structural formula (D), when R ¹ is β-D-glucopyranosyl and R ² is a methyl group.
Structural formula (54): In the above structural formula (D), R is β-D-glucuronopyranosyl and R ² is a methyl group.
Structural formula (55): In the above structural formula (D), R ¹ is α-L-rhamnopyranosyl (1 → 6) β-D-glucopyranosyl, and R ² is a methyl group.
Structural formula (56): In the above structural formula (D), R ¹ is α-L-rhamnopyranosyl (1 → 6) β-D-galactopyranosyl, and R ² is a methyl group.

  The present inventor further examined the olive oil-induced blood neutral lipid elevation inhibitory action and lipase inhibitory action using mice as an index for evaluating the activity of some of the compounds as neutral lipid absorption inhibitory action. did. As a result, it has been found that some of the novel saponin compounds and the known saponin compounds exhibit a neutral lipid absorption inhibitory action, and the second and third aspects of the present invention are obtained. completed.

  That is, the present invention provides, as a second aspect thereof, the following novel compounds obtained by extracting daisy flowers and exhibiting neutral lipid absorption inhibitory action.

A saponin compound represented by the structural formula (1) (claim 2).
A saponin compound represented by the structural formula (2) (claim 3).
A saponin compound represented by the structural formula (26) (claim 4).
A saponin compound represented by the structural formula (27) (claim 5).
A saponin compound represented by the structural formula (28) (claim 6).
A saponin compound represented by the structural formula (29) (claim 7).
A saponin compound represented by the structural formula (32) (claim 8).
A saponin compound represented by the structural formula (37) (claim 9).

  In addition, the present invention provides, as a third aspect, a saponin compound represented by the structural formula (1), a saponin compound represented by the structural formula (2), a saponin compound represented by the structural formula (26), and a structure. The saponin compound represented by formula (27), the saponin compound represented by structural formula (28), the saponin compound represented by structural formula (29), the saponin compound represented by structural formula (32), the structural formula ( A neutral lipid absorption inhibitor comprising as an active ingredient at least one compound selected from the group consisting of a saponin compound represented by 37) and a saponin compound represented by Structural Formula (42) To do. That is, it is a neutral lipid absorption inhibitor characterized by containing, as an active ingredient, a saponin compound that is obtained by extracting daisy flowers and exhibits neutral lipid absorption inhibitory action.

  Since the neutral lipid absorption inhibitor of the first aspect and the third aspect has a neutral lipid absorption inhibitory action, a medicament having a neutral lipid absorption inhibitory action is obtained by containing this in the medicine. be able to. That is, the present invention provides, as a fourth aspect thereof, a human or animal pharmaceutical comprising the neutral lipid absorption inhibitor described in the first aspect and the third aspect.

  Moreover, the foodstuff which has neutral lipid absorption inhibitory action, for example, health food, can be obtained by making the foodstuff contain the neutral lipid absorption inhibitor of the said 1st aspect and 3rd aspect. That is, this invention provides the foodstuff containing the neutral lipid absorption inhibitor of the said 1st aspect and 3rd aspect as the 5th aspect.

  A first aspect of the present invention, that is, an extract obtained by extracting a daisy flower, a daisy flower with water, a lower aliphatic alcohol, or the like, or an extract obtained by concentrating the extract Neutral lipid absorption inhibitor characterized by containing as an active ingredient is an olive oil-induced blood neutral lipid absorption inhibitory effect using mice, implemented as an index for evaluating activity as a neutral lipid absorption inhibitor, or / And it shows activity in the lipase inhibitory action, and has excellent activity as a neutral lipid absorption inhibitor.

  Moreover, the neutral lipid absorption inhibitor characterized by including the novel saponin compound of the 2nd aspect of this invention or / and the known saponin compound represented by said Structural formula (42) as an active ingredient, ie, The neutral lipid absorption inhibitor of the third aspect of the present invention is an olive oil-induced blood neutral lipid absorption inhibitory effect using mice, implemented as an index for evaluating the activity as a neutral lipid absorption inhibitor, or / and It exhibits activity in lipase inhibitory action and has excellent activity as a neutral lipid absorption inhibitor.

  Therefore, the medicine (fourth aspect) containing the neutral lipid absorption inhibitor of the first aspect and the third aspect of the present invention is a medicine exhibiting an excellent neutral lipid absorption inhibitory action, The food (5th aspect) which contained the neutral lipid absorption inhibitor of the 1st aspect of this invention and the 3rd aspect is a foodstuff which shows the neutral lipid absorption inhibitory effect outstanding.

  Next, although the best mode for carrying out the present invention will be described, the scope of the present invention is not limited only to this embodiment.

As the neutral lipid absorption inhibitor of the first aspect of the present invention,
1) Contains daisy flowers (without extraction or other processing) as an active ingredient,
2) An extract obtained by extracting the flower part of daisy with water, a lower aliphatic alcohol or a hydrate of lower aliphatic alcohol as an active ingredient, and 3) obtained by concentrating the extract. Containing extract as an active ingredient,
Can be mentioned.

  1) In the case of containing a daisy flower part (without processing such as extraction) as an active ingredient, the daisy flower part can be used as it is, or a shape by crushing, crushing, cutting, grinding, etc. What changed, or what gave preparations, such as drying, can also be used.

  2) In the case of containing an extract obtained by extracting daisy flowers as an active ingredient, the extract is selected from water, a lower aliphatic alcohol and a lower aliphatic alcohol hydrate, as it is. However, the method of extracting the daisy flower part with a shape change by crushing, crushing, cutting, grinding, etc., and extracting with the extraction solvent is effective for extraction efficiency. In terms of surface.

  Examples of the alcohol used as the extraction solvent include lower alcohols having 1 to 4 carbon atoms, specifically, methanol, ethanol, propanol, isopropanol, n-butanol, isobutanol, t-butanol, or these. A mixed solution is mentioned. Although water is also used as the extraction solvent, these alcohols or hydrous alcohols containing up to 30% by volume of water in these alcohols are preferably used. Of the above alcohols, methanol or ethanol is preferred. These extraction solvents are preferably used in an amount of about 1 to 50 times (weight), more preferably about 10 to 30 times the extraction material.

  Although extraction temperature can be arbitrarily set between room temperature and the boiling point of a solvent, the range of the boiling point of 50 degreeC-extraction solvent is preferable, for example. Extraction is performed by shaking, non-shaking, or refluxing the above-mentioned material to be extracted, that is, the daisy flower part as it is, or the daisy flower part is changed in shape by crushing, crushing, cutting, grinding, etc. Etc. are suitably performed by immersing in the above-mentioned extraction solvent.

  The preferred extraction time varies depending on the extraction temperature and the presence or absence of shaking during extraction, and is not particularly limited. For example, when the extraction material is immersed under shaking at 50 ° C. or higher, it is appropriate to perform for about 30 minutes to 10 hours, and when immersed for non-shaking, it is performed for about 1 hour to 20 days. Is appropriate. Moreover, when extracting under reflux of an extraction solvent, it is preferable to heat to reflux for 30 minutes to several hours. In addition, although it is possible to extract by immersing at a temperature lower than 50 ° C., in that case, it is preferable to immerse for a longer time than the above time. The extraction operation may be performed only once for the same material, but is preferably repeated a plurality of times, for example, about 2 to 5 times.

  In the extract obtained by the extraction step, the daisy flower component is eluted. The neutral lipid absorption inhibitor of the present invention may be used as an active ingredient by adding the extract thus obtained as it is, but the extract may be concentrated to make an extract. Concentration is preferably carried out at a low temperature and under reduced pressure. The filtrate may be concentrated by filtration before concentration. The extract can be used in a concentrated state, but the concentration may be performed until it is solidified, or may be used as a powder or lyophilized product. As a method of concentrating, a method of forming a powder and a lyophilized product, a method known in the art can be used.

  The extract or extract thus obtained can be subjected to a purification treatment and separated into each component contained. The separated components contain the above-mentioned novel saponin compounds having a neutral lipid absorption inhibitory action and known saponin compounds, and these can also be used as neutral lipid absorption inhibitors (the present invention). 3rd aspect).

  As the purification treatment, for example, a chromatographic method, an elution method using an ion exchange resin, partition extraction with a solvent, or the like can be employed alone or in combination. Examples of the chromatographic method include normal phase chromatography, reverse phase chromatography, thin layer chromatography, centrifugal liquid chromatography, high performance liquid chromatography, and the like, and include a method of performing any of these or a combination thereof. It is done. In this case, purification conditions such as a carrier and an elution solvent can be appropriately selected according to various chromatographies.

  A novel saponin compound represented by any one of the structural formulas (1) to (39) or the structural formula (40) is obtained by subjecting the extract or the extract to purification treatment and separating it into each component. A known saponin compound represented by any one of () to (58) can be obtained.

  As described above, daisy flower part, daisy flower part is extracted with water, a lower aliphatic alcohol or a hydrate of lower aliphatic alcohol, an extract obtained by concentrating the extract, Saponin compound represented by structural formula (1), saponin compound represented by structural formula (2), saponin compound represented by structural formula (26), saponin compound represented by structural formula (27), structural formula The saponin compound represented by (28), the saponin compound represented by structural formula (29), the saponin compound represented by structural formula (32), the saponin compound represented by structural formula (37), and the structural formula ( 42) The saponin compound represented by 42) is an olive oil-induced blood neutral lipid absorption inhibitory action using mice and / or lipase carried out as an index for evaluating activity as a neutral lipid absorption inhibitor. Activity was found in adverse effects. Therefore, they can be used as neutral lipid absorption inhibitors.

  Further, the neutral lipid absorption inhibitor (first and third aspects) of the present invention can be applied to medicines and foods, and is excellent in neutrality by containing this neutral lipid absorption inhibitor. A medicine or food having a lipid absorption inhibitory effect, such as a health food, can be produced.

  For example, a daisy flower part, the extract of a daisy flower part, an extract obtained by concentrating the extract, or structural formula (1), structural formula (2), structural formula (26), structural formula (27), the structural formula (28), the structural formula (29), the structural formula (32), the structural formula (37), and the saponin compound represented by the structural formula (42) are each independently Or as a mixture, diluted as it is or with a suitable medium, and used as a medicine in various forms such as powders, granules, tablets, capsules or liquids by known methods in the field of pharmaceuticals be able to.

  Suitable vehicles include pharmaceutically acceptable excipients such as binders (eg syrup, gum arabic, gelatin, sorbitol, tragacanth or polyvinylpyrrolidone), fillers (eg lactose, sugar, corn starch, calcium phosphate, sorbitol). Or glycine), a lubricant for tablets (for example, magnesium stearate, talc, polyethylene glycol or silica), a disintegrant (for example, potato starch) or a wetting agent (for example, sodium lauryl sulfate).

  In the case of a tablet, it may be coated by a well-known method in ordinary pharmaceuticals. In the case of a liquid preparation, it may be in the form of, for example, an aqueous or oily suspension, solution, emulsion, syrup or elixir. It may also be provided as a dry product that is mixed with water or other suitable excipients prior to use.

  These liquid preparations contain usual additives such as suspending agents such as sorbitol, syrup, methylcellulose, glucose syrup, gelatin water-added edible fat, and emulsifiers such as lecithin, sorbitan monooleate and gum arabic (including edible fat). Or non-aqueous excipients such as almond oil, fractionated coconut oil or glycerin, oily esters such as propylene glycol or ethylene glycol, preservatives such as methyl or propyl p-hydroxybenzoate or sorbic acid Moreover, a coloring agent or a fragrance may be further contained as desired.

  Daisy flower part, daisy flower part extract, extract obtained by concentrating the extract, or structural formula (1), structural formula (2), structural formula (26), structural formula (27 ), The structural formula (28), the structural formula (29), the structural formula (32), the structural formula (37), and the structural formula (42), each of the saponin compounds may be used alone or as a mixture. As described above, it can be contained in foods to give neutral lipid absorption inhibitory effects to foods such as health foods.

  Here, the health food means a food that has a more positive meaning than normal food and is intended for health, health maintenance / promotion, and the like. As the form of food or health food, for example, liquid or semi-solid, solid products, specifically powders, granules, tablets, capsules or liquids, cookies, rice crackers, jelly, yokan, yogurt, manju And other forms of confectionery, soft drinks, teas, nutritional drinks, soups and the like. In the production process of these foods, or in the final product, the flower part, the extract, the extract, and / or the saponin compound can be added by mixing, coating, spraying, or the like to obtain a health food.

  The amount of the flower part, extract, extract extract and / or saponin compound used in the medicament or food of the present invention is the concentration, the degree of purification, the strength of activity, the purpose of use, the target disease and subjective symptoms. It is appropriately adjusted depending on the degree of the user, the weight of the user, age, etc. For example, when used for adults as a medicine, for each administration, the extract or extract is used in the range of about 1 mg to 20 g, and adjusted within this range according to the degree of purification, water content, etc. Is often appropriate. Moreover, when using the said saponin compound, 1 mg-about 1g are suitable in many cases.

  When used as a health food, the amount of the extract or extract and the saponin compound is about 1 mg to 20 g per 1 kg of the target food, for example, in an amount that does not adversely affect the taste and appearance of the food. It is often appropriate to add in the range of.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the examples do not limit the scope of the present invention. In the following examples, the following various solvents, filter paper, chromatography carrier and HPLC column were used unless otherwise specified.

[solvent]
Methanol: Nacalai Tesque, first grade Chloroform: Nacalai Tesque, first grade Methanol for HPLC: Kanto Chemical Co., special grade Acetonitrile for HPLC: Kanto Chemical Co., special grade

[Filter paper] Advantech: No. 2

[Chromatographic carrier]
Normal phase silica gel column chromatograph carrier: BW-200, 150-300 mesh (Fuji Silysia)
Carrier for reverse phase ODS column chromatography: Chromatorex ODS1020T, 100-200 mesh (Fuji Silysia)
Carrier for porous polymer column chromatography: Diaion HP-20 (manufactured by Nippon Nersui Co., Ltd.)

[HPLC column]
Cosmosil 5C18-MS-II, 20 mm (id) × 250 mm (manufactured by Nacalai Tesque),
Cosmosil HILIC, 20 mm (id) × 250 mm (manufactured by Nacalai Tesque) and Wakopak Navi 30C-5, 20 mm (id) × 250 mm (manufactured by Wako Pure Chemical Industries, Ltd.)

Example 1 Preparation of Methanol Extract Extract of Daisy Flower Part 3.0 kg of dried daisy flower part was pulverized, and about 10 times the amount of methanol (30 L) was added thereto, followed by extraction with heating under reflux for 3 hours. After extraction, the mixture was filtered with a fold-fold filter paper, and about 10 times the amount of methanol (30 L) was newly added to the extraction residue, and the same extraction operation was performed three times. When the extract obtained by each extraction operation was combined, the solvent was distilled off under reduced pressure using a rotary evaporator. As a result, 775 g of a daisy flower part methanol extract (yield from dried raw material 25.8%) was obtained. Obtained.

Example 2 Separation and Purification of Methanol Extract Extract Water (about 6 L) was added to the methanol extract (720 g) and suspended, and the same volume of ethyl acetate (about 6 L) was added to the suspension, followed by a liquid separation operation. Carried out and partitioned between ethyl acetate and water transition. About the obtained water transfer part, the same volume ethyl acetate was newly added and the same liquid separation operation was implemented 3 times in total. About what combined the ethyl acetate transfer part obtained by each liquid separation operation, when the solvent was distilled off under reduced pressure using a rotary evaporator, 187.1 g of ethyl acetate transfer parts of the methanol extract of a daisy flower part were obtained. Obtained.

Example 3 Preparation of water transfer part The water transfer part obtained in Example 2 was subjected to porous polymer column chromatography (Diaion HP-20: 4.0 kg, mobile phase: first eluted with water and then into methanol. And then eluting with water, the water-eluting portion (concentrated portion eluted with water. 350.0 g) and the methanol-eluting portion (concentrated portion eluted with methanol). 0 g) was obtained.

Example 4 Separation and Purification of Methanol Elution Part The methanol elution part (140.0 g) was subjected to normal phase silica gel column chromatography (3.0 kg, mobile phase: chloroform / methanol / water (lower layer of 20/3/1) → 10/3/1 lower layer → 6/4/1) → methanol (elution was carried out by changing the composition of the mobile phase in the order described here, that is, elution in the mobile phase of the first composition was almost complete. At that time, the composition was changed to the mobile phase having the following composition, and the composition was changed in the same manner as described in the following examples and reference examples. In this case, the elution fractions 1 (0.85 g), 2 (5.67 g), 3 (2.41 g), 4 (1.24 g), 5 (7.73 g), 6 (96.05 g), 7 (10.11 g) and 8 (16.09 g) was obtained (the elution fraction was composed of elution fractions 1 to 8, and the number given to the elution fraction represents the elution order. The amount in parentheses is the amount of each elution fraction. Represents.)

Reference example 1
Among these, the elution fraction 2 (5.67 g) was subjected to reverse phase ODS column chromatography (200 g, mobile phase: methanol / water (40/60 → 50/50 → 70/30 → 80/20) → methanol). Thus, apigenin (48) (76.2 mg) was obtained (the number in parentheses after the compound name represents the number of the structural formula representing the structure of the compound. Examples below) The same applies to the reference examples.)

Reference example 2
Elution fraction 3 (2.41 g) was separated by reverse phase ODS column chromatography (100 g, mobile phase: methanol / water (40/60 → 50/50 → 70/30 → 80/20) → methanol), Purification gave apigenin 7-O-β-D-glucuronide (50) (20.0 mg) and methylsyrin gate 4-O-β-D-glucopyranoside (57) (54.6 mg).

Reference example 3
Elution fraction 4 (1.24 g) was separated by reverse phase ODS column chromatography (60 g, mobile phase: methanol / water (40/60 → 50/50 → 70/30 → 80/20) → methanol). Among the obtained fractions (elution fractions 4-1 to 4-12), the elution fraction 4-4 (151.9 mg) was further subjected to HPLC (column: Cosmosil 5C18-MS-II, mobile phase: acetonitrile. / Methanol / water (10/16/74)) and purified to give (Z) -3-hexenyl β-D-glucopyranoside (58) (28.9 mg). The eluted fraction 4-5 (179.6 mg) was further separated and purified by HPLC (column: Cosmosil 5C18-MS-II, mobile phase: acetonitrile / methanol / water (20/16/64)), Apigenin 7-O-β-D-glucuronic acid methyl ester (51) (16.3 mg) was obtained.

Reference example 4
Elution fraction 5 (7.73 g) was separated by reverse phase ODS column chromatography (300 g, mobile phase: methanol / water (40/60 → 50/50 → 70/30 → 80/20) → methanol). Among the obtained fractions (elution fractions 5-1 to 5-12), elution fraction 5-4 (2217.0 mg) was identified as apigenin 7-O-β-D-glucopyranoside (49). It was. Further, about 750.0 mg of the eluted fraction 5-5 (1190.8 mg), HPLC (column: Cosmosil 5C18-MS-II, mobile phase: acetonitrile / methanol / 1% acetic acid aqueous solution (26/16/58) ) And was purified to give isorhamnetin 3-O-β-D-glucopyranoside (53) (360.0 mg). Further, the eluted fraction 5-7 (182.7 mg) was further separated by HPLC (column: Cosmosil 5C18-MS-II, mobile phase: acetonitrile / methanol / 1% acetic acid aqueous solution (35/16/49)). Purification gave compound 20 (9.1 mg). Of the eluted fraction 5-8 (762.8 mg), 250.3 mg was further separated and purified by HPLC (column: Cosmosil HILIC, mobile phase: acetonitrile / water (90/10)), and compound 21 (36.6 mg) was obtained.

Example 5
Elution fraction 6 (96.05 g) was subjected to reverse phase ODS column chromatography (1.5 kg, mobile phase: methanol / water (30/70 → 40/60 → 50/50 → 70/30) → methanol). Separated and eluted in sequence, elution fractions 6-1 (1.398 g), 6-2 (3.418 g), 6-3 (1.148 g), 6-4 (1.290 g), 6-5 ( 0.800 g), 6-6 (3.179 g), 6-7 (1.680 g), 6-8 (2.317 g), 6-9 (1.216 g), 6-10 (1.682 g), 6-11 (4.850 g), 6-12 (50.269 g), 6-13 (13.375 g), 6-14 (2.208 g), and 6-15 (1.888 g) were obtained. Consists of elution fractions 6-1 to 6-15, and the number assigned to elution fraction 6 indicates the elution order. The amount of to. In parentheses represents the amount of each elution fraction.).

  The obtained eluted fraction 6-6 (205.8 mg) was further separated and purified by HPLC (column: Cosmosil 5C18-MS-II, mobile phase: acetonitrile / water (20/80)), and rutin (52 ) (24.8 mg) was obtained. Further, the eluted fraction 6-7 (345.0 mg) was further separated and purified by HPLC (column: Cosmosil 5C18-MS-II, mobile phase: acetonitrile / water (19/81)), and isorhamnetin 3-O. -Β-D-glucuronide (54) (105.7 mg) was obtained. The eluted fraction 6-8 (343.9 mg) was further separated and purified by HPLC (column: Cosmosil 5C18-MS-II, mobile phase: acetonitrile / water (18/82)), and isorhamnetin 3-O-β. -D-glucopyranoside (53) (8.6 mg), isorhamnetin 3-O-rutinoside (55) (31.8 mg) and isorhamnetin 3-O-robinobioside (56) (32.5 mg) were obtained.

For the eluted fraction 6-11 (1820 mg), HPLC (column: Wakopak Navi 30C-5, mobile phase: acetonitrile / methanol / 1% acetic acid aqueous solution (32/16/52) and column: Cosmosil 5C18-MS-II. , Mobile phase: acetonitrile / methanol / water (25/16/59), acetonitrile / methanol / 1% aqueous acetic acid (32/16/52) or column: Cosmosil HILIC, mobile phase: acetonitrile / water (90/10)) According to the expected suitability, some fractions were separated by Cosmosil 5C18-MS-II, and other fractions were separated by Cosmosil HILIC. (Eluted fraction separated by Wakopak Navi 30C-5) ) Separated. Also in the following examples and reference examples, when similar operations are performed, they are represented in the same manner as described above. ). The obtained elution fraction contained an elution fraction consisting of Bernaldioside B ₂ (46) (112.2 mg). The other elution fractions were Compound 3a (10.5 mg), Compound 26a (42.0 mg), Compound 28 (33.1 mg), Compound 31 (24.3 mg), and Compound 39 (23.9 mg).

  For the eluted fraction 6-12 (2015 mg), HPLC (column: Wakopak Navi 30C-5, mobile phase: acetonitrile / 1% aqueous acetic acid solution (40/60) and column: Cosmosil 5C18-MS-II, mobile phase: Separation and purification with acetonitrile / methanol / 1% acetic acid aqueous solution (30/16/54, 32/16/52 or 35/16/49) or column: Cosmosil HILIC, mobile phase: acetonitrile / water (90/10)) did. The obtained elution fraction contains an elution fraction consisting of belisoside E (42) (68.7 mg), belisoside F (43) (25.0 mg) or beridioside A (44) (35.3 mg). It was. Other elution fractions were compound 22 (9.3 mg), compound 23 (8.1 mg), compound 24 (10.4 mg), compound 25 (28.5 mg), compound 26 (109.2 mg), compound 27 ( 125.8 mg), Compound 29 (45.3 mg), Compound 30 (25.6 mg), Compound 32 (31.7 mg), Compound 33 (17.6 mg), Compound 34 (27.7 mg), Compound 35 (2. 5 mg) and Compound 36 (2.5 mg).

  The eluted fraction 6-13 (782.5 mg) was separated and purified by HPLC (column: Wakopak Navi 30C-5, mobile phase: acetonitrile / 1% aqueous acetic acid solution (33/67 and 40/60)). . The obtained elution fraction includes an elution fraction consisting of berizoside D (41) (35.8 mg), belideoside E (42) (153.7 mg) and beridioside A (44) (26.0 mg). It was. The other elution fraction was Compound 7 (12.2 mg). The eluted fraction 6-14 (946.3 mg) was separated and purified by HPLC (column: Cosmosil 5C18-MS-II, mobile phase: acetonitrile / 1% aqueous acetic acid solution (40/60)). The obtained elution fractions were Compound 1 (122.4 mg), Compound 2 (110.2 mg), Compound 8 (75.7 mg) and Compound 9 (75.7 mg).

Example 6
For elution fraction 7 (10.11 g), reverse phase ODS column chromatography (300 g, mobile phase: methanol / water (20/80 → 30/70 → 40/60 → 50/50 → 70/30) → methanol) And eluted fractions 7-1 (796.8 mg), 7-2 (2520.6 mg), 7-3 (641.1 mg), 7-4 (713.4 mg), 7-5 (1910. 7 mg), 7-6 (3098.7 mg), 7-7 (257.8 mg), 7-8 (286.5 mg) and 7-9 (361.2 mg) were eluted. (The elution fraction consists of elution fractions 7-1 to 7-9, and the number given to elution fraction 7 represents the elution order. The amount in parentheses represents the amount of each elution fraction). The obtained eluted fraction 7-4 (713.4 mg) was separated and purified by HPLC (column: Cosmosil 5C18-MS-II, mobile phase: acetonitrile / methanol / water (22/16/62)), and astor Batanoside D (45) (174.5 mg) was obtained.

  For elution fraction 7-5 (1910.7 mg), HPLC (column: Cosmosil 5C18-MS-II, mobile phase: acetonitrile / methanol / water (26/16/58) and column: Cosmosil HILIC, mobile phase: acetonitrile / Separation and purification with water (90/10)), compound 3 (31.5 mg), compound 4 (26.4 mg), compound 12 (150.2 mg), compound 13 (51.1 mg), compound 18 (272) .3 mg) and compound 19 (10.9 mg) were obtained.

  The eluted fraction 7-6 (450.5 mg) was separated and purified by HPLC (column: Cosmosil 5C18-MS-II, mobile phase: acetonitrile / methanol / water (30/16/54)), and compound 5 ( 14.2 mg), compound 6 (20.4 mg), compound 10 (42.4 mg) and compound 11 (44.7 mg) and compound 37 (81.4 mg), compound 38 (28.2 mg) and belisaponin BS1 (40) (11.9 mg) was obtained.

Reference Example 5
For elution fraction 8 (16.09 g), reverse phase ODS column chromatography (300 g, mobile phase: methanol / water (20/80 → 30/70 → 40/60 → 50/50 → 70/30) → methanol) And eluted fractions 8-1 (3987.2 mg), 8-2 (759.6 mg), 8-3 (774.2 mg), 8-4 (5033.2 mg), 8-5 (427. 2 mg), 8-6 (946.7 mg), 8-7 (2280.8 mg), 8-8 (2189.0 mg) and 8-9 (710.1 mg).

  The obtained eluted fraction 8-7 (960.0 mg) was separated and purified by HPLC (column: Cosmosil 5C18-MS-II, mobile phase: acetonitrile / methanol / water (22/16/62)), Compound 15 (7.1 mg), compound 16 (133.1 mg), compound 18a (46.7 mg) and belisaponin BS6 (47) (43.6 mg) were obtained. The eluted fraction 8-8 (506.5 mg) was separated and purified by HPLC (column: Cosmosil 5C18-MS-II, mobile phase: acetonitrile / methanol / 1% acetic acid aqueous solution (20/16/64)), Perenisoside XIV (14) (19.4 mg) and XVII (17) (70.4 mg) and belisaponin BS6 (47) (102.4 mg) were obtained.

  About each of the compounds 1-39 obtained in the said Example, the property was observed and the optical rotation, the high resolution mass spectrometry, the infrared absorption spectrum, the mass analysis, and the nuclear magnetic resonance spectrum were measured. The results are shown below. From the structural analysis based on this measurement result, each compound was recognized as a novel saponin compound represented by any one of the structural formulas (1) to (39) as shown below. (Note that the same measurement was performed for Compound 3a, Compound 18a, and Compound 26a, and it was recognized that they were the same as Compound 3, Compound 18, and Compound 26, respectively.)

  In addition, with respect to the known compounds of the structural formulas (40) to (58) obtained in the above examples, physical properties such as nuclear magnetic resonance spectrum and mass spectrometry (MS spectrum) are measured, and various physicochemical data are obtained. -It was identified as a known compound represented by the structural formula of the number shown in the said Example by comparison with the literature value of data.

  In addition, the optical rotation was measured using Horiba high sensitive SEPA-300 digital polarimeter (l = 0.5). The infrared absorption spectrum was measured using a Shimadzu FTIR-8100 spectrumometer. High-resolution mass spectrometry (High-resolution FAB-MS) and mass spectrometry (FAB-MS) were measured using a JEOL JMS-SX102A mass spectrometer. Hydrogen and carbon nuclear magnetic resonance spectra (1H and 13C-NMR) spectra were measured using a JEOL JNM ECA-600 spectrometer, and TMS was used as an internal standard. High performance liquid chromatography (HPLC) was measured using a suggested refraction detector, Shimadzu RID-6A and pump Shimadzu LC-6AD.

The numbering used for the following structural analysis by ¹ H-NMR and ¹³ C-NMR is based on general triterpene saponins. Moreover, the symbol in Table 1-Table 44 which shows the measurement result of < ¹ > H-NMR and < ¹³ > C-NMR represents the group shown below, respectively.
Glc: β-D-glucopyranosyl group t-Glc: β-D-glucopyranosyl group at the end t-Glc-I: terminal β-D-glucopyranosyl group t-Glc- bonded to the 3-position of β-D-glucopyranosyl group II: Terminal β-D-glucopyranosyl group bonded to the 6-position of β-D-glucopyranosyl group Rha: α-L-rhamnopyranosyl group i-Rha: α-L-rhamnopyranosyl group inside t-Rha: α at terminal -L-rhamnopyranosyl group Gal: β-D-galactopyranosyl group Fuc: fucopyranosyl group Xyl: xylopyranosyl group Ara (f): arabinofuranosyl Api: apiofuranosyl

  Properties of Compounds 1 to 39 obtained in the Examples: All are amorphous powders

Physical property measurement value and optical rotation of compound 1: [α] _D ²⁶ : + 16.1 ° (c = 3.06, MeOH)
High-resolution mass spectrometry (High-resolution positive-ion FAB-MS: The same applies to the following compounds 2 to 39):
Theoretical value C ₅₂ H ₈₂ O ₂₁ Na (M + Na) ⁺ : 1065.5246
Actual value: 1065.5253
Infrared absorption spectrum (KBr, cm ⁻¹ ): 3442, 1744, 1655, 1254, 1075
-Mass spectrometry positive-ion FAB-MS: m / z 1065 (M + Na) ⁺
negative-ion FAB-MS: m / z 1041 (M-H) ⁻ , 879 (M-C ₆ H ₁₁ O ₅ ) ⁻ , 529 (M-C ₂₀ H ₃₃ O ₁₅ ) ⁻
Nuclear magnetic resonance ^spectrum: 1 H-NMR (600MHz, pyridine -d ₅₎ of [delta] of Table 1 ^shows 13 C-NMR (150MHz, pyridine -d ₅₎ of [delta] _C of Table 2.
From the above results, it was found that it was perenisoside I represented by the structural formula (1).

Physical property measurement value and optical rotation of compound 2: [α] _D ²⁶ : + 19.5 ° (c = 2.76, MeOH)
・ High resolution mass spectrometry:
Theoretical value C ₅₂ H ₈₂ O ₂₁ Na (M + Na) ⁺ : 1065.5246
Actual value: 1065.5242
Infrared absorption spectrum (KBr, cm ⁻¹ ): 3445, 1745, 1655, 1252, 1070
-Mass spectrometry positive-ion FAB-MS: m / z 1065 (M + Na) ⁺
negative-ion FAB-MS: m / z 1041 (M-H) ⁻ , 879 (M-C ₆ H ₁₁ O ₅ ) ⁻ , 529 (M-C ₂₀ H ₃₃ O ₁₅ ) ⁻
Nuclear magnetic resonance ^spectrum: 1 H-NMR (600MHz, pyridine -d ₅₎ of [delta] of Table 1 ^shows 13 C-NMR (150MHz, pyridine -d ₅₎ of [delta] _C of Table 2.
From the above results, it was found that perenisoside II represented by the structural formula (2).

Measured physical properties of compound 3 and optical rotation: [α] _D ²⁷ : + 1.7 ° (c = 2.09, MeOH)
・ High resolution mass spectrometry:
Theoretical value C ₅₆ H ₉₀ O ₂₅ Na (M + Na) ⁺ : 1185.5669
Actual value: 1185.5660
Infrared absorption spectrum (KBr, cm ⁻¹ ): 3445, 1736, 1655, 1256, 1074
-Mass spectrometry positive-ion FAB-MS: m / z 1185 (M + Na) ⁺
negative-ion FAB-MS: m / z 1161 (M−H) ⁻ , 999 (M−C ₆ H ₁₁ O ₅ ) ⁻ , 649 (M−C ₂₀ H ₃₃ O ₁₅ ) ⁻
Nuclear magnetic resonance ^spectrum: 1 H-NMR (600MHz, pyridine -d ₅₎ of [delta] of Table 1 ^shows 13 C-NMR (150MHz, pyridine -d ₅₎ of [delta] _C of Table 2.
From the above results, it was found that it was perenisoside III represented by the structural formula (3).

Physical property measurement value and optical rotation of compound 4: [α] _D ²⁷ : + 4.6 ° (c = 1.80, MeOH)
・ High resolution mass spectrometry:
Theoretical value C ₅₆ H ₉₀ O ₂₅ Na (M + Na) ⁺ : 1185.5669
Actual measurement value: 1185.5665
Infrared absorption spectrum (KBr, cm ⁻¹ ): 3440, 1736, 1655, 1256, 1076
-Mass spectrometry positive-ion FAB-MS: m / z 1185 (M + Na) ⁺
negative-ion FAB-MS: m / z 1161 (M−H) ⁻ , 999 (M−C ₆ H ₁₁ O ₅ ) ⁻ , 649 (M−C ₂₀ H ₃₃ O ₁₅ ) ⁻
Nuclear magnetic resonance ^spectrum: 1 H-NMR (600MHz, pyridine -d ₅₎ of [delta] of Table 1 ^shows 13 C-NMR (150MHz, pyridine -d ₅₎ of [delta] _C of Table 2.
From the above results, it was found that it was perenisoside IV represented by the structural formula (4).

Physical property measurement value and optical rotation of compound 5: [α] _D ²⁵ : + 7.1 ° (c = 0.95, MeOH)
・ High resolution mass spectrometry:
Theoretical value C ₅₆ H ₉₀ O ₂₅ Na (M + Na) ⁺ : 1185.5669
Actual value: 1185.5663
Infrared absorption spectrum (KBr, cm ⁻¹ ): 3440, 1736, 1655, 1260, 1074
-Mass spectrometry positive-ion FAB-MS: m / z 1185 (M + Na) ⁺
negative-ion FAB-MS: m / z 1161 (M-H) ⁻ , 999 (M-C ₆ H ₁₁ O ₅ ) ⁻ , 691 (M-C ₁₈ H ₃₁ O ₁₄ ) ⁻ , 529 (M-C ₂₄ H ₄₁ O ₁₉ ) ⁻
Nuclear magnetic resonance ^spectrum: 1 H-NMR (600MHz, pyridine -d ₅₎ of [delta] of Table 1 ^shows 13 C-NMR (150MHz, pyridine -d ₅₎ of [delta] _C of Table 2.
From the above results, it was found that it was perenisoside V represented by the structural formula (5).

Measured physical properties and optical rotation of compound 6: [α] _D ²⁵ : + 8.8 ° (c = 1.35, MeOH)
・ High resolution mass spectrometry:
Theoretical value C ₅₆ H ₉₀ O ₂₅ Na (M + Na) ⁺ : 1185.5669
Actual measurement value: 1185.5567
Infrared absorption spectrum (KBr, cm ⁻¹ ): 3440, 1736, 1656, 1256, 1075
-Mass spectrometry positive-ion FAB-MS: m / z 1185 (M + Na) ⁺
negative-ion FAB-MS: m / z 1161 (M−H) ⁻ , 999 (M−C ₆ H ₁₁ O ₅ ) ⁻ , 837 (M−C ₁₂ H ₂₂ O ₁₀ ) ⁻
Nuclear magnetic resonance ^spectrum: 1 H-NMR (600MHz, pyridine -d ₅₎ of [delta] of Table 1 ^shows 13 C-NMR (150MHz, pyridine -d ₅₎ of [delta] _C of Table 2.
From the above results, it was found that it was perenisoside VI represented by the structural formula (6).

Physical property measured value and optical rotation of compound 7: [α] _D ²⁵ : + 14.3 ° (c = 0.71, MeOH)
・ High resolution mass spectrometry:
Theoretical value C ₅₈ H ₉₂ O ₂₆ Na (M + Na) ⁺ : 1227.5775
Actual value: 1227.5780
Infrared absorption spectrum (KBr, cm ⁻¹ ): 3445, 1736, 1655, 1256, 1077
-Mass spectrometry positive-ion FAB-MS: m / z 1227 (M + Na) ⁺
negative-ion FAB-MS: m / z 1203 (M-H) ⁻ , 1041 (M-C ₆ H ₁₁ O ₅ ) ⁻ , 691 (M-C ₂₀ H ₃₃ O ₁₅ ) ⁻ , 529 (M-C ₂₆ H ₄₃ O ₂₀ ) ⁻
Nuclear magnetic resonance ^spectrum: 1 H-NMR (600MHz, pyridine -d ₅₎ a [delta] in Table 3 and Table ^{4, 13 C-NMR (150MHz} , pyridine -d ₅₎ of [delta] _C in Table 5 and Table 6 Show.
From the above results, it was found that perenisoside VII represented by the structural formula (7).

Physical property measurement value and optical rotation of compound 8: [α] _D ²⁷ : + 11.9 ° (c = 3.09, MeOH)
・ High resolution mass spectrometry:
Theoretical value C ₅₈ H ₉₂ O ₂₅ Na (M + Na) ⁺ : 1211.5825
Actual measurement value: 1211.5833
Infrared absorption spectrum (KBr, cm ⁻¹ ): 3440, 1744, 1655, 1256, 1069
-Mass spectrometry positive-ion FAB-MS: m / z 1211 (M + Na) ⁺
negative-ion FAB-MS: m / z 1187 (M-H) ⁻ , 1041 (M-C ₆ H ₁₁ O ₄ ) ⁻ , 1025 (M-C ₆ H ₁₁ O ₅ ) ⁻ , 879 (M-C ₁₂ H ₂₁ O ₉ ) ⁻ , 675 (M-C ₂₀ H ₃₃ O ₁₅ ) ⁻ , 529 (M-C ₂₆ H ₄₃ O ₁₉ ) ⁻
Nuclear magnetic resonance ^spectrum: 1 H-NMR (600MHz, pyridine -d ₅₎ a [delta] in Table 3 and Table ^{4, 13 C-NMR (150MHz} , pyridine -d ₅₎ of [delta] _C in Table 5 and Table 6 Show.
From the above results, it was found that perenisoside VIII represented by the structural formula (8).

Physical property measured value and optical rotation of compound 9: [α] _D ²⁷ : + 13.9 ° (c = 3.79, MeOH)
・ High resolution mass spectrometry:
Theoretical value C ₅₈ H ₉₂ O ₂₅ Na (M + Na) ⁺ : 1211.5825
Actual measurement value: 1211.5819
Infrared absorption spectrum (KBr, cm ⁻¹ ): 3440, 1736, 1656, 1256, 1065
-Mass spectrometry positive-ion FAB-MS: m / z 1211 (M + Na) ⁺
negative-ion FAB-MS: m / z 1187 (M-H) ⁻ , 1041 (M-C ₆ H ₁₁ O ₄ ) ⁻ , 1025 (M-C ₆ H ₁₁ O ₅ ) ⁻ , 879 (M-C ₁₂ H ₂₁ O ₉ ) ⁻ , 675 (M-C ₂₀ H ₃₃ O ₁₅ ) ⁻ , 529 (M-C ₂₆ H ₄₃ O ₁₉ ) ⁻
Nuclear magnetic resonance ^spectrum: 1 H-NMR (600MHz, pyridine -d ₅₎ a [delta] in Table 3 and Table ^{4, 13 C-NMR (150MHz} , pyridine -d ₅₎ of [delta] _C in Table 5 and Table 6 Show.
From the above results, it was found that it was perenisoside IX represented by the structural formula (9).

Physical property measured value and optical rotation of compound 10: [α] _D ²⁵ : + 11.3 ° (c = 1.97, MeOH)
・ High resolution mass spectrometry:
Theoretical value C ₆₄ H ₁₀₂ O ₃₁ Na (M + Na) ⁺ : 1389.6303
Actual value: 1389.6296
Infrared absorption spectrum (KBr, cm ⁻¹ ): 3440, 1736, 1656, 1256, 1077
-Mass spectrometry positive-ion FAB-MS: m / z 1389 (M + Na) ⁺
negative-ion FAB-MS: m / z 1365 (M-H) ⁻ , 1203 (M-C ₆ H ₁₁ O ₅ ) ⁻ , 1041 (M-C ₁₂ H ₂₁ O ₁₀ ) ⁻ , 733 (M-C ₂₄ H ₄₁ O ₁₉ ) ⁻ , 691 (M-C ₂₆ H ₄₃ O ₂₀ ) ⁻ , 529 (M-C ₃₂ H ₅₃ O ₂₅ ) ⁻
Nuclear magnetic resonance ^spectrum: 1 H-NMR (600MHz, pyridine -d ₅₎ a [delta] in Table 3 and Table ^{4, 13 C-NMR (150MHz} , pyridine -d ₅₎ of [delta] _C in Table 5 and Table 6 Show.
From the above results, it was found that perenisoside X represented by the structural formula (10).

Measured physical properties of compound 11 and optical rotation: [α] _D ²⁵ : + 31.2 ° (c = 2.98, MeOH)
・ High resolution mass spectrometry:
Theoretical value C ₆₄ H ₁₀₂ O ₃₁ Na (M + Na) ⁺ : 1389.6303
Actual value: 1389.6298
Infrared absorption spectrum (KBr, cm ⁻¹ ): 3445, 1736, 1655, 1251, 1075
-Mass spectrometry positive-ion FAB-MS: m / z 1389 (M + Na) ⁺
negative-ion FAB-MS: m / z 1365 (M-H) ⁻ , 1203 (M-C ₆ H ₁₁ O ₅ ) ⁻ , 1041 (M-C ₁₂ H ₂₁ O ₁₀ ) ⁻ , 733 (M-C ₂₄ H ₄₁ O ₁₉ ) ⁻ , 691 (M-C ₂₆ H ₄₃ O ₂₀ ) ⁻ , 529 (M-C ₃₂ H ₅₃ O ₂₅ ) ⁻
Nuclear magnetic resonance ^spectrum: 1 H-NMR (600MHz, pyridine -d ₅₎ a [delta] in Table 3 and Table ^{4, 13 C-NMR (150MHz} , pyridine -d ₅₎ of [delta] _C in Table 5 and Table 6 Show.
From the above results, it was found that perenisoside XI represented by the structural formula (11).

Measured physical properties and optical rotation of compound 12: [α] _D ²⁷ : + 0.9 ° (c = 2.53, MeOH)
・ High resolution mass spectrometry:
Theoretical value C ₅₄ H ₈₈ O ₂₄ Na (M + Na) ⁺ : 1143.5563
Actual value: 1143.5569
Infrared absorption spectrum (KBr, cm ⁻¹ ): 3440, 1736, 1655, 1260, 1075
-Mass spectrometry positive-ion FAB-MS: m / z 1143 (M + Na) ⁺
negative-ion FAB-MS: m / z 1119 (M-H) ⁻ , 957 (M-C ₆ H ₁₁ O ₅ ) ⁻ , 811 (M-C ₁₂ H ₂₁ O ₉ ) ⁻ , 649 (M-C ₁₈ H ₃₁ O ₁₄ ) ⁻ , 487 (M-C ₂₄ H ₄₁ O ₁₉ ) ⁻
Nuclear magnetic resonance ^spectrum: 1 H-NMR (600MHz, pyridine -d ₅₎ in Table 7 and Table 8 [delta] ^of, 13 C-NMR ^(150MHz, pyridine -d ₅₎ of [delta] _C of Table 9 and Table 10 Show.
From the above results, it was found that perenisoside XII represented by the structural formula (12).

Physical property measured value and optical rotation of compound 13: [α] _D ²⁷ : + 7.6 ° (c = 3.11, MeOH)
・ High resolution mass spectrometry:
Theoretical _{C 58 H 94 O 26 Na (} M + Na) +: 1229.5931
Actual measurement value: 129.5924
Infrared absorption spectrum (KBr, cm ⁻¹ ): 3440, 1736, 1655, 1075
-Mass spectrometry positive-ion FAB-MS: m / z 1229 (M + Na) ⁺
negative-ion FAB-MS: m / z 1205 (M−H) ⁻ , 1119 (M C ₄ H ₇ O ₂ ) ⁻ , 957 (M C ₁₀ H ₁₇ O ₇ ) ⁻ , 811 (M C _{16 ^{H 27 O 11) -, 649}} (M-C 22 H 37 O 16) -, 487 (M-C 28 H 46 O 21) -
Nuclear magnetic resonance ^spectrum: 1 H-NMR (600MHz, pyridine -d ₅₎ in Table 7 and Table 8 [delta] ^of, 13 C-NMR ^(150MHz, pyridine -d ₅₎ of [delta] _C of Table 9 and Table 10 Show.
From the above results, it was found that perenisoside XIII represented by the structural formula (13).

Physical property measurement value and optical rotation of compound 14: [α] _D ²³ : −2.3 ° (c = 1.30, MeOH)
・ High resolution mass spectrometry:
Theoretical value C ₆₀ H ₉₈ O ₂₉ Na (M + Na) ⁺ : 1305.5609
Actual measurement value: 1305.5608
Infrared absorption spectrum (KBr, cm ⁻¹ ): 3440, 1736, 1656, 1260, 1075
-Mass spectrometry positive-ion FAB-MS: m / z 1305 (M + Na) ⁺
negative-ion FAB-MS: m / z 1281 (M-H) ⁻ , 1119 (M-C ₆ H ₁₁ O ₅ ) ⁻ , 957 (M-C ₁₂ H ₂₁ O ₁₀ ) ⁻ , 811 (M-C ₁₈ H ₃₁ O ₁₄ ) ⁻ , 649 (M-C ₂₄ H ₄₁ O ₁₉ ) ⁻ , 487 (M-C ₃₀ H ₅₀ O ₂₄ ) ⁻
Nuclear magnetic resonance ^spectrum: 1 H-NMR (600MHz, pyridine -d ₅₎ in Table 7 and Table 8 [delta] ^of, 13 C-NMR ^(150MHz, pyridine -d ₅₎ of [delta] _C of Table 9 and Table 10 Show.
From the above results, it was found that perenisoside XIV represented by the structural formula (14).

Physical property measurement value and optical rotation of compound 15: [α] _D ²⁵ : −2.5 ° (c = 0.56, MeOH)
・ High resolution mass spectrometry:
Theoretical value C ₆₂ H ₁₀₀ O ₃₀ Na (M + Na) ⁺ : 1347.6197
Actual measurement value: 1347.6190
Infrared absorption spectrum (KBr, cm ⁻¹ ): 3445, 1736, 1655, 1260, 1076
-Mass spectrometry positive-ion FAB-MS: m / z 1347 (M + Na) ⁺
negative-ion FAB-MS: m / z 1323 (M-H) ⁻ , 1161 (M-C ₆ H ₁₁ O ₅ ) ⁻ , 999 (M-C ₁₂ H ₂₁ O ₁₀ ) ⁻ , 649 (M-C ₂₆ H ₄₃ O ₂₂ ) ⁻ , 487 (M-C ₃₂ H ₅₃ O ₂₇ ) ⁻
Nuclear magnetic resonance ^spectrum: 1 H-NMR (600MHz, pyridine -d ₅₎ in Table 7 and Table 8 [delta] ^of, 13 C-NMR ^(150MHz, pyridine -d ₅₎ of [delta] _C of Table 9 and Table 10 Show.
From the above results, it was found that perenisoside XV represented by the structural formula (15).

Physical property measurement value and optical rotation of compound 16: [α] _D ²⁷ : + 1.5 ° (c = 3.58, MeOH)
・ High resolution mass spectrometry:
Theoretical value C ₆₄ H ₁₀₄ O ₃₁ Na (M + Na) ⁺ : 1391.6459
Actual value: 1391.6454
Infrared absorption spectrum (KBr, cm ⁻¹ ): 3440, 1736, 1655, 1260, 1076
-Mass spectrometry positive-ion FAB-MS: m / z 1391 (M + Na) ⁺
negative-ion FAB-MS: m / z 1367 (M−H) ⁻ , 1281 (M C ₄ H ₇ O ₂ ) ⁻ , 1119 (M C ₁₀ H ₁₇ O ₇ ) ⁻ , 811 (M C ₂₂ H ₃₇ O ₁₆ ) ⁻ , 649 (M-C ₂₈ H ₄₇ O ₂₁ ) ⁻ , 487 (M-C ₃₄ H ₅₇ O ₂₆ ) ⁻
Nuclear magnetic resonance ^spectrum: 1 H-NMR (600MHz, pyridine -d ₅₎ in Table 7 and Table 8 [delta] ^of, 13 C-NMR ^(150MHz, pyridine -d ₅₎ of [delta] _C of Table 9 and Table 10 Show.
From the above results, it was found that perenisoside XVI represented by the structural formula (16).

Physical property measurement value and optical rotation of compound 17: [α] _D ²⁷ : −1.1 ° (c = 3.27, MeOH)
・ High resolution mass spectrometry:
Theoretical value C ₆₀ H ₉₈ O ₂₉ Na (M + Na) ⁺ : 1305.5609
Actual value: 1305.5097
Infrared absorption spectrum (KBr, cm ⁻¹ ): 3440, 1736, 1655, 1230, 1075
-Mass spectrometry positive-ion FAB-MS: m / z 1305 (M + Na) ⁺
negative-ion FAB-MS: m / z 1281 (M-H) ⁻ , 1119 (M-C ₆ H ₁₁ O ₅ ) ⁻ , 957 (M-C ₁₂ H ₂₁ O ₁₀ ) ⁻ , 811 (M-C ₁₈ H ₃₁ O ₁₄ ) ⁻ , 649 (M-C ₂₄ H ₄₁ O ₁₉ ) ⁻ , 487 (M-C ₃₀ H ₅₁ O ₂₄ ) ⁻
Nuclear magnetic resonance ^spectrum: 1 H-NMR (600MHz, pyridine -d ₅₎ a [delta] in Table 11 and Table ^{12, 13 C-NMR (150MHz} , pyridine -d ₅₎ of [delta] _C in Table 13 and Table 14 Show.
From the above results, it was found that perenisoside XVII represented by the structural formula (17).

Physical property measured value / optical rotation of compound 18: [α] _D ²⁶ : −3.8 ° (c = 3.39, MeOH)
・ High resolution mass spectrometry:
Theoretical value C ₅₄ H ₈₈ O ₂₃ Na (M + Na) ⁺ : 1127.5614
Actual value: 1127.5619
Infrared absorption spectrum (KBr, cm ⁻¹ ): 3440, 1736, 1655, 1260, 1063
-Mass spectrometry positive-ion FAB-MS: m / z 1127 (M + Na) ⁺
negative-ion FAB-MS: m / z 1103 (M-H) ⁻ , 957 (M-C ₆ H ₁₁ O ₄ ) ⁻ , 941 (M-C ₆ H ₁₁ O ₅ ) ⁻ , 633 (M-C ₁₈ H ₃₁ O ₁₄ ) ⁻ , 487 (M-C ₂₄ H ₄₁ O ₁₈ ) ⁻
Nuclear magnetic resonance ^spectrum: 1 H-NMR (600MHz, pyridine -d ₅₎ a [delta] in Table 11 and Table ^{12, 13 C-NMR (150MHz} , pyridine -d ₅₎ of [delta] _C in Table 13 and Table 14 Show.
From the above results, it was found that perenisoside XVIII represented by the structural formula (18).

Physical property measured value and optical rotation of compound 19: [α] _D ²⁴ : −7.5 ° (c = 0.57, MeOH)
・ High resolution mass spectrometry:
Theoretical value C ₅₄ H ₈₈ O ₂₃ Na (M + Na) ⁺ : 1127.5614
Actual value: 1127.5619
Infrared absorption spectrum (KBr, cm ⁻¹ ): 3451, 1741, 1655, 1260, 1061
-Mass spectrometry positive-ion FAB-MS: m / z 1127 (M + Na) ⁺
negative-ion FAB-MS: m / z 1103 (M−H) ⁻ , 649 (M−C ₁₈ H ₃₃ O ₁₃ ) ⁻ , 633 (M−C ₁₈ H ₃₃ O ₁₄ ) ⁻
Nuclear magnetic resonance ^spectrum: 1 H-NMR (600MHz, pyridine -d ₅₎ a [delta] in Table 11 and Table ^{12, 13 C-NMR (150MHz} , pyridine -d ₅₎ of [delta] _C in Table 13 and Table 14 Show.
From the above results, it was found that perenisoside XIX represented by the structural formula (19).

Physical property measurement value and optical rotation of compound 20: [α] _D ²⁴ : −7.8 ° (c = 0.76, MeOH)
・ High resolution mass spectrometry:
Theoretical value C ₅₇ H ₉₀ O ₂₄ Na (M + Na) ⁺ : 1181.5720
Actual measurement value: 1181.5724
Infrared absorption spectrum (KBr, cm ⁻¹ ): 3445, 1736, 1655, 1260, 1050
-Mass spectrometry positive-ion FAB-MS: m / z 1181 (M + Na) ⁺
negative-ion FAB-MS: m / z 1157 (M−H) ⁻ , 1115 (M C ₂ H ₃ O) ⁻ , 1011 (M C ₆ H ₁₁ O ₄ ) ⁻ , 649 (M C ₂₁ H ₃₃ O ₁₄ ) ⁻ , 503 (M-C ₂₇ H ₄₃ O ₁₈ ) ⁻
Nuclear magnetic resonance ^spectrum: 1 H-NMR (600MHz, pyridine -d ₅₎ in Table 15 and Table 16 [delta] ^of, 13 C-NMR ^(150MHz, pyridine -d ₅₎ of [delta] _C in Table 17 and Table 18 Show.
From the above results, it was found that it was perenisaponin A represented by the structural formula (20).

Physical property measurement value and optical rotation of compound 21: [α] _D ²⁶ : −13.4 ° (c = 1.68, MeOH)
・ High resolution mass spectrometry:
Theoretical value C ₇₁ H ₁₁₂ O ₃₁ Na (M + Na) ⁺ : 1483.7085
Actual value: 1483.7076
Infrared absorption spectrum (KBr, cm ⁻¹ ): 3445, 1736, 1655, 1260, 1080
-Mass spectrometry positive-ion FAB-MS: m / z 1483 (M + Na) ⁺
negative-ion FAB-MS: m / z 1459 (M-H) ⁻ , 1115 (M-C ₁₆ H ₂₅ O ₈ ) ⁻ , 649 (M-C ₃₅ H ₅₅ O ₂₁ ) ⁻ , 503 (M-C ₄₁ H ₆₅ O ₂₅ ) ⁻
Nuclear magnetic resonance ^spectrum: 1 H-NMR (600MHz, pyridine -d ₅₎ in Table 15 and Table 16 [delta] ^of, 13 C-NMR ^(150MHz, pyridine -d ₅₎ of [delta] _C in Table 17 and Table 18 Show.
From the above results, it was found that it was perenisaponin B represented by the structural formula (21).

Physical property measured value and optical rotation of compound 22: [α] _D ²⁶ : −13.3 ° (c = 0.83, MeOH)
・ High resolution mass spectrometry:
Theoretical value C ₆₅ H ₁₀₄ O ₂₈ Na (M + Na) ⁺ : 1355.6612
Actual measurement value: 13555.6617
Infrared absorption spectrum (KBr, cm ⁻¹ ): 3445, 1735, 1655, 1261, 1051
-Mass spectrometry positive-ion FAB-MS: m / z 1355 (M + Na) ⁺
negative-ion FAB-MS: m / z 1331 (M-H) ⁻ , 1119 (M-C ₅ H ₉ O ₄ ) ⁻ , 649 (M-C ₂₉ H ₄₇ O ₁₈ ) ⁻ , 503 (M-C ₃₅ H ₅₇ O ₂₂ ) ⁻
Nuclear magnetic resonance ^spectrum: 1 H-NMR (600MHz, pyridine -d ₅₎ in Table 15 and Table 16 [delta] ^of, 13 C-NMR ^(150MHz, pyridine -d ₅₎ of [delta] _C in Table 17 and Table 18 Show.
From the above results, it was found that it was perenisaponin C represented by the structural formula (22).

Measured physical properties and optical rotation of compound 23: [α] _D ²⁶ : −14.9 ° (c = 0.68, MeOH)
・ High resolution mass spectrometry:
Theoretical value C ₆₅ H ₁₀₄ O ₂₉ Na (M + Na) ⁺ : 1371.6561
Actual value: 1371.6558
Infrared absorption spectrum (KBr, cm ⁻¹ ): 3445, 1736, 1656, 1261, 1049
-Mass spectrometry positive-ion FAB-MS: m / z 1371 (M + Na) ⁺
negative-ion FAB-MS: m / z 1347 (M-H) ⁻ , 1219 (M-C ₆ H ₉ O ₃ ) ⁻ , 1073 (M-C ₁₂ H ₁₉ O ₇ ) ⁻ , 941 (M-C ₁₇ H ₂₇ O ₁₁ ) ⁻ , 649 (M-C ₂₉ H ₄₉ O ₁₉ ) ⁻
Nuclear magnetic resonance ^spectrum: 1 H-NMR (600MHz, pyridine -d ₅₎ in Table 19 and Table 20 to [delta] ^of, 13 C-NMR ^(150MHz, pyridine -d ₅₎ of [delta] _C in Table 21 and Table 22 Show.
From the above results, it was found that it was perenisaponin D represented by the structural formula (23).

Measured physical properties and optical rotation of compound 24: [α] _D ²⁶ : −13.7 ° (c = 0.83, MeOH)
・ High resolution mass spectrometry:
Theoretical value C ₇₅ H ₁₂₀ O ₃₄ Na (M + Na) ⁺ : 15875.7559
Actual value: 15877.556
Infrared absorption spectrum (KBr, cm ⁻¹ ): 3445, 1736, 1656, 1250, 1051
-Mass spectrometry positive-ion FAB-MS: m / z 1587 (M + Na) ⁺
negative-ion FAB-MS: m / z 1563 (M-H) ⁻ , 1417 (M-C ₆ H ₁₁ O ₄ ) ⁻ , 1287 (M-C ₁₁ H ₁₇ O ₈ ) ⁻ , 1141 (M-C ₁₇ H ₂₇ O ₁₂ ) ⁻ , 649 (M-C ₃₉ H ₆₃ O ₂₄ ) ⁻
Nuclear magnetic resonance ^spectrum: 1 H-NMR (600MHz, pyridin -d ₅₎ in Table 19 and Table 20 to [delta] ^a, 13 C-NMR ^(150MHz, pyridin -d ₅₎ of [delta] _C in Table 21 and Table 22 Show.
From the above results, it was found that it was Perenisaponin E represented by Structural Formula (24).

Physical property measured value and optical rotation of compound 25: [α] _D ²⁴ : −21.2 ° (c = 1.97, MeOH)
・ High resolution mass spectrometry:
Theoretical value C ₇₁ H ₁₁₄ O ₃₂ Na (M + Na) ⁺ : 1501.7191
Actual measurement value: 1501.7188
Infrared absorption spectrum (KBr, cm ⁻¹ ): 3445, 1736, 1655, 1260, 1049
-Mass spectrometry positive-ion FAB-MS: m / z 1501 (M + Na) ⁺
negative-ion FAB-MS: m / z 1477 (M-H) ⁻ , 1219 (M-C ₁₂ H ₁₉ O ₆ ) ⁻ , 795 (M-C ₂₉ H ₄₇ O ₁₈ ) ⁻ , 649 (M-C ₃₅ H ₇₇ O ₂₂ ) ⁻
Nuclear magnetic resonance ^spectrum: the 1 H-NMR (600MHz, pyridine -d ₅₎ Table 23 [delta] of the ^shows 13 C-NMR (150MHz, pyridine -d ₅₎ of [delta] _C of Table 24.
From the above results, it was found that it was perenisaponin F represented by the structural formula (25).

Measured physical properties and optical rotation of compound 26: [α] _D ²⁵ : −19.5 ° (c = 4.48, MeOH)
・ High resolution mass spectrometry:
Theoretical value C ₆₃ H ₁₀₀ O ₂₈ Na (M + Na) ⁺ : 1327.6299
Actual value: 1327.6296
Infrared absorption spectrum (KBr, cm ⁻¹ ): 3450, 1736, 1638, 1255, 1049
-Mass spectrometry positive-ion FAB-MS: m / z 1327 (M + Na) ⁺
negative-ion FAB-MS: m / z 1303 (M−H) ⁻ , 1157 (M−C ₆ H ₁₁ O ₄ ) ⁻
Nuclear magnetic resonance spectrum: δ of ¹ H-NMR (600 MHz, pyridine-d ₅ ) in Table 25 and Table 26, δ _C of ¹³ C-NMR (150 MHz, pyridine-d ₅ ) in Table 27 and Table 28 Show.
From the above results, it was found that it was perenisaponin G represented by the structural formula (26).

Measured physical property value and optical rotation of compound 27: [α] _D ²⁵ : −30.9 ° (c = 4.43, MeOH)
・ High resolution mass spectrometry:
Theoretical value C ₆₃ H ₁₀₀ O ₂₈ Na (M + Na) ⁺ : 1327.6299
Actual value: 1327.6305
Infrared absorption spectrum (KBr, cm ⁻¹ ): 3450, 1734, 1638, 1252, 1049
-Mass spectrometry positive-ion FAB-MS: m / z 1327 (M + Na) ⁺
negative-ion FAB-MS: m / z 1303 (M-H) ⁻ , 1157 (M-C ₆ H ₁₁ O ₄ ) ⁻ , 649 (M-C ₂₇ H ₄₃ O ₁₈ ) ⁻
Nuclear magnetic resonance spectrum: δ of ¹ H-NMR (600 MHz, pyridine-d ₅ ) in Table 25 and Table 26, δ _C of ¹³ C-NMR (150 MHz, pyridine-d ₅ ) in Table 27 and Table 28 Show.
From the above results, it was found that it was perenisaponin H represented by the structural formula (27).

Measured physical properties and optical rotation of compound 28: [α] _D ²⁵ : −22.0 ° (c = 2.28, MeOH)
・ High resolution mass spectrometry:
Theoretical value C ₆₅ H ₁₀₄ O ₂₉ Na (M + Na) ⁺ : 1371.6561
Actual value: 1371.6558
Infrared absorption spectrum (KBr, cm ⁻¹ ): 3450, 1738, 1638, 1256, 1049
-Mass spectrometry positive-ion FAB-MS: m / z 1371 (M + Na) ⁺
negative-ion FAB-MS: m / z 1347 (M−H) ⁻ , 1157 (M−C ₁₀ H ₁₇ O ₆ ) ⁻ , 649 (M−C ₂₉ H ₄₇ O ₁₉ ) ⁻
Nuclear magnetic resonance spectrum: δ of ¹ H-NMR (600 MHz, pyridine-d ₅ ) in Table 25 and Table 26, δ _C of ¹³ C-NMR (150 MHz, pyridine-d ₅ ) in Table 27 and Table 28 Show.
From the above results, it was found that it was perenisaponin I represented by the structural formula (28).

Measured physical properties and optical rotation of compound 29: [α] _D ²⁵ : −48.5 ° (c = 1.20, MeOH)
・ High resolution mass spectrometry:
Theoretical value C ₆₇ H ₁₀₆ O ₃₀ Na (M + Na) ⁺ : 1413.6667
Actual value: 1413.6664
Infrared absorption spectrum (KBr, cm ⁻¹ ): 3445, 1734, 1638, 1260, 1049
-Mass spectrometry positive-ion FAB-MS: m / z 1413 (M + Na) ⁺
negative-ion FAB-MS: m / z 1389 (M−H) ⁻ , 649 (M−C ₃₁ H ₄₉ O ₂₀ ) ⁻
Nuclear magnetic resonance ^spectrum: 1 H-NMR (600MHz, pyridine -d ₅₎ a [delta] in Table 29 and Table ^{30, 13 C-NMR (150MHz} , pyridine -d ₅₎ of [delta] _C in Table 31 and Table 32 Show.
From the above results, it was found that it was Perenisaponin J represented by Structural Formula (29).

Measured physical properties and optical rotation of compound 30: [α] _D ²⁵ : −47.1 ° (c = 1.00, MeOH)
・ High resolution mass spectrometry:
Theoretical value C ₆₇ H ₁₀₆ O ₃₀ Na (M + Na) ⁺ : 1413.6667
Actual value: 1413.6667
Infrared absorption spectrum (KBr, cm ⁻¹ ): 3445, 1734, 1638, 1260, 1049
-Mass spectrometry positive-ion FAB-MS: m / z 1413 (M + Na) ⁺
negative-ion FAB-MS: m / z 1389 (M−H) ⁻ , 649 (M−C ₃₁ H ₄₉ O ₂₀ ) ⁻
Nuclear magnetic resonance ^spectrum: 1 H-NMR (600MHz, pyridine -d ₅₎ a [delta] in Table 29 and Table ^{30, 13 C-NMR (150MHz} , pyridine -d ₅₎ of [delta] _C in Table 31 and Table 32 Show.
From the above results, it was found that it was perenisaponin K represented by the structural formula (30).

Measured value and optical rotation of compound 31: [α] _D ²⁶ : −22.4 ° (c = 1.64, MeOH)
・ High resolution mass spectrometry:
Theoretical value C ₆₉ H ₁₁₀ O ₃₁ Na (M + Na) ⁺ : 14577.6929
Actual value: 14577.6937
Infrared absorption spectrum (KBr, cm ⁻¹ ): 3450, 1736, 1655, 1260, 1049
-Mass spectrometry positive-ion FAB-MS: m / z 1457 (M + Na) ⁺
negative-ion FAB-MS: m / z 1433 (M-H) ⁻ , 1115 (M-C ₁₄ H ₂₃ O ₈ ) ⁻ , 649 (M-C ₃₃ H ₅₃ O ₂₁ ) ⁻ , 503 (M-C ₃₉ H ₆₂ O ₂₅ ) ⁻
Nuclear magnetic resonance ^spectrum: 1 H-NMR (600MHz, pyridine -d ₅₎ a [delta] in Table 29 and Table ^{30, 13 C-NMR (150MHz} , pyridine -d ₅₎ of [delta] _C in Table 31 and Table 32 Show.
From the above results, it was found that it was perenisaponin L represented by the structural formula (31).

Physical property measurement value and optical rotation of compound 32: [α] _D ²⁵ : −24.2 ° (c = 1.42, MeOH)
・ High resolution mass spectrometry:
Theoretical value C ₇₃ H ₁₁₆ O ₃₃ Na (M + Na) ⁺ : 1543.7297
Actual value: 1543.7290
Infrared absorption spectrum (KBr, cm ⁻¹ ): 3445, 1736, 1655, 1260, 1049
-Mass spectrometry positive-ion FAB-MS: m / z 1543 (M + Na) ⁺
negative-ion FAB-MS: m / z 1519 (M−H) ⁻ , 649 (M−C ₃₇ H ₅₉ O ₂₃ ) ⁻
Nuclear magnetic resonance spectrum: δ of ¹ H-NMR (600 MHz, pyridine-d ₅ ) in Table 33 and Table 34, δ _C of ¹³ C-NMR (150 MHz, pyridine-d ₅ ) in Table 35 and Table 36 Show.
From the above results, it was found that it was perenisaponin M represented by the structural formula (32).

Measured physical properties and optical rotation of compound 33: [α] _D ²⁵ : −26.6 ° (c = 1.22, MeOH)
・ High resolution mass spectrometry:
Theoretical value C ₇₃ H ₁₁₆ O ₃₃ Na (M + Na) ⁺ : 1543.7297
Actual value: 1543.7294
Infrared absorption spectrum (KBr, cm ⁻¹ ): 3445, 1736, 1656, 1261, 1051
-Mass spectrometry positive-ion FAB-MS: m / z 1543 (M + Na) ⁺
negative-ion FAB-MS: m / z 1519 (M−H) ⁻ , 649 (M−C ₃₇ H ₅₉ O ₂₃ ) ⁻
Nuclear magnetic resonance spectrum: δ of ¹ H-NMR (600 MHz, pyridine-d ₅ ) in Table 33 and Table 34, δ _C of ¹³ C-NMR (150 MHz, pyridine-d ₅ ) in Table 35 and Table 36 Show.
From the above results, it was found that it was perenisaponin N represented by the structural formula (33).

Measured physical properties and optical rotation of compound 34: [α] _D ²⁵ : −24.2 ° (c = 1.80, MeOH)
・ High resolution mass spectrometry:
Theoretical value C ₇₇ H ₁₂₂ O ₃₅ Na (M + Na) ⁺ : 1629.664
Actual value: 1629,659
Infrared absorption spectrum (KBr, cm ⁻¹ ): 3445, 1736, 1638, 1261, 1049
-Mass spectrometry positive-ion FAB-MS: m / z 1629 (M + Na) ⁺
negative-ion FAB-MS: m / z 1605 (M−H) ⁻ , 649 (M−C ₄₁ H ₆₅ O ₂₅ ) ⁻
Nuclear magnetic resonance spectrum: δ of ¹ H-NMR (600 MHz, pyridine-d ₅ ) in Table 33 and Table 34, δ _C of ¹³ C-NMR (150 MHz, pyridine-d ₅ ) in Table 35 and Table 36 Show.
From the above results, it was found that it was perenisaponin O represented by the structural formula (34).

Measured physical properties and optical rotation of compound 35: [α] _D ²⁶ : −10.6 ° (c = 0.25, MeOH)
・ High resolution mass spectrometry:
Theoretical value C ₆₇ H ₁₀₆ O ₃₀ Na (M + Na) ⁺ : 1413.6667
Actual value: 1413.6653
Infrared absorption spectrum (KBr, cm ⁻¹ ): 3445, 1736, 1655, 1256, 1049
-Mass spectrometry positive-ion FAB-MS: m / z 1413 (M + Na) ⁺
negative-ion FAB-MS: m / z 1389 (M−H) ⁻
Nuclear magnetic resonance spectrum: δ of ¹ H-NMR (600 MHz, pyridine-d ₅ ) in Table 37 and Table 38, δ _C of ¹³ C-NMR (150 MHz, pyridine-d ₅ ) in Table 39 and Table 40 Show.
From the above results, it was found that it was perenisaponin P represented by the structural formula (35).

Physical property measured value / optical rotation of compound 36: [α] _D ²⁶ : −12.5 ° (c = 0.23, MeOH)
・ High resolution mass spectrometry:
Theoretical value C ₆₇ H ₁₀₆ O ₃₀ Na (M + Na) ⁺ : 1413.6667
Actual value: 1413.6670
Infrared absorption spectrum (KBr, cm ⁻¹ ): 3445, 1736, 1655, 1260, 1051
-Mass spectrometry positive-ion FAB-MS: m / z 1413 (M + Na) ⁺
negative-ion FAB-MS: m / z 1389 (M−H) ⁻ , 649 (M−C ₃₁ H ₄₉ O ₂₀ ) ⁻
Nuclear magnetic resonance spectrum: δ of ¹ H-NMR (600 MHz, pyridine-d ₅ ) in Table 37 and Table 38, δ _C of ¹³ C-NMR (150 MHz, pyridine-d ₅ ) in Table 39 and Table 40 Show.
From the above results, it was found that it was perenisaponin Q represented by the structural formula (36).

Physical property measurement value and optical rotation of compound 37: [α] _D ²⁷ : −28.5 ° (c = 3.88, MeOH)
・ High resolution mass spectrometry:
Theoretical value C ₆₆ H ₁₀₆ O ₃₁ Na (M + Na) ⁺ : 1417.6616
Actual value: 1417.6611
Infrared absorption spectrum (KBr, cm ⁻¹ ): 3450, 1736, 1655, 1260, 1048
-Mass spectrometry positive-ion FAB-MS: m / z 1417 (M + Na) ⁺
negative-ion FAB-MS: m / z 1393 (M-H) ⁻ , 1247 (M-C ₆ H ₁₁ O ₄ ) ⁻ , 1115 (M-C ₁₁ H ₁₉ O ₈ ) ⁻ , 649 (M-C ₃₀ H ₄₉ O ₂₁ ) ⁻
Nuclear magnetic resonance spectrum: δ of ¹ H-NMR (600 MHz, pyridine-d ₅ ) in Table 41 and Table 42, δ _C of ¹³ C-NMR (150 MHz, pyridine-d ₅ ) in Table 43 and Table 44 Show.
From the above results, it was found that it was Perenisaponin R represented by Structural Formula (37).

Physical property measurement value and optical rotation of compound 38: [α] _D ²⁶ : + 10.8 ° (c = 1.85, MeOH)
・ High resolution mass spectrometry:
Theoretical value C ₆₆ H ₁₀₆ O ₃₁ Na (M + Na) ⁺ : 1417.6616
Actual value: 1417.6610
Infrared absorption spectrum (KBr, cm ⁻¹ ): 3445, 1736, 1655, 1251, 1048
-Mass spectrometry positive-ion FAB-MS: m / z 1417 (M + Na) ⁺
negative-ion FAB-MS: m / z 1393 (M-H) ⁻ , 1247 (M-C ₆ H ₁₁ O ₄ ) ⁻ , 1115 (M-C ₁₁ H ₁₉ O ₈ ) ⁻ , 649 (M-C ₃₀ H ₄₉ O ₂₁ ) ⁻
Nuclear magnetic resonance spectrum: δ of ¹ H-NMR (600 MHz, pyridine-d ₅ ) in Table 41 and Table 42, δ _C of ¹³ C-NMR (150 MHz, pyridine-d ₅ ) in Table 43 and Table 44 Show.
From the above results, it was found that it was perenisaponin S represented by the structural formula (38).

Measured physical properties and optical rotation of compound 39: [α] _D ²⁶ : −28.8 ° (c = 1.27, MeOH)
・ High resolution mass spectrometry:
Theoretical value C ₆₈ H ₁₀₈ O ₃₂ Na (M + Na) ⁺ : 1459.6721
Actual value: 145.97.624
Infrared absorption spectrum (KBr, cm ⁻¹ ): 3445, 1736, 1655, 1258, 1049
-Mass spectrometry positive-ion FAB-MS: m / z 1459 (M + Na) ⁺
negative-ion FAB-MS: m / z 1435 (M−H) ⁻ , 1393 (M−C ₂ H ₃ O) ⁻ , 1115 (M−C ₁₃ H ₂₁ O ₉ ) ⁻
Nuclear magnetic resonance spectrum: δ of ¹ H-NMR (600 MHz, pyridine-d ₅ ) in Table 41 and Table 42, δ _C of ¹³ C-NMR (150 MHz, pyridine-d ₅ ) in Table 43 and Table 44 Show.
From the above results, it was found that it was perenisaponin T represented by the structural formula (39).

Example 7 Inhibitory effect test 1 on olive oil-induced blood neutral lipid elevation using mice
As an index of the neutral lipid absorption inhibitory effect, Daisy's flower part extract extract was subjected to an inhibitory effect test on olive oil-induced blood neutral lipid elevation using mice. The test method is shown below.

  The following test substances were administered to ddY male rats (body weight about 25 to 30 g) fasted for 24 to 26 hours, and the administration dose shown in Table 45 (value in mg per 1 kg body weight of the rat. Mg). / Kg, po) was orally administered, and 30 minutes later, olive oil (5 mL per kg body weight of the rat) was orally administered. 2, 4, and 6 hours after the administration of olive oil, blood was collected from the orbital vein, and the amount of neutral lipid in the serum was measured using a commercially available kit “Triglyceride E Test Wako (manufactured by Wako Pure Chemical Industries)” (Yoshikawa M. et al. , J. Nat. Prod., 68, 1360-1365 (2005)).

[Test substance]
Daisy flower part: The daisy flower part is crushed.
MeOH extract: Methanol extract obtained in Example 1.
EtOAc transition: Ethyl acetate transition obtained in Example 2.
H ₂ O elution part: The water elution part obtained in Example 3.
MeOH elution part: Methanol elution part obtained in Example 3.
Clofibrate: Commercially available antihyperlipidemic agent (Reagent manufactured by Tocris Cookson)

  The results are shown in Table 45 and Table 46. In addition, the value which measured the amount of neutral lipids in serum similarly except not administering olive oil was shown in Table 45 and Table 46 as Control. Table 45 shows the values obtained by measuring the amount of neutral lipid in serum in the same manner except that olive oil administration and test substance administration were not performed.

  The values in the table (the amount of neutral lipid in the blood) are the mean (mean value) ± S. E. M.M. (*) Mark means 0.05%, and (**) mark means 0.01% and there is a significant difference.

  From the results of Table 45, it can be seen that the methanol extract of daisy flowers has a significant neutral lipid absorption inhibitory action in a suppressive action test against olive oil-induced blood neutral lipid elevation using mice. Was observed to be concentrated in the methanol elution part. The activity intensity of the methanol elution part was stronger than that of the commercially available antihyperlipidemic drug clofibrate.

Example 8 Inhibitory effect test 2 on olive oil-induced blood neutral lipid elevation using mice
Since the activity intensity of the methanol elution part was stronger than that of clofibrate, the following saponin compound (test substance) obtained from the methanol elution part was used in olive oil-induced blood using a mouse in the same manner as in Example 7. An inhibitory effect test on the increase in neutral lipid was performed. The results are shown in Table 46.

[Test substance]
Perenisoside I of Structural Formula (1): Compound 1 obtained in Example 5.
Perenisoside II of Structural Formula (2): Compound 2 obtained in Example 5.
Perenisoside XII of the structural formula (12): Compound 12 obtained in Example 6.
Berisoside E of the structural formula (42) obtained in Example 5.
Bernaldioside B ₂ of the structural formula (46) obtained in Example 5.
Berisaponin BS6 of the structural formula (47) obtained in Reference Example 5.

  From the results shown in Table 46 above, the new saponin compounds Perenisoside I (1) and Perensoside II (2) and the known saponin compound Berisoside E (42) found from the flower part of Daisy were found in olive oil-induced blood in mice. In the inhibitory action test on the increase in the neutral lipid, it was found to have a significant neutral lipid absorption inhibitory action.

Example 9 Lipase Inhibitory Action Test 1
A lipase inhibitory effect test was conducted on the extract from the daisy flower as an index of the neutral lipid absorption inhibitory action. The test method is shown below.

  As a substrate, 9 mL of 0.1 M Tris buffer (containing 0.1 M NaCl, pH 7.0) was added to a triolein-lecithin solution (80 mg triolein, 10 mg phosphatidylcholine and 5 mg sodium taurocholate), and sonicated for 10 minutes. Solution) 5 μL of the following test substance (sample solution) was added to 100 μL, and further 95 μL of Tris buffer was added to make the total volume 200 μL. After preheating at 37 ° C. for 3 minutes, 50 μL of lipase solution (EC 3.1.1.3 Type II, derived from porcine pancreas) was added and allowed to react for 30 minutes, and then placed in a boiling water bath for 2 minutes to stop the reaction. Separately, for each sample, 50 μL of Tris buffer was added instead of the lipase solution, and the same operation was performed as a blind test (Conc .: 0 μg / mL or 0 μM). The amount of oleic acid produced was measured by the NEFA C kit method (NEFA C-Test Wako), and the reduction rate (inhibition%) of the amount of oleic acid produced relative to the blind case was calculated. The results are shown in Table 47. In addition, the numerical value in a table | surface is shown by an average value (N = 2-4). The lipase was dissolved in Tris buffer and prepared to a concentration at which about 0.7 mmol / L oleic acid was produced under the experimental conditions. The test sample was dissolved and diluted with DMSO.

[Test substance]
MeOH extract: Methanol extract obtained in Example 1.
EtOAc transition: Ethyl acetate transition obtained in Example 2.
MeOH elution part: Methanol elution part obtained in Example 3.
H ₂ O elution part: The water elution part obtained in Example 3.

  From the results in Table 47 above, it can be seen that the methanol extract obtained from the daisy flower part has a lipase inhibitory action, and its activity is concentrated in the ethyl acetate transfer part and the methanol elution part. It was.

Example 10 Lipase inhibitory action test 2
Therefore, the following saponin compounds were used as test substances, and lipase inhibitory action tests were similarly conducted on these compounds. The results are shown in Table 48.

[Test substance]
Perenisoside I of Structural Formula (1): Compound 1 obtained in Example 5.
Perenisoside II of Structural Formula (2): Compound 2 obtained in Example 5.
Perenisoside XII of the structural formula (12): Compound 12 obtained in Example 6.
Perenisaponin G of the structural formula (26): Compound 26 obtained in Example 5.
Perenisaponin H of Structural Formula (27): Compound 27 obtained in Example 5.
Perenisaponin I of Structural Formula (28): Compound 28 obtained in Example 5.
Perenisaponin J of Structural Formula (29): Compound 29 obtained in Example 5.
Perenisaponin M of Structural Formula (32): Compound 32 obtained in Example 5.
Perenisaponin R of the structural formula (37): Compound 37 obtained in Example 6.
Berisoside E of the structural formula (42) obtained in Example 5.
Astelbatanoside D of structural formula (45) obtained in Example 6.
Bernaldioside B ₂ of the structural formula (46) obtained in Example 5.
Berisaponin BS6 of the structural formula (47) obtained in Reference Example 5.

  From the results of Table 48 above, the new saponins pereniside I (1) and II (2) perenisaponins G (26), H (27), I (28), M (32) and R ( 37) and the known saponin beryside E (42) was found to have a lipase inhibitory action.

Claims (12)

  Daisy flower part, daisy flower part extracted with water, a lower aliphatic alcohol or a water content of a lower aliphatic alcohol, or an extract obtained by concentrating the extract as an active ingredient A neutral lipid absorption inhibitor characterized by comprising. A saponin compound represented by the following structural formula (1):

A saponin compound represented by the following structural formula (2):

A saponin compound represented by the following structural formula (26):

A saponin compound represented by the following structural formula (27):

A saponin compound represented by the following structural formula (28):

A saponin compound represented by the following structural formula (29):

A saponin compound represented by the following structural formula (32):

A saponin compound represented by the following structural formula (37):

It contains at least one compound selected from the group consisting of the saponin compound according to any one of claims 2 to 9 and the saponin compound represented by the following structural formula (42) as an active ingredient. A neutral lipid absorption inhibitor.

[Wherein, R ¹ represents a substituent represented by the following formula II. ]

  A human or veterinary pharmaceutical comprising the neutral lipid absorption inhibitor according to claim 1 or 10.   A food comprising the neutral lipid absorption inhibitor according to claim 1 or 10.